WO2004032834A2 - Thrombin inhibitors - Google Patents

Abstract

Compounds of the invention (Formula I) are useful in inhibiting thrombin and associated thrombotic occlusions having the following structure: I.The compounds are 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.

Description

TITLE OF THE INVENTION THROMBIN J-NHLBITORS

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 60/415,976, filed October 4, 2002.

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analogs of peptidase substrates in which the nitrogen atom of the scissile amide group of the substrate peptide has been replaced by hydrogen or a substituted carbonyl moiety.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.

The present invention concerns proline-based compounds having heterobiaryl substituents.

SUMMARY OF THE INVENTION

The invention includes compounds of Formula I,_and pharmaceutically acceptable salts thereof, and compositions comprising the compounds and additionally including a pharmaceutically acceptable carrier, useful 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, and 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. 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. The compositions may additionally include anticoagulants, antiplatelet agents, and thrombolytic agents.

DETAILED DESCRIPTION OF THE INVENTION AND

PREFERRED EMBODIMENTS

Compounds of the invention are useful as thrombin inhibitors and have therapeutic value in for example, preventing coronary artery disease. The invention includes compounds of Formula I:

or a pharmaceutically acceptable salt thereof, wherein

wherein Z¹ is hydrogen or fluorine and Z² is fluorine, N₃, NH₂,

OH or C_M alkyl, or

1 - R .1 - is selected from the group consisting of

1)

wherein

R .4 a _„ndj _π R5 are independently selected from the group consisting of hydrogen, halogen, C ι.₄ alkoxy, C ι_ alkyl, -OH, and cyano,

2)

wherein

R⁶ is selected from the group consisting of a) hydrogen, b) -OH, and c) -NR⁹R¹⁰, where R⁹ and R¹⁰ are independently selected from the group consisting of

1) hydrogen, and

2) C ι_₆ alkyl, unsubstituted or substituted with one or more of -OH, -COOH, C₃.₇ cycloalkyl, or COOR¹¹, where R¹¹ is C ι_₄ alkyl,

3) C₃_ cycloalkyl,

4) C(O)OR¹²,

5) C(O)R¹²,

6) C(O)NHR 1^l2, 7) SO₂R¹²,

8) C(O)NH₂, and 9) CN, wherein R¹² is selected from the group consisting of C₁.₄alkyl, aryl, and C ₃-₇ cycloalkyl, and

R⁷ and R⁸ are independently selected from the group consisting of a) hydrogen, b) -CF₃, c) unsubstituted C ι_₆ alkyl, d) a rin sleeted from the group consisting

wherein R¹³ and R¹⁴ are independently selected from the group consisting of 1) hydrogen,

2) halogen,

3) C ι_₄ alkoxy,

4) C ι_₄ alkyl,

5) hydroxy, 6) CF₃, and

7) cyano, e) C ₃_₆ cycloalkyl, f) C ι_₆ alkyl substituted with one of the group consisting of

1) C ₃_₆ cycloalkyl, unsubstituted or substituted with halogen,

2) -COOH,

3) -OH,

wherein R¹⁵ and R¹⁶ are independently selected from the group consisting of aa) hydrogen, bb) halogen, cc) C ι_₄ alkoxy, dd) C ι_₄ alkyl, ee) hydroxy, ff) CF₃ and gg) cyano, and

wherein y is 0, 1 or 2, and

T7 1 R

R and R are independently selected from the group consisting of hydrogen, halogen and C ι_₆ alkyl;

R and R are independently selected from the group consisting of 1) hydrogen,

2) halogen,

3) C ₁.₄ alkyl,

4) C ₃.₇ cycloalkyl,

5) CF₃,

6) OCF₃,

7) C ι_₄ alkoxy, and

8) cyano; and

³ is

1) a 5-membered heteroaryl ring having 2, 3, or 4 heteroatoms, provided that at least 1 heteroatom is N, and at most 1 heteroatom is S or O, said ring being unsubstituted or substituted, at any one ring atom, with Cι_₆ alkyl or halogen, or 2) a 6-membered heteroaryl ring with 1-2 nitrogen atoms, said ring being unsubstituted or substituted with with Cι_₆ alkyl or halogen.

In a class of compounds of the invention, R2 is CI, R¹⁹ is hydrogen or F, and R3 is selected from the group consisting of

In a subclass of the class of compounds, Q is independently selected from the group c

In a group of this subclass of compounds, R¹ is selected from the group consisting of

g:

and pharmaceutically acceptable salts thereof. These exemplary compounds correspond with the following compound names:

1 4-methyl-D-leucyl-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-4,4- difluoroprolinamide

2 3-methyl-D-valyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide 3 4-methyl-D-leucyl-(4S)-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-4- fluoroprolinamide

4 (4i?)-l-[(2R)-2-amino-2-cyclohexylethanoyl]-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide

5 4-methyl-D-leucyl-(4i?)-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-4- fluoroprolinamide 6 (4Λ)-l-[(2i?)-2-arnino-2-cycloρentylethanoyl]-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide

7 3 -( 1 -methylcyclopropyl)-D-alanyl-N- [5-chloro-2-( lH-tetraazol- 1 -yl)benzyl] -4,4- difluoroprolinamide 8 3-cyclohexyl-D-alanyl-(4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide

9 3-methyl-D-valyl-(4R)-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-4- fluoroprolinamide

103-cyclopropyl-D-alanyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide

11 4-methyl-D-leucyl-(4Λ)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide

12 3-(l-chlorocyclopropyl)-D-alanyl-(4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide 13 3-(l-methylcyclopropyl)-D-alanyl-(4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide

143-cyclobutyl-D-alanyl-(42?)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide i5 3-(l-methylcyclopropyl)-D-alanyl-(4S)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide

16 3-(3,3-difluorocyclobutyl)-D-alanyl-(4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide

17 3-(l-methylcycloρroρyl)-D-alanyl-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]- 4,4-difluoroprolinamide 18 3-cyclopropyl-D-valyl-(47?)-N-[5-chloro-2-(lH-l ,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide

19 D-prolyl-(4i?)-N-[5-chloro-2-(lH-l ,2,4-triazol- l-yl)benzyl] -4-fluoroprolinamide

204-methyl-D-leucyl-(3i?)-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-3- methylprolinamide 21 3,3-dimethyl-D-prolyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide

224-methyl-D-leucyl-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-5- methylprolinamide

23 (4i?)-N-[5-chloro-2-(l,3-oxazoi-5-yl)benzyl]-l-[(2R)-2-(3-chlorophenyl)-2- hydroxyethanoyl] -4-fluoroprolinamide 24 (2i?)-N¹-[2-({[5-chloro-2-(lH-tetraazol-l-yl)benzyl]amino}carbonyl)-l-(4-methyl-

D-leucyl)-2,5-dihydro-lH-pyrrol-2-yl]-N¹-[5-({[5-chloro-2-(lH-tetraazol-l- yl)benzyl]amino}carbonyl)-l-(4-methyl-D-leucyl)-2,5-dihydro-lH-pyrrol-2-yl]-4- methyl-D-leucinamide 25 (4S)-4-fluoro-D-prolyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide

27 (42?)-4-fluoro-D-prolyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide

29 4,4-difluoro-D-prolyl-(4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide

30 (45)-4-azido-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-l-[(9-hydroxy-9H- fluoren-9-yl)carbonyl]prolinamide

31 (4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoro-l-[(2R)-2-hydroxy-2- phenylethanoyl]prolinamide 32 (4S)-4-amino-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-l-[(9-hydroxy-9H- fluoren-9-yl)carbonyl]prolinamide

33 3-(l-methylcyclopropyl)-D-alanyl-(4i?)-N-[3-chloro-2-fluoro-6-(lH-l,2,4-triazol-

1 -yl)benzyl] -4-fluoroprolinamide

34 (4S)-4-amino-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-l-[(2R)-2-cyclohexyl- 2-hydroxyethanoyl]prolinamide

35 3-methyl-D-valyl-(4_JR)-N-[5-chloro-2-(l,2,5-thiadiazol-3-yl)benzyl]-4- fluoroprolinamide

364-methyl-D-leucyl-(4R)-N-(5-chloro-2-pyrazin-2-ylbenzyl)-4-fluoroprolinamide 3 (45)-4-amino-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-l-[(2R,3i?)-2-hydroxy- 3-methylpentanoyl]prolinamide

38 (4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoro-l-[(2R)-piperidin-2- ylcarbonyl]prolinamide

The compounds of the present invention, may have chiral centers and occur as racemates, racemic mixtures and as individual diastereomers, or enantiomers with all isomeric forms being included in the present invention. The compounds of the present invention may also have polymorphic crystalline forms, with all polymorphic crystalline forms being included in the present invention. The compounds of the present invention also include pharmaceutically acceptable salts of the compounds of Formula I. 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

Designation

BuLi butyl lithium

CH₂C1₂ dichloromethane

DIEA diisopropylethylamine

DMF dimethylformamide

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

EtOAc ethyl acetate

EtOH ethanol

HCI hydrochloric acid

HATU hexafluorophosphate

HOAT 1 -hydroxy-7-azabenzotriazole

LiCl lithium chloride

LiOH lithium hydroxide

MeOH methanol

MgSO₄ magnesium sulfate

NK_tOH ammonium hydroxide

NaHCO₃ sodium hydrogen carbonate

Na₂SO sodium sulfate

Pd-C palladium on activated carbon catalyst

THF tetrahydrofuran

As used herein except where noted, "alkyl" is intended to include both branched- and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms (Me is methyl, Et is ethyl, Pr is propyl, Bu is butyl); "alkoxy" represents a linear or branched alkyl group of indicated number of carbon atoms attached through an oxygen bridge; "halogen", as used herein, means fluoro, chloro, bromo and iodo; and "counterion" is used to represent a small, single negatively-charged species, such as chloride, bromide, hydroxide, acetate, trifluoroacetate, perchlorate, nitrate, benzoate, maleate, sulfate, tartrate, hemitartrate, benzene sulfonate, and the like.

The term "cycloC3-7alkyl" is intended to include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, and the like. The term "aryl" as used herein except where noted, represents 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 Cι_4 lower alkyl; hydroxy; alkoxy; halogen; amino.

A 5-membered heteroaryl ring is a stable, unsaturated, monocyclic ring containing 2, 3, or 4 heteroatoms, provided that at least 1 heteroatom is N, and at miost 1 heteroatom is S or O, either unsubstituted or substituted, at any one ring atom, with Cι_₆ alkyl or halogen. A 6-membered heteroaryl ring is a stable, unsaturated, monocyclic ring containing 1-2 nitrogen atoms, either unsubstituted or substituted with Cι_₆ alkyl or halogen. The heteroaryl rings may be attached at any heteroatom or carbon atom, provided that attachment results in the creation of a stable structure.

When the heteroaryl ring has substituents, it is understood that the substituents may be attached to any heteratom or carbon atom in the ring, provided that a stable chemical structure results.

The pyridyl N-oxide portion of the compounds of the invention are structurally depicted using conventional representations

o which have equivalent meanings.

In this specification methyl substituents may be represented by l-CH₃ or § —

' ^ς . For example, the structures

HN^ ^CH- _HN. and have equivalent meanings.

The pharmaceutically-acceptable salts of the compounds of Formula I (in the form of water- or oil-soluble or dispersible products) include the conventional non-toxic salts such as those derived from inorganic acids, e.g. hydrochloric, hydrobromoic, sulfuric, sulfamic, phosphoric, nitric and the like, or the quaternary ammonium salts which are formed, e.g., from inorganic or organic acids or bases. Examples of acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, 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.

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 are 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 DI 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 are 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 are 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 non- toxic amount of the compound desired can be employed as an anti-aggregation agent. For treating ocular build up of fibrin, the compounds may be administered 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 about 0.025-7.5 mg/kg/day, more preferably about 0.1-2.5 mg/kg/day, and most preferably about 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 about 2- 600 mg/day, more preferably about 8-200 mg/day, and most preferably about 8-40 mg/kg/day. A suitably prepared medicament for once a day administration would thus contain between about 0.8 mg and 2.4 g, preferably between about 2 mg and 600 mg, more preferably between about 8 mg and 200 mg, and most preferably between about 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 about 0.4 mg and 4 g, preferably between about 1 mg and 300 mg, more preferably between about 4 mg and 100 mg, and most preferably between about 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 about 0.025-7.5 mg/kg/day, preferably about 0.1-2.5 mg/kg/day, and more preferably about 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 about 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. Consideration should be given to the solubility of the drug in choosing an appropriate buffer. 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 invention also includes a method for treating an inflammatory disease in a patient which comprises treating the patient with a composition comprising a compound of the present invention. Such diseases include but are not limited to nephritis, systemic lupus erythematosus, rheumatoid arthritis, glomerulonephritis, and sacoidosis.

The invention is also a method for treating an inflammatory disease in a patient that comprises treating the patient with a combination comprising a compound of the invention and an NSAID, e.g., a COX-2 inhibitor. Such diseases include but are not limited to nephritis, systemic lupus, erythematosus, rheumatoid arthritis, glomerulonephritis, vasculitis and sacoidosis.

The present invention is a method for relieving pain, fever and inflammation of a variety of conditions including nephritis, systemic lupus erythematosus, rheumatoid arthritis, glomerulonephritis, sacoidosis, rheumatic fever, symptoms associated with influenza or other viral infections, common cold, low back and neck pain, dysmenorrhea, headache, toothache, sprains and strains, myositis, neuralgia, synovitis, arthritis, including rheumatoid arthritis degenerative joint diseases (osteoarthritis), gout and ankylosing spondylitis, bursitis, burns, injuries, following surgical and dental procedures in a patient by administering to the patient a therapeutically effective amount of a compound of the invention. Thrombin inhibitors may also be useful for the treatment of dementia including pre-senile and senile dementia, and in particular, dementia associated with Alzheimer Disease.

In inflammatory diseases wherein fibrin formation is prominent, the fibrin may be a determinant of the pathology. Fibrin serves as a matrix onto which inflammatory cells can migrate and adhere, (see Sherman et al., 1977 J. Exp. Med.

145:76-85; Altieri et al., 1986 J. Clin. Invest. 78:968-976; Wright et al., 1983 Proc.

Natl. Acad. Sci. 85:7734-7738; Altieri et al., 1993 J. Biol. Chem. 268;1847-1853).

Fibrin also enhances expression of the inflammatory cytokine IL-lbeta and decreases expression of IL-1 receptor antagonist by human peripheral blood mononuclear cells

(see Perez 1995 J. Immunol. 154:1879-1887). The anticoagulants warfarin and heparin attenuate delayed-type hypersensitivity reactions and experimental nephritis in animals, (see Jasain et al., Immunopathogenesis of Rheumatoid Arthritis Eds. G.S.

Panayi et al., Surrey, UK, Reedbooks, Ltd. and Halpern et al., 1965 Nature 205:257- 259). Enzymatic defibrination with ancrod diminishes the degree of experimental nephritis (Naish et al., 1972 Clin. Sci. 42:643-646) , systemic lupus erythematosus (Cole et al., 1990 Kidney Int. 37:29-35, and rheumatoid arthritis (see Busso et al., 1998 I. Clin. Invest. 102:41-50) in animals, and glomerulonephritis in man (see Kim et al., 1988 O. I. Med. 69:879-905). Additionally, intra articular injection of fibrin induces arthritis in rabbits immunized with fibrin Dumonde et al., 1961 British Journal of Experimental Pathology XLDI:373-383), and antigen-induced arthritis in mice is exacerbated in urokinase-deficient mice wherein fibrinolysis synovial fibrin is compromised (see Busso et al., 1998 J. Clin. Invest. 102:41-50).

In diseases where fibrin deposition is prominent such as, but not limited to, rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, vasculitis and sacoidosis, lowering the steady state concentration of fibrin by administration of a compound of the invention will, according to the instant invention, diminish the pathological inflammatory responses associated with these diseases. Similarly, compounds of the invention will be useful as a partial or complete substitute for conventional NSAIDs in preparations wherein they are presently co-administered with other agents or ingredients. Thus in further aspects, the invention encompasses pharmaceutical compositions for treating inflammatory diseases as defined above comprising a non-toxic therapeutically effective amount of a compound of the invention as defined above and one or more ingredients such as another pain reliever including acetominophen or phenacetin; a potentiator including caffeine; an H2-antagonist, aluminum or magnesium hydroxide, simethicone, a decongestant including phenylephrine, phenylpropanolamine, pseudophedrine, oxymetazoline, ephinephrine, naphazoline, xylometazoline, propylhexedrine, or levo- desoxyephedrine; an antiitussive including codeine, hydrocodone, caramiphen, carbetapentane, or dextramethorphan; a diuretic; a sedating or non-sedating antihistamine. In addition the invention encompasses a method of treating inflammatory diseases comprising administration to a patient in need of such treatment a non-toxic therapeutically effect amount of a compound of the invention, optionally co-administered with one or more of such ingredients as listed immediately above.

The instant invention also involves a novel combination therapy comprising the administration of a therapeutically effective amount of an NSAID such as a COX-2 inhibitor in combination with a therapeutically effective amount of a compound of the invention to a mammal, and more particularly, to a human. The combination therapy is used to treat inflammatory diseases. The instant pharmaceutical combinations comprising a compound of the invention in combination with an NSAID such as a COX-2 inhibitor include administration of a single pharmaceutical dosage formulation which contains both a compound of the invention and the NSAID, as well as administration of each active agent in its own separate pharmaceutical dosage formulation. Where separate dosage formulations are used, the compund of the invention and the NSAID can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e, sequentially. The "instant pharmaceutical combination" is understood to include all these regimens. Administration in these various ways are suitable for the present invention as long as the beneficial pharmaceutical effect of the compound of the invention and the NSAID are realized by the patient at substantially the same time. Such beneficial effect is preferably achieved when the target blood level concentrations of each active drug are maintained at substantially the same time. It is preferred that the compound of the invention and the NSAID be co-administered concurrently on a once-a-day dosing schedule; however, varying dosing schedules, such as the compound of the invention once per day and the NSAID once, twice or more times per day, or the NSAID once per day and the compound of the invention once, twice or more times per day, is also encompassed herein. A single oral dosage formulation comprised of both the compound of the invention and the NSAID is preferred. A single dosage formulation will provide convenience for the patient. The instant invention also provides pharmaceutical compositions comprised of a therapeutically effective amount of an NSAID, or a pharmaceutically acceptable salt thereof, in combination with a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. One embodiment of the instant compositions is a single composition adapted for oral administration comprised of a therapeutically effective amount of a COX-2 inhibitor in combination with a therapeutically effective amount of a compound of the invention and a pharmaceutically acceptable carrier. The combination can also be administered in separate dosage forms, each having one of the active agents. If administered in separate dosage forms, the separate dosage forms are administered such that the beneficial effect of each active agent is realized by the patient at substantially the same time.

Common NSAIDs include salicylates such as aspirin, sodium salicylate, choline salicylate, salicylsalicylic acid, diflunisal, and salsalate; indoleacetic acids such as indomethacin and sulindac; pyrazoles such as phenylbutazone, oxyphenbutazone; pyrrolealkanoic acids such as tolmetin; phenylacetic acids such as ibuprofen, feroprofen, flurbiprofen, and ketoprofen; fenamates such as mefanamic acid, and meclofenamate; oxicams such as piroxicam; and naphthaleneacetic acids such as naproxen. Cyclo-oxygenase inhibitors such as COX-1 and COX-2 inhibitors are also NSAIDs.

Employing the human whole blood COX-1 assay and the human whole blood COX-2 assay described in C. Brideau et al, Inflamm. Res. 45: 68-74 (1996), herein incorporated by reference, preferably, the compounds have a cyclooxygenase-2 IC50 of less than about 2 μM in the human whole blood COX-2 assay, yet have a cyclooxygenase-1 IC50 of greater than about 5 μM in the human whole blood COX-1 assay. Also preferably, the compounds have a selectivity ratio of cyclooxygenase-2 inhibition over cyclooxygenase-1 inhibition of at least 10, and more preferably of at least 40. The resulting selectivity may indicate an ability to reduce the incidence of common NSAJD-induced side effects. The inhibitor of cyclooxygenase-2 may be administered at a dosage level up to conventional dosage levels for NSAIDs. Suitable dosage levels will depend upon the antiinflammatory effect of the chosen inhibitor of cyclooxygenase-2, but typically suitable levels will be about 0.001 to 50 mg kg per day, preferably about 0.005 to 30mg/kg per day, and especially about 0.05 to lOmg/kg per day. The compound may be administered on a regimen of up to 6 times per day, preferably 1 to 4 times per day, and especially once per day.

The dosage regimen utilizing a compound of the invention in combination with the NSAID 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 or ester thereof employed. Since two different active agents are being used together in a combination therapy, the potency of each of the agents and the interactive effects achieved by combining them together must also be taken into account. A consideration of these factors is well within the purview of the ordinarily skilled clinician for the purpose of determining the therapeutically effective or prophylactically effective dosage amounts needed to prevent, counter, or arrest the progress of the condition.

Administration of the drug combination to the patient includes both self-administration and administration to the patient by another person. Additional active agents may be used in combination with the compound of the invention in a single dosage formulation, or may be administered to the patient in a separate dosage formulation, which allows for concurrent or sequential administration. Examples of additional active agents which may be employed include HMG-CoA synthase inhibitors; squalene epoxidase inhibitors; squalene synthetase inhibitors (also known as squalene synthase inhibitors), acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitors; probucol; niacin; fibrates such as clofibrate, fenofibrate, and gemfibrizol; cholesterol absorption inhibitors; bile acid sequestrants; LDL (low density lipoprotein) receptor inducers; vitamin B6 (also known as pyridoxine) and the pharmaceutically acceptable salts thereof such as the HCI salt; vitamin B12 (also known as cyanocobalamin); β-adrenergic receptor blockers; folic acid or a pharmaceutically acceptable salt or ester thereof such as the sodium salt and the methylglucamine salt; and anti-oxidant vitamins such as vitamin C and E and beta carotene. The thrombin inhibitors can also be co-administered with suitable anti- platelet agents, including, but not limited to, fibrinogen receptor antagonists (e.g. to treat or prevent unstable angina or to prevent reocclusion after angioplasty and restenosis), anticoagulants such as aspirin, thrombolytic agents such as plasminogen activators or streptokinase to achieve synergistic effects in the treatment of various vascular pathologies, or lipid lowering agents including antihypercholesterolemics (e.g. HMG CoA reductase inhibitors such as lovastatin and simvastatin, HMG CoA synthase inhibitors, etc.) to treat or prevent atherosclerosis. 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.

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.

Unless otherwise stated, all NMR determinations were made using 400 MHz field strength.

SCHEMES

The compounds of the present invention are essentially comprised of three subunits, referred to as the PI, P2 and P3 subunits, which are connected together by two amide bonds. The central subunit, the P2 subunit, is an amino acid. This P2 amino acid is connected at its carboxy end via an amide bond to the PI subunit, and this P2 amino acid is connected at its amino end via an amide bond to the P3 subunit. Variables R², R³, R⁶, R⁷, R⁸, Z¹ and Z² have the meanings defined above.

Scheme 1 The (2S, 4R)-4-fluoroproline is prepared from commercial (2S, 4S)-4- hydroxyproline using published literature procedures. For methyl ester formation, see Issartel, V.; Spehner, V.; Bahaji, H.; Seilles, E.; Couquelet, J. Eur. J. Med. Chem. 1996, 31(9), 717-723. For fluorination of hydroxy and ketoprolines, see Demange, L.; Menez, A.; Dugave, C. Tetrahedron Lett. 1998, 39, 1169-1172.

H 1. SOCI₂, MeOH

C0₂H 2. Boo₂0, Et3N, _rC0₂H

H CH₂CI₂

(commercial) (also commercial)

or R⁸ = NHBoc

Scheme 2 The (4S)-fluoroepimer is prepared in similar fashion from commercial (2S, 4R)-4-hydroxyproline:

Scheme 3 The 4,4-difluoroproline is prepared from the 4-ketoproline intermediate, (For methyl ester formation, see Issartel, V.; Spehner, V.; Bahaji, H.; Seilles, E.; Couquelet, J. Eur. J. Med. Chem. 1996, 31(9), 717-723. For fluorination of hydroxy and ketoprolines, see Demange, L.; Menez, A.; Dugave, C. Tetrahedron Lett. 1998, 39, 1169-1172) which is itself prepared via oxidation of either of the Boc- 4-hydroxyproline isomers:

Scheme 4 The corresponding P3-D-proline acids are prepared in similar fashion from the commercial 4-hydroxy-D-proline starting materials:

As above O _N--"'C0₂

(4 steps) Boc

2. HCI, EtOAc or TFA,

CH2CI

As above N X ""^C°² 1. EDC, HOAt,

(5 steps ^Boc Hunig's Base, DMF

via ketone) 2. HCI, EtOAc or TFA,

CH2CI

Scheme 5 The 4-amino and 4-azidoprolines are prepared from 4-hydroxyprolines:

N-(tert-butoxycarbonyl)-3,4-dehydro-L-proline is commercially available and incorporated essentially according to the protocols shown above (i.e., couple to PI, deprotect, couple to P3, deprotect). Racemic 5-methylproline is commercially available and is Boc- protected and incorporated essentially according to the protocols shown above.

Scheme 6 The 3-methylproline P2 is prepared according to chemistry described in Karoyan, P.; Chassaing, G. Tetrahedron: Asymm. 1997, 8, 2025-2032:

As above

(3-4 steps)

EXAMPLE 1 Preparation of 4-methyl-D-leucyl-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-4,4- difluoroprolinamide

Step A: 5 -chloro-2-( lH-tetraazol- 1 -yPbenzoic acid

A suspension of 2-amino-5-chloro-benzoic acid (5.0 g, 0.029 mol), trimethyl orthoformate (9.5 mL, 0.087 mol) and sodium azide (5.6 g, 0.087 mol) in glacial acetic acid (105 mL) was stirred at room temperature for 2 h. Filtration and concentration from toluene gave 5-chloro-2-(lH-tetraazol-l-yl)benzoic acid (4.0 g, 62%); 1H NMR (400 MHz, CD₃OD): δ 9.47 (s, 1H), 8.16 (d, J = 2.5 Hz, 1H), 7.83 (dd, J= 2.5, 8.5 Hz, 1H), 7.62 (d, J= 8.5 Hz, 1H).

Step B: 5-chloro-2-( lH-tetraazol- 1 -vDbenzamide

A solution of 5-chloro-2-(lH-tetraazol-l-yl)benzoic acid (2.0 g, 8.9 mmol), ammonium chloride (0.95 g, 17.8 mmol), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (3.4 g, 17.8 mmol), ΗOAt (2.4 g, 17.8 mmol) and DIEA (6.2 mL, 35.6 mmol) in DMF (26 mL) was stirred at room temperature overnight. Water was added and the reaction mixture was extracted with ethyl acetate. The combined organic layers were washed with brine. Drying and solvent evaporation gave 5-chloro-2-(lH-tetrazol-l-yl)benzamide (2.3 g); 1H NMR (400 MHz, CD₃OD): δ 9.44 (s, 1H), 7.82 (d, J = 2.3 Hz, 1H), 7.75 (dd, / = 2.3, 8.5 Hz, lH), 7.66 (d, /= 8.5 Hz, 1H).

Step C: 5-chloro-2-(lH-tetraazol-l-yl benzonitrile

To a solution of 5-chloro-2-(lH-tetrazol-l-yl)benzamide (1.0 g, 4.5 mmol) in TΗF (29 mL) was added (methoxycarbonylsulfamoyl)ammonium hydroxide, inner salt (2.1 g, 8.8 mmol) in five portions over 5 h. Water was added and the reaction mixture was extracted with ethyl acetate. The combined organic layers were washed with brine. Drying and solvent evaporation gave 5-chloro-2-tetrazol-l-yl-benzonitrile (0.83 g, 90%); 1H NMR (400 MHz, CDC1₃): δ 9.26 (s, 1H), 7.90 (d, J= 1.0 Hz, 1H,), 7.85 (m, 2H).

Step D: 1-T5 -chloro-2-( lH-tetraazol- 1 -vDphenyllmethanamine

A solution of 5-chloro-2-(lH-tetraazol-l-yl)benzonitrile (113 mg, 0.55 mmol) in ethanol saturated with ammonia (20 mL) was stirred in the presence of Raney nickel (50% slurry in water, washed with ethanol, catalytic amount) under a hydrogen atmosphere for 1 h. The reaction mixture was filtered through Celite and concentrated to give l-[5-chloro-2-(lH-tetraazol-l-yl)phenyl]methana ine (58 mg, 50%); 1H NMR (400 MHz, CDC1₃): δ 9.24 (s, 1H), 7.64 (d, J = 1.1 Hz, 1H), 7.46 ( , 1H), 7.38 (m, 1H), 3.68 (s, 2H).

Step E: Methyl (4- )-4-hvdroxy-L-prolinate hydrochloride The title compound was prepared from (4i?)-4-hydroxy-L-proline essentially according to the procedure described in Issartel, N.; Spehner, N.; Bahaji, H.; Seilles, E.; Couquelet, J. Eur. J. Med. Chem. 1996, 31 (9), 717-723.

Step F: l-tert-Butyl-2-methyl (2S, 4R)-4-hvdroxypyrrolidine-1.2-dicarboxylate To a stirred suspension of methyl (4-R)-4-hydroxy-L-prolinate hydrochloride

(9.08 g, 50.0 mmol) in CH₂C1₂ (180 mL) at room temperature was added Et₃Ν (17.4 mL, 125 mmol) and di-tert-butyl dicarbonate (12.0 g, 55.0 mmol). The mixture was stirred for 16 h at room temperature. The solvent was removed in vacuo and the residue partitioned between EtOAc and H₂O. The layers were separated and the aqueous layer was extracted once more with EtOAc. The combined organic layers were washed successively with 10% aqueous citric acid solution, saturated aqueous NaHCO₃ solution and brine. The organic solution was dried (Na₂SO₄), filtered and concentrated to give a colorless, viscous oil. 1H NMR (400 MHz, CDC1₃): δ 4.50 (br m, 1 H), 4.46 - 4.38 (m, 1 H), 3.74 (s, 3 H), 3.65 (dd, = 4.4, 11.7 Hz, 1 H), 3.57 - 3.44 (m, 1 H), 2.33 - 2.28 (m, 1 H), 2.11 - 2.05 (m, 1 H), 1.68 (br m, 1 H), 1.46 and 1.41 (s, 9 H, Boc rotamers).

Step G: 1-tert-Butyl 2-methyl (2S)-4-oxopyrrolidine- 2-dicarboxylate

A solution of l-tert-butyl-2-methyl (2S, 4R)-4-hydroxypyιτolidine-l,2- dicarboxylate (50 mmol from previous step) in anhydrous CH₂C1₂ (375 mL) was stirred with 4A molecular sieves at room temperature for 10 min. Pyridinium dichromate (22.6 g, 60.0 mmol) was added, followed by Celite. The rapidly darkening mixture was stirred under nitrogen atmosphere for 6 h. Additional pyridinium dichromate (22.6 g, 60.0 mmol) was added and the mixture was stirred for an additional 16 h. The mixture was then diluted with Et₂O and filtered through Celite. The filtrate was concentrated to a brown oil, which was purified by silica gel chromatography (50% EtOAc-hexanes) to afford the title compound as a nearly colorless oil. 1H NMR (400 MHz, CDC1₃): δ 4.77 (dd, 7 = 9.3, 39.7 Hz, 1 H), 3.96 - 3.89 (m, 2 H), 3.77 (s, 3 H), 2.96 - 2.89 (m, 1 H), 2.59 (d, 7 = 18.8 Hz, 1 H), 1.47 (s, 9 H).

Step H: 1-tβrt-Butyl 2-methyl (2S)-4,4-difluoropyrrolidine-l,2-dicarboxylate

DAST (3.4 mL, 25.6 mmol, 5 equiv.) was added rapidly dropwise to 1 -tert- butyl 2-methyl (2S)-4-oxopyrrolidine-l,2-dicarboxylate (1.24 g, 5.11 mmol) at 0 °C. The resulting orange solution was stirred while warming the mixture to room temperature. After 4 h the mixture was diluted with CH₂C1₂ (40 mL) and transferred to a separatory funnel. The solution was added slowly dropwise from the funnel to a cold, saturated aqueous solution of Na₂CO₃. The layers were separated and the aqueous layer was further extracted with CH₂C1₂. The combined organic layers were washed with brine, dried (anhydrous Na₂SO₄) and concentrated to a brown oil. Silica gel chromatography (20% EtOAc-hexanes) afforded the title compound as a light orange oil after drying in vacuo. 1H NMR (400 MHz, CDC1₃): δ 4.50 (ddd, J = 5.2, 8.8, 39.6 Hz, 1 H), 3.92 - 3.75 (m, 5 H), 2.78 - 2.62 ( , 1 H), 2.46 (qd, 7= 5.2, 14.0 Hz, 1 H), 1.47 and 1.42 (s, 9 H, Boc rotamers).

Step I: l-(tert-Butoxycarbonyl -4.4-difluoro-L-proline

Lithium hydroxide monohydrate (127 mg, 3.03 mmol) was added to a stirred solution of 1-tert-butyl 2-methyl (2S)-4,4-difluoropyrrolidine-l,2-dicarboxylate (618 mg, 2.33 mmol) in THF (50 mL). Water (50 mL) was added and the resulting yellow solution was stirred at room temperature for 16 h. The volatiles were removed in vacuo and the resulting cloudy aqueous residue was cooled to 0 °C and acidified to pH 2 by dropwise addition of 1 N HCI (3.2 mL). The cold mixture was extracted three times with EtOAc, saturating the aqueous layer with NaCl before each extraction. The organic extract was dried (anhydrous Na₂SO₄) and concentrated to afford the title compound as a pale yellow solid. See also Demange, L.; Menez, A.; Dugave, C. Tetrahedron Lett. 1998, 39, 1169 - 1172 for spectral data for this compound. 1H NMR (400 MHz, CD₃OD): δ 4.52 - 4.45 (m, 1 H), 3.87 - 3.70 (m, 2 H), 2.92 - 2.76 (m, 1 H), 2.55 - 2.44 (m, 1 H), 1.48 and 1.44 (s, 9 H, Boc rotamers).

Step J: l-(tert-Butoxycarbonyl -N-r5-chloro-2-(lH-tetraazol-l-yl)benzyll-4.4- difluoro-L-prolinamide

A mixture of l-(tert-butoxycarbonyl)-4,4-difluoro-L-proline (200 mg, 0.80 mmol), l-[5-chloro-2-(lH-tetraazol-l-yl)phenyl]methanamine (Step D above, 179 mg, 0.85 mmol), EDC (229 mg, 1.19 mmol) and ΗOAt (54 mg, 0.40 mmol) in DMF (4.4 mL) was stirred at room temperature for 2 h. The DMF was removed in vacuo and the residue was partitioned between EtOAc and saturated aqueous K₂CO₃. The layers were separated and the organic layer was washed with brine. The combined aqueous layers were extracted once with CΗ₂C1₂ and the combined organic extract was then dried (anhydrous Νa₂SO₄) and concentrated to an orange oil. Silica gel chromatography (60% EtOAc-hexanes) afforded the title compound as a white foam. 1H NMR (300 MHz, CDC1₃): δ 8.98 (s, 1 H), 7.65 (br s, 1 H), 7.48 -7.26 (m, 3 H), 4.45 (br m, 1 H), 4.32 - 4.20 (m, 2 H), 3.88 - 3.69 (m, 2 H), 2.90 (br m, 1 H), 2.55 (br m, 1 H), 1.54 (br s, 9 H). LCMS (M+H): 443.1.

Step K: N- \5 -chloro-2-( lH-tetraazol- 1 -yDbenzyll -4.4-difluoro-L-prolinamide

To a solution of l-(tert-butoxycarbonyl)-N-[5-chloro-2-(lH-tetraazol-l- yl)benzyl]-4,4-difluoro-L-prolinamide (309 mg, 0.70 mmol) in EtOAc (2 mL) at 0 °C was added a freshly-prepared saturated solution of ΗC1 in EtOAc (10 mL). The mixture was warmed to room temperature over 30 minutes, and was then stirred at room temperature for an additional 30 minutes. Another 5 mL of the saturated ΗC1- EtOAc solution was added and the mixture was stirred at room temperature for 16 h. The solvent was removed in vacuo, Et₂O was added and the solvent was again removed in vacuo to afford the title compound as a white solid. 1H ΝMR (400 MHz, CD₃OD): δ 9.56 (s, 1 H), 8.85 (s, 1 H), 7.70 (d, 7 = 2.3 Hz, 1 H), 7.61 (dd, 7 = 2.3, 8.5 Hz, 1 H), 7.54 (d, 7 = 8.5 Hz, 1 H), 4.57 (apparent t, 7 = 8.7 Hz, 1 H), 4.34 (br s, 2 H), 3.88 - 3.74 (m, 2 H), 3.14 - 2.88 (m, 1 H), 2.65 - 2.51 (m, 1 H). LCMS (M+H): 343.0.

Step L : N-(tert-butoxycarbonyl)-4-methyl-D-leucyl-N-r5-chloro-2-dH- tetraazol- 1 -vDbenzyll -4.4-difluoro-L-prolinamide

The title compound was prepared from N-[5-chloro-2-(lH-tetraazol-l- yl)benzyl]-4,4-difluoro-L-prolinamide (234 mg, 0.56 mmol), N-(tert-butoxycarbonyl)- 4-methyl D- leucine (145 mg, 0.59 mmol), EDC (162 mg, 0.84 mmol) and ΗOAt (77 mg, 0.56 mmol) in DMF (4 mL) essentially according to the procedure described in Step J above, with the addition of Hunig's Base (206 mL, 1.18 mmol). The title compound was isolated by silica gel chromatography (70% EtOAc-hexanes) as a white foam. 1H ΝMR (400 MHz, CDC1₃): δ 9.04 (s, 1 H), 7.71 (br s, 1 H), 7.58 (d, 7 = 2.2 Hz, 1 H), 7.41 (dd, 7= 2.4, 8.4 Hz, 1 H), 7.24 (m, 1 H), 5.00 (d, 7= 5.1 Hz, 1 H), 4.84 (dd, 7= 2.4, 9.7 Hz, 1 H), 4.33 - 4.10 (m, 3 H), 3.95 - 3.85 (m, 1 H), 2.88 - 2.79 (m, 1 H), 2.67 - 2.52 (m, 1 H), 1.64 (m, 2 H), 1.31 (br s, 9 H), 1.02 (br s, 9 H). LCMS (M+H): 570.0.

Step M: 4-Methyl-D-leucyl-N-{5-chloro-2-flH-tetraazol-l-yl')benzyll-4.4- difluoro-L-prolinamide Trifluoroacetic acid (3 mL) was added to a stirred solution of N-(tert- butoxycarbonyl)-4-methyl-D-leucyl-N-{5-chloro-2-(lH-tetraazol-l-yl)benzyl]-4,4- difluoro-L-prolinamide (174 mg, 0.31 mmol) in CΗ₂C1₂ (9 mL). The solution was stirred for 30 min at room temperature. The solvent was removed in vacuo to give an oily solid which was taken up in DMF and purified by reverse phase HPLC (Example 37). The product fractions were concentrated to afford the title compound as an oily residue which was suspended in Et₂O. The solvent was removed in vacuo, and the process of suspension/evaporation repeated until the compound was obtained as a white foamy solid. 1H ΝMR (400 MHz, CD₃OD): δ 9.52 (s, 1 H), 7.74 (d, 7 = 2.0 Hz, 1 H), 7.60 - 7.57 (m, 1 H), 7.52 - 7.50 (m, 1 H), 4.55 - 4.47 (m, 1 H), 4.36 (d, 7 = 15.6 Hz, 1 H), 4.21 - 4.00 (m, 4 H), 2.87 - 2.74 (m, 1 H), 2.47 - 2.35 (m, 1 H), 1.95 (dd, 7 = 6.0, 14.8 Hz, 1 H), 1.67 (dd, 7 = 6.8, 14.8 Hz, 1 H), 1.01 (s, 9 H). HRMS (ESI, M+H): 470.1872 (found); 470.1883 (calculated).

EXAMPLE 2 Preparation of 4-methyl-D-leucyl-(4S)-N- [5 -chloro-2-( lH-tetraazol- 1 -yl)benzyl] -4- fluoro-L-prolinamide

Step A: 1-tert-Butyl 2-methyl (2S. 4S)-4-fluoropyrrolidine-L2-dicarboxylate

A solution of l-tert-butyl-2-methyl (2S,4R)-4-hydroxypyrrolidine-l,2- dicarboxylate (l.Olg, 4.12 mmol, 1 equiv., Example 1, Step F) in anhydrous CΗ₂C1₂ was cooled to -78 °C and treated with DAST (0.57 mL, 4.32 mmol, 1.05 equiv.). The resulting orange solution was stirred under nitrogen while warming to room temperature overnight. The mixture was then diluted with CH₂C1₂ and washed with saturated aqueous ΝaHCO₃. The aqueous layer was extracted once with CH C1₂ and the combined organics were then washed with brine, dried (Na₂SO₄) and concentrated to an orange oil. The product was purified by normal phase MPLC with a solvent gradient of 89:10:1 to 75:10:15 hexane:CH₂Cl₂:iPrOH over 17 minutes to separate the starting material from the product and another impurity. The mixture was then further purified by silica gel chromatography eluting with 25% EtOAc-hexanes to give the title compound as a clear, colorless oil. 1H NMR (400 MHz, CDC1₃): δ 5.20 (app d, 7 = 52.7 Hz, 1 H), 4.54 and 4.42 (app d, 7 = 9.7 Hz, 1 H, rotamers), 3.93 - 3.56 (br m, 5 H), 2.53 - 2.24 (br m, 2 H), 1.48 and 1.43 (br s, 9 H, Boc rotamers). The relevant chemistry is also described in Demange, L.; Menez, A.; Dugave, C. Tetrahedron Lett. 1998, 39, 1169-1172. Step B: (4S)-l-(tert-butoxycarbonyl -4-fluoro-L-proline

The title compound was prepared from 1 -tert-butyl 2-methyl (2S, 4S)-4- fluoropyrrolidine-l,2-dicarboxylate essentially according to the procedure described in Example 1, Step I. 1H NMR (500 MHz, CDC1₃): δ 5.28 - 5.16 (dt, 7 = 4.0, 48.5 Hz, 1 H), 4.55 - 4.48 (br m, 1 H), 3.79 - 3.56 (br m, 2 H), 2.90 - 2.84 (br m, 1 H), 2.56 - 2.23 (br m, 1 H), 1.50 and 1.45 (br s, 9 H, Boc rotamers).

Step C : (4S -N-r5-chloro-2-(lH-tetraazol-l-yl)benzyll-4-fluoro-L-prolinamide The title compound was prepared from (4S)-l-(tert-butoxycarbonyl)-4-fluoro-

L-proline (212 mg, 0.91 mmol), l-[5-chloro-2-(lH-tetraazol-l- yl)phenyl]methanamine (190 mg, 0.91 mmol, 1.0 equiv.), EDC (261 mg, 1.36 mmol, 1.5 equiv.) and ΗOAt (62 mg, 0.45 mmol, 0.5 equiv.) in DMF (2 mL) followed by deprotection in TFA-CΗ₂C1₂ essentially according to the procedures described in Example 1, Steps J and M . Purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CΝ (+0.1% TFA) to 50:50 water (+0.1% TFA)/CH₃CN (+0.1% TFA)] followed by lyophilization afforded the title compound as a white powder. 1H NMR (400 MHz, CD₃OD): δ 9.53 (s, 1 H), 7.63 (d, 7 = 2.0 Hz, 1 H), 7.55 (dd, 7= 2.4, 8.4 Hz, 1 H), 7.49 (d, 7= 8.8 Hz, 1 H), 5.46-5.32 (m, 1 H), 4.48 (dd, 7 = 3.6, 10.0 Hz, 1 H), 4.30 (s, 2 H), 3.76-3.67 (m, 1 H), 3.57-3.43 (m, 1 H), 2.74-2.64 (m, 1 H), 2.48-2.42 (m, 1 H). LCMS (M+H): 325.0.

Step D: 4-Methyl-D-leucyl-(^'4SVN-r5-chloro-2-αH-tetraazol-l-yl benzyll-4- fluoro-L-prolinamide The title compound was prepared from N-(tert-butoxycarbonyl)-4-methyl-D- leucine (28 mg, 0.11 mmol), (4S)-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-4-fluoro- L-prolinamide (50 mg, 0.11 mmol, 1.0 equiv.), EDC (33 mg, 0.17 mmol, 1.5 equiv.) and ΗOAt (8 mg, 0.06 mmol, 0.5 equiv.) in DMF (1 mL) followed by deprotection in TFA-CΗ₂C1₂ essentially according to the procedure described in Example 1, Steps J and M. Purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CN (+0.1% TFA) to 5:95 water (+0.1% TFA)/CH₃CN (+0.1% TFA)] afforded the title compound as a white powder. 1H NMR (300 MHz, CD₃OD): δ 9.51 (s, 1 H), 7.69 (d, 7= 1.8 Hz, 1 H), 7.55 (dd, 7= 2.1, 8.4 Hz, 1 H), 7.49 (d, 7= 8.4 Hz,

1 H), 5.45-5.25 (m, 1 H), 4.55 (dd, 7= 9.0 Hz, 1 H), 4.35-4.30 (m, 1 H), 4.20-4.06 (m,

2 H), 3.97-3.81 (m, 1 H), 2.59-2.33 (m, 2 H), 2.03-1.92 (m, 2 H), 1.70-1.63 (m, 1 H), 1.02 (s, 9 H). LCMS (M+H): 452.2.

EXAMPLE 3 Preparation of 4-methyl-D-leucyl-(4R)-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-4- fluoro-L-prolinamide

Step A; 1-tert-butyl 2-methyl (2R,4i?V4-fluoropyrrolidine-L2-dicarboxylate

DAST (0.81 mL, 6.12 mmol, 1.5 equiv.) was added to a solution of 1-tert- butyl-2-methyl (2S,4S)-4-hydroxypyrrolidine-l,2-dicarboxylate (Chem-Impex, l.OOg, 4.08 mmol, 1 equiv.) in anhydrous CΗ₂C1₂ (20 mL) at -78 °C under nitrogen. The solution was warmed to room temperature with stirring overnight. The mixture was diluted with CH₂C1₂, washed with saturated aqueous ΝaHCO₃ and brine and the organic layer was dried (Na₂SO₄), filtered and concentrated to an orange oil. Silica gel chromatography eluting with 25% EtOAc-hexanes afforded the title compound as a nearly colorless oil. 1H NMR (400 MHz, CDC1₃): δ 5.21 (dt, 7 = 3.2, 52.8 Hz, 1 H), 4.47 and 4.40 (dd, 7 = 8.0, 9.2 Hz, 1 H, rotamers), 3.96 - 3.75 (br m, 4 H), 3.67 and 3.56 (dd, 7 = 3.6, 13.2 Hz, 1 H, rotamers), 2.63 - 2.54 (br m, 1 H), 2.18 - 2.03 (br m, 1 H), 1.47 and 1.42 (s, 9 H, Boc rotamers).

Ste B: (4R)-l-(tert-butoxycarbonylV4-fluoro-L-proline A solution of 1-tert-butyl 2-methyl (2R,4- )-4-fluoropyrrolidine-l,2- dicarboxylate (696 mg, 2.81 mmol, 1 equiv.) in CH₃CN (93 mL) and water (31 mL) was treated with LiOH.H₂O (236 mg, 5.63 mmol, 2.0 equiv.). The slightly cloudy, pale yellow solution was stirred at room temperature for 4 h. The CH₃CN was removed in vacuo and the residual aqueous mixture was cooled to 0 °C and treated with aqueous 1 N HCI (5.63 mL) dropwise. The mixture was extracted with EtOAc (three times, saturating the aqueous layer with NaCl before each extraction) and the combined organic extracts were dried (Na₂SO₄) and concentrated to a viscous oil which gave a white foam after prolonged drying under high vacuum. 1H NMR (400 MHz, CD₃OD): δ 5.49 (app dd, 3.2, 52.8 Hz, 1 H), 4.37 - 4.29 (m, 1 H), 3.83 - 3.72 (m, 1 H), 3.63 - 3.50 (m, 1 H), 2.66 - 2.56 (m, 1 H), 2.24 - 2.07 (m, 1 H), 1.48 and 1.42 (s, 9 H, Boc rotamers). The relevant fluorination chemistry and spectral data for this intermediate are also described in Demange, L.; Menez, A.; Dugave, C. Tetrahedron Lett. 1998, 39, 1169-1172.

Step C: (4RVN- \5 -chloro-2-( lH-tetraazol- 1 -yDbenzyl] -4-fluoro-L-prolinamide

The title compound was prepared from (4R)-l-(tert-butoxycarbonyl)-4-fluoro-

L-proline (from Step B above) and l-[5-chloro-2-(lH-tetraazol-l- yl)phenyl]methanamine (Example 1, Step D) essentially according to the procedures described in Example 1, Steps J and K, with the addition of diisopropylethylamine (1 equiv.) in the coupling step J. The title compound was isolated as the bis-ΗCl salt.

Step D: 4-methyl-D-leucyl-(4R -N-r5-chloro-2-(^,lH-tetraazol-l-yl)benzyl1-4- fluoro-L-prolinamide A mixture of N-(tert-butoxycarbonyl)-4-methyl-D-leucine (42 mg, 0.17 mmol) , (4R)-N- [5-chloro-2-( lH-tetraazol- 1 -yl)benzyl] -4-fluoro-L-prolinamide (Example 3, Step C, 75 mg, 0.17 mmol, 1.0 equiv), EDC (49 mg, 0.26 mmol, 1.5 equiv) and ΗOAt (12 mg, 0.09 mmol, 0.5 equiv) in DMF (1 mL) was brought to pΗ 8 by dropwise addition of Hunig's base and stirred at room temperature for 18 h. The reaction was diluted with water (10 mL), and the resulting precipitate was filtered and washed with more water. The filtrate was extracted into EtOAc twice. The organic layers were combined, dried over Na₂SO₄ and concentrated in vacuo to give a solid which was then combined with the filtered precipitate. Purification by silica gel chromatography (70% EtOAc/hexanes to EtOAc) afforded the intermediate as a clear oil. The oil was dissolved in CH₂C1₂, and the protecting group was removed by stirring in the presence of concentrated HCI for 3 h. The solvent was removed in vacuo, and purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CN (+0.1% TFA) to 5:95 water (+0.1% TFA)/CH₃CN (+0.1% TFA)] afforded the title compound as a white solid. 1H NMR (400 MHz, CD₃OD): δ 9.52 (s, 1 H), 7.75 (d, 7 = 2.4 Hz, 1 H), 7.57 (dd, 7 = 2.4, 8.4 Hz, 1 H), 7.50 (d, 7 = 8.4 Hz, 1 H), 5.37 (d, 7 = 52 Hz, 1 H), 4.42-4.32 (m, 2 H), 4.22-4.12 (m, 2 H), 4.00-3.81 (m, 2 H), 2.58-2.48 (m, 1 H), 2.15-1.96 (m, 2 H), 1.65 (dd, 7= 6.0, 14.4 Hz, 1 H), 0.99 (s, 9 H). HRMS (ESI, M+H): 452.1983 (found); 452.1972 (calculated).

EXAMPLE 4 Preparation of 3-(l-methylcyclopropyl)-D-alanyl-N-[5-chloro-2-(iH-tetraazol-l- yl)benzyl]-4,4-difluoro-L-prolinamide

Step A: tert-Butyl (3R, 5R,6S)-3-(^'2-methylprop-2-envD-2-oxo-5 ,6-diphenyl- morpholine-4-carboxylate

To a stirred solution of tert-butyl (5R,6S)-2-oxo-5,6-diphenylmorpholine-4- carboxylate (1.5 g, 4.24 mmol) in dry TΗF (30 mL) was added dropwise a 1 M solution of ΝaΗMDS in TΗF (4.67 mL, 4.67 mmol) at -78°C under Ar. The mixture was stirred at this temperature for 30 min, then treated with l-bromo-2-methyl-3- propene (0.43 mL, 4.24 mmol) dropwise via syringe. The mixture was stirred at -78 °C for 30 min and the bath was then removed and the mixture stirred at room temperature overnight. The reaction was quenched by the addition of aqueous 20% NH₄CI and the THF was removed at reduced pressure. The residue was partitioned between EtOAc and H₂O, and the aqueous layer extracted with EtOAc. The combined organic layers were washed with brine, dried over Na₂SO₄ and concentrated at reduced pressure to give a residue that was dissolved in CHC1₃ and chromatographed on a 35 g RediSep column using a gradient elution of 0-50% EtOAc-hexane, 40 min at a flow rate of 40 mlJmin. The pure fractions were combined and concentrated to give 785 mg of the title compound as a colorless solid: 1H NMR (300 MHz, DMSO-d₆) mixture of rotamers: δ 7.0-7.35 (m, 8H), 6.52 (t, 7 = 6.3 Hz, 2H), 6.28-6.35 (m, 1H), 4.82-5.15 (m, 4H), 2.7-2.95 (m, 2H), 1.87 (s, 3H), 1.41 (s, 3H), 1.03 (s, 6H).

Step B: tert-ButvH3R,5R.6S)-3-rd-methylcvclopropynmethyll-2-oxo-5.6- diphenylmorpholine-4-carboxylate

To a stirred mixture of 8 mL of ether and 2.15 mL 25% KOH at 0°C was added 1 -methyl-3 -nitro- 1-nitrosoguanidine (637 mg, 4.33 mmol) in small portions. After 5 min, the aqueous layer was removed via pipette, and the yellow organic layer was added to a solution of tert-butyl (3R, 5R,6S)-3-(2-methylprop-2-enyl)-2-oxo-5,6- diphenylmorpholine-4-carboxylate (785 mg, 1.93 mmol) in THF (10 mL) at 0° C under Ar. To this stirred solution was added a very small portion of Pd(OAc) , resulting in mild gas evolution. After 10 min, a second portion of Pd(OAc)₂ was added. After 20 min, the reaction mixture was filtered through a pad of SiO₂ eluting with THF and the solvents removed at reduced pressure to give a white solid. NMR analysis indicated that this material contained approximately 50% of the starting alkene. This material was treated with double the amount of diazomethane solution prepared as described above. The reaction was allowed to stir overnight under Ar and was then quenched by the addition of a few drops of HO Ac, then filtered as above to give 900 mg of an almost colorless solid that was chromatographed on a 35 g Redi- Sep column, (loaded in CHC1₃, eluted with 0-50% EtOAc-hexane). The pure fractions were combined to give 701 mg of the title compound as a colorless solid. 1H NMR (300 MHz, DMSO-d₆) mixture of rotamers: δ 7.0-7.35 (m, 8H), 6.52 (t, 7 = 6.3 Hz, 2H), 6.28-6.35 (m, 1H), 4.85-5.18 (m, 2H), 2.5-1.95 (m, 2H), 1.40 (s, 4H), 1.22 (s, 3H), 1.01 (s, 5H).

Step C: N-(tert-Butoxycarbonyl)-3-( 1 -methylcvclopropyO-D-alanine

To a stirred solution containing ethanol (0.96 mL, 16.6 mmol), liquid ammonia (-40 mL), dry THF (20 mL) and tert-butyl (3R, 5R,6S)-3-[(l- methylcyclopropyl)methyl]-2-oxo-5,6-diphenylmorpholine-4-carboxylate (701 mg, 1.66 mmol), was added lithium wire (230 mg, 33.3 mg-atom) in small pieces until a deep blue color persisted for 10 min. The reaction was quenched by careful addition of 20% aqueous ΝH₄CI, the cold bath removed and the ammonia allowed to evaporate overnight. The reaction mixture was diluted with water and extracted with three portions of ether. The aqueous phase was cooled in ice and acidified with 2M HCI and immediately extracted with three portions of EtOAc. The combined organic layers were washed with brine, dried over Na₂SO₄ and concentrated at reduced pressure to give 347 mg of the title compound as a colorless solid: 1H NMR (300 MHz, CDC1₃): δ 4.99 (br d, 7 = 6.1 Hz, 1H), 4.35-4.45 (m, 1H), 1.84 (dd, 7 = 5.6, 14.2 Hz, 1H), 1.55 (dd, 7= 8.5, 14.2 Hz, 1H), 1.46 (s, 9H), 1.11 (s, 3H), 0.25-0.4 (m, 4H).

Step D: N-ftert-butoxycarbonvD-S-d-methylcvclopropyD-D-alanyl-N-lS- chloro-2-(iH-tetraazol-l-vDbenzyl1-4,4-difluoro-L-prolinamide

A mixture of N-(tert-butoxycarbonyl)-3-(l-methylcyclopropyl)-D-alanine (61 mg, 0.25 mmol), N-[5-chloro-2-(2H-tetraazol-l-yl)benzyl]-4,4-difluoro-L-prolinamide hydrochloride (Example 1, Step K, 91 mg, 0.24 mmol), ΗOAT (33 mg, 0.24 mmol), EDC-ΗC1 (69 mg, 0.36 mmol), and Hunig's base (88 μL, 0.50 mmol) in 1.7 mL of DMF was stirred at rt for 3.5 h. Solvent was removed in vacuo to afford the title compound, which was carried on directly to deprotection. LCMS (M + H): 568.1. Step E: 3-(l-methylcyclopropyl -D-alanyl-N-[5-chloro-2-(iH-tetraazol-l- yl)benzyl1-4.4-difluoro-L-prolinamide

To a stirred solution of N-(tert-butoxycarbonyl)-3-(l-methylcyclopropyl)-D- alanyl-N-[5-chloro-2-(iH-tetraazol-l-yl)benzyl]-4,4-difluoro-L-prolinamide (136 mg, 0.24 mmol) in CΗ₂C1₂ (2.0 mL) at 0°C was added TFA (1.0 mL, excess). The reaction was allowed to warm to rt while stirring overnight. Solvent was removed in vacuo. The remaining residue was taken up in DMF and purified by reverse phase HPLC (Example 37). After lyophilization, the TFA salt of the title compound was isolated as a white fluffy solid. LCMS (M + H): 468.0. 1H ΝMR (400 MHz, CD₃OD): δ 9.52 (s, 1 H), 8.76 - 8.75 (m, 1 H), 7.73 (d, J = 2.0 Hz, 1 H), 7.57 (dd, 7 = 2.0 Hz, 8.4 Hz, 1 H), 7.51 (d, 7= 8.4 Hz, 1 H), 4.54 (dd, J = 6.4 Hz, 9.2 Hz, 1 H), 4.39 - 4.33 (m, 1 H), 4.28 - 4.03 (br m, 4 H), 2.86 - 2.74 (m, 1 H), 2.48 - 2.36 (m, 1 H), 2.04 (dd, 7 = 6.0 Hz, 14.4 Hz, 1 H), 1.50 (dd, 7= 8.2 Hz, 14.4 Hz, 1 H), 1.16 (s, 3 H), 0.51 - 0.29 (br m, 4 H).

EXAMPLE 5 Preparation of 3 -methyl-D- valyl-(4i?)-N- [5-chloro-2-( lH-tetraazol- 1 -yl)benzyl] -4- fluoroprolinamide

A mixture of N-(tert-butoxycarbonyl)-3-methyl-D-valine (26 mg, 0.11 mmol), (4R)-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-4-fluoro-L-prolinamide (Example 3, Step C, 50 mg, 0.11 mmol, 1.0 equiv), EDC (33 mg, 0.17 mmol, 1.5 equiv) and ΗOAt (16 mg, 0.11 mmol, 1.0 equiv) in DMF (1 mL) was brought to pΗ 8 by dropwise addition of Hunig's base and stirred at room temperature for 18 h. The solvent was removed in vacuo. The resulting residue was dissolved in dichloromethane (5 mL), and the solution was saturated with hydrogen chloride gas. After stirring for 1 h, the solvent was again removed in vacuo. Purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CN (+0.1% TFA) to 50:50 water (+0.1% TFA)/CH₃CN (+0.1 % TFA)] afforded the title compound as a white solid. 1H NMR (400 MHz, CD₃OD): δ 9.54 (s, 1H), 7.77 (d, 7 = 2.4 Hz, 1 H), 7.59 (dd, 7 = 2.4, 8.4 Hz, 1 H), 7.52 (d, 7 = 8.8 Hz, 1 H), 5.35 (br, d, 7 = 52.0 Hz, 1 H), 4.47 (app t, 7 = 8.4 Hz, 1 H), 4.36-4.31 (m, 1 H), 4.26-4.12 (m, 2 H), 4.01 (s, 1 H), 3.84-3.71 ( , 1 H), 2.61-2.50 (m, 1 H), 2.15-1.99 (m, 1 H), 1.11 (s, 9 H). HRMS (ESI, M+H): 438.1813 (found); 438.1815 (calculated).

EXAMPLE 6 Preparation of 4-methyl-D-leucyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]- 4-fluoroprolinamide

Step A: 5-chloro-2-( 1H- 1 ,2,4-triazol- l-yl benzonitrile

To a solution of 2,5-dichlorobenzonitrile (10 g, 58.1 mmol) in DMF (100 ml) is added cesium carbonate (22.7 g, 69.8 mmol) and 1,2,4-triazole (4.8 g, 69.8 mmol) and the reaction mixture is stirred at 65 °C for 5.5 h, at 75 °C for 16 h, at 85 °C for 7 h. More 1,2,4-triazole (5 g) is added and the reaction mixture is stirred at 85 °C for 18 h and at 100 °C for 4h. After cooling to room temperature, the mixture is diluted with water and extracted with EtOAc 3 times. The combined organic layer is washed with aqueous LiCl, dried on sodium sulfate, concentrated in vacuo to give 5-Chloro-2- [l,2,4]triazol-l-yl-benzonitrile as a white solid which is used in the next step without further purification. Step B: 1 -f5-chloro-2-( IH- 1 ,2,4-triazol- 1 -yDphenyllmethanamine

To a suspension of 5-chloro-2-(lH-l,2,4-triazol-l-yl)benzonitrile (11.87 g, 58 mmol) in EtOΗ saturated with NΗ₃ (500 ml) was added Raney Nickel (ca. 5 pipets of suspension in water, washed / decanted with EtOH several times). The mixture was hydrogenated at 1 atmosphere for 26 h. The reaction mixture was filtered on Celite under a flow of argon and the filtrate was concentrated in vacuo. The crude product was purified by flash chromatography (silica gel, 5% MeOH containing 10% NH OH in CH₂C1₂ to 10%) to give 5-Chloro-2-[l,2,4]triazol-l-yl-benzylamine as a white solid. 1H NMR (CDCI₃, 400 MHz) δ 8.47 (s, IH); 8.14 (s, IH); 7.58 (d, J = 2.3 Hz, 1 H); 7.38 (dd, J = 2.3, 7.9 Hz, 1 H); 7.30 (d, J = 7.9 Hz, 1 H); 3.70 (s, 2 H).

Step C: (4J? -N-[5-chloro-2-(lH-1.2.4-triazol-l-yl benzyll-4-fluoroρrolinamide

The title compound was prepared from (4R)-l-(tert-butoxycarbonyl)-4-fluoro- L-proline (Example 3, Step B) and l-[5-chloro-2-(lH-l,2,4-triazol-l- yl)phenyl]methanamine essentially according to the procedures described in Example 1, Steps J and K, with the addition of diisopropylethylamine (1 equiv.) in the coupling step J. The title compound was isolated as the bis-ΗCl salt. 1H ΝMR (400 MHz, CD₃OD): δ 9.37 (s, 1 H), 8.84 (m, 1 H), 8.57 (s, 1 H), 7.66 (d, 7= 2.0 Hz, 1 H), 7.58 - 7.52 (m, 2 H), 5.47 (app dt, 7 = 3.2, 51.6 Hz, 1 H), 4.52 - 4.45 (m, 1 H), 4.42 - 4.37 (m, 2 H), 3.71 - 3.57 (m, 2 H), 2.80 - 2.69 (m, 1 H), 2.26 - 2.09 (m, 1 H). HRMS (ES, M+H): 324.1040.

Step D: 4-methyl-D-leucyl-(4J? -N-[5-chloro-2-dH-l,2.4-triazol-l-vnbenzyll- 4-fluoroprolinamide

A mixture of N-(tert-butoxycarbonyl)-4-methyl-D-leucine (62 mg, 0.25 mmol), (4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoroprolinamide (100 mg, 0.25 mmol, 1.0 equiv.), EDC (73 mg, 0.38 mmol, 1.5 equiv.) and ΗOAt (17 mg, 0.13 mmol, 0.5 equiv.) in DMF (1 mL) was brought to pΗ 8 by dropwise addition of Hunig's base and stirred at room temperature for 18 h. The solvent was removed in vacuo. The resulting residue was dissolved in dichloromethane (5 mL), and the solution was saturated with hydrogen chloride gas. After stirring for 1 h, the solvent was again removed in vacuo. Purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CN (+0.1% TFA) to 50:50 water (+0.1% TFA)/CH₃CN (+0.1 % TFA)] afforded the title compound as a white solid. 1H NMR (400 MHz, CD₃OD): 8.80 (s, 1 H), 8.22 (s, 1 H), 7.69 (d, 7 = 2.4 Hz, 1 H), 7.51 (dd, 7 = 2.4, 8.4 Hz, 1 H), 7.46 (d, 7 = 8.4 Hz, 1 H), 5.39 (br d, 7 = 52.0 Hz, 1 H), 4.47-4.38 (m, 2 H), 4.26-4.14 (m, 2 H), 4.02-3.83 (m, 2 H), 2.62-2.51 (m, 1 H), 2.18-2.01 (m, 1 H), 1.95 (dd, 7= 6.8, 14.8 Hz, 1 H), 1.67 (dd, 7= 6.0, 14.4 Hz, 1 H), 1.00 (s, 9 H). HRMS (ESI, M+H): 451.2010 (found); 451.2019 (calculated).

EXAMPLE 7 Preparation of 3-(l-methylcyclopropyl)-D-alanyl-(4R)-N-[5-chloro-2-(lH-l,2,4- triazol- 1 -yl)benzyl] -4-fluoroprolinamide

A mixture of N-(tert-butoxycarbonyl)-3-(l-methylcyclopropyl)-D-alanine (Example 4, Step C, 33 mg, 0.14 mmol), (4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide (Example 6, Step C, 60 mg, 0.14 mmol, 1.0 equiv.), EDC (39 mg, 0.21 mmol, 1.5 equiv.) and ΗOAt (9 mg, 0.07 mmol, 0.5 equiv.) in DMF (1 mL) was brought to pΗ 8 by dropwise addition of Hunig's base and stirred at room temperature for 18 h. The solvent was removed in vacuo. The resulting residue was dissolved in dichloromethane (5 mL), and the solution was saturated with hydrogen chloride gas. After stirring for 1 h, the solvent was again removed in vacuo: Purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CΝ (+0.1% TFA) to 50:50 water (+0.1% TFA)/CH₃CN (+0.1% TFA)] afforded the title compound as a white solid. 1H NMR (400 MHz, CD₃OD): δ 8.79 (s, IH), 8.21 (s, IH), 7.68 (d, 7 = 2.4 Hz, IH), 7.49 (dd, 7 = 2.4, 8.4 Hz, 1 H), 7.45 (d, 7 = 8.4 Hz, 1 H), 4.88 (br d, 7 = 52.4 Hz, 1 H), 4.47-4.38 (m, 2 H), 4.26-4.20 (m, 2 H), 4.03-3.83 (m, 2 H), 2.59-2.51 (m, 1 H), 2.18-1.97 (m, 2 H), 1.52 (dd, 7 = 8.0, 14.8, 1 H), 1.14 (s, 3 H), 0.49-0.29 (m, 4 H). HRMS (APCI, M+H): 449.1855 (found); 449.1868 (calculated).

EXAMPLE 8

Preparation of 3-(l-methylcyclopropyl)-D-alanyl-(4S)-N-[5-chloro-2-(lH-l,2,4- triazol-l-yl)benzyl]-4-fluoroprolinamide

Step A: (4S)-N-r5-Chloro-2-dH-1.2.4-triazol-l-vnbenzyl1-4-fluoro-L- prolinamide

A mixture of (4S)-l-(tert-butoxycarbonyl)-4-fluoro-L-proline (Example 2, Step B, 995 mg, 4.27 mmol), 5-chloro-2-(lH-l,2,4-triazol-l-yl)benzylamine (Example 6, Step B, 890 mg, 4.27 mmol, 1.0 equiv), EDC (1.23 g, 6.40 mmol, 1.5 equiv) and ΗOAt (290 mg, 2.13 mmol, 0.5 equiv) in DMF (3 mL) was brought to pΗ 8 by dropwise addition of Hunig's base and stirred at room temperature for 18 h. The solvent was removed in vacuo. Purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CΝ (+0.1% TFA) to 5:95 water (+0.1% TFA)/CH₃CN (+0.1% TFA) over 30 min] afforded a solid. The product was dissolved in EtOAc, and the solution was washed with saturated aqueous K₂CO₃ solution three times. The organic layer was then washed with water and brine, dried over Na₂SO₄, and concentrated in vacuo to afford the free base. The intermediate was dissolved in CH C1₂ (10 mL), and the solution was saturated with hydrogen chloride gas. After stirring for 2 h, the solvent was removed in vacuo to afford the title compound as a white solid. 1H NMR (400 MHz, CD₃OD): δ 9.64 (s, 1 H), 8.74 (s, 1 H), 7.66-7.65 (m, 1 H), 7.57-7.56 (m, 2 H), 5.42 (br d, 7= 51.2 Hz, 1 H), 4.89-4.38 (m, 3 H), 3.78- 3.70 (m, 1 H), 3.62-3.50 (m, 1 H), 2.80-2.65 (m, 1 H), 2.51-2.45 (m 1 H). HRMS (ESI, M+H): 324.1030 (found); 324.1022 (calculated).

Step B: 3-d-methylcvclopropyl -D-alanyl-(45 -N-[5-chloro-2-(lH-1.2,4- tri azol- 1 -ypbenz yl] -4-fluoroprolinamide A mixture of N-(tert-butoxycarbonyl)-3-(l-methylcyclopropyl)-D-alanine

(Example 4, Step C, 30 mg, 0.12 mmol), (4S)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl]-4-fluoro-L-prolinamide (49 mg, 0.12 mmol, 1.0 equiv.), EDC (36 mg, 0.18 mmol, 1.5 equiv.) and ΗOAt (8 mg, 0.06 mmol, 0.5 equiv.) in DMF (1 mL) was brought to pΗ 8 by dropwise addition of Hunig's base and stirred at room temperature for 18 h. The solvent was removed in vacuo. The resulting residue was dissolved in dichloromethane (5 mL), and the solution was saturated with hydrogen chloride gas. After stirring for 2 h, the solvent was again removed in vacuo. Purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CΝ (+0.1% TFA) to 50:50 water (+0.1% TFA)/CH₃CN (+0.1% TFA)] afforded the title compound as a white powder. 1H NMR (400 MHz, CD₃OD): 8.77(s, 1 H), 8.21 (s, 1 H), 7.61 (d, 7 = 2.0 Hz, 1 H), 7.47 (dd, 7 = 2.0, 8.4 Hz, 1 H), 7.42 (d, 7 = 8.4 Hz, 1 H), 5.37 (br d, 7 = 53.4 Hz, 1 H), 4.59 (d, 7 = 10.0 Hz, 1 H), 4.38-4.10 (m, 4 H), 3.99-3.87 (m, 1 H), 2.60-2.33 (m, 2 H), 2.11-2.06 (m, 1 H), 1.50-1.41 (m, 1 H), 1.16 (s, 3 H), 0.50-0.27 (m, 4 H). HRMS (APCI, M+H): 449.1857 (found); 449.1868 (calculated).

EXAMPLE 9 Preparation of 3-(l-methylcyclopropyl)-D-alanyl-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl]-4,4-difluoroprolinamide

Step A: N-r5-Chloro-2-dH-1.2,4-triazol-l-yl benzyl]-4,4-difluoro-L- prolinamide A mixture of l-(tert-butoxycarbonyl)-4,4-difluoro-L-proline (Example 1, Step

1, 1.00 g, 3.98 mmol), 5-chloro-2-(lH-l,2,4-triazol-l-yl)benzylamine (Example 6, Step B, 831 mg, 3.98 mmol, 1.0 equiv), EDC (1.14 g, 5.97 mmol, 1.5 equiv) and ΗOAt (271 mg, 1.99 mmol, 0.5 equiv) in DMF (3 mL) was brought to pΗ 8 by dropwise addition of Hunig's base and stirred at room temperature for 18 h. The solvent was removed in vacuo. Purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CΝ (+0.1% TFA) to 5:95 water (+0.1% TFA)/CH₃CN (+0.1% TFA) over 30 min] afforded a solid. The product was dissolved in EtOAc, and the solution was washed with saturated aqueous K₂CO₃ solution three times. The organic layer was then washed with water and brine, dried over Na₂SO , and concentrated in vacuo to afford the free base. The intermediate was dissolved in CH C1₂ (10 mL), and the solution was saturated with hydrogen chloride gas. After stirring for 2 h, the solvent was removed in vacuo to afford the title compound as a white solid. 1H NMR (400 MHz, CD₃OD): δ 9.48 (s, 1 H), 8.64 (s, 1 H), 7.68-7.67 (m, 1 H), 7.58-7.55 (m, 2 H), 4.67-4.62 (m, 1 H), 4.45-4.42 (m, 2 H), 3.86-3.79 (m, 2 H), 3.02-2.95 (m, 1 H), 2.63-2.57 (m, 1 H). HRMS (ESI, M+H): 342.0937 (found); 342.0928 (calculated).

Step B: 3-d-methylcvclopropyl -D-alanyl-N-r5-chloro-2-dH-L2.4-triazol-l- y benzyl] -4,4-difluoroprolinamide A mixture of N-(tert-butoxycarbonyl)-3-(l-methylcyclopropyl)-D-alanine

(Example 4, Step C, 30 mg, 0.12 mmol), N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl]-4,4-difluoro-L-prolinamide (51 mg, 0.12 mmol, 1.0 equiv.), EDC (36 mg, 0.18 mmol, 1.5 equiv.) and HOAt (8 mg, 0.06 mmol, 0.5 equiv.) in DMF (1 mL) was brought to pH 8 by dropwise addition of Hunig's base and stirred at room temperature for 18 h. The solvent was removed in vacuo. The resulting residue was dissolved in dichloromethane (5 mL), and the solution was saturated with hydrogen chloride gas. After stirring for 1 h, the solvent was again removed in vacuo. Purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CN (+0.1% TFA) to 50:50 water (+0.1% TFA)/CH₃CN (+0.1% TFA)] afforded the title compound as a white powder. 1H NMR (400 MHz, CD₃OD): 8.77 (s, 1 H), 8.21 (s, 1 H), 7.64 (s, 1 H), 7.50-7.43 (m, 2 H), 4.85-4.55 (m, 1 H), 4.43-4.37 (m, 1 H), 4.28-4.15 (m, 3 H), 4.08- 4.03 (m, 1 H), 2.83-2.77 (m, 1 H), 2.45-2.40 (m, 1 H), 2.06-2.01 (m, 1 H), 1.53-1.47 (m, 1 H), 1.14 (s, 3 H), 0.48-0.29 (m, 4 H). HRMS (ESI, M+H): 467.1760 (found); 467.1769 (calculated).

EXAMPLE 10 Preparation of 3-methyl-D-valyl-(4i?)-N-[5-chloro-2-(lH-l ,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide

A mixture of N-(tert-butoxycarbonyl)-3-methyl-D-valine (32 mg, 0.14 mmol),

(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoroprolinamide (Example 6, Step C, 60 mg, 0.14 mmol, 1.0 equiv.), EDC (39 mg, 0.21 mmol, 1.5 equiv.) and ΗOAt (9 mg, 0.07 mmol, 0.5 equiv.) in DMF (1 mL) was brought to pΗ 8 by dropwise addition of Hunig's base and stirred at room temperature for 18 h. The solvent was removed in vacuo. The resulting residue was dissolved in dichloromethane (5 mL), and the solution was saturated with hydrogen chloride gas. After stirring for 1 h, the solvent was again removed in vacuo. Purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CN (+0.1% TFA) to 50:50 water (+0.1% TFA)/CH₃CN (+0.1% TFA)] afforded the title compound as a white solid. ^!H NMR (400 MHz, CD₃OD): δ 8.82 (s, IH), 8.23 (s, IH), 7.70 (d, 7 = 2.4 Hz, IH), 7.52 (dd, 7 = 2.4, 8.4 Hz, 1 H), 7.47 (d, 7 = 8.4 Hz, 1 H), 5.36 (br d, 7 = 52.4 Hz, 1 H), 4.51 (app t, 7= 8.4 Hz, 1 H), 4.40 (d, 7 = 15.6 Hz, 1 H), 4.26 (d, 7= 16.0 Hz, 1 H), 4.21-4.13 (m, 1 H), 4.02 (s, 1 H), 3.85-3.71 (m, 1 H), 2.63-2.52 (m, 1 H), 2.18- 2.01 (m, 1 H), 1.11 (s, 9 H). HRMS (APCI, M+H): 437.1858 (found); 437.1863 (calculated).

EXAMPLE 11 Preparation of (4i?)-l-[(22?)-2-amino-2-cyclohexylethanoyl]-N-[5-chloro-2-(lH-l,2,4- triazol- 1 -yl)benzyl]-4-fluoroprolinamide

A mixture of (2R)-[(tert-butoxycarbonyl)amino](cyclohexyl)ethanoic acid (46 mg, 0.18 mmol), (4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide (Example 6, Step C, 78 mg, 0.18 mmol, 1.0 equiv.), EDC (51 mg, 0.27 mmol, 1.5 equiv.) and ΗOAt (12 mg, 0.09 mmol, 0.5 equiv.) in DMF (1 mL) was brought to pΗ 8 by dropwise addition of Hunig's base and stirred at room temperature for 18 h. The solvent was removed in vacuo. The resulting residue was dissolved in dichloromethane (5 mL), and the solution was saturated with hydrogen chloride gas. After stirring for 1 h, the solvent was again removed in vacuo. Purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CΝ (+0.1% TFA) to 50:50 water (+0.1% TFA)/CH₃CN (+0.1% TFA)] afforded the title compound as a white solid. 1H NMR (400 MHz, CD₃OD): 8.80 (s, 1 H), 8.22 (s, 1 H), 7.69 (d, 7 = 2.4 Hz, 1 H), 7.51 (dd, 7= 2.4, 8.4 Hz, 1 H), 7.46 (d, 7= 8.4 Hz, 1 H), 5.36 (br d, 7= 52.0 Hz, 1 H), 4.50 (app t, 7 = 8.4 Hz, 1 H), 4.44-4.38 (m, 1 H), 4.27-4.23 (m, 1 H), 4.09-3.95 (m , 2H), 3.85-3.72 (m, 1 H), 2.62-2.51 (m, 1 H), 2.18-1.99 (m, 1 H), 1.86-1.71 (m, 6 H), 1.33-1.13 (m, 5 H). HRMS (APCI, M+H): 463.2010 (found); 463.2019 (calculated).

EXAMPLE 12 Preparation of (4R)-l-[(2i?)-2-amino-2-cyclopentylethanoyl]-N-[5-chloro-2-(lH-l,2,4- triazol-l-yl)benzyl]-4-fluoroprolinamide

Step A: (4R)- 1-1 (2RV2- [(tert-butoxycarbonyl aminol -2-cvclopentylethanoyl I -

Ν-[5-chloro-2-(lΗ-l,2.4-triazol-l-yl benzyl]-4-fluoroprolinamide

To a stirred solution of (4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide (Example 6, Step C, 84 mg, 0.21 mmol) in DMF (5 mL) at ambient temperature under nitrogen atmosphere was added (2R)-[(tert- butoxycarbonyl)amino](cyclopentyl)ethanoic acid (51 mg, 0.21 mmol), 1- hydroxybenzotriazole hydrate (31 mg, 0.23 mmol), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (60 mg, 0.31 mmol), and diisopropylethylamine (120 μL, 0.63 mmol). The mixture was stirred at ambient temperature for 72 h and was then concentrated in vacuo. The residue was partitioned between EtOAc and saturated sodium bicarbonate solution. The organic layer was separated, dried over anhydrous sodium sulfate, and filtered. The solvent was removed under reduced pressure and the residue was purified by preparative layer plate silica gel chromatography developed with 5% MeOH in dichloromethane. The desired band was removed and eluted with 5% MeOH in dichloromethane and the solvent was removed under reduced pressure to afford the title compound as a clear colorless oil. (HPLC RT = 3.31 min, Method A; LC-MS m/z = 549.23).

Step B: (^'4R^')-l-r(2R -2-amino-2-cvclopentylethanoyll-N-r5-chloro-2-dH- 1.2,4-triazol-l-yl benzyl1-4-fluoroprolinamide

HCI gas was bubbled through a stirred solution at 0 °C of (4R)-l-{(2R)-2- [(tert-butoxycarbonyl)amino]-2-cyclopentylethanoyl}-N-[5-chloro-2-(lH-l,2,4- triazol-l-yl)benzyl]-4-fluoroprolinamide from the previous step (100 mg, 0.18 mmol) in 10 mL of EtOAc for 5 minutes. The mixture was stirred at 0 °C for 15 minutes and was then concentrated in vacuo. The title compound was obtained as a white solid. HPLC RT = 2.49 min, Method A; LC-MS m/z = 449.27; 1H NMR (400 MHz, CD₃OD): δ 9.03 (s, 1 H), 8.37 (s, 1 H), 7.71 (d, 7 = 1.7 Hz, 1 H), 7.52 (d, 7 = 8.4 Hz, 1 H), 7.48 (d, 7 = 8.4 Hz, 1 H), 5.43 and 5.30 (br s, 1 H), 4.5 (t, 7 = 8.4 Hz, 1 H), 4.42 (d, 7= 15.7 Hz, 1 H), 4.26 (d, 7= 15.7 Hz, 1 H), 4.1 (m, 2 H), 3.85 and 3.76 (dd, 7 = 2.4, 12.4 Hz, 1 H), 2.58 (m, 1 H), 2.28 (m, 1 H), 2.16 (m, 1 H), 2.06 (m, 1 H), 1.56- 1.92 (m, 6 H), 1.43 (m, 1 H).

EXAMPLE 13 , Preparation of 3-cyclohexyl-D-alanyl-(4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide

A mixture of N-(tert-butoxycarbonyl)-3-cyclohexyl-D-alanine (75 mg, 0.28 mmol) , (4R)-N- [5 -chloro-2-( IH- 1 ,2,4-triazol- 1 -yl)benzyl] -4-fluoroprolinamide (Example 6, Step C, 100 mg, 0.28 mmol, 1.0 equiv), EDC (80 mg, 0.42 mmol, 1.5 equiv) and ΗOAt (19 mg, 0.14 mmol, 0.5 equiv) in DMF (1 mL) was brought to pΗ 8 by dropwise addition of Hunig's base and stirred at room temperature for 18 h. The solvent was removed in vacuo. The resulting residue was dissolved in dichloromethane (5 mL), and the solution was saturated with hydrogen chloride gas. After stirring for 1 h, the solvent was again removed in vacuo. Purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CN (+0.1% TFA) to 50:50 water (+0.1% TFA)/CH₃CN (+0.1% TFA)] afforded the title compound as a white powder. 1H NMR (400 MHz, CD₃OD): δ 8.78 (s, 1 H), 8.21 (s, 1 H), 7.69 (d, 7 = 2.0 Hz, 1 H), 7.49 (dd, 7 = 2.0, 8.4 Hz, 1 H), 7.44 (d, 7 = 8.4 Hz, 1 H), 5.37 (br d, 7 = 53.2 Hz, 1 H), 4.49-4.38 (m, 2 H), 4.23-4.13 (m, 2 H), 3.86-3.79 (m, 2 H), 2.57-2.52 (m, 1 H), 2.13-1.98 (m, 1 H), 1.86-1.83 (m, 1 H), 1.73-1.58 (m, 6 H), 1.41-1.18 (m, 4 H), 1.03-0.96 (m, 2 H). HRMS (ESI, M+H): 477.2176 (found); 477.2176 (calculated).

EXAMPLE 14 Preparation of 3-cyclopropyl-D-alanyl-(4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide

Step A: (2R -2-[(tgrt-Butoxycarbonyl)amino1pent-4-enoic acid.

To a cold (0 °C) stirred solution of (2R)-2-aminopent-4-enoic acid (3.7 g, 32.1 mmol) in IM ΝaOΗ (34 mL, 34 mmol) was added dropwise a solution of BOC anhydride (7.72 g, 35.4 mmol) in dioxane (11 mL). Shortly after the start of the addition, the cold bath was removed. The progress of the reaction was monitored by removing aliquots, concentrating at reduced pressure and analyzing by ΝMR. After 4h, additional BOC anhydride (770 mg, 3.54 mmol) and IM ΝaOΗ (3.2 mL, 3.2 mmol) was added and stirring continued for 2.5 h. The dioxane was removed at reduced pressure, the residue cooled by the addition of ice, and acidified by the addition of 2M HCI (25 mL). The resulting mixture was extracted with two portions of EtOAc and the combined organic extracts washed with water, brine, dried over Na SO and concentrated at reduced pressure to give the title compound as slightly colored oil (4.27 g): 1H NMR (300 MHz, CDC1₃) δ 9.15 (br s, IH), 5.65-5.85 (m, IH), 5.10-5.20 (m, 2H), 5.06 (d, 7 = 8.1 Hz, 2H), 4.36-4.47 (m, IH), 2.40-2.68 (m, 2H), 1.45 (s, 9H).

Step B: Methyl N-(tgrt-butoxycarbonyl)-3-cyclopropyl-D-alaninate

To a stirred solution of (2R)-2-[(tert-butoxycarbonyl)amino]pent-4-enoic acid (2.15 g, 10.0 mmol) in CH₂C1₂ (20 mL) and MeOH (1.5 mL) was added a 2.0 M solution of trimethylsilyldiazomethane in hexane (10 L) dropwise. After the yellow color persisted and gas evolution ceased, the solution was stirred for 15 min, and then quenched with two drops of HO Ac. The reaction mixture was washed with sat. ΝaHCO₃, the aqueous layer extracted with CH C1₂, the combined organic layers dried over Na₂SO₄, treated with activated carbon and concentrated at reduced pressure to give 2.2 g of methyl (2R)-2-[(tert-butoxycarbonyl)amino]pent-4-enoate as a yellow oil. This material was dissolved in 20 mL ether, and treated with a solution of diazomethane (prepared by the portionwise treatment of a stirred mixture of 35 mL ether and 10 mL 25% KOH at 0° C with 3.32 g (22.5 mmol) 1 -methyl-3 -nitro- 1- nitrosoguanidine). The resulting cold (0 °C) stirred solution was treated with a very small portion of Pd(OAc)₂ which caused vigorous gas evolution. The cold bath was removed, and stirring continued for lh. The reaction mixture was filtered through a 1 cm pad of SiO and eluted with ether. The filtrate was concentrated to give a yellow oil that was chromatographed on a 110 g Redi-Sep column using a 0-40% EtOAc- hexane gradient over 40 min, 40 mL min. The pure fractions were combined and concentrated at reduced pressure to give 1.0 g of the title compound as a colorless oil: 1H NMR (300 MHz, CDC1₃): δ 5.10-5.20 (br d, 7= 6.1 Hz, IH), 4.34-4.43 (m, IH), 3.74 (s, 3H), 1.66 (t, 7 = 6.5 Hz, 2H), 1.45 (s, 9H), 0.65-0.75 (m, IH), 0.4-0.55 (m, 2H), 0.01-0.14 (m, 2H). Step C: N-(tert-Butoxycarbonyl -3-cyclopropyl-D-alanine

To a stirred solution of methyl N-(tert-butoxycarbonyl)-3-cyclopropyl-D- alaninate (1.0 g, 4.1 mmol) in MeOH (25 mL) at 0°C was added IM ΝaOH (5.0 mL, 5.0 mmol) dropwise. After lh, the cold bath was removed, and the reaction followed by HPLC. After 5.5 h, the methanol was removed at reduced pressure, and the residue diluted with 15 mL water and washed with ether. The aqueous layer was acidified with cold 2M HCI and extracted with three portions of EtOAc. The combined organic layers were washed with water, brine, dried over Νa₂SO₄ and concentrated at reduced pressure to give the title compound as a colorless oil (929 mg): 1H NMR (300 MHz, CDC1₃): δ 5.10-5.22 (br d, 7 = 6.1 Hz, IH), 4.35-4.46 (m, IH), 1.72 (t, 7 = 6.2 Hz, 2H), 1.39 (s, 9H), 0.70-0.85 (m, IH), 0.42-0.60 (m, 2H), 0.03-0.21 (m, 2H).

Step D: 3-cvclopropyl-D-alanyl-(4i? -N-[5-chloro-2-dH-L2,4-triazol-l- vPbenzyl] -4-fluoroprolinamide A mixture of N-(tert-butoxycarbonyl)-3-cyclopropyl-D-alanine (64 mg, 0.28 mmol), (4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoroprolinamide (Example 6, Step C, 100 mg, 0.28 mmol, 1.0 equiv), EDC (80 mg, 0.42 mmol, 1.5 equiv) and ΗOAt (19 mg, 0.14 mmol, 0.5 equiv) in DMF (1 mL) was brought to pΗ 8 by dropwise addition of Hunig's base and stirred at room temperature for 18 h. The solvent was removed in vacuo. The resulting residue was dissolved in dichloromethane (5 mL), and the solution was saturated with hydrogen chloride gas. After stirring for 1 h, the solvent was again removed in vacuo. Purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CΝ (+0.1% TFA) to 50:50 water (+0.1% TFA)/CH₃CN (+0.1% TFA)] afforded the title compound as a white powder. 1H NMR (400 MHz, CD₃OD): δ 8.71 (s, 1 H), 8.13 (s, 1 H), 7.61 (d, 7 = 2.0 Hz, 1 H), 7.41 (dd, 7= 2.0, 8.8 Hz, 1 H), 7.36 (d, 7= 8.4 Hz, 1 H), 5.28 (br d, 7= 52.0 Hz, 1 H), 4.40 (app t, 7= 9.2 Hz, 1 H), 4.34-4.30 (m, 1 H), 4.16-4.11 (m, 2 H), 4.04- 3.96 (m, IH), 3.81-3.69 (m, 1 H), 2.53-2.43 ( , 1 H), 2.09-1.95 (m, 1 H), 1.70-1.58 (m, 2 H), 0.72-0.68 (m, 1 H), 0.48-0.46 (m, 2 H), 0.13-0.04 (m, 2 H). HRMS (ESI, M+H): 435.1706 (found); 435.1706 (calculated). EXAMPLE 15 Preparation of 3-(l-chlorocyclopropyl)-D-alanyl-(4R)-N-[5-chloro-2-(lH-l,2,4- triazol- 1 -yl)benzyl] -4-fluoroprolinamide

Step A: 3-Chloro-4.5-dihydro-3H-pyrazole-3-carbonitrile

MΝΝG (18.39 g, 125 mmol) was added in portions to a stirred 2 phase mixture of ether (180 mL) and 40% potassium hydroxide solution (55 mL) at 0 C. After 15 min the ether layer was decanted into a stirred solution of 2- chloroacrylonitrile (7.98 mL, 100 mmol) in ether (300 mL) at 0 C. After 15 min acetic acid (1.50 mL) was added dropwise and the solution was dried over potassium hydroxide pellets, filtered and evaporated in vacuo (200 mm Ηg) to give the title compound as a clear oil: 1H ΝMR (CDC1₃) δ 2.40 (m, 2 Η), 4.74 (m, 1Η), 5.02 (m, 2Η).

Step B: 1 -Chlorocyclopropanecarbonitrile

A stirred solution of 3-chloro-4,5-dihydro-3H-pyrazole-3-carbonitrile from the previous step in benzene (100 mL) was heated at reflux for 16 h and then was distilled collecting the fraction boiling at 92 C (200 mm Hg) to give the title compound as an oil: 1H ΝMR (CDC1₃) δ 1.49 (m, 2 H), 1.67 (m, 2H).

Step C: Methyl (2E)-3-d-chlorocyclopropyDprop-2-enoate

DIB AL (33 mL of a 1 M solution in toluene) was added to a stirred solution of 1-chlorocyclopropanecarbonitrile (3.02 g, 29.74 mmol) in toluene (30 mL) at -78 C under nitrogen. The solution was warmed to 0 C and after 30 min was quenched with 1 M hydrochloric acid (50 mL). Concentrated hydrochloric acid (6 mL) was added to give a cloudy mixture and sodium chloride was added to saturate the aqueous layer. The layers were separated and the organic layer was dried (Na₂SO₄) and filtered, washing the solids with a minimal volume of toluene. (Carbomethoxymethylene)triphenyl-phosρhorane (9.94 g, 29.74 mmol) was added to the filtrate and the solution was stirred for one hour. The volatiles were evaporated in vacuo (12 mm Hg, rt) and hexanes were added to the residue. The solids were removed by filtration and the filtrate was evaporated in vacuo. The residue was purified by flash column chromatography on silica (1:1 methylene chloride/hexanes) to give the title compound as a crystalline solid: 1H NMR (CDC1₃) δ 1.27 (m, 2 H), 1.51 (m, 2H), 3.75 (s, 3H), 6.17 (d, J=14.9 Hz, IH), 6.53 (d, J=14.9 Hz, IH).

Step D: 3-(l-Chlorocvclopropyl)propanoic acid

A suspension of 10% palladium on carbon (180 mg) in a solution of methyl (2E)-3-(l-chlorocyclopropyl)prop-2-enoate (3.50 g, 21.8 mmol) in methanol (60 mL) was stirred under an atmosphere of hydrogen (balloon). After 16 h the mixture was filtered through Celite washing with ethyl acetate, and evaporated in vacuo. Lithium hydroxide (1.03 g, 24.5 mmol) was added to a solution of the residual oil in 1:1:1 THF/methanol/water (60 mL). After 30 min the volatiles were evaporated in vacuo and the remaining solution was washed with ether. The aqueous solution was acidified with IM hydrochloric acid and was extracted with ether. The extract was dried (Na₂SO₄) and evaporated in vacuo to give the title compound as an oil: 1H NMR (CDC1₃) δ 0.81 (m, 2 H), 1.08 (m, 2H), 1.99 (t, J=7.8 Hz, 2H), 2.69 (t, J=7.8 Hz, IH).

Step E: (4J?)-4-Benzyl-3-[3-d-chlorocyclopropyl')propanoyl1-L3-oxazolidin-2- one

Triethylamine (3.89 mL, 27.93 mmol) was added to a stirred solution of 3-(l- chlorocyclopropyl)propanoic acid (1.66 g, 11.17 mmol) in THF (130 mL) and the solution was cooled to -20 C (carbon tetrachloride/dry ice bath). Pivaloyl chloride (1.38 mL, 11.17 mmol) was added to give a precipitate. After 2 h lithium chloride (0.525 g, 12.29 mmol) and (4R)-4-benzyl-l,3-oxazolidin-2-one (1.98 g, 11.17 mmol) were added and the mixture was warmed to rt. After 16 h the mixture was filtered through a glass frit and the filtrate was evaporated in vacuo. The residual oil was partitioned between ethyl acetate and dilute hydrochloric acid. The organic layer was washed with water, sodium hydrogen carbonate solution and brine, dried (Na₂SO₄) and evaporated to an oil. The crude product was purified by flash column chromatography on silica (ethyl acetate/hexanes gradient, 20-30% ethyl acetate) to give the title compound as a heavy oil: 1H NMR (CDC1₃) δ 0.85 (m, 2 H), 1.09 (m, 2H), 2.07 (t, J=7.7 Hz, 2H), 2.77 (dd, J=9.7 and 13.4 Hz, IH), 3.21-3.33 (m, 3H), 4.20 (m, 2H), 4.68 (m, IH), 7.20-7.36 (m, 5H).

Step F: (2R -2- Azido-3 -d -chlorocvclopropyDpropanoic acid

A solution of (4R)-4-benzyl-3-[3-(l-chlorocyclopropyl)propanoyl]-l,3- oxazolidin-2-one_(1.01 g, 3.29 mmol) in THF (10 mL) was added to a stirred solution of potassium bis(trimethylsilyl)amide (7.25 mL of a 0.5 M solution in toluene, 3.62 mmol) in THF (10 mL) at -78 C under nitrogen. After 30 min a solution of 2,4,6- triisopropylbenzenesulfonyl azide (1.27 g, 4.11 mmol) in THF (10 mL) was added and followed after 1 min by acetic acid (0.866 mL, 15.13 mmol). The resulting mixture was warmed to 30 C and after 1 h was diluted with ethyl acetate and washed with brine which was basified with sodium hydrogen carbonate. The organic layer was dried (Na₂SO₄) and evaporated to an oil. Lithium hydroxide monohydrate (276 mg, 6.58 mmol) was added to a stirred solution of this crude material in 2:2:1 THF/methanol/water (25 mL) at 0 C and the reaction was warmed to rt. After 16 h excess saturated sodium hydrogen carbonate solution was added and the volatiles were evaporated in vacuo. Water was added to the mixture to dissolve the salts and the solution was washed with methylene chloride (4 times). The aqueous solution was acidified with concentrated hydrochloric acid and extracted with ether. The extract was dried (Na SO ) and evaporated in vacuo to an oil which was purified by flash column chromatography on silica (1% acetic acid/hexanes/ethyl acetate gradient, 9-29% ethyl acetate) to give the title compound as an oil: 1H NMR (CDC1₃) δ 0.80- 1.24 (m, 4H), 1.76 (dd, J=9.5 and 14.8 Hz, IH), 2.55 (dd, J=4.2 and 14.8 Hz, IH), 4.43 (dd, 1=4.4 and 9.5 Hz, IH), 8.05 (br s, IH).

Step G: (4R)- 1 - [(2R -2- Azido-3 -( 1 -chlorocyclopropyPpropanoyl] -N- [5-chloro-

2-dH-L2,4-triazol-l-yl)benzyll-4-fluoro-L-prolinamide

A mixture of (2R)-2-azido-3-(l-chlorocyclopropyl)propanoic acid (105 mg, 0.56 mmol), (4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoroprolinamide (Example 6, Step C, 200 mg, 0.56 mmol, 1.0 equiv.), EDC (160 mg, 0.83 mmol, 1.5 equiv.) and ΗOAt (38 mg, 0.28 mmol, 0.5 equiv.) in DMF (1 mL) was brought to pΗ 8 by dropwise addition of Hunig's base and stirred at room temperature for 18 h. The solvent was removed in vacuo. Purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CΝ (+0.1% TFA) to 5:95 water (+0.1% TFA)/CH₃CN (+0.1% TFA)] afforded an oil. The product was dissolved in EtOAc, and the solution was washed with saturated aqueous NaHCO₃ solution. The aqueous layer was extracted twice into EtOAc. The organic layers were combined, dried over Na SO , and concentrated in vacuo to afford the title compound as the free base. 1H NMR (400 MHz, CDC1₃): δ 8.74 (s, 1 H), 8.32 (s, 1 H), 7.60 (d, 7 = 2 Hz, 1 H), 7.43 (dd, 7 = 2.0, 8.4 Hz, 1 H), 7.29 (d, 7 = 8.8 Hz, 1 H), 5.34 (br d, 7 = 52.4 Hz, 1 H), 4.64 (app t, 7= 8.0 Hz, 1 H), 4.32 (d, 7 = 6.0 Hz, 2 H), 4.25 (dd, 7= 6.0, 7.6 Hz, 1 H), 4.16-4.08 (m, 1 H), 3.83-3.71 (m, 1 H), 2.66-2.06 (m, 3 H), 2.05-1.98 (m, 1 H), 1.20-0.77 (m, 4 H). LCMS (M+H): 495.3.

Step H: 3-(l-Chlorocyclopropyl alanyl-(^'4R)-N-[5-chloro-2-dH-1.2,4-triazol-l- yl .benzyl] -4-fluoro-L-prolinamide

To a solution of (4R)-l-[(2R)-2-azido-3-(l-chlorocyclopropyl)propanoyl]-N- [5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoro-L-prolinamide (156 mg, 0.31 mmol) in TΗF (2 mL) at 0 ° C was added triphenylphosphine (87 mg, 0.33 mmol, 1.05 equiv.). After stirring for 1 h at 0 °C, water (114 μL, 6.30 mmol, 20 eq) was added. The reaction was warmed to 40 °C for 2 h then allowed to stir at 25 °C for 66 h. Solvent was removed in vacuo, and the product was purified by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CN (+0.1% TFA) to 50:50 water (+0.1% TFA)/CH₃CN (+0.1% TFA)] to afford a diastereomeric mixture. The components were separated by chiral HPLC (Chiralcel OD 5x50 column, eluting with 1:1 A/B, where A = 0.1% diethylamine/hexanes and B = 2-propanol) affording the Diastereomer A (earlier eluting) as a white solid. Diastereomer B (later eluting) was further purified by reverse phase chromatography [95:5 water (+0.1% TFA)/CH₃CN (+0.1% TFA) to 50:50 water (+0.1% TFA)/CH₃CN (+0.1% TFA)] to an oil. Diastereomer A: 1H NMR (400 MHz, CD₃OD): 8.80 (s, H), 8.21 (s, 1 H), 7.72 (d, 7 = 1.6 Hz, 1 H), 7.48 (dd, 7= 2.0, 8.4 Hz, 1 H), 7.43 (d, 7= 8.4 Hz, 1 H), 5.37 (br d, 7 = 52.4 Hz, 1 H), 4.50-4.45 (m ,1 H), 4.38-4.27 (m, 3 H), 4.03-3.85 (m, 2 H), 2.61-2.51 (m, 1 H), 2.20-2.03 (m, 2 H), 1.94-1.88 (m, 1 H), 1.09-0.88 (m, 3 H), 0.78-0.73 (m, 1 H). HRMS (ESI, M+H): 469.1288 (found); 469.1317 (calculated). Diastereomer B: 1H NMR (300 MHz, CD₃OD): 8.82 ( s, 1 H), 8.23 (s, 1 H), 7.69 (d, 7 = 2.4 Hz, 1 H), 7.51 (dd, 7 = 2.1, 8.4 Hz, 1 H), 7.46 (d, 7 = 8.7 Hz, 1 H), 5.41 (br d, 7 = 52.2 Hz, 1 H), 4.61-4.56 (m, 2 H), 4.44-4.37 (m, 1 H), 4.25-4.10 (m, 2 H), 3.99-3.82 (m, 1 H), 2.64- 2.41 (m, 2H), 2.25-1.99 (m, 2 H), 1.21-1.11 (m, 2 H), 1.01-0.96 (m, 2 H). LCMS (M+H):469.2.

EXAMPLE 16 Preparation of 3-cyclobutyl-D-alanyl-(4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide

Step A: 3-cyclobutylalanine hydrochloride

The title compound was prepared essentially according to the procedures described in Burger, A. et al; J Med Chem.; 6; 1963; 221 - 227.

Step B: N-(tert-butoxycarbonyl -3-cyclobutylalanine

To a solution of 3-cyclobutylalanine hydrochloride (7.95 g, 44.29 mmol) in dioxane (90.0 mL), water (44.30 mL), and 1 Ν ΝaOH (48.70 mL) at 0°C was added (BOC)₂O (10.63 g, 48.72 mmol) in portions. The reaction was allowed to warm to rt over 7.5 h. The pH was determined to be 5 - 6 using short range pH paper. The pH was adjusted to 9 - 10 using 1 Ν ΝaOH and the mixture was stirred overnight.

Dioxane was removed in vacuo. The remaining aqueous portion was acidified to pH 3 using 1 Ν HCI and immediately extracted with EtOAc. The organic layer was washed with brine. The combined aqueous layers were saturated with ΝaCl and extracted with EtOAc. The combined organics were dried over Νa₂SO₄ and concentrated to give the title compound as a yellow oil. 1H NMR (CD₃OD, 400

MHz): δ 4.09 - 3.96 (m, 1 H), 2.45 - 2.39 (m, 1 H), 2.08 - 1.99 (m, 2 H), 1.92 - 1.62 (m, 6 H), 1.43 (s, 9 H). HRMS (APCI) M+H: calculated for (Cι₂H₂₁NO₄)⁺ 244.1543, found 244.1548.

Step C: N-(tert-butoxycarbonyl^')-3-cvclobutylalanyl-(4R)-N-[5-chloro-2-dH-

L2,4-triazol-l-yl)benzvn-4-fluoro-L-prolinamide

The title compound was prepared from N-(tert-butoxycarbonyl)-3- cyclobutylalanine (0.100 g, 0.41 mmol), (4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide hydrochloride (Example 6, Step C, 0.163 g, 0.41 mmol), ΗOAT (0.056 g, 0.41 mmol), EDC-ΗCl (0.118 g, 0.62 mmol), and Et₃Ν (114 μL, 0.82 mmol) essentially according to the coupling procedure described in Example 1, Step L. The product was purified by preparative reverse phase ΗPLC [gradient elution with 95:5 water (+0.1% TFA) / CΗ₃CN (+0.1% TFA) to 5:95 water (+0.1% TFA) / CH₃CN (+0.1% TFA)] to afford a pale yellow gum. 1H NMR (CD₃OD, 400 MHz): δ 8.82 (s, 1 H), 8.22 (s, 1 H), 7.69 - 7.67 (m, 1 H), 7.50 - 7.41 (m, 2 H), 5.40 - 5.27 (m, 1 H), 4.52 - 4.46 (m, 1 H), 4.30 - 4.29 (m, 2 H), 4.24 - 4.15 (m, 1 H), 3.98 - 3.70 (m, 2 H), 2.60 - 2.38 (m, 2 H), 2.17 - 2.03 (m, 3 H), 1.89 - 1.56 (m, 6 H), 1.42 and 1.36 (BOC rotamers, s, 9 H). The diastereomers were separated on Chiralpak AD 2 x 25 column with a flow of 8.0 mL / min and detection at 240 nM with the following solvent system: A = 0.1 % DEA/hexane, B = EtOH, C = MeOH and eluting with 50/25/25 : A/B/C gradient to 20/40/40 : A/B/C. Diastereomer A: LCMS (M+H): 549.4. Diastereomer B: LCMS (M+H): 549.4.

Step D: 3-cvclobutylalanyl-(4R -N-[5-chloro-2-dH-1.2.4-triazol-l-vDbenzvn-4- fluoro-L-prolinamide

Diastereomer A: To a solution of Ν-(tert-butoxycarbonyl)-3-cyclobutylalanyl- (4R)-N-[5-chloro-2-(lΗ-l,2,4-triazol-l-yl)benzyl]-4-fluoro-L-prolinamide (single diastereomer, early peak; 23 mg, 0.042 mmol) in CH₂C1₂ (1.0 mL) was added TFA (0.4 mL, excess) and stirred overnight at rt. Solvent was removed in vacuo, azeotroping with Et₂O, to give the TFA salt of the title compound as a pale yellow gum. 1H NMR (CD₃OD, 400 MHz): δ 8.82 (s, 1 H), 8.79 (br s, 1 H), 8.23 (s, 1 H), 7.52 (dd, 7 = 2.2 MHz, 8.6 MHz, 1 H), 7.47 (d, 7 = 8.4 MHz, 1 H), 5.46 - 5.33 (m, 1 H), 4.45 - 4.39 (m, 2 H), 4.26 - 4.22 (m, 1 H), 4.07 - 3.76 (m, 3 H), 2.57 - 2.42 (m, 2 H), 2.18- 2.12 (m, 2 H), 2.08 - 1.83 (m, 4 H), 1.74 - 1.68 (m, 2 H). HRMS (ES) M+H: calculated for (C₂₁H₂₆CIFN₆O₂)⁺ 449.1863, found 449.1864.

Diastereomer B (28 mg, 0.051 mmol) was deprotected with TFA as described above to afford a pale yellow gum. 1H NMR (CD₃OD, 400 MHz): δ 8.83 (s, 1 H), 8.69 (br s, 1 H), 8.23 (s, 1 H), 7.59 (d, 7= 2.0 MHz, 1 H), 7.51 (dd, 7= 2.4 MHz, 8.8 MHz, 1 H), 7.46 (d, 7 = 8.4 MHz, 1 H), 5.46 - 5.32 (m, 1 H), 4.59 - 4.55 (m, 1 H), 4.41 - 4.36 (m, 1 H), 4.27 - 4.22 (m, 1 H), 4.15 - 4.02 (m, 2 H), 3.88 - 3.76 (m, 1 H), 2.60 - 2.45 (m, 2 H), 2.21 - 1.83 (m, 6 H), 1.76 - 1.70 (m, 2 H). HRMS (ES) M+H: calculated for (C₂₁H₂₆ClFN₆O₂)⁺ 449.1863, found 449.1866. EXAMPLE 17 Preparation of 3-(3,3-difluorocyclobutyl)-D-alanyl-(4R)-N-[5-chloro-2-(lH-l,2,4- triazol-l-yl)benzyl]-4-fluoroprolinamide

Step A: 3-Oxocyclobutanecarboxylic acid

The title compound was prepared according to the procedures described in Pigou, P. E.; Schiesser, C. Η. 7. Org. Chem. 1988, 53, 3841-3843.

Step B: Ethyl 3-oxocyclobutanecarboxylate DCC (26.6 g, 129 mmol, 1.1 equiv.) was added in portions to a stirred solution of 3-oxocyclobutanecarboxylic acid (13.4 g, 117 mmol, 1 equiv.) andDMAP (11.5 g, 93.9 mmol, 0.8 equiv.) in absolute ethanol under nitrogen at 0 °C. The mixture was stirred at rt overnight and was then filtered. The orange-brown filtrate was washed successively with 0.5 M ΗC1, saturated ΝaΗCO₃ and water. The organic layer was dried (MgSO₄) and concentrated to an orange-brown oil. Silica gel chromatography (20% EtOAc-hexanes) afforded the title compound as a nearly colorless oil (product is somewhat volatile). 1H NMR (400 MHz, CDC1₃): δ 4.22 (q, 7 = 7.1 H, 2 H), 3.46 - 3.18 (br m, 5 H), 1.30 (t, 7 = 7.2 Hz, 3 H).

Step C: Ethyl 3 ,3-difluorocyclobutanecarboxylate

DAST (11.4 mL, 86.1 mmol, 3.0 equiv.) was added to a cold (0 °C) stirred solution of ethyl 3-oxocyclobutanecarboxylate (4.08 g, 28.7 mmol, 1 equiv.) in anhydrous CH₂C1₂ (150 mL) under nitrogen. The solution was allowed to warm to room temperature while stirring overnight, and was then poured over crushed ice. The layers were separated and the organic layer was washed successively with brine, saturated aqueous NaHCO₃ and more brine. The organic layer was dried (MgSO₄), filtered and concentrated to give the title compound as a red-orange oil. 1H NMR (400 MHz, CDC1₃): δ 4.19 (q, 7 = 7.2 Hz, 2 H), 2.97 - 2.77 (br m, 5 H), 1.28 (t, 7 = 7.2 Hz, 3 H).

Step D: (3,3 -DifluorocvclobutyDmethanol

A solution of ethyl 3,3-difluorocyclobutanecarboxylate (4.74 g, 28.9 mmol, 1 equiv.) in anhydrous THF (29 mL) was added via cannula to a stirred suspension of LAH (2.41 g, 63.5 mmol, 2.2 equiv.) in anhydrous THF (145 mL) at -45 °C. The resulting gray-green mixture was allowed to warm to -30 °C over 1 h, then to -10 °C over another hour while stirring under nitrogen. The reaction was quenched by the sequential addition of water (2.5 mL), 10% aqueous NaOH (2.5 mL) and more water (7.5 mL). The mixture was then diluted with EtOAc (200 mL) and stirred vigorously while warming to room temperature. After 1 h, the mixture was filtered through Celite, washing well with EtOAc. The filtrate was dried (Na SO ) and concentrated to a brown oil. Silica gel chromatography (5% MeOH-CH₂Cl₂) afforded the title compound as a dark orange oil. 1H NMR (400 MHz, CDC1₃): δ 3.68 (m, 2 H), 2.69 - 2.60 (br m, 2 H), 2.41 - 2.29 (br m, 3 H).

Step E: (3.3 -DifluorocyclobutyDmethyl 4-methylbenzenesulf onate Tosyl chloride (1.59 g, 8.36 mmol, 1 equiv.) was dissolved in ice-cold, anhydrous pyridine (3 mL). A solution of (3,3-difluorocyclobutyl)methanol (1.01 g, 8.28 mmol, 1 equiv.) in anhydrous pyridine (1.5 mL) was added via cannula and the sides of the flask were rinsed with an additional 0.5 mL pyridine. The mixture was then stirred at room temperature overnight under nitrogen. The mixture^' was then poured over crushed ice (12 g) and the flask was rinsed with water and the rinsate added to the ice slurry. Concentrated HCI solution (5 mL) was added and the mixture was extracted twice with ether. The aqueous layer was saturated with NaCl and extracted once more with ether. The combined organic extracts were washed successively with saturated aqueous NaHCO₃, water and brine. The organic layer was dried (MgSO₄), filtered and concentrated to an orange oil. Silica gel chromatography (30% EtOAc-hexanes) afforded the title compound as a yellow oil. 1H NMR (400 MHz, CDC1₃): δ 7.79 (d, 7 = 8.0 Hz, 2 H), 7.37 (d, 7 = 8.0 Hz, 2 H), 4.06 (d, 7 = 8.5 Hz, 2 H), 2.69 - 2.58 (br m, 2 H), 2.52 - 2.48 (m, 1 H), 2.46 (s, 3 H), 2.35 - 2.23 (br m, 2 H).

Step F: Diethyl 2-(acetylamino)-2-[(3,3-difluorocyclobutyl methyllmalonate

See also Burger, A. et al; J Med Chem.; 6; 1963; 221 - 227 for the preparation of the desfluoro derivative of this compound.

To a suspension of NaH (60% dispersion in mineral oil, 92 mg, 2.30 mmol) in DMF (5.0 mL) at 0°C was added diethyl acetamidomalonate (0.424 g, 1.95 mmol) in portions, allowing for foaming to subside between portions. The reaction was allowed to warm to rt over 1 h. To the reaction was added (3,3- difluorocyclobutyl)methyl-4-methylbenzenesulfonate (0.566 g, 2.05 mmol) as a solution in 2 mL of DMF via cannula and heated to 130 - 135°C for 1.5 h then an additional 1.25 h at rt. Solvent was removed in vacuo and the remaining residue was partitioned between H₂O and Et₂O. Combined organics were washed with saturated aqueous NaHCO₃ and water, dried over MgSO₄, and concentrated. Silica gel chromatography (20% - 50% EtOAc/hexanes) afforded the title compound as a yellow oil. 1H NMR (CDC1₃, 400 MHz): δ 6.75 (br s, 1 H), 4.31 - 4.19 (m, 4 H), 2.65 - 2.52 (m, 4 H), 2.22 - 2.09 (m, 2 H), 2.05 - 1.98 (m, 5 H), 1.57 - 1.55 (m, 1 H), 1.30 - 1.24 (m, 6 H). LCMS (M+H): 322.2.

Step G: 3 ,3 -difluoroc yclobutylalanine hydrochloride

A mixture of diethyl 2-(acetylamino)-2-[(3,3- difluorocyclobutyl)methyl]malonate (0.184 g, 0.57 mmol) and 18% HCI (5 mL) was heated to reflux overnight. After cooling to rt, the HCI solution was washed with Et₂O and concentrated in vacuo to afford the HCI salt as a yellow solid. The solid was washed with additional Et₂O. 1H NMR (CD₃OD, 400 MHz): δ 3.91 (t, 7 = 6.6 MHz, 1 H), 2.77 - 2.71 (m, 2 H), 2.35 - 2.24 (m, 3 H), 2.18 - 2.13 (m, 1 H), 2.08 - 2.03 (m, 1 H). Step H: N-(tert-butoxycarbonyl -3-(3,3-difluorocvclobutyl alanine

To a solution of 3,3-difluorocyclobutylalanine hydrochloride (0.100 g, 0.46 mmol) in dioxane (2.0 mL), water (0.47 mL), and 1 Ν ΝaOH (0.51 mL) at 0°C was added (BOC)₂O (0.111 g, 0.51 mmol). The reaction was allowed to warm to rt overnight then concentrated to dryness. The remaining orange residue was taken up in water and acidified to pH 3 using 1 Ν HCI and immediately extracted with EtOAc (x2). The aqueous layer was saturated with ΝaCl and extracted once with THF. The combined organics were dried over Νa₂SO₄ and concentrated to give a pale orange solid. 1H NMR (CD₃OD, 400 MHz): δ 4.07 - 4.03 (m, 1 H), 2.66 - 2.60 (m, 2 H), 2.25 - 2.19 (m, 3 H), 2.01 - 1.96 (m, 1 H), 1.88 - 1.85 (m, 1 H), 1.44 and 1.40 (BOC rotamers, s, 9 H). LCMS (M+H-BOC): 179.9.

Step I: N-(tert-butoxycarbonyl)-3-(3,3-difluorocvclobutyl^')alanyl-(4R^')-N-[5- chloro-2-(lH-L2,4-triazol-l-yl benzyl14-fluoro-L-prolinamide

The title compound was prepared from N-(tert-butoxycarbonyl)-3-(3,3- difluorocyclobutyl)alanine (50 mg, 0.18 mmol), (4R)-N-[5-chloro-2-(lH-l,2,4-triazol- l-yl)benzyl] -4-fluoroprolinamide hydrochloride (Example 6, Step C, 71 mg, 0.18 mmol), ΗOAT (24 mg, 0.18 mmol), EDC-ΗCl (51 mg, 0.27 mmol), and Hunig's base (62 μL, 0.36 mmol) essentially according to the coupling procedure described above. The product was purified by preparative reverse phase HPLC [gradient elution with 95:5 water (+0.1% TFA) / CH₃CN (+0.1% TFA) to 5:95 water (+0.1% TFA) / CH₃CN (+0.1% TFA)] to afford a foamy white solid. 1H NMR (CD₃OD, 400 MHz): δ 8.83 - 8.81 (m, 1 H), 8.22 (s, 1 H), 7.70 - 7.69 (m, 1 H), 7.50 - 7.41 (m, 2 H), 5.40 - 5.27 (m, 1 H), 4.51 - 4.47 (m, 1 H), 4.36 - 3.72 (m, 5 H), 2.66 - 2.46 (m, 3 H), 2.26 - 1.77 (m, 6 H). LCMS (M + H) = 585.4. Diastereomers were separated on Chiralpak AD 2 x 25 column with a flow of 8.0 mL / min and the following solvent system: A = 0.1% DEA/hexane, B = 2-propanol and eluting with a gradient of 60 - 100% over 45 min. Diastereomer A: LCMS (M+H): 585.4. Diastereomer B: (M+H): 585.4. Step J: 3-(3,3-difluorocvclobutvnalanyl-(4RVN-r5-chloro-2-dH-1.2.4-triazol-

1 -yl)benzyl]4-fluoro-L-prolinamide

Diastereomer A: To a solution of the earlier diastereomer N-(tert- butoxycarbonyl)-3-(3,3-difluorocyclobutyl)alanyl-(4R)-N-[5-chloro-2-(lH-l,2,4- triazol-l-yl)benzyl]4-fluoro-L-prolinamide (44 mg, 0.075 mmol) in CΗ₂C1₂ (1.0 mL) at rt was added TFA (0.4 mL, excess). After 2 h, the solvent was removed in vacuo. The remaining yellow oil was taken up in water and treated with saturated aqueous K₂CO₃. The aqueous layer was extracted with EtOAc (x3). The combined organics were dried over Νa₂SO₄ and filtered. The EtOAc layer was cooled to 0°C and through it was bubbled HCI (g). The solvent was removed in vacuo , azeotroping with EtOAc and CHC1₃ to give an off white solid. The product was further purified by preparative reverse phase HPLC [gradient elution with 95:5 water (+0.1% TFA) / CH₃CN (+0.1% TFA) to 5:95 water (+0.1% TFA) / CH₃CN (+0.1% TFA)] to afford the TFA salt of the title compound. 1H NMR (CD₃OD, 400 MHz): δ 8.82 (s, 1 H), 8.23 (s, 1 H), 7.70 (d, 7 = 2.0 MHz, 1 H), 7.52 (dd, 7 = 2.4 MHz, 8.4 MHz, 1 H), 7.47 (d, 7 = 8.8 MHz, 1 H), 5.47 - 5.34 (m, 1 H), 4.49 - 4.40 (m, 2 H), 4.27 - 4.18 (m, 2 H), 3.99 - 3.77 (m, 2 H), 2.74 - 2.58 (m, 3 H), 2.29 - 2.02 (m, 6 H). HRMS (ES) M+H: calculated for (C₂₁H₂₄ClF₃N₆O₂)⁺ 485.1674, found 485.1673. Diastereomer B: The later diastereomer B (26 mg, 0.044 mmol) was deprotected according to the TFA procedure described above to afford a yellow oil. 1H NMR (CD₃OD, 400 MHz): δ 8.83 (s, 1 H), 8.74 - 8.72 (m, 1 H), 8.23 (s, 1 H), 7.69 (d, 7= 2.4 MHz, 1 H), 7.51 (dd, 7= 2.2 MHz, 8.6 MHz, 1 H), 7.46 (d, 7= 8.4 MHz, 1 H), 5.46 - 5.33 (m, 1 H), 4.60 - 4.55 (m, 1 H), 4.42 - 4.36 (m, 1 H), 4.27 - 4.21 (m, 2 H), 4.13 - 4.05 (m, 1 H), 3.86 - 3.74 (m, 1 H), 2.77 - 2.53 (m, 3 H), 2.32 - 1.99 (m, 6 H). HRMS (ES) M+H: calculated for (C₂₁H₂₄ClF₃N₆O₂)⁺ 485.1674, found 485.1671.

EXAMPLE 18 Preparation of 3-cyclopropyl-D-valyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide

Step A: 3-Methylbut-2-enyl N-(tert-butoxycarbonyl glvcinate DCC (1 M in CΗ₂C1₂, 26.2 mL, 26.2 mmol, 1.05 equiv.) was added to a stirred solution of Boc-Glycine (4.24 g, 24.2 mmol, 0.97 equiv.), 3-methyl-2-buten-l-ol (2.15 g, 25.0 mmol, 1 equiv.) and DMAP (152 mg, 1.25 mmol, 0.05 equiv.) in anhydrous CH₂C1₂ (155 mL) at 0 °C. The mixture was then warmed to room temperature while stirring overnight. The mixture was filtered twice to remove the precipitated dicyclohexylurea, washing the solids well with CH₂C1₂. The filtrate was concentrated to a slightly colored oil. Silica gel chromatography (15% EtOAc-hexanes) afforded the title compound as a colorless oil. IH ΝMR (400 MHz, CDC1₃): δ 5.34 (t, 7 = 7.2 Hz, 1 H), 5.00 (br s, 1 H), 4.65 (d, 7 = 7.2 Hz, 2 H), 3.91 (d, 7 = 5.2 Hz, 2 H), 1.76 (s, 3 H), 1.72 (s, 3 H), 1.45 (s, 9 H).

Step B: 2-[(tert-butoxycarbonyl^')amino1-3.3-dimethylpent-4-enoic acid

LDA was prepared by the dropwise addition of BuLi (2.5 M in hexanes, 7.73 mL, 19.3 mmol, 2.1 equiv) to a solution of anhydrous diisopropylamine (2.8 mL, 20.3 mmol, 2,2 equiv) in anhydrous THF (65 mL) at 0°C under nitrogen. The solution was stirred for 15 min at 0°C and was then cooled to -78°C. A solution of 3-methylbut-2- enyl N-(tert-butoxycarbonyl)glycinate (2.24 g, 9.2 mmol, 1 equiv) in anhydrous THF

(5 mL) was added dropwise over 2 min. The mixture was stirred for 15 min at -78°C and then treated with TMSC1 (2.5 mL, 19.3 mmol, 2.1 equiv). The mixture was then stirred at -78°C for 5 min, then at room temperature for 30 min, then at 60°C for one hour. TLC after one hour (50% EtOAc/hexanes, KMnθ4 stain) showed that most of the starting material had been consumed. After 1 hr and 45 min, the reaction mixture was cooled to room temperature and quenched with MeOH (5 mL). The THF was removed in vacuo and the aqueous residue was extracted once with ether to remove any remaining starting material. The aqueous layer was cooled to 0°C and adjusted to pH 3 by the dropwise addition of IN HCI. The mixture was saturated with solid NaCl and extracted immediately with EtOAc three times. The combined organic extracts were dried (Na SO₄), filtered and concentrated in vacuo to give a beige, oily solid, which was further dried overnight under high vacuum. 1H NMR (400 MHz, CDC1₃): δ 5.90 - 5.84 (m, 1 H), 5.14 - 4.92 (m, 2 H), 4.12 (br s, 1 H), 1.42 (s, 9 H), 1.10 (s, 6 H).

Step C: Methyl N-(tert-butoxycarbonyl)-3 -cyclopropylvalinate

To a mixture of 5Ν NaOH solution (31 mL) and Et₂O (40 mL) at 0 °C was added 1 -methyl-3 -nitro- 1 -nitrosoguanidine (3.05 g, 20.71 mmol, 12.0 equiv.), portionwise over 15 min. The mixture was stirred vigorously for 30 min, then allowed to partition. The organic layer was transferred by pipet to a solution of 2- [(tert-butoxycarbonyl)amino]-3,3-dimethylpent-4-enoic acid (420 mg, 1.73 mmol) in Et₂O (40 mL) at 0 °C. imediately following transfer, a catalytic amount of Pd(OAc)₂ was added. The reaction was stirred at 25 °C for 18 h. Acetic acid was added dropwise until the yellow reaction color diminished, and the solvent was then removed in vacuo. Purification by silica gel chromatography (hexanes to 20% EtOAc/hexanes) afforded a pale yellow oil. The entire procedure was repeated in order to convert any unreacted material to the title compound. 1H NMR (400 MHz, CDC1₃): δ 5.25-5.21 (br m, 1 H), 4.21 (d, 7= 9.6 Hz, 1 H), 3.73 (s, 3 H), 1.44(s, 9 H), 0.85 (s, 3 H), 0.81-0.77 (m, 1 H), 0.75 (s, 3 H), 0.34-0.32 (m, 2 H), 0.21-0.19 (m, 2 H). LCMS (M+H): 272.2.

Step D: N-(tert-butoxycarbonyl)-3-cvclopropylvaline

To a stirred solution of methyl N-(tert-butoxycarbonyl)-3-cyclopropylvalinate (248 mg, 0.91 mmol) in CH₃CΝ (15 mL) was added LiOH/H₂O (77 mg, 1.82 mmol, 2.0 equiv.) in water (5 mL). The reaction was stirred at 25 °C for 3 h then was warmed to 70 °C for 2 h. Acetonitrile was removed in vacuo, and the remaining aqueous solution was cooled to 0 °C and acidified to pH 3 by dropwise addition of IN HCI solution. Following saturation with NaCl, the solution was extracted into EtOAc three times. The organic layers were combined, dried over Na₂SO₄, and concentrated in vacuo to afford the title compound as a clear oil. 1H NMR (400 MHz, CDC1₃): δ 6.41 and 5.64 (rotamers, s, 1 H), 5.23 (d, 7= 8.8 Hz, IH), 4.19 (d, 7= 9.2 Hz, 1 H), 1.45 (s, 9 H), 0.91 (s, 3 H), 0.88-0.84 (m, 1 H), 0.82 (s, 3 H), 0.36-0.34 (m, 2 H), 0.26-0.23 (m, 2 H). LCMS (M+H): 258.1.

Step E: 3-Cvclopropylvalyl-(^,4R')-N-r5-chloro-2-dH-L2,4-triazol-l-vnbenzyll-

4-fluoro-L-prolinamide

A mixture of N-(tert-butoxycarbonyl)-3-cyclopropylvaline (200 mg, 0.78 mmol, 2.0 equiv.), (4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide (Example 6, Step C, 154 mg, 0.39 mmol), EDC (112 mg, 0.58 mmol, 1.5 equiv.) and ΗOAt (53 mg, 0.39 mmol, 1.0 equiv.) in DMF (1 mL) was brought to pΗ 8 by dropwise addition of Hunig's base and stirred at room temperature for 18 h. The solvent was removed in vacuo, and the resulting oil was dissolved in EtOAc and washed with water. The aqueous layer was saturated with ΝaCl and extracted into EtOAc twice more. The organic layers were combined, dried over Νa₂SO , and concentrated in vacuo. The diastereomers were separated by reverse phase chromatography (DeltaPak C-18 column, eluting with 40:60 A/B to 35:65 A/B to 30:70 A/B, where A = 0.1% NILOAc solution and B = MeOH). Each diastereomer was dissolved in CH₂C1₂ (5 mL), and the solutions were saturated with hydrogen chloride gas. After stirring for 18 h, the solvent was removed in vacuo to afford Diastereomer A (more polar before deprotection) as a pale yellow powder and Diastereomer B (less polar before deprotection) as a white powder. Diastereomer A: 1H NMR (400 MHz, CD₃OD): δ 9.17 (s, 1 H), 8.44 (s, 1 H), 7.74 (s, 1 H), 7.51-7.46 (m, 2 H), 5.36 (br d, 7 = 52.0 Hz, 1 H), 4.63 (dd, 7 = 7.2, 9.6 Hz, 1 H), 4.42 (d, 7 = 16.0 Hz, 1 H), 4.24-4.15 (m, 3 H), 3.88-3.75 (m, 1 H), 2.60-2.49 (m, 1 H), 2.21-2.01 (m, 1 H), 0.99 (s, 3 H), 0.97-0.91 (m, 1 H), 0.84 (s, 3 H), 0.45-0.41 (m, 2 H), 0.37- 0.33 (m, 2 H). HRMS (ESI, M+H): 463.2025 (found); 463.2012 (calculated). Diastereomer B: 1H NMR (400 MHz, CD₃OD): δ 8.91 (s, 1 H), 8.29 (s, 1 H), 7.72 (d, 7 = 2.0 Hz, 1 H), 7.53 (dd, 7 = 2.4, 8.8 Hz, 1 H), 7.48 (d, 7 = 8.8 Hz, 1 H), 5.36 (br d, 7 = 51.6 Hz, 1 H), 4.50 (app t, 7= 8.8 Hz, 1 H), 4.42 (d, 7= 15.2 Hz, 1 H), 4.28-4.22 (m, 2 H), 4.10 (s, 1 H), 3.88-3.76 (m, 1 H), 2.52-2.20 (m, 1 H), 2.18-1.97 (m, 1 H), 0.99 (s, 3 H), 0.91 (s, 3 H), 0.90-0.86 (m, 1 H), 0.46-0.41 (m, 2 H), 0.34-0.31 (m, 2 H). HRMS (ESI, M+H): 463.2023 (found); 463.2019 (calculated).

EXAMPLE 19 Preparation of D-prolyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide

Step A: l-(tert-Butoxycarbonyl -D-prolyl-f4J? -N-[5-chloro-2-dH-l,2.4- triazol- 1 -yPbenzyll -4-fluoroprolinamide

EDC (59 mg, 0.31 mmol, 2.0 equiv) was added to a stirred solution of (4R)-Ν- [5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoroprolinamide hydrochloride (Example 6, Step C, 61 mg, 0.15 mmol, 1 equiv), l-(tert-butoxycarbonyl)-D-proline (35 mg, 0.16 mmol, 1.05 equiv) and ΗOAt (21 mg, 0.15 mmol, 1 equiv) in DMF (1.2 mL). Hunig's base was added dropwise to adjust the pH to 8 (15 drops required). The resulting clear, yellow solution was stirred over the weekend at RT. The crude product was purified by preparative reverse phase HPLC (Example 37) to give the title compound as a clear, colorless oil, which was carried on directly to the deprotection step below. LCMS (M+H) = 521.2. Step B: D-prolyl-(4RVN-r5-chloro-2-dH-L2.4-triazol-l-yl)benzyll-4- fluoroprolinamide

The title compound was prepared from l-(tert-butoxycarbonyl)-D-prolyl-(4R)- N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoroprolinamide essentially according to the deprotection procedure described in Example 1, Step M. 1H ΝMR (500 MHz, CD₃OD): δ 8.80 (s, 1 H), 8.74 (br m, 1 H), 8.22 (s, 1 H), 7.68 (d, 7= 2.0 Hz, 1 H), 7.51 (dd, 7 = 2.0, 8.5 Hz, 1 H), 7.46 (d, 7= 8.5 Hz, 1 H), 5.38 (app d, 7 = 52.0 Hz, 1 H), 4.52 - 4.44 (m, 1 H), 4.40 (dd, 7 = 3.5, 15.5 Hz, 1 H), 4.24 (dd, 7 = 3.5, 15.0 Hz, 1 H), 3.96 -3.90 (m, 1 H), 3.85 - 3.75 (m, 1 H), 3.48 - 3.43 (m, 1 H), 3.34 - 3.32 (m, 1 H), 2.62 - 2.53 (m, 2 H), 2.17 - 2.04 (m, 3 H), 1.97 - 1.90 (m, 1 H). LCMS (M+H) = 421.3.

EXAMPLE 20 Preparation of (4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoro-l-[(2R)- piperidin-2-ylcarbonyl]prolinamide

The title compound was prepared from (2i?)-l-(tert- butoxycarbonyl)piperidine-2-carboxylic acid and (4R)-Ν-[5-chloro-2-(lH-l,2,4- triazol-l-yl)benzyl] -4-fluoroprolinamide hydrochloride (Example 6, Step C) essentially according to the procedures described in Example 19, Steps A and B. 1H NMR (500 MHz, CD₃OD): δ 8.80 (s, 1 H), 8.76 (br t, 7 = 5.5 Hz, 1 H), 8.22 (s, 1 H), 7.69 (d, 7 = 2.5 Hz, 1 H), 7.50 (dd, 7 = 2.5 Hz, 8.5 Hz, 1 H), 7.46 (d, 7 = 8.5 Hz, 1 H), 5.38 (app d, 7 = 52.0 Hz, 1 H), 4.47 (app t, 7 = 8.5 Hz, 1 H), 4.41 (dd, 7 = 6.3, 15.0 Hz, 1 H), 4.21 (dd, 7 = 5.0, 16.0 Hz, 1 H), 4.09 - 4.06 (m, 1 H), 3.97 - 3.91 (m, 1 H), 3.81 (ddd, 7 = 2.5, 12.5, 36.0 Hz, 1 H), 3.42 - 3.40 (m, 1 H), 3.09 - 3.04 (m, 1 H), 2.60 -2.52 (m, 1 H), 2.16 - 2.11 (m, 1 H), 2.09 - 2.03 (m, 1 H), 1.95 - 1.88 (m, 2 H), 1.79 - 1.61 (m, 3 H). HRMS (ES, M+H): calculated 435.1706; found 435.1721.

EXAMPLE 21 Preparation of 3,3-dimethyl-D-prolyl-(4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide

Step A: 3 ,3-Dimethyl-dl-proline The procedure for the preparation of 3,3-dimethyl-dl-proline as described in

Morgan, B. A.; Schafer, D. J. US Patent 4,060,603, Nov. 29, 1977, was followed, with the following modifications: the intermediate 2,2-dicarboethoxy-3,3-dimethyl- pyrrolidine was chromatographed on a silica gel column eluting with 50% EtOAc/hexanes. Acid hydrolysis of this intermediate as described in US 4,060,603 afforded the crude 3,3-dimethyl-dl-proline as a brown solid. 1H NMR (CD₃OD, 400 MHz): δ 3.97 (s, 1 H), 3.40 (m, 2 H), 2.00 (m, 2 H), 1.37 (s, 3 H), 1.07 (s, 3 H).

Step B: l-(tert-Butoxycarbonyl -3,3-dimethyl-dl-proline

To a stirred solution of 3,3-dimethyl-dl-proline (1.00 g, 6.98 mmol) in H₂O (7.0 mL), 1 N NaOH (7.6 mL), and dioxane (21.0 mL) at 0°C was added BOC anhydride (1.68 g, 7.7 mmol) in portions. The reaction was allowed to warm to room temperature and stirred for 72 h. The solvent was removed in vacuo. The remaining residue was taken up in H₂O and acidified to pH 2 using 1 N HCI. The aqueous layer was extracted with EtOAc. The organic layer was washed with brine, dried (Na₂SO₄), and concentrated to a yellow oil. Silica gel chromatography (gradient elution with

30% EtOAc / hexanes - 100% EtOAc - 5% AcOH / EtOAc) afforded the product as a brown solid. LCMS (M+H): 244.1. 1HNMR (CD₃OD, 400 MHz): δ 3.81 and 3.78 (m, rotamers, 1 H), 3.50 (m, 1 H), 3.40 (m, 1 H), 1.85 (m, IH), 1.62 (m, 1 H), 1.45 and 1.42 (s, Boc rotamers, 9 H), 1.16 (s, 3 H), 1.07 (s, 3 H). The proline diastereomers were resolved by chiral HPLC under the following conditions: Chiralpak AD 5 x 50 cm column, eluting with 95:5 A:B where A = hexane + 0.1% TFA and B = 2-propanol at a flow rate of 70 mlJmin with UV detection at 220 nm. By analytical chiral HPLC (Chiralpak AD column 250 x 4.6 mm, eluting with 93:7 A:B where A = hexane + 0.1% TFA and B = 2-propanol at a flow-rate 1.0 m min with UV detection at 215, 220 and 230 nm), the early peak eluted at 3.85 min and the later peak eluted at 5.35 min.

Step C: l-(tert-butoxycarbonyl)-3.3-dimethyl-L-prolyl-(4RVN-[5-chloro-2-

(1H- 1 ,2,4-triazol- 1 -yPbenzyl] -4-fluoro-L-prolinamide

The title compound was prepared from l-(tert-butoxycarbonyl)-3,3-dimethyl- proline (single enantiomer, early peak from previous step, 45 mg, 0.18 mmol), (4R)- Ν- [5 -chloro-2-( IH- 1 ,2,4-triazol- 1 -yl)benzyl] -4-fluoroprolinamide hydrochloride (Example 6, Step C, 73 mg, 0.18 mmol), ΗOAT (25 mg, 0.18 mmol), EDC-ΗC1 (53 mg, 0.28 mmol), and Hunig's base (64 μL, 0.37 mmol) essentially according to the coupling procedure described in Example 1, Step L. The product was purified by preparative reverse phase HPLC [gradient elution with 95:5 water (+0.1% TFA) / CH₃CΝ (+0.1% TFA) to 5:95 water (+0.1% TFA) / CH₃CN (+0.1% TFA)]. LCMS (M+H): 549.3.

Step D: 3,3-dimethyl-L-prolyl-(^'4RVN-r5-chloro-2-dH-l,2,4-triazol-l- yDbenzyl] -4-fluoro-L-prolinamide

To a solution of l-(tert-butoxycarbonyl)-3,3-dimethyl-L-prolyl-(4R)-N-[5- chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoro-L-prolinamide (14 mg, 0.03 mmol) in CΗ₂C1₂ (1.0 mL) at rt was added TFA (0.5 mL, excess). After 3 h, the solvent was removed in vacuo to give the TFA salt of the title compound as a pale brown gum.' 1H NMR (CD₃OD, 400 MHz): δ 8.81 (s, 1 H), 8.80 - 8.78 (m, 1 H), 8.23 (s, 1 H), 7.72 (d, 7 = 2.0 MHz, 1 H), 7.52 (dd, 7 = 2.2 MHz, 8.6 MHz, 1 H), 7.47 (d, 7 = 8.4 MHz, 1 H), 5.46 - 5.33 (m, 1 H), 4.52 (t, 7 = 8.6 MHz, 1 H), 4.45 - 4.39 (m, 1 H), 4.27 - 4.23 (m, 2 H), 4.05 - 3.97 (m, 1 H), 3.88 - 3.77 (m, 1 H), 3.58 - 3.51 (m, 1 H), 3.43 - 3.36 (m, 1 H), 2.60 - 2.57 (m, 1 H), 2.20 - 1.91 (m, 3 H), 1.34 (s, 3 H), 1.11 (s, 3 H). HRMS (ES) M+H: calculated for (C₂₁H₂₆ClFN₆O₂)⁺ 449.1863, found 449.1869.

EXAMPLE 22 Preparation of 4,4-difluoro-D-prolyl-(4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl]-4-fluoroprolinamide

Step A: Methyl-(4R -4-hydroxy-D-prolinate hydrochloride

To a stirred suspension of (4R)-4-hydroxy-D-proline hydrochloride (4.84 g,

28.9 mmol) in dry MeOΗ (32.0 mL) at 0°C under Ν₂ was added SOCl₂ (2.10 mL, 28.9 mmol) dropwise. The mixture was stirred vigorously while warming to room temperature. After 4 h, the reaction mixture was evaporated to dryness, azeotroping with benzene and Et₂O to afford the title compound as a light gray solid. 1H NMR

(CD₃OD, 400 MHz): δ 4.56 - 4.50 (m, 2 H), 3.85 (s, 3 H), 3.40 - 3.34 (m, 2 H), 2.46

- 2.38 (m, 2 H).

Step B: 1 -tert-butyl 2-methyl (2R,4R)-4-hydroxypyrrolidine-l,2-dicarboxylate

To a stirred suspension of methyl (4R)-4-hydroxy-D-prolinate chloride (4.98 g, 27.4 mmol) in CH₂C1₂ (100 mL) at room temperature was added Et₃N (9.55 mL,

68.6 mmol) followed by 5 mL of MeOH to aid in dissolution. To the solution was added BOC anhydride (6.58 g, 30.2 mmol) and gas evolution was contained in two balloons as the reaction continued to stir at rt. After 2 h, LCMS showed complete reaction. The yellow solution was washed with 10% aqueous citric acid, saturated aqueous NaHCO₃, water, and brine. The combined organic extracts were dried (MgSO ) and concentrated to a yellow oil. Silica gel chromatography (5% MeOH/CHCl₃) afforded the title compound as a colorless oil. 1H NMR (CDC1₃, 400 MHz): δ 4.39 - 4.28 (m, 2 H), 3.80 and 3.78 (rotamers, s, 3 H), 3.71 and 3.64 (rotamers, d, 7 = 12.0 Hz, 1 H), 3.57 - 3.25 (m, 2 H), 2.36 - 2.28 (m, 1 H), 2.12 - 2.06 (m, 1 H), 1.47 and 1.42 (BOC rotamers, s, 9 H).

Step C: l-tert-butyl-2-methyl (2R -4-oxopyrrolidine-l,2-dicarboxylate To a stirred solution of l-tert-butyl-2-methyl (2R)-4-oxopyrrolidine-l,2- dicarboxylate (5.43 g, 22.1 mmol) in CH C1₂ (170 mL) at room temperature was added PDC and 4A molecular sieves. The solution was stirred for 10 min and Celite was added. The mixture was stirred under N₂ overnight. The mixture was diluted with Et₂O and filtered through Celite, washing with Et₂O and CHC1₃. The filtrate was concentrated to a brown oil. Silica gel chromatography (50% EtOAc/hexanes) afforded the title compound as a yellow oil. 1H NMR (CD₃OD, 400 MHz): δ 4.83 - 4.70 (m, 1 H), 3.91 - 3.88 (m, 2 H), 3.77 - 3.74 (m, 3 H), 3.02 - 2.89 (m, 1 H), 2.61 - 2.56 (m, I H), 1.48 (s, 9 H).

Step D: 1 -tert-butyl 2-methyl (2R -4,4-difluoropyrrolidine-l,2-dicarboxylate

To a stirred solution of l-tert-butyl-2-methyl (2R)-4-oxopyrrolidine-l,2- dicarboxylate (200 mg, 0.82 mmol) in dry CH₂C1₂ (3.5 mL) at room temperature was added DAST (0.43 mL, 3.29 mmol) dropwise. After 4 h, the reaction was quenched by the slow addition of saturated aqueous NaHCO . The aqueous layer was extracted with CH₂C1₂ (x3). The combined organics were dried over Na SO and concentrated to give an orange oil. Silica gel chromatography (20% - 50% EtOAc/hexanes) afforded the title compound as a sticky orange solid. (See also Demange, L.; Menez, A.; Dugave, C. Tetrahedron Lett. 1998, 39, 1169-1172 which describes the preparation of the isomeric 1-tert-butyl 2-methyl (2S)-4,4-difluoropyrrolidine-l,2- dicarboxylate). 1H NMR (CDCI₃, 400 MHz): δ 4.56 - 4.43 (m, 1 H), 3.89 - 3.81 (m, 2 H), 3.77 (s, 3 H), 2.74 - 2.66 (m, 1 H), 2.52 - 2.41 (m, 1 H), 1.43 (s, 9 H).

Step E: l-(tert-butoxycarbonylV4.4-difluoro-D-proline To a stirred solution of 1 -tert-butyl 2-methyl (2R)-4,4-difluoropyrrolidine-l,2- dicarboxylate (212 mg, 0.80 mmol) in 3:1 MeCN/H₂O (32.0 mL) at room temperature was added LiOH-H₂O (67 mg, 1.60 mmol). The mixture was stirred for 5 h and the MeCN was removed in vacuo. The remaining aqueous mixture was acidified to pH 3 using 1 N HCI and concentrated in vacuo to afford the title compound as an orange solid. 1H NMR (CD₃OD, 400 MHz): δ 4.34 - 4.24 (m, 1 H), 3.82 - 3.75 (m, 2 H), 2.78 - 2.68 (m, 1 H), 2.46 - 2.33 (m, 1 H), 1.45 (s, 9 H).

Step F: 4,4-difluoro-D-prolyl-(4R')-N-[5-chloro-2-(lH-l,2,4-triazol-l- yPbenzyl] -4-fluoroprolinamide The title compound was prepared from l-(tert-butoxycarbonyl)-4,4-difluoro-

D-proline and (4R)-Ν-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoroprolinamide hydrochloride (Example 6, Step C) essentially according to the procedures described in Example 19, Steps A and B. 1H NMR (500 MHz, CD₃OD): δ 8.80 (s, 1 H), 8.22 (s, 1 H), 7.69 (d, 7 = 2.0 Hz, 1 H), 7.51 (dd, 7= 2.0, 8.5 Hz, 1 H), 7.47 (d, 7= 8.5 Hz), 5.39 (app d, 7 = 52.5 Hz, 1 H), 4.84 (app t, 7 = 9.0 Hz, 1 H), 4.53 (app t, 7 = 8.0 Hz, 1 H), 4.41 (d, 7 = 16.0 Hz, 1 H), 4.24 (d, 7 = 15.5 Hz, 1 H), 3.93 - 3.77 (m, 4 H), 3.22 - 3.13 (m, 1 H), 2.62 - 2.50 (m, 2 H), 2.18 - 2.01 (m, 1 H). LCMS (M+H) = 457.4.

EXAMPLE 23

Preparation of (4S)-4-fluoro-D-prolyl-(4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide

Step A: 1 -tert-butyl-2-methyl (2R.4S -4-fluoropyrrolidine- 1 ,2-dicarboxylate

To a stirred solution of l-tert-butyl-2-methyl (2R,4R)-4-hydroxypyrrolidine- 1,2-dicarboxylate (Example 22, Step B, 485 mg, 1.98 mmol) in CH₂C1₂ (8.5 mL) at room temperature was added DAST (0.47 mL, 3.56 mmol) dropwise. After 2.5 h the reaction was quenched as described in example 22, Step D. Silica gel chromatography (30% EtOAc/hexanes) afforded the title compound as an orange oil. LCMS (M + H -BOC): 148.0. 1H NMR (CD₃OD, 400 MHz): δ 5.30 - 5.18 (m, 1 H), 4.39 - 4.34 (m, 1 H), 3.82 - 3.76 (m, 1 H), 3.75 (s, 3 H), 3.63 - 3.50 (m, 1 H), 2.60 - 2.54 (m, 1 H), 2.19 - 2.01 (m, 1 H), 1.41 (m, 9 H).

Step B: (4S)- 1 -(tert-butoxycarbonyl -4-fluoro-D-proline l-tert-butyl-2-methyl (2R,4S)-4-fluoropynolidine-l,2-dicarboxylate (257 mg, 1.04 mmol) was hydrolyzed according to the procedure described in Example 22, Step E. 1H NMR (CD₃OD, 400 MHz): δ 5.29 - 5.16 (m, 1 H), 4.33 - 4.24 (m, 1 H), 3.81 - 3.70 (m, 1 H), 3.65 - 3.52 (m, 1 H), 2.62 - 2.52 (m, 1 H), 2.20 - 2.03 (m, 1 H), 1.45 (s, 9 H).

Step C: (4S -4-fluoro-D-prolyl-(4J?)-N-15-chloro-2-dH-l,2.4-triazol-l- yPbenzyl] -4-fluoroprolinamide

The title compound was prepared from (4S)-l-(tert-butoxycarbonyl)-4-fluoro- D-proline and (4R)-Ν-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoroprolinamide hydrochloride (Example 6, Step C) essentially according to the procedures described in Example 19, Steps A and B. 1H NMR (500 MHz, CD₃OD): δ 8.80 (s, 1 H), 8.23 (s, 1 H), 7.68 (d, 7 = 2.0 Hz, 1 H), 7.52 (dd, 7 = 2.0, 8.5 Hz, 1 H), 7.47 (d, 7 = 8.5 Hz, 1 H), 5.51 (app dt, 7 = 3.3, 51.5 Hz, 1 H), 5.38 (app d, 7 = 52.0 Hz, 1 H), 4.73 (dd, 7 = 8.0, 9.5 Hz, 1 H), 4.51 (app t, 7= 8.0 Hz, 1 H), 4.40 (d, 7= 15.5 Hz, 1 H), 4.25 (d, 7 = 15.0 Hz, 1 H), 3.98 - 3.92 (m, 1 H), 3.83 (ddd, 7= 2.5, 10.0, 36.0 Hz, 1 H), 3.72 (ddd, 7= 2.0, 13.5, 19.0 Hz, 1 H), 3.61 (ddd, 7= 3.5, 13.5, 36.5 Hz, 1 H), 3.00 - 2.92 (m, 1 H), 2.62 - 2.54 (m, 1 H), 2.29 - 2.04 (m, 2 H). LCMS (M+H) = 439.4.

EXAMPLE 24 Preparation of (4R)-4-fluoro-D-ρrolyl-(42?)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide

Step A: Methyl (4S -4-hydroxy-D-prolinate chloride

To a stirred solution of trans-4-hydroxy-D-proline (300 mg, 1.79 mmol) in dry MeOΗ (4.0 mL) at 0°C under Ν₂ was added SOCl₂ dropwise. The resulting thick mixture was stirred while warming to rt. After 4 h, the reaction was evaporated to dryness, azeotroping with benzene and Et₂O to afford the title compound as a gray solid. 1H NMR (400 MHz, CD₃OD): δ 4.62 - 4.57 (m, 2 H), 3.86 (s, 3 H), 3.44 - 3.40 (m, 1 H), 3.31 - 3.30 (m, 1 H), 2.45 - 2.39 (m, 1 H), 2.23 - 2.15 (m, 1 H).

Step B: l-tert-butyl-2-methyl (2R,4S -4-hvdroxypyrrolidine-l,2-dicarboxylate To a stirred suspension of methyl (4S)-4-hydroxy-D-prolinate chloride (350 mg, 1.93 mmol) in CH₂C1₂ (9.0 mL) at rt was added Et₃N (0.67 mL, 4.82 mmol) followed by 0.1 mL of MeOH to aid in dissolution, and BOC anhydride (463 mg, 2.12 mmol). Gas evolution was contained in two balloons as the reaction continued to stir at rt for 72 h. The yellow solution was washed with 10% aqueous citric acid, saturated aqueous NaHCO₃, water, and brine. The organic layer was dried over MgSO₄ and concentrated to afford the title compound as a yellow oil. LCMS (M + H): 246.6. 1H NMR (400 MHz, CD₃OD): δ 4.39 - 4.33 (m, 2 H), 3.74 and 3.72 (rotamers, s, 3 H), 3.54 - 3.50 (m, 1 H), 3.47 - 3.43 (m, 1 H), 3.32 - 2.22 (m, 1 H), 2.05 - 2.00 (m, 1 H), 1.46 and 1.41 (BOC rotamers, s, 9 H).

Step C: l-tert-butyl-2-methyl (2R,4R -4-fluoropyrrolidine-l,2-dicarboxylate

To a stirred solution of l-tert-butyl-2-methyl (2R,4S)-4-hydroxypyrrolidine- 1 ,2-dicarboxylate (200 mg, 0.82 mmol) in CH₂C1₂ at rt was added DAST (0.19 mL, 1.47 mmol) dropwise. After 2.5 h, LCMS showed no remaining starting material. The reaction was quenched with the slow addition of saturated aqueous NaHCO₃ and extracted with CH₂C1₂ (x3). The combined organics were washed with brine, dried over MgSO₄, and concentrated to an orange oil. Silica gel chromatography (30% EtOAc/hexanes) afforded the title compound as an orange oil. LCMS (M + H - BOC): 148.0. 1H NMR (400 MHz, CD₃OD): δ 5.29 - 5.15 (m, 1 H), 4.48 - 4.45 (m, 1 H), 3.73 - 3.67 (m, 3 H), 3.64 - 3.60 (m, 2 H), 2.43 - 2.38 (m, 2 H), 1.48 and 1.43 (BOC rotamers, s, 9 H).

Step D: (4R -l-(tert-butoxycarbonyl -4-fluoro-D-proline

To a stirred solution of l-tert-butyl-2-methyl (2R,4R)-4-fluoropyrrolidine-l,2- dicarboxylate (109 mg, 0.44 mmol) in 18 mL of 3 : 1 MeCN/H₂O at rt was added LiOH-H₂O (37 mg, 0.88 mmol) and stirred overnight. The MeCN was removed in vacuo and remaining aqueous portion was acidified to pH 3 using 1 N HCI. The solution was concentrated in vacuo to give the LiCl salt of the title compound as a white solid. 1H NMR (400 MHz, CD₃OD): δ 5.29 - 5.15 (m, 1 H), 4.45 - 4.40 (m, 1 H), 3.75 - 3.57 (m, 2 H), 2.59 - 2.41 (m, 2 H), 1.49 and 1.44 (BOC rotamers, s, 9 H).

Step E: (4J?V4-fluoro-D-prolyl-(4E)-N-r5-chloro-2-dH-l,2.4-triazol-l- yPbenzyll -4-fluoroprolinamide

The title compound was prepared from (4R)-l-(tert-butoxycarbonyl)-4-fluoro- D-proline and (4R)-Ν-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoroprolinamide hydrochloride (Example 6, Step C) essentially according to the procedures described in Example 19, Steps A and B. 1H NMR (500 MHz, CD₃OD): δ 8.81 (s, 1 H), 8.22 (s, 1 H), 7.67 (d, 7= 2.0 Hz, 1 H), 7.51 (dd, 7= 2.5, 9.0 Hz, 1 H), 7.47 (d, 7= 8.5 Hz, 1 H), 5.44 (app dt, 7= 4.0, 51.5 Hz, 1 H), 5.39 (app d, 7= 52.0 Hz), 4.62 (dd, 7 = 5.0, 11.0 Hz, 1 H), 4.53 (app t, 7 = 8.0 Hz, 1 H), 4.38 (d, 7= 15.5 Hz, 1 H), 4.26 (d, 7 = 15.5 Hz, 1 H), 3.91 - 3.74 (m, 3 H), 3.55 - 3.44 (m, 1 H), 3.01 - 2.86 (m, 1 H), 2.63 - 2.54 (m, 1 H), 2.32 - 2.05 (m, 2 H). LCMS (M+H) = 439.4.

EXAMPLE 25

Preparation of (4i?)-N-[5-chloro-2-(lH-l ,2,4-triazol- l-yl)benzyl] -4-fluoro-l- [(2i?)-2- hydroxy-2-phenylethanoyl]prolinamide

The title compound was prepared from (R)-mandelic acid and (4R)-Ν-[5- chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoroprolinamide hydrochloride (Example 6, Step C) essentially according to the procedure described in Example 1, Step L. The product was purified by reverse phase ΗPLC [gradient elution with 95:5 water (+0.1% TFA) / CΗ₃CN (+0.1% TFA) to 5:95 water (+0.1% TFA) / CH₃CN (+0.1% TFA)] to give the title compound as a white foam. 1H NMR (500 MHz, CD₃OD): δ 8.84 (s, 1 H), 8.64 (t, 7 = 5.5 Hz, 1 H), 8.24 (s, 1 H), 7.74 (d, 7 = 2.5 Hz, 1 H), 7.50 (dd, 7 = 2.0, 8.0 Hz, 1 H), 7.45 (d, 7 = 8.5 Hz, 1 H), 7.42 - 7.33 (5 H), 5.24 (s, 1 H), 5.18 (d, 7 = 55.5 Hz, 1 H), 4.48 (app t, 7 = 8.5 Hz, 1 H), 4.38 - 4.29 (m, 2 H), 3.76 - 3.60 (m, 2 H), 2.46 - 2.38 (m, 1 H), 2.08 - 1.94 (m, 1 H).

EXAMPLE 26 Preparation of 3-(l-methylcyclopropyl)-D-alanyl-(4/?)-N-[3-chloro-2-fluoro-6-(lH- l,2,4-triazol-l-yl)benzyl]-4-fluoroprolinamide

Step A: 3-Chloro-2-fluoro-6-dΗ-l,2,4-triazol-l-vDbenzamide To an 80 °C stirred slurry of 3-chloro-2,6-difluorobenzamide (1.5 g, 7.84 mmol) and K₂CO₃ (795 mg, 5.75 mmol) in 20 mL of DMF was added dropwise a solution of 1,2,4-triazole (361 mg, 5.23 mmol) in 5 mL of DMF under Ar. After stirring at 80°C for two hours, the reaction was allowed to stand at RT overnight. The reaction mixture was concentrated at reduced pressure and triturated with 50 mL of CHC1₃. The triturate was concentrated and chromatographed on a 35 g RediSep column using CHC1₃ to 9: lCHCl₃-MeOH over 40 min, 30 mlJmin. The product containing fractions were combined and concentrated at reduced pressure to give 180 mg of a colorless solid. This material was purified by preparative HPLC, Waters Delta-Prep C-18, 95:5 to 60:40 0.1% aqueous TFA-CH₃CΝ over 1 h at a flow rate of 50 mlJmin. The pure fractions were combined and concentrated at reduced pressure to give the title compound as a colorless film: 1H NMR (d6-DMSO) δ 8.85 (s, IH), 8.24 (s, IH), 8.21 (br s, IH), 7.82-7.95 (m, 2H), 7.56 (dd, J=8.8, 1.5 Hz, IH).

Step B: 3-Chloro-2-fluoro-6-(lH- 1 ,2,4-triazol- l-yl benzonitrile To a stirred solution of 3-chloro-2-fluoro-6-(lH-l,2,4-triazol-l-yl)benzamide

(84 mg, 0.35 mmol) in 3 mL of THF was added

(methoxycarbonylsulfamoyl)triethylammonium hydroxide (Burgess Reagent, 125 mg, 0.52 mmol) in three portions over a 15 min. period. Reaction progress was monitored by TLC. Additional Burgess Reagent (20 mg) was added after 1.25 h, and another 30 mg after 1 h more. The reaction mixture was concentrated at reduced pressure and partitioned between EtOAc and water. The aqueous layer was extracted twice with EtOAc and the combined organic layers washed with brine, dried over Na₂SO₄ and concentrated at reduced pressure to give a semi-solid that was dissolved in EtOAc and filtered through a pad of SiO₂ eluting with EtOAc. The filtrate was concentrated at reduced pressure to give the title compound as a colorless oil: 1H CDC1₃ δ 8.81 (s, IH), 8.20 (s, IH), 7.82 (dd, J=8.79, 7.57 Hz, IH), 7.60 (dd, J=8.92, 1.6 Hz, IH).

Step C: l-[3-Chloro-2-fluoro-6-(lH-l,2,4-triazol-l-yl phenyllmethanamine

A solution of 3-chloro-2-fluoro-6-(lH-l,2,4-triazol-l-yl)benzonitrile (87 mg, 0.39 mmol) in 10 mL of absolute ethanol was added to approx. 200 mg of ethanol- washed Raney Ni under Ar. Into the resulting slurry was bubbled NH₃ gas for 3 min, then the flask was fitted with an H₂ balloon and stirred overnight. The flask was purged with Ar, the contents filtered under Ar through a glass fiber filter, and washed with ethanol. The filtrate was concentrated at reduced pressure and chromatographed on a 10 g RediSep column using CHC1₃ to 90:10:1 CHCls-MeOH-NH^OH over 40 min, 20 mL/min. The pure fractions were combined and concentrated at reduced pressure to give the title compound as a colorless oil: 1H NMR (dg-DMSO) δ 9.05 (s, IH), 8.30 (s, IH), 7.71 (t, 8.05 Hz, IH), 7.56 (dd, 1=8.6, 1.58 Hz, IH), 3.56 (d, 1.71 Hz, 2H), 1.90 (br s, 2H).

Step D: f4R -N-tert-butoxycarbonyl-N-r3-chloro-2-fluoro-6-dH-1.2,4-triazol-

4-ium- 1 -vDbenzyl] -4-fluoro-L-prolinamide

To a stirred solution of l-[3-chloro-2-fluoro-6-(lH-l,2,4-triazol-l- yl)phenyl]methanamine from previous step (0.066 g, 0.22 mmol), (4R)-l-(tert- butoxycarbonyl)-4-fluoro-L-proline (Example 3, Step B, 0.057 g, 0.24 mmol), and HOBT hydrate (0.036g, 0.26 mmol) in DMF (2 mL) was added EDC (0.063 g, 0.33 mmol). The pH of the solution was slowly raised to pH 6 (as measured on wetted E. Merck pH indicator strips) by the gradual addition of diisopropylethylamine (- 0.15 mL). Reaction was stirred for 3 days. The solvent was removed on a rotary evaporator (bath temp 30°C, -0.5 torr) and the residue was partitioned between EtOAc and 10% Na₂CO₃. The organic phase was washed with water and brine, dried (MgSO₄), filtered, and the solvent was removed on a rotary evaporator (bath temp 30°C, -50 torr). (4R)-N-tert-butoxycarbonyl-N-[3-chloro-2-fluoro-6-(lH-l,2,4- triazol-4-ium-l-yl)benzyl]-4-fluoro-L-prolinamide was obtained as an oil (LC-MS m/z = 442, 444).

Step E: (4R)-N-[3-chloro-2-fluoro-6-(lH-l,2,4-triazol-4-ium-l-vnbenzyll-4- fluoro-L-prolinamide dichloride

(4R)-N-tert-butoxycarbonyl-N-[3-chloro-2-fluoro-6-(lH-l,2,4-triazol-4-ium- l-yl)benzyl] -4-fluoro-L-prolinamide from the previous step (0.106 g, 0.24 mmol) was dissolved in EtOAc (1 mL) and cooled with stirring to 0°C. HCl/EtOAc (3.55M, 2 mL) was added. After 0.75 hours at 0°C, the bath was removed and stirred for 1 hour. HPLC analysis indicated completion and the solvent was removed under reduced pressure. (4R)-N-[3-chloro-2-fluoro-6-(lH-l,2,4-triazol-4-ium-l-yl)benzyl]-4-fluoro- L-prolinamide dichloride was obtained as a solid (HPLC RT = 0.77 min, LC-MS (M+H) =331, 333)

Step F: N-(tert-butoxycarbonyl -3-(l-methylcyclopropyD-D-alanyl-(4R -N-[3- chloro-2-fluoro-6-dH- 1 ,2,4-triazol- 1 -vDbenzyl] -4-fluoro-L-prolinamide

To a stirred solution of (4R)-Ν-[3-chloro-2-fluoro-6-(lΗ-l,2,4-triazol-4-ium- l-yl)benzyl] -4-fluoro-L-prolinamide dichloride from the previous step (0.048 g, 0.12 mmol), N-(tert-butoxycarbonyl)-3-(l-methylcyclopropyl)-D-alanine (Example 4, Step C, 0.028 g, 0.12 mmol), and HOBT hydrate (0.019 g, 0.14 mmol) in DMF (1 mL) was added EDC (0.033 g, 0.17 mmol). Diisopropylethylamine (0.045 mL, 0.35 mmol) was added and the mixture was stirred at ambient temperature overnight, at which time HPLC analysis indicated complete consumption of the proline starting material. The DMF was removed under reduced pressure and the residue was partitioned between EtOAc (50 mL) and 10% aqueous Na₂CO₃ (20 mL). The EtOAc layer was separated, washed with water and brine, dried over anhydrous MgSO , and filtered. The filtrate solvent was removed under reduced pressure and the residue was purified by preparative HPLC on a Gilson prep system using 10-100% CH₃CN H₂O with 0.1%TFA as eluant over 10 min. Combine product peak and remove organics under reduced pressure. The aqueous residue was basified with 10% Na₂CO₃, extracted with 4x20mL CH₂C1₂, dried over Na₂SO and filtered. The filtrate solvent was removed under reduced pressure to afford N-(tert-butoxycarbonyl)-3-(l- methylcyclopropyl)-D-alanyl-(4R)-N-[3-chloro-2-fluoro-6-(lH-l,2,4-triazol-l- yl)benzyl]-4-fluoro-L-prolinamide as a semi-solid. HPLC RT = 2.4 min; LC-MS (M+H) = 567, 569.

Step G: 3-d-methylcvclopropyl -D-alanyl-(4R -N-[3-chloro-2-fluoro-6-dH- l,2,4-triazol-4-ium-l-yl)benzyl]-4-fluoro-L-prolinamide dichloride

N-(tert-butoxycarbonyl)-3-(l-methylcyclopropyl)-D-alanyl-(4R)-N-[3-chloro- 2-fluoro-6-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoro-L-prolinamide from the previous step (0.021 g, 0.04 mmol, HPLC RT = 2.4 min) was dissolved in EtOAc (1 mL) and cooled with stirring to 0°C. HCl EtOAc (3.55M, 1 mL) was added. After 1.5 hours at 0°C, HPLC analysis indicated completion and the solvent was removed under reduced pressure to give 3-(l-methylcyclopropyl)-D-alanyl-(4R)-N-[3-chloro-2-fluoro-6-(lH- l,2,4-triazol-4-ium-l-yl)benzyl]-4-fluoro-L-prolinamide dichloride as a glass. HPLC RT = 1.4 min; LC-MS (M+H) = 467, 469; 1H NMR, 400 MHz, CD₃OD, 8.93, (br s, IH); 8.29, (br s, IH); 7.68, (t, J=8.2Hz, IH); 7.38, (t, J=8.6Hz, IH); 5.49 (s, 2H); 4.33-4.45, (m, 2H); 4.25 (t, J=8Hz, IH); 3.90-4.03, (m, IH); 3.80-3.88 (m, IH); 2.45- 2.59 (m, IH); 1.91-2.14, (m, 2H); 1.50-1.58 (m, IH); 0.83-0.95 (m, 3H); 0.26-0.50 (m, 4H).

EXAMPLE 27 Preparation of 3-methyl-D-valyl-(4i?)-N-[5-chloro-2-(l ,2,5-thiadiazol-3-yl)benzyl]-4- fluoroprolinamide

Step A: 3-(4-Chloro-2-methylphenyl -1.2,5-thiadiazole

To a stirred solution of 3-chloro-4-(4-chloro-2-methylphenyl)-l,2,5-thiadiazole (0.503 g, 2.05 mmol) in THF (5.0 mL) was added 2M lithium borohydride in THF (2.05 mL, 4.10 mmol). The reaction was stirred at ambient temperature for 15 min then was quenched with water and extracted into EtOAc. The organic phase was dried (Na₂SO₄) and reduced in vacuo. The residue was purified by flash chromatography (eluting with CH₂Cl₂/hexanes gradient, 1-15% CH₂C1₂). 3-(4-Chloro-2-methylphenyl)-l,2,5- thiadiazole was isolated as a solid. 1H NMR (CDCI₃, 400 MHz) δ 2.49 (s, 3H), 7.30 (d, 7= 8.2 Hz, 2H), 7.34 (s, IH), 7.53 (d, 7= 8.2 Hz, IH), 8.71 (s, IH); MS m/z = 211.4.

Step B: 3-r4-Chloro-2-(dibromomethv phenyl]-l,2,5-thiadiazole

A mixture of 3-(4-chloro-2-methylphenyl)-l,2,5-thiadiazole (0.278 g, 1.32 mmol), NBS (0.235 g, 1.32 mmol) and benzoyl peroxide (16.0 mg, 0.07 mmol) in carbon tetrachloride (5.0 mL) was refluxed for 1 h. More benzoyl peroxide (16.0 mg, 0.07 mmol) was added and the solution was refluxed for 16 h. More NBS (0.235 g, 1.32 mmol) and benzoyl peroxide (32.0 mg, 0.14 mmol) were added and the solution was refluxed for a further 16 h. The resulting suspension was filtered, rinsing with ether. The filtrate was washed with 10% aqueous sodium sulfite, saturated aqueous sodium bicarbonate and brine. The organic layer was separated, dried (Na₂SO₄) and reduced in vacuo. 3-[4-Chloro-2-(dibromomethyl)phenyl]-l,2,5-thiadiazole was isolated as a yellow solid. 1H NMR (CDCI_3.400 MHz) δ 7.42 (dd, 7 = 2.1, 8.5 Hz, IH), 7.49 (d, 7 = 8.5 Hz, IH), 7.53 (s, IH), 8.20 (s, IH), 8.80 (s, IH); MS m/z = 287.7.

Step C: 5-Chloro-2-(l,2,5-thiadiazol-3-yl benzaldehyde A stirred solution of 3-[4-chloro-2-(dibromomethyl)phenyl]-l,2,5-thiadiazole (0.452 g, 1.23 mmol) and potassium sulfate (0.334, 2.45 mmol) in concentrated H₂SO (20.0 mL) was heated to 90°C for 1.5 h. The solution was poured into ice water, basified with saturated aqueous sodium carbonate and extracted into EtOAc. The organic phase was dried (Na₂SO₄) and reduced in vacuo. 5-Chloro-2-(l,2,5-thiadiazol- 3-yl)benzaldehyde was isolated as a solid. 1H NMR (CDC1₃, 400 MHz) δ 7.70 (s, 2H), 8.05 (s, lH), 8.82 (s, IH), 10.28 (s, IH).

Step D: N-[5-Chloro-2-d,2,5-thiadiazol-3-v benzyl]-N-(4-methoxybenzyl amine To a stirred solution of 5-chloro-2-(l,2,5-thiadiazol-3-yl)benzaldehyde (0.155 g,

0.69 mmol), 4-methoxybenzylamine (0.09 mL, 0.69 mmol) and acetic acid (0.05 mL, , 0.83 mmol) in 1,2-dichloroethane (5.2 mL) was added sodium triacetoxyborohydride (0.731 g, 3.45 mmol). The solution was stirred at ambient temperature for 16 h then partitioned between CH₂C1₂ and saturated aqueous sodium bicarbonate. The organic phase was dried (Νa₂SO₄) and reduced in vacuo. The residue was purified by automated preparative chromatography on silica (eluting with 5% CH₂Cl₂/EtOAc/hexanes gradient, 5-50% EtOAc). N-[5-Chloro-2-(l,2,5-thiadiazol-3- yl)benzyl]-N-(4-methoxybenzyl)amine was isolated as an oil. 1H ΝMR (CDCl_3ι 400 MHz) δ 3.74 (s, 2H), 3.81 (s, 3H), 3.82 (s, 2H), 6.86 (d, 7 = 8.6 Hz, IH), 7.22 (d, 7 = 8.4 Hz, IH), 7.55 (d, 7 = 8.4 Hz, IH), 8.84 (s,lH); MS m/z = 346.5.

Step E: 5-Chloro-2-(l,2,5-thiadiazol-3-yl)benzylammohium trifluoroacetate

To a stirred 0°C solution of N-[5-chloro-2-(l,2,5-thiadiazol-3-yl)benzyl]-N-(4- methoxybenzyl)amine (0.104 g, 0.30 mmol) in CH CΝ (5.0 mL) was added a solution of CAN (0.990 g, 1.81 mmol) in water (10.0 mL). The solution was stirred at ambient temperature for 1 h then was partitioned between EtOAc and 10% aqueous NB iOH. The resulting suspension was filtered, and the EtOAc layer was separated, washed with 10% sodium sulfite and brine, dried (Na₂SO₄) and reduced in vacuo. The resulting solid was purified by HPLC (Cι₈ eluting with an acetonitrile/water/0.1% TFA gradient). 5- Chloro-2-(l,2,5-thiadiazol-3-yl)benzylammonium trifluoroacetate was isolated as a solid. 1H NMR (CD₃OD, 400 MHz) δ 4.36 (s, 2H), 6.67 (dd, 7 = 2.3, 8.3 Hz, IH), 7.72 (s, IH), 8.06 (d, 7= 8.4 Hz, IH), 9.17 (s,lH); MS m/z = 226.5.

Step F: l-(tert-butoxycarbonyl -(4R -N-r5-chloro-2-d,2,5-thiadiazol-3- yPbenzyll -4-fluoro-L-prolinamide

A mixture of (4R)-l-(tert-butoxycarbonyl)-4-fluoro-D-proline (Example 3,

Step B, 43 mg, 0.19 mmol), l-[5-chloro-2-(l,2,5-thiadiazol-3-yl)phenyl]methanamine

(42 mg, 0.19 mmol) HOAT (13 mg, 0.09 mmol), and EDC-HC1 (54 mg, 0.28 mmol) in DMF (1.0 mL) was stirred at rt for 3 h. Solvent was removed in vacuo. Silica gel chromatography (50% EtOAc/hexanes) afforded the title compound as a brown oil.

1H NMR (CD₃OD, 400 MHz): δ 9.00 - 8.99 (m, 1 H), 7.77 - 7.62 (m, 2 H), 7.48 -

7.44 (m, 1 H), 5.29 - 5.16 (m, 1 H), 4.71 - 4.52 (m, 2 H), 4.35 - 4.31 (m, 1 H), 3.85 -

3.76 (m, IH), 3.66 - 3.50 (m, 1 H), 2.58 - 2.44 (m, 1 H), 2.34 - 1.96 (m, 1 H), 1.48 and 1.28 (BOC rotamers, s, 9 H). LCMS (M+H-BOC): 341.1.

Step G: (4R)-N-r5-chloro-2-d,2,5-thiadiazol-3-yl benzyll-4-fluoro-L- prolinamide chloride

To a solution of l-(tert-butoxycarbonyl)-(4R)-N-[5-chloro-2-(l,2,5-thiadiazol-

3-yl)benzyl]-4-fluoro-L-prolinamide(84 mg, 0.19 mmol) in EtOAc (1.0 mL) was added 4.0 M HCI in dioxane (0.5 mL, excess) and stirred overnight at rt. Solvent was removed in vacuo to give the title compound as a brown solid. LCMS (M + H) =

341.0.

Step H: N-(tert-butoxycarbonyl)-3-methyl-D-valyl-(4RVN-[5-chloro-2-d,2.5- thiadiazol-3-yl)benzyl1-4-fluoro-L-prolinamide

The title compound was prepared from (4R)-N-[5-chloro-2-(l,2,5-thiadiazol- 3-yl)benzyl]-4-fluoro-L-prolinamide chloride (45 mg, 0.19 mmol), N-(tert- butoxycarbonyl)-3-methyl-D-valine (66 mg, 0.28 mmol), HOAT (13 mg, 0.10 mmol), EDC-HC1 (56 mg, 0.29 mmol), andEt₃N (27 μL, 0.19 mmol) essentially according to the coupling procedure described in Example 1, Step L. The product was purified by preparative reverse phase HPLC [gradient elution with 95:5 water (+0.1% TFA) / CH₃CN (+0.1% TFA) to 5:95 water (+0.1% TFA) / CH₃CN (+0.1% TFA)] to afford the TFA salt as a pale yellow gum. 1H NMR (CD₃OD, 400 MHz): δ 9.05 - 8.99 (m, 1 H), 8.43 (m, 1 H), 7.81 - 7.69 (m, 2 H), 7.45 - 7.43 (m, 1 H), 5.39 - 5.26 (m, 1 H), 4.65 - 4.64 (m, 1 H), 4.55 - 4.51 (m, 1 H), 4.23 - 4.14 (m, 2 H), 3.93 - 3.80 (m, 2 H), 2.60 - 2.46 (m, 1 H), 2.18 - 1.98 (m, 1 H), 1.37 (s, 9 H), 1.03 (s, 9 H). LCMS (M+H): 554.1.

Step I: 3-methyl-D-valyl-(4R -N-[5-chloro-2-d.2.5-thiadiazol-3-yl benzvn-4- fluoro-L-prolinamide

To a solution of N-(tert-butoxycarbonyl)-3-methyl-D-valyl-(4R)-N-[5-chloro- 2-(l,2,5-thiadiazol-3-yl)benzyl]-4-fluoro-L-prolinamide (38 mg, 0.07 mmol) in CH₂C1 (1.5 mL) at rt was added TFA (0.4 mL, excess) and stirred overnight. Solvent was removed in vacuo to give the TFA salt of the title compound as a pale orange gum. 1H NMR (CD₃OD, 400 MHz): δ 9.01 (s, 1 H), 8.84 (br s, 1 H), 7.76 (d, 7 = 8.4 MHz, 1 H), 7.68 (d, 7 = 2.4 Hz, 1 H), 7.48 (dd, 7 = 2.0 MHz, 8.4 MHz, 1 H), 5.43 - 5.04 (m, 1 H), 4.66 (m, 2 H), 4.58 (t, 7 = 8.6 MHz, 1 H), 4.22 - 4.13 (m, 1 H), 4.03 (s, 1 H), 3.80 (m, 1 H), 2.71 - 2.53 (m, 1 H), 2.14 - 2.04 (m, 1 H), 1.12 (s, 9 H). LCMS (M+H): 454.2.

EXAMPLE 28 Preparation of (4S)-4-azido-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-l-[(9- hydroxy-9H-fluoren-9-yl)carbonyl]prolinamide

Step A: l-tert-butyl 2-methyl (2S,4R -4-[(^'methylsulfonyl oxylpyrrolidine-l,2- dicarboxylate

To a stirred solution of 1-tert-butyl 2-methyl (2S,4R)-4-hydroxypyrrolidine- 1,2-dicarboxylate (2.5 g, 10.2 mmol) in CH₂C1₂ (15 mL) was added Et₃N (1.7 mL, 12.3 mmol) and the mixture cooled to 0 °C under a nitrogen atmosphere.

Methanesulfonyl chloride (0.9 mL, 11.3 mmol) was added and the mixture allowed to stir for an additional 5 hours. The reaction mixture was then diluted with CH₂C1₂, washed with three aliquots of water and dried to yield 1-tert-butyl 2-methyl (2S,4R)- 4-[(methylsulfonyl)oxy]pyrrolidine-l,2-dicarboxylate (HPLC RT = 2.95 min, Method A; LCMS m/z = 268). Note: The t-butyl group was removed under LCMS conditions, so actual mass was 323.

Step B: 1-tert-butyl 2-methyl (2S,4S -4-azidopyrrolidine-l,2-dicarboxylate

To a stirred solution of 1-tert-butyl 2-methyl (2S,4R)-4- [(methylsulfonyl)oxy]pyrrolidine-l,2-dicarboxylate (3.3 g, 10.2 mmol) in degassed DMF (20 mL) was added sodium azide (1.2 g, 19.3 mmol) and the mixture was warmed to 35 °C under a nitrogen atmosphere. The reaction was followed to completion by thin layer chromatography (1:1 EtOAc to hexane to develop and phosphomolybdic acid (PMA) to visualize). Upon completion, the reaction mixture was evaporated in vacuo and purified on silica gel using a 1 :3 mixture of EtOAc to hexane as mobile phase and PMA to visualize. Pure fractions were combined and dried to yield 1-tert-butyl 2-methyl (2S,4S)-4-azidopyrrolidine-l,2-dicarboxylate (HPLC RT = 3.12 min, Method A; LCMS m/z = 271).

Step C: (4SV4-azido-l-(tert-butoxycarbonyl)-L-proline

1-tert-Butyl 2-methyl (2S,4S)-4-azidopyrrolidine-l,2-dicarboxylate (0.2 g, 0.88 mmol) was dissolved in ethyl alcohol (2 mL) with IN sodium hydroxide solution (1.06 mL, 0.96 mmol) and stirred at room temperature for 72 hours. Upon completion, the mixture was neutralized with IN HCI solution (1.06 mL, 0.96 mmol) and evaporated to yield (4S)-4-azido-l-(tert-butoxycarbonyl)-L-proline (HPLC RT = 2.64 min, Method A).

Step D: (4S -4-azido- 1 -(tert-butoxycarbonyl)-N-[5-chloro-2-dH- 1 ,2,4-triazol- 1 - vDbenzyll -L-prolinamide

To a stirred solution of (4S)-4-azido-l-(tert-butoxycarbonyl)-L-proline (0.2 g, 0.88 mmol) in degassed DMF (2 mL) was added ΗOBt hydrate (0.15 g, 1.14 mmol), EDC (0.25 g, 1.27 mmol), l-[5-chloro-2-(lH-l,2,4-triazol-l-yl)phenyl]methanamine (Example 6, Step B, 0.2 g, 1.05 mmol) and N-methylmorpholine to pΗ = 7. The reaction was complete after 1.5 hours, evaporated in vacuo and partitioned between EtOAc and water. The organic layer was washed with 15 volumes of water, evaporated in vacuo and dried to yield (4S)-4-azido-l-(tert-butoxycarbonyl)-N-[5- chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-L-prolinamide (ΗPLC RT = 3.11 min, Method A).

Step E: (4S -4-Azido-N-[5-Chloro-2-dH-l,2,4-triazol-l-yl benzyl1-L- prolinamide

(4S)-4-Azido-l-(tert-butoxycarbonyl)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -L-prolinamide (0.32 g, 0.72 mmol) was dissolved in EtOAc (20 mL), cooled to 0 °C and saturated with anhydrous ΗC1 gas. After 3 hours the mixture was evaporated in vacuo and dried to yield (4S)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl]-4-azido-L-prolinamide (ΗPLC RT = 2.32 min, Method A).

Step F: (4S)-4-Azido-N-r5-chloro-2-dH-l,2,4-triazol-l-vPbenzyll-l-r(9- hydroxy-9H-fluoren-9-yl)carbonyl]-L-prolinamide

(4S)-4-Azido-N-[5-Chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-L-prolinamide (0.25 g, 0.65 mmol) was dissolved in degassed DMF (2 mL). To this solution was added ΗATU (0.36 g, 0.94 mmol), 9-hydroxy-9H-fluorene-9-carboxylic acid (0.17 g, 0.76 mmol) and DIEA (0.26 mL) and the mixture stirred at room temperature for 1 hour. The reaction mixture was then filtered and purified on a preparative ΗPLC. Pure fractions were combined and dried to yield (4S)-4-azido-N-[5-chloro-2-(lH- l,2,4-triazol-l-yl)benzyl]-l-[(9-hydroxy-9H-fluoren-9-yl)carbonyl]-L-prolinamide (ΗPLC RT = 3.23 min, Method A; LCMS m/z = 555).

EXAMPLE 29 Preparation of (4S)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-l-[(9-hydroxy-9H- fluoren-9-yl)carbonyl]-4-methylprolinamide

To a stirred solution of (4S)-4-azido-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl]-l-[(9-hydroxy-9H-fluoren-9-yl)carbonyl]-L-prolinamide (Example 28, Step F, 0.28 g, 0.51 mmol) in freshly distilled TΗF (3.5 mL) was added distilled water (0.18 mL, 10.2 mmol) and triphenylphosphine (0.15 g, 0.59 mmol). The reaction was stirred at room temperature overnight then evaporated in vacuo and purified by preparative ΗPLC. Pure fractions were combined and dried to give the TFA salt of (4S)-4-amino-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-l-[(9-hydroxy-9H- fluoren-9-yl)carbonyl]-L-prolinamide (ΗPLC RT = 2.67 min, Method A; LCMS m/z = 529). 1H ΝMR (400 MHz, CD₃OD): δ 8.84 (s, IH), 8.24 (s, IH), 7.82-7.80 (m, 3H), 7.56-7.53 (m, IH), 7.50-7.43 (m, 3H), 7.42-7.38 (m, IH), 7.36-7.31 (m, 3H), 4.75-4.63 (d, IH), 4.48-4.44 (d, IH), 4.29-4.25 (d,lH), 3.56 (s, IH), 2.86-2.69 (m, 2H), 2.40-2.32 (m, IH), 1.85-1.81 (d, IH).

EXAMPLE 30 Preparation of (4S)-4-amino-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-l-[(2i?,3R)- 2-hydroxy-3-methylpentanoyl]prolinamide

Step A: (4R -4-hvdroxy-l-(tert-butoxycarbonyl -N-[5-chloro-2-dH-1.2.4- triazol- 1 -vDbenzyl] -L-prolinamide

To a stirred solution of l-[5-chloro-2-(iH-l,2,4-triazol-l- yl)phenyl]methanamine (Example 6, Step B, 0.80 g, 3.8 mmol, ΗPLC RT = 1.81 min, Method A), (4R)-4-hydroxy-l-(tert-butoxycarbonyl)-L-proline (Example 3, Step B, 0.89 g, 3.8 mmol), and ΗOBt hydrate (0.58 g, 3.8 mmol) in DMF (15 mL) was added EDC (0.94 g, 4.9 mmol). The pΗ of the solution was slowly raised to pΗ 6 (as measured on wetted E. Merck pΗ indicator strips) by the gradual addition of DIEA (~ 0.7 mL). At 4 hours reaction time, ΗPLC analysis indicated complete consumption of the phenylmethanamine starting material. The solvent was removed on a rotary evaporator (bath temp 30°C, -0.5 torr) and the residue was partitioned between EtOAc (100 mL) and water (50 mL). The organic phase was dried (MgSO₄), filtered, and the solvent was removed on a rotary evaporator. The residue was purified by flash silica gel column chromatography using a gradient elution of 2%, 4%, 8% MeOΗ in dichloromethane. Product-containing fractions were combined, and removal of solvent under reduced pressure gave (4R)-4-hydroxy-l-(tert- butoxycarbonyl)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-L-prolinamide as a foam (TLC R_f = 0.3 (95:5 CΗ₂Cl₂:MeOΗ); HPLC RT = 2.62 min, method A; LCMS m/z = 422).

Ste B: (4R)-4-[(methylsulfonyDoxy1-l-(tert-butoxycarbonvD-N-r5-chloro-2- dH-l,2,4-triazol-l-yDbenzyl1-L-ρrolinamide A stirred solution of (4R)-4-hydroxy-l-(tert-butoxycarbonyl)-N-[5-chloro-2- (lH-l,2,4-triazol-l-yl)benzyl]-L-prolinamide from the previous step (1.5 g, 3.5 mmol) and triethylamine (0.98 mL, 7.0 mmol) in dichloromethane (20 mL) was cooled to 0 °C. To the solution was added methanesulfonyl chloride (0.29 mL, 3.8 mmol) and the resulting solution was stirred at 0 °C for 1 h and then at ambient temperature for 3 h. The mixture was diluted with CΗ₂C1₂ (30 mL) and washed with water (50 mL). The organic phase was dried (MgSO₄), filtered, and the solvent was removed on a rotary evaporator to give (4R)-4-[(methylsulfonyl)oxy]-l-(tert-butoxycarbonyl)-N-[5-chloro- 2-(lH-l,2,4-triazol-l-yl)benzyl]-L-prolinamide as a foam (ΗPLC RT = 2.99 min, method A).

Step C: (4S -4-azido-l-(tert-butoxycarbonvD-N-r5-chloro-2-dH-1.2,4-triazol-

1-vDbenzyll-L-prolinamide

To a stirred solution of (4R)-4-[(methylsulfonyl)oxy]-l-(tert-butoxycarbonyl)- N-[5-chloro-2~(lH-l,2,4-triazol-l-yl)benzyl]-L-prolinamide (1.7 g, 3.4 mmol) from the previous step in DMF (15 mL) was added sodium azide (1.1 g, 17 mmol). The mixture was warmed to 50 °C under an inert atmosphere and stirred for 18 h. The DMF was removed under reduced pressure and the residue was partitioned between EtOAc (100 mL) and water (50 mL). The organic phase was dried (MgSO₄), filtered, and the solvent was removed under reduced pressure to give (4S)-4-azido-l-(tert- butoxycarbonyl)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-L-prolinamide as a foam (ΗPLC RT = 3.10 min, method A; LCMS m/z = 447).

Step D: (4S -4-Azido-N-[5-chloro-2-dH-l,2,4-triazol-l-vDbenzyll-L- prolinamide

Into a stirred solution of (4S)-4-azido-l-(tert-butoxycarbonyl)-N-[5-chloro-2- (lH-l,2,4-triazol-l-yl)benzyl]-L-prolinamide from the previous step (1.4 g, 10.6 mmol, 3.1 mmol) in EtOAc (25 mL) cooled to 0 °C was bubbled ΗC1 gas for 10 minutes. The mixture was stirred for 1.5 h at 0 °C and the EtOAc was removed under reduced pressure. The resulting solid was triturated in a small volume of EtOAc and collected by filtration to give 4(S)-4-azido-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -L-prolinamide as a solid (ΗPLC RT = 2.32 min, method A; LCMS m/z =

347).

Step E: 4(S)-4-Azido-N-r5-chloro-2-(iH-l,2.4-triazol-l-vDbenzyl1-l-

[(2R,3R>2-hvdroxy-3 -methylpentanoyl] -L-prolinamide

To a solution of 4(S)-4-azido-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-L- prolinamide from the previous step (0.25 g, 0.65 mmol), 2(R), 3(R)-2-hydroxy-3- methylpentanoic acid (95 mg, 0.72 mmol), and ΗOBt hydrate (99 mg, 0.65 mmol) in DMF (5 mL) was added EDC (165 mg, 0.85 mmol). Diisopropylethylamine was then added slowly in portions (-0.1 mL total) to bring the pΗ of the solution to 6-7 as measured on wetted E. Merck pΗ indicator strips. The mixture was stirred at ambient temperature for 18 h, and the solvent was removed under reduced pressure. The residue was partitioned between EtOAc (50 mL) and saturated aqueous ΝaΗCO₃ (20 mL). The EtOAc layer was separated, dried over anhydrous MgSO₄, and filtered.

The filtrate solvent was removed under reduced pressure and the residue was purified by flash chromatography using a gradient elution of 2%, 3%, 4% MeOH in dichloromethane. Product-containing fractions were combined and the solvent was removed under reduced pressure to give 4(S)-4-azido-N-[5-chloro~2-(iH-l,2,4-triazol- l-yl)benzyl]-l-[(2R,3R)-2-hydroxy-3-methylpentanoyl]-L-prolinamide as a foam

(TLC R_f = 0.3 (97:3 CΗ₂Cl₂:MeOΗ); HPLC RT = 2.86 min, method A; LCMS m/z = 461).

Step F: 4(^'S -4-Amino-N-r5-chloro-2-(^'iH-1.2.4-triazol-l-vDbenzyl1-l- r(2R,3R)-2-hvdroxy-3-methylpentanoyll-L-prolinamide

To a stirred solution of 4(S)-4-azido-N-[5-chloro-2-(iH-l,2,4-triazol-l- yl)benzyl]-l-[(2R,3R)-2-hydroxy-3-methylpentanoyl]-L-prolinamide from the previous step (0.29 g, 0.63 mmol) in TΗF (6 mL) was added triphenylphosphine (0.20 g, 0.76 mmol) and water (1 mL). The mixture was heated to 60 °C for 4 h, at which time ΗPLC analysis indicated complete consumption of the starting azide (ΗPLC RT = 2.86 min) and an intermediate which had formed (HPLC RT = 3.22 min, presumably the imino phosphorane). The solvent was removed under reduced pressure and the residue was purified by pressurized silica gel column chromatography using a gradient elution of 5%, 8%, 12%, 15% A in CH₂C1₂ (A = 95:5 MeOH÷NHiOH). Product-containing fractions were combined and the solvent was removed in vacuo to give the title compound as an amorphous solid (TLC Rf = 0.2 (92:8 CH₂Cl₂:MeOH); HPLC RT = 2.41 min, method A; LCMS m/z = 435).

EXAMPLE 31

Preparation of (4S)-4-amino-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-l-[(27?)-2- cyclohexyl-2-hydroxyethanoyl]-L-prolinamide

Step A; (4S)-4-azido-N-r5-chloro-2-dH-1.2.4-triazol-l-vDbenzyll-l-[(2J?)-2- cyclohexyl-2-hvdroxyethanoyll-L-prolinamide

To a stirred solution of (4S)-4-Azido-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl]-L-prolinamide (Example 30, Step D, 120 mg, 0.38 mmol, ΗPLC RT = 2.28 min, method A), R-(-)-hexahydromandelic acid (60 mg, 0.38 mmol), ΗOBT hydrate (58 mg, 0.38 mmol), and EDC (94 mg, 0.49 mmol) in DMF (3 mL) was added diisopropylethylamine (approximately 60 microliters) until a reading of pΗ 6 on wetted E. Merck pΗ indicator strips was obtained. The mixture was stirred at ambient temperature for 3 h, and then the DMF was removed under reduced pressure. The residue was partitioned between EtOAc (25 mL) and saturated aqueous sodium bicarbonate solution (10 mL). The organic phase was washed with water (10 mL), dried (MgSO₄), filtered, and the solvent was removed under reduced pressure to give the title compound as a gum (HPLC RT = 2.98 min, method A; LC-MS RT = 2.07 min, m/z = 487.2).

Step B: (4S)-4-amino-N-[5-chloro-2-dH-1.2,4-triazol-l-vDbenzvn-l-[(2J? -2- cyclohexyl-2-hvdroxyethanoyl]-L-prolinamide

To a stirred solution of (4S)-4-azido-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl]-l-[(2R)-2-cyclohexyl-2-hydroxyethanoyl]-L-prolinamide (160 mg, 0.33 mmol) in TΗF (5 mL) was added water (0.5 mL) and triphenylphosphine (260 mg, 1.0 mmol). The mixture was warmed to 50 °C and stirred for 24 h. The reaction mixture was purified by preparative reverse phase ΗPLC using an acetonitrile/water/TFA gradient elution (Example 37). Product-containing fractions were combined and the solvents were removed in vacuo to give the TFA of the title compound as an amorphous solid (ΗPLC RT = 2.51 min, method A; LC-MS RT = 1.34 min, m z = 461.27; Exact mass C₂₂Η₂₉ClΝ₆O₃ + H = 461.2063, found 461.2064).

EXAMPLE 32 Preparation of 4-methyl-D-leucyl-(3i?)-N- [5 -chloro-2-( lH-tetraazol- 1 -ypbenzyl] -3- methylprolinamide

Step A: (S -rBut-3-enyl-(l-phenyl-ethyD-aminol-acetic acid benzyl ester

The title compound was prepared essentially according to the procedure described in Karoyan, P.; Chassaing, G. Tetrahedron: Asymm. 1997, 8, 2025-2032 for the preparation of (S)-[but-3-enyl-(l-phenyl-ethyl)-amino]-acetic acid ethyl ester.

Ste B: Benzyl (3R)-3-methyl-l-d-phenylethvD-L-prolinate The title compound was prepared from (S)-[but-3-enyl-(l-phenyl-ethyl)- amino]-acetic acid benzyl ester essentially according to the cyclization protocol described in Karoyan, P.; Chassaing, G. Tetrahedron: Asymm. 1997, 8, 2025-2032 with the following modifications: LDA was added at -40 °C, then the solution was warmed to 0 °C for 10 min, then re-coόled to -40 °C for the zinc bromide addition. The transmetallation step was omitted and the anion was quenched directly with 2:1 saturated NH₄CI: ammonium hydroxide. Extractive workup (Et O) afforded an orange oil which was purified by silica gel chromatography (5 - 10% EtOAc-hexanes) to give the title compound as a yellow oil. 1H NMR (400 MHz, CDC1₃): δ 7.38 - 7.18 (m, 10 H), 5.10 (d, 7= 12.3 Hz, 1 H), 5.02 (d, 7 = 12.1 Hz, 1 H), 3.69 (q, 7 = 6.6 Hz, 1 H), 3.38 (d, 7 = 8.2 Hz, 1 H), 3.07 - 3.02 (m, 1 H), 2.92 - 2.86 (m, 1 H), 2.45 - 2.39 (m, 1 H), 2.01 - 1.93 (m, 1 H), 1.68 -1.60 (m, 1 H), 1.33 (d, 7 = 6.6 Hz, 3 H), 0.88 (d, 7 = 7.0 Hz, 3 H).

Step C: GRy3-methyl-L-proιine

Pearlman's catalyst (325 mg) was added to a solution of benzyl (3R)-3- methyl-l-(l-phenylethyl)-L~prolinate (1.61 g, 4.98 mmol) in absolute MeOH (60 mL). The mixture was degassed and purged with Argon and was then shaken on a Parr hydrogenation apparatus under 50 psi hydrogen atmosphere for 16 h. The mixture was then filtered through Celite. The filter cake was washed with MeOH and H₂O and the filtrate was concentrated to give the title compound as a white solid. 1H NMR (400 MHz, CD₃OD): δ 3.93 (d, 7 = 7.6 Hz, 1 H), 3.48 - 3.42 (m, 1 H), 3.25 - 3.18 (m, 1 H), 2.72 - 2.61 (m, 1 H), 2.22 - 2.13 (m, 1 H), 1.79 - 1.71 (m, 1 H),1.06 (d, 7 = 7.1 Hz, 3 H).

Step D: (3R)- 1 -(tert-butoxycarbonyD-3 -methyl-L-proline

To a stirred solution of (3R)-3 -methyl-L-proline (315 mg, 2.44 mmol) in dioxane (6.4 mL) and H₂O (2.2 mL) at 0 C was added 1 N NaOH solution (2.6 mL, 2.6 mmol) and BOC anhydride (576 mg, 2.64 mmol). The mixture was stirred at room temperature for 16 h. The dioxane was removed in vacuo and the aqueous residue was cooled to 0 °C and adjusted to pH 3 by the dropwise addition of 1 N HCI (2.7 mL). The cold mixture was extracted three times with EtOAc and once with THF, saturating the aqueous layer with NaCl before each extraction. The combined organic extracts were dried (Na₂SO₄), filtered and concentrated to give a pale yellow oil. The product slowly solidified after azeotroping with CHC1₃ and drying under high vacuum to give the title compound as a foam. 1H NMR (400 MHz, CDC1₃): δ 4.16 (d, 7= 8.2 Hz, 1 H), 3.63 - 3.56 (m, 1 H), 3.36 - 3.32 (m, 1 H), 2.60 - 2.48 (m, 1 H), 2.00 - 1.94 (m, 1 H), 1.76 -1.61 (m, 1 H), 1.46 and 1.42 (s, rotamers of Boc group, 9 H), 1.06 (d, 7 = 7.0 Hz, 3 H).

Step E: (3J?)-l-(tert-butoxycarbonvD-N-r5-chloro-2-dH-tetraazol-l- yPbenzyl] -3 -methyl-L-prolinamide

A mixture of (3R)-l-(tert-butoxycarbonyl)-3-methyl-L-proline (305 mg, 1.33 mmol), l-[5-chloro-2-(lH-tetraazol-l-yl)phenyl]methanamine (Example 1, Step D, 298 mg, 1.42 mmol), EDC (383 mg, 2.00 mmol) and ΗOAt (91 mg, 0.66 mmol) in DMF (7.0 mL) was stirred at room temperature for 16 h. The DMF was removed in vacuo and the residue was partitioned between EtOAc and saturated aqueous K CO₃. The layers were separated and the organic layer was washed with brine. The combined aqueous layers were extracted once with CΗ₂C1₂ and the combined organic extracts were then dried (Νa₂SO₄), filtered and concentrated to an orange oil. Silica gel chromatography afforded the title compound as a white foam. 1H NMR (400 MHz, CDC1₃): δ 9.01 (s, 1 H), 7.76 (d, 7= 1.8 Hz, 1 H), 7.44 (br m, 1 H), 6.62 - 6.51 (br m, 2 H), 4.25 - 4.11 (m, 2 H), 4.08 (d, 7= 8.4 Hz, 1 H), 3.63 (br m, 1 H), 3.35 (br m, 1 H), 2.45 (br m, 1 H), 1.96 (br m, 1 H), 1.77 - 1.61 (br m, 1 H), 1.59 (s, 9 H), 0.97 (d, 7 = 6.8 Hz, 3 H). LCMS (M+H): 420.9.

Step F: (3R -N-[5-chloro-2-dH-tetraazol-l-vPbenzvn-3-methyl-L-prolinamide

To a stirred solution of (3E)-l-(tert-butoxycarbonyl)-N-[5-chloro-2-(lH- tetraazol-l-yl)benzyl]-3-methyl-L-prolinamide (415 mg, 0.99 mmol) in MeOΗ (20 mL) was added 1 M ΗCl-Et₂O solution (2.5 mL, 2.5 mmol). After 16 h, additional 1 M HCl-Et₂O (3 mL) was added, and after 5 h another 5.5 mL was added. After 16 h the solvent was evaporated to give the hydrochloride salt of the title compound as a white solid. 1H NMR (400 MHz, CD₃OD): δ 9.58 (s, 1 H), 8.84 (br m, 1 H), 7.72 (d, 7 = 2.1 Hz, 1 H), 7.60 - 7.52 (m, 2 H), 4.39 - 4.24 (m, 2 H), 4.19 (d, 7 = 7.7 Hz, 1 H), 3.51 - 3.44 (m, 1 H), 3.34 - 3.24 (m, 1 H), 2.74 - 2.67 (m, 1 H), 2.26 - 2.18 (m, 1 H), 1.80 - 1.71 (m, 1 H), 0.91 (d, 7 = 7.1 Hz, 3 H). LCMS (M+H): 321.0.

Step G: 4-Methyl-D-leucyl-(3R -N-[5-chloro-2-dH-tetraazol-l-vPbenzyl]-3- methyl-L-prolinamide The title compound was prepared from N-(tert-butoxycarbonyl)-4-methyl-D- leucine (62 mg, 0.25 mmol), (3R)-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-3-methyl- L-prolinamide (100 mg, 0.25 mmol, 1.0 equiv.), EDC (73 mg, 0.38 mmol, 1.5 equiv.) and ΗOAt (17 mg, 0.13 mmol, 0.5 equiv.) in DMF (2 mL) followed by deprotection with TFA essentially according to the procedure described in Example 1, Steps L and M. Purification by reverse phase chromatography [95:5 water (+0.1% TFA)/CΗ₃CΝ (+0.1% TFA) to 5:95 water (+0.1% TFA)/CH₃CN (+0.1% TFA)] afforded the compound as a white powder. 1H NMR (400 MHz, CDC1₃): δ 9.16 (s, 1 H), 8.30 (br s, 2 H), 8.07 (br s, 1 H), 7.64 (d, 7 = 2.0 Hz, 1 H), 7.40 (dd, 7 = 2.0, 8.4 Hz, 1 H), 7.24 (d, 7 = 8.4 Hz, 1 H), 4.27-4.25 (m, 2 H), 4.16 (dd, 7 = 4.8, 15.2 Hz, 1 H), 4.09 (dd, 7 = 5.6, 15,2 Hz, 1 H), 4.01-3.97 (m, 1 H), 3.52-3.44 (m, 1 H), 2.46-2.39 (m, 1 H), 1.92- 1.77 (m, 4 H), 1.02 (d, 7 = 6.8 Hz, 3 H), 0.98 (s, 9 H). HRMS (APCI) M+H: calculated for (C₂ιH₃₀N₇O₂Cl)⁺ 448.2222, found 448.2215.

EXAMPLE 33

Preparation of 4-methyl-D-leucyl-N-[5-chloro-2-(lH-l ,2,4-triazol-l-yl)benzyl]-5- methylprolinamide

Step A: l-(tert-Butoxycarbonyp-5-methyl-L-proline

A solution of 5-methyl-L-proline (mixture of diastereomers at C-5, 249 mg, 1.93 mmol) in water (1.8 mL) and dioxane (5.5 mL) at 0 °C was treated with 1 N NaOH (2.12 mL, 2.12 mmol, 1.1 equiv.) and Boc₂O (463 mg, 2.12 mmol, 1.1 equiv.). The mixture was stirred at room temperature for 105 min. The dioxane was removed in vacuo and the aqueous residue was diluted with water (2 mL) and cooled to 0 °C. The cold mixture was treated slowly dropwise with 1 N HCI (2.2 mL, to pH 3). The mixture was saturated with NaCl and extracted with EtOAc. The saturation/extraction process was repeated twice more and the combined organic extracts were dried

(Na₂SO ), filtered and concentrated to give the title compound as a sticky white solid. 1H NMR (400 MHz, CD₃OD): δ 4.27 - 4.19 (m, 1 H), 4.08 - 3.96 (br m, 1 H), 2.31 - 2.24 (br m, 1 H), 2.13 - 1.93 (brm, 2 H), 1.67 - 1.60 (m, 1 H), 1.52 and 1.47 (br s, 9 H, Boc rotamers), 1.27 (d, 7 = 6.4 Hz, 3 H).

Step B: N-[5-chloro-2-(lH-l,2,4-triazol-l-vPbenzyll-5-methyl-L-prolinamide

A mixture of l-(tert-Butoxycarbonyl)-5-methyl-L-proline (177 mg, 0.77 mmol, 1 equiv.), l-[5-chloro-2-(lH-l,2,4-triazol-l-yl)phenyl]methanamine (Example 6, Step B, 169 mg, 0.81 mmol, 1.05 equiv.), EDC (222 mg, 1.16 mmol, 1.5 equiv.) and ΗOAt (53 mg, 0.39 mmol, 0.5 equiv.) in DMF (5.3 mL) was stirred at room temperature for 2 h. The crude mixture was purified by reverse phase ΗPLC (Example 37) and the product fractions were combined and concentrated in vacuo. The aqueous residue was basified by the addition of saturated aqueous K₂CO₃. The mixture was saturated with ΝaCl and extracted with EtOAc. The saturation/extraction process was repeated twice and the combined organic extracts were then dried

(Νa₂SO₄), filtered and concentrated to a white foam. LCMS (M+Η) = 420.2. The solid was dissolved in EtOAc (6.5 mL), cooled to 0 °C and treated with a 4 M solution of HCI in dioxane (1 mL). Some precipitation occurred and the precipitate was dissolved by the addition of a minimal volume of MeOH. The mixture was allowed to warm to room temperature and additional 4 M HCl-dioxane (2.5 mL) was added after 3 h and again at 6 h. The mixture was stirred for an additional 1.5 h and was then concentrated in vacuo. The residue was stripped from ether to afford the bis- HC1 salt of the title compound as a light yellow foam. LCMS (M+H) = 320.1. 1H NMR (400 MHz, CD₃OD): δ 9.39 (s, 1 H), 8.79 (br m, 1 H), 8.58 (s, 1 H), 7.64 (d, 7 = 1.2 Hz, 1 H), 7.58 - 7.52 (m, 2 H), 4.40 (app d, 7 = 5.2 Hz, 2 H), 4.32 - 4.23 (m, 1 H), 3.86 - 3.63 (br m, 1 H), 2.43 - 2.35 (m, 1 H), 2.28 - 2.19 (m, 1 H), 2.09 - 2.04 (m, 1 H), 1.70 - 1.59 (m, 1 H), 1.43 (d, 7= 6.8 Hz, 3 H).

Step C: N-(tert-butoxycarbonyP-4-methyl-D-leucyl-N-[5-chloro-2-dH-l,2.4- triazol- 1 -yPbenzyll -5 -methyl-L-prolinamide A mixture of N-[5-chloro-2-(lH-l ,2,4-triazol- l-yl)benzyl]-5-methyl-L- prolinamide hydrochloride (100 mg, 0.28 mmol, 1 equiv.), N-(tert-butoxycarbonyl)-4- methyl-D-leucine (83 mg, 0.34 mmol, 1.2 equiv.), EDC (81 mg, 0.42 mmol, 1.5 equiv.), ΗOAt (38 mg, 0.28 mmol, 1 equiv.) and Hunig's base (98 μL, 0.56 mmol, 2.0 equiv.) in DMF was stirred at room temperature for 2 h. The crude mixture was purified by reverse phase HPLC (Example 37) to give a mixture of diastereomers of the title compound as a white foam. LCMS (M+H) = 547.4. The diastereomers were separated on a Chiralpak AD column (5x50 cm) eluting with 80:20 (to elute first diastereomer) then 60:40 (to elute second diastereomer) of A:B where A = 0.1% diethylamine in hexanes and B = 2-propanol and carried on to the deprotection step below.

Step D: 4-methyl-D-leucyl-N-r5-chloro-2-dH-l,2,4-triazol-l-vPbenzyl1-5- methyl-L-prolinamide

Diastereomer A (the late eluting peak, major diastereomer) of N-(tert- butoxycarbonyl)-4-methyl-D-leucyl-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-5- methyl-L-prolinamide was dissolved in CH₂C1₂ (3 mL) and treated with TFA (1 mL). After 30 min, the solvent was removed in vacuo. The oily residue was taken up in DMF (1 mL) and purified by reverse phase HPLC (Example 37). The product fractions were concentrated and azeotroped with ether to give the TFA salt of the title compound as a white solid. HRMS (ESI, M+H) = 447.2264. IH NMR (400 MHz, CD₃OD): δ 8.83 (s, 1 H), 8.68 (m, 1 H), 8.23 (s, 1 H), 7.70 (d, 7= 2.2 Hz, 1 H), 7.60 - 7.46 (br m, 2 H), 4.44 - 4.38 (m, 1 H), 4.30 - 4.06 (br m, 4 H), 2.31 - 2.24 (m, 1 H), 2.16 - 2.10 (m, 1 H), 2.09 - 1.82 (br m, 3 H), 1.73 (dd, 7= 6.8, 14.8 Hz, 1 H), 1.37 (d, 7 = 6.4 Hz, 3 H), 1.03 (s, 9 H).

Diastereomer B (the early eluting peak, minor diastereomer) of N-(tert- butoxycarbonyl)-4-methyl-D-leucyl-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-5- methyl-L-prolinamide was deprotected and isolated in similar fashion. ΗRMS (ESI, M+Η) = 447.2266. 1Η ΝMR (400 MHz, CD₃OD): δ 8.80 (s, 1 H), 8.22 (s, 1 H), 7.68 (d, 7 = 2.2 Hz, 1 H), 7.56 - 7.45 (br m, 2 H), 4.46 - 4.33 (br m, 3 H), 4.24 - 4.16 (br m, 2 H), 2.45 - 2.38 (m, 1 H), 2.37 - 2.24 (m, 1 H), 2.07 - 1.98 (m, 1 H), 1.88 - 1.83 (m, 1 H), 1.76 - 1.61 (br m, 2 H), 1.31 (d, 7= 6.6 Hz, 3 H), 1.03 (s, 9 H).

EXAMPLE 34

Preparation of 4-methyl-D-leucyl-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-L-3,4- dehydroprolinamide

Step A: tert-butyl-(2SV2-(( r5-chloro-2-dH-tetraazol-l- yPbenzyl] amino 1 carbonvP-3 ,4-dehydro- lH-pyrrole- 1 -carboxylate The title compound was prepared from l-[5-chloro-2-(lH-tetraazol-l- yl)phenyl]methanamine (Example 1, Step D, 58 mg, 0.28 mmol) and Boc-3,4- dehydro-L-proline (59 mg, 0.28 mmol) essentially according to the EDC coupling procedure described in Example 1, Step J. The crude product was purified by silica gel chromatography (100% CΗC1₃ - 5% MeOH/CHCl₃) to afford the title compound as a pale orange oil. LCMS (M + H): 405.1. 1H NMR (CDC1₃, 400 MHz): δ 9.00 (br s, 1 H), 7.66 (s, 1 H), 7.65 (m, 1 H), 6.77 (br s, 1 H), 5.94 - 5.83 (m, 2 H), 4.99 - 4.93 (m, 1 H), 4.26 - 4.16 (m, 4 H), 1.49 and 1.33 (BOC rotamers, s, 9 H).

Step B: (2SV 2-( { [5-chloro-2-( lH-tetraazol- 1 -vPbenzyll amino ) carbonvP-3.4- dehydro-lH-pyrrolium chloride

To a stirred solution of tert-butyl-(2S)-2-({[5-chloro-2-(lH-tetraazol-l- yl)benzyl] amino }carbonyl)-3,4-dehydro-lH-pyrrole-l -carboxylate (122 mg, 0.30 mmol) in EtOAc (3.0 mL) was added excess 4.0 N ΗC1 in dioxane (1.5 mL) and the mixture was stirred overnight at room temperature. The solvent was removed in vacuo to give the title compound as a yellow solid. LCMS (M + Η): 305.0. 1H NMR (CD₃OD, 400 MHz): δ 9.55 (s, 1 H), 8.90 (br s, 1 H), 7.68 (d, J = 2.4 Hz, 1 H), 7.61 - 7.52 (m, 2 H), 6.08 - 6.05 (m, 1 H), 5.93 - 5.89 (m, 1 H), 5.01 (br s, 1 H), 4.34 - 4.33 (m, 2 H), 4.20 - 4.06 (m, 2 H).

Step C: l-(tert-butoxycarbonyP-4-methyl-D-leucyl-N-[5-chloro-2-dH- tetraazol- 1 -vDbenzyl] -L-3 ,4-dehydroprolinamide

The title compound was prepared from (2S)-2-({[5-chloro-2-(lH-tetraazol-l- yl)benzyl]amino}carbonyl)-3,4-dehydro-lH-pyrrolium chloride (54 mg, 0.16 mmol) and N-(te; -butoxycarbonyl)-4-methyl-D-leucine (39 mg, 0.16 mmol) essentially according to the EDC coupling procedure described in Example 1, Step L. The solvent was removed in vacuo and the residue was purified by silica gel chromatography (70% EtOAc/hexanes) to afford the title compound as a yellow oil. LCMS (M + Η): 532.7. Step D: 4-methyl-D-leucyl-N-[5-chloro-2-dH-tetraazol-l-vPbenzyll-L-3,4- dehvdroprolinamide

To a stirred solution of l-(tert-butoxycarbonyl)-4-methyl-D-leucyl-N-[5- chloro-2-(lH-tetraazol-l-yl)benzyl]-L-3,4-dehydroprolinamide (66 mg, 0.12 mmol) in CΗ₂C1₂ (2.0 mL) was added excess TFA (0.5 mL). The mixture was stirred at room temperature for 2 h. The solvent was removed in vacuo and the residue was purified by reverse phase HPLC (Example 37) to afford the TFA salt of the title compound as a foamy white solid. LCMS (M + H): 432.2. 1H ΝMR (CD₃OD, 400 MHz): δ 9.53 (s, 1 H), 7.75 (d, 7 = 2.4 Hz, 1 H), 7.59 (dd, 7 = 2.4 Hz, 8.4 Hz, 1 H), 7.51 (d, 7 = 8.4 Hz, 1 H), 6.12 - 6.10 (m, 1 H), 5.81 - 5.79 (m, 1 H), 5.04 - 5.02 (m, 1 H), 4.86 - 4.56 (m, 1 H), 4.47 - 4.35 (m, 2 H), 4.22 - 4.18 (m, 2 H), 2.02 - 1.94 (m, 1 H), 1.71 - 1.61 (m, 1 H), 1.02 (s, 9 H).

EXAMPLE 35

Preparation of 4-methyl-D-leucyl-(4i?)-N-(5-chloro-2-pyrazin-2-ylbenzyl)-4-fluoro-L- prolinamide

Step A: (2-Bromo-5-chlorophenyPmethanol Borane-dimethylsulfide (17.3 mL, 180 mmol, 5.8 equiv.) was added in portions to a stirred solution of 2-bromo-5-chlorobenzoic acid (7,35 g, 31.2 mmol, 1 equiv.) in anhydrous THF (204 mL) at 0 °C under nitrogen. The mixture was then allowed to warm to room temperature overnight. The mixture was then cooled to 0 °C and quenched by the slow addition of MeOH (100 mL). The mixture was stirred at room temperature for 20 min and was then concentrated in vacuo. MeOH (50 mL) was added to the residue and the mixture was again concentrated in vacuo to give the title compound as a white solid. 1H NMR (400 MHz, CDC1₃): δ 7.51 (d, 7 = 2.4 Hz, 1 H), 7.45 (d, 7 = 8.4 Hz, 1 H), 7.14 (dd, 7 = 2.6, 8.6 Hz, 1 H), 4.71 (br s, 2 H), 2.13 (br s, l H).

Step B: r(2-Bromo-5-chlorobenzvDoxy1(triisopropyPsilane

A mixture of (2-Bromo-5-chlorophenyl)methanol (2.22 g, 10 mmol), triisopropylsilyl choride (2.89 g, 15 mmol, 1.5 equiv.) and imidazole (1.02 g, 15 mmol, 1.5 equiv.) in anhydrous DMF (15 mL) was stirred at room temperature overnight. The mixture was partitioned between hexanes and water. The layers were separated and the organic layer was washed with water. The combined aqueous washes were extracted once with hexanes and the combined organic extracts were then dried (Na₂SO₄), filtered and concentrated to an oil. Silica gel chromatography (5% CH₂Cl₂-hexanes) afforded the title compound as a clear, colorless oil. 1H NMR (400 MHz, CDC1₃): δ 7.61 (app t, 7= 1.2 Hz, 1 H), 7.40 (d, 7= 8.4 Hz, 1 H), 7.11 (dd, 7= 0.8, 2.0 Hz, 1 H), 4.77 (s, 2 H), 1.26 - 1.17 (br m, 3 H), 1.11 (d, 7= 6.0 Hz, 18 H).

Step C: 4-Chloro-2-l [(triisopropylsilypoxylmethyl Iphenylboronic acid

BuLi (1.6 M, 3.8 mL, 6.08 mmol, 1.2 equiv.) was added dropwise to a stirred solution of [(2-Bromo-5-chlorobenzyl)oxy](triisopropyl)silane (1.92 g, 5.07 mmol, 1 equiv.) in ether (9.2 mL) at -78 °C under nitrogen. The pale yellow mixture was stirred at -78 °C for 30 min and was then treated with trimethylborate (1.58 g, 15.2 mmol, 3.0 equiv.). After 15 min, the bath was removed and the mixture allowed to warm to room temperature while stirring overnight. The mixture was then cooled to 0 °C and quenched by the addition of aqueous 1 M KHSO₄ solution (9 mL). The mixture was diluted with ether and water and the layers were separated. The aqueous layer was extracted with ether and the combined organic layers were washed with brine, dried (MgSO₄), filtered and concentrated to a yellow oily solid. Silica gel chromatography (20% EtOAc-hexanes) afforded the title compound as a white solid. LCMS (M+H) = 343.2 Step D: 2-(4-Chloro-2-|[(triisopropylsilvPoxylmethyllphenvPpyrazine

A solution of Pd(PPh₃) (35 mg, 0.03 mmol, 0.03 equiv.) and chloropyrazine (126 mg, 1.10 mmol, 1.1 equiv.) in anhydrous DME (5.4 mL) was stirred at room temperature under nitrogen for 20 min. A solution of Na₂CO₃ (106 mg, 1.00 mmol, 1.0 equiv.) in water (1.5 mL) was added, resulting in a red-orange mixture. 4-Chloro- 2-{[(triisopropylsilyl)oxy]methyl}phenylboronic acid (343 mg, 1.00 mmol, 1 equiv.) was added and the red solution was heated to reflux under nitrogen. After 2 h and 45 min, the mixture was cooled to room temperature and the DME was removed in vacuo. The residue was diluted with water and CH₂C1₂, the layers were separated and the aqueous layer was extracted with CH₂C1₂. The combined organic extracts were dried (Na₂SO ), filtered and concentrated to a red-orange oily solid. Silica gel chromatography (10% EtOAc-hexanes) afforded the title compound as a clear, colorless oil. 1H NMR (400 MHz, CDC1₃): δ 8.77 (m, 1 H), 8.62 (m, 1 H), 8.55 (m, 1 H), 7.78 (m, 1 H, 7.42 - 7.36 (m, 2 H), 4.93 (s, 2 H), 1.16 - 1.07 (m, 3 H), 1.04 (d, 7 = 6.4 Hz, 18 H).

Step E: (5-Chloro-2-pyrazin-2-ylphenyPmethanol

TBAF (1 M in THF, 2 mL, 2.0 mmol, 2.0 equiv.) was added to a stirred solution of 2-(4-chloro-2-{[(triisopropylsilyl)oxy]methyl}phenyl)pyrazine (375 mg, 1.0 mmol, 1 equiv.) in THF (20 mL) at room temperature. After 30 min, the solvent was removed in vacuo and the residue was partitioned between EtOAc and saturated aqueous NKUCl. The layers were separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried (MgSO ), filtered and concentrated to a light yellow solid. Silica gel chromatography (70% EtOAc-hexanes) afforded the title compound as a white solid. LCMS (M+H) = 221.0. 1H NMR (400 MHz, CDC1₃): δ 8.93 (d, 7= 1.2 Hz, 1 H), 8.66 - 8.67 (m, 1 H), 8.62 - 8.61 (m, 1 H), 7.56 - 7.54 (m, 2 H), 7.47 - 7.44 (m, 1 H), 5.09 (t, 7 = 7.2 Hz, 1 H), 4.46 (d, 7 = 7.6 Hz, 2 H),

Step F: 2-[2-(AzidomethvD-4-chlorophenyl]pyrazine DPPA (251 mg, 197 μL, 0.91 mmol, 1.2 equiv.) was added dropwise to a stirred solution of (5-chloro-2-pyrazin-2-ylphenyl)methanol (168 mg, 0.76 mmol, 1 equiv.) in anhydrous THF (2 mL) at 0 °C under nitrogen. DBU (127 mg, 0.84 mmol, 1.1 equiv.) was then added and the mixture was allowed to warm to room temperature while stirring overnight. More DPPA (100 μL) and DBU (70 μL) were added and the mixture was heated to 60 °C under nitrogen for 8 h. The mixture was then stirred at room temperature overnight and was then diluted with EtOAc and washed with water. The aqueous layer was extracted once more with EtOAc and the combined organic layers were washed successively with 10% aqueous citric acid, water, saturated NaHCO₃ and brine. The organic layer was dried (Na₂SO₄), filtered and concentrated to an orange oil. Silica gel chromatography (40% EtOAc-hexanes) afforded the title compound as an off-white solid. 1H NMR (400 MHz, CDC1₃): δ 8.79 (s, 1 H), 8.67 - 8.66 (m, 1 H), 8.60 (d, 7 = 2.4 Hz, 1 H), 7.55 (m, 1 H), 7.49 - 7.44 (m, 2 H), 4.60 (s, 2 H).

Step G: l-(5-Chloro-2-pyrazin-2-ylphenvDmethanamine

Triphenylphosphine (227 mg, 0.87 mmol, 1.5 equiv.) was added to a stirred solution of 2-[2-(Azidomethyl)-4-chlorophenyl]pyrazine (142 mg, 0.58 mmol, 1 equiv.) in THF (12 mL) at room temperature. Water (220 μL) was added after 20 min and the resulting clear, pale yellow solution was heated to 60 C for 4 h. The solvent was removed in vacuo to give an orange oil which was dissolved in EtOAc (1.5 mL) and treated with excess 1 M HCl-ether solution. The resulting light yellow precipitate was collected by filtration and washed with EtOAc. The sticky solid was dried in vacuo for three days to afford the title compound as the bis-HCl salt (powdery solid after drying). 1H NMR (400 MHz, CD₃OD): δ 9.05 (d, 7 = 1.6 Hz, 1 H), 8.76 - 8.75 (m, 1 H), 8.73 - 8.68 (m, 1 H), 7.86 - 7.84 (m, 1 H), 7.76 - 7.72 (m, 1 H), 7.69 - 7.66 (m, l H), 4.15 (s, 2 H).

Step H: (4-RVN-(5-chloro-2-pyrazin-2-ylbenzvD-4-fluoro-L-prolinamide The title compound was prepared from l-(5-Chloro-2-pyrazin-2- ylphenyl)methanamine and (4R)- 1 -(tert-butoxycarbonyl)-4-fluoro-L-proline essentially according to the procedures described in Example 34, Steps A and B and was isolated as a hydrochloride salt. LCMS (M+H) = 335.2.

Step I: N-(tert-butoxycarbonyD-4-Methyl-D-leucyl-(4J?)-N-(5-chloro-2- pyrazin-2-ylbenzyD-4-fluoro-L-prolinamide

A mixture of N-(tert-butoxycarbonyl)-4-methyl-D-leucine (47 mg, 0.19 mmol, 1.05 equiv.), (4-R)-N-(5-chloro-2-pyrazin-2-ylbenzyl)-4-fluoro-L-prolinamide hydrochloride (75 mg, 0.18 mmol, 1 equiv.), EDC (291 mg, 1.52 mmol, 8.4 equiv.), HOAt (13 mg, 0.092 mmol, 0.50 equiv.) and Hunig's base (100 μL) in DMF (4 mL) was stirred at room temperature for 1 h. The crude reaction mixture was purified by reverse phase HPLC (Example 37) and the product fractions were combined and concentrated in vacuo to give an oily solid. The solid was dissolved in saturated aqueous K₂CO₃ and the mixture was extracted with EtOAc (x3), saturating the aqueous layer with ΝaCl after each extraction. The combined organic extracts were dried (Νa₂SO₄) and concentrated to give the title compound as a clear oil which slowly formed a white foamy solid on prolonged drying under high vacuum. LCMS (M+H) = 562.5.

Step J: 4-Methyl-D-leucyl-(4-R -N-(5-chloro-2-pyrazin-2-ylbenzvD-4-fluoro-L- prolinamide

TFA (2 mL) was added to a stirred solution of N-(tert-butoxycarbonyl)-4- Methyl-D-leucyl-(4i?)-N-(5-chloro-2-pyrazin-2-ylbenzyl)-4-fluoro-L-prolinamide (69 mg, 0.12 mmol) in CH₂C1₂ (4 mL) at 0 C. After 1 h, the solvent was removed in vacuo to give a pale yellow oil, which was dissolved in DMF and purified by reverse phase HPLC (Example 37) to afford the title compound as a pale pink foam (trifluoroacetate salt). LCMS (M+H) = 462.3. 1H ΝMR (500 MHz, CD₃OD): δ 8.83 (d, 7= 1.5 Hz, 1 H), 8,78 (br t, 7= 5.5 Hz, 1 H), 8.73 (dd, 7 = 1.5, 2.5 Hz, 1 H), 8.61 (d, 7 = 2.5 Hz, 1 H), 7.64 (d, 7 = 2.0 Hz, 1 H), 7.52 (d, 7 = 8.0 Hz, 1 H), 7.47 (dd, 7 = 2.0, 8.0 Hz, 1 H), 5.38 (app d, 7 = 52 Hz, 1 H), 4.57 - 4.45 (br m, 2 H), 4.15 (app t, 7 = 6.2 Hz, 1 H), 4.00 - 3.83 (br m, 2 H), 2.57 - 2.49 (br m, 1 H), 2.11 - 1.97 (br m, 1 H), 1.94 (dd, 7 = 6.5, 14.5 Hz, 1 H), 1.66 (dd, 7 = 6.5, 15.0 Hz, 1 H), 1.00 (s, 9 H).

EXAMPLE 36

Preparation of (4R)-N-[5-chloro-2-(l,3-oxazol-5-yl)benzyl]-l-[(2R)-2-(3- chlorophenyl)-2-hydroxyethanoyl]-4-fluoroprolinamide

Step A: [(5-chloro-2-iodobenzvDoxy1(triisopropyDsilane To a stirred solution of 2-iodo-5-chlorobenzyl alcohol (2.7 g, 10.1 mmol) in

DMF (5 mL) at ambient temperature under nitrogen atmosphere was added triisopropylsilyl chloride (2.4 mL, 11.1 mmol) and imidazole (1.0 g, 15.1 mmol). The mixture was stirred at ambient temperature for 18 h and was concentrated in vacuo. The residue was purified by filtration through silica gel using dichloromethane as an eluent to afford the title compound as a clear colorless oil. (HPLC RT = 4.94 min, Method A).

Step B: 4-Chloro-2-{ [(triisopropylsilyDoxylmethyl Ibenzaldehyde

To a stirred solution at -78 °C under nitrogen atmosphere of [(5-chloro-2- iodobenzyl)oxy](triisopropyl)silane from the previous step (4.39 g, 10.3 mmol) in 10 mL of Et₂O was added 2.5 M n-BuLi in hexanes solution (4.5 mL, 11.4 mmol). The mixture was stirred at -78 °C for 15 minutes and DMF was added (1 mL, 12.4 mmol).

The mixture was allowed to equilibrate to ambient temperature and stirred for 1 h.

The reaction was quenched with saturated ΝH₄CI solution and partitioned between Et O and water. The organic layer was separated, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the title compound as a yellow oil. (HPLC RT = 4.63 min, Method A).

Step C: 5-(4-Chloro-2-([(triisopropylsilyDoxy1methyllphenvD-l,3-oxazole To a stirred solution of 4-chloro-2-

{ [(triisopropylsilyl)oxy]methylbenzaldehyde from the previous step (2.2 g, 6.7 mmol) in 60 mL of MeOH was added tosylmethyl isocyanide (1.33 g, 11.4 mmol), and K₂CO₃ (1.0 g, 7.4 mmol). The mixture was refluxed for 2 h and concentrated in vacuo. The residue was partitioned between EtOAc and water. The organic layer was separated, dried over anhydrous sodium sulfate, and filtered. The solvent was removed under reduced pressure to afford the title compound as a light brown oil. (HPLC RT = 4.75 min, Method A: LC-MS m z = 366.28).

Step D: 5-Chloro-2-d ,3-oxazol-5-yl)benzyl alcohol To a stirred solution of 5-(4-chloro-2-{ [(triisopropylsilyl)oxy]methyl}phenyl)-

1,3-oxazole from the previous step (1.8 g, 4.9 mmol) in 60 mL of MeOH was added CBr₄ (0.21 g, 0.62 mmol). The mixture was refluxed for 12 days and was then concentrated in vacuo. The residue was partitioned between EtOAc and water. The organic layer was separated, dried over anhydrous sodium sulfate, and filtered. The solvent was removed under reduced pressure. The residue was filtered through silica gel eluting with CH₂C1₂ then 5% MeOH in CH₂C1₂ to afford the title compound as an oil. (HPLC RT = 2.77 min, Method A: LC-MS m z = 210.16).

Step E: 5-F2-(AzidomethvD-4-chlorophenvH-l,3-oxazole To a stirred solution of 5-chloro-2-(l,3-oxazol-5-yl)benzyl alcohol from the previous step (0.516 g, 2.46 mmol) in 60 mL of THF at ambient temperature under nitrogen atmosphere was added DPPA (0.58 mL, 2.71 mmol), and DBU (0.41 mL, 2.71 mmol). The mixture was stirred at ambient temperature for 17 h and was then partitioned between EtOAc and water. The organic layer was separated, dried over anhydrous sodium sulfate, and filtered. The solvent was removed under reduced pressure and the residue was purified by flash silica gel chromatography using gradient elution from 5% EtOAc- hexanes to 20% EtOAc-hexanes in 5% increasing increments of EtOAc. The title compound was obtained as an oil. (HPLC RT = 3.43 min, Method A: LC-MS m/z = 235.19).

Step F: 5-Chloro-2-( 1 ,3-oxazol-5-yPbenzylamine

To a stirred solution of 5-[2-(azidomethyl)-4-chlorophenyl]-l,3-oxazole from the previous step (0.24 g, 1.03 mmol) in 60 mL of 10/1-THF/water at rt was added triphenylphosphine (0.41 g, 1.55 mmol). The mixture was heated at 50 °C for 17 h and was then concentrated in vacuo. The residue was purified by flash silica gel chromatography eluting with 5% MeOH in CH₂C1₂ to afford the title compound as an oil. (HPLC RT = 2.05 min, Method A: LC-MS m/z = 209.2).

Step G: 1 -(tert-ButoxycarbonvD-(2S ,4R)-2-( I F5-chloro-2-( 1 ,3-oxazol-5- vDbenzyl] amino lcarbonyP-4-fluoroprolinamide

To a stirred solution of (4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide (Example 6, Step C, 12 mg, 0.05 mmol) in 5 mL of DMF at ambient temperature under nitrogen atmosphere was added 5-chloro-2-(l,3-oxazol-5- yl)benzylamine from the previous step (10 mg, 0.05 mmol), 1-hydroxybenzotriazole hydrate (8 mg, 0.05 mmol), and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (14 mg, 0.07 mmol). The mixture was stirred at ambient temperature for 15 minutes and was concentrated in vacuo. The residue was partitioned between EtOAc and saturated sodium bicarbonate solution. The organic layer was separated, dried over anhydrous sodium sulfate, and filtered. The solvent was removed under reduced pressure to afford the title compound as a clear colorless oil. (HPLC RT = 3.19 min, Method A; LC-MS m/z = 424.2).

Step H: (2S, 4J? -N-[5-chloro-2-(l,3-oxazol-5-vPbenzyl1-4-fluoroprolinamide

HCI gas was bubbled through a stirred solution at 0 °C of l-(tert- butoxycarbonyl)-(2S,4R)-2-({[5-chloro-2-(l,3-oxazol-5-yl)benzyl]amino}carbonyl)- 4-fluoroprolinamide from the previous step (19 mg, 0.05 mmol) in 10 mL EtOAc for 5 minutes. The mixture was stirred at 0 °C for 15 minutes and concentrated in vacuo to afford the title compound as a white solid. (HPLC RT = 2.35 min, Method A; LCMS m/z = 324.2).

Step I: (2S, 4J?)-N-r5-chloro-2-d.3-oxazol-5-vPbenzyll-l-[(2R)-2-(3- chlorophenyP-2-hydroxyethanoyl1-4-fluoroprolinamide

To a stirred solution of (2S,4R)-Ν-[5-chloro-2-(l,3-oxazol-5-yl)benzyl]-4- fluoroprolinamide from the previous step (15 mg, 0.05 mmol) in 5 mL of DMF at ambient temperature under nitrogen atmosphere was added R-3-chloromandelic acid (9.5 mg, 0.05 mmol), 1-hydroxybenzotriazole hydrate (7.8 mg, 0.05 mmol), and l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (13.3 mg, 0.07 mmol). The mixture was stirred at ambient temperature for 72 h and was concentrated in vacuo. The residue was partitioned between EtOAc and saturated sodium bicarbonate solution. The organic layer was separated, dried over anhydrous sodium sulfate, and filtered. The solvent was removed under reduced pressure and the residue was purified by reverse phase HPLC using a C-8 stationary phase and a gradient of 95/5- H₂θ/acetonitrile to 100% acetonitrile over 30 minutes with a flow rate of 16 mlJmin. The product fractions were concentrated in vacuo to afford the title compound as a white solid. HPLC RT = 3.12 min, Method A; HRMS m= 492.09; 1H NMR (400

MHz, CDC1₃): δ 8.01 (s, 1 H), 7.58 (d, 7= 8.4 Hz, 1 H), 7.30-7.38 (m, 4 H), 7.24-7.30 (m, 2 H), 7.20 (d, 7= 8.4 Hz, 1 H), 5.28 (m, 1 H), 5.12 (br s, 1 H), 5.05 (s, 1 H), 4.72 (t, 7 = 7.7 Hz, 1 H), 4.50-4.70 (m, 2 H), 4.12 (q, 7 = 7.1 Hz, 1 H), 4.00 (m, 1 H), 3.55 (m, 1 H), 2-55-2.62 (m, 1 H), 2.3 (m, 1 H).

EXAMPLE 37

HPLC Methods

HPLC Method A: Stationary Phase: Hewlett-Packard Zorbax SB-C8 column 75 x 4.6 mm, 3.5 micron Mobile Phase: A = H₂O containing 0.1 % by volume TFA

B = CH₃CN containing 0.1% by volume TFA Gradient: 95:5 A:B to 0:100 A:B over 4.5 minutes Flow Rate: 3.0 mlJmin

UN Detection at 215 nm

Preparative Reverse Phase HPLC Method: Stationary Phase: YMC-Pack Pro C 18 Column 150 x 20 mm, 5 micron

Mobile Phase: A = H₂O containing 0.1 % by volume TFA

B = CH₃CΝ containing 0.1% by volume TFA Gradient: 95:5 to 5:95 A:B over 30 minutes Flow Rate: 15 mlJmin Dual UV Detection at 215 nm and 254 nm

LC-MS HPLC Method:

Stationary Phase: YMC Pro C18 5 micron 120 A 3.0x50 mm Mobile Phase: A = H₂O containing 0.05% by volume TFA B = CH₃CN containing 0.0425% by volume TFA

Gradient: 92:8 A:B to 100:0 A:B over 3.6 minutes Flow Rate: 1.5 to 2.0 mL/min over 3.6 min UV Detection at 215 nm Electrospray ionization for mass detector.

Typical tablet cores suitable for administration of thrombin inhibitors are comprised of, but not limited to, the following amounts of standard ingredients: Excipient General Range Preferred Range Most Preferred Range

(%) (%) (%) mannitol 10-90 25-75 30-60 microcrystalline 10-90 25-75 30-60 cellulose magnesium stearate 0.1-5.0 0.1-2.5 0.5-1.5

Mannitol, microcrystalline cellulose and magnesium stearate may be substituted with alternative pharmaceutically acceptable excipients.

In Vitro Assay For Determining Proteinase Inhibition

Assays of human α-thrombin and human trypsin were performed by the methods substantially as described in Thrombosis Research, Issue No. 70, page 173 (1993) by S.D. Lewis et al. 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 - thrombin (K_m=125 μM) and bovine trypsin (Km=125 μM). p-Nitroanilide substrate concentration was determined from measurements of absorbance at 342 nm using an extinction coefficient of 8270 cm^~ M" .

In certain studies with potent inhibitors (Ki < 10 nM) where the degree of inhibition of thrombin was high, a more sensitive activity assay was employed. In this assay the rate of thrombin catalyzed hydrolysis of the fluorogenic substrate Z- GPR-afc (K_m=27 μM) was determined from the increase in fluorescence at 500 nm

(excitation at 400 nm) associated with production of 7-amino-4-trifluoromethyl coumarin. Concentrations of stock solutions of Z-GPR-afc were determined from measurements of absorbance at 380 nm of the 7-amino-4-trifluoromethyl coumarin produced upon complete hydrolysis of an aliquot of the stock solution by thrombin. Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration < 0.1 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 (Ki) 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 = l + [I]/Ki

The activities shown by this assay indicate that the compounds of the invention are 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 38 Tablet Preparation

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 compound 4-methyl-D-leucyl-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-4 ,4- difluoroprolinamide.

Amount-(mε)

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 39 Tablet Preparation

Exemplary compositions of compound 4-methyl-D-leucyl-N-[5-chloro-2-(lH- tetraazol-l-yl)benzyl]-4,4-difluoroprolinamide (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 were film-coated with an aqueous dispersion of hydroxypropyl cellulose, hydroxypropyl methylcellulose and titanium dioxide, providing a nominal weight gain of 2.4%.

Tablet preparation via direct compression

Active I, mannitol and microcrystalline cellulose were sieved through mesh screens of specified size (generally 250 to 750 μm) and combined in a suitable blender. The mixture was subsequently blended (typically 15 to 30 min) until the drug was uniformly distributed in the resulting dry powder blend. Magnesium stearate was 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 was 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 were dedusted and film-coated with an aqueous dispersion of water-soluble polymers and pigment.

Tablet preparation via dry granulation

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 40

Intravenous Formulations Intravenous formulations of compound 4-methyl-D-leucyl-N-[5-chloro-2-(lH- tetraazol-l-yl)benzyl]-4,4-difluoroprolinamide (Active I) were prepared according to general intravenous formulation procedures.

Component Estimated range Active I 0.12 - 0.61 mg

D-glucuronic acid* 0.5 - 5 mg

Mannitol ΝF 50-53 mg

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

Water for injection q.s. 1.0 mL

Exemplary compositions A-C are as follows:

Component A B C

Active I 0.61 mg* 0.30** 0.15***

D-glucuronic acid* 1.94 mg 1.94 mg 1.94 mg

Mannitol ΝF 51.2 mg 51.2 mg 51.2 mg

1 Ν Sodium Hydroxide q.s. pH 4.0 q.s. pH 4.0 q.s. pH 4.0

Water for injection q.s. 1.0 mL q.s. 1.0 mL q.s. 1.0 mL

* 0.50 mg free base ** 0.25 mg free base *** 0.12 mg free base

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:

1. A compound of the general formula I,

or a pharmaceutically acceptable salt thereof, wherein

wherein Z 1 is hydrogen or fluorine and Z 2 is fluorine, N₃, NH₂, OH or Ci_₄ alkyl, or

R¹ is selected from the group consisting of

1)

wherein R and R are independently selected from the group consisting of hydrogen, halogen, C _t_₄ alkoxy, C ι_₄ alkyl, -OH, and cyano,

2)

wherein

R⁶ is selected from the group consisting of d) hydrogen, e) -OH, and f) -NR⁹R¹⁰, where R⁹ and R¹⁰ are independently selected from the group consisting of

1) hydrogen, and

2) C ι-₆ alkyl, unsubstituted or substituted with one or more of -OH, -COOH, C₃_₇ cycloalkyl, or COOR¹¹, where R¹¹ is C _M alkyl, 3) C₃_ cycloalkyl,

4) C(O)OR¹²,

5) C(O)R¹²,

6) C(O)NHR¹²,

7) SO₂R¹², 8) C(O)NH₂, and

9) CN, wherein R¹² is selected from the group consisting of Cι.₄alkyl, aryl, and C ₃_ cycloalkyl, and

R and R are independently selected from the group consisting of a) hydrogen, b) -CF₃, c) unsubstituted C ι_₆ alkyl, d) a ring sleeted from the group consisting of

wherein R¹³ and R¹⁴ are independently selected from the group consisting of

1) hydrogen,

2) halogen,

3) C ι_₄ alkoxy,

4) C ι.₄ alkyl,

5) hydroxy,

6) CF₃, and 7) cyano, e) C ₃_₆ cycloalkyl, f) C ι_₆ alkyl substituted with one of the group consisting of

1) C ₃_₆ cycloalkyl, unsubstituted or substituted with halogen,

2) -COOH,

3) -OH,

5)

R¹

wherein R¹⁵ and R¹⁶ are independently selected from the group consisting of aa) hydrogen, bb) halogen, cc) C ι_₄ alkoxy, dd) C alkyl, ee) hydroxy, ff) CF₃ and gg) cyano, and

wherein y is 0, 1 or 2, and

R¹⁷ and R¹⁸ are independently selected from the group consisting of hydrogen, halogen and C ι_₆ alkyl;

R² and R¹⁹ are independently selected from the group consisting of

1) hydrogen,

2) halogen, 3) C ₁.₄ alkyl,

4) C ₃_ cycloalkyl,

5) CF₃,

6) OCF₃,

7) C ι_₄ alkoxy, and 8) cyano; and

R³ is

1) a 5-membered heteroaryl ring having 2, 3, or 4 heteroatoms, provided that at least 1 heteroatom is N, and at most 1 heteroatom is S or O, said ring being unsubstituted or substituted, at any one ring atom, with Cι_₆ alkyl or halogen, or 2) a 6-membered heteroaryl ring with 1-2 nitrogen atoms, said ring being unsubstituted or substituted with with Cι_₆ alkyl or halogen.

2. The compound of Claim 1, or pharmaceutically acceptable salt thereof, wherein R2 is CI, R¹⁹ is hydrogen or F, and R is selected from the group consisting of

3. The compound of Claim 2, or pharmaceutically acceptable salt thereof, wherein Q is indepen group consisting of

4. The compound of Claim 3, or pharmaceutically acceptable salt thereof, wherein R¹ is selected from the group consisting of

5. The compound of Claim 4, or pharmaceutically acceptable salt thereof, selected from the group consisting of

4-methyl-D-leucyl-N- [5 -chloro-2-( lH-tetraazol- 1 -yl)benzyl] -4,4-difluoroprolinamide,

3-methyl-D-valyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide, 4-methyl-D-leucyl-(4S)-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-4- fluoroprolinamide,

(4R)-l-[(2^)-2-amino-2-cyclohexylethanoyl]-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide,

4-methyl-D-leucyl-(4E)-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-4- fluoroprolinamide, (4R)-l-[(2R)-2-amino-2-cyclopentylethanoyl]-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl]-4-fluoroprolinamide,

3-(l-methylcyclopropyl)-D-alanyl-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-4,4- difluoroprolinamide, 3-cyclohexyl-D-alanyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide,

3-methyl-D-valyl-(4R)-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-4-fluoroprolinamide,

3-cyclopropyl-D-alanyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide, 4-methyl-D-leucyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide,

3-(l-chlorocyclopropyl)-D-alanyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-

4-fluoroprolinamide,

3-(l-methylcyclopropyl)-D-alanyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]- 4-fluoroprolinamide,

3-cyclobutyl-D-alanyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide,

3-(l-methylcyclopropyl)-D-alanyl-(4S)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-

4-fluoroprolinamide, 3-(3,3-difluorocyclobutyl)-D-alanyl-(4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l- yl)benzyl]-4-fluoroprolinamide,

3-(l-methylcyclopropyl)-D-alanyl-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4,4- difluoroprolinamide,

3-cyclopropyl-D-valyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide,

D-prolyl-(4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoroprolinamide,

4-methyl-D-leucyl-(3R)-N-[5-chloro-2-(lH-tetraazol-l-yl)benzyl]-3- methylprolinamide,

3,3-dimethyl-D-ρrolyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide,

4-methyl-D-leucyl-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-5-methylprolinamide,

(4R)-N-[5-chloro-2-(l,3-oxazol-5-yl)benzyl]-l-[(2R)-2-(3-chlorophenyl)-2- hydroxyethanoyl]-4-fluoroprolinamide,

(2i?)-N¹-[2-({ [5-chloro-2-(lH-tetraazol-l-yl)benzyl]amino}carbonyl)-l-(4-methyl-D- leucyl)-2,5-dihydro-lH-pyrτol-2-yl]-N¹-[5-({[5-chloro-2-(lH-tetraazol-l- yl)benzyl]amino}carbonyl)-l-(4-methyl-D-leucyl)-2,5-dihydro-lH-pyrrol-2-yl]-4- methyl-D-leucinamide,

(4S)-4-fluoro-D-prolyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide, (4R)-4-fluoro-D-prolyl-(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide,

4,4-difluoro-D-prolyl-(4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4- fluoroprolinamide,

(4S)-4-azido-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-l-[(9-hydroxy-9H-fluoren- 9-yl)carbonyl]prolinamide,

(4i?)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoro-l-[(2i?)-2-hydroxy-2- phenylethanoyl]prolinamide,

(4S)-4-amino-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-l-[(9-hydroxy-9H-fluoren-

9-yl)carbonyl]prolinamide, 3-(l-methylcyclopropyl)-D-alanyl-(4R)-N-[3-chloro-2-fluoro-6-(lH-l,2,4-triazol-l- yl)benzyl] -4-fluoroprolinamide,

(4S)-4-amino-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-l-[(2R)-2-cyclohexyl-2- hydroxyethanoyl]prolinamide,

3-methyl-D-valyl-(4R)-N-[5-chloro-2-(l,2,5-thiadiazol-3-yl)benzyl]-4- fluoroprolinamide,

4-methyl-D-leucyl-(4-R)-N-(5-chloro-2-pyrazin-2-ylbenzyl)-4-fluoroprolinamide,

(4S)-4-amino-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-l-[(2i?,3R)-2-hydroxy-3- methylpentanoyl]prolinamide, and

(4R)-N-[5-chloro-2-(lH-l,2,4-triazol-l-yl)benzyl]-4-fluoro-l-[(2i?)-piperidin-2- ylcarbonyl]prolinamide.

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

7. A method for inhibiting thrombus formation in a patient comprising administering to the patient a therapeutically effective amount of the compound of Claim 5 or a pharmaceutically acceptable salt thereof.

8. A method for inhibiting thrombin in blood comprising adding to the blood an effective amount of the compound of Claim 1 or a pharmaceutically acceptable salt thereof.

9. A method for inhibiting formation of blood platelet aggregates in a pateient comprising administering to the patient a therapeutically effective amount of the compound of Claim 1 or a pharmaceutically acceptable salt thereof.

10. A method for inhibiting thrombus formation in a patient comprising administering to the patient a therapeutically effective amount of the compound of Claim 1 or a pharmaceutically acceptable salt thereof.

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 therapeutically effective amount of the compound of Claim 1 or a pharmaceutically acceptable salt thereof.

13. A method for treating or preventing deep vein thrombosis in a mammal comprising administering to the mammal a therapeutically effective amount of the compound of Claim 1 or a pharmaceutically acceptable salt thereof.

14. A method for treating or preventing thromboembolic stroke in humans and other mammals comprising administering to the mammal a therapeutically effective amount of the compound of Claim 1 or a pharmaceutically acceptable salt thereof.