WO2014025658A1

WO2014025658A1 - Pyrrolidine thrombin inhibitors

Info

Publication number: WO2014025658A1
Application number: PCT/US2013/053551
Authority: WO
Inventors: Libo Xu; Harry Chobanian; Brian T. Campbell; Yan Guo; Barbara Pio; Rui Zhang
Original assignee: Merck Sharp & Dohme Corp.
Priority date: 2012-08-08
Filing date: 2013-08-05
Publication date: 2014-02-13

Abstract

Compounds of the invention, which may be useful in inhibiting thrombin and associated thrombotic occlusions, have the following structure: (I) or a pharmaceutically acceptable salt thereof, wherein R is a heterocycle or -(CR⁴R⁵)_1-2NH₂, wherein R⁴ and R⁵, each time in which they occur, are independently H, C_1-6 alkyl, -CH₂F, -CHF₂, CF3 or -CH₂OH; R¹ is C_1-6alkyl; R² is NH₂, OH, NHSO₂C_1-6alkyl, or NHC(O)C_1-6alkyl; and R³ is C_3-8 carbocycle, aryl, or CR⁶R⁷R⁸, where R⁶ is hydrogen or CH₃, R⁷is hydrogen or CH₃, and R⁸ is CH₃, aryl, or 5-7-membered heteroaryl, wherein aryl and 5-7-membered heteroaryl, in each instance in which they occur, are independently unsubstituted or mono-, di- tri- or tetra- substituted with halogen, said heteroaryl having 1, 2, 3, or 4 nitrogen atoms.

Description

TITLE OF THE INVENTION

PYRROLIDINE 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 cc-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analogs of peptidase substrates in which the nitrogen atom of the scissile amide group of the substrate peptide has been replaced by hydrogen or a substituted carbonyl moiety.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 a-keto carboxyl derivatives. R. J. Brown et al., J. Med. Chem., Vol. 37, pages 1259-1261 (1994) describes orally active, non-peptidic inhibitors of human leukocyte elastase which contain trifluoromethylketone and pyridinone moieties. H. Mack et al., J. Enzyme Inhibition, Vol. 9, pages 73-86 (1995) describes rigid amidino-phenylalanine thrombin inhibitors which contain a pyridinone moiety as a central core structure.

SUMMARY OF THE INVENTION

The invention includes compounds 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 compounds may optionally include anticoagulants, antiplatelet agents, and thrombolytic agents. The compounds can be added to blood, blood products, or mammalian organs in order to effect the desired inhibitions. The invention also includes a compound 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 compounds 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 are thrombin inhibitors and may have therapeutic value in, for example, preventing coronary artery disease. The invention includes compounds of formula I:

or a pharmaceutically acceptable salt thereof, wherein

4 5 4 5

R is a heterocycle or -(CR R )i_2NH2, wherein R and R , each time in which they occur, are independently H, Ci_6 alk l, -CH2F, -CHF2, CF3 or -CH2OH;

R^ is Ci_6 alkyl;

R is NH₂, OH, NHS02Ci_6alk l, or NHC(0)Ci_6alk l; and

3 6 7 8 6 7

R is C3.8 carbocycle, aryl, or CR R R , where R is hydrogen or CH3, R is

g

hydrogen or CH3, and R is CH3, aryl, or 5-7-membered heteroaryl, wherein aryl and 5-7-membered heteroaryl, in each instance in which they occur, are independently unsubstituted or mono-, di- tri- or tetra- substituted with halogen, said heteroaryl having 1, 2, 3, or 4 nitrogen atoms.

In one embodiment of the invention, R is -CH2NH2.

In another embodiment of the invention, R is CH3 or CH2CH3.

2

In another embodiment of the invention, R is NI¾, OH, NHSO2CH3, or NHC(0)CH₃.

In another embodiment of the invention, R is -C(CH3)3, C6Hn, '

In another embodiment of the invention, the compound

(S)-l-((R)-2-amino-3,3-dimethylbutanoyl)-N-(2-(aminomethyl)-5-chlorobenzyl)- 2-methylpyrrolidine-2-carboxamide,

(S)-N-(2-(aminomethyl)-5-chlorobenzyl)-l-((R)-2-hydroxy-3,3-dimethylbutanoyl)- 2-methylpyrrolidine-2-carboxamide,

(S)-N-(2-(aminomethyl)-5-chlorobenzyl)-l-((R)-2-cyclohexyl-2-hydroxyacetyl)- 2-methylpyrrolidine-2-carboxamide,

(S)-N-(2-(aminomethyl)-5-chlorobenzyl)-l-((R)-2-(3-chlorophenyl)-2- hydroxyacetyl)-2-methylpyrrolidine-2-carboxamide,

(S)-N-(2-(aminomethyl)-5-chlorobenzyl)-l-((R)-3,3-dimethyl-2- (methylsulfonamido)butanoyl)-2-methylpyrrolidine-2-carboxamide,

N-(2-(aminomethyl)-5-chlorobenzyl)-l-((R)-2-cyclohexyl-2-hydroxyacetyl)-2- ethylpyrrolidine-2-carboxamide,

(S)-l-((R)-2-acetamido-3,3-dimethylbutanoyl)-N-(2-(aminomethyl)-5- chlorobenzyl)-2-methylpyrrolidine-2-carboxamide, (S)-l-((R)-2-amino-3-(4-fluorophenyl)propanoyl)-N-(2-(aminomethyl)-5- chlorobenzyl)-2-methylpyrrolidine-2-carboxamide,

(S)-l-((R)-2-amino-2-cyclohexylacetyl)-N-(2-(aminomethyl)-5- chlorobenzyl)-2-methylpyrrolidine-2-carboxamide,

(S)-l-((R)-2-amino-3-(pyridin-2-yl)propanoyl)-N-(2-(aminomethyl)-5- chlorobenzyl)-2-methylpyrrolidine-2-carboxamide,

(S)-N-(2-(aminomethyl)-5-chlorobenzyl)-l-((S)-2-(3-chlorophenyl)-2- hydroxyacetyl)-2-methylpyrrolidine-2-carboxamide,

(2S)-l-(2-amino-2-(3-chlorophenyl)acetyl)-N-(2-(aminomethyl)-5-chlorobenzyl)-2- methylpyrrolidine-2-carboxamide,

or a pharmaceutically acceptable salt thereof.

Table 1 shows structures and names of compounds of the invention:

Table 1

The present invention encompasses all stereoisomeric forms of the compounds of Formula I. Centers of asymmetry that are present in the compounds of Formula I can all independently of one another have (R) configuration or (S) configuration. When bonds to the chiral carbon are depicted as straight lines in the structural Formulas of the invention, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the Formula. Similarly, when a compound name is recited without a chiral designation for a chiral carbon, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence individual enantiomers and mixtures thereof, are embraced by the name. The production of specific stereoisomers or mixtures thereof may be identified in the Examples where such stereoisomers or mixtures were obtained, but this in no way limits the inclusion of all stereoisomers and mixtures thereof from being within the scope of this invention.

The invention includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, for example mixtures of enantiomers and/or diastereomers, in all ratios. Thus, enantiomers are a subject of the invention in enantiomerically pure form, both as levorotatory and as dextrorotatory antipodes, in the form of racemates and in the form of mixtures of the two enantiomers in all ratios. In the case of a cis/trans isomerism the invention includes both the cis form and the trans form as well as mixtures of these forms in all ratios. The preparation of individual stereoisomers can be carried out, if desired, by separation of a mixture by customary methods, for example by chromatography or crystallization, by the use of stereochemically uniform starting materials for the synthesis or by stereoselective synthesis. Optionally a derivatization can be carried out before a separation of stereoisomers. The separation of a mixture of stereoisomers can be carried out at an intermediate step during the synthesis of a compound of Formula I or it can be done on a final racemic product. Absolute stereochemistry may be determined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing a stereogenic center of known configuration. Where compounds of this invention are capable of tautomerization, all individual tautomers as well as mixtures thereof are included in the scope of this invention. The present invention includes all such isomers, as well as salts, solvates (including hydrates) and solvated salts of such racemates, enantiomers, diastereomers and tautomers and mixtures thereof.

Furthermore, compounds of the present invention may exist in amorphous form and/or one or more crystalline forms, and as such all amorphous and crystalline forms and mixtures thereof of the compounds of Formula I are intended to be included within the scope of the present invention. In addition, some of the compounds of the instant invention may form solvates with water (i.e., a hydrate) or common organic solvents. Such solvates and hydrates, particularly the

pharmaceutically acceptable solvates and hydrates, of the instant compounds are likewise encompassed within the scope of this invention, along with un-solvated and anhydrous forms.

Reference to the compounds of this invention as those of a specific formula or embodiment, e.g., Formula I or any other generic structural formula or specific compound described or claimed herein, is intended to encompass the specific compound or compounds falling within the scope of the formula or embodiment, including salts thereof, particularly pharmaceutically acceptable salts, solvates of such compounds and solvated salt forms thereof, where such forms are possible unless specified otherwise.

In the compounds of Formula I, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present invention is meant to include all suitable isotopic variations of the compounds of Formula I. For example, different isotopic forms of hydrogen (H) include protium (iH) and deuterium (¾I). Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples. Isotopically-enriched compounds within Formula I can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Schemes and Examples herein using appropriate isotopically-enriched reagents and/or intermediates.

It will be understood that, as used herein, references to the compounds of structural Formula I are meant to also include the pharmaceutically acceptable salts, and also salts that are not pharmaceutically acceptable when they are used as precursors to the free compounds or their pharmaceutically acceptable salts or in other synthetic manipulations.

The compounds of the present invention may be administered in the form of a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts of basic compounds encompassed within the term "pharmaceutically acceptable salt" refer to non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid. Representative salts of basic compounds of the present invention include, but are not limited to, the following: acetate, ascorbate, adipate, alginate, aspirate,

benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, clavulanate, citrate, cyclopentane propionate, diethylacetic, digluconate, dihydrochloride, dodecylsulfanate, edetate, edisylate, estolate, esylate, ethanesulfonate, formic, fumarate, gluceptate, glucoheptanoate, gluconate, glutamate, glycerophosphate, glycollylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate,

hydrabamine, hydrobromide, hydrochloride, 2-hydroxyethanesulfonate,

hydroxynaphthoate, iodide, isonicotinic, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, methanesulfonate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N- methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, phosphate/diphosphate, pimelic, phenylpropionic, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, thiocyanate, tosylate, triethiodide, trifluoroacetate, undeconate, valerate and the like. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof include, but are not limited to, salts derived from inorganic bases including aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, mangamous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, cyclic amines, dicyclohexyl amines and basic ion- exchange resins, such as arginine, betaine, caffeine, choline, N,N- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, ethylamine, ethylenediamine, N- ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. Also, included are the basic nitrogen- containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, 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.

Also, in the case of a carboxylic acid (-COOH) or alcohol group being present in the compounds of the present invention, pharmaceutically acceptable esters of carboxylic acid derivatives, such as methyl, ethyl, or pivaloyloxymethyl, or acyl derivatives of alcohols, such as O-acetyl, O-pivaloyl, O-benzoyl, and O-aminoacyl, can be employed. Included are those esters and acyl groups known in the art for modifying the solubility or hydrolysis characteristics for use as sustained-release or prodrug formulations.

If the compounds of Formula I simultaneously contain acidic and basic groups in the molecule the invention also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions). Salts can be obtained from the compounds of Formula I by customary methods which are known to the person skilled in the art, for example by combination with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange from other salts. The present invention also includes all salts of the compounds of Formula I which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of physiologically acceptable salts.

Any pharmaceutically acceptable pro-drug modification of a compound of this invention which results in conversion in vivo to a compound within the scope of this invention is also within the scope of this invention. For example, esters can optionally be made by esterification of an available carboxylic acid group or by formation of an ester on an available hydroxy group in a compound. Similarly, labile amides can be made. Pharmaceutically acceptable esters or amides of the compounds of this invention may be prepared to act as pro-drugs which can be hydrolyzed back to an acid (or -COO- depending on the pH of the fluid or tissue where conversion takes place) or hydroxy form particularly in vivo and as such are encompassed within the scope of this invention. Examples of pharmaceutically acceptable pro-drug modifications include, but are not limited to, -C_{1 6}alkyl esters and -C_{1 6}alkyl substituted with phenyl esters.

Accordingly, the compounds within the generic structural formulas, embodiments and specific compounds described and claimed herein encompass salts, all possible stereoisomers and tautomers, physical forms (e.g., amorphous and crystalline forms), solvate and hydrate forms thereof and any combination of these forms, as well as the salts thereof, pro-drug forms thereof, and salts of pro-drug forms thereof, where such forms are possible unless specified otherwise.

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.

Some abbreviations that may appear in this application are as follows:

ABBREVIATIONS

ACN acetonitrile

Boc t-Butyloxycarbonyl

DBU 1.8-diazabicyclo[5.4.0]undec-7-ene

DIPEA N,N-Diisopropylethylamine (Hiinig's base)

DMF dimethylformamide

DPP A diphenyl phosphorazidate

EDC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide

EtOAc ethyl acetate

Fmoc 9-fluorenylmethoxycarbonyl

HATU 0-(7-Azabenzotriazole- 1 -yl)- 1 , 1 ,3,3-tetramethyluronium

hexafluoropho sphate

Hex hexane HOBt 1-hydroxybenzotriazole

HPLC high pressure liquid chromatography

LAH lithium aluminum hydroxide

LC/MS liquid chromatography/mass spectrometry

MeOH methanol

OAc acetoxy group

PR Pro-Arg

PEG polyethylene glycol

pna p-nitroanilide

PyBOP (Benzotriazol-l-yloxy)tripyrrolidinophosphonium

hexafluoropho sphate

RT room temperature

sar sarcosine

THF tertahydrofuran

TFA trifluoroacetate

TRIS tris (hydroxymethyl)aminomethane

Z-GPR-afc Z-Gly-Pro-Arg-7-amino-4-trifluoromethylcoumarin

Except where noted, the term "alkyl" refers to 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), unsubstituted or substituted with Cl-4 alkyl or halogen.

Except where noted, the term "halogen" means fluorine, chlorine, bromine or iodine.

Except where noted, the term "C3-8 cycloalkyl" refers to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, and the like, unsubstituted or substituted with Cl-4 alkyl or halogen.

Except where noted, the term "carbocycle" (and variations thereof such as "carbocyclic" or "carbocyclyl") as used herein, unless otherwise indicated, refers to a C₃ to C_g monocyclic saturated ring. Saturated carbocyclic rings are also referred to as cycloalkyl rings, e.g., cyclopropyl, cyclobutyl, etc. Except where noted, the term "aryl" refers to a stable 6- to 10- membered mono- or bicyclic ring system such as phenyl, or naphthyl. The aryl ring can be unsubstituted or substituted with one or more of Cl-4 alkyl, hydroxyl, alkoxy, halogen, or amino.

Except where noted, the term "heterocycle" or "heterocyclic ring refers to a stable 5- to 7-membered mono- or bicyclic or stable 7- to 10-membered bicyclic heterocyclic ring system unsubstituted or substituted with Cl-4 alkyl or halogen, any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to four 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 four 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, pyridyl N-oxide, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, thiadiazoyl, benzopyranyl, benzothiazolyl, benzoxazolyl, furyl, tetrahydrofuryl, tetrahydropyranyl, tetrazole, thienyl, benzothienyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, and oxadiazolyl. Morpholino is the same as morpholinyl.

In this specification methyl substituents may be represented by

. For example, the structures

and

have equivalent meanings. Thrombin Inhibitors - Therapeutic Uses- Method of Using

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, the 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 prosthesis, cardiac prosthesis, and extracorporeal circulation systems.

Compounds of the invention may be useful for treating or preventing venous thromboembolism (e.g. obstruction or occlusion of a vein by a detached thrombus; obstruction or occlusion of a lung artery by a detached thrombus), cardiogenic thromboembolism (e.g. obstruction or occlusion of the heart by a detached thrombus), arterial thrombosis (e.g. formation of a thrombus within an artery that may cause infarction of tissue supplied by the artery), atherosclerosis (e.g.

arteriosclerosis characterized by irregularly distributed lipid deposits) in mammals, and for lowering the propensity of devices that come into contact with blood to clot blood.

Examples of venous thromboembolism which may be treated or prevented with compounds of the invention include obstruction of a vein, obstruction of a lung artery (pulmonary embolism), deep vein thrombosis, thrombosis associated with cancer and cancer chemotherapy, thrombosis inherited with thrombophilic diseases such as Protein C deficiency, Protein S deficiency, antithrombin ΙΠ deficiency, and Factor V Leiden, and thrombosis resulting from acquired

thrombophilic disorders such as systemic lupus erythematosus (inflammatory connective tissue disease). Also with regard to venous thromboembolism, compounds of the invention may be useful for maintaining patency of indwelling catheters.

Examples of cardiogenic thromboembolism which may be treated or prevented with compounds of the invention include thromboembolic stroke (detached thrombus causing neurological affliction related to impaired cerebral blood supply), cardiogenic thromboembolism associated with atrial fibrillation (rapid, irregular twitching of upper heart chamber muscular fibrils), cardiogenic thromboembolism associated with prosthetic heart valves such as mechanical heart valves, and cardiogenic thromboembolism associated with heart disease.

Examples of arterial thrombosis include unstable angina (severe constrictive pain in chest of coronary origin), myocardial infarction (heart muscle cell death resulting from insufficient blood supply), ischemic heart disease (local anemia due to obstruction (such as by arterial narrowing) of blood supply), reocclusion during or after percutaneous transluminal coronary angioplasty, restenosis after percutaneous transluminal coronary angioplasty, occlusion of coronary artery bypass grafts, and occlusive cerebrovascular disease. Also with regard to arterial thrombosis, compounds of the invention may be useful for maintaining patency in arteriovenous cannulas.

Examples of atherosclerosis include arteriosclerosis.

Examples of devices that come into contact with blood include vascular grafts, stents, orthopedic prosthesis, 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 nontoxic amount of the compound desired may be employed as an anti-aggregation agent. For treating ocular build up of fibrin, the compounds may be administered

intraocularly 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, 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 hydro gels.

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 0.01 mg per kg of body weight per day (mg/kg/day) to about 30 mg/kg/day, preferably 0.025-7.5 mg/kg/day, more preferably 0.1-2.5 mg/kg/day, and most preferably 0.1-0.5 mg/kg/day (unless specificed otherwise, amounts of active ingredients are on free base basis). For example, an 80 kg patient would receive between about 0.8 mg/day and 2.4 g/day, preferably 2-600 mg/day, more preferably 8-200 mg/day, and most preferably 8-40 mg/kg/day. A suitably prepared medicament for once a day administration would thus contain between 0.8 mg and 2.4 g, preferably between 2 mg and 600 mg, more preferably between 8 mg and 200 mg, and most preferably 8 mg and 40 mg, e.g., 8 mg, 10 mg, 20 mg and 40 mg. Advantageously, the thrombin inhibitors may be administered in divided doses of two, three, or four times daily. For administration twice a day, a suitably prepared medicament would contain between 0.4 mg and 4 g, preferably between 1 mg and 300 mg, more preferably between 4 mg and 100 mg, and most preferably 4 mg and 20 mg, e.g., 4 mg, 5 mg, 10 mg and 20 mg.

Intravenously, the patient would receive the active ingredient in quantities sufficient to deliver between 0.025-7.5 mg/kg/day, preferably 0.1-2.5 mg/kg/day, and more preferably 0.1-0.5 mg/kg/day. Such quantities may be administered in a number of suitable ways, e.g. large volumes of low concentrations of active ingredient during one extended period of time or several times a day, low volumes of high concentrations of active ingredient during a short period of time, e.g. once a day. Typically, a conventional intravenous formulation may be prepared which contains a concentration of active ingredient of between about 0.01-1.0 mg/ml, e.g. 0.1 mg/ml, 0.3 mg/ml, and 0.6 mg/ml, and administered in amounts per day of between 0.01 ml/kg patient weight and 10.0 ml/kg patient weight, e.g. 0.1 ml/kg, 0.2 ml/kg, 0.5 ml/kg. In one example, an 80 kg patient, receiving 8 ml twice a day of an intravenous formulation having a concentration of active ingredient of 0.5 mg/ml, receives 8 mg of active ingredient per day. Glucuronic acid, L-lactic acid, acetic acid, citric acid or any pharmaceutically acceptable acid/conjugate base with reasonable buffering capacity in the pH range acceptable for intravenous administration may be used as buffers. The choice of appropriate buffer and pH of a formulation, depending on solubility of the drug to be administered, is readily made by a person having ordinary skill in the art.

The compounds can also 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.

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, elixers, 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, distintegrating 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, carboxymethylcellulose, 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 anticoagulants, including, but not limited to, other thrombin inhibitors, thrombin receptor antagonists, factor Vila inhibitors, factor IXa inhibitors, factor Xa inhibitors, factor XIa inhibitors, adenosine diphosphate antiplatelet agents (e.g., P2Y12 antagonists), fibrinogen receptor antagonists (e.g. to treat or prevent unstable angina or to prevent reocclusion after angioplasty and restenosis), other anticoagulants such as aspirin, and thrombolytic agents such as plasminogen activators or streptokinase to achieve synergistic effects in the treatment of various vascular pathologies. Such anticoagulants include, for example, apixaban, dabigatran, cangrelor, ticagrelor, vorapaxar, clopidogrel, edoxaban, mipomersen, prasugrel, rivaroxaban, and semuloparin. For example, patients suffering from coronary artery disease, and patients subjected to angioplasty procedures, would benefit from coadministration of fibrinogen receptor antagonists and thrombin inhibitors. Also, 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.

Alternatively or additionally, one or more additional pharmacologically active agents may be administered in combination with a compound of Formula I. The additional active agent (or agents) is intended to mean a pharmaceutically active agent (or agents) that is active in the body, including pro-drugs that convert to pharmaceutically active form after administration, which is different from the compound of Formula I, and also includes free-acid, free-base and pharmaceutically acceptable salts of said additional active agents when such forms are sold

commercially or are otherwise chemically possible. Generally, any suitable additional active agent or agents, including but not limited to anti-hypertensive agents, additional diuretics, anti-atherosclerotic agents such as a lipid modifying compound, anti- diabetic agents and/or anti-obesity agents may be used in any combination with the compound of Formula I in a single dosage formulation (a fixed dose drug

combination), or may be administered to the patient in one or more separate dosage formulations which allows for concurrent or sequential administration of the active agents (co-administration of the separate active agents). Examples of additional active agents which may be employed include but are not limited to angiotensin converting enzyme inhibitors (e.g, alacepril, benazepril, captopril, ceronapril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, imidapril, lisinopril, moveltipril, perindopril, quinapril, ramipril, spirapril, temocapril, or trandolapril); angiotensin Π receptor antagonists also known as angiotensin receptor blockers or ARBs (e.g., losartan i.e., COZAAR®, valsartan, candesartan, olmesartan, telmesartan, eprosartan, irbesartan and any of these drugs used in combination with hydrochlorothiazide such as HYZAAR®); diuretics, e.g. hydrochlorothiazide (HCTZ); potassium sparing diuretics such as amiloride HCl, spironolactone, epleranone, triamterene, each with or without HCTZ; neutral endopeptidase inhibitors (e.g., thiorphan and

phosphoramidon); aldosterone antagonists; aldosterone synthase inhibitors; renin inhibitors; enalkrein; RO 42-5892; A 65317; CP 80794; ES 1005; ES 8891; SQ 34017; aliskiren (2(S),4(S),5(S),7(S)-N-(2-carbamoyl-2-methylpropyl)-5-amino-4- hydroxy-2,7-diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy)-phenyl]-octanamid hemifumarate) SPP600, SPP630 and SPP635); endothelin receptor antagonists;

vasodilators (e.g. nitroprusside); calcium channel blockers (e.g., amlodipine, nifedipine, verapamil, diltiazem, , felodipine, gallopamil, niludipine, nimodipine, nicardipine); potassium channel activators (e.g., nicorandil, pinacidil, cromakalim, minoxidil, aprilkalim, loprazolam); sympatholitics; beta-adrenergic blocking drugs (e.g., acebutolol, atenolol, betaxolol, bisoprolol, carvedilol, metoprolol, metoprolol tartate, nadolol, propranolol, sotalol, timolol); alpha adrenergic blocking drugs (e.g., doxazocin, prazocin or alpha methyldopa); central alpha adrenergic agonists;

peripheral vasodilators (e.g. hydralazine); lipid lowering agents, e.g., HMG-CoA reductase inhibitors such as simvastatin and lovastatin which are marketed as

ZOCOR® and MEVACOR® in lactone pro-drug form and function as inhibitors after administration, and pharmaceutically acceptable salts of dihydroxy open ring acid HMG-CoA reductase inhibitors such as atorvastatin (particularly the calcium salt sold in LIPITOR®), rosuvastatin (particularly the calcium salt sold in CRESTOR®), pravastatin (particularly the sodium salt sold in PRAVACHOL®), and fluvastatin (particularly the sodium salt sold in LESCOL®); a cholesterol absorption inhibitor such as ezetimibe (ZETIA®), and ezetimibe in combination with any other lipid lowering agents such as the HMG-CoA reductase inhibitors noted above and particularly with simvastatin (VYTORIN®) or with atorvastatin calcium; niacin in immediate-release or controlled release forms, and particularly niacin in combination with a DP antagonist such as laropiprant (TREDAPTrVE®) and/or with an HMG- CoA reductase inhibitor; niacin in immediate-release or controlled release forms, and particularly niacin in combination with a DP antagonist such as laropiprant

(TREDAPTIVE®) and/or with an HMG-CoA reductase inhibitor; niacin receptor agonists such as acipimox and acifran, as well as niacin receptor partial agonists; metabolic altering agents including insulin sensitizing agents and related compounds for the treatment of diabetes such as biguanides (e.g., metformin), meglitinides (e.g., repaglinide, nateglinide), sulfonylureas (e.g., chlorpropamide, glimepiride, glipizide, glyburide, tolazamide, tolbutamide), thiazolidinediones also referred to as glitazones (e.g., pioglitazone, rosiglitazone), alpha glucosidase inhibitors (e.g., acarbose, miglitol), dipeptidyl peptidase inhibitors, (e.g., sitagliptin (JANUVIA®), alogliptin, vildagliptin, saxagliptin, linagliptin, dutogliptin, gemigliptin), ergot alkaloids (e.g., bromocriptine), combination medications such as JANUMET® (sitagliptin with metformin), and injectable diabetes medications such as exenatide and pramlintide acetate; or with other drugs beneficial for the prevention or the treatment of the above- mentioned diseases including but not limited to diazoxide; and including the free-acid, free-base, and pharmaceutically acceptable salt forms of the above active agents where chemically possible.

Typical doses of thrombin inhibitors of the invention in combination with other suitable anti-platelet agents, anticoagulation agents, or thrombolytic agents may be the same as those doses of thrombin inhibitors administered without coadministration of additional anti-platelet agents, anticoagulation agents, or thrombolytic agents, or may be substantially less that those doses of thrombin inhibitors administered without coadministration of additional anti-platelet agents, anticoagulation agents, or thrombolytic agents, depending on a patient's therapeutic needs.

General Procedure

Compounds of the present invention may be prepared according to the methodology outlined in the following general synthetic scheme.

Intermediate 2, prepared from either optically pure 2-alkyl proline or racemic 2-alkyl proline (1) via protection of the secondary amino group under standard conditions, is coupled to intermediate 3 in the presence of a peptide coupling reagent such as EDC in a solvent such as DMF to form intermediate 4. After Fmoc group is removed with a base such as piperidine, intermediate 5 is subject to amide coupling with an carboxylic acid reagent 6 using an amide coupling reagent such as HATU in a solvent such as DMF. For some compounds in which R is amino group, it requires protection of the amino group in reagent 6 with a carbamate such as Boc, and deprotection with an acid such as TFA to get to the final compound.

Scheme 1

Preparation of iert-Butyl 2-(aminomethyl)-4-chlorobenzylcarbamate (Intermediate 3a, where R is -CH₂NHBoc)

Intermediate 3a

Step A: Preparation of 2-bromo-5-chlorobenzoate

Through a solution of 2-bromo-5-chlorobenzoic acid (l lg, 46.7 mmol) in methanol (250ml) was bubbled HCl gas. The reaction was allowed to warm to room temperature and stirred overnight. The reaction mixture is concentrated in vacuo to give an orange oil, which is purified by flash chromatography (silica gel, hexane) to give the title compound as a colorless oil.

1H NMR (CDCI3, 400MHz): δ 7.78 (d, 1 H, J= 2.6 Hz); 7.59 (d, 1H, J= 12.81 Hz);7.30 (dd, 1 H, J= 8.6, 2.5 Hz); 3.94 (s, 3H) Step B: Preparation of Methyl 5-chloro-2-cyanobenzoate

To a solution of methyl 2-bromo-5-chlorobenzoate (1.15g, 4.6 mmol) in degassed DMF was added zinc cyanide (282 mg, 2.40 mmol) and palladium tetrakis triphenylphosphine (lOOmg, 0.086 mmol) and the reaction is stirred at 90°C over night. The reaction was partitioned between ethyl acetate and water. The organic was concentrated in vacuo and purified by flash chromatography eluting a gradient to 10 to 25% ethyl acetate in hexane yielding a white solid (methyl 5-chloro- 2-cyanobenzoate.

H NMR (CDCI₃, 400 MHz): δ 8.13 (d, 1 H, J= 1.83 Hz); 3.09 (d, 1 H, J= 8.24 Hz); 7.29 (dd, 1 H, J= 8.34, 2.10 Hz); 4.02 (s, 3 H)

Step C: Preparation of 2-(aminomethyl)-5-chlorophenyl]methanol

To LAH (1 M/Et₂0, 104.4 ml, 104.4 mmol) in anhydrous THF (300 ml) at 0C was added methyl 5-chloro-2-cyanobenzoate (9.28g, 0.512 mmol) maintaining the temperature below 20 °C. After one half hour, quenched at 0°C with water (3.97 ml), NaOH (IN, 11.9 ml, 11.9 mmol) and water (3.97 ml). A precipitate was filtered out and washed with THF. The filtrate was concentrated in vacuo and was used immediately in the next step.

H NMR (CDCI₃, 400 MHz): δ 7.17-7.36 (m, 3 H); 4.60 (s, 2 H); 3.98 (s, 2 H);

Step D: Preparation of iert-butyl 4-chloro-2-(hydroxymethyl)benzylcarbamate

To a solution of [2-(aminomethyl)-5-chlorophenyl]methanol in dichloromethane (200ml), was added di-tert-butyl-dicarbonate (11.38 g, 52.18 mmol) at room temperature. After one hour, the reaction was partitioned. The organic layer was concentrated in vacuo and purified by flash chromatography eluting a gradient of ethyl acetate/hexane which gave a brown oil, which was taken up in dichloromethane (500 ml) and treated with activated charcoal yielding a pink solid.

H NMR (CDCI₃, 400 MHz): δ 7.36 (s, 1 H); 7.2-7.5 (m, 2 H); 4.69 (b s, 2 H); 4.32 (d, 2 H, J= 6.04 Hz); 1.43 (s, 9 H). Step E: Preparation of ie/t-Butyl 2-(azidomethyl)-4-chlorobenzylcarbamate

To a solution of iert-butyl 4-chloro-2-(hydroxymethyl)benzylcarbamate (10 g, 36.8 mmol) in anhydrous THF (100 ml) was added DPPA (8.3 ml, 38.6 mmol) and DBU (5.79 ml, 38.6 mmol). The mixture was stirred overnight and then was partitioned between ethyl acetate and water. The organic layer was washed with brine, and was concentrated in vacuo to a crude oil (14.6 g). Purification was accomplished by silica gel chromatography, eluting a gradient of ethyl acetate-hexane (10, 15, 20, 25, 50%) to give iert-butyl 2-(aminomethyl)-4-chlorobenzylcarbamate. H NMR (CDC1₃, 400 MHz): δ 7.25-7.39 (m, 3 H); 4.41 (s, 2 H), 4.32 (d, 2 H, J= 5.86 Hz); 1.45 (s, 9 H).

Step F: Preparation of ie/t-Butyl 2-(aminomethyl)-4-chlorobenzylcarbamate

To a solution of iert-butyl 2-(azidomethyl)-4-chlorobenzylcarbamate

(10.9 g, 36.73 mmol) in THF (60 ml) and water (6 ml) was added triphenylpho spine (10.59 g, 40.40 mmol). The reaction was heated to 65 °C and stirred overnight at room temperature. The reaction was concentrated in vacuo and flashed with 4%

(10%NH₄OH/MeOH)/ dichlor-omethane. A second purification using silica gel column chromatography with a careful gradient of 3 to 5% (10%NH₄OH /MeOH)/ dichloro methane gave the title compound.

H NMR (CDCI₃, 400 MHz) δ 7.21-7.52 (m, 3 H); 4.32 (b d, 2 H); 3.90 (s, 2 H); 1.44

(s, 9 H).

Separation of diastereomers can be carried out at various stages in the preparation of the desired final compounds; however, it is typically carried out on intermediate 7 before removal of the protective group using reversed phase HPLC or supercritical fluid chromatography. Separation of enantiomeric pairs is achieved by supercritical fluid chromatography using various chiral columns. The absolute configuration is not determined.

Analytical HPLC mass spectrometry conditions:

Column: Waters Xterra MS C-18, 3.5 μιη, 3.0 x 50 mm

Temperature: 50 °C

Eluent: 10:90 to 98:2 v/v acetonitrile/water + 0.05% TFA over 3.75 min. Flow Rate: 1.0 mL/min, Injection 10 L

Detection: PDA, 200-600 nm

MS: mass range 150-750 amu; positive ion electrospray ionization

Preparative reverse phase HPLC (RP-HPLC) conditions:

Column: Xterra MS, 5μΜ, 30 x 100 mm

Flow Rate: 40.0 mL/min

Eluent: acetonitrile/water + 0.1% TFA

Gradient: 10 to 100 v/v acetonitrile/water + 0.1% TFA over 20.0min.

Temperature: ambient

Detection: PDA, 254nm

Preparative thin layer chromatography (PTLC) was performed on 20 20cm plates (500 μπι - 1500 μπι thick silica gel) using hexanes/ethyl acetate as eluent. Silica gel chromatography was conducted on a Biotage SP-1 or Isco flash chromatography system using a hexanes/ethyl acetate or DCM/hexanes gradient.

The following examples are provided so that the invention might be more fully

understood. They should not be construed as limiting the invention in any way.

EXAMPLE 1

(_lS')-l-((R)-2-amino-3,3-dimethylbutanoyl)-N-(2-(aminomethyl)-5-chlorobenzyl)-2- methylpyrrolidine-2-carboxamide (1-5)

Step A: (S)- l-(((9H-fluoren-9-yl)methoxy)carbonyl)-2-methylpyrrolidine-2- carboxylic acid (1-1)

At 0 °C, 9-fluorenylmethyl chloroformate (2.10 g, 8.13 mmol) was added portion wise to a solution of alpha- methyl-L-proline (1 g, 7.74 mmol) and potassium carbonate (2.68 g, 19.36 mmol) in water (20 ml) and 1,4-dioxane (6 ml). After stirring at room temperature overnight, it was extracted with Et₂0 (20 mL X 2), and the aqueous phase was acidified with aqueous IN HC1 and extracted with CH₂C1₂ (40 mL X 3). The combined organic layer was dried over MgS0₄, filtered and concentrated to afford IA as a colorless oil. LCMS: m/z 352.09, retention time 2.02 min. 1H NMR (CDC1₃, 500 MHz) ppm 1.60 (s, 3H), 1.88 (m, 4H), 3.50-3.59 (m, 2H), 4.24 (t, J = 7.2 Hz, 1H), 4.36 (m, 1H), 4.44 (m, 1H), 7.30 (m, 2H), 7.39 (m, 2H), 7.57 (m, 2H), 7.75 (d, J = 1.6 Hz, 2H).

Step B: (£)-(9H-fruoren-9-yl)methyl 2-((5-(((fer?-butoxycarbonyl)amino)methyl)-2- chlorobenzyl)carbamoyl)-2-methylpyrrolidine- 1 -carboxylate ( 1 -2)

(_lS')-l-(((9H-fluoren-9-yl)methoxy)carbonyl)-2-methylpyrrolidine-2- carboxylic acid (2.56 g, 6.70 mmol) was dissolved in a mixture of CH₂C1₂ (10.8 mL) and DMF (0.38 mL). 7¾rt-butyl 2-(aminomethyl)-4-chlorobenzylcarbamate (1.65 g, 6.09 mmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiidide hydrochloride (1.75 g, 9.14 mmol), and HOBt (0.82 g, 6.09 mmol) were added to the above reaction mixture. After stirring at ambient temperature overnight, volatiles were removed and the residue was purified with silica gel chromatography, eluting with 0-70% EtOAc/hex. to afford 1^2 as light yellow foam. LCMS: m/z 604.42, retention time 2.46 min. 1H NMR (CDC1₃, 500 MHz) ppm 1.41 (s, 9H), 1.64 (s, 2H), 1.76-1.83 (m, 3H), 2.01 (s, 2H), 2.59 (s, 1H), 3.48-3.54 (m, 2H), 4.20-4.27 (m, 3H), 4.36-4.47 (m, 3H), 5.30 (s, 2H), 7.17 (m, 1H), 7.23 (m, 2H), 7.31 (m, 2H), 7.39 (q, J = 7.7 Hz, 2H), 7.57 (m, 2H), 7.76 (d, J = 7.58 Hz, 2H). Step C. (S)-tert-butyl 4-chloro-2-((2-methylpyrrolidine-2-carboxamido)methyl) Benzylcarbamate (1-3)

To a solution of (S)-(9H-fluoren-9-yl)methyl 2-((5-(((fert- butoxycarbonyl)amino)methyl)-2-chlorobenzyl)carbamoyl)-2-methylpyrrolidine- l- carboxylate (11.02 g, 18.24 mmol) in DMF (73.0 ml) was addedpiperidine (18.06 ml, 182 mmol) and the mixture was stirred at r.t.for 40 min. Volatiles were removed under reduced pressure, and the residue was purified with silica gel chromatography, eluting with 0-50% EtOAc/hexanes, then 0-6% MeOH/CH₂Cl₂ to afford L3 as light brown oil. LCMS: m/z 382.20, retention time 1.55 min. 1H NMR (CDC1₃, 500 MHz) < ppm 1.42 (s, 3H), 1.43 (s, 9H), 1.62- 1.76 (m, 3H), 2.26 (m, 1H), 2.83 (m, 1H), 3.08 (m, 1H), 4.28 (d, J = 6.00 Hz, 2H), 4.41 (m, 2H), 5.26 (s, 1H), 7.22 (m, 2H), 7.27- 7.29 (m, 1H), 8.32 (s, 1H).

Step D. N, N-Diboc-(_tS' - l-(^'(R -2-amino-3,3-dimethylbutanoyl -N-(^'2-(^'aminomethyl -5- chlorobenzyl)-2-methylpyrrolidine-2-carboxamide ( 1 -4)

To a solution of 1^3 (6.61 g, 17.32 mmol) in a mixture of anhydrous CH₂C1₂ (22.48 ml) and DMF (0.926 ml), were added Boc-D-cc-i-butylglycine (6.01 g, 26.0 mmol), HATU (9.88 g, 26.0 mmol), and triethylamine (4.83 ml, 34.6 mmol). After stirring at r.t. over night, the reaction mixture was partitioned between saturated aqueous NaHC0₃ (100 mL) and CH₂CI₂ (200 mL). The aqueous layer was extracted with CH₂CI₂ (2 X 200 mL). The combined organic layer was dried over MgS0₄, filtered and concentrated. The residue was purified with silica gel chromatography, eluting with 0-70% EtOAc/hex. to afford the desired product as light brown oil.

LCMS: m/z 595.39, retention time 1.28 min. 1H NMR (CDC1₃, 500 MHz) ppm 1.03 (s, 9H), 1.32 (s, 9H), 1.43 (s, 9H), 1.64 (s, 3H), 1.90-2.04 (m, 2H), 2.21 (m, 1H), 3.73- 3.80 (m, 1H), 4.10 (m, 1H), 4.16 (d, J = 6.32 Hz, 1H), 4.23 (m, 2H), 4.32-4.46 (m, 2H), 6.48 (s, 1H), 7.05 (s, 1H), 7.17-7.26 (m, 2H), 7.39 (s, 1H), 7.92 (s, 1H). Step E. (_tS')-l-((R)-2-amino-3,3-dimethylbutanoyl)-N-(^'2-(^'aminomethyl)-5- chlorobenzyl)-2-methylpyrrolidine-2-carboxamide (1-5)

1-4 (4 g, 6.72 mmol) was dissolved in a mixture of trifluoroacetic acid (6.72 ml, 87 mmol) and CH₂CI₂ (13.44 ml), and the mixture was stirred at r.t. over night. Volatiles were removed under reduced pressure and the residue was purified with reversed-phase HPLC, eluting with 10-70% ACN/H₂0 with 0.1% TFA.

Approriate fractions were lyophilized to afford the desired product (5)- 1-((Λ)-2- amino-3,3-dimethylbutanoyl)-N-(2-(aminomethyl)-5-chlorobenzyl)-2- methylpyrrolidine-2-carboxamide. LCMS: m/z 395.15, retention time 1.21 min. 1H NMR (CD₃OD, 500 MHz) ppm 1.11 (s, 9H), 1.64 (s, 3H), 1.93-2.04 (m, 3H), 2.09- 2.15 (m, 1H), 3.69-3.76 (m, 1H), 3.86-3.92 (m, 1H), 4.03 (s, 1H), 4.26 (m, 2H), 4.36 (d, J = 15.25 Hz, 1H), 4.47 (d, J = 15.24 Hz, 1H), 7.33-7.40 (2 H, m), 7.52 (d, J = 2.07 Hz, 1H).

EXAMPLE 2 (_lS')-N-(2-(aminomethyl)-5-chlorobenzyl)- l-((R)-2-hydroxy-3,3-dimethylbutanoyl)-2- methylpyrrolidine-2-carboxamide (2-2)

Step A. Tert-butyl 4-chloro-2-((^'(^'S)-l-(^'(^'R)-2-hvdroxy-3,3-dimethylbutanoyl)-2- methylpyrrolidine-2-carboxamido)methyl)benzylcarbamate (2- 1 )

(S)-tert-b ty\ 4-chloro-2-((2-methylpyrrolidine-2- carboxamido)methyl)benzylcarbamate (65 mg, 0.170 mmol) was dissolved in anhydrous DMF (340 μΐ), then (R)-2-hydroxy-3,3-dimethylbutanoic acid (45.0 mg, 0.340 mmol), HATU (129 mg, 0.340 mmol) and DIPEA (1 19 μΐ, 0.681 mmol) were added to the above mixture. It was stirred at r.t. for 2h. The reaction mixture was purified with preparative HPLC, 10- 100% ACN/H₂0 with 0.1% TFA. Lyophilized to afford 24, as off-white solid. LCMS: mJz 496.32, retention time 2.11 min. 1H NMR (CDC1₃, 500 MHz) < ppm 0.98 (s, 9H), 1.44 (s, 9H), 1.65 (s, 3H), 1.77-1.83 (m, 1H), 1.99-2.01 (m, 2H), 2.43-2.48 (m, 1H), 3.58-3.63 (m, 1H), 4.03 (s, 2H), 4.24-4.27 (m, 2H), 4.33-4.48 (m, 2H), 7.21-7.23 (m, 2H), 7.27 (m, 1H).

Step B. (_tS')-N-(2-(aminomethyl)-5-chlorobenzyl)- l-((R)-2-hvdroxy-3,3- dimethylbutanoyl)-2-methylp rolidine-2-carboxamide (2-2) reri-butyl 4-chloro-2-(((S)-l-((R)-2-hydroxy-3,3-dimethylbutanoyl)-2- methylpyrrolidine-2-carboxamido)methyl)benzylcarbamate (11.7mg, 0.024 mmol) was dissolved in CH₂C1₂ (118 μΐ) and 4.0M HC1 in dioxane (41.0 μΐ, 1.179 mmol) was added. It was stirred at r.t. for 2 h. Volatiles were removed and the residue was purified with preparative reversed-phase HPLC, eluting with 10-70% ACN/H₂0 with 0.1% TFA. Lyophilized to afford 7 2 as product. LCMS: m/z 396.26, retention time 1.49 min. 1H NMR (CD₃OD, 500 MHz) ppm 0.96 (s, 9H), 1.55 (s, 3H), 1.88-2.12 (m, 4H), 3.70 (m, IH), 3.87 (m, IH), 4.05 (s, IH), 4.25 (s, 2H), 4.36-4.45 (m, 2H), 7.35-7.42 (m, 2H), 7.48 (d, J = 2.08 Hz, IH).

EXAMPLE 3

(_lS')-N-(2-(aminomethyl)-5-chlorobenzyl)-l-((R)-2-cyclohexyl-2-hydroxyacetyl)-2- methylpyrrolidine-2-carboxamide (3-1)

This compound was made similarly as EXAMPLE 2 as the TFA salt. LCMS: m/z 422.24, retention time 1.62 min. 1H NMR (CD₃OD, 500 MHz) ppm

1.03-1.31 (m, 6H), 1.40- 1.79 (m, 6H), 1.74 (d, J = 11.30 Hz, 2H), 1.88-2.03 (m, 3H), 2.09 (m, IH), 3.65 (m, IH), 3.81 (m, IH), 4.06 (d, J = 5.07 Hz, IH), 4.19-4.30 (m, 2H), 4.34-4.47 (m, 2H), 7.34-7.41 (m, 2H), 7.47 (s, IH). EXAMPLE 4

(_lS')-N-(2-(aminomethyl)-5-chlorobenzyl)-l-((R)-2-(3-chlorophenyl)-2-hydroxyacetyl)- 2-methylpyrrolidine-2-carboxamide (4- 1)

OH 4

This compound was made similarly as EXAMPLE 2 the TFA salt. LCMS: m/z 450.18, retention time 1.63 min. 1H NMR (CD₃OD, 500 MHz) ppm 1.48 (s, 3H), 1.68-1.94 (m, 5H), 2.00-2.06 (m, IH), 3.11-3.19 (m, 3H), 3.72-3.78 (m, IH), 4.22-4.32 (m, 3H), 4.37 (d, J = 15.05 Hz, IH), 4.49 (d, J = 15.00 Hz, IH), 5.23 (s, IH), 7.25-7.30 (m, IH), 7.32-7.43 (m, 5H), 7.48 (d, J = 2.10 Hz, IH). EXAMPLE 5

(_lS^,)-N-(2-(aminomethyl)-5-chlorobenzyl)- l-((R)-3,3-dimethyl-2- (methylsulfonamido)butanoyl)-2-methylpyrrolidine-2-carboxamide (5-3)

Step A. Tert-butyl 2-(((^)-l-((R)-2-(2-(9H-fluoren-9-yl)acetamido)-3,3-dimethyl- butanoyl)-2-methylpyrrolidie-2-carboxamido)methyl)-4-chlorobenzylcarbamate (5- 1 )

To a suspension of (S)-tert-butyl 4-chloro-2-((2-methylpyrrolidine-2- carboxamido)methyl)benzylcarbamate (100 mg, 0.262 mmol), Fmoc-D-alpha-t- butylglycine (102 mg, 0.288 mmol), and l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (55.2 mg, 0.288 mmol) in CH₂C1₂ (1500 μΐ) and DMF (300 μΐ) was added pyridine (63.5 μΐ, 0.786 mmol), and the mixture was stirred at r.t. for lh. It was then diluted with CH₂C1₂, washed with saturated aqueous LiCl solution and brine. The organic layer was dried over magnesium sulfate, filtered and concentrated. The crude material was purified via prep. TLC plate (50% EtOAc/hex.) to afford 5Λ as product. LCMS: mJz 717.50, retention time 2.63 min. 1H NMR (CD₃OD, 500 MHz) ppm 1.07 (s, 9H), 1.41 (s, 9H), 1.65 (s, 3H), 1.90-2.04 (m, 4H), 2.20 (m, 1H), 3.76 (d, J = 10.52 Hz, 1H), 3.90 (t, J = 8.36 Hz, 1H), 4.04 (t, J = 7.58 Hz, 1H), 4.10 (m, 2H), 4.12-4.29 (m, 3H), 4.31-4.38 (m, 1H), 6.85-7.17 (m, 2H), 7.24-7.31 (m, 3H), 7.32-7.42 (m, 2H), 7.50-7.74 (m, 2H), 7.77 (m, 2H).

Step B. Tert-butyl 2-(((_tS')-l-((R)-2-amino-3,3-dimethylbutanoyl)-2-methylpyrrolidine- 2-carboxamido)methyl)-4-chlorobenzylcarbamate (5-2)

5-2 was prepared similarly as described for the coupling step in EXAMPLE 2. The reaction mixture was purified with prep. TLC plate (5% MeOH/CH₂Cl₂), to afford the desired product as colorless film. LCMS: m/z 495.30, retention time 1.76 min. 1H NMR (CD₃OD, 500 MHz) ppm 1.00 (s, 9H), 1.43 (s, 9H), 1.61 (s, 3H), 1.91-2.04 (m, 4H), 2.18 (t, J = 1.65 Hz, IH), 2.85 (s, IH), 2.98 (s, IH), 3.34-3.45 (m, IH), 3.78-3.68 (m, IH), 3,86-3.93 (m, IH), 4.25 (s, 2H), 4.37 (d, J = 15.80 Hz, IH), 4.46 (d, J = 15.78 Hz, IH), 7.18-7.32 (m, 2H), 7.34-7.42 (m, IH).

Step C. (_tS^,)-N-(2-(^'aminomethyl)-5-chlorobenzyl)- l-(^'(^'R)-3,3-dimethyl-2- (methylsulfonamido)butanoyl)-2-methylpyrrolidine-2-carboxamide (5-3) To a solution of 5^2 (2.4 mg, 4.85 μιηοΐ) in anhydrous CH₂C1₂ (100 μν> 0°C was added methanesulfonyl chloride (5 μί, 0.065 mmol) and DIPEA (5 μί, 0.029 mmol). The mixture was allowed to warm to ambient temperature and stirred till reaction was complete. Volatiles were removed under reduced pressure. It was used in the next step without further purification. LCMS: m/z 573.35, retention time 2.15 min. 1H NMR (CD₃OD, 500 MHz) ppm 1.04 (s, 9H), 1.44 (s, 9H), 1.64 (s, 3H), 1.90-2.04 (m, 4H), 2.19 (m, IH), 2.69 (s, 3H), 3.77 (m, IH), 3.94 (m, IH), 3.99 (m, IH), 4.23-4.31 (m, 2H), 4.36-4.51 (m, 3H), 7.18-7.27 (m, 2H), 7.36 (m, IH).

The residue was treated with TFA as described in EXAMPLE 1 and yielded (_lS')-N-(2-(aminomethyl)-5-chlorobenzyl)-l-((R)-3,3-dimethyl-2-(methyl- sulfonamido)butanoyl)-2-methylpyrrolidine-2-carboxamide. LCMS: m/z 473.19, retention time 1.54 min. 1H NMR (CD₃OD, 500 MHz) ppm 1.04 (s, 9H), 1.61 (s, 3H), 1.88-2.02 (m, 4H), 2.05-2.12 (m, IH), 2.99 (s, 3H), 3.77 (dt, J = 10.19, 6.53 Hz, IH), 3.94-4.02 (m, 2H), 4.24 (s, 2H), 4.36 (d, J = 5.26 Hz, 3H), 7.32-7.39 (m, 2H), 7.46 (d, J = 2.09 Hz, IH), 7.92 (s, IH).

EXAMPLE 6

N- (2- (aminomethyl)- 5 -chlorobenzyl)- 1 - ( (R) -2-cyclohexyl-2-hydroxyacetyl) -2- ethylpyrrolidine-2-carboxamide (6-4)

Step A. (R)-2-acetoxy-2-cyclohexylacetic acid (6-1)

(R)-(-)-hexahydromandelic acid (200 mg, 1.264 mmol) was mixed with dry pyridine (307 μΐ, 3.79 mmol) at 0°C, and to it was added acetic anhydride (239 μΐ, 2.53 mmol). The mixture was stirred at RT overnight. Volatiles were removed under reduced pressure. The residue was partitioned between water (5 mL) and CH₂CI₂ (10 mL), organic phase was washed with aqueous IN HC1 (5 mL) and brine, dried and concentrated to afford 6Λ as colorless oil. LCMS: m/z 223.12 [M+Na⁺], retention time 1.45 min. 1H NMR (CD₃OD, 600 MHz) ppm 1.13-1.35 (m, 6H), 1.63-1.82 (m, 4H), 1.84-1.94 (m, 1H), 2.09 (s, 3H), 4.76 (d, J = 4.4 Hz, 1H).

Step B. l-((R)-2-acetoxy-2-cyclohexylacetyl)-2-ethylpyrrolidine-2-carboxylic acid (6- 2) To a solution of (R)-2-acetoxy-2-cyclohexylacetic acid (0.29 g, 1.264 mmol) in anhydrous CH₂CI₂ (4.5 mL) at 0°C was added oxalyl chloride (0.553 ml, 6.32 mmol) dropwise followed by addition of 2 drops of DMF. The reaction mixture was stirred at r.t. for 1 h. Volatiles were removed under reduced pressure. The residue was azotroped with toluene (20 mL), dissolved in anhydrous CH₂CI₂ (2.480 ml) and added to a solution of 2-ethylproline (0.181 g, 1.264 mmol) and triethylamine (0.529 ml, 3.79 mmol) in anhydrous CH₂CI₂ (2.48 ml). It was stirred at r.t. overnight. The mixture was diluted with CH₂C1₂ (10 mL), washed with 5% HC1 (10 mL), water (10 mL) and brine (10 mL), dried over Na₂S0₄ and concentrated to afford 6^2 as a off- white solid , which was used in the next step without further purification.

Step C. re/t-butyl 4-chloro-2-((l-((R)-2-cyclohexyl-2-hydroxyacetyl)-2- ethylpyrrolidine-2-carboxamido)methyl)benzylcarbamate (6-3)

6-2 (260 mg, 23% pure, 0.184 mmol) was dissolved in DMF (368 μΐ), and to this were added ie/t-butyl 2-(aminomethyl)-4-chlorobenzylcarbamate (100 mg, 0.368 mmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (70.5 mg, 0.368 mmol), and 1-hydroxybenzotriazole (24.83 mg, 0.184 mmol). It was stirred at r.t. overnight. The reaction mixture was partitioned between sat. NaHC0₃ (3 mL) and EtOAc (5 mL). The aqueous layer was extracted with EtOAc (5 mL X 3). The combined organic layers was dried over MgS0₄, filtered and concentrated. The residue was purified with silica gel flash column chromatography, eluting with 0- 100% EtOAc/hexanes to afford a & mixture of the two diastereomers, which were separated with preparative reversed-phase HPLC, eluting with 10-100% ACN/H₂0 with 0.1% TFA. Lyophilized to afford the desired products.

A: Fast-eluting isomer. LCMS: m/z 478.25, retention time 2.38 min. 1H

NMR (CD₃OD, 500 MHz) ppm 0.84 (m, 3H), 1.09-1.23 (m, 6H), 1.43 (s, 9H), 1.61- 1.74 (m, 6H), 1.87-2.05 (m, 6H), 2.07 (s, 3H), 2.17-2.21 (m, IH), 2.42-2.48 (m, IH), 3.71-3.82 (m, 2H), 4.23 (m, 2H), 4.41 (m, 2H), 4.91 (d, J = 6.3 Hz, IH), 7.20-7.27 (m, 2H), 7.30 (d, / = 2.10 Hz, IH), 7.94 (m, IH).

B: Slow-eluting isomer. LCMS: m/z 478.28, retention time 2.47 min.

1H NMR (CD₃OD, 500 MHz) ppm 0.85 (m, 3H), 1.16-1.32 (m, 6H), 1.43 (s, 9H), 1.68-1.88 (m, 6H), 1.91 (s, 3H), 1.97 (m, IH), 2.06-2.16 (m, 3H), 2.40 (m, IH), 3.54- 3.61 (m, IH), 4.10 (m, IH), 4.18-4.32 (m, 3H), 4.47 (m, IH), 7.19-7.30 (m, 3H). Step D. N-(2-(aminomethyl)-5-chlorobenzyl)-l-((R)-2-cyclohexyl-2-hydroxyacetyl)-2- ethylpyrrolidine-2-carboxamide (6-4)

The Fast-eluting isomer from Step C (32.54 mg, 0.056 mmol) was treated with aqueous saturated potassium carbonate (20.2 μί, 0.056 mmol) in MeOH (225 ίΌ) at r.t. overnight. The reaction mixture was partitioned between water (5 mL) and EtOAc (5 mL) and the aqueous layer was extracted with EtOAc (5 mL X 3). The combined organic layers was dried over MgS0₄, filtered and concentrated. To the residue was added CH₂C1₂ (225 and trifluoroacetic acid (112 μί, 1.460 mmol), and the mixture was stirred at r.t. for 2 h. Volatiles were removed under reduced pressure. The residue was purified with preparative reversed-phase HPLC, eluting with 10-100% ACN/H₂0 with 0.1% TFA. Lyophilized to afford &4 as product.

LCMS: m/z 436.15, retention time 1.59 min. 1H NMR (CD₃OD, 500 MHz) ppm 0.85 (t, / = 7.44 Hz, 3H), 0.93-1.19 (m, 6H), 1.63 (m, 5H), 1.40 (m, IH), 1.94-2.00 (m, 2H), 2.03-2.08 (m, 2H), 2.35-2.43 (m, IH), 3.60-3.65 (m, IH), 3.61-3.84 (m, IH), 3.95 (d, / = 6.27 Hz, IH), 4.15-4.21 (m, 2H), 4.34 (d, / = 13.9 Hz, IH), 4.64 (d, / = 15.0 Hz, IH), 7.35-7.40 (m, 2H), 7.44 (d, / = 2.10 Hz, IH).

EXAMPLE 7

N-(2-(aminomethyl)-5-chlorobenzyl)-2-ethyl-l-((R)-2-hydroxy-3,3- dimethylbutanoly)pyrrolidine-2-carboxamide (7-5)

Step A. Methyl 2-ethylpyrrolidine-2-carboxylate (7-1) solution of 2-ethylproline (1 g, 5.57 mmol) in anhydrous methanol (50 ml, 1.236 mol) at -5 °C under an atmosphere of nitrogen, was added thionyl chloride (9.75 ml, 134 mmol) dropwise. The reaction mixture was heated under reflux for 24 h, and the resultant pale yellow-colored solution was concentrated to dryness in vacuo. The residue was dissolved in a 1: 1 mixture of methanol and toluene (50 mL) and concentrated to dryness to remove residual thionyl chloride. This procedure was repeated twice more, yielding the methyl ester as a hygroscopic, spectroscopically pure, light brown solid. It was basified with NEt₃, and purified with silica gel flash column chromatography, eluting with 0-8% MeOH/CH₂Cl₂, to afford 7-1 as product. LCMS: m/z 158.09, retention time 0.12 min. 1H NMR (CD₃OD, 500 MHz) < ppm 0.99 (t, J = 7.49 Hz, 3H), 1.89-2.14 (m, 4H), 2.17-2.24 (m, 1H), 2.44- 2.52 (m, 1H), 3.41 (t, J = 7.05 Hz, 3H), 3.88 (s, 3H).

Step B. Methyl 2-ethyl-l-((R)-2-hvdroxy-3,3-dimethylbutanoly)pyrrolidine-2- carboxylate (7-2)

To a solution of methyl 2-ethylpyrrolidine-2-carboxylate (120 mg,

0.763 mmol) and (R)-2-hydroxy-3,3-dimethylbutanoic acid (151 mg, 1.145 mmol) in DMF (0.76 ml) was added l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (293 mg, 1.527 mmol) and 1-hydroxybenzotriazole (51.6 mg, 0.382 mmol), and the mixture was stirred at r.t. overnight. Volatiles were removed under reduced pressure. The residue was purified with silica gel flash column

chromatography, eluting with 0-8% MeOH/CH₂Cl₂, to afford 1^1 as product. LCMS: m/z 272.27, retention time 1.45 min. 1H NMR (CD₃OD, 500 MHz) ppm 0.86 (m, 3H), 1.01 (s, 9H), 1.89-2.11 (m, 4H), 2.18 (m, 1H), 2.34-2.41 (m, 1H), 3.30 (m, 3H), 3.65 (s, 3H). Step C. Lithium 2-ethyl-l-((R)-2-hydroxy-3,3-dimethylbutanoly)pyrrolidine-2- carboxylate (7-3)

To a mixture of methyl 2-ethyl-l-((R)-2-hydroxy-3,3- dimethylbutanoly)pyrrolidine-2-carboxylate (28.1 mg, 0.104 mmol) in THF (1020 μΐ) and water (270 μΐ) was added 1.5 N aq. lithium hydroxide (69.0 μΐ, 0.104 mmol). After stirring at r.t. for 3 h, it was concentrated to dryness to provide 73 as light brown solid. Step D. Tert-butyl 4-chloro-2-((2-ethyl- l-((R)-2-hvdroxy-3,3-dimethylbutanolv) pyrrolidine-2-carboxamido)methyl)benzylcarbamate (7-4)

To a solution of lithium 2-ethyl-l-((R)-2-hydroxy-3,3- dimethylbutanoly)pyrrolidine-2-carboxylate (22.5 mg, 0.085 mmol) were added tert- butyl 2-(aminomethyl)-4-chlorobenzylcarbamate (23.14 mg, 0.085 mmol), l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (19.66 mg, 0.103 mmol), and 1-hydroxybenzotriazole (13.86 mg, 0.103 mmol). The reaction mixture was stirred at r.t. overnight. It was purified with preparative HPLC, 10-100% ACN/H₂0 with 0.1% TFA. Lyophilized to afford 74. LCMS: mJz 510.26, retention time 2.12 min. 1H NMR (CD₃OD, 500 MHz) ppm 0.87 (t, J = 7.42 Hz, 3H), 0.95 (s, 9H), 1.43 (s, 9H), 1.88-2.07(m, 5H), 2.18-2.24 (m, IH), 2.45-2.55 (m, IH), 3.57-3.67 (m, IH), 3.86-3.95 (m, IH), 4.02 (s, IH)), 4.24 (s, 2H), 4.35-4.49 (m, 2H), 7.20-7.26 (m, 2H), 7.34 (m, IH). Step E. N-(2-(aminomethyl)-5-chlorobenzyl)-2-ethyl-l-((R)-2-hydroxy-3,3- dimethylbutanoly)pyrrolidine-2-carboxamide (7-5)

7-5 was made in the same method as EXAMPLE 2. LCMS: m/z 410.27, retention time 1.44 min. 1H NMR (CD₃OD, 500 MHz) ppm 0.87 (m, 10H), 1.89- 1.99 (m, 3H), 2.01-2.08 (m, 2H), 2.34-2.43 (m, IH), 3.62 (dt, J = 10.30, 7.65 Hz, IH), 3.85-3.92 (m, IH), 3.99 (s, IH), 4.14-4.28 (m, 2H), 4.36 (d, J = 13.69 Hz, IH), 4.59 (m, IH), 7.32-7.40 (m, 2H), 7.46 (d, J = 2.16 Hz, IH).

In Vitro Assay For Determining Proteinase Inhibition Relevant in vitro assays are referenced in Morris sette, et al., Bioorg. Med. Chem. Lett. 2004, 14, 4161-4164 and described in Lewis, et al. Thromb. Res. 1993, 70, 173 (assays of human -thrombin and human trypsin), and Lewis, et al. Thromb. Haemostasis 1995, 74, 1107-1112. The assays were carried out 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-nitroaniline. sar-PR-pna was used to assay human a-thrombin (K_m=125 μΜ) and bovine trypsin (K_m=125 μΜ). p- Nitroanilide substrate concentration was determined from measurements of absorbance at 342 nm using an extinction coefficient of 8270 cm^"

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 benzyloxycarbonyl-Gly-Pro-Arg-7-amino-4-trifluoromethylcoumarin (Z-GPR-afc, Lewis S.D. et al. (1998) J. Biol. Chem. 273, pp. 4843-4854) (K_m=27 μΜ) 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 K_m 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 (V₀) or presence of inhibitor (Vi) were measured. Assuming competitive inhibition, and that unity is negligible compared K_m/[S], [I]/e, and [I]/e (where [S], [I], and e respectively represent the total concentrations, of substrate, inhibitor and enzyme), the equilibrium constant (¾) for dissociation of the inhibitor from the enzyme can be obtained from the dependence of V₀/Vi on [I] shown in the following equation.

V₀/Vi = 1 + [I]/Ki

The activities shown by this assay indicate that the compounds of the invention may be therapeutically 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.

EXAMPLE 8

Tablets containing 25, 50, and 100 mg., respectively, of the following active compounds are prepared as illustrated below (compositions A-C). Active I is (_lS')-l-((R)-2-amino-3,3-dimethylbutanoyl)-N-(2-(aminomethyl)-5-chlorobenzyl)-2- methylpyrrolidine-2-carboxamide.

Amount- (mg)

Component A B C

Active I 25 50 100

Microcrystalline cellulose 37.25 100 200

Modified food corn starch 37.25 4.25 8.5

Magnesium stearate 0.5 0.75 1.5

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, 50, and 100 mg, respectively, of active ingredient per tablet. EXAMPLE 9

Tablet Preparation

Exemplary compositions of (5')-l-((R)-2-amino-3,3-dimethylbutanoyl)-N-(2- (aminomethyl)-5-chlorobenzyl)-2-methylpyrrolidine-2-carboxamide (Active I) tablets are shown below:

Component 0.25 mg 2 mg 10 mg 50 mg

Active I 0.500% 1.000% 5.000% 14.29% mannitol 49.50% 49.25% 47.25% 42.61% microcrystalline cellulose 49.50% 49.25% 47.25% 42.61% magnesium stearate 0.500% 0.500% 0.500% 0.500%

0.25, 2, 10 and 50 mg tablets are film-coated with an aqueous dispersion of hydroxypropyl cellulose, hydroxypropyl methylcellulose and titanium dioxide, providing a nominal weight gain of 2.4%.

Active I, mannitol and microcrystalline cellulose are sieved through mesh screens of specified size (generally 250 to 750 μιη) and combined in a suitable blender. The mixture is subsequently blended (typically 15 to 30 min) until the drug was uniformly distributed in the resulting dry powder blend. Magnesium stearate is screened and added to the blender, after which a precompression tablet blend was achieved upon additional mixing (typically 2 to 10 min). The precompression tablet blend is then compacted under an applied force, typically ranging from 0.5 to 2.5 metric tons, sufficient to yield tablets of suitable physical strength with acceptable disintegration times (specifications will vary with the size and potency of the compressed tablet). In the case of the 2, 10 and 50 mg potencies, the tablets are dedusted and film-coated with an aqueous dispersion of water-soluble polymers and pigment.

Alternatively, a dry powder blend is compacted under modest forces and remilled to afford granules of specified particle size. The granules are then mixed with magnesium stearate and tabletted as stated above. EXAMPLE 10

Intravenous (_lS')-l-((R)-2-amino-3,3-dimethylbutanoyl)-N-(2- (aminomethyl)-5-chlorobenzyl)-2-methylpyrrolidine-2-carboxamide (Active I) prepared according to general intravenous formulation procedures.

Component Estimated range

Active I 0.12 - 0.50 mg

D-glucuronic acid 0.5 - 5 mg

Mannitol NF 50-53 mg

1 N Sodium Hydroxide q.s. pH 3.9 - 4.1

Water for injection q.s. 1.0 mL

Various other buffer acids, such as L-lactic acid, acetic acid, citric acid or any pharmaceutically acceptable acid/conjugate base with reasonable buffering capacity in the pH range acceptable for intravenous administration may be substituted for glucuronic acid.

Claims

WHAT IS CLAIMED IS:

A compound of the formula I

or a pharmaceutically acceptable salt thereof, wherein

4 5 4 5

R is a heterocycle or -(CR R )i_2NH2, wherein R and R , each time in which they occur, are independently H, Ci_6 alkyl, -CH2F, -CHF2, CF3 or -CH2OH; R¹ is Ci_6 alkyl;

R² is NH₂, OH, NHS02Ci_6alk l, or NHC(0)Ci_6alk l; and

3 6 7 8 6 7

R is C3.8 carbocycle, aryl, or CR R R , where R is hydrogen or CH3, R is

g

hydrogen or CH3, and R is CH3, aryl, or 5-7-membered heteroaryl,

wherein aryl and 5-7-membered heteroaryl, in each instance in which they occur, are independently unsubstituted or mono-, di- tri- or tetra- substituted with halogen, said heteroaryl having 1, 2, 3, or 4 nitrogen atoms.

2. A compound of Claim 1, or a pharmaceutically acceptable salt thereof, wherein R is -CH2NH2.

3. A compound of Claim 1, or a pharmaceutically acceptable salt thereof, wherein R is CH3 or CH2CH3.

4. A compound of Claim 1, or a pharmaceutically acceptable salt thereof, wherein R² is NH₂, OH, NHSO2CH3, or NHC(0)CH₃.

5. A compound of Claim 1, or a pharmaceutically acceptable salt 3

thereof, wherein R is -C(CH3)3, C6H1 1 , '

6. A compound of Claim 1, or pharmaceutically acceptable salt thereof, which is

(S)-l-((R)-2-acetamido-3,3-dimethylbutanoyl)-N-(2-(aminomethyl)-5- chlorobenzyl)-2-methylpyrrolidine-2-carboxamide,

(S)-l-((R)-2-amino-3-(4-fluorophenyl)propanoyl)-N-(2-(aminomethyl)-5- chlorobenzyl)-2-methylpyrrolidine-2-carboxamide,

(S)-N-(2-(aminomethyl)-5-chlorobenzyl)-l-((S)-2-(3-chlorophenyl)-2- hydroxyacetyl)-2-methylpyrrolidine-2-carboxamide, or

(2S)-l-(2-amino-2-(3-chlorophenyl)acetyl)-N-(2-(aminomethyl)-5-chlorobenzyl)-2- methylpyrrolidine-2-carboxamide.

7. A composition for inhibiting thrombus formation in blood comprising a compound of Claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

8. A method for inhibiting thrombin in blood comprising adding to the blood a composition of Claim 7.

9. A method for inhibiting formation of blood platelet aggregates in blood comprising adding to the blood a composition of Claim 7.

10. A method for inhibiting thrombus formation in blood comprising adding to the blood a composition of Claim 7.

11. The use of a compound of Claim 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting thrombin, inhibiting thrombus formation, treating thrombus formation, or preventing thrombus formation in a mammal.

12. A method for treating or preventing venous thromboembolism and pulmonary embolism in a mammal comprising administering to the mammal a composition of Claim 7.

13. A method for treating or preventing deep vein thrombosis in a mammal comprising administering to the mammal a composition of Claim 7.

14. A method for treating or preventing thromboembolic stroke in humans and other mammals comprising administering to the mammal a composition of Claim 7.