U.S. patent application number 11/537482 was filed with the patent office on 2007-02-15 for serine protease inhibitors.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Ignacio Aliagas-Martin, Dean R. Artis, Michael S. Dina, John A. Flygare, Richard A. Goldsmith, Regina A. Munroe, Alan G. Olivero, Richard Pastor, Thomas E. Rawson, Kirk D. Robarge, Daniel P. Sutherlin, Kenneth J. Weese, Aihe Zhou, Yan Zhu.
Application Number | 20070037813 11/537482 |
Document ID | / |
Family ID | 26813550 |
Filed Date | 2007-02-15 |
United States Patent
Application |
20070037813 |
Kind Code |
A1 |
Aliagas-Martin; Ignacio ; et
al. |
February 15, 2007 |
Serine Protease Inhibitors
Abstract
Compounds having the structure shown below are useful to inhibit
serine protease enzymes, such as TF/factor VIIa, factor Xa,
thrombin and kallikrein. These compounds may be used in methods of
preventing and/or treating clotting disorders. ##STR1##
Inventors: |
Aliagas-Martin; Ignacio;
(San Francisco, CA) ; Artis; Dean R.; (Kensington,
CA) ; Dina; Michael S.; (Daly City, CA) ;
Flygare; John A.; (Burlingame, CA) ; Goldsmith;
Richard A.; (Daly City, CA) ; Munroe; Regina A.;
(San Mateo, CA) ; Olivero; Alan G.; (Half Moon
Bay, CA) ; Pastor; Richard; (San Francisco, CA)
; Rawson; Thomas E.; (Mountain View, CA) ;
Robarge; Kirk D.; (San Francisco, CA) ; Sutherlin;
Daniel P.; (San Carlos, CA) ; Weese; Kenneth J.;
(So. San Francisco, CA) ; Zhou; Aihe; (San Jose,
CA) ; Zhu; Yan; (Foster City, CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
26813550 |
Appl. No.: |
11/537482 |
Filed: |
September 29, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11088511 |
Mar 24, 2005 |
|
|
|
11537482 |
Sep 29, 2006 |
|
|
|
10224114 |
Aug 19, 2002 |
6919369 |
|
|
11088511 |
Mar 24, 2005 |
|
|
|
09509104 |
Mar 21, 2000 |
6472393 |
|
|
PCT/US00/00673 |
Jan 11, 2000 |
|
|
|
10224114 |
Aug 19, 2002 |
|
|
|
60115772 |
Jan 13, 1999 |
|
|
|
60152029 |
Sep 1, 1999 |
|
|
|
Current U.S.
Class: |
514/247 ;
514/352; 514/620; 544/224; 546/306 |
Current CPC
Class: |
C07C 257/18 20130101;
A61P 9/10 20180101; C07C 311/13 20130101; C07C 311/29 20130101;
C07C 311/51 20130101; C07C 311/18 20130101; A61P 7/02 20180101;
C07C 311/08 20130101; C07C 311/05 20130101 |
Class at
Publication: |
514/247 ;
514/352; 514/620; 544/224; 546/306 |
International
Class: |
A61K 31/50 20070101
A61K031/50; A61K 31/44 20070101 A61K031/44; A61K 31/165 20060101
A61K031/165; C07D 237/02 20070101 C07D237/02 |
Claims
1. A compound having the structure shown below: ##STR1034## where A
and B are independently CH, CR.sub.3 or N; X is C.dbd.O or
(CR.sub.4aR.sub.4b).sub.m where m=1 or 2; Y is S(O).sub.n--R.sub.1
where n=1 or 2, S(O).sub.n--NR.sub.2R.sub.2 where n=1 or 2,
S(O).sub.n--OR.sub.2 where n=1 or 2, C(O)R.sub.1, C(S)R.sub.1,
C(O)--OR.sub.1, C(O)--NR.sub.2R.sub.2; N.sub.1 and N.sub.2 are
nitrogen atoms; Q and R.sub.1 are independently (1) optionally
substituted alkyl having 1 to about 10 carbon atoms; (2) optionally
substituted aralkyl containing an aryl moiety having 6 to about 10
ring carbon atoms bonded to an alkyl moiety containing 1 to about
10 carbon atoms; (3) optionally substituted heteroaralkyl
containing a heteroaryl moiety having 5 to about 10 ring atoms
bonded to an alkyl moiety having 1 to about 10 carbon atoms; (4)
optionally substituted carbocycloalkyl containing a carbocyclyl
moiety having 3 to about 10 ring carbon atoms bonded to an alkyl
moiety having 1 to about 10 carbon atoms; (5) optionally
substituted heterocycloalkyl containing a heterocyclyl moiety
having 3 to about 10 ring atoms bonded to an alkyl moiety having 1
to about 10 carbon atoms; (6) optionally substituted alkenyl having
2 to about 10 carbon atoms; (7) optionally substituted aralkenyl
containing an aryl moiety having 5 to about 10 ring atoms bonded to
an alkenyl moiety having 2 to about 10 carbon atoms; (8) optionally
substituted heteroaralkenyl containing a heteroaryl moiety having 5
to about 10 ring atoms bonded to an alkenyl moiety having 2 to
about 10 carbon atoms; (9) optionally substituted carbocycloalkenyl
containing a carbocyclyl moiety having 3 to about 10 ring carbon
atoms bonded to an alkenyl moiety having 2 to about 10 carbon
atoms; (10) optionally substituted heterocycloalkenyl containing a
heterocyclyl moiety having 3 to about 10 ring atoms bonded to an
alkenyl moiety having 2 to about 10 carbon atoms; (11) optionally
substituted aryl having 6 to about 10 ring carbon atoms; (12)
optionally substituted heteroaryl having 5 to about 10 ring atoms
with ring atoms selected from carbon atoms and heteroatoms, where
the heteroatoms are nitrogen, oxygen or sulfur; (13) optionally
substituted carbocyclyl having 3 to about 10 ring carbon atoms;
(14) optionally substituted heterocyclyl having 3 to about 10 ring
atoms with ring atoms selected from carbon atoms and heteroatoms,
where the heteroatoms are nitrogen, oxygen or sulfur; each R.sub.2
is, independently, H, alkyl, substituted alkyl, C(O)R.sub.7 or
C(NH)R.sub.7, or N.sub.1R.sub.2 and N.sub.2R.sub.2 are together
form the group N.sub.1--CO--N.sub.2; R.sub.3 is H, C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.6 alkoxy, halogen or OH; R.sub.4aand R.sub.5
are, independently, a member selected from the group consisting of
H, unsubstituted or substituted alkyl, unsubstituted or substituted
alkoxyalkyl, unsubstituted or substituted haloalkyl, unsubstituted
or substituted aryl, alkyl-OR.sub.7, alkyl-NR.sub.7R.sub.8,
alkyl-OC(O)R.sub.7, alkyl-C(O)OR.sub.7, alkyl-C(O)R.sub.7,
OC(O)R.sub.7, C(O)OR.sub.7, C(O)R.sub.7 and members in which the
alkyl, R.sub.7 or R.sub.8 is substituted with 1-3 F, Cl, Br, I,
OR.sub.7, SR.sub.7, NR.sub.7R.sub.8, OC(OR.sub.7), C(O)OR.sub.7,
C(O)R.sub.7, C(O)NR.sub.7R.sub.8, NHC(NH)NH.sub.2, PO.sub.3,
unsubstituted or substituted indolyl or unsubstituted or
substituted imidazolyl groups; R.sub.4bis H, alkyl, or substituted
alkyl; R.sub.6 is selected from the group selected from H,
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkyl-OR.sub.7,
C.sub.1-C.sub.6 alkyl-N R.sub.7R.sub.8, C.sub.1-C.sub.6 haloalkyl,
halo, cyano, OR.sub.7, SR.sub.7, NR.sub.7R.sub.8, C(O)OR.sub.7,
C(O)R.sub.7 and OC(O)R.sub.7; R.sub.7 and R.sub.8 are independently
H or C.sub.1-C.sub.6 alkyl; and acid and base addition salts and
prodrugs thereof.
2. The compound of claim 1, wherein Q is phenyl optionally
substituted with 1-5, preferably 2-4, more preferably 2-3,
substituents selected from the group consisting of halo, nitro,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, NR.sub.7R.sub.8, OR.sub.7, SR.sub.7, C.sub.1-C.sub.6
alkyl-C(O)OR.sub.7, OC.sub.1-C.sub.6 alkyl-C(O)OR.sub.7,
C.sub.1-C.sub.6 alkyl-OR.sub.7, OC.sub.1-C.sub.6 alkyl-OR.sub.7,
C.sub.1-C.sub.6 alkyl-NR.sub.7R.sub.8, OC.sub.1-C.sub.6
alkyl-NR.sub.7R.sub.8, C.sub.1-C.sub.6 alkyl-C(O)NR.sub.7R.sub.8,
OC.sub.1-C.sub.6 alkyl-C(O)NR.sub.7R.sub.8, C.sub.1-C.sub.6
alkyl-C(O)R.sub.7, OC.sub.1-C.sub.6 alkyl-C(O)R.sub.7,
C.sub.1-C.sub.6 haloalkyl, O-aralkyl (e.g. benzyloxy),
C(O)OR.sub.7, C(O)NR.sub.7R.sub.8, OC(O)NR.sub.7R.sub.8,
NHC(O)R.sub.7, NHC(O)NR.sub.7R.sub.8, NR.sub.7S(O).sub.nR.sub.1,
NR.sub.7S(O).sub.nR.sub.7, S(O).sub.nR.sub.7, S(O).sub.nNR.sub.7,
where R.sub.7 and R.sub.8 independently are H or C.sub.1-C.sub.6
alkyl.
3. The compound of any one of claims 1-2, wherein Q has the
structure ##STR1035## wherein R.sub.9 is H, C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6
alkoxy, hydroxy, NR.sub.7R.sub.8, SR.sub.7 or OR.sub.7, where
R.sub.7 and R.sub.8, independently, are H or unsubstituted or
substituted C.sub.1-C.sub.6 alkyl; R.sub.10, R.sub.11 and Z.sub.2,
independently, are each selected from the group consisting of H,
halo, nitro, cyano, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl,
NR.sub.7R.sub.8, OR.sub.7, SR.sub.7, C.sub.1-C.sub.6
alkyl-C(O)R.sub.7, C.sub.1-C.sub.6 alkyl-C(O)NR.sub.7R.sub.8,
C.sub.1-C.sub.6 alkyl-C(O)OR.sub.7, C.sub.1-C.sub.6
alkyl-OC(O)R.sub.7, C.sub.1-C.sub.6 alkyl-OR.sub.7,
OC.sub.1-C.sub.6 alkyl-C(O)R.sub.7, OC.sub.1-C.sub.6
alkyl-C(O)OR.sub.7, OC.sub.1-C.sub.6 alkyl-OC(O)R.sub.7,
O--C.sub.1-C.sub.6 alkyl-OR.sub.7, OC.sub.1-C.sub.6
alkyl-C(O)NR.sub.7R.sub.8, C.sub.1-C.sub.6 haloalkyl, OR.sub.12,
C.sub.1-C.sub.6 alkyl-R.sub.12, O--C.sub.1-C.sub.6 alkyl-R.sub.12,
C(O)OR.sub.7, C(O)OR.sub.12, C(O)NR.sub.7R.sub.8,
OC(O)NR.sub.7R.sub.8, NR.sub.7C(O)R.sub.7, NR.sub.7C(O)R.sub.12,
NR.sub.7C(O)--NR.sub.7R.sub.8, NR.sub.7C(O)OR.sub.7,
NR.sub.7C(O)OR.sub.12, NR.sub.7S(O)n-R.sub.1, NR.sub.7S(O)n-R.sub.7
and NR.sub.7S(O)n-R.sub.12, where R.sub.7 and R.sub.8,
independently, are H or unsubstituted or substituted
C.sub.1-C.sub.6 alkyl, R.sub.12 is unsubstituted or substituted
C.sub.6-C.sub.10 aryl or heterocycl as defined above and n is 1 or
2; Z.sub.1 is H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
halogen or nitro.
4. The compound of any one of claims 1-3, wherein R.sub.10 is
selected from the group consisting of C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 aminoalkyl, C.sub.1-C.sub.6
haloalkyl, C.sub.1-C.sub.6 hydroxyalkyl, phenyl, phenoxy, benzyl,
benzyloxy, as well as phenoxy- and benzyloxy-substituted with
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, halo,
C.sub.1-C.sub.6 haloalkyl, C.sub.1-C.sub.6 hydroxyalkyl,
C.sub.1-C.sub.6 aminoalkyl, OC(O)--C.sub.1-C.sub.6 alkyl,
C(O)O--C.sub.1-C.sub.6 alkyl and C(O)OH.
5. The compound of any one of claims 3-4, wherein R.sub.11 is
NR.sub.7C.sub.1-C.sub.6 alkyl-C(O)NR.sub.7R.sub.8,
NR.sub.7S(O)n-R.sub.7 or NR.sub.7S(O)n-R.sub.12, n is 1 or 2 and/or
where Z, =Z.sub.2=H and/or where R.sub.10 is OR.sub.7, OR.sub.12,
OC.sub.7-C.sub.10-aralkyl, OC.sub.1-C.sub.6 alkyl-OR.sub.7 or
OC.sub.1-C.sub.6 alkyl-OR.sub.12.
6. The compound of any one of claims 3-5, wherein Z.sub.1 and
Z.sub.2 are hydrogen; Z.sub.1, Z.sub.2 and R.sub.11 are hydrogen;
or Z.sub.1, R.sub.10 and R.sub.11 are hydrogen.
7. The compound of any one of claims 3-6, wherein Z.sub.1 or
Z.sub.2 is C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, halogen
or nitro.
8. The compound of any one of claims 3-7, wherein R.sub.10 is
OR.sub.7, OR.sub.12, C.sub.1-C.sub.6 alkyl-OR.sub.7 or
C.sub.1-C.sub.6 alkyl-OR.sub.12.
9. The compound of any one of claims 3-8, wherein Y is
S(O).sub.nR.sub.1 where n=1 or 2 and R.sub.11 is
NR.sub.7S(O).sub.n--R.sub.7 or NR.sub.7S(O).sub.n--R.sub.12, n is 1
or 2.
10. The compound of any one of claims 3-9, wherein R.sub.10 and
Z.sub.2 or Z.sub.2 and R.sub.11 are bonded together to form a fused
unsubstituted or substituted, carbocyclic or hetercyclic ring.
11. The compound of any one of claims 1-10, wherein X is a carbonyl
group;
12. The compound of any one of claims 1-11, wherein R.sub.1 is
selected from the group consisting of C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6
cycloalkyl, phenyl, naphthyl, benzyl and heteroaryl having 5-6 ring
atoms selected from carbon atoms and 1-2 heteroatoms, where the
heteroatoms are N, S, or O, and R.sub.1 optionally substituted with
1-3 substituents selected from the group consisting of halo, nitro,
C.sub.1-C.sub.6 alkyl, NR.sub.7R.sub.8, OR.sub.7, SR.sub.7,
C.sub.1-C.sub.6 alkyl-C(O)OR.sub.7, C.sub.1-C.sub.6
alkyl-OC(O)R.sub.7, C.sub.1-C.sub.6 alkyl-C(O)R.sub.7,
C.sub.1-C.sub.6 alkyl-OR.sub.7, C.sub.1-C.sub.6 haloalkyl,
C.sub.1-C.sub.6 alkyl-NR.sub.7R.sub.8, C(O)OR.sub.7, OC(O)R.sub.7,
C(O)NR.sub.7R.sub.8, OC(O)NR.sub.7R.sub.8, NHC(O)R.sub.7, and
NHC(O)NR.sub.7R.sub.8, where R.sub.7 and R.sub.8 independently are
H or C.sub.1-C.sub.6 alkyl.
13. The compound of any one of claims 1-12, wherein A and B are
CH.
14. The compound of any one of claims 1-13, wherein R.sub.6 is
H.
15. The compound of any one of claims 1-14, wherein Y is
S(O).sub.nR.sub.1 where n=1 or 2.
16. A composition, comprising the compound of any one of claims
1-15 and a carrier or excipient.
17. A method of inhibiting TF/factor VIIa, factor Xa, thrombin or
kallikrein activity, comprising contacting TF/factor VIIa factor
Xa, thrombin or kallikrein with an effective amount of the
composition of claim 16.
18. A method of treating a TF/factor VIIa, factor Xa, thrombin or
kallikrein mediated disorder, comprising administering to a mammal
in need thereof an effective amount of the composition of claim
16.
19. A method of preventing thrombosis or treating abnormal
thrombosis, comprising administering to a mammal in need thereof an
effective amount of the composition of claim 16.
Description
DISCUSSION OF THE BACKGROUND
[0001] 1. Field of the Invention
[0002] In one aspect, the invention relates to novel compounds
which are inhibitors of Tissue Factor (TF)/factor VIIa, factor
VIIa, factor Xa, thrombin and/or kallikrein, as well as
compositions containing these compounds. The compounds are useful
for inhibiting these factors and for treating disorders mediated
thereby. For example, the compounds are useful for preventing
thrombosis or treating abnormal thrombosis in a mammal by
inhibiting TF/factor VIIa, factor Xa, thrombin and/or
kallikrein.
[0003] 2. Background of the Invention
[0004] Normal haemeostasis is the result of a complex balance
between the processes of clot initiation, formation and clot
dissolution. The complex interactions between blood cells, specific
plasma proteins and the vascular surface, maintain the fluidity of
blood unless injury and blood loss occurs.
[0005] Many significant disease states are related to abnormal
haemeostasis. For example, local thrombus formation due to the
rupture of atherosclerotic plaque is a major cause of acute
myocardial infarction and unstable angina. Treatment of an
occlusive coronary thrombus by either thrombolytic therapy or
percutaneous angioplasty may be accompanied by acute thrombolytic
reclosure of the affected vessel. Furthermore, a high percentage of
patients undergoing surgery, particularly in the lower extremities,
suffer thrombus formation in the venous vascular system which
results in reduced blood flow to the affected area.
[0006] There continues to be a need for safe and effective
therapeutic anticoagulants to limit or prevent thrombus
formation.
[0007] Blood coagulation is vital for the containment of bodily
fluids upon tissue injury and is an important component of host
defense mechanisms. Coagulation or clotting involves the sequential
activation of multiple zymogens in a process leading to thrombin
generation and the conversion of fibrinogen to an impermeable
cross-linked fibrin clot. Thrombin production is the result of a
blood coagulation cascade which has been intensively studied and
increasingly characterized. See for example, Lawson, J. H., et al.
(1994) J. Biol. Chem. 269:23357. The coagulation reactions of this
cascade involve initiation, amplification and propagation phases.
Additionally, the cascade has been divided into extrinsic and
intrinsic pathways. The intrinsic pathway involves factors XII, XI,
and 1.times. and leads to the formation of a complex of factor IXa
with its cofactor, factor VIIIa. This complex converts factor X to
Xa. Factor Xa is an enzyme which forms a complex with its cofactor,
factor Va, and rapidly converts prothrombin to thrombin. Thrombin
converts fibrinogen to fibrin monomers which polymerize to form a
clot. The extrinsic pathway involves factor VIIa and tissue factor,
which form a complex (TF/factor VIIa), and convert factor X to Xa.
As in the intrinsic pathway, factor Xa converts prothrombin to
thrombin.
[0008] Thrombin (factor IIa), as noted above, occupies a central
position in the coagulation cascade by converting fibrinogen to
fibrin. Consequently, substantial synthetic efforts have been
directed to the development of thrombin inhibitors. See, for
example, U.S. Pat. No. 5,656,600; U.S. Pat. No. 5, 656, 645; U.S.
Pat. No. 5,670,479; U.S. Pat. No. 5,646,165; U.S. Pat. No.
5,658,939; U.S. Pat. No. 5,658,930 and WO 97/30073. Additional
compounds which have been prepared as synthetic thrombin inhibitors
are N-arylsulfinated phenylalanine amides.
[0009] Known inhibitors of factor Xa include bisamidine compounds
(Katakura, S. (1993) Biochem. Biophys. Res. Commun., 197:965) and
compounds based on the structure of arginine (WO 93/15756; WO
94/13693). Phenyl and naphthylsulfonamides have also been shown to
be factor Xa inhibitors (WO 96/10022; WO 96/16940; WO
96/40679).
[0010] TF/factor VIIa is a serine protease complex that
participates in blood coagulation by activating factor X and/or
factor IX. Factor VIIa is produced from its precursor, factor VII,
which is synthesized in the liver and secreted into the blood where
it circulates as a single chain glycopeptide. The cDNA sequence for
factor VII has been characterized (Hagen et al., 1986, Proc. Natl.
Acad. Sci. U.S.A., 83:2412-2416).
[0011] A variety of natural and synthetic inhibitors of TF/factor
VIIa are known and have varying potency and selectivity. Tissue
factor pathway inhibitor (TFPI; Broze, 1995, Thromb. Haemostas.,
74:90) and nematode anticoagulant peptide c2 (NAPc2; Stanssens et
al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93:2149) bind factor Xa
prior to the formation of a quaternary inhibitory complex with the
TF/factor VIIa complex. Small protein direct inhibitors (Dennis et
al, 1994, J. Biol. Chem., 35:22137) and inactive forms of TF/factor
VIIa are also known (Kirchhofer et al, 1995, Arteriosclerosis,
Thrombosis and Vascular Biol., 15:1098; Jang et al, 1995,
Circulation, 92:3041). Additionally, synthetic peptides and soluble
forms of mutant TF which retain binding affinity but have reduced
cofactor activity have been prepared (Roenning et al, 1996, Thromb.
Res., 82:73; Kelley et al, 1997, Blood, 89:3219). U.S. Pat. No.
5,679,639 describes polypeptides and antibodies which inhibit
serine protease activity. U.S. Pat. No. 5,580,560 describes a
mutant factor VIIa which has an improved half-life. U.S. Pat. No.
5,504,067 and U.S. Pat. No. 5,504,064 describe a truncated TF for
the treatment of bleeding. Kunitz domain-tissue factor fusion
proteins have also been shown to be bifunctional anticoagulants
(Lee et al, 1997, Biochemistry, 36:5607-5611). The TF/factor VIIa
complex has been indicated as an attractive target for the
development of inhibitors based on a dissociation between surgical
bleeding and prevention of intravascular thrombosis (Harker et al,
1995, Thromb. Haemostas., 74:464).
[0012] Compounds which block or inhibit enzymes in the coagulation
cascade are therapeutically useful in treating or preventing
thrombosis in a mammal suspected of having a condition
characterized by abnormal thrombosis. For example, with respect to
arterial vasculature, abnormal thrombus formation due to
deterioration of an established atherosclerotic plaque is a major
cause of acute myocardial infarction and unstable angina. Treatment
of an occlusive coronary thrombus by thrombolytic therapy or
percutaneous transluminal coronary angioplasty (PTCA) may be
accompanied by reclosure of the vessel. In the venous vasculature,
many patients undergoing surgery, particularly in the abdominal and
lower body regions, experience thrombus formation which reduces
blood flow and can lead to a pulmonary embolism. Disseminated
intravascular coagulopathy in both the venous and arterial systems
occurs commonly during septic shock, some viral infections, and
cancer and may lead to rapid and widespread thrombus formation and
organ failure.
[0013] PTCA and recanalization are favored procedures for treating
occluded vessels. However, arterial thrombosis following these
procedures remains a leading cause of failure. Heparin, the most
widely used anticoagulant, has not been shown to be entirely
effective in the treatment and prevention of acute arterial
thrombosis or rethrombosis.
[0014] The synthesis and development of small molecule inhibitors
based on the known three-dimensional structure of proteins is a
challenge of modern drug development. Many thrombin inhibitors have
been designed to have a hirudin-type structure. Stubbs and Bode,
Current Opinion in Structural Biology 1994, 4:823-832. New
synthetic thrombin inhibitors, as well as inhibitors of factor Xa
and TF/factor VIIa, are reported. See, for example, Annual Reports
in Medicinal Chemistry, 1995-1997, Academic Press, San Diego,
Calif.
[0015] U.S. Pat. No. 5,589,173 describes the use of a tissue factor
antagonist and a thrombolytic agent to treat myocardial
infarction.
[0016] U.S. Pat. No. 5,399,487 describes naphthalenesulfonamides
which are useful for determining proteolytic enzyme activity or as
enzyme inhibitors.
[0017] A need continues to exist for compounds which are effective
inhibitors of enzymes in the coagulation cascade and which exhibit
improved inhibitory activity and/or selectivity towards selected
enzymes in the cascade.
SUMMARY OF THE INVENTION
[0018] Accordingly, one object of the present invention is to
provide novel compounds which inhibit factors/enzymes in the
coagulation cascade and which are useful to prevent or treat
thrombus formation in arterial or venous vessels. These compounds
are useful as coagulation factor inhibitors and as anticoagulants
in general.
[0019] In one embodiment, an object of the invention is to provide
inhibitors which inhibit factor VIIa, TF/factor VIIa selectively
relative to factor Xa, thrombin or kallikrein. The compounds of
this embodiment preferably inhibit TF/factor VIIa about one order
of magnitude (10.times.), more preferably about two orders of
magnitude (100.times.), even more preferably about three orders of
magnitude (1000.times.), better than they inhibit factor Xa,
thrombin and/or kallikrein.
[0020] In another embodiment, an object of the invention is to
provide compounds which specifically inhibit factor Xa relative to
the inhibition of factor VIIa, TF/factor VIIa, thrombin or
kallikrein. The compounds of this embodiment preferably inhibit
factor Xa about one order of magnitude (10.times.), more preferably
about two orders of magnitude (100.times.), even more preferably
about three orders of magnitude (1000.times.), better than they
inhibit TF/factor VIIa, thrombin and/or kallikrein.
[0021] In another embodiment, a specific object of the invention is
to provide compounds which inhibit thrombin relative to inhibition
of factor VIIa, TF/factor VIIa, Xa, or kallikrein. The compounds of
this embodiment preferably inhibit factor thrombin about one order
of magnitude (10.times.), more preferably about two orders of
magnitude (100.times.), even more preferably about three orders of
magnitude (1000.times.), better than they inhibit TF/factor VIIa,
factor Xa and/or kallikrein.
[0022] A further object of the invention is to provide a method of
inhibiting TF/factor VIIa, Xa or thrombin activity by contacting
these enzymes with an effective inhibitory amount of the novel
inhibitors of the present invention or a composition containing
these compounds. A further object is to provide a method of
treating a TF/factor VIIa, Xa or thrombin mediated disorder by
administering to a mammal in need of such treatment an effective
amount of one of the compounds of the invention or a composition
containing the compound. An additional object is to provide a
method of preventing thrombosis or treating abnormal thrombosis by
administering to a mammal in need of such treatment an effective
amount of one of the compounds of the invention or a composition
containing the compound and a carrier or excipient.
[0023] These and other objects which will become apparent in the
course of the following description have been achieved by the
compounds of the present invention having the structure shown
below: ##STR2## where
[0024] A and B are independently CH, CR.sub.3 or N;
[0025] X is C.dbd.O or (CR.sub.4aR.sub.4b).sub.m where m=1 or
2;
[0026] Y is S(O).sub.n--R.sub.1 where n=1 or 2,
S(O).sub.n--NR.sub.2R.sub.2 where n=1 or 2, S(O).sub.n--OR.sub.2
where n=1 or 2, C(O)R.sub.1, C(S)R.sub.1, C(O)--OR.sub.1,
C(O)--NR.sub.2R.sub.2;
[0027] N.sub.1 and N.sub.2 are nitrogen atoms;
[0028] Q and R.sub.1 are independently [0029] (1) optionally
substituted alkyl having 1 to about 10 carbon atoms; [0030] (2)
optionally substituted aralkyl containing an aryl moiety having 6
to about 10 ring carbon atoms bonded to an alkyl moiety containing
1 to about 10 carbon atoms; [0031] (3) optionally substituted
heteroaralkyl containing a heteroaryl moiety having 5 to about 10
ring atoms bonded to an alkyl moiety having 1 to about 10 carbon
atoms; [0032] (4) optionally substituted carbocycloalkyl containing
a carbocyclyl moiety having 3 to about 10 ring carbon atoms bonded
to an alkyl moiety having 1 to about 10 carbon atoms; [0033] (5)
optionally substituted heterocycloalkyl containing a heterocyclyl
moiety having 3 to about 10 ring atoms bonded to an alkyl moiety
having 1 to about 10 carbon atoms; [0034] (6) optionally
substituted alkenyl having 2 to about 10 carbon atoms; [0035] (7)
optionally substituted aralkenyl containing an aryl moiety having 5
to about 10 ring atoms bonded to an alkenyl moiety having 2 to
about 10 carbon atoms; [0036] (8) optionally substituted
heteroaralkenyl containing a heteroaryl moiety having 5 to about 10
ring atoms bonded to an alkenyl moiety having 2 to about 10 carbon
atoms; [0037] (9) optionally substituted carbocycloalkenyl
containing a carbocyclyl moiety having 3 to about 10 ring carbon
atoms bonded to an alkenyl moiety having 2 to about 10 carbon
atoms; [0038] (10) optionally substituted heterocycloalkenyl
containing a heterocyclyl moiety having 3 to about 10 ring atoms
bonded to an alkenyl moiety having 2 to about 10 carbon atoms;
[0039] (11) optionally substituted aryl having 6 to about 10 ring
carbon atoms; [0040] (12) optionally substituted heteroaryl having
5 to about 10 ring atoms with ring atoms selected from carbon atoms
and heteroatoms, where the heteroatoms are nitrogen, oxygen or
sulfur; [0041] (13) optionally substituted carbocyclyl having 3 to
about 10 ring carbon atoms; [0042] (14) optionally substituted
heterocyclyl having 3 to about 10 ring atoms with ring atoms
selected from carbon atoms and heteroatoms, where the heteroatoms
are nitrogen, oxygen or sulfur;
[0043] each R.sub.2 is, independently, H, alkyl, substituted alkyl,
C(O)R.sub.7 or C(NH)R.sub.7, or N.sub.1R.sub.2 and N.sub.2R.sub.2
are together form the group N.sub.1--CO--N.sub.2;
[0044] R.sub.3 is H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
halogen or OH;
[0045] R.sub.4a and R.sub.5 are, independently, a member selected
from the group consisting of H, unsubstituted or substituted alkyl,
unsubstituted or substituted alkoxyalkyl, unsubstituted or
substituted haloalkyl, unsubstituted or substituted aryl,
alkyl-OR.sub.7, alkyl-NR.sub.7R.sub.8, alkyl-OC(O)R.sub.7,
alkyl-C(O)OR.sub.7, alkyl-C(O)R.sub.7, OC(O)R.sub.7, C(O)OR.sub.7,
C(O)R.sub.7 and members in which the alkyl, R.sub.7 or R.sub.8 is
substituted with 1-3 F, Cl, Br, I, OR.sub.7, SR.sub.7,
NR.sub.7R.sub.8, OC(OR.sub.7), C(O)OR.sub.7, C(O)R.sub.7,
C(O)NR.sub.7R.sub.8, NHC(NH)NH.sub.2, PO.sub.3, unsubstituted or
substituted indolyl or unsubstituted or substituted imidazolyl
groups;
[0046] R.sub.4b is H, alkyl, or substituted alkyl;
[0047] each R.sub.6 is independently selected from the group
selected from H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkyl-OR.sub.7, C.sub.1-C.sub.6 alkyl-NR.sub.7R.sub.8,
C.sub.1-C.sub.6 haloalkyl, halo, cyano, OR.sub.7, SR.sub.7,
NR.sub.7R.sub.8, C(O)OR.sub.7, C(O)R.sub.7 and OC(O)R.sub.7;
[0048] R.sub.7 and R.sub.8 are independently H or C.sub.1-C.sub.6
alkyl; and acid and base addition salts and prodrugs thereof.
[0049] Additionally, the objects of the invention are achieved by
compositions containing these compounds and the methods described
below.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
[0050] The term "factor VIIa, TF/factor VIIa, factor Xa, thrombin
or kallikrein mediated disorder" means a disease or physiological
condition involving clotting of the blood and in which inhibition
of one or more of these enzymes reduces or eliminates at least one
of the physiological symptoms of the disease or condition.
[0051] The term "thrombosis" means the development of or formation
of a blood clot or thrombus in a blood vessel of a mammal or in a
synthetic vessel, such as a plastic or glass tube or vial. A
thrombus which has detached from its original site and is found in
another site is called a thrombotic embolus.
[0052] The term "abnormal thrombosis" means thrombosis occurring in
a mammal which is contrary to the good health of the mammal.
[0053] The term "alkyl", used alone or as part of another term,
means a branched or unbranched, saturated aliphatic hydrocarbon
group, having the number of carbon atoms specified, or if no number
is specified, having up to and including 12 carbon atoms. Examples
of alkyl groups include methyl, ethyl, n-propyl, isopropyl,
n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl,
2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 2,2-dimethylbutyl,
n-heptyl, 3-heptyl, 2-methylhexyl, and the like. The terms "lower
alkyl" "C.sub.1-C.sub.6 alkyl" and "alkyl of 1 to 6 carbon atoms"
are synonymous and used interchangeably. Preferred "C.sub.1-C.sub.6
alkyl" groups are methyl, ethyl, 1-propyl, isopropyl, 1-butyl or
sec-butyl.
[0054] The terms "substituted alkyl" or "substituted
C.sub.n-C.sub.m alkyl" where m and n are integers identifying the
range of carbon atoms contained in the alkyl group, denotes the
above alkyl groups that are substituted by one, two or three
halogen (F, Cl, Br, I), trifluoromethyl, hydroxy, unsubstituted and
substituted C.sub.1-C.sub.7 alkoxy, protected hydroxy, amino
(including alkyl and dialkyl amino), protected amino, unsubstituted
and substituted C.sub.1-C.sub.7 acyloxy, unsubstituted and
substituted C.sub.3-C.sub.7 heterocyclyl, unsubstituted and
substituted phenoxy, nitro, carboxy, protected carboxy,
unsubstituted and substituted carboalkoxy, unsubstituted and
substituted acyl, carbamoyl, carbamoyloxy, cyano,
methylsulfonylamino, unsubstituted and substituted benzyloxy,
unsubstituted and substituted C.sub.3-C.sub.6 carbocyclyl or
C.sub.1-C.sub.4 alkoxy groups. The substituted alkyl groups may be
substituted once (preferably), twice or three times with the same
or with different substituents.
[0055] Examples of the above substituted alkyl groups include, but
are not limited to; cyanomethyl, nitromethyl, hydroxymethyl,
trityloxymethyl, propionyloxymethyl, aminomethyl, carboxymethyl,
carboxyethyl, trifuoroethyl, trifluoropropyl, carboxypropyl,
2-aminopropyl, alkyloxycarbonylmethyl, allyloxycarbonylaminomethyl,
carbamoyloxymethyl, methoxymethyl, ethoxymethyl, t-butoxymethyl,
acetoxymethyl, chloromethyl, bromomethyl, iodomethyl,
trifluoromethyl, 6-hydroxyhexyl, 2,4-dichloro(n-butyl),
2-amino(iso-propyl), 2-carbamoyloxyethyl and the like. The alkyl
group may also be substituted with a carbocyclo group. Examples
include cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and
cyclohexylmethyl groups, as well as the corresponding ethyl,
-propyl, -butyl, -pentyl, -hexyl groups, etc. A preferred group of
examples within the above group includes the substituted methyl
group, e.g. a methyl group substituted by the same substituents as
the "substituted C.sub.n-C.sub.m alkyl" group. Examples of the
substituted methyl group include groups such as hydroxymethyl,
protected hydroxymethyl (e.g. tetrahydropyranyloxymethyl),
acetoxymethyl, carbamoyloxymethyl, trifluoromethyl, chloromethyl,
carboxymethyl, bromomethyl and iodomethyl.
[0056] The term "alkoxy" denotes groups having the number of carbon
atoms specified such as methoxy, ethoxy, n-propoxy, isopropoxy,
n-butoxy, s-butoxy, t-butoxy and like groups. The term "substituted
alkoxy" means these alkoxy groups substituted by the same
substituents as the "substituted C.sub.n-C.sub.m alkyl" group, for
example, 2,2,2-trifluoroethoxy, 2,2,2-trifluoropropoxy, etc.
[0057] The term "acyloxy" denotes herein carboacyloxy groups having
the specified number of carbon atoms such as formyloxy, acetoxy,
propionyloxy, butyryloxy, pentanoyloxy, hexanoyloxy, heptanoyloxy,
and the like. The term "substituted acyloxy" means these acyloxy
groups substituted by the same substituents as the "substituted
C.sub.n-C.sub.m alkyl" group.
[0058] The term "alkylcarbonyl", "alkanoyl" and "acyl" are used
interchangeably herein encompass groups having the specified number
of carbon atoms such as formyl, acetyl, propionyl, butyryl,
pentanoyl, hexanoyl, heptanoyl, benzoyl and the like.
[0059] The terms "carbocyclyl", "carbocyclylic" and "carbocyclo"
alone and when used as a moiety in a complex group such as a
carbocycloalkyl group, refers to a mono-, bi-, or tricyclic
aliphatic ring having 3 to 14 carbon atoms and preferably 3 to 7
carbon atoms. Preferred carbocyclic groups include cyclopropyl,
cyclobutyl, cyclopentyl and cyclohexyl groups. The terms
"substituted carbocyclyl" and "carbocyclo" mean these groups
substituted by the same substituents as the "substituted
C.sub.n-C.sub.m alkyl" group.
[0060] A "carbocycloalkyl" group is a carbocyclo group as defined
above covalently bonded to an alkyl group as defined above.
[0061] The term "alkenyl" means a branched or unbranched
hydrocarbon group having the number of carbon atoms designated
containing one or more carbon-carbon double bonds, each double bond
being independently cis, trans, or a nongeometric isomer. The term
"substituted alkenyl" means these alkenyl groups substituted by the
same substituents as the "substituted C.sub.n-C.sub.m alkyl"
group.
[0062] The term "alkynyl" means a branched or unbranched
hydrocarbon group having the number of carbon atoms designated
containing one or more carbon-carbon triple bonds. The term
"substituted alkynyl" means these alkynyl groups substituted by the
same substituents as the "substituted C.sub.n-C.sub.m alkyl"
group.
[0063] The terms "alkylthio" and "C.sub.1-C.sub.12 substituted
alkylthio" denote C.sub.1-C.sub.12 alkyl and C.sub.1-C.sub.12
substituted alkyl groups, respectively, attached to a sulfur which
is in turn the point of attachment for the alkylthio or substituted
alkylthio group to the group or substituent designated.
[0064] The term "aryl" when used alone or as part of another term
means a homocyclic aromatic group whether or not fused having the
number of carbon atoms designated or if no number is designated, up
to 14 carbon atoms. Preferred aryl groups include phenyl, naphthyl,
biphenyl, phenanthrenyl, naphthacenyl, and the like (see e.g.
Lang's Handbook of Chemistry (Dean, J. A., ed) 13.sup.th ed. Table
7-2 [1985]).
[0065] The term "substituted phenyl" or "substituted aryl" denotes
a phenyl group or aryl group substituted with one, two, three, four
or five, preferably 1-2, 1-3 or 1-4 substituents chosen from
halogen (F, Cl, Br, I), hydroxy, protected hydroxy, cyano, nitro,
alkyl (preferably C.sub.1-C.sub.6 alkyl), alkoxy (preferably
C.sub.1-C.sub.6 alkoxy), benzyloxy, carboxy, protected carboxy,
carboxymethyl, protected carboxymethyl, hydroxymethyl, protected
hydroxymethyl, aminomethyl, protected aminomethyl, trifluoromethyl,
alkylsulfonylamino, arylsulfonylamino, heterocyclylsulfonylamino,
heterocyclyl, aryl, or other groups specified. One or methyne (CH)
and/or methylene (CH.sub.2) groups in these substituents may in
turn be substituted with a similar group as those denoted above.
Examples of the term "substituted phenyl" includes but is not
limited to a mono- or di(halo)phenyl group such as 4-chlorophenyl,
2,6-dichlorophenyl, 2,5-dichlorophenyl, 3,4-dichlorophenyl,
3-chlorophenyl, 3-bromophenyl, 4-bromophenyl, 3,4-dibromophenyl,
3-chloro-4-fluorophenyl, 2-fluorophenyl and the like; a mono- or
di(hydroxy)phenyl group such as 4-hydroxyphenyl, 3-hydroxyphenyl,
2,4-dihydroxyphenyl, the protected-hydroxy derivatives thereof and
the like; a nitrophenyl group such as 3- or 4-nitrophenyl; a
cyanophenyl group, for example, 4-cyanophenyl; a mono- or di(lower
alkyl)phenyl group such as 4-methylphenyl, 2,4-dimethylphenyl,
2-methylphenyl, 4-(iso-propyl)phenyl, 4-ethylphenyl,
3-(n-propyl)phenyl and the like; a mono or di(alkoxy)phenyl group,
for example, 3,4-dimethoxyphenyl, 3,4-diethoxyphenyl,
3-ethoxy-4-isopropoxyphenyl, 3-ethoxy-s-butoxyphenyl,
3-methoxy-4-benzyloxyphenyl,
3-methoxy-4-(1-chloromethyl)benzyloxy-phenyl, 3-ethoxyphenyl,
4-(isopropoxy)phenyl, 4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl
and the like; 3- or 4-trifluoromethylphenyl; a mono- or
dicarboxyphenyl or (protected carboxy)phenyl group such
4-carboxyphenyl; a mono- or di(hydroxymethyl)phenyl or (protected
hydroxymethyl)phenyl such as 3-(protected hydroxymethyl)phenyl or
3,4-di(hydroxymethyl)phenyl; a mono- or di(aminomethyl)phenyl or
(protected aminomethyl)phenyl such as 2-(aminomethyl)phenyl or
2,4-(protected aminomethyl)phenyl; or a mono- or
di(N-(methylsulfonylamino))phenyl such as
3-(N-methylsulfonylamino))phenyl. Also, the term "substituted
phenyl" represents disubstituted phenyl groups where the
substituents are different, for example, 3-methyl-4-hydroxyphenyl,
3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,
4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,
2-hydroxy-4-chlorophenyl, and the like, as well as trisubstituted
phenyl groups where 1, 2, or 3 of the substituents are different,
for example 3-methoxy-4-benzyloxy-6-methyl sulfonylamino,
3-methoxy-4-benzyloxy-6-phenyl sulfonylamino, and tetrasubstituted
phenyl groups where the substituents are different such as
3-methoxy-4-benzyloxy-5-methyl-6-phenyl sulfonylamino. Preferred
substituted phenyl groups include the 3-methoxyphenyl,
3-ethoxy-phenyl, 4-benzyloxyphenyl, 4-methoxyphenyl,
3-ethoxy-4-benzyloxyphenyl, 3,4-diethoxyphenyl,
3-methoxy-4-benzyloxyphenyl,
3-methoxy-4-(1-chloromethyl)benzyloxy-phenyl,
3-methoxy-4-(1-chloromethyl)benzyloxy-6-methyl sulfonyl aminophenyl
groups. Also, the term "substituted phenyl" represents phenyl
groups having an aryl, phenyl or heteroaryl group fused thereto.
The fused ring may also be substituted with any, preferably 1, 2 or
3, of the substituents identified above for "substituted alkyl
"groups.
[0066] The term "aralkyl" means one, two, or three aryl groups
having the number of carbon atoms designated, appended to an alkyl
group having the number of carbon atoms designated including but
not limited to; benzyl, napthylmethyl, phenethyl, benzhydryl
(diphenylmethyl), trityl, and the like. A preferred arylalkyl group
is the benzyl group.
[0067] The term "substituted aralkyl" denotes an alkyl group,
preferably a C.sub.1-C.sub.8alkyl group, substituted at any carbon
with an aryl group, preferably a C.sub.6-C.sub.10aryl group, bonded
to the alkyl group through any aryl ring position and substituted
on the alkyl portion with one, two or three groups chosen from
halogen (F, Cl, Br, I), hydroxy, protected hydroxy, amino,
protected amino, C.sub.1-C.sub.7acyloxy, nitro, carboxy, protected
carboxy, carbamoyl, carbamoyloxy, cyano, C.sub.1-C.sub.6alkylthio,
N-(methylsulfonylamino) or C.sub.1-C.sub.4alkoxy. Optionally the
aryl group may be substituted with one, two, three, four or five
groups chosen from halogen, hydroxy, protected hydroxy, nitro,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy, carboxy, protected
carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl,
protected hydroxymethyl, aminomethyl, protected aminomethyl, or an
N-(methylsulfonylamino) group. As before, when either the
C.sub.1-C.sub.8 alkyl portion or the aryl portion or both are
disubstituted, the substituents can be the same or different. This
group may also appear as the substituted aralkyl moiety of a
substituted aralkoxy group.
[0068] Examples of the term "substituted aralkyl" and this group
when it occurs in a "substituted aralkoxy" group include groups
such as 2-phenyl-1-chloroethyl, 1-phenyl-1-chloromethyl,
1-phenyl-1-bromomethyl, 2-(4-methoxyphenyl)ethyl,
2,6-dihydroxy-4-phenyl(n-hexyl),
5-cyano-3-methoxy-2-phenyl(n-pentyl),
3-(2,6-dimethylphenyl)n-propyl, 4-chloro-3-aminobenzyl,
6-(4-methoxyphenyl)-3-carboxy(n-hexyl), 5-(4-aminomethyl
phenyl)-3-(aminomethyl)(n-pentyl), and the like.
[0069] The term "carboxy-protecting group" as used herein refers to
one of the ester derivatives of the carboxylic acid group commonly
employed to block or protect the carboxylic acid group while
reactions are carried out on other functional groups on the
compound. Examples of such carboxylic acid protecting groups
include 4-nitrobenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl,
2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl, 2,4,6-trimethylbenzyl,
pentamethylbenzyl, 3,4-methylenedioxybenzyl, benzhydryl,
4,4'-dimethoxybenzhydryl, 2,2',4,4'-tetramethoxybenzhydryl, alkyl
such as methyl, ethyl, isopropyl, t-butyl or t-amyl, trityl,
4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4''-trimethoxytrityl,
2-phenylprop-2-yl, trimethylsilyl, t-butyldimethylsilyl, phenacyl,
2,2,2-trichloroethyl, beta-(trimethylsilyl)ethyl,
beta-(di(n-butyl)methylsilyl)ethyl, p-toluenesulfonylethyl,
4-nitrobenzylsulfonylethyl, allyl, cinnamyl,
1-(trimethylsilylmethyl)prop-1-en-3-yl, and like moieties. The
species of carboxy-protecting group employed is not critical so
long as the derivatized carboxylic acid is stable to the condition
of subsequent reaction(s) on other positions of the molecule and
can be removed at the appropriate point without disrupting the
remainder of the molecule. In particular, it is important not to
subject a carboxy-protected molecule to strong nucleophilic bases
or reductive conditions employing highly activated metal catalysts
such as Raney nickel. (Such harsh removal conditions are also to be
avoided when removing amino-protecting groups and
hydroxy-protecting groups, discussed below.) Preferred carboxylic
acid protecting groups are the allyl and p-nitrobenzyl groups.
Similar carboxy-protecting groups used in the cephalosporin,
penicillin and peptide arts can also be used to protect a carboxy
group substituents. Further examples of these groups are found in
E. Haslam, "Protective Groups in Organic Chemistry", J. G. W.
McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, and T.
W. Greene, "Protective Groups in Organic Synthesis", John Wiley and
Sons, New York, N.Y., 1981, Chapter 5. The term "protected carboxy"
refers to a carboxy group substituted with one of the above
carboxy-protecting groups.
[0070] As used herein the term "amide-protecting group" refers to
any group typically used in the peptide art for protecting the
peptide nitrogens from undesirable side reactions. Such groups
include p-methoxyphenyl, 3,4-dimethoxybenzyl, benzyl,
O-nitrobenzyl, di-(p-methoxyphenyl)methyl, triphenylmethyl,
(p-methoxyphenyl)diphenylmethyl, diphenyl-4-pyridylmethyl,
m-2-(picolyl)-N'-oxide, 5-dibenzosuberyl, trimethylsilyl, t-butyl
dimethylsilyl, and the like. Further descriptions of these
protecting groups can be found in "Protective Groups in Organic
Synthesis", by Theodora W. Greene, 1981, John Wiley and Sons, New
York.
[0071] The terms "heterocyclic group", "heterocyclic",
"heterocyclyl", or "heterocyclo" alone and when used as a moiety in
a complex group such as a heterocycloalkyl group, are used
interchangeably and refer to any mono-, bi-, or tricyclic saturated
or non-aromatically unsaturated ring having the number of atoms
designated, generally from 3 to about 10 ring atoms, where the ring
atoms are carbon and 1,2,3 or 4 nitrogen, sulfur or oxygen atoms.
Typically, a 5-membered ring has 0 to 2 double bonds and 6- or
7-membered ring has 0 to 3 double bonds and the nitrogen or sulfur
heteroatoms may optionally be oxidized, and any nitrogen heteroatom
may optionally be quaternized. Examples include pyrrolidinyl,
oxiranyl, oxetanyl, tetrahydrofuranyl, 2,3-dihydrofuranyl,
2H-pyranyl, tetrahydropyranyl, thiiranyl, thietanyl,
tetrahydrothietanyl, aziridinyl, azetidinyl, 1-methyl-2-pyrrolyl,
piperidinyl, and 3,4,5,6-tetrahydropiperidinyl.
[0072] A "heterocycloalkyl" or a "heterocycloalkenyl" group is a
heterocyclo group as defined above covalently bonded to an alkyl or
alkenyl group as defined above.
[0073] Unless otherwise specified, "heteroaryl" alone and when used
as a moiety in a complex group such as a heteroaralkyl group,
refers to any mono-, bi-, or tricyclic aromatic ring system having
the number of atoms designated where at least one ring is a 5-, 6-
or 7-membered ring containing from one to four heteroatoms selected
from the group nitrogen, oxygen, and sulfur, and preferably at
least one heteroatom is nitrogen (Lang's Handbook of Chemistry,
supra). Included in the definition are any bicyclic groups where
any of the above heteroaryl rings are fused to a benzene ring.
Heteroaryls in which nitrogen or oxygen is the heteroatom are
preferred.
[0074] The following ring systems are examples of the heteroaryl
(whether substituted or unsubstituted) groups denoted by the term
"heteroaryl": thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl,
isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl,
oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl,
pyrimidyl, pyrazinyl, pyridazinyl, thiazinyl, oxazinyl, triazinyl,
thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl,
tetrazinyl, thiatriazinyl, oxatriazinyl, dithiadiazinyl,
imidazolinyl, dihydropyrimidyl, tetrahydropyrimidyl,
tetrazolo[1,5-b]pyridazinyl and purinyl, as well as benzo-fused
derivatives, for example benzoxazolyl, benzofuryl, benzothiazolyl,
benzothiadiazolyl, benzotriazolyl, benzoimidazolyl and indolyl.
[0075] Heterocyclic 5-membered ring systems containing a sulfur or
oxygen atom and one to three nitrogen atoms are also suitable for
use in the instant invention. Examples of such preferred groups
include thiazolyl, in particular thiazol-2-yl and thiazol-2-yl
N-oxide, thiadiazolyl, in particular 1,3,4-thiadiazol-5-yl and
1,2,4-thiadiazol-5-yl, oxazolyl, preferably oxazol-2-yl, and
oxadiazolyl, such as 1,3,4-oxadiazol-5-yl, and
1,2,4-oxadiazol-5-yl. A group of further preferred examples of
5-membered ring systems with 2 to 4 nitrogen atoms include
imidazolyl, preferably imidazol-2-yl; triazolyl, preferably
1,3,4-triazol-5-yl; 1,2,3-triazol-5-yl, 1,2,4-triazol-5-yl, and
tetrazolyl, preferably 1H-tetrazol-5-yl. A preferred group of
examples of benzo-fused derivatives are benzoxazol-2-yl,
benzthiazol-2-yl and benzimidazol-2-yl.
[0076] Further suitable specific examples of the above heterocylic
ring systems are 6-membered ring systems containing one to three
nitrogen atoms. Such examples include pyridyl, such as pyrid-2-yl,
pyrid-3-yl, and pyrid-4-yl; pyrimidyl, preferably pyrimid-2-yl and
pyrimid-4-yl; triazinyl, preferably 1,3,4-triazin-2-yl and
1,3,5-triazin-4-yl; pyridazinyl, in particular pyridazin-3-yl, and
pyrazinyl. The pyridine N-oxides and pyridazine N-oxides and the
pyridyl, pyrimid-2-yl, pyrimid-4-yl, pyridazinyl and the
1,3,4-triazin-2-yl groups, are a preferred group.
[0077] The substituents for the optionally substituted heterocyclic
ring systems, and further examples of the 5- and 6-membered ring
systems discussed above can be found in W. Druckheimer et al., U.S.
Pat. No. 4,278,793.
[0078] A particularly preferred group of "heteroaryl" include;
1,3-thiazol-2-yl, 4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl,
4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl sodium salt,
1,2,4-thiadiazol-5-yl, 3-methyl-1,2,4-thiadiazol-5-yl,
1,3,4-triazol-5-yl, 2-methyl-1,3,4-triazol-5-yl,
2-hydroxy-1,3,4-triazol-5-yl, 2-carboxy-4-methyl-1,3,4-triazol-5-yl
sodium salt, 2-carboxy-4-methyl-1,3,4-triazol-5-yl,
1,3-oxazol-2-yl, 1,3,4-oxadiazol-5-yl,
2-methyl-1,3,4-oxadiazol-5-yl,
2-(hydroxymethyl)-1,3,4-oxadiazol-5-yl, 1,2,4-oxadiazol-5-yl,
1,3,4-thiadiazol-5-yl, 2-thiol-1,3,4-thiadiazol-5-yl,
2-(methylthio)-1,3,4-thiadiazol-5-yl, 2-amino
-1,3,4-thiadiazol-5-yl, 1H-tetrazol-5-yl,
1-methyl-1H-tetrazol-5-yl, 1-(1-(dimethylamino)eth-2-yl)
-1H-tetrazol-5-yl, 1-(carboxymethyl)-1H-tetrazol-5-yl,
1-(carboxymethyl)-1H-tetrazol-5-yl sodium salt, 1-(methylsulfonic
acid)-1H-tetrazol-5-yl, 1-(methylsulfonic acid)-1H-tetrazol-5-yl
sodium salt, 2-methyl-1H-tetrazol -5-yl, 1,2,3-triazol-5-yl,
1-methyl-1,2,3-triazol-5-yl, 2-methyl-1,2,3-triazol-5-yl,
4-methyl-1,2,3-triazol -5-yl, pyrid-2-yl N-oxide,
6-methoxy-2-(n-oxide)-pyridaz-3-yl, 6-hydroxypyridaz-3-yl,
1-methylpyrid -2-yl, 1-methylpyrid-4-yl, 2-hydroxypyrimid-4-yl,
1,4,5,6-tetrahydro-5,6-dioxo-4-methyl-as-triazin -3-yl,
1,4,5,6-tetrahydro-4-(formylmethyl)-5,6-dioxo-as-triazin-3-yl,
2,5-dihydro-5-oxo-6-hydroxy-astriazin -3-yl,
2,5-dihydro-5-oxo-6-hydroxy-as-triazin-3-yl sodium salt,
2,5-dihydro-5-oxo-6-hydroxy-2-methyl-astriazin -3-yl sodium salt,
2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl,
2,5-dihydro-5-oxo -6-methoxy-2-methyl-as-triazin-3-yl,
2,5-dihydro-5-oxo-as-triazin-3-yl,
2,5-dihydro-5-oxo-2-methyl-as-triazin -3-yl,
2,5-dihydro-5-oxo-2,6-dimethyl-as-triazin-3-yl,
tetrazolo[1,5-b]pyridazin-6-yl and
8-aminotetrazolo[1,5-b]-pyridazin-6-yl.
[0079] An alternative group of "heteroaryl" includes;
4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl, 4-(carboxymethyl)
-5-methyl-1,3-thiazol-2-yl sodium salt, 1,3,4-triazol-5-yl,
2-methyl-1,3,4-triazol-5-yl, 1H-tetrazol -5-yl,
1-methyl-1H-tetrazol-5-yl,
1-(1-(dimethylamino)eth-2-yl)-1H-tetrazol-5-yl, 1-(carboxymethyl)
-1H-tetrazol-5-yl, 1-(carboxymethyl)-1H-tetrazol-5-yl sodium salt,
1-(methylsulfonic acid)-1H-tetrazol-5-yl, 1-(methylsulfonic
acid)-1H-tetrazol-5-yl sodium salt, 1,2,3-triazol-5-yl,
1,4,5,6-tetrahydro -5,6-dioxo-4-methyl-as-triazin-3-yl,
1,4,5,6-tetrahydro-4-(2-formylmethyl)-5,6-dioxo-as-triazin -3-yl,
2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl sodium salt,
2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin -3-yl,
tetrazolo[1,5-b]pyridazin-6-yl, and
8-aminotetrazolo[1,5-b]pyridazin-6-yl.
[0080] A "heteroaralkyl" or a "heteroaralkenyl" group is a
heteroaryl group as defined above covalently bonded to an alkyl
group or to an alkenyl group as defined above.
[0081] "Pharmaceutically acceptable salts" include both acid and
base addition salts. "Pharmaceutically acceptable acid addition
salt" refers to those salts which retain the biological
effectiveness and properties of the free bases and which are not
biologically or otherwise undesirable, formed with inorganic acids
such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, carbonic acid, phosphoric acid and the like, and organic
acids may be selected from aliphatic, cycloaliphatic, aromatic,
araliphatic, heterocyclic, carboxylic, and sulfonic classes of
organic acids such as formic acid, acetic acid, propionic acid,
glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic
acid, malic acid, maleic acid, maloneic acid, succinic acid,
fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic
acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid,
mandelic acid, embonic acid, phenylacetic acid, methanesulfonic
acid, ethanesulfonic acid, p-toluenesulfonic acid, salicyclic acid
and the like.
[0082] "Pharmaceutically acceptable base addition salts" include
those derived from inorganic bases such as sodium, potassium,
lithium, ammonium, calcium, magnesium, iron, zinc, copper,
manganese, aluminum salts and the like. Particularly preferred are
the ammonium, potassium, sodium, calcium and magnesium salts. Salts
derived from pharmaceutically acceptable organic nontoxic bases
includes salts of primary, secondary, and tertiary amines,
substituted amines including naturally occurring substituted
amines, cyclic amines and basic ion exchange resins, such as
isopropylamine, trimethylamine, diethylamine, triethylamine,
tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,
dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,
hydrabamine, choline, betaine, ethylenediamine, glucosamine,
methylglucamine, theobromine, purines, piperizine, piperidine,
N-ethylpiperidine, polyamine resins and the like. Particularly
preferred organic non-toxic bases are isopropylamine, diethylamine,
ethanolamine, trimethamine, dicyclohexylamine, choline, and
caffeine.
[0083] The term "prodrug" as used herein means a derivative of a
parent drug molecule that enhances pharmaceutically desirable
characteristics or properties (e.g. transport, bioavailability,
pharmacodynamics, etc.) and that requires biotransformation, either
spontaneous or enzymatic, within the organism to release the active
parent drug.
EMBODIMENTS
[0084] The invention is generally directed to compounds having the
structure shown below. ##STR3## In this structure R.sub.2, R.sub.5,
R.sub.6, A, B, N1, N2, Q, X, and Y have the meanings described
above. In these meanings, alkyl is preferably unsubstituted or
substituted C.sub.1-C.sub.6 alkyl; alkenyl is preferably
unsubstituted or substituted C.sub.2-C.sub.6 alkenyl; alkynyl is
preferably unsubstituted or substituted C.sub.2-C.sub.6 alkynyl;
aryl is preferably unsubstituted or substituted naphthyl or phenyl,
more preferably phenyl; aralkyl is preferably unsubstituted or
substituted benzyl. The variable m is preferably 1.
[0085] The group Y is preferably S(O).sub.n--R.sub.1 where n=1 or 2
or the group S(O).sub.n--NR.sub.2R.sub.2 where n=1 or 2, more
preferably S(O).sub.n--R.sub.1.
[0086] In one preferred embodiment, R.sub.1, for example when Y is
S(O).sub.n--R.sub.1, is selected from the group consisting of
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.6 cycloalkyl, phenyl, naphthyl, benzyl and
heteroaryl having 5-6 ring atoms selected from carbon atoms and 1-2
heteroatoms, where the heteroatoms are N, S, or O, and R.sub.1
optionally substituted with 1-3 substituents selected from the
group consisting of halo, nitro, C.sub.1-C.sub.6 alkyl,
NR.sub.7R.sub.8, OR.sub.7, SR.sub.7, C.sub.1-C.sub.6
alkyl-C(O)OR.sub.7, C.sub.1-C.sub.6 alkyl-OC(O)R.sub.7,
C.sub.1-C.sub.6 alkyl-C(O)R.sub.7, C.sub.1-C.sub.6 alkyl-OR.sub.7,
C.sub.1-C.sub.6 haloalkyl, C.sub.1-C.sub.6 alkyl-NR.sub.7R.sub.8,
C(O)OR.sub.7, OC(O)R.sub.7, C(O)NR.sub.7R.sub.8,
OC(O)NR.sub.7R.sub.8, NHC(O)R.sub.7, and NHC(O)NR.sub.7R.sub.8,
where R.sub.7 and R.sub.8 independently are H or C.sub.1-C.sub.6
alkyl. In this embodiment, each of the remaining variables R.sub.2,
R.sub.5, R.sub.6, A, B, Q, X, and Y may be independently selected
to be any of the groups in the respective definitions described
above.
[0087] In a second preferred embodiment, Q is phenyl optionally
substituted with 1-5, preferably 2-4, more preferably 2-3,
substituents selected from the group consisting of halo, nitro,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, NR.sub.7R.sub.8, OR.sub.7, SR.sub.7, C.sub.1-C.sub.6
alkyl-C(O)OR.sub.7, OC.sub.1-C.sub.6 alkyl-C(O)OR.sub.7,
C.sub.1-C.sub.6 alkyl-OR.sub.7, OC.sub.1-C.sub.6 alkyl-OR.sub.7,
C.sub.1-C.sub.6 alkyl-NR.sub.7R.sub.8, OC.sub.1-C.sub.6
alkyl-NR.sub.7R.sub.8, C.sub.1-C.sub.6 alkyl-C(O)NR.sub.7R.sub.8,
OC.sub.1-C.sub.6 alkyl-C(O)NR.sub.7R.sub.8, C.sub.1-C.sub.6
alkyl-C(O)R.sub.7, OC.sub.1-C.sub.6 alkyl-C(O)R.sub.7,
C.sub.1-C.sub.6 haloalkyl, O-aralkyl (e.g. benzyloxy),
C(O)OR.sub.7, C(O)NR.sub.7R.sub.8, OC(O)NR.sub.7R.sub.8,
NHC(O)R.sub.7, NHC(O)NR.sub.7R.sub.8, NR.sub.7S(O).sub.nR.sub.1,
NR.sub.7S(O).sub.nR.sub.7, S(O).sub.nR.sub.7, S(O).sub.nNR.sub.7,
where R.sub.7 and R.sub.8 independently are H or C.sub.1-C.sub.6
alkyl. In this embodiment, each of the remaining variables R.sub.2,
R.sub.5, R.sub.6, A, B, X, and Y (and R.sub.1) may be independently
selected to have any of the definitions described above. Each
alkyl, alkenyl and alkynyl moiety may also be substituted as
defined above.
[0088] In a third preferred embodiment, Q has the structure
##STR4##
[0089] where
[0090] R.sub.9 is H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 alkoxy, hydroxy,
NR.sub.7R.sub.8, SR.sub.7 or OR.sub.7, where R.sub.7 and R.sub.8,
independently, are H or unsubstituted or substituted
C.sub.1-C.sub.6 alkyl;
[0091] R.sub.10, R.sub.11, and Z.sub.2, independently, are each
selected from the group consisting of H, halo, nitro, cyano,
C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, NR.sub.7R.sub.8,
OR.sub.7, SR.sub.7, C.sub.1-C.sub.6 alkyl-C(O)R.sub.7,
C.sub.1-C.sub.6 alkyl-C(O)NR.sub.7R.sub.8, C.sub.1-C.sub.6
alkyl-C(O)OR.sub.7, C.sub.1-C.sub.6 alkyl-OC(O)R.sub.7,
C.sub.1-C.sub.6 alkyl-OR.sub.7, OC.sub.1-C.sub.6 alkyl-C(O)R.sub.7,
OC.sub.1-C.sub.6 alkyl-C(O)OR.sub.7, OC.sub.1-C.sub.6
alkyl-OC(O)R.sub.7, O--C.sub.1-C.sub.6 alkyl-OR.sub.7,
OC.sub.1-C.sub.6 alkyl-C(O)NR.sub.7R.sub.8, C.sub.1-C.sub.6
haloalkyl, OR.sub.12, C.sub.1-C.sub.6 alkyl-R.sub.12,
O--C.sub.1-C.sub.6 alkyl-R.sub.12, C(O)OR.sub.7, C(O)OR.sub.12,
C(O)NR.sub.7R.sub.8, OC(O)NR.sub.7R.sub.8, NR.sub.7C(O)R.sub.7,
NR.sub.7C(O)R.sub.12, NR.sub.7C(O)--NR.sub.7R.sub.8,
NR.sub.7C(O)OR.sub.7, NR.sub.7C(O)OR.sub.12, NR.sub.7S(O)n-R.sub.1,
NR.sub.7S(O)n-R.sub.7 and NR.sub.7S(O)n-R.sub.12, where R.sub.7 and
R.sub.8, independently, are H or unsubstituted or substituted
C.sub.1-C.sub.6 alkyl, R.sub.12 is unsubstituted or substituted
C.sub.6-C.sub.10 aryl or heterocycl as defined above and n is 1 or
2;
[0092] Z.sub.1 is H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
halogen or nitro. In this embodiment, each of the remaining
variables R.sub.2, R.sub.5, R.sub.6, A, B, X, and Y may be
independently selected to have any of the definitions described
above. Each alkyl, alkenyl and alkynyl moiety may also be
substituted as defined above.
[0093] In various aspects of the invention, Z.sub.1 and Z.sub.2 may
be hydrogen; Z.sub.1, Z.sub.2 and R.sub.11 may be hydrogen; or
Z.sub.1, R.sub.10 and R.sub.11 may be hydrogen; and the remaining
ring substituents are as defined above.
[0094] In another embodiment, the substituents at the 4- and
5-positions or at the 5- and 6-positions of the ring when Q is
substituted phenyl may be bonded together to form an unsubstituted
or substituted carbocyclic or heterocyclic ring. Examples of such
compounds are shown below, where the symbol ##STR5## is preferably
a 5-membered or a 6-membered carbocyclic or heterocyclic ring which
is fused to the phenyl ring in the positions shown below.
##STR6##
[0095] Examples of suitable 5-membered or a 6-membered carbocyclic
or heterocyclic rings which may be fused to the phenyl ring include
the ring systems shown below, where R.sub.6 is as defined above.
##STR7##
[0096] In another preferred embodiment, Y is S(O).sub.n--R.sub.1
where n is 1 or 2, preferably 2. In this embodiment, R.sub.1 may be
as defined above and each of the remaining variables may be
independently selected to have any of the definitions described
above.
[0097] Compounds in which Q is substituted phenyl and R.sub.10 is
selected from the group consisting of C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 aminoalkyl, C.sub.1-C.sub.6
haloalkyl, C.sub.1-C.sub.6 hydroxyalkyl, phenyl, phenoxy, benzyl,
benzyloxy, as well as phenoxy- and benzyloxy-substituted with
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, halo,
C.sub.1-C.sub.6 haloalkyl, C.sub.1-C.sub.6 hydroxyalkyl,
C.sub.1-C.sub.6 aminoalkyl, OC(O)--C.sub.1-C.sub.6 alkyl,
C(O)O--C.sub.1-C.sub.6 alkyl and C(O)OH are also preferred, where
each of the remaining variables may be independently selected to
have any of the definitions described above.
[0098] Also of interest are compounds in which R.sub.11 is
NR.sub.7C.sub.1-C.sub.6 alkyl-C(O)NR.sub.7R.sub.8,
NR.sub.7S(O)n-R.sub.7 or N R.sub.7S(O)n-R.sub.12, n is 1 or 2
and/or where Z.sub.1=Z.sub.2=H and/or where R.sub.10 is OR.sub.7,
OR.sub.12, OC.sub.7-C.sub.10-aralkyl, OC.sub.1-C.sub.6
alkyl-OR.sub.7 or OC.sub.1-C.sub.6 alkyl-OR.sub.12 where R.sub.7
and R.sub.12 are unsubstituted or substituted as defined above.
Suitable substituted R.sub.7 and R.sub.12 include these groups
substituted as described above, for example, having 1 or 2
C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkoxy-C.sub.1-C.sub.6
alkoxy, halo, C.sub.1-C.sub.6 haloalkyl, C.sub.1-C.sub.6
hydroxyalkyl, C.sub.1-C.sub.6 aminoalkyl, OC(O)--C.sub.1-C.sub.6
alkyl, C(O)O--C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkyl
C(O)OR.sub.7, C.sub.1-C.sub.6 alkyl OC(O)R.sub.7 or C(O)OH. In
these compounds, each of the remaining variables may be
independently selected to have any of the definitions described
above. These compounds are also interesting where, Y is
S(O).sub.n--R.sub.1 where n is 1 or 2, that is, disulfonamide
compounds.
[0099] In another embodiment, A and B are independently CH or
CR.sub.3, where R.sub.3 is H, C.sub.1-6 alkyl or OH, where the
remaining variables may be independently selected to have any of
the definitions described above.
[0100] In another embodiment, R.sub.6 is H or R.sub.3 is CH, where
the remaining variables may be independently selected to have any
of the definitions described above.
[0101] In another preferred embodiment, X is a carbonyl group
(C.dbd.O), where the remaining variables may be independently
selected to have any of the definitions described above. In this
embodiment, preferably m=1.
[0102] Table 1, setting forth examples of some preferred groups at
various positions of some compounds of the invention, is shown
below. A group of specific compounds is disclosed in this table and
is obtained by selecting all unique combinations of substituents,
one from each column of the table, for each variable and combining
these groups with the structure disclosed above Table 1.
TABLE-US-00001 TABLE 1 ##STR8## X R9 R10 Z2 R11 R1 Z3 CH2 OEt OEt H
H Me H C.dbd.O OMe OH OEt NMeSO2Me Et OH CH2CH3 OMe OMe Ph Pr Cl
CH.dbd.CH2 OiPr Ph Naphthyl Bu F CCH OCH2Ph OiPr iPr CH2CCH
CH(CH3)Ph OPr NHSO2Me iBu H CH(CH2Cl)Ph CH(CH2Cl)Ph NPrSO2Me sBu Pr
OCH2CH2CF3 OCH2CH2CF3 N(CH2CO2H)SO2Me Ph Cl OCH2CF3 OCH2CF3
NMeSO2CH2CO2H O-tolyl SCH3 CH(CO2H)Ph CH(CO2H)Ph NHSO2CH2CO2H
CH2CH2CO2H SCH2CH3 CH(CO2Me)Ph CH(CO2Me)Ph NHCOCH3 CH2CH2CONH2
NHCH3 Ph Ph NHCOCH2CO2H CH2CH2CO2Me NHCH2CH3 OPh OPh
NHSO2-thiophene p-tolyl H Cl NHSO2CH2CO2H 4-chlorophenyl Cl Br
NHSO2CH2CO2Me 4-aminomethyl- phenyl Br F OCH2CO2H 4-aminophenyl F
OCH2Ph pyridyl 2-chlorophenyl H NCH2CH3 3-nitrophenyl NHCH2CH3 SCH3
1-naphthyl 2-thiophene 3-thiophene 2-furan 3-furan CH2CH(NH2)CH3
pyridyl 2-naphthyl
Methods of Making
[0103] Compounds of the present invention can be prepared by
methods employing standard chemical methodologies described and
referenced in standard textbooks (e.g. March, J. "Advanced Organic
Chemistry" McGraw-Hill, New York, 1977; Collman, J. P., Hegedus, L.
S., Norton, J. R., Finke, R. G. "Principles and Applications of
Organotransition Metal Chemistry" University Science, Mill Valley,
1987; Larock, R. C. "Comprehensive Organic Transformations" Verlag,
New York, 1989).
[0104] A key intermediate in the synthesis of compounds of the
invention has the formula shown below ##STR9## In this formula, A,
B, R.sub.2, R.sub.4a, R.sub.4b, R.sub.5, R.sub.6, m and Q have the
meanings and preferred meanings described above. This compound can
be prepared using several alternative synthetic routes. After
preparation, the cyano group may be converted into an amidino group
(C(NH)NH.sub.2), for example, using known procedures, such as the
Pinner reaction. A cyano compound having the formula shown above
may be reacted with hydroxyl amine, preferably in an alcohol
solvent, followed by reduction with Raney Ni, preferably in an
alcohol solvent, or may be reacted first with ethanolic HCl and
then with alcoholic ammonia to yield the corresponding amidino
compounds. Alternatively, a modified Pinner reaction using
pyridine/diethylamine (1/1)/hydrogen sulfide followed by methyl
iodide/acetonitrile and then ammonium acetate/ethanol will provide
the desired amidino product.
[0105] One synthetic route to compounds having the formula shown
above is a condensation reaction using appropriately substituted
precursors as shown in the scheme below. ##STR10##
[0106] This condensation is performed in the presence of a
catalyst, preferably a Lewis acid catalyst, and an alkyl alcohol
(ROH), preferably a lower alkyl alcohol such as methanol, ethanol,
i-propanol, etc., followed by hydrolysis of the intermediate,
preferably with an excess of water, generally up to about 10
equivalents of water. Suitable Lewis Acids include BF.sub.3
etherate, AlCl.sub.3, etc. W-NC is an isonitrile in which W may be
any suitable hydrocarbon group, generally an alkyl,
carbocycloalkyl, or aralkyl group, preferably having no more than
about 12 carbon atoms. A particularly preferred isonitrile is
benzyl isonitrile. The ester product may be purified by standard
techniques, including high pressure liquid chromatography (HPLC),
column chromatography, recrystallization, etc.
[0107] Reduction of the resulting ester to an alcohol can be
accomplished using any known reducing agent ([H]) which will
preferentially reduce an ester before a nitrile. Suitable reducing
agents and procedures are well known in the art. See, for example,
Modern Synthetic Reactions, H. O. House, W. A. Benjami, Inc.,
Second Ed., 1972. A useful reducing agent is lithium borohydride.
The alcohol may then be converted to an amine using known chemical
reactions. Suitable conditions include first reacting the alcohol
with hydrogen azide, DEAD, and triphenyl phosphine (PPh.sub.3),
following by PPh.sub.3 and water or first with phthalimide, DEAD
and PPh.sub.3, followed by hydrazine. These reactions are shown in
the scheme below. Alternatively, the ester may be reacted with a
reagent having a nucleophilic carbon atom to introduce suitable
R.sub.4a groups. Such reagents may include an activated methylene
carbon, for example a methylene which is adjacent to one or more
strong electron withdrawing groups such as nitro (NO.sub.2),
carboalkoxy (COOR.sub.4a), etc., Grignard reagents (R.sub.4aMgHal,
where Hal is a halogen), etc. and then converted to the alcohol and
to the amine. ##STR11##
[0108] Conversion of the amine functional group to a sulfonamide
and the conversion of the nitrile functional group to an amidine
may be performed in any desired order. A preferred reaction scheme
is shown in the scheme below. ##STR12##
[0109] These conversions are accomplished using known chemical
reactions, purification and separation procedures. The amine may be
converted to a sulfonamide by reaction with an appropriately
substituted sulfonyl chloride (ClSO.sub.2R.sub.1) in the presence
of a base. The nitrile may be reacted with hydroxylamine in an
alcohol solvent followed by reduction, for example, with Raney
nickel and hydrogen, or by reaction with HCl/alcohol and then
ammonia/alcohol.
[0110] An example of a suitable reaction sequence is shown below.
##STR13## In this sequence, a=BF.sub.3OEt.sub.2/EtOH,
b=LiBH.sub.4/DME, c=phthalimide, DIAD/PPh.sub.3/THF,
d=H.sub.2NNH.sub.2/EtOH, e=R.sub.1SO.sub.2Cl, f=H.sub.2/Pt/C/EtOH,
and g=R.sub.7SO.sub.2Cl/NEt.sub.3, NH.sub.2OH--HCl/NEt.sub.3,
H.sub.2/Ra--Ni/MeOH.
[0111] An analogous related synthetic scheme may be used to prepare
the corresponding compounds in which X is a carbonyl as shown
below. ##STR14##
[0112] Compounds in which m=2 can be prepared using according to
the scheme shown below which provides an alcohol which is
homologous to the alcohol shown in the scheme above and which can
be converted to an amine (and further elaborated compounds) in an
analogous manner. In the scheme below, (a) is a base and (b) is a
reducing agent such as LiBH.sub.4. ##STR15##
[0113] Compounds in which Y is C(O)--R.sup.1; C(O)--OR.sup.1;
C(O)--NR.sup.1R.sup.2 are prepared as described above using the
corresponding acyl halide (preferably an acyl chloride), alkyl
haloformate (preferably a chloroformate) or isocyanate as shown in
the scheme below: ##STR16##
[0114] An example of a suitable reaction sequence is shown below.
##STR17##
[0115] The esters resulting from the condensation reactions shown
above can also function as intermediates in the synthesis of
compounds in which X is a carbonyl group. Conversion of the ester
to a carboxylic acid is easily performed by saponification with an
alkali-metal hydroxide such as lithium, sodium, or potassium
hydroxide. Coupling of a sulfonamide to the acid is accomplished by
first activating the carboxylate for coupling using, for example,
carbonyl diimidazole or other routine activating agents used in
peptide synthesis. The second part of the coupling is done by
mixing an alkyl or aryl sulfonamide with a strong base such as DBU
or sodium hydride, preferably in an anhydrous solvent, such as a
hydrocarbon or ether solvent, e.g. tetrahydrofuran. The nitrile is
converted to an amidine by methods already described. ##STR18## In
a more preferred variation of this embodiment, Q is a substituted
phenyl having substituents Z.sub.1, Z.sub.2, and R.sub.9-R.sub.11
as described below.
[0116] A further method of preparing intermediate compounds useful
in preparing the compounds of the invention is shown below and
involves the synthesis of imine compounds from readily available
aldehydes and ketones followed by nucleophilic addition of a
nucleophilic carbon atom containing reagent, i.e. in general "Nu".
"Nu" may be a moiety such as CHR.sub.4aNO.sub.2, CHR.sub.4aCOOR,
CH(NO.sub.2)(COOR), etc., which are generated using well known
Grignard reactions, reactions in which a base is used to remove a
proton from the carbon atom adjacent to an electron withdrawing
group (CO, COO, NO.sub.2), etc. ##STR19##
[0117] "Nu" can be converted into a group such as
CHR.sub.4aNH.sub.2 or CHR.sub.4aCH.sub.2OH or
CHR.sub.4aNH.sub.2CH.sub.2OH by known reduction reactions as shown
below. In these intermediates, an amino group can be further
sulfonated or otherwise acylated as described above. An example of
a suitable reaction sequence is shown below. ##STR20##
[0118] An alternative synthetic procedure can be used to prepare
the alcohol intermediates described above. As shown in the scheme
below, reaction of an initial styrene derivative with a peracid
usually produces a mixture of products containing non-hydrogen
R.sub.4aand/or R.sub.5 substituents as shown below which can be
converted without separation to the alcohol by reaction with a
cyano-aniline or corresponding cyano-pyridine. ##STR21## The
alcohol can then be used to prepare compounds of the invention as
described above.
[0119] When the corresponding compounds in which A and B are
nitrogen are desired, the aniline or substituted aniline used in
the reactions described above is replaced with the corresponding
amino-pyridine or substituted amino-pyridine compounds.
[0120] Compounds in which the sulfonamide nitrogen bears a
substituent can be prepared by conventional alkylation of the
nitrogen atom using known reactions, for example, alkylation with
dialkyl sulfate, alkyl halide etc, according to known
procedures.
[0121] In a preferred embodiment, Q is a substituted aryl, and more
preferably, a substituted phenyl group and has the structure shown
below. ##STR22##
[0122] In this structure, Z.sub.1, Z.sub.2, R.sub.9-R.sub.11 are as
defined above both generally and in preferred embodiments.
Compounds of this embodiment are prepared as described in scheme 1
above using an appropriately substituted benzaldehyde having
structure Q-CHO(R.sub.5 is H). These substituted benzaldehydes are
readily available from commercial sources or can be easily prepared
from known benzaldehydes using well known synthetic chemistry.
[0123] In one embodiment, Q is substituted with a nitro group. A
preferred position for the nitro group is at R.sub.11, (where
Z.sub.1, Z.sub.2, R.sub.9 and R.sub.10 are as defined above
generally and in preferred embodiments), which nitro group can be
further reduced to an amino group using a suitable reducing agent.
Generally, the cyano-amine compound or the cyano-sulfonamide
compound shown in scheme 3 will be reacted with a reducing agent
which will preferentially reduce the nitro group at R.sub.11 over
the cyano group. Any reducing agent having these properties may be
used, for example, hydrogen and a Pt/C catalyst. The aniline
resulting from the reduction can then be reacted with a sulfonyl
chloride (ClSO.sub.2W where W is as defined above) to produce a
disulfonamide compound.
[0124] The preparation of cyclic urea derivatives in which
N.sub.1--R.sub.2 and N.sub.2--R.sub.2 together form a urea linkage,
i.e. N.sub.1--C(O)--N.sub.2, provides additional compounds of the
invention and provides an additional method of preparing
enantiomerically pure compounds of the invention. The cyclic urea
compounds can be used, for example, to prepare dialkoxy
bis-sulfonamides and other compounds of the invention as shown in
the scheme below.
[0125] Alternatively, nitric acid can be replaced by sulfuric acid
in the scheme below to give sulfonic acid derivatives which can be
further converted to sulfonamides and sulfones by known reactions.
##STR23## Other compounds of the invention, including heterocyclic
compounds, are readily prepared from simple starting materials
which can be used in the synthetic schemes described above. For
example, beginning with simple nitro and hydroxy substituted
aldehydes, condensation as described above provides the
corresponding esters which can be converted directly to cyclic
urethane or oxazole compounds which can then be further elaborated
as already described to provide compounds of the invention. These
reactions are shown schematically below for rings fused in the
5-position and 6-position. ##STR24##
[0126] Compounds in which the ring is fused to the 4-position and
the 5-position of the phenyl ring are prepared by analogous methods
stating with the appropriately substituted aldehyde as shown below.
##STR25##
[0127] Other fused heterocyclic compounds are prepared using
conventional synthetic chemical reactions and appropriately
substituted starting materials which are well known in the art of
chemical synthesis to provide additional compounds of the
invention. For example, fused furan ring systems can be prepared
from the corresponding halo and hydroxy substituted aldehydes as
shown below. ##STR26##
[0128] Also included in the scope of this invention are prodrugs of
the compounds described above. Suitable prodrugs include known
amino-protecting and carboxy-protecting groups which are released,
for example hydrolyzed, to yield the parent compound under
physiologic conditions. A preferred class of prodrugs are compounds
in which a nitrogen atom in an amino, amidino, aminoalkyleneamino,
iminoalkyleneamino or guanidino group is substituted with a hydroxy
(OH) group, an alkylcarbonyl (--CO-W) group, an alkoxycarbonyl
(--CO--OW), an acyloxyalkyl-alkoxycarbonyl (--CO--O-W-O--CO-W)
group where W is a monovalent or divalent group and as defined
above or a group having the formula --C(O)--O--CP1P2-haloalkyl,
where P1 and P2 are the same or different and are H, lower alkyl,
lower alkoxy, cyano, halo lower alkyl or aryl. Preferably the
nitrogen atom is one of the nitrogen atoms of the amidino group of
the compounds of the invention. These prodrug compounds are
prepared reacting the compounds of the invention described above
with an activated acyl compound to bond a nitrogen atom in the
compound of the invention to the carbonyl of the activated acyl
compound. Suitable activated carbonyl compounds contain a good
leaving group bonded to the carbonyl carbon and include acyl
halides, acyl amines, acyl pyridinium salts, acyl alkoxides, in
particular acyl phenoxides such as p-nitrophenoxy acyl,
dinitrophenoxy acyl, fluorophenoxy acyl, and defluorophenoxy acyl.
The reactions are generally exothermic and are carried out in inert
solvents at reduced temperatures such as -78 to about 50 C. The
reactions are usually also carried out in the presence of an
inorganic base such as potassium carbonate or sodium bicarbonate,
or an organic base such as an amine, including pyridine,
triethylamine, etc. One manner of preparing prodrugs is described
in WO98/46576, published 22 Oct. 1998.
[0129] Using the synthetic methods described above, the following
exemplary compounds of the invention shown in Table 2 below can be
prepared (m=1). For each entry in the table, X may be carbonyl or
(CR.sub.4aR.sub.4b).sub.m where m=1 or 2; and the benzamidine ring
may bear a halogen, hydroxy or alkyl substituent. TABLE-US-00002
TABLE 2 ##STR27## Com- pound No. Z.sup.2 R.sup.1 R.sup.9 R.sup.10
R.sup.11 1 --H ##STR28## OCH.sub.3 ##STR29## H (benzyloxy) 2 --H
##STR30## OCH.sub.3 ##STR31## H 3 --H ##STR32## OCH.sub.3 ##STR33##
H 4 --H ##STR34## OCH.sub.3 ##STR35## H 5 --H ##STR36## OCH.sub.3
##STR37## H 6 --H ##STR38## OCH.sub.3 ##STR39## H 7 --H ##STR40##
OCH.sub.3 ##STR41## H 8 --H ##STR42## OCH.sub.3 ##STR43## H 9 --H
##STR44## OCH.sub.3 ##STR45## ##STR46## 10 --H ##STR47## OCH.sub.3
OCH.sub.3 Br 11 --H ##STR48## OCH.sub.3 ##STR49## H 12 --H
--CH(CH.sub.3).sub.2 OCH.sub.3 ##STR50## H 13 --H
--CH.sub.2CH.sub.2CH.sub.3 OCH.sub.3 ##STR51## CH.sub.3SO.sub.2--NH
14 --H ##STR52## OCH.sub.3 ##STR53## H 15 --H ##STR54## OCH.sub.3
##STR55## H 16 --H ##STR56## OCH.sub.3 ##STR57## H 17 --H
--CH.sub.2CH.sub.3 OCH.sub.3 ##STR58## H 18 --H ##STR59## OCH.sub.3
##STR60## H 19 --H ##STR61## OCH.sub.3 ##STR62## H 20 --H ##STR63##
OCH.sub.3 ##STR64## H 21 --H ##STR65## OCH.sub.3 ##STR66## H 22 --H
##STR67## OCH.sub.3 ##STR68## H 23 --H ##STR69## OCH.sub.3
##STR70## H 24 --H --CH.sub.3 OCH.sub.3 ##STR71## H 25 --H
##STR72## OCH.sub.3 ##STR73## H 26 --H ##STR74## OCH.sub.3
##STR75## H 27 --H ##STR76## OCH.sub.3 ##STR77## H 28 --H ##STR78##
OCH.sub.3 ##STR79## H 29 --H ##STR80## OCH.sub.3 ##STR81## H 30 --H
##STR82## OCH.sub.3 ##STR83## H 31 --H ##STR84## OCH.sub.3
##STR85## H 32 --H ##STR86## OCH.sub.3 ##STR87## H 33 --H
--CH.sub.2CH.sub.2CH.sub.3 OCH.sub.3 ##STR88## H 34 --H
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3 OCH.sub.3 ##STR89## H 35 --H
##STR90## OCH.sub.3 ##STR91## H 36 --H ##STR92## OCH.sub.3
##STR93## H 37 --H --CH.sub.2CH.sub.2CH.sub.3 OCH.sub.3 ##STR94## H
38 --H --CH.sub.2CO.sub.2CH.sub.2CH.sub.3 OCH.sub.3 ##STR95## H 39
--H --CH.sub.2CO.sub.2H OCH.sub.3 ##STR96## H 40 --H
--(CH.sub.2).sub.6CH.sub.3 OCH.sub.3 ##STR97## H 41 --H
--CH.dbd.CH.sub.2 OCH.sub.3 ##STR98## H 42 --H --CH.sub.2--C.dbd.CH
OCH.sub.3 ##STR99## H 43 --H ##STR100## OCH.sub.3 ##STR101## H 44
--H ##STR102## OCH.sub.3 ##STR103## H 45 --H ##STR104## OCH.sub.3
##STR105## H 46 --H ##STR106## OCH.sub.3 ##STR107## H 47 --H
##STR108## OCH.sub.3 ##STR109## H 48 --H ##STR110## OCH.sub.3
##STR111## H 49 --H ##STR112## OCH.sub.3 ##STR113## H 50 --H
##STR114## OCH.sub.3 ##STR115## H 51 --H ##STR116## OCH.sub.3
##STR117## H 52 --H ##STR118## OCH.sub.3 ##STR119## H 53 --H
##STR120## OCH.sub.3 ##STR121## H 54 --H ##STR122## OCH.sub.3
##STR123## H 55 --H ##STR124## OCH.sub.3 ##STR125## H 56 --H
##STR126## OCH.sub.3 ##STR127## H 57 --H ##STR128## OCH.sub.3
##STR129## H 58 --H ##STR130## OCH.sub.3 ##STR131## H 59 --H
##STR132## OCH.sub.3 ##STR133## H 60 --H ##STR134##
OCH.sub.2CH.sub.3 OCH.sub.2CH.sub.3 H 61 --H ##STR135##
OCH.sub.2CH.sub.2CH.sub.3 ##STR136## H 62 --H ##STR137##
OCH.sub.2CH.sub.2CH.sub.3 ##STR138## H 63 --H ##STR139##
OCH.sub.2CH.sub.3 ##STR140## H 64 --H ##STR141## OCH.sub.2CH.sub.3
##STR142## H 65 --H ##STR143## OCH.sub.2CH.sub.3 ##STR144## H 66
--H ##STR145## OCH.sub.2(CH.sub.2).sub.4CH.sub.3 ##STR146## H 67
--H ##STR147## OCH.sub.2CH.sub.3 ##STR148## H 68 --H ##STR149##
OCH.sub.2CH.sub.3 ##STR150## ##STR151## 69 --H ##STR152##
OCH.sub.2CH.sub.3 OCH.sub.3 Br 70 --H ##STR153## OCH.sub.2CH.sub.3
##STR154## H 71 --H --CH(CH.sub.3).sub.2 OCH.sub.2CH.sub.3
##STR155## H 72 --H --CH.sub.2CH.sub.2CH.sub.3 OCH.sub.2CH.sub.3
##STR156## CH.sub.3SO.sub.2--NH 73 --H ##STR157## OCH.sub.2CH.sub.3
##STR158## H 74 --H --CH.sub.2CH.sub.2CH.sub.3 OCH.sub.2CH.sub.3
##STR159## CH.sub.3CH.sub.2--O.sub.2CCH.sub.2SO.sub.2NH-- 75 --H
--CH.sub.2CH.sub.2CH.sub.3 OCH.sub.2CH.sub.3 ##STR160##
HO.sub.2CCH.sub.2SO.sub.2NH-- 76 --H --CH.sub.2CH.sub.2CH.sub.3
OCH.sub.2CH.sub.3 ##STR161## ##STR162## 77 --H
--CH.sub.2CH.sub.2CH.sub.3 OCH.sub.2CH.sub.3 ##STR163## ##STR164##
78 --H ##STR165## OCH.sub.2CH.sub.3 ##STR166## H 79 --H ##STR167##
OCH.sub.2CH.sub.3 ##STR168## H 80 --H --CH.sub.2CH.sub.3
OCH.sub.2CH.sub.3 ##STR169## H 81 --H ##STR170## OCH.sub.2CH.sub.3
##STR171## H 82 --H ##STR172## OCH.sub.2CH.sub.3 ##STR173## H 83
--H ##STR174## OCH.sub.2CH.sub.3 ##STR175## H 84 --H ##STR176##
OCH.sub.2CH.sub.3 ##STR177## H 85 --H ##STR178## OCH.sub.2CH.sub.3
##STR179## H 86 --H ##STR180## OCH.sub.2CH.sub.3 ##STR181## H 87
--H --CH.sub.3 OCH.sub.2CH.sub.3 ##STR182## H 88 --H ##STR183##
OCH.sub.2CH.sub.3 ##STR184## H 89 --H ##STR185## OCH.sub.2CH.sub.3
##STR186## H 90 --H ##STR187## OCH.sub.2CH.sub.3 ##STR188## H 91
--H ##STR189## OCH.sub.2CH.sub.3 ##STR190## H 92 --H ##STR191##
OCH.sub.2CH.sub.3 ##STR192## H 93 --H ##STR193## OCH.sub.2CH.sub.3
##STR194## H 94 --H ##STR195## OCH.sub.2CH.sub.3 ##STR196## H 95
--H ##STR197## OCH.sub.2CH.sub.3 ##STR198## H 96 --H
--CH.sub.2CH.sub.2CH.sub.3 OCH.sub.2CH.sub.3 ##STR199## H 97 --H
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3 OCH.sub.2CH.sub.3 ##STR200## H
98 --H ##STR201## OCH.sub.2CH.sub.3 ##STR202## H 99 --H ##STR203##
OCH.sub.2CH.sub.3 ##STR204## H 100 --H --CH.sub.2CH.sub.2CH.sub.3
OCH.sub.2CH.sub.3 ##STR205## H 101 --H --(CH.sub.2).sub.6CH.sub.3
OCH.sub.2CH.sub.3 ##STR206## H 102 --H --CH.dbd.CH.sub.2
OCH.sub.2CH.sub.3 ##STR207## H 103 --H --CH.sub.2--C.ident.CH
OCH.sub.2CH.sub.3 ##STR208## H 104 --H ##STR209## OCH.sub.2CH.sub.3
##STR210## H 105 --H ##STR211## OCH.sub.2CH.sub.3 ##STR212## H 106
--H ##STR213## OCH.sub.2CH.sub.3 ##STR214## H 107 --H ##STR215##
OCH.sub.2CH.sub.3 ##STR216## H 108 --H ##STR217## OCH.sub.2CH.sub.3
##STR218## H 109 --H ##STR219## OCH.sub.2CH.sub.3 ##STR220## H 110
--H ##STR221## OCH.sub.2CH.sub.3 ##STR222## H 111 --H ##STR223##
OCH.sub.2CH.sub.3 ##STR224## H 112 --H ##STR225## OCH.sub.2CH.sub.3
##STR226## H 113 --H ##STR227## OCH.sub.2CH.sub.3 ##STR228## H 114
--H ##STR229## OCH.sub.2CH.sub.3 ##STR230## H
115 --H ##STR231## OCH.sub.2CH.sub.3 ##STR232## H 116 --H
##STR233## OCH.sub.2CH.sub.3 ##STR234## H 117 --H ##STR235##
OCH.sub.2CH.sub.3 ##STR236## H 118 --H ##STR237## OCH.sub.2CH.sub.3
##STR238## H 119 --H ##STR239## OCH.sub.2CH.sub.3 ##STR240## H 120
--H ##STR241## OCH.sub.2CH.sub.3 ##STR242## H 121 --H ##STR243## OH
##STR244## H 122 --H ##STR245## OH ##STR246## H 123 --H ##STR247##
OH ##STR248## H 124 --H ##STR249## OH ##STR250## H 125 --H
##STR251## OH ##STR252## H 126 --H ##STR253## OH ##STR254## H 127
--H ##STR255## OH ##STR256## H 128 --H ##STR257## OH ##STR258##
##STR259## 129 --H ##STR260## OH OCH.sub.3 Br 130 --H ##STR261## OH
##STR262## H 131 --H --CH(CH.sub.3).sub.2 OH ##STR263## H 132 --H
--CH.sub.2CH.sub.2CH.sub.3 OH ##STR264## CH.sub.3SO.sub.2--NH-- 133
--H ##STR265## OH ##STR266## H 134 --H ##STR267## OH ##STR268## H
135 --H ##STR269## OH ##STR270## H 136 --H --CH.sub.2CH.sub.3 OH
##STR271## H 137 --H ##STR272## OH ##STR273## H 138 --H ##STR274##
OH ##STR275## H 139 --H ##STR276## OH ##STR277## H 140 --H
##STR278## OH ##STR279## H 141 --H ##STR280## OH ##STR281## H 142
--H ##STR282## OH ##STR283## H 143 --H --CH.sub.3 OH ##STR284## H
144 --H ##STR285## OH ##STR286## H 145 --H ##STR287## OH ##STR288##
H 146 --H ##STR289## OH ##STR290## H 147 --H ##STR291## OH
##STR292## H 148 --H ##STR293## OH ##STR294## H 149 --H ##STR295##
OH ##STR296## H 150 --H ##STR297## OH ##STR298## H 151 --H
##STR299## OH ##STR300## H 152 --H --CH.sub.2CH.sub.2CH.sub.3 OH
##STR301## H 153 --H --CH.sub.2CH.sub.2CH.sub.2CH.sub.3 OH
##STR302## H 154 --H ##STR303## OH ##STR304## H 155 --H ##STR305##
OH ##STR306## H 156 --H --CH.sub.2CH.sub.2CH.sub.3 OH ##STR307## H
157 --H --(CH.sub.2).sub.6CH.sub.3 OH ##STR308## H 158 --H
--CH.dbd.CH.sub.2 OH ##STR309## H 159 --H --CH.sub.2--C.ident.CH OH
##STR310## H 160 --H ##STR311## OH ##STR312## H 161 --H ##STR313##
OH ##STR314## H 162 --H ##STR315## OH ##STR316## H 163 --H
##STR317## OH ##STR318## H 164 --H ##STR319## OH ##STR320## H 165
--H ##STR321## OH ##STR322## H 166 --H ##STR323## OH ##STR324## H
167 --H ##STR325## OH ##STR326## H 168 --H ##STR327## OH ##STR328##
H 169 --H ##STR329## OH ##STR330## H 170 --H ##STR331## OH
##STR332## H 171 --H ##STR333## OH ##STR334## H 172 --H ##STR335##
OH ##STR336## H 173 --H ##STR337## OH ##STR338## H 174 --H
##STR339## OH ##STR340## H 175 --H ##STR341## OH ##STR342## H 176
--H ##STR343## OH ##STR344## H 177 --H ##STR345## --CH.sub.3
##STR346## H 178 --H ##STR347## --CH.sub.2CH.sub.3 ##STR348## H 179
--H ##STR349## --CH.sub.2CH.sub.3 ##STR350## H 180 --H ##STR351##
--CH.sub.2CH.sub.3 ##STR352## H 181 --H ##STR353##
--CH.sub.2CH.sub.2CH.sub.3 ##STR354## H 182 --H ##STR355##
--CH.sub.3 ##STR356## H 183 --H ##STR357## --CH(CH.sub.3).sub.2
##STR358## H 184 --H ##STR359## --CH.sub.3 ##STR360## H 185 --H
##STR361## --CH.sub.2(CH.sub.2).sub.3CH.sub.3 ##STR362## ##STR363##
186 --H ##STR364## --CH.sub.3 OCH.sub.3 Br 187 --H ##STR365##
--CH.sub.3 ##STR366## H 188 --H --CH(CH.sub.3).sub.2 --CH.sub.3
##STR367## H 189 --H --CH.sub.2CH.sub.2CH.sub.3 --CH.sub.3
##STR368## CH.sub.3SO.sub.2--NH-- 190 --H ##STR369## --CH.sub.3
##STR370## H 191 --H ##STR371## --CH.sub.3 ##STR372## H 192 --H
##STR373## --CH.sub.3 ##STR374## H 193 --H --CH.sub.2CH.sub.3
--CH.sub.3 ##STR375## H 194 --H ##STR376## --CH.sub.3 ##STR377## H
195 --H ##STR378## --CH.sub.3 ##STR379## H 196 --H ##STR380##
--CH.sub.3 ##STR381## H 197 --H ##STR382## --CH.sub.3 ##STR383## H
198 --H ##STR384## --CH.sub.3 ##STR385## H 199 --H ##STR386##
--CH.sub.3 ##STR387## H 200 --H --CH.sub.3 --CH.sub.3 ##STR388## H
201 --H ##STR389## --CH.sub.3 ##STR390## H 202 --H ##STR391##
--CH.sub.3 ##STR392## H 203 --H ##STR393## --CH.sub.3 ##STR394## H
204 --H ##STR395## --CH.sub.3 ##STR396## H 205 --H ##STR397##
--CH.sub.3 ##STR398## H 206 --H ##STR399## --CH.sub.3 ##STR400## H
207 --H ##STR401## --CH.sub.3 ##STR402## H 208 --H ##STR403##
--CH.sub.3 ##STR404## H 209 --H --CH.sub.2CH.sub.2CH.sub.3
--CH.sub.3 ##STR405## H 210 --H --CH.sub.2CH.sub.2CH.sub.2CH.sub.3
--CH.sub.3 ##STR406## H 211 --H ##STR407## --CH.sub.3 ##STR408## H
212 --H ##STR409## --CH.sub.3 ##STR410## H 213 --H
--CH.sub.2CH.sub.2CH.sub.3 --CH.sub.3 ##STR411## H 214 --H
--(CH.sub.2).sub.6CH.sub.3 --CH.sub.3 ##STR412## H 215 --H
--CH.dbd.CH.sub.2 --CH.sub.3 ##STR413## H 216 --H
--CH.sub.2--C.ident.CH --CH.sub.3 ##STR414## H 217 --H ##STR415##
--CH.sub.3 ##STR416## H 218 --H ##STR417## --CH.sub.3 ##STR418## H
219 --H ##STR419## --CH.sub.3 ##STR420## H 220 --H ##STR421##
--CH.sub.3 ##STR422## H 221 --H ##STR423## --CH.sub.3 ##STR424## H
222 --H ##STR425## --CH.sub.3 ##STR426## H 223 --H ##STR427##
--CH.sub.3 ##STR428## H 224 --H ##STR429## --CH.sub.3 ##STR430## H
225 --H ##STR431## --CH.sub.3 ##STR432## H 226 --H ##STR433##
--CH.sub.3 ##STR434## H 227 --H ##STR435## --CH.sub.3 ##STR436## H
228 --H ##STR437## --CH.sub.3 ##STR438## H 229 --H ##STR439##
--CH.sub.3 ##STR440## H 230 --H ##STR441## --CH.sub.3 ##STR442## H
231 --H ##STR443## --CH.sub.3 ##STR444## H 232 --H ##STR445##
--CH.sub.3 ##STR446## H 233 --H ##STR447## --CH.sub.3 ##STR448## H
234 --H ##STR449## --CH.dbd.CH.sub.2 ##STR450## H 235 --H
##STR451## --CH.dbd.CH.sub.2 ##STR452## H 236 --H ##STR453##
--CH.dbd.CH.sub.2 ##STR454## H 237 --H ##STR455## --CH.dbd.CH.sub.2
##STR456## H 238 --H ##STR457## --CH.dbd.CH.sub.2 ##STR458## H
239 --H ##STR459## --CH.dbd.CHCH.sub.3 ##STR460## H 240 --H
##STR461## --CH.dbd.CH.sub.2 ##STR462## H 241 --H ##STR463##
--CH.dbd.CH.sub.2 ##STR464## H 242 --H ##STR465## --CH.dbd.CH.sub.2
##STR466## ##STR467## 243 --H ##STR468## --CH.dbd.CH.sub.2
OCH.sub.3 Br 244 --H ##STR469## --CH.dbd.CH.sub.2 ##STR470## H 245
--H --CH(CH.sub.3).sub.2 --CH.dbd.CHCH.sub.2CH.sub.3 ##STR471## H
246 --H --CH.sub.2CH.sub.2CH.sub.3 --CH.dbd.CH.sub.2 ##STR472##
CH.sub.3SO.sub.2--NH-- 247 --H ##STR473## --CH.dbd.CH.sub.2
##STR474## H 248 --H ##STR475## --CH.dbd.CH.sub.2 ##STR476## H 249
--H ##STR477## --CH.dbd.CH.sub.2 ##STR478## H 250 --H
--CH.sub.2CH.sub.3 --CH.dbd.CH.sub.2 ##STR479## H 251 --H
##STR480## --CH.dbd.CH.sub.2 ##STR481## H 252 --H ##STR482##
--CH.sub.2CH.dbd.CH.sub.2 ##STR483## H 253 --H ##STR484##
--CH.dbd.CH.sub.2 ##STR485## H 254 --H ##STR486## --CH.dbd.CH.sub.2
##STR487## H 255 --H ##STR488## --CH.dbd.CH.sub.2 ##STR489## H 256
--H ##STR490## --CH.dbd.CH.sub.2 ##STR491## H 257 --H --CH.sub.3
--CH.sub.2CH.dbd.CH.sub.2 ##STR492## H 258 --H ##STR493##
--CH.dbd.CH.sub.2 ##STR494## H 259 --H ##STR495## --CH.dbd.CH.sub.2
##STR496## H 260 --H ##STR497## --CH.dbd.CH.sub.2 ##STR498## H 261
--H ##STR499## --CH.dbd.CH.sub.2 ##STR500## H 262 --H ##STR501##
--CH.dbd.CH.sub.2 ##STR502## H 263 --H ##STR503## --CH.dbd.CH.sub.2
##STR504## H 264 --H ##STR505## --CH.dbd.CH.sub.2 ##STR506## H 265
--H --CH.sub.2CH.sub.2CH.sub.3 --CH.dbd.CH.sub.2 ##STR507## H 266
--H --CH.sub.2CH.sub.2CH.sub.2CH.sub.3 --CH.dbd.CH.sub.2 ##STR508##
H 267 --H ##STR509## --CH.dbd.CH.sub.2 ##STR510## H 268 --H
##STR511## --CH.dbd.CH.sub.2 ##STR512## H 269 --H
--CH.sub.2CH.sub.2CH.sub.3 --CH.dbd.CH.sub.2 ##STR513## H 270 --H
--(CH.sub.2).sub.6CH.sub.3 --CH.dbd.CH.sub.2 ##STR514## H 271 --H
--CH.dbd.CH.sub.2 --CH.dbd.CH.sub.2 ##STR515## H 272 --H
--CH.sub.2--C.ident.CH --CH.dbd.CH.sub.2 ##STR516## H 273 --H
##STR517## --CH.dbd.CH.sub.2 ##STR518## H 274 --H ##STR519##
--CH.dbd.CH.sub.2 ##STR520## H 275 --H ##STR521## --CH.dbd.CH.sub.2
##STR522## H 276 --H ##STR523## --CH.dbd.CH.sub.2 ##STR524## H 277
--H ##STR525## --CH.dbd.CH.sub.2 ##STR526## H 278 --H ##STR527##
--CH.dbd.CH.sub.2 ##STR528## H 279 --H ##STR529## --CH.dbd.CH.sub.2
##STR530## H 280 --H ##STR531## --CH.dbd.CH.sub.2 ##STR532## H 281
--H ##STR533## --CH.dbd.CH.sub.2 ##STR534## H 282 --H ##STR535##
--CH.dbd.CH.sub.2 ##STR536## H 283 --H ##STR537## --CH.dbd.CH.sub.2
##STR538## H 284 --H ##STR539## --CH.dbd.CH.sub.2 ##STR540## H 285
--H ##STR541## --CH.dbd.CH.sub.2 ##STR542## H 286 --H ##STR543##
--CH.dbd.CH.sub.2 ##STR544## H 287 --H ##STR545## --CH.dbd.CH.sub.2
##STR546## H 288 --H ##STR547## --CH.dbd.CH.sub.2 ##STR548## H 289
--H ##STR549## --CH.sub.2C.ident.CH ##STR550## H 290 --H ##STR551##
--CH.sub.2C.ident.CH ##STR552## H 291 --H ##STR553##
--CH.sub.2C.ident.CH ##STR554## H 292 --H ##STR555##
--CH.sub.2C.ident.CH ##STR556## H 293 --H ##STR557##
--CH.sub.2C.ident.CH ##STR558## H 294 --H ##STR559##
--CH.sub.2C.ident.CH ##STR560## H 295 --H ##STR561##
--CH.sub.2C.ident.CH ##STR562## H 296 --H ##STR563##
--CH.sub.2C.ident.CH ##STR564## H 297 --H ##STR565##
--CH.sub.2C.ident.CH ##STR566## ##STR567## 298 --H ##STR568##
--CH.sub.2C.ident.CH OCH.sub.3 Br 299 --H ##STR569##
--CH.sub.2C.ident.CH ##STR570## H 300 --H --CH(CH.sub.3).sub.2
--CH.sub.2C.ident.CH ##STR571## H 301 --H
--CH.sub.2CH.sub.2CH.sub.3 --CH.sub.2C.ident.CH ##STR572##
CH.sub.3SO.sub.2--NH-- 302 --H ##STR573## --CH.sub.2C.ident.CH
##STR574## H 303 --H ##STR575## --CH.sub.2C.ident.CH ##STR576## H
304 --H ##STR577## --CH.sub.2C.ident.CH ##STR578## H 305 --H
--CH.sub.2CH.sub.3 --CH.sub.2C.ident.CH ##STR579## H 306 --H
##STR580## --CH.sub.2C.ident.CH ##STR581## H 307 --H ##STR582##
--CH.sub.2C.ident.CH ##STR583## H 308 --H ##STR584##
--CH.sub.2C.ident.CH ##STR585## H 309 --H ##STR586##
--CH.sub.2C.ident.CH ##STR587## H 310 --H ##STR588##
--CH.sub.2C.ident.CH ##STR589## H 311 --H ##STR590##
--CH.sub.2C.ident.CH ##STR591## H 312 --H --CH.sub.3
--CH.sub.2C.ident.CH ##STR592## H 313 --H ##STR593##
--CH.sub.2C.ident.CH ##STR594## H 314 --H ##STR595##
--CH.sub.2C.ident.CH ##STR596## H 315 --H ##STR597##
--CH.sub.2C.ident.CH ##STR598## H 316 --H ##STR599##
--CH.sub.2C.ident.CH ##STR600## H 317 --H ##STR601##
--CH.sub.2C.ident.CH ##STR602## H 318 --H ##STR603##
--CH.sub.2C.ident.CH ##STR604## H 319 --H ##STR605##
--CH.sub.2C.ident.CH ##STR606## H 320 --H
--CH.sub.2CH.sub.2CH.sub.3 --CH.sub.2C.ident.CH ##STR607## H 321
--H --CH.sub.2CH.sub.2CH.sub.2CH.sub.3 --CH.sub.2C.ident.CH
##STR608## H 322 --H ##STR609## --CH.sub.2C.ident.CH ##STR610## H
323 --H ##STR611## --CH.sub.2C.ident.CH ##STR612## H 324 --H
--CH.sub.2CH.sub.2CH.sub.3 --CH.sub.2C.ident.CH ##STR613## H 325
--H --(CH.sub.2).sub.6CH.sub.3 --CH.sub.2C.ident.CH ##STR614## H
326 --H --CH.dbd.CH.sub.2 --CH.sub.2C.ident.CH ##STR615## H 327 --H
--CH.sub.2--C.ident.CH --CH.sub.2C.ident.CH ##STR616## H 328 --H
##STR617## --CH.sub.2C.ident.CH ##STR618## H 329 --H ##STR619##
--CH.sub.2C.ident.CH ##STR620## H 330 --H ##STR621##
--CH.sub.2C.ident.CH ##STR622## H 331 --H ##STR623##
--CH.sub.2C.ident.CH ##STR624## H 332 --H ##STR625##
--CH.sub.2C.ident.CH ##STR626## H 333 --H ##STR627##
--CH.sub.2C.ident.CH ##STR628## H 334 --H ##STR629##
--CH.sub.2C.ident.CH ##STR630## H 335 --H ##STR631##
--CH.sub.2C.ident.CH ##STR632## H 336 --H ##STR633##
--CH.sub.2C.ident.CH ##STR634## H 337 --H ##STR635##
--CH.sub.2C.ident.CH ##STR636## H 338 --H ##STR637##
--CH.sub.2C.ident.CH ##STR638## H 339 --H ##STR639##
--CH.sub.2C.ident.CH ##STR640## H 340 --H ##STR641##
--CH.sub.2C.ident.CH ##STR642## H 341 --H ##STR643##
--CH.sub.2C.ident.CH ##STR644## H 342 --H ##STR645##
--CH.sub.2C.ident.CH ##STR646## H 343 --H ##STR647##
--CH.sub.2C.ident.CH ##STR648## H 344 --H ##STR649## OCH.sub.3
OCH.sub.3 H 345 --H ##STR650## OCH.sub.3 OCH.sub.3 H 346 --H
##STR651## OCH.sub.3 OCH.sub.3 H 347 --H ##STR652## OCH.sub.3
OCH.sub.3 H 348 --H ##STR653## OCH.sub.3 OCH.sub.3 H 349 --H
##STR654## OCH.sub.3 OCH.sub.3 H 350 --H ##STR655## OCH.sub.3
OCH.sub.3 H 351 --H ##STR656## OCH.sub.3 OCH.sub.3 H 352 --H
##STR657## OCH.sub.3 OCH.sub.3 ##STR658## 353 --H ##STR659##
OCH.sub.3 OCH.sub.3 Br 354 --H ##STR660## OCH.sub.3 OCH.sub.3 H 355
--H --CH(CH.sub.3).sub.2 OCH.sub.3 OCH.sub.3 H 356 --H
--CH.sub.2CH.sub.2CH.sub.3 OCH.sub.3 OCH.sub.3
--CH.sub.3SO.sub.2--NH-- 357 --H ##STR661## OCH.sub.3 OCH.sub.3 H
358 --H ##STR662## OCH.sub.3 OCH.sub.3 H 359 --H ##STR663##
OCH.sub.3 OCH.sub.3 H 360 --H --CH.sub.2CH.sub.3 OCH.sub.3
OCH.sub.3 H 361 --H ##STR664## OCH.sub.3 OCH.sub.3 H
362 --H ##STR665## OCH.sub.3 OCH.sub.3 H 363 --H ##STR666##
OCH.sub.3 OCH.sub.3 H 364 --H ##STR667## OCH.sub.3 OCH.sub.3 H 365
--H ##STR668## OCH.sub.3 OCH.sub.3 H 366 --H ##STR669## OCH.sub.3
OCH.sub.3 H 367 --H --CH.sub.3 OCH.sub.3 OCH.sub.3 H 368 --H
##STR670## OCH.sub.3 OCH.sub.3 H 369 --H ##STR671## OCH.sub.3
OCH.sub.3 H 370 --H ##STR672## OCH.sub.3 OCH.sub.3 H 371 --H
##STR673## OCH.sub.3 OCH.sub.3 H 372 --H ##STR674## OCH.sub.3
OCH.sub.3 H 373 --H ##STR675## OCH.sub.3 OCH.sub.3 H 374 --H
##STR676## OCH.sub.3 OCH.sub.3 H 375 --H --CH.sub.2CH.sub.2CH.sub.3
OCH.sub.3 OCH.sub.3 H 376 --H --CH.sub.2CH.sub.2CH.sub.2CH.sub.3
OCH.sub.3 OCH.sub.3 H 377 --H ##STR677## OCH.sub.3 OCH.sub.3 H 378
--H ##STR678## OCH.sub.3 OCH.sub.3 H 379 --H
--CH.sub.2CH.sub.2CH.sub.3 OCH.sub.3 OCH.sub.3 H 380 --H
--(CH.sub.2).sub.6CH.sub.3 OCH.sub.3 OCH.sub.3 H 381 --H
--CH.dbd.CH.sub.2 OCH.sub.3 OCH.sub.3 H 382 --H
--CH.sub.2--C.ident.CH OCH.sub.3 OCH.sub.3 H 383 --H ##STR679##
OCH.sub.3 OCH.sub.3 H 384 --H ##STR680## OCH.sub.3 OCH.sub.3 H 385
--H ##STR681## OCH.sub.3 OCH.sub.3 H 386 --H ##STR682## OCH.sub.3
OCH.sub.3 H 387 --H ##STR683## OCH.sub.3 OCH.sub.3 H 388 --H
##STR684## OCH.sub.3 OCH.sub.3 H 389 --H ##STR685## OCH.sub.3
OCH.sub.3 H 390 --H ##STR686## OCH.sub.3 OCH.sub.3 H 391 --H
##STR687## OCH.sub.3 OCH.sub.3 H 392 --H ##STR688## OCH.sub.3
OCH.sub.3 H 393 --H ##STR689## OCH.sub.3 OCH.sub.3 H 394 --H
##STR690## OCH.sub.3 OCH.sub.3 H 395 --H ##STR691## OCH.sub.3
OCH.sub.3 H 396 --H ##STR692## OCH.sub.3 OCH.sub.3 H 397 --H
##STR693## OCH.sub.3 OCH.sub.3 H 398 --H ##STR694## OCH.sub.3
OCH.sub.3 H 399 --H ##STR695## OCH.sub.3 OCH.sub.2CH.sub.3 H 400
--H ##STR696## OCH.sub.3 OCH.sub.2CH.sub.3 H 401 --H ##STR697##
OCH.sub.3 OCH.sub.2CH.sub.3 H 402 --H ##STR698## OCH.sub.3
OCH.sub.2CH.sub.3 H 403 --H ##STR699## OCH.sub.3 OCH.sub.2CH.sub.3
H 404 --H ##STR700## OCH.sub.3 OCH.sub.2CH.sub.3 H 405 --H
##STR701## OCH.sub.3 OCH.sub.2CH.sub.3 H 406 --H ##STR702##
OCH.sub.3 OCH.sub.2CH.sub.3 H 407 --H ##STR703## OCH.sub.3
OCH.sub.2CH.sub.3 ##STR704## 408 --H ##STR705## OCH.sub.3
OCH.sub.2CH.sub.3 Br 409 --H ##STR706## OCH.sub.3 OCH.sub.2CH.sub.3
H 410 --H --CH(CH.sub.3).sub.2 OCH.sub.3 OCH.sub.2CH.sub.3 H 411
--H --CH.sub.2CH.sub.2CH.sub.3 OCH.sub.3 OCH.sub.2CH.sub.3
CH.sub.3SO.sub.2--NH-- 412 --H ##STR707## OCH.sub.3
OCH.sub.2CH.sub.3 H 413 --H ##STR708## OCH.sub.3 OCH.sub.2CH.sub.3
H 414 --H ##STR709## OCH.sub.3 OCH.sub.2CH.sub.3 H 415 --H
--CH.sub.2CH.sub.3 OCH.sub.3 OCH.sub.2CH.sub.3 H 416 --H ##STR710##
OCH.sub.3 OCH.sub.2CH.sub.3 H 417 --H ##STR711## OCH.sub.3
OCH.sub.2CH.sub.3 H 418 --H ##STR712## OCH.sub.3 OCH.sub.2CH.sub.3
H 419 --H ##STR713## OCH.sub.3 OCH.sub.2CH.sub.3 H 420 --H
##STR714## OCH.sub.3 OCH.sub.2CH.sub.3 H 421 --H ##STR715##
OCH.sub.3 OCH.sub.2CH.sub.3 H 422 --H --CH.sub.3 OCH.sub.3
OCH.sub.2CH.sub.3 H 423 --H ##STR716## OCH.sub.3 OCH.sub.2CH.sub.3
H 424 --H ##STR717## OCH.sub.3 OCH.sub.2CH.sub.3 H 425 --H
##STR718## OCH.sub.3 OCH.sub.2CH.sub.3 H 426 --H ##STR719##
OCH.sub.3 OCH.sub.2CH.sub.3 H 427 --H ##STR720## OCH.sub.3
OCH.sub.2CH.sub.3 H 428 --H ##STR721## OCH.sub.3 OCH.sub.2CH.sub.3
H 429 --H ##STR722## OCH.sub.3 OCH.sub.2CH.sub.3 H 430 --H
--CH.sub.2CH.sub.2CH.sub.3 OCH.sub.3 OCH.sub.2CH.sub.3 H 431 --H
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3 OCH.sub.3 OCH.sub.2CH.sub.3 H
432 --H ##STR723## OCH.sub.3 OCH.sub.2CH.sub.3 H 433 --H ##STR724##
OCH.sub.3 OCH.sub.2CH.sub.3 H 434 --H --CH.sub.2CH.sub.2CH.sub.3
OCH.sub.3 OCH.sub.2CH.sub.3 H 435 --H --(CH.sub.2).sub.6CH.sub.3
OCH.sub.3 OCH.sub.2CH.sub.3 H 436 --H --CH.dbd.CH.sub.2 OCH.sub.3
OCH.sub.2CH.sub.3 H 437 --H --CH.sub.2--C.ident.CH OCH.sub.3
OCH.sub.2OH.sub.3 H 438 --H ##STR725## OCH.sub.3 OCH.sub.2CH.sub.3
H 439 --H ##STR726## OCH.sub.3 OCH.sub.2CH.sub.3 H 440 --H
##STR727## OCH.sub.3 OCH.sub.2CH.sub.3 H 441 --H ##STR728##
OCH.sub.3 OCH.sub.2CH.sub.3 H 442 --H ##STR729## OCH.sub.3
OCH.sub.2CH.sub.3 H 443 --H ##STR730## OCH.sub.3 OCH.sub.2CH.sub.3
H 444 --H ##STR731## OCH.sub.3 OCH.sub.2CH.sub.3 H 445 --H
##STR732## OCH.sub.3 OCH.sub.2CH.sub.3 H 446 --H ##STR733##
OCH.sub.3 OCH.sub.2CH.sub.3 H 447 --H ##STR734## OCH.sub.3
OCH.sub.2CH.sub.3 H 448 --H ##STR735## OCH.sub.3 OCH.sub.2CH.sub.3
H 449 --H ##STR736## OCH.sub.3 OCH.sub.2CH.sub.3 H 450 --H
##STR737## OCH.sub.3 OCH.sub.2CH.sub.3 H 451 --H ##STR738##
OCH.sub.3 OCH.sub.2CH.sub.3 H 452 --H ##STR739## OCH.sub.3
OCH.sub.2CH.sub.3 H 453 --H ##STR740## OCH.sub.3 OCH.sub.2CH.sub.3
H 454 --H ##STR741## OCH.sub.3 OH H 455 --H ##STR742## OCH.sub.3 OH
H 456 --H ##STR743## OCH.sub.3 OH H 457 --H ##STR744## OCH.sub.3 OH
H 458 --H ##STR745## OCH.sub.3 OH H 459 --H ##STR746## OCH.sub.3 OH
H 460 --H ##STR747## OCH.sub.3 OH H 461 --H ##STR748## OCH.sub.3 OH
H 462 --H ##STR749## OCH.sub.3 OH ##STR750## 463 --H ##STR751##
OCH.sub.3 OH Br 464 --H ##STR752## OCH.sub.3 OH H 465 --H
--CH(CH.sub.3).sub.2 OCH.sub.3 OH H 466 --H
--CH.sub.2CH.sub.2CH.sub.3 OCH.sub.3 OH CH.sub.3SO.sub.2--NH-- 467
--H ##STR753## OCH.sub.3 OH H 468 --H ##STR754## OCH.sub.3 OH H 469
--H ##STR755## OCH.sub.3 OH H 470 --H --CH.sub.2CH.sub.3 OCH.sub.3
OH H 471 --H ##STR756## OCH.sub.3 OH H 472 --H ##STR757## OCH.sub.3
OH H 473 --H ##STR758## OCH.sub.3 OH H 474 --H ##STR759## OCH.sub.3
OH H 475 --H ##STR760## OCH.sub.3 OH H 476 --H ##STR761## OCH.sub.3
OH H 477 --H ##STR762## OCH.sub.3 OH H 478 --H ##STR763## OCH.sub.3
OH H 479 --H --CH.sub.2CH.sub.3 OCH.sub.3 OH H 480 --H ##STR764##
OCH.sub.3 OH H 481 --H ##STR765## OCH.sub.3 OH H 482 --H ##STR766##
OCH.sub.3 OH H 483 --H ##STR767## OCH.sub.3 OH H 484 --H ##STR768##
OCH.sub.3 OH H 485 --H ##STR769## OCH.sub.3 OH H 486 --H --CH.sub.3
OCH.sub.3 OH H 487 --H ##STR770## OCH.sub.3 OH H 488 --H ##STR771##
OCH.sub.3 OH H 489 --H ##STR772## OCH.sub.3 OH H 490 --H ##STR773##
OCH.sub.3 OH H 491 --H ##STR774## OCH.sub.3 OH H
492 --H ##STR775## OCH.sub.3 OH H 493 --H ##STR776## OCH.sub.3 OH H
494 --H --CH.sub.2CH.sub.2CH.sub.3 OCH.sub.3 OH H 495 --H
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3 OCH.sub.3 OH H 496 --H
##STR777## OCH.sub.3 OH H 497 --H ##STR778## OCH.sub.3 OH H 498 --H
--CH.sub.2CH.sub.2CH.sub.3 OCH.sub.3 OH H 499 --H
--(CH.sub.2).sub.6CH.sub.3 OCH.sub.3 OH H 500 --H --CH.dbd.CH.sub.2
OCH.sub.3 OH H 501 --H --CH.sub.2--C.ident.CH OCH.sub.3 OH H 502
--H ##STR779## OCH.sub.3 OH H 503 --H ##STR780## OCH.sub.3 OH H 504
--H ##STR781## OCH.sub.3 OH H 505 --H ##STR782## OCH.sub.3 OH H 506
--H ##STR783## OCH.sub.3 OH H 507 --H ##STR784## OCH.sub.3 OH H 508
--H ##STR785## OCH.sub.3 OH H 509 --H ##STR786## OCH.sub.3 OH H 510
--H ##STR787## OCH.sub.3 OH H 511 --H ##STR788## OCH.sub.3 OH H 512
--H ##STR789## OCH.sub.3 OH H 513 --H ##STR790## OCH.sub.3 OH H 514
--H ##STR791## OCH.sub.3 OH H 515 --H ##STR792## OCH.sub.3 OH H 516
--H ##STR793## OCH.sub.3 OH H 517 --H ##STR794## OCH.sub.3 OH H 518
--H ##STR795## OCH.sub.3 OH CH.sub.3CH.sub.2SO.sub.2NH-- 519 --H
##STR796## OCH.sub.3 OH CH.sub.3(CH.sub.2).sub.2SO.sub.2NH-- 520
--H ##STR797## OCH.sub.3 OH CH.sub.3(CH.sub.2).sub.3SO.sub.2NH--
521 --H ##STR798## OCH.sub.3 OH (CH.sub.3).sub.3CSO.sub.2NH-- 522
--H ##STR799## OCH.sub.3 OH (CH.sub.3).sub.2CHSO.sub.2NH-- 523 --H
##STR800## OCH.sub.3 OH ##STR801## 524 --H ##STR802## OCH.sub.3
OCH.sub.3 CH.sub.3CH.sub.2SO.sub.2NH-- 525 --H ##STR803## OCH.sub.3
OCH.sub.3 CH.sub.3(CH.sub.2).sub.2SO.sub.2NH-- 526 --H ##STR804##
OCH.sub.3 OCH.sub.3 CH.sub.3(CH.sub.2).sub.3SO.sub.2NH-- 527 --H
##STR805## OCH.sub.3 OCH.sub.3 (CH.sub.3).sub.3CSO.sub.2NH-- 528
--H ##STR806## OCH.sub.3 OCH.sub.3 (CH.sub.3).sub.2CHSO.sub.2NH--
529 --H ##STR807## OCH.sub.3 OCH.sub.3 ##STR808## 530 --H
##STR809## OCH.sub.3 OCH.sub.2CH.sub.3 CH.sub.3CH.sub.2SO.sub.2NH--
531 --H ##STR810## OCH.sub.3 OCH.sub.2CH.sub.3
CH.sub.3(CH.sub.2).sub.2SO.sub.2NH-- 532 --H ##STR811## OCH.sub.3
OCH.sub.2CH.sub.3 CH.sub.3(CH.sub.2).sub.3SO.sub.2NH-- 533 --H
##STR812## OCH.sub.3 OCH.sub.2CH.sub.3
(CH.sub.3).sub.3CSO.sub.2NH-- 534 --H ##STR813## OCH.sub.3
OCH.sub.2CH.sub.3 (CH.sub.3).sub.2CHSO.sub.2NH-- 535 --H ##STR814##
OCH.sub.3 OCH.sub.2CH.sub.3 ##STR815## 536 --H ##STR816## OCH.sub.3
OH CH.sub.3CH.sub.2SO.sub.2NH-- 537 --H ##STR817## OCH.sub.3 OH
CH.sub.3(CH.sub.2).sub.2SO.sub.2NH-- 538 --H ##STR818## OCH.sub.3
OH CH.sub.3(CH.sub.2).sub.3SO.sub.2NH-- 539 --H ##STR819##
OCH.sub.3 OH (CH.sub.3).sub.3CSO.sub.2NH-- 540 --H ##STR820##
OCH.sub.3 OH (CH.sub.3).sub.2CHSO.sub.2NH-- 541 --H ##STR821##
OCH.sub.3 OH ##STR822## 542 --H --CH.sub.2CH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCH.sub.2CO.sub.2H 543
--H --CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCH.sub.2Ph
--OCH.sub.2CO.sub.2H 544 --H ##STR823## --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --OCH.sub.2CO.sub.2H 545 --H ##STR824##
--OCH.sub.2CH.sub.3 --OCH.sub.2Ph --OCH.sub.2CO.sub.2H 546 --H
##STR825## --OCH.sub.2CH.sub.3 --OCH.sub.2Ph --OCH.sub.2CO.sub.2H
547 --H ##STR826## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--OCH.sub.2CO.sub.2H 548 --H -n-Butyl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --OCH.sub.2CO.sub.2H 549 --H -isopropyl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCH.sub.2CO.sub.2H 550
--H -isobutyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--OCH.sub.2CO.sub.2H 551 --H -sec-butyl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --OCH.sub.2CO.sub.2H 552 --H
--CH.sub.2CH.sub.2Cl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--OCH.sub.2CO.sub.2H 553 --H --CH.sub.2CH.sub.2CH.sub.2Cl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCH.sub.2CO.sub.2H 554
--H --CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR827## 555 --H --CH.sub.2CH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --OCH.sub.2Ph ##STR828## 556 --H ##STR829##
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR830## 557 --H
##STR831## --OCH.sub.2CH.sub.3 --OCH.sub.2Ph ##STR832## 558 --H
##STR833## --OCH.sub.2CH.sub.3 --OCH.sub.2Ph ##STR834## 559 --H
##STR835## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR836## 560
--H -n-Butyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR837## 561
--H -isopropyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR838##
562 --H -isobutyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
##STR839## 563 --H -sec-butyl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR840## 564 --H --CH.sub.2CH.sub.2Cl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR841## 565 --H
--CH.sub.2CH.sub.2CH.sub.2Cl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR842## 566 --H --CH.sub.2CH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --OCH(CH.sub.3).sub.2 --NHSO.sub.2CH.sub.3 567
--H ##STR843## --OCH.sub.2CH.sub.3 --OCH(CH.sub.3).sub.2
--NHSO.sub.2CH.sub.3 568 --H ##STR844## --OCH.sub.2CH.sub.3
--OCH(CH.sub.3).sub.2 --NHSO.sub.2CH.sub.3 569 --H -n-Butyl
--OCH.sub.2CH.sub.3 --OCH(CH.sub.3).sub.2 --NHSO.sub.2CH.sub.3 570
--H -isopropyl --OCH.sub.2CH.sub.3 --OCH(CH.sub.3).sub.2
--NHSO.sub.2CH.sub.3 571 --H -isobutyl --OCH.sub.2CH.sub.3
--OCH(CH.sub.3).sub.2 --NHSO.sub.2CH.sub.3 572 --H -sec-butyl
--OCH.sub.2CH.sub.3 --OCH(CH.sub.3).sub.2 --NHSO.sub.2CH.sub.3 573
--H --CH.sub.2CH.sub.2Cl --OCH.sub.2CH.sub.3 --OCH(CH.sub.3).sub.2
--NHSO.sub.2CH.sub.3 574 --H --CH.sub.2CH.sub.2CH.sub.2Cl
--OCH.sub.2CH.sub.3 --OCH(CH.sub.3).sub.2 --NHSO.sub.2CH.sub.3 575
--H --CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--OCH(CH.sub.3)Et --NHSO.sub.2CH.sub.3 576 --H -isobutyl
--OCH.sub.2CH.sub.3 --OCH(CH.sub.3)Et --NHSO.sub.2CH.sub.3 577 --H
-sec-butyl --OCH.sub.2CH.sub.3 --OCH(CH.sub.3)Et
--NHSO.sub.2CH.sub.3 578 --H --CH.sub.2CH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR845## 579 --H
--CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCH.sub.2Ph
##STR846## 580 --H ##STR847## --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR848## 581 --H ##STR849##
--OCH.sub.2CH.sub.3 --OCH.sub.2Ph ##STR850## 582 --H ##STR851##
--OCH.sub.2CH.sub.3 --OCH.sub.2Ph ##STR852## 583 --H ##STR853##
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR854## 584 --H -n-Butyl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR855## 585 --H
-isobutyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR856## 586
--H -sec-butyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR857##
587 --H --CH.sub.2CH.sub.2Cl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR858## 588 --H --CH.sub.2CH.sub.2CH.sub.2Cl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR859## 589 --H
--CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NHSO.sub.2CH.sub.2CO.sub.2Et 590 --H --CH.sub.2CH.sub.2CH.sub.3
--OCH.sub.2OH.sub.3 --OCH.sub.2Ph --NHSO.sub.2CH.sub.2CO.sub.2Et
591 --H ##STR860## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NHSO.sub.2CH.sub.2CO.sub.2H 592 --H ##STR861##
--OCH.sub.2CH.sub.3 --OCH.sub.2Ph --NHSO.sub.2CH.sub.2CO.sub.2Et
593 --H ##STR862## --OCH.sub.2CH.sub.3 --OCH.sub.2Ph
--NHSO.sub.2CH.sub.2CO.sub.2H 594 --H ##STR863##
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NHSO.sub.2CH.sub.2CO.sub.2Et 595 --H -n-Butyl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --NHSO.sub.2CH.sub.2CO.sub.2Et 596 --H -propyl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NHSO.sub.2CH.sub.2CO.sub.2H 597 --H -isobutyl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --NHSO.sub.2CH.sub.2CO.sub.2Et 598 --H
-sec-butyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
NHSO.sub.2CH.sub.2CO.sub.2Et 599 --H --CH.sub.2CH.sub.2Cl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NHSO.sub.2CH.sub.2CO.sub.2Et 600 --H --CH.sub.2CH.sub.2CH.sub.2Cl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NHSO.sub.2CH.sub.2CO.sub.2Et 601 --H --CH.sub.2CH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --NCH.sub.3SO.sub.2CH.sub.3
602 --H --CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--OCH.sub.2Ph --NCH.sub.3SO.sub.2CH.sub.3 603 --H ##STR864##
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NCH.sub.3SO.sub.2CH.sub.3 604 --H ##STR865## --OCH.sub.2CH.sub.3
--OCH.sub.2Ph --NCH.sub.3SO.sub.2CH.sub.3 605 --H ##STR866##
--OCH.sub.2CH.sub.3 --OCH.sub.2Ph --NCH.sub.3SO.sub.2CH.sub.3 606
--H ##STR867## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NCH.sub.3SO.sub.2CH.sub.3 607 --H -n-Butyl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --NCH.sub.3SO.sub.2CH.sub.3 608 --H -isopropyl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --NCH.sub.3SO.sub.2CH.sub.3
609 --H -isobutyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NCH.sub.3SO.sub.2CH.sub.3 610 --H -sec-butyl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --NCH.sub.3SO.sub.2CH.sub.3 611 --H
--CH.sub.2CH.sub.2Cl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NCH.sub.3SO.sub.2CH.sub.3 612 --H --CH.sub.2CH.sub.2CH.sub.2Cl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --NCH.sub.3SO.sub.2CH.sub.3
613 --H --CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --NHSO.sub.2i-Pr 614 --H
--CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCH.sub.2Ph
--NHSO.sub.2i-Pr 615 --H ##STR868## --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --NHSO.sub.2i-Pr 616 --H ##STR869##
--OCH.sub.2CH.sub.3 --OCH.sub.2Ph --NHSO.sub.2i-Pr 617 --H
##STR870## --OCH.sub.2CH.sub.3 --OCH.sub.2Ph --NHSO.sub.2i-Pr 618
--H ##STR871## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NHSO.sub.2i-Pr 619 --H -n-Butyl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --NHSO.sub.2i-Pr 620 --H -isopropyl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --NHSO.sub.2i-Pr 621 --H
-isobutyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --NHSO.sub.2i-Pr
622 --H -sec-butyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NHSO.sub.2i-Pr 623 --H --CH.sub.2CH.sub.2Cl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --NHSO.sub.2i-Pr 624 --H
--CH.sub.2CH.sub.2CH.sub.2Cl --OCH.sub.2CH.sub.3 --OCH2CH.sub.3
--NHSO.sub.2i-Pr 625 --H --CH.sub.2CH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --NHSO.sub.2Ph 626 --H
--CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCH.sub.2Ph
--NHSO.sub.2Ph 627 --H ##STR872## --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --NHSO.sub.2Ph 628 --H ##STR873##
--OCH.sub.2CH.sub.3 --OCH.sub.2Ph --NHSO.sub.2Ph 629 --H ##STR874##
--OCH.sub.2CH.sub.3 --OCH.sub.2Ph --NHSO.sub.2Ph 630 --H ##STR875##
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --NHSO.sub.2Ph 631 --H
-n-Butyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --NHSO.sub.2Ph 632
--H -isopropyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NHSO.sub.2Ph 633 --H -isobutyl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --NHSO.sub.2Ph 634 --H -sec-butyl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --NHSO.sub.2Ph 635 --H
--CH.sub.2CH.sub.2Cl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NHSO.sub.2Ph 636 --H --CH.sub.2CH.sub.2CH.sub.2Cl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --NHSO.sub.2Ph 637 --H
--CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
##STR876## 638 --H --CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--OCH.sub.2Ph ##STR877## 639 --H ##STR878## --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR879## 640 --H ##STR880##
--OCH.sub.2CH.sub.3 --OCH.sub.2Ph ##STR881## 641 --H ##STR882##
--OCH.sub.2CH.sub.3 --OCH.sub.2Ph ##STR883## 642 --H ##STR884##
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR885## 643 --H -n-Butyl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR886## 644 --H
-isopropyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR887## 645
--H -isobutyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR888##
646 --H -sec-butyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
##STR889## 647 --H --CH.sub.2CH.sub.2Cl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR890## 648 --H --CH.sub.2CH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR891## 649 --H
--CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCH.sub.2Ph
##STR892## 650 --H ##STR893## --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR894## 651 --H ##STR895##
--OCH.sub.2CH.sub.3 --OCH.sub.2Ph ##STR896## 652 --H ##STR897##
--OCH.sub.2CH.sub.3 --OCH.sub.2Ph ##STR898## 653 --H ##STR899##
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR900## 654 --H -n-Butyl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR901## 655 --H
-isopropyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR902## 656
--H -isobutyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR903##
657 --H -sec-butyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
##STR904## 658 --H --CH.sub.2CH.sub.2Cl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR905## 659 --H --CH.sub.2CH.sub.2CH.sub.2Cl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR906## 660 --H
--CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--OCHMeCO.sub.2H 661 --H --CH.sub.2CH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --OCH.sub.2Ph --OCHMeCO.sub.2H 662 --H
##STR907## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCHMeCO.sub.2H
663 --H ##STR908## --OCH.sub.2CH.sub.3 --OCH.sub.2Ph
--OCHMeCO.sub.2H 664 --H ##STR909## --OCH.sub.2CH.sub.3
--OCH.sub.2Ph --OCHMeCO.sub.2H 665 --H ##STR910##
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCHMeCO.sub.2H 666 --H
-n-Butyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCHMeCO.sub.2H
667 --H -isopropyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--OCHMeCO.sub.2H 668 --H -isobutyl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --OCHMeCO.sub.2H 669 --H -sec-butyl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCHMeCO.sub.2H 670 --H
--CH.sub.2CH.sub.2Cl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--OCHMeCO.sub.2H 671 --H --CH.sub.2CH.sub.2CH.sub.2Cl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCHMeCO.sub.2H 672 --H
--CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NHSO.sub.2CH.sub.3 673 --H ##STR911## --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --NHSO.sub.2CH.sub.3 674 --H ##STR912##
--OCH.sub.2CH.sub.3 --OCH.sub.2Ph --NHSO.sub.2CH.sub.3 675 --H
##STR913## --OCH.sub.2CH.sub.3 --OCH.sub.2Ph --NHSO.sub.2CH.sub.3
676 --H ##STR914## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NHSO.sub.2CH.sub.3 677 --H -propyl --OCH.sub.2CH.sub.3 --OH
--NHSO.sub.2CH.sub.3 678 --H -isopropyl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --NHSO.sub.2CH.sub.3 679 --H -isobutyl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --NHSO.sub.2CH.sub.3 680
--H -sec-butyl --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--NHSO.sub.2CH.sub.3 681 --H --CH.sub.2CH.sub.2Cl
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --NHSO.sub.2CH.sub.3 682
--H --CH.sub.2CH.sub.2CH.sub.2Cl --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --NHSO.sub.2CH.sub.3 683 --OCH.sub.2CH.sub.3
--CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --H --H 684
--OCH.sub.2CH.sub.3 --CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --H --H
685 --OCH.sub.2CH.sub.3 --CH.sub.2CH.sub.2CH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --H --H 686 --OCH.sub.2CH.sub.3 ##STR915##
--OCH.sub.2CH.sub.3 --H --H 687 --OCH.sub.2CH.sub.3 ##STR916##
--OCH.sub.2CH.sub.3 --H --H 688 --OCH(CH.sub.3).sub.2
--CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --H --H 689
--OCH(CH.sub.3).sub.2 --CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --H
--H 690 --OCH(CH.sub.3).sub.2 --CH.sub.2CH.sub.2CH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 --H --H 691 --OCH(CH.sub.3).sub.2 ##STR917##
--OCH.sub.2CH.sub.3 --H --H 692 --OCH(CH.sub.3).sub.2 ##STR918##
--OCH.sub.2CH.sub.3 --H --H 693 ##STR919## ##STR920##
--OCH.sub.2CH.sub.3 --H --H 694 ##STR921##
--CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --H --H 695 --H
##STR922## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR923## 696
--H ##STR924## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR925##
697 --H ##STR926## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
##STR927## 698 --H ##STR928## --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR929## 699 --H ##STR930##
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR931## 700 --H
--CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
##STR932## 701 --H --CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR933## 702 --H
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR934## 703 --H ##STR935##
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR936## 704 --H
--CH.sub.2CH.sub.2CO.sub.2H --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
##STR937## 705 --H --CH.sub.2CH.sub.2CONH.sub.2 --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR938## 706 --H --CH.sub.2CH.sub.2CH.sub.2OH
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR939## 707 --H
##STR940## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR941## 708
--H ##STR942## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR943##
709 --H ##STR944## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
##STR945## 710 --H ##STR946## --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR947## 711 --H ##STR948##
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR949## 712 --H
##STR950## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR951## 713
--H ##STR952## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR953##
714 --H ##STR954## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
##STR955## 715 --H ##STR956## --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR957##
716 --H ##STR958## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
##STR959## 717 --H ##STR960## --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR961## 718 --H ##STR962##
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR963## 719 --H
##STR964## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR965## 720
--H ##STR966## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR967##
721 --H ##STR968## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
##STR969## 722 --H ##STR970## --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR971## 723 --H ##STR972##
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR973## 724 --H
##STR974## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR975## 725
--H ##STR976## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR977##
726 --H ##STR978## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
##STR979## 727 --H ##STR980## --OCH.sub.2CH.sub.3
--OCH.sub.2CH.sub.3 ##STR981## 728 --H ##STR982##
--OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR983## 729 --H
##STR984## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3 ##STR985## 730
--H ##STR986## --OCH.sub.2CH.sub.3 --OCH.sub.2CH.sub.3
##STR987##
[0130] The compounds of the invention contain one or more
asymmetric carbon atoms. Accordingly, the compounds may exist as
diastereomers, enantiomers or mixtures thereof. The syntheses
described above may employ racemates, diastereomers or enantiomers
as starting materials or as intermediates. Diastereomeric compounds
may be separated by chromatographic or crystallization methods.
Similarly, enantiomeric mixtures may be separated using the same
techniques or others known in the art. Each of the asymmetric
carbon atoms may be in the R or S configuration and both of these
configurations are within the scope of the invention.
UTILITY
[0131] It has been discovered that the compounds of the invention
when made and selected as disclosed herein are inhibitors of serine
protease enzymes, for example, factor VIIa, TF/factor VIIa, factor
Xa, kallikrein and/or thrombin. These compounds are capable of
inhibiting the catalytic activity of these enzymes and as such
function to inhibit the coagulation cascade and prevent or limit
coagulation and/or the formation of thrombi or emboli in blood
vessels and/or increase the time of coagulation of blood. The
compounds of the present invention, therefore, inhibit the ability
of TF/factor VIIa to convert factor X to factor Xa, inhibit the
ability of factor Xa to convert prothrombin to thrombin (factor
IIa); and/or the ability of thrombin to convert fibrinogen to
fibrin monomers.
[0132] The selectivity of the compounds of the invention as
inhibitors of these enzymes can be determined using Ki values as
described in the examples below. Representative selectivities are
shown in the tables below. TABLE-US-00003 ##STR988## Ki(TFVIIa) R1
X R9 R10 Z2 R11 uM Ph C.dbd.O OEt OiPr H H 0.003 Pr CH2 OEt OCH2Ph
H NHSO2Me 0.004 Ph C.dbd.O OEt OEt H H 0.005 Ph C.dbd.O OEt H OEt H
0.007 Ki(IIa) R1 X R9 R10 Z2 R11 uM Ph CH2 OMe OCH(CH2Cl)Ph H H
0.001 2-thiophene CH2 OMe OCH2Ph H H 0.016 Ph C.dbd.O OEt OiPr H H
0.113 Pr CH2 OMe OCH(CH2Cl)Ph H H 0.001 Ki(Kallikrein) R1 X R9 R10
Z2 R11 uM Pr CH2 OEt OiBu H NHSO2Pr 0.001 Pr CH2 OEt OiPr H NHSO2Me
0.001 Et C.dbd.O OEt OiPr H H 0.011 Ph C.dbd.O OEt OEt H H 0.002
Ki(Xa) R1 X R9 R10 Z2 R11 uM Et C.dbd.O OEt OiPr H H 0.565 Bu
C.dbd.O OEt OiPr H H 0.624 Ph C.dbd.O OEt OiPr H H 0.898 Pr CH2 OMe
OCH(CH2Cl)Ph H H 0.140
[0133] The anti-coagulant activity of the compounds of the
invention can be tested using assays. Prothrombin time (PT) and
activated partial thromboplastin time (APTT) clotting time assays
can be performed in pooled normal plasmas (human or various animal
species) following addition of increasing concentrations of
inhibitors to the plasma. Clotting times are determined using an
ACL 300 Automated Coagulation Analyzer (Coulter Corp., Miami, Fla.)
and commercially available reagents as follows.
[0134] PT assay: Aqueous solutions of inhibitor at various
concentrations are added to pooled normal plasma in a ratio of 1
part inhibitor to 9 parts plasma. These mixtures are then added to
the analyzer's sample cups. Innovin.RTM. (Dade International Inc.,
Miami, Fla.), a mixture of human relipidated tissue factor and
Ca.sup.++ ions is added to the reagent cup. Precise volumes of
sample and Innovin.RTM. are automatically transferred to cells of
an acrylic rotor that is pre-equilibrated to 37 C. Following a 2
minute incubation period, coagulation is initiated when the two
components are mixed together by centrifugation. Coagulation is
monitored optically and clotting time is reported in seconds. In
agreement with Janson et al. (Janson, T. L., et al., 1984,
Haemostasis 14: 440-444) relipidated human tissue factor is a
potent initiator of coagulation in all species tested. In this
system, the clotting time of control plasmas (plasma plus inhibitor
diluent) is typically 8 to 10 seconds. A curve is fit to the
clotting time versus inhibitor concentration data and the
concentration at which the PT is doubled compared to control plasma
is determined for each inhibitor.
[0135] APTT assay: Inhibitor and plasma are mixed together and
transferred to the ACL 300 sample cups as described above. Actin
FS.RTM. and CaCl.sub.2 (Dade International Inc., Miami, Fla.), are
added to reagent cups 1 and 2 respectively. Precise volumes of
sample and activator (Actin FS.RTM.) are automatically transferred
to cells of a pre-equilibrated rotor (37 C) and mixed by
centrifugation. Following a 2 minute activation period, coagulation
is initiated by the addition of CaCl.sub.2. Coagulation is
monitored and data calculated as described in the PT method. APTT
of plasma controls is typically 12 to 32 seconds, depending on the
species of plasma used in the assay.
[0136] Representative PT and APTT assay results are shown in Table
3 below. TABLE-US-00004 TABLE 3 Compound No. 2 .times. PT (.mu.M) 2
.times. APTT (.mu.M) 7 14 8 13 16 57 33 5.5 11 72 30 60 589 22 40
596 8 140 628 125 90 672 34 78
[0137] The compounds of the invention are useful as diagnostic
reagents in vitro for inhibiting clotting in blood drawing tubes.
The use of stoppered test tubes having a vacuum therein as a means
to draw blood is well know. Kasten, B. L., "Specimen Collection",
Laboratory Test Handbook, 2nd Ed., Lexi-Comp Inc., Cleveland, PP
16-17, Eds. Jacobs, D. S. et al, 1990. Such vacuum tubes may be
free of clot-inhibiting additives, in which case, they are useful
for the isolation of mammalian serum from the blood. They may also
contains clot-inhibiting additives, such as heparin salts, citrate
salts or oxalate salts, in which case they are useful for the
isolation of mammalian plasma from the blood. The compounds of the
invention may be incorporation into blood collection tubes and
function to inhibit TF/factor VIIa, factor Xa, thrombin and/or
kallikrein and to prevent clotting of the mammalian blood drawn
into the tubes.
[0138] When used in blood collection tubes, the compounds of the
invention may be used alone, as mixtures or in combination with
other clotting inhibiting compounds known in this art. The amount
of the compound of the invention should be an amount sufficient to
prevent or inhibit the formation of a clot when blood is drawn into
the tube. These compounds may be introduced into the tubes in the
same manner as known clot-inhibiting compounds such as heparin
salts. Liquids are usually lyophilized using known methods.
Typically, the tubes will contain about 2 to about 10 ml of
mammalian blood and the compounds are added in an amount sufficient
to prevent coagulation of this amount of blood. A suitable
concentration is about 10-1000 nM.
[0139] These compounds also inhibit the formation of emboli and
thrombi in the circulatory system in mammals and therefor are
useful in vivo. Thromboembolic disorders have been shown to be
directly related to the susceptibility of the mammal to formation
of emboli and thrombi. For example, the formation of a thrombus in
a veinous vessel results in thrombophlebitis, which is typically
treated with rest and the administration of anticoagulants. Other
conditions which can be treated with the anticoagulant compounds of
the invention include, thrombolymphangitis, thrombosinusitis,
thromboendocarditis, thromboangiitis, and thromboarteritis.
[0140] Mammals exposed to medical procedures such as angioplasty
and thrombolytic therapy are particularly susceptible to thrombus
formation. The compounds of the present invention can be used to
inhibit thrombus formation following angioplasty. They may also be
used in combination with antithrombolytic agents such as tissue
plasminogen activator and its derivatives (U.S. Pat. Nos.
4,752,603; 4,766,075; 4,777,043; EP 199 574; EP 238 304; EP 228
862; EP 297 860; PCT WO89/04368; PCT WO89/00197), streptokinase and
its derivatives, or urokinase and its derivatives to prevent
arterial reocclusion following thrombolytic therapy. When used in
combination with the above thrombolytic agents, the compounds of
the present invention may be administered prior to, simultaneously
with, or subsequent to the antithrombolytic agent.
[0141] Mammals exposed to renal dialysis, blood oxygenation,
cardiac catheterization and similar medical procedures as well as
mammals fitted with certain prosthetic devices are also susceptible
to thromboembolic disorders. Physiologic conditions, with or
without known cause may also lead to thromboembolic disorders.
[0142] Thus, the compounds described herein may be useful in
treating thromboembolic disorders in mammals. The compounds
described herein may also be used as adjuncts to anticoagulant
therapy, for example in combination with aspirin, heparin or
warfarin and other anticoagulant agents. The various coagulation
disorders described above are treated with the compounds of the
invention in such a fashion as to prevent bleeding as a result of
the disorder. The application of the compounds described herein for
these and related disorders will be apparent to those skilled in
the art.
[0143] Compounds of this invention are also useful as intermediates
generally, or as precursors of coagulation serine protease
inhibitors and thus in addition to treating cardiovascular disease,
these compounds may be usefully employed in metastatic disease, or
for any disease where inhibition of coagulation is indicated.
[0144] Typically, the inhibitors used in the method of this
invention is formulated by mixing it at ambient temperature at the
appropriate pH, and at the desired degree of purity, with
physiologically acceptable carriers, i.e., carriers that are
non-toxic to recipients at the dosages and concentrations employed.
The pH of the formulation depends mainly on the particular use and
the concentration of compound, but preferably ranges anywhere from
about 3 to about 8. Formulation in an acetate buffer at pH 5 is a
suitable embodiment.
[0145] The inhibitory compound for use herein is preferably
sterile. The compound ordinarily will be stored as a solid
composition, although lyophilized formulations or aqueous solutions
are acceptable.
[0146] The composition of the invention will be formulated, dosed,
and administered in a fashion consistent with good medical
practice. Factors for consideration in this context include the
particular disorder being treated, the particular mammal being
treated, the clinical condition of the individual patient, the
cause of the disorder, the site of delivery of the agent, the
method of administration, the scheduling of administration, and
other factors known to medical practitioners. The "therapeutically
effective amount" of the compound to be administered will be
governed by such considerations, and is the minimum amount
necessary to prevent, ameliorate, or treat the coagulation factor
mediated disorder. Such amount is preferably below the amount that
is toxic to the host or renders the host significantly more
susceptible to bleeding.
[0147] As a general proposition, the initial pharmaceutically
effective amount of the inhibitor administered parenterally per
dose will be in the range of about 0.01-100 mg/kg, preferably about
0.1 to 20 mg/kg of patient body weight per day, with the typical
initial range of compound used being 0.3 to 15 mg/kg/day.
[0148] The compound of the invention is administered by any
suitable means, including oral, topical, transdermal, parenteral,
subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and,
if desired for local immunosuppressive treatment, intralesional
administration (including perfusing or otherwise contacting the
graft with the inhibitor before transplantation). Parenteral
infusions include intramuscular, intravenous, intraarterial,
intraperitoneal, or subcutaneous administration.
[0149] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention. All patent and literature
citations are herein incorporated by reference in their
entirety.
EXAMPLES
[0150] The compounds of the invention can be prepared generally by
the reaction scheme shown below. Compounds other than the specific
product shown are prepared as described above using corresponding
starting materials. For example, additional compounds can be
prepared by using different starting styrene compounds, which are
readily prepared from commercially available starting compounds and
standard reactions which are well known in this art. ##STR989##
Example 1
[0151] ##STR990##
[0152] 4-benzyloxy-3-methoxy-styrene (10 g, 42 mmoles) was
dissolved in dichloromethane (400 ml). Solid potassium bicarbonate
(11 g, 110 mmoles) was added and the reaction cooled to zero
degrees Celsius. Meta-chloroperbenzoic acid (12 g, ca. 42 mmoles)
was added and the reaction allowed to warm to room temperature and
stirred for 16 hours. The reaction was monitored by thin-layer
chromatography. An additional amount of meta-chloroperbenzoic acid
(4 g) was added and the reaction stirred for an additional 4 hours
to completely consume starting material. The reaction was poured
into a separatory funnel and washed first with water, then with
sodium bicarbonate and finally with NaOH. The organic layer was
separated and dried over anhydrous sodium sulfate. The solution was
filtered and the solvent removed in vacuo to yield approximately 11
g of crude product.
[0153] The crude product was then dissolved in acetonitrile (60 ml)
and lithium perchlorate (8.5 g, 80 mmoles) added. The suspension
was stirred for five minutes at which time the reaction became
homogeneous. 4-amino-benzonitrile (9.5 g, 80 mmoles) was added and
the reaction heated to 60 degrees C. for 12 hours. Thin layer
chromatography showed the presence of a new product at lower Rf.
The solvent was removed in vacuo and the residue taken up in ethyl
acetate, washed with water an dried over anhydrous sodium sulfate.
The crude product was then submitted to flash chromatography
(hexanes:ethyl acetate 1:1) to yield 6 grams of
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-hydroxy-ethylamino]-benzonitrile
A. .sup.1HNMR(CDCl.sub.3): 7.3-7.45, (m, 7H), 6.8 (m, 3 H), 6.5 (d,
2H), 5.18 (s, 2H), 4.42 (m, 1H), 3.95 (dd, 1H), 3.85 (s, 3H), 3.8
(dd, 1H). ##STR991##
[0154]
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-hydroxy-ethylamino]-benzonit-
rile A (470 mg, 1.25 mmoles), phthalimide (1.47 g, 10 mmoles), and
triphenylphosphine (787 mg, 3 mmoles) were added to 40 ml of
tetrahydrofuran. The mixture was stirred for 10 minutes and then
cooled to zero degrees Celsius. Diisopropylazodiacarboxylate (DIAD,
0.6 ml, 3 mmoles) was then added slowly. The reaction was allowed
to stir 1 hour. TLC indicated new product. The solvent was removed
in vacuo and the residue taken up in 50 ml of ethyl acetate. The
solution was washed three times with 2N sodium hydroxide and twice
with water. The organic layer was separated, dried over anhydrous
sodium sulfate and filtered. The solvent was removed in vacuo and
the residue submitted to flash chromatography (hexanes:ethyl
acetate, 1:1) to yield 478 mg of product
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(1,3-dioxo-1,3-dihydro-isoindol-2-y-
l)-ethylamino]-benzonitrile B (76% yield). .sup.1HNMR(CDCl.sub.3):
7.85 (m, 2H), 7.75 (m, 2H), 7.23-7.45 (m, 9H), 6.9 (m, 3H), 6.42
(d, 2H), 5.45 (d, 1H), 5.15 (s, 2H), 4.62 (m, 1H), 4.0 (m, 2H),
3.83 (s, 3H). ##STR992##
[0155]
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(1,3-dioxo-1,3-dihydro-isoin-
dol-2-yl)-ethylamino]-benzonitrile B was then dissolved in ethanol
(60 ml) and hydrazine hydrate (2 g) was added. The solution was
heated to 60-70 degrees C. for 1.5 hours. TLC showed reaction was
complete. The suspension was filtered to remove by-product and the
ethanol removed in vacuo. The residue was submitted to flash
chromatography on silica gel (ethyl acetate: 2N NH3 in methanol,
9:1) to yield 372 mg of 4-[2-Amino-1-(4-benzyloxy
-3-methoxy-phenyl)-ethylamino]-benzonitrile D (100%).
.sup.1HNMR(CDCl.sub.3): 7.3-7.45 (m, 7H), 6.32 (m, 3H), 6.5 (d,
2H), 5.52 (d, 1H), 4.3 (q, 1H), 3.83 (s, 3H), 3.08 (m, 2H), 1.95
(s, 2H). ##STR993##
[0156]
4-[2-Amino-1-(4-benzyloxy-3-methoxy-phenyl)-ethylamino]-benzonitri-
le D (300 mg, 0.8 mmoles) was dissolved in ethanol (3 ml) and
hydroxylamine-hydrochloride (350 mg, 5 mmoles) and triethylamine (1
ml, 5.7 mmoles) were added. The reaction was heated to 65-70
degrees C. for 2 hours. The residue was taken up in ethyl acetate
and water. The organic layer was separated, dried over anhydrous
sodium sulfate and filtered. The solvent was removed in vacuo and
replaced with 4 ml methanol with 0.5 ml acetic acid. Raney nickel
(ca. 300 ul suspension in sodium hydroxide, Aldrich) was added and
the reaction placed under a hydrogen atmosphere. The reaction was
stirred vigorously for 3 hours, the catalyst filtered off and the
solvent removed in vacuo. The crude product was purified by flash
chromatography on silica gel (ethyl
acetate:acetone:methanol:ammonia, 2:1:1:0.05) to yield 160 mgs of
4-[2-Amino-1-(4-benzyloxy-3-methoxy-phenyl)-ethylamino]-benzamidine
E. MS (M+H)=391. ##STR994##
[0157]
4-[2-Amino-1-(4-benzyloxy-3-methoxy-phenyl)-ethylamino]-benzamidin-
e E (20 mg, 0.03 mmoles) was dissolved in acetonitrile (2 ml)
containing triethylamine (17 ul, 0.12 mmoles) and water (0.3 ml).
To this was added the desired sulfonyl chloride having a formula
ClSO.sub.2R (0.03 mmoles) and the reaction stirred for 4 hours. The
solvent was removed in vacuo and the compounds purified by
reverse-phase preparative HPLC (gradient acetonitrile/water with
0.1% trifluoroacetic acid) to yield the final product upon
lyophilization.
Examples 2a-2dd
[0158] Using an analogous procedure, other compounds of the
invention were prepared, including: [0159] a)
4-[Benzenesulfonylamino-1-(4-benzyloxy-3-methoxy-phenyl)-ethylamino]-benz-
amidine: MS (M+H)=531, [0160] b)
N-{4-[2-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-carbamimidoyl-phenylamino)-et-
hylsulfamoyl]-phenyl}-acetamide: MS (M+H)=588, [0161] c)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-nitro-benzenesulfonylamino)-ethy-
lamino]-benzamidine: MS (M+H)=576, [0162] d)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-fluoro-benzenesulfonylamino)-eth-
ylamino]-benzamidine: MS (M+H)=549, [0163] e)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-fluoro-benzenesulfonylamino)-eth-
ylamino]-benzamidine: MS (M+H)=565, [0164] f)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(3-nitro-benzenesulfonylamino)-ethy-
lamino]-benzamidine: MS (M+H)=576, [0165] g)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(2,5-dichloro-benzenesulfonylamino)-
-ethylamino]-benzamidine: MS (M+H)=599, [0166] h)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(2-bromo-benzenesulfonylamino)-ethy-
lamino]-benzamidine: MS (M+H)=609, 611, [0167] i)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-bromo-benzenesulfonylamino)-ethy-
lamino]-benzamidine: MS (M+H)=609, [0168] j)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-isopropyl-benzenesulfonylamino)--
ethylamino]-benzamidine: MS (M+H)=573, [0169] k)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-phenylmethanesulfonylamino-ethylami-
no]-benzamidine: MS (M+H)=545, [0170] l)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-carboxy-benzenesulfonylamino)-et-
hylamino]-benzamidine: MS (M+H)=575, [0171] m)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(3-carboxy-benzenesulfonylamino)-et-
hylamino]-benzamidine: MS (M+H)=575, [0172] n)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(2,4-dinitro-benzenesulfonylamino)--
ethylamino]-benzamidine: MS (M+H)=621, [0173] o)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(2,3,5,6-tetramethyl-benzenesulfony-
lamino)-ethylamino]-benzamidine: MS (M+H)=587, [0174] p)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(3,5-dichloro-2-hydroxy-benzenesulf-
onylamino)-ethylamino]-benzamidine: MS (M+H)=615, [0175] q)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(3,4-dimethoxy-benzenesulfonylamino-
)-ethylamino]-benzamidine: MS (M+H)=591, [0176] r)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(thiophene-2-sulfonylamino)-ethylam-
ino]-benzamidine: MS(M+H)=537, [0177] s)
N-{5-[2-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-carbamimidoyl-phenylamino)-et-
hylsulfamoyl]-4-methyl-thiazol-2-yl}-acetamide: MS(M+H)=595, [0178]
t)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(naphthalene-2-sulfonylamino)-ethyl-
amino]-benzamidine: MS (M+H)=581, [0179] u)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(naphthalene-1-sulfonylamino)-ethyl-
amino]-benzamidine: MS (M+H)=581, [0180] v)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(2-phenyl-ethenesulfonylamino)-ethy-
lamino]-benzamidine: MS (M+H)=557, [0181] w)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(3-trifluoromethyl-benzenesulfonyla-
mino)-ethylamino]-benzamidine: MS (M+H)=599, [0182] x)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(2,3,4,5,6-pentafluoro-benzenesulfo-
nylamino)-ethylamino]-benzamidine: MS (M+H)=521, [0183] y)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-methanesulfonylamino-ethylamino]-be-
nzamidine: MS(M+H)=469, [0184] z)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-ethanesulfonylamino-ethylamino]-ben-
zamidine: MS(M+H)=483, [0185] aa)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-propanesulfonylamino-ethylamino]-be-
nzamidine: MS(M+H)=497, [0186] bb)
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-butanesulfonylamino-ethylamino]-ben-
zamidine: MS(M+H)=511, [0187] cc)
[2-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-carbamimidoyl-phenylamino)-ethylsu-
lfamoyl]-acetic acid ethyl ester MS (M+H)=541, [0188] dd)
[2-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-carbamimidoyl-phenylamino)-ethylsu-
lfamoyl]-acetic acid.
Example 3
[0189] ##STR995##
[0190]
4-[1-(4-Benzyloxy-3-methoxy-phenyl)-2-(1,3-dioxo-1,3-dihydro-isoin-
dol-2-yl)-ethylamino]-benzonitrile B (1.8 g) was dissolved in a
mixture of ethanol (50 ml), acetic acid (3 ml), methanol (5 ml),
and ethyl acetate (5 ml). This solution was added to a Parr flask
containing 10% Pd/C (500 mg) and hydrogenated at 35 psi for 16
hours. The catalyst was removed by filtration through Celite and
the solvent removed in vacuo to provide 1 g of the product
4-[2-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-1-(4-hydroxy-3-methoxy-phenyl)-
-ethylamino]-benzonitrile (68%). ##STR996##
[0191]
4-[2-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-1-(4-hydroxy-3-methoxy--
phenyl)-ethylamino]-benzonitrile (1 g, 2.43 mmoles) was dissolved
in tetrahydrofuran (30 ml) and triphenylphosphine (1.27 g, 4.84
mmoles), (S)-2-chloro-1-phenyl-ethanol (1.13 g, 7.26 mmoles) added.
The reaction was cooled to 0 .sup.oC and diethylazodicarboxylate
(0.842 g, 4.8 mmoles) added. The reaction temperature was allowed
to come to room temperature and the reaction stirred for 2.5 hours.
The solvent was removed in vacuo and the residue taken up in ethyl
acetate, washed with 0.5 N sodium hydroxide several times, washed
once with brine and dried over anhydrous sodium sulfate. The crude
product was purified by flash chromatography on silica gel (30%
ethyl acetate in hexanes) to yield 1.1 g of
4-[1-[4-(2-chloro-1-phenyl-ethoxy)-3-methoxy-phenyl]-2-(1,3-dioxo-1,3-dih-
ydro-isoindol-2-yl)-ethylamino]-benzonitrile, (82%). ##STR997##
[0192]
4-[1-[4-(2-Chloro-1-phenyl-ethoxy)-3-methoxy-phenyl]-2-(1,3-dioxo--
1,3-dihydro-isoindol-2-yl)-ethylamino]-benzonitrile (0.5 g) was
dissolved in ethanol (40 ml) and hydrazine hydrate (0.14 g) added.
The reaction was heated at 65 .sup.oC for 2 hours. The solvent was
removed and replaced with ethyl acetate. The solution was washed
twice with water and once with brine. The solution was dried over
anhydrous sodium sulfate and the solvent removed in vacuo to yield
318 mg of desired amine
4-{2-Amino-1-[4-(2-chloro-1-phenyl-ethoxy)-3-methoxy-phenyl]-ethylamino}--
benzonitrile, (83%). ##STR998##
[0193]
4-{2-Amino-1-[4-(2-chloro-1-phenyl-ethoxy)-3-methoxy-phenyl]-ethyl-
amino}-benzonitrile, (0.1 g, 0.237 mmoles) was dissolved in
dichloromethane (6 ml) and triethylamine (34 ul, 0.3 mmoles) added.
Phenyl sulfonylchloride (46 ul, 0.26 mmoles) was added and the
reaction stirred for 90 minutes. The reaction was diluted with
dichloromethane and washed once with sat. sodium bicarbonate and
once with water. The solution was dried over sodium sulfate and the
solvent removed in vacuo. The product was purified by silica gel to
yield 100 mg of desired product
N-[2-[4-(2-chloro-1-phenyl-ethoxy)-3-methoxy-phenyl]-2-(4-cyano-phenylami-
no)-ethyl]-benzenesulfonamide. ##STR999##
[0194]
N-[2-[4-(2-chloro-1-phenyl-ethoxy)-3-methoxy-phenyl]-2-(4-cyano-ph-
enylamino)-ethyl]-benzenesulfonamide (100 mg 0.18 mmoles) was
dissolved in ethanol (3 ml) and hydroxylamine-hydrochloride (62 mg,
0.89 mmoles) added. To this was added triethylamine (90 mg, 0.89
mmoles) and later potassium carbonate (62 mg). The reaction was
heated to 80 .sup.oC for 48 hours. The reaction was cooled and the
solvent removed in vacuo. The residue was taken up in ethyl acetate
and washed twice with water and once with brine. The solution was
dried over sodium sulfate and the solvent removed. The crude
intermediate was dissolved in methanol (4 ml) and a few drops of
acetic acid added. Approximately 50-100 mg of Raney nickel
suspended in sodium hydroxide (Aldrich) was added and the reaction
placed under an atmosphere of hydrogen. The suspension was stirred
vigorously for 8 hours, the catalyst filtered off and the solvent
removed in vacuo. The crude product was purified by reverse-phase
chromatography to yield
4-{2-benzenesulfonylamino-1-[4-(2-chloro-1-phenyl-ethoxy)-3-meth-
oxy-phenyl]-ethylamino}-benzamidine (32 mg). MS(M+H)=579.
Example 4
[0195]
4-{2-propanesulfonylamino-1-[4-(2-chloro-1-phenyl-ethoxy)-3-methox-
y-phenyl]-ethylamino}-benzamidine was prepared similarly to Example
2, except propanesulfonyl chloride was substituted for
benzenesulfonyl chloride in the reaction with
4-{2-amino-1-[4-(2-chloro-1-phenyl-ethoxy)-3-methoxy-phenyl]-ethylamino}--
benzonitrile. MS(M+H)=545.
Example 5
[0196] ##STR1000## 4-Benzyloxy-5-methoxy-2-nitrobenzaldehyde (12.2
g 42 mmoles) and 4-aminobenzonitrile (5 g, 42 mmoles) were
dissolved in methanol (165 ml) and stirred for two hours and then
heated to 60.sup.oC for 30 minutes. The reaction was allowed to
cool to room temperature and benzyl isonitrile (5 g, 42 mmoles)
added. The reaction was cooled to 0.sup.oC and boron
trifluoroetherate (16 ml, 126 mmoles) added dropwise over five
minutes. The reaction was stirred at 0.sup.oC for 20 minutes and
then allowed to come to room temperature and then stirred at
ambient temperature for two hours. Water (4 ml) was added and the
mixture stirred at room temperature overnight. A yellow precipitate
was evident the next morning and the solid filtered off. The solid
was washed with methanol and air dried to yield 8 grams of the
desired product. The solvent from the filtrate was removed in vacuo
and replaced with ethyl acetate. The solution was washed with water
and saturated sodium bicarbonate, dried over anhydrous magnesium
sulfate and the solvent removed. The crude material was submitted
to flash chromatography (hexanes:ethyl acetate, 1:1) to yield an
additional 7 g of the desired product
(4-Benzyloxy-5-methoxy-2-nitro-phenyl)-(4-cyano-phenylamino)-acetic
acid methyl ester. .sup.1HNMR(CDCl.sub.3): 7.68 (s, 1H), 7.4 (m,
7H), 7.0 (s, 1H), 6.61 (d, 2H), 6.2 (s, 1H), 5.2 (s,2H), 3.87 (s,
3H), 3.75 (s, 3H). ##STR1001##
[0197]
(4-Benzyloxy-5-methoxy-2-nitro-phenyl)-(4-cyano-phenylamino)-aceti-
c acid methyl ester (4.5 g, 10 mmole) was dissolved in
dimethoxyethane and lithium borohydride (0.210 g, 10 mmole) added.
The reaction was heated to reflux for three hours and cooled to
room temperature. The reaction was quenched with water containing
acetic acid and diluted with ethyl acetate. After transferring to a
separatory funnel, the organic layer was washed with water several
times. The organic layer was dried over anhydrous sodium sulfate,
filtered and the solvent removed. The crude material was then
submitted to flash chromatography to yield 3.1 g of
4-[1-(4-Benzyloxy-5-methoxy-2-nitro-phenyl)-2-hydroxy-ethylamino]-benzoni-
trile (74%). .sup.1HNMR(CDCl.sub.3):7.77 (s,1H), 7.3-7.5 (m, 7H),
7.15 (s, 1H), 6.42 (d, 2H), 5.4 (bs, 1H), 5.18 (dd AB syst., 2H),
4.15 (dd, 1H), 3.83 (s, 3H), 3.79-3.86 (dd, 1H). ##STR1002##
[0198]
4-[1-(4-Benzyloxy-5-methoxy-2-nitro-phenyl)-2-hydroxy-ethylamino]--
benzonitrile (3.1 g, 7.4 mmoles) was dissolved in tetrahydrofuran
(120 ml) and triphenylphosphine (5.9 g, 22 mmoles) and phthalimide
(5.4 g, 37 mmoles) added. The reaction was cooled to 0 C and
diisopropylazadicarboxylate (DIAD, 4.6 g) added dropwise. The
reaction was allowed to come to room temperature and stirred
overnight. The solvent was removed in vacuo and replaced with ethyl
acetate. The solution was washed with 1 N NaOH several times and
dried over anhydrous sodium sulfate. Flash chromatography
(hexanes:ethyl acetate, 1:1) provided the desired material with
some DIAD still present. The solid was washed several times with
ethanol to yield 3.2 g of the desired phthalimide
4-[1-(4-Benzyloxy-5-methoxy-2-nitro-phenyl)-2-(1,3-dioxo-1,3-dihydro-isoi-
ndol-2-yl)-ethylamino]-benzonitrile (3.2 g).
.sup.1HNMR(CDCl.sub.3): 7.83 (m, 2H), 7.75 (m, 2H), 7.37 (m, 7H),
6.91 (s, 1H), 6.41 (d, 2H), 6.17 (d, 1H), 5.65 (m, 1H), 5.18 (s,
2H), 4.27 (m, 2H), 3.61 (s, 3H). ##STR1003##
[0199]
4-[1-(4-Benzyloxy-5-methoxy-2-nitro-phenyl)-2-(1,3-dioxo-1,3-dihyd-
ro-isoindol-2-yl)-ethylamino]-benzonitrile (2.7 g, 5 mmoles) was
dissolved in ethanol (100 ml) and hydrazine hydrate (0.65 ml, 20
mmoles) added. The reaction was heated to 60 .sup.oC for 3 hours
then at room temperature for 48 hours. The solids that precipitated
were filtered off and the residue submitted to flash chromatography
to yield
4-[2-Amino-1-(4-benzyloxy-5-methoxy-2-nitro-phenyl)-ethylamino]-benzonitr-
ile (1.5 g). .sup.1HNMR(CDCl.sub.3): 7.75 (s,1H), 7.3-7.5 (m, 7H),
7.05 (s,1H), 6.45 (d, 2H), 5.80 (bs, 1H), 5.32 (m, 1H), 5.19 (s,
2H), 3.81 (s, 3H), 3.25 (dd, 1H), 3.0 (dd, 1H), 1.65 (bs, 2H).
##STR1004##
[0200]
4-[2-Amino-1-(4-benzyloxy-5-methoxy-2-nitro-phenyl)-ethylamino]-be-
nzonitrile (0.227 g, 0.66 mmoles) was dissolved in dichloromethane
(4 ml) and triethylamine (0.14 ml, 1 mmoles). The reaction was
cooled to 0 C and 1-propanesulfonyl chloride (0.085 ml, 0.75
mmoles). The reaction was stirred for 20 minutes and the product
purified by flash chromatography (hexanes:ethyl acetate 1:1) to
yield 260 mg of desired product--propane-1-sulfonic acid
[2-(4-benzyloxy-5-methoxy-2-nitro-phenyl)-2-(4-cyano-phenylamino)-ethyl]--
amide. .sup.1HNMR(CDCl.sub.3): 7.77 (s, 1H), 7.3-7.5 (m, 7H), 7.12
(s, 1H), 6.41 (d, 2H), 6.0 (d, 1H), 5.3 (m, 1H), 5.17 (dd, A-B,
2H), 4.75 (t, 1H), 3.85 (s, 3H), 3.65 (m, 1H), 3.5 (m, 1H), 3.04
(m, 2H), 1.83 (m, 2H), 1.05 (t, 3H). ##STR1005##
[0201] Propane-1-sulfonic acid
[2-(4-benzyloxy-5-methoxy-2-nitro-phenyl)-2-(4-cyano-phenylamino)-ethyl]--
amide (0.250 g) was dissolved in ethanol (10 ml) and added to Pt/C
(5%). The reaction was placed under a hydrogen atmosphere and
stirred vigorously for 3 hours. The catalyst was filtered off and
the product chromatographed (hexanes:ethyl acetate 1:2) to yield
133 mg of Propane-1-sulfonic acid
[2-(2-amino-4-benzyloxy-5-methoxy-phenyl)-2-(4-cyano-phenylamino)-ethyl]--
amide. .sup.1HNMR(CDCl.sub.3): 7.3-7.45 (m, 7H), 6.71 (s, 1H), 6.52
(d, 21H), 6.3 (s, 11H), 5.33 (2, 1H), 5.08 (s, 2H), 5.0 (t, 1H),
4.42 (q, 1H), 3.75 (s, 3H), 3.70 (bs, 2H), 3.45 (t, 2H), 2.97 (m,
2H), 1.80 (m, 2H), 1.03 (t, 3H). ##STR1006##
[0202] Propane-1-sulfonic acid
[2-(2-amino-4-benzyloxy-5-methoxy-phenyl)-2-(4-cyano-phenylamino)-ethyl]--
amide (133 mg, 0.27 mmoles) was dissolved in dichloromethane and
triethylamine added (0.05 ml, 0.35 mmoles). The reaction was cooled
and methanesulfonyl chloride (0.023 ml, 0.3 mmoles) added dropwise.
The reaction was stirred for two hours and the product purified by
flash chromatography (hexanes:ethyl acetate, 1:1). The product was
then taken-up in ethanol and hydroxylamine hydrochloride (35 mg,
0.5 mmoles) added. Sodium ethoxide (48 mg, 0.7 mmoles was added and
the reaction heated for 48 hours. The ethanol was removed and water
(4 ml) added. The solid was filtered off and washed with water. The
crude product was then taken up in 4 ml of methanol with 0.5 ml
acetic acid. Raney Nickel (ca. 50 mg as a suspension in sodium
hydroxide, Aldrich) was added and the reaction placed under a
hydrogen atmosphere. The reaction was stirred vigorously for 3
hours and the catalyst filtered off. The crude product was
submitted to reverse-phase preparative chromatography to yield the
final product 4-[1-(4-Benzyloxy-2-methanesulfonylamino
-5-methoxy-phenyl)-2-(propane-1-sulfonylamino)-ethylamino]-benzamidine
(12 mg): MS (M+H)=590.
Example 6a-6g
[0203] Using a procedure analogous to Example 5, the following
compounds were prepared: [0204] a)
4-[2-Benzenesulfonylamino-1-(2-benzenesulfonylamino-4-benzyloxy-5-methoxy-
-phenyl)-ethylamino]-benzamidine. The procedure was the same as
above except phenyl sulfonyl chloride was used instead of
propanesulfonyl chloride and methane sulfonyl chloride. MS:
(M+H)=686. [0205] b)
4-[2-Benzenesulfonylamino-1-(2-benzenesulfonylamino-4-benzyloxy-5-ethoxy--
phenyl)-ethylamino]-benzamidine. The procedure was the same as
above except starting with 3-ethoxy, 4-benzyloxy,
6-nitro-benzaldehyde. MS: (M+H)=700. [0206] c)
4-[1-(4-Benzyloxy-5-ethoxy-2-methanesulfonylamino-phenyl)-2-(propane-1-su-
lfonylamino)-ethylamino]-benzamidine. The procedure was the same as
above except starting with 3-ethoxy, 4-benzyloxy,
6-nitro-benzaldehyde. MS (M+H)=604. [0207] d)
4-[1-(4,5-Diethoxy-2-methanesulfonylamino-phenyl)-2-(propane-1-sulfonylam-
ino)-ethylamino]-benzamidine. The procedure was the same except
starting with 3,4-diethoxy, 6-nitrobenzaldehyde. MS (M+H)=542.
[0208] e)
{5-Benzyloxy-2-[1-(4-carbamimidoyl-phenylamino)-2-(propane-1-sulfonylamin-
o)-ethyl]-4-methoxy-phenylsulfamoyl}-acetic acid ethyl ester. The
procedure was the same except using chlorosulfonyl-acetic acid
ethyl ester instead of methanesulfonyl chloride. MS: (M+H)=676.
[0209] f)
{5-Benzyloxy-2-[1-(4-carbamimidoyl-phenylamino)-2-(propane-1-sulfonylamin-
o)-ethyl]-4-methoxy-phenylsulfamoyl}-acetic acid.
{5-Benzyloxy-2-[1-(4-carbamimidoyl-phenylamino)-2-(propane-1-sulfonylamin-
o)-ethyl]-4-methoxy-phenylsulfamoyl}-acetic acid ethyl ester (10
mg) was dissolved in water (2 ml) and tetrahydrofuran (2 ml) and
LiOH added (3 mg). Allowed to stir overnight. Product purified by
reverse-phase preparative HPLC. 3 mg MS (M+H)=648. [0210] g)
4-[1-(3,4-Dimethoxy-2-methanesulfonylaminophenyl)-2-(propane-1-sulfonylam-
ino)-ethylamino]-benzamidine. This compound was prepared with a
similar procedure as described above except 2-bromo
-3,4-dimethoxybenzaldehyde was used instead of
4-benzyloxy-5-methoxy-2-nitrobenzaldehyde. MS(M+H)=499.
Examples 7a-7g
[0211] The compounds 7a-7g were generally prepared as follows.
Compound E (20 mg, 0.03 mmoles) was dissolved in acetonitrile (2
ml) containing triethylamine (17 ul, 0.12 mmoles) and water (0.3
ml). To this was added the respective acyl chloride, alkyl
chloroformate, or isocyanate (0.03 mmoles) and the reaction stirred
for 4 hours. The solvent was removed in vacuo and the compounds
purified by reverse-phase preparative HPLC (acetonitrile/water with
0.1% trifluoroacetic acid) to yield the final product upon
lyophilization. [0212] 7a:
N-[2-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-carbamimidoyl-phenylamino)-ethyl-
]-2,2,2-trifluoro-acetamide, MS (M+H)=487. [0213] 7b:
N-[2-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-carbamimidoyl-phenylamino)-ethyl-
]-acetamide, MS (M+H)=433. [0214] 7c:
N-[2-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-carbamimidoyl-phenylamino)-ethyl-
]-butyramide, MS (M+H)=461. [0215] 7d:
N-[2-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-carbamimidoyl-phenylamino)-ethyl-
]-2-chloro-acetamide, MS (M+H)=467. [0216] 7e:
[2-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-carbamimidoyl-phenylamino)-ethyl]--
carbonic acid methyl ester, MS (M+H)=449. [0217] 7f:
[2-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-carbamimidoyl-phenylamino)-ethyl]--
carbonic acid isobutyl ester, MS (M+H)=491. [0218] 7g:
[2-(4-Benzyloxy-3-methoxy-phenyl)-2-(4-carbamimidoyl-phenylamino)-ethyl]--
carbonic acid 2,2,2-trichloro-ethyl ester, MS (M+H)=565
Example 8
[0219] ##STR1007##
[0220] The methyl ester of the acid shown above (920 mg 2.85
mmoles) was suspended in 3/1 THF/water (40 ml) and cooled to
0.degree. C. The solution was treated with 1 N LiOH (7.1 ml, 7.1
mmoles) and allowed to stir overnight. The reaction was acidified
with trifluoroacetic acid until pH=4.0 was obtained. The solvent
was removed in vacuo and the crude material purified by flash
chromatography (ethyl acetate with 0.5% acetic acid) to yield 1 g
of carboxylic acid. ##STR1008##
[0221] Carbonyl diimidazole (131 mg, 0.8 mmoles) was dissolved in
anhydrous THF (1.6 ml) and the carboxylic acid prepared above (251
mg, 0.8 mmoles) added dropwise as a solution in THF (1.6 ml). The
reaction was allowed to stir at room temperature for 30 minutes,
refluxed for 30 minutes and then cooled to room temperature again.
n-Propylsulfonamide (100 mg) was added and stirred for 10 minutes.
DBU (123 mg) was added as a solution in THF (1.6 ml). The reaction
was worked up by acidification and extraction into ethyl acetate.
The solvent was removed and the crude product purified by flash
chromatography (SiO2, ethyl acetate) to yield 195 mg of the acyl
sulfonamide shown above. ##STR1009##
[0222] The nitrile prepared above (90 mg, 0.2 mmoles) was dissolved
in ethanol (2.5 ml). Diisopropylethylamine (202 mg, 1.56 mmoles)
was added followed by hydroxylamine hydrochloride (83 mg, 1.2
mmoles). The reaction was heated to 70.degree. C. for 21 hours. The
reaction was cooled and the solvent removed in vacuo. The residue
was taken up in 30% acetonitrile/water (4 ml) and purified by
preparative reverse-phase chromatography (water/acetonitrile 0.1%
TFA gradient) to yield 14 mg of the hydroxyamidine shown above.
##STR1010##
[0223] The hydroxyamidine product was then taken up in ethanol (2
ml) and acetic acid (8 drops). Raney Ni (ca. 100 mg) was added and
the reaction stirred vigorously under a hydrogen atmosphere for 2
hours 45 minutes. The product was filtered through Celite and the
Celite rinsed first with 30% acetonitrile/water containing 0.1% TFA
and then with acetonitrile. The solvent was removed in vacuo and
the crude product purified by preparative reverse-phase
chromatography (water/acetonitrile 0.1% TFA gradient) to yield the
desired product (6 mg). M+H=435.
Example 9
Synthesis of Enantiomerically Pure 6-alkylsulfonylamino
sulfonamides
[0224] ##STR1011##
[0225] 3-Ethoxy-4-hydroxybenzaldehyde (40 g) was added to
dimethylformamide (600 mL) followed by potassium carbonate (40 g,
1.2 Equiv.). Ethyl iodide (28.87 mL, 1.5 Equiv.) was added and the
solution was heated to 60.degree. C. for six hours. The solution
was cooled to room temperature and the solvent was removed under
reduced pressure. The solution was diluted with ethyl acetate (500
mL), and washed with water, brine, dried with magnesium sulfate,
and evaporated to yield the crude product 3,4-ethoxybenzaldehyde
(49 g, 104%). The 3,4-ethoxybenzaldehyde (45 g) was dissolved in
ethanol (300 mL), and the solution was cooled to 0.degree. C. In a
separate flask, potassium hydroxide (19.5 g, 1.5 Equiv.) was added
to ethanol (300 mL) followed by nitromethane (26 g, 1.5 Equiv.) and
the solution was stirred at room temperature for ten minutes and
cooled to 0.degree. C. This solution was added to the
3,4-ethoxybenzaldehyde and stirred for 20 minutes and poured onto
concentrated hydrochloric acid (200 mL) at 0.degree. C. The ethanol
was removed under reduced pressure, and the solution was diluted
with water (300 mL) and the reaction mixture was extracted with
ethyl acetate. The organic layer was dried with magnesium sulfate
and the solvent removed to yield crude product. The crude product
was purified by recrystallization with ethyl acetate (47 g, 87%).
MS (M+H)=238. ##STR1012##
[0226] The 1-nitro-2-(3,4-diethoxyphenyl)ethylene (16.64 g) and
4-aminobenzonitrile (9.12 g, 1.1 Equiv.) were added to
tetrahydrofuran (350 mL), and cooled to 0.degree. C. Lithium
diisopropylamide (47.8 mL, 1.02 Equiv.) was added slowly until a
persistent purple color was formed. Zinc (50 g) was added in one
portion, followed by acetic acid (35 mL). The solution was warmed
to room temperature and stirred for 2 hours followed by the
addition of acetic acid (35 mL) and zinc (10 g). After an
additional 2 hours, acetic acid (25 mL) was added and the reaction
mixture was stirred for one hour. Concentrated hydrochloric acid
(15 mL) was added and the solution was stirred an additional hour.
The solution was filtered through a pad of celite and water (250
mL) was added. The solution was concentrated to 300 mL, under
reduced pressure and added to citric acid (0.5 M, 500 mL) and ethyl
acetate/hexane (500 mL). The citric acid layer was collected and
ammonium hydroxide was added until the solution became basic. This
solution was extracted with ethyl acetate (3.times.200 mL), and the
combined organics were dried with magnesium sulfate, and evaporated
under reduced pressure to yield the crude product. The crude
product was diluted with dichloromethane (350 mL) and cooled to
0.degree. C. Phosgene (40.88 mL of a 20% solution in toluene, 1.1
Equiv.) was added followed by Hunigs base (24.46 mL, 2 Equiv.). The
solution was stirred for ten minutes and water (200 mL) was added.
The dichloromethane was collected, dried with magnesium sulfate,
and purified by flash chromatography on silica gel (80% ethyl
acetate/20% hexane) to yield the product as a white solid (9.76 g,
40%). MS (M+H)=352. ##STR1013##
[0227] The
4-[5-(3,4-diethoxyphenyl)-2-oxo-imidazolidin-1-yl-benzonitrile
(2.10 g) was added to tetrahydrofuran (200 mL) and cooled to
-78.degree. C. n-Butyllithium (3.74 mL, 1 Equiv.) was added
dropwise, and the solution stirred for ten minutes.
(S)-(+)-2-(6-methoxy-2-naphthyl)propionyl chloride (1.49 g, 1
Equiv.) was added in one portion as a solid and the reaction was
stirred for one hour. The reaction mixture was warmed to room
temperature, and evaporated under reduced pressure to 50 mL. The
solution was diluted with ethyl acetate (300 mL), and washed with
citric acid (0.5M), water, brine, and dried with magnesium sulfate.
The solution was evaporated under reduced pressure and purified by
flash chromatography on silica gel (50% ethyl acetate/50% hexane)
to yield the product
4-{5-(3,4-diethoxyphenyl)-3-[2-(6-methoxynaphthalen-2-yl)-propionyl]-2-ox-
o-imidazolidin-1-yl}-benzonitrile as one diastereomer (1.20 g,
71%). This product was added to methanol (200 mL) followed by
lithium hydroxide (1 mL of a 10% aqueous solution) and stirred for
fifteen minutes. Acetic acid (10 drops was added, the methanol was
removed under reduced pressure and the product was purified by
flash chromatography on silica gel (80% ethyl acetate/20% hexane)
to yield the product (0.71 g, 95%) as a white solid.
[.alpha.].sub.Na-55.0 (c 2.20, acetone). MS (M+H)=352.
##STR1014##
[0228] The
(R)-(-)-4-[5-(3,4-diethoxyphenyl)-2-oxo-imidazolidin-1-yl-benzonitrile
(0.710 g) was added to tetrahydrofuran (20 mL) and cooled to
-78.degree. C. n-Butyllithium (1.27 mL of a 1.6 M solution in
hexane, 1 Equiv.) was added dropwise and the solution was stirred
for ten minutes. 1-Propylsulfonylchloride (0.275 mL, 1.2 Equiv.)
was added and the solution was stirred for fifteen minutes and
warmed to room temperature. Acetic acid (10 drops) was added, the
solvent removed under reduced pressure, and the product was
purified by flash chromatography on silica gel (50% ethyl
acetate/50% hexane) to yield the product (0.740 g, 80%). This
material (0.300 g) was diluted with dichloroethane (10 mL) and
cooled to 0.degree. C. Nitric acid (0.137 mL, 5 Equiv.) was added
dropwise and the solution was stirred for one hour. The solution
was diluted with dichloroethane (100 mL), and washed with water,
saturated sodium bicarbonate, dried with magnesium sulfate, and the
solvent was evaporated under reduced pressure. This material was
diluted with methanol (50 mL), and acetic acid (1 mL), and platinum
(0.050 g, 5% on carbon) was added. The solution was hydrogenated
for one hour, filtered through a pad of celite, and the solvent was
evaporated under reduced pressure. This material was dissolved in
dichloromethane (5 mL), and Hunigs base (0.177 mL, 1.5 Equiv.) and
chlorosulfonylacetic acid ethyl ester (0.189 g, 11.5 Equiv.) was
added and the solution was stirred for two hours. The solution was
purified by direct flash chromatography on silica gel (50% ethyl
acetate/50% hexane) to yield the product (0.160 g)
(R)-{2-[3-(4-cyanophenyl)-2-oxo-1-(propane-1-sulfonyl)-imidazolidin
-4-yl]-4,5-diethoxyphenylsulfamoyl}-acetic acid ethyl ester. MS
(M+H)=624. ##STR1015##
[0229]
(R)-{2-[3-(4-cyanophenyl)-2-oxo-1-(propane-1-sulfonyl)-imidazolidi-
n-4-yl]-4,5-diethoxyphenylsulfamoyl}-acetic acid ethyl ester (0.160
g) was diluted with ethanol (5 mL) followed by lithium hydroxide (1
mL of a 10% aqueous solution, 10 Equiv.) and the solution was
stirred for forty eight hours. The solution was purified by direct
flash chromatography on silica gel (20% methanol/80%
dichloromethane) to yield the product. This product was diluted
with ethanol (1 mL) and hydroxylamine (0.035 g, 10 Equiv.) and
Hunigs base (0.088 mL, 10 Equiv.) was added and the solution was
heated to 60.degree. C. for six hours. The reaction mixture was
cooled to room temperature and stirred for twelve hours. Ethanol (3
mL) and acetic acid (0.5 mL), and Raney nickel (0.025 g) was added
and the solution was hydrogenated for one hour. The reaction
mixture was filtered through a pad of celite and the solvent was
removed under reduced pressure. The product was purified by
reverse-phase preparative chromatography to yield
{2-[1-(4-carbamimidoylphenylamino)-2-(propane-1-sulfonylamino)-ethyl]-4,5-
-diethoxyphenylsulfamoyl}acetic acid (52 mg). [.alpha.].sub.Na-42.1
(c 1.01, methanol) MS (M+H)=587.
Example 10
6-alkoxy substituted sulfonamides
[0230] ##STR1016##
[0231] Aminoacetonitrile (14.25 g, 92.5 mmoles) was dissolved in
1,2-dichloroethane (150 ml) and the reaction was placed under
N.sub.2. Triethylamine (32.76 g, 324 mmoles) was added and the
reaction was cooled to zero degrees Celsius. A solution of
propane-1-sulfonyl chloride (13.19 g, 92.5 mmoles) in
1,2-dichloroethane (20 ml) was added dropwise and the reaction was
allowed to warm to room temperature and was stirred for 16 hours.
Thin-layer chromatography showed the presence of a new product with
a higher R.sub.f. The solvent was removed in vacuo and the crude
product was submitted to flash chromatography (methylene
chloride:ethyl acetate, 9:1) to yield 10.56 g of propane-1-sulfonic
acid cyanomethyl-amide. .sup.1HNMR (CDCl.sub.3): 5.40 (s, 1H), 4.11
(s, 2H), 3.15 (m, 2H), 1.89 (q, 2H), 1.10 (t, 3H). ##STR1017##
[0232] 3-Ethoxy-4-hydroxy-benzaldehyde (Aldrich, 100 g, 0.602 mole)
was dissolved in N,N-dimethylformamide (1 L). The reaction was
placed under N.sub.2. Solid potassium carbonate (103 g, 0.662
moles) was added and the reaction was stirred 10 minutes.
Iodoethane (175 g, 1.26 moles) was added and the reaction was
stirred for 16 hours at room temperature. The reaction was
monitored by thin-layer chromatography which indicated complete
consumption of the phenol to give a new product. The reaction was
filtered to remove the potassium carbonate and solvent removed in
vacuo. The residue was dissolved in methylene chloride and
filtered. Silica gel (200 g) was added and the dichloromethane
removed in vacuo. The crude product absorbed on silica gel was
submitted to flash chromatography (hexane, 100%, 2 L, then ethyl
acetate:hexane, 1:3, 2 L, then ethyl acetate:hexane, 1:1) to yield
109.36 g of 3,4-diethoxy-benzaldehyde .sup.1HNMR (CDCl.sub.3): 9.83
(s, 1H), 7.41 (m, 2H), 6.96 (d, 1H), 4.17 (m, 4H), 1.50 (t, 3H)
1.47 (t, 3H). ##STR1018##
[0233] 3,4-Diethoxy-benzaldehyde (31.45 g, 0.162 moles) was
dissolved in methylene chloride (500 ml). 3-Chloroperbenzoic acid
was added and the reaction was heated to reflux for 4 hours.
Thin-layer chromatography showed consumption of the aldehyde. The
reaction was cooled to room temperature, diluted with methylene
chloride and quenched with saturated aqueous potassium carbonate.
The layers were separated and the aqueous solution extracted with
methylene chloride. The methylene chloride extracts were combined,
washed with water, brine, and dried over anhydrous sodium sulfate,
filtered and the solvent was removed in vacuo to yield a crude
oil.
[0234] The crude oil was dissolved in methanol (300 ml) and a
solution of 10% aqueous KOH (60 ml) was added. The reaction was
stirred at room temperature for 16 hours. Thin-layer chromatography
showed consumption of the intermediate which had formed. The
methanol was removed in vacuo and the aqueous solution was
acidified with 1.2 N HCl. The solution was extracted with ethyl
acetate. The ethyl acetate extracts were washed with water and
brine, dried over anhydrous sodium sulfate, filtered, and the
solvent removed in vacuo. The residue was submitted to flash
chromatography (hexane:ethyl acetate, 3:1) to yield 22.08 g of
3,4-diethoxy-phenol. .sup.1HNMR (CDCl.sub.3): 6.56 (d, 1H), 6.25
(d, 1H), 6.10 (dd, 1H), 3.82 (m, 4H), 1.22 (t, 3H), 1.20 (t, 3H).
##STR1019##
[0235] Nitrobenzene (100 ml) was cooled to zero degrees Celsius and
saturated with HCl gas. To this solution was added of
3,4-diethoxy-phenol (11.64 g, 64 mmoles), propane-1-sulfonic acid
cyanomethyl-amide (10.36 g, 64 mmoles) and zinc chloride (17.45 g,
128 mmoles). The reaction was stirred for 1 hour at zero degrees
Celsius and then allowed to warm to room temperature. Stirring was
continued for 2 hours. Thin-layer chromatography showed consumption
of 3,4-diethoxy-phenol. Water (100 ml) was added cautiously and the
reaction was heated to 100 degrees Celsius for 1 hour. The reaction
was cooled, and extracted with methylene chloride. The extracts
were washed with water, brine, dried over anhydrous sodium sulfate,
filtered, and the solvent was removed in vacuo. The residue was
submitted to flash chromatography (2.5-5% ethyl acetate in
methylene chloride). The purified product was triturated with
ether, filtered and air dried to yield 17.3 g of propane-1-sulfonic
acid [2-(4,5-diethoxy-2-hydroxy-phenyl)-2-oxo-ethyl]-amide.
.sup.1HNMR (CDCl.sub.3): 11.86 (s, 1H), 6.93 (s, 1H), 6.46 (s, 1H),
5.26 (t, 1H), 4.55 (d, 2H), 4.14 (q, 2H), 4.03 (q, 2H), 3.05 (m,
2H), 1.90 (m, 2H), 1.45 (m, 6H), 1.07 (t, 3H). ##STR1020##
[0236] Propane-1-sulfonic acid
[2-(4,5-diethoxy-2-hydroxy-phenyl)-2-oxo-ethyl]-amide (200 mg,
0.579 mmoles) was dissolved in tetrahydrofuran (5 ml), placed under
N.sub.2, and cooled to zero degrees Celsius. Borane:THF complex
(1.74 ml, 1.0 M in THF, 1.74 mmoles) was added dropwise. The
reaction was stirred 15 minutes and the allowed to warm to room
temperature over 2 hours. Thin-layer chromatography showed
consumption of starting material and formation of a new product
with a lower R.sub.f. The reaction was quenched in 1.2 N aqueous
HCl (2.5 ml), diluted with water and extracted with ethyl acetate.
The extracts were washed with water and brine, dried over anhydrous
sodium sulfate, filtered, and the solvent removed in vacuo to yield
190 mg of a crude oil.
[0237] The crude oil was dissolved in acetonitrile (5 ml).
4-Aminobenzonitrile (194 mg, 1.64 mmoles) and lithium perchlorate
(233 mg, 2.19 mmoles) were added. The reaction was heated to 90
degrees Celsius for 3.5 hours then stirred at room temperature for
18 hours. Thin-layer chromatography showed the presence of a new
product with a higher R.sub.f. versus the product from the
reduction. The reaction was quenched in water and extracted with
ethyl acetate. The ethyl acetate extracts were washed with water
and brine, dried over anhydrous sodium sulfate, filtered, and the
solvent removed in vacuo. The residue was submitted to flash
chromatography (ethyl acetate:methylene chloride, 1:9) to yield 134
mg of propane-1-sulfonic acid [2-(4-cyano-phenylamino)
-2-(4,5-diethoxy-2-hydroxy-phenyl)-ethyl]-amide. .sup.1HNMR
(CDCl.sub.3): 7.34 (d, 2H), 6.71 (s, 1H), 6.61 (d, 2H), 6.41 (s,
1H), 5.58 (d, 1H), 4.79 (m,1H), 4.57 (m, 1H), 3.99 (m, 4H), 3.48
(t, 2H), 3.00 (m, 2H), 1.80 (q, 2H), 1.41 (t, 3H), 1.34 (t, 3H),
1.03 (t, 3H). ##STR1021##
[0238] Propane-1-sulfonic acid
[2-(4-cyano-phenylamino)-2-(4,5-diethoxy-2-hydroxy-phenyl)-ethyl]-amide
(100 mg, 0.223 mmoles) was dissolved in dimethylformamide (1.5 ml)
and treated with solid potassium bicarbonate (22 mg, 0.223 mmoles)
followed by ethyl bromoacetate (0.37 ml, 0.223 mmoles). The
reaction was stirred under N.sub.2 for 67 hours. Thin-layer
chromatography showed presence of a new product with a higher
R.sub.f. The reaction was quenched in water and extracted with
ethyl acetate. The ethyl acetate extracts were washed with water
and brine, dried over anhydrous sodium sulfate, filtered and the
solvent removed in vacuo. The residue was submitted to flash
chromatography (ethyl acetate:methylene chloride 1:9) to yield 78
mg of
{2-[1-(4-cyano-phenylamino)-2-(propane-1-sulfonylamino)-ethyl]-4,5-dietho-
xy-phenoxy}-acetic acid ethyl ester. .sup.1HNMR (CHCl.sub.3): 7.35
(d, 2H), 6.81 (s, 1H), 6.57 (d, 2H), 6.43 (s, 1H), 4.81 (t, 1H),
4.68 (ABq, 2H), 4.56 (t, 1H), 4.26 (q, 2H), 4.05 (q, 2H), 3.95 (m,
2H), 3.54 (t, 2H), 2.98 (m, 2H) 1.80 (m, 2H) 1.42 (t, 3H), 1.32 (t,
6H), 1.02 (t, 3H). MS (M+H): 534. ##STR1022##
[0239]
{2-[1-(cyano-phenylamino)-2-(propane-1-sulfonylamino)-ethyl]-4,5-d-
iethoxy-phenoxy}-acetic acid ethyl ester (76 mg, 0.142 mmoles) was
dissolved in tetrahydrofuran (3 ml). Water (1 ml) was added and the
reaction was cooled to zero degrees Celsius. Aqueous LiOH (1.0 M,
0.42 ml, 0.42 mmol) was added to the reaction. After stirring for 5
minutes, the reaction was allowed to warm to room temperature and
was stirred for 16 hours at room temperature. The disappearance of
the ester was monitored by analytical high pressure liquid
chromatography. Additional LiOH (1.0 M. 0.14 ml) was added. The
reaction was stirred another 8 hours at room temperature.
Consumption of the ester was not complete so freshly prepared LiOH
(1.0 M, 0.14 ml), was added and the reaction stirred 24 hours. More
LiOH (1.0 M, 0.28 ml) was added and after 67 hours, analytical RP
HPLC showed consumption of the ester (total 1.0 M LiOH=0.98 mL, 6.9
equiv.). The reaction was acidified with acetic acid and the THF
was allowed to evaporate under a stream of N.sub.2. The solution
was clarified by addition of acetonitrile and then purified by
preparative reverse-phase HPLC (gradient acetonitrile/water with
0.1% trifluoroacetic acid) to yield after lyophilization 44 mg of
the mono-TFA salt of
{2-[1-(cyano-phenylamino)-2-(propane-1-sulfonylamino)-ethyl]-4,5-diethoxy-
-phenoxy}-acetic acid. .sup.1NMR (CD.sub.3OD): 7.15 (d, 2H), 6.70
(s, 1H), 6.48 (d, 2H), 6.45 (s, 1H), 4.76 (t, 1H), 4.60 (s, 2H),
3.85 (q, 2H), 3.73 (m, 2H), 3.43 (dd, 1H), 3.26 (dd, 1H partially
obscured by the CH3OH solvent peak), 2.76 (m, 2H), 1.52 (m, 2H),
1.18 (t, 3H), 1.07 (t, 3H), 0.77 (t, 3H). MS (M+H): 506.
##STR1023##
[0240]
{2-[1-(cyano-phenylamino)-2-(propane-1-sulfonylamino)-ethyl]-4,5-d-
iethoxy-phenoxy}-acetic acid, mono-TFA salt form (44 mg, 0.071
mmoles) was dissolved in ethanol (1 ml) and treated with
diisopropylethylamine (0.89 ml, 0.515 mmoles) and solid
hydroxylamine hydrochloride (25 mg, 0.355 mmoles). The reaction was
placed under N.sub.2 and heated to 60 degrees Celsius for 3 hours.
Analytical high pressure liquid chromatography analysis after 3
hours revealed the reaction had not gone to completion. The
reaction mixture was cooled to room temperature and stirred at room
temperature for 16 hours and the progress of the conversion
assessed by HPLC. The reaction mixture was heated at 60 degrees
Celsius for 8 hours, stirred at room temperature for 16 hours,
heated at 70 degrees for 8 hours and stirred at room temperature
for 16 hours. HPLC showed consumption of starting material. The
reaction was acidified with acetic acid, Raney nickel added and the
reaction was hydrogenated at approximately 1 atm under a balloon of
hydrogen for 3 hours. Analytical HPLC showed consumption of the
hydroxy amidine intermediate. The Raney nickel catalyst was
filtered and the solvent was removed in vacuo. The residue purified
by preparative reverse-phase HPLC (gradient acetonitrile/water with
0.1% trifluoroacetic acid) to yield after lyophilization 5.6 mg of
{2-[1-(carbamimidoyl-phenylamino)-2-(propane-1-sulfonylamino)-ethyl]-4,5--
diethoxy-phenoxy}-acetic acid, bis-TFA salt form. MS (M+H):
523.
Example 11
6-alkylsulfonyl-alkyl-amino substituted acylsulfonamide
synthesis
[0241] ##STR1024##
[0242] 4,5 diethoxy-2-nitrobenzaldehyde (55.5 g 206 mmoles) and
4-aminobenzonitrile (23 g, 195 mmoles) were dissolved in methanol
(700 ml) and stirred at 60.sup.oC for 2 hours. The reaction was
allowed to cool to 0.degree. C. and tosylmethylisonitrile (45 g.
230 mmoles) added. Boron trifluoroetherate (78 ml, 620 mmoles) was
added dropwise over 10 minutes. The reaction was stirred at
0.sup.oC for 30 minutes, allowed to come to room temperature and
then stirred at ambient temperature for 1.5 hours. Water (18 ml)
was added and the mixture stirred at room temperature overnight.
The following day the methanol was removed in vacuo and the residue
taken up in ethyl acetate. The organic layer was washed with water
and then dried over anhydrous sodium sulfate. The sodium sulfate
was filtered off and the ethyl acetate removed in vacuo. The crude
material was submitted to flash chromatography (hexanes:ethyl
acetate, 2:1 then 1:1) to yield 46 g of the desired product
(4-ethoxy-5-ethoxy-2-nitro-phenyl)-(4-cyano-phenylamino)-acetic
acid methyl ester. ##STR1025##
[0243]
(4-ethoxy-5-ethoxy-2-nitro-phenyl)-(4-cyano-phenylamino)-acetic
acid methyl ester (11 g, 27.5 mmole) was dissolved in ethyl acetate
(300 m) and added to a flask containing 5% Pt/C (3 g) under a
nitrogen atmosphere. The nitrogen was removed and replaced by
hydrogen (balloon) and the reaction stirred vigorously for 6 hours.
The catalyst was filtered off and the solvent removed in vacuo. The
residue was taken up in dichloromethane (ca. 300 ml) and pyridine
(5.6 ml, 70 mmole) added. The reaction was cooled to 0.degree. C.
and methanesulfonyl chloride (2.5 ml, 33 mmole) added dropwise. The
reaction was stirred overnight. The solution was washed with water
and the solvent removed in vacuo. The crude product was
chromatographed on silica using flash chromatography (Hexane:ethyl
acetate 1:1) to yield 5 g of desired material
--(4-cyano-phenylamino)-[4,5-diethoxy-2-(methanesulfonylamino)-phenyl]-ac-
etic acid methyl ester. The product from about
(4-cyano-phenylamino)-4,5-diethoxy-2-methanesulfonylamino-phenyl)-acetic
acid methyl ester (5 g, 10.7 mmoles) was dissolved in dry DMF (100
ml) and cesium carbonate (7.25 g, 22 mmoles) and iodomethane (1 ml,
16 mmoles) added. The reaction was stirred at room temperature for
3 hours and the solvent removed in vacuo. The residue was taken up
in ethyl acetate, acidified with 1N hydrochloric acid and the
organic layer washed once with water. The material was dried over
anhydrous sodium sulfate and the solvent removed in vacuo. The
residue was flash chromatographed (hexane:ethyl acetate, 1:1) to
yield 2.6 g of desired material
--(4-cyano-phenylamino)-[4,5-diethoxy-2-(methanesulfonyl-methyl-amino)-ph-
enyl]-acetic acid methyl ester. ##STR1026##
[0244] The
(4-cyano-phenylamino)-4,5-diethoxy-2-methanesulfonyl-methyl-amino-phenyl)-
-acetic acid methyl ester obtained above (2.6 g, 5 mmole) was
dissolved in methanol. 1 N LiOH was added (25 ml) and the reaction
stirred at room temperature for 5 hours. The methanol was removed
in vacuo and the reaction was acidified with 1 N hydrochloric acid.
The product was extracted into ethyl acetate and washed with water.
Flash chromatography (Ethyl acetate with 5% acetic acid) yielded
1.9 g the desired
acid--(4-cyano-phenylamino)-[4,5-diethoxy-2-(methanesulfonyl-methyl-amino-
)-phenyl]-acetic acid.
[0245] The
(4-cyano-phenylamino)-4,5-diethoxy-2-methanesulfonyl-methyl-amino-phenyl)-
-acetic acid obtained above (350 mg, 0.75 mmole) was combined with
carbonyl diimidazole (610 mg, 3.77 mmole) in dry THF (6 ml). The
reaction was heated at 60.degree. C. for 1 hour and cooled to room
temperature. To this solution was added phenylsulfonamide (650 mg,
4.14 mmole) and DBU (5 mmole) as a solution in 5 ml THF. The
reaction was stirred for 3 hours and the THF removed in vacuo. The
residue was taken up in ethyl acetate and acidified with 1 N
hydrochloric acid. The organic layer was separated, washed with
water and dried over anhydrous sodium sulfate. The crude product
was purified by flash chromatography (Hexanes:ethyl acetate 1:1
then ethyl acetate with 5% acetic acid) to yield 302 mg of desired
product
--N-{(4-cyano-phenylamino)-[4,5-diethoxy-2-(methanesulfonyl-methy-
l-amino)-phenyl]-acetyl}benzenesulfonamide. ##STR1027##
[0246] The
N-{(4-cyano-phenylamino)-[4,5-diethoxy-2-(methanesulfonyl-methyl-amino)-p-
henyl]-acetyl}benzenesulfonamide obtained above (126 mg, 0.21
mmole) was dissolved in ethanol (1.8 ml) and heated to 60.degree.
C. Diisopropylethylamine (DIPEA --260 ul, 1.5 mmole) was added
followed by hydroxylamine-hydrochloride (74 mg, 1.04 mmole). The
reaction was stirred at 60.degree. C. under a nitrogen atmosphere
for 6 hours. The reaction was then allowed to cool to room
temperature. The solution was diluted with methanol (5 ml) and
acetic acid (2 ml) and Raney Nickel 2800 (ca. 50 mg) added as a
suspension. The reaction was then stirred vigorously under a
hydrogen atmosphere for 1 hour. The catalyst was filtered off and
the solvent removed. The crude product was purified by preparative
reverse-phase HPLC using a water-acetonitrile (0.1% TFA) gradient
to yield 40 mg of desired
4-{2-benzenesulfonylamino-1-[4,5-diethoxy-2-(methanesulfonyl-methyl-amino-
)-phenyl]-2-oxo-ethylamino}benzamidine as its trifluoroacetic acid
salt. MS: (M+H)=604.
Example 12
[0247] ##STR1028##
[0248] 4-isopropoxy-5-ethoxy-benzaldehyde (10.6 g 50 mmoles) and
4-aminobenzonitrile (5.9 g, 50 mmoles) were dissolved in methanol
(150 ml) and stirred at 60.sup.oC for 1.6 hours. The reaction was
allowed to cool to 0.degree. C. and tosylmethylisonitrile (9.75 g.
50 mmoles) added. Boron trifluoroetherate (19 ml, 150 mmoles) was
added dropwise over 10 minutes. The reaction was stirred at
0.sup.oC for 30 minutes, allowed to come to room temperature and
then stirred at ambient temperature for 1.5 hours. Water (4.5 ml)
was added and the mixture stirred at room temperature 2 days. The
methanol was removed in vacuo and the residue taken up in ethyl
acetate. The organic layer was washed with water and then dried
over anhydrous sodium sulfate. The sodium sulfate was filtered off
and the ethyl acetate removed in vacuo. The crude material was
submitted to flash chromatography (hexanes:ethyl acetate, 1:1) to
yield 12.5 g of the desired product
(4-isopropoxy-5-ethoxy-phenyl)-(4-cyano-phenylamino)-acetic acid
methyl ester.
[0249] The product from above,
(4-isopropoxy-5-ethoxy-phenyl)-(4-cyano-phenylamino)-acetic acid
methyl ester, (6 g, 16.3 mmole) was treated with 1 N LiOH (ca. 50
ml) in THF (ca. 150 ml). The reaction was stirred at room
temperature for 6 hours and acidified with 1 N hydrochloric acid.
The THF was removed in vacuo and the product extracted into ethyl
acetate. The crude material was purified by reverse phase
chromatography (ethyl acetate 3% acetic acid) to yield 4.85 g of
desired
acid--(4-isopropoxy-5-ethoxy-phenyl)-(4-cyano-phenylamino)-acetic
acid. ##STR1029##
[0250] The
(4-isopropoxy-5-ethoxy-phenyl)-(4-cyano-phenylamino)-acetic acid
obtained above (200 mg, 0.57 mmole) was combined with carbonyl
diimidazole (200 mg, 1.2 mmole) in dry THF (4 ml). The reaction was
allowed to stir at room temperature for 1 hour. To this solution
was added the corresponding alkyl or arylsulfonamide (2.2 mmole)
and DBU (2.2 mmole) as a solution in 3 ml THF. The reaction was
stirred overnight and the THF removed in vacuo. The residue was
taken up in ethyl acetate and acidified with acetic acid. The
organic layer was separated, washed with water and dried over
anhydrous sodium sulfate. The crude product was purified by flash
chromatography (Hexanes:ethyl acetate 1:2) to yield desired product
--N-{(4-cyano-phenylamino)-[4
isopropoxyl,5-ethoxy-phenyl]-acetyl}(alkyl or aryl)
sulfonamide.
[0251] The N-{(4-cyano-phenylamino)-[4
isopropoxyl-5-ethoxy-phenyl]-acetyl}(alkyl or aryl) sulfonamide
obtained above (ca. 0.24 mmole) was dissolved in ethanol (1-3 ml)
and heated to 60.degree. C. Diisopropylethylamine (DIPEA --260 ul,
1.5 mmole, 6 eq.) was added followed by hydroxylamine-hydrochloride
(84 mg, 1.25 mmole, 5 eq.). The reaction was stirred at 60.degree.
C. under a nitrogen atmosphere for ca. 6 hours. The reaction was
then allowed to cool to room temperature. The solution was diluted
with methanol (5 ml) and acetic acid (2 ml) and Raney Nickel 2800
(ca. 50 mg) added as a suspension. The reaction was then stirred
vigorously under a hydrogen atmosphere for 1-6 hours. The catalyst
was filtered off and the solvent removed. The crude products were
purified by preparative reverse-phase HPLC using a
water-acetonitrile (0.1% TFA) gradient or by flash chromatography
(ethyl acetate:acetone:water:acetic acid, 6:2:1:1) to yield the
desired 4-{2-(alkyl or aryl)
sulfonylamino-1-[4-isopropoxy,5-ethoxy)-phenyl]-2-oxo-ethylamino}benzamid-
ine as its trifluoroacetic acid or acetic acid salt.
[0252] Using an analogous procedure, the following compounds having
different R.sub.1 groups were prepared: [0253] R.sub.1=ethyl:
4-{2-ethylsulfonylamino
1-[4-isopropoxy,5-ethoxy)-phenyl]-2-oxo-ethylamino}benzamidine:
MS(M+H)=463. [0254] R.sub.1=n-propyl:
4-{2-propylsulfonylamino-1-[4-isopropoxy,5-ethoxy)-phenyl]-2-oxo-ethylami-
no}benzamidine: MS(M+H)=477. [0255] R.sub.1=n-butyl:
4-{2-butylsulfonylamino-1-[4-isopropoxy,5-ethoxy)-phenyl]-2-oxo-ethylamin-
o}benzamidine: MS(M+H)=491. [0256]
R.sub.1=CH.sub.2CH.sub.2CO.sub.2Me:
3-[(4-carbamimidoyl-phenylamino)-(3-ethoxy-4-isopropoxy-phenyl)-acetylsuf-
amoyl]-propionic acid methyl ester: MS(M+H)=521. [0257]
R.sub.1=phenyl:
4-{2-benzenesulfonylamino-1-[4-isopropoxy,5-ethoxy)-phenyl]-2-oxo-ethylam-
ino}benzamidine: MS(M+H)=511.
Example 13
Acylsulfonamide with Substitution on the Aminobenzamidine Ring
[0258] ##STR1030##
[0259] 2-Hydroxy-4-nitro-benzonitrile (11.2 g, 68 mmole) was
dissolved in DMF (200 ml). Potassium carbonate (11 g. 80 mmole) and
benzyl bromide (9 ml, 75 mmole) were added. The reaction was
stirred at room temperature overnight. The DMF was removed in vacuo
and the residue taken up in ethyl acetate and water. The organic
layer was separated, washed with 1 N NaOH, then with water, and
dried over sodium sulfate. The crude product (5 g) was dissolved in
ethyl acetate (75 ml) and added to a flask containing 5% Pt/C (500
mg). The reaction was placed under a hydrogen atmosphere (balloon)
and stirred vigorously for several hours until the reaction was
done (TLC). The catalyst was filtered off and the solvent removed.
The product was purified by flash chromatography to yield 4.12 g of
4-amino, 2-benzyloxybenzonitrile. ##STR1031##
[0260] 4,5-diethoxy-benzaldehyde (3.6 g, 17.8 mmole) and
4-amino-2-benzyloxybenzonitrile (3.7 g, 17.8 mmole) were dissolved
in methanol (40 ml) and stirred for 2 hours. Tosylmethylisonitrile
(3.48 g. 17.8 mmoles) was added. The reaction was cooled to
0.degree. C. and boron trifluoroetherate (6.7 ml, 54 mmoles) was
added dropwise. The reaction was stirred at 0.sup.oC for 30
minutes, allowed to come to room temperature and then stirred at
ambient temperature for 3.5 hours. Water (1.6 ml) was added and the
mixture stirred at room temperature 2 days. The methanol was
removed in vacuo and the residue taken up in ethyl acetate. The
organic layer was washed with water and then dried over anhydrous
sodium sulfate. The sodium sulfate was filtered off and the ethyl
acetate removed in vacuo. The crude material was submitted to flash
chromatography (hexanes:ethyl acetate, 4:1) to yield 4.2 g of the
desired product
(3-benzyloxy-4-cyano-phenylamino)-3,4-ethoxy-phenyl-)-acetic acid
methyl ester.
[0261] The product from above was treated with LiOH (1.96 g) in
water (50 ml) methanol (100 m), and THF (50 ml). The reaction was
stirred at room temperature for 3 hours and acidified with acetic
acid. The solvent was removed in vacuo and the product extracted
into ethyl acetate. The crude material was purified by reverse
phase chromatography (ethyl acetate 3% acetic acid) to yield 5 g of
desired acid--product (3-benzyloxy
-4-cyano-phenylamino)-3,4-ethoxy-phenyl-)-acetic acid.
##STR1032##
[0262] (3-benzyloxy-4-cyano-phenylamino)-3,4-ethoxy-phenyl-)-acetic
acid (750 mg, 1.68 mmoles) was dissolved in dry THF (3 ml) and
carbonyl diimidizole (CDI--545 mg, 3.36 mmoles) was added. The
reaction was heated to 40 degrees for 1 hour then cooled to room
temperature. To this was added a solution of benzenesulfonamide
(1.05 g, 6.7 mmoles), DBU (1.02 g 6.72 mmoles) in THF (3 ml). The
reaction was stirred overnight and then 1 N hydrochloric acid was
added. The THF was removed in vacuo and the product purified by
flash chromatography (ethyl acetate 2% acetic acid) to yield the
desired product. This material (215 mg) was dissolved in ethanol (5
ml) containing acetic acid (1 drop) and added to 5% Pd/C (100 mg).
The reaction was placed under a hydrogen atmosphere and stirred
vigorously until the reaction was complete. The catalyst was
filtered off and than solvent removed in vacuo. The crude product
was purified by flash chromatography to yield 150 mg of desired
product--N-[(3-hydroxy-4-cyano-phenylamino)-(3,4-diethoxy-phenyl-acetyl]b-
enzenesulfonamide. ##STR1033##
[0263] The
N-[(3-hydroxy-4-cyano-phenylamino)-(3,4-diethoxy-phenyl-acetyl]benzenesul-
fonamide obtained above (75 mg, 0.15 mmoles) was dissolved in
ethanol (2 ml) and heated to 60.degree. C. Diisopropylethylamine
(DIPEA--185 ul, 1 mmole) was added followed by
hydroxylamine-hydrochloride (53 mg, 0.75 mmole). The reaction was
stirred at 60.degree. C. under a nitrogen atmosphere for ca. 6
hours. The reaction was then allowed to cool to room temperature
overnight. The solution was diluted with acetic acid (1 ml) and
methanol (1 ml). Raney Nickel 2800 (ca. 50 mg) was added as a
suspension. The reaction was then stirred vigorously under a
hydrogen atmosphere for 0.5 hours. The catalyst was filtered off
and the solvent removed. The crude products were purified by
preparative reverse-phase HPLC using a water-acetonitrile (0.1%
TFA) gradient to yield the desired amidine
product--4-[2-benzenesulfonylamino-1-(3,4
diethoxy-phenyl)-2-oxo-ethylamino]-2-hydroxybenzamidine. MS
(M+H)=513.
[0264] Using an analogous procedure, the corresponding halogen
containing compounds can be made from 2-chloro-4-nitro-benzonitrile
and 2-bromo-4-nitro-benzonitrile.
Example 14
Tissue Factor/Factor VIIa Antagonist Assay
[0265] This procedure can be used to determine the constant of
inhibition (Ki) for a sample compound of the invention.
Materials:
[0266] Assay Buffer: 100 mM Hepes pH 7.8, 140 mM NaCl, 0.1%
PEG-8000, 0.02% Tween-80, 5 mM CaCl.sub.2 Coagulation Factor:
recombinant human factor VIIa (NB #25942-16) Cofactor: soluble
Tissue Factor (1-219) [0267] Substrate: Chromozym-tPA (Boehringer
Mannheim, Cat. #1093 037) Reconstitute at 20 mM in H.sub.2O. Dilute
to 4 mM in assay buffer with CaCl.sub.2 prior to use. Samples:
Dilute samples to 3% DMSO in assay buffer (lacking CaCl.sub.2).
Procedure: [0268] 1. Prepare a solution of 2 .mu.g/mL (90 nM)
tissue factor and 1.5 .mu.g/mL (30 nM) factor VIIa in assay buffer
with CaCl.sub.2. [0269] 2. Incubate for 15 minutes at room
temperature. [0270] 3. Add 50 .mu.L sample to each well. [0271] 4.
Add 50 .mu.L tissue factor/factor VIIa solution to each well.
[0272] 5. Incubate for 15 minutes at room temperature with gentle
agitation. [0273] 6. Add 50 .mu.L substrate to each well. [0274] 7.
Agitate plate for 20-25 sec. [0275] 8. Monitor absorbance at 405 nM
every 10 sec for a total of 5 minutes at room temperature. [0276]
9. Calculate Vmax over 10 points.
Example 15
Factor Xa, Thrombin, and Plasma Kallikrein Assays
[0277] These procedures can be used to determine the constant of
inhibition (Ki) for a sample compound of the invention.
Materials:
Assay Buffer: 100 mM Hepes pH 7.8, 140 mM NaCl, 0.1% PEG-8000,
0.02% Tween-80
Coagulation human Factor Xa, Thrombin, or Plasma Kallikrein
(Hematologic Technologies)
Factor: Dilute to 0.45 .mu.g/mL (9.8 nM) in assay buffer.
[0278] Substrate: S-2222, S2366 or S2302-(See below--Chromogenix
Inc,) Reconstitute at 5 mM in H.sub.2O. Dilute to 1.5 mM in assay
buffer prior to use. Samples: Dilute samples to 3% DMSO in assay
buffer. Procedure: [0279] 1. Add 50 .mu.L sample to each well.
[0280] 2. Add 50 .mu.L appropriately diluted coagulation factor to
each well. [0281] 3. Incubate for 5 minutes at room temperature
with gentle agitation. [0282] 4. Add 50 mL appropriately diluted
substrate to each well. [0283] 5. Agitate plate for 20-25 sec.
[0284] 6. Monitor absorbance at 405 nM every 10 sec for a total of
5 minutes at room temperature. [0285] 7. Calculate Vmax over 10
points.
[0286] Assay--Enzyme, Substrate and Final Concentrations
TABLE-US-00005 Assay TF/FVIIa FXa Thrombin PlasmaKallikrein Coag
Factor 10 nM FVIIa 3.3 nM 8.2 nM 1.5 nM Final 30 nM TF concen-
tration Substrate Chromozyme S-2222 S-2366 S-2302 tPA Final Conc.
1.33 mM 0.5 mM 0.3 mM 0.3 mM of Substrate
* * * * *