U.S. patent application number 16/372255 was filed with the patent office on 2020-02-27 for azaindoline compounds as granzyme b inhibitors.
This patent application is currently assigned to viDA Therapeutics Inc.. The applicant listed for this patent is viDA Therapeutics Inc.. Invention is credited to Dale R. Cameron.
Application Number | 20200062803 16/372255 |
Document ID | / |
Family ID | 55216535 |
Filed Date | 2020-02-27 |
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United States Patent
Application |
20200062803 |
Kind Code |
A1 |
Cameron; Dale R. |
February 27, 2020 |
AZAINDOLINE COMPOUNDS AS GRANZYME B INHIBITORS
Abstract
Azaindoline compounds as granzyme B inhibitors, compositions
that include the compounds, and methods for using the compounds.
Methods for treating cutaneous scleroderma, epidermolysis bullosa,
radiation dermatitis, alopecia areata, and discoid lupus
erythematosus are provided.
Inventors: |
Cameron; Dale R.; (Richmond,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
viDA Therapeutics Inc. |
North Vancouver |
|
CA |
|
|
Assignee: |
viDA Therapeutics Inc.
North Vancouver
CA
|
Family ID: |
55216535 |
Appl. No.: |
16/372255 |
Filed: |
April 1, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15500781 |
Jan 31, 2017 |
10246487 |
|
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PCT/CA2015/050724 |
Jul 31, 2015 |
|
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16372255 |
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62032471 |
Aug 1, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/06 20130101;
A61Q 7/00 20130101; C07K 5/0812 20130101; A61K 8/64 20130101; A61K
38/00 20130101; C07K 5/101 20130101; C07K 5/0202 20130101; C07K
5/06104 20130101; C07K 5/0808 20130101; A61P 17/00 20180101; A61K
38/05 20130101 |
International
Class: |
C07K 5/103 20060101
C07K005/103; A61K 38/06 20060101 A61K038/06; A61K 38/05 20060101
A61K038/05; C07K 5/02 20060101 C07K005/02; C07K 5/087 20060101
C07K005/087; C07K 5/083 20060101 C07K005/083; C07K 5/072 20060101
C07K005/072; A61K 8/64 20060101 A61K008/64; A61Q 7/00 20060101
A61Q007/00 |
Claims
1. A compound having Formula (I): ##STR00087## its stereoisomers,
tautomers, and pharmaceutically acceptable salts thereof, wherein:
R.sub.1 is a heteroaryl group selected from (a) 1,2,3-triazolyl,
and (b) 1,2,3,4-tetrazolyl; n is 1 or 2; R.sub.2a and R.sub.2b are
independently selected from hydrogen and C1-C6 alkyl; R.sub.2c at
each occurrence is independently selected from (a) hydrogen, (b)
halogen, (c) C.sub.1-C.sub.6 alkyl, (d) --XR.sub.11, wherein X is
selected from O, C(.dbd.O), S, S.dbd.O, or S(.dbd.O).sub.2, (e)
--C(.dbd.O)N(R.sub.12)(R.sub.13), (f)
--N(R.sub.11)(R.sub.12)(R.sub.13), (g) --N--C(.dbd.O)--R.sub.11,
and (h) --N--C(.dbd.O)O--R.sub.11, wherein R.sub.11, R.sub.12, and
R.sub.13 are independently selected from the group consisting of
hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.6-C.sub.10 aryl, aralkyl, and
C.sub.3-C.sub.10 heteroaryl; m is 1, 2, or 3; R.sub.3 is selected
from (a) hydrogen, (b) C.sub.1-C.sub.4 alkyl optionally substituted
with a carboxylic acid, carboxylate, or carboxylate C.sub.1-C.sub.8
ester group (--CO.sub.2H, --CO.sub.2.sup.-,
--C(.dbd.O)OC.sub.1-C.sub.8), an amide optionally substituted with
an alkylheteroaryl group, or a heteroaryl group; Z is an acyl group
selected from the group (a) ##STR00088## and (b) ##STR00089##
wherein Y is hydrogen, heterocycle, --NH.sub.2, or C.sub.1-C.sub.4
alkyl; R.sub.4 is selected from (i) C.sub.1-C.sub.12 alkyl, (ii)
C.sub.1-C.sub.6 heteroalkyl optionally substituted with
C.sub.1-C.sub.6 alkyl, (iii) C.sub.3-C.sub.6 cycloalkyl, (iv)
C.sub.6-C.sub.10 aryl, (v) heterocyclyl, (vi) C.sub.3-C.sub.10
heteroaryl, (vii) aralkyl, and (viii) heteroalkylaryl; R.sub.5 is
heteroaryl or --C(.dbd.O)--R.sub.10, wherein R.sub.10 is selected
from (i) C.sub.1-C.sub.12 alkyl optionally substituted with
C.sub.6-C.sub.10 aryl, C.sub.1-C.sub.10 heteroaryl, amino, or
carboxylic acid, (ii) C.sub.1-C.sub.10 heteroalkyl optionally
substituted with C.sub.1-C.sub.6 alkyl or carboxylic acid, (iii)
C.sub.3-C.sub.6 cycloalkyl optionally substituted with
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.6-C.sub.10
aryl, optionally substituted C.sub.3-C.sub.10 heteroaryl, amino, or
carboxylic acid, (iv) C.sub.6-C.sub.10 aryl optionally substituted
with C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.6-C.sub.10
aryl, optionally substituted C.sub.3-C.sub.10 heteroaryl, amino, or
carboxylic acid, (v) heterocyclyl, (vi) C.sub.3-C.sub.10
heteroaryl, (vii) aralkyl, and (viii) heteroalkylaryl.
2. The compound of claim 1, its stereoisomers, tautomers, and
pharmaceutically acceptable salts thereof, wherein: R.sub.1 is a
heteroaryl group selected from (a) 1,2,3-triazolyl, and (b)
1,2,3,4-tetrazolyl; n is 1; R.sub.2a, R.sub.2b, and R.sub.2c are
hydrogen; R.sub.3 is selected from (a) hydrogen, (b)
C.sub.1-C.sub.4 alkyl optionally substituted with a carboxylic
acid, carboxylate, or carboxylate C.sub.1-C.sub.8 ester group
(--CO.sub.2H, --CO.sub.2.sup.-, --C(.dbd.O)OC.sub.1-C.sub.8), an
amide optionally substituted with an alkylheteroaryl group, or a
heteroaryl group; Z is an acyl group selected from the group (a)
##STR00090## and (b) ##STR00091##
3. The compound of claim 1, its stereoisomers, tautomers, and
pharmaceutically acceptable salts thereof, wherein: R.sub.1 is
tetrazole or triazole; n is 1; R.sub.3 is hydrogen, C.sub.1-C.sub.4
alkyl substituted with a carboxylic acid or carboxylate group,
C.sub.1-C.sub.4 alkyl substituted with an amide optionally
substituted with an alkylheteroaryl group, or a heteroaryl group;
and Z is ##STR00092##
4. The compound of claim 1, its stereoisomers, tautomers, and
pharmaceutically acceptable salts thereof, wherein: R.sub.1 is
tetrazole or triazole; n is 1; R.sub.3 is hydrogen, or
C.sub.1-C.sub.4 alkyl substituted with a carboxylic acid or
carboxylate group, an amide optionally substituted with an
alkylheteroaryl group, or a heteroaryl group; and Z is ##STR00093##
wherein R.sub.4 is selected from (i) C.sub.1-C.sub.12 alkyl, (ii)
C.sub.3-C.sub.6 cycloalkyl, (iii) C.sub.6-C.sub.10 aryl, and (iv)
C.sub.3-C.sub.10 heteroaryl; R.sub.5 is --C(.dbd.O)--R.sub.10,
wherein R.sub.10 is selected from (i) C.sub.1-C.sub.12 alkyl
optionally substituted with C.sub.6-C.sub.10 aryl, C.sub.1-C.sub.10
heteroaryl, amino, or carboxylic acid, (ii) C.sub.1-C.sub.10
heteroalkyl optionally substituted with C.sub.1-C.sub.6 alkyl or
carboxylic acid, (iii) C.sub.3-C.sub.6 cycloalkyl optionally
substituted with C.sub.1-C.sub.6 alkyl, optionally substituted
C.sub.6-C.sub.10 aryl, optionally substituted C.sub.3-C.sub.10
heteroaryl, amino, or carboxylic acid, (iv) C.sub.6-C.sub.10 aryl
optionally substituted with C.sub.1-C.sub.6 alkyl, optionally
substituted C.sub.6-C.sub.10 aryl, optionally substituted
C.sub.3-C.sub.10 heteroaryl, amino, or carboxylic acid, (v)
C.sub.3-C.sub.10 heteroaryl; and Y is hydrogen, C.sub.1-C.sub.4
alkyl, or --NH.sub.2.
5. A compound having Formula (II): ##STR00094## its stereoisomers,
tautomers, and pharmaceutically acceptable salts thereof, wherein:
R.sub.1 is a heteroaryl group selected from (a) 1,2,3-triazolyl,
and (b) 1,2,3,4-tetrazolyl; R.sub.3 is selected from (a) hydrogen,
(b) C.sub.1-C.sub.4 alkyl optionally substituted with a carboxylic
acid, carboxylate, or carboxylate C.sub.1-C.sub.8 ester group
(--CO.sub.2H, --CO.sub.2--, --C(.dbd.O)OC.sub.1-C.sub.8), an amide
optionally substituted with an alkylheteroaryl group, or a
heteroaryl group; R.sub.4 is selected from (i) C.sub.1-C.sub.12
alkyl, (ii) C.sub.1-C.sub.6 heteroalkyl optionally substituted with
C.sub.1-C.sub.6 alkyl, (iii) C.sub.3-C.sub.6 cycloalkyl, (iv)
C.sub.6-C.sub.10 aryl, (v) heterocyclyl, (vi) C.sub.3-C.sub.10
heteroaryl, (vii) aralkyl, and (viii) heteroalkylaryl; and R.sub.10
is selected from (i) C.sub.1-C.sub.12 alkyl optionally substituted
with C.sub.6-C.sub.10 aryl, C.sub.1-C.sub.10 heteroaryl, amino, or
carboxylic acid, (ii) C.sub.1-C.sub.10 heteroalkyl optionally
substituted with C.sub.1-C.sub.6 alkyl or carboxylic acid, (iii)
C.sub.3-C.sub.6 cycloalkyl optionally substituted with
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.6-C.sub.10
aryl, optionally substituted C.sub.3-C.sub.10 heteroaryl, amino, or
carboxylic acid, (iv) C.sub.6-C.sub.10 aryl optionally substituted
with C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.6-C.sub.10
aryl, optionally substituted C.sub.3-C.sub.10 heteroaryl, amino, or
carboxylic acid, (v) heterocyclyl, (vi) C.sub.3-C.sub.10
heteroaryl, (vii) aralkyl, and (viii) heteroalkylaryl.
6. The compound of claim 5, its stereoisomers, tautomers, and
pharmaceutically acceptable salts thereof, wherein: R.sub.10, when
defined as C.sub.1-C.sub.12 alkyl substituted with a carboxylic
acid or carboxylate group, is: --(CH.sub.2).sub.n--CO.sub.2H, where
n is 2, 3, 4, 5, or 6; optionally wherein one or more single
methylene carbons are substituted with a fluoro, hydroxy, amino,
C.sub.1-C.sub.3 alkyl, or C.sub.6-C.sub.10 aryl group; optionally
wherein one or more single methylene carbons are substituted with
two fluoro or C.sub.1-C.sub.3 alkyl groups; optionally wherein one
or more single methylene carbons are substituted with two alkyl
groups that taken together with the carbon to which they are
attached form a 3, 4, 5, or 6-membered carbocyclic ring; and
optionally wherein adjacent carbon atoms from an unsaturated
carbon-carbon bond or taken form a benzene ring; or
7. The compound of claim 5, its stereoisomers, tautomers, and
pharmaceutically acceptable salts thereof, wherein: wherein
R.sub.10, when defined as C.sub.3-C.sub.6 cycloalkyl substituted
with a carboxylic acid or carboxylate group, is: ##STR00095##
wherein n is 1, 2, 3, or 4; and optionally, for n=3 or 4, wherein
adjacent carbon atoms from an unsaturated carbon-carbon bond.
8. The compound of claim 5, its stereoisomers, tautomers, and
pharmaceutically acceptable salts thereof, wherein: R.sub.1 is
tetrazole or triazole; R.sub.3 is hydrogen; C.sub.1-C.sub.4 alkyl
optionally substituted with a carboxylic acid, carboxylate, or a
carboxylate ester group; or C.sub.1-C.sub.4 alkyl optionally
substituted with an amide, which may be optionally substituted with
an alkylheteroaryl group; R.sub.4 is C.sub.1-C.sub.12 alkyl,
C.sub.3-C.sub.6 cycloalkyl, C.sub.6-C.sub.10 aryl, C.sub.3-C.sub.10
heteroaryl, or heterocyclyl; and R.sub.10 is C.sub.1-C.sub.12 alkyl
optionally substituted with C.sub.6-C.sub.10 aryl, C.sub.1-C.sub.10
heteroaryl, amino, or carboxylic acid.
9. The compound of claim 5, its stereoisomers, tautomers, and
pharmaceutically acceptable salts thereof, wherein: R.sub.1 is
tetrazole or triazole; R.sub.3 is C.sub.1-C.sub.4 alkyl optionally
substituted with a carboxylic acid, carboxylate, or a carboxylate
ester group; R.sub.4 is C.sub.1-C.sub.8 alkyl or C.sub.3-C.sub.6
cycloalkyl; and R.sub.10 is selected from: (a) C.sub.1-C.sub.3
alkyl substituted with C.sub.6-C.sub.10 aryl (e.g., phenyl) or
C.sub.1-C.sub.10 heteroaryl (e.g., triazolyl or tetrazolyl); (b)
--(CH.sub.2).sub.n--CO.sub.2H, where n is 2, 3, 4, 5, or 6; (c)
##STR00096## wherein n is 1, 2, 3, or 4.
10. A compound having Formula (III): ##STR00097## its
stereoisomers, tautomers, and pharmaceutically acceptable salts
thereof, wherein R.sub.1 is a heteroaryl group selected from (a)
1,2,3-triazolyl, and (b) 1,2,3,4-tetrazolyl; R.sub.3 is selected
from (a) hydrogen, (b) C.sub.1-C.sub.4 alkyl optionally substituted
with a carboxylic acid, carboxylate, or carboxylate C.sub.1-C.sub.8
ester group (--CO.sub.2H, --CO.sub.2--,
--C(.dbd.O)OC.sub.1-C.sub.8), an amide optionally substituted with
an alkylheteroaryl group, or a heteroaryl group; Y is hydrogen,
heterocycle, --NH.sub.2, or C.sub.1-C.sub.4 alkyl; and R.sub.4 is
selected from (i) C.sub.1-C.sub.12 alkyl, (ii) C.sub.1-C.sub.6
heteroalkyl optionally substituted with C.sub.1-C.sub.6 alkyl,
(iii) C.sub.3-C.sub.6 cycloalkyl, (iv) C.sub.6-C.sub.10 aryl, (v)
heterocyclyl, (vi) C.sub.3-C.sub.10 heteroaryl, (vii) aralkyl, and
(viii) heteroalkylaryl.
11. The compound of claim 10, its stereoisomers, tautomers, and
pharmaceutically acceptable salts thereof, wherein: R.sub.1 is
tetrazole or triazole; R.sub.3 is hydrogen; C.sub.1-C.sub.4 alkyl
optionally substituted with a carboxylic acid, carboxylate, or a
carboxylate ester group; or C.sub.1-C.sub.4 alkyl optionally
substituted with an amide, which may be optionally substituted with
an alkylheteroaryl group; R.sub.4 is C.sub.1-C.sub.12 alkyl,
C.sub.3-C.sub.6 cycloalkyl, C.sub.6-C.sub.10 aryl, C.sub.3-C.sub.10
heteroaryl, or heterocyclyl; and Y is hydrogen, C.sub.1-C.sub.4
alkyl, or --NH.sub.2.
12. The compound of claim 10, its stereoisomers, tautomers, and
pharmaceutically acceptable salts thereof, wherein: R.sub.1 is
tetrazole or triazole; R.sub.3 is C.sub.1-C.sub.4 alkyl optionally
substituted with a carboxylic acid, carboxylate, or a carboxylate
ester group; R.sub.4 is selected from (i) C.sub.1-C.sub.8 alkyl,
(ii) C.sub.3-C.sub.6 cycloalkyl, (iii) C.sub.6-C.sub.10 aryl, (iv)
C.sub.3-C.sub.10 heteroaryl, and (v) heterocyclyl; and Y is
hydrogen.
13. A compound as shown in Table 1.
14. A pharmaceutical composition, comprising a compound of any one
of claims 1-13 and a pharmaceutically acceptable carrier.
15. A method for inhibiting Granzyme B in a subject, comprising
administering an effective amount of a compound of any one of
claims 1-13 or a pharmaceutical composition of claim 14 to a
subject in need thereof.
16. A method for treating a disease, disorder, or condition
treatable by inhibiting Granzyme B, comprising administering a
therapeutically effective amount of a compound of any one of claims
1-13 or a pharmaceutical composition of claim 14 to a subject in
need thereof.
17. The method of claim 16, wherein the disease, disorder, or
condition treatable by inhibiting Granzyme B is selected from
treating dissection, aneurysm, and atherosclerosis.
18. The method of claim 16, wherein the condition treatable by
inhibiting Granzyme B is a wound and administering the compound or
composition promotes wound healing.
19. The method of any one of claims 15-18, wherein administering
the compound or composition comprises topical administration, oral
administration, and administration by injection.
20. A method for treating cutaneous scleroderma, epidermolysis
bullosa, radiation dermatitis, alopecia areata, or discoid lupus
erythematosus, comprising administering a therapeutically effective
amount of a compound of any one of claims 1-13 or a pharmaceutical
composition of claim 14 to a subject in need thereof.
21. The method of claim 20, wherein administering the compound or
compositin comprises topical administration.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application in a continunation of U.S. application Ser.
No. 15/500,781, filed Jan. 31, 2017, which is a National Stage of
International Application No. PCT/CA2015/050724, filed Jul. 31,
2015, which claims the benefit of U.S. Provisional Application No.
62/032,471, filed Aug. 1, 2014, the disclosures of which are
expressly incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention disclosure relates generally to agents
for treating diseases, disorders, and conditions treatable by
inhibiting Granzyme B, and more specifically to azaindoline
compounds that are inhibitors of Granzyme B.
BACKGROUND OF THE INVENTION
[0003] Granzyme B is a pro-apoptotic serine protease found in the
granules of cytotoxic lymphocytes (CTL) and natural killer (NK)
cells. Granzyme B is released towards target cells, along with the
pore-forming protein, perforin, resulting in its perforin-dependent
internalization into the cytoplasm and subsequent induction of
apoptosis (see, for e.g., Medema et al., Eur. J. Immunol.
27:3492-3498, 1997). However, during aging, inflammation and
chronic disease, Granzyme B can also be expressed and secreted by
other types of immune (e.g., mast cell, macrophage, neutrophil, and
dendritic cells) or non-immune (keratinocyte, chondrocyte) cells
and has been shown to possess extracellular matrix remodeling
activity (Choy et al., Arterioscler. Thromb. Vasc. Biol.
24(12):2245-2250, 2004 and Buzza et al., J. Biol. Chem.
280:23549-23558, 2005).
[0004] Inhibitors of Granzyme B in humans have been limited to (a)
relatively weak, nonspecific inhibitors such as isocoumarins (Odake
et al., (1991), Biochemistry, 30(8), 2217-2227); (b) biological
inhibitors such as serpinB9 (Sun et al., (1996), J. Biol. Chem.,
271(44), 27802-27809); (c) covalently coupled inhibitors such as
aldehydes (Willoughby et al., (2002), Bioorg. Med. Chem. Lett.,
12(16), 2197), halomethyl ketones (Kam et al., (2000), Biochim.
Biophy. Acta, 1477(1-2), 307-323), and phosphonates (Mahrus and
Craik, (2005), Chem. & Biol., 12, 567-77 and Kam et al.,
(2000)); and (d) tricyclic inhibitors (Willoughby et al.,
(2002)).
[0005] Nonspecific inhibitors (such as isocoumarins) are not
sufficiently potent or specific to be effective treatments for
Granzyme-B-related diseases, disorders, and conditions. Likewise,
the use of biological inhibitors such as serpins is limited by the
ability to deliver the inhibitor to the target mammal, the cost of
manufacturing the biological agents, and other, off-target
activities, such as inhibition of other serine proteases such as
human neutrophil elastase (Dahlen et al., (1999), Biochim. Biophys.
Acta, 1451(2-3), 233-41), Caspase-1 (Annaud et al., (1999),
Biochem. J., September 15; 342 Pt3, 655-65; Krieg et al., (2001),
Mol. Endocrinol., 15(11), 1971-82; and Young et al., (2000), J.
Exp. Med., 191(9), 1535-1544); Caspase-4 and Caspase-8 (Annaud et
al., (1999)).
[0006] The tricyclic inhibitors (Willoughby et al. (2001)) also
suffer from synthetic complexity/high manufacturing cost due to the
complex core and accompanying low water solubility.
[0007] Despite the advances in development of Granzyme B
inhibitors, there exists a need for compounds that inhibit Granzyme
B with selectivity, that are relatively simple to manufacture at
low cost, and that do not present drug delivery challenges. The
present invention seeks to fulfill this need and provides further
related advantages.
SUMMARY OF THE INVENTION
[0008] The present invention provides Granzyme B inhibitor
compounds, compositions that include the compounds, and methods for
using the compounds.
[0009] In one aspect of the invention, the invention provides
Granzyme B inhibitor compounds.
[0010] In one embodiment, the invention provides the compounds
having Formula (I):
##STR00001##
[0011] its stereoisomers, tautomers, and pharmaceutically
acceptable salts thereof, wherein:
[0012] R.sub.1 is a heteroaryl group selected from
[0013] (a) 1,2,3-triazolyl, and
[0014] (b) 1,2,3,4-tetrazolyl;
[0015] n is 1 or 2;
[0016] R.sub.2a and R.sub.2b are independently selected from
hydrogen and C1-C6 alkyl;
[0017] R.sub.2c at each occurrence is independently selected
from
[0018] (a) hydrogen,
[0019] (b) halogen,
[0020] (c) C.sub.1-C.sub.6 alkyl,
[0021] (d) --XR.sub.11, wherein X is selected from O, C(.dbd.O), S,
S.dbd.O, or S(.dbd.O).sub.2,
[0022] (e) --C(.dbd.O)N(R.sub.12)(R.sub.13),
[0023] (f) --N(R.sub.11)(R.sub.12)(R.sub.13),
[0024] (g) --N--C(.dbd.O)--R.sub.1, and
[0025] (h) --N--C(.dbd.O)O--R.sub.11,
[0026] wherein R.sub.11, R.sub.12, and R.sub.13 are independently
selected from the group consisting of hydrogen, C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.6-C.sub.10 aryl, aralkyl, and C.sub.3-C.sub.10
heteroaryl;
[0027] m is 1, 2, or 3;
[0028] R.sub.3 is selected from
[0029] (a) hydrogen,
[0030] (b) C.sub.1-C.sub.4 alkyl optionally substituted with a
carboxylic acid, carboxylate, or carboxylate C.sub.1-C.sub.8 ester
group (--CO.sub.2H, --CO.sub.2--, --C(.dbd.O)OC.sub.1-C.sub.8), an
amide optionally substituted with an alkylheteroaryl group, or a
heteroaryl group;
[0031] Z is an acyl group selected from the group
[0032] (a)
##STR00002##
and
[0033] (b)
##STR00003##
[0034] wherein
[0035] Y is hydrogen, heterocycle, --NH.sub.2, or C.sub.1-C.sub.4
alkyl;
[0036] R.sub.4 is selected from
[0037] (i) C.sub.1-C.sub.12 alkyl,
[0038] (ii) C.sub.1-C.sub.6 heteroalkyl optionally substituted with
C.sub.1-C.sub.6 alkyl,
[0039] (iii) C.sub.3-C.sub.6 cycloalkyl,
[0040] (iv) C.sub.6-C.sub.10 aryl,
[0041] (v) heterocyclyl,
[0042] (vi) C.sub.3-C.sub.10 heteroaryl,
[0043] (vii) aralkyl, and
[0044] (viii) heteroalkylaryl;
[0045] R.sub.5 is heteroaryl or --C(.dbd.O)--R.sub.10,
[0046] wherein R.sub.10 is selected from
[0047] (i) C.sub.1-C.sub.12 alkyl optionally substituted with
C.sub.6-C.sub.10 aryl, C.sub.1-C.sub.10 heteroaryl, amino, or
carboxylic acid,
[0048] (ii) C.sub.1-C.sub.10 heteroalkyl optionally substituted
with C.sub.1-C.sub.6 alkyl or carboxylic acid,
[0049] (iii) C.sub.3-C.sub.6 cycloalkyl optionally substituted with
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.6-C.sub.10
aryl, optionally substituted C.sub.3-C.sub.10 heteroaryl, amino, or
carboxylic acid,
[0050] (iv) C.sub.6-C.sub.10 aryl optionally substituted with
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.6-C.sub.10
aryl, optionally substituted C.sub.3-C.sub.10 heteroaryl, amino, or
carboxylic acid,
[0051] (v) heterocyclyl,
[0052] (vi) C.sub.3-C.sub.10 heteroaryl,
[0053] (vii) aralkyl, and
[0054] (viii) heteroalkylaryl.
[0055] In another embodiment, the invention provides compounds
having Formula (II):
##STR00004##
[0056] its stereoisomers, tautomers, and pharmaceutically
acceptable salts thereof, wherein:
[0057] R.sub.1, R.sub.3, R.sub.4, and R.sub.10 are as above for
Formula (I).
[0058] In a further embodiment, the invention provides compounds
having Formula (III):
##STR00005##
[0059] its stereoisomers, tautomers, and pharmaceutically
acceptable salts thereof, wherein R.sub.1, R.sub.3, R.sub.4, and Y
are as defined above for Formula (I).
[0060] In another aspect, the invention provides pharmaceutical
compositions comprising a Granzyme B inhibitor compound of the
invention and a pharmaceutically acceptable carrier.
[0061] In a further aspect of the invention, a method for
inhibiting Granzyme B is provided. In one embodiment, the method
comprises administering an effective amount of a Granzyme B
inhibitor compound of the invention or a pharmaceutical composition
of the invention to a subject in need thereof.
[0062] In a further aspect of the invention, methods for treating a
disease, disorder, or condition treatable by inhibiting Granzyme B
is provided. In one embodiment, the method comprises administering
a therapeutically effective amount of a Granzyme B inhibitor
compound of the invention or a pharmaceutical composition of the
invention to a subject in need thereof. Representative routes of
administration include topical administration, oral administration,
and administration by injection.
[0063] In one embodiment, the invention provides a method for
treating discoid lupus erythematosus (DLE) comprising administering
a therapeutically effective amount of a Granzyme B inhibitor
compound of the invention or a pharmaceutical composition of the
invention to a subject in need thereof. In certain embodiments, the
Granzyme B inhibitor compound of the invention or pharmaceutical
composition is administered topically.
[0064] Cosmetic compositions comprising a Granzyme B inhibitor
compound of the invention and a cosmetically acceptable carrier are
also provided, as are methods for using the compositions to treat,
reduce, and/or inhibit the appearance of ageing in the skin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] FIG. 1 is a schematic illustration of a representative
synthetic pathway for the preparation of representative compounds
of the invention P5-P4-P3-P2-P1 starting from P1.
[0066] FIG. 2 is a schematic illustration of another representative
synthetic pathway for the preparation of representative compounds
of the invention P5-P4-P3-P2-P1 starting from P5.) FIG. 3 is a
schematic illustration of a further representative synthetic
pathway for the preparation of representative compounds of the
invention P5-P4-P3-P2-P1 starting from a component other than P1 or
P5.
DETAILED DESCRIPTION OF THE INVENTION
[0067] The present invention provides Granzyme B inhibitor
compounds, compositions that include the compounds, and methods for
using the compounds. The compounds of the invention effectively
inhibit Granzyme B.
[0068] In one aspect of the invention, the invention provides
Granzyme B inhibitor compounds.
[0069] In one embodiment, the invention provides the compounds
having Formula (I):
##STR00006##
[0070] its stereoisomers, tautomers, and pharmaceutically
acceptable salts thereof, wherein:
[0071] R.sub.1 is a heteroaryl group selected from
[0072] (a) 1,2,3-triazolyl, and
[0073] (b) 1,2,3,4-tetrazolyl;
[0074] n is 1 or 2;
[0075] R.sub.2a and R.sub.2b are independently selected from
hydrogen and C1-C6 alkyl;
[0076] R.sub.2c at each occurrence is independently selected
from
[0077] (a) hydrogen,
[0078] (b) halogen,
[0079] (c) C.sub.1-C.sub.6 alkyl,
[0080] (d) --XR.sub.11, wherein X is selected from O, C(.dbd.O), S,
S.dbd.O, or S(.dbd.O).sub.2,
[0081] (e) --C(.dbd.O)N(R.sub.12)(R.sub.13),
[0082] (f) --N(R.sub.11)(R.sub.12)(R.sub.13),
[0083] (g) --N--C(.dbd.O)--R.sub.1, and
[0084] (h) --N--C(.dbd.O)O--R.sub.11,
[0085] wherein R.sub.11, R.sub.12, and R.sub.13 are independently
selected from the group consisting of hydrogen, C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.6 heteroalkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.6-C.sub.10 aryl, aralkyl, and C.sub.3-C.sub.10
heteroaryl;
[0086] m is 1, 2, or 3;
[0087] R.sub.3 is selected from
[0088] (a) hydrogen,
[0089] (b) C.sub.1-C.sub.4 alkyl optionally substituted with a
carboxylic acid, carboxylate, or carboxylate C.sub.1-C.sub.8 ester
group (--CO.sub.2H, --CO.sub.2.sup.-, --C(.dbd.O)OC.sub.1-C.sub.8),
an amide optionally substituted with an alkylheteroaryl group, or a
heteroaryl group;
[0090] Z is an acyl group selected from the group
[0091] (a)
##STR00007##
and
[0092] (b)
##STR00008##
[0093] wherein
[0094] Y is hydrogen, heterocycle, --NH.sub.2, or C.sub.1-C.sub.4
alkyl;
[0095] R.sub.4 is selected from
[0096] (i) C.sub.1-C.sub.12 alkyl,
[0097] (ii) C.sub.1-C.sub.6 heteroalkyl optionally substituted with
C.sub.1-C.sub.6 alkyl,
[0098] (iii) C.sub.3-C.sub.6 cycloalkyl,
[0099] (iv) C.sub.6-C.sub.10 aryl,
[0100] (v) heterocyclyl,
[0101] (vi) C.sub.3-C.sub.10 heteroaryl,
[0102] (vii) aralkyl, and
[0103] (viii) heteroalkylaryl;
[0104] R.sub.5 is heteroaryl or --C(.dbd.O)--R.sub.10,
[0105] wherein R.sub.10 is selected from
[0106] (i) C.sub.1-C.sub.12 alkyl optionally substituted with
C.sub.6-C.sub.10 aryl, C.sub.1-C.sub.10 heteroaryl, amino, or
carboxylic acid,
[0107] (ii) C.sub.1-C.sub.10 heteroalkyl optionally substituted
with C.sub.1-C.sub.6 alkyl or carboxylic acid,
[0108] (iii) C.sub.3-C.sub.6 cycloalkyl optionally substituted with
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.6-C.sub.10
aryl, optionally substituted C.sub.3-C.sub.10 heteroaryl, amino, or
carboxylic acid,
[0109] (iv) C.sub.6-C.sub.10 aryl optionally substituted with
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.6-C.sub.10
aryl, optionally substituted C.sub.3-C.sub.10 heteroaryl, amino, or
carboxylic acid, (v) heterocyclyl,
[0110] (vi) C.sub.3-C.sub.10 heteroaryl,
[0111] (vii) aralkyl, and
[0112] (viii) heteroalkylaryl.
[0113] In another embodiment, the invention provides compounds
having Formula (I), its stereoisomers, tautomers, and
pharmaceutically acceptable salts thereof, wherein:
[0114] R.sub.1 is a heteroaryl group selected from
[0115] (a) 1,2,3-triazolyl, and
[0116] (b) 1,2,3,4-tetrazolyl;
[0117] n is 1;
[0118] R.sub.2a, R.sub.2b, and R.sub.2c are hydrogen;
[0119] R.sub.3 is selected from
[0120] (a) hydrogen,
[0121] (b) C.sub.1-C.sub.4 alkyl optionally substituted with a
carboxylic acid, carboxylate, or carboxylate C.sub.1-C.sub.8 ester
group (--CO.sub.2H, --CO.sub.2.sup.-, --CO.sub.2C.sub.1-C.sub.8),
an amide optionally substituted with an alkylheteroaryl group, or a
heteroaryl group;
[0122] Z is an acyl group selected from the group
[0123] (a)
##STR00009##
and
[0124] (b)
##STR00010##
[0125] wherein R.sub.4, R.sub.5, and Y are as described above.
[0126] In further embodiments, the invention provides compounds
having Formula (I), its stereoisomers, tautomers, and
pharmaceutically acceptable salts thereof, wherein:
[0127] R.sub.1 is tetrazole or triazole; n is 1; R.sub.3 is
hydrogen, C.sub.1-C.sub.4 alkyl substituted with a carboxylic acid
or carboxylate group, C.sub.1-C.sub.4 alkyl substituted with an
amide optionally substituted with an alkylheteroaryl group, or a
heteroaryl group; and Z is
##STR00011##
and
[0128] R.sub.1 is tetrazole or triazole; n is 1; R.sub.3 is
hydrogen, or C.sub.1-C.sub.4 alkyl substituted with a carboxylic
acid or carboxylate group, an amide optionally substituted with an
alkylheteroaryl group, or a heteroaryl group; and Z is
##STR00012##
[0129] wherein
[0130] R.sub.4 is selected from
[0131] (i) C.sub.1-C.sub.12 alkyl,
[0132] (ii) C.sub.3-C.sub.6 cycloalkyl,
[0133] (iii) C.sub.6-C.sub.10 aryl, and
[0134] (iv) C.sub.3-C.sub.10 heteroaryl;
[0135] R.sub.5 is --C(.dbd.O)--R.sub.10, wherein R.sub.10 is
selected from
[0136] (i) C.sub.1-C.sub.12 alkyl optionally substituted with
C.sub.6-C.sub.10 aryl, C.sub.1-C.sub.10 heteroaryl, amino, or
carboxylic acid,
[0137] (ii) C.sub.1-C.sub.10 heteroalkyl optionally substituted
with C.sub.1-C.sub.6 alkyl or carboxylic acid,
[0138] (iii) C.sub.3-C.sub.6 cycloalkyl optionally substituted with
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.6-C.sub.10
aryl, optionally substituted C.sub.3-C.sub.10 heteroaryl, amino, or
carboxylic acid,
[0139] (iv) C.sub.6-C.sub.10 aryl optionally substituted with
C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.6-C.sub.10
aryl, optionally substituted C.sub.3-C.sub.10 heteroaryl, amino, or
carboxylic acid,
[0140] (v) C.sub.3-C.sub.10 heteroaryl; and
[0141] Y is hydrogen, C.sub.1-C.sub.4 alkyl, or --NH.sub.2.
[0142] In another embodiment, the invention provides compounds
having Formula (II):
##STR00013##
[0143] its stereoisomers, tautomers, and pharmaceutically
acceptable salts thereof, wherein:
[0144] R.sub.1, R.sub.3, R.sub.4, and R.sub.10 are as above for
Formula (I).
[0145] In certain embodiments, R.sub.10, when defined as
C.sub.1-C.sub.12 alkyl substituted with a carboxylic acid or
carboxylate group, is:
[0146] --(CH.sub.2).sub.n--CO.sub.2H, where n is 2, 3, 4, 5, or
6;
[0147] optionally wherein one or more single methylene carbons are
substituted with a fluoro, hydroxy, amino, C.sub.1-C.sub.3 alkyl
(e.g., methyl), or C.sub.6-C.sub.10 aryl group;
[0148] optionally wherein one or more single methylene carbons are
substituted with two fluoro (e.g., difluoro, perfluoro) or
C.sub.1-C.sub.3 alkyl (e.g., gem-dimethyl) groups;
[0149] optionally wherein one or more single methylene carbons are
substituted with two alkyl groups that taken together with the
carbon to which they are attached form a 3, 4, 5, or 6-membered
carbocyclic ring (e.g., spiro groups such as cyclopropyl,
cyclobutyl, cyclopentyl, and cyclohexyl); and
[0150] optionally wherein adjacent carbon atoms from an unsaturated
carbon-carbon bond (e.g., alkenyl such as --CH.dbd.CH--) or taken
form a benzene ring (e.g., 1,2-, 1,3-, and 1,4-phenylene); or
[0151] wherein R.sub.10, when defined as C.sub.3-C.sub.6 cycloalkyl
substituted with a carboxylic acid or carboxylate group, is:
##STR00014##
wherein n is 1, 2, 3, or 4; and optionally, for n=3 or 4, wherein
adjacent carbon atoms from an unsaturated carbon-carbon bond (e.g.,
cyclopentenyl or cyclohexenyl).
[0152] In certain embodiments, the invention provides compounds
having Formula (II), its stereoisomers, tautomers, and
pharmaceutically acceptable salts thereof, wherein:
[0153] R.sub.1 is tetrazole or triazole;
[0154] R.sub.3 is hydrogen; C.sub.1-C.sub.4 alkyl optionally
substituted with a carboxylic acid, carboxylate, or a carboxylate
ester group; or C.sub.1-C.sub.4 alkyl optionally substituted with
an amide, which may be optionally substituted with an
alkylheteroaryl group;
[0155] R.sub.4 is C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.6
cycloalkyl, C.sub.6-C.sub.10 aryl, C.sub.3-C.sub.10 heteroaryl, or
heterocyclyl; and
[0156] R.sub.10 is C.sub.1-C.sub.12 alkyl optionally substituted
with C.sub.6-C.sub.10 aryl, C.sub.1-C.sub.10 heteroaryl, amino, or
carboxylic acid.
[0157] In further embodiments, the invention provides compounds
having Formula (II), its stereoisomers, tautomers, and
pharmaceutically acceptable salts thereof, wherein:
[0158] R.sub.1 is tetrazole or triazole;
[0159] R.sub.3 is C.sub.1-C.sub.4 alkyl optionally substituted with
a carboxylic acid, carboxylate, or a carboxylate ester group;
[0160] R.sub.4 is C.sub.1-C.sub.8 alkyl or C.sub.3-C.sub.6
cycloalkyl; and
[0161] R.sub.10 is selected from:
[0162] (a) C.sub.1-C.sub.3 alkyl substituted with C.sub.6-C.sub.10
aryl (e.g., phenyl) or C.sub.1-C.sub.10 heteroaryl (e.g., triazolyl
or tetrazolyl);
[0163] (b) --(CH.sub.2).sub.n--CO.sub.2H, where n is 2, 3, 4, 5, or
6;
[0164] (c)
##STR00015##
wherein n is 1, 2, 3, or 4.
[0165] Representative compounds of Formula (II) include A1, C1-C24,
C26, C28-C40.
[0166] In a further embodiment, the invention provides compounds
having Formula (III):
##STR00016##
[0167] its stereoisomers, tautomers, and pharmaceutically
acceptable salts thereof, wherein R.sub.1, R.sub.3, R.sub.4, and Y
are as defined above for Formula (I).
[0168] In certain embodiments, the invention provides compounds
having Formula (III), its stereoisomers, tautomers, and
pharmaceutically acceptable salts thereof, wherein:
[0169] R.sub.1 is tetrazole or triazole;
[0170] R.sub.3 is hydrogen; C.sub.1-C.sub.4 alkyl optionally
substituted with a carboxylic acid, carboxylate, or a carboxylate
ester group; or C.sub.1-C.sub.4 alkyl optionally substituted with
an amide, which may be optionally substituted with an
alkylheteroaryl group;
[0171] R.sub.4 is C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.6
cycloalkyl, C.sub.6-C.sub.10 aryl, C.sub.3-C.sub.10 heteroaryl, or
heterocyclyl; and
[0172] Y is hydrogen, C.sub.1-C.sub.4 alkyl, or --NH.sub.2.
[0173] In further embodiments, the invention provides compounds
having Formula (III), its stereoisomers, tautomers, and
pharmaceutically acceptable salts thereof, wherein:
[0174] R.sub.1 is tetrazole or triazole;
[0175] R.sub.3 is C.sub.1-C.sub.4 alkyl optionally substituted with
a carboxylic acid, carboxylate, or a carboxylate ester group;
[0176] R.sub.4 is selected from
[0177] (i) C.sub.1-C.sub.8 alkyl (e.g., methyl, ethyl, n-propyl,
i-propyl),
[0178] (ii) C.sub.3-C.sub.6 cycloalkyl (i.e., cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl),
[0179] (iii) C.sub.6-C.sub.10 aryl (e.g., phenyl),
[0180] (iv) C.sub.3-C.sub.10 heteroaryl (e.g., thiophenyl), and
[0181] (v) heterocyclyl (e.g., morpholinyl); and
[0182] Y is hydrogen.
[0183] Representative compounds of Formula (III) include C20, C25,
C27, and C31-C40.
[0184] For the compounds of Formulae (I), (II), or (III),
representative substituents R.sub.3 include the following:
##STR00017##
[0185] For the compounds of Formulae (I), (II), or (III),
representative substituents R.sub.4 include the following:
##STR00018##
[0186] For the compounds of Formulae (I), (II), or (III),
representative substituents R.sub.5 include the following:
##STR00019## ##STR00020## ##STR00021## ##STR00022##
[0187] Each of the inhibitor compounds of the invention contain
asymmetric carbon centers and give rise to stereoisomers (i.e.,
optical isomers such as diastereomers and enantiomers). It will be
appreciated that the present invention includes such diastereomers
as well as their racemic and resolved enantiomerically pure forms.
It will also be appreciated that in certain configurations, the
relative stereochemistry of certain groups may be depicted as "cis"
or "trans" when absolute stereochemistry is not shown.
[0188] Some of the compounds described herein contain olefinic
double bonds, and unless specified otherwise, are meant to include
both E and Z geometric isomers.
[0189] Certain of the compounds of the invention may exist in one
or more tautomeric forms (e.g., acid or basic forms depending on pH
environment). It will be appreciated that the compounds of the
invention include their tautomeric forms (i.e., tautomers).
[0190] When the compounds of the present invention are basic, salts
may be prepared from pharmaceutically acceptable non-toxic acids,
including inorganic and organic acids. Examples of such acids
include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric,
ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic,
hydrochloric, isethionic, lactic, maleic, malic, mandelic,
methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric,
succinic, sulfuric, tartaric, and p-toluenesulfonic acids.
[0191] The invention is described using the following definitions
unless otherwise indicated.
[0192] As used herein, the term "alkyl" refers to a saturated or
unsaturated, branched, straight-chain or cyclic monovalent
hydrocarbon group derived by the removal of one hydrogen atom from
a single carbon atom of a parent alkane, alkene, or alkyne.
Representative alkyl groups include methyl; ethyls such as ethanyl,
ethenyl, ethynyl; propyls such as propan-1-yl, propan-2-yl,
cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl
(allyl), cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl,
and prop-2-yn-1-yl; butyls such as butan-1-yl, butan-2-yl,
2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,
but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,
but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl,
buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl,
cyclobuta-1,3-dien-1-yl, but-1-yn-1-yl, but-1-yn-3-yl, and
but-3-yn-1-yl; and the like. Where a specific level of saturation
is intended, the expressions "alkanyl," "alkenyl," and "alkynyl"
are used. Alkyl groups include cycloalkyl groups. The term
"cycloalkyl" refers to mono-, bi-, and tricyclic alkyl groups
having the indicated number of carbon atoms. Representative
cycloalkyl groups include cyclopropyl, cyclopentyl, cycloheptyl,
adamantyl, cyclododecylmethyl, and 2-ethyl-1-bicyclo[4.4.0]decyl
groups. The alkyl group may be unsubstituted or substituted as
described below.
[0193] "Alkanyl" refers to a saturated branched, straight-chain, or
cyclic alkyl group. Representative alkanyl groups include methanyl;
ethanyl; propanyls such as propan-1-yl, propan-2-yl(isopropyl), and
cyclopropan-1-yl; butanyls such as butan-1-yl, butan-2-yl
(sec-butyl), 2-methyl-propan-1-yl(isobutyl),
2-methyl-propan-2-yl(t-butyl), and cyclobutan-1-yl; and the like.
The alkanyl group may be substituted or unsubstituted.
Representative alkanyl group substituents include
[0194] --R.sub.14, --OR.sub.14, --SR.sub.14,
--NR.sub.14(R.sub.15),
[0195] --X, --CX.sub.3, --CN, --NO.sub.2,
[0196] --C(.dbd.O)R.sub.14, --C(.dbd.O)OR.sub.14,
--C(.dbd.O)NR.sub.14(R.sub.15), --C(.dbd.O)SR.sub.14,
[0197] --C(.dbd.NR.sub.14)R.sub.14, --C(.dbd.NR.sub.14)OR.sub.14,
--C(.dbd.NR.sub.14)NR.sub.14(R.sub.15),
--C(.dbd.NR.sub.14)SR.sub.14,
[0198] --C(.dbd.S)R.sub.14, --C(.dbd.S)OR.sub.14,
--C(.dbd.S)NR.sub.14(R.sub.15), --C(.dbd.S)SR.sub.14,
[0199] --NR.sub.14C(.dbd.O)NR.sub.14(R.sub.15),
--NR.sub.14(.dbd.NR.sub.14)NR.sub.14(R.sub.15),
--NR.sub.14C(.dbd.S)NR.sub.14(R.sub.15),
[0200] --S(.dbd.O).sub.2R.sub.14, --S(.dbd.O).sub.2OR.sub.14,
--S(.dbd.O).sub.2NR.sub.14(R.sub.15),
[0201] --OC(.dbd.O)R.sub.14, --OC(.dbd.O)OR.sub.14,
--OC(.dbd.O)NR.sub.14(R.sub.15), --OC(.dbd.O)SR.sub.14,
[0202] --OS(.dbd.O).sub.2OR.sub.14,
--OS(.dbd.O).sub.2NR.sub.14(R.sub.15), and
[0203] --OP(.dbd.O).sub.2(OR.sub.14),
[0204] wherein each X is independently a halogen; and R.sub.14 and
R.sub.15 are independently hydrogen, C1-C6 alkyl, C6-C14 aryl,
arylalkyl, C3-C10 heteroaryl, and heteroarylalkyl, as defined
herein.
[0205] In certain embodiments, two hydrogen atoms on a single
carbon atom can be replaced with .dbd.O, .dbd.NR.sub.12, or
.dbd.S.
[0206] "Alkenyl" refers to an unsaturated branched, straight-chain,
cyclic alkyl group, or combinations thereof having at least one
carbon-carbon double bond derived by the removal of one hydrogen
atom from a single carbon atom of a parent alkene. The group may be
in either the cis or trans conformation about the double bond(s).
Representative alkenyl groups include ethenyl; propenyls such as
prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl),
prop-2-en-2-yl, and cycloprop-1-en-1-yl; cycloprop-2-en-1-yl;
butenyls such as but-1-en-1-yl, but-1-en-2-yl,
2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl,
but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,
cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, and
cyclobuta-1,3-dien-1-yl; and the like. The alkenyl group may be
substituted or unsubstituted. Representative alkenyl group
substituents include
[0207] --R.sub.14,
[0208] --X, --CX.sub.3, --CN,
[0209] --C(.dbd.O)R.sub.14, --C(.dbd.O)OR.sub.14,
--C(.dbd.O)NR.sub.14(R.sub.15), --C(.dbd.O)SR.sub.14,
[0210] --C(.dbd.NR.sub.14)R.sub.14, --C(.dbd.NR.sub.14)OR.sub.14,
--C(.dbd.NR.sub.14)NR.sub.14(R.sub.15),
--C(.dbd.NR.sub.14)SR.sub.14,
[0211] --C(.dbd.S)R.sub.14, --C(.dbd.S)OR.sub.14,
--C(.dbd.S)NR.sub.14(R.sub.15), --C(.dbd.S)SR.sub.14,
[0212] wherein each X is independently a halogen; and R.sub.14 and
R.sub.15 are independently hydrogen, C1-C6 alkyl, C6-C14 aryl,
arylalkyl, C3-C10 heteroaryl, and heteroarylalkyl, as defined
herein.
[0213] "Alkynyl" refers to an unsaturated branched, straight-chain,
or cyclic alkyl group having at least one carbon-carbon triple bond
derived by the removal of one hydrogen atom from a single carbon
atom of a parent alkyne. Representative alkynyl groups include
ethynyl; propynyls such as prop-1-yn-1-yl and prop-2-yn-1-yl;
butynyls such as but-1-yn-1-yl, but-1-yn-3-yl, and but-3-yn-1-yl;
and the like. The alkynyl group may be substituted or
unsubstituted. Representative alkynyl group substituents include
those as described above for alkenyl groups.
[0214] The term "haloalkyl" refers to an alkyl group as defined
above having the one or more hydrogen atoms replaced by a halogen
atom. Representative haloalkyl groups include halomethyl groups
such as chloromethyl, fluoromethyl, and trifluoromethyl groups; and
haloethyl groups such as chloroethyl, fluoroethyl, and
perfluoroethyl groups.
[0215] The term "heteroalkyl" refers to an alkyl group having the
indicated number of carbon atoms and where one or more of the
carbon atoms is replaced with a heteroatom selected from O, N, or
S. Where a specific level of saturation is intended, the
expressions "heteroalkanyl," "heteroalkenyl," and "heteroalkynyl"
are used. Representative heteroalkyl groups include ether, amine,
and thioether groups. Heteroalkyl groups include heterocyclyl
groups. The term "heterocyclyl" refers to a 5- to 10-membered
non-aromatic mono- or bicyclic ring containing 1-4 heteroatoms
selected from O, S, and N. Representative heterocyclyl groups
include pyrrolidinyl, piperidinyl, piperazinyl, tetrahydrofuranyl,
tetrahydropuranyl, and morpholinyl groups. The heteroalkyl group
may be substituted or unsubstituted. Representative heteroalkyl
substituents include
[0216] --R.sub.14, --OR.sub.14, --SR.sub.14,
--NR.sub.14(R.sub.15),
[0217] --X, --CX.sub.3, --CN, --NO.sub.2,
[0218] --C(.dbd.O)R.sub.14, --C(.dbd.O)OR.sub.14,
--C(.dbd.O)NR.sub.14(R.sub.15), --C(.dbd.O)SR.sub.14,
[0219] --C(.dbd.NR.sub.14)R.sub.14, --C(.dbd.NR.sub.14)OR.sub.14,
--C(.dbd.NR.sub.14)NR.sub.14(R.sub.15),
--C(.dbd.NR.sub.14)SR.sub.14,
[0220] --C(.dbd.S)R.sub.14, --C(.dbd.S)OR.sub.14,
--C(.dbd.S)NR.sub.14(R.sub.15), --C(.dbd.S)SR.sub.14,
[0221] --NR.sub.14C(.dbd.O)NR.sub.14(R.sub.15),
--NR.sub.14(.dbd.NR.sub.14)NR.sub.14(R.sub.15),
--NR.sub.14C(.dbd.S)NR.sub.14(R.sub.15),
[0222] --S(.dbd.O).sub.2R.sub.14, --S(.dbd.O).sub.2OR.sub.14,
--S(.dbd.O).sub.2NR.sub.14(R.sub.15),
[0223] --OC(.dbd.O)R.sub.14, --OC(.dbd.O)OR.sub.14,
--OC(.dbd.O)NR.sub.14(R.sub.15), --OC(.dbd.O)SR.sub.14,
[0224] --OS(.dbd.O).sub.2OR.sub.14,
--OS(.dbd.O).sub.2NR.sub.14(R.sub.15), and
[0225] --OP(.dbd.O).sub.2(OR.sub.14),
[0226] wherein each X is independently a halogen; and R.sub.14 and
R.sub.15 are independently hydrogen, C1-C6 alkyl, C6-C14 aryl,
arylalkyl, C3-C10 heteroaryl, and heteroarylalkyl, as defined
herein.
[0227] In certain embodiments, two hydrogen atoms on a single
carbon atom can be replaced with .dbd.O, .dbd.NR.sub.12, or
.dbd.S.
[0228] The term "alkoxy" refers to an alkyl group as described
herein bonded to an oxygen atom. Representative C1-C3 alkoxy groups
include methoxy, ethoxy, propoxy, and isopropoxy groups.
[0229] The term "alkylamino" refers an alkyl group as described
herein bonded to a nitrogen atom. The term "alkylamino" includes
monoalkyl- and dialkylaminos groups. Representative C1-C6
alkylamino groups include methylamino, dimethylamino, ethylamino,
methylethylamino, diethylamino, propylamino, and isopropylamino
groups.
[0230] The term "alkylthio" refers an alkyl group as described
herein bonded to a sulfur atom. Representative C1-C6 alkylthio
groups include methylthio, propylthio, and isopropylthio
groups.
[0231] The term "aryl" refers to a monovalent aromatic hydrocarbon
group derived by the removal of one hydrogen atom from a single
carbon atom of a parent aromatic ring system. Suitable aryl groups
include groups derived from aceanthrylene, acenaphthylene,
acephenanthrylene, anthracene, azulene, benzene, chrysene,
coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene,
as-indacene, s-indacene, indane, indene, naphthalene, octacene,
octaphene, octalene, ovalene, penta-2,4-diene, pentacene,
pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,
pleiadene, pyrene, pyranthrene, rubicene, triphenylene,
trinaphthalene, and the like. In certain embodiments, the aryl
group is a C5-C14 aryl group. In other embodiments, the aryl group
is a C5-C10 aryl group. The number of carbon atoms specified refers
to the number of carbon atoms in the aromatic ring system.
Representative aryl groups are phenyl, naphthyl, and
cyclopentadienyl. The aryl group may be substituted or
unsubstituted. Representative aryl group substituents include
[0232] --R.sub.14, --OR.sub.14, --SR.sub.14,
--NR.sub.14(R.sub.15),
[0233] --X, --CX.sub.3, --CN, --NO.sub.2,
[0234] --C(.dbd.O)R.sub.14, --C(.dbd.O)OR.sub.14,
--C(.dbd.O)NR.sub.14(R.sub.15), --C(.dbd.O)SR.sub.14,
[0235] --C(.dbd.NR.sub.14)R.sub.14, --C(.dbd.NR.sub.14)OR.sub.14,
--C(.dbd.NR.sub.14)NR.sub.14(R.sub.15),
--C(.dbd.NR.sub.14)SR.sub.14,
[0236] --C(.dbd.S)R.sub.14, --C(.dbd.S)OR.sub.14,
--C(.dbd.S)NR.sub.14(R.sub.15), --C(.dbd.S)SR.sub.14,
[0237] --NR.sub.14C(.dbd.O)NR.sub.14(R.sub.15),
--NR.sub.14(.dbd.NR.sub.15)NR.sub.14(R.sub.15),
--NR.sub.14C(.dbd.S)NR.sub.14(R.sub.15),
[0238] --S(.dbd.O).sub.2R.sub.14, --S(.dbd.O).sub.2OR.sub.14,
--S(.dbd.O).sub.2NR.sub.14(R.sub.15),
[0239] --OC(.dbd.O)R.sub.14, --OC(.dbd.O)OR.sub.14,
--OC(.dbd.O)NR.sub.14(R.sub.15), --OC(.dbd.O)SR.sub.14,
[0240] --OS(.dbd.O).sub.2OR.sub.14,
--OS(.dbd.O).sub.2NR.sub.14(R.sub.15), and
[0241] --OP(.dbd.O).sub.2(OR.sub.14),
[0242] wherein each X is independently a halogen; and R.sub.14 and
R.sub.15 are independently hydrogen, C1-C6 alkyl, C6-C14 aryl,
arylalkyl, C3-C10 heteroaryl, and heteroarylalkyl, as defined
herein.
[0243] The term "aralkyl" refers to an alkyl group as defined
herein with an aryl group, optionally substituted, as defined
herein substituted for one of the alkyl group hydrogen atoms.
Suitable aralkyl groups include benzyl, 2-phenylethan-1-yl,
2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,
2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, and
the like. Where specific alkyl moieties are intended, the terms
aralkanyl, aralkenyl, and aralkynyl are used. In certain
embodiments, the aralkyl group is a C6-C20 aralkyl group, (e.g.,
the alkanyl, alkenyl, or alkynyl moiety of the aralkyl group is a
C1-C6 group and the aryl moiety is a C5-C14 group). In other
embodiments, the aralkyl group is a C6-C13 aralkyl group (e.g., the
alkanyl, alkenyl, or alkynyl moiety of the aralkyl group is a C1-C3
group and the aryl moiety is a C5-C10 aryl group. In certain
embodiments, the aralkyl group is a benzyl group.
[0244] The term "heteroaryl" refers to a monovalent heteroaromatic
group derived by the removal of one hydrogen atom from a single
atom of a parent heteroaromatic ring system, which may be
monocyclic or fused ring (i.e., rings that share an adjacent pair
of atoms). A "heteroaromatic" group is a 5- to 14-membered aromatic
mono- or bicyclic ring containing 1-4 heteroatoms selected from O,
S, and N. Representative 5- or 6-membered aromatic monocyclic ring
groups include pyridine, pyrimidine, pyridazine, furan, thiophene,
thiazole, oxazole, and isooxazole. Representative 9- or 10-membered
aromatic bicyclic ring groups include benzofuran, benzothiophene,
indole, pyranopyrrole, benzopyran, quionoline, benzocyclohexyl, and
naphthyridine. Suitable heteroaryl groups include groups derived
from acridine, arsindole, carbazole, .beta.-carboline, chromane,
chromene, cinnoline, furan, imidazole, indazole, indole, indoline,
indolizine, isobenzofuran, isochromene, isoindole, isoindoline,
isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole,
oxazole, perimidine, phenanthridine, phenanthroline, phenazine,
phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,
pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine,
quinazoline, quinoline, quinolizine, quinoxaline, tetrazole,
thiadiazole, thiazole, thiophene, triazole, xanthene, and the like.
In certain embodiments, the heteroaryl group is a 5-14 membered
heteroaryl group. In other embodiments, the heteroaryl group is a
5-10 membered heteroaryl group. Preferred heteroaryl groups are
those derived from thiophene, pyrrole, benzothiophene, benzofuran,
indole, pyridine, quinoline, imidazole, oxazole, and pyrazine. The
heteroaryl group may be substituted or unsubstituted.
Representative heteroaryl group substituents include those
described above for aryl groups.
[0245] The term "heteroarylalkyl" refers to an alkyl group as
defined herein with a heteroaryl group, optionally substituted, as
defined herein substituted for one of the alkyl group hydrogen
atoms. Where specific alkyl moieties are intended, the terms
heteroarylalkanyl, heteroarylalkenyl, or heteroarylalkynyl are
used. In certain embodiments, the heteroarylalkyl group is a 6-20
membered heteroarylalkyl (e.g., the alkanyl, alkenyl or alkynyl
moiety of the heteroarylalkyl is a C1-C6 group and the heteroaryl
moiety is a 5-14-membered heteroaryl group. In other embodiments,
the heteroarylalkyl group is a 6-13 membered heteroarylalkyl (e.g.,
the alkanyl, alkenyl or alkynyl moiety is C1-C3 group and the
heteroaryl moiety is a 5-10-membered heteroaryl group).
[0246] The term "acyl" group refers to the --C(.dbd.O)--R' group,
where R' is selected from optionally substituted alkyl, optionally
substituted aryl, and optionally substituted heteroaryl, as defined
herein.
[0247] The term "halogen" or "halo" refers to fluoro, chloro,
bromo, and iodo groups.
[0248] The term "substituted" refers to a group in which one or
more hydrogen atoms are each independently replaced with the same
or different substituent(s).
[0249] Representative compounds of the invention and related
intermediates were prepared from commercially available starting
materials or starting materials prepared by conventional synthetic
methodologies. Representative compounds of the invention were
prepared according to Methods A to J as described below and
illustrated in FIGS. 1-3. The preparations of certain intermediates
(I-1 to I-12) useful in the preparation of compounds of the
invention are described in the Synthetic Intermediate section
below.
[0250] FIGS. 1-3 present schematic illustrations of representative
synthetic pathways for the preparation of representative compounds
of the invention P5-P4-P3-P2-P1. As used herein, "P5-P4-P3-P2-P1"
refers to compounds of the invention prepared from five (5)
components: P1, P2, P3, P4, and P5. Protected version of the
components useful in the preparation of the compounds of the
invention are designated as, for example, "PG-P2," "PG-P2-P1,"
"PG-P3," and "PG-P3-P2-P1," where "PG" is refers to a protecting
group that allows for the coupling of, for example, P1 to P2 or P3
to P1-P2, and that is ultimately removed to provide, for example,
P1-P2 or P1-P2-P3.
[0251] FIG. 1 is a schematic illustration of another representative
synthetic pathway for the preparation of representative compounds
of the invention P5-P4-P3-P2-P1 starting from P5. In this pathway,
compound P5-P4-P3-P2-P1 is prepared in a stepwise manner starting
with P5 by sequential coupling steps, separated as appropriate by
deprotection steps and other chemical modifications. As shown in
FIG. 1, P5 is coupled with PG-P4 to provide P5-P4-PG, which is then
deprotected to provide P5-P4 and ready for coupling with the next
component, P3-PG. The process is continued with subsequent
couplings PG-P2 with P5-P4-P3 and PG-P1 with P5-P4-P3-P2 to
ultimately provide P5-P4-P3-P2-P1. Example A1 was prepared by this
method.
[0252] FIG. 2 is a schematic illustration of a representative
synthetic pathway for the preparation of representative compounds
of the invention P5-P4-P3-P2-P1 starting from P1. In this pathway,
compound P5-P4-P3-P2-P1 is prepared in a stepwise manner starting
with P1 by sequential coupling steps, separated as appropriate by
deprotection steps and other chemical modifications. As shown in
FIG. 2, P1 is coupled with PG-P2 to provide PG-P2-P1, which is then
deprotected to provide P2-P1 and ready for coupling with the next
component, PG-P3. The process is continued with subsequent
couplings PG-P4 with P3-P2-P1 and PG-P5 with P4-P3-P2-P1 to
ultimately provide P5-P4-P3-P2-P1.
[0253] FIG. 3 is a schematic illustration of a further
representative synthetic pathway for the preparation of
representative compounds of the invention P5-P4-P3-P2-P1 starting
from a component other than P1 or P5. In this pathway, compound
P5-P4-P3-P2-P1 is prepared in a stepwise manner starting with P2 by
sequential coupling steps, separated as appropriate by deprotection
steps and other chemical modifications. As shown in FIG. 3, there
are multiple pathways to P5-P4-P3-P2-P1. Examples C1-C41 were
prepared by this method.
[0254] The preparation of representative compounds and their
characterization are described in Examples A1 and C1-C41. The
structures of representative compounds are set forth in Table 1
TABLE-US-00001 TABLE 1 Representative Compounds. Cmpd # Structure
A1 ##STR00023## C1 ##STR00024## C2 ##STR00025## C3 ##STR00026## C4
##STR00027## C5 ##STR00028## C6 ##STR00029## C7 ##STR00030## C8
##STR00031## C9 ##STR00032## C10 ##STR00033## C11 ##STR00034## C12
##STR00035## C13 ##STR00036## C14 ##STR00037## C15 ##STR00038## C16
##STR00039## C17 ##STR00040## C18 ##STR00041## C19 ##STR00042## C20
##STR00043## C21 ##STR00044## C22 ##STR00045## C23 ##STR00046## C24
##STR00047## C25 ##STR00048## C26 ##STR00049## C27 ##STR00050## C28
##STR00051## C29 ##STR00052## C30 ##STR00053## C31 ##STR00054## C32
##STR00055## C33 ##STR00056## C34 ##STR00057## C35 ##STR00058## C36
##STR00059## C37 ##STR00060## C38 ##STR00061## C39 ##STR00062## C40
##STR00063## C41 ##STR00064##
[0255] A general kinetic enzyme assay useful for determining the
inhibitory activity of the compounds of the invention is described
in Examples D1 and D4.
[0256] A Granzyme B enzymatic inhibition assay is described in
Example D2 and Example D5. The compounds of the invention
identified in Table 1 exhibited Granzyme B inhibitory activity. In
certain embodiments, select compounds exhibited IC.sub.50<50,000
nM. In other embodiments, select compounds exhibited
IC.sub.50<10,000 nM. In further embodiments, select compounds
exhibited IC.sub.50<1,000 nM. In still further embodiments,
select compounds exhibited IC.sub.50<100 nM. In certain
embodiments, select compounds exhibited IC.sub.50 from 10 nM to 100
nM, preferably from 1 nM to 10 nM, more preferably from 0.1 nM to 1
nM, and even more preferably from 0.01 nM to 0.1 nM.
[0257] A caspase enzymatic inhibition assay is described in Example
D3 and Example D6. None of the compounds of the invention tested
demonstrated an ability to significantly inhibit any of the
caspases evaluated at a concentration of 50 .mu.M. In certain
embodiments, the compounds exhibited less than 50% inhibition at 50
.mu.M. In other embodiments, the compounds exhibited greater than
50% inhibition at 50 .mu.M, but less than 10% inhibition at 25
.mu.M. The results demonstrate that select compounds of the
invention selectively inhibit Granzyme B without significantly
inhibiting caspases.
[0258] A cell detachment assay is described in Example D7.
[0259] A fibronectin cleavage assay is described in Example D8.
[0260] A cell adhesion based on fibronectin cleavage assay is
described in Example D9.
[0261] Pharmaceutical Compositions
[0262] The pharmaceutical compositions of the present invention
include an inhibitor compound of the invention (e.g., a compound of
Formulae (I), (II), or (III)) as an active ingredient or a
pharmaceutically acceptable salt thereof in combination with a
pharmaceutically acceptable carrier, and optionally other
therapeutic ingredients.
[0263] The term "pharmaceutically acceptable salts" refers to salts
prepared from pharmaceutically acceptable bases including inorganic
bases and organic bases. Representative salts derived from
inorganic bases include aluminum, ammonium, calcium, copper,
ferric, ferrous, lithium, magnesium, manganic, manganous, ammonium,
potassium, sodium, and zinc salts. Representative salts derived
from pharmaceutically acceptable organic bases include salts of
primary, secondary and tertiary amines, substituted amines
including naturally occurring substituted amines, cyclic amines,
and basic ion exchange resins, such as arginine, betaine, caffeine,
choline, N,N'-dibenzylethylenediamine, diethylamine,
2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,
ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine,
glucosamine, histidine, hydrabamine, isopropylamine, lysine,
methylglucamine, morpholine, piperazine, piperidine, polyamine
resins, procaine, purines, theobromine, triethylamine,
trimethylamine, tripropylamine, and trimethamine.
[0264] Compositions can include one or more carriers acceptable for
the mode of administration of the preparation, be it by topical
administration, lavage, epidermal administration, sub-epidermal
administration, dermal administration, subdermal administration,
transdermal administration, subcutaneous administration, systemic
administration, injection, inhalation, oral, or any other mode
suitable for the selected treatment. Topical administration
includes administration to external body surfaces (e.g., skin) as
well as to internal body surfaces (e.g., mucus membranes for
vaginal or rectal applications by, for example, suppositories).
Suitable carriers are those known in the art for use in such modes
of administration.
[0265] Suitable compositions can be formulated by means known in
the art and their mode of administration and dose determined by a
person of skill in the art. For parenteral administration, the
compound can be dissolved in sterile water or saline or a
pharmaceutically acceptable vehicle used for administration of
non-water soluble compounds. For enteral administration, the
compound can be administered in a tablet, capsule, or dissolved or
suspended in liquid form. The tablet or capsule can be enteric
coated, or in a formulation for sustained release. Many suitable
formulations are known including, polymeric or protein
microparticles encapsulating a compound to be released, ointments,
pastes, gels, hydrogels, foams, creams, powders, lotions, oils,
semi-solids, soaps, medicated soaps, shampoos, medicated shampoos,
sprays, films, or solutions which can be used topically or locally
to administer a compound. A sustained release patch or implant may
be employed to provide release over a prolonged period of time.
Many techniques known to one of skill in the art are described in
Remington: the Science & Practice of Pharmacy by Alfonso
Gennaro, 20th ed., Williams & Wilkins, (2000). Formulations can
contain excipients, polyalkylene glycols such as polyethylene
glycol, oils of vegetable origin, or hydrogenated naphthalenes.
Biocompatible, biodegradable lactide polymer, lactide/glycolide
copolymer, or polyoxyethylene-polyoxypropylene copolymers can be
used to control the release of a compound. Other potentially useful
delivery systems for a modulatory compound include ethylene-vinyl
acetate copolymer particles, osmotic pumps, implantable infusion
systems, and liposomes. Formulations can contain an excipient, for
example, lactose, or may be aqueous solutions containing, for
example, polyoxyethylene-9-lauryl ether, glycocholate, and
deoxycholate, or can be an oily solution for administration in the
form of drops, as a gel, or for other semi-solid formulation.
[0266] Compounds or pharmaceutical compositions in accordance with
this invention or for use in the methods disclosed herein can be
administered in combination with one or more other therapeutic
agents as appropriate. Compounds or pharmaceutical compositions in
accordance with this invention or for use in the methods disclosed
herein can be administered by means of a medical device or
appliance such as an implant, graft, prosthesis, stents, and wound
dressings. Also, implants can be devised that are intended to
contain and release such compounds or compositions. An example
would be an implant made of a polymeric material adapted to release
the compound over a period of time.
[0267] One skilled in the art will appreciate that suitable methods
of administering a Granzyme B inhibitor directly to the eye are
available (i.e., invasive and noninvasive methods). Although more
than one route can be used to administer the Granzyme B inhibitor,
a particular route can provide a more immediate and more effective
reaction than another route. The present use is not dependent on
the mode of administering the agent to an animal, preferably a
human, to achieve the desired effect, and the described routes of
administration are exemplary. As such, any route of administration
is appropriate so long as the agent contacts an ocular cell. Thus,
the Granzyme B inhibitor can be appropriately formulated and
administered in the form of an injection, eye lotion, ointment, and
implant.
[0268] The Granzyme B inhibitor can be applied, for example,
systemically, topically, intracamerally, subconjunctivally,
intraocularly, retrobulbarly, periocularly (e.g., subtenon
delivery), subretinally, or suprachoroidally. In certain cases, it
can be appropriate to administer multiple applications and employ
multiple routes to ensure sufficient exposure of ocular cells to
the Granzyme B inhibitor (e.g., subretinal and intravitreous).
Multiple applications of the Granzyme B inhibitor can also be
required to achieve the desired effect.
[0269] Depending on the particular case, it may be desirable to
non-invasively administer the Granzyme B inhibitor to a patient.
For instance, if multiple surgeries have been performed, the
patient displays low tolerance to anesthetic, or if other
ocular-related disorders exist, topical administration of the
Granzyme B inhibitor may be most appropriate. Topical formulations
are well known to those of skill in the art. Such formulations are
suitable in the context of the use described herein for application
to the skin or to the surface of the eye. The use of patches,
corneal shields (see, U.S. Pat. No. 5,185,152), and ophthalmic
solutions (see, e.g., U.S. Pat. No. 5,710,182) and ointments is
within the skill in the art.
[0270] The Granzyme B inhibitor also can be present in or on a
device that allows controlled or sustained release, such as an
ocular sponge, meshwork, mechanical reservoir, or mechanical
implant. Implants (see U.S. Pat. Nos. 5,443,505, 4,853,224 and
4,997,652), devices (see U.S. Pat. Nos. 5,554,187, 4,863,457,
5,098,443 and 5,725,493), such as an implantable device (e.g., a
mechanical reservoir, an intraocular device or an extraocular
device with an intraocular conduit, or an implant or a device
comprised of a polymeric composition are particularly useful for
ocular administration of the expression vector). The Granzyme B
inhibitor also can be administered in the form of sustained-release
formulations (see U.S. Pat. No. 5,378,475) comprising, for example,
gelatin, chondroitin sulfate, a polyphosphoester, such as
bis-2-hydroxyethyl-terephthalate, or a polylactic-glycolic
acid.
[0271] When used for treating an ocular disease the Granzyme B
inhibitor is administered via an ophthalmologic instrument for
delivery to a specific region of an eye. Use of a specialized
ophthalmologic instrument ensures precise administration while
minimizing damage to adjacent ocular tissue. Delivery of the
Granzyme B inhibitor to a specific region of the eye also limits
exposure of unaffected cells to the Granzyme B inhibitor. A
preferred ophthalmologic instrument is a combination of forceps and
subretinal needle or sharp bent cannula.
[0272] Alternatively, the Granzyme B inhibitor can be administered
using invasive procedures, such as, for instance, intravitreal
injection or subretinal injection, optionally preceded by a
vitrectomy, or periocular (e.g., subtenon) delivery. The
pharmaceutical composition of the invention can be injected into
different compartments of the eye (e.g., the vitreal cavity or
anterior chamber).
[0273] While intraocular injection is preferred, injectable
compositions can also be administered intramuscularly,
intravenously, intraarterially, and intraperitoneally.
Pharmaceutically acceptable carriers for injectable compositions
are well-known to those of ordinary skill in the art (see
Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co.,
Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982),
and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages
622-630 (1986)).
[0274] An "effective amount" of a Granzyme B inhibitor or a
pharmaceutical composition of the invention as described herein
includes a therapeutically effective amount or a prophylactically
effective amount. A "therapeutically effective amount" refers to an
amount effective, at dosages and for periods of time necessary, to
achieve the desired therapeutic result, such as reduced levels of
Granzyme B activity. A therapeutically effective amount of a
compound may vary according to factors such as the disease state,
age, sex, and weight of the subject, and the ability of the
compound to elicit a desired response in the subject. Dosage
regimens can be adjusted to provide the optimum therapeutic
response. A therapeutically effective amount is also one in which
any toxic or detrimental effects of the compound are outweighed by
the therapeutically beneficial effects. A "prophylactically
effective amount" refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired prophylactic
result, such as Granzyme B activity. Typically, a prophylactic dose
is used in subjects prior to or at an earlier stage of disease, so
that a prophylactically effective amount may be less than a
therapeutically effective amount.
[0275] It is to be noted that dosage values can vary with the
severity of the condition to be alleviated. For any particular
subject, specific dosage regimens can be adjusted over time
according to the individual need and the professional judgment of
the person administering or supervising the administration of the
compositions. Dosage ranges set forth herein are exemplary only and
do not limit the dosage ranges that can be selected by a medical
practitioner. The amount of active compound(s) in the composition
can vary according to factors such as the disease state, age, sex,
and weight of the subject. Dosage regimens can be adjusted to
provide the optimum therapeutic response. For example, a single
bolus can be administered, several divided doses can be
administered over time or the dose can be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It may be advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage.
[0276] In general, compounds of the invention should be used
without causing substantial toxicity. Toxicity of the compounds of
the invention can be determined using standard techniques, for
example, by testing in cell cultures or experimental animals and
determining the therapeutic index (i.e., the ratio between the
LD.sub.50, the dose lethal to 50% of the population, and the
LD.sub.100, the dose lethal to 100% of the population). In some
circumstances however, such as in severe disease conditions, it may
be necessary to administer substantial excesses of the
composition.
[0277] Methods of Use
[0278] In a further aspect, the invention provides methods of using
the compounds of the invention as Granzyme B inhibitors.
[0279] In one embodiment, the invention provides a method for
inhibiting Granzyme B in a subject. In the method, an effective
amount of a compound of the invention (e.g., a compound of Formulae
(I), (II), or (III) is administered to a subject in need
thereof.
[0280] In another embodiment, the invention provides a method for
treating a disease, disorder, or condition treatable by inhibiting
Granzyme B. In the method, a therapeutically effective amount of a
compound of the invention (e.g., a compound of Formulae (I), (II),
or (III)) is administered to a subject in need thereof.
[0281] As used herein, the term "disease, disorder, or condition
treatable by inhibiting Granzyme B" refers to a disease, disorder,
or condition in which Granzyme B is involved in the pathway related
to for the disease, disorder, or condition, and that inhibiting
Granzyme B results in the treatment or prevention of the disease,
disorder, or condition.
[0282] Representative methods of treatment using the compounds of
the invention include those described for Granzyme B inhibitors in
WO 2007/101354 (Methods of Treating, Reducing, and Inhibiting the
Appearance of Ageing in the Skin), WO 2009/043170 (Treatment of
Dissection, Aneurysm, and Atherosclerosis Using Granzyme B
Inhibitors), WO 2012/076985 (Granzyme B Inhibitor Compositions,
Methods and Uses for Promoting Wound Healing), each expressly
incorporated herein by reference in its entirety. The compounds of
the invention are useful for treating, reducing, and inhibiting the
appearance of aging of the skin; treating dissection, aneurysm, and
atherosclerosis; and promoting wound healing.
[0283] Other disease and disorders described as treatable using the
Granzyme B inhibitors are disclosed in WO 2003/065987 (Granzyme B
Inhibitors), expressly incorporated herein by reference in its
entirety. Disease and disorders described as treatable by Granzyme
B inhibitors in this reference include autoimmune or chronic
inflammatory diseases, such as systemic lupus erythematosis,
chronic rheumatoid arthritis, type I diabetes mellitus,
inflammatory bowel disease, biliary cirrhosis, uveitis, multiple
sclerosis, Crohn's disease, ulcerative colitis, bullous pemphigoid,
sarcoidosis, psoriasis, autoimmune myositis, Wegener's
granulomatosis, ichthyosis, Graves ophthalmopathy, asthma,
schleroderma and Sjogren's syndrome. The Granzyme B inhibitors
described in the reference are noted as more particularly useful to
treat or prevent diseases or disorders including diseases or
disorders resulting from transplantation of organs or tissue,
graft-versus-host diseases brought about by transplantation,
autoimmune syndromes including rheumatoid arthritis, systemic lupus
erythematosus, Hashimoto's thyroiditis, multiple sclerosis,
myasthenia gravis, type I diabetes, uveitis, posterior uveitis,
allergic encephalomyelitis, glomerulonephritis, post-infectious
autoimmune diseases including rheumatic fever and post-infectious
glomerulonephritis, inflammatory and hyperproliferative skin
diseases, psoriasis, atopic dermatitis, contact dermatitis,
eczematous dermatitis, seborrhoeic dermatitis, lichen planus,
pemphigus, bullous pemphigoid, epidermolysis bullosa, urticaria,
angioedemas, vasculitis, erythema, cutaneous eosinophilia, lupus
erythematosus, acne, alopecia areata, keratoconjunctivitis, vernal
conjunctivitis, uveitis associated with Behcet's disease,
keratitis, herpetic keratitis, conical cornea, dystrophia
epithelialis corneae, corneal leukoma, ocular pemphigus, Mooren's
ulcer, scleritis, Graves' opthalmopathy, Vogt-Koyanagi-Harada
syndrome, sarcoidosis, pollen allergies, reversible obstructive
airway disease, bronchial asthma, allergic asthma, intrinsic
asthma, extrinsic asthma, dust asthma, chronic or inveterate
asthma, late asthma and airway hyper-responsiveness, bronchitis,
gastric ulcers, vascular damage caused by ischemic diseases and
thrombosis, ischemic bowel diseases, inflammatory bowel diseases,
necrotizing enterocolitis, intestinal lesions associated with
thermal burns, coeliac diseases, proctitis, eosinophilic
gastroenteritis, mastocytosis, Crohn's disease, ulcerative colitis,
migraine, rhinitis, eczema, interstitial nephritis, Goodpasture's
syndrome, hemolytic-uremic syndrome, diabetic nephropathy, multiple
myositis, Guillain-Barre syndrome, Meniere's disease, polyneuritis,
multiple neuritis, mononeuritis, radiculopathy, hyperthyroidism,
Basedow's disease, pure red cell aplasia, aplastic anemia,
hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune
hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic
anemia, anerythroplasia, osteoporosis, sarcoidosis, fibroid lung,
idiopathic interstitial pneumonia, dermatomyositis, leukoderma
vulgaris, ichthyosis vulgaris, photoallergic sensitivity, cutaneous
T cell lymphoma, arteriosclerosis, atherosclerosis, aortitis
syndrome, polyarteritis nodosa, myocardosis, scleroderma, Wegener's
granuloma, Sjogren's syndrome, adiposis, eosinophilic fascitis,
lesions of gingiva, periodontium, alveolar bone, substantia ossea
dentis, glomerulonephritis, male pattern alopecia or alopecia
senilis by preventing epilation or providing hair germination
and/or promoting hair generation and hair growth, muscular
dystrophy, pyoderma and Sezary's syndrome, Addison's disease,
ischemia-reperfusion injury of organs which occurs upon
preservation, transplantation or ischemic disease, endotoxin-shock,
pseudomembranous colitis, colitis caused by drug or radiation,
ischemic acute renal insufficiency, chronic renal insufficiency,
toxinosis caused by lung-oxygen or drugs, lung cancer, pulmonary
emphysema, cataracta, siderosis, retinitis pigmentosa, senile
macular degeneration, vitreal scarring, corneal alkali burn,
dermatitis erythema multiforme, linear IgA ballous dermatitis and
cement dermatitis, gingivitis, periodontitis, sepsis, pancreatitis,
diseases caused by environmental pollution, aging, carcinogenesis,
metastasis of carcinoma and hypobaropathy, disease caused by
histamine or leukotriene-C4 release, Behcet's disease, autoimmune
hepatitis, primary biliary cirrhosis, sclerosing cholangitis,
partial liver resection, acute liver necrosis, necrosis caused by
toxin, viral hepatitis, shock, or anoxia, B-virus hepatitis,
non-A/non-B hepatitis, cirrhosis, alcoholic cirrhosis, hepatic
failure, fulminant hepatic failure, late-onset hepatic failure,
"acute-on-chronic" liver failure, augmentation of chemotherapeutic
effect, cytomegalovirus infection, HCMV infection, AIDS, cancer,
senile dementia, trauma, and chronic bacterial infection. To the
extent that the diseases and disorders noted in the reference are
treatable by the Granzyme B inhibitors described in the reference,
the Granzyme B inhibitors of the present invention are also useful
in treating and/or ameliorating a symptom associated with these
diseases and conditions.
[0284] Elevated Granzyme B levels have been identified in cells and
tissues from subjects suffering from a variety of diseases and
conditions including Rasmussen encephalitis, amyotrophic lateral
sclerosis (ALS), chronic inflammation, Stevens-Johnson syndrome
(SJS), toxic epidermal necrolysis (TEN), Kawasaki disease,
idiopathic pulmonary fibrosis, chronic obstructive pulmonary
disease (COPD), coronary artery disease (CAD), transplant vascular
disease (TVD), restenosis, acute respiratory distress syndrome
(ARDS), chronic obstructive sialadentis (associated with
sialolithiasis), vitiligo, allergic contact dermatitis (ACD),
atopic dermatitis (AD), pityriasis rosea (PR), rheumatoid arthritis
(RA), osteoarthritis (OA), vasculitic neuropathy, sensory
perineuritis, ischemic stroke, spinal cord injury, myasthenia
gravis (MG), lymphocytic gastritis, autoimmune cholangitis (AIC),
nodular regenerative hyperplasia (NRH) of the liver, achalasia,
esophagitis, eosinophilic fasciitis, cryptorchidism, necrotizing
lymphadenitis, Duchenne muscular dystrophy, facioscapulo humeral
muscular dystrophy, and Higashi syndrome. Other diseases and
conditions in which elevated Granzyme B levels have been identified
include those described in WO 2009/043167 (Granzyme A and Granzyme
B Diagnostics), expressly incorporated herein by reference in its
entirety. The Granzyme B inhibitors of the invention may be useful
for treating, alleviating or ameliorating a symptom of, diminishing
the extent of, stabilizing, or ameliorating or palliating the
diseases and conditions noted above in which elevated Granzyme B
levels have been identified. A description of intracellular versus
extracellular Granzyme B in immunity and disease is provided in
Granville et al., Laboratory Investigation, 2009, 1-26, expressly
incorporated herein by reference in its entirety. The reference
provides a listing of conditions in which the pathogenic role of
Granzyme B has been identified.
[0285] The compounds of the invention are useful in treating
cutaneous scleroderma, epidermolysis bullosa, radiation dermatitis,
alopecia areata, and discoid lupus erythematosus.
[0286] Cutaneous Scleroderma.
[0287] Scleroderma refers to a heterogeneous group of autoimmune
fibrosing disorders. Limited cutaneous systemic sclerosis (CREST
syndrome or LcSSc) develop sclerosis of the skin distal to their
elbows and knees and have facial involvement. Patients with diffuse
cutaneous systemic sclerosis (DcSSc) develop proximal, in addition
to distal, skin sclerosis. Both groups of patients are also at high
risk of developing internal organ involvement. Patients with LcSSc
and DcSSc suffer from Raynaud's phenomenon (excessively reduced
blood flow in response to cold or emotional stress, causing
discoloration of the fingers, toes, and occasionally other areas
believed to be the result of vasospasms that decrease blood supply
to the respective regions) with high frequencies. Management of
progressive skin involvement is dependent on additional
comorbidities. In patients with skin involvement only,
mycophenolate mofetil (Cellsept, immunomodulator) or methotrexate
(T cell modulator) have been recommended.
[0288] Epidermolysis Bullosa.
[0289] Epidermolysis bullosa acquisita (EBA) is a chronic
mucocutaneous autoimmune skin blistering disease. EBA patients can
be classified into two major clinical subtypes: noninflammatory
(classical or mechanobullous) and inflammatory EBA, which is
characterized by cutaneous inflammation. In patients with
inflammatory EBA, widespread vesiculobullous eruptions are
observed, typically involving the trunk, central body, extremities,
and skin folds. Usually the patients suffer from pruritus (rashes).
Autoantibodies targeting type VII collagen (COL7) has been
implicated in the pathogenesis. Therefore, EBA is a prototypical
autoimmune disease with a well-characterized pathogenic relevance
of autoantibody binding to the target antigen. EBA is a rare
disease with an incidence of 0.2-0.5 new cases per million and per
year. The current treatment of EBA relies on general
immunosuppressive therapy, which does not lead to remission in all
cases.
[0290] Radiation Dermatitis.
[0291] Radiation Dermatitis (acute skin reaction) ranges from a
mild rash to severe ulceration. Approximately 85-90% of patients
treated with radiation therapy will experience a moderate-to-severe
skin reaction. Acute radiation-induced skin reactions often lead to
itching and pain, delays in treatment, and diminished aesthetic
appearance--and subsequently to a decrease in quality of life. Skin
reactions related to radiation therapy usually manifest within 1-4
weeks of radiation start, persist for the duration of radiation
therapy, and may require 2-4 weeks to heal after completion of
therapy. The severity of the skin reaction ranges from mild
erythema (red rash) and dry desquamation (itchy, peeling skin) to
more severe moist desquamation (open wound) and ulceration.
Treatments that have been assessed for the management of
radiation-induced skin reactions include topical steroid creams,
nonsteroidal creams, dressings, and herbal remedies. Only three
trials have showed a significant difference: one in favor of a
corticosteroid cream, one favoring a nonsteroidal cream, and one
for a dressing. However, all three of these trials were small and
had limitations, thus there is still an unmet medical need.
[0292] Late effects of radiation therapy, typically months to years
post exposure, occur at doses greater than a single dose of 20-25
Gy or fractionated doses of 70 Gy or higher. The major underlying
histopathological findings at the chronic stage include
telangiectasia, dense dermal fibrosis (round fibrosis), sebaceous
and sweat gland atrophy, loss of hair follicles, and with higher
doses, increased melanin deposition or depigmentation and skin
ulcers.
[0293] Ramipril was very effective in reducing the late effects of
skin injury, whereas its mitigating effects on the acute and
sub-acute injury were modest. However, the dose required to
mitigate these late effects may be pharmacologically too high to be
clinically relevant. More recently, it has been shown that
significant mitigation of acute skin injury using an
adeno-associated virus encoding the manganese SOD gene, when
injected subcutaneously shortly after irradiation. However,
difficulties in delivery, application and cost limit the utility of
this treatment strategy.
[0294] Alopecia Aerata.
[0295] Alopecia areata (AA) is a CD8+ T-cell dependent autoimmune
disease of the hair follicle (HF) in which the collapse of HF
immune privilege (IP) plays a key role. Mast cells (MCs) are
crucial immunomodulatory cells implicated in the regulation of T
cell-dependent immunity, IP, and hair growth. Many of these
infiltrating immune cells express GzmB, suggesting it may be a key
mediator in immune cell-mediated follicular attack. The peptide
substance P was shown to increase the CD8+ cells expressing GzmB in
the intrafollicular dermis, co-relating to a regression of
follicles into the catagen stage of follicle growth cessation
(Siebenhaar et al., J Invest Dermatol, 2007, 127: 1489-1497).
[0296] In mice fed a diet with excess vitamin A, AA was accelerated
and GzmB expressing cells were found in excess surrounding hair
follicles, including in the isthmus (the region of the follicle
containing stem cells) (Duncan et al., J Invest Dermatol 2013, 133:
334-343). As GzmB is expressed in the immune cell infiltrate within
and surrounding growing follicles, it may be a key protease
involved in hair loss through autoimmunity, apoptosis and ECM
degradation.
[0297] No drug is currently approved by the US FDA for the
treatment of alopecia areata. A number of treatments have been
found to be effective using the American College of Physician's
criteria, for example, topical and oral corticosteroids and the
sensitizing agents diphenylcyclopropenone and dinitrochlorobenzene.
However, there is no cure for alopecia areata, nor is there any
universally proven therapy that induces and sustains remission.
[0298] Discoid Lupus Erythematosus.
[0299] Granzyme B is a serine protease found in cytoplasmic
granules of cytotoxic lymphocytes and natural killer cells that
plays an important role in inducing apoptotic changes in target
cells during granule exocytosis-induced cytotoxicity. When Granzyme
B is secreted into the cytoplasm of a target cell through the pore
formed by perforin, it triggers cytotoxic-induced cell death (Shah
et al., Cell Immunology 2011, 269:16-21).
[0300] Lupus erythematosus (LE) is a chronic, autoimmune,
multisystem disease that displays many diverse symptoms in which
localized cutaneous LE (CLE) is on one end of the spectrum and
severe systemic LE (SLE) on the other end. CLE is a disfiguring,
chronic skin disease, with a significant impact on the patients'
everyday life. CLE are further divided into four main subsets:
Acute CLE (ACLE), subacute CLE (SCLE) and chronic CLE (CCLE), where
classic discoid LE (DLE) is the most common form. There is also a
drug-induced form of the disease. The disease often has a chronic
and relapsing course that can be induced or aggravated by UV light.
CLE patients display well-defined skin lesions, often in
sun-exposed areas. Discoid LE is the most common subtype of CLE,
60-80% is localized above the neck and 20-40% is generalized
(lesions both above and below the neck). 70-90% of the DLE patients
suffer from photosensitivity and sun exposed areas such as the
scalp, ears and cheeks, which are most commonly involved areas. The
lesions start as erythematosus maculae or papules with a scaly
surface and then grow peripherally into larger discoid plaques that
heal with atrophic scar and pigmentary changes. DLE often results
in scarring and alopecia. Mutilation with tissue loss can be seen
when the lesions affect the ears and tip of the nose. CLE can be
managed but so far, not cured. Avoidance of trigger factors is of
utmost importance, such as, cessation of smoking and avoidance of
sun exposure. The treatment is about the same for the different CLE
subsets where first-line of treatment is sun-protection and local
therapy with corticosteroids or calcineurin inhibitors.
Antimalarial are the first choice of systemic treatment.
[0301] Strong co-expression of Granzyme B and the skin-homing
molecule, cutaneous lymphocyte antigen (CLA) was found in lesional
lymphocytes of patients with scarring localized chronic DLE and
disseminated chronic DLE, which was enhanced compared with
nonscarring subacute CLE and healthy controls (Wenzel et al.,
British Journal of Dermatology 2005, 153: 1011-1015). Wenzel et al.
conclude that skin-homing cytotoxic Granzyme B-positive lymphocytes
play an important role in the pathophysiology of scarring chronic
DLE and that the potentially autoreactive cytotoxic lymphocytes
targeting adnexal structures may lead to scarring lesions in
chronic DLE.
[0302] Correlation between Granzyme B-positive lymphocytes and the
presence of CLE was shown by Grassi (Grassi et al., Clinical and
Experimental Dermatology 2009, 34:910-914). Granzyme B is an
apoptosis immunological mediator that, once synthesized and free
from activated cytotoxic lymphocytes, enters the target cell and
starts apoptotic mechanisms involved at different levels in all
apoptotic pathways. In CLE, apoptosis is characterized by the
presence of colloid or Civatte bodies, which are evident in the
epidermis and papillary dermis of CLE lesions, and since Granzyme B
is mainly expressed in CLE lesions, Grassi et al. conclude that
Granzyme B could play a role in the induction of apoptotic
mechanisms in CLE.
[0303] The expression of Granzyme B and perforin was correlated
with clinicopathological features in patients with DLE, where both
Granzyme B and perforin were expressed in DLE, with absent
expression in normal skin (Abdou et al., Ultrastructural Pathology
2013, Early Online 1-9). Abdou et al. concluded that cytotoxicity
in dermal lymphocytic inflammation was due to expression of both
Granzyme B and perforin.
[0304] Extracellular Granzymes B is also reported to play a role in
DLE by Grassi et al. Further, UV light increases Granzyme B
expression in keratinocytes as well as mast cells
(Hernandez-Pigeon, J. Biol. Chem., 2007, 282:8157-8164). As
Granzymes B is in abundance at the dermal-epidermal junction (DEJ),
where many key extracellular matrix substrates are present (for
example, laminin, fibronectin, decorin), it follows that Granzymes
B may also be damaging the DEJ, as is observed in DLE. Given its
expression in adnexal structures, Granzyme B may also be
contributing to alopecia, as reduced Granzymes B is associated with
reduced hair loss in a murine model of skin aging. Similarly,
reduced extracellular Granzyme B activity is associated with
improved collagen organization and reduced scarring in the skin and
aorta.
[0305] In view of the established connection between Granzyme B and
DLE, by virtue of their ability to inhibit Granzyme B, the
compounds of the invention are useful in methods for treating lupus
erythematosus (LE) including severe systemic LE (SLE) and localized
cutaneous LE (CLE) (e.g., acute CLE (ACLE), subacute CLE (SCLE),
chronic CLE (CCLE) and the most common form classic discoid LE
(DLE)). In one embodiment, the invention provides a method for
treating DLE comprising administering a therapeutically effective
amount of a compound of the invention to a subject suffering from
DLE.
[0306] Administration.
[0307] In the above methods, the administration of the Granzyme B
inhibitor can be a systemic administration, a local administration
(e.g., administration to the site, an inflamed microenvironment, an
inflamed joint, an area of skin, a site of a myocardial infarct, an
eye, a neovascularized tumor), or a topical administration to a
site (e.g., a site of inflammation or a wound).
[0308] The term "subject" or "patient" is intended to include
mammalian organisms. Examples of subjects or patients include
humans and non-human mammals, e.g., nonhuman primates, dogs, cows,
horses, pigs, sheep, goats, cats, mice, rabbits, rats, and
transgenic non-human animals. In specific embodiments of the
invention, the subject is a human.
[0309] The term "administering" includes any method of delivery of
a Granzyme B inhibitor or a pharmaceutical composition comprising a
Granzyme B inhibitor into a subject's system or to a particular
region in or on a subject. In certain embodiments, a moiety is
administered topically, intravenously, intramuscularly,
subcutaneously, intradermally, intranasally, orally,
transcutaneously, intrathecal, intravitreally, intracerebral, or
mucosally.
[0310] As used herein, the term "applying" refers to administration
of a Granzyme B inhibitor that includes spreading, covering (at
least in part), or laying on of the inhibitor. For example, a
Granzyme B inhibitor may be applied to an area of inflammation on a
subject or applied to, for example the eye or an area of
inflammation by spreading or covering the surface of the eye with
an inhibitor, by injection, oral or nasal administration.
[0311] As used herein, the term "contacting" includes contacting a
cell or a subject with a Granzyme B inhibitor. Contacting also
includes incubating the Granzyme B inhibitor and the cell together
in vitro (e.g., adding the inhibitor to cells in culture) as well
as administering the inhibitor to a subject such that the inhibitor
and cells or tissues of the subject are contacted in vivo.
[0312] As used herein, the terms "treating" or "treatment" refer to
a beneficial or desired result including, but not limited to,
alleviation or amelioration of one or more symptoms, diminishing
the extent of a disorder, stabilized (i.e., not worsening) state of
a disorder, amelioration or palliation of the disorder, whether
detectable or undetectable. "Treatment" can also mean prolonging
survival as compared to expected survival in the absence of
treatment.
[0313] Cosmetic Compositions and Related Methods
[0314] In further aspects, the invention provides cosmetic
compositions that include one or more granzyme B inhibitors of the
invention and methods for using the compositions to treat, reduce,
and/or inhibit the appearance of ageing of the skin.
[0315] This aspect of the invention is based, in part, on the
observation that granzyme B expression is induced in keratinocytes
and immune cells, such as mast cells in the skin during aging. When
released by these cells, granzyme B cleaves extracellular matrix
proteins such as decorin which can result in collagen
disorganization. This invention is also based in part on the
observation that granzyme B cleaves decorin, in addition to other
extracellular matrix proteins, in the interstitial space
surrounding cells.
[0316] Skin is comprised of three main layers: the epidermis, the
dermis and subcutaneous layers. Each of these three layers has
individual compositions. The functions and structures of these
layers are known to a person of skill in the art. The epidermis is
the outermost layer of skin and includes both living and dead cell
layers. The dermis is the middle layer of skin and is comprised of
arrangements of collagen fibers, which surround many specialized
cells and structures. Hair follicles are found within the dermis,
and produce the hair shaft which grows out through layers of the
dermis and epidermis to become visible as hair. The lowermost layer
of the skin is the subcutaneous layer, often called the sub-dermis.
The subcutaneous layer is comprised largely of fat and connective
tissue and houses larger blood vessels and nerves. Collagen may be
found in all layers of the skin, but is most prominently in the
dermis layer.
[0317] A youthful appearance is achieved by not having at least one
of the characteristic signs of age. This is often achieved by being
young. Nevertheless, there are circumstances in which being young
does not confer a youthful appearance as a disease or disorder or
other non-time related event has conferred the characteristics
associated with age. A youthful appearance is often characterized
by the condition of the skin and the following skin qualities are
typically associated with, but not limited to, a youthful
appearance: small pore size, healthy skin tone, radiance, clarity,
tautness, firmness, plumpness, suppleness, elasticity, softness,
healthy skin texture, healthy skin contours, such as few or no
wrinkles, shallow wrinkle depth, few or no fine lines, healthy skin
luster and brightness, moisturized skin, healthy skin thickness and
resilient skin. If a skin of a subject comprises any one or more of
these characteristics then a youthful appearance is achieved.
[0318] The appearance of ageing can occur for a variety of reasons,
but typically happens at a normal rate associated with the passage
of time. A rate of appearance of ageing will be different for
different subjects, depending on a variety of factors including
age, gender, diet and lifestyle. An appearance of ageing is often
characterized by the condition of the skin. Characteristics
associated with an appearance of ageing in the skin include, but
are not limited to, skin fragility, skin atrophy, skin wrinkles,
fine lines, skin discoloration, skin sagging, skin fatigue, skin
stress, skin inelasticity, skin fragility, skin softening, skin
flakiness, skin dryness, enlarged pore size, skin thinning, reduced
rate of skin cell turnover, deep and deepening of skin wrinkles.
The rate of appearance of ageing can be measured by measuring the
rate at which any one or more of the above characteristics appear.
An appearance of ageing may be inhibited, reduced, or treated by
reducing or maintaining a state of any one or more of these skin
characteristics.
[0319] In many circumstances a reduction in the appearance of
ageing of skin occurs when the rate of collagen cleavage exceeds
the rate of collagen formation. In many other circumstances, a
youthful appearance of skin is maintained when the rate of collagen
formation is equal to the rate of collagen cleavage. In many other
circumstances, a reduction in a rate of appearance of ageing of
skin is achieved when the rate of decorin cleavage and collagen
disorganization and cleavage is slowed such that the rate of
collagen fibrillogenesis exceeds the rate of collagen cleavage and
the ratio of the rate of collagen fibrillogenesis to the rate of
collagen cleavage is greater after application of granzyme B
inhibitor compound compared to the ratio before application of the
compound. In many other circumstances, an extracellular protein,
other than decorin, is also cleaved by granzyme B, and the
beneficial effects of inhibiting granzyme B can be enhanced beyond
what is realized by inhibiting decorin cleavage alone.
[0320] In one aspect, the invention provides a cosmetic
composition. The composition comprises a cosmetically acceptable
carrier and one or more compounds of the invention (e.g., a
compound of Formulae (I), (II), or (III), or stereoisomers,
tautomers, and cosmetically acceptable salts thereof, as described
herein).
[0321] As used herein, the term "cosmetically acceptable salt"
refers to a salt prepared from a cosmetically acceptable base, such
as an inorganic base and an organic base, or a salt prepared from a
cosmetically acceptable acid. Representative salts derived from
inorganic bases include aluminum, ammonium, calcium, copper,
ferric, ferrous, lithium, magnesium, manganic, manganous, ammonium,
potassium, sodium, and zinc salts. Representative salts derived
from cosmetically acceptable organic bases include salts of
primary, secondary and tertiary amines, substituted amines
including naturally occurring substituted amines, cyclic amines,
and basic ion exchange resins, such as arginine, betaine, caffeine,
choline, N,N'-dibenzylethylenediamine, diethylamine,
2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,
ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine,
glucosamine, histidine, hydrabamine, isopropylamine, lysine,
methylglucamine, morpholine, piperazine, piperidine, polyamine
resins, procaine, purines, theobromine, triethylamine,
trimethylamine, tripropylamine, and trimethamine.
[0322] The cosmetic compositions can be formulated by means known
in the art and their mode of administration and the amount of
granzyme B inhibitor compound as described herein can be determined
by a person of skill in the art. Compositions for use in the
methods described herein can comprise one of more of a granzyme B
inhibitor compound or a cosmetically acceptable salt thereof as an
active ingredient, in combination with a cosmetically acceptable
carrier.
[0323] The cosmetic compositions can include diluents, excipients,
solubilizing agents, emulsifying agents, and salts known to be
useful for cosmetic compositions. Examples of suitable agents
include thickeners, buffers, preservatives, surface active agents,
neutral or cationic lipids, lipid complexes, liposomes, and
penetration enhancers. In certain embodiments, the cosmetic
compositions further include other cosmetic ingredients known in
the art.
[0324] In certain embodiments, the cosmetic composition can include
one or more penetration enhancers. Numerous types of penetration
enhancers are known, such as fatty acids, bile salts, chelating
agents, surfactants and non-surfactants (Lee et al., Critical
Reviews in Therapeutic Drug Carrier Systems 8:91-192, 1991;
Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems
7:1-33, 1990). Fatty acids and their derivatives which act as
penetration enhancers include, for example, cabrylic acid, oleic
acid, lauric acid, capric acid, caprylic acid, hexanoic acid,
myristic acid, palmitic acid, valeric acid, stearic acid, linoleic
acid, linolenic acid, arachidonic acid, oleic acid, elaidic acid,
erucic acid, nervonic acid, dicaprate, tricaprate, recinleate,
monoolein (also known as 1-monooleoyl-rac-glycerol), dilaurin,
arachidonic acid, glyceryll-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono-
and di-glycerides and physiologically acceptable salts thereof
(e.g., oleate, laurate, caprate, myristate, palmitate, stearate,
linoleate) (Lee et al., Critical Reviews in Therapeutic Drug
Carrier Systems page 92, 1991; Muranishi, Critical Reviews in
Therapeutic Drug Carrier Systems 7:1, 1990; El-Hariri et al., J.
Pharm. Pharmacol. 44:651-654, 1992).
[0325] In certain embodiments, the cosmetic composition further
includes other cosmetic ingredients known in the art to be useful
for cosmetic, skincare, and/or dermatological applications (e.g.,
anti-wrinkle active ingredients including flavone glycosides such
as alpha-glycosylrutin; coenzyme Q10; vitamin E and derivatives; as
well as sunblock ingredients, moisturizers, and perfumes).
[0326] The cosmetic compositions of the invention can be
administered for "cosmetic" or "skincare" (e.g., dermatologic)
applications, either alone or as an "additive" in combination with
other suitable agents or ingredients. As used herein, "cosmetic"
and "skincare" applications includes, for example, preventive
and/or restorative applications in connection with dermatological
changes in the skin, such as, for example, during pre-mature skin
aging; dryness; roughness; formation of dryness wrinkles; itching;
reduced re-fatting (e.g., after washing); visible vascular
dilations (e.g., telangiectases, cuperosis); flaccidity; formation
of wrinkles and lines; local hyperpigmentation; hypopigmentation;
incorrect pigmentation (e.g., age spots); increased susceptibility
to mechanical stress (e.g., cracking); skin-sagging (e.g., lack of
firmness) and the appearance of dry or rough skin surface
features.
[0327] The cosmetic compositions of the invention can be formulated
for topical administration. Such compositions can be administered
topically in any of a variety of forms. Such compositions are
suitable in the context of the use described herein for application
to the skin or to the surface of the eye. The use of patches,
corneal shields (see, U.S. Pat. No. 5,185,152), and ophthalmic
solutions (see, for example, U.S. Pat. No. 5,710,182) and ointments
is within the skill in the art.
[0328] Compositions for topical administration include dermal
patches, ointments, lotions, serums, creams, gels, hydrogels,
pastes, foams, oils, semi-solids, shampoos, soaps, drops, sprays,
films, liquids, and powders. Examples of such compositions include
those in which a cosmetically effective amount of a compound of the
invention is encapsulated in a vehicle selected from
macro-capsules, micro-capsules, nano-capsules, liposomes,
chylomicrons and microsponges. Another example of such a
composition includes absorption of a compound of the invention on
or to a material selected from powdered organic polymers, talcs,
bentonites, and other mineral supports. A third example of such a
composition or formulation includes a mixture of a cosmetically
effective amount of a compound of the invention with other
ingredients selected from extracted lipids, vegetable extracts,
liposoluble active principles, hydrosoluble active principles,
anhydrous gels, emulsifying polymers, tensioactive polymers,
synthetic lipids, gelifying polymers, tissue extracts, marine
extracts, vitamin A, vitamin C, vitamin D, vitamin E, solar filter
compositions, and antioxidants. Other examples of suitable
composition ingredients can be found in US2005/0249720.
[0329] In the cosmetic compositions, the compounds of the invention
can be incorporated into any gelanic form, such as oil/water
emulsions and water/oil emulsions, milks, lotions, gelifying and
thickening tensioactive and emulsifying polymers, pomades, lotions,
capillaries, shampoos, soaps, powders, sticks and pencils, sprays,
and body oils.
[0330] Regardless of the compound or formulation described herein,
application/administration to a subject as a colloidal dispersion
system can be used as a delivery vehicle to enhance the in vivo
stability of the compound and/or to target the granzyme B inhibitor
compound to a particular skin layer, tissue or cell type. Colloidal
dispersion systems include, but are not limited to, macromolecule
complexes, nanocapsules, microspheres, beads and lipid-based
systems including oil-in-water emulsions, micelles, mixed micelles,
liposomes and lipid:inhibitor complexes of uncharacterized
structure. An example of a colloidal dispersion system is a
plurality of liposomes. Liposomes are microscopic spheres having an
aqueous core surrounded by one or more outer layers made up of
lipids arranged in a bilayer configuration (see, generally, Chonn
et al., Current Op. Biotech. 6:698-708, 1995). Sustained-release
dosage forms of the compounds described herein can also be
used.
[0331] The amount of the granzyme B inhibitor compound administered
or applied to a subject is not critical, except that it should be
an amount sufficient to effect improvement of the condition for
which the composition is administered/applied. Application can be
dependent on a number of factors, including severity and
responsiveness of the condition to be treated, and with the course
of treatment lasting from several days to several months, or until
improvement of a condition is effected or a diminution of a symptom
is achieved.
[0332] A "cosmetically effective amount" of a granzyme B inhibitor
compound includes a cosmetically effective amount or a
prophylactically effective amount. A "cosmetically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired cosmetic result, such as
improved skin elasticity, skin durability, skin firming, skin
texture, decrease the appearance or decrease rate of appearance of
aging, and the like. A cosmetically effective amount of a compound
may vary according to factors such as the skin state, age, sex, and
weight of the subject, and the ability of the compound to elicit a
desired response in the subject. Dosage regimens can be adjusted to
provide the optimum cosmetic response. A cosmetically effective
amount is also one in which any toxic or detrimental effects of the
compound are outweighed by the cosmetically beneficial effects. A
"prophylactically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired prophylactic result, such as improved skin elasticity, skin
durability, skin firming, skin texture, a decrease appearance or a
decrease in the rate of appearance of aging, and the like.
Typically, a prophylactic dose is used in subjects prior to or at
an earlier stage of skin deterioration, so that a prophylactically
effective amount may be less than a cosmetically effective
amount.
[0333] The amount of granzyme B inhibitor administered/applied may
vary with the severity of the appearance, or rate of appearance, of
age of the skin. For any particular subject, specific dosage
regimens may be adjusted over time according to the individual need
and the judgment of the person applying or supervising the applying
of the compositions. Dosage ranges set forth herein are exemplary
only and do not limit the dosage ranges that may be selected. The
amount of granzyme B inhibitor compound(s) in the composition or
formulation can vary according to factors such as the skin state,
age, sex, and weight of the subject. Dosage regimens can be
adjusted to provide the optimum response. For example, a single
application can be administered/applied, several divided doses can
be administered/applied over time or the amount of the composition
administered/applied can be proportionally reduced or increased as
indicated by the exigencies of the situation. It can be
advantageous to formulate the granzyme B inhibitor compounds in a
composition into a dosage unit form for ease of administration and
uniformity of application.
[0334] By way of example, a granzyme B inhibitor compound of the
cosmetic composition can be administered/applied to achieve from
about 0.01 micrograms per milliliter (.mu.g/mL) to about 10
milligrams per milliliter, from about 0.1 .mu.g/mL to about 500
.mu.g/mL, from about 0.1 .mu.g/mL to about 1500 .mu.g/mL, from
about 1 .mu.g/mL to about 2000 .mu.g/mL, and from about 0.1
.mu.g/mL to about 5000 .mu.g/mL, including any range within these
ranges, final concentrations at a target site.
[0335] Appropriate dosage values can depend on the characteristics
of the site to which the composition is to be applied/administered
and on the form of the granzyme B inhibitor compound used. Guidance
as to particular dosages and methods of delivery is provided in the
literature and generally available to practitioners in the art.
Those skilled in the art will employ different formulations for
different uses and the granzyme B inhibitor compound used. Persons
of ordinary skill in the art can easily estimate repetition rates
for dosing based on measured residence times and concentrations of
the granzyme B inhibitor compound in, for example, a bodily fluid
or a tissue. Following successful treatment, it can be desirable to
have the subject undergo maintenance therapy to prevent the
recurrence of the condition, wherein a selected compound is
administered/applied in maintenance doses applied, for example,
once or more daily, to once every few days. In certain embodiments,
granzyme B inhibitor compounds are administered/applied in an
amount to achieve ex vivo concentrations from about 1 micromolar to
about 10 millimolar, from about 10 micromolar to about 5000
micromolar, or from about 30 micromolar to about 3000 micromolar,
and from about 25 micromolar to about 3000 micromolar final
concentration over a site of interest, and including, about 25
micromolar, or about 1600 micromolar, or about 3000 micromolar
final concentration over the site, and still more typically between
about 1 micromolar to about 1000 micromolar.
[0336] Compounds or compositions of granzyme B inhibitors can be
administered/applied by means of a device or appliance such as an
implant, graft, prosthesis, garment of clothing, stent, and the
like. Also, implants can be devised which are intended to contain
and release such compounds or compositions. An example would be an
implant made of a polymeric material adapted to release the
compound over a period of time. Such implants can be placed into a
garment to be worn by a subject, for example a glove, shirt, mask
or hat.
[0337] The cosmetic compositions of the invention can be used to
inhibit or reduce the appearance of ageing. Ageing is a natural
phenomenon that cannot be reversed per se, but the appearance of
ageing, such as skin deterioration including, but not limited to,
skin inelasticity, skin fragility, skin softening, skin flakiness,
skin dryness, enlarged pore size, skin thinning, reduced rate of
skin cell turnover, skin wrinkling, deepening of skin wrinkles,
skin sagging, fine lines, and skin discoloration may be inhibited
or reduced.
[0338] The cosmetic compositions can be used to increase or
decrease a rate of increasing or a rate of decreasing occurrences
of a particular skin characteristic. In other words, the
composition, when applied to the skin or a portion of the skin of a
subject delays the onset of an appearance of aging. For example, in
a population of subjects where half of the population applies a
granzyme B inhibitor to their skin and another half of the
population does not apply a granzyme B inhibitor to their skin, the
half which applied a granzyme B inhibitor would not appear as aged
as the half which did not apply the granzyme B inhibitor after a
period of time had elapsed. The half of the population which
applied a granzyme B inhibitor to the skin would also have
maintained a youthful appearance.
[0339] The rate at which a particular subject experiences a change
in the rate of appearance of a particular skin characteristic,
i.e., an increasing or decreasing rate of the appearance of a
particular skin characteristic, will depend on a variety of
factors, including, but not limited to age, weight, sex and
lifestyle of the subject. As such, rates are not necessarily
constant, but a normal rate of increase or of decrease of an
appearance of a characteristic, defined as being the new occurrence
of a particular characteristic over a predetermined period of time
under a set of conditions that do not include the presence of a
granzyme B inhibitor applied by a method or use of this invention,
is increased or decreased by applying a granzyme B inhibitor in
accordance with a method or use of this invention. Methods of
measuring skin characteristics, rates of increasing appearance of
skin characteristics and rates of decreasing appearance of skin
characteristics are known to a person of skill in the art, see for
example, Measuring the Skin by Agache et al., Springer (2004).
[0340] Surprisingly, granzyme B inhibitors can also be used to
increase the density of hair follicles of a skin of a subject and
may be used to reduce the occurrences of cutaneous xanthomas of a
skin of a subject. Actively growing hair follicles contain
melanocytes that transfer pigment to matrix keratinocytes,
imparting color to hair. Additionally, sebum, produced in sebaceous
glands, is often secreted via hair follicles. Increased density of
hair follicles results in increased pigment production and
increased sebum secretion resulting in improved hair appearance
(e.g., hair that is less grey in color or not grey at all) as well
as healthier hair and skin. Granzyme B inhibitors also cause hair
follicles to appear deeper in the skin which provide stronger hair
that is less susceptible to mechanical damage. Additionally, a
characteristic sign of ageing is the reduction in hair follicle
density. It is known in the art that age and follicular
miniaturization are weak predictors of total hair count (see
Chapman et al., Brit. J. Dermatol. 152:646-649, 2005).
Consequently, the characteristic sign of age associated with hair
follicle density is not predictive of hair density.
[0341] The cosmetic composition may be applied to a portion of the
skin of a subject or to the whole of the skin of the subject. For
example, the composition may be applied to the skin, only on the
face, only on the scalp, on the whole head or to each part of the
body.
INCORPORATION BY REFERENCE
[0342] Each reference cited is incorporated herein by reference in
its entirety.
Abbreviations
[0343] As used herein, the following abbreviations have the
indicated meanings.
[0344] .sup.1H NMR: proton nuclear magnetic resonance
[0345] .sup.19F NMR: fluorine-19 nuclear magnetic resonance
[0346] % Inh: Percent inhibition
[0347] Ac-IEPD-AMC:
acetyl-isoleucyl-glutamyl-prolyl-aspartyl-(7-amino-4-methylcoumarin)
substrate
[0348] ACN: acetonitrile
[0349] BHET: bis-2-hydroxyethyl-terephthalate
[0350] Boc: tert-butoxycarbonyl
[0351] BSA: Bovine serum albumin
[0352] CHAPS:
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
[0353] DAPI: 4',6-diamidino-2-phenylindole
[0354] DCM: dichloromethane
[0355] DIPEA: diisopropylethylamine
[0356] DMAP: 4-dimethylaminopyridine
[0357] DMF: dimethylformamide
[0358] DMSO: dimethylsulfoxide
[0359] DMSO-d6: dimethylsulfoxide-d6
[0360] DTT: dithiothreitol
[0361] EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
[0362] EDTA:
2-({2-[Bis(carboxymethyl)amino]ethyl}(carboxymethyl)amino)acetic
acid
[0363] ESI: Electrospray ionization
[0364] EtOAc: ethyl acetate
[0365] eq.: equivalent(s)
[0366] GzmB: Granzyme B
[0367] HATU:
2-(7-aza-1H-benzotriazole-1-yl)-1,1,1,1-tetramethyluronium
hexafluorophosphate
[0368] HCl: hydrochloric acid
[0369] HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid
[0370] hGzmB: human Granzyme B
[0371] HPLC: high performance liquid chromatography
[0372] HOBt: 1-hydroxy-benzotriazol
[0373] IC.sub.50: inhibitory concentration that provides 50%
inhibition
[0374] LC/MS: liquid chromatography/mass spectrometry
[0375] MeOH: methanol
[0376] mGzmB: murine Granzyme B
[0377] MS: mass spectrometry
[0378] m/z: mass to charge ratio.
[0379] Oxyma: ethyl 2-cyano-2-(hydroxyimino)acetate
[0380] PBS: phosphate buffered saline (pH 7.4)
[0381] RPM: revolution per minute
[0382] RT: room temperature
[0383] tert-BuOH: tert-butyl alcohol
[0384] THF: tetrahydrofuran
[0385] TFA: trifluoroacetic acid
[0386] wt %: weight percent
General Methods A-J
[0387] Representative compounds of the invention were prepared
according to Methods A to J as described below and illustrated in
FIGS. 1-3.
[0388] It will be appreciated that in the following general methods
and preparation of synthetic intermediates, reagent levels and
relative amounts or reagents/intermediates can be changed to suit
particular compounds to be synthesized, up or down by up to 50%
without significant change in expected results.
[0389] Method A: General Method for Deprotection Followed by
Coupling Reaction Using EDC/HOBt/DIPEA.
##STR00065##
[0390] HCl Solution in dioxane (4M, 5 ml) was added to respective
carbamate compound (0.125 mmol) and stirred for 2 hrs at RT. The
reaction mixture was concentrated to dryness under vacuum and
swapped with MeOH (5 ml) three times. The resulting residue was
dried well under vacuum and subjected to next reaction as it was.
The residue obtained above, respective acid moiety (0.125 mmol),
EDC (0.19 mmol), HOBt (0.16 mmol) and DIPEA (0.5 mmol) were stirred
in anhydrous DCM (5 ml) for 16 hrs. The reaction mixture was
concentrated under vacuum to give the crude product which was
purified on a C18 column using 10-50% MeOH in water to yield
product as an off-white solid (35-55%).
[0391] Method B: General Method for Deprotection Followed by
Reaction with Anhydride.
##STR00066##
[0392] HCl Solution in dioxane (4M, 5 ml) was added to a
representative Boc-protected compound (0.125 mmol) and stirred for
2 hrs at RT. The reaction mixture was concentrated to dryness under
vacuum and washed with MeOH (5 ml) three times. The resulting
residue was dried well under vacuum and subjected to next reaction
as it was. The residue obtained above, the respective anhydride
moiety (0.125 mmol), and triethylamine (0.5 mmol) were added to
anhydrous DCM (5 mL) and stirred for 16 hrs. The mixture was
concentrated under vacuum to give the crude product which was
purified on a C18 column using 10-50% MeOH in water to yield
product as an off-white solid (40-60%).
[0393] Method C: General Method of Coupling Reaction Using
HATU/DIPEA.
##STR00067##
[0394] The respective acid moiety (0.125 mmol), HATU (0.17 mmol),
DIPEA (0.5 mmol) and respective amine moiety (0.125 mmol) were
stirred in anhydrous DCM (5 ml) for 16 hrs. The reaction mixture
was concentrated under vacuum to give the crude product which was
purified on a C18 column using 10-50% MeOH in water (or similar
ratio as needed) to yield product as an off-white solid
(35-55%).
[0395] Method D: General Method of Hydrolysis Using LiOH.
##STR00068##
[0396] To the stirring solution of the ester compound (0.08 mmol)
in ethanol (1 ml) was added solution of lithium hydroxide
monohydrate (0.4 mmol) in water (0.5 ml). After stirring the
reaction mixture for 5 hrs at RT, the mixture was acidified using
citric acid (saturated solution) and concentrated under vacuum to
give the crude product which was purified on a C18 column using
10-40% MeOH in water to yield product as an off-white solid
(50-65%).
[0397] Method E: General Method for Boc Deprotection.
##STR00069##
[0398] HCl Solution in dioxane (4M, 0.5 ml) was added to the
respective carbamate compound (0.06 mmol) and stirred for 3 hrs at
RT. The reaction mixture was concentrated under vacuum to give the
crude product which was purified on a C18 column using 10-40% MeOH
in water to yield product as an off-white solid (50-60%).
[0399] Method F: General Method for Deprotection Followed by
Reaction with Anhydride).
##STR00070##
[0400] This method is an improved procedure for the method B. HCl
Solution in dioxane (4M, 5 ml) was added to a representative
Boc-protected compound (0.125 mmol) and stirred for 2 hrs at RT.
The reaction mixture was concentrated to dryness under vacuum and
swapped with MeOH (5 ml) three times. The resulting residue was
dried well under vacuum and subjected to next reaction as it was.
The residue obtained above, the respective anhydride moiety (0.19
mmol, 1.5 eq.), and triethylamine (0.5 mmol, 4 eq.) were added to
anhydrous DCM (5 mL) and stirred for 16 hrs. The mixture was
acidified with formic acid and then concentrated under vacuum to
give the crude product which was purified on a C18 column using
25-65% MeOH in water to yield product as an off-white solid
(30-80%).
[0401] Method G: General Method for Boc Protection.
##STR00071##
[0402] To respective amine compound (6.1 mmol) in dioxane (6 ml)
and NaOH solution (0.5M, 12 ml) was added slowly solution of
di-tert-butyl dicarbonate (6.7 mmol) in dioxane (6 ml) at 0.degree.
C. The reaction mixture was warmed to RT and stirred overnight. The
reaction mixture was then washed with hexanes (10 ml). The
separated water layer was acidified using saturated solution of
citric acid and extracted with ethyl acetate (3.times.15 ml). The
organic layer was washed with brine, separated, dried over sodium
sulfate and concentrated to give Boc protected amine compound as
off-white solid (65-90%).
[0403] Method H: General Method for EDC/HOBt/DIPEA Coupling of an
Intermediate Existing as an HCl Salt and a Free Carboxylic
Acid.
##STR00072##
[0404] To an intermediate collected as an HCl salt (0.125 mmol)
were added the carboxylic acid (0.125 mmol), EDC (0.19 mmol), HOBt
(0.16 mmol), and anhydrous DCM (5 ml). The flask was purged with
N.sub.2, sonicated for 20 s and DIPEA (0.5 mmol) was added. The
reaction was stirred at room temperature for 6 hrs then
concentrated under reduced pressure. The residue was purified on a
C18 column using 10-80% MeOH in water to yield the product as an
off-white solid (40-90%).
[0405] Method I: General Method for Coupling
(2H-Tetrazol-5-Yl)Methylamine and a Free Carboxylic Acid.
##STR00073##
[0406] To the carboxylic acid (0.18 mmol), were added the
(2H-tetrazol-5-yl)methylamine (0.22 mmol), EDC (0.275 mmol), HOBt
(0.22 mmol), and anhydrous DMF (15 ml). The flask was purged with
N.sub.2, sonicated for 20 s and DIPEA (0.73 mmol) was added. The
reaction was stirred at room temperature for 16 hrs. Analysis of
the reaction by LC/MS showed approximately 75% conversion of the
acid. An additional one half of the portion of the amine, EDC,
HOBt, and DIPEA were added and the reaction was heated at
45.degree. C. for another 6 hrs then concentrated under reduced
pressure. The residue was purified on a C18 column using 10-70%
MeOH in water to yield the product as an off-white solid
(40-95%).
[0407] Method J: General Method for Hydrogenate Deprotection of
Benzyl Esters or Benzyl Carbamates.
##STR00074##
[0408] To a flask containing the respective benzylated compound
(1.0 eq.) under a nitrogen atmosphere was added palladium on carbon
(10 wt %, wetted, 0.2 eq.) then MeOH (0.05 M). The atmosphere was
changed to hydrogen (vacuum+H.sub.2 backfill.times.3) and the
suspension of black solids was stirred for 3 hrs, then filtered
over a pad of CELITE.TM. and washed with excess MeOH. The reaction
mixture was concentrated under vacuum to give the crude product
which was purified on a C18 column using 10-50% MeOH in water to
yield the product (50-95%).
[0409] The following examples are provided for the purpose of
illustrating, not limiting, the invention.
EXAMPLES
Synthetic Intermediates
[0410] The following is a description of synthetic intermediates
(I-1 to I-12) useful for making representative compounds of the
invention.
[0411] Intermediate I-1
##STR00075##
2-Bromo-3-(bromomethyl)pyridine (I-1)
[0412] This intermediate was generated by a modified procedure
based on that disclosed in Rebek, J., et al., J. Am. Chem. Soc.,
107, 7487 (1985)). A three-neck round bottom flask with a stir bar
was flame dried, cooled under vacuum and purged with N.sub.2. To
the flask were added 2-bromo-3-methylpyridine (5.2 mL, 29.1 mmol),
N-bromosuccinimide (5.5 g, 32.0 mmol), and degassed benzene (126
mL). The flask was fitted with a condenser, heated to 40.degree. C.
and AIBN (0.24 g, 1.5 mmol) was added in several portions. The
reaction was irradiated using a sun lamp as it was stirred at
40.degree. C. The reaction was monitored using TLC and HPLC and was
stopped after 80% conversion of the pyridine reagent (approximately
8 hrs). The reaction was concentrated under reduced pressure, then
redissolved in 4:1 DCM/EtOAc (120 mL) and extracted once with 50 mL
of a saturated solution of NaHCO.sub.3(aq), water and a saturated
solution of NaCl.sub.(aq). The organic phase was dried over
anhydrous sodium sulphate, filtered and concentrated. Upon standing
the residue could not be fully redissolved in DCM and the resultant
suspension was filtered to remove the insoluble solid. The filtrate
was concentrated to near dryness and the residue was purified by
normal phase flash chromatography (EtOAc/Hexanes) to give the title
compound I-1 (3.0 g, 11.9 mmol, 41%) as a yellow solid. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 8.33 (1H, dd, J=5.2 Hz), 7.78 (1H,
dd, J=7.2 Hz), 7.28 (1H, dd, J=5.4 Hz), 4.57 (2H, s), MS (LC/MS)
m/z observed 249.97, expected 249.89 [M+H]
[0413] Intermediate I-2
##STR00076##
tert-butyl 2-((diphenylmethylene)amino)acetate (I-2)
[0414] This intermediate was generated by a generic procedure based
on that disclosed in US2010/0189644 and O'Donnell, Acc. Chem. Res.,
37, 506 (2004). A round bottom flask was charged with a stir-bar,
diphenylmethanimine (8.6 g, 47.5 mmol), tert-butyl 2-bromoacetate
(9.3 g, 47.5 mmol), and acetonitrile (40 mL). The reaction was
heated to 70.degree. C. and DIPEA (8.3 mL, 47.5 mmol) was added
slowly. The flask was fitted with a reflux condenser and heated at
70.degree. C. for 16 hrs. Analysis of the reaction by HPLC and TLC
showed complete conversion of the reactants and the reaction was
cooled to room temperature. A solution of 5:3 water/formic acid (1
mL) was added the reaction was concentrated under reduced pressure.
The resultant solid was filtered and washed 2.times.60 mL of a cold
solution of 3:1 water/ethanol and once with 30 mL of a cold
solution of 1:1 water/ethanol. The solid was dried under high
vacuum to give tert-butyl 2-((diphenylmethylene)amino)acetate (I-2)
as a white solid (14.9 g, 47.0 mmol, 99%). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.66 (2H, m), 7.47 (3H, m), 7.41 (1H, t, J=8
Hz), 7.34 (1H, t, J=8 Hz), 7.20 (2H, m), 4.13 (2H, s), 1.48 (9H,
s), MS (LC/MS) m/z observed 295.93, expected 296.16 [M+H].
[0415] Intermediate I-3
##STR00077##
(1S,2S,4S,5R)-1-(anthracen-9-ylmethyl)-2-(hydroxy(quinolin-4-yl)methyl)-5-
-vinylquinuclidin-1-ium Chloride (I-3)
[0416] This intermediate was generated by a procedure based on that
disclosed in Corey, E. J., et al., J. Am. Chem. Soc., 119, 12414
(1997). A round bottom flask was charged with a stir-bar,
quinolin-4-yl((1S,2S,4S,5R)-5-vinylquinuclidin-2-yl)methanol (1.5
g, 5.10 mmol) also known as cinchonine, 9-(chloromethyl)anthracene
(1.21 g, 5.35 mmol) and toluene (15 mL). The flask was fitted with
a condenser and heated for 2 hrs at 110.degree. C. Conversion of
the amine was confirmed by LCMS and the reaction was cooled to room
temperature and poured into 100 mL of diethyl ether. The formed
yellow precipitate was filtered and washed with 2.times.10 mL of
cold DCM. The solid was set aside and the filtrate was concentrated
and suspended overnight in 10% Et.sub.2O/DCM at 0.degree. C. The
cold suspension was filtered. The solids were pooled together and
dried on high vacuum to give the title compound
(1S,2S,4S,5R)-1-(anthracen-9-ylmethyl)-2-(hydroxy(quinolin-4-yl)methyl)-5-
-vinylquinuclidin-1-ium chloride (I-3) as a bright yellow solid
(2.6 g, 5.0 mmol, 98%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
9.06 (1H, d, J=8 Hz), 8.84 (2H, d, J=4 Hz), 8.73 (1H, d, J=8 Hz),
8.20 (1H, d, J=4 Hz), 8.03 (1H, d, J=4 Hz), 7.99 (1H, s), 7.70-7.55
(3H, m), 7.40 (1H, d, J=8 Hz), 7.30-7.15 (6H, m), 7.15-7.05 (2H,
m), 6.83 (1H, t, J=14 Hz), 6.68 (1H, t, J=14 Hz), 5.44 (1H, m),
4.91 (1H, dd, J=10.4 Hz), 4.74 (2H, m), 6.83 (1H, d, J=14 Hz), 6.68
(1H, d, J=14 Hz), 5.44 (1H, m), 5.27 (1H, d, J=16 Hz), 6.68 (1H,
dd, J=8.3 Hz), 4.74 (2H, m), 2.59 (1H, dd, J=14.12 Hz), 2.42 (1H,
m), 2.36 (2H, s), 2.13 (1H, m), 1.90-1.75 (3H, m), 1.70 (1H, m),
2.42 (1H, m), 1.12 (1H, m), 1.01 (1H, m), MS (LC/MS) m/z observed
485.08, expected 485.26 [M-C1].
[0417] Intermediate I-4
##STR00078##
(1S,2S,4S,5R)-2-((allyloxy)(quinolin-4-yl)methyl)-1-(anthracen-9-ylmethyl-
)-5-vinylquinuclidin-1-ium bromide (I-4)
[0418] This catalyst was generated by a procedure based on that
disclosed in Corey, E. J., et al., J. Am. Chem. Soc., 119, 12414
(1997). A round bottom flask with a stir bar was flame dried,
cooled under vacuum and purged with N.sub.2. To the flask were
added I-3 (1.0 g, 1.92 mmol), allyl bromide (0.5 mL 5.76 mmol) and
DCM (8 mL). To the yellow foamy mixture was added a solution of 50%
w/w KOH (2 mL, 9.60 mmol) at RT. A slight exotherm was observed.
Analysis of the reaction by HPLC after 4 hrs showed complete
conversion of I-3 and the reaction was diluted with 30 mL of DCM
and water and transferred to a separatory funnel. The organic phase
was collected, then extracted 2.times.20 mL of water and washed
with a saturated solution of NaCl.sub.(aq). The organic phase was
dried over anhydrous sodium sulphate, filtered and concentrated. To
the residue was added 8 mL of methanol, which produced a clear red
solution with a small amount of precipitate. Diethyl ether was
slowly added to the solution at 0.degree. C. and the solution
became cloudy. After the addition of 50 mL of ether the precipitate
was filtered, washed once with cold ether (10 mL) and dried under
high vacuum to give the title compound
(1S,2S,4S,5R)-2-((allyloxy)(quinolin-4-yl)methyl)-1-(anthracen-9-ylmethyl-
)-5-vinylquinuclidin-1-ium bromide (I-4) (0.63 g, 1.03 mmol, 54%).
MS (LC/MS) m/z observed 525.08, expected 525.29 [M+H]. Compound was
confirmed using LC/MS and moved to next step as it was.
[0419] Intermediate I-5
##STR00079##
(S)-tert-butyl 3-(2-bromopyridin-3-yl)-2-((diphenylmethylene)amino)
propanoate (I-5)
[0420] This intermediate was generated by a modified procedure
based on that disclosed in Viswanathan, R., et al., J. Am. Chem.
Soc., 125, 163 (2003) and Synthesis 2, 330 (2005). A three neck
round bottom flask with a stir bar was charged with I-2 (40.1 g,
135.7 mmol), I-4 (8.2 g, 13.6 mmol), powdered KOH (69.1 g, 1221.4
mmol), and DCM (600 mL). The opaque yellow suspension was cooled to
-78.degree. C. and the flask fitted with a dropping funnel. A
suspension of I-1 (152.0 g, 610.7 mmol) in 400 mL DCM was
transferred to the dropping funnel and added to the reaction at
-78.degree. C. over about 1 hr. The suspension in the dropping
funnel would occasionally settle and the solid would be
resuspended. After the end of the addition the funnel was rinsed
with an additional 200 mL of DCM and the rinse was added to the
reaction. After 10 hrs at -78.degree. C. the reaction was allowed
to stir overnight as it warmed to room temperature. Analysis of the
reaction by HPLC and TLC showed complete conversion of I-2. The
reaction was diluted with 3 L of DCM, transferred to a 15 L reactor
and extracted 2.times.1 L of water. During the separation the
organic phase appeared cloudy due to a solid formed from I-1. The
organic phase was collected, then washed with a saturated aqueous
solution of NaCl, dried over anhydrous sodium sulphate, filtered
and concentrated to near dryness and purified by normal phase flash
chromatography. A three solvent mobile phase was used for the
separation; initially DCM/hexanes to elute the excess I-1, followed
by EtOAc/Hexanes to elute the title compound (S)-tert-butyl
3-(2-bromopyridin-3-yl)-2-((diphenylmethylene)amino) propanoate
(I-5) obtained as a yellow solid (23.1 g, 226.0 mmol, 37%). .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 8.20 (1H, dd, J=4.2 Hz), 7.60
(2H, d, J=8 Hz), 7.56 (1H, dd, J=4.2 Hz), 7.45-7.25 (6H, m), 7.12
(1H, dd, J=8.4 Hz), 6.67 (1H, d, J=d Hz), 4.39 (1H, dd, J=8.4 Hz),
3.39 (1H, dd, J=12.4 Hz), 3.21 (1H, dd, J=12.4 Hz), 1.46 (9H, s),
MS (LC/MS) m/z observed 464.87, expected 465.12 [M+H].
[0421] Intermediate I-6
##STR00080##
(S)-tert-butyl
1-benzhydryl-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate
(I-6)
[0422] This intermediate was generated by a modified procedure
based on that disclosed in Viswanathan, R., et al., J. Am. Chem.
Soc., 125, 163 (2003) and Synthesis 2, 330 (2005). A three-neck
round bottom flask with a stir bar was flame dried, cooled under
vacuum and purged with N.sub.2. To the flask were added I-5 (3.0 g,
6.46 mmol), tri-n-butyltin hydride (3.8 mL, 14.2 mmol), and
degassed toluene (646 mL). The flask was fitted with a condenser,
and a dropping funnel and heated to 85.degree. C. A solution of
AIBN (1.27 g, 7.8 mmol) in 40 mL toluene was prepared in the
dropping funnel and added to the reaction over the course of 1 hr.
After 2 hrs the reaction was monitored by LCMS and approximately
50% conversion. Another portion of tri-n-butyltin hydride was added
and the reaction was heated at 85.degree. C. for another 4 hrs.
Analysis of the reaction by TLC, HPLC and LCMS showed complete
conversion of I-5. The reaction was concentrated to near dryness
and to the residue was added 250 mL of diethyl ether and 100 mL of
a saturated solution of KF.sub.(aq). The biphasic mixture was
stirred vigorously at room temperature for 3 hrs during with time a
white solid formed at the interface and on the flask wall. The
mixture was filtered through CELITE.TM. and the cake washed with
200 mL of diethyl ether. The filtrate was transferred to a
separatory funnel, the organic phase was collected, dried over
anhydrous sodium sulphate, filtered and concentrated to near
dryness. The residue was purified by normal phase flash
chromatography (EtOAc/Hexanes) to give the title compound I-6 as an
off-white solid (1.37 g, 3.5 mmol, 55%). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.87 (1H, d, J=4 Hz), 7.41 (2H, d, J=8 Hz),
7.35-7.15 (8H, m), 6.55-6.45 (2H, m), 4.21 (1H, dd, J=10.6 Hz),
3.43 (1H, dd, J=18.10 Hz), 3.21 (1H, dd, J=18.6 Hz), 1.73 (9H, s),
MS (LC/MS) m/z observed 487.04, expected 487.21 [M+H].
[0423] Intermediate I-7
##STR00081##
(2S)-2-Carboxy-2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-ium (I-7)
[0424] This intermediate was generated by a modified procedure
based on that disclosed in Viswanathan, R., et al., J Am. Chem.
Soc., 125, 163 (2003) and Synthesis 2, 330 (2005). A round bottom
flask was charged with I-6 (670 mg, 1.7 mmol), DCM (5 mL) and
triethylsilane (1 mL, 8.65 mmol). To the clear yellow solution was
added TFA (3.3 mL) at room temperature and the yellow/orange
reaction was stirred at room temperature for 16 hrs. Analysis of
the reaction by HPLC showed complete conversion of I-6 and the
reaction was concentrated to approximately one quarter of the
volume. Diethyl ether (60 mL) was added slowly to the residue,
which resulted in the precipitation of a fine white solid. The
mixture was cooled to 0.degree. C. for 10 min then sonicated and
filtered. The white solid was washed with 10 mL of cold diethyl
ether to give
(2S)-2-carboxy-2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-ium
trifluoroacetate (I-7) (263 mg, 0.93 mmol, 54%). .sup.1H NMR (400
MHz, DMSO) .delta. 8.52 (1H, bs), 7.69 (1H, d, J=4 Hz), 7.55 (1H,
d, J=4 Hz), 6.68 (2H, dd, J=8.4 Hz), 4.59 (1H, dd, J=12.4 Hz), 3.45
(1H, dd, J=16.12 Hz), 3.15 (1H, dd, J=20.4 Hz), MS (LC/MS) m/z
observed 165.02, expected 165.07 [M-C1].
[0425] Intermediate I-8
##STR00082##
2-Phenylacetic acid (I-8)
[0426] A solution of methyl 2-phenylacetate (10 g, 64 mmol) in
methanol (60 ml) was treated with solution of sodium hydroxide (5.1
g, 127 mmol) in water (40 ml) at 70.degree. C. for 3 hrs. The
resulting mixture was concentrated under vacuum to remove the
methanol. The residue was diluted with water (40 ml) and washed
with diethyl ether (40 ml). The separated water layer was acidified
to pH 2 using a mixture of water and HCl (1:1) and extracted with
DCM (3.times.80 ml). Combined organic extracts were washed with
brine, 80 ml, separated, dried over sodium sulfate and concentrated
to give 2-phenylacetic acid (I-8) as a white solid (9 g, 96%) used
without further characterization. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 3.64 (2H, s), 7.27-7.35 (5H, m), 11.5 (1H, bs).
[0427] Intermediate I-9
##STR00083##
(2S,3S)-3-Methyl-2-(2-phenylacetamido)pentanoic acid (I-9)
[0428] I-8 (2.0 g, 14.7 mmol) and thionyl chloride (6.6 ml, 90.3
mmol) were stirred together for 1 hr at RT. Thionyl chloride was
removed by distillation under vacuum. The acid chloride was added
to the stirring solution of L-isoleucine (1.75 g, 13.4 mmol) in
NaOH (2N, 17 ml) at 0.degree. C. The resulting mixture was warmed
to RT and stirred overnight. The mixture was washed with diethyl
ether (20 ml) and acidified to pH 4-5 by adding citric acid
(aqueous, saturated solution). The precipitated solid was filtered,
washed with diethyl ether and dried to yield
(2S,3S)-3-methyl-2-(2-phenylacetamido)pentanoic acid (I-9) as a
white solid (1.6 g, 44%). .sup.1H NMR (400 MHz, DMSO-d6) .delta.
0.78-0.82 (6H, t, J=8 Hz), 1.12-1.18 (1H, m), 1.34-1.40 (1H, m),
1.72-1.78 (1H, m), 3.41-3.53 (2H, q, J=16 Hz), 4.13-4.16 (1H, dd,
J=4.12 Hz), 7.15-7.19 (1H, m), 7.22-7.28 (1H, m), 8.19-8.21 (1H, d,
J=8 Hz), 12.54 (1H, s), MS (LC/MS) m/z observed 250.02, expected
250.14 [M+H].
[0429] Intermediate I-10
##STR00084##
(S)-3-methyl-2-(2-phenylacetamido)butanoic (I-10)
[0430] I-8 (0.5 g, 3.67 mmol) and thionyl chloride (1.6 ml, 22
mmol) were stirred together for 1 hr at room temperature. Thionyl
chloride was removed by distillation under vacuum. The acid
chloride was added to the stirring solution of L-valine (0.39 g,
3.31 mmol) in NaOH (2N, 4.2 ml) at 0.degree. C. The resulting
reaction mixture was warmed to RT and stirred overnight. The
mixture was washed with diethyl ether (5 ml) and acidified to pH
4-5 by adding citric acid (aqueous, saturated solution). The
precipitated solid was filtered, washed with diethyl ether and
dried to yield (S)-3-methyl-2-(2-phenylacetamido)butanoic acid
(I-10) as a white solid (0.64 g, 74%). .sup.1H NMR (400 MHz,
DMSO-d6) .delta. 0.82-0.84 (3H, d, J=8 Hz), 0.85-0.87 (3H, d, J=8
Hz), 1.99-2.06 (1H, m), 3.44-3.55 (2H, q, J=12 Hz), 4.10-4.14 (1H,
dd, J=8.12 Hz), 7.16-7.21 (1H, m), 7.24-7.29 (4H, m), 7.19-7.21
(1H, d, J=8 Hz), 12.55 (1H, s), MS (LC/MS) m/z observed 236.04,
expected 236.13 [M+H].
[0431] Intermediate I-11
##STR00085##
Ethyl
2-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanamido)aceta-
te (I-11)
[0432] Boc-L-Isoleucine and glycine ethyl ester hydrochloride were
combined using method A except the purification was performed on
normal phase using 0% to 30% ethyl acetate in hexanes as the eluent
to yield ethyl
2-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanamido)aceta-
te (I-11). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.91 (3H, t,
J=7 Hz), 0.96 (3H, d, J=7 Hz), 1.14 (1H, m), 1.28 (3H, t, J=7 Hz),
1.45 (9H, s), 1.51 (1H, m), 1.92 (1H, m), 3.95-4.12 (3H, m), 4.22
(2H, q, J=7 Hz), 5.55 (1H, d, J=9 Hz), 6.52 (1H, bs), MS (LC/MS)
m/z observed 317.42, expected 317.21 [M+H].
[0433] Intermediate I-12
##STR00086##
Ethyl
2-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanamido)aceta-
te (I-12)
[0434] Intermediate I-12 was prepared from I-11 using method D with
2 eq. of LiOH.H.sub.2O. .sup.1H NMR (400 MHz, DMSO-d6) .delta.
0.74-0.85 (6H, m), 1.08 (1H, m), 1.31-1.41 (10H, m), 1.71 (1H, m),
3.38-4.50 (2H, m), 3.80 (1H, t, J=8 Hz), 6.85 (1H, d, J=9 Hz), 7.50
(1H, bs), MS (LC/MS) m/z observed 288.88, expected 289.18
[M+H].
Representative Granzyme B Inhibitor Compounds
[0435] The following is a description of the preparation of
representative Granzyme B inhibitor compounds of the invention.
[0436] Example A1 was prepared by the representative synthetic
pathway illustrated schematically in FIG. 1.
Example A1
(S)-1-(2-((2S,3S)-2-(2-(2H-TETRAZOL-5-YL)ACETAMIDO)-3-METHYLPENTANAMIDO)AC-
ETYL)-N-((2H-TETRAZOL-5-YL)METHYL)-2,3-DIHYDRO-1H-PYRROLO[2,3-B]PYRIDINE-2-
-CARBOXAMIDE
[0437] Ethyl
2-((2S,3S)-2-(2-(1H-tetrazol-5-yl)acetamido)-3-methylpentanamido)
acetate (620 mg, 1.64 mmol, 57%) was collected as an off-white
solid from I-12 (0.91 g, 2.88 mmol) and 2-(2H-tetrazol-5-yl)acetic
(307 mg, 2.4 mmol) using method A in DMF. MS (LC/MS) m/z observed
326.86, expected 327.18 [M+H]. Compound was confirmed using LC/MS
and moved to next step as it was.
[0438] A round bottom flask was charged with a stir bar, ethyl
2-((2S,3S)-2-(2-(1H-tetrazol-5-yl)acetamido)-3-methylpentanamido)
acetate (290 mg, 0.89 mmol), LiOH (94 mg, 2.23 mmol), tert-BuOH
(6.6 ml), and water (3.3 mL). The reaction was stirred at room
temperature for 2 hrs. Analysis of the reaction by LC/MS showed
complete conversion and concentrated HCl (aqueous) was added to
reach pH 2. The reaction was concentrated under reduced pressure
and reconcentrated from tert-BuOH. The remaining off white solid
contained
2-((2S,3S)-2-(2-(1H-tetrazol-5-yl)acetamido)-3-methylpentanamido)acetic
acid. (MS (LC/MS) m/z observed 298.89, expected 299.15 [M+H].
Compound was confirmed using LC/MS and moved to next step as it
was.
[0439]
(S)-1-(2-((2S,3S)-2-(2-(1H-Tetrazol-5-yl)acetamido)-3-methylpentana-
mido)acetyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic
acid was prepared from I-7 and
2-((2S,3S)-2-(2-(1H-tetrazol-5-yl)acetamido)-3-methylpentanamido)acetic
acid (3 eq.) using method C in DMF. (LC/MS) m/z observed 445.04,
expected 445.19 [M+H]. Compound was confirmed using LC/MS and moved
to next step as it was.
[0440]
(S)-1-(2-((2S,3S)-2-(2-(1H-Tetrazol-5-yl)acetamido)-3-methylpentana-
mido)acetyl)-N-((2H-tetrazol-5-yl)methyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyr-
idine-2-carboxamide (A1) was prepared from
(S)-1-(2-((2S,3S)-2-(2-(1H-tetrazol-5-yl)acetamido)-3-methylpentanamido)a-
cetyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid and
(2H-tetrazol-5-yl)methyl-amine using method A in DMF but without
HCl treatment. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.82 (3H, t,
J=7 Hz), 0.90 (3H, d, J=7 Hz), 1.12 (1H, m), 1.48 (1H, m), 1.75
(1H, m), 2.99 (1H, dd, J=4, 17 Hz), 3.46 (1H, dd, J=11, 18 Hz),
3.93-4.04 (2H, m), 4.31 (1H, t, J=8 Hz), 4.45-4.53 (2H, m), 4.62
(1H, dd, J=6, 16 Hz), 4.71 (1H, dd, J=4, 18 Hz), 4.96 (1H, dd, J=4,
11 Hz), 7.03 (1H, dd, J=5, 7 Hz), 7.65 (1H, d, J=7 Hz), 8.15 (1H,
d, J=5 Hz), 8.35 (1H, t, J=6 Hz), 8.47 (1H, d, J=9 Hz), 8.95 (1H,
t, J=5 Hz), (MS (LC/MS) m/z observed 526.06, expected 526.24
[M+H].
[0441] Examples C1-C41 were prepared by the representative
synthetic pathway illustrated schematically in FIG. 3.
Example C1
3-{[(1S,2S)-2-METHYL-1-({2-OXO-2-[(2S)-2-[(2H-1,2,3,4-TETRAZOL-5-YLMETHYL)-
CARBAMOYL]-2,3-DIHYDRO-1H-PYRROLO[2,3-B]PYRIDIN-1-YL]ETHYL}CARBAMOYL)BUTYL-
]CARBAMOYL}PROPANOIC ACID
[0442] Intermediate I-7 (600 mg, 2.160 mmol) was suspended in EtOH
(40 mL) at 0.degree. C. and thionyl chloride (0.313 mL, 4.320 mmol,
2 eq.) was added dropwise. The resulting clear mixture was allowed
to come to RT and stirred for 16 hours. The reaction mixture was
then concentrated to dryness and swapped with EtOH (2.times.25 mL).
The solid obtained was dried well under reduced pressure to give
(S)-ethyl 2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate as a
white solid (494 mg, quantitative). MS (LC/MS) m/z observed 193.52,
expected 193.10 [M+H]. Compound was confirmed using LC/MS and moved
to next step as it was.
[0443] (S)-Ethyl
1-(2-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanamido)acetyl)--
2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate was prepared
from (S)-ethyl 2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate
and I-12 using method C. (LC/MS) m/z observed 463.05, expected
463.26 [M+H]. Compound was confirmed using LC/MS and moved to next
step as it was.
[0444]
(S)-1-(2-((2S,3S)-2-((tert-Butoxycarbonyl)amino)-3-methylpentanamid-
o)acetyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid
was prepared from (S)-ethyl
1-(2-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanamido)acetyl)--
2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate using method D
with 2 eq of LiOH.H.sub.2O. MS (LC/MS) m/z observed 435.05,
expected 435.22 [M+H]. Compound was confirmed using LC/MS and moved
to next step as it was.
[0445] tert-Butyl
((2S,3S)-1-((2-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1-
H-pyrrolo[2,3-b]pyridin-1-yl)-2-oxoethyl)amino)-3-methyl-1-oxopentan-2-yl)-
carbamate was prepared from
(S)-1-(2-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanamido)acet-
yl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid and
(2H-tetrazol-5-yl)methyl-amine using method A in DMF but without
HCl treatment. MS (LC/MS) m/z observed 516.04, expected 516.27
[M+H]. Compound was confirmed using LC/MS and moved to next step as
it was.
[0446]
3-{[(1S,2S)-2-Methyl-1-({2-oxo-2-[(2S)-2-[(2H-1,2,3,4-tetrazol-5-yl-
methyl)carbamoyl]-2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-yl]ethyl}carbamoy-
l)butyl]carbamoyl}propanoic acid (C1) was prepared from tert-butyl
((2S,3S)-1-((2-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1-
H-pyrrolo[2,3-b]pyridin-1-yl)-2-oxoethyl)amino)-3-methyl-1-oxopentan-2-yl)-
carbamate and succinic anhydride using method I. .sup.1H NMR (400
MHz, DMSO-d6) .delta. 0.81 (3H, t, J=7 Hz), 0.87 (3H, d, J=7 Hz),
1.10 (1H, m), 1.46 (1H, m), 1.72 (1H, m), 2.34-2.46 (4H, m), 3.00
(1H, dd, J=4, 17 Hz), 3.42 (1H, dd, J=11, 18 Hz), 4.24 (1H, t, J=8
Hz), 4.43-4.52 (2H, m), 4.58-4.74 (2H, m), 4.96 (1H, dd, J=4, 11
Hz), 7.00 (1H, dd, J=5, 7 Hz), 7.64 (1H, d, J=7 Hz), 7.91 (1H, d,
J=9 Hz), 8.12-8.18 (2H, m), 8.92 (1H, t, J=6 Hz), MS (LC/MS) m/z
observed 516.11, expected 516.23 [M+H].
Example C2
(S)-N-((1H-1,2,3-TRIAZOL-4-YL)METHYL)-1-(2-((2S,3S)-3-METHYL-2-(2-PHENYLAC-
ETAMIDO)PENTANAMIDO)ACETYL)-2,3-DIHYDRO-1H-PYRROLO[2,3-B]PYRIDINE-2-CARBOX-
AMIDE
[0447]
(S)-1-(2-((tert-Butoxycarbonyl)amino)acetyl)-2,3-dihydro-1H-pyrrolo-
[2,3-b]pyridine-2-carboxylic acid was prepared from I-7 and
Boc-glycine (3 eq.) using method C in DMF. (LC/MS) m/z observed
322.63, expected 322.14 [M+H]. Compound was confirmed using LC/MS
and moved to next step as it was.
[0448] (S)-tert-Butyl
(2-(2-(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2-
,3-b]pyridin-1-yl)-2-oxoethyl)carbamate was prepared from
(S)-1-(2-((tert-butoxycarbonyl)amino)acetyl)-2,3-dihydro-1H-pyrrolo[2,3-b-
]pyridine-2-carboxylic acid and (2H-1,2,3-triazol-4-yl)methyl-amine
using method A but without HCl treatment. MS (LC/MS) m/z observed
402.55, expected 402.19 [M+H]. Compound was confirmed using LC/MS
and moved to next step as it was.
[0449]
(S)-N-((1H-1,2,3-Triazol-4-yl)methyl)-1-(2-((2S,3S)-3-methyl-2-(2-p-
henylacetamido)pentanamido)acetyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-
-carboxamide (C2) was prepared from (S)-tert-butyl
(2-(2-(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2-
,3-b]pyridin-1-yl)-2-oxoethyl)carbamate and I-9 using method A.
.sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.76-0.87 (6H, m), 1.10 (1H,
m), 1.44 (1H, m), 1.74 (1H, m), 2.93 (1H, dd, J=4, 17 Hz),
3.40-3.62 (3H, m), 4.27 (1H, m), 4.32-4.37 (2H, m), 4.47 (1H, dd,
J=5, 18 Hz), 4.72 (1H, dd, J=6, 18 Hz), 4.94 (1H, dd, J=4, 11 Hz),
7.02 (1H, dd, J=5, 7 Hz), 7.20 (1H, m), 7.24-7.30 (4H, m),
7.60-7.70 (2H, m), 8.10-8.18 (2H, m), 8.25 (1H, t, J=6 Hz), 8.72
(1H, t, J=6 Hz), MS (LC/MS) m/z observed 533.11, expected 533.26
[M+H].
Example C3
(S)-N-((2H-TETRAZOL-5-YL)METHYL)-1-(2-((2S,3S)-3-METHYL-2-(2-PHENYLACETAMI-
DO)PENTANAMIDO)ACETYL)-2,3-DIHYDRO-1H-PYRROLO[2,3-B]PYRIDINE-2-CARBOXAMIDE
[0450] (S)-tert-Butyl
(2-(2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-b]-
pyridin-1-yl)-2-oxoethyl)carbamate was prepared from
(S)-1-(2-((tert-butoxycarbonyl)amino)acetyl)-2,3-dihydro-1H-pyrrolo[2,3-b-
]pyridine-2-carboxylic acid (from Example C2) and
(2H-tetrazol-5-yl)methyl-amine using method A in DMF but without
HCl treatment. MS (LC/MS) m/z observed 403.35, expected 403.18
[M+H]. Compound was confirmed using LC/MS and moved to next step as
it was.
[0451]
(S)-N-((2H-Tetrazol-5-yl)methyl)-1-(2-((2S,3S)-3-methyl-2-(2-phenyl-
acetamido)pentanamido)acetyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carb-
oxamide (C3) was prepared from (S)-tert-butyl
(2-(2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-b]-
pyridin-1-yl)-2-oxoethyl)carbamate and I-9 using method A. .sup.1H
NMR (400 MHz, DMSO-d6) .delta. 0.76-0.87 (6H, m), 1.08 (1H, m),
1.35 (1H, m), 1.78 (1H, m), 2.98 (1H, dd, J=4, 17 Hz), 3.40-3.62
(3H, m), 4.42 (1H, m), 4.48-4.54 (2H, m), 4.58-4.75 (2H, m), 4.97
(1H, m), 7.02 (1H, dd, J=5, 7 Hz), 7.20 (1H, m), 7.24-7.30 (4H, m),
7.64 (1H, d, J=7 Hz), 7.99 (1H, d, J=9 Hz), 8.10-8.18 (2H, m), 8.95
(1H, t, J=6 Hz), MS (LC/MS) m/z observed 556.15, expected 556.24
[M+Na].
Example C4
(S)-5-((S)-2(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-PYRROLO[2-
,3-B]PYRIDIN-1-YL)-4-((2S,3S)-2-(3-CARBOXYPROPANAMIDO)-3-METHYLPENTANAMIDO-
)-5-OXOPENTANOIC ACID
[0452] To a suspension of Boc-L-glutamic acid y-benzyl ester (2.54
g, 7.2 mmol, 4.0 eq.) in a mixture of DCM:DMF (29 ml, 5:1 (v/v))
was added HATU (1.0 g, 2.7 mmol, 1.5 eq.), then DIPEA (1.6 ml, 9.0
mmol, 5.0 eq.) in that order. The reaction mixture was stirred for
20 minutes whereupon the reaction mixture became a yellow solution.
Intermediate I-7 (0.5 g, 1.8 mmol, 1.0 eq.) was added and the
reaction mixture was stirred and additional 30 minutes. The
reaction mixture was concentrated under vacuum and was purified on
a C18 column using 10-65% MeOH in water to yield the
(S)-1-((S)-5-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-5-oxopentanoyl)-2-
,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid as an
off-orange solid. MS (LC/MS) m/z observed 484, expected 484.20
[M+H].sup.+. Compound was confirmed using LC/MS and moved to next
step as it was.
[0453] (S)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate was
prepared from
(S)-1-((S)-5-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-5-oxopentanoyl)-2-
,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid via method O
but without the second amine addition. MS (LC/MS) m/z observed 565,
expected 565.24 [M+H].sup.+. Compound was confirmed using LC/MS and
moved to next step as it was.
[0454] (S)-Benzyl
5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3--
b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanam-
ido)-5-oxopentanoate was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate and
Boc-L-isoleucine via method A but without swapping with MeOH; and
DMF as the solvent of the coupling step. MS (LC/MS) m/z observed
678; expected 678.33 [M+H].sup.+. Compound was confirmed using
LC/MS and moved to next step as it was.
[0455] To a solution of
(S)-5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[-
2,3-b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpent-
anamido)-5-oxopentanoate benzyl ester (327 mg, 0.48 mmol, 1.0 eq.)
in dioxane (3 ml) was added hydrochloric acid (2 ml, 4.0 M solution
in dioxane, 8.0 mmol, 16 eq.), then stirred at RT for 2 h whereupon
a white paste forms upon the walls of the reaction vessel. The
reaction mixture was concentrated under vacuum to dryness, then
methanol (6 ml), DIPEA (0.67 ml, 3.8 mmol, 8.0 eq.), and succinic
anhydride (236.9 mg, 2.4 mmol, 5.0 eq.) were added in that order.
The reaction mixture was stirred at RT for 1 hr, then acidified to
pH<4 with formic acid, then concentrated under vacuum to
dryness. The reaction vessel was purged with nitrogen, then
palladium on carbon (98 mg, 10 wt %, wetted) and MeOH (10 ml) were
added in that order. The atmosphere changed to hydrogen
(vacuum+H.sub.2 backfill.times.3) and the suspension of black
solids was stirred for 3 hr, then filtered over a pad of CELITE.TM.
and washed with excess MeOH. The reaction mixture was concentrated
under vacuum and purified on a C18 column using 10-60% MeOH in
water to yield the title compound
(S)-5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[-
2,3-b]pyridin-1-yl)-4-((2S,3S)-2-(3-carboxypropanamido)-3-methylpentanamid-
o)-5-oxopentanoic acid (C4) as a white solid (166 mg, 59% over 3
steps). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.72-0.91 (m, 6H),
1.08 (ddd, J=14.4, 10.7, 4.7 Hz, 1H), 1.35-1.47 (m, 1H), 1.59-1.73
(m, 1H), 1.77-1.92 (m, 1H), 1.94-2.07 (m, 1H), 2.25-2.47 (m, 6H),
2.89-3.01 (m, 1H), 3.36-3.53 (m, 1H), 4.20 (t, J=8.2 Hz, 1H), 4.47
(dd, J=15.8, 5.0 Hz, 1H), 4.69 (dd, J=15.9, 6.0 Hz, 1H), 4.99 (dd,
J=11.1, 3.9 Hz, 1H), 5.84 (s, 1H), 7.02 (dd, J=7.3, 5.0 Hz, 1H),
7.65 (d, J=7.4 Hz, 1H), 7.83 (d, J=8.8 Hz, 1H), 8.10-8.22 (m, 2H),
8.97 (t, J=5.5 Hz, 1H). MS (LC/MS) m/z observed 588, expected
588.25 [M+H].sup.+.
Example C5
(S)-5-((S)-2-(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-PYRROLO[-
2,3-B]PYRIDIN-1-YL)-4-((2S,3S)-2-ACETAMIDO-3-METHYLPENTANAMIDO)-5-OXOPENTA-
NOIC ACID
[0456] (S)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((2S,3S)-2-acetamido-3-methylpentanamido)-5-oxopentanoa-
te was prepared from (S)-benzyl
5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3--
b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanam-
ido)-5-oxopentanoate from Example C4 and acetic acid via method A
but with DMF as the solvent. MS (LC/MS) m/z observed 620, expected
620.29 [M+H].sup.+. Compound was confirmed using LC/MS and moved to
next step as it was.
[0457]
(S)-5-((S)-2-(((2H-Tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-p-
yrrolo[2,3-b]pyridin-1-yl)-4-((2S,3S)-2-acetamido-3-methylpentanamido)-5-o-
xopentanoic acid (C5) was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((2S,3S)-2-acetamido-3-methylpentanamido)-5-oxopentanoa-
te via Method Q. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.72-0.87
(m, 6H), 0.99-1.13 (m, 1H), 1.33-1.46 (m, 1H), 1.62 (q, J=8.4, 7.9
Hz, 1H), 1.75-1.88 (m, 4H), 1.91-2.04 (m, 1H), 2.37 (td, J=11.5,
5.1 Hz, 2H), 2.93 (dd, J=17.6, 3.9 Hz, 1H), 3.42 (dd, J=17.4, 11.3
Hz, 1H), 4.15 (t, J=8.3 Hz, 1H), 4.44 (dd, J=15.8, 5.0 Hz, 1H),
4.65 (dd, J=15.9, 6.0 Hz, 1H), 4.96 (dd, J=11.3, 3.9 Hz, 1H), 5.81
(ddd, J=10.9, 7.2, 3.7 Hz, 1H), 7.00 (dd, J=7.4, 5.1 Hz, 1H), 7.63
(d, J=7.4 Hz, 1H), 7.82 (d, J=8.7 Hz, 1H), 8.07-8.22 (m, 2H), 8.92
(t, J=5.5 Hz, 1H). MS (LC/MS) m/z observed 503, expected 530.24
[M+H].sup.+.
Example C6
3-{[(1S,2S)-2-METHYL-1-{[(2S)-1-OXO-1-[(2S)-2-[(2H-1,2,3,4-TETRAZOL-5-YLME-
THYL)CARBAMOYL]-1H,2H,3H-PYRROLO[2,3-B]PYRIDIN-1-YL]PROPAN-2-YL]CARBAMOYL}-
BUTYL]CARBAMOYL}PROPANOIC ACID
[0458]
(S)-1-((S)-2-((tert-Butoxycarbonyl)amino)propanoyl)-2,3-dihydro-1H--
pyrrolo[2,3-b]pyridine-2-carboxylic acid was prepared from I-7 and
Boc-L-alanine (3 eq.) using method C in DMF. (LC/MS) m/z observed
335.85, expected 336.16 [M+H]. Compound was confirmed using LC/MS
and moved to next step as it was.
[0459] tert-Butyl
((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-1-oxopropan-2-yl)carbamate was prepared from
(S)-1-((S)-2-((tert-butoxycarbonyl)amino)propanoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridine-2-carboxylic acid and
(2H-tetrazol-5-yl)methyl-amine using method A in DMF but without
HCl treatment. MS (LC/MS) m/z observed 416.85, expected 417.20
[M+H]. Compound was confirmed using LC/MS and moved to next step as
it was.
[0460] tert-Butyl
((2S,3S)-1-(((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihyd-
ro-1H-pyrrolo[2,3-b]pyridin-1-yl)-1-oxopropan-2-yl)amino)-3-methyl-1-oxope-
ntan-2-yl)carbamate was prepared from tert-butyl
((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-1-oxopropan-2-yl)carbamate and
Boc-L-Isoleucine using method A. MS (LC/MS) m/z observed 529.91,
expected 530.28 [M+H]. Compound was confirmed using LC/MS and moved
to next step as it was.
[0461] Title compound
3-{[(1S,2S)-2-methyl-1-{[(2S)-1-oxo-1-[(2S)-2-[(2H-1,2,3,4-tetrazol-5-ylm-
ethyl)carbamoyl]-1H,2H,3H-pyrrolo[2,3-b]pyridin-1-yl]propan-2-yl]carbamoyl-
}butyl]carbamoyl}propanoic acid (C6) was prepared from tert-butyl
((2S,3S)-1-(((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihyd-
ro-1H-pyrrolo[2,3-b]pyridin-1-yl)-1-oxopropan-2-yl)amino)-3-methyl-1-oxope-
ntan-2-yl)carbamate and succinic anhydride using method I. .sup.1H
NMR (400 MHz, DMSO-d6) .delta. 0.76 (3H, t, J=7 Hz), 0.80 (3H, d,
J=7 Hz), 1.05 (1H, m), 1.28 (3H, d, J=7 Hz), 1.40 (1H, m), 1.63
(1H, m), 2.29-2.42 (4H, m), 2.93 (1H, dd, J=4, 17 Hz), 3.42 (1H,
dd, J=11, 18 Hz), 4.16 (1H, t, J=8 Hz), 4.44 (1H, dd, J=5, 16 Hz),
4.64 (1H, dd, J=6, 16 Hz), 4.96 (1H, dd, J=4, 11 Hz), 5.82 (1H, m),
6.99 (1H, dd, J=5, 7 Hz), 7.63 (1H, d, J=7 Hz), 7.79 (1H, d, J=9
Hz), 8.12 (1H, d, J=6 Hz), 8.18 (1H, d, J=7 Hz), 8.90 (1H, t, J=6
Hz), MS (LC/MS) m/z observed 529.97, expected 530.25 [M+H].
Example C7
(3S)-3-[(2S,3S)-2-(3-CARBOXYPROPANAMIDO)-3-METHYLPENTANAMIDO]-4-OXO-4-[(2S-
)-2-[(2H-1,2,3,4-TETRAZOL-5-YLMETHYL)CARBAMOYL]-1H,2H,3H-PYRROLO[2,3-B]PYR-
IDIN-1-YL]BUTANOIC ACID
[0462]
(S)-1-((S)-4-(tert-Butoxy)-2-((tert-butoxycarbonyl)amino)-4-oxobuta-
noyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-yl)-2-carboxylic acid
was prepared in the same manner as
(S)-1-((S)-5-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-5-oxopentanoyl)-2-
,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid in Example
C4, starting from Boc-L-aspartic acid .beta.-tert-butyl ester. MS
(LC/MS) m/z observed 436; expected 436.20 [M+H].sup.+. Compound was
confirmed using LC/MS and moved to next step as it was.
[0463] (S)-tert-Butyl
4-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3--
b]pyridin-1-yl)-3-((tert-butoxycarbonyl)amino)-4-oxobutanoate was
prepared from
(S)-1-((S)-4-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutan-
oyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-yl)-2-carboxylic acid
via method O but without the second amine addition. MS (LC/MS) m/z
observed 517; expected 517.24 [M+H].sup.+. Compound was confirmed
using LC/MS and moved to next step as it was.
[0464]
(S)-4-((S)-2(((2H-Tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-py-
rrolo[2,3-b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-meth-
ylpentanamido)-4-oxobutanoic acid was prepared from (S)-tert-butyl
4-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3--
b]pyridin-1-yl)-3-((tert-butoxycarbonyl)amino)-4-oxobutanoate and
Boc-L-isoleucine (4 eq.) via method A but without swapping with
MeOH; and DMF as the solvent of the coupling step. MS (LC/MS) m/z
observed 574; expected 574.27 [M+H].sup.+. Compound was confirmed
using LC/MS and moved to next step as it was.
[0465] Title compound
(3S)-3-[(2S,3S)-2-(3-carboxypropanamido)-3-methylpentanamido]-4-oxo-4-[(2-
S)-2-[(2H-1,2,3,4-tetrazol-5-ylmethyl)carbamoyl]-1H,2H,3H-pyrrolo[2,3-b]py-
ridin-1-yl]butanoic acid (C7) was prepared from
(S)-4-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-H-pyrrolo[2-
,3-b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpenta-
namido)-4-oxobutanoic acid via method I but with MeOH as the
solvent for anhydride opening. .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 0.72-0.89 (m, 6H), 1.10-1.24 (m, 1H), 1.32-1.48 (m, 1H),
1.67-1.82 (m, 1H), 2.26-2.47 (m, 6H), 2.71-2.81 (m, 1H), 2.97 (d,
J=17.3 Hz, 1H), 3.45 (dd, J=17.4, 11.2 Hz, 1H), 4.12-4.26 (m, 3H),
4.46 (dd, J=15.9, 5.2 Hz, 1H), 4.62 (td, J=14.5, 13.2, 6.2 Hz, 1H),
4.96 (dd, J=11.1, 3.5 Hz, 1H), 5.97-6.08 (m, 1H), 7.00 (dd, J=7.4,
5.2 Hz, 1H), 7.64 (d, J=7.4 Hz, 1H), 7.75 (d, J=9.3 Hz, 1H),
7.95-8.07 (m, 2H), 8.25 (d, J=6.7 Hz, 1H), 8.86 (t, J=5.8 Hz, 1H).
MS (LC/MS) m/z observed 574, expected 574.23 [M+H].sup.+.
Example C8
(S)-5-((S)-2-(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-PYRROLO[-
2,3-B]PYRIDIN-1-YL)-4-((S)-2-(3-CARBOXYPROPANAMIDO)-2-CYCLOPENTYLACETAMIDO-
)-5-OXOPENTANOIC ACID
[0466] (S)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((S)-2-((tert-butoxycarbonyl)amino)-2-cyclopentylacetam-
ido)-5-oxopentanoate was prepared from (S)-benzyl
5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3--
b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanam-
ido)-5-oxopentanoate (from Example C4) and boc-L-cyclopentylglycine
dicyclohexylammonium salt via method A but with DMF as the solvent.
MS (LC/MS) m/z observed 690, expected 690.33 [M+H].sup.+. Compound
was confirmed using LC/MS and moved to next step as it was.
[0467]
4-(((S)-2-(((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3--
dihydro-1H-pyrrolo[2,3-b]pyridin-1-yl)-5-(benzyloxy)-1,5-dioxopentan-2-yl)-
amino)-1-cyclopentyl-2-oxoethyl)amino)-4-oxobutanoic acid was
prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((S)-2-((tert-butoxycarbonyl)amino)-2-cyclopentylacetam-
ido)-5-oxopentanoate via method I. MS (LC/MS) m/z observed 690,
expected 690.29 [M+H].sup.+. Compound was confirmed using LC/MS and
moved to next step as it was.
[0468] Title compound
(S)-5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo-
[2,3-b]pyridin-1-yl)-4-((S)-2-(3-carboxypropanamido)-2-cyclopentylacetamid-
o)-5-oxopentanoic acid (C8) was prepared from
4-(((S)-2-(((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydr-
o-1H-pyrrolo[2,3-b]pyridin-1-yl)-5-(benzyloxy)-1,5-dioxopentan-2-yl)amino)-
-1-cyclopentyl-2-oxoethyl)amino)-4-oxobutanoic acid via Method Q.
.sup.1H NMR (400 MHz, DMSO-d6) .delta. 1.15-1.63 (m, 8H), 1.92-2.01
(m, 1H), 2.23-2.44 (m, 6H), 2.91 (d, J=17.6 Hz, 1H), 4.13-4.26 (m,
2H), 4.35-4.51 (m, 1H), 4.92-5.04 (m, 1H), 5.75-5.89 (m, 1H),
6.73-6.85 (m, 1H), 6.95-7.02 (m, 1H), 7.62 (d, J=7.3 Hz, 1H), 7.88
(d, J=8.6 Hz, 1H), 8.07-8.17 (m, 2H), 8.31-8.41 (m, 1H). MS (LC/MS)
m/z observed 600, expected 600.25 [M+H].sup.+.
Example C9
(S)-5-((S)-2-(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-PYRROLO[-
2,3-B]PYRIDIN-1-YL)-4-((2S,3S)-3-METHYL-2-(2-PHENYLACETAMIDO)PENTANAMIDO)--
5-OXOPENTANOIC ACID
[0469] (S)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((2S,3S)-3-methyl-2-(2-phenylacetamido)pentanamido)-5-o-
xopentanoate was prepared from (S)-benzyl
5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3--
b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanam-
ido)-5-oxopentanoate (from Example C4) and intermediate I-9 via
method A but without swapping with MeOH; and DMF as the solvent of
the coupling step. MS (LC/MS) m/z observed 696, expected 696.32
[M+H].sup.+. Compound was confirmed using LC/MS and moved to next
step as it was.
[0470] Title compound
(S)-5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo-
[2,3-b]pyridin-1-yl)-4-((2S,3S)-3-methyl-2-(2-phenylacetamido)pentanamido)-
-5-oxopentanoic acid (C9) was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((2S,3S)-3-methyl-2-(2-phenylacetamido)pentanamido)-5-o-
xopentanoate via Method Q. C9 exists as a mixture of rotamers in a
1.5:1 ratio; only the major signals are reported. .sup.1H NMR (400
MHz, DMSO-d6) .delta. 0.71-0.88 (m, 7H), 0.98-1.16 (m, 1H),
1.33-1.47 (m, 1H), 1.74-1.92 (m, 1H), 1.95-2.07 (m, 1H), 2.20-2.45
(m, 2H), 2.96 (d, J=17.1 Hz, 1H), 3.39-3.67 (m, 4H), 4.20 (t, J=8.3
Hz, 1H), 4.48 (dt, J=15.8, 4.8 Hz, 1H), 4.67 (dt, J=14.7, 7.0 Hz,
1H), 4.99 (dd, J=11.2, 3.8 Hz, 1H), 5.85 (tt, J=7.5, 3.6 Hz, 1H),
7.03 (dd, J=7.3, 5.2 Hz, 1H), 7.17-7.23 (m, 1H), 7.23-7.31 (m, 5H),
7.66 (d, J=7.3 Hz, 1H), 8.02 (d, J=8.9 Hz, 1H), 8.14-8.18 (m, 1H),
8.21 (dd, J=7.5, 3.2 Hz, 1H), 8.96 (q, J=5.2 Hz, 1H). MS (LC/MS)
m/z observed 606, expected 606.27 [M+H].sup.+.
Example C10
(S)-5-((S)-2-(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-PYRROLO[-
2,3-B]PYRIDIN-1-YL)-4-((S)-3-METHYL-2-(2-PHENYLACETAMIDO)BUTANAMIDO)-5-OXO-
PENTANOIC ACID
[0471] To a solution of (S)-benzyl
5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3--
b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanam-
ido)-5-oxopentanoate (from Example C4) (85 mg, 0.15 mmol, 1.0 eq.)
in dioxane (1 ml) was added hydrochloric acid (1.5 ml, 4.0 M
solution in dioxane, 6.0 mmol, 40 eq.), then stirred at RT for 2 h
whereupon a white paste forms upon the walls of the reaction
vessel. The reaction mixture was concentrated under vacuum to
dryness and coupled to intermediate I-10 via method C but with DMF
as the solvent to obtain the (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((S)-3-methyl-2-(2-phenylacetamido)butanamido)-5-oxopen-
tanoate. MS (LC/MS) m/z observed 682, expected 682.30 [M+H].sup.+.
Compound was confirmed using LC/MS and moved to next step as it
was.
[0472] Title compound
(S)-5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo-
[2,3-b]pyridin-1-yl)-4-((S)-3-methyl-2-(2-phenylacetamido)butanamido)-5-ox-
opentanoic acid (C10) was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((S)-3-methyl-2-(2-phenylacetamido)butanamido)-5-oxopen-
tanoate via Method Q. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.82
(dd, J=11.7, 6.7 Hz, 6H), 1.74-2.05 (m, 3H), 2.37 (q, J=13.9, 11.9
Hz, 1H), 2.96 (d, J=17.7 Hz, 1H), 3.21-3.62 (m, 4H), 4.20 (t, J=7.9
Hz, 1H), 4.46 (dd, J=15.8, 5.0 Hz, 1H), 4.67 (dd, J=15.9, 6.0 Hz,
1H), 4.93-5.04 (m, 1H), 5.84 (s, 1H), 7.03 (dd, J=7.3, 5.1 Hz, 1H),
7.21 (d, J=6.7 Hz, 1H), 7.27 (d, J=6.4 Hz, 4H), 7.66 (d, J=7.3 Hz,
1H), 7.98 (d, J=8.8 Hz, 1H), 8.16 (d, J=5.2 Hz, 1H), 8.21 (d, J=7.4
Hz, 1H), 8.94 (t, J=5.4 Hz, 1H). MS (LC/MS) m/z observed 592,
expected 592.26 [M+H].sup.+.
Example C11
(S)-5-((S)-2(((1H-1,2,3-TRIAZOL-4-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-PYRR-
OLO[2,3-B]PYRIDIN-1-YL)-4-((2S,3S)-2-(3-CARBOXYPROPANAMIDO)-3-METHYLPENTAN-
AMIDO)-5-OXOPENTANOIC ACID
[0473] (S)-Benzyl
5-((S)-2-(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate
was prepared from
(S)-1-((S)-5-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-5-oxopentanoyl)-2-
,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (from
Example C4) via method O but with 1H-1,2,3-triazole methylamine
hydrochloride in place of (2H-tetrazol-5-yl)methylamine and without
the second amine addition. MS (LC/MS) m/z observed 564, expected
564.25 [M+H].sup.+. Compound was confirmed using LC/MS and moved to
next step as it was.
[0474] (S)-Benzyl
5-((S)-2(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo-
[2,3-b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpen-
tanamido)-5-oxopentanoate was prepared from (S)-benzyl
5-((S)-2-(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate
and Boc-L-isoleucine via method A but without swapping with MeOH;
and DMF as the solvent for the coupling step described therein. MS
(LC/MS) m/z observed 677; expected 677.33 [M+H].sup.+. Compound was
confirmed using LC/MS and moved to next step as it was.
[0475] Title compound
(S)-5-((S)-2(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyr-
rolo[2,3-b]pyridin-1-yl)-4-((2S,3S)-2-(3-carboxypropanamido)-3-methylpenta-
namido)-5-oxopentanoic acid (C11) was prepared in the same manner
as
(S)-5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[-
2,3-b]pyridin-1-yl)-4-((2S,3S)-2-(3-carboxypropanamido)-3-methylpentanamid-
o)-5-oxopentanoic acid (from Example C4) starting from (S)-benzyl
5-((S)-2(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo-
[2,3-b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpen-
tanamido)-5-oxopentanoate. .sup.1H NMR (400 MHz, DMSO-d6) .delta.
0.73-0.88 (m, 6H), 1.01-1.15 (m, 1H), 1.33-1.46 (m, 1H), 1.59-1.74
(m, 1H), 1.78-1.95 (m, 1H), 1.95-2.08 (m, OH), 2.27-2.45 (m, 7H),
2.88 (d, J=17.2 Hz, 1H), 3.40-3.49 (m, 2H), 4.21 (t, J=8.1 Hz, 1H),
4.30 (dd, J=15.3, 5.4 Hz, 1H), 4.39 (dd, J=15.4, 5.7 Hz, 1H), 4.95
(dd, J=11.3, 4.1 Hz, 1H), 5.79-5.90 (m, 1H), 6.99-7.06 (m, 1H),
7.63 (d, J=7.5 Hz, 1H), 7.67 (s, 1H), 7.84 (d, J=8.8 Hz, 1H),
8.09-8.21 (m, 2H), 8.64-8.74 (m, 1H). MS (LC/MS) m/z observed 587,
expected 587.25 [M+H].sup.+.
Example C12
(4S)-4-[(2S,3S)-3-METHYL-2-[2-(2H-1,2,3,4-TETRAZOL-5-YL)ACETAMIDO]PENTANAM-
IDO]-5-OXO-5-[(2S)-2-[(2H-1,2,3,4-TETRAZOL-5-YLMETHYL)CARBAMOYL]-1H,2H,3H--
PYRROLO[2,3-B]PYRIDIN-1-YL]PENTANOIC ACID
[0476]
(4S)-4-[(2S,3S)-3-Methyl-2-[2-(2H-1,2,3,4-tetrazol-5-yl)acetamido]p-
entanamido]-5-oxo-5-[(2S)-2-[(2H-1,2,3,4-tetrazol-5-ylmethyl)carbamoyl]-1H-
,2H,3H-pyrrolo[2,3-b]pyridin-1-yl]pentanoic acid benzyl ester was
prepared from (S)-benzyl
5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3--
b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanam-
ido)-5-oxopentanoate from Example C4 and 2H-tetrazole-5-acetic acid
via method A. MS (LC/MS) m/z observed 688, expected 688.30
[M+H].sup.+. Compound was confirmed using LC/MS and moved to next
step as it was.
[0477] Title compound
(4S)-4-[(2S,3S)-3-methyl-2-[2-(2H-1,2,3,4-tetrazol-5-yl)acetamido]pentana-
mido]-5-oxo-5-[(2S)-2-[(2H-1,2,3,4-tetrazol-5-ylmethyl)carbamoyl]-1H,2H,3H-
-pyrrolo[2,3-b]pyridin-1-yl]pentanoic acid (C12) was prepared from
(S)-benzyl
4-((2S,3S)-2-(2-(1H-tetrazol-5-yl)methyl)acetamido)-3-methylpentamamido)--
5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-b-
]pyridin-1-yl)-5-oxopentanoate via Method Q. .sup.1H NMR (400 MHz,
DMSO-d6) .delta. 0.81 (t, J=7.4 Hz, 3H), 0.86 (d, J=6.8 Hz, 3H),
0.99-1.23 (m, 1H), 1.44 (s, 1H), 1.69 (s, 1H), 1.85 (d, J=10.9 Hz,
1H), 1.94-2.06 (m, 1H), 2.27-2.46 (m, 2H), 2.96 (d, J=17.4 Hz, 1H),
3.40-3.52 (m, 1H), 3.88-4.03 (m, 2H), 4.25 (t, J=8.1 Hz, 1H), 4.48
(dd, J=15.9, 5.1 Hz, 1H), 4.69 (dd, J=16.0, 6.0 Hz, 1H), 4.94-5.05
(m, 1H), 5.79-5.91 (m, 1H), 6.98-7.06 (m, 1H), 7.66 (d, J=7.4 Hz,
1H), 8.15 (d, J=5.1 Hz, 1H), 8.29-8.39 (m, 2H), 8.97 (bs, 1H). MS
(LC/MS) m/z observed 598, expected 598.25 [M+H].sup.+.
Example C13
(S)-BENZYL
5-((S)-2-(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-P-
YRROLO[2,3-B]PYRIDIN-1-YL)-4-((2S,3S)-2-ACETAMIDO-3-METHYLPENTANAMIDO)-5-O-
XOPENTANOATE
[0478] To a solution of (S)-benzyl
5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3--
b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanam-
ido)-5-oxopentanoate (from Example C4) (85 mg, 0.15 mmol, 1.0 eq.)
in dioxane (1 ml) was added hydrochloric acid (1.5 ml, 4.0 M
solution in dioxane, 6.0 mmol, 40 eq.), then stirred at RT for 2 h
whereupon a white paste forms upon the walls of the reaction
vessel. The reaction mixture was concentrated under vacuum to
dryness and coupled to intermediate N-acetyl-L-valine via method C
but with DMF as the solvent to obtain the title compound (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((2S,3S)-2-acetamido-3-methylpentanamido)-5-oxopentanoa-
te (C13). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.79-0.82 (d,
J=6.7 Hz, 3H), 0.85 (d, J=6.7 Hz, 3H), 1.84 (s, 3H), 1.86-1.96 (m,
1H), 2.96 (d, J=17.2 Hz, 1H), 3.17 (s, 1H), 3.38-3.50 (m, 2H), 4.15
(t, J=8.0 Hz, 1H), 4.44 (d, J=16.9 Hz, 1H), 4.65 (dd, J=15.8, 6.1
Hz, 1H), 4.99 (d, J=10.7 Hz, 1H), 5.08 (s, 2H), 5.79-5.92 (m, 1H),
7.01 (t, J=6.3 Hz, 1H), 7.25-7.42 (m, 5H), 7.65 (d, J=7.5 Hz, 1H),
7.81 (d, J=8.7 Hz, 1H), 8.14 (d, J=5.1 Hz, 1H), 8.18 (d, J=7.4 Hz,
1H), 8.87 (s, 1H). MS (LC/MS) m/z observed 606, expected 606.27
[M+H].sup.+.
Example C14
(S)-N-((2H-TETRAZOL-5-YL)METHYL)-1-((S)-2-((2S,3S)-3-METHYL-2-(2-PHENYLACE-
TAMIDO)PENTANAMIDO)PROPANOYL)-2,3-DIHYDRO-1H-PYRROLO[2,3-B]PYRIDINE-2-CARB-
OXAMIDE
[0479] Title compound
(S)-N-((2H-tetrazol-5-yl)methyl)-1-((S)-2-((2S,3S)-3-methyl-2-(2-phenylac-
etamido)pentanamido)propanoyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-car-
boxamide (C14) was prepared tert-butyl
((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-1-oxopropan-2-yl)carbamate from Example C6
and intermediate I-9 using method A. .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 0.71-0.87 (6H, m), 1.05 (1H, m), 1.28 (3H, d, J=7 Hz), 1.40
(1H, m), 1.63 (1H, m), 2.95 (1H, dd, J=4, 17 Hz), 3.38-3.54 (3H,
m), 4.17 (1H, t, J=8 Hz), 4.45 (1H, dd, J=5, 16 Hz), 4.65 (1H, dd,
J=6, 16 Hz), 4.98 (1H, dd, J=4, 11 Hz), 5.84 (1H, m), 7.01 (1H, dd,
J=5, 7 Hz), 7.16-7.30 (5H, m), 7.64 (1H, d, J=7 Hz), 7.99 (1H, d,
J=9 Hz), 8.13 (1H, d, J=6 Hz), 8.24 (1H, d, J=7 Hz), 8.88 (1H, t,
J=6 Hz), MS (LC/MS) m/z observed 547.96, expected 548.27 [M+H].
Example C15
(2S)-1-[(2R)-2-[(2S,3S)-3-METHYL-2-(3-CARBOXYPROPANAMIDO)PENTANAMIDO]PROPA-
NOYL]-N-(2H-1,2,3,4-TETRAZOL-5-YLMETHYL)-2,3-DIHYDRO-1H-PYRROLO[2,3-B]PYRI-
DINE-2-CARBOXAMIDE
[0480]
(S)-1-((R)-2-((tert-Butoxycarbonyl)amino)propanoyl)-2,3-dihydro-1H--
pyrrolo[2,3-b]pyridine-2-carboxylic acid was prepared from I-7 and
Boc-D-alanine (3 eq.) using method C in DMF. (LC/MS) m/z observed
335.97, expected 336.16 [M+H]. Compound was confirmed using LC/MS
and moved to next step as it was.
[0481] tert-Butyl
((R)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-1-oxopropan-2-yl)carbamate was prepared from
(S)-1-((R)-2-((tert-butoxycarbonyl)amino)propanoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridine-2-carboxylic acid and
(2H-tetrazol-5-yl)methyl-amine using method A in DMF but without
HCl treatment. MS (LC/MS) m/z observed 416.86, expected 417.20
[M+H]. Compound was confirmed using LC/MS and moved to next step as
it was.
[0482] tert-Butyl
((3S)-1-(((R)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro--
1H-pyrrolo[2,3-b]pyridin-1-yl)-1-oxopropan-2-yl)amino)-3-methyl-1-oxopenta-
n-2-yl)carbamate was prepared from tert-butyl
((R)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-1-oxopropan-2-yl)carbamate and
Boc-L-Isoleucine using method A. MS (LC/MS) m/z observed 530.96,
expected 530.28 [M+H]. Compound was confirmed using LC/MS and moved
to next step as it was.
[0483] Title compound (2S)-1-[(2R)-2-[(2S,3
S)-3-methyl-2-(3-carboxypropanamido)pentanamido]propanoyl]-n-(2H-1,2,3,4--
tetrazol-5-ylmethyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxamide
(C15) was prepared from tert-butyl
((3S)-1-(((R)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro--
1H-pyrrolo[2,3-b]pyridin-1-yl)-1-oxopropan-2-yl)amino)-3-methyl-1-oxopenta-
n-2-yl)carbamate and succinic anhydride using method I. .sup.1H NMR
(400 MHz, DMSO-d6) .delta. 0.76-0.85 (6H, m), 1.07 (1H, m), 1.19
(3H, d, J=7 Hz), 1.41 (1H, m), 1.70 (1H, m), 2.30-2.43 (4H, m),
2.96 (1H, dd, J=4, 17 Hz), 3.42 (1H, dd, J=11, 18 Hz), 4.31 (1H, t,
J=8 Hz), 4.46 (1H, dd, J=5, 16 Hz), 4.59 (1H, dd, J=6, 16 Hz), 4.93
(1H, dd, J=4, 11 Hz), 6.11 (1H, m), 7.03 (1H, dd, J=5, 7 Hz), 7.63
(1H, d, J=7 Hz), 7.91 (1H, d, J=9 Hz), 8.12-8.19 (2H, m), 8.75 (1H,
t, J=6 Hz), MS (LC/MS) m/z observed 530.00, expected 530.25
[M+H].
Example C16
(S)-6-((S)-2-(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-PYRROLO[-
2,3-B]PYRIDIN-1-YL)-5-((2S,3S)-2-(3-CARBOXYPROPANAMIDO)-3-METHYLPENTANAMID-
O)-6-OXOHEXANOIC ACID
[0484]
(S)-1-((S)-6-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-6-oxohexa-
noyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid was
prepared in the same manner as
(S)-1-((S)-5-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-5-oxopentanoyl)-2-
,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid in Example
C4, starting from boc-L-.alpha.-aminoadipic acid .delta.-tert-butyl
ester (prepared from L-a-aminoadipic acid .delta.-tert-butyl ester
hydrochloride via method K). MS (LC/MS) m/z observed 464; expected
464.23 [M+H].sup.+. Compound was confirmed using LC/MS and moved to
next step as it was.
[0485] (S)-tert-Butyl
6-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-5-((tert-butoxycarbonyl)amino)-6-oxohexanoate was
prepared from
(S)-1-((S)-6-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-6-oxohexanoyl)--
2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid via method
O but without the second amine addition. MS (LC/MS) m/z observed
545; expected 545.28 [M+H].sup.+. Compound was confirmed using
LC/MS and moved to next step as it was.
[0486]
(S)-6-((S)-2-(((2H-Tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-p-
yrrolo[2,3-b]pyridin-1-yl)-5-((2 S,3
S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanamido)-6-oxohexanoic
acid was prepared from (S)-tert-butyl
6-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-5-((tert-butoxycarbonyl)amino)-6-oxohexanoate and
boc-L-isoleucine via method A. MS (LC/MS) m/z observed 602;
expected 602.30 [M+H].sup.+. Compound was confirmed using LC/MS and
moved to next step as it was.
[0487] Title compound
(S)-6-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo-
[2,3-b]pyridin-1-yl)-5-((2S,3
S)-2-(3-carboxypropanamido)-3-methylpentanamido)-6-oxohexanoic acid
(C16) was prepared from
(S)-6-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo-
[2,3-b]pyridin-1-yl)-5-((2S,3
S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanamido)-6-oxohexanoic
acid via method I. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.72-0.90
(m, 6H), 1.03-1.15 (m, 1H), 1.33-1.47 (m, 1H), 1.47-1.59 (m, 1H),
1.59-1.74 (m, 3H), 1.77-1.89 (m, 1H), 2.21-2.46 (m, 6H), 2.88-2.99
(m, 1H), 3.43 (dd, J=17.7, 11.2 Hz, 1H), 4.21 (t, J=8.2 Hz, 1H),
4.43 (dd, J=15.8, 5.0 Hz, 1H), 4.64 (dd, J=15.8, 6.0 Hz, 1H), 4.98
(dd, J=11.1, 3.9 Hz, 1H), 5.86 (t, J=7.8 Hz, 1H), 7.01 (dd, J=7.4,
5.1 Hz, 1H), 7.64 (d, J=7.4 Hz, 1H), 7.81 (d, J=8.9 Hz, 1H), 8.03
(d, J=7.7 Hz, 1H), 8.13 (d, J=5.0 Hz, 1H), 8.85 (t, J=5.4 Hz, 1H).
MS (LC/MS) m/z observed 602, expected 602.26 [M+H].sup.+.
Example C17
(S)-5-((S)-2-(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-PYRROLO[-
2,3-B]PYRIDIN-1-YL)-4-((S)-2-(3-CARBOXYPROPANAMIDO)-3-METHYLBUTANAMIDO)-5--
OXOPENTANOIC ACID
[0488] (S)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-
-5-oxopentanoate was prepared from (S)-benzyl
5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3--
b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanam-
ido)-5-oxopentanoate (from Example C4) and boc-L-valine via method
A but with DMF as the solvent. MS (LC/MS) m/z observed 664,
expected 663.31 [M+H].sup.+. Compound was confirmed using LC/MS and
moved to next step as it was.
[0489]
4-(((S)-1-(((S)-1-((S)-2-(((2H-Tetrazol-5-yl)methyl)carbamoyl)-2,3--
dihydro-1H-pyrrolo[2,3-b]pyridin-1-yl)-5-(benzyloxy)-1,5-dioxopentan-2-yl)-
amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid was
prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-
-5-oxopentanoate via method I. MS (LC/MS) m/z observed 664,
expected 663.28 [M+H].sup.+. Compound was confirmed using LC/MS and
moved to next step as it was.
[0490] Title compound
(S)-5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo-
[2,3-b]pyridin-1-yl)-4-((S)-2-(3-carboxypropanamido)-3-methylbutanamido)-5-
-oxopentanoic acid (C17) was prepared from
4-(((S)-1-(((S)-1-((S)-2-(((2H-Tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydr-
o-1H-pyrrolo[2,3-b]pyridin-1-yl)-5-(benzyloxy)-1,5-dioxopentan-2-yl)amino)-
-3-methyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid via Method Q.
.sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.82 (d, J=6.8 Hz, 3H), 0.86
(d, J=6.8 Hz, 3H), 1.77-2.07 (m, 3H), 2.27-2.48 (m, 6H), 2.95 (dd,
J=17.5, 3.9 Hz, 1H), 3.45 (dd, J=17.4, 11.3 Hz, 1H), 4.19 (dd,
J=8.8, 6.9 Hz, 1H), 4.46 (dd, J=15.9, 5.1 Hz, 1H), 4.68 (dd,
J=15.9, 6.0 Hz, 1H), 4.98 (dd, J=11.2, 3.9 Hz, 1H), 5.78-5.90 (m,
1H), 7.02 (dd, J=7.3, 5.1 Hz, 1H), 7.65 (d, J=7.4 Hz, 1H), 7.79 (d,
J=8.8 Hz, 1H), 8.14 (dd, J=7.6, 3.3 Hz, 2H), 8.94 (t, J=5.6 Hz,
1H). MS (LC/MS) m/z observed 574, expected 573.23 [M+H].sup.+.
Example C18
(S)-5-((S)-2-(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-PYRROLO[-
2,3-B]PYRIDIN-1-YL)-4-((S)-2-CYCLOPENTYL-2-(2-PHENYLACETAMIDO)ACETAMIDO)-5-
-OXOPENTANOIC ACID
[0491] (S)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((S)-2-cyclopentyl-2-(2-phenylacetamido)acetamido)-5-ox-
opentanoate was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((S)-2-((tert-butoxycarbonyl)amino)-2-cyclopentylacetam-
ido)-5-oxopentanoate (from Example C8) and phenylacetic acid via
method A. MS (LC/MS) m/z observed 708, expected 708.32 [M+H].sup.+.
Compound was confirmed using LC/MS and moved to next step as it
was.
[0492] Title compound
(S)-5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo-
[2,3-b]pyridin-1-yl)-4-((S)-2-cyclopentyl-2-(2-phenylacetamido)acetamido)--
5-oxopentanoic acid (C18) was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((S)-2-cyclopentyl-2-(2-phenylacetamido)acetamido)-5-ox-
opentanoate via Method Q. .sup.1H NMR (400 MHz, DMSO-d6) .delta.
1.10-1.65 (m, 9H), 1.77-1.90 (m, 1H), 1.94-2.18 (m, 2H), 2.29-2.47
(m, 2H), 2.96 (dd, J=17.6, 3.8 Hz, 1H), 3.39-3.56 (m, 3H), 4.21 (t,
J=8.6 Hz, 1H), 4.48 (dd, J=15.9, 5.1 Hz, 1H), 4.69 (dd, J=15.9, 6.0
Hz, 1H), 5.00 (dd, J=11.3, 3.9 Hz, 1H), 5.84 (td, J=8.2, 3.7 Hz,
1H), 7.03 (dd, J=7.4, 5.1 Hz, 1H), 7.15-7.35 (m, 5H), 7.66 (dd,
J=7.4, 1.7 Hz, 1H), 8.10 (d, J=8.5 Hz, 1H), 8.16 (dd, J=5.2, 1.6
Hz, 1H), 8.22 (d, J=7.5 Hz, 1H), 8.97 (t, J=5.6 Hz, 1H). MS (LC/MS)
m/z observed 618, expected 618.27 [M+H].sup.+.
Example C19
(4S)-4-[(2S)-2-ACETAMIDO-3-METHYLBUTANAMIDO]-5-OXO-5-[(2S)-2-[(2H-1,2,3,4--
TETRAZOL-5-YLMETHYL)CARBAMOYL]-1H,2H,3H-PYRROLO[2,3-B]PYRIDIN-1-YL]PENTANO-
IC ACID
[0493] Title compound
(4S)-4-[(2S)-2-acetamido-3-methylbutanamido]-5-oxo-5-[(2S)-2-[(2H-1,2,3,4-
-tetrazol-5-ylmethyl)carbamoyl]-1H,2H,3H-pyrrolo[2,3-b]pyridin-1-yl]pentan-
oic acid (C19) was prepared from C13 via Method Q. .sup.1H NMR (400
MHz, DMSO-d6) .delta. 0.82 (d, J=6.8 Hz, 3H), 0.86 (d, J=6.8 Hz,
3H), 1.85 (s, 3H), 1.88-2.07 (m, 1H), 2.39 (td, J=10.9, 5.2 Hz,
1H), 2.90-3.01 (m, 1H), 3.39-3.49 (m, 2H), 4.16 (t, J=7.9 Hz, 1H),
4.43 (dd, J=15.6, 4.9 Hz, 1H), 4.65 (dd, J=15.8, 5.9 Hz, 1H), 4.98
(dd, J=11.3, 3.9 Hz, 1H), 5.82 (d, J=9.6 Hz, 1H), 7.02 (dd, J=7.4,
5.1 Hz, 1H), 7.65 (d, J=7.3 Hz, 1H), 7.81 (d, J=8.8 Hz, 1H),
8.11-8.19 (m, 2H), 8.87 (t, J=5.6 Hz, 1H). MS (LC/MS) m/z observed
516, expected 516.22 [M+H].sup.+.
Example C20
(S)-5-((S)-2-(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-PYRROLO[-
2,3-B]PYRIDIN-1-YL)-4-((S)-2-AMINO-3-METHYLBUTANAMIDO)-5-OXOPENTANOIC
ACID HYDROCHLORIDE
[0494] Title compound
(S)-5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo-
[2,3-b]pyridin-1-yl)-4-((S)-2-amino-3-methylbutanamido)-5-oxopentanoic
acid hydrochloride (C20) was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-
-5-oxopentanoate (from Example C17) via Method Q immediately
followed by method E. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.92
(t, J=5.9 Hz, 6H), 1.80-2.15 (m, 3H), 2.44 (t, J=8.3 Hz, 2H), 2.94
(dd, J=17.5, 3.9 Hz, 1H), 3.37 (dd, J=17.3, 11.1 Hz, 1H), 3.56 (d,
J=5.8 Hz, 1H), 4.27 (dd, J=14.9, 4.5 Hz, 1H), 4.45 (dd, J=14.9, 5.7
Hz, 1H), 5.00 (dd, J=11.2, 4.0 Hz, 1H), 5.87-6.00 (m, 1H), 7.03
(dd, J=7.4, 5.1 Hz, 1H), 7.63 (d, J=7.3 Hz, 1H), 8.16 (d, J=5.0 Hz,
1H), 8.46 (t, J=5.3 Hz, 1H), 8.64 (d, J=7.6 Hz, 1H). MS (LC/MS) m/z
observed 510, expected 509.19 [M+H].sup.+.
Example C21
(R)-5-((S)-2(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-PYRROLO[2-
,3-B]PYRIDIN-1-YL)-4-((2S,3S)-2-(3-CARBOXYPROPANAMIDO)-3-METHYLPENTANAMIDO-
)-5-OXOPENTANOIC ACID
[0495]
(S)-1-((R)-5-(Benzyloxy)-2-((tert-butoxycarbonyl)amino)-5-oxopentan-
oyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid was
prepared in the same manner as
(S)-1-((S)-5-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-5-oxopentanoyl)-2-
,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid in Example
C4, starting from Boc-D-glutamic acid y-benzyl ester (365 mg). MS
(LC/MS) m/z observed 484, expected 484.20 [M+H].sup.+. Compound was
confirmed using LC/MS and moved to next step as it was.
[0496] (R)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate was
prepared from
(S)-1-((R)-5-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-5-oxopentanoyl)-2-
,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid via method O
but without the second amine addition. MS (LC/MS) m/z observed 565,
expected 565.24 [M+H].sup.+. Compound was confirmed using LC/MS and
moved to next step as it was.
[0497] (R)-Benzyl
5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3--
b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanam-
ido)-5-oxopentanoate was prepared from (R)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate via
method A but without swapping with MeOH; and DMF as the solvent for
the coupling step described therein. MS (LC/MS) m/z observed 678;
expected 678.33 [M+H].sup.+. Compound was confirmed using LC/MS and
moved to next step as it was.
[0498] Title compound
(R)-5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[-
2,3-b]pyridin-1-yl)-4-((2S,3S)-2-(3-carboxypropanamido)-3-methylpentanamid-
o)-5-oxopentanoic acid (C21) was prepared in the same manner as
(S)-5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[-
2,3-b]pyridin-1-yl)-4-((2S,3S)-2-(3-carboxypropanamido)-3-methylpentanamid-
o)-5-oxopentanoic acid (from Example C4) starting from
(R)-5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[-
2,3-b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpent-
anamido)-5-oxopentanoate benzyl ester. C21 exists as a mixture of
rotamers in a 4:1 ratio; only the major signals are reported.
.sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.73-0.88 (m, 6H), 1.00-1.15
(m, 1H), 1.42 (d, J=13.7 Hz, 1H), 1.63-1.93 (m, 2H), 2.13-2.47 (m,
6H), 2.95 (dd, J=17.6, 3.4 Hz, 1H), 3.21-3.51 (m, 7H), 4.32-4.40
(m, 1H), 4.45 (dd, J=15.5, 5.3 Hz, 1H), 4.59 (dd, J=15.6, 5.8 Hz,
1H), 4.91 (d, J=10.5 Hz, 1H), 6.16 (s, 1H), 6.97-7.10 (m, 1H), 7.66
(d, J=7.3 Hz, 1H), 7.90 (d, J=9.1 Hz, 1H), 8.19 (dd, J=10.4, 6.5
Hz, 2H), 8.77 (t, J=5.6 Hz, 1H). MS (LC/MS) m/z observed 588,
expected 588.25 [M+H].sup.+.
Example C22
4-(((S)-1-(((S)-1-((S)-2-(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-
-1H-PYRROLO[2,3-B]PYRIDIN-1-YL)-5-AMINO-1,5-DIOXOPENTAN-2-YL)AMINO)-3-METH-
YL-1-OXOBUTAN-2-YL)AMINO)-4-OXOBUTANOIC ACID
[0499]
(S)-1-((S)-5-Amino-2-((tert-butoxycarbonyl)amino)-5-oxopentanoyl)-2-
,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid was prepared
in the same manner as
(S)-1-((S)-5-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-5-oxopentanoyl)-2-
,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid in Example
C4, starting from boc-L-glutamine. MS (LC/MS) m/z observed 393;
expected 393.17 [M+H].sup.+. Compound was confirmed using LC/MS and
moved to next step as it was.
[0500] tert-Butyl
((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-5-amino-1,5-dioxopentan-2-yl)carbamate was
prepared from
(S)-1-((S)-5-amino-2-((tert-butoxycarbonyl)amino)-5-oxopentanoyl)-2,-
3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid via method O
but without the second amine addition. MS (LC/MS) m/z observed 474;
expected 474.21 [M+H].sup.+. Compound was confirmed using LC/MS and
moved to next step as it was.
[0501] tert-Butyl
((S)-1-(((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1-
H-pyrrolo[2,3-b]pyridin-1-yl)-5-amino-1,5-dioxopentan-2-yl)amino)-3-methyl-
-1-oxobutan-2-yl)carbamate was prepared from tert-butyl
((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-5-amino-1,5-dioxopentan-2-yl)carbamate and
boc-L-valine via method A. MS (LC/MS) m/z observed 573; expected
573.28 [M+H].sup.+. Compound was confirmed using LC/MS and moved to
next step as it was.
[0502] Title compound
4-(((S)-1-(((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydr-
o-1H-pyrrolo[2,3-b]pyridin-1-yl)-5-amino-1,5-dioxopentan-2-yl)amino)-3-met-
hyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid (C22) was prepared
from tert-butyl
((S)-1-(((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1-
H-pyrrolo[2,3-b]pyridin-1-yl)-5-amino-1,5-dioxopentan-2-yl)amino)-3-methyl-
-1-oxobutan-2-yl)carbamate via method I. .sup.1H NMR (400 MHz,
DMSO-d6) .delta. 0.82 (d, J=6.9 Hz, 3H), 0.85 (d, J=6.8 Hz, 3H),
1.71-1.86 (m, 1H), 1.87-2.05 (m, 2H), 2.13-2.44 (m, 6H), 2.94 (dd,
J=17.7, 3.9 Hz, 1H), 3.38-3.51 (m, 1H), 3.60-3.85 (m, 4H),
4.02-4.33 (m, 3H), 4.47 (dd, J=15.8, 5.2 Hz, 1H), 4.67 (dd, J=15.9,
5.9 Hz, 1H), 4.98 (dd, J=11.3, 3.9 Hz, 1H), 5.74-5.87 (m, 1H), 6.75
(s, 1H), 7.02 (dd, J=7.3, 5.1 Hz, 1H), 7.24 (s, 1H), 7.65 (d, J=7.3
Hz, 1H), 7.78 (d, J=8.9 Hz, 1H), 8.10-8.23 (m, 2H), 8.93 (t, J=5.8
Hz, 1H). MS (LC/MS) m/z observed 573, expected 573.25
[M+H].sup.+.
Example C23
(S)-METHYL
5-((S)-2-(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-P-
YRROLO[2,3-B]PYRIDIN-1-YL)-4-((S)-3-METHYL-2-(2-PHENYLACETAMIDO)BUTANAMIDO-
)-5-OXOPENTANOATE
[0503] (S)-Methyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((S)-2-amino-3-methylbutanamido)-5-oxopentanoate
hydrochloride was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-
-5-oxopentanoate (from Example C17) via method E but with MeOH as a
co-solvent in a 1:1 (v/v) ratio. MS (LC/MS) m/z observed 488,
expected 487.23 [M+H].sup.+. Compound was confirmed using LC/MS and
moved to next step as it was.
[0504] Title compound (S)-methyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((S)-3-methyl-2-(2-phenylacetamido)butanamido)-5-oxopen-
tanoate (C23) was prepared from (S)-Methyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((S)-2-amino-3-methylbutanamido)-5-oxopentanoate
hydrochloride and phenylacetic acid via method M but with DMF as
the solvent. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.80 (d, J=6.9
Hz, 3H), 0.83 (d, J=6.6 Hz, 3H), 1.56-1.66 (m, 1H), 1.83-2.07 (m,
4H), 2.86-3.08 (m, 3H), 3.36-3.45 (m, 2H), 3.57 (s, 3H), 4.19 (dd,
J=8.9, 6.9 Hz, 1H), 4.29 (dd, J=15.2, 4.3 Hz, 1H), 4.51 (dd,
J=15.0, 5.9 Hz, 1H), 5.01 (dd, J=11.2, 3.9 Hz, 1H), 5.81-5.89 (m,
1H), 7.01 (dd, J=7.4, 5.0 Hz, 1H), 7.16-7.23 (m, 1H), 7.23-7.33 (m,
4H), 7.65 (d, J=7.3 Hz, 1H), 7.99 (d, J=8.9 Hz, 1H), 8.14 (d, J=5.1
Hz, 1H), 8.23 (d, J=7.6 Hz, 1H), 8.54 (t, J=5.4 Hz, 1H). MS (LC/MS)
m/z observed 606, expected 606.27 [M+H].sup.+.
Example C24
METHYL
(4S)-4-[(2S)-2-ACETAMIDO-3-METHYLBUTANAMIDO]-5-OXO-5-[(2S)-2-[(2H-1-
,2,3,4-TETRAZOL-5-YLMETHYL)CARBAMOYL]-1H,2H,3H-PYRROLO[2,3-B]PYRIDIN-1-YL]-
PENTANOATE
[0505] Title compound methyl
(4S)-4-[(2S)-2-acetamido-3-methylbutanamido]-5-oxo-5-[(2S)-2-[(2H-1,2,3,4-
-tetrazol-5-ylmethyl)carbamoyl]-1H,2H,3H-pyrrolo[2,3-b]pyridin-1-yl]pentan-
oate (C24) was prepared from (S)-methyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((S)-2-amino-3-methylbutanamido)-5-oxopentanoate
hydrochloride (from Example C23) and acetic acid via method M but
with DMF as a solvent. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.81
(d, J=6.8 Hz, 3H), 0.84 (d, J=6.8 Hz, 3H), 1.83 (s, 3H), 1.89 (dd,
J=13.5, 7.4 Hz, 2H), 1.96-2.06 (m, 1H), 2.93 (d, J=16.4 Hz, 1H),
3.37-3.46 (m, 1H), 3.56 (s, 3H), 4.13 (t, J=7.9 Hz, 1H), 4.40 (dd,
J=15.5, 4.9 Hz, 1H), 4.61 (dd, J=15.6, 6.0 Hz, 1H), 4.97 (dd,
J=11.2, 4.0 Hz, 1H), 5.79-5.88 (m, 1H), 7.00 (dd, J=7.3, 5.1 Hz,
1H), 7.64 (d, J=7.3 Hz, 1H), 7.79 (d, J=8.7 Hz, 1H), 8.13 (d, J=5.5
Hz, 1H), 8.16 (d, J=7.5 Hz, 1H), 8.81 (s, 1H). MS (LC/MS) m/z
observed 530, expected 530.24 [M+H]+.
Example C25
(S)-5-((S)-2-(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-PYRROLO[-
2,3-B]PYRIDIN-1-YL)-4-(3-METHYLBUTANAMIDO)-5-OXOPENTANOIC ACID
[0506] (S)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-(3-methylbutanamido)-5-oxopentanoate was
prepared from (S)-benzyl
5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3--
b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanam-
ido)-5-oxopentanoate (from Example C4) and and isovaleric acid
using method A but the solvent was DMF for the coupling reaction.
MS (LC/MS) m/z observed 549.98, expected 549.26 [M+H]. Compound was
confirmed using LC/MS and moved to next step as it was.
[0507] Title compound
(S)-5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo-
[2,3-b]pyridin-1-yl)-4-(3-methylbutanamido)-5-oxopentanoic acid
(C25) was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-(3-methylbutanamido)-5-oxopentanoate using
Method Q. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.83-0.89 (6H, d,
J=7 Hz), 1.77 (1H, m), 1.88-2.05 (4H, m), 2.30-2.45 (2H, m), 2.95
(1H, d, J=16 Hz), 3.45 (1H, dd, J=11, 17 Hz), 4.45 (1H, dd, J=5, 16
Hz), 4.65 (1H, dd, J=6, 16 Hz), 4.98 (1H, dd, J=4, 11 Hz), 5.83
(1H, m), 7.03 (1H, dd, J=5, 7 Hz), 7.66 (1H, d, J=7 Hz), 8.05 (1H,
d, J=8 Hz), 8.17 (1H, d, J=4 Hz), 8.93 (1H, t, J=6 Hz), MS (LC/MS)
m/z observed 458.83, expected 459.21 [M+H].
Example C26
4-(((S)-1-(((S)-1-((S)-2-(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-
-1H-PYRROLO[2,3-B]PYRIDIN-1-YL)-4-AMINO-1,4-DIOXOBUTAN-2-YL)AMINO)-3-METHY-
L-1-OXOBUTAN-2-YL)AMINO)-4-OXOBUTANOIC ACID
[0508]
(S)-1-((S)-2-((tert-Butoxycarbonyl)amino)-4-oxo-4-(tritylamino)buta-
noyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid was
prepared from I-7 and Na-Boc-N.sup..gamma.-trityl-L-asparagine (3
eq.) using method C in DMF. MS (LC/MS) m/z observed 620.77,
expected 621.27 [M+H]. Compound was confirmed using LC/MS and moved
to next step as it was.
[0509] tert-Butyl
((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-1,4-dioxo-4-(tritylamino)butan-2-yl)carbamate
was prepared from
(S)-1-((S)-2-((tert-butoxycarbonyl)amino)-4-oxo-4-(tritylamino)butanoyl)--
2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid and
(2H-tetrazol-5-yl)methyl-amine using method A in DMF but without
HCl treatment. MS (LC/MS) m/z observed 701.77, expected 702.32
[M+H]. Compound was confirmed using LC/MS and moved to next step as
it was.
[0510] tert-Butyl
((S)-1-(((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1-
H-pyrrolo[2,3-b]pyridin-1-yl)-1,4-dioxo-4-(tritylamino)butan-2-yl)amino)-3-
-methyl-1-oxobutan-2-yl)carbamate was prepared from tert-butyl
((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-1,4-dioxo-4-(tritylamino)butan-2-yl)carbamate
and Boc-L-valine using method A but without swapping with MeOH. DMF
was used as the solvent of the coupling step. MS (LC/MS) m/z
observed 800.79, expected 801.38 [M+H].sup.+. Compound was
confirmed using LC/MS and moved to next step as it was.
[0511] tert-Butyl
((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-1,4-dioxo-4-(tritylamino)butan-2-yl)carbamate
(217 mg, 0.271 mmol, 1 eq.) was treated with HCl in dioxane (20 mL)
for 1 h at rt. The solvent was then concentrated to dryness. The
residue and succinic anhydride (41 mg, 0.406 mmol, 1.5 eq.) were
suspended in dry DCM (10 mL) under N.sub.2 and DIPEA (0.151 mL,
1.08 mmol, 4 eq.) was added to the mixture. The reaction mixture
was left at rt for 2 hrs and trifluoroacetic acid (30 mL) was
added. The reaction was left at rt for 1 h until full deprotection
of the trityl group. The solvent were then evaporated and the
product was purified by preparative HPLC using a gradient from 20%
to 32% of methanol in water in 10 minutes to give the title
compound
4-(((S)-1-(((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydr-
o-1H-pyrrolo[2,3-b]pyridin-1-yl)-4-amino-1,4-dioxobutan-2-yl)amino)-3-meth-
yl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid (C26) as a white solid
(56 mg, 37%). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.81 (3H, t,
J=7 Hz), 0.84 (3H, d, J=7 Hz), 1.92 (1H, m), 2.32-2.50 (5H, m),
2.66 (1H, dd, J=5, 15 Hz), 3.01 (1H, dd, J=4, 17 Hz), 3.48 (1H, dd,
J=11, 18 Hz), 4.22 (1H, dd, J=7, 9 Hz), 4.51 (1H, dd, J=5, 16 Hz),
4.62 (1H, dd, J=6, 16 Hz), 4.97 (1H, dd, J=4, 11 Hz), 6.10 (1H, m),
6.97 (1H, s), 7.03 (1H, dd, J=5, 7 Hz), 7.31 (1H, s), 7.66 (1H, d,
J=7 Hz), 7.77 (1H, d, J=9 Hz), 8.12 (1H, d, 5 Hz), 8.25 (1H, d, J=7
Hz), 8.97 (1H, t, J=6 Hz), MS (LC/MS) m/z observed 558.98, expected
559.24 [M+H].
Example C27
(S)-5-((S)-2-(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-PYRROLO[-
2,3-B]PYRIDIN-1-YL)-4-(2-CYCLOPENTYLACETAMIDO)-5-OXOPENTANOIC
ACID
[0512] (S)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-(2-cyclopentylacetamido)-5-oxopentanoate was
prepared from (S)-benzyl
5-((S)-2(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3--
b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanam-
ido)-5-oxopentanoate (from Example C4) and cyclopentylacetic acid
using method A but the solvent was DMF for the coupling reaction.
MS (LC/MS) m/z observed 574.92, expected 575.27 [M+H]. Compound was
confirmed using LC/MS and moved to next step as it was.
[0513] Title compound
(S)-5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo-
[2,3-b]pyridin-1-yl)-4-(2-cyclopentylacetamido)-5-oxopentanoic acid
(C27) was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-(2-cyclopentylacetamido)-5-oxopentanoate using
Method Q. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 1.06-1.18 (2H, m),
1.41-1.50 (2H, m), 1.52-1.58 (22H, m), 1.62-1.70 (2H, m), 1.80 (1H,
m), 1.96-2.14 (4H, m), 2.30-2.47 (2H, m), 2.95 (1H, dd, J=4, 17
Hz), 3.45 (1H, dd, J=l 1, 17 Hz), 4.47 (1H, dd, J=5, 16 Hz), 4.67
(1H, dd, J=6, 16 Hz), 4.98 (1H, dd, J=4, 11 Hz), 5.83 (1H, m), 7.03
(1H, dd, J=5, 7 Hz), 7.66 (1H, d, J=7 Hz), 8.03 (1H, d, J=8 Hz),
8.17 (1H, d, J=4 Hz), 8.95 (1H, t, J=6 Hz), MS (LC/MS) m/z observed
484.89, expected 485.23 [M+H].
Example C28
(S)-5-((S)-2-((2-(2H-TETRAZOL-5-YL)ETHYL)CARBAMOYL)-2,3-DIHYDRO-1H-PYRROLO-
[2,3-B]PYRIDIN-1-YL)-4-((2S,3S)-2-(3-CARBOXYPROPANAMIDO)-3-METHYLPENTANAMI-
DO)-5-OXOPENTANOIC ACID
[0514] (S)-Benzyl
5-((S)-2-((2-(2H-tetrazol-5-yl)ethyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,-
3-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate
was prepared from
(S)-1-((S)-5-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-5-oxopentanoyl)-2-
,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (from
Example C4) and (2H-tetrazol-5-yl)ethyl-amine using method A in DMF
but without HCl treatment. MS (LC/MS) m/z observed 578.91, expected
579.27 [M+H]. Compound was confirmed using LC/MS and moved to next
step as it was.
[0515] (S)-Benzyl
5-((S)-2-((2-(2H-tetrazol-5-yl)ethyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,-
3-b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentan-
amido)-5-oxopentanoate was prepared from (S)-benzyl
5-((S)-2-((2-(2H-tetrazol-5-yl)ethyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,-
3-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate
and Boc-L-Isoleucine using method A but the solvent was DMF for the
coupling reaction. MS (LC/MS) m/z observed 691.95, expected 692.35
[M+H]. Compound was confirmed using LC/MS and moved to next step as
it was.
[0516]
4-(((2S,3S)-1-(((S)-1-((S)-2-((2-(2H-tetrazol-5-yl)ethyl)carbamoyl)-
-2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-yl)-5-(benzyloxy)-1,5-dioxopentan--
2-yl)amino)-3-methyl-1-oxopentan-2-yl)amino)-4-oxobutanoic acid was
prepared from (S)-benzyl
5-((S)-2-((2-(2H-tetrazol-5-yl)ethyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,-
3-b]pyridin-1-yl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentan-
amido)-5-oxopentanoate and succinic anhydride using method I. MS
(LC/MS) m/z observed 691.97, expected 692.32 [M+H]. Compound was
confirmed using LC/MS and moved to next step as it was.
[0517] Title compound
(S)-5-((S)-2-((2-(2H-tetrazol-5-yl)ethyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-4-((2S,3S)-2-(3-carboxypropanamido)-3-methylpentanam-
ido)-5-oxopentanoic acid (C28) was prepared from
4-(((2S,3S)-1-(((S)-1-((S)-2-((2-(2H-tetrazol-5-yl)ethyl)carbamoyl)-2,3-d-
ihydro-1H-pyrrolo[2,3-b]pyridin-1-yl)-5-(benzyloxy)-1,5-dioxopentan-2-yl)a-
mino)-3-methyl-1-oxopentan-2-yl)amino)-4-oxobutanoic acid using
Method Q. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.76-0.85 (6H, m),
1.08 (1H, m), 1.40 (1H, m), 1.65 (1H, m), 1.85 (1H, m), 2.00 (1H,
m), 2.25-2.48 (6H, m), 2.77 (1H, dd, J=4, 17 Hz), 2.95-3.10 (2H,
m), 3.28-3.43 (2H, m), 3.56 (1H, m), 4.20 (1H, m), 4.87 (1H, dd,
J=4, 11 Hz), 5.85 (1H, m), 7.03 (1H, dd, J=5, 7 Hz), 7.63 (1H, d,
J=7 Hz), 7.82 (1H, d, J=9 Hz), 8.12-8.18 (2H, m), 8.36 (1H, t, J=6
Hz), MS (LC/MS) m/z observed 601.96, expected 602.27 [M+H].
Example C29
3-{[(1S,2S)-2-METHYL-1-{[(2S)-1-OXO-3-[(2H-1,2,3,4-TETRAZOL-5-YLMETHYL)CAR-
BAMOYL]-1-[(2S)-2-[(2H-1,2,3,4-TETRAZOL-5-YLMETHYL)CARBAMOYL]-1H,2H,3H-PYR-
ROLO[2,3-B]PYRIDIN-1-YL]PROPAN-2-YL]CARBAMOYL}BUTYL]CARBAMOYL}PROPANOIC
ACID
[0518]
(S)-1-((S)-4-(Benzyloxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutano-
yl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid was
prepared from I-7 and Boc-L-aspartic acid .beta.-benzyl ester (3
eq.) using method C in DMF. MS (LC/MS) m/z observed 469.90,
expected 470.19 [M+H]. Compound was confirmed using LC/MS and moved
to next step as it was.
[0519] (S)-Benzyl
4-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-3-((tert-butoxycarbonyl)amino)-4-oxobutanoate was
prepared from
(S)-1-((S)-4-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutanoyl)-2,-
3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid and
(2H-tetrazol-5-yl)methyl-amine using method A in DMF but without
HCl treatment. MS (LC/MS) m/z observed 550.87, expected 551.24
[M+H]. Compound was confirmed using LC/MS and moved to next step as
it was.
[0520] (S)-Benzyl
4-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-3-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentana-
mido)-4-oxobutanoate was prepared from (S)-benzyl
4-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-3-((tert-butoxycarbonyl)amino)-4-oxobutanoate and
Boc-L-Isoleucine using method A but the solvent was DMF for the
coupling reaction. MS (LC/MS) m/z observed 663.87, expected 664.32
[M+H]. Compound was confirmed using LC/MS and moved to next step as
it was.
[0521]
(S)-4-((S)-2-(((2H-Tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-p-
yrrolo[2,3-b]pyridin-1-yl)-3-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-met-
hylpentanamido)-4-oxobutanoic acid was prepared from (S)-benzyl
4-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-3-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentana-
mido)-4-oxobutanoate using Method Q. MS (LC/MS) m/z observed
573.89, expected 574.27 [M+H]. Compound was confirmed using LC/MS
and moved to next step as it was.
[0522] tert-Butyl
((2S,3S)-1-(((S)-4-(((2H-tetrazol-5-yl)methyl)amino)-1-((S)-2-(((2H-tetra-
zol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-yl)-1,4--
dioxobutan-2-yl)amino)-3-methyl-1-oxopentan-2-yl)carbamate was
prepared from
(S)-4-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-py-
rrolo[2,3-b]pyridin-1-yl)-3-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-meth-
ylpentanamido)-4-oxobutanoic acid and
(2H-tetrazol-5-yl)methyl-amine using method A in DMF but without
HCl treatment. MS (LC/MS) m/z observed 654.83, expected 655.32
[M+H]. Compound was confirmed using LC/MS and moved to next step as
it was.
[0523] Title compound
3-{[(1S,2S)-2-methyl-1-{[(2S)-1-oxo-3-[(2H-1,2,3,4-tetrazol-5-ylmethyl)ca-
rbamoyl]-1-[(2S)-2-[(2H-1,2,3,4-tetrazol-5-ylmethyl)carbamoyl]-1H,2H,3H-py-
rrolo[2,3-b]pyridin-1-yl]propan-2-yl]carbamoyl}butyl]carbamoyl}propanoic
acid (C29) was prepared from tert-butyl
((2S,3S)-1-(((S)-4-(((2H-tetrazol-5-yl)methyl)amino)-1-((S)-2-(((2H-tetra-
zol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridin-1-yl)-1,4--
dioxobutan-2-yl)amino)-3-methyl-1-oxopentan-2-yl)carbamate and
succinic anhydride using method I. .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 0.70-0.84 (6H, m), 1.04 (1H, m), 1.36 (1H, m), 1.62 (1H,
m), 2.26-2.47 (4H, m), 2.58 (1H, dd, J=9, 15 Hz), 2.77 (1H, dd,
J=5, 16 Hz), 3.00 (1H, dd, J=4, 17 Hz), 3.45 (1H, dd, J=11, 17 Hz),
4.16 (1H, t, J=8 Hz), 4.45-4.65 (4H, m), 4.96 (1H, dd, J=4, 11 Hz),
6.11 (1H, m), 7.03 (1H, dd, J=5, 7 Hz), 7.63 (1H, d, J=7 Hz), 7.82
(1H, d, J=9 Hz), 8.00 (1H, d, J=4 Hz), 8.22 (1H, d, J=7 Hz), 8.58
(1H, t, J=6 Hz), 8.93 (1H, t, J=6 Hz), MS (LC/MS) m/z observed
654.95, expected 655.28 [M+H].
Example C30
(S)-5-((S)-2-(((2H-TETRAZOL-5-YL)METHYL)CARBAMOYL)-2,3-DIHYDRO-1H-PYRROLO[-
2,3-B]PYRIDIN-1-YL)-4-((2S,3S)-3-METHYL-2-(PYRIMIDIN-2-YLAMINO)PENTANAMIDO-
)-5-OXOPENTANOIC ACID
[0524] I-7 (200 mg, 0.719 mmol) was dissolved in a mixture of allyl
alcohol and HCl in dioaxane (4M) (20 mL, 1:1 (v/v)) and the
reaction mixture was stirred at rt for 3 hours. The reaction
mixture was then concentrated to dryness and swapped with allyl
alcohol (2.times.25 mL). The solid obtained was dried well under
reduced pressure to give (S)-allyl
2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate hydrochloride
as a white solid (173 mg, quantitative). .sup.1H NMR (400 MHz,
DMSO-d6) 3.23 (1H, dd, J=5, 18 Hz), 3.55 (1H, dd, J=11, 18 Hz),
4.65 (2H, d, J=5 Hz), 4.86 (1H, dd, J=5, 11 Hz), 5.25 (1H, d, J=10
Hz), 5.36 (1H, d, J=17 Hz), 5.94 (1H, m), 6.80 (1H, t, J=7 Hz),
7.68-7.74 (2H, m), 9.29 (1H, bs), MS (LC/MS) m/z observed 204.98,
expected 205.10 [M+H].
[0525] (S)-Allyl
1-((S)-5-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-5-oxopentanoyl)-2,3-d-
ihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate was prepared from
(S)-allyl 2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate
hydrochloride and Boc-L-glutamic acid y-benzyl ester (1.2 eq.)
using method C in DMF. MS (LC/MS) m/z observed 523.95, expected
524.24 [M+H]. Compound was confirmed using LC/MS and moved to next
step as it was.
[0526] (S)-Allyl
1-((S)-5-(benzyloxy)-2-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpe-
ntanamido)-5-oxopentanoyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxy-
late was prepared from (S)-allyl
1-((S)-5-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-5-oxopentanoyl)-2,3-d-
ihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate and Boc-L-Isoleucine
using method A but without swapping with MeOH. MS (LC/MS) m/z
observed 636.97, expected 637.32 [M+H]. Compound was confirmed
using LC/MS and moved to next step as it was.
[0527] (S)-Allyl
1-((S)-5-(benzyloxy)-2-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpe-
ntanamido)-5-oxopentanoyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxy-
late (218 mg, 0.342 mmol, 1 eq.) and 2-bromopyrimidine (136 mg,
0.855 mmol, 2.5 eq.) were added in a microwave vial. DMF (8 mL) and
DIPEA (0.174 mL, 1.710 mmol, 5 eq.) were then added. The reaction
mixture was irradiated (microwave) at 145.degree. C. for 4 hours.
The solvent was then evaporated and the product was purified by
column chromatography using 15% to 80% ethyl acetate in hexanes as
the eluent to give (S)-allyl
1-((S)-5-(benzyloxy)-2-((2S,3S)-3-methyl-2-(pyrimidin-2-ylamino)pentanami-
do)-5-oxopentanoyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate
as an orange glass (40 mg, 19%). MS (LC/MS) m/z observed 614.98,
expected 615.29 [M+H]. Compound was confirmed using LC/MS and moved
to next step as it was.
[0528] (S)-Allyl
1-((S)-5-(benzyloxy)-2-((2S,3S)-3-methyl-2-(pyrimidin-2-ylamino)pentanami-
do)-5-oxopentanoyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate
(40 mg, 0.0651 mmol, 1 equiv) and Pd(PPh.sub.3).sub.4(15 mg,
0.0130, 0.2 equiv) were dissolved in CH.sub.2Cl.sub.2 (10 mL) under
N.sub.2. Morpholine (0.017 mL, 0.195 mmol, 3 equiv) was then added
and the reaction was left at rt for 1 h. The solvent was then
evaporated and the product was purified by column chromatography
reverse phase using 10% to 50% methanol in water as the eluent to
give
(S)-1-((S)-5-(benzyloxy)-2-((2S,3S)-3-methyl-2-(pyrimidin-2-ylamino)penta-
namido)-5-oxopentanoyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic
acid as a colorless glass 37 mg, quantitative). MS (LC/MS) m/z
observed 574.94, expected 575.26 [M+H]. Compound was confirmed
using LC/MS and moved to next step as it was.
[0529] (S)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((2S,3S)-3-methyl-2-(pyrimidin-2-ylamino)pentanamido)-5-
-oxopentanoate was prepared from (S)-allyl
1-((S)-5-(benzyloxy)-2-((2S,3S)-3-methyl-2-(pyrimidin-2-ylamino)pentanami-
do)-5-oxopentanoyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate
and (2H-tetrazol-5-yl)methyl-amine using method A in DMF but
without HCl treatment. MS (LC/MS) m/z observed 655.98, expected
656.31 [M+H]. Compound was confirmed using LC/MS and moved to next
step as it was.
[0530] Title compound
(S)-5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo-
[2,3-b]pyridin-1-yl)-4-((2S,3S)-3-methyl-2-(pyrimidin-2-ylamino)pentanamid-
o)-5-oxopentanoic acid (C30) was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((2S,3S)-3-methyl-2-(pyrimidin-2-ylamino)pentanamido)-5-
-oxopentanoate using Method Q. MS (LC/MS) m/z observed 565.95,
expected 566.26 [M+H].
Example C31
(4S)-4-(2-CYCLOHEXYLACETAMIDO)-5-OXO-5-[(2S)-2-[(2H-1,2,3,4-TETRAZOL-5-YLM-
ETHYL)CARBAMOYL]-1H,2H,3H-PYRROLO[2,3-B]PYRIDIN-1-YL]PENTANOIC
ACID
[0531] (S)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-(2-cyclohexylacetamido)-5-oxopentanoate was
prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate
(from Example C4) and 2-cyclohexylacetic acid using method A but
the solvent was DMF for the coupling reaction. MS (LC/MS) m/z
observed 588.97, expected 589.29 [M+H]. Compound was confirmed
using LC/MS and moved to next step as it was.
[0532] Title compound
(4S)-4-(2-cyclohexylacetamido)-5-oxo-5-[(2S)-2-[(2H-1,2,3,4-tetrazol-5-yl-
methyl)carbamoyl]-1H,2H,3H-pyrrolo[2,3-b]pyridin-1-yl]pentanoic
acid (C31) was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-(2-cyclohexylacetamido)-5-oxopentanoate using
Method Q. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.78-0.92 (2H, m),
1.02-1.22 (3H, m), 1.52-1.65 (6H, m), 1.75 (1H, m), 1.93-2.02 (3H,
m), 2.25-2.45 (2H, m), 2.93 (1H, d, J=18 Hz), 3.43 (1H, m), 4.47
(1H, d, J=16 Hz), 4.67 (1H, d, J=16 Hz), 4.95 (1H, d, J=11 Hz),
5.81 (1H, s), 7.00 (1H, s), 7.62 (1H, d, J=7 Hz), 8.03 (1H, d, J=8
Hz), 8.15 (1H, s), 8.92 (1H, s), MS (LC/MS) m/z observed 498.96,
expected 498.23 [M+H].
Example C32
(4S)-5-OXO-4-(2-PHENYLACETAMIDO)-5-[(2S)-2-[(2H-1,2,3,4-TETRAZOL-5-YLMETHY-
L)CARBAMOYL]-1H,2H,3H-PYRROLO[2,3-B]PYRIDIN-1-YL]PENTANOIC ACID
[0533] (S)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-5-oxo-4-(2-phenylacetamido)pentanoate was prepared
from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate
(from Example C4) and 2-phenylacetic acid acid using method A but
the solvent was DMF for the coupling reaction. MS (LC/MS) m/z
observed 582.92, expected 583.24 [M+H]. Compound was confirmed
using LC/MS and moved to next step as it was.
[0534] Title compound
(4S)-5-oxo-4-(2-phenylacetamido)-5-[(2S)-2-[(2H-1,2,3,4-tetrazol-5-ylmeth-
yl)carbamoyl]-1H,2H,3H-pyrrolo[2,3-b]pyridin-1-yl]pentanoic acid
(C32) was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-5-oxo-4-(2-phenylacetamido)pentanoate using Method
Q. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 1.82 (1H, m), 2.05 (1H,
m), 2.25-2.45 (2H, m), 2.95 (1H, d, J=17 Hz), 3.25-3.50 (3H, m),
4.47 (1H, d, J=16 Hz), 4.67 (1H, d, J=16 Hz), 4.98 (1H, d, J=11
Hz), 5.87 (1H, m), 7.03 (1H, m), 7.17-7.35 (5H, m), 7.66 (1H, d,
J=7 Hz), 8.15 (1H, s), 8.40 (1H, d, J=8 Hz), 8.96 (1H, s), MS
(LC/MS) m/z observed 492.92, expected 493.19 [M+H].
Example C33
(4S)-5-OXO-4-[(2R)-2-PHENYLPROPANAMIDO]-5-[(2S)-2-[(2H-1,2,3,4-TETRAZOL-5--
YLMETHYL)CARBAMOYL]-1H,2H,3H-PYRROLO[2,3-B]PYRIDIN-1-YL]PENTANOIC
ACID
[0535] (S)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-5-oxo-4-((R)-2-phenylpropanamido)pentanoate was
prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate
(from Example C4) and (R)-2-phenylpropanoic acid using method A but
the solvent was DMF for the coupling reaction. MS (LC/MS) m/z
observed 596.93, expected 597.26 [M+H]. Compound was confirmed
using LC/MS and moved to next step as it was.
[0536] Title compound
(4S)-5-oxo-4-[(2R)-2-phenylpropanamido]-5-[(2S)-2-[(2H-1,2,3,4-tetrazol-5-
-ylmethyl)carbamoyl]-1H,2H,3H-pyrrolo[2,3-b]pyridin-1-yl]pentanoic
acid was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-5-oxo-4-((R)-2-phenylpropanamido)pentanoate using
Method Q. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 1.28 (3H, d, J=8
Hz), 1.78 (1H, m), 1.99 (1H, m), 2.18 (1H, m), 2.33 (1H, m), 2.95
(1H, d, J=17 Hz), 3.45 (1H, m), 3.73 (1H, m), 4.47 (1H, d, J=16
Hz), 4.67 (1H, dd, J=8, 16 Hz), 5.00 (1H, d, J=11 Hz), 5.77 (1H,
m), 7.03 (1H, m), 7.20 (1H, m), 7.25-7.36 (4H, m), 7.66 (1H, d, J=7
Hz), 8.12 (1H, s), 8.28 (1H, d, J=8 Hz), 8.96 (1H, s), MS (LC/MS)
m/z observed 506.94, expected 507.21 [M+H].
Example C34
(4S)-5-OXO-4-[(2S)-2-PHENYLPROPANAMIDO]-5-[(2S)-2-[(2H-1,2,3,4-TETRAZOL-5--
YLMETHYL)CARBAMOYL]-1H,2H,3H-PYRROLO[2,3-B]PYRIDIN-1-YL]PENTANOIC
ACID
[0537] (S)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-5-oxo-4-((S)-2-phenylpropanamido)pentanoate was
prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate
(from Example C4) and (S)-2-phenylpropanoic acid using method A but
the solvent was DMF for the coupling reaction. MS (LC/MS) m/z
observed 596.94, expected 597.26 [M+H]. Compound was confirmed
using LC/MS and moved to next step as it was.
[0538] Title compound
(4S)-5-oxo-4-[(2S)-2-phenylpropanamido]-5-[(2S)-2-[(2H-1,2,3,4-tetrazol-5-
-ylmethyl)carbamoyl]-1H,2H,3H-pyrrolo[2,3-b]pyridin-1-yl]pentanoic
acid (C34) was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-5-oxo-4-((S)-2-phenylpropanamido)pentanoate using
Method Q. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 1.32 (3H, d, J=8
Hz), 1.80 (1H, m), 2.01 (1H, m), 2.30-2.48 (2H, m), 2.95 (1H, d,
J=17 Hz), 3.43 (1H, m), 3.75 (1H, m), 4.47 (1H, d, J=16 Hz), 4.67
(1H, m), 4.95 (1H, dd, J=11.18 Hz), 5.90 (1H, m), 7.03 (1H, m),
7.18 (1H, m), 7.25-7.35 (4H, m), 7.66 (1H, d, J=7 Hz), 8.12 (1H,
m), 8.28 (1H, d, J=8 Hz), 8.96 (1H, s), MS (LC/MS) m/z observed
506.94, expected 507.21 [M+H].
Example C35
(4S)-4-(2-CYCLOBUTYLACETAMIDO)-5-OXO-5-[(2S)-2-[(2H-1,2,3,4-TETRAZOL-5-YLM-
ETHYL)CARBAMOYL]-1H,2H,3H-PYRROLO[2,3-B]PYRIDIN-1-YL]PENTANOIC
ACID
[0539] (S)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-(2-cyclobutylacetamido)-5-oxopentanoate was
prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate
(from Example C4) and cyclobutylacetic acid using method A but the
solvent was DMF for the coupling reaction. MS (LC/MS) m/z observed
561.00, expected 561.26 [M+H]. Compound was confirmed using LC/MS
and moved to next step as it was.
[0540] Title compound
(4S)-4-(2-cyclobutylacetamido)-5-oxo-5-[(2S)-2-[(2H-1,2,3,4-tetrazol-5-yl-
methyl)carbamoyl]-1H,2H,3H-pyrrolo[2,3-b]pyridin-1-yl]pentanoic
acid (C35) was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-(2-cyclobutylacetamido)-5-oxopentanoate using
Method Q. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 1.60-1.71 (2H, m),
1.72-1.82 (3H, m), 1.93-2.04 (3H, m), 2.17-2.25 (2H, m), 2.31-2.43
(2H, m), 2.53 (1H, m), 2.95 (1H, d, J=18 Hz), 3.45 (1H, dd, J=11,
18 Hz), 4.46 (1H, dd, J=5, 16 Hz), 4.66 (1H, dd, J=6, 16 Hz), 4.98
(1H, dd, J=4, 11 Hz), 5.83 (1H, m), 7.02 (1H, dd, J=5, 7 Hz), 7.65
(1H, d, J=7 Hz), 8.01 (1H, d, J=8 Hz), 8.17 (1H, d, J=5 Hz), 8.92
(1H, m), MS (LC/MS) m/z observed 470.98, expected 471.21 [M+H].
Example C36
(4S)-4-(2-CYCLOPROPYLACETAMIDO)-5-OXO-5-[(2S)-2-[(2H-1,2,3,4-TETRAZOL-5-YL-
METHYL)CARBAMOYL]-1H,2H,3H-PYRROLO[2,3-B]PYRIDIN-1-YL]PENTANOIC
ACID
[0541] (S)-Benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-(2-cyclopropylacetamido)-5-oxopentanoate was
prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate
(from Example C4) and 2-cyclopropylacetic acid using method A but
the solvent was DMF for the coupling reaction. MS (LC/MS) m/z
observed 546.98, expected 547.24 [M+H]. Compound was confirmed
using LC/MS and moved to next step as it was.
[0542] Title compound
(4S)-4-(2-cyclopropylacetamido)-5-oxo-5-[(2S)-2-[(2H-1,2,3,4-tetrazol-5-y-
lmethyl)carbamoyl]-1H,2H,3H-pyrrolo[2,3-b]pyridin-1-yl]pentanoic
acid (C36) was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-(2-cyclopropylacetamido)-5-oxopentanoate using
Method Q. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.08-0.15 (2H, m),
0.37-0.44 (2H, d, J=9 Hz), 0.95 (1H, m), 1.80 (1H, m), 1.96-2.06
(3H, m), 2.30-2.45 (2H, m), 2.96 (1H, d, J=18 Hz), 3.43 (1H, dd,
J=11, 18 Hz), 4.47 (1H, dd, J=5, 16 Hz), 4.67 (1H, dd, J=6, 16 Hz),
5.00 (1H, dd, J=4, 11 Hz), 5.88 (1H, m), 7.02 (1H, dd, J=5, 7 Hz),
7.65 (1H, d, J=7 Hz), 7.99 (1H, d, J=8 Hz), 8.17 (1H, d, J=5 Hz),
8.92 (1H, m), MS (LC/MS) m/z observed 456.92, expected 457.19
[M+H].
Example C37
(4S)-5-OXO-5-[(2S)-2-[(2H-1,2,3,4-TETRAZOL-5-YLMETHYL)CARBAMOYL]-1H,2H,3H--
PYRROLO[2,3-B]PYRIDIN-1-YL]-4-[2-(THIOPHEN-3-YL)ACETAMIDO]PENTANOIC
ACID
[0543]
(S)-1-((S)-5-(tert-Butoxy)-2-((tert-butoxycarbonyl)amino)-5-oxopent-
anoyl)-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid was
prepared from I-7 and Boc-L-glutamic acid tert-butyl ester (3 eq.)
using method C in DMF. MS (LC/MS) m/z observed 449.98, expected
450.22 [M+H]. Compound was confirmed using LC/MS and moved to next
step as it was.
[0544] (S)-tert-Butyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate was
prepared from
(S)-1-((S)-5-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-5-oxopentanoyl)-
-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid and
(2H-tetrazol-5-yl)methyl-amine using method A in DMF but without
HCl treatment. MS (LC/MS) m/z observed 530.96, expected 531.27
[M+H]. Compound was confirmed using LC/MS and moved to next step as
it was.
[0545] (S)-tert-Butyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate
(49.7 mg, 0.0880 mmol) was dissolved in 4M HCl in dioxane (10 mL)
and the reaction was heated to 50.degree. C. for 3 hours. Both
tert-butyl ester and Boc group were removed. The solvent was
evaporated to give a residue as a colorless glass. In a separated
flask were dissolved 2-(thiophen-3-yl)acetic acid (125.1 mg, 0.880
mmol, 10 eq.), HOBt (16.1 mg, 0.106 mmol, 1.2 eq.) and EDC (30.2
mg, 0.106 mmol, 1.2 eq.) in DMF (5 mL). DIPEA was then added (0.230
mL, 1.320 mmol, 15 eq.) and the mixture was stirred at RT for 10
minutes. A solution in DMF (2 mL) of the residue obtained
previously was slowly added to the mixture containing the
2-(thiophen-3-yl)acetic acid and the reaction was left at RT for 10
minutes. The solvent was then evaporated and the product was
purified by first a preparative reverse phase HPLC purification
using a 10 minutes gradient from 40% to 52% methanol in water. The
excess 2-(thiophen-3-yl)acetic acid and the desired product
co-eluted on this column. The product was then repurified by normal
phase chromatography using 5% methanol in DCM as the eluent to
remove the excess acid and then 10% methanol, 1% HCOOH and 89% DCM
to elute desired the title compound
(4S)-5-oxo-5-[(2S)-2-[(2H-1,2,3,4-tetrazol-5-ylmethyl)carbamoyl]-1H,2H,3H-
-pyrrolo[2,3-b]pyridin-1-yl]-4-[2-(thiophen-3-yl)acetamido]pentanoic
acid (C37) that was obtained as a white solid (12.5 mg, 27%).
.sup.1H NMR (400 MHz, DMSO-d6) .delta. 1.82 (1H, m), 2.03 (1H, m),
2.30-2.45 (2H, m), 2.48-2.53 (2H, m), 2.96 (1H, d, J=18 Hz), 3.43
(1H, dd, J=11, 18 Hz), 4.47 (1H, dd, J=5, 16 Hz), 4.67 (1H, dd,
J=6, 16 Hz), 5.00 (1H, dd, J=4, 11 Hz), 5.88 (1H, m), 6.98-7.04
(2H, m), 7.23 (1H, s), 7.43 (1H, m), 7.65 (1H, d, J=7 Hz), 8.14
(1H, d, J=5 Hz), 8.35 (1H, d, J=8 Hz), 8.92 (1H, m), MS (LC/MS) m/z
observed 498.96, expected 499.15 [M+H].
Example C38
(4S)-4-[2-(MORPHOLIN-2-YL)ACETAMIDO]-5-OXO-5-[(2S)-2-[(2H-1,2,3,4-TETRAZOL-
-5-YLMETHYL)CARBAMOYL]-1H,2H,3H-PYRROLO[2,3-B]PYRIDIN-1-YL]PENTANOIC
ACID
[0546] (9H-Fluoren-9-yl)methyl
2-(2-(((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H--
pyrrolo[2,3-b]pyridin-1-yl)-5-(benzyloxy)-1,5-dioxopentan-2-yl)amino)-2-ox-
oethyl)morpholine-4-carboxylate was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate
(from Example C4) and
2-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)morpholin-2-yl)acetic acid
using method A but the solvent was DMF for the coupling reaction.
MS (LC/MS) m/z observed 813.99, expected 814.33 [M+H]. Compound was
confirmed using LC/MS and moved to next step as it was.
[0547] (9H-Fluoren-9-yl)methyl
2-(2-(((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H--
pyrrolo[2,3-b]pyridin-1-yl)-5-(benzyloxy)-1,5-dioxopentan-2-yl)amino)-2-ox-
oethyl)morpholine-4-carboxylate (41.3 mg, 0.0516 mmol) was
dissolved in a 1:1 mixture methanol/DCM (20 mL) and palladium on
charcoal 10% by wt (10 mg) was added to the solution under N.sub.2.
The flask was then flushed with H.sub.2 and H.sub.2 was bubbled
into the reaction mixture for 4 hrs. The flask was flushed with
N.sub.2 and the reaction mixture was filtered over CELITE.TM.. The
solids were washed with methanol (3.times.10 mL) and
CH.sub.2Cl.sub.2 (3.times.10 mL) and the filtrate and washings were
then concentrated to give a light brown solid that was dissolved in
DMF (5 mL) and morpholine (5 mL). The reaction was left at RT for 1
h and the solvent was evaporated. The product was then purified by
reverse phase preparative HPLC using a 10 minutes gradient from 0%
to 15% methanol in water (containing 0.1% HCOOH) to give title
compound
(4S)-4-[2-(morpholin-2-yl)acetamido]-5-oxo-5-[(2S)-2-[(2H-1,2,3,4-tetrazo-
l-5-ylmethyl)carbamoyl]-1H,2H,3H-pyrrolo[2,3-b]pyridin-1-yl]pentanoic
acid as a light brown solid (15.5 mg, 56%). .sup.1H NMR (400 MHz,
DMSO-d6) .delta. 1.80 (1H, m), 2.02 (1H, m), 2.27-2.45 (4H, m),
2.76 (1H, m), 2.87-3.02 (2H, m), 3.06-3.25 (2H, m), 3.45 (1H, dd,
J=11, 18 Hz), 3.66 (1H, m), 3.90 (1H, m), 4.02 (1H, m), 4.47 (1H,
dd, J=5, 16 Hz), 4.67 (1H, dd, J=6, 16 Hz), 5.00 (1H, m), 5.88 (1H,
m), 7.04 (1H, m), 7.65 (1H, d, J=7 Hz), 8.14 (1H, d, J=5 Hz), 8.35
(1H, m), 9.01 (1H, m), 9.23-9.37 (2H, m), MS (LC/MS) m/z observed
502.01, expected 502.22 [M+H].
Example C39
(4S)-4-[2-(MORPHOLIN-3-YL)ACETAMIDO]-5-OXO-5-[(2S)-2-[(2H-1,2,3,4-TETRAZOL-
-5-YLMETHYL)CARBAMOYL]-1H,2H,3H-PYRROLO[2,3-B]PYRIDIN-1-YL]PENTANOIC
ACID
[0548] tert-Butyl
3-(2-(((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H--
pyrrolo[2,3-b]pyridin-1-yl)-5-(benzyloxy)-1,5-dioxopentan-2-yl)amino)-2-ox-
oethyl)morpholine-4-carboxylate was prepared from (S)-benzyl
5-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2,3-
-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate
(from Example C4) and
2-(4-(tert-butoxycarbonyl)morpholin-3-yl)acetic acid using method A
but the solvent was DMF for the coupling reaction. MS (LC/MS) m/z
observed 691.89, expected 692.32 [M+H]. Compound was confirmed
using LC/MS and moved to next step as it was.
[0549] tert-Butyl
3-(2-(((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H--
pyrrolo[2,3-b]pyridin-1-yl)-5-(benzyloxy)-1,5-dioxopentan-2-yl)amino)-2-ox-
oethyl)morpholine-4-carboxylate (41.3 mg, 0.0597 mmol) was
dissolved in methanol (10 mL) and palladium on charcoal 10% by wt
(10 mg) was added to the solution under N.sub.2. The flask was then
flushed with H.sub.2 and H.sub.2 was bubbled into the reaction
mixture for 4 hrs. The flask was flushed with N.sub.2 and the
reaction mixture was filtered over CELITE.TM.. The solids were
washed with methanol (3.times.10 mL) and the filtrate and washings
were then concentrated to give a light brown solid that was
dissolved 4M HCl in dioxane (5 mL). The reaction was left at RT for
1 h and the solvent was evaporated. The product was then purified
by reverse phase preparative HPLC using a 10 minutes gradient from
0% to 15% methanol in water (containing 0.1% HCOOH) to give title
compound
(4S)-4-[2-(morpholin-3-yl)acetamido]-5-oxo-5-[(2S)-2-[(2H-1,2,3,4-tetrazo-
l-5-ylmethyl)carbamoyl]-1H,2H,3H-pyrrolo[2,3-b]pyridin-1-yl]pentanoic
acid (C39) as white solid (12.1 mg, 29%). .sup.1H NMR (400 MHz,
DMSO-d6) .delta. 1.90 (1H, m), 2.02 (1H, m), 2.30-2.45 (4H, m),
2.91-3.06 (3H, m), 3.25-3.32 (2H, m), 3.35-3.55 (2H, m), 2.75-3.85
(2H, m), 4.33 (1H, d, J=16 Hz), 4.50 (1H, d, J=16 Hz), 5.00 (1H, d,
J=11 Hz), 5.92 (1H, s), 7.02 (1H, m), 7.65 (1H, d, J=7 Hz), 8.14
(1H, s), 8.50 (1H, m), 8.60 (1H, m), MS (LC/MS) m/z observed
502.02, expected 502.22 [M+H].
Example C40
(4S)-4-(2-CYCLOPENTYLACETAMIDO)-5-OXO-5-[(2S)-2-[(1H-1,2,3-TRIAZOL-4-YLMET-
HYL)CARBAMOYL]-1H,2H,3H-PYRROLO[2,3-B]PYRIDIN-1-YL]PENTANOIC
ACID
[0550] (S)-Benzyl
5-((S)-2-(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-4-(2-cyclopentylacetamido)-5-oxopentanoate
was prepared from (S)-benzyl
5-((S)-2-(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate
(from Example C11) and cyclopentylacetic acid using method A but
the solvent was DMF for the coupling reaction. MS (LC/MS) m/z
observed 574.05, expected 574.28 [M+H]. Compound was confirmed
using LC/MS and moved to next step as it was.
[0551] Title compound
(4S)-4-(2-cyclopentylacetamido)-5-oxo-5-[(2S)-2-[(1H-1,2,3-triazol-4-ylme-
thyl)carbamoyl]-1H,2H,3H-pyrrolo[2,3-b]pyridin-1-yl]pentanoic acid
(C40) was prepared from (S)-benzyl
5-((S)-2-(((1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrol-
o[2,3-b]pyridin-1-yl)-4-(2-cyclopentylacetamido)-5-oxopentanoate
using Method Q. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 1.06-1.18
(2H, m), 1.41-1.50 (2H, m), 1.52-1.58 (2H, m), 1.62-1.70 (2H, m),
1.80 (1H, m), 1.96-2.14 (4H, m), 2.30-2.47 (2H, m), 2.95 (1H, dd,
J=4, 17 Hz), 3.45 (1H, dd, J=11, 17 Hz), 4.28-4.41 (2H, m), 4.96
(1H, dd, J=4, 11 Hz), 5.83 (1H, m), 7.03 (1H, dd, J=5, 7 Hz),
7.60-7.70 (2H, m), 8.05 (1H, d, J=8 Hz), 8.17 (1H, d, J=4 Hz), 8.70
(1H, m), MS (LC/MS) m/z observed 483.93, expected 484.23 [M+H].
Example C41
3-{[(1S,2S)-2-METHYL-1-{[(2S)-1-OXO-3-(2H-1,2,3,4-TETRAZOL-5-YL)-1-(2S)-2--
[(2H-1,2,3,4-TETRAZOL-5-YLMETHYL)CARBAMOYL]-1H,2H,3H-PYRROLO[2,3-B]PYRIDIN-
-1-YL]PROPAN-2-YL]CARBAMOYL}BUTYL]CARBAMOYL}PROPANOIC ACID
[0552] (S)-2-(Fmoc-amino)-3-(2H-tetrazol-5-yl) propanoic acid (400
mg, 1.054 mmol, 1 eq.) was dissolved in DMF (5 mL). Morpholine (5
mL) was then added and the reaction was left at RT for 10 minutes,
where it went to completion. The solvents were concentrated and the
residue was dissolved in dioxane (10 mL) and Boc.sub.2O (276.1 mg,
1.265 mmol, 1.2 eq.) was added, followed by triethylamine (0.365
mL, 2.635 mmol, 2.5 eq.). The reaction was left at RT for 2 hrs and
was then acidified to pH 4 with a saturated solution of citric
acid. The solvent was evaporated and the product was purified by
reverse phase C18 column chromatography using 10% methanol in water
as the eluent.
(S)-2-((tert-Butoxycarbonyl)amino)-3-(2H-tetrazol-5-yl)propanoic
acid was obtained as a colorless glass (202 mg, 75%). MS (LC/MS)
m/z observed 257.87, expected 258.12 [M+H].sup.+. Compound was
confirmed using LC/MS and moved to next step as it was.
[0553] Allyl
1-((S)-2-((tert-butoxycarbonyl)amino)-3-(2H-tetrazol-5-yl)propanoyl)-2,3--
dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate was prepared from
(S)-2-((tert-butoxycarbonyl)amino)-3-(2H-tetrazol-5-yl)propanoic
acid and (S)-allyl
2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate hydrochloride
(from Example C30) using method C in DMF. MS (LC/MS) m/z observed
443.82, expected 444.20 [M+H]. Compound was confirmed using LC/MS
and moved to next step as it was.
[0554] Allyl
1-((S)-2-((tert-butoxycarbonyl)amino)-3-(2H-tetrazol-5-yl)propanoyl)-2,3--
dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (90 mg, 0.203 mmol,
1 equiv) and Pd(PPh.sub.3).sub.4(46.9 mg, 0.0406, 0.2 equiv) were
dissolved in CH.sub.2Cl.sub.2 (15 mL) under N.sub.2. Morpholine
(0.053 mL, 0.609 mmol, 3 equiv) was then added and the reaction was
left at RT for 1 h. The solvent was then evaporated and the product
was purified by column chromatography reverse phase using 10% to
50% methanol in water as the eluent to give
1-((S)-2-((tert-butoxycarbonyl)amino)-3-(2H-tetrazol-5-yl)propanoyl)-2,3--
dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid as a colorless
glass 72 mg, 88%). MS (LC/MS) m/z observed 403.83, expected 404.17
[M+H]. Compound was confirmed using LC/MS and moved to next step as
it was.
[0555] tert-Butyl
((2S)-1-(2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2-
,3-b]pyridin-1-yl)-1-oxo-3-(2H-tetrazol-5-yl)propan-2-yl)carbamate
was prepared from
1-((S)-2-((tert-butoxycarbonyl)amino)-3-(2H-tetrazol-5-yl)propanoyl)-2,3--
dihydro-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid and
(2H-tetrazol-5-yl)methyl-amine using method A in DMF but without
HCl treatment. MS (LC/MS) m/z observed 484.83, expected 485.21
[M+H]. Compound was confirmed using LC/MS and moved to next step as
it was.
[0556] tert-Butyl
((2S,3S)-1-(((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihyd-
ro-1H-pyrrolo[2,3-b]pyridin-1-yl)-1-oxo-3-(2H-tetrazol-5-yl)propan-2-yl)am-
ino)-3-methyl-1-oxopentan-2-yl)carbamate was prepared from
tert-butyl
((2S)-1-(2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihydro-1H-pyrrolo[2-
,3-b]pyridin-1-yl)-1-oxo-3-(2H-tetrazol-5-yl)propan-2-yl)carbamate
and Boc-L-Isoleucine using method A but the solvent was DMF for the
coupling reaction. MS (LC/MS) m/z observed 597.88, expected 598.30
[M+H]. Compound was confirmed using LC/MS and moved to next step as
it was.
[0557] Title compound
3-{[(1S,2S)-2-methyl-1-{[(2S)-1-oxo-3-(2H-1,2,3,4-tetrazol-5-yl)-1-[(2S)--
2-[(2H-1,2,3,4-tetrazol-5-ylmethyl)carbamoyl]-1H,2H,3H-pyrrolo[2,3-b]pyrid-
in-1-yl]propan-2-yl]carbamoyl}butyl]carbamoyl}propanoic acid (C41)
was prepared from tert-butyl
((2S,3S)-1-(((S)-1-((S)-2-(((2H-tetrazol-5-yl)methyl)carbamoyl)-2,3-dihyd-
ro-1H-pyrrolo[2,3-b]pyridin-1-yl)-1-oxo-3-(2H-tetrazol-5-yl)propan-2-yl)am-
ino)-3-methyl-1-oxopentan-2-yl)carbamate and succinic anhydride
using method I. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 0.76-0.85
(6H, m), 1.05 (1H, m), 1.39 (1H, m), 1.65 (1H, m), 2.35-2.45 (4H,
m), 2.60-2.70 (2H, m), 2.75 (1H, m), 3.25 (1H, m), 4.15 (1H, m),
4.51-4.61 (2H, m), 5.02 (1H, m), 6.25 (1H, s), 7.03 (1H, m), 7.63
(1H, d, J=7 Hz), 7.90 (1H, d, J=9 Hz), 8.08 (1H, m), 8.36 (1H, m),
9.25 (1H, s), MS (LC/MS) m/z observed 597.91, expected 598.26
[M+H].
Example D1
General Kinetic Enzyme Assay Protocol
[0558] A specific 2.times. assay buffer was prepared for the enzyme
to be tested (see Table 2 for final 1.times. assay buffer
compositions). If the assay buffer included DTT, it was added
immediately prior to running the assay. A 2.times. enzyme mix was
prepared (see Table 3 for enzyme assay conditions) at 80 uL per
well. Compounds were screened at one or two appropriate
concentrations (to determine the percent inhibition at those
concentrations) and/or a full dose response curve (typically 8
points, to identify the IC.sub.50) in duplicate, triplicate, or
higher replicates as needed. An appropriate control was also
assessed in full dose response, in duplicate for each assay/plate.
Background control wells consisted of 1.times. assay buffer, DMSO
(5% v/v) and substrate. Positive control wells consisted of enzyme,
DMSO (5% v/v) and substrate. Test compounds and control compounds
were diluted in DMSO to 40.times. the final desired concentration.
For example, a test compound may be tested in dose response, in
serial, tripling dilution condition starting at 20 uM and ending at
9.1 nM (or any appropriate concentration range and dilution
scheme). Control compounds were prepared similarly. Diluted
compounds were prepared in a dilution plate and transferred to the
reaction plate (96-well medium binding plate (Greiner Bio-One
FLUOTRAC.TM.)) to allow for the desired final concentrations when
added to the enzyme with AB. After mixing, the reaction plate was
placed on a shaker (at 300 RPM) for 5 min, followed by incubation
(covered) on the bench, for 20 min. Plates were warmed to reaction
temperature (see Table 3) for a total incubation time of 30 min.
Plates so prepared were ready for addition of substrate and the
subsequent reaction.
[0559] An appropriate substrate for each assay was prepared in
advance at 2.times. the final desired concentration (see Table 2)
in DMSO. The appropriate substrate mix was added to each
appropriate well on the reaction plate, and the plate was read
immediately in the TECAN plate reader (TECAN INFINITE.RTM. M1000
Pro), set to the correct wavelength as needed for each assay (see
Table 3) using 25 cycles, kinetic interval of 1 min, number of
reads per well of 20 with shaking set to is, double orbital, 2 mm
amplitude. For fluorescent assays the gain was set to optimal
(50%).
TABLE-US-00002 TABLE 2 Assay Buffer Composition. Enzyme Assay
Buffer Composition Caspase 1, 3, 4, 5, 7, 8*, 9 & 10/a 50 mM
HEPES pH 7.2 (General caspase assay buffer) 50 mM NaCl 0.1% (w/v)
CHAPS 10 mM EDTA 5% (v/v) Glycerol 10 mM DTT GzmB & Caspase 8
50 mM HEPES pH 7.5 10% (w/v) sucrose 0.2% (w/v) CHAPS 5 mM DTT *Can
also use GzmB assay buffer for the Caspase-8 assay; Assay buffer
components were sourced as follows: HEPES, DTT, Glycerol and
sucrose: Sigma-Aldrich, St. Louis, MO, USA, NaCl and EDTA: Fisher
Scientific, Pittsburgh, PA, USA, CHAPS: Calbiochem, Billerica, MA,
USA.
TABLE-US-00003 TABLE 3 Enzyme assay conditions. Substrate Assay
Enzyme Conc. Ex/Em .lamda.* Temp Control Name Conc. Name (.mu.M)
(nm) (.degree. C.) Inhibitor hGzmB 10 nM Ac-IEPD-AMC 150 380/460 30
Ac-IEPD-CHO Caspase-1 6.25 mU/.mu.l YVAD-AFC 25 400/505 37
Z-VAD-FMK Caspase-3 and 6.25 mU/.mu.l Ac-DEVD-AMC 20 380/460 37
Z-VAD-FMK Caspase 7 Caspase-4 and 3.125 mU/ul Ac-WEHD-AFC 100
400/505 37 Z-WEHD-FMK Caspase-5 Caspase-8 3.125 mU/ul Ac-IEPD-AMC
75 380/460 30 Ac-IEPD-CHO Caspase-9 3.125 mU/ul LEHD-AFC 50 400/505
37 Q-LEHD-Oph Caspase-10/a 6.25 mU/.mu.l Ac-IETD-AMC 100 400/505 30
Ac-AEVD-CHO *Ex/Em .lamda. is the excitation and emission
wavelengths at which to measure fluorescence. Enzyme and substrate
concentrations are the final concentrations in the well. Note that
most protocols require preparing 2X enzyme and substrate mixes, as
they are diluted 2-fold in the well.
[0560] Enzymes were sourced as follows: hGzmB, Froelich Lab,
Northshore University Health Systems Research Institute, Evanston,
Ill., USA; Caspases, Biovision Inc., Milpitas, Calif., USA.
Substrates were sourced as follows: Ac-IEPD-AMC, California Peptide
Research Inc., Napa, Calif., USA; YVAD-AFC, Biovision Inc.,
Milpitas, Calif., USA; Ac-DEVD-AMC, LEHD-AFC, AC-WEHD-AFC and
Ac-IETD-AMC, Enzo Life Sciences Inc, Farmingdale, N.Y., USA.
Control inhibitors were sourced as follows: Ac-IEPD-CHO,
Ac--WEHD-FMK and Q-LEHD-Oph, Biovision Inc., Milpitas, Calif., USA;
Z-VAD-FMK, R&D Systems, Minneapolis, Minn., USA; and
Ac-AEVD-CHO, Enzo Life Sciences Inc, Farmingdale, N.Y., USA.
Example D2
Human Granzyme B Enzymatic Inhibition Assay
[0561] An in vitro fluorogenic detection assay for assessing the
IC.sub.50 and/or percent inhibition at a given concentration of
inhibitors against human Granzyme B (hGzmB) enzyme was performed as
described in Example D1. When appropriate, percent inhibition data
was collected and fitted to generate IC.sub.50 data using GraphPad
Prism 5 (GraphPad Software, La Jolla Calif. USA, www.graphpad.com)
and its non-linear regression analysis tools or other equivalent
tools.
[0562] Select compounds of Examples A1 and C1-C41 exhibited
inhibitory activity against hGzmB. Each of the compounds of the
invention identified in Table 1 exhibited Granzyme B inhibitory
activity.
[0563] In certain embodiments, select compounds exhibited
IC.sub.50<50,000 nM. In other embodiments, select compounds
exhibited IC.sub.50<10,000 nM. In further embodiments, select
compounds exhibited IC.sub.50<1,000 nM. In still further
embodiments, select compounds exhibited IC.sub.50<100 nM. In
certain embodiments, select compounds exhibited IC.sub.50 from 10
nM to 100 nM, preferably from 1 nM to 10 nM, more preferably from
0.1 nM to 1 nM, and even more preferably from 0.01 nM to 0.1
nM.
Example D3
Human Caspase Enzymatic Inhibition Assay
[0564] In vitro fluorogenic detection assays for assessing the
IC.sub.50 and/or percent inhibition at a given concentration of
inhibitors, against a set of human Caspase enzymes, was performed
as described in Example D1. Representative compounds of the
invention do not significantly inhibit any caspase enzyme tested at
a concentration of 50 .mu.M.
[0565] In certain embodiments, the compounds exhibited less than
50% inhibition at 50 .mu.M. In other embodiments, the compounds
exhibited greater than 50% inhibition at 50 .mu.M, but less than
10% inhibition at 25 .mu.M.
Example D4
General Kinetic Enzyme Assay Protocol (384 Well)
[0566] A specific 2.times. assay buffer was prepared for the enzyme
to be tested (see Table 4 for final 1.times. assay buffer
compositions). If the assay buffer included DTT, it was added
immediately prior to running the assay. A 2.times. enzyme mix was
prepared (see Table 3 for enzyme assay conditions) at 26 uL per
well. Compounds were screened at one or two appropriate
concentrations (to determine the percent inhibition at those
concentrations) and/or a full dose response curve (typically 12
points, to identify the IC.sub.50) in duplicate, triplicate, or
higher replicates as needed. An appropriate control was also
assessed in full dose response, in duplicate for each assay/plate.
Background control wells consisted of 1.times. assay buffer and
substrate. Positive control wells consisted of enzyme (no DMSO) and
substrate. Test compounds and control compounds were diluted in
1.times. Assay Buffer to 15.times. the final desired concentration.
For example, a test compound may be tested in dose response, in
serial, tripling dilution condition starting at 20 uM and ending at
0.1 nM (or any appropriate concentration range and dilution
scheme). Control compounds were prepared similarly. Diluted
compounds were prepared in a dilution plate and transferred to the
reaction plate (384-well medium binding plate (Greiner Bio-One
FLUOTRAC.TM.)) to allow for the desired final concentrations when
added to the enzyme with AB. After mixing, the reaction plate was
placed on a shaker (at 300 RPM) for 5 min, followed by incubation
(covered) on the bench, for 20 min. Plates were warmed to reaction
temperature (see Table 5) for 5 mins for a total incubation time of
30 min. Plates so prepared were ready for addition of substrate and
the subsequent reaction.
[0567] An appropriate substrate for each assay was prepared in
advance at 2.times. the final desired concentration (see Table 4)
in assay buffer. 30 uL of the appropriate substrate mix was added
to each appropriate well on the reaction plate, and the plate was
read immediately in the TECAN plate reader (TECAN INFINITE.RTM.
M1000 Pro), set to the correct wavelength as needed for each assay
(see Table 5) using 15 cycles, kinetic interval of 1 min, number of
reads per well of 20 with shaking set to is, double orbital, 2 mm
amplitude. For fluorescent assays the gain was set to optimal (100%
with gain regulation) for all assays except human GzmB which was
set to 85 (with the z set at 23000 um).
TABLE-US-00004 TABLE 4 Assay Buffer Composition. Enzyme Assay
Buffer Composition Caspase 1, 3, 4, 5, 7, 8*, 9 & 10/a 50 mM
HEPES pH 7.2 (General caspase assay buffer) 50 mM NaCl 0.1% (w/v)
CHAPS 10 mM EDTA 5% (v/v) Glycerol 10 mM DTT GzmB & Caspase 8
50 mM HEPES pH 7.5 0.2% (w/v) CHAPS 5 mM DTT Cathepsin G 320 mM
Tris-HCL pH 7.4 3.2M NaCl *Can also use GzmB assay buffer for the
Caspase-8 assay; Assay buffer components were sourced as follows:
HEPES, DTT, Glycerol and sucrose: Sigma-Aldrich, St. Louis, MO,
USA, NaCl and EDTA: Fisher Scientific, Pittsburgh, PA, USA, CHAPS:
Calbiochem, Billerica, MA, USA.
TABLE-US-00005 TABLE 5 Enzyme assay conditions. Substrate Enzyme
Conc. Ex/Em .lamda.* Assay Control Name Conc. Name (.mu.M) (nm)
Temp (.degree. C.) Inhibitor hGzmB 10 nM Ac-IEPD- 50 380/460 30
V2248 AMC Caspase-1 12.5 mU/.mu.L YVAD- 5 400/505 37 Z-VAD- AFC FMK
Caspase-3 and 0.8 mU/.mu.L Ac-DEVD- 40 380/460 37 Z-VAD- Caspase 7
&1.5 mU/.mu.L AMC &5 FMK Caspase-4 and 3.125mU/uL &
Ac-WEHD- 40 400/505 37 Z-WEHD- Caspase-5 1.5mU/uL AFC & 100 FMK
Caspase-8 4 mU/uL Ac-IEPD- 80 380/460 37 Ac-IEPD- AMC CHO Caspase-9
2mU/uL LEHD-AFC 50 400/505 37 Q-LEHD- Oph Caspase-10/a 3 mU/.mu.L
Ac-IETD- 10 400/505 37 Ac-AEVD- AMC CHO Cathepsin G 200 nM
Suc-AAPF- 200 uM 410 25 Cat G pNA absorbance inhibitor Human 0.125
ug/mL MeOSuc- 50 384/500 37 Sivelestat Neutrophil AAPF-AFC Elastase
*Ex/Em .lamda. is the excitation and emission wavelengths at which
to measure fluorescence. Enzyme and substrate concentrations are
the final concentrations in the well. Note that most protocols
require preparing 2X enzyme and substrate mixes, as they are
diluted 2-fold in the well.
[0568] Enzymes were sourced as follows: hGzmB, Froelich Lab,
Northshore University Health Systems Research Institute, Evanston,
Ill., USA; Caspases and Elastase, Biovision Inc., Milpitas, Calif.,
USA; Cathepsin G, Athens Research and Technologies, Athens, Ga.,
USA. Substrates were sourced as follows: Ac-IEPD-AMC, California
Peptide Research Inc., Napa, Calif., USA; YVAD-AFC and
MeOSuc-AAPF-AFC Biovision Inc., Milpitas, Calif., USA; LEHD-AFC and
Suc-AAPF-pNA Millipore, Billerica Mass., USA. Ac-DEVD-AMC,
AC-WEHD-AFC and Ac-IETD-AMC, Enzo Life Sciences Inc, Farmingdale,
N.Y., USA. Control inhibitors were sourced as follows: Ac-IEPD-CHO,
Ac--WEHD-FMK, Q-LEHD-Oph and CatG inhibito,r Biovision Inc.,
Milpitas, Calif., USA; Z-VAD-FMK, R&D Systems, Minneapolis,
Minn., USA; and Ac-AEVD-CHO, Enzo Life Sciences Inc, Farmingdale,
N.Y., USA. Sivelestat, Tocris Bioscience, Bristol, UK.
Example D5
Human Granzyme B Enzymatic Inhibition Assay
[0569] An in vitro fluorogenic detection assay for assessing the
IC.sub.50 and/or percent inhibition at a given concentration of
inhibitors against human Granzyme B (hGzmB) enzyme was performed as
described in Example D4. When appropriate, percent inhibition data
was collected and fitted to generate IC.sub.50 data using GraphPad
Prism 5 (GraphPad Software, La Jolla Calif. USA, www.graphpad.com)
and its non-linear regression analysis tools or other equivalent
tools.
[0570] Select compounds of Examples A1 and C1 to C41 exhibited
inhibitory activity against hGzmB. Each of the compounds of the
invention identified in Table 1 exhibited Granzyme B inhibitory
activity.
[0571] In certain embodiments, select compounds exhibited
IC.sub.50<50,000 nM. In other embodiments, select compounds
exhibited IC.sub.50<10,000 nM. In further embodiments, select
compounds exhibited IC.sub.50<1,000 nM. In still further
embodiments, select compounds exhibited IC.sub.50<100 nM. In
certain embodiments, select compounds exhibited IC.sub.50 from 10
nM to 100 nM, preferably from 1 nM to 10 nM, more preferably from
0.1 nM to 1 nM, and even more preferably from 0.01 nM to 0.1
nM.
Example D6
Human Caspase Enzymatic Inhibition Assay
[0572] In vitro fluorogenic detection assays for assessing the
IC.sub.50 and/or percent inhibition at a given concentration of
inhibitors, against a set of human Caspase enzymes, was performed
as described in Example D4. Representative compounds of the
invention do not significantly inhibit any caspase enzyme tested at
a concentration of 50 .mu.M.
[0573] In certain embodiments, the compounds exhibited less than
50% inhibition at 50 .mu.M. In other embodiments, the compounds
exhibited greater than 50% inhibition at 50 .mu.M, but less than
10% inhibition at 25 .mu.M.
Example D7
Inhibition of Cell Detachment by GzmB Assay
[0574] HDFa primary human fibroblasts were plated at 10 k/well in
200 ul, approximately hrs before treatment. The next day, controls
and 100 nM GzmB (recombinant, human) plus or minus inhibitor
treatments were prepared in serum-free media. GzmB and inhibitor
were incubated for 20 minutes at RT before adding to cells. Before
addition, media and serum was removed from the cells and the cells
were washed with PBS (1.times.), using pipettes to prevent
disturbing the cells. Treatment preparations (100 ul) were added to
the wells and incubated for 7 hours in a tissue culture incubator.
After 7 hours, media and treatments were removed and the cells were
washed with PBS (1.times.) to removed detached cells, using
pipettes only. Phase pictures were taken then the PBS was removed
and replaced with 100 uL of serum-free media and 20 uL of MTS and
the cells were allowed to incubate for 3 hours in a cell culture
incubator. After 3 hours the absorbance was read at 490 nm and a
percent inhibition value for the treatments with inhibitors was
determined from the control wells. The resulting date is shown in
Table 6.
TABLE-US-00006 TABLE 6 Inhibition of Cell Detachment by GzmB
Results. Compound Percent Inhibition of cell detachment at 50 uM C4
100%
Example D8
Inhibition of Fibronectin Cleavage by GzmB
[0575] Black, 96 well high-binding assay plates (Griener Bio-one)
were treated overnight at 4.degree. C. with 40 uL of 8 ug/mL Hilyte
Fluor 488 labeled Fibronectin (Cytoskeleton, Inc). After
fibronectin coating, plates were washed 3 times in buffer (20 mM
Tris-HCl, pH 7.4, 20 mM NaCl) then once with granzyme B assay
buffer (50 mM HEPES, pH 7.5, 0.1% CHAPS). After washing, 50 uL of
granzyme B assay buffer was added to each fibronectin-coated well.
In a separate non-binding 96 well assay plate 5 uL of 20.times.
inhibitor serial dilution stocks were added to 45 uL of
2.22.times.GzmB mix to establish inhibition (enzyme/inhibitor mixes
were all prepared in granzyme B assay buffer and were incubated
first at room temperature for 20 minutes, then at 30.degree. C. for
another 10 minutes). After incubation, 50 uL of this 2.times.
enzyme/inhibitor mix was added to the corresponding coated well to
initiate fibronectin cleavage (20 nM final granzyme B
concentration, 8-point inhibitor dilution series starting at 50
uM). The assay was conducted at 30.degree. C. in the TECAN plate
reader (TECAN INFINITE.RTM. M1000 Pro), which was programmed to
monitor the kinetic fluorescence polarization signal (filter set
Ex/Em 470 nm/527 nm) with readings taken every minute, for 1 hour.
Proteolytic activity was evaluated as the rate of fluorescence
enhancement in the parallel emission over the linear range of the
reaction. % Inhibition values were calculated from assay controls
and the resulting date is shown in Table 7.
TABLE-US-00007 TABLE 7 Inhibition of Fibronectin Cleavage by GzmB
Results. Percent Inhibition at Inhibitor Concentration Compound 50
uM 5.56 uM 0.62 uM A1 88% 82% 66 C4 98% 79% 45% C9 91% 81% 58% C17
94% 77% 56%
Example D9
Inhibition of Cell Adhesion by GzmB Cleavage of Fibronectin
[0576] Black, 96 well high-binding clear-bottom assay plates
(Griener Bio-one) were treated overnight at 4.degree. C. with 40 uL
of 5 ug/mL Fibronectin (Sigma-Aldrich). After fibronectin coating,
wells were washed 3 times in Tris wash buffer (20 mM Tris-HCl, pH
7.4, 20 mM NaCl) then once with granzyme B assay buffer (HEPES, (50
mM, pH 7.5), CHAPS (0.1%)). After washing, 50 uL of granzyme B
assay buffer was added to each fibronectin-coated well. In a
separate non-binding 96 well assay plate 5 uL of relevant 20.times.
inhibitor dilution stocks were added to 45 uL of 2.22.times.GzmB
mix to establish inhibition (enzyme/inhibitor mixes were all
prepared in granzyme B assay buffer and were incubated first at
room temperature for 20 minutes, then at 30.degree. C. for another
10 minutes). After incubation, 50 uL of this 2.times.
enzyme/inhibitor mix was added to the corresponding coated well to
initiate fibronectin cleavage (20 nM final granzyme B
concentration, 3 final inhibitor concentrations--0.1 uM, 10 uM and
100 uM). The assay was conducted at 30.degree. C. in a plate warmer
for 2 hours. After incubation, wells were washed 3 times with PBS,
and then blocked with 2% BSA in PBS for 1 hour at room temperature.
After sufficient blocking, wells were washed an additional 3 times
with PBS to remove residual BSA. 3T3 fibroblasts, harvested from
sub-confluent conditions, were prepared in serum free DMEM and
introduced into the treated wells at 10,000 cells/well. Cells were
allowed to adhere for 90 minutes or until appropriate attached
phenotype was detected. After attachment, wells were agitated with
gentle repeat pipetting 3 times, gently aspirated manually and
washed once with PBS. Wells were then fixed with 4%
paraformaldehyde in PBS for 1 hr, washed twice with PBS and stained
with the nuclear dye DAPI. Microscopic detection and counting of
stained nuclei was performed using IMAGE-PRO.RTM. Plus software.
Cell count was normalized to % Cell Adhesion. The results are shown
in Table 8.
TABLE-US-00008 TABLE 8 Inhibition of Cell Adhesion by GzmB Results.
Percent Cell Adhesion at Inhibitor Concentration Compound 100 uM 10
uM 0.1 uM C4 146% 88% 13%
* * * * *
References