AU698911B2 - Thrombin inhibitors - Google Patents

Thrombin inhibitors

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AU698911B2
AU698911B2 AU54385/96A AU5438596A AU698911B2 AU 698911 B2 AU698911 B2 AU 698911B2 AU 54385/96 A AU54385/96 A AU 54385/96A AU 5438596 A AU5438596 A AU 5438596A AU 698911 B2 AU698911 B2 AU 698911B2
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alkyl
ring
hydrogen
heterocyclic ring
bicyclic heterocyclic
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AU5438596A (en
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Mark G. Bock
Bruce D Dorsey
Dong-Mei Feng
Roger M. Freidinger
Joseph P. Vacca
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Merck and Co Inc
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Merck and Co Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/022—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -X-C(=O)-(C)n-N-C-C(=O)-Y-; X and Y being heteroatoms; n being 1 or 2
    • C07K5/0222—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -X-C(=O)-(C)n-N-C-C(=O)-Y-; X and Y being heteroatoms; n being 1 or 2 with the first amino acid being heterocyclic, e.g. Pro, Trp
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • General Health & Medical Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Description

TITLE OF THE INVENTION
THROMBIN INHIBITORS
BACKGROUND OF THE INVENTION
Thrombin is a serine protease present in blood plasma in the form of a precursor, prothrombin. Thrombin plays a central role in the mechanism of blood coagulation by converting the solution plasma protein, fibrinogen, into insoluble fibrin.
Edwards et al., J. Amer. Chem. Soc., ( 1992) vol. 114, pp. 1854-63, describes peptidyl a-ketobenzoxazoles which are reversible inhibitors of the serine proteases human leukocyte elastase and porcine pancreatic elastase.
European Publication 363 284 describes analogs of peptidase substrates in which the nitrogen atom of the scissile amide group of the substrate peptide has been replaced by hydrogen or a substituted carbonyl moiety.
Australian Publication 86245677 also describes peptidase inhibitors having an activated electrophilic ketone moiety such as fluoromethylene ketone or α-keto carboxyl derivatives.
Thrombin inhibitors described in prior publications contain sidechains of arginine and lysine. These structures show low selectivity for thrombin over other trypsin-like enzymes. Some of them show toxicity of hypotension and liver toxicity.
European Publication 601 459 describes sulfonamido heterocyclic thrombin inhibitors, such as N-[4-f(aminoimino- methyl)amino]butyl]- 1 -[N-(2-naphthalenyIsulfonyl)-L-phenylalanyl]-L- prolinamide.
WO 94/29336 describes compounds which are useful as thrombin inhibitors. SUMMARY OF THE INVENTION
Compounds of the invention have the following structure:
wherein
A is C or N:
X 1 , X2 and X3, each independently attached to a ring carbon atom, are independently selected from the group consisting of
hydrogen. C 1 -4 alkyl, and C1 -4 alkoxy;
Y, attached to a ring carbon atom, is H, NH2 or OH:
Z is -(CH2) 1 -3-;
R 1 , R2, and R2' are independently
hydrogen.
phenyl,
mono- or di-halogenated phenyl,
naphthyl.
biphenyl.
a 5- to 10-membered mono- or bicyclic heterocyclic ring or 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,
C1 -4 alkyl,
branched C 1 -4 alkyl,
C3-7 cycloalkyl,
C5- 1 2 bicyclic alkyl,
C11- 1 6 tricyclic alkyl,
R4(CH2)n,
(R4)2(CH), (R4)(OR4)CH,
R4O(CH2)n, or
R 1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N, O and S, where n is 1 , 2, 3 or 4;
R3 is hydrogen,
(R2)2N, wherein R2 is the same or different,
R2'OCONH, provided R2' is not hydrogen.
R2CONH.
HO(CH2)p, where p is 0, 1 , 2, 3 or 4,
R2'SO2NH, provided R2' is not hydrogen, or
(R2)mNCONH, where m is 1 or 2, wherein R2 is the same or different; R4 is independently
phenyl,
mono- or di-halogenated phenyl,
naphthyl,
biphenyl,
a 5- to 10-membered mono- or bicyclic heterocyclic ring or 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,
-COR5,
-OR6,
C1 -4 alkyl,
branched C1 -4 alkyl,
C1 -4 alkoxy, C3-7 cycloalkyl,
C5- 12 bicyclic alkyl, or
C1 1 - 16 tricyclic alkyl; R5 is
-OH,
-OR6,
-N(R7)2, where R7 is same or different, and
where D is -CH2CH2-, -CH2-O-CH2-, or -CH2-NH-CH2-
R6 is C1 -4 alkyl; R7 is hydrogen or C1 -4 alkyl;
G is (CH2)q where q is 1 or 2; or
NR 1CH2; and
Q is SCH2, or
(CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.
These compounds show selectivity for thrombin inhibition over inhibition of trypsin and other trypsin-like enzymes. Trypsin-like enzymes (such as trypsin, thrombin, factor xa, kallikrein, plasmin, urokinase, and plasminogen activator) are serine dependent enzymes that catalyze hydrolysis at arginyl and lysyl peptide bonds.
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.
The invention also includes a composition for preventing or treating unstable angina, refractory angina, myocardial infarction, transient ischemic attacks, atrial fibrillation, thrombotic stroke, embolic stroke, deep vein thrombosis, disseminated intravascular coagulation. ocular build up of fibrin, and reocclusion or restenosis of recanalized vessels, 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 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.
DETAILED DESCRIPTION OF THE INVENTION
Compounds of the invention have the following structure:
wherein
A is C or N;
X 1 , X2 and X3, each independently attached to a ring carbon atom, are independently selected from the group consisting of
hydrogen, C1 -4 alkyl, and C1 -4 alkoxy;
Y, attached to a ring carbon atom, is H, NH2 or OH;
Z is -(CH2)1 -3-;
R 1 , R2, and R2' are independently
hydrogen,
phenyl. mono- or di-halogenated phenyl,
naphthyl,
biphenyl,
a 5- to 10-membered mono- or bicyclic heterocyclic ring or 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,
C 1 -4 alkyl.
branched C 1 -4 alkyl,
C3-7 cycloalkyl,
C5- 1 2 bicyclic alkyl,
C1 1 - 1 6 tricyclic alkyl,
R4(CH2)n,
(R4)2(CH),
(R4)(OR4)CH,
R4O(CH2)n, or
R 1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N, O and S, where n is 1 , 2, 3 or 4;
R3 is hydrogen,
(R2)2N, wherein R2 is the same or different.
R2'OCONH, provided R2' is not hydrogen.
R2CONH,
HO(CH2)p, where p is 0, 1 , 2, 3 or 4,
R2'SO2NH, provided R2' is not hydrogen, or
(R2)mNCONH, where m is 1 or 2, wherein R2 is the same or different;
R4 is independently phenyl,
mono- or di-halogenated phenyl,
naphthyl,
biphenyl,
a 5- to 10-membered mono- or bicyclic heterocyclic ring or 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, OandS,
-COR5,
-OR6,
C1-4 alkyl,
branched C1-4 alkyl,
C1-4 alkoxy.
C3-7 cycloalkyl,
C5-12 bicyclic alkyl, or
C11-16 tricyclic alkyl;
R5 is
-OH,
-OR6,
-N(R7)2, where R7 is same or different, and
where D is -CH2CH2-, -CH2-O-CH2-, or -CH2-NH-CH2-;
R6 is C1-4 alkyl;
R7 is hydrogen or C1-4 alkyl; G is (CH2)q where q is 1 or 2; or
NR1CH2; and
Q is SCH2, or (CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.
In one class, compounds of the invention have the following structure:
wherein
A is C or N;
X 1 , X2 and X3, each independently attached to a ring carbon atom, are independently selected from the group consisting of
hydrogen and C1 -4 alkyl;
Y, attached to a ring carbon atom, is hydrogen, NH2 or OH;
R 1 , R2, and R2' are independently
hydrogen,
phenyl,
mono- or di-halogenated phenyl,
naphthyl,
biphenyl,
a 5- to 7-membered mono- or bicyclic heterocyclic ring or 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,
C1 -4 alkyl,
branched C1 -4 alkyl,
C3-7 cycloalkyl,
C5- 1 2 bicyclic alkyl,
C1 1 - 1 6 tricyclic alkyl,
R4(CH2)n, (R4)2(CH),
(R4)(OR4)CH,
R4O(CH2)n , or
R 1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N, O and S. where n is 1 , 2, 3 or 4; R3 is
hydrogen.
(R2)2N, wherein R2 is the same or different.
R2'OCONH, provided R2' is not hydrogen,
R2CONH,
HO(CH2)p, where p is 0, 1 , 2, 3 or 4,
R2'SO2NH, provided R2' is not hydrogen, or
(R2)rnNCONH, where m is 1 or 2, wherein R2 is the same or different; R4 is independently
phenyl,
mono- or di-halogenated phenyl,
naphthyl,
biphenyl,
a 5- to-7- membered mono- or bicyclic heterocyclic ring or 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,
COOH,
C1 -4 alkyl,
branched C1 -4 alkyl,
C3-7 cycloalkyl,
C5- 1 2 bicyclic alkyl, or C1 1 - 16 tricyclic alkyl;
G is (CH2)q where q is 1 or 2; or
NR 1 CH2; and
Q is SCH2, or
(CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.
In a subclass of this class, the compounds have the following structure:
wherein
A is C or N:
X 1 and X2, each independently attached to a ring carbon atom, are independently selected from the group consisting of
H and C1 -4 alkyl;
Y, attached to a ring carbon atom, is hydrogen, NH2 or OH;
R 1 , R2, and R2' are independently
hydrogen,
phenyl.
mono- or di-halogenated phenyl,
naphthyl,
biphenyl,
a 5- to 7-membered mono- or bicyclic heterocyclic ring or 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,
C1 -4 alkyl,
branched C1 -4 alkyl,
C3-7 cycloalkyl,
C5- 12 bicyclic alkyl,
C1 1 - 1 6 tricyclic alkyl,
R4(CH2)n,
(R4)2(CH),
(R4)(OR4)CH,
R4O(CH2)n, or
R 1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N, O and S, where n is 1 , 2, 3 or 4;
R3 is
hydrogen,
(R2)2N, wherein R2 is the same or different,
R2'OCONH, provided R2' is not hydrogen,
R2CONH,
HO(CH2)p, where p is 0, 1 , 2, 3 or 4,
R2' SO2NH, provided R2' is not hydrogen, or
(R2)mNCONH, where m is 1 or 2, wherein R2 is the same or different;
R4 is independently
phenyl,
mono- or di-halogenated phenyl,
naphthyl,
biphenyl,
a 5- to-7- membered mono- or bicyclic heterocyclic ring or 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,
COOH,
C1-4 alkyl,
branched C1-4 alkyl,
C3-7 cycloalkyl,
C5-12 bicyclic alkyl, or
C11-16 tricyclic alkyl; G is (CH2)q where q is 1 or 2; or
NR1CH2;and
Q is SCH2, or
(CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.
In a group of this subclass, compounds of the invention have the following structure:
wherein
X1 and X2, each independently attached to a ring carbon atom, are
independently selected from the group consisting of
hydrogen and C1-4 alkyl;
Y, attached to a ring carbon atom, is hydrogen or NH2;
R1, R2, and R2' are independently
hydrogen,
phenyl, mono- or di-halogenated phenyl,
naphthyl,
biphenyl.
a 5- to 7-membered mono- or bicyclic heterocyclic ring or 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,
C1 -4 alkyl,
branched C1 -4 alkyl.
C3-7 cycloalkyl,
C5 - 12 bicylic alkyl,
C1 1 - 1 6 tricylic alkyl,
R4(CH2)n,
(R4)2CH, wherein R4 is the same or different,
(R4)(OR4)CH,
R4O(CH2)n, or
R 1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N, O and S, where n is 1 , 2, 3 or 4;
R3 is
hydrogen,
(R2)2N, wherein R2 is the same or different.
R2'OCONH, provided R2' is not hydrogen,
R2CONH,
HO(CH2)p, where p is 0, 1 , 2, 3 or 4,
R2'SO2NH, provided R2' is not hydrogen, or
(R2)mNCONH, where m is 1 or 2, wherein R2 is the same or different;
R4 is independently phenyl,
mono- or di-halogenated phenyl,
naphthyl,
biphenyl,
a 5- to-7- membered mono- or bicyclic heterocyclic ring or 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,
COOH,
C1 -4 alkyl.
branched C 1 -4 alkyl.
C3-7 cycloalkyl,
C5- 1 2 bicyclic alkyl, or
C 1 1 - 16 tricyclic alkyl;
G is (CH2)q where q is 1 or 2, or
NR 1 CH2; and Q is SCH2, or
(CH2)r where r is 1 or 2. and pharmaceutically acceptable salts thereof. In another group of this subclass, compounds of the invention have the following structure:
wherein Y, attached to a ring carbon atom, is H or NH2; R 1 , R2, and R2' are independently
hydrogen,
phenyl,
mono- or di-halogenated phenyl,
naphthyl,
biphenyl,
a 5- to 7-membered mono- or bicyclic heterocyclic ring or 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 croup consisting of N, O and S,
C 1 -4 alkyl.
branched C1 -4 alkyl.
C3-7 cycloalkyl.
C5- 1 2 bicyclic alkyl,
C 1 1 - 1 6 tricyclic alkyl,
R4(CH2)n,
(R4)2CH, wherein R4 is the same or different,
(R4)(OR4)CH,
R4O(CH2)n, or
R 1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N, O and S, where n is 1 , 2, 3 or 4;
R3 is
hydrogen,
(R2)2N, wherein R2 is the same or different,
R2'OCONH, provided R2' is not hydrogen,
R2CONH,
HO(CH2)p, where p is 0, 1 , 2, 3 or 4,
R2'SO2NH, provided R2' is not hydrogen, or
(R2)mNCONH, where m is 1 or 2, wherein R2 is the same or different;
R4 is independently
phenyl,
mono- or di-halogenated phenyl,
naphthyl,
biphenyl,
a 5- to-7- membered mono- or bicyclic heterocyclic ring or 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 co nsisting of N, O and S,
COOH,
C 1 -4 alkyl,
branched C1 -4 alkyl,
C3-7 cycloalkyl,
C5- 12 bicyclic alkyl, or
C 1 1 - 16 tricyclic alkyl;
G is (CH2)q where q is 1 or 2, or
NR 1CH2; and
Q is SCH2, or
(CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.
In another group of this subclass, compounds of the invention have the following structure:
wherein Y, attached to a ring carbon atom, is hydrogen or NH2;
R 1 , R2, and R2' are independently
hydrogen,
phenyl,
mono- or di-halogenated phenyl,
naphthyl,
biphenyl,
a 5- to 7-membered mono- or bicyclic heterocyclic ring or 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,
C 1 -4 alkyl,
branched C 1 -4 alkyl,
C3-7 cycloalkyl,
C5- 1 2 bicyclic alkyl,
C 1 1 - 1 6 tricyclic alkyl,
R4(CH2)n,
(R4)2CH, wherein R4 is the same or different.
(R4)(OR4)CH,
R4O(CH2)n, or
R 1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N, O and S, where n is 1 , 2, 3 or 4;
R3 is
hydrogen,
(R2)2N, wherein R2 is the same or different.
R2'OCONH. provided R2' is not hydrogen,
R2CONH,
HO(CH2)p, where p is 0, 1, 2, 3 or 4, R2'SO2NH, provided R2' is not hydrogen, or
(R2)mNCONH, where m is 1 or 2, wherein R2 is the same or different; R4 is independently
phenyl,
mono- or di-halogenated phenyl,
naphthyl,
biphenyl,
a 5- to-7- membered mono- or bicyclic heterocyclic ring or 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,
COOH,
C 1 -4 alkyl,
branched C 1 -4 alkyl,
C3-7 cycloalkyl.
C5- 1 2 bicyclic alkyl, or
C 1 1 - 1 6 tricyclic alkyl;
G is (CH2)q where q is 1 or 2, or
NR 1 CH2; and Q is SCH2, or
(CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.
Some abbreviations that may appear in this application are as follows.
The compounds of the present invention may have chiral centers and occur as racemates, racemic mixtures and as individual diastereomers, or enantiomers with all isomeric forms being included in the present invention. A racemate or racemic mixture does not imply a 50:50 mixture of stereoisomers.
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.
As used herein except where noted, "alkyl" is intended to include both branched- and straight-chain saturated aliphatic
hydrocarbon groups having the specified number of carbon atoms (Me is methyl, Et is ethyl, Pr is propyl. Bu is butyl); "alkoxy" represents an alkyl group of indicated number of carbon atoms attached through an oxygen bridge; "Halo", as used herein, means fluoro, chloro, bromo and iodo; and "counterion" is used to represent a small, single negatively- charged species, such as chloride, bromide, hydroxide, acetate, trifluroacetate, perchlorate. nitrate, benzoate, maleate, tartrate, hemitartrate, benzene sulfonate, and the like.
Cyclic alkyl, bicyclic alkyl, and tricyclic alkyl refer to saturated ring systems, including spiro systems, fused systems, and bridged systems, unsubstituted or substituted with oxygen to form carbonyl carbon systems, or C1 -2 alkyl.
The term heterocycle or heterocyclic, 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. 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 elements 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.
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, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate. 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. Also, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides. such as methyl, ethyl, propyi, and butyl chloride, 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.
Amide couplings used to form the compounds of this invention are typically performed by the carbodiimide method with reagents such as dicyclohexylcarbodiimide, or 1-ethyl-3-(3-dimethyl- aminopropyl) carbodiimide. Other methods of forming the amide or peptide bond include, but are not limited to the synthetic routes via an acid chloride, azide, mixed anhydride or activated ester, Typically, solution phase amide coupling are performed, but solid-phase synthesis by classical Merrifield techniques may be employed instead. The addition and removal of one or more protecting groups is also typical practice.
The compounds shown in the tables below are exemplary compounds of the present invention, having Ki (uM) for human thrombin IIa in the range between 0.00005 and 100:
Compounds of the invention can be prepared according to the general procedures outlined below:
EXAMPLE 1
Preparation of BOC-D-3,3-Dicyclohexylalanyl-6-(aminopyridin-3- yl)methyl-L-prolineamide
Step A: Preparation of 6-Amino-3-aminomethylpyridine
A 300-ml flask was dried in an oven and cooled down in a dry nitrogen atmosphere. The flask was equipped with a rubber syringe cap and a magnetic stirring bar. The flask was immersed in a ice-water bath , and 21 ml (21 mmole) of 1.0M borane solution in THF was introduced into the reaction flask, followed by 0.3 ml of THF. Then 6- Aminonicotinamide (420 mg, 3.06 mmole) in 10 ml ot THF was introduced. The resulting mixture was stirred tor 10 min, and 5 ml of 6N HCl was added slowly, and then 15.0 ml of H2O and 100 ml of MeOH was introduced, and stirred continually over night, and filtered, evaporated in vac uo to give product as a white solid which was further purified by chromatography using two columns of 40 g silica gel 60 (E. Merck) each and eluting with n-Butanol-HOAC-H2O (4: 1 :2): Fractions containing product were combined to give 285 mg (76% yield) of product.
EI+: 123
TLC: Rf=0.51. silica gel, n-Butanol-HOAC-H2O (4: 1 :2);
Step B: Boc-D-3,3-Dicha-OH (1 -2)
A solution of BOC-D-3,3-Dιphe-OH (2.0 g. 5.8 mmol) in
50 ml acetic acid/10 ml H2O was hydrogenated at 62 psi on a Parr apparatus over 400 mg of Ir black catalyst. After 24 h. a second portion of catalyst was added and the reaction continued for a second 24 h interval. The reaction was filtered through a Celite pad, and the filtrate was added with 150 ml of H2O and filtrated again to give 2.0 g of BOC-D-3,3-Dicha as white solid (97% yield).
FABMS: 354
HPLC: retention time 24.3 min; C1 8, 95%A to 5%A over 30 min,
A=0.1 %TFA-H2O, B=0.1 % TFA-CH3CN Step C: Boc-D-3,3-Dicha-Pro-OMe ( 1 -3)
To a solution of Boc-D-3,3-Dicha-OH (1.77 g, 5.0 mmol) and H-Pro-OMe•HCl (0.91 g, 5.5 mmol) in 12 ml of DMF was added 4.6 g (6.0 mmol) of HOBt•H2O, the pH of the solution was adjusted to 8 (moist narrow pH paper), and EDC (6.47 g, 6.76 mmol) was added with magnetic stirring. After 3.5 hrs the reaction was quenched by the addition of 10 ml of water. After keeping the mixture at room
temperature for 5 hrs, the solvents were evaporated at reduced pressure and the residue was dissolved in EtOAc-H2O. Aqueous KHSO4 was added to this two-phase mixture and the layers were separated. The organic layer was extracted with NaHCO3, saturated NaCl, and dried over MgSO4. The solvent was evaporated to give product as a white solid which was further purified by chromatography using two columns of 600 g silica gel 60 (E. Merck) each and eluting with EtOAc-hexane (2:8). Fractions containing product were combined to give 2.26 g (97% yield) of product.
In a similar manner are prepared the following:
N-(benzylsulfonyl)-D-3,4-Dichloro-Phe-Pro-OMe (2-2), by coupling of N-(benzylsulfonyl)-D-3,4-Dichloro-Phe-OH (2- 1 ) with H- Pro-OMe-HCl.
BOC-D-3, 3-Diphe-Pro-OMe (3- 1 ), by coupling of Boc-D- 3,3-Diphe-OH with H-Pro-OMe-HCl.
Step D: Boc-D-3,3-Dicha-Pro-OH
A sample of Boc-D-3,3-Dicha-Pro-OMe ( 1.76 g, 3.8 mmol ) dissolved in 100 ml of 1 : 1 (v/v) MeOH/H2O was treated with 2.2 N LiOH (2.2 ml) in portions over 1.5 hrs. keeping the pH at 12- 13. After 3.5 hrs, the reaction solution was adjusted to pH 7 with dilute KHSO4 solution, 100 ml of EtOAc and 50 ml of H2O were added, and the aqueous layer was further adjusted to pH 2 with KHSO4 solution. The organic layer was separated and washed twice with 50% saturated NaCl solution, dried over Na2SO4, and evaporated in vacuo to give 1.64 g, (96% yield).
FABMS: 451 HPLC: retention time 26.4 min; C1 8, 95%A to 5%A over 30 min,
A=0.1 %TFA-H2O, B=0.1 %TFA-CH3CN
In a similar manner are prepared the following: N-(benzylsulfonyl)-D-3,4-Dichloro-Phe-Pro-OH, by hydrolysis of N-(benzylsulfonyl)-D-3,4-Dichloro-Phe-OMe with LiOH.
BOC-D-3,3-Diphe-Pro-OH, by hydrolysis of Boc-D-3,3- Diphe-OMe with LiOH Step E: Preparation of BOC-D-3,3-Dicyclohexylalanyl-6-
(aminopyridin-3-yl )methyl-L-prolineamide
A solution of 1 13 mg (0.25 mmol) of Boc-D-3.3-Dicha- Pro-OH, 62 mg (0.50 mmol) of 6-Amino-3-aminomethylpyridine. 43 mg (0.28 mmol) of HOBT, 54 mg (0.28 mmol) of EDC in 1.7 ml anh. NMP was treated with DIEA to PH 8.5, and the resulting solution stirred at room temp, in an N2 atmosphere for 8 h. The reaction was diluted with 3X its volume of water, and the suspension stirred vigorously at room temp, for 15 min. The suspension was filtered, the residue purified by preparative HPLC using a trifluroacetic acid
(0.1 %)-CH3CN gradient. Lyophilization of pure fractions gave 135 mg (97%) of product as a trifluoroacetic acid hydrate salt.
Anal. CHN: C3 1 H49N5O4•1.75 CF3CO2H•0.90 H2O.
FAB MS: M+1 = 556.
HPLC: 99% pure @ 214, retention times=22.7 min, (Vydac C1 8, gradient of 95%A/B to 5%A/B over 30 min, A=0.1 %TFA-H2O, B=0.1 %TFA-CH3CN
EXAMPLE 2 Preparation of D-3,3-Dicyclohexylalanyl-N-(6-aminopyridin-3- yl)methyl-L-prolineamide
A solution of 122 mg (0.22 mmol) BOC-D-3,3- dicyclohexylalanyl-N-(6-aminopyridin-3-yl)methyl-L-prolineamide in 10 ml of 50% TFA/CH2CI2 was stirred for 20 min. and the TFA was removed under reduced pressure and the product purified by
preparative HPLC using a TFA(0.1 %)-CH3CN gradient. Lyophilization of pure fractions gave 96 mg (96%) of the title compound as a
trifluoroacetic acid hydrate salt.
Anal. CHN: C26H41N5O2•2.70 CF3CO2H•0.55 H2O.
FAB MS: M+1 = 456.
HPLC: 99% pure @214. retention times= 16.5 min, (Vydac C1 8, gradient of 95%A/B to 5%A/B over 30 min, A=0.1 %TFA-H2O,
B=0.1 %TFA-CH3CN
EXAMPLE 3
Preparation of N-(benzylsulfonyl)-D-3,4-dichlorophenylalanyl-N-(6- aminopyridin-3-yl)methyl-L-prolineamide
Step A: Preparation of N-(benzylsulfonyl)-D-3,4 -Cl2Phe-OH(2- 1 )
(D)-3,4 -Cl2PheOH (1.4 g, 6.0 mmol) was dissolved in 48 mL dioxane by addition of 6 ml 1 N NaOH. The resulting solution was treated dropwise with phenylmethanesulfonyl chloride with rapid stirring at room temperature. After 2.5 hour, the aqueous layer was further adjusted to pH 2 with KHSO4 solution. 150 ml of EtOAc were added. The organic layer was separated and washed twice with saturated NaCl solution, dried over Na2Sθ4, and evaporated in vacuo to give 2.31 g (80% yield).
FAB MS: M+1 = 485
HPLC: 97% pure @ 214, retention times=20.1 min. (Vydac C 1 8, gradient of 95%A/B to 5%A/B over 30 min, A=0.1 %TFA-H2O,
B=0.1 %TFA-CH3CN Step B: Preparation of N-(benzylsulfonyl)-D-3,4-dichlorophenyl- alanyl-N-(6-aminopyridin-3-yl)methyl-L-prolineamide
A solution of 121 mg (0.25 mmol) of N-(benzylsulfonyl)- D-3,4-Cl2Phe-Pro-OH, 62 mg (0.50 mmol) of 6-Amino-3- aminomethylpyridine, 43 mg (0.28 mmol) of HOBT. 54 mg (0.28 mmol) of EDC in 1.7 ml anh. NMP was treated with DIEA to pH 8.5, and the resulting solution stirred at room temp, in an N2 atmosphere for 8 h. The reaction was diluted with 3X its volume of water, and the suspension stirred vigorously at room temp, for 15 min. The
suspension was filtered, the residue purified by preparative HPLC using a trifluroacetic acid (0.1 %)-CH3CN gradient. Lyophilization of pure fractions gave 140 mg (95%) of product as a trifluoroacetic acid hydrate salt.
Anal. CHN: C27H29N5O4Sl Cl2•2.35 CF3CO2H•0.95 H2O.
FAB MS: M+ 1 = 590.
HPLC: 99% pure @214. retention times= 18.8 mm. ( Vydac C1 8, gradient of 95%A/B to 5%A/B over 30 min. A=0.1 %TFA-H2O,
B=0.1 %TFA-CH3CN EXAMPLE 4
Preparation of N-BOC-D-3,3-diphenylaIanyl-N-(6-aminopyridin-3- yl)methyl-L-prolineamide
1 10 mg. 0.25 mmol of N-BOC-D-3,3-Diphe-Pro-OH and 130 mg, 0.50 mmol of 6-Amino-3-aminomethylpyridine were coupled with hydroxybenztriazole hydrate (43 mg, 0.28 mmol) and EDC-HCl (54 mg. 0.28 mmol) in 1.5 mL DMF at pH 8.5 with DIEA. The mixture was stirred under N2 at room temperature overnight, then diluted with 10 mL of 10% aqueous citric acid and extracted with
CH2CI2. The CH2CI2 extracts were washed with aqueous Na2CO3, dried (Na2SO4), filtered and concentrated in vacuo to give the crude Boc derivative of the title compound, and the product purified by preparative HPLC using a TFA(0.1 %)-CH3CN gradient. Lyophilization of pure fractions gave 122 mg (90%) of the white powder as a
trifluoroacetic acid hydrate salt.
Anal.CHN: C3 1H37N5O4•1.75 CF3CO2H•1.05 H2O.
FAB MS: M+1 = 544. HPLC: 99% pure @214, retention times=18.8 min, (Vydac C1 8, gradient of 95%A/B to 5%A/B over 30 min, A=0.1 %TFA-H2O.
B=0.1 %TFA-CH3CN EXAMPLE 5
Preparation of D-3,3-diphenylaIanyl-N-(6-aminopyridin-3-yl)methyl-
L-prolineamide
A solution of ( 109 mg, 0.20 mmol) N-BOC-D-3.3- diphenylalanyI-N-(6-aminopyridin-3-yl)methyl-L-prolineamide in 10 ml of 50%- TFA/CH2CI2 was stirred for 20 min, the TFA was removed under reduced pressure, and the product purified by preparative HPLC using a TFA(0.1 %)-CH3CN gradient. Lyophilization of pure fractions gave 87 mg (98%) of the title compound as a trifluoroacetic acid hydrate salt.
Anal.CHN: C26H29N5O2•2.80 CF3CO2H•1.35 H2O.
FAB MS: M+1 = 444.
HPLC: 99% pure @214, retention times= 13.9 min, (Vydac C1 8, gradient of 95%A/B to 5%A/B over 30 min. A=0.1 %TFA-H2O,
B=0.J %TFA-CH3CN
EXAMPLE 6
Preparation of BOC-D-3,3-Dicyclohexylalanyl-N-(6-amino-2,4- dimethylpyridin-3-yl)methyl-L-prolineamide
Step A: Preparation of 6-Amino-2,4-dimethyl-3-amino- methylpyridine
A 300-ml flask was dried in an oven and cooled down in a dry nitrogen atmosphere. The flask was equipped with a rubber syringe cap and a magnetic stirring bar. The flask was immersed in a ice-water bath, and 21 ml (21 mmole) of 1.0M borane solution in THF was introduced into the reaction flask, followed by 0.3 ml of THF. Then 6- Amino-2,4-dimethyI-3-pyridinecarbonitrile (442 mg, 3.0 mmole) in 10 ml of THF was introduced. The resulting mixture was stirred for 10 min, and 5 ml of 6N HCl was added slowly, and then 15.0 ml of H2O and 100 ml of MeOH was introduced, and stirred continually over night, and filtered, filtrate was evaporated in vacuo to give product as a white solid which was further purified by chromatography using a columns of 40 g silica gel 60 (E. Merck) and eluting with n-Butanol-HOAC-H2O (4:1 :2). Fractions containing product were combined to give 407 mg (90% yield).
EI+: 51
TLC: Rf=0.73, silica gel, n-Butanol-HOAC-H2O (4: 1 :2)
Step B: Preparation of BOC-D-3,3-Dicyclohexylalanyl-N-(6- amino-2,4-dimethylpyridin-3-yl)methyl-L-prolineamide A solution of 1 13 mg (0.25 mmol) of Boc-D-3,3-Dicha- Pro-OH. 75.5 mg (0.50 mmol) of 6-Amino-2,4-dimethyl-3- aminomethylpyridine ( 1 - 1 ), 43 mg (0.28 mmol) of HOBT. 54 mg (0.28 mmol) of EDC in 1.7 ml anh. NMP was treated with DIEA to PH 8.5. and the resulting solution stirred at room temp, in an N2 atmosphere for 8 h. The reaction was diluted with 3X its volume of water, and the suspension stirred vigorously at room temp, for 15 min. The
suspension was filtered, the residue purified by preparative HPLC using a trifluroacetic acid (0.1 %)-CH3CN gradient. Lyophilization of pure fractions gave 135 mg (93%) of product as a trifluoroacetic acid hydrate salt.
Anal. CHN: C33H53N5O4•1.55 CF3CO2H•0.80 H2O.
FAB MS: M+1 = 584.
HPLC: 99% pure @ 214, retention times=23.2 min, (Vydac C1 8, gradient of 95%A/B to 5%A/B over 30 min, A=0.1 %TFA-H2O,
B=0.1 %TFA-CH3CN EXAMPLE 7
Preparation of D-3,3-DicyclohexyIaIanyl-N-(6-amino-2,4-dimethyl- pyridin-3-yl )methyl-L-prolineamide
A solution of 128 mg (0.22 mmol) N-BOC-D-3,3-
Dicyclohexylalanyl-N-(6-amino-2.4-dimethylpyridin-3-yl)methyl-L- prolineamide in 10 ml of 50% TFA/CH2CI2 was stirred for 20 min, and the TFA was removed under reduced pressure and the product purified by preparative HPLC using a TFA(0.1 %)-CH3CN gradient.
Lyophilization of pure fractions gave 102 mg (96% ) of the title compound as a trifluoroacetic acid hydrate salt.
Anal.CHN: C28H45N5O2•2.50 CF3CO2H• 1.35 H2O.
FAB MS: M+1 = 484.
HPLC: 99% pure @ 214, retention times= 16.7 min, (Vydac C1 8, gradient of 95%A/B to 5%A/B over 30 min. A=0.1 %TFA-H2O,
B=0.1 %TFA-CH3CN
EXAMPLE 8 Preparation of N-(benzylsulfonyl)-D-3,4-dichlorophen ylalanyl-N-(6- amino-2,4-dimethylpyridin-3-yl )methyl-L-proline amide
A solution of 121 mg (0.25 mmol) of N-(benzylsulfonyl)- D-3,4-Cl2Phe-Pro-OH, 75.5 mg (0.50 mmol) of 6-Amino-2,4-dimethyl- 3-aminomethylpyridine, 43 mg (0.28 mmol) of HOBT. 54 mg (0.28 mmol) of EDC in 1.7 ml anh. NMP was treated with DIEA to pH 8.5, and the resulting solution stirred at room temp, in an N2 atmosphere for 8 h. The reaction was diluted with 3X its volume of water, and the suspension stirred vigorously at room temp, for 15 min. The
suspension was filtered, the residue purified by preparative HPLC using a trifluroacetic acid (0.1 %)-CH3CN gradient. Lyophilization of pure fractions gave 140 mg (95%) of white solid as a trifluoroacetic acid hydrate salt.
Anal. CHN: C29H33N5O4S1 CI2•1.65 CF3CO2H•0.60 H2O.
FAB MS: M+1 = 618. HPLC: 99% pure @214, retention times=19.5 min. (Vydac C1 8, gradient of 95%A/B to 5%A/B over 30 min. A=0.1 %TFA-H2O, B=0.1 %TFA-CH3CN EXAMPLE 9
Preparation of N-BOC-D-3,3-diphenylalanyl-N-(6-amino-2,4- dimethylpyridin-3-yl )methyl-L-proline amide
108 mg, 0.25 mmol of N-BOC-D-3,3-Diphe-Pro-OH and 75.5 mg. 0.50 mmol of 6-Amino-2,4-dimethyl-3-aminomethylpyridine were coupled with hydroxybenztriazoie hydrate (43 mg. 0.28 mmol) and EDC-HCl (54 mg, 0.28 mmol) in 1.5 mL DMF at PH 8.5 with DIEA. The mixture was stirred under N2 at room temperature overnight, then diluted with 10 mL of 10% aqueous citric acid and extracted with CH2CI2. The CH2CI2 extracts were washed with aqueous Na2CO3, dried (Na2SO4), filtered and concentrated in vacuo to give the crude Boc derivative of the title compound, and the product purified by preparative HPLC using a TFA(0.1 %)-CH3CN gradient. Lyophilization of pure fractions gave 128 mg (90%) of the white powder as a trifluoroacetic acid hydrate salt.
Anal. CHN: C33H4 1 N5O4•1.35 CF3CO2H•1.55 H2O.
FAB MS: M+1 = 572.
HPLC: 99% pure @ 214, retention times= 19.3min, (Vydac C 1 8, gradient of 95%A/B to 5%A/B over 30 min. A=0.1 %TFA-H2O, B=0.1 %TFA-CH3CN EXAMPLE 10
Preparation of D-3,3-diphenylalanyl-N-(6-amino-2,4-dimethylpyridin- 3-yl)methyl-L-proline amide
A solution of (1 14 mg, 0.20 mmol) N-BOC-D-3,3- diphenylalanyl-N-(6-amino-2,4-dimethylpyridin-3-yl)methyl-L-proIine amide in 10 ml of 50% TFA/CH2CI2 was stirred for 20 min, and the TFA was removed under reduced pressure and the product purified by preparative HPLC using a TFA(0.1 %)-CH3CN gradient. Lyophilization of pure fractions gave 92 mg (98%) of the title compound as a
trifluoroacetic acid hydrate salt.
Anal.CHN: C28H33N5O2•2.75 CF3CO2H•2.90 H2O.
FAB MS: M+1 = 472.
HPLC: 99% pure @214, retention times= 14.2 min, (Vydac C1 8, gradient of 95%A/B to 5%A/B over 30 min. A=0.1 %TFA-H2O.
B=0.1 %TFA-CH3CN EXAMPLE 1 1
Preparation of N-(t-butyloxy-carboxymethyl)-D-4-chlorophenyl- alanyl-N-(6-aminopyridin-3-yl )methyl-L-proline amide Step A: Preparation of 6-amino-3-(aminomethyl)pyridine
To 6-aminonicotinamide ( 1 1.0 g. 80.21 mmol) suspended in 500 mL of THF was added solid lithium aluminum hydride (6.35 g, 167.3 mmol) portionwise. This mixture was refluxed for 72 h. cooled to RT and quenched by the addition of water (4.71 mL). 1 N NaOH (4.71 mL) and then water (19 mL). After 1 h of vigorous stirring the mixture was filtered through celite and washed with 500 mL of
THF:MeOH (85:15). The volatiles were removed in vacuo and the solid was purified by flash column chromatography (60 x 150 mm column of Siθ2, EtOH/NH4OH gradient elution 99:1 , 98:2 97:3) to afford 3.70 g (37% yield) of a white solid: TLC (EtOH:NH4OH, 99: 1 ) Rf = 0.16: 1 H NMR (400 MHz. CDCI3) δ 1 .54 (br s, 2H), 3.73 (s, 2H), 4.50 (br s, 2H), 6.48 (d, J = 8.4 Hz, 1 H), 7.42 (dd, J = 8.3 and 2.3 Hz, 1 H), 7.99 (d, J = 1.6 Hz, 1H). Step B: Preparation of N-Boc-D-4-chlorophenylalanyl-L-proline methyl ester
To a solution of N-Boc-D-4-chlorophenylalanine (2.72 g, 9.07 mmol), L-proline methyl ester hydrochloride (1.50 g, 9.07 mmol), EDC (2.08 g, 10.884 mmol), and HOBt (1.51 g, 10.884 mmol) in 15 mL of DMF at 0°C was added triethylamine (3.0 mL. 21.76 mmol). This stirred 1 h at 0°C then 15 h at RT and was diluted with 200 mL of ethyl acetate and washed with sat'd NaHCO3 ( 1 x 25 mL), water (5 x 25 mL), and brine ( 1 x 20 mL). The solution was dried over MgSO4, filtered and concentrated to an oil which was purified by flash column chromatography (40 x 150 mm column of SiO2, EtOAc:Hex gradient elution 1 :2. to 1 : 1 ,) to afford 3.24 g (87% yield) of a white foam: TLC (EtOAc:Hex. 2: 1 ) Rf = 0.68; 1 H NMR (400 MHz, CDCI3) δ 1.42 (s, 9H), 1.61 - 1.66 (m, 1 H), 1.88-2.02 (m, 3H), 2.84-3.02 (m, 3H), 3.58- 3.67 (m, 1 H), 3.72 and 3.72 rotomers (s, 3H), 4.31 (dd, J = 7.7 and 3.5 Hz, 1 H), 4.62 (dd, J = 14.6 and 8.3 Hz, 1 H), 5.31 (d, J = 8.4 Hz, 1 H), 7.08-7.15 (m, 2H), 7.23-7.26 (m, 2H).
Step C: Preparation of N-Boc-D-4-chlorophenylalanyI-L-proline carboxylic acid
To a solution of N-Boc-D-4-chlorophenylalanyl-L-proline methyl ester (4.98 g, 12.12 mmol) in 45 mL of DME was added LiOH ( 1.16 g) dissolved in 15 mL of water. After 0.5h the solution was concentrated to 15 mL acidified to pH 3 with 10% HCl and extracted with ethyl acetate (3 x 100 mL). The combined organic layer was washed with brine ( 1 x 15 mL), dried over MgSO4, filtered and concentrated in vacuo to provide 5.05 g of a white foam : TLC
( EtOAc:Hex. 2: 1 ) Rf = 0.05; 1 H NMR (400 MHz, CDCI3) δ 1.41 (s, 9H), 1.69-1.80 (m, 2H), 1.88-1.94 (m, 1H), 2.09 (s, 1 H), 2.20-2.25 (m, 1 H), 2.77-2.83 (m, 1 H), 2.92-2.99 (m, 2H), 3.55-3.63 (m, 1 H), 4.37 (d, J = 5.8 Hz, 1 H), 4.62-4.64 (m. 1 H), 5.38 (d, J = 6.2 Hz, 1 H), 7.09-7.18 (m, 2H), 7.20-7.27 (m, 2H).
Step D: Preparation of 6-amino-3-(aminomethyl)pyridine-N-Boc- D-4-chlorophenylalanyl-L-proline amide
To a solution of N-Boc-D-4-chlorophenyIalanine-L-proline carboxylic acid (1.22 g, 3.07 mmol) in 5 mL DMF was added 6-amino- 3-(aminomethyl)pyridine (0.434 g, 3.53 mmol), HOBt (0.501 g, 3.684 mmol), and EDC (0.706 g, 3.684 mmol), cooled to 0°C and added triethylamine (0.51 mL, 3.68 mmol). After 18 h the reaction mixture was concentrated, diluted into ethyl acetate (150 mL) and washed with saturated NaHCO3 ( 1x20 mL), water (5x20 mL) and brine (1 x15 mL), dried over MgSO4, filtered and concentrated to an oil. Flash column chromatography (30x 150 mm column of SiO2, CH2CI2/CH2CI2 saturated with NH3/MeOH gradient elution 60:39: 1 to 60:38:2 to
60:35:5) provided 1.30 g of a yellowish foam: TLC (CH2CI2/CH2CI2 saturated NH3/MeOH, 60:35:5) Rf = 0.34; 1 H NMR (400 MHz, CDCI3) d1.36 (s. 9H), 1.64-1 .74 (m, 2H), 1.85 (br s, 1 H), 2.25-2.27 (m, 1 H), 2.74-2.87 (m, 1 H), 2.89-2.99 (m, 2H), 3.64-3.66 ( m, 1 H), 4.17-4.31 (m, 2H), 4.42-4.50 (m, 4H), 5.25 (d, J = 3.8 Hz, 1 H), 6.45 (dd, J = 8.3 and 1.9 Hz, 1 H), 7.12 = 7.14 (m, 2H), 7.25 = 7.37 (m, 4H), 7.91 (s, 1 H). Anal.CHN: C25H32N5O4CI• 0.2 H2O.
Calc. C 59.38: H 6.46; N 13.85.
Found C 59.02; H 6.27: N 13.58.
Step E: Preparation of N-D-4-chlorophenyl-alanyl-N-(6- aminopyridin-3-yl)methyl-L-proline amide
To a solution of N-BOC-D-4-chlorophenyl-alanyl-N-(6- aminopyridin-3-yl)methyl-L-proline amide ( 1.30 g. 2.59 mmol) dissolved in ethyl acetate ( 10 mL). cooled to 0°C was bubbled in HCl (g) for 10 min. Reaction stirred an additional 0.5 h and was concentrated in vacuo. The residue was tritrated with ethyl acetate and filtered to provide 1.19 g of a white solid: mp 198-205°C. LRMS (M+) 402.
Step F: Preparation of N-(t-butyloxy-carboxymethyl)-D-4- chlorophenyl-alanyl-N-(6-aminopyridin-3-yl)methyl-L- proline amide
To N-D-4-chlorophenyl-alanyl-N-(6-aminopyridin-3- yl)methyl-L-proline amide (0.80 g, 1.68 mmol) dissolved in 7 mL of DMF was added tert-butyl bromoacetate (0.30 mL, 1.84 mmol) and triethylamine (0.86 mL, 6.21 mmol). After 48 h the solution was diluted with 100 mL of ethyl acetate and washed with saturated NaHCO3 ( 1 x20 mL) water (4x20 mL), brine (1 x20 mL), dried over MgSO4 filtered and concentrated to a yellow oil. This was purified by flash column chromatography (30x 150 mm column of SiO2, CH2CI2/CH2CI2 saturated NH3/MeOH 60:39: 1 , 60:38:2, 60:37:3, 500 mL each) to provide a white solid. Recrystallization from ethyl acetate/hexanes afforded 0.232 g of a crystalline solid, mp 176-178°C. Anal. CHN:
C26H3SN5O4CI•0.1 H2O
calc C 60.30: H 6.66; N 13.53.
found C 59.96: N 6.65, N 13.25. EXAMPLE 12
Preparation of N-( N',N'diethyl-carboxymethyl)-D-4-chlorophenyl- alanyl-N-(6-amino-pyridin-3-yl)methyl-L-proline amide
As above in Example 12, Step F, replacement of tert-butyl bromoacetate with N, N-diethyl-2-bromoacetamide afforded the desired product. After flash column chromatography the product was isolated as a foam: TLC (CH2CI2/CH2CI2 saturated NH3/MeOH, 60:35:5) Rf = 0.36; 1 H NMR (400 MHz. CDCI3), δ 1.01 (t, J = 1.0 Hz, 3H), 1.09 (t, J = 7.0 Hz, 3H); 1.60- 1.65 (m, 1 H), 1.69- 1.83 (m, 2H), 2.00 (br s, 1 H), 2.14-2.18 (m, 1 H), 2.64-2.71 (m, 1 H), 2.82 (d, J=7.5Hz. 2H), 3.09-3.18 (m, 3H), 3.25-3.33 (m, 4H), 3.34-3.49 (m, 3H), 4.27 (d, J=5.8Hz, 1 H), 4.37 (br s, 1 H), 4.52 (d. J=5.5Hz, 1 H), 6.44 (d, J=8.5Hz, 1 H), 7.12 (d, J =8.2Hz, 2H), 7.22=7.26 (m, 2H), 7.43 (dd, J=8.3 and 2.1Hz, 1H), 7.90 (d, J=14Hz, 1H), 8.14 (dd, J=10.8, 5.4Hz, 1H).
Anal. CHN: C26H35N6O3CI• 0.35 CH2CI2:
Calc C 58.37: H 6.68; N 15.30
Found C 58.73; H 6.87; N 14.93 EXAMPLE 13
Preparation of N-(2R)-azido-3-(3,4-methylenedioxyphenyl)alanyl-N-
(6-amino-2-methylpyridin-3-yl)methyl- L-proline amide
Step A: Preparation of 6-amino-3-cyano-2-methylpyridine
To a solution of 6-amino-3-bromo-2-methylpyridine (20.0g 0.1069 mol) in 40 mL of DMF was added copper(l ) cynanide ( 1 1.50 g, 0.128 mol). The reaction mixture was refluxed under argon for 10 h, cooled to 80°C and poured into a solution of NaCN (21.50 g) dissolved in 70 mL water. This stirred for 1 h and was extracted with ethyl acetate (4 x 100 mL). The combined organic layer was washed with 10% NaCN ( 1 x 75 mL), water ( 1 x 75 mL) and brine ( 1 x 50 mL) and dried over MgSO4. The solution was filtered, concentrated and the residue was tritrated with ethyl acetate and hexanes to provide the product as a tan solid (8.90 g): 1 H NMR (300 MHz, CDCI3) δ 2.55 (s, 3H), 5.39 (br s, 2H), 6.35 (d, J = 8.55 Hz, 1 H), 7.54 (d, J = 8.54 Hz, 1 H). Step B: Preparation of the dihydrochloride of 6-amino-3-
(aminomethyl)-2-methylpyridine
To a solution of 6-amino-3-cyano-2-methylpyridine ( 10.38 g 78.04 mmol) in 300 mL of ethanol/ methanol (3: 1 ) and 30 mL of 6N HCl was added 10% Pd/C (5.00 g). The solution was flushed with hydrogen and vigorously stirred. After 14 h the mixture was filtered through celite and washed with 500 mL of methanol. The volatiles were removed in vacuo to provide a yellow solid : 1 H NMR (300 MHz, CDCI3) δ 2.59 (s, 3H), 4.12 (s, 2H), 4.89 (s, 6H), 6.94 (d, J = 9.1 Hz, 1H), 7.96 (d, J = 9.2 Hz, 1H).
Step C: Preparation of (4R)-3-(3-(3,4-methylenedioxyphenyl)-
1 -oxopropyl)-4-(phenylmethyl)-2-oxazolidinone
To a solution of 3-(3,4-methylenedioxyphenyl)propionic acid ( 10.93 g, 0.0563 mol) dissolved in THF was added triethylamine (8.22 mL, 0.0589 mol) and cooled to -78°C. Trimethylacetylchloride (6.93 mL, 0.0563 mol) was added dropwise and the reaction was warmed to 0°C for 0.5 h and recooled to -78°C. To this was added a solution of (4R)-(3-phenylmethyl)-2-oxazolidinone (9.49 g, 0.0536 mol) and n-BuLi (22.5 mL, 2.5 M in hexanes. 0.563 mol) in 150 mL THF after stirring for 1 h at -78°C via cannula. After 1 h the reaction was warmed to 0°C and stirred an additional 2 h then quenched with 100 mL of 1/2 sat'd NH4CI. The aqueous layer was extracted with ethyl acetate (3 x 100 mL) and the combined organic layer was washed with sat'd NaHCO3 ( 1 x 100 mL), water ( 1 x 100 mL). and brine ( 1 x 100 mL ), dried over MgSO4, filtered and concentrated to an oil. Recrystallization from ethyl acetate and hexanes afforded 15.17 g (76% yield) of white crystalline product: mp 86-87°C. Step D: Preparation of (3(2R),4R)-3-(2-azido-3-(3,4-methylene- dioxyphenyl)-1 -oxopropyl)-4-(phenylmethyl)-2- oxazolidinone
To a solution of N-(4R)-3-(3,4-methylenedioxyphenyl)-1 - oxopropyl)-4-(phenylmethyl)-2-oxazoIidinone (7.59 g, 21.5 mmol) dissolved in THF and cooled to -78°C was added KHMDS (46.0 mL. 0.5 M in toluene. 23.0 mmol) dropwise. After 1 h a precooled solution ( -78°C) of trisyl azide (7.46 g, 24.15 mmol) dissolved in THF (50 mL) was added via cannula. The reaction was quench with glacial acetic acid (7.0 mL) after 3 minutes and stirred at RT for 6 h. Saturated NH4CI (50 mL) was added and the aqueous was extracted with ethyl acetate (2 x 75 mL). The combined organic layer was washed with sat'd
NaHCO3 (2 x 50 mL), water (1 x 50 mL), brine (1 x 50 mL), dried over MgSO4, filtered and concentrated in vacuo. The solid was recrystallized from ethyl acetate and hexanes to afford 5.74 g of a yellow white solid, mp 125-132; 1 H NMR (400 MHz, CDCI3) δ 2.83 (dd, J= 13.4 and 9.5Hz, 1 H), 2.94 (dd, J=13.6 and 9.2Hz, 1H), 3.13 (dd, J=13.7 and 5.1Hz, 1H), 3.33 (dd, J=13.4 and 3.0Hz, 1H), 4.15-4.23 (m, 2H), 4.62-4.66 (m, 1H), 5.19 (dd, J=9.1 and 5.1Hz, 1H), 5.94 (s, 2H), 6.76-6.82 (m, 2H), 7.20-7.36 (m, 5H). Step E: Preparation of N-(2R)-azido-3-(3,4-methyIene- dioxyphenyl)- 1 -propanoic acid
To a solution of (3(2R),4R)-3-(2-azido-3-(3.4-methylene- dioxyphenyl)-1 -oxopropyl)-4-(phenylmethyl)-2-oxazolidinone (5.743 g, 14.57 mmol) dissolved in 75 mL of dioxane cooled to 0°C was added a solution of LiOH (0.419 g, 17.49 mmol) dissolved in water (25 mL) and 30% H2O2 (8.27 mL). After 0.5 h ethyl acetate was added ( 15 mL) and the solution was concentrated to 25 mL in vacuo. The aqueous layer was acidified to pH 2 with 10% HCl and extracted with ethyl acetate (3x75 mL), the combined organic layer was washed with water ( 1 x20 mL), brine ( 1x20 mL). dried over MgSO4, filtered and concentrated to an oil. This material was used directly in the next step.
Step F: Preparation of N-(2R)-azido-3-(3,4-methylene- dioxyphenyl)alanyl-L-proline methyl ester
To a solution of (2R)-azido-3-(3,4-methylenedioxyphenyl)- 1 -propanoic acid (from above) dissolved in 30 mL of DMF was added L-proIine methyl ester hydrochloride (2.53 g. 15.30 mmol), HOBt (2.06 g, 15.30 mmol). EDC (2.93 g, 15.30 mmol). cooled to 0°C and then added triethylamine (4.46 mL, 32.06 mmol). After 1 h the reaction was warmed to RT and stirred 18 h, then diluted with ethyl acetate (300 mL) and washed with sat'd NaHCO3 ( 1 x50 mL), water (3x50 mL), brine ( 1 x50 mL), dried over MgSO4, filtered and concentrated to an oil. The residue was purified by flash column chromatography (40 x 150 mm column of SiO2, EtOAc:Hex gradient elution 1 :2 1000 mL, to 1 : 1 500 mL) to afford 3.26 g of waxy solid. TLC (EtOAc:Hex. 1 :2) Rf = 0.17; 1 H NMR (400 MHz, CDCI3) δ 1.78-1 .82 (m, 1 H), 1.97-2.40 (m, 2H), 3.02-3.15 (m, 3H), 3.64-3.74 (m, 4H), 3.84-3.89 (m, 1H), 4.40-4.52 (m, 1 H), 5.92 and 5.93 rotamers (s, 2H), 6.68-6.75 (m, 3H). Step G: Preparation of N-(2R)-azido-3-(3,4-methylene- dioxyphenyl)alanyl-L-proline carboxylic acid
To N-(2R)-azido-3-(3,4-methylenedioxyphenyl)alanyl-L- proline methyl ester (0.84 g, 2.43 mmol) dissolved in 6 mL of THF cooled to 0°C was added a solution of LiOH (0.62 g, 2.58 mmol) in 2 mL of water. After 0.5 h reaction was quenched by the addition of ethyl acetate ( 10 mL) and concentrated to a 3 mL volume. The solution was acidified to pH 2 with 10% HCl and extracted with ethyl acetate (3x 25mL), the organic layer was washed with water ( 1 x 10 mL). and brine ( 1 x 5 mL). dried over MgSO4, filtered and concentrated to a white solid (0.748 g, 93% yield). 1H NMR (400 MHz, CDCI3) δ 1.81 - 1.98 (m, 3H ), 2.25-2.28 ( m, 1 H), 2.96-3.19 (m, 3H), 3.55-3.62 (m, 2H), 3.85 (dd, J = 8.1 and 6.9 Hz, 1 H), 4.49 (dd, J = 7.9 and 2.9 Hz, 1 H), 5.94 (s, 2H), 6.68-6.76 (m, 3H).
Step H: Preparation of N-(2R)-azido-3-(3,4-methylene- dioxyphenyl)alanyl-N-(6-amino-2-methylpyridin-3- yl)methyl- L-proline amide
To a solution of (2R)-azido-3-(3,4-methylenedioxy- phenyl)alanyl-L-proline carboxylic acid (0.319 g, 0.961 mmol) dissolved in 4 mL of DMF was added 6-amino-3-(aminomethyl)-2- methylpyridine dihydrochloride (0.222 g, 1.05 mmol). EDC (0.202 g, 1.05 mmol), and HOBt (0.142 g, 1.05 mmol), cooled to 0°C and added the triethylamine (0.428 mL, 3.074 mmol). The reaction was warmed to RT after 1 h and quenched after 16 h by diluting into 100 mL of ethyl acetate and washed with sat'd NaHCO3 ( 1 x25 mL), water (4x25 mL), brine ( 1 x20 mL), dried over MgSO4, filtered and concentrated to a foam. Flash column chromatography (25x 150 mm column of SiO2, CH2CI2/CH2Cl2 saturated with NH3/MeOH gradient elution 60:38:2 to 60:37:3) provided 0.324 g of an oil: TLC (CH2CI2/CH2CI2 saturated NH3/MeOH, 60:30: 10) Rf = 0.34; 1 H NMR (400 MHz, CDCI3) δ 1.61 - 1.79 (m, 2H), 2.01 -2.07 (m, 2H), 2.35 (s, 3H), 2.85-2.90 (m, 1H), 3.01 (dd, J=13.4 and 6.6Hz, 1H), 3.14 (dd, J=13.4 and 8.4Hz, 1 H), 3.50 (t, J=2.7Hz, 1 H), 3.83 (dd, J=8.4 and 6.6Hz, 1 H), 4.20-4.31 (m, 2H), 4.42 (s, 2H), 4.53 (dd, J=7.8 and 1.8Hz, 1H), 5.93 (dd, J=2.0 and 1.3 Hz, 1H), 6.29 (d, J=8.2Hz, 1H), 6.65-6.74 (m, 4H), 7.27 (d, J=3.7Hz, 1 H).
Step I: N-(2R)-amino-3-(3,4-methylenedioxyphenyl)alanyl-N- (6-amino-2-methylpyridin-3-yl)methyl- L-proline amide
To N-(2R)-azido-3-(3,4-methylenedioxyphenyl)alanyl-N-
(6-amino-2-methylpyridin-3-yl)methyl- L-proline amide dissolved in 10 mL of ethanol and 2.99 mL of 10% HCl was added 10% Pd/C (0.30 g) and charged with hydrogen. After 2 h the reaction was flushed with argon and filtered through celite washing with absolute ethanol. The solution was concentrated in vacuo and the oil was tritrated with ethyl acetate to afford 0.21 g white solid, mp 1 78- 188°C;
Analysis for• C22H27N5O4• 2HCl 0.95• H2O
calc C, 51 .26: H, 6.04; N, 13.59
found C, 51.22; H. 6.14; N, 13.28
EXAMPLE 14
Preparation of (2R)-N-(N-morphilino-2-acetamide)-3-(3,4-methylene- dioxyphenyl)alanyl-N-(6-amino-2-methylpyridin-3-yl)methyl-L- prolineamide
To a solution of N-(2R)-amino-3-(3,4-methylenedioxy- phenyl)alanyl-N-(6-amino-2-methylpyridin-3-yl)methyl-L-proline amide dihydrochloride (0.95 g, 0.184 mmol) in DMF (2 mL) cooled to 0°C was added N-morphilino-2-bromoacetamide (0.038 g, 0.184 mmol) and triethylamine (0.028 mL, 0.202 mmol). The reaction was warmed to RT over 3 h and after 19 h the volatiles were removed in vacuo. The residue was purified by flash column chromatography ( 15 x 150 mm column of SiO2, CH2CI2/CH2CI2 saturated with NH3/MeOH gradient elution 60:39: 1 ; 60:38:2; 60:37:3; 60:36:4) to provide 0.009 g of a white solid: TLC (CH2CI2/CH2CI2 saturated NH3/MeOH, 60:30: 10) Rf = 0.15; 1 H NMR (400 MHz, CDCI3) δ 1.78- 1.83 (m, 2H), 2.20-2.25 (m, 1 H), 2.35 (s, 3H), 2.71 (dd, J=16.1 and 7.3Hz, 1H), 2.77 (d, J=7.3 Hz, 2H), 3.15 (d, J=14.5 Hz, 1H), 3.29 (d, J=14.5 Hz, 1H), 3.30-3.36 (m, 2H), 3.41-3.55 (m, 6H), 3.62-3.65 (m, 2H), 4.21 -4.38 (m, 4H), 4.57 (dd, J=7.8 and 2.6Hz, 1H), 5.92 (dd, J=3.3 and 1.3Hz, 2H), 6.28 (d, J=8.2Hz, 1H), 6.62-6.73 (m, 4H), 7.26-7.28 (m, 2H). EXAMPLE 15
Preparation of (2R)-N-(N',N'-diethyl-2-acetamide)-3-(3.4-methylene- dioxyphenyl)alanyl-N-(6-amino-2-methylpyridin-3-yl)methyl-L- prolineamide
The titled compound was prepared Tin a manner similar to that described in Example 16 using N, N-diethyI-2-bromoacetamide in place of N-morphilino-2-bromoacetamide to afford an amphorous solid: mp 85- 100°C; TLC (CH2CI2/CH2CI2 saturated NH3/MeOH, 60:30: 10) Rf = 0.39;
Analysis for C28H38N6O5• 0.45 H2O
calc C, 61.62; H, 7.00; N, 15.40
found C, 61.65; H, 7.20; N, 15.12
EXAMPLE 16
(2R)-N-(N'-pyrrolidine-2-acetamide)-3-(3,4-methylenedioxy- phenyl)alanyl-N-(6-amino-2-methylpyridin-3-yl)methyl-L- prolineamide
This was prepared in a manor similar to that described in Example 16 using N-pyrrolidine-2-bromoacetamide in place of N- morphilino-2-bromoacetamide to afford an amphorous solid; mp 78- 82°C; TLC (CH2CI2/CH2CI2 saturated NH3/MeOH. 60:30: 10) Rf = 0.32;
Analysis for C28H36N6O5• 0.50 H2O
calc. C, 61.64; H. 6.84; N, 15.40
found C, 61.62; H. 6.77; N, 15.01 EXAMPLE 17
Preparation of N-BOC-D-3,3-diphenylaIanyl-N-(6-amino-2-methyl- pyridin-3-yl)methyl-L-proline amide
To a solution of N-BOC-D-3,3-diphen\ialanyl-L-Pro-OH
(0.48 g, 1.096 mmol) in DMF (5 mL) cooled to 0°C was added EDC (0.23 g, 1.205 mmol), HOBt (0.162 g, 1.205 mmol), the dihydrochloride of 6-amino-3-(aminomethyl)-2-methylpyridine (0.253 g, 1.205 mmol) and then triethylamine (0.61 1 mL, 4.38 mmol). The reaction was warmed to RT after 1 h and quenched after 16 h by diluting into 100 mL of ethyl acetate and washed with sat'd NaHCO3 ( 1 x25 mL), water (4x25 mL), brine ( 1 x20 mL), dried over MgSO4, filtered and
concentrated to a foam. Flash column chromatography (25x 150 mm column of SiO2, CH2CI2/CH2CI2 saturated with NH3/MeOH gradient elution 60:38:2 to 60:37:3) provided 0.525 g of a foam. The foam was dissolved in EtOAc (5 mL) and prepcipitated with hexanes (20 mL) to afford a white solid: TLC (CH2CI2/CH2CI2 saturated NH3/MeOH, 65:30:5) Rf = 0.47; 1 H NMR (400 MHz, CDCl3) δ 1.24 (s, 9H), 1 ,42- 1.48 (m, 1 H), 1.57 (m, 2H), 2.10-2.14 (m, 1 H), 2.32- (s, 3H), 2.50-2.57 (m, 1 H), 3.67 (dd, J=8.9 and 8.6Hz, 1 H), 4.19-4.30 (m, 3H), 4.36 (dd, J=1 1.3 and 8.6Hz, 2H), 4.84-4.90 (m, 2H), 6.32 (d, J=8.24Hz, 1 H), 7.16-7.41 (m, 13H).
CHN anal, for C32H39N5O4• 0.45 H2O.
Calc C. 67.93; H, 7.1 1 ; N, 12.38.
Found C. 67.92; H, 7.02; N, 12.42. EXAMPLE 18
Preparation of N-D-3,3-diphenylalanyl-N-(6-amino-2-methylpyridin- 3-yl)methyl-L-proline amide
To a solution of (0.452 g, 0.81 mmol) dissolved in ethyl acetate (10 mL), cooled to -78°C was bubbled in HCl (g) for 10 min. Reaction stirred an additional 4 h at 0°C and was concentrated in vacuo. The residue was tritrated with ethyl acetate and filtered to provide 0.407 g of a white solid: mp 21 1-216°C,
CHN anal, for C27H31 N5O2• 0.70H2O and 1.85 HCl
Calc. C, 60.32; H. 6.42; N, 13.03.
Found C, 60.32; H, 6.41 ; N, 12.78.
Ki for thrombin is the inhibition constant for the tested compound with human thrombin. Ki for trypsin is the inhibition constant for the tested compound with bovine trypsin. Rate constants were determined using the following in vitro procedures.
In vitro assay for determining proteinase inhibition
Assays of human a-thrombin and bovine trypsin were performed at 25°C in 0.05 M TRIS buffer pH 7.4, 0.15 M NaCl, 0.1 % PEG. Trypsin assays also contained 1 mM CaCl2.
In assays wherein rates of hydrolysis of a p-nitroanilide (pna) substrate were determined, a Thermomax 96-well plate reader was used was used to measure (at 405 nm) the time dependent
appearance of p -nitroaniIine. sar-PR-pna (sarcosine-Pro-Arg-p- nitroanilide) was used to assay human a-thrombin (Km= 125 μM) and bovine trypsin (Km= 125 μM). p -Nitroanilide substrate concentration was determined from measurements of absorbance at 342 nm using an extinction coefficient of 8270 cm- 1 M- 1.
In certain studies with potent inhibitors (Ki < 10 nM) where the degree of inhibition of thrombin was high, a more sensitive activity assay was employed. In this assay the rate of thrombin
catalyzed hydrolysis of the fluorogenic substrate Z-GPR-afc (Cbz-Gly- Pro-Arg-7-amino-4-trifluoromethyl coumarin) (Km =27 μM) was determined from the increase in fluorescence at 500 nm (excitation at 400 nm) associated with production of 7-amino-4-trifluoromethyl coumarin. Concentrations of stock solutions of Z-GPR-afc were determined from measurements of absorbance at 380 nm of the 7- amino-4-trifluoromethyl coumarin produced upon complete hydrolysis of an aliquot of the stock solution by thrombin.
Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration≤ 0.1 Km into a solution containing enzyme or enzyme equilibrated with inhibitor.
Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. Initial velocities of product formation in the absence (Vo) or presence of inhibitor (Vi) were measured. Assuming competitive inhibition, and that unity is negligible compared Km/[S], [I]/e, and [l]/e (where [S], [I], and e respectively represent the total concentrations, of substrate, inhibitor and enzyme), the equilibrium constant (Ki) for dissociation of the inhibitor from the enzyme can be obtained from the dependence of Vo/Vi on [I] shown in equation 1.
Vo/Vi = 1 + [I]/Ki ( 1 )
The activities shown by this assay indicate that the compounds of the invention are therapeutical ly useful for treating various conditions in patients suffering from unstable angina, refractory angina, myocardial infarction, transient ischemic attacks, atrial fibrillation, thrombotic stroke, embolic stroke, deep vein thrombosis, disseminated intravascular coagulation, and reocclusion or restenosis of recanalized vessels.
Thrombin Inhibitors - Therapeutic Uses
Anticoagulant therapy is indicated for the treatment and prevention of a variety of thrombotic conditions, particularly coronary artery and cerebrovascular disease. Those experienced in this field are readily aware of the circumstances requiring anticoagulant therapy. The term "patient" used herein is taken to mean mammals such as primates, including humans, sheep, horses, cattle, pigs, dogs, cats, rats, and mice. Thrombin inhibition is useful not only in the anticoagulant therapy of individuals having thrombotic conditions, but is useful whenever inhibition of blood coagulation is required such as to prevent coagulation of stored whole blood and to prevent coagulation in other biological samples for testing or storage. Thus, thrombin inhibitors can be added to or contacted with any medium containing or suspected of containing thrombin and in which it is desired that blood coagulation be inhibited, e.g. when contacting the mammal's blood with material selected from the group consisting of vascular grafts, stents. orthopedic prothesis. cardiac prosthesis, and extracorporeal circulation systems
The thrombin inhibitors of the invention can be administered in such oral forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixers, tinctures, suspensions, syrups, and emulsions.
Likewise, they may be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be
employed as an anti-aggregation agent. For treating ocular build up of fibrin, the compounds may be administered intraoculariy or topically as well as orally or parenterally.
The thrombin inhibitors can be administered in the form of a depot injection or implant preparation which may be formulated in such a manner as to permit a sustained release of the active ingredient. The active ingredient can be compressed into pellets or small cylinders and implanted subcutaneously or intramuscularly as depot injections or implants. Implants may employ inert materials such as biodegradable polymers or synthetic silicones, for example, Silastic, silicone rubber or other polymers manufactured by the Dow-Corning Corporation.
The thrombin inhibitors can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol,
stearylamine or phosphatidylcholines. The thrombin inhibitors may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The thrombin inhibitors may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinlypyrrolidone, pyran copolymer. polyhydroxy-propyl- methacrylamide-phenol, polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysine 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, poly lactic acid, polyglycolic acid, copolymers of poly lactic and polyglycolic acid, polyepsilon caprolactone. polyhydroxy butyric acid, polyorthoesters. polyacetals, polydihydropyrans.
polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels.
The dosage regimen utilizing the thrombin inhibitors is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient: the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.
Oral dosages of the thrombin inhibitors, when used for the indicated effects, will range between about 0.1 mg per kg of body weight per day (mg/kg/day) to about 100 mg/kg/day and preferably 1.0- 100 mg/kg/day and most preferably I -20 mg/kg/day. Intravenously, the most preferred doses will range from about 0.01 to about 10
mg/kg/minute during a constant rate infusion. Advantageously, the thrombin inhibitors may be administered in divided doses of two, three, or four times daily. Furthermore, they can be administered in
intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, or course, be continuous rather than intermittent throughout the dosage regime.
For example, oral tablets can be prepared which contain an amount of active compound of between 100 and 500 mg. typically between 200 and 250 mg. Typically, a patient in need of thrombin inhibitor compound, depending on weight and metabolism of the patient, would be administered between about 100 and 1000 mg active
compound per day. For a patient requiπng 1000 mg per day, two tablets containing 250 mg of active compound can be administered in the morning and two tablets containing 250 mg of active compound can again be administered in the evening. For a patient requiring 500 mg per day, one tablet containing 250 mg of active compound can be administered in the morning and one tablet containing 250 mg of active compound can again be administered in the evening.
The thrombin inhibitors are typically administered as active ingredients in admixture with suitable pharmaceutical diluents,
excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is. oral tablets, capsules, ehxers, syrups and the like, and consistent with convention pharmaceutical practices.
For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate,
dicalcium phosphate, calcium sulfate. mannitol, sorbitol and the like; for oral administration in liquid form, the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta- lactose, corn-sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulo.se, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate. sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch methyl cellulose, agar, bentonite. xanthan gum and the like.
The thrombin inhibitors can also be co-administered with suitable anti-coagulation agents or thrombolytic agents such as plasminogen activators or streptokinase to achieve synergistic effects in the treatment of various ascular pathologies. For example, thrombin inhibitors enhance the efficiency of tissue plasminogen activator- mediated thrombolytic reperfusion. Thrombin inhibitors may be administered first following thrombus formation, and tissue
plasminogen activator or other plasminogen activator is administered thereafter. They may also be combined with heparin, aspirin, or warfarin.

Claims (1)

  1. WHAT IS CLAIMED IS:
    1 . A compound having the following structure:
    wherein
    A is C or N;
    X 1 , X2 and X3, each independently attached to a ring carbon atom, are independently selected from the group consisting of
    hydrogen, C1 -4 alkyl, and C1 -4 alkoxy;
    Y, attached to a ring carbon atom, is H, NH2 or OH;
    Z is -(CH2)1 -3-;
    R1, R2, and R2' are independently
    hydrogen,
    phenyl,
    mono- or di-halogenated phenyl,
    naphthyl,
    biphenyl,
    a 5- to 10-membered mono- or bicyclic heterocyclic ring or 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,
    C 1 -4 alkyl,
    branched C1 -4 alkyl,
    C3-7 cycloalkyl,
    C5- 1 2 bicyclic alkyl,
    C1 1 - 16 tricyclic alkyl,
    R4(CH2)n,
    (R4)2(CH), (R4)(OR4)CH,
    R4O(CH2)n, or
    R 1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N, O and S, where n is 1 , 2, 3 or 4;
    R3 is hydrogen,
    (R2)2N, wherein R2 is the same or different,
    R2'OCONH, provided R2' is not hydrogen,
    R2CONH,
    HO(CH2)p, where p is 0, 1 , 2, 3 or 4.
    R2' SO2NH, provided R2' is not hydrogen, or
    (R2)m NCONH, where m is 1 or 2, wherein R2 is the same or different; R4 is independently
    phenyl,
    mono- or di-halogenated phenyl,
    naphthyl,
    biphenyl,
    a 5- to 10-membered mono- or bicyclic heterocyclic ring or 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,
    -COR5 ,
    -OR6,
    C1 -4 alkyl,
    branched C1 -4 alkyl,
    C1 -4 alkoxy, C3-7 cycloalkyl.
    C5-12 bicyclic alkyl, or
    C11-16 tricyclic alkyl; R5 is
    -OH,
    -OR6,
    -N(R7)2, where R7 is same or different, and
    where D is -CH2CH2-, -CH2-O-CH2-, or -CH2-NH-CH2-;
    R6 is C1-4 alkyl;
    R7 is hydrogen or C1-4 alkyl;
    G is (CH2)q where q is 1 or 2; or
    NR1CH2; and
    Q is SCH2, or
    (CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.
    2. A compound of claim 1 having the following structure:
    wherein
    A is C or N; X1, X2 and X3, each independently attached to a ring carbon atom, are independently selected from the group consisting of
    Hand C1-4 alkyl;
    Y, attached to a ring carbon atom, is H, NH2 or OH;
    R1, R2, and R2' are independently
    hydrogen,
    phenyl,
    mono- or di-halogenated phenyl,
    naphthyl,
    biphenyl,
    a 5- to 7-membered mono- or bicyclic heterocyclic ring or 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 ofN,OandS,
    C1-4 alkyl,
    branched C1-4 alkyl,
    C3-7 cycloalkyl,
    C5-12 bicyclic alkyl,
    C11-16 tricyclic alkyl,
    R4(CH2)n,
    (R4)2(CH),
    (R4)(OR4)CH,
    R4O(CH2)n, or
    R1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N, O and S, where n is 1,2,3 or 4;
    R3 is
    H,
    (R2)2N, wherein R2 is the same or different,
    R2'OCONH, provided R2' is not hydrogen, R2CONH,
    HO(CH2)p, where p is 0, 1, 2, 3 or 4,
    R2'SO2NH, provided R2' is not hydrogen, or
    (R2)mNCONH, where m is 1 or 2, wherein R2 is the same or different;
    R4 is independently
    phenyl,
    mono- or di-halogenated phenyl,
    naphthyl,
    biphenyl.
    a 5- to-7- membered mono- or bicyclic heterocyclic ring or 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 ofN, OandS,
    COOH,
    C1-4 alkyl,
    branched C1-4 alkyl,
    C3-7 cycloalkyl,
    C5-12 bicyclic alkyl, or
    C11-16 tricyclic alkyl;
    G is (CH2)q where q is 1 or 2; or
    NR1CH2;and
    Q is SCH2, or
    (CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.
    3. A compound of Claim 2 which has the structure: -85
    wherein
    A is C or N;
    X1 and X2, each independently attached to a ring carbon atom, are
    independently selected from the group consisting of
    Hand C1-4 alkyl:
    Y, attached to a ring carbon atom, is H or NH2;
    R1, R2, and R2' are independently
    hydrogen,
    phenyl,
    mono- or di-halogenated phenyl,
    naphthyl,
    biphenyl,
    a 5- to 7-membered mono- or bicyclic heterocyclic ring or 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 ofN,OandS,
    C1-4 alkyl.
    branched C1-4 alkyl,
    C3-7 cycloalkyl,
    C5-12 bicyclic alkyl,
    C11-16 tricyclic alkyl,
    R4(CH2)n,
    (R4)2(CH),
    (R4)(OR4)CH,
    R4O(CH2)n, or
    R1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N, O and S, where n is 1,2,3 or 4;
    R3 is
    H,
    (R2)2N, wherein R2 is the same or different,
    R2'OCONH, provided R2' is not hydrogen,
    R2CONH,
    HO(CH2)p, where p is 0, 1, 2, 3 or 4,
    R2'SO2NH, provided R2' is not hydrogen, or
    (R2)mNCONH, where m is 1 or 2, wherein R2 is the same or different;
    R4 is independently
    phenyl,
    mono- or di-halogenated phenyl,
    naphthyl,
    biphenyl,
    a 5- to-7- membered mono- or bicyclic heterocyclic ring or 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 ofN, OandS,
    COOH,
    C1-4 alkyl,
    branched C1-4 alkyl,
    C3-7 cycloalkyl,
    C5-12 bicyclic alkyl, or
    C11-16 tricyclic alkyl; G is (CH2)q where q is 1 or 2; or
    NR1CH2;and
    Q is SCH2, or
    (CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.
    4. A compound of Claim 3 having the structure:
    wherein
    X 1 and X2, each independently attached to a ring carbon atom, are independently selected from the group consisting of
    H and C1 -4 alkyl;
    Y, attached to a ring carbon atom, is H or NH2;
    R 1 , R2, and R2' are independently
    hydrogen,
    phenyl.
    mono- or di-halogenated phenyl,
    naphthyl,
    biphenyl,
    a 5- to 7-membered mono- or bicyclic heterocyclic ring or 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,
    C1 -4 alkyl,
    branched C1 -4 alkyl,
    C3-7 cycloalkyl,
    C5- 12 bicyclic alkyl,
    C1 1 - 1 6 tricyclic alkyl,
    R4(CH2)n,
    (R4)2CH, wherein R4 is the same or different,
    (R4)(OR4)CH, R4O(CH2)n, or
    R 1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N. O and S, where n is 1 , 2, 3 or 4;
    R3 is
    H,
    (R2)2N, wherein R2 is the same or different.
    R2'OCONH, provided R2 is not hydrogen.
    R2CONH.
    HO(CH2)p, where p is 0, 1 , 2, 3 or 4,
    R2'SO2NH, provided R2 is not hydrogen, or
    (R2)mNCONH, where m is 1 or 2, wherein R2 is the same or different;
    R4 is independently
    phenyl,
    mono- or di-halogenated phenyl,
    naphthyl.
    biphenyl.
    a 5- to-7- membered mono- or bicyclic heterocyclic ring or 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,
    COOH,
    C1 -4 alkyl,
    branched C1 -4 alkyl,
    C3-7 cycloalkyl,
    C5- 12 bicyclic alkyl, or
    C1 1 - 16 tricyclic alkyl; G is (CH2)q where q is 1 or 2, or
    NR 1CH2; and
    Q is SCH2, or
    (CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.
    5. A compound of Claim 3 having the structure:
    wherein
    Y, attached to a ring carbon atom, is H or NH2: R 1 , R2, and R2' are independently
    hydrogen,
    phenyl.
    mono- or di-halogenated phenyl,
    naphthyl,
    biphenyl,
    a 5- to 7-membered mono- or bicyclic heterocyclic ring or 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,
    C1 -4 alkyl,
    branched C1 -4 alkyl,
    C3 -7 cycloalkyl,
    C5- 1 2 bicyclic alkyl,
    C1 1 - 16 tricyclic alkyl, R4(CH2)n,
    (R4)2CH, wherein R4 is the same or different,
    (R4)(OR4)CH,
    R4O(CH2)n, or
    R 1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N, O and S, where n is 1 , 2, 3 or 4;
    R3 i s
    H,
    (R2)2N, wherein R2 is the same or different,
    R2'OCONH, provided R2' is not hydrogen,
    R2CONH,
    HO(CH2)p, where p is 0, 1 , 2, 3 or 4,
    R2'SO2NH, provided R2 is not hydrogen, or
    (R2)mNCONH, where m is 1 or 2, wherein R2 is the same or different;
    R4 is independently
    phenyl,
    mono- or di-halogenated phenyl,
    naphthyl,
    biphenyl,
    a 5- to-7- membered mono- or bicyclic heterocyclic ring or 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,
    COOH,
    C1 -4 alkyl,
    branched C1 -4 alkyl,
    C3-7 cycloalkyl, C5- 12 bicyclic alkyl, or
    C1 1 - 1 6 tricyclic alkyl;
    G is (CH2)q where q is 1 or 2, or
    NR11H2; and
    Q is SCH2, or
    (CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.
    6. A compound of Claim 3 having the structure:
    wherein Y, attached to a ring carbon atom, is H or NH2;
    R 1 , R2, and R2' are independently
    hydrogen,
    phenyl.
    mono- or di-halogenated phenyl,
    naphthyl,
    biphenyl,
    a 5- to 7-membered mono- or bicyclic heterocyclic ring or 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,
    C1 -4 alkyl,
    branched C1 -4 alkyl,
    C3-7 cycloalkyl, C5- 12 bicyclic alkyl,
    C1 1 - 1 6 tricyclic alkyl,
    R4(CH2)n,
    (R4)2CH, wherein R4 is the same or different.
    (R4)(OR4)CH,
    R4O(CH2)n, or
    R 1 may be joined with R2 to form a four- to seven membered carbon ring in which zero to two carbon atoms may be substituted with heteroatoms independently selected from the list N. O and S. where n is 1 , 2, 3 or 4;
    R3 is
    H,
    (R2)2N, wherein R2 is the same or different.
    R2'OCONH, provided R2' is not hydrogen,
    R2CONH,
    HO(CH2)p, where p is 0, 1 , 2, 3 or 4,
    R2'SO2NH, provided R2' is not hydrogen, or
    (R2)mNCONH, where m is 1 or 2, wherein R2 is the same or different;
    R4 is independently
    phenyl,
    mono- or di-halogenated phenyl,
    naphthyl,
    biphenyl,
    a 5- to-7- membered mono- or bicyclic heterocyclic ring or 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,
    COOH,
    C1 -4 alkyl,
    branched C1 -4 alkyl,
    C3-7 cycloalkyl, C5-12 bicyclic alkyl, or
    C11-16 tricyclic alkyl;
    G is (CH2)q where q is 1 or 2, or
    NR1CH2;and
    Q is SCH2, or
    (CH2)r where r is 1 or 2, and pharmaceutically acceptable salts thereof.
    7. A compound of Claim 3 selected from the group consisting of:
    and pharmaceutically acceptable salts thereof.
    8. A composition for inhibiting thrombin in blood comprising a compound of Claim 1 and a pharmaceutically acceptable carrier.
    9. A composition for inhibiting formation of blood platelet aggregates in blood comprising a compound of Claim 1 and a pharmaceutically acceptable carrier.
    10. A composition for inhibiting formation of fibrin in blood comprising a compound of Claim 1 and a pharmaceutically acceptable carrier. 1 1. A composition for inhibiting thrombus formation in blood comprising a compound of Claim 1 and a pharmaceutically acceptable carrier.
    12. A method for inhibiting thrombin in blood in a mammal comprising administering to the mammal a composition of
    Claim 8.
    13. A method for inhibiting formation of blood platelet aggregates in blood in a mammal comprising administering to the mammal a composition of Claim 9.
    14. A method for inhibiting formation of fibrin in blood in a mammal comprising administering to the mammal a composition of Claim 10.
    15. A method for inhibiting thrombus formation in blood in a mammal comprising administering to the mammal a composition of Claim 1 1 . 16. A method for inhibiting thrombin in stored blood comprising administering to the mammal a composition of Claim 8.
    17. A method for inhibiting formation of blood platelet aggregates in stored blood comprising administering to the mammal a composition of Claim 9.
    18. A method for inhibiting formation of fibrin in stored blood comprising administering to the mammal a composition of Claim 10.
    19. A method for inhibiting thrombus formation in stored blood comprising administering to the mammal a composition of Claim 1 1. 20. The use of a compound of Claim 1 , or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting thrombus formation, preventing thrombus formation, inhibiting thrombin, inhibiting formation of fibrin, and inhibiting formation of blood platelet aggregates, in a mammal.
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