U.S. patent application number 16/850866 was filed with the patent office on 2020-10-29 for glucocorticoid receptor agonist and immunoconjugates thereof.
This patent application is currently assigned to AbbVie Inc.. The applicant listed for this patent is AbbVie Inc.. Invention is credited to Christian Goess, Martin E. Hayes, Axel Hernandez, JR., Adrian D. Hobson, Christopher C. Marvin, Michael J. McPherson, Jason Z. Oh, John T. Randolph, Diana Schmidt, Wendy Waegell.
Application Number | 20200338208 16/850866 |
Document ID | / |
Family ID | 1000004957419 |
Filed Date | 2020-10-29 |
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United States Patent
Application |
20200338208 |
Kind Code |
A1 |
McPherson; Michael J. ; et
al. |
October 29, 2020 |
GLUCOCORTICOID RECEPTOR AGONIST AND IMMUNOCONJUGATES THEREOF
Abstract
Provided herein are glucocorticoid receptor agonist
immunoconjugates, glucocorticoid receptor agonists, and methods of
using the same, e.g., to treat autoimmune or inflammatory
diseases.
Inventors: |
McPherson; Michael J.;
(Ashby, MA) ; Hobson; Adrian D.; (Shrewsbury,
MA) ; Hayes; Martin E.; (Pepperell, MA) ;
Marvin; Christopher C.; (Grayslake, IL) ; Schmidt;
Diana; (Antioch, IL) ; Waegell; Wendy;
(Brookfield, MA) ; Goess; Christian; (Sturbridge,
MA) ; Oh; Jason Z.; (Shrewsbury, MA) ;
Hernandez, JR.; Axel; (Charlton, MA) ; Randolph; John
T.; (Libertyville, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie Inc. |
North Chicago |
IL |
US |
|
|
Assignee: |
AbbVie Inc.
North Chicago
IL
|
Family ID: |
1000004957419 |
Appl. No.: |
16/850866 |
Filed: |
April 16, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16408602 |
May 10, 2019 |
10668167 |
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16850866 |
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15611037 |
Jun 1, 2017 |
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16408602 |
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62371134 |
Aug 4, 2016 |
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62344948 |
Jun 2, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/73 20130101;
A61K 47/6803 20170801; C07K 2318/20 20130101; C07K 2317/31
20130101; A61K 2039/505 20130101; C07K 16/241 20130101; C07K
2319/30 20130101; A61P 37/06 20180101; A61K 47/6849 20170801; C07K
2317/21 20130101; C07K 2317/55 20130101; A61K 47/6889 20170801;
C07K 2317/24 20130101; C07K 16/18 20130101; A61K 47/6845 20170801;
C07K 2317/569 20130101; C07J 71/0031 20130101; C07K 2317/92
20130101 |
International
Class: |
A61K 47/68 20060101
A61K047/68; C07K 16/24 20060101 C07K016/24; A61P 37/06 20060101
A61P037/06; C07J 71/00 20060101 C07J071/00 |
Claims
1-23. (canceled)
24. A compound having Formula VII: ##STR01057## or a
pharmaceutically acceptable salt or solvate thereof, wherein:
R.sup.1 is selected from the group consisting of hydrogen and halo;
R.sup.2 is selected from the group consisting of hydrogen, halo,
and hydroxy; R.sup.3 is selected from the group consisting of
--CH.sub.2OH, --CH.sub.2SH, --CH.sub.2Cl, --SCH.sub.2Cl,
--SCH.sub.2F, --SCH.sub.2CF.sub.3, --CH.sub.2OS(.dbd.O).sub.2OH,
hydroxy, --OCH.sub.2CN, --OCH.sub.2Cl, --OCH.sub.2F, --OCH.sub.3,
--OCH.sub.2CH.sub.3, --SCH.sub.2CN, ##STR01058## R.sup.3a is
selected from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.3b is selected from the group consisting of C.sub.1-4 alkyl
and C.sub.1-4 alkoxy; R.sup.3c is selected from the group
consisting of hydrogen, C.sub.1-4 alkyl, --CH.sub.2OH, C.sub.1-4
alkoxy, --CH.sub.2(amino), and
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.3f; R.sup.3d and R.sup.3e are
independently selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.3f is selected from the group consisting of
hydrogen and C.sub.1-4 alkyl; X is selected from the group
consisting of --(CR.sup.4aR.sup.4b)--, --O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--, --CH.sub.2O--,
--N(H)C(R.sup.8a)(R.sup.8b)--, --CR.sup.4c--CR.sup.4d--,
--C.ident.C--, --N(R.sup.5)C(.dbd.O)--, and --OC(.dbd.O)--; or X is
absent; t is 1 or 2; Z is selected from the group consisting of
.dbd.CR.sup.11a-- and .dbd.N--; each R.sup.4a and R.sup.4b are
independently selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; or R.sup.4a and R.sup.4b taken together with the
carbon atom to which they are attached form a 3- to 6-membered
cycloalkyl; R.sup.4c and R.sup.4d are independently selected from
the group consisting of hydrogen and C.sub.1-4 alkyl; R.sup.5 is
selected from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.6a, R.sup.6b, R.sup.6c, and R.sup.6d are each independently
selected from the group consisting of hydrogen, halo, C.sub.1-4
alkyl, haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and
alkoxy; R.sup.7a is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl; R.sup.7b is selected from the group consisting
of hydrogen, -L-H, -L-PG, ##STR01059## or R.sup.7a and R.sup.7b
taken together with the nitrogen atom to which they are attached
form: or ##STR01060## R.sup.7a and R.sup.7b taken together with the
nitrogen atom to which they are attached form a nitro group; m is
1, 2, 3, 4, 5, or 6; L is a linker; PG is a protecting group;
R.sup.9f is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.8a and R.sup.8b are independently selected
from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.11a and R.sup.11b are independently selected from the group
consisting of hydrogen, halo, C.sub.1-4 alkyl, C.sub.1-4haloalkyl,
cyano, hydroxy, thiol, amino, alkylthio, and alkoxy; and represents
a single or double bond.
25. A compound having Formula VII-A or Formula VII-B: ##STR01061##
or a pharmaceutically acceptable salt or solvate thereof, wherein:
R.sup.1 is selected from the group consisting of hydrogen and halo;
R.sup.2 is selected from the group consisting of hydrogen, halo,
and hydroxy; R.sup.3 is selected from the group consisting of
--CH.sub.2OH, --CH.sub.2SH, --CH.sub.2Cl, --SCH.sub.2Cl,
--SCH.sub.2F, --SCH.sub.2CF.sub.3, --CH.sub.2OS(.dbd.O).sub.2OH,
hydroxy, --OCH.sub.2CN, --OCH.sub.2Cl, --OCH.sub.2F, --OCH.sub.3,
--OCH.sub.2CH, --SCH.sub.2CN, ##STR01062## R.sup.3a is selected
from the group consisting of hydrogen and C.sub.1-4 alkyl; R.sup.3b
is selected from the group consisting of C.sub.1-4 alkyl and
C.sub.1-4 alkoxy; R.sup.3c is selected from the group consisting of
hydrogen, C.sub.1-4 alkyl, --CH.sub.2OH, C.sub.1-4 alkoxy,
--CH.sub.2(amino), and --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.3f;
R.sup.3d and R.sup.3e are independently selected from the group
consisting of hydrogen and C.sub.1-4 alkyl; R.sup.3f is selected
from the group consisting of hydrogen and C.sub.1-4 alkyl; X is
selected from the group consisting of
--(CR.sup.4aR.sup.4b).sub.t--, --O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--, --CH.sub.2O--,
--N(H)C(R.sup.8a)(R.sup.8b)--, --CR.sup.4c.dbd.CR.sup.4d--,
--C.ident.C--, --N(R.sup.5)C(.dbd.O)--, and --OC(.dbd.O)--; or X is
absent; t is 1 or 2; Z is selected from the group consisting of
.dbd.CR.sup.11a-- and .dbd.N--; each R.sup.4a and R.sup.4b are
independently selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; or R.sup.4a and R.sup.4b taken together with the
carbon atom to which they are attached form a 3- to 6-membered
cycloalkyl; R.sup.4c and R.sup.4d are independently selected from
the group consisting of hydrogen and C.sub.1-4 alkyl; R.sup.5 is
selected from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.6a, R.sup.6b, and R.sup.6c are each independently selected
from the group consisting of hydrogen, halo, C.sub.1-4 alkyl,
haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and alkoxy;
R.sup.7a is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.7b is selected from the group consisting of
hydrogen, -L-H, -L-PG, ##STR01063## or R.sup.7a and R.sup.7b taken
together with the nitrogen atom to which they are attached form:
##STR01064## or R.sup.7a and R.sup.7b taken together with the
nitrogen atom to which they are attached form a nitro group; m is
1, 2, 3, 4, 5, or 6; L is a linker; PG is a protecting group;
R.sup.9f is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.8a and R.sup.8b are independently selected
from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.11a and R.sup.11b are independently selected from the group
consisting of hydrogen, halo, C.sub.1-4 alkyl, C.sub.1-4haloalkyl,
cyano, hydroxy, thiol, amino, alkylthio, and alkoxy; and represents
a single or double bond.
26. The compound of claim 24 or 25, or a pharmaceutically
acceptable salt or solvate thereof, wherein: R.sup.7b is selected
from the group consisting of: ##STR01065## m is 1, 2, 3, 4, 5, or
6; and R.sup.10a and R.sup.10b are each independently selected from
the group consisting of hydrogen and optionally substituted
C.sub.1-6 alkyl.
27. The compound of claim 24 or 26, or a pharmaceutically
acceptable salt or solvate thereof, having Formula VIII-a:
##STR01066##
28. The compound of any one of claims 24-27, or a pharmaceutically
acceptable salt or solvate thereof, wherein: represents a double
bond; R.sup.1 is selected from the group consisting of hydrogen and
fluoro; R.sup.2 is selected from the group consisting of hydrogen
and fluoro; R.sup.3 is selected from the group consisting of
--CH.sub.2OH, --CH.sub.2Cl, --SCH.sub.2Cl, --SCH.sub.2F, and
##STR01067## R.sup.3d and R.sup.3e are independently selected from
the group consisting of hydrogen, methyl, and ethyl; Z is
.dbd.CH--; R.sup.6a, R.sup.6b, R.sup.6c, and R.sup.6d are hydrogen;
R.sup.7a is hydrogen; X is selected from the group consisting of
--CH.sub.2--, --O--, --S--, --S(.dbd.O)--, --S(.dbd.O).sub.2--,
--CH.sub.2S--, and --N(H)CH.sub.2--; R.sup.9f is hydrogen; and
R.sup.11b is hydrogen.
29. The compound of any one of claims 24-28, or a pharmaceutically
acceptable salt or solvate thereof, wherein R.sup.7b is
hydrogen.
30. The compound of any one of claims 24-28, or a pharmaceutically
acceptable salt or solvate thereof, wherein R.sup.7b is
R.sup.7b-1.
31. The compound of any one of claims 24-28, or a pharmaceutically
acceptable salt or solvate thereof, wherein R.sup.7b is R.sup.7b-2,
and PG is BOC.
32. The compound of any one of claims 24-28, or a pharmaceutically
acceptable salt or solvate thereof, wherein R.sup.7b is
R.sup.7b-3.
33. The compound of claim 29, or a pharmaceutically acceptable salt
or solvate thereof, which is any one or more of the compounds of
Table VI.
34. The compound of claim 29, or a pharmaceutically acceptable salt
or solvate thereof, which is any one of the compounds of Table
VII.
35. The compound of claim 33, or a pharmaceutically acceptable salt
or solvate thereof, which is: ##STR01068##
36. The compound of claim 24, or a pharmaceutically acceptable salt
or solvate thereof, which is any one or more of the compounds of
Table VIII, wherein R.sup.7b is selected from the group consisting
of: ##STR01069##
37. The compound of claim 24, or a pharmaceutically acceptable salt
or solvate thereof, which is any one or more of the compounds of
Table X.
38. The compound of claim 37, or a pharmaceutically acceptable salt
or solvate thereof, which is: ##STR01070##
39-40. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/344,948, filed Jun. 2, 2016, and U.S.
Provisional Application No. 62/371,134, filed Aug. 4, 2016, each of
which is herein incorporated by reference in its entirety.
REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA
EFS-WEB
[0002] The content of the electronically submitted sequence listing
(Name: 3685.0100002_SeqListing_ST25.txt, Size: 95,162 bytes; and
Date of Creation: Jun. 1, 2017), filed with the application is
herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The field of the invention generally relates to
glucocorticoid receptor agonist immunoconjugates, and methods of
making and using the same, e.g., to treat autoimmune or
inflammatory diseases.
BACKGROUND OF THE INVENTION
[0004] Tumor Necrosis Factor alpha (TNFa) plays a central role in
the pathophysiology of several human disorders, and anti-TNFa
agents (e.g., adalimumab, etanercept, and infliximab) have
clinically validated therapeutic utility in the treatment of
autoimmune and inflammatory disorders, such as rheumatoid
arthritis, psoriasis and inflammatory bowel disease. Despite their
success in the clinic, anti-TNFa biologics are still limited in the
maximal efficacy they can achieve in patients, necessitating the
identification and development of more potent and effective
therapeutics. Patients treated with anti-TNFa biologics may also
develop an immunogenic response to the therapeutic thus limiting
its effectiveness. Therefore anti-TNFa therapies with lower
immunogenicity and high efficacy would be useful for further
controlling disease.
[0005] Synthetic glucocorticoid receptor agonists (e.g.,
dexamethasone, prednisolone, and budesonide) are a potent class of
small molecules used in the treatment of inflammatory disorders,
but their utility in the chronic treatment of disease is limited
due to severe side effects. Several approaches to retain the
anti-inflammatory efficacy of synthetic glucocorticoids while
sparing the unwanted toxicities have been described (Rosen, J and
Miner, J N Endocrine Reviews 26: 452-64 (2005)). However these
methodologies have met with little success. There is a need in the
field of autoimmune and inflammatory disease therapeutics to
develop therapeutics with enhanced efficacy and longer duration of
action compared to anti-TNF antibodies and with minimal unwanted
effects.
BRIEF SUMMARY OF THE INVENTION
[0006] In one aspect, the present disclosure provides a
glucocorticoid receptor agonist immunoconjugate represented by
Formulae I-a and I-b, below, and the pharmaceutically acceptable
salts, solvates, or prodrugs thereof. In another aspect, the
present disclosure provides a glucocorticoid receptor agonist
immunoconjugate represented by Formulae I-a and I-b, below.
Glucocorticoid receptor agonist immunoconjugates having Formulae
I-a and I-b are useful for treating autoimmune diseases such as,
but not limited to, rheumatoid arthritis, juvenile idiopathic
arthritis, psoriatic arthritis, ankylosing spondylitis, adult
Crohn's disease, pediatric Crohn's disease, ulcerative colitis,
plaque psoriasis, hidradenitis suppurativa, uveitis, Behcets
disease, a spondyloarthropathy, or psoriasis. In one aspect,
glucocorticoid receptor agonist immunoconjugates having Formulae
I-a and I-b are useful for treating rheumatoid arthritis. In one
aspect, glucocorticoid receptor agonist immunoconjugates having
Formulae I-a and I-b are useful for treating juvenile idiopathic
arthritis. In one aspect, glucocorticoid receptor agonist
immunoconjugates having Formulae I-a and I-b are useful for
treating psoriatic arthritis. In one aspect, glucocorticoid
receptor agonist immunoconjugates having Formulae I-a and I-b are
useful for treating ankylosing spondylitis. In one aspect,
glucocorticoid receptor agonist immunoconjugates having Formulae
I-a and I-b are useful for treating adult Crohn's disease. In one
aspect, glucocorticoid receptor agonist immunoconjugates having
Formulae I-a and I-b are useful for treating pediatric Crohn's
disease. In one aspect, glucocorticoid receptor agonist
immunoconjugates having Formulae I-a and I-b are useful for
treating ulcerative colitis. In one aspect, glucocorticoid receptor
agonist immunoconjugates having Formulae I-a and I-b are useful for
treating plaque psoriasis. In one aspect, glucocorticoid receptor
agonist immunoconjugates having Formulae I-a and I-b are useful for
treating hidradenitis suppurativa. In one aspect, glucocorticoid
receptor agonist immunoconjugates having Formulae I-a and I-b are
useful for treating uveitis. In one aspect, glucocorticoid receptor
agonist immunoconjugates having Formulae I-a and I-b are useful for
treating Behcets disease. In one aspect, glucocorticoid receptor
agonist immunoconjugates having Formulae I-a and I-b are useful for
treating a spondyloarthropathy. In one aspect, glucocorticoid
receptor agonist immunoconjugates having Formulae I-a and I-b are
useful for treating psoriasis.
[0007] In another aspect, the present disclosure provides a
glucocorticoid receptor agonist represented by Formulae VII, VII-A,
VII-B, VIII, VIII-a, VIII-b, IX, IX-a, and IX-b, or by Formulae
VII', VII-A', VII-B', VIII', VIII-a', VIII-b', IX', IX-a', IX-b',
VII'', VII-A'', VII-B'', VIII'', VIII-a'', VIII-b'', IX'', IX-a'',
and IX-b'', below, (wherein R.sup.7b is hydrogen) and the
pharmaceutically acceptable salts, solvates, or prodrugs thereof.
In another aspect, the present disclosure provides a glucocorticoid
receptor agonist represented by Formulae VII, VII-A, VII-B, VIII,
VIII-a, VIII-b, IX, IX-a, and IX-b, or by Formulae VII', VII-A',
VII-B', VIII', VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'',
VII-B'', VIII'', VIII-a'', VIII-b'', IX'', IX-a'', and IX-b'',
below, (wherein R.sup.7b is hydrogen). Compounds having Formulae
VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX, IX-a, and IX-b, or
Formulae VII', VII-A', VII-B', VIII', VIII-a', VIII-b', IX', IX-a',
IX-b', VII'', VII-A'', VII-B'', VIII'', VIII-a'', VIII-b'', IX'',
IX-a'', and IX-b'', are useful for treating autoimmune diseases
such as, but not limited to, rheumatoid arthritis, juvenile
idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis,
adult Crohn's disease, pediatric Crohn's disease, ulcerative
colitis, plaque psoriasis, hidradenitis suppurativa, uveitis,
Behcets disease, a spondyloarthropathy, or psoriasis. In one
aspect, compounds having Formulae VII, VII-A, VII-B, VIII, VIII-a,
VIII-b, IX, IX-a, and IX-b, or Formulae VII', VII-A', VII-B',
VIII', VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'',
VII-B'', VIII'', VIII-a'', VIII-b'', IX'', IX-a'', and IX-b'', are
useful for treating rheumatoid arthritis. In one aspect, compounds
having Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX, IX-a,
and IX-b, or Formulae VII', VII-A', VII-B', VIII', VIII-a',
VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'', VIII'',
VIII-a'', VIII-b'', IX'', IX-a'', and IX-b'', are useful for
treating juvenile idiopathic arthritis. In one aspect, compounds
having Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX, IX-a,
and IX-b, or Formulae VII', VII-A', VII-B', VIII', VIII-a',
VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'', VIII'',
VIII-a'', VIII-b'', IX'', IX-a'', and IX-b'', are useful for
treating psoriatic arthritis. In one aspect, compounds having
Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX, IX-a, and
IX-b, or Formulae VII', VII-A', VII-B', VIII', VIII-a', VIII-b',
IX', IX-a', IX-b', VII'', VII-A'', VII-B'', VIII'', VIII-a'',
VIII-b'', IX'', IX-a'', and IX-b'', are useful for treating
ankylosing spondylitis. In one aspect, compounds having Formulae
VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX, IX-a, and IX-b, or
Formulae VII', VII-A', VII-B', VIII', VIII-a', VIII-b', IX', IX-a',
IX-b', VII'', VII-A'', VII-B'', VIII'', VIII-a'', VIII-b'', IX'',
IX-a'', and IX-b'', are useful for treating adult Crohn's disease.
In one aspect, compounds having Formulae VII, VII-A, VII-B, VIII,
VIII-a, VIII-b, IX, IX-a, and IX-b, or Formulae VII', VII-A',
VII-B', VIII', VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'',
VII-B'', VIII'', VIII-a'', VIII-b'', IX'', IX-a'', and IX-b'', are
useful for treating pediatric Crohn's disease. In one aspect,
compounds having Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b,
IX, IX-a, and IX-b, or Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', and IX-b'', are useful
for treating ulcerative colitis. In one aspect, compounds having
Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX, IX-a, and
IX-b, or Formulae VII', VII-A', VII-B', VIII', VIII-a', VIII-b',
IX', IX-a', IX-b', VII'', VII-A'', VII-B'', VIII'', VIII-a'',
VIII-b'', IX'', IX-a'', and IX-b'', are useful for treating plaque
psoriasis. In one aspect, compounds having Formulae VII, VII-A,
VII-B, VIII, VIII-a, VIII-b, IX, IX-a, and IX-b, or Formulae VII',
VII-A', VII-B', VIII', VIII-a', VIII-b', IX', IX-a', IX-b', VII'',
VII-A'', VII-B'', VIII'', VIII-a'', VIII-b'', IX'', IX-a'', and
IX-b'', are useful for treating hidradenitis suppurativa. In one
aspect, compounds having Formulae VII, VII-A, VII-B, VIII, VIII-a,
VIII-b, IX, IX-a, and IX-b, or Formulae VII', VII-A', VII-B',
VIII', VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'',
VII-B'', VIII'', VIII-a'', VIII-b'', IX'', IX-a'', and IX-b'', are
useful for treating uveitis. In one aspect, compounds having
Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX, IX-a, and
IX-b, or Formulae VII', VII-A', VII-B', VIII', VIII-a', VIII-b',
IX', IX-a', IX-b', VII'', VII-A'', VII-B'', VIII'', VIII-a'',
VIII-b'', IX'', IX-a'', and IX-b'', are useful for treating Behcets
disease. In one aspect, compounds having Formulae VII, VII-A,
VII-B, VIII, VIII-a, VIII-b, IX, IX-a, and IX-b, or Formulae VII',
VII-A', VII-B', VIII', VIII-a', VIII-b', IX', IX-a', IX-b', VII'',
VII-A'', VII-B'', VIII'', VIII-a'', VIII-b'', IX'', IX-a'', and
IX-b'', are useful for treating a spondyloarthropathy. In one
aspect, compounds having Formulae VII, VII-A, VII-B, VIII, VIII-a,
VIII-b, IX, IX-a, and IX-b, or Formulae VII', VII-A', VII-B',
VIII', VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'',
VII-B'', VIII'', VIII-a'', VIII-b'', IX'', IX-a'', and IX-b'', are
useful for treating psoriasis.
[0008] In another aspect, the present disclosure provides compounds
represented by Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b,
IX, IX-a, and IX-b, or by Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', and IX-b'', as synthetic
intermediates that can be used to prepare glucocorticoid receptor
agonist immunoconjugates having Formulae I-a and I-b.
[0009] In another aspect, the present disclosure provides a
pharmaceutical composition comprising a glucocorticoid receptor
agonist immunoconjugate represented by Formulae I-a and I-b, or a
glucocorticoid receptor agonist represented by Formulae VII, VII-A,
VII-B, VIII, VIII-a, VIII-b, IX, IX-a, and IX-b, or by Formulae
VII', VII-A', VII-B', VIII', VIII-a', VIII-b', IX', IX-a', IX-b',
VII'', VII-A'', VII-B'', VIII'', VIII-a'', VIII-b'', IX'', IX-a'',
and IX-b'', and an excipient and/or a pharmaceutically acceptable
carrier.
[0010] In another aspect, the present disclosure provides a
glucocorticoid receptor agonist immunoconjugate represented by
Formulae I-a and I-b, or a glucocorticoid receptor agonist
represented by Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b,
IX, IX-a, and IX-b or by Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', and IX-b'', for use in
treatment of autoimmune diseases.
[0011] In another aspect, the present disclosure provides a use of
a glucocorticoid receptor agonist immunoconjugates represented by
Formulae I-a and I-b, or a glucocorticoid receptor agonist
represented by Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b,
IX, IX-a, and IX-b, or by Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', and IX-b'', for the
manufacture of a medicament for treating autoimmune diseases.
[0012] In another aspect, the present disclosure provides methods
of preparing glucocorticoid receptor agonist immunoconjugates
represented by Formulae I-a and I-b.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0013] FIG. 1 shows the proteolytic stability of an ADC containing
a steroid and an ADC containing MMAE (monomethyl auristatin E).
(See Example 76.)
[0014] FIG. 2 shows the kinetics of drug linker loss of steroid ADC
in mice. (See Example 77.)
[0015] FIG. 3 shows the activity of a single therapeutic dose
response of anti-mTNFa steroid ADC in a mouse model of arthritis.
(See Example 85.)
[0016] FIG. 4 shows the activity of anti-human TNFa steroid in
huTNFa Tg CAIA mouse model of arthritis. (See Example 87.)
[0017] FIG. 5 is a HIC chromatogram showing a heterogenous mixture
containing antibodies having zero SM-L-Q- molecules attached ("E0"
peak), two SM-L-Q- molecules attached ("E2" peak), four SM-L-Q-
molecules attached ("E4" peak), SM-L-Q- moieties attached ("E6"
peak), and eight SM-L-Q-molecules attached ("E8" peak), depending
upon the number of interchain disulfide bonds that have been
reduced. (SM is a radical of a glucocorticosteroid; L is a linker,
and Q is a heterobifunctional group or heterotrifunctional group;
or Q is absent.) (See Example 74.)
[0018] FIG. 6 is a SEC chromatogram of adalimumab conjugated with a
glucocorticosteroid. (See Example 74.)
[0019] FIG. 7 is a line graph showing raw MS data of adalimumab
conjugated with a glucocorticosteroid. (See Example 74.)
[0020] FIG. 8 is a line graph showing deconvoluted MS data of
adalimumab conjugated with a glucocorticosteroid. Black square and
circle represent the ADC with succinimide hydrolyzed and
unhydrolyzed, respectively. The relative abundance of hydrolyzed
and unhydrolyzed ADC is used to determine hydrolysis conversion.
(See Example 74.)
[0021] FIG. 9 shows that an anti-TNF steroid ADC is significantly
more effective in reducing ear inflammation in mice than the
concurrent combination of the anti-TNF antibody and the steroid or
than the anti-TNF antibody alone. (See Example 84.)
[0022] FIG. 10 shows that a single dose of an anti-TNF steroid ADC
is as effective in reducing paw swelling as 21 days of daily dosing
of a steroid. (See Example 85.)
[0023] FIG. 11 shows the change in weights of animals treated with
steroid, an anti-TNF antibody, an anti-TNF ADC, or an isotype ADC.
(See Example 85.)
[0024] FIG. 12 shows that a single dose of an anti-TNF steroid ADC
can reduce established paw swelling, whereas a single dose of an
anti-TNF antibody had a minimal effect. (See Example 88.)
[0025] FIG. 13 shows the effect of treatment with an anti-TNF
steroid ADC on tarsal bone loss as measured by Micro-Computed
Tomography (.mu.CT). (The individual data points (e.g., circles,
squares, or triangles) represent individual animals.) (See Example
88.)
[0026] FIG. 14 shows the effect of treatment with an anti-TNF
steroid ADC on inflammation. (The individual data points (e.g.,
circles, squares, or triangles) represent individual animals.) (See
Example 88.)
[0027] FIG. 15 shows the effect of treatment with an anti-TNF
steroid ADC on pannus formation. (The individual data points (e.g.,
circles, squares, or triangles) represent individual animals.) (See
Example 88.)
[0028] FIG. 16 shows the effect of treatment with an anti-TNF
steroid ADC on bone erosion. (The individual data points (e.g.,
circles, squares, or triangles) represent individual animals.) (See
Example 88.)
[0029] FIG. 17 shows the effect of treatment with an anti-TNF
steroid ADC on cartilage damage. (The individual data points (e.g.,
circles, squares, or triangles) represent individual animals.) (See
Example 88.)
[0030] FIG. 18 shows effect of treatment with an anti-TNF steroid
ADC on white blood cells in peripheral blood. (The individual data
points (e.g., circles, squares, or diamonds) represent individual
animals.) (See Example 88.)
[0031] FIG. 19 shows effect of treatment with an anti-TNF steroid
ADC on neutrophils in peripheral blood. (The individual data points
(e.g., circles, squares, or diamonds) represent individual
animals.) (See Example 88.)
[0032] FIG. 20 shows effect of treatment with an anti-TNF steroid
ADC on lymphocytes in peripheral blood. (The individual data points
(e.g., circles, squares, or diamonds) represent individual
animals.) (See Example 88.)
[0033] FIG. 21 shows effect of treatment with an anti-TNF steroid
ADC on monocytes in peripheral blood. (The individual data points
(e.g., circles, squares, or diamonds) represent individual
animals.) (See Example 88.)
[0034] FIG. 22 shows effect of treatment with an anti-TNF steroid
ADC on eosinophils in peripheral blood. (See Example 88.)
[0035] FIG. 23 shows effect of treatment with an anti-TNF steroid
ADC on basophils in peripheral blood. (See Example 88.)
[0036] FIG. 24 shows the activity of an anti-TNF steroid ADC and an
anti-CD163 steroid ADC in mouse collagen-induced arthritis. (See
Example 89.)
DETAILED DESCRIPTION OF THE INVENTION
[0037] Provided herein are glucocorticoid receptor agonist
immunoconjugates, glucocorticoid receptor agonists, and methods of
making and using the same.
I. Definitions
[0038] To facilitate an understanding of the present disclosure, a
number of terms and phrases are defined below.
[0039] The term "anti-TNF alpha protein" refers to proteins that
are capable of (i) binding to TNF alpha and (ii) inhibiting binding
of soluble TNF-alpha to cell surface TNF receptors (p55 and/or p75)
and/or lysing surface TNF alpha or TNF alpha receptor expressing
cells in vitro in the presence of complement. Anti-TNF alpha
proteins include, for example, anti-TNF antibodies or
antigen-binding fragments thereof (e.g., adalimumab or infliximab)
as well as soluble TNF receptors (e.g., etanercept).
[0040] As used herein, the terms "antibody" and "antibodies" are
terms of art and can be used interchangeably herein and refer to a
molecule with an antigen-binding site that specifically binds an
antigen.
[0041] The term "antibody" means an immunoglobulin molecule that
recognizes and specifically binds to a target, such as a protein,
polypeptide, peptide, carbohydrate, polynucleotide, lipid, or
combinations of the foregoing through at least one antigen
recognition site within the variable region of the immunoglobulin
molecule. As used herein, the term "antibody" encompasses intact
polyclonal antibodies, intact monoclonal antibodies, chimeric
antibodies, humanized antibodies, human antibodies, fusion proteins
comprising an antibody, and any other modified immunoglobulin
molecule so long as the antibodies exhibit the desired biological
activity. An antibody can be of any the five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses
(isotypes) thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2),
based on the identity of their heavy-chain constant domains
referred to as alpha, delta, epsilon, gamma, and mu, respectively.
The different classes of immunoglobulins have different and well
known subunit structures and three-dimensional configurations.
Antibodies can be naked or conjugated to other molecules such as
toxins, radioisotopes, etc. As used herein, the term "antibody"
encompasses bispecific and multispecific antibodies.
[0042] The term "antibody fragment" refers to a portion of an
intact antibody. An "antigen-binding fragment" refers to a portion
of an intact antibody that binds to an antigen. An antigen-binding
fragment can contain the antigenic determining variable regions of
an intact antibody. Examples of antibody fragments include, but are
not limited to Fab, Fab', F(ab')2, and Fv fragments, linear
antibodies, and single chain antibodies. An "antigen-binding
fragment" can be a bispecific or multispecific antigen-binding
fragment.
[0043] A "blocking" antibody or an "antagonist" antibody is one
which inhibits or reduces biological activity of the antigen it
binds, such as TNF-alpha. In some embodiments, blocking antibodies
or antagonist antibodies substantially or completely inhibit the
biological activity of the antigen. The biological activity can be
reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even
100%.
[0044] The term "anti-TNF-alpha antibody" or "an antibody that
binds to TNF-alpha" refers to an antibody that is capable of
binding TNF-alpha with sufficient affinity such that the antibody
is useful as a diagnostic and/or therapeutic agent in targeting
TNF-alpha. The extent of binding of an anti-TNF-alpha antibody to
an unrelated, non-TNF-alpha protein can be less than about 10% of
the binding of the antibody to TNF-alpha as measured, e.g., by a
radioimmunoassay (RIA). In certain embodiments, an antibody that
binds to TNF-alpha has a dissociation constant (Kd) of .ltoreq.1 M,
.ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, or .ltoreq.0.1 nM.
[0045] A "monoclonal" antibody or antigen-binding fragment thereof
refers to a homogeneous antibody or antigen-binding fragment
population involved in the highly specific recognition and binding
of a single antigenic determinant, or epitope. This is in contrast
to polyclonal antibodies that typically include different
antibodies directed against different antigenic determinants. The
term "monoclonal" antibody or antigen-binding fragment thereof
encompasses both intact and full-length monoclonal antibodies as
well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single
chain (scFv) mutants, fusion proteins comprising an antibody
portion, and any other modified immunoglobulin molecule comprising
an antigen recognition site. Furthermore, "monoclonal" antibody or
antigen-binding fragment thereof refers to such antibodies and
antigen-binding fragments thereof made in any number of manners
including but not limited to by hybridoma, phage selection,
recombinant expression, and transgenic animals.
[0046] The term "humanized" antibody or antigen-binding fragment
thereof refers to forms of non-human (e.g. murine) antibodies or
antigen-binding fragments that are specific immunoglobulin chains,
chimeric immunoglobulins, or fragments thereof that contain minimal
non-human (e.g., murine) sequences. Typically, humanized antibodies
or antigen-binding fragments thereof are human immunoglobulins in
which residues from the complementary determining region (CDR) are
replaced by residues from the CDR of a non-human species (e.g.
mouse, rat, rabbit, hamster) that have the desired specificity,
affinity, and capability ("CDR grafted") (Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);
Verhoeyen et al., Science 239:1534-1536 (1988)). In some instances,
the Fv framework region (FR) residues of a human immunoglobulin are
replaced with the corresponding residues in an antibody or fragment
from a non-human species that has the desired specificity,
affinity, and capability. The humanized antibody or antigen-binding
fragment thereof can be further modified by the substitution of
additional residues either in the Fv framework region and/or within
the replaced non-human residues to refine and optimize antibody or
antigen-binding fragment thereof specificity, affinity, and/or
capability. In general, the humanized antibody or antigen-binding
fragment thereof will comprise substantially all of at least one,
and typically two or three, variable domains containing all or
substantially all of the CDR regions that correspond to the
non-human immunoglobulin whereas all or substantially all of the FR
regions are those of a human immunoglobulin consensus sequence. The
humanized antibody or antigen-binding fragment thereof can also
comprise at least a portion of an immunoglobulin constant region or
domain (Fc), typically that of a human immunoglobulin. Examples of
methods used to generate humanized antibodies are described in U.S.
Pat. No. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA,
91(3):969-973 (1994), and Roguska et al., Protein Eng.
9(10):895-904 (1996). In some embodiments, a "humanized antibody"
is a resurfaced antibody.
[0047] A "variable region" of an antibody refers to the variable
region of the antibody light chain or the variable region of the
antibody heavy chain, either alone or in combination. The variable
regions of the heavy and light chain each consist of four framework
regions (FR) connected by three complementarity determining regions
(CDRs) also known as hypervariable regions. The CDRs in each chain
are held together in close proximity by the FRs and, with the CDRs
from the other chain, contribute to the formation of the
antigen-binding site of antibodies. There are at least two
techniques for determining CDRs: (1) an approach based on
cross-species sequence variability (i.e., Kabat et al. Sequences of
Proteins of Immunological Interest, (5th ed., 1991, National
Institutes of Health, Bethesda Md.)); and (2) an approach based on
crystallographic studies of antigen-antibody complexes (Al-lazikani
et al (1997) J. Molec. Biol. 273:927-948)). In addition,
combinations of these two approaches are sometimes used in the art
to determine CDRs.
[0048] The Kabat numbering system is generally used when referring
to a residue in the variable domain (approximately residues 1-107
of the light chain and residues 1-113 of the heavy chain) (e.g.,
Kabat et al., Sequences of Immunological Interest. 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)). Unless explicitly indicated otherwise, the numbering
system used herein is the Kabat numbering system.
[0049] The amino acid position numbering as in Kabat, refers to the
numbering system used for heavy chain variable domains or light
chain variable domains of the compilation of antibodies in Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991). Using this numbering system, the actual linear amino acid
sequence can contain fewer or additional amino acids corresponding
to a shortening of, or insertion into, a FR or CDR of the variable
domain. For example, a heavy chain variable domain can include a
single amino acid insert (residue 52a according to Kabat) after
residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and
82c, etc. according to Kabat) after heavy chain FR residue 82. The
Kabat numbering of residues can be determined for a given antibody
by alignment at regions of homology of the sequence of the antibody
with a "standard" Kabat numbered sequence. Chothia refers instead
to the location of the structural loops (Chothia and Lesk J. Mol.
Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when
numbered using the Kabat numbering convention varies between H32
and H34 depending on the length of the loop (this is because the
Kabat numbering scheme places the insertions at H35A and H35B; if
neither 35A nor 35B is present, the loop ends at 32; if only 35A is
present, the loop ends at 33; if both 35A and 35B are present, the
loop ends at 34). The AbM hypervariable regions represent a
compromise between the Kabat CDRs and Chothia structural loops, and
are used by Oxford Molecular's AbM antibody modeling software.
TABLE-US-00001 Loop Kabat AbM Chothia L1 L24-L34 L24-L34 L24-L34 L2
L50-L56 L50-L56 L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B
H26-H35B H26-H32 . . . 34 (Kabat Numbering) H1 H31-H35 H26-H35
H26-H32 (Chothia Numbering) H2 H50-H65 H50-H58 H52-H56 H3 H95-H102
H95-H102 H95-H102
[0050] In certain aspects, the CDRs of an antibody or
antigen-binding fragment thereof can be determined according to the
Chothia numbering scheme, which refers to the location of
immunoglobulin structural loops (see, e.g., Chothia C & Lesk A
M, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J
Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227:
799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and
U.S. Pat. No. 7,709,226). Typically, when using the Kabat numbering
convention, the Chothia CDR-H1 loop is present at heavy chain amino
acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at
heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is
present at heavy chain amino acids 95 to 102, while the Chothia
CDR-L1 loop is present at light chain amino acids 24 to 34, the
Chothia CDR-L2 loop is present at light chain amino acids 50 to 56,
and the Chothia CDR-L3 loop is present at light chain amino acids
89 to 97. The end of the Chothia CDR-H1 loop when numbered using
the Kabat numbering convention varies between H32 and H34 depending
on the length of the loop (this is because the Kabat numbering
scheme places the insertions at H35A and H35B; if neither 35A nor
35B is present, the loop ends at 32; if only 35A is present, the
loop ends at 33; if both 35A and 35B are present, the loop ends at
34).
[0051] In certain aspects, the CDRs of an antibody or
antigen-binding fragment thereof can be determined according to the
IMGT numbering system as described in Lefranc M-P, (1999) The
Immunologist 7: 132-136 and Lefranc M-P et al., (1999) Nucleic
Acids Res 27: 209-212. According to the IMGT numbering scheme,
VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57,
VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to
32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions
89 to 97.
[0052] In certain aspects, the CDRs of an antibody or
antigen-binding fragment thereof can be determined according to
MacCallum R M et al., (1996) J Mol Biol 262: 732-745. See also,
e.g., Martin A. "Protein Sequence and Structure Analysis of
Antibody Variable Domains," in Antibody Engineering, Kontermann and
Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin
(2001).
[0053] In certain aspects, the CDRs of an antibody or
antigen-binding fragment thereof can be determined according to the
AbM numbering scheme, which refers AbM hypervariable regions which
represent a compromise between the Kabat CDRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody
modeling software (Oxford Molecular Group, Inc.).
[0054] The term "human" antibody means an antibody produced by a
human or an antibody having an amino acid sequence corresponding to
an antibody produced by a human made using any technique known in
the art. This definition of a human antibody includes intact or
full-length antibodies, fragments thereof, and/or antibodies
comprising at least one human heavy and/or light chain polypeptide
such as, for example, an antibody comprising murine light chain and
human heavy chain polypeptides.
[0055] The term "chimeric" antibodies refers to antibodies wherein
the amino acid sequence of the immunoglobulin molecule is derived
from two or more species. Typically, the variable region of both
light and heavy chains corresponds to the variable region of
antibodies derived from one species of mammals (e.g. mouse, rat,
rabbit, etc.) with the desired specificity, affinity, and
capability while the constant regions are homologous to the
sequences in antibodies derived from another (usually human) to
avoid eliciting an immune response in that species.
[0056] The term "epitope" or "antigenic determinant" are used
interchangeably herein and refer to that portion of an antigen
capable of being recognized and specifically bound by a particular
antibody. When the antigen is a polypeptide, epitopes can be formed
both from contiguous amino acids and noncontiguous amino acids
juxtaposed by tertiary folding of a protein. Epitopes formed from
contiguous amino acids are typically retained upon protein
denaturing, whereas epitopes formed by tertiary folding are
typically lost upon protein denaturing. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation.
[0057] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Low-affinity antibodies generally
bind antigen slowly and tend to dissociate readily, whereas
high-affinity antibodies generally bind antigen faster and tend to
remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for
purposes of the present disclosure. Specific illustrative
embodiments are described in the following.
[0058] "Or better" when used herein to refer to binding affinity
refers to a stronger binding between a molecule and its binding
partner. "Or better" when used herein refers to a stronger binding,
represented by a smaller numerical Kd value. For example, an
antibody which has an affinity for an antigen of "0.6 nM or
better", the antibody's affinity for the antigen is <0.6 nM,
i.e. 0.59 nM, 0.58 nM, 0.57 nM etc. or any value less than 0.6
nM.
[0059] By "specifically binds," it is generally meant that an
antibody binds to an epitope via its antigen binding domain, and
that the binding entails some complementarity between the antigen
binding domain and the epitope. According to this definition, an
antibody is said to "specifically bind" to an epitope when it binds
to that epitope, via its antigen binding domain more readily than
it would bind to a random, unrelated epitope. The term
"specificity" is used herein to qualify the relative affinity by
which a certain antibody binds to a certain epitope. For example,
antibody "A" may be deemed to have a higher specificity for a given
epitope than antibody "B," or antibody "A" may be said to bind to
epitope "C" with a higher specificity than it has for related
epitope "D."
[0060] By "preferentially binds," it is meant that the antibody
specifically binds to an epitope more readily than it would bind to
a related, similar, homologous, or analogous epitope. Thus, an
antibody which "preferentially binds" to a given epitope would more
likely bind to that epitope than to a related epitope, even though
such an antibody may cross-react with the related epitope.
[0061] An antibody is said to "competitively inhibit" binding of a
reference antibody to a given epitope if the antibody
preferentially binds to that epitope or an overlapping epitope to
the extent that it blocks, to some degree, binding of the reference
antibody to the epitope. Competitive inhibition may be determined
by any method known in the art, for example, competition ELISA
assays. An antibody may be said to competitively inhibit binding of
the reference antibody to a given epitope by at least 90%, at least
80%, at least 70%, at least 60%, or at least 50%.
[0062] The phrase "substantially similar," or "substantially the
same", as used herein, denotes a sufficiently high degree of
similarity between two numeric values (generally one associated
with an antibody of the disclosure and the other associated with a
reference/comparator antibody) such that one of skill in the art
would consider the difference between the two values to be of
little or no biological and/or statistical significance within the
context of the biological characteristic measured by said values
(e.g., Kd values). The difference between said two values can be
less than about 50%, less than about 40%, less than about 30%, less
than about 20%, or less than about 10% as a function of the value
for the reference/comparator antibody.
[0063] A polypeptide, antibody, polynucleotide, vector, cell, or
composition which is "isolated" is a polypeptide, antibody,
polynucleotide, vector, cell, or composition which is in a form not
found in nature. Isolated polypeptides, antibodies,
polynucleotides, vectors, cell or compositions include those which
have been purified to a degree that they are no longer in a form in
which they are found in nature. In some embodiments, an antibody,
polynucleotide, vector, cell, or composition which is isolated is
substantially pure.
[0064] As used herein, "substantially pure" refers to material
which is at least 50% pure (i.e., free from contaminants), at least
90% pure, at least 95% pure, at least 98% pure, or at least 99%
pure.
[0065] The term "immunoconjugate," "conjugate," "antibody-drug
conjugate," or "ADC" as used herein refers to a compound or a
derivative thereof that is linked to protein such as a cell binding
agent (e.g., an anti-TNF-alpha antibody or fragment thereof) and is
defined by a generic formula: (SM-L-Q).sub.n-A, wherein SM=radical
derived from a small-molecule glucocorticoid receptor agonist,
e.g., a glucocorticosteroid, L=linker, Q=heterobifunctional group,
a heterotrifunctional group, or is absent, and A=a protein (e.g.,
an antibody or antigen-binding fragment thereof, an anti-TNF
protein, an anti-TNF-alpha antibody or fragment thereof, a soluble
receptor, or a soluble TNF receptor), and n=1-10. Immunoconjugates
can also be defined by the generic formula in reverse order:
A-(Q-L-SM).sub.n. By way of illustration, the following generic
formula shows a immunoconjugate having a dipeptide (Ala-Ala) linker
and succinimide thioether-based heterobifunctional group:
##STR00001##
[0066] In the present disclosure, the term "linker" refers to any
chemical moiety capable of linking a protein, e.g., antibody,
antibody fragment (e.g., antigen binding fragments) or functional
equivalent to a glucocorticosteroid. Linkers may be susceptible to
cleavage (a "cleavable linker") thereby facilitating release of the
glucocorticosteroid. For example, such cleavable linkers may be
susceptible to acid-induced cleavage, photo-induced cleavage,
peptidase-induced cleavage, esterase-induced cleavage, and
disulfide bond cleavage, at conditions under which the
glucocorticosteroid and/or the antibody remains active.
Alternatively, linkers may be substantially resistant to cleavage
(a "noncleavable linker").
[0067] In the present disclosure, non-cleavable linkers are any
chemical moiety capable of linking a glucocorticosteroid to an
antibody in a stable, covalent manner and does not fall off under
the categories listed above for cleaveable linkers. Thus,
non-cleavable linkers are substantially resistant to acid-induced
cleavage, photo-induced cleavage, peptidase-induced cleavage,
esterase-induced cleavage and disulfide bond cleavage. Furthermore,
non-cleavable refers to the ability of the chemical bond in the
linker or adjoining to the linker to withstand cleavage induced by
an acid, photolabile-cleaving agent, a peptidase, an esterase, or a
chemical or physiological compound that cleaves a disulfide bond,
at conditions under which a glucocorticosteroid and/or the antibody
does not lose its activity.
[0068] Some cleavable linkers are cleaved by peptidases ("peptidase
cleavable linkers"). Only certain peptides are readily cleaved
inside or outside cells, see e.g. Trout et al., 79 Proc. Natl.
Acad. Sci. USA, 626-629 (1982) and Umemoto et al. 43 Int. J.
Cancer, 677-684 (1989). Furthermore, peptides are composed of
.alpha.-amino acid units and peptidic bonds, which chemically are
amide bonds between the carboxylate of one amino acid and the amino
group of a second amino acid. Other amide bonds, such as the bond
between a carboxylate and the .alpha.-amino acid group of lysine,
are understood not to be peptidic bonds and are considered
non-cleavable.
[0069] Some linkers are cleaved by esterases ("esterase cleavable
linkers"). Only certain esters can be cleaved by esterases present
inside or outside of cells. Esters are formed by the condensation
of a carboxylic acid and an alcohol. Simple esters are esters
produced with simple alcohols, such as aliphatic alcohols, and
small cyclic and small aromatic alcohols.
[0070] In some embodiments, the cleavable linker component may
comprise a peptide comprising one to ten amino acid residues. In
these embodiments, the peptide allows for cleavage of the linker by
a protease, thereby facilitating release of the glucocorticosteroid
upon exposure to intracellular proteases, such as lysosomal enzymes
(Doronina et al. (2003) Nat. Biotechnol. 21:778-784). Exemplary
peptides include, but are not limited to, dipeptides, tripeptides,
tetrapeptides, and pentapeptides. Exemplary dipeptides include, but
are not limited to, alanine-alanine (ala-ala), valine-citrulline
(vc or val-cit), alanine-phenylalanine (af or ala-phe);
phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine
(phe-homolys); and N-methyl-valine-citrulline (Me-val-cit).
Exemplary tripeptides include, but are not limited to,
glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine
(gly-gly-gly).
[0071] A peptide may comprise naturally-occurring and/or
non-natural amino acid residues. The term "naturally-occurring
amino acid" refer to Ala, Asp, Cys, Glu, Phe, Gly, His, He, Lys,
Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr.
"Non-natural amino acids" (i.e., amino acids do not occur
naturally) include, by way of non-limiting example, homoserine,
homoarginine, citrulline, phenylglycine, taurine, iodotyrosine,
seleno-cysteine, norleucine ("Nle"), norvaline ("Nva"),
beta-alanine, L- or D-naphthalanine, ornithine ("Orn"), and the
like. Peptides can be designed and optimized for enzymatic cleavage
by a particular enzyme, for example, a tumor-associated protease,
cathepsin B, C and D, or a plasmin protease.
[0072] Amino acids also include the D-forms of natural and
non-natural amino acids. "D-" designates an amino acid having the
"D" (dextrorotary) configuration, as opposed to the configuration
in the naturally occurring ("L-") amino acids. Natural and
non-natural amino acids can be purchased commercially (Sigma
Chemical Co., Advanced Chemtech) or synthesized using methods known
in the art.
[0073] In the present disclosure, the term "glucocorticosteroid"
refers to naturally-occurring or synthetic steroid hormones that
interact with glucocorticoid receptors. Non-limiting exemplary
glucocorticosteroids include:
##STR00002## ##STR00003##
By way of example, the A-, B-, C-, and D-rings of the steroid
skeleton are marked for budesonide. Glucocorticosteroids are
described in WO 2009/069032.
[0074] A "radical of a glucocorticosteroid" is derived from the
removal of one or more hydrogen atoms from a parent
glucocorticosteroid. The removal of hydrogen atom(s) facilitates
the attachment of the parent glucocorticosteroid to a linker. In
one embodiment, the hydrogen atom is removed from any suitable
--NH.sub.2 group of the parent glucocorticosteroid. In another
embodiment, the hydrogen atom is removed from any suitable --OH
group of the parent glucocorticosteroid. In another embodiment, the
hydrogen atom is removed from any suitable a --SH group of the
parent glucocorticosteroid. In another embodiment, the hydrogen
atom is removed from any suitable --N(H)-- group of the parent
glucocorticosteroid. In another embodiment, the hydrogen atom is
removed from any suitable --CH.sub.3, --CH.sub.2-- or --CH=group of
the parent glucocorticosteroid. In one embodiment, the "radical of
a glucocorticosteroid" is a monovalent radical derived from the
removal of one hydrogen atom from a parent glucocorticosteroid.
[0075] In the present disclosure, the term "heterobifunctional
group" or the term "heterotrifunctional group" refers to a chemical
moiety that connects a linker and protein, e.g., an antibody.
Heterobi- and tri-functional groups are characterized as having
different reactive groups at either end of the chemical moiety.
Non-limiting exemplary heterobifunctional groups include:
##STR00004##
A non-limiting exemplary heterotrifunctional group is:
##STR00005##
[0076] The term "drug antibody ratio" or "DAR" refers to the number
of SMs (i.e., radical derived from a small-molecule glucocorticoid
receptor agonist, e.g., a glucocorticosteroid) linked to A (i.e., a
protein, e.g., an antibody or antigen-binding fragment thereof, an
anti-TNF protein, an anti-TNF-alpha antibody or fragment thereof, a
soluble receptor, or a soluble TNF receptor). Thus, in the
immunoconjugate having the generic formula (SM-L-Q).sub.n-A, the
DAR is defined by the variable "n."
[0077] When referring to a compound having formula (SM-L-Q).sub.n-A
representing an individual immunoconjugate, the DAR refers to the
number of SMs linked to the individual A (e.g., n is an integer of
1 to 10).
[0078] When referring to a compound having formula (SM-L-Q).sub.n-A
representing a plurality of immunoconjugates, the DAR refers to the
average number of SMs linked to the As (e.g., n is an integer or
fraction of 1 to 10). Thus, by way of an example, a compound having
formula (SM-L-Q).sub.n-A comprising a first immunoconjugate with 3
SM per A and a second immunoconjugate with 4 SM per A would have a
DAR (i.e., an "n") of 3.5.
[0079] The term "subject" refers to any animal (e.g., a mammal),
including, but not limited to humans, non-human primates, rodents,
and the like, which is to be the recipient of a particular
treatment. Typically, the terms "subject" and "patient" are used
interchangeably herein in reference to a human subject.
[0080] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of the active ingredient to be effective, and which
contains no additional components which are unacceptably toxic to a
subject to which the formulation would be administered. The
formulation can be sterile.
[0081] An "effective amount" of an immunoconjugate or
glucocorticoid receptor agonist as disclosed herein is an amount
sufficient to carry out a specifically stated purpose. An
"effective amount" can be determined in relation to the stated
purpose.
[0082] The term "therapeutically effective amount" refers to an
amount of an immunoconjugate or glucocorticoid receptor agonist
effective to "treat" a disease or disorder in a subject or mammal.
A "prophylactically effective amount" refers to an amount effective
to achieve the desired prophylactic result.
[0083] Terms such as "treating" or "treatment" or "to treat" or
"alleviating" or "to alleviate" refer to therapeutic measures that
cure, slow down, lessen symptoms of, and/or halt progression of a
diagnosed pathologic condition or disorder. Thus, those in need of
treatment include those already diagnosed with or suspected of
having the disorder. Prophylactic or preventative measures refer to
measures that prevent and/or slow the development of a targeted
pathological condition or disorder. Thus, those in need of
prophylactic or preventative measures include those prone to have
the disorder and those in whom the disorder is to be prevented.
[0084] "Polynucleotide," or "nucleic acid," as used interchangeably
herein, refer to polymers of nucleotides of any length, and include
DNA and RNA. The nucleotides can be deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their
analogs, or any substrate that can be incorporated into a polymer
by DNA or RNA polymerase. A polynucleotide can comprise modified
nucleotides, such as methylated nucleotides and their analogs. If
present, modification to the nucleotide structure can be imparted
before or after assembly of the polymer. The sequence of
nucleotides can be interrupted by non-nucleotide components. A
polynucleotide can be further modified after polymerization, such
as by conjugation with a labeling component. Other types of
modifications include, for example, "caps", substitution of one or
more of the naturally occurring nucleotides with an analog,
internucleotide modifications such as, for example, those with
uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoamidates, cabamates, etc.) and with charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), those containing
pendant moieties, such as, for example, proteins (e.g., nucleases,
toxins, antibodies, signal peptides, ply-L-lysine, etc.), those
with intercalators (e.g., acridine, psoralen, etc.), those
containing chelators (e.g., metals, radioactive metals, boron,
oxidative metals, etc.), those containing alkylators, those with
modified linkages (e.g., alpha anomeric nucleic acids, etc.), as
well as unmodified forms of the polynucleotide(s). Further, any of
the hydroxyl groups ordinarily present in the sugars can be
replaced, for example, by phosphonate groups, phosphate groups,
protected by standard protecting groups, or activated to prepare
additional linkages to additional nucleotides, or can be conjugated
to solid supports. The 5' and 3' terminal OH can be phosphorylated
or substituted with amines or organic capping group moieties of
from 1 to 20 carbon atoms. Other hydroxyls can also be derivatized
to standard protecting groups. Polynucleotides can also contain
analogous forms of ribose or deoxyribose sugars that are generally
known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl,
2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs,
.alpha.-anomeric sugars, epimeric sugars such as arabinose, xyloses
or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses,
acyclic analogs and abasic nucleoside analogs such as methyl
riboside. One or more phosphodiester linkages can be replaced by
alternative linking groups. These alternative linking groups
include, but are not limited to, embodiments wherein phosphate is
replaced by P(O)S ("thioate"), P(S)S ("dithioate"), "(O)NR.sub.2
("amidate"), P(O)R, P(O)OR', CO or CH.sub.2 ("formacetal"), in
which each R or R' is independently H or substituted or
unsubstituted alkyl (1-20 C) optionally containing an ether (--O--)
linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not
all linkages in a polynucleotide need be identical. The preceding
description applies to all polynucleotides referred to herein,
including RNA and DNA.
[0085] The term "vector" means a construct, which is capable of
delivering, and optionally expressing, one or more gene(s) or
sequence(s) of interest in a host cell. Examples of vectors
include, but are not limited to, viral vectors, naked DNA or RNA
expression vectors, plasmid, cosmid or phage vectors, DNA or RNA
expression vectors associated with cationic condensing agents, DNA
or RNA expression vectors encapsulated in liposomes, and certain
eukaryotic cells, such as producer cells.
[0086] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer can be linear or branched, it can comprise
modified amino acids, and it can be interrupted by non-amino acids.
The terms also encompass an amino acid polymer that has been
modified naturally or by intervention; for example, disulfide bond
formation, glycosylation, lipidation, acetylation, phosphorylation,
or any other manipulation or modification, such as conjugation with
a labeling component. Also included within the definition are, for
example, polypeptides containing one or more analogs of an amino
acid (including, for example, unnatural amino acids, etc.), as well
as other modifications known in the art. It is understood that,
because the polypeptides of this disclosure are based upon
antibodies, in certain embodiments, the polypeptides can occur as
single chains or associated chains.
[0087] The terms "identical" or percent "identity" in the context
of two or more nucleic acids or polypeptides, refer to two or more
sequences or subsequences that are the same or have a specified
percentage of nucleotides or amino acid residues that are the same,
when compared and aligned (introducing gaps, if necessary) for
maximum correspondence, not considering any conservative amino acid
substitutions as part of the sequence identity. The percent
identity can be measured using sequence comparison software or
algorithms or by visual inspection. Various algorithms and software
are known in the art that can be used to obtain alignments of amino
acid or nucleotide sequences. One such non-limiting example of a
sequence alignment algorithm is the algorithm described in Karlin
et al, Proc. Natl. Acad. Sci., 87:2264-2268 (1990), as modified in
Karlin et al., Proc. Natl. Acad. Sci., 90:5873-5877 (1993), and
incorporated into the NBLAST and XBLAST programs (Altschul et al.,
Nucleic Acids Res., 25:3389-3402 (1991)). In certain embodiments,
Gapped BLAST can be used as described in Altschul et al., Nucleic
Acids Res. 25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul et
al., Methods in Enzymology, 266:460-480 (1996)), ALIGN, ALIGN-2
(Genentech, South San Francisco, Calif.) or Megalign (DNASTAR) are
additional publicly available software programs that can be used to
align sequences. In certain embodiments, the percent identity
between two nucleotide sequences is determined using the GAP
program in GCG software (e.g., using a NWSgapdna.CMP matrix and a
gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3,
4, 5, or 6). In certain alternative embodiments, the GAP program in
the GCG software package, which incorporates the algorithm of
Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) can be
used to determine the percent identity between two amino acid
sequences (e.g., using either a Blossum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments,
the percent identity between nucleotide or amino acid sequences is
determined using the algorithm of Myers and Miller (CABIOS, 4:11-17
(1989)). For example, the percent identity can be determined using
the ALIGN program (version 2.0) and using a PAM120 with residue
table, a gap length penalty of 12 and a gap penalty of 4.
Appropriate parameters for maximal alignment by particular
alignment software can be determined by one skilled in the art. In
certain embodiments, the default parameters of the alignment
software are used. In certain embodiments, the percentage identity
"X" of a first amino acid sequence to a second sequence amino acid
is calculated as 100.times.(Y/Z), where Y is the number of amino
acid residues scored as identical matches in the alignment of the
first and second sequences (as aligned by visual inspection or a
particular sequence alignment program) and Z is the total number of
residues in the second sequence. If the length of a first sequence
is longer than the second sequence, the percent identity of the
first sequence to the second sequence will be longer than the
percent identity of the second sequence to the first sequence.
[0088] As a non-limiting example, whether any particular
polynucleotide has a certain percentage sequence identity (e.g., is
at least 80% identical, at least 85% identical, at least 90%
identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or
99% identical) to a reference sequence can, in certain embodiments,
be determined using the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, 575 Science Drive, Madison, Wis. 53711).
Bestfit uses the local homology algorithm of Smith and Waterman
(Advances in Applied Mathematics 2: 482 489 (1981)) to find the
best segment of homology between two sequences. When using Bestfit
or any other sequence alignment program to determine whether a
particular sequence is, for instance, 95% identical to a reference
sequence according to the present disclosure, the parameters are
set such that the percentage of identity is calculated over the
full length of the reference nucleotide sequence and that gaps in
homology of up to 5% of the total number of nucleotides in the
reference sequence are allowed.
[0089] In some embodiments, two nucleic acids or polypeptides of
the disclosure are substantially identical, meaning they have at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide
or amino acid residue identity, when compared and aligned for
maximum correspondence, as measured using a sequence comparison
algorithm or by visual inspection. Identity can exist over a region
of the sequences that is at least about 10, about 20, about 40-60
residues in length or any integral value there between, and can be
over a longer region than 60-80 residues, for example, at least
about 90-100 residues, and in some embodiments, the sequences are
substantially identical over the full length of the sequences being
compared, such as the coding region of a nucleotide sequence for
example.
[0090] A "conservative amino acid substitution" is one in which one
amino acid residue is replaced with another amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art, including basic
side chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid, glutamic acid), uncharged polar side chains
(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). For example, substitution of a phenylalanine for a
tyrosine is a conservative substitution. In some embodiments,
conservative substitutions in the sequences of the polypeptides and
antibodies of the disclosure do not abrogate the binding of the
antibody containing the amino acid sequence, to the antigen(s),
e.g., the TNF-alpha to which the antibody binds. Methods of
identifying nucleotide and amino acid conservative substitutions
which do not eliminate antigen binding are well-known in the art
(see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993);
Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et
al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).
[0091] In the present disclosure, the term "halo" as used by itself
or as part of another group refers to --Cl, --F, --Br, or --I. In
one embodiment, the halo is --Cl or --F.
[0092] In the present disclosure, the term "hydroxy" as used by
itself or as part of another group refers to --OH.
[0093] In the present disclosure, the term "thiol" or the term
"sulfhydryl" as used by itself or as part of another group refers
to --SH.
[0094] In the present disclosure, the term "alkyl" as used by
itself or as part of another group refers to unsubstituted
straight- or branched-chain aliphatic hydrocarbons containing from
one to twelve carbon atoms, i.e., C.sub.1-12 alkyl, or the number
of carbon atoms designated, e.g., a C.sub.1 alkyl such as methyl, a
C.sub.2 alkyl such as ethyl, a C.sub.3 alkyl such as propyl or
isopropyl, a C.sub.1-3 alkyl such as methyl, ethyl, propyl, or
isopropyl, and so on. In one embodiment, the alkyl is a C.sub.1-10
alkyl. In another embodiment, the alkyl is a C.sub.1-6 alkyl. In
another embodiment, the alkyl is a C.sub.1-4 alkyl. In another
embodiment, the alkyl is a straight chain C.sub.1-10 alkyl. In
another embodiment, the alkyl is a branched chain C.sub.3-10 alkyl.
In another embodiment, the alkyl is a straight chain C.sub.1-6
alkyl. In another embodiment, the alkyl is a branched chain
C.sub.3-6 alkyl. In another embodiment, the alkyl is a straight
chain C.sub.1-4 alkyl. In another embodiment, the alkyl is a
branched chain C.sub.3-4 alkyl. In another embodiment, the alkyl is
a straight or branched chain C.sub.3-4alkyl. Non-limiting exemplary
C.sub.1-10 alkyl groups include methyl, ethyl, propyl, isopropyl,
butyl, sec-butyl, tert-butyl, iso-butyl, 3-pentyl, hexyl, heptyl,
octyl, nonyl, and decyl. Non-limiting exemplary C.sub.1-4 alkyl
groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,
tert-butyl, and iso-butyl.
[0095] In the present disclosure, the term "optionally substituted
alkyl" as used by itself or as part of another group refers to an
alkyl that is either unsubstituted or substituted with one, two, or
three substituents independently selected from the group consisting
of nitro, hydroxy, cyano, haloalkoxy, aryloxy, alkylthio,
sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl,
arylsulfonyl, carboxy, carboxamido, alkoxycarbonyl, thiol,
--N(H)C(.dbd.O)NH.sub.2, and --N(H)C(.dbd.NH)NH.sub.2, optionally
substituted aryl, and optionally substituted heteroaryl. In one
embodiment, the optionally substituted alkyl is substituted with
two substituents. In another embodiment, the optionally substituted
alkyl is substituted with one substituent. In another embodiment,
the optionally substituted alkyl is unsubstituted. Non-limiting
exemplary substituted alkyl groups include --CH.sub.2OH,
--CH.sub.2SH, --CH.sub.2Ph, --CH.sub.2(4-OH)Ph,
--CH.sub.2(imidazolyl), --CH.sub.2CH.sub.2CO.sub.2H,
--CH.sub.2CH.sub.2SO.sub.2CH.sub.3, --CH.sub.2CH.sub.2COPh, and
--CH.sub.2OC(.dbd.O)CH.sub.3.
[0096] In the present disclosure, the term "cycloalkyl" as used by
itself or as part of another group refers to unsubstituted
saturated or partially unsaturated, e.g., containing one or two
double bonds, cyclic aliphatic hydrocarbons containing one to three
rings having from three to twelve carbon atoms, i.e., C.sub.3-12
cycloalkyl, or the number of carbons designated. In one embodiment,
the cycloalkyl has two rings. In another embodiment, the cycloalkyl
has one ring. In another embodiment, the cycloalkyl is saturated.
In another embodiment, the cycloalkyl is unsaturated. In another
embodiment, the cycloalkyl is a C.sub.3-8 cycloalkyl. In another
embodiment, the cycloalkyl is a C.sub.3-6 cycloalkyl. The term
"cycloalkyl" is meant to include groups wherein a ring --CH.sub.2--
is replaced with a --C(.dbd.O)--. Non-limiting exemplary cycloalkyl
groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, cyclooctyl, norbornyl, decalin, adamantyl,
cyclohexenyl, cyclopentenyl, and cyclopentanone.
[0097] In the present disclosure, the term "optionally substituted
cycloalkyl" as used by itself or as part of another group refers to
a cycloalkyl that is either unsubstituted or substituted with one,
two, or three substituents independently selected from the group
consisting of halo, nitro, cyano, hydroxy, alkylcarbonyloxy,
cycloalkylcarbonyloxy, amino, haloalkyl, hydroxyalkyl, alkoxy,
haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido,
sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl,
arylsulfonyl, carboxy, carboxyalkyl, optionally substituted alkyl,
optionally substituted cycloalkyl, alkenyl, alkynyl, optionally
substituted aryl, optionally substituted heteroaryl, optionally
substituted heterocyclo, alkoxyalkyl, (amino)alkyl,
(carboxamido)alkyl, (heterocyclo)alkyl, and --OC(.dbd.O)-amino, The
term optionally substituted cycloalkyl includes cycloalkyl groups
having a fused optionally substituted aryl, e.g., phenyl, or fused
optionally substituted heteroaryl, e.g., pyridyl. An optionally
substituted cycloalkyl having a fused optionally substituted aryl
or fused optionally substituted heteroaryl group may be attached to
the remainder of the molecule at any available carbon atom on the
cycloalkyl ring. In one embodiment, the optionally substituted
cycloalkyl is substituted with two substituents. In another
embodiment, the optionally substituted cycloalkyl is substituted
with one substituent. In another embodiment, the optionally
substituted cycloalkyl is unsubstituted.
[0098] In the present disclosure, the term "aryl" as used by itself
or as part of another group refers to unsubstituted monocyclic or
bicyclic aromatic ring systems having from six to fourteen carbon
atoms, i.e., a C.sub.6-14 aryl. Non-limiting exemplary aryl groups
include phenyl (abbreviated as "Ph"), naphthyl, phenanthryl,
anthracyl, indenyl, azulenyl, biphenyl, biphenylenyl, and fluorenyl
groups. In one embodiment, the aryl group is phenyl or
naphthyl.
[0099] In the present disclosure, the term "optionally substituted
aryl" as used herein by itself or as part of another group refers
to an aryl that is either unsubstituted or substituted with one to
five substituents independently selected from the group consisting
of halo, nitro, cyano, hydroxy, thiol, amino, alkylamino,
dialkylamino, optionally substituted alkyl, haloalkyl,
hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio,
carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl,
alkylsulfonyl, haloalkylsulfonyl cycloalkylsulfonyl,
(cycloalkyl)alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl,
heterocyclosulfonyl, carboxy, carboxyalkyl, optionally substituted
cycloalkyl, alkenyl, alkynyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted
heterocyclo, alkoxycarbonyl, alkoxyalkyl, (amino)alkyl,
(carboxamido)alkyl, and (heterocyclo)alkyl.
[0100] In one embodiment, the optionally substituted aryl is an
optionally substituted phenyl. In another embodiment, the
optionally substituted phenyl has four substituents. In another
embodiment, the optionally substituted phenyl has three
substituents. In another embodiment, the optionally substituted
phenyl has two substituents. In another embodiment, the optionally
substituted phenyl has one substituent. In another embodiment, the
optionally substituted phenyl is unsubstituted. Non-limiting
exemplary substituted aryl groups include 2-methylphenyl,
2-methoxyphenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl,
3-methylphenyl, 3-methoxyphenyl, 3-fluorophenyl, 3-chlorophenyl,
4-methylphenyl, 4-ethylphenyl, 4-methoxyphenyl, 4-fluorophenyl,
4-chlorophenyl, 2,6-di-fluorophenyl, 2,6-di-chlorophenyl, 2-methyl,
3-methoxyphenyl, 2-ethyl, 3-methoxyphenyl, 3,4-di-methoxyphenyl,
3,5-di-fluorophenyl 3,5-di-methylphenyl, 3,5-dimethoxy,
4-methylphenyl, 2-fluoro-3-chlorophenyl, 3-chloro-4-fluorophenyl,
4-(pyridin-4-ylsulfonyl)phenyl The term optionally substituted aryl
includes phenyl groups having a fused optionally substituted
cycloalkyl or fused optionally substituted heterocyclo group. An
optionally substituted phenyl having a fused optionally substituted
cycloalkyl or fused optionally substituted heterocyclo group may be
attached to the remainder of the molecule at any available carbon
atom on the phenyl ring.
[0101] In the present disclosure, the term "alkenyl" as used by
itself or as part of another group refers to an alkyl containing
one, two or three carbon-to-carbon double bonds. In one embodiment,
the alkenyl has one carbon-to-carbon double bond. In another
embodiment, the alkenyl is a C.sub.2-6 alkenyl. In another
embodiment, the alkenyl is a C.sub.2-4 alkenyl. Non-limiting
exemplary alkenyl groups include ethenyl, propenyl, isopropenyl,
butenyl, sec-butenyl, pentenyl, and hexenyl.
[0102] In the present disclosure, the term "optionally substituted
alkenyl" as used herein by itself or as part of another group
refers to an alkenyl that is either unsubstituted or substituted
with one, two or three substituents independently selected from the
group consisting of halo, nitro, cyano, hydroxy, amino, alkylamino,
dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy,
aralkyloxy, alkylthio, carboxamido, sulfonamido, alkylcarbonyl,
arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxy, carboxyalkyl,
optionally substituted alkyl, optionally substituted cycloalkyl,
alkenyl, alkynyl, optionally substituted aryl, heteroaryl, and
optionally substituted heterocyclo.
[0103] In the present disclosure, the term "alkynyl" as used by
itself or as part of another group refers to an alkyl containing
one to three carbon-to-carbon triple bonds. In one embodiment, the
alkynyl has one carbon-to-carbon triple bond. In another
embodiment, the alkynyl is a C.sub.2-6 alkynyl. In another
embodiment, the alkynyl is a C.sub.2-4 alkynyl. Non-limiting
exemplary alkynyl groups include ethynyl, propynyl, butynyl,
2-butynyl, pentynyl, and hexynyl groups.
[0104] In the present disclosure, the term "optionally substituted
alkynyl" as used herein by itself or as part refers to an alkynyl
that is either unsubstituted or substituted with one, two or three
substituents independently selected from the group consisting of
halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino,
haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy,
alkylthio, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl,
alkylsulfonyl, arylsulfonyl, carboxy, carboxyalkyl, optionally
substituted alkyl, cycloalkyl, alkenyl, alkynyl, optionally
substituted aryl, optionally substituted heteroaryl, and
heterocyclo.
[0105] In the present disclosure, the term "haloalkyl" as used by
itself or as part of another group refers to an alkyl substituted
by one or more fluorine, chlorine, bromine and/or iodine atoms. In
one embodiment, the alkyl group is substituted by one, two, or
three fluorine and/or chlorine atoms. In another embodiment, the
haloalkyl group is a C.sub.4 haloalkyl group. Non-limiting
exemplary haloalkyl groups include fluoromethyl, 2-fluoroethyl,
difluoromethyl, trifluoromethyl, pentafluoroethyl,
1,1-difluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl,
3,3,3-trifluoropropyl, 4,4,4-trifluorobutyl, and trichloromethyl
groups.
[0106] In the present disclosure, the term "alkoxy" as used by
itself or as part of another group refers to an optionally
substituted alkyl, optionally substituted cycloalkyl, optionally
substituted alkenyl, or optionally substituted alkynyl attached to
a terminal oxygen atom. In one embodiment, the alkoxy is an
optionally substituted alkyl attached to a terminal oxygen atom. In
one embodiment, the alkoxy group is a C.sub.1-6 alkyl attached to a
terminal oxygen atom. In another embodiment, the alkoxy group is a
C.sub.1-4 alkyl attached to a terminal oxygen atom. Non-limiting
exemplary alkoxy groups include methoxy, ethoxy, and
tert-butoxy.
[0107] In the present disclosure, the term "alkylthio" as used by
itself or as part of another group refers to an optionally
substituted alkyl attached to a terminal sulfur atom. In one
embodiment, the alkylthio group is a C.sub.1-4 alkylthio group.
Non-limiting exemplary alkylthio groups include --SCH.sub.3 and
--SCH.sub.2CH.sub.3.
[0108] In the present disclosure, the term "haloalkoxy" as used by
itself or as part of another group refers to a haloalkyl attached
to a terminal oxygen atom. Non-limiting exemplary haloalkoxy groups
include fluoromethoxy, difluoromethoxy, trifluoromethoxy, and
2,2,2-trifluoroethoxy.
[0109] In the present disclosure, the term "heteroaryl" refers to
unsubstituted monocyclic and bicyclic aromatic ring systems having
5 to 14 ring atoms, i.e., a 5- to 14-membered heteroaryl, wherein
at least one carbon atom of one of the rings is replaced with a
heteroatom independently selected from the group consisting of
oxygen, nitrogen and sulfur. In one embodiment, the heteroaryl
contains 1, 2, 3, or 4 heteroatoms independently selected from the
group consisting of oxygen, nitrogen and sulfur. In one embodiment,
the heteroaryl has three heteroatoms. In another embodiment, the
heteroaryl has two heteroatoms. In another embodiment, the
heteroaryl has one heteroatom. In another embodiment, the
heteroaryl is a 5- to 10-membered heteroaryl. In another
embodiment, the heteroaryl is a 5- or 6-membered heteroaryl. In
another embodiment, the heteroaryl has 5 ring atoms, e.g., thienyl,
a 5-membered heteroaryl having four carbon atoms and one sulfur
atom. In another embodiment, the heteroaryl has 6 ring atoms, e.g.,
pyridyl, a 6-membered heteroaryl having five carbon atoms and one
nitrogen atom. Non-limiting exemplary heteroaryl groups include
thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl,
furyl, benzofuryl, pyranyl, isobenzofuranyl, benzooxazonyl,
chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl,
pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, isoindolyl,
3H-indolyl, indolyl, indazolyl, purinyl, isoquinolyl, quinolyl,
phthalazinyl, naphthyridinyl, cinnolinyl, quinazolinyl, pteridinyl,
4aH-carbazolyl, carbazolyl, .beta.-carbolinyl, phenanthridinyl,
acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, thiazolyl,
isothiazolyl, phenothiazolyl, isoxazolyl, furazanyl, and
phenoxazinyl. In one embodiment, the heteroaryl is selected from
the group consisting of thienyl (e.g., thien-2-yl and thien-3-yl),
furyl (e.g., 2-furyl and 3-furyl), pyrrolyl (e.g., 1H-pyrrol-2-yl
and 1H-pyrrol-3-yl), imidazolyl (e.g., 2H-imidazol-2-yl and
2H-imidazol-4-yl), pyrazolyl (e.g., 1H-pyrazol-3-yl,
1H-pyrazol-4-yl, and 1H-pyrazol-5-yl), pyridyl (e.g., pyridin-2-yl,
pyridin-3-yl, and pyridin-4-yl), pyrimidinyl (e.g., pyrimidin-2-yl,
pyrimidin-4-yl, and pyrimidin-5-yl), thiazolyl (e.g., thiazol-2-yl,
thiazol-4-yl, and thiazol-5-yl), isothiazolyl (e.g.,
isothiazol-3-yl, isothiazol-4-yl, and isothiazol-5-yl), oxazolyl
(e.g., oxazol-2-yl, oxazol-4-yl, and oxazol-5-yl), isoxazolyl
(e.g., isoxazol-3-yl, isoxazol-4-yl, and isoxazol-5-yl), and
indazolyl (e.g., 1H-indazol-3-yl). The term "heteroaryl" is also
meant to include possible N-oxides. A non-limiting exemplary
N-oxide is pyridyl N-oxide.
[0110] In one embodiment, the heteroaryl is a 5- or 6-membered
heteroaryl. In one embodiment, the heteroaryl is a 5-membered
heteroaryl, i.e., the heteroaryl is a monocyclic aromatic ring
system having 5 ring atoms wherein at least one carbon atom of the
ring is replaced with a heteroatom independently selected from
nitrogen, oxygen, and sulfur. Non-limiting exemplary 5-membered
heteroaryl groups include thienyl, furyl, pyrrolyl, oxazolyl,
pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, and isoxazolyl. In
another embodiment, the heteroaryl is a 6-membered heteroaryl,
e.g., the heteroaryl is a monocyclic aromatic ring system having 6
ring atoms wherein at least one carbon atom of the ring is replaced
with a nitrogen atom. Non-limiting exemplary 6-membered heteroaryl
groups include pyridyl, pyrazinyl, pyrimidinyl, and
pyridazinyl.
[0111] In the present disclosure, the term "optionally substituted
heteroaryl" as used by itself or as part of another group refers to
a heteroaryl that is either unsubstituted or substituted with one
two, three, or four substituents, independently selected from the
group consisting of halo, nitro, cyano, hydroxy, amino, alkylamino,
dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy,
aralkyloxy, alkylthio, carboxamido, sulfonamido, alkylcarbonyl,
arylcarbonyl, alkylsulfonyl, haloalkylsulfonyl cycloalkylsulfonyl,
(cycloalkyl)alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl,
carboxy, carboxyalkyl, optionally substituted alkyl, optionally
substituted cycloalkyl, alkenyl, alkynyl, optionally substituted
aryl, optionally substituted heteroaryl, optionally substituted
heterocyclo, alkoxyalkyl, (amino)alkyl, (carboxamido)alkyl, and
(heterocyclo)alkyl. In one embodiment, the optionally substituted
heteroaryl has one substituent. In another embodiment, the
optionally substituted heteroaryl is unsubstituted. Any available
carbon or nitrogen atom can be substituted. The term optionally
substituted heteroaryl includes heteroaryl groups having a fused
optionally substituted cycloalkyl or fused optionally substituted
heterocyclo group. An optionally substituted heteroaryl having a
fused optionally substituted cycloalkyl or fused optionally
substituted heterocyclo group may be attached to the remainder of
the molecule at any available carbon atom on the heteroaryl
ring.
[0112] In the present disclosure, the term "heterocyclo" as used by
itself or as part of another group refers to unsubstituted
saturated and partially unsaturated, e.g., containing one or two
double bonds, cyclic groups containing one, two, or three rings
having from three to fourteen ring members, i.e., a 3- to
14-membered heterocyclo, wherein at least one carbon atom of one of
the rings is replaced with a heteroatom. Each heteroatom is
independently selected from the group consisting of oxygen, sulfur,
including sulfoxide and sulfone, and/or nitrogen atoms, which can
be oxidized or quaternized. The term "heterocyclo" includes groups
wherein a ring --CH.sub.2 is replaced with a --C(.dbd.O)--, for
example, cyclic ureido groups such as 2-imidazolidinone and cyclic
amide groups such as .beta.-lactam, .gamma.-lactam, .delta.-lactam,
.epsilon.-lactam, and piperazin-2-one. The term "heterocyclo" also
includes groups having fused optionally substituted aryl groups,
e.g., indolinyl or chroman-4-yl. In one embodiment, the heterocyclo
group is a C.sub.4-6 heterocyclo, i.e., a 4-, 5- or 6-membered
cyclic group, containing one ring and one or two oxygen and/or
nitrogen atoms. In one embodiment, the heterocyclo group is a
C.sub.4-6 heterocyclo containing one ring and one nitrogen atom.
The heterocyclo can be optionally linked to the rest of the
molecule through any available carbon or nitrogen atom.
Non-limiting exemplary heterocyclo groups include azetidinyl,
dioxanyl, tetrahydropyranyl, 2-oxopyrrolidin-3-yl, piperazin-2-one,
piperazine-2,6-dione, 2-imidazolidinone, piperidinyl, morpholinyl,
piperazinyl, pyrrolidinyl, and indolinyl.
[0113] In the present disclosure, the term "optionally substituted
heterocyclo" as used herein by itself or part of another group
refers to a heterocyclo that is either unsubstituted or substituted
with one, two, three, or four substituents independently selected
from the group consisting of halo, nitro, cyano, hydroxy, amino,
alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy,
haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido,
sulfonamido, alkylcarbonyl, cycloalkylcarbonyl, alkoxycarbonyl,
CF.sub.3C(.dbd.O)--, arylcarbonyl, alkylsulfonyl, arylsulfonyl,
carboxy, carboxyalkyl, alkyl, optionally substituted cycloalkyl,
alkenyl, alkynyl, optionally substituted aryl, optionally
substituted heteroaryl, optionally substituted heterocyclo,
alkoxyalkyl, (amino)alkyl, (carboxamido)alkyl, or
(heterocyclo)alkyl. Substitution may occur on any available carbon
or nitrogen atom, or both.
[0114] In the present disclosure, the term "amino" as used by
itself or as part of another group refers to a radical of the
formula --NR.sup.22aR.sup.22b, wherein R.sup.22a and R.sup.22b are
each independently selected from the group consisting of hydrogen,
optionally substituted alkyl, and aralkyl, or R.sup.22a and
R.sup.22b are taken together to form a 3- to 8-membered optionally
substituted heterocyclo. Non-limiting exemplary amino groups
include --NH.sub.2 and --N(H)(CH.sub.3).
[0115] In the present disclosure, the term "carboxamido" as used by
itself or as part of another group refers to a radical of formula
--C(.dbd.O)NR.sup.23aR.sup.23b, wherein R.sup.23a and R.sup.23b are
each independently selected from the group consisting of hydrogen,
optionally substituted alkyl, hydroxyalkyl, and optionally
substituted aryl, optionally substituted heterocyclo, and
optionally substituted heteroaryl, or R.sup.23a and R.sup.23b taken
together with the nitrogen to which they are attached form a 3- to
8-membered optionally substituted heterocyclo group. In one
embodiment, R.sup.23a and R.sup.23b are each independently hydrogen
or optionally substituted alkyl. In one embodiment, R.sup.23a and
R.sup.23b are taken together to taken together with the nitrogen to
which they are attached form a 3- to 8-membered optionally
substituted heterocyclo group. Non-limiting exemplary carboxamido
groups include --CONH.sub.2, --CON(H)CH.sub.3, and
--CON(CH.sub.3).sub.2.
[0116] In the present disclosure, the term "alkoxycarbonyl" as used
by itself or as part of another group refers to a carbonyl group,
i.e., --C(.dbd.O)--, substituted with an alkoxy. In one embodiment,
the alkoxy is a C.sub.1-4 alkoxy. Non-limiting exemplary
alkoxycarbonyl groups include --C(.dbd.O)OMe, --C(.dbd.O)OEt, and
--C(.dbd.O)OtBu.
[0117] In the present disclosure, the term "carboxy" as used by
itself or as part of another group refers to a radical of the
formula --CO.sub.2H.
[0118] In the present disclosure, the term "maleimide" as used by
itself or as part of another group refers to:
##STR00006##
[0119] In the present disclosure, the term "succinimide" as used as
part of a cleavable linker refers to:
##STR00007##
[0120] In the present disclosure, the term "hydrolyzed succinimide"
as used as part of a cleavable linker refers to:
##STR00008##
[0121] In the present disclosure, the term "amide" as used as part
of a cleavable linker refers to:
##STR00009##
[0122] In the present disclosure, the term "thiourea" as used as
part of a cleavable linker refers to:
##STR00010##
[0123] In the present disclosure, the term "thioether" as used as
part of a cleavable linker refers to:
##STR00011##
[0124] In the present disclosure, the term "oxime" as used as part
of a cleavable linker refers to:
##STR00012##
[0125] In the present disclosure, the term "self-immolative group"
as used as part of a cleavable linker refers to bifunctional
chemical moiety that is capable of covalently linking two spaced
chemical moieties into a normally stable tripartite molecule, can
release one of the spaced chemical moieties from the tripartite
molecule by means of enzymatic cleavage; and following enzymatic
cleavage, can spontaneously cleave from the remainder of the
molecule to release the other of the spaced chemical moieties,
e.g., a glucocorticosteroid. In some embodiments, a self-immolative
group comprises a p-aminobenzyl unit. In some such embodiments, a
p-aminobenzyl alcohol is attached to an amino acid unit via an
amide bond, and a carbamate, methylcarbamate, or carbonate is made
between the benzyl alcohol and the drug (Hamann et al. (2005)
Expert Opin. Ther. Patents (2005) 15:1087-1103). In some
embodiments, the self-immolative group is p-aminobenzyloxycarbonyl
(PAB).
[0126] In the present disclosure, the term "protecting group" or
"PG" refers to group that blocks, i.e., protects, the amine
functionality while reactions are carried out on other functional
groups or parts of the molecule. Those skilled in the art will be
familiar with the selection, attachment, and cleavage of amine
protecting groups, and will appreciate that many different
protective groups are known in the art, the suitability of one
protective group or another being dependent on the particular the
synthetic scheme planned. Treatises on the subject are available
for consultation, such as Wuts, P. G. M.; Greene, T. W., "Greene's
Protective Groups in Organic Synthesis", 4th Ed., J. Wiley &
Sons, N Y, 2007. Suitable protecting groups include the
carbobenzyloxy (Cbz), tert-butyloxycarbonyl (BOC),
9-fluorenylmethyloxycarbonyl (FMOC), and benzyl (Bn) group. In one
embodiment, the protecting group is the BOC group.
[0127] The compounds disclosed herein contain asymmetric centers
and thus give rise to enantiomers, diastereomers, and other
stereoisomeric forms. The present disclosure is meant to encompass
the use of all such possible forms, as well as their racemic and
resolved forms and mixtures thereof. The individual enantiomers can
be separated according to methods known in the art in view of the
present disclosure. When the compounds described herein contain
olefinic double bonds or other centers of geometric asymmetry, and
unless specified otherwise, it is intended that they include both E
and Z geometric isomers. All tautomers are also intended to be
encompassed by the present disclosure.
[0128] The present disclosure encompasses the preparation and use
of solvates of the compounds disclosed herein. Solvates typically
do not significantly alter the physiological activity or toxicity
of the compounds, and as such may function as pharmacological
equivalents. The term "solvate" as used herein is a combination,
physical association and/or solvation of a compound of the present
disclosure with a solvent molecule such as, e.g. a disolvate,
monosolvate or hemisolvate, where the ratio of solvent molecule to
compound of the present disclosure is about 2:1, about 1:1 or about
1:2, respectively. This physical association involves varying
degrees of ionic and covalent bonding, including hydrogen bonding.
In certain instances, the solvate can be isolated, such as when one
or more solvent molecules are incorporated into the crystal lattice
of a crystalline solid. Thus, "solvate" encompasses both
solution-phase and isolatable solvates. Compounds disclosed herein
can be present as solvated forms with a pharmaceutically acceptable
solvent, such as water, methanol, ethanol, and the like, and it is
intended that the disclosure includes both solvated and unsolvated
forms of compounds disclosed herein. One type of solvate is a
hydrate. A "hydrate" relates to a particular subgroup of solvates
where the solvent molecule is water. Solvates typically can
function as pharmacological equivalents. Preparation of solvates is
known in the art. See, for example, M. Caira et al, J. Pharmaceut.
Sci., 93(3):601-611 (2004), which describes the preparation of
solvates of fluconazole with ethyl acetate and with water. Similar
preparation of solvates, hemisolvates, hydrates, and the like are
described by E. C. van Tonder et al., AAPS Pharm. Sci. Tech.,
5(1):Article 12 (2004), and A. L. Bingham et al., Chem. Commun.
603-604 (2001). A typical, non-limiting, process of preparing a
solvate would involve dissolving a compound disclosed herein in a
desired solvent (organic, water, or a mixture thereof) at
temperatures above 20.degree. C. to about 25.degree. C., then
cooling the solution at a rate sufficient to form crystals, and
isolating the crystals by known methods, e.g., filtration.
Analytical techniques such as infrared spectroscopy can be used to
confirm the presence of the solvent in a crystal of the
solvate.
[0129] The present disclosure encompasses the preparation and use
of salts of the compounds disclosed herein, including non-toxic
pharmaceutically acceptable salts. Examples of pharmaceutically
acceptable addition salts include inorganic and organic acid
addition salts and basic salts. The pharmaceutically acceptable
salts include, but are not limited to, metal salts such as sodium
salt, potassium salt, cesium salt and the like; alkaline earth
metals such as calcium salt, magnesium salt and the like; organic
amine salts such as triethylamine salt, pyridine salt, picoline
salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine
salt, N,N'-dibenzylethylenediamine salt and the like; inorganic
acid salts such as hydrochloride, hydrobromide, phosphate, sulphate
and the like; organic acid salts such as citrate, lactate,
tartrate, maleate, fumarate, mandelate, acetate, dichloroacetate,
trifluoroacetate, oxalate, formate and the like; sulfonates such as
methanesulfonate, benzenesulfonate, p-toluenesulfonate and the
like; and amino acid salts such as arginate, asparginate, glutamate
and the like.
[0130] Acid addition salts can be formed by mixing a solution of
the particular compound disclosed with a solution of a
pharmaceutically acceptable non-toxic acid such as hydrochloric
acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric
acid, tartaric acid, carbonic acid, phosphoric acid, oxalic acid,
dichloroacetic acid, or the like. Basic salts can be formed by
mixing a solution of the compound of the present disclosure with a
solution of a pharmaceutically acceptable non-toxic base such as
sodium hydroxide, potassium hydroxide, choline hydroxide, sodium
carbonate and the like.
[0131] As used in the present disclosure and claims, the singular
forms "a," "an," and "the" include plural forms unless the context
clearly dictates otherwise.
[0132] It is understood that wherever embodiments are described
herein with the language "comprising," otherwise analogous
embodiments described in terms of "consisting of" and/or
"consisting essentially of" are also provided.
[0133] The term "and/or" as used in a phrase such as "A and/or B"
herein is intended to include both "A and B," "A or B," "A," and
"B." Likewise, the term "and/or" as used in a phrase such as "A, B,
and/or C" is intended to encompass each of the following
embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and
C; A and B; B and C; A (alone); B (alone); and C (alone).
II. Proteins for Linkage to Glucocorticoid Receptor Agonists
[0134] The present disclosure provides agents immunoconjugates
containing glucocorticoid receptor agonists linked to proteins, for
example, antibodies or antigen-binding fragments thereof and
soluble receptor proteins. In some embodiments, the antibody or
antigen-binding fragment thereof is human, humanized, chimeric, or
murine. In some embodiments, the protein, e.g., antibody,
antigen-binding fragment thereof, or soluble receptor protein, can
bind to a target on the surface of a cell and become
internalized.
[0135] The present disclosure also provides immunoconjugates
containing glucocorticoid receptor agonists linked to anti-TNF
alpha proteins. In certain embodiments, the anti-TNF alpha proteins
are antibodies or antigen-binding fragments thereof. In certain
embodiments, the anti-TNF alpha proteins are antibodies or
antigen-binding fragments thereof that bind to TNF alpha (e.g.,
soluble TNF alpha and/or membrane bound TNF alpha). In certain
embodiments, the anti-TNF alpha proteins are soluble TNF receptor
proteins, e.g., soluble TNF receptor proteins fused to a heavy
chain constant domain or fragment thereof such as an Fc. In some
embodiments, the anti-TNF alpha protein, e.g., anti-TNF antibody,
antigen-binding fragment thereof, or soluble TNF receptorcan bind
to TNF alpha on the surface of a cell and become internalized. For
example, US 2014/0294813, which is herein incorporated by reference
in its entirety, discloses anti-TNF proteins that exhibit cellular
internalization upon binding to cell surface human TNF.
[0136] In certain embodiments, the antibodies or antigen-binding
fragments thereof bind to human and/or mouse TNF-alpha. Antibodies
and antigen-binding fragments that bind to TNF-alpha are known in
the art.
[0137] The full-length amino acid sequence for membrane bound human
TNF alpha is:
TABLE-US-00002 (SEQ ID NO: 1)
MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGAT
TLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVA
HVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIY
SQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRE
TPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQ VYFGIIAL.
Soluble human TNF alpha contains amino acids 77-233 of SEQ ID NO:1.
The full-length amino acid sequence for membrane bound murine
TNF-alpha is:
TABLE-US-00003 (SEQ ID NO: 2)
MSTESMIRDVELAEEALPQKMGGFQNSRRCLCLSLFSFLLVAGAT
TLFCLLNFGVIGPQRDEKFPNGLPLISSMAQTLTLRSSSQNSSDK
PVAHVVANHQVEEQLEWLSQRANALLANGMDLKDNQLVVPADGLY
LVYSQVLFKGQGCPDYVLLTHTVSRFAISYQEKVNLLSAVKSPCP
KDTPEGAELKPWYEPIYLGGVFQLEKGDQLSAEVNLPKYLDFAES GQVYFGVIAL.
Soluble murine TNF alpha contains amino acids 80-235 of SEQ ID
NO:2.
[0138] In some embodiments, the anti-TNF-alpha antibody or
antigen-binding fragment thereof binds to human TNF-alpha. In some
embodiments, the anti-TNF-alpha antibody or antigen-binding
fragment thereof is human, humanized, or chimeric.
[0139] In some embodiments, the anti-TNF-alpha antibody or
antigen-binding fragment thereof binds to murine TNF-alpha. In some
embodiments, the anti-TNF-alpha antibody or antigen-binding
fragment thereof is murine.
[0140] In certain embodiments, the anti-TNF-alpha antibody or
antigen-binding fragment has one or more of the following effects:
neutralizes human TNF-alpha cytotoxicity in a in vitro L929 assay
with an IC50 of 1.times.10.sup.-7 M or less; blocks the interaction
of TNF-alpha with p55 and p75 cell surface receptors; and/or lyses
surface TNF expressing cells in vitro in the presence of
complement.
[0141] In certain embodiments, the anti-TNF-alpha antibody or
antigen-binding fragment does not bind to TNF-beta.
[0142] Anti-TNF-alpha antibodies and antigen-binding fragments
thereof include, for example, adalimumab, infliximab, certolizumab
pegol, afelimomab, nerelimomab, ozoralizumab, placulumab, and
golimumab. Additional anti-TNF-alpha antibodies and antigen-binding
fragments are provided, for example, in WO 2013/087912, WO
2014/152247 and WO 2015/073884, each of which is herein
incorporated by reference in its entirety.
[0143] Adalimumab is described in U.S. Pat. No. 6,258,562, which is
herein incorporated by reference in its entirety. Infliximab is
described in U.S. Pat. No. 5,656,272, which is herein incorporated
by reference in its entirety. Certolizumab is discussed in WO
01/94585, which is herein incorporated by reference in its
entirety. Afelimomab (also known as MAK195) is discussed in
Vincent, Int. J. Clin. Pract. 54: 190-193 (2000), which is herein
incorporated by reference in its entirety. Ozoralizumab (also known
as ATN-103) is a nanobody. It contains three heavy chain variable
regions fused by GlySer linkers. Variable regions 1 and 3 are
identical, and ozoralizumab does not contain a heavy chain.
Ozoralizumab is discussed in WO 2012/131053, which is herein
incorporated by reference in its entirety. Placulumab (also known
as CEP-37247) is a domain antibody consisting of a dimer of
VL-pCH1-CH2-CH3 or [V-kappa]2-Fc and is discussed in Gay et al.,
Mabs 2: 625-638 (2010), which is herein incorporated by reference
in its entirety. Golimumab (also known as CNTO 148) is discussed in
WO2013/087912, and sequences are provided in GenBank: DI496971.1
and GenBank DI 496970.1, each of which is herein incorporated by
reference in its entirety.
[0144] Anti-TNF-alpha antibodies and antigen-binding fragments
thereof also include antibodies and antigen-binding fragments
thereof that competitively inhibit binding of adalimumab,
infliximab, certolizumab pegol, afelimomab, nerelimomab,
ozoralizumab, placulumab, or golimumab to TNF-alpha. Anti-TNF-alpha
antibodies and antigen-binding fragments thereof also include
antibodies and antigen-binding fragments that bind to the same
TNF-alpha epitope as adalimumab, infliximab, certolizumab pegol,
afelimomab, nerelimomab, ozoralizumnab, placulumab, or
golimumab.
[0145] In certain embodiments, the anti-TNF-alpha antibody or
antigen-binding fragment thereof competitively inhibits binding of
adalimumab to TNF-alpha. In certain embodiments, the anti-TNF-alpha
antibody orantigen-binding fragment thereof binds to the same
TNF-alpha epitope as adalimumab. In certain embodiments, the
anti-TNF-alpha antibody or antigen-binding fragment thereof is
adalimumab or an antigen-binding fragment thereof. In certain
embodiments, the anti-TNF-alpha antibody or antigen-binding
fragment thereof is adalimumab.
[0146] In certain embodiments, an anti-TNF-alpha antibody or
antigen-binding fragment thereof comprises sequences of
(adalimumab, infliximab, certolizuab pegol, afelimomab,
nerelimomab, ozoralizumab, placulumab, or golimumab, e.g., the
complementarity-determining regions (CDRs),the variable heavy
domain (VH), and/or the variable light domain (VL). Sequences of
exemplary anti-TNF-alpha antibodies or antigen-binding fragments
thereof are provided in Tables 1-6.
TABLE-US-00004 TABLE 1 Variable heavy chain CDR amino acid
sequences: Antibody VH-CDR1 VH-CDR2 VH-CDR3 adalimumab DYAMH
AITWNSGHIDYADSVEG VSYLSTASS (SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID
NO: 5) or VSYLSTASSLDY GFTFDDYAMH (SEQ ID NO: 94) (SEQ ID NO: 6)
infliximab GFIFSNHWMN EIRSKSINSATHYAESVKG NYYGSTYDY (SEQ ID NO: 7)
(SEQ ID NO: 8) (SEQ ID NO: 9) certolizumab DYGMN WINTYIGEPIYADSVKG
GYRSYAMDY (SEQ ID NO: 10) (SEQ ID NO: 11) (SEQ ID NO: 12) or
GYVFTDYGMN (SEQ ID NO: 13) afelimomab DYGVN M1WGDGSTDYDSTLKS
EWHHGPVAY (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16)
nerelimomab DYNVD NINPNNGGTIYNQKFKG SAFYNNYEYFDV (SEQ ID NO: 17)
(SEQ ID NO: 18) (SEQ ID NO: 19) ozoralizumab V1: DYWMY V1: V1:
SPSGFNR (SEQ ID NO: 20) EINTNGLITKYPDSVKG (SEQ ID NO: 22) V2: SFGMS
(SEQ ID NO: 21) V2: GGSLSRSS (SEQ ID NO: 23) V2: (SEQ ID NO: 25)
V3: DYWMY SISGSGSDTLYADSVKG V3: SPSGFNR (SEQ ID NO: 26) (SEQ ID NO:
24) (SEQ ID NO: 28) V3: EINTNGLITKYPDSVKG (SEQ ID NO: 27) golimumab
GFIFSSYAMH FMSYDGSNKKY ADSVKG DRGIAAGGNYYYYGMDV (SEQ ID NO: 29)
(SEQ ID NO: 30) (SEQ ID NO: 31) placulumab RASQAIDSYLH SASNLET
QQVVWRPFT (SEQ ID NO: 88) (SEQ ID NO: 89) (SEQ ID NO: 90)
TABLE-US-00005 TABLE 2 Variable light chain CDR amino acid
sequences Antibody VL-CDR1 VL-CDR2 VL-CDR3 adalimumab RASQGIRNYLA
AASTLQS QRYNRAPYT (SEQ ID NO: 32) (SEQ ID NO: 33) (SEQ ID NO: 34)
infliximab RASQFVGSSIH YASESMS QQSHSWPFT (SEQ ID NO: 35) (SEQ ID
NO: 36) (SEQ ID NO: 37) certolizumab KASQNVGTNVA SASFLYS QQYNIYPLT
(SEQ ID NO: 38) (SEQ ID NO: 39) (SEQ ID NO: 40) afelimomab
KASQAVSSAVA WASTRHT QQHYSTPFT (SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ
ID NO: 43) nerelimomab KSSQSLLYSNNQKNYLA WASTRES QQYYDYPWT (SEQ ID
NO: 44) (SEQ ID NO: 45) (SEQ ID NO: 46) ozoralizumab N/A N/A N/A
golimumab RASQSVYSYLA DASNRAT QQRSNWPPFT (SEQ ID NO: 47) (SEQ ID
NO: 48) (SEQ ID NO: 49)
TABLE-US-00006 TABLE 3 Variable heavy chain amino acid sequences
Antibody VH Amino Acid Sequence (SEQ ID NO) adalimumab
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSA
ITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSY
LSTASSLDYWGQGTLVTVSS(SEQ ID NO: 50) infliximab
EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAE
IRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSRNY
YGSTYDYWGQGTTLTVSS(SEQ ID NO: 91)
EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAE
IRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMNSLRTEDTGVYYCSRNY
YGSTYDYWGQGTTLTVS(SEQ ID NO: 51) certolizumab
EVQLVESGGGLVQPGGSLRLSCAASGYVFTDYGMNWVRQAPGKGLEWMG
WINTYIGEPIYADSVKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARGY
RSYAMDYWGQGTLVTVSS(SEQ ID NO: 52) afelimomab
QVQLKESGPGLVAPSQSLSITCTVSGFSLTDYGVNWVRQPPGKGLEWLGMI
WGDGSTDYDSTLKSRLSISKDNSKSQIFLKNNSLQTDDTARYYCAREWHHG PVAYWGQGTLVTVSA
(SEQ ID NO: 53) nerelimomab
QVQLVQSGAEVVKPGSSVKVSCKASGYTFTDYNVDWVKQAPGQGLQWIG
NINPNNGGTIYNQKFKGKGTLTVDKSTSTAYMELSSLTSEDTAVYYCARSAF
YNNYEYFDVWGQGTTVTVSS(SEQ ID NO: 54) ozoralizumab V1:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSE
INTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSG FNRGQGTLVTVSS
(SEQ ID NO: 55) V2:
EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGK.GLEWVSSI
SGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLS RSSQGTLVTVSS
(SEQ ID NO: 56) V3:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSE
INTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSG FNRGQGTLVTVSS
(SEQ ID NO: 57) golimumab
QVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGNGLEWVAF
MSYDGSNKKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDR
GIAAGGNYYYYGMDVWGQGTTVTVSS(SEQ ID NO: 58)
TABLE-US-00007 TABLE 4 Variable light chain amino acid sequences
Antibody VL Amino Acid Sequence (SEQ ID NO) adalimumab
DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAAS
TLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKV EIK (SEQ ID
NO: 59) infliximab
D1LLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASES
MSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVK (SEQ ID NO:
60) certolizumab
DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSA
SFLYSGVPYRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPLTFGQGTKV EIK (SEQ ID
NO: 61) afelimomab
DIVMTQSHKFMSTTVGDRVSITCKASQAVSSAVAWYQQKPGQSPKLLIYWA
STRHTGVPDRFTGSGSVTDFTLTIHNLQAEDLALYYCQQHYSTPFTFGSGTK LEIK (SEQ ID
NO: 62) nerelimomab
DIMMTQSPSTLSASVGDRVTITCKSSQSLLYSNNQKNYLAWYQQKPGQAPK
LLISWASTRESGVPSRFIGSGSGTEFTLTISSLQPDDVATYYCQQYYDYPWTF GQGTKVEIK
(SEQ ID NO: 92) DIMMTQSPSTLSASVGDRVTITCKSSQSLLYSNNQKNYLAWYQQKPGQAPK
LLISWASTRESGVPSRFIGSGSGTEFTLTISSLQPDDVATYYCQQYYDYPWTF GQGTKVEIKR
(SEQ ID NO: 63) placulumab
DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYSAS
NLETGVPSRFSGSGSGTDFTLTISSLLPEDFATYYCOQVVWRPFTFGQGTKV EIK (SEQ ID
NO: 64) golimumab
EIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQKPGQAPRLLIYDAS
NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKV DIK (SEQ ID
NO: 65)
TABLE-US-00008 TABLE 5 Full-length heavy chain amino acid sequences
Antibody Full-Length Heavy Chain Amino Acid Sequence (SEQ ID NO)
Adalimumab EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSA
(D2E7) ITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSY
LSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 66) infliximab
EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAE
IRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSRNY
YGSTYDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
HKPSNTKVDKKVEPKSCDK.THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT
PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL
TKNQVSLTCLVKGFYPSD1AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 67) certolizumab
EVQLVESGGGLVQPGGSLRLSCAASGYVFTDYGMNWVRQAPGKGLEWMG
WINTYIGEPIYADSVKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARGY
RSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKKVEPKSCDKTHTCAA (SEQ ID NO: 68) afelimomab
QVQLKESGPGLVAPSQSLSITCTVSGFSLTDYGVNWVRQPPGKGLEWLGMI
WGDGSTDYDSTLKSRLSISKDNSKSQIFLKNNSLQTDDTARYYCAREWHHG
PVAYWGQGTLVTVSAATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEP
VTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPA
SKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTC
VVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQ
DWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKK
VSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDT
DSWLQGEIFTCSVVHEALHNHHTQKNLSRSPGK (SEQ ID NO: 69) ozoralizumab
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSE
INTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSG
FNRGQGTLVTVSSggggsgggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSSF
GMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQM
NSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSggggsgggsEVQLVESGGGLVQP
GGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSV
KGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSGFNRGQGTLVTVSS (SEQ ID NO: 70)
placulumab VEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 93)
RVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALPAP1EKTISKAKGQPREPQVYTLPPS
RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 71)
golimumab QVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGNGLE
WVAFMSYDGSNKKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA
VYYCARDRGIAAGGNYYYYGMDVWGQGTTVTVSSASTKGPSVFPL
APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSD1AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 72)
TABLE-US-00009 TABLE 6 Full-length light chain amino acid sequences
Antibody Full-length Light Chain Amino Acid Sequence (SEQ ID NO)
Adalimumab DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAAS
(D2E7) TLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKV
EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC (SEQ ID
NO: 73) infliximab
DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASES
MSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID
NO: 74) certolizumab
DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSA
SFLYSGVPYRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPLTFGQGTKV
EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC (SEQ ID
NO: 75) afelimomab
DIVMTQSHKFMSTTVGDRVSITCKASQAVSSAVAWYQQKPGQSPKLLIYWA
STRHTGVPDRFTGSGSVTDFTLTIHNLQAEDLALYYCQQHYSTPFTFGSGTK
LEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ
NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKS FNRNEC (SEQ ID
NO: 76) placulumab
DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYSAS
NLETGVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQGTKV EIKR (SEQ ID
NO: 77) golimumab
EIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQKPGQAPRLLIYDAS
NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKV
DIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC (SEQ ID
NO: 78)
[0147] Also provided are antibodies or antigen-binding fragments
that comprise a VH and a VL having at least 80% sequence identity
to SEQ ID NOs: 50 and 59, 51 and 60, 52 and 61, 53 and 62, 54 and
63, or 58 and 65, respectively. Also provided are antibodies or
antigen-binding fragments that comprise a VH and a VL having at
least 85% sequence identity to SEQ ID NOs: 50 and 59, 51 and 60, 52
and 61, 53 and 62, 54 and 63, or 58 and 65, respectively. Also
provided are antibodies or antigen-binding fragments that comprise
a VH and a VL having at least 85% sequence identity to SEQ ID NOs:
91 and 60, or 54 and 92, respectively. Also provided are antibodies
or antigen-binding fragments that comprise a VH and a VL having at
least 90% sequence identity to SEQ ID NOs: 50 and 59, 51 and 60, 52
and 61, 53 and 62, 54 and 63, or 58 and 65, respectively. Also
provided are antibodies or antigen-binding fragments that comprise
a VH and a VL having at least 90% sequence identity to SEQ ID NOs:
91 and 60, or 54 and 92, respectively. Also provided are antibodies
or antigen-binding fragments that comprise a VH and a VL having at
least 95% sequence identity to SEQ ID NOs: 50 and 59, 51 and 60, 52
and 61, 53 and 62, 54 and 63, or 58 and 65, respectively. Also
provided are antibodies or antigen-binding fragments that comprise
a VH and a VL having at least 95% sequence identity to SEQ ID NOs:
91 and 60, or 54 and 92, respectively. Also provided are antibodies
or antigen-binding fragments that comprise a VH and a VL having at
least 96% sequence identity to SEQ ID NOs: 50 and 59, 51 and 60, 52
and 61, 53 and 62, 54 and 63, or 58 and 65, respectively. Also
provided are antibodies or antigen-binding fragments that comprise
a VH and a VL having at least 96% sequence identity to SEQ ID NOs:
91 and 60, or 54 and 92, respectively. Also provided are antibodies
or antigen-binding fragments that comprise a VH and a VL having at
least 97% sequence identity to SEQ ID NOs: 50 and 59, 51 and 60, 52
and 61, 53 and 62, 54 and 63, or 58 and 65, respectively. Also
provided are antibodies or antigen-binding fragments that comprise
a VH and a VL having at least 97% sequence identity to SEQ ID NOs:
91 and 60, or 54 and 92, respectively. Also provided are antibodies
or antigen-binding fragments that comprise a VH and a VL having at
least 98% sequence identity to SEQ ID NOs: 50 and 59, 51 and 60, 52
and 61, 53 and 62, 54 and 63, or 58 and 65, respectively. Also
provided are antibodies or antigen-binding fragments that comprise
a VH and a VL having at least 98% sequence identity to SEQ ID NOs:
91 and 60, or 54 and 92, respectively. Also provided are antibodies
or antigen-binding fragments that comprise a VH and a VL having at
least 99% sequence identity to SEQ ID NOs: 50 and 59, 51 and 60, 52
and 61, 53 and 62, 54 and 63, or 58 and 65, respectively. Also
provided are antibodies or antigen-binding fragments that comprise
a VH and a VL having at least 99% sequence identity to SEQ ID NOs:
91 and 60, or 54 and 92, respectively.
[0148] Also provided are antibodies or antigen-binding fragments
that comprise a VH and a VL having at least 80% sequence identity
to SEQ ID NOs: 50 and 59, 51 and 60, 52 and 61, 53 and 62, 54 and
63, or 58 and 65, respectively, and contain the CDRs of SEQ ID NOs:
3 or 6, 4, 5, and 32-34; 7-9 and 35-37; 10 or 13, 11, 12, and
38-40; 14-16, and 41-43; 17-19 and 44-46; or 29-31 and 47-49,
respectively. Also provided are antibodies or antigen-binding
fragments that comprise a VH and a VL having at least 80% sequence
identity to SEQ ID NOs: 50 and 59, 91 and 60, or 54 and 92
respectively, and contain the CDRs of SEQ ID NOs: 3 or 6, 4, 94,
and 32-34; 7-9 and 35-37; or 17-19 and 44-46, respectively. Also
provided are antibodies or antigen-binding fragments that comprise
a VH and a VL having at least 85% sequence identity to SEQ ID NOs:
50 and 59, 51 and 60, 52 and 61, 53 and 62, 54 and 63, or 58 and
65, respectively and contain the CDRs of SEQ ID NOs: 3 or 6, 4, 5,
and 32-34; 7-9 and 35-37; 10 or 13, 11, 12, and 38-40; 14-16, and
41-43; 17-19 and 44-46; or 29-31 and 47-49, respectively. Also
provided are antibodies or antigen-binding fragments that comprise
a VH and a VL having at least 85% sequence identity to SEQ ID NOs:
50 and 59, 91 and 60, or 54 and 92 respectively, and contain the
CDRs of SEQ ID NOs: 3 or 6, 4, 94, and 32-34; 7-9 and 35-37; or
17-19 and 44-46, respectively. Also provided are antibodies or
antigen-binding fragments that comprise a VH and a VL having at
least 90% sequence identity to SEQ ID NOs: 50 and 59, 51 and 60, 52
and 61, 53 and 62, 54 and 63, or 58 and 65, respectively and
contain the CDRs of SEQ ID NOs: 3 or 6, 4, 5, and 32-34; 7-9 and
35-37; 10 or 13, 11, 12, and 38-40; 14-16, and 41-43; 17-19 and
44-46; or 29-31 and 47-49, respectively. Also provided are
antibodies or antigen-binding fragments that comprise a VH and a VL
having at least 90% sequence identity to SEQ ID NOs: 50 and 59, 91
and 60, or 54 and 92 respectively, and contain the CDRs of SEQ ID
NOs: 3 or 6, 4, 94, and 32-34; 7-9 and 35-37; or 17-19 and 44-46,
respectively. Also provided are antibodies or antigen-binding
fragments that comprise a VH and a VL having at least 95% sequence
identity to SEQ ID NOs: 50 and 59, 51 and 60, 52 and 61, 53 and 62,
54 and 63, or 58 and 65, respectively and contain the CDRs of SEQ
ID NOs: 3 or 6, 4, 5, and 32-34; 7-9 and 35-37; 10 or 13, 11, 12,
and 38-40; 14-16, and 41-43; 17-19 and 44-46; or 29-31 and 47-49,
respectively. Also provided are antibodies or antigen-binding
fragments that comprise a VH and a VL having at least 95% sequence
identity to SEQ ID NOs: 50 and 59, 91 and 60, or 54 and 92
respectively, and contain the CDRs of SEQ ID NOs: 3 or 6, 4, 94,
and 32-34; 7-9 and 35-37; or 17-19 and 44-46, respectively. Also
provided are antibodies or antigen-binding fragments that comprise
a VH and a VL having at least 96% sequence identity to SEQ ID NOs:
50 and 59, 51 and 60, 52 and 61, 53 and 62, 54 and 63, or 58 and
65, respectively and contain the CDRs of SEQ ID NOs: 3 or 6, 4, 5,
and 32-34; 7-9 and 35-37; 10 or 13, 11, 12, and 38-40; 14-16, and
41-43; 17-19 and 44-46; or 29-31 and 47-49, respectively. Also
provided are antibodies or antigen-binding fragments that comprise
a VH and a VL having at least 96% sequence identity to SEQ ID NOs:
50 and 59, 91 and 60, or 54 and 92 respectively, and contain the
CDRs of SEQ ID NOs: 3 or 6, 4, 94, and 32-34; 7-9 and 35-37; or
17-19 and 44-46, respectively. Also provided are antibodies or
antigen-binding fragments that comprise a VH and a VL having at
least 97% sequence identity to SEQ ID NOs: 50 and 59, 51 and 60, 52
and 61, 53 and 62, 54 and 63, or 58 and 65, respectively and
contain the CDRs of SEQ ID NOs: 50 and 59, 51 and 60, 52 and 61, 53
and 62, 54 and 63, or 58 and 65, respectively. Also provided are
antibodies or antigen-binding fragments that comprise a VH and a VL
having at least 97% sequence identity to SEQ ID NOs: 50 and 59, 91
and 60, or 54 and 92 respectively, and contain the CDRs of SEQ ID
NOs: 3 or 6, 4, 94, and 32-34; 7-9 and 35-37; or 17-19 and 44-46,
respectively. Also provided are antibodies or antigen-binding
fragments that comprise a VH and a VL having at least 98% sequence
identity to SEQ ID NOs: 50 and 59, 51 and 60, 52 and 61, 53 and 62,
54 and 63, or 58 and 65, respectively and contain the CDRs of SEQ
ID NOs: 3 or 6, 4, 5, and 32-34; 7-9 and 35-37; 10 or 13, 11, 12,
and 38-40; 14-16, and 41-43; 17-19 and 44-46; or 29-31 and 47-49,
respectively. Also provided are antibodies or antigen-binding
fragments that comprise a VH and a VL having at least 98% sequence
identity to SEQ ID NOs: 50 and 59, 91 and 60, or 54 and 92
respectively, and contain the CDRs of SEQ ID NOs: 3 or 6, 4, 94,
and 32-34; 7-9 and 35-37; or 17-19 and 44-46, respectively. Also
provided are antibodies or antigen-binding fragments that comprise
a VH and a VL having at least 99% sequence identity to SEQ ID NOs:
50 and 59, 51 and 60, 52 and 61, 53 and 62, 54 and 63, or 58 and
65, respectively and contain the CDRs of SEQ ID NOs: 3 or 6, 4, 5,
and 32-34; 7-9 and 35-37; 10 or 13, 11, 12, and 38-40; 14-16, and
41-43; 17-19 and 44-46; or 29-31 and 47-49, respectively. Also
provided are antibodies or antigen-binding fragments that comprise
a VH and a VL having at least 99% sequence identity to SEQ ID NOs:
50 and 59, 91 and 60, or 54 and 92 respectively, and contain the
CDRs of SEQ ID NOs: 3 or 6, 4, 94, and 32-34; 7-9 and 35-37; or
17-19 and 44-46, respectively.
[0149] In certain embodiments, the anti-TNF alpha antibody or
antigen-binding fragment thereof comprises the CDRs of SEQ ID NOs:
3-5 and 32-34 or of SEQ ID NOs: 6, 4, 5, and 32-34. In certain
embodiments, the anti-TNF alpha antibody or antigen-binding
fragment thereof comprises the CDRs of SEQ ID NOs: 3, 4, 94 and
32-34 or of SEQ ID NOs: 6, 4, 94, and 32-34. In certain
embodiments, the anti-TNF alpha antibody or antigen-binding
fragment thereof comprises the VH of SEQ ID NO:50 and/or the VL of
SEQ ID NO:59. In certain embodiments, the anti-TNF alpha antibody
comprises the heavy chain of SEQ ID NO: 66 and/or the light chain
of SEQ ID NO:75.
[0150] F alpha antibody comprises the heavy chain of SEQ ID NO:74
and/or the light chain of SEQ ID NO:82.
[0151] In certain aspects, provided herein are antibodies or
antigen-binding fragments thereof that specifically bind to
TNF-alpha and comprise the Chothia VL CDRs of a VL of adalimumab,
infliximab, certolizumab pegol, afelimomab, nerelimomab,
ozoralizumab, placulumab, or golimumab. In certain aspects,
provided herein are antibodies or antigen-binding fragments thereof
that specifically bind to TNF-alpha and comprise the Chothia VH
CDRs of a VH of adalimumab, infliximab, certolizumab pegol,
afelimomab, nerelimomab, ozoralizumab, placulumab, or golimumab. In
certain aspects, provided herein are antibodies or antigen-binding
fragments thereof that specifically bind to TNF-alpha and comprise
the Chothia VL CDRs of a VL of adalimumab, infliximab, certolizumab
pegol, afelimomab, nerelimomab, ozoralizumab, placulumab, or
golimumab and comprise the Chothia VH CDRs of a VH of adalimumab,
infliximab, certolizumab pegol, afelimomab, nerelimomab,
ozoralizumab, placulumab, or golimumab. In certain embodiments,
antibodies or antigen-binding fragments that specifically bind to
TNF-alpha comprise one or more CDRs, in which the Chothia and Kabat
CDRs have the same amino acid sequence. In certain embodiments,
provided herein are antibodies and antigen-binding fragments
thereof that specifically bind to TNF-alpha and comprise
combinations of Kabat CDRs and Chothia CDRs.
[0152] In a particular embodiment, provided herein are antibodies
or antigen-binding fragments thereof that specifically bind to
TNF-alpha and comprise CDRs of adalimumab, infliximab, certolizumab
pegol, afelimomab, nerelimomab, ozoralizumab, placulumab, or
golimumab as determined by the IMGT numbering system, for example,
as described in Lefranc M-P (1999) supra and Lefranc M-P et al.,
(1999) supra).
[0153] In a particular embodiment, provided herein are antibodies
that specifically bind to TNF-alpha and comprise CDRs of
adalimumab, infliximab, certolizumab pegol, afelimomab,
nerelimomab, ozoralizumab, placulumab, or golimumab as determined
by the method in MacCallum R M et al.
[0154] In a particular embodiment, provided herein are antibodies
or antigen-binding fragments thereof that specifically bind to
TNF-alpha and comprise CDRs of adalimumab, infliximab, certolizumab
pegol, afelimomab, nerelimomab, ozoralizumab, placulumab, or
golimumab as determined by the AbM numbering scheme.
[0155] In a particular embodiment, provided herein are antibodies
or antigen-binding fragments thereof that specifically bind to
CD163.
[0156] Monoclonal antibodies can be prepared using hybridoma
methods, such as those described by Kohler and Milstein (1975)
Nature 256:495. Using the hybridoma method, a mouse, hamster, or
other appropriate host animal, is immunized to elicit the
production by lymphocytes of antibodies that will specifically bind
to an immunizing antigen. Lymphocytes can also be immunized in
vitro. Following immunization, the lymphocytes are isolated and
fused with a suitable myeloma cell line using, for example,
polyethylene glycol, to form hybridoma cells that can then be
selected away from unfused lymphocytes and myeloma cells.
Hybridomas that produce monoclonal antibodies directed specifically
against a chosen antigen as determined by immunoprecipitation,
immunoblotting, or by an in vitro binding assay (e.g.,
radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA))
can then be propagated either in vitro culture using standard
methods (Goding, Monoclonal Antibodies: Principles and Practice,
Academic Press, 1986) or in vivo as ascites tumors in an animal.
The monoclonal antibodies can then be purified from the culture
medium or ascites fluid as described for polyclonal antibodies.
[0157] Alternatively monoclonal antibodies can also be made using
recombinant DNA methods as described in U.S. Pat. No. 4,816,567.
The polynucleotides encoding a monoclonal antibody are isolated
from mature B-cells or hybridoma cells, such as by RT-PCR using
oligonucleotide primers that specifically amplify the genes
encoding the heavy and light chains of the antibody, and their
sequence is determined using conventional procedures. The isolated
polynucleotides encoding the heavy and light chains are then cloned
into suitable expression vectors, which when transfected into host
cells such as E. coli cells, simian COS cells, Chinese hamster
ovary (CHO) cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, monoclonal antibodies are generated by the
host cells. Also, recombinant monoclonal antibodies or fragments
thereof of the desired species can be isolated from phage display
libraries expressing CDRs of the desired species as described
(McCafferty et al., 1990, Nature, 348:552-554; Clackson et al.,
1991, Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol.,
222:581-597).
[0158] The polynucleotide(s) encoding a monoclonal antibody can
further be modified in a number of different manners using
recombinant DNA technology to generate alternative antibodies. In
some embodiments, the constant domains of the light and heavy
chains of, for example, a mouse monoclonal antibody can be
substituted 1) for those regions of, for example, a human antibody
to generate a chimeric antibody or 2) for a non-immunoglobulin
polypeptide to generate a fusion antibody. In some embodiments, the
constant regions are truncated or removed to generate the desired
antibody fragment of a monoclonal antibody. Site-directed or
high-density mutagenesis of the variable region can be used to
optimize specificity, affinity, etc. of a monoclonal antibody.
[0159] In some embodiments, the monoclonal antibody against the
TNF-alpha is a humanized antibody. In certain embodiments, such
antibodies are used therapeutically to reduce antigenicity and HAMA
(human anti-mouse antibody) responses when administered to a human
subject.
[0160] Methods for engineering, humanizing or resurfacing non-human
or human antibodies can also be used and are well known in the art.
A humanized, resurfaced or similarly engineered antibody can have
one or more amino acid residues from a source that is non-human,
e.g., but not limited to, mouse, rat, rabbit, non-human primate or
other mammal. These non-human amino acid residues are replaced by
residues that are often referred to as "import" residues, which are
typically taken from an "import" variable, constant or other domain
of a known human sequence.
[0161] Such imported sequences can be used to reduce immunogenicity
or reduce, enhance or modify binding, affinity, on-rate, off-rate,
avidity, specificity, half-life, or any other suitable
characteristic, as known in the art. In general, the CDR residues
are directly and most substantially involved in influencing
TNF-alpha binding. Accordingly, part or all of the non-human or
human CDR sequences are maintained while the non-human sequences of
the variable and constant regions can be replaced with human or
other amino acids.
[0162] Antibodies can also optionally be humanized, resurfaced,
engineered or human antibodies engineered with retention of high
affinity for the antigen e.g., TNF-alpha, and other favorable
biological properties. To achieve this goal, humanized (or human)
or engineered antibodies and resurfaced antibodies can be
optionally prepared by a process of analysis of the parental
sequences and various conceptual humanized and engineered products
using three-dimensional models of the parental, engineered, and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen, such as TNF-alpha. In this way,
framework (FR) residues can be selected and combined from the
consensus and import sequences so that the desired antibody
characteristic, such as increased affinity for the target
antigen(s), is achieved.
[0163] Humanization, resurfacing or engineering of antibodies of
the present disclosure can be performed using any known method,
such as but not limited to those described in, Winter (Jones et
al., Nature 321:522 (1986); Riechmann et al., Nature 332:323
(1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J.
Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901
(1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285
(1992); Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat. Nos.
5,639,641, 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476;
5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370;
5,693,762; 5,530,101; 5,585,089; 5,225,539; 4,816,567; PCT/:
US98/16280; US96/18978; US91/09630; US91/05939; US94/01234;
GB89/01334; GB91/01134; GB92/01755; WO90/14443; WO90/14424;
WO90/14430; EP 229246; 7,557,189; 7,538,195; and 7,342,110, each of
which is entirely incorporated herein by reference, including the
references cited therein.
[0164] In certain alternative embodiments, the antibody (e.g., an
anti-TNFalpha antibody) is a human antibody. Human antibodies can
be directly prepared using various techniques known in the art.
Immortalized human B lymphocytes immunized in vitro or isolated
from an immunized individual that produce an antibody directed
against a target antigen can be generated (See, e.g., Cole et al.,
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77
(1985); Boemer et al., 1991, J. Immunol., 147 (1):86-95; and U.S.
Pat. No. 5,750,373). Also, the human antibody can be selected from
a phage library, where that phage library expresses human
antibodies, as described, for example, in Vaughan et al., 1996,
Nat. Biotech., 14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad.
Sci., 95:6157-6162, Hoogenboom and Winter, 1991, J. Mol. Biol.,
227:381, and Marks et al., 1991, J. Mol. Biol., 222:581).
Techniques for the generation and use of antibody phage libraries
are also described in U.S. Pat. Nos. 5,969,108, 6,172,197,
5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081;
6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et al.,
2007, J. Mol. Bio., doi:10.1016/j.jmb.2007.12.018 (each of which is
incorporated by reference in its entirety). Affinity maturation
strategies and chain shuffling strategies (Marks et al., 1992,
Bio/Technology 10:779-783, incorporated by reference in its
entirety) are known in the art and can be employed to generate high
affinity human antibodies.
[0165] Humanized antibodies can also be made in transgenic mice
containing human immunoglobulin loci that are capable upon
immunization of producing the full repertoire of human antibodies
in the absence of endogenous immunoglobulin production. This
approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; and 5,661,016.
[0166] In certain embodiments are provided an antibody fragment to,
for example, increase tumor penetration. Various techniques are
known for the production of antibody fragments. Traditionally,
these fragments are derived via proteolytic digestion of intact
antibodies (for example Morimoto et al., 1993, Journal of
Biochemical and Biophysical Methods 24:107-117; Brennan et al.,
1985, Science, 229:81). In certain embodiments, antibody fragments
are produced recombinantly. Fab, Fv, and scFv antibody fragments
can all be expressed in and secreted from E. coli or other host
cells, thus allowing the production of large amounts of these
fragments. Such antibody fragments can also be isolated from
antibody phage libraries. The antibody fragment can also be linear
antibodies as described in U.S. Pat. No. 5,641,870. Other
techniques for the production of antibody fragments will be
apparent to the skilled practitioner.
[0167] For the purposes of the present disclosure, it should be
appreciated that modified antibodies can comprise any type of
variable region that provides for the association of the antibody
with the antigen (e.g., TNF alpha). In this regard, the variable
region can comprise or be derived from any type of mammal that can
be induced to mount a humoral response and generate immunoglobulins
against the desired tumor associated antigen. As such, the variable
region of the modified antibodies can be, for example, of human,
murine, non-human primate (e.g., cynomolgus monkeys, macaques,
etc.) or lupine origin. In some embodiments both the variable and
constant regions of the modified immunoglobulins are human. In
other embodiments the variable regions of compatible antibodies
(usually derived from a non-human source) can be engineered or
specifically tailored to improve the binding properties or reduce
the immunogenicity of the molecule. In this respect, variable
regions useful in the present disclosure can be humanized or
otherwise altered through the inclusion of imported amino acid
sequences.
[0168] In certain embodiments, the variable domains in both the
heavy and light chains are altered by at least partial replacement
of one or more CDRs and, if necessary, by partial framework region
replacement and sequence changing. Although the CDRs can be derived
from an antibody of the same class or even subclass as the antibody
from which the framework regions are derived, it is envisaged that
the CDRs will be derived from an antibody of different class and in
certain embodiments from an antibody from a different species. It
may not be necessary to replace all of the CDRs with the complete
CDRs from the donor variable region to transfer the antigen-binding
capacity of one variable domain to another. Rather, it may only be
necessary to transfer those residues that are necessary to maintain
the activity of the antigen-binding site. Given the explanations
set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it
will be well within the competence of those skilled in the art,
either by carrying out routine experimentation or by trial and
error testing to obtain a functional antibody with reduced
immunogenicity.
[0169] Anti-TNF alpha proteins include soluble TNF receptor
proteins. The anti-TNF alpha protein can be a soluble p75 TNF
receptor. The anti-TNF alpha protein can be a soluble p55 TNF
receptor.
[0170] The soluble TNF receptor can bind to both TNF alpha and TNF
beta. The soluble TNF receptor can bind to TNF alpha, but not to
TNF beta.
[0171] The soluble TNF receptor can inhibit binding of TNF alpha
(and optionally TNF beta) to cell surface TNF receptors.
[0172] The soluble TNF receptor can be etanercept.
[0173] An anti-TNF alpha protein, e.g., a soluble TNF receptor, can
be fused to a heavy chain constant domain or fragment thereof or an
Fc region or fragment thereof. The heavy chain constant domain
fragment or Fc fragment can be a portion of the constant domain or
Fc that is capable of binding to Fc receptor. The heavy chain
constant domain fragment or Fc fragment can be a portion of the
constant domain or Fc that is capable of inducing cell lysis in
vitro in the presence of complement. The heavy chain constant
domain fragment or Fc fragment can be a portion of the constant
domain or Fc that is capable of inducing ADCC.
[0174] The heavy chain constant domain or fragment thereof or Fc
region or fragment thereof can be a human heavy chain constant
domain or fragment thereof or human Fc region or fragment thereof.
The heavy chain constant domain or fragment thereof or Fc region or
fragment thereof can be an IgG1 heavy chain constant domain or
fragment thereof or an IgG1 Fc region or fragment thereof. The
heavy chain constant domain or fragment thereof or Fc region or
fragment thereof can be a human IgG1 heavy chain constant domain or
fragment thereof or human IgG1 Fc region or fragment thereof.
[0175] Those skilled in the art will appreciate that the antibodies
and antigen-binding fragments thereof of this disclosure and the
anti-TNF proteins of this disclosure include antibodies,
antigen-binding fragments thereof, and anti-TNF proteins (e.g.,
full-length antibodies, antigen-binding fragments of antibodies, or
soluble TNF receptor proteins) comprising one or more of constant
region domains, including domains that have been altered so as to
provide desired biochemical characteristics such as reduced serum
half-life when compared with an antibody, antigen-binding fragment
thereof, or anti-TNF protein of approximately the same
immunogenicity comprising a native or unaltered constant region. In
some embodiments, the constant region of the antibody,
antigen-binding fragment thereof, or anti-TNF protein (e.g.,
full-length antibodies, antigen-binding fragments of antibodies, or
soluble TNF receptor proteins) will comprise a human constant
region. Modifications to the constant region compatible with this
disclosure comprise additions, deletions, or substitutions of one
or more amino acids in one or more domains. That is, the antibody,
antigen-binding fragment thereof, or anti-TNF proteins (e.g.,
full-length antibodies, antigen-binding fragments of antibodies, or
soluble TNF receptor proteins) disclosed herein can comprise
alterations or modifications to one or more of the three heavy
chain constant domains (CH1, CH2 or CH3) and/or to the light chain
constant domain (CL). In some embodiments, modified constant
regions wherein one or more domains are partially or entirely
deleted are contemplated. In some embodiments, the antibodies,
antigen-binding fragments thereof, or anti-TNF proteins (e.g.,
full-length antibodies, antigen-binding fragments of antibodies, or
soluble TNF receptor proteins) will comprise domain deleted
constructs or variants wherein the entire CH2 domain has been
removed (.DELTA.CH2 constructs). In some embodiments, the omitted
constant region domain will be replaced by a short amino acid
spacer (e.g., 10 residues) that provides some of the molecular
flexibility typically imparted by the absent constant region.
[0176] It will be noted that in certain embodiments, the
antibodies, antigen-binding fragments thereof, or anti-TNF proteins
(e.g., full-length antibodies, antigen-binding fragments of
antibodies, or soluble TNF receptor proteins) can be engineered to
fuse the CH3 domain directly to the hinge region of the respective
antibodies, antigen-binding fragments thereof, or anti-TNF proteins
(e.g., full-length antibodies, antigen-binding fragments of
antibodies, or soluble TNF receptor proteins). In other constructs
it can be desirable to provide a peptide spacer between the hinge
region and the modified CH2 and/or CH3 domains. For example,
compatible constructs could be expressed wherein the CH2 domain has
been deleted and the remaining CH3 domain (modified or unmodified)
is joined to the hinge region with a 5-20 amino acid spacer. Such a
spacer can be added, for instance, to ensure that the regulatory
elements of the constant domain remain free and accessible or that
the hinge region remains flexible. However, it should be noted that
amino acid spacers can, in some cases, prove to be immunogenic and
elicit an unwanted immune response against the construct.
Accordingly, in certain embodiments, any spacer added to the
construct will be relatively non-immunogenic, or even omitted
altogether, so as to maintain the desired biochemical qualities of
the antibodies, antigen-binding fragments thereof, or anti-TNF
proteins (e.g., full-length antibodies, antigen-binding fragments
of antibodies, or soluble TNF receptor proteins).
[0177] It will be appreciated that the antibodies, antigen-binding
fragments thereof, and anti-TNF proteins (e.g., full-length
antibodies, antigen-binding fragments of antibodies, or soluble TNF
receptor proteins) of the present disclosure can be provided by the
partial deletion or substitution of a few or even a single amino
acid. For example, the mutation of a single amino acid in selected
areas of the CH2 domain can be enough to substantially reduce Fc
binding and thereby increase tumor localization. Similarly, it may
be desirable to simply delete that part of one or more constant
region domains that control the effector function (e.g., complement
C1Q binding) to be modulated. Such partial deletions of the
constant regions can improve selected characteristics of the
antibody (serum half-life) while leaving other desirable functions
associated with the subject constant region domain intact.
Moreover, as alluded to above, the constant regions of the
disclosed antibodies, antigen-binding fragments thereof, and
anti-TNF proteins (e.g., full-length antibodies, antigen-binding
fragments of antibodies, or soluble TNF receptor proteins) can be
modified through the mutation or substitution of one or more amino
acids that enhances the profile of the resulting construct. In this
respect it can be possible to disrupt the activity provided by a
conserved binding site (e.g., Fc binding) while substantially
maintaining the configuration and immunogenic profile of the
antibodies, antigen-binding fragments thereof, and anti-TNF
proteins (e.g., full-length antibodies, antigen-binding fragments
of antibodies, or soluble TNF receptor proteins). Certain
embodiments can comprise the addition of one or more amino acids to
the constant region to enhance desirable characteristics such as
decreasing or increasing effector function or provide for more
glucocorticoid receptor agonist attachment. In such embodiments it
can be desirable to insert or replicate specific sequences derived
from selected constant region domains.
[0178] It will be appreciated that the antibodies, antigen-binding
fragments thereof, and anti-TNF proteins (e.g., full-length
antibodies, antigen-binding fragments of antibodies, or soluble TNF
receptor proteins) of the present disclosure can be modified to
reduce immunogenicity, i.e., to reduce the anti-drug immune
response (ADA). Methods of doing so are disclosed, for example, in
WO 2015/073884, which is herein incorporated by reference in its
entirety.
[0179] The present disclosure further embraces variants and
equivalents which are substantially homologous to antibodies,
antigen-binding fragments thereof, and anti-TNF proteins (e.g.,
full-length antibodies, antigen-binding fragments of antibodies, or
soluble TNF receptor proteins) set forth herein. These can contain,
for example, conservative substitution mutations, i.e., the
substitution of one or more amino acids by similar amino acids. For
example, conservative substitution refers to the substitution of an
amino acid with another within the same general class such as, for
example, one acidic amino acid with another acidic amino acid, one
basic amino acid with another basic amino acid or one neutral amino
acid by another neutral amino acid. What is intended by a
conservative amino acid substitution is well known in the art.
[0180] The polypeptides of the present disclosure can be
recombinant polypeptides, natural polypeptides, or synthetic
polypeptides of an antibody, antigen-binding fragment thereof, or
anti-TNF protein. It will be recognized in the art that some amino
acid sequences of the disclosure can be varied without significant
effect of the structure or function of the protein. Thus, the
disclosure further includes variations of the polypeptides which
show substantial activity or which include regions of an antibody,
antigen-binding fragment thereof, or anti-TNF alpha protein. Such
mutants include deletions, insertions, inversions, repeats, and
type substitutions.
[0181] The polypeptides and analogs can be further modified to
contain additional chemical moieties not normally part of the
protein. Those derivatized moieties can improve the solubility, the
biological half life or absorption of the protein. The moieties can
also reduce or eliminate any desirable side effects of the proteins
and the like. An overview for those moieties can be found in
REMINGTON'S PHARMACEUTICAL SCIENCES, 20th ed., Mack Publishing Co.,
Easton, Pa. (2000).
[0182] The isolated polypeptides described herein can be produced
by any suitable method known in the art. Such methods range from
direct protein synthetic methods to constructing a DNA sequence
encoding isolated polypeptide sequences and expressing those
sequences in a suitable transformed host. In some embodiments, a
DNA sequence is constructed using recombinant technology by
isolating or synthesizing a DNA sequence encoding a wild-type
protein of interest. Optionally, the sequence can be mutagenized by
site-specific mutagenesis to provide functional analogs thereof.
See, e.g., Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066
(1984) and U.S. Pat. No. 4,588,585.
[0183] In some embodiments a DNA sequence encoding a polypeptide of
interest would be constructed by chemical synthesis using an
oligonucleotide synthesizer. Such oligonucleotides can be designed
based on the amino acid sequence of the desired polypeptide and
selecting those codons that are favored in the host cell in which
the recombinant polypeptide of interest will be produced. Standard
methods can be applied to synthesize an isolated polynucleotide
sequence encoding an isolated polypeptide of interest. For example,
a complete amino acid sequence can be used to construct a
back-translated gene. Further, a DNA oligomer containing a
nucleotide sequence coding for the particular isolated polypeptide
can be synthesized. For example, several small oligonucleotides
coding for portions of the desired polypeptide can be synthesized
and then ligated. The individual oligonucleotides typically contain
5' or 3' overhangs for complementary assembly.
[0184] Once assembled (by synthesis, site-directed mutagenesis or
another method), the polynucleotide sequences encoding a particular
isolated polypeptide of interest will be inserted into an
expression vector and operatively linked to an expression control
sequence appropriate for expression of the protein in a desired
host. Proper assembly can be confirmed by nucleotide sequencing,
restriction mapping, and expression of a biologically active
polypeptide in a suitable host. As is well known in the art, in
order to obtain high expression levels of a transfected gene in a
host, the gene must be operatively linked to transcriptional and
translational expression control sequences that are functional in
the chosen expression host.
[0185] In certain embodiments, recombinant expression vectors are
used to amplify and express DNA encoding antibodies,
antigen-binding fragments thereof, or anti-TNF proteins (e.g.,
full-length antibodies, antigen-binding fragments of antibodies, or
soluble TNF receptor proteins). Recombinant expression vectors are
replicable DNA constructs which have synthetic or cDNA-derived DNA
fragments encoding a polypeptide chain of an antibody,
antigen-binding fragment thereof, or anti-TNF protein (e.g.,
full-length antibodies, antigen-binding fragments of antibodies, or
soluble TNF receptor proteins), operatively linked to suitable
transcriptional or translational regulatory elements derived from
mammalian, microbial, viral or insect genes. A transcriptional unit
generally comprises an assembly of (1) a genetic element or
elements having a regulatory role in gene expression, for example,
transcriptional promoters or enhancers, (2) a structural or coding
sequence which is transcribed into mRNA and translated into
protein, and (3) appropriate transcription and translation
initiation and termination sequences. Such regulatory elements can
include an operator sequence to control transcription. The ability
to replicate in a host, usually conferred by an origin of
replication, and a selection gene to facilitate recognition of
transformants can additionally be incorporated. DNA regions are
operatively linked when they are functionally related to each
other. For example, DNA for a signal peptide (secretory leader) is
operatively linked to DNA for a polypeptide if it is expressed as a
precursor which participates in the secretion of the polypeptide; a
promoter is operatively linked to a coding sequence if it controls
the transcription of the sequence; or a ribosome binding site is
operatively linked to a coding sequence if it is positioned so as
to permit translation. Structural elements intended for use in
yeast expression systems include a leader sequence enabling
extracellular secretion of translated protein by a host cell.
Alternatively, where recombinant protein is expressed without a
leader or transport sequence, it can include an N-terminal
methionine residue. This residue can optionally be subsequently
cleaved from the expressed recombinant protein to provide a final
product.
[0186] The choice of expression control sequence and expression
vector will depend upon the choice of host. A wide variety of
expression host/vector combinations can be employed. Useful
expression vectors for eukaryotic hosts, include, for example,
vectors comprising expression control sequences from SV40, bovine
papilloma virus, adenovirus and cytomegalovirus. Useful expression
vectors for bacterial hosts include known bacterial plasmids, such
as plasmids from Escherichia coli, including pCR 1, pBR322, pMB9
and their derivatives, wider host range plasmids, such as M13 and
filamentous single-stranded DNA phages.
[0187] Suitable host cells for expression of antibodies,
antigen-binding fragments thereof, and anti-TNF proteins (e.g.,
full-length antibodies, antigen-binding fragments of antibodies, or
soluble TNF receptor proteins) include prokaryotes, yeast, insect
or higher eukaryotic cells under the control of appropriate
promoters. Prokaryotes include gram negative or gram positive
organisms, for example E. coli or bacilli. Higher eukaryotic cells
include established cell lines of mammalian origin. Cell-free
translation systems could also be employed. Appropriate cloning and
expression vectors for use with bacterial, fungal, yeast, and
mammalian cellular hosts are described by Pouwels et al. (Cloning
Vectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevant
disclosure of which is hereby incorporated by reference. Additional
information regarding methods of protein production, including
antibody production, can be found, e.g., in U.S. Patent Publication
No. 2008/0187954, U.S. Pat. Nos. 6,413,746 and 6,660,501, and
International Patent Publication No. WO 04009823, each of which is
hereby incorporated by reference herein in its entirety.
[0188] Various mammalian or insect cell culture systems are also
advantageously employed to express recombinant protein. Expression
of recombinant proteins in mammalian cells can be performed because
such proteins are generally correctly folded, appropriately
modified and completely functional. Examples of suitable mammalian
host cell lines include HEK-293 and HEK-293T, the COS-7 lines of
monkey kidney cells, described by Gluzman (Cell 23:175, 1981), and
other cell lines including, for example, L cells, C127, 3T3,
Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalian
expression vectors can comprise nontranscribed elements such as an
origin of replication, a suitable promoter and enhancer linked to
the gene to be expressed, and other 5' or 3' flanking
nontranscribed sequences, and 5' or 3' nontranslated sequences,
such as necessary ribosome binding sites, a polyadenylation site,
splice donor and acceptor sites, and transcriptional termination
sequences. Baculovirus systems for production of heterologous
proteins in insect cells are reviewed by Luckow and Summers,
Bio/Technology 6:47 (1988).
[0189] The proteins produced by a transformed host can be purified
according to any suitable method. Such standard methods include
chromatography (e.g., ion exchange, affinity and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for protein purification. Affinity tags
such as hexahistidine, maltose binding domain, influenza coat
sequence and glutathione-S-transferase can be attached to the
protein to allow easy purification by passage over an appropriate
affinity column. Isolated proteins can also be physically
characterized using such techniques as proteolysis, nuclear
magnetic resonance and x-ray crystallography.
[0190] For example, supernatants from systems which secrete
recombinant protein into culture media can be first concentrated
using a commercially available protein concentration filter, for
example, an Amicon or Millipore Pellicon ultrafiltration unit.
Following the concentration step, the concentrate can be applied to
a suitable purification matrix. Alternatively, an anion exchange
resin can be employed, for example, a matrix or substrate having
pendant diethylaminoethyl (DEAE) groups. The matrices can be
acrylamide, agarose, dextran, cellulose or other types commonly
employed in protein purification. Alternatively, a cation exchange
step can be employed. Suitable cation exchangers include various
insoluble matrices comprising sulfopropyl or carboxymethyl groups.
Finally, one or more reversed-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify anti-TNF proteins (e.g.,
full-length antibodies, antigen-binding fragments of antibodies, or
soluble TNF receptor proteins). Some or all of the foregoing
purification steps, in various combinations, can also be employed
to provide a homogeneous recombinant protein.
[0191] Recombinant protein produced in bacterial culture can be
isolated, for example, by initial extraction from cell pellets,
followed by one or more concentration, salting-out, aqueous ion
exchange or size exclusion chromatography steps. High performance
liquid chromatography (HPLC) can be employed for final purification
steps. Microbial cells employed in expression of a recombinant
protein can be disrupted by any convenient method, including
freeze-thaw cycling, sonication, mechanical disruption, or use of
cell lysing agents.
[0192] Methods known in the art for purifying antibodies,
antigen-binding fragments thereof, and anti-TNF alpha proteins also
include, for example, those described in U.S. Patent Publication
Nos. 2008/0312425, 2008/0177048, and 2009/0187005, each of which is
hereby incorporated by reference herein in its entirety.
III. Immunoconjugates Containing Glucocorticoid Receptor
Agonists
[0193] Immunoconjugates containing glucocorticoid receptor agonists
are provided herein. In some embodiments, an immunoconjugate
provided herein binds to Fc gamma receptor. In some embodiments, an
immunoconjugate provided herein is active in the GRE transmembrane
TNF-alpha reporter assay (as used herein the "GRE transmembrane
TNF-alpha reporter assay" refers to the assay used in Example 79
below). In some embodiments, an immunoconjugate provided herein is
active in the L929 assay (as used herein, the "L929 assay" refers
to the assay used in Example 82 below). In some embodiments, an
immunoconjugate provided herein shows reduced immunogenicity
(reduced anti-drug immune response (ADA)) as compared to the
protein in the immunoconjugate (e.g., the antibody, antigen-binding
fragment thereof, or soluble receptor) alone.
[0194] In one embodiment, disclosed herein is a compound having
Formula I-a:
(SM-L-Q).sub.n-A.sup.1 I-a
or a pharmaceutically acceptable salt thereof, wherein:
[0195] A.sup.1 is an anti-tumor necrosis factor (TNF) alpha
protein;
[0196] L is a linker;
[0197] Q is a heterobifunctional group or heterotrifunctional
group; or
[0198] Q is absent;
[0199] n is 1-10; and
[0200] SM is a radical of a glucocorticosteroid.
[0201] In one embodiment, disclosed herein is a compound having
Formula I-a:
(SM-L-Q).sub.n-A.sup.1 I-a
or a pharmaceutically acceptable salt thereof, wherein:
[0202] A.sup.1 is an anti-tumor necrosis factor (TNF) alpha
antibody, an anti-TNF alpha monoclonal antibody, or adalimumab;
[0203] L is a linker;
[0204] Q is a heterobifunctional group or heterotrifunctional
group; or
[0205] Q is absent;
[0206] n is 1-10; and
[0207] SM is a radical of a glucocorticosteroid.
[0208] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, wherein
SM is a monovalent radical of a glucocorticosteroid.
[0209] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, wherein
SM is a monovalent radical of a glucocorticosteroid selected from
the group consisting of:
##STR00013##
[0210] wherein the sulfur, oxygen, or nitrogen atom is attached
directly or indirectly to the C- or D-ring of the
glucocorticosteroid, and R is C.sub.4 alkyl. In another embodiment,
the sulfur, oxygen, or nitrogen atom is attached directly or
indirectly to the D-ring of the glucocorticosteroid.
[0211] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, wherein
SM is a monovalent radical of a glucocorticosteroid having Formula
II-a:
##STR00014##
[0212] wherein:
[0213] R.sup.1 is selected from the group consisting of hydrogen
and halo;
[0214] R.sup.2 is selected from the group consisting of hydrogen,
halo, and hydroxy;
[0215] R.sup.3 is selected from the group consisting of
--CH.sub.2H, --CH.sub.2SH, --CH.sub.2Cl, --SCH.sub.2Cl,
--SCH.sub.2F, --SCH.sub.2CF.sub.3, --OH (or hydroxy),
--OCH.sub.2CN, --OCH.sub.2Cl, --OCH.sub.2F, --OCH.sub.3,
--OCH.sub.2CH.sub.3, --SCH.sub.2CN,
##STR00015##
[0216] R.sup.3a is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0217] R.sup.3b is selected from the group consisting of C.sub.1-4
alkyl and C.sub.1-4 alkoxy;
[0218] R.sup.3c is selected from the group consisting of hydrogen,
C.sub.1-4 alkyl, --CH.sub.2OH, and C.sub.1-4 alkoxy;
[0219] R.sup.3d and R.sup.3e are independently selected from
hydrogen and C.sub.1-4 alkyl;
[0220] R.sup.9a is selected from the group consisting of optionally
substituted alkyl, optionally substituted cycloalkyl, optionally
substituted aryl, and optionally substituted heteroaryl;
[0221] R.sup.9b is selected from the group consisting of hydrogen
and alkyl; or
[0222] R.sup.9a is:
##STR00016##
and
[0223] R.sup.9b is hydrogen or methyl;
[0224] X is selected from the group consisting of
--(CR.sup.4aR.sup.4b).sub.t--, --O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--, --CH.sub.2O--,
--N(H)C(R.sup.8a)(R.sup.8b)--, --CR.sup.4c.dbd.CR.sup.4d--, and
--C.ident.C--; or
[0225] X is absent;
[0226] t is 1 or 2;
[0227] Z is selected from the group consisting of .dbd.CH--,
.dbd.C(OH)--, and .dbd.N--;
[0228] each R.sup.4a and R.sup.4b are independently selected from
the group consisting of hydrogen and C.sub.1-4 alkyl; or
[0229] R.sup.4a and R.sup.4b taken together with the carbon atom to
which they are attached form a 3- to 6-membered cycloalkyl;
[0230] R.sup.4c and R.sup.4d are independently selected from the
group consisting of hydrogen and C.sub.1-4 alkyl;
[0231] R.sup.5 is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0232] R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d, and R.sup.6e are
each independently selected from the group consisting of hydrogen,
halo, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, cyano, hydroxy, thiol,
amino, alkylthio, and alkoxy;
[0233] R.sup.8a and R.sup.8b are independently selected from the
group consisting of hydrogen and C.sub.1-4 alkyl;
[0234] R.sup.11 is selected from the group consisting of hydrogen,
halo, C.sub.1-4 alkyl, hydroxy, thiol, amino, alkylthio, and
alkoxy; and
[0235] represents a single or double bond.
[0236] In another embodiment, disclosed herein is a compound having
Formula I-a, wherein SM is a monovalent radical of a
glucocorticosteroid having Formula II-a, wherein R.sup.9a is:
##STR00017##
[0237] In another embodiment, disclosed herein is a compound having
Formula I-a, wherein SM is a monovalent radical of a
glucocorticosteroid having Formula II-a':
##STR00018##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.9a, R.sup.9b and areas
defined in connection with Formula II-a.
[0238] In another embodiment, disclosed herein is a compound having
Formula I-a, wherein SM is a monovalent radical of a
glucocorticosteroid having Formula II-b:
##STR00019##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.9a, R.sup.9b and are as
defined in connection with Formula II-a.
[0239] In another embodiment, disclosed herein is a compound having
Formula I-a, wherein SM is a monovalent radical of a
glucocorticosteroid having Formula II-b':
##STR00020##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.9a, R.sup.9b, and are as
defined in connection with Formula II-a.
[0240] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, wherein
SM is a monovalent radical of a glucocorticosteroid having Formula
II-c:
##STR00021##
[0241] wherein R.sup.1, R.sup.2, R.sup.9a, R.sup.9b, and are as
defined in connection with Formula II-a; and
[0242] W is selected from the group consisting of --O-- and --S--.
In another embodiment, W is --O--. In another embodiment, W is
--S--.
[0243] In another embodiment, disclosed herein is a compound having
Formula I-a, wherein SM is a monovalent radical of a
glucocorticosteroid having Formula II-c':
##STR00022##
wherein R.sup.1, R.sup.2, R.sup.9a, R.sup.9b, W, and are as defined
in connection with Formula II-c.
[0244] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, wherein
SM is a monovalent radical of a glucocorticosteroid having Formula
II-d:
##STR00023##
[0245] wherein R.sup.1, R.sup.2, R.sup.9a, R.sup.9b, W, and are as
defined in connection with Formula II-c.
[0246] In another embodiment, disclosed herein is a compound having
Formula I-a, wherein SM is a monovalent radical of a
glucocorticosteroid having Formula II-d':
##STR00024##
wherein R.sup.1, R.sup.2, R.sup.9a, R.sup.9b, W, and are as defined
in connection with Formula II-c.
[0247] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, wherein
SM is a monovalent radical of a glucocorticosteroid having Formula
II-e:
##STR00025##
[0248] wherein:
[0249] R.sup.1, R.sup.2, W, and are as defined in connection with
Formula II-c;
[0250] R.sup.9c is selected from the group consisting of hydrogen,
C.sub.1-4 alkyl, and --C(.dbd.O)R.sup.9e;
[0251] R.sup.9d is selected from the group consisting of hydrogen,
optionally substituted alkyl, optionally substituted cycloalkyl,
optionally substituted aryl, and optionally substituted heteroaryl;
and
[0252] R.sup.9e is selected from the group consisting of hydrogen,
optionally substituted alkyl, optionally substituted cycloalkyl,
optionally substituted aryl, and optionally substituted
heteroaryl.
[0253] In another embodiment, disclosed herein is a compound having
Formula I-a, wherein SM is a monovalent radical of a
glucocorticosteroid having Formula II-e':
##STR00026##
wherein R.sup.1, R.sup.2, W, R.sup.9c, R.sup.9d, and are as defined
in connection with Formula II-e.
[0254] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, wherein
SM is a monovalent radical of a glucocorticosteroid having Formula
II-f:
##STR00027##
[0255] wherein:
[0256] R.sup.1, R.sup.2, R.sup.9c, R.sup.9d, W, and are as defined
in connection with Formula II-e.
[0257] In another embodiment, disclosed herein is a compound having
Formula I-a, wherein SM is a monovalent radical of a
glucocorticosteroid having Formula II-f':
##STR00028##
wherein R.sup.1, R.sup.2, R.sup.9c, R.sup.9d, W, and are as defined
in connection with Formula II-e.
[0258] In another embodiment, disclosed herein is a compound having
Formula I-b:
(SM-L-Q).sub.n-A.sup.2 I-b
or a pharmaceutically acceptable salt thereof, wherein:
[0259] A.sup.2 is a protein;
[0260] L is a linker;
[0261] Q is a heterobifunctional group or heterotrifunctional
group; or
[0262] Q is absent;
[0263] n is 1-10; and
[0264] SM is a monovalent radical of a glucocorticosteroid having
any one of:
##STR00029## ##STR00030##
[0265] wherein:
[0266] R' is selected from the group consisting of hydrogen and
halo;
[0267] R.sup.2 is selected from the group consisting of hydrogen,
halo, and hydroxy;
[0268] R.sup.3 is selected from the group consisting of
--CH.sub.2OH, --CH.sub.2SH, --CH.sub.2Cl, --SCH.sub.2Cl,
--SCH.sub.2F, --SCH.sub.2CF.sub.3, --OH, --OCH.sub.2CN,
--OCH.sub.2Cl, --OCH.sub.2F, --OCH.sub.3, --OCH.sub.2CH.sub.3,
--SCH.sub.2CN,
##STR00031##
[0269] R.sup.3a is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0270] R.sup.3b is selected from the group consisting of C.sub.1-4
alkyl and C.sub.1-4 alkoxy;
[0271] R.sup.3c is selected from the group consisting of hydrogen,
C.sub.1-4 alkyl, --CH.sub.2OH, and C.sub.1-4 alkoxy;
[0272] R.sup.3d and R.sup.3e are independently selected from
hydrogen and C.sub.1-4 alkyl;
[0273] R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d, and R.sup.6e are
each independently selected from the group consisting of hydrogen,
halo, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, cyano, hydroxy, thiol,
amino, alkylthio, and alkoxy;
[0274] X is selected from the group consisting of
--(CR.sup.4aR.sup.4b).sub.t--, --O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--, --CH.sub.2O--,
--N(H)C(R.sup.8a)(R.sup.8b)--, --CR.sup.4c.dbd.CR.sup.4d--
(including both E and Z isomers), and --C.ident.C--; (wherein when
X is --CH.sub.2S--, --CH.sub.2O--, or
--N(H)C(R.sup.8a)(R.sup.8b)--, the hetereoatom of --CH.sub.2S--,
--CH.sub.2O--, or --N(H)C(R.sup.8a)(R.sup.8b)-- can be attached to
either 6-membered ring, i.e., --CH.sub.2S-- is equivalent to
--SCH.sub.2--, --CH.sub.2O-- is equivalent to --OCH.sub.2--, and
--N(H)C(R.sup.8a)(R.sup.8b)-- is equivalent to
--C(R.sup.8a)(R.sup.8b)N(H)--); or
[0275] X is absent, i.e., X represents a chemical bond;
[0276] Y.sup.2 is selected from the group consisting of --O--,
--S--, and --N(R.sup.7a)--; or
[0277] Y.sup.2 is absent, i.e., Y.sup.2 represents a chemical
bond;
[0278] t is 1 or 2;
[0279] Z is selected from the group consisting of .dbd.CR.sup.11a--
and .dbd.N--;
[0280] each R.sup.4a and R.sup.4b are independently selected from
the group consisting of hydrogen and C.sub.1-4 alkyl; or
[0281] R.sup.4a and R.sup.4b taken together with the carbon atom to
which they are attached form a 3- to 6-membered cycloalkyl;
[0282] R.sup.4c and R.sup.4d are independently selected from the
group consisting of hydrogen and C.sub.1-4 alkyl;
[0283] R.sup.5 is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0284] R.sup.7a is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0285] R.sup.8a and R.sup.8b are independently selected from the
group consisting of hydrogen and C.sub.1-4 alkyl;
[0286] R.sup.9f is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0287] R.sup.11a and R.sup.11b are independently selected from the
group consisting of hydrogen, halo, C.sub.1-4 alkyl, C.sub.1-4
haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and alkoxy;
and
[0288] represents a single or double bond.
[0289] In another embodiment, disclosed herein is a compound having
Formula I-b:
(SM-L-Q).sub.n-A.sup.2 I-b
wherein:
[0290] A.sup.2 is a protein;
[0291] L is a linker;
[0292] Q is a heterobifunctional group or heterotrifunctional
group; or
[0293] Q is absent;
[0294] n is 1-10; and
[0295] SM is a monovalent radical having any one of:
##STR00032## ##STR00033##
[0296] wherein R.sup.1, R.sup.2, R.sup.3, , R.sup.6, R.sup.6a,
R.sup.6d, R.sup.6e, R.sup.9f, R.sup.11b, Y.sup.2, X, and Z are as
defined in connection with Formula II-l.
[0297] In another embodiment, disclosed herein is a compound having
Formula I-b:
(SM-L-Q).sub.n-A.sup.2 I-b
wherein:
[0298] A.sup.2 is a protein;
[0299] L is a linker;
[0300] Q is a heterobifunctional group or heterotrifunctional
group; or
[0301] Q is absent;
[0302] n is 1-10; and
[0303] SM is a monovalent radical having any one of:
##STR00034## ##STR00035##
[0304] wherein R.sup.1, R.sup.2, R.sup.3, , R.sup.6a, R.sup.6c,
R.sup.6d, R.sup.6e, R.sup.9f, R.sup.11b, Y.sup.2, and X are as
defined in connection with Formula II-l, and the carbon atom marked
with an "*" is either the R-isomer or the S-isomer when R.sup.2 is
halo or hydroxyl. In one embodiment, the carbon atom marked with an
"*" is the R-isomer. In another embodiment, the carbon atom marked
with an "*" is the S-isomer.
[0305] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
Formula II-l:
##STR00036##
[0306] wherein:
[0307] R.sup.1 is selected from the group consisting of hydrogen
and halo;
[0308] R.sup.2 is selected from the group consisting of hydrogen,
halo, and hydroxy;
[0309] R.sup.3 is selected from the group consisting of
--CH.sub.2OH, --CH.sub.2SH, --CH.sub.2Cl, --SCH.sub.2Cl,
--SCH.sub.2F, --SCH.sub.2CF.sub.3, --OH, --OCH.sub.2CN,
--OCH.sub.2Cl, --OCH.sub.2F, --OCH.sub.3, --OCH.sub.2CH.sub.3,
--SCH.sub.2CN,
##STR00037##
[0310] R.sup.3a is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0311] R.sup.3b is selected from the group consisting of C.sub.1-4
alkyl and C.sub.1-4 alkoxy;
[0312] R.sup.3c is selected from the group consisting of hydrogen,
C.sub.1-4 alkyl, --CH.sub.2OH, and C.sub.1-4 alkoxy;
[0313] R.sup.3d and R.sup.3e are independently selected from
hydrogen and C.sub.1-4 alkyl;
[0314] X is selected from the group consisting of
--(CR.sup.4aR.sup.4b).sub.t--, --O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--, --CH.sub.2O--,
--N(H)C(R.sup.8a)(R.sup.8b)--, --CR.sup.4c.dbd.CR.sup.4--, and
--C.ident.C--; or
[0315] X is absent;
[0316] t is 1 or 2;
[0317] Z is selected from the group consisting of .dbd.CR.sup.11a--
and .dbd.N--;
[0318] each R.sup.4a and R.sup.4b are independently selected from
the group consisting of hydrogen and C.sub.1-4 alkyl; or
[0319] R.sup.4a and R.sup.4b taken together with the carbon atom to
which they are attached form a 3- to 6-membered cycloalkyl;
[0320] R.sup.4c and R.sup.4d are independently selected from the
group consisting of hydrogen and C.sub.1-4 alkyl;
[0321] R.sup.5 is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0322] R.sup.6b, R.sup.6c, R.sup.6d, and R.sup.6e are each
independently selected from the group consisting of hydrogen, halo,
C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, cyano, hydroxy, thiol, amino,
alkylthio, and alkoxy;
[0323] Y.sup.2 is selected from the group consisting of --O--,
--S--, and --N(R.sup.7a)--; or
[0324] Y.sup.2 is absent;
[0325] R.sup.7a is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0326] R.sup.8a and R.sup.8b are independently selected from the
group consisting of hydrogen and C.sub.1-4 alkyl;
[0327] R.sup.9f is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0328] R.sup.7a and R.sup.7b are independently selected from the
group consisting of hydrogen, halo, C.sub.1-4 alkyl, C.sub.1-4
haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and alkoxy;
and
[0329] represents a single or double bond.
[0330] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
Formula II-m:
##STR00038##
[0331] wherein R.sup.1, R.sup.2, R.sup.3, , R.sup.6a, R.sup.6c,
R.sup.6d, R.sup.6e, R.sup.9f, R.sup.11b, Y.sup.2, X, and Z are as
defined in connection with Formula II-l.
[0332] In another embodiment, disclosed herein is a compound having
Formula I-a or Ib, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
Formula II-n:
##STR00039##
[0333] wherein R.sup.1, R.sup.2, R.sup.3, , R.sup.6a, R.sup.6c,
R.sup.6d, R.sup.6e, R.sup.9f, R.sup.11b, Y.sup.2, X, and Z are as
defined in connection with Formula II-l.
[0334] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
Formula II-o:
##STR00040##
[0335] wherein R.sup.1, R.sup.2, R.sup.3, , R.sup.6a, R.sup.6d,
R.sup.6e, R.sup.9f, R.sup.11b, Y.sup.2, X, and Z are as defined in
connection with Formula II-l; and R.sup.6b is selected from the
group consisting of hydrogen, halo, C.sub.1-4 alkyl, C.sub.1-4
haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and alkoxy.
[0336] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
Formula II-p:
##STR00041##
[0337] wherein R.sup.1, R.sup.2, R.sup.3, , R.sup.6a, R.sup.6d,
R.sup.6e, R.sup.9f, R.sup.11b, Y.sup.2, X, and Z are as defined in
connection with Formula II-o.
[0338] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
Formula II-q:
##STR00042##
[0339] wherein R.sup.1, R.sup.2, R.sup.3, , R.sup.6a, R.sup.6d,
R.sup.6e, R.sup.9f, R.sup.11b, Y.sup.2, X, and Z are as defined in
connection with Formula II-o.
[0340] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-a, II-b, II-c, II-d, II-e, II-f, II-l, II-m,
II-n, II-o, II-p, or II-q, or any one of Formulae II-a', II-b',
II-c', II-d', II-e', II-f, II-l', II-m', II-n', II-o', II-p',
II-q', II-l'', II-m'', II-n'', II-o'', II-p'', or II-q'', wherein
represents a double bond.
[0341] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-a, II-b, II-c, II-d, II-e, II-f, II-l, II-m,
II-n, II-o, II-p, or II-q, or any one of Formulae II-a', II-b',
II-c', II-d', II-e', II-f, II-l', II-m', II-n', II-o', II-p',
II-q', II-l'', II-m'', II-n'', II-o'', II-p'', or II-q'', wherein
R.sup.1 is selected from the group consisting of hydrogen and
fluoro.
[0342] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-a, II-b, II-c, II-d, II-e, II-f, II-l, II-m,
II-n, II-o, II-p, or II-q, or any one of Formulae II-a', II-b',
II-c', II-d', II-e', II-l', II-nm', II-n', II-o', II-p', II-q',
II-l'', II-m'', II-n'', II-o'', II-p'', or II-q'', wherein R.sup.2
is selected from the group consisting of hydrogen and fluoro.
[0343] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-a, II-b, II-l, II-m, II-n, II-o, II-p, or
II-q, or any one of Formulae II-a', II-b', II-l', II-m', II-n',
II-o', II-p', II-q', II-l', II-m'', II-n'', II-o'', II-p'', or
II-q'', wherein R.sup.3 is selected from the group consisting of
--CH.sub.2OH, --CH.sub.2Cl, --SCH.sub.2Cl, --SCH.sub.2F, and
--OH.
[0344] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-a, II-b, II-l, II-m, II-n, II-o, II-p, or
II-q, or any one of Formulae II-a', II-b', II-l', II-m', II-n',
II-o', II-p', II-q', II-l', II-m'', II-n'', II-o'', II-p'', or
II-q'',wherein:
[0345] R.sup.3 is selected from the group consisting of:
##STR00043##
[0346] R.sup.3a is selected from the group consisting of hydrogen
and methyl;
[0347] R.sup.3b is selected from the group consisting of methyl,
ethyl, isopropyl, isobutyl, methoxy, ethoxy, isopropoxy, and
isobutoxy;
[0348] R.sup.3c is selected from the group consisting of hydrogen,
methyl, ethyl, --CH.sub.2OH, methoxy, ethoxy, and isopropoxy;
[0349] R.sup.3d and R.sup.3e are independently selected from the
group consisting of hydrogen, methyl, and ethyl.
[0350] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-a, II-b, II-c, II-d, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-l', II-m', II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'',
II-o'', II-p'', or II-q'', wherein R.sup.5 and R.sup.8a are
independently selected from the group consisting of hydrogen and
methyl.
[0351] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-a, II-b, II-c, II-d, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-l', II-m', II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'',
II-o'', II-p'', or II-q'', wherein Z is .dbd.CH--.
[0352] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-a, II-b, II-c, II-d, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-l', II-m', II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'',
II-o'', II-p'', or II-q'', wherein Z is .dbd.N--.
[0353] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-a, II-b, II-c, II-d, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-l', II-m', II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'',
II-o'', II-p'', or II-q'', wherein R.sup.6a, R.sup.6d, and R.sup.6e
are hydrogen.
[0354] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein Y.sup.2 is
--N(R.sup.7a)--. In another embodiment, R.sup.7a is selected from
the group consisting of hydrogen and methyl. In another embodiment,
R.sup.7a is hydrogen. In another embodiment, R.sup.7a is
methyl.
[0355] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-a, II-b, II-c, II-d, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-l', II-m', II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'',
II-o'', II-p'', or II-q'', wherein:
[0356] X is selected from the group consisting of
--(CR.sup.4aR.sup.4b).sub.t--, --O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --CH.sub.2S--, and --N(H)CH(R.sup.8a)--;
[0357] t is 1;
[0358] R.sup.4a and R.sup.4b are independently selected from the
group consisting of hydrogen and methyl; or
[0359] R.sup.4a and R.sup.4b taken together with the carbon atom to
which they are attached form a 3-membered cycloalkyl; and
[0360] R.sup.8a is selected from the group consisting of hydrogen
and methyl. In another embodiment, X is --CH.sub.2--. In another
embodiment, X is selected from the group consisting of:
##STR00044##
[0361] In another embodiment, X is --O--. In another embodiment, X
is --S--. In another embodiment, X is --CH.sub.2S--. In another
embodiment, X is --N(H)CH.sub.2--. In another embodiment, X is
selected from the group consisting of:
##STR00045##
[0362] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-a, II-b, II-c, II-d, II-l, II-m, or II-n, or
any one of Formulae II-a', II-b', II-c', II-d', II-l', II-m',
II-n', II-l'', II-m'', or II-n'', wherein R.sup.6c is selected from
the group consisting of hydrogen, --Cl, --OMe (or --OCH.sub.3), and
--OH.
[0363] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-a, II-b, II-c, II-d, II-o, II-p, or II-q, or
any one of Formulae II-a', II-b', II-c', II-d', II-o', II-p',
II-q', II-o'', II-p'', or II-q'' wherein R.sup.6b is selected from
the group consisting of hydrogen, --Cl, --OMe (or --OCH.sub.3), and
--OH.
[0364] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein R.sup.9f is
hydrogen.
[0365] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein R.sup.9f is
methyl.
[0366] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein R.sup.7a is
selected from the group consisting of hydrogen and --OH.
[0367] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein R.sup.11b is
hydrogen.
[0368] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f, II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein L is a cleavable
linker. In another embodiment, the cleavable linker comprises a
succinimide, amide, thiourea, thioether, oxime, or self-immolative
group, or a combination thereof. In another embodiment, the
cleavable linker comprises a peptide. In another embodiment, the
cleavable linker comprises a tripeptide. In another embodiment, the
cleavable linker comprises a dipeptide. In another embodiment, the
cleavable linker comprises phosphate ester. In another embodiment,
the cleavable linker comprises a pyrophosphate diester.
[0369] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f, II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein Q is absent.
[0370] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f, II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein Q is a
heterobifunctional group.
[0371] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f, II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein Q is a
heterobifunctional group selected from the group consisting of:
##STR00046##
wherein m is 1, 2, 3, 4, 5, or 6. In another embodiment, Q is
selected from the group consisting of Q-1, Q-2, Q-3, and Q-4. In
another embodiment, Q is selected from the group consisting of Q-3
and Q-4. In another embodiment, m is 2.
[0372] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f, II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein Q is a
heterotrifunctional group.
[0373] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f, II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein Q is a
heterotrifunctional group that is:
##STR00047##
[0374] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f, II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein -L-Q- is:
##STR00048##
[0375] m is 2 or 3; and
[0376] R.sup.10a and R.sup.10b are independently selected from the
group consisting of hydrogen and optionally substituted C.sub.1-6
alkyl. In another embodiment, m is 2. In another embodiment, m is
1. In another embodiment, -L-Q- is:
##STR00049##
In another embodiment, -L-Q- is:
##STR00050##
In another embodiment, -L-Q- is:
##STR00051##
In another embodiment, -L-Q- is:
##STR00052##
[0377] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f, II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein -L-Q- is:
##STR00053##
[0378] m is 2 or 3; and
[0379] R.sup.10a and R.sup.10b are independently selected from the
group consisting of hydrogen and optionally substituted C.sub.1-6
alkyl. In another embodiment, m is 2. In another embodiment, -L-Q-
is:
##STR00054##
In another embodiment, -L-Q- is:
##STR00055##
In another embodiment, -L-Q- is:
##STR00056##
In another embodiment, -L-Q- is:
##STR00057##
[0380] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f, II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein L is a
noncleavable linker. In another embodiment, the linker comprises
one or more polyethylene glycol units.
[0381] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f, II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein -L-Q- is:
##STR00058##
[0382] m is 2 or 3; and
[0383] x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or
15. In another embodiment, m is 2.
[0384] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f, II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein -L-Q- is:
##STR00059##
[0385] m is 2 or 3; and
[0386] x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or
15. In another embodiment, m is 2.
[0387] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f, II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein -L-Q- is:
##STR00060##
and
[0388] x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or
15.
[0389] The compound of any one of claims 1-47, or a
pharmaceutically acceptable salt or solvate thereof, wherein -L-Q-
is:
##STR00061##
and
[0390] x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or
15.
[0391] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f', II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein -L-Q- is:
##STR00062##
m is 1 or 2; x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
or 15; and R.sup.10a and R.sup.10b are independently selected from
the group consisting of hydrogen and optionally substituted
C.sub.1-6 alkyl. In another embodiment, -L-Q- is:
##STR00063##
In another embodiment, -L-Q- is:
##STR00064##
In another embodiment, -L-Q- is:
##STR00065##
In another embodiment, -L-Q- is:
##STR00066##
[0392] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f, II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein -L-Q- is:
##STR00067##
m is 1 or 2; x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
or 15; and R.sup.10a and R.sup.10b are independently selected from
the group consisting of hydrogen and optionally substituted
C.sub.1-6 alkyl.
[0393] In another embodiment, -L-Q- is:
##STR00068##
In another embodiment, -L-Q- is:
##STR00069##
In another embodiment, -L-Q- is:
##STR00070##
In another embodiment, -L-Q- is:
##STR00071##
[0394] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f', II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein -L-Q- is:
##STR00072##
[0395] x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or
15; and R.sup.10a and R.sup.10b are independently selected from the
group consisting of hydrogen and optionally substituted C.sub.1-6
alkyl.
[0396] In another embodiment, -L-Q- is:
##STR00073##
[0397] In another embodiment, -L-Q- is:
##STR00074##
[0398] In another embodiment, -L-Q- is:
##STR00075##
[0399] In another embodiment, -L-Q- is:
##STR00076##
[0400] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f', II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein -L-Q- is:
##STR00077##
[0401] x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or
15; and R.sup.10a and R.sup.10b are independently selected from the
group consisting of hydrogen and optionally substituted C.sub.1-6
alkyl.
[0402] In another embodiment, -L-Q- is:
##STR00078##
[0403] In another embodiment, -L-Q- is:
##STR00079##
[0404] In another embodiment, -L-Q- is:
##STR00080##
[0405] In another embodiment, -L-Q- is:
##STR00081##
[0406] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f', II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein -L-Q- is any one
of the chemical structures of Table I:
TABLE-US-00010 TABLE I ##STR00082## ##STR00083## ##STR00084##
##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089##
##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094##
##STR00095## ##STR00096## ##STR00097## ##STR00098## ##STR00099##
##STR00100## ##STR00101## ##STR00102##
[0407] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
e.g. a compound having Formula I-a or I-b wherein SM is a
monovalent radical of a glucocorticosteroid having any one of
Formulae II-a, II-b, II-c, II-d, II-e, I-f, II-l, II-m, II-n, II-o,
II-p, or II-q, or any one of Formulae II-a', II-b', II-c', II-d',
II-e', II-f', II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein n is 2-8. In
another embodiment, n is 1-5. In another embodiment, n is 2-5. In
another embodiment, n is 1. In another embodiment, n is 2. In
another embodiment n is 3. In another embodiment, n is 4. In
another embodiment, n is 5. In another embodiment, n is 6. In
another embodiment, n is 7. In another embodiment, n is 8.
[0408] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid which
is any one of the chemical structures of Table II.
TABLE-US-00011 TABLE II ##STR00103## ##STR00104## ##STR00105##
##STR00106## ##STR00107## ##STR00108## ##STR00109## ##STR00110##
##STR00111## ##STR00112## ##STR00113## ##STR00114## ##STR00115##
##STR00116## ##STR00117## ##STR00118## ##STR00119## ##STR00120##
##STR00121## ##STR00122## ##STR00123## ##STR00124## ##STR00125##
##STR00126## ##STR00127## ##STR00128## ##STR00129## ##STR00130##
##STR00131## ##STR00132## ##STR00133## ##STR00134## ##STR00135##
##STR00136## ##STR00137## ##STR00138## ##STR00139## ##STR00140##
##STR00141## ##STR00142## ##STR00143## ##STR00144## ##STR00145##
##STR00146## ##STR00147## ##STR00148## ##STR00149## ##STR00150##
##STR00151## ##STR00152## ##STR00153## ##STR00154## ##STR00155##
##STR00156## ##STR00157## ##STR00158## ##STR00159## ##STR00160##
##STR00161## ##STR00162## ##STR00163## ##STR00164## ##STR00165##
##STR00166## ##STR00167## ##STR00168## ##STR00169##
[0409] In another embodiment, disclosed herein is a compound having
Formula I-a or I-b, or a pharmaceutically acceptable salt thereof,
wherein SM is a monovalent radical of a glucocorticosteroid
selected from the group consisting of:
##STR00170##
[0410] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, II-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', II-f', II-4', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-4, II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1 is an
antibody or antigen-binding fragment thereof or wherein A.sup.2 is
an antibody or antigen-binding fragment thereof.
[0411] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', I-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1 is an
anti-tumor necrosis factor (TNF) alpha protein that binds to human
TNF alpha and/or murine TNF alpha or wherein A.sup.2 is protein
that binds to human TNF alpha and/or murine TNF alpha.
[0412] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-l',II-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1 is an
anti-tumor necrosis factor (TNF) alpha protein that binds to
soluble TNF alpha or wherein A.sup.2 is protein that binds to
soluble TNF alpha.
[0413] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-l', II-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1 is an
anti-tumor necrosis factor (TNF) alpha protein that binds to
membrane-bound TNF alpha or wherein A.sup.2 is a protein that binds
to membrane-bound TNF alpha.
[0414] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', I-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1 is an
anti-tumor necrosis factor (TNF) alpha protein comprising an
anti-TNF antibody or wherein A.sup.2 is protein comprising an
anti-TNF antibody.
[0415] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d',II-e', I-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1 is an
anti-tumor necrosis factor (TNF) alpha protein comprising an
anti-TNF receptor antibody or wherein A.sup.2 is a protein
comprising an anti-TNF receptor antibody.
[0416] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d',II-e', I-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1 is an
anti-tumor necrosis factor (TNF) alpha protein comprising an
antigen-binding fragment of an anti-TNF antibody or wherein A.sup.2
is a protein comprising an antigen-binding fragment of an anti-TNF
antibody.
[0417] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d',II-e', I-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1 is an
anti-tumor necrosis factor (TNF) alpha protein comprising an
antigen-binding fragment of an anti-TNF receptor antibody or
wherein A.sup.2 is an anti-tumor necrosis factor (TNF) alpha
protein comprising an antigen-binding fragment of an anti-TNF
receptor antibody.
[0418] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', II-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'',wherein the
antigen-binding fragment is selected from the group consisting of
Fab, Fab', F(ab').sub.2, single chain Fv or scFv, disulfide linked
Fv, V-NAR domain, IgNar, intrabody, IgG.DELTA.CH2, minibody,
F(ab')3, tetrabody, triabody, diabody, single-domain antibody,
DVD-Ig, Fcab, mAb2, (scFv).sub.2, or scFv-Fc.
[0419] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', II-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, I-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein the antibody or
antigen-binding fragment thereof is murine, chimeric, humanized, or
human.
[0420] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', II-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'',wherein A.sup.1 is an
anti-tumor necrosis factor (TNF) alpha protein comprising a soluble
TNF receptor or wherein A.sup.2 is a protein comprising a soluble
TNF receptor. In another embodiment, the soluble TNF receptor is a
soluble p75 TNF receptor.
[0421] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', II-f, II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1
comprises a heavy chain constant domain or a fragment thereof or
wherein or A.sup.2 comprises a heavy chain constant domain or a
fragment thereof. In another embodiment, the heavy chain constant
domain or fragment thereof comprises a constant domain selected
from the group consisting of: (a) an IgA constant domain; (b) an
IgD constant domain; (c) an IgE constant domain; (d) an IgG
constant domain; (e) an IgG2 constant domain; (f) an IgG3 constant
domain; (g) an IgG4 constant domain; and (h) an IgM constant domain
or is a fragment thereof. In another embodiment, the heavy chain
constant domain comprises a human IgG1 heavy chain constant domain
or fragment thereof. In another embodiment, the heavy chain
constant domain comprises a human IgG1 Fc domain.
[0422] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-c',II-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, wherein
A.sup.1 comprises a light chain constant domain or a fragment
thereof or wherein A.sup.2 comprises a light chain constant domain
or a fragment thereof. In another embodiment, the light chain
constant domain or fragment thereof comprises a constant domain
selected group consisting of (a) an Ig kappa constant domain and
(b) an Ig lambda constant domain or is a fragment thereof.
[0423] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', II-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1
competitively inhibits binding of an antibody selected from the
group consisting of adalimumab, infliximab, certolizumab pegol, and
golimumab to TNF-alpha or wherein A.sup.2 competitively inhibits
binding of an antibody selected from the group consisting of
adalimumab, infliximab, certolizumab pegol, and golimumab to
TNF-alpha.
[0424] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', I-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1 binds to
the same TNF-alpha epitope as an antibody selected from the group
consisting of adalimumab, infliximab, certolizumab pegol,
afelimomab, nerelimomab, ozoralizumab, placulumab, and golimumab or
wherein A.sup.2 binds to the same TNF-alpha epitope as an antibody
selected from the group consisting of adalimumab, infliximab,
certolizumab pegol, afelimomab, nerelimomab, ozoralizumab,
placulumab, and golimumab.
[0425] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', I-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein the anti-TNF
alpha protein is selected from the group consisting of adalimumab,
infliximab, certolizumab pegol, afelimomab, nerelimomab,
ozoralizumab, placulumab, and golimumab.
[0426] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d',II-e', II-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1
comprises the variable heavy chain CDR1, CDR2, and CDR3 sequences
of SEQ ID NO:3 or 6, SEQ ID NO:4, and SEQ ID NO:5, respectively and
the variable light chain CDR1, CDR2, and CDR3 sequences of SEQ ID
NO:32, SEQ ID NO:33, and SEQ ID NO:34, respectively or wherein
A.sup.2 comprises the variable heavy chain CDR1, CDR2, and CDR3
sequences of SEQ ID NO:3 or 6, SEQ ID NO:4, and SEQ ID NO:5
respectively and the variable light chain CDR1, CDR2, and CDR3
sequences of SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34,
respectively.
[0427] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', I-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1
comprises the variable heavy chain sequence of SEQ ID NO:50 and the
variable light chain sequence of SEQ ID NO:59 or wherein A.sup.2
comprises the variable heavy chain sequence of SEQ ID NO:50 and the
variable light chain sequence of SEQ ID NO:59.
[0428] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', II-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1 does not
bind to TNF beta or wherein A.sup.2 does not bind to TNF beta.
[0429] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', II-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1 binds to
TNF beta or wherein A.sup.1 binds to TNF beta.
[0430] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', II-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1
neutralizes human TNF-alpha cytotoxicity in a in vitro L929 assay
with an IC50 of 1.times.10.sup.-7 M or less or wherein A.sup.2
neutralizes human TNF-alpha cytotoxicity in a in vitro L929 assay
with an IC50 of 1.times.10.sup.-7 M or less.
[0431] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', I-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1 blocks
the interaction of TNF-alpha with p55 and p75 cell surface
receptors or wherein A.sup.2 blocks the interaction of TNF-alpha
with p55 and p75 cell surface receptors.
[0432] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', I-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein A.sup.1 lyses
surface TNF expressing cells in vitro in the presence of complement
or wherein A.sup.2 lyses surface TNF expressing cells in vitro in
the presence of complement.
[0433] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e',II-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, II-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein the soluble p75
TNF receptor is etanercept.
[0434] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, e.g., a
compound having Formula I-a, wherein SM is a monovalent radical of
a glucocorticosteroid having any one of Formulae II-a, II-b, II-c,
II-d, II-e, I-f, II-l, II-m, II-n, II-o, II-p, or II-q, or any one
of Formulae II-a', II-b', II-c', II-d', II-e', II-f', II-l', II-m',
II-n', II-o', II-p', II-q', II-l'', II-m'', II-n'', II-o'', II-p'',
or II-q'', or a compound having Formula I-b, or a pharmaceutically
acceptable salt thereof, e.g., a compound having Formula I-b,
wherein SM is a monovalent radical of a glucocorticosteroid having
any one of Formulae II-l, I-m, II-n, II-o, II-p, or II-q, or any
one of Formulae II-l', II-m', II-n', II-o', II-p', II-q', II-l'',
II-m'', II-n'', II-o'', II-p'', or II-q'', wherein the antibody is
adalimumab.
[0435] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, or a
compound having Formula I-b, or a pharmaceutically acceptable salt
thereof, which is any one of the chemical structures of Table
III:
TABLE-US-00012 TABLE III ##STR00171## ##STR00172## ##STR00173##
##STR00174## ##STR00175## ##STR00176## ##STR00177## ##STR00178##
##STR00179## ##STR00180## ##STR00181## ##STR00182## ##STR00183##
##STR00184## ##STR00185## ##STR00186## ##STR00187## ##STR00188##
##STR00189## ##STR00190## ##STR00191## ##STR00192## ##STR00193##
##STR00194## ##STR00195## ##STR00196## ##STR00197## ##STR00198##
##STR00199## ##STR00200## ##STR00201## ##STR00202## ##STR00203##
##STR00204## ##STR00205## ##STR00206## ##STR00207## ##STR00208##
##STR00209## ##STR00210## ##STR00211## ##STR00212## ##STR00213##
##STR00214## ##STR00215## ##STR00216## ##STR00217## ##STR00218##
##STR00219## ##STR00220## ##STR00221## ##STR00222## ##STR00223##
##STR00224## ##STR00225## ##STR00226## ##STR00227##
##STR00228##
wherein n is 1-5 and A is A.sup.1 or A.sup.Z. In another
embodiment, A is adalimumab.
[0436] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, or a
compound having Formula I-b, or a pharmaceutically acceptable salt
thereof, which is any one of the chemical structures of Table
IV:
TABLE-US-00013 TABLE IV Structure n ##STR00229## 4.5 ##STR00230##
4.4 ##STR00231## 2 ##STR00232## 2 ##STR00233## 4 ##STR00234## 2
##STR00235## 4 ##STR00236## 2 ##STR00237## 4 ##STR00238## 2
##STR00239## 4 ##STR00240## 4 ##STR00241## 1.3 ##STR00242## 2
##STR00243## 4 ##STR00244## 2 ##STR00245## 2 ##STR00246## 4
##STR00247## 4 ##STR00248## 4 ##STR00249## 4 ##STR00250## 4
##STR00251## 4 ##STR00252## 2 ##STR00253## 4 ##STR00254## 2
##STR00255## 4 ##STR00256## 4 ##STR00257## 2 ##STR00258## 4
##STR00259## 2 ##STR00260## 4 ##STR00261## 4 ##STR00262## 2
##STR00263## 4 ##STR00264## 2 ##STR00265## 4 ##STR00266## 4
##STR00267## 4 ##STR00268## 4 ##STR00269## 2 ##STR00270## 4
##STR00271## 2 ##STR00272## 4 ##STR00273## 4 ##STR00274## 4
##STR00275## 4 ##STR00276## 2 ##STR00277## 4 ##STR00278## 3.7
##STR00279## 4.1 ##STR00280## 3.9 ##STR00281## 2 ##STR00282## 4
##STR00283## 4 ##STR00284## 3.9 ##STR00285## 3.5 ##STR00286## 3.5
##STR00287## 2 ##STR00288## 4 ##STR00289## 2 ##STR00290## 3.6
##STR00291## 3.6 ##STR00292## 3.5 ##STR00293## 2 ##STR00294## 4
##STR00295## 3.8 ##STR00296##
wherein A is A.sup.1 or A.sup.2. In another embodiment, A is
adalimumab.
[0437] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, or a
compound having Formula I-b, or a pharmaceutically acceptable salt
thereof, which is any one of the chemical structures of Table
IV-A:
TABLE-US-00014 TABLE IV-A ##STR00297## 2 ##STR00298## 2
##STR00299## 2 ##STR00300## 4 ##STR00301## 4 ##STR00302## 4
##STR00303##
wherein A is A.sup.1 or A.sup.2. In another embodiment, A is
adalimumab.
[0438] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, or a
compound having Formula I-b, or a pharmaceutically acceptable salt
thereof, which is any one of the chemical structures of Table
V:
TABLE-US-00015 TABLE V ##STR00304## ##STR00305## ##STR00306##
wherein n is 1-5 and A is A.sup.1 or A.sup.2. In another
embodiment, A is adalimumab.
[0439] In another embodiment, disclosed herein is a compound having
Formula I-a, or a pharmaceutically acceptable salt thereof, or a
compound having Formula I-b, or a pharmaceutically acceptable salt
thereof, selected from the group consisting of:
##STR00307## ##STR00308##
wherein n is 2-4, A is A.sup.1 or A.sup.2. In another embodiment, A
is adalimumab. In another embodiment, n is 2 or 4. In another
embodiment, n is 2. In another embodiment, n is 4.
IV. Glucocorticoid Receptor Agonists
[0440] In another embodiment, disclosed herein is a compound having
Formula VII:
##STR00309##
[0441] or a pharmaceutically acceptable salt thereof, wherein:
[0442] R.sup.1 is selected from the group consisting of hydrogen
and halo;
[0443] R.sup.2 is selected from the group consisting of hydrogen,
halo, and hydroxy;
[0444] R.sup.3 is selected from the group consisting of
--CH.sub.2OH, --CH.sub.2SH, --CH.sub.2Cl, --SCH.sub.2Cl,
--SCH.sub.2F, --SCH.sub.2CF.sub.3, --CH.sub.2OS(.dbd.O).sub.2-0H,
--OH, --OCH.sub.2CN, --OCH.sub.2Cl, --OCH.sub.2F, --OCH.sub.3,
--OCH.sub.2CH.sub.3, --SCH.sub.2CN,
##STR00310##
[0445] R.sup.3a is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0446] R.sup.3b is selected from the group consisting of C.sub.1-4
alkyl and C.sub.1-4 alkoxy;
[0447] R.sup.3c is selected from the group consisting of hydrogen,
C.sub.1-4 alkyl, --CH.sub.2OH, C.sub.1-4 alkoxy, --CH.sub.2(amino),
and --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.3f;
[0448] R.sup.3d and R.sup.3e are independently selected from the
group consisting of hydrogen and C.sub.1-4 alkyl;
[0449] R.sup.3f is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0450] X is selected from the group consisting of
--(CR.sup.4aR.sup.4b).sub.t--, --O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--, --CH.sub.2O--,
--N(H)C(R.sup.8a)(R.sup.8b)--, --CR.sup.4c.dbd.CR.sup.4d--
(including both E and Z isomers), --C.ident.C--,
--N(R')C(.dbd.O)--, and --OC(.dbd.O)--; (wherein when X is
--CH.sub.2S--, --CH.sub.2O--, --N(H)C(R.sup.8a)(R.sup.8b)--,
--N(R')C(.dbd.O)--, or --OC(.dbd.O)--; the hetereoatom of
--CH.sub.2S--, --CH.sub.2O--, --N(H)C(R.sup.8a)(R.sup.8b)--,
--N(R')C(.dbd.O)--, or --OC(.dbd.O)--; can be attached to either
6-membered ring, i.e., --CH.sub.2S-- is equivalent to
--SCH.sub.2--, --CH.sub.2-- is equivalent to --OCH.sub.2--,
--N(H)C(R.sup.8a)(R.sup.8b)-- is equivalent to
--C(R.sup.8a)(R.sup.8b)N(H)--), --N(R')C(.dbd.O)-- is equivalent to
--C(.dbd.O)N(R.sup.5)C.dbd.O)-- and --OC(.dbd.O)-- is equivalent to
--C(.dbd.O)O--; or
[0451] X is absent, i.e., X represents a chemical bond;
[0452] t is 1 or 2;
[0453] Z is selected from the group consisting of .dbd.CR.sup.11a--
and .dbd.N--;
[0454] each R.sup.4a and R.sup.4b are independently selected from
the group consisting of hydrogen and C.sub.1-4 alkyl; or
[0455] R.sup.4a and R.sup.4b taken together with the carbon atom to
which they are attached form a 3- to 6-membered cycloalkyl;
[0456] R.sup.4c and R.sup.4d are independently selected from the
group consisting of hydrogen and C.sub.1-4 alkyl;
[0457] R.sup.5 is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0458] R.sup.6a, R.sup.6b, R.sup.6c, and R.sup.6d are each
independently selected from the group consisting of hydrogen, halo,
C.sub.1-4 alkyl, haloalkyl, cyano, hydroxy, thiol, amino,
alkylthio, and alkoxy;
[0459] R.sup.7a is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0460] R.sup.7b is selected from the group consisting of hydrogen,
-L-H, -L-PG,
##STR00311##
or
[0461] R.sup.7a and R.sup.7b taken together with the nitrogen atom
to which they are attached form:
##STR00312##
or
[0462] R.sup.7a and R.sup.7b taken together with the nitrogen atom
to which they are attached form a nitro (--NO.sub.2) group;
[0463] m is 1, 2, 3, 4, 5, or 6;
[0464] L is a linker;
[0465] PG is a protecting group, e.g., Boc, FMOC;
[0466] R.sup.9f is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0467] R.sup.8a and R.sup.8b are independently selected from the
group consisting of hydrogen and C.sub.1-4 alkyl;
[0468] R.sup.11a and R.sup.11b are independently selected from the
group consisting of hydrogen, halo, C.sub.1-4 alkyl, C.sub.1-4
haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and alkoxy;
and
[0469] represents a single or double bond. In another embodiment,
R.sup.7b is hydrogen. In another embodiment, R.sup.7b is selected
from the group consisting of:
##STR00313##
[0470] m is 1, 2, 3, 4, 5, or 6; and
[0471] R.sup.10a and R.sup.10b are each independently selected from
the group consisting of hydrogen and optionally substituted
C.sub.1-6 alkyl.
[0472] In another embodiment, disclosed herein is a compound having
Formula VII':
##STR00314##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, X, and Z are as defined in
connection with Formula VII.
[0473] In another embodiment, disclosed herein is a compound having
Formula VII'':
##STR00315##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, and X are as defined in
connection with Formula VII, and the carbon atom marked with an "*"
is either the R-isomer or the S-isomer when R.sup.2 is halo or
hydroxyl. In one embodiment, the carbon atom marked with an "*" is
the R-isomer. In another embodiment, the carbon atom marked with an
"*" is the S-isomer.
[0474] In another embodiment, disclosed herein is a compound having
Formula VII-A or Formula VII-B:
##STR00316##
[0475] or a pharmaceutically acceptable salt or solvate thereof,
wherein:
[0476] R.sup.1 is selected from the group consisting of hydrogen
and halo;
[0477] R.sup.2 is selected from the group consisting of hydrogen,
halo, and hydroxy;
[0478] R.sup.3 is selected from the group consisting of
--CH.sub.2OH, --CH.sub.2SH, --CH.sub.2Cl, --SCH.sub.2Cl,
--SCH.sub.2F,
--SCH.sub.2CF.sub.3,--CH.sub.2OS(.dbd.O).sub.2-0H,
[0479] hydroxy, --OCH.sub.2CN, --OCH.sub.2Cl, --OCH.sub.2F,
--OCH.sub.3, --OCH.sub.2CH.sub.3, --SCH.sub.2CN,
##STR00317##
[0480] R.sup.3a is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0481] R.sup.3b is selected from the group consisting of C.sub.1-4
alkyl and C.sub.1-4 alkoxy;
[0482] R.sup.3c is selected from the group consisting of hydrogen,
C.sub.1-4 alkyl, --CH.sub.2OH, C.sub.1-4 alkoxy,
--CH--.sub.2(amino), and --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.3f;
[0483] R.sup.3d and R.sup.3e are independently selected from the
group consisting of hydrogen and C.sub.1-4 alkyl;
[0484] R.sup.3f is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl; X is selected from the group consisting of
--(CR.sup.4aR.sup.4b).sub.t--, --O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--, --CH.sub.2O--,
--N(H)C(R.sup.8a)(R.sup.8b), --CR.sup.4c.dbd.CR.sup.4d--,
--C.ident.C--, --N(R.sup.5)C(.dbd.O)--, and --OC(.dbd.O)--; or
[0485] X is absent;
[0486] t is 1 or 2;
[0487] Z is selected from the group consisting of .dbd.CR.sup.11a--
and .dbd.N--;
[0488] each R.sup.4a and R.sup.4b are independently selected from
the group consisting of hydrogen and C.sub.1-4 alkyl; or
[0489] R.sup.4a and R.sup.4b taken together with the carbon atom to
which they are attached form a 3- to 6-membered cycloalkyl;
[0490] R.sup.4c and R.sup.4d are independently selected from the
group consisting of hydrogen and C.sub.1-4 alkyl;
[0491] R.sup.5 is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0492] R.sup.6a, R.sup.6b, and R.sup.6c are each independently
selected from the group consisting of hydrogen, halo, C.sub.1-4
alkyl, haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and
alkoxy;
[0493] R.sup.7a is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0494] R.sup.7b is selected from the group consisting of hydrogen,
-L-H, -L-PG,
##STR00318##
or
[0495] R.sup.7a and R.sup.7b taken together with the nitrogen atom
to which they are attached form:
##STR00319##
or
[0496] R.sup.7a and R.sup.7b taken together with the nitrogen atom
to which they are attached form a nitro (--NO.sub.2) group;
[0497] m is 1, 2, 3, 4, 5, or 6;
[0498] L is a linker;
[0499] PG is a protecting group;
[0500] R.sup.9f is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0501] R.sup.8a and R.sup.8b are independently selected from the
group consisting of hydrogen and C.sub.1-4 alkyl;
[0502] R.sup.11a and R.sup.11b are independently selected from the
group consisting of hydrogen, halo, C.sub.1-4 alkyl, C.sub.1-4
haloalkyl, cyano hydroxy, thiol, amino, alkylthio, and alkoxy;
and
[0503] represents a single or double bond. In another embodiment,
R.sup.7b is hydrogen. In another embodiment, R.sup.7b is selected
from the group consisting of:
##STR00320##
[0504] m is 1, 2, 3, 4, 5, or 6; and
[0505] R.sup.10a and R.sup.10b are each independently selected from
the group consisting of hydrogen and optionally substituted
C.sub.1-6 alkyl.
[0506] In another embodiment, disclosed herein is a compound having
Formula VII-A.sup.1 or Formula VII-B':
##STR00321##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, X, and Z are as defined in
connection with Formula VII-A.
[0507] In another embodiment, disclosed herein is a compound having
Formula VII-A'' or Formula VII-B'':
##STR00322##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, and X, are as defined in
connection with Formula VII-A, and the carbon atom marked with an
"*" is either the R-isomer or the S-isomer when R.sup.2 is halo or
hydroxyl. In one embodiment, the carbon atom marked with an "*" is
the R-isomer. In another embodiment, the carbon atom marked with an
"*" is the S-isomer.
[0508] In another embodiment, disclosed herein is a compound having
Formula VIII:
##STR00323##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, X, and Z are as defined in
connection with Formula VII.
[0509] In another embodiment, disclosed herein is a compound having
Formula VIII':
##STR00324##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, X, and Z are as defined in
connection with Formula VII.
[0510] In another embodiment, disclosed herein is a compound having
Formula VIII'':
##STR00325##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, and X are as defined in
connection with Formula VII, and the carbon atom marked with an "*"
is either the R-isomer or the S-isomer when R.sup.2 is halo or
hydroxyl. In one embodiment, the carbon atom marked with an "*" is
the R-isomer. In another embodiment, the carbon atom marked with an
"*" is the S-isomer.
[0511] In another embodiment, disclosed herein is a compound having
Formula VIII-a:
##STR00326##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, X, and Z are as defined in
connection with Formula VII.
[0512] In another embodiment, disclosed herein is a compound having
Formula VIII-a':
##STR00327##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, X, and Z are as defined in
connection with Formula VII.
[0513] In another embodiment, disclosed herein is a compound having
Formula VIII-a'':
##STR00328##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, and X are as defined in
connection with Formula VII, and the carbon atom marked with an "*"
is either the R-isomer or the S-isomer when R.sup.2 is halo or
hydroxyl. In one embodiment, the carbon atom marked with an "*" is
the R-isomer. In another embodiment, the carbon atom marked with an
"*" is the S-isomer.
[0514] In another embodiment, disclosed herein is a compound having
Formula VIII-b:
##STR00329##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, X, and Z are as defined in
connection with Formula VII.
[0515] In another embodiment, disclosed herein is a compound having
Formula VIII-b':
##STR00330##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, X, and Z are as defined in
connection with Formula VII.
[0516] In another embodiment, disclosed herein is a compound having
Formula VIII-b'':
##STR00331##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, and X are as defined in
connection with Formula VII, and the carbon atom marked with an "*"
is either the R-isomer or the S-isomer when R.sup.2 is halo or
hydroxyl. In one embodiment, the carbon atom marked with an "*" is
the R-isomer. In another embodiment, the carbon atom marked with an
"*" is the S-isomer.
[0517] In another embodiment, disclosed herein is a compound having
Formula IX:
##STR00332##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, X, and Z are as defined in
connection with Formula VII.
[0518] In another embodiment, disclosed herein is a compound having
Formula IX':
##STR00333##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, X, and Z are as defined in
connection with Formula VII.
[0519] In another embodiment, disclosed herein is a compound having
Formula IX'':
##STR00334##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, and X are as defined in
connection with Formula VII, and the carbon atom marked with an "*"
is either the R-isomer or the S-isomer when R.sup.2 is halo or
hydroxyl. In one embodiment, the carbon atom marked with an "*" is
the R-isomer. In another embodiment, the carbon atom marked with an
"*" is the S-isomer.
[0520] In another embodiment, disclosed herein is a compound having
Formula IX-a:
##STR00335##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, X, and Z are as defined in
connection with Formula VII.
[0521] In another embodiment, disclosed herein is a compound having
Formula IX-a':
##STR00336##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, X, and Z are as defined in
connection with Formula VII.
[0522] In another embodiment, disclosed herein is a compound having
Formula IX-a'':
##STR00337##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, and X are as defined in
connection with Formula VII, and the carbon atom marked with an "*"
is either the R-isomer or the S-isomer when R.sup.2 is halo or
hydroxyl. In one embodiment, the carbon atom marked with an "*" is
the R-isomer. In another embodiment, the carbon atom marked with an
"*" is the S-isomer.
[0523] In another embodiment, disclosed herein is a compound having
Formula IX-b:
##STR00338##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, X, and Z are as defined in
connection with Formula VII.
[0524] In another embodiment, disclosed herein is a compound having
Formula IX-b':
##STR00339##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, X, and Z are as defined in
connection with Formula VII.
[0525] In another embodiment, disclosed herein is a compound having
Formula IX-b'':
##STR00340##
or a pharmaceutically acceptable salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, , R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d,
R.sup.7a, R.sup.7b, R.sup.9f, R.sup.11b, and X are as defined in
connection with Formula VII, and the carbon atom marked with an "*"
is either the R-isomer or the S-isomer when R.sup.2 is halo or
hydroxyl. In one embodiment, the carbon atom marked with an "*" is
the R-isomer. In another embodiment, the carbon atom marked with an
"*" is the S-isomer.
[0526] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein represents a
single or double bond. In another embodiment, represents a double
bond.
[0527] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.1 is
selected from the group consisting of hydrogen and fluoro.
[0528] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.2 is
selected from the group consisting of hydrogen and fluoro.
[0529] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.3 is
selected from the group consisting of --CH.sub.2OH, --CH.sub.2Cl,
--SCH.sub.2Cl, --SCH.sub.2F, and --OH.
[0530] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein:
[0531] R.sup.3 is selected from the group consisting of:
##STR00341##
[0532] R.sup.3a is selected from the group consisting of hydrogen
and methyl;
[0533] R.sup.3b is selected from the group consisting of methyl,
ethyl, isopropyl, isobutyl, methoxy, ethoxy, isopropoxy, and
isobutoxy;
[0534] R.sup.3c is selected from the group consisting of hydrogen,
methyl, ethyl, --CH.sub.2OH, methoxy, ethoxy, and isopropoxy;
[0535] R.sup.3d and R.sup.3e are independently selected from the
group consisting of hydrogen, methyl, and ethyl.
[0536] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.5 and
R.sup.8a are independently selected from the group consisting of
hydrogen and methyl.
[0537] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', or IX-b',or a pharmaceutically
acceptable salt thereof, wherein Z is .dbd.CH--.
[0538] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', or a pharmaceutically
acceptable salt thereof, wherein Z is .dbd.N--.
[0539] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.7a is
selected from the group consisting of hydrogen and methyl. In
another embodiment, R.sup.7a is hydrogen. In another embodiment,
R.sup.7a is methyl.
[0540] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein:
[0541] X is selected from the group consisting of
--(CR.sup.4aR.sup.4b).sub.t,--O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --CH.sub.2S--, and --N(H)CH(R.sup.8a)--;
[0542] t is 1; and
[0543] R.sup.4a and R.sup.4b are independently selected from the
group consisting of hydrogen and methyl; or
[0544] R.sup.4a and R.sup.4b taken together with the carbon atom to
which they are attached form a 3-membered cycloalkyl. In another
embodiment, X is --CH.sub.2--. In another embodiment, X is selected
from the group consisting of:
##STR00342##
In another embodiment, X is --O--. In another embodiment, X is
--S--. In another embodiment, X is --CH.sub.2S--. In another
embodiment, X is --N(H)CH.sub.2--. In another embodiment, X is
selected from the group consisting of:
##STR00343##
[0545] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.11b is
hydrogen.
[0546] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.7b is
hydrogen.
[0547] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.7a and
R.sup.7b are hydrogen.
[0548] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, R.sup.6b is selected from
the group consisting of hydrogen, --Cl, --OMe (or --OCH.sub.3), and
--OH.
[0549] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.9f is
hydrogen.
[0550] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.9f is
methyl.
[0551] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.7a is
selected from the group consisting of hydrogen and --OH.
[0552] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.7b is
hydrogen.
[0553] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.7b is
R.sup.7b-1. In another embodiment, R.sup.10a and R.sup.10b are
independently optionally substituted C.sub.1-6 alkyl. In another
embodiment, R.sup.10a and R.sup.10b are independently optionally
substituted C.sub.1-4 alkyl.
[0554] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.7b is
R.sup.7b-2, and PG is BOC. In another embodiment, R.sup.10a and
R.sup.10b are independently optionally substituted C.sub.1-6 alkyl.
In another embodiment, R.sup.10a and R.sup.10b are independently
optionally substituted C.sub.1-4 alkyl.
[0555] In another embodiment, disclosed herein is a compound having
any one of Formulae VII, VII-A, VII-B, VIII, VIII-a, VIII-b, IX,
IX-a, or IX-b, or any one of Formulae VII', VII-A', VII-B', VIII',
VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'', VII-B'',
VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.7b is
R.sup.7b-3. In another embodiment, m is 2 or 3, and R.sup.10a and
R.sup.10b are each optionally substituted C.sub.1-6 alkyl. In
another embodiment, m is 2. In another embodiment, R.sup.10a and
R.sup.10b are independently optionally substituted C.sub.1-4
alkyl.
[0556] In another embodiment, disclosed herein is a compound having
Formulae VIII, or a pharmaceutically acceptable salt thereof, which
is any one of the compounds of Table VI.
TABLE-US-00016 TABLE VI ##STR00344## ##STR00345## ##STR00346##
##STR00347## ##STR00348## ##STR00349## ##STR00350## ##STR00351##
##STR00352## ##STR00353## ##STR00354## ##STR00355## ##STR00356##
##STR00357## ##STR00358## ##STR00359## ##STR00360## ##STR00361##
##STR00362## ##STR00363## ##STR00364## ##STR00365## ##STR00366##
##STR00367## ##STR00368## ##STR00369## ##STR00370## ##STR00371##
##STR00372## ##STR00373## ##STR00374## ##STR00375## ##STR00376##
##STR00377##
[0557] In another embodiment, disclosed herein is a compound having
Formulae VIII, or a pharmaceutically acceptable salt thereof, which
is any one of the compounds of Table VI-A.
TABLE-US-00017 TABLE VI-A ##STR00378## ##STR00379## ##STR00380##
##STR00381## ##STR00382## ##STR00383## ##STR00384## ##STR00385##
##STR00386## ##STR00387## ##STR00388## ##STR00389## ##STR00390##
##STR00391## ##STR00392## ##STR00393## ##STR00394## ##STR00395##
##STR00396## ##STR00397## ##STR00398## ##STR00399## ##STR00400##
##STR00401## ##STR00402## ##STR00403## ##STR00404## ##STR00405##
##STR00406## ##STR00407## ##STR00408## ##STR00409## ##STR00410##
##STR00411## ##STR00412## ##STR00413## ##STR00414## ##STR00415##
##STR00416## ##STR00417## ##STR00418## ##STR00419## ##STR00420##
##STR00421## ##STR00422## ##STR00423## ##STR00424## ##STR00425##
##STR00426##
[0558] In another embodiment, disclosed herein is a compound having
Formula VII-A or Formula VII-B, or a pharmaceutically acceptable
salt thereof, which is any one of the compounds of Table VI-B.
TABLE-US-00018 TABLE VI-B ##STR00427## ##STR00428## ##STR00429##
##STR00430## ##STR00431## ##STR00432##
[0559] In another embodiment, disclosed herein is a compound having
Formulae VIII, or a pharmaceutically acceptable salt thereof,
selected from the group consisting of:
##STR00433##
[0560] In another embodiment, disclosed herein is a compound having
Formula IX, or a pharmaceutically acceptable salt thereof, which is
any one of the compounds of Table VII.
TABLE-US-00019 TABLE VII ##STR00434## ##STR00435## ##STR00436##
##STR00437## ##STR00438## ##STR00439## ##STR00440## ##STR00441##
##STR00442## ##STR00443## ##STR00444## ##STR00445## ##STR00446##
##STR00447## ##STR00448## ##STR00449## ##STR00450## ##STR00451##
##STR00452## ##STR00453## ##STR00454## ##STR00455## ##STR00456##
##STR00457## ##STR00458## ##STR00459## ##STR00460## ##STR00461##
##STR00462## ##STR00463## ##STR00464##
[0561] In another embodiment, disclosed herein is a compound having
Formula IX-a, or a pharmaceutically acceptable salt thereof,
selected from the group consisting of:
##STR00465##
V. Methods of Making Immunoconjugates and Synthetic
Intermediates
[0562] The general synthesis of the immunoconjugates of the
disclosure is described in General Scheme 1.
##STR00466##
[0563] In General Scheme 1, SM-N(R.sup.7a)(R.sup.7b) is a
glucocorticosteroid having an --NH(R.sup.7a) group (wherein
R.sup.7a is hydrogen or C.sub.1-4 alky), or a compound having any
one of Formulae VII, VIII, VIII-a, VIII-b, IX, IX-a, or IX-b, or
any one of Formulae VII', VII-A', VII-B', VIII', VIII-a', VIII-b',
IX', IX-a', IX-b', VII'', VII-A'', VII-B'', VIII'', VIII-a'',
VIII-b'', IX'', IX-a'', or IX-b'', or a compound of Table 9; HS-A
is an antibody or antigen binding fragment having a limited number
of reduced interchain disulfide bonds, R' and R'' are independently
any side chain found in a naturally-occurring, e.g., methyl,
isopropyl, and/or non-natural amino acid, e.g.,
--CH.sub.2CH.sub.2CH.sub.2N(H)C(.dbd.O)NH.sub.2, m is 1, 2, 3, 4,
5, or 6, and PG is a protecting group, e.g., BOC. For the purpose
of illustration, General Scheme 1 shows only one sulfhydryl as
being available for conjugation in the antibody or antigen binding
fragment.
[0564] In another embodiment, disclosed herein is a method of
making a compound having Formula I-c:
##STR00467##
or a pharmaceutically acceptable salt thereof, wherein:
[0565] A is A.sup.1 or A;
[0566] A.sup.1 is an anti-tumor necrosis factor (TNF) alpha
protein;
[0567] A.sup.2 is a protein;
[0568] L is a linker;
[0569] n is 1-10; and
[0570] SM is a radical of a glucocorticosteroid, e.g., a compound
having any one of Formulae II-a-q; the method comprising:
[0571] a) conjugating a compound having Formula X:
##STR00468##
[0572] with an anti-tumor necrosis factor (TNF) alpha protein or a
protein; and
[0573] b) isolating the compound having Formula I-c, or a
pharmaceutically acceptable salt thereof. In another embodiment,
the method further comprises hydrolyzing the compound having
Formula I-c to give a compound having Formula I-d:
##STR00469##
[0574] In another embodiment, disclosed herein is a method of
making a compound having Formula I-e:
##STR00470##
[0575] or a pharmaceutically acceptable salt thereof, wherein:
[0576] A is A.sup.1 or A.sup.2;
[0577] A.sup.1 is an anti-tumor necrosis factor (TNF) alpha
protein;
[0578] A.sup.2 is a protein;
[0579] L is a linker;
[0580] R.sup.7a is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0581] n is 1-10;
[0582] m is 1, 2, 3, 4, 5, or 6; and
[0583] SM is a radical of a glucocorticosteroid, e.g., a compound
having any one of Formulae II-a-e or 1-q;
the method comprising:
[0584] a) conjugating a compound having Formula XI:
##STR00471##
[0585] with an anti-tumor necrosis factor (TNF) alpha protein or a
protein; and
[0586] b) isolating the compound having Formula I-e, or a
pharmaceutically acceptable salt thereof. In another embodiment,
the method further comprises hydrolyzing the compound having
Formula I-e to give a compound having Formula I-f:
##STR00472##
[0587] In another embodiment, disclosed herein is a method of
making a compound having Formula I-G:
##STR00473##
wherein:
[0588] A is adalimumab; and
[0589] n is 1-10, the method comprising:
[0590] a) conjugating Cpd. No. 88:
##STR00474##
[0591] with partially-reduced adalimumab; and
[0592] b) isolating, e.g., by chromatography, the compound having
Formula I-G.
[0593] In another embodiment, disclosed herein is a method of
making a compound having Formula I-H:
##STR00475##
wherein:
[0594] A is adalimumab; and
[0595] n is 1-10,
the method comprising hydrolyzing the compound having Formula I-G
to give Formula I-H.
[0596] In another embodiment, disclosed herein is a method of
making a compound having Formula I-G or Formula I-H, wherein n is
1-7. In another embodiment, n is 1-5. In another embodiment, n is
2-4. In another embodiment, n is 1. In another embodiment, n is
1.5. In another embodiment, n is 2. In another embodiment, n is
2.5. In another embodiment, n is 3. In another embodiment, n is
3.5. In another embodiment, n is 4. In another embodiment, n is
4.5. In another embodiment, n is 5.
[0597] In another embodiment, disclosed herein is a compound having
Formula I-H:
##STR00476##
wherein:
[0598] A is adalimumab; and
[0599] n is 1-10.
[0600] In another embodiment, disclosed herein is a compound having
Formula I-H, wherein n is 1-7. In another embodiment, n is 1-5. In
another embodiment, n is 2-4. In another embodiment, n is 1. In
another embodiment, n is 1.5. In another embodiment, n is 2. In
another embodiment, n is 2.5. In another embodiment, n is 3. In
another embodiment, n is 3.5. In another embodiment, n is 4. In
another embodiment, n is 4.5. In another embodiment, n is 5. In
another embodiment, n is 5.5. In another embodiment, n is 6. In
another embodiment, n is 6.5. In another embodiment, n is 7. In
another embodiment, n is 7.5. In another embodiment, n is 8.
[0601] According to the present disclosure, glucorticoid receptor
agonists can be linked to the antibody, antigen-binding fragment
thereof, or anti-TNF alpha proteins via any method and at any
location that does not prevent the antibody, antigen-binding
fragment thereof, or anti-TNF alpha protein from binding antigen
(e.g., TNF alpha) or prevent activity of the glucorticoid receptor
agonist. Methods for achieving such a linkage have been discussed,
for example, in Panowski et al., mAbs 6: 34-45 (2014), Jain et al.,
Pharm. Res. 32: 3526-3540 (2015), Mack et al., Seminars in Oncology
41: 637-652 (2014), U.S. Published Application No. 2008/0305044,
and U.S. Published Application No. 2011/0097322 each of which is
herein incorporated by reference in its entirety.
[0602] The glucorticoid receptor agonists can be linked to the
antibodies, antigen-binding fragments thereof, or anti-TNF alpha
proteins via a natural amino acid, e.g., an amino acid that has a
side-chain with a nucleophilic group.
[0603] For example, the glucorticoid receptor agonist can be linked
to a lysine residue. Methods for conjugation via lysine are known.
Such methods include a two-step process in which a linker is
attached to the antibody, antigen-binding fragment thereof, or
anti-TNF alpha protein in a first chemical reaction and then the
linker is reacted with the glucocorticoid receptor agonist in a
second chemical reaction. In another method, a one-step reaction
with a preformed linker-glucocorticoid receptor agonist to form the
conjugate containing the glucocorticoid receptor agonist linked to
the antibody, antigen-binding fragment thereof, or anti-TNF alpha
protein.
[0604] The glucorticoid receptor agonist can also be linked to a
cysteine residue. Methods for conjugation via cysteine are know.
IgG1 antibodies contain four inter-chain disulfide bonds, and
conjugation via cysteine can occur after reduction of these bonds
creates sulfhydryls available for conjugation.
[0605] The glucorticoid receptor agonists can be linked to the
antibody, antigen-binding fragment thereof, or anti-TNF alpha
proteins via site-specific conjugation.
[0606] One method of site-specific conjugation is cysteine-based
site-specific conjugation. An example of this method has been
reported by Junutula et al., Nat. Biotechnol 26: 925-935 (2008);
see also Junutula et al., J. Immunol. Methods 332: 41-52 (2008),
each of which is herein incorporated by reference in its entirety.
Using this method, antibodies, antigen-binding fragments thereof or
anti-TNF alpha proteins can be engineered with additional cysteines
that provide reactive thiol groups to conjugate glucocorticoid
receptor agonist. These publications also provide guidance
regarding the selection of reactive cysteins that do not interfere
with antigen binding.
[0607] Another method of site-specific conjugation makes use of
selenocysteine. Selenocysteine is similar to cysteine but contains
a more reactive selenium atom in place of the sulfur atom in
cysteine. Conditions can be used in which selenocysteines are
selectively activated. Hofer et al., Biochemistry 48: 12047-12057
(2009), which is herein incorporated by reference in its entirety,
has exemplified this technique.
[0608] Another method of site-specific conjugation makes use of
unnatural amino acids, e.g., acetylphenylalanine (pAcPhe) or
para-azido phenylalanine (pAF). Wang et al. Proc. Natl. Acad. Sci.
100: 56-61 (2003), Axup et al., Proc. Natl. Acad. Sci.
109:16101-16106 (2012), and Kern et al., JACS 138: 1430-1445
(2016), each of which is herein incorporated by reference in its
entirety, have exemplified this technique.
[0609] Another method of site-specific conjugation makes use of
enzymatic approaches, e.g., via glycotransferases or
transglutaminases. Mutant glycotransferases can be used to attach a
chemically active sugar moiety to a glycosylation site on an
antibody, antigen-binding fragment thereof, or anti-TNF alpha
protein. Human IgG antibodies contain an N-glycosylation site at
residue Asn-297 of the Fc fragment. The glycans attached at this
residue can be degalactosylated so that a mutant glycotransferase
is capable of transferring thereto. Boeggeman et al., Bioconjug.
Chem. 20: 1228-1236 (2009), which is herein incorporated by
reference in its entirety, has exemplified this technique.
Transglutaminases, e.g., from Streptoverticillium mobaranse,
recognize a glutamine tag, e.g., LLQG, that can be engineered into
an anti-TNF alpha protein. Jeger et al., Angew Chem. Int. Ed. Engl.
49: 9995-9997 (2010), which is herein incorporated by reference in
its entirety, has exemplified this technique.
[0610] C-terminal attachment via expressed protein ligation can
also be used. For example, intein mediated C-terminal thioester
formation can be used for chemoselective ligation with an anti-TNF
alpha protein containing an N-terminal cysteine peptide. Chiang et
al., J. Am. Chem. Soc. 136: 3370-3373 (2014), which is herein
incorporated by reference in its entirety, has exemplified this
technique.
[0611] Also provided herein are synthetic intermediates, e.g.,
compounds having Formula X and XI, that useful for the preparation
of immunoconjugates.
[0612] In one embodiment, the synthetic intermediate disclosed
herein is a compound having any one of Formulae VII, VIII, VIII-a,
VIII-b, IX, IX-a, or IX-b, or any one of Formulae VII', VII-A',
VII-B', VIII', VIII-a', VIII-b', IX', IX-a', IX-b', VII'', VII-A'',
VII-B'', VIII'', VIII-a'', VIII-b'', IX'', IX-a'', or IX-b'', or a
pharmaceutically acceptable salt thereof, wherein R.sup.7b is
selected from the group consisting of -L-H, -L-PG,
##STR00477##
[0613] In another embodiment, the synthetic intermediate disclosed
herein is a compound having Formula VIII, or a pharmaceutically
acceptable salt thereof, which is any one or more of the compounds
of Table VIII:
TABLE-US-00020 TABLE VIII ##STR00478## ##STR00479## ##STR00480##
##STR00481## ##STR00482## ##STR00483## ##STR00484## ##STR00485##
##STR00486## ##STR00487## ##STR00488## ##STR00489## ##STR00490##
##STR00491## ##STR00492## ##STR00493## ##STR00494## ##STR00495##
##STR00496## ##STR00497## ##STR00498## ##STR00499## ##STR00500##
##STR00501## ##STR00502## ##STR00503## ##STR00504## ##STR00505##
##STR00506## ##STR00507## ##STR00508## ##STR00509## ##STR00510##
##STR00511## ##STR00512##
wherein R.sup.7b is selected from the group consisting of -L-H,
-L-PG,
##STR00513##
In another embodiment, R.sup.7b is selected from the group
consisting of:
##STR00514##
In another embodiment, R.sup.7b is R.sup.7b-4. In another
embodiment, R.sup.7b is R.sup.7b-5. In another embodiment, R.sup.7b
is R.sup.7b-6. In another embodiment, R.sup.7b is any one of the
structures of Table IX.
TABLE-US-00021 TABLE IX ##STR00515## ##STR00516## ##STR00517##
##STR00518## ##STR00519## ##STR00520## ##STR00521## ##STR00522##
##STR00523## ##STR00524## ##STR00525## ##STR00526## ##STR00527##
##STR00528## ##STR00529## ##STR00530## ##STR00531## ##STR00532##
##STR00533##
[0614] In another embodiment, the synthetic intermediate disclosed
herein is a compound having Formulae VIII, or a pharmaceutically
acceptable salt thereof, which is any one of the compounds of Table
X.
TABLE-US-00022 TABLE X ##STR00534## ##STR00535## ##STR00536##
##STR00537## ##STR00538## ##STR00539## ##STR00540## ##STR00541##
##STR00542## ##STR00543## ##STR00544## ##STR00545## ##STR00546##
##STR00547## ##STR00548## ##STR00549## ##STR00550## ##STR00551##
##STR00552## ##STR00553## ##STR00554## ##STR00555## ##STR00556##
##STR00557## ##STR00558## ##STR00559## ##STR00560## ##STR00561##
##STR00562## ##STR00563## ##STR00564## ##STR00565## ##STR00566##
##STR00567## ##STR00568## ##STR00569## ##STR00570## ##STR00571##
##STR00572## ##STR00573##
[0615] In another embodiment, the synthetic intermediate disclosed
herein is a compound having Formulae VIII, or a pharmaceutically
acceptable salt thereof, which is any one of the compounds of Table
X-A.
TABLE-US-00023 TABLE X-A ##STR00574## ##STR00575## ##STR00576##
##STR00577## ##STR00578## ##STR00579## ##STR00580## ##STR00581##
##STR00582## ##STR00583## ##STR00584## ##STR00585## ##STR00586##
##STR00587##
[0616] In another embodiment, the synthetic intermediate disclosed
herein is a compound having Formula IX, or a pharmaceutically
acceptable salt thereof, which is any one of the chemical
structures of Table XI.
TABLE-US-00024 TABLE XI ##STR00588## ##STR00589## ##STR00590##
##STR00591## ##STR00592## ##STR00593## ##STR00594## ##STR00595##
##STR00596## ##STR00597## ##STR00598## ##STR00599## ##STR00600##
##STR00601## ##STR00602## ##STR00603## ##STR00604## ##STR00605##
##STR00606## ##STR00607## ##STR00608## ##STR00609## ##STR00610##
##STR00611## ##STR00612## ##STR00613## ##STR00614## ##STR00615##
##STR00616## ##STR00617## ##STR00618##
wherein R.sup.7b is selected from the group consisting of -L-H,
-L-PG,
##STR00619##
In another embodiment, R is selected from the group consisting
of:
##STR00620##
In another embodiment, R.sup.7b is R.sup.7b-4. In another
embodiment, R.sup.7b is R.sup.7b-5. In another embodiment, R.sup.7b
is R.sup.7b-6. In another embodiment, R.sup.7b any one of the
chemical structures of Table IX.
[0617] In another embodiment, the synthetic intermediate disclosed
herein is a compound having Formula IX, or a pharmaceutically
acceptable salt thereof, which is any one of the compounds of Table
XII.
TABLE-US-00025 TABLE XII ##STR00621## ##STR00622## ##STR00623##
##STR00624## ##STR00625## ##STR00626## ##STR00627## ##STR00628##
##STR00629## ##STR00630## ##STR00631## ##STR00632## ##STR00633##
##STR00634## ##STR00635## ##STR00636## ##STR00637##
##STR00638##
VI. Methods of Use and Pharmaceutical Compositions
[0618] Provided herein are conjugates having Formulae I-a and I-b,
and glucocorticoid receptor agonists having Formulae VII, VII-A,
VII-B, VIII, VIII-a, VIII-b, IX, IX-a, or IX-b, or any one of
Formulae VII', VII-A', VII-B', VIII', VIII-a', VIII-b', IX', IX-a',
IX-b', VII'', VII-A'', VII-B'', VIII'', VIII-a'', VIII-b'', IX'',
IX-a'', or IX-b'' (wherein R.sup.7b is hydrogen) that can be used
in vitro or in vivo. Accordingly, also provided herein are
compositions, e.g., pharmaceutical compositions for certain in vivo
uses, comprising a conjugate or a glucocorticoid receptor agonist
described herein having the desired degree of purity in a
physiologically acceptable carrier, excipient or stabilizer
(Remington's Pharmaceutical Sciences (1990) Mack Publishing Co.,
Easton, Pa.). Acceptable carriers, excipients, or stabilizers are
nontoxic to recipients at the dosages and concentrations
employed.
[0619] The compositions (e.g., pharmaceutical compositions) to be
used for in vivo administration can be sterile. This is readily
accomplished by filtration through, e.g., sterile filtration
membranes. The compositions (e.g., pharmaceutical compositions) to
be used for in vivo administration can comprise a preservative.
[0620] A pharmaceutical composition comprising a glucocorticoid
receptor agonist provided herein can be formulated, for example, as
a nasal spray, an inhalation aerosol (e.g., for oral inhalation),
or a capsule, tablet, or pill (e.g., for oral administration).
[0621] The glucocorticoid receptor agonists provided herein (e.g.,
an anti-TNF ADC) are compounds, wherein the average number of
glucocorticosteroids per antibody (DAR) in the composition is about
1 to about 10. In some embodiments, the average number of
glucocorticosteroids per antibody (DAR) in the composition is about
2 to about 6. In some embodiments, the average number of
glucocorticosteroids per antibody (DAR) in the composition is about
3 to about 4. In some embodiments, the average number of
glucocorticosteroids per antibody (DAR) in the composition is about
3.1. In some embodiments, the average number of
glucocorticosteroids per antibody (DAR) in the composition is about
3.2. In some embodiments, the average number of
glucocorticosteroids per antibody (DAR) in the composition is about
3.3. In some embodiments, the average number of
glucocorticosteroids per antibody (DAR) in the composition is about
3.4. In some embodiments, the average number of
glucocorticosteroids per antibody (DAR) in the composition is about
3.5. In some embodiments, the average number of
glucocorticosteroids per antibody (DAR) in the composition is about
3.6. In some embodiments, the average number of
glucocorticosteroids per antibody (DAR) in the composition is about
3.7. In some embodiments, the average number of
glucocorticosteroids per antibody (DAR) in the composition is about
3.8. In some embodiments, the average number of
glucocorticosteroids per antibody (DAR) in the composition is about
3.9.
[0622] Glucocorticoid receptor agonists and pharmaceutical
compositions comprising a glucocorticoid receptor agonist described
herein can be useful in inhibiting cytokine release (in vitro or in
vivo) and/or for the treatment of autoimmune or inflammatory
diseases. Glucocorticoid receptor agonists and pharmaceutical
compositions comprising a glucocorticoid receptor agonist described
herein can be used for the treatment of asthma (e.g., bronchial
asthma), Crohn's disease (e.g., mild to moderate active Crohn's
disease involving the ileum and/or the ascending colon and/or the
maintenance of clinical remission of mild to moderate Crohn's
disease involving the ileum and/or the ascending colon for up to 3
months), ulcerative colitis (e.g., for the induction of remission
in patients with active, mild to moderate ulcerative colitis),
allergic rhinitis (e.g. nasal symptoms associated with seasonal
allergic rhinitis and/or perennial allergic rhinitis).
[0623] For administration to human patients, the total daily dose
of glucocorticoid receptor agonists provided herein is typically in
the range of 0.001 mg to 5000 mg, or in the range of 0.01 mg to
1000 mg, depending on the mode of administration. For example, oral
administration or intravenous, intramuscular, intra-articular, or
peri-articular administration can require a total daily dose of
from 0.01 mg to 1000 mg, or from 0.1 mg to 100 mg. The total daily
dose can be administered in single or divided doses.
[0624] A pharmaceutical composition comprising a conjugate provided
herein can be formulated, for example, for intravenous
administration or infusion.
[0625] Conjugates and pharmaceutical compositions comprising
conjugates described herein can be useful in lysing a cell
expressing surface TNF-alpha (in vitro or in vivo), for the
treatment of diseases or disorders characterized by increased
TNF-alpha (e.g., increased TNF-alpha in synovial fluid), and/or for
the treatment of an autoimmune or inflammatory disease.
[0626] A pharmaceutical composition comprising a glucocortic
receptor agonist or a conjugate described herein is used for the
treatment of rheumatoid arthritis (RA), juvenile idiopathic
arthritis (JIA), psoriatic arthritis (PsA), a spondyloarthropathy
such as ankylosing spondylitis (AS) or axial spondyloarthritis
(axSpA), adult Crohns' disease (CD), pediatric Crohn's disease,
ulcerative colitis (UC), plaque psoriasis (Ps), hidradenitis
suppurativa (HS), uveitis, Behcets disease, or psoriasis, including
plaque psoriasis.
[0627] For administration to human patients, the total daily dose
of conjugate provided herein is typically in the range of from 0.01
.mu.g to 100 mg per kg of body weight, and can be given once or
more daily, weekly, monthly or yearly.
[0628] The disclosure also provides Embodiments (Embs) 1-209 as
particular embodiments. The Formulae and Tables referred to these
particular embodiments that are not shown in the embodiment are set
forth in the description above.
Embodiment (Emb) 1
[0629] A compound having Formula I-a:
(SM-L-Q).sub.n-A.sup.1 I-a
or a pharmaceutically acceptable salt or solvate thereof,
wherein:
[0630] A.sup.1 is an anti-tumor necrosis factor (TNF) alpha
protein; L is a linker; Q is a heterobifunctional group or
heterotrifunctional group; or Q is absent; n is 1-10; and SM is a
radical of a glucocorticosteroid.
Emb 2
[0631] The compound of Emb 1, or a pharmaceutically acceptable salt
or solvate thereof, wherein SM is a monovalent radical of a
glucocorticosteroid.
Emb 3
[0632] The compound of Emb 2, or a pharmaceutically acceptable salt
or solvate thereof, wherein SM is a monovalent radical of a
glucocorticosteroid selected from the group consisting of:
##STR00639##
wherein the sulfur, oxygen, or nitrogen atom is attached directly
or indirectly to the C- or D-ring of the glucocorticosteroid, and R
is C.sub.1-4 alkyl.
Emb 4
[0633] The compound of Emb 3, or a pharmaceutically acceptable salt
or solvate thereof, wherein the sulfur, oxygen, or nitrogen atom is
attached directly or indirectly to the D-ring of the
glucocorticosteroid.
Emb 5
[0634] The compound of Emb 2 or Emb 3, or a pharmaceutically
acceptable salt or solvate thereof, wherein SM is a monovalent
radical of a glucocorticosteroid having Formula II-a, wherein:
[0635] R.sup.1 is selected from the group consisting of hydrogen
and halo; R.sup.2 is selected from the group consisting of
hydrogen, halo, and hydroxy; R.sup.3 is selected from the group
consisting of --CH.sub.2OH, --CH.sub.2SH, --CH.sub.2Cl,
--SCH.sub.2Cl, --SCH.sub.2F, --SCH.sub.2CF.sub.3, hydroxy,
--OCH.sub.2CN, --OCH.sub.2Cl, --OCH.sub.2F, --OCH.sub.3,
--OCH.sub.2CH.sub.3, --SCH.sub.2CN,
##STR00640##
[0636] R.sup.3a is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl; R.sup.3b is selected from the group consisting
of C.sub.1-4 alkyl and C.sub.1-4 alkoxy; R.sup.3c is selected from
the group consisting of hydrogen, C.sub.1-4 alkyl, --CH.sub.2OH,
and C.sub.1-4 alkoxy; R.sup.3d and R.sup.3e, are independently
selected from the group consisting of hydrogen and C.sub.1-4
alkyl;
[0637] R.sup.9a is selected from the group consisting of optionally
substituted alkyl, optionally substituted cycloalkyl, optionally
substituted aryl, and optionally substituted heteroaryl; R.sup.9b
is selected from the group consisting of hydrogen and alkyl; or
R.sup.9a is:
##STR00641##
and
[0638] R.sup.9b is hydrogen or methyl;
[0639] X is selected from the group consisting of
--(CR.sup.4aR.sup.4b).sub.t--, --O--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--, --CH.sub.2O--,
--N(H)C(R.sup.8a)(R.sup.8b)--, --CR.sup.4c.dbd.CR.sup.4d--, and
--C.ident.C--; or X is absent; t is 1 or 2;
[0640] Z is selected from the group consisting of .dbd.CH--,
.dbd.C(OH)--, and .dbd.N--; each R.sup.4a and R.sup.4b are
independently selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; or R.sup.4a and R.sup.4b taken together with the
carbon atom to which they are attached form a 3- to 6-membered
cycloalkyl; R.sup.4c and R.sup.4d are independently selected from
the group consisting of hydrogen and C.sub.1-4 alkyl; R.sup.5 is
selected from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.6a, R.sup.6b, R.sup.6c, R.sup.6d, and R.sup.6e are each
independently selected from the group consisting of hydrogen, halo,
C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, cyano, hydroxy, thiol, amino,
alkylthio, and alkoxy; R.sup.8a and R.sup.8b are independently
selected from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.11 is selected from the group consisting of hydrogen, halo,
C.sub.1-4 alkyl, hydroxy, thiol, amino, alkylthio, and alkoxy; and
represents a single or double bond.
Emb 6
[0641] The compound of Emb 5, or a pharmaceutically acceptable salt
or solvate thereof, wherein SM is a monovalent radical of a
glucocorticosteroid having Formula II-b.
Emb 7
[0642] The compound of any one of Embs 2-4, or a pharmaceutically
acceptable salt or solvate thereof, wherein SM is a monovalent
radical of a glucocorticosteroid having Formula II-c, wherein:
R.sup.1 is selected from the group consisting of hydrogen and
halo;
[0643] R.sup.2 is selected from the group consisting of hydrogen,
halo, and hydroxy; R.sup.9a is selected from the group consisting
of optionally substituted alkyl, optionally substituted cycloalkyl,
optionally substituted aryl, and optionally substituted heteroaryl;
and R.sup.9b is selected from the group consisting of hydrogen and
alkyl; or R.sup.9a is:
##STR00642##
and
[0644] R.sup.9b is hydrogen;
[0645] W is selected from the group consisting of --O-- and --S--;
X is selected from the group consisting of
--(CR.sup.4aR.sup.4b).sub.t--O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--, --CH.sub.2O--,
--N(H)C(R.sup.8a)(R.sup.8b), --CR.sup.4c.dbd.CR.sup.4d--, and
--C.ident.C--; or X is absent; t is 1 or 2; Z is selected from the
group consisting of .dbd.CH--, .dbd.C(OH)--, and .dbd.N--; each
R.sup.4a and R.sup.4b are independently selected from the group
consisting of hydrogen and C.sub.1-4 alkyl; or R.sup.4a and
R.sup.4b taken together with the carbon atom to which they are
attached form a 3- to 6-membered cycloalkyl; R.sup.4c and R.sup.4d
are independently selected from the group consisting of hydrogen
and C.sub.1-4 alkyl; R is selected from the group consisting of
hydrogen and C.sub.1-4 alkyl; R.sup.6a, R.sup.6b, R.sup.6c,
R.sup.6d, and R.sup.6e are each independently selected from the
group consisting of hydrogen, halo, C.sub.1-4 alkyl, C.sub.1-4
haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and alkoxy;
R.sup.8a and R.sup.8b are independently selected from the group
consisting of hydrogen and C.sub.1-4 alkyl; R.sup.11 is selected
from the group consisting of hydrogen, halo, C.sub.1-4 alkyl,
C.sub.1-4 haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and
alkoxy; and =represents a single or double bond.
Emb 8
[0646] The compound of Emb 7, or a pharmaceutically acceptable salt
or solvate thereof, wherein SM is a monovalent radical of a
glucocorticosteroid having Formula II-d.
Emb 9
[0647] The compound of any one of Embs 2-4, or a pharmaceutically
acceptable salt or solvate thereof, wherein SM is a monovalent
radical of a glucocorticosteroid having Formula II-e, wherein:
R.sup.1 is selected from the group consisting of hydrogen and halo;
R.sup.2 is selected from the group consisting of hydrogen, halo,
and hydroxy; R.sup.9c is selected from the group consisting of
hydrogen, C.sub.1-4 alkyl, and --C(.dbd.O)R.sup.9e; R.sup.9d is
selected from the group consisting of hydrogen, optionally
substituted alkyl, optionally substituted cycloalkyl, optionally
substituted aryl, and optionally substituted heteroaryl; R.sup.9e
is selected from the group consisting of hydrogen, optionally
substituted alkyl, optionally substituted cycloalkyl, optionally
substituted aryl, and optionally substituted heteroaryl; W is
selected from the group consisting of --O-- and --S--; and
=represents a single or double bond.
Emb 10
[0648] The compound of Emb 9, or a pharmaceutically acceptable salt
or solvate thereof, wherein SM is a monovalent radical of a
glucocorticosteroid having Formula II-f.
Emb 11
[0649] The compound of any one of Embs 7-10, or a pharmaceutically
acceptable salt or solvate thereof, wherein W is --S--.
Emb 12
[0650] The compound of any one of Embs 7-10, or a pharmaceutically
acceptable salt or solvate thereof, wherein W is --O--.
Emb 13
[0651] A compound having Formula I-b:
(SM-L-Q).sub.n-A.sup.2 I-b
or a pharmaceutically acceptable salt or solvate thereof, wherein:
A.sup.2 is a protein; L is a linker; Q is a heterobifunctional
group or heterotrifunctional group; or Q is absent; n is 1-10; and
SM is a monovalent radical of a glucocorticosteroid having any one
of: Formula II-l, Formula II-m, Formula II-n, Formula II-o, Formula
II-p or Formula II-q, wherein: R.sup.1 is selected from the group
consisting of hydrogen and halo; R.sup.2 is selected from the group
consisting of hydrogen, halo, and hydroxy; R.sup.3 is selected from
the group consisting of --CH.sub.2OH, --CH.sub.2SH, --CH.sub.2Cl,
--SCH.sub.2Cl, --SCH.sub.2F, --SCH.sub.2CF.sub.3, hydroxy,
--OCH.sub.2CN, --OCH.sub.2Cl, --OCH.sub.2F, --OCH.sub.3,
--OCH.sub.2CH.sub.3, --SCH.sub.2CN,
##STR00643##
R.sup.3a is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.3b is selected from the group consisting of
C.sub.1-4 alkyl and C.sub.1-4 alkoxy; R.sup.3c is selected from the
group consisting of hydrogen, C.sub.1-4 alkyl, --CH.sub.2OH, and
C.sub.1-4 alkoxy; R.sup.3d and R.sup.3e are independently selected
from hydrogen and C.sub.1-4 alkyl; R.sup.6a, R.sup.6b, R.sup.6c,
R.sup.6d, and R.sup.6e are each independently selected from the
group consisting of hydrogen, halo, C.sub.1-4 alkyl, C.sub.1-4
haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and alkoxy; X
is selected from the group consisting of
--(CR.sup.4aR.sup.4b)--,--O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--, --CH.sub.2O--,
--N(H)C(R.sup.8a)(R.sup.8b)--, --CR.sup.4c.dbd.CR.sup.4d--, and
--C.ident.C--; or X is absent; Y.sup.2 is selected from the group
consisting of --O--, --S--, and --N(R.sup.7a)--; or Y.sup.2 is
absent; t is 1 or 2; Z is selected from the group consisting of
.dbd.CR.sup.11a-- and .dbd.N--; each R.sup.4a and R.sup.4b are
independently selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; or R.sup.4a and R.sup.4b taken together with the
carbon atom to which they are attached form a 3- to 6-membered
cycloalkyl; R.sup.4c and R.sup.4d are independently selected from
the group consisting of hydrogen and C.sub.1-4 alkyl; R.sup.1 is
selected from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.7a is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.8a and R.sup.8b are independently selected
from the group consisting of hydrogen and C.sub.1-4 alkyl; R.sup.9f
is selected from the group consisting of hydrogen and C.sub.1-4
alkyl; R.sup.11a and R.sup.11b are independently selected from the
group consisting of hydrogen, halo, C.sub.1-4 alkyl, C.sub.1-4
haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and alkoxy; and
represents a single or double bond.
Emb 14
[0652] The compound of any one of Embs 2-4 or 13, or a
pharmaceutically acceptable salt or solvate thereof, wherein SM is
a monovalent radical of a glucocorticosteroid having Formula II-l,
wherein: R.sup.1 is selected from the group consisting of hydrogen
and halo; R.sup.2 is selected from the group consisting of
hydrogen, halo, and hydroxy; R.sup.3 is selected from the group
consisting of --CH.sub.2OH, --CH.sub.2SH, --CH.sub.2Cl,
--SCH.sub.2Cl, --SCH.sub.2F, --SCH.sub.2CF.sub.3, hydroxy,
--OCH.sub.2CN, --OCH.sub.2Cl, --OCH.sub.2F, --OCH.sub.3,
--OCH.sub.2CH.sub.3, --SCH.sub.2CN,
##STR00644##
R.sup.3a is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.3b is selected from the group consisting of
C.sub.1-4 alkyl and C.sub.1-4 alkoxy; R.sup.3c is selected from the
group consisting of hydrogen, C.sub.1-4 alkyl, --CH.sub.2OH, and
C.sub.1-4 alkoxy; R.sup.3d and R.sup.3e are independently selected
from hydrogen and C.sub.1-4 alkyl; X is selected from the group
consisting of --(CR.sup.4aR.sup.4b).sub.t--, --O--, --S--,
--S(.dbd.O)--, --S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--,
--CH.sub.2O--, --N(H)C(R.sup.8a)(R.sup.8b)--,
--CR.sup.4.dbd.CR.sup.4--, and --C.ident.C--; or X is absent; t is
1 or 2; Z is selected from the group consisting of
.dbd.CR.sup.11a-- and .dbd.N--; each R.sup.4a and R.sup.4b are
independently selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; or R.sup.4a and R.sup.4b taken together with the
carbon atom to which they are attached form a 3- to 6-membered
cycloalkyl; R.sup.4c and R.sup.4d are independently selected from
the group consisting of hydrogen and C.sub.1-4 alkyl; R.sup.1 is
selected from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.6a, R.sup.6c, R.sup.6d, and R.sup.6e are each independently
selected from the group consisting of hydrogen, halo, C.sub.1-4
alkyl, C.sub.1-4 haloalkyl, cyano, hydroxy, thiol, amino,
alkylthio, and alkoxy; Y.sup.2 is selected from the group
consisting of --O--, --S--, and --N(R.sup.7a)--; or Y.sup.2 is
absent; R.sup.7a is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl; R.sup.8a and R.sup.8b are independently
selected from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.9f is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.11a and R.sup.11b are independently selected
from the group consisting of hydrogen, halo, C.sub.1-4 alkyl,
C.sub.1-4 haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and
alkoxy; and represents a single or double bond.
Emb 15
[0653] The compound of Emb 14, or a pharmaceutically acceptable
salt or solvate thereof, wherein SM is a monovalent radical of a
glucocorticosteroid having Formula II-m.
Emb 16
[0654] The compound of Emb 14, or a pharmaceutically acceptable
salt or solvate thereof, wherein SM is a monovalent radical of a
glucocorticosteroid having Formula II-n.
Emb 17
[0655] The compound of any one of Embs 2-4 or 13, or a
pharmaceutically acceptable salt or solvate thereof, wherein SM is
a monovalent radical of a glucocorticosteroid having Formula II-o,
wherein: R.sup.1 is selected from the group consisting of hydrogen
and halo; R.sup.2 is selected from the group consisting of
hydrogen, halo, and hydroxy; R.sup.3 is selected from the group
consisting of --CH.sub.2OH, --CH.sub.2SH, --CH.sub.2Cl,
--SCH.sub.2Cl, --SCH.sub.2F, --SCH.sub.2CF.sub.3, hydroxy,
--OCH.sub.2CN, --OCH.sub.2Cl, --OCH.sub.2F, --OCH.sub.3,
--OCH.sub.2CH.sub.3, --SCH.sub.2CN,
##STR00645##
R.sup.3a is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.3b is selected from the group consisting of
C.sub.1-4 alkyl and C.sub.1-4 alkoxy; R.sup.3c is selected from the
group consisting of hydrogen, C.sub.1-4 alkyl, --CH.sub.2OH, and
C.sub.1-4 alkoxy; R.sup.3d and R.sup.3e are independently selected
from hydrogen and C.sub.1-4 alkyl; X is selected from the group
consisting of --(CR.sup.4aR.sup.4b)--, --O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--, --CH.sub.2O--,
--N(H)C(R.sup.8a)(R.sup.8b), --CR.sup.4c.dbd.CR.sup.4d--, and
--C.ident.C--; or X is absent; t is 1 or 2; Z is selected from the
group consisting of .dbd.CR.sup.11a-- and .dbd.N--; each R.sup.4a
and R.sup.4b are independently selected from the group consisting
of hydrogen and C.sub.1-4 alkyl; or R.sup.4a and R.sup.4b taken
together with the carbon atom to which they are attached form a 3-
to 6-membered cycloalkyl; R.sup.4c and R.sup.4d are independently
selected from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.5 is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.6a, R.sup.6b, R.sup.6d, and R.sup.6e are
each independently selected from the group consisting of hydrogen,
halo, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, cyano, hydroxy, thiol,
amino, alkylthio, and alkoxy; Y.sup.2 is selected from the group
consisting of --O--, --S--, and --N(R.sup.7a)--; or Y.sup.2 is
absent; R.sup.7a is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl; R.sup.8a and R.sup.8b are independently
selected from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.9f is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.11a and R.sup.11b are independently selected
from the group consisting of hydrogen, halo, C.sub.1-4 alkyl,
C.sub.1-4 haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and
alkoxy; and =represents a single or double bond.
Emb 18
[0656] The compound of Emb 17, or a pharmaceutically acceptable
salt or solvate thereof, wherein SM is a monovalent radical of a
glucocorticosteroid having Formula II-p.
Emb 19
[0657] The compound of Emb 17, or a pharmaceutically acceptable
salt or solvate thereof, wherein SM is a monovalent radical of a
glucocorticosteroid having Formula II-q.
Emb 20
[0658] The compound of any one of Embs 5-19, or a pharmaceutically
acceptable salt or solvate thereof, wherein represents a double
bond.
Emb 21
[0659] The compound of any one of Embs 5-20, or a pharmaceutically
acceptable salt or solvate thereof, wherein R.sup.1 is selected
from the group consisting of hydrogen and fluoro.
Emb 22
[0660] The compound of any one of Embs 5-21, or a pharmaceutically
acceptable salt or solvate thereof, wherein R.sup.2 is selected
from the group consisting of hydrogen and fluoro.
Emb 23
[0661] The compound of any one of Embs 5, 6, or 13-22, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.3 is selected from the group consisting of --CH.sub.2OH,
--CH.sub.2Cl, --SCH.sub.2Cl, --SCH.sub.2F, and hydroxy.
Emb 24
[0662] The compound of any one of Embs 5, 6, or 13-22, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.3 is selected from the group consisting of:
##STR00646##
R.sup.3a is selected from the group consisting of hydrogen and
methyl; R.sup.3b is selected from the group consisting of methyl,
ethyl, isopropyl, isobutyl, methoxy, ethoxy, isopropoxy, and
isobutoxy; R.sup.3c is selected from the group consisting of
hydrogen, methyl, ethyl, --CH.sub.2OH, methoxy, ethoxy, and
isopropoxy; R.sup.3d and R.sup.3e are independently selected from
the group consisting of hydrogen, methyl, and ethyl.
Emb 25
[0663] The compound of any one of Embs 5-8 or 11-24, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.5 and R.sup.8a are independently selected from the group
consisting of hydrogen and methyl.
Emb 26
[0664] The compound of any one of Embs 5-8, 11-25, or a
pharmaceutically acceptable salt or solvate thereof, wherein Z is
.dbd.CH--.
Emb 27
[0665] The compound of any one of Embs 5-8 or 11-25, or a
pharmaceutically acceptable salt or solvate thereof, wherein Z is
.dbd.N--.
Emb 28
[0666] The compound of any one of Embs 5-8 or 11-27, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.6a, R.sup.6d, and R.sup.6e are hydrogen.
Emb 29
[0667] The compound of any one of Embs 13-28, or a pharmaceutically
acceptable salt or solvate thereof, wherein Y.sup.2 is
--N(R.sup.7a)--.
Emb 30
[0668] The compound of Emb 29, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.7a is selected from the
group consisting of hydrogen and methyl.
Emb 31
[0669] The compound of Emb 30, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.7a is hydrogen.
Emb 32
[0670] The compound of Emb 30, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.7a is methyl.
Emb 33
[0671] The compound of any one of Embs 5-8 or 13-32, or a
pharmaceutically acceptable salt or solvate thereof, wherein: X is
selected from the group consisting of
--(CR.sup.4aR.sup.4b).sub.t--, --O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --CH.sub.2S--, and --N(H)CH(R.sup.8a)--; t is
1; R.sup.4a and R.sup.4b are independently selected from the group
consisting of hydrogen and methyl; or R.sup.4a and R.sup.4b taken
together with the carbon atom to which they are attached form a
3-membered cycloalkyl; and R.sup.8a is selected from the group
consisting of hydrogen and methyl.
Emb 34
[0672] The compound of Emb 33, or a pharmaceutically acceptable
salt or solvate thereof, wherein X is --CH.sub.2--.
Emb 35
[0673] The compound of Emb 33, or a pharmaceutically acceptable
salt or solvate thereof, wherein X is selected from the group
consisting of:
##STR00647##
Emb 36
[0674] The compound of Emb 33, or a pharmaceutically acceptable
salt or solvate thereof, wherein X is --O--.
Emb 37
[0675] The compound of Emb 33, or a pharmaceutically acceptable
salt or solvate thereof, wherein X is --S--.
Emb 38
[0676] The compound of Emb 33, or a pharmaceutically acceptable
salt or solvate thereof, wherein X is --CH.sub.2S--.
Emb 39
[0677] The compound of Emb 33, or a pharmaceutically acceptable
salt or solvate thereof, wherein X is --N(H)CH.sub.2--.
Emb 40
[0678] The compound of Emb 33, or a pharmaceutically acceptable
salt or solvate thereof, wherein X is selected from the group
consisting of:
##STR00648##
Emb 41
[0679] The compound of any one of Embs 13-16 or 20-40, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.6c is selected from the group consisting of hydrogen, --Cl,
--OCH.sub.3, and hydroxy.
Emb 42
[0680] The compound of any one of Embs 13 or 17-40, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.6b is selected from the group consisting of hydrogen, --Cl,
--OCH.sub.3, and hydroxy.
Emb 43
[0681] The compound of any one of Embs 13-42, or a pharmaceutically
acceptable salt or solvate thereof, wherein R.sup.9f is
hydrogen.
Emb 44
[0682] The compound of any one of Embs 13-42, or a pharmaceutically
acceptable salt or solvate thereof, wherein R.sup.9f is methyl.
Emb 45
[0683] The compound of any one of Embs 13-44, or a pharmaceutically
acceptable salt or solvate thereof, wherein R.sup.11a is selected
from the group consisting of hydrogen and hydroxy.
Emb 46
[0684] The compound of any one of Embs 13-44, or a pharmaceutically
acceptable salt or solvate thereof, wherein R.sup.11b is
hydrogen.
Emb 47
[0685] The compound of any one of Embs 1-46, or a pharmaceutically
acceptable salt or solvate thereof, wherein L is a cleavable
linker.
Emb 48
[0686] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein the cleavable linker
comprises a succinimide, amide, thiourea, thioether, oxime, or
self-immolative group, or a combination thereof.
Emb 49
[0687] The compound of any one of Embs 1-48, or a pharmaceutically
acceptable salt or solvate thereof, wherein the cleavable linker
comprises a peptide.
Emb 50
[0688] The compound of Emb 49, or a pharmaceutically acceptable
salt or solvate thereof, wherein the cleavable linker comprises a
tripeptide.
Emb 51
[0689] The compound of Emb 49, or a pharmaceutically acceptable
salt or solvate thereof, wherein the cleavable linker comprises a
dipeptide.
Emb 52
[0690] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein the cleavable linker
comprises phosphate ester.
Emb 53
[0691] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein the cleavable linker
comprises a pyrophosphate diester.
Emb 54
[0692] The compound of any one of Embs 1-53, or a pharmaceutically
acceptable salt or solvate thereof, wherein Q is absent.
Emb 55
[0693] The compound of any one of Embs 1-53, or a pharmaceutically
acceptable salt or solvate thereof, wherein Q is a
heterobifunctional group selected from the group consisting of Q-1,
Q-2, Q-3, Q-4, Q-5, and Q-6, wherein m is 1, 2, 3, 4, 5, or 6.
Emb 56
[0694] The compound of any one of Embs 1-53, or a pharmaceutically
acceptable salt or solvate thereof, wherein Q is a
heterotrifunctional group that is Q-7.
Emb 57
[0695] The compound of Emb 55, or a pharmaceutically acceptable
salt or solvate thereof, wherein Q is selected from the group
consisting of Q-1, Q-2, Q-3, and Q-4.
Emb 58
[0696] The compound of Emb 57, or a pharmaceutically acceptable
salt or solvate thereof, wherein Q is selected from the group
consisting of Q-3 and Q-4.
Emb 59
[0697] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein -L-Q- is LQ-1; m is 1
or 2; and R.sup.10a and R.sup.10b are independently selected from
the group consisting of hydrogen and optionally substituted
C.sub.1-6 alkyl.
Emb 60
[0698] The compound of Emb 59, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-2.
Emb 61
[0699] The compound of Emb 59, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-3.
Emb 62
[0700] The compound of Emb 59, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-4.
Emb 63
[0701] The compound of Emb 59, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-5.
Emb 64
[0702] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein -L-Q- is LQ-6; m is 1
or 2; and R.sup.10a and R.sup.10b are independently selected from
the group consisting of hydrogen and optionally substituted
C.sub.1-6 alkyl.
Emb 65
[0703] The compound of Emb 64, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-7.
Emb 66
[0704] The compound of Emb 64, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-8.
Emb 67
[0705] The compound of Emb 64, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-9.
Emb 68
[0706] The compound of Emb 64, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is: LQ-10.
Emb 69
[0707] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein L is a noncleavable
linker.
Emb 70
[0708] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein the linker comprises
one or more polyethylene glycol units.
Emb 71
[0709] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein -L-Q- is LQ-11; m is 1
or 2; and x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or
15.
Emb 72
[0710] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein -L-Q- is LQ-12; m is 1
or 2; and x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or
15.
Emb 73
[0711] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein -L-Q- is LQ-14; m is 1
or 2; x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15;
and R.sup.10a and R.sup.10b are independently selected from the
group consisting of hydrogen and optionally substituted C.sub.1-6
alkyl.
Emb 74
[0712] The compound of Emb 73, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-15.
Emb 75
[0713] The compound of Emb 73, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-16.
Emb 76
[0714] The compound of Emb 73, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-17.
Emb 77
[0715] The compound of Emb 73, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-18.
Emb 78
[0716] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein -L-Q- is LQ-19; m is 1
or 2; x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15;
and R.sup.10a and R.sup.10b are independently selected from the
group consisting of hydrogen and optionally substituted C.sub.1-6
alkyl.
Emb 79
[0717] The compound of Emb 78, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-20.
Emb 80
[0718] The compound of Emb 78, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-21.
Emb 81
[0719] The compound of Emb 78, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-22.
Emb 82
[0720] The compound of Emb 78, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-23.
Emb 83
[0721] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein -L-Q- is LQ-13; and x
is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
Emb 84
[0722] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein -L-Q- is LQ-29; and x
is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
Emb 85
[0723] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein -L-Q- is LQ-24; x is 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; and R.sup.10a
and R.sup.10b are independently selected from the group consisting
of hydrogen and optionally substituted C.sub.1-6 alkyl.
Emb 86
[0724] The compound of Emb 85, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-25.
Emb 86A
[0725] The compound of Emb 85, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-26.
Emb 87
[0726] The compound of Emb 85, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-27.
Emb 88
[0727] The compound of Emb 85, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-28.
Emb 89
[0728] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein -L-Q- is LQ-30; x is 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; and R.sup.10a
and R.sup.10b are independently selected from the group consisting
of hydrogen and optionally substituted C.sub.1-6 alkyl.
Emb 90
[0729] The compound of Emb 89, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-31.
Emb 91
[0730] The compound of Emb 89, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-32.
Emb 92
[0731] The compound of Emb 89, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-33.
Emb 93
[0732] The compound of Emb 89, or a pharmaceutically acceptable
salt or solvate thereof, wherein -L-Q- is LQ-34.
Emb 94
[0733] The compound of any one of Embs 55, 59-68, or 71-82, or a
pharmaceutically acceptable salt or solvate thereof, wherein m is
2.
Emb 95
[0734] The compound of any one of Embs 1-47, or a pharmaceutically
acceptable salt or solvate thereof, wherein -L-Q- is any one the
chemical structures of Table I.
Emb 96
[0735] The compound of any one of Embs 1-95, or a pharmaceutically
acceptable salt or solvate thereof, wherein n is 2-8.
Emb 97
[0736] The compound of Emb 96, or a pharmaceutically acceptable
salt or solvate thereof, wherein n is 2-5.
Emb 98
[0737] The compound of any one of Embs 1-95, or a pharmaceutically
acceptable salt or solvate thereof, wherein n is 2.
Emb 99
[0738] The compound of any one of Embs 1-95, or a pharmaceutically
acceptable salt or solvate thereof, wherein n is 4.
Emb 100
[0739] The compound of any one of Embs 1 or 47-99, or a
pharmaceutically acceptable salt or solvate thereof, wherein SM is
a monovalent radical of a glucocorticosteroid which is any one of
the chemical structures of Table II.
Emb 101
[0740] The compound of Emb 100, or a pharmaceutically acceptable
salt or solvate thereof, wherein SM is a monovalent radical of a
glucocorticosteroid selected from the group consisting of:
##STR00649##
Emb 102
[0741] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 is an antibody
or antigen-binding fragment thereof or wherein A.sup.2 is an
antibody or antigen-binding fragment thereof.
Emb 103
[0742] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 is an
anti-tumor necrosis factor (TNF) alpha protein that binds to human
TNF alpha and/or murine TNF alpha or wherein A.sup.2 is protein
that binds to human TNF alpha and/or murine TNF alpha.
Emb 104
[0743] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 is an
anti-tumor necrosis factor (TNF) alpha protein that binds to
soluble TNF alpha or wherein A.sup.2 is a protein that binds to
soluble TNF alpha.
Emb 105
[0744] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 is an
anti-tumor necrosis factor (TNF) alpha protein that binds to
membrane-bound TNF alpha or wherein A.sup.2 is a protein that binds
to membrane-bound TNF alpha.
Emb 106
[0745] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 is an
anti-tumor necrosis factor (TNF) alpha protein comprising an
anti-TNF antibody or wherein A.sup.2 is protein comprising an
anti-TNF antibody.
Emb 107
[0746] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A is an anti-tumor
necrosis factor (TNF) alpha protein comprising an antigen-binding
fragment of an anti-TNF antibody or wherein A.sup.2 is a protein
comprising an antigen-binding fragment of an anti-TNF antibody.
Emb 108
[0747] The compound of any one of Embs 102-105 or 107, or a
pharmaceutically acceptable salt or solvate thereof, wherein the
antigen-binding fragment is selected from the group consisting of
Fab, Fab', F(ab').sub.2, single chain Fv or scFv, disulfide linked
Fv, V-NAR domain, IgNar, intrabody, IgG.DELTA.CH2, minibody,
F(ab')3, tetrabody, triabody, diabody, single-domain antibody,
DVD-g, Fcab, mAb2, (scFv)2, or scFv-Fc.
Emb 109
[0748] The compound of any one of Embs 1-108, or a pharmaceutically
acceptable salt or solvate thereof, wherein the antibody or
antigen-binding fragment thereof is murine, chimeric, humanized, or
human.
Emb 110
[0749] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 is an
anti-tumor necrosis factor (TNF) alpha protein comprising a soluble
TNF receptor or wherein A.sup.2 is a protein comprising a soluble
TNF receptor.
Emb 111
[0750] The compound of Emb 110, or a pharmaceutically acceptable
salt or solvate thereof, wherein the soluble TNF receptor is a
soluble p75 TNF receptor.
Emb 112
[0751] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 comprises a
heavy chain constant domain or a fragment thereof or wherein or
A.sup.2 comprises a heavy chain constant domain or a fragment
thereof.
Emb 113
[0752] The compound of Emb 112, or a pharmaceutically acceptable
salt or solvate thereof, wherein the heavy chain constant domain or
fragment thereof comprises a constant domain selected from the
group consisting of: (a) an IgA constant domain; (b) an IgD
constant domain; (c) an IgE constant domain; (d) an IgG1 constant
domain; (e) an IgG2 constant domain; (f) an IgG3 constant domain;
(g) an IgG4 constant domain; and (h) an IgM constant domain or is a
fragment thereof.
Emb 114
[0753] The compound of Emb 113, or a pharmaceutically acceptable
salt or solvate thereof, wherein the heavy chain constant domain
comprises a human IgG1 heavy chain constant domain or fragment
thereof.
Emb 115
[0754] The compound of Emb 114, or a pharmaceutically acceptable
salt or solvate thereof, wherein the heavy chain constant domain
comprises a human IgG1 Fc domain.
Emb 116
[0755] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 comprises a
light chain constant domain or a fragment thereof or wherein
A.sup.2 comprises a light chain constant domain or a fragment
thereof.
Emb 117
[0756] The compound of Emb 116, or a pharmaceutically acceptable
salt or solvate thereof, wherein the light chain constant domain or
fragment thereof comprises a constant domain selected group
consisting of (a) an Ig kappa constant domain and (b) an Ig lambda
constant domain or is a fragment thereof.
Emb 118
[0757] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 competitively
inhibits binding of an antibody selected from the group consisting
of adalimumab, infliximab, certolizumab pegol, and golimumab to
TNF-alpha or wherein A.sup.2 competitively inhibits binding of an
antibody selected from the group consisting of adalimumab,
infliximab, certolizumab pegol, and golimumab to TNF-alpha.
Emb 119
[0758] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 binds to the
same TNF-alpha epitope as an antibody selected from the group
consisting of adalimumab, infliximab, certolizumab pegol,
afelimomab, nerelimomab, ozoralizumab, placulumab, and golimumab or
wherein A.sup.2 binds to the same TNF-alpha epitope as an antibody
selected from the group consisting of adalimumab, infliximab,
certolizumab pegol, afelimomab, nerelimomab, ozoralizumab,
placulumab, and golimumab.
Emb 120
[0759] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein the anti-TNF alpha
protein or protein is selected from the group consisting of
adalimumab, infliximab, certolizumab pegol, afelimomab,
nerelimomab, ozoralizumab, placulumab, and golimumab.
Emb 121
[0760] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 comprises the
variable heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:3
or 6, SEQ ID NO:4, and SEQ ID NO:5, respectively and the variable
light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:32, SEQ ID
NO:33, and SEQ ID NO:34, respectively or wherein A.sup.2 comprises
the variable heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID
NO:3 or 6, SEQ ID NO:4, and SEQ ID NO:5 respectively and the
variable light chain CDR1, CDR2, and CDR3 sequences of SEQ ID
NO:32, SEQ ID NO:33, and SEQ ID NO:34, respectively.
Emb 122
[0761] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 comprises the
variable heavy chain sequence of SEQ ID NO:50 and the variable
light chain sequence of SEQ ID NO:59 or wherein A.sup.2 comprises
the variable heavy chain sequence of SEQ ID NO:50 and the variable
light chain sequence of SEQ ID NO:59.
Emb 123
[0762] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 does not bind
to TNF beta or wherein A.sup.2 does not bind to TNF beta.
Emb 124
[0763] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 binds to TNF
beta or wherein A.sup.2 binds to TNF beta.
Emb 125
[0764] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 neutralizes
human TNF-alpha cytotoxicity in a standard in vitro L929 assay with
an IC50 of 1.times.10.sup.-7 M or less or wherein A.sup.2
neutralizes human TNF-alpha cytotoxicity in a standard in vitro
L929 assay with an IC50 of 1.times.10.sup.-7 M or less.
Emb 126
[0765] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 blocks the
interaction of TNF-alpha with p55 and p75 cell surface receptors or
wherein A.sup.2 blocks the interaction of TNF-alpha with p55 and
p75 cell surface receptors.
Emb 127
[0766] The compound of any one of Embs 1-101, or a pharmaceutically
acceptable salt or solvate thereof, wherein A.sup.1 lyses surface
TNF expressing cells in vitro in the presence of complement or
wherein A.sup.2 lyses surface TNF expressing cells in vitro in the
presence of complement.
Emb 128
[0767] The compound of Emb 111, or a pharmaceutically acceptable
salt or solvate thereof, wherein the soluble p75 TNF receptor is
etanercept.
Emb 129
[0768] The compound of Emb 102, or a pharmaceutically acceptable
salt or solvate thereof, wherein the antibody is adalimumab.
Emb 130
[0769] The compound of any one of Embs 1-101, wherein A.sup.1 binds
to Fc gamma receptor or wherein A.sup.2 binds to Fc gamma
receptor.
Emb 131
[0770] The compound of any one of Embs 1-101, wherein A.sup.1 is
active in the GRE transmembrane TNF-alpha reporter assay and/or the
L929 assay or wherein A.sup.2 is active in the GRE transmembrane
TNF-alpha reporter assay and/or the L929 assay.
Emb 132
[0771] The compound of any one of Embs 1 or 102-131, or a
pharmaceutically acceptable salt or solvate thereof, which is any
one of the chemical structures of Table III, wherein n is 1-5 and A
is A.sup.1 or A.sup.2.
Emb 133
[0772] The compound of Emb 132, or a pharmaceutically acceptable
salt or solvate thereof, which is any one of the chemical
structures of Table IV, wherein A is A or A.sup.2.
Emb 134
[0773] The compound of any one of Embs 1 or 102-131, or a
pharmaceutically acceptable salt or solvate thereof, which is any
one of the chemical structures of Table V, wherein n is 1-5 and A
is A.sup.1 or A.sup.2.
Emb 135
[0774] The compound of Emb 134, or a pharmaceutically acceptable
salt or solvate thereof, wherein n is 2 or 4.
Emb 136
[0775] A pharmaceutical composition comprising the compound of any
one of Embs 1-135, or a pharmaceutically acceptable salt or solvate
thereof, and a pharmaceutically acceptable carrier.
Emb 137
[0776] A pharmaceutical composition comprising a plurality of the
compounds of any one of Embs 1-135 or a pharmaceutically salt or
solvate thereof, wherein the compounds in the pharmaceutical
composition have an average of 1 to 10 SM-L-Q per A.sup.1 or
A.sup.2, i.e., n is 1-10, 2 to 6 SM-L-Q per A or A.sup.2, 3 to 4
SM-L-Q per A or A.sup.2, about 2 SM-L-Q per A.sup.1 or A.sup.2,
about 3 SM-L-Q per A.sup.1 or A.sup.2, or about 4 SM-L-Q per
A.sup.1 or A.sup.2.
Emb 138
[0777] A method for lysing a cell expressing surface TNF-alpha
comprising contacting the cell with the compound of any one of Embs
1-135 or the pharmaceutical composition of Embs 136 or 137.
Emb 139
[0778] A method for treating an autoimmune disease in a patient in
need thereof comprising administering to said patient the compound
of any one of Embs 1-135 or the pharmaceutical composition of Embs
136 or 137.
Emb 140
[0779] The method of Emb 139, wherein said autoimmune disease is
rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic
arthritis, ankylosing spondylitis, adult Crohn's disease, pediatric
Crohn's disease, ulcerative colitis, plaque psoriasis, hidradenitis
suppurativa, uveitis, Behcets disease, a spondyloarthropathy, or
psoriasis.
Emb 141
[0780] A method for treating a disease or disorder characterized by
increased TNF-alpha in synovial fluid in a patient in need thereof
comprising administering to said patient the compound of any one of
Embs 1-135 or the pharmaceutical composition of Embs 136 or
137.
Emb 142
[0781] A compound having Formula VII, or a pharmaceutically
acceptable salt or solvate thereof, wherein: R.sup.1 is selected
from the group consisting of hydrogen and halo; R.sup.2 is selected
from the group consisting of hydrogen, halo, and hydroxy; R.sup.3
is selected from the group consisting of --CH.sub.2OH,
--CH.sub.2SH, --CH.sub.2Cl, --SCH.sub.2Cl, --SCH.sub.2F,
--SCH.sub.2CF.sub.3, hydroxy, --OCH.sub.2CN, --OCH.sub.2Cl,
--OCH.sub.2F, --OCH.sub.3, --OCH.sub.2CH.sub.3, --SCH.sub.2CN,
##STR00650##
R.sup.3a is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.3b is selected from the group consisting of
C.sub.1-4 alkyl and C.sub.1-4 alkoxy; R.sup.3c is selected from the
group consisting of hydrogen, C.sub.1-4 alkyl, --CH.sub.2OH, and
C.sub.1-4 alkoxy; R.sup.3d and R.sup.3e are independently selected
from hydrogen and C.sub.1-4 alkyl; X is selected from the group
consisting of --(CR.sup.4aR.sup.4b).sub.t--, --O--, --S--,
--S(.dbd.O)--, --S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--,
--CH.sub.2O--, --N(H)C(R.sup.8a)(R.sup.8b)--,
--CR.sup.4c.dbd.CR.sup.4d--, and --C.ident.C--; or X is absent; t
is 1 or 2; Z is selected from the group consisting of
.dbd.CR.sup.11a-- and .dbd.N--; each R.sup.4a and R.sup.4b are
independently selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; or R.sup.4a and R.sup.4b taken together with the
carbon atom to which they are attached form a 3- to 6-membered
cycloalkyl; R.sup.4 and R.sup.4d are independently selected from
the group consisting of hydrogen and C.sub.1-4 alkyl; R.sup.5 is
selected from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.6a, R.sup.6b, R.sup.6c, and R.sup.6d are each independently
selected from the group consisting of hydrogen, halo, C.sub.1-4
alkyl, v haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and
alkoxy; R.sup.7a is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl; R.sup.7b is selected from the group consisting
of hydrogen, -L-H, -L-PG,
##STR00651##
m is 1, 2, 3, 4, 5, or 6; L is a linker; PG is a protecting group;
R.sup.9f is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.8a and R.sup.8b are independently selected
from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.11a and R.sup.11b are independently selected from the group
consisting of hydrogen, halo, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl,
cyano, hydroxy, thiol, amino, alkylthio, and alkoxy; and represents
a single or double bond.
Emb 143
[0782] The compound of Emb 142, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.7b is selected from the
group consisting of R.sup.7b-1, R.sup.7b-2, and R.sup.7b-3; m is 1,
2, 3, 4, 5, or 6; and R.sup.10a and R.sup.10b are each
independently selected from the group consisting of hydrogen and
optionally substituted C.sub.1-6 alkyl.
Emb 144
[0783] The compound of Embs 142 or 143, or a pharmaceutically
acceptable salt or solvate thereof, having Formula VIII.
Emb 145
[0784] The compound of Emb 144, or a pharmaceutically acceptable
salt or solvate thereof, having Formula VIII-a.
Emb 146
[0785] The compound of Emb 144, or a pharmaceutically acceptable
salt or solvate thereof, having Formula VIII-b.
Emb 147
[0786] The compound of Emb 142 or 143, or a pharmaceutically
acceptable salt or solvate thereof, having Formula IX.
Emb 148
[0787] The compound of Emb 147, or a pharmaceutically acceptable
salt or solvate thereof, having Formula IX-a.
Emb 149
[0788] The compound of Emb 147, or a pharmaceutically acceptable
salt or solvate thereof, having Formula IX-b.
Emb 150
[0789] The compound of any one of Embs 142-149, or a
pharmaceutically acceptable salt or solvate thereof, wherein
represents a double bond.
Emb 151
[0790] The compound of any one of Embs 142-150, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.1 is selected from the group consisting of hydrogen and
fluoro.
Emb 152
[0791] The compound of any one of Embs 142-151, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.2 is selected from the group consisting of hydrogen and
fluoro.
Emb 153
[0792] The compound of any one of Embs 142-152, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.3 is selected from the group consisting of --CH.sub.2OH,
--CH.sub.2Cl, --SCH.sub.2Cl, --SCH.sub.2F, and hydroxy.
Emb 154
[0793] The compound of any one of Embs 142-152, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.3 is selected from the group consisting of:
##STR00652##
[0794] R.sup.3a is selected from the group consisting of hydrogen
and methyl; R.sup.3b is selected from the group consisting of
methyl, ethyl, isopropyl, isobutyl, methoxy, ethoxy, isopropoxy,
and isobutoxy; R.sup.3c is selected from the group consisting of
hydrogen, methyl, ethyl, --CH.sub.2OH, methoxy, ethoxy, and
isopropoxy; R.sup.3d and R.sup.3e are independently selected from
the group consisting of hydrogen, methyl, and ethyl.
Emb 155
[0795] The compound of any one of Embs 142-154, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.5 and R.sup.8a are independently selected from the group
consisting of hydrogen and methyl.
Emb 156
[0796] The compound of any one of Embs 142-155, or a
pharmaceutically acceptable salt or solvate thereof, wherein Z is
.dbd.CH--.
Emb 157
[0797] The compound of any one of Embs 142-155, or a
pharmaceutically acceptable salt or solvate thereof, wherein Z is
.dbd.N--.
Emb 158
[0798] The compound of any one of Embs 142-155, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.7a is selected from the group consisting of hydrogen and
methyl.
Emb 159
[0799] The compound of Emb 158, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.7a is hydrogen.
Emb 160
[0800] The compound of Emb 158, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.7a is methyl.
Emb 161
[0801] The compound of any one of Embs 142-160, or a
pharmaceutically acceptable salt or solvate thereof, wherein:
[0802] X is selected from the group consisting of
--(CR.sup.4aR.sup.4b).sub.t, --O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --CH.sub.2S--, and --N(H)CH(R.sup.8a)--; t is
1; and R.sup.4a and R.sup.4b are independently selected from the
group consisting of hydrogen and methyl; or R.sup.4a and R.sup.4b
taken together with the carbon atom to which they are attached form
a 3-membered cycloalkyl.
Emb 162
[0803] The compound of Emb 161, or a pharmaceutically acceptable
salt or solvate thereof, wherein X is --CH.sub.2--.
Emb 163
[0804] The compound of Emb 161, or a pharmaceutically acceptable
salt or solvate thereof, wherein X is selected from the group
consisting of:
##STR00653##
Emb 164
[0805] The compound of Emb 161, or a pharmaceutically acceptable
salt or solvate thereof, wherein X is --O--.
Emb 165
[0806] The compound of Emb 161, or a pharmaceutically acceptable
salt or solvate thereof, wherein X is --S--.
Emb 166
[0807] The compound of Emb 161, or a pharmaceutically acceptable
salt or solvate thereof, wherein X is --CH.sub.2S--.
Emb 167
[0808] The compound of Emb 161, or a pharmaceutically acceptable
salt or solvate thereof, wherein X is --N(H)CH.sub.2--.
Emb 168
[0809] The compound of Emb 161, or a pharmaceutically acceptable
salt or solvate thereof, wherein X is selected from the group
consisting of:
##STR00654##
Emb 169
[0810] The compound of any one of Embs 142-168, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.11b is hydrogen.
Emb 170
[0811] The compound of any one of Embs 142-169, or a
pharmaceutically acceptable salt or solvate thereof, wherein R is
hydrogen.
Emb 171
[0812] The compound of any one of Embs 142-170, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.6b is selected from the group consisting of hydrogen, --Cl,
--OCH.sub.3, and hydroxy.
Emb 172
[0813] The compound of any one of Embs 142-171, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.9f is hydrogen.
Emb 173
[0814] The compound of any one of Embs 142-171, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.9f is methyl.
Emb 174
[0815] The compound of any one of Embs 142-173, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.7a is selected from the group consisting of hydrogen and
hydroxy.
Emb 175
[0816] The compound of any one of Embs 142-174, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.11b is hydrogen.
Emb 176
[0817] The compound of any one of Embs 143-175, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.7b is R.sup.7b-1.
Emb 177
[0818] The compound of Emb 176, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.10a and R.sup.10b are
independently optionally substituted C.sub.1-6 alkyl.
Emb 178
[0819] The compound of any one of Embs 143-175, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.7b is R.sup.7b-2, and PG is BOC.
Emb 179
[0820] The compound of Emb 178, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.10a and R.sup.10b are
independently optionally substituted C.sub.1-6 alkyl.
Emb 180
[0821] The compound of any one of Embs 143-175, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.7b is R.sup.7b-3.
Emb 181
[0822] The compound of Emb 180, or a pharmaceutically acceptable
salt or solvate thereof, wherein m is 1 or 2, and R.sup.10a and
R.sup.10b are each optionally substituted C.sub.1-6 alkyl.
Emb 182
[0823] The compound of Emb 142, or a pharmaceutically acceptable
salt or solvate thereof, which is any one or more of the compounds
of Table VI.
Emb 183
[0824] The compound of Emb 182, or a pharmaceutically acceptable
salt or solvate thereof, selected from the group consisting of:
##STR00655##
Emb 184
[0825] The compound of Emb 142, or a pharmaceutically acceptable
salt or solvate thereof, which is any one or more of the chemical
structures of Table VIII, wherein R.sup.7b is selected from the
group consisting of R.sup.7b-4, R.sup.7b-5, and R.sup.7b-6.
Emb 185
[0826] The compound of Emb 184, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.7b is R.sup.7b-4.
Emb 186
[0827] The compound of Emb 184, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.1 is R.sup.7b-5.
Emb 187
[0828] The compound of Emb 184, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.7b is R.sup.7b-6.
Emb 188
[0829] The compound of Emb 142, or a pharmaceutically acceptable
salt or solvate thereof, which is any one or more of the chemical
structures of Table VIII,
[0830] wherein R.sup.7b is any one of the chemical structures of
Table IX.
Emb 189
[0831] The compound of Emb 142, or a pharmaceutically acceptable
salt or solvate thereof, which is any one of the compounds of Table
X.
Emb 190
[0832] The compound of Emb 142, or a pharmaceutically acceptable
salt or solvate thereof, which is any one of the compounds of Table
VII.
Emb 191
[0833] The compound of Emb 190, or a pharmaceutically acceptable
salt or solvate thereof, selected from the group consisting of:
##STR00656##
Emb 192
[0834] The compound of Emb 142, or a pharmaceutically acceptable
salt or solvate thereof, which is any one of the compounds of Table
XI,
[0835] wherein R.sup.7b is selected from the group consisting of
R.sup.7b-4, R.sup.7b-5 and R.sup.7b-6.
Emb 193
[0836] The compound of Emb 192, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.7b is R.sup.7b-4.
Emb 194
[0837] The compound of Emb 192, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.7 is R.sup.7b-5.
Emb 195
[0838] The compound of Emb 192, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.7b is R.sup.7b-6.
Emb 196
[0839] The compound of Emb 142, or a pharmaceutically acceptable
salt or solvate thereof, which is any one of the chemical
structures of Table XI,
[0840] wherein R.sup.7b any one of the structures of Table IX.
Emb 197
[0841] The compound of Emb 142, or a pharmaceutically acceptable
salt or solvate thereof, which is any one of the compounds of Table
XII.
Emb 198
[0842] A pharmaceutical composition comprising the compound of any
one of Embs 142-197, or a pharmaceutically acceptable salt or
solvate thereof, wherein R.sup.7b is hydrogen, and a
pharmaceutically acceptable carrier.
Emb 199
[0843] A method for treating an autoimmune or inflammatory disease
in a patient in need thereof, the method comprising administering
to said patient the compound of any one of Embs 142-197, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.7b is hydrogen, or the pharmaceutical composition of Emb
198.
Emb 200
[0844] The method of Emb 199, wherein said autoimmune disease is
rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic
arthritis, ankylosing spondylitis, adult Crohn's disease, pediatric
Crohn's disease, ulcerative colitis, plaque psoriasis, or
hidradenitis suppurativa.
Emb 201
[0845] A method of making a compound having Formula I-c, or a
pharmaceutically acceptable salt or solvate thereof, wherein:
A.sup.1 is an anti-tumor necrosis factor (TNF) alpha protein; L is
a linker; n is 1-10; and SM is a radical of a glucocorticosteroid,
the method comprising: a) conjugating a compound having Formula X
with an anti-tumor necrosis factor (TNF) alpha protein; and b)
isolating the compound having Formula I-c, or a pharmaceutically
acceptable salt or solvate thereof.
Emb 202
[0846] The method of Emb 201 further comprising hydrolyzing the
compound having Formula Ic to give a compound having Formula
I-d.
Emb 203
[0847] A method of making a compound having Formula I-e, or a
pharmaceutically acceptable salt or solvate thereof, wherein:
A.sup.1 is an anti-tumor necrosis factor (TNF) alpha protein; L is
a linker; R.sup.7a is selected from the group consisting of
hydrogen and C.sub.4 alkyl; n is 1-10; m is 1, 2, 3, 4, 5, or 6;
and SM is a radical of a glucocorticosteroid, the method
comprising: a) conjugating a compound having Formula XI, with an
anti-tumor necrosis factor (TNF) alpha protein; and b) isolating
the compound having Formula I-e, or a pharmaceutically acceptable
salt or solvate thereof.
Emb 204
[0848] The method of Emb 203 further comprising hydrolyzing the
compound having Formula I-e to give a compound having Formula
I-f.
Emb 205
[0849] The compound of Emb 182, which is
##STR00657##
or a pharmaceutically acceptable salt or solvate thereof.
Emb 206
[0850] The compound of Emb 182, which is
##STR00658##
or a pharmaceutically acceptable salt or solvate thereof.
Emb 207
[0851] The compound of Emb 182, which is
##STR00659##
or a pharmaceutically acceptable salt or solvate thereof.
Emb 208
[0852] The compound of Emb 189, which is
##STR00660##
or a pharmaceutically acceptable salt or solvate thereof.
Emb 209
[0853] The compound of Emb 132, or a pharmaceutically acceptable
salt or solvate thereof, which is any one of the chemical
structures of Table IV, wherein A is A.sup.1 or A.sup.2.
[0854] The disclosure also provides Embs I-XXXIII as particular
embodiments. The Formulae and Tables referred to these particular
embodiments that are not shown in Embs I-XXXII are set forth in the
description above.
Emb I
[0855] A compound having Formula I-a:
(SM-L-Q).sub.n-A.sup.1 I-a
wherein: A.sup.1 is an anti-tumor necrosis factor (TNF) alpha
protein; L is a linker; Q is a heterobifunctional group or
heterotrifunctional group; or Q is absent; n is 1-10; and SM is a
monovalent radical of a glucocorticosteroid.
Emb II
[0856] A compound having Formula I-b:
(SM-L-Q).sub.n-A.sup.2 I-b
wherein A.sup.2 is a protein; L is a linker; Q is a
heterobifunctional group or heterotrifunctional group; or Q is
absent; n is 1-10; and SM is a radical of a glucocorticosteroid
having Formula II-m or Formula II-p; R.sup.1 is selected from the
group consisting of hydrogen and halo; R.sup.2 is selected from the
group consisting of hydrogen, halo, and hydroxy; R.sup.3 is
selected from the group consisting of --CH.sub.2OH, --CH.sub.2SH,
--CH.sub.2Cl, --SCH.sub.2Cl, --SCH.sub.2F, --SCH.sub.2CF.sub.3,
hydroxy, --OCH.sub.2CN, --OCH.sub.2Cl, --OCH.sub.2F, --OCH.sub.3,
--OCH.sub.2CH.sub.3, --SCH.sub.2CN,
##STR00661##
R.sup.3a is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.3b is selected from the group consisting of
C.sub.1-4 alkyl and C.sub.1-4 alkoxy; R.sup.3c is selected from the
group consisting of hydrogen, C.sub.1-4 alkyl, --CH.sub.2OH, and
C.sub.1-4 alkoxy; R.sup.3d and R.sup.3e are independently selected
from hydrogen and C.sub.1-4 alkyl; R.sup.6a, R.sup.6b, R.sup.6c,
R.sup.6d, and R.sup.6e are each independently selected from the
group consisting of hydrogen, halo, C.sub.1-4 alkyl, C.sub.1-4
haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and alkoxy; X
is selected from the group consisting of
--(CR.sup.4aR.sup.4b).sub.t, --O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--, --CH.sub.2O--,
--N(H)C(R.sup.8a)(R.sup.8b)--, --CR.sup.4c=CR.sup.4d--, and
--C.ident.C--; or X is absent; Y.sup.2 is selected from the group
consisting of --O--, --S--, and --N(R.sup.7a)--; or Y.sup.2 is
absent; t is 1 or 2; Z is selected from the group consisting of
.dbd.CR.sup.11a-- and .dbd.N--; each R.sup.4a and R.sup.4b are
independently selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; or R.sup.4a and R.sup.4b taken together with the
carbon atom to which they are attached form a 3- to 6-membered
cycloalkyl; R.sup.4 and R.sup.4d are independently selected from
the group consisting of hydrogen and C.sub.1-4 alkyl; R.sup.5 is
selected from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.7a is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.7a and R are independently selected from the
group consisting of hydrogen and C.sub.1-4 alkyl; R.sup.9f is
selected from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.11a and R.sup.11b are independently selected from the group
consisting of hydrogen, halo, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl,
cyano, hydroxy, thiol, amino, alkylthio, and alkoxy; and represents
a single or double bond.
Emb III
[0857] The compound of Embs I or II, wherein SM is a radical of a
glucocorticosteroid having Formula II-m; R.sup.1 is selected from
the group consisting of hydrogen and halo; R.sup.2 is selected from
the group consisting of hydrogen, halo, and hydroxy; R.sup.3 is
selected from the group consisting of --CH.sub.2OH, --CH.sub.2SH,
--CH.sub.2Cl, --SCH.sub.2Cl, --SCH.sub.2F, --SCH.sub.2CF.sub.3,
hydroxy, --OCH.sub.2CN, --OCH.sub.2Cl, --OCH.sub.2F, --OCH.sub.3,
--OCH.sub.2CH.sub.3, --SCH.sub.2CN,
##STR00662##
R.sup.3a is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.3b is selected from the group consisting of
C.sub.1-4 alkyl and C.sub.1-4 alkoxy; R.sup.3c is selected from the
group consisting of hydrogen, C.sub.1-4 alkyl, --CH.sub.2OH, and
C.sub.1-4 alkoxy; R.sup.3d and R.sup.3e are independently selected
from hydrogen and C.sub.1-4 alkyl; R.sup.6a, R.sup.6c, R.sup.6d,
and R.sup.6e are each independently selected from the group
consisting of hydrogen, halo, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl,
cyano, hydroxy, thiol, amino, alkylthio, and alkoxy; X is selected
from the group consisting of --(CR.sup.4aR.sup.4b)--, --O--, --S--,
--S(.dbd.O)--, --S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--,
--CH.sub.2O--, --N(H)C(R.sup.8a)(R.sup.8b),
--CR.sup.4c.dbd.CR.sup.4d--, and --C.ident.C--; or X is absent;
Y.sup.2 is selected from the group consisting of --O--, --S--, and
--N(R.sup.7a)--; or Y.sup.2 is absent; t is 1 or 2; Z is .dbd.CH--;
each R.sup.4a and R.sup.4b are independently selected from the
group consisting of hydrogen and C.sub.1-4 alkyl; or R.sup.4a and
R.sup.4b taken together with the carbon atom to which they are
attached form a 3- to 6-membered cycloalkyl; R.sup.4c and R.sup.4d
are independently selected from the group consisting of hydrogen
and C.sub.1-4 alkyl; R.sup.1 is selected from the group consisting
of hydrogen and C.sub.1-4 alkyl; R.sup.7a is selected from the
group consisting of hydrogen and C.sub.1-4 alkyl; R.sup.8a and
R.sup.8b are independently selected from the group consisting of
hydrogen and C.sub.1-4 alkyl; R.sup.9f is selected from the group
consisting of hydrogen and C.sub.1-4 alkyl; R.sup.11b is selected
from the group consisting of hydrogen, halo, C.sub.1-4 alkyl,
C.sub.1-4 haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and
alkoxy; and represents a single or double bond.
Emb IV
[0858] The compound of Embs II or III, wherein represents a double
bond; R.sup.1 is selected from the group consisting of hydrogen and
fluoro; R.sup.2 is selected from the group consisting of hydrogen
and fluoro; R.sup.3 is selected from the group consisting of
--CH.sub.2OH, --CH.sub.2Cl, --SCH.sub.2Cl, --SCH.sub.2F, and
##STR00663##
R.sup.3a and R.sup.3e are independently selected from the group
consisting of hydrogen, methyl, and ethyl; R.sup.6a, R.sup.6c,
R.sup.6d, and R.sup.6e are hydrogen; X is selected from the group
consisting of --CH.sub.2--, --O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --CH.sub.2S--, and --N(H)CH.sub.2--; Y.sup.2
is --N(H)--; Z is .dbd.CH--; R.sup.9f is hydrogen; and R.sup.11b is
hydrogen.
Emb V
[0859] The compound of any one of Embs I-IV, wherein L is a linker
comprising a dipeptide.
Emb VI
[0860] The compound of any one of Embs I-V, wherein Q is a
heterobifunctional group selected from the group consisting of Q-3
and Q-4 and m is 1, 2, 3, or 4.
Emb VII
[0861] The compound of any one of Embs I-VII, wherein -L-Q- is
LQ-7; m is 2 or 3; and R.sup.10a and R.sup.10b are independently
selected from the group consisting of hydrogen and C.sub.1-4
alkyl.
Emb VIII
[0862] The compound of any one of Embs I-VII, wherein n is 2-5.
Emb IX
[0863] The compound of Embs I or II, wherein SM is a monovalent
radical of a glucocorticosteroid which is any one of the compounds
of Table II.
Emb X
[0864] The compound of any one of Embs I or III-IX, wherein A.sup.1
is (i) an antibody or antigen-binding fragment thereof that binds
to human TNF alpha or (ii) a soluble TNF receptor.
Emb XI
[0865] The compound of any one of Embs I or III-X, wherein A is
selected from the group consisting of adalimumab, infliximab,
certolizumab pegol, afelimomab, nerelimomab, ozoralizumab,
placulumab, and golimumab.
Emb XII
[0866] The compound of Emb I, which is any one or more of the
compounds of Table III, wherein n is 1-5; A is A.sup.2; and A is
selected from the group consisting of adalimumab, infliximab,
certolizumab pegol, afelimomab, nerelimomab, ozoralizumab,
placulumab, and golimumab.
Emb XIII
[0867] The compound of Emb II, which is any one or more of the
compounds of Table III, wherein n is 1-5; A is A.sup.2; and A.sup.2
is selected from the group consisting of antibody, an
antigen-binding fragment thereof, or a soluble receptor
protein.
Emb XIV
[0868] A compound selected from the group consisting of:
##STR00664## ##STR00665##
wherein n is 1-5 and A is an antibody comprising the heavy and
light chain sequences of SEQ ID NO:66 and SEQ ID NO:73,
respectively.
Emb XV
[0869] The compound of Emb XIV selected from the group consisting
of:
##STR00666## ##STR00667##
Emb XVI
[0870] The compound of Emb XIV, wherein the compound is
##STR00668##
Emb XVII
[0871] The compound of Emb XIV, wherein the compound is
##STR00669##
Emb XVIII
[0872] The compound of Emb XIV, wherein the compound is
##STR00670##
Emb XIX
[0873] The compound of Emb XIV, wherein the compound is
##STR00671##
Emb. XX
[0874] The compound of Emb XIV, wherein the compound is
##STR00672##
Emb. XXI
[0875] The compound of Emb XIV, wherein the compound is
##STR00673##
Emb XXII
[0876] A pharmaceutical composition comprising the compound of any
one of Embs I-XXI, and a pharmaceutically acceptable carrier.
Emb XXIII
[0877] A method for treating an autoimmune disease in a patient in
need thereof comprising administering to said patient the compound
of any one of Embs I-XXI or the pharmaceutical composition of Emb
XXII, optionally wherein said autoimmune disease is rheumatoid
arthritis, juvenile idiopathic arthritis, psoriatic arthritis,
ankylosing spondylitis, adult Crohn's disease, pediatric Crohn's
disease, ulcerative colitis, plaque psoriasis, hidradenitis
suppurativa, uveitis, Behcets disease, a spondyloarthropathy, or
psoriasis.
Emb XXIV
[0878] A compound having Formula VII, or a pharmaceutically
acceptable salt or solvate thereof, wherein R.sup.1 is selected
from the group consisting of hydrogen and halo; R.sup.2 is selected
from the group consisting of hydrogen, halo, and hydroxy; R.sup.3
is selected from the group consisting of --CH.sub.2OH,
--CH.sub.2SH, --CH.sub.2Cl, --SCH.sub.2Cl, --SCH.sub.2F,
--SCH.sub.2CF.sub.3, --CH.sub.2OS(.dbd.O).sub.2-0H, hydroxy,
--OCH.sub.2CN, --OCH.sub.2Cl, --OCH.sub.2F, --OCH.sub.3,
--OCH.sub.2CH.sub.3, --SCH.sub.2CN,
##STR00674##
R.sup.3a is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.3b is selected from the group consisting of
C.sub.1-4 alkyl and C.sub.1-4 alkoxy; R.sup.3c is selected from the
group consisting of hydrogen, C.sub.1-4 alkyl, --CH.sub.2OH,
C.sub.1-4 alkoxy, --CH.sub.2(amino), and
--CH.sub.2CH.sub.2C(.dbd.O)OR.sup.3f; R.sup.3d and R.sup.3e are
independently selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.3e is selected from the group consisting of
hydrogen and C.sub.1-4 alkyl; X is selected from the group
consisting of --(CR.sup.4aR.sup.4b).sub.t--, --O--, --S--,
--S(.dbd.O)--, --S(.dbd.O).sub.2--, --NR.sup.5--, --CH.sub.2S--,
--CH.sub.2O--, --N(H)C(R.sup.8a)(R.sup.8b)--,
--CR.sup.4c.dbd.CR.sup.4d--, --C.ident.C--, --N(R')C(.dbd.O)--, and
--OC(.dbd.O)--; or X is absent; t is 1 or 2; Z is selected from the
group consisting of .dbd.CR.sup.11a-- and .dbd.N--; each R.sup.4a
and R.sup.4b are independently selected from the group consisting
of hydrogen and C.sub.1-4 alkyl; or R.sup.4a and R.sup.4b taken
together with the carbon atom to which they are attached form a 3-
to 6-membered cycloalkyl; R.sup.4c and R.sup.4d are independently
selected from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.5 is selected from the group consisting of hydrogen and
C.sub.1-4 alkyl; R.sup.6a, R.sup.6b, R.sup.6c, and R.sup.6d are
each independently selected from the group consisting of hydrogen,
halo, C.sub.1-4 alkyl, haloalkyl, cyano, hydroxy, thiol, amino,
alkylthio, and alkoxy; R.sup.7a is selected from the group
consisting of hydrogen and C.sub.1-4 alkyl; R.sup.7b is selected
from the group consisting of hydrogen, -L-H, -L-PG,
##STR00675##
or R.sup.7a and R.sup.7b taken together with the nitrogen atom to
which they are attached form:
##STR00676##
or R.sup.7a and R.sup.7b taken together with the nitrogen atom to
which they are attached form a nitro (--NO.sub.2) group; m is 1, 2,
3, 4, 5, or 6; L is a linker; PG is a protecting group; R.sup.9f is
selected from the group consisting of hydrogen and C.sub.1-4 alkyl;
R.sup.8a and R.sup.8b are independently selected from the group
consisting of hydrogen and C.sub.1-4 alkyl; R.sup.11a and R.sup.11b
are independently selected from the group consisting of hydrogen,
halo, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, cyano, hydroxy, thiol,
amino, alkylthio, and alkoxy; and represents a single or double
bond
Emb XXV
[0879] A compound having Formula VII-A or Formula VII-B:
##STR00677##
[0880] or a pharmaceutically acceptable salt or solvate thereof,
wherein:
[0881] R.sup.1 is selected from the group consisting of hydrogen
and halo;
[0882] R.sup.2 is selected from the group consisting of hydrogen,
halo, and hydroxy;
[0883] R.sup.3 is selected from the group consisting of
--CH.sub.2OH, --CH.sub.2SH, --CH.sub.2Cl, --SCH.sub.2Cl,
--SCH.sub.2F, --SCH.sub.2CF.sub.3, --CH.sub.2OS(.dbd.O).sub.2-0H,
hydroxy, --OCH.sub.2CN, --OCH.sub.2Cl, --OCH.sub.2F, --OCH.sub.3,
--OCH.sub.2CH.sub.3, --SCH.sub.2CN,
##STR00678##
[0884] R.sup.3a is selected from the group consisting of hydrogen
and C.sub.1-4alkyl;
[0885] R.sup.3b is selected from the group consisting of
C.sub.1-4alkyl and C.sub.1-4alkoxy;
[0886] R.sup.3c is selected from the group consisting of hydrogen,
C.sub.1-4alkyl, --CH.sub.2OH, C.sub.1-4alkoxy, --CH.sub.2(amino),
and --CH.sub.2CH.sub.2C(.dbd.O)OR.sup.3f;
[0887] R.sup.3d and R.sup.3e are independently selected from the
group consisting of hydrogen and C.sub.1-4alkyl;
[0888] R.sup.3f is selected from the group consisting of hydrogen
and C.sub.1-4alkyl; X is selected from the group consisting of
(CR.sup.4aR.sup.4b).sub.t--, --O--, --S--,--S.dbd.--S(.dbd.O)--,
--S(.dbd.O).sub.2--, --NR.sup.5--,--CH.sub.2S--,--CH.sub.2--,
--N(H)C(R.sup.8a)(R.sup.8b)--, --CR.sup.4=CR.sup.4d, --C.ident.C--,
--N(R)C(.dbd.)--, and --OC(.dbd.O)--; or
[0889] X is absent;
[0890] t is 1 or 2;
[0891] Z is selected from the group consisting of .dbd.CR.sup.11a--
and .dbd.N--;
[0892] each R.sup.4a and R.sup.4b are independently selected from
the group consisting of hydrogen and C.sub.1-4alkyl; or
[0893] R.sup.4a and R.sup.4b taken together with the carbon atom to
which they are attached formula 3- to 6-membered cycloalkyl;
[0894] R.sup.4c and R.sup.4d are independently selected from the
group consisting of hydrogen and C.sub.1-4alkyl;
[0895] R.sup.5 is selected from the group consisting of hydrogen
and C.sub.1-4alkyl;
[0896] R.sup.6a, R.sup.6b, and R.sup.6c are each independently
selected from the group consisting of hydrogen, halo,
C.sub.1-4alkyl, haloalkyl, cyano, hydroxy, thiol, amino, alkylthio,
and alkoxy;
[0897] R.sup.7a is selected from the group consisting of hydrogen
and C.sub.1-4alkyl;
[0898] R.sup.7b is selected from the group consisting of hydrogen,
-L-H, -L-PG,
##STR00679##
or
[0899] R.sup.7a and R.sup.7b taken together with the nitrogen atom
to which they are attached form:
##STR00680##
[0900] m is 1, 2, 3, 4, 5, or 6;
[0901] L is a linker;
[0902] PG is a protecting group;
[0903] R.sup.9f is selected from the group consisting of hydrogen
and C.sub.1-4 alkyl;
[0904] R.sup.8a and R.sup.8b are independently selected from the
group consisting of hydrogen and C.sub.1-4 alkyl;
[0905] R.sup.11a and R.sup.11b are independently selected from the
group consisting of hydrogen, halo, C.sub.1-4 alkyl, C.sub.1-4
haloalkyl, cyano, hydroxy, thiol, amino, alkylthio, and alkoxy;
and
[0906] represents a single or double bond.
Emb XXVI
[0907] The compound of Embs XXIV or XXV, or a pharmaceutically
acceptable salt or solvate thereof, wherein R.sup.7b is selected
from the group consisting of R.sup.7b-1, R.sup.7b-2, and
R.sup.7b-3; m is 1, 2, 3, 4, 5, or 6; and R.sup.10a and R.sup.10b
are each independently selected from the group consisting of
hydrogen and optionally substituted C.sub.1-6 alkyl.
Emb XXVII
[0908] The compound of Embs XXIV or XXVI, or a pharmaceutically
acceptable salt or solvate thereof, having Formula VIII-a.
Emb XXVIII
[0909] The compound of any one of Embs XXIV-XXVII, or a
pharmaceutically acceptable salt or solvate thereof, wherein
represents a double bond; R.sup.1 is selected from the group
consisting of hydrogen and fluoro; R is selected from the group
consisting of hydrogen and fluoro; R.sup.3 is selected from the
group consisting of --CH.sub.2OH, --CH.sub.2Cl, --SCH.sub.2Cl,
--SCH.sub.2F, and
##STR00681##
R.sup.3d and R.sup.3e are independently selected from the group
consisting of hydrogen, methyl, and ethyl; Z is .dbd.CH--;
R.sup.6a, R.sup.6b, R.sup.6c, and R.sup.6d are hydrogen; R.sup.7a
is hydrogen; X is selected from the group consisting of
--CH.sub.2--, --O--, --S--, --S(.dbd.O)--, --S(.dbd.O).sub.2--,
--CH.sub.2S--, and --N(H)CH.sub.2--; R.sup.9f is hydrogen; and
R.sup.7b is hydrogen.
Emb XXIX
[0910] The compound of any one of Embs XXIV-XXVIII, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.7b is hydrogen.
Emb XXX
[0911] The compound of any one of Embs XXIV-XXVIII, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.7b is RP-1.
Emb XXXI
[0912] The compound of any one of Embs XXIV-XXVIII, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.7b is R.sup.7b-2, and PG is BOC.
Emb XXXII
[0913] The compound of any one of Embs XXIV-XXVIII, or a
pharmaceutically acceptable salt or solvate thereof, wherein
R.sup.7b is R.sup.7b-3.
Emb XXXIII
[0914] The compound of Emb XXIX, or a pharmaceutically acceptable
salt or solvate thereof, which is any one or more of the compounds
of Table VI.
Emb XXXIV
[0915] The compound of Emb XXIX, or a pharmaceutically acceptable
salt or solvate thereof, which is any one of the compounds of Table
VII.
Emb XXXV
[0916] The compound of Emb XXXIII, or a pharmaceutically acceptable
salt or solvate thereof, which is:
##STR00682##
Emb XXXVI
[0917] The compound of Emb XXIV, or a pharmaceutically acceptable
salt or solvate thereof, which is any one or more of the compounds
of Table VIII, wherein R.sup.7b is selected from the group
consisting of R.sup.7b-4, R.sup.7b-5, and R.sup.7b-6.
Emb XXXVII
[0918] The compound of Emb XXIV, or a pharmaceutically acceptable
salt or solvate thereof, which is any one or more of the compounds
of Table X.
Emb XXXVIII
[0919] The compound of Emb XXXVII, or a pharmaceutically acceptable
salt or solvate thereof, which is:
##STR00683##
Emb XXXIX
[0920] A method of making a compound having Formula I-e:
##STR00684##
or a pharmaceutically acceptable salt or solvate thereof, wherein A
is A.sup.1 or A.sup.2; A.sup.1 is an anti-tumor necrosis factor
(TNF) alpha protein; A.sup.2 is a protein; L is a linker; R.sup.7a
is selected from the group consisting of hydrogen and C.sub.1-4
alkyl; n is 1-10; m is 1, 2, 3, 4, 5, or 6; and SM is a radical of
a glucocorticosteroid,
[0921] the method comprising:
[0922] a) conjugating a compound having Formula XI:
##STR00685##
[0923] with an anti-tumor necrosis factor (TNF) alpha protein or a
protein; and
[0924] b) isolating the compound having Formula I-e, or a
pharmaceutically acceptable salt or solvate thereof.
Emb XL
[0925] The method of Emb XXXIV further comprising hydrolyzing the
compound having Formula I-e to give a compound having Formula
I-f:
##STR00686##
or a pharmaceutically acceptable salt or solvate thereof.
Emb XLI
[0926] A compound which is:
##STR00687##
wherein A is adalimumab.
Emb XLII
[0927] A composition comprising the compound of Emb XLI.
[0928] Embodiments of the present disclosure can be further defined
by reference to the following non-limiting examples, which describe
in detail preparation of certain antibodies of the present
disclosure and methods for using antibodies of the present
disclosure. It will be apparent to those skilled in the art that
many modifications, both to materials and methods, can be practiced
without departing from the scope of the present disclosure.
EXAMPLES
[0929] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this disclosure.
Analytical Methods for Compound Synthesis and Characterization
[0930] Analytical data is included within the procedures below, in
the illustrations of the general procedures, or in the tables of
examples. Unless otherwise stated, all .sup.1H and .sup.13CNMR data
were collected on a Varian Mercury Plus 400 MHz or a Bruker AVIII
300 MHz instrument; hemical shifts are quoted in parts per million
(ppm). HPLC analytical data are either detailed within the
experimental or referenced to the table of LC/MS and HPLC
conditions, using the method provided in Table 7.
TABLE-US-00026 TABLE 7 List of LC/MS and GC/MS Methods Method
Conditions a The gradient was 10-100% B in 3.4 min with a hold at
100% B for 0.45 min, 100-10% B in 0.01 min, and then held at 10% B
for 0.65 min (0.8 mL/min flow rate). Mobile phase A was 0.0375%
trifluoroactic acid in water, mobile phase B was 0.018% TFA in
MeCN. The column used for the chromatography was a 2.0 .times. 50
mm phenomenex Luna-C18 column (5 .mu.m particles). Detection
methods are diode array (DAD) and evaporative light scattering
(ELSD) detection as well as positive electrospray ionization(MS). b
The gradient was 1-90% B in 3.4 min, 90-100% B in 0.45 min, 100-1%
B in 0.01 min, and then held at 1% B for 0.65 min (0.8 mL/min flow
rate). Mobile phase A was 0.0375% CF.sub.3CO.sub.2H in water,
mobile phase B was 0.018% CF.sub.3CO.sub.2H in CH.sub.3CN. The
column used for the chromatography was a 2.0 .times. 50 mm
phenomenex Luna-C18 column (5 .mu.m particles). Detection methods
are diode array (DAD) and evaporative light scattering (ELSD)
detection as well as positive electrospray ionization (MS). c The
gradient was 10-100% B in 3.4 min with a hold at 100% B for 0.45
min, 100-10% B in 0.01 min, and then held at 10% B for 0.65 min
(0.8 mL/min flow rate). Mobile phase A was 0.0375%
CF.sub.3CO.sub.2H in water, mobile phase B was 0.018%
CF.sub.3CO.sub.2H in CH.sub.3CN. The column used for the
chromatography was a 2.0 .times. 50 mm phenomenex Luna-C18 column
(5 .mu.m particles). Detection methods are diode array (DAD) and
evaporative light scattering (ELSD) detection as well as positive
electrospray ionization (MS). d The gradient was 5% B for 0.2 min,
and to 95% B within 1.7 min then with a hold at 95% B for 1.3 min,
back to 5% B within 0.01 min (2.3 mL/min flow rate). Mobile phase A
was 0.01% TFA in water, and mobile phase B was 0.01% TFA in HPLC
grade MeCN. The column used for the chromatography was an XBridge
C18 column (4.6 .times. 50 mm, 3.5 .mu.m particles). Detection
methods are diode array (DAD) and evaporative light scattering
(ELSD) detection as well as positive/negative electrospray
ionization e The gradient was 5% B to 95% B within 1.5 min then
with a hold at 95% B for 1.5 min, back to 5% B within 0.01 min (2.3
mL/min flow rate). Mobile phase A was 10 mM NH.sub.4HCO.sub.3 in
water, and mobile phase B was HPLC grade MeCN. The column used for
the chromatography was a XBridge C18 column (4.6 .times. 50 mm, 3.5
.mu.m particles). Detection methods are diode array (DAD) and
evaporative light scattering (ELSD) detection as well as
positive/negative electrospray ionization. f Mobile Phase: A: Water
(0.01% TFA); B: MeCN (0.01% TFA). Gradient: 5% B increase to 95% B
within 1.2 min, 95% B for 1.3 min, back to 5% B within 0.01 min, at
a flow rate of 2.0 mL/min. Column: SunFire C18 (4.6 .times. 50 mm,
3.5 .quadrature.m). Column Temp: 50.degree. C. Detection: UV (214,
254 nm) and MS (ESI, Pos mode, 110 to 1000 amu) g The gradient was
5% B for 0.1 min, and to 95% B within 1.0 min then with a hold at
95% B for 0.9 min, back to 5% B within 0.01 min (3.0 mL/min flow
rate). Mobile phase A was 0.05% TFA in water, and mobile phase B
was 0.05% TFA in HPLC grade MeCN. The column used for the
chromatography was a Zorbax SB-C18 Rapid Resolution HT column (4.6
.times. 30 mm, 1.8 .mu.m particles). Detection methods are diode
array (DAD) and evaporative light scattering (ELSD) detection as
well as positive/negative electrospray ionization. h Mobile Phase:
A: water (0.1% TFA); B: MeCN (0.1% TFA). Gradient: 5% B increase to
95% B within 1.3 min, 95% B for 1.5 min, back to 5% B within 0.01
min, at a flow rate of 2 mL/min. Column: Sunfire C18 (4.6 .times.
50 mm, 3.5 .mu.m). Column Temperature: 50.degree. C. i Mobile
Phase: A: water (0.01% TFA); B: MeCN (0.01% TFA). Gradient: 5% B
for 0.2 min, increase to 95% B within 1.5 min, 95% B for 1.5 min,
back to 5% B within 0.01 min, at a flow rate of 2 mL/min. Column:
Sunfire (50 .times. 4.6 mm, 3.5 .mu.m). Column Temperature:
50.degree. C. j Mobile phase: A: water (0.05% TFA); B: MeCn (0.05%
TFA). Gradient: 5% increase to 100% of B in 1.3 min, at a flow rate
of 2 mL/min. Column: SunFire C18 (4.6 .times. 50 mm, 3.5 .mu.m).
Detection: UV (214, 254 nm) and MS (ESI, Pos mode, 110 to 1000
amu). Column Temperature: 50.degree. C. k Mobile Phase: A: water
(10 mM NH.sub.4HCO.sub.3); B: MeCN. Gradient: 5% increase to 95% B
in 1.5 min, at a flow rate of 1.8 mL/min. Column: XBridge C18 (4.6
.times. 50 mm, 3.5 .mu.m). Column Temperature: 50.degree. C. l
Mobile phase: A: water (10 mM NH.sub.4HCO.sub.3); B: MeCN.
Gradient: 10% increase to 95% of B in 1.5 min, at a flow rate of
1.8 mL/min. Column: Xbridge C18(2) (4.6 .times. 50 mm, 3.5 .mu.m).
Column Temperature: 50.degree. C. Detection: UV (214, 254 nm) and
MS (ESI, Pos mode, 103 to 800 amu) m Mobile Phase: A: Water (0.01%
TFA) B: MeCN (0.01% TFA). 5% B increase to 95% B within 1.2 min,
95% B for 1.3 min, back to 5% B within 0.01 min. Flow Rate: 2.0
mL/min. Column: SunFire C18, 4.6*50 mm, 3.5.quadrature.m. Column
Temperature: 50.degree. C. Detection: UV (214, 4 nm) and MS (ESI,
Pos mode,110 to 1000 amu). n The gradient was 10-100% B in 3.4 min
with a hold at 100% B for 0.45 min, 100-10% B in 0.01 min, and then
held at 10% B for 0.65 min (0.8 mL/min flow rate). Mobile phase A
was 0.0375% TFA in water. Mobile phase B was 0.018% TFA in
CH.sub.3CN. The column used for the chromatography was a Phenomenex
Luna-C18 column (2.0 .times. 50 mm, 5 .mu.m particles). Detection
methods are diode array (DAD) and evaporative light scattering
(ELSD) detection as well as positive electrospray ionization (MS).
o A gradient of 5-100% MeCN (A) and 10 mM ammonium acetate in water
(B) was used, at a flow rate of 1.5 mL/min (0-0.05 min 5% A,
0.05-1.2 min 5-100% A, 1.2-1.4 min 100% A, 1.4-1.5 min 100-5% A.
0.25 min post-run delay). q 2-coupled C8 5 um 100 .ANG. Waters
Sunfire columns (30 mm .times. 75 mm each). A gradient of MeCN (A)
and 10 mM ammonium acetate in water (B) was used, at a flow rate of
50 mL/min (0-0.5 min 5% A, 0.5-8.5 min linear gradient X to Y % A,
8.7-10.7 min 100% A, 10.7-11 min linear gradient 100-05% A). Linear
gradient is stated in the synthetic procedure of the compound. r A
gradient of 5-100% MeCN (A) and 0.1% TFA in water (B) was used, at
a flow rate of 1.5 mL/min (0-0.05 min 5% A, 0.05-1.2 min 5-100% A,
1.2-1.4 min 100% A, 1.4-1.5 min 100-5% A. 0.25 min post-run delay).
s Analytical UPLC-MS was performed on a Waters SQD mass
spectrometer and Acquity UPLC system running MassLynx 4.1 and
Openlynx 4.1 software. The SQD mass spectrometer was operated under
positive APCI ionization conditions. The column used was a Waters
BEH C8, 1.7 .mu.m (2.1 mm .times. 30 mm) at a temperature of
55.degree. C. A gradient of 10-100% acetonitrile (A) and 10 mM
ammonium acetate in water (B) was used, at a flow rate of 1.0
mL/min (0-0.1 min 10% A, 0.1-1.1 min 10-100% A, 1.1-1.3 min 100% A,
1.3-1.4 min 100-10% A).
[0931] Abbreviations used in the examples that follow are:
TABLE-US-00027 APCI Atmospheric pressure chemical ionization Bn
Benzyl BOC tert-butyloxycarbonyl BSA Bovine serum albumin Cbz
Carbobenzyloxy CuCN Copper cyanide D.sub.2O Deuterated water DAD
Diode array DCM Dichloromethane DIAD Diisopropyl azodicarboxylate
DIPEA N, N-Diisopropylethylamine DMA Dimethylacetamide DMF Dimethyl
formamide DMSO Dimethyl sulfoxide EIC Extracted ion chromatogram
ELSD Evaporative light scattering detector Eq Equivalent Et.sub.2O
Diethyl ether EtOAc Ethyl acetate FMOC 9-Fluorenylmethyloxycarbony1
H Hour(s) H.sub.2SO.sub.4 Sulfuric acid HATU
1-[Bis(dimethylamino)methylene]-1H-1,2,3- triazolo[4,5-b]pyridinium
3-oxid hexafluoro- phosphate HCl Hydrochloric acid HEPES
4-(2-hydroxyethyl)-1-piperazineethane- sulfonic acid HIC
Hydrophobic Interaction Chromatography HPLC High performance liquid
chromatography IBX 2-Iodoxybenzoic acid MeCN Acetontrile MeOH
Methanol MgSO.sub.4 Magnesium sulfate Min Minute(s) MP-NaCNBH.sub.3
Sodium cynaoborohydride on solid support MTBE Dimethyl methyl
tert-butyl ether NaCN Sodium cyanide NaHCO.sub.3 Sodium hydrogen
carbonate NaHSO.sub.3 Sodium hydrogen sulfate Na.sub.2SO.sub.4
Sodium sulfate NMR Nuclear magnetic resonance Pd.sub.2dba.sub.3
tris(dibenzylideneacetone)dipalladium(0) PBST Phosphate Buffered
Saline with Tween 20 PE Petroleum ether PPh.sub.3 Triphenyl
phosphine RP Reverse phase R.sub.t Retention time TBAF
Tetrabutylammonium flouride TBS-Cl tert-Butylchlorodimethylsilane
TFA Trifluoroacetic acid TLC Thin layer chromatography
Example 1: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(4-aminophenoxy)phenyl)--
2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,-
8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]di-
oxol-4-one
Step 1: Synthesis of Tert-Butyl
(4-(4-formylphenoxy)phenyl)carbamate
##STR00688##
[0933] To a solution of tert-butyl (4-hydroxyphenyl)carbamate (10
g, 47.8 mmol) and 4-fluorobenzaldehyde (11.86 g, 96 mmol) in
N,N-dimethylformamide (100 mL) was added potassium carbonate (39.6
g, 287 mmol). The mixture was stirred at 90.degree. C. for 5 hours.
One additional vial was set up as described above. All two reaction
mixtures were combined and diluted with DCM (300 mL), then
extracted with water (3.times.100 mL). The organic layer was washed
with brine (100 mL) and dried over Na.sub.2SO.sub.4, filtered and
concentrated under reduced pressure. The residue was purified by
column chromatography (eluted with PE:EtOAc=30:1 to 5:1) to obtain
the target compound (20 g, 63.8 mmol, 66.7% yield) as a yellow
solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.91 (s, 1H),
9.45 (s, 1H), 7.90 (d, J=8.6 Hz, 2H), 7.54 (d, J=8.8 Hz, 2H),
7.11-7.02 (m, 4H), 1.48 (s, 9H).
Step 2: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(4-aminophenoxy)phenyl)--
2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,-
8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]di-
oxol-4-one
##STR00689##
[0935] A suspension of
(6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-
-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-
-3H-cyclopenta[a]phenanthren-3-one (4.5 g, 10.91 mmol) and
magnesium sulfate (6.57 g, 54.6 mmol) in MeCN (100 mL) was allowed
to stir at 20.degree. C. for 1 hours A solution of tert-butyl
(4-(4-formylphenoxy)phenyl)carbamate (3.42 g, 10.91 mmol) in MeCN
(100 mL) was added in one portion. Trifluoromethanesulfonic acid
(4.84 mL, 54.6 mmol) was added dropwise via syringe while
maintaining an internal temperature of 25.degree. C. using an ice
bath. After the addition, the mixture was stirred at 20.degree. C.
for 2 hours. Three additional vials were set up as described above.
All four reaction mixtures were combined and filtered, the filtrate
was concentrated under reduced pressure to give a residue, which
was purified by Prep-HPLC to obtain the target compound (7.5 g,
12.34 mmol, 28.8% yield) as a yellow solid. LCMS (Method a, Table
7) R.sub.t=2.21 min; MS m/z=608.3 (M+H)+; .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 7.36 (d, J=8.6 Hz, 2H), 7.27 (d, J=10.1 Hz,
1H), 6.85 (d, J=8.6 Hz, 2H), 6.75 (d, J=8.6 Hz, 2H), 6.58 (d, J=8.6
Hz, 2H), 6.29 (dd, J=1.3, 10.1 Hz, 1H), 6.13 (s, 1H), 5.76-5.65 (m,
1H), 5.62-5.57 (m, 1H), 5.54 (d, J=3.1 Hz, 1H), 5.44 (s, 1H), 5.12
(t, J=5.8 Hz, 1H), 5.00 (s, 2H), 4.94 (d, J=4.9 Hz, 1H), 4.53 (dd,
J=6.4, 19.4 Hz, 1H), 4.26-4.14 (m, 2H), 2.72-2.58 (m, 1H),
2.34-2.17 (m, 2H), 2.04 (d, J=13.7 Hz, 1H), 1.77-1.62 (m, 3H), 1.49
(s, 3H), 0.86 (s, 3H). Prep-HPLC Method: Instrument: Gilson 281
semi-preparative HPLC system, Mobile phase: A:
CF.sub.3CO.sub.2H/H.sub.2O=0.075% v/v; B: CH.sub.3OH; Column:
Phenomenex Luna C18 250*50 mm*10 um; Flow rate: 80 mL/min; Monitor
wavelength: 220 & 254 nm.
TABLE-US-00028 Time 0.0 20.0 20.1 20.2 30.2 30.3 31.5 B % 28 58 58
100 100 28 28
Example 2: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2-
,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8-
a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dio-
xol-4-one
Step 1: Synthesis of 4-(bromomethyl)benzaldehyde
##STR00690##
[0937] Diisobutylaluminum hydride (153 mL, 153 mmol, 1 M in
toluene) was added drop-wise to a 0.degree. C. solution of
4-(bromomethyl)benzonitrile (20 g, 102 mmol) in toluene (400 mL
over 1 hour Two additional vials were set up as described above.
All three reaction mixtures were combined. The mixture solution was
added 10% aqueous HCl (1.5 L). The mixture was extracted with DCM
(3.times.500 mL). The organic layer was dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure.
The residue was purified by column chromatography on silica gel
(eluted with PE/EtOAc=10/1) to obtain the target compound (50 g,
yield 82%) as white solid. .sup.1H NMR (400 MHz, chloroform-d)
.delta. 10.02 (s, 1H), 7.91-7.82 (m, 2H), 7.56 (d, J=7.9 Hz, 2H),
4.55-4.45 (m, 2H).
Step 2: Synthesis of
3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)aniline
##STR00691##
[0939] To a solution of 3-bromoaniline (40 g, 233 mmol) in
1,4-dioxane (480 mL) was added
4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (94 g,
372 mmol), potassium acetate (45.6 g, 465 mmol),
2-dicyclohexylphosphino-2',4',6'-tri-i-propyl-1,1'-biphenyl
(X-phos) (8.07 g, 13.95 mmol),
tris(dibenzylideneacetone)dipalladium(0) (8.52 g, 9.30 mmol). Then
the mixture was heated at 80.degree. C. for 4 hours under nitrogen.
Another additional vial was set up as described above. Two reaction
mixtures were combined, concentrated and the residue was purified
by column chromatography on silica gel (eluted with PE/EtOAc=10/1)
to obtain the target compound (60 g, yield 55.4%) as light yellow
solid. .sup.1H NMR (400 MHz, chloroform-d) .delta. 7.23-7.13 (m,
3H), 6.80 (d, J=7.5 Hz, 1H), 3.82-3.38 (m, 2H), 1.34 (s, 12H).
Step 3: Synthesis of Tert-Butyl
(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)
carbamate
##STR00692##
[0941] 3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (30
g, 137 mmol) and di-tert-butyl dicarbonate (38.9 g, 178 mmol) were
mixed in toluene (600 mL) at 100.degree. C. for 24 hours. Another
additional vial was set up as described above. Two reaction
mixtures were combined. The brown mixture was evaporated, dissolved
in EtOAc (1.5 L), washed with 0.1 N HCl (3.times.2 L) and brine (3
L), dried over Na.sub.2SO.sub.4, filtered and concentrated under
reduced pressure to give the title compound (50 g, yield 57%) as
red solid. .sup.1H NMR (400 MHz, chloroform-d) .delta. 7.63 (br.
s., 2H), 7.48 (d, J=7.1 Hz, 1H), 7.37-7.28 (m, 1H), 1.52 (s, 9H),
1.34 (s, 12H).
Step 4: Synthesis of Tert-Butyl
(3-(4-formylbenzyl)phenyl)carbamate
##STR00693##
[0943] A mixture of 4-(bromomethyl)benzaldehyde (24.94 g, 125
mmol), 1,1'-bis(diphenylphosphino) ferrocenedichloro palladium(II)
DCM complex (13.75 g, 18.80 mmol), tert-butyl
(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate
(20 g, 62.7 mmol) and potassium carbonate (43.3 g, 313 mmol) in
tetrahydrofuran (400 mL) was heated to 80.degree. C. for 12 hours.
Another additional vial was set up as described above. Two reaction
mixtures were combined. The reaction mixture was diluted with water
(500 mL). The aqueous layer was extracted with EtOac (3.times.500
mL). The organic layer was dried over Na.sub.2SO.sub.4, filtered
and concentrated under reduced pressure. The residue was purified
by column chromatography on silica gel (eluted with PE/EtOAc=10/1)
to obtain the title compound (15 g, yield 38.4%) as white solid.
.sup.1H NMR (400 MHz, chloroform-d) .delta. 9.95 (s, 1H), 7.78 (d,
J=7.9 Hz, 2H), 7.33 (d, J=7.9 Hz, 2H), 7.27-7.13 (m, 3H), 6.82 (d,
J=7.1 Hz, 1H), 6.47 (br. s., 1H), 4.00 (s, 2H), 1.48 (s, 9H).
Step 5: Synthesis of
(6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-
-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-
-3H-cyclopenta[a]phenanthren-3-one
##STR00694##
[0945]
(2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(-
2-hydroxyacetyl)-6a,8a,10,10-tetramethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12-
b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one
(20 g, 44.2 mmol) was suspended in 40% aqueous HBF.sub.4 (440 mL)
and the mixture was stirred at 25.degree. C. for 48 hours. After
the reaction was complete, 2 L of H.sub.2O was added and the solid
was collected by filtration to give a white solid. This solid was
washed with H.sub.2O (1 L) and then MeOH (200 mL) to give the title
compound (11 g, yield 60.3%) as a white solid. .sup.1H NMR (400
MHz, DMSO-d6) .delta. 7.25 (d, J=10.1 Hz, 1H), 6.28 (d, J=10.1 Hz,
1H), 6.10 (s, 1H), 5.73-5.50 (m, 1H), 5.39 (br. s., 1H), 4.85-4.60
(m, 2H), 4.50 (d, J=19.4 Hz, 1H), 4.20-4.04 (m, 2H), 2.46-2.06 (m,
6H), 1.87-1.75 (m, 1H), 1.56-1.30 (m, 6H), 0.83 (s, 3H).
Step 6: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2-
,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8-
a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dio-
xol-4-one
##STR00695##
[0947] A suspension of
(6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-
-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-
-3H-cyclopenta[a]phenanthren-3-one (4.4 g, 10.67 mmol) and
magnesium sulfate (6.42 g, 53.3 mmol) in MeCN (100 mL) was allowed
to stirred at 20.degree. C. for 1 hour A solution of tert-butyl
(3-(4-formylbenzyl)phenyl)carbamate (3.65 g, 11.74 mmol) in MeCN
(100 mL) was added in one portion. Trifluoromethanesulfonic acid
(9.01 mL, 53.3 mmol) was added drop wise while maintaining an
internal temperature below 25.degree. C. using an ice bath. After
the addition, the mixture was stirred at 20.degree. C. for 2 hours.
Three additional vials were set up as described above. All four
reaction mixtures were combined. The mixture solution was
concentrated and the residue was purification by Prep-HPLC to give
the title compound (4.5 g, yield 14.2%) as yellow solid. LCMS
(Method b, Table 7) R.sub.t=2.65 min; MS m/z=606.2 (M+H)+; .sup.1H
NMR (400 MHz, DMSO-d6) .delta. 7.44-7.17 (m, 5H), 6.89 (t, J=7.7
Hz, 1H), 6.44-6.25 (m, 4H), 6.13 (br. s., 1H), 5.79-5.52 (m, 2H),
5.44 (s, 1H), 5.17-4.89 (m, 3H), 4.51 (d, J=19.4 Hz, 1H), 4.25-4.05
(m, 2H), 3.73 (s, 2H), 3.17 (br. s., 1H), 2.75-2.55 (m, 1H),
2.36-1.97 (m, 3H), 1.76-1.64 (m, 3H), 1.59-1.39 (m, 4H), 0.94-0.78
(m, 3H). Prep-HPLC Method: Instrument: Gilson 281 semi-preparative
HPLC system; Mobile phase: A: Formic Acid/H.sub.2O=0.01% v/v; B:
CH3CN; Column: Luna C18 150*25 5 micron; Flow rate: 25 mL/min;
Monitor wavelength: 220 and 254 nm.
TABLE-US-00029 Time 0.0 10.5 10.6 10.7 13.7 13.8 15.0 B % 15 35 35
100 100 10 10
Example 2A: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-7-hy-
droxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,1-
2b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one
(Cpd. No. 41)
Step 1: Synthesis of 4-(bromomethyl)benzaldehyde
##STR00696##
[0949] To a solution of 4-(bromomethyl)benzonitrile (50 g, 255
mmol) in toluene (1 L) was added diisobutylaluminum hydride (383
mL, 383 mmol, 1 M in toluene) dropwise at 0.degree. C. The mixture
was stirred for 1 hour Two additional vials were set up as
described above. All three reaction mixtures were combined. 10%
aqueous HCl (1.5 L) was added and then extracted with DCM
(3.times.1.5 L). The organic layer was dried over Na.sub.2SO.sub.4,
filtered and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel (eluted with
petroleum ether/ethyl acetate=10/1) to afford the title compound
(120 g, 82%) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 10.01 (s, 1H), 7.86 (d, J=8.4 Hz, 2H), 7.55 (d, J=7.9 Hz,
2H), 4.51 (s, 2H).
Step 2: Synthesis of
3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline
##STR00697##
[0951] To a solution of 3-bromoaniline (80 g, 465 mmol) in
1,4-dioxane (960 mL) was added
4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (177 g,
698 mmol), potassium acetate (91 g, 930 mmol),
2-dicyclohexylphosphino-2',4',6'-tri-i-propyl-1,1'-biphenyl (13.45
g, 23.25 mmol) and tris(dibenzylideneacetone)dipalladium(0) (17.03
g, 18.60 mmol). The mixture was heated at 80.degree. C. for 4 hours
under nitrogen. Two additional vials were set up as described
above. Three reaction mixtures were combined, concentrated and the
residue purified by column chromatography on silica gel (eluted
with petroleum ether/ethyl acetate=10/1) to afford the title
compound (150 g, 46.6%) as a light yellow solid. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 7.23-7.13 (m, 3H), 6.80 (d, J=7.5 Hz, 1H),
3.82-3.38 (m, 2H), 1.34 (s, 12H).
Step 3: Synthesis of Tert-Butyl
(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate
##STR00698##
[0953] 3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (50
g, 228 mmol) and di-tert-butyl dicarbonate (64.8 g, 297 mmol) were
mixed in toluene (500 mL) and the mixture stirred at 100.degree. C.
for 24 hours. Two additional vials were set up as described above.
The three reaction mixtures were combined. The brown mixture was
concentrated and the residue was washed with PE to afford the title
compound (120 g, 49.5%) as a white solid. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.62 (s, 2H), 7.48 (d, J=7.5 Hz, 1H), 7.35-7.29
(m, 1H), 6.46 (br. s., 1H), 1.52 (s, 9H), 1.34 (s, 12H).
Step 4: Synthesis of Tert-Butyl
(3-(4-formylbenzyl)phenyl)carbamate
##STR00699##
[0955] A mixture of 4-(bromomethyl)benzaldehyde (29.9 g, 150 mmol),
1,1'-bis(diphenylphosphino) ferrocenedichloro palladium(II) (20.63
g, 28.2 mmol), tert-butyl
(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate
(30 g, 94 mmol) and potassium carbonate (64.9 g, 470 mmol) in THF
(600 mL) was heated to 80.degree. C. for 12 hours. Three additional
vials were set up as described above. All four reaction mixtures
were combined. The reaction mixture was diluted with water (1 L).
The aqueous layer was extracted with EtOAc (3.times.800 mL). The
organic layer was dried over Na.sub.2SO.sub.4, filtered and
concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel (eluted with PE/EtOAc=10/1) to
afford the title compound (35.5 g, 27.3%) as a white solid. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 9.97 (s, 1H), 7.80 (d, J=7.9 Hz,
2H), 7.35 (d, J=7.9 Hz, 2H), 7.26 (s, 2H), 7.24-7.13 (m, 2H), 6.84
(d, J=7.1 Hz, 1H), 6.43 (br. s., 1H), 4.02 (s, 2H), 1.50 (s,
9H).
Step 5: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-7-hy-
droxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,1-
2b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one
##STR00700##
[0957] To a solution of
(8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)--
10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a-
]phenanthren-3-one (6 g, 15.94 mmol) and tert-butyl
(3-(4-formylbenzyl)phenyl)carbamate (4.96 g, 15.94 mmol) in MeCN
(50 mL) was added perchloric acid (4.79 mL, 80 mmol) dropwise while
maintaining an internal temperature below 25.degree. C. using an
ice bath. After the addition, the mixture was stirred at 20.degree.
C. for 2 hours. Three additional vials were set up as described
above. All four reaction mixtures were combined. The reaction
mixture was quenched with saturated NaHCO.sub.3 aqueous (500 mL)
and extracted with dichloromethane (3.times.800 mL). The organic
phase was concentrated and the residue was purified by Prep-HPLC to
afford the title compound (10 g, 27.0%) as a yellow solid. .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 7.36 (d, J=7.9 Hz, 2H), 7.31
(d, J=10.1 Hz, 1H), 7.20 (d, J=7.9 Hz, 2H), 6.89 (t, J=7.9 Hz, 1H),
6.39-6.28 (m, 3H), 6.16 (dd, J=1.5, 9.9 Hz, 1H), 5.93 (s, 1H), 5.39
(s, 1H), 5.08 (t, J=5.7 Hz, 1H), 4.98-4.87 (m, 3H), 4.78 (d, J=3.1
Hz, 1H), 4.49 (dd, J=6.2, 19.4 Hz, 1H), 4.29 (br. s., 1H), 4.17
(dd, J=5.5, 19.6 Hz, 1H), 3.74 (s, 2H), 2.61-2.53 (m, 1H),
2.36-2.26 (m, 1H), 2.11 (d, J=11.0 Hz, 1H), 2.07 (s, 1H), 2.02 (d,
J=12.8 Hz, 1H), 1.83-1.54 (m, 5H), 1.39 (s, 3H), 1.16-0.96 (m, 2H),
0.85 (s, 3H). LCMS: t.sub.R=2.365 min, 98% purity, m/z=570.2
(M+H).sup.+ LC/MS (Table 7, method a)
[0958] Method of Prep-HPLC: Instrument: Gilson 281 semi-preparative
HPLC system, Mobile phase: A: CF.sub.3COOH/H.sub.2O=0.075% v/v; B:
CH.sub.3CN, Column: Phenomenex Luna(2) C18 250*50 10 u, Flow rate:
80 mL/min, Monitor wavelength: 220&254 nm, Time B %, 0.0 28,
20.0 45, 20.1 45, 20.2 100, 30.2 100, 0.3 28, 31.5 28.
Example 2B
Synthesis of
(2R,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-Aminobenzyl)phenyl)-6-
b-fluoro-2,7-dihydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b-
,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(-
2H)-one
##STR00701##
[0960] Trifluoromethane sulfonic acid (1.34 ml, 15.11 mmol) was
added drop-wise to a -10.degree. C. suspension of
(6R,8S,9R,10S,11S,13S,14S,16R,17S)-9-fluoro-6,11,16,17-tetrahydroxy-17-(2-
-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-
-3H-cyclopenta[a]phenanthren-3-one (1.55 g, 3.78 mmol), tert-butyl
(3-(4-formylbenzyl)phenyl)carbamate (from Example 2, step 4) (1.176
g, 3.78 mmol), and MgSO.sub.4 (2.273 g, 18.89 mmol) in MeCN (15.1
mL). After 20 min, the reaction was quenched by addition of a
saturated aqueous solution of NaHCO.sub.3 (15 mL), followed by
water (60 mL) and EtOAc (100 mL). The organic layer was washed
sequentially with water (60 mL), brine (60 mL), dried
(Na.sub.2SO.sub.4), and solvent was removed under reduced pressure.
Purification by chromatography (silica, 40 g) eluting with a
gradient of 40-100% EtOAc/heptanes provided the title compound as a
foam (880 mg, 1.458 mmol, 39% yield) in 90% purity. The product
could be further purified by reverse phase HPLC on a Waters
XBridge.TM. RP18 5 micron column (30.times.100 mm). A gradient of
MeCN (A) and 0.1 mM NH.sub.4CO.sub.3 in water (B) was used, at a
flow rate of 40 mL/min (0-5.0 min 5% A, 5.0-19.0 min linear
gradient 15-55% A). LC-MS (Method r, Table 7) Rt=0.72 min,
m/z=604.3 [M+H.sup.+]. .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta.
7.37-7.31 (m, 2H), 7.28 (d, J=10.1 Hz, 1H), 7.24-7.19 (m, 2H),
6.93-6.85 (m, 1H), 6.36 (d, J=2.1 Hz, 2H), 6.35 (p, J=1.1 Hz, 1H),
6.23 (dd, J=10.1, 1.9 Hz, 1H), 6.10 (d, J=1.9 Hz, 1H), 5.45 (s,
1H), 5.38 (s, 1H), 5.10 (s, 1H), 4.96-4.91 (m, 3H), 4.51 (d, J=19.4
Hz, 1H), 4.38 (s, 1H), 4.28-4.16 (m, 2H), 3.74 (s, 2H), 2.76-2.60
(m, 1H), 2.20 (td, J=12.5, 6.3 Hz, 1H), 2.08 (s, 2H), 1.86 (d,
J=11.8 Hz, 1H), 1.75-1.58 (m, 7H), 0.89 (s, 3H).
Example 3: Synthesis of
(6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-aminophenyl)thio)phenyl-
)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a-
,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]diox-
ol-4-one
Step 1: Synthesis of Tert-Butyl (3-mercaptophenyl)carbamate
##STR00702##
[0962] To a mixture of zinc perchlorate (0.422 g, 1.598 mmol) and
3-aminobenzenethiol (10 g, 80 mmol) was added di-tert-butyl
dicarbonate (22.66 g, 104 mmol) drop wise. The solution was stirred
at 25.degree. C. for 12 hours. Three additional vials were set up
as described above. Four reaction mixtures were combined. The
mixture was dissolved in EtOAc (200 mL) and washed with water (500
mL). The organic layer was dried over Na.sub.2SO.sub.4, filtered
and concentrated under reduced pressure to give a residue. The
residue was purified by column chromatography on silica gel (eluted
with PE/EtOAc=5/1) to obtain the target compound (50 g, yield
69.4%) as white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
7.45 (br. s., 1H), 7.16-7.09 (m, 1H), 7.06-7.01 (m, 1H), 6.92 (d,
J=7.4 Hz, 1H), 6.55 (br. s., 1H), 3.46 (s, 1H), 1.52 (s, 9H).
Step 2: Synthesis of Tert-Butyl
(3-((4-formylphenyl)thio)phenyl)carbamate
##STR00703##
[0964] To a solution of tert-butyl
(3-((4-formylphenyl)thio)phenyl)carbamate (10 g, 44.4 mmol) in DMF
(300 mL) was added triphenylphosphine (11.64 g, 44.4 mmol) and
N-ethyl-N-isopropylpropan-2-amine (11.47 g, 89 mmol) at 25.degree.
C. The reaction mixture was stirred at 25.degree. C. for 30 minutes
under N.sub.2. The mixture was added 4-fluorobenzaldehyde (8.26 g,
66.6 mmol) at 100.degree. C. and the mixture was stirred at
100.degree. C. for 12 hours. Four additional vials were set up as
described above. The five reaction mixtures were combined. The
mixture was diluted with water (2 L) and extracted with EtOAc
(3.times.1 L). The organic layer was dried over Na.sub.2SO.sub.4,
filtered and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel (eluted with
PE/EtOAc=10/1) to obtain the target compound (55 g, yield 75%) as
yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 9.90 (s, 1H),
7.71 (d, J=8.4 Hz, 2H), 7.58 (s, 1H), 7.48-7.41 (m, 1H), 7.33 (t,
J=7.9 Hz, 1H), 7.25 (d, J=8.4 Hz, 2H), 7.17 (d, J=7.9 Hz, 1H), 6.72
(br. s., 1H), 1.50 (s, 9H).45 (br. s., 1H), 7.16-7.09 (m, 1H),
7.06-7.01 (m, 1H), 6.92 (d, J=7.4 Hz, 1H), 6.55 (br. s., 1H), 3.46
(s, 1H), 1.52 (s, 9H).
Step 3: Synthesis of Sodium
(4-((3-((tert-butoxycarbonyl)amino)phenyl)thio)phenyl)
(hydroxy)methanesulfonate
##STR00704##
[0966] To a solution of the aldehyde (15 g, 45.5 mmol) in
CH.sub.3CN (30 mL) was added a solution of sodium metabisulfite
(11.25 g, 59.2 mmol) in water (90 mL) at 25.degree. C. The mixture
was stirred at 25.degree. C. for 48 hours. Another additional vial
was set up as described above. Two reaction mixtures were combined.
The solution was filtered and the solid was washed with water (150
mL), CH.sub.3CN (150 mL) and dried under reduced pressure to give
the target compound (32 g, yield 81%) as white solid. .sup.1H NMR
(400 MHz, DMSO-d6) .delta. 9.45 (br. s., 1H), 7.54-7.49 (m, 1H),
7.47-7.35 (m, 3H), 7.33-7.17 (m, 3H), 6.85 (d, J=7.9 Hz, 1H), 5.97
(d, J=4.9 Hz, 1H), 4.98 (d, J=4.9 Hz, 1H), 1.45 (s, 9H).
Step 4: Synthesis of
(6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-aminophenyl)thio)phenyl-
)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a-
,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]diox-
ol-4-one
##STR00705##
[0968] To a solution of
(8S,9R,10S,11S,13S,14S,16R,17S)-9-fluoro-11,16,17-trihydroxy-17-(2-hydrox-
yacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyc-
lopenta[a]phenanthren-3-one (6 g, 15.21 mmol) and sodium
(4-((3-((tert-butoxycarbonyl)amino)phenyl)thio)phenyl)(hydroxy)methanesul-
fonate (4.74 g, 15.21 mmol) in THF (50 mL) was added perchloric
acid (4.58 mL, 76 mmol) drop wise while maintaining an internal
temperature below 25.degree. C. using an ice bath. After the
addition, the mixture was stirred at 20.degree. C. for 2 hours.
Three additional vials were set up as described above. All four
reaction mixtures were combined. The reaction mixture was quenched
with sat. NaHCO.sub.3 aqueous (500 mL) and extracted with DCM
(3.times.800 mL). The organic phase was concentrated and the
residue was purification by Prep-HPLC to give the target compound
(9.5 g, 25.8%) as yellow solid. LCMS (Method b, Table 7)
R.sub.t=2.68 min, m/z=588.1 (M+H)+; .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 7.37-7.26 (m, 3H), 7.21 (d, J=7.9 Hz, 2H), 6.89 (t, J=7.7
Hz, 1H), 6.43-6.30 (m, 3H), 6.23 (d, J=10.1 Hz, 1H), 6.04 (s, 1H),
5.75 (s, 1H), 5.44 (s, 2H), 5.09 (t, J=5.7 Hz, 1H), 4.93 (br. s.,
3H), 4.50 (dd, J=6.2, 19.4 Hz, 1H), 4.28-4.09 (m, 2H), 3.74 (s,
2H), 2.73-2.54 (m, 2H), 2.35 (d, J=13.2 Hz, 1H), 2.25-2.12 (m, 1H),
2.05 (d, J=15.0 Hz, 1H), 1.92-1.77 (m, 1H), 1.74-1.58 (m, 3H), 1.50
(s, 3H), 1.45-1.30 (m, 1H), 0.87 (s, 3H). Prep-HPLC Method:
Instrument: Gilson 281 semi-preparative HPLC system; Mobile phase:
A: CF.sub.3CO.sub.2H/H.sub.2O=0.075% v/v; B: CH.sub.3CN; Column:
Phenomenex Luna C18 250.times.*50 mm*10 micron; Flow rate: 80
mL/min; Monitor wavelength: 220 and 254 nm.
TABLE-US-00030 Time 0.0 20.0 20.1 20.2 30.2 30.3 31.5 B % 10 42 42
100 100 10 10
Example 4: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-aminophenyl)thio)phenyl-
)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12-
,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one
##STR00706##
[0970] To a solution of
(8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)--
10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a-
]phenanthren-3-one (6 g, 15.94 mmol) and sodium
(4-((3-((tert-butoxycarbonyl)amino)phenyl)thio)phenyl)(hydroxy)methanesul-
fonate (4.96 g, 15.94 mmol) in MeCN (50 mL) was added perchloric
acid (4.79 mL, 80 mmol) drop wise while maintaining an internal
temperature below 25.degree. C. using an ice bath. After the
addition, the mixture was stirred at 20.degree. C. for 2 hours.
Three additional vials were set up as described above. All four
reaction mixtures were combined. The reaction mixture was quenched
with sat. aqueous NaHCO.sub.3 (500 mL) and extracted with DCM
(3.times.800 mL). The organic phase was concentrated and the
residue was purification by Prep-HPLC to give the target compound
(10 g, 27.0%) as yellow solid. LCMS (Method b, Table 7)
R.sub.t=2.36 min, m/z=570.2 (M+H)+; .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 7.36 (d, J=7.9 Hz, 2H), 7.31 (d, J=10.1 Hz, 1H), 7.20 (d,
J=7.9 Hz, 2H), 6.89 (t, J=7.9 Hz, 1H), 6.39-6.28 (m, 3H), 6.16 (dd,
J=1.5, 9.9 Hz, 1H), 5.93 (s, 1H), 5.39 (s, 1H), 5.08 (t, J=5.7 Hz,
1H), 4.98-4.87 (m, 3H), 4.78 (d, J=3.1 Hz, 1H), 4.49 (dd, J=6.2,
19.4 Hz, 1H), 4.29 (br. s., 1H), 4.17 (dd, J=5.5, 19.6 Hz, 1H),
3.74 (s, 2H), 2.61-2.53 (m, 1H), 2.36-2.26 (m, 1H), 2.11 (d, J=11.0
Hz, 1H), 2.07 (s, 1H), 2.02 (d, J=12.8 Hz, 1H), 1.83-1.54 (m, 5H),
1.39 (s, 3H), 1.16-0.96 (m, 2H), 0.85 (s, 3H). Prep-HPLC Method:
Instrument: Gilson 281 semi-preparative HPLC system; Mobile phase:
A: CF.sub.3CO.sub.2H/H.sub.2-=0.075% v/v; B: CH.sub.3CN; Column:
Phenomenex Luna C18 250.times.*50 mm*10 micron; Flow rate: 80
mL/min; Monitor wavelength: 220 and 254 nm.
TABLE-US-00031 Time 0.0 20.0 20.1 20.2 30.2 30.3 31.5 B % 28 45 45
100 100 28 28
Example 5: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-aminophenyl)
thio)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl--
1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno-
[1,2-d][1,3]dioxol-4-one
##STR00707##
[0972] To a solution of steroid (10 g, 24.25 mmol) in CH.sub.3CN
(200 mL) was added magnesium sulfate (10.21 g, 85 mmol) at
25.degree. C. The mixture was stirred at 25.degree. C. for 4 hours.
Then to the above solution was added sodium
(4-((3-((tert-butoxycarbonyl)amino)phenyl)thio)
phenyl)(hydroxy)methanesulfonate (10.51 g, 24.25 mmol) and
trifluoromethanesulfonic acid (20.48 mL, 121 mmol) at 0.degree. C.
The resulting mixture was stirred at 25.degree. C. for 1 hour. Two
additional vials were set up as described above. Three reaction
mixtures were combined. The mixture was diluted with 1 N NaOH (300
mL) and extracted with EtOAc (3.times.600 mL). The organic layer
was concentrated under reduced pressure to give a residue. The
residue was dissolve in EtOAc (60 mL) and added 2-butanone (180
mL). After stirring for 30 minutes, the solid was collected by
filtration and purified by Prep-HPLC to give the title compound
(8.4 g, yield 17.52%) as yellow solid. LCMS (Method c, Table 7)
R.sub.t=2.66 min; MS m/z=624.1 (M+H)+; .sup.1H NMR (400 MHz,
DMSO-d6) .delta. 7.39 (d, J=8.4 Hz, 2H), 7.25 (d, J=8.4 Hz, 3H),
7.03 (t, J=7.7 Hz, 1H), 6.61 (s, 1H), 6.53 (t, J=8.2 Hz, 2H), 6.29
(dd, J=1.5, 9.9 Hz, 1H), 6.12 (s, 1H), 5.76-5.49 (m, 2H), 5.46 (s,
1H), 4.96 (d, J=4.9 Hz, 1H), 4.52 (d, J=19.4 Hz, 1H), 4.21 (d,
J=19.4 Hz, 2H), 2.74-2.53 (m, 2H), 2.34-2.13 (m, 2H), 2.09-1.96 (m,
1H), 1.79-1.62 (m, 3H), 1.57-1.43 (m, 4H), 0.86 (s, 3H). Prep-HPLC
method: Instrument: Shimadzu LC-8A preparative HPLC; Column:
Phenomenex Luna C18 250.times.*50 mm*10 micron; Mobile phase: A for
H.sub.2O (0.09% CF.sub.3CO.sub.2H) and B for CH.sub.3CN; Gradient:
B from 22% to 52% in 20 min; Flow rate: 80 mL/min; Wavelength:
220&254 nm.
Example 6: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-Amino-4-hydroxybenzyl)ph-
enyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12-
,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(2H)-one
##STR00708##
[0974] Triflic acid (0.2 mL, 2.183 mmol) was added drop-wise to a
0.degree. C. slurry of
(8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)--
10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a-
]phenanthren-3-one (0.164 g, 0.437 mmol), tert-butyl
(2-((tert-butyldimethylsilyl)oxy)-5-(4-formylbenzyl)phenyl)carbamate
(0.193 g, 0.437 mmol) and MgSO.sub.4 (0.189 g, 1.572 mmol) in MeCN
(1.8 mL). After 40 min the reaction was diluted with EtOAc (15 mL),
and then washed sequentially with a saturated aqueous solution of
NaHCO.sub.3 (10 mL.times.2), and with a saturated aqueous solution
of brine (5 mL). The organic phase was dried (Na.sub.2SO.sub.4) and
solvent was removed under reduced pressure. Purification by
chromatography (silica, 12 g) eluting with a gradient of 0-10%
MeOH/DCM gave the title compound (163 mg, 0.278 mmol, 64% yield) as
a waxy solid. A portion of this material (ca. 48.9 mg) was further
purified by reverse phase HPLC on a Phenomenex C18(2) 10 micron
column (250.times.50 mm column). A gradient of MeCN (A) and 0.1%
TFA in water (B) was used, at a flow rate of 90 mL/min (0-5.0 min
15% A, 5.0-20 min linear gradient 15-70% A, hold 2 min). Combined
fractions were concentrated under reduced pressure to remove
volatile solvents, and the resulting solution was frozen and
lyophilized to give a off-white solid (11.9 mg). LCMS (Method r,
Table 7) R.sub.t=0.75 min, m/z=586.26 [M+H.sup.+]. .sup.1H NMR (400
MHz, DMSO-d6) .delta. 10.27 (s, 1H), 9.04 (s, 2H), 7.34 (d, J=8.0
Hz, 2H), 7.28 (d, J=10.1 Hz, 1H), 7.18 (d, J=8.0 Hz, 2H), 6.94 (dd,
J=8.1, 2.1 Hz, 1H), 6.90 (d, J=2.1 Hz, 1H), 6.82 (d, J=8.2 Hz, 1H),
6.17-6.07 (m, 1H), 5.90 (d, J=1.6 Hz, 1H), 5.37 (s, 1H), 4.89 (d,
J=4.9 Hz, 1H), 4.75 (s, 1H), 4.46 (d, J=19.4 Hz, 1H), 4.26 (q,
J=3.3 Hz, 1H), 4.14 (d, J=19.5 Hz, 1H), 3.80 (s, 2H), 2.58-2.46 (m,
1H), 2.36-1.92 (m, 3H), 1.76-1.56 (m, 4H), 1.36 (s, 3H), 1.10-0.90
(m, 2H), 0.83 (s, 3H).
Example 7: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-Aminobenzyl)-3-hydrox-
yphenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b-
,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3-
]dioxol-4(2H)-one
Step 1: Synthesis of
4-((3-bromophenyl)(hydroxy)methyl)-3-methoxybenzonitrile
##STR00709##
[0976] Isopropylmagnesium chloride lithium chloride complex (1.3 M
in THF, 8.34 mL, 10.85 mmol) was added drop-wise to a 0-5.degree.
C. solution of 4-bromo-3-methoxybenzonitrile (2 g, 9.43 mmol) in
THF (21 mL). The reaction was stirred for 5 h, whereupon a solution
of 3-bromobenzaldehyde (1.979 g, 10.38 mmol) in THF (10.5 mL) was
added drop-wise, maintaining a temperature of <10.degree. C. The
reaction was permitted to slowly warm to room temperature
overnight. The reaction quenched with a saturated aqueous solution
of NH.sub.4Cl (25 mL) and extracted with MTBE (50 mL.times.3). The
combined organics were washed with brine (20 mL), dried
(Na.sub.2SO.sub.4), and solvents were removed under reduced
pressure. Purification by chromatography (80 g silica) eluting with
a gradient of 0-10% MTBE/heptanes gave the title compound (1.77 g,
5.56 mmol, 59% yield) as a beige syrup/oil. LCMS (Method r, Table
7) R.sub.t=0.86 min; MS (ESI-) m/z=315.7 [M-H.sup.+]. .sup.1H NMR
(501 MHz, DMSO-d6) .delta. 7.67 (d, J=7.8 Hz, 1H), 7.47 (t, J=1.8
Hz, 1H), 7.43 (dd, J=7.8, 1.5 Hz, 1H), 7.41 (d, J=1.4 Hz, 1H),
7.40-7.36 (m, 1H), 7.28 (dt, J=7.8, 1.5 Hz, 1H), 7.23 (d, J=7.8 Hz,
1H), 6.10 (d, J=4.4 Hz, 1H), 5.94 (d, J=4.1 Hz, 1H), 3.80 (s,
3H).
Step 2: Synthesis of 4-(3-bromobenzyl)-3-methoxybenzonitrile
##STR00710##
[0978] Chlorotrimethylsilane (3.63 g, 33.4 mmol) was added to a
room temperature solution of sodium iodide (5.00 g, 33.4 mmol) in
MeCN (18.5 mL), which resulted in the immediate precipitation of a
white solid. A solution of
4-((3-bromophenyl)(hydroxy)methyl)-3-methoxybenzonitrile (1.77 g,
5.56 mmol) in MeCN (18.5 mL) was then added, whereupon the reaction
mixture was heated to 55.degree. C. for 60 min. After cooling to
room temperature the reaction was partitioned between MTBE (50 mL)
and water (50 mL). After separating the layers the aqueous phase
was extracted with MTBE (50 mL.times.2). The combined organics were
washed sequentially with a 1 M aqueous solution of
Na.sub.2S.sub.2O.sub.3 (50 mL.times.2), followed by a saturated
aqueous solution of brine (30 mL), dried (Na.sub.2SO.sub.4), and
solvents were removed under reduced pressure. Purification by
chromatography (silica, 80 g) eluting with a gradient of 5-40%
MTBE/heptanes gave the title compound (1.58 g, 5.23 mmol, 94%
yield) as an off-white solid. LCMS (Method r, Table 7) R.sub.t=1.02
min; MS m/z=not observed. .sup.1H NMR (501 MHz, DMSO-d6) .delta.
7.42 (d, J=1.5 Hz, 1H), 7.39-7.30 (m, 4H), 7.22 (td, J=7.6, 0.6 Hz,
1H), 7.18 (dt, J=7.7, 1.4 Hz, 1H), 3.94 (s, 2H), 3.82 (s, 3H).
Step 3: Synthesis of 4-(3-bromobenzyl)-3-methoxybenzaldehyde
##STR00711##
[0980] Diisobutylaluminum hydride (4.9 mL, 1.0 M solution in
hexanes, 4.9 mmol) was added drop-wise over 5 min to a 0.degree. C.
solution of 4-(3-bromobenzyl)-3-methoxybenzonitrile (0.99 g, 3.28
mmol) in toluene (16 mL), maintaining a temperature of
<6.degree. C. After 10 min the reaction was quenched by careful
addition of a 1 N aqueous solution of HCl (100 mL) at 0.degree. C.
It was then extracted with DCM (50 mL.times.4), washed with a
saturated aqueous solution of brine (30 mL), and solvent was
removed under reduced pressure. Purification by chromatography
(silica, 40 g) eluting with a gradient of 0-40% MTBE/heptanes gave
the title compound (780 mg, 2.56 mmol, 78% yield) as a colorless
oil. LCMS (Method r, Table 7) R.sub.t=0.95 min, MS (DCI+)
m/z=303.9, 305.9 (M). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 9.93
(s, 1H), 7.47 (dd, J=7.5, 1.5 Hz, 1H), 7.44 (d, J=1.5 Hz, 1H),
7.42-7.33 (m, 3H), 7.25-7.17 (m, 2H), 3.96 (s, 2H), 3.85 (s,
3H).
Step 4: Synthesis of 4-(3-bromobenzyl)-3-hydroxybenzaldehyde
##STR00712##
[0982] Boron tribromide (1.0 M in methylene chloride, 6.4 mL, 6.4
mmol) was added drop-wise to a 0-3.degree. C. solution of
4-(3-bromobenzyl)-3-methoxybenzaldehyde (0.78 g, 2.56 mmol) in DCM
(7.8 mL). The reaction was stirred at 0.degree. C. for 30 min; then
was stirred for 90 min at room temperature. Solvent was removed
under reduced pressure and the resulting dark oil was treated with
MeOH (20 mL) and water (15 mL), which gave a heterogeneous mixture.
MeCN was added until a homogeneous solution was obtained (ca. 10
mL) and the solution was stirred overnight. Volatile solvents were
removed under reduced pressure and the resulting aqueous suspension
was extracted with DCM (25 mL.times.3). The combined organics were
washed with brine (20 mL), dried over Na.sub.2SO.sub.4 and solvent
was removed under reduced pressure. Purification by chromatography
(silica, 40 g) eluting with a gradient of 10-50% MTBE/heptanes gave
4-(3-bromobenzyl)-3-hydroxybenzaldehyde (660 mg, 2.267 mmol, 89%
yield) as a white solid. LCMS (Method r, Table 7) R.sub.t=0.85 min;
MS (DCI+) m/z=307.98, 309.97 [M+NH.sub.4]. .sup.1H NMR (400 MHz,
DMSO-d6) .delta. 10.07 (s, 1H), 9.83 (s, 1H), 7.39 (q, J=1.3 Hz,
1H), 7.33 (ddt, J=6.5, 4.4, 2.0 Hz, 1H), 7.30 (d, J=0.9 Hz, 2H),
7.25 (s, 1H), 7.25-7.15 (m, 2H), 3.92 (s, 2H).
Step 5: Synthesis of
4-(3-bromobenzyl)-3-((tert-butyldimethylsilyl)oxy)benzaldehyde
##STR00713##
[0984] Imidazole (0.231 g, 3.40 mmol) and
tert-butyldimethylchlorosilane (0.410 g, 2.72 mmol) were added to a
room temperature suspension of
4-(3-bromobenzyl)-3-hydroxybenzaldehyde (0.660 g, 2.267 mmol) in
DCM (7.6 mL), which was stirred for 3 h. MeOH (0.5 mL) was added
and stirring continued for 10 min, whereupon the reaction was
diluted with DCM (100 mL), washed sequentially with water (25 mL),
a 1 N aqueous solution of HCl (25 mL), and with a saturated aqueous
solution of brine (20 mL). The organic phase was dried
(Na.sub.2SO.sub.4) and solvent was removed under reduced pressure
to give a syrup. Purification by chromatography (silica, 40 g)
eluting with a gradient of 0-10% MTBE/heptanes gave the target
compound (820 mg, 2.023 mmol, 89% yield) as a colorless oil. LCMS
(Method r, Table 7) R.sub.t=1.18 min, MS (DCI+) m/z=422.07, 424.09
[M+NH.sub.4+]. .sup.1H NMR (500 MHz, DMSO-d6) .delta. 9.94 (s, 1H),
7.50 (dd, J=7.7, 1.6 Hz, 1H), 7.42-7.36 (m, 2H), 7.36-7.32 (m, 2H),
7.25 (t, J=7.8 Hz, 1H), 7.17 (ddd, J=7.7, 1.7, 1.0 Hz, 1H), 4.01
(s, 2H), 0.92 (s, 9H), 0.26 (s, 6H).
Step 6: Synthesis of Tert-Butyl
(3-(4-formyl-2-hydroxybenzyl)phenyl)carbamate
##STR00714##
[0986] Nitrogen was sparged through a mixture of
4-(3-bromobenzyl)-3-((tert-butyldimethylsilyl)oxy)benzaldehyde
(0.820 g, 2.023 mmol), tert-butyl carbamate (0.3027 g, 2.58 mmol),
Cs.sub.2CO.sub.3 (1.006 g, 3.09 mmol) in p-dioxane (16 mL) for 30
min. Added the 2nd generation XPhos precatalyst (0.0937 g, 0.119
mmol) and continued sparging for 5 min, whereupon the reaction was
heated to 100.degree. C. for 4 h. The reaction was cooled to room
temperature, treated with a 1 N aqueous solution of HCl (25 mL),
and was extracted with MTBE (25 mL.times.3). The combined organics
were washed with brine (30 mL), dried over Na.sub.2SO.sub.4 and
solvent was removed under reduced pressure. The residue was
redissolved in THF (16 mL, 0.125 M) and treated with TBAF/SiO.sub.2
(1.0-1.5 mmol/g, 4.1338 g, 4.13-6.2 mmol) for 45 min, whereupon
solvent was removed under reduced pressure. Purification by
chromatography (silica, 40 g) eluting with a gradient of 0-75%
MTBE/heptanes gave tert-butyl
(3-(4-formyl-2-hydroxybenzyl)phenyl)carbamate (380 mg, 1.161 mmol,
57% yield) was isolated as a gummy foam. LCMS (Method r, Table 7)
R.sub.t=0.85 min; MS (DCI+) m/z=345.0 [M+NH.sub.4+]. .sup.1H NMR
(500 MHz, DMSO-d6) .delta. 10.04 (s, 1H), 9.86 (s, 1H), 9.25 (s,
1H), 7.37 (s, 1H), 7.34-7.28 (m, 2H), 7.27-7.20 (m, 2H), 7.14 (t,
J=7.8 Hz, 1H), 6.82 (dt, J=7.7, 1.2 Hz, 1H), 3.89 (s, 2H), 1.45 (s,
9H).
Step 7: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)-3-hydrox-
yphenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b-
,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3-
]dioxol-4(2H)-one
##STR00715##
[0988] Triflic acid (0.060 mL, 0.680 mmol) was added drop-wise to a
0.degree. C. slurry of
(6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-
-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-
-3H-cyclopenta[a]phenanthren-3-one (0.056 g, 0.136 mmol),
tert-butyl (3-(4-formyl-2-hydroxybenzyl)phenyl)carbamate (0.049 g,
0.150 mmol) and MgSO.sub.4 (0.049 g, 0.408 mmol) in MeCN (1.5 mL),
maintaining a reaction temperature of <5.degree. C. After 30 min
the reaction mixture was diluted with EtOAc (15 mL), and was washed
sequentially with a saturated aqueous solution of NaHCO.sub.3 (5
mL.times.2), and then with a saturated aqueous solution of brine (3
mL). The organic phase was dried (Na.sub.2SO.sub.4) and solvent was
removed under reduced pressure. Purification by reverse phase HPLC
on a Phenomenex C18(2) 10 micron column (250.times.30 mm column). A
gradient of MeCN (A) and 0.1% formic acid in water (B) was used, at
a flow rate of 60 mL/min (0-5.0 min 15% A, 5.0-20.0 min linear
gradient 15-80% A, hold 5 min). Combined fractions were
concentrated under reduced pressure to remove volatile solvents,
and were then lyophilized to give the title compound as a white
amorphous solid (6.7 mg). LCMS (Method r, Table 7) R.sub.t=0.70
min; MS m/z=622.39 [M+H.sup.+]. .sup.1H NMR (501 MHz, DMSO-d6)
.delta. 9.51 (s, 1H), 7.25 (d, J=10.2 Hz, 1H), 6.96 (d, J=7.7 Hz,
1H), 6.89-6.81 (m, 2H), 6.75 (d, J=7.7 Hz, 1H), 6.37-6.24 (m, 4H),
6.11 (s, 1H), 5.63 (ddd, J=49.2, 11.2, 6.4 Hz, 1H), 5.50 (d, J=4.3
Hz, 1H), 5.30 (s, 1H), 5.07 (s, 1H), 4.91 (d, J=4.8 Hz, 1H), 4.85
(s, 2H), 4.47 (d, J=19.3 Hz, 1H), 4.21-4.14 (m, 2H), 3.70-3.60 (m,
2H), 2.69-2.50 (m, 1H), 2.26 (s, 1H), 2.31-2.16 (m, 1H), 2.07-1.94
(m, 1H), 1.68 (q, J=10.2, 8.9 Hz, 2H), 1.64-1.50 (m, 1H), 1.48 (s,
3H), 0.84 (s, 3H).
Example 8: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)-3-hydroxyph-
enyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12-
,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(2H)-one
##STR00716##
[0990] Triflic acid (0.35 mL1, 3.83 mmol) was added drop-wise to a
0.degree. C. slurry of
(8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)--
10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a-
]phenanthren-3-one (0.296 g, 0.786 mmol), tert-butyl
(3-(4-formyl-2-hydroxybenzyl)phenyl)carbamate (0.251 g, 0.767 mmol)
and MgSO.sub.4 (0.332 g, 2.76 mmol) in MeCN (3.0 mL), maintaining a
reaction temperature of <5.degree. C. After 40 min the reaction
was diluted with EtOAc (15 mL), and was washed sequentially with a
saturated aqueous solution of NaHCO.sub.3 (10 mL.times.2), and then
with a saturated aqueous solution of brine (5 mL). The organic
layer was dried over Na.sub.2SO.sub.4 and solvent was removed under
reduced pressure. Purification by chromatography (silica, 12 g)
eluting with a gradient of 0-10% MeOH/DCM gave the title compound
(238.4 mg, 0.407 mmol, 53% yield) as a white solid. A portion of
this material (ca. 79.1 mg) was further purified by reverse phase
HPLC on a Phenomenex C18(2) 10 micron column (250.times.30 mm
column). A gradient of MeCN (A) and 0.1% TFA in water (B) was used,
at a flow rate of 60 mL/min (0-5.0 min 15% A, 5.0-20 min linear
gradient 15-60% A, hold 2 min). Combined fractions were
concentrated under reduced pressure to remove volatile solvents,
and the resulting solution was frozen and lyophilized to give the
title compound as an off-white solid (43.4 mg). LCMS (Method r,
Table 7) R.sub.t=0.73 min; MS m/z=586.2 [M+H.sup.+]. .sup.1H NMR
(501 MHz, DMSO-d6) .delta. 9.61 (s, 1H), 7.30 (d, J=10.1 Hz, 1H),
7.27-7.20 (m, 1H), 7.04 (dd, J=7.7, 2.9 Hz, 2H), 6.95-6.91 (m, 2H),
6.90 (d, J=1.6 Hz, 1H), 6.79 (dd, J=7.7, 1.6 Hz, 1H), 6.15 (dd,
J=10.1, 1.9 Hz, 1H), 5.92 (d, J=1.6 Hz, 1H), 5.29 (s, 1H), 4.88 (d,
J=5.1 Hz, 1H), 4.79 (s, 1H), 4.45 (d, J=19.4 Hz, 1H), 4.28 (q,
J=3.3 Hz, 1H), 4.15 (d, J=19.4 Hz, 1H), 3.82 (s, 2H), 2.59-2.49 (m,
1H), 2.30 (dt, J=13.0, 3.8 Hz, 1H), 2.16-2.05 (m, 1H), 2.07-1.98
(m, 1H), 1.75 (d, J=3.0 Hz, 2H), 1.73-1.54 (m, 3H), 1.38 (s, 3H),
1.05 (qd, J=12.9, 4.8 Hz, 1H), 0.97 (dd, J=11.2, 3.6 Hz, 1H), 0.84
(s, 3H).
Example 9: Synthesis of
(S)-N-(3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-Difluoro-7-h-
ydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,1-
2,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)-
benzyl)phenyl)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanami-
do)propanamido)propanamide
Step 1: Synthesis of (9H-Fluoren-9-yl)methyl
((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-di-
fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8-
a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dio-
xol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)ca-
rbamate
##STR00717##
[0992] HATU (1.3 g, 3.41 mmol) and 2,6-lutidine (0.4 mL, 3.43 mmol)
were added to a room temperature suspension of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2-
,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b-
,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(-
2H)-one (1.0327 g, 1.705 mmol), and
(S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)propa-
noic acid (0.782 g, 2.046 mmol) in THF (11.5 mL). After 3 hours at
room temperature, the reaction was diluted with EtOAc (16 mL), then
washed sequentially with a 1N aqueous solution of HCl (4
mL.times.3), and a saturated aqueous solution of brine (4 mL).
Purification by chromatography (silica, 40 g) eluting with a
gradient of 75-100% EtOAc/heptanes gave the title compound (0.926
g, 0.955 mmol, 56% yield). LC-MS (Method r, Table 7) R.sub.t=1.01
min, m/z=970.18 [M+H.sup.+]. .sup.1H NMR (500 MHz, DMSO-d6) .delta.
9.85 (d, J=5.6 Hz, 1H), 8.08 (d, J=7.3 Hz, 1H), 7.89 (dd, J=7.5,
1.0 Hz, 2H), 7.76-7.69 (m, 2H), 7.55 (d, J=7.4 Hz, 1H), 7.49-7.16
(m, 13H), 6.94-6.88 (m, 1H), 6.30 (ddd, J=10.1, 3.7, 1.9 Hz, 1H),
6.14 (dt, J=2.6, 1.2 Hz, 1H), 5.74-5.55 (m, 1H), 5.53 (dt, J=5.0,
2.5 Hz, 1H), 5.12 (t, J=6.0 Hz, 1H), 4.95 (d, J=5.1 Hz, 1H), 4.52
(dd, J=19.4, 6.2 Hz, 1H), 4.38 (p, J=7.0 Hz, 1H), 4.32-4.16 (m,
5H), 4.09 (p, J=6.9 Hz, 1H), 3.88 (d, J=10.9 Hz, 2H), 2.65-2.60 (m,
1H), 2.33-2.20 (m, 1H), 2.05 (d, J=13.5 Hz, 1H), 1.77-1.63 (m, 3H),
1.50 (s, 3H), 1.28 (d, J=7.1 Hz, 3H), 1.23 (d, J=7.1 Hz, 4H), 0.88
(d, J=12.6 Hz, 3H).
Step 2: Synthesis of
(S)-2-amino-N-((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2-
,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b-
,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1-
,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)propanamide
##STR00718##
[0994] Diethylamine (0.5 mL, 4.79 mmol) was added to a room
temperature solution of (9H-fluoren-9-yl)methyl
((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-di-
fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8-
a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dio-
xol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)ca-
rbamate (1.18 g, 1.216 mmol) in THF (6.0 mL). After 2 h, MTBE (10
mL) was added, which resulted in the immediate precipitation of a
yellow solid. This slurry was stirred for 90 min, filtered, and
washed with MTBE (5 mL.times.3) to give a yellow solid (802.7 mg).
This material was purified further by reverse phase HPLC on a
Phenomenex C18(2) 10 micron column (250.times.50 mm column). A
gradient of MeCN (A) and 0.1% formic acid in water (B) was used, at
a flow rate of 90 mL/min (0-5.0 min 15% A, 5.0-20.0 min linear
gradient 15-75% A, hold 2 min, 22.0-22.5 min linear gradient from
75-95%, hold for 4 min). Combined fractions were concentrated under
reduced pressure to dryness and then dried overnight in the vacuum
oven at 50.degree. C. LC-MS (Method r, Table 7) R.sub.t=0.76 min,
m/z=748.5 [M+H.sup.+]. .sup.1H NMR indicates that the title
compound is an approximately 1:1 mixture with
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-aminopr-
opanamido)
propanamido)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimeth-
yl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4-
,5]indeno[1,2-d][1,3]dioxole-8b-carboxylic acid. (0.170 g total,
0.115 mol and 10% yield of each compound). .sup.1H NMR (500 MHz,
DMSO-d) .delta. 10.00 (d, J=3.3 Hz, 2H), 8.42 (d, J=34.2 Hz, 2H),
8.30 (s, 1H), 7.48-7.37 (m, 3H), 7.38-7.29 (m, 5H), 7.31-7.15 (m,
8H), 6.92 (d, J=7.6 Hz, 2H), 6.28 (ddd, J=10.3, 6.6, 1.9 Hz, 3H),
6.12 (d, J=3.7 Hz, 3H), 5.77-5.53 (m, 3H), 5.45 (d, J=7.7 Hz, 3H),
5.04-4.99 (m, 1H), 4.94 (d, J=5.1 Hz, 1H), 4.50 (d, J=19.4 Hz, 1H),
4.40 (s, 3H), 4.23-4.12 (m, 2H), 3.54 (dq, J=17.6, 6.9 Hz, 1H),
2.71-2.56 (m, 1H), 2.30-2.15 (m, 1H), 2.03 (d, J=14.2 Hz, 2H), 1.94
(d, J=14.3 Hz, 1H), 1.84 (d, J=14.1 Hz, 1H), 1.76-1.59 (m, 7H),
1.49 (d, J=2.6 Hz, 8H), 1.39-1.10 (m, 13H), 1.00 (s, 4H), 0.86 (s,
3H).
Step 3: Synthesis of
(S)-N-(3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-Difluoro-7-h-
ydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,1-
2,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)-
benzyl)phenyl)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanami-
do)propanamido)propanamide
##STR00719##
[0996] Diisopropylethylamine (0.1 mL, 0.573 mmol) was added to a
room temperature solution of
(S)-2-amino-N-((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2-
,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b-
,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho [2',1':
4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)
amino)-1-oxopropan-2-yl)propanamide (0.170 g, 0.227 mmol) and
N-succinimidyl 3-maleimidopropionate (0.0691 g, 0.260 mmol) in DMF
(2.5 mL). After 30 min, the pH of the reaction mixture was adjusted
to 4-5 by drop-wise addition of a 7% solution of TFA in water (1.2
mL). The crude mixture was purified by reverse phase HPLC on a
Phenomenex C18(2) 10 micron column (250.times.50 mm column). A
gradient of MeCN (A) and 0.1% TFA in water (B) was used, at a flow
rate of 90 mL/min (0-5.0 min 15% A, 5.0-20 min linear gradient
15-85% A, hold 2 min). Combined fractions were concentrated under
reduced pressure to remove volatile solvents, and the resulting
solution was frozen and lyophilized to give a white solid (85.2 mg,
0.0473 mmol, 21% yield). LC-MS (Method R, Table 7) R.sub.t=0.82
min, m/z=899.92 [M+H.sup.+]. .sup.1H NMR data was consistent with a
1:1 mixture of the title compound
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-(3-(2,5-
-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)propanamido)propanamido)benz-
yl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a-
,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-
e-8b-carboxylic acid (see example 10 for an alternative preparation
of the title compound, which avoids this mixture). MS analysis
confirms that this material is a mixture of two compounds with
m/z=899.1 [M+H*] and m/z=885.0 [M+H.sup.+]. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 9.71 (s, 2H), 8.16 (d, J=7.1 Hz, 2H), 8.03
(d, J=7.3 Hz, 2H), 7.49-7.29 (m, 9H), 7.30-7.13 (m, 9H), 6.96 (s,
3H), 6.92-6.85 (m, 2H), 6.27 (dt, J=10.1, 1.9 Hz, 2H), 6.11 (d,
J=2.3 Hz, 2H), 5.74-5.53 (m, 2H), 5.46 (d, J=23.9 Hz, 4H), 4.93 (d,
J=5.0 Hz, 1H), 4.32 (p, J=7.1 Hz, 2H), 4.27-4.13 (m, 3H), 4.17 (s,
3H), 3.59 (t, J=7.3 Hz, 4H), 2.69-2.53 (m, 2H), 2.38 (t, J=7.3 Hz,
4H), 2.28 (s, 3H), 2.22 (s, 1H), 2.08-1.98 (m, 1H), 1.98-1.90 (m,
1H), 1.83-1.68 (m, 2H), 1.69 (s, 2H), 1.66 (s, 2H), 1.48 (d, J=3.7
Hz, 8H), 1.25 (d, J=7.0 Hz, 6H), 1.15 (d, J=7.1 Hz, 6H), 0.99 (s,
3H), 0.84 (s, 3H).
Example 10: Synthesis of
(S)-N-(3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-Difluoro-7-h-
ydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,1-
2,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)-
benzyl)phenyl)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanami-
do)propanamido)propanamide
Step 1: Synthesis of Tert-Butyl
((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,
8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)--
6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-nap-
htho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopr-
opan-2-yl)amino)-1-oxopropan-2-yl)carbamate
##STR00720##
[0998] HATU (610 mg, 1.605 mmol) and 2,6-lutidine (0.3 mL, 2.58
mmol) were added to a room temperature mixture of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2-
,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b-
,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(-
2H)-one (648.1 mg, 1.070 mmol), and
(S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanoic acid
(334 mg, 1.284 mmol) in THF (11.5 mL). After 9 hours the reaction
was diluted with EtOAc (16 mL), then washed with a 1N aqueous
solution of HCl (4 mL.times.3), followed by a saturated aqueous
solution of brine (4 mL). Purification by chromatography (silica,
40 g) eluting with a gradient of 0-10% MeOH/DCM gave the title
compound as a yellow foam (773.7 mg, 0.912 mmol, 85% yield). LC-MS
(Method r, Table 7) R.sub.t=0.92 min, m/z=848.53 [M+H.sup.+].
Step 2: Synthesis of
(S)-2-amino-N-((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2-
,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b-
,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1-
,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)propanamide
##STR00721##
[1000] TFA (1.97 mL, 25.6 mmol) was added drop-wise to a room
temperature solution of tert-butyl
((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-di-
fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8-
a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dio-
xol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)ca-
rbamate (0.7683 g, 0.906 mmol) in DCM (6.0 mL). After 50 min
solvent was removed under reduced pressure to give a brown syrup.
The residue was dissolved in 1:1 DMSO:MeOH (12 mL) and purified by
reverse phase HPLC on a Phenomenex C18(2) 10 micron column
(250.times.50 mm column). A gradient of MeCN (A) and 0.1% TFA in
water (B) was used, at a flow rate of 90 mL/min (0-5.0 min 15% A,
5.0-20 min linear gradient 15-75% A, hold 2 min, 22.0-22.5 min
linear gradient 75-95% A, hold 4 min). Combined fractions were
concentrated under reduced pressure to dryness and the residue was
dried overnight in the vacuum oven at 50.degree. C. to give the
title compound (230 mg, 0.308 mmol, 34% yield. LC-MS (Method r,
Table 7) major acetal isomer R.sub.t=0.73 min, m/z=748.78
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 10.01 (s,
1H), 8.62 (d, J=7.2 Hz, 1H), 8.04 (d, J=5.4 Hz, 3H), 7.46-7.31 (m,
4H), 7.31-7.13 (m, 4H), 6.91 (d, J=7.6 Hz, 1H), 6.27 (dd, J=10.2,
1.9 Hz, 1H), 6.11 (s, 1H), 5.76-5.47 (m, 2H), 5.43 (s, 1H), 4.93
(d, J=4.6 Hz, 1H), 4.49 (d, J=19.5 Hz, 1H), 4.42 (q, J=7.1 Hz, 1H),
4.23-4.13 (m, 2H), 2.72-2.54 (m, 1H), 2.33-2.16 (m, 2H), 2.02 (dt,
J=13.6, 3.6 Hz, 1H), 1.69 (h, J=5.9, 5.1 Hz, 3H), 1.48 (s, 4H),
1.33 (d, J=7.0 Hz, 3H), 1.30 (d, J=7.1 Hz, 3H), 0.85 (s, 3H).
Step 3: Synthesis of
(S)-N-(3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-Difluoro-7-h-
ydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,1-
2,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)-
benzyl)phenyl)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanami-
do)propanamido)propanamide
##STR00722##
[1002] Diisopropylethylamine (0.1 mL, 0.573 mmol) was added to a
room temperature solution of
(S)-2-amino-N-((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2-
,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b-
,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1-
,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)propanamide
(0.220 g, 0.294 mmol) and N-succinimidyl 3-maleimidopropionate
(0.086 g, 0.324 mmol) in DMF (2.8 mL). After 30 min the pH of the
reaction mixture was adjusted to 4-5 by drop-wise addition of a 7%
solution of TFA in water (1.0 mL). The crude mixture was purified
by reverse phase HPLC on a Phenomenex C18(2) 10 micron column
(250.times.50 mm column). A gradient of MeCN (A) and 0.1% TFA in
water (B) was used, at a flow rate of 90 mL/min (0-5.0 min 15% A,
5.0-20 min linear gradient 15-85% A, hold 2 min). Combined
fractions were concentrated under reduced pressure to remove
volatile solvents, and the resulting solution was frozen and
lyophilized to give a white solid (175.2 mg, 0.195 mmol, 66%
yield). LC-MS (Method r, Table 7) R.sub.t=0.82 min, m/z=899.87
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.70 (s,
1H), 8.14 (d, J=7.0 Hz, 1H), 8.01 (d, J=7.2 Hz, 1H), 7.47-7.35 (m,
2H), 7.32 (d, J=8.1 Hz, 2H), 7.26-7.10 (m, 4H), 6.95 (s, 1H), 6.87
(dt, J=7.6, 1.3 Hz, 1H), 6.26 (dd, J=10.2, 1.9 Hz, 1H), 6.09 (d,
J=2.0 Hz, 1H), 5.72-5.51 (m, 1H), 5.48 (s, 1H), 5.41 (s, 1H), 4.91
(d, J=4.9 Hz, 1H), 4.47 (d, J=19.4 Hz, 1H), 4.30 (p, J=7.1 Hz, 1H),
4.25-4.11 (m, 3H), 3.85 (s, 2H), 3.57 (t, J=7.3 Hz, 2H), 2.71-2.48
(m, 1H), 2.36 (dd, J=8.0, 6.7 Hz, 2H), 2.23 (ddt, J=25.1, 12.2, 6.6
Hz, 2H), 2.01 (dt, J=13.7, 3.7 Hz, 1H), 1.75-1.57 (m, 3H), 1.48 (p,
J=11.9 Hz, 1H), 1.46 (s, 3H), 1.24 (d, J=7.2 Hz, 3H), 1.13 (d,
J=7.2 Hz, 3H), 0.83 (s, 3H).
Example 11: Synthesis of
(S)-N-((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2-
,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b-
,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1-
,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-
-yl)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-(1H-imidazo-
l-4-yl)propanamide
Step 1: Synthesis of
(S)-2-((tert-Butoxycarbonyl)amino)-3-(1H-imidazol-5-yl)propanoic
Acid, 2 Hydrochloric Acid
##STR00723##
[1004] To a solution of (S)-2-amino-3-(1H-imidazol-5-yl)propanoic
acid (1.55 g, 9.99 mmol) in water (40 mL) and 1,4-dioxane (10 mL)
at 0.degree. C. were added NaOH (10 mL, 19.98 mmol) and
BOC-anhydride (2.319 mL, 9.99 mmol). The resulting mixture was
stirred at 23.degree. C. for 4 h. Then the mixture was acidified
with HCl solution to pH 5, and washed with EtOAc (3.times.30 mL).
Then the inorganic layer was freeze-dried to give the title
compound (including NaCl)) (4.449 g, 9.90 mmol, 99% yield) as a
white solid. LCMS (Method m, Table 7) R.sub.t=1.22 min, m/z 256.2
(M+1)+.
Step 2: Synthesis of Tert-Butyl
((S)-1-(((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,
8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)--
6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-nap-
htho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopr-
opan-2-yl)amino)-1-oxopropan-2-yl)amino)-3-(1H-imidazol-5-yl)-1-oxopropan--
2-yl)carbamate
##STR00724##
[1006] To a solution of
(S)-2-((tert-butoxycarbonyl)amino)-3-(1H-imidazol-5-yl)propanoic
acid, 2hydrochloric acid (170 mg, 0.197 mmol),
(S)-2-((tert-butoxycarbonyl)amino)-3-(1H-imidazol-5-yl)propanoic
acid, 2 hydrochloric acid (443 mg, 0.986 mmol) in THF (20 mL) at
0.degree. C. were added DIPEA (0.345 mL, 1.973 mmol) and HATU (90
mg, 0.237 mmol), DMAP (31.3 mg, 0.256 mmol) and the resulting
mixture was stirred at 0.degree. C. for 10 min, and gradually
warmed to 25.degree. C. for 16 h. After that, the mixture was
concentrated to give the residue, which was purified by
DCM/MeOH(10:1) by silica gel to obtain the title compound (194 mg,
0.138 mmol, 69.9% yield) as a yellow solid. LCMS (Method m, Table
7) R.sub.t=1.72 min, m/z 985.3 (M+1).sup.+; .sup.1H NMR: (400 MHz,
DMSO-d6) .delta. ppm: 0.82-0.89 (m, 10H), 1.12-1.18 (m, 9H), 1.23
(s, 9H), 1.68-1.71 (m, 2H), 2.20-2.33 (m, 2H), 3.86-3.88 (m, 2H),
4.18-4.29 (m, 4H), 4.36-4.39 (m, 1H), 4.49-4.54 (m, 1H), 4.94 (d,
J=4.4 Hz, 1H), 5.13 (bs, 1H), 5.45 (s, 1H), 5.57-5.74 (m, 2H), 6.12
(s, 1H), 6.29 (d, J=10.0 Hz, 1H), 6.91 (d, J=8.0 Hz, 1H), 7.01 (d,
J=8.0 Hz, 1H), 7.10-7.25 (m, 4H), 7.29-7.37 (m, 3H), 7.44-7.49 (m,
2H), 8.06 (d, J=6.0 Hz, 1H).
Step 3: Synthesis of
(S)-2-amino-N-((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,
10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-di-
methyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',-
1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-y-
l)amino)-1-oxopropan-2-yl)-3-(1H-imidazol-5-yl)propanamide,
3trifluoroacetic Acid
##STR00725##
[1008] To a solution of tert-butyl
((S)-1-(((S)-1-(((S)-1-((3-(4-((2S,6aS, 6bR,7S,8aS, 8bS,10R,1l
aR,12aS,12b S)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxy acetyl)-6a,
8a-dimethyl-4-oxo-2,4,6a, 6b, 7,8,8a,
8b,11a,12,12a,12b-dodecahydro-1H-naphtho
[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropa-
n-2-yl)amino)-1-oxopropan-2-yl)amino)-3-(1H-imidazol-5-yl)-1-oxopropan-2-y-
l)carbamate (120 mg, 0.122 mmol) in DCM (3 mL) was added TFA (0.6
mL, 7.79 mmol), and the reaction mixture was stirred at 20.degree.
C. for 1 hour. After that, the mixture was diluted with DCM,
concentrated in vacuo at about 25.degree. C. to give the title
compound (149 mg, 0.103 mmol, 84.90% yield) as a yellow solid. LCMS
(Method m, Table 7) R.sub.t=1.64 min, m/z 885.3 (M+1)+.
Step 4: Synthesis of
(S)-N-((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2-
,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b-
,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1-
,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-
-yl)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-(1H-imidazo-
l-4-yl)propanamid
##STR00726##
[1010] DIPEA (0.106 mL, 0.607 mmol) was added to the solution of
(S)-2-amino-N-((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS-
,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2-
,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[-
1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxo-
propan-2-yl)-3-(1H-imidazol-5-yl)propanamide, 3trifluoroacetic acid
(149 mg, 0.121 mmol) and 2,5-dioxopyrrolidin-1-yl
3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (48.5 mg, 0.182
mmol) in DMF (2 mL) at 0.degree. C., and then the mixture was
stirred at room temperature for 2 h. The reaction mixture was
purified by prep-HPLC (Mobile Phase:A=0.05% TFA in water, B=MeCN;
Flow Rate: 2 mL/min) to afford the title compound (11.4 mg, 9.02
mmol, 7.43% yield) as a white solid. LCMS (Method m, Table 7)
R.sup.7b=1.62 min, m/z 1058.3 (M+Na).sup.+; .sup.1H NMR: (400 MHz,
DMSO-d) 6 ppm: 0.86 (s, 3H), 1.24-1.29 (m, 9H), 1.46-1.54 (m, 3H),
1.68-1.76 (m, 2H), 1.98-2.06 (m, 2H), 2.20-2.33 (m, 2H), 2.40-2.44
(m, 2H), 2.60-2.68 (m, 1H), 2.88-2.94 (m, 1H), 3.00-3.05 (m, 1H),
3.57 (t, J=7.4 Hz, 2H), 3.89 (s, 2H), 4.18-4.39 (m, 4H), 4.49-4.60
(m, 2H), 4.95 (d, J=4.8 Hz, 1H), 5.46 (s, 1H), 5.54-5.74 (m, 2H),
6.13 (s, 1H), 6.30 (d, J=10.4 Hz, 1H), 6.92 (d, J=7.6 Hz, 1H), 7.00
(s, 2H), 7.20-7.28 (m, 4H), 7.35-7.46 (m, 5H), 8.16 (d, J=6.4 Hz,
1H), 8.23 (d, J=7.2 Hz, 1H), 8.31 (d, J=8.4 Hz, 1H), 8.96 (s, 1H),
9.89 (s, 1H).
Example 12: Synthesis of
(S)-5-(((S)-1-((4-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-di-
fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8-
a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dio-
xol-10-yl)phenyl)thio)phenyl)amino)-1-oxopropan-2-yl)amino)-4-(3-(2,5-diox-
o-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-5-oxopentanoic Acid (Cpd.
No. 81)
Step 1: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((4-Aminophenyl)thio)phe-
nyl)-8b-(2-((tert-butyldimethylsilyl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6-
a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,-
5]indeno[1,2-d][1,3]dioxol-4(2H)-one
##STR00727##
[1012] To a stirred solution of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((4-aminophenyl)thio)phe-
nyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8-
,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dio-
xol-4(2H)-one (62.4 mg, 0.1 mmol) and imidazole (34.0 mg, 0.500
mmol) in DCM was added TBS-Cl (45.2 mg, 0.300 mmol) at 0.degree.
C., After stirring was continued for 30 min at the same
temperature, the mixture was allowed to warm to room temperature
and stirred for 2 h. the reaction mixture was diluted with EtOAc(10
mL), washed with water (2.times.10 mL) and brine (1.times.1 mL),
dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo.
The residue was purified by column (EA:PE=1:10-1:1) to give the
product
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((4-aminophenyl)thio)
phenyl)-8b-(2-((tert-butyldimethylsilyl)oxy)acetyl)-2,6b-difluoro-7-hydro-
xy-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1-
:4,5]indeno[1,2-d][1,3]dioxol-4(2H)-one (50 mg, 0.068 mmol, 67.8%
yield). LCMS (Method m, Table 7) R.sub.t=2.144 min, m/z 738
(M+H).
Step 2: Synthesis of Tert-Butyl
((S)-1-((4-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((tert-b-
utyldimethylsilyl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-
-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]inden-
o[1,2-d][1,3]dioxol-10-yl)phenyl)thio)phenyl)amino)-1-oxopropan-2-yl)carba-
mate
##STR00728##
[1014] To a stirred solution of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((4-aminophenyl)thio)phe-
nyl)-8b-(2-((tert-butyldimethylsilyl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6-
a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,-
5] indeno[1,2-d][1,3]dioxol-4(2H)-one (0.148 g, 0.2 mmol) and
(S)-2-((tert-butoxycarbonyl) amino)propanoic acid (0.076 g, 0.400
mmol) in DCM (3 mL) was added pyridine (0.162 mL, 2.000 mmol),
followed by POCl.sub.3 (0.075 mL, 0.800 mmol) in dropwise. The
reaction mixture was stirred for 1 hour at ambient temperature,
then concentrated in vacuo, and the residue was purified by column
(EA:PE=1:10-9:1) to give tert-butyl
((S)-1-((4-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((tert-b-
utyldimethylsilyl)
oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a-
,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]diox-
ol-10-yl)phenyl)thio)phenyl)amino)-1-oxopropan-2-yl)carbamate
(0.073 g, 0.080 mmol, 40% yield) as a semi-solid. LCMS (Method m,
Table 7) R.sub.t=2.156 min, m/z 909 (M+H).
Step 3: Synthesis of
(S)-2-amino-N-(4-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-dif-
luoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a-
,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]diox-
ol-10-yl)phenyl)thio)phenyl)propanamide
##STR00729##
[1016] To a stirred solution of tert-butyl
((S)-1-((4-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,
12aS,12bS)-8b-(2-((tert-butyldimethylsilyl)oxy)acetyl)-2,6b-difluoro-7-hy-
droxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro--
1H-naphtho[2',1':4,5]
indeno[1,2-d][1,3]dioxol-10-yl)phenyl)thio)phenyl)amino)-1-oxopropan-2-yl-
)carbamate (0.091 g, 0.1 mmol) in methylene chloride (1 mL) was
added TFA (1 mL, 12.98 mmol), and the solution was stirred for 2
hours at ambient temperature, then concentrated in vacuo to give
the product
(S)-2-amino-N-(4-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-dif-
luoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a-
,
8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dio-
xol-10-yl)phenyl)thio)phenyl)propanamide (7.21 g, 10.38 mmol, 80%
yield). LCMS (Method m, Table 7) R.sub.t=1.653 min, m/z 695
(M+H).
Step 4: Synthesis of
(S)-5-(tert-Butoxy)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamid-
o)-5-oxopentanoic Acid
##STR00730##
[1018] To a stirred solution of
(S)-2-amino-5-(tert-butoxy)-5-oxopentanoic acid (406 mg, 2 mmol)
and 2,5-dioxopyrrolidin-1-yl
3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (532 mg, 2.000
mmol) in dimethyl formamide (2 mL) was added DIPEA (0.524 mL, 3.00
mmol). After stirring was continued for 2 h at room temperature,
the reaction mixture was diluted with EtOAc (10 mL), washed with
water (2.times.10 mL) and brine (.times.10 mL), dried over
Na.sub.2SO.sub.4, filtered and concentrated in vacuo, and the
residue was purified by column (MeOH/DCM=0:10-1:10) to give the
title compound (209 mg, 0.590 mmol, 29.5% yield) as a yellow oil.
LCMS (Method m, Table 7) R.sub.t=1.490 min, m/z 377 (M+Na).
Step 5: Synthesis of (S)-tert-butyl
5-(((S)-1-((4-((4-((2S,6aS,6bR,7S,8aS,
8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8-
a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho-
[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenyl)thio)phenyl)amino)-1-oxop-
ropan-2-yl)amino)-4-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)--
5-oxopentanoate
##STR00731##
[1020] A solution of
(S)-2-amino-N-(4-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-dif-
luoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a-
,8b,
11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dio-
xol-10-yl)phenyl) thio)phenyl)propanamide (40 mg, 0.058 mmol),
(S)-5-(tert-butoxy)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamid-
o)-5-oxopentanoic acid (30.6 mg, 0.086 mmol), HATU (32.8 mg, 0.086
mmol) and DIPEA (0.030 mL, 0.173 mmol) in dimethyl formamide (2 mL)
was stirred overnight at room temperature, and diluted with EtOAc
(10 mL), washed with water (2.times.10 mL) and brine (1.times.10
mL), dried over Na.sub.2SO.sub.4, filtered and evaporated in
reduced pressure. The residue was purified by column chromatography
(MeOH/DCM=0:10; 1:10) to give the title compound (30 mg, 0.029
mmol, 50.5% yield). LCMS (Method m, Table 7) R.sub.t=2.051 min, m/z
1031 (M+H).
Step 6: Synthesis of
(S)-5-(((S)-1-((4-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-Di-
fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8-
a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dio-
xol-10-yl)phenyl)thio)phenyl)amino)-1-oxopropan-2-yl)amino)-4-(3-(2,5-diox-
o-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-5-oxopentanoic Acid
##STR00732##
[1022] To a stirred solution of (S)-tert-butyl
5-(((S)-1-((4-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,
11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimeth-
yl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4-
,5]indeno[1,2-d][1,3]dioxol-10-yl)phenyl)thio)phenyl)amino)-1-oxopropan-2--
yl)amino)-4-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-5-oxopen-
tanoate (10.31 mg, 0.01 mmol) in DCM (0.5 mL) was added TFA (0.5
mL, 6.49 mmol). After stirring was continued for 2 h, the reaction
mixture was concentrated in vacuo to give the title compound (6.83
mg, 7.00 .mu.mol, 70% yield). LCMS (Method m, Table 7)
R.sub.t=1.875 min, m/z 975 (M+H).
Example 13: Synthesis of
N-(4-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydro-
xy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12-
a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phen-
oxy)phenyl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,-
12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-amide
Step 1: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(4-Aminophenoxy)phenyl)--
8b-(2-((tert-butyldimethylsilyl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a--
dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]ind-
eno[1,2-d][1,3]dioxol-4(2H)-one
##STR00733##
[1024] To a stirred solution of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(4-aminophenoxy)phenyl)--
2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8-
b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]
dioxol-4(2H)-one (290 mg, 0.477 mmol) and imidazole (162 mg, 2.386
mmol) in CH.sub.2Cl.sub.2 (10 mL) was added TBS-Cl (216 mg, 1.432
mmol) at 0.degree. C., After stirring was continued for 30 min at
the same temperature, the mixture was allowed to warm to room
temperature and stirred for 2 h. the reaction mixture was diluted
with EtOAc(10 mL), washed with water (2.times.10 mL) and brine
(1.times.10 mL), dried over Na.sub.2SO.sub.4, filtered and
concentrated in vacuo. The residue was purified by column
(EA:PE=1:10-9:1) to give title compound (300 mg, 0.416 mmol, 87%
yield). LCMS (Method m, Table 7) R.sub.t=1.812 min, m/z 722
(M+H).
Step 2: Synthesis of Tert-Butyl
(39-((4-(4-((2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-8b-(2-((tert-Butyldime-
thylsilyl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,-
6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d]-
[1,3]dioxol-10-yl)phenoxy)phenyl)amino)-39-oxo-3,6,9,12,15,18,21,24,27,30,-
33,36-dodecaoxanonatriacontyl)carbamate
##STR00734##
[1026] To a stirred solution of
(2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-10-(4-(4-aminophenoxy)phenyl)-8b-(-
2-((tert-butyldimethylsilyl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-6a,8a-dime-
thyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]
indeno[1,2-d][1,3]dioxol-4(2H)-one (144 mg, 0.2 mmol) and
2,2-dimethyl-4-oxo-3,8,11,14,17,20,23,26,29,32,35,38,41-tridecaoxa-5-azat-
etratetracontan-44-oic acid (144 mg, 0.200 mmol) in
CH.sub.2Cl.sub.2 (3 mL) was added pyridine (0.162 mL, 2.000 mmol),
followed by POCl.sub.3 (0.037 mL, 0.400 mmol) in dropwise. The
reaction mixture was stirred for 1 hour at ambient temperature,
then concentrated in vacuo, and the residue was purified by column
chromatography (MeOD:DCM=0:10-1:10) to give the title compound (120
mg, 0.084 mmol, 42.2% yield) as a semi-solid. LCMS (Method m, Table
7) R.sub.t=2.065 min, m/z 1422 (M+H-100).
Step 3: Synthesis of
1-amino-N-(4-(4-((2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-Difluoro-7-h-
ydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,1-
2,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)-
phenoxy)phenyl)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-3-
9-amide
##STR00735##
[1028] To a stirred solution of tert-butyl
(39-((4-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,
12bS)-8b-(2-((tert-butyldimethylsilyl)oxy)acetyl)-2,6b-difluoro-7-hydroxy-
-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-na-
phtho[2',1':4,5]
indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)phenyl)amino)-39-oxo-3,6,9,12,15,1-
8,21,24,27,30,33,36-dodecaoxanonatriacontyl)carbamate (190 mg,
0.134 mmol) in methylene chloride (0.5 mL) was added TFA (0.1 mL,
1.298 mmol), and the solution was stirred for 2 hours at ambient
temperature, then concentrated in vacuo to give the title compound
(100 mg, 0.083 mmol, 62.0% yield). LCMS (Method m, Table 7)
R.sub.t=1.521 min, m/z 1208 (M+H).
Step 4: Synthesis of
N-(4-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydro-
xy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12-
a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phen-
oxy)phenyl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,-
12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-amide
##STR00736##
[1030] DIPEA (6.99 .mu.L, 0.040 mmol) was added to a solution of
1-amino-N-(4-(4-((2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-h-
ydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,1-
2,12a,12b-dodecahydro-1H-naphtho
[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phenoxy)phenyl)-3,6,9,12,15,18,-
21,24,27,30,33,36-dodecaoxanonatriacontan-39-amide (0.024 g, 0.02
mmol) and 2,5-dioxopyrrolidin-1-yl
3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (7.99 mg, 0.030
mmol) in N,N-dimethylformamide (1 mL), and the mixture was stirred
at room temperature for 2 h. The reaction mixture was diluted with
EtOAc (10 mL) and washed with water (2.times.10 mL),
brine(1.times.10 mL), dried over Na.sub.2SO.sub.4, filtered and
concentrated in vacuo. The residue was purified by column
chromatography (MeOH/DCM=0:100-10:100) to give the title compound
(0.011 g, 8.20 .mu.mol, 41% yield). LCMS (Method m, Table 7)
R.sub.t=1.679 min, m/z 1359 (M+H).
Example 14: Synthesis of 2,5-Dioxopyrrolidin-1-yl
6-(((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-
-difluoro-7-hydroxy-8b-(2-hydroxy
acetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahyd-
ro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino-
)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-6-oxohexanoate
(Cpd. No. 78)
##STR00737##
[1032] A solution of
(S)-2-amino-N-((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2-
,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b-
,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1-
,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)propanamide
(0.060 g, 0.080 mmol) and N,N-diisopropylethylamine (0.14 mL, 0.802
mmol) in DMSO (1 mL) was added drop-wise to a room temperature
solution of bis(2,5-dioxopyrrolidin-1-yl) adipate (0.273 g, 0.802
mmol) in DMSO (3.5 mL). After 60 min the reaction was quenched by
addition of a 7 wt % solution of TFA in water to bring the reaction
mixture to a pH of 4-5. The crude reaction mixture was purified by
reverse phase HPLC on a Phenomenex C18(2) 5 micron column
(250.times.21 mm column). A gradient of MeCN (A) and 0.1% formic
acid in water (B) was used, at a flow rate of 30 mL/min (0-1.0 min
15% A, 1.0-11 min linear gradient 15-80% A, hold 1 min). Combined
fractions were concentrated under reduced pressure to remove
volatile solvents, and the resulting solution was frozen and
lyophilized to give the title compound as a white solid (21.2 mg,
0.022 mmol, 27% yield). LCMS (Method r, Table 7) R.sub.t=0.80 min,
m/z=1005.1 [M+MeOH+H.sup.+]. .sup.1H NMR (DMSO) .delta. 0.84 (s,
3H), 1.17 (d, J=7.1 Hz, 3H), 1.25 (d, J=7.1 Hz, 3H), 1.48 (s, 4H),
1.57 (q, J=6.2 Hz, 4H), 1.68 (dq, J=13.7, 6.3 Hz, 3H), 1.99-2.06
(m, 1H), 2.09-2.18 (m, 2H), 2.18-2.36 (m, 2H), 2.55-2.72 (m, 3H),
2.78 (s, 4H), 3.87 (s, 2H), 4.14-4.22 (m, 2H), 4.26 (p, J=7.1 Hz,
1H), 4.33 (p, J=7.1 Hz, 1H), 4.49 (d, J=19.4 Hz, 1H), 4.93 (d,
J=5.1 Hz, 1H), 5.43 (s, 1H), 5.49 (d, J=5.4 Hz, 1H), 5.54-5.75 (m,
1H), 6.11 (s, 1H), 6.28 (dd, J=10.2, 2.0 Hz, 1H), 6.89 (d, J=7.6
Hz, 1H), 7.17 (t, J=7.9 Hz, 1H), 7.23 (t, J=9.7 Hz, 3H), 7.34 (d,
J=7.8 Hz, 2H), 7.39 (s, 1H), 7.44 (d, J=8.1 Hz, 1H), 7.99 (d, J=7.2
Hz, 1H), 8.02 (d, J=7.3 Hz, 1H), 9.77 (s, 1H); MS (ESI-)
m/z=971.
Example 15: Synthesis of Tert-Butyl
((S)-1-(((S)-1-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-di-
fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8-
a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dio-
xol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-
-2-yl)carbamate
##STR00738##
[1034] HATU (106 mg, 0.280 mmol) and 2,6-lutidine (0.1 mL, 0.859
mmol) were added to a room temperature suspension of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2-
,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b-
,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(-
2H)-one (113 mg, 0.187 mmol) and
(tert-butoxycarbonyl)-L-valyl-L-alanine (53.8 mg, 0.187 mmol) in
THF (1.25 mL). After 8 h the reaction was diluted with EtOAc (16
mL), then washed sequentially with a 1M aqueous solution of HCl (4
mL.times.3), a saturated aqueous solution of NaHCO.sub.3 (4 mL),
and then a saturated aqueous solution of brine (4 mL). Solvent was
removed under reduced pressure and the product was purified by
chromatography (12 g silica), eluting with a gradient of 0-10%
MeOH/DCM to give the title compound (148.6 mg, 0.170 mmol, 91%
yield). LCMS (Method r, Table 7) R.sub.t=0.94 min, m/z=875.9
[M+H.sup.+]. .sup.1H NMR (DMSO-d) .delta. 9.85 (s, 1H), 7.99 (d,
J=7.1 Hz, 1H), 7.43 (dd, J=8.0, 1.7 Hz, 1H), 7.36-7.31 (m, 3H),
7.27-7.15 (m, 5H), 6.89 (d, J=7.5 Hz, 1H), 6.67 (d, J=8.8 Hz, 1H),
6.27 (dd, J=10.2, 1.9 Hz, 1H), 6.11 (s, 1H), 5.73-5.52 (m, 1H),
5.50 (dd, J=4.5, 1.7 Hz, 1H), 5.43 (s, 1H), 5.07 (t, J=5.9 Hz, 1H),
4.93 (d, J=4.8 Hz, 1H), 4.49 (dd, J=19.5, 6.4 Hz, 1H), 4.37 (t,
J=7.0 Hz, 1H), 4.25-4.12 (m, 2H), 3.87 (s, 2H), 3.80 (t, J=7.7 Hz,
1H), 2.73-2.53 (m, 1H), 2.23 (ddd, J=18.7, 11.9, 6.0 Hz, 2H),
2.08-1.99 (m, 1H), 1.93 (q, J=7.0 Hz, 1H), 1.77-1.59 (m, 3H), 1.48
(s, 3H), 1.35 (s, 9H), 1.25 (d, J=7.0 Hz, 3H), 0.89-0.74 (m,
9H).
Example 16: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((4-Aminophenyl)thio)phe-
nyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10-trimethyl-6a,6b-
,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3-
]dioxol-4(2H)-one and
(2S,6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((4-Aminophenyl)thio)phe-
nyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10-trimethyl-6a,6b-
,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3-
]dioxol-4(2H)-one
Step 1: Synthesis of 1-(4-((4-bromophenyl)thio)phenyl)ethanone
##STR00739##
[1036] 1-(4-Fluorophenyl)ethanone (2.19 mL, 18.04 mmol) was added
to a stirred solution of 4-bromobenzenethiol (3.1 g, 16.40 mmol)
and K.sub.2CO.sub.3 (2.72 g, 19.67 mmol) in DMF (45 mL), whereupon
the reaction was heated to 100.degree. C. for 20 min. The reaction
was cooled to ambient temperature, diluted with water (50 mL) and
extracted with EtOAc (3.times.50 mL). The combined organics were
dried (MgSO.sub.4) and solvents were removed under reduced
pressure. Purification by chromatography (silica, 120 g) eluting
with a gradient of 0-60% EtOAc/heptanes gave the title compound
(3.24 g, 10.55 mmol, 64% yield) as a yellow solid. LCMS (Method r,
Table 7) R.sub.t=0.95 min; m/z=307.0 [M+H.sup.+]. .sup.1H NMR (400
MHz, DMSO-d6) .delta. 7.87 (d, J=8.7 Hz, 2H), 7.62 (d, J=8.6 Hz,
2H), 7.38 (d, J=8.6 Hz, 2H), 7.28 (d, J=8.7 Hz, 2H), 2.50 (s,
3H).
Step 2: Synthesis of Tert-Butyl
(4-((4-acetylphenyl)thio)phenyl)carbamate
##STR00740##
[1038] Nitrogen was sparged through a mixture of
1-(4-((4-bromophenyl)thio)phenyl)ethanone (3.24 g, 10.55 mmol),
tert-butyl carbamate (1.483 g, 12.66 mmol), Cs.sub.2CO.sub.3 (5.15
g, 15.82 mmol), and
dicyclohexyl(2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphine
(0.503 g, 1.055 mmol) in 1,4-dioxane for 30 min. The flask was
evacuated and back filled with N.sub.2 (3.times.).
Pd.sub.2dba.sub.3 (0.290 g, 0.316 mmol) was added and the reaction
was evacuated and back filled with N.sub.2 (3.times.). The reaction
mixture was heated to 100.degree. C. for 18 h. The reaction was
cooled to ambient temperature, treated with water (75 mL), then
extracted with EtOAc (3.times.50 mL), dried (MgSO.sub.4), and
solvents were removed under reduced pressure. Purification by
chromatography (silica, 120 g) eluting with a gradient of 0-60%
EtOAc/heptanes gave the title compound (2.0 g, 5.82 mmol, 55%
yield) as a yellow solid. LCMS (Method r, Table 7) R.sub.t=0.96
min; m/z=344.0 [M+H.sup.+]. .sup.1H NMR (501 MHz, DMSO-d6) .delta.
9.62 (s, 1H), 7.82 (d, J=8.7 Hz, 2H), 7.58 (d, J=8.7 Hz, 2H), 7.43
(d, J=6.7 Hz, 2H), 7.11 (d, J=8.7 Hz, 2H), 2.49 (s, 3H), 1.47 (s,
9H).
Step 3: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((4-Aminophenyl)thio)phe-
nyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10-trimethyl-6a,6b-
,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3-
]dioxol-4(2H)-one and
(2S,6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((4-Aminophenyl)thio)phe-
nyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10-trimethyl-6a,6b-
,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3-
]dioxol-4(2H)-one
##STR00741##
[1040] Triflic acid (0.431 mL, 4.85 mmol) was added drop-wise to a
0.degree. C. slurry of
(6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-
-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-
-3H-cyclopenta[a]phenanthren-3-one (0.400 g, 0.970 mmol),
tert-butyl (4-((4-acetylphenyl)thio)phenyl)carbamate (0.366 g,
1.067 mmol), and MgSO.sub.4 (0.350 g, 2.91 mmol) in MeCN (4.0 mL).
After 30 min the reaction was diluted with EtOAc (25 mL), washed
sequentially with a saturated aqueous solution of NaHCO.sub.3 (20
mL), with a saturated aqueous solution of brine (25 mL), dried
(MgSO.sub.4), and then solvent was removed under reduced pressure
to give a yellow foam. Purification by chromatography (silica, 40
g) eluting with a gradient of 0-10% MeOH/DCM gave the product as a
mixture of the ketal isomers (460 mg, 0.721 mmol, 74% yield). A
portion of this material was purified by reverse phase HPLC on a
Phenomenex C18(2) 10 micron column (250.times.50 mm). A gradient of
MeCN (A) and 0.1% TFA in water (B) was used, at a flow rate of 90
mL/min (0-5.0 min 15% A, 5.0-18 min linear gradient 15-75% A, then
hold 5 min). Combined fractions were concentrated to remove
volatile solvents under reduced pressure, and the resulting
solutions were frozen and lyophilized to give the ketal isomers as
yellow solids. Minor ketal isomer:
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((4-aminophenyl)thio)phe-
nyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10-trimethyl-6a,6b-
,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3-
]dioxol-4(2H)-one. Yellow powder (10.0 mg). LCMS (Method r, Table
7) R.sub.t=0.80 min; m/z=638.2 [M+H*]. .sup.1H NMR (500 MHz,
DMSO-d) .delta. 7.33 (d, J=8.5 Hz, 2H), 7.23 (dd, J=10.1, 1.5 Hz,
1H), 7.19-7.12 (m, 2H), 6.96 (d, J=8.5 Hz, 2H), 6.76-6.63 (m, 2H),
6.29 (dd, J=10.2, 1.9 Hz, 1H), 6.10 (s, 1H), 5.66-5.45 (m, 2H),
5.14 (d, J=5.8 Hz, 1H), 4.65 (d, J=19.3 Hz, 1H), 4.22-4.07 (m, 2H),
2.48-2.35 (m, 1H), 2.14-2.04 (m, 1H), 2.02-1.91 (m, 1H), 1.77-1.64
(m, 2H), 1.63-1.56 (m, 1H), 1.50 (dd, J=13.2, 6.3 Hz, 1H), 1.44 (s,
3H), 1.36 (s, 3H), 1.14-0.98 (m, 1H), 0.80 (s, 3H). Major ketal
isomer:
(2S,6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((4-aminophenyl)thio)phe-
nyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10-trimethyl-6a,6b-
,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3-
]dioxol-4(2H)-one. Yellow powder (18.1 mg). LCMS (Method r, Table
7) R.sub.t=0.85 min; m/z=638.2 [M+H.sup.+]. .sup.1H NMR (500 MHz,
DMSO-d.sub.6) .delta. 7.28 (d, J=10.2 Hz, 1H), 7.20 (dd, J=8.4, 7.2
Hz, 4H), 6.95 (d, J=8.4 Hz, 2H), 6.73 (d, J=8.5 Hz, 2H), 6.31 (d,
J=12.0 Hz, 1H), 6.13 (s, 1H), 5.75-5.57 (m, 1H), 5.53 (s, 1H), 5.00
(d, J=5.1 Hz, 1H), 4.22 (d, J=7.2 Hz, 1H), 4.06-3.80 (m, 4H),
2.72-2.55 (m, 1H), 2.39-2.27 (m, 1H), 2.17-2.02 (m, 2H), 1.79-1.56
(m, 3H), 1.50 (d, J=12.4 Hz, 6H), 0.73 (s, 3H).
Example 17: Synthesis of
(6aR,6bS,7S,8aS,8bS,1R,1aR,12aS,12bS)-10-(4-((4-aminophenyl)thio)phenyl)--
7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10-trimethyl-6a,6b,7,8,8a,8b,11a,12,1-
2a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(2H)-one
and
(6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((4-aminophenyl)thio)ph-
enyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10-trimethyl-6a,6b,7,8,8a,8b,11-
a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(2H)-
-one
##STR00742##
[1042] Triflic acid (0.24 mL, 2.66 mmol) was added drop-wise to a
0.degree. C. slurry of
(8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)--
10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a-
]phenanthren-3-one (0.200 g, 0.531 mmol), tert-butyl
(4-((4-acetylphenyl)thio)phenyl)carbamate (0.201 g, 0.584 mmol),
and MgSO.sub.4 (0.192 g, 1.59 mmol) in MeCN (2.0 mL). After 30 min
the reaction was diluted with EtOAc (15 mL), washed sequentially
with a saturated aqueous solution of NaHCO.sub.3 (10 mL), and then
with a saturated aqueous solution of brine (10 mL), dried
(MgSO.sub.4), and solvent was removed under reduced pressure to
give a yellow foam. Purification by chromatography (silica, 24 g)
eluting with a gradient of 0-10% MeOH/DCM gave the product as a
mixture of the ketal isomers (198 mg, 0.329 mmol, 62% yield). A
portion of this material was purified by reverse phase HPLC on a
Phenomenex C18(2) 10 micron column (250.times.50 mm). A gradient of
MeCN (A) and 0.1% TFA in water (B) was used, at a flow rate of 90
mL/min (0-5.0 min 15% A, 5.0-18 min linear gradient 15-75% A, then
hold 5 min). Combined fractions were concentrated to remove
volatile solvents under reduced pressure, and the resulting
solution was frozen and lyophilized to give both ketal isomers as
white solids. Major ketal isomer:
(6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((4-aminophenyl)thi-
o)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10-trimethyl-6a,6b,7,8,8a,8-
b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-
(2H)-one. White powder (14.6 mg). LCMS (Method r; Table 7)
R.sub.t=0.83 min; m/z=602.1 [M+H.sup.+]. .sup.1H NMR (501 MHz,
DMSO-d6) .delta. 7.30 (d, J=10.1 Hz, 1H), 7.22-7.12 (m, 4H), 6.91
(d, J=8.5 Hz, 2H), 6.68 (d, J=8.5 Hz, 2H), 6.16 (dd, J=10.1, 1.9
Hz, 1H), 5.91 (s, 1H), 4.93 (d, J=4.6 Hz, 1H), 4.74 (brs, 2H), 4.30
(d, J=2.9 Hz, 1H), 4.02-3.79 (m, 4H), 2.53 (dt, J=14.7, 7.6 Hz,
1H), 2.30 (d, J=14.8 Hz, 1H), 2.16-1.95 (m, 2H), 1.85 (d, J=3.6 Hz,
1H), 1.78-1.67 (m, 2H), 1.55 (td, J=15.2, 13.3, 7.7 Hz, 2H), 1.50
(s, 3H), 1.37 (s, 3H), 1.13-0.97 (m, 2H), 0.70 (s, 3H). Minor ketal
isomer:
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((4-aminophenyl)thio)phenyl-
)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10-trimethyl-6a,6b,7,8,8a,8b,11a,12-
,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(2H)-one-
. White powder (12.0 mg). LCMS (Method r, Table 7) R.sub.t=0.80
min; m/z=602.1[M+H*]. .sup.1H NMR (501 MHz, DMSO-d6) .delta. 7.32
(d, J=6.8 Hz, 2H), 7.25 (d, J=10.1 Hz, 1H), 7.13 (d, J=8.5 Hz, 2H),
6.95 (d, J=8.5 Hz, 2H), 6.66 (d, J=8.5 Hz, 2H), 6.13 (dd, J=10.1,
1.9 Hz, 1H), 5.87 (s, 1H), 5.09 (d, J=6.1 Hz, 1H), 4.71 (brs, 1H),
4.62 (d, J=19.3 Hz, 1H), 4.22 (d, J=2.9 Hz, 1H), 4.11 (d, J=19.2
Hz, 2H), 2.47-2.37 (m, 1H), 2.25-2.07 (m, 1H), 1.94 (qd, J=11.3,
3.8 Hz, 1H), 1.87-1.75 (m, 1H), 1.70 (s, 2H), 1.59-1.44 (m, 2H),
1.32 (d, J=5.1 Hz, 6H), 1.18-1.03 (m, 1H), 0.78 (s, 3H), 0.61 (dd,
J=11.2, 3.5 Hz, 1H), 0.50 (qd, J=12.9, 4.8 Hz, 1H).
Example 18: Synthesis of
2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(4-Aminophenoxy)-3-hydrox-
yphenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b-
,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3-
]dioxol-4(2H)-one
Step 1: Synthesis of 3-Methoxy-4-(4-nitrophenoxy)benzaldehyde
##STR00743##
[1044] Vanillin (2.5 g, 16.43 mmol), 4-Fluoronitrobenzene (2.61 mL,
24.65 mmol), and potassium carbonate (4.54 g, 32.9 mmol) were
dissolved in DMF (15 mL) and stirred at 80.degree. C. overnight.
After cooling, the mixture was treated with water, and extracted
with EtOAc (.times.2). The combined organic layers were washed with
water and brine, dried (Na.sub.2SO.sub.4), and concentrated.
Purification by chromatography (silica, 120 g) eluting with a
gradient of 0-40% EtOAc in heptanes afforded the title compound as
a slightly yellow solid (3.37 g, 75%). LCMS (Method r, Table 7)
R.sub.t=0.88 min; m/z not observed. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 10.00 (s, 1H), 8.26-8.17 (m, 2H), 7.72-7.60
(m, 2H), 7.42 (d, J=8.1 Hz, 1H), 7.12-7.03 (m, 2H), 3.82 (s,
3H).
Step 2: Synthesis of 3-hydroxy-4-(4-nitrophenoxy)benzaldehyde
##STR00744##
[1046] Tribromoborane (110 mL, 110 mmol) was added to a -78.degree.
C. solution of 3-methoxy-4-(4-nitrophenoxy)benzaldehyde (6.02 g,
22.03 mmol) in DCM (100 mL). The reaction was stirred at
-78.degree. C. for 1 h, then stirred at 0.degree. C. for 5 hours.
The mixture was mixed with ice, and extracted with DCM. The
combined organic layers were washed with water and brine, dried
(Na.sub.2SO.sub.4), and concentrated. Purification by
chromatography (silica, 120 g) eluting with a gradient of 0-30%
EtOAc in heptanes afforded the title compound as a purplish oil
(5.55 g, 97% yield). LCMS (Method r, Table 7) R.sub.t=0.80 min; m/z
not observed. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 10.24 (s, 1H),
8.22-8.14 (m, 2H), 7.36 (s, 1H), 7.30 (d, J=2.1 Hz, 1H), 7.15-7.07
(m, 2H), 7.02-6.96 (m, 2H).
Step 3: Synthesis of 4-(4-aminophenoxy)-3-hydroxybenzaldehyde
##STR00745##
[1048] Stannous chloride (18.29 g, 96 mmol) was added to a solution
of added 3-hydroxy-4-(4-nitrophenoxy)benzaldehyde (5 .g, 19.29
mmol), stannous chloride (18.29 g, 96 mmol) in ethanol (60 mL),
which was heated to 80.degree. C. for 2 h. The mixture was cooled
and mixed carefully with ice, and saturated sodium bicarbonate
aqueous solution, then extracted with EtOAc multiple times. The
combined organic layers were washed with brine, dried
(Na.sub.2SO.sub.4), filtered through Celite.RTM., and the filtrate
was concentrated to afford the title compound as a yellow solid
(1.18 g, 27% yield). LCMS (Method r, Table 7) R.sub.t=0.48 min;
m/z=not observed. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 9.90-9.87
(m, 1H), 10.90-9.26 (m, 2H), 8.66-8.56 (m, 1H), 7.66-7.61 (m, 1H),
7.50-7.46 (m, 1H), 7.46-7.38 (m, 2H), 7.38-7.29 (m, 4H), 7.16-6.99
(m, 6H); MS(ESI-) m/z=227.9 (M-H).
Step 4: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(4-Aminophenoxy)-3-hydro-
xyphenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6-
b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,-
3]dioxol-4(2H)-one
##STR00746##
[1050] Perchloric acid (2.64 mL, 24.25 mmol) was added to a room
temperature solution of 4-(4-aminophenoxy)-3-hydroxybenzaldehyde
(0.611 g, 2.67 mmol) and
(6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-
-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-
-3H-cyclopenta[a]phenanthren-3-one (1 g, 2.425 mmol) in THF (70
mL). After 16 hours the reaction was treated with water and
extracted twice with EtOAc. The combined organic layers were washed
with a saturated aqueous solution of sodium bicarbonate, a
saturated aqueous solution of sodium thiosulfate solution, then a
saturated aqueous solution of brine, dried (Na.sub.2SO.sub.4), and
solvent was removed under reduced pressure. The material was
purified by reverse phase HPLC on a Phenomenex C18(2) 5 micron
column (250.times.21 mm column). A gradient of MeCN (A) and 0.1%
TFA in water (B) was used at a flow rated of 30 mL/min (0.0-1.0 min
15% A, 1.0-10 min linear gradient to 65% A, hold 1 min). Combined
fractions were concentrated under reduced pressure to remove
volatile solvents, and the resulting solution frozen and
lyophilized to give the title product as a yellow solid (338.9 mg,
23% yield). LCMS (Method r, Table 7) R.sub.t=0.72 min; MS (ESI+)
624.2 (M+H); .sup.1H NMR (400 MHz, DMSO-d6) .delta. 9.88 (s, 2H),
7.28 (dd, 1H), 7.27-7.22 (m, 2H), 7.07 (d, 1H), 7.00 (d, 1H),
6.96-6.88 (m, 3H), 6.30 (dd, 1H), 6.18-6.08 (m, 1H), 5.78-5.67 (m,
1H), 5.65-5.52 (m, 1H), 5.42 (s, 1H), 5.00-4.95 (m, 1H), 4.53 (d,
1H), 4.27-4.18 (m, 2H), 2.79-2.57 (m, 1H), 2.36-2.28 (m, 1H), 2.24
(td, 1H), 2.13-2.01 (m, 1H), 1.80-1.66 (m, 3H), 1.65-1.52 (m, 1H),
1.51 (s, 3H), 0.88 (s, 3H).
Example 19: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((4-Aminophenyl)sulfonyl)ph-
enyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12-
,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(2H)-one
Step 1: Synthesis of 4-((4-Bromophenyl)thio)benzonitrile
##STR00747##
[1052] Potassium carbonate (4.39 g, 31.7 mmol) was added to a
solution of 4-bromobenzenethiol (5.0 g, 26.4 mmol) and
4-fluorobenzonitrile (3.20 g, 26.4 mmol) in DMF (50 mL), which was
heated to 120.degree. C. for 3 h. The reaction was cooled to
0.degree. C. water was added (100 mL) and the mixture was extracted
with EtOAc (3.times.50 mL). The combined organics were dried
(MgSO.sub.4) and solvent was removed under reduced pressure.
Purification by chromatography (80 g silica) eluting with a
gradient of 0-60% EtOAc/heptanes gave the title compound (6.82 g,
23.5 mmol, 89% yield) as a yellow solid. LCMS (Method r, Table 7)
R.sub.t=0.95 min; m/z=291.2 [M+H.sup.+]. .sup.1H NMR (400 MHz,
DMSO-d6) .delta. 7.72 (d, J=8.7 Hz, 2H), 7.65 (d, J=8.5 Hz, 2H),
7.43 (d, J=8.5 Hz, 2H), 7.26 (d, J=8.7 Hz, 2H).
Step 2: Synthesis of Tert-Butyl
(4-((4-cyanophenyl)thio)phenyl)carbamate
##STR00748##
[1054] Nitrogen was sparged through a mixture of
4-((4-bromophenyl)thio)benzonitrile (6.0 g, 20.68 mmol), tert-butyl
carbamate (2.91 g, 24.81 mmol),
diisopropyl(2',4',5'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphine
(0.820 g, 2.068 mmol), and Cs.sub.2CO.sub.3 (10.11 g, 31.0 mmol) in
1,4-dioxane (207 mL) for 30 min. The flask was evacuated and back
filled with N.sub.2 (3.times.). Pd.sub.2dba.sub.3 (0.568 g, 0.620
mmol) was added and the reaction was evacuated and back filled with
N.sub.2 (3.times.) times. The reaction mixture was heated to
100.degree. C. for 28 h. The reaction was cooled to room
temperature, whereupon it was treated with water (200 mL),
extracted with EtOAc (3.times.75 mL), dried (MgSO.sub.4) and
solvents were removed under reduced pressure. Purification by
chromatography (silica, 120 g) eluting with a gradient of 0-30%
EtOAc/heptanes gave the title compound (3.20 g, 9.80 mmol, 47%
yield) as a yellow solid. LCMS (Method r, Table 7) R.sub.t=1.0 min;
m/z=344.1 [M+NH.sub.4]. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 9.67
(s, 1H), 7.69 (d, J=8.7 Hz, 2H), 7.61 (d, J=8.7 Hz, 2H), 7.47 (d,
J=8.7 Hz, 2H), 7.14 (d, J=8.6 Hz, 2H), 1.49 (s, 9H).
Step 3: Synthesis of Tert-Butyl
(4-((4-cyanophenyl)sulfonyl)phenyl)carbamate
##STR00749##
[1056] 3-Chloroperoxybenzoic acid (639 mg, 3.71 mmol) was added
portion-wise to a room temperature solution of tert-butyl
(4-((4-cyanophenyl)thio)phenyl)carbamate (480 mg, 1.471 mmol) in
CH.sub.2C12 (15 mL). After 30 min, the reaction was portioned
between water (20 mL) and EtOAc (10 mL). The layers were separated
and the aqueous phase was extracted with EtOAc (2.times.25 mL). The
combined organics were washed with a saturated aqueous solution of
brine (50 mL), dried over MgSO.sub.4, and solvents were removed
under reduced pressure. Purification by chromatography (silica, 40
g) eluting with a gradient of 0-60% EtOAc/heptanes gave the title
compound (372 mg, 1.04 mmol, 71% yield) as a yellow solid. LCMS
(Method r, Table 7) R.sub.t=0.86 min; m/z=376.0 [M+NH.sub.4-].
.sup.1H NMR (400 MHz, DMSO-d6) .delta. 9.92 (s, 1H), 8.06 (s, 4H),
7.87 (d, J=8.9 Hz, 2H), 7.66 (d, J=9.0 Hz, 2H), 1.45 (s, 9H).
Step 4: Synthesis of Tert-Butyl
(4-((4-formylphenyl)sulfonyl)phenyl)carbamate
##STR00750##
[1058] Diisobutylaluminum hydride (6.53 mL, 1.0 M in toluene, 6.53
mmol) was added drop-wise over 5 minutes to a 0.degree. C. solution
of tert-butyl (4-((4-cyanophenyl)sulfonyl)phenyl)carbamate (0.780
g, 2.176 mmol) in THF (20 mL). After 30 min diisobutylaluminum
hydride (1.0 M in toluene) (2.176 mL, 2.176 mmol) was added and the
reaction was stirred at 0.degree. C. for an additional 1 h. The
reaction was quenched at 0.degree. C. by slow addition of al N
aqueous solution of HCl (120 mL) and the aqueous phase was
extracted with EtOAc (2.times.75 mL). The combined organics were
washed with a saturated aqueous solution of brine (50 mL), dried
over MgSO.sub.4 and solvents were removed under reduced pressure.
Purification by chromatography (silica, 80 g) eluting with a
gradient of 0-10% CH.sub.2Cl.sub.2/MeOH gave the title compound
(0.275 g, 0.761 mmol, 35% yield) as a yellow oil. LCMS (Method r,
Table 7) R.sub.t=0.83 min; m/z=359.9 [M-H.sup.-]. .sup.1H NMR (400
MHz, DMSO-d6) .delta. 10.04 (s, 1H), 9.89 (s, 1H), 8.18-7.97 (m,
4H), 7.85 (d, J=8.9 Hz, 2H), 7.64 (d, J=8.9 Hz, 2H), 1.43 (s,
9H).
Step 5: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((4-Aminophenyl)sulfonyl)ph-
enyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12-
,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(2H)-one
##STR00751##
[1060] Triflic acid (0.12 mL, 1,328 mmol) was added drop-wise to a
0.degree. C. slurry of
(8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)--
10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a-
]phenanthren-3-one (0.100 g, 0.266 mmol), tert-butyl
(4-((4-formylphenyl)sulfonyl)phenyl)carbamate (0.106 g, 0.292
mmol), and MgSO.sub.4 (0.096 g, 0.797 mmol) in MeCN (1.0 mL). After
30 minutes the reaction was diluted with EtOAc (15 mL), and then
washed with a saturated aqueous solution of NaHCO.sub.3 (10 mL)
followed by a saturated aqueous solution of brine (10 mL), and
dried (MgSO.sub.4). Removal of solvent under reduced pressure gave
a light yellow foam, which was purified by chromatography (silica,
24 g) eluting with a gradient of 0-10% CH.sub.2Cl.sub.2/MeOH to
give a colorless glass. The acetal isomers were separated by
preparative reverse phase HPLC on a Phenomenex C18 (2) 10 micron
column, (250.times.30 mm). A gradient of MeCN (A) and 0.1% TFA in
water (B) was used, at a flow rate of 60 mL/min (0-3.0 min 15% A,
3.0-18 min linear gradient 15-80% A, then hold 5 min). Combined
fractions were concentrated to remove volatile solvents under
reduced pressure, and the resulting solution was frozen and
lyophilized to give the title compound as a white solid (8.0 mg,
18% yield). LCMS (Method r, Table 7) R.sub.t=0.76 min; MS m/z=620.0
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 7.81 (d, J=8.4
Hz, 2H), 7.61 (d, J=8.4 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H), 7.27 (d,
J=10.0 Hz, 1H), 6.56 (d, J=8.8 Hz, 2H), 6.12 (dd, J=10.1, 1.9 Hz,
1H), 5.89 (s, 1H), 5.47 (s, 1H), 4.91 (d, J=4.6 Hz, 1H), 4.73 (s,
1H), 4.48 (d, J=19.4 Hz, 1H), 4.24 (s, 1H), 4.13 (d, J=19.5 Hz,
1H), 2.51 (s, 2H), 2.32-2.22 (m, 1H), 2.13-2.01 (m, 1H), 2.02-1.88
(m, 1H), 1.78-1.56 (m, 5H), 1.35 (s, 3H), 1.11-0.96 (m, 2H), 0.82
(s, 3H).
Example 20:
N-(3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-Difluoro-7-hydro-
xy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12-
a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benz-
yl)phenyl)-3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)et-
hoxy)ethoxy)propanamide
Step 1: Synthesis of Tert-Butyl (2-(2-(3-((3-(4-((2S,6aS,6bR,7S,
8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)--
6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-nap-
htho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-3-oxopr-
opoxy)ethoxy)ethyl)carbamate
##STR00752##
[1062] HATU (0.125 g, 0.328 mmol) was added to a room temperature
solution of 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatetradecan-14-oic
acid (0.100 g, 0.361 mmol),
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2-
,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b-
,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(-
2H)-one (0.199 g, 0.328 mmol) and 2,6-dimethylpyridine (0.12 mL,
0.983 mmol) in THF (2.0 mL). After 24 hours solvents were removed
under reduced pressure and the reaction mixture was purified by
chromatography (silica, 24 g) eluting with a gradient of 0-10%
MeOH/CH.sub.2Cl.sub.2 to give the title compound as a light yellow
foam (226 mg, 0.261 mmol, 80% yield). LCMS (Method r, Table 7)
R.sub.t=0.91 min, m/z=865.5 [M+H.sup.+]. .sup.1H NMR (DMSO-d)
.delta. 0.86 (s, 3H), 1.36 (s, 9H), 1.50 (s, 4H), 1.71 (ddt,
J=17.9, 13.3, 5.8 Hz, 3H), 1.94-2.14 (m, 2H), 2.18-2.39 (m, 1H),
2.55-2.74 (m, 1H), 3.03 (q, J=6.0 Hz, 2H), 3.48 (hept, J=3.1, 2.7
Hz, 4H), 3.66 (t, J=6.3 Hz, 2H), 3.88 (s, 2H), 4.13-4.26 (m, 2H),
4.51 (d, J=19.4 Hz, 1H), 4.94 (d, J=5.1 Hz, 1H), 5.45 (s, 1H), 5.52
(dd, J=4.3, 1.7 Hz, 1H), 5.65 (dddd, J=48.5, 11.4, 6.7, 2.0 Hz,
1H), 6.13 (d, J=2.1 Hz, 1H), 6.73 (t, J=5.8 Hz, 1H), 6.80-6.97 (m,
1H), 7.18 (t, J=7.8 Hz, 1H), 7.25 (td, J=9.1, 8.2, 1.6 Hz, 3H),
7.32-7.39 (m, 3H), 7.45 (dd, J=8.4, 2.0 Hz, 1H), 7.63 (d, J=7.8 Hz,
1H), 8.11-8.85 (m, 1H), 9.83 (s, 1H).
Step 2:
3-(2-(2-Aminoethoxy)ethoxy)-N-(3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11-
aR,
12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-
-4-oxo-2,4,6a,6b,7,8,8a,8b,
11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol--
10-yl)benzyl)phenyl)propanamide
##STR00753##
[1064] TFA (1.0 mL, 12.98 mmol) was added to a room temperature
solution of tert-butyl
(2-(2-(3-((3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-
-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,1-
1a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-
-yl)benzyl)phenyl)amino)-3-oxopropoxy)ethoxy)ethyl)carbamate (226
mg, 0.261 mmol) in CH.sub.2Cl.sub.2 (3.0 mL). After 45 min
volatiles were removed under vacuum and the crude product was
carried on to the next step without further purification, assuming
100% yield. LCMS (Method r, Table 7) R.sub.t=0.80 min, m/z=765.4
[M+H.sup.+].
Step 3: Synthesis of
N-(3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-Difluoro-7-hydro-
xy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12-
a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benz-
yl)phenyl)-3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)et-
hoxy)ethoxy)propanamide
##STR00754##
[1066] N,N-Diisopropylethylamine (0.155 mL, 0.88 mmol) was added to
a room temperature solution of
3-(2-(2-aminoethoxy)ethoxy)-N-(3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS-
,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2-
,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[-
1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)propanamide (0.226 g, 0.295
mmol) and 2,5-dioxopyrrolidin-1-yl
3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (0.087 g, 0.325
mmol) in DMF (2.0 mL). After 45 min, the crude reaction mixture was
purified by reverse phase HPLC on a Phenomenex C18(2) 10 micron
column (250.times.50 mm column). A gradient of MeCN (A) and 0.1%
formic acid in water (B) was used, at a flow rate of 80 mL/min
(0-5.0 min 18% A, 5.0-25.0 min linear gradient 15-80% A, hold 5
min). Combined fractions were concentrated under reduced pressure
to remove volatile solvents, and the resulting solution was frozen
and lyophilized to give the title compound as a white solid (48 mg,
0.052 mmol, 18% yield). LCMS (Method r, Table 7) R.sub.t=0.84 min,
m/z=916.4 [M+H.sup.+]. .sup.1H NMR (DMSO-d.sub.6) .delta. 0.84 (s,
3H), 1.48 (s, 4H), 1.59-1.76 (m, 3H), 2.03 (d, J=13.9 Hz, 1H),
2.17-2.38 (m, 4H), 2.54-2.72 (m, 1H), 3.11 (q, J=5.8 Hz, 2H),
3.31-3.35 (m, 4H), 3.42-3.51 (m, 4H), 3.57 (dd, J=7.8, 6.8 Hz, 2H),
3.64 (t, J=6.3 Hz, 2H), 3.86 (s, 2H), 4.10-4.25 (m, 2H), 4.49 (dd,
J=19.5, 6.0 Hz, 1H), 4.93 (d, J=5.1 Hz, 1H), 5.07 (t, J=5.9 Hz,
1H), 5.43 (s, 1H), 5.51 (s, 1H), 5.53-5.74 (m, 1H), 6.11 (s, 1H),
6.28 (dd, J=10.2, 1.9 Hz, 1H), 6.88 (d, J=7.5 Hz, 1H), 6.97 (s,
2H), 7.16 (t, J=7.8 Hz, 1H), 7.20-7.28 (m, 3H), 7.30-7.39 (m, 3H),
7.38-7.48 (m, 1H), 7.96 (t, J=5.6 Hz, 1H), 9.81 (s, 1H).
Example 21:
N-(3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-Difluoro-7-hydro-
xy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12-
a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benz-
yl)phenyl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,1-
2-tetraoxapentadecan-15-amide
##STR00755##
[1068] Prepared by the same procedure as Example 20. White solid
(17 mg, 0.017 mmol, 9% yield). LCMS (Method r, Table 7)
R.sub.t=0.82 min, m/z=1026 [M+Na.sup.+]. H NMR (DMSO-d.sub.6)
.delta. 0.85 (s, 3H), 1.22 (s, 8H), 1.49 (s, 3H), 1.61-1.77 (m,
2H), 2.03 (d, J=13.9 Hz, 1H), 2.12-2.40 (m, 3H), 2.55-2.66 (m, 1H),
3.12 (q, J=5.8 Hz, 2H), 3.33 (s, 1H), 3.41-3.51 (m, 11H), 3.58 (t,
J=7.3 Hz, 2H), 3.65 (t, J=6.3 Hz, 2H), 3.87 (s, 2H), 4.18 (d,
J=14.1 Hz, 2H), 4.42-4.61 (m, 1H), 4.93 (d, J=5.2 Hz, 1H), 5.07 (s,
1H), 5.44 (s, 1H), 5.50 (s, 1H), 5.6-5.7 (m, 1H), 6.28 (dd, J=10.2,
1.9 Hz, 1H), 6.88 (d, J=7.8 Hz, 1H), 6.98 (s, 2H), 7.17 (t, J=7.9
Hz, 1H), 7.24 (t, J=9.8 Hz, 3H), 7.32-7.38 (m, 3H), 7.43 (d, J=8.3
Hz, 1H), 7.98 (s, 1H), 9.81 (s, 1H).
Example 22:
N-(3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-Difluoro-7-hydro-
xy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12-
a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benz-
yl)phenyl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,1-
2,15,18-hexaoxahenicosan-21-amide
##STR00756##
[1070] Prepared by the same procedure as Example 20. White solid
(23.2 mg, 0.021 mmol, 22% yield). LCMS (Method r, Table 7)
R.sub.t=0.83 min, m/z=1092.3 [M+H.sup.+]. .sup.1H NMR
(DMSO-d.sub.6) .delta. 0.84 (s, 3H), 1.48 (s, 4H), 1.58-1.76 (m,
3H), 2.02 (dt, J=14.0, 3.6 Hz, 1H), 2.17-2.37 (m, 4H), 2.62 (dtd,
J=24.1, 11.9, 4.4 Hz, 1H), 3.12 (q, J=5.8 Hz, 2H), 3.40-3.52 (m,
23H), 3.57 (t, J=7.3 Hz, 2H), 3.64 (t, J=6.3 Hz, 2H), 3.86 (s, 2H),
4.10-4.25 (m, 2H), 4.49 (d, J=19.4 Hz, 1H), 4.92 (d, J=5.0 Hz, 1H),
5.08 (s, 1H), 5.43 (s, 1H), 5.49-5.73 (m, 2H), 6.11 (s, 1H), 6.27
(dd, J=10.1, 1.9 Hz, 1H), 6.87 (d, J=7.6 Hz, 1H), 6.97 (s, 2H),
7.16 (t, J=7.8 Hz, 1H), 7.23 (dd, J=13.9, 9.0 Hz, 3H), 7.30-7.38
(m, 3H), 7.43 (d, J=8.1 Hz, 1H), 7.98 (t, J=5.6 Hz, 1H), 9.81 (s,
1H).
Example 23:
N-(3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-Difluoro-7-hydro-
xy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12-
a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benz-
yl)phenyl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,1-
2,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-amide
##STR00757##
[1072] Prepared by the same procedure as Example 20. Isolated as a
colorless glass (20 mg, 0.015 mmol, 18% yield). LCMS (Method r,
Table 7) R.sub.t=0.85 min, m/z=1356.4 [M+H.sup.+]. H NMR
(DMSO-d.sub.6) .delta. 0.84 (s, 3H), 1.48 (s, 4H), 1.67 (d, J=14.3
Hz, 3H), 2.03 (d, J=14.0 Hz, 1H), 2.30 (q, J=9.8, 8.5 Hz, 4H), 2.65
(s, 1H), 3.13 (q, J=5.8 Hz, 2H), 3.34 (t, J=6.2 Hz, 2H), 3.39-3.54
(m, 46H), 3.57 (t, J=7.3 Hz, 2H), 3.64 (t, J=6.2 Hz, 2H), 3.86 (s,
2H), 4.18 (d, J=14.6 Hz, 2H), 4.49 (d, J=19.2 Hz, 1H), 4.93 (d,
J=4.8 Hz, 1H), 5.07 (s, 1H), 5.43 (s, 1H), 5.50 (s, 1H), 5.62 (d,
J=41.1 Hz, 1H), 6.11 (s, 1H), 6.20-6.36 (m, 1H), 6.87 (d, J=7.5 Hz,
1H), 6.98 (s, 2H), 7.16 (t, J=7.8 Hz, 1H), 7.23 (t, J=9.0 Hz, 3H),
7.34 (d, J=8.4 Hz, 3H), 7.43 (d, J=8.4 Hz, 1H), 7.97 (s, 1H), 9.80
(s, 1H).
Example 24:
N-(3-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydr-
oxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,1-
2a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phe-
nyl)thio)phenyl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide
##STR00758##
[1074] In a 4 mL vial
3-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)propanoic acid was added
(43.5 mg, 0.26 mmol), followed by HATU (148 mg, 0.39 mmol)
dissolved in DMA (1.0 mL), followed by N,N-Diisopropylethylamine
neat (67 ul, 0.39 mmol). Then a solution of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-aminophenyl)thio)phe-
nyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8-
,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dio-
xol-4(2H)-one(80.83 mg, 0.13 mmol)(80.83 mg, 0.13 mmol) dissolved
in DMA(0.5 mL) was added. The reaction was shaken at room
temperature for 2 hours. The reaction was checked by LC/MS and
purified by reverse phase HPLC (Method q, linear gradient 45-75%),
to provide the title compound. LCMS (Method s, Table 7)
R.sub.t=0.78 min; MS m/z=775.3 (M+H).sup.+; .sup.1H NMR (400 MHz,
DMSO-d6/D.sub.2-0, Temp=27.degree. C.) .delta. 7.61-7.57 (m, 1H),
7.49-7.44 (m, 1H), 7.43-7.37 (m, 2H), 7.34-7.22 (m, 4H), 7.06-7.02
(m, 1H), 6.92 (s, 2H), 6.29 (dd, J=10.2, 1.9 Hz, 1H), 6.14-6.09 (m,
1H), 5.72-5.52 (m, 1H), 5.46 (s, 1H), 4.98-4.93 (m, 1H), 4.52 (d,
J=19.4 Hz, 1H), 4.26-4.14 (m, 2H), 3.73-3.71 (m, 2H), 3.69-3.65 (m,
2H), 2.73-2.55 (m, 1H), 2.35-2.26 (m, 1H), 2.25-2.12 (m, 1H),
2.03-1.95 (m, 1H), 1.79-1.62 (m, 3H), 1.55-1.39 (m, 4H), 0.85 (s,
3H).
Example 25:
N-(3-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydr-
oxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,1-
2a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phe-
nyl)thio)phenyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide
##STR00759##
[1076] Prepared as described in example 24 from
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid. Purified by
reverse phase HPLC (Method s, linear gradient 50-80%). LCMS (Method
c, Table 7) R.sub.t=0.82 min; MS m/z=817.3 (M+H).sup.+; .sup.1H NMR
(400 MHz, DMSO-d6/D.sub.2-0, Temp=27.degree. C.) .delta. 7.68-7.65
(m, 1H), 7.53-7.49 (m, 1H), 7.42-7.38 (m, 2H), 7.33-7.24 (m, 4H),
7.04-7.01 (m, 1H), 6.91 (s, 2H), 6.29 (dd, J=10.1, 1.9 Hz, 1H),
6.14-6.10 (m, 1H), 5.72-5.53 (m, 1H), 5.46 (s, 1H), 4.96-4.92 (m,
1H), 4.51 (d, J=19.4 Hz, 1H), 4.24-4.15 (m, 2H), 3.38 (t, J=7.0 Hz,
2H), 2.70-2.54 (m, 1H), 2.35-2.11 (m, 5H), 2.03-1.96 (m, 1H),
1.76-1.61 (m, 3H), 1.59-1.41 (m, 8H), 1.24-1.13 (m, 2H), 0.85 (s,
3H).
Example 26:
N-(3-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydr-
oxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,1-
2a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phe-
nyl)thio)phenyl)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamide
##STR00760##
[1078] Prepared as described in example 24 from
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic acid. Purified by
reverse phase HPLC (Method s, linear gradient 50-80%). LCMS (Method
c, Table 7) R.sub.t=0.83 min; MS m/z=823.2 (M+H).sup.+; .sup.1H NMR
(400 MHz, DMSO-d6/D.sub.2-0, Temp=27.degree. C.) .delta. 8.02-7.97
(m, 2H), 7.88-7.85 (m, 1H), 7.77-7.73 (m, 1H), 7.52-7.47 (m, 2H),
7.44-7.36 (m, 3H), 7.35-7.30 (m, 2H), 7.29-7.23 (m, 1H), 7.15 (s,
2H), 7.14-7.10 (m, 1H), 6.29 (dd, J=10.2, 1.9 Hz, 1H), 6.15-6.09
(m, 1H), 5.71-5.54 (m, 1H), 5.47 (s, 1H), 4.97-4.94 (m, 1H), 4.52
(d, J=19.4 Hz, 1H), 4.24-4.14 (m, 2H), 2.70-2.57 (m, 1H), 2.37-2.27
(m, 1H), 2.24-2.12 (m, 1H), 2.03-1.97 (m, 1H), 1.75-1.64 (m, 3H),
1.54-1.42 (m, 4H), 0.85 (s, 3H).
Example 27:
N-(3-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydr-
oxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,1-
2a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phe-
nyl)thio)phenyl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexa-
necarboxamide
##STR00761##
[1080] Prepared as described in example 24 from
4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylic
acid. Purified by reverse phase HPLC (Method q, linear gradient
50-80%). LCMS (Method s, Table 7) R.sub.t=0.85 min; MS m/z=843.3
(M+H).sup.+; .sup.1H NMR (400 MHz, DMSO-d6/D.sub.2-0,
Temp=27.degree. C.) .delta. 7.68 (t, J=2.0 Hz, 1H), 7.54-7.49 (m,
1H), 7.42-7.37 (m, 2H), 7.35-7.22 (m, 4H), 7.04-7.01 (m, 1H), 6.95
(s, 2H), 6.29 (dd, J=10.1, 1.9 Hz, 1H), 6.12 (s, 1H), 5.71-5.53 (m,
1H), 5.46 (s, 1H), 4.99-4.93 (m, 1H), 4.51 (d, J=19.4 Hz, 1H),
4.25-4.15 (m, 2H), 3.26 (d, J=7.0 Hz, 2H), 2.73-2.58 (m, 1H),
2.35-2.14 (m, 3H), 2.03-1.96 (m, 1H), 1.83-1.62 (m, 7H), 1.59-1.40
(m, 5H), 1.37-1.24 (m, 2H), 0.98-0.87 (m, 2H), 0.85 (s, 3H).
Example 28:
N-(3-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydr-
oxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,1-
2a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phe-
nyl)thio)phenyl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-
,6,9,12-tetraoxapentadecan-15-amide
##STR00762##
[1082] Prepared as described in example 24 from
1-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)-3-oxo-7,10,13,16-tetraoxa-4-azano-
nadecan-19-oic acid. Purified by reverse phase HPLC (Method s,
linear gradient 45-75%). LCMS (Method c, Table 7) R.sub.t=0.76 min;
MS m/z=1022.4 (M+H).sup.+; .sup.1H NMR (400 MHz, DMSO-d6/D.sub.2-0,
Temp=27.degree. C.) .delta. 7.70-7.66 (m, 1H), 7.55-7.50 (m, 1H),
7.43-7.37 (m, 2H), 7.34-7.23 (m, 4H), 7.06-7.01 (m, 1H), 6.92 (s,
2H), 6.29 (dd, J=10.2, 1.9 Hz, 1H), 6.14-6.11 (m, 1H), 5.72-5.53
(m, 1H), 5.46 (s, 1H), 5.00-4.92 (m, 1H), 4.51 (d, J=19.4 Hz, 1H),
4.26-4.15 (m, 2H), 3.66 (t, J=6.1 Hz, 2H), 3.59 (t, J=7.2 Hz, 2H),
3.51-3.40 (m, 11H), 3.33 (t, J=5.8 Hz, 2H), 3.12 (t, J=5.8 Hz, 2H),
2.70-2.58 (m, 1H), 2.51-2.47 (m, 3H), 2.36-2.25 (m, 3H), 2.24-2.13
(m, 1H), 2.04-1.97 (m, 1H), 1.75-1.65 (m, 3H), 1.56-1.42 (m, 4H),
0.85 (s, 3H).
Example 29:
N-(3-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydr-
oxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,1-
2a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phe-
nyl)thio)phenyl)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraox-
apentadecan-15-amide
##STR00763##
[1084] Prepared as described in example 24 from
1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-o-
ic acid. Purified by reverse phase HPLC (Method s, linear gradient
45-75%). LCMS (Method c, Table 7) R.sub.t=0.80 min; MS m/z=951.3
(M+H).sup.+; .sup.1H NMR (400 MHz, DMSO-d6/D.sub.2-0,
Temp=27.degree. C.) .delta. 7.69-7.66 (m, 1H), 7.54-7.49 (m, 1H),
7.42-7.37 (m, 2H), 7.35-7.24 (m, 4H), 7.06-7.01 (m, 1H), 6.93 (s,
2H), 6.29 (dd, J=10.2, 1.9 Hz, 1H), 6.17-6.10 (m, 1H), 5.71-5.55
(m, 1H), 5.46 (s, 1H), 4.98-4.93 (m, 1H), 4.51 (d, J=19.4 Hz, 1H),
4.24-4.16 (m, 2H), 3.66 (t, J=6.1 Hz, 2H), 3.56-3.51 (m, 2H),
3.50-3.36 (m, 14H), 2.71-2.60 (m, 1H), 2.51-2.48 (m, 2H), 2.33-2.27
(m, 1H), 2.18 (q, J=10.5 Hz, 1H), 2.03-1.94 (m, 1H), 1.74-1.66 (m,
3H), 1.56-1.44 (m, 4H), 0.85 (s, 3H).
Example 30:
N-(3-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydr-
oxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,1-
2a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phe-
nyl)thio)phenyl)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18-h-
exaoxahenicosan-21-amide
##STR00764##
[1086] Prepared as described in example 24 from
1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)
3,6,9,12,15,18-hexaoxahenicosan-21-oic acid. Purified by reverse
phase HPLC (Method q, linear gradient 10-100%). LCMS (Method s,
Table 7) R.sub.t=0.80 min; MS m/z did not ionize; .sup.1H NMR (400
MHz, DMSO-d6/D.sub.2-0, Temp=27.degree. C.) .delta. 7.69-7.66 (m,
1H), 7.55-7.50 (m, 1H), 7.44-7.37 (m, 2H), 7.34-7.24 (m, 4H),
7.06-7.01 (m, 1H), 6.94 (s, 2H), 6.29 (dd, J=10.2, 1.9 Hz, 1H),
6.13 (s, 1H), 5.69-5.55 (m, 1H), 5.46 (s, 1H), 4.97-4.93 (m, 1H),
4.51 (d, J=19.4 Hz, 1H), 4.23-4.16 (m, 2H), 3.66 (t, J=6.1 Hz, 2H),
3.56-3.38 (m, 22H), 2.70-2.63 (m, 1H), 2.54-2.53 (m, 2H), 2.51-2.48
(m, 2H), 2.33-2.26 (m, 1H), 2.18 (q, J=10.3 Hz, 1H), 2.03-1.97 (m,
1H), 1.73-1.65 (m, 3H), 1.55-1.44 (m, 4H), 0.85 (s, 3H).
Example 31:
N-(3-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydr-
oxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,1-
2a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phe-
nyl)thio)phenyl)-3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)e-
thoxy)ethoxy)propanamide
##STR00765##
[1088] Prepared as described in example 24 from
3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)pro-
panoic acid. Purified by reverse phase HPLC (Method q, linear
gradient 45-75%). LCMS (Method s, Table 7) R.sub.t=0.80 min; MS
m/z=908.1 (M+H).sup.+; .sup.1H NMR (400 MHz, DMSO-d6/D.sub.2-0,
Temp=27.degree. C.) .delta. 7.69-7.66 (m, 1H), 7.54-7.51 (m, 1H),
7.42-7.38 (m, 2H), 7.34-7.24 (m, 4H), 7.06-7.02 (m, 1H), 6.93 (s,
2H), 6.29 (dd, J=10.2, 1.9 Hz, 1H), 6.12 (s, 1H), 5.68-5.55 (m,
1H), 5.46 (s, 1H), 4.98-4.94 (m, 1H), 4.51 (d, J=19.4 Hz, 1H),
4.24-4.16 (m, 2H), 3.64 (t, J=6.1 Hz, 2H), 3.55-3.50 (m, 2H),
3.47-3.37 (m, 9H), 2.69-2.66 (m, 1H), 2.54-2.53 (m, 1H), 2.50-2.47
(m, 2H), 2.32-2.25 (m, 1H), 2.21-2.14 (m, 1H), 2.03-1.97 (m, 1H),
1.74-1.65 (m, 3H), 1.54-1.43 (m, 4H), 0.85 (s, 3H).
Example 32:
N-(3-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydr-
oxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,1-
2a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phe-
nyl)thio)phenyl)-3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanam-
ido)ethoxy)ethoxy)propanamide
##STR00766##
[1090] Prepared as described in example 24 from
3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)propanamido)ethoxy)ethoxy-
)propanoic acid. Purified by reverse phase HPLC (Method q, linear
gradient 45-75%). LCMS (Method s, Table 7) R.sub.t=0.76 min; MS
m/z=934.4 (M+H).sup.+; .sup.1H NMR (400 MHz, DMSO-d6/D.sub.2-0,
Temp=27.degree. C.) .delta. 7.69-7.66 (m, 1H), 7.54-7.49 (m, 1H),
7.42-7.38 (m, 2H), 7.34-7.23 (m, 4H), 7.05-7.01 (m, 1H), 6.91 (s,
2H), 6.29 (dd, J=10.2, 1.9 Hz, 1H), 6.13 (s, 1H), 5.68-5.56 (m,
1H), 5.46 (s, 1H), 4.97-4.93 (m, 1H), 4.51 (d, J=19.4 Hz, 1H),
4.24-4.15 (m, 2H), 3.66 (t, J=6.2 Hz, 2H), 3.58 (t, J=7.2 Hz, 2H),
3.51-3.43 (m, 4H), 3.33 (t, J=5.8 Hz, 2H), 3.10 (t, J=5.7 Hz, 2H),
2.63-2.58 (m, 1H), 2.55-2.53 (m, 1H), 2.50-2.49 (m, 2H), 2.32-2.27
(m, 2H), 2.18 (q, J=10.3 Hz, 1H), 2.03-1.97 (m, 1H), 1.73-1.63 (m,
3H), 1.54-1.42 (m, 4H), 0.85 (s, 3H).
Example 33:
N-(3-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydr-
oxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,1-
2a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phe-
nyl)thio)phenyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide
##STR00767##
[1092] Prepared as described in example 24 from
2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid. Purified by
reverse phase HPLC (Method s, linear gradient 45-75%). LCMS (Method
c, Table 7) R.sub.t=0.95 min; MS m/z=761.7 (M+H).sup.+; .sup.1H NMR
(400 MHz, DMSO-d6/D.sub.2-0, Temp=27.degree. C.) .delta. 7.65-7.61
(m, 1H), 7.52-7.47 (m, 1H), 7.44-7.39 (m, 2H), 7.36 (t, J=7.9 Hz,
1H), 7.33-7.25 (m, 3H), 7.11-7.08 (m, 1H), 7.07 (s, 2H), 6.32 (dd,
J=10.1, 1.9 Hz, 1H), 6.15 (s, 1H), 5.72-5.55 (m, 1H), 5.47 (s, 1H),
5.00-4.92 (m, 1H), 4.53 (d, J=19.5 Hz, 1H), 4.30-4.17 (m, 4H),
2.72-2.61 (m, 1H), 2.38-2.28 (m, 1H), 2.19 (q, J=10.3 Hz, 1H),
2.05-1.98 (m, 1H), 1.78-1.64 (m, 3H), 1.60-1.42 (m, 4H), 0.87 (s,
3H).
Example 34:
N-(3-((4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydr-
oxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,1-
2a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)phe-
nyl)thio)phenyl)-3-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)etho-
xy)propanamide
##STR00768##
[1094] Prepared as described in example 24 from
3-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)propanoic
acid. Purified by reverse phase HPLC (Method q, linear gradient
40-75%). LCMS (Method c, Table 7) R.sub.t=0.95 min; MS m/z=863.9
(M+H).sup.+; .sup.1H NMR (400 MHz, DMSO-d6/D.sub.2-0,
Temp=27.degree. C.).sub..delta. 7.71-7.66 (m, 1H), 7.55-7.51 (m,
1H), 7.44-7.38 (m, 2H), 7.36-7.25 (m, 4H), 7.08-7.03 (m, 1H), 6.91
(s, 2H), 6.31 (dd, J=10.1, 1.9 Hz, 1H), 6.14 (s, 1H), 5.72-5.55 (m,
1H), 5.47 (s, 1H), 4.98-4.94 (m, 1H), 4.53 (d, J=19.4 Hz, 1H),
4.27-4.14 (m, 2H), 3.63 (t, J=6.1 Hz, 2H), 3.53-3.43 (m, 8H),
2.72-2.61 (m, 1H), 2.48 (t, J=6.2 Hz, 2H), 2.35-2.24 (m, 1H), 2.20
(q, J=10.4 Hz, 1H), 2.05-1.96 (m, 1H), 1.76-1.65 (m, 3H), 1.57-1.41
(m, 4H), 0.87 (s, 3H).
Example 34A: Synthesis of
3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-(4-((6aR,6b-
S,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dime-
thyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1'-
:4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)-
amino)-1-oxopropan-2-yl)propanamide
Step 1: Synthesis of
(S)-2-amino-N-((S)-1-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hy-
droxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12-
,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)b-
enzyl)phenyl)amino)-1-oxopropan-2-yl)propanamide
##STR00769##
[1096] HATU (601 mg, 1.580 mmol) and 2,6-lutidine (0.37 mL, 3.16
mmol) were added to a 0.degree. C. solution of
(S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)propa-
noic acid (765 mg, 2.00 mmol),
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-7-hy-
droxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-d-
ecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(2H)-one
(600 mg, 1.053 mmol) in DCM (6 mL) and DMF (12 mL). After 30 min,
the mixture was warmed to room temperature and stirred overnight.
Diethylamine (2.18 mL, 21.06 mmol) was added to the reaction
mixture, and stirring continued at room temp for 3 h, whereupon
volatile solvents were removed under reduced pressure. The residue
was dissolved in 1:1 DMSO:MeOH (12 mL) and purified by reverse
phase HPLC on a Phenomenex C18(2) 10 micron column (250.times.50 mm
column). A gradient of MeCN (A) and 0.1% TFA in water (B) was used,
at a flow rate of 90 mL/min (0-5.0 min 15% A, 5.0-20 min linear
gradient 15-85% A, hold 5 min). Combined product fractions were
lyophilized to give the title compound as an off-white solid (447
mg, 0.628 mmol, 60% yield). LC-MS (Method r, Table 7) R.sub.t=0.78
min, m/z=711.9 [M+H]. .sup.1H NMR (501 MHz, DMSO-d.sub.6) .delta.
10.03 (s, 1H), 8.63 (d, J=7.2 Hz, 1H), 8.07 (d, J=5.4 Hz, 3H),
7.44-7.38 (m, 2H), 7.38-7.34 (m, 2H), 7.29 (d, J=10.1 Hz, 1H),
7.23-7.16 (m, 3H), 6.90 (dt, J=7.7, 1.3 Hz, 1H), 6.14 (dd, J=10.1,
1.9 Hz, 1H), 5.90 (t, J=1.6 Hz, 1H), 5.38 (s, 1H), 4.90 (d, J=5.3
Hz, 1H), 4.52-4.37 (m, 2H), 4.27 (q, J=3.3 Hz, 1H), 4.16 (d, J=19.4
Hz, 1H), 3.87 (s, 2H), 2.58-2.49 (m, 1H), 2.28 (ddd, J=13.4, 4.5,
2.1 Hz, 1H), 2.09 (dtd, J=17.0, 10.6, 5.0 Hz, 1H), 2.00 (dd,
J=12.2, 5.7 Hz, 1H), 1.78-1.54 (m, 5H), 1.37 (s, 3H), 1.35 (s, 3H),
1.30 (d, J=7.1 Hz, 3H), 1.01 (ddd, J=22.1, 11.9, 4.2 Hz, 2H), 0.84
(s, 3H).
Step 2: Synthesis of
3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-(4-((6aR,6b-
S,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dime-
thyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1'-
:4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)-
amino)-1-oxopropan-2-yl)propanamide
##STR00770##
[1098] N,N-Diisopropylethylamine (0.33 mL, 1.875 mmol) was added to
a room temperature solution of N-succinimidyl 3-maleimidopropionate
(250 mg, 0.938 mmol) and
(S)-2-amino-N-((S)-1-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hy-
droxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12-
,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)b-
enzyl)phenyl)amino)-1-oxopropan-2-yl)propanamide (445 mg, 0.625
mmol) in DMF (12 mL). After 30 min at room temperature, the
volatile solvents were removed under reduced pressure. The residue
was diluted with 1:1 DMSO:MeOH (12 mL) and purified by reverse
phase HPLC on a Phenomenex C18(2) 10 micron column (250.times.50 mm
column). A gradient of MeCN (A) and 0.1% TFA in water (B) was used,
at a flow rate of 90 mL/min (0-5.0 min 25% A, 5.0-20 min linear
gradient 25-90% A, hold 5 min). Combined product fractions were
lyophilized to give the title compound as an off-white solid (295.1
mg, 0.342 mmol, 55% yield). LC-MS (Method r, Table 7) R.sub.t=0.85
min, m/z=863.4 [M+H]. .sup.1H NMR (501 MHz, DMSO-d.sub.6) .delta.
9.71 (s, 1H), 8.17 (d, J=7.0 Hz, 1H), 8.03 (d, J=7.3 Hz, 1H), 7.43
(dd, J=7.8, 1.1 Hz, 2H), 7.38-7.32 (m, 2H), 7.29 (d, J=10.1 Hz,
1H), 7.22-7.15 (m, 3H), 6.96 (s, 2H), 6.88 (dt, J=7.8, 1.3 Hz, 1H),
6.13 (dd, J=10.1, 1.9 Hz, 1H), 5.90 (t, J=1.6 Hz, 1H), 5.37 (s,
1H), 4.90 (d, J=5.4 Hz, 1H), 4.48 (d, J=19.4 Hz, 1H), 4.32 (p,
J=7.1 Hz, 1H), 4.27 (q, J=3.3 Hz, 1H), 4.21 (p, J=7.1 Hz, 1H), 4.16
(d, J=19.4 Hz, 1H), 3.87 (s, 2H), 3.59 (t, J=7.3 Hz, 2H), 2.57-2.49
(m, 1H), 2.38 (dd, J=8.0, 6.6 Hz, 2H), 2.32-2.24 (m, 1H), 2.15-2.04
(m, 1H), 2.04-1.95 (m, 1H), 1.80-1.54 (m, 5H), 1.37 (s, 3H), 1.26
(d, J=7.1 Hz, 3H), 1.15 (d, J=7.1 Hz, 3H), 1.02 (ddd, J=21.2, 12.1,
4.2 Hz, 2H), 0.84 (s, 3H).
Example 35
[1099] The following compounds were prepared using the methods
described above.
TABLE-US-00032 Cpd. No. Structure 70 ##STR00771## 71 ##STR00772##
72 ##STR00773## 72 ##STR00774## 73 ##STR00775## 74 ##STR00776## 75
##STR00777## 76 ##STR00778## 77 ##STR00779## 78 ##STR00780## 79
##STR00781## 80 ##STR00782## 81 ##STR00783## 82 ##STR00784## 83
##STR00785## 84 ##STR00786## 85 ##STR00787## 86 ##STR00788## 87
##STR00789## 89 ##STR00790## 90 ##STR00791## 99 ##STR00792## 100
##STR00793## 101 ##STR00794## 102 ##STR00795## 103 ##STR00796## 104
##STR00797## 105 ##STR00798## 106 ##STR00799## 107 ##STR00800## 108
##STR00801## 109 ##STR00802## 110 ##STR00803## 111 ##STR00804## 112
##STR00805## 113 ##STR00806## 114 ##STR00807## 115 ##STR00808## 116
##STR00809## 117 ##STR00810## 118 ##STR00811## 119 ##STR00812## 120
##STR00813## 121 ##STR00814## 122 ##STR00815## 123 ##STR00816## 124
##STR00817## 125 ##STR00818## 126 ##STR00819## 127 ##STR00820## 128
##STR00821## 129 ##STR00822## 130 ##STR00823## 131 ##STR00824## 132
##STR00825## 133 ##STR00826##
TABLE-US-00033 Cpd. No. LC-MS Data 1H NMR Data 70 Method a LC-MS,
.sup.1H NMR (DMSO-d.sub.6) .delta.: 0.79-0.87 (m, 10H), 0.95-1.13
(m, 1H), 1.29 (d, J = 7.1 Hz, 3H), Table 7 1.38 (s, 3H), 1.57-1.76
(m, 2H), 1.76 (s, 2H), 1.90-2.01 (m, 2H), 2.05 (s, 2H), R.sub.t =
1.28 min; 2.28 (s, 2H), 2.43 (dd, J = 14.4, 7.0 Hz, 2H), 3.58 (p, J
= 6.8 Hz, 2H), m/z = 894.0 4.11 (dd, J = 8.4, 6.7 Hz, 1H), 4.16 (d,
J = 19.4 Hz, 1H), 4.28 (s, 1H), [M + H.sup.+] 4.36 (q, J = 6.7 Hz,
1H), 4.49 (d, J = 19.4 Hz, 1H), 4.75 (s, 1H), 4.90 (d, J = 5.1 Hz,
1H), 5.40 (s, 1H), 5.90 (s, 1H), 6.14 (dd, J = 10.1, 1.9 Hz, 1H),
6.89-7.02 (m, 6H), 7.29 (d, J = 10.1 Hz, 1H), 7.42 (d, J = 8.7 Hz,
2H), 7.56-7.63 (m, 2H), 7.98 (d, J = 8.4 Hz, 1H), 8.13 (d, J = 6.9
Hz, 1H), 9.86 (s, 1H) 71 Method r, .sup.1H NMR (DMSO-d.sub.6)
.delta.: 0.85 (s, 3H), 1.16 (d, J = 7.1 Hz, 3H), 1.29 (d, J = 7.1
Hz, 3H), Table 7 1.48 (s, 3H), 1.53 (d, J = 12.6 Hz, 1H), 1.60-1.77
(m, 3H), 1.98-2.09 (m, 1H), R.sub.t = 0.79 min; 2.24 (dd, J = 21.2,
8.6 Hz, 2H), 2.39 (dd, J = 8.0, 6.5 Hz, 2H), 2.53-2.72 (m, 1H), m/z
= 900.91 3.59 (dd, J = 8.1, 6.5 Hz, 2H), 4.13-4.27 (m, 2H), 4.34
(p, J = 7.1 Hz, 1H), [M + H.sup.+] 4.51 (dd, J = 19.5, 6.4 Hz, 1H),
4.93 (d, J = 5.0 Hz, 1H), 5.07 (t, J = 6.0 Hz, 1H), 5.45 (s, 1H),
5.50 (dd, J = 4.5, 1.7 Hz, 1H), 5.53-5.75 (m, 1H), 6.07-6.12 (m,
1H), 6.27 (dd, J = 10.2, 1.9 Hz, 1H), 6.92-7.00 (m, 6H), 7.24 (dd,
J = 10.2, 1.4 Hz, 1H), 7.36-7.43 (m, 2H), 7.58-7.66 (m, 2H), 8.08
(d, J = 7.3 Hz, 1H), 8.18 (d, J = 7.0 Hz, 1H), 9.83 (s, 1H) 72
Method m LC-MS, .sup.1H NMR (MeOH-d.sub.4) .delta.: 1.00 (s, 3H),
1.37 (dd, J = 12.2, 7.1 Hz, 3H), 1.48 (t, J = 7.2 Hz, 3H), Table 7
1.59 (s, 4H), 1.69 (dd, J = 27.0, 13.1 Hz, 2H), 1.79 (dd, J = 13.7,
5.8 Hz, 2H), R.sub.t = 1.71 min; 2.26 (d, J = 13.6 Hz, 1H), 2.38
(d, J = 8.0 Hz, 3H), 2.56 (td, J = 12.5, 11.2, 6.8 Hz, 3H), m/z =
917 2.60-2.81 (m, 1H), 3.80 (dt, J = 12.7, 6.8 Hz, 2H), 4.24 (dd, J
= 11.9, 7.0 Hz, 1H), [M + H.sup.+] 4.32 (s, 2H), 4.43-4.51 (m, 1H),
4.64 (d, J = 19.4 Hz, 1H), 5.07 (d, J = 4.6 Hz, 1H), 5.47 (s, 1H),
5.57 (d, J = 42.9 Hz, 1H), 6.27-6.38 (m, 3H), 6.73 (d, J = 3.0 Hz,
2H), 7.16-7.25 (m, 2H), 7.36 (dt, J = 16.7, 8.0 Hz, 6H), 7.70 (dd,
J = 22.7, 8.4 Hz, 2H) 72 Method m LC-MS, 1H NMR (MeOH-d.sub.4)
.delta.: 1.00 (s, 3H), 1.49 (d, J = 7.2 Hz, 3H), 1.59 (s, 3H),
1.60-1.89 (m, 3H), Table 7 2.04 (d, J = 52.2 Hz, 1H), 2.27 (d, J =
13.5 Hz, 1H), 2.31-2.52 (m, 4H), 2.58 (t, J = 6.7 Hz, 2H), R.sub.t
= 1.88 min; 3.81 (t, J = 6.7 Hz, 2H), 4.34 (d, J = 19.7 Hz, 3H),
4.42-4.53 (m, 1H), 4.64 (d, J = 19.4 Hz, 1H), m/z = 975 5.07 (d, J
= 4.6 Hz, 1H), 5.47 (s, 1H), 5.58 (d, J = 40.9 Hz, 1H), 6.25-6.47
(m, 2H), 6.76 (s, 2H), [M + H.sup.+] 7.20 (d, J = 8.1 Hz, 2H),
7.28-7.44 (m, 5H), 7.67 (d, J = 8.5 Hz, 2H) 73 Method a LC-MS,
.sup.1H NMR (DMSO-d.sub.6) .delta.: 0.84 (s, 3H), 1.16 (d, J = 7.1
Hz, 3H), 1.27 (d, J = 7.1 Hz, 3H), 1.48 (s, 4H), Table 7 1.59-1.77
(m, 3H), 1.96-2.08 (m, 1H), 2.13-2.33 (m, 2H), 2.39 (dd, J = 7.9,
6.7 Hz, 2H), 2.52 (s, 1H), R.sub.t = 2.08 min; 2.53-2.72 (m, 1H),
3.59 (t, J = 7.3 Hz, 2H), 4.08-4.26 (m, 5H), 4.32 (p, J = 7.0 Hz,
1H), m/z = 931.30 4.50 (d, J = 19.4 Hz, 1H), 4.93 (d, J = 5.0 Hz,
1H), 5.44 (s, 1H), 5.45-5.51 (m, 1H), [M + H.sup.+] 5.63 (dt, J =
48.4, 9.3 Hz, 1H), 6.11 (d, J = 2.1 Hz, 1H), 6.28 (dd, J = 10.2,
1.9 Hz, 1H), 6.93-7.02 (m, 3H), 7.19 (t, J = 8.0 Hz, 1H), 7.24 (dd,
J = 10.1, 1.4 Hz, 1H), 7.30-7.44 (m, 5H), 7.65 (t, J = 1.9 Hz, 1H),
8.05 (d, J = 7.2 Hz, 1H), 8.16 (d, J = 7.0 Hz, 1H), 9.77 (s, 1H) 74
Method r, .sup.1H NMR (DMSO-d.sub.6) .delta.: 0.84 (s, 3H), 1.15
(d, J = 7.1 Hz, 3H), 1.26 (d, J = 7.2 Hz, 4H), Table 7 1.48 (s,
4H), 1.59-1.79 (m, 3H), 1.94-2.10 (m, 1H), 2.10-2.31 (m, 2H), 2.37
(t, J = 7.3 Hz, 2H), R.sub.t = 0.82 min; 2.51-2.77 (m, 1H), 3.58
(t, J = 7.3 Hz, 2H), 4.10-4.25 (m, 3H), 4.31 (p, J = 7.1 Hz, 1H),
m/z = 918.60 4.51 (d, J = 19.4 Hz, 1H), 4.94 (d, J = 5.0 Hz, 1H),
5.45 (s, 1H), 5.50 (s, 1H), [M + H.sup.+] 5.62 (dt, J = 48.6, 9.4
Hz, 1H), 6.10 (s, 1H), 6.27 (dd, J = 10.1, 1.9 Hz, 1H), 6.96 (s,
2H), 7.02 (dd, J = 7.3, 1.7 Hz, 1H), 7.20-7.34 (m, 4H), 7.40 (d, J
= 8.3 Hz, 2H), 7.58 (dd, J = 7.9, 2.1 Hz, 1H), 7.69 (d, J = 2.1 Hz,
1H), 8.07 (d, J = 7.2 Hz, 1H), 8.16 (d, J = 7.0 Hz, 1H), 9.89 (s,
1H) 75 Method r, .sup.1H NMR (DMSO-d.sub.6) .delta.: 0.83 (s, 3H),
1.13 (d, J = 7.1 Hz, 3H), 1.24 (d, J = 7.2 Hz, 3H), Table 7 1.46
(s, 4H), 1.57-1.77 (m, 3H), 2.01 (dt, J = 13.9, 3.7 Hz, 1H),
2.13-2.32 (m, 2H), R.sub.t = 0.82 min; 2.36 (dd, J = 8.0, 6.7 Hz,
2H), 2.51-2.73 (m, 1H), 3.56 (d, J = 7.3 Hz, 2H), m/z = 899.87 3.85
(s, 2H), 4.10-4.25 (m, 3H), 4.30 (p, J = 7.1 Hz, 1H), 4.47 (d, J =
19.4 Hz, 1H), [M + H.sup.+] 4.91 (d, J = 4.9 Hz, 1H), 5.41 (s, 1H),
5.48 (s, 1H), 5.51-5.71 (m, 1H), 6.09 (d, J = 2.0 Hz, 1H), 6.26
(dd, J = 10.2, 1.9 Hz, 1H), 6.87 (dt, J = 7.6, 1.3 Hz, 1H), 6.95
(s, 2H), 7.16 (t, J = 7.8 Hz, 1H), 7.18-7.27 (m, 3H), 7.32 (d, J =
8.1 Hz, 2H), 7.39 (d, J = 1.9 Hz, 1H), 7.43 (dd, J = 8.5, 1.9 Hz,
1H), 8.01 (d, J = 7.2 Hz, 1H), 8.14 (d, J = 7.0 Hz, 1H), 9.70 (s,
1H) 76 Method r, .sup.1H NMR (DMSO-d.sub.6) .delta.: 0.88 (d, J =
7.4 Hz, 3H), 1.19 (dt, J = 33.9, 7.1 Hz, 11H), Table 7 1.35-1.63
(m, 10H), 1.61-1.85 (m, 2H), 2.06 (q, J = 7.4 Hz, 3H), 2.16-2.35
(m, 1H), R.sub.t = 1.00 min; 2.38 (t, J = 7.3 Hz, 1H), 3.87 (d, J =
8.1 Hz, 2H), 4.10-4.42 (m, 3H), 4.67-5.15 (m, 2H), m/z = not
5.51-5.73 (m, 3H), 6.11 (s, 1H), 6.28 (dd, J = 10.1, 2.1 Hz, 1H),
6.89 (d, J = 7.3 Hz, 1H), observed 6.92-7.02 (m, 3H), 7.13-7.29 (m,
5H), 7.30-7.41 (m, 2H), 7.42-7.57 (m, 1H), 7.88-8.34 (m, 2H), 9.74
(s, 1H) 77 Method m, .sup.1H NMR (DMSO-d.sub.6) .delta.: 0.86 (s,
3H), 1.26 (dd, J = 15.9, 6.5 Hz, 9H), 1.50 (s, 4H), Table 7 1.70
(t, J = 8.4 Hz, 3H), 1.90-2.10 (m, 2H), 2.14-2.35 (m, 1H),
2.35-2.45 (m, 1H), R.sub.t = 1.62 min; 2.54-2.77 (m, 1H), 2.96
(ddd, J = 47.6, 15.3, 6.6 Hz, 2H), 3.57 (t, J = 7.3 Hz, 2H), m/z =
1058.3 3.89 (s, 2H), 4.20 (d, J = 19.0 Hz, 1H), 4.32 (dt, J = 27.9,
7.0 Hz, 2H), [M + Na.sup.+] 4.45-4.64 (m, 2H), 4.94 (d, J = 4.7 Hz,
1H), 5.07 (d, J = 39.6 Hz, 1H), 5.45 (s, 1H), 5.49-5.79 (m, 2H),
6.12 (s, 1H), 6.29 (dd, J = 10.2, 1.8 Hz, 1H), 6.92 (d, J = 7.6 Hz,
1H), 6.99 (s, 2H), 7.20 (t, J = 7.9 Hz, 1H), 7.25 (t, J = 8.6 Hz,
3H), 7.33-7.42 (m, 4H), 7.45 (d, J = 8.1 Hz, 1H), 8.31 (d, J = 8.0
Hz, 1H), 8.95 (s, 1H), 9.88 (s, 1H), 14.10 (s, 2H) 78 Method r,
.sup.1H NMR (DMSO-d.sub.6) .delta.: 0.84 (s, 3H), 1.17 (d, J = 7.1
Hz, 3H), 1.25 (d, J = 7.1 Hz, 3H), 1.48 (s, 4H), Table 7 1.57 (q, J
= 6.2 Hz, 4H), 1.68 (dq, J = 13.7, 6.3, 5.6 Hz, 3H), 1.99-2.06 (m,
1H), 2.09-2.18 (m, 2H), R.sub.t = 0.80 min; 2.18-2.36 (m, 2H),
2.55-2.72 (m, 3H), 2.78 (s, 4H), 3.87 (s, 2H), 4.14-4.22 (m, 2H),
4.26 (p, J = 7.1 Hz, 1H), m/z = 1005.1 4.33 (p, J = 7.1 Hz, 1H),
4.49 (d, J = 19.4 Hz, 1H), 4.93 (d, J = 5.1 Hz, 1H), 5.43 (s, 1H),
[M + MeOH + H.sup.+1 5.49 (d, J = 5.4 Hz, 1H), 5.54-5.75 (m, 1H),
6.11 (s, 1H), 6.28 (dd, J = 10.2, 2.0 Hz, 1H), 6.89 (d, J = 7.6 Hz,
1H), 7.17 (t, J = 7.9 Hz, 1H), 7.23 (t, J = 9.7 Hz, 3H), 7.34 (d, J
= 7.8 Hz, 2H), 7.39 (s, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.99 (d, J =
7.2 Hz, 1H), 8.02 (d, JJ = 7.3 Hz, 1H), 9.77 (s, 1H) 79 Method r,
.sup.1H NMR (DMSO-d.sub.6) .delta.: 0.85 (s, 3H), 1.14 (d, J = 7.1
Hz, 3H), 1.26 (d, J = 7.1 Hz, 3H), 1.48 (s, 4H), Table 7 1.61-1.80
(m, 3H), 2.04 (d, J = 13.1 Hz, 1H), 2.25 (ddd, J = 18.6, 14.9, 8.4
Hz, 2H), R.sub.t = 0.80 min; 2.37 (dd, J = 8.0, 6.5 Hz, 2H),
2.53-2.74 (m, 1H), 3.57 (t, J = 7.3 Hz, 2H), 4.09-4.24 (m, 2H), m/z
= 901.81 4.30 (p, J = 7.1 Hz, 1H), 4.52 (dd, J = 19.5, 6.4 Hz, 1H),
4.94 (d, J = 5.0 Hz, 1H), [M + H.sup.+] 5.08 (t, J = 5.9 Hz, 1H),
5.46 (s, 1H), 5.48-5.53 (m, 1H), 5.63 (dt, J = 48.9, 9.1 Hz, 1H),
6.10 (s, 1H), 6.27 (dd, J = 10.1, 1.9 Hz, 1H), 6.69 (ddd, J = 7.9,
2.6, 1.1 Hz, 1H), 6.96 (s, 2H), 6.98-7.06 (m, 2H), 7.22-7.32 (m,
2H), 7.32-7.40 (m, 2H), 7.39-7.51 (m, 2H), 8.06 (d, J = 7.2 Hz,
1H), 8.15 (d, J = 7.0 Hz, 1H), 9.87 (s, 1H) 80 Method m, .sup.1H
NMR (MeOH-d.sub.4) .delta.: 1.00 (s, 3H), 1.36 (dd, J = 11.5, 7.1
Hz, 4H), 1.46 (t, J = 6.9 Hz, 3H), Table 7 1.53-1.76 (m, 5H),
1.75-1.89 (m, 2H), 2.28 (d, J = 13.8 Hz, 1H), 2.33-2.48 (m, 1H),
2.48-2.62 (m, 2H), R.sub.t = 1.64 min; 2.61-2.84 (m, 1H), 3.72-3.88
(m, 2H), 3.95 (s, 2H), 4.18-4.40 (m, 3H), 4.46 (q, J = 6.9 Hz, 1H),
m/z = 899 4.65 (d, J = 19.4 Hz, 1H), 5.07 (d, J = 4.6 Hz, 1H),
5.43-5.69 (m, 2H), 6.30-6.39 (m, 2H), 6.69 (s, 2H), [M + H.sup.+]
7.14 (dd, J = 8.2, 5.6 Hz, 2H), 7.23 (dd, J = 7.9, 3.4 Hz, 3H),
7.34 (d, J = 10.1 Hz, 1H), 7.38 (d, J = 7.8 Hz, 2H), 7.51 (d, J =
8.2 Hz, 2H), 7.55 (d, J = 8.2 Hz, 1H), 8.55 (s, 1H) 81 Method m,
.sup.1H NMR (MeOH-d.sub.4) .delta.: 0.88 (s, 3H), 1.21 (d, J = 11.6
Hz, 3H), 1.35 (d, J = 7.1 Hz, 3H), 1.48 (s, 3H), Table 7 1.57 (dd,
J = 25.8, 13.5 Hz, 2H), 1.68 (dd, J = 13.4, 5.5 Hz, 2H), 1.83 (dd,
J = 14.4, 7.5 Hz, 1H), R.sub.t = 1.526 1.88-2.05 (m, 1H), 2.12-2.21
(m, 1H), 2.30 (q, J = 12.7, 10.3 Hz, 3H), 2.44 (t, J = 6.7 Hz, 2H),
min; 2.49-2.73 (m, 1H), 3.68 (t, J = 6.7 Hz, 2H), 3.82 (s, 2H),
4.11-4.28 (m, 3H), 4.34 (q, J = 7.1 Hz, 1H), m/z = 957 4.53 (d, J =
19.4 Hz, 1H), 4.95 (d, J = 4.6 Hz, 1H), 5.30-5.59 (m, 2H), 6.23
(dd, J = 13.2, 3.0 Hz, 2H), [M + H.sup.+] 6.60 (s, 2H), 7.02 (d, J
= 8.2 Hz, 2H), 7.10 (d, J = 7.9 Hz, 2H), 7.24 (dd, J = 15.1, 8.9
Hz, 3H), 7.37 (d, J = 8.2 Hz, 2H) 82 Method r, .sup.1H NMR
(DMSO-d.sub.6) .delta.: 0.84 (s, 3H), 0.96 (s, 2H), 1.01-1.19 (m,
4H), 1.19-1.35 (m, 2H), 1.48 (s, 3H), Table 7 1.67 (d, J = 14.3 Hz,
2H), 2.03 (d, J = 19.7 Hz, 1H), 2.13-2.42 (m, 5H), 2.64 (d, J = 8.4
Hz, 2H), R.sub.t = 0.77 min; 3.08 (s, 3H), 3.38-3.61 (m, 1H), 3.93
(s, 2H), 4.17 (d, J = 18.9 Hz, 3H), 4.49 (d, J = 19.3 Hz, 1H),
913.27 4.92 (d, J = 4.8 Hz, 1H), 5.43 (s, 1H), 5.49 (s, 1H),
5.53-5.77 (m, 1H), 6.11 (s, 1H), [M + H.sup.+] 6.28 (dd, J = 10.2,
1.8 Hz, 1H), 6.96 (d, J = 6.1 Hz, 1H), 7.10-7.41 (m, 8H), 7.83-8.20
(m, 1H) 83 Method r, .sup.1H NMR (DMSO-d.sub.6) .delta.: 0.84 (s,
3H), 0.93 (dd, J = 23.5, 6.9 Hz, 3H), 1.04-1.16 (m, 3H), 1.48 (s,
4H), Table 7 1.60-1.76 (m, 3H), 2.03 (d, J = 20.7 Hz, 1H),
2.17-2.26 (m, 1H), 2.24-2.40 (m, 2H), 2.55-2.72 (m, 2H), R.sub.t =
0.88 min; 2.96(d, J = 13.0 Hz, 3H), 3.55 (t, J = 7.3 Hz, 1H), 3.72
(d, J = 57.6 Hz, 3H), m/z = 943.52 3.93 (d, J = 4.7 Hz, 2H),
4.10-4.28 (m, 4H), 4.49 (d, J = 19.5 Hz, 1H), 4.93 (d, J =5.0 Hz,
1H), [M + H.sup.+] 5.44 (d, J = 2.9 Hz, 1H), 5.50 (d, J = 4.2 Hz,
1H), 5.63 (dt, J = 48.7, 9.8 Hz, 1H), 6.11 (s, 1H), 6.28 (dd, J =
10.2, 1.9 Hz, 1H), 6.82 (t, J = 7.1 Hz, 1H), 6.93-7.00 (m, 1H),
7.02-7.07 (m, 1H), 7.19 (d, J = 7.9 Hz, 1H), 7.24 (d, J = 10.0 Hz,
1H), 7.29-7.41 (m, 4H), 7.93 (d, J = 7.1 Hz, 1H) 84 Method r,
.sup.1H NMR (DMSO-d.sub.6) .delta.: 0.83 (s, 3H), 1.17 (d, J = 7.1
Hz, 3H), 1.25 (d, J = 7.1 Hz, 3H), 1.46 (s, 4H), Table 7 1.58-1.77
(m, 3H), 2.01 (dt, J = 13.8, 4.1 Hz, 1H), 2.23 (dtd, J = 25.2,
12.3, 10.9, 5.8 Hz, 2H), R.sub.t = 0.82 min; 2.31-2.40 (m, 2H),
2.49-2.73 (m, 1H), 3.56 (t, J = 7.3 Hz, 2H), 3.74 (s, 3H), 3.84 (s,
2H), m/z = 929.45 4.11-4.20 (m, 2H), 4.27 (p, J = 7.1 Hz, 1H), 4.39
(p, J = 7.2 Hz, 1H), 4.47 (d, J = 19.4 Hz, 1H), [M + H.sup.+] 4.91
(d, J = 4.8 Hz, 1H), 5.41 (s, 1H), 5.42-5.50 (m, 1H), 5.50-5.78 (m,
1H), 6.09 (s, 1H), 6.26 (dd, J = 10.1, 1.9 Hz, 1H), 6.70 (dd, J =
8.3, 1.8 Hz, 1H), 6.89 (d, J = 1.8 Hz, 1H), 6.95 (s, 2H), 7.23 (d,
J = 8.5 Hz, 3H), 7.31 (d, J = 7.9 Hz, 2H), 7.87 (d, J = 8.2 Hz,
1H), 8.14 (d, J = 7.5 Hz, 1H), 8.23 (d, J = 7.2 Hz, 1H), 8.81 (s,
1H) 85 Method r, .sup.1H NMR (DMSO-d.sub.6) .delta.: 0.84 (s, 3H),
1.16 (d, J = 7.1 Hz, 3H), 1.27 (d, J = 7.1 Hz, 3H), Table 7 1.39
(qd, J = 13.1, 5.2 Hz, 1H), 1.48 (s, 3H), 1.55-1.72 (m, 3H),
1.78-1.90 (m, 1H), R.sub.t = 0.73 min; 2.03 (d, J = 25.4 Hz, 1H),
2.13 (td, J = 12.2, 6.8 Hz, 1H), 2.29-2.41 (m, 3H), m/z = 897.3
2.48 (p, J = 1.9 Hz, 1H), 2.58-2.69 (m, 1H), 3.59 (t, J = 7.3 Hz,
2H), 4.09-4.26 (m, 3H), [M + H.sup.+] 4.29-4.44 (m, 3H), 4.47 (d, J
= 19.4 Hz, 1H), 4.82-4.91 (m, 1H), 5.32 (s, 1H), 5.41 (s, 1H), 6.02
(d, J = 1.7 Hz, 1H), 6.22 (dd, J = 10.1, 1.9 Hz, 1H), 6.47 (d, J =
8.7 Hz, 1H), 6.97 (s, 2H), 7.18-7.21 (m, 2H), 7.23 (t, J = 6.2 Hz,
1H), 7.27 (d, J = 10.1 Hz, 1H), 7.33 (dd, J = 8.7, 2.3 Hz, 1H),
7.44-7.57 (m, 2H), 7.95 (d, J = 2.3 Hz, 1H), 8.04 (d, J = 7.3 Hz,
1H), 8.10-8.23 (m, 2H), 9.73 (s, 1H) 86 Method r, .sup.1H NMR
(DMSO-d.sub.6) .delta.: 0.83 (s, 3H), 1.15 (d, J = 7.2 Hz, 3H),
1.26 (d, J = 7.1 Hz, 3H), Table 7 1.36 (d, J = 6.7 Hz, 4H), 1.48
(s, 3H), 1.63 (t, J = 11.1 Hz, 3H), 1.74-1.90 (m, 1H), R.sub.t =
0.73 min; 1.93-2.19 (m, 2H), 2.26-2.41 (m, 3H), 2.48 (p, J =
1.8 Hz, 1H), 2.58-2.70 (m, 1H), m/z = 911.46 3.59 (t, J = 7.3 Hz,
2H), 4.09-4.27 (m, 3H), 4.33 (p, J = 7.2 Hz, 1H), [M + H.sup.+]
4.46 (d, J = 19.4 Hz, 1H), 4.85 (d, J =4.1 Hz, 1H), 4.93 (t, J =
6.9 Hz, 1H), 5.04 (s, 1H), 5.30 (s, 1H), 5.40 (dd, J = 4.5, 1.7 Hz,
1H), 6.02 (s, 1H), 6.21 (dd, J = 10.1, 1.9 Hz, 1H), 6.48 (s, 1H),
6.97 (s, 2H), 7.26 (t, J = 9.3 Hz, 3H), 7.32 (d, J = 8.0 Hz, 1H),
7.48 (d, J = 8.3 Hz, 2H), 7.90 (d, J = 2.2 Hz, 1H), 8.03 (d, J =
7.3 Hz, 1H), 8.16 (d, J = 7.0 Hz, 1H), 9.71 (s, 1H) 87 Method r,
.sup.1H NMR (DMSO-d.sub.6) .delta.: 0.83 (s, 3H), 1.15 (d, J = 7.1
Hz, 3H), 1.27 (d, J = 7.1 Hz, 3H), Table 7 1.35 (d, J = 6.8 Hz,
4H), 1.48 (s, 3H), 1.55-1.70 (m, 3H), 1.77-1.88 (m, 1H), R.sub.t =
0.72 min; 1.99 (d, J = 13.5 Hz, 1H), 2.12 (td, J = 12.3, 6.6 Hz,
1H), 2.28-2.40 (m, 3H), m/z = 911.64 2.48 (p, J = 1.8 Hz, 1H),
2.56-2.69 (m, 1H), 3.59 (t, J = 7.3 Hz, 2H), 4.06-4.28 (m, 3H), [M
+ H.sup.+] 4.33 (p, J = 7.2 Hz, 1H), 4.45 (dd, J = 19.4, 6.4 Hz,
1H), 4.85 (d, J = 4.9 Hz, 1H), 4.93 (t, J = 7.2 Hz, 1H), 5.03 (t, J
= 6.0 Hz, 1H), 5.28 (s, 1H), 5.40 (dd, J = 4.4, 1.9 Hz, 1H), 6.02
(d, J = 2.1 Hz, 1H), 6.22 (dd, J = 10.1, 1.9 Hz, 1H), 6.43 (d, J =
8.6 Hz, 1H), 6.97 (s, 2H), 7.16 (d, J = 7.8 Hz, 1H), 7.20-7.33 (m,
4H), 7.44-7.49 (m, 2H), 7.90 (d, J = 2.3 Hz, 1H), 8.03 (d, J = 7.3
Hz, 1H), 8.17 (d, J = 7.1 Hz, 1H), 9.71 (s, 1H) 88 Method r,
.sup.1H NMR (DMSO-d.sub.6) .delta.: 0.84 (s, 3H), 1.02 (ddd, J =
21.3, 12.1, 4.2 Hz, 2H), 1.15 (d, J = 7.1 Hz, 3H), Table 7 1.26 (d,
J = 7.1 Hz, 3H), 1.37 (s, 3H), 1.53-1.81 (m, 4H), 2.00 (dd, J =
12.2, 5.5 Hz, 1H), R.sub.t = 0.87 min; 2.04-2.15 (m, 1H), 2.23-2.33
(m, 1H), 2.38 (dd, J = 8.0, 6.6 Hz, 2H), 2.51 (d, J = 18.2 Hz, 1H),
m/z = 863.32 3.59 (t, J = 7.3 Hz, 2H), 3.87 (s, 2H), 4.16 (d, J =
19.4 Hz, 1H), 4.21 (p, J = 7.1 Hz, 1H), [M + H.sup.+] 4.27 (q, J =
3.3 Hz, 1H), 4.32 (p, J = 7.1 Hz, 1H), 4.48 (d, J = 19.4 Hz, 1H),
4.72 (s, 2H), 4.90 (d, J = 5.4 Hz, 1H), 5.37 (s, 1H), 5.90 (t, J =
1.6 Hz, 1H), 6.13 (dd, J = 10.1, 1.8 Hz, 1H), 6.88 (dt, J =7.9, 1.3
Hz, 1H), 6.96 (s, 2H), 7.14-7.22 (m, 3H), 7.29 (d, J = 10.1 Hz,
1H), 7.33-7.38 (m, 2H), 7.43 (dd, J = 7.8, 1.1 Hz, 2H), 8.03 (d, J
= 7.2 Hz, 1H), 8.17 (d, J = 7.0 Hz, 1H), 9.71 (s, 1H) 89 Method m,
.sup.1H NMR (MeOH-d.sub.4) .delta.: 1.01 (d, J = 8.5 Hz, 3H),
1.10-1.30 (m, 2H), 1.34 (dd, J = 10.5, 7.1 Hz, 3H), Table 7 1.45
(dd, J = 7.2, 3.6 Hz, 3H), 1.52 (s, 3H), 1.80 (t, J = 13.0 Hz, 1H),
1.90 (p, J = 8.3, 7.3 Hz, 1H), R.sub.t = 1.99 min; 2.04 (d, J =
12.4 Hz, 1H), 2.12-2.32 (m, 2H), 2.42 (d, J = 11.2 Hz, 1H),
2.46-2.58 (m, 2H), 2.60-2.78 (m, 1H), m/z = 863 3.68-3.84 (m, 2H),
3.96 (d, J = 6.0 Hz, 2H), 4.12 (d, J = 19.3 Hz, 1H), 4.16-4.27 (m,
1H), 4.27-4.38 (m, 1H), [M + H.sup.+] 4.43 (d, J = 6.2 Hz, 2H),
5.40 (d, J = 6.3 Hz, 1H), 6.05 (s, 1H), 6.12 (d, J = 4.4 Hz, 1H),
6.28 (dd, J = 9.9, 1.8 Hz, 1H), 6.75 (d, J = 3.3 Hz, 2H), 6.96 (d,
J = 7.7 Hz, 1H), 7.15-7.30 (m, 5H), 7.43 (d, J = 18.7 Hz, 3H), 7.56
(d, J = 8.2 Hz, 3H) 90 Method r, .sup.1H NMR (DMSO-d.sub.6)
.delta.: 0.79 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H), 0.84
(s, 2H), 0.95-1.12 (m, 2H), Table 7 1.26 (d, J = 7.1 Hz, 3H), 1.38
(d, J = 4.7 Hz, 3H), 1.54-1.77 (m, 4H), 1.91 (h, J = 6.8 Hz, 1H),
R.sub.t = 0.91 min; 1.96-2.05 (m, 1H), 2.04-2.17 (m, 1H), 2.23-2.34
(m, 1H), 2.37-2.47 (m, 2H), 2.49-2.58 (m, 1H), m/z = 891.36
3.51-3.67 (m, 2H), 3.87 (s, 2H), 4.11 (s, 1H), 4.16 (d, J = 19.4
Hz, 1H), 4.27 (q, J = 3.4 Hz, 1H), [M + H.sup.+] 4.32 (p, J = 7.1
Hz, 1H), 4.48 (d, J = 19.4 Hz, 1H), 4.73 (s, 1H), 4.90 (d, J = 5.3
Hz, 1H), 5.38 (s, 1H), 5.90 (d, J = 1.6 Hz, 1H), 6.13 (dt, J =
10.1, 1.8 Hz, 1H), 6.88 (dt, J = 7.7, 1.3 Hz, 1H), 6.96 (s, 2H),
7.18 (dd, J = 16.7, 8.1 Hz, 3H), 7.29 (d, J = 10.1 Hz, 1H),
7.32-7.40 (m, 3H), 7.43 (ddd, J = 8.1, 2.2, 1.0 Hz, 1H), 7.99 (d, J
= 8.4 Hz, 1H), 8.10 (d, J = 7.0 Hz, 1H), 9.74 (s, 1H) 99 Method r,
.sup.1H NMR (DMSO-d.sub.6) .delta.: 0.83 (s, 3H), 1.13 (d, J = 7.1
Hz, 3H), 1.24 (d, J = 7.1 Hz, 3H), Table 7 1.35 (qd, J = 13.3,
12.8, 5.1 Hz, 1H), 1.46 (s, 3H), 1.63 (q, J = 9.7, 8.5 Hz, 3H),
1.73-1.88 (m, 1H), R.sub.t = 0.85 min; 2.01 (dt, J = 13.7, 3.5 Hz,
1H), 2.14 (td, J = 11.8, 7.2 Hz, 1H), 2.26-2.40 (m, 3H), 2.48-2.69
(m, 2H), m/z = 881.46 3.57 (t, J = 7.3 Hz, 2H), 3.85 (s, 2H), 4.17
(ddd, J = 17.5, 11.7, 6.2 Hz, 3H), 4.30 (p, J = 7.2 Hz, 1H), [M +
H.sup.+] 4.47 (d, J = 19.4 Hz, 1H), 4.83-4.95 (m, 1H), 5.40 (s,
2H), 5.99 (d, J = 1.6 Hz, 1H), 6.20 (dd, J = 10.1, 1.9 Hz, 1H),
6.87 (d, J = 7.5 Hz, 1H), 6.95 (s, 2H), 7.16 (t, J = 7.9 Hz, 1H),
7.20 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 10.1 Hz, 1H), 7.31 (d, J =
8.0 Hz, 2H), 7.38 (d, J = 1.9 Hz, 1H), 7.43 (dd, J = 8.0, 2.0 Hz,
1H), 8.01 (d, J = 7.3 Hz, 1H), 8.14 (d, J = 7.1 Hz, 1H), 9.70 (s,
1H) 100 Method r, .sup.1H NMR (DMSO-d.sub.6) .delta.: 0.93 (s, 3H),
1.15 (d, J = 7.2 Hz, 3H), 1.26 (d, J = 7.1 Hz, 3H), 1.48 (s, 4H),
Table 7 1.73 (dd, J = 25.4, 11.3 Hz, 3H), 2.00 (d, J = 14.1 Hz,
1H), 2.12-2.26 (m, 1H), 2.27 (s, 1H), R.sub.t = 0.87 min; 2.37 (q,
J = 8.3, 7.8 Hz, 2H), 2.65 (d, J = 33.3 Hz, 1H), 3.59 (t, J = 7.3
Hz, 2H), 3.88 (s, 2H), m/z = 933.0 4.14-4.27 (m, 2H), 4.32 (t, J =
7.2 Hz, 1H), 4.92 (d, J = 3.5 Hz, 1H), 5.50 (s, 1H), [M + H.sup.+]
5.55 (s, 1H), 5.54-5.72 (m, 1H), 5.79-6.04 (m, 2H), 6.11 (s, 1H),
6.28 (dd, J = 10.1, 1.9 Hz, 1H), 6.90 (d, J = 7.7 Hz, 1H), 6.97 (s,
2H), 7.18 (t, J = 7.8 Hz, 1H), 7.24 (t, J = 9.5 Hz, 3H), 7.34 (d, J
= 7.8 Hz, 2H), 7.40 (s, 1H), 7.46 (d, J = 8.2 Hz, 1H), 8.03 (d, J =
7.3 Hz, 1H), 8.16 (d, J = 7.1 Hz, 1H), 9.72 (s, 1H) 101 Method r,
.sup.1H NMR (DMSO-d.sub.6) .delta.: 0.99 (s, 3H), 1.15 (d, J = 7.1
Hz, 3H), 1.25 (d, J = 7.1 Hz, 3H), 1.49 (s, 4H), Table 7 1.60-1.75
(m, 2H), 1.79 (d, J = 14.0 Hz, 1H), 1.94 (dt, J = 14.4, 3.5 Hz,
1H), R.sub.t = 0.84 min; 2.20 (q, J = 10.4 Hz, 1H), 2.24-2.33 (m,
1H), 2.38 (dd, J = 8.0, 6.5 Hz, 2H), m/z = 885.41 2.62 (dtd, J =
30.0, 12.0, 11.5, 4.1 Hz, 1H), 3.59 (t, J = 7.3 Hz, 2H), 3.87 (s,
2H), [M + H.sup.+] 4.16 (d, J = 9.1 Hz, 1H), 4.21 (p, J = 7.2 Hz,
1H), 4.32 (p, J = 7.2 Hz, 1H), 5.00 (t, J = 2.9 Hz, 1H), 5.40-5.47
(m, 1H), 5.48 (s, 1H), 5.54-5.72 (m, 1H), 6.11 (s, 1H), 6.27 (dd, J
= 10.2, 1.9 Hz, 1H), 6.89 (d, J = 7.6 Hz, 1H), 6.96 (s, 2H), 7.17
(t, J = 7.9 Hz, 1H), 7.21 (d, J = 8.0 Hz, 2H), 7.24 (dd, J = 10.3,
1.5 Hz, 1H), 7.32 (d, J = 7.9 Hz, 2H), 7.39 (t, J = 1.9 Hz, 1H),
7.46 (dd, J = 8.1, 2.1 Hz, 1H), 8.04 (d, J = 7.3 Hz, 1H), 8.16 (d,
J = 7.1 Hz, 1H), 9.73 (s, 1H) 102 Method r, .sup.1H NMR
(DMSO-d.sub.6) .delta.: 0.85 (s, 3H), 1.16 (d, J = 7.1 Hz, 3H),
1.28 (d, J = 7.1 Hz, 3H), 1.48 (s, 3H), Table 7 1.56 (p, J = 12.4,
12.0 Hz, 1H), 1.62-1.76 (m, 3H), 1.98-2.10 (m, 1H), 2.22 (td, J =
12.3, 6.6 Hz, 1H), R.sub.t = 0.77 min; 2.24-2.33 (m, 1H), 2.38 (td,
J = 7.0, 1.0 Hz, 2H), 2.54-2.72 (m, 1H), 3.59 (t, J = 7.3 Hz, 2H),
4.15-4.25 (m, 3H), m/z = 917.22 4.33 (p, J = 7.1 Hz, 1H), 4.50 (d,
J = 19.4 Hz, 1H), 4.93 (d, J = 5.0 Hz, 1H), 5.36 (s, 1H), [M +
H.sup.+] 5.50 (s, 1H), 5.55-5.73 (m, 1H), 6.11 (q, J = 1.5 Hz, 1H),
6.27 (dd, J = 10.2, 1.9 Hz, 1H), 6.78-6.83 (m, 2H), 6.84 (d, J =
1.8 Hz, 2H), 6.97 (s, 2H), 7.01 (d, J = 1.6 Hz, 1H), 7.25 (dd, J =
10.1, 1.5 Hz, 1H), 7.45-7.61 (m, 2H), 8.04 (d, J = 7.3 Hz, 1H),
8.17 (d, J = 7.0 Hz, 1H), 9.73 (s, 1H) 103 Method r, .sup.1H NMR
(DMSO-d.sub.6) .delta.: 0.84 (s, 3H), 1.15 (d, J = 7.3 Hz, 3H),
1.25 (d, J = 7.1 Hz, 3H), 1.48 (s, 3H), Table 7 1.51 (d, J =7.3 Hz,
4H), 1.67 (d, J = 14.2 Hz, 2H), 2.02 (d, J = 13.7 Hz, 1H),
2.13-2.34 (m, 2H), R.sub.t = 0.84 min; 2.38 (t, J = 7.3 Hz, 2H),
2.65 (s, 1H), 3.59 (t, J = 7.3 Hz, 2H), 4.08 (d, J = 7.1 Hz, 1H),
m/z = 935.4 4.12-4.24 (m, 2H), 4.32 (t, J = 7.2 Hz, 1H), 4.48 (dd,
J = 19.6, 6.3 Hz, 1H), [M + Na.sup.+] 4.92 (d, J =4.8 Hz, 1H), 5.07
(t, J = 5.8 Hz, 1H), 5.72-5.54 (m, 1H), 5.41 (s, 1H), 5.49 (s, 1H),
6.11 (s, 1H), 6.28 (d, J = 9.9 Hz, 1H), 6.88-6.95 (m, 1H), 6.97 (s,
2H), 7.17 (t, J = 7.9 Hz, 1H), 7.25 (d, J = 8.1 Hz, 3H), 7.33 (d, J
= 7.9 Hz, 2H), 7.45 (d, J = 12.0 Hz, 2H), 8.03 (d, J = 7.2 Hz, 1H),
8.16 (d, J =6.9 Hz, 1H), 9.71 (s, 1H) 104 Method r, .sup.1H NMR
(DMSO-d.sub.6) .delta.: 0.85 (s, 3H), 1.16 (d, J = 7.1 Hz, 3H),
1.26 (d, J = 7.1 Hz, 3H), 1.49 (s, 3H), Table 7 1.52 (d, J = 7.3
Hz, 3H), 1.69 (t, J = 12.7 Hz, 3H), 2.04 (d, J = 13.9 Hz, 1H),
2.18-2.33 (m, 2H), R.sub.t = 0.84 min; 2.33-2.42 (m, 2H), 2.56-2.74
(m, 1H), 3.60 (t, J = 7.3 Hz, 2H), 4.09 (q, J = 7.2 Hz, 1H), m/z =
935.4 4.20 (tq, J = 13.0, 6.3, 5.6 Hz, 3H), 4.32 (p, J = 7.1 Hz,
1H), 4.49 (dd, J = 19.5, 6.3 Hz, 1H), [M + Na.sup.+] 4.93 (d, J =
5.1 Hz, 1H), 5.08 (t, J = 6.0 Hz, 1H), 5.42 (s, 1H), 5.51 (d, J =
4.2 Hz, 1H), 5.64 (dt, J = 48.9, 8.9 Hz, 1H), 6.12 (s, 1H), 6.29
(dd, J = 10.0, 1.9 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 6.97 (s, 2H),
7.18 (t, J = 7.9 Hz, 1H), 7.23-7.29 (m, 3H), 7.34 (d, J = 8.2 Hz,
2H), 7.44 (d, J = 2.1 Hz, 1H), 7.44-7.49 (m, 1H), 8.03 (d, J = 7.3
Hz, 1H), 8.17 (d, J = 7.1 Hz, 1H), 9.72 (s, 1H) 105 Method r,
.sup.1H NMR (DMSO-d.sub.6) .delta.: 0.84 (s, 3H), 1.15 (d, J = 7.2
Hz, 3H), 1.26(d, J = 7.1 Hz, 3H), 1.44-1.58 (m, 7H), Table 7 1.67
(d, J = 13.8 Hz, 2H), 2.03 (d, J = 14.0 Hz, 1H), 2.18-2.34 (m, 2H),
2.38 (t, J = 7.3 Hz, 2H), R.sub.t = 0.80 min; 2.50-2.72 (m, 1H),
3.58 (t, J = 7.3 Hz, 2H), 4.07 (p, J = 7.3 Hz, 1H), 4.11-4.26 (m,
2H), m/z = 913.75 4.33 (d, J = 7.2 Hz, 1H), 4.48 (d, J = 19.3 Hz,
1H), 4.92 (d, J = 4.8 Hz, 1H), 5.41 (s, 1H), [M + H.sup.+]
5.45-5.53 (m, 1H), 5.62 (dd, J = 48.5, 9.8 Hz, 1H), 6.11 (s, 1H),
6.28 (d, J = 10.0 Hz, 1H), 6.97 (s, 2H), 7.11-7.20 (m, 2H),
7.20-7.29 (m, 3H), 7.32 (d, J = 8.0 Hz, 2H), 7.47 (d, J = 8.3 Hz,
2H), 8.03 (d, J =7.2 Hz, 1H), 8.16 (d, J = 7.0 Hz, 1H), 9.71 (s,
1H) 106 Method r, .sup.1H NMR (DMSO-d.sub.6) .delta.: 0.84 (s, 3H),
1.15 (d, J = 7.1 Hz, 3H), 1.26 (d, J = 7.1 Hz, 3H), 1.48 (s, 3H),
Table 7 1.48-1.54 (m, 4H), 1.60-1.75 (m, 2H), 2.03 (dt, J = 13.8,
3.6 Hz, 1H), 2.08-2.18 (m, 1H), R.sub.t = 0.84 min; 2.18-2.25 (m,
1H), 2.25-2.32 (m, 1H), 2.38 (dd, J = 8.1, 6.4 Hz, 2H), 2.54-2.72
(m, 1H), m/z = 913.46 3.59 (t, J = 7.3 Hz, 2H), 4.06 (dq, J = 14.9,
7.0 Hz, 1H), 4.11-4.28 (m, 3H), [M + H.sup.+] 4.33 (p, J = 7.1 Hz,
1H), 4.48 (d, J = 19.6 Hz, 1H), 4.92 (d, J = 5.0 Hz, 1H), 5.42 (s,
1H), 5.45-5.54 (m, 1H), 5.54-5.73 (m, 1H), 6.12 (d, J = 2.2 Hz,
1H), 6.26-6.33 (m, 1H), 6.97 (d, J = 1.3 Hz, 2H), 7.10-7.18 (m,
3H), 7.20-7.28 (m, 3H), 7.29-7.36 (m, 2H), 7.47 (dd, J = 8.6, 4.4
Hz, 2H), 8.04 (d, J = 7.3 Hz, 1H), 8.17 (d, J = 7.1 Hz, 1H), 9.71
(d, J = 2.9 Hz, 1H) 107 Method r, .sup.1H NMR (DMSO-d.sub.6)
.delta.: 0.84 (s, 3H), 0.94-1.11 (m, 2H), 1.16 (d, J = 7.1 Hz, 3H),
Table 7 1.27 (d, J = 7.1 Hz, 3H), 1.37 (s, 3H), 1.51-1.82 (m, 5H),
1.94-2.03 (m, 1H), 2.08 (d, J = 19.3 Hz, 1H), R.sub.t = 0.88 min;
2.30 (t, J = 8.2 Hz, 1H), 2.38 (t, J = 7.3 Hz, 2H), 2.48-2.59 (m,
1H), 3.60 (t, J = 7.3 Hz, 2H), m/z = 895.30 4.11-4.25 (m, 4H), 4.27
(d, J = 3.6 Hz, 1H), 4.32 (p, J = 7.2 Hz, 1H), 4.48 (d, J = 19.5
Hz, 1H), [M + H.sup.+] 4.90 (d, J = 5.0 Hz, 1H), 5.38 (s, 1H), 5.91
(d, J = 1.6 Hz, 1H), 6.14 (dd, J = 10.1, 1.8 Hz, 1H), 6.97 (s, 2H),
6.94-7.01 (m, 1H), 7.19 (t, J = 8.0 Hz, 1H), 7.25-7.37 (m, 2H),
7.37 (s, 4H), 7.66 (t, J = 1.9 Hz, 1H), 8.10 (d, J = 7.2 Hz, 1H),
8.20 (d, J = 6.9 Hz, 1H), 9.80 (s, 1H) 108 Method r, .sup.1H NMR
(DMSO-d.sub.6) .delta.: 0.85 (s, 3H), 1.16 (d, J = 7.1 Hz, 3H),
1.27 (d, J = 7.1 Hz, 3H), Table 7 1.37 (qd, J = 12.8, 5.2 Hz, 1H),
1.48 (s, 3H), 1.57-1.71 (m, 3H), 1.83 (dt, J = 11.7, 5.4 Hz, 1H),
R.sub.t = 0.83 min; 2.03 (dt, J = 13.8, 3.6 Hz, 1H), 2.14 (td, J =
12.1, 6.8 Hz, 1H), 2.30-2.36 (m, 1H), m/z = 913.0 2.38 (t, J =7.3
Hz, 2H), 2.42-2.57 (m, 1H), 2.63 (td, J = 13.3, 5.8 Hz, 1H), 3.59
(t, J = 7.3 Hz, 2H), [M + H.sup.+] 4.13-4.26 (m, 5H), 4.32 (p, J =
7.2 Hz, 1H), 4.49 (d, J = 19.4 Hz, 1H), 4.91 (d, J = 4.7 Hz, 1H),
5.42 (s, 2H), 6.02 (s, 1H), 6.21 (dd, J = 10.1, 1.9 Hz, 1H), 6.98
(dd, J = 7.9, 1.8 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 7.27 (d, J =
10.2 Hz, 1H), 7.29-7.44 (m, 5H), 7.61-7.70 (m, 1H), 8.07 (d, J =7.2
Hz, 1H), 8.18 (d, J = 7.0 Hz, 1H), 9.79 (s, 1H) 109 Method r,
.sup.1H NMR (DMSO-d.sub.6) .delta.: 0.85 (s, 3H), 1.19 (d, J = 7.1
Hz, 3H), 1.28 (d, J = 7.1 Hz, 3H), 1.49 (s, 4H), Table 7 1.61-1.78
(m, 3H), 2.04 (d, J = 13.7 Hz, 1H), 2.22 (dd, J = 12.3, 6.3 Hz,
1H), 2.26-2.32 (m, 1H), R.sub.t = 0.80 min; 2.37 (td, J = 7.0, 2.0
Hz, 2H), 2.55-2.73 (m, 1H), 3.58 (t, J = 7.3 Hz, 2H), 3.78 (s, 2H),
m/z = 915.54 4.18 (d, J = 19.4 Hz, 1H), 4.29 (p, J = 7.1 Hz, 1H),
4.39 (p, J = 7.1 Hz, 1H), [M + H.sup.+] 4.50 (d, J = 19.4 Hz, 1H),
4.93 (d, J = 5.2 Hz, 1H), 5.43 (s, 1H), 5.51 (dd, J = 4.5, 1.7 Hz,
1H), 5.54-5.75 (m, 1H), 6.12 (s, 1H), 6.29 (dd, J = 10.1, 1.9 Hz,
1H), 6.72-6.78 (m, 2H), 6.98 (s, 2H), 7.19 (d, J = 7.9 Hz, 2H),
7.25 (dd, J = 10.1, 1.5 Hz, 1H), 7.29-7.37 (m, 2H), 7.76 (d, J =
1.8 Hz, 1H), 8.18 (d, J = 7.5 Hz, 1H), 8.30 (d, J = 7.1 Hz, 1H),
8.92 (s, 1H), 9.72 (s, 1H) 110 Method r, .sup.1H NMR (DMSO-d.sub.6)
.delta.: 0.83 (s, 3H), 1.13 (d, J = 7.2 Hz, 3H), 1.25 (d, J = 7.2
Hz, 3H), Table 7 1.36 (qd, J = 12.7, 5.4 Hz, 1H), 1.46 (s, 3H),
1.55-1.72 (m, 3H), 1.75-1.87 (m, 1H), R.sub.t = 0.84 min; 1.93-2.05
(m, 1H), 2.05-2.18 (m, 1H), 2.25-2.40 (m, 3H), 2.47 (p, J = 1.9 Hz,
1H),
m/z = 899.0 2.53-2.69 (m, 1H), 3.56 (t, J = 7.3 Hz, 2H), 4.09-4.23
(m, 3H), 4.29 (p, J = 7.1 Hz, 1H), [M + H.sup.+] 4.49 (d, J = 19.5
Hz, 1H), 4.87-4.95 (m, 1H), 5.40 (s, 1H), 5.43 (s, 1H), 5.99 (d, J
= 1.7 Hz, 1H), 6.19 (dd, J = 10.1, 1.9 Hz, 1H), 6.95 (s, 2H), 7.00
(dt, J = 7.8, 1.3 Hz, 1H), 7.21-7.33 (m, 4H), 7.33-7.41 (m, 2H),
7.56 (ddd, J =8.2, 2.1, 1.0 Hz, 1H), 7.67 (t, J = 2.0 Hz, 1H), 8.05
(d, J = 7.1 Hz, 1H), 8.14 (d, J = 7.0 Hz, 1H), 9.87 (s, 1H) 111
Method r, .sup.1H NMR (DMSO-d.sub.6) .delta.: 0.84 (s, 3H), 1.04
(ddd, J = 14.7, 11.7, 4.1 Hz, 2H), 1.14 (d, J = 7.1 Hz, 3H), Table
7 1.26 (d, J = 7.1 Hz, 3H), 1.37 (s, 3H), 1.54-1.85 (m, 4H),
1.93-2.03 (m, 1H), 2.09 (d, J = 11.6 Hz, 1H), R.sub.t = 0.86 min;
2.23-2.33 (m, 1H), 2.36 (q, J = 6.4, 5.5 Hz, 2H), 2.50 (d, J = 9.7
Hz, 1H), 3.57 (t, J = 7.3 Hz, 2H), m/z = 881.38 4.12-4.24 (m, 2H),
4.24-4.34 (m, 2H), 4.48 (d, J = 19.5 Hz, 1H), 4.76 (s, 1H), 4.90
(d, J = 5.1 Hz, 1H), [M + H.sup.+] 5.39 (s, 1H), 5.89 (s, 1H), 6.14
(dd, J = 10.1, 1.9 Hz, 1H), 6.97 (s, 2H), 7.03 (d, J = 7.7 Hz, 1H),
7.22-7.35 (m, 4H), 7.42 (d, J = 8.2 Hz, 2H), 7.57 (dd, J = 8.2, 2.0
Hz, 1H), 7.71 (d, J = 2.0 Hz, 1H), 8.09 (d, J = 7.1 Hz, 1H), 8.17
(d, J = 6.9 Hz, 1H), 9.89 (s, 1H) 112 Method r, .sup.1H NMR
(DMSO-d.sub.6) .delta.: 0.84 (s, 3H), 1.02 (ddd, J = 21.5, 11.7,
4.1 Hz, 2H), 1.18 (d, J = 7.1 Hz, 3H), Table 7 1.27 (d, J = 7.1 Hz,
3H), 1.37 (s, 3H), 1.54-1.81 (m, 5H), 1.95-2.03 (m, 1H), 2.09 (dd,
J = 11.1, 4.0 Hz, 1H), R.sub.t = 0.85 min; 2.29 (d, J = 12.8 Hz,
1H), 2.36 (td, J =7.1, 2.0 Hz, 2H), 2.49-2.59 (m, 1H), 3.57 (d, J =
7.3 Hz, 2H), m/z = 879.30 3.77 (s, 2H), 4.15 (d, J = 19.4 Hz, 1H),
4.23-4.34 (m, 2H), 4.38 (p, J = 7.1 Hz, 1H), 4.47 (d, J = 19.5 Hz,
1H), [M + H.sup.+] 4.74 (s, 1H), 4.89 (d, J = 5.3 Hz, 1H), 5.37 (s,
1H), 5.90 (t, J = 1.6 Hz, 1H), 6.13 (dd, J = 10.1, 1.9 Hz, 1H),
6.73 (d, J = 1.8 Hz, 2H), 6.97 (s, 2H), 7.17 (d, 2H), 7.28 (d, J =
10.1 Hz, 1H), 7.31-7.36 (m, 2H), 7.76 (d, J = 1.5 Hz, 1H), 8.17 (d,
J = 7.6 Hz, 1H), 8.29 (d, J = 7.1 Hz, 1H), 8.90 (s, 1H), 9.72 (s,
1H) 113 Method r, .sup.1H NMR (DMSO-d.sub.6) .delta.: 0.84 (s, 3H),
1.04 (ddd, J = 34.7, 11.8, 4.1 Hz, 2H), 1.14 (d, J = 7.1 Hz, 3H),
Table 7 1.25 (d, J = 7.1 Hz, 3H), 1.38 (s, 3H), 1.54-1.81 (m, 4H),
1.95-2.04 (m, 1H), 2.10 (tt, J = 10.9, 5.9 Hz, 1H), R.sub.t = 0.80
min; 2.25-2.33 (m, 1H), 2.37 (dd, J = 8.0, 6.5 Hz, 2H), 2.49-2.59
(m, 1H), 3.58 (t, J = 7.3 Hz, 2H), m/z = 880.26 3.78 (s, 2H), 4.15
(d, J = 19.4 Hz, 1H), 4.21 (p, J = 7.1 Hz, 1H), 4.25-4.36 (m, 2H),
[M + H.sup.+] 4.45 (d, J = 19.4 Hz, 1H), 4.76 (s, 1H), 4.87 (d, J =
5.3 Hz, 1H), 5.27 (s, 1H), 5.92 (t, J = 1.6 Hz, 1H), 6.14 (dd, J =
10.1, 1.9 Hz, 1H), 6.77 (dd, J = 7.7, 1.6 Hz, 1H), 6.84-6.91 (m,
2H), 6.97 (s, 2H), 6.99 (d, J = 7.7 Hz, 1H), 7.11-7.17 (m, 1H),
7.30 (d, J = 10.1 Hz, 1H), 7.39-7.45 (m, 2H), 8.03 (d, J = 7.3 Hz,
1H), 8.16 (d, J = 7.2 Hz, 1H), 9.55 (s, 1H), 9.72 (s, 1H)
Example 36: Synthesis of N-(3-Aminophenyl)-4-((6aR, 6bS, 7S, 8aS,
8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,
8a-dimethyl-4-oxo-2, 4, 6a, 6b, 7,8,8a, 8b, 11a, 12, 12a,
12b-dodecahydro-1H-naphtho[2',
1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzamide
Step 1: Synthesis of 4-Formylbenzoyl Chloride
##STR00827##
[1101] Oxalyl chloride (17.51 mL, 200 mmol) was added drop-wise to
a 0.degree. C. solution of 4-formylbenzoic acid (15.01 g, 100 mmol)
in THF (100 mL), followed by N,N-dimethylformamide (0.387 mL, 5.00
mmol) in drop-wise addition. The mixture was allowed to warm to
room temperature and then stirred for an additional 2 h. The
mixture was concentrated in vacuo to give 4-formylbenzoyl chloride
(16.86 g, 100 mmol, 100% yield), which was used without further
purification.
Step 2: Synthesis of Tert-Butyl
(3-(4-formylbenzamido)phenyl)carbamate
##STR00828##
[1103] Triethylamine (63.4 mL, 455 mmol) was added drop-wise to a
0.degree. C. solution of 4-formylbenzoyl chloride (16.86 g, 100
mmol) in THF (100 mL), followed by addition of tert-butyl
(3-aminophenyl)carbamate (18.93 g, 91 mmol). After stirring at room
temperature for 2 h, the mixture was diluted with EtOAc (200 mL),
washed with water (2.times.100 mL) and brine (100 mL), dried over
Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was
triturated in 20 mL of EtOAc/PE (1:1), and the resulting
precipitate was collected to give tert-butyl
(3-(4-formylbenzamido)phenyl)carbamate (27.8 g, 82 mmol, 90% yield)
as a yellow solid. LCMS (Method e Table 7)R,=2.00 min; MS m/z=285
[M-t-Bu].
Step 3: Synthesis of N-(3-Aminophenyl)-4-((6aR, 6bS, 7S, 8aS, 8bS,
10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,
8a-dimethyl-4-oxo-2, 4, 6a, 6b, 7, 8,8a, 8b, 11a, 12, 12a,
12b-dodecahydro-1H-naphtho[2', 1':4, 5]indeno[1, 2-d][1,
3]dioxol-10-yl)benzamide
##STR00829##
[1105] To a stirred solution of (8S, 9S, 10R, 11S, 13S, 14S, 16R,
17S)-11, 16, 17-trihydroxy-17-(2-hydroxyacetyl)-10, 13-dimethyl-6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (9.42 g, 25.02
mmol), tert-butyl (3-(4-formylbenzamido)phenyl) carbamate, made in
a manner similar to Example 2, Step 5, (8.515 g, 25.02 mmol and
MgSO.sub.4 (12.04 g, 100 mmol) in MeCN(250 mL) was added drop-wise
trifluoromethanesulfonic acid (11.11 ml, 125 mmol) at 0.degree. C.
The mixture was stirred at 0.degree. C. for 2 hours and then warmed
to room temperature and stirred for additional 16 h. The mixture
was filtered and washed with THF, and the filtrate was concentrated
in vacuo. The residue was dissolved in THF (100 mL) and then
neutralized with 1 M NaOH aqueous solution to pH=8. The mixture was
extracted with EtOAc (200 mL), washed with water (2.times.100 mL)
and brine (100 mL), dried over Na.sub.2SO.sub.4, and concentrated
in vacuo. Purification by chromatography (silica) eluting with 5%
MeOH/DCM gave crude product, which was further purified by reverse
phase HPLC on a Sunfire C18 10 micron (250.times.19 mm column). A
gradient of MeCN (A) and 0.05% TFA in water (B) was used, at a flow
rate of 30 mL/min (0-10.0 min linear gradient22-32%, hold 5 min).
Combined fractions were frozen and lyophilized to give
N-(3-aminophenyl)-4-((6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS,
12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a, 8a-dimethyl-4-oxo-2, 4,
6a, 6b, 7, 8, 8a, 8b, 11a, 12, 12a, 12b-dodecahydro-1H-naphtho[2',
1':4, 5]indeno[1, 2-d][1, 3]dioxol-10-yl)benzamide (1.972 g, 3.29
mmol, 13% yield) as a white solid. LCMS (Method f, Table 7)R,=1.37
min; MS m/z=599 [M+H.sup.+]. .sup.1H NMR (400 MHz,
Methanol-d.sub.4) .delta. 8.01-7.92 (m, 3H), 7.64 (d, J=8.0 Hz,
2H), 7.55-7.40 (m, 3H), 7.05 (d, J=7.8 Hz, 1H), 6.27 (dd, J=10.2,
1.8 Hz, 1H), 6.03 (s, 1H), 5.60 (s, 1H), 5.13 (d, J=4.1 Hz, 1H),
4.68 (d, J=19.4 Hz, 1H), 4.45 (d, J=3.3 Hz, 1H), 4.37 (d, J=19.4
Hz, 1H), 2.68 (dt, J=14.5, 7.0 Hz, 1H), 2.41 (dd, J=13.7, 10.2 Hz,
1H), 2.29 (d, J=10.5 Hz, 1H), 2.18 (d, J=12.8 Hz, 1H), 1.99 (dd,
J=13.8, 3.5 Hz, 1H), 1.94-1.80 (m, 2H), 1.82-1.69 (m, 2H), 1.52 (s,
3H), 1.14 (m, J=16.8, 8.0 Hz, 2H), 1.02 (s, 3H).
[1106] Minor acetal isomer:
N-(3-Aminophenyl)-4-((6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-7-hydroxy-8b-
-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-
-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzamide
(112 mg, 0.176 mmol, 0.9% yield) as a yellow solid. LCMS (Method e,
Table 7) R.sub.t=1.53 min; MS m/z=599 [M+H.sup.+]. .sup.1H NMR (400
MHz, MeOH-d.sub.4) .delta. 7.90 (d, J=7.9 Hz, 2H), 7.48 (dd, J=8.1,
3.5 Hz, 3H), 7.15 (d, J=2.2 Hz, 1H), 7.09 (t, J=8.0 Hz, 1H), 6.96
(d, J=8.1 Hz, 1H), 6.59-6.51 (m, 1H), 6.28 (dd, J=10.1, 1.9 Hz,
1H), 6.25 (s, 1H), 6.05 (s, 1H), 5.51-5.37 (m, 1H), 4.45 (s, 1H),
4.30 (d, J=19.2 Hz, 1H), 4.14 (d, J=19.2 Hz, 1H), 2.70 (t, J=13.6
Hz, 1H), 2.43 (d, J=13.3 Hz, 1H), 2.22 (dd, J=23.3, 12.5 Hz, 2H),
2.07 (d, J=13.5 Hz, 1H), 1.93 (q, J=5.1, 3.5 Hz, 2H), 1.80 (d,
J=14.0 Hz, 2H), 1.53 (d, J=1.7 Hz, 3H), 1.21 (dd, J=41.7, 12.1 Hz,
2H), 1.03 (s, 3H).
Example 37: Synthesis of
N-(3-Aminophenyl)-4-((6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-6b-fluoro-7--
hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,-
12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl-
)benzamide
##STR00830##
[1108] Synthesized using the same procedure as Example 36 above.
Major acetal isomer:
N-(3-Aminophenyl)-4-((6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-6b-fluoro-7--
hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,-
12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl-
)benzamide. LCMS (Method f, Table 7)R,=1.35 min; MS m/z=617
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 10.29 (d,
J=7.4 Hz, 1H), 8.00-7.93 (m, 2H), 7.73 (s, 1H), 7.59 (d, J=8.1 Hz,
2H), 7.43 (s, OH), 7.38 (s, 1H), 7.29 (dd, J=10.0, 5.3 Hz, 2H),
6.81 (s, 1H), 6.24 (dd, J=10.1, 1.9 Hz, 1H), 6.05 (d, J=1.6 Hz,
1H), 5.62 (s, 1H), 5.49 (s, 1H), 5.03-4.96 (m, 1H), 4.58 (d, J=19.5
Hz, 1H), 4.23 (d, J=19.6 Hz, 1H), 2.73-2.52 (m, 1H), 2.40-2.32 (m,
1H), 2.25-2.12 (m, 1H), 2.11-2.02 (m, 1H), 1.92-1.84 (m, 1H),
1.76-1.67 (m, 3H), 1.51 (s, 3H), 1.40 (tt, J=14.3, 7.1 Hz, 1H),
0.90 (s, 3H).
[1109] Minor acetal isomer:
N-(3-Aminophenyl)-4-((6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-6b-fluoro-7--
hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,-
12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl-
)benzamide. LCMS (Method B, Table 7)R,=1.45 min; MS m/z=617
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm: 8.06
(s, 1H), 7.95 (d, J=8.4 Hz, 2H), 7.54-7.41 (m, 5H), 7.09 (d, J=6.8
Hz, 1H), 6.34 (d, J=10 Hz, 1H), 6.28 (s, 1H), 6.13 (s, 1H), 5.49
(d, J=6.4 Hz, 1H), 4.34-4.13 (m, 3H), 2.79-2.24 (m, 5H), 1.74-1.63
(m, 2H), 1.60 (s, 3H), 1.04 (s, 3H).
Example 38: Synthesis of
N-(3-Aminophenyl)-4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difl-
uoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,-
8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxo-
l-10-yl)benzamide
##STR00831##
[1111] Synthesized using the same procedure as Example 36 above.
Major acetal isomer:
N-(3-Aminophenyl)-4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difl-
uoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,-
8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxo-
l-10-yl)benzamide. LCMS (Method f, Table 7)R,=1.376 min, MS m/z=635
[M+H.sup.+]. .sup.1H NMR (400 MHz, MeOH-d.sub.4) .delta. 7.96 (d,
J=8.0 Hz, 2H), 7.88 (d, J=2.1 Hz, 1H), 7.64 (dd, J=8.3, 1.5 Hz,
2H), 7.48-7.42 (m, 1H), 7.39 (t, J=7.9 Hz, 1H), 7.34 (d, J=10.0 Hz,
1H), 6.98 (dt, J=7.7, 1.6 Hz, 1H), 6.41-6.26 (m, 2H), 5.71-5.45 (m,
2H), 5.14 (d, J=4.1 Hz, 1H), 4.69 (d, J=19.4 Hz, 1H), 4.44-4.28 (m,
2H), 2.73 (dt, J=25.9, 12.1 Hz, 1H), 2.41 (td, J=11.7, 6.9 Hz, 2H),
2.29 (dt, J=14.0, 3.6 Hz, 1H), 1.91-1.67 (m, 4H), 1.60 (s, 4H),
1.02 (s, 3H).
[1112] Minor acetal isomer:
N-(3-Aminophenyl)-4-((2S,6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-2,6b-difl-
uoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,-
8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxo-
l-10-yl)benzamide. LCMS (Method e, Table 7) R.sub.t=1.506 min, MS
m/z=635 [M+H.sup.+]. .sup.1H NMR (400 MHz, MeOH-d.sub.4) .delta.
7.90 (d, J=8.0 Hz, 2H), 7.49 (d, J=7.9 Hz, 2H), 7.35 (d, J=10.0 Hz,
1H), 7.16 (d, J=2.2 Hz, 1H), 7.09 (t, J=8.0 Hz, 1H), 6.96 (d, J=8.2
Hz, 1H), 6.65-6.48 (m, 1H), 6.41-6.27 (m, 3H), 5.76-5.39 (m, 2H),
4.42-4.22 (m, 2H), 4.15 (d, J=19.4 Hz, 1H), 2.66 (dd, J=27.8, 13.4
Hz, 1H), 2.47-2.24 (m, 3H), 2.08-1.85 (m, 2H), 1.75 (t, J=14.9 Hz,
2H), 1.61 (s, 3H), 1.03 (s, 3H).
Example 39: Synthesis of 3-Aminophenyl
4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b--
(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b--
dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzoate
Step 1: Synthesis of Tert-Butyl (3-hydroxyphenyl)carbamate
##STR00832##
[1114] To a solution of 3-aminophenol (10 g, 92 mmol) in THF(450
mL) was added Boc anhydride (23.40 mL, 101 mmol). The mixture was
heated at 85.degree. C. for 16 h, monitored by LCMS. After that,
the mixture was concentrated to obtain a residue, which was
dissolved in EtOAc (150 mL) and washed with water (100 mL),
saturated NaHCO.sub.3 (100 mL) and brine (100 mL), dried over
Na.sub.2SO.sub.4, and concentrated in vacuo. The crude material was
washed with PE (50 mL.times.2) to give the title compound (16.5 g,
76 mmol, 82% yield) as a white solid. LCMS (Method g, Table 7)
R.sub.t=1.66 min, MS m/z=232.1 [M+Na*].
Step 2: Synthesis of 3-((tert-Butoxycarbonyl)amino)phenyl
4-formylbenzoate
##STR00833##
[1116] To a solution of tert-butyl (3-hydroxyphenyl)carbamate (5 g,
23.90 mmol) in DCM (60 mL) at 0.degree. C. was added
4-formylbenzoic acid (3.59 g, 23.90 mmol), N,
N'-dicyclohexylcarbodiimide (7.40 g, 35.8 mmol) and
4-dimethylaminopyridine (0.584 g, 4.78 mmol). The resulting mixture
was stirred at that temperature for 10 min under an atmosphere of
argon. Then the mixture was warmed to room temperature and stirring
was continued for 16 h. The mixture was cooled in an ice bath. The
side product N, N'-dicyclohexylurea was filtered off as a
precipitate and the clear filtrate was concentrated under vacuum.
The crude material was purified by silica gel chromatography
eluting with DCM/EtOAc (100%30:1) to give the title compound (7.0
g, 18.54 mmol, 78% yield) as a white solid. LCMS (Method d, Table
7)R,=2.17 min, MS m/z=364.0 [M+Na*].
Step 3: Synthesis of 3-Aminophenyl
4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b--
(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b--
dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzoate
##STR00834##
[1118] To a stirred solution of (6S, 8S, 9R, 10S, 11S, 13S, 14S,
16R, 17S)-6, 9-difluoro-11, 16,
17-trihydroxy-17-(2-hydroxyacetyl)-10, 13-dimethyl-6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16,
17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (Step 5, Example
2) (7.248 g, 17.58 mmol) and 3-((tert-butoxycarbonyl)amino)phenyl
4-formylbenzoate (6 g, 17.58 mmol) in anhydrous MeCN (40 mL) and
THF (40 mL) at 0.degree. C. under nitrogen was added drop-wise
trifluoromethanesulfonic acid (7.8 mL, 87.9 mmol). The mixture was
stirred at 0.degree. C. for 1 h, then poured onto ice water (30 mL)
and extracted with EtOAc (2.times.45 mL). The combined organic
layers were washed with cooled water (2.times.30 mL), brine (30
mL), saturated NaHCO.sub.3 (30 mL) and additional water (30 mL),
concentrated in vacuo affording a yellow solid. The crude material
was purified by silica gel column chromatography (200-300 mesh),
eluting with DCM/MmeOH (100%.about.40:1) and then further purified
by prep HPLC to give the title compound (major acetal isomer)
(2.166 g, 3.32 mmol, 19% yield) as a white solid. LCMS (Method d,
Table 7)R,=1.54 min; MS m/z=636.3 [M+H.sup.+]. .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. 8.13 (d, J=8.2 Hz, 2H), 7.66 (d, J=8.2
Hz, 2H), 7.27 (dd, J=10.1, 1.4 Hz, 1H), 7.06 (t, J=8.0 Hz, 1H),
6.48 (dd, J=8.3, 2.1 Hz, 1H), 6.40 (t, J=2.2 Hz, 1H), 6.34 (dd,
J=7.8, 2.2 Hz, 1H), 6.30 (dd, J=10.1, 1.9 Hz, 1H), 6.12 (s, 1H),
5.82-5.47 (m, 3H), 5.31 (s, 2H), 5.15 (t, J=5.9 Hz, 1H), 5.03 (d,
J=5.1 Hz, 1H), 4.60 (dd, J=19.5, 6.4 Hz, 1H), 4.33-4.12 (m, 2H),
2.66 (ddd, J=26.2, 13.7, 9.3 Hz, 1H), 2.31 (d, J=10.9 Hz, 1H), 2.21
(td, J=12.4, 6.3 Hz, 1H), 2.12-1.98 (m, 1H), 1.84-1.64 (m, 3H),
1.50 (s, 4H), 0.89 (s, 3H).
[1119] 3-Aminophenyl 4-((2S, 6aS, 6bR, 7S, 8aS, 8bS, 10S, 11aR,
12aS, 12bS)-2, 6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,
8a-dimethyl-4-oxo-2, 4, 6a, 6b, 7, 8, 8a, 8b, 11a, 12, 12a,
12b-dodecahydro-1H-naphtho[2', 1':4, 5] indeno[1, 2-d][1,
3]dioxol-10-yl)benzoate, the minor acetal isomer was also isolated
(1.073 g, 1.676 mmol, 10% yield) as a white solid. LCMS (Method d,
Table 7) R.sub.t=1.58 min; MS m/z=636.3 [M+H.sup.+]. .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. 8.13-8.06 (m, 2H), 7.51 (d, J=8.2
Hz, 2H), 7.28 (dd, J=10.1, 1.4 Hz, 1H), 7.06 (t, J=8.0 Hz, 1H),
6.52-6.44 (m, 1H), 6.43-6.28 (m, 4H), 6.14 (s, 1H), 5.80-5.48 (m,
2H), 5.39 (d, J=6.8 Hz, 1H), 5.31 (s, 2H), 5.04 (t, J=6.1 Hz, 1H),
4.26-4.15 (m, 2H), 4.05 (dd, J=19.2, 5.9 Hz, 1H), 2.67-2.51 (m,
1H), 2.29 (d, J=6.9 Hz, 1H), 2.27-2.14 (m, 1H), 2.11 (d, J=13.5 Hz,
1H), 1.96-1.59 (m, 4H), 1.51 (s, 3H), 0.90 (s, 3H).
Example 40: Synthesis of 3-Aminophenyl
4-((6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-6b-fluoro-7-hydroxy-8b-(2-hydr-
oxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecah-
ydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzoate
##STR00835##
[1121] Synthesized using the same procedure as Example 39 above.
Major acetal isomer: 3-aminophenyl
4-((6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-6b-fluoro-7-hydroxy-8b-(2-hydr-
oxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecah-
ydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzoate.
LCMS (Method d, Table 7)R,=1.54 min; MS m/z=618.3 [M+H.sup.+].
.sup.1H NMR (400 MHz, MeOH-d.sub.4) .delta. 8.19 (dd, J=7.8, 1.9
Hz, 2H), 7.67 (d, J=8.0 Hz, 2H), 7.41 (d, J=10.1 Hz, 1H), 7.35 (t,
J=8.0 Hz, 1H), 6.97-6.84 (m, 3H), 6.32 (dd, J=10.1, 1.9 Hz, 1H),
6.12 (s, 1H), 5.63 (s, 1H), 5.13 (d, J=5.0 Hz, 1H), 4.69 (d, J=19.4
Hz, 1H), 4.43-4.30 (m, 2H), 2.76 (td, J=13.6, 5.8 Hz, 1H),
2.70-2.54 (m, 1H), 2.43 (d, J=13.6 Hz, 1H), 2.31 (ddd, J=14.9,
11.6, 4.5 Hz, 2H), 2.01-1.92 (m, 1H), 1.89-1.69 (m, 3H), 1.62 (s,
4H), 1.03 (s, 3H).
[1122] Minor acetal isomer: 3-aminophenyl
4-((6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-6b-fluoro-7-hydroxy-8b-(2-hydr-
oxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecah-
ydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzoate.
LCMS (Method d, Table 7) R.sub.t=1.58 min; MS m/z=618.2
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.10 (d,
J=8.1 Hz, 2H), 7.52 (d, J=8.1 Hz, 2H), 7.30 (d, J=10.1 Hz, 1H),
7.16 (dd, J=9.8, 6.0 Hz, 1H), 6.64 (d, J=8.2 Hz, 1H), 6.61-6.51 (m,
2H), 6.31-6.19 (m, 2H), 6.05 (s, 1H), 5.47 (s, 1H), 5.38 (d, J=6.7
Hz, 1H), 4.19 (d, J=18.7 Hz, 2H), 4.04 (d, J=19.2 Hz, 1H), 2.66
(td, J=13.9, 6.3 Hz, 1H), 2.48-2.33 (m, 1H), 2.17-2.05 (m, 2H),
1.87 (dt, J=13.8, 7.0 Hz, 2H), 1.84-1.69 (m, 2H), 1.51 (s, 4H),
0.90 (s, 3H).
Example 41: Synthesis of 3-Aminophenyl
4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-
-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-na-
phtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzoate
##STR00836##
[1124] Synthesized using the same procedure as Example 39 above.
Major acetal isomer: 3-aminophenyl
4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-
-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-na-
phtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzoate. LCMS
(Method d, Table 7) R.sub.t=1.86 min; MS m/z=599.8 [M+H.sup.+].
.sup.1H NMR (400 MHz, MeOH-d.sub.4) .delta. 8.14 (d, J=8.2 Hz, 2H),
7.64 (d, J=8.1 Hz, 2H), 7.44 (d, J=10.0 Hz, 1H), 7.14 (t, J=8.0 Hz,
1H), 6.63 (dd, J=8.1, 2.2 Hz, 1H), 6.54 (q, J=2.6 Hz, 1H), 6.48
(dd, J=8.1, 2.2 Hz, 1H), 6.24 (dd, J=10.0, 2.0 Hz, 1H), 6.00 (s,
1H), 5.59 (s, 1H), 5.13 (d, J=4.4 Hz, 1H), 4.69 (d, J=19.4 Hz, 1H),
4.43 (q, J=3.3 Hz, 1H), 4.37 (d, J=19.4 Hz, 1H), 2.66 (td, J=13.4,
5.3 Hz, 1H), 2.38 (dd, J=13.7, 4.1 Hz, 1H), 2.32-2.19 (m, 1H), 2.14
(d, J=12.7 Hz, 1H), 2.06-1.93 (m, 1H), 1.94-1.85 (m, 1H), 1.89-1.68
(m, 3H), 1.50 (s, 3H), 1.22-1.01 (m, 2H), 1.02 (s, 3H).
[1125] Minor acetal isomer: 3-aminophenyl
4-((6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-
-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-na-
phtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzoate. LCMS
(Method d, Table 7)R,=1.89 min; MS m/z=599.8 [M+H.sup.+]. .sup.1H
NMR (400 MHz, MeOH-d.sub.4) .delta. 8.22-8.11 (m, 2H), 7.53 (d,
J=8.2 Hz, 2H), 7.49 (d, J=10.0 Hz, 1H), 7.14 (t, J=8.0 Hz, 1H),
6.63 (dd, J=8.1, 2.1 Hz, 1H), 6.55 (t, J=2.2 Hz, 1H), 6.53-6.45 (m,
1H), 6.33-6.24 (m, 2H), 6.05 (t, J=1.6 Hz, 1H), 5.46 (t, J=3.8 Hz,
1H), 4.46 (q, J=3.3 Hz, 1H), 4.30 (d, J=19.2 Hz, 1H), 4.15 (d,
J=19.2 Hz, 1H), 2.70 (td, J=13.5, 5.4 Hz, 1H), 2.48-2.38 (m, 1H),
2.23 (ddd, J=24.3, 12.6, 6.6 Hz, 2H), 2.12-2.03 (m, 1H), 2.02-1.89
(m, 2H), 1.89-1.77 (m, 2H), 1.53 (s, 3H), 1.26 (tt, J=12.4, 6.3 Hz,
1H), 1.17 (dd, J=11.1, 3.6 Hz, 1H), 1.03 (s, 3H).
Example 42: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-Aminophenoxy)methyl)phe-
nyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a-
,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-on-
e
Step 1: Synthesis of Tert-Butyl
(3-((4-formylbenzyl)oxy)phenyl)carbamate
##STR00837##
[1127] K.sub.2CO.sub.3 (47.8 g, 346 mmol) and tert-butyl
(3-hydroxyphenyl)carbamate (36.2 g, 173 mmol) were added
sequentially to a solution of 4-(bromomethyl)benzaldehyde (34.4 g,
173 mmol) in dimethyl formamide (200 mL). The yellow suspension was
then heated at 80.degree. C. in an oil bath for 2 h. The reaction
was quenched with water (200 mL) and extracted with EtOAc
(2.times.200 mL). The combined organic layers were washed with
brine (100 mL), dried over Na.sub.2SO.sub.4, and were concentrated
under reduced pressure. The residue obtained was purified by
chromatography (silica gel; petroleum ether to 80:20 PE/EtOAc;
gradient elution) to provide tert-butyl
(3-((4-formylbenzyl)oxy)phenyl)carbamate (47.27 g, 144 mmol, 83%
yield) as a white solid. LCMS (Method h Table 7)R,=1.92 min; MS
m/z=272 [M-t-Bu+H.sup.+].
Step 2: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-Aminophenoxy)methyl)phe-
nyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a-
,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-on-
e
##STR00838##
[1129] Trifluoromethanesulfonic acid (17.76 mL, 200 mmol) was added
drop-wise to a stirred 0.degree. C. suspension of
(8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)--
10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a-
]phenanthren-3-one (15.06 g, 40 mmol) and tert-butyl
(3-((4-formylbenzyl)oxy)phenyl) carbamate (13.75 g, 42.0 mmol), and
MgSO.sub.4 (19.26 g, 160 mmol) in MeCN (400 mL). The reaction
mixture was then warmed to 20.degree. C. and stirred for an
additional 2 h. The mixture was filtered and washed with THF, and
the filtrate was concentrated in vacuo. The residue was dissolved
in THF (100 mL), neutralized with 1 M aqueous NaOH to pH 8, diluted
with EtOAc (200 mL), washed with water (2.times.200 mL) and brine
(200 mL), dried (Na.sub.2SO.sub.4), and concentrated in vacuo. The
residue was purified by flash column (MeOH:DCM=1:20). The resulting
material was purified further by prep HPLC on a Sunfire C18 10
micron (250.times.19 mm column). A gradient of MeCN (A) and 0.05%
TFA in water (B) was used, at a flow rate of 30 mL/min (0-10.0 min
linear gradient 22-32% A, hold 5 min) to give the title compound
(7.338 g, 12.15 mmol, 30% yield) as a yellow solid. LCMS (Method i,
Table 7)R,=1.47 min; MS m/z=586 [M+H.sup.+]. .sup.1H NMR (400 MHz,
MeOD-d.sub.4) .delta. 7.502-7.446 (m, 5H), 7.389-7.349 (m, 1H),
7.009, 6.988 (dd, J1=2 Hz, J2=8.4 Hz, 1H), 6.890-6.859 (m, 2H),
6.275, 6.250 (dd, J1=1.2 Hz, J2=8.8 Hz, 1H), 6.027 (s, 1H), 5.501
(s, 1H), 5.147 (s, 2H), 5.107, 5.078 (dd, J1=6.8 Hz, J2=11.6 Hz,
1H), 4.672 (d, J=19.6 Hz, 1H), 4.436 (s, 1H), 4.370 (d, J=19.2 Hz,
1H), 2.706-2.671 (m, 1H), 2.652-2.265 (m, 3H), 2.002-1.700 (m, 5H),
1.512 (s, 3H), 1.151-1.112 (m, 1H), 1.054-1.009 (m, 4H).
[1130] Minor acetal isomer:
(6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((3-aminophenoxy)methyl)phe-
nyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a-
,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-on-
e, the minor acetal isomer was also isolated (354 mg, 0.604 mmol,
2% yield) as a yellow solid. LCMS (Method i, Table 7)R,=1.51 min;
MS m/z=586 [M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
7.416 (d, J=8 Hz, 2H), 7.332-7.276 (m, 3H), 6.879 (t, J=8 Hz, 1H),
6.185-6.115 (m, 5H), 5.948 (s, 1H), 5.319 (d, J=6.8 Hz, 1H),
5.041-5.014 (m, 3H), 4.980 (s, 2H), 4.791 (d, J=3.2 Hz, 1H),
4.302-4.239 (m, 2H), 4.056, 4.008 (dd, J1=6 Hz, J2=19.6 Hz, 1H),
2.552-2.540 (m, 1H), 2.337-2.304 (m, 1H), 2.075-2.005 (m, 2H),
1.884-1.736 (m, 5H), 1.395 (s, 3H), 1.219-1.045 (m, 2H), 0.892 (s,
3H).
Example 43: Synthesis of
(6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-aminophenoxy)methyl)phe-
nyl)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8-
,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]d-
ioxol-4-one
##STR00839##
[1132] Synthesized using the same procedure as Example 42 above.
Major acetal isomer:
(6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-aminophenoxy)methyl)phe-
nyl)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8-
,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]d-
ioxol-4-one. LCMS (Method i, Table 7)R,=1.74 min; MS m/z=604
[M+H.sup.+]. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.48-7.49
(m, 4H), 7.44-7.33 (m, 2H), 7.02 (dd, J=8.3, 1.9 Hz, 1H), 6.96-6.84
(m, 2H), 6.32 (dd, J=10.1, 1.8 Hz, 1H), 6.13 (s, 1H), 5.52 (s, 1H),
5.16 (s, 2H), 5.08 (d, J=4.9 Hz, 1H), 4.65 (d, J=19.4 Hz, 1H),
4.46-4.27 (m, 2H), 2.84-2.50 (m, 2H), 2.45-2.27 (m, 3H), 2.01-1.90
(m, 1H), 1.80-1.70 (m, 3H), 1.62 (s, 3H), 1.55 (dd, J=12.8, 4.8 Hz,
1H), 1.02 (s, 3H).
[1133] Minor acetal isomer:
(6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((3-aminophenoxy)methyl)phe-
nyl)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8-
,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]d-
ioxol-4-one. LCMS (Method i, Table 7)R,=1.77 min; MS m/z=604
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO) .delta. 7.42 (d, J=8.1 Hz,
2H), 7.36-7.25 (m, 3H), 7.01 (t, J=8.1 Hz, 1H), 6.43-6.30 (m, 3H),
6.24 (dd, J=10.1, 1.5 Hz, 1H), 6.12 (s, 1H), 6.04 (s, 1H), 5.47 (s,
1H), 5.35 (d, J=7.1 Hz, 1H), 5.02 (s, 2H), 4.31-4.14 (m, 2H), 4.04
(d, J=19.2 Hz, 1H), 2.72-2.58 (m, 1H), 2.18-1.98 (m, 2H), 1.85 (d,
J=6.9 Hz, 2H), 1.77-1.63 (m, 2H), 1.58-1.40 (m, 4H), 0.90 (s,
3H).
Example 44: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-Aminophenoxy)methyl)-
phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a-
,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d-
][1,3]dioxol-4-one
##STR00840##
[1135] Synthesized using the same procedure as Example 42 above.
Major acetal isomer:
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-aminophenoxy)methyl)-
phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a-
,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d-
][1,3]dioxol-4-one. LCMS (Method f, Table 7)R,=1.45 min; MS m/z=622
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO) .delta. 7.44 (s, 4H), 7.27
(d, J=10.1 Hz, 1H), 6.87 (t, J=8.0 Hz, 1H), 6.30 (dd, J=10.1, 1.5
Hz, 1H), 6.24-6.04 (m, 4H), 5.81-5.39 (m, 3H), 5.13 (t, J=5.9 Hz,
1H), 5.09-4.91 (m, 5H), 4.55 (dd, J=19.5, 6.4 Hz, 1H), 4.32-4.09
(m, 2H), 3.60 (t, J=6.3 Hz, 2H), 2.81-2.55 (m, 1H), 2.40-2.14 (m,
2H), 2.06 (d, J=13.6 Hz, 1H), 1.85-1.63 (m, 6H), 1.58-1.43 (m, 4H),
0.88 (s, 3H).
[1136] Minor acetal isomer:
(2S,6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((3-aminophenoxy)methyl)-
phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a-
,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d-
][1,3]dioxol-4-one. LCMS (Method f, Table 7)R,=1.49 min; MS m/z=622
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO) .delta. 7.41 (d, J=8.1 Hz,
3H), 7.35-7.20 (m, 3H), 6.88 (t, J=8.0 Hz, 1H), 6.31 (dd, J=10.1,
1.6 Hz, 1H), 6.16 (dd, J=13.9, 5.0 Hz, 6H), 5.77-5.45 (m, 2H), 5.36
(d, J=7.1 Hz, 1H), 4.35-4.13 (m, 2H), 4.05 (dd, J=18.9, 4.9 Hz,
1H), 2.70-2.53 (m, 1H), 2.29 (s, 1H), 2.24-2.13 (m, 1H), 2.12-2.04
(m, 1H), 1.96-1.81 (m, 1H), 1.81-1.63 (m, 3H), 1.50 (s, 4H), 0.89
(s, 3H).
Example 45: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-Aminobenzyl)oxy)phenyl)-
-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,-
12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one
Step 1: Synthesis of Tert-Butyl
(3-(hydroxymethyl)phenyl)carbamate
##STR00841##
[1138] To a solution of (3-aminophenyl)methanol (88.5 g, 719 mmol)
in THF (80 mL) was added di-tert-butyl dicarbonate (184 mL, 790
mmol). The mixture was stirred at 25.degree. C. overnight. The
mixture was then concentrated to dryness and the residue was
purified by silica gel column chromatography (eluted with
EtOAc/hexanes=1:9, v/v) to afford the title compound (161.1 g, 722
mmol, 100% yield), as a white solid. LCMS (Method j, Table 7)
R.sub.t=1.77 min; MS m/z=246 [M+Na.sup.+]. Step 2: Synthesis of
Tert-Butyl (3-(bromomethyl)phenyl)carbamate
##STR00842##
[1139] To a solution of tert-butyl
(3-(hydroxymethyl)phenyl)carbamate (120 g, 484 mmol) in THF (50 mL)
at -20.degree. C. was added triphenylphosphine (254 g, 967 mmol)
followed by N-bromosuccinimide (103 g, 580 mmol). After stirring
for 3 h, the solvent was removed in vacuo, and the residue was
purified by silica gel column chromatography (eluted with
hexane:EtOAc=100:1) to provide the title compound (125 g, 437 mmol,
90% yield) as a white solid. LCMS (Method j, Table 7) R.sub.t=2.10
min; MS m/z=230, 232 [M-t-Bu+H.sup.+].
Step 3: Synthesis of Tert-Butyl
(3-((4-formylphenoxy)methyl)phenyl)carbamate
##STR00843##
[1141] A mixture of 4-hydroxybenzaldehyde (25.6 g, 210 mmol) and
potassium carbonate (29.0 g, 210 mmol) in dimethyl formamide (300
mL) was stirred for 15 min. Then tert-butyl
(3-(bromomethyl)phenyl)carbamate (60 g, 210 mmol) was added. The
mixture was heated to 60.degree. C. and stirred for 2 hours at this
temperature. The mixture was poured into 50 mL of water, extracted
with EtOAc (3.times.50 mL). The combined organic layers was washed
with water (1.times.100 mL) and brine (1.times.100 mL),
concentrated in vacuum. The crude material was purified by silica
gel column chromatography (eluted with
dichloromethane/methanol=500:1) to afford the title compound (72 g,
209 mmol, 100% yield) as a white solid. LCMS (Method j, Table
7)R,=2.08 min; MS m/z=272 [M-t-Bu+H.sup.+].
Step 4: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-aminobenzyl)oxy)phenyl)-
-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,-
12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one
##STR00844##
[1143] Trifluoromethanesulfonic acid (12.38 mL, 139 mmol) was added
drop-wise to a mixture of magnesium sulfate (13.43 g, 112 mmol),
tert-butyl (3-((4-formylphenoxy)methyl)phenyl)carbamate (10.96 g,
33.5 mmol) and (8S,9S,10R,11S,13S,14S,16R,17S)-11, 16,
17-trihydroxy-17-(2-hydroxyacetyl)-10,
13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]p-
henanthren-3-one (10.5 g, 27.9 mmol) in MeCN (150 mL) at 0.degree.
C. The mixture was warmed to room temperature and stirred for 2
hours at this temperature. The mixture was filtered and the
filtrate was poured into 500 mL of saturated sodium bicarbonate
solution, extracted with EtOAc (250 mL). The organic layer was
washed with brine (200 mL) and water (200 mL), concentrated in
vacuo. The crude material was purified by silica gel column
chromatography (eluted with DCM-MeOH=50:1, v/v), and the resulting
product was purified further by prep-HPLC to afford the title
compound (6.04 g, 10.31 mmol, 37% yield) as a white solid. LCMS
(Method k, Table 7)R,=1.91 min; MS m/z=586 [M+H.sup.+]. .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. 7.35 (dd, J=19.6, 9.4 Hz, 3H),
7.09-6.89 (m, 3H), 6.61-6.33 (m, 3H), 6.18 (dd, J=10.1, 1.7 Hz,
1H), 5.95 (s, 1H), 5.38 (s, 1H), 5.16-5.01 (m, 3H), 5.02-4.85 (m,
3H), 4.80 (d, J=3.0 Hz, 1H), 4.50 (dd, J=19.5, 6.3 Hz, 1H), 4.31
(s, 1H), 4.18 (dd, J=19.4, 5.5 Hz, 1H), 2.33 (d, J=10.5 Hz, 1H),
2.17-1.98 (m, 2H), 1.90-1.53 (m, 5H), 1.40 (s, 3H), 1.13-0.96 (m,
2H), 0.87 (s, 3H).
Example 46: Synthesis of
(6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-aminobenzyl)oxy)phenyl)-
-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,-
8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxo-
l-4-one
##STR00845##
[1145] Synthesized using the same procedure as Example 45 above.
LCMS (Method k, Table 7) R,=1.89 min; MS m/z=604 [M+H.sup.+].
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.32 (dd, J=17.3, 9.4
Hz, 3H), 7.10-6.94 (m, 3H), 6.65-6.35 (m, 3H), 6.25 (dd, J=10.1,
1.7 Hz, 1H), 6.05 (s, 1H), 5.58-5.32 (m, 2H), 5.22-5.03 (m, 3H),
5.01-4.86 (m, 3H), 4.52 (dd, J=19.5, 6.4 Hz, 1H), 4.20 (dd, J=19.4,
5.5 Hz, 2H), 2.78-2.56 (m, 1H), 2.44-2.31 (m, 1H), 2.19 (td,
J=12.0, 6.8 Hz, 1H), 2.06 (d, J=13.7 Hz, 1H), 1.95-1.81 (m, 1H),
1.68 (dd, J=15.4, 9.7 Hz, 3H), 1.57-1.30 (m, 4H), 0.88 (s, 3H).
Example 47: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-aminobenzyl)oxy)phen-
yl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,-
7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,-
3]dioxol-4-one
##STR00846##
[1147] Synthesized using the same procedure as Example 45 above.
LCMS (Method C, Table 7) R,=1.45 min; MS m/z=622 [M+H.sup.+].
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.57-7.29 (m, 6H), 7.24
(d, J=7.7 Hz, 1H), 7.02 (d, J=8.6 Hz, 2H), 6.45-6.23 (m, 2H),
5.69-5.49 (m, 1H), 5.46 (s, 1H), 5.16 (s, 2H), 5.06 (d, J=3.7 Hz,
1H), 4.64 (d, J=19.5 Hz, 1H), 4.43-4.15 (m, 2H), 2.89-2.56 (m, 1H),
2.52-2.32 (m, 2H), 2.28 (d, J=13.8 Hz, 1H), 1.87-1.62 (m, 4H), 1.60
(s, 3H), 1.00 (s, 3H).
[1148] Minor acetal isomer:
(2S,6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((3-aminobenzyl)oxy)phen-
yl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,-
7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,-
3]dioxol-4-one. LCMS (Method C, Table 7)R,=1.48 min; MS m/z=622
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO) .delta. 7.41-7.13 (m, 3H),
7.08-6.90 (m, 3H), 6.61 (s, 1H), 6.52 (dd, J=17.3, 7.5 Hz, 2H),
6.31 (d, J=10.2 Hz, 1H), 6.11 (d, J=18.4 Hz, 2H), 5.79-5.56 (m,
1H), 5.53 (d, J=3.3 Hz, 1H), 5.34 (d, J=7.2 Hz, 1H), 5.18-5.00 (m,
3H), 4.93 (s, 2H), 4.28 (dd, J=19.1, 6.2 Hz, 1H), 4.19 (d, J=5.9
Hz, 1H), 4.05 (dd, J=19.1, 5.9 Hz, 1H), 3.60 (t, J=6.2 Hz, 3H),
2.72-2.51 (m, 1H), 2.29 (s, 1H), 2.22-2.11 (m, 1H), 2.06 (d, J=13.4
Hz, 1H), 1.93-1.80 (m, 1H), 1.80-1.60 (m, 6H), 1.50 (s, 3H), 1.36
(s, 1H), 0.89 (s, 3H).
Example 48: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-Aminophenyl)ethynyl)phe-
nyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a-
,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-on-
e
Step 1: Synthesis of Tert-Butyl (3-ethynylphenyl)carbamate
##STR00847##
[1150] Di-tert-butyl dicarbonate (123 ml, 531 mmol) was added to a
stirred solution of 3-ethynylaniline (56.6 g, 483 mmol) in THF (300
mL). The mixture was heated to reflux for overnight. The mixture
was then cooled to ambient temperature and taken up in ethyl
acetate (500 mL) and washed sequentially with 1N aqueous HCl (200
mL), saturated aqueous Na.sub.2CO.sub.3 (200 mL) and brine (200
mL). The organic layer was dried over Na.sub.2SO.sub.4,
concentrated in vacuo, and purified by silica gel column
chromatography (eluted with 15% EtOAc/PE) to give tert-butyl
(3-ethynylphenyl)carbamate (94 g, 435 mmol, 90% yield). LCMS
(Method f, Table 7)R,=1.80 min; MS m/z=162 [M-t-Bu+H.sup.+].
Step 2: Synthesis of Tert-Butyl
(3-((4-formylphenyl)ethynyl)phenyl)carbamate
##STR00848##
[1152] In a 500 mL of round bottom flask 4-iodobenzaldehyde (30.2
g, 130 mmol), bis(triphenylphosphine)palladium(II) chloride (4.56
g, 6.50 mmol), copper(I) iodide (2.476 g, 13.00 mmol) and
triphenylphosphine (3.41 g, 13.00 mmol) were dissolved in THF (200
mL) and triethylamine (181 mL, 1300 mmol) followed by addition of
tert-butyl (3-ethynylphenyl)carbamate (28.2 g, 130 mmol). The
mixture was stirred at 75.degree. C. under nitrogen atmosphere for
16 h. After completion of the reaction, the volatile solvents were
completely removed. The crude material was purified by silica gel
column chromatography (eluted with PE/CH.sub.2Cl.sub.2=1:3) to
obtain tert-butyl (3-((4-formylphenyl)ethynyl)phenyl)carbamate
(35.5 g, 111 mmol, 85% yield) as an off-white solid. LCMS (Method
f, Table 7)R,=2.08 min; MS m/z=322 [M+H.sup.+].
Step 3: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-Aminophenyl)ethynyl)phe-
nyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a-
,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-on-
e
##STR00849##
[1154] Trifluoromethanesulfonic acid (4.44 ml, 50.0 mmol) was added
drop-wise to a 0.degree. C. suspension of
(8S,9S,10R,11S,13S,14S,16R,17S)-11, 16,
17-trihydroxy-17-(2-hydroxyacetyl)-10,
13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]p-
henanthren-3-one (3.76 g, 10.00 mmol), tert-butyl
(3-((4-formylphenyl)ethynyl)phenyl) carbamate (3.21 g, 10 mmol) and
MgSO.sub.4 (4.81 g, 40.0 mmol) in MeCN (100 ml). The mixture was
stirred for additional 2 h. The mixture was filtered and washed
with THF. The filtrate was concentrated in vacuo. The residue was
dissolved in THF (50 mL), neutralized with 1 M aqueous NaOH aqueous
solution to pH=8, extracted with EtOAc (200 mL), washed with water
(2.times.100 mL) and brine (100 mL), dried over (Na.sub.2SO.sub.4,
and concentrated in vacuo. The residue was purified by column
chromatography (silica gel, eluted with MeOH:DCM=1:40) to give 2.5
g of the crude product which was further purified by prep-HPLC to
afford the title compound (1.449 g, 2.500 mmol, 25% yield) as a
yellow solid. LCMS (Method 1, Table 7)R,=1.86 min; MS m/z=580
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d) .delta. 7.65-7.41 (m,
4H), 7.32 (d, J=9.9 Hz, 2H), 7.05 (dd, J=9.8, 5.7 Hz, 1H),
6.85-6.49 (m, 4H), 6.29-6.05 (m, 1H), 6.01-5.83 (m, 1H), 5.63-5.40
(m, 1H), 5.26 (s, 2H), 5.12 (t, J=5.8 Hz, 1H), 4.96 (d, J=4.3 Hz,
1H), 4.82 (d, J=3.0 Hz, 1H), 4.63-4.41 (m, 1H), 4.37-4.08 (m, 2H),
2.40-1.91 (m, 5H), 1.87-1.52 (m, 6H), 1.40 (s, 4H), 1.14-0.95 (m,
2H), 0.88 (s, 3H).
[1155] The minor acetal isomer, (6aR, 6bS, 7S, 8aS, 8bS, 10S, 11aR,
12aS,
12bS)-10-(4-((3-aminophenyl)ethynyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl-
)-6a, 8a-dimethyl-6a, 6b, 7, 8, 8a, 8b, 11a, 12, 12a,
12b-decahydro-1H-naphtho[2', 1':4, 5]indeno[1, 2-d][1,
3]dioxol-4(2H)-one, also was isolated (85 mg, 0.147 mmol, 1.5%
yield) as a yellow solid. LCMS (Method i, Table 7)R,=1.93 min; MS
m/z=580 [M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
7.51 (d, J=7.6 Hz, 2H), 7.42-7.21 (m, 3H), 7.05 (t, J=7.7 Hz, 1H),
6.83-6.45 (m, 3H), 6.29-6.07 (m, 2H), 5.95 (s, 1H), 5.47-5.14 (m,
3H), 4.82 (s, 1H), 4.38-4.14 (m, 2H), 4.03 (d, J=19.3 Hz, 1H), 2.33
(d, J=10.3 Hz, 2H), 2.15-1.96 (m, 1H), 1.93-1.68 (m, 5H), 1.40 (s,
3H), 1.33-0.97 (m, 3H), 0.89 (s, 3H).
Example 49: Synthesis of
(6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-Aminophenyl)ethynyl)phe-
nyl)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8-
,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]d-
ioxol-4-one
##STR00850##
[1157] Synthesized using the same procedure as Example 48 above.
Major acetal isomer:
(6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-aminophenyl)ethynyl)phe-
nyl)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8-
,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]d-
ioxol-4-one. LCMS (Method f, Table 7)R,=1.57 min; MS m/z=598
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.55 (d,
J=8.1 Hz, 2H), 7.46 (d, J=8.1 Hz, 2H), 7.30 (d, J=10.1 Hz, 1H),
7.05 (t, J=7.8 Hz, 1H), 6.71 (s, 1H), 6.66 (d, J=7.5 Hz, 1H), 6.60
(d, J=7.8 Hz, 1H), 6.24 (d, J=8.9 Hz, 1H), 6.04 (s, 1H), 5.51 (d,
J=15.2 Hz, 2H), 5.26 (s, 2H), 4.97 (d, J=4.4 Hz, 1H), 4.55 (d,
J=19.5 Hz, 1H), 4.22 (d, J=19.5 Hz, 2H), 2.74-2.56 (m, 1H), 2.36
(d, J=9.7 Hz, 1H), 2.24-2.10 (m, 1H), 2.06 (d, J=14.5 Hz, 1H),
1.92-1.78 (m, 1H), 1.78-1.58 (m, 3H), 1.50 (s, 3H), 1.45-1.31 (m,
1H), 0.88 (s, 3H).
[1158] Minor acetal isomer:
(6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((3-aminophenyl)ethynyl)phe-
nyl)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8-
,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]d-
ioxol-4-one. LCMS (Method f, Table 7)R,=1.61 min; MS m/z=598
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.51 (d,
J=8.1 Hz, 2H), 7.39-7.22 (m, 3H), 7.05 (t, J=7.8 Hz, 1H), 6.72 (s,
1H), 6.67 (d, J=7.6 Hz, 1H), 6.60 (d, J=8.1 Hz, 1H), 6.24 (d,
J=10.1 Hz, 1H), 6.18 (s, 1H), 6.05 (s, 1H), 5.49 (s, 1H), 5.35 (d,
J=6.9 Hz, 1H), 5.26 (s, 2H), 4.19 (d, J=18.9 Hz, 2H), 4.04 (d,
J=19.1 Hz, 1H), 2.75-2.55 (m, 1H), 2.37 (d, J=10.2 Hz, 1H), 2.09
(d, J=7.1 Hz, 2H), 1.84 (d, J=6.5 Hz, 2H), 1.78-1.62 (m, 2H), 1.50
(s, 4H), 0.89 (s, 3H).
Example 50: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-Aminophenyl)ethynyl)-
phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a-
,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d-
][1,3]dioxol-4-one
##STR00851##
[1160] Synthesized using the same procedure as Example 48 above.
Major acetal isomer:
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-aminophenyl)ethynyl)-
phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a-
,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d-
][1,3]dioxol-4-one. LCMS (Method f, Table 7)R,=1.57 min; MS m/z=616
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.55 (d,
J=8.1 Hz, 2H), 7.47 (d, J=8.2 Hz, 2H), 7.27 (d, J=10.1 Hz, 1H),
7.05 (t, J=7.8 Hz, 1H), 6.71 (s, 1H), 6.66 (d, J=7.6 Hz, 1H), 6.60
(d, J=8.1 Hz, 1H), 6.30 (dd, J=10.1, 1.4 Hz, 1H), 6.13 (s, 1H),
5.80-5.58 (m, 1H), 5.55 (d, J=7.1 Hz, 2H), 5.26 (s, 2H), 5.14 (t,
J=5.9 Hz, 1H), 4.99 (d, J=5.1 Hz, 1H), 4.56 (dd, J=19.5, 6.4 Hz,
1H), 4.23 (dd, J=19.5, 5.4 Hz, 2H), 2.79-2.56 (m, 1H), 2.31 (s,
1H), 2.26-2.14 (m, 1H), 2.12-1.99 (m, 1H), 1.83-1.62 (m, 3H),
1.61-1.40 (m, 4H), 0.88 (s, 3H).
[1161] Minor acetal isomer:
(2S,6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((3-aminophenyl)ethynyl)-
phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a-
,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d-
][1,3]dioxol-4-one. LCMS (Method f, Table 7)R,=1.61 min; MS m/z=616
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.52 (d,
J=8.3 Hz, 2H), 7.33 (d, J=8.2 Hz, 2H), 7.28 (d, J=10.0 Hz, 1H),
7.05 (t, J=7.8 Hz, 1H), 6.73 (s, 1H), 6.68 (d, J=7.5 Hz, 1H), 6.61
(d, J=8.1 Hz, 1H), 6.32 (dd, J=10.1, 1.7 Hz, 1H), 6.21 (s, 1H),
6.15 (s, 1H), 5.78-5.58 (m, 1H), 5.55 (d, J=2.7 Hz, 1H), 5.36 (t,
J=7.9 Hz, 1H), 5.27 (s, 2H), 5.08 (t, J=5.8 Hz, 1H), 4.33-4.12 (m,
2H), 4.06 (dd, J=19.1, 5.0 Hz, 1H), 2.72-2.53 (m, 1H), 2.29 (s,
1H), 2.23-2.02 (m, 2H), 1.92-1.82 (m, 1H), 1.82-1.61 (m, 3H), 1.51
(s, 4H), 0.90 (s, 3H).
Example 51: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((E)-3-Aminostyryl)pheny-
l)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7-
,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3-
]dioxol-4-one
Step 1: Synthesis of Tert-Butyl
(E)-(3-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)phenyl)carba-
mate
##STR00852##
[1163] 4,4,5,5-Tetramethyl-1, 3, 2-dioxaborolane (16.70 mL, 115
mmol) and
carbonylchlorohydridotris(triphenylphosphine)ruthenium(II) (2.63 g,
2.76 mmol) were added to a nitrogen-purged solution of tert-butyl
(3-ethynylphenyl)carbamate (10 g, 46.0 mmol) in toluene (150 mL).
The mixture was heated at 50.degree. C. for 16 h, whereupon it was
concentrated under reduced pressure. Purification by chromatography
(silica) eluting with PE/EtOAc (100%10:1) gave the title compound
(13.25 g, 36.8 mmol, 80% yield) as a white solid. LCMS (Method d
Table 7)R,=2.19 min; MS m/z=290.1 [M-tBu].sup.+. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 1.33 (s, 12H), 1.54 (s, 9H), 6.17 (d,
J=18.4 Hz, 1H), 6.49 (bs, 1H), 7.18 (d, J=7.6 Hz, 1H), 7.26-7.29
(m, 1H), 7.35-7.40 (m, 2H), 7.47 (s, 1H).
Step 2: Synthesis of Tert-Butyl
(E)-(3-(4-formylstyryl)phenyl)carbamate
##STR00853##
[1165] To a solution of tert-Butyl
(E)-(3-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)phenyl)carba-
mate (6 g, 17.38 mmol) and 4-bromobenzaldehyde (3.38 g, 18.25 mmol)
in THF (85 mL) at 20.degree. C. under N.sub.2 were added
K.sub.2CO.sub.3 (4.80 g, 34.8 mmol) and Pd(Ph.sub.3P).sub.4 (1.607
g, 1.390 mmol). The mixture was stirred at 80.degree. C. for 32 h.
The mixture was concentrated to give the residue, which was
dissolved with EtOAc (50 mL) and filtered. The filtrate was
concentrated. The residue was purified by silica gel column
chromatography eluting with PE/EtOAc (10:1-6:1) to give the
product, which was further washed with PE (10 mL) to obtain the
title compound (3.43 g, 10.49 mmol, 60% yield) as a green solid.
LCMS (Method d, Table 7)R,=2.08 min; MS m/z=324.1 [M+H*].
Step 3: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((E)-3-Aminostyryl)pheny-
l)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7-
,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3-
]dioxol-4-one
##STR00854##
[1167] Trifluoromethanesulfonic acid (5.38 mL, 60.6 mmol) was added
drop-wise to a 0.degree. C. stirred suspension of
(6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-
-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-
-3H-cyclopenta[a]phenanthren-3-one (Example 2, Step 5) (5.0 g,
12.12 mmol) and (E)-tert-butyl (3-(4-formylstyryl)phenyl)carbamate
(4.612 g, 12.12 mmol) in anhydrous MeCN (30 mL) and THF (30 mL)
under nitrogen. The mixture was stirred at 0.degree. C. for 1 h,
then poured onto ice water (30 mL) and extracted with EtOAc
(2.times.50 mL). The combined organic layers were washed with
cooled water (30 mL), brine (30 mL), saturated NaHCO.sub.3 (30 mL)
and water (30 mL) again, and concentrated in vacuo affording a
yellow solid. The crude material was purified by silica gel column
chromatography (200-300 mesh), eluting with DCM/MeOH (100%40:1) and
then further purified by prep-HPLC to give the title compound (1.45
g, 2.328 mmol, 19% yield). LCMS (Method d, Table 7) R.sub.t=1.47
min; MS m/z=618.3 [M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 7.61 (d, J=8.2 Hz, 2H), 7.43 (d, J=8.3 Hz, 2H), 7.28 (d,
J=10.9 Hz, 1H), 7.15 (d, J=16.4 Hz, 1H), 7.03 (dd, J=15.5, 7.6 Hz,
2H), 6.75 (dd, J=8.0, 4.7 Hz, 2H), 6.50 (dd, J=7.9, 1.3 Hz, 1H),
6.31 (dd, J=10.1, 1.8 Hz, 1H), 6.15 (s, 1H), 5.79-5.46 (m, 3H),
5.13 (dd, J=14.7, 8.7 Hz, 3H), 4.97 (d, J=5.1 Hz, 1H), 4.55 (dd,
J=19.5, 6.4 Hz, 1H), 4.23 (dd, J=19.4, 5.5 Hz, 2H), 2.73-2.56 (m,
1H), 2.40-2.21 (m, 2H), 2.15-2.02 (m, 1H), 1.82-1.64 (m, 3H),
1.61-1.44 (m, 4H), 0.88 (s, 3H).
[1168] The minor acetal isomer,
(2S,6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((E)-3-aminostyryl)pheny-
l)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7-
,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3-
]dioxol-4-one (0.30 g, 0.456 mmol, 4% yield), also was isolated as
a white solid. LCMS (Method d, Table 7) R.sub.t=1.51 min; MS
m/z=618.3 [M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
7.57 (d, J=8.2 Hz, 2H), 7.27 (d, J=7.9 Hz, 3H), 7.18-6.97 (m, 3H),
6.75 (d, J=7.8 Hz, 2H), 6.50 (d, J=7.4 Hz, 1H), 6.31 (dd, J=10.2,
1.6 Hz, 1H), 6.15 (d, J=10.3 Hz, 2H), 5.78-5.67 (m, 1H), 5.63-5.49
(m, 2H), 5.37 (d, J=7.0 Hz, 1H), 5.07 (dd, J=12.0, 5.8 Hz, 3H),
4.33-4.15 (m, 2H), 4.06 (dd, J=19.2, 5.7 Hz, 1H), 2.69-2.54 (m,
1H), 2.36-2.08 (m, 3H), 1.94-1.60 (m, 4H), 1.50 (s, 3H), 0.90 (s,
3H).
Example 52: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((E)-3-Aminostyryl)phenyl)--
7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,1-
2a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one
##STR00855##
[1170] Synthesized using the same procedure as Example 51 above.
Major acetal isomer:
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((E)-3-aminostyryl)phenyl)--
7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,1-
2a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one.
LCMS (Method d, Table 7)R,=1.48 min; MS m/z=582.3 [M+H.sup.+].
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 0.88 (s, 3H), 1.00-1.09
(m, 2H), 1.40 (s, 3H), 1.63-1.79 (m, 5H), 2.04-2.15 (m, 2H),
2.32-2.34 (m, 1H), 2.55-2.60 (m, 1H), 4.20 (dd, J=20.2 Hz, 5.0 Hz,
1H), 4.31 (s, 1H), 4.54 (dd, J=19.2 Hz, 6.0 Hz, 1H), 4.82 (s, 1H),
4.94-4.95 (m, 1H), 5.10-5.14 (m, 3H), 5.46 (s, 1H), 5.95 (s, 1H),
6.18 (d, J=10.0 Hz, 1H), 6.50 (d, J=7.6 Hz, 1H), 6.73-6.76 (m, 2H),
7.00-7.14 (m, 3H), 7.32 (d, J=10.0 Hz, 1H), 7.45 (d, J=8.4 Hz, 2H),
7.59 (d, J=8.0 Hz, 2H).
[1171] Minor acetal isomer:
(6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((E)-3-aminostyryl)phenyl)--
7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,1-
2a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one.
LCMS (Method d, Table 7)R,=1.52 min; MS m/z=582.3 [M+H.sup.+].
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 0.89 (s, 3H), 1.05-1.31
(m, 3H), 1.40 (s, 3H), 1.74-1.89 (m, 5H), 2.05-2.07 (m, 2H),
2.31-2.34 (m, 1H), 2.54-2.59 (m, 1H), 4.00-4.06 (m, 1H), 4.23-4.31
(m, 2H), 4.80 (s, 1H), 5.05-5.09 (m, 3H), 5.31-5.32 (m, 1H), 5.95
(s, 1H), 6.12 (s, 1H), 6.18 (d, J=9.6 Hz, 1H), 6.49-6.50 (m, 1H),
6.74-6.76 (m, 2H), 7.00-7.14 (m, 3H), 7.26 (d, J=7.6 Hz, 2H), 7.32
(d, J=10.0 Hz, 1H), 7.57 (d, J=7.6 Hz, 2H).
Example 53: Synthesis of
(6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((E)-3-aminostyryl)phenyl)--
6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8-
b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-
-4-one
##STR00856##
[1173] Synthesized using the same procedure as Example 51 above.
Major acetal isomer:
(6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((E)-3-aminostyryl)phenyl)--
6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8-
b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-
-4-one. LCMS (Method d, Table 7)R,=1.45 min; MS m/z=600.3
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.61 (d,
J=8.1 Hz, 2H), 7.42 (d, J=8.1 Hz, 2H), 7.30 (d, J=10.1 Hz, 1H),
7.15 (d, J=16.4 Hz, 1H), 7.03 (dd, J=15.6, 7.6 Hz, 2H), 6.79-6.70
(m, 2H), 6.50 (d, J=7.7 Hz, 1H), 6.25 (dd, J=10.1, 1.4 Hz, 1H),
6.06 (s, 1H), 5.54-5.43 (m, 2H), 5.13 (t, J=6.0 Hz, 3H), 4.95 (d,
J=4.5 Hz, 1H), 4.55 (dd, J=19.5, 6.4 Hz, 1H), 4.22 (dd, J=19.3, 5.4
Hz, 2H), 2.62 (m, 2H), 2.42-2.02 (m, 3H), 1.92-1.80 (m, 1H),
1.77-1.61 (m, 3H), 1.51 (s, 3H), 1.47-1.32 (m, 1H), 0.89 (s,
3H).
[1174] Minor acetal isomer:
(6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((E)-3-aminostyryl)phenyl)--
6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8-
b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-
-4-one.
[1175] LCMS (Method d, Table 7)R,=1.48 min; MS m/z=600.3
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.57 (d,
J=8.1 Hz, 2H), 7.28 (dd, J=12.2, 9.3 Hz, 3H), 7.18-6.97 (m, 3H),
6.75 (d, J=7.7 Hz, 2H), 6.50 (d, J=7.3 Hz, 1H), 6.25 (dd, J=10.1,
1.2 Hz, 1H), 6.13 (s, 1H), 6.05 (s, 1H), 5.46 (d, J=2.8 Hz, 1H),
5.35 (d, J=6.9 Hz, 1H), 5.06 (dd, J=14.0, 7.9 Hz, 3H), 4.24 (dd,
J=19.3, 6.3 Hz, 2H), 4.05 (dd, J=19.1, 5.8 Hz, 1H), 2.73-2.58 (m,
1H), 2.47-2.30 (m, 2H), 2.09 (d, J=10.1 Hz, 2H), 1.85 (d, J=6.5 Hz,
2H), 1.78-1.65 (m, 2H), 1.50 (s, 4H), 0.90 (s, 3H).
Example 54: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminophenethyl)phenyl-
)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,-
8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]-
dioxol-4-one
Step 1: Synthesis of Tert-Butyl
(E)-(3-(4-(hydroxymethyl)styryl)phenyl)carbamate
##STR00857##
[1177] NaBH.sub.4 (0.936 g, 24.74 mmol) was added to a 0.degree. C.
solution of (E)-tert-butyl (3-(4-formylstyryl)phenyl)carbamate)Step
2, Example. 51)(4.0 g, 12.37 mmol) in MeOH (60 mL) and THF (60 mL)
and stirred at 0.degree. C. for 1 h. The mixture was quenched with
saturated aqueous NH.sub.4Cl (20 mL), concentrated to obtain a
residue, which was partitioned between EtOAc (100 mL) and water
(100 mL). The organic layer was concentrated under reduced
pressure, and was purified by silica gel column chromatography,
eluting with DCM/EtOAc (10:15:1) to give the title compound (3.23
g, 7.08 mmol, 57% yield) as a light red solid. LCMS (Method d,
Table 7) R.sub.t=1.98 min; MS m/z=348.1 [M+Na.sup.+].
Step 2: Synthesis of Tert-Butyl
(3-(4-(hydroxymethyl)phenethyl)phenyl)carbamate
##STR00858##
[1179] A suspension of Pd/C (0.657 g, 0.618 mmol) and
(E)-tert-butyl (3-(4-(hydroxymethyl)styryl) phenyl)carbamate(pure)
7 (3.35 g, 10.29 mmol) in EtOAc (50 mL) and THF (50 mL) was treated
with hydrogen balloon and stirred at 0.degree. C. for 1.5 h,
monitored by LCMS. The mixture was filtered. Additional Pd/C (0.657
g, 0.618 mmol) was added to the filtrate. The mixture was stirred
for additional 1 h under an atmosphere of hydrogen and monitored by
LCMS. The mixture was filtered and washed with EtOAc (15 mL). The
filtrate was concentrated to give a residue, which was purified by
silica gel column chromatography, eluting with PE/EtOAc (10:12:1)
to give the title compound (1.2 g, 3.49 mmol, 34% yield) as a white
solid. LCMS (Method d, Table 7)R,=2.0 min; MS m/z=350.0
[M+Na.sup.+].
Step 3: Synthesis of Tert-Butyl
(3-(4-formylphenethyl)phenyl)carbamate
##STR00859##
[1181] A suspension of MnO.sub.2 (9.24 g, 106 mmol) and tert-butyl
(3-(4-(hydroxymethyl)phenethyl) phenyl)carbamate (2.9 g, 8.86 mmol)
in DCM (40 mL) was treated with nitrogen balloon and stirred at
30.degree. C. for 2 h, monitored by LCMS. Additional MnO.sub.2 (0.8
g, 9.2 mmol) was added to the above mixture, stirred at 30.degree.
C. for additional 1 h. The mixture was filtered and washed with DCM
(20 mL). The filtrate was concentrated to obtain the title compound
(2.9 g, 8.58 mmol, 97% yield) as a yellow solid. LCMS (Method d,
Table 7)R,=2.14 min; MS m/z=226.0 [M-Boc]*. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 9.90 (s, 1H), 7.71 (d, J=7.8 Hz, 2H), 7.25 (s,
2H), 7.19-6.97 (m, 3H), 6.73 (d, J=7.3 Hz, 1H), 6.48 (s, 1H),
2.98-2.87 (m, 2H), 2.86-2.78 (m, 2H), 1.44 (s, 9H).
Step 4: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-Aminophenethyl)phenyl-
)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,-
8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]-
dioxol-4-one
##STR00860##
[1183] Trifluoromethanesulfonic acid (5.61 mL, 64.2 mmol) was added
drop-wise to a stirred 0.degree. C. suspension of tert-butyl
(3-(4-formylphenethyl)phenyl)carbamate (4.18 g, 12.85 mmol) and
(6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-
-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-
-3H-cyclopenta[a]phenanthren-3-one (5.3 g, 12.85 mmol) in anhydrous
MeCN (30 mL) and THF (30 mL) under. The resulting mixture was
stirred at 0.degree. C. for 1 h, then poured onto ice water (20 mL)
and extracted with EtOAc (2.times.25 mL). The combined organic
layers were washed with cooled water (20 mL), brine (10 mL),
saturated aqueous NaHCO.sub.3 (20 mL) and water (20 mL),
concentrated in vacuo affording a yellow solid. The crude material
was purified by silica gel column chromatography (200-300 mesh),
eluting with dichloromethane/methanol (100%40:1) to obtain the
product, which was further purified by prep-HPLC to give the title
compound (2.21 g, 3.57 mmol, 28% yield) as a white solid. LCMS
(Method d, Table 7)R,=1.75 min; MS m/z=619.8 [M+H.sup.+]. .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 7.34 (d, J=8.1 Hz, 2H), 7.27
(t, J=8.0 Hz, 3H), 6.89 (t, J=7.7 Hz, 1H), 6.43 (s, 1H), 6.36 (d,
J=7.9 Hz, 2H), 6.31 (dd, J=10.2, 1.8 Hz, 1H), 6.14 (s, 1H),
5.75-5.56 (m, 1H), 5.54 (d, J=2.9 Hz, 1H), 5.46 (s, 1H), 5.12 (t,
J=6.0 Hz, 1H), 4.95 (d, J=5.1 Hz, 1H), 4.92 (s, 2H), 4.53 (dd,
J=19.5, 6.4 Hz, 1H), 4.21 (dd, J=19.4, 5.6 Hz, 2H), 2.83-2.79 (m,
2H), 2.73-2.57 (m, 3H), 2.275-2.25 (m, 2H), 2.08-2.04 (m, 1H),
1.79-1.62 (m, 3H), 1.67-1.50 (m, 4H), 0.87 (s, 3H).
[1184] The minor acetal isomer,
(2S,6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-(3-aminophenethyl)phenyl-
)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,-
8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]-
dioxol-4-one (0.45 g, 0.667 mmol, 5% yield) also was isolated as a
white solid. LCMS (Method d, Table 7)R,=1.79 min; MS m/z=619.8
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.33-7.17
(m, 5H), 6.89 (t, J=7.7 Hz, 1H), 6.46-6.27 (m, 4H), 6.12 (d, J=8.8
Hz, 2H), 5.75-5.55 (m, 1H), 5.53 (s, 1H), 5.34 (d, J=7.1 Hz, 1H),
5.06 (t, J=5.9 Hz, 1H), 4.92 (s, 2H), 4.31-4.15 (m, 2H), 4.05 (dd,
J=19.2, 5.6 Hz, 1H), 2.83-2.79 (m, 2H), 2.72-2.54 (m, 3H), 2.29 (s,
1H), 2.21-2.13 (m, 1H), 2.09-2.05 (m, 1H), 1.93-1.81 (m, 1H),
1.79-1.60 (m, 3H), 1.50 (s, 3H), 0.88 (s, 3H).
Example 55: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminophenethyl)phenyl)-7-
-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12-
a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one
##STR00861##
[1185] =16.7, 9.1 Hz, 3H), 7.24 (d, J=8.0 Hz, 2H), 6.89 (t, J=7.7
Hz, 1H), 6.42 (s, 1H), 6.36 (dd, J=7.7, 1.6 Hz, 2H), 6.17 (dd,
J=10.1, 1.7 Hz, 1H), 5.95 (s, 1H), 5.41 (s, 1H), 5.11 (t, J=5.9 Hz,
1H), 4.93 (d, J=5.4 Hz, 3H), 4.81 (d, J=3.0 Hz, 1H), 4.52 (dd,
J=19.5, 6.4 Hz, 1H), 4.30 (s, 1H), 4.19 (dd, J=19.5, 5.6 Hz, 1H),
2.87-2.77 (m, 2H), 2.73-2.64 (m, 2H), 2.62-2.52 (m, 1H), 2.32 (d,
J=11.0 Hz, 1H), 2.18-1.98 (m, 2H), 1.83-1.58 (m, 5H), 1.40 (s, 3H),
1.24-0.97 (m, 2H), 0.87 (s, 3H).
[1186] Minor acetal isomer,
(6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-(3-aminophenethyl)phenyl)-7-
-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12-
a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one:
LCMS (Method d, Table 7) RM=1.77 min; MS m/z=583.9 [M+H]. .sup.1H
NMR (400 MHz, DMSO-d) .delta. 7.32 (d, J=10.1 Hz, 1H), 7.19 (q,
J=8.2 Hz, 4H), 6.89 (t, J=7.7 Hz, 1H), 6.44-6.29 (m, 3H), 6.17 (dd,
J=10.1, 1.8 Hz, 1H), 6.07 (s, 1H), 5.95 (s, 1H), 5.29 (d, J=6.9 Hz,
1H), 5.03 (t, J=6.1 Hz, 1H), 4.92 (s, 2H), 4.78 (d, J=3.1 Hz, 1H),
4.34-1.19 (m, 2H), 4.02 (dd, J=19.2, 5.9 Hz, 1H), 2.81 (dd, J=9.5,
6.1 Hz, 2H), 2.68 (dd, J=9.6, 6.0 Hz, 2H), 2.61-2.52 (m, 1H), 2.32
(d, J=10.4 Hz, 1H), 2.03 (d, J=7.8 Hz, 2H), 1.91-1.67 (m, 5H), 1.39
(s, 3H), 1.27-1.01 (m, 2H), 0.89 (s, 3H).
Example 56: Synthesis of
(6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-Aminophenethyl)phenyl)-6-
b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b-
,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol--
4-one
##STR00862##
[1188] Synthesized using the same procedure as Example 54 above.
Major acetal isomer: LCMS (Method d, Table 7)R,=1.74 min; MS
m/z=601.9 [M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
7.32 (t, J=7.2 Hz, 2H), 7.26 (t, J=8.0 Hz, 2H), 6.89 (t, J=7.7 Hz,
1H), 6.43 (s, 1H), 6.36 (d, J=7.7 Hz, 2H), 6.24 (dd, J=10.1, 1.7
Hz, 1H), 6.05 (s, 1H), 5.45 (s, 2H), 5.10 (t, J=5.9 Hz, 1H),
4.97-4.85 (m, 3H), 4.52 (dd, J=19.5, 6.4 Hz, 1H), 4.20 (dd, J=19.2,
5.6 Hz, 2H), 2.85-2.76 (m, 2H), 2.72-2.54 (m, 3H), 2.36 (d, J=10.4
Hz, 1H), 2.20-2.18 (m, 1H), 2.04 (s, 1H), 1.91-1.80 (m, 1H),
1.73-1.61 (m, 3H), 1.50 (s, 3H), 1.40-1.38 (m, 1H), 0.87 (s,
3H).
[1189] Minor acetal isomer,
(6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-(3-aminophenethyl)phenyl)-6-
b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,b,7,8,8a,b,1-
1a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4--
one: LCMS (Method d, Table 7)R,=1.77 min; MS m/z=601.9 [M+H].
.sup.1H NMR (400 MHz, DMSO-d) .delta. 7.13 (m, 5H), 6.89 (t, J=7.7
Hz, 1H), 6.41 (s, 1H), 6.36 (d, J=7.6 Hz, 2H), 6.24 (dd, J=10.1,
1.7 Hz, 1H), 6.06 (d, J=13.8 Hz, 2H), 5.44 (d, J=2.6 Hz, 1H), 5.33
(d, J=7.0 Hz, 1H), 5.04 (t, J=6.0 Hz, 1H), 4.91 (s, 2H), 4.27-4.21
(m, 2H), 4.04 (dd, J=19.2, 5.9 Hz, 1H), 2.85-2.76 (m, 2H),
2.70-2.66 (m, 3H), 2.37-2.35 (m, 2H), 2.07-2.06 (m, 2H), 1.84 (d,
J=7.1 Hz, 2H), 1.71 (t, J=10.3 Hz, 2H), 1.50 (s, 4H), 0.90 (s,
3H).
Example 57: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-Aminophenyl)amino)pheny-
l)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,1-
2,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one
Step 1: Synthesis of Tert-Butyl
(3-((4-formylphenyl)amino)phenyl)carbamate
##STR00863##
[1191] A mixture tert-butyl (3-aminophenyl)carbamate (31.2 g, 150
mmol), 4-bromobenzaldehyde (33.3 g, 180 mmol), Pd(OAc).sub.2 (1.684
g, 7.50 mmol), BINAP
((RS).sub.2,2'-bis(diphenylphosphino)-1,1'-binaphthyl) (9.34 g,
15.00 mmol), Cs.sub.2CO.sub.3 (98 g, 300 mmol) was refluxed in
toluene (300 mL) under nitrogen for 16 h. After cooling to room
temperature, the mixture was partitioned between water and EtOAc.
The organic layer was concentrated and purified by column
chromatography eluting with PE:EtOAc (5:1) to give the title
compound (32.8 g, 105 mmol, 70% yield) as a yellow oil. LCMS
(Method j, Table 7)R,=1.94 min; MS m/z=313 [M+H.sup.+].
Step 2: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-Aminophenyl)amino)pheny-
l)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,1-
2,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one
##STR00864##
[1193] Trifluoromethanesulfonic acid (14.21 ml, 160 mmol) was added
drop-wise to a 0.degree. C. suspension of
(8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)--
10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a-
]phenanthren-3-one (12.05 g, 32.0 mmol) and tert-butyl
(3-((4-formylphenyl)amino)phenyl)carbamate (10 g, 32.0 mmol) in THF
(50.00 ml) and MeCN (50 ml). The reaction mixture was stirred for
additional 2 hours at the same temperature. The mixture was diluted
with EtOAc (200 mL), washed with water (100 mL), saturated
NaHCO.sub.3 solution (1.times.100 mL), and brine (1.times.100 mL),
dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The residue
was purified by column chromatography (MeOH:DCM=1:40), and the
resulting material was purified further by prep-HPLC to afford the
title compound (1.729 g, 3.03 mmol, 10% yield) as a white solid.
LCMS (Method k, Table 7)R,=1.50 min; MS m/z=571[M+H.sup.+]. .sup.1H
NMR (400 MHz, DMSO) .delta. 8.01 (s, 1H), 7.33 (d, J=10.1 Hz, 1H),
7.27 (d, J=8.5 Hz, 2H), 7.00 (d, J=8.5 Hz, 2H), 6.87 (t, J=7.9 Hz,
1H), 6.36 (s, 1H), 5.94 (s, 1H), 5.32 (s, 1H), 5.10 (s, 1H),
5.02-4.87 (m, 3H), 4.80 (d, J=2.8 Hz, 1H), 4.51 (d, J=16.4 Hz, 1H),
4.31 (s, 1H), 4.20 (d, J=17.8 Hz, 1H), 2.62-2.52 (m, 1H), 2.32 (d,
J=11.0 Hz, 1H), 2.20-1.98 (m, 2H), 1.86-1.69 (m, 4H), 1.69-1.55 (m,
1H), 1.41 (s, 3H), 1.18-0.97 (m, 2H), 0.87 (s, 3H).
[1194] The minor acetal isomer,
(6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((3-aminophenyl)amino)pheny-
l)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,1-
2,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one(-
78 mg, 0.137 mmol, 0.4% yield) as a white solid. LCMS (Method k,
Table 7) R.sub.t=1.53 min; MS m/z=571[M+H.sup.+]. .sup.1H NMR (400
MHz, DMSO) .delta. 8.00 (s, 1H), 7.32 (d, J=10.1 Hz, 1H), 7.07 (d,
J=8.5 Hz, 2H), 6.96 (d, J=8.5 Hz, 2H), 6.87 (t, J=7.9 Hz, 1H), 6.35
(s, 1H), 6.24 (d, J=7.9 Hz, 1H), 6.17 (d, J=10.0 Hz, 1H), 6.10 (d,
J=7.9 Hz, 1H), 6.00 (s, 1H), 5.95 (s, 1H), 5.27 (d, J=7.0 Hz, 1H),
5.02 (t, J=5.9 Hz, 1H), 4.97 (s, 2H), 4.78 (d, J=2.7 Hz, 1H), 4.30
(s, 2H), 4.03 (dd, J=19.1, 5.8 Hz, 1H), 2.65-2.52 (m, 1H), 2.32 (d,
J=10.2 Hz, 1H), 2.14-1.95 (m, 2H), 1.89-1.63 (m, 5H), 1.39 (s, 3H),
1.28-1.11 (m, 1H), 1.05 (d, J=10.7 Hz, 1H), 0.89 (s, 3H).
Example 58: Synthesis of
(6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-Aminophenyl)amino)pheny-
l)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8-
a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dio-
xol-4-one
##STR00865##
[1196] Synthesized using the same procedure as Example 57 above.
Major acetal isomer: LCMS (Method k, Table 7) R.sub.t=1.49 min; MS
m/z=589 [M+H.sup.+]. H NMR (400 MHz, DMSO) .delta. 8.02 (s, 1H),
7.30 (d, J=10.1 Hz, 1H), 7.23 (d, J=8.5 Hz, 2H), 6.99 (d, J=8.5 Hz,
2H), 6.87 (t, J=7.9 Hz, 1H), 6.36 (s, 1H), 6.31-6.16 (m, 2H), 6.10
(d, J=7.8 Hz, 1H), 6.04 (s, 1H), 5.45 (d, J=2.6 Hz, 1H), 5.35 (s,
1H), 5.11 (t, J=5.9 Hz, 1H), 4.97 (s, 2H), 4.91 (d, J=4.6 Hz, 1H),
4.51 (dd, J=19.5, 6.3 Hz, 1H), 4.20 (dd, J=19.2, 5.5 Hz, 2H),
2.74-2.58 (m, 1H), 2.36 (d, J=10.2 Hz, 1H), 2.27-2.13 (m, 1H), 2.06
(d, J=9.5 Hz, 2H), 1.93-1.78 (m, 1H), 1.78-1.57 (m, 3H), 1.51 (s,
3H), 1.42 (dd, J=12.4, 4.5 Hz, 1H), 0.87 (s, 3H).
[1197] Minor acetal isomer,
(6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((3-aminophenyl)amino)pheny-
l)-6b-fluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8-
a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dio-
xol-4-one: LCMS (Method k, Table 7) R.sub.t=1.50 min; MS m/z=589
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO) .delta. 8.01 (s, 1H), 7.29
(d, J=10.1 Hz, 1H), 7.08 (d, J=8.5 Hz, 2H), 6.97 (d, J=8.5 Hz, 2H),
6.87 (t, J=7.9 Hz, 1H), 6.35 (s, 1H), 6.24 (d, J=8.6 Hz, 2H), 6.10
(d, J=7.7 Hz, 1H), 6.02 (d, J=18.4 Hz, 2H), 5.44 (s, 1H), 5.30 (d,
J=7.1 Hz, 1H), 4.97 (s, 3H), 4.30 (d, J=19.1 Hz, 1H), 4.19 (d,
J=9.2 Hz, 1H), 4.05 (d, J=19.1 Hz, 1H), 2.78-2.56 (m, 1H), 2.36 (d,
J=13.3 Hz, 1H), 2.06 (d, J=10.7 Hz, 2H), 1.83 (dd, J=16.3, 10.0 Hz,
2H), 1.76-1.61 (m, 2H), 1.50 (s, 4H), 0.89 (s, 3H).
Example 59: Synthesis of
(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-Aminophenyl)amino)ph-
enyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6-
b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][-
1,3]dioxol-4-one
##STR00866##
[1199] Synthesized using the same procedure as Example 57 above.
Major acetal isomer: LCMS (Method 1, Table 7) R.sub.t=1.62 min; MS
m/z=607 [M+H.sup.+]. H NMR (400 MHz, DMSO) .delta. 8.02 (s, 1H),
7.25 (dd, J=18.2, 9.4 Hz, 3H), 6.99 (d, J=8.6 Hz, 2H), 6.87 (t,
J=7.9 Hz, 1H), 6.36 (t, J=1.9 Hz, 1H), 6.30 (dd, J=10.1, 1.8 Hz,
1H), 6.25-6.19 (m, 1H), 6.14 (s, 1H), 6.09 (dd, J=7.9, 1.3 Hz, 1H),
5.77-5.55 (m, 1H), 5.53 (d, J=2.8 Hz, 1H), 5.35 (s, 1H), 5.11 (t,
J=6.0 Hz, 1H), 4.96 (s, 2H), 4.92 (d, J=5.1 Hz, 1H), 4.51 (dd,
J=19.5, 6.4 Hz, 1H), 4.21 (dd, J=19.3, 5.6 Hz, 2H), 2.76-2.53 (m,
1H), 2.28 (dd, J=12.6, 5.9 Hz, 2H), 2.06 (d, J=12.0 Hz, 3H), 1.70
(dt, J=20.2, 6.0 Hz, 3H), 1.60-1.40 (m, 4H), 0.86 (s, 3H).
[1200] Minor acetal isomer,
(2S,6aS,6bR,7S,8aS,8bS,10S,11aR,12aS,12bS)-10-(4-((3-aminophenyl)amino)ph-
enyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6-
b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][-
1,3]dioxol-4-one: LCMS (Method 1, Table 7)R,=1.65 min; MS m/z=607
[M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO) .delta. 8.01 (s, 1H), 7.27
(d, J=9.8 Hz, 1H), 7.09 (d, J=7.9 Hz, 2H), 6.97 (d, J=8.0 Hz, 2H),
6.88 (t, J=7.7 Hz, 1H), 6.41-6.19 (m, 3H), 6.20-6.07 (m, 2H), 6.03
(s, 1H), 5.65 (d, J=46.4 Hz, 1H), 5.52 (s, 1H), 5.32 (d, J=6.8 Hz,
1H), 5.06 (s, 1H), 4.97 (s, 2H), 4.32 (dd, J=19.1, 5.3 Hz, 1H),
4.19 (s, 1H), 4.06 (dd, J=18.7, 4.8 Hz, 1H), 2.59 (d, J=13.8 Hz,
1H), 2.29 (s, 1H), 2.17 (d, J=7.2 Hz, 1H), 2.07 (s, 1H), 1.87 (d,
J=6.7 Hz, 1H), 1.69 (dd, J=23.7, 12.4 Hz, 3H), 1.50 (s, 4H), 0.89
(s, 3H).
Example 60: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-Aminobenzyl)thio)phenyl-
)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12-
,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one
Step 1: Synthesis of 4-((3-Nitrobenzyl)thio)benzaldehyde
##STR00867##
[1202] To a solution of (3-nitrophenyl)methanethiol (35 g, 282
mmol) and 4-fluorobenzaldehyde (52.5 g, 310 mmol) in dry dimethyl
sulfoxide (220 mL) was added potassium carbonate (78 g, 564 mmol).
The reaction mixture was heated to 100.degree. C. for 4 hours. One
additional vial was set up as described above. The two reactions
were combined and diluted with water (2 L) and then extracted with
EtOAc (3.times.600 mL). The combined organic layer was dried over
Na.sub.2SO.sub.4, and concentrated to give a residue, which was
purified by column chromatography (eluted with PE/EtOAc=20/1 to
5/1) to give the title compound (62 g, 80% yield) as slight brown
solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.54 (s, 2H)
7.51 (d, J=8.33 Hz, 2H) 7.59 (s, 1H) 7.77 (d, J=8.33 Hz, 2H) 7.87
(d, J=7.89 Hz, 1H) 8.05-8.10 (m, 1H) 8.30 (s, 1H) 9.87 (s, 1H).
Step 2: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-Hydroxy-8b-(2-hydroxyacetyl)-6a-
,8a-dimethyl-10-(4-((3-nitrobenzyl)thio)phenyl)-1,2,6a,6b,7,8,8a,8b,11a,12-
,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one
##STR00868##
[1204] Trifluoromethanesulfonic acid (21.23 mL, 239 mmol) was added
drop-wise to a 0.degree. C. solution of
(8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)--
10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a-
]phenanthren-3-one (9 g, 23.91 mmol) and
4-((3-nitrobenzyl)thio)benzaldehyde (7.19 g, 26.3 mmol) in MeCN
(500 mL). The reaction was stirred for 1 hour at 0.degree. C. Two
additional vials were set up as described above. All three
reactions were combined and poured into water (2 L). The resulting
mixture was extracted with EtOAc (3.times.500 mL). The combined
organic layer was dried over Na.sub.2SO.sub.4, and concentrated to
give a residue, which was purified by prep-HPLC to give the title
compound (5.57 g, 16% yield) as white solid. LCMS (Method n Table
7): R,=3.20 min; m/z=632.0 [M+H.sup.+]. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 0.84 (s, 3H) 0.93-1.08 (m, 2H) 1.37 (s, 3H)
1.52-1.76 (m, 5H) 1.94-2.15 (m, 2H) 2.29 (br d, J=11.91 Hz, 1H)
2.50-2.58 (m, 1H) 4.15 (dd, J=19.40, 5.51 Hz, 1H) 4.27 (br d,
J=2.87 Hz, 1H) 4.39 (s, 2H) 4.48 (dd, J=19.40, 6.39 Hz, 1H) 4.77
(d, J=3.09 Hz, 1H) 4.89 (d, J=4.63 Hz, 1H) 5.07 (t, J=5.95 Hz, 1H)
5.38 (s, 1H) 5.91 (s, 1H) 6.15 (dd, J=10.14, 1.76 Hz, 1H) 7.25-7.38
(m, 5H) 7.55 (t, J=7.94 Hz, 1H) 7.79 (d, J=7.72 Hz, 1H) 8.04 (dd,
J=8.16, 1.54 Hz, 1H) 8.19 (d, J=1.76 Hz, 1H)
[1205] The minor acetal isomer,
(6aR,6bS,7S,8aS,8bS,10S,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a-
,8a-dimethyl-10-(4-((3-nitrobenzyl)thio)phenyl)-1,2,6a,6b,7,8,8a,8b,11a,12-
,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one
(0.34 g, 1% yield) also was obtained as a white solid. LCMS (Method
n, Table 7): R,=3.28 min; MS m/z=631.8 [M+H.sup.+]. .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. 0.86 (s, 3H) 0.98-1.05 (m, 1H)
1.10-1.21 (m, 1H) 1.37 (s, 3H) 1.66-1.88 (m, 5H) 1.94-2.08 (m, 2H)
2.29 (br dd, J=13.23, 2.87 Hz, 1H) 2.50-2.56 (m, 1H) 3.99 (dd,
J=19.18, 5.95 Hz, 1H) 4.20 (dd, J=19.07, 6.28 Hz, 1H) 4.27 (br s,
1H) 4.39 (s, 2H) 4.77 (d, J=3.09 Hz, 1H) 4.99 (s, 1H) 5.26 (d,
J=6.84 Hz, 1H) 5.92 (s, 1H) 6.04 (s, 1H) 6.15 (dd, J=10.03, 1.87
Hz, 1H) 7.16 (d, J=8.38 Hz, 2H) 7.26-7.34 (m, 3H) 7.55 (t, J=7.94
Hz, 1H) 7.75 (d, J=7.72 Hz, 1H) 8.05 (dd, J=8.16, 1.54 Hz, 1H) 8.21
(t, J=1.76 Hz, 1H).
Step 3: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-Aminobenzyl)thio)phenyl-
)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12-
,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one
##STR00869##
[1207] A mixture of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a-
,8a-dimethyl-10-(4-((3-nitrobenzyl)thio)phenyl)-1,2,6a,6b,7,8,8a,8b,11a,12-
,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4-one
(138 mg, 0.22 mmol), zinc (214 mg, 3.28 mmol), and acetic acid (0.4
ml, 6.99 mmol) in EtOAc (2 mL) was stirred at 40.degree. C. for 2
hours. LCMS showed partial conversion to the desired aniline
product. Added more zinc (71 mg, 1.09 mml) and stirred at
40.degree. C. for an additional 2 hours. The solution was cooled to
room temperature and partitioned between saturated aqueous
NaHCO.sub.3 and EtOAc (3.times.). The combined organic layers were
dried over Na.sub.2SO.sub.4 and purified by chromatography (silica
gel) eluting with 0-5% MeOH in DCM to give the title compound (64
mg, 0.106 mmol, 49% yield). LCMS (Method r, Table 7)R,=0.77 min; MS
m/z=601.9 [M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
7.35 (d, J=8.5 Hz, 2H), 7.32-7.24 (m, 3H), 6.89 (t, J=7.7 Hz, 1H),
6.56 (q, J=2.3 Hz, 1H), 6.47 (d, J=7.4 Hz, 1H), 6.40 (ddd, J=7.6,
2.6, 1.4 Hz, 1H), 6.15 (dd, J=10.1, 1.8 Hz, 1H), 5.95-5.89 (m, 1H),
5.38 (s, 1H), 5.03 (d, J=14.0 Hz, 3H), 4.90 (d, J=4.8 Hz, 1H), 4.77
(d, J=3.5 Hz, 1H), 4.54-4.44 (m, 1H), 4.28 (s, 1H), 4.16 (d, J=20.6
Hz, 1H), 4.06 (d, J=2.3 Hz, 2H), 2.59-2.50 (m, 1H), 2.30 (d, J=11.5
Hz, 1H), 2.14-2.03 (m, 1H), 1.97 (s, 2H), 1.88-1.67 (m, 4H), 1.63
(td, J=11.9, 10.4, 5.1 Hz, 1H), 1.37 (d, J=1.9 Hz, 3H), 1.10-0.92
(m, 2H), 0.84 (s, 3H).
Example 61: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((2-Aminopyridin-4-yl)methy-
l)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8-
b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-
-4-one
Step 1: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(Bromomethyl)phenyl)-7-hydr-
oxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-dec-
ahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(2H)-one
##STR00870##
[1209] 4-(Bromomethyl)benzaldehyde (0.539 g, 2.71 mmol) was added
to a 0.degree. C. suspension of
(8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)--
10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a-
]phenanthren-3-one (1.0738 g, 2.85 mmol),
4-(bromomethyl)benzaldehyde (0.539 g, 2.71 mmol), and MgSO.sub.4
(1.33 g, 11.05 mmol) in MeCN (18 ml). Trifluoromethanesulfonic acid
(2.0 g, 13.5 mmol) was added in a drop-wise manner, so as to
maintain a temperature of less than 7.degree. C. The reaction was
stirred for 4 min, whereupon it was quenched by addition of
saturated aqueous NaHCO.sub.3 (20 mL) and extracted with EtOAc (60
mL). The combined organics were washed with brine (10 mL) and
solvent was removed under reduced pressure. Purification by
chromatography (silica, 40 g) eluting with a gradient of 0-5%
MeOH/DCM gave the title compound (1.59 g, 2.85 mmol, 100% yield) as
an off-white foam (9:1 mixture of acetal diastereomers.
Characterization is provided for the major acetal isomer: LCMS
(Method r, Table 7)R,=1.04 min; MS m/z=557.2, 559.2 [M+H]. 1H NMR
(501 MHz, DMSO-d6) .delta. 7.44 (s, 4H), 7.30 (dd, J=10.1, 2.2 Hz,
1H), 6.15 (ddd, J=10.1, 4.8, 1.9 Hz, 1H), 5.91 (t, J=1.7 Hz, 1H),
5.43 (s, 1H), 5.07 (s, 1H), 4.93 (d, J=5.1 Hz, 1H), 4.77 (dd,
J=3.6, 0.9 Hz, 1H), 4.67 (s, 2H), 4.51 (dd, J=19.4, 4.1 Hz, 1H),
4.31-4.26 (m, 1H), 4.17 (d, J=19.5 Hz, 1H), 2.58-2.49 (m, 1H), 2.30
(dd, J=12.9, 4.7 Hz, 1H), 2.16-2.05 (m, 1H), 1.99 (d, J=23.9 Hz,
1H), 1.89-1.71 (m, 2H), 1.75-1.65 (m, 1H), 1.67-1.57 (m, 1H), 1.38
(s, 3H), 1.11-0.91 (m, 2H), 0.85 (s, 3H).
Step 2: Synthesis of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((2-Aminopyridin-4-yl)methy-
l)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8-
b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-
-4-one
##STR00871##
[1211] A 20 mL vial was charged with
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(bromomethyl)phenyl)-7-hydr-
oxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-dec-
ahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(2H)-one
(0.100 g, 0.179 mmol),
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine
(0.039 g, 0.179 mmol), and K.sub.2C03 (0.099 g, 0.718 mmol) in
degassed dioxane (2.0 ml)/water (0.200 mL) solution. The suspension
was evacuated and back filled with dry N.sub.2 (3.times.).
Pd(dppf)C12 (0.012 g, 0.016 mmol) was added and the vial was once
again evacuated and back filled with dry N2. The reaction mixture
was heated to 90.degree. C. After 1.5 hours the starting material
was consumed. The reaction was allowed to cool to room temperature,
diluted with EtOAc (20 mL) and washed with water (25 mL) then brine
(25 mL), dried over MgSO.sub.4, and solvent was removed under
reduced pressure. Purification by chromatography (silica, 40 g)
eluting with a gradient of 0-10% MeOH/CH.sub.2Cl.sub.2 gave a light
tan solid. Further purification by reverse phase prep HPLC on a
Phenomenex C18(2) 10 micron column (250.times.50 mm). A gradient of
MeCN (A) and 0.1% TFA in water (B) was used, at a flow rate of 80
mL/min (0-5.0 min 15% A, 5.0-20 min linear gradient 15-85% A, 20-25
min hold). Combined fractions were frozen and lyophilized to give
the title compound (27 mg, 0.047 mmol, 26% yield) as a white solid.
LCMS (Method r, Table 7) R.sub.t=0.90 min; MS m/z=571.3
[M+H.sup.+]. 1H NMR (501 MHz, DMSO-d6) .delta. 7.93 (s, 2H), 7.82
(d, J=6.6 Hz, 1H), 7.44 (d, J=8.1 Hz, 2H), 7.39-7.22 (m, 3H), 6.73
(d, J=8.1 Hz, 1H), 6.69 (s, 1H), 6.24-6.09 (m, 1H), 5.93 (s, 1H),
5.44 (s, 1H), 4.94 (d, J=5.1 Hz, 1H), 4.80 (s, 1H), 4.50 (d, J=19.4
Hz, 1H), 4.30 (s, 1H), 4.19 (d, J=19.4 Hz, 1H), 3.99 (s, 2H),
2.61-2.51 (m, 1H), 2.35-2.27 (m, 1H), 2.19-2.08 (m, 1H), 2.08-1.99
(m, 1H), 1.82-1.59 (m, 5H), 1.40 (s, 3H), 1.02 (ddd, J=27.9, 11.7,
3.2 Hz, 2H), 0.87 (s, 3H).
[1212] The following examples were synthesized using the same
procedure as Example 61 (above).
TABLE-US-00034 TABLE 8 Example Structure and Name LCMS and .sup.1H
NMR 62 ##STR00872## (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12b LCMS
(Method r, Table 7) R.sub.t = 0.87 min; MS m/z = 571.4 [M + H].
.sup.1H NMR (500 MHz, DMSO-d6) .delta. 7.84 (d, J = 2.1 Hz, 2H),
7.75 (dd, J = 9.1, 2.1 Hz, 1H),7.44- 7.37 (m, 2H), 7.31 (d, J =
10.1 Hz, 1H), 7.26 (d, J = 8.2 Hz, 2H), 6.90 (d, J = 9.0 Hz, 1H),
6.17 (dd, J = 10.1, 1.9 Hz, 1H), 5.93 (t, J = 1.6 Hz, 1H), 5.42 (s,
1H), 5.10 (s, 1H), 4.92 (d, J = 4.9 Hz, 1H), 4.80 (d, J = 3.4 Hz,
1H), 4.50 (d, J = 19.4 Hz, 1H), 4.29 (s, 1H), 4.17 (d, J = 19.5 Hz,
1H), 3.84 (s, 2H), 2.61-2.52 (m, 2H), 2.31 (d, J = 12.3 Hz, 1H),
2.13 (d, J = 10.9 Hz, 1H), 2.08-1.98 (m, 1H), 1.81-1.58 (m, 5H),
1.40 (s, 3H), 1.00 (ddd, J = 32.4, 11.7, 4.1 Hz, 2H), 0.86 (s, 3H).
S)-10-(4-((6-aminopyridin-3- yl)methyl)phenyl)-7-hydroxy-8b-(2-
hydroxyacetyl)-6a,8a-dimethyl- 1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-
dodecahydro-4H- naphtho[2',1':4,5]indeno[1,2- d][1,3]dioxol-4-one
63 ##STR00873## LCMS (Method r, Table 7) R.sub.t = 0.87 min; MS m/z
= 571.4 [M + H]. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 7.88 (s,
1H), 7.81 (d, J = 2.2 Hz, 1H), 7.36 (d, J = 8.1 Hz, 2H), 7.32-7.18
(m, 3H), 6.10 (dd, J = 10.1, 1.8 Hz, 1H), 5.87 (s, 1H), 5.36 (s,
1H), 4.86 (d, J = 4.8 Hz, 1H), 4.74 (s, 1H), 4.43 (d, J = 19.5 Hz,
1H), 4.23 (s, 1H), 4.11 (d, J = 19.4 Hz, 1H), 3.93 (s, 2H),
2.54-2.45 (m, 1H), 2.31-2.17 (m, 1H), 2.14-1.90 (m, 2H), 1.76-1.50
(m, 5H), 1.33 (s, 3H), 1.05-0.85 (m, 2H), 0.80 (s, 3H)
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12b S)-10-(4-((5-aminopyridin-3-
yl)methyl)phenyl)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-
1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-4H-
naphtho[2',1':4,5]indeno[1,2- d][1,3]dioxol-4-one 64 ##STR00874##
LCMS (Method r, Table 7) R.sub.t = 0.86 min; MS m/z = 571.3 [M +
H]. .sup.1H NMR (501 MHz, DMSO-d6) .delta. 7.89 (s, 2H), 7.82 (d, J
= 6.2 Hz, 1H), 7.58 (d, J = 6.1 Hz, 1H), 7.36 (d, J = 8.1 Hz, 2H),
7.23 (dd, J = 20.2, 9.1 Hz, 3H), 6.85-6.71 (m, 1H), 6.10 (d, J =
11.9 Hz, 1H), 5.86 (s, 1H), 5.36 (s, 1H), 4.87 (d, J = 5.0 Hz, 1H),
4.75 (s, 1H), 4.44 (d, J = 19.4 Hz, 1H), 4.23 (s, 1H), 4.12 (d, J =
19.4 Hz, 1H), 3.87 (s, 2H), 2.55-2.45 (m, 1H), 2.24 (d, J = 11.0
Hz, 1H), 2.05 (d, J = 22.0 Hz, 1H), 2.01- 1.91 (m, 1H), 1.77-1.49
(m, 5H), 1.33 (s, 3H), 1.04-0.87 (m, 2H), 0.80 (s, 3H).
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12b S)-10-(4-((2-aminopyridin-3-
yl)methyl)phenyl)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-
1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-4H-
naphtho[2',1':4,5]indeno[1,2- d[[1,3]dioxol-4-one 65 ##STR00875##
LCMS (Method r, Table 7) R.sub.t = 0.85 min; MS m/z = 607.4 [M +
H]. .sup.1H NMR (500 MHz, DMSO-d6) .delta. 7.80 (d, J = 6.5 Hz,
1H), 7.59 (s, 2H), 7.41 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 7.9 Hz,
2H), 7.27 (d, J = 10.8 Hz, 1H), 6.69 (d, J = 6.8 Hz, 1H), 6.64 (s,
1H), 6.30 (dd, J = 10.2, 1.9 Hz, 1H), 6.13 (s, 1H), 5.55 (d, J =
5.8 Hz, 1H), 5.49 (s, 1H), 5.13 (s, 1H), 4.96 (d, J = 4.5 Hz, 1H),
4.26-4.13 (m, 2H), 3.95 (s, 2H), 2.62-2.53 (m, 1H), 2.35- 2.28 (m,
1H), 2.28-2.18 (m, 1H), 2.09-1.99 (m, 1H), 1.77-1.66 (m, 3H), 1.50
(s, 4H), 0.87 (s, 3H). (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,
12bS)-10-(4-((2-aminopyridin-4- yl)methyl)phenyl)-2,6b-difluoro-7-
hydroxy-8b-(2-hydroxyacetyl)-6a,8a- dimethyl-
1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-4H-
naphtho[2',1':4,5]indeno[1,2- d][1,3]dioxol-4-one 66 ##STR00876##
LCMS (Method r, Table 7) R.sub.t = 0.85 min; MS m/z = 607.4 [M +
H]. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 7.76 (s, 1H), 7.65 (d, J
=9.5 Hz, 1H), 7.59 (s, 1H), 7.31 (d, J = 8.2 Hz, 2H), 7.25-7.15 (m,
3H), 6.79 (d, J = 9.2 Hz, 1H), 6.23 (d, J = 10.2 Hz, 1H), 6.06 (s,
1H), 5.69-5.49 (m, 1H), 5.46 (d, J = 4.0 Hz, 1H), 5.40 (s, 1H),
5.03 (s, 1H), 4.88 (d, J = 4.2 Hz, 1H), 4.44 (d, J = 19.4 Hz, 1H),
4.21-4.05 (m, 2H), 3.76 (s, 2H), 2.67-2.51 (m, 1H), 2.32- 2.09 (m,
2H), 1.96 (d, J = 13.1 Hz, 1H), 1.71- 1.57 (m, 3H), 1.50-1.35 (m,
4H), 0.80 (s, 3H). (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,
12bS)-10-(4-((6-aminopyridin-3- yl)methyl)phenyl)-2,6b-difluoro-7-
hydroxy-8b-(2-hydroxyacetyl)-6a,8a- dimethyl-
1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-4H-
naphtho[2',1':4,5]indeno[1,2- d][1,3]dioxol-4-one
Example 67
Synthesis of
1-(3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-Hydroxy-8b-(2-hydroxya-
cetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-
-1H-naphtho
[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)-1H-pyrrole-2,5-d-
ione
##STR00877##
[1213] Step 1: Synthesis of
(Z)-4-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-Hydroxy-8b-(2-hyd-
roxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodeca-
hydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)am-
ino)-4-oxobut-2-enoic Acid
##STR00878##
[1215] Maleic anhydride (46.5 mg, 0.474 mmol) was added to a room
temperature solution of
(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-7-hy-
droxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-d-
ecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-4(2H)-one
(239 mg, 0.420 mmol) in THF (3.0 mL). After 75 min, solvent was
removed under reduced pressure to give the title compound as an
off-white foam. This was used without further purification in the
next step (100% yield was assumed). LCMS (Method o, Table 7)R,=0.86
min; MS m/z=668.5 [M+H.sup.+]. .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 12.99 (s, 1H), 10.29 (s, 1H), 7.45-7.38 (m, 2H), 7.38-7.31
(m, 2H), 7.27 (d, J=10.1 Hz, 1H), 7.24-7.15 (m, 3H), 6.92 (dt,
J=7.8, 1.3 Hz, 1H), 6.38 (d, J=12.1 Hz, 1H), 6.25 (d, J=12.0 Hz,
1H), 6.12 (dd, J=10.1, 1.9 Hz, 1H), 5.89 (d, J=1.5 Hz, 1H), 5.36
(s, 1H), 5.03 (s, 1H), 4.88 (d, J=5.1 Hz, 1H), 4.73 (d, J=3.3 Hz,
1H), 4.46 (d, J=19.4 Hz, 1H), 4.26 (p, J=3.2 Hz, 1H), 4.14 (d,
J=19.4 Hz, 1H), 3.87 (s, 2H), 2.52 (dd, J=13.6, 5.3 Hz, 1H),
2.32-2.23 (m, 1H), 2.07 (tt, J=10.8, 6.2 Hz, 1H), 2.02-1.94 (m,
1H), 1.84-1.51 (m, 5H), 1.36 (s, 3H), 1.09 0.93 (m, 2H), 0.82 (s,
3H).
Step 2: Synthesis of
1-(3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-Hydroxy-8b-(2-hydroxya-
cetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-
-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)-1H-pyr-
role-2,5-dione
##STR00879##
[1217] Bis(trimethylsilyl)amine (HMDS) (63.4 .mu.L, 0.306 mmol) was
added to a solution of zinc bromide (75.0 mg, 0.333 mmol) and
(Z)-4-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hyd-
roxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodeca-
hydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)am-
ino)-4-oxobut-2-enoic acid (171 mg, 0.256 mmol) in tetrahydrofuran
(2.0 mL). The mixture was heated to 50.degree. C. for 2.5 h. LCMS
indicated incomplete conversion, so another aliquot of
bis(trimethylsilyl)amine (HMDS) (63.4 .mu.L, 0.306 mmol) was added.
The reaction was complete after an additional 90 min at 50.degree.
C. The mixture was cooled to room temperature, diluted with EtOAc
(20 mL), then washed sequentially with 1 N aqueous HCl (2.times.10
mL), saturated aqueous NaHCO.sub.3 (10 mL), brine (10 mL), dried
over Na.sub.2SO.sub.4, and solvent was removed under reduced
pressure. Purification by chromatography (silica, 12 g) eluting
with a gradient of 0-10% MeOH/DCM gave the title compound (82.6 mg,
0.127 mmol, 50% yield) as an off-white solid. LCMS (Method r, Table
7)R,=1.02 min; MS m/z=650.5 [M+H.sup.+]. .sup.1H NMR (400 MHz,
DMSO-d6) .delta. 7.27 (dt, J=7.8, 3.7 Hz, 3H), 7.19 (d, J=10.1 Hz,
1H), 7.17-7.06 (m, 4H), 7.06-7.01 (m, 3H), 6.04 (dd, J=10.1, 1.9
Hz, 1H), 5.81 (t, J=1.5 Hz, 1H), 5.64 (s, 1H), 5.29 (s, 1H), 4.95
(t, J=5.9 Hz, 1H), 4.80 (d, J=5.1 Hz, 1H), 4.65 (d, J=3.2 Hz, 1H),
4.38 (dd, J=19.4, 6.4 Hz, 1H), 4.18 (t, J=3.4 Hz, 1H), 4.06 (dd,
J=19.5, 5.7 Hz, 1H), 3.86 (s, 2H), 2.45 (dd, J=13.5, 5.4 Hz, 1H),
2.30-2.11 (m, 1H), 2.11-1.81 (m, 1H), 1.76-1.44 (m, 4H), 1.28 (s,
3H), 1.02-0.83 (m, 2H), 0.75 (s, 3H).
Example 68: Synthesis of
2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(2,5-Dioxo-2,5-dihydr-
o-1H-pyrrol-1-yl)benzyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7-
,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3-
]dioxol-8b-yl)-2-oxoethyl dihydrogen phosphate
##STR00880##
[1219] Diphosphoryl chloride (158 mg, 0.609 mmol) was added
drop-wise to a -51.degree. C. solution of
1-(3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxya-
cetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-
-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)-1H-pyr-
role-2,5-dione (82 mg, 0.126 mmol) in tetrahydrofuran (0.5 mL). The
reaction was slowly warmed to -10.degree. C. over an hour then
quenched with water at -5.degree. C. The mixture was treated with a
saturated aqueous solution of NaHCO.sub.3 to give a solution with a
pH of 8. Treatment with EtOAc (5 mL) gave a milky emulsion.
Adjusting the pH to 1 by addition of 1 N aqueous HCl improved the
emulsion. Extracted with EtOAc (4.times.5 mL), then washed the
combined organics with brine (5 mL), dried (Na.sub.2SO.sub.4), and
removed solvent under reduced pressure. The product was purified by
reverse phase prep HPLC on a Phenomenex C18(2) 10 micron column
(250.times.50 mm). A gradient of MeCN (A) and 0.1% TFA in water (B)
was used, at a flow rate of 90 mL/min (0-5.0 min 15% A, 5.0-20.0
min linear gradient 15-95% A). Combined fractions were frozen and
lyophilized to give the title compound (3.6 mg, 4.93 mmol, 4%
yield) as a white solid. LCMS (Method r, Table 7)R,=0.95 min; MS
m/z=730.5 [M+H.sup.+]. .sup.1H NMR (501 MHz, DMSO-d6) .delta. 7.38
(dt, J=7.8, 3.7 Hz, 3H), 7.31 (d, J=10.1 Hz, 1H), 7.29-7.26 (m,
2H), 7.25-7.22 (m, 1H), 7.19 (t, J=1.9 Hz, 1H), 7.17-7.12 (m, 3H),
6.16 (dd, J=10.1, 1.9 Hz, 1H), 5.93 (t, J=1.6 Hz, 1H), 5.48 (s,
1H), 4.96-4.86 (m, 2H), 4.84 (s, 1H), 4.56 (dd, J=18.1, 8.1 Hz,
1H), 4.30 (q, J=3.3 Hz, 1H), 3.97 (s, 2H), 2.59-2.52 (m, 1H), 2.31
(d, J=12.0 Hz, 1H), 2.17-2.07 (m, 1H), 2.05-1.98 (m, 1H), 1.85-1.56
(m, 5H), 1.39 (s, 3H), 1.03 (ddd, J=18.5, 11.8, 4.1 Hz, 2H), 0.88
(s, 3H).
Example 69: Synthesis of
2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-((S)-2-((S)-2-(2-(2,-
5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)pheno-
xy)methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,1-
2,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-
-2-oxoethyl dihydrogen phosphate
##STR00881##
[1220] Step 1: Synthesis of Tert-Butyl
((S)-1-(((S)-1-((3-((4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((d-
i-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,-
6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d]-
[1,3]dioxol-10-yl)benzyl)oxy)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopr-
opan-2-yl)carbamate
##STR00882##
[1222] Di-tert-butyl N,N-diethylphosphoramidite (0.226 ml, 0.811
mmol) was added to a room temperature solution of tert-butyl
((S)-1-(((S)-1-((3-((4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-
-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,-
12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl-
)oxy)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate
(463 mg, 0.559 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.97 ml,
2.237 mmol) in dimethyl acetamide (2 ml). Additional di-tert-butyl
N,N-diethylphosphoramidite (0.2 mL) was charged after 4.5 hours and
stirring was continued overnight. The reaction was cooled to
0.degree. C., whereupon a 30% solution of hydrogen peroxide in
water (0.17 mL, 1.67 mmol) was added drop-wise. Oxidation to the
phosphate was complete within 1.5. The reaction was cooled to
0.degree. C., and the reaction was quenched by addition of a 1M aq.
solution of Na.sub.2S.sub.2O.sub.3 (8 mL). The mixture was
extracted with EtOAc (2.times.30 mL), the combined organic layers
were washed with brine, dried over Na.sub.2SO.sub.4, and solvent
removed under reduced pressure. Purification by chromatography
(silica) using 100% EtOAc as eluent provided the title compound
(366 mg, 0.359 mmol, 64% yield) as white solid. LCMS (Method r,
Table 7)R,=1.08 min; MS m/z=1020.5 [M+H*].
Step 2: Synthesis of
2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-((S)-2-((S)-2-Aminop-
ropanamido)propanamido)phenoxy)methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-o-
xo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]ind-
eno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl Dihydrogen Phosphate
##STR00883##
[1224] TFA (0.95 mL) was added to a room temperature solution of
tert-butyl
((S)-1-(((S)-1-((3-((4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((d-
i-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,-
6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d]-
[1,3]dioxol-10-yl)benzyl)oxy)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopr-
opan-2-yl)carbamate (364 mg, 0.357 mmol) in DCM (2 mL). The
reaction was complete within 2 h, whereupon solvent was removed
under reduced pressure. The title compound was obtained as a foamy
light yellow solid and was used without further purification. LCMS
(Method r, Table 7) major acetal isomer: R,=0.77 min; MS m/z=808.3
[M+H.sup.+], minor acetal isomer: R,=0.79 min; MS m/z=808.3
[M+H.sup.+].
Step 3: Synthesis of
2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-((S)-2-((S)-2-(2-(2,-
5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)pheno-
xy)methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,1-
2,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-
-2-oxoethyl Dihydrogen Phosphate
##STR00884##
[1226] N,N-Diisopropylethylamine (0.37 mL, 2.12 mmol) and
maleimidoacetic acid N-hydroxysuccinimide ester (89 mg, 0.353 mmol)
were added sequentially to a room temperature solution of
2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3-((S)-2-((S)-2-aminop-
ropanamido)propanamido)phenoxy)methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-o-
xo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]ind-
eno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl dihydrogen phosphate (285
mg, 0.353 mmol) in dimethyl formamide (1.5 mL) and was stirred
overnight. The reaction mixture was diluted with DMSO and was
purified by preparative reverse phase HPLC on a Phenomenex C18(2)
10 micron column (250.times.50 mm). A gradient of MeCN (A) and 0.1%
TFA in water (B) was used, at a flow rate of 30 mL/min (0-3.0 min
15% A, 3.0-19.0 min linear gradient 15-60% A, then 19.0-23.0 min
linear gradient to 85% A). Combined fractions were concentrated to
remove volatile solvents under reduced pressure, and the resulting
solution was frozen and lyophilized to give the title compound (93
mg, 0.098 mmol, 28% yield) as a white solid. Major acetal isomer:
LCMS (Method r, Table 7) R.sub.t=0.83 min; MS m/z=945.4
[M+H.sup.+]. 1H NMR (400 MHz, DMSO-d6) .delta. 9.78 (s, 1H), 8.39
(d, J=7.2 Hz, 1H), 8.13 (d, J=7.2 Hz, 1H), 7.49-7.37 (m, 4H), 7.33
(t, J=2.2 Hz, 1H), 7.28 (d, J=10.1 Hz, 1H), 7.14 (t, J=8.1 Hz, 1H),
7.10-7.05 (m, 1H), 7.03 (s, 2H), 6.64 (dd, J=8.0, 2.4 Hz, 1H), 6.13
(dd, J=10.1, 1.9 Hz, 1H), 5.89 (d, J=1.5 Hz, 1H), 5.50 (s, 1H),
5.04 (s, 2H), 4.96-4.85 (m, 2H), 4.81 (s, 1H), 4.55 (dd, J=18.1,
8.2 Hz, 1H), 4.38-4.21 (m, 3H), 4.13-3.98 (m, 2H), 2.53 (dd,
J=13.2, 5.2 Hz, 1H), 2.28 (d, J=16.1 Hz, 1H), 2.09 (d, J=11.2 Hz,
1H), 2.08-1.95 (m, 1H), 1.70 (dddd, J=29.9, 25.9, 14.4, 6.4 Hz,
5H), 1.36 (s, 3H), 1.26 (d, J=7.0 Hz, 3H), 1.18 (d, J=7.1 Hz, 3H),
1.02 (ddd, J=14.7, 11.6, 4.0 Hz, 2H), 0.86 (s, 3H).
Example 70: Synthesis of
2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-(2-(2,5-
-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)benzyl-
)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-
-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoeth-
yl 2-(dimethylamino)acetate 2,2,2-trifluoroacetate
##STR00885##
[1227] Step 1: Synthesis of
2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-((tert--
Butoxycarbonyl)amino)propanamido)propanamido)benzyl)phenyl)-7-hydroxy-6a,8-
a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho-
[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl
2-(dimethylamino)acetate 2,2,2-trifluoroacetate
##STR00886##
[1229] To a solution of tert-butyl
((2S)-1-(((2S)-1-((3-(4-((6aR,7S,8aS,8bS,10R,11aR,12aS,12bS)-7
hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a-
,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-y-
l)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate
(Prepared in same manner as Example 10, Step 1)(78 mg, 0.096 mmol),
2-(dimethylamino)acetic acid (10.9 mg, 0.106 mmol), and
2,6-dimethylpyridine (0.022 mL, 0.192 mmol) in anhydrous
N,N-dimethylformamide (2.0 mL) was added HATU (43.8 mg, 0.115
mmol), and the resulting solution was stirred at room temperature
for 45 minutes. The crude product was purified by C18 HPLC, eluting
with a solvent gradient of 5-95% MeCN in 0.1M aqueous TFA.
Fractions containing the pure product were concentrated by
lyophilization to afford the title compound (82 mg, 89% yield).
LCMS (Method r, Table 7)R,=0.80 min, MS m/z=898.2 [M+H.sup.+].
Step 2: Synthesis of
2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-(2-(2,5-
-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)benzyl-
)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-
-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoeth-
yl 2-(dimethylamino)acetate 2,2,2-trifluoroacetate
##STR00887##
[1231] A solution of
2-((6aR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-((tert-
butoxycarbonyl)amino)propanamido)propanamido)benzyl)phenyl)-7-hydroxy-6a,-
8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphth-
o[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl
2-(dimethylamino)acetate (82 mg, 0.074 mmol) in DCM (4 mL) and TFA
(1 mL) was stirred at room temperature for 20 minutes, and then
concentrated in vacuo. To a solution of this compound in anhydrous
N,N-dimethylformamide (1 mL) was added Hunig's base (0.20 mL, 1.15
mmol) and 2,5-dioxopyrrolidin-1-yl
2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (27.8 mg, 0.11
mmol). The resulting mixture was stirred at room temperature for 15
minutes, and TFA (0.106 mL, 1.376 mmol) was added. The crude
product was purified by C18 HPLC, eluting with a solvent gradient
of 5-95% MeCN in 0.1M aqueous TFA. Fractions containing the pure
product were concentrated by lyophilization to afford the title
compound as a colorless solid (46 mg, 0.0439 mmol, 59% yield). LCMS
(Method r, Table 7) major acetal isomer R,=0.82 min, MS m/z=934
[M+H*]; minor acetal isomer R.sub.t==0.81 min, MS m/z=934
[M+H.sup.+]. .sup.1H NMR (501 MHz, DMSO-d.sub.6) .delta. 10.12 (s,
2H), 9.75 (s, 1H), 8.40 (d, J=7.3 Hz, 1H), 8.11 (d, J=7.1 Hz, 1H),
7.45-7.42 (m, 1H), 7.38 (dd, J=8.2, 2.0 Hz, 2H), 7.31 (d, J=10.1
Hz, 1H), 7.22 (d, J=8.2 Hz, 2H), 7.17 (t, J=7.8 Hz, 1H), 7.06 (s,
1H), 6.89 (d, J=7.7 Hz, 1H), 6.50 (s, 1H), 6.15 (dd, J=10.1, 1.9
Hz, 1H), 5.93-5.90 (m, 1H), 5.52 (s, 1H), 5.30 (d, J=17.7 Hz, 1H),
5.00 (d, J=17.7 Hz, 1H), 4.86 (t, J=5.0 Hz, 2H), 4.36-4.25 (m, 4H),
4.12-4.02 (m, 2H), 3.87 (s, 1H), 2.82 (s, 3H), 2.56-2.51 (m, 1H),
2.50 (s, OH), 2.50 (d, J=1.8 Hz, 0H), 2.33-2.26 (m, 2H), 2.15-2.06
(m, 2H), 2.04-1.97 (m, 2H), 1.84-1.80 (m, 1H), 1.77-1.60 (m, 4H),
1.37 (s, 3H), 1.26 (d, J=7.1 Hz, 3H), 1.19 (d, J=7.1 Hz, 3H),
1.10-0.98 (m, 3H), 0.89 (s, 3H).
Example 71
Synthesis of
4-(2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-(2-(-
2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)ben-
zyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,-
12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxo-
ethoxy)-4-oxobutanoic Acid
##STR00888##
[1232] Step 1: Synthesis of
2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-((tert--
butoxycarbonyl)amino)propanamido)propanamido)benzyl)phenyl)-7-hydroxy-6a,8-
a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho-
[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl tert-butyl
Succinate
##STR00889##
[1234] The title compound was prepared using the method described
for Example 70, substituting 4-(tert-butoxy)-4-oxobutanoic acid for
2-(dimethylamino)acetic acid. LCMS (Method r, Table 7) R.sub.t=1.03
min; MS m/z=968 [M+H*].
Step 2: Synthesis of
4-(2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-(2-(-
2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)ben-
zyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,-
12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxo-
ethoxy)-4-oxobutanoic Acid
##STR00890##
[1236] The title compound was prepared using the method described
in Step 2, Example 69. It was isolated as a colorless solid (49 mg,
43%). LCMS (Method r, Table 7)R,=0.88 min; MS m/z=948.9
[M+H.sup.+]. H NMR (400 MHz, DMSO-d.sub.6) .delta. 12.23 (s, 1H),
9.74 (s, 1H), 8.37 (d, J=7.3 Hz, 1H), 8.09 (d, J=7.2 Hz, 1H),
7.42-7.33 (m, 3H), 7.31-7.25 (m, 1H), 7.17 (dd, J=20.7, 7.9 Hz,
3H), 6.90-6.84 (m, 1H), 6.15-6.09 (m, 1H), 5.90-5.87 (m, 1H), 5.48
(s, 1H), 5.07 (d, J=17.7 Hz, 1H), 4.86-4.79 (m, 2H), 4.37-4.23 (m,
3H), 4.12-3.98 (m, 2H), 3.85 (s, 2H), 2.65-2.58 (m, 2H), 2.52-2.47
(m, 2H), 2.32-2.24 (m, 2H), 2.09 (d, J=10.8 Hz, 2H), 2.02-1.94 (m,
2H), 1.85-1.56 (m, 6H), 1.36 (s, 3H), 1.24 (d, J=7.1 Hz, 3H), 1.17
(d, J=7.1 Hz, 3H), 1.10-0.95 (m, 3H), 0.85 (s, 3H).
Example 72
Synthesis of
2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-(2-(2,5-
-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)benzyl-
)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-
-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoeth-
yl Hydrogen Sulfate
##STR00891##
[1237] Step 1: Synthesis of
2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-((tert--
Butoxycarbonyl)amino)propanamido)propanamido)benzyl)phenyl)-7-hydroxy-6a,8-
a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho-
[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl hydrogen
sulfate
##STR00892##
[1239] To a solution of tert-butyl
((S)-1-(((S)-1-((3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy--
8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,1-
2b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-
phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate,
prepared in a similar manner to Example 10, Step 1, (53 mg, 0.065
mmol) in MeCN (2 mL) was added pyridine sulfur trioxide complex (42
mg, 0.26 mmol). The mixture was stirred at room temperature for 2
hours. The crude product was purified by C18 HPLC, eluting with a
solvent gradient of 5-95% MeCN in 0.1M aqueous TFA. Fractions
containing the pure product were concentrated by lyophilization to
afford the title compound. LCMS (Method r, Table 7)R,=0.83 min; MS
m/z=892.0 [M+H.sup.+].
Step 2: Synthesis of
2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((S)-2-((S)-2-(2-(2,5-
-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamido)propanamido)benzyl-
)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-
-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoeth-
yl Hydrogen Sulfate
##STR00893##
[1241] The title compound was prepared using the method described
in Step 2, Example 69. It was isolated as a colorless solid (27 mg,
28% yield). LCMS (Method r, Table 7)R,=0.77 min; MS m/z=928.9
[M+H.sup.+]. H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.75 (s, 1H),
8.37 (d, J=7.3 Hz, 1H), 8.10 (d, J=7.2 Hz, 1H), 7.44 (d, J=8.3 Hz,
1H), 7.40-7.32 (m, 2H), 7.33-7.29 (m, 1H), 7.27 (d, J=10.1 Hz, 1H),
7.23-7.12 (m, 3H), 7.04 (s, 1H), 6.93-6.83 (m, 2H), 6.12 (dd,
J=10.1, 1.9 Hz, 1H), 5.91-5.86 (m, 1H), 5.42 (s, 1H), 4.87 (d,
J=5.1 Hz, 1H), 4.84 (s, 1H), 4.74 (d, J=18.3 Hz, 1H), 4.45 (d,
J=18.3 Hz, 1H), 4.36-4.24 (m, 3H), 4.11-3.99 (m, 2H), 3.86 (s, 2H),
2.58-2.48 (m, 1H), 2.32-2.22 (m, 1H), 2.08 (d, J=11.1 Hz, 1H), 1.98
(s, 1H), 1.77 (s, 2H), 1.75-1.56 (m, 4H), 1.36 (s, 3H), 1.24 (d,
J=7.1 Hz, 3H), 1.17 (d, J=7.0 Hz, 3H), 1.10-0.95 (m, 2H), 0.83 (s,
3H).
Example 73: Conjugation Protocols
General Cysteine Conjugation Protocol
[1242] An approximate 10 mg/mL solution of the desired antibody was
prepared in PBS buffer, pH 7.4 as well as a 10 mM TCEP solution in
PBS (Pierce Bond-Breaker, cat. 77720). Antibodies (anti-hTNF hIgG1
(D2E7) or anti-mTNF mIgG2a (8C11; McRae B L et al. J Crohns Colitis
10 (1): 69-76 (2016)) were then partially reduced by adding
approximately two molar eq of 10 mM TCEP, briefly mixing, and
incubating for 60 min at 37.degree. C. DMSO was then added to the
partially reduced antibodies in sufficient quantity to 15% total
DMSO. For the conjugations, 8 molar eq of a 10 mM D-L-maleimide
solution (wherein SM is a radical of a glucocorticosteroid and L is
a linker) were then added and incubated for 30 min at room
temperature. Excess combo and DMSO were then removed using NAP-5
desalting columns (GE Healthcare, cat. 17-0853-02) previously
equilibrated with PBS buffer, pH 7.4. Desalted samples were then
analyzed by size exclusion chromatography (SEC), hydrophobic
interaction chromatography (HIC), and reduced mass
spectrometry.
Thiosuccinimide Hydrolysis
##STR00894##
[1244] Hydrolysis of the thiosuccinimide ring of ADCs of the
disclosure was accomplished by incubating the ADCs at an elevated
pH. Briefly, a 0.7 M arginine, pH 9.0 solution was prepared and
added to each ADC in PBS buffer to bring the total arginine
concentration to 50 mM (pH 8.9). The material was then incubated at
25.degree. C. for 72 hours. Hydrolysis of the succinimide ring was
then confirmed by reduced mass spectrometry, after which,
hydrolysis was quenched with the addition of a 0.1 M acetic acid
solution to 12.5 mM total acetic acid (pH 7.1).
[1245] General Lysine Conjugation Protocol
[1246] An approximate 10 mg/mL solution of the desired antibody was
initially prepared in PBS buffer, pH 7.4. Eight molar eq of the
D-L-N-hydroxysuccinimide (wherein SM is a radical of a
glucocorticosteroid and L is a linker) was then added to the
antibody and incubated at 23.degree. C. for up to 24 hours in the
presence of 15% DMSO. Conjugated samples were then desalted to
remove excess combo and DMSO using NAP-5 desalting columns (GE
Healthcare, cat. 17-0853-02) equilibrated with PBS buffer, pH 7.4.
Desalted samples were then analyzed by size exclusion
chromatography (SEC), hydrophobic interaction chromatography (HIC),
and reduced mass spectrometry.
[1247] ADC Analytical Procedures
[1248] Hydrophobic Interaction Chromatography. ADCs were profiled
by hydrophobic interaction chromatography (HIC) to determine degree
of conjugation and to calculate approximate drug to antibody drug
ratios (DARs). Briefly, 100 .mu.g of the ADCs were loaded onto an
Ultimate 3000 Dual LC system (Thermo Scientific) equipped with a
4.6.times.35 mm butyl-NPR column (Tosoh Bioscience, cat. 14947).
ADCs were loaded onto the column equilibrated in 100% buffer A and
eluted using a linear gradient from 100% buffer A to 100% buffer B
over 12 min at 0.8 mL/min, where buffer A is 25 mM sodium
phosphate, 1.5 M ammonium sulfate, pH 7.25 and buffer B is 25 mM
sodium phosphate, 20% isopropanol, pH 7.25. The DAR was determined
by taking the sum of each peak percent area multiplied by their
corresponding drug load and dividing the weighted sum by 100.
[1249] Size Exclusion Chromatography. Size distributions of the
ADCs were profiled by size exclusion chromatography (SEC) using an
Ultimate 3000 Dual LC system (Thermo Scientific) equipped with a
7.8.times.300 mm TSK-gel 3000SW.sub.XL column (Tosoh Bioscience,
cat. 08541). 20 ug of each of the ADCs were loaded onto the column
and eluted over 17 min using an isocratic gradient at 1 mL/min of
100 mM sodium sulfate, 100 mM sodium phosphate, pH 6.8 at 0.8
mL/min.
Example 74: Preparation of Adalimumab Conjugated with a
Glucocorticosteroid to Give an ADC
[1250] Adalimumab MP-ala-ala steroid ADC having an average DAR 3.5
was prepared by a two-step chemical process: disulfide reduction of
adalimumab followed by alkylation (conjugation) with
maleimidopropyl alanine-alanine steroid Cpd. No. 88.
##STR00895##
[1251] In the first step, a limited number of interchain disulfide
bonds of adalimumab are reduced with tris(2-carboxyethyl) phosphine
("TCEP") (.gtoreq.1.8 equiv). Partially-reduced adalimumab is then
conjugated to Cpd. No. 88 (.gtoreq.5 equiv) in DMSO.
[1252] Referring to FIG. 5 which shows a chromatographic resolution
of the resultant ADC preparation, the ADC is a heterogenous mixture
containing antibodies having zero drug linker molecules attached
("E0" peak), two drug linker molecules attached ("E2" peak), four
drug linker molecules attached ("E4" peak), six drug linker
molecules attached ("E6" peak) and eight drug linker molecules
attached ("E8" peak), depending upon the number of interchain
disulfide bonds reduced. Methods of chromatographically separating
and isolating the homogenous E2 and E4 peaks are described by
Hamblett et al., Clin Cancer Res 2004; 10:7063-7070. The HIC
conditions used in FIG. 5 were as follows:
[1253] The column was TOSOH Tskgel Butyl-NPR, 4.6 mm.times.3.5 cm,
2.5 g and the column temperature was 30.degree. C. Wavelength was
280 nm, run time was 22 minutes, injection, volume was
[1254] 40 L, flow rate was 0.5 mL/minute. Mobile Phase A: 25 mM
Na.sub.2HPO.sub.4, pH 7.0 and 1.5M (NH.sub.4).sub.2SO.sub.4, Mobile
Phase B: 25 mM Na.sub.2HPO.sub.4, pH 7.0/IPA=75/25. Gradient
Profile:
TABLE-US-00035 Time (minutes) Mobile Phase A Mobile Phase B 0 90 10
2 85 15 18 5 95 18.1 90 10 22 90 10
[1255] Methods of chromatographically separating and isolating the
homogenous E2 and E4 peaks are described by Hamblett et al., Clin
Cancer Res 2004; 10:7063-7070. Briefly, after hydrolysis and
adjustment to pH<7.4, the broad distribution mixture was treated
with 3 M ammonium sulfate/50 mM phosphate buffer to bring the
overall solution concentration of ammonium sulfate to approximately
0.8 M. A pre-packed Hydrophobic Interaction Chromatography (HIC)
column (resin butyl sepharose HP) was prepared by sanitizing with
0.5 N NaOH solution (4 CV), rinsing with water for injection (WFI,
0.5 CV) and equilibration with 0.8 M ammonium sulfate/25 mM
phosphate buffer (4 CV). The broad distribution/ammonium sulfate
buffered solution was loaded on the HIC column (approximate
loading, 30 mg protein per mL of resin) followed by a wash with 0.8
M ammonium sulfate/25 mM phosphate buffer (2.5 CV). Elution of the
product was as follows: 0.72 M ammonium sulfate/25 mM phosphate
buffer (3 CV), unconjugated mAb; 0.56 M ammonium sulfate/25 mM
phosphate buffer (4.5 CV), DAR2 ADC; 0.32 M ammonium sulfate/25 mM
phosphate buffer (6.5 CV), DAR4 ADC. The DAR 2 and DAR4 product
fractions were then separately concentrated to approximately 30
mg/mL via ultrafiltration (Millipore Ultracel, 30 kD cutoff)
followed by diafiltration into WFI (8 CV).
[1256] The succinimide of the purified E4 conjugate was hydrolyzed
to provide the stabilized attachment by adjusting the pH of the
product solution to .gtoreq.9 using an arginine buffer. The
solution was held at ambient temperature for .gtoreq.2 days at
which time LC-MS analysis determined the hydrolysis was >90%
complete. See FIG. 6 for a portion of the LC-MS chromatogram. The
SEC conditions used in FIG. 6 were as follows:
[1257] The column was TOSOH TSK-gel G3000SW.sub.xL, 5 .mu.l, 250
.ANG., 7.8.times.300 mm, the column was ambient temperature,
Wavelength was 214 nm, Run Time was 55 minutes, Injection Volume
was 10 L, Flow Rate was 0.25 mL/minute, Autosampler Temp. was
4.degree. C. Mobile Phase: 100 mM Na.sub.2HPO.sub.4 & 100 mM
Na.sub.2SO.sub.4, pH 6.8/IPA=90/10.
[1258] Raw (FIG. 7) and deconvoluted (FIG. 8) MS data of adalimumab
conjugated with MP-ala-ala-steroid Cpd. No. 88. Black square and
circle represent the ADC with succinimide hydrolyzed and
unhydrolyzed, respectively. The relative abundance of hydrolyzed
and unhydrolyzed ADC is used to determine hydrolysis
conversion.
[1259] Hydrolysis
[1260] Hydrolysis of succinimide ring after conjugation was
conducted with borate buffer at pH 8.0, pH 8.5 and pH 9.0 and
arginine buffer at pH 8.0 and pH 9.0 to study the rate of ring
hydrolysis. The results are shown in Table 9 below.
TABLE-US-00036 TABLE 9 Succinimide ring hydrolysis Hydrolysis after
Hydrolysis after Hydrolysis after 1 day/% (pH) 2 days/% (pH) 3
days/% (pH) Cpd. Borate Borate Borate Arg. Arg. Borate Borate
Borate Arg. Arg. Borate Borate Borate Arg. Arg. No. (8.0) (8.5)
(9.0) (8.0) (9.0) (8.0) (8.5) (9.0) (8.0) (9.0) (8.0) (8.5) (9.0)
(8.0) (9.0) 121 28 37.6 49.7 24.5 63.4 42.6 55.9 71.8 33.8 84.4 56
72.1 85.4 46.5 100 122 -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
123 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 124
-- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 125 38.2 49.8 64.5
29.8 75.7 56.4 71.5 88.6 42.2 100 73.6 86.2 100 58 100 126 30.5
37.7 50.3 23.5 59.3 44.5 57.1 72.2 33.4 82.4 58.7 74.9 90.3 45.8
100 127 32.2 40.8 53.9 24.6 63 46.4 60.1 75.1 33.7 84.7 61.2 77
90.3 48.5 100 128 28.6 35.6 48.4 20.7 56.9 40.8 53.6 69.9 30.1 82.5
54.9 71.5 88.9 42.6 100 129 39.1 49.6 65.7 30 76.6 57.5 71.9 88.4
42.1 100 73.9 100 100 58.7 100 130 84.3 100 100 74.4 100 100 100
100 94.2 100 100 100 100 100 100 131 28.6 35.2 49.5 23.9 55.4 40.9
51.4 67.7 29.8 79.2 53.4 68.1 85.6 41.7 100
Example 75: In Vitro Activity of Small Molecule Steroids
Glucocorticoid Receptor Binding Assay
[1261] Small molecules were tested for glucocorticoid receptor (GR)
binding using the Polarscreen.TM. Glucocorticoid Receptor Assay
Kit, Red (ThermoFisher A 15898) according to the manufacturer's
protocol. Briefly, compounds were serially diluted in DMSO then
transferred into assay kit buffer at a 1:10 dilution. Compounds
were further diluted 1:5 in assay kit buffer, and 10 .mu.l was
transferred to a 384 well low volume black walled plate (Corning
4514). 5 .mu.l of 4.times. Fluormone GS Red stock solution and 5 ul
of 4.times.GR full length stock solution were added to each well
containing test compound, and plates were incubated protected from
light at room temperature for 4 hours. Fluorescence Polarization
(mP) was measured for each plate using an EnVision Multilabel Plate
Reader (Perkinelmer #2104-0010), and data were analyzed using a
four parameter curve fit to generate EC50 values. The results are
shown in Table 10 below.
Mineralcorticoid Receptor Cell Assay
[1262] Small molecules were tested for mineralcorticoid receptor
(MR) agonist activity using the PathHunter.RTM. NHRPRO CHO-K1 MR
cell line (DiscoveRx cat #93-0451C2) according to the
manufacturer's protocol. Briefly, 20,000 cells/well in culture
medium were plated in a 96 half-well plate (Costar cat #3885)
overnight at 37.degree. C. Media was removed and replaced with
serially diluted small molecules in assay medium (30 .mu.l; 0.3%
DMSO final). Plates were incubated overnight at 37.degree. C. Media
was removed, replaced with detection reagent (DiscoveRx cat
#93-0001; 12 l/well), and incubated at room temperature (R.sup.7b)
for 60 minutes. Luminescence was measured for each plate using an
EnVision Multilabel Plate Reader (Perkinelmer #2104-0010), and data
were analyzed using a four parameter curve fit to generate EC50
values. The results are shown in Table 10 below.
Progesterone Receptor Binding Assay
[1263] Small molecules were tested for progersterone receptor (PR)
binding using a modification of the LanthaScreen.RTM. TR-FRET
Progesterone Receptor Coactivator Assay (Thermofisher cat # A15903)
where the fluorescein-labeled coactivator peptide was replaced with
Fluormone AL-Red (Thermofisher cat # PV4294) to improve assay
signal. Briefly, compounds were serially diluted in DMSO, then
transferred into assay buffer (Thermofisher cat # PV4301+5 mM DTT)
at a 1:10 dilution. 10 .mu.l of compound was transferred to a 96
half-area black well plate (Corning cat #3694) in duplicate. 5
.mu.l of PR-LBD protein (4 nM stock in assay buffer; Thermofisher
cat # P2899) was added to each well. In addition 5 1 of a prepared
mixture of Fluormone AL-Red (12 nM) and terbium-labeled anti-GST
monoclonal antibody (mAb) (20 nM; Thermofisher cat # PV3550) in
assay buffer was also added to each well. Plates were incubated at
room temperature (RT) for 2 hours, and then TR-FRET emission ratio
was measured using an EnVision Multilabel Plate Reader (Perkinelmer
#2104-0010). Data were analyzed using a four parameter curve fit to
generate EC50 values. The results are shown in Table 10 below.
Androgen Receptor Binding Assay
[1264] Small molecules were tested for androgen receptor (AR)
binding using a modification of the LanthaScreen.RTM. TR-FRET
Androgen Receptor Coactivator Assay (Thermofisher cat # A15878)
where the fluorescein-labeled coactivator peptide was replaced with
Fluormone AL-Red (Thermofisher cat # PV4294) to improve assay
signal. Briefly, compounds were serially diluted in DMSO then
transferred into assay buffer (Thermofisher cat # PV4295+5 mM DTT)
at a 1:10 dilution. 10 of compound was transferred to a 96
half-area black well plate (Corning cat #3694) in duplicate. 5
.mu.l of AR-LBD protein (5 nM stock in assay buffer; Thermofisher
cat #3009) was added to each well. In addition 5 .mu.l of a
prepared stock of Fluormone AL-Red (20 nM) and terbium-labeled
anti-GST monoclonal antibody (mAb) (30 nM; Thermofisher cat #
PV3550) in assay buffer was also added to each well. Plates were
incubated at room temperature (RT) for 6 hours then TR-FRET
emission ratio was measured using an EnVision Multilabel Plate
Reader (Perkinelmer #2104-0010). Data were analyzed using a four
parameter curve fit to generate EC50 values. The results are shown
in Table 10 below.
GRE Reporter Assay
[1265] K562 parental GRE (pGL4.36[luc2P/MMTV/Hygro]) cells
described in Example 78 were plated onto 96 well tissue culture
treated white plates (Costar: 3917) at 50,000 cells per well in 50
.mu.L of assay medium (RPMI, 1% CSFBS, 1% L-glutamine, 1% Na
Pyruvate and 1% MEAA). Small molecule GR agonist compounds were
serial diluted at a starting concentration of 100 .mu.M and serial
diluted 4 fold in 100% DMSO. The small molecule compounds were
diluted further in assay medium by transferring 2 .mu.l of serial
diluted compounds into 248 .mu.l assay medium into a secondary
dilution plate (1:125 dilution). The cells were then treated with
25 .mu.L of 1:125 diluted GR agonist compound for a final starting
concentration of 266.7 nM (1:3) or media alone and incubated for 24
hours at 37.degree., 5% CO.sub.2. After 24 hours incubation, cells
were treated with 75 .mu.L of Dual-Glo Luciferase Assay System
(Promega-E2920) for 10 minutes and analyzed for luminescence using
the TopCount or MicroBeta2 (PerkinElmer).
Estrogen Receptor Binding Assay
[1266] Small molecules were tested for estrogen receptor (ER) alpha
binding using a modification of the LanthaScreen.RTM. TR-FRET
Estrogen Receptor Alpha Coactivator Assay (Thermofisher cat #
A15885) where the fluorescein-labeled coactivator peptide was
replaced with Fluormone ES2 Green (Thermofisher cat # PV6045) to
improve assay signal. Briefly, compounds were serially diluted in
DMSO then transferred into assay buffer (Thermofisher cat #
PV4295+5 mM DTT) at a 1:10 dilution. 10 .mu.l of compound was
transferred to a 96 half-area black well plate (Corning cat #3694)
in duplicate. 5 .mu.l of ER-LBD protein (5 nM stock in assay
buffer; Thermofisher cat #4542) was added to each well. In addition
5 .mu.l of a prepared stock of Fluormone ES2 Green (12 nM) and
terbium-labeled anti-GST monoclonal antibody (mAb) (8 nM;
Thermofisher cat # PV3550) in assay buffer was also added to each
well. Plates were incubated at room temperature (R.sup.7b) for 4
hours, and then TR-FRET emission ratio was measured using an
EnVision Multilabel Plate Reader (Perkinelmer #2104-0010). Data
were analyzed using a four parameter curve fit to generate EC50
values. The results are shown in Table 10 below.
TABLE-US-00037 TABLE 10 in vitro activity GR GRE MR PR ER AR
binding Reporter (Agonist) Binding Binding Binding Cpd. IC.sub.50
EC.sub.50 EC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 No. Chemical
Structure (.mu.M) (.mu.M) (.mu.M) (.mu.M) (.mu.M) (.mu.M) 2 (TFA)
##STR00896## 0.0066 NT NT NT NT NT 3 ##STR00897## 0.0036 0.0002
0.0846 0.0026 30++ >30 4 (TFA) ##STR00898## 0.0095 0.0003 0.836
0.957 0.0198 >30 3.99 5 ##STR00899## 0.0120 0.0184 NT NT NT NT 6
(TFA) ##STR00900## 0.0641 0.0396 NT NT NT NT 7 ##STR00901## 0.0155
0.0005 0.515 0.300 0.0157 >30 >30 8 ##STR00902## 0.0201
0.0151 NT NT NT NT 9 ##STR00903## 0.0094 0.0001 2.61 0.0116 30++
>30 10 ##STR00904## 0.0156 0.0001 0.305 0.0105 5.13 >30 11
##STR00905## 0.0139 0.0185 NT NT NT NT 12 ##STR00906## 0.0247
0.0161 NT NT NT NT 13 ##STR00907## 0.0157 0.0009 10.1 >30 0.0154
>30 >30 14 ##STR00908## 0.0255 0.0001 0.119 0.0222 2.71
>30 15 ##STR00909## 0.0149 0.0006 0.141 0.0165 3.77 >30 16
##STR00910## 0.0537 0.0188 NT NT NT NT 17 ##STR00911## 0.0278
0.0656 NT NT NT NT 18 ##STR00912## 0.0437 0.0299 NT NT NT NT 19
(TFA) ##STR00913## 0.0101 0.0152 NT NT NT NT 20 ##STR00914## 0.0194
0.0002 0.521 0.0186 0.814 >30 20 (TFA) ##STR00915## 0.0086
0.0003 1.64 1.64 0.0171 30++ >30 21 ##STR00916## 0.0094 0.0002
0.149 4.42 0.0073 >30 >30 22 ##STR00917## 0.0283 0.0075 NT NT
NT NT 22 (TFA) ##STR00918## 0.0082 0.0233 NT NT NT NT 23
##STR00919## 0.0108 0.0051 NT NT NT NT 24 (TFA) ##STR00920## 0.0138
0.0023 1.06 0.0089 30++ >30 25 ##STR00921## 0.0216 0.0002 3.09
0.0133 30++ >30 26 ##STR00922## 0.0256 0.0167 NT NT NT NT 27
##STR00923## 0.0102 NV NT NT NT NT 28 ##STR00924## 0.0146 0.0419 NT
NT NT NT 29 ##STR00925## 0.0132 0.0011 NT NT NT NT 30 ##STR00926##
0.0177 0.0078 NT NT NT NT 31 ##STR00927## 0.0107 0.0003 NT NT NT NT
32 ##STR00928## 0.0074 0.0001 3.56 0.013 30++ >30 33
##STR00929## 0.0169 0.0306 NT NT NT NT 34 ##STR00930## 0.0122
0.0034 NT NT NT NT 35 ##STR00931## 0.0034 0.0214 NT NT NT NT 35
(TFA) ##STR00932## 0.0067 0.0178 0.407 0.407+ 0.256 30++ >30 36
(TFA) ##STR00933## 0.0034 0.0002 0.129 >30 0.052 >30 >30
37 ##STR00934## 0.0131 0.1320 2.12 5.05 1.09 >30 >30 38
##STR00935## 0.0128 0.266++ 0.876 1.47 1.19 >30 9.28 39
##STR00936## 0.0156 0.0322 0.422 0.137 30++ >30 40 ##STR00937##
0.0111 0.0007 >30 >30 0.106 >30 >30 41 ##STR00938##
0.0028 0.0014 30++ 0.076 30++ >30 42 ##STR00939## 0.0124 0.0004
10.1 0.0873 9.75 >30 45 (TFA) ##STR00940## 0.0661 0.0013 30++
>10 0.08 6.48 >14.42 46 (TFA) ##STR00941## 0.0267 0.0541 30++
2.13 30++ >30 47 ##STR00942## 0.0065 0.0042 3.86 0.171 >30 NT
48 ##STR00943## 0.0118 0.0871 >30 0.108 >30 NT 49
##STR00944## 0.0056 0.0183 >30 2.7 >30 NT 50 ##STR00945##
0.0080 0.0009 10.1 0.751 0.0345 30++ >30 51 (TFA) ##STR00946##
0.0042 0.0006 4.98 0.0136 30++ >30 52 ##STR00947## NT NT NT NT
NT NT 53 ##STR00948## 0.0065 0.0009 NT NT NT NT 54 ##STR00949##
0.0095 0.0003 0.269 6.23 0.0527 >30 >30 55 (TFA) ##STR00950##
0.0114 0.0004 10.5 >30 0.0933 >30 >30 57 ##STR00951##
0.0063 0.0015 10.1 0.0914 30++ >30 58 ##STR00952## 0.0078 0.0013
0.830 0.0341 5.98 NT 59 ##STR00953## 0.0127 0.0004 0.179 0.0134
>30 NT 60 ##STR00954## 0.0121 0.0017 >30 0.0699 >30 NT 61
##STR00955## 0.0302 0.0191 >30 0.839 >30 NT 62 ##STR00956##
0.0210 0.0030 0.475 0.0248 >30 NT 63 ##STR00957## 0.0038 0.0067
>30 0.447 >30 NT 64 ##STR00958## 0.0116 0.0214 30++ 0.121
>30 NT 65 ##STR00959## NT NT NT NT NT NT 66 ##STR00960## NT NT
NT NT NT NT 67 ##STR00961## 0.00749 0.00313 30++ 0.181 30++ NT 68
##STR00962## NT NT NT NT NT NT 69 ##STR00963## 0.012 0.00244 30++
0.0096 30++ NT 70 ##STR00964## 0.0198 0.00932 NT NT NT NT ++
indicates that the reported data is an average of multiple data
points and the reported data can be read as "greater than" the
reported data NT indicates Not Tested
Example 76: Stability of Anti-TNF-Alpha Immunoconjugates
Matrix Stability
[1267] Anti-TNF.alpha. steroid ADCs were tested for their
susceptibility to prematurely release small molecule payload under
physiological conditions. In these experiments, ADCs were diluted
in plasma (human, monkey, mouse, or rat) or buffer in duplicate and
incubated for 6 days at 37.degree. C., 5% CO.sub.2. Each sample was
quenched at time 0 minutes and at various time points over the
6-day period. Samples were then analyzed using LC/MS/MS and
compared with standard curves for the corresponding small molecule.
The % maximum release of small molecule payload over time was
calculated. The results are summarized in Table 11 below.
TABLE-US-00038 TABLE 11 Stability of anti-TNF.alpha. steroid ADCs
Matrix stability (% max SM release) Cynomolgous Cpd. PBS Human
Monkey Mouse No. buffer plasma Plasma plasma 136 0.00864 8.34E-04
0.0165 0.0327 137 0 0 0 0 138 0.00954 0.00471 0.00444 0.0215 139 0
0 0 0 142 0.00306 0 0.00766 0.0564 144 0 0 0 0 145 0.00451 0 0 0
146 0.031 0.016 0.0306 0.0744 147 0 0 0 0.00565 148 4.51E-04 0.0032
0.0188 0.0162 149 0 0 0 0.00648 150 0.00809 0 0.0153 0.074 152
0.00513 0 5.42E-04 0.0392 153 0 4.01E-04 0 0.132 154 0.00E+00 0 0 0
155 0 0 0 0 156 0 0 0 0.152 157 0.0134 0.00559 0 0.026
[1268] These results demonstrate that anti-TNF.alpha. steroid ADCs
are stable in buffer and plasma of multiple species and that
minimal small molecule release is observed.
Proteolytic Stability
[1269] The susceptibility of steroid ADCs to release their payload
through protease treatment was compared with an ADC generated using
the vcmcMMAE drug linker conjugated to a murine CD-19 antibody.
ADCs (average DAR of 4) were incubated with either cathepsin B or
proteinase K, and payload release was analyzed by LC-MS at various
timepoints (0, 1, 4, 7 and 24 hours).
[1270] The results are shown in FIG. 1 and demonstrate that the
steroid ADCs are resistant to exogenous cathepsin-mediated release
of payload from the ADC. This is in contrast to a known payload
linker (mcvcMMAE) ADC, where MMAE is released in significant
amounts upon cathepsin treatment. This data indicates that steroid
ADCs are much less susceptible to premature payload release that
results from cathepsin activity in circulation than known ADCs.
Indeed, steroid release is only observed with proteinase K, a
serine protease that displays broad cleavage specificity. This
indicates that the antibody portion of the steroid ADC needs to be
significantly catabolized prior to steroid linker cleavage and that
payload release can be restricted to an environment where digestion
of the antibody scaffold of the ADC can occur, such as the
lysosome.
Cathepsin B Digestion
[1271] A 0.2 mg/mL stock solution of cathepsin B (Sigma) was
prepared in buffer (25 mM Tris, 50 mM NaCl and 5% glycerol). To
generate a 10 g/mL working solution of cathepsin B, 5 .mu.l of 0.2
mg/mL cathepsin B stock was mixed with 95 .mu.l of activation
buffer (50 mM sodium acetate pH5, 1 mM EDTA, and 5 mM DTT) and
incubated at 37.degree. C. for 15 minutes. For ADC digestion, 20
.mu.l of 100 ug/mL ADC and 20 .mu.l of cathepsin B working solution
were mixed with 160 .mu.l dilution buffer (50 mM sodium acetate, 1
mM EDTA). The sample was incubated at 37.degree. C. with shaking,
and 40 .mu.l aliquots were removed after 0, 1, 4, 7, and 24 hours.
To each aliquot was added 160 .mu.l of quench solution (0.1% formic
acid; 1:1 MeOH:MeCN; 100 nM carbutamide), and released small
molecule was detected by LC-MS/MS as previously described.
Proteinase K Digestion
[1272] A 5 mg/mL stock of proteinase K (Sigma) was prepared in
deionized (DI) water. A 0.25 mg/mL working solution of proteinase K
was prepared by mixing 50 .mu.L of 5 mg/mL proteinase K with 950
.mu.l dilution buffer (1x HBSS and 1 mM EDTA). For ADC digestion,
20 .mu.L of 100 ug/mL ADC and 40 .mu.l of proteinase K working
solution were mixed with 140 .mu.l dilution buffer. The sample was
incubated at 37.degree. C. with shaking, and 40 .mu.L aliquots were
removed after 0, 1, 4, 7, and 24 hours. To each aliquot was added
160 .mu.l of quench solution (0.1% formic acid; 1:1 MeOH:MeCN; 100
nM carbutamide), and released small molecule was detected by
LC-MS/MS as previously described.
Example 77: In Vivo Stability of Anti-TNF-Alpha
Immunoconjugates
[1273] The susceptibility of the steroid ADC to undergo drug linker
loss was assessed in mice. MP-Ala-Ala-steroid was conjugated to
human IgG1 mAb (av. DAR 4) and incubated at pH 9 to catalyze
ring-opening hydrolysis of the thiosuccinimide ring. After
neutralization, the steroid ADC was injected in mice, and the
kinetics of drug linker loss were monitored over 7 days by
LC-MS.
[1274] In these experiments, ADC formulated in phosphate buffer
saline was dosed intravenously to 15 male DBA/1 mice at 5 mg/kg.
Three mice were sacrificed at 1 hr, 24 hr, 72 hr, 168 hr and 240 hr
post-dose. EDTA whole blood was collected and serum was prepared
for in vivo DAR analysis by mass spectrometry.
Serum Sample Pre-Dilution
[1275] Serum samples were diluted in horse serum (Life
technologies, 16050-122) based on total antibody concentrations of
ADC measured by total antibody ligand binding assay. Dilutions were
based on estimations of final concentration to a range of 10-30
.mu.g/mL, which is suitable for the magnetic beads upper limit of
binding capacity.
Immunoaffinity Affinity Purification
[1276] In a protein LoBind tube (Eppendorf North America), 350
.mu.L horse serum was added to 100 .mu.L of each pre-diluted ADC
serum sample to a total volume of 450 .mu.L, followed by addition
of 4 .mu.g of biotin-anti-human Fc antibody (2 .mu.L of biotin-anti
human at 2 mg/mL solution). Samples were incubated for 2 hours (hr)
at room temperature by shaking at 900 rpm on an orbital shaker. For
each serum sample, 50 .mu.L slurry of streptavidin coated magnetic
beads (Pierce, Cat #88817) was equilibrated with 0.1% Tween in PBS
buffer (PBST) in a LoBind tube. Phosphate Buffered Saline with
Tween 20 (PBST) buffer was removed by a pipette after pulling the
magnetic beads to the side of the LoBind tube by placing the LoBind
tube on a magnetic rack. Serum samples after 2 hr incubation with
anti-human capturing reagent were transferred to the LoBind tubes
containing equilibrated magnetic beads, and incubated at room
temperature for 1 hr at 900 rpm on an orbital shaker. Serum was
removed after magnetic bead incubation, and the magnetic bead was
washed thoroughly with 500 .mu.L PBST (3 times) followed by 500
.mu.L 5% MeOH in MilliQ water (3 times). Magnetic bead bound ADC
was released by incubating the magnetic beads with 100 .mu.L 0.5%
formic acid in 50% MeOH/MilliQ water for 15 minutes.
Reduction of Purified ADC
[1277] Released ADC was reduced by adding 10 .mu.L reducing reagent
(10 mM TCEP freshly prepared from powder purchased from Thermo
Scientific, with 10 mM EDTA in 2M pH7.5 Tris buffer) to 100 .mu.L
of sample and incubated at 37.degree. C. for 30 minutes.
LC/MS Analysis
[1278] Reduced samples (10 .mu.L) were injected into an Agilent
6550 QT of LC/MS system through a temperature controlled (5.degree.
C.) CTC autosampler. Sample elution was achieved on a Waters C-4,
3.5 .mu.m, 300 .ANG., 2.1.times.50 mm i.d. HPLC column. The mobile
phases were: A: 0.1% formic acid in water, and B: 0.1% formic acid
in MeCN; the flow rate was 0.45 mL/min; and the column compartment
was maintained at 40.degree. C.
[1279] The HPLC gradient was as follows:
TABLE-US-00039 Time (min) % A % B 0 95 5 0.6 95 5 1.1 10 90 2.2 10
90 2.4 95 5 3.5 95 5
[1280] High resolution MS analysis of reduced ADC was performed on
an Agilent 6550 quadruple time-of-flight (Agilent Technology, San
Clara, Calif.) equipped with a Dual Agilent Jet Stream electrospray
ionization (ESI) source operated in the positive ion mode. Mass
spectrometer was operated in the extended dynamic range (2G Hz)
mode with a MS range up to 3,200 m/z. The primary ESI source was
used for LC/MS analysis, and the secondary ESI probe was used for
infusing calibration solution at 922.009798 m/z to achieve real
time MS calibration. The mass spectrometer was calibrated on a
daily basis. Typical mass errors of analytes relative to
theoretical masses were less than 5 parts per million in daily
operations. MS data were processed using MassHunter Qual Browser
Build 5.0.
MS Spectrum Deconvolution
[1281] Maximum entropy method in the MassHunter Bioconfirm software
package was used to deconvolute the multiple charged ion mass
spectra to derive neutral molecular weight spectra. The intensity
of the deconvoluted peak was used to calculate DAR.
DAR Value Calculation from De-Convoluted MS Spectrum
[1282] DAR values were calculated using de-convoluted MS peak
intensity based on the following equations:
DAR value from light chain(LC):LC DAR=(2.times.peak intensity of
LC{circumflex over ( )})((peak intensity of LC+peak intensity of
LC{circumflex over ( )}))
LC and LC{circumflex over ( )} are light chains with zero and one
drug linker, respectively. DAR value from heavy chain (HC):
HC DAR=2.times.(peak intensity of HC{circumflex over (
)}+2.times.peak intensity of HC{circumflex over ( )}{circumflex
over ( )}+3.times.peak intensity of HC{circumflex over (
)}{circumflex over ( )}{circumflex over ( )})/(peak intensity of
HC+peak intensity of HC{circumflex over ( )}+peak intensity of
HC{circumflex over ( )}{circumflex over ( )}+peak intensity of
HC{circumflex over ( )}{circumflex over ( )}{circumflex over (
)})
HC, HC{circumflex over ( )}, HC{circumflex over ( )}{circumflex
over ( )} and HC{circumflex over ( )}{circumflex over (
)}{circumflex over ( )} are heavy chains with zero, one, two and
three drug linkers, respectively.
Total DAR=LC DAR+HC DAR
Results
[1283] The results are shown in FIG. 2. This example demonstrates
that minimal loss of drug linker is observed from steroid ADC over
7 days.
Example 78: Generation of Human and Mouse Transmembrane TNF-Alpha
GRE Reporter Cell Lines
[1284] In order to create a parental cell line, K562 cells were
seeded onto a 6 well dish (Costar: 3516) with 2 mL of complete
growth medium (RPMI, 10% FBS, 1% L-glutamine, 1% Na Pyruvate and 1%
MEM NEAA) at 500,000 cells per well for 24 hours at 37.degree., 5%
CO.sub.2. The next day, 1.5 g of pGL4.36[Luc2P/MMTV/Hygro]
(Promega: E316),1.5 ug pG14.75 [hRLuc/CMV] (Promega: E639A), and 3
g of PLUS reagent (Invitrogen: 10964-021) were diluted into 244 uL
Opti-MEM (Gibco: 31985-070) and incubated at room temperature for
15 minutes. The pGL4.36[luc2P/MMTV/Hygro] vector contains MMTV LTR
(Murine Mammary Tumor Virus Long Terminal Repeat) that drives the
transcription of the luciferase reporter gene luc2P in response to
activation of several nuclear receptors such as glucocorticoid
receptor and androgen receptor. The pGL4.75[hRluc/CMV] Vector
encodes the luciferase reporter gene hRluc (Renilla renformis) and
is designed for high expression and reduced anomalous
transcription. After incubation, diluted DNA solution was
pre-incubated with 1:1 Lipofectamine LTX solution (Invitrogen:
94756) (13.2 l+256.8 g Opti-MEM) and incubated at room temperature
for 25 minutes to form DNA-Lipofectamine LTX complexes. After
incubation, 500 of DNA-Lipofectamine complexes were added directly
to the well containing cells. K562 cells were transfected for 24
hours at 37.degree., 5% CO.sub.2. After incubation, cells were
washed with 3 mL of PBS and selected with complete growth medium
containing 125 g/mL of hygromycin B (Invitrogen: 10687-010) for two
weeks. "K562 pGL4.36[Luc2P/MMTV/Hygro]_pGL4.75[hRLuc/CMV]" cells
were produced.
[1285] In order to create a murine transmembrane TNF-alpha GRE
reporter cell line, the parental cells, K562
pGL4.36[Luc2P/MMTV/Hygro]_pGL4.75[hRLuc/CMV], were seeded onto 6
well dish (Costar: 3516) with 2 mL of complete growth medium (RPMI,
10% FBS, 1% L-glutamine, 1% Na Pyruvate and 1% MEM NEAA) at 500,000
cells per well for 24 hours at 37.degree., 5% CO.sub.2. The next
day, 3 g of mFL_TNFa DNA (Origene: MC208048), which encodes
untagged mouse TNF, and 3 .mu.l of PLUS reagent (Invitrogen:
10964-021) were diluted into 244 uL Opti-MEM (Gibco: 31985-070) and
incubated at room temperature for 15 minutes. After incubation,
diluted DNA solution was pre-incubated with 1:1 Lipofectamine LTX
solution (Invitrogen: 94756) (13.2 uL+256.8 uL Opti-MEM) and
incubated at room temperature for 25 minutes to form
DNA-Lipofectamine LTX complexes. After incubation, 500 .mu.l of
DNA-Lipofectamine complexes were added directly to the well
containing cells. The parental K562
pGL4.36[Luc2P/MMTV/Hygro]_pGL4.75[hRLuc/CMV] cells were transfected
for 24 hours at 37.degree., 5% CO.sub.2. After incubation, cells
were washed with 3 mL of PBS and selected with complete growth
medium containing 125 g/mL of hygromycin B (Invitrogen: 10687-010)
and 250 g/mL G418 (Gibco: 10131-027) for two weeks. "K562 mouse
FL-TNFa GRE (pGL4.36[luc2P/MMTV/Hygro])" cells were produced.
[1286] In order to create a human transmembrane TNF-alpha GRE
reporter cell line, the parental cells, K562
pGL4.36[Luc2P/MMTV/Hygro]_pGL4.75[hRLuc/CMV], were transfected with
the plasmid hTNF delta 1-12 C-Myc pcDNA3.1 (-) plasmid construct.
This plasmid is pcDNA 3.1 (Thermofisher cat # V79020) encoding tace
resistant transmembrane TNF (i.e., SEQ ID NO:1 lacking amino acids
77-88). (See Perez C et al. Cell 63 (2): 251-8 (1990) discussing
tace resistant transmembrane TNF.) These cell lines were then used
in the TNF-alpha reporter assays described in the subsequent
examples.
Example 79: Activity of Anti-TNF-Alpha Immunoconjugates in GRE
Transmembrane TNF-Alpha Reporter Assays
[1287] K562 parental GRE (pGL4.36[luc2P/MMTV/Hygro]) cells and K562
mFL-TNF-a or hTNF delta 1-12 GRE (pGL4.36[luc2P/MMTV/Hygro]) cells
were plated onto 96 well tissue culture treated white plates
(Costar: 3917) at 50,000 cells per well in 50 .mu.L of assay medium
(RPMI, 1% CSFBS, 1% L-glutamine, 1% Na Pyruvate and 1% MEAA). The
cells were treated with 25 .mu.L of 3.times. serial diluted murine
or human anti-TNF-a antibody drug conjugates in assay medium,
steroid compound, or media alone and incubated for 48 hours at
37.degree., 5% CO.sub.2. After 48 hours of incubation, cells were
treated with 75 .mu.L of Dual-Glo Luciferase Assay System
(Promega-E2920) for 10 minutes and analyzed for luminescence using
the TopCount (PerkinElmer). Data were analyzed using a four
parameter curve fit to generate EC50 values. % maximum activation
was normalized to 100 nM dexamethasone, which was considered
maximum activation. The results using the murine TNF-alpha cell
line are shown in Table 12 below, and the results using the human
TNF-alpha cell line are shown in Table 13 below. In Table 12 below,
A refers to 8C11. In Table 13 below, A refers to adalimumab (SEQ ID
NOs: 66 and 73). Percent (%) monomer was determined by SEC as
previously described (see ADC analytical procedures).
TABLE-US-00040 TABLE 12 In vitro activity of anti-murine TNFa
antibody drug conjugates in mouse transmembrane TNFa GRE reporter
assay (A refers to the anti-murine TNFa antibody 8C11) K562 GRE
K562 mTNFa mTNFa EC.sub.50 GRE Cpd. % GRE EC50 GRE (% (ug/m (% No.
Structure n monomer (ug/mL) max) L) max) 134 ##STR00965## 4.5 91.4
0.00519 118 1.61 74 135 ##STR00966## 4.4 95.4 1.27 106 36.3 68 136
##STR00967## 2 99.9 0.0108 95 9.3 46 137 ##STR00968## 4 99.9 0.0105
114 5.27 93 138 ##STR00969## 2 98.02 0.0297 108 28.7 53 139
##STR00970## 4 96.6 0.0239 92 15.2 61 140 ##STR00971## 2 98.8 0.179
112 50 23 141 ##STR00972## 4 98 0.144 96 >50 43 142 ##STR00973##
2 99.1 0.0515 96 >50 57 143 ##STR00974## 2 97.7 0.0795 82 >50
24 144 ##STR00975## 4 94.25 0.0406 116 14.7 74 145 ##STR00976## 4
98 0.0393 95 24.7 36 146 ##STR00977## 2 98.5 0.0399 118 27.5 73 147
##STR00978## 4 97.6 0.0259 113 7.89 80 148 ##STR00979## 2 97.8
0.0384 120 23.6 77 149 ##STR00980## 4 91.8 0.0314 113 10.7 78 150
##STR00981## 2 98 0.02092 102 10.99 84 151 ##STR00982## 4 90.1
0.0098 104 2.85 87 152 ##STR00983## 2 98.3 0.0247 96 3.44 70 153
##STR00984## 4 93.8 0.0185 75 2.77 94 154 ##STR00985## 2 96.6 2.00E
- 04 104 12.1 91 155 ##STR00986## 4 96 0.0171 94 2.8 79 156
##STR00987## 2 98.2 0.039 107 15.8 73 157 ##STR00988## 4 92.5
0.0198 115 3.63 83 158 ##STR00989## 2 99.4 >50 0.05 >50 0.2
159 ##STR00990## 4 98.4 >50 0.075 >50 0.7 160 ##STR00991## 2
98.2 0.0242 91 >50 63 161 ##STR00992## 4 95.8 0.0203 94 16.9 77
162 ##STR00993## 4 95 0.0072 119 12.4 88 163 ##STR00994## 2 97.7
>50 6 >50 0.4 164 ##STR00995## 4 97.8 >50 24 >50 0.8
165 ##STR00996## 2 96.3 0.0179 93.6 >50 94 166 ##STR00997## 2 98
0.0136 107 14.6 75 167 ##STR00998## 4 94.4 0.0108 97 11.4 73 168
##STR00999## 2 97.9 0.146 81 50 71 169 ##STR01000## 4 92.25 0.0551
117 18.4 88 170 ##STR01001## 2 99.5 0.463 18.4 >50 0.5 171
##STR01002## 4 97.6 0.276 35 >50 5 172 ##STR01003## 4 94.6
0.0319 89 2.9 64 173 ##STR01004## 1.3 98 0.0959 78 >50 35 174
##STR01005## 2 98 0.0607 143 4.23 14 175 ##STR01006## 4 93.4 0.0464
92 >50 55 176 ##STR01007## 4 97.4 0.0262 113 41.4 60 177
##STR01008## 4 95.6 0.00998 105 7.94 66 178 ##STR01009## 4 88.3
8.00E-04 93 3.88 48 179 ##STR01010## 2 97.7 0.0249 89 11.7 84 180
##STR01011## 4 96.8 0.0118 84 2.75 78 181 ##STR01012## 4 95.3
0.0593 83.8 50 35 182 ##STR01013## 4 97.6 >50 8 >50 4 183
##STR01014## 4 94.7 0.0144 87 12 49 184 ##STR01015## 2 98.9 0.0525
75 46.7 36 185 ##STR01016## 4 96.6 0.0294 64 5.02 76 186
##STR01017## 4 97.5 0.0479 143 >50 31 187 ##STR01018## 4 91.8
0.0185 103 >50 73 188 ##STR01019## 4 94 0.0107 126 >50 43 189
##STR01020## 4 99.9 0.0215 149 >50 59 190 ##STR01021## 4 99.9
0.0112 109 >50 90 191 ##STR01022## 3.7 88.6 0.0692 122 14.4 90
192 ##STR01023## 4.1 74.4 0.0225 1.23 99 193 ##STR01024## 3.9 68
0.149 124 6.4 104 194 ##STR01025## 3.9 67.7 0.0517 95.3 5.01 85 195
##STR01026## 3.5 92.2 0.123 156 23.6 75 196 ##STR01027## 3.5 92.9
0.0331 96 24.2 63 197 ##STR01028## 3.6 94.1 0.0626 143 39.3 60 198
##STR01029## 3.6 93 0.0614 96 12.7 91 199 ##STR01030## 3.5 93
0.0654 93 23.3 73 200 ##STR01031## 3.8 90.1 0.0114 114 4.76 92 207
##STR01032## 4 208 ##STR01033## 4 99.3 0.0154 123 50.0 98.7 209
##STR01034## 3.84 99.5 0.16 116 50.0 70.0 210 ##STR01035## 4 100
0.154 129 50.0 61.7 211 ##STR01036## 4 99.4 0.0341 130 18.7 91.7
212 ##STR01037## 3.99 99.2 0.00633 101 1.9 348 213 ##STR01038##
4.08 99.2 0.0267 140 27.6 146 214 ##STR01039## 3.8 99.2 0.362 248
50.0 97.1 215 ##STR01040## 3.8 99.3 0.0166 126 1.93 103 216
##STR01041## 3.74 99.4 0.351 108 50.0 27.9 217 ##STR01042## 3.7
98.8 0.0147 101 18.3 97.2 218 ##STR01043## 3.7 98.9 0.023 96.0 22.9
102 219 ##STR01044## 4.03 99.1 0.0371 140 50.0 91.2 220
##STR01045## 1.7 99.5 0.00329 97.7 9.96 112 221 ##STR01046## 3.6 99
0.011 131 4.28 112 222 ##STR01047## 0 3.8 99 0.114 102 43.4
86.6
TABLE-US-00041 TABLE 13 In vitro activity of anti-human TNFa
antibody drug conjugates in human transmembrane TNFa GRE reporter
assay (A refers to the anti- human TNFa antibody adalimumab (SEQ ID
NOs: 66 and 73) Cpd. No. Structure 201 ##STR01048## 202
##STR01049## 203 ##STR01050## 204 ##STR01051## 205 ##STR01052## 206
##STR01053## hTNFa hTNFa K562 K562 GRE GRE GRE GRE Cpd. No. n %
monomer EC.sub.50 (ug/mL) (% max) EC.sub.50 (ug/mL) (% max) 201 4
97.6 0.0179 100 43 66 202 2 99.1 0.0318 121 >50 51 203 4 98.2
0.0482 118 >50 35 204 2 99.4 0.0767 103 >50 21 205 4 96.9
0.0035 97 17.5 103 206 2 99.4 0.0082 101 >50 97
Example 80: Activity of Various Anti-Human TNF-Alpha
Immunoconjugates in GRE Transmembrane TNF-Alpha Reporter Assays
Preparation of Anti-Human TNF Alpha Immunoconjugates
[1288] All proteins were conjugated to Cpd. No. 99 using conditions
highlighted under the general cysteine conjugation protocol in
Example 36. Where indicated in Table 14 below, a cysteine addition
(underlined) was engineered into the anti-TNF sequence to allow
conjugation.
TABLE-US-00042 TABLE 14 Amino acid sequences of anti-human TNF
alpha antibodies used in immunoconjugates Antibody Sequence (SEQ ID
NO) Infliximab HC SEQ ID NO: 67 Infliximab LC SEQ ID NO: 74
Golimumab HC SEQ ID NO: 72 Golimumab LC SEQ ID NO: 78 etanercept
LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDT
VCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGW
YCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSS
TDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQH
TQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ
ID NO: 79) ABT-122 HC
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAI
TWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYL
STASSLDYWGQGTLVTVSSGGGGSGGGGSEVQLVQSGAEVKKPGSSVKVSC
KASGGSFGGYGIGWVRQAPGQGLEWMGGITPFFGFADYAQKFQGRVTITAD
ESTTTAYMELSGLTSDDTAVYYCARDPNEFWNGYYSTHDFDSWGQGTTVTV
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 80) ABT-122 LC
DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAA
STLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGT
KVEIKRGGSGGGGSGEIVLTQSPDFQSVTPKEKVTITCRASQDIGSELHWY
QQKPDQPPKLLIKYASHSTSGVPSRFSGSGSGTDFTLTINGLEAEDAGTYY
CHQTDSLPYTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN
FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 81) certolizumab SEQ ID NO:
68 pegol HC certolizumab SEQ ID NO: 75 pegol LC Certolizumab HC
EVQLVESGGGLVQPGGSLRLSCAASGYVFTDYGMNWVRQAPGKGLEWMGWI
NTYIGEPIYADSVKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARGYRS
YAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
HKPSNTKVDKKVEPKSCDKTHTCAAHHHHHH (SEQ ID NO: 82) Certolizumab LC
DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSA
SFLYSGVPYRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPLTFGQGT
KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ
ID NO: 83) Adalimumab Fab
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAI HC
TWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYL
STASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHTCAAHHHHHH (SEQ ID NO: 84) Adalimumab Fab
DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAA LC
STLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGT
KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ
ID NO: 85) Affibody.sup.1
GVDNKFNKENIAAMTEITRLPNLNPYQRAAFIWSLSDDPSQSANLLAEAKK LNDAQAPKC (SEQ
ID NO: 86) Ozoralizumab
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEI Nanobody
NTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSG
FNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTF
SSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLY
LQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVES
GGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLIT
KYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSGFNRGQGT
LVTVSSGSEQKLISEEDLCHHHHHH (SEQ ID NO: 87) .sup.1Kronqvist J et al.
Protein Engineering, Design & Selection 21 (4):247-255
(2008)
Activity of Anti-Human TNF Alpha Immunoconjugates in GRE Reporter
Assay
[1289] Anti-human TNF alpha immunoconjugates (also referred to as
anti-human TNF alpha ADCs, or anti-hTNF alpha steroid ADCs) were
tested for activity in the K562 parental GRE
(pGL4.36[luc2P/MMTV/hydgro]) and K562 hTNF delta 1-12 GRE
(pGL4.36[luc2P/MMTV/hydgro]) cell lines under conditions as
described in Example 79. The results shown in Table 15 indicate
that all anti-hTNF alpha steroid ADCs tested demonstrate potent
antigen-dependent activity dissociated from their activity on the
parental cell line.
TABLE-US-00043 TABLE 15 In vitro activity of anti-human TNF alpha
ADCs in human transmembrane TNFa GRE reporter assay (ADC
concentrations were normalized for MW and DAR) ##STR01054## hTNFa
hTNFa K562 K562 GRE GRE GRE GRE Cpd. No. A.sup.1 SEQ ID n % monomer
EC50 (nM) (% max) EC50 (nM) (% max) 233 infliximab 67/74 3.3 99.99
1.3 122 374 55.3 234 golumumab 72/78 4.9 99 3.3 144 1633 67.5 235
etanercept 79 2.3 87.6 0.6 105.5 164 104 236 ABT-122 80/81 4.1
99.98 1.02 116.9 608 61.3 237 certolizumab pegol 68/75 2 95.9 0.44
97 1111 39 238 certolizumab 82/83 1 ND 0.4 95 266 60 239 adalimumab
Fab 84/85 1 ND 1 118 190 137 240 affibody 86 1 100 9.1 98 26 84 241
ozoralizumab (nanobody) 87 1 98 0.5 131 875 99
Binding of Anti-Human TNF Alpha Immunoconjugates to Human TNF
Alpha
[1290] Binding kinetics of anti-h TNF alpha steroid ADCs to
recombinant soluble TNF.alpha. trimer were determined by surface
plasmon resonance-based measurements made on Biacore T200
instrument (GE Healthcare) at 25.degree. C. using either anti-human
Fc/anti-human F(ab').sub.2 capture (used for all ADCs except
affibody and ozoralizumab ADCs) or direct NHS/EDC mediated amine
coupling approach (used only for ozoralizumab ADC). Approximately
10000 RU of goat anti-human IgG Fc polyclonal antibody (Thermo
Fisher Scientific Inc., cat. No. 31125) or goat anti-human
F(ab').sub.2 polyclonal antibody (Jackson Immunoresearch
Laboratories, Inc. cat. No. 109-006-006) was diluted to 5 .mu.g/mL
in 10 mM sodium acetate (pH 4.5) and was immobilized across a CM5
biosensor chip using a standard amine coupling kit according to
manufacturer's instructions and procedures. Unreacted moieties on
the biosensor surface were blocked with 1M ethanolamine. For direct
amine coupling approach, approximately 750 RU of ozoralizumab
steroid conjugate was directly immobilized onto CM5 chip. Chip
preparation and binding kinetic measurements were made in the assay
buffer HBS-EP+(10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05%
Tween 20). For binding kinetic measurements in capture format, each
assay cycle consisted of the following steps: 1) capture of test
ADC on test surface at a concentration of 0.5 .mu.g/mL and at a
flow rate of 5 .mu.L/min for 60 s; 2) analyte injection (human
TNF.alpha. or buffer only) over both reference and test surface for
300s at 50 .mu.l/min, after which the dissociation was monitored
for 600 seconds at 50 .mu.l/min; 3) regeneration of capture surface
by 10 mM Glycine-HCl, pH 1.5 or 100 mM HCl (for directly coupled
ADC) injections over both reference and test surface. For binding
kinetics measurements in direct amine coupling format, only step 2)
and step 3) were performed. During the assay, all measurements were
referenced against the blank surface alone (i.e. with no captured
test antibody or immobilized nanobody) and buffer-only injections
were used for double referencing. TNF.alpha. injections ranged in
concentration from 50 nM to 0.39 nM in a 2-fold dilution series,
respectively. Data were processed and fitted globally to a 1:1
binding model using Biacore T200 Evaluation software to determine
the binding kinetic rate constants, k.sub.a (M.sup.-1s.sup.-1) and
k.sub.d (s.sup.-1), and the equilibrium dissociation constant
K.sub.D (M). Two independent experiments were conducted. Reported
values in Table 16 are averages from these experiments.
TABLE-US-00044 TABLE 16 Binding affinities of anti-hTNF alpha
steroid ADCs to human TNF alpha Cpd anti-TNF k.sub.a k.sub.d
K.sub.D No. ADC (1/Ms) (1/s) (M) 233 infliximab 9.90E+05 3.10E-04
3.10E-10 234 golimumab 9.70E+05 2.40E-04 2.50E-10 235 etanercept
6.30E+06 9.80E-05 1.60E-11 236 ABT-122 4.00E+05 6.90E-05 1.80E-10
237 certolizumab 2.30E+06 1.70E-04 7.30E-11 pegol 238 certolizumab
2.70E+06 1.60E-04 6.00E-11 239 adalimumab 1.50E+06 1.80E-04
1.30E-10 Fab 240 affibody ND ND ND 241 ozoralizumab 1.30E+06
7.00E-05 5.20E-11 (nanobody) (ND = not determined)
Example 81: Activity of Anti-hTNF Alpha Steroid ADCs in
Lipopolysacharride Stimulated Human PBMC Cytokine Release Assay
[1291] Primary human peripheral blood mononuclear cells (PBMCs)
were purchased from Biological Specialty Corporation (cat
#214-00-10), washed in 50 mL PBS, re-suspended in FBS with 5% DMSO,
aliquoted and cryopreserved in liquid nitrogen until use. The PBMCs
were thawed, re-suspended in RPMI supplemented with 2% FBS, and 1%
Penicillin-Streptomycin, and plated into a cell assay plate (Costar
#3799). The cells were incubated with varying concentration of
anti-hTNF alpha steroid ADCs at 37.degree. C. and 5% CO.sub.2 for 4
hours. Cells were then stimulated with 100 ng/ml LPS for overnight.
On the following day, plate was spun for five minutes at 1000 rpm,
and 100 .mu.L of supernatant media was directly transferred to an
additional 96-well plate and analyzed for IL-6 (MSD, # K151AKB) and
IL-1 beta(MSD, # K151AGB) concentrations. The dose response data
were fitted to a sigmoidal curve using nonlinear regression, and
the IC50 values calculated with the aid of GraphPad 5.0 (GraphPad
Software, Inc.). The results shown in Table 17 demonstrate that the
anti-hTNF alpha steroid ADCs have potent activity in inhibiting the
release of pro-inflammatory cytokines IL-6 and IL-1beta from
activated primary immune cells.
TABLE-US-00045 TABLE 17 In vitro activity of anti-human TNF alpha
ADCs in LPS-stimulated human PBMC cytokine release assay Cpd
Cytokine Release IC50 (ng/ml) No. IL-6 IL-1beta 203 14.3 .+-. 3.5
3.6 .+-. 1.2 201 86.8 .+-. 69.6 25.5 .+-. 21.3 205 42.4 .+-. 27.9
22 .+-. 18 (n = 2)
Example 82: Activity of Anti-TNF-Alpha Immunoconjugate in
TNFa-Induced Cytotoxicity Assay in L929 Cells
[1292] L929 is a murine aneuploid fibrosarcoma cell line that is
sensitized by pretreatment with actinomycin D. Treatment with TNFa
initiates apoptosis and subsequent cell death. L929 cells in log
phase were harvested using trypsin 0.05%, washed twice with D-PBS
and counted by CEDEX. The cells were resuspended at 1E6 cell/mL in
assay media containing 4 .mu.g/mL actinomycin D and 50 .mu.L was
added to all wells. Anti-murine TNF alpha steroid ADC (anti-murine
TNF alpha 8C11 conjugated to Cpd 71; also referred to as anti-mTNF
alpha steroid ADC) and anti-murine TNF mAb (8C11) were diluted to a
4.times. concentration in assay media and serial 1:3 dilutions were
performed. Mouse TNF.alpha. was diluted to a 4.times. concentration
of 600 .mu.g/mL. The anti-mTNF steroid ADC and anti-mTNF mAb (125
.mu.L) were added to the mTNFa (125 .mu.L) in a 1:2 dilution scheme
and allowed to incubate for 1 hour at room temperature, gently
shaking. The antibody/mTNF.alpha. (or ADC/mTNFa) mixture was added
to wells at 50 .mu.L/well in triplicate. The plates were incubated
for 20 hours at 37.degree. C., 5% CO.sub.2. To quantify viability,
10 .mu.L of WST-1 reagent (Roche cat #11644807001) was added to
wells. Plates were incubated under assay conditions for 3.5 hours,
centrifuged at 500.times.g and 75 .mu.L supernatant transferred to
an ELISA plate (Costar cat #3369). The plates were read at OD
420-600 nm using a Spectromax 190 ELISA plate reader. Data was
analyzed and IC.sub.50 values calculated using a sigmoidal dose
response (variable slope) fit in GraphPad Prism 5.
[1293] Anti-mTNF alpha steroid ADC had comparable neutralizing
potency (IC.sub.50 1.9 nM) to unconjugated anti-mTNF alpha mAb
(IC.sub.50 1.5 nM).
[1294] Anti-human TNF alpha immunoconjugates were tested for
neutralizing activity under conditions described above. The results
are shown in Table 18 and indicate the anti-human TNF alpha
immunoconjugates tested demonstrate potent neutralization of human
TNF alpha.
TABLE-US-00046 TABLE 18 Neutralization potencies of anti-hTNF alpha
steroid ADCs to human TNF alpha-induced cytoxicity in L929 cells
SEQ ADC % IC50 Compound anti-TNF ID DAR monomer (nM) 233 infliximab
67/74 3.3 99.99 ND 234 golimumab 72/78 4.9 99 0.050 235 etanercept
79 2.3 87.6 0.002 236 ABT-122 80/81 4.1 99.98 0.074 237
certolizumab 68/75 2 95.9 0.046 pegol 238 certolizumab 82/83 1 ND
0.085 239 adalimumab 84/85 1 ND 0.6 Fab 240 affibody 86 1 100 ND
241 ozoralizumab 87 1 98 0.018 (nanobody) (ND = not determined)
Example 83: Binding of Anti-mTNF-Alpha Steroid ADC to Mouse Fcgamma
Receptors
[1295] SPR (surface plasmon resonance) based Biacore T200
instrument (GE Healthcare) was used to evaluate binding of
anti-mTNF-alpha steroid ADC (anti-mTNF 8C11 conjugated to Cpd 71)
and anti-mTNF-alpha mAb to recombinant mouse FcgRs (all R&D
Systems). The FcgRs were directly immobilized on the surface of the
flow cells two, three and/or four of the CM5 type S Biacore chip(s)
to achieve densities of 1000-2000 RU (resonance units). Blank
modified surface of the flow cell one of each Biacore chip was used
as a reference surface. Each experiment consisted of association
and dissociation phases. Association phase consisted of titrating
parental mAb and ADC over all flow cells at a flow rate of 50
ul/min and concentrations of 4000, 2000, 1000, 500, 250, 125, 62.5,
31.25 and 0 nM for FcgRIIB and FcgRIII and 100, 50, 25, 12.5, 6.25,
3.13, 1.56 and 0 nM for receptors I and IV. Dissociation phase
consisted of the continuous flow of the running buffer (HBS-EP+, pH
7.4, GE Healthcare) at the flow rate of 50 ul/min. Association and
dissociation phases were monitored for 5 min each (receptors I and
IV) or 1 min (receptors II and III). Chip surfaces were regenerated
with a 5s pulse of 100 mM HCl at a flow rate of 100 ul/min after
each binding cycle. Biacore Evaluation software was used to fit the
raw data to 1:1 (FcgRI and IV) or Steady State (receptors IIB and
III) binding models. Results are shown in Table 19. k.sub.a is the
association rate constant (1/Ms); k.sub.d is the dissociation rate
constant (1/s); K.sub.D is the equilibrium dissociation constant
(M).
TABLE-US-00047 TABLE 19 Binding affinities of anti-TNF-alpha
immunoconjugate to mouse Fcgamma receptors muFcgRI muFcgRIIb
muFcgRIII muFcgRIV k.sub.a k.sub.d K.sub.D K.sub.D K.sub.D k.sub.a
k.sub.d K.sub.D (1/Ms) (1/s) (M) (M) (M) (1/Ms) (1/s) (M) Anti-mTNF
7.4E+05 1.6E-02 2.1E-08 3.7E-06 3.1E-06 1.2E+05 5.3E-03 4.6E-08 mAb
Anti-mTNF 3.2E+05 8.7E-03 2.8E-08 5.9E-06 3.3E-06 8.4E+04 4.6E-03
5.5E-08 ADC
Example 84: Activity of Anti mTNF-Alpha Steroid ADCs in Contact
Hypersensitivity Model
[1296] Anti-mTNF alpha steroid ADCs were evaluated in an acute
contact hypersensitivity model, an elicitation of acute skin
inflammation using delayed-type hypersensitivity (DTH) response
(T-cell driven) via application of a sensitizing agent (fluorescein
isothiocyanate (FITC)). The efficacy of anti-mTNF alpha steroid
ADCs was measured by the ability to reduce ear swelling. The
steroid biomarkers corticosterone and procollagen type 1 N-terminal
propeptide (P1NP) were included as readouts to assess the putative
impact of anti-mTNF alpha steroid ADC treatment on the
Hypothalamus-Pituitary-Adrenal (HPA) axis and bone turnover
respectively.
Ear Swelling
[1297] On day 0 mice were placed under general anesthesia and the
abdomens were shaved. Using a micropipettor, mice were sensitized
by epicutaneous application of 400 uL of FITC solution (1.5%
solution in 1:1 acetone:DBP) on the abdomen. 6 days later mice were
dosed with vehicle or therapeutic agent 1 hour prior to ear
challenge with FITC. For ear challenge, mice were placed under
general anesthesia and were challenged with 201 FITC applied onto
right ear. 24 hours after challenge mice were placed under general
anesthesia and their ear thickness is measured by caliper.
Difference between challenged and unchallenged ears was calculated.
72 hours after ear challenge, mice were injected with ACTH at 1 mpk
IP, and terminally bled at 30 min post-ACTH. Plasma is collected
and analyzed P1NP, corticosterone, free steroid, and large molecule
levels.
Quantification of Released Free Steroid and Endogenous
Corticosterone
[1298] Calibration curve of steroid was prepared in mouse plasma
with final concentrations from 0.03 nM to 0.1 .mu.M at 8 different
concentration levels. Corticosterone calibration curve ranging from
0.3 nM to 1 .mu.M final corticosterone concentrations was prepared
in 70 mg/mL bovine serum albumin solution in PBS buffer. A solution
of 160 .mu.L MeCN with 0.1% formic acid was added to 40 .mu.L study
plasma samples or calibration standards. Supernatants were diluted
with distilled water and 30 .mu.L final sample solution was
injected for LC/MS analysis.
[1299] Quantification of released free steroid and corticosterone
was conducted on an AB Sciex 5500 triple quadruple mass
spectrometer connected to a Shimadzu AC20 HPLC system interfaced
with an electrospray ionization source operating in positive mode.
A Waters XBridge BEH C18, 2.1.times.30 mm, 3.5 .mu.m column was
used for chromatography separation. The mobile phase A was 0.1%
formic acid in Milli Q HPLC water, and mobile phase B was 0.1%
formic acid in MeCN. A linear gradient from 2% of mobile phase B to
98% mobile phase B was applied from 0.6 to 1.2 minutes. The total
run time was 2.6 min at a flow rate of 0.8 mL/min. The mass
spectrometer was operated in positive MRM mode at source
temperature of 700.degree. C.
Quantification of Plasma P1NP
[1300] Quantification of plasma P1NP was conducted on a LC/MS
platform based on protein trypsin digestion. Plasma samples were
partially precipitated and fully reduced by adding MeCN/0.1M
ammonium bicarbonate/DTT mixture. Supernatant was collected and
alkylated by adding iodoacetic acid. The alkylated proteins were
digested by trypsin and resulting tryptic peptides were analyzed by
LC/MS. Calibration curve were generated by using synthetic tryptic
peptide spiked into horse serum (non-interfering surrogate matrix).
Stable isotope labeled flanking peptide (3-6 amino acids extension
on both termini of the tryptic peptide) was used as internal
standard added in the MeCN/DTT protein precipitation mixture to
normalize both digestion efficiency and LC/MS injection.
[1301] A Columnex Chromenta BB-C18, 2.1.times.150 mm, 5 .mu.m
column was used for chromatography separation. The mobile phase A
was 0.1% formic acid in Milli Q HPLC water and mobile phase B was
0.1% formic acid in MeCN. A linear gradient from 2% of mobile phase
B to 65% mobile phase B was applied from 0.6 to 3 min. The total
run time was 8 min at a flow rate of 0.45 mL/min. An AB Sciex
4000Qtrap mass spectrometer was used in positive MRM mode to
quantify P1NP peptides, at source temperature of 700.degree. C.
Quantification of Total ADC in Plasma
[1302] Concentrations of total antibody (ADC and backbone mAb) were
measured by ligand binding assay using Mesoscale Discovery (MSD)
platform. Biotin labeled mouse TNF was used as the capture reagent
for anti-mTNF alpha steroid ADCs and Sulfo-TAG conjugated goat
anti-mouse detection antibody was used for detection. A calibration
curve was generated by serial dilution of the ADC molecule in
matching matrix and QC samples were used to qualify the assay
Results
[1303] The results are shown in Table 20 below:
TABLE-US-00048 TABLE 20 Comparison of anti-mTNF alpha steroid ADC
activity on ear swelling and steroid biomarkers in CHS model of
inflammation Ear swelling (% P1NP (% Corticosterone(% ADC inhib @
10 mpk) SEM inhib. @ 10 mpk) SEM inhib @ 10 mpk) SEM Cpd. No. 151
87.8 3.5 32.3 3.9 71.5 5.6 Cpd. No. 145 87.8 3.4 19.2 6.3 15.1 9.9
Cpd. No. 169 90.2 2.2 38.3 2.8 60.1 4.7 Cpd. No. 167 86.1 2.4 26.1
6.9 48.3 5.1 Cpd. No. 162 76.3 2.7 25.9 6.5 50.4 5.5 Cpd. No. 161
64.4 4.6 1.4 7.2 37.1 4.1 Cpd. No. 172 79.8 3.9 14.6 4.9 6.3 6.8
Cpd. No. 176 81.4 3.9 20.0 7.9 15.0 9.4 Cpd. No. 177 94.3 1.2 27.0
6.0 17.1 9.0 Cpd. No. 180 80.8 2.1 45.7 6.0 39.9 3.8 Cpd. No. 149
92.4 2.3 52.8 2.8 74.4 3.7 Cpd. No. 175 66.5 4.7 12.1 4.5 49.3 3.5
Cpd. No. 207 87.3 3.6 44.0 5.4 54.6 5.2 Cpd. No. 178 94.4 1.8 58.0
2.4 73.8 4.7 Cpd. No. 181 78.8 4.6 -13.2 7.2 29.4 8.0 Cpd. No. 182
60.1 5.6 -15.1 11.5 3.4 4.8 Cpd. No. 185 85.0 4.0 51.6 7.0 43.9 9.6
Cpd. No. 186 70.5 3.9 1.5 9.4 19.1 3.9
[1304] These results demonstrate that anti-mTNF alpha steroid ADCs
can obtain an efficacious response equivalent to small molecule
steroid treatment while sparing the undesired effects on
corticosterone and P1NP.
[1305] An additional contact hypersensitivity (CHS) study was
conducted to address whether conjugation of the steroid payload to
anti-TNF mAb was required for enhanced efficacy. Mice were dosed
i.p. once according to the protocol described above with either
vehicle, anti-mTNF alpha mAb (10 mpk), anti-mTNF alpha steroid ADC
(10 mpk) (cpd no 139) or a mixture of anti-mTNF alpha mAb co-dosed
(concurrently delivered in a single i.p. injection) with an
equivalent amount of small molecule steroid to match the ADC
stoichiometry. For a 10 mpk dose of anti-mTNF alpha steroid ADC
with a DAR of 4, this was calculated to be 4 g of small molecule
steroid (Cpd. No. 42). The results shown in FIG. 9 demonstrate that
anti-mTNF alpha steroid ADC treatment had significantly increased
efficacy in reducing ear inflammation when compared to the
combination of anti-mTNF alpha mAb and small molecule steroid or
anti-mTNF alpha mAb alone.
Example 85: Activity of Anti-mTNF-Alpha Steroid ADCs in
Collagen-Induced Arthritis
[1306] The ability of anti-mTNF alpha steroid ADC (Cpd. No. 137) to
impact disease was assessed in the collagen-induced arthritis (CIA)
model of arthritis.
[1307] In these experiments, male DBA/1J mice were obtained from
Jackson Labs (Bar Harbor, Me.). Mice were used at 6 to 12 weeks of
age. All animals were maintained at constant temperature and
humidity under a 12-hour light/dark cycle and fed with rodent chow
(Lab Diet 5010 PharmaServ, Framingham, Mass.) and water ad libitum.
AbbVie is AAALAC (Association for Assessment and Accreditation of
Laboratory Animal Care) accredited, and all procedures were
approved by the Institutional Animal Care and Use Committee (IACUC)
and monitored by an attending veterinarian. Body weight and
condition were monitored, and animals were euthanized if exhibiting
>20% weight loss.
[1308] The male DBA/J mice were immunized intradermally (i.d.) at
the base of the tail with 100 .mu.L of emulsion containing 100
.mu.g of type II bovine collagen (MD Biosciences) dissolved in 0.1
N acetic acid and 200 .mu.g of heat-inactivated Mycobacterium
tuberculosis H37R.sup.7a (Complete Freund's Adjuvant, Difco,
Laurence, Kans.). Twenty-one days after immunization with collagen,
mice were boosted IP with 1 mg of Zymosan A (Sigma, St. Louis, Mo.)
in PBS. Following the boost, mice were monitored 3 to 5 times per
week for arthritis. Rear paws were evaluated for paw swelling using
Dyer spring calipers (Dyer 310-115)
[1309] Mice were enrolled between days 24 and 28 at the first
clinical signs of disease and distributed into groups of equivalent
arthritic severity. Early therapeutic treatment began at the time
of enrollment.
[1310] Animals were dosed once orally (p.o.) with steroid (Cpd. No.
3) (10 mpk) in a 0.5% HPMC/0.02% Tween80 vehicle [v/] or
intraperitoneal (i.p.) with anti-mTNF alpha mAb (10 mpk) (8C11) or
anti-mTNF alpha steroid ADC (10 mpk) (Cpd. No. 137) in 0.9% saline.
Blood was collected for antibody exposure by tail nick at 24 and 72
hours after dose. Paws were collected at the terminal timepoint for
histopathology. Blood was collected at the terminal timepoint by
cardiac puncture for complete blood counts (Sysmex XT-2000iV).
Statistical significance was determined by ANOVA.
[1311] The results are shown in FIG. 3 and demonstrate that a
single dose of anti-mTNF alpha steroid ADC can exhibit an extended
duration of action through amelioration of paw swelling for 6 weeks
compared to anti-mTNF alpha mAb or small molecule steroid
alone.
[1312] In a separate study designed to address the TNF-targeting
functionality of the anti-mTNF alpha steroid ADC, animals were
dosed once i.p. with anti-mTNF alpha mAb (0 mpk) or anti-mTNF alpha
steroid ADC (10 mpk) (Cpd. No. 145) or with isotype steroid ADC (10
mpk) (Cpd. No. 224):
##STR01055##
which recognizes the hen egg protein ovalbumin, an antigen not
expressed in mice. Both ADCs had equivalent drug load. Small
molecule steroid (3 mpk) was dosed orally once daily (q.d). The
results are shown in FIG. 10 and demonstrate that a single dose of
anti-mTNF alpha steroid ADC has equivalent efficacy to small
molecule steroid dosed daily over a 21 day period. A single dose of
the non-targeted isotype steroid ADC had only partial efficacy,
similar to anti-mTNF mAb alone over the same time-frame.
Percentages denote % inhibition compared to vehicle. An evaluation
of the animals' body weights throughout the course of this study
(FIG. 11) revealed all the treatment groups with the exception of
the anti-mTNF alpha steroid ADC group lost weight. In contrast, the
anti-mTNF alpha steroid ADC treated mice exhibited normal weight
gain throughout the 21 day study.
Example 86: Activity of Various Anti-mTNF Alpha Steroid ADCs in
Collagen-Induced Arthritis
[1313] Several anti-mTNF alpha steroid ADCs with different steroid
payloads or drug:antibody ratios (DARs) were tested for efficacy in
a mouse model of arthritis. The studies were conducted according to
the procedure outlined in Example 85. The results are shown in
Table 21 below.
TABLE-US-00049 TABLE 21 Efficay of anti-mTNF alpha steroid ADCs in
model of arthritis % inhibition of paw swelling vs vehicle Cpd DAR
(AUC.sub.0-21 d) No. (n) (at 10 mpk) SEM 136 2 75 3.7 137 4 91 6.5
139 4 93 2.9 143 2 96 3.3 145 4 95 4 151 4 101 2.5 172 4 74 9.3 176
4 85 8.3 177 4 99 4.2
Example 87: Activity of Anti-hTNF-Alpha Immunoconjugates in Human
TNF Transgenic Tg1278TNF Knock-Out Mouse Model of Collagen Antibody
Induced Arthritis
[1314] The efficacy of anti-human TNF alpha ADCs was assessed in a
human TNFa transgenic mouse model of arthritis.
[1315] The Collagen Antibody-Induced Arthritis (CAIA) model (Moore,
A R J Transl Med 12:285 (2014)) was performed using the human TNF
transgenic Tg1278TNF knock-out mice as previously described (Moore
A et al. J Transl Med 12 (1): 285 (2014)). Eight mgs of a cocktail
of monoclonal antibodies that target different epitopes of collagen
type II (ArthritoMab.TM.) were administered intraperitoneally
(i.p.) to the mice on day 0. On day 3, the mice were injected i.p.
with 10 g LPS to boost the disease pathology. Animals were
evaluated for arthritic score daily starting from day 3 until day
14 of the study. Eight male mice were used per group and test
articles or PBS vehicle were administered i.p. twice a week for two
weeks.
[1316] The results are shown in FIG. 4 and demonstrate that
anti-human TNF alpha ADCs can significantly reduce disease score
compared to an anti-human TNF alpha mAb (adalimumab).
Example 88: Activity of Anti-mTNF Alpha Steroid ADCs on Peak
Inflammation
[1317] A mouse CIA experiment was conducted to establish the
efficacy of anti-mTNF alpha steroid ADC on animals with peak
inflammation. For late therapeutic dosing, mice were enrolled in
the study at first clinical signs of disease and dosed 6 days after
enrollment. A group of animals was sacrificed at day 7 of disease
to provide a baseline for arthritic changes by micro-computed
tomography (.mu.CT) and histologic analysis at the time all other
groups were dosed. All animals were dosed once on day 6 with either
vehicle (0.9% saline), anti-mTNF alpha mAb (10 mpk) (8C11) or
anti-mTNF alpha steroid ADC (10 mpk) (Cpd. No. 145) and sacrificed
on day 21. Arthritic hind paws were collected and .mu.CT analysis
was performed. The same paws were then used for histologic
evaluation. At the termination of the experiment, whole blood was
collected by cardiac puncture to evaluate complete blood counts
(CBCs).
Micro-Computed Tomography (.mu.CT)
[1318] Rear paws were removed intact at the tibia/fibula and fixed
in 10% Formalin. Paws were scanned by .mu.CT (Scanco Medical AG,
Micro-CT40) at 55 kVp at 145 .mu.A at High Resolution setting (1000
Projections/180.degree. at 2048.times.2048 Pixel Reconstruction)
using Isotropic Voxels and 300 millisecond integration time. A
cylindrical contour was manually drawn around region of interest
from the tibiotalar junction and extending into the ankle for 100
slices (1.8 mm). Evaluation was performed by Scanco software
utilizing 0.8 sigma gauss, with an upper threshold of 1000 and a
lower threshold of 320.
Histologic Evaluation
[1319] Rear paws from treated mice were immersion fixed in 10%
neutral buffered formalin and partially decalcified in Calrite
solution for 48 hours to allow trimming of the lateral and medial
edges of the tarsus. Paws were then placed back into Calrite for 48
hours to complete decalcification. Samples were routinely
processed, embedded in paraffin in the sagittal plane, sectioned at
5 microns and stained with hematoxylin and eosin. Slides were
evaluated microscopically for the presence of inflammation/pannus
formation, neutrophil infiltration, bone erosion and cartilage
damage using a 0-4 scale: 0=none present, 1=mild, 2=moderate,
3=marked, 4=severe.
[1320] The results shown in FIG. 12 demonstrate that a single dose
of anti-mTNF alpha steroid ADC can reverse established disease and
reduce paw swelling to near baseline. In contrast, a single dose of
anti-mTNF alpha mAb had a minimal effect on inflammation.
[1321] The effect of treatment on tarsal bone loss as measured by
.mu.CT is shown in FIG. 13. The results demonstrate that a single
dose of anti-mTNF alpha steroid ADC administered at peak of
inflammation is able to significantly inhibit disease-mediated
joint bone erosion compared to anti-mTNF alpha mAb alone.
[1322] The results of histological evaluation of the joints of
treated CIA mice are shown in FIGS. 14-17. They demonstrate that a
single dose of anti-mTNF alpha steroid ADC administered at peak
disease resulted in a significant decrease in inflammation, pannus
formation, bone erosion and cartilage damage by day 21 relative to
age-matched vehicle controls (p<0.001), and levels of disease
were equivalent to the levels observed in controls at baseline
(vehicle d6). In two of six paws evaluated, no disease was
detectable in the tarsus/phalangeal joints of anti-mTNF alpha
steroid ADC treated animals at day 21, as compared to 100%
incidence in mice at day 6 baseline (prior to treatment) and day 21
vehicle treated mice.
[1323] In contrast, a single dose of anti-mTNF alpha mAb at peak
disease did not inhibit inflammation, bone erosion, pannus
formation, or cartilage destruction, relative to age-matched
vehicle controls at d21. Levels of disease were more severe than
baseline controls, and a mild trend for improved inflammation was
observed.
[1324] Whole blood was analyzed to evaluate changes in peripheral
blood cell subsets with treatment. The results shown in FIG. 18-23
demonstrate that the increase in some peripheral blood cell
populations observed in diseased animals can be resolved with a
single dose of anti-mTNF alpha steroid ADC. Statistically
significant reductions in overall white blood cells, neutrophils
and monocytes were observed with anti-mTNF alpha steroid ADC
treatment.
Example 89: Comparison of Anti-mTNF-Alpha Steroid ADCs and
Anti-CD163 ADCs
[1325] To demonstrate the enhanced therapeutic efficacy of an
anti-TNF immunoconjugate in the treatment of inflammatory disease,
we compared its activity to an ADC targeting the hemoglobin
scavenger receptor CD163, a glucocorticoid immunoconjugate approach
described in the literature to have targeted anti-inflammatory
functionality (PCT Int. Appl. WO2011039510A2 by Graversen N J H, et
al.; Graversen J H et al., Mol. Ther. 20 (8): 1550-8 (2012)).
Generation of a Mouse CD163 GRE Report Cell Line
[1326] A parental cell line was created similar to that described
in Example 78 but with CHO-K1 cells instead of K562 cells. The
resulting parental cell line CHO
pGL4.36[Luc2P/MMTV/Hygro]_PGL4.75[hRLuc/CMV] was then transfected
with a plasmid which encodes mouse CD163 (Origene cat.no. MR216798)
under conditions described in Example 78. The resulting cell line
CHO mCD163 GRE (pGL4.36[luc2P/MMTV/Hygro]) was used to test the in
vitro activity of both anti-mTNF-alpha and anti-mouse CD163
immunoconjugates (also referred to as anti-mCD163 immunoconjugates
or anti-mCD163 steroid ADC).
Preparation of an Anti-Mouse CD163 Immunoconjugate
[1327] A chimeric rat anti-mouse CD163 mIgG2a/k antibody was
generated from the VH and VL sequence for clone 3E10B10 as
described (SEQ ID NO: 87/88 from PCT Int. Appl. WO
2011/039510A.sup.2). This antibody was conjugated to Cpd. No. 99
using conditions highlighted under the general cysteine conjugation
protocol in Example 36 to give a drug:antibody ratio (DAR) of
4.
Activity of an Anti-Mouse CD163 Immunoconjugate in Mouse CD163 GRE
Reporter Assay
[1328] The anti-mouse CD163 immunoconjugate was tested for activity
on the CHO mCD163 GRE (pGL4.36[luc2P/MMTV/Hygro]) cell line under
conditions described in Example 79. An anti-mTNF alpha steroid ADC
(Cpd. No. 145) was included as a negative control. The results in
Table 22 demonstrate the anti-mouse CD163 immunoconjugate (Cpd. No.
223):
##STR01056##
shows antigen-dependent activity dissociated from the anti-mTNF
alpha steroid ADC on the mouse CD163 GRE cell line.
TABLE-US-00050 TABLE 22 % mCD163 mCD163 CHO GRE CHO Cpd mono- GRE
EC50 GRE EC50 GRE No. DAR mer (ug/ml) (% max) (ug/ml) (% max) 223 4
93 0.2 92 >50 55 145 4 97 >20 70 >50 39
Activity of Anti-Mouse CD163 Immunoconjugate in Mouse
Collagen-Induced Arthritis
[1329] The ability of anti-mouse CD163 steroid immunoconjugate to
impact paw swelling was assessed in the collagen-induced arthritis
(CIA) model of RA. A control anti-mTNF alpha steroid ADC (cpd 139)
with the same drug-linker and DAR as the anti-mCD163 steroid ADC
was also evaluated in the same study and the parental mAbs for both
ADCs were also included as treatment groups. The experiment was
conducted according to the procedure outlined in Example 85. The
results are shown in FIG. 24 and demonstrate that while the
anti-mCD163 steroid ADC initially reduces paw swelling in the first
few days after single dose treatment, this effect is transient. In
comparison, a single dose of anti-mTNF alpha steroid ADC is
sufficient to completely suppress inflammation through the duration
of the study
[1330] It is to be appreciated that the Detailed Description
section, and not the Summary and Abstract sections, is intended to
be used to interpret the claims. The Summary and Abstract sections
sets forth one or more, but not all, exemplary embodiments of the
present disclosure as contemplated by the inventor(s), and thus,
are not intended to limit the present disclosure and the appended
claims in any way.
[1331] The present disclosure has been described above with the aid
of functional building blocks illustrating the implementation of
specified functions and relationships thereof. The boundaries of
these functional building blocks have been arbitrarily defined
herein for the convenience of the description. Alternate boundaries
can be defined so long as the specified functions and relationships
thereof are appropriately performed.
[1332] The foregoing description of the specific embodiments will
so fully reveal the general nature of the disclosure that others
can, by applying knowledge within the skill of the art, readily
modify and/or adapt for various applications such specific
embodiments, without undue experimentation, without departing from
the general concept of the present disclosure. Therefore, such
adaptations and modifications are intended to be within the meaning
and range of equivalents of the disclosed embodiments, based on the
teaching and guidance presented herein. It is to be understood that
the phraseology or terminology herein is for the purpose of
description and not of limitation, such that the terminology or
phraseology of the present specification is to be interpreted by
the skilled artisan in light of the teachings and guidance.
[1333] The breadth and scope of the present disclosure should not
be limited by any of the above-described exemplary embodiments, but
should be defined only in accordance with the following claims and
their equivalents.
Sequence CWU 1
1
941233PRThomo sapiens 1Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu
Leu Ala Glu Glu Ala1 5 10 15Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly
Ser Arg Arg Cys Leu Phe 20 25 30Leu Ser Leu Phe Ser Phe Leu Ile Val
Ala Gly Ala Thr Thr Leu Phe 35 40 45Cys Leu Leu His Phe Gly Val Ile
Gly Pro Gln Arg Glu Glu Phe Pro 50 55 60Arg Asp Leu Ser Leu Ile Ser
Pro Leu Ala Gln Ala Val Arg Ser Ser65 70 75 80Ser Arg Thr Pro Ser
Asp Lys Pro Val Ala His Val Val Ala Asn Pro 85 90 95Gln Ala Glu Gly
Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 100 105 110Leu Ala
Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 115 120
125Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly
130 135 140Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg
Ile Ala145 150 155 160Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser
Ala Ile Lys Ser Pro 165 170 175Cys Gln Arg Glu Thr Pro Glu Gly Ala
Glu Ala Lys Pro Trp Tyr Glu 180 185 190Pro Ile Tyr Leu Gly Gly Val
Phe Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205Ser Ala Glu Ile Asn
Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210 215 220Gln Val Tyr
Phe Gly Ile Ile Ala Leu225 2302235PRTmurine 2Met Ser Thr Glu Ser
Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala1 5 10 15Leu Pro Gln Lys
Met Gly Gly Phe Gln Asn Ser Arg Arg Cys Leu Cys 20 25 30Leu Ser Leu
Phe Ser Phe Leu Leu Val Ala Gly Ala Thr Thr Leu Phe 35 40 45Cys Leu
Leu Asn Phe Gly Val Ile Gly Pro Gln Arg Asp Glu Lys Phe 50 55 60Pro
Asn Gly Leu Pro Leu Ile Ser Ser Met Ala Gln Thr Leu Thr Leu65 70 75
80Arg Ser Ser Ser Gln Asn Ser Ser Asp Lys Pro Val Ala His Val Val
85 90 95Ala Asn His Gln Val Glu Glu Gln Leu Glu Trp Leu Ser Gln Arg
Ala 100 105 110Asn Ala Leu Leu Ala Asn Gly Met Asp Leu Lys Asp Asn
Gln Leu Val 115 120 125Val Pro Ala Asp Gly Leu Tyr Leu Val Tyr Ser
Gln Val Leu Phe Lys 130 135 140Gly Gln Gly Cys Pro Asp Tyr Val Leu
Leu Thr His Thr Val Ser Arg145 150 155 160Phe Ala Ile Ser Tyr Gln
Glu Lys Val Asn Leu Leu Ser Ala Val Lys 165 170 175Ser Pro Cys Pro
Lys Asp Thr Pro Glu Gly Ala Glu Leu Lys Pro Trp 180 185 190Tyr Glu
Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp 195 200
205Gln Leu Ser Ala Glu Val Asn Leu Pro Lys Tyr Leu Asp Phe Ala Glu
210 215 220Ser Gly Gln Val Tyr Phe Gly Val Ile Ala Leu225 230
23535PRTartificial sequenceadalimumab VH-CDR1 3Asp Tyr Ala Met His1
5417PRTartificial sequenceadalimumab VH-CDR2 4Ala Ile Thr Trp Asn
Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu1 5 10
15Gly59PRTartificial sequenceadalimumab VH-CDR3 5Val Ser Tyr Leu
Ser Thr Ala Ser Ser1 5610PRTartificial sequenceadalimumab VH-CDR1
6Gly Phe Thr Phe Asp Asp Tyr Ala Met His1 5 10710PRTartificial
sequenceinfliximab VH-CDR1 7Gly Phe Ile Phe Ser Asn His Trp Met
Asn1 5 10819PRTartificial sequenceinfliximab VH-CDR2 8Glu Ile Arg
Ser Lys Ser Ile Asn Ser Ala Thr His Tyr Ala Glu Ser1 5 10 15Val Lys
Gly99PRTartificial sequenceinfliximab VH-CDR3 9Asn Tyr Tyr Gly Ser
Thr Tyr Asp Tyr1 5105PRTartificial sequencecertolizumab VH-CDR1
10Asp Tyr Gly Met Asn1 51117PRTartificial sequencecertolizumab
VH-CDR2 11Trp Ile Asn Thr Tyr Ile Gly Glu Pro Ile Tyr Ala Asp Ser
Val Lys1 5 10 15Gly129PRTartificial sequencecertolizumab VH-CDR3
12Gly Tyr Arg Ser Tyr Ala Met Asp Tyr1 51310PRTartificial
sequencecertolizumab VH-CDR1 13Gly Tyr Val Phe Thr Asp Tyr Gly Met
Asn1 5 10145PRTartificial sequenceafelimomab VH-CDR1 14Asp Tyr Gly
Val Asn1 51516PRTartificial sequenceafelimomab VH-CDR2 15Met Ile
Trp Gly Asp Gly Ser Thr Asp Tyr Asp Ser Thr Leu Lys Ser1 5 10
15169PRTartificial sequenceafelimomab VH-CDR3 16Glu Trp His His Gly
Pro Val Ala Tyr1 5175PRTartificial sequencenerelimomab VH-CDR1
17Asp Tyr Asn Val Asp1 51817PRTartificial sequencenerelimomab
VH-CDR2 18Asn Ile Asn Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys
Phe Lys1 5 10 15Gly1912PRTartificial sequencenerelimomab VH-CDR3
19Ser Ala Phe Tyr Asn Asn Tyr Glu Tyr Phe Asp Val1 5
10205PRTartificial sequenceozoralizumab VH-CDR1 20Asp Tyr Trp Met
Tyr1 52117PRTartificial sequenceozoralizumab VH-CDR2 21Glu Ile Asn
Thr Asn Gly Leu Ile Thr Lys Tyr Pro Asp Ser Val Lys1 5 10
15Gly227PRTartificial sequenceozoralizumab VH-CDR3 22Ser Pro Ser
Gly Phe Asn Arg1 5235PRTartificial sequenceozoralizumab VH-CDR1
23Ser Phe Gly Met Ser1 52417PRTartificial sequenceozoralizumab
VH-CDR2 24Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser
Val Lys1 5 10 15Gly258PRTartificial sequenceozoralizumab VH-CDR3
25Gly Gly Ser Leu Ser Arg Ser Ser1 5265PRTartificial
sequenceozoralizumab VH-CDR1 26Asp Tyr Trp Met Tyr1
52717PRTartificial sequenceozoralizumab VH-CDR2 27Glu Ile Asn Thr
Asn Gly Leu Ile Thr Lys Tyr Pro Asp Ser Val Lys1 5 10
15Gly287PRTartificial sequenceozoralizumab VH-CDR3 28Ser Pro Ser
Gly Phe Asn Arg1 52910PRTartificial sequencegolimumab VH-CDR1 29Gly
Phe Ile Phe Ser Ser Tyr Ala Met His1 5 103017PRTartificial
sequencegolimumab VH-CDR2 30Phe Met Ser Tyr Asp Gly Ser Asn Lys Lys
Tyr Ala Asp Ser Val Lys1 5 10 15Gly3117PRTartificial
sequencegolimumab VH-CDR3 31Asp Arg Gly Ile Ala Ala Gly Gly Asn Tyr
Tyr Tyr Tyr Gly Met Asp1 5 10 15Val3211PRTartificial
sequenceadalimumab VL-CDR1 32Arg Ala Ser Gln Gly Ile Arg Asn Tyr
Leu Ala1 5 10337PRTartificial sequenceadalimumab VL-CDR2 33Ala Ala
Ser Thr Leu Gln Ser1 5349PRTartificial sequenceadalimumab VL-CDR3
34Gln Arg Tyr Asn Arg Ala Pro Tyr Thr1 53511PRTartificial
sequenceinfliximab VL-CDR1 35Arg Ala Ser Gln Phe Val Gly Ser Ser
Ile His1 5 10367PRTartificial sequenceinfliximab VL-CDR2 36Tyr Ala
Ser Glu Ser Met Ser1 5379PRTartificial sequenceinfliximab VL-CDR3
37Gln Gln Ser His Ser Trp Pro Phe Thr1 53811PRTartificial
sequencecertolizumab VL-CDR1 38Lys Ala Ser Gln Asn Val Gly Thr Asn
Val Ala1 5 10397PRTartificial sequencecertolizumab VL-CDR2 39Ser
Ala Ser Phe Leu Tyr Ser1 5409PRTartificial sequencecertolizumab
VL-CDR3 40Gln Gln Tyr Asn Ile Tyr Pro Leu Thr1 54111PRTartificial
sequenceafelimomab VL-CDR1 41Lys Ala Ser Gln Ala Val Ser Ser Ala
Val Ala1 5 10427PRTartificial sequenceafelimomab VL-CDR2 42Trp Ala
Ser Thr Arg His Thr1 5439PRTartificial sequenceafelimomab VL-CDR3
43Gln Gln His Tyr Ser Thr Pro Phe Thr1 54417PRTartificial
sequencenerelimomab VL-CDR1 44Lys Ser Ser Gln Ser Leu Leu Tyr Ser
Asn Asn Gln Lys Asn Tyr Leu1 5 10 15Ala457PRTartificial
sequencenerelimomab VL-CDR2 45Trp Ala Ser Thr Arg Glu Ser1
5469PRTartificial sequencenerelimomab VL-CDR3 46Gln Gln Tyr Tyr Asp
Tyr Pro Trp Thr1 54711PRTartificial sequencegolimumab VL-CDR1 47Arg
Ala Ser Gln Ser Val Tyr Ser Tyr Leu Ala1 5 10487PRTartificial
sequencegolimumab VL-CDR2 48Asp Ala Ser Asn Arg Ala Thr1
54910PRTartificial sequencegolimumab VL-CDR3 49Gln Gln Arg Ser Asn
Trp Pro Pro Phe Thr1 5 1050121PRTartificial sequenceadalimumab VH
50Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp
Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala
Asp Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val Ser Tyr Leu Ser Thr Ala
Ser Ser Leu Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12051119PRTartificial sequenceinfliximab VH 51Glu Val
Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Met Lys Leu Ser Cys Val Ala Ser Gly Phe Ile Phe Ser Asn His 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45Ala Glu Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr His Tyr Ala
Glu 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys
Ser Ala65 70 75 80Val Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp
Thr Gly Val Tyr 85 90 95Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser Thr Tyr
Asp Tyr Trp Gly Gln 100 105 110Gly Thr Thr Leu Thr Val Ser
11552118PRTartificial sequencecertolizumab VH 52Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Tyr Val Phe Thr Asp Tyr 20 25 30Gly Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Asn Thr Tyr Ile Gly Glu Pro Ile Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Tyr Arg Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 11553117PRTartificial
sequenceafelimomab VH 53Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu
Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly
Phe Ser Leu Thr Asp Tyr 20 25 30Gly Val Asn Trp Val Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Met Ile Trp Gly Asp Gly Ser
Thr Asp Tyr Asp Ser Thr Leu Lys 50 55 60Ser Arg Leu Ser Ile Ser Lys
Asp Asn Ser Lys Ser Gln Ile Phe Leu65 70 75 80Lys Asn Asn Ser Leu
Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala 85 90 95Arg Glu Trp His
His Gly Pro Val Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr
Val Ser Ala 11554121PRTartificial sequencenerelimomab VH 54Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ser1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25
30Asn Val Asp Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Gln Trp Ile
35 40 45Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys
Phe 50 55 60Lys Gly Lys Gly Thr Leu Thr Val Asp Lys Ser Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Ser Ala Phe Tyr Asn Asn Tyr Glu Tyr
Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser
115 12055115PRTartificial sequenceozoralizumab VH V1 55Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30Trp
Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Pro Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Ser Pro Ser Gly Phe Asn Arg Gly Gln Gly
Thr Leu Val Thr 100 105 110Val Ser Ser 11556115PRTartificial
sequenceozoralizumab VH V2 56Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser
Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ile
Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105
110Val Ser Ser 11557115PRTartificial sequenceozoralizumab VH V3
57Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp
Tyr 20 25 30Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Pro
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Pro Ser Gly Phe Asn Arg
Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser
11558126PRTartificial sequencegolimumab VH 58Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Tyr 20 25 30Ala Met His
Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Val 35 40 45Ala Phe
Met Ser Tyr Asp Gly Ser Asn Lys Lys Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Arg Gly Ile Ala Ala Gly Gly Asn Tyr Tyr Tyr Tyr
Gly 100 105 110Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser 115 120 12559107PRTartificial sequenceadalimumab VL 59Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn
Arg Ala Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys
100 10560107PRTartificial sequenceinfliximab VL 60Asp Ile Leu Leu
Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Val
Ser Phe Ser Cys Arg Ala Ser Gln Phe Val Gly Ser Ser 20 25 30Ile His
Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45Lys
Tyr Ala Ser Glu Ser Met Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Thr Val Glu Ser65
70 75 80Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser His Ser Trp Pro
Phe 85 90 95Thr Phe Gly Ser Gly Thr Asn Leu Glu Val Lys 100
10561107PRTartificial sequencecertolizumab VL 61Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile 35 40 45Tyr Ser
Ala Ser Phe Leu Tyr Ser Gly Val Pro Tyr Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10562107PRTartificial sequenceafelimomab VL 62Asp Ile Val Met Thr
Gln Ser His Lys Phe Met Ser Thr Thr Val Gly1 5 10 15Asp Arg Val Ser
Ile Thr Cys Lys Ala Ser Gln Ala Val Ser Ser Ala 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Trp
Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser
Gly Ser Val Thr Asp Phe Thr Leu Thr Ile His Asn Leu Gln Ala65 70 75
80Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Phe
85 90 95Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100
10563114PRTartificial sequencenerelimomab VL 63Asp Ile Met Met Thr
Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30Asn Asn Gln
Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ala Pro
Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro
Ser Arg Phe Ile Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr65 70 75
80Ile Ser Ser Leu Gln Pro Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln
85 90 95Tyr Tyr Asp Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile 100 105 110Lys Arg64107PRTartificial sequenceplaculumab VL
64Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Asp Ser
Tyr 20 25 30Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Leu Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Val Val Trp Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 10565108PRTartificial sequencegolimumab VL 65Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Tyr Ser Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Trp Pro Pro 85 90 95Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile
Lys 100 10566451PRTartificial sequenceAdalimumab (D2E7) Full-Length
Heavy Chain 66Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Thr Trp Asn Ser Gly His Ile
Asp Tyr Ala Asp Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val Ser Tyr Leu
Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135
140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250
255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375
380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
45067450PRTartificial sequenceinfliximab Full-Length Heavy Chain
67Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Ile Phe Ser Asn
His 20 25 30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu
Trp Val 35 40 45Ala Glu Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr His
Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Ser Ala65 70 75 80Val Tyr Leu Gln Met Thr Asp Leu Arg Thr
Glu Asp Thr Gly Val Tyr 85 90 95Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser
Thr Tyr Asp Tyr Trp Gly Gln 100 105 110Gly Thr Thr Leu Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155
160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280
285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395
400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His 420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 445Gly Lys 45068229PRTartificial
sequencecertolizumab Full-Length Heavy Chain 68Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Tyr Val Phe Thr Asp Tyr 20 25 30Gly Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Asn Thr Tyr Ile Gly Glu Pro Ile Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Tyr Arg Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220His Thr Cys Ala Ala22569447PRTartificial
sequenceafelimomab Full-Length Heavy Chain 69Gln Val Gln Leu Lys
Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser Ile
Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr 20 25 30Gly Val Asn
Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Met
Ile Trp Gly Asp Gly Ser Thr Asp Tyr Asp Ser Thr Leu Lys 50 55 60Ser
Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Ile Phe Leu65 70 75
80Lys Asn Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala
85 90 95Arg Glu Trp His His Gly Pro Val Ala Tyr Trp Gly Gln Gly Thr
Leu 100 105 110Val Thr Val Ser Ala Ala Thr Thr Thr Ala Pro Ser Val
Tyr Pro Leu 115 120 125Val Pro Gly Cys Ser Asp Thr Ser Gly Ser Ser
Val Thr Leu Gly Cys 130 135 140Leu Val Lys Gly Tyr Phe Pro Glu Pro
Val Thr Val Lys Trp Asn Tyr145 150 155 160Gly Ala Leu Ser Ser Gly
Val Arg Thr Val Ser Ser Val Leu Gln Ser 165 170 175Gly Phe Tyr Ser
Leu Ser Ser Leu Val Thr Val Pro Ser Ser Thr Trp 180 185 190Pro Ser
Gln Thr Val Ile Cys Asn Val Ala His Pro Ala Ser Lys Thr 195 200
205Glu Leu Ile Lys Arg Ile Glu Pro Arg Ile Pro Lys Pro Ser Thr Pro
210 215 220Pro Gly Ser Ser Cys Pro Pro Gly Asn Ile Leu Gly Gly Pro
Ser Val225 230 235 240Phe Ile Phe Pro Pro Lys Pro Lys Asp Ala Leu
Met Ile Ser Leu Thr 245 250 255Pro Lys Val Thr Cys Val Val Val Asp
Val Ser Glu Asp Asp Pro Asp 260 265 270Val His Val Ser Trp Phe Val
Asp Asn Lys Glu Val His Thr Ala Trp 275 280 285Thr Gln Pro Arg Glu
Ala Gln Tyr Asn Ser Thr Phe Arg Val Val Ser 290 295 300Ala Leu Pro
Ile Gln His Gln Asp Trp Met Arg Gly Lys Glu Phe Lys305 310 315
320Cys Lys Val Asn Asn Lys Ala Leu Pro Ala Pro Ile Glu Arg Thr Ile
325 330 335Ser Lys Pro Lys Gly Arg Ala Gln Thr Pro Gln Val Tyr Thr
Ile Pro 340 345 350Pro Pro Arg Glu Gln Met Ser Lys Lys Lys Val Ser
Leu Thr Cys Leu 355 360 365Val Thr Asn Phe Phe Ser Glu Ala Ile Ser
Val Glu Trp Glu Arg Asn 370 375 380Gly Glu Leu Glu Gln Asp Tyr Lys
Asn Thr Pro Pro Ile Leu Asp Ser385 390 395 400Asp Gly Thr Tyr Phe
Leu Tyr Ser Lys Leu Thr Val Asp Thr Asp Ser 405 410 415Trp Leu Gln
Gly Glu Ile Phe Thr Cys Ser Val Val His Glu Ala Leu 420 425 430His
Asn His His Thr Gln Lys Asn Leu Ser Arg Ser Pro Gly Lys 435 440
44570363PRTartificial sequenceozoralizumab Full-Length Heavy Chain
70Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp
Tyr 20 25 30Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Pro
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Pro Ser Gly Phe Asn Arg
Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly Ser Glu Val Gln Leu 115 120 125Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu 130 135 140Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp145 150 155
160Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile
Ser
165 170 175Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly
Arg Phe 180 185 190Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr
Leu Gln Met Asn 195 200 205Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Thr Ile Gly Gly 210 215 220Ser Leu Ser Arg Ser Ser Gln Gly
Thr Leu Val Thr Val Ser Ser Gly225 230 235 240Gly Gly Gly Ser Gly
Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly 245 250 255Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 260 265 270Ser
Gly Phe Thr Phe Ser Asp Tyr Trp Met Tyr Trp Val Arg Gln Ala 275 280
285Pro Gly Lys Gly Leu Glu Trp Val Ser Glu Ile Asn Thr Asn Gly Leu
290 295 300Ile Thr Lys Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile
Ser Arg305 310 315 320Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met
Asn Ser Leu Arg Pro 325 330 335Glu Asp Thr Ala Val Tyr Tyr Cys Ala
Arg Ser Pro Ser Gly Phe Asn 340 345 350Arg Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 355 36071234PRTartificial sequenceplaculumab
Full-Length Heavy Chain 71Arg Val Glu Pro Lys Ser Ser Asp Lys Thr
His Thr Cys Pro Pro Cys1 5 10 15Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 20 25 30Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 35 40 45Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp 50 55 60Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu65 70 75 80Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 85 90 95His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 100 105 110Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120
125Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
130 135 140Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr145 150 155 160Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn 165 170 175Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe 180 185 190Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215 220Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys225 23072456PRTartificial
sequencegolimumab Full-Length Heavy Chain 72Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Tyr 20 25 30Ala Met His Trp
Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Val 35 40 45Ala Phe Met
Ser Tyr Asp Gly Ser Asn Lys Lys Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Arg Gly Ile Ala Ala Gly Gly Asn Tyr Tyr Tyr Tyr
Gly 100 105 110Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser Ala Ser 115 120 125Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr 130 135 140Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro145 150 155 160Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 165 170 175His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 180 185 190Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 195 200
205Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
210 215 220Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala225 230 235 240Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro 245 250 255Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val 260 265 270Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val 275 280 285Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 290 295 300Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln305 310 315
320Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
325 330 335Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro 340 345 350Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr 355 360 365Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser 370 375 380Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr385 390 395 400Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 405 410 415Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 420 425 430Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 435 440
445Ser Leu Ser Leu Ser Pro Gly Lys 450 45573214PRTartificial
sequenceAdalimumab (D2E7) Full-Length Light Chain 73Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro
Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val
Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21074214PRTartificial sequenceinfliximab
Full-Length Light Chain 74Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile
Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Val Ser Phe Ser Cys Arg Ala
Ser Gln Phe Val Gly Ser Ser 20 25 30Ile His Trp Tyr Gln Gln Arg Thr
Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Glu Ser Met
Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Ser Ile Asn Thr Val Glu Ser65 70 75 80Glu Asp Ile Ala
Asp Tyr Tyr Cys Gln Gln Ser His Ser Trp Pro Phe 85 90 95Thr Phe Gly
Ser Gly Thr Asn Leu Glu Val Lys Arg Thr Val Ala Ala 100 105 110Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 21075214PRTartificial sequencecertolizumab Full-Length Light
Chain 75Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly
Thr Asn 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Ala Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Tyr
Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Tyr Asn Ile Tyr Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21076214PRTartificial sequenceafelimomab Full-Length Light Chain
76Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Thr Val Gly1
5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Ala Val Ser Ser
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu
Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg
Phe Thr Gly 50 55 60Ser Gly Ser Val Thr Asp Phe Thr Leu Thr Ile His
Asn Leu Gln Ala65 70 75 80Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln
His Tyr Ser Thr Pro Phe 85 90 95Thr Phe Gly Ser Gly Thr Lys Leu Glu
Ile Lys Arg Ala Asp Ala Ala 100 105 110Pro Thr Val Ser Ile Phe Pro
Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125Gly Ala Ser Val Val
Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140Asn Val Lys
Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu145 150 155
160Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn
Ser Tyr 180 185 190Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
Ile Val Lys Ser 195 200 205Phe Asn Arg Asn Glu Cys
21077108PRTartificial sequenceplaculumab Full-Length Light Chain
77Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Asp Ser
Tyr 20 25 30Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Leu Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Val Val Trp Arg Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 10578215PRTartificial sequencegolimumab Full-Length
Light Chain 78Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu
Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Tyr Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile
Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95Phe Thr Phe Gly Pro Gly
Thr Lys Val Asp Ile Lys Arg Thr Val Ala 100 105 110Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135
140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn Arg Gly Glu
Cys 210 21579467PRTArtificial Sequenceetanercept 79Leu Pro Ala Gln
Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser1 5 10 15Thr Cys Arg
Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys 20 25 30Ser Lys
Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr 35 40 45Ser
Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 50 55
60Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser65
70 75 80Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile
Cys 85 90 95Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu
Gly Cys 100 105 110Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly
Phe Gly Val Ala 115 120 125Arg Pro Gly Thr Glu Thr Ser Asp Val Val
Cys Lys Pro Cys Ala Pro 130 135 140Gly Thr Phe Ser Asn Thr Thr Ser
Ser Thr Asp Ile Cys Arg Pro His145 150 155 160Gln Ile Cys Asn Val
Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala 165 170 175Val Cys Thr
Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val 180 185 190His
Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr 195 200
205Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly
210 215 220Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys
Ser Cys225 230 235 240Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 245 250 255Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 260 265 270Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 275 280 285Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr305 310 315
320Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
325 330 335Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile 340
345 350Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val 355 360 365Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser 370 375 380Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu385 390 395 400Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455
460Pro Gly Lys46580587PRTArtificial SequenceABT-122 Heavy Chain
80Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp
Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala
Asp Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val Ser Tyr Leu Ser Thr Ala
Ser Ser Leu Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Gly Gly Ser Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 130 135 140Pro Gly Ser
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Ser Phe145 150 155
160Gly Gly Tyr Gly Ile Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
165 170 175Glu Trp Met Gly Gly Ile Thr Pro Phe Phe Gly Phe Ala Asp
Tyr Ala 180 185 190Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp
Glu Ser Thr Thr 195 200 205Thr Ala Tyr Met Glu Leu Ser Gly Leu Thr
Ser Asp Asp Thr Ala Val 210 215 220Tyr Tyr Cys Ala Arg Asp Pro Asn
Glu Phe Trp Asn Gly Tyr Tyr Ser225 230 235 240Thr His Asp Phe Asp
Ser Trp Gly Gln Gly Thr Thr Val Thr Val Ser 245 250 255Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 260 265 270Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 275 280
285Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
290 295 300Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr305 310 315 320Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln 325 330 335Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp 340 345 350Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro 355 360 365Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 370 375 380Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr385 390 395
400Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
405 410 415Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg 420 425 430Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val 435 440 445Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser 450 455 460Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys465 470 475 480Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 485 490 495Glu Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 500 505 510Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 515 520
525Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
530 535 540Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly545 550 555 560Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr 565 570 575Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 580 58581331PRTArtificial SequenceABT-122 Light Chain 81Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr
Asn Arg Ala Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Gly Gly Ser Gly 100 105 110Gly Gly Gly Ser Gly Glu Ile Val
Leu Thr Gln Ser Pro Asp Phe Gln 115 120 125Ser Val Thr Pro Lys Glu
Lys Val Thr Ile Thr Cys Arg Ala Ser Gln 130 135 140Asp Ile Gly Ser
Glu Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Pro145 150 155 160Pro
Lys Leu Leu Ile Lys Tyr Ala Ser His Ser Thr Ser Gly Val Pro 165 170
175Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
180 185 190Asn Gly Leu Glu Ala Glu Asp Ala Gly Thr Tyr Tyr Cys His
Gln Thr 195 200 205Asp Ser Leu Pro Tyr Thr Phe Gly Pro Gly Thr Lys
Val Asp Ile Lys 210 215 220Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu225 230 235 240Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe 245 250 255Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 260 265 270Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 275 280 285Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 290 295
300Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser305 310 315 320Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 325
33082235PRTArtificial SequenceCertolizumab Heavy Chain 82Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Val Phe Thr Asp Tyr 20 25
30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45Gly Trp Ile Asn Thr Tyr Ile Gly Glu Pro Ile Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr
Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Gly Tyr Arg Ser Tyr Ala Met Asp Tyr
Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr 210 215 220His Thr Cys Ala Ala His His His His His
His225 230 23583214PRTArtificial SequenceCertolizumab Light Chain
83Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr
Asn 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala
Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Tyr Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Asn Ile Tyr Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21084238PRTArtificial SequenceAdalimumab Fab Heavy Chain 84Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25
30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser
Val 50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser
Leu Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Ala Ala His His
His His His His225 230 23585214PRTArtificial SequenceAdalimumab Fab
Light Chain 85Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr
Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
2108660PRTArtificial SequenceAffibody 86Gly Val Asp Asn Lys Phe Asn
Lys Glu Asn Ile Ala Ala Met Thr Glu1 5 10 15Ile Thr Arg Leu Pro Asn
Leu Asn Pro Tyr Gln Arg Ala Ala Phe Ile 20 25 30Trp Ser Leu Ser Asp
Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu 35 40 45Ala Lys Lys Leu
Asn Asp Ala Gln Ala Pro Lys Cys 50 55 6087382PRTArtificial
SequenceOzoralizumab Nanobody 87Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30Trp Met Tyr Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Glu Ile Asn Thr Asn
Gly Leu Ile Thr Lys Tyr Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Ser Pro Ser Gly Phe Asn Arg Gly Gln Gly Thr Leu Val Thr 100 105
110Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu
115 120 125Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu
Arg Leu 130 135 140Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe
Gly Met Ser Trp145 150 155 160Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val Ser Ser Ile Ser 165 170 175Gly Ser Gly Ser Asp Thr Leu
Tyr Ala Asp Ser Val Lys Gly Arg Phe 180 185 190Thr Ile Ser Arg Asp
Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn 195 200 205Ser Leu Arg
Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly 210 215 220Ser
Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser Gly225 230
235 240Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser
Gly 245 250 255Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
Cys Ala Ala 260 265 270Ser Gly Phe Thr Phe Ser Asp Tyr Trp Met Tyr
Trp Val Arg Gln Ala 275 280 285Pro Gly Lys Gly Leu Glu Trp Val Ser
Glu Ile Asn Thr Asn Gly Leu 290 295 300Ile Thr Lys Tyr Pro Asp Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg305 310 315 320Asp Asn Ala Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro 325 330 335Glu Asp
Thr Ala Val Tyr Tyr Cys Ala Arg Ser Pro Ser Gly Phe Asn 340 345
350Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Glu Gln Lys
355 360 365Leu Ile Ser Glu Glu Asp Leu Cys His His His His His His
370 375 3808811PRTArtificial Sequenceplaculumab VH-CDR1 88Arg Ala
Ser Gln Ala Ile Asp Ser Tyr Leu His1 5 10897PRTArtificial
Sequenceplaculumab VH-CDR2 89Ser Ala Ser Asn Leu Glu Thr1
5909PRTArtificial Sequenceplaculumab VH-CDR3 90Gln Gln Val Val Trp
Arg Pro Phe Thr1 591120PRTArtificial
Sequenceinfliximab VH 91Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Met Lys Leu Ser Cys Val Ala Ser Gly
Phe Ile Phe Ser Asn His 20 25 30Trp Met Asn Trp Val Arg Gln Ser Pro
Glu Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile Arg Ser Lys Ser Ile
Asn Ser Ala Thr His Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Ser Ala65 70 75 80Val Tyr Leu Gln Met
Thr Asp Leu Arg Thr Glu Asp Thr Gly Val Tyr 85 90 95Tyr Cys Ser Arg
Asn Tyr Tyr Gly Ser Thr Tyr Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Thr Leu Thr Val Ser Ser 115 12092113PRTArtificial
Sequencenerelimomab VL 92Asp Ile Met Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ser
Ser Gln Ser Leu Leu Tyr Ser 20 25 30Asn Asn Gln Lys Asn Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ala Pro Lys Leu Leu Ile Ser
Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Ser Arg Phe Ile Gly
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu
Gln Pro Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Asp
Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110Lys93233PRTArtificial Sequenceplaculumab Full-Length Heavy Chain
93Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro1
5 10 15Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys 20 25 30Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val 35 40 45Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr 50 55 60Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu65 70 75 80Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His 85 90 95Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys 100 105 110Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln 115 120 125Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 130 135 140Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro145 150 155
160Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu 180 185 190Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val 195 200 205Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln 210 215 220Lys Ser Leu Ser Leu Ser Pro Gly
Lys225 2309412PRTArtificial Sequenceadalimumab VH-CDR3 94Val Ser
Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr1 5 10
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