U.S. patent application number 11/032297 was filed with the patent office on 2005-07-21 for aminopyridinyl-, aminoguanidinyl- and alkoxyguanidinyl-substituted phenyl acetamides as protease inhibitors.
Invention is credited to Lu, Tianbao, Markotan, Thomas P., Pan, Wenxi, Tomczuk, Bruce E..
Application Number | 20050159457 11/032297 |
Document ID | / |
Family ID | 22896640 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050159457 |
Kind Code |
A1 |
Pan, Wenxi ; et al. |
July 21, 2005 |
Aminopyridinyl-, aminoguanidinyl- and alkoxyguanidinyl-substituted
phenyl acetamides as protease inhibitors
Abstract
Phenyl acetamide compounds are described, including compounds of
Formula I: 1 or a solvate, hydrate or pharmaceutically acceptable
salt thereof; wherein R.sup.3-R.sup.6, R.sup.11, B, Y and W are set
forth in the specification. The compounds of the invention are
potent inhibitors of proteases, especially trypsin-like serine
proteases, such as thrombin and factor Xa. Compositions for
inhibiting loss of blood platelets, inhibiting formation of blood
platelet aggregates, inhibiting formation of fibrin, inhibiting
thrombus formation, and inhibiting embolus formation are described.
Other uses of compounds of the invention are as anticoagulants
either embedded in or physically linked to materials used in the
manufacture of devices used in blood collection, blood circulation,
and blood storage, such as catheters, blood dialysis machines,
blood collection syringes and tubes, blood lines and stents.
Additionally, the compounds can be detectably labeled and employed
for in vivo imaging of thrombi.
Inventors: |
Pan, Wenxi; (Exton, PA)
; Lu, Tianbao; (Kennett Square, PA) ; Markotan,
Thomas P.; (Morgantown, PA) ; Tomczuk, Bruce E.;
(Collegeville, PA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE
46TH FLOOR
PHILADELPHIA
PA
19103
US
|
Family ID: |
22896640 |
Appl. No.: |
11/032297 |
Filed: |
January 10, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11032297 |
Jan 10, 2005 |
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10262871 |
Oct 3, 2002 |
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6900231 |
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10262871 |
Oct 3, 2002 |
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09971000 |
Oct 5, 2001 |
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6521663 |
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60238132 |
Oct 6, 2000 |
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Current U.S.
Class: |
514/352 ;
514/602; 514/614; 546/309; 564/147; 564/86 |
Current CPC
Class: |
C07C 311/13 20130101;
A61P 29/00 20180101; A61P 9/08 20180101; A61K 31/155 20130101; A61P
43/00 20180101; C07C 279/00 20130101; A61P 7/02 20180101; C07C
311/21 20130101; A61K 31/44 20130101; C07D 213/75 20130101; C07D
213/73 20130101; A61P 9/10 20180101; A61P 7/00 20180101; C07C
309/73 20130101; A61K 31/155 20130101; A61K 2300/00 20130101; A61K
31/44 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/352 ;
514/614; 514/602; 546/309; 564/086; 564/147 |
International
Class: |
A61K 031/44; A61K
031/165; C07C 311/45; C07C 279/20 |
Claims
What is claimed is:
1. A compound of Formula I: 108or a solvate, hydrate or
pharmaceutically acceptable salt thereof; wherein: W is hydrogen,
R.sup.1, R.sup.1OC(O), R.sup.1C(O), R.sup.1(CH.sub.2).sub.sNHC(O),
R.sup.1S(O).sub.2, or (R.sup.1).sub.2CH(CH.sub.2).sub.sNHC(O),
wherein s is 0-4; R.sup.1 is R.sup.2,
R.sup.2(CH.sub.2).sub.1C(R.sup.12).sub.2, where t is 0-3, and each
R.sup.12 can be the same or different,
(R.sup.2)(OR.sup.12)CH(CH.sub- .2).sub.p, where p is 1-4,
(R.sup.2).sub.2(OR.sup.12)C(CH.sub.2).sub.p, where p is 1-4,
R.sup.2C(R.sup.12).sub.2(CH.sub.2).sub.1, wherein t is 0-3, and
each R.sup.12 can be the same or different, wherein
(R.sup.12).sub.2 can also form a ring with C represented by
C.sub.3-9 cycloalkyl,
R.sup.2CF.sub.2C(R.sup.12).sub.2(CH.sub.2).sub.q, wherein q is 0-2,
and each R.sup.12 can be the same or different, wherein
(R.sup.12).sub.2 can also form a ring with C represented by
C.sub.3-9cycloalkyl,
R.sup.2CH.sub.2C(R.sup.12).sub.2(CH.sub.2).sub.q, wherein q is 0-2,
and each R.sup.12 can be the same or different, wherein
(R.sup.12).sub.2 can also form a ring with C represented by
C.sub.3-9 cycloalkyl, (R.sup.2).sub.2CH(CH.sub.2).sub.r, where r is
0-4 and each R.sup.2 can be the same or different, and wherein
(R.sup.2).sub.2 can also form a ring with CH represented by
C.sub.3-9 cycloalkyl, C.sub.7-12 bicylic alkyl, C.sub.10-16
tricylic alkyl, or a 5- to 7-membered mono- or bicyclic
heterocyclic ring which can be saturated or unsaturated, and which
contains from one to three heteroatoms selected from the group
consisting of N, O and S, R.sup.2O(CH.sub.2).sub.p, wherein p is
2-4, (R.sup.2).sub.2CF(CH.sub.2).sub.r, wherein r is 0-4 and each
R.sup.12 can be the same different, wherein (R.sup.12).sub.2 can
also form a ring with C represented by C.sub.3-9 cycloalkyl,
C.sub.7-12 bicyclic alkyl, C.sub.10-16 tricyclic alkyl, or a 5- to
7-membered mono- or bicyclic heterocyclic ring which can be
saturated or unsaturated, and which contains from one to three
heteroatoms selected from the group consisting of N, O and S,
109where s is 0 or 1, or R.sup.2CF.sub.2C(R.sup.12).sub.2- ;
R.sup.2 is phenyl, naphthyl, or biphenyl, each of which is
unsubstituted or substituted with one or more of C.sub.1-4 alkyl,
C.sub.1-4 alkoxy, halogen, hydroxy, CF.sub.3, OCF.sub.3, COOH,
CONH.sub.2, or SO.sub.2NH.sub.2, a 5- to 7-membered mono- or a 9-
to 10-membered bicyclic heterocyclic ring or non-heterocyclic ring
which can be saturated or unsaturated, wherein the heterocyclic
ring contains from one to four heteroatoms selected from the group
consisting of N, O and S, and wherein the heterocyclic or
non-heterocyclic ring is unsubstituted or substituted with halogen
or hydroxy, C.sub.1-12 alkyl, unsubstituted or substituted with one
or more of hydroxy, COOH, amino, optionally C.sub.1-3 alkyl
substituted aryl, C.sub.3-9 cycloalkyl, CF.sub.3,
N(CH.sub.3).sub.2, heteroaryl, or heterocycloalkyl, CF.sub.3,
C.sub.3-9 cycloalkyl, unsubstituted or substituted with aryl,
C.sub.7-12 bicyclic alkyl, or C.sub.10-16 tricyclic alkyl; Y is
--NH-- or --O--; R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are
independently hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl,
optionally substituted aryl, optionally substituted aralkyl,
optionally substituted heteroaryl, haloalkyl, hydroxy, alkoxy,
aryloxy, heteroaryloxy, halogen, haloalkoxy, hydroxyalkyl, cyano,
nitro, --CO.sub.2R.sup.x, --CH.sub.2OR.sup.x or --OR.sup.x, where
R.sup.x, in each instance, is independently one of hydrogen,
C.sub.1-12 alkyl or C.sub.3-9 cycloalkyl wherein said C.sub.1-12
alkyl or C.sub.3-9 cycloalkyl groups may optionally have one or
more unsaturations; R.sup.11 is hydrogen, alkyl, or alkenyl;
R.sup.12 is hydrogen or halogen, phenyl, naphthyl, or biphenyl,
each of which is unsubstituted or substituted with one or more of
C.sub.1-4 alkyl, C.sub.1-4 alkoxy, halogen, hydroxy, CF.sub.3,
OCF.sub.3, COOH, or CONH.sub.2, a 5- to 7-membered mono- or a 9- to
10-membered bicyclic heterocyclic ring which can be saturated or
unsaturated, and which contains from one to four heteroatoms
selected from the group consisting of N, O and S, C.sub.1-12 alkyl,
unsubstituted or substituted with one or more of hydroxy, COOH,
amino, C.sub.6-14 aryl, heteroaryl, or heterocycloalkyl, CF.sub.3,
C.sub.3-9 cycloalkyl, C.sub.7-12 bicyclic alkyl, or C.sub.10-16
tricyclic alkyl; B is selected from the group consisting of:
110wherein R.sup.7, R.sup.8, R.sup.9, and R.sup.10 are
independently hydrogen, alkyl, aralkyl, aryl, hydroxyalkyl,
aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl or carboxyalkyl;
or R.sup.7 and R.sup.8 are taken together to form
--(CH.sub.2).sub.u--, where u is 2 to 7, preferably 2 to 5, while
R.sup.9 and R.sup.10 are defined as above; or R.sup.9 and R.sup.10
are taken together to form --(CH.sub.2).sub.v--, where v is 2 to 7,
preferably 2 to 5, while R.sup.7 and R.sup.8 are defined as above;
or R.sup.7 and R.sup.9 are taken together to form
--(CH.sub.2).sub.y--, where y is 0 (a bond) or 1 to 7, preferably
0-4, while R.sup.8 and R.sup.10 are defined as above; X is --O--,
--N.sup.18--, or --CH.dbd.N-- (where N is bonded to NR.sup.13)
where R.sup.18 is hydrogen, alkyl, cycloalkyl or aryl, wherein said
alkyl, cycloalkyl or aryl are optionally substituted with amino,
monoalkylamino, dialkylamino, alkoxy, hydroxy, carboxy,
alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, aryl,
heteroaryl, acylamino, cyano or trifluoromethyl; R.sup.a, R.sup.b
and R.sup.c are independently hydrogen, alkyl, hydroxy, alkoxy,
aryloxy, aralkoxy, alkoxycarbonyloxy, cyano or --CO.sub.2R.sup.w,
where R.sup.w is C.sub.1-12 alkyl, C.sub.3-9 cycloalkyl, C.sub.6-14
aryl, C.sub.6-14ar(C.sub.1-12) alkyl, 111where R.sup.e and R.sup.f
are independently hydrogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl or
C.sub.6-14 aryl, R.sup.g is hydrogen, C.sub.1-6 alkyl, C.sub.2-6
alkenyl or C.sub.6-14 aryl, R.sup.h is hydrogen, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl or C.sub.6-14 aryl, and R.sup.i is C.sub.6-14
ar(C.sub.1-12)alkyl or C.sub.1-6 alkyl; n is from zero to 8; and m
is from zero to 6; R.sup.13 is hydrogen, alkyl, alkenyl, aralkyl,
aryl, hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl,
dialkylaminoalkyl or carboxyalkyl; R.sup.14 and R.sup.15 are
independently hydrogen, alkyl, cycloalkyl, halogen or alkoxy; and
R.sup.16 and R.sup.17 are independently hydrogen, alkyl, hydroxy,
alkoxy, aryloxy, alkoxycarbonyl, cyano or --CO.sub.2R.sup.j, where
R.sup.j is C.sub.1-12 alkyl, C.sub.3-9 cycloalkyl, C.sub.6-14 aryl,
C.sub.6-14ar(C.sub.1-12)alkyl, halo(C.sub.1-12)alkyl or 112where
R.sup.e, R.sup.f and R.sup.g are independently hydrogen or
C.sub.1-12 alkyl.
2. A compound of claim 1, wherein R.sup.3, R.sup.4, R.sup.5 and
R.sup.6 are independently hydrogen, C.sub.1-12 alkyl, C.sub.3-9
cycloalkyl, halogen, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl,
optionally substituted C.sub.6-14 aryl, optionally substituted
C.sub.6-14ar(C.sub.1-12)alkyl, optionally substituted heteroaryl,
halo(C.sub.1-12)alkyl, C.sub.1-12 alkoxy, C.sub.6-14 aryloxy,
heteroaryloxy, halo(C.sub.1-20)alkoxy or hydroxy(C.sub.1-12)alkyl;
R.sup.11 is hydrogen, C.sub.1-12 alkyl or C.sub.2-20 alkenyl;
R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are independently hydrogen,
C.sub.1-12 alkyl, C.sub.6-14ar(C.sub.1-12)alkyl, C.sub.6-14aryl,
hydroxy(C.sub.1-12)alkyl, amino(C.sub.1-12)alkyl,
mono(C.sub.1-12)alkylamino(C.sub.1-12)alkyl,
di(C.sub.1-12)alkylamino(C.s- ub.1-12)alkyl, or
carboxy(C.sub.1-12)alkyl; R.sup.18 is C.sub.1-12 alkyl, C.sub.3-9
cycloalkyl or C.sub.6-14 aryl, each of which is optionally
substituted with amino, mono(C.sub.1-12)alkylamino,
di(C.sub.1-12)alkylamino, C.sub.1-20 alkoxy, hydroxy, carboxy,
C.sub.1-20alkoxycarbonyl, C.sub.6-14 aryloxycarbonyl,
C.sub.6-14ar(C.sub.1-20)alkoxycarbonyl, C.sub.6-14 aryl, C.sub.5-10
heteroaryl, acylamino, cyano or trifluoromethyl; R.sup.a, R.sup.b
and R.sup.c are independently C.sub.1-12 alkyl, C.sub.1-20 alkoxy,
C.sub.6-14 aryloxy, C.sub.6-14ar(C.sub.1-20)alkoxy, or C.sub.1-20
alkoxycarbonyloxy; R.sup.13 is C.sub.1-12 alkyl, C.sub.1-20 alkoxy,
C.sub.6-14 aryloxy or C.sub.1-20 alkoxycarbonyl; R.sup.14 and
R.sup.15 are independently C.sub.1-12 alkyl, C.sub.3-9 cycloalkyl
or C.sub.1-20 alkoxy; and R.sup.16 and R.sup.17 are independently
C.sub.1-12 alkyl, C.sub.1-20 alkoxy, C.sub.6-14 aryloxy or
C.sub.1-20 alkoxycarbonyl.
3. A compound according to claim 1, wherein B is 113
4. A compound according to claim 1, wherein B is 114
5. A compound according to claim 1, wherein Y is --NH--.
6. A compound according to claim 1, wherein W is R.sup.1 or
R.sup.1S(O).sub.2, where R.sup.1 is R.sup.2 and R.sup.2 is either
optionally substituted aryl or C.sub.1-7 alkyl substituted with
aryl.
7. A compound according to claim 1, wherein W is R.sup.1, where
R.sup.1 is R.sup.2 or
R.sup.2CF.sub.2C(R.sup.12).sub.2(CH.sub.2).sub.q, and R.sup.2 is
either optionally substituted aryl or C.sub.1-7 alkyl substituted
with aryl; R.sup.12 is hydrogen; and q is zero.
8. A compound according to claim 1, wherein R.sup.6 is C.sub.1-6
alkyl or halogen.
9. A compound according to claim 8, wherein R.sup.6 is methyl,
chloro or fluoro.
10. A compound according to claim 9, wherein R.sup.6 is chloro
while R.sup.3 is fluoro or hydroxy.
11. A compound according to claim 1, wherein R.sup.11 is
hydrogen.
12. A compound according to claim 1, wherein R.sup.a, R.sup.b,
R.sup.c and R.sup.13 are each hydrogen.
13. A compound according to claim 1, wherein each of R.sup.7,
R.sup.8, R.sup.9 and R.sup.10 are hydrogen.
14. A compound of claim 1, which is one of:
N-[2-Amidinoaminooxy)ethyl]-2--
{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}acetamide;
N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-{3-[(2,2-difluoro-2-phenylethyl-
)amino]-6-chloro-2-fluorophenyl}acetamide;
N-(6-Amino-2,4-dimethyl(3-pyrid-
yl))methyl]-2-{3-[(2,2-difluoro-2-phenylethyl}acetamide;
N-[2-(Amidinoaminooxy)ethyl]-2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethy-
l]amino}-6-chloro-2-fluorophenyl)acetamide;
N-[(6-Amino-2-methyl(3-pyridyl-
))methyl]-2-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-flu-
orophenyl)acetamide; N-2-(guanidinooxy)ethyl]-2-(3
{[benzylsulfonyl]amino}- phenyl)acetamide;
N-[2-(Guanidinooxy)ethyl]-2-(2-chloro-5-{[benzylsulfonyl-
]amino}phenyl)acetamide;
N-[2-(Guanidinooxy)ethyl]-2-(2-methyl-5-{[benzyls-
ulfonyl]amino}phenyl)acetamide;
N-[2-(Guanidinooxy)ethyl]-2-(2-hydroxy-6-m-
ethyl-3-{[(3-methylphenyl)sulfonyl]amino}phenyl)acetamide;
N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-(2-hydroxy-6-methyl-3-{[(3-meth-
ylphenyl)sulfonyl]amino}phenyl)acetamide; or
N-({N-[2-(Guanidinooxy)ethyl]-
carbamoyl}methyl)-2-hydroxy-4-methylphenyl
3-methylbenzenesulfonate; or a solvate, hydrate or pharmaceutically
acceptable salt thereof
15. A pharmaceutical composition, comprising a compound of claim 1
and a pharmaceutically-acceptable carrier.
16. A pharmaceutical composition, comprising a compound of claim 3
and a pharmaceutically-acceptable carrier.
17. A pharmaceutical composition, comprising a compound of claim 4
and a pharmaceutically-acceptable carrier.
18. A pharmaceutical composition, comprising a compound of claim 14
and a pharmaceutically-acceptable carrier.
19. A pharmaceutical composition according to claim 15, further
comprising at least one of an anticoagulant, an antiplatelet agent
or a thrombolytic agent.
20. A pharmaceutical composition according to claim 15, wherein
said compound is present in an amount between about 0.1 and about
500 mg.
21. A method of inhibiting or treating aberrant proteolysis,
thrombosis, ischemic, stroke, restenosis or inflammation in a
mammal in need thereof, comprising administering to said mammal an
effective amount of a compound of claim 1.
22. A method of inhibiting or treating aberrant proteolysis,
thrombosis, ischemic, stroke, restenosis or inflammation in a
mammal in need thereof, comprising administering to said mammal an
effective amount of a compound of claim 3.
23. A method of inhibiting or treating aberrant proteolysis,
thrombosis, ischemic, stroke, restenosis or inflammation in a
mammal in need thereof, comprising administering to said mammal an
effective amount of a compound of claim 4.
24. A method of inhibiting or treating aberrant proteolysis,
thrombosis, ischemic, stroke, restenosis or inflammation in a
mammal in need thereof, comprising administering to said mammal an
effective amount of a compound of claim 14.
25. A method for the treatment or prophylaxis of states
characterized by abnormal venous or arterial thrombosis involving
either thrombin production or action in a mammal in need thereof,
comprising administering to said mammal a composition of claim
15.
26. A method for the treatment or prophylaxis of states
characterized by abnormal venous or arterial thrombosis involving
either thrombin production or action in a mammal in need thereof,
comprising administering to said mammal a composition of claim
16.
27. A method for the treatment or prophylaxis of states
characterized by abnormal venous or arterial thrombosis involving
either thrombin production or action in a mammal in need thereof,
comprising administering to said mammal a composition of claim
17.
28. A method for the treatment or prophylaxis of states
characterized by abnormal venous or arterial thrombosis involving
either thrombin production or action in a mammal in need thereof,
comprising administering to said mammal a composition of claim
18.
29. A method for the treatment or prophylaxis of states
characterized by abnormal venous or arterial thrombosis involving
either thrombin production or action in a mammal in need thereof,
comprising administering to said mammal a composition of claim
19.
30. A method for the treatment or prophylaxis of states
characterized by abnormal venous or arterial thrombosis involving
either thrombin production or action in a mammal in need thereof,
comprising administering to said mammal a composition of claim
20.
31. A medical device for use in blood collection, blood storage or
blood circulation, comprising a compound of claim 1 embedded in or
physically attached to said medical device.
32. A medical device for use in blood collection, blood storage or
blood circulation, comprising a compound of claim 3 embedded in or
physically attached to said medical device.
33. A medical device for use in blood collection, blood storage or
blood circulation, comprising a compound of claim 4 embedded in or
physically attached to said medical device.
34. A medical device according to claim 31, which is a catheter,
stent, blood dialysis machine, blood collection syringe or tube, or
a blood line.
35. A method of inhibiting the action of a proteolytic enzyme,
comprising contacting said enzyme with a compound of claim 1.
36. A method of inhibiting the action of a proteolytic enzyme,
comprising contacting said enzyme with a compound of claim 3.
37. A method of inhibiting the action of a proteolytic enzyme,
comprising contacting said enzyme with a compound of claim 4.
38. A method according to claim 35, wherein said enzyme is
leukocyte neutrophil elastase, chymotrypsin, trypsin, urokinase,
plasminogen activator, pancreatic elastase, cathepsin G, thrombin
or factor Xa.
39. A method according to claim 36, wherein said enzyme is
leukocyte neutrophil elastase, chymotrypsin, trypsin, urokinase,
plasminogen activator, pancreatic elastase, cathepsin G, thrombin
or factor Xa.
40. A method according to claim 37, wherein said enzyme is
leukocyte neutrophil elastase, chymotrypsin, trypsin, urokinase,
plasminogen activator, pancreatic elastase, cathepsin G, thrombin
or factor Xa.
41. A pharmaceutical composition according to claim 15 adapted for
oral administration.
42. A pharmaceutical composition according to claim 16 adapted for
oral administration.
43. A pharmaceutical composition according to claim 17 adapted for
oral administration.
44. A pharmaceutical composition according to claim 18 adapted for
oral administration.
45. A pharmaceutical composition according to claim 19 adapted for
oral administration.
46. A pharmaceutical composition according to claim 20 adapted for
oral administration.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to novel compounds that
function as proteolytic enzyme inhibitors, and particularly to a
new class of thrombin inhibitors.
[0003] 2. Related Art
[0004] Proteases are enzymes that cleave proteins at single,
specific peptide bonds. Proteases can be classified into four
generic classes: serine, thiol or cysteinyl, acid or aspartyl, and
metalloproteases (Cuypers et al., J. Biol. Chem. 257: 7086 (1982)).
Proteases are essential to a variety of biological activities, such
as digestion, formation and dissolution of blood clots,
reproduction and the immune reaction to foreign cells and
organisms. Aberrant proteolysis is associated with a number of
disease states in man and other mammals. The human neutrophil
proteases, elastase and cathepsin G, have been implicated as
contributing to disease states marked by tissue destruction. These
disease states include emphysema, rheumatoid arthritis, corneal
ulcers and glomerular nephritis. (Barret, in Enzyme Inhibitors as
Drugs, Sandler, ed., University Park Press, Baltimore, (1980)).
Additional proteases such as plasmin, C-1 esterase, C-3 convertase,
urokinase, plasminogen activator, acrosin, and kallikreins play key
roles in normal biological functions of mammals. In many instances,
it is beneficial to disrupt the function of one or more proteolytic
enzymes in the course of therapeutically treating a mammal.
[0005] Serine proteases include such enzymes as elastase (human
leukocyte), cathepsin G, plasmin, C-1 esterase, C-3 convertase,
urokinase, plasminogen activator, acrosin, chymotrypsin, trypsin,
thrombin, factor Xa and kallikreins.
[0006] Human leukocyte elastase is released by polymorphonuclear
leukocytes at sites of inflammation and thus is a contributing
cause for a number of disease states. Cathepsin G is another human
neutrophil serine protease. Compounds with the ability to inhibit
the activity of these enzymes are expected to have an
anti-inflammatory effect useful in the treatment of gout,
rheumatoid arthritis and other inflammatory diseases, and in the
treatment of emphysema. Chymotrypsin and trypsin are digestive
enzymes. Inhibitors of these enzymes are useful in treating
pancreatitis. Inhibitors of urokinase and plasminogen activator are
useful in treating excessive cell growth disease states, such as
benign prostatic hypertrophy, prostatic carcinoma and
psoriasis.
[0007] The serine protease thrombin occupies a central role in
hemostasis and thrombosis, and as a multifactorial protein, induces
a number of effects on platelets, endothelial cells, smooth muscle
cells, leukocytes, the heart, and neurons. Activation of the
coagulation cascade through either the intrinsic pathway (contact
activation) or the extrinsic pathway (activation by exposure of
plasma to a non-endothelial surface, damage to vessel walls or
tissue factor release) leads to a series of biochemical events that
converge on thrombin. Thrombin cleaves fibrinogen ultimately
leading to a hemostatic plug (clot formation), potently activates
platelets through a unique proteolytic cleavage of the cell surface
thrombin receptor (Coughlin, Seminars in Hematology 31(4): 270-277
(1994)), and autoamplifies its own production through a feedback
mechanism. Thus, inhibitors of thrombin function have therapeutic
potential in a host of cardiovascular and non-cardiovascular
diseases.
[0008] Factor Xa is another serine protease in the coagulation
pathway. Factor Xa associates with factor Va and calcium on a
phospholipid membrane thereby forming a prothrombinase complex.
This prothrombinase complex then converts prothrombin to thrombin
(Claeson, Blood Coagulation and Fibrinolysis 5: 411-436 (1994);
Harker, Blood Coagulation and Fibrinolysis 5 (Suppl 1): S47-S58
(1994)). Inhibitors of factor Xa are thought to offer an advantage
over agents that directly inhibit thrombin since direct thrombin
inhibitors still permit significant new thrombin generation
(Lefkovits and Topol, Circulation 90(3): 1522-1536 (1994); Harker,
Blood Coagulation and Fibrinolysis 5 (Suppl 1): S47-S58
(1994)).
[0009] In vivo diagnostic imaging methods for intravascular thrombi
have been previously reported. These imaging methods use compounds
that are detectably labeled with radioactive or paramagnetic atoms.
For example, platelets labeled with the gamma emitter, In-111, can
be employed as an imaging agent for detecting thrombi (Thakur, M.
L. et al., Thromb Res. 9: 345 (1976); Powers et al., Neurology 32:
938 (1982)). The thrombolytic enzyme streptokinase labeled with
Tc-99m has been proposed as an imaging agent (Wong, U.S. Pat. No.
4,418,052 (1983)). The fibrin-binding domains of Staphylococcus
aureus derived protein A labeled with the gamma emitters, I-125 and
I-131, have been proposed as imaging agents (Pang, U.S. Pat. No.
5,011,686 (1991)). Monoclonal antibodies having specificity for
fibrin (in contrast to fibrinogen) and labeled with Tc-99m have
been proposed as imaging agents (Berger et al., U.S. Pat. No.
5,024,829 (1991); Dean et al., U.S. Pat. No. 4,980,148 (1990)). The
use of the paramagnetic contrasting agent, gadolinium
diethylenetriaminepentaacetic acid in magnetic resonance imaging of
patients treated by thrombolysis for acute myocardial infarction
has been reported (De Roos, A. et al., Int. J. Card. Imaging 7: 133
(1991)). Radiolabeled and paramagnetically labeled alpha-ketoamide
derivatives have also been proposed as thrombus imaging agents
(Abelman et al., U.S. Pat. No. 5,656,600).
[0010] A need continues to exist for non-peptidic compounds that
are potent and selective protease inhibitors, and which possess
greater bioavailability and fewer side-effects than currently
available protease inhibitors. Accordingly, new classes of potent
protease inhibitors, characterized by potent inhibitory capacity
and low mammalian toxicity, are potentially valuable therapeutic
agents for a variety of conditions, including treatment of a number
of mammalian proteolytic disease states.
SUMMARY OF THE INVENTION
[0011] The present invention is directed to novel aminopyridinyl-,
aminoguanidinyl-, and alkoxyguanidinyl-substituted phenyl
acetamides having Formula I (below). Also provided are processes
for preparing compounds of Formula I. The novel compounds of the
present invention are potent inhibitors of proteases, especially
trypsin-like serine proteases, such as chymotrypsin, trypsin,
thrombin, plasmin and factor Xa. Certain of the compounds exhibit
antithrombotic activity via direct, selective inhibition of
thrombin, or are intermediates useful for forming compounds having
antithrombotic activity. Also provided are methods of inhibiting or
treating aberrant proteolysis in a mammal and methods of treating
thrombosis, ischemia, stroke, restenosis or inflammation in a
mammal by administering an effective amount of a compound of
Formula I.
[0012] The invention includes a composition for inhibiting loss of
blood platelets, inhibiting formation of blood platelet aggregates,
inhibiting formation of fibrin, inhibiting thrombus formation, and
inhibiting embolus formation in a mammal, comprising a compound of
the invention in a pharmaceutically acceptable carrier. These
compositions may optionally include anticoagulants, antiplatelet
agents, and thrombolytic agents. The compositions can be added to
blood, blood products, or mammalian organs in order to effect the
desired inhibitions.
[0013] Also provided are methods of inhibiting or treating aberrant
proteolysis in a mammal, and methods for treating myocardial
infarction; unstable angina; stroke; restenosis; deep vein
thrombosis; disseminated intravascular coagulation caused by
trauma, sepsis or tumor metastasis; hemodialysis; cardiopulmonary
bypass surgery; adult respiratory distress syndrome; endotoxic
shock; rheumatoid arthritis; ulcerative colitis; induration;
metastasis; hypercoagulability during chemotherapy; Alzheimer's
disease; Down's syndrome; fibrin formation in the eye; and wound
healing. Other uses of compounds of the invention are as
anticoagulants either embedded in or physically linked to materials
used in the manufacture of devices used in blood collection, blood
circulation, and blood storage, such as catheters, blood dialysis
machines, blood collection syringes and tubes, blood lines and
stents.
[0014] The invention also includes a method for reducing the
thrombogenicity of a surface in a mammal by attaching to the
surface, either covalently or noncovalently, a compound of the
invention.
[0015] In another aspect, the present invention includes
compositions which are useful for in vivo imaging of thrombi in a
mammal, comprising a compound of the present invention which is
capable of being detected outside the body. Preferred are
compositions comprising a compound of the present invention and a
detectable label, such as a radioactive or paramagnetic atom.
[0016] In another aspect, the present invention provides diagnostic
compositions which are useful for in vivo imaging of thrombi in a
mammal, comprising a pharmaceutically acceptable carrier and a
diagnostically effective amount of a compound or composition of the
present invention.
[0017] In another aspect, the present invention includes methods
which are useful for in vivo imaging of thrombi in a mammal.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0018] Compounds of the present invention include compounds of
Formula I: 2
[0019] or a solvate, hydrate or pharmaceutically acceptable salt
thereof; wherein:
[0020] W is hydrogen, R.sup.1, R.sup.1OC(O), R.sup.1C(O),
R.sup.1(CH.sub.2).sub.sNHC(O), R.sup.1S(O).sub.2, or
(R.sup.1).sub.2CH(CH.sub.2).sub.sNHC(O), wherein s is 0-4;
[0021] R.sup.1 is
[0022] R.sup.2,
[0023] R.sup.2(CH.sub.2).sub.tC(R.sup.12).sub.2, where t is 0-3,
and each R.sup.12 can be the same or different,
[0024] (R.sup.2)(OR.sup.12)CH(CH.sub.2).sub.p, where p is 1-4,
[0025] (R.sup.2).sub.2(OR.sup.12)C(CH.sub.2).sub.p, where p is
1-4,
[0026] R.sup.2C(R.sup.12).sub.2(CH.sub.2).sub.t, wherein t is 0-3,
and each R.sup.12 can be the same or different, wherein
(R.sup.12).sub.2 can also form a ring with C represented by
C.sub.3-9 cycloalkyl,
[0027] R.sup.2CF.sub.2C(R.sup.12).sub.2(CH.sub.2).sub.q, wherein q
is 0-2, and each R.sup.12 can be the same or different, wherein
(R.sup.12).sub.2 can also form a ring with C represented by
C.sub.3-9cycloalkyl,
[0028] R.sup.2CH.sub.2C(R.sup.12).sub.2(CH.sub.2).sub.q, wherein q
is 0-2, and each R.sup.12 can be the same or different, wherein
(R.sup.12).sub.2 can also form a ring with C represented by
C.sub.3-9 cycloalkyl,
[0029] (R.sup.2).sub.2CH(CH.sub.2).sub.r, where r is 0-4 and each
R.sup.2 can be the same or different, and wherein (R.sup.2).sub.2
can also form a ring with CH represented by C.sub.3-9 cycloalkyl,
C.sub.7-12 bicylic alkyl, C.sub.10-16 tricylic alkyl, or a 5- to
7-membered mono- or bicyclic heterocyclic ring which can be
saturated or unsaturated, and which contains from one to three
heteroatoms selected from the group consisting of N, O and S,
[0030] R.sup.2O(CH.sub.2).sub.p, wherein p is 2-4,
[0031] (R.sup.2).sub.2CF(CH.sub.2).sub.r, wherein r is 0-4 and each
R.sup.2 can be the same different, wherein (R.sup.2).sub.2 can also
form a ring with C represented by C.sub.3-9 cycloalkyl, C.sub.7-12
bicyclic alkyl, C.sub.10-16 tricyclic alkyl, or a 5- to 7-membered
mono- or bicyclic heterocyclic ring which can be saturated or
unsaturated, and which contains from one to three heteroatoms
selected from the group consisting of N, O and S, 3
[0032] where s is 0 or 1, or
[0033] R.sup.2CF.sub.2C(R.sup.12).sub.2;
[0034] R.sup.2 is
[0035] phenyl, naphthyl, or biphenyl, each of which is
unsubstituted or substituted with one or more of C.sub.1-4 alkyl,
C.sub.1-4 alkoxy, halogen, hydroxy, CF.sub.3, OCF.sub.3, COOH,
CONH.sub.2, or SO.sub.2NH.sub.2,
[0036] a 5- to 7-membered mono- or a 9- to 10-membered bicyclic
heterocyclic ring or non-heterocyclic ring which can be saturated
or unsaturated, wherein the heterocyclic ring contains from one to
four heteroatoms selected from the group consisting of N, O and S,
and wherein the heterocyclic or non-heterocyclic ring is
unsubstituted or substituted with halogen or hydroxy,
[0037] C.sub.1-12 alkyl, unsubstituted or substituted with one or
more of hydroxy, COOH, amino, optionally C.sub.1-3 alkyl
substituted aryl, C.sub.3-9 cycloalkyl, CF.sub.3,
N(CH.sub.3).sub.2, heteroaryl, or heterocycloalkyl,
[0038] CF.sub.3,
[0039] C.sub.3-9 cycloalkyl, unsubstituted or substituted with
aryl,
[0040] C.sub.7-12 bicyclic alkyl, or
[0041] C.sub.10-16 tricyclic alkyl;
[0042] Y is --NH-- or --O--;
[0043] R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are independently
hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, optionally
substituted aryl, optionally substituted aralkyl, optionally
substituted heteroaryl, haloalkyl, hydroxy, alkoxy, aryloxy,
heteoraryloxy, halogen, haloalkoxy, hydroxyalkyl, cyano, nitro,
--CO.sub.2R.sup.x, --CH.sub.2OR.sup.x or --OR.sup.x,
[0044] where R.sup.x, in each instance, is independently one of
hydrogen, C.sub.1-12 alkyl or C.sub.3-9 cycloalkyl wherein said
C.sub.1-12 alkyl or C.sub.3-9 cycloalkyl groups may optionally have
one or more unsaturations;
[0045] R.sup.11 is hydrogen, alkyl, or alkenyl;
[0046] R.sup.12 is
[0047] hydrogen or halogen,
[0048] phenyl, naphthyl, or biphenyl, each of which is
unsubstituted or substituted with one or more of C.sub.1-4 alkyl,
C.sub.1-4 alkoxy, halogen, hydroxy, CF.sub.3, OCF.sub.3, COOH, or
CONH.sub.2,
[0049] a 5- to 7-membered mono- or a 9- to 10-membered bicyclic
heterocyclic ring which can be saturated or unsaturated, and which
contains from one to four heteroatoms selected from the group
consisting of N, O and S,
[0050] C.sub.1-12 alkyl, unsubstituted or substituted with one or
more of hydroxy, COOH, amino, C.sub.6-14 aryl, heteroaryl, or
heterocycloalkyl,
[0051] CF.sub.3,
[0052] C.sub.3-9 cycloalkyl,
[0053] C.sub.7-12 bicyclic alkyl, or
[0054] C.sub.10-16 tricyclic alkyl;
[0055] B is selected from the group consisting of: 4
[0056] wherein
[0057] R.sup.7, R.sup.8, R.sup.9, and R.sup.10 are independently
hydrogen, alkyl, aralkyl, aryl, hydroxyalkyl, aminoalkyl,
monoalkylaminoalkyl, dialkylaminoalkyl or carboxyalkyl;
[0058] or R.sup.7 and R.sup.8 are taken together to form
--(CH.sub.2).sub.u--, where u is 2 to 7, preferably 2 to 5, while
R.sup.9 and R.sup.10 are defined as above;
[0059] or R.sup.9 and R.sup.10 are taken together to form
--(CH.sub.2).sub.v--, where v is 2 to 7, preferably 2 to 5, while
R.sup.7 and R.sup.8 are defined as above;
[0060] or R.sup.7 and R.sup.9 are taken together to form
--(CH.sub.2).sub.y--, where y is 0 (a bond) or 1 to 7, preferably
0-4, while R.sup.8 and R.sup.10 are defined as above;
[0061] X is --O--, --NR.sup.18--, or --CH.dbd.N-- (where N is
bonded to NR.sup.13) where R.sup.18 is hydrogen, alkyl, cycloalkyl
or aryl, wherein said alkyl, cycloalkyl or aryl are optionally
substituted with amino, monoalkylamino, dialkylamino, alkoxy,
hydroxy, carboxy, alkoxycarbonyl, aryloxycarbonyl,
aralkoxycarbonyl, aryl, heteroaryl, acylamino, cyano or
trifluoromethyl;
[0062] R.sup.a, R.sup.b and R.sup.c are independently hydrogen,
alkyl, hydroxy, alkoxy, aryloxy, aralkoxy, alkoxycarbonyloxy, cyano
or --CO.sub.2R.sup.w, where R.sup.w is C.sub.1-12 alkyl, C.sub.3-9
cycloalkyl, C.sub.6-14 aryl, C.sub.6-14ar(C.sub.1-12) alkyl, 5
[0063] where R.sup.e and R.sup.f are independently hydrogen,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.6-14 aryl, R.sup.g is
hydrogen, C.sub.1-4 alkyl, C.sub.2-6 alkenyl or C.sub.6-14 aryl,
R.sup.h is hydrogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl or
C.sub.6-14 aryl, and R.sup.j is C.sub.6-14ar(C.sub.1-12)alkyl or
C.sub.1-12 alkyl;
[0064] n is from zero to 8; and
[0065] m is from zero to 6;
[0066] R.sup.13 is hydrogen, alkyl, alkenyl, aralkyl, aryl,
hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl or
carboxyalkyl;
[0067] R.sup.14 and R.sup.15 are independently hydrogen, alkyl,
cycloalkyl, halogen or alkoxy; and
[0068] R.sup.16 and R.sup.17 are independently hydrogen, alkyl,
hydroxy, alkoxy, aryloxy, alkoxycarbonyl, cyano or
--CO.sub.2R.sup.j, where R.sup.j is C.sub.1-12 alkyl, C.sub.3-9
cycloalkyl, C.sub.6-14 aryl, C.sub.6-14ar(C.sub.1-12)alkyl,
halo(C.sub.1-12)alkyl or 6
[0069] where R.sup.e, R.sup.f and R.sup.g are independently
hydrogen or C.sub.1-12 alkyl.
[0070] Compounds within the scope of the present invention include
those for which:
[0071] R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are independently
hydrogen, C.sub.1-12 alkyl, C.sub.3-9 cycloalkyl, halogen,
C.sub.2-20 alkenyl, C.sub.2-20 alkynyl, optionally substituted
C.sub.6-14 aryl, optionally substituted
C.sub.6-14ar(C.sub.1-12)alkyl, optionally substituted heteroaryl,
halo(C.sub.1-12)alkyl, C.sub.1-12 alkoxy, C.sub.6-14 aryloxy,
heteroaryloxy, halo(C.sub.1-20)alkoxy or
hydroxy(C.sub.1-12)alkyl;
[0072] R.sup.11 is hydrogen, C.sub.1-12 alkyl or C.sub.2-20
alkenyl;
[0073] R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are independently
hydrogen, C.sub.1-12 alkyl, C.sub.6-14ar(C.sub.1-12)alkyl,
C.sub.6-14 aryl, hydroxy(C.sub.1-12)alkyl, amino(C.sub.1-12)alkyl,
mono(C.sub.1-12)alkylam- ino(C.sub.1-12)alkyl,
di(C.sub.1-12)alkylamino(C.sub.1-12)alkyl, or
carboxy(C.sub.1-12)alkyl;
[0074] R.sup.18 is C.sub.1-12 alkyl, C.sub.3-9 cycloalkyl or
C.sub.6-14 aryl, each of which is optionally substituted with
amino, mono(C.sub.1-12)alkylamino, di(C.sub.1-12)alkylamino,
C.sub.1-20 alkoxy, hydroxy, carboxy, C.sub.1-20 alkoxycarbonyl,
C.sub.6-14 aryloxycarbonyl, C.sub.6-14ar(C.sub.1-20)alkoxycarbonyl,
C.sub.6-14aryl, C.sub.5-10heteroaryl, acylamino, cyano or
trifluoromethyl;
[0075] R.sup.a, R.sup.b and R.sup.c are independently C.sub.1-12
alkyl, C.sub.1-20 alkoxy, C.sub.6-14 aryloxy,
C.sub.6-14ar(C.sub.1-20)alkoxy, or C.sub.1-20
alkoxycarbonyloxy;
[0076] R.sup.13 is C.sub.1-12 alkyl, C.sub.1-20 alkoxy, C.sub.6-14
aryloxy or C.sub.1-20 alkoxycarbonyl;
[0077] R.sup.14 and R.sup.15 are independently C.sub.1-12 alkyl,
C.sub.3-9 cycloalkyl or C.sub.1-20 alkoxy; and
[0078] R.sup.16 and R.sup.17 are independently C.sub.1-12 alkyl,
C.sub.1-20 alkoxy, C.sub.6-14 aryloxy or C.sub.1-20
alkoxycarbonyl.
[0079] Preferred compounds of Formula I above are those for which Y
is --NH-- or --SO.sub.2NH--.
[0080] A preferred subgenus of compounds of Formula I above are
those for which B is 7
[0081] where R.sup.7-R.sup.10, R.sup.13 and R.sup.a-R.sup.c are as
defined above.
[0082] Another preferred subgenus of compounds of Formula I above
are those for which B is 8
[0083] where R.sup.9, R.sup.10 and R.sup.14-R.sup.17 are as defined
above.
[0084] Preferred compounds of Formula I above are those for which W
is R.sup.1, where R.sup.1 is R.sup.2 and R.sup.2 is either
optionally substituted phenyl, optionally substituted naphthyl or
C.sub.1-7 alkyl substituted with aryl.
[0085] Preferred compounds of Formula I above are those for which
R.sup.1 is R.sup.2CF.sub.2C(R.sup.12).sub.2(CH.sub.2).sub.q.
[0086] Preferred compounds of Formula I above are those for which
R.sup.6 is C.sub.1-6 alkyl or halogen. More preferred compounds
within the third preferred subgenus are those for which R.sup.6 is
methyl or chloro, including compounds for which R.sup.6 is chloro
while R.sup.3 is fluoro.
[0087] Preferred compounds of Formula I above are those for which
R.sup.11 is hydrogen.
[0088] Preferred values of R.sup.a, R.sup.b and R.sup.c in Formula
I are independently hydrogen, hydroxy, C.sub.1-6alkyl, C.sub.1-6
alkoxy, cyano or --CO.sub.2R.sup.w, where R.sup.w, in each
instance, is preferably one of C.sub.1-4alkyl, C.sub.4-7cycloalkyl
or benzyloxycarbonyl. Suitable values of R.sup.a, R.sup.b and
R.sup.c include hydrogen, methyl, ethyl, propyl, n-butyl, hydroxy,
methoxy, ethoxy, cyano, --CO.sub.2CH.sub.3,
--CO.sub.2CH.sub.2CH.sub.3 and --CO.sub.2CH.sub.2CH.sub.2CH.sub.3.
In the most preferred embodiments, R.sup.a, R.sup.b and R.sup.c are
each hydrogen.
[0089] Also preferred at R.sup.a, R.sup.b and R.sup.c is the group
--CO.sub.2R.sup.w, where R.sup.w is one of 9
[0090] where R.sup.e-R.sup.i are defined as above. When R.sup.a,
R.sup.b and R.sup.c are --CO.sub.2R.sup.w, where R.sup.w is one of
one of these moieties, the resulting compounds are prodrugs that
possess desirable formulation and bioavailability characteristics.
A preferred value for each of R.sup.e, R.sup.f and R.sup.h is
hydrogen, R.sup.g is methyl, and preferred values for R.sup.i
include benzyl and tert-butyl.
[0091] Preferred compounds are those of Formula I, where R.sup.7,
R.sup.8, R.sup.9 and R.sup.10 are independently one of hydrogen,
C.sub.1-6 alkyl, C.sub.6-10 ar(C.sub.1-6)alkyl, C.sub.6-10 aryl,
C.sub.2-10 hydroxyalkyl or C.sub.2-7 carboxyalkyl. Useful values of
R.sup.7, R.sup.8, R.sup.9 and R.sup.10 include hydrogen, methyl,
ethyl, propyl, n-butyl, benzyl, phenylethyl, 2-hydroxyethyl,
3-hydroxypropyl, 4-hydroxybutyl, 2-carboxymethyl, 3-carboxyethyl
and 4-carboxypropyl. Additional preferred compounds are those where
R.sup.7 and R.sup.8 or R.sup.9 and R.sup.10 are taken together to
form --(CH.sub.2).sub.y-- where y is 2.
[0092] Preferred compounds when X is NR.sup.18 are those wherein
R.sup.18 is hydrogen or C.sub.1-6 alkyl, optionally substituted by
one, two or three, preferably one, of amino, monoalkylamino,
dialkylamino, alkoxy, hydroxy, alkoxycarbonyl, aryloxycarbonyl,
aralkoxycarbonyl, carboalkoxy, phenyl, cyano, trifluoromethyl,
acetylamino, pyridyl, thiophenyl, furyl, pyrrolyl or
imidazolyl.
[0093] Suitable values of R.sup.18 include hydrogen, methyl, ethyl,
propyl, n-butyl, benzyl, phenethyl, 2-hydroxyethyl,
3-hydroxypropyl, 4-hydroxybutyl, carboxymethyl and
carboxyethyl.
[0094] Most preferred compounds are those where X is oxygen.
[0095] R.sup.6 can represent hydrogen, C.sub.1-3 alkyl, halogen, or
C.sub.1-2 alkoxy. R.sup.6 is preferably C.sub.1-3 alkyl, e.g.,
methyl, or halogen, e.g., chlorine, bromine or fluorine.
[0096] R.sup.3, R.sup.4, R.sup.5 and R.sup.6 can independently
represent hydrogen, hydroxy, C.sub.1-3 alkyl, halogen, or C.sub.1-2
alkoxy. Preferably R.sup.3 is fluorine and hydroxy.
[0097] Preferred values of n in Formula I include from zero to 6,
more preferably from zero to 4, and most preferably zero, 1 or 2.
Preferred values of m include from zero to 4, more preferably zero,
1, 2 or 3.
[0098] It is also to be understood that the present invention is
considered to include stereoisomers as well as optical isomers,
e.g. mixtures of enantiomers as well as individual enantiomers and
diastereomers, which arise as a consequence of structural asymmetry
in selected compounds of the present series. The compounds of the
present invention may also have polymorphic crystalline forms, with
all polymorphic crystalline forms being included in the present
invention.
[0099] The compounds of Formula I may also be solvated, especially
hydrated. Hydration may occur during manufacturing of the compounds
or compositions comprising the compounds, or the hydration may
occur over time due to the hygroscopic nature of the compounds.
[0100] Certain compounds within the scope of Formula I are
derivatives referred to as prodrugs. The expression "prodrug"
denotes a derivative of a known direct acting drug, which
derivative has enhanced delivery characteristics and therapeutic
value as compared to the drug, and is transformed into the active
drug by an enzymatic or chemical process. Useful prodrugs are those
where R.sup.a, R.sup.b, R.sup.c and/or R.sup.d are
--CO.sub.2R.sup.w, where R.sup.w is defined above. See, U.S. Pat.
No. 5,466,811 and Saulnier et al., Bioorg. Med. Chem. Lett. 4:
1985-1990 (1994).
[0101] When any variable occurs more than one time in any
constituent or in Formula I, its definition on each occurrence is
independent of its definition at every other occurrence. Also,
combinations of substituents and/or variables are permissible only
if such combinations result in stable compounds.
[0102] In another aspect, the present invention includes
compositions which are useful for in vivo imaging of thrombi in a
mammal, comprising a compound of the present invention which is
capable of being detected outside the body. Preferred are
compositions comprising a compound of the present invention and a
detectable label, such as a radioactive or paramagnetic atom.
[0103] In another aspect, the present invention provides diagnostic
compositions which are used for in vivo imaging of thrombi in a
mammal, comprising a pharmaceutically acceptable carrier and a
diagnostically effective amount of a compound or composition of the
present invention.
[0104] In another aspect, the present invention includes methods
which are useful for in vivo imaging of thrombi in a mammal.
[0105] According to a preferred aspect, useful compounds are those
wherein the R.sup.1 substituent is substituted with a detectable
label, such as a radioactive iodine atom, such as I-125, I-131 or
I-123. In this aspect, R.sup.1 is preferably phenyl, having a para
I-123, para I-125 or para I-131 substitution, or benzyl, having a
meta 1-123, meta I-125 or meta I-131 substitution.
[0106] The detectable label can also be a radioactive or
paramagnetic chelate in which a suitable ligand (L) is attached to
an R.sup.1 substituent, either directly or via a divalent linking
group A". Alternatively, the group -A"-L substitutes for the group
W in Formula I. By suitable ligand is meant an organic moiety that
is capable of chelating a radioactive or paramagnetic metal
ion.
[0107] In these compounds, the divalent linking group A" includes
groups that are capable of covalently bonding with a free amino
group and the chelating means. For example, A" may be
--C(.dbd.S)--, --C(.dbd.O)--,
--(.dbd.NH)--(CH.sub.2).sub.6--C(.dbd.NH)--,
--C(.dbd.O)--(CH.sub.2).sub.- 6--C(.dbd.O)--, 10
[0108] and the like.
[0109] Also, in the compounds represented by Formula I, the
chelating ligand, L, includes groups capable of covalently bonding
to or noncovalently binding to either a radioactive or paramagnetic
atom. The chelating means including those which are customarily
used for complexing radioactive or paramagnetic atoms. These
include chelating means containing 3 to 12, preferably 3 to 8,
methylene phosphonic acid groups, methylene carbohydroxamic acid
groups, carboxyethylidene groups, or especially carboxymethylene
groups, which are bonded to a nitrogen atom. If only one or two of
the acid groups are bonded to a nitrogen atom, then that nitrogen
is bonded to another nitrogen atom having such groups by an
optionally substituted ethylene group or by up to four separated
ethylene units separated by a nitrogen or oxygen or sulfur atom.
Preferred as a completing means is
diethylenetrimine-N,N,N',N",N"-pentaacetic acid (DTPA). DTPA is
well known in the art as a chelating means for the radioactive
atoms indium-111 (In-111), technetium-99m (Tc-99m), and the
paramagnetic atom gadolinium (Gd). Khaw, et al., Science 209: 295
(1980); Paik C. H. et al., U.S. Pat. No. 4,652,440 (1987); Gries,
H. et al., U.S. Pat. No. 4,957,939 (1990). A preferred chelating
ligand, L, is 1-(p-aminobenzyl)-diethylenetriaminepentaacetic acid.
Also included as chelating means are compounds which contain
sulfhdryl or amine moieties, the total of which in any combination
is at least four. These sulfhydryl or amine moieties are separated
from each other by at least two atoms which can be either carbon,
nitrogen, oxygen, or sulfur. Especially preferred for chelating
means, L, is metallothionein which is well known in the art as a
chelating means for Tc-99m.
[0110] The term "alkyl" as employed herein by itself or as part of
another group refers to both straight and branched chain radicals
of up to 12 carbons, such as methyl, ethyl, propyl, isopropyl,
butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl,
4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl,
undecyl, dodecyl. Preferably, alkyl is 1 to 6 carbon atoms.
[0111] The term "alkenyl" is used herein to mean a straight or
branched chain radical of 2-20 carbon atoms, unless the chain
length is limited thereto, including, but not limited to, ethenyl,
1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl,
and the like. Preferably, the alkenyl chain is 2 to 10 carbon atoms
in length, more preferably, 2 to 8 carbon atoms in length most
preferably from 2 to 4 carbon atoms in length.
[0112] The term "alkynyl" is used herein to mean a straight or
branched chain radical of 2-20 carbon atoms, unless the chain
length is limited thereto, wherein there is at least one triple
bond between two of the carbon atoms in the chain, including, but
not limited to, acetylene, 1-propylene, 2-propylene, and the like.
Preferably, the alkynyl chain is 2 to 10 carbon atoms in length,
more preferably, 2 to 8 carbon atoms in length, most preferably
from 2 to 4 carbon atoms in length.
[0113] In all instances herein where there is an alkenyl or alkynyl
moiety as a substituent group, the unsaturated linkage, i.e., the
vinylene or acetylene linkage, is preferably not directly attached
to a nitrogen, oxygen or sulfur moiety.
[0114] The term "alkoxy" is used herein to mean a straight or
branched chain radical of 1 to 20 carbon atoms, unless the chain
length is limited thereto, bonded to an oxygen atom, including, but
not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, and the
like. Preferably the alkoxy chain is 1 to 10 carbon atoms in
length, more preferably 1 to 8 carbon atoms in length.
[0115] The term "aryl" as employed herein by itself or as part of
another group refers to monocyclic or bicyclic aromatic groups
containing from 6 to 14 carbons in the ring portion, preferably
6-10 carbons in the ring portion, such as phenyl, naphthyl or
tetrahydronaphthyl.
[0116] The term "heteroaryl" as employed herein refers to groups
having 5 to 14 ring atoms; 6, 10 or 14 .pi. electrons shared in a
cyclic array; and containing carbon atoms and 1, 2 or 3 oxygen,
nitrogen or sulfur heteroatoms (where examples of heteroaryl groups
are: thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl,
furyl, pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl,
xanthenyl, phenoxathlinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl,
pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,
indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl,
4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl,
naphthyridinyl, quinazolinyl, cinnolinyl, pteridinyl,
4.alpha.H-carbazolyl, carbazolyl, .beta.-carbolinyl,
phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl,
phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl and
phenoxazinyl groups).
[0117] The term "aralkyl" or "arylalkyl" as employed herein by
itself or as part of another group, refers to C.sub.1-12 alkyl,
preferably C.sub.1-6 alkyl, groups as discussed above having an
aryl substituent, such as benzyl, phenylethyl or
2-naphthylmethyl.
[0118] The term "cycloalkyl" as employed herein by itself or as
part of another group, refers to cycloalkyl groups containing 3 to
9 carbon atoms, preferably 3 to 7 carbon atoms. Typical examples
are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclooctyl and cyclononyl.
[0119] The term "C.sub.7-12 bicyclic alkyl" is intended to include
bicyclo[2.2.1]heptyl (norbornyl), bicyclo[2.2.2]octyl,
1,1,3-trimethylbicyclo[2.2.1]heptyl (bornyl), and the like.
[0120] The term "C.sub.10-16 tricyclic alkyl" is intended to
include tricyclo[5,2,1,0.sup.2,6]decyl, adamantyl, and the
like.
[0121] The term "halogen" or "halo" as employed herein by itself or
as part of another group refers to chlorine, bromine, fluorine or
iodine with chlorine and fluorine being preferred.
[0122] The term "monoalkylamine" as employed herein by itself or as
part of another group refers to an amino group which is substituted
with one alkyl group having from 1 to 12, preferably 1 to 6, carbon
atoms.
[0123] The term "dialkylamine" as employed herein by itself or as
part of another group refers to an amino group which is substituted
with two alkyl groups, each having from 1 to 12, preferably 1 to 6,
carbon atoms.
[0124] The term "hydroxyalkyl" as employed herein refers to any of
the above alkyl groups substituted by one or more hydroxyl
moieties.
[0125] The term "carboxyalkyl" as employed herein refers to any of
the above alkyl groups substituted by one or more carboxylic acid
moieties.
[0126] The term "heterocycle" or "heterocyclic ring", as used
herein except where noted, represents a stable 5- to 7-membered
mono- or bicyclic or stable 7- to 10-membered bicyclic heterocyclic
ring system, any ring of which may be saturated or unsaturated, and
which consists of carbon atoms and from one to three heteroatoms
selected from the group consisting of N, O and S, and wherein the
nitrogen and sulfur heteroatoms may optionally be oxidized, and the
nitrogen heteroatom may optionally be quaternized, and including
any bicyclic group in which any of the above-defined heterocyclic
rings is fused to a benzene ring. Especially useful are rings
containing one oxygen or sulfur, one to three nitrogen atoms, or
one oxygen or sulfur combined with one or two nitrogen atoms. The
heterocyclic ring may be attached at any heteroatom or carbon atom
which results in the creation of a stable structure. Examples of
such heterocyclic groups include piperidinyl, piperazinyl,
2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl,
2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl,
pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl,
pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl,
oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl,
thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl,
indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, thiadiazoyl,
benzopyranyl, benzothiazolyl, benzoxazolyl, furyl, tetrahydrofuryl,
tetrahydropyranyl, thienyl, benzothienyl, thiamorpholinyl,
thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, and
oxadiazolyl. Morpholino is the same as morpholinyl.
[0127] The term "heteroatom" is used herein to mean an oxygen atom
("O"), a sulfur atom ("S") or a nitrogen atom ("N"). It will be
recognized that when the heteroatom is nitrogen, it may form an
NR.sup.aR.sup.b moiety, wherein R.sup.a and R.sup.b are,
independently from one another, hydrogen or C.sub.1 to C.sub.8
alkyl, or together with the nitrogen to which they are bound, form
a saturated or unsaturated 5-, 6-, or 7-membered ring.
[0128] Schemes 1-8 outline a synthetic route to compounds of
Formula I. 11
[0129] In Scheme 1, an acetic acid side chain is introduced onto a
benzene ring by reaction of a fluorinated nitrobenzene 1, such as
1,2,3-trifluoro-4-nitrobenzene, with the metal salt of a
substituted or non-substituted malonate ester, such as diethyl
malonate, in a suitable solvent such as tetrahydrofuran (THF),
followed by acid hydrolysis and subsequent decarboxylation upon
heating, to produce compound 2 (Yokomoto, M, W., et al., EP
published application No. 0 470 578 A1 (1991)). The carboxyl group
of 2 is converted to a hydroxyl group under typical reducing
conditions, such as borane (BH.sub.3)-THF complex and sodium
borohydride (NaBH.sub.4), in a suitable solvent such as THF, to
give alcohol 3 (Yokomoto, M, W., et al., ibid). Introduction of a
suitable functionality para to the nitro group in the ring is
achieved by aromatic nucleophilic substitution of the fluoride in
compound 3 with a suitable nucleophile, such as tert-butyl amine,
in suitable solvents such as dimethyl sulfoxide (DMSO) and toluene
under reflux, to afford compound 4 (Yokomoto, M, W., et al., ibid).
The nitrogen protecting group, such as tert-butyl, in compound 4 is
removed under standard conditions, such as concentrated
hydrochloric acid (HCl) under reflux, to give compound 5 (Yokomoto,
M, W., et al., ibid). The hydroxyl group of compound 5 is masked
with a suitable protecting group, such as acetyl, under standard
conditions well known in the art (Greene, T. W., and Wuts, P. G.
M., Protecting Groups in Organic Synthesis, 2.sup.nd ed., John
Wiley and Sons, Inc., New York (1991)), such as acetyl chloride in
dichloromethane (DCM) in the presence of base such as triethylamine
or diisopropylethylamine (DIEA), to give compound 6. Coupling of an
activated carbonyl compound ACOCl with compound 6 in a suitable
solvent, such: as DCM, produces compound 7. 12
[0130] In Scheme 2, reduction of arylnitro compound 7 under typical
conditions, such as catalytic hydrogenation with hydrogen in the
presence of palladium on activated carbon in ethanol or methanol,
gives arylamine 8. The acetyl protecting group of compound 8 is
removed (in order to increase the solubility of the compound prior
to the amino group manipulation) by hydrolysis under basic
conditions, such as aqueous potassium carbonate (K.sub.2CO.sub.3)
solution in methanol, to free the protected hydroxyl group, giving
compound 9. The desired R.sup.6 is introduced into the center
scaffold of compound 9 by a Sandmeyer-type reaction ((a) Gunstone,
F. D., et al., Org. Syn. Collect Vol. 1, Wiley, New York, N.Y.
(1941), p. 170; (b) Yokomoto, M, W., et al., EP published
application No. 0 470 578 A1 (1991)) with suitable reagents, such
as sodium nitrite (NaNO.sub.2) and HCl followed by copper (I)
chloride (CuCl), or by substitutive deamination (Doyle, M. P., et
al. J. Org. Chem. 42: 2426 (1977)) with suitable reagents, such as
tert-butylnitrite (t-BuONO) and copper (II) chloride (CuCl.sub.2),
to give compound 10. The amino group of arylamine 9 can be
converted to a methyl group under carbon-carbon coupling conditions
in the presence of a palladium catalyst through an arenediazonium
salt intermediate (Kikukawa, K., et al., J. Org. Chem. 48: 1333
(1983)). Compound 10 in turn, is reduced with a suitable reducing
agent, such as BH.sub.3, to generate desired fragment WY of
compound 11 where Y is --NH--. Oxidation of 11 with an oxidizing
agent, such as sulfur trioxide pyridine complex (SO.sub.3 pyridine)
in DCM, yields aldehyde 12. Construction of the center and left
fragment of the target compound is finally achieved by further
oxidation of the aldehyde 12 to carboxylic acid 13 under suitable
oxidation conditions, such as sodium chlorite (NaClO.sub.2) in the
presence of sodium dihydrogenphosphate (NaH.sub.2PO.sub.4) and DMSO
(Dalcanale, E., et al., J. Org. Chem. 51: 567 (1986)). 13
[0131] In Scheme 3, acid 13 is coupled with a suitable amine 14,
such as protected O-guanidinyl amine (Tianbao Lu, et al., WO
99/26926 (1999)), or aminopyridinyl amine (Sanderson, P. E., et
al., WO 97/01338 (1997)) in the presence of a typical peptide
coupling reagent, such as Castro's reagent (BOP), and a base, such
as DIEA, in a suitable solvent, such as N,N-dimethylformamide
(DMF), to produce amide 15. Optionally, the protecting groups, such
as tert-(butoxy)carbonyl (Boc), can be removed under typical
deprotection conditions, such as trifluoroacetic acid (TFA)
solution in DCM when B is O-guanidine, or HCl solution in
1,4-dioxane when B is aminopyridine, to generate free O-guanidine,
or aminopyridine, respectively. 14
[0132] In Scheme 4, the phenylacetic acid derivative 16 is nitrated
in the meta position of the benzene ring using standard conditions,
such as 96% nitric acid in conc. sulfuric acid (Sindelar et al.,
Coll. Czechoslov. Chem. Commun. 42: 2231 (1977)), to give the nitro
compound 17. The carboxylic acid group of compound 17 is then
protected using standard conditions well known in the art (Greene,
T. W. and Wuts, P. G. M., Protective Groups in Organic Synthesis,
2.sup.nd ed., John Wiley and Sons, New York (1991)), such as
conversion to the ester by reaction with oxalyl chloride followed
by alcohol POH, to afford ester 18 (where P is a typical carboxylic
acid protecting group). Reduction of the nitro group is
accomplished using a suitable reagent, such as tin (II) chloride,
in an appropriate solvent, such as ethanol, and the resulting amine
19 is reacted with an acylating agent (W.dbd.R.sup.1C(O)) or a
sulfonylating agent (W.dbd.R.sup.1S(O).sub.2), such as
benzylsulfonyl chloride, and a suitable base, such as
N-methylmorpholine, in a solvent, such as DCM, to provide the
N-substituted-aminophenylacetate 20 (Y=--NH--). The carboxylic acid
group is deprotected using standard conditions well known in the
art (Greene, T. W. and Wuts, P. G. M., Protective Groups in Organic
Synthesis, 2.sup.nd edition, John Wiley and Sons, New York (1991)),
such as hydrolysis with aqueous hydroxide, to give acid 13
(Y=--NH--). This is then coupled with amine 14 and deprotected, as
in Scheme 3, to produce phenylacetamide 15 (Y=NH). 15
[0133] In Scheme 5, nitrophenylacetic acid 17 is coupled to an
aminoalcohol 21, such as ethanolamine, using a standard peptide
coupling procedure, such as in Scheme 3, to give alcohol 22. The
alcohol is converted to the protected alkoxyamine by coupling to
N-hydroxyphthalimide using standard reagents (Mitsunobu, O.,
Synthesis 1: 1 (1981)), such as triphenylphosphine and
diethylazodicarboxylate, in a suitable solvent, such as THF, to
afford compound 23, which is then converted to aniline 24 under
typical reducing conditions, such as hydrogenation over
palladium(0) on carbon, in a suitable solvent, such as ethanol. The
amine is then acylated or sulfonylated as in Scheme 4 to give
intermediate 25, and the alkoxyamine deprotected using standard
conditions well known in the art (Greene, T. W. and Wuts, P. G. M.,
Protective Groups in Organic Synthesis, 2nd edition, John Wiley and
Sons, New York (1991)), such as aqueous methylamine in ethanol/THF.
Guanidinylation of the resulting alkoxyamine 26 is accomplished
with a standard guanidinylation reagent, such as
N,N'-bis(tert-butoxycarbonyl)-S- -methylthiourea (Bergeron, R. J.
and McManis, J. S., J. Org. Chem. 52: 1700 (1987)) or
N--R.sup.a--N'--R.sup.b,R.sup.c-1H-pyrazole-1-carboxamidi- ne
(Bernatowicz, M. S. et al. Tetrahedron Lett. 34: 3389 (1993)), and
the guanidine optionally deprotected as in Scheme 3, to provide
final target 27. 16
[0134] In Scheme 6, the ketone, aldehyde (R.sup.11=H), or
carboxylic acid (R.sup.11=OH) starting material 28 is reduced with
a suitable reagent, such as borane-THF, to give alcohol 29, which
is then converted to a better leaving group by reaction with a
sulfonyl chloride, such as methanesulfonyl chloride, in a suitable
solvent, such as DCM, to produce compound 30. The sulfonate is
displaced by cyanide under standard conditions, such as potassium
cyanide in refluxing acetonitrile, to give nitrile 31, which is
then hydrolyzed with a typical reagent, such as aqueous hydroxide.
Coupling of the resulting acid 17 with amine 14 is accomplished as
in Scheme 3 to give intermediate 32, and the nitro group is reduced
as in Scheme 4 or 5 to afford aniline 33. This is acylated or
sulfonylated as in Scheme 4 and the guanidine optionally
deprotected as in Scheme 3 to give the final target 15 (Y=NH).
1718
[0135] In Scheme 7, nitrophenol 34 is alkylated with allylic halide
35 and a suitable base, such a cesium carbonate, in a polar aprotic
solvent, such as DMF, giving intermediate 36, which is then
converted to compound 37 via the aromatic Claisen rearrangement by
heating. The phenol is protected using typical reagents, such as
benzyl bromide and cesium carbonate, in a solvent, such as DMF, to
give 38 (where P is a typical hydroxyl protecting group) and the
nitro group is reduced as in Scheme 4 or 5 to produce aniline 39.
Aniline 39 is converted to intermediate 40 as in Scheme 4 and the
alkene is oxidatively cleaved using standard conditions, such as
sodium periodate and osmium tetraoxide in dioxane/water followed by
Jones reagent, to provide acid 41. This is then coupled to amine
14, the guanidine optionally deprotected as in Scheme 3, and the
phenol group optionally deprotected using standard conditions, such
as hydrogenation over palladium (0) on carbon, in a suitable
solvent, such as ethanol, to produce the target compound 42.
1920
[0136] In Scheme 8, the mono-protected catechol 43 is sulfonylated
with a reagent W--Cl, such as meta-toluenesulfonyl chloride, in a
solvent, such as DCM, in the presence of a base, such as
triethylamine, giving compound 44. The protecting group is removed
using standard conditions, such as boron tribromide in DCM, and the
resulting phenol 45 is alkylated with allylic halide 35 to give 46,
rearranged to phenol 47, and protected to afford intermediate 48 as
in Scheme 7. The alkene is oxidatively cleaved using standard
conditions, such as sodium periodate and ruthenium(III) chloride in
acetonitrile/water (Ashby, E. C. and Goel, A. B., J. Org. Chem. 46:
3936 (1981)) followed by Jones reagent, giving acid 49, which is
then coupled with amine 14 and optionally deprotected as in Schemes
3 and 7 to afford target compound 50.
[0137] The pharmaceutically-acceptable salts of the compounds of
Formula I (in the form of water- or oil-soluble or dispersible
products) include the conventional non-toxic salts or the
quaternary ammonium salts which are formed, e.g., from inorganic or
organic acids or bases. Examples of such acid addition salts
include acetate, adipate, alginate, aspartate, benzoate,
benzenesulfonate, bisulfate, butyrate, citrate, camphorate,
camphorsulfonate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,
glycerophosphate, hemisulfate, heptanoate, hexanoate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,
lactate, maleate, methanesulfonate, 2-naphthalenesulfonate,
nicotinate, nitrate, oxalate, pamoate, pectinate, persulfate,
3-phenylpropionate, picrate, pivalate, propionate, succinate,
sulfate, tartrate, thiocyanate, tosylate, trifluoroacetate, and
undecanoate. Base salts include ammonium salts, alkali metal salts
such as sodium and potassium salts, alkaline earth metal salts such
as calcium and magnesium salts, salts with organic bases such as
dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino
acids such as arginine, lysine, and so forth, including salts with
a guanidinyl moiety. Also, the basic nitrogen-containing groups may
be quaternized with such agents as lower alkyl halides, such as
methyl, ethyl, propyl, and butyl chlorides, bromides and iodides;
dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl
sulfates; long chain halides such as decyl, lauryl, myristyl and
stearyl chlorides, bromides and iodides; aralkyl halides like
benzyl and phenethyl bromides and others. Preferred acids for
forming acid addition salts include HCl, acetic acid and
trifluoroacetic acid.
[0138] The compounds of the present invention represent a novel
class of potent inhibitors of metallo, acid, thiol and serine
proteases. Examples of the serine proteases inhibited by compounds
within the scope of the invention include leukocyte neutrophil
elastase, a proteolytic enzyme implicated in the pathogenesis of
emphysema; chymotrypsin and trypsin, digestive enzymes; pancreatic
elastase, and cathepsin G, a chymotrypsin-like protease also
associated with leukocytes; thrombin and factor Xa, proteolytic
enzymes in the blood coagulation pathway. Inhibition of
thermolysin, a metalloprotease, and pepsin, an acid protease, are
also contemplated uses of compounds of the present invention. The
compounds of the present invention are preferably employed to
inhibit trypsin-like proteases.
[0139] For their end-use application, the potency and other
biochemical parameters of the enzyme-inhibiting characteristics of
the compounds of the present invention are readily ascertained by
standard biochemical techniques known to those of skill in the art.
For example, an end use application of the compounds that inhibit
chymotrypsin and trypsin is in the treatment of pancreatitis.
Actual dose ranges for their specific end-use application will, of
course, depend upon the nature and severity of the disease state of
the patient or animal to be treated, as determined by the attending
diagnostician. It is expected that a useful dose range will be
about 0.01 to 10 mg per kg per day for an effective therapeutic
effect.
[0140] Compounds of the present invention that are distinguished by
their ability to inhibit thrombin may be employed for a number of
therapeutic purposes. As thrombin inhibitors, compounds of the
present invention inhibit thrombin production. Therefore, these
compounds are useful for the treatment or prophylaxis of states
characterized by abnormal venous or arterial thrombosis involving
either thrombin production or action. These states include, but are
not limited to, deep vein thrombosis; disseminated intravascular
coagulopathy which occurs during septic shock, viral infections and
cancer; myocardial infarction; stroke; coronary artery bypass;
fibrin formation in the eye; hip replacement; and thrombus
formation resulting from either thrombolytic therapy or
percutaneous transluminal coronary angioplasty (PCTA). Other uses
include the use of said thrombin inhibitors as anticoagulants
either embedded in or physically linked to materials used in the
manufacture of devices used in blood collection, blood circulation,
and blood storage, such as catheters, blood dialysis machines,
blood collection syringes and tubes, and blood lines. The compounds
of the present invention may also be used as an anticoagulant in
extracorporeal blood circuits.
[0141] Stents have been shown to reduce restenosis, but are
thrombogenic. A strategy for reducing the thrombogenicity of stents
is to coat, embed, adsord or covalently attach a
thrombin-inhibiting agent to the stent surface. The compounds of
the present invention can be employed for this purpose. Compounds
of the invention can be attached to, or embedded within soluble
and/or biodegradeable polymers as and thereafter coated onto stent
materials. Such polymers can include polyvinylpyrrolidone,
polyhydroxy-propylmethacrylamide-phenol,
polyhydroxyethyl-aspartamide-phe- nol, or
polyethyleneoxide-polylysine substituted with palmitoyl residues,
polylactic acid, polyglycolic acid, copolymers of polylactic and
polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric
acid, polyorthoesters, polyacetals, polydihydropyrans,
polycyanoacrylates and cross linked or amphipathic block copolymers
of hydrogels. See European Application 761 251, European
Application 604,022, Canadian Patent No. 2,164,684 and PCT
Published Applications Nos. WO 96/11668, WO 96/32143 and WO
96/38136.
[0142] By virtue of the effects of thrombin on a host of cell
types, such as smooth muscle cells, endothelial cells and
neutrophils, the compounds of the present invention find additional
use in the treatment or prophylaxis of adult respiratory distress
syndrome; inflammatory responses; wound healing; reperfusion
damage; atherosclerosis; and restenosis following an injury such as
balloon angioplasty, atherectomy, and arterial stent placement.
[0143] The compounds of the present invention may be useful in
treating neoplasia and metastasis as well as neurodegenerative
diseases, such as Alzheimer's disease and Parkinson's disease.
[0144] When employed as thrombin inhibitors, the compounds of the
present invention may be administered in an effective amount within
the dosage range of about 0.1 to about 500 mg/kg, preferably
between 0.1 to 10 mg/kg body weight, on a regimen in single or 2-4
divided daily doses.
[0145] When employed as inhibitors of thrombin, the compounds of
the present invention may be used in combination with thrombolytic
agents such as tissue plasminogen activator, streptokinase, and
urokinase. Additionally, the compounds of the present invention may
be used in combination with other antithrombotic or anticoagulant
drugs such as, but not limited to, fibrinogen antagonists and
thromboxane receptor antagonists.
[0146] The thrombin inhibitors may also be coupled with soluble
polymers as targetable drug carriers. Such polymers can include
polyvinylpyrrolidone, pyran copolymer,
polyhydroxy-propylmethacrylamide-p- henol,
polyhydroxyethyl-aspartamide-phenol, or
polyethyleneoxide-polylysin- e substituted with palmitoyl residues.
Furthermore, the thrombin inhibitors may be coupled to a class of
biodegradable polymers useful in achieving controlled release of a
drug, for example, polylactic acid, polyglycolic acid, copolymers
of polylactic and polyglycolic acid, polyepsilon caprolactone,
polyhydroxy butyric acid, polyorthoesters, polyacetals,
polydihydropyrans, polycyanoacrylates and cross linked or
amphipathic block copolymers of hydrogels.
[0147] Human leucocyte elastase is released by polymorphonuclear
leukocytes at sites of inflammation and thus is a contributing
cause for a number of disease states. Compounds of the present
invention are expected to have an anti-inflammatory effect useful
in the treatment of gout, rheumatoid arthritis and other
inflammatory diseases, and in the treatment of emphysema. The
leucocyte elastase inhibitory properties of compounds of the
present invention are determined by the method described below.
Cathepsin G has also been implicated in the disease states of
arthritis, gout and emphysema, and in addition, glomerulonephritis
and lung infestations caused by infections in the lung. In their
end-use application the enzyme inhibitory properties of the
compounds of Formula I are readily ascertained by standard
biochemical techniques that are well-known in the art.
[0148] The Cathepsin G inhibitory properties of compounds within
the scope of the present invention are determined by the following
method. A preparation of partially purified human Cathepsin G is
obtained by the procedure of Baugh et al., Biochemistry 15: 836
(1979). Leukocyte granules are a major source for the preparation
of leukocyte elastase and cathepsin G (chymotrypsin-like activity).
Leukocytes are lysed and granules are isolated. The leukocyte
granules are extracted with 0.20 M sodium acetate, pH 4.0, and
extracts are dialyzed against 0.05 M Tris buffer, pH 8.0 containing
0.05 M NaCl overnight at 4.degree. C. A protein fraction
precipitates during dialysis and is isolated by centrifugation.
This fraction contains most of the chymotrypsin-like activity of
leukocyte granules. Specific substrates are prepared for each
enzyme, namely N-Suc-Ala-Ala-Pro-Val-p-nitroanilide and
Suc-Ala-Ala-Pro-Phe-p-nit- roanilide. The latter is not hydrolyzed
by leukocyte elastase. Enzyme preparations are assayed in 2.00 mL
of 0.10 M Hepes buffer, pH 7.5, containing 0.50 M NaCl, 10%
dimethylsulfoxide and 0.0020 M Suc-Ala-Ala-Pro-Phe-p-nitroanilide
as a substrate. Hydrolysis of the p-nitroanilide substrate is
monitored at 405 nm and at 25.degree. C.
[0149] Useful dose range for the application of compounds of the
present invention as neutrophil elastase inhibitors and as
Cathepsin G inhibitors depend upon the nature and severity of the
disease state, as determined by the attending diagnostician, with a
range of 0.01 to 10 mg/kg body weight, per day, being useful for
the aforementioned disease states.
[0150] Compounds of the present invention that inhibit urokinase or
plasminogen activator are potentially useful in treating excessive
cell growth disease state. As such compounds of the present
invention may also be useful in the treatment of benign prostatic
hypertrophy and prostatic carcinoma, the treatment of psoriasis,
and as abortifacients. For their end-use application, the potency
and other biochemical parameters of the enzyme inhibiting
characteristics of compounds of the present invention are readily
ascertained by standard biochemical techniques well known in the
art. Actual dose ranges for this application will depend upon the
nature and severity of the disease state of the patient or animal
to be treated as determined by the attending diagnostician. It is
to be expected that a general dose range will be about 0.01 to 10
mg per kg per day for an effective therapeutic effect.
[0151] Additional uses for compounds of the present invention
include analysis of commercial reagent enzymes for active site
concentration. For example, chymotrypsin is supplied as a standard
reagent for use in clinical quantitation of chymotrypsin activity
in pancreatic juices and feces. Such assays are diagnostic for
gastrointestinal and pancreatic disorders. Pancreatic elastase is
also supplied commercially as a reagent for quantitation of
.alpha..sub.1-antitrypsin in plasma. Plasma
.alpha..sub.1-antitrypsin increases in concentration during the
course of several inflammatory diseases, and
.alpha..sub.1-antitrypsin deficiencies are associated with
increased incidence of lung disease. Compounds of the present
invention can be used to enhance the accuracy and reproducibility
of these assays by titrametric standardization of the commercial
elastase supplied as a reagent. See, U.S. Pat. No. 4,499,082.
[0152] Protease activity in certain protein extracts during
purification of particular proteins is a recurring problem which
can complicate and compromise the results of protein isolation
procedures. Certain proteases present in such extracts can be
inhibited during purification steps by compounds of the present
invention, which bind tightly to various proteolytic enzymes.
[0153] The pharmaceutical compositions of the invention can be
administered to any animal that can experience the beneficial
effects of the compounds of the invention. Foremost among such
animals are humans, although the invention is not intended to be so
limited.
[0154] The pharmaceutical compositions of the present invention can
be administered by any means that achieve their intended purpose.
For example, administration can be by parenteral, subcutaneous,
intravenous, intramuscular, intraperitoneal, transdermal, buccal,
or ocular routes. Alternatively, or concurrently, administration
can be by the oral route. The dosage administered will be dependent
upon the age, health, and weight of the recipient, kind of
concurrent treatment, if any, frequency of treatment, and the
nature of the effect desired.
[0155] In addition to the pharmacologically active compounds, the
new pharmaceutical preparations can contain suitable
pharmaceutically acceptable carriers comprising excipients and
auxiliaries that facilitate processing of the active compounds into
preparations that can be used pharmaceutically.
[0156] The pharmaceutical preparations of the present invention are
manufactured in a manner that is, itself, known, for example, by
means of conventional mixing, granulating, dragee-making,
dissolving, or lyophilizing processes. Thus, pharmaceutical
preparations for oral use can be obtained by combining the active
compounds with solid excipients, optionally grinding the resulting
mixture and processing the mixture of granules, after adding
suitable auxiliaries, if desired or necessary, to obtain tablets or
dragee cores.
[0157] For compositions of the present invention suitable for
administration to a human, the term "excipient" is meant to
include, but not be limited by, those excipients described in the
Handbook of Pharmaceutical Excipients, American Pharmaceutical
Association, 2n Ed. (1994), which is herein incorporated by
reference in its entirety. Suitable excipients are, in particular,
fillers such as saccharides, for example, lactose or sucrose,
mannitol or sorbitol, cellulose preparations and/or calcium
phosphates, for example, tricalcium phosphate or calcium hydrogen
phosphate, as well as binders, such as, starch paste, using, for
example, maize starch, wheat starch, rice starch, potato starch,
gelatin, tragacanth, methyl cellulose,
hydroxy-propylmethylcellulose, sodium carboxymethylcellulose,
and/or polyvinyl pyrrolidone. If desired, disintegrating agents can
be added, such as, the above-mentioned starches and also
carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or
alginic acid or a salt thereof, such as, sodium alginate.
Auxiliaries are, above all, flow-regulating agents and lubricants,
for example, silica, talc, stearic acid or salts thereof, such as,
magnesium stearate or calcium stearate, and/or polyethylene glycol.
Dragee cores are provided with suitable coatings that, if desired,
are resistant to gastric juices. For this purpose, concentrated
saccharide solutions can be used, which may optionally contain gum
arabic, talc, polyvinyl pyrrolidone, polyethylene glycol, and/or
titanium dioxide, lacquer solutions and suitable organic solvents
or solvent mixtures. In order to produce coatings resistant to
gastric juices, solutions of suitable cellulose preparations, such
as, acetylcellulose phthalate or hydroxypropylmethyl-cellulose
phthalate, are used. Dye stuffs or pigments can be added to the
tablets or dragee coatings, for example, for identification or in
order to characterize combinations of active compound doses.
[0158] Other pharmaceutical preparations which can be used orally
include push-fit capsules made of gelatin, as well as soft, sealed
capsules made of gelatin and a plasticizer, such as, glycerol or
sorbitol. The push-fit capsules can contain the active compounds in
the form of granules that may be mixed with fillers such as
lactose, binders such as starches, and/or lubricants such as talc
or magnesium stearate and, optionally, stabilizers. In soft
capsules, the active compounds are preferably dissolved or
suspended in suitable liquids, such as, fatty oils or liquid
paraffin. In addition, stabilizers may be added.
[0159] Suitable formulations for parenteral administration include
aqueous solutions of the active compounds in water-soluble form,
for example, water-soluble salts, alkaline solutions and
cyclodextrin inclusion complexes. Especially preferred alkaline
salts are ammonium salts prepared, for example, with Tris, choline
hydroxide, Bis-Tris propane, N-methylglucamine, or arginine. One or
more modified or unmodified cyclodextrins can be employed to
stabilize and increase the water solubility of compounds of the
present invention. Useful cyclodextrins for this purpose are
disclosed in U.S. Pat. Nos. 4,727,064, 4,764,604, and
5,024,998.
[0160] In addition, suspensions of the active compounds as
appropriate oily injection suspensions can be administered.
Suitable lipophilic solvents or vehicles include fatty oils, for
example, sesame oil, or synthetic fatty acid esters, for example,
ethyl oleate or triglycerides or polyethylene glycol-400 (the
compounds are soluble in PEG-400). Aqueous injection suspensions
can contain substances that increase the viscosity of the
suspension, for example, sodium carboxymethyl cellulose, sorbitol,
and/or dextran. Optionally, the suspension may also contain
stabilizers.
[0161] Compounds of Formula I can be labeled with radioactive
iodine by using an exchange reaction. Exchange of hot iodine for
cold iodine is well known in the art. Alternatively, a radio iodine
labeled compound can be prepared from the corresponding bromo
compound via a tributylstannyl intermediate. See, U.S. Pat. No.
5,122,361, herein incorporated by reference.
[0162] The present invention also includes compositions which are
useful for in vivo imaging of thrombi in a mammal, wherein the
compositions are comprised of a compound of Formula I complexed
with a radioactive atom.
[0163] For the compounds of Formula I, suitable radioactive atoms
include Co-57, Cu-67, Ga-67, Ga-68, Ru-97, Tc-99m, In-111, In-113m,
Hg-197, Au-198, and Pb-203. Some radioactive atoms have superior
properties for use in radiochemical imaging techniques. In
particular, technetium-99m (Tc-99m) is an ideal radioactive atom
for imaging because of its nuclear properties. Rhenium-186 and -188
also have gamma emission which allows it to be imaged. Preferred
compositions contain the radioactive atom, Tc-99m.
[0164] The compounds of Formula I can be labeled by any of the many
techniques known in the art to provide a composition of the present
invention. For example, these compounds can be labeled through a
chelating agent such as diethylene-triaminepentaacetic acid (DTPA)
or metallothionein, both of which can be covalently attached to the
compound of Formula I.
[0165] In general, the compositions of the present invention
containing technetium-99m are prepared by forming an aqueous
mixture of technetium-99m and a reducing agent and a water-soluble
ligand, and then contacting the mixture with a compound of the
present invention represented by Formula I. For example, the
imaging compounds of this invention are made by reacting
technetium-99m (in an oxidized state) with the compounds of the
present invention having a chelating means in the presence of a
reducing agent to form a stable complex between technetium-99m in a
reduced state (IV or V valence state).
[0166] One embodiment of the composition of the present invention
is prepared by labeling a compound of Formula I having a DTPA
chelating means with technetium-99m. This may be accomplished by
combining a predetermined amount (as 5 .mu.g to 0.5 mg) of compound
of the present invention with an aqueous solution containing
citrate buffer and stannous reducing agent, then adding freshly
eluted sodium pertechnetate containing a predetermined level of
radioactivity (as 15 mCi). After allowing an incubation of the
mixture at room temperature, the reaction mixture is loaded into a
shielded syringe through a sterile filter (0.2-0.22 micron), then
is dispensed into 0.9% saline for injection, if desired.
[0167] Another embodiment of the compositions of the present
invention is prepared by labeling a compound of Formula I having a
metallothionein chelating means with technetium-99m. This may be
accomplished by combining aqueous sodium pertechnetate-99m with
aqueous stannous glucoheptonate to form a soluble complex of
technetium-99m (in reduced state) with two glucoheptonate
molecules, then combining this solution with a compound of the
Formula I having a metallothionein attached thereto. After
incubating the mixture for a period of time and under conditions
which allow for an exchange of the technetium-99m from the
glucoheptonate complex to the metallothionein of the compound of
Formula I, the technetium-labeled composition of the present
invention is formed.
[0168] Reducing agents for use in the method are physiologically
acceptable for reducing technetium-99m from its oxidized state to
the IV or V valence state or for reducing rhenium from its oxidized
state. Reducing agents which can be used are stannous chloride,
stannous fluoride, stannous glucoheptonate, stannous tartarate, and
sodium dithionite. The preferred agents are stannous reducing
agents, especially stannous chloride or stannous glucoheptonate.
The amount of reducing agent is that amount necessary to reduce the
technetium-99m to provide for the binding to the chelating means of
a compound of Formula I in this radioisotope's reduced state. For
example, stannous chloride (SnCl.sub.2) is the reducing agent and
can be used in range from 1-1,000 .mu.g/mL.
[0169] Citric acid complexes with technetium-99m quickly to form a
stable technetium-99m-citrate complex. Upon contact with a compound
of Formula I, substantially quantitative transfer of technetium-99m
from its citrate complex to the chelating means of the compound of
Formula I is achieved rapidly and under mild conditions. The amount
of citric acid (as sodium citrate) can range from about 0.5 mg/ml
up to the amount maximally soluble in the medium. Preferred amounts
of citric acid range from 15 to 30 .mu.g/ml.
[0170] The amount of compound of Formula I having a chelating means
can range from 0.001 to about 3 mg/mL, preferably about 0.017 to
about 0.15 mg/mL. Finally, technetium-99m in the form of
pertechnetate can be used in amounts of preferably about 1-50 mCi.
The amount of mCi per mg of compound of the present invention is
preferably about 30-150.
[0171] The reaction between the compound of Formula I and the metal
ion-transfer ligand complex is preferably carried out in a aqueous
solution at a pH at which the compound of Formula I is stable. By
"stable", it is meant that the compound remains soluble and retains
its inhibitory activity against .alpha.-thrombin. Normally, the pH
for the reaction will be from about 5 to 9, the preferred pH being
above 6-8. The technetium-99m-citrate complex and a compound of
Formula I are incubated, preferably at a temperature from about
20.degree. C. to about 60.degree. C., most preferably from about
20.degree. C. to about 37.degree. C., for a sufficient amount of
time to allow transfer of the metal ion from the citrate complex to
the chelating means of the compound of Formula I. Generally, less
than one hour is sufficient to complete the transfer reaction under
these conditions.
[0172] Alternative compositions of the present invention include an
In-111 labeled compound of the present invention.
[0173] The present invention also includes compositions of the
compounds of the present invention which are useful for in vivo
imaging of thrombi in a mammal, comprised of a compound represented
by Formula I complexed to a paramagnetic atom.
[0174] Preferred paramagnetic atoms are divalent or trivalent ions
of elements with an atomic number of 21 to 29, 42, 44 and 58 to 70.
Suitable ions include chromium(III), manganese(II), iron(III),
iron(II), cobalt(II), nickel(II), copper(II), praseodymium(III),
neodymium(III), samarium(III) and ytterbium(III). Because of their
very strong magnetic moments, gadolinium(III), terbium(III),
dysoprosium(III), holmium(III), and erbium(III) are preferred.
Especially preferred for the paramagnetic atom is
gadolinium(III).
[0175] The compositions of the present invention may be prepared by
combining a compound of Formula I with a paramagnetic atom. For
example, the metal oxide or a metal salt (for example, nitrate,
chloride or sulfate) of a suitable paramagnetic atom is dissolved
or suspended in a medium comprised of water and an alcohol, such as
methyl, ethyl or isopropyl alcohol. This mixture is added to a
solution of an equimolar amount of the compound of Formula I in a
similar aqueous medium and stirred. The reaction mixture may be
heated moderately until the reaction is completed. Insoluble
compositions formed may be isolated by filtering, while soluble
compositions may be isolated by evaporation of the solvent. If acid
groups on the chelating means are still present in the composition
of the present invention, inorganic or organic bases, and even
amino acids, may be added to convert the acidic complex into a
neutral complex to facilitate isolation or purification of
homogenous composition. Organic bases or basic amino acids may be
used as neutralizing agents, as well as inorganic bases such as
hydroxides, carbonates or bicarbonates of sodium, potassium or
lithium.
[0176] The present invention also include diagnostic compositions
which are useful for in vivo imaging of thrombi in a mammal,
comprising a pharmaceutically acceptable carrier and a
diagnostically effective amount of compositions derived from the
compounds of Formula I.
[0177] The "diagnostically effective amount" of the composition
required as a dose will depend on the route of administration, the
type of mammal being treated, and the physical characteristics of
the specific mammal under consideration. These factors and their
relationship to determining this dose are well known to skilled
practitioners in the medial diagnostic arts. Also, the
diagnostically effective amount and method of administration can be
tailored to achieve optimal efficacy but will depend on such
factors as weight, diet, concurrent medication and other factors
which those skilled in the medical arts will recognize. In any
regard, the dose for imaging should be sufficient for detecting the
presence of the imaging agent at the site of a thrombus in
question. Typically, radiologic imaging will require that the dose
provided by the pharmaceutical composition position of the present
invention be about 5 to 20 .mu.Ci, preferably about 10 .mu.Ci.
Magnetic resonance imaging will require that the dose provided be
about 0.001 to 5 mmole/kg, preferably about 0.005 to 0.5 mmole/kg
of a compound of Formula I complexed with paramagnetic atom. In
either case, it is known in the art that the actual dose will
depend on the location of the thrombus.
[0178] "Pharmaceutically acceptable carriers" for in vivo use are
well known in the pharmaceutical art, and are described, for
example, in Remington's Pharmaceutical Sciences, Mack Publishing
Co. (A. R. Gennaro edit. 1985). The pharmaceutical compositions of
the present invention may be formulated with a pharmaceutically
acceptable carrier to provide sterile solutions or suspensions for
injectable administration. In particular, injectables can be
prepared in conventional forms, either as liquid solutions or
suspensions, solid forms suitable for solution or suspensions in
liquid prior to injection, or as emulsions. Suitable excipients
are, for example, water, saline, dextrose, mannitol, lactose,
lecithin, albumin, sodium glutamate, cysteine hydrochloride, or the
like. In addition, if desired, the injectable pharmaceutical
compositions may contain minor amounts of nontoxic auxiliary
substances, such as wetting agents, pH buffering agents, and the
like. If desired, absorption enhancing preparations (e.g.,
liposomes) may be utilized.
[0179] The present invention also encompasses diagnostic
compositions prepared for storage or administration. These would
additionally contain preservatives, stabilizers and dyes. For
example, sodium benzoate, sorbic acid and esters of
p-hydroxybenzoic acid may be added as preservatives. Id. at 1449.
In addition, antioxidants and suspending agents may be used.
[0180] The in vivo imaging methods of the present invention also
offer several advantages over previous imaging techniques for the
detection or monitoring of the presence, size, regression or
increase of a thrombus. In particular, the present invention
provides compounds, compositions and diagnostic compositions that
bind tightly to the thrombin associated with a thrombus and thereby
reduce "background" due to circulating radioactivity or
paramagnetism arising from unbound imaging agent. Furthermore, in
vivo imaging by intracoronary injection of the compounds,
compositions or diagnostic compositions of the present invention,
is expected to be almost instantaneous since these imaging agents
would saturate the thrombin bound to the thrombus immediately.
[0181] Accordingly, the present invention also includes methods for
in vivo imaging of a thrombus in a mammal, comprising the steps of:
(1) administering to a mammal a diagnostically acceptable amount of
a compound, composition, or diagnostic composition of the present
invention and (2) detecting a thrombus in a blood vessel.
[0182] The term "in vivo imaging" as used herein relates to methods
of the detection of a thrombus in a mammal, as well as the
monitoring of the size, location and number of thrombi in a mammal,
as well as dissolution or growth of the thrombus.
[0183] In employing the compounds, compositions or diagnostic
compositions in vivo by this method, "administering" is
accomplished parenterally, in either a systemic or local targeted
manner. Systemic administration is accomplished by injecting the
compounds, compositions by diagnostic compositions of the present
invention into a convenient and accessible vein or artery. This
includes but is not limited to administration by the ankecubutal
vein. Local targeted administration is accomplished by injecting
the compounds, compositions or diagnostic compositions of the
present invention proximal in flow to a vein or artery suspected to
contain thrombi distal to the injection site. This includes but is
not limited to direct injection into the coronary arterial
vasculature to image coronary thrombi, into the carotid artery to
image thrombi in the cerebral vasculature, or into a pedal vein to
image deep vein thrombosis of the leg.
[0184] Also, the manner of delivery of a composition of the present
invention to the site of a thrombus is considered within the scope
of the term "administering". For example, a compound represented by
Formula I having a chelating means attached thereto may be injected
into the mammal, followed at a later time by the radioactive atom
thereby forming in vivo at the site of the thrombus the composition
comprising the compound of formula complexed to radioactive atom.
Alternatively, a composition comprising the compound of formula
complexed to radioactive atom may be injected into the mammal.
[0185] The "diagnostically effective amount" of the compounds,
compositions or diagnostic compositions used in the methods of the
present invention will, as previously mentioned, depend on the
route of administration, the type of mammal being treated, and the
physical characteristics of the specific mammal under treatment.
These factors and their relationship to determining this dose are
well known to skilled practitioners in the medical diagnostic arts.
In any regard, the dose for in vivo imaging should be sufficient
for detecting the presence of the imaging agent at the site of a
thrombus in question. Typically, radiologic imaging will require
that the dose provided by the diagnostic composition of the present
invention be about 5 to 20 .mu.Ci, preferably about 10 .mu.Ci.
Magnetic resonance imaging will require that the dose provided by
the diagnostic composition be about 0.001 to 5 mmole/kg, preferably
about 0.005 to 0.5 mmole/kg of a compound of Formula I complexed
with paramagnetic atom. In either case, it is known in the art that
the actual dose will depend on the location of the thrombus.
[0186] The detecting of a thrombus by imaging is made possible by
the presence of radioactive or paramagnetic atoms localized at such
thrombus.
[0187] The radioactive atoms associated with the compositions and
diagnostic compositions of the present invention are preferably
imaged using a radiation detection means capable of detecting gamma
radiation, such as a gamma camera or the like. Typically, radiation
imaging cameras employ a conversion medium (wherein the high energy
gamma ray is absorbed, displacing an electron which emits a photon
upon its return to the orbital state), photoelectric detectors
arranged in a spatial detection chamber (to determine the position
of the emitted photons), and circuitry to analyze the photons
detected in the chamber and produce an image.
[0188] The paramagnetic atoms associated with the compositions and
diagnostic compositions of the present invention are detected in
magnetic resonance imaging (MRI) systems. In such systems, a strong
magnetic field is used to align the nuclear spin vectors of the
atoms in a patient's body. The field is disturbed by the presence
of paramagnetic atoms localized at a thrombus and an image of the
patient is read as the nuclei return to their equilibrium
alignments.
[0189] The following examples are illustrative, but not limiting,
of the method and compositions of the present invention. Other
suitable modifications and adaptations of the variety of conditions
and parameters normally encountered and obvious to those skilled in
the art are within the spirit and scope of the invention.
EXAMPLES
Example 1
N-[2-(Amidinoaminooxy)ethyl]-2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-ch-
loro-2-fluorophenyl}acetamide trifluoroacetate Salt
[0190] 21
1. Ethyl 2,2-difluoro-2-phenylacetate (Middleton, W., et al. J.
Org. Chem. 42: 2883 (1980)). PhCF.sub.2CO.sub.2Et
[0191] A mixture of ethyl benzoylformate (12.5 g, 70.0 mmol) and
(diethylamino)sulfur trifluoride (DAST, 18.5 mL, 140 mmol) was
stirred for 48 hours at ambient temperature, and then poured over
ice. The oil formed was taken up into dichloromethane (DCM), washed
with H.sub.2O, dried over Na.sub.2SO.sub.4, concentrated, and
filtered through a short column of silica gel eluting with 50%
DCM/hexane. The filtrate was concentrated to give the title
compound (12.3 g, 88% yield) as a brown liquid. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 7.65-7.63 (m, 2H), 7.52-7.43 (m, 3H), 4.30
(q, J=7.1 Hz, 2H), 1.30 (t, J=7.1 Hz, 3H).
2. 2,2-Difluoro-2-phenylacetic Acid
PhCF.sub.2CO.sub.2H
[0192] A suspension of ethyl 2,2-difluoro-2-phenylacetate (6.0 g,
30 mmol), as prepared in the preceding step, in 1 N NaOH (36 mL, 36
mmol) was stirred at ambient temperature. After 36 hours, the
reaction became almost homogeneous. The mixture was acidified with
1N HCl (36 mL), and extracted with DCM twice. The extracts were
combined, washed with H.sub.2O, dried over Na.sub.2SO.sub.4, and
concentrated to give the title compound (3.85 g, 81% yield) as a
pale yellow solid, that was used without further purification.
3. 2,2-Difluoro-2-phenylacetyl Chloride PhCF.sub.2COCl
[0193] To a flask charged with 2,2-difluoro-2-phenylacetic acid
(0.8 g, 5.06 mmol), as prepared in the preceding step, under argon
in an ice-bath was added oxalyl chloride (5 mL), and the reaction
mixture stirred for 15 min. A solution of dimethylformamide (DMF)
(37 mg, 0.506 mmol) in DCM (0.5 mL) was added. After 2 hours, the
ice-bath was removed, and the mixture continued to stir for 1 hour.
The solvents were evaporated, DCM was added, and then evaporated in
vacuo giving the title compound (0.88 g, 98% yield), that was used
immediately without further purification.
4. 2-(2,3-Difluoro-6-nitrophenyl)acetic Acid (Yokomoto, M, W., et
al. 1991, EP 0 470 578 A1)
[0194] 22
[0195] To a suspension of NaH (11.3 g, 60% oil dispersion, 282
mmol) in tetrahydrofuran (THF) (35 mL) in an ice-bath was added a
solution of diethyl malonate (45.2 g, 42.9 mL, 282 mmol) in THF (70
mL) over a period of an hour so that the reaction temperature was
kept below 20.degree. C. Some white solid precipitated during the
addition. To the above reaction mixture was added a solution of
1,2,3-trifluoro-4-nitrobenzene (25.0 g, 141 mmol) in THF (35 mL)
over a period of 1 hour so that the reaction temperature was kept
below 10.degree. C. The ice-bath was removed and the mixture was
stirred at ambient temperature for 2 hours. Acetic acid (18 mL) was
added to the reaction solution, and THF was evaporated under
reduced pressure. Chloroform (200 mL), H.sub.2O (250 mL), and
concentrated HCl (18 mL) were added. The organic layer was
separated, concentrated, mixed with 4N HCl (45 mL) and acetic acid
(35 mL), and refluxed for 14 hours. The reaction mixture was
allowed to cool to room temperature. The solid precipitated upon
cooling was filtered off, washed with diisopropyl ether, and
dissolved in MeOH (70 mL). After treating with active carbon, the
solvent was evaporated, and the crystalline residue washed with
isopropyl ether, and filtered off to give the title compound (17.6
g, 58% yield) as a white solid. .sup.1H-NMR (400 MHz, CDCl.sub.3)
.delta. 8.05-8.01 (m, 1H), 7.47 (dd, J=17.4, 8.9 Hz, 1H), 4.10 (s,
2H).
5. 2-(2,3-Difluoro-6-nitrophenyl)ethanol (Yokomoto, M, W., et al.,
EP 0 470 578 A1 (1991))
[0196] 23
[0197] To a mixture of NaBH.sub.4 (3.60 g, 95.4 mmol) in THF (12
mL) cooled below 10.degree. C. was added a solution of
2-(2,3-difluoro-6-nitrophenyl)acetic acid (10.9 g, 50.2 mmol), as
prepared in the preceding step, in THF (4 mL) over a period of 1
hour. To this mixture was added a solution of boron trifluoride
diethyl etherate complex (16.5 mL, 131 mmol) in THF (24 mL) over a
period of 1 hour, keeping the reaction temperature below 10.degree.
C. After the addition the reaction was continued to stir on ice for
15 minutes, and then at ambient temperature for 20 minutes. To a
mixture of DCM (180 mL) and H.sub.2O (140 mL) was added NaHCO.sub.3
(15 g, 179 mmol). The reaction mixture was slowly added to the
above NaHCO.sub.3 solution and stirred overnight. The organic layer
was separated, dried over Na.sub.2SO.sub.4, and concentrated to
give the title compound (10.1 g, 99% yield) as light brown oil.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.82 (dd, J=9.1, 4.4 Hz,
1H), 7.27-7.18 (m, 1H), 3.95 (t, J=6.3 Hz, 2H), 3.30-3.27 (m, 2H),
1.82 (s, 1H).
6. 2-{3-[(tert-Butyl)amino]-2-fluoro-6-nitrophenyl}ethanol
(Yokomoto, M, W., et al. EP 0 470 578 A1 (1991)).
[0198] 24
[0199] A mixture of 2-(2,3-difluoro-6-nitrophenyl)ethanol (6.00 g,
29.6 mmol), as prepared in the preceding step, tert-butylamine
(18.6 mL, 1.77 mmol), DMSO (30 mL), and toluene (5 mL) was heated
at reflux for 16 hours. After cooling to ambient temperature the
brown solution was poured into H.sub.2O (300 mL), and the deposited
yellow crystals were filtered and washed with H.sub.2O twice. The
yellow solid was dissolved in CHCl.sub.3 (70 mL), dried over
Na.sub.2SO.sub.4, concentrated, and crystallized from hexane to
give the title compound (4.70 g, 62% yield) as a yellow solid.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.92 (dd, J=9.3, 1.5 Hz,
1H), 6.79 (t, J=8.7 Hz, 1H), 4.69 (br s, 1H), 3.96 (dd, J=11.4, 5.9
Hz, 2H), 3.32 (dt, J=6.5, 3.1 Hz, 2H), 1.75 (t, J=5.3 Hz, 1H), 1.46
(s, 9H).
7. 2-(3-Amino-2-fluoro-6-nitrophenyl)ethanol (Yokomoto, M, W., et
al. EP 0 470 578 A1 (1991))
[0200] 25
[0201] A solution of
2-{3-[(tert-butyl)amino]-2-fluoro-6-nitrophenyl}ethan- ol (3.9 g,
15 mmol), as prepared in the preceding step, in concentrated HCl
(40 mL) was refluxed for 2 hours. After cooling to room
temperature, the mixture was extracted with ethyl acetate
(6.times.50 mL). The extracts were combined, washed with saturated
NaHCO.sub.3 (2 times) and brine, dried over Na.sub.2SO.sub.4, and
concentrated to give the crude product as a solid. The solid was
triturated in hexane, filtered, and dried in high vacuum to produce
the title compound (2.8 g, 93% yield) as a yellow solid.
.sup.1H-NMR (400 MHz, CD.sub.3OD) .delta. 7.80 (dd, J=9.1, 1.5 Hz,
1H), 6.70 (t, J=9.0 Hz, 1H), 3.77 (t, J=7.1 Hz, 2H), 3.26 (dt,
J=7.3, 2.8 Hz, 2H).
8. 2-(3-Amino-2-fluoro-6-nitrophenyl)ethyl Acetate
[0202] 26
[0203] To a solution of DIEA (1.80 mL, 10.6 mmol) and
2-(3-amino-2-fluoro-6-nitrophenyl)ethanol (0.88 g, 4.40 mmol), as
prepared in the preceding step, in THF (10 mL) in an ice-bath was
added a solution of acetyl chloride (319 .mu.L, 4.49 mmol) in THF
(5 mL). After stirring for 1.5 hour, the ice-bath was removed and
the mixture was continued to stir at ambient temperature overnight.
Additional acetyl chloride (63 .mu.L, 0.88 mmol) was added, and the
mixture was stirred for another 16 hours. The solvents were
removed, and the mixture was partitioned between DCM and H.sub.2O.
The organic layer was separated, and the aqueous layer was
back-extracted with DCM. The organic layers were combined, washed
with H.sub.2O (twice), dried over Na.sub.2SO.sub.4, concentrated,
and flash chromatographed on silica gel eluting with EtOAc/DCM (0,
1, 2, and 5%) to give the title compound (0.73 g, 69% yield) as a
yellow solid. .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 7.85 (dd,
J=9.0, 1.5 Hz, 1H), 6.68 (t, J=8.9 Hz, 1H), 4.38-4.35 (m, 4H), 3.38
(dt, J=6.6, 2.8 Hz, 2H), 2.03 (s, 3H).
9.
2-[3-(2,2-Difluoro-2-phenylacetylamino)-2-fluoro-6-nitrophenyl]ethyl
acetate
[0204] 27
[0205] To a solution of DIEA (1.49 mL, 8.55 mmol) and
2-(3-amino-2-fluoro-6-nitrophenyl)ethyl acetate (690 mg, 2.85
mmol), as prepared in the preceding step, in DCM (6 mL) was added a
solution of 2,2-difluoro-2-phenylacetyl chloride (0.99 g, 5.20
mmol), as prepared according to the procedure of step 3 of Example
1, in DCM (3 mL). After stirring for 24 hours, the mixture was
concentrated, and partitioned between DCM and H.sub.2O. The organic
layer was separated, and the aqueous layer extracted with DCM. The
organic layers were combined, washed with H.sub.2O and brine, dried
over Na.sub.2SO.sub.4, concentrated, and flash chromatographed on
silica gel eluting with EtOAc/DCM (0, 2.5, and 5%) to give the
title compound (1.04 g, 92% yield) as an orange oil. .sup.1H-NMR
(400 MHz, CDCl.sub.3) .delta. 8.49-8.43 (m, 2H), 7.87 (d, J=9.2 Hz,
1H), 6.68 (d, J=7.1 Hz, 2H), 7.55-7.49 (m, 3H), 4.35 (t, J=6.4 Hz,
2H), 3.37 (dt, J=6.3, 2.3 Hz, 2H), 2.01 (s, 3H).
10.
2-[2-Amino-5-(2,2-difluoro-2-phenylacetylamino)-6-fluorophenyl]ethyl
Acetate
[0206] 28
[0207] A mixture of
2-[3-(2,2-difluoro-2-phenylacetylamino)-2-fluoro-6-nit-
rophenyl]ethyl acetate (0.84 g, 2.12 mmol), as prepared in the
preceding step, and palladium catalyst (226 mg, 10% on activated
carbon, 0.212 mmol) in ethanol (17 mL) was hydrogenated under a
hydrogen balloon for 3.5 hours. The mixture was filtered through
Celite (diatomaceous earth) and washed with MeOH. The filtrate and
washings were combined, concentrated, and flash chromatographed on
silica gel eluting with EtOAc/DCM (5, 10, and 20%) to give the
title compound (0.713 g, 92% yield) as a white solid. .sup.1H-NMR
(400 MHz, CDCl.sub.3) .delta. 8.10 (bs, 1H), 7.81 (t, J=8.7 Hz,
1H), 7.68-7.66 (m, 2H), 7.52-7.44 (m, 3H), 6.45 (dd, J=8.8, 1.2 Hz,
1H), 4.18 (t, J=7.4 Hz, 2H), 4.07 (bs, 2H), 2.90 (dt, J=7.4, 1.9
Hz, 2H), 2.07 (s, 3H). Mass spectrum (LCMS, ESI) calc'd for
C.sub.18H.sub.18F.sub.3N.sub.2O.sub.3 (M+H): 367.1. Found:
367.1.
11.
N-[4-Amino-2-fluoro-3-(2-hydroxyethyl)phenyl]-2,2-difluoro-2-phenylace-
tamide
[0208] 29
[0209] To a solution of
2-[2-amino-5-(2,2-difluoro-2-phenylacetylamino)-6--
fluorophenyl]ethyl acetate (0.67 g, 1.84 mmol), as prepared in the
preceding step, in MeOH (19 mL) was added dropwise a solution of
K.sub.2CO.sub.3 (280 mg, 2.03 mmol) in H.sub.2O (4.8 mL). The
mixture was stirred for 45 minutes, and then neutralized with 1N
HCl. The MeOH was evaporated, and the mixture was extracted with
EtOAc twice. The extracts were combined, washed with H.sub.2O,
dried over Na.sub.2SO.sub.4, and concentrated to give the title
compound (0.55 g, 92% yield) as a pale yellow solid. .sup.1H-NMR
(400 MHz, CD.sub.3OD) .delta. 7.70-7.68 (m, 2H), 7.54-7.48 (m, 3H),
7.01 (t, J=8.5 Hz, 1H), 6.53 (dd, J=8.6, 1.3 Hz, 1H), 3.70 (t,
J=6.7 Hz, 2H), 2.80 (dt, J=6.7, 2.0 Hz, 2H). Mass spectrum (LCMS,
ESI) calc'd for C.sub.16H.sub.16F.sub.3N.sub.2O.sub.2 (M+H): 325.1.
Found: 325.3.
12.
N-[4-Chloro-2-fluoro-3-(2-hydroxyethyl)phenyl]-2,2-difluoro-2-phenylac-
etamide (Yokomoto, M, W., et al. 1991, EP 0470 578 A1).
[0210] 30
[0211] A suspension of
N-[4-amino-2-fluoro-3-(2-hydroxyethyl)phenyl]-2,2-d-
ifluoro-2-phenylacetamide (1.63 g, 5.00 mmol), as prepared
according to the procedure of the preceding step, in 6N HCl (9 mL)
was cooled in an ice-bath, and then a solution of NaNO.sub.2 (434
mg, 6.30 mmol) in H.sub.2O (2.4 mL) was added over a period of 5
minutes. After 30 minutes, acetic acid (2.9 mL) and concentrated
HCl (2.9 mL) were added and the reaction mixture stirred for 1
hour. To this mixture was added a solution of CuCl (848 mg, 8.55
mmol) in concentrated HCl (5 mL) over a period of 20 minutes. After
stirring in an ice-bath for 3 hours, the reaction mixture was
extracted with EtOAc (200 mL.times.3). The extracts were combined,
washed with H.sub.2O (2 times) and brine, dried over
Na.sub.2SO.sub.4, concentrated, and flash chromatographed on silica
gel eluting with EtOAc/DCM (0, 2.5, and 5%) to deliver the title
compound (0.845 g, 48% yield) as a pale orange oil. .sup.1H-NMR
(400 MHz, CDCl.sub.3) .delta. 8.31 (s, 1H), 8.14 (t, J=8.6 Hz, 1H),
7.68-7.66 (m, 2H), 7.54-7.46 (m, 3H), 7.19 (dd, J=8.9, 1.7 Hz, 1H),
3.86 (dd, J=12.6, 6.5 Hz, 2H), 3.09 (dt, J=6.7, 2.3 Hz, 2H), 1.58
(t, J=5.7 Hz, 1H). Mass spectrum (LCMS, ESI) calc'd for
C.sub.16H.sub.14ClF.sub.3NO.sub.2 (M+H): 344.1. Found: 344.2.
13.
2-{3-[(2,2-Difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}ethan-
ol
[0212] 31
[0213] To a solution
N-[4-chloro-2-fluor-3-(2-hydroxyethyl)phenyl]-2,2-dif-
luoro-2-phenylacetamide (1.05 g, 3.06 mmol), as prepared according
to the procedure in the preceding step, in THF (12 mL) at 0.degree.
C. under argon was added a solution of borane-THF complex in THF
(12.3 mL, 12.3 mmol, 1.0 M) over a period of 10 minutes, and the
reaction mixture continued to stir until the ice-bath expired. The
reaction mixture was heated at reflux for 20 hours, and allowed to
cool to room temperature. A solution of K.sub.2CO.sub.3 (1.7 g, 12
mmol) in H.sub.2O (12 mL) was added, the THF was removed in vacuo,
and the mixture was extracted with DCM (3 times). The extracts were
combined, washed with brine, dried over Na.sub.2SO.sub.4,
concentrated, and flash chromatographed on silica gel eluting with
EtOAC/DCM (0 and 2.5%) to give the title compound (815 mg, 81%
yield) as a colorless oil. .sup.1H-NMR (400 MHz, CDCl.sub.3)
.delta. 7.52-7.43 (m, 5H), 6.97 (dd, J=8.8, 1.7 Hz, 1H), 6.51 (t,
J=8.9 Hz, 1H), 4.17 (bs, 1H), 3.83 (t, J=6.9 Hz, 2H), 3.74 (dt,
J=13.4, 6.6 Hz, 2H), 3.04 (dt, J=6.9, 2.4 Hz, 2H), 1.43 (s, 1H).
Mass spectrum (LCMS, ESI) calc'd for C.sub.16H.sub.16ClF.sub.3NO
(M+H): 330.1. Found: 330.3.
14.
2-{3-[(2,2-Difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}ethan-
al
[0214] 32
[0215] To a solution of DMSO (1.03 mL, 14.5 mmol), DIEA (1.99 mL,
11.4 mmol), and
2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophen-
yl}ethanol (1.45 g, 4.4 mmol), as prepared according to the
procedure in the preceding step, in DCM (140 mL) in an ice-bath was
added sulfur trioxide pyridine complex (1.82 g, 11.4 mmol) and
stirred at the same temperature for 3.5 hours. The mixture was
diluted with DCM (300 mL), washed with 10% citric acid (3 times),
H.sub.2O, and brine, dried over Na.sub.2SO.sub.4, and concentrated
to give the title compound (1.43 g, 99% yield) as an orange oil,
that was used without further purification.
15. 2-{3-[(2,2-Difluoro-2-phenylethyl)amino]-6-chloro-2-
fluorophenyl}acetic Acid (Dalcanale, E., et al. J. Org. Chem.,
51:567 (1986)).
[0216] 33
[0217] A solution of sodium chlorite (692 mg, 6.11 mmol) in
H.sub.2O (6.1 mL) was added over a period of 30 minutes to a
stirred mixture of
2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}ethanal
(1.43 g, 4.37 mmol), as prepared in the preceding step, in DMSO
(4.5 mL) and of NaH.sub.2PO.sub.4 (141 mg, 1.18 mmol) in H.sub.2O
(1.7 mL). After the addition, the mixture was stirred at ambient
temperature overnight, acidified with 10 M HCl to pH 1, and
extracted with DCM (3 times). The extracts were combined, washed
with H.sub.2O and brine, dried over Na.sub.2SO.sub.4, and
concentrated in vacuo. The resulting residue was flash
chromatographed on silica gel eluting with MeOH/DCM (0, 2, and 4%)
to give the title compound (0.77 g, 51% yield) as a pale brown
solid. .sup.1H-NMR (400 MHz, CD.sub.3OD) .delta. 7.53-7.51 (m, 2H),
7.44-7.41 (m, 3H), 6.93 (dd, J=8.9, 1.8 Hz, 11H), 6.62 (t, J=9.1
Hz, 11H), 3.80 (t, J=13.7 Hz, 2H), 3.74 (d, J=2.2 Hz, 2H). Mass
spectrum (LCMS, ESI) calc'd for C.sub.16H.sub.14ClF.sub.3NO.sub.2
(M+H): 344.1. Found: 344.4.
16. Tert-Butyl
2-aza-3-{[2-(2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chl-
oro-2-fluorophenyl}acetylamino)ethoxy]amino}-3-[(tert-butoxy)carbonylamino-
]prop-2-enate
[0218] 34
[0219] To a solution of
2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro--
2-fluorophenyl}acetic acid (28 mg, 82 .mu.mol), as prepared in the
preceding step, in DMF (0.3 mL) in an ice-bath was added BOP (58
mg, 130 .mu.mol), HCl salt of
[N,N'-di(tert-butoxycarbonyl)]-2-aminoethoxyguanidi- ne (36 mg, 102
.mu.mmol) (Tianbao Lu, et al., WO 99/26926 (1999)), and a solution
of DIEA (42 mg, 33 .mu.mol) in DMF (0.1 mL). After the ice-bath
expired, the reaction mixture continued to stir at ambient
temperature overnight. The solvents were evaporated, and the
resulting residue was partitioned between saturated NaHCO.sub.3 and
DCM. The aqueous phase was extracted with DCM, and the organic
layers were combined, washed with 10% KHSO.sub.4, H.sub.2O, and
brine, dried over Na.sub.2SO.sub.4, concentrated, and flash
chromatographed on silica gel eluting with MeOH/DCM (1%) to give
the title compound (44 mg, 83% yield) as a colorless oil.
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 7.54-7.50 (m, 2H),
7.45-7.42 (m, 3H), 6.93 (dd, J=8.8, 1.7 Hz, 1H), 6.63 (t, J=9.0 Hz,
1H), 4.03 (t, J=4.8 Hz, 2H), 3.81 (t, J=13.7 Hz, 2H), 3.71 (d,
J=1.9 Hz, 2H), 3.47 (t, J=5.1 Hz, 2H), 1.50 (s, 9H), 1.48 (s, 9H).
Mass spectrum (LCMS, ESI) calc'd for
C.sub.29H.sub.38ClF.sub.3N.sub.5O.sub.6 (M+H): 644.2. Found:
644.1.
17.
N-[2-(Amidinoaminooxy)ethyl]-2-{3-[(2,2-difluoro-2-phenylethyl)amino]--
6-chloro-2-fluorophenyl}acetamide Trifluoroacetate Salt
[0220] 35
[0221] A solution of tert-butyl
2-aza-3-{[2-(2-{3-[(2,2-difluoro-2-phenyle-
thyl)amino]-6-chloro-2-fluorophenyl}acetylamino)ethoxy]amino}-3-[(tert-but-
oxy) carbonylamino]prop-2-enoate (44 mg, 68 .mu.mol), as prepared
in the preceding step, in TFA/DCM (2 mL, 2/3) was stirred at room
temperature for 4 hours. The solvents were evaporated, and the
resulting residue was flash chromatographed on silica gel eluting
with 0.05% TFA in MeOH/DCM (5 and 10%) to deliver the title
compound (37 mg, 98% yield) as a white solid. .sup.1H-NMR (400 MHz,
CD.sub.3OD) .delta. 7.67-7.52 (m, 2H), 7.47-7.42 (m, 3H), 6.96 (dd,
J=8.9, 1.7 Hz, 1H), 6.66 (t, J=9.1 Hz, 1H), 3.93 (t, J=5.4 Hz, 2H),
3.82 (t, J=13.8 Hz, 2H), 3.71 (d, J=2.0 Hz, 2H), 3.50 (t, J=5.4 Hz,
2H). Mass spectrum (LCMS, ESI) calc'd for
C.sub.19H.sub.22ClF.sub.3N.sub.5O.sub.2 (M+H): 444.1. Found:
444.2.
Example 2
N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-(3-[(2,2-difluoro-2-phenylethyl)-
amino]-6-chloro-2-fluorophenyl)acetamide Hydrochloride Salt
[0222] 36
1.
2-{3-[(2,2-Difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}-N-({6-
-[(tert-butoxy)carbonylamino]-2-methyl(3-pyridyl)}methyl)acetamide
[0223] 37
[0224] To a solution of
2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro--
2-fluorophenyl}acetic acid (516 mg, 1.5 mmol), as prepared in step
15 of Example 1, in DMF (8.0 mL) was added
N-[5-(aminomethyl)-6-methyl (2-pyridyl)](tert-butoxy)carboxamide
(498 mg, 2.1 mmol) (Sanderson, P. E., et al., WO 97/01338 (1997)),
BOP (1.06 g, 2.4 mmol), and DIEA (0.78 mL, 4.5 mmol). After
stirring for 18 hours, additional amine (107 mg, 450 .mu.mol) was
added, and the mixture continued to stir for 18 hours. The solvents
were evaporated, and the reaction mixture was partitioned between
DCM and saturated NaHCO.sub.3. The organic layer was separated, and
the aqueous layer extracted with DCM. The organic layers were
combined, washed with 10% KHSO.sub.4 (2 times), H.sub.2O, and
brine, dried over Na.sub.2SO.sub.4, concentrated, and flash
chromatographed on silica gel eluting with MeOH/DCM (0, 1.5, and
2.5%) to give the title compound (770 mg, 91% yield) as a pale
brown foam. .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 7.66 (d,
J=8.4 Hz, 1H), 7.51-7.41 (m, 6H), 7.23 (bs, 1H), 7.01 (dd, J=8.8,
1.5 Hz, 1H), 6.57 (t, J=9.0 Hz, 1H), 5.64 (br s, 1H), 4.37 (d,
J=5.6 Hz, 2H), 4.26-4.22 (m, 1H), 3.79-3.70 (m, 4H), 2.35 (s, 3H),
1.50 (s, 9H). Mass spectrum (LCMS, ESI) calc'd for
C.sub.28H.sub.31ClF.sub.3N.sub.4O.sub.3 (M+H): 563.2. Found:
562.9.
2.
N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-{3-[(2,2-difluoro-2-phenyleth-
yl)amino]-6-chloro-2-fluorophenyl}acetamide Hydrochloride Salt
[0225] 38
[0226] To a flask charged with
2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6--
chloro-2-fluorophenyl}-N-({6-[(tert-butoxy)carbonylamino]-2-methyl(3-pyrid-
yl)}methyl)acetamide (770 mg, 1.37 mmol), as prepared in the
preceding step, was added a solution of HCl in 1,4-dioxane (5 mL,
20 mmol, 4.0 M). After stirring at ambient temperature for 1.5
hours, some solid precipitated. A solution of MeOH (1 mL) in DCM (3
mL) was added to dissolve the solid, and the mixture was stirred
for additional 4 hours. The solvents were removed, and the
resulting residue was washed with DCM (5 mL.times.2), ether (8
mL.times.2), and dried in high vacuum to give the title compound
(620 mg, 91% yield) as a pale brown solid. .sup.1H-NMR (400 MHz,
CD.sub.3OD) .delta. 7.81 (d, J=9.1 Hz, 1H), 7.54-7.51 (m, 2H),
7.45-7.42 (m, 3H), 6.94 (dd, J=8.9, 1.6 Hz, 1H), 6.80 (d, J=9.1 Hz,
1H), 6.65 (t, J=9.1 Hz, 1H), 4.25 (s, 2H), 3.81 (t, J=13.8 Hz, 2H),
3.70 (d, J=1.8 Hz, 2H), 2.50 (s, 3H). Mass spectrum (LCMS, ESI)
calcd for C.sub.23H.sub.23ClF.sub.3N.sub.4O (M+H): 463.1. Found:
463.7.
Example 3
N-[(6-Amino-2,4-dimethyl(3-pyridyl))methyl]-2-{3-[(2,2-difluoro-2-phenylet-
hyl)amino]-6-chloro-2-fluorophenyl}acetamide Hydrochloride Salt
[0227] 39
1.
2-{3-[(2,2-Difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}-N-({6-
-[(tert-butoxy)carbonylamino]-2,4-dimethyl(3-pyridyl)}methyl)acetamide
[0228] 40
[0229] To a solution of
2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro--
2-fluorophenyl}acetic acid (25 mg, 73 .mu.mol), as prepared in step
15 of Example 1, in DMF (0.25 mL) was added BOP (52 mg, 116
.mu.mol), a solution of DIEA (38 mg, 295 mol) in DMF (0.1 mL), and
N-[5-(aminomethyl)-4,6-dimethyl(2-pyridyl)](tert-butoxy)carboxamide
(23 mg, 91 .mu.mol) (Sanderson, P. E., et al. WO 97/01338 (1997)).
After stirring at ambient temperature for 2 days, additional amine
(7 mg, 28 .mu.mol) BOP (16 mg, 36 .mu.mol), and DIEA (9 mg, 70
.mu.mol) were added, and the mixture was stirred for another 16
hours. The solvents were evaporated, and the resulting residue was
partitioned between saturated NaHCO.sub.3 and DCM. The organic
layer was separated, and the aqueous layer was extracted with DCM.
The organic layers were combined, washed with 10% citric acid,
H.sub.2O, and brine, dried over Na.sub.2SO.sub.4, concentrated, and
flash chromatographed on silica gel eluting with MeOH/DCM (0, 1,
2%) to produce the title compound (18.5 mg, 44% yield) as a pale
brown solid. .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 7.58 (s,
1H), 7.50-7.41 (m, 5H), 7.24 (s, 1H), 6.98 (dd, J=8.8, 1.1 Hz, 1H),
6.55 (t, J=9.0 Hz, 1H), 5.33 (bs, 1H), 4.40 (d, J=4.7 Hz, 2H),
4.24-4.20 (m, 1H), 3.78-3.70 (m, 4H), 2.39 (s, 3H), 2.28 (s, 3H),
1.50 (s, 9H). Mass spectrum (LCMS, ESI) calcd for
C.sub.29H.sub.33ClF.sub.3N.sub.4O.sub.3 (M+H): 577.0. Found:
577.1.
2.
N-[(6-Amino-2,4-dimethyl(3-pyridyl))methyl]-2-{3-[(2,2-difluoro-2-pheny-
lethyl)amino]-6-chloro-2-fluorophenyl}acetamide Hydrochloride
Salt
[0230] 41
[0231] A solution of HCl in 1,4-dioxane (4.0 M, 0.5 mL, 2 mmol) was
added to
2-{3-[(2,2-difluoro-2-phenylethyl)amino]-6-chloro-2-fluorophenyl}-N-({-
6-[(tert-butoxy)carbonylamino]-2,4-dimethyl(3-pyridyl)}methyl)acetamide
(18.5 mg, 32 .mu.mol), as prepared in the preceding step. After
stirring at ambient temperature for 3 hours, solid precipitated. A
solution of MeOH (0.1 mL) in DCM (1 mL) was added to dissolve the
solid. After stirring for another 2 hours the reaction was
concentrated to give a brown solid, that was washed with ether and
DCM and dried in vacuo to produce the title compound (12.6 mg, 77%
yield) as a pale brown solid. .sup.1H-NMR (400 MHz, CD.sub.3OD)
.delta. 8.40 (bs, 1H), 7.53-7.51 (m, 2H), 7.47-7.40 (m, 3H), 6.93
(dd, J=8.8, 1.3 Hz, 1H), 6.68 (s, 1H), 6.64 (t, J=9.1 Hz, 1H), 4.31
(d, J=4.6 Hz, 2H), 3.81 (t, J=13.8 Hz, 2H), 3.67 (d, J=1.2 Hz, 2H),
2.54 (s, 3H), 2.42 (s, 3H). Mass spectrum (LCMS, ESI) calc'd for
C.sub.24H.sub.25ClF.sub.3N.sub.4O (M+H): 477.2. Found: 477.5.
Example 4
N-[2-(Amidinoaminooxy)ethyl]-2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl-
]amino}-6-chloro-2-fluorophenyl)acetamide Trifluoroacetate Salt
[0232] 42
1. Ethyl 2-(4-fluoronaphthyl)-2-oxoacetate
[0233] 43
[0234] A solution of n-butyllithium (2.5 M in THF, 20 mL, 50 mmol)
was cooled to -78.degree. C., and a solution of
1-bromo-4-fluoronaphthalene (11.25 g, 50 mmol) in THF (40 mL) was
added slowly and the mixture was stirred for 1 hour. The reaction
mixture was warmed to -20.degree. C., then added to a solution of
diethyl oxalate (29.2 g, 200 mmol) in THF (40 mL) at -78.degree. C.
After slowly warming-up to room temperature, EtOAc (100 mL), 10%
HCl (50 mL) and water (50 mL) were added and the phases were
separated. The aqueous layer was extracted with EtOAc (2.times.100
mL), and the organic layers were combined, washed with brine (50
mL), and dried over Na.sub.2SO.sub.4. After evaporating the solvent
and the excess diethyl oxalate under high vacuum, the residue was
purified by flash column chromatography (1:1 DCM:hexane) to give
the title compound (9.4 g, 76% yield) as a white solid. .sup.1H-NMR
(400 MHz, CDCl.sub.3) o (9.13 (d, J=8.6 Hz, 1H), 8.20 (d, J=8.4 Hz,
1H), 8.01 (dd, J=8.2, 5.4 Hz, 1H), 7.76(t, J=7.2 Hz, 1H), 7.67 (t,
J=8.1 Hz, 1H), 7.21 (t, J=8.5 Hz, 1H), 4.49 (q, J=7.1 Hz, 2H), 1.45
(t, J=7.1 Hz, 3H).
2. Ethyl 2,2-difluoro-2-(4-fluoronaphthyl)acetate
[0235] 44
[0236] To a solution of ethyl 2-(4-fluoronaphthyl)-2-oxoacetate
(9.4 g, 38.2 mmol), as prepared in the preceding step, in DCM (60
mL) was added DAST (16.1 g, 100 mmol). The mixture was stirred at
room temperature overnight, poured into ice slowly, and extracted
with DCM (3.times.50 mL). The organic layers were combined, washed
with brine, and dried over Na.sub.2SO.sub.4. After evaporating the
solvent, the residue was purified by flash column chromatography
(1:1 DCM:hexane) to give the title compound (9.7 g, 95% yield) as a
light brown oil. .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.20 (m,
2H), 7.82 (dd, J=8.2, 5.3 Hz, 1H), 7.63 (m, 2H), 7.20 (t, J=8.4 Hz,
1H), 4.28 (q, J=7.1 Hz, 2H), 1.24 (t, J=7.1 Hz, 3H).
3. 2,2-Difluoro-2-(4-fluoronaphthyl)acetic Acid
[0237] 45
[0238] To a solution of ethyl
2,2-difluoro-2-(4-fluoronaphthyl)acetate (9.6 g, 35.8 mmol), as
prepared in the preceding step, in methanol (20 mL) and THF (20 mL)
was added a solution of NaOH (2.0 g, 50 mmol) in water (40 mL). The
reaction mixture was stirred at room temperature for 2 hours. After
evaporating the methanol and THF in vacuo, the aqueous phase was
acidified to pH 2 using 10% HCl, and extracted with DCM (3.times.50
mL). The extracts were combined, washed with brine, dried over
Na.sub.2SO.sub.4, and concentrated to give the title compound (8.1
g, 94% yield) as an off white solid. .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta.9.68 (br s, 1H), 8.18 (m, 2H), 7.83 (dd, J=8.1,
5.3 Hz, 1H), 7.62 (m, 2H), 7.19 (t, =8.3 Hz, 1H).
4.
2-{3-[2,2-Difluoro-2-(4-fluoronaphthyl)acetylamino]-2-fluoro-6-nitrophe-
nyl}ethyl Acetate
[0239] 46
[0240] To a solution of DIEA (7.8 mL) and
2-(3-amino-2-fluoro-6-nitropheny- l)ethyl acetate (4.6 g, 19 mmol),
prepared as in step 8 of Example 1, in DCM (60 mL) was added
2,2-difluoro-2-(4-fluoronaphthyl)acetyl chloride (prepared by
refluxing 2,2-difluoro-2-(4-fluoronaphthyl)acetic acid, as prepared
in the preceding step, with oxalyl chloride) (7.8 g, 30 mmol) in
DCM (40 mL). The mixture was stirred at room temperature for 1
hour. Additional DCM (100 mL) was added, and the resulting mixture
was washed with 10% citric acid (3.times.40 mL) and brine, and
dried over Na.sub.2SO.sub.4. After evaporating the solvent, the
residue was purified by flash column chromatography eluting with
DCM to give the title compound (5.3 g, 61%) as a yellow oil.
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.45 (m, 2H), 8.21 (d,
J=7.0 Hz, 2H), 7.87 (s, 1H), 7.85 (t, J=4.1 Hz, 1H), 7.66 (m, 2H),
7.22 (t, J=8.3 Hz, 1H), 4.34 (t, J=6.4 Hz, 2H), 3.36 (t, J=6.4 Hz,
2H), 2.00 (s, 3H).
5.
2-{2-Amino-5-[2,2-difluoro-2-(4-fluoronaphthyl)acetylamino]-6-fluorophe-
nyl}ethyl Acetate
[0241] 47
[0242] A mixture of
2-{3-[2,2-difluoro-2-(4-fluoronaphthyl)acetylamino]-2--
fluoro-6-nitrophenyl}ethyl acetate (4.9 g, 10.5 mmol), as prepared
in the preceding step, and Pd/C (10%, 500 mg) in ethanol (50 mL)
and THF (50 mL) was stirred under hydrogen for 5 hours. The
reaction mixture was filtered through Celite, and washed with THF
and MeOH. The filtrate and washings were combined, concentrated in
vacuo, and flash chromatographed on silica gel eluting with
EtOAc/DCM (0 to 2%) to produce the title compound (3.8 g, 83%) as
an off white solid. .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.27
(d, J=8.2 Hz, 1H), 8.19 (d, J=8.6 Hz, 1H), 8.10 (s, 1H), 7.82 (m,
2H), 7.63 (m, 2H), 7.19 (t, J=8.6 Hz, 1H), 6.46 (d, J=8.7 Hz, 1H),
4.19 (t, J=7.4 Hz, 2H), 4.08 (s, 2H), 2.91(t, J=7.3 Hz, 2H), 2.08
(s, 3H).
6.
N-[4-Amino-2-fluoro-3-(2-hydroxyethyl)phenyl]-2,2-difluoro-2-(4-fluoron-
aphthyl)acetamide
[0243] 48
[0244] To a solution of
2-{2-amino-5-[2,2-difluoro-2-(4-fluoronaphthyl)ace-
tylamino]-6-fluorophenyl}ethyl acetate (3.8 g, 8.8 mmol), as
prepared in the preceding step, in MeOH (40 mL) and THF (20 mL) was
added a solution of K.sub.2CO.sub.3 (1.68 g, 12 mmol) in water (30
mL). The mixture was stirred at room temperature for 3 hours.
Additional water (50 mL) was added, and the resulting mixture was
extracted with EtOAc (3.times.50 mL). The extracts were combined,
washed with brine, dried over Na.sub.2SO.sub.4, and concentrated in
vacuo to give the title compound (3.3 g, 96%) as an off white
solid. .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.27 (d, J=8.4 Hz,
1H), 8.19 (d, J=8.2 Hz, 1H), 8.13 (s, 1H), 7.81 (m, 2H), 7.64 (m,
2H), 7.19 (t, J=8.7 Hz, 1H), 6.47 (d, J=8.7 Hz, 1H), 4.07 (s, 2H),
3.89 (t, J=5.6 Hz, 2H), 2.85(t, J=5.5 Hz, 2H).
7.
N-[4-Chloro-2-fluoro-3-(2-hydroxyethyl)phenyl]-2,2-difluoro-2-(4-fluoro-
naphthyl)acetamide (Doyle, M. P., et al. J. Org. Chem., 42: 2426
(1977))
[0245] 49
[0246] To a flask charged with copper(II) chloride (1.84 g, 13.7
mmol) was added a solution of tert-butylnitrite (1.46 g, 12.8 mmol,
90%, Aldrich) in acetonitrile (35 mL) under argon atmosphere. The
resulting green reaction mixture was cooled in an ice-bath to
0.degree. C., and a solution of N-[4-amino-2-fluor-3-
(2-hydroxyethyl)phenyl]-2,2-difluoro-2-- (4-fluoronaphthyl)
acetamide (3.58 g, 9.13 mmol), as prepared according to the
procedure of the preceding step, in acetonitrile (60 mL) was added
over a period of 45 minutes. After stirring for an additional 6
hours at 0.degree. C., the resulting brown mixture was allowed to
warm up to ambient temperature, then poured into 20% aqueous HCl
(160 mL), and extracted with DCM (3 times). The extracts were
combined, washed with 20% HCl, H.sub.2O, and brine, dried over
Na.sub.2SO.sub.4, concentrated, and flash chromatographed on silica
gel eluting with EtOAc/DCM (0 and 2.5%) to deliver the title
compound (2.1 g, 56% yield) as a white solid. .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 8.31 (br s, 1H), 8.24-8.12 (m, 3H), 7.84 (dd,
J=8.2, 5.3 Hz, 1H). 7.68-7.60 (m, 2H), 7.22-7.20 (m, 2H), 3.87 (dd,
J=12.6, 6.5 Hz, 2H), 3.09 (dt, J=6.7, 2.2 Hz, 2H), 1.47 (t, J=5.6
Hz, 2H). Mass spectrum (LCMS, ESI) calc'd for
C.sub.20H.sub.15ClF.sub.4NO.sub.2 (M+H): 412.1. Found: 412.6.
8.
2-(3-{[2,2-Difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorop-
henyl)ethanol
[0247] 50
[0248] To a solution of
N-[4-chloro-2-fluoro-3-(2-hydroxyethyl)phenyl]-2,2-
-difluoro-2-(4-fluoronaphthyl)acetamide (1.9 g, 4.6 mmol), as
prepared in the preceding step, in THF (19 mL) at 0.degree. C. was
added dropwise a solution of BH.sub.3-THF complex (19.4 mL, 19.4
mmol, 1.0 M in THF) over a period of 20 minutes, and the reaction
mixture continued to stir until the ice bath expired. The mixture
was then heated at reflux in an oil bath at 75 to 80.degree. C. for
3 hours, and continued to stir at ambient temperature overnight. A
solution of NaHCO.sub.3 (1.63 g, 19.4 mmol) in H.sub.2O (20 mL) was
added, the THF was evaporated, and the resulting mixture was
extracted with DCM twice. The extracts were combined, washed with
H.sub.2O and brine, dried over Na.sub.2SO.sub.4, concentrated, and
flash chromatographed on silica gel eluting with EtOAc/DCM (0, 1,
1.5%) to give the title compound (805 mg, 44% yield) as a white
solid. .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.24-8.19 (m, 2H),
7.70-7.61 (m, 3H), 7.14 (t, J=9.3 Hz, 1H), 6.90 (dd, J=8.7, 1.6 Hz,
1H), 6.42 (t, J=8.9 Hz, 1H), 4.22-4.16 (m, 1H), 4.00 (dt, J=13.4,
6.8 Hz, 2H), 3.82 (dd, J=12.1, 6.4 Hz, 2H), 3.02 (dt, J=6.8, 2.3
Hz, 2H), 1.39 (br s, 1H). Mass spectrum (LCMS, ESI) calc'd for
C.sub.20H.sub.17ClF.sub.4NO (M+H): 398.1. Found: 398.3.
9.
2-(3-{[2,2-Difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorop-
henyl)ethanal
[0249] 51
[0250] To a solution of DMSO (470 mg, 6.0 mmol), DIEA (823 .mu.L,
4.74 mmol) and
2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-
-fluorophenyl)ethanol (723 mg, 1.82 mmol), as prepared in the
preceding step, in DCM (55 mL) in an ice-bath was added sulfur
trioxide pyridine complex (754 mg, 4.74 mmol). After stirring for
3.5 hours the reaction mixture was diluted with DCM (110 mL). The
organic layer was separated, and the aqueous layer extracted with
DCM (100 mL). The organic layers were combined, washed with 10%
citric acid (3 times), H.sub.2O, and brine, dried over
Na.sub.2SO.sub.4, and concentrated to give the title compound (722
mg, quantitative yield) as an orange oil. .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 9.69 (d, J=1.1 Hz, 1H), 8.22-8.18 (m, 2H),
7.67-7.60 (m, 3H), 7.14 (t, J=9.2 Hz, 1H), 6.94 (d, J=8.8 Hz, 1H),
6.49 (t, J=8.7 Hz, 1H), 4.20 (bs, 1H), 4.00 (dt, J=13.3, 6.7 Hz,
2H), 3.83 (s, 2H).
10.
2-(3-{[2,2-Difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluoro-
phenyl)acetic Acid
[0251] 52
[0252] A solution of sodium chlorite (309 mg, 2.73 mmol, 80%) in
H.sub.2O (3.0 mL) was added dropwise over a period of 30 minutes to
a stirred mixture of
2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro--
2-fluorophenyl)ethanal (722 mg, 1.82 mmol), as prepared in the
preceding step, in DMSO (3.6 mL) and of NaH.sub.2PO.sub.4 (74 mg,
0.55 mmol) in H.sub.2O (0.9 mL). After the addition, the mixture
was stirred at ambient temperature for 48 hours, then acidified
with 10 M HCl to pH 1, and extracted with DCM (3 times). The
extracts were combined, washed with H.sub.2O and brine, dried over
Na.sub.2SO.sub.4, and concentrated in vacuo. The resulting residue
was flash chromatographed on silica gel eluting with MeOH/DCM (0,
1, 1.5, and 2%) to give the starting aldehyde
2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorophe-
nyl)ethanal (165 mg, 23% yield) and the title compound (570 mg, 76%
yield) as a solid. .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.32
(d, J=8.4 Hz, 1H), 8.15 (d, J=8.0 Hz, 1H), 7.71-7.61 (m, 3H), 7.18
(dd, J=9.8, 8.5 Hz, 1H), 6.78 (dd, J=8.8, 1.5 Hz, 1H), 6.44 (t,
J=9.1 Hz, 1H), 4.05 (t, J=13.5 Hz, 2H), 3.69 (d, J=2.1 Hz, 2H).
11. tert-Butyl
2-aza-3-({2-[2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]-
amino}-6-chloro-2-fluorophenyl)acetylamino]ethoxy}amino)-3-[(tert-butoxy)c-
arbonylamino]prop-2-enoate
[0253] 53
[0254] To a solution of
2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amin-
o}-6-chloro-2-fluorophenyl)acetic acid (570 mg, 1.39 mmol), as
prepared in the preceding step, in DMF (7.5 mL) in an ice-bath was
added BOP (981 mg, 2.22 mmol), HCl salt of
[N,N'-di(tert-butoxycarbonyl)]-2-aminoethoxyguani- dine (689 mg,
1.94 mmol), and DIEA (0.96 mL, 5.55 mmol). After the ice-bath
expired, the mixture continued to stir at ambient temperature
overnight. Additional BOP (123 mg, 0.28 mmol) and the HCl salt of
[N,N'-di(tert-butoxycarbonyl)]-2-aminoethoxyguanidine (98 mg, 0.28
mmol) were added, and the reaction mixture was stirred for 24
hours. The solvents were evaporated, and the resulting residue was
partitioned between saturated NaHCO.sub.3 and DCM. The organic
layer was separated, and the aqueous layer was extracted with DCM.
The organic layers were combined, washed with 10% KHSO.sub.4,
H.sub.2O, and brine, dried over Na.sub.2SO.sub.4, concentrated, and
flash chromatographed on silica gel eluting with MeOH/DCM (0,0,5,
and 1%) to give the title compound (720 mg, 73% yield) as a white
foam. .sup.1H-NMR (400 MHz, CD.sub.3OD) .delta. 9.12 (s, 1H),
8.24-8.19 (m, 2H), 7.78-7.81 (m, 1H), 7.70-7.60 (m, 4H), 7.14 (dd,
J=9.6, 8.4 Hz, 1H), 6.91 (dd, J=8.8, 1.6 Hz, 1H), 6.44 (t, J=8.8
Hz, 1H), 4.20-4.15 (m, 1H), 4.13-4.10 (m, 2H), 3.98 (dt, J=13.4,
6.7 Hz, 2H), 3.78 (d, J=1.9 Hz, 2H), 3.60 (dd, J=8.6, 4.9 Hz, 2H),
1.51 (s, 9H), 1.48 (s, 9H). Mass spectrum (LCMS, ESI) calc'd for
C.sub.33H.sub.39ClF.sub.4N.sub.5O.sub.6 (M+H): 712.2. Found:
712.3.
12.
N-[2-(Amidinoaminooxy)ethyl]-2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)e-
thyl]amino}-6-chloro-2-fluorophenyl)acetamide Trifluoroacetate
Salt
[0255] 54
[0256] A solution of tert-butyl
2-aza-3-({2-[2-(3-{[2,2-difluoro-2-(4-fluo-
ronaphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)acetylamino]ethoxy}amino)--
3-[(tert-butoxy)carbonylamino]prop-2-enoate (720 mg, 1.01 mmol), as
prepared in the preceding step, in TFA/DCM (2: 3, 30 mL) was
stirred at ambient temperature for 4 hours. The solvents were
evaporated, and the resulting residue was flash chromatographed on
silica gel eluting with 0.05% TFA in MeOH/DCM (5 and 10%) to give
the title compound (626 mg, 99% yield) as a pale brown foam.
.sup.1H-NMR (400 MHz, CD.sub.3OD) .delta. 8.33 (d, J=8.5 Hz, 1H),
8.17-8.15 (m, 1H), 7.73-7.63 (m, 3H), 7.20 (dd, J=10.0, 8.3 Hz,
1H), 6.82 (dd, J=8.8, 1.6 Hz, 1H), 6.92 (t, J=9.1 Hz, 1H), 4.07 (t,
J=13.7 Hz, 2H), 3.92 (t, J=5.4 Hz, 2H), 3.67 (d, J=1.8 Hz, 2H),
3.52-3.51 (m, 2H). Mass spectrum (LCMS, ESI) calc'd for
C.sub.23H.sub.23ClF.sub.4N.sub.5O.sub.2 (M+H): 512.1. Found:
512.2.
Example 5
N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-(3-{[2,2-difluoro-2-(4-fluoronap-
hthyl)ethyl]amino}-6-chloro-2-fluorophenyl)acetamide Hydrochloride
Salt
[0257] 55
1.
2-(3-{[2,2-Difluoro-2-(4-fluoronaphthyl)ethyl]amino}-6-chloro-2-fluorop-
henyl)-N-({6-[(tert-butoxy)carbonylamino]-2-methyl(3-pyridyl)}methyl)aceta-
mide
[0258] 56
[0259] To a solution of
2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amin-
o}-6-chloro-2-fluorophenyl)acetic acid (15 mg, 37 .mu.mol), as
prepared according to the procedure of step 10 of Example 4, in DMF
(0.3 mL) was added BOP (26 mg, 58 .mu.mol),
N-[5-(aminomethyl)-6-methyl(2-pyridyl)](te- rt-butoxy)carboxamide
(12 mg, 51 .mu.mol), and a solution of DIEA (19 mg, 146 mmol) in
DMF (0.1 mL) (Sanderson, P. E., et al., WO 97/01338 (1997)). The
mixture was stirred overnight, the solvents evaporated, and the
resulting mixture was partitioned between saturated NaHCO.sub.3 and
DCM. The organic layer was separated, and the aqueous layer
extracted with DCM. The organic layers were combined, washed with
10% KHSO.sub.4, H.sub.2O, and brine, dried over Na.sub.2SO.sub.4,
concentrated, and flash chromatographed on silica gel eluting with
MeOH/DCM (0.3, 0.6, and 1%) to give the title compound (11 mg, 49%
yield) as a white solid. .sup.1H-NMR (400 MHz, CD.sub.3OD) .delta.
8.33 (d, J=8.4 Hz, 1H), 8.17-8.14 (m, 1H), 7.72-7.61 (m, 4H), 7.55
(d, J=8.5 Hz, 1H), 7.19 (dd, J=10.0, 8.3 Hz, 1H), 6.81 (dd, J=8.8,
1.7 Hz, 1H), 6.47 (t, J=9.1 Hz, 1H), 4.32 (s, 2H), 4.12-4.03 (m,
2H), 3.67 (d, J=2.0 Hz, 2H), 2.40 (s, 3H), 1.50 (s, 9H). Mass
spectrum (LCMS, ESI) calc'd for
C.sub.32H.sub.32ClF.sub.4N.sub.4O.su- b.3 (M+H): 631.2. Found:
631.1.
2.
N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-(3-{[2,2-difluoro-2-(4-fluoro-
naphthyl)ethyl]amino}-6-chloro-2-fluorophenyl)acetamide
Hydrochloride Salt
[0260] 57
[0261] A solution of
2-(3-{[2,2-difluoro-2-(4-fluoronaphthyl)ethyl]amino}--
6-chloro-2-fluorophenyl)-N-({6-[(tert-butoxy)carbonylamino]-2-methyl(3-pyr-
idyl)}methyl)acetamide (10 mg, 16 .mu.mol), as prepared in the
preceding step, in HCl (0.5 mL, 4.0 M in 1,4-dioxane) was stirred
for 2 hours at ambient temperature. A solution of MeOH/DCM (25%,
0.4 mL) was added, and the mixture continued to stir overnight. The
solvents were evaporated, and the resulting brown residue was flash
chromatographed on silica gel eluting with MeOH/DCM (2.5, 5, and
10%) to give a solid product. It was treated with HCl solution
(0.01 mL, 4.0 M in 1,4-dioxane, 40 .mu.mol) in DCM (0.5 mL),
stirred for 5 minutes, and the solvents evaporated to give the
title compound (6.2 mg, 69% yield) as a white solid. .sup.1H-NMR
(400 MHz, CD.sub.3OD) .delta. 8.26 (d, J=8.4 Hz, 1H), 8.17-8.14 (m,
1H), 7.80 (d, J=9.1 Hz, 1H), 7.73-7.64 (m, 4H), 7.20 (dd, J=10.0,
8.3 Hz, 1H), 6.82-6.79 (m, 1H), 6.49 (t, J=9.1 Hz, 1H), 4.24 (s,
2H), 4.10-4.02 (m, 2H), 3.66 (d, J=2.3 Hz, 2H), 2.49 (s, 3H). Mass
spectrum (LCMS, ESI) calc'd for C.sub.27H.sub.24ClF.sub.4N.sub.4O
(M+H): 531.1. Found: 531.6.
Example 6
N-[2-(Guanidinooxy)ethyl]-2-(3-{[benzylsulfonyl]amino}phenyl)acetamide
Trifluoroacetate Salt
[0262] 58
1. N-(2-Hydroxyethyl)-2-(3-nitrophenyl)acetamide
[0263] 59
[0264] To a solution of 3-nitrophenylacetic acid (3.21 g, 17.7
mmol), ethanolamine (2.8 g, 46 mmol), and triethylamine (3.0 mL, 22
mmol) in anhydrous DMF (110 mL) was added a solution of
benzotriazole-1-yl-oxy-tri- s-pyrrolidino-phosphonium
hexafluorophosphate (PyBOP, 9.37 g, 18.0 mmol) in anhydrous DMF (80
mL). After stirring 16 hours at ambient temperature (under
nitrogen), the reaction mixture was concentrated in vacuo,
dissolved in DCM and filtered. The filtrate was washed with 10%
aqueous citric acid, saturated aqueous NaHCO.sub.3, pH 7 buffer,
and brine, dried over Na.sub.2SO.sub.4 and filtered. The evaporated
filtrate was then purified by flash chromatography (10% methanol in
DCM) giving the title compound (1.02 g, 26%) as a light yellow
solid. .sup.1H NMR (300 MHz, CDCl.sub.3/CD.sub.3OD) .delta. 8.18
(m, 1H), 8.11 (ddd, 1H, J=8.1 Hz, 2.4 Hz, 1.1 Hz), 7.72 (m, 1H),
7.60 (t, 1H, J=7.8 Hz), 3.62 (s, 2H), 3.44 (t, 2H, J=5.9 Hz), 3.16
(t, 2H, J=5.9 Hz).
2.
N-[2-(N'-Phthalimidyl)hydroxyethyl]-2-(3-nitrophenyl)acetamide
[0265] 60
[0266] To a solution of the product of the preceding step (1.02 g,
4.55 mmol), N-hydroxyphthalimide (0.76 g, 4.64 mmol), and
triphenylphosphine (1.22 g, 4.65 mmol) in anhydrous THF (100 mL)
was added diethylazadicarboxylate (0.75 mL, 4.77 mmol) via syringe.
After stirring overnight at ambient temperature (under nitrogen),
the reaction was concentrated in vacuo and purified by flash
chromatography (40% ethyl acetate in DCM) giving an impure product
that was dissolved in DCM, cooled, and filtered. The evaporated
filtrate was then purified by flash chromatography (66%-100% ethyl
acetate in hexane) giving the title compound (0.86 g, 51%) as a
white solid. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.26 (t, 1H,
J=1.7 Hz), 8.15 (ddd, 1H, J=8.3 Hz, 2.3 Hz, 1.0 Hz), 7.82 (m, 4H),
7.74 (m, 1H), 7.53 (t, 1H, J=7.9 Hz), 7.03 (br s, 1H), 4.26 (m,
2H), 3.76 (s, 2H), 3.57 (dd, 2H, J=9.8 Hz, 5.7 Hz).
3.
N-[2-(N'-Phthalimidyl)hydroxyethyl]-2-(3-aminophenyl)acetamide
[0267] 61
[0268] A solution of the product of the preceding step (0.66 g,
1.80 mmol) and 10% palladium on carbon (15 mg) in degassed 1:1
ethanol:THF (40 mL) was stirred under hydrogen at ambient
temperature. After 6 hours the reaction was filtered over Celite
and the filtrate evaporated and purified by flash chromatography
(5% methanol in DCM) giving the title compound (0.20 g, 33%) as a
yellow solid. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.81 (m,
4H), 7.14 (t, 1H, J=8.0 Hz), 6.72 (m, 2H), 6.61 (ddd, 1H, J=8.0 Hz,
2.2 Hz, 1.0 Hz), 4.23 (m, 2H), 3.55 (m, 4H). Mass spectrum
(MALDI-TOF, .alpha.-cyano-4-hydroxycinnamic acid matrix) calc'd.
for C.sub.18H.sub.17N.sub.3O.sub.4: 362.1 (M+Na), 340.1 (M+H).
Found: 362.2, 340.3.
4.
N-[2-(N'-Phthalimidyl)hydroxyethyl]-2-(3-{[benzylsulfonyl]-amino}phenyl-
)acetamide
[0269] 62
[0270] To an ice-cold solution of the product of the preceding step
(0.20 g, 0.58 mmol) in anhydrous DCM (50 mL) was added a solution
of .alpha.-toluenesulfonyl chloride (0.11 g, 0.58 mmol) in
anhydrous DCM (20 mL) followed by N-methylmorpholine (0.10 mL, 0.91
mmol). After stirring 16 hours at ambient temperature, more
.alpha.-toluenesulfonyl chloride (0.07 g, 0.36 mmol) and
N-methylmorpholine (0.10 mL, 0.91 mmol) were added and the reaction
stirred an additional 4 hours and evaporated in vacuo. The residue
was dissolved in DCM, washed with 10% aqueous citric acid, pH 7
buffer and brine, dried over Na.sub.2SO.sub.4, filtered, and the
filtrate evaporated giving the title compound (0.20 g, 69%) as a
light yellow solid. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.77
(m, 4H), 7.35 (m, 1H), 7.22 (s, 5H), 7.17 (m, 1H), 7.11 (m, 1H),
7.05 (s, 1H), 6.71 (br m, 1H), 4.37 (s, 2H), 4.25 (m, 2H), 3.64 (s,
2H), 3.64 (dd, 2H, J=10 Hz, 5.5 Hz).
5.
N-[2-(Aminooxy)ethyl]-2-(3-{[benzylsulfonyl]amino}phenyl)acetamide
[0271] 63
[0272] The product of the preceding step (0.19 g, 0.39 mmol) was
dissolved in 1:1 ethanol:THF (20 mL) and reacted with 40% aqueous
methylamine (10 mL) for 1 hour at ambient temperature. The reaction
was evaporated in vacuo and purified on a Waters Sep-Pak (5 g
silica, 1:1 DCM:ethyl acetate) giving an impure yellow solid. This
was then purified by preparative thin layer chromatography (10%
methanol in DCM) giving the title compound (0.10 g, 72%) as a white
solid. .sup.1H NMR (300 MHz, CDCl.sub.3/CD.sub.3OD) .delta. 7.34
(m, 3H), 7.28 (m, 3H), 7.10 (m, 1H), 7.05 (m, 2H), 4.35 (s, 2H),
3.69 (t, 2H, J=5 Hz), 3.51 (s, 2H), 3.43 (t, 2H, J=5 Hz). Mass
spectrum (MALDI-TOF, .alpha.-cyano-4-hydroxycinnamic acid matrix)
calc'd. for C.sub.17H.sub.21N.sub.3O.sub.4S: 386.1 (M+Na). Found:
386.6.
6. N-[2-({N,N'-Di[tert-butoxycarbonyl]}guanidinooxy)ethyl]-2-
(3-{[benzylsulfonyl]amino}phenyl)acetamide
[0273] 64
[0274] A solution of the product of the preceding step (89 mg, 0.24
mmol) and [N,N'-di(tert-butoxycarbonyl)]amidinopyrazole (86 mg,
0.28 mmol) in DMF (5 mL) was stirred for 4 days at ambient
temperature. The reaction was evaporated in vacuo and the crude
product purified by flash chromatography (5% methanol in DCM)
giving an impure yellow oil. This was then purified by preparative
thin layer chromatography (5% methanol in DCM) giving the title
compound (72 mg, 49%) as a colorless solid. .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 9.19 (s, 1H), 8.22 (br t, 1H, J=5.0 Hz), 7.62
(s, 1H), 7.23 (m, 10H (Ar.sup.+ NH)), 4.27 (s, 2H), 4.08 (m, 2H),
3.57 (m, 4H), 1.51 (s, 9H), 1.49 (s, 9H).
7.
N-[2-(Guanidinooxy)ethyl]-2-(3-{[benzylsulfonyl]amino}phenyl)acetamide
Trifluoroacetate Salt
[0275] 65
[0276] The product of the preceding step (72 mg, 0.12 mmol) was
dissolved in DCM (5 mL) and reacted with trifluoroacetic acid (2
mL) for 4 hours at ambient temperature. The reaction was
concentrated in vacuo and the crude product purified by preparative
thin layer chromatography (20% methanol in DCM) giving the title
compound (44 mg, 71%) as a pale yellow wax. .sup.1H NMR (300 MHz,
CDCl.sub.3/CD.sub.3OD) .delta. 7.34 (m, 3H), 7.28 (m, 3H), 7.11 (m,
1H), 7.05 (m, 2H), 4.35 (s, 2H), 3.90 (t, 2H, J=4.9 Hz), 3.52 (s,
2H), 3.47 (t, 2H, J=4.8 Hz). Mass spectrum (MALDI-TOF,
.alpha.-cyano-4-hydroxycinnamic acid matrix) calc'd. for
C.sub.18H.sub.23N.sub.5O.sub.4S: 428.1 (M+Na), 406.2 (M+H). Found:
428.4, 406.4.
Example 7
N-[2-(Guanidinooxy)ethyl]-2-(2-chloro-5-{[benzylsulfonyl]amino}phenyl)acet-
amide Trifluoroacetate Salt
[0277] 66
1. 2-Chloro-5-nitrophenylacetic Acid Monohydrate
[0278] 67
[0279] A solution of 2-chlorophenylacetic acid (10.0 g, 58.6 mmol)
in concentrated sulfuric acid (40 mL) was cooled to -10.degree. C.
and slowly reacted with a solution of fuming nitric acid (2.80 mL,
66.7 mmol) in concentrated sulfuric acid (7.2 mL). After 2.5 hours
the reaction was slowly poured over ice water (400 mL), filtered
over a coarse filter frit, washed once with cold water, and dried
on the frit overnight giving the title compound (13.5 g, 98%) as a
white solid. Integration of the proton NMR spectrum showed the
product contained about 0.2 equivalents of 2-chlorophenylacetic
acid, but thin-layer chromatography showed this to be inseparable
from the product. .sup.1H NMR (300 MHz, CDCl.sub.3/CD.sub.3OD)
.delta. 8.25 (d, 1H, J=2.7 Hz), 8.13 (dd, 1H, J=8.7 Hz, 2.7 Hz),
7.61 (d, 1H, J=8.8 Hz), 3.89 (s, 2H).
2. Ethyl 2-(3-amino-6-chlorophenyl)acetate
[0280] 68
[0281] The product of the preceding step (4.14 g, 17.7 mmol) was
suspended in DCM (70 mL) and reacted with oxalyl chloride (4.0 mL,
46 mmol) and a few drops of DMF. After stirring 1 hour at ambient
temperature the reaction became homogeneous, reagent grade ethanol
(30 mL) was added and the reaction stirred another 30 minutes. The
crude product was evaporated in vacuo and purified by flash
chromatography (10% to 15% ethyl acetate in hexane) giving the
title compound (4.6 g) as a pale yellow oil. Proton NMR showed the
product contained about 0.8 equivalents of diethyloxalate that
could not be located by thin-layer chromatography. .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 8.21 (d, 1H, J=2.7 Hz), 8.11 (dd, 1H,
J=8.8 Hz, 2.7 Hz), 7.57 (d, 1H, J=8.8 Hz), 4.21 (q, 2H, J=7.2 Hz),
3.87 (s, 2H), 1.28 (t, 3H, J=7.2 Hz).
3. Ethyl 2-(3-amino-6-chlorophenyl)acetate
[0282] 69
[0283] A solution of the product of the preceding step (2.00 g,
8.21 mmol) in reagent grade ethanol (50 mL) was reacted with
tin(II)chloride dihydrate (9.40 g, 41.7 mmol) at ambient
temperature. After 16 hours the reaction was concentrated in vacuo,
dissolved in DCM and filtered. The filtrate was washed with water
and brine, dried over Na.sub.2SO.sub.4, and filtered. The
evaporated filtrate was then purified by flash chromatography (40%
ethyl acetate in hexane) giving the title compound (0.53 g, 30%) as
a pale yellow oil. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.12
(d, 1H, J=8.5 Hz), 6.60 (d, 1H, J=2.8 Hz), 6.53 (dd, 1H, 8.5 Hz,
2.9 Hz), 4.17 (q, 2H, J=7.1 Hz), 3.65 (s, 2H), 1.26 (t, 3H, J=7.1
Hz).
4. Ethyl 2-(2-chloro-5-{[benzylsulfonyl]amino}phenyl)acetate
[0284] 70
[0285] A solution of the product of the preceding step (0.50 g,
2.32 mmol) and .alpha.-toluenesulfonyl chloride (0.74 g, 3.88 mmol)
in DCM (40 mL) and N-methylmorpholine (0.80 mL, 7.3 mmol) was
stirred at ambient temperature for 3 hours, washed with dilute
aqueous HCl, water, and brine, dried over Na.sub.2SO.sub.4, and
filtered. The evaporated filtrate was purified by flash
chromatography (5% ethyl acetate in DCM) giving the title compound
(0.693 g, 81%) as a pale yellow solid. .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 7.36 (m, 4H), 7.26 (m, 2H), 7.04 (d, 1H, J=2.7
Hz), 6.99 (dd, 1H, J=8.5 Hz, 2.8 Hz), 6.47 (br s, 1H), 4.33 (s,
2H), 4.20 (q, 2H, J=7.1 Hz), 3.73 (s, 2H), 1.29 (t, 3H, J=7.1 Hz).
Mass spectrum (MALDI-TOF, .alpha.-cyano-4-hydroxycinnamic acid
matrix) calc'd. for C.sub.17H.sub.18NO.sub.4S Cl: 390.1 (M+Na).
Found: 390.7.
5. 2-(2-Chloro-5-{[benzylsulfonyl]amino}phenyl)acetic Acid
[0286] 71
[0287] A solution of the product of the preceding step (0.69 g,
1.87 mmol) in 1:1 water/THF (20 mL) was reacted with potassium
hydroxide (0.52 g, 9.32 mmol) at ambient temperature for 20 hours.
After evaporating the THF in vacuo, the remaining aqueous layer was
acidified to pH 3 with 1N HCl and extracted with DCM and ether. The
combined organic layers were washed with brine, dried over
Na.sub.2SO.sub.4, and filtered. The filtrate was then evaporated in
vacuo to give the title compound (0.586 g, 92%) as a white solid.
.sup.1H NMR (300 MHz, CDCl.sub.3/CD.sub.3OD) .delta. 7.34 (m, 4H),
7.28 (m, 2H), 7.07 (m, 2H), 4.34 (s, 2H), 3.73 (s, 2H). Mass
spectrum (MALDI-TOF, .alpha.-cyano-4-hydroxycinnamic acid matrix)
calc'd. for C.sub.15H.sub.14NO.sub.4S Cl: 378.0 (M+K), 362.0
(M+Na). Found: 378.8,362.9.
6.
N-[2-{(N,N'-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]-2-(2-chloro-5--
{[benzylsulfonyl]amino}phenyl)acetamide
[0288] 72
[0289] A solution of the product of the preceding step (0.21 g,
0.60 mmol) and
[N,N'-di(tert-butoxycarbonyl)]-2-aminoethoxyguanidine (Tianbao Lu,
et al., WO 99/26926 (1999)) (0.19 g, 0.60 mmol), in anhydrous THF
(50 mL) was reacted with BOP (0.33 g, 0.75 mmol) and triethylamine
(0.25 mL, 1.8 mmol) at ambient temperature for 16 hours. The
reaction was evaporated in vacuo, dissolved in DCM, washed with pH
7 buffer and brine, dried over Na.sub.2SO.sub.4, and filtered. The
evaporated filtrate was purified by flash chromatography (5%
methanol in DCM) giving the title compound (0.380 g, 98%) as an
orange solid. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 9.14 (s,
1H), 8.22 (br t, 1H, J=5.0 Hz), 7.60 (s, 1H), 7.34 (m, 3H), 7.28
(m, 3H), 7.10 (d, 1H, J=2.6 Hz), 7.03 (dd, 1H, J=8.6 Hz, 2.7 Hz),
6.84 (br s, 1H), 4.29 (s, 2H), 4.13 (m, 2H), 3.75 (s, 2H), 3.62
(dd, 2H, J=8.8 Hz, 5.1 Hz), 1.51 (s, 9H), 1.46 (s, 9H). Mass
spectrum (MALDI-TOF, gentisic acid matrix) calc'd. for
C.sub.28H.sub.38N.sub.5O.su- b.8S Cl: 662.2 (M+Na), 440.1 (M-2
Boc+H). Found: 661.7, 439.9.
7.
N-[2-(Guanidinooxy)ethyl]-2-(2-chloro-5-{[benzylsulfonyl]-amino}phenyl)-
acetamide Trifluoroacetate Salt
[0290] 73
[0291] A solution of the product of the preceding step (0.375 g,
0.586 mmol) in DCM (10 mL) was reacted with trifluoroacetic acid (5
mL) at ambient temperature for 16 hours. The reaction was
evaporated in vacuo, and the crude product purified by flash
chromatography (20% methanol in DCM) giving the title compound
(0.326 g, 100%) as a pale yellow solid. .sup.1H NMR (300 MHz,
CDCl.sub.3/CD.sub.3OD) .delta. 7.34 (m, 3H), 7.29 (m, 3H), 7.06 (m,
2H), 4.35 (s, 2H), 3.94 (br t, 2H, J=5 Hz), 3.65 (s, 2H), 3.49 (br
t, 2H, J=5 Hz). Mass spectrum (MALDI-TOF,
.alpha.-cyano-4-hydroxycinnamic acid matrix) calc'd. for
C.sub.18H.sub.22N.sub.5O.sub.4S Cl: 462.1 (M+Na), 440.1 (M+H).
Found: 461.9,439.9.
Example 8
N-[2-(Guanidinooxy)ethyl]-2-(2-methyl-5-{[benzylsulfonyl]amino}phenyl)acet-
amide Trifluoroacetate Salt
[0292] 74
1. (2-Methyl-5-nitrophenyl)methanol
[0293] 75
[0294] 2-Methyl-5-nitrobenzoic acid (2.00 g, 11.0 mmol) was warmed
under nitrogen, dissolved in anhydrous THF (25 mL), and treated
with a 1N solution of borane in THF (16.5 mL). After stirring 18
hours at ambient temperature the reaction was quenched with a
solution of potassium carbonate (1.8 g, 13 mmol) in water (50 mL),
and the THF removed in vacuo. The remaining aqueous solution was
extracted with DCM, and the organic layer washed with pH 7 buffer
and brine, dried over sodium sulfate, and filtered. The evaporated
filtrate gave the title compound as a pale yellow solid (1.76 g,
95%). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.29 (d, 1H, J=2.5
Hz), 8.04 (dd, 1H, J=8.3 Hz, 2.5 Hz), 7.31 (d, 1H, J=8.31 Hz), 4.77
(d, 2H, J=5.5 Hz), 2.41 (s, 3H), 2.09 (t, 1H, J=5.6 Hz).
2. (2-Methyl-5-nitrophenyl)methyl Methylsulfonate
[0295] 76
[0296] A solution of the product of the preceding step (1.74 g,
10.4 mmol) in DCM (50 mL) was cooled to 0.degree. C. and treated
with methanesulfonyl chloride (0.90 mL, 11.6 mmol) and
triethylamine (1.75 mL, 12.6 mmol). After stirring 30 minutes the
reaction was warmed to ambient temperature, stirred another 30
minutes, and poured onto pH 7 buffer solution. The phases were
separated and the organic layer washed with brine, dried over
sodium sulfate, and filtered. The evaporated filtrate gave the
title compound as a pale yellow oil (2.53 g, 99%). .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 8.26 (d, 1H, J=2.5 Hz), 8.16 (dd, 1H,
J=8.4 Hz, 2.4 Hz), 7.42 (d, 1H, J=8.4 Hz), 5.31 (s, 2H), 3.08 (s,
3H), 2.51 (s, 3H).
3. 2-(2-Methyl-5-nitrophenyl)ethanenitrile
[0297] 77
[0298] The product of the preceding step (2.48 g, 10.1 mmol) and
potassium cyanide (2.00 g, 30.7 mmol) were refluxed in acetonitrile
(100 mL) for 8 hours, then cooled to ambient temperature and
stirred overnight. The reaction was evaporated in vacuo, dissolved
in DCM, and filtered. The filtrate was washed with pH 7 buffer and
brine, evaporated, and purified by flash column chromatography (1:1
hexane:ethyl acetate eluant) giving the title compound (1.27 g,
71%) as a yellow solid. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
8.26 (d, 1H, J=2.4 Hz), 8.13 (dd, 1H, J=8.3 Hz, 2.4 Hz), 7.42 (d,
1H, J=8.3 Hz), 3.78 (s, 2H), 2.48 (s, 3H).
4. 2-(2-Methyl-5-nitrophenyl)acetic Acid
[0299] 78
[0300] To a solution of the product of the preceding step (1.27 g,
7.21 mmol) in methanol (30 mL) was added a solution of potassium
hydroxide (4.06 g, 72.4 mmol) in water (30 mL). The reaction was
heated at reflux overnight and the methanol removed in vacuo. The
remaining aqueous layer was acidified with 3N HCl and filtered, the
solid washed with diethyl ether, and the filtrate separated. The
aqueous layer was washed with DCM and diethyl ether, and the
combined organic layers washed with brine, dried over sodium
sulfate, and filtered. The evaporated filtrate gave the title
compound (0.84 g, 60%) as an orange solid. .sup.1H NMR (300 MHz,
acetone-d.sub.6) .delta. 8.17 (d, 1H, J=2.3 Hz), 8.06 (dd, 1H,
J=8.4 Hz, 2.5 Hz), 7.29 (d, 1H, J=8.4 Hz), 3.88 (s, 2H), 2.45 (s,
3H).
5.
N-[2-({N,N'-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]-2-(2-methyl-5--
nitrophenyl)acetamide
[0301] 79
[0302] A solution of the product of the preceding step (0.27 g,
1.40 mmol), BOP (0.70 g, 1.58 mmol), triethylamine (0.50 mL, 3.60
mmol), and [N,N'-di(tert-butoxycarbonyl)] 2-aminoethoxyguanidine
(Tianbao Lu, et al., WO 99/26926 (1999)) (0.44 g, 1.38 mmol), in
anhydrous DMF were stirred at ambient temperature overnight. The
reaction was concentrated in vacuo and the crude product purified
by flash column chromatography (5% methanol in DCM eluant) giving
the title compound as a pale orange solid (0.63 g, 92%). .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 9.21 (s, 1H), 8.42 (m, 1H), 8.17
(d, 1H, J=2.4 Hz), 7.99 (dd, 1H, J=8.4 Hz, 2.5 Hz), 7.59 (s, 1H),
7.29(d, 1H, J=8.4 Hz), 4.12(m, 2H), 3.74 (s, 2H), 3.63 (m, 2H),
2.48 (s, 3H), 1.52 (s, 9H), 1.47 (s, 9H).
6. N-[2-
({N,N'-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]-2-(3-amino-6--
methylphenyl)acetamide
[0303] 80
[0304] The product of the preceding step (0.29 g, 0.58 mmol) and
10% palladium (0) on carbon (0.06 g) were dissolved in reagent
ethanol (50 mL), degassed with nitrogen and vacuum, and stirred
under a hydrogen balloon at ambient temperature. After 4 hrs the
reaction was filtered over Celite, the frit washed with methanol,
and the filtrate evaporated in vacuo giving the title compound
(0.27 g, 100%). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 9.10 (s,
1H), 7.61 (s, 1H), 7.30 (m, 1H), 6.94 (d, 1H, J=8.0 Hz), 6.63 (d,
1H, J=2.4 Hz), 6.51 (dd, 1H, J=8.0 Hz, 2.5 Hz), 4.09 (m, 2H), 3.58
(m, 2H), 3.53 (s, 2H), 2.21 (s, 3H), 1.52 (s, 9H), 1.47 (s,
9H).
7. N-[2-({N,N'-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]-2-
(2-methyl-5-([benzylsulfonyl]amino)phenyl)acetamide
[0305] 81
[0306] The product of the preceding step (0.27 g, 0.58 mmol),
.alpha.-toluenesulfonyl chloride (0.18 g, 0.96 mmol), and
N-methylmorpholine (0.20 mL, 1.82 mmol) were stirred at ambient
temperature in DCM (20 mL). After 2 hours the reaction was diluted
with additional DCM and washed with dilute aqueous HCl, saturated
aqueous sodium bicarbonate, pH 7 buffer, and brine. The organic
layer was dried over sodium sulfate, filtered, and the filtrate
concentrated in vacuo giving the title compound as a pale yellow
solid (0.35 g, 97%). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 9.15
(s, 1H), 8.04 (br t, 1H, J=5 Hz), 7.60 (s, 1H), 7.34 (m, 5H), 7.12
(d, 1H, J=8.0 Hz), 7.03 (dd, 1H, J=11 Hz, 2.3 Hz), 6.26 (s, 1H),
4.31 (s, 2H), 4.12 (m, 2H), 3.63 (m, 4H), 2.33 (s, 3H), 1.51 (s,
9H), 1.45 (s, 9H).
8.
N-[2-(Guanidinooxy)ethyl]-2-(2-methyl-5-{[benzylsulfonyl]-amino}phenyl)-
acetamide Trifluoroacetate Salt
[0307] 82
[0308] The product of the preceding step (0.35 g, 0.56 mmol) was
dissolved in DCM (10 mL) and treated with trifluoroacetic acid (3
mL) at ambient temperature. After 16 hours the reaction was
concentrated in vacuo and the crude product purified on a 10 g
Waters silica Sep-Pak.RTM. (5 to 20% methanol in DCM gradient
elution) giving the title compound as a pale yellow solid (0.27 g,
89%). .sup.1H NMR (300 MHz, CDCl.sub.3/CD.sub.3OD) .delta. 7.34 (m,
3H), 7.29 (m, 2H), 7.14 (d, 1H, J=9.0 Hz), 7.02 (dd, 1H, J=6.5 Hz,
2.3 Hz), 7.00 (s, 1H), 4.33 (s, 2H), 3.92 (br t, 2H, J=5 Hz), 3.55
(s, 2H), 3.48 (br t, 2H, J=5 Hz), 2.28 (s, 3H). Mass spectrum
(MALDI-TOF, gentisic acid matrix) calc'd. for
C.sub.19H.sub.25N.sub.5O.su- b.4S: 442.2 (M+Na), 420.2 (M+H).
Found: 442.5, 420.6.
Example 9
N-[2-(Guanidinooxy)ethyl]-2-(2-hydroxy-6-methyl-3-{[(3-methylphenyl)sulfon-
yl]amino}phenyl)acetamide Hydrochloride Salt
[0309] 83
1. 4-Methyl-1-nitro-2-prop-2-enyloxybenzene
[0310] 84
[0311] A solution of 5-methyl-2-nitrophenol (2.00 g, 13.1 mmol),
allyl bromide (1.30 mL, 15.0 mmol), and cesium carbonate (5.5 g, 17
mmol) in DMF (100 mL) was stirred at ambient temperature. After 20
hrs the reaction was filtered, frit washed with methanol, and the
filtrate evaporated in vacuo at 50.degree. C. The residue was
purified by flash column chromatography (4:1 then 2:1 hexane:ethyl
acetate eluant) giving the title compound as a yellow oil (2.39 g,
95%) that crystallized after sitting 3 days at ambient temperature.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.79 (d, 1H, J=8.2 Hz),
6.86 (s, 1H), 6.82 (m, 1H), 6.05 (ddt, 1H, J=17.3 Hz, 10.6 Hz, 5.0
Hz), 5.50 (ddd, 1H, J=17.3 Hz, 3.3 Hz, 1.7 Hz), 5.33 (ddd, 1H,
J=10.6 Hz, 2.9 Hz, 1.5 Hz), 4.67 (dt, 2H, J=4.9 Hz, 1.6 Hz), 2.40
(s, 3H).
2. 3-Methyl-6-nitro-2-prop-2-enylphenol
[0312] 85
[0313] 4-Methyl-1-nitro-2-prop-2-enyloxybenzene (7.11 g, 36.8
mmol), prepared as in the preceding step, was heated neat at
200.degree. C. under nitrogen for 3 hours, cooled to ambient
temperature, and purified by flash column chromatography (1:1
hexane:DCM eluant) giving the title compound as an orange oil (5.04
g, 71%). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 11.07 (s, 1H),
7.90 (d, 1H, J=8.7 Hz), 6.80 (d, 1H, J=8.7 Hz), 5.93 (m, 1H), 5.03
(m, 1H), 4.96 (m, 1H), 3.50 (dt, 2H, J=5.9 Hz, 1.7 Hz), 2.36 (s,
3H).
3. 1-Methyl-4-nitro-3-(phenylmethoxy)-2-prop-2-enylbenzene
[0314] 86
[0315] A solution of the product of the preceding step (5.02 g,
26.0 mmol), benzyl bromide (3.40 mL, 28.6 mmol), and cesium
carbonate (17.2 g, 52.8 mmol) in DMF (100 mL) was stirred at
ambient temperature for 20 hours. The solution was filtered, the
filtrate concentrated in vacuo, and the crude product adsorbed onto
silica. This was poured onto a short bed of silica and eluted with
DCM, and the eluate evaporated to give the title compound (7.20 g,
98%) as a yellow oil. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
7.72 (d, 1H, J=8.4 Hz), 7.46 (m, 2H), 7.37 (m, 3H), 7.06 (d, 1H,
J=8.5 Hz), 5.93 (m, 1H), 5.08 (m, 1H), 4.98 (s, 2H), 4.88 (m, 1H),
3.51 (dt, 2H, J=5.4 Hz, 1.9 Hz), 2.36 (s, 3H).
4. 4-Methyl-2-(phenylmethoxy)-3-prop-2-enylphenylamine
[0316] 87
[0317] The product of the preceding step (0.55 g, 1.94 mmol) and
tin(II) chloride dihydrate (2.89 g, 12.8 mmol) were stirred in
reagent grade ethanol (40 mL) at ambient temperature. After 20
hours the reaction was concentrated in vacuo and the residue
partitioned between saturated sodium bicarbonate and DCM. The
resulting emulsion was filtered, the solids and aqueous layer
washed with additional DCM, and the combined organic layers washed
with brine, dried over sodium sulfate, and filtered. The filtrate
was then evaporated giving the title compound as an orange oil
(0.51 g, 90%). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.47 (m,
2H), 7.38 (m, 3H), 6.78 (d, 1H, J=8.1 Hz), 6.60 (d, 1H, J=8.0 Hz),
5.97 (m, 1H), 5.02 (m, 1H), 4.95 (m, 1H), 4.85 (s, 2H), 3.64 (br s,
2H), 3.47 (dt, 2H, J=5.7 Hz, 1.8 Hz), 2.20 (s, 3H).
5.
[4-Methyl-2-(phenylmethoxy)-3-prop-2-enylphenyl][(3-methylphenyl)
sulfonyl]amine
[0318] 88
[0319] The product of the preceding step (0.49 g, 1.92 mmol) was
dissolved in DCM (10 mL) and treated with m-toluenesulfonyl
chloride (0.37 g, 1.96 mmol) and N-methylmorpholine (0.25 mL, 2.27
mmol) at ambient temperature. After 18 hours the reaction was
concentrated in vacuo and the residue purified by flash column
chromatography (DCM eluant) giving the title compound as a pale
yellow oil (0.72 g, 92%). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
7.54 (m, 2H), 7.35 (m, 8H), 6.89 (d, 1H, J=8.3 Hz), 6.80 (s, 1H),
5.87 (m, 1H), 4.99 (m, 1H), 4.79 (m, 1H), 4.43 (s, 2H), 3.36 (dt,
2H, J=5.3 Hz, 1.9 Hz), 2.31 (s, 3H), 2.19 (s, 3H).
6. 2-(6-Methyl-3-{[(3-methylphenyl)sulfonyl]amino}-2-
(phenylmethoxy)phenyl)ethanol
[0320] 89
[0321] The product of the preceding step (0.71 g, 1.74 mmol) was
dissolved in 1,4-dioxane (25 mL) and treated with a solution of
sodium periodate (1.50 g, 7.01 mmol) in water (12 mL) and a 2.5 wt
% solution of osmium tetraoxide (0.25 mL, 0.02 mmol) in
2-methyl-2-propanol. After stirring 4 hours at ambient temperature
the reaction was diluted with DCM, washed with 5% aqueous sodium
bisulfite, water, and brine, dried over sodium sulfate, and
filtered. The filtrate was concentrated in vacuo giving a pale
yellow oil that was used without further purification.
7.
2-(6-Methyl-3-{[(3-methylphenyl)sulfonyl]amino}-2-(phenylmethoxy)phenyl-
)acetic Acid
[0322] 90
[0323] A solution of sodium dichromate (0.79 g, 2.64 mmol) and
concentrated sulfuric acid (1.5 mL, 28 mmol) in water (25 mL) was
added to a solution of the product of the preceding step in acetone
(25 mL), and the reaction stirred at ambient temperature for 3
days. After adding methanol (3 mL) and stirring an additional 15
minutes, the organic solvents were removed in vacuo and the
remaining aqueous layer extracted with DCM. The DCM layer was
washed with brine, dried over sodium sulfate, filtered, and the
filtrate concentrated and purified by flash column chromatography
(10% methanol in DCM) giving the title compound as a pale yellow
solid (0.51 g, 69% from Step 5). .sup.1H NM (300 MHz, CDCl.sub.3)
.delta. 7.56 (m, 2H), 7.39 (m, 4H), 7.31 (m, 4H), 6.93 (d, 1H,
J=8.4 Hz), 6.78 (s, 1H), 4.52 (s, 2H), 3.67 (s, 2H), 2.33 (s, 3H),
2.21 (s, 3H). Mass spectrum (LCMS, ESI pos.) calc'd. for
C.sub.23H.sub.23NO.sub.5S: 448.1 (M+Na), 425.1 (M+H). Found: 448.1,
425.9.
8.
N-[2-({N,N'-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]-2-(6-methyl-3--
{[(3-methylphenyl)sulfonyl]amino}-2-(phenylmethoxy)phenyl)acetamide
[0324] 91
[0325] A solution of the product of the preceding step (0.32 g,
0.75 mmol), BOP (0.34 g, 0.77 mmol), triethylamine (0.25 mL, 1.80
mmol), and [N,N'-di(tert-butoxycarbonyl)]-2-aminoethoxyguanidine
(Tianbao Lu, et al. WO 99/26926 (1999)) (0.27 g, 0.76 mmol), in DMF
(15 mL) was stirred at ambient temperature overnight. The reaction
was concentrated in vacuo, the residue dissolved in DCM, washed
with saturated sodium bicarbonate, water, and brine, dried over
sodium sulfate, and filtered. The filtrate was concentrated in
vacuo and the crude product purified by flash column chromatography
(7% methanol in DCM eluant) giving the title compound as a pale
yellow solid (0.41 g, 76%). .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 9.11 (s, 1H), 7.93 (m, 1H), 7.55 (m, 3H), 7.36 (m, 8H),
6.90 (d, 1H, J=8.3 Hz), 6.84 (s, 1H), 4.60 (s, 2H), 4.06 (m, 2H),
3.71 (s, 2H), 3.56 (dd, 2H, J=8.8 Hz, 5.2 Hz), 2.34 (s, 3H), 2.23
(s, 3H), 1.51 (s, 9H), 1.43 (s, 9H).
9.
N-[2-({N,N'-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]-2-(2-hydroxy-6-
-methyl-3-{[(3-methylphenyl)sulfonyl]amino}phenyl)acetamide
[0326] 92
[0327] The product of the preceding step (0.41 g, 0.57 mmol) and
10% palladium (0) on carbon (60 mg) were dissolved in reagent grade
ethanol (20 mL), degassed with nitrogen and vacuum, and stirred
under a hydrogen balloon at ambient temperature for 4 hours. The
reaction was filtered over Celite, the frit washed with methanol,
the filtrate evaporated, and the residue purified by preparative
thin-layer chromatography (5% methanol in DCM eluant) giving the
title compound as pale yellow solid (13.0 mg, 4%). .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 9.25 (s, 1H), 8.62 (m, 1H), 7.61 (m, 3H),
7.29 (m, 3H), 6.61 (d, 1H, J=8.5 Hz), 4.06 (m, 2H), 3.64 (s, 2H),
3.54 (dd, 2H, J=8.7 Hz, 5.0 Hz), 2.34 (s, 3H), 2.30 (s, 3H), 1.51
(s, 9H), 1.49 (s, 9H).
10.
N-[2-(Guanidinooxy)ethyl]-2-(2-hydroxy-6-methyl-3-{[(3-methylphenyl)su-
lfonyl]amino}phenyl)acetamide Hydrochloride Salt
[0328] 93
[0329] The product of the preceding step (13.0 mg, 0.02 mmol) was
dissolved in DCM (5 mL) and treated with trifluoroacetic acid (1
mL) at ambient temperature. After 16 hours the reaction was
concentrated in vacuo and the crude product purified by preparative
thin-layer chromatography (12% methanol in DCM eluant, saturated
with ammonia). The resulting product was treated with 4N HCl in
ethanol, filtered, the filtrate evaporated, and the solid washed
with diethyl ether and vacuum-dried giving the title compound (5.0
mg, 52%) as a tan solid. .sup.1H NMR (300 MHz, CD.sub.3OD) .delta.
7.51 (m, 2H), 7.38 (m, 2H), 6.56 (m, 2H), 3.91 (t, 2H, J=5.3 Hz),
3.61 (s, 2H), 3.47 (t, 2H, J=5.3 Hz), 2.36 (s, 3H), 2.27 (s, 3H).
Mass spectrum (LCMS, ESI pos.) calc'd. for
C.sub.19H.sub.25N.sub.5O.sub.5S: 436.1 (M+H). Found: 436.2.
Example 10
N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-(2-hydroxy-6-methyl-3-{[(3-methy-
lphenyl)sulfonyl]amino}phenyl)acetamide Hydrochloride Salt
[0330] 94
1N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-(6-ethyl-3-{[(3
methylphenyl)sulfonyl]amino}-2-(phenylmethoxy)phenyl)acetamide
[0331] 95
[0332] The product of Example 9, step 7 (0.18 g, 0.42 mmol), BOP
(0.21 g, 0.47 mmol), triethylamine (0.25 mL, 1.80 mmol), and
2-amino-5-aminomethyl-6-methylpyridine dihydrochloride (Sanderson,
P. E., et al. WO 97/01338 (1997)) (0.10 g, 0.48 mmol), were
dissolved in DMF (10 mL) and stirred at ambient temperature for 2
hours. The reaction was concentrated in vacuo and the crude product
purified by flash column chromatography (gradient elution: 10% to
15% methanol in DCM) giving an impure product that was dissolved in
DCM, washed with saturated sodium bicarbonate, water, and brine,
dried over sodium sulfate, and filtered. The evaporated filtrate
then gave the title compound as a pale yellow solid (0.23 g, 99%).
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.64 (m, 1H), 7.56 (m,
1H), 7.35 (m, 10H), 7.11 (d, 1H, J=8.3 Hz), 6.94 (d, 1H, J=8.5 Hz),
6.22 (d, 1H, J=8.1 Hz), 5.60 (br t, 1H, J=5.3 Hz), 4.49 (s, 2H),
4.37 (br s, 2H), 4.19 (d, 2H, J=5.5 Hz), 3.56 (s, 2H), 2.34 (s,
3H), 2.23 (s, 3H), 2.22 (s, 3H). Mass spectrum (LCMS, ESI pos.)
calc'd. for C.sub.30H.sub.32N.sub.4O.sub.4S: 545.2 (M+H). Found:
545.2.
2.
N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-(2-hydroxy-6-methyl-3-{[(3-me-
thylphenyl)sulfonyl]amino}phenyl)acetamide Hydrochloride Salt
[0333] 96
[0334] The product of the preceding step (0.22 g, 0.41 mmol) and
10% palladium (0) on carbon (0.03 g) were dissolved in 2:1
ethanol:THF (30 mL), degassed with nitrogen and vacuum, and stirred
under a hydrogen balloon at ambient temperature. After 7 hours the
reaction was filtered over Celite, the frit washed with methanol,
and the filtrate concentrated in vacuo. The residue was treated
with 4N HCl in ethanol (ca. 3 mL), evaporated under high vacuum,
dissolved in DCM, filtered, and the filtrate evaporated under high
vacuum again giving the title compound (0.14 g, 71%) as a pale
beige solid. .sup.1H NMR (300 MHz, CDCl.sub.3/CD.sub.3OD) .delta.
7.60 (m, 3H), 7.33 (m, 2H), 6.82 (d, 1H, J=8.2 Hz), 6.67 (d, 1H,
J=9.0 Hz), 6.58 (d, 1H, J=8.3 Hz), 4.18 (s, 2H), 3.58 (s, 2H), 2.45
(s, 3H), 2.37 (s, 3H), 2.25 (s, 3H). Mass spectrum (LCMS, ESI pos.)
calc'd. for C.sub.23H.sub.26N.sub.4O.sub.4S: 455.2 (M+H). Found:
455.2.
Example 11
3-({N-[2-(Guanidinooxy)ethyl]carbamoyl}methyl)-2-hydroxy-4-methylphenyl
3-methylbenzenesulfonate Hydrochloride Salt
[0335] 97
1. 2-Methoxy-4-methylphenyl 3-methylbenzenesulfonate
[0336] 98
[0337] A solution of m-toluenesulfonyl chloride (0.53 g, 2.78 mmol)
and 2-methoxy-4-methylphenol (0.38 g, 2.75 mmol) in DCM (10 mL) was
treated with triethylamine (0.5 mL, 3.6 mmol) and stirred at
ambient temperature. After 18 hours the reaction was concentrated
in vacuo, the residue dissolved in 1:1 hexane:DCM, filtered, and
the filtrate evaporated under high vacuum giving the title compound
as a white solid (0.79 g, 99%). .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.72 (br s, 1H), 7.66 (br d, 1H, J=7.2 Hz), 7.45 (br d, 1H,
J=7.2 Hz), 7.38 (t, 1H, J=7.6 Hz), 7.00 (d, 1H, J=8.1 Hz), 6.67 (m,
1H), 3.54 (s, 3H), 2.42 (s, 3H), 2.31 (s, 3H).
2. 2-Hydroxy-4-methylphenyl 3-methylbenzenesulfonate
[0338] 99
[0339] The product of the preceding step (0.79 g, 2.72 mmol) was
dissolved in DCM (10 mL), cooled to -78.degree. C., and treated
with 1N boron tribromide in DCM (3.0 mL) under nitrogen. After 10
min the dry ice bath was removed, and the reaction stirred another
hour while warming to ambient temperature. After slowly quenching
with water, the reaction was diluted with additional DCM, washed
with brine, dried over sodium sulfate, and filtered. The filtrate
was concentrated in vacuo and the residue purified by flash column
chromatography (gradient elution: 50% to 33% to 0% hexane in DCM)
giving the title compound as a white crystalline solid (0.51 g,
68%). .sup.1H NMR (300 MWz, CDCl.sub.3) .delta. 7.71 (br s, 1H),
7.67 (br d, 1H, J=7.7 Hz), 7.51 (brd, 1H, J=8.1 Hz), 7.43 (t, 1H,
J=7.7 Hz), 6.81 (d, 1H J=1.7 Hz), 6.63(d, 1H, J=8.3 Hz), 6.55 (m,
1H), 5.87(s, 1H), 2.43 (s, 3H), 2.26 (s, 3H).
3. 4-Methyl-2-prop-2-enyloxyphenyl 3-methylbenzenesulfonate
[0340] 100
[0341] A solution of the product of the preceding step (0.51 g,
1.83 mmol), allyl bromide (0.20 mL, 2.30 mmol), and cesium
carbonate (0.77 g, 2.40 mmol) in DMF (25 mL) was stirred for 16
hours at ambient temperature. The reaction was concentrated in
vacuo, the residue dissolved in DCM, filtered, and the filtrate
washed with 1N aqueous KOH, water, and brine, dried over sodium
sulfate, and filtered. The evaporated filtrate then gave the title
compound as a pale yellow oil (0.54 g, 93%). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.72 (s, 1H), 7.64 (d, 1H, J=7.7 Hz), 7.42 (d,
1H, J=7.7 Hz), 7.35 (t, 1H, J=7.7 Hz), 7.04 (d, 1H, J=8.2 Hz), 6.68
(m, 2H), 5.80 (ddt, 1H, J=17.3 Hz, 10.6 Hz, 5.1 Hz), 5.28 (ddd, 1H,
J=17.3 Hz, 3.1 Hz, 1.6 Hz), 5.20 (ddd, 1H, J=10.6 Hz, 2.8 Hz, 1.3
Hz), 4.29 (dt, 2H, J=5.1 Hz, 1.5 Hz), 2.40 (s, 3H), 2.30 (s,
3H).
4. 2-Hydroxy-4-methyl-3-prop-2-enylphenyl
3-methylbenzenesulfonate
[0342] 101
[0343] The product of the preceding step (0.54 g, 1.70 mmol) was
heated neat at 200.degree. C. for 6 hrs, cooled to ambient
temperature, and purified twice by flash column chromatography
(first with 2:1 DCM:hexane, then with 4:1 hexane:ethyl acetate
eluant) giving the title compound as a colorless oil (84 mg, 16%).
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.74 (s, 1H), 7.67 (d,
1H, J=7.8 Hz), 7.44 (d, 1H, J=7.6 Hz), 7.36 (t, 1H, J=7.7 Hz), 6.86
(d, 1H, J=8.3 Hz), 6.55 (d, 1H, J=8.3 Hz), 4.74 (m, 1H), 3.15 (dd,
1H, J=15.5 Hz, 8.9 Hz), 2.61 (dd, 1H, J=15.5 Hz, 7.6 Hz), 2.41 (s,
3H), 2.16 (s, 3H), 1.24 (d, 2H, J=6.3 Hz).
5.
4-Methyl-2-(phenylmethoxy)-3-prop-2-enylphenyl3-methylbenzenesulfonate
[0344] 102
[0345] The product of the preceding step (68 mg, 0.21 mmol) and
cesium carbonate (0.19 g, 0.58 mmol) were dissolved in DMF (5 mL)
and treated with benzyl bromide (0.05 mL, 0.42 mmol) at ambient
temperature. After 3 days the reaction was concentrated in vacuo
and the residue purified by flash column chromatography (DCM
eluant) giving the title compound as a pale yellow oil (50 mg,
58%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.60 (br s, 1H),
7.54 (br d, 1H, J=7.8 Hz), 7.34 (m, 4H), 7.27 (m, 3H), 7.10 (d, 1H,
J=8.4 Hz), 6.91 (d, 1H, J3=8.4 Hz), 5.74 (ddt, 1H, J=17.1 Hz, 10.2
Hz, 5.7 Hz), 4.93 (dq, 1H, J=10.2 Hz, 1.7 Hz), 4.81 (s, 2H), 4.72
(dq, 1H, J=17.1 Hz, 1.8 Hz), 3.30 (dt, 2H, J=5.7 Hz, 1.7 Hz), 2.24
(s, 3H), 2.23 (s, 3H).
6.
4-Methyl-3-(2-oxoethyl)-2-(phenylmethoxy)phenyl-3-methylbenzenesulfonat-
e
[0346] 103
[0347] To a solution of the product of the preceding step (50 mg,
0.12 mmol) and sodium periodate (0.12 g, 0.56 mmol) in 5:1
acetonitrile:water (12 mL) was added ruthenium(III) chloride
hydrate (8 mg, 0.04 mmol). The reaction was stirred 6 hours at
ambient temperature, diluted with DCM, and washed with 5% aqueous
sodium bisulfite, water and brine. The organic solution was dried
over sodium sulfate, filtered, and the filtrate evaporated giving
the title compound as a crude oil that was used without further
purification.
7.
2-{6-Methyl-3-[(3-methylphenyl)sulfonyloxy]-2-(phenylmethoxy)phenyl}ace-
tic Acid
[0348] 104
[0349] The product of the preceding step was dissolved in acetone
(5 mL) and treated with a solution of sodium dichromate (65 mg,
0.22 mmol) and concentrated sulfuric acid (1 mL) in water (4 mL) at
ambient temperature. After stirring 3 days the acetone was removed
in vacuo, and the remaining aqueous layer extracted with DCM. The
organic phase was then washed with brine, dried over sodium
sulfate, filtered, and the evaporated filtrate purified by flash
column chromatography (8% methanol in DCM eluant) giving the title
compound (45 mg, 88% from step 5) as a white solid. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 7.61 (br s, 1H), 7.53 (brd, 1H, J=7.8
Hz), 7.33 (brd, 1H, J=7.6 Hz), 7.27 (m, 6H), 7.12 (d, 1H, J=8.4
Hz), 6.92 (d, 1H, J=8.4 Hz), 4.87 (s, 2H), 3.60 (s, 2H), 2.24 (s,
3H), 2.23 (s, 3H).
8.
3-({N-[2-({N,N'-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]carbamoyl}m-
ethyl)-4-methyl-2-(phenylmethoxy)phenyl
3-methylbenzenesulfonate
[0350] 105
[0351] To a solution of the product of the preceding step (45 mg,
0.11 mmol), BOP (48 mg, 0.11 mmol), and
[N,N'-di(tert-butoxycarbonyl)]-2-amino- ethoxyguanidine (Tianbao
Lu, et al., WO 99/26926 (1999)) (39 mg, 0.11 mmol), in DMF (5 mL)
was added triethylamine (0.2 mL, 1.4 mmol). After stirring 18 hours
at ambient temperature, the reaction was concentrated in vacuo and
the residue purified by flash column chromatography (3:1 DCM:ethyl
acetate eluant) giving the title compound as a colorless oil (61
mg, 79%). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 9.11 (s, 1H),
7.65 (br s, 1H), 7.58 (br d, 1H, J=8.5 Hz), 7.48 (m, 1H), 7.36 (br
d, 1H, J=7.6 Hz), 7.29 (m, 7H), 7.03 (d, 1H, J=8.4 Hz), 6.90 (d,
1H, J=8.5 Hz), 4.92 (s, 2H), 4.02 (m, 2H), 3.66 (s, 2H), 3.50 (dd,
2H, J=9.2 Hz, 5.2 Hz), 2.28 (s, 3H), 2.26 (s, 3H), 1.51 (s, 9H),
1.45 (s, 9H).
9.
3-({N-[2-({N,N'-Di-[tert-butoxycarbonyl]}guanidinooxy)ethyl]carbamoyl}m-
ethyl)-2-hydroxy-4-methylphenyl3-methylbenzenesulfonate
[0352] 106
[0353] The product of the preceding step (61 mg, 0.08 mmol) and 10%
palladium (0) on carbon (20 mg) were dissolved in a 1:1:1 mixture
of THF, methanol, and water (50 mL), degassed with nitrogen and
vacuum, and stirred vigorously under a hydrogen balloon at ambient
temperature. After 18 hours the reaction was filtered over Celite,
the frit washed with methanol, and the evaporated filtrate purified
by flash column chromatography (10% ethyl acetate in DCM eluant)
giving the title compound as a colorless solid (40 mg, 74%).
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 9.25 (s, 1H), 8.50 (br t,
1H, J=4.9 Hz), 7.79 (s, 1H), 7.57 (d, 1H, J=7.7 Hz), 7.59 (br s,
1H), 7.43 (d, 1H, J=7.7 Hz), 7.38 (t, 1H, J=7.6 Hz), 6.90 (d, 1H,
J=8.3 Hz), 6.62 (d, 1H, J=8.4 Hz), 4.08 (m, 2H), 3.69 (s, 2H), 3.56
(dd, 2H, J=8.7 Hz, 5.0 Hz), 2.41 (s, 3H), 2.35 (s, 3H), 1.51 (s,
9H), 1.50 (s, 9H).
10.
3-({N-[2-(Guanidinooxy)ethyl]carbamoyl}methyl)-2-hydroxy-4-methylpheny-
l 3-methylbenzenesulfonate Hydrochloride Salt
[0354] 107
[0355] The product of the preceding step (40 mg, 0.06 mmol) was
dissolved in DCM (4 mL) and treated with neat trifluoroacetic acid
(1.5 mL) at ambient temperature. After 3 hours the reaction was
concentrated in vacuo and the residue purified by preparative
thin-layer chromatography (20% methanol in DCM saturated with
ammonia gas as eluant), treated with 4N HCl in ethanol, and
filtered. The evaporated filtrate was washed with diethyl ether and
dried under high vacuum giving the title compound as a pale yellow
solid (17 mg, 57%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
10.87 (s, 1H), 9.76 (s, 1H), 7.67 (m, 5H), 7.60 (d, 1H, J=7.6 Hz),
7.51 (t, 1H, J=7.7 Hz), 6.70 (d, 1H, J=8.3 Hz), 6.60 (d, 1H, J=8.5
Hz), 3.79 (t, 2H, J=5.5 Hz), 3.45 (m, 4H), 2.39 (s, 3H), 2.17 (s,
3H). Mass spectrum (LCMS, ESI pos.) calcd. for
C.sub.19H.sub.24N.sub.4O.sub.6S- : 437.1 (M+H). Found: 437.3.
Example 12
Tablet Preparation
[0356] Tablets containing 25.0, 50.0, and 100.0 mg, respectively,
of the following active compounds are prepared as illustrated
below:
[0357] a.
N-[2-(Amidinoaminooxy)ethyl-2-{3-[(2,2-difluoro-2-phenylethyl)am-
ino]-6-chloro-2-fluorophenyl}acetamide trifluoroacetate salt;
and
[0358] b.
N-[(6-Amino-2-methyl(3-pyridyl))methyl]-2-{3-[(2,2-difluoro-2-ph-
enylethyl)amino]-6-chloro-2-fluorophenyl}acetamide hydrochloride
salt.
1 TABLET FOR DOSES CONTAINING FROM 25-100 MG OF THE ACTIVE COMPOUND
Amount-mg Active Compound 25.0 50.0 100.00 Microcrystalline
cellulose 37.25 100.0 200.0 Modified food corn starch 37.25 4.25
8.5 Magnesium stearate 0.50 0.75 1.5
[0359] All of the active compound, cellulose, and a portion of the
corn starch are mixed and granulated to 10% corn starch paste. The
resulting granulation is sieved, dried and blended with the
remainder of the corn starch and the magnesium stearate. The
resulting granulation is then compressed into tablets containing
25.0, 50.0, and 100.0 mg, respectively, of active ingredient per
tablet.
Example 13
Intravenous Solution Preparation
[0360] An intravenous dosage form of the above-indicated active
compounds of Examples 1 and 2 is prepared as follows:
2 Active Compound 0.5-10.0 mg Sodium Citrate 5-50 mg Citric Acid
1-15 mg Sodium Chloride 1-8 mg Water for Injection (USP) q.s. to 1
ml
[0361] Utilizing the above quantities, the active compound is
dissolved at room temperature in a previously prepared solution of
sodium chloride, citric acid, and sodium citrate in Water for
Injection (USP, see page 1636 of United States
Pharmacopeia/National Formulary for 1995, published by United
States Pharmacopeial Convention, Inc., Rockville, Md. (1994).
Example 14
In Vitro Inhibition of Purified Enzymes
[0362] Reagents: All buffer salts were obtained from Sigma Chemical
Company (St. Louis, Mo.), and were of the highest purity available.
The enzyme substrates, N-benzoyl-Phe-Val-Arg-p-nitroanilide (Sigma
B7632), N-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide hydrochloride
(Sigma B2291), N-p-Tosyl-Gly-Pro-Lys-p-nitroanilide (Sigma T6140),
N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma S7388) and
N-CBZ-Val-Gly-Arg-p-nitroanilide (Sigma C7271) were obtained from
sigma. N-succinyl-Ala-Ala-Pro-Arg-p-nitroanilide (BACHEM L-1720)
and N-succinyl-Ala-Ala-Pro-Val-p-nitroanilide (BACHEM L-1770) were
obtained from BACHEM (King of Prussia, Pa.).
[0363] Human .alpha.-thrombin, human factor Xa and human plasmin
were obtained from Enzyme Research Laboratories (South Bend, Ind.).
Bovine .alpha.-chymotrypsin (Sigma C4129), bovine trypsin (Sigma
T8642) and human kidney cell urokinase (Sigma U5004) were obtained
from Sigma. Human leukocyte elastase was obtained from Elastin
Products (Pacific, Mo.).
[0364] K.sub.i Determinations: All assays are based on the ability
of the test compound to inhibit the enzyme catalyzed hydrolysis of
a peptide p-nitroanilide substrate. In a typical K.sub.i
determination, substrate is prepared in DMSO, and diluted into an
assay buffer consisting of 50 mM HEPES, 200 mM NaCl, pH 7.5. The
final concentrations for each of the substrates is listed below. In
general, substrate concentrations are lower than the experimentally
determined value for K.sub.m. Test compounds are prepared as a 1.0
mg/ml solution in DMSO. Dilutions are prepared in DMSO yielding 8
final concentrations encompassing a 200 fold concentration range.
Enzyme solutions are prepared at the concentrations listed below in
assay buffer.
[0365] In a typical K.sub.i determination, into each well of a 96
well plate is pipetted 280 mL of substrate solution, 10 mL of test
compound solution, and the plate allowed to thermally equilibrate
at 37.degree. C. in a Molecular Devices plate reader for >15
minutes. Reactions were initiated by the addition of a 10 mL
aliquot of enzyme and the absorbance increase at 405 nm is recorded
for 15 minutes. Data corresponding to less than 10% of the total
substrate hydrolysis were used in the calculations. The ratio of
the velocity (rate of change in absorbance as a function of time)
for a sample containing no test compound is divided by the velocity
of a sample containing test compound, and is plotted as a function
of test compound concentration. The data are fit to a linear
regression, and the value of the slope of the line calculated. The
inverse of the slope is the experimentally determined K.sub.i
value.
[0366] Thrombin: Thrombin activity was assessed as the ability to
hydrolyze the substrate N-succinyl-Ala-Ala-Pro-Arg-p-nitroanilide.
Substrate solutions were prepared at a concentration of 32 mM (32
mM<<Km=180 mM) in assay buffer. Final DMSO concentration was
4.3%. Purified human .alpha.-thrombin was diluted into assay buffer
to a concentration of 15 nM. Final reagent concentrations were:
[thrombin]=0.5 nM, [substrate
N-succinyl-Ala-Ala-Pro-Arg-p-nitroanilide]=32 mM.
[0367] Factor X [FXa]: FXa activity was assessed as the ability to
hydrolyze the substrate N-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide
hydrochloride. Substrate solutions were prepared at a concentration
of 51 mM (51<<K.sub.m=1.3 mM) in assay buffer. Final DMSO
concentration was 4.3%. Purified activated human Factor X was
diluted into assay buffer to a concentration of 300 nM. Final
reagent concentration were: [FXa]=10 nM,
[N-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide hydrochloride]=51 mM.
[0368] Plasmin: Plasmin activity was assessed as the ability to
hydrolyze the N-p-Tosyl-Gly-Pro-Lys-p-nitroanilide. Substrate
solutions were prepared at a concentration of 37 mM (37
mM<<K.sub.m=243 mM) in assay buffer. Final DMSO concentration
was 4.3%. Purified human plasmin was diluted into assay buffer to a
concentration of 240 nM. Final reagent concentrations were:
[Plasmin]=8 nM, [N-p-Tosyl-Gly-Pro-Lys-p-nitroanilid- e]=37 mM.
[0369] Chymotrypsin: Chymotrypsin activity was assessed as the
ability to hydrolyze N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide.
Substrate solutions were prepared at a concentration of 14 mM (14
mM<<K.sub.m=62 mM) in assay buffer. Final DMSO concentration
was 4.3%. Purified bovine chymotrypsin was diluted into assay
buffer to a concentration of 81 nM. Final reagent concentrations
were: [Chymotrypsin]=2.7 nM,
[N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide]=14 mM.
[0370] Trypsin: Trypsin activity was assessed as the ability to
hydrolyze N-benzoyl-Phe-Val-Arg-p-nitroanilide. Substrate solutions
were prepared at a concentration of 13 mM (13 mM<<K.sub.m=291
mM) in assay buffer. Final DMSO concentration was 4.3%. Purified
bovine trypsin was diluted into assay buffer to a concentration of
120 nM. Final reagent concentrations were: [Trypsin]=4 nM,
[N-benzoyl-Phe-Val-Arg-p-nitroanilid- e]=13 mM.
[0371] Elastase: Elastase activity was assessed as the ability to
hydrolyze N-succinyl-Ala-Ala-Pro-Val-p-nitroanilide. Substrate
solutions were prepared at a concentration of 19 mM (19
mM<<K.sub.m=89 mM) in assay buffer. Final DMSO concentration
was 4.3%. Purified human leukocyte elastase was diluted into assay
buffer to a concentration of 750 nM. Final reagent concentrations
were: [Elastase]=25 nM,
[N-succinyl-Ala-Ala-Pro-Val-p-nitroanilide]=19 mM.
[0372] Urokinase: Urokinase activity was assessed as the ability to
hydrolyze N-CBZ-Val-Gly-Arg-p-nitroanilide. Substrate solutions
were prepared at a concentration of 100 mM (100 mM<K.sub.m=1.2
mM) in assay buffer. Final DMSO concentration was 4.3%. Purified
human kidney urokinase was diluted into assay buffer to a
concentration of 1.2 mM. Final reagent concentrations were:
[Urokinase]=40 nM, and [N-CBZ-Val-Gly-Arg-p-nitroanilide]=100
mM.
[0373] The results indicate that the compounds of Examples 1
through 11 have Ki values for human thrombin of between 0.0028 and
20 .mu.M. The compound of Example 5 has a Ki of 0.0028 .mu.M.
[0374] Having now fully described this invention, it will be
understood to those of ordinary skill in the art that the same can
be performed within a wide and equivalent range of conditions,
formulations, and other parameters without affecting the scope of
the invention or any embodiment thereof. All patents and
publications cited herein are fully incorporated by reference
herein in their entirety.
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