U.S. patent application number 11/706925 was filed with the patent office on 2007-07-05 for pharmaceutically active compounds and methods of use thereof.
This patent application is currently assigned to Tanox, Inc.. Invention is credited to Jin-An Jiao, Lawrence K. Luepschen, Esperanza Nieves, Dean P. Taylor, Hing C. Wong.
Application Number | 20070155703 11/706925 |
Document ID | / |
Family ID | 22286985 |
Filed Date | 2007-07-05 |
United States Patent
Application |
20070155703 |
Kind Code |
A1 |
Jiao; Jin-An ; et
al. |
July 5, 2007 |
Pharmaceutically active compounds and methods of use thereof
Abstract
The invention includes pharmaceutically active compounds and
methods of treatment and pharmaceutical compositions that utilize
or comprise one or more such compounds. Compounds of the invention
are particularly useful for treatment or prophylaxis of undesired
thrombosis.
Inventors: |
Jiao; Jin-An; (Weston,
FL) ; Luepschen; Lawrence K.; (Miami, FL) ;
Nieves; Esperanza; (Newark, DE) ; Wong; Hing C.;
(Weston, FL) ; Taylor; Dean P.; (Weston,
FL) |
Correspondence
Address: |
FOLEY HOAG, LLP;PATENT GROUP, WORLD TRADE CENTER WEST
155 SEAPORT BLVD
BOSTON
MA
02110
US
|
Assignee: |
Tanox, Inc.
Houston
TX
|
Family ID: |
22286985 |
Appl. No.: |
11/706925 |
Filed: |
February 15, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11005871 |
Dec 6, 2004 |
7199113 |
|
|
11706925 |
Feb 15, 2007 |
|
|
|
09406269 |
Sep 24, 1999 |
6828312 |
|
|
11005871 |
Dec 6, 2004 |
|
|
|
60101887 |
Sep 25, 1998 |
|
|
|
Current U.S.
Class: |
514/89 ; 514/114;
514/90 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 9/08 20180101; A61P 9/10 20180101; C07F 9/3873 20130101; A61P
29/00 20180101; C07F 9/386 20130101; A61P 7/02 20180101; A61K 31/66
20130101; A61K 31/675 20130101; A61K 31/663 20130101; A61P 7/04
20180101; A61P 37/02 20180101; A61P 9/00 20180101 |
Class at
Publication: |
514/089 ;
514/090; 514/114 |
International
Class: |
A61K 31/675 20060101
A61K031/675; A61K 31/66 20060101 A61K031/66 |
Claims
1. A method for treating a mammal susceptible to, suffering from,
suspected of having or recovering from a disease impacted by tissue
factor (TF), the method comprising administering to the mammal a
therapeutically effective amount of at least one TF blocking
compound to treat the disease.
2. The method of claim 1, wherein the disease is selected from the
group consisting of a cardiovascular disease, a blood coagulation
disorder, a cell proliferation disorder, post-operative
complication, an immune disorder, atherosclerosis, inflammation or
cancer.
3. A method of blocking or inhibiting tissue factor-dependent
activation of factor X and/or factor IX, comprising contacting
tissue factor with a TF blocking compound to thereby inhibit
formation of a functional complex of factor X or factor IX with
tissue factor or TF VIIA.
4. The method of claim 3 wherein the TF blocking compound binds to
tissue factor to thereby inhibit formation of the functional
complex.
5. The method of any one of claims 1-4 wherein the tissue factor
blocking compound exhibits an IC.sub.50 of less than about 100
.mu.M in a standard assay for measuring TF/VIIa-dependent factor X
activation.
6. The method of any one of claims 1-4 wherein the tissue factor
blacking compound exhibits an IC.sub.50 of about 200 .mu.M or less
in a standard assay for measuring TF/VIIa-dependent factor IX
activation.
7. The method of any one of claims 1-4 wherein the tissue factor
compound comprises at least one phosphonate group.
8. The method of any one of claims 1-4 wherein the tissue factor
compound comprises at least one bis-phosphonate group.
9. The method of claim 7 or 8 wherein the tissue factor compound
further comprises an optionally substituted carbocyclic aryl group,
or an optionally substituted heteroaryl group.
10. The method of claim 7 or 8 wherein the tissue factor compound
further comprises an optionally substituted phenyl group.
11. The method of any one of claims 1-4 wherein the TF blocking
compound is of the following Formula I: Ar-(CXY).sub.m-(Het).sub.0
or 1-(CX.sup.1Y.sup.1).sub.n--C(Z).sub.p-(PO.sub.3).sub.3-p I
wherein Ar is optionally substituted carbocyclic aryl or optionally
substituted heteroaryl; Het is optionally substituted N, O, S, S(O)
or S(O.sub.2); each X, each Y, each X', each Y' and each Z are each
independently hydrogen; halogen; hydroxyl; sulfhydryl; amino;
optionally substituted alkyl preferably; optionally substituted
alkenyl; optionally substituted alkynyl; optionally substituted
alkoxy; optionally substituted alkylthio; optionally substituted
alkylsulfinyl; optionally substituted alkylsulfonyl; or optionally
substituted alkylamino; m and n each is independently an integer of
from 0 to 4; p is 1 or 2; and pharmaceutically acceptable salts
thereof.
12. The method of any one of claims 1-4 wherein the TF blocking
compound is of the following Formula II: ##STR5## wherein Ar is
optionally substituted carbocyclic aryl or optionally substituted
heteroaryl; Het is optionally substituted N, O, S, S(O) or
S(O.sub.2); each X, each Y, each X', each Y' and each Z are each
independently hydrogen; halogen; hydroxyl; sulfhydryl; amino;
optionally substituted alkyl preferably; optionally substituted
alkenyl; optionally substituted alkynyl; optionally substituted
alkoxy; optionally substituted alkylthio; optionally substituted
alkylsulfinyl; optionally substituted alkylsulfonyl; or optionally
substituted alkylamino; each R.sup.1 is independently halogen;
amino; hydroxy; nitro; carboxy; sulfhydryl; optionally substituted
alkyl; optionally substituted alkenyl; optionally substituted
alkynyl; optionally substituted alkoxy; optionally substituted
alkylthio; optionally substituted alkylsulfinyl; optionally
substituted alkylsulfonyl; optionally substituted alkylamino;
optionally substituted alkanoyl; optionally substituted carbocyclic
aryl; or optionally substituted aralkyl; m and n each is
independently an integer of from 0 to 4; p is 1 or 2; q is an
integer of from 0 to 5; and pharmaceutically acceptable salts
thereof.
13. A method for identifying a candidate TF blocking compound, the
method comprising screening the compound in at least one primary
screening assay using purified tissue factor; and selecting the TF
blocking compound.
14. The method of claim 13 further comprising screening the
candidate TF blocking compound in at least one secondary screening
assay.
15. The method of claim 13 or 14 comprising screening the candidate
TF blocking compound in a standard in vitro assay for measuring
TF/VIIa-dependent factor X activation.
16. The method of claim 13 or 14 comprising screening the candidate
TF blocking compound in a standard in vitro assay for measuring
TF/VIIa dependent factor IX activation.
17. The method of claim 13 wherein the screening assay is a
standard prothrombin time (PT) assay.
18. The method of any of claims 13-17 wherein the purified tissue
factor is lipidated recombinant human tissue factor.
19. A tissue factor (TF) blocking compound exhibiting an IC.sub.50
of less than about 100 .mu.M in a standard assay for measuring
TF/VIIa-dependent factor X activation.
20. The compound of claim 19 further exhibiting equivalent or
greater than about 70% inhibition in the assay.
21. The compound of claim 19 further exhibiting an IC.sub.50 of
about 200 .mu.M or less in a standard assay for measuring
TF/VIIa-dependent factor IX activation.
22. The compound of claim 19 further exhibiting at least about a 5%
to 20% increase in plasma clotting time relative to a control in a
standard prothrombin time (PT) assay.
23. The compound of claim 19 further exhibiting at least 70%
inhibition in the assay for measuring TF/VIIa-dependent factor X
activation and at least of the following activities: 1) an
IC.sub.50 of less than about 100 .mu.M in a standard assay for
measuring TF/VIIa-dependent factor IX activation, and 2) at least
about a 5% to 20% increase in plasma clotting time relative to a
control in a standard prothrombin time (PT) assay.
24. The compound of claim 19 wherein the compound is represented by
the following Formula I: Ar-(CXY).sub.m-(Het).sub.0 or
1-(CX.sup.1Y.sup.1).sub.n--C(Z).sub.p-(PO.sub.3).sub.3-p I wherein
Ar is optionally substituted carbocyclic aryl or optionally
substituted heteroaryl; Het is optionally substituted N, O or S;
each X, each Y, each X', each Y' and each Z are each independently
hydrogen; halogen; hydroxyl; sulfhydryl; amino; optionally
substituted alkyl preferably; optionally substituted alkenyl;
optionally substituted alkynyl; optionally substituted alkoxy;
optionally substituted alkylthio; optionally substituted
alkylsulfinyl; optionally substituted alkylsulfonyl; or optionally
substituted alkylamino; m and n each is independently an integer of
from 0 to 4; p is 1 or 2; and pharmaceutically acceptable salts
thereof.
25. The compound of claim 19 wherein the compound is represented by
the following Formula II: ##STR6## wherein Ar is optionally
substituted carbocyclic aryl or optionally substituted heteroaryl;
Het is optionally substituted N, O, S, S(O) or S(O.sub.2); each X,
each Y, each X', each Y' and each Z are each independently
hydrogen; halogen; hydroxyl; sulfhydryl; amino; optionally
substituted alkyl preferably; optionally substituted alkenyl;
optionally substituted alkynyl; optionally substituted alkoxy;
optionally substituted alkylthio; optionally substituted
alkylsulfinyl; optionally substituted alkylsulfonyl; or optionally
substituted alkylamino; each R.sup.1 is independently halogen;
amino; hydroxy; nitro; carboxy; sulfhydryl; optionally substituted
alkyl; optionally substituted alkenyl; optionally substituted
alkynyl; optionally substituted alkoxy; optionally substituted
alkylthio; optionally substituted alkylsulfinyl; optionally
substituted alkylsulfonyl; optionally substituted alkylamino;
optionally substituted alkanoyl; optionally substituted carbocyclic
aryl; or optionally substituted aralkyl; m and n each is
independently an integer of from 0 to 4; p is 1 or 2; q is an
integer of from 0 to 5; and pharmaceutically acceptable salts
thereof.
26. The compound of claim 19 wherein the compound is represented by
the following Formula III: ##STR7## wherein each X, each Y, each
X', each Y' and each Z are each independently hydrogen; halogen;
hydroxyl; sulfhydryl; amino;.optionally substituted alkyl
preferably; optionally substituted alkenyl; optionally substituted
alkynyl; optionally substituted alkoxy; optionally substituted
alkylthio; optionally substituted alkylsulfinyl; optionally
substituted alkylsulfonyl; or optionally substituted alkylamino; m
and n each is independently an integer of from 0 to 4; p is 1 or 2;
q is an integer of from 0 to 5; W is hydrogen, optionally
substituted alkyl; optionally substituted alkenyl; optionally
substituted alkynyl; optionally substituted alkoxy; optionally
substituted alkylthio; optionally substituted alkylsulfinyl;
optionally substituted alkylsulfonyl; optionally substituted
alkylamino; optionally substituted alkanoyl; optionally substituted
carbocyclic aryl; or optionally substituted aralkyl; R.sup.1 is
independently halogen; amino; hydroxy; nitro; carboxy; sulfhydryl;
optionally substituted alkyl; optionally substituted alkenyl;
optionally substituted alkynyl; optionally substituted alkoxy;
optionally substituted alkylthio; optionally substituted
alkylsulfinyl; optionally substituted alkylsulfonyl; optionally
substituted alkylamino; optionally substituted alkanoyl; optionally
substituted carbocyclic aryl; or optionally substituted aralkyl; q
is an integer of from 0 to 5; and pharmaceutically acceptable salts
thereof.
27. The compound of claim 19 wherein the compound is represented by
the following Formula IIIa: ##STR8## wherein R.sup.1 is
independently halogen; amino; hydroxy; nitro; carboxy; sulfhydryl;
optionally substituted alkyl; optionally substituted alkenyl;
optionally substituted alkynyl; optionally substituted alkoxy;
optionally substituted alkylthio; optionally substituted
alkylsulfinyl; optionally substituted alkylsulfonyl; optionally
substituted alkylamino; optionally substituted alkanoyl; optionally
substituted carbocyclic aryl; or optionally substituted aralkyl;
and q is an integer of from 0 to 5; and pharmaceutically acceptable
salts thereof.
28. The compound of claim 19 wherein the compound is represented by
the following Formula IV: ##STR9## wherein each X, each Y, each X',
each Y' and each Z are each independently hydrogen; halogen;
hydroxyl; sulfhydryl; amino; optionally substituted alkyl
preferably; optionally substituted alkenyl; optionally substituted
alkynyl; optionally substituted alkoxy; optionally substituted
alkylthio; optionally substituted alkylsulfinyl; optionally
substituted alkylsulfonyl; or optionally substituted alkylamino; m
and n each is independently an integer of from 0 to 4; p is 1 or 2;
q is an integer of from 0 to 5; R.sup.1 is independently halogen;
amino; hydroxy; nitro; carboxy; sulfhydryl; optionally substituted
alkyl; optionally substituted alkenyl; optionally substituted
alkynyl; optionally substituted alkoxy; optionally substituted
alkylthio; optionally substituted alkylsulfinyl; optionally
substituted alkylsulfonyl; optionally substituted alkylamino;
optionally substituted alkanoyl; optionally substituted carbocyclic
aryl; or optionally substituted aralkyl; q is an integer of from 0
to 5; and pharmaceutically acceptable salts thereof.
29. The compound of claim 19 wherein the compound is represented by
the following Formula IVA: ##STR10## wherein each X' and each Y' is
independently hydrogen; halogen; hydroxyl; sulfhydryl; amino;
optionally substituted alkyl preferably; optionally substituted
alkenyl; optionally substituted alkynyl; optionally substituted
alkoxy; optionally substituted alkylthio; optionally substituted
alkylsulfinyl; optionally substituted alkylsulfonyl; or optionally
substituted alkylamino; R.sup.1 is independently halogen; amino;
hydroxy; nitro; carboxy; sulfhydryl; optionally substituted alkyl;
optionally substituted alkenyl; optionally substituted alkynyl;
optionally substituted alkoxy; optionally substituted alkylthio;
optionally substituted alkylsulfinyl; optionally substituted
alkylsulfonyl; optionally substituted alkylamino; optionally
substituted alkanoyl; optionally substituted carbocyclic aryl; or
optionally substituted aralkyl; n is an integer of from 0 to 4; q
is an integer of from 0 to 5; and pharmaceutically acceptable salts
thereof.
30. A compound of any one of claims 24 through 27 wherein at least
one R.sup.1 is hydroxy and m is 1 or 2.
31. A pharmaceutical composition comprising a compound of any one
of claims 19 to 30 and a pharmaceutically acceptable carrier.
32. A method for treating a mammal suffering from or susceptible to
a disease impacted by tissue factor, comprising administering to
the mammal an effective amount of a compound or composition of any
one of claims 19-31.
33. A method for treating a mammal suffering from or susceptible to
a cardiovascular disease, a blood coagulation disorder, a cell
proliferation disorder, post-operative complication, an immune
disorder, atherosclerosis, inflammation, or cancer, comprising
administering to the mammal an effective amount of a compound or
composition of any one of claims 19-31.
34. A method of inhibiting blood coagulation in a mammal,
comprising administering to the mammal an effective amount of a
compound or composition of any one of claims 19 to 31.
35. The method of claim 34 wherein the mammal is suffering from,
suspected of having, or recovering from a thrombosis.
36. The method of claim 34 wherein the mammal is suffering from,
susceptible to, or recovering from restenosis associated with an
invasive medical procedure.
37. The method of claim 34 wherein the invasive medical procedure
is angioplasty, endarterectomy, deployment of a stent, use of
catheter, graft implantation or use of an arteriovenous shunt.
38. The method of claim 34 wherein the mammal is suffering from, at
risk of developing, or recovering from a thromboembolic condition
associated with cardiovascular disease, an infectious disease, a
neoplastic disease or use of a thrombolytic agent.
39. The method of any one of claims 34-38 wherein at least one of
an anti-platelet composition, thrombolytic composition or an
anti-coagulant composition is administered in combination with a
tissue factor blocking compound exhibiting an IC.sub.50 of less
than about 100 .mu.M in a standard assay for measuring
TF/VIIa-dependent factor X activation.
40. A method of treating or preventing a thromboembolic disorder in
a mammal, comprising administering to the mammal an effective
amount of the TF blocking compound of any one of claims 19-31.
41. The method of claim 40 wherein the mammal is suffering from, at
risk developing, or is recovering from a thromboembolic condition
associated with cardiovascular disease, an infectious disease, a
neoplastic disease or use of a thrombolytic agent or an
anti-platelet agent.
42. The method of claim 40 wherein at least one of an
anti-platelet, thrombolytic, or an anti-coagulant composition is
administered in combination with a tissue factor blocking compound
exhibiting an IC.sub.50 of less than about 100 .mu.M in a standard
assay for measuring TF/VIIa-dependent factor X activation.
43. The method of any one of claims 32-42 wherein the mammal is a
human.
44. A compound of the following Formula I:
Ar-(CXY).sub.m-(Het).sub.0 or
1-(CX.sup.1Y.sup.1).sub.n--C(Z).sub.p-(PO.sub.3).sub.3-p I wherein
Ar is optionally substituted carbocyclic aryl or optionally
substituted heteroaryl; Het is optionally substituted N, O, S, S(O)
or S(O).sub.2; each X, each Y, each X', each Y' and each Z are each
independently hydrogen; halogen; hydroxyl; sulfhydryl; amino;
optionally substituted alkyl preferably; optionally substituted
alkenyl; optionally substituted alkynyl; optionally substituted
alkoxy; optionally substituted alkylthio; optionally substituted
alkylsulfinyl; optionally substituted alkylsulfonyl; or optionally
substituted alkylamino; m and n each is independently an integer of
from 0 to 4; p is 1 or 2; and pharmaceutically acceptable salts
thereof.
45. A compound of claim 44 having the following Formula II:
##STR11## wherein Ar is optionally substituted carbocyclic aryl or
optionally substituted heteroaryl; Het is optionally substituted N,
O, S, S(O) or S(O.sub.2); each X, each Y, each X', each Y' and each
Z are each independently hydrogen; halogen; hydroxyl; sulfhydryl;
amino; optionally substituted alkyl preferably; optionally
substituted alkenyl; optionally substituted alkynyl; optionally
substituted alkoxy; optionally substituted alkylthio; optionally
substituted alkylsulfinyl; optionally substituted alkylsulfonyl; or
optionally substituted alkylamino; each R.sup.1 is independently
halogen; amino; hydroxy; nitro; carboxy; sulfhydryl; optionally
substituted alkyl; optionally substituted alkenyl; optionally
substituted alkynyl; optionally substituted alkoxy; optionally
substituted alkylthio; optionally substituted alkylsulfinyl,
optionally substituted alkylsulfonyl; optionally substituted
alkylamino; optionally substituted alkanoyl; optionally substituted
carbocyclic aryl; or optionally substituted aralkyl; m and n each
is independently an integer of from 0 to 4; p is 1 or 2; q is an
integer of from 0 to 5; and pharmaceutically acceptable salts
thereof.
46. A compound of claim 44 having the following Formula III:
##STR12## wherein each X, each Y, each X', each Y' and each Z are
each independently hydrogen; halogen; hydroxyl; sulfhydryl; amino;
optionally substituted alkyl preferably; optionally substituted
alkenyl; optionally substituted alkynyl; optionally substituted
alkoxy; optionally substituted alkylthio; optionally substituted
alkylsulfinyl; optionally substituted alkylsulfonyl; or optionally
substituted alkylamino; m and n each is independently an integer of
from 0 to 4; p is 1 or 2; q is an integer of from 0 to 5; W is
hydrogen, optionally substituted alkyl; optionally substituted
alkenyl; optionally substituted alkynyl; optionally substituted
alkoxy; optionally substituted alkylthio; optionally substituted
alkylsulfinyl; optionally substituted alkylsulfonyl; optionally
substituted alkylamino; optionally substituted alkanoyl; optionally
substituted carbocyclic aryl; or optionally substituted aralkyl;
R.sup.1 is independently halogen; amino; hydroxy; nitro; carboxy;
sulfhydryl; optionally substituted alkyl; optionally substituted
alkenyl; optionally substituted alkynyl; optionally substituted
alkoxy; optionally substituted alkylthio; optionally substituted
alkylsulfinyl; optionally substituted alkylsulfonyl; optionally
substituted alkylamino; optionally substituted alkanoyl; optionally
substituted carbocyclic aryl; or optionally substituted aralkyl; q
is an integer of from 0 to 5; and pharmaceutically acceptable salts
thereof.
47. A compound of claim 44 having the following Formula IIIa:
##STR13## wherein R.sup.1 is independently halogen; amino; hydroxy;
nitro; carboxy; sulfhydryl; optionally substituted alkyl;
optionally substituted alkenyl; optionally substituted alkynyl;
optionally substituted alkoxy; optionally substituted alkylthio;
optionally substituted alkylsulfinyl; optionally substituted
alkylsulfonyl; optionally substituted alkylamino; optionally
substituted alkanoyl; optionally substituted carbocyclic aryl; or
optionally substituted aralkyl; and q is an integer of from 0 to 5;
and pharmaceutically acceptable salts thereof.
48. A compound of claim 44 having the following Formula IV:
##STR14## wherein wherein each X, each Y, each X', each Y' and each
Z are each independently hydrogen; halogen; hydroxyl; sulfhydryl;
amino; optionally substituted alkyl preferably; optionally
substituted alkenyl; optionally substituted alkynyl; optionally
substituted alkoxy; optionally substituted alkylthio; optionally
substituted alkylsulfinyl; optionally substituted alkylsulfonyl; or
optionally substituted alkylamino; m and n each is independently an
integer of from 0 to 4; p is 1 or 2; q is an integer of from 0 to
5; R.sup.1 is independently halogen; amino; hydroxy; nitro;
carboxy; sulfhydryl; optionally substituted alkyl; optionally
substituted alkenyl; optionally substituted alkynyl; optionally
substituted alkoxy; optionally substituted alkylthio; optionally
substituted alkylsulfinyl; optionally substituted alkylsulfonyl;
optionally substituted alkylamino; optionally substituted alkanoyl;
optionally substituted carbocyclic aryl; or optionally substituted
aralkyl; q is an integer of from 0 to 5; and pharmaceutically
acceptable salts thereof.
49. A compound of claim 44 having the following Formula IVa:
##STR15## wherein each X' and each Y' is independently hydrogen;
halogen; hydroxyl; sulfhydryl; amino; optionally substituted alkyl
preferably; optionally substituted alkenyl; optionally substituted
alkynyl; optionally substituted alkoxy; optionally substituted
alkylthio; optionally substituted alkylsulfinyl; optionally
substituted alkylsulfonyl; or optionally substituted alkylamino;
R.sup.1 is independently halogen; amino; hydroxy; nitro; carboxy;
sulfhydryl; optionally substituted alkyl; optionally substituted
alkenyl; optionally substituted alkynyl; optionally substituted
alkoxy; optionally substituted alkylthio; optionally substituted
alkylsulfinyl; optionally substituted alkylsulfonyl; optionally
substituted alkylamino; optionally substituted alkanoyl; optionally
substituted carbocyclic aryl;,or optionally substituted aralkyl;
and q is an integer of from 0 to 5; and pharmaceutically acceptable
salts thereof.
50. A compound of any one of claims 44 through 46 wherein at least
one R.sup.1 is hydroxy and m is 1 or 2.
51. A compound of claim 44, that is: ##STR16## or a
pharmaceutically acceptable salt thereof.
52. A method of inhibiting blood coagulation in a mammal,
comprising administering to the mammal an effective amount of a
compound of any one of claims 44 to 51.
53. The method of claim 52 wherein the mammal is suffering from or
suspected of having a thrombosis.
54. The method of claim 52 wherein the mammal is suffering from or
susceptible to restenosis associated with an invasive medical
procedure.
55. The method of claim 54 wherein the invasive medical procedure
is angioplasty, endarterectomy, deployment of a stent, use of
catheter, graft implantation or use of an arteriovenous shunt.
56. The method of claim 52 wherein the mammal is suffering from or
at risk of developing a thromboembolic condition associated with
cardiovascular disease, an infectious disease, a neoplastic disease
or use of a thrombolytic agent.
57. The method of any one of claims 52 to 56 wherein an
anti-platelet composition, a thrombolytic composition or an
anti-coagulant composition is administered in combination with a
compound of Formula I.
58. A method of treating or preventing a thromboembolic disorder in
a mammal, comprising administering to the mammal an effective
amount of a compound of any one of claims 44 to 51.
59. The method of claim 58 wherein the mammal is suffering from or
at risk developing a thromboembolic condition associated with
cardiovascular disease, an infectious disease, a neoplastic disease
or use of a thrombolytic agent or an anti-platelet agent.
60. The method of claim 58 or 59 wherein an anti-platelet
composition, a thrombolytic composition or an anti-coagulant
composition is administered in combination with a compound of
Formula I.
61. A method of treating a mammal suffering from or susceptible to
atherosclerosis, comprising administering to the mammal an
effective amount of a compound of any one of claims 44 to 51.
62. The method of any one of claims 52 to 61 wherein the mammal is
a human.
63. A pharmaceutical composition comprising a compound of any one
of claims 44 through 51 and a pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application claims the benefit of U.S.
Provisional Application No. 60/101,887 filed on Sep. 25, 1998, the
disclosure of which is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to pharmaceutically active
compounds and more particularly to pharmaceutical compositions that
utilize or comprise one or more of such compounds. Preferred
compounds are especially useful for the treatment or prophylaxis of
undesired thrombosis. Also included are methods for treating
thrombosis. The invention has a wide spectrum of applications
including use in screening candidate compounds for the treatment or
prophylaxis of thrombosis.
[0004] 2. Background
[0005] Blood clotting assists hemostasis by minimizing blood loss.
Generally, blood clotting is initiated by vessel damage and
requires platelet aggregation, coagulation factors and inhibition
of fibrinolysis. The coagulation factors act through a cascade that
relates the vessel damage to formation of a blood clot (see
generally L. Stryer, Biochemistry, 3rd Ed, W.H. Freeman Co., New
York; and A. G. Gilman et al., The Pharmacological Basis of
Therapeutics, 8th Edition, McGraw Hill Inc., New York, pp. 1311-
1331).
[0006] Tissue factor (TF), an integral membrane protein of 263
amino acids, is responsible for initiating the coagulation protease
cascade. Vascular damage exposes blood to tissue factor expressed
on subendothelial cell surfaces, leading to the formation of a
calcium-dependent, high-affinity complex with the plasma factor VII
(FVII) or activated factor VII (FVIIa). Binding to TF promotes
rapid proteolytic cleavage of the zymogen FVII to the active serine
protease FVIIa by a number of proteases such as factor Xa, or
thrombin. TF also functions as an essential cofactor for FVIIa by
dramatically enhancing the catalytic efficiency of FVIIa for its
protein substrates factors IX and X. TF/VIIa complex activates
factors IX (FIX) and X (FX) via limited proteolysis, ultimately
leading to thrombin generation and fibrin deposition. Under
pathological conditions such as atherosclerosis or following
invasive surgical procedures such as microvascular graft,
angioplasty, deployment of an implanted device (e.g., a stent,
catheter or arteriovenous shunt), or endarterectomy, TF-initiated
coagulation can lead to thrombotic disorders that can result e.g.
in heart attack, stroke, unstable angina or other coronary
disorder.
[0007] Thrombosis also may accompany various thromboembolic
disorders and coagulopathies such as a pulmonary embolism (e.g.,
atrial fibrillation with embolization, deep vein thrombosis, etc.)
and disseminated intravascular coagulation, respectively.
Manipulation of body fluids can also result in an undesirable
thrombus, particularly in blood transfusions or fluid sampling, as
well as procedures involving extracorporeal circulation (e.g.,
cardiopulmonary bypass surgery) and dialysis.
[0008] Certain anti-coagulants have been used to alleviate or avoid
blood clots associated with thrombosis. Blood clotting often can be
minimized or eliminated by administering a suitable anti-coagulant
or mixture thereof, including one or more of a coumarin derivative
(e.g., warfarin and dicumarol) or a charged polymer (e.g., heparin,
hirudin or hirulog). See e.g., Gilman et al., supra, R. J.
Beigering et al., Ann. Hemathol., 72:177 (1996); J. D. Willerson,
Circulation, 94:866 (1996).
[0009] Certain antibodies with anti-platelet activity have also
been used to alleviate various thromboses. For example, ReoPro.TM.
is a therapeutic antibody that is routinely administered to
alleviate various thromboembolic disorders such as those arising
from angioplasty, myocardial infarction, unstable angina and
coronary artery stenoses. Additionally, ReoPro.TM. can be used as a
prophylactic to reduce the risk of myocardial infarction and angina
(J. T. Willerson, Circulation, 94:866 (1996); M. L. Simmons et al.,
Circulation, 89:596 (1994)).
[0010] However, use of prior anti-coagulants is often associated
with side effects such as hemorrhaging, re-occlusion, "white-clot"
syndrome, irritation, birth defects, thrombocytopenia and hepatic
dysfunction. Long-term administration of anti-coagulants can
particularly increase risk of life-threatening illness (see e.g.,
Gilman et al., supra).
[0011] Protein-based agents are potentially safer, but generally
are limited to treatment of acute conditions and often are
restricted to parenteral administration. Such agents are considered
less suitable for long-term therapies for chronic diseases (such as
atherosclerosis, a major cause of heart attack) due to the
increased probability of immune response to a protein therapeutic,
relatively high production cost and/or limited oral
bioavailability.
[0012] It would thus be desirable to have new anti-coagulant
agents. It would be particularly desirable to have new
anti-coagulant agents that could be administered over a relatively
long period to treat chronic conditions such as
atherosclerosis.
SUMMARY OF TH INVENTION
[0013] We have now discovered pharmaceutically active compounds and
compositions that are useful to treat or prevent undesired
thrombosis. Preferred compounds of the invention are tissue factor
(TF) antagonists that preferably specifically block human factor X
and IX activation catalyzed by human tissue factor/factor VIIa
complex. Also discovered are methods for treating or preventing
thrombosis that use the compounds and compositions disclosed
herein.
[0014] More particular methods of this invention include
administering a therapeutically effective amount of at least one
compound or composition of this invention. The compound or
composition is typically given to a mammal in need of such
treatment such as a human patient who is susceptible to, suffering
from, or recovering from undesired thrombosis, or mammal that is
suffering from, recovering from or susceptible to other disease or
disorder impacted by tissue factor such as a cardiovascular
disease, cell proliferation disorder, postoperative complication,
or an immune disorder. Preferred compounds and compositions may
also be used to treat or prevent recognized disorders impacted by
various thromboses such as those particular disorders disclosed
herein.
[0015] The invention also includes methods for blocking or
inhibiting tissue factor-dependent activation of factor X and/or
factor IX. These methods in general include contacting tissue
factor with a TF blocking compound to thereby inhibit formation of
a functional complex of factor X or factor IX with tissue factor or
TF/VIIa. Preferably the TF blocking compound binds to tissue factor
to thereby inhibit formation of the functional complex. Inhibition
or prevention of formation of such a functional complex can have
quite broad application, including for treatment of the
above-mentioned diseases or disorders in mammals, particularly
humans suffering from or susceptible to such diseases or
disorders.
[0016] Preferred compounds of the invention generally exhibit good
blocking activity in at least one test for detecting and preferably
measuring TF-mediated blood clotting. More particular tests are
standard in vitro assays for measuring activity of a specific blood
coagulation factor in which the assay is recognized as providing
optimal results in the presence of TF or a TF-associated complex
such as the human TF/VIIa complex. The TF can be provided in the
assay as a recombinant molecule or molecule purified from natural
sources depending usually on the specific assay selected.
[0017] A more particular in vitro assay detects and measures
activity of a specific blood coagulation factor which has a
recognized activity enhanced in the presence of human TF or the
human TF/VIIa complex. Of preferred interest are standard in vitro
assays for measuring TF-dependent activation of factor X to FXa and
factor IX to FIXa. Sometimes these assays will be referred to
herein as a "primary screening assay" or related term or phrase
such as "method of discovery" to denote preferred use of the assay
in screening compounds.
[0018] For example, a particularly preferred compound of the
invention will exhibit good blocking activity in the primary
screening assay for measuring TF-dependent activation of factor X
to FXa. Additionally preferred compounds will exhibit good blocking
activity in the primary screening assay for measuring TF-dependent
activation of factor IX to FIXa.
[0019] It will be appreciated that by the phrase "good blocking
activity" or related phrase is meant preferred use of a compound of
this invention to reduce or inhibit TF/VIIa-dependent activation of
factor X to FXa and/or factor IX to FIXa. A preferred compound is a
synthetic or semi-synthetic compound such as those small molecule
compounds disclosed below. More particular disclosure relating to
the primary screening assays is provided as follows.
[0020] Preferred compounds of this invention will exhibit an
IC.sub.50 (concentration required to inhibit factor X activation by
about 50% relative to a suitable control) of about 100 .mu.M or
less and preferably about 10 .mu.M or less. Additionally preferred
compounds will exhibit equivalent or greater than about 70%
inhibition of TF- or TF/VIIa dependent FX activation in the assay.
In a preferred embodiment, the primary screening assay includes all
of the following steps: [0021] 1) admixing in a suitable assay
solution TF/VIIa complex and factor X under conditions conducive to
forming factor Xa, [0022] 2) contacting the solution with a
detectably-labeled factor Xa substrate; and [0023] 3) detecting
labeled product in the solution as being indicative of the factor X
activation.
[0024] Preferred use of this primary screening assay effectively
measures the capability of a candidate compound to decrease or
eliminate TF- or TF/VIIa dependent factor X activation. The assay
is generally flexible and can be manipulated as necessary to test a
compound for capability to block factor X activation. For example,
the candidate compound can be added at any one or more of the steps
shown above with addition of the compound at step 1) being
preferred for many screening applications.
[0025] A-preferred TF/VIIa complex for use in the method includes
TF which has been exposed to conditions conducive to exposing good
TF blocking sites. More specific conditions for isolating and using
the TF are provided below.
[0026] As mentioned above, another primary screening assay is a
standard in vitro assay for measuring factor IX activation by TF or
TF/VIIa. In this example, a preferred compound will exhibit an
IC.sub.50 (concentration required to inhibit factor IX activation
in the assay by about 50% relative to a suitable control) in the
assay of about 200 .mu.M or less, and preferably about 10 .mu.M or
less. In a preferred embodiment, the standard assay for measuring
the factor IX activation includes all of the following steps:
[0027] 1) admixing in a suitable assay solution TF/VIIa complex
with factor IX under conditions conducive to forming factor FIXa,
[0028] 2) contacting the solution with FX and detectably-labeled
FXa substrate; and [0029] 3) detecting labeled product in the
solution as being indicative of the factor IXa activation by
TF/VIIa
[0030] In preferred embodiments, this screening assay effectively
measures capacity or capability of the candidate compound to
decrease or eliminate factor IX activation. The assay is generally
sensitive to TF- or TF/VIIa-dependent formation of FIXa and can be
used in several ways to test a desired compound for capacity or
capability to block the factor IX activation. For example, a
compound to be further tested can be added at one or more of the
steps shown above with addition of the compound at step 1) being
preferred for most screening applications. Typically preferred
compounds of this invention will exhibit good blocking activity in
this example of the primary screening assay.
[0031] A further preferred primary screen of the invention is the
Prothrombin Time (PT) test or assay which measures extrinsic
pathway clotting. This test is standard in the field and is
routinely used to measure clotting in biological samples such as
blood plasma.
[0032] More particularly preferred compounds of this invention will
exhibit good inhibitory activity in the PT assay. A typically
preferred compound will increase plasma clotting time in the PT
assay relative to a suitable control by at least about 5% to about
10% (seconds). Preferred use of the PT assay measures TF-mediated
blood plasma clot time and is performed as follows: [0033] 1)
providing citrated plasma in a suitable assay solution under
conditions conducive to plasma coagulation, [0034] 2) admixing a
suitable tissue factor preparation and calcium in the solution
under conditions suitable for initiating plasma clotting; and
[0035] 3) measuring the clot time in the solution to determine the
prothrombin clot time (PT).
[0036] Preferred use of the PT assay measures capability of the
compound tested to prolong the prothrombin clot time. The PT assay
is well known in this field and can be employed in one or a
combination of ways to test the compound for capacity or capability
to increase or block the prothrombin clot time.
[0037] Especially preferred compounds of this invention exhibit
good activity in at least one of the primary assays mentioned above
(factor X, factor IX activation and/or PT tests).
[0038] Good inhibition of the TF- or TF/VIIa-dependent activation
in any one or more of the above primary screening assays at least
in many cases can be attributed to effects of the compound on
TF/VIIa and/or FXa activities. As discussed, preferred compounds of
the invention are TF-antagonists and generally exhibit good
blocking activity in preferred in vitro assays for measuring
TF-mediated blood coagulation. Thus it will usually be desirable to
further test compounds giving good blocking activity in one or more
of the above primary screening assays and in at least one and
preferably more than one of the "secondary screening assays"
discussed below. Such secondary assays can facilitate further
identification and selection of candidate compounds having desired
TF-antagonist activity, e.g., by eliminating from consideration
compounds having activity other than desired activity such as
compounds impacting protease activity.
[0039] A variety of secondary assays can be conducted in accord
with this invention to further evaluate compounds identified in a
primary assay, e.g. to further evaluate activity identified in a
primary assay or to determine the presence of a certain undesired
activity. For example, additionally preferred compounds of this
invention will exhibit substantially reduced or negligible activity
in other secondary screening assays which are not optimized to
measure TF-antagonism. That is, these secondary assays may not be
TF dependent. Particular examples of such assays include those
formatted to measure thrombin, trypsin, or activated factors such
as FXa, FIXa, or FVIIa. Also, preferred compounds exhibit
negligible activity in an Activated Partial Thromoplastin Time
(APTT) test or assay. More specific examples of such secondary
screening assays are provided in the discussion and Examples which
follow.
[0040] In any one or all of the assays disclosed herein including
the primary screens and secondary tests discussed above, the
candidate compound can be provided in the assay as the sole active
agent or it can be combined with other agents to be tested
including other compounds or compositions of this invention. In
this embodiment, the screening assays are particularly useful for
detecting and preferably quantifying synergism between the
compounds, agents or compositions.
[0041] A variety of inhibitors against human tissue factor are
disclosed herein. These compounds can be used in the screening
assays described herein as well as the treatment and prevention
methods of this invention.
[0042] For example, disclosed herein are phosphonate compounds that
are sometimes referenced herein as "TF antagonists", "TF blocking
compounds" or similar phrase. Preferred compounds of the invention
are small molecules and do not include peptide linkage groups. More
particular compounds consist of a phosphonate group and a
"headpiece". Typically, the headpiece is covalently bound to the
phosphonate group and will include or consist of an amine group or
a cyclic ring such as an aromatic group. In embodiments in which
the headpiece includes the aromatic group, the headpiece will
preferably be linked to a phosphonate (preferably bisphosphonate)
group by a nitrogen or oxygen atom. Particular aromatic groups are
phenyl groups which can be substituted with one or more other
groups as discussed below. In embodiments in which the headpiece is
an amine group, it will be appreciated that the compound will be
representative of a primary or further substituted amine
compound.
[0043] More specifically, preferred compounds of the invention
include those of the following Formula I:
Ar-(CXY).sub.m-(Het).sub.0 or
1-(CX.sup.1Y.sup.1).sub.n--C(Z).sub.p-(PO.sub.3).sub.3-p I
[0044] Ar is optionally substituted carbocyclic aryl or optionally
substituted heteroaryl;
[0045] Het is optionally substituted N, O, S, S(O) or
S(O.sub.2);
[0046] each X, each Y, each X', each Y' and each Z are each
independently hydrogen; halogen; hydroxyl; sulfhydryl; amino;
optionally substituted alkyl preferably having 1 to about 12
carbons, more preferably 1 to about 6 carbons; optionally
substituted alkenyl preferably having from about 2 to 12 carbon
atoms, more preferably about 2 to 6 carbons; optionally substituted
alkynyl preferably having from about 2 to 12 carbon atoms, more
preferably about 2 to 6 carbon atoms; optionally substituted alkoxy
preferably having 1 to about 12 carbon atoms, more preferably 1 to
about 6 carbon atoms; optionally substituted alkylthio preferably
having from about 1 to 12 carbon atoms, more preferably about 1 to
6 carbon atoms; optionally substituted alkylsulfinyl preferably
having from about 1 to 12 carbon atoms, more preferably about 1 to
6 carbon atoms; optionally substituted alkylsulfonyl preferably
having from about 1 to 12 carbon atoms, more preferably about 1 to
6 carbon atoms; or optionally substituted alkylamino preferably
having from about 1 to 12 carbon atoms, more preferably about 1 to
6 carbon atoms;
[0047] m is an integer of from 0 (where the hetero atom is directly
substituted on the aryl group) to 4, and preferably is 0, 1 or
2;
[0048] n is an integer of from 0 to 4, and preferably n is 1 or
2;
[0049] p is 1 (where the compound is a bisphosphonate) or 2 (where
the compound has a single terminal PO.sub.3 group); and
pharmaceutically acceptable salts thereof.
[0050] It is understood that in Formula I above, and elsewhere the
designation of "(Het).sub.0 or 1" specifies that the Het group may
be absent (i.e. where the Het subscript is zero) or present in a
single occurrence (i.e. where the Het subscript is one).
[0051] Additional preferred compounds include those of the above
formula where Ar is a carbocyclic aryl group, particularly phenyl,
such as compounds of the following Formula II: ##STR1##
[0052] wherein X, Y, Het, X', Y', Z, m, n, and p are the same as
defined in Formula I above;
[0053] wherein each R.sup.1 is independently halogen (F, Cl, Br,
I); amino; hydroxy; nitro; carboxy; sulfhydryl; optionally
substituted alkyl preferably having 1 to about 20 carbon atoms,
more preferably 1 to about 10 carbon atoms, still more preferably 1
to about 6 carbon atoms; optionally substituted alkenyl preferably
having 2 to about 20 carbon atoms, more preferably 2 to about 10
carbon atoms, still more preferably 2 to about 6 carbon atoms;
optionally substituted alkynyl preferably having 2 to about 20
carbon atoms, more preferably 2 to about 10 carbon atoms, still
more preferably 2 to about 6 carbon atoms; optionally substituted
alkoxy preferably having 1 to about 20 carbon atoms, more
preferably 1 to about 10 carbon atoms, still more preferably 1 to
about 6 carbon atoms; optionally substituted alkylthio preferably
having 1 to about 20 carbon atoms, more preferably 1 to about 10
carbon atoms, still more preferably 1 to about 6 carbon atoms;
optionally substituted alkylsulfinyl preferably having 1 to about
20 carbon atoms, more preferably 1 to about 10 carbon atoms, still
more preferably 1 to about 6 carbon atoms; optionally substituted
alkylsulfonyl preferably having 1 to about 20 carbon atoms, more
preferably 1 to about 10 carbon atoms, still more preferably 1 to
about 6 carbon atoms; optionally substituted alkylamino preferably
having 1 to about 20 carbon atoms, more preferably 1 to about 10
carbon atoms, still more preferably 1 to about 6 carbon atoms;
optionally substituted alkanoyl preferably having 1 to about 20
carbon atoms, more preferably 1 to about 10 carbon atoms, still
more preferably 1 to about 6 carbon atoms; optionally substituted
carbocyclic aryl; or optionally substituted aralkyl;
[0054] q is an integer of from 0 (where the phenyl ring positions
are fully hydrogen substituted) to 5, and preferably m is 0, 1 2 or
3; and pharmaceutically acceptable salts thereof.
[0055] Of the compounds of the above Formulae I and II, additional
compounds include those where the group Het is optionally
substituted nitrogen or oxygen, such as compounds of the following
Formulae III and IV: ##STR2##
[0056] wherein in each of Formula III and IV, R.sup.1, X, Y, X',
Y', Z, q, m, n, and p are the same as defined in Formulae I and II
above; and W is hydrogen, optionally substituted alkyl, preferably
having 1 to about 8 carbon atoms, more preferably 1 to about 6
carbon atoms; optionally substituted alkenyl, preferably having 2
to about 8 carbon atoms, more preferably 2 to about 6 carbon atoms;
optionally substituted alkynyl, preferably having 2 to about 8
carbon atoms, more preferably 2 to about 6 carbon atoms; optionally
substituted alkoxy, preferably having 1 to about 8 carbon atoms,
more preferably 1 to about 6 carbon atoms; optionally substituted
alkylthio, preferably having 1 to about 8 carbon atoms, more
preferably 1 to about 6 carbon atoms; optionally substituted
alkylsulfinyl, preferably having 1 to about 8 carbon atoms, more
preferably 1 to about 6 carbon atoms; optionally substituted
alkylsulfonyl; optionally substituted alkylamino; optionally
substituted alkanoyl, preferably having 1 to about 8 carbon atoms,
more preferably 1 to about 6 carbon atoms; optionally substituted
carbocyclic aryl; or optionally substituted aralkyl; and
pharmaceutically acceptable salts thereof.
[0057] Additional compounds of Formula III include those where the
nitrogen group is a direct (no interposed carbon or other atoms)
phenyl ring substituent, and particularly preferred compounds of
Formula IV include those where the oxygen is a direct ring
substituent or a single methylene group is present, such as
compounds of the following Formulae IIIa and IVa: ##STR3##
[0058] wherein R.sup.1, X', Y', n and q are the same as defined in
Formulae I and II above; and pharmaceutically acceptable salts of
those compounds.
[0059] Additional compounds of the invention bind tissue factor
(TF) so that FX does not effectively bind to the TF/factor VIIa
complex whereby FX is not effectively converted to its activated
form (FXa). Preferred compounds of the invention can inhibit TF
function by effectively blocking FX binding or access to TF
molecules. See, for instance, the results of Example 2 which
follows. As used herein, references herein to "compounds of the
invention" are inclusive of compounds of Formulae I, II, III, IIA,
IV and IVA above.
[0060] In preferred aspects, the invention provides methods for
inhibiting blood coagulation and blood clot formation in a mammal,
methods for inhibiting thrombin generation in a mammal, and methods
for treating or preventing thromboembolic disorders in a mammal.
The methods of the invention in general comprise administering to a
mammal, such as a primate particularly a human, a therapeutically
effective amount of a compound of the invention.
[0061] Compounds of the invention are particularly useful to
alleviate various diseases impacted by tissue factor (TF). By the
term "impacted" is meant that the severity or duration of the
disease is increased by presence of the TF according to the
recognized assays or tests. Particular diseases include thromboses,
especially to prevent or inhibit restenosis, or other thromboses
following an invasive medical procedure such as arterial or cardiac
surgery (e.g., angioplasty or endartectomy), including for
prophylaxis of deep vein thrombosis associated with orthopedic or
other surgery. Compounds of the invention also can be employed to
reduce or even effectively eliminate blood coagulation arising from
use of medical implementation (e.g., a catheter, stent,
arteriovenous shunt or other medical device). Compounds of the
invention also will be useful for prophylaxis for long term risk
for myocardial infarction. Compounds of the invention also will be
useful for treatment of thrombotic conditions that may be
associated with acute promyelocytic leukemia, diabetes, multiple
myelomas, disseminated intravascular coagulation associated with
septic shock, purpura filminanas associated infection, adult
respiratory distress syndrome, unstable angina, and thrombotic
complications associated with aortic valve or vascular
prosthesis.
[0062] Additional uses for the present compounds include use in the
treatment of atherosclerosis, inflammation, and as an
anti-angiogenic agent, especially to treat cancers, particularly
solid cancers such as cancers residing in the lung, breast, liver,
brain or other tissue.
[0063] Compounds of the invention also can be employed as an
anti-coagulant in extracorporeal circulation of a mammal,
particularly a human subject. In such methods, one or more
compounds of the invention is administered to the mammal in an
amount sufficient to inhibit blood coagulation prior to or during
extracorporeal circulation such as may be occur with
cardiopulmonary bypass surgery, organ transplant surgery or other
prolonged surgeries.
[0064] Compounds of the invention also can be employed in in vivo
diagnostic methods including in vivo diagnostic imaging of a
patient.
[0065] Compounds of the invention also can be used in in vitro
assays, e.g. to selectively inhibit factor X activation. Such
assays of the invention will be useful to determine the presence or
likelihood of a patient having blood coagulation or a blood
clot.
[0066] Pharmaceutical compositions also are provided comprising an
effective amount of one or more compounds of the invention and a
pharmaceutically acceptable carrier.
[0067] Other aspects of the invention are discussed infra.
BREIF DESCRIPTION OF THE DRAWINGS
[0068] FIG. 1 is a flow chart of a screening assay employed in
examples below.
[0069] FIGS. 2(a), 2(b), 2(c) and 2(d) disclose results of examples
which follow.
[0070] FIG. 3 is a table showing IC.sub.50 values and inhibition of
protease activities by certain TF antagonists of this invention,
i.e. compounds 1, 2, 3, 4, and 6. The compound labeled "Fosamax" is
represented by the formula NH.sub.2-bisphosphonate.
[0071] FIG. 4 is a table showing the effect of specific TF
antagonists (compounds 1 and 2) in the prothrombin time (PT)
assay.
[0072] FIG. 5 is a table showing effects of compound 1 on K.sub.m
values for FX in TF/VIIa-dependent activation assay.
[0073] FIG. 6 is a graph showing inhibition of TF/VIIa-dependent
activation by compound 1. FX concentration was 30 nM in the
assay.
[0074] FIG. 7 is a graph showing inhibition of TF/VIIa-dependent FX
activation by compound 1. FX concentration was 5 nM in the
assay.
DETAILED DESCRIPTION OF THE INVENTION
[0075] As discussed, the present invention features compounds such
as pharmaceutically active compounds and especially pharmaceutical
compositions that utilize or comprise one or more of such
compounds. Preferred compounds are effective TF antagonists as
determined by standard in vitro screening assays disclosed herein.
Especially preferred compounds are very useful for the treatment or
prophylaxis of undesired thrombosis. The invention has a wide
spectrum of applications including use in screening candidate
compounds having significant TF-antagonistic activity.
[0076] As discussed, preferred compounds and compositions of this
invention are good TF antagonists that exhibit significant blocking
activity in at least one of and preferably all of the primary
screening assays (TF- or TF/VIIa-dependent activation of factor X
or factor IX, and PT assay). Especially preferred compounds do not
exhibit significant blocking activity in the Activated Partial
Thromoplastin Time (APTT) assay discussed previously. Further
preferred are those compounds of this invention showing
insignificant activity in other secondary assays such as those for
measuring trypsin, thrombin, factor Xa, factor IXa, and factor VIIa
activity as discussed below.
[0077] The standard in vitro assays disclosed herein are well-known
in the field and are generally flexible. Moreover, the assays can
be conveniently manipulated to detect and quantify TF-antagonistic
activity as needed. The assays are typically compatible with
testing compounds or compositions of this invention in the presence
of other therapeutic or experimental agents giving good
anti-platelet, anti-thrombolytic, or anti-coagulant activity. In
addition, the assays can be used to test effects with recognized
anti-TF antibodies. In these embodiments, the standard in vitro
assays are especially useful for detecting and preferably measuring
significant co-operative or synergistic effects exhibited by the
compounds or compositions of this invention.
[0078] A more particular example of the primary screening assay
discussed previously is as follows. The assay is standard for
measuring TF/VIIa-dependent factor X activation. A preferred
compound exhibits an IC.sub.50 in the assay of less than about 100
.mu.M and preferably less than about 10 .mu.M exemplifying good
blocking activity in this assay. In a more preferred embodiment,
the primary screening preferably includes the following steps.
[0079] 1) admixing in a suitable assay solution about 0.1 nM of
human recombinant TF/VIIa complex (lipidated), about 180 nM human
FX, and between from about 0.5 .mu.l to about 10 .mu.l of at least
one compound to be tested (optionally dissolved in an appropriate
vehicle such as water or dimethylsulfoxide (DMSO)) and incubating
the reaction at 37.degree. C. for a few minutes up to about an hour
or more, [0080] 2) contacting the solution with a suitable
chelating agent such as ethylenediaminetetra acetic acid (EDTA) to
reduce or stop factor X activation, [0081] 3) contacting the
solution with a detectable amount of a chromogenic substrate
specific for FXa (e.g., Spectrozyme FXa or S-2765) and incubating
same at 37.degree. C.; and [0082] 4) detecting chromophore produced
in the solution as being indicative of the factor X activation.
[0083] Reference herein to a "standard assay for measuring
TF/VIIa-dependent factor X activation" or similar phrase will
preferably refer to the above steps 1)-4). More specific disclosure
relating to the assay can be found in Example 2 below in which the
standard assay for measuring TF/VIIa-dependent factor X activation
is specifically adapted for spectrophotometric detection of FXa
produced chromophores at 405 nm.
[0084] A preferred TF/VIIa complex for use in the method includes
TF that has been exposed to conditions suitable for exhibiting good
TF blocking sites. Such TF molecules can be obtained by one or a
combination of approaches. In one method, human TF is obtained from
an overproducing immortalized cell line or an acetone powder
derived from human brain. TF is preferably isolated in the presence
of at least one non-ionic detergent such as TRITON.RTM. X-100
(polyoxyethylene (10) isooctylphenyl ether) under moderate
conditions of salt and pH, e.g., 100 mM NaCl and pH 8.0. Preferred
amounts of the non-ionic detergent will vary depending on intended
use but will generally be in an amount of from between about 0.05%
to about 0.5% (w/v). See the General Comments of the examples below
for more specific information about isolating human TF.
[0085] Additionally preferred TF is exposed to conditions in the
standard assay for measuring TF/VIIa-dependent factor X activation.
See Example 2 below for more specific disclosure about that
standard assay.
[0086] Additionally preferred compounds of this invention exhibit
good blocking activity in the other primary screening assay for
measuring TF/VIIa-dependent factor IX activation. Preferred
compounds exhibit an IC.sub.50 in the assay of less than about 200
.mu.M with preferably less than about 10 .mu.M exemplifying good
blocking activity in this assay. In a more particular embodiment,
the standard assay preferably includes the following steps: [0087]
1) admixing in a suitable assay solution about 0.7 nM TF/VIIa
complex with 300 nM factor IX and 1000 nM factor X, and from
between about 0.5 .mu.l to about 10 .mu.l of at least one compound
to be tested (optionally dissolved in an appropriate vehicle such
as water or dimethylsulfoxide (DMSO)) and incubating the solution
at 37.degree. C. from between about a few minutes up to about an
hour under conditions suitable for forming FIXa and FXa; [0088] 2)
contacting the solution with a suitable chelating agent such as
EDTA to stop FIX activation; [0089] 3) contacting the solution with
a chromogenic substrate specific for the FXa (e.g., Spectrozyme
FXa) and incubating same at 37.degree. C.; and p1 4) detecting
chromophore in the solution as being indicative of the factor IX
activation.
[0090] Reference herein to a "standard assay for measuring TF/VIIa
dependent factor IX activation" or similar term or phrase will
specifically refer to the above steps 1)-4). See Example 2 below
for a more specific illustration of the standard assay adapted for
spectrophotometric detection of preferred chromophore at 405
nm.
[0091] The table in FIG. 3 below shows specific IC.sub.50 values
for specific TF antagonists of the invention, i.e. compound 1,
compound 2, compound 3, compound 4, and compound 6, as well as
Fosamax. The values were determined in the standard assays for
measuring TF/VIIa-dependent factor X activation and
TF/VIIa-dependent factor IX activation. As can be seen from the
table in FIG. 3, these compounds give good blocking activity in
these assays.
[0092] As discussed, additionally preferred compounds of this
invention exhibit good clot time inhibition in the PT assay,
preferably an increase in clotting time from between about 20% to
at least 100%, and more preferably from between about 20% to at
least 500% relative to a suitable control. Clot times are generally
measured in seconds. Preferred PT assays are typically performed by
adding a suitable amount (e.g. about 1 to 3 nM) of lipidated tissue
factor to an assay solution that includes conventionally citrated
plasma. The PT assay measures TF-mediated blood plasma clot time
and is preferably performed by conducting the following steps:
[0093] 1) providing about 0.1 ml of citrated human plasma in a
suitable assay solution, and combining same with between from about
0.5 .mu.l to 10 .mu.l of at least one compound to be tested
(optionally dissolved in vehicle such as water or dimethylsulfoxide
(DMSO)) and incubating same at room temperature for about 3 to 10
minutes, [0094] 2) admixing into the solution from between about
0.2 ml (ca. 1-3 nM recombinant human tissue factor) and about 5-10
mM of calcium to initiate plasma clotting; and [0095] 3) measuring
the plasma clot time to determine the prothrombin clot time
(PIT).
[0096] Reference herein to a "standard PT assay " or similar phrase
or term will specifically refer to the above steps 1)-3). See also
Williams Hematology, 5.sup.th Ed. (Beutler, E. et al. Eds.)
McGraw-Hill, Inc. Health Professions Div., New York, for more
specific disclosure relating to conducting the PT assay.
[0097] As mentioned, the present invention provides a variety of
assays for detecting and preferably measuring capability of
preferred compounds of this invention to antagonize good TF
activity. As has also been discussed, certain standard in vitro
screening assays are sometimes referred to herein as "secondary
screening assays" to denote preferred use with one or more or all
of the primary screening assays mentioned previously. Practice of
such particular secondary screening assays in conjunction with one
or more of the primary screening assays will provide a wide
spectrum of useful compounds featuring good anti-TF activity.
[0098] Secondary screening assays are disclosed herein and include
those optimized to detect and preferably measure the catalytic
activities of factor VIIa (FVIIa), factor IXa (FIXa), factor Xa
(FXa), thrombin, trypsin, or Russell's viper venom (RVV). In most
instances, optimal practice of these assays does not require added
TF. Preferred compounds of this invention are specific
TF-antagonists and will generally exhibit substantially reduced or
negligible activity in these assays. Practice of the secondary
screening tests in conjunction with the primary and preferred
secondary screening assays discussed previously will facilitate
selection of preferred compounds exhibiting highly specific anti-TF
activity. Reference herein to "reduced" or "negligible" activity
with respect to these secondary screening assays is meant to denote
between from about 2% to about 10% of the activity exhibited by a
suitable control such as water or DMSO.
[0099] As discussed above, the invention provides a wide spectrum
of pharmaceutically active compounds and compositions that are
useful to treat or prevent undesired thrombosis. Preferred
compounds are tissue factor (TF) antagonists and preferably can
specifically block human factor X and IX activation catalyzed by
human tissue factor/factor VIIa complex. Illustrative compounds of
the invention include the anti-coagulant phosphonate of the
above-defined Formula I, II, III, IIIA, IV, and IVA.
[0100] Illustrative compounds of the invention are bisphosphonate,
i.e. compounds of the above formulae where p is 1 and two
--PO.sub.3 groups are present. Preferred R.sup.1 ring substituents
of the above formulae include hydroxy, halogen, alkyl such as
C.sub.1-6 alkyl, amino, and alkylamino such as mono-or
di-(C.sub.1-4)alkyl. Preferred W groups (optional amino
substituent) include hydrogen, and optionally substituted alkyl,
particularly C.sub.1-6 optionally substituted alkyl. Preferred X,
Y, X', Y' and Z groups include hydrogen and optionally substituted
alkyl, particularly C.sub.1-6 optionally substituted alkyl.
[0101] Additional compounds of the invention include the following
compounds 1 through 6 and pharmaceutically acceptable salts of
those compounds. Compounds 1, 2 and 6 below are particularly
preferred. Those compound designations 1 through 6 are used
throughout the present disclosure and refer to the specified
compounds of the structures shown immediately below. ##STR4##
[0102] Suitable halogen substituent groups of compounds of the
invention (which includes e.g. compounds of Formulae I, II, III,
IIIA, IV and/or IVA as those formulae are defined above) are F, Cl,
Br and I. As used herein, the term alkyl unless otherwise modified
refers to both cyclic and noncyclic groups, although cyclic groups
will comprise at least three carbon ring atoms. Alicyclic alkyl
groups are generally preferred. Alkenyl and alkynyl groups of
compounds of the invention have one or more unsaturated linkages,
typically 1 to about 3 or 4 unsaturated linkages. Also, the terms
alkenyl and alkynyl as used herein refer to both cyclic and
noncyclic groups, although straight or branched noncyclic groups
are generally more preferred. Alkoxy groups of compounds of the
invention have one or more oxygen linkages, typically 1 to about 5
or 6 oxygen linkages. Alkylthio groups of compounds of the
invention have one or more thioether linkages, typically 1 to about
5 or 6 thioether linkages. Alkylsulfinyl groups of compound of the
invention have one or more sulfinyl (SO) linkages, typically 1 to
about 5 or 6 sulfinyl linkages. Alkylsulfonyl groups of compounds
of the invention have one or more sulfonyl (SO.sub.2) linkages,
typically 1 to about 5 or 6 sulfonyl linkages. Preferred alkylamino
groups of compounds of the invention include those groups having
one or more primary, secondary and/or tertiary amine groups,
preferably 1 to about 3 or 4 amine groups. Suitable alkanoyl groups
have one or more carbonyl groups, typically 1 to about 4 or 5
carbonyl groups. Alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,
alkanoyl and other groups may be suitably either linear or
branched. Carbocyclic aryl as used herein refers to non-hetero
aromatic groups that have 1 to 3 separate or fused rings and 6 to
about 18 carbon ring members and may include e.g. phenyl, naphthyl,
biphenyl, acenaphthyl, phenanthracyl, and the like. Phenyl and
naphthyl are often preferred. Suitable heteroaromatic or heteroaryl
groups will have 1 to 3 rings, 3 to 8 ring members in each ring and
from 1 to about 3 hetero atoms (N, O or S). Specifically suitable
heteroaromatic or heteroaryl groups include e.g. courmarinyl,
quinolinyl, pyridyl, pyrazinyl, pyrimdinyl, furyl, pyrrolyl,
thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl,
and benzothiazol.
[0103] Compounds of Formulae II, III, IIIA, IV and/or IVA as those
formulae are defined above preferably have a R.sup.1 group present
as a para substituent on the phenyl ring.
[0104] As discussed above, R.sup.1, W, X, Y, X', Y', nitrogen "Het"
groups, and Z groups are optionally substituted. Suitable groups
that may be present on a "substituted" R.sup.1, W, X, Y, X', Y',
Het and Z substituent include e.g. halogen such as fluoro, chloro,
bromo and iodo; cyano; hydroxyl; nitro; azido; sulfhydryl; alkanoyl
e.g. C.sub.1-6 alkanoyl group such as acetyl and the like;
carboxamido; alkyl groups including those groups having 1 to about
12 carbon atoms, preferably from 1 to about 6 carbon atoms; alkenyl
and alkynyl groups including groups having one or more unsaturated
linkages and from 2 to about 12 carbon atoms, preferably from 2 to
about 6 carbon atoms; alkoxy groups having one or more oxygen
linkages and from 1 to about 12 carbon atoms, preferably 1 to about
6 carbon atoms; aryloxy such as phenoxy; alkylthio groups including
those moieties having one or more thioether linkages and from 1 to
about 12 carbon atoms, preferably from 1 to about 6 carbon atoms;
alkylsulfinyl groups including those moieties having one or more
sulfinyl linkages and from 1 to about 12 carbon atoms, preferably
from 1 to about 6 carbon atoms; alkylsulfonyl groups including
those moieties having one or more sulfonyl linkages and from 1 to
about 12 carbon atoms, preferably from 1 to about 6 carbon atoms;
aminoalkyl groups such as groups having one or more N atoms and
from 1 to about 12 carbon atoms, preferably from 1 to about 6
carbon atoms; carbocyclic aryl having 6 or more carbons,
particularly phenyl; aryloxy such as phenoxy; aralkyl having 1 to 3
separate or fused rings and from 6 to about 18 carbon ring atoms,
with benzyl being a preferred group; aralkoxy having 1 to 3
separate or fused rings and from 6 to about 18 carbon ring atoms,
with O-benzyl being a preferred group; or a heteroaromatic or
heteroalicyclic group having 1 to 3 separate or fused rings with 3
to about 8 members per ring and one or more N, O or S atoms, e.g.
coumarinyl, quinolinyl, pyridyl, pyrazinyl, pyrimidyl, furyl,
pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl,
benzofuranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl,
piperidinyl, morpholino and pyrrolidinyl. A "substituted" R.sup.1,
W, X, Y and Z substituent of a compound of the invention may be
substituted at one or more available positions, typically 1, 2 or 3
positions, by one or more suitable groups such as those listed
immediately above.
[0105] Compounds of the invention can be prepared by procedures
generally known in the art. For example, phosphonate acids of
Formula I can be prepared by formation of the corresponding alkyl
diester followed by conversion to the diacid, e.g. by treatment of
the diester with bromotrimethylsilane, and then further reaction of
that intermediate to provide a compound of the invention. See, for
instance, C. R. Degenhardt et al., J. Org. Chem., 51:3488-3490
(1986); I. S. Alfer et al., Izv. Akad Nauk SSSR, 1122-1126 (1984);
I. S. Alfer et al., Izv. Akad Nauk SSSR, 2802-2806 (1983); and U.S.
Pat. No. 5,728,650. See also Example 1 which follows.
[0106] Reference herein to a "TF blocking compound," "TF
antagonist" or related term generally includes those compounds
disclosed herein exhibiting good blocking activity in at least one
of the primary screening assays such as the PT assay. More
particular TF blocking compounds specifically bind TF. Without
wishing to be bound to theory, the compounds are believed to block
FX or FIX from binding TF in a way sufficient to reduce or block
activation to FX or FIX, respectively.
[0107] Reference to a "therapeutically effective amount" of a
composition is such as to produce a desired effect in a host such
as a mammal and especially a primate such as a human patient.
Preferably the effect can be monitored using several end-points
known to those of skill in the field. For example, one desired
effect is an increase or stabilization of cardiovascular function
as measured, e.g., by enhanced heart function and especially blood
flow within subject vessels. Such impact can be monitored and
usually measured in terms of a therapeutic effect, e.g., improved
cardiovascular function, alleviation of one or more symptoms
indicative of compromised heart function or function of related
vasculature, or other particularized physiological assays. These
specific methods are not intended to be inclusive and further
methods intended to suit a specific application such as thrombosis,
cancer, or atherosclerosis will be apparent to the skilled worker
in the field.
[0108] As discussed above, a compound of the invention can be
administered to a mammal, preferably a primate such as a human, to
prevent or reduce thromboses. Therapies in which compounds of the
invention will be useful include treatment or prophylaxis of venous
thrombosis and pulmonary embolism, arterial thrombosis e.g.
myocardial ischemia, myocardial infarction, unstable angina, stroke
associated with thrombosis, and peripheral arterial thrombosis.
Compounds of the invention also may be useful for treatment or
prophylaxis of atherosclerotic diseases e.g. coronary arterial
disease, cerebral arterial disease and peripheral arterial disease.
See e.g., Wilde, R. G. et al. Bioinorganic & Medicinal
Chemistry Letters 167-172 (1995). Compounds of the invention also
will be useful for anticoagulation treatment involving artificial
organs, cardiac valves, medical implementation (e.g. an indwelling
device such as a catheter, stent, etc.) and the like. Compounds of
the invention also will be useful for therapy in other disorders or
diseases where blood coagulation may be involved as a related
disorder, e.g. cancer, inflammatory diseases particularly
arthritis, and diabetes.
[0109] One or more compounds also may be administered as the sole
therapeutic agent(s) in a particular protocol, or the compound(s)
of the invention may be administered together with other
therapeutic agents, e.g. a pharmaceutical targeted for interaction
in the blood clotting mechanism such as streptokinase, tPA,
urokinase and other agents that lyse clots. A compound of the
invention also can be administered with other agents such as one or
more other anti-coagulants (e.g., heparin, hirudin, or hirulog), or
an anti-platelet (e.g., ReoPro or aspirin). In such combination
therapy, a compound of the invention may be administered prior to,
or after administration of one or more other suitable
anti-coagulant, anti-platelet, thrombolytic or other agents to
boost or prolong desired anti-coagulation activity.
[0110] Compounds of this invention can be administered
intranasally, orally or by injection, e.g., intramuscular,
intraperitoneal, subcutaneous or intravenous injection, or by
transdermal, intraocular or enteral means. Intravenous or
parenteral administration includes e.g. sub-cutaneous,
intraperitoneal or intramuscular administration. Generally
preferred is oral administration. The optimal dose can be
determined by conventional means. Compounds of the present
invention are suitably administered to a subject in the protonated
and water-soluble form, e.g., as a pharmaceutically acceptable salt
of an organic or inorganic acid, e.g., hydrochloride, sulfate,
hemi-sulfate, phosphate, nitrate, acetate, oxalate, citrate,
maleate, mesylate, etc.
[0111] Compounds of the invention can be employed, either alone or
in combination with one or more other therapeutic agents as
discussed above, as a pharmaceutical composition in mixture with
conventional excipient, i.e., pharmaceutically acceptable organic
or inorganic carrier substances suitable for parenteral, enteral or
intranasal application which do not deleteriously react with the
active compounds and are not deleterious to the recipient
thereof.
[0112] Suitable pharmaceutically acceptable carriers include but
are not limited to water, salt solutions, alcohol, vegetable oils,
polyethylene glycols, gelatin, lactose, amylose, magnesium
stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty
acid monoglycerides and diglycerides, petroethral fatty acid
esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc. The
pharmaceutical preparations can be sterilized and if desired mixed
with auxiliary agents, e.g., lubricants, preservatives,
stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic pressure, buffers, colorings, flavorings and/or aromatic
substances and the like which do not deleteriously react with the
active compounds.
[0113] For parenteral application, particularly suitable are
solutions, preferably oily or aqueous solutions as well as
suspensions, emulsions, or implants, including suppositories.
Ampules are convenient unit dosages.
[0114] For enteral application, particularly suitable are tablets,
dragees or capsules having talc and/or carbohydrate carrier binder
or the like, the carrier preferably being lactose and/or corn
starch and/or potato starch. A syrup, elixir or the like can be
used wherein a sweetened vehicle is employed. Sustained release
compositions can be formulated including those wherein the active
component is protected with differentially degradable coatings,
e.g., by microencapsulation, multiple coatings, etc.
[0115] See, in general, Remington's Pharmaceutical Sciences, (Mack
Publishing Co., Easton Pa., (1980)), for a discussion of suitable
administration formulations.
[0116] It will be appreciated that the actual preferred amounts of
active compounds used in a given therapy will vary according to the
specific compound being utilized, the particular compositions
formulated, the mode of application, the particular site of
administration, etc. Optimal administration for a given protocol of
administration can be readily ascertained by those skilled in the
art using conventional dosage determination tests conducted with
regard to the foregoing guidelines. In general, a suitable
effective dose of one or more compounds of Formula I will be in the
range of about 0.01 to 100 milligrams per kilogram of bodyweight of
recipient per day, preferably in the range of from about 0.01 to 20
to 50 milligrams per kilogram or bodyweight of recipient per
day.
[0117] All documents mentioned herein are fully incorporated by
reference in their entirety.
[0118] The following non-limiting examples are illustrative of the
invention.
General Comments
[0119] In the examples below, purified human factors VIIa, IX and
X, thrombin, and Russell's viper venom were obtained from Enzyme
Research Laboratories Inc. Trypsin was from Boehriger Mannheim.
Chromogenic substrates S-2222, S-2288, S-2238, and S-2765 were from
DiaPharma Group Inc., and Spectrozyme FXa was from American
Diagnostica Inc. Truncated recombinant human tissue factor (e.g.
composed of 243 amino acids) is expressed in E. coli and purified
by immunoaffinity chromatography. A preferred truncated recombinant
human tissue factor lacks the cytomplasmic domain. Native human TF
was extracted from human carcinoma cell line J82 with 50 mM
Tris-HCl, pH 8.0, containing 0.1 M NaCl, 1 mM EDTA, 0.3% Triton
X-100. Native TF from other sources is extracted with the same
buffer from animal brain acetone powders. All other reagents were
from Sigma
EXAMPLE 1
Preparation of 1-(bisphosponate)-2-amino(3-hydroxyphenyl)ethyl
(Compound 1 Above)
[0120] The method of Degenhardt et al., J. Org. Chem., 51:
3488-3490 (1986) can be followed to produce the compound. Briefly,
paraformaldehyde (104.2 g, 3.47 mol) and di-ethylamine (50.8 g,
0.69 mol) are combined in 2 liters of methanol and the mixture
warmed until clear. The heat is removed and
CH.sub.2(PO.sub.3(CH.sub.2CH.sub.3).sub.2).sub.2 (200 g, 0.69 mol)
is added. The mixture is refluxed for 24 hours, and then an
additional 2 liters of methanol is added, and the solution
concentrated under reduced pressure at 35.degree. C. 1 liter of
toluene is added to the concentrate, and the resulting solution
concentrated, and the toluene addition and concentration repeated.
The resulting intermediate is then dissolved in 1 liter of dry
toluene, p-toluenesulfonic acid monohydrate (0.50 g) is added and
the mixture is refluxed. Resulting methanol is removed, e.g. via a
Dean-Stark trap or molecular sieves. After 14 hours the solution
can be concentrated, diluted in chloroform, washed with water
(2.times.150 ml), dried over MgSO.sub.4 and concentrated. The
resulting compound,
CH.sub.2.dbd.C(PO.sub.3(CH.sub.2CH.sub.3).sub.2).sub.2, can be
purified if desired such as distillation. The compound
CH.sub.2.dbd.C(PO.sub.3(CH.sub.2CH.sub.3).sub.2).sub.2 can then be
reacted as desired to provide compounds of the invention. In
particular, to provide the title compound,
CH.sub.2.dbd.C(PO.sub.3(CH.sub.2CH.sub.3).sub.2).sub.2, can be
reacted with NH.sub.2(3-hydroxyphenyl) in a Michael reaction. The
phosphono di-ester can be converted to the di-acid by treatment
with bromotrimethylsilane (see, e.g. Morita et al., Bull. Chem.
Soc. Jpn., 54:267 (1981)).
EXAMPLE 2
Screening
[0121] The primary screening for compounds that inhibit tissue
factor/factor VIIa (TF/VIIa) is based on TF/VIIa-dependent FX
activation assay (See flow chart in FIG. 1 of the drawings). In
this assay, the ability of TF/VIIa complex to activate FX is
determined in two discontinuous stages. In the first stage (FX
activation), the inactive FX is converted to an active enzyme form,
FXa, by TF/VIIa in the presence of phospholipids and calcium. In
the second stage (FXa activity assay), EDTA is added at indicated
times to the FX activation mixture to chelate calcium, thus leading
to the termination of FX.fwdarw.FXa conversion. Calcium is required
for TF/VIIa activity. The activity of FXa is then measured by
FXa-specific chromogenic substrates such as S-2222, S-2765, or
Spectrozyme FXa In the primary screening, compounds from a
previously prepared chemical library are first tested at relatively
high concentrations (.about.0.833 mM) in TF/VIIa-dependent FX
activation assay to identify hits of potential TF pathway
antagonists (see FIG. 1). However, it is evident that the
inhibition of TF/VIIa-dependent FX activation by a compound in this
enzyme-coupled assay can be attributed to effects of the compound
on TF/VIIa and/or FXa activities. Thus, secondary screening tests
are designed to determine how inhibition takes place and the
inhibition mechanism. In secondary screening experiments, effects
of those compounds identified from primary screening are tested on
catalytic activities of factor VIIa (FVIIa), factor IXa (FIXa),
factor Xa (FXa), thrombin, Russell's viper venom (RVV), and
trypsin. Additional tests such as the TF/VIIa-dependent factor IX
activation assay and the prothrombin time (PT) assay were conducted
to confirm desired activity, and secondary tests were conducted to
further select compounds with good TF-antagonistic activity and
that did not exhibit undesired activity.
[0122] A. Primary Screening: TF/VIIa-Dependent FX Activation
[0123] Primary screening was done in duplicate in 96-well plates
using the TF/VIIa-dependent FX activation assay. All compounds to
be screened were dissolved in dimethyl sulfoxide (DMSO), other
reagents were prepared or diluted in 25 mM HEPES-NaOH, 5 mM
CaCl.sub.2, 150 mM NaCl, 0.1% BSA, pH 7.5. For assays where TF was
used, purified human recombinant TF (100 nM) was first lipidated
with phosphatidylcholine (0.07 mg/ml) and phosphatidylserine (0.03
mg/ml) in 50 mM Tris-HCl, pH 7.5, 0.1% bovine serum albumin (BSA)
for 30 minutes at 37.degree. C. A stock solution of TF/VIIa complex
was then prepared by combining equal volumes of 100 nM lipidated TF
and 100 nM FVIIa The complex was incubated at 37.degree. C. for 30
minutes and then was aliquoted and stored at -70.degree. C. for
future uses.
[0124] For screening assays, 5 .mu.l of each compound (about 10 mM
in DMSO) or DMSO were placed in each well of a 96-well plate,
followed by adding 45 .mu.l of TF/VIIa complex (0.1 nM). The
components in each well were mixed either with pipette tips or by
shaking the plate on a Lab-Line titer plate shaker for 30 seconds.
After 15 minutes incubation of the plate at a 37.degree. C., 10
.mu.l of human FX (180 nM) was added to each well and mixed as
above. The plate was then incubated at 37.degree. C. for 3 to 15
minutes, followed by addition of 10 .mu.l of EDTA (400 mM in 144 mM
HEPES, 864 mM NaCl, 0.576% BSA, pH adjusted to 7.5) to each well to
terminate FX activation. Ten microliters of FXa substrate (5 mM
Spectrozyme FXa, or 3.2 mM S-2765) was added to each well to
measure FXa activity. The plate was mixed as above, and after about
a 15 minute incubation at 37.degree. C., FXa activity was quenched
with 20 .mu.l of 50% acetic acid. Absorbance at 405 nm was then
read by an ELISA reader. The OD.sub.405 nm values were transferred
to a Microsoft Excel file and the percent inhibition of
TF/VIIa-dependent FX activation was calculated by the following
formula: % Inhibition=100-(100.times.A/B) where A and B are the OD
values in the presence and absence of a compound, respectively. Any
compound showing equivalent or greater than 70% inhibition of
TF/VIIa-dependent FX activation was designated as a candidate for
secondary screening test.
[0125] B. Secondary Screening
[0126] Those compounds identified in primary screening were
retested in TF/VIIa-dependent FX activation assay at 10-, 50- or
100-fold diluted concentrations (see flow chart of FIG. 1 of the
drawings). Compounds that failed to show significant inhibition at
diluted concentrations, indicating that the inhibition is either
non-specific or very weak, were not tested further. Compounds that
inhibited TF/VIIa-dependent FX activation at diluted concentrations
were further tested for their ability to inhibit activities of the
following proteases, trypsin, RVV, thrombin, FXa, FVIIa, and FIXa.
A target was to identify compounds that specifically prevent FX
(and FIX) binding to TF/VIIa complex or interfere with TF and VIIa
interaction so that FX (and FIX) activation is blocked. However,
those compounds that have broad ability (non-specific) to inhibit
several protease activities were not further investigated.
Compounds that met the specified criteria, that is, to inhibit
TF/VIIa-dependent FX activation at lower concentrations (<0.1
mM) but without significant effects on protease activities, were
selected, including compounds 1 and 2. whose structures are shown
above, identified as strong TF antagonists and investigated
further.
EXAMPLE 3
Effects of Compounds of the Invention on FVIIa, FXa, Thrombin, and
Trypsin
[0127] To test whether compounds 1 and 2 inhibit coagulation
proteases and trypsin, the following assays were conducted.
[0128] A. FVIIa Activity Assay
[0129] Factor VIIa (FVIIa) activity, or the effect of TF and FVIIa
interactions, can be determined in the presence of TF using FX and
a small peptide (chromogenic) substrate or in the absence of TF
using FX as substrate. Assays using FVIIa-specific chromogenic
substrate S-2288 directly measures the effect of a compound on
FVIIa catalytic activity. In this assay, 55 .mu.l of TF/FVIIa
complex (containing 10 nM TF and 10 nM VIIa) was first incubated
with 5 .mu.l of DMSO (or diluted DMSO) or compound, in a 96-well
plate for 15 minutes at 37.degree. C., then admixed with 20 .mu.l
of 8 mM S-2288. The reaction was incubated for 1-2 hours at
37.degree. C. Absorbance at 405 nm was then measured after the
reaction was quenched with 20 .mu.l of 50% acetic acid. The percent
inhibition of TF/VIIa activity was calculated from OD.sub.405 nm
values in the absence and presence of a compound. Results are shown
in Table 1 (FIG. 2(a)) and show that compounds 1 and 2 do not have
significant effect on TF/VIIa catalytic activity toward S-2288,
indicating that these compounds do not bind to the active site of
FVIIa, nor do they interfere with TF and VIIa interactions.
Inhibition of TF/VIIa activity toward S-2288 by the two compounds
would be expected if they were to bind to VIIa active site or
prevent TF and VIIa from forming an active complex.
[0130] B. FXa Activity Assay
[0131] FX is converted to FXa by TF/VIIa complex in the absence of
any compound. To do that, 54 .mu.l of TF/VIIa (50 nM) was added to
27 ml of buffer in a 50-ml tube. Then 6 ml of FX (180 nM) was added
and incubated at 37.degree. C. for 15 minutes. Six ml of EDTA was
added to stop FX activation. Five .mu.l of compound or DMSO was
placed in each well of a 96-well plate in duplicate, then 65 .mu.l
of FXa generated above was added to each well and mixed. After
incubation for 15 minutes at 37.degree. C., 10 .mu.l of FXa
substrate Spectrozyme FXa was added and incubated for 20 minutes at
37.degree. C. Absorbance at 405 nm was then measured following
addition of 20 .mu.l of 50% acetic acid. The percent inhibition of
FXa activity was calculated from OD.sub.405 nm values in the
absence and presence of a compound. Results shown in Table 1 (FIG.
2(a)) indicate that compounds 1 and 2 do not inhibit FXa activity,
suggesting that inhibition of TF/VIIa-dependent FX activation is
not due to the inhibition of FXa activity by these two
compounds.
[0132] C. Thrombin Activity Assay
[0133] For thrombin activity assay, 55 .mu.l of buffer was mixed
with 5 .mu.l of DMSO or compound, followed by addition of 10 .mu.l
of thrombin (20 nM). Mix and incubate at 37.degree. C. for 10
minutes. Then 10 .mu.l of substrate S-2238 was added and allowed to
incubate at 37.degree. C. for 15-20 minutes. Absorbance at 405 nm
was then measured and the percent inhibition of thrombin activity
was calculated from OD.sub.405 nm values in the absence and
presence of a compound. Results shown Table 1 (FIG. 2(a)) indicated
that compounds 1 and 2 (structures shown above) do not inhibit
thrombin activity.
[0134] D. Trypsin Activity
[0135] For trypsin activity assay, 4 .mu.l of trypsin (100 nM) was
first mixed with 61 .mu.l of buffer, 5 .mu.l of DMSO or 5 .mu.l of
compound (in DMSO), followed by 15 minute incubation at 37.degree.
C. Then 10 .mu.l of substrate S-2222 (4.8 mM) was added to start
the reaction. After a 15 minute incubation at 37.degree. C., 20
.mu.l of 50% acetic acid was added to quench the reaction.
Absorbance at 405 nm was then measured and the percent inhibition
of trypsin activity was calculated from OD.sub.405 nm values in the
absence and presence of a compound. Results in Table 1 (FIG. 2(a))
showed that the compounds 1 and 2 (structures shown above) do not
inhibit trypsin activity.
[0136] See also FIG. 3 showing percent inhibition of factor Xa and
factor VIIa activity using 83 .mu.M compound 1 or compound 2. FIG.
3 also shows percent inhibition of thrombin activity at 63 .mu.M
compound 1 or compound 2. Also shown in the figure is percent
inhibition of trypsin activity at 71 .mu.M compound 1 or compound
2.
Example 4
Effects of TF Antagonists on FX Activation Catalyzed by RVV, FIXa,
and FVIIa
[0137] In addition to TF/VIIa complex, RVV, FIXa, and FVIIa are
also able to activate FX in vitro. The following assays were
conducted to examine the effects of 1 and 2 (structures shown
above) on FX activation catalyzed by RVV, FIXa and VIIa. Data from
these assays will help understand where these compounds may bind
and the inhibitory mechanism.
[0138] A. RVV-Dependent FX Activation
[0139] 45 .mu.l of RVV (0.1 nM) was added into each well of a
96-well plate that contains 5 .mu.l of diluted DMSO (or buffer) or
compound, then mixed and incubated at 37.degree. C. for 15 minutes.
Then 10 .mu.l of FX (180 nM) was added and incubated for 15 minutes
at 37.degree. C. After adding 10 .mu.l of EDTA (400 mM) and 10
.mu.l of Spectrozyme FXa (5 mM), the reaction was incubated for 20
minutes at 37.degree. C. Absorbance at 405 nm was then measured
following addition of 20 .mu.l of 50% acetic acid. The percent
inhibition of RVV-dependent FX activation was calculated from
OD.sub.405 nm values in the absence and presence of a compound. The
data shown in Table 2 (FIG. 2(b)) indicated that the compounds 1
and 2 (structures shown above) do not inhibit RVV catalytic
activity, and they do not bind to the cleavage site of FX. It has
been established independently that all FX-activating enzymes
(TF/VIIa, FVIIa, FIXa, and RVV) cleave the same site on FX.
[0140] B. FIXa-Dependent FX Activation Assay:
[0141] FIX was first converted to FIXa by TF/VIIa in the absence of
compound. This was done in a 50-ml tube. After incubating TF/VIIa
(0.67 nM, 10 ml) at 37.degree. C. for 15 minutes, FIX (300 nM,
0.123 ml) was added and incubated at 37.degree. C. for 30 minutes.
Then 1.54 ml of EDTA (400 mM) was added to stop the FIX activation.
Then 65 .mu.l of the above FIXa sample was transferred into wells
of a 96-well plate that contain 5 .mu.l of diluted DMSO (or buffer)
or compound (in diluted DMSO or buffer). Ten .mu.l of FX (1000 nM),
10 .mu.l of polylysine (300 nM), and 10 .mu.l of Spectrozyme FXa (5
mM) were added to each well and incubate at 37.degree. C. until an
OD.sub.405 nm value of .about.0.8 was reached. Absorbance at 405 nm
was then measured following addition of 20 .mu.l of 50% acetic
acid. The percent inhibition of FIXa activity was calculated from
OD.sub.405 nm values in the absence and presence of a compound.
Again, compounds 1 and 2 do not inhibit FIXa activity, nor do they
bind to FX in such a way that FX can not be activated by FIXa
(Table 2, FIG. 2(b)).
[0142] C. FVIIa-Dependent FX Activation:
[0143] FVIIa alone in the presence of phospholipids and calcium is
also able to activate FX, although at a very low rate. This
experiment was designed to examine whether compounds 1 and 2
inhibit FVIIa-dependent FX activation if TF is omitted. Inhibition
of FVIIa-dependent-FX activation means that the two compounds may
bind to FVIIa, while no inhibition indicates that the two compounds
will not bind to FVIIa or FX. This assay was done at relatively
high FVIIa concentrations. Six microliters of compound 1(1 mM in
10% DMSO) or 10% DMSO was mixed with40 .mu.l of 25 mM HEPES-NaOH, 5
mM CaCl.sub.2, 150 mM NaCl, 0.1% BSA, pH 7.5 containing
phosphatidylcholine (0.07 mg/ml) and phosphatidylserine (0.03
mg/ml). Then 4 .mu.l of FVIIa (1.5 .mu.M) and 10 .mu.l of FX (180
nM) were added and mixed. The mixture was incubated for 1 hour at
37.degree. C. Then 10 .mu.l of EDTA (400 mM) was added to stop FVII
activity by removing the calcium required for factor VII activity.
Then 10 .mu.l of FXa substrate S-2765 (3.2 mM) was added to measure
the FXa activity generated by FVIIa. After 16 minutes incubation at
37.degree. C., 20 .mu.l of 50% acetic acid was added to quench the
reaction. Absorbance at 405 nm was read and the percent inhibition
of FVIIa-dependent FX activation was calculated from OD.sub.405 nm
values in the absence and presence of compound 1. The data shown in
Table 2 (FIG. 2(b)) indicates that compound 1 does not inhibit
FVIIa-dependent FX activation, indicating that it does not bind to
FVIIa or to FX.
[0144] D. Inhibition of TF/VIIa-Dependent FX Activation
[0145] To determine the inhibition of TF/VIIa-dependent FX
activation by compounds 1 and 2 at lower concentrations, compounds
in DMSO were diluted with 10 mM HEPES-NaOH, pH 7.5 and the assays
were then performed as previously described in the primary
screening method. Table 3 (FIG. 2 (c)) is the titration results of
TF/VIIa-dependent FX activation for compounds 1 and 2. In some
experiments, both compounds were dissolved in 0.1 M NaOH, then
diluted by water to 16.5 mM NaOH. From the data in Table 3, the
compounds 1 and 2 (structures shown above) inhibit
TF/VIIa-dependent FX activation, with IC.sub.50 (inhibitor
concentrations at which 50% of TF/VIIa-dependent FX activation is
inhibited) values of 19.0 .mu.M for compound 1 and 9.7 .mu.M for
2.
[0146] E. Inhibition of TF/VIIa-Dependent FIX Activation
[0147] TF/VIIa is not only able to activate FX, but is also able to
convert FIX to FIXa. To examine whether the two antagonists 1 and 2
were able to block FIX activation catalyzed by TF/VIIa. FIX
activation experiment was conducted. To each well of a 96-well
plate, 5 .mu.l of diluted DMSO or compound was added, followed by
45 .mu.l of TF/VIIa (0.67 nM). After mixing and incubating for 15
minutes at 37.degree. C., 10 .mu.l of FIX (300 nM) was added and
incubated for 10 minutes at 37.degree. C. 10 .mu.l of EDTA (400 nM
in 144 mM HEPES, 864 nM NaCl, 0.576% BSA, pH adjusted to 7.5) was
added, followed by addition of 10 .mu.l of FX (1000 nM), 10 .mu.l
of polylysine, and 10 .mu.l of Spectrozyme FXa (6 mM). After 3 hour
incubation at 37.degree. C., absorbance at 405 nm was measured
following addition of 20 .mu.l of 50% acetic acid. The percent
inhibition of TF/VIIa-dependent FIX activation was calculated from
OD.sub.405 nm values in the absence and presence of a compound. The
data in Table 4 (FIG. 2(d)) shows that compounds 1 and 2 (structure
shown above) inhibit TF/VIIa-dependent FIX activation similarly as
seen in TF/VIIa-dependent FX activation.
EXAMPLE 5
Inhibition Mechanism of TF Antagonists
[0148] Examples 3 and 4 above showed that the compounds of the
invention are TF specific antagonists. To elucidate the inhibition
mechanism of TF antagonists, the following experiments were
conducted. Compound 1 was titrated from 0 to 84 .mu.M under two
assay conditions using two different FX concentrations. Under one
condition, compound 1 was preincubated with TF/VIIa for 15 minutes
at 37.degree. C. prior to addition of FX. Under another condition,
TF/VIIa, FX, and compound 1 were added and mixed simultaneously.
One set of experiments was conducted at 5 nM FX, the other set at
30 nM FX. From results shown in FIGS. 6 and 7, IC.sub.50 values
from preincubation experiments are somewhat lower than those from
the simultaneous addition expeiments (compare 8.3 .mu.M with 18.1
.mu.M when FX was 5 nM, and 12.5 .mu.M with 33.2 .mu.M when FX was
30 nM). Furthermore, IC.sub.50 values increased with increasing FX
concentration. For example, IC.sub.50 values increased from 8.3
.mu.M and 18.1 .mu.M to 12.5 .mu.M and 33.2 .mu.M, respectively,
when FX increased from 5 nM to 30 nM under the two assay
conditions. These data suggest that compound 1 compete each other
for binding to TF.
[0149] Further kinetic analysis of TF-VIIa-dependent FX activation
showed that Compound 1 significantly increased the apparent Km
values for FX substrate (see FIG. 5). This indicates that (1),
compound 1 is a competitive inhibitor for TF/VIIa complex, (2), the
binding of compound 1 to TF/VIIa blocks FX binding to TF/VIIa
complex. The binding of compound 1 to TF (243 form lipidated or 219
form unlipidated) also was directly observed by isothermal
calorimetry analysis.
EXAMPLE 6
Effects of Compounds of the Invention in the Prothrombin Time (PT)
Assay
[0150] The prothrombin time (PT) test was conducted as follows:
[0151] The PT assay was performed at 37.degree. C. with an Electra
800 (Medical Automation, Inc.). The PT reaction was initiated by
adding 0.2 ml of lipidated recombinant human tissue factor into
0.105 ml of human plasma (Ci-Trol Control Level I, from VWR, Cat.
No. 68100-336). 1 ml purified water was added to each vial of
Ci-Trol and mixed to solubilize. If more than one vial was used, it
was often helpful to combine them into one container. 5 .mu.l of
DMSO or 5 .mu.l of compound was added to each well of the twin-well
cuvette that contains 0.1 ml of Ci-Trol. It is helpful to use a
pipet with 0.1 ml tip to mix each well. Make sure no air bubbles
are in the well. Following mixing the compound (or DMSO) with
plasma (Ci-Trol), 0.2 ml of lipidated reocombinant tissue factor
(1-3 nM) is added to the plasma and clotting times were measured
within 10 min. The data in FIG. 4 indicate compounds 1 and 2
prolonged TF-initiated PT times significantly.
[0152] The invention has been described in detail with reference to
preferred embodiments thereof. However, it will be appreciated that
those skilled in the art, upon consideration of the disclosure, may
make modification and improvements within the spirit and scope of
the invention.
* * * * *