TITLE OF THE INVENTION

SUBSTITUTED 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 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analogs of peptidase substrates in which the nitrogen atom of the scissile amide group of the substrate peptide has been replaced by hydrogen or a substituted carbonyl moiety.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 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 that may be useful for inhibiting loss of blood platelets, inhibiting formation of blood platelet aggregates, inhibiting fomiation of fibrin, inhibiting thrombus formation, and inhibiting embolus foraiation 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 compounds of the invention may also be useful 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:

(I)

or a pharmaceutically acceptable salt thereof, wherein

Q is C or Si;

4 5 4 5

R is a heterocycle or -(CR R )i-2 H2, wherein R and R , each time in which they occur, are independently H, Cj.g alkyl, -CH2F, -CHF2, CF3 or -CH20H;

R is hydrogen, or together with R , forms C3.8 carbocycle;

2 3

R is halogen, CF3, Ci _6 alkyl, or together with R , forms C3.8 carbocycle, or together with R , forms C3.8 carbocycle; and R is hydrogen, halogen, OH, Cj.g alkyl, or together with R , forms C3.8 carbocycle.

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

In another embodiment of the invention, R is hydrogen, or together

2

with R , forms cyclopropyl.

2

In another embodiment of the invention, R is F, CF3, CH3, or

3 1

together with R , forms cyclopropyl, or together with R , forms cyclopropyl.

2

In another embodiment of the invention, R is F, CF3, CH3, or 3

together with R , forms cyclopropyl.

In another embodiment of the invention, R^ is hydrogen.

3

In another embodiment of the invention, R is hydrogen, F, OH, CH3, 2

or together with R , forms cyclopropyl

In another embodiment of the invention, the compound is

(S)-N-(2-(aminomethyl)-5-chlorobenzyl)-4,4-difluoro-l-((R )-2-hydroxy-3,3- dimethylbutanoyl)pyrrolidine-2-carboxamide,

(2S)-N-(2-(aminomethyl)-5-chlorobenzyl)-4-hydroxy-l-((S)- 2-hydroxy-3,3- dimethylbutanoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxami de,

(R)-N-(2-(aminomethyl)-5-chlorobenzyl)-l -((R)-2-hydroxy-3,3-dimethylbutanoyl)- 3,3-dimethyl-l ,3-azasilolidine-5-carboxamide, (R)-N-(5-chloro-2-(lH-tetrazol-l-yl)benzyl)-l-((R)-2-hydroxy -3,3-dimethylbutanoyl)- 3,3-dimethyl-l ,3-azasilolidine-5-carboxamide, (2S,4S)-N-(2-(aminomethyl)-5-chlorobenzyl)-l -((S)-2-hydroxy-3,3- dimethylbutanoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxami de, (2S,4R)-N-(2-(aminomethyl)-5-chlorobenzyl)-l-((R)-2-hydroxy- 3,3- dimethylbutanoyl)-4-methylpyrrolidine-2-carboxamide, or

(S)-N-(2-(aminomethyl)-5-chlorobenzyl)-5-((R)-2-hydroxy-3 ,3-dimethylbutanoyl)-5- azaspiro[2.4]heptane-6-carboxamide.

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 stereo genie 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 (2H). 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 phamiaceutically 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 quatemized 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, ( -benzoyl, and ( -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 _j _ ₆ alkyl esters and -C _j _ 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

BOC (Boc) tert-butyloxycarbonyl

(BOC) ₂ 0 di-t-butyl dicarbonate

CBZ benzyloxycarbonyl

CBZ-C1 benzyl chloroformate

Celite® Celite® (Fluka) diatomite is diatomaceous earth

COD 1,5-cyclooctadiene

DAST diethylaminosulfur trifluoride DCC Ν,Ν'-dicyclohexylcarbodiimide

DIPEA diisopropylethylamine

(i-Pr2Net;

Hunig's base)

DMAP dimethylaminopyridine

DME dimethoxyethane

DMF dimethylformamide

DMP Dess-Martin Periodinane

EDC (EDCI) 1 -ethyl-3 -(3 -dimethylaminopropyl)carbodiimide

EtOAc ethyl acetate

Et ₃ N triethylamine

Fmoc 9-fluorenylmethoxycarbonyl

Fmoc-Cl 9-Fluorenylmethyl chloroformate

HATU 0-(7- Azabenzotriazol- 1 -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

HOBt 1-hydroxybenzotriazole

MeCN acetonitrile

MeOH methanol

MTBE methyl tert-butyl ether

NMM N-methylmo holine

pna p-nitroanilide

OAc acetoxy group

Pd-C palladium on carbon

PEG polyethylene glycol

Ph3PHCH3Br methyltriphenylphosphonium bromide

Py pyridine

PyBOP (Benzotriazol- 1 -yloxy)tripyrrolidinophosphonium

hexafluorophosphate

rt room temperature

TBAF tetrabutylammonium fluoride TBDMSC1 tert-butyldimethylsilyl chloride

TBS tert-butyldimethylsilyl

TEMPO (2,2,6,6-Tetramethylpiperidin- 1 -yl)oxyl

TFA trifluoroacetic acid

THF tetrahydrofuran

TRIS tris(hydroxymethyl)aminomethane

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

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 C1 -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 C]-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 C ₃ to C ₈ 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 Ci-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 C] _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, fhiamorpholinyl 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 may be useful not only in the anticoagulant therapy of individuals having thrombotic conditions, but may be 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, and may be useful for inhibiting thrombin.

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 III 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 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 0.01 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 earners (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, phannaceutically 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, 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, antidiabetic 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 II 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 drags 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)-ph enyl]-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-drag 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 (TREDAPTIVE®) 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

(T EDAPTIVE®) 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 Procedures

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

Intermediate 1, prepared by various methods, is coupled to intermediate 2 in the presence of a peptide coupling reagent such as EDC in a solvent such as DMF to form intermediate 3. After Fmoc group is removed with a base such as piperidine, intermediate 4 is subject to amide coupling with an carboxylic acid reagent 5 using an amide coupling reagent such as HATU in a solvent such as DMF. For some compounds in which R has an amino group, it requires protection of the amino group in reagent 2 with a carbamate such as Boc, and deprotection with an acid such as TFA to get to the final compound. Celite® (Fluka) diatomite is diatomaceous earth, and can be referred to as "celite".

Scheme 1

Alternatively, intermediate 7 can be acylated first with intermediate 8 to form intermediate 9 in a solvent such as THF in the presence of base such as triethylamine. After sopanofication with a base such as LiOH, the carboxylic acid intermediate 10 is coupled to intermediate 2 in the presence of a peptide coupling reagent such as EDC in a solvent such as DMF to form intermediate 6. For some compounds in which R has an amino group, it requires protection of the amino group in reagent 2 with a carbamate such as Boc, and deprotection with an acid such as TFA to get to the final compound.

Scheme 2

Separation of diastereomers can be earned out at various stages in the preparation of the desired final compounds; however, it is typically carried out on intermediate 6 before removal of the protective group using 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 μιτι, 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 μΐ.

Detection: Programmed Data Acquisition, 200-600 nm

MS: mass range 150-750 amu; positive ion electrospray ionization Preparative thin layer chromatography (PTLC) was performed on 20 x

20cm plates (500 μιτι - 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

Preparation of (S)-N- {2-(Aminomethyl)-5-chlorobenzyl}-4,4-difluoro-l - {(R)-2- hydroxy-3,3-dimethylbutanoyl}pyrrolidine-2-carboxamide-2,2,2 -trifluoroacetate CL 13)

(2S, 4i?)-Methyl-4-hydroxypyrrolidine-2-carboxylate hydrochloride (1 -2)

Thionyl chloride (5.50 mL, 76.0 mmol) was added to the solution of 4- hydroxy proline LI (5.00 g, 38.0 mmol) in CH ₃ OH (120 mL) at 0 °C over a period of 10 min and reaction mixture was stirred at room temperature for 15 h. The solvent was removed at reduced pressure and residue was azeotroped with toluene (2 x 50 mL). The residue was washed with hexanes, filtered and dried over high vacuum to provide ester 2 (7.10 g, quant.) as an off-white hygroscopic solid.

(2S, 4i?)-l-tert-Butyl-2-methyl 4-hydroxypyrrolidine-l ,2-dicarboxylate (1-3)

Boc anhydride (3.92 g, 18.1 mmol) was added to the solution of ester h½ (3.0 g, 16.5 mmol) in THF (50 mL) and H ₂ 0 (50 mL) followed by addition of solid Na ₂ C0 ₃ (3.67 g, 34.6 mmol). The reaction mixture stirred at room temperature overnight. Solvent was removed under reduced pressure. The residue dissolved in water (20 mL) and neutralized to pH 6-7 with 2M aqueous HC1 and extracted with EtOAc (2 x 100 mL). The combined organic layer was washed with brine (100 mL), dried over anhydrous Na ₂ S0 ₄ . and, concentrated to provide Boc protected hydroxyl proline ester 1^3 (3.90 g) as a colorless oil.

(5)-l-iert-Butyl 2-methyl 4-oxopyrrolidine-l,2-dicarboxylate (1-4)

Dess-Martin periodinane (8.30 g, 19.5 mmol) was added to the solution of hydroxy proline L3 (3.90 g, 15.9 mmol) in CH ₂ C1 ₂ (150 mL) at 0 °C and reaction mixture was stirred at room temperature for 4 h. Upon completion, mixture of saturated aqueous Na ₂ S ₂ 0 ₃ and NaHC0 ₃ solution (1 :1, 100 mL) was added and mixture was stirred until clear organic layer was observed (ca. 10-15 min). Organic layer was separated and dried over anhydrous Na ₂ S0 ₄ and concentrated to provide keto ester L (3.00 g) as a colorless oil:

(S)-l -teri-Butyl-2-methyl-4,4-difluoropyrrolidine-l ,2-dicarboxylate (1-5)

DAST (3.80 mL, 30.8 mmol) was added dropwise to the solution of ketone L4 (3.00 g, 12.3 mmol) in CH ₂ C1 ₂ (60 mL) at -78 °C over a period of 10 min and mixture was wanned to room temperature and stirred at same temperature for 15 h. Reaction mixture was diluted with CH ₂ C1 ₂ (100 mL) and poured into saturated aqueous NaHC0 ₃ solution (200 mL). Organic layer was separated and washed with brine solution (1 x 100 mL) and water (2 ^χ 100 mL), dried over anhydrous Na ₂ S0 ₄ and concentrated to afford difluoro proline intermediate L5 (2.81 g) as brown oil. (S)-l -(iert-Butoxycarbonyl)-4,4-difluoropyrrolidine-2-carboxylic acid (1-6)

A solution of ΙΛΟΗ·Η ₂ 0 (0.63 g, 15.0 mmol) in water (4 mL) was added to the solution of ester 1^5 (2.00 g, 7.54 mmol) in THF (6 mL) at 0 °C and reaction mixture was stirred at room temperature for 2 h. Solvent was removed at reduced pressure and obtained residue was dissolved in water (10 mL) and acidified with 2M aqueous HC1 to pH 4-5. The aqueous layer extracted with EtOAc (3 * 40 mL). The combined organic layer was dried over anhydrous Na ₂ S0 ₄ , filtered and and concentrated to obtain acid 1^5 (1.78 g) as a pale yellow solid

(■S)-l-[ {(9H-Fluoren-9-yl)methoxy}carbonyl]-4,4-difluoropyrrolidine- 2-carboxylic acid ( i)

Trifluoro acetic acid (12 mL, 50% solution in CH ₂ C1 ₂ ) was added to the solution of Boc proline1^6 (1.78 g, 7.08 mmol) in CH ₂ C1 ₂ (6 mL) and reaction mixture was stirred under nitrogen atmosphere for 3 h. The solvent was removed at reduced pressure and the residue was azeotroped with toluene (2 10 mL) and dried under high vacuum for 1 h. The crude residue (2.06 g, 7.76 mmol) was dissolved in 1 ,4-dioxane (8 mL) and aqueous solution of Na ₂ C0 ₃ (2.00 g, 1.90 mmol, 10 mL) was added to it at 0 °C. Solution of Fmoc-Cl (2.00 g, 7.76 mmol) in 1,4-dioxane (10 mL) was added drop wise and mixture was stirred at room temperature for 16 h. Organic solvent was removed at reduced pressure and residual was diluted with water (30 mL). The mixture was washed with MTBE (2 ^χ 50 mL), the aqueous layer was acidified with 2 M aqueous HC1 to pH 2-3 and extracted with EtOAc (3 x 50 mL). The combined organic layer was washed with water (50 mL), brine solution (50 mL), dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure to obtain Fmoc protected proline JV7 (1.10 g) as an off-white solid.

(5)-(9H-Fluoren-9-yl)methyl-2- {(2-[{(tert-butoxycarbonyl)amino}methyl]-5- chlorobenzyl)carbamoyl}-4,4-difluoropyrrolidine-l -carboxylate (1-9) A mixture of acid 7 (0.50 g, 1.33 mmol), amine S (0.49 g, 1.33 mmol), EDCI (0.28 g, 1.46 mmol and HOBt (0.04 g, 0.33 mmol) in DMF (10 mL) was stirred under nitrogen for 3 h at room temperature. The reaction was quenched with water (100 mL) and extracted with EtOAc (3 x 50 mL). The combined organic layer was washed with water (2 x 100 mL), brine solution (100 mL), dried over anhydrous Na ₂ S0 ₄ , and concentrated under reduced pressure. The residue was purified by reverse phase combiflash chromatography (CI 8, 2:3 H ₂ 0/CH ₃ CN) to afford amide 1^9 (0.40 g) as beige solid. (S)-teri-Butyl-4-chloro-2- {(4,4-difluoropyrrolidine-2- carboxamido)methyl } benzyl carbamate (1 -10)

Piperidine (10 mL, 20% solution in CH ₂ C1 ₂ ) was added to the solution of Fmoc proline 9 (0.40 g, 0.63 mmol) in CH ₂ C1 ₂ (5 mL) under nitrogen and reaction mixture was stirred for 30 min at room temperature. The reaction mixture was washed with water (2 x 50 mL), dried over anhydrous Na ₂ S0 ₄ filtered, and concentrated. The residue was purified by reverse phase combiflash chromatography (CI 8, 1 :4 H ₂ 0/CH ₃ CN) to obtain amine LTO (0.11 g) as brown oil. tert-Butyl-4-chloro-2-[ {(5)-4,4-difluoro-l- {(i?)-2-hydroxy-3,3- dimethylbutanoyl } pyrrolidine-2-carboxamido } methyl]benzyl carbamate (1-12)

A mixture of amine 140 (0.1 1 g, 0.28 mmol), acid TT l (0.02 g, 0.31 mmol), EDCI (0.06 g, 0.31 mmol), HOBt (0.04 g, 0.28 mmol), and DIPEA (0.16 mL, 0.85 mmol) in DMF (6 mL) was stirred under nitrogen for 18 h. The reaction mixture was quenched with water (20 mL) and extracted with CH ₂ C1 ₂ (2 ^χ 20 mL). The combined organic layer washed with saturated NaHC0 ₃ solution (30 mL), water (3 ^χ 50 mL), brine solution (50 mL), dried over anhydrous Na ₂ S0 ₄ and concentrated under reduced pressure. The residue was purified by reverse phase combiflash

chromatography (CI 8, 3:2 H ₂ 0/CH ₃ CN) to afford Boc protected M2 (0.05 g) as colorless oil. (5)-N-{2-(aminomethyl)-5-chlorobenzyl}-4,4-difluoro-l -{(7?)-2-hydroxy-3,3- dimethylbutanoyl}pyrrolidine-2-carboxamide (1 -13)

Trifluoroacetic acid (4 mL, 50% solution in CH ₂ C1 ₂ ) was added to the solution of Boc protected 1-12 (0.06 g, 0.16 mmol) in CH ₂ C1 ₂ (4 mL) and reaction mixture was stirred under nitrogen for 2 h. The solvent was removed at reduced pressure and the residue was azeotroped with toluene (5 mL). Hygroscopic solid was dissolved in 1 :1 acetonitrile/water (3 mL) and lyophilized for 24 h to obtain 1-13 as a white hygroscopic solid. Ή NMR (300 MHz, MeOD) δ 7.49 (d, J= 1.5 Hz, 1H), 7.40 (d, J= 2.4 Hz, 2H), 4.62 (d, J= 15.0 Hz, 1H), 4.56 (dd, J= 9.30, 6.60 Hz, 1H), 4.44^1.35 (m, 2H), 4.33 (d, J= 6.60 Hz, 2 H), 4.20^1.05 (m, 1H), 3.98 (s, 1H), 2.72- 2.55 (m, 1H), 2.49-2.38 (m, 1H), 0.99 (s, 9H)

EXAMPLE 2

Preparation of (2S)-jV-{2-(Aminomethyl)-5-chlorobenzyl}-4-hydroxy-l-{(S)-2- hydroxy-3,3-dimethylbutanoyl}-4-(trifluoromethyl)pyrrolidine -2-carboxamide-2,2,2- trifluoroacetate (2-10)

(2<S)- 1 -[ {(9H-Fluoren-9-yl)methoxy} carbonyl]-4-hydroxy-4- (trifluoromethyl)pyrrolidine-2-carboxylic acid (2-3)

A 50% solution of TFA in CH ₂ C1 ₂ (3 mL) was added o the solution of Boc proline intermediate 2-2 (0.35 g, 1.17 mmol) in CH ₂ C1 ₂ (1 mL) and reaction mixture was stirred at room temperature for 3 h. The solvent was removed under reduced pressure, residue was azeotroped with toluene (2 x 10 mL) and dried over high vacuum pump for 2 h. A solution of Fmoc-Cl (0.36 g, 3.51 mmol) in 1 ,4- dioxane (4 mL) was added dropwise to the mixture of above residue and Na ₂ C0 ₃ (368 mg, 3.51 mmol) in 1 : 1 dioxane/water (8 mL) and mixture was stirred at room temperature for 16 h. The solvent was removed at reduced pressure and residue was suspended in water (20 mL). The aqueous layer was washed with MTBE (20 mL), acidified with 10% KHS0 ₄ to pH 2 and extracted with EtOAc (3 x 30 mL). The combined organic extract was dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated to provide Fmoc protected acid 2^3 (0.41 g) as white solid.

(2S)-(9H-Fluoren-9-yl)methyl 2- {(2-[ {(tert-butoxycarbonyl)amino}methyl]-5- chlorobenzyl)carbamoyl} -4-hydroxy-4-(trifluoromethyl)pyrrolidine- 1 -carboxylate (2^ 5)

The mixture of acid 2^3 (0.41 g, 0.97 mmol), amine A (0.32 g, 1.17 mmol), EDC (0.22 g, 1.17 mmol), and HOBt (0.16 g, 1.17 mmol) in DMF (10 mL) was stirred at room temperature for 4 h. The reaction was quenched with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic extract was dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated. The residue was purified by combiflash (silica gel; 30% EtOAc/hexanes) to provide amide 2^5 (0.60 g) as white solid. fert-Butyl 4-chloro-2-[ {(25 -4-hydroxy-4-(trifluoromethyl)pyrrolidine-2- carboxamido }methyl]benzyl carbamate (2-6)

A solution of 50% piperidine in CH ₂ C1 ₂ (0.4 mL) was added to the stirred solution of Fmoc proline 2^5 (0.60 g, 0.89 mmol) in CH ₂ C1 ₂ (5 mL) and reaction mixture was stirred at room temperature for 1 h. The solvent was removed and residue was purified by reverse phase combiflash (CI 8; 10-100%

acetonitrile/water) to provide proline intermediate 2 ₂ 6 (0.21 g) as colorless gum.

(25)- 1 - {(25)-2- {(2-[ {(tert-Butoxycarbonyl)amino}methyl]-5- chlorobenzyl)carbamoyl}-4-hydroxy-4-(trifluoromethyl)pyrroli din-l-yl)-3,3-dimethyl- 1 -oxobutan-2-yl acetate (2-8)

A solution of acid chloride 2 7 (0.17 mg, 0.93 mmol) in THF (3 mL) was added dropwise to the mixture of amine 2^6 (0.21 g, 0.46 mmol) and Et ₃ N (0.12 mL, 0.93 mmol) in CH ₂ C1 ₂ (10 mL) at 0 °C and reaction mixture was stirred at same temperature for 1 h. The reaction was quenched with water (20 mL) and extracted with CH ₂ C1 ₂ (3 x 20 mL). The combined organic extract was dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated. The residue was purified by combiflash (silica gel; 30% EtOAc/hexanes) to provide amide 2^8 (0.13 g) as off white solid. tert-Butyl-4-chloro-2-[ {(25)-4-hydroxy-l- {(5)-2-hydroxy-3,3-dimethylbutanoyl}-4- (trifluoromethyl)pyrrolidine-2-carboxamido } methyljbenzylcarbamate (2-9)

A solid K2CO3 (14 mg, 0.10 mmol) was added to the stirred solution of amide 2^8 (0.12 g, 0.20 mmol) in eOH (3 mL) and mixture was stirred at room temperature for 1 h. Reaction was quenched by saturated aqueous NH ₄ C1 (10 mL), and extracted with EtOAc (2 x 25 mL). The combined organic extract was dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated. The residue was purified by reverse phase combiflash (CI 8; 60-70% acetonitrile/water) to provide alcohol 2^9 (0.098 g) as white solid.

(2,S)-N- {2-(Aminomethyl)-5-chlorobenzyl}-4-hydroxy-l- {(5)-2-hydroxy-3,3- dimethylbutanoyl} -4-(trifluoromethyl)pyrrolidine-2-carboxamide-2,2,2- trifluoroacetate (2-10)

A 50% solution of TFA in CH ₂ C1 ₂ (5 mL) was added to the stirred solution of Boc protected amino alcohol intermediate 2^9 (0.098 mg, 0.17 mmol) and reaction mixture was stirred at room temperature for 3 h. The solvent was removed at reduced pressure and residue was azeotrope with toluene (2 x 20 mL) and dried over high vacuum pump for 2 h. The crude product was purified by reverse phase combiflash (30% acetonitrile/water) to provide 2-10 as white solid. 1H NMR (MeOD , 400 MHz) (δ) ppm: 7.51 (d, J= 1.6 Hz, 1H), 7.43-7.38 (m, 2 H), 4.60 (d, J= 15.2 Hz, 1H), 4.55 (dd, J = 12 Hz, J= 9.2 Hz, 1H), 4.34-4.13 (m, 4H), 4.00 (s, 1 H), 3.95 (d, J = 1 1.6 Hz, 1 H), 2.62 (dd, J= 9.6 Hz, J= 13.6 Hz, 1 H), 2.13 (dd, J= 3.6 Hz, J = 14 Hz, 1H), 0.94 (s, 9H).

EXAMPLE 3 Preparation of (i?)-7V-{2-(Aminomethyl)-5-chlorobenzyl}-l -{(i?)-2-hydroxy-3,3- dimethylbutanoyl}-3,3-dimethyl-l,3-azasilolidine-5-carboxami de-2,2,2- trifluoroacetate (3-14)

Bis(iodomethyl)dimethylsilane (3-2)

The mixture of bis(chloromethyl)dimethylsilane (6.0 g, 38.0 mmol) and sodium iodide (28.6 g, 191 mmol) in dry acetone (100 mL) was refluxed for 3 h. The reaction mixture was cooled to room temperature, filtered and solvent was removed under reduced pressure. MTBE (150 mL) was added and solid was filtered. The filtrate was washed with water (200 mL), dried over anhydrous Na ₂ S0 ₄ and concentrated under reduced pressure to provide bis-iodo intermediate 3-2 (8.7 g) as oil.

(2i?,5i?)-2-[{(Iodomethyl)dimethylsilyl}methyl)-5-isoprop yl-3,6-dimethoxy-2,5- dihydropyrazine (3-4) To a solution of (i?)-2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine 3- 3 (2.6 g, 14.1 mmol) in THF (50 mL), slowly added rc-BuLi (1.6 M in hexane, 10.0 mL) at -70 °C and the mixture was stirred for 15 min at this temperature and then solution of bis(iodomethyl)dimethylsilane (7.10 g, 21.0 mmol) in THF (4 mL) was added dropwise. After 3 h, the mixture was allowed to warm to room temperature. The reaction mixture was diluted with MTBE (50 mL), washed with H ₂ 0 (100 mL) and brine solution (100 mL). The organic layer was dried over anhydrous Na2S04, filtered and concentrated under reduced pressure. The residue was purified by combiflash column chromatography (silica gel; eluent: 0-10% MTBE/hexanes) to provide bis lactim ether 3^4 (1.7 g) as oil.

(i?)-N-(fert-Butoxycarbonyl)silaproline Methyl Ester (3-7)

The alkylated bis lactim ether intermediate 3 (1.5 g, 3.78 mmol), freshly purified by chromatography, was stirred in MeOH/10% aqueous HC1 (3 : 1 , 8 mL) at room temperature for 4 h. The solvent was removed at reduced pressure and residue was co-evaporated with MeOH (3 x 10 mL) and the residue was dissolved in CH ₂ C1 ₂ /Et ₂ 0 (4:6, 20 mL). DIPEA (1.0 mL, 8.36 mmol) was added and reaction mixture was stirred at room temperature for 4 h. Boc ₂ 0 (180 mg, 8.36 mmol) was added and the mixture was stirred overnight at room temperature. The solvent was removed at reduced pressure and the residue diluted with EtOAc (50 mL). The organic phase was washed with 1M HS0 ₄ (3 ^χ 25 mL), H ₂ 0 (15 mL), dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated under reduced pressure. The residue was purified by combiflash column chromatography (silica gel; eluent: 15%

EtOAc/hexanes) to give silaproline ester 3^7 (132 mg) as oil.

(i?)-Methyl 1 - {(i?)-2-acetoxy-3 ,3 -dimethylbutanoyl } -3 ,3 -dimethyl- 1 ,3 -azasilolidine-5 - carboxylate (3-10)

The solution of TFA (50% in CH ₂ C1 ₂ , 2 mL) was added to the solution of Boc silaproline 3 7 (158 mg, 0.57 mmol) in CH ₂ C1 ₂ (1 mL) and reaction was stirred at room temperature for 4 h. The solvent was removed at reduced pressure and residue was azeotroped with toluene (3 x 10 mL) and dried over high vacuum pump for 1 h. The crude residue was dissolved in CH2CI2 (5 mL) and acid chloride 3^9 (231 mg, 1.15 mmol) and DIPEA (0.35 mL, 2.85 mmol) were added at 0 °C and mixture was allowed to stir at same temperature for 30 min. The reaction mixture was diluted with CH ₂ C1 ₂ (20 mL) and washed with water (20 mL), brine solution (20 mL). The organic layer was dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated under reduced pressure. The residue was purified by combiflash column chromatography (silica gel; eluent: 20%-25% EtOAc/hexanes) to provide ester 3-10 (135 mg) as white solid.

(R)- 1 - {(i?)-2-Hydroxy-3 ,3 -dimethylbutanoyl } -3 ,3 -dimethyl- 1 ,3 -azasilolidine-5- carboxylic acid (3-1 1)

The solution of LiOH*H ₂ 0 (51 mg, 1.23 mmol) in water (1 mL) was added drop wise to the solution of silaproline ester 3-10 (135 mg, 0.41 mmol) in MeOH (3 mL) and mixture was stirred at room temperature for 4 h. The solvent was removed at room temperature and residue was dissolved in water (10 mL). The mixture was acidified with 10% aqueous KHSO ₄ solution to pH 2 and extracted with ethyl acetate (3 ^χ 25 mL). The combined organic extract was washed with water (50 mL), brine solution (50 mL), dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated under reduced pressure to provide silaproline acid 3-1 1 (98 mg, quant) as white solid. tert-Butyl 2-(4-chloro-2-[ {(i? l - {(i?)-2-hydiOxy-3,3-dimethylbutanoyl}-3,3-dimethyl- 1 ,3-azasilolidine-5-carboxamido)methyl}phenyl]acetate (3-13)

The mixture of silaproline acid 3-1 1 (97 mg, 0.36 mmol), amine 3-12 (1 18 mg, 0.43 mmol), EDCI (83 mg, 0.43 mmol) and HOBt (57 mg, 0.43 mmol) in DMF/CH ₂ C1 ₂ (3 :2, 5 mL) was stirred at room temperature for 16 h. The solvent was removed at reduced pressure and residue was purified by reverse phase combiflash column chromatography (CI 8; eluent: 60-70% acetonitrile/water) to provide Boc protected 3-13 (103 mg) as white solid. (i?)-N-{2-(Aminomethyl)-5-chlorobenzyl}-l -{(i?)-2-hydroxy-3,3-dimethylbutanoyl}- 3,3-dimethyl-l ,3-azasilolidine-5-carboxamide-2,2,2-trifluoroacetate (3-14)

The solution of TFA (50% in CH ₂ C1 ₂ , 4 mL) was added to the solution of Boc protected intermediate 3-13 (103 mg, 0.19 mmol) and reaction mixture was stirred at room temperature for 2 h. The solvent was removed at reduced pressure and residue was azeotroped with toluene (2 x 10 mL). The crude product was purified by reverse phase combiflash column chromatography (CI 8; eluent: 30-40%

water/acetonitrile) to provide 3-14 as white solid. Ή NMR (MeOD _, 400 MHz) (δ) ppm: 7.47 (d, J= 2 Hz, 1H), 7.43-7.34 (m, 2H), 4.76 (dd, J= 4 Hz, J= 10.4 Hz, 2H), 4.60 (d, J= 14.8 Hz, 1H), 4.41 (s, 1H), 4.25-4.15 (m, 3H), 3.14-3.10 (m, 1H), 2.97 (d, J= 13.6 Hz, 1H), 1.27 (dd, J= 10.4, J= 16.2 Hz, 1H), 1.03 (dd, J= 3.6 Hz, J= 15 Hz, 1H), 0.96 (s, 9H), 0.23 (d, J= 6.4 Hz, 6H).

EXAMPLE 4

Preparation of (i?)-N-{5-Chloro-2-(lH-tetrazol-l -yl)benzyl}-l - {(i?)-2-hydroxy-3,3- dimethylbutanoyl}-3,3-dimethyl-l,3-azasilolidine-5-carboxami de (4-4)

(6i?,8ai?)-6-(te^Butyl)-2,2-dimethyldihydro-lH-[l ,3]azasilolo[5,l-c][l ,4]oxazine-

5,8(6H, 8aH)-dione (4-2)

EDCI (54 mg, 0.28 mol) and HOBt (39 mg, 0.28 mmol) were added to the stirred solution of hydroxyl acid (65 mg, 0.23 mmol) in DMF (5 mL) and reaction mixture was stirred at room temperature for 16 h. The solvent was removed at reduced pressure and residue was purified by reverse phase combiflash column chromatography (CI 8; eluent: 90% acetonitrile-water) to provide lactone 4^2 (60 mg, quant) as colorless gum. (R)-N- { 5-Chloro-2-( 1 H-tetrazol- 1 -yl)benzyl } - 1 - {(i?)-2-hydroxy-3 ,3 - dimethylbutanoyl}-3,3-dimethyl-l ,3-azasilolidine-5-carboxamide (4-4)

The mixture of lactone 4^2 (60 mg, 0.23 mmol), amine 4^3 (88 mg, 0.47 mmol) and Et ₃ N (0.071 mL, 0.47 mL) in THF (3 mL) was stirred at room temperature for 4 h. The solvent was removed at reduced pressure and residue was purified by reverse phase combiflash column chromatography (CI 8; eluent: 60% acetonitrile- water) to provide as light yellow solid. 1H NMR (MeOD , 400 MH _Z ) (δ) ppm: 9.54 (s, 1H), 7.70 (d, J= 2.4 Hz, 1H), 7.54-7.46 (m, 2H), 4.86 (dd, J = 2.8 Hz, J= 10.8 Hz, 1H), 4.38 (s, 1H), 4.18 (s, 2H), 3.13 (dd, J= 13.6 Hz, J= 68.4 Hz, 2H), 1.22-1.15 (m, 1H), 1.04 (d, J = 3.2 Hz, 15.2 Hz, 1H), 0.91 (s, 9H), 0.24 (d, J = 10.4 Hz, 6H)

EXAMPLE 5

Preparation of (2S,4S)-N- {2-(Aminomethyl)-5-chlorobenzyl} -1 - {( )-2-hydroxy-3,3- dimethylbutanoyl}-4-(trifluoromethyl)pyrrolidine-2-carboxami de-2,2,2- trifluoroacetate (5-10)

(2S,4S)- 1 -(teri-Butoxycarbonyl)-4-(trifluoromethyl)pyrrolidine-2-carb oxylic acid (5_; 2)

Pd/C (80 mg, 10% by wt) was added to the stirred solution of benzyl ester 1 (800 mg, 2.15 mmol) in MeOH (20 mL) and reaction mixture was stirred under H ₂ atmosphere (1 atm) at room temperature for 16 h. The reaction mixture was diluted with EtOAc (20 mL) and filtered through pad of Celite®. The filtrate was concentrated under reduced pressure to provide acid 5-2 (550 mg) as off white solid.

(25,45)-l-[{(9H-Fluoren-9-yl)methoxy}carbonyl]-4-(trifluo romethyl)pyrrolidine-2- carboxylic acid (5-3)

A 50% solution of TFA in CH ₂ C1 ₂ (4 mL) was added o the solution of Boc proline intermediate 5^2 (550 mg, 1.94 mmol) in CH ₂ C1 ₂ (1 mL) and reaction mixture was stirred at room temperature for 3 h. The solvent was removed at reduced pressure and the residue was azeotroped with toluene (2 x 10 mL) and dried over high vacuum pump for 2 h. A solution of Fmoc-Cl (552 mg, 2.13 mmol) in 1 ,4-dioxane (4 mL) was added drop wise to the mixture of the above residue and Na ₂ C0 ₃ (61 1 mg, 5.82 mmol) in 1 : 1 dioxane/water (8 mL) and the reaction mixture was stirred at room temperature for 16 h. The solvent was removed at reduced pressure and the residue was suspended in water (20 mL). The aqueous layer was washed with MTBE (20 mL), acidified with 10% aqueous KHS0 ₄ to pH 2 and extracted with EtOAc (3 ^χ 30 mL). The combined organic extract was dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated under reduced pressure to provide Fmoc protected acid 5^3 (650 mg) as white solid.

(2,S ^, ,45)-(9H-Fluoren-9-yl)methyl 2- {(2-[ {(tert-butoxycarbonyl)amino}methyl]-5- chlorobenzyl)carbamoyl } -4-(trifluoromethyl)pyrrolidine- 1 -carboxylate (5-5)

The mixture of acid 5^3 (650 mg, 1.60 mmol), amine 5^ (516 mg, 1.92 mmol), EDCI (366 mg, 1.92 mmol), and HOBt (259 mg, 1.92 mmol) in DMF (15 mL) was stirred at room temperature for 4 h. The reaction was quenched with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic extract was dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated. The residue was purified by combiflash column chromatography (silica gel; 40% EtOAc/hexanes) to provide amide 5^5 (600 mg) as white solid. fer/-Butyl-4-chloro-2-[ {(2»S,45)-4-(trifluoromethyl)pyrrolidine-2- carboxamido } methyl]benzyl carbamate (5-6)

A solution of 50% piperidine in CH ₂ C1 ₂ (0.4 mL) was added to the stirred solution of Fmoc proline 5-5 (600 mg, 0.91 mmol) in CH ₂ C1 ₂ (5 mL) and reaction mixture was stirred at room temperature for 1 h. The solvent was removed and residue was purified by combiflash column chromatography (silica gel; 0-100%o EtOAc/hexanes and 0-1% MeOH/CH ₂ Cl ₂ ) to provide proline intermediate 5^6 (350 mg) as colorless gum. (S 1 - {(2S,4S)-2- {(2-[ {(tert-Butoxycarbonyl)amino}methyl]-5- chlorobenzyl)carbamoyl } -4-(trifluoromethyl)pyrrolidin- 1 -yl} -3 ,3 -dimethyl- 1 - oxobutan-2-yl acetate (5-8)

A solution of acid chloride 5 7 (185 mg, 0.96 mmol) in THF (3 mL) was added drop wise to the mixture of amine 5^6 (350 mg, 0.80 mmol) and Et ₃ N

(0.144 mL, 0.96 mmol) in CH2CI2 (10 mL) at 0 °C and reaction mixture was stirred at same temperature for 1 h. The reaction was quenched with water (20 mL) and extracted with CH ₂ C1 ₂ (3 ^χ 20 mL). ). The combined organic extract was dried over anhydrous Na ₂ S0 , filtered and concentrated. The residue was purified by combiflash column chromatography (silica gel; 40% EtOAc/hexanes) to provide amide 5^8 (285 mg) as colorless gum. te -Butyl-4-chloro-2- [ {(25,46)- 1 - {(,S)-2-hydroxy-3 ,3 -dimethylbutanoyl } -4- (trifluoromethyl)pyrrolidine-2-carboxamido}methyl]benzylcarb amate (5-9)

A solid K ₂ C0 ₃ (32 mg, 0.23 mmol) was added to the stirred solution of amide 5^8 (275 mg, 0.46 mmol) in MeOH (5 mL) and mixture was stirred at room temperature for 1 h. Reaction was quenched by saturated aqueous NH ₄ C1 (10 mL), and extracted with EtOAc (2 25 mL). The combined organic extract was dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated under reduced pressure. The residue was purified by reverse phase combiflash column chromatography (CI 8; 60-70% acetonitrile/water) to provide alcohol 5^ (245 mg) as colorless gum.

(2S,4S)-N- {2-(Aminomethyl)-5-chlorobenzyl}-l - {(S)-2-hydroxy-3,3- dimethylbutanoyl}-4-(trifluoiOmethyl)pyrrolidine-2-carboxami de-2,2,2- trifluoroacetate (5-10)

A 50% solution of TFA in CH ₂ C1 ₂ (6 mL) was added to the stirred solution of Boc protected amino alcohol intermediate 5^ (230 mg, 0.41 mmol) and reaction mixture was stirred at room temperature for 3 h. The solvent was removed at reduced pressure and residue was azeotrope with toluene (2 20 mL) and dried over high vacuum pump for 2 h. The crude product was purified by reverse phase combiflash (CI 8; 0-30% acetonitrile/water) to provide 5-10 as white solid. Ή NMR (MeOD , 400 MHz) (5) ppm: 7.51 (d, J = 1.4 Hz, 1H), 7.43-7.38 (m, 2H), 4.64 (d, J= 15.2 Hz, 1 H), 4.34 (dd, J= 8 Hz, J = 10.6 Hz, 1 H), 4.29-4.19 (m, 3H), 4.04 (s, 1 H), 3.65 (t, J= 10.8 Hz, 1 H), 3.25-3.18 (m, 2H), 2.56-2.49 (m, 1H), 1.99-1.91 (m, 1 H), 0.97 (s, 9H)

EXAMPLE 6

Preparation of (2,S',4i?)-N- {2-(Aminomethyl)-5-chlorobenzyl)-l- {(i?)-2-hydroxy -3,3- dimethylbutanoyl}-4-methylpyrrolidine-2-carboxamide-2,2,2-tr ifluoroacetate (6-16)

6-1 70% 50% 6-3 trichloroisocyanuric acid

TEMPO, CH ₂ C1 ₂

80% 6-4 94% 6-5 quant.

(25',4 ?)-N-ter Butyloxycarbonyl-4-hydroxy-2-hydro yrnethyl yrrolidine (6-2)

A solution of (25',4i?)-l -(ieri-butoxycarbonyl)-4-hydroxyproline & (4.95 g, 21.4 mmol) in 1 ,2-dimethoxy ethane (20 mL) was added to

isobutylchloroformate (2.92 mL, 24.75 mmol) and NMM (2.47 mL, 24.75 mmol) at - 20 °C. The resulting mixture was stirred at same temperature for 1 h. The precipitate was removed by rapid filtration under nitrogen. The filtrate was collected into a dry two-necked flask and solution of NaBH ₄ (0.91 g, 47.13 mmol) in water (30 mL) was added drop wise at 0 °C over a period of 30 min. The mixture was stirred at 0 °C for 1 h and concentrated under reduced pressure. The residue was purified by combiflash column chromatography (Si0 ₂ ; eluent: EtOAc/hexanes 7:3) to obtain the prolinol 6=2 (3.26 g) as a colorless solid.

(2 ',4i?)-2-teri-Butyldimethylsilyloxymethyl-N-teri-butyloxycar bonyl-4- hydroxypyrrolidine (6-3)

TBDMSC1 (2.49 g, 16.51 mmol) was added in portions to a mixture of the prolinol 6=2 (3.26 g, 15.01 mmol), Et ₃ N (2.51 mL, 18.01 mmol), and DMAP (183 mg, 1.50 mmol) in CH ₂ C1 ₂ (50 mL) and the mixture was stirred at room temperature for 18 h. Additional Et ₃ N (1 ml) and TBDMSC1 (0.45 g) was added and reaction mixture was further stirred at room temperature for 24 h. The mixture was diluted with CH ₂ C1 ₂ (100 mL) and washed with water (2 x 100 mL). The combined organic phase was dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated under reduced pressure. The residue was purified by combiflash column chromatography (Si0 ₂ ; eluent: EtOAc/hexanes 3 :7) to obtain the protected prolinol 6=3 (2.47 g) as a colorless oil.

(S)- 2-ieri-Butyldimethylsilyloxyiriethyl-7V-iert-butyloxycarbony lpyrrolidin-4-one (6; 4)

Trichloroisocyanuric acid (1.70 g, 7.43 mmol) was added in portions to a solution of hydroxy proline 6=3 (2.46 g, 7.43 mmol) in CH ₂ C1 ₂ (30 mL) and reaction mixture was stirred at room temperature for 10 min. with occasional sonication to facilitate dissolution. The suspension was then added TEMPO (1 15 mg, 0.74 mmol) at 0 °C, and the resulting orange colored mixture was stirred at 0 °C for 1.5 h. The mixture was diluted with CH ₂ C1 ₂ (150 mL) and washed successively with saturated aqueous NaHC0 ₃ ( 100 mL), IN aqueous HC1 (100 mL) and brine (100 mL). The colorless organic phase was dried over anhydrous MgS0 , filtered and concentrated under reduced pressure. The residue was purified by combiflash column chromatography (Si0 ₂ ; eluent: EtOAc/hexanes 1 :4) to obtain the ketone 6=4 (1.94 g) as colorless oil.

(5)-2-tert-Butyldimethylsilyloxymethyl-N-iert-butyloxycar bonyl-4- methylenepyrrolidine (6-5)

Potassium fert-butoxide (1.65 g, 14.72 mmol) was added to the suspension of methyltriphenylphosphonium bromide (5.26 g, 14.72 mmol) in THF (30 mL) and the bright yellow mixture obtained was stirred at room temperature for 1.5 h. A solution of the ketone 6=4 (1.94 g, 5.89 mmol) in THF (20 mL) was added drop wise to the above ylide. After stirring at room temperature for 1 h, the reaction was quenched by slow addition of saturated aqueous NH ₄ C1 (30 mL). The solvent was removed under reduced pressure; the residue was diluted with EtOAc (150 mL) and washed successively with water (100 mL) and brine (100 mL). The organic phase was dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated under reduced pressure. The residue was purified by combiflash column chromatography (Si0 ₂ ; eluent:

EtOAc/hexanes 1 :9) to obtain 6-5 (1.81 g) as a colorless oil.

(2,S)-2-Hydroxymethyl-N-teri-butyloxycarbonyl-4-methylene pyrrolidine (6-6)

A solution of the silyl ether 6=5 (1.81 g, 5.53 mmol) in THF (40 mL) was treated with TBAF (1M in THF; 1 1.0 mL, 11.0 mmol) and stirred at room temperature for 20 h. Solvent was removed under reduced pressure and the residue was diluted with EtOAc (100 mL) and washed successively with water (3 x 100 mL) and brine (100 mL). The organic phase was dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated under reduced pressure. The residue was purified by Combiflash column chromatography (Si0 ₂ ; eluent: EtOAc/hexanes 3 :7) to obtain 6=6 (1.1 g, quant.) as a colorless oil.

(25,4i?)-N-fert-Butyloxycarbonyl-2-hydroxymethyl-4-methyl pyrrolidine (6-7)

A solution of the olefin 6=6 (1.10 g, 4.69 mmol) in CH ₂ C1 ₂ (24 mL) was purged with N ₂ for 1 min. and treated with Ir(COD)PyPCy ₃ PF ₆ (1 13 mg, 3 mol%). The mixture was immediately placed under H ₂ (1 atm.) and stirred at room temperature for 36 h. After removing the solvent under reduced pressure the residue was purified by combiflash column chromatography (Si0 ₂ ; eluent: EtOAc/hexanes 1 :4) to obtain methyl proline &J_ (0.94 g) as a pale orange oil.

(25, 4i?)-N-tert-Butyloxycarbonyl-4-methylproline (6-8)

A solution of 6^7 (0.94 g, 4.36 mmol) in 3 :2 MeCN/NaH ₂ P0 buffer pH 6.6 (50 mL) was added TEMPO (103 mg, 0.66 mmol) and wanned to 45 °C. Solutions of NaC10 ₂ (1.19 g, 13.1 mmol) in H ₂ 0 (5.64 mL) and NaClO (Clorox; 384 μί,) in H ₂ 0 (5.64 mL) were simultaneously added drop wise into the above reaction mixture over 1 h. After stirring at 45 °C for 56 h, the reaction mixture was cooled to room temperature, added saturated aqueous Na ₂ S0 ₃ solution u drop wise until it became colorless. After removing MeCN under reduced pressure the residue was basified to pH > 10 with IN aqueous NaOH, and washed with 1 :4 EtOAc/hexanes (2 x 50 mL). The aq. phase was acidified to pH < 3with 1 N HCl and extracted with 1 : 1 EtOAc/hexanes (5 x 50 mL). The combined organic phases were dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated under reduced pressure to obtain proline 6-8 (0.85 g) as a colorless oil which was used for next step without further purification.

(2S,4R)-4-Methylproline ( )

A 50% solution TFA in CH ₂ C1 ₂ (20 mL) was added drop wise to the solution of Boc protected methyl proline 6^8 (0.85 g, 3.71 mmol) in CH ₂ C1 ₂ (1 mL) and reaction mixture was stirred at room temperature for 3.5 h. The volatiles were removed under reduced pressure. The residue was azeotroped with toluene (3 x 20 mL) and dried under high vacuum overnight to obtain the amine 6^9 (479 mg, quant) as pale yellow oil which was used without purification.

(2S,4R)-N-(9H-Fluoren-9-yl)methyl-4-methylproline (6-10) A solution of Fmoc-Cl (952 mg, 3.68 mmol) in 1,4-dioxane (10 mL) was added drop wise to the mixture of amine &9 (475 g, 3.68 mmol) and K ₂ C0 ₃ (1.52 g, 1 1.0 mmol) in 1 ,4-dioxane/H ₂ 0 (1 :3, 40 mL) and mixture was stirred at room temperature for 18 h. The organic solvent was evaporated and the aqueous phase was washed with 1 :4 EtOAc/hexanes (2 x 20 mL). The aqueous layer was acidified to pH 2 with IN aqueous HC1 and extracted with CH ₂ C1 ₂ (4 x 200 mL). The combined organic phases were dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated under reduced pressure to provide acid 6-10 (1.09 g) as colorless solid which was used without purification

(2S,4i?)-(9H-Fluoren-9-yl)methyl 2-[ {(2-[ {(iert-butoxycarbonyl)amino}methyl]-5 chlorobenzyl } carbamoyl] -4-methylpyrrolidine- 1 -carboxylate (6-12)

A mixture of the acid 6-10 (955 mg, 2.71 mmol) and the amine 6-1 1 (734 mg, 2.71 mmol) in DMF (10 mL) was treated with EDC ^« HC1 (1.56 g, 8.15 mmol) and HOBt (367 mg, 2.71 mmol) and was stirred at room temperature for 1 h. The reaction mixture was quenched with H ₂ 0 (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic extracts were washed with saturated aqueous NaHC0 ₃ (2 x 100 mL), water (100 mL) and brine, dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated under reduced pressure to obtain amide 6-12 (1.65 g, quant) as a colorless solid which was used without purification. iert-Butyl-4-chloro-2-[{(25',4i?)-4-methylpyrrolidine-2-carb oxamido}methyl]benzyl carbamate (6-13)

Piperidine (0.2 mL) was added to the solution of the Fmoc-protected amine 6-12 (158 mg, 0.26 mmol) in CH ₂ C1 ₂ (3 mL) and the mixture was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure and residue was purified by reverse phase combiflash column chromatography (CI 8; eluent: 10-100% acetonitrile: water) to obtain the amine 6-13 (53 mg) as a colorless gum. (R)- 1 - {(25,4R)-2- {(2-[ {(/er/-butoxycarbonyl)amino}methyl]-5- chlorobenzyl}carbonyl]-4-methylpyrrolidin-l -yl}-3,3-dimethyl-l-oxobutan-2-yl acetate (6-15)

The mixture of acid 6 4 (0.07 g, 0.52 mmol), amine &1A (0.1 g, 0.26 mmol), HATU (0.11 g, 0.29 mmol), DIPEA (0.07 mL, 0.52 mmol) in THF (5 mL) was stirred at room temperature for 16 h. The reaction mixture was diluted with EtOAc (50 mL) and washed with saturated aqueous NaHC0 ₃ solution (20 mL), citric acid (10% aqueous solution, 20 mL), and brine solution (2 x 20 mL). The organic layer was separated, dried over anhydrous Na ₂ S0 ₄ , filtered and concentrated under reduced pressure. The residue was purified by reverse phase combiflash column chromatography (eluent: 0-80% acetonitrile: water) to obtain amide 6-15 (0.055 g) as off-white solid.

(25, 47?)-N-{2-(aminomethyl)-5-chlorobenzyl}-l -((i?)-2-hydroxy-3,3- dimethylbutanoyl)-4-methylpyrrolidine-2-carboxamide-bis(2,2, 2-trifluoroacetate) (6- 16)

Trifiuoro acetic acid (0.30 mL, 3.92 mmol) was added to the solution of Boc protected amine 6-15 (0.055 g, 0.1 1 mmol) in CH ₂ C1 ₂ (3 mL) and the reaction mixture was stirred at room temperature for 4 h. Solvent and volatile by-products were removed under reduced pressure. The residue was purified by reverse phase combiflash column chromatography (CI 8; eluent: 10-100% acetonitrile/water) to obtain 6 6 as off-white solid. Ή NMR (MeOD-d _{4 ,} 400 MH _Z ) (5) ppm: 7.47 (d, J = 1.6 Hz 1H), 7.38-7.43 (m, 2H), 4.64 (d, J= 14.8 Hz, 1H), 4.33-4.36 (dd, J= 8.8 Hz, J= 2.8 Hz, 1H), 4.16-4.27 (m, 3H), 4.07 (s, 1H), 3.94-9.98 (m, 1H), 3.18-3.22 (m, 1H), 2.45-2.55 (m, 1H), 1.97-2.02 (m, 1H), 1.78-1.86 (m, 1H), 1.05(d, J= 6.8 Hz, 3H), 0.97 (s, 9H).

EXAMPLE 7

Preparation of (S)-N-(2-(aminomethyl)-5-chlorobenzyl)-5-((R)-2-hydroxy-3,3- dimethylbutanoyl)-5-azaspiro[2.4]heptane-6-carboxamide (7-5) Following the procedure described for preparing Example 6, but

Preparation of Compound 7-4.

To a cooled (0°C) THF (20 mL) slurry of MePPreBr (1.05 g, 2.96 mmol) was added KOtBu (2.96 mL, 1M in THF) via syringe. The orange solution was stirred for 30 min and then N-Boc-4-oxo-L-proline methyl ester ΊΛ _. (719 mg, 2.96 mmol) was added dropwise as a THF solution. The reaction then turned brownish over the next 30 min while stirring at 0°C and was then warmed to room temperature. The reaction was quenched with NH4CI (sat, aq, 20 mL) and poured into a sepratory funnel. The mixture was extracted with ethyl acetate (2 x 20 mL) and dried over MgS0 ₄ . The combined organics were concentrated and purified by HPLC (ISCO, 80 g, 0 to 20% EA/Hex) to give the product 1 1 as a clear oil that crystallized after several days o

7-2 Zr3

Diethylzinc (13.2 mL, 13.2 mmol, 1M in hexanes) was added dropwise 20 minutes to a cooled (-20°C) toluene (25mL) solution of the ester 7^2 (1.06 g, 4.40 mmol). Chloroiodomethane (1.92 mL, 26.4 mmol)) was added dropwise over 10 minutes and the reaction was stirred at -20°C. After 16 h, the reaction was quenched with NaHC0 ₃ (25 mL, saturated, aqueous) and then warmed to room temperature. The mixture was filtered to remove solid and the precipitate was washed with EtOAc. The layers of the filtrate were separated and the organic phase was dried over MgS04, filtered and concentrated. The oil 7^3 was purified via HPLC using a Biotage on a 100 g SNAP column with a 0 to 50% gradient EA/Hex).

a THF (1 ml) slurry of ester 7 (510 mg, 2.0 mmol) and LiOH

(480 mg, 20 mmol) was added MeOH (1 mL) and water (1 mL). The resulting mixture was heated to 60°C. After 16 h, the reaction mixture was poured into IN HCl (20 mL) and extracted with ethyl acetate (2 x lOmL). The combined organics were dried ( gS0 ₄ ), concentrated, and purified by ISCO (0 to 40% MeOH/DCM) to form 74·

In Vitro Assay For Determining Proteinase Inhibition

Relevant in vitro assays are referenced in Morrissette, et al., Bioorg. Med. Chem. Lett. 2004, 14, 4161 164 and described in Lewis, et al. Thromb. Res. 1993, 70, 173 (assays of human a-thrombin and human trypsin), and Lewis, et al. Thromb. Haemostasis 1995, 74, 1 107-1 1 12. 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 ^" l]vH . In certain studies with potent inhibitors (¾ < 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-trifluoromethylcouma rin (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 (V ₀ ) 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 V ₀ /Vi on [I] shown in the following equation.

V ₀ /Vi = l + [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 25.0, 50.0, and 100.0 mg., respectively, of the following active compounds are prepared as illustrated below (compositions A-C). Active I is (S)-N- {2-(Aminomethyl)-5-chlorobenzyl}-4,4-difluoro-l - {(/?)-2-hydroxy- 3,3-dimethylbutanoyl}pyrrolidine-2-carboxamide-2,2,2-trifluo roacetate.

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 25.0, 50.0, and 100.0 mg, respectively, of active ingredient per tablet.

EXAMPLE 9

Tablet Preparation

Exemplary compositions of (5)-iV- {2-(Aminomethyl)-5-chlorobenzyl}-4,4-difluoro-l - {(7?)-2-hydroxy-3,3-dimethylbutanoyl}pyrrolidine-2-carboxami de-2,2,2- trifluoroacetate (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%

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 μηι) and combined in a suitable blender. The mixture is subsequently blended (typically 15 to 30 min) until the drug is uniformly distributed in the resulting dry powder blend. Magnesium stearate is screened and added to the blender, after which a precompression tablet blend is 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 formulations (5)-N-{2-(Aminomethyl)-5-chlorobenzyl}- 4,4-difluoro-l-{(i?)-2-hydroxy-3,3-dimethylbutanoyl}pyrrolid ine-2-carboxamide- 2,2,2-trifluoroacetate (Active I) are 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.