U.S. patent application number 10/157791 was filed with the patent office on 2004-01-08 for immunomodulatory compounds and method of use thereof.
Invention is credited to Hawkins, Lynn D., Ishizaka, Sally T..
Application Number | 20040006242 10/157791 |
Document ID | / |
Family ID | 29582541 |
Filed Date | 2004-01-08 |
United States Patent
Application |
20040006242 |
Kind Code |
A1 |
Hawkins, Lynn D. ; et
al. |
January 8, 2004 |
Immunomodulatory compounds and method of use thereof
Abstract
The present invention is directed to methods of treating
diseases and disorders related to immune responses by administering
one or more immunomodulatory compounds. In particular, the
invention is directed to methods of stimulating and reducing immune
responses, treating autoimmune conditions, treating allergic
reactions and asthma, and preventing ischemic damage and asthma by
administering one or more immunomodulatory compounds.
Inventors: |
Hawkins, Lynn D.; (Concord,
MA) ; Ishizaka, Sally T.; (Weston, MA) |
Correspondence
Address: |
HALE AND DORR, LLP
60 STATE STREET
BOSTON
MA
02109
|
Family ID: |
29582541 |
Appl. No.: |
10/157791 |
Filed: |
May 28, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10157791 |
May 28, 2002 |
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09918849 |
Jul 31, 2001 |
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6551600 |
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09918849 |
Jul 31, 2001 |
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09496152 |
Feb 1, 2000 |
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6290973 |
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60118131 |
Feb 1, 1999 |
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Current U.S.
Class: |
554/78 |
Current CPC
Class: |
A61K 2039/55511
20130101; A61P 1/00 20180101; A61P 29/00 20180101; A61P 37/04
20180101; A61K 31/16 20130101; A61K 31/6615 20130101; C07F 9/10
20130101; A61P 3/10 20180101; A61P 27/02 20180101; A61P 7/06
20180101; A61K 31/17 20130101; A61P 37/06 20180101; C07F 9/091
20130101; A61P 19/06 20180101; A61P 17/00 20180101; A61P 31/04
20180101; A61P 37/08 20180101; A61P 9/10 20180101; A61P 31/12
20180101; A61P 25/00 20180101; A61P 1/16 20180101; A61P 19/02
20180101; A61P 11/06 20180101; A61P 21/00 20180101; A61P 37/02
20180101; A61P 9/00 20180101; A61K 39/39 20130101; A61K 2039/55555
20130101; A61P 17/02 20180101; C07F 9/65515 20130101; A61P 1/04
20180101; A61P 11/02 20180101 |
Class at
Publication: |
554/78 |
International
Class: |
C07F 009/02 |
Claims
What is claimed is:
1. A method of inducing or stimulating an immune response in a
subject comprising administering an effective amount of a compound
of the formula I, II, or III, as the active ingredient: 291Wherein
for each of formula I, II, or III: R.sup.1 is selected from the
group consisting of (a) C(O); (b) C(O)--C.sub.1-14 alkyl-C(O),
wherein said C.sub.1-14 alkyl is optionally substituted with
hydroxy, C.sub.1-5 alkoxy, C.sub.1-5 alkylenedioxy, C.sub.1-5
alkylamino, or C.sub.1-5-alkyl-aryl, wherein said aryl moiety of
said C.sub.1-15-alkyl-aryl is optionally substituted with C.sub.1-5
alkoxy, C.sub.1-5 alkyl amino, C.sub.1-5 alkoxy-amino, C.sub.1-5
alkylamino-C.sub.1-5 alkoxy, --O--C.sub.1-5 alkylamino-C.sub.1-5
alkoxy, --O--C.sub.1-5 alkylamino-C(O)--C.sub.1-5 alkyl C(O)OH,
--O--C-.sub.1-5 alkylamino-C(O)--C.sub.1-5 alkyl-C(O)--C.sub.1-5
alkyl; (c) C.sub.2 to C.sub.15 straight or branched chain alkyl
optionally substituted with hydroxy or alkoxy; and (d)
--C(O)--C.sub.6-12 arylene-C(O)-- wherein said arylene is
optionally substituted with hydroxy, halogen, nitro or armno; a and
b are independently 0, 1, 2, 3 or 4; d, d', d", e, e' and e" are
independently an integer from 1 to 4; X.sup.1, X.sup.2, Y.sup.1 and
Y.sup.2 are independently selected from the group consisting of
null, oxygen, NH and N(C(O)C.sub.1-4 alkyl), and N(C.sub.1-4
alkyl).sub.2; W.sup.1 and W.sup.2 are independently selected from
the group consisting of carbonyl, methylene, sulfone and sulfoxide;
R.sup.2 and R.sup.5 are independently selected from the group
consisting of: (a) C.sub.2 to C.sub.20 straight chain or branched
chain alkyl which is optionally substituted with oxo, hydroxy or
alkoxy, (b) C.sub.2 to C.sub.20 straight chain or branched chain
alkenyl or dialkenyl which is optionally substituted with oxo,
hydroxy or alkoxy; (c) C.sub.2 to C.sub.20 straight chain or
branched chain alkoxy which is optionally substituted with oxo,
hydroxy or alkoxy; (d) --NH--C.sub.2 to C.sub.20 straight chain or
branched chain alkyl, wherein said alkyl group is optionally
substituted with oxo, hydroxy or alkoxy; and (e) 292wherein Z is
selected from the group consisting of O and NH, and M and N are
independently selected from the group consisting of C.sub.2 to
C.sub.20 straight chain or branched chain alkyl, alkenyl, alkoxy,
acyloxy, alkylamino, and acylamino; R.sup.1 and R.sup.6 are
independently selected from the group consisting of C.sub.2 to
C.sub.20 straight chain or branched chain alkyl or alkenyl
optionally substituted with oxo or fluoro; R.sup.4 and R.sup.7 are
independently selected from the group consisting of C(O)C.sub.2 to
C.sub.20 straight chain or branched chain alkyl or alkenyl; C.sub.2
to C.sub.20 straight chain or branched chain alkyl; C.sub.2 to
C.sub.20 straight chain or branched chain alkoxy; C.sub.2 to
C.sub.20 straight chain or branched chain alkenyl; wherein said
alkyl, alkenyl or alkoxy groups can be independently and optionally
substituted with hydroxy, fluoro or C.sub.1 to C.sub.5 alkoxy;
G.sup.1, G.sup.2, G.sup.3 and G.sup.4 are independently selected
from the group consisting of oxygen, methylene, amino, thiol,
--NHC(O)--, and --N(C(O)C.sub.1-4 alkyl)--; or G.sup.2R.sup.4 or
G.sup.4R.sup.7 may together be a hydrogen atom or hydroxyl; or a
pharmaceutically acceptable salt thereof; and wherein for Formula
II: a' and b' are independently 2, 3, 4, 5, 6, 7, or 8, preferably
2; Z.sup.1 is selected from the group consisting of
--OP(O)(OH).sub.2, --P(O)(OH).sub.2,--OP(O)(OR.sup.8)(OH) where
R.sup.8is a C1-C4 alkyl chain,
--OS(O).sub.2OH,--S(O).sub.2OH--,--CO.sub.2H, --OB(OH).sub.2, --OH,
--CH.sub.3, --NH.sub.2, NR.sup.9.sub.3 where R.sup.9 is a C1-C4
alkyl chain; Z.sup.2 is --OP(O)(OH).sub.2, --P(O)(OH).sub.2',
--OP(O)(OR.sup.10)(OH) where R.sup.10 is a C1-C4 alkyl chain, --OS
(O).sub.2OH,--S(O).sub.2OH, CO.sub.2H, --OB(OH).sub.2, --OH,
CH.sub.3, --NH.sub.2, --NR.sup.11, where R.sup.11 is a C1-C4 alkyl
chain; and wherein for Formula 3: R.sup.12 is selected from H and a
C1-C4 alkyl chain; or a pharmaceutical salt thereof, with the
proviso that the compounds of formula I, II, or III are not 293
2. The method of claim 1, wherein the compounds bind a Toll-like
receptor.
3. The method of claim 2, wherein the Toll-like receptor is
Toll-like receptor 4.
4. The method of claim 1, wherein the subject is a human.
5. The method of claim 1, wherein the subject is a mammal, fish, or
reptile.
6. The method of claim 1, wherein the immune response stimulates
the production of at least one cytokine selected from the group
consisting of IL-1.alpha., IL-1.beta., IL-6, IL-10, IL12,
interferon-.alpha., interferon-.gamma., and GM-CSF.
7. A method for upregulation of the immune system comprising
administering a therapeutically effective amount of a compound of
formula I, II, or III, 294Wherein for each of formula I, II, or
III: R.sup.1 is selected from the group consisting of (a) C(O); (b)
C(O)--C.sub.1-14 alkyl-C(O), wherein said C.sub.1-14 alkyl is
optionally substituted with hydroxy, C.sub.1-5 alkoxy, C.sub.1-5
alkylenedioxy, C.sub.1-5 alkylamino, or C.sub.1-5-alkyl-aryl,
wherein said aryl moiety of said C.sub.1-15-alkyl-aryl is
optionally substituted with C.sub.1-5 alkoxy, C.sub.1-5 alkyl
amino, C.sub.1-5 alkoxy-amino, C.sub.1-5 alkylamino-C.sub.1-5
alkoxy, --O--C.sub.1-5 alkylamino-C.sub.1-5 alkoxy, --O--C.sub.1-5
alkylamino-C(O)--C.sub.1-5 alkyl C(O)OH, --O--C.sub.1-5
alkylamino-C(O)--C.sub.1-5 alkyl-C(O)--C.sub.1-5 alkyl; (c) C.sub.2
to C.sub.15 straight or branched chain alkyl optionally substituted
with hydroxy or alkoxy; and (d) --C(O)--C.sub.6-12 arylene-C(O)--
wherein said arylene is optionally substituted with hydroxy,
halogen, nitro or amino; a and b are independently 0, 1, 2, 3 or 4;
d, d', d", e, e' and e" are independently an integer from 1 to 4;
X.sup.1, X.sup.2, Y.sup.1 and Y.sup.2 are independently selected
from the group consisting of null, oxygen, NH and N(C(O)C.sub.1-4
alkyl), and N(C.sub.1-4 alkyl).sub.2; W.sup.1 and W.sup.2 are
independently selected from the group consisting of carbonyl,
methylene, sulfone and sulfoxide; R.sup.2 and R.sup.5 are
independently selected from the group consisting of: (a) C.sub.2 to
C.sub.20 straight chain or branched chain alkyl which is optionally
substituted with oxo, hydroxy or alkoxy, (b) C.sub.2 to C.sub.20
straight chain or branched chain alkenyl or dialkenyl which is
optionally substituted with oxo, hydroxy or alkoxy; (c) C.sub.2 to
C.sub.20 straight chain or branched chain alkoxy which is
optionally substituted with oxo, hydroxy or alkoxy; (d)
--NH--C.sub.2 to C.sub.20 straight chain or branched chain alkyl,
wherein said alkyl group is optionally substituted with oxo,
hydroxy or alkoxy; and (e) 295wherein Z is selected from the group
consisting of O and NH, and M and N are independently selected from
the group consisting of C.sub.2 to C.sub.20 straight chain or
branched chain alkyl, alkenyl, alkoxy, acyloxy, alkylamino, and
acylamino; R.sup.1 and R.sup.6 are independently selected from the
group consisting of C.sub.2 to C.sub.20 straight chain or branched
chain alkyl or alkenyl optionally substituted with oxo or fluoro;
R.sup.4 and R.sup.7 are independently selected from the group
consisting of C(O)C.sub.2 to C.sub.20 straight chain or branched
chain alkyl or alkenyl; C.sub.2 to C.sub.20 straight chain or
branched chain alkyl; C.sub.2 to C.sub.20 straight chain or
branched chain alkoxy; C.sub.2 to C.sub.20 straight chain or
branched chain alkenyl; wherein said alkyl, alkenyl or alkoxy
groups can be independently and optionally substituted with
hydroxy, fluoro or C, to C.sub.5 alkoxy; G.sup.1, G.sup.2, G.sup.3
and G.sup.4 are independently selected from the group consisting of
oxygen, methylene, amino, thiol, --NHC(O)--, and --N(C(O)C.sub.1-4
alkyl)--; or G.sup.2R.sup.4 or G.sup.4R.sup.7 may together be a
hydrogen atom or hydroxyl; or a pharmaceutically acceptable salt
thereof; and wherein for Formula II: a' and b' are independently 2,
3, 4, 5, 6, 7, or 8, preferably 2; Z.sup.1 is selected from the
group consisting of
--OP(O)(OH).sub.2,--P(O)(OH).sub.2,--OP(O)(OR.sup.8)(OH) where
R.sup.8 is a C1-C4 alkyl chain,
--OS(O).sub.2OH,--S(O).sub.2OH--,--CO.sub.2H, --OB(OH).sub.2, --OH,
--CH.sub.3, --NH.sub.2, NR.sup.9.sub.3 where R.sup.9 is a C1-C4
alkyl chain; Z.sup.2 is --OP(O)(OH).sub.2, --P(O)(OH).sub.2',
--OP(O)(OR.sup.10) (OH) where R.sup.10is a C1-C4 alkyl chain, --OS
(O).sub.2OH,--S(O).sub.2OH, CO.sub.2H, --OB(OH).sub.2, --OH,
CH.sub.3, --NH.sub.2, --NR.sup.11, where R.sup.11 is a C1-C4 alkyl
chain; and wherein for Formula 3: R.sup.12 is selected from H and a
C1-C4 alkyl chain; or a pharmaceutical salt thereof, with the
proviso that the compounds of formula I, II, or III are not
296wherein said compound has immunostimulatory activity.
8. A method of reducing an immune response in a subject, the method
comprising administering to the subject a compound of formula I,
II, or III 297Wherein for each of formula I, IT, or III: R.sup.1 is
selected from the group consisting of (a) C(O); (b)
C(O)--C.sub.1-14 alkyl-C(O), wherein said C.sub.1-14 alkyl is
optionally substituted with hydroxy, C.sub.1-5 alkoxy, C.sub.1-5
alkylenedioxy, C.sub.1-5alkylamino, or C.sub.1-5-alkyl-aryl,
wherein said aryl moiety of said C.sub.1-15-alkyl-aryl is
optionally substituted with C.sub.1-5 alkoxy, C.sub.1-5 alkyl
amino, C.sub.1-5 alkoxy-amino, C.sub.1-5 alkylamino-C.sub.1-5
alkoxy, --O--C.sub.1-5 alkylamino-C.sub.5 alkoxy, --O--C.sub.1-5
alkylamino-C (O)--C.sub.1-5 alkyl C(O)OH, --O--C.sub.1-5
alkylamino-C(O)--C.sub.1-5 alkyl-C(O)--C.sub.1-5 alkyl; (c) C.sub.2
to C.sub.15 straight or branched chain alkyl optionally substituted
with hydroxy or alkoxy; and (d) --C(O)--C.sub.6-12 arylene-C(O)--
wherein said arylene is optionally substituted with hydroxy,
halogen, nitro or amino; a and b are independently 0, 1, 2, 3 or 4;
d, d', d", e, e' and e" are independently an integer from 1 to 4;
X.sup.1, X.sup.2, Y.sup.1 and Y.sup.2 are independently selected
from the group consisting of null, oxygen, NH and N(C(O)C.sub.1-4
alkyl), and N(C.sub.1-4 alkyl).sub.2; W.sup.1 and W.sup.2 are
independently selected from the group consisting of carbonyl,
methylene, sulfone and sulfoxide; R.sup.2 and R.sup.5 are
independently selected from the group consisting of: (a) C.sub.2 to
C.sub.20 straight chain or branched chain alkyl which is optionally
substituted with oxo, hydroxy or alkoxy, (b) C.sub.2 to C.sub.20
straight chain or branched chain alkenyl or dialkenyl which is
optionally substituted with oxo, hydroxy or alkoxy; (c) C.sub.2 to
C.sub.20 straight chain or branched chain alkoxy which is
optionally substituted with oxo, hydroxy or alkoxy; (d)
--NH--C.sub.2 to C.sub.20 straight chain or branched chain alkyl,
wherein said alkyl group is optionally substituted with oxo,
hydroxy or alkoxy; and (e) 298wherein Z is selected from the group
consisting of O and NH, and M and N are independently selected from
the group consisting of C.sub.2 to C.sub.20 straight chain or
branched chain alkyl, alkenyl, alkoxy, acyloxy, alkylamino, and
acylamino; R.sup.1 and R.sup.6 are independently selected from the
group consisting of C.sub.2 to C.sub.20 straight chain or branched
chain alkyl or alkenyl optionally substituted with oxo or fluoro;
R.sup.4 and R.sup.7 are independently selected from the group
consisting of C(O)C.sub.2 to C.sub.20 straight chain or branched
chain alkyl or alkenyl; C.sub.2 to C.sub.20 straight chain or
branched chain alkyl; C.sub.2 to C.sub.20 straight chain or
branched chain alkoxy; C.sub.2 to C.sub.20 straight chain or
branched chain alkenyl; wherein said alkyl, alkenyl or alkoxy
groups can be independently and optionally substituted with
hydroxy, fluoro or C.sub.1 to C.sub.5 alkoxy; G.sup.1, G.sup.2,
G.sup.3 and G.sup.4 are independently selected from the group
consisting of oxygen, methylene, amino, thiol, --NHC(O)--, and
--N(C(O)C.sub.1-4 alkyl)-; or G.sup.2R.sup.4 or G.sup.4R.sup.7 may
together be a hydrogen atom or hydroxyl; or a pharmaceutically
acceptable salt thereof; and wherein for Formula II: a' and b' are
independently 2, 3, 4, 5, 6, 7, or 8, preferably 2; Z.sup.1 is
selected from the group consisting of
--OP(O)(OH).sub.2,--P(O)(OH).sub.2,--OP(O)(OR.sup.8)(OH) where
R.sup.8 is a C1-C4 alkyl chain,
--OS(O).sub.2OH,--S(O).sub.2OH--,--CO.sub.2H, --OB(OH).sub.2, --OH,
--CH.sub.3, --NH.sub.2, NR.sup.9.sub.3 where R.sup.9 is a C1-C4
alkyl chain; Z.sup.2 is --OP(O)(OH).sub.2, --P(O)(OH).sub.2',
--OP(O)(OR.sup.10)(OH) where R.sup.10is a C1-C4 alkyl chain, --OS
(O).sub.2OH,--S(O).sub.2OH, CO.sub.2H, --OB(OH).sub.2, --OH,
CH.sub.3, --NH.sub.2, --NR.sup.11, where R.sup.11 is a C1-C4 alkyl
chain; and wherein for Formula 3: R.sup.12 is selected from H and a
C1-C4 alkyl chain; or a pharmaceutical salt thereof, with the
proviso that the compounds of formula I, II, or III are not 299in
an amount sufficient to reduce an immune response in the subject
and thereby induce a therapeutic effect.
9. A method of desensitizing a subject against the occurrence of an
allergic reaction in response to contact with a particular antigen
or allergen, comprising administering to the subject an effective
amount of an immunostimulatory compound of the formula I, II, or
III 300Wherein for each of formula I, II, or III: R.sup.1 is
selected from the group consisting of (a) C(O); (b)
C(O)--C.sub.1-14 alkyl-C(O), wherein said C.sub.1-14 alkyl is
optionally substituted with hydroxy, C.sub.1-5 alkoxy, C.sub.1-5
alkylenedioxy, C.sub.1-5 alkylamino, or C .sub.1-5-alkyl-aryl,
wherein said aryl moiety of said C.sub.1-15-alkyl-aryl is
optionally substituted with C.sub.1-5 alkoxy, C.sub.1-5 alkyl
amino, C.sub.1-5 alkoxy-amino, C.sub.1-5 alkylamino-C.sub.1-5
alkoxy, --O--C.sub.1-5 alkylamino-C.sub.1-5 alkoxy, --O--C.sub.1-5
alkylamino-C(O)--C.sub.1-5 alkyl C(O)OH, --O--C.sub.1-5
alkylamino-C(O)--C.sub.1-5 alkyl-C(O)--C.sub.1-5 alkyl; (c) C.sub.2
to C.sub.15 straight or branched chain alkyl optionally substituted
with hydroxy or alkoxy; and (d) --C(O)--C.sub.6-12 arylene-C(O)--
wherein said arylene is optionally substituted with hydroxy,
halogen, nitro or amino; a and b are independently 0, 1, 2, 3 or 4;
d, d', d", e, e' and e" are independently an integer from 1 to 4;
X.sup.1, X.sup.2, Y.sup.1 and Y.sup.2 are independently selected
from the group consisting of null, oxygen, NH and N(C(O)C.sub.1-4
alkyl), and N(C.sub.1-4 alkyl).sub.2; W.sup.1 and W.sup.2 are
independently selected from the group consisting of carbonyl,
methylene, sulfone and sulfoxide; R.sup.2 and R.sup.5 are
independently selected from the group consisting of: (a) C.sub.2 to
C.sub.20 straight chain or branched chain alkyl which is optionally
substituted with oxo, hydroxy or alkoxy, (b) C.sub.2 to C.sub.20
straight chain or. branched chain alkenyl or dialkenyl which is
optionally substituted with oxo, hydroxy or alkoxy; (c) C.sub.2 to
C.sub.20 straight chain or branched chain alkoxy which is
optionally substituted with oxo, hydroxy or alkoxy; (d)
--NH--C.sub.2 to C.sub.20 straight chain or branched chain alkyl,
wherein said alkyl group is optionally substituted with oxo,
hydroxy or alkoxy; and (e) 301wherein Z is selected from the group
consisting of O and NH, and M and N are independently selected from
the group consisting of C.sub.2 to C.sub.20 straight chain or
branched chain alkyl, alkenyl, alkoxy, acyloxy, alkylamino, and
acylamino; R.sup.1 and R.sup.6 are independently selected from the
group consisting of C.sub.2 to C.sub.20 straight chain or branched
chain alkyl or alkenyl optionally substituted with oxo or fluoro;
R.sup.4 and R.sup.7 are independently selected from the group
consisting of C(O)C.sub.2 to C.sub.20 straight chain or branched
chain alkyl or alkenyl; C.sub.2 to C.sub.20 straight chain or
branched chain alkyl; C.sub.2 to C.sub.20 straight chain or
branched chain alkoxy; C.sub.2 to C.sub.20 straight chain or
branched chain alkenyl; wherein said alkyl, alkenyl or alkoxy
groups can be independently and optionally substituted with
hydroxy, fluoro or C.sub.1 to C.sub.5 alkoxy; G.sup.1, G.sup.2,
G.sup.3 and G.sup.4 are independently selected from the group
consisting of oxygen, methylene, amino, thiol, --NHC(O)--, and
--N(C(O)C.sub.1-4alkyl)--; or G.sup.2R.sup.4 or G.sup.4R.sup.7 may
together be a hydrogen atom or hydroxyl; or a pharmaceutically
acceptable salt thereof; and wherein for Formula II: a' and b' are
independently 2, 3, 4, 5, 6, 7, or 8, preferably 2; Z.sup.1 is
selected from the group consisting of
--OP(O)(OH.sub.2,--P(O)(OH).sub.2,--OP(O)(OR.sup.8)(OH) where
R.sup.8 is a C1-C4 alkyl chain,
--OS(O).sub.2OH,--S(O).sub.2OH--,--CO.sub.2H, --OB(OH).sub.2, --OH,
--CH.sub.3, --NH.sub.2, NR.sup.9.sub.3 where R.sup.9 is a C1-C4
alkyl chain; Z.sup.2 is --OP(O)(OH).sub.2, --P(O)(OH).sub.2',
--OP(O)(OR.sup.10)(OH) where R.sup.10is a C1-C4 alkyl chain,
--OS(O).sub.2OH,--S(O).sub.2OH, CO.sub.2H, --OB(OH).sub.2, --OH,
CH.sub.3, --NH.sub.2, --NR.sup.11, where R.sup.11 is a C1-C4 alkyl
chain; and wherein for Formula 3: R.sup.12 is selected from H and a
C1-C4 alkyl chain; or a pharmaceutical salt thereof, with the
proviso that the compounds of formula I, II, or III are not 302as
the active ingredient.
10. The method of claim 9, wherein the subject suffers from asthma,
atopic dermatitis, or allergic rhinitis.
11. A method of treating an autoimmune disease, the method
comprising administering to a subject diagnosed as having an
autoimmune disease a pharmaceutical composition comprising (a) a
compound of formula I, II, or III, 303Wherein for each of formula
I, II, or III: R.sup.1 is selected from the group consisting of (a)
C(O); (b) C(O)--C.sub.1-14 alkyl-C(O), wherein said C.sub.1-14
alkyl is optionally substituted with hydroxy, C.sub.1-5 alkoxy,
C.sub.1-5 alkylenedioxy, C.sub.1-5 alkylamino, or
C.sub.1-5-alkyl-aryl, wherein said aryl moiety of said
C.sub.1-15-alkyl-aryl is optionally substituted with C.sub.1-5
alkoxy, C.sub.1-5 alkyl amino, C.sub.1-5 alkoxy-amino, C.sub.1-5
alkylamino-C.sub.1-5 alkoxy, --O--C.sub.1-5 alkylamino-C1-5 alkoxy,
--O--C.sub.1-5 alkylamino-C(O)--C.sub.1-5 alkyl C(O)OH,
--O--C.sub.1-5 alkylamino-C(O)--C.sub.1-5 alkyl-C(O)--C.sub.1-5
alkyl; (c) C.sub.2 to C.sub.15 straight or branched chain alkyl
optionally substituted with hydroxy or alkoxy; and (d)
--C(O)--C.sub.6-12 arylene-C(O)-- wherein said arylene is
optionally substituted with hydroxy, halogen, nitro or amino; a and
b are independently 0, 1, 2, 3 or 4; d, d', d", e, e' and e" are
independently an integer from 1 to 4; X.sup.1, X.sup.2, Y.sup.1 and
Y.sup.2 are independently selected from the group consisting of
null, oxygen, NH and N(C(O)C.sub.1-4 alkyl), and N(C.sub.1-4
alkyl).sub.2; W.sup.1 and W.sup.2 are independently selected from
the group consisting of carbonyl, methylene, sulfone and sulfoxide;
R.sup.2 and R.sup.5 are independently selected from the group
consisting of: (a) C.sub.2 to C.sub.20 straight chain or branched
chain alkyl which is optionally substituted with oxo, hydroxy or
alkoxy, (b) C.sub.2 to C.sub.20 straight chain or branched chain
alkenyl or dialkenyl which is optionally substituted with oxo,
hydroxy or alkoxy; (c) C.sub.2 to C.sub.20 straight chain or
branched chain alkoxy which is optionally substituted with oxo,
hydroxy or alkoxy; (d) --NH--C.sub.2 to C.sub.20 straight chain or
branched chain alkyl, wherein said alkyl group is optionally
substituted with oxo, hydroxy or alkoxy; and (e) 304wherein Z is
selected from the group consisting of O and NH, and M and N are
independently selected from the group consisting of C.sub.2 to
C.sub.20 straight chain or branched chain alkyl, alkenyl, alkoxy,
acyloxy, alkylamino, and acylamino; R.sup.1 and R.sup.6 are
independently selected from the group consisting of C.sub.2 to
C.sub.20 straight chain or branched chain alkyl or alkenyl
optionally substituted with oxo or fluoro; R.sup.4 and R.sup.7 are
independently selected from the group consisting of C(O)C.sub.2 to
C.sub.20 straight chain or branched chain alkyl or alkenyl; C.sub.2
to C.sub.20 straight chain or branched chain alkyl; C.sub.2 to
C.sub.20 straight chain or branched chain alkoxy; C.sub.2 to
C.sub.20 straight chain or branched chain alkenyl; wherein said
alkyl, alkenyl or alkoxy groups can be independently and optionally
substituted with hydroxy, fluoro or C.sub.1 to C.sub.5 alkoxy;
G.sup.1, G.sup.2, G.sup.3 and G.sup.4 are independently selected
from the group consisting of oxygen, nethylene, amino, thiol,
--NHC(O)--, and --N(C(O)C.sub.1-4 alkyl)--; or G.sup.2R.sup.4 or
G.sup.4R.sup.7 may together be a hydrogen atom or hydroxyl; or a
pharmaceutically acceptable salt thereof; and wherein for Formula
II: a' and b' are independently 2, 3, 4, 5, 6, 7, or 8, preferably
2; Z.sup.1 is selected from the group consisting of
--OP(O)(OH).sub.2,--P(O)(OH).sub.2,--OP(O)(OR.sup.8)(OH) where
R.sup.8 is a C1-C4 alkyl chain,
--OS(O).sub.2OH,--S(O).sub.2OH--,--CO.sub.2H, --OB(OH).sub.2, --OH,
--CH.sub.3, --NH.sub.2, NR.sup.9.sub.3 where R.sup.9 is a C1-C4
alkyl chain; Z.sup.2 is --OP(O)(OH).sub.2, --P(O)(OH).sub.2',
--OP(O)(OR.sup.10)(OH) where R.sup.10is a C1-C4 alkyl chain,
--OS(O).sub.2OH,--S(O).sub.2OH, CO.sub.2H,--OB(OH).sub.2, --OH,
CH.sub.3, --NH.sub.2, --NR.sup.11, where R.sup.11 is a C1-C4 alkyl
chain; and wherein for Formula 3: R.sup.12 is selected from H and a
C1-C4 alkyl chain; or a pharmaceutical salt thereof, with the
proviso that the compounds of formula I, II, or III are not 305or a
salt thereof, and (b) a pharmaceutically acceptable carrier
therefor, wherein the compound is an immunoinhibitory compound.
12. The method of claim 11, wherein the autoimmune disease is
systemic lupus erythematosis, sceleroderma, Sjogren's syndrome,
multiple sclerosis and other demyelinating diseases, rheumatoid
arthritis, juvenile arthritis, systemic lupus erythamatosus,
myocarditis, Graves'disease, uveitis, Reiter's syndrome, gout,
osteoarthritis, polymyositis, myocarditis, primary biliary
cirrhosis, Crohn's disease, ulcerative colitis, aplastic anemia,
Addison's disease, or insulin-dependent diabetes mellitus.
13. A method of treating an inflammmatory condition in a subject
comprising administering to the subject an effective amount of a
compound of the formula I, II, or III 306Wherein for each of
formula I, II, or III: R.sup.1 is selected from the group
consisting of (a) C(O); (b) C(O)--C.sub.1-14 alkyl-C(O), wherein
said C.sub.1-14 alkyl is optionally substituted with hydroxy,
C.sub.1-5 alkoxy, C.sub.1-5 alkylenedioxy, C.sub.1-5 alkylamino, or
C.sub.1-5-alkyl-aryl, wherein said aryl moiety of said
C.sup.1-15-alkyl-aryl is optionally substituted with C.sub.1-5
alkoxy, C.sub.1-5 alkyl amino, C.sub.1-5 alkoxy-amino, C.sub.1-5
alkylamino-C.sub.1-5 alkoxy, --O-C.sub.1-5 alkylamino-C.sub.1-5
alkoxy, --O--C.sub.1-5 alkylamino-C (O)--C.sub.1-5 alkyl C(O)OH,
--O--C.sub.1-5 alkylamino-C(O)--C.sub.1-5 alkyl-C(O)--C.sub.1-5
alkyl; (c) C.sub.2 to C.sub.15 straight or branched chain alkyl
optionally substituted with hydroxy or alkoxy; and (d)
--C(O)--C.sub.6-12 arylene-C(O)-- wherein said arylene is
optionally substituted with hydroxy, halogen, nitro or amino; a and
b are independently 0, 1, 2, 3 or 4; d, d', d", e, e' and e" are
independently an integer from 1 to 4; X.sup.1, X.sup.2, Y.sup.1 and
Y.sup.2 are independently selected from the group consisting of
null, oxygen, NH and N(C(O)C.sub.1-4 alkyl), and N(C.sub.1-4
alkyl).sub.2; W.sup.1 and W.sup.2 are independently selected from
the group consisting of carbonyl, methylene, sulfone and sulfoxide;
R.sup.2 and R.sup.5 are independently selected from the group
consisting of: (a) C.sub.2 to C.sub.20 straight chain or branched
chain alkyl which is optionally substituted with oxo, hydroxy or
alkoxy, (b) C.sub.2 to C.sub.20 straight chain or branched chain
alkenyl or dialkenyl which is optionally substituted with oxo,
hydroxy or alkoxy; (c) C.sub.2 to C.sub.20 straight chain or
branched chain alkoxy which is optionally substituted with oxo,
hydroxy or alkoxy; (d) --NH--C.sub.2 to C.sub.20 straight chain or
branched chain alkyl, wherein said alkyl group is optionally
substituted with oxo, hydroxy or alkoxy; and (e) 307wherein Z is
selected from the group consisting of O and NH, and M and N are
independently selected from the group consisting of C.sub.2 to
C.sub.20 straight chain or branched chain alkyl, alkenyl, alkoxy,
acyloxy, alkylamino, and acylamino; R.sup.1 and R.sup.6 are
independently selected from the group consisting of C.sub.2 to
C.sub.20 straight chain or branched chain alkyl or alkenyl
optionally substituted with oxo or fluoro; R.sup.4 and R.sup.7 are
independently selected from the group consisting of C(O)C.sub.2 to
C.sub.20 straight chain or branched chain alkyl or alkenyl; C.sub.2
to C.sub.20 straight chain or branched chain alkyl; C.sub.2 to
C.sub.20 straight chain or branched chain alkoxy; C.sub.2 to
C.sub.20 straight chain or branched chain alkenyl; wherein said
alkyl, alkenyl or alkoxy groups can be independently and optionally
substituted with hydroxy, fluoro or C.sub.1 to C.sub.5 alkoxy;
G.sup.1, G.sup.2, G.sup.3 and G.sup.4 are independently selected
from the group consisting of oxygen, methylene, amino, thiol,
--NHC(O)--, and --N(C(O)C.sub.1-4 alkyl)--; or G.sup.2R.sup.4 or
G.sup.4R.sup.7 may together be a hydrogen atom or hydroxyl; or a
pharmaceutically acceptable salt thereof; and wherein for Formula
II: a' and b' are independently 2, 3, 4, 5, 6, 7, or 8, preferably
2; Z.sup.1 is selected from the group consisting of
--OP(O)(OH).sub.2,--P(O)(OH).sub.2,--OP(O)(OR.sup.8)(OH) where
R.sup.8is a C1-C4 alkyl chain,
--OS(O).sub.2OH,--S(O).sub.2OH--,--CO.sub.2H, --OB(OH).sub.2, --OH,
--CH.sub.3, --NH.sub.2, NR.sup.9.sub.3 where R.sup.9 is a C1-C4
alkyl chain; Z.sup.2 is --OP(O)(OH).sub.2, --P(O)(OH).sub.2',
--OP(O)(OR.sup.10)(OH) where R.sup.10is a C1-C4 alkyl chain,
--OS(O).sub.2OH,--S(O).sub.2OH, CO.sub.2H, --OB(OH).sub.2, --OH,
CH.sub.3, --NH.sub.2, --NR.sup.11, where, R.sup.11 is a C1-C4 alkyl
chain; and wherein for Formula 3: R.sup.12 is selected from H and a
C1-C4 alkyl chain; or a pharmaceutical salt thereof, with the
proviso that the compounds of formula I, II, or III are not 308as
the active ingredient.
14. The method of claim 13, wherein the inflammatory condition is
selected from the group consisting of inflammatory bowel disease,
multiple sclerosis and autoimmune diabetes.
15. An immunostimulatory remedy containing, as the active
ingredient, a compound of the formula I, II, or III: 309Wherein for
each of formula I, II, or III: R.sup.1 is selected from the group
consisting of (a) C(O); (b) C(O)--C.sub.1-4 alkyl-C(O), wherein
said C.sub.1-4 alkyl is optionally substituted with hydroxy,
C.sub.1-5 alkoxy, C.sub.1-5 alkylenedioxy, C.sup.1-5 alkylamino, or
C.sub.1-5-alkyl-aryl, wherein said aryl moiety of said
C.sub.1-15-alkyl-aryl is optionally substituted with C.sub.1-5
alkoxy, C.sub.1-5 alkyl amino, C.sub.1-5 alkoxy-amino, C.sub.1-5
alkylamino-C.sub.1-5 alkoxy, --O--C.sub.1-5 alkylamino-C.sub.1-5
alkoxy, --O--C.sub.1-5 alkylamino-C(O)--C.sub.1-5 alkyl C(O)OH,
--O--C.sub.1-5 alkylamino-C(O)--C.sub.1-5 alkyl-C(O)--C.sub.1-5
alkyl; (c) C.sub.2 to C.sub.15 straight or branched chain alkyl
optionally substituted with hydroxy or alkoxy; and (d)
--C(O)--C.sub.6-12 arylene-C(O)-- wherein said arylene is
optionally substituted with hydroxy, halogen, nitro or amino; a and
b are independently 0, 1, 2, 3 or 4; d, d', d", e, e' and e" are
independently an integer from 1 to 4; X.sup.1, X.sup.2, Y.sup.1 and
Y.sup.2 are independently selected from the group consisting of
null, oxygen, NH and N(C(O)C.sub.1-4 alkyl), and N(C.sub.1-4
alkyl).sub.2; W.sup.1 and W.sup.2 are independently selected from
the group consisting of carbonyl, methylene, sulfone and sulfoxide;
R.sup.2 and R.sup.5 are independently selected from the group
consisting of: (a) C.sub.2 to C.sub.20 straight chain or branched
chain alkyl which is optionally substituted with oxo, hydroxy or
alkoxy, (b) C.sub.2 to C.sub.20 straight chain or branched chain
alkenyl or dialkenyl which is optionally substituted with oxo,
hydroxy or alkoxy; (c) C.sub.2 to C.sub.20 straight chain or
branched chain alkoxy which is optionally substituted with oxo,
hydroxy or alkoxy; (d) --NH--C.sub.2 to C.sub.20 straight chain or
branched chain alkyl, wherein said alkyl group is optionally
substituted with oxo, hydroxy or alkoxy; and (e) 310wherein Z is
selected from the group consisting of O and NH, and M and N are
independently selected from the group consisting of C.sub.2 to
C.sub.20 straight chain or branched chain alkyl, alkenyl, alkoxy,
acyloxy, alkylamino, and acylamino; R.sup.1 and R.sup.6 are
independently selected from the group consisting of C.sub.2 to
C.sub.20 straight chain or branched chain alkyl or alkenyl
optionally substituted with oxo or fluoro; R.sup.4 and R.sup.7 are
independently selected from the group consisting of C(O)C.sub.2 to
C.sub.20 straight chain or branched chain alkyl or alkenyl; C.sub.2
to C.sub.20 straight chain or branched chain alkyl; C.sub.2 to
C.sub.20 straight chain or branched chain alkoxy; C.sub.2 to
C.sub.20 straight chain or branched chain alkenyl; wherein said
alkyl, alkenyl or alkoxy groups can be independently and optionally
substituted with hydroxy, fluoro or C.sub.1 to C.sub.5 alkoxy;
G.sup.1, G.sup.2, G.sup.3 and G.sup.4 are independently selected
from the group consisting of oxygen, methylene, amino, thiol,
--NHC(O)--, and --N(C(O)C.sub.1-4 alkyl)--; or G.sup.2R.sup.4 or
G.sup.4R.sup.7 may together be a hydrogen atom or hydroxyl; or a
pharmaceutically acceptable salt thereof; and wherein for Formula
II: a' and b' are independently 2, 3, 4, 5, 6, 7, or 8, preferably
2; Z.sup.1 is selected from the group consisting of
--OP(O)(OH).sub.2,--P(O)(OH).sub.2,--OP(O)(OR.sup.8)(OH) where
R.sup.8 is a C1-C4 alkyl chain,
--OS(O).sub.2OH,--S(O).sub.2OH--,--CO.sub.2H, --OB(OH).sub.2, --OH,
--CH.sub.3, --NH.sub.2, NR.sup.9.sub.3 where R.sup.9 is a C1-C4
alkyl chain; Z.sup.2 is --OP(O)(OH).sub.2, --P(O)(OH).sub.2',
'OP(O)(OR.sup.10)(OH) where R.sup.10 is a C1-C4 alkyl chain, --OS
(O).sub.2OH,--S(O).sub.2OH, CO.sub.2H, --OB(OH).sub.2, --OH,
CH.sub.3, --NH.sub.2, --NR.sup.11, where R.sup.11 is a C1-C4 alkyl
chain; and wherein for Formula 3: R.sup.2 is selected from H and a
C1-C4 alkyl chain; or a pharmaceutical salt thereof, with the
proviso that the compounds of formula I, II, or III are not 311
16. A method of preventing or reducing ischemic damage in a
subject, the method comprising administering to the subject an
effective amount of a compound of the formula I, II, or III prior
to performing surgery on the patient.
17. A method of preventing, ameliorating, or delaying asthma in a
subject, the method comprising administering to the subject an
effective amount of a compound of the formula I, II, or III
312Wherein for each of formula I, II, or III: R.sup.1 is selected
from the group consisting of (a) C(O); (b) C(O)--C.sub.1-14
alkyl-C(O), wherein said C.sub.1-14 alkyl is optionally substituted
with hydroxy, C.sub.1-5 alkoxy, C.sub.1-5 alkylenedioxy, C.sub.1-5
alkylamino, or C.sub.1-5-alkyl-aryl, wherein said aryl moiety of
said C.sub.1-15-alkyl-aryl is optionally substituted with C.sub.1-5
alkoxy, C.sub.1-5 alkyl amino, C.sub.1-5 alkoxy-amino, C.sub.1-5
alkylamino-C.sub.1-5 alkoxy, --O--C.sub.1-5 alkylamino-C.sub.1-5
alkoxy, --O--C.sub.1-5 alkylamino-C(O)--C.sub.1-5 alkyl C(O)OH,
--O--C.sub.1-5 alkylamino-C(O)--C.sub.1-5 alkyl-C(O)--C.sub.1-5
alkyl; (c) C.sub.2 to C.sub.15 straight or branched chain alkyl
optionally substituted with hydroxy or alkoxy; and (d)
--C(O)--C.sub.6-12 arylene-C(O)-- wherein said arylene is
optionally substituted with hydroxy, halogen, nitro or amino; a and
b are independently 0, 1, 2, 3 or 4; d, d', d", e, e' and e" are
independently an integer from 1 to 4; X.sup.1, X.sup.2, Y.sup.1 and
Y.sup.2 are independently selected from the group consisting of
null, oxygen, NH and N(C(O)C.sub.1-4 alkyl), and N(C.sub.1-4
alkyl).sub.2; W.sup.1 and W.sup.2 are independently selected from
the group consisting of carbonyl, methylene, sulfone and sulfoxide;
R.sup.2 and R.sup.5 are independently selected from the group
consisting of: (a) C.sub.2 to C.sub.20 straight chain or branched
chain alkyl which is optionally substituted with oxo, hydroxy or
alkoxy, (b) C.sub.2 to C.sub.20 straight chain or branched chain
alkenyl ordialkenyl which is optionally substituted with oxo,
hydroxy or alkoxy; (c) C.sub.2 to C.sub.20 straight chain or
branched chain alkoxy which is optionally substituted with oxo,
hydroxy or alkoxy; (d) --NH--C.sub.2 to C.sub.20 straight chain or
branched chain alkyl, wherein said alkyl group is optionally
substituted with oxo, hydroxy or alkoxy; and (e) 313wherein Z is
selected from the group consisting of O and NH, and M and N are
independently selected from the group consisting of C.sub.2 to
C.sub.20 straight chain or branched chain alkyl, alkenyl, alkoxy,
acyloxy, alkylamino, and acylamino; R.sup.1 and R.sup.6 are
independently selected from the group consisting of C.sub.2 to
C.sub.20 straight chain or branched chain alkyl or alkenyl
optionally substituted with oxo or fluoro; R.sup.4 and R.sup.7 are
independently selected from the group consisting of C(O)C.sub.2 to
C.sub.20 straight chain or branched chain alkyl or alkenyl; C.sub.2
to C.sub.20 straight chain or branched chain alkyl; C.sub.2 to
C.sub.20 straight chain or branched chain alkoxy; C.sub.2 to
C.sub.20 straight chain or branched chain alkenyl; wherein said
alkyl, alkenyl or alkoxy groups can be independently and optionally
substituted with hydroxy, fluoro or C.sub.1 to C.sub.5 alkoxy;
G.sup.1, G.sup.2, G.sup.3 and G.sup.4 are independently selected
from the group consisting of oxygen, methylene, amino, thiol,
--NHC(O)--, and --N(C(O)C.sub.1-4 alkyl)--; or G.sup.2R.sup.4 or
G.sup.4R.sup.7 may together be a hydrogen atom or hydroxyl; or a
pharmaceutically acceptable salt thereof; and wherein for Formula
II: a' and b' are independently 2, 3, 4, 5, 6, 7, or 8, preferably
2; Z.sup.1 is selected from the group consisting of
--OP(O)(OH).sub.2,--P(O)(OH).sub.2,--OP(O)(OR.sup.8)(OH) where
R.sup.8 is a C1-C4 alkyl chain,
--OS(O).sub.2OH,--S(O).sub.2OH--,--CO.sub.2H, --OB(OH).sub.2, --OH,
--CH.sub.3, --NH.sub.2, NR.sup.9.sub.3 where R.sup.9 is a C1-C4
alkyl chain; Z.sup.2 is --OP(O)(OH).sub.2, --P(O)(OH).sub.2',
--OP(O)(OR.sup.10)(OH) where R.sup.10is a C1-C4 alkyl chain,
--OS(O).sub.2OH,--S(O).sub.2OH, CO.sub.2H, --OB(OH).sub.2, --OH,
CH.sub.3, --NH.sub.2, --NR.sup.11, where R.sup.11 is a C1-C4 alkyl
chain; and wherein for Formula 3: R.sup.12 is selected from H and a
C1-C4 alkyl chain; or a pharmaceutical salt thereof, with the
proviso that the compounds of formula I, II, or III are not 314as
the active ingredient.
Description
BACKGROUND OF THE INVENTION
[0001] The immune system provides vital defenses against invading
pathogens, such as bacteria, viruses, parasites, helminthes, and
other foreign invaders, as well as providing protection against the
proliferation of neoplastic cells. The elimination of pathogens and
neoplastic cells requires stimulation of the immune system.
However, in certain cases immune responses and immune stimulation
can also cause or contribute to diseases and pathologies such as
autoimmune disease, inflammation, allergy, anaphylaxis, and septic
shock.
[0002] The generation of effective treatments for these diseases
and pathologies has proved elusive. For example, broad spectrum
immunosuppressants such as cyclosporine A and steroids can be used
to treat autoimmune diseases, allergies, and other pathologies, but
these treatments can present severe side effects. Similarly,
current treatments for inflammatory conditions such as chronic
adrenocortical disorder and hyperfunction, allergies, rheumatoid
arthritis, lupus, inflammatory bowel disease, pneumonia, bronchial
asthma, hematological disorders, dermatitis and eczema can present
undesired side effects of these agents including hypertension,
atherosclerosis, diabetes, hyperglycemia, bone thinning and
electrolyte imbalance.
[0003] Improved treatments for diseases and pathologies associated
with the immune system and immune responses requires the ability to
modulate and redirect certain immune responses so as to suppress
harmful responses without compromising an individual's ability to
eliminate infections.
SUMMARY OF THE INVENTION
[0004] In one aspect, the present invention provides a method for
inducing or stimulating an immune response by administering an
effective amount of a compound of the formula I, II, or III.
[0005] In another aspect, the invention provides a method for
upregulating an immune response by administering an effective
amount of a compound of the formula I, II, or III.
[0006] In another aspect, the invention provides a method for
reducing an immune response in a subject, the method comprising
administering to the subject a compound of the formula I, II, or
III.
[0007] In another aspect, the invention provides a method for
desensitizing a subject against the occurrence of an allergic
reaction in response to contact with a particular allergen or
antigen, comprising administering to the subject an effective
amount of a compound of the formula I, II, or III.
[0008] In another aspect, the invention provides a method for
treating a subject having an autoimmune disease, comprising
administering to the subject an effective amount of a compound of
the formula I, II, or III.
[0009] In another aspect, the invention provides a method for
treating a subject having an inflammatory condition, comprising
administering to the subject an effective amount of a compund of
the formula I, II, or III.
[0010] In another aspect, the invention provides a method for
preventing or reducing ischemic damage in a subject requiring
surgery, comprising administering to the subject an effective
amount of a compound of the formula I, II, or III.
[0011] In another aspect, the invention provides a method for
preventing, ameliorating, or delaying the onset of asthma in a
subject, comprising administering to the subject an effective
amount of a compound of the formula I, II, or III.
[0012] In another aspect, the invention provides an
immunostimulatory remedy containing as the active ingredient a
compound of the formula I, II, or III.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a graph that shows the results of an in vitro
assay for induction of TNF-alpha cytokine release by compounds 100,
184 or 186 of the invention.
[0014] FIG. 2 is a graph that shows the results of an in vitro
assay for induction of IL-10 and IL-12 release by compounds
ER803022, ER803702, ER804053, ER804057, ER804058, and ER804059.
[0015] FIG. 3 is a graph that shows stimulation of alkaline
phosphatase expression from an inducible reporter construct with
the TNF promoter (TNF-PLAP) in THP-1 cells by compounds 106 and 126
in the absence and presence of 10% serum.
[0016] FIG. 4 is a graph showing stimulation of IL-10 release from
normal mouse splenocytes by compounds 104, 106, 124, 126, 160, and
162 of the invention.
[0017] FIG. 5 is a graph showing stimulation of interferon-gamma
release from normal mouse splenocytes by compounds 104, 106, 124,
126, 160, and 162 of the invention.
[0018] FIG. 6 is a graph showing stimulation of TNF-alpha release
in response to TLR2, TLR4, and TLR9 ligands by untreated mouse
macrophage cells and mouse macrophage cells pretreated with the
immunomodulatory compound ER803022.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0019] The present invention is directed in part to methods of
using immunomodulatory compounds of the formula I, II, or III:
1
[0020] Wherein for each of formula I, II, or III:
[0021] R.sup.1 is selected from the group consisting of
[0022] (a) C(O);
[0023] (b) C(O)--C.sub.1-14 alkyl-C(O), wherein said C.sub.1-14
alkyl is optionally substituted with hydroxy, C.sub.1-5 alkoxy,
C.sub.1-5 alkylenedioxy, C.sub.1-5 alkylamino, or
C.sub.1-5-alkyl-aryl, wherein said aryl moiety of said
C.sub.1-15-alkyl-aryl is optionally substituted with C.sub.1-5
alkoxy, C.sub.1-5 alkyl amino, C.sub.1-5 alkoxy-amino, C.sub.1-5
alkylamino-C.sub.1-5 alkoxy, --O--C.sub.1-5 alkylamino-C.sub.1-5
alkoxy, --O--C.sub.1-5 alkylamino-C (O)--C.sub.1-5 alkyl C(O)OH,
--O--C.sub.1-5 alkylamino-C(O)--C.sub.1-5 alkyl-C(O)--C.sub.1-5
alkyl;
[0024] (c) C.sub.2 to C.sub.5 straight or branched chain alkyl
optionally substituted with hydroxy or alkoxy; and
[0025] (d) --C(O)--C.sub.6-12 arylene-C(O)-- wherein said arylene
is optionally substituted with hydroxy, halogen, nitro or
amino;
[0026] a and b are independently 0, 1, 2, 3 or 4;
[0027] d, d', d", e, e' and e" are independently an integer from 1
to 4;
[0028] X.sup.1, X.sup.2, Y.sup.1 and Y.sup.2 are independently
selected from the group consisting of null, oxygen, NH and
N(C(O)C.sub.1-4 alkyl), and N(C.sub.1-4 alkyl).sub.2;
[0029] W.sup.1 and W.sup.2 are independently selected from the
group consisting of carbonyl, methylene, sulfone and sulfoxide;
[0030] R.sup.2 and R.sup.5 are independently selected from the
group consisting of:
[0031] (a) C.sub.2 to C.sub.20 straight chain or branched chain
alkyl which is optionally substituted with oxo, hydroxy or
alkoxy,
[0032] (b) C.sub.2 to C.sub.20 straight chain or branched chain
alkenyl or dialkenyl which is optionally substituted with oxo,
hydroxy or alkoxy;
[0033] (c) C.sub.2 to C.sub.20 straight chain or branched chain
alkoxy which is optionally substituted with oxo, hydroxy or
alkoxy;
[0034] (d) --NH--C.sub.2 to C.sub.20 straight chain or branched
chain alkyl, wherein said alkyl group is optionally substituted
with oxo, hydroxy or alkoxy; and
[0035] (e) 2
[0036] wherein Z is selected from the group consisting of O and NH,
and M and N are independently selected from the group consisting of
C.sub.2 to C.sub.20 straight chain or branched chain alkyl,
alkenyl, alkoxy, acyloxy, alkylamino, and acylamino; R.sup.1 and
R.sup.6 are independently selected from the group consisting of
C.sub.2 to C.sub.20 straight chain or branched chain alkyl or
alkenyl optionally substituted with oxo or fluoro;
[0037] R.sup.4 and R.sup.7 are independently selected from the
group consisting of C(O)C.sub.2 to C.sub.20 straight chain or
branched chain alkyl or alkenyl; C.sub.2 to C.sub.20 straight chain
or branched chain alkyl; C.sub.2 to C.sub.20 straight chain or
branched chain alkoxy; C.sub.2 to C.sub.20 straight chain or
branched chain alkenyl; wherein said alkyl, alkenyl or alkoxy
groups can be independently and optionally substituted with
hydroxy, fluoro or C.sub.1 to C.sub.5 alkoxy;
[0038] G.sup.1, G.sup.2, G.sup.3 and G.sup.4 are independently
selected from the group consisting of oxygen, methylene, amino,
thiol, --NHC(O)--, and --N(C(O)C.sub.1-4 alkyl)--;
[0039] or G.sup.2R.sup.4 or G.sup.4R.sup.7 may together be a
hydrogen atom or hydroxyl;
[0040] or a pharmaceutically acceptable salt thereof;
[0041] and wherein for Formula II:
[0042] a' and b' are independently 2, 3, 4, 5, 6, 7, or 8,
preferably 2;
[0043] Z.sup.1 is selected from the group consisting of
--OP(O)(OH).sub.2,--P(O)(OH).sub.2,--OP(O)(OR.sup.8)(OH) where
R.sup.8 is a C1-C4 alkyl chain,
--OS(O).sub.2OH,--S(O).sub.2OH--,--CO.sub.2H, --OB(OH).sub.2, --OH,
--CH.sub.3, --NH.sub.2, NR.sup.9.sub.3 where R.sup.9 is a C1-C4
alkyl chain;
[0044] Z.sup.2 is --OP(O)(OH).sub.2, --P(O)(OH).sub.2',
--OP(O)(OR.sup.10)(OH) where R.sup.10 is a C1-C4 alkyl chain,
--OS(O) .sub.2OH,--S(O).sub.2OH, CO.sub.2H, --OB(OH).sub.2, --OH,
CH.sub.3, --NH.sub.2, --NR.sup.11, where R.sup.11 is a C1-C4 alkyl
chain;
[0045] and wherein for Formula 3:
[0046] R.sup.12is selected from H and a C1-C4 alkyl chain;
[0047] or a pharmaceutical salt thereof,
[0048] with the proviso that the compounds of formula I, II, or III
are not 3
[0049] In preferred compounds of the invention, one or more of the
following is present: each of a and b is 2; each of X.sup.1 and
y.sup.1 is NH; R.sup.1 is C(O) or C(O)--C.sub.1-14 alkyl-C(O); each
of d' and e' is 1; each of d" and e" is 1; X is O or NH, more
preferably NH; and W is C(O); or each of d' and e' are 2.
[0050] In further preferred embodiments, R' is a C(O)C.sub.1-14
alkyl-C(O), wherein said C.sub.1-14 alkyl is substituted, for
example with a C.sub.1-5 alkoxy group;
[0051] In a most preferred embodiment, the invention is directed to
compounds ER 803022, ER 803058, ER 803732, ER 804053, ER 804057, ER
804058, ER 804059, ER 80442, ER 804680 and ER 804764, and
compositions containing these compounds.
[0052] Definitions
[0053] Carbonyl, as used herein, is a (C.dbd.O) moiety.
[0054] Dicarbonyl, as used herein, is a moiety with the structure
(C.dbd.O)-alkyl-(C.dbd.O) or (C.dbd.O)-aryl-(C.dbd.O), which is
bonded to a molecule through the carbon atoms of both of the
terminal carbonyl moieties.
[0055] Oxo, as used herein, is a .dbd.O group.
[0056] Alkyl ester, as used herein, is a moiety with the structure
O--(C.dbd.O)-alkyl, which is bonded to a molecule through the
non-double bonded oxygen of the ester group.
[0057] Alkenyl ester, as used herein, is a moiety with the
structure O--(C.dbd.O)-carbon chain, where the carbon chain
contains a carbon-to-carbon double bond, which is bonded to a
molecule through the non-double bonded oxygen of the ester
group.
[0058] The term "alkylene" means a bivalent straight chain or
branched alkyl hydrocarbon group.
[0059] The term "alkenylene" means a bivalent straight chain or
branched hydrocarbon group having a single carbon to carbon double
bond.
[0060] The term "dialkenylene" means a bivalent unsaturated
straight chain or branched chain hydrocarbon group having two
carbon to carbon double bonds.
[0061] The term "arylene" refers to a bivalent aromatic group.
[0062] The abbreviation "Boc" as used herein means
t-butyloxycarbonyl.
[0063] As used herein with reference to compounds and compositions
of the invention, the term "type 1" refers to those compounds of
the invention corresponding to formula I above where the values of
a and b are the same; the values of d and e are the same; the
values of d' and e' are the same; the values of d" and e" are the
same; X.sup.1 and Y.sup.1 are the same; X.sup.2 and Y.sup.2 are the
same; W.sup.1 and W.sup.2 are the same; R.sup.2 and R.sup.5 are the
same; G.sup.1 and G.sup.3 are the same; R.sup.3 and R.sup.6 are the
same; G.sup.2 and G.sup.4 are the same; and R.sup.4 and R.sup.7 are
the same. "Type 2", as used herein, refers to compounds or
compositions corresponding to formula I where any one or more of
the following applies: the values of a and b are different, the
values of d and e are the same, the values of d' and e' are
different; the values of d" and e" are the same; X.sup.1 and
Y.sup.1 are different; X.sup.2 and Y.sup.2 are different; W.sup.1
and W.sup.2 are different; R.sup.2 and R.sup.5 are different;
G.sup.1 and G.sup.3 are different; R.sup.3 and R.sup.6 are
different; G.sup.2 and G.sup.4 are different; or R.sup.4 and
R.sup.7 are different.
[0064] All patents, patent applications, and publications referred
to herein are incorporated by reference in their entirety. In case
of a conflict in terminology, the present specification is
controlling.
[0065] Methods of Modulating Immune Responses
[0066] The present invention is directed to methods of modulating
immune responses by administering immunomodulatory compounds that
elicit cytokines and activate immune cells. In particular, the
present invention is directed to methods of using immunomodulatory
compounds to stimulate immune responses directed against pathogens
or neoplastic cells, or to suppress immune responses associated
with inflammation, allergy, and anaphylaxis.
[0067] As used herein "immunomodulatory compounds" describes
compounds which, when administered to a subject, stimulate the
production of cytokines and elicit particular responses by immune
cells. The stimulation of cytokines is known to enhance the
activity of some immune cells and to suppress the activity of other
immune cells.
[0068] The immunomodulatory compounds used in the methods of the
invention are ligands for the TLR4 receptor. TLR4 is a member of
the Toll-like receptor (TLR) family of receptors. In humans, the
TLR family comprises ten known receptors, designated TLR1-10. TLR
receptors are associated with innate immune recognition of
pathogens, and known TLR ligands are associated with pathogens or
tissue damage. For example, other known TLR4 ligands include
bacterial endotoxin (also known as lipopolysaccharide, or LPS),
parasite lipoproteins, human heat shock protein 70, and human
necrotic cell debris. Ligands to other known TLRs are also
associated with pathogens and tissue damage, and include
peptidoglycan, which is recognized by TLR2, flagellin, which is
recognized by TLR5, and unmethylated bacterial CpG DNA sequences,
which are recognized by TLR9.
[0069] Recognition of ligand by TLR4 results in the secretion of
cytokines and activation of various pathways and behaviors in
immune cells. TLR4 ligands typically elicit a set of cytokines that
includes IL-1.beta., IL-6, IL-10, IL-12, and TNF.alpha.. Many of
the cytokines elicited by TLR4 ligands have known immunomodulatory
or immunoprotective effects. At least two of these cytokines, IL-10
and IL-12, play a role in regulating inflammatory responses. For
example, IL-10 has anti-inflammatory properties, and is associated
with T cell populations that down-regulate inflammatory reactions.
IL-10 may also be involved in down-regulating responses mediated by
the Th1 subset of T helper cells, which are associated with many
forms of inflammatory disease such as rheumatoid arthritis and
Crohn's disease. Thus, TLR4 ligands can be useful for modulating
responses mediated by Th1 cells. As another example, IL-12 is
associated with Th1 related functions involved in suppressing
certain aspects of allergic disease, including B cell synthesis of
IgE, which are mediated through the Th2 subset of T helper cells.
Thus, TLR4 agonists and related compounds may be useful for
down-regulating Th2 responses and resolving conditions dependent on
IL-4 or other Th2 associated cytokines.
[0070] As described in more detail in the Examples provided below,
the immunomodulatory compounds used in the methods of the invention
can elicit production of cytokines, including IL-1.alpha.,
IL-1.beta., IL-6, IL-10, IL12, interferon-.alpha.,
interferon-.gamma., and GM-CSF.
[0071] Thus, in one aspect, the invention provides a method of
inducing or stimulating an immune response in an animal by
administering a compound of the formula I, II, or III. As used
herein, inducing or stimulating an immune response means
stimulating the production of cytokines, stimulating the
proliferation of immune cells, stimulating the activation of immune
cells, or stimulating the lytic activity of immune cells. Examples
of immune responses stimulated by the methods of the invention are
the secretion of cytokines, the activation of NK cells, the
proliferation of B cells, T cells, macrophages, monocytes, and
other immune cells, and other immune responses. These responses may
in turn enhance or down-regulate other immune functions.
[0072] The methods of the invention can be used to stimulate immune
responses to treat a variety of infections, including, but not
limited to, gram-positive and gram-negative bacterial infections,
viral infections, fungal infections, and parasitic infections. The
methods of the invention can also be used to treat neoplastic
conditions, including, but not limited to biliary tract cancers,
brain cancer, breast cancer, cervical cancer, choriocarcinoma,
colon cancer, endometrial cancer, esophageal cancer, gastric
cancer, intraepithelial neoplasms, lymphomas, liver cancer, lung
cancer, melanoma, neuroblastomas, oral cancer, ovarian cancer,
pancreatic cancer, prostate cancer, rectal cancer, sarcomas, skin
cancer, testicular cancer, thyroid cancer, renal cancer, and other
carcinomas and sarcomas.
[0073] In another aspect, the invention provides a method for
upregulating an immune response in a subject by administering an
immunomodulatory compound of the formula I, II, or III. As used
herein, upregulating an immune response means to increase an
existing immune response or a component of an existing immune
response. Methods for upregulating immune responses can be used to
treat any of the infections or neoplastic disorders described above
herein. Methods for upregulating immune responses can also be used
to treat autoimmune, inflammatory, or allergic disorders by
altering the balance of Th1 and Th2 responses. For example,
administration of the immunomodulatory compounds of the invention
can be used to alter the balance of Th1 and Th2 responses, thereby
reducing a subject's immune response to animal danders, pollen,
dust mites, hymenoptera venoms, and other antigens or allergens.
Administration of the immunomodulatory compounds of the invention
can also be used to treat conditions such as asthma, atopic
dermatitis, allergic rhinitis, eczema, urticaria, and food
allergies.
[0074] In another aspect, the invention provides a method for
reducing an immune response in a subject by administering an
immunomodulatory compound of the formula I, II, or III. As used
herein, reducing an immune response in a subject means to cause a
decrease in the production of cytokines, the proliferation of
lymphocytes, monocytes, macrophages, dendritic cells, or natural
killer cells, to cause a decrease in the lytic activity of natural
killer cells, or to cause a decrease in the lytic activity of
cytotoxic T cells.
[0075] In another aspect, the invention provides a method of
desensitizing a subject against the occurrence of an allergic
reaction in response to contact with a particular antigen or
allergen, comprising administering an immunostimulatory compound of
formula I, II, or III as the active ingredient. As used herein,
desensitizing a subject means to reduce the immune response of the
subject to exposure to particular allergens or antigens. For
example, the subject may display decreased production of IgE,
decreased production of IgE producing B cells, decreased production
of histamine, or decreased release of cytokines in response to
exposure to an allergen or antigen.
[0076] In another aspect, the invention provides a method of
preventing, ameliorating, or delaying the onset of asthma in a
subject by administering a compound of formula I, II, or III to the
subject.
[0077] Recent studies have indicated that exposure to microbial
agents during childhood can confer a protective benefits, such as
providing protection against development of asthma and other
allergic conditions. It has been suggested that providing safe and
effective antigens that mimic the protective effects of microbial
pathogens without the associated risks could confer similar
protection against development of allergy and asthma (Liu, A. H.
(2002) J. Allergy Clin Immunol. 109:379-92).
[0078] In the methods of the invention, at least one of the
immunostimulatory compounds according to the formula I, II, or III
is administered to a subject at risk of developing allergies or
asthma. Preferably, the subject is a juvenile subject.
[0079] The TLR ligand endotoxin is associated with a phenomenon
known as endotoxin tolerance. Endotoxin (also known as
lipopolysaccharide, or LPS) is a glycolipid found in the cell
membranes of Gram-negative bacteria. Endotoxin is one of the most
potent known stimulators of immune responses, and exposure to
endotoxin induces cytokine production by monocytes and macrophages.
Endotoxin tolerance refers to the observation that an initial low
or sublethal dose of endotoxin results in a decreased immune
response to a later, high dose of endotoxin, and can protect
against lethal subsequent doses of endotoxin. The decreased immune
response is manifested in the down-regulation of macrophage
responsiveness, and decreased levels of cytokine release compared
to individuals who were not pretreated, or tolerized, with low
doses of endotoxin.
[0080] Similarly, administration of the immunomodulatory compounds
described herein can suppress subsequent responses to TLR4 ligands
such as endotoxin. Moreover, administration of the immunomodulatory
compounds described herein can also down regulate responses
mediated by subsequent exposure to other TLR ligands, including
TLR2 ligands, such as lipoprotein, and TLR9 ligands, such as
unmethylated CpG nucleic acids, as described in more detail in
Example 7.
[0081] Administration of the immunomodulatory compounds of the
invention can be used to suppress the ability of TLR ligands such
as bacterial DNA, viral RNA, endogenous human heat shock proteins,
parasitic or bacterial lipids, glycolipids or lipoproteins to
stimulate immune responses.
[0082] TLR ligands such as endotoxin are shown to elicit or
exacerbate a number of immune-based diseases. For example,
intestinally derived endotoxin is released during graft-versus-host
disease after bone marrow grafting, and increases the severity of
post-graft symptoms. Endotoxin is the active principle in eliciting
lung responses to environmental or occupational irritants such as
grain dust, cotton dust, or poultry processing dusts. Environmental
endotoxin is found to enhance bronchial responses in pre-existing
asthma. A TLR9 ligand is found to enhance proliferation of B cells
associated with secretion of autoantibodies in systemic lupus
erythematosus. Tolerization of TLRs may prevent TLR ligands from
exacerbating or causing diseases or syndromes such as systemic
lupus erythematosus, asthma, atherosclerosis, graft-versus-host
disease, grain dust fever, inflammatory bowel disease, rheumatoid
arthritis, mucositis, and others.
[0083] In another aspect, the invention provides a method of
preventing or reducing ischemic damage in a subject requiring
surgery by administering a compound of formula I, II, or III prior
to performing surgery on the subject.
[0084] Ischemia and reperfusion result in tissue injury in a number
of organs, including heart, brain, kidney, and gastrointestinal
tract. Ischemia/reperfusion injury is associated with a number of
surgical procedures, including transplantation of organs such as
kidney, liver and heart, procedures that require periods of
hyperperfusion, and revascularization procedures.
[0085] The administration of the TLR4 ligand endotoxin has been
shown to induce cross-tolerance to insults other than endotoxemia.
Pretreatment of subjects with endotoxin has been shown to provide
protection against ischemia-reperfusion injury in the myocardium,
liver, and kidney (Meldrum, et al. (1996) Arch. Surg.
131:1203-1208, Colletti, et al. (1994) J. Surg. Res. 57:337-343,
and Heemann et al. (2000) Am. J. Path. 156:287-293).
[0086] In the methods of the invention, at least one of the
immunostimulatory compounds according to the formula I, II, or III,
is administered to a subject prior to surgery. The compound or
compounds can be administered from several hours up to three days
prior to surgery.
[0087] In another aspect, the invention provides a method of
treating autoimmune condition by administering an immunostimulatory
compound of formula I, II, or III.
[0088] Innate immunity has been implicated in the development and
progression of autoimmune conditions including type I diabetes
mellitus, systemic lupus erythamatosus, and others. The modulation
of autoimmune responses is mediated by toll-like receptor-ligand
interactions, and recent evidence indicates that TLR ligands can be
used to modulate autoimmune responses. A TLR-9 mediated mechanism
enhances proliferation of B cells associated with secretion of
autoantibodies in systemic lupus erythematosus. B cells activated
by endotoxin have been shown to prevent the onset of autoimmune
diabetes in nonobese diabetic mice (Tian, J., et al. (2001) J.
Immunol. 167: 1081-9). Thus, modulation of immune responses by TLR
ligands can provide treatments for autoimmune conditions.
[0089] In the methods of the invention, at least one
immunomodulatory compound defined by the general formula I, II, or
III is administered to a subject suffering from an autoimmune
disorder or other disorder having an autoimmune component.
Administration of an immunomodulatory compound can modulate the
release of cytokines and suppress T-cell subsets involved in
autoimmune disease. Many autoimmune disorders are associated with
Th1 cytokine patterns. Redirection of T help to Th2 may treat these
diseases. Alternatively, tolerization of TLRs using these
immunostimulatory compounds may suppress aspects of the disease
that are mediated by TLR stimulation.
[0090] Diseases and conditions that can be treated by the methods
of the invention include, but are not limited to, systemic lupus
erythematosis, sceleroderma, Sjogren's syndrome, multiple sclerosis
and other demyelinating diseases, rheumatoid arthritis, juvenile
arthritis, myocarditis, Graves' disease, uveitis, Reiter's
syndrome, gout, osteoarthritis, polymyositis, primary biliary
cirrhosis, Crohn's disease, ulcerative colitis, aplastic anemia,
Addison's disease, insulin-dependent diabetes mellitus, and other
diseases.
[0091] In another aspect, the invention provides a method of
treating an inflammatory condition by administering an
immunomodulatory compound of formula I, II, or III.
[0092] As discussed herein, the immunomodulatory compounds used in
the methods of the invention elicit the production of cytokines
such as IL-1.alpha., IL-1.beta., IL-6, IL-10, IL12,
interferon-.alpha., interferon-.gamma., and GM-CSF. The cytokines
IL-1.alpha., IL-1.beta., and IL-10 are associated with the
suppression of inflammatory responses, and agents that stimulate
the production of these cytokines can be used to treat inflammatory
conditions.
[0093] Inflammatory conditions that can be treated according to the
methods of the invention include, but are not limited to,
inflammatory bowel disease, multiple sclerosis, autoimmune
diabetes, atopic dermatitis, urticaria, contact sensitivity,
cutaneous allergic conditions, psoriasis, chronic adrenocortical
disorder and hyperfunction, rheumatoid arthritis, lupus, pneumonia,
bronchial asthma, hematological disorders, dermatitis and
eczema.
[0094] The invention also provides immunostimulatory remedies
comprising as the active ingredient a compound of the formula I,
II, or III.
[0095] The host animals to which the immunomodulatory compounds of
the present invention are usefully administered include human as
well as non-human mammals, birds, fish, reptiles, etc.
[0096] The specific formulation of therapeutically effective
compositions of the present invention may thus be carried out in
any suitable manner which will render the immunomodulatory compound
or compounds bioavailable, safe and effective in the subject to
whom the formulation is administered.
[0097] The modes of administration may comprise the use of any
suitable means and/or methods for delivering the immunomodulatory
compound to one or more corporeal loci of the host animal where the
adjuvant and associated antigens are immumostimulatively effective.
Delivery modes may include, without limitation, parenteral
administration methods, such as subcutaneous (SC) injection,
transmucosal, intranasal (IN), ophthalmic, transdermal,
intramuscular (IM), intradermal (ID), intraperitoneal (IP),
intravaginal, pulmonary, and rectal administration, as well as
non-parenteral, e.g., oral, administration.
[0098] The dose rate and suitable dosage forms for the
immunomodulatory compounds of the present invention may be readily
determined by those of ordinary skill in the art without undue
experimentation, by use of conventional antibody titer
determination techniques and conventional
bioefficacy/biocompatibility protocols, and depending on the
desired therapeutic effect, and the desired time span of
bioactivity.
[0099] The immunomodulatory compounds of the present invention may
be usefully administered to the host animal with any other suitable
pharmacologically or physiologically active agents, e.g., antigenic
and/or other biologically active substances.
[0100] Formulations of the invention can include additional
components such as saline, oil, squalene, oil-water dispersions,
liposomes, and the like.
[0101] General Synthetic Methods
[0102] 1. Synthesis of Diamide Compounds
[0103] In general, a 2-amino-1,3-dihydroxypropane or (.+-.) serinol
is transformed into the 2-azido compound by reaction with
trifluoromethanesulfonyl azide followed by protection as the
per-acetate for easy manipulation. The resulting compound is
deacetylated, followed by reaction with an appropriately activated
primary alcohol of a diol moiety. The primary alcohol moiety of the
product of this reaction is then protected, e.g., by using TBDPSCL,
followed by reaction with phosgene and then allyl alcohol, to yield
a fully protected diol. The protected diol is then treated to
cleave the protecting group from the primary alcohol. The
unprotected alcohol is reacted with a properly functionalized
phosphorylating reagent with formula (11) as indicated in the
Examples, to form a phosphate ester compound. The azido moiety of
the product is reduced, and then reacted with an activated acyl
acid to form an amide. The protected terminal amine on the
functionalized phosphate is deprotected, and subsequently reacted
with a phosgene or a dicarboxylic acid in the presence of a
dehydrating agent, such as EDC. The phosphate groups of the
resulting compound are then deprotected, yielding a racemic
amide.
[0104] 2. Synthesis of Chiral Diamide Compounds of Type 1
[0105] In general, a chiral amino acid ester with the desired
structure is protected with a benzimidate ester. The protected
compound is reacted with a reducing agent, e.g., DIBAL or the like,
to reduce the acid moiety of the amino acid to an alcohol. The
resulting alcohol compound is reacted with an appropriately
activated primary alcohol of a diol moiety, followed by cleavage of
the benzimidate protecting group, yielding an amino-diol. The diol
is then reacted with an appropriate acid chloride to yield a
diol-amide.
[0106] The diol-amide is then reacted with a properly
functionalized phosphorylating reagent at the free primary hydroxyl
group. The resulting compound is esterified at the secondary
alcohol group with an appropriate acyl moiety. The N-BOC group is
then cleaved from the amino group introduced by phosphorylating
reagent (11), yielding a phosphate ester compound with a free
primary amine. This product is then reacted with phosgene or a
dicarboxylic acid in the presence of a dehydrating agent, to yield
a diamide product. The protected phosphate groups of the diamide
product are then deprotected, typically with palladium(0) and
phenylsilane.
[0107] 3. Synthesis of Chiral Diamide Compounds of Type 2
[0108] Chiral diamide compounds of Type 2 are synthesized
essentially as described for chiral diamide compounds of Type 1, up
to the point just after cleavage of the protecting group from the
primary amine group of the phosphate ester compound. At this point,
a dicarboxylic acid which has one of the acid moieties protected is
reacted with the primary amine group, to yield a monoamide. The
protecting group on the other carboxylic acid is then cleaved,
providing a free carboxylic acid which can then be reacted with a
primary amine from an alternative, appropriately substituted
phosphate system, in the presence of a dehydrating agent to yield a
diamide of type 2, which can then be treated to deprotect the
phosphate group or groups to yield a desired compound of the
invention.
[0109] In the special case of chiral urea compounds of type 2 of
the invention, the primary amino group of the N-BOC amino group of
the phosphate ester is deprotected and then reacted with
trichloromethyl chloroformate or the like, in order to form an
isocyanate compound. The isocyanate is then reacted with a primary
amine from an alternative, appropriately substituted phosphate
system to yield a urea product of type 2. This product can then be
treated to deprotect the phosphate group or groups.
[0110] 4. Glycerol Diamide Analogs
[0111] These compounds of the invention have an ester moiety
attached to the carbon which is beta to the phosphate group,
instead of an amide moiety.
[0112] In general, these compounds are prepared by the
etherification of a protected chiral glycerol with an activated
primary alcohol of a diol moiety, followed by esterification of the
secondary alcohol moiety and subsequent deprotection of the
glycerol moiety, to yield a new diol. The primary hydroxyl group of
the diol is then protected, and the secondary hydroxyl group is
condensed with an acyl moiety to yield a diester. The primary
hydroxyl is deprotected, followed by esterification with a
phosphorylating agent, of which compound (11), below is exemplary.
Following deprotection of the amine group introduced by the
phosphorylating agent, the product is reacted with phosgene or a
dicarboxylic acid using a dehydrating agent such as EDC. Subsequent
deprotection of the phosphate groups yield compounds of the
invention.
[0113] In the synthesis described generally above, the substituent
at R.sup.1 of the compounds of the invention can easily be varied
by utilizing different dicarboxylic acid compounds. Such acids can
be coupled to the amine group of the phosphate ester intermediate
of the reaction scheme outlined above, either using a dehydrating
agent such as EDC, or by activating the dicarboxylic acid by
synthesizing, e.g., the corresponding diacid chloride.
[0114] The substituents represented by variables R.sup.2 and
R.sup.5 in formula I above can easily be varied by utilizing an
appropriate activated acid or acid chloride in the amidation or
esterification reaction of the heteroatom represented by X or Y in
formula I.
[0115] The substituents represented by variables R.sup.3 and
R.sup.6 of formula I can be varied by using an intermediate
containing the desired number of carbon atoms which also contains
an activated carbon functionality, e.g., a halogen or sulfonate
(OSO.sub.2CH.sub.3, OSO.sub.2CF.sub.3,
OSO.sub.2CH.sub.2C.sub.6H.sub.4--p--CH.sub.3) which can be reacted
with the azido diol, amino alcohol, or glycerol starting
materials.
[0116] The substituents represented by variables R.sup.4 and
R.sup.7 in formula I above can be varied by using an appropriate
activated acid or acid chloride in the esterification of the
secondary hydroxyl group used in the reaction schemes outlined
above.
[0117] The values of a and b in compounds of formula I can be
varied by using the appropriate compound (11) below. The values of
variables d and e in compounds of formula I can be modified by
using the appropriate 2-aminodiol or 2-hydroxydiol starting
materials
SYNTHETIC EXAMPLES
[0118] All reaction products in the synthetic methods described
below gave satisfactory NMR spectra and thin layer chromatography
profiles on silica gel. All chromatography was performed on silica
gel and the elution monitored by thin layer chromatography. All
completed reactions were determined by thin layer chromatographic
analysis. All reactions were run under nitrogen at room temperature
unless otherwise specified. All reaction solvents were anhydrous
unless otherwise noted. The typical work-up for the chemical
reactions described below includes aqueous washings, drying over
anhydrous sodium sulfate and removal of solvent under reduced
pressure.
Example 1
Succinate-1
[0119] 4
[0120] To a solution of sodium azide (107.67 g) in 250 mL of water
was added 300 mL of methylene chloride. The mixture was cooled to
0.degree. C. and trifluoromethanesulfonic anhydride (57 mL) was
added dropwise at a 0.32 mL/minute rate. The mixture was stirred
for an additional 6 hours at 0.degree. C. and stored at -20.degree.
C. for 72 hours. The mixture was warmed to 10.degree. C. followed
by extraction with methylene chloride in a Teflon.RTM. separatory
funnel. The combined organic layers were dried (magnesium sulfate).
The above suspension was slowly filtered into a stirred solution of
(.+-.)-2-amino-1,3-dihydroxypropane (1) (9.89 g) in methanol (200
mL) and 4-N,N-dimethylaminopyridine (DMAP, 54 g) at 10.degree. C.
The resultant reaction mixture was stirred for 17 hours at room
temperature.
[0121] The solvent was removed under reduced pressure and the
residue dissolved in pyridine (200 mL) and cooled to 0.degree. C.
Acetic anhydride (50 mL) was added dropwise and the mixture stirred
for 20 hours at room temperature. Additional acetic anhydride (20
mL) was added and after 4 hours, the mixture was poured onto ice
and worked up in the usual manner. Chromatography gave 16 g of
diacetate (2) as an oil. 5
[0122] The diacetate (2) (16 g) was dissolved in methanol (150 mL)
and sodium metal (2.0 g) was slowly added. The mixture was stirred
for 90 minutes and Dowex.RTM. 50-8 resin was added until the pH was
less than or equal to 7. The mixture was filtered followed by
concentration of the filtrate and chromatography to give 6.73 g of
the diol (3). 6
[0123] To a suspension of sodium hydride (1.24 g of a 60% oil
dispersion washed three times with hexanes and dried under
nitrogen) in dimethylformamide (DMF, 200 mL) was added dropwise the
azido-diol (3) (6.73 g) in THF (100 mL), followed by the dropwise
addition of 3-R-hydroxy-1-O-tosyl-1-decanol (4) (tosylate, 9.44 g)
in THF (100 mL). The mixture was stirred for 16 hours, diluted with
methanol (200 mL), stirred with Amberlite.RTM. 25 H.sup.+ for 25
minutes and concentrated to dryness. Chromatography gave 4.37 g of
(5). 7
[0124] To a solution of the diol (5) (5.32 g) in methylene chloride
(30 mL) was added triethylamine (TEA, 6 mL) and DMAP (trace),
followed by t-butyldiphenylsilyl chloride (TBDPSC1, 5 mL) and the
mixture was stirred overnight. The mixture was worked up as usual.
Chromatography gave 3.6 g of secondary alcohol (6) as an oil. 8
[0125] To a solution of the secondary alcohol (6) (2.95 g) in
toluene (30 mL) was added pyridine (1.8 mL) followed by a slow
addition of phosgene (4.5 mL of a 1.93 M solution in toluene) at
0.degree. C. After stirring at 0.degree. C. for 20 minutes, allyl
alcohol (3.1 mL) was added dropwise. After an additional stirring
for 60 minutes at room temperature, the reaction was worked up in
the usual way. Chromatography gave 3.24 g of protected alcohol (7)
as an oil. 9
[0126] To a solution of protected alcohol (7) (1.29 g) in methylene
chloride (3 mL) was added hydrofluoric acid (HF, 4 mL) in
acetonitrile (12 mL). The mixture was stirred overnight and worked
up in the usual way. Chromatography gave 150 mg of the alcohol (8)
as an oil. 10
[0127] To a solution of alcohol (8) (150 mg) in methylene chloride
(0.6 mL) was added tetrazole (74 mg) and the phosphorylating
reagent (11) (175 mg). After 30 minutes, oxone (323 mg) in a cooled
THF (0.5 mL)-water (0.5 mL) solution was added to the cooled
reaction mixture. After 3 hours, the reaction was worked up in the
usual way. Chromatography gave 242 mg of (9) as an oil. 11
[0128] To make phosphorylating reagent 11, to a solution of
distilled diisopropylamine (9.0 mL) in methylene chloride was added
tetrazole (4.51 g) at room temperature followed by stirring for 1.5
hours. Allyl phosphorodiamidite (10) (20.5 mL) was added dropwise
at a 6.5 mL/hour rate followed by stirring for an additional 3
hours. N-Boc-2-aminoethanol (10.36 g) in methylene chloride (50 mL)
was added to the above reaction mixture dropwise at a 8.4 mL/hour
rate followed by stirring for an additional 18 hours. The white
suspension was filtered through Celite 545 with two 20 mL washings
with methylene chloride. The filtrate was concentrated followed by
the suspension and filtering of the residue with hexanes (200 mL).
The resulting hexanes filtrate was concentrated to dry and
azeotroped with 2,10-mL portions of toluene to provide the crude
product (11) (21.54 g) as an oil. 12
[0129] To a suspension of dithiophenol tin (1.3 g) in methylene
chloride (7.8 mL) was added thiophenol (400 .mu.L) followed by TEA
(543 .mu.L). The reaction mixture was stirred at room temperature
for 15 minutes followed by stopping the stirring and allowing the
residue to settle to the bottom of the flask. 1.0 mL of the above
solution was added to a solution of the azide (9) (242 mg) in
methylene chloride (0.5 mL) and allowed to stir for 30 minutes.
Quenching with 0.1 N NaOH followed by the usual work-up afforded
193.1 mg of the amine (12) as an oil. 13
[0130] To a dried solution of the amine (12) (193 mg) and acyl acid
(which can be made according to Christ et al., U.S. Pat. No.
5,530,113) (132 mg) in methylene chloride was added
1-(3-dimethylaminopropyl)-3-ethylcarbodii- mide hydrochloride (EDC,
93 mg). After stirring at room temperature for 90 minutes the
reaction was quenched and processed in the usual way to provide 232
mg of protected phosphate (13) as an oil. 14
[0131] To a solution of the protected phosphate (13) (232 mg) in
methylene chloride (1 mL) was added triethylsilane (TES, 120 .mu.L)
and trifluoroacetic acid (TFA, 1.2 mL) followed by stirring for 30
minutes. The TFA was removed under reduced pressure followed by
azeotroping with 3, 5-mL portions of toluene. 20 mL of methylene
chloride was added and the mixture was worked up in the usual
manner to give 174 mg of free amine (14) as an oil. 15
[0132] To a dried solution of the free amine (14) (174 mg) in
methylene chloride (0.5 mL) was added succinic acid (12.1 mg) and
EDC (59 mg). After 1 hour, the reaction was worked up in the usual
manner. Chromatography gave 143.1 mg of blocked diphosphate (15) as
an oil. 16
[0133] To a solution of blocked diphosphate (15) (177.9 mg) in
degassed chloroform (1.7 mL) in a dry box was first added
phenylsilane (PhSiH.sub.3, 50 .mu.L) and then
tetrakis-triphenylphosphine palladium (0) (Pd(PPH.sub.3).sub.4, 70
mg). After 1 hour, the reaction was removed from the box and
chloroform: methanol: water, 2:3:1, was added and the mixture
stirred for 1 hour. It was poured onto a diethylamino-ethylcellul-
ose (DEAE) chromatography column. Elution of the column with a
linear gradient of 0.0 M to 0.1 M ammonium acetate in chloroform:
methanol: water, 2:3:1, extraction of the desired fractions with an
equal volume of chloroform, concentration to dryness and the
addition of 0.1 N NaOH (175 .mu.L) followed by lyopholization gave
136.2 mg of (16) as a white solid.
Example 2
Chiral Malonate-Type 1
[0134] 17
[0135] To a cooled solution of potassium carbonate (165 g) in water
(575 mL) was added methylene chloride (200 mL) followed by ethyl
benzimidate hydrochloride (17) (100 g) after which time the mixture
was stirred for 8 minutes. The layers were separated and the
aqueous layer extracted with methylene chloride. The organic layers
were combined, dried and the solvent removed under reduced pressure
to give 83 g of (18). 18
[0136] To a solution of L-serine methyl ester hydrochloride (19)
(41.6 g) in 1,2-dichloroethane (450 mL) was added ethyl benzimidate
(18) (36 g). The mixture was heated to reflux for 20 hours, cooled,
filtered through diatomaceous earth, and concentrated to dryness to
give 56 g of ethyl ester (21) as a white solid. 19
[0137] To an ice cold solution of ethyl ester (21) (56 g) in THF
(500 mL) was added dropwise diisobutylaluminum hydride (DIBAL, 545
mL of a 1 M solution in hexane). The mixture was allowed to warm to
room temperature overnight and then carefully poured onto an
aqueous solution of Rochelle's salt (500 g in 1.0 L water). The
mixture stirred for 1 hour and worked up in the usual manner.
Chromatography gave 25.5 g of alcohol (22) as a white solid. 20
[0138] To a suspension of washed sodium hydride (5.3 g of a 60% oil
dispersion) in DMF (200 mL) was added the alcohol (22) (24 g) in
250 mL of THF After 30 minutes, the tosylate (4) was added in 250
mL of THF over 2.5 hours and stirred overnight. The mixture was
cooled in ice, methanol was added, the solvent removed under
reduced pressure and chromatographed to give 4.32 g of alcohol (24)
as an oil. 21
[0139] The alcohol (24) (4.3 g) was dissolved in 4 N aqueous
hydrochloric acid and heated to reflux for 20 hours. The mixture
was cooled, filtered, extracted with ether, made basic with sodium
hydroxide and extracted twice with chloroform. The combined
chloroform layers were dried and the solvent removed to give 2.88 g
of diol (25) as an oil. 22
[0140] The diol (25) (2.88 g) was dissolved in saturated aqueous
sodium bicarbonate (45 mL) and THF (25 mL) and allowed to stir for
five minutes. Myristoyl chloride (3.4 mL) was added dropwise over a
25-minute period after which time the reaction mixture was allowed
to stir for an additional hour. The reaction was worked up in the
usual manner and chromatographed to give 3.07 g of alcohol (26) as
an oil. 23
[0141] To a solution of the alcohol (26) (1.78 g) in methylene
chloride (140 mL) was added tetrazole (683 mg), followed by the
phosphorylating reagent (11) (1.6 mL). After 30 minutes, the
mixture was cooled in ice, and THF (105 mL) was added followed by
an oxone solution (3 g in 90 mL of water). After 5 minutes, the ice
bath was removed and the mixture stirred for 30 minutes. The
reaction was worked up in the usual manner and chromatographed to
give 2.99 g of alcohol (27) as an oil. 24
[0142] To a solution of the alcohol (27) (3.9 g) in methylene
chloride (126 mL) was added EDC (10.8 g), DMAP (66 mg) and dodecyl
acid (1.62 g) and stirred overnight. Additional acid (1.6 g), EDC
(1 g) and DMAP (0.5 g) was added. After 3 hours, the reaction was
worked up in the usual manner and chromatographed to give 2.07 g of
N-BOC-protected amine (28). 25
[0143] To a solution of the N-BOC-protected amine (28) (187.3 mg)
in methylene chloride (1.5 mL) was added TES (240 .mu.L) and TFA
(300 .mu.L) and the mixture stirred for 45 minutes. Toluene was
added and the solvent removed under reduced pressure. The residue
was dissolved in methylene chloride and worked up in the usual
manner to give 154 mg of amine (29) as an oil. 26
[0144] To an ice cold solution of the amine (29) (75.6 mg) and
malonic acid (4.8 mg) in methylene chloride (0.5 mL) was added EDC
(27 mg), followed by removal of the cooling bath. After 1 hour, a
trace of DMAP was added. After 2.5 hours, the mixture was directly
chromatographed to give 54.1 mg of dimer product (30). 27
[0145] To a solution of the dimer (30) (54 mg) in degassed
chloroform (2 mL) was added PhSiH.sub.3 (14 .mu.L) and
Pd(PPh.sub.3).sub.4 (18 mg) followed by removal of the cooling
bath. After 1 hour, the reaction was quenched with chloroform:
methanol:water (2:3:1) and poured onto a DEAE cellulose
chromatography column and chromatographed to give a semi-solid. The
solid was dissolved in sterile water and 0.1 N aqueous sodium
hydroxide (306 .mu.L) was added and the mixture lyophilized to
yield white solid (31) (25 mg).
Example 3
Chiral Malonate-Type 2
[0146] 28
[0147] To a solution of D-serine methyl ester hydrochloride (32)
(25 g) in dichloroethane (270 mL) was added ethyl benzimidate (20)
(24 g). After 20 hours at reflux, the mixture was cooled to room
temperature, filtered through diatomaceous earth and the solvent
removed under reduced pressure to give 34 g of methyl ester (33) as
a white solid. 29
[0148] To a ice cold solution of the methyl ester (33) (34 g) in
THF (300 mL) was added dropwise a solution of DIBAL in hexane (1.0
M, 322 mL). The mixture was allowed to warm to room temperature
overnight and then carefully poured into an aqueous solution of
Rochelle's salt. The mixture was then stirred for 1 hour and worked
up. Chromatography gave 18.6 g of alcohol (36) as a white solid.
30
[0149] To a suspension of washed sodium hydride (4 g of a 60% oil
suspension) in DMF (100 mL) was added a solution of the alcohol
(36) (8.6 g) in THF (40 mL). The mixture was stirred for 1 hour and
a solution of the tosylate (23) (17.5 g) in THF (40 mL) was added.
The mixture was stirred overnight and then additional tosylate (23)
was added (5 g) and stirred for another 4 hours. Methanol was added
to the cooled reaction mixture, the solvent was removed under
reduced pressure, and the residue was chromatographed to give 1.03
g of alcohol (37) as a solid. 31
[0150] The alcohol (37) (1.03) was dissolved in 4 N aqueous
hydrochloric acid (25 mL) and the mixture was heated to 100.degree.
C. for 20 hours. Additional hydrochloric acid (5 nmL) was added and
the reflux continued for 6 hours. The mixture was cooled, washed
with ether, made basic with 1 N aqueous sodium hydroxide, and
extracted (3.times.) with chloroform. The combined chloroform
layers were dried and the solvent removed under reduced pressure to
give 553 mg of amino-diol (39). 32
[0151] To a solution of amino-diol (39) (553 mg) in THF (3 mL) was
added saturated aqueous sodium bicarbonate (6 mL) followed by
myristoyl chloride (628 .mu.L). After 50 minutes, the reaction was
worked up in the usual way. Chromatography gave 389 mg of
amide-diol (41) as an oil. 33
[0152] To a solution of the amide-diol (41) (531 mg) in methylene
chloride (40 mL) was added tetrazole (203 mg) and the mixture was
stirred for 5 minutes. Then phosphorylating reagent (11) (482 mg)
was added. After 20 minutes, additional phosphorylating reagent
(11) (100 mg) was added and after an additional 20 minutes, 100 mg
more was added. After an additional 20 minutes, 50 mg more was
added. After 20 minutes, the mixture was poured into a cold
solution of THF (30 mL)/oxone (1.07 g) /water (30 mL). The mixture
was stirred at 0.degree. C. for 5 minutes, and then 20 minutes at
room temperature after which time the reaction was worked up in the
usual manner. Chromatography gave 852 mg of phosphate alcohol (42)
as an oil. 34
[0153] To a solution of the phosphate alcohol (42) (3.9 g) in
methylene chloride (126 mL) was added EDC (10.8 g), DMAP (66 mg)
and dodecyl acid (1.62 g). The reaction mixture was stirred
overnight. Additional acid (1.6 g), EDC (1 g) and DMAP (0.5 g) was
added. After 3 hours, the reaction was worked up in the usual
manner and chromatographed to give 2.07 g of protected amine (43).
35
[0154] To a solution of the protected amine (43) (194 mg) in
methylene chloride (1.5 mL) was added TES (240 .mu.L), and TFA (300
.mu.L). The mixture was stirred for 20 minutes and additional TES
(50 .mu.L) and TFA (50 .mu.L) was added. After 1 hour, toluene was
added and the solvent removed under reduced pressure and then the
mixture was worked up in the usual manner. The crude free amine
product (44) was used immediately in the next reaction. 36
[0155] To an ice cold solution of the free amine (29) (43.5 mg) in
methylene chloride (250 .mu.L) was added mono-t-butyl malonate (8.3
.mu.L), EDC (12.4 mg) and a trace of DMAP. The ice bath was removed
and after 2 hours, the reaction was worked up in the usual manner.
Chromatography gave 44 mg of amide (45). 37
[0156] To a solution of the amide (45) (44 mg) in methylene
chloride (0.5 mL) was added TES (90 .mu.L) and TFA (100 .mu.L).
After 2 hours, toluene was added and the solvent removed under
reduced pressure. The mixture was worked up in the usual manner to
give 44.2 mg of free acid (46). 38
[0157] To an ice cold solution of the free acid (46) (41 mg) and
the free amine (44) (26.3 mg) in methylene chloride (500 .mu.L) was
added EDC (13 mg) and the mixture was stirred for 30 minutes.
Additional EDC (5 mg) and DMAP (2 mg) was added and after 1 hour,
the reaction was worked up in the usual manner. Chromatography gave
32.7 mg of coupled compound 47 as an oil. 39
[0158] To a solution of the coupled compound (47) (32.7 mg) in
degassed chloroform (1.5 mL) in a dry box was added PhSiH.sub.3
(8.5 .mu.L) and Pd(PPH.sub.3).sub.4 (11 mg). The mixture was
removed from the box and cooled in ice. After 5 minutes, the ice
bath was removed and after 1 hour, chloroform:methanol:water
(2:3:1) was added and the mixture stirred for 15 minutes and stored
in the freezer overnight. The mixture was then poured onto a DEAE
chromatography column. Chromatography gave 13.9 mg of a compound
(48) as a white powder after 0.1 N NaOH treatment followed by
lyophilization.
Example 4
Chiral Urea-Type 1
[0159] 40
[0160] To a solution of amine (44) (46.1 mg) in toluene was added
saturated sodium bicarbonate (0.5 mL) followed by phosgene (15
.mu.L of a 1.93 M solution in toluene). After 30 minutes,
additional phosgene (10 .mu.L) was added. After 2 hours, additional
phosgene (5 .mu.L) was added. The reaction was quenched with
aqueous sodium bicarbonate and worked up in the usual manner to
give 29.6 mg of urea (49) with protected phosphates. 41
[0161] To a solution of the urea with protected phosphates (49)
(29.6 mg) in degassed chloroform (1.5 mL) in a dry box was added
PhSiH.sub.3 (8 .mu.L). The reaction vessel was removed from the dry
box and placed on ice. Pd(PPh.sub.3).sub.4 (10 mg) was added and
after minutes the ice bath removed. After 1 hour, the reaction was
quenched by addition of chloroform:methanol:water. The mixture was
stirred for 15 minutes and stored overnight in the freezer. It was
chromatographed on DEAE to give 24.1 mg of (50) as a white powder
after 0.1 N NaOH treatment followed by lyophilization.
Example 5
Chiral Urea-Type 2
[0162] 42
[0163] To an ice-cold solution of trichloromethyl chloroformate
(2.6 .mu.L) in methylene chloride (200 .mu.L) was added a solution
of free amine (29) (35 mg) and 1,8-bis-(dimethylamino)-naphthalene
in methylene chloride (200 .mu.L). After 5 minutes, the ice bath
was removed. After 15 minutes, additional methylene chloride was
added and the mixture worked up in the usual manner. Chromatography
gave 9.4 mg of isocyanate (51) as an oil. 43
[0164] To a solution of the isocyanate (51) (9.4 mg) in methylene
chloride (0.2 mL) was added a solution of the amine (44) (10.3 mg)
in methylene chloride. After 15 minutes, the reaction was worked up
in the usual manner. Chromatography gave 5.5 mg of coupled compound
(61) as an oil. 44
[0165] To a solution of the coupled compound (61) (25.2 mg) in
degassed chloroform (0.5 mL) in a dry box was added PhSiH.sub.3
(6.6 .mu.L) and Pd(PPH.sub.3).sub.4 (8.8 mg). The mixture was
removed from the box and cooled in ice. After 5 minutes, the ice
bath was removed and after I hour, chloroform:methanol:water
(2:3:1) was added and the mixture stirred for 15 minutes and stored
in the freezer overnight. The mixture was then poured onto a DEAE
chromatography column. Chromatography gave 7.5 mg of (62) as a
white powder after 0.1 N NaOH treatment followed by
lyophilization.
Example 6
Chiral Glycerol Analogue of Type 1
[0166] 45
[0167] To a stirred suspension of sodium hydride (145.5 mg of 60%
oil dispersion washed with hexanes) in DMF (12 mL) was added
(S)-(+)-alcohol (63) (0.41 mL) in 8 mL of THF dropwise over a 1
hour period. The mixture was stirred for an additional 30 minutes
followed by a dropwise addition of the tosylate (4) (0.789 g) in 10
mL of THF over a 10-minute period. The resulting reaction mixture
was stirred overnight. The usual work up gave 0.56 mg of the
desired adduct (65). 46
[0168] A solution of lauric acid (1.40 g), EDC (1.35 g), DMAP (0.04
g) and the alcohol adduct (65) (0.564 g) in 4 mL of methylene
chloride was stirred for 15 hours at room temperature. Brine and
saturated aqueous sodium bicarbonate (1:1) were added and the
mixture extracted with methylene chloride. The mixture was worked
up in the usual manner and chromatographed. The desired fraction
was dissolved in 20 mL of 4:1 acetic acid: water and stirred for 15
hours. The solvent was removed under reduced pressure and the
residue chromatographed to give 0.77 g of semi-solid diol (66).
47
[0169] A solution of the diol (66) (0.22 g), DMAP (6.3 mg), TEA
(100 .mu.L) and TBDPSCl (164 .mu.L) was stirred for 24 hours at
room temperature. Methanol (2 mL) and a trace of aqueous
hydrochloric acid was added followed by extraction with methylene
chloride. The mixture was worked up in the usual way. The residue
was chromatographed to give 0.3 g of alcohol (67) as an oil. 48
[0170] A solution of the alcohol (67) (0.3 g), DMAP (5.5 mg), EDC
(258 mg), myristic acid (308 mg) in methylene chloride (4 mL) was
stirred for 18 hours at room temperature followed by the addition
of brine and saturated sodium bicarbonate. The mixture was worked
up in the usual way and chromatographed to give 0.4 g of silyl
protected ether product (68). 49
[0171] To a solution of the silyl protected ether (68) (195 mg) in
acetonitrile (2.7 mL) was added 48% hydrofluoric acid (0.756 ml).
After 30 hours, saturated sodium bicarbonate was added and the
mixture worked up in the usual way. Chromatography gave 94.7 mg of
free alcohol (69). 50
[0172] To a solution of the free alcohol (69) (57 mg) in methylene
chloride (0.5 mL) was added tetrazole (15.6 mg) and phosphorylating
reagent (11) (40 mg) at room temperature. After four hours, the
mixture was cooled to 0.degree. C. followed by the addition of
oxone (82 mg) in THF (0.5 mL): water (0.6 mL). The mixture was
warmed to room temperature and stirred for 80 minutes. The final
reaction mixture was worked up in the usual manner. Chromatography
gave 72 mg of protected phosphate (70). 51
[0173] To a solution of the protected phosphate (70) (72 mg) in
methylene chloride (1 mL) was added TES (120 .mu.L) and
trifluoroacetic acid (0.6 mL) followed by stirring for 1 hour. The
TFA was removed under reduced pressure followed by azeotroping with
10 mL of toluene. 20 mL of methylene chloride was added and the
mixture was worked up in the usual manner to give 0.52 g of an
oil.
[0174] The crude amine was dissolved in methylene chloride (0.7 mL)
followed by the addition of malonic acid (4.5 mg) and EDC (25.6
mg). The mixture was stirred overnight and worked up in the usual
way to give 32.5 mg of the dimer product (71). 52
[0175] The protected dimer (71) (32.5 mg) from the preceding
reaction was dissolved in degassed chloroform (2 mL) and
PhSiH.sub.3 (8.6 .mu.L) was added in a dry box. The mixture was
removed from the dry box and Pd(PPh.sub.3).sub.4 (22.6 mg),
previously weighed in the dry box, was added. After 2 hours, the
mixture was chromatographed on DEAE to give 27.9 mg of white solid
(72) after the addition of IN sodium hydroxide (34.2 .mu.L)
followed by lyophilization.
[0176] Preparation of ER-804253 53
[0177] The alcohol (558 mg) was dissolved in methylene chloride (5
mL) cooled to 0.degree. C. and triethylamine (0.466 mL) was added
under a nitrogen atmosphere. After stirring for 5 minutes
methanesulfonyl chloride (0.142 mL) was added dropwise. The mixture
was stirred for an additional 5 minutes at 0.degree. C. and then
warmed to room temperature. After stirring for an additional hour,
the mixture was worked up with sat. sodium bicarbonate, extracted
with ethyl acetate and the extract washed with water, dilute
aqueous hydrochloric acid, water, brine, dried and the solvent
removed to give 630 mg. 54
[0178] The mesylate (630 mg) and sodium azide (299 mg) were
dissolved in DMSO (6 mL) and heated to 60.degree. C. for 90
minutes. After cooling to room temperature the reaction mixture was
diluted with methylene chloride, washed with water and brine. After
extracting the aqueous washes the combined organic was dried,
concentrated, purified over silica gel using a 4:1 ratio of hexanes
to ethyl acetate and the dried product fractions give 420 mg.
55
[0179] The azide (295 mg) was dissolved in ethanol (5 mL) and
Lindlar catalyst (200 mg) was added. After stirring under an
atmosphere of hydrogen gas at atmospheric pressure, the filtered
solution was dried to give 274 mg. 56
[0180] The amine (930 mg) was dissolved in THF (10 mL) and sat.
sodium bicarbonate (22 mL). After stirring for 5 minutes, lauroyl
chloride (0.712 mL) was added dropwise over 20 minutes. The final
mixture was extracted with chloroform, dried to give 1.45 g. 57
[0181] The amide (1.11 g) was dissolved in methanol (14 mL) and 4 N
hydrochloric acid (8 mL) added. The mixture was stirred for 1 our
at 50.degree. C. and then concentrated. Methanol (16 mL) and 40%
sodium hydroxide (8 mL) was added and the mixture refluxed for I
hour. It was cooled, extracted with methylene chloride and the
extract washed with water, dried and the solvent removed to give
930 mg. 58
[0182] The amino alcohol was dissolved in THF (6 mL) and saturated
sodium bicarbonate added (13 mL). After 5 minutes the mixture was
cooled to 0.degree. C. and myristoyl chloride ( 300 .mu.L) added.
After 30 minutes, the mixture was worked up in the usual way to
give 430 mg. 59
[0183] The alcohol (101.7 mg) was dissolved in ice cold methylene
chloride and phosphorylating reagent 11 (90 .mu.L) was added and
the mixture stirred for 30 minutes. Ice cold oxone (166.3 mg) was
added and the mixture stirred for 30 minutes. The reaction was
quenched with thiosulfate. The mixture was worked up the usual way
and chromatographed to give 174 mg (not purified) 60
[0184] The protected amine from the above reaction was dissolved in
ice cold methylene chloride (1 mL), trifluoroacetic acid (1 mL) was
added and the mixture stirred for 1 hour. The TFA was removed and
the mixture purified to give 106.7 mg. 61
[0185] The amine was dissolved in methylene chloride (3 mL) and
saturated. sodium bicarbonate solution (3 mL) was added. The
mixture was cooled in ice and phosgene in toluene (0.55 equiv.) was
added dropwise. The mixture was stirred for 20 minutes and worked
up to give 112.3 mg. 62
[0186] To a solution of the blocked phosphate (40.5 mg) in ice cold
chloroform (2.6 mL) was added phenylsilane (10.7 mg) and
tetrakis(triphenylphosphine)palladium [0] (28.7 mg) and the mixture
stirred for 1 hour. The mixture was chromatographed on a DEAE
column to give 27.7 mg of ER-804253.
[0187] Preparation of ER-804130 63
[0188] To a solution of the amide (3 g) in THF (65 mL) at
-78.degree. C. was added an equivalent of butyllithium, followed by
a solution of nonanoyl chloride in THF (6 mL). Aqueous ammonium
chloride was added and the mixture worked up in the usual manner to
give 5.35 g. 64
[0189] To a solution of the alcohol (5 g) in ice cold methylene
chloride (100 mL) was added triethylamine (4.1 mL) and mesyl
chloride (2.1 mL). The mixture was stirred for 4 hours and worked
up in the usual manner to give 6.99 g. 65
[0190] The a solution of the mesylate (6.99 g) in ice cold DMF (100
mL) was added potassium bromide. The mixture was allowed to warm to
room temperature and stirred for five hours. It was worked up in
the usual manner to give 4.63 g of clear oil. 66
[0191] A solution of the amide (2.8 g) in THF (15 mL) was added to
a -78.degree. C. solution of sodium his-trimethylsilylamide in THF
(15 mL). After 1 hour, the bromide was added and the mixture
allowed to warm to room temperature and worked up in the usual
manner to give 1.02 g. 67
[0192] To a solution of the olefin (1.02 g) in EtOAc was added
palladium on carbon (126 mg) and the mixture placed under hydrogen.
After 4 hours, the mixture was worked up in the usual manner to
give 1.0 g. 68
[0193] To a solution of the amide (1.0 g) in ice cold THF (20 mL)
was added water, hydrogen peroxide and lithium hydroxide. The next
day, the mixture was worked up in the usual manner to give 590 mg
of acid. 69
[0194] To a solution of the acid in ice cold THF (10 mL) was added
diborane:THF complex and the mixture allowed to warm slowly. After
seven hours, dilute hydrochloric acid was added carefully and the
mixture worked up in the usual manner. The crude material was
dissolved in ice cold ether and LAH solution (2 mL, of 1M) added.
After 5 minutes, the mixture was worked up in the usual manner to
give 556 mg of the alcohol. 70
[0195] To a -78.degree. C. solution of oxalyl chloride (2.2 mL) in
methylene chloride (10 mL) was added DMSO (1.1 mL) and after 2
minutes the alcohol (556 mg) was added in methylene chloride (5
mL). After 20 minutes, triethylamine (1 mL) was added and the
mixture warmed to 0.degree. C. The mixture was diluted with ether
and worked up in the usual manner to give 567 mg. 71
[0196] The Wittig reagent (679 mg) was suspended in THF (10 mL) and
KHMDS solution (4 mL of 0.5 M) added. After 20 minutes, the mixture
was cooled to -78.degree. C. and the aldehyde (567 mg) in THF (5
mL) was added. After 15 minutes, the mixture was worked up in the
usual manner to give 342 mg. 72
[0197] To a solution of the enol ether (342 mg) in acetonitrile
(3.5 mL) and water (0.15 mL) was added hydroiodic acid. After 4
hours, the mixture was worked up in the usual manner to give 325
mg. 73
[0198] To a solution of the aldehyde (325 mg) in methanol (10 mL)
was added sodium borohydride (38 mg). After 3 hours, the reaction
was worked up in the usual manner to give 303 mg of the alcohol.
74
[0199] To an ice cold solution of the alcohol (303 mg) in methylene
chloride (10 mL) was added triethylamine (150 .mu.L) and mesyl
chloride (76 .mu.L). After 4 hours, the reaction was worked up in
the usual manner to give 352 mg. 75
[0200] To a solution of the oxazoline (1 mL) in ice cold THF (5 mL)
was added potassium t-butoxide solution (2.2 mL of 1M). After 30
minutes, the mesylate was added in THF (5 mL) and the mixture
stirred for 8 hours. The usual work-up gave 318.5 mg. 76
[0201] A solution of the oxazoline in methanol (8 mL) and
hydrochloric acid (4 mL of 4M) was warmed to 50.degree. C. for 90
minutes. Additional methanol was added and the solvent removed. The
residue was dissolved in methanol (8 mL) and sodium hydroxide
solution (4 mL) and briefly warmed to 50.degree. C. The mixture was
cooled and extracted with chloroform. The usual work-up gave 114
mg. 77
[0202] To a solution of the amine (114 mg) in THF (2 mL) and
saturated aqueous sodium bicarbonate (2 mL) was added the acid
chloride. After 30 minutes additional acid chloride was added.
After 30 minutes, the reaction was worked up in the usual manner to
give 146 mg. 78
[0203] To a solution of tetrazole (48 mg), the phosphorylating
reagent (122 mg) in ice cold methylene chloride (2 mL) was added
the alcohol (146 mg). Oxone (230 mg) in water ( 1 mL) and THF (2
mL) was added. After 90 minutes, thiosulfate was used to quench the
reaction. Standard work up gave 140 mg. 79
[0204] To a solution of the substrate in ice cold methylene
chloride was added triethylsilane (370 .mu.L) and trifluoroacetic
acid (110 .mu.L). After 5 minutes, the volatiles were removed to
give 148 mg. 80
[0205] To a solution of the amine (66.9 mg) in ice cold methylene
chloride (0.8 mL) was added saturated aqueous sodium bicarbonate
(0.8 mL) and phosgene solution (20 .mu.L of 1.93 M). After one
hour, additional phosgene (10 .mu.L) was added. After 30 minutes,
the usual work-up gave 47.7 mg. 81
[0206] To a solution of phenylsilane (15 uL),
tetrakis(triphenylphosphine) palladium [0] (24.8 mg) in ice cold
chloroform under an inert atmosphere was added the substrate. After
5 minutes, the mixture was applied to a DEAE column and
chromatographed to give 31 mg of ER-804130.
[0207] Preparation of ER-804558 82
[0208] To a solution of the amine (325 mg) in methylene chloride
was added triethylamine (321 .mu.L) and 1-dodecanesulfonyl
chloride. After 3 hours, the usual work up gave 384 mg. 83
[0209] To a solution of the protected alcohol (384 mg) in THF (4
mL) was added tetrabutylammonium fluoride (123 mg) and acetic acid
(29 .mu.L). After 2 hours, the usual work up gave 180 mg.
[0210] The remainder of the synthesis was completed as outlined
above for other compounds of the present invention, i.e.
phosphorylating, deblocking, coupling with phosgene, and
deprotecting with phenylsilane and palladium.
[0211] Preparation of ER-804442
[0212] The diol amine was mono-protected as its
t-butyl-diphenylsilylether outlined above. 84
[0213] The amine (2.6 g) and benzophenone imine (1.1 mL) were mixed
and heated to 40.degree. C. for 4 days to give after chromatography
3.3 g. 85
[0214] To a solution of the imine (3.3 g) was in ice cold methylene
chloride was added lauric acid (1.5 g), EDC (1.7 g) and DMAP (155
mg). The next day, the reaction was worked up in the usual manner
to give 3.15 g. 86
[0215] To a solution of the imine (3.14 g) in ether was added 1 N
aqueous hydrochloric acid. The next day, the reaction was worked up
in the usual manner to give 2.81 g. 87
[0216] To a solution of trichloromethylchloroformate (12 .mu.L) in
ice cold methylene chloride (250.mu.L) was added dodecylamine (18
.mu.L) and diisopropylethylamine (27 .mu.L). After 30 minutes, the
solvent was removed. The residue was dissolved in ice cold
methylene chloride, to which was added the amine (55.6 mg) and
additional diisopropylethylamine (13 .mu.L). After 2 hours, the
usual work up gave, after chromatography, 60.9 mg.
[0217] This product was de-protected with fluoride, phosphorylated,
de-protected with TFA, dimerized with phosgene, and un-blocked with
phenylsilane and palladium as outlined above to give ER-804442.
[0218] Preparation of ER-804221 88
[0219] To an ice cold solution of glycine (8.26 g) in aqueous
sodium hydroxide (4.4 g in 60 mL) was added lauroyl chloride (21.8
g). After 1 hour, acid was added and the mixture worked up in the
usual way. Recrystallization from ethyl acetate gave 9.7 g. 89
[0220] To an ice cold solution of the amine (1.4 g) in methanol was
added triflic azide (20 mg). The next day, additional azide was
added. After 2 hours, the reaction was worked up in the usual
manner to give after chromatography 1.14 g. 90
[0221] To a solution of the alcohol (1.14 g) in methylene chloride
was added t-butyl-diphenylsilyl chloride (1.09 mL), triethylamine
(1.8 mL) and DMAP (50 mg). After 3 hours, the usual work up gave
1.4 g. 91
[0222] To a solution of the alcohol (1.4 g) in ice cold methylene
chloride was added lauric acid (826 mg), EDC (1.05 g) and DMAP (33
mg). The next day, the usual work up gave after chromatography 778
mg. 92
[0223] To a solution of the azide (778 mg) in THF was added acetic
acid (77 .mu.L) and TBAF (323 mg). The next day, the usual work up
gave, after chromatography, 428 mg. 93
[0224] To a solution of the azide (460 mg) in methylene chloride
was added tetrazole (165 mg), the phosphorylating reagent (390 mg),
and after 30 minutes, oxone in water (722 mg in 3 mL). The reaction
was quenched with thiosulfate. Usual work up, after chromatography,
gave 392 mg. 94
[0225] The protected amine (460 mg) was dissolved in methylene
chloride and trifluoroacetic acid (394 .mu.L) and triethylsilane
(308 .mu.L). After 1.5 hour, the usual work up gave, 392 mg. 95
[0226] To an ice cold solution of the amine in methylene chloride
(5.5 mL) was added saturated sodium bicarbonate (5.5 mL), and
phosgene (164 .mu.L of a 1.93 M solution in toluene). After 15
minutes, the usual work up, after chromatography gave 342 mg.
96
[0227] To a ice cold solution of the azide (187 mg) in methylene
chloride was added the tin reagent (1.5 mL) which was prepared as
outlined in U.S. Pat. No. 5,756,718 incorporated herein by
reference. After 30 minutes, the mixture was chromatographed to
give 187 mg. 97
[0228] To a ice cold solution of the urea (55 mg) in methylene
chloride was added the acid (59 mg) (prepared as above) and EDC (44
mg). The next day, additional EDC (5 mg) and acid (5 mg) was added.
After 2 hours, the normal work up provided 45.7 mg.
[0229] Normal removal of the protecting groups with phenylsilane
and palladium gave ER-804221.
[0230] ER-804222 was prepared in a similar manner except that the
condensation product between lauryl chloride and glycine,
15-methylmyristic acid was used.
[0231] Preparation of ER-804281 98
[0232] To a ice cold solution of the protected alcohol (8.3 g) in
acetonitrile: water was added CAN (41.4 g). After 1 hour, the usual
work up gave 5.7 g. 99
[0233] A solution of the alcohol (5.63 g) in 4 N HCl solution was
heated to reflux for 1 hour, cooled, neutralized with sodium
hydroxide and worked up in the usual manner to give 2.1 g. 100
[0234] To an ice cold solution of the alcohol (2.2 g) in
rmiethylene chloride was added imidazole (0.7 g),
t-butyl-diphenylsilyl chloride in 15 mL of methylene chloride. The
next day, the usual work up gave 1.54 g. 101
[0235] To a solution of the alcohol (1.93 g) in methylene chloride
(40 mL) was added benzophenone imine (0.8 mL). After 1 day, the
mixture was heated to reflux overnight. The usual work up gave 1.67
g. 102
[0236] To an ice cold solution of the alcohol (1.67 g) in methylene
chloride was added DMAP (159 mg), EDC (0.99 g) and lauric acid
(1.04 g). After one day, the usual work up gave 74% yield. 103
[0237] To an ice cold solution of the imine (2.9 g) in ether (50
mL) was added 1 N HCl (50 mL). The next day, the usual work up gave
2.09 g. 104
[0238] To a solution of the amine (1.24 g) in dichloroethane was
added sodium cyanoborohydride (178 mg) and tetradecanal (411 mg).
The next day, the usual work up gave 1.5 g. 105
[0239] To a ice cold solution of the amine (221 mg) in dioxane was
added allyl chloroformate (40 mg) and 308 .mu.L of 1 N NaOH
solution. After 2 hours, the usual work up gave 200 mg. 106
[0240] To an ice cold solution of the protected alcohol (365 mg) in
THF was added TBAF (1924 .mu.L) and acetic acid (122 .mu.L). The
next day, the usual work up gave 271 mg.
[0241] This material was phosphorylated, deblocked with TFA,
dimerized with phosgene and the allyl protecting groups removed
with phenylsilane and palladium as described above to give
ER-804281.
[0242] Preparation of ER-804339
[0243] ER-804281.fwdarw.ER-804339
[0244] To a ice cold solution of ER-804281 (7 mg) in methylene
chloride was added triethylamine (5 .mu.L), DMAP (0.6 mg) and
acetyl chloride (1.8 .mu.L). After 4 days, the usual work-up gave
1.1 mg.
[0245] Preparation of ER-804674
[0246] ER-804281.fwdarw.ER-804674
[0247] To a solution of ER-804281 (12.7 mg) in THF (1.0 mL) was
added methyl iodide (9.2 mg) and sodium bicarbonate (6.8 mg). The
mixture was stirred for 5 days and sodium bicarbonate (14 mg) and
additional methyl iodide (8 mL) was added. After an additional 3
days, additional bicarbonate (28 mg) and Mei (16 .mu.L). After an
additional 6 days, the mixture was worked up to give 9.1 mg of
product.
[0248] Preparation of ER-804596 107
[0249] To a solution of the alcohol (393 mg) in methylene chloride
(2 mL) was added diisopropylamine (210 .mu.L), tetrazole (105 mg)
and phosphorylating reagent (as described above) (488 mg). After
21/5 hours, the usual work up gave the desired product. 108
[0250] To a solution of the diol (73 mg) in acetonitrile was added
tetrazole (175 mg), the azide (1 equivalent). After 3 hours, the
mixture was cooled and ozone (1229 mg) added. The next day, usual
work up gave the desired product. 109
[0251] To an ice cold solution of the protected alcohol (92.9 mg)
in acetonitrile:water (6 mL: 1.5 mL) was added CAN(358 mg). After 1
hour, the usual work up provided 68.5 mg. 110
[0252] To an ice cold solution of the diol (68.5 mg) in methylene
chloride was added lauric acid (76.5 mg), DMAP (4.7 mg) and EDC (73
mg). The next day, the usual work up gave 76.5 mg.
[0253] The azides were reduced using the tin reagent described
above. The diamine was acylated with dodecanoyl chloride, and the
protecting groups removed with phenylsilane and palladium as
described above to give ER-804596.
[0254] Preparation of ER-804732 111
[0255] The alcohol (7.04 g) was dissolved in methylene chloride
(300 mL) with triethylamine (11.13 mL) and then cooled to 0.degree.
C. under a nitrogen atmosphere. Methanesulfonyl chloride (3.69 mL)
was added dropwise after which time the reaction was stirred at
room temperature for 1 hour. The usual work up gave 5.551 g.
112
[0256] The mesylated (1.114 g) was dissolved in DMF (30 mL)
followed by sodium azide (0.9337 g). The reaction mixture was
warmed to 57.degree. C. and stirred for 16 hours and then to
104.degree. C. for and additional 3 hours. After cooling to room
temperature the mixture was worked up in the usual manner and gave
0.466 g. 113
[0257] The protected aminoalcohol (0.466 g) was hydrolyzed using 4
N HCl (15 mL) at 107.degree. C. for 3 hours. After cooling to room
temperature, the reaction mixture was filtered and extracted with
ethyl ether, dried, concentrated and used in the next reaction.
114
[0258] The crude aminoalcohol was dissolved in THF (5 mL) with
saturated sodium bicarbonate (6 mL) and cooled to 0.degree. C.
Myristoyl chloride (0.79 mL) was added dropwise after which time
the reaction was warmed to room temperature and stirred for 2
hours. The reaction mixture was worked up using the usual methods
and gave 0.751 g. 115
[0259] The alcohol (0.185 g) was dissolved in DMF (3.0 mL) with
imidazole (0.077 g) and tert-butyldiphenylsilyl chloride (0.197
mL). The reaction mixture was stirred at room temperature for 16
hours after which time the usual work up gave 0.320 g. 116
[0260] The azide (0.975 g) was dissolved in methanol (20 mL) with
10% palladium on carbon (0.180 g). The mixture was stirred under an
atmosphere of hydrogen gas under atmospheric pressure for 2 hours
after which time the gas was evacuated and the mixture filtered
over Celite 545 and concentrated. Purification using the usual
methods gave 0.873 g. 117
[0261] DMSO (1.5 mL) was added dropwise to oxalyl chloride (0.92
mL) in methylene chloride (30 mL) at 78.degree. C. After stirring
for 15 minutes the alcohol (1.727 g) in methylene chloride (30 mL)
was added dropwise and stirred for an additional 30 minutes.
Triethylamine (4.90 mL) was added dropwise, the reaction was warmed
to 0.degree. C. and quenched using saturated ammonium chloride.
Purification of the crude product using silica gel chromatography
with 20% ethyl acetate in hexanes gave 1.653 g. 118
[0262] The primary amine (0.135 g) and aldehyde (0.077 g) were
dissolved in 1,2-dichloroethane (5 mL) followed by the addition of
sodium cyanoborohydride (0.032 g). The reaction was stirred for 20
hours after which time acetic acid (0.02 mL) was added and the
reaction worked up in the usual manner to give 0.103 g. 119
[0263] The secondary amine was dissolved in 1,4-dioxane (15 mL) and
cooled to 0.degree. C. followed by the slow addition of 1 M sodium
hydroxide (3.0 mL). After stirring for 10 minutes allyl
chloroformate (0.236 mL) was added dropwise after which time the
reaction was warmed to room temperature and stirred for 16 hours.
Work up in the usual manner gave 0.613 g. 120
[0264] The para-methoxybenzyl ether (0.613 g) was dissolved in a 4
to 1 ratio of acetonitrile to water (15 mL), cooled to 0.degree. C.
and then CAN (1.525 g) was added. The reaction mixture was stirred
at 0.degree. C. for 2 hours and then worked up in the usual manner
to give 0.357 g. 121
[0265] The alcohol (0.357 g) was dissolved in methylene chloride (5
mL) with lauric acid (0.184 g), EDC (0.175 g) and cooled to
0.degree. C. 4-Dimethylaminopyridine (0.012 g) was added and the
resulting mixture was stirred at room temperature for 2 hours. Work
up in the usual manner gave 0.436 g. 122
[0266] The silyl protected alcohol (0.211 g) was dissolved in THF
(5 mL) with acetic acid (0.03 mL). Tetrabutylammonium fluoride
(0.115 g) was added in one portion and the reaction mixture was
stirred at room temperature for 16 hours. A normal work up gave
0.150 g.
[0267] This material was phosphorylated, deblocked with TFA,
dimerized with phosgene and the allyl protecting groups removed
with phenylsilane and palladium as descried above to give
ER-804732.
[0268] Preparation of ER-804680 123
[0269] The aldehyde (1.54 g) was dissolved in THF (28 mL) and
cooled to 0.degree. C. after which time 2-methyl-2-butene (14 mL)
and tert-butyl alcohol (28 mL) was added. A stirred suspension of
sodium chlorite (3.70 g) and sodium trihydrogen phosphate (4.09 g)
in water (42.7 mL) was added to the above mixture and stirred at
0.degree. C. for 1.5 hours. The completed reaction was diluted with
ethyl acetate (100 mL) and washed with 10% sodium bisulfite, brine,
dried, concentrated and silica gel chromatographed to give 1.55 g.
124
[0270] The amine (0.553 g) and acid (0.381 g) were mixed in
methylene chloride (8 mL) and cooled to 0.degree. C. after which
time EDC (0.230 g) was added and the reaction mixture was stirred
at room temperature for 72 hours. The usual work up gave 0.567 g.
125
[0271] The methoxybenzyl ether (0.567 g) was dissolved in a 1 to 1
ratio of acetonitrile to water (16 mL) with methylene chloride (8
mL) and cooled to 0.degree. C. CAN (1.53 g) was added and the
reaction mixture was stirred for 1 hour after which time it was
worked up in the usual manner to give the crude alcohol. 126
[0272] The crude alcohol from above was dissolved in methylene
chloride (15 mL) with lauric acid (0.280 g) and 4
dimethylaminopyridine (0.017 g). The reaction mixture was cooled to
0.degree. C. and EDC (0.267 g) was added in one portion after which
time the reaction mixture was warmed to room temperature and
stirred for 16 hours. Normal work up procedures provided 0.622 g.
127
[0273] The silyl ether (0.563 g) was dissolved in THF (10 mL) with
acetic acid (0.087 mL). Tert-butylammonium fluoride (0.330 g) was
added and the reaction was stirred at room temperature for 16
hours. Work up in the usual manner gave 0.384 g.
[0274] This material was phosphorylated, deblocked with TFA,
dimerized with phosgene and the allyl protecting groups removed
with phenylsilane and palladium as described above to give
ER-804780.
[0275] Preparation of ER-804679 128
[0276] The protected secondary amine (0.071 g) was dissolved in
degassed chloroform (3 mL) with phenylsilane (0.017 mL) and acetic
anhydride (0.014 mL). The reaction mixture was cooled to 0.degree.
C. followed by the addition of tetrakistriphenylphosphine palladium
(0) (0.002 g). The reaction mixture was warmed to room temperature
and allowed to stir for 30 minutes. The completed reaction was
diluted with methylene chloride, washed with water, dried,
concentrated, and chromatographed to give 0.068 g. 129
[0277] The silyl ether was deprotected in THF (5 mL) with acetic
acid (0.025 mL) with the addition of tert-butylammonium fluoride
(0.092 g). After stirring at room temperature for 16 hours the
reaction was worked up in the usual manner to give 0.120 g.
[0278] This material was phosphorylated, deblocked with TFA,
dimerized with phosgene and the allyl protecting groups removed
with phenylsilane and palladium as described above to give
ER-804679.
[0279] Preparation of ER-804764 130
[0280] DMSO (0.33 mL) was added dropwise to oxalyl chloride (0.203
mL) in methylene chloride (10 mL) at 78.degree. C. After stirring
for 15 minutes the alcohol (0.993 g) in methylene chloride (3 mL)
was added dropwise and stirred for an additional 30 minutes.
Triethylamine (1.08 mL) was added dropwise, the reaction was warmed
to 0.degree. C. and quenched using saturated ammonium chloride.
Purification of the crude product using silica gel chromatography
with 20% ethyl acetate in hexanes gave 0.743 g. 131
[0281] The 1.6 M n-butyl lithium in hexanes (1.5 mL) was added
dropwise to the phosphonium salt (0.797 g) in THF (10 mL) at
0.degree. C. After stirring for 30 minutes the aldehyde (0.734 g)
in THF (15 mL) was added dropwise. After stirring at room
temperature for one hour the reaction was worked up in the usual
manner to give 0.193 g. 132
[0282] The enol ether (0.193 g) was hydrolyzed with 57% hydrogen
iodide (0.114 L) in acetonitrile (2 mL). After stirring at room
temperature for 2 hours the reaction was quenched with saturated
sodium bicarbonate, extracted with methylene chloride, and dried to
give 0.211 g crude aldehyde. 133
[0283] The crude aldehyde (0.211 g) was dissolved in methanol (3
mL) and sodium borohydride (0.033 g) was added at 0.degree. C.
After stirring for 30 minutes the reaction was diluted with water,
extracted with methylene chloride, dried, concentrated and purified
by silica gel chromatography to give 0.148 g.
[0284] This material was phosphorylated, deblocked with TFA,
dimerized with phosgene and the allyl protecting groups removed
with phenylsilane and palladium as described above to give
ER-804764.
[0285] Preparation of ER-804772 134
[0286] The commercially available diol (1.486 g) was mixed with the
acetal (1.864 g) and para-toluenesulfonic acid (0.195 g) in DMF (10
mL). After stirring for 20 hours at room temperature under a
nitrogen atmosphere, the reaction was quenched with sat. sodium
bicarbonate, extracted with methylene chloride, dried and
concentrated via high vacuum. Silica gel chromatography of the
resultant crude product using 10% ethyl acetate in hexanes gave
2.084 g. 135
[0287] The acetal (2.084 g) was cooled to 78.degree. C. under a
nitrogen atmosphere in methylene chloride (30 mL) followed by the
dropwise addition of 1.0 M DIBAL in hexanes (14.3 mL). After
additional DIBAL (14 mL) was added, the reaction mixture was
stirred for 1 hour, warmed to room temperature and quenched with
sodium, potassium tartarate. The normal work up gave 2.1 g. 136
[0288] The alcohol (1.286 g) was mixed with triethylamine (0.883 g)
in methylene chloride (15 mL) and cooled to 0.degree. C.
Methanesulfonyl chloride (0.575 g) was added dropwise followed by
stirring for 20 minutes at 0.degree. C. and room temperature for 2
hours. The normal work up gave 1.496 g. 137
[0289] The alcohol (1.495 g) in DMF (10 mL) was added dropwise to a
stirring suspension of washed 60% sodium hydride (0.257 g) in DMF
(20 mL) at 0.degree. C. After stirring for 3 hours the mesylate
(0.925 g) in DMF (10 mL) was added dropwise. After stirring for an
additional 3 days, the reaction was quenched and worked up in the
usual manner gave 0.905 g.
[0290] As with examples provided above, the para-methoxybenzyl
protecting group was hydrolyzed with CAN, the protected amino
alcohol hydrolyzed using aqueous HCl then KOH, acylation of the
amine with tetradecanoyl chloride, silylation of the primary
alcohol with TBDPS, acylation of the secondary alcohol with
dodecanoyl chloride, and hydrolysis of the silyl protecting group
using TBAF to give the primary alcohol. This material was
phosphorylated, deblocked with TFA, dimerized with phosgene and the
allyl protecting groups removed with phenylsilane and palladium as
described above to give ER-804772.
[0291] Preparation of ER-804947 138
[0292] The alcohol (0.263 g) in THF (5 mL) was added dropwise to
washed 60% sodium hydride (0.216 g) in DMF (2.0 mL) at room
temperature under a nitrogen atmosphere. The reaction mixture was
stirred for 30 minutes after which time benzyl bromide (0.272 mL)
with a catalytic amount (0.05 g) of tetrabutylammonium iodide. The
final reaction mixture was stirred for an additional hour after
which time the mixture was quenched and worked up in the usual
manner to give 0.365 g. 139
[0293] The protected aminoalcohol (0.189 g) was hydrolyzed using 4
N hydrochloric acid (2.5 mL) followed by 40% sodium hydroxide (2.5
mL) as described previously to provide 0.121 g. 140
[0294] The aminoalcohol (0.121 g) was dissolved in methylene
chloride (2 mL) with saturated sodium bicarbonate (2 mL). After
cooling to 0.degree. C., myristoyl chloride (0.199 mL) was added
dropwise. After continued stirring for 2 hours the mixture was
worked up in the usual manner and gave 0.181 g. 141
[0295] The alcohol (0.181 g) was dissolved in methylene chloride (5
mL) with the acid (0.180 g) and
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimi- de (EDC 0.133 g).
The mixture was cooled to 0.degree. C. and 4-dimethylaminopyridine
was added follow by stirring for 16 hours at room temperature. The
usual work up gave 0.310 g. 142
[0296] The para-methoxybenzyl ether (0.305 g) was dissolved in
acetonitrile (8 mL) with water (2 mL) and cooled to 0.degree. C.
Cerium ammonium nitrate (1.110 g) was added and the reaction
mixture was stirred for 2 hours after which time using the normal
work up gave crude alcohol. 143
[0297] The crude alcohol was dissolved in methylene chloride (8 mL)
with lauric acid (0.126 g) and 4-dimethylaminopyridine (0.011 g).
After cooling to 0.degree. C., EDC (0.119 g) was added and the
mixture was stirred at room temperature for 16 hours. The usual
work up gave 0.355 g. 144
[0298] The benzyl ether (0.355 g) was dissolved in ethyl acetate
(50 mL) with palladium hydroxide (0.048 g) and acetic acid (0.25
mL). The reaction mixture was placed under 50 psi of a hydrogen
atmosphere and shaken for 10 hours. Work up in the usual manner
gave 0.255 g.
[0299] This material was phosphorylated, deblocked with TFA,
dimerized with phosgene and the allyl protecting groups removed
with phenylsilane and palladium as described above to give
ER-804947.
[0300] Reaction Scheme for Quarternary Amine Analogue
[0301] Persons familiar with the art can easily envision the
preparation of quaternary amine compounds. As exemplified in the
Scheme below, oxidation of an alcohol to an aldehyde, reductive
animation with the appropriately functionalized amine, followed by
protection of the ensuing secondary amine with a protecting group
such as Fmoc provides the desired protected intermediate. Selective
deprotection of the Boc-group on the primary amine followed by
condensation with the appropriate linker such as phosgene provides
the protected dimer. The final desired product can be produced by
the deprotection of the secondary amine followed by dialkylation of
the amine in the presence of an simple alkyl halide, such as methyl
iodide. The product is purified by cation exchange chromatography
using CM-Sephadex using dilute HCl as the eluting counter ion,
followed by silica gel chromatography, and then anion exchange with
SP-Sephadex containing the appropriate anionic counter ion using
similar elution solvents as described in the previous
experimentals. 145
BIOLOGICAL EXAMPLES
Example 7
Induction of Cytokines (In Vitro)
[0302] E. Assays in Human Whole Blood
[0303] The most readily available human system to test compound
activity on monocytes/macrophages is in whole blood. Various
concentrations of compounds of the invention were added as
10.times. stocks in 50 .mu.l of Ca.sup.++, Mg.sup.++-free Hank's
balanced salt solution (HBSS) followed by 50 .mu.l of HBSS into 400
.mu.l of heparinized whole blood obtained from normal volunteers
(18-51 years old; 110-230 lb.) into the wells of plastic assay
plates, for a total volume of 500 .mu.l/well (final concentration
of whole blood was 80%). After a 3-hour incubation with gentle
shaking at 37.degree. C. in a 5% CO.sub.2 atmosphere, the assay
plates were centrifuged at 1000.times. g for 10 min. at 4.degree.
C. and plasma was drawn off and frozen at -80.degree. C. Plasma
samples were analyzed for TNF-alpha, IL-10, and IL-12 by ELISA
(Genzyme Corp., Cambridge, Mass.). Each assay point was tested in
triplicate.
[0304] As shown in FIG. 1, compounds such as 100, 184 and 186
stimulate blood-borne cells to release TNF-alpha. This stimulatory
activity can be compared to that of 10 ng/ml endotoxin (or LPS)
present in similar incubations in the same assay. As shown in Table
1, activity of compounds (tested at 10 .mu.M) ranges from inactive
(such as compound 110) to compounds demonstrating greater activity
than the LPS standard.
[0305] As shown in FIG. 2, compounds such as 803022, 804057, and
804058 stimulate blood-borne cells to release IL-10. Compounds
803022, 804057, 804058, and 804059 stimulate blood-borne cells to
release IL-12.
[0306] As shown in Table 2, a variety of cytokines are secreted by
nonadherent and adherent peripheral blood mononuclear cells (PBMC)
when treated with compound 804057, including IL1-.alpha.,
IL-1.beta., IL-6, IL-10, IL-12, Interferon-.alpha.,
Interferon-.gamma., GM-CSF, and TNF.alpha..
[0307] B. Cultured Human Cell Lines
[0308] Similar results can be obtained when compounds of the
invention are tested in a cell-culture model. In this assay,
compounds of the invention are tested for their ability to
stimulate secretion of alkaline phosphatase from THP-1 cells that
have been transfected with the gene for secreted alkaline
phosphatase under the control of the TNF-alpha promoter, as
described in detail in Goto et al., Molecular Pharmacology 49;
860-873 (1996). In this assay, however, the effects of removing
serum.sup.1--a condition that may more-likely mimic a subcutaneous
environment--can be evaluated. As shown in FIG. 2 and described in
Table 1, results from these assays indicate that compounds of the
invention stimulate induction of genes under the control of the
TNF-alpha promoter when added to cells in the absence as well as
the presence of serum.
1TABLE 1 Stimulation of cytokine release by compounds in vitro
THP-1 cell Stimulation Whole Blood (% of compound (% of LPS 100 at
10 .mu.M).sup.(1) ER # Compound at 10 .mu.M) +serum -serum MPL
Standard 29.sup.(2) 146 112022 147 131 + 10.2 (n = 6) 111230 148 49
111231 149 17 111232 150 158 155 225 111233 151 141 112043 152 0
112044 153 0 112047 154 0 112048 155 0 24 112049 156 0 112063 157 0
112064 158 50 112065 159 86 112066 160 162 330 112071 161 0 112072
162 0 112091 163 0 112092 164 0 112093 165 0 112098 166 0 112049
167 0 112100 168 0 112859 169 0 112860 170 0 112861 171 0 113634
172 0 113635 173 0 113643 174 0 113644 175 0 113651 176 133 .+-.
4.4 (N = 4) 215 254 113665 177 113666 178 118023 179 63 019772 180
69 118989 181 159 118999 182 105 119000 183 60 119001 184 113
118949 185 138 119327 186 165 .+-. 33 (n = 3) 119328 187 181 .+-.
42 (n = 3) 119329 188 2 .+-. 2 (n = 2) 119521 189 103 119522 190
129 119523 191 176 803022 192 164 803045 193 65 803056 194 151 .+-.
42 803058 195 149 .+-. 37 (n = 2) 803059 196 2 803592 197 15
.sup.1This is important to determine if serum components such as
lipopolysacharide binding protein are necessary for drug activity.
.sup.(1)Response in each assay was compared to 10 .mu.M compound
100 internal standard which typically induced 2-3 fold increase in
TNF-alpha PLAP expression over basal. .sup.(2)Tested at @ 5.8
.mu.M.
[0309]
2TABLE 2 Cytokines Resulting from Stimualtion of Human PBMC by
ER804057 Cytokine Length of Production (pg/mL) Stimulation ER804057
Cytokine Cell population (hrs) Medium (50 nM) IL-1.alpha.
Nonadherent PBMC 24 hrs 4 108 Adherent PBMC 4 hrs 0 8 IL-1.beta.
Nonadherent PBMC 24 hrs 4 431 Adherent PBMC 4 hrs 0 55 IL-6
Adherent PBMC 4 hrs <2 551 IL-10 Adherent PBMC 24 hrs 17 175
IL-12p70 Whole blood 24 hrs 0 1332 Interferon-.alpha. Adherent PBMC
4 hrs 61 345 Adherent PBMC 48 hrs 5 175 Interferon-.gamma.
Nonadherent PBMC 24 hrs 4 331 GM-CSF Nonadherent PBMC 24 hrs 14 353
TNF-.alpha. Adherent PBMC 4 hrs 7 3627
[0310] C. Murine Splenocytes
[0311] The ability of compounds to stimulate cytokine release from
splenocytes can be assessed in a mouse model. Spleen cells
harvested from C57BL/6 mice are cultured for 24 hours in RPMI 1640
cell culture medium containing 5% FBS, 1 mM sodium pyruvate, 2 mM
L-10 glutamine, 100 U/ml penicillin/streptomycin and 50 .mu.M
beta-mercaptoethanol, various concentrations of test compound for
20-24 hours, after which the cell culture supernatant is tested for
the presence of cytokines.
[0312] Spleen cells harvested from mice were cultured for 24 house
with test compound and the supernatant was tested for release of
cytokines. As shown in FIGS. 3 and 4, the release of cytokines such
as IL-10 and interferon-gamma from splenocytes is stimulated by
compounds such as 104, 106, 124, 160, and 162.
[0313] These assays utilized a heterogeneous population of cells
derived from the spleen. This makes it possible that cytokine
induction can be caused both by direct effects of test compounds on
cells and through more indirect stimulation of cytokine "cascades"
where the release of a cytokine by one type of cell can induce
release of other cytokines in other cells present in the same
media. It is possible that this cytokine "milieu" is responsible
for part of this robust immune responses.
[0314] D. Tolerization to TLR Family Ligands
[0315] Preliminary experiments were carried out to determine dosage
ranges for the different ligands so as to assay for comparable
levels of secreted TNF.alpha.. Cells of the mouse macrophage line
RAW 264.7 were plated in RPMI 1640 complete culture medium (10%
fetal bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 100
U/mL penicillin/streptomycin and 50 .mu.M .beta.-mercaptoethanol).
Cells were treated for 24 hours with the TLR4 ligand ER-803022 at
0.1 .mu.g/mL by addition of a concentrated stock solution. Negative
control cells were treated with an equivalent volume of PBS. After
24 hours, cells were washed twice in RPMI 1640 complete medium and
incubated in medium alone for 3 hours. Cultures were then
restimulated with the following synthetic TLR ligands: lipopeptide
(S-lipo, a ligand for TLR2) at 0.1 .mu.g/mL, ER-803022 at 0.1
.mu.g/mL, a mouse CpG oligonucleotide of the sequence
TCCATGACGTTCCTGATGCT (a ligand of murine TLR9) at 0.5 .mu.g/mL, or
PBS. Supernatants were taken at 3 and 4 hours nad cytokine levels
were determined by ELISA. As shown in FIG. 6, mouse macrophage
cells that were treated with an initial exposure to the
immunomodulatory compound ER803022 released less TNF-alpha upon
subsequent exposure to ER803022 compared to those that were not
pretreated. In addition, cells that were pretreated with ER803022
displayed diminished release of TNF-alpha in response to exposure
to the TLR2 ligand S-lipoprotein, and in response to the CpG
oligonucleotide, a TLR9 ligand.
3TABLE 3 WB ED.sub.50 vs. % of LPS at 10 ng/ml WB ED.sub.50 vs ER #
Structure LPS @ 10 ng/ml MPL Standard 198 >>10 .mu.m 112022
199 0.696 .mu.M 111230 200 111231 201 0.29 .mu.M 111232 202 111233
203 112043 204 112044 205 112047 206 112048 207 >>10 .mu.M
112049 208 112063 209 112064 210 112065 211 0.25 .mu.M 112066 212
0.04 .mu.M 112071 213 112072 214 112091 215 112092 216 112093 217
112098 218 112099 219 112100 220 112859 221 112860 222 112861 223
113634 224 113635 225 113643 226 113644 227 113651 228 0.70 .mu.M
113665 229 113666 230 118023 231 019772 232 118989 233 0.1 .mu.M
118999 234 119000 235 119001 236 1.23 .mu.M 118949 237 119327 238
0.015 .mu.M 119328 239 >>10 .mu.M 119329 240 119521 241
119522 242 119523 243 803022 244 0.06 .mu.M 803028 245 803045 246
803056 247 803058 248 0.022 .mu.M 803059 249 0.89 .mu.M 803592 250
803596 251 803597 252 803598 253 803599 254 803613 255 803731 256
>10 .mu.M 803732 257 0.85 .mu.M 803733 258 0.70 .mu.M 803751 259
803783 260 803784 261 803789 262 0.10 .mu.M 804053 263 1.34 .mu.M
804057 264 0.008 .mu.M 804058 265 0.03 .mu.M 804059 266 >10
.mu.M 804061 267 2.5 .mu.M 804097 268 0.3 .mu.M 804121 269 0.46
.mu.M 804130 270 0.66 .mu.M 804221 271 2.2 .mu.M 804222 272 0.008
.mu.M 804252 273 400 nm (576-021) +EtOH 804253 274 >10 .mu.M
804281 275 0.45 .mu.M 804313 276 0.014 .mu.M 804339 277 1.06 .mu.M
804372 278 0.4 .mu.M 804442 279 0.007 .mu.M 804503 280 0.35 .mu.M
804558 281 0.16 .mu.M 804596 282 >10 .mu.M 804674 283 1.2 .mu.M
804678 284 0.018 .mu.M 804679 285 0.53 .mu.M 804680 286 0.015 .mu.M
804732 287 <0.001 .mu.M 804764 288 0.015 .mu.M 804772 289 0.008
.mu.M 804947 290 >>10 .mu.M
[0316] Table 4 below contains the compound number as referenced
herein to the corresponding ER number.
4TABLE 4 Correspondence of Compound Nos. to ER Nos. Compound # ER #
16 112048 31 803058 48 803733 50 803022 62 803789 72 803592 100
112022 102 111230 104 111231 106 111232 108 111233 110 112043 112
112047 114 112047 116 112048 118 112049 120 112063 122 112064 124
112065 126 112066 128 112071 130 112072 132 112091 134 112092 136
112093 138 112098 140 112099 142 112100 146 112859 148 112860 150
112861 152 113634 154 113635 156 113643 158 113644 160 113651 164
113665 166 113666 168 118023 170 019772 172 118989 176 118999 178
119000 180 119001 182 118949 184 119327 186 119328 188 119329 190
119521 192 119522 194 119523 196 803022 198 803045 200 803056 202
803058 204 803059 206 803592
* * * * *