U.S. patent application number 15/633894 was filed with the patent office on 2017-10-12 for plasminogen activator inhibitor-1 inhibitors and methods of use thereof.
The applicant listed for this patent is EASTERN MICHIGAN UNIVERSITY, THE REGENTS OF THE UNIVERSITY OF MICHIGAN. Invention is credited to Gregory Abernathy, Cory Emal, Daniel A. Lawrence, Shih-Hon Li, Ashley Reinke, Mark Warnock.
Application Number | 20170291893 15/633894 |
Document ID | / |
Family ID | 50628040 |
Filed Date | 2017-10-12 |
United States Patent
Application |
20170291893 |
Kind Code |
A1 |
Lawrence; Daniel A. ; et
al. |
October 12, 2017 |
PLASMINOGEN ACTIVATOR INHIBITOR-1 INHIBITORS AND METHODS OF USE
THEREOF
Abstract
The invention relates to plasminogen activator-1 (PAI-1)
inhibitor compounds and uses thereof in the treatment of any
disease or disorder associated with elevated PAI-1. The invention
includes, but is not limited to, the use of such compounds to
prevent or reduce thrombosis and fibrosis, to promote thrombolysis,
and to modulate lipid metabolism and treat diseases or disorders
associated with elevated PAI-1, cholesterol, or lipid levels.
Inventors: |
Lawrence; Daniel A.; (Ann
Arbor, MI) ; Emal; Cory; (Ann Arbor, MI) ;
Reinke; Ashley; (Ann Arbor, MI) ; Li; Shih-Hon;
(Ypsilanti, MI) ; Warnock; Mark; (Brighton,
MI) ; Abernathy; Gregory; (Washington Township,
OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF MICHIGAN
EASTERN MICHIGAN UNIVERSITY |
Ann Arbor
Ypsilanti |
MI
MI |
US
US |
|
|
Family ID: |
50628040 |
Appl. No.: |
15/633894 |
Filed: |
June 27, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14439843 |
Apr 30, 2015 |
9718760 |
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PCT/US13/67695 |
Oct 31, 2013 |
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15633894 |
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61720838 |
Oct 31, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 209/34 20130101;
C07C 237/20 20130101; C07C 311/37 20130101; C07C 69/84 20130101;
C07C 251/76 20130101; C07C 311/48 20130101; C07D 277/34 20130101;
C07C 311/49 20130101; C07D 261/18 20130101; C07D 209/08 20130101;
C07C 233/13 20130101; C07C 323/60 20130101; C07D 417/04 20130101;
C07C 233/56 20130101; C07C 271/16 20130101; C07C 311/29 20130101;
C07C 235/46 20130101; C07D 249/08 20130101; C07C 311/17 20130101;
C07C 259/06 20130101; C07D 271/113 20130101; C07C 243/26 20130101;
C07C 235/34 20130101; C07C 235/80 20130101; C07D 213/42 20130101;
C07C 69/94 20130101; C07D 207/48 20130101; C07D 417/10 20130101;
C07C 275/64 20130101 |
International
Class: |
C07D 417/10 20060101
C07D417/10; C07C 275/64 20060101 C07C275/64; C07C 235/80 20060101
C07C235/80; C07C 237/20 20060101 C07C237/20; C07C 243/26 20060101
C07C243/26; C07D 249/08 20060101 C07D249/08; C07D 277/34 20060101
C07D277/34; C07D 209/34 20060101 C07D209/34; C07C 311/48 20060101
C07C311/48; C07C 311/29 20060101 C07C311/29; C07D 207/48 20060101
C07D207/48; C07D 209/08 20060101 C07D209/08; C07C 311/37 20060101
C07C311/37; C07C 271/16 20060101 C07C271/16; C07C 235/34 20060101
C07C235/34; C07C 323/60 20060101 C07C323/60; C07C 69/94 20060101
C07C069/94; C07D 271/113 20060101 C07D271/113 |
Goverment Interests
STATEMENT OF GOVERNMENT RIGHTS
[0001] This invention was made with government support under
HL.sup.089407 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A compound of formula I or a salt, ester, or prodrug thereof:
##STR00267## wherein: R.sup.1 is selected from the group consisting
of C.sub.1 to C.sub.12 alkyl, -L.sup.1-C.sub.3-C.sub.6 cycloalkyl,
-L.sup.2-C.sub.2-C.sub.6 heterocycloalkyl, benzyl, -L.sup.3-aryl,
and -L.sup.4-heteroaryl; R.sup.2 is selected from the group
consisting of -L.sup.5-C(.dbd.O)R.sup.3, -L.sup.6-R.sup.4, and
NHR.sup.5; R.sup.3 is selected from the group consisting of
OR.sup.6, NR.sup.7R.sup.8, and NHNHR.sup.9; R.sup.5 is selected
from the group consisting of OR.sup.10, C.sub.1 to C.sub.12 alkyl,
-L.sup.7-C.sub.3-C.sub.6 cycloalkyl, -L.sup.8-C.sub.2-C.sub.6
heterocycloalkyl, benzyl, -L.sup.9-aryl, and -L.sup.10-heteroaryl;
R.sup.8 is selected from the group consisting of OR.sup.11,
N.dbd.R.sup.12R.sup.13, -L.sup.11R.sup.14, NHSO.sub.2R.sup.15, and
NHR.sup.16; R.sup.6, R.sup.7, R.sup.9, R.sup.10, R.sup.11,
R.sup.12, R.sup.13, and R.sup.17 are independently selected from
the group consisting of H and C.sub.1 to C.sub.12 alkyl; R.sup.4,
R.sup.14, R.sup.15, R.sup.16 are independently selected from the
group consisting of -L.sup.12-C.sub.3-C.sub.6 cycloalkyl,
-L.sup.13-C.sub.2-C.sub.6 heterocycloalkyl, benzyl, -L.sup.14-aryl,
and -L.sup.15-heteroaryl; and L.sup.1, L.sup.2, L.sup.3, L.sup.4,
L.sup.5, L.sup.6, L.sup.7, L.sup.8, L.sup.9, L.sup.10, L.sup.11,
L.sup.12, L.sup.13, L.sup.14, and L.sup.15 are independently
selected from the group consisting of null, C.sub.1 to C.sub.12
alkylene, and C.sub.1 to C.sub.12 alkenylene.
2. The compound of claim 1 having a formula selected from the group
consisting of II, III, IV, and salts, esters, or prodrugs thereof:
##STR00268##
3. The compound of claim 1 having a formula selected from the group
consisting of V, VI, VII, and salts, esters, or prodrugs thereof:
##STR00269##
4. The compound of claim 1, wherein R.sup.1 is selected from the
group consisting of: ##STR00270## ##STR00271## pentyl, and
butyl.
5. The compound of claim 1, wherein R.sup.4 is selected from the
group consisting of: ##STR00272##
6. The compound of claim 1, wherein R.sup.5 is OH.
7. The compound of claim 1, wherein R.sup.8 is selected from the
group consisting of: ##STR00273##
8. The compound of claim 1 having a formula selected from the group
consisting of C256, C259, C265, C267, C276, C277, C288, C309, C311,
C280, C300, C313, C314, C320, C323, C326, C328, C334, C342, C240,
C241, C246, C248, C251, C255, C260, C261, C262, C263, C264, C266,
C268, C278, C281, C282, C287, C289, C295, C296, C297, C301, C304,
C305, C307, C310, C322, C336, C339, C340, C341, C362, C279, C285,
C286, C299, C306, C330, C344, C345, C346, C347, C348, C356, C357,
C358, C359, C360, C361, C363, C364, C284, and salts, esters, or
prodrugs thereof: ##STR00274## ##STR00275## ##STR00276##
##STR00277## ##STR00278## ##STR00279## ##STR00280## ##STR00281##
##STR00282## ##STR00283## ##STR00284## ##STR00285##
9. A compound of formula VIII or a salt, ester, or prodrug thereof:
##STR00286## wherein: W is C or N; X.sub.1, X.sub.2, X.sub.3,
X.sub.4, and X.sub.5 are independently selected from the group
consisting of --H, --OH, --OR, --F, --Cl, --Br, --I, --NO.sub.2,
--NO, --N(R).sub.2, --N(R).sub.3.sup.+, --C(O)R, --C(O)OR, --CHO,
--C(O)NH.sub.2, --C(O)SR, --CN, --S(O).sub.2R, --SO.sub.3R,
--SO.sub.3H, --SO.sub.2N(R).sub.2, --S.dbd.O, C.sub.1 to C.sub.12
alkyl, aryl, substituted aryl, heteroaryl, and substituted
heteroaryl; R is selected from the group consisting of C.sub.1 to
C.sub.6 alkyl, C.sub.3 to C.sub.6 cycloalkyl,
CH.sub.2--C.sub.3-C.sub.6 cycloalkyl, phenyl, tolyl, and benzyl;
R.sup.a is selected from the group consisting of C.sub.1 to
C.sub.12 alkyl, C.sub.3 to C.sub.6 cycloalkyl,
(CH.sub.2).sub.m--C.sub.3-C.sub.6 cycloalkyl, C.sub.2 to C.sub.6
heterocycloalkyl, (CH.sub.2).sub.m--C.sub.2-C.sub.6
heterocycloalkyl, C.sub.2 to C.sub.6 heterocycloalkyl,
(CH.sub.2).sub.m--C.sub.2-C.sub.6 heterocycloalkyl, benzyl, aryl,
(CH.sub.2).sub.m-aryl, heteroaryl, (CH.sub.2).sub.m-heteroaryl, and
substituted derivatives thereof; and m is 1, 2, 3, 4, 5, or 6; with
the proviso that at most two of X.sub.1, X.sub.2, X.sub.3, X.sub.4,
and X.sub.5 are OH; and excluding a compound of formula
##STR00287##
10. The compound of claim 9, wherein at most three of X.sub.1,
X.sub.2, X.sub.3, X.sub.4, and X.sub.5 are H.
11. The compound of claim 9 having a formula selected from the
group consisting of C152, C155, C173, C189, C191, C197, C224, C292,
C293, C294, and salts, esters, or prodrugs thereof: ##STR00288##
##STR00289## ##STR00290##
12. A compound of formula IX or a salt, ester, or prodrug thereof:
##STR00291## wherein R.sup.a is selected from the group consisting
of aryl, heteroaryl, (CH.sub.2).sub.m-heteroaryl, and substituted
derivatives thereof; and m is 1, 2, 3, 4, 5, or 6.
13. The compound of claim 12, wherein R.sup.a is selected from the
group consisting of: ##STR00292## wherein: W is C or N; and R.sup.1
and R.sup.2 are independently selected from the group consisting of
--H, --F, --Cl, --Br, --I, --CF.sub.3, C.sub.1 to C.sub.12 alkyl,
and phenyl.
14. The compound of claim 12 having a formula selected from the
group consisting of C153, C162, C163, C165, C188, C195, and salts,
esters, or prodrugs thereof: ##STR00293## ##STR00294##
15. A compound having a formula selected from the group consisting
of C157, C158, C182, C183, and salts, esters, or prodrugs thereof:
##STR00295##
16. A compound of formula X or a salt, ester, or prodrug thereof:
##STR00296## wherein R.sup.a is C.sub.1 to C.sub.12 alkyl, R.sup.b
is selected from the group consisting of C.sub.1 to C.sub.12 alkyl,
aryl, heteroaryl, (CH.sub.2).sub.m--R, and ##STR00297## or R.sup.a
and R.sup.b taken together with the N atom to which they are bonded
form an optionally substituted 3-, 4-, 5-, 7-, or 8-membered
heterocyclic ring; m is 1, 2, 3, 4, 5, or 6; n is 0, 1, 2, 3, 4, 5,
or 6; Y is selected from the group consisting of NH.sub.2 and OH;
and R selected from the group consisting of ##STR00298##
substituted phenyl and heteroaryl.
17. The compound of claim 16, wherein R.sup.a is selected from the
group consisting of butyl, pentyl, hexyl, heptyl, octyl, nonyl, and
decyl.
18. The compound of claim 16, wherein R.sup.b is selected from the
group consisting of butyl, pentyl, hexyl, heptyl, octyl, nonyl,
decyl, fluorophenyl, chlorophenyl, bromophenyl, iodophenyl,
trifluoromethylphenyl, and dichlorohydroxyphenyl.
19. The compound of claim 16 having a formula selected from the
group consisting of C170, C171, C172, C175, C177, C179, C180, C186,
C193, C205, and salts, esters, or prodrugs thereof: ##STR00299##
##STR00300## ##STR00301##
20. A compound of formula XI or a salt, ester, or prodrug thereof:
##STR00302## wherein R.sup.b is selected from the group consisting
of aryl, heteroaryl, (CH.sub.2).sub.m--R, and ##STR00303## m is 1,
2, 3, 4, 5, or 6; n is 0, 1, 2, 3, 4, 5, or 6; Y is selected from
the group consisting of NH.sub.2 and OH; and R selected from the
group consisting of ##STR00304## CO.sub.2H, phenyl, substituted
phenyl and heteroaryl.
21. The compound of claim 20 having a formula selected from the
group consisting of C160, C187, C190, C198, C232, C233, C249, C270,
C271, C272, C273, C274, C275, C303, and salts, esters, or prodrugs
thereof: ##STR00305## ##STR00306## ##STR00307##
22. A compound of formula XII or a salt, ester, or prodrug thereof:
##STR00308## wherein R.sup.b is selected from the group consisting
of aryl, heteroaryl, (CH.sub.2).sub.m--R, and ##STR00309## m is 1,
2, 3, 4, 5, or 6; n is 0, 1, 2, 3, 4, 5, or 6; Y is selected from
the group consisting of NH.sub.2 and OH; and R selected from the
group consisting of ##STR00310## CO.sub.2H, phenyl, substituted
phenyl and heteroaryl.
23. The compound of claim 22 having a formula selected from the
group consisting of C210 and salts, esters, or prodrugs thereof:
##STR00311##
24. A compound having a formula selected from the group consisting
of C168, C176, C184, C185, C196, and salts, esters, or prodrugs
thereof: ##STR00312## ##STR00313##
25. A compound having a formula selected from the group consisting
of C156, C161, C200, C204, C236, and salts, esters, or prodrugs
thereof: ##STR00314##
26. A compound of formula XIII or a salt, ester, or prodrug
thereof: ##STR00315## wherein n is 0 or 1; R.sup.a and R.sup.b are
independently selected from the group consisting of C.sub.1 to
C.sub.12 alkyl, C.sub.3 to C.sub.6 cycloalkyl,
(CH.sub.2).sub.m--C.sub.3-C.sub.6 cycloalkyl, C.sub.2 to C.sub.6
heterocycloalkyl, (CH.sub.2).sub.m--C.sub.2-C.sub.6
heterocycloalkyl, C.sub.2 to C.sub.6 heterocycloalkyl,
(CH.sub.2).sub.m--C.sub.2-C.sub.6 heterocycloalkyl, benzyl, aryl,
(CH.sub.2).sub.m-aryl, heteroaryl, (CH.sub.2).sub.m-heteroaryl, and
substituted derivatives thereof; and m is 1, 2, 3, 4, 5, or 6.
27. The compound of claim 26, wherein R.sup.a and R.sup.b are
independently selected from the group consisting of butyl, pentyl,
cyclopropyl, phenyl, difluorophenyl, and hydroxyphenyl.
28. The compound of claim 26 having a formula selected from the
group consisting of C201, C208, C213, C216, C220, C221, C222, C223,
and salts, esters, or prodrugs thereof: ##STR00316##
##STR00317##
29. A compound of formula XIV or a salt, ester, or prodrug thereof:
##STR00318## wherein R is selected from the group consisting of
phenyl and substituted biphenyl.
30. The compound of claim 29 having a formula selected from the
group consisting of C199, C207, and salts, esters, or prodrugs
thereof: ##STR00319##
31. A compound of formula XV or a salt, ester, or prodrug thereof:
##STR00320## wherein: V is selected from the group consisting of
(CH.sub.2).sub.n, C.sub.3 to C.sub.8 cycloalkyl,
(CH.sub.2).sub.n--C.sub.3-C.sub.8 cycloalkyl-(CH.sub.2).sub.p,
aryl, (CH.sub.2).sub.n-aryl-(CH.sub.2).sub.p, heteroaryl,
(CH.sub.2).sub.n-heteroaryl-(CH.sub.2).sub.p, ##STR00321## and
substituted derivatives thereof; n and p are independently 0, 1, 2,
3, 4, 5, or 6; X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5 and
X.sub.6 are independently selected from the group consisting of H,
--OH, --OR, --F, --Cl, --Br, --I, --NO.sub.2, --NO, --N(R).sub.2,
--N(R).sub.3.sup.+, --C(O)R, --C(O)OR, --CHO, --C(O)NH.sub.2,
--C(O)SR, --CN, --S(O).sub.2R, --SO.sub.3R, --SO.sub.3H,
--SO.sub.2N(R).sub.2, --S.dbd.O, C.sub.1 to C.sub.12 alkyl, aryl,
substituted aryl, heteroaryl, and substituted heteroaryl; R is
selected from the group consisting of C.sub.1 to C.sub.6 alkyl,
C.sub.3 to C.sub.6 cycloalkyl, CH.sub.2--C.sub.3-C.sub.6
cycloalkyl, phenyl, tolyl, and benzyl; Y.sub.1 is selected from the
group consisting of O, NH, NR.sup.a, S, and CH.sub.2; Y.sub.2 is
selected from the group consisting of O, NH, NR.sup.b, S, and
CH.sub.2; R.sup.a and R.sup.b are independently selected from the
group consisting of C.sub.1 to C.sub.12 alkyl, C.sub.3 to C.sub.6
cycloalkyl, (CH.sub.2).sub.m--C.sub.3-C.sub.6 cycloalkyl, C.sub.2
to C.sub.6 heterocycloalkyl, (CH.sub.2).sub.m--C.sub.2-C.sub.6
heterocycloalkyl, benzyl, aryl, (CH.sub.2).sub.m-aryl, heteroaryl,
(CH.sub.2).sub.m-heteroaryl, and substituted derivatives thereof; m
is 1, 2, 3, 4, 5, or 6; and Z.sub.1, Z.sub.2, Z.sub.3, and Z.sub.4
are independently selected from the group consisting of C, P--OH,
S, and S.dbd.O.
32. The compound of claim 31 having a formula XVI or a salt, ester,
or prodrug thereof: ##STR00322##
33. The compound of claim 31 having a formula selected from the
group consisting of C225, and salts, esters, or prodrugs thereof:
##STR00323##
34. A compound of formula XVII or a salt, ester, or prodrug
thereof: ##STR00324## wherein: X.sub.1, X.sub.2, X.sub.3, X.sub.4,
X.sub.5 and X.sub.6 are independently selected from the group
consisting of H, --OH, --OR, --F, --Cl, --Br, --I, --NO.sub.2,
--NO, --N(R).sub.2, --N(R).sub.3.sup.+, --C(O)R,--C(O)OR, --CHO,
--C(O)NH.sub.2, --C(O)SR, --CN, --S(O).sub.2R, --SO.sub.3R,
--SO.sub.3H, --SO.sub.2N(R).sub.2, --S.dbd.O, C.sub.1 to C.sub.12
alkyl, aryl, substituted aryl, heteroaryl, and substituted
heteroaryl; R is selected from the group consisting of C.sub.1 to
C.sub.6 alkyl, C.sub.3 to C.sub.6 cycloalkyl,
CH.sub.2--C.sub.3-C.sub.6 cycloalkyl, phenyl, tolyl, and benzyl;
R.sup.b is selected from the group consisting of H, C.sub.1 to
C.sub.12 alkyl, C.sub.3 to C.sub.6 cycloalkyl,
(CH.sub.2).sub.m--C.sub.3-C.sub.6 cycloalkyl, C.sub.2 to C.sub.6
heterocycloalkyl,
(CH.sub.2).sub.m--C.sub.2-C.sub.6heterocycloalkyl, benzyl, aryl,
(CH.sub.2).sub.m-aryl, heteroaryl, (CH.sub.2).sub.m-heteroaryl, and
substituted derivatives thereof; m is 1, 2, 3, 4, 5, or 6; and
Z.sub.4 is selected from the group consisting of C, P--OH, S, and
S.dbd.O.
35. The compound of claim 34 having a formula XVIII or a salt,
ester, or prodrug thereof: ##STR00325##
36. The compound of claim 34, wherein X.sub.1 and X.sub.2 are
independently selected from the group consisting of --OH and
--OR.
37. The compound of claim 34 having a formula selected from the
group consisting of C227, C228, and salts, esters, or prodrugs
thereof: ##STR00326##
38. A compound of formula XIX or a salt, ester, or prodrug thereof:
##STR00327## wherein: X.sub.3, X.sub.4, and X.sub.6 are
independently selected from the group consisting of --H, --OH,
--OR, --F, --Cl, --Br, --I, --NO.sub.2, --NO, --N(R).sub.2,
--N(R).sub.3.sup.+, --C(O)R, --C(O)OR, --CHO, --C(O)NH.sub.2,
--C(O)SR, --CN, --S(O).sub.2R, --SO.sub.3R, --SO.sub.3H,
--SO.sub.2N(R).sub.2, --S.dbd.O, aryl, substituted aryl,
heteroaryl, and substituted heteroaryl; R is selected from the
group consisting of C.sub.1 to C.sub.6 alkyl, C.sub.3 to C.sub.6
cycloalkyl, CH.sub.2--C.sub.3-C.sub.6 cycloalkyl, phenyl, tolyl,
and benzyl; Y.sub.1 is selected from the group consisting of
CHR.sup.aR.sup.b, OR.sup.a, NHR.sup.a, NR.sup.aR.sup.b, and
SR.sup.a; R.sup.a and R.sup.b are independently selected from the
group consisting of C.sub.1 to C.sub.12 alkyl, C.sub.3 to C.sub.6
cycloalkyl, (CH.sub.2).sub.m--C.sub.3-C.sub.6 cycloalkyl, C.sub.2
to C.sub.6 heterocycloalkyl, (CH.sub.2).sub.m--C.sub.2-C.sub.6
heterocycloalkyl, benzyl, aryl, (CH.sub.2).sub.m-aryl, heteroaryl,
(CH.sub.2).sub.m-heteroaryl, --U, (CH.sub.2).sub.m--U, and
substituted derivatives thereof, or R.sup.a and R.sup.b taken
together with the N atom to which they are bonded form a 3- to
8-membered heterocyclic ring; U is selected from the group
consisting of --NR.sup.cR.sup.d, --NR.sup.cC(O)R.sup.e,
--NR.sup.cC(O)OR.sup.e, --NR.sup.cC(O)NR.sup.fR.sup.e,
--NR.sup.cC(O)SR.sup.e, --NR.sup.cP(O)(OH)R.sup.e,
--NR.sup.cP(O)(OH)OR.sup.e, --NR.sup.cP(O)(OH)NR.sup.fR.sup.c,
--NR.sup.cP(O)(OH)SR.sup.c, --NR.sup.cS(O)R.sup.c,
--NR.sup.cS(O)OR.sup.c, --NR.sup.cS(O)NR.sup.fR.sup.e,
--NR.sup.cS(O)SR.sup.e, --NR.sup.cS(O).sub.2R.sup.e,
--NR.sup.cS(O).sub.2OR.sup.e, --NR.sup.cS(O).sub.2NR.sup.fR.sup.e,
--NR.sup.cS(O).sub.2SR.sup.e, --OR.sup.f, --OC(O)R.sup.e,
--OC(O)OR.sup.e, --OC(O)NR.sup.fR.sup.e, --OC(O)SR.sup.e,
--OP(O)(OH)R.sup.c, --OP(O)(OH)OR.sup.c,
--OP(O)(OH)NR.sup.dR.sup.c, --OP(O)(OH)SR.sup.c, --OS(O)R.sup.c,
--OS(O)OR.sup.e, --OS(O)NR.sup.dR.sup.e, --OS(O)SR.sup.e,
--OS(O).sub.2R.sup.e, --OS(O) .sub.2OR.sup.e,
--OS(O).sub.2NR.sup.dR.sup.e, --OS(O).sub.2SR, --C(O)OR.sup.c,
--C(O)NR.sup.cR.sup.d, --C(O)SR.sup.c, and --C(O)R.sup.c; R.sup.c
and R.sup.d are independently selected from the group consisting of
H, C.sub.1 to C.sub.12 alkyl, C.sub.1 to C.sub.12 haloalkyl,
C.sub.3 to C.sub.6 cycloalkyl, (CH.sub.2).sub.m--C.sub.3-C.sub.6
cycloalkyl, C.sub.2 to C.sub.6 heterocycloalkyl,
(CH.sub.2).sub.m--C.sub.2-C.sub.6heterocycloalkyl, benzyl, aryl,
(CH.sub.2).sub.m-aryl, heteroaryl, (CH.sub.2).sub.m-heteroaryl, and
substituted derivatives thereof, or R.sup.c and R.sup.d taken
together with the N atom to which they are bonded form a 3- to
8-membered heterocyclic ring; R.sup.e, R.sup.f, and R.sup.g are
independently selected from the group consisting of H, C.sub.1 to
C.sub.12 alkyl, C.sub.1 to C.sub.12 haloalkyl, C.sub.3 to C.sub.6
cycloalkyl, (CH.sub.2).sub.m--C.sub.3-C.sub.6 cycloalkyl, C.sub.2
to C.sub.6 heterocycloalkyl,
(CH.sub.2).sub.m--C.sub.2-C.sub.6heterocycloalkyl, benzyl, aryl,
(CH.sub.2).sub.m-aryl, heteroaryl, (CH.sub.2).sub.m-heteroaryl, and
substituted derivatives thereof; m is 1, 2, 3, 4, 5, or 6; and
Z.sub.1 and Z.sub.4 are independently selected from the group
consisting of C, P--OH, S, and S.dbd.O.
39. The compound of claim 38 having a formula selected from the
group consisting of C229, and salts, esters, or prodrugs thereof:
##STR00328##
40. A composition comprising an isolated compound according to any
one of Formulas I to XIX, C256, C259, C265, C267, C276, C277, C288,
C309, C311, C280, C300, C313, C314, C320, C323, C326, C328, C334,
C342, C240, C241, C246, C248, C251, C255, C260, C261, C262, C263,
C264, C266, C268, C278, C281, C282, C287, C289, C295, C296, C297,
C301, C304, C305, C307, C310, C322, C336, C339, C340, C341, C362,
C279, C285, C286, C299, C306, C330, C344, C345, C346, C347, C348,
C356, C357, C358, C359, C360, C361, C363, C364, C284, C152, C155,
C173, C189, C191, C197, C224, C292, C293, C294, C153, C162, C163,
C165, C188, C195, C157, C158, C182, C183, C170, C171, C172, C175,
C177, C179, C180, C186, C193, C205, C160, C187, C190, C198, C232,
C233, C249, C270, C271, C272, C273, C274, C275, C303, C210, C168,
C176, C184, C185, C196, C156, C161, C200, C204, C236, C201, C208,
C213, C216, C220, C221, C222, C223, C199, C207, C225, C227, C228,
or C229, or a salt, ester, or prodrug thereof, wherein said
compound is present in an amount effective to inhibit PAI-1.
41. A composition comprising the compound according to any one of
Formulas I to XIX, C256, C259, C265, C267, C276, C277, C288, C309,
C311, C280, C300, C313, C314, C320, C323, C326, C328, C334, C342,
C240, C241, C246, C248, C251, C255, C260, C261, C262, C263, C264,
C266, C268, C278, C281, C282, C287, C289, C295, C296, C297, C301,
C304, C305, C307, C310, C322, C336, C339, C340, C341, C362, C279,
C285, C286, C299, C306, C330, C344, C345, C346, C347, C348, C356,
C357, C358, C359, C360, C361, C363, C364, C284, C152, C155, C173,
C189, C191, C197, C224, C292, C293, C294, C153, C162, C163, C165,
C188, C195, C157, C158, C182, C183, C170, C171, C172, C175, C177,
C179, C180, C186, C193, C205, C160, C187, C190, C198, C232, C233,
C249, C270, C271, C272, C273, C274, C275, C303, C210, C168, C176,
C184, C185, C196, C156, C161, C200, C204, C236, C201, C208, C213,
C216, C220, C221, C222, C223, C199, C207, C225, C227, C228, or
C229, or a salt, ester, or prodrug thereof and a pharmaceutically
acceptable carrier.
42. A method of treating or preventing a disease or disorder
associated with an elevated level of PAI-1 in a subject comprising
administering an effective amount of a composition comprising the
compound according to any one of Formulas I to XXIX, C256, C259,
C265, C267, C276, C277, C288, C309, C311, C280, C300, C313, C314,
C320, C323, C326, C328, C334, C342, C240, C241, C246, C248, C251,
C255, C260, C261, C262, C263, C264, C266, C268, C278, C281, C282,
C287, C289, C295, C296, C297, C301, C304, C305, C307, C310, C322,
C336, C339, C340, C341, C362, C279, C285, C286, C299, C306, C330,
C344, C345, C346, C347, C348, C356, C357, C358, C359, C360, C361,
C363, C364, C284, C152, C155, C173, C189, C191, C197, C224, C292,
C293, C294, C153, C162, C163, C165, C188, C195, C157, C158, C182,
C183, C170, C171, C172, C175, C177, C179, C180, C186, C193, C205,
C160, C187, C190, C198, C232, C233, C249, C270, C271, C272, C273,
C274, C275, C303, C210, C168, C176, C184, C185, C196, C156, C161,
C200, C204, C236, C201, C208, C213, C216, C220, C221, C222, C223,
C199, C207, C225, C227, C228, or C229, or a salt, ester, or prodrug
thereof and a pharmaceutically acceptable carrier to decrease the
elevated level of PAI-1 in the subject.
43. The method of claim 42, wherein the disease or disorder is
cancer, septicemia, obesity, insulin resistance, a disease or
disorder associated with dysregulation of lipid metabolism, a
disease or disorder associated with an elevated level of VLDL or
LDL, high cholesterol, a proliferative disease or disorder,
fibrosis and fibrotic disease, coagulation homeostasis,
cerebrovascular disease, microvascular disease, hypertension,
dementia, atherosclerosis, osteoporosis, osteopenia, arthritis,
asthma, heart failure, arrhythmia, angina, hormone insufficiency,
Alzheimer's disease, hypertension, inflammation, sepsis,
fibrinolytic disorder, stroke, dementia, coronary heart disease,
myocardial infarction, stable and unstable angina, vascular
disease, peripheral arterial disease, acute vascular syndrome,
thrombosis, prothrombosis, deep vein thrombosis, pulmonary
embolism, cerebrovascular disease, microvascular disease,
hypertension, diabetes, hyperglycemia, hyperinsulinemia, malignant
lesions, premalignant lesions, gastrointestinal malignancies,
liposarcoma, epithelial tumor, and psoriasis, an extracellular
matrix accumulation disorder, neoangiogenesis, myelofibrosis,
fibrinolytic impairment, polycystic ovary syndrome, bone loss
induced by estrogen deficiency, angiogenesis, neoangiogenesis,
myelofibrosis, or fibrinolytic impairment.
44. The method of claim 43, wherein the disease or disorder
involving thrombosis or prothrombosis is formation of
atherosclerotic plaques, venous thrombosis, arterial thrombosis,
myocardial ischemia, atrial fibrillation, deep vein thrombosis, a
coagulation syndrome, pulmonary thrombosis, cerebral thrombosis, a
thromboembolic complication of surgery, and peripheral arterial
occlusion.
45. The method of claim 43, wherein the disease or disorder is
fibrosis.
46. The method of claim 43, wherein the extracellular matrix
accumulation disorder is renal fibrosis, chronic obstructive
pulmonary disease, polycystic ovary syndrome, restenosis,
renovascular disease, diabetic nephropathy, or organ transplant
rejection.
47. A method of modulating cholesterol, lipid clearance, and/or
lipid uptake in a subject with an elevated level of PAI-1
comprising administering an effective amount of a composition
comprising the compound according to any one of Formulas Ito XXIX,
C256, C259, C265, C267, C276, C277, C288, C309, C311, C280, C300,
C313, C314, C320, C323, C326, C328, C334, C342, C240, C241, C246,
C248, C251, C255, C260, C261, C262, C263, C264, C266, C268, C278,
C281, C282, C287, C289, C295, C296, C297, C301, C304, C305, C307,
C310, C322, C336, C339, C340, C341, C362, C279, C285, C286, C299,
C306, C330, C344, C345, C346, C347, C348, C356, C357, C358, C359,
C360, C361, C363, C364, C284, C152, C155, C173, C189, C191, C197,
C224, C292, C293, C294, C153, C162, C163, C165, C188, C195, C157,
C158, C182, C183, C170, C171, C172, C175, C177, C179, C180, C186,
C193, C205, C160, C187, C190, C198, C232, C233, C249, C270, C271,
C272, C273, C274, C275, C303, C210, C168, C176, C184, C185, C196,
C156, C161, C200, C204, C236, C201, C208, C213, C216, C220, C221,
C222, C223, C199, C207, C225, C227, C228, or C229, or a salt,
ester, or prodrug thereof and a pharmaceutically acceptable carrier
in an amount effective to decrease the elevated level of PAI and
modulate cholesterol, lipid clearance, and/or lipid uptake in the
subject.
48. The method of claim 47, wherein the composition increases
circulating high density lipoprotein (HDL) and/or decreases
circulating very low density lipoprotein (VLDL) in the subject.
49. The method of claim 47, wherein the composition inhibits
apolipoprotein E (ApoE) or apolipoprotein A (ApoA) binding to
VLDL-R.
50. The method of claim 47, wherein the composition affects HDL or
apolipoprotein E (ApoE) or apolipoprotein A (ApoA) binding to an
ApoA receptor.
51. The method of claim 47, wherein the composition decreases PAI-1
binding to apolipoprotein E (ApoE).
52. The method of claim 47, wherein the composition decreases PAI-1
binding to apolipoprotein A (ApoA).
53. The method of claim 47, wherein the composition decreases PAI-1
binding to VLDL.
54. The method of claim 47, wherein the composition binds to PAI-1
in the presence of vitronectin.
55. The method of claim 47, wherein the composition binds to PAI-1
in the presence of urokinase type plasminogen activator (uPA).
56. The method of any one of claims 42-55 wherein the subject is
human.
Description
FIELD OF THE INVENTION
[0002] The invention generally relates to methods and compositions
for modulating plasminogen activator inhibitor-1 (PAI-1) activity.
More particularly, the invention is directed to methods of
identifying inhibitors of PAI-1 and the uses of such inhibitors in
regulating PAI-1 activity. The invention also relates to uses of
these inhibitors for the treatment of many diseases or disorders
associated with PAI-1 activity. Such diseases or disorders include,
but are not limited to, dysregulation of lipid metabolism, obesity,
diabetes, polycystic ovary syndrome, bone loss induced by estrogen
deficiency, fibrosis and fibrotic disease, inflammation, cell
migration and migration-driven proliferation of cells,
angiogenesis, and thrombosis. Such inhibitors are also contemplated
to be useful for modulation of endogenous fibrinolysis, and in
conjunction with pharmacologic thrombolysis.
BACKGROUND OF THE INVENTION
[0003] Plasminogen activator inhibitor-1 (PAI-1) is a 50 kDa
single-chain glycoprotein that is the principal inhibitor of both
urokinase type plasminogen activator (uPA) and tissue type PA
(tPA). PAI-1 inhibits tPA and uPA with second-order rate constants
.about.10.sup.7 M.sup.-1s.sup.-1, a value that is 10-1000 times
faster than the rates of PA inhibition by other PAIs. Moreover,
approximately 70% of the total tPA in carefully collected normal
human plasma is detected in complex with PAI-1, suggesting that
inhibition of tPA by PAI-1 is a normal, ongoing process. PAI-1 can
also directly inhibit plasmin. Thus, PAI-1 is the chief regulator
of plasmin generation in vivo, and as such it appears to play an
important role in both fibrotic and thrombotic disease. PAI-1 has
three potential N-linked glycosylation sites and contains between
15 and 20% carbohydrate.
[0004] PAI-1 belongs to the Serine Protease Inhibitor super family
(SERPIN), which is a gene family that includes many of the protease
inhibitors found in blood, as well as other proteins with unrelated
or unknown functions. Serpins are consumed in the process of
protease inactivation and thus act as "suicide inhibitors." The
association between a serpin and its target protease occurs at an
amino acid residue, referred to as the "bait" residue, located on a
surface loop of the serpin called the reactive center loop (RCL).
The "bait" residue is also called the P1 residue, and is thought to
mimic the normal substrate of the enzyme. Upon association of the
P1 residue with the S1 site of a target protease, cleavage of the
RCL occurs. This is coupled to a large conformational change in the
serpin which involves rapid insertion of the RCL into the major
structural feature of a serpin, (.beta.-sheet A. This results in
tight docking of the protease to the serpin surface and to
distortion of the enzyme structure, including its active site. RCL
insertion also produces a large increase in serpin structural
stability making the complex rigid and thus trapping the protease
in a covalent acyl-enzyme complex with the serpin.
[0005] Native PAI-1 exists in at least two distinct conformations,
an active form that is produced by cells and secreted, and an
inactive or latent form that accumulates in cell culture medium
over time. In blood and tissues, most of the PAI-1 is in the active
form; however, in platelets both active and latent forms of PAI-1
are found. In active PAI-1, the RCL is exposed on the surface of
the molecule, but upon reaction with a protease, the cleaved RCL
integrates into the center of .beta. sheet A. In the latent form,
the RCL is intact, but instead of being exposed, the entire amino
terminal side of the RCL is inserted as the central strand into the
.beta. sheet A. This accounts for the increased stability of latent
PAI-1 as well as its lack of inhibitory activity.
[0006] Active PAI-1 spontaneously converts to the latent form with
a half-life of one to two hours at 37.degree. C., and latent PAI-1
can be converted back into the active form by treatment with
denaturants. Negatively charged phospholipids can also convert
latent PAI-1 to the active form, suggesting that cell surfaces may
modulate PAI-1 activity. The observation that latent PAI-1 infused
into rabbits is apparently converted to the active form is
consistent with this hypothesis. The spontaneous reversible
interconversion between the active and latent structures is unique
for PAI-1 and distinguishes it from other serpins; however, the
biological significance of the latent conformation remains
unknown.
[0007] Other non-inhibitory forms of PAI-1 have also been
identified. The first form results from oxidation of one or more
critical methionine residues within active PAI-1. This form differs
from latent PAI-1 in that it can be partially reactivated by an
enzyme that specifically reduces oxidized methionine residues.
Oxidative inactivation of PAI-1 may be an additional mechanism for
the regulation of PAI-1, and oxygen radicals produced locally by
neutrophils or other cells may inactivate PAI-1 and thus facilitate
the generation of plasmin at sites of infection or in areas of
tissue remodeling. PAI-1 also exists in two different cleaved
forms. As noted above, PAI-1 in complex with a protease is cleaved
in its RCL. Uncomplexed PAI-1 can also be found with its RCL
cleaved, which can arise from dissociation of PAI-1-PA complexes or
from cleavage of the RCL by a non-target protease at a site other
than the P1. None of these forms of PAI-1 are able to inhibit
protease activity; however, they may interact with other
ligands.
[0008] The interaction of PAI-1 with non-protease ligands plays an
essential role in PAI-1 function. PAI-1 binds with high affinity to
heparin, the cell adhesion protein vitronectin, and members the
endocytic low-density lipoprotein receptor (LDL-R) family, such as
the lipoprotein receptor-related protein (LRP), and the very low
density lipoprotein receptor (VLDL-R). These non-protease
interactions are important for both PAI-1 localization and
function, and they are largely conformationally controlled through
structural changes associated with RCL insertion. In blood, most of
the active PAI-1 circulates in complex with the glycoprotein
vitronectin. The PAI-1 binding site for vitronectin has been
localized to a region on the edge of .beta.-sheet A in the PAI-1
structure. The binding site for LDL-R family members is less well
characterized, but has been identified, in a region of PAI-1
associated with alpha helix D that is adjacent to the vitronectin
binding domain. The heparin binding domain on PAI-1 has also been
mapped. This site also localizes to alpha helix D in a region
homologous to the heparin binding domain of antithrombin III, and
may overlap with the binding site for LDL-R family members.
[0009] Vitronectin circulates in plasma and is present in the
extracellular matrix primarily at sites of injury or remodeling.
PAI-1 and vitronectin appear to have a significant functional
interdependence. Vitronectin stabilizes PAI-1 in its active
conformation, thereby increasing its biological half-life.
[0010] Vitronectin also enhances PAI-1 inhibitory efficiency for
thrombin approximately 300-fold. In turn, PAI-1 binding to
vitronectin alters its conformation from the native plasma form,
which does not support cell adhesion, to an "activated" form that
is competent to bind integrins. However, integrin binding is
blocked by the presence of PAI-1. As noted above, the association
of PAI-1 with vitronectin is conformationally controlled and upon
inhibition of a protease, the conformational change in PAI-1
associated with RCL insertion results in a loss of high affinity
for vitronectin and a gain in affinity for LDL-R family members.
This is due to RCL insertion in PAI-1, disrupting the vitronectin
binding site, while simultaneously exposing a cryptic receptor
binding site that is revealed only when PAI-1 is in a complex with
a protease, which results in an approximately 100,000-fold shift in
the relative affinity of PAI-1 from vitronectin to LDL-R family
members and a subsequent shift in PAI-1 localization from
vitronectin to the cellular receptor. Thus, PAI-1 association with
vitronectin and LDL-R is conformationally controlled.
[0011] High PAI-1 levels are associated with various diseases and
disorders. For example, high PAI-1 levels are associated with acute
diseases, such as sepsis and myocardial infarction, and chronic
disorders, such as cancer, atherosclerosis, and type 2 diabetes. In
addition, high PAI-1 levels are associated with cardiovascular
disease, wherein PAI-1 expression is significantly increased in
severely atherosclerotic vessels, and PAI-1 protein levels rise
consistently during disease progression from normal vessels to
fatty streaks to atherosclerotic plaques. Increased PAI-1 levels
are also linked to obesity, and insulin resistance.
[0012] In addition, elevated plasma levels of PAI-1 have been
associated with thrombotic events, and antibody neutralization of
PAI-1 activity resulted in promotion of endogenous thrombolysis and
reperfusion. Elevated levels of PAI-1 have also been implicated in
polycystic ovary syndrome and bone loss induced by estrogen
deficiency.
[0013] PAI-1 is synthesized in both murine and human adipocytes.
There is also a strong correlation between the amount of visceral
fat and plasma levels of PAI-1 in humans and mice. This dramatic
up-regulation of PAI-1 in obesity has lead to the suggestion that
adipose tissue itself may directly contribute to elevated systemic
PAI-1, which in-turn increases the probability of vascular disease
through increased thrombosis, and accelerated atherosclerosis.
Notably, very recent data suggests that PAI-1 may also play a
direct role in obesity.
[0014] In one study, genetically obese and diabetic ob/ob mice
crossed into a PAI-1 deficient background had significantly reduced
body weight and improved metabolic profiles compared to ob/ob mice
with PAI-1. Likewise, nutritionally-induced obesity and insulin
resistance were dramatically attenuated in mice genetically
deficient in PAI-1 and in mice treated with an orally active PAI-1
inhibitor. The improved adiposity and insulin resistance in
(PAI-1)-deficient mice may be related to the observation that PAI-1
deficient mice on a high fat diet had increased metabolic rates and
total energy expenditure compared to wild-type mice, and peroxysome
proliferator-activated receptor (PPAR.gamma.) and adiponectin were
maintained. However, the precise mechanism involved was not shown
and may be complex, since the over-expression of PAI-1 in mice also
impaired adipose tissue formation. Taken together, these
observations suggest that PAI-1 plays a previously unrecognized
direct role in obesity and insulin resistance that involves
interactions beyond its identified activities of modulating
fibrinolysis and tissue remodeling.
[0015] Indeed, if PAI-1 positively regulates adipose tissue
development, then the association of increased PAI-1 expression
with developing obesity may constitute a positive feedback loop
promoting adipose tissue expansion and dysregulation of normal
cholesterol homeostasis. Thus, there exists a need in the art for a
greater understanding of how PAI-1 is involved in metabolism,
obesity and insulin resistance. The invention provides methods of
identifying and using inhibitors of PAI-1.
SUMMARY OF THE INVENTION
[0016] The invention provides plasminogen activator inhibitor-1
(PAI-1) inhibitors and uses thereof in the treatment of any disease
or disorder associated with elevated PAI-1 levels in a subject.
Such uses include, but are not limited to, the treatment of many
diseases or disorders associated with elevated PAI-1 levels or
activity as discussed herein below. The invention further provides
compositions comprising isolated PAI-1 inhibitors and a
pharmaceutically acceptable carrier, wherein the PAI-1 inhibitors
are present in an amount effective to inhibit PAI-1.
[0017] Such PAI-1 inhibitors include, but are not limited to, any
of the compounds of Formulas Ito XXIX or any of the compounds
depicted in Tables 1, 3, 5, 7, 9, 11, 12, and 14, including C256,
C259, C265, C267, C276, C277, C288, C309, C311, C280, C300, C313,
C314, C320, C323, C326, C328, C334, C342, C240, C241, C246, C248,
C251, C255, C260, C261, C262, C263, C264, C266, C268, C278, C281,
C282, C287, C289, C295, C296, C297, C301, C304, C305, C307, C310,
C322, C336, C339, C340, C341, C362, C279, C285, C286, C299, C306,
C330, C344, C345, C346, C347, C348, C356, C357, C358, C359, C360,
C361, C363, C364, C284, C152, C155, C173, C189, C191, C197, C224,
C292, C293, C294, C153, C162, C163, C165, C188, C195, C157, C158,
C182, C183, C170, C171, C172, C175, C177, C179, C180, C186, C193,
C205, C160, C187, C190, C198, C232, C233, C249, C270, C271, C272,
C273, C274, C275, C303, C210, C168, C176, C184, C185, C196, C156,
C161, C200, C204, C236, C201, C208, C213, C216, C220, C221, C222,
C223, C199, C207, C225, C227, C228, and C229 as set out herein.
[0018] In a further embodiment, methods of treating or preventing a
disease or disorder associated with elevated levels of PAI-1 or
elevated PAI-1 activity are provided. The methods comprise
administering a PAI-1 inhibitor to the subject in an amount
effective to treat the disease or disorder. In one aspect, the
disease or disorder includes, but is not limited to, cancer,
septicemia, a disorder associated with a dysregulation of lipid
metabolism, a proliferative disease or disorder, psoriasis,
fibrosis and fibrotic disease, coagulation homeostasis,
cerebrovascular disease, vascular disease, microvascular disease,
hypertension, dementia, atherosclerosis, osteoporosis, osteopenia,
arthritis, asthma, heart failure, arrhythmia, angina, hormone
insufficiency, Alzheimer's disease, inflammation, sepsis,
fibrinolytic disorder, stroke, dementia, coronary heart disease,
myocardial infarction, stable and unstable angina, peripheral
arterial disease, acute vascular syndrome, thrombosis,
prothrombosis, pulmonary embolism, insulin resistance, non-insulin
dependent diabetes mellitus, Type 1 and 2 diabetes and related
diabetic diseases, obesity, hyperglycemia, hyperinsulinemia,
malignant lesions, premalignant lesions, gastrointestinal
malignancies, liposarcoma, epithelial tumor, an extracellular
matrix accumulation disorder, neoangiogenesis, myelofibrosis,
fibrinolytic impairment, polycystic ovary syndrome, bone loss
induced by estrogen deficiency, angiogenesis, neoangiogenesis,
myelofibrosis, or fibrinolytic impairment.
[0019] In some aspects, the disease or disorder involving
thrombosis or prothrombosis includes, but is not limited to,
formation of atherosclerotic plaques, venous and/or arterial
thrombosis, deep vein thrombosis, arterial thrombosis, myocardial
ischemia, atrial fibrillation, deep vein thrombosis, a coagulation
syndrome, pulmonary thrombosis, cerebral thrombosis, a
thromboembolic complication of surgery, and peripheral arterial
occlusion.
[0020] In some aspects, the disease or disorder involving
microvascular disease includes, but is not limited to, nephropathy,
neuropathy, retinopathy and nephrotic syndrome.
[0021] In some aspects, the disease or disorder involving fibrosis
or an extracellular matrix accumulation includes, but is not
limited to, renal fibrosis, chronic obstructive pulmonary disease,
polycystic ovary syndrome, restenosis, renovascular disease,
diabetic nephropathy, or organ transplant rejection.
[0022] In some aspects, the disease or disorder involving
dysregulation of lipid metabolism includes, but is not limited to,
high cholesterol, elevated triglycerides, elevated levels of VLDL
or LDL, and low levels of HDL. In various aspects, therefore, the
PAI-1 inhibitor compounds of the invention are used in methods of
modulating cholesterol and/or lipid uptake and/or lipid clearance.
In some aspects, the PAI-1 inhibitor compounds decrease PAI-1
binding to ApoE, ApoA, VLDL, VLDL-R, ApoA-R, or LDL. In yet another
aspect, the PAI-1 inhibitor compounds bind to PAI-1 in the presence
of vitronectin and/or uPA. In one aspect, the PAI-1 inhibitor is
administered to a subject in an amount effective to inhibit VLDL or
ApoE or ApoA binding to VLDL-R. In one aspect, the PAI-1 inhibitor
is administered to a subject in an amount effective to affect HDL
or ApoE or ApoA binding to an ApoA receptor. In particular aspects,
the PAI-1 inhibitor is used to increase HDL and/or decrease VLDL in
a subject.
[0023] In another embodiment, the PAI-1 inhibitor compounds of the
invention are useful for modulation of endogenous fibrinolysis and
for use in pharmacologic thrombolysis.
[0024] In some aspects, the subject is human.
[0025] Uses of compounds of the invention for the production of a
medicament for the treatment or prevention of any disease or
disorder discussed herein are also provided. The compounds of the
invention are inhibitors of the serine protease inhibitor PAI-1,
and are therefore useful in the treatment or prophylaxis of those
processes which involve the production and/or action of PAI-1.
[0026] The foregoing summary is not intended to define every aspect
of the invention, and additional aspects are described in other
sections, such as the Detailed Description. The entire document is
intended to be related as a unified disclosure, and it should be
understood that all combinations of features described herein are
contemplated, even if the combination of features are not found
together in the same sentence, or paragraph, or section of this
document.
[0027] In addition to the foregoing, the invention includes, as an
additional aspect, all embodiments of the invention narrower in
scope in any way than the variations specifically mentioned above.
With respect to aspects of the invention described as a genus, all
individual species are individually considered separate aspects of
the invention. Additional features and variations of the invention
will be apparent to those skilled in the art from the entirety of
this application, and all such features are intended as aspects of
the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The invention describes materials and methods for the
inhibition of plasminogen activator inhibitor-1 (PAI-1). In
exemplary aspects, the invention describes PAI-1 inhibitor
compounds.
[0029] PAI-1 Inhibitor Compounds of the Invention
[0030] As used herein, the term "haloalkyl" refers to a hydrocarbon
group substituted with one or more halogens selected from F, Cl,
Br, and I.
[0031] As used herein, the term "cycloalkyl" refers to a cyclic
hydrocarbon group, e.g., cyclopropyl, cyclobutyl, cyclohexyl,
cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. As used
herein, the term "heterocycloalkyl" or "heterocyclic ring" refers
to a cyclic hydrocarbon group having one or more heteroatoms, for
example, one to three heteroatoms, independently selected from the
group consisting of oxygen, nitrogen, and sulfur.
[0032] As used herein, the term "aryl" refers to a monocyclic or
polycyclic aromatic group, preferably a monocyclic or bicyclic
aromatic group. Unless otherwise indicated, an aryl group can be
unsubstituted or substituted with one or more, and in particular
one to four, groups independently selected from, for example, --OH,
--OR (including --OCH.sub.3), --F, --Cl, --Br, --I, --CF.sub.3,
--NO.sub.2, --NO, --N(R).sub.2, --N(R).sub.3.sup.+, --C(O)R,
--C(O)OR, --CHO, --C(O)NH.sub.2, --C(O)SR, --CN, --S(O).sub.2R,
--SO.sub.3R, --SO.sub.3H, --SO.sub.2N(R).sub.2, --S.dbd.O, aryl,
and heteroaryl. Exemplary aryl groups include, but are not limited
to, phenyl, tolyl, naphthyl, tetrahydronaphthyl, chlorophenyl,
methylphenyl, methoxyphenyl, trifluoromethylphenyl, nitrophenyl,
2,4-methoxychlorophenyl, and the like.
[0033] As used herein, the term "heteroaryl" refers to a monocyclic
or polycyclic ring system containing one or more aromatic rings and
containing at least one nitrogen, oxygen, or sulfur atom in an
aromatic ring. Unless otherwise indicated, a heteroaryl group can
be unsubstituted or substituted with one or more, and in particular
one to four, substituents selected from, for example, --OH, --OR
(including --OCH.sub.3), --F, --Cl, --Br, --I, --CF.sub.3,
--NO.sub.2, --NO, --N(R).sub.2, --N(R).sub.3.sup.+, --C(O)R,
--C(O)OR, --CHO, --C(O)NH.sub.2, --C(O)SR, --CN, --S(O).sub.2R,
--SO.sub.3R, --SO.sub.3H, --SO.sub.2N(R).sub.2, --S.dbd.O, aryl,
and heteroaryl. Examples of heteroaryl groups include, but are not
limited to, thienyl, furyl, pyridyl, oxazolyl, quinolyl,
thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl,
isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl,
pyrimidinyl, thiazolyl, and thiadiazolyl.
[0034] As used herein, the term "substituted benzyl" refers to a
benzyl group substituted with one or more, and in particular one to
four, groups independently selected from, for example, --OH, --OR
(including --OCH.sub.3), --F, --Cl, --Br, --I, --CF.sub.3,
--NO.sub.2, --NO, --N(R).sub.2, --N(R).sub.3.sup.+, --C(O)R,
--C(O)OR, --CHO, --C(O)NH.sub.2, --C(O)SR, --CN, --S(O).sub.2R,
--SO.sub.3R, --SO.sub.3H, --SO.sub.2N(R).sub.2, --S.dbd.O, aryl,
and heteroaryl.
[0035] As used herein, the term "amino acid" refers to naturally
occurring and non-natural amino acids, as well as amino acid
analogs. Naturally encoded amino acids include the 20 common amino
acids (alanine, arginine, asparagine, aspartic acid, cysteine,
glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, proline, serine, threonine,
tryptophan, tyrosine, and valine), pyrrolysine, and selenocysteine.
Amino acid analogs refers to compounds that have the same basic
chemical structure as a naturally occurring amino acid, i.e., a
carbon that is bound to a hydrogen, a carboxyl group, an amino
group, and an R group, for example, 3-nitrotyrosine, homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (such as 3-nitrotyrosine) or
modified peptide backbones, but retain the same basic chemical
structure as a naturally occurring amino acid. Amino acid analogs
also include amino acid esters (e.g., amino acid alkyl esters, such
as amino acid methyl esters) and acylated amino acids (e.g.,
acetylated amino acids).
[0036] It will also be appreciated that certain of the compounds of
present invention can exist in free form for treatment, or where
appropriate, as a pharmaceutically acceptable derivative thereof.
According to the present invention, a pharmaceutically acceptable
derivative includes, but is not limited to, pharmaceutically
acceptable salts, esters, salts of such esters, prodrugs, salts of
such prodrugs, or any other adduct or derivative which upon
administration to a patient in need is capable of providing,
directly or indirectly, a compound as otherwise described
herein.
[0037] As used herein, the term "pharmaceutically acceptable salt"
refers to those salts which are, within the scope of sound medical
judgment, suitable for use in contact with the tissues of humans
and lower animals without undue toxicity, irritation, allergic
response and the like, and are commensurate with a reasonable
benefit/risk ratio. A "pharmaceutically acceptable salt" means any
salt or salt of an ester of a compound of this invention that, upon
administration to a recipient, is capable of providing, either
directly or indirectly, a compound of this invention.
[0038] Pharmaceutically acceptable salts are well known in the art.
For example, S. M. Berge et al. describe pharmaceutically
acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66,
1-19, which is incorporated herein by reference. Pharmaceutically
acceptable salts of the compounds of this invention include those
derived from suitable inorganic and organic acids and bases.
Examples of pharmaceutically acceptable, nontoxic acid addition
salts are salts of an amino group formed with inorganic acids such
as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric
acid and perchloric acid or with organic acids such as acetic acid,
trifluoroacetic acid, oxalic acid, maleic acid, tartaric acid,
citric acid, succinic acid or malonic acid or by using other
methods used in the art such as ion exchange. Other
pharmaceutically acceptable salts include adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,
borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, glutamate, hemisulfate, heptanoate,
hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,
lactate, laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Salts of compounds containing a carboxylic acid or other acidic
functional group can be prepared by reacting with a suitable base.
Such salts include, but are not limited to, alkali metal, alkaline
earth metal, aluminum salts, ammonium,
N.sup.+(C.sub.1-4alkyl).sub.4 salts, and salts of organic bases
such as trimethylamine, triethylamine, morpholine, pyridine,
piperidine, picoline, dicyclohexylamine,
N,N'-dibenzylethylenediamine, 2-hydroxyethylamine,
bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine,
dibenzylpiperidine, dehydroabietylamine,
N,N'-bisdehydroabietylamine, glucamine, N-methylglucamine,
collidine, quinine, quinoline, and basic amino acids such as lysine
and arginine. This invention also envisions the quaternization of
any basic nitrogen-containing groups of the compounds disclosed
herein. Water or oil-soluble or dispersible products may be
obtained by such quaternization. Representative alkali or alkaline
earth metal salts include sodium, lithium, potassium, calcium,
magnesium, and the like. Further pharmaceutically acceptable salts
include, when appropriate, nontoxic ammonium, quaternary ammonium,
and amine cations formed using counterions such as halide,
hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl
sulfonate and aryl sulfonate.
[0039] The term "prodrug" as used herein refers to compounds that
are rapidly converted to an active form (i.e., drug) within the
body or cells thereof by the action of endogenous enzymes or other
chemicals and/or conditions. Prodrug design is discussed generally
in Hardma et al. (Eds.), Goodman and Gilman's The Pharmacological
Basis of Therapeutics, 9th ed., pp. 11-16 (1996). Prodrugs of the
compounds disclosed herein include, but are not limited to, esters
formed from available hydroxyl or carboxyl groups (also referred to
as ester prodrugs or prodrug esters), amides formed from available
amino, amido, or carboxyl groups, thioesters formed from available
thiol or carboxyl groups, carbonates formed from available hydroxyl
or carboxyl groups, carbamates formed from available hydroxyl,
amino, or amido groups, carbamides formed from available amido or
amino groups, sulfonate esters and sulfate esters formed from
available hydroxyl groups, sulfonamides formed from available amino
groups, and phosphonamides formed from available amino groups.
Suitable ester prodrugs include, but are not limited to, aliphatic
esters, aryl esters, benzyl esters, and derivatives thereof.
[0040] Compounds of the invention include those of formula I or a
salt, ester, or prodrug thereof:
##STR00001##
[0041] wherein:
[0042] R.sup.1 is selected from the group consisting of C.sub.1 to
C.sub.12 alkyl, -L.sup.1-C.sub.3-C.sub.6 cycloalkyl,
L.sup.2-C.sub.2-C.sub.6 heterocycloalkyl, benzyl, -L.sup.3-aryl,
and -L.sup.4-heteroaryl;
[0043] R.sup.2 is selected from the group consisting of
-L.sup.5-C(.dbd.O)R.sup.3, -L.sup.6-R.sup.4, and NHR.sup.5;
[0044] R.sup.3 is selected from the group consisting of OR.sup.6,
NR.sup.7R.sup.8, and NHNHR.sup.9;
[0045] R.sup.5 is selected from the group consisting of OR.sup.10,
C.sub.1 to C.sub.12 alkyl, -L.sup.7-C.sub.3-C.sub.6 cycloalkyl,
-L.sup.8-C.sub.2-C.sub.6 heterocycloalkyl, benzyl, -L.sup.9-aryl,
and -L.sup.10-heteroaryl;
[0046] R.sup.8 is selected from the group consisting of OR.sup.11,
N.dbd.R.sup.12R.sup.13, -L.sup.11-R.sup.14, NHSO.sub.2R.sup.15, and
NHR.sup.16;
[0047] R.sup.6, R.sup.7, R.sup.9, R.sup.10, R.sup.11, R.sup.12,
R.sup.13, and R.sup.17 are independently selected from the group
consisting of H and C.sub.1 to C.sub.12 alkyl;
[0048] R.sup.4, R.sup.14, R.sup.15, and R.sup.16 are independently
selected from the group consisting of -L.sup.12-C.sub.3-C.sub.6
cycloalkyl, -L.sup.13-C.sub.2-C.sub.6 heterocycloalkyl, benzyl,
-L.sup.14-aryl, and -L.sup.15-heteroaryl; and
[0049] L.sup.1, L.sup.2, L.sup.3, L.sup.4, L.sup.5, L.sup.6,
L.sup.7, L.sup.8, L.sup.9, L.sup.10, L.sup.11, L.sup.12, L.sup.13,
L.sup.14, and L.sup.15 are independently selected from the group
consisting of null, C.sub.1 to C.sub.12 alkylene, and C.sub.1 to
C.sub.12 alkenylene.
[0050] Compounds of the invention include those of formula II or a
salt, ester, or prodrug thereof:
##STR00002##
wherein R.sup.1, L.sup.5, and R.sup.3 are as defined above for
formula I.
[0051] Compounds of the invention include those of formula III or a
salt, ester, or prodrug thereof:
##STR00003##
wherein R.sup.1, L.sup.6, and R.sup.4 are as defined above for
formula I.
[0052] Compounds of the invention include those of formula IV or a
salt, ester, or prodrug thereof:
##STR00004##
wherein R.sup.1 and R.sup.5 are as defined above for formula I.
[0053] Compounds of the invention include those of formula V or a
salt, ester, or prodrug thereof:
##STR00005##
wherein R.sup.1, L.sup.5, and R.sup.6 are as defined above for
formula I.
[0054] Compounds of the invention include those of formula VI or a
salt, ester, or prodrug thereof:
##STR00006##
wherein R.sup.1, L.sup.5, R.sup.7, and R.sup.8 are as defined above
for formula I.
[0055] Compounds of the invention include those of formula VII or a
salt, ester, or prodrug thereof:
##STR00007##
wherein R.sup.1, L.sup.5, and R.sup.9 are as defined above for
formula I.
[0056] In formulas I to VII, R.sup.1 is selected from C.sub.1 to
C.sub.12 alkyl, -L.sup.1-C.sub.3-C.sub.6 cycloalkyl,
-L.sup.2-C.sub.2-C.sub.6 heterocycloalkyl, benzyl, -L.sup.3-aryl,
and -L.sup.4-heteroaryl, and is optionally substituted with one,
two, three, or more substituent groups that are the same or
different. Suitable substituent groups include, but are not limited
to, F, Cl, Br, I, CF.sub.3, CH.sub.3, OCF.sub.3, OCH.sub.3, and
--CN. Additional R.sup.1 groups include, but are not limited to,
optionally substituted phenyl, halophenyl (e.g., fluorophenyl,
chlorophenyl, bromophenyl, iodophenyl), dihalophenyl (e.g.,
difluorophenyl, dichlorophenyl, dibromophenyl, diiodophenyl),
trihalophenyl (e.g., trifluorophenyl, trichlorophenyl,
tribromophenyl, triiodophenyl), (trifluoromethyl)phenyl,
fluoro(trifluoromethyl)phenyl, chloro(trifluoromethyl)phenyl,
bromo(trifluoromethyl)phenyl, iodo(trifluoromethyl)phenyl, tolyl,
xylyl, fluorotolyl, chlorotolyl, bromotolyl, iodotolyl,
fluoroxylyl, chloroxylyl, bromoxylyl, iodoxylyl, methoxyphenyl,
dimethoxyphenyl, (trifluoromethoxy)phenyl, cyanophenyl,
dimethoxybenzyl, methylisoxazolyl, 3H-1,3,4-oxadiazol-2-one-5-yl,
methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,
decyl, undecyl, and dodecyl. Further R.sup.1 groups include
##STR00008## ##STR00009##
[0057] pentyl, and butyl.
[0058] R.sup.4, R.sup.14, R.sup.15, and R.sup.16 are independently
selected from -L.sup.12-C.sub.3-C.sub.6 cycloalkyl,
-L.sup.13-C.sub.2-C.sub.6 heterocycloalkyl, benzyl, -L.sup.14-aryl,
and -L.sup.15-heteroaryl, and are optionally substituted with one,
two, three, or more substituent groups that are the same or
different. Suitable substituent groups include, but are not limited
to, F, Cl, Br, I, CF.sub.3, CH.sub.3, OCF.sub.3, OCH.sub.3, and
--CN. Additional R.sup.4, .sub.R.sup.14, R.sup.15, and R.sup.16
groups include, but are not limited to, optionally substituted
phenyl, halophenyl (e.g., fluorophenyl, chlorophenyl, bromophenyl,
iodophenyl), dihalophenyl (e.g., difluorophenyl, dichlorophenyl,
dibromophenyl, diiodophenyl), trihalophenyl (e.g., trifluorophenyl,
trichlorophenyl, tribromophenyl, triiodophenyl),
(trifluoromethyl)phenyl, fluoro(trifluoromethyl)phenyl,
chloro(trifluoromethyl)phenyl, bromo(trifluoromethyl)phenyl,
iodo(trifluoromethyl)phenyl, tolyl, xylyl, fluorotolyl,
chlorotolyl, bromotolyl, iodotolyl, fluoroxylyl, chloroxylyl,
bromoxylyl, iodoxylyl, methoxyphenyl, dimethoxyphenyl,
(trifluoromethoxy)phenyl, cyanophenyl, dimethoxybenzyl,
methylisoxazolyl, and 3H-1,3,4-oxadiazol-2-one-5-yl. Further
R.sup.4 groups include
##STR00010##
[0059] R.sup.5 is selected from OR.sup.10, C.sub.1 to C.sub.12
alkyl, -L.sup.7-C.sub.3-C.sub.6 cycloalkyl,
-L.sup.8-C.sub.2-C.sub.6 heterocycloalkyl, benzyl, -L.sup.9-aryl,
and -L.sup.10-heteroaryl, and is optionally substituted with one,
two, three, or more substituent groups that are the same or
different. Suitable substituent groups include, but are not limited
to, F, Cl, Br, I, CF.sub.3, CH.sub.3, OCF.sub.3, OCH.sub.3, and CN.
Additional R.sup.5 groups include, but are not limited to,
optionally substituted phenyl, halophenyl (e.g., fluorophenyl,
chlorophenyl, bromophenyl, iodophenyl), dihalophenyl (e.g.,
difluorophenyl, dichlorophenyl, dibromophenyl, diiodophenyl),
trihalophenyl (e.g., trifluorophenyl, trichlorophenyl,
tribromophenyl, triiodophenyl), (trifluoromethyl)phenyl,
fluoro(trifluoromethyl)phenyl, chloro(trifluoromethyl)phenyl,
bromo(trifluoromethyl)phenyl, iodo(trifluoromethyl)phenyl, tolyl,
xylyl, fluorotolyl, chlorotolyl, bromotolyl, iodotolyl,
fluoroxylyl, chloroxylyl, bromoxylyl, iodoxylyl, methoxyphenyl,
dimethoxyphenyl, (trifluoromethoxy)phenyl, cyanophenyl,
dimethoxybenzyl, methylisoxazolyl, 3H-1,3,4-oxadiazol-2-one-5-yl,
methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,
decyl, undecyl, and dodecyl. Further R.sup.5 groups include OH.
[0060] R.sup.6, R.sup.7, R.sup.9, R.sup.10, R.sup.11, R.sup.12,
R.sup.13, and R.sup.17 are independently selected from H and
C.sub.1 to C.sub.12 alkyl, including C.sub.1 alkyl, C.sub.2 alkyl,
C.sub.3 alkyl, C.sub.4 alkyl, C.sub.5 alkyl, C.sub.6 alkyl, C.sub.7
alkyl, C.sub.8 alkyl, C.sub.9 alkyl, C.sub.10 alkyl, C.sub.11
alkyl, and C.sub.12 alkyl.
[0061] R.sup.8 is selected from OR.sup.11, N.dbd.R.sup.12R.sup.13,
-L.sup.11 --R.sup.14, NHSO.sub.2R.sup.15, and NHR.sup.16. Exemplary
R.sup.8 groups include, but are not limited to, OH, OCH.sub.3,
N.dbd.(CH.sub.3).sub.2,
##STR00011##
[0062] L.sup.1, L.sup.2, L.sup.3, L.sup.4, L.sup.5, L.sup.6,
L.sup.7, L.sup.8, L.sup.9, L.sup.10, L.sup.11, L.sup.12, L.sup.13,
L.sup.14, and L.sup.15 are independently selected from null (a
bond), C.sub.1 to C.sub.12 alkylene, including C.sub.1 alkylene,
C.sub.2 alkylene, C.sub.3 alkylene, C.sub.4 alkylene, C.sub.5
alkylene, C.sub.6 alkylene, C.sub.7 alkylene, C.sub.8 alkylene,
C.sub.9 alkylene, C.sub.10 alkylene, C.sub.11 alkylene, and
C.sub.12 alkylene, and C.sub.1 to C.sub.12 alkenylene, including
C.sub.1 alkenylene, C.sub.2 alkenylene, C.sub.3 alkenylene, C.sub.4
alkenylene, C.sub.5 alkenylene, C.sub.6 alkenylene, C.sub.7
alkenylene, C.sub.8 alkenylene, C.sub.9 alkenylene, C.sub.10
alkenylene, C.sub.11 alkenylene, and C.sub.12 alkenylene.
[0063] Exemplary compounds of the invention have a formula selected
from C256, C259, C265, C267, C276, C277, C288, C309, C311, C280,
C300, C313, C314, C320, C323, C326, C328, C334, C342, C240, C241,
C246, C248, C251, C255, C260, C261, C262, C263, C264, C266, C268,
C278, C281, C282, C287, C289, C295, C296, C297, C301, C304, C305,
C307, C310, C322, C336, C339, C340, C341, C362, C279, C285, C286,
C299, C306, C330, C344, C345, C346, C347, C348, C356, C357, C358,
C359, C360, C361, C363, C364, C284, and salts, esters, or prodrugs
thereof. These compounds are depicted in Table 1 herein below.
TABLE-US-00001 TABLE 1 Synthesized PAI-1 Inhibitor Compounds PAI-1/
uPA PAI-1/ IC.sub.50 in uPA IC.sub.50 PAI-1/ 1.5% in 10% uPA
IC.sub.50 BSA plasma in buffer buffer buffer No. Structure (.mu.M)
(.mu.M) ( .mu.M) C256 ##STR00012## 1040, 1353 204, 235 214 C259
##STR00013## 2892, 3141 625, 760 526 C265 ##STR00014## 4018 C267
##STR00015## 3794 C276 ##STR00016## 1438 428 476 C277 ##STR00017##
750.6, 719 165.4, 158 148, 142 C288 ##STR00018## 2909 1061 752 C309
##STR00019## 1200 C311 ##STR00020## C280 ##STR00021## 207, 203 166,
159 168 C300 ##STR00022## 5491 C313 ##STR00023## 1196 532 490 C314
##STR00024## C320 ##STR00025## 329 513 693 C323 ##STR00026## C326
##STR00027## C328 ##STR00028## 713 345 318 C334 ##STR00029## 189
C342 ##STR00030## C240 ##STR00031## 3766, 3912 2221, 1973 1208 C241
##STR00032## 1933 C246 ##STR00033## 2559 C248 ##STR00034## 1190,
1313 652, 646 478 C251 ##STR00035## 68.6, 76 110, 123 108 C255
##STR00036## 2064, 1780 221, 292 279 C260 ##STR00037## 147, 187 43,
545 525 C261 ##STR00038## 102, 103 68, 86 69 C262 ##STR00039## 170,
187 63, 84 86 C263 ##STR00040## 2837 1685 1356 C264 ##STR00041##
2121 1175 1248 C266 ##STR00042## 568, 549 126 219 C268 ##STR00043##
135, 134 65 77 C278 ##STR00044## 640, 642 118, 130 142 C281
##STR00045## 2285 C282 ##STR00046## 225, 219 163, 154 121 C287
##STR00047## 1187 363 304 C289 ##STR00048## 2725 1099 733 C295
##STR00049## 1337 776 736 C296 ##STR00050## 112 69 64 C297
##STR00051## 136 111 87 C301 ##STR00052## 173 128 106 C304
##STR00053## 162 90 74 C305 ##STR00054## 224 81 62 C307
##STR00055## 465 99 101 C310 ##STR00056## C322 ##STR00057## 4315
C336 ##STR00058## 5469 892 851 C339 ##STR00059## 282 247 303 C340
##STR00060## 337 249 272 C341 ##STR00061## 900 1460 1868 C362
##STR00062## 533 353 229 C279 ##STR00063## 539, 593 545, 504 680
C285 ##STR00064## 422, 388 265 306 C286 ##STR00065## 580 C299
##STR00066## 238 739 628 C306 ##STR00067## 1159 403 405 C330
##STR00068## 365, 423* 112 115 C344 ##STR00069## 1002 830 866 C345
##STR00070## 1469 673 539 C346 ##STR00071## 353 C347 ##STR00072##
888 309 293 C348 ##STR00073## 99 454 240 C356 ##STR00074## 1100
1241 544 C357 ##STR00075## 1370 1227 567 C358 ##STR00076## 1501
1229 619 C359 ##STR00077## 3373 2084 974 C360 ##STR00078## 164 1670
771 C361 ##STR00079## 232 2000 851 C363 ##STR00080## 199 751 309
C364 ##STR00081## 475 898 533 C284 ##STR00082## 2481 *pH 7.8 was
used.
[0064] Comparative compounds that do not demonstrate PAI-1
inhibitory activity in buffer (40 mM HEPES, 100 mM NaCl, 0.05%
Tween-20, 10% DMSO, pH 7.4) in the assay described in Example 11
herein are depicted in Table 2.
TABLE-US-00002 TABLE 2 Synthesized Comparative Compounds No.
Structure E242 ##STR00083## E243 ##STR00084## E244 ##STR00085##
E245 ##STR00086## E247 ##STR00087## E254 ##STR00088## E257
##STR00089## E258 ##STR00090## E290 ##STR00091## E319 ##STR00092##
E308 ##STR00093## E312 ##STR00094## E324 ##STR00095## E325
##STR00096## E329 ##STR00097## E331 ##STR00098## E332 ##STR00099##
E335 ##STR00100## E343 ##STR00101## E338 ##STR00102## E234
##STR00103## E235 ##STR00104## E237 ##STR00105## E238 ##STR00106##
E252 ##STR00107## E253 ##STR00108## E269 ##STR00109## E291
##STR00110## E327 ##STR00111## E333 ##STR00112## E321 ##STR00113##
E349 ##STR00114## E350 ##STR00115## E351 ##STR00116## E352
##STR00117## E353 ##STR00118## E354 ##STR00119## E355 ##STR00120##
E283 ##STR00121## E298 ##STR00122##
[0065] Compounds of the invention include those of formula VIII or
a salt, ester, or prodrug thereof:
##STR00123##
[0066] wherein:
[0067] W is C or N;
[0068] X.sub.1, X.sub.2, X.sub.3, X.sub.4, and X.sub.5 are
independently selected from the group consisting of --H, --OH,
--OR, --F, --Cl, --Br, --I, --NO.sub.2, --NO, --N(R).sub.2,
--N(R).sub.3.sup.+, --C(O)R, --C(O)OR, --CHO, --C(O)NH.sub.2,
--C(O)SR, --CN, --S(O).sub.2R, --SO.sub.3R, --SO.sub.3H,
--SO.sub.2N(R).sub.2, --S.dbd.O, C.sub.1 to C.sub.12 alkyl, aryl,
substituted aryl, heteroaryl, and substituted heteroaryl;
[0069] R is selected from the group consisting of C.sub.1 to
C.sub.6 alkyl, C.sub.3 to C.sub.6 cycloalkyl,
CH.sub.2--C.sub.3-C.sub.6 cycloalkyl, phenyl, tolyl, and
benzyl;
[0070] R.sup.a is selected from the group consisting of C.sub.1 to
C.sub.12 alkyl, C.sub.3 to C.sub.6 cycloalkyl,
(CH.sub.2).sub.m-C.sub.3-C.sub.6 cycloalkyl, C.sub.2 to C.sub.6
heterocycloalkyl, (CH.sub.2).sub.m-C.sub.2-C.sub.6heterocycloalkyl,
C.sub.2 to C.sub.6 heterocycloalkyl,
(CH.sub.2).sub.m-C.sub.2-C.sub.6heterocycloalkyl, benzyl, aryl,
(CH.sub.2).sub.m-aryl, heteroaryl, (CH.sub.2).sub.m-heteroaryl, and
substituted derivatives thereof; and
[0071] m is 1, 2, 3, 4, 5, or 6.
[0072] In some embodiments, compounds of the invention include
those of formula VIII as defined above with the proviso that at
most two of X.sub.1, X.sub.2, X.sub.3, X.sub.4, and X.sub.5 are
OH.
[0073] In some embodiments, compounds of the invention include
those of formula VIII as defined above excluding compounds having a
formula
##STR00124##
[0074] In some embodiments, compounds of the invention include
those of formula VIII as defined above with the proviso that at
most three of X.sub.1, X.sub.2, X.sub.3, X.sub.4, and X.sub.5 are
H.
[0075] Compounds of the invention include those of formula IX or a
salt, ester, or prodrug thereof:
##STR00125##
[0076] wherein R.sup.a is selected from the group consisting of
benzyl, aryl, (CH.sub.2).sub.m-aryl, heteroaryl,
(CH.sub.2).sub.m-heteroaryl, and substituted derivatives thereof;
and
[0077] m is 1, 2, 3, 4, 5, or 6.
[0078] In some embodiments, compounds of the invention include
those of formula VIII or IX as defined above wherein R.sup.a is
selected from the group consisting of:
##STR00126##
[0079] wherein: W is C or N; and
[0080] R.sup.1 and R.sup.2 are independently selected from the
group consisting of --H, --F, --Cl, --Br, --I, --CF.sub.3, C.sub.1
to C.sub.12 alkyl, and phenyl.
[0081] Exemplary compounds of the invention have a formula selected
from C152, C155, C173, C189, C191, C197, C224, C292, C293, C294,
C153, C162, C163, C165, C188, C195, C157, C158, C182, C183, and
salts, esters, or prodrugs thereof. These compounds are depicted in
Table 3 herein below.
TABLE-US-00003 TABLE 3 Synthesized PAI-1 Inhibitor Compounds PAI-1/
PAI-1/ ATIII/ uPA tPA .alpha.IIa IC.sub.50 IC.sub.50 IC.sub.50 No.
Structure (.mu.M) (.mu.M) (.mu.M) C152 ##STR00127## 1.57 1.71 87
C155 ##STR00128## 30.44 34.84 2326 C173 ##STR00129## 19.2 C189
##STR00130## 2.29 C191 ##STR00131## 1.28 C197 ##STR00132## 0.173
C224 ##STR00133## 4.34 C292 ##STR00134## 4.94, 68* C293
##STR00135## 77.6, 123* C294 ##STR00136## 120.5, 234* C153
##STR00137## 0.70 1.02 C162 ##STR00138## 3.90 7.59 C163
##STR00139## 0.288 0.611 C165 ##STR00140## 0.35 0.51 C188
##STR00141## 0.12 C195 ##STR00142## 0.92 C157 ##STR00143## 0.25
0.98 44.37 C158 ##STR00144## 2.60 1.44 547 C182 ##STR00145## 0.033
C183 ##STR00146## 0.18 *pH 7.4 was used.
[0082] Comparative compounds that do not demonstrate PAI-1
inhibitory activity in buffer (40 mM HEPES, 100 mM NaCl, 0.05%
Tween-20, 10% DMSO, pH 7.8) in the assay described in Example 12
herein are depicted in Table 4.
TABLE-US-00004 TABLE 4 Synthesized Comparative Compounds No.
Structure E174 ##STR00147##
[0083] Compounds of the invention include those of formula X or a
salt, ester, or prodrug thereof:
##STR00148##
[0084] wherein
[0085] R.sup.a is C.sub.1 to C.sub.12 alkyl,
[0086] R.sup.b is selected from the group consisting of C.sub.1 to
C.sub.12 alkyl, aryl, heteroaryl, (CH.sub.2).sub.m-R, and
##STR00149##
or
[0087] R.sup.a and R.sup.b taken together with the N atom to which
they are bonded form an optionally substituted 3- to 8-membered
heterocyclic ring;
[0088] m is 1, 2, 3, 4, 5, or 6;
[0089] n is 0, 1, 2, 3, 4, 5, or 6;
[0090] Y is selected from the group consisting of NH.sub.2 and OH;
and
[0091] R selected from the group consisting of
##STR00150##
substituted phenyl and heteroaryl.
[0092] In some embodiments, compounds of the invention include
those of formula X as defined above wherein R.sup.a is selected
from the group consisting of butyl, pentyl, hexyl, heptyl, octyl,
nonyl, and decyl. In some embodiments, compounds of the invention
include those of formula X as defined above wherein R.sup.b is
selected from the group consisting of butyl, pentyl, hexyl, heptyl,
octyl, nonyl, decyl, fluorophenyl, chlorophenyl, bromophenyl,
iodophenyl, trifluoromethylphenyl, and dichlorohydroxyphenyl.
[0093] Compounds of the invention include those of formula XI or a
salt, ester, or prodrug thereof:
##STR00151##
[0094] wherein
[0095] R.sup.b is selected from the group consisting of aryl,
heteroaryl, (CH.sub.2).sub.m-R, and
##STR00152##
[0096] m is 1, 2, 3, 4, 5, or 6;
[0097] n is 0, 1, 2, 3, 4, 5, or 6;
[0098] Y is selected from the group consisting of NH.sub.2 and OH;
and
[0099] R selected from the group consisting of
##STR00153##
CO.sub.2H, phenyl, substituted phenyl and heteroaryl.
[0100] Compounds of the invention include those of formula XII or a
salt, ester, or prodrug thereof:
##STR00154##
[0101] wherein
[0102] R.sup.b is selected from the group consisting of aryl,
heteroaryl, (CH.sub.2).sub.m-R, and
##STR00155##
[0103] m is 1, 2, 3, 4, 5, or 6;
[0104] n is 0, 1, 2, 3, 4, 5, or 6;
[0105] Y is selected from the group consisting of NH.sub.2 and OH;
and
[0106] R selected from the group consisting of
##STR00156##
CO.sub.2H, phenyl, substituted phenyl and heteroaryl.
[0107] Exemplary compounds of the invention have a formula selected
from C170, C171, C172, C175, C177, C179, C180, C186, C193, C205,
C160, C187, C190, C198, C232, C233, C249, C270, C271, C272, C273,
C274, C275, C303, C210, and salts, esters, or prodrugs thereof.
These compounds are depicted in Table 5 herein below.
TABLE-US-00005 TABLE 5 Synthesized PAI-1 Inhibitor Compounds PAI-1/
ATIII/ PAI-1/ tPA .alpha.IIa uPA IC.sub.50 IC.sub.50 IC.sub.50 No.
Structure (.mu.M) (.mu.M) (.mu.M) C170 ##STR00157## 5.49 C171
##STR00158## 1.67 C172 ##STR00159## 1.98 C175 ##STR00160## 6.74
C177 ##STR00161## 5.11 C179 ##STR00162## 2.40 C180 ##STR00163##
1.34 C186 ##STR00164## 6.98 C193 ##STR00165## 552 C205 ##STR00166##
5.05 C160 ##STR00167## 39.04 58.16 2326 C187 ##STR00168## 0.91 C190
##STR00169## 9.70 C198 ##STR00170## 0.051 C232 ##STR00171## 13.3
C233 ##STR00172## 74.8 C249 ##STR00173## 663 C270 ##STR00174## 29.5
C271 ##STR00175## 1.82 C272 ##STR00176## 16.4 C273 ##STR00177##
55.6 C274 ##STR00178## 73.6 C275 ##STR00179## 30.5 C303
##STR00180## 12.6*, 13.6 C210 ##STR00181## 189.6 *pH 7.4 was
used.
[0108] Comparative compounds that do not demonstrate PAI-1
inhibitory activity in buffer (40 mM HEPES, 100 mM NaCl, 0.05%
Tween-20, 10% DMSO, pH 7.8) in the assay described in Example 12
herein are depicted in Table 6.
TABLE-US-00006 TABLE 6 Synthesized Comparative Compounds No.
Structure E169 ##STR00182## E178 ##STR00183## E192 ##STR00184##
E194 ##STR00185##
[0109] A comparative compound that does not demonstrate PAI-1
inhibitory activity in buffer (40 mM HEPES, 100 mM NaCl, 0.05%
Tween-20, 10% DMSO, pH 7.4) in the assay described in Example 11
herein is depicted below.
##STR00186##
[0110] Exemplary compounds of the invention have a formula selected
from C168, C176, C184, C185, C196, C156, C161, C200, C204, C236,
and salts, esters, or prodrugs thereof. These compounds are
depicted in Table 7 herein below.
TABLE-US-00007 TABLE 7 Synthesized PAI-1 Inhibitor Compounds PAI-1/
ATIII/ PAI-1/ tPA .alpha.IIa uPA IC.sub.50 IC.sub.50 IC.sub.50 No.
Structure (.mu.M) (.mu.M) (.mu.M) C168 ##STR00187## 1.98 C176
##STR00188## 0.62 C184 ##STR00189## 0.59 C185 ##STR00190## 0.42
C196 ##STR00191## 0.45 C156 ##STR00192## 188.7 581.2 C161
##STR00193## 3.70 2.21 1670 C200 ##STR00194## 58.37 C204
##STR00195## 0.035 C236 ##STR00196## 174
[0111] Comparative compounds that do not demonstrate PAI-1
inhibitory activity in buffer (40 mM HEPES, 100 mM NaCl, 0.05%
Tween-20, 10% DMSO, pH 7.4) in the assay described in Example 11
herein are depicted in Table 8.
TABLE-US-00008 TABLE 8 Synthesized Comparative Compounds No.
Structure E164 ##STR00197## E250 ##STR00198## E181 ##STR00199##
E167 ##STR00200## E166 ##STR00201##
[0112] Compounds of the invention include those of formula XIII or
a salt, ester, or prodrug thereof:
##STR00202##
[0113] wherein
[0114] n is 0 or 1;
[0115] R.sup.a and R.sup.b are independently selected from the
group consisting of C.sub.1 to C.sub.12 alkyl, C.sub.3 to C.sub.6
cycloalkyl, (CH.sub.2).sub.m-C.sub.3-C.sub.6 cycloalkyl, C.sub.2 to
C.sub.6 heterocycloalkyl, (CH.sub.2).sub.m-C.sub.2-C.sub.6
heterocycloalkyl, C.sub.2 to C.sub.6 heterocycloalkyl,
(CH.sub.2).sub.m-C.sub.2-C.sub.6 heterocycloalkyl, benzyl, aryl,
(CH.sub.2).sub.m-aryl, heteroaryl, (CH.sub.2).sub.m-heteroaryl, and
substituted derivatives thereof; and
[0116] m is 1, 2, 3, 4, 5, or 6.
[0117] In some embodiments, compounds of the invention include
those of formula XIII as defined above wherein R.sup.a and R.sup.b
are independently selected from the group consisting of butyl,
pentyl, cyclopropyl, phenyl, difluorophenyl, and hydroxyphenyl.
[0118] Exemplary compounds of the invention have a formula selected
from C201, C208, C213, C216, C220, C221, C222, C223, and salts,
esters, or prodrugs thereof. These compounds are depicted in Table
9 herein below.
TABLE-US-00009 TABLE 9 Synthesized PAI-1 Inhibitor Compounds PAI-1/
uPA IC.sub.50 in buffer No. Structure (.mu.M) C201 ##STR00203## 181
C208 ##STR00204## 77.2 C213 ##STR00205## C216 ##STR00206## C220
##STR00207## 116.46 C221 ##STR00208## 1608 C222 ##STR00209## 236
C223 ##STR00210## 124.6
[0119] Comparative compounds that do not demonstrate PAI-1
inhibitory activity in buffer (40 mM HEPES, 100 mM NaCl, 0.05%
Tween-20, 10% DMSO, pH 7.8) in the assay described in Example 12
herein are depicted in Table 10.
TABLE-US-00010 TABLE 10 Synthesized Comparative Compounds No.
Structure E202 ##STR00211## E209 ##STR00212## E211 ##STR00213##
E212 ##STR00214## E215 ##STR00215## E217 ##STR00216## E218
##STR00217## E219 ##STR00218## E226 ##STR00219##
[0120] Compounds of the invention include those of formula XIV or a
salt, ester, or prodrug thereof:
##STR00220##
[0121] wherein R is selected from the group consisting of phenyl
and substituted biphenyl.
[0122] Exemplary compounds of the invention have a formula selected
from C199, C203, C206, C207, and salts, esters, or prodrugs
thereof. These compounds are depicted in Table 11 herein below.
TABLE-US-00011 TABLE 11 Synthesized PAI-1 Inhibitor Compounds
PAI-1/ uPA IC.sub.50 in buffer No. Structure (.mu.M) C199
##STR00221## 5.36 C203 ##STR00222## 17.07 C206 ##STR00223## 4.96
C207 ##STR00224## 16.83
[0123] Compounds of the invention include those of formula XV or a
salt, ester, or prodrug thereof:
##STR00225##
[0124] wherein:
[0125] V is selected from the group consisting of (CH.sub.2).sub.n,
C.sub.3 to C.sub.8 cycloalkyl, (CH.sub.2).sub.n--C.sub.3-C.sub.8
cycloalkyl-(CH.sub.2).sub.p, aryl,
(CH.sub.2).sub.n-aryl-(CH.sub.2).sub.p, heteroaryl,
(CH.sub.2).-heteroaryl-(CH.sub.2).sub.p,
##STR00226##
and substituted derivatives thereof;
[0126] n and p are independently 0, 1, 2, 3, 4, 5, or 6;
[0127] X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5 and X.sub.6 are
independently selected from the group consisting of --H, --OH,
--OR, --F, --Cl, --Br, --I, --NO.sub.2, --NO, --N(R).sub.2,
--N(R).sub.3.sup.+, --C(O)R,-- C(O)OR, --CHO, --C(O)NH.sub.2,
--C(O)SR, --CN, --S(O).sub.2R, --SO.sub.3R, --SO.sub.3H,
--SO.sub.2N(R).sub.2, --S.dbd.O, C.sub.1 to C.sub.12 alkyl, aryl,
substituted aryl, heteroaryl, and substituted heteroaryl;
[0128] R is selected from the group consisting of C.sub.1 to
C.sub.6 alkyl, C.sub.3 to C.sub.6 cycloalkyl,
CH.sub.2--C.sub.3-C.sub.6 cycloalkyl, phenyl, tolyl, and
benzyl;
[0129] Y.sub.1 is selected from the group consisting of O, NH,
NR.sup.a, S, and CH.sub.2;
[0130] Y.sub.2 is selected from the group consisting of O, NH,
NR.sup.b, S, and CH.sub.2;
[0131] R.sup.a and R.sup.b are independently selected from the
group consisting of C.sub.1 to C.sub.12 alkyl, C.sub.3 to C.sub.6
cycloalkyl, (CH.sub.2).sub.m--C.sub.3-C.sub.6 cycloalkyl, C.sub.2
to C.sub.6 heterocycloalkyl, (CH.sub.2).sub.m--C.sub.2-C.sub.6
heterocycloalkyl, benzyl, aryl, (CH.sub.2).sub.m-aryl, heteroaryl,
(CH.sub.2).sub.m-heteroaryl, and substituted derivatives
thereof;
[0132] m is 1, 2, 3, 4, 5, or 6; and
[0133] Z.sub.1, Z.sub.2, Z.sub.3, and Z.sub.4 are independently
selected from the group consisting of C, P--OH, S, and S.dbd.O.
[0134] Compounds of the invention include those of formula XVI or a
salt, ester, or prodrug thereof:
##STR00227##
wherein V, n, p, X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5,
X.sub.6, .sup.Y1, Y.sub.2, R.sup.a, R.sup.b, Z.sub.1, Z.sub.2,
Z.sub.3, and Z.sub.4 are as defined above for formula XV.
[0135] Compounds of the invention include those of formula XVII or
a salt, ester, or prodrug thereof:
##STR00228##
[0136] wherein:
[0137] X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5 and X.sub.6 are
independently selected from the group consisting of --H, --OH,
--OR, --F, --Cl, --Br, --I, --NO.sub.2, --NO, --N(R).sub.2,
--N(R).sub.3.sup.+, --C(O)R,--C(O)OR, --CHO, --C(O)NH.sub.2,
--C(O)SR, --CN, --S(O).sub.2R, --SO.sub.3R, --SO.sub.3H,
SO.sub.2N(R).sub.2, --S.dbd.O, C.sub.1 to C.sub.12 alkyl, aryl,
substituted aryl, heteroaryl, and substituted heteroaryl;
[0138] R is selected from the group consisting of C.sub.1 to
C.sub.6 alkyl, C.sub.3 to C.sub.6 cycloalkyl,
CH.sub.2--C.sub.3-C.sub.6 cycloalkyl, phenyl, tolyl, and
benzyl;
[0139] R.sup.b is selected from the group consisting of H, C.sub.1
to C.sub.12 alkyl, C.sub.3 to C.sub.6 cycloalkyl,
(CH.sub.2).sub.m--C.sub.3-C.sub.6 cycloalkyl, C.sub.2 to C.sub.6
heterocycloalkyl,
(CH.sub.2).sub.m--C.sub.2-C.sub.6heterocycloalkyl, benzyl, aryl,
(CH.sub.2).sub.m-aryl, heteroaryl, (CH.sub.2).sub.m-heteroaryl, and
substituted derivatives thereof;
[0140] m is 1, 2, 3, 4, 5, or 6; and
[0141] Z.sub.4 is selected from the group consisting of C, P--OH,
S, and S.dbd.O.
[0142] Compounds of the invention include those of formula XVIII or
a salt, ester, or prodrug thereof:
##STR00229##
wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6,
R.sup.b, and Z.sub.4 are as defined above for formula XVII.
[0143] In some embodiments, compounds of the invention include
those of formula XVII or XVIII as defined above wherein X.sub.1 and
X.sub.2 are independently selected from --OH and --OR.
[0144] Compounds of the invention include those of formula XIX or a
salt, ester, or prodrug thereof:
##STR00230##
[0145] wherein:
[0146] X.sub.3, X.sub.4, and X.sub.6 are independently selected
from the group consisting of --H, --OH, --OR, --F, --Cl, --Br, --I,
--NO.sub.2, --NO, --N(R).sub.2, --N(R).sub.3.sup.+, --C(O)R,
--C(O)OR, CHO, --C(O)NH.sub.2, --C(O)SR, --CN, --S(O).sub.2R,
--SO.sub.3R, --SO.sub.3H, --SO.sub.2N(R).sub.2, --S.dbd.O, aryl,
substituted aryl, heteroaryl, and substituted heteroaryl;
[0147] R is selected from the group consisting of C.sub.1 to
C.sub.6 alkyl, C.sub.3 to C.sub.6 cycloalkyl,
CH.sub.2--C.sub.3-C.sub.6 cycloalkyl, phenyl, tolyl, and
benzyl;
[0148] Y.sub.1 is selected from the group consisting of
CHR.sup.aR.sup.b, OR.sup.a, NHR.sup.a, NR.sup.aR.sup.b, and
SR.sup.a;
[0149] R.sup.a and R.sup.b are independently selected from the
group consisting of C.sub.1 to C.sub.12 alkyl, C.sub.3 to C.sub.6
cycloalkyl, (CH.sub.2).sub.m--C.sub.3-C.sub.6 cycloalkyl, C.sub.2
to C.sub.6 heterocycloalkyl, (CH.sub.2).sub.m--C.sub.2-C.sub.6
heterocycloalkyl, benzyl, aryl, (CH.sub.2).sub.m-aryl, heteroaryl,
(CH.sub.2).sub.m-heteroaryl, --U, (CH.sub.2).sub.m--U, and
substituted derivatives thereof, or R.sup.a and R.sup.b taken
together with the N atom to which they are bonded form a 3- to
8-membered heterocyclic ring;
[0150] U is selected from the group consisting of
--NR.sup.cR.sup.d, NR.sup.cC(O)R.sup.e, NR.sup.cC(O)OR.sup.e,
--NR.sup.cC(O)NR.sup.fR.sup.e, --NR.sup.cC(O)SR.sup.e,
--NR.sup.cP(O)(OH)R.sup.e, --NR.sup.cP(O)(OH)OR.sup.e,
--NR.sup.cP(O)(OH)NR.sup.fR.sup.e, --NR.sup.cP(O)(OH)SR.sup.e,
--NR.sup.cS(O)R.sup.e, --NR.sup.cS(O)OR.sup.e,
--NR.sup.cS(O)NR.sup.fR.sup.e, --NR.sup.cS(O)SR.sup.e,
--NR.sup.eS(O).sub.2R.sup.e, --NR.sup.cS(O).sub.2OR.sup.e,
--NR.sup.cS(O).sub.2NR.sup.fR.sup.e, NR.sup.cS(O).sub.2SR.sup.e,
--OR.sup.f, --OC(O)R.sup.e, --OC(O)OR.sup.e,
--OC(O)NR.sup.fR.sup.e, --OC(O)SR.sup.e, --OP(O)(OH)R.sup.e,
OP(O)(OH)OR.sup.e, --OP(O)(OH)NR.sup.dR.sup.e, --OP(O)(OH)SR.sup.e,
--OS(O)R.sup.e, --OS(O)OR.sup.e, --OS(O)NR.sup.dR.sup.e,
--OS(O)SR.sup.e, --OS(O).sub.2R.sup.e, --OS(O).sub.2OR.sup.e,
--OS(O).sub.2NR.sup.dR.sup.e, --OS(O).sub.2SR, --C(O)OR.sup.c,
--C(O)NR.sup.cR.sup.d, --C(O)SR.sup.c, and --C(O)R.sup.c;
[0151] R.sup.c and R.sup.d are independently selected from the
group consisting of H, C.sub.1 to C.sub.12 alkyl, C.sub.1 to
C.sub.12 haloalkyl, C.sub.3 to C.sub.6 cycloalkyl,
(CH.sub.2).sub.m--C.sub.3-C.sub.6 cycloalkyl, C.sub.2 to C.sub.6
heterocycloalkyl,
(CH.sub.2).sub.m--C.sub.2-C.sub.6heterocycloalkyl, benzyl, aryl,
(CH.sub.2).sub.m-aryl, heteroaryl, (CH.sub.2).sub.m-heteroaryl, and
substituted derivatives thereof, or R.sup.c and R.sup.d taken
together with the N atom to which they are bonded form a 3- to
8-membered heterocyclic ring;
[0152] R.sup.e, R.sup.f, and R.sup.g are independently selected
from the group consisting of H, C.sub.1 to C.sub.12 alkyl, C.sub.1
to C.sub.12 haloalkyl, C.sub.3 to C.sub.6 cycloalkyl,
(CH.sub.2).sub.m--C.sub.3-C.sub.6 cycloalkyl, C.sub.2 to C.sub.6
heterocycloalkyl, (CH.sub.2).sub.m--C.sub.2-C.sub.6
heterocycloalkyl, benzyl, aryl, (CH.sub.2).sub.m-aryl, heteroaryl,
(CH.sub.2).sub.mheteroaryl, and substituted derivatives
thereof;
[0153] m is 1, 2, 3, 4, 5, or 6; and
[0154] Z.sub.1 and Z.sub.4 are independently selected from the
group consisting of C, P--OH, S, and S.dbd.O.
[0155] Exemplary compounds of the invention have a formula selected
from C225, C227, C228, C229, and salts, esters, or prodrugs
thereof. These compounds are depicted in Table 12 herein below.
TABLE-US-00012 TABLE 12 Synthesized PAI-1 Inhibitor Compounds
PAI-1/ uPA IC.sub.50 in buffer No. Structure (.mu.M) C225
##STR00231## 163 C227 ##STR00232## 205 C228 ##STR00233## 14.8 C229
##STR00234## 1288
[0156] Comparative compounds that do not demonstrate PAI-1
inhibitory activity in buffer (40 mM HEPES, 100 mM NaCl, 0.05%
Tween-20, 10% DMSO, pH 7.4) in the assay described in Example 11
herein are depicted in Table 13.
TABLE-US-00013 TABLE 13 Synthesized Comparative Compounds No.
Structure E230 ##STR00235## E231 ##STR00236## E315 ##STR00237##
E316 ##STR00238## E317 ##STR00239## E318 ##STR00240##
[0157] Compounds for use in the methods of the invention include
those of formula XX to XXIX or a salt, ester, or prodrug thereof.
Compounds of formula XX to XXIX that demonstrate PAI-1 inhibitory
activity are depicted in Table 14.
TABLE-US-00014 TABLE 14 PAI-1 Inhibitor Compounds Compound No.
Structure Supplier XX ##STR00241## Chembridge XXI ##STR00242##
Synthon Labs XXII ##STR00243## Vitas M Labs XXIII ##STR00244##
Chembridge XXIV ##STR00245## Chembridge XXV ##STR00246## Chembridge
XXVI ##STR00247## Chembridge XXVII ##STR00248## Synthon Labs XXVIII
##STR00249## Synthon Labs XXIX ##STR00250## Enamine
[0158] Methods of Making Inhibitors of PAI-1 Activity
[0159] The compounds of the present invention can be readily
prepared according to the following reaction schemes or
modification thereof using readily available starting materials,
reagents and conventional synthetic procedures. It is also possible
to make use of variants of these process steps, which in themselves
are known to and well within the preparatory skill of the medicinal
chemist.
[0160] Derivatives of PAI-1 inhibitors are also included herein.
Such derivatives include molecules modified by one or more water
soluble polymer molecules, such as polyethylene glycol, or by the
addition of polyamino acids, including fusion proteins (procedures
for which are well-known in the art). Such derivatization may occur
singularly or there may be multiple sites of derivatization.
[0161] Primary High-Throughput Screen for PAI-1 Inhibition
[0162] High-throughput screening was carried out using the protocol
described below. All screening was performed in the Center for
Chemical Genomics in the Life Sciences Institute at the University
of Michigan. For assay validation, the Microsource Spectrum 2000
was screened both in HEPES-buffered saline (HBS) and in HBS
containing 15 mg/mL BSA. All other libraries were screened in HBS
with 15 mg/mL BSA only. Purified compound libraries screened were
the NIH Clinical Collection, Chemical Methodologies Libraries
Development (Boston University), Maybridge, Chembridge, ChemDiv,
National Cancer Institute-Development Therapeutics Program Library,
the Cayman Cannabinoid and Epigenetics collections, EMD Protein
Kinase collection, and the Enzo Autophagy, Protease, Natural
products, REDOX, and Wnt Pathway libraries. Together, these
libraries totaled approximately 152,899 purified compounds. For
primary screening, all reagents except for compounds were added
using a Thermo Scientific Multidrop Combi.
[0163] Briefly, 6 .mu.L of 15 nM PAI-1 in 15 mg/mL BSA was added
per well, followed by addition of 200 nL of compound using a
Beckman Biomek FX with a pin-tool attachment liquid handling
system. One compound was added per well, yielding a compound
concentration of approximately 32 .mu.M (3.2% DMSO) in the presence
of PAI-1. Following a 15-minute incubation, 3 .mu.L of 15 nM uPA in
HEPES-buffered saline (HBS) was added per well, for final
concentrations of PAI-1 and uPA of 10 nM and 5 nM, respectively. A
2:1 PAI-1:uPA ratio was chosen to enrich for the most active
compounds, as greater than half of the PAI-1 must be inactivated
before a signal is generated. After an additional 15-minute
incubation, 3 .mu.L of the pNA/AMC substrate mixture was added in
HBS to yield final concentrations of 200 .mu.M and 100 .mu.M,
respectively. The mixture of uPA and substrates in the absence of
PAI-1 served as a positive control, while the negative control
consisted of the mixture uPA and substrates along with PAI-1.
Following a 90-minute incubation to allow for substrate turnover,
quenching of WPF by pNA (ex/em 430/470 nm) and AMC fluorescence
(ex/em 380/470) were recorded using a BMG Labtech Pherastar plate
reader. All data was analyzed using Tripos Benchware Dataminer.
[0164] Using this HTS protocol, 152,899 purified compounds were
screened from 15 different collections in the Center for Chemical
Genomics (CCG) at the University of Michigan. Compounds were
considered PAI-1 inactivators if a change in both the pNA and AMC
signals was observed that was greater than three standard
deviations (>3SD) from the negative control. Compounds that
displayed a change in only one of the reporter signals were
classified as false-positives and not considered for further
evaluation. The average Z-factor values (Z'), which serve as a
statistical gauge for the quality of the HTS assay, for pNA and AMC
were 0.72 and 0.68, respectively, indicating that the pNA/AMC
dual-reporter system is a statistically excellent assay.
[0165] Because the pNA and AMC fluorescence reads were recorded
individually, the data from each signal was also compared
separately to explore the hit rates using pNA or AMC versus the
dual-reporter system. Interestingly, a significantly high hit rate
was observed for each reporter alone. Analysis of the AMC reporter
alone revealed that 20.3% of compounds displayed a change in signal
greater than 3SD from the negative control, exemplifying an high
hit rate for a primary screen and portraying the difficulty in
triaging compounds for confirmation and follow-up. For the pNA
reporter only, the hit rate was lower relative to AMC, but also
high at 8.7%. Upon applying both signals for the hit criteria, the
overall hit rate was significantly reduced to 1.5% (2,363
compounds), indicating a reduction in the false-positive rate of
7.2-18.8%, depending upon which single reporter was used. Together,
these HTS results demonstrate the usefulness and efficiency of
applying a dual-reporter system for ruling out false-positives
compared to a single reporter assay.
[0166] Confirmation and Dose-Response Testing for PAI-1
Inhibition
[0167] Confirmation testing for purified compounds was performed as
described for the primary assay except that compounds were stamped
in triplicate. In addition, a `pre-read` was recorded after
addition of PAI-1, compound and uPA, but prior to substrate mixture
addition. Compounds that showed a change in pNA and AMC signals
>3SD in at least 2 out of 3 wells were further examined. Of this
subset, only compounds that showed <3SD change in signal in 2 or
less wells relative to the negative control in the pre-read were
chosen for dose-response testing. Based on these criteria, 300
compounds were chosen for dose-response testing in the CCG. For
this analysis, varying volumes (29-600 nL) of compound were stamped
in duplicate using a TTP Labtech Mosquito X1 liquid handling
system, resulting in an approximate final concentration of 12-250
.mu.M. Development was carried out as described for the primary
screen.
[0168] Methods of Using PAI-1 Inhibitors
[0169] As mentioned herein above, it is contemplated that methods
of the invention include treating a disease or disorder associated
with elevated levels of PAI-1 comprising administering a PAI-1
inhibitor. In one aspect, the subject is a mammal. In a preferred
aspect, the mammalian subject is human.
[0170] In one embodiment, the invention includes PAI-1 inhibitor
compounds and methods of using the compounds in the treatment of
many diseases or disorders associated with PAI-1 activity. Such
conditions, e.g., diseases or disorders, include, but are not
limited to, dysregulation of lipid metabolism, obesity, diabetes,
polycystic ovary syndrome, bone loss induced by estrogen
deficiency, fibrosis and fibrotic disease, inflammation, cell
migration and migration-driven proliferation of cells, and
angiogenesis or thrombosis. In one aspect, such inhibitors are also
contemplated to be useful for modulation of endogenous
fibrinolysis, and in conjunction with pharmacologic thrombolysis.
In another aspect, the invention includes PAI-1 inhibitor compounds
and methods of using the compounds in the treatment of acute
diseases associated with high PAI-1 levels, such as, but not
limited to, sepsis, myocardial infarction, and thrombosis, compared
to PAI-1 levels in normal subjects known not to suffer from sepsis,
myocardial infarction, or thrombosis. In another aspect, the PAI-1
inhibitor compounds of the invention are used in methods for
treating diseases and disorders associated with high PAI-1 levels,
such as, but not limited to, cancer, atherosclerosis, insulin
resistance, type 2 diabetes, and fibrotic diseases compared to
PAI-1 levels in normal subjects known not to suffer from these
diseases or disorders. In another aspect, the invention includes
PAI-1 inhibitor compounds for regulating lipid metabolism,
including increasing circulating HDL and/or decreasing circulating
VLDL in a subject.
[0171] In various aspects, a PAI-1 inhibitor is useful in the
treatment of any condition, including a disease or disorder,
wherein the lowering of PAI-1 levels will provide benefits. The
PAI-1 inhibitor is useful alone, or in combination with other
compounds, which may act as to promote the reduction of PAI-1
levels.
[0172] One of the therapeutic embodiments of the invention is the
provision, to a subject in need thereof, compositions comprising
one or more PAI-1 inhibitor. In one aspect, the PAI-1 inhibitor is
isolated from a known compound or is chemically synthesized. In
another aspect, the PAI-1 inhibitor formulation for therapy in a
subject is selected based on the route of administration and in
certain aspects includes liposome and micelle formulations as well
as classic pharmaceutical preparations.
[0173] The PAI-1 inhibitor is formulated into an appropriate
preparation and administered to one or more sites within the
subject in a therapeutically effective amount. In one embodiment,
the PAI-1 inhibitor-based therapy is effected via continuous or
intermittent intravenous administration. In one aspect, the PAI-1
inhibitor-based therapy is effected via continuous or intermittent
intramuscular or subcutaneous administration. In another aspect,
the PAI inhibitor-based therapy is effected via oral or buccal
administration. By "effective amount" the invention refers to an
amount of PAI-1 inhibitor compound that is sufficient to support an
observable change in the level of one or more biological activities
of PAI-1, plasminogen activator, HDL, LDL, or VLDL and/or an
observable change in an indication for which the method of
treatment is intended. The change may be reduced level of PAI-1
activity. In one aspect, the change is an increase in plasminogen
activator, and/or HDL and/or a reduction in LDL and VLDL.
[0174] In various aspects, administration of the compositions is
systemic or local, and in still other aspects comprises a single
site injection of a therapeutically-effective amount of the PAI-1
inhibitor composition. Any route known to those of skill in the art
for the administration of a therapeutic composition of the
invention is contemplated including, for example, intravenous,
intramuscular, subcutaneous, oral, or a catheter for long-term
administration.
[0175] Alternatively, it is contemplated that the therapeutic
composition is delivered to the patient at multiple sites. The
multiple administrations are rendered simultaneously or are
administered over a period of several hours. It is likewise
contemplated that the therapeutic composition is taken on a regular
basis via oral administration. In certain cases, it is beneficial
to provide a continuous flow of the therapeutic composition.
Additional therapy is administered on a period basis, for example,
daily, weekly, or monthly.
[0176] In addition to therapies based solely on the delivery of the
PAI-1 inhibitor composition, combination therapy is specifically
contemplated. In the context of the invention, it is contemplated
that the PAI-1 inhibitor composition therapy is used similarly in
conjunction with other agents commonly used for the treatment of
elevated levels of PAI-1, LDL and VLDL.
[0177] To achieve the appropriate therapeutic outcome, using the
methods and compositions of the invention, one would generally
provide a composition comprising a PAI-1 inhibitor and at least one
other therapeutic agent (second therapeutic agent). In one aspect
of the invention, it is contemplated that methods include
administration or inclusion of at least one additional factor or
other drug. Such drugs include drugs used to manage cardiovascular
disease including, but not limited to, cholesterol lowering drugs,
such as statins, anti-inflammatories, and ACE inhibitors. Such
drugs also include drugs targeting neurological disorders
including, but not limited to drugs for targeting stroke, seizures,
and Alzheimer's Disease. In another aspect, the additional drugs
include, but are not limited to, drugs targeting diabetes. These
are all disorders associated with elevated levels of PAI-1 and,
therefore, it is contemplated that combination therapy may be used
with PAI-1 inhibitors and other known therapies.
[0178] The combination therapy compositions are provided in a
combined amount effective to produce the desired therapeutic
outcome in the treatment of increased levels of PAI-1, VLDL, or LDL
and/or make a detectable change in an indication as described
herein. This process involves administering the PAI-1 inhibitor and
the second agent(s) or factor(s) at the same time. Methods thus
include administering a single composition or pharmacological
formulation that includes both agents, or administering two
distinct compositions or formulations, at the same time, wherein
one composition includes the PAI-1 inhibitor therapeutic
composition and the other includes the second therapeutic
agent.
[0179] Alternatively, the PAI-1 inhibitor treatment precedes or
follows the second therapeutic agent treatment by intervals ranging
from minutes to weeks. In embodiments where the second therapeutic
agent and the PAI-1 inhibitor are administered separately, one
generally ensures that a significant period of time did not expire
between the times of each delivery, such that the second
therapeutic agent and the PAI-1 inhibitor are able to exert an
advantageously combined effect. In such instances, it is
contemplated that one administers both modalities within about
12-24 hours of each other, or alternately, within about 6-12 hours
of each other, or alternately, with a delay time of only about 12
hours. In some situations, it is desirable to extend the time
period for treatment significantly, however, where several days (2,
3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse
between the respective administrations.
[0180] Systemic delivery of PAI-1 inhibitors to patients is a very
efficient method for delivering a therapeutically effective amount
of the compound to counteract the immediate clinical manifestations
of a disease or disorder. Alternatively, local delivery of the
PAI-1 inhibitor and/or the second therapeutic agent is appropriate
in certain circumstances. In a certain embodiment, it is
contemplated that the PAI-1 inhibitor is delivered to a patient for
an extended period of time. It is further contemplated that the
PAI-1 inhibitor is taken throughout a patient's lifetime to lower
PAI-1, VLDL and/or LDL levels.
[0181] Pharmaceutical Compositions
[0182] As mentioned herein above, the invention also comprehends
methods using pharmaceutical compositions comprising effective
amounts of PAI-1 inhibitor together with pharmaceutically
acceptable diluents, preservatives, solubilizers, emulsifiers,
adjuvants and/or carriers useful in PAI-1 inhibitor therapy. Such
compositions include diluents of various buffer content (e.g.,
Tris-HCl, acetate, phosphate), pH and ionic strength; additives
such as detergents and solubilizing agents (e.g., Tween 80,
Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium
metabisulfite), preservatives (e.g., thimersol, benzyl alcohol),
and bulking substances (e.g., lactose, mannitol); incorporation of
the material into particulate preparations of polymeric compounds,
such as polylactic acid, polyglycolic acid, etc., or in association
with liposomes or micelles. Such compositions will influence the
physical state, stability, rate of in vivo release, and rate of in
vivo clearance of the PAI-1 inhibitor. See, e.g., Remington's
Pharmaceutical Sciences, 18th Ed. (1990) Mack Publishing Co.,
Easton, Pa., pages 1435-1712, which are herein incorporated by
reference.
[0183] Sterile liquid compositions include solutions, suspensions,
emulsions, syrups and elixirs. The compounds of this invention may
be dissolved or suspended in the pharmaceutically acceptable
carrier, such as sterile water, sterile organic solvent or a
mixture of both. In one aspect, the liquid carrier is one suitable
for parental injection. Where the compounds are sufficiently
soluble they can be dissolved directly in normal saline with or
without the use of suitable organic solvents, such as propylene
glycol or polyethylene glycol. If desired, dispersions of the
finely divided compounds can be made-up in aqueous starch or sodium
carboxymethyl cellulose solution, or in a suitable oil, such as
arachis oil. Liquid pharmaceutical compositions, which are sterile
solutions or suspensions, can be utilized by intramuscular,
intraperitoneal or subcutaneous injection. In many instances a
liquid composition form may be used instead of the preferred solid
oral method of administration.
[0184] It is preferred to prepare unit dosage forms of the
compounds for standard administration regimens. In this way, the
composition can be subdivided readily into smaller doses at the
physician's direction. For example, unit dosages may be made up in
packeted powders, vials or ampoules and, in one aspect, in capsule
or tablet form. The active compound present in these unit dosage
forms of the composition may be present in an amount of from about
one gram to about fifteen grams or more, for single or multiple
daily administration, according to the particular need of the
patient. The daily dose of active compound will vary depending upon
the route of administration, the size, age and sex of the patient,
the severity of the disease state, and the response to the therapy
as traced by blood analysis and the patient's recovery rate.
[0185] The precise dosage to be employed depends upon several
factors including the host, whether in veterinary medicine or human
medicine, the nature and severity of the condition, e.g., disease
or disorder, being treated, the mode of administration and the
particular active substance employed. The compounds may be
administered by any conventional route, in particular enterally,
and, in one aspect, orally in the form of tablets or capsules.
Administered compounds can be in the free form or pharmaceutically
acceptable salt form as appropriate, for use as a pharmaceutical,
particularly for use in the prophylactic or curative treatment of
atherosclerosis and sequelae (angina pectoris, myocardial
infarction, arrhythmias, heart failure, kidney failure, stroke,
peripheral arterial occlusion, and related disease states). These
measures will slow the rate of progress of the disease state and
assist the body in reversing the process direction in a natural
manner.
[0186] PAI-1 inhibitors or derivatives thereof may be formulated
for injection, or oral, nasal, pulmonary, topical, or other types
of administration as one skilled in the art will recognize. The
formulation may be liquid or may be solid, such as lyophilized, for
reconstitution.
[0187] PAI-1 inhibitor or derivatives thereof are useful in the
treatment of any of the acute or chronic diseases or disorders
associated with increased levels of PAI-1, LDL, or VLDL. Conditions
(e.g., diseases or disorders) alleviated or modulated by the
administration of PAI-1 inhibitor, in some aspects, are those
characterized by increased levels of VLDL and LDL. Such conditions
may be induced as a course of therapy for other purposes, such as
chemotherapy or radiation therapy. It is contemplated that such
conditions may result from genetic inheritance or be the side
effect of another condition or medication.
[0188] The phrase "pharmaceutically or pharmacologically
acceptable" refers to molecular entities and compositions that do
not produce adverse, allergic, or other untoward reactions when
administered to an animal or a human. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutically active substances
is well-known in the art. Except insofar as any conventional media
or agent is incompatible with the vectors or cells of the
invention, its use in therapeutic compositions is contemplated.
Supplementary active ingredients also can be incorporated into the
compositions.
[0189] The active compositions used in the methods of the invention
include classic pharmaceutical preparations. Administration of
these compositions according to the invention will be via any
common route so long as the target tissue is available via that
route. The pharmaceutical compositions may be introduced into the
subject by any conventional method, e.g., by intravenous,
intradermal, intramusclar, intramammary, intraperitoneal,
intrathecal, retrobulbar, intrapulmonary (e.g., term release); by
oral, sublingual, nasal, anal, vaginal, or transdermal delivery, or
by surgical implantation at a particular site. The treatment may
consist of a single dose or a plurality of doses over a period of
time.
[0190] The active compounds may be prepared for administration as
solutions of free base or pharmacologically acceptable salts in
water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions also can be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0191] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to include
isotonic agents (for example, sugars or sodium chloride). Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption (for
example, aluminum monostearate and gelatin).
[0192] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with several of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle that contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques that
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0193] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well-known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients also can be
incorporated into the compositions.
[0194] For oral administration of the compositions used in the
methods of the invention, a PAI-1 inhibitor may be incorporated
with excipients and used in the form of non-ingestible mouthwashes
and dentifrices. A mouthwash may be prepared incorporating the
active ingredient in the required amount in an appropriate solvent,
such as a sodium borate solution (Dobell's Solution).
Alternatively, the active ingredient may be incorporated into an
antiseptic wash containing sodium borate, glycerin and potassium
bicarbonate. The active ingredient may also be dispersed in
dentifrices, including: gels, pastes, powders and slurries. The
active ingredient may be added in a therapeutically effective
amount to a paste dentifrice that may include water, binders,
abrasives, flavoring agents, foaming agents, and humectants.
[0195] The compositions used in the methods of the invention may be
formulated in a neutral or salt form. Pharmaceutically-acceptable
salts include the acid addition salts (formed with the free amino
groups of the protein) and which are formed with inorganic acids
such as, for example, hydrochloric or phosphoric acids, or such
organic acids as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed with the free carboxyl groups also can be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0196] The compositions used in the methods of the invention may be
formulated in micelles or liposomes. Such formulations include
sterically stabilized micelles or liposomes and sterically
stabilized mixed micelles or liposomes. Such formulations can
facilitate intracellular delivery, since lipid bilayers of
liposomes and micelles are known to fuse with the plasma membrane
of cells and deliver entrapped contents into the intracellular
compartment.
[0197] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms such as injectable solutions, drug
release capsules and the like. For parenteral administration in an
aqueous solution, for example, the solution should be suitably
buffered if necessary and the liquid diluent first rendered
isotonic with sufficient saline or glucose. These particular
aqueous solutions are especially suitable for intravenous,
intramuscular, subcutaneous and intraperitoneal administration.
[0198] Generally, an effective amount of a PAI-1 inhibitor, or
derivatives thereof, will be determined by the age, weight, and
condition or severity of disease or disorder of the recipient. See,
Remington's Pharmaceutical Sciences, supra, pages 697-773, herein
incorporated by reference. Typically, a dosage of between about
0.001 .mu.g/kg body weight/day to about 1000 .mu.g/kg body
weight/day, may be used, but more or less, as a skilled
practitioner will recognize, may be used. Dosing may be one or more
times daily, or less frequently, and may be in conjunction with
other compositions as described herein. It should be noted that the
invention is not limited to the dosages recited herein.
[0199] By initiating the treatment regimen with a minimal daily
dose of about one gram, the blood levels of PAI-1 and the patient's
symptomatic relief analysis may be used to determine whether a
larger dose is indicated. One skilled in the art will appreciate
that the appropriate dosage levels for treatment will thus vary
depending, in part, upon the molecule delivered, the indication for
which the PAI-1 inhibitor compound is being used, the route of
administration, and the size (body weight, body surface or organ
size) and condition (the age and general health) of the patient.
Accordingly, the clinician may titer the dosage and may modify the
route of administration to obtain the optimal therapeutic effect. A
typical dosage may range from about 0.1 .mu.g/kg to up to about 100
mg/kg or more, depending on the factors mentioned above. In other
embodiments, the dosage may range from 0.1 .mu.g/kg up to about 100
mg/kg; or 1 .mu.g/kg up to about 100 mg/kg; or 5 .mu.g/kg up to
about 100 mg/kg.
[0200] "Unit dose" is defined as a discrete amount of a therapeutic
composition dispersed in a suitable carrier. Parenteral
administration may be carried out with an initial bolus followed by
continuous infusion to maintain therapeutic circulating levels of
drug product. Those of ordinary skill in the art will readily
optimize effective dosages and administration regimens as
determined by good medical practice and the clinical condition of
the individual patient.
[0201] The frequency of dosing will depend on the pharmacokinetic
parameters of the agents and the routes of administration. The
optimal pharmaceutical formulation will be determined by one of
skill in the art depending on the route of administration and the
desired dosage. See, for example, Remington's Pharmaceutical
Sciences, supra, pages 1435-1712, incorporated herein by reference.
Such formulations may influence the physical state, stability, rate
of in vivo release and rate of in vivo clearance of the
administered agents. Depending on the route of administration, a
suitable dose may be calculated according to body weight, body
surface areas or organ size. Further refinement of the calculations
necessary to determine the appropriate treatment dose is routinely
made by those of ordinary skill in the art without undue
experimentation, especially in light of the dosage information and
assays disclosed herein, as well as the pharmacokinetic data
observed in animals or human clinical trials.
[0202] Appropriate dosages may be ascertained through the use of
established assays for determining level of myocardial infarct in
conjunction with relevant dose-response data. The final dosage
regimen will be determined by the attending physician, considering
factors that modify the action of drugs, e.g., the drug's specific
activity, severity of the damage and the responsiveness of the
patient, the age, condition, body weight, sex and diet of the
patient, the severity of any infection, time of administration and
other clinical factors. As studies are conducted, further
information will emerge regarding appropriate dosage levels and
duration of treatment.
[0203] It will be appreciated that the pharmaceutical compositions
and treatment methods of the invention are useful in fields of
human medicine and veterinary medicine. Thus the subject to be
treated is in one aspect a mammal. In another aspect, the mammal is
a human.
[0204] In addition, the invention contemplates a kit containing
components comprising a composition comprising a PAI-1 inhibitor;
and optionally, at least one additional factor useful in the
treatment of the acute and chronic diseases and disorders discussed
herein.
[0205] Uses of Compounds of the Invention in the Treatment of
Diseases or Disorders
[0206] The invention includes the use of compounds of the invention
for the production of a medicament for the treatment or prevention
of any disease or disorder discussed herein.
[0207] The compounds of the invention are inhibitors of the serine
protease inhibitor PAI-1, and are therefore useful in the treatment
or prophylaxis of those processes which involve the production
and/or action of PAI-1. Thus, the compounds of the invention, in
various aspects, are useful in preventing or reducing thrombosis,
promoting thrombolysis, reducing fibrosis regulating lipid
metabolism as described herein. In one aspect, the compounds of the
invention are useful in treating high cholesterol and diseases or
disorders associated with elevated levels of PAI-1. In another
aspect, the compounds of the invention are useful in treating
elevated levels of VLDL or LDL. In another aspect, the compounds of
the invention are useful in elevating HDL.
[0208] In one aspect, the invention includes the uses of these
inhibitors for the treatment of many diseases or disorders
associated with PAI-1 activity. Such diseases or disorders include,
but are not limited to, inflammation, cell migration and
migration-driven proliferation of cells, and angiogenesis or
thrombosis. Such inhibitors are also contemplated to be useful for
modulation of endogenous fibrinolysis, and in conjunction with
pharmacologic thrombolysis.
[0209] The compounds of the invention are useful in the treatment
or prevention of insulin resistance, obesity, non-insulin dependent
diabetes mellitus, cardiovascular disease, thrombotic events
associated with coronary artery and cerebrovascular disease. The
compounds of the invention are also useful for inhibiting the
disease process involving the thrombotic and prothrombotic states
which include, but are not limited to, formation of atherosclerotic
plaques, venous and arterial thrombosis, myocardial ischemia,
atrial fibrillation, deep vein thrombosis, coagulation syndromes,
pulmonary thrombosis, cerebral thrombosis, thromboembolic
complications of surgery (such as joint replacement), and
peripheral arterial occlusion. These compounds are also useful in
treating stroke associated with or resulting from atrial
fibrillation.
[0210] The compounds of the invention are also used in the
treatment or prophylaxis of high cholesterol and diseases or
disorders associated with such a condition.
[0211] The compounds of the invention may also be used in the
treatment of diseases or disorders associated with extracellular
matrix accumulation, including, but not limited to, renal fibrosis,
chronic obstructive pulmonary disease, polycystic ovary syndrome,
restenosis, renovascular disease and organ transplant
rejection.
[0212] The compounds of the invention may also be used in the
treatment of malignancies, and diseases or disorders associated
with neoangiogenesis (such as diabetic retinopathy).
[0213] The compounds in the invention may also be used in
conjunction with and following processes or procedures involving
maintaining blood vessel patency, including vascular surgery,
vascular graft and stent patency, organ, tissue and cell
implantation and transplantation.
[0214] The compounds of the invention may also be used in the
treatment of Alzheimer's disease. This method may also be
characterized as the inhibition of plasminogen activator by PAI-1
in a mammal, particularly a human, experiencing or subject to
Alzheimer's disease. This method may also be characterized as a
method of increasing or normalizing levels of plasmin concentration
in a mammal, particularly those experiencing or subject to
Alzheimer's disease.
[0215] The compounds of the invention may be used for the treatment
of myelofibrosis with myeloid metaplasia by regulating stromal cell
hyperplasia and increases in extracellular matrix proteins.
[0216] The compounds of the invention may also be used in
conjunction with protease inhibitor-containing highly active
antiretroviral therapy (HAART) for the treatment of diseases or
disorders which originate from fibrinolytic impairment and
hypercoagulability of HIV-1 infected patients receiving such
therapy.
[0217] The compounds of the invention may be used for the treatment
of diabetic nephropathy and renal dialysis associated with
nephropathy.
[0218] The compounds of the invention may be used to treat cancer,
septicemia, proliferative diseases, such as psoriasis, improving
coagulation homeostasis, cerebrovascular diseases, microvascular
disease, hypertension, dementia, atherosclerosis, osteoporosis,
arthritis, asthma, heart failure, arrhythmia, angina, and as a
hormone replacement agent, treating, preventing or reversing
progression of atherosclerosis, Alzheimer's disease, osteoporosis,
osteopenia; reducing inflammatory markers, fibrinolytic disorder,
reducing C-reactive protein, or preventing or treating low grade
vascular inflammation, stroke, dementia, coronary heart disease,
primary and secondary prevention of myocardial infarction, stable
and unstable angina, primary prevention of coronary events,
secondary prevention of cardiovascular events, peripheral vascular
disease, peripheral arterial disease, acute vascular syndromes,
deep vein thrombosis, pulmonary embolism, reducing the risk of
undergoing a myocardial revascularization procedure, microvascular
diseases such as nephropathy, neuropathy, retinopathy and nephrotic
syndrome, hypertension, Type 1 and 2 diabetes and related diseases,
obesity, insulin resistance, hyperglycemia, hyperinsulinemia,
malignant lesions, premalignant lesions, gastrointestinal
malignancies, liposarcomas and epithelial tumors, proliferative
diseases such as psoriasis, improving coagulation homeostasis,
and/or improving endothelial function, and all forms of
cerebrovascular diseases.
[0219] The compounds of the invention may be used for the topical
applications in wound healing for prevention of scarring.
[0220] The compounds in the invention can be used in the treatment
of inflammatory diseases, septic shock and the vascular damage
associated with infections and for the treatment of blood and blood
products used in dialysis, blood storage in the fluid phase,
especially ex vivo platelet aggregation. The compounds in the
present invention may also be used in combination with
prothrombolytic, fibrinolytic and anticoagulant agents. The present
compounds may also be added to human plasma during the analysis of
blood chemistry in hospital settings to determine the fibrinolytic
capacity thereof.
[0221] This invention further comprises methods for treating,
preventing, ameliorating or inhibiting each of the maladies
mentioned herein in a mammal, in one aspect, in a human, the
method(s) each comprising administering to a mammal in need of such
treatment, prevention, amelioration or inhibition a
pharmaceutically or therapeutically effective amount of a compound
of this invention, or a pharmaceutically acceptable salt, ester, or
prodrug form thereof.
[0222] The compounds of the invention may also be used to treat
cancer including, but not limited to, breast and ovarian cancer,
and as imaging agents for the identification of metastatic
cancers.
[0223] It will be understood that a pharmaceutically or
therapeutically effective amount of a compound herein refers to an
amount of the compound in question which will sufficiently inhibit
the serine protease inhibitor PAI-1 in the mammal in need thereof
to a sufficient extent to provide a desirable improvement in the
condition in question or provide sufficient inhibition of the
serine protease inhibitor PAI-1 to prevent, inhibit or limit the
onset of the physiological basis for the malady or condition in
question.
EXAMPLES
[0224] The invention is described in more detail with reference to
the following non-limiting examples, which are offered to more
fully illustrate the invention, but are not to be construed as
limiting the scope thereof. Those of skill in the art will
understand that the techniques described in these examples
represent techniques described by the inventors to function well in
the practice of the invention, and as such constitute preferred
modes for the practice thereof. However, it should be appreciated
that those of skill in the art should in light of the present
disclosure, appreciate that many changes can be made in the
specific methods that are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
[0225] As discussed herein above, elevated levels of PAI-1 have
been implicated in a variety of diseases and disorders. The
development of therapeutic agents that act as selective inhibitors
of PAI-1 may provide an approach to treat these diseases and
disorders. The design and synthesis of a variety of compounds and
their structure:activity relationship with PAI-1 is described.
Additional synthetic methods for obtaining PAI-1 inhibitors are
disclosed in US 2010/0137194, which is incorporated by reference in
its entirety.
Example 1
Synthesis of Compounds of Formula III
[0226] General Procedure A
[0227] Compounds of formula III were synthesized according to the
procedure described below and as shown in the following scheme:
##STR00251##
[0228] To a 0.degree. C. solution of an appropriately-substituted
carboxylic acid (1 equiv.), an appropriately-substituted amine (1.2
equiv.), N-methylmorpholine (1 equiv.), and HOBT.H.sub.2O (1
equiv.) in 2.5:1 dry CH.sub.2Cl.sub.2 and DMF, EDC.HCl (1 equiv.)
was added in portions. The resulting slurry was allowed to warm to
room temperature overnight with stirring. The reaction mixture was
concentrated by rotary evaporation, diluted with a 4:1 solution of
EtOAc and hexane, washed with 0.1N HCl (2.times.), saturated NaHCO3
(2.times.), and brine (2.times.). The organic phase was dried with
anhydrous MgSO.sub.4 and concentrated in vacuo. The resultant solid
was triturated with chloroform and the solid obtained after
filtration was dried in vacuo, which afforded compounds of formula
III.
Synthesis of
N-(4-chloro-3-(trifluoromethyl)benzyl)-3-(3,4-dihydroxyphenyl)propanamide
(compound C330)
[0229]
N-(4-chloro-3-(trifluoromethyl)benzyl)-3-(3,4-dihydroxyphenyl)propa-
namide (compound C330) was synthesized according to General
Procedure A. To a 0.degree. C. solution of 3,4-dihydroxyhydro
cinnamic acid (205.8 mg, 1.13 mmol), 4-chloro-3-(trifluoromethyl)
benzylamine (0.2 mL, 1.36 mmol), N-methylmorpholine (0.12 mL, 1.13
mmol) and HOBT.H.sub.2O (173.0 mg, 1.13 mmol) in 5 mL of dry
CH.sub.2Cl.sub.2 and 2 mL of DMF, EDC.HCl (216.6 mg, 1.13 mmol) was
added in portions. The resulting slurry was allowed to warm to room
temperature overnight with stirring. The reaction mixture was
concentrated by rotary evaporation, diluted with 30 mL of a 4:1
solution of EtOAc and hexane, washed with 0.1N HCl (2.times.),
saturated NaHCO3 (2.times.), and brine (2.times.). The organic
phase was dried with anhydrous MgSO.sub.4 and concentrated in
vacuo. The resultant solid was triturated with chloroform and the
solid obtained after filtration was dried in vacuo, which afforded
235 mg (48%) of product as a pale yellow crystalline solid. .sup.1H
NMR (DMSO-d.sub.6, 400 MHz) .delta. 8.66 (s, 1H), 8.59 (s, 1H),
8.35 (t, J=6.0 Hz, 1H), 7.66 (s, 1H), 7.57 (d, J=8.2 Hz, 1H), 7.31
(d, J=8.2 Hz, 1H), 6.57 (d, J=8.2 Hz, 1H), 6.54 (d, J=1.8 Hz, 1H),
6.39 (dd, J=1.8, 8.2 Hz, 1H), 4.27 (d, J=6.0 Hz, 2H), 2.62 (t,
J=7.8 Hz, 2H), 2.33 (t, J=7.7 Hz, 2H). .sup.13C NMR (DMSO-d.sub.6,
100 MHz) .delta. 172.32, 145.53, 143.89, 140.46, 133.24, 132.45,
131.99, 129.25, 126.99 (q, J=5.7 Hz), 126.53 (q, J=30.5 Hz),
125.96, 123.41 (q, J=270.8 Hz), 119.30, 116.29, 115.92, 41.54,
37.90. HRMS, DART calcd. for C.sub.17H.sub.15F.sub.3NO.sub.3Cl
[M+H].sup.+374.07707, found: 374.07880.
[0230] The following compounds also were synthesized according to
General Procedure A: C279, C286, C330, C344, C345, C346, C347,
C348, C356, C357, C358, C359, C360, C361, C363, C364.
Synthesis of
N-(4-chloro-3-(trifluoromethyl)benzyl)-5-oxo-4,5-dihydro-1,3,4-oxadiazole-
-2-carboxamide (compound C285)
[0231] N-(4-Chloro-3
-(trifluoromethyl)benzyl)-5-oxo-4,5-dihydro-1,3,4-oxadiazole-2-carboxamid-
e (C285) was synthesized according to the procedure described below
and as shown in the following scheme:
##STR00252##
[0232] To a solution of compound C251 (0.125 g, 0.422 mmol) in 6 mL
tetrahydrofuran was added 1,1'-carbonyldiimidazole (0.082 g, 0.506
mmol). The mixture was allowed to stir for 18 hr at room
temperature, at which time the reaction was quenched with 1N HCl,
taken up in 30 mL ethyl acetate, and washed with brine. The organic
layer was dried, filtered, and concentrated in vacuo. The resulting
residue was triturated with chloroform and filtered to provide
0.087 g (64%) of product (compound C285) as a solid.
[0233] Compounds C299 and C306 were prepared according to above
procedure except that compound C282 and compound C305,
respectively, were used in place of compound C251.
Example 2
Synthesis of Compounds of Formula IV
[0234] General Procedure B
[0235] Compounds of formula IV were synthesized according to the
procedure described below and as shown in the following scheme:
##STR00253##
[0236] Step 1: To a 0.degree. C. solution of triphosgene (1 equiv.)
in dichloromethane, an appropriately-substituted amine (2.5 equiv.)
was added dropwise with stirring. A solution of triethylamine (4.7
equiv.) in dichloromethane was then added. The mixture was allowed
to stir for 5 min at 0.degree. C., then overnight at 25.degree. C.
The reaction mixture was concentrated in vacuo, triturated with
ethyl acetate, and filtered. The filtrate was concentrated in vacuo
to provide the isocyanate product.
[0237] Step 2: An ethanolic solution of hydroxylamine (1 equiv.)
was added to a --60.degree. C. solution of an
appropriately-substituted isocyanate (1 equiv.) in dry
dichloromethane. The mixture was allowed to warm to room
temperature and stir for 18 hr. The reaction solution was chilled
in an ice bath and filtered cold. The filtrate was concentrated in
vacuo and the resulting residue was taken up in ethyl acetate and
washed with brine (1.times.). The organic layer was separated,
dried, filtered, and concentrated in vacuo. The resulting residue
was triturated with chloroform and filtered to provide compounds of
formula IV.
Synthesis of 4-chloro-3-trifluoromethylbenzyl isocyanate
[0238] To a 0.degree. C. solution of triphosgene (0.630 g, 2.12
mmol) in 10 mL dichloromethane, 4-chloro-3-trifluoromethylbenzyl
amine (0.800 mL, 5.23 mmol) was added dropwise with stirring. A
solution of triethylamine (1.39 mL, 10 mmol) in 5 mL
dichloromethane was then added. A white precipitate formed
immediately. The mixture was allowed to stir for 5 min at 0.degree.
C., then overnight at 25.degree. C. The reaction mixture was
concentrated in vacuo, triturated with ethyl acetate, and filtered.
The filtrate was concentrated in vacuo to provide 1.19 g (97%) of
4-chloro-3-trifluoromethylbenzyl isocyanate as a clear oil.
Synthesis of 1-(4-chloro-3-(trifluoromethyl)benzyl)-3-hydroxyurea
(compound C284)
[0239] An ethanolic hydroxylamine solution was prepared by addition
at 0.degree. C. of a solution of 25 mmol NaOH in 55 mL absolute
ethanol to a stirring suspension of 25 mmol hydroxylamine
hydrochloride in 60 mL of absolute ethanol. 8.9 mL of the ethanolic
solution of hydroxylamine (1.93 mmol) was added to a --60.degree.
C. solution of 4-chloro-3-trifluoromethylbenzyl isocyanate (0.455
g, 1.93 mmol) in 10 mL dry dichloromethane. The mixture was allowed
to warm to room temperature and stir for 18 hr. The reaction
solution was chilled in an ice bath and filtered cold. The filtrate
was concentrated in vacuo and the resulting residue was taken up in
30 mL ethyl acetate and washed with brine (1.times.). The organic
layer was separated, dried, filtered, and concentrated in vacuo.
The resulting residue was triturated with chloroform and filtered
to provide 0.140 g (27%) of compound C284 as a white solid.
Example 3
Synthesis of Compounds of Formula V
[0240] General Procedure C
[0241] Compounds of formula V were synthesized according to the
procedure described below and as shown in the following scheme:
##STR00254##
[0242] To a 0.degree. C. solution of an appropriately-substituted
amine (1 equiv.) and pyridine (3.7 equiv.) in dichloromethane, an
appropriately-substituted chlorocarbonyl ester (1 equiv.) was added
dropwise. The solution was removed from the ice bath and left to
react for 30 minutes. The reaction mixture was washed with 0.2 N
HCl (2.times.), saturated NaHCO.sub.3 (2.times.), and brine
(2.times.), and was then dried with MgSO.sub.4, filtered and
concentrated in vacuo to afford compounds of formula V.
Synthesis of ethyl
2-((4-chloro-3-(trifluoromethyl)benzyl)amino)-2-oxoacetate
(compound C256)
[0243] Ethyl 2-((4-chloro-3
-(trifluoromethyl)benzyl)amino)-2-oxoacetate (compound C256) was
synthesized according to General Procedure C. To a 0.degree. C.
solution of 4-chloro-3-trifluoromethylbenzylamine (187 .mu.L, 1.23
mmol) and pyridine (370 .mu.L, 4.6 mmol) in dichloromethane (6 ml),
ethyl 2-chloro-2-oxoacetate (170 .mu.L, 1.25 mmol) was added
dropwise. The solution was removed from the ice bath and left to
react for 30 minutes. The reaction mixture was washed with 0.2 N
HCl (2.times.), saturated NaHCO.sub.3 (2.times.), and brine
(2.times.), and was then dried with MgSO.sub.4, filtered and
concentrated in vacuo to afford 0.381 g of compound C256 as a white
solid (74%). .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 7.48 (m,
3H), 4.53 (d, J=6.4 Hz, 2H), 4.35 (q, J=7.3 Hz, 6.9 Hz, 2H), 1.38
(t, J=6.9 Hz, 3H); .sup.13C NMR (CDCl.sub.3, 100 MHz) .delta.
160.48, 156.87, 136.25, 132.48, 131.94, 131.85, 128.77 (q, J=31.5
Hz), 127.15 (q, J=4.8 Hz), 122.69 (q, J=271.7 Hz), 63.53, 42.89,
13.97.
[0244] The following compounds also were synthesized according to
General Procedure C: C256, C259, C265, C267, C276, C277, C288.
[0245] General Procedure D
[0246] Carboxylic acids were synthesized according to the procedure
described below and as shown in the following scheme:
##STR00255##
[0247] An appropriately-substituted ester (1 equiv.) was dissolved
in a 2:1 solution of ethanol and water. 1.0 N NaOH (4 equiv.) was
added to the reaction. After TLC analysis indicated that the
starting material had been completely consumed, approximately 3
equivalents of 1.0 N HCl were used to quench the reaction. The
reaction was cooled over ice as a precipitate formed and was then
concentrated via filtration to afford the product carboxylic
acid.
Synthesis of
2-((4-chloro-3-(trifluoromethyl)benzyl)amino)-2-oxoacetic acid
(compound C309)
[0248] 2-((4-Chloro-3-(trifluoromethyl)benzyl)amino)-2-oxoacetic
acid (compound C309) was synthesized according to General Procedure
D. Ethyl 2-((4-chloro-3-(trifluoromethyl)benzyl)amino)-2-oxoacetate
(324.7 mg, 1.0 mmol) was dissolved in 12 ml of a 2:1 solution of
ethanol and water. 4 ml of 1.0 N NaOH (4.0 mmol) was added to the
reaction. After approximately 5 minutes, TLC analysis indicated
that the starting material had been completely consumed.
Approximately 3 equivalents of 1.0 N HCl (12 ml) were used to
quench the reaction. The reaction was cooled over ice as a
precipitate formed and was then concentrated via filtration to
afford 143 mg (50%) of product as a white solid. .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta. 9.42 (t, J=6.0 Hz, 1H), 7.65 (d,
J=8.3 Hz, 1H), 7.73 (d, J=1.8 Hz, 1H), 7.54 (dd, J=8.3 Hz, 1H),
4.34 (d, J=6.44 Hz, 2H); .sup.13C NMR (DMSO-d.sub.6, 100 MHz)
.delta. 162.38, 159.10, 139.26, 133.70, 132.15, 129.70 (q, J=1.9
Hz), 127.39 (q, J=4.8 Hz), 126.95 (q, J=30.5 Hz), 123.38 (q,
J=271.7 Hz), 42.09. Percent Yield: 50.74%. HRMS, DART calcd. for
C.sub.10H.sub.8ClF.sub.3NO.sub.3 282.01448, found: 282.01169.
[0249] The following compound also was synthesized according to
General Procedure D: C311.
Example 4
Synthesis of Compounds of Formula VI
[0250] General Procedure E
[0251] Compounds of formula VI were synthesized according to the
procedure described below and as shown in the following scheme:
##STR00256##
[0252] To a mixture of an appropriately-substituted carboxylic acid
(1 equiv.), an appropriately-substituted amine (1.15 equiv.),
1-hydroxybenzotriazole [HOBt.H.sub.2O] (1.15 equiv.),
N-methylmorpholine [NMM] (1.15 equiv.) in methylene chloride,
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDC.HCl) (1.15 equiv.) was added and stirred for overnight. The
resulting solution was diluted with ethyl acetate and washed with
1N HCl (2.times.), saturated NaHCO.sub.3 (2.times.), and brine
solution, dried with MgSO.sub.4, filtered, and concentrated in
vacuo to obtain compounds of formula VI.
Synthesis of
N-(4-chloro-3-(trifluoromethyl)benzyl)-N'-methoxyoxalamide
(compound C320)
[0253] N-(4-chloro-3-(trifluoromethyl)benzyl)-N'-methoxyoxalamide
(compound C320) was synthesized according to General Procedure E.
To a mixture of
2-((4-chloro-3-(trifluoromethyl)benzyl)amino)-2-oxoacetic acid
(103.8 mg, 0.369 mmol), methoxyamine hydrochloride (36.9 mg, 0.426
mmol), 1-hydroxybenzotriazole [HOBt.H.sub.2O] (65.8 mg, 0.426
mmol), N-methylmorpholine [NMM] (47.0 .mu.l, 0.426 mmol) in
methylene chloride (4 ml),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC.HCl) (82.4 mg, 0.426 mmol) was added and stirred for
overnight. The resulting solution was diluted with .about.20 ml of
ethyl acetate and washed with 1N HCl (2.times.), saturated
NaHCO.sub.3 (2.times.), and brine solution, dried with MgSO.sub.4,
filtered, and concentrated in vacuo to obtain 0.0541 g (47.2%) of
the product as a white solid. .sup.1H NMR (DMSO-d.sub.6, 400 MHz)
.delta. 12.11 (bs, 1H), 9.45 (t, J=5.9 Hz, 1H), 7.73 (d, J=1.8 Hz,
1H), 7.65 (d, J=8.2 Hz, 1H), 7.53 (dd, J=6.8 Hz, J=1.4 Hz, 1H),
4.33 (d, J=6.4 Hz, 2H), 3.59 (s, 3H); .sup.13C NMR (DMSO-d.sub.6,
100 MHz) .delta. 160.17, 156.99, 139.26, 133.78, 132.16, 129.70,
127.52 (q, J=4.77 Hz), 126.92 (q, J=31.46 Hz), 223.38 (q, J=270.8
Hz), 63.67, 41.83; HRMS, DART calcd. for
C.sub.11H.sub.11CIF.sub.3N.sub.2O.sub.3 [M+H].sup.+311.04103,
found: 311.03891.
[0254] The following compounds also were synthesized according to
General Procedure E: C280, C300, C313, C314, C320, C323, C326,
C328, C334, C342.
Example 5
Synthesis of Compounds of Formula VII
[0255] General Procedure F
[0256] Compounds of formula VII were synthesized according to the
procedure described below and as shown in the following scheme:
##STR00257##
[0257] An appropriately-substituted compound of formula V prepared
according to General Procedure C in Example 3 (1 equiv.) was
dissolved in ethanol. An appropriately-substituted aqueous
hydrazine (2 equiv.) was then added dropwise. The mixture was
allowed to stir at room temperature for 48 hours. The solution was
filtered, which afforded compounds of formula VII.
[0258] Synthesis of Compound C301
[0259] Compound C301 was synthesized according to General Procedure
F. Ethyl 2-(2,3-dichlorobenzylamino)-2-oxoacetate (88.5 mg, 0.32
mmol, according to General Procedure C) was dissolved in 5 mL
ethanol. 50% aqueous hydrazine hydrate (41 .mu.L, 0.64 mmol) was
then added dropwise. The mixture was allowed to stir at room
temperature for 48 hours. The solution was filtered, which afforded
0.2045 g (quantitative yield) of product as a white solid.
.sup.1H-NMR (DMSO-d.sub.6, 400 MHz) .delta. 10.06 (bs, 1H), 9.29
(t, J=6.0 Hz, 1H), 7.52 (dd, J=1.4, 7.8 Hz, 1H), 7.31 (t, J=7.8 Hz,
1H), 7.17 (dd, J=1.4, 7.8 Hz, 1H), 4.52 (bs, 2H), 4.38 (d, J=6.4
Hz, 2H); .sup.13C-NMR (DMSO-d.sub.6, 100 MHz) .delta. 160.67,
158.24, 138.79, 132.21, 130.29, 129.59, 128.59, 127.48, 41.31;
HRMS, DART calcd. for C.sub.9H.sub.10N.sub.3O.sub.2Cl.sub.2
[M+H.sup.+] 262.01500, found: 262.00989.
[0260] The following compounds also were synthesized according to
General Procedure F: C240, C241, C246, C248, C251, C255, C260,
C261, C262, C263, C264, C266, C268, C278, C281, C282, C287, C289,
C295, C296, C297, C301, C304, C305, C307, C310, C322, C336, C339,
C340, C341, C362.
Example 6
Synthesis of Triazole Compounds of Formula XIII
[0261] General Procedure G
[0262] Compounds of formula XIII were synthesized according to the
procedure described below and as shown in the following scheme:
##STR00258##
[0263] An appropriately-substituted carboxylic acid (1 equiv.), an
appropriately-substituted amidine (1.4 equiv.), HATU (1.1 equiv.),
diisopropylethylamine (2.8 equiv.), and DMF were added to a round
bottom flask, stirred at 25.degree. C. and monitored via TLC (20%
methanol in CH.sub.2Cl.sub.2 with 2 drops of acetic acid) for
consumption of acid and formation of intermediate. Upon consumption
of the acid, an appropriately-substituted hydrazine (1.5 equiv.)
and acetic acid (13.8 equiv.) were added, and the mixture was
stirred at 80.degree. C. and monitored via TLC (20% methanol in
CH.sub.2Cl.sub.2) for formation of product. The mixture was taken
up in 4:1 ethyl acetate/hexane, washed with saturated NaHCO.sub.3
solution (3.times.), dried with MgSO.sub.4, and concentrated by
rotary evaporation. Flash chromatography (10% methanol in DCM) was
used to isolate compounds of formula XIII. The above procedure was
adapted from that described in Castanedo, G. M. et al., J. Org.
Chem. 2011, 76, 1177-1179, which is incorporated by reference in
its entirety.
Synthesis of
4-(1-(tert-butyl)-3-phenyl-1H-1,2,4-triazol-5-yl)benzene-1,2-diol
(compound C201)
[0264]
4-(1-(Tert-butyl)-3-phenyl-1H-1,2,4-triazol-5-yl)benzene-1,2-diol
(compound C201) was synthesized according to General Procedure G.
3,4-Dihydroxybenzoic acid (265 mg, 1.72 mmol), benzamidine.HCl (292
mg, 2.43 mmol), HATU (707 mg, 1.86 mmol), diisopropylethylamine
(850 .mu.L, 4.86 mmol), and DMF (6 mL) were added to a 25 mL round
bottom flask, stirred at 25.degree. C. and monitored via TLC (20%
methanol in CH.sub.2Cl.sub.2 with 2 drops of acetic acid) for
consumption of acid and formation of intermediate. Upon consumption
of the acid, t-butylhydrazine.HCl (189 mg, 2.55 mmol) and acetic
acid (1 mL, 23.8 mmol) were added, and the mixture was stirred at
80.degree. C. and monitored via TLC (20% methanol in
CH.sub.2Cl.sub.2) for formation of product. The mixture was taken
up in 100 mL of 4:1 ethyl acetate/hexane, washed with saturated
NaHCO.sub.3 solution (3.times.), dried with MgSO.sub.4, and
concentrated by rotary evaporation. Flash chromatography (10%
methanol in DCM) was used to isolate 14.6 mg of product (3%).
.sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 8.10 (dd, J=1.36, 8.24
Hz, 2H), 7.5-7.36 (m, 3H), 6.77 (d, J=1.84 Hz, 1H), 6.72 (d, J=7.8
Hz, 1H), 6.66 (dd, J=1.84, 7.76 Hz, 1H), 1.51 (s, 9H); .sup.13C
(CDCl.sub.3, 100 MHz) .delta. 157.86, 155.02, 147.25, 143.99,
130.51, 129.37, 128.82, 128.71, 126.35, 121.92, 117.84, 114.90,
61.83, 30.76.
[0265] The following compounds also were synthesized according to
General Procedure G: C208, C213, C214, C216, C220, C221, C222,
C223.
Example 7
Synthesis of Thiazolidinedione Compounds of Formula XIV
3-(3-Bromobenzyl)thiazolidine-2,4-dione
[0266] 3-(3-Bromobenzyl)thiazolidine-2,4-dione was synthesized
according to the procedure described below and as shown in the
following scheme:
##STR00259##
[0267] To a solution of 2,4-thiazolidinedione (0.50 g, 4.27 mmol)
and 3-bromobenzyl bromide (1.03 g, 4.12 mmol) in 12.5 mL absolute
ethanol, 1.27 mL 5N NaOH was added and the reaction mixture was
heated to reflux. After 26 h the reaction was diluted with 15 mL
H.sub.2O and 20 mL ethyl acetate. The organic layer was washed with
H.sub.2O (3.times.15 mL) and brine (3.times.15 mL), dried over
sodium sulfate, filtered, and concentrated in vacuo. The resulting
residue was purified by flash column chromatography on silica
(20:80 ethyl acetate:hexane) to provide 0.254 g (21%) of
product.
Synthesis of
(Z)-3-(3-bromobenzyl)-5-(3,4-dihydroxybenzylidene)thiazolidine-2,4-dione
(compound C206)
[0268]
(Z)-3-(3-bromobenzyl)-5-(3,4-dihydroxybenzylidene)thiazolidine-2,4--
dione (compound C206) was synthesized according to the procedure
described below and as shown in the following scheme:
##STR00260##
[0269] A solution of 3-(3-bromobenzyl)thiazolidine-2,4-dione (0.123
g, 0.43 mmol), 3,4-dihydroxybenzaldehyde (0.060 g, 0.43 mmol),
piperidine (6 drops), acetic acid (6 drops), and 8 mL toluene was
heated at reflux for 40 min. The reaction mixture was concentrated
and purified by column chromatography (40:60 ethyl acetate:hexane)
to provide 0.039 g (22%) of product (compound C206).
[0270] Compounds C199 and C203 were prepared according to above
procedure except that 3-(benzyl)thiazolidine-2,4-dione and
3-(4-bromobenzyl)thiazolidine-2,4-dione, respectively, were used in
place of 3-(3-bromobenzyl)thiazolidine-2,4-dione.
(Z)-5-(3,4-dihydroxybenzylidene)-3-((4'-(trifluoromethoxy)-[1,1'-biphenyl]-
-3-yl)methyl)thiazolidine-2,4-dione (compound C207)
[0271]
(Z)-5-(3,4-dihydroxybenzylidene)-3-((4'-(trifluoromethoxy)-[1,1'-bi-
phenyl]-3-yl)methyl)thiazolidine-2,4-dione (compound C207) was
synthesized according to the procedure described below and as shown
in the following scheme:
##STR00261##
[0272] To a mixture of compound C206 (0.0365 g, 0.090 mmol),
4-trifluoromethoxy phenylboronic acid (0.028 g, 0.131 mmol), 1.3 mL
tetrahydrofuran, and 0.15 mL of 2M Na.sub.2CO.sub.3 was added
tetrakis(triphenylphosphine) palladium (0.0029 g, 0.0025 mmol). The
mixture was heated to reflux for 3 h. The reaction mixture was
diluted with 20 mL H.sub.2O and acidified to pH 3 by dropwise
addition of 1 N HC1. The solution was extracted with ethyl acetate
(3.times.15 mL), and the combined organics were washed with
H.sub.2O (3.times.15 mL), dried over MgSO.sub.4, filtered, and
concentrated in vacuo. The resulted residue was recrystallized from
ethanol to provide 0.034 g (78%) of product (compound C207).
[0273] Additional synthetic methods for obtaining PAI-1 inhibitors
are disclosed in EP 166469 A1, which is incorporated by reference
in its entirety.
Example 8
Synthesis of Oxindole Compounds of Formula XV
Synthesis of 5-(2-chloroacetyl)indolin-2-one
[0274] 5-(2-Chloroacetyl)indolin-2-one was synthesized according to
the procedure described below and as shown in the following
scheme:
##STR00262##
[0275] To a 0.degree. C. round bottom flask containing a suspension
of 7.53 g AlCl.sub.3 (56.3 mmol) in 7 mL 1,2-dichloroethane, 3.6 mL
(45.1 mmol) chloroacetyl chloride was added dropwise, resulting in
a color change from yellow to dark red. The reaction was allowed to
run for 1 hour after which a solution of 3.00 g oxindole (22.5
mmol) in 16 mL 1,2-dichloroethane was added. The reaction was
allowed to run for an additional 2 hours at 0.degree. C., and
thereafter for another 3 hours at 45.degree. C. The reaction was
stopped by pouring into ice cold water, in which a tan precipitate
formed immediately. The precipitate was filtered to provide 0.788 g
(77%) of product as a beige solid.
Synthesis of
5,5'-(2,2'-(ethane-1,2-diylbis(azanediyl))bis(acetyl))bis(indolin-2-one)
(compound C225)
[0276]
5,5'-(2,2'-(Ethane-1,2-diylbis(azanediyl))bis(acetyl))bis(indolin-2-
-one) (compound C225) was synthesized according to the procedure
described below and as shown in the following scheme:
##STR00263##
[0277] To a round bottom flask, 7.2 mL of acetonitrile, 320 mg
potassium iodide (1.93 mmol), 250 mg potassium carbonate (1.79
mmol), and 24 .mu.L of 1,2-ethylenediamine were added. T hereafter,
150 mg of 5-(2-chloroacetyl)indolin-2-one was added to the reaction
flask and the reaction was allowed to run overnight (under
nitrogen, with stirring) at reflux at 85.degree. C. The reaction
flask contents were concentrated in vacuo and transferred into a
separatory funnel using ethyl acetate. The product was then washed
with a saturated salt solution (3.times.). The organic layer was
dried, filtered, and concentrated in vacuo to provide 0.291 g
(6.6%) of product (compound C225).
Example 9
Synthesis of Oxindole Compounds of Formula XVII
[0278] Synthesis of
5-(2-(3,4-dimethoxyphenyl)thiazol-4-yl)indolin-2-one (C227)
[0279] 5-(2-(3,4-dimethoxyphenyl)thiazol-4-yl)indolin-2-one
(compound C227) was synthesized according to the procedure
described below and as shown in the following scheme:
##STR00264##
[0280] To a round bottom flask containing a solution of 60 mg
(0.286 mmol) 5-(2-chloroacetyl)indolin-2-one, 1.5 mL THF, and 1.5
mL absolute ethanol was added 57 mg (0.286 mmol)
3,4-dimethoxythiobenazmide. The reaction was stirred overnight
under nitrogen at reflux (80.degree. C.). The reaction flask
contents were then concentrated in vacuo and the resulting residue
was purified by trituration in acetone to provide 50 mg (50%) of
product (compound C227).
[0281] Compound C228 was prepared according to above procedure
except that 3,4-dihydroxythiobenazmide was used in place of
3,4-dimethoxythiobenazmide.
Example 10
Synthesis of Oxindole Compounds of Formula XIX
Synthesis of 2-oxoindoline-5-sulfonyl chloride
[0282] 2-Oxoindoline-5-sulfonyl chloride was synthesized according
to the procedure described below and as shown in the following
scheme:
##STR00265##
[0283] To a round bottom flask, 2.00 g oxindole (15.0 mmol) was
added in small portions to 4.0 mL (61.2 mmol) chlorosulfonic acid
at 30.degree. C. while stirring. After all of the oxindole was
added, the temperature was reduced to 25.degree. C. and was left to
react for 1.5 hours under nitrogen gas. The reaction heated to
70.degree. C. for an additional 1.5 hr. The reaction mixture was
then placed in an ice bath and quenched by dropwise addition of
deionized water to the reaction mixture. Upon addition of water, a
pink precipitate was formed, which was isolated by suction
filtration to provide 1.47 g (42%) of product as a dusty rose
solid.
Synthesis of 2-oxo-N-phenethylindoline-5-sulfonamide (compound
C229)
[0284] 2-Oxo-N-phenethylindoline-5-sulfonamide (compound C229) was
synthesized according to the procedure described below and as shown
in the following scheme:
##STR00266##
[0285] To a round bottom flask containing a solution of 100 mg
2-oxoindoline-5-sulfonyl chloride (0.432 mmol) in 9.5 mL methylene
chloride, 0.119 mL (0.950 mmol) of phenethylamine was added. The
reaction was stirred for 12 hours at room temperature under
nitrogen gas. The reaction mixture was then diluted with 45 mL of
ethyl acetate. This solution was washed with 1M HCl (3.times.15 mL)
and a saturated salt solution (1.times.15 mL). The organic layer
was collected and dried over magnesium sulfate, filtered, and
concentrated in vacuo to provide 0.110 g (67%) of product (compound
C229).
Example 11
Fluorometric PAI-1/uPA IC.sub.50 Plate Assay at pH 7.4
[0286] To determine the efficacy of various synthesized compounds
as PAI-1 inhibitors, a fluorometric plate assay was carried out to
measure the half maximal inhibitory concentration (IC.sub.50) of
these compounds on recombinant active human PAI-1 in vitro. An
IC.sub.50 is a measure of the effectiveness of a compound in
inhibiting biological or biochemical function. Stated another way,
IC.sub.50 represents the concentration of a drug that is required
for 50% inhibition in vitro. The IC.sub.50 of various compounds was
measured using a fluorometric plate assay as set out below, and the
results are shown in Tables 1 and 12.
[0287] PAI-1 inhibitor compounds were dissolved in DMSO to a final
concentration of (10-50 mM), depending upon solubility. Compounds
were then diluted in physiologic buffer (40 mM HEPES, 100 mM NaCl,
0.05% Tween-20, pH7.4) containing 10% DMSO and a dilution series
(from 0 to 1000 uM depending on solubility) was prepared. 80 .mu.L
of compound was added per well to a 96-well black, opaque
microplate in duplicate. 10 .mu.L of 20 nM Recombinant active human
PAI-1 (Molecular Innovations) in physiologic buffer (40 mM HEPES,
100 mM NaCl, 0.05% Tween-20, 10% DMSO, pH 7.4), or physiologic
buffer with 15 mg/mL, or physiologic buffer with 10% human plasma,
was added and the mixture was agitated for 15 minutes at room
temperature. 10 .mu.L of 25 nM uPA (Rheotromb.RTM.) was added to
each reaction well and the plate was agitated for an additional 30
minutes at room temperature. Tripeptide aminomethylcoumarin
Gly-Gly-Arg-AMC (Calbiochem) fluorogenic substrate (100 .mu.L of
100 .mu.M) was then added and residual uPA activity was determined
based upon cleavage of this substrate. The rate of AMC release by
uPA (fluorescence) was measured at an excitation wavelength of 370
nm and an emission wavelength of 440 nm. Controls included PAI-1
and uPA in the absence of compound and uPA alone. Percent PAI-1
inhibition was calculated using the following formula: [(uPA
alone-uPA/PAI-1+compound)/(uPA alone-PAI-1/uPA)]*100%. The
IC.sub.50 was calculated using Graphit (IC.sub.50 0-100%).
Example 12
Fluorometric PAI-1/uPA I.sub.C50 Plate Assay at pH 7.8
[0288] To determine the efficacy of various synthesized compounds
as PAI-1 inhibitors at pH 7.8, additional fluorometric plate assays
were carried out to measure the half maximal inhibitory
concentration (IC.sub.50) of these compounds on recombinant active
human PAI-1 in vitro. The IC.sub.50 of each of these various
compounds was measured using an assay as set out below, and the
results are shown in Tables 3, 5, 7, 9, and 11.
[0289] PAI-1 inhibitor compounds were dissolved in DMSO to a final
concentration of (10-50 mM), depending upon solubility. Compounds
were then diluted in physiologic buffer (40 mM HEPES, 100 mM NaCl,
0.05% Tween-20, pH7.8) containing (10% DMSO and a dilution series
(from 0 to 1000 uM depending on solubility) was prepared. 80 .mu.L
of compound was added per well to a 96-well black, opaque
microplate in duplicate. 10 .mu.L of 20 nM PAI-1 in physiologic
buffer (pH 7.8) was added and the mixture was agitated for 15
minutes at room temperature. 10 .mu.L of 25 nM uPA was added and
the plate was agitated for an additional 30 minutes at room
temperature. 100 .mu.L of 100 .mu.M fluorogenic, tripeptide
aminomethylcoumarin Gly-Gly-Arg-AMC (Calbiochem) fluorogenic
substrate was then added and residual uPA activity was determined
based upon cleavage of this substrate. Fluorescence, as a measure
of the rate of AMC release by uPA, was read at the following
wavelengths; excitation 370 nm, emission 440 nm. Controls included
PAI-1 and uPA in the absence of compound and uPA alone. Percent
PAI-1 inhibition was calculated using the following formula: [(uPA
alone-uPA/PAI-1+compound)/(uPA alone-PAI-1/uPA)]*100%. The
IC.sub.50 was calculated using Graphit (IC.sub.50 0-100%).
Example 13
Fluorometric PAI-1/tPA I.sub.C50 Plate Assay at pH 7.8
[0290] Additional fluorometric plate assays were carried out, as
set out below, to determine the IC.sub.50 of various synthesized
compounds as PAI-1 inhibitors. Results are shown in Tables 3, 5, 7,
9, and 11.
[0291] PAI-1 inhibitor compounds were dissolved in DMSO to a final
concentration of (10-50 mM), depending upon solubility. Compounds
were then diluted in physiologic buffer (40 mM HEPES, 100 mM NaCl,
0.05% Tween-20, pH7.8) containing (10% DMSO and a dilution series
(from 0 to 1000 .mu.M depending on solubility) was prepared. 80
.mu.L of compound was added per well to a 96-well black, opaque
microplate in duplicate. 10 .mu.L of 20 nM recombinant active human
PAI-1 (Molecular Innovations) in physiologic buffer, as set out
above, was added per well and the mixture was agitated for 15
minutes at room temperature. 10 uL of 25 nM human tissue type PA
(tPA) (Activase.RTM. (alteplase), Genentech) was added per well and
the plate was agitated for an additional 30 minutes at room
temperature. Tissue type PA activity in each reaction mixture was
determined by adding Phe-Gly-Arg-AMC fluorogenic substrate (100
.mu.L of 100 .mu.M) (Centerchem). The rate of AMC release by tPA
was measured at an excitation wavelength of 370 nm and an emission
wavelength of 440 nm. Controls included PAI-1 and tPA in the
absence of compound and tPA alone. Percent PAI-1 inhibition was
calculated using the following formula: [(tPA
alone-tPA/PAI-1+compound)/(tPA alone-PAI-1/tPA)]*100%. The IC50 is
calculated using Graphit (IC50 0-100%).
Example 14
Fluorometric ATIII/.alpha.IIa I.sub.C50 Plate Assay at pH 7.8
[0292] Additional fluorometric plate assays were carried out, as
set out below, to determine the IC.sub.50 of various synthesized
compounds as PAI-1 inhibitors. Results are shown in Tables 3, 5,
and 7.
[0293] Alpha-thrombin is an active enzyme, related to uPA or tPA.
Alpha-thrombin is inhibited by the serpin ATIII, which is closely
related to PAI-1. This assay was used therefore as a control in
testing for specificity of the PAI-1 inhibitor compounds. Thus, any
compound that is specific for inhibiting PAI-1 should not inhibit
ATIII.
[0294] PAI-1 inhibitor compounds were dissolved in DMSO to a final
concentration of (10-50 mM), depending upon solubility. Compounds
were then diluted in physiologic buffer (40 mM HEPES, 100 mM NaCl,
0.05% Tween-20, pH7.8) containing (10% DMSO and a dilution series
(from 0 to 1000 .mu.M depending on solubility) was prepared. 80
.mu.L of compound was added per well to a 96-well black, opaque
microplate in duplicate. 10 .mu.L of 20 nM recombinant active
anti-thrombin III (ATIII) (Molecular Innovations), a
(PAI-1)-related protein, in physiologic buffer, as set out above,
was added and the mixture was agitated for 15 minutes at room
temperature. 10 .mu.L of 25 nM human .alpha.-Thrombin (.alpha.IIa)
(Haematologic Technologies) was added to each reaction well and the
plate was agitated for an additional 30 minutes at room
temperature. 100 .mu.L of 100 .mu.M fluorogenic, tripeptide
aminomethylcoumarin benzoyl Phe-Val-Arg-AMC substrate was then
added and residual aIIa activity was determined based upon cleavage
of this substrate. The rate of AMC release by .alpha.IIa was
measured at an excitation wavelength of 370 nm and an emission
wavelength of 440 nm. Controls included PAI-1 and .alpha.IIa in the
absence of compound and .alpha.IIa alone. Percent PAI-1 inhibition
is calculated using the following formula: [(.alpha.IIa
alone-.alpha.IIa/PAI-1+compound)/(.alpha.IIa
alone-PAI-1/.alpha.IIa)]*100%. The IC50 is calculated using Graphit
(IC50 0-100%).
[0295] The invention has been described in terms of particular
embodiments found or proposed to comprise preferred modes for the
practice of the invention. It will be appreciated by those of
ordinary skill in the art that, in light of the disclosure,
numerous modifications and changes can be made in the particular
embodiments exemplified without departing from the intended scope
of the invention. Therefore, it is intended that the appended
claims cover all such equivalent variations which come within the
scope of the invention as claimed.
* * * * *