WO2001038323A1 - Pyrazinone thrombin inhibitors - Google Patents

Pyrazinone thrombin inhibitors Download PDF

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Publication number
WO2001038323A1
WO2001038323A1 PCT/US2000/031787 US0031787W WO0138323A1 WO 2001038323 A1 WO2001038323 A1 WO 2001038323A1 US 0031787 W US0031787 W US 0031787W WO 0138323 A1 WO0138323 A1 WO 0138323A1
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WO
WIPO (PCT)
Prior art keywords
mmol
pyridyl
nmr
oxide
difluoro
Prior art date
Application number
PCT/US2000/031787
Other languages
French (fr)
Inventor
Christopher Burgey
Richard C. Isaacs
Bruce D. Dorsey
Kyle A. Robinson
Donnette Staas
Philip E. Sanderson
James Barrow
Original Assignee
Merck & Co., Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to JP2001540086A priority Critical patent/JP2003514905A/en
Priority to EP00983727A priority patent/EP1259506A1/en
Priority to CA002391012A priority patent/CA2391012A1/en
Priority to AU20444/01A priority patent/AU2044401A/en
Publication of WO2001038323A1 publication Critical patent/WO2001038323A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings

Definitions

  • 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.
  • 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.
  • the invention includes compounds for inhibiting loss of blood platelets, inhibiting formation of blood platelet aggregates, inhibiting formation of fibrin, inhibiting thrombus formation, and inhibiting embolus formation in a mammal, comprising a compound of the invention in a pharmaceutically acceptable carrier.
  • These compounds may optionally include anticoagulants, antiplatelet agents, and thrombolytic agents.
  • the compounds can be added to blood, blood products, or mammalian organs in order to effect the desired inhibitions.
  • the invention also includes a compound for preventing or treating unstable angina, refractory angina, myocardial infarction, transient ischemic attacks, atrial fibrillation, thrombotic stroke, embolic stroke, deep vein thrombosis, disseminated intravascular coagulation, ocular build up of fibrin, and reocclusion or restenosis of recanalized vessels, in a mammal, comprising a compound of the invention in a pharmaceutically acceptable carrier.
  • These compounds may optionally include anticoagulants, antiplatelet agents, and thrombolytic agents.
  • the invention also includes a method for reducing the thrombogenicity of a surface in a mammal by attaching to the surface, either covalently or noncovalently, a compound of the invention.
  • Compounds of the invention are useful as thrombin inhibitors and have therapeutic value in for example, preventing coronary artery disease, and have the following stmcture:
  • n 0-l
  • R is hydrogen, C M alkyl unsubstituted or substituted with halogen, OR 9 , N(R 9 ) 2 , COOR 9 ; CON(R 9 ) 2 aryl, or a heterocyclic ring, wherein R 9 is independently hydrogen or C 1 alkyl; R 2 is hydrogen,
  • R 3 , R 7 , R 8 , and R 13 are independently chosen from hydrogen, halogen, C M alkyl;
  • R 14 and R 15 are independently chosen from hydrogen, C ,_ 2 alkyl, and C ,_ 2 alkyl substituted with OR 16 or COOR 16 , wherein R 16 is hydrogen or C,_ 4 alkyl;
  • R 4 , R 5 and R 6 are independently chosen from hydrogen, halogen, hydroxy,
  • R 10 is C , .4 alkyl unsubstituted or substituted with C(CH 3 ) 2 NH 2 , C(CH 3 ) 2 OH, C(CH 3 ) 2 NHCOCF 3 , or CF 3
  • R" is phenyl unsubstituted or substituted with one or more of C M alkyl, C alkoxy, halogen, hydroxy, COOH, CONH2, CH2OH, or CO2R 12 , wherein R 12 is C ,. 4 alkyl.
  • methyl substituents are conventionally indicated as bonds attached to an atom, Me, or CH 3 .
  • A is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • R 2 is Cl, CH 3 or CN; R and R are independently selected from the group consisting of hydrogen, C alkyl, SMe,
  • R 7 is hydrogen or fluoro
  • R 13 is hydrogen or fluoro
  • R and R are independently selected from the group consisting of hydrogen, CH , Cl, F, SMe, SOMe, SO 2 Me,
  • OCH 2 CF 3 OCH 3 SCH 2 C(CH 3 ) 2 NH 2 , OCF 3 , SCH 2 CH 3 , SOCH 2 CH 3 , SO 2 CH 2 CH 3 , SCH 2 CF 3 , SOCH 2 CF 3 , and SO 2 CH 2 CF 3 .
  • A is selected from the group consisting of
  • A is selected from the group consisting of
  • the compound is selected from the group consisting of
  • the compound is selected from the group consisting of
  • 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.
  • 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.
  • cycloC3-7alkyl is intended to include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, and the like.
  • 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 Ci-4 lower alkyl; hydroxy; alkoxy; halogen; amino.
  • the pharmaceutically-acceptable salts of the compounds of Formula I 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.
  • 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, tart
  • 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.
  • the basic nitrogen-containing groups may be quatemized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides
  • dialkyl sulfates like dimethyl, diethyl, dibutyl
  • diamyl sulfates long chain halides
  • 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.
  • 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.
  • 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 extraco ⁇ oreal 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. obstmction 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 i ⁇ egularly distributed lipid deposits) in mammals, and for lowering the propensity of devices that come into contact with blood to clot blood.
  • 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
  • Examples of venous thromboembolism which may be treated or prevented with compounds of the invention include obstruction of a vein, obstruction of a lung artery (pulmonary embolism), deep vein thrombosis, thrombosis associated with cancer and cancer chemotherapy, thrombosis inherited with thrombophilic diseases such as Protein C deficiency, Protein S deficiency, antithrombin III deficiency, and Factor V Leiden, and thrombosis resulting from acquired thrombophilic disorders such as systemic lupus erythematosus (inflammatory connective tissue disease). Also with regard to venous thromboembolism, compounds of the invention are useful for maintaining patency of indwelling catheters.
  • cardiogenic thromboembolism examples include thromboembolic stroke (detached thrombus causing neurological affliction related to impaired cerebral blood supply), cardiogenic thromboembolism associated with atrial fibrillation (rapid, i ⁇ egular 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.
  • arterial thrombosis examples 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 obstmction (such as by arterial na ⁇ owing) 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.
  • atherosclerosis examples 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-Coming 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 ca ⁇ iers to which the compound molecules are coupled.
  • the thrombin inhibitors may also be coupled with soluble polymers as targetable dmg carriers.
  • Such polymers can include polyvinlypy ⁇ olidone, pyran copolymer, polyhydroxy-propyl-methacrylamide-phenol, polyhydroxyethyl- aspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues.
  • 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.
  • biodegradable polymers useful in achieving controlled release of a drug
  • 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 a ⁇ est the progress of the condition.
  • Oral dosages of the thrombin inhibitors when used for the indicated effects, will range between about 0.01 mg per kg of body weight per day (mg/kg/day) to about 30 mg/kg/day, preferably 0.025-7.5 mg/kg/day, more preferably 0.1-2.5 mg/kg/day, and most preferably 0.1-0.5 mg/kg/day (unless specificed otherwise, amounts of active ingredients are on free base basis).
  • an 80 kg patient would receive between about 0.8 mg/day and 2.4 g/day, preferably 2-600 mg/day, more preferably 8-200 mg/day, and most preferably 8-40 mg/kg/day.
  • a suitably prepared medicament for once a day administration would thus contain between 0.8 mg and 2.4 g, preferably between 2 mg and 600 mg, more preferably between 8 mg and 200 mg, and most preferably 8 mg and 40 mg, e.g., 8 mg, 10 mg, 20 mg and 40 mg.
  • the thrombin inhibitors may be administered in divided doses of two, three, or four times daily.
  • a suitably prepared medicament would contain between 0.4 mg and 4 g, preferably between 1 mg and 300 mg, more preferably between 4 mg and 100 mg, and most preferably 4 mg and 20 mg, e.g., 4 mg, 5 mg, 10 mg and 20 mg.
  • the patient would receive the active ingredient in quantities sufficient to deliver between 0.025-7.5 mg/kg/day, preferably 0.1-2.5 mg/kg/day, and more preferably 0.1-0.5 mg/kg/day.
  • Such quantities may be administered in a number of suitable ways, e.g. large volumes of low concentrations of active ingredient during one extended period of time or several times a day, low volumes of high concentrations of active ingredient during a short period of time, e.g. once a day.
  • a conventional intravenous formulation may be prepared which contains a concentration of active ingredient of between about 0.01-1.0 mg/ml, e.g.
  • 0.1 mg/ml, 0.3 mg/ml, and 0.6 mg/ml and administered in amounts per day of between 0.01 ml/kg patient weight and 10.0 ml/kg patient weight, e.g. 0.1 ml/kg, 0.2 ml/kg, 0.5 ml/kg.
  • 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 dmg in choosing an The choice of appropriate buffer and pH of a formulation, depending on solubility of the dmg 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.
  • the dosage administration will, or course, be continuous rather than intermittent throughout the dosage regime.
  • thrombin inhibitors are typically administered as active ingredients in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively refe ⁇ ed 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.
  • carrier suitable pharmaceutical diluents, excipients or carriers
  • the active dmg 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 dmg components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • 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, com-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.
  • R, R 2 , R , R 7 , R 8 , R 13 , m and n have the above-described meanings and A is fluoropyridyl, can be prepared by reacting an acid such as
  • the pyridine is oxidized, and then the azide is reduced to give amine 7.
  • the pyrazinone IA is prepared by reacting ethylchlorooxalate 8 with ethyl glycinate 9 to afford K) which is reacted with amine 11 to give compound 12.
  • Compound 2 is cyclized with acid and reacted with phosphorous oxybromide to afford 14.
  • Amine 7 is then reacted with pyrazinone 14 as shown to give the ester 1_5.
  • the ring of 1_5 is chlorinated with n-chlorosuccinimide, and the ester group is hydrolyzed to afford acid ⁇ 6 which is coupled to 3-fluoro-2-aminomethyl-pyridine to afford the final product 17
  • solution phase amide couplings may be used to form the final product, but solid-phase synthesis by classical Merrifield techniques may be employed instead.
  • the addition and removal of one or more protecting groups is also typical practice.
  • Step B Ethyl 2-pyridinoylformate (3).
  • Step F 2,2-Difluoro-2-(2-pyridyl)ethylazide (6).
  • Triphenylphosphine (7.72 g, 29.5 mmol) was added to a water bath cooled solution of 2,2-difluoro-2-(2-pyridyl-N-oxide)ethylazide (5.61 g, 28.1 mmol) in THF (90 mL). After 1 h water (10 mL) was added and the mixture was heated to 55 °C. Two hours after the addition of water, the heating bath was removed and the solution was allowed to stir overnight. The reaction was subsequently concentrated, diluted with EtOAc (250 mL), and HCI (25 mL, 2.6M in EtOAc) was added dropwise.
  • EtOAc 250 mL
  • HCI 25 mL, 2.6M in EtOAc
  • Step A Ethyl 6-formyl-3 -hydroxypyrazin( 1 H)-2-one- 1 -acetate.
  • Step B Ethyl 6-formoximyl-3-hydroxypyrazin(lH)-2-one-l -acetate.
  • Step F 3-(2,2-Difluoro-2-(2-pyridyl-N-oxide-ethylamino)-6-cyanopyrazin(lH)-2-one-l-acetic acid.
  • Step B 2 Aminomethyl-3 -(2,2,2 -trifluoroethoxy)pyridine dihy drochloride .
  • Trifluoroacetic anhydride (0.50 mL, 3.53 mmol) was added dropwise to a sti ⁇ ed mixture of 2-amino-2-methylpropanethiol hydrochloride (500 mg, 3.53 mmol) and triethylamine (0.98 mL, 7.06 mmol) in methylene chloride (25 mL) at 0 C and the resulting mixture was warmed to room temperature.
  • Step B 2-Aminomethyl-3-(2-hydroxy-2-methylpropanethio)pyridine
  • the catalyst was removed by filtration and the solvents concentrated at reduced pressure.
  • 2-methylpropanethio)pyridine dihydrochoride 1.3 g (6.0 mmol), di-tert-butyl- dicarbonate, and 1.5 g (18.0 mmol) sodium hydrogencarbonate in 150 mL of tetrahydrofuran and 50 mL water was heated to 50 °C for 2 hours. The reaction was partitioned between water and methylene chloride. The organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure.
  • Step E 2-Aminomethyl-3-(2-hydroxy-2-methylpropanesulfonyl)pyridine
  • 121 mg (0.35 mmol) 2-tert-butoxycarbonylaminomethyl- 3-(2-hydroxy-2-methylpropanesulfonyl)pyridine,10 mL 4M HCl/dioxane and 5 mL methanol was sti ⁇ ed at room temperature for 2 hours. The reaction was evaporated to dryness.
  • Step F 3-(2-hydroxy-2-methylpropanesulfonyl)-2-pyridylmethyl 3-(2,2-difluoro-2-(2- pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide
  • the title compound was prepared from 2-cyano-5-methoxy- benzylamine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin- 2-one-l -acetic acid according to the general EDC mediated coupling procedure.
  • the DMF was removed under reduced pressure.
  • the resulting residue was then sti ⁇ ed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water.
  • the product was allowed to dry and then it was further purified by preparative HPLC and characterized as the trifluoroacetate salt: l K NMR (CD3OD) ⁇ 8.86 (m, IH), 8.37 (d,
  • the title compound was prepared from 2,2-difluoro-2-(2- pyridyl)ethylamine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6- chloropyrazin-2-one-l -acetic acid according to the general EDC mediated coupling procedure.
  • the DMF was removed under reduced pressure.
  • the resulting residue was then sti ⁇ ed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water.
  • the title compound was prepared from 3-fluorophenethyl-amine and 3- [2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin-2-one-l-acetic acid according to the general EDC mediated coupling procedure.
  • the DMF was removed under reduced pressure.
  • the resulting residue was then sti ⁇ ed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added.
  • the title compound was prepared from 3-trifluoromethoxy- benzylamine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin- 2-one-l -acetic acid according to the general EDC mediated coupling procedure.
  • the DMF was removed under reduced pressure.
  • the resulting residue was then sti ⁇ ed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added.
  • the title compound was prepared from 2,2-difluorophenethyl-amine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin-2-one-l-acetic acid according to the modified general EDC mediated coupling procedure.
  • the DMF was removed under reduced pressure.
  • the resulting residue was then sti ⁇ ed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water.
  • the product was allowed to dry and then it was added to a 1 :1 mixture of EtOAc and MeCN.
  • the trifluorethoxy triflate thus prepared was added to a mixture of 5-chlorosalicylaldehyde (5 g, 32 mmol) and cesium carbonate (21 g, 64 mmol) in DMF (50 mL) at room temperature. After sti ⁇ ing at room temperature overnight the solids were filtered off and the filtrate concentrated under reduced pressure. The residue was diluted with ether and washed well with water.
  • Step B 2-(2,2,2-Trifluoroethoxy)-5-chlorobenzyl alcohol
  • Step C 2-(2,2,2-Trifluoroethoxy)-5-chlorobenzyl azide
  • 2-(2,2,2-trifluoroethoxy)-5-chlorobenzyl alcohol 1.9 g, 7.9 mmol
  • THF 20 mL
  • DBU diphenyl phosphoryl azide
  • the reaction mixture was allowed to warm to room temperature overnight following which the solvent was removed under reduced pressure.
  • the residue was partitioned between ether and water and the organic layer dried over sodium sulfate.
  • the title compound was prepared from 2-(2,2,2-trifluoroethoxy)-5- chlorobenzylamine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6- chloropyrazin-2-one-l -acetic acid according to the general EDC mediated coupling procedure.
  • the DMF was removed under reduced pressure.
  • the resulting residue was then sti ⁇ ed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water.
  • Step A Ethyl 3-[2-(2-pyridyl)ethylamino1pyrazin-2-one-l -acetate
  • Step C Ethyl 3 - [2-(2-pyridyl-N-oxide)ethylamino] -6-chloropyrazin-2-one- 1 -acetate m-Chloroperbenzoic acid (70-75%, 1.2 g, ⁇ 4.85 mmol) was added to a mixture of ethyl 3-[2-(2-pyridyl)ethylamino]-6-chloropyrazin-2-one-l-acetate (1.4 g, 4.2 mmol) in dichloroethane (30 mL) and 10% sodium carbonate (8 mL) at room temperature. After stirring for 24 h, the reaction mixture was diluted with EtOAc and washed with cold 10% sodium carbonate and then saturated sodium bicarbonate.
  • Step B 2-Methyl-5-chlorobenzyl 3-[2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin-2- one-1 -acetamide
  • the title compound was prepared from 2-methyl-5-chlorobenzylamine and 3-[2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin-2-one- 1 -acetic acid according to the general EDC mediated coupling procedure.
  • the DMF was removed under reduced pressure.
  • the resulting residue was then sti ⁇ ed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water.
  • the product was allowed to dry and then it was added to a 1 :1 mixture of EtOAc and MeCN. Addition of a 1 M solution of HCI in ether gave the hydrochloride which was then collected via filtration.:
  • the title compound was prepared from 2-methyl-5-chloro-benzylamine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin-2-one-l-acetic acid according to the general EDC mediated coupling procedure.
  • the DMF was removed under reduced pressure.
  • the resulting residue was then sti ⁇ ed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration, repeated washing with water and subsequent drying.: ⁇ H NMR (CDCI3) ⁇
  • the title compound was prepared from 2-trifluoromethoxy- benzylamine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin- 2-one-l -acetic acid according to the general EDC mediated coupling procedure.
  • the DMF was removed under reduced pressure.
  • the resulting residue was then sti ⁇ ed while water (which had been rendered mildly basic by the addition of 10%> by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water.
  • the title compound was prepared from 2,5-dichlorobenzyl-amine and 3-[2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin-2-one-l-acetic acid according to the general EDC mediated coupling procedure.
  • the DMF was removed under reduced pressure.
  • the resulting residue was then sti ⁇ ed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Sti ⁇ ing was continued until the product solidified whereupon it was collected via filtration and repeated washing with water.
  • the product was allowed to dry and then it was added to a 1 :1 mixture of EtOAc and MeCN. Addition of a 1 M solution of
  • Step A 3-[2,2-Difluoro-2-(2-pyridyl-N-oxide)ethylamino]pyrazin-2-one-l-acetic acid
  • Step B 2-(2,2,2-Trifluoroethoxy)-5-chlorobenzyl 3-[2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino]pyrazin-2-one- 1 -acetamide
  • the title compound was prepared from 2-(2,2,2-trifluoroethoxy)-5- chlorobenzylamine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]pyrazin-2- one-1 -acetic acid according to the general EDC mediated coupling procedure.
  • the DMF was removed under reduced pressure.
  • the resulting residue was then sti ⁇ ed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water.
  • the product was allowed to dry and then it was added to a 1 : 1 mixture of EtOAc and
  • 3-Fluorobenzyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2- one-1 -acetamide.
  • To a sti ⁇ ed solution of 90 mg (0.125 mmol, 50% by weight) of 3- (2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin(lH)-2-one-l -acetic acid and 22 ⁇ L (0.19 mmol) 3-fluorobenzylamine in 1 mL of DMF was added 36 mg (0.19 mmol) of EDC, 20 mg (0.15 mmol) of HOAT and 0.52 mL (0.375 mmol) triethylamine.
  • 3-Chlorobenzyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2- one-1 -acetamide.
  • To a sti ⁇ ed solution of 90 mg (0.125 mmol, 50%> by weight) of 3- (2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin(lH)-2-one-l -acetic acid and 23 ⁇ L (0.19 mmol) 3-chlorobenzylamine in 1 mL of DMF was added 36 mg (0.19 mmol) of EDC, 20 mg (0.15 mmol) of HOAT and 0.52 mL (0.375 mmol) triethylamine.
  • 3-Chlorobenzyl 3-(2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one- 1 - acetamide.
  • To a sti ⁇ ed solution of 60 mg (0.133 mmol, 72% by weight) of 3-(2-(2- pyridyl-N-oxide)ethylamino)-6-chloropyrazin(lH)-2-one-l -acetic acid and 25 ⁇ L (0.20 mmol) 3-chlorobenzylamine in 1 mL of DMF was added 38 mg (0.20 mmol) of EDC, 20 mg (0.15 mmol) of HOAT and 0.60 mL (0.532 mmol) N-methylmorpholine.
  • N-Phthalimido-3-(methylthio)pyridyl-2-methylamine was oxidized according to the procedure of Example 11.
  • the cmde product was purified by silica gel chromatography (gradient elution with 60% EtOAc-hexanes to 100% EtOAc to 5% MeOH-CHCl 3 ) to afford the title compound as a mixture of enantiomers which were inseparable by chiral HPLC:
  • Step C N-Phthalimido-3-(methylsulfonyl)pyridyl-2-methylamine
  • the title compound was prepared according to a procedure which was modified from that described in Giam, C. S.; Kikukawa, K.; Trujillo, D. A.; Org. Prep. Proced. Int. 1981, 13 (2), 137-140 as follows: A mixture of sodium tungstate dihydrate (14 mg, 0.042 mmol), water (4 mL), and acetic acid (1 drop) was heated to reflux for 10 min.
  • the cmde reaction mixture was purified directly by reverse phase HPLC (gradient elution with 95:5 water/0.1% trifluoroacetic acid:acetonitrile/0.1% trifluoroacetic acid to 5:95 water/0.1% trifluoroacetic acid:acetonitrile/0.1% trifluoroacetic acid) to afford the title compound as a white powder:
  • Enantiomer A (from the amine prepared from the faster-moving isomer of 2-
  • Step B 2-(Ethylsulfonyl)benzylamine hydrochloride To a sti ⁇ ed solution of 2-(ethylsulfonyl)benzyltrifluoroacetamide (88 mg, 0.30 mmol) in methanol (11 mL) and water (0.74 mL) was added potassium carbonate (124 mg, 0.90 mmol). The mixture was heated to 65 °C and sti ⁇ ed under Ar overnight. The mixture was then concentrated to a colorless oil which was taken up in EtOAc and washed with a small amount of brine. The aqueous layer was extracted twice with CHC1 3 and was then saturated with sodium chloride and further extracted with THF.
  • Methylthiosalicylate (5.0 mL, 36 mmol) was added dropwise to a sti ⁇ ed suspension of NaH (60%> oil dispersion, 1.43 g, 36 mmol) in dry DMF (30 mL) at room temperature. The mixture was sti ⁇ ed in the dark under Ar for 30 min and was then cooled to 0 °C and treated with a solution of 1,1,1 -trifluoro-2-iodoethane
  • Step D 2-Aminomethyl-(2,2,2-trifluoroethylthio)benzene hydrochloride
  • the title compound was prepared from 2-azidomethyl-(2,2,2- trifluoroethylthio)benzene according to the procedure of Example 34, Step D, and was isolated as a white solid:
  • the biphasic mixture was cooled to 0°C and treated with MCPBA (7.28 g, 31.6 mmol) in portions over 5 min. The mixture was sti ⁇ ed under N 2 overnight and was then transfe ⁇ ed to a separatory funnel and the aqueous layer was determined to be basic (pH 8-9). The mixture was diluted with CHC1 3 and the layers were separated. The aqueous layer was further extracted with CHC1 3 (x3). The combined organic extracts were dried over MgSO 4 and concentrated in vacuo to afford a yellow oil.
  • Step D 3 (ethylthio)pyridine-2-methylamine dihydrochloride
  • 2-cyano-3-(ethylthio)pyridine (613 mg, 3.73 mmol) and concentrated HCI (0.93 mL, 11.2 mmol) in degassed ethanol (18 mL) was hydrogenated over 10%> palladium on carbon (184 mg) at 60 psi overnight. Additional 10%> palladium on carbon (180 mg) and cone. HCI (1 mL) were added to the reaction and hydrogenation was resumed at 61 psi overnight.
  • the reaction mixture was filtered through Celite and washed with methanol. The filtrate was concentrated at reduced pressure to an orange solid which was taken up in methanol (12 mL).
  • Step E 3-(ethylthio)-2-pyridylmethyl-3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6- chloropyrazin-2-one- 1 -acetamide dihydrochloride
  • Step B ( ⁇ )-N-Phthalimido-3-(ethylsulfoxy)pyridyl-2-methylamine
  • N-Phthalimido-3-(Ethylsulfonyl)pyridyl-2-methylamine The title compound was prepared according to a procedure which was modified from that described in Giam, C. S.; Kikukawa, K.; Trujillo, D. A.; Org. Prep. Proced. Int.
  • Example 43 The title compound was prepared from 3-(2,2,2- trifluoroethylthio)pyridyl-2-methylamine dihydrochloride (Example 43, Step B) according to the procedure described in Example 39, Step A and was purified by silica gel chromatography (isocratic elution with 30%> EtOAc-hexanes) to afford a pale yellow oil:
  • Step A N-Boc-3-(2,2,2-trifluoroethylsulfonyl)pyridyl-N-oxide-2-methylamine
  • the sulfoxide enantiomers were separated on a Chiralpak AD column under the following conditions: 95:5 to 85:15 hexanes/0.1%> diethylamine:ethanol over 60 min, followed by 80: hexanes/0.1%> diethylamine: ethanol for 1 min, followed by 65:35 hexanes/0.1%) diethylamine:ethanol until the separated isomers eluted.
  • Potassium carbonate 120 mg, 0.871 mmol was added to a degassed solution of the faster moving enantiomer of 2-(methylsulfoxy) benzyltrifluoroacetamide in methanol (9.7 mL) and water (0.65 mL) and the mixture was heated at 65 °C overnight under Ar atmosphere. Additional potassium carbonate (100 mg) was added and heating was resumed at 70 °C overnight.
  • Silica gel was added to the cmde mixture and the solvent was removed at reduced pressure. The residual powder was loaded onto a pre-packed silica gel column and the product was eluted with 9:0.9:0.1 CHC13-MeOH-NH4OH.
  • (+)- and (-)-2-(Methylsulfoxy)benzyl-3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate The faster-moving enantiomer (on a Chiralpak AD column eluting with 30:70 0.1%>TFA-hexanes:ethanol) of the title compound was prepared according to the general EDC coupling procedure described in Example 33, Step E using the pure enantiomer of 2-(methylsulfoxy)benzylamine which came from the faster- moving isomer of 2-(methylsulfoxy)benzyltrifluoroacetamide.
  • Mannitol, microcrystalline cellulose and magnesium stearate may be substituted with alternative pharmaceutically acceptable excipients.
  • the thrombin inhibitors can also be co-administered with suitable antiplatelet 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, HMG CoA synthase inhibitors, etc.) to treat or prevent atherosclerosis.
  • 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
  • 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.
  • 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.
  • p-Nitroanilide substrate concentration was determined from measurements of absorbance at 342 nm using an extinction coefficient of 8270 cm ⁇ lM ⁇ 0
  • Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration ⁇ 0.1 K m into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. Initial velocities of product formation in the absence (V 0 ) or presence of inhibitor (Vi) were measured.
  • V 0 /Vi 1 + [I]/Kj
  • 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.
  • Active I is compound 3-Fluoro-2-pyridylmethyl 3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one-l -acetamide (Example 1).
  • All of the active compound, cellulose, and a portion of the com starch are mixed and granulated to 10% com starch paste.
  • the resulting granulation is sieved, dried and blended with the remainder of the com 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.
  • compositions of compound 3-Fluoro-2-pyridylmethyl 3- (2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one-l-acetamide (Active I) tablets are shown below:
  • Active I, mannitol and microcrystalline cellulose were sieved through mesh screens of specified size (generally 250 to 750 ⁇ m) and combined in a suitable blender. The mixture was subsequently blended (typically 15 to 30 min) until the dmg 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.
  • Intravenous Formulations Intravenous formulations of compound 3-Fluoro-2-pyridylmethyl 3-
  • compositions A-C are as follows:
  • 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.

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Abstract

Compounds of the invention are useful in inhibiting thrombin and associated thrombotic occlusions having the structure (I) or a pharmaceutically acceptable salt thereof, wherein A is (a), (b), (c) or (d).

Description

TITLE OF THE INVENTION PYRAZINONE THROMBIN INHIBITORS
BACKGROUND OF THE INVENTION Thrombin is a serine protease present in blood plasma in the form of a precursor, prothrombin. Thrombin plays a central role in the mechanism of blood coagulation by converting the solution plasma protein, fibrinogen, into insoluble fibrin.
Edwards et al., J, Amer. Chem. Soc, (1992) vol. 114, pp. 1854-63, describes peptidyl a-ketobenzoxazoles which are reversible inhibitors of the serine proteases human leukocyte elastase and porcine pancreatic elastase.
European Publication 363 284 describes analogs of peptidase substrates in which the nitrogen atom of the scissile amide group of the substrate peptide has been replaced by hydrogen or a substituted carbonyl moiety. Australian Publication 86245677 also describes peptidase inhibitors having an activated electrophilic ketone moiety such as fluoromethylene ketone or a- keto carboxyl derivatives.
R. J. Brown et al., J. Med. Chem., Vol. 37, pages 1259-1261 (1994) describes orally active, non-peptidic inhibitors of human leukocyte elastase which contain trifluoromethylketone and pyridinone moieties.
H. Mack et al., J. Enzyme Inhibition, Vol. 9, pages 73-86 (1995) describes rigid amidino-phenylalanine thrombin inhibitors which contain a pyridinone moiety as a central core structure.
SUMMARY OF THE INVENTION
The invention includes compounds for inhibiting loss of blood platelets, inhibiting formation of blood platelet aggregates, inhibiting formation of fibrin, inhibiting thrombus formation, and inhibiting embolus formation in a mammal, comprising a compound of the invention in a pharmaceutically acceptable carrier. These compounds may optionally include anticoagulants, antiplatelet agents, and thrombolytic agents. The compounds can be added to blood, blood products, or mammalian organs in order to effect the desired inhibitions.
The invention also includes a compound for preventing or treating unstable angina, refractory angina, myocardial infarction, transient ischemic attacks, atrial fibrillation, thrombotic stroke, embolic stroke, deep vein thrombosis, disseminated intravascular coagulation, ocular build up of fibrin, and reocclusion or restenosis of recanalized vessels, in a mammal, comprising a compound of the invention in a pharmaceutically acceptable carrier. These compounds may optionally include anticoagulants, antiplatelet agents, and thrombolytic agents.
The invention also includes a method for reducing the thrombogenicity of a surface in a mammal by attaching to the surface, either covalently or noncovalently, a compound of the invention.
DETAILED DESCRIPTION OF THE INVENTION AND
PREFERRED EMBODIMENTS
Compounds of the invention are useful as thrombin inhibitors and have therapeutic value in for example, preventing coronary artery disease, and have the following stmcture:
Figure imgf000003_0001
wherein A is
Figure imgf000003_0002
n = 0-l;
R is hydrogen, C M alkyl unsubstituted or substituted with halogen, OR9, N(R9)2, COOR9; CON(R9)2 aryl, or a heterocyclic ring, wherein R9 is independently hydrogen or C 1 alkyl; R2 is hydrogen,
C M alkyl, CF3, halogen, cyano, or cyclo C 3_7 alkyl;
R3, R7, R8, and R13 are independently chosen from hydrogen, halogen, C M alkyl;
R14 and R15 are independently chosen from hydrogen, C ,_2 alkyl, and C ,_2 alkyl substituted with OR16 or COOR16, wherein R16 is hydrogen or C,_4 alkyl; and
R4, R5 and R6 are independently chosen from hydrogen, halogen, hydroxy,
C M alkyl,
C M alkoxy, cyano, CF3O,
CHF2O,
CF3CH2O,
SR10,
SOR10, SO2R10, OR", SR", NHR11
wherein
R10 is C ,.4 alkyl unsubstituted or substituted with C(CH3)2NH2, C(CH3)2OH, C(CH3)2NHCOCF3, or CF3, and R" is phenyl unsubstituted or substituted with one or more of C M alkyl, C alkoxy, halogen, hydroxy, COOH, CONH2, CH2OH, or CO2R12, wherein R12 is C ,.4 alkyl. Note that methyl substituents are conventionally indicated as bonds attached to an atom, Me, or CH3.
In a class of compounds, A is
Figure imgf000005_0001
In a subclass of the class of compounds, R2 is Cl, CH3 or CN; R and R are independently selected from the group consisting of hydrogen, C alkyl, SMe,
SOMe, SO2Me, CN, OCH2CF3, OCH3 SCH2C(CH3)2NH2, OCF3, SCH2CH3, SOCH2CH3, SO2CH2CH3, SCH2CF3, SOCH2CF3, SO2CH2CF3, and halogen, R7 is hydrogen or fluoro; and R13 is hydrogen or fluoro.
4 5
In a family of the subclass of compounds, R and R are independently selected from the group consisting of hydrogen, CH , Cl, F, SMe, SOMe, SO2Me,
CN, OCH2CF3, OCH3 SCH2C(CH3)2NH2, OCF3, SCH2CH3, SOCH2CH3, SO2CH2CH3, SCH2CF3, SOCH2CF3, and SO2CH2CF3.
In a first subfamily of the family, A is selected from the group consisting of
Figure imgf000006_0001
Figure imgf000006_0002
Figure imgf000006_0003
Figure imgf000006_0004
Figure imgf000006_0005
In a second subfamily of the family, A is selected from the group consisting of
Figure imgf000007_0001
Figure imgf000007_0002
Figure imgf000007_0003
In a collection of the first subfamily, the compound is selected from the group consisting of
Figure imgf000008_0001
Figure imgf000008_0002
Figure imgf000008_0003
Figure imgf000008_0004
Figure imgf000008_0005
Figure imgf000009_0001
Figure imgf000009_0002
Figure imgf000009_0003
Figure imgf000009_0004
Figure imgf000009_0005
Figure imgf000010_0001
Figure imgf000010_0002
O
Figure imgf000010_0003
Figure imgf000010_0004
O
Figure imgf000011_0001
Figure imgf000011_0002
O
Figure imgf000011_0003
O
Figure imgf000011_0004
Figure imgf000011_0005
Figure imgf000012_0001
Figure imgf000012_0002
Figure imgf000012_0003
O
Figure imgf000012_0004
Figure imgf000012_0005
Figure imgf000013_0001
O
Figure imgf000013_0002
O
Figure imgf000013_0003
O
Figure imgf000013_0004
O
Figure imgf000013_0005
O
Figure imgf000014_0001
Figure imgf000014_0002
O
Examples of this collection include
Figure imgf000014_0003
and pharmaceutically acceptable salts thereof. In a collection of the second subfamily, the compound is selected from the group consisting of
Figure imgf000015_0001
Figure imgf000015_0002
Figure imgf000015_0003
Figure imgf000015_0004
Figure imgf000015_0005
Figure imgf000016_0001
Figure imgf000016_0002
O
Figure imgf000016_0003
O
Figure imgf000016_0004
O
Figure imgf000017_0001
O
Figure imgf000017_0002
O
Figure imgf000017_0003
O
Figure imgf000017_0004
O
Figure imgf000017_0005
Figure imgf000018_0001
Examples of this collection include
Figure imgf000018_0002
and pharmaceutically acceptable salts thereof.
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.
When any variable occurs more than one time in any constituent or in formula I, its definition on each occuπence is independent of its definition at every other occuπence. 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
BH3 borane
CH3CH2OTf ethyl triflate
Cs2CO3 cesium carbonate
DAST diethylaminosulfurtri fluoride
DBU l,8-diazabicyclo[5.4.0]undec-7-ene
DCE 1 ,2-dichloroethane
DMAP dimethylaminopyridine
DMF dimethylformamide
DPPA diphenylphosphoryl azide
EDC 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
EtOH ethanol
Et3N (TEA) triethylamine
HCI hydrochloric acid
HOAc acetic acid
HOAT 1 -hydroxy-7-azabenzotriazole iPrOH isopropyl alcohol
KOH potassium hydroxide
K2CO3 potassium carbonate
LAH lithium aluminum hydride
LDA lithium diisopropylamide
LiAlH4 lithium aluminum hydride
MCPBA meta-chloroperbenzoic acid
Mel iodomethane
MeOH methanol
NaBH4 sodium borohydride
NaN3 sodium azide nBuLi n-butyllithium
NH4OH ammonium hydroxide
NCS N-chlorosuccinimide Pd-C palladium on activated carbon catalyst
Pd(O) palladium oxide
PhB(OH)2 phenylboronic acid
POBr3 phosphorous oxybromide
PPh3 triphenylphosphine
TBAF tetrabutylammonium fluoride
TBSC1 tert-butyldimethylsilyl chloride
TFN trifluoroacetic acid
Tf2O trifluoromethanesulfonic anhydride
THF tetrahydrofuran
TMSCΝ trimethylsilyl cyanide
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 Ci-4 lower alkyl; hydroxy; alkoxy; halogen; amino.
The pyridyl Ν-oxide portion of the compounds of the invention are structurally depicted using conventional representations
Figure imgf000020_0001
O which 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 quatemized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others.
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 extracoφoreal 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. obstmction 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 iπegularly distributed lipid deposits) in mammals, and for lowering the propensity of devices that come into contact with blood to clot blood. Examples of venous thromboembolism which may be treated or prevented with compounds of the invention include obstruction of a vein, obstruction of a lung artery (pulmonary embolism), deep vein thrombosis, thrombosis associated with cancer and cancer chemotherapy, thrombosis inherited with thrombophilic diseases such as Protein C deficiency, Protein S deficiency, antithrombin III deficiency, and Factor V Leiden, and thrombosis resulting from acquired thrombophilic disorders such as systemic lupus erythematosus (inflammatory connective tissue disease). Also with regard to venous thromboembolism, compounds of the invention 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, iπegular 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 obstmction (such as by arterial naπowing) 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-Coming 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 caπiers to which the compound molecules are coupled. The thrombin inhibitors may also be coupled with soluble polymers as targetable dmg carriers. Such polymers can include polyvinlypyπolidone, 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 aπest the progress of the condition.
Oral dosages of the thrombin inhibitors, when used for the indicated effects, will range between about 0.01 mg per kg of body weight per day (mg/kg/day) to about 30 mg/kg/day, preferably 0.025-7.5 mg/kg/day, more preferably 0.1-2.5 mg/kg/day, and most preferably 0.1-0.5 mg/kg/day (unless specificed otherwise, amounts of active ingredients are on free base basis). For example, an 80 kg patient would receive between about 0.8 mg/day and 2.4 g/day, preferably 2-600 mg/day, more preferably 8-200 mg/day, and most preferably 8-40 mg/kg/day. A suitably prepared medicament for once a day administration would thus contain between 0.8 mg and 2.4 g, preferably between 2 mg and 600 mg, more preferably between 8 mg and 200 mg, and most preferably 8 mg and 40 mg, e.g., 8 mg, 10 mg, 20 mg and 40 mg. Advantageously, the thrombin inhibitors may be administered in divided doses of two, three, or four times daily. For administration twice a day, a suitably prepared medicament would contain between 0.4 mg and 4 g, preferably between 1 mg and 300 mg, more preferably between 4 mg and 100 mg, and most preferably 4 mg and 20 mg, e.g., 4 mg, 5 mg, 10 mg and 20 mg.
Intravenously, the patient would receive the active ingredient in quantities sufficient to deliver between 0.025-7.5 mg/kg/day, preferably 0.1-2.5 mg/kg/day, and more preferably 0.1-0.5 mg/kg/day. Such quantities may be administered in a number of suitable ways, e.g. large volumes of low concentrations of active ingredient during one extended period of time or several times a day, low volumes of high concentrations of active ingredient during a short period of time, e.g. once a day. Typically, a conventional intravenous formulation may be prepared which contains a concentration of active ingredient of between about 0.01-1.0 mg/ml, e.g. 0.1 mg/ml, 0.3 mg/ml, and 0.6 mg/ml, and administered in amounts per day of between 0.01 ml/kg patient weight and 10.0 ml/kg patient weight, e.g. 0.1 ml/kg, 0.2 ml/kg, 0.5 ml/kg. In one example, an 80 kg patient, receiving 8 ml twice a day of an intravenous formulation having a concentration of active ingredient of 0.5 mg/ml, receives 8 mg of active ingredient per day. Glucuronic acid, L-lactic acid, acetic acid, citric acid or any pharmaceutically acceptable acid/conjugate base with reasonable buffering capacity in the pH range acceptable for intravenous administration may be used as buffers. Consideration should be given to the solubility of the dmg in choosing an The choice of appropriate buffer and pH of a formulation, depending on solubility of the dmg 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 refeπed 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 dmg 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 dmg 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, com-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.
Compounds having the general stmcture
Figure imgf000026_0001
wherein, R, R2, R , R7, R8, R13, m and n have the above-described meanings and A is fluoropyridyl, can be prepared by reacting an acid such as
Figure imgf000026_0002
with an amine such as
Figure imgf000026_0003
under conditions suitable for forming amide bond between the acid and the amine wherein R4, R5 and R6 have the above described meanings. The title compounds may be prepared according to the following procedures described in Example I and illustrated in the scheme below. Bromopyridine (1) is reacted with n-BuLi to afford an anion intermediate which is reacted with diethyl oxalate (2) to afford ketoester (3). Ester 3 is reacted with diethylaminosulfurtrifluoride (DAST) to afford difluoride 4 and the ester is reduced with sodium borohydride in ethanol to give alcohol 5. The alcohol is converted to a triflate leaving group which is reacted with sodium azide to afford the azide 6. The pyridine is oxidized, and then the azide is reduced to give amine 7. The pyrazinone IA is prepared by reacting ethylchlorooxalate 8 with ethyl glycinate 9 to afford K) which is reacted with amine 11 to give compound 12. Compound 2 is cyclized with acid and reacted with phosphorous oxybromide to afford 14. Amine 7 is then reacted with pyrazinone 14 as shown to give the ester 1_5. The ring of 1_5 is chlorinated with n-chlorosuccinimide, and the ester group is hydrolyzed to afford acid ^6 which is coupled to 3-fluoro-2-aminomethyl-pyridine to afford the final product 17
Figure imgf000028_0001
Figure imgf000028_0002
Figure imgf000028_0003
Figure imgf000028_0004
O O TEA
,CI
EtO' Y- ι2ι Et
O DCE 8 9
iPrOH
Figure imgf000029_0001
Figure imgf000029_0002
12
Figure imgf000029_0003
Figure imgf000030_0001
Figure imgf000030_0002
Figure imgf000030_0003
Synthesis of 2-aminomethyl-3-fluoropyridine begins with catalytic reduction of 2- cyano-3-fluoropyridine (Sakamoto et al., Chem. Pharm. Bull. 33(2) 565-571 (1985)) using palladium on carbon which provides 2-aminomethyl-3-fluoropyridine B_as the dihydrochloride salt.
Typically, solution phase amide couplings may be used to form the final product, but solid-phase synthesis by classical Merrifield techniques may be employed instead. The addition and removal of one or more protecting groups is also typical practice.
Compounds having different groups at variable A can be prepared by coupling alternative commercially available amino derivatives
Figure imgf000031_0001
where Yl and γ2 are defined above, using the coupling procedure described for coupling
Figure imgf000031_0002
to the carboxylic acid. Alternative amino derivatives and methods for preparing amino derivatives are known to those skilled in the art and described below.
1)
Figure imgf000031_0003
is commercially available.
2)
reduction
Figure imgf000032_0001
Figure imgf000032_0002
for example
M 1)LDA, -78°
2) Mel, -78°C
Figure imgf000032_0003
Figure imgf000032_0004
and also for example
LiAIH4
Figure imgf000032_0006
Figure imgf000032_0005
Unless otherwise stated, all NMR determinations were made using 400 MHz field strength.
EXAMPLE 1 Preparation of 3 -Fluoro-2-pyridy Imethy 1 3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide (17)
Figure imgf000033_0001
Step A
2-Aminomethyl-3-fluoropyridine (B) as a dihydrochloride salt
A stiπed solution of 6.11 g (50.1 mmol) of 2-cyano-3-fluoropyridine in 250 mL of ethanol and 12.5 mL (150 mmol) of cone. HCI was hydrogenated over 1.90 g of 10% palladium on carbon at 40 psi for 16 h. The catalyst was removed by filtration and the solvents removed at reduced pressure. The resulting solid was diluted with acetonitrile and filtered to give the title compound as an off-white solid: Η NMR (CD3OD) δ 8.48 (d, 1H, 4.8 Hz), 7.69 (td,lH, 9.2, 1.1 Hz), 7.68 (ddd, 1H, 8.8, 4.4, 4.4 Hz), 4.34 (s, 2 H).
Step B Ethyl 2-pyridinoylformate (3).
To a stiπed solution of 20 mL (210 mmol) of 2-bromopyridine in 500 mL of dry ether at -78°C under Ar was added 85 mL of a 2.5 M solution of n- butyllithium in hexane in a slow stream. After stiπing in the cold for 30 min, the solution was transfeπed over a 5 min period via two cannula into a 0°C stiπed solution of 100 mL (736 mmol) of diethyl oxalate in 1.0 L of dry ether under Ar.
After stirring for 2h in the cold, the reaction mixture was washed with 600 mL of sat. NaHCO3, water, and brine. The solution was dried over MgSO4 and the solvents concentrated at reduced pressure to give a red oil that was purified by SiO2 chromatography (10 x 15 cm) using 1 :4 to 35:65 EtOAc-hexanes. The product- containing fractions were concentrated at reduced pressure to afford 3 as a reddish oil: 'H NMR (CDC13) δ 1.42 (t, 3H), 4.45-4.55 (m, 2H), 7.55-7.6 (m, IH), 7.9-7.95 (m, IH), 8.11 (d, IH), 8.78 (d, IH).
Step C
Ethyl difluoro-2-pyridylacetate (4).
A stiπed solution of 22 g (123 mmol) of ethyl 2-pyridinoylformate 3 and 75 g (465 mmol) of diethylaminosulfurtrifluoride (DAST) were heated to 55°C under Ar overnight. Because the reaction was not complete, 5 g additional DAST was added, and the reaction heated for an additional 24 h. The reaction mixture was cooled to rt, and poured very slowly into a stiπed mixture of 1 kg of ice, 400 mL of ethyl acetate and 500 mL of sat. NaHCO3. After the addition, the mixture was basifϊed by the addition of solid NaHCO3. The aqueous layer was extracted with EtOAc, and the combined organic layers washed with sat. NaHCO3, brine, dried over Na2SO4 and the solvents concentrated at reduced pressure to give 4 as a brown oil: 'H NMR (CDC13) δ 1.35 (t, 3H), 4.35-4.4 (m, 2H), 7.4-7.45 (m, IH), 7.75 (d, IH), 7.95 (d, IH), 8.45 (d, IH).
Step D 2,2-Difluoro-2-(2-pyridyl)ethanol (5).
To a stiπed solution of 19.5 g (97 mmol) of ethyl difluoro-2- pyridylacetate 4 in 200 mL of absolute ethanol at 0°C was added 4.42 g (116 mmol) of sodium borohydride in small portions. After 30 min, the reaction was quenched by the addition of 50 mL of sat. NH4C1. The reaction mixture was concentrated at reduced pressure and the residue partitioned between 500 mL of ethyl acetate and sat. NaHCO3. The organic layer was washed with water, brine, and dried over Na^O,, and concentrated at reduced pressure to give a brown oil that was purified on SiO2 (10 x 17 cm) using 1 :1 EtOAc-hexane. After re-chromatographing the mixed fractions, all clean fractions were combined and concentrated at reduced pressure, giving 5 as a beige crystalline solid: 'H NMR (CDC13) δ 3.6 (t, IH), 4.17-4.3 (m, 2H), 7.4-7.45 (m, IH), 7.73 (d, IH), 7.84-7.91 (m, IH), 8.61 (d, IH).
Step E
2,2-Difluoro-2-(2-pyridyl)ethyl trifluoromethanesulfonate (5a). To a stiπed solution of 5 g (31.4 mmol) of 2,2-difluoro-2-(2- pyridyl)ethanol 5 and 9.69 g (47.2 mmol) of 2,6-di-t-butyl-4-methylpyridine in 110 mL of methylene chloride at -78°C under Ar was added 7.93 mL (47.2 mmol) of triflic anhydride dropwise. After lh, the reaction was diluted with 100 mL of pentane and filtered. The filtrate was concentrated and treated again with pentane and filtered. Concentration of the filtrate gave 5a as a brown oil, contaminated with 2,6-di-t-butyl- 4-methylpyridine: 'H NMR (CDC13) δ 5.12 (t, 2H), 7.45-7.5 (m, IH), 7.75 (d, IH), 7.86-7.94 (m, IH), 8.65 (d, IH).
Step F 2,2-Difluoro-2-(2-pyridyl)ethylazide (6).
To a stiπed solution of 5.5 g of 2,2-difluoro-2-(2-pyridyl)ethyl trifluoromethanesulfonate 5a in 70 mL of DMF was added 6.74 g (104 mmol) of sodium azide under Ar. The mixture was heated to 60°C overnight. A second batch was mn in the same manner, and after cooling to rt, both reactions were poured into 600 mL of water, and extracted with 3 x 500 mL of ether. The combined extracts were washed with brine, dried over Na,SO4 and concentrated at reduced pressure to give an oil that was purified by SiO2 (10 x 6 cm) using hexane 1 :3 EtOAc-hexane and 1 : 1 EtO Ac -hexane. The product-containing fractions were concentrated at reduced pressure to give 6 as a yellow oil: Η NMR (CDC13) δ 4.05 (t, 2H), 7.4-7.45 (m, IH), 7.73 (d, IH), 7.83-7.89 (m, IH), 8.67 (d, IH).
Figure imgf000035_0001
6a
Step G 2,2-Difluoro-2-(2-pyridyl-N-oxide)ethylazide (6a).
To a stiπed solution of 2,2-difluoro-2-(2-pyridyl-N-oxide)ethylazide (5.75 g, 31.3 mmol ) in 1 ,2-dichloroethane (100 mL) was added 3- chloroperoxybenzoic acid (10.26 g, 41.6 mmol) and 3-tert-butyl-4-hydroxy-5- methylphenyl sulfide (1.12 g, 3.13 mmol) under Ar. The mixture was heated at 55 °C overnight. In the morning, the solution was poured into a sat. aq. NaHCO3/Na2S2O3 solution (200 mL). The layers were separated and the aqueous phase was backwashed with dichloromethane (3 x 150 mL). The combined organic layers were dried over MgSO4, concentrated and chromatographed on a short SiO2 column using 100% EtOAc to give the title compound as a white solid: Η NMR (CDC13) δ 4.38 (t, 2H, 13.5 Hz), 7.36-7.44 (m, 2H), 7.72 (dd, IH, 2.3 Hz, 7.6 Hz), 8.26 (d, IH, 6.1 Hz).
Figure imgf000036_0001
Step H
2,2-Difluoro-2-(2-pyridyl-N-oxide)ethylamine (7).
Triphenylphosphine (7.72 g, 29.5 mmol) was added to a water bath cooled solution of 2,2-difluoro-2-(2-pyridyl-N-oxide)ethylazide (5.61 g, 28.1 mmol) in THF (90 mL). After 1 h water (10 mL) was added and the mixture was heated to 55 °C. Two hours after the addition of water, the heating bath was removed and the solution was allowed to stir overnight. The reaction was subsequently concentrated, diluted with EtOAc (250 mL), and HCI (25 mL, 2.6M in EtOAc) was added dropwise. Stirring was continued for 20 min, after which time the mixture was filtered and rinsed with EtOAc (150 mL). To a stiπed suspension of this solid in dichloromethane (300 mL) was added NaOH (3.33 g in 15 mL H2O) dropwise. After 15 min the mixture was poured into a separatory funnel and the organic phase was separated. The aqueous phase was saturated with solid NaCI and extracted with CH2C12 (3 x 100 mL). The combined organic extracts were dried over Na2SO4 and concentrated to an oil which solidified upon storage in the freezer (white solid): 'H NMR (CDC13) δ 8.25 (br d, IH, 6.2 Hz), 7.69 (dd, IH, 2.8, 7.3 Hz), 7.32-7.39 (m, 2H), 3.76 (t, 2 H, 15.2 Hz), 1.29 (br s, 2H).
Step I
Ethyl N-(ethyl carboxymethyl)oxamate (10)
To a suspension of ethyl glycine»HCl (38.4 g, 275 mmol ) in 1,2- dichloroethane (360 mL) was added triethylamine (77.0 mL, 550 mmol) at room temperature. After stirring for 30 minutes the heterogenous mixture was cooled to 0 °C and ethyl oxalyl chloride (30.3 mL, 275 mol) was added dropwise over the course of 1 h. Upon completion of the addition, the cooling bath was removed and the reaction was stiπed at room temperature overnight. The reaction was diluted with water (250 mL) and the layers separated. The aqueous layer was backwashed with 2 portions of dichloromethane (250 mL). The combined organic layers were washed with water (250 mL), followed by brine (250 mL), dried over MgSO4 and concentrated to give an oil K) that was taken directly onto the next step.
Step J
N-(Ethyl carboxymethyl)-N'-(2,2-dimethoxyethyl)oxamide (12)
To a solution of the oxamate (84.0 g, 414 mmol) K) in 2-propanol (500 mL) was added aminoacetaldehyde dimethyl acetal (45.7 g, 435 mmol) in one portion. After stirring overnight at room temperature, the reaction mixture was concentrated to a thick orange oil. This thick slurry was diluted with 2-propanol (300 mL) and the solid was broken up with a spatula. Filtration afforded a solid which was further rinsed with an additional portion of 2-propanol. Removal of residual 2- propanol was accomplished via high vacuum to afford a light orange solid 12: 'H NMR (CDClj) δ7.82 (br s, IH), 7.50 (br s, IH), 4.41 (t, IH, 5.3 Hz), 4.24 (q, 2H, 7.1 Hz), 4.09 (d, 2H, 5.9 Hz), 3.47 (dd, 2H, 5.3, 6.2 Hz), 3.40 (s, 6H), 1.30 (t, 3 H, 7.1 Hz).
Step K
Ethyl 3 -hydroxypyrazin(lH)-2-one-l -acetate (13) A solution of the oxamide (89.8 g, 343 mmol) 12, acetic acid (400 mL), and cone. HCI (2 mL) was heated to reflux. After 1 h the black reaction was concentrated to a thick oil (high vacuum employed to ensure complete removal of AcOH) which was diluted with EtOH (150 mL) and MeOH (150 mL). Scraping the thick black oil with a spatula induced precipitation of the product. The MeOH was removed via rotary evaporation and the remaining slurry was filtered and rinsed with EtOH (200 mL) to deliver a tan solid. Recrystallization from refluxing EtOH (300 mL) afforded an off-white powder 03: Η NMR (CD3OD) δ 6.50 (d, IH, 5.9 Hz), 6.36 (d, IH, 5.9 Hz), 4.58 (s, 2H), 4.23 (q, 2H, 7.1 Hz), 1.28 (t, 3 H, 7.1 Hz). Further cmde dione could be obtained upon concentration of the mother liquor. Step L
Ethyl 3-bromopyrazin(lH)-2-one-l -acetate (14)
A solution of the hydroxypyrazinone (25.0 g, 126 mmol) 2C3_ and phosphorous oxybromide (37.9 g, 132 mmol) in 1 ,2-dichloroethane (250 mL) was heated to reflux. After 8 h the reaction mixture was treated with sat. aq. NajCO-, (250 mL) and stiπed for lh. The mixture was diluted with water (100 mL) and dichloromethane (100 mL), the layers were separated and the aqueous layer was backwashed with EtOAc (3 x 200mL). The combined organics were dried (MgSO4), and concentrated to give an oil which was stored on a high vacuum line ovemite to afford brown solid 2^4: Η NMR (CDC13) δ 7.17 (d, IH, 4.2 Hz), 7.07 (d, IH, 4.2 Hz), 4.65 (s, 2H), 4.27 (q, 2H, 7.2 Hz), 1.31 (t, 3 H, 7.2 Hz).
Figure imgf000038_0001
Step M
Ethyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide-ethylamino)pyrazin(lH)-2-one-l -acetate
(151
A mixture of 3.0 g (17.2 mmol) of 2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamine, 2.72 mL (19.5 mmol) of triethylamine and 4.5 g (17.2 mmol) of ethyl 3-bromopyrazin(lH)-2-one-l-acetate in 9 mL of toluene and 3 mL of ethanol was heated to 120 °C in a sealed tube for 24 h. The reaction was concentrated and the residue was partitioned between EtOAc (200 mL) and sat. aq. NaHCO3 (200 mL). The aqueous layer was backwashed with EtOAc (5 x 150 mL). The combined organic layers were dried over MgSO4 and the solvents removed at reduced pressure to give a brown solid. This cmde material was diluted with EtOAc (50 mL), filtered, and rinsed with EtOAc (2 x 50 mL) to afford the title compound as a tan powder: 'H NMR (CDC13) δ 8.26 (d, IH, 6.4 Hz), 7.61 (br d, IH, 7.9 Hz), 7.34 (dd, IH, 6.6, 6.6 Hz), 7.26 (dd, IH), 6.78 (d, IH, 4.6 Hz), 6.39 (br t, IH, 6.6 Hz), 6.37 (d, IH, 4.6 Hz), 4.66 (td, 2H, 13.8, 7.0 Hz), 4.52 (s, 2H), 4.23 (q, 2H, 7.1 Hz), 1.28 (t, 3 H, 7.1 Hz).
Figure imgf000039_0001
Figure imgf000039_0002
Step N
Ethyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide-ethylamino)-6-chloropyrazin(lH)-2-one-l- acetate (15a).
A stiπed solution of 4.96 g (14.0 mmol) of ethyl 3-(2,2-difluoro-2-(2- pyridyl-N-oxide-ethylamino)pyrazin(lH)-2-one-l -acetate and 1.86 g (14.0 mmol) of N- chlorosuccinimide in 200 mL of 1 ,2-dichloroethane was heated to 70 °C. After 3 h the solution was cooled to room temperature and partitioned between dichloromethane (150 mL) and sat. aq. NaHCO3 (200 mL). The layers were separated and the aqueous phase was backwashed with dichloromethane (4 x 200 mL) and EtOAc (2 x 200 mL). The combined organic layers were dried over NaSO4 and the solution concentrated. This cmde solid was purified on a SiO2 column with 100 % EtOAc to 10:90 MeOH:EtOAc to give the title compound as a white solid: 'H NMR (CDC13) δ 8.26 (d, IH, 6.4 Hz), 7.62 (dd, IH, 2.2, 7.9 Hz), 7.35 (ddd, IH, 2.1, 7.7, 7.7 Hz), 7.2 (dd, IH, 7.7 Hz), 6.86 (s, IH), 6.35 (br t, IH, 6.7 Hz), 4.85 (s, 2H), 4.64 (td, 2H, 13.8, 6.9 Hz), 4.24 (q, 2H, 7.1 Hz), 1.29 (t, 3 H, 7.1 Hz).
Figure imgf000040_0001
Step O
3 -(2,2-Difluoro-2-(2-pyridyl-N-oxide-ethylamino)-6-chloropyrazin( 1 H)-2-one- 1 -acetic acid (16).
To a stiπed solution of 4.88 g (12.6 mmol) of ethyl 3-(2,2-difluoro-2-(2- pyridyl-N-oxide-ethylamino)-6-chloropyrazin(lH)-2-one-l -acetate in methanol (100 mL) was added 5.0 g potassium hydroxide (89.1 mmol dissolved in 20 mL water). After 1 h the solution was concentrated, diluted with 50 mL of water and acidified to pH = 7 using cone. HCI. Concentration at reduced pressure (azeotrope with PhCH3) afforded an off-white solid containing potassium chloride and the title compound: Η NMR (CD3OD) δ 8.36 (d, IH, 6.2 Hz), 7.69 (dd, IH, 7.7, 2.2 Hz), 7.51- 7.59 (m, 2H), 6.67 (s, IH), 4.62 (s, 2H), 4.55 (t, 2H, 13.1 Hz).
Figure imgf000041_0001
Step R
3-Fluoro-2-pyridylmethyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6- chloropyrazin-2-one-l-acetamide (17).
A stiπed solution of 522 mg (1.45 mmol) of 3-(2,2-difluoro-2-(2- pyridyl-N-oxide)ethylamino)-6-chloropyrazin(lH)-2 -one- 1 -acetic acid and 430 mg (2.18 mmol) 2-aminomethyl-3-fluoropyridine dihydrochloride in 10 mL of DMF was added 415 mg (2.17 mmol) of EDC, 295 mg (2.17 mmol) of HOAT and 1.60 mL (14.5 mmol) N-methylmorpholine. After stirring overnight, the volatiles were removed en vacuo. The residue was diluted with water, filtered, and rinsed with water to afford a solid. This material was suspended in MeOH and treated with cone. HCI until the solution became homogeneous. Concentration afforded a gummy solid, which was diluted with EtOH and filtered to give 500 mg of the product as a white solid: Η NMR (CD3OD) δ 8.63 (d, IH, 5.5 Hz), 8.50 (br d, IH), 8.33 (dd, IH, 8.8, 8.8 Hz), 7.96-7.93 (m, IH), 7.84 (m, IH), 7.71 (m, 2H), 6.90 (s, IH), 4.93 (s, 2H), 4.75 (s, 2H), 4.61 (t, 2H, 13.8 Hz).
EXAMPLE 2
Figure imgf000042_0001
Preparation of 3-Fluoro-4-methyl-2-pyridylmethyl 3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide.
A stiπed solution of 90 mg (0.125 mmol) of 3-(2,2-difluoro-2-(2- pyridyl-N-oxide)ethylamino)-6-chloropyrazin(lH)-2-one-l -acetic acid and 40 mg (0.188 mmol) 2-aminomethyl-3-fluoro-4-methylpyridine dihydrochloride in 2 mL of DMF was added 36 mg (0.19 mmol) of EDC, 25 mg (0.19 mmol) of HOAT and 0.20 mL (1.25 mmol) N-methylmorpholine. After stirring overnight, the volatiles were removed en vacuo. The residue was diluted with water, filtered, and rinsed with water to afford a solid. This material was suspended in MeOH and treated with cone. HCI until the solution became homogeneous. Concentration afforded the title compound as a solid: 'H NMR (CD3OD) δ 8.53 (m, 2H), 7.96 (dd, IH, 6.2, 6.2 Hz), 7.86 (m, IH), 7.73 (m, 2H), 6.93 (s, IH), 4.94 (s, 2H), 4.76 (br s, 2H), 4.62 (t, 2H, 13.8 Hz), 2.61 (d, 3H, 1.7 Hz)
EXAMPLE 3
Figure imgf000043_0001
Preparation of 2-Fluorobenzyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6- chloropyrazin-2-one- 1 -acetamide.
A stiπed solution of 1.23 g (2.28 mmol, 67% by weight) of 3-(2,2- difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin(lH)-2-one-l-acetic acid and 0.391 mL (3.42 mmol) 2-fluorobenzylamine in 7 mL of DMF was added 657 mg (3.42 mmol) of EDC, 313 mg (2.30 mmol) of HOAT and 1.25 mL (11.4 mmol) N- methylmorpholine. After stirring overnight, the volatiles were removed en vacuo. The residue was diluted with 25 mL sat. aq. NaHCO3, filtered and rinsed with 25 mL water. This solid was further rinsed with dichloromethane (2 x 15 mL) to afford the title compound as a white powder (0.98 g): 'H NMR (CDC13) D 8.24 (d, IH, 6.3 Hz), 7.62 (dd, IH, 2.1, 7.8 Hz), 7.52-7.26 (m, 4H), 7.11 (dd, IH, 7.5, 7.5), 7.03 (m, IH), 6.88 (s, IH), 6.43 (br t, IH, 6.4 Hz), 6.22 (br t, IH), 4.80 (s, 2H), 4.63 (td, 2H, 13.9, 6.9 Hz), 4.51 (d, 2H, 5.6 Hz). This material was suspended in 50 mL of MeOH and treated with 2.0 mL of 4.0M solution of HCI in dioxane. Concentration afforded the product as a white solid: Η NMR (CD3OD) δ 8.52 (dd, IH, 2.7, 4.7), 7.88 (m, IH), 7.76-7.70 (m, 2H), 7.36 (ddd, IH, 1.5, 7.6, 7.6), 7.33-7.27 (m, IH), 7.13 (ddd, IH, 0.9, 7.6, 7.6), 7.08 (dd, IH, 9.3, 9.3), 6.95 (s, IH), 4.89 (s, 2H), 4.64 (t, 2H, 13.8 Hz). 4.46 (s, 2H). EXAMPLE 4 Preparation of 3-Fluoro-2-pyridylmethyl 3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-cyanopyrazin-2-one- 1 -acetamide hydrochloride
Figure imgf000044_0001
Figure imgf000044_0002
Step A Ethyl 6-formyl-3 -hydroxypyrazin( 1 H)-2-one- 1 -acetate.
A suspension of the hydroxypyrazinone (5.0 g, 23.6 mmol) and selenium(IV) oxide (2.62g, 23.6 mmol), in 1,4-dioxane (100 mL) was heated to reflux for 24 h. The dark reaction mixture was cooled and filtered through a pad of Celite with MeOH. Concentration and purification of the residue on a SiO2 column with 3:97 to 10:90 MeOH:CH2Cl2 afforded the title compound as an orange solid: Η NMR (CD3OD) δ 9.11 (s, IH), 7.39 (s, IH), 5.12 (s, 2H), 4.22 (q, 2H, 7.1 Hz), 1.29 (t, 3 H, 7.1 Hz).
Figure imgf000044_0003
Step B Ethyl 6-formoximyl-3-hydroxypyrazin(lH)-2-one-l -acetate.
To a suspension of the formylpyrazinone (5.43 g, 24.0 mmol) and hydroxylamine hydrochloride (1.67 g, 24.0 mmol) in ethanol (100 mL) was added pyridine (1.90 mL, 24.0 mmol). After 2 h at reflux, the reaction mixture was cooled and concentrated. This cmde solid was recrystallized from ethanol (100 mL) to deliver 2.80 g of the title compound as a solid. An additional 1.90 g batch of product was obtained by concentration of the filtrate and trituration with water (50 mL): Η NMR (DMSO) δ 11.85 (d, IH), 11.19 (s, IH), 7.82 (s, IH), 6.79 (d, IH, 5.9 Hz), 5.05 (s, 2H), 4.12 (q, 2H, 7.1 Hz), 1.20 (t, 3 H, 7.1 Hz).
Figure imgf000045_0001
Step C
Ethyl 6-cyano-3 -hydroxypyrazin( 1 H)-2-one- 1 -acetate. A slurry of the hydroxypyrazinone (2.70 g, 11.2 mmol) and polymer- bound triphenylphosphine (1.55 mmol g resin: 15.1 g, 23.5 mmol) in 1,2-dichloroethane (90 mL) and carbon tetrachloride (9 mL) was heated to reflux for 1.5 h. The reaction mixture was cooled, filtered, and the resin rinsed with of 1 :1 MeOH:CH2Cl2(200 mL). Concentration of the filtrate yielded the product as a tan solid: Η NMR (CDC13) δ 7.06 (s, IH), 4.73 (s, 2H), 4.29 (q, 2H, 7.1 Hz), 1.33 (t, 3 H, 7.1 Hz).
Figure imgf000045_0002
Step D
Ethyl 3 -chloro-6-cyanopyrazin( 1 H)-2-one- 1 -acetate.
A suspension of the hydroxypyrazinone (450 mg, 2.02 mmol) and ammonium chloride (237 mg, 4.44 mmol) in phosphorous oxychloride (2 mL) was heated at reflux for 1.5 h. The reaction mixture was cooled, and the volatiles were removed via rotary evaporation. The residue was quenched with water and solid Na^O-j was added until the mixture was basic. This aqueous mixture was extracted with dichloromethane (3x), and the combined organics were dried (NajSO , and concentrated to give the product as an amber oil: 'H NMR (CDC13) δ 7.60 (s, IH), 4.87 (s, 2H), 4.32 (q, 2H, 7.1 Hz), 1.31 (t, 3 H, 7.1 Hz).
Figure imgf000046_0001
Figure imgf000046_0002
Step E
Ethyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide-ethylamino)-6-cyanopyrazin( 1 H)-2-one- 1 - acetate.
A mixture of 0.415 g (1.72 mmol) of 2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamine, 0.24 mL (1.72 mmol) of triethylamine and 0.33 g (1.89 mmol) of ethyl 3-chloro-6-cyanopyrazin(lH)-2-one-l-acetate in 6 mL of toluene and 2 mL of ethanol was heated to 45°C in a sealed tube for 0.5 h. The reaction was concentrated and chromatographed on silica gel, using 3-5% methanol/chloroform saturated with ammonia to afford the title compound as a tan powder: ]H NMR (CD3OD) δ 8.48 (d, IH, 7 Hz), 7.91 (s, IH), 7.76-7.65 (m, IH), 7.65-7.52 (m, 2H), 4.78 (s, 2H), 4.62 (t, 2H, 16 Hz), 4.23 (q, 2H, 7.1 Hz), 1.28 (t, 3 H, 7.1 Hz).
Step F 3-(2,2-Difluoro-2-(2-pyridyl-N-oxide-ethylamino)-6-cyanopyrazin(lH)-2-one-l-acetic acid.
To a stiπed solution of 0.36 g (0.95 mmol) of ethyl 3-(2,2-difluoro-2-(2- pyridyl-N-oxide-ethylamino)-6-cyanopyrazin( 1 H)-2-one- 1 -acetate in dimethoxyethane (5 mL) at 0 °C was added 0.95 mL lithium hydroxide solution (2.0M in water). After 0.5 h, warm to room temperature and stir 20 min. Add 0.95 mL water and stir for 15 min. The solution was neutralized using 1.9 mL IM HCI. Concentration at reduced pressure (azeotrope with PhCH3) afforded an off-white solid containing lithium chloride and the title compound: Η NMR (CD3OD) δ 8.36 (d, IH, 6.2 Hz), 7.69 (dd, IH, 7.7, 2.2 Hz), 7.51- 7.62 (m, 2H), 7.22 (s, IH), 4.74 (s, 2H), 4.62 (t, 2H, 16 Hz).
Step G
3-Fluoro-2-pyridylmethyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6- cyanopyrazin-2-one- 1 -acetamide hydrochloride.
Prepared in the same manner as above for 3-Fluoro-2-pyridylmethyl 3- (2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one-l-acetamide. 'H NMR (CD3OD) δ 8.67 (d, IH, 5.6 Hz), 8.50-8.42 (m, 2H), 8.07-8.01 (m, IH), 7.79-7.76 (m, IH), 7.70-7.64 (m, 2H), 7.23 (s, IH), 4.79 (s, 2H), 4.58 (s, 2H), 4.62 (t, 2H, 13.1 Hz).
EXAMPLE 5 Preparation of 2-Fluorobenzyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6- methylpyrazin-2-one- 1 -acetamide.
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000048_0002
Step A
Ethyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide-ethylamino)-6-methylpyrazin( 1 H)-2-one- 1 - acetate.
A mixture of 500 mg (2.87 mmol) of 2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamine, 0.52 mL (3.00 mmol) of N,N-diisopropylethylamine and 608 mg (2.39 mmol) of ethyl 3-bromo-6-methylpyrazin(lH)-2-one-l -acetate in 10 mL of toluene and 5 mL of ethanol was heated at 110 °C for 3 d. The reaction was cooled, concentrated and the cmde residue was chromatographed on SiO2 using 5:95 MeOH- CH2C12 to give the title compound as an orange oil: Η NMR (CDC13) δ 8.25 (d, IH, 6.4 Hz), 7.61 (dd, IH, 2.0, 7.9 Hz), 7.33 (ddd, IH, 2.2, 7.0, 7.0 Hz), 7.28-7.24 (m, IH), 6.63 (d, IH, 1.1 Hz), 6.15 (br t, IH, 6.6 Hz), 4.67 (s, 2H), 4.63 (td, 2H, 13.8, 6.9 Hz), 4.23 (q, 2H, 7.1 Hz), 2.08 (d, 3H, 1.1 Hz), 1.28 (t, 3 H, 7.1 Hz).
Figure imgf000049_0001
O
Figure imgf000049_0002
O
Step B
3-(2,2-Difluoro-2-(2-pyridyl-N-oxide-ethylamino)-6-methylpyrazin(lH)-2-one-l-acetic acid.
To a stiπed solution of 137 mg (0.372 mmol) of ethyl 3-(2,2-difluoro-2- (2-pyridyl-N-oxide-ethylamino)-6-methylpyrazin(lH)-2-one-l -acetate in methanol (20 mL) was added 167 mg potassium hydroxide (3.0 mmol dissolved in 5 mL water). After 2 h the solution was concentrated, diluted with 5 mL of water and acidified to pH = 6 using 1.5M HCI. Concentration at reduced pressure (azeotrope with PhCH3) afforded a yellow solid containing potassium chloride and the title compound. This mixture was used directly the amide coupling reactions.
Figure imgf000049_0003
Step C
2-Fluorobenzyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-methylpyrazin-2- one-1 -acetamide. To a stiπed solution of 60 mg (0.18 mmol, remainder KC1) of 3-(2,2- difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-methylpyrazin( 1 H)-2-one- 1 -acetic acid and 66 mg (0.53 mmol) 2-fluorobenzylamine in 1.5 mL of DMF was added 48 mg (0.25 mmol) of EDC, and 35 mg (0.25 mmol) of HOAT. After stirring for 3 d, the volatiles were removed en vacuo. The residue was partitioned between EtOAc and sat. aq. K2CO3, the layers were separated and the aqueous phase extracted with EtOAc (2x). The organics were combined, concentrated and chromatographed on SiO2 using 5:95 to 10:90 MeOH-CH2Cl2 to give the free base as a white solid This material was suspended in 5 mL of MeOH and treated with 2.0 mL of 4.0M solution of HCI in dioxane. Concentration afforded the product as a white solid: Η NMR (CD3OD) δ 8.52 (br d, IH, 3.5), 7.93 (ddd, IH, 3.3, 3.3, 6.5), 7.74 (m, 2H), 7.38 (br t, IH, 7.2), 7.31 (m, IH), 7.16-7.06 (m, 2H), 6.72 (d, IH, 2.2 Hz), 4.81 (d, 2H, 3.1 Hz), 4.67 (td, 2H, 2.9, 14.4 Hz), 4.48 (d, 2H, 2.2 Hz), 2.22 (d, 3H, 2.4 Hz).
EXAMPLE 6 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one-l- acetamide.
Figure imgf000051_0001
O
Figure imgf000051_0002
Step A 2-Cyano-3-(2,2,2-trifluoroethoxy)pyridine.
A stiπed solution of 680 mg (6.80 mmol) of 2,2,2-trifluoroethanol in 25 mL of THF was added 273 mg (6.83 mmol) of sodium hydride (60% dispersion). After 30 min at room temperature, 822 mg (6.73 mmol) of 2-cyano-3-fluoropyridine in 5 mL THF was added via syringe. The reaction was quenched after lh, by the addition of 15% K2CO3 aqueous solution. Concentration gave an oil that was partitioned between water and EtOAc. The layers were separated, the aqueous phase was backwashed with EtOAc (2x), and the combined organic layers were dried (MgSO4) and concentrated to an orange oil. The resultant cmde residue was chromatographed on SiO2 using 25:75 EtOAc-hexanes to afford the title compound as a yellow oil: 'H NMR (CDC13) δ 8.43 (d,lH, 4.6 Hz), 7.55 (dd, IH, 4.6, 8.7 Hz), 7.42 (d, IH, 8.7 Hz), 4.55 (q, 2H, 7.8 Hz).
Figure imgf000051_0003
Step B 2 - Aminomethyl-3 -(2,2,2 -trifluoroethoxy)pyridine dihy drochloride .
A solution of 0.53 g (2.62 mmol) of 2-cyano-3-(2,2,2- trifluoroethoxy)pyridine and 0.66 mL (7.87 mmol) of cone. HCI in 50 mL of ethanol was hydrogenated over 0.27 g of 10% palladium on carbon at 45 psi for 1.5 h. The catalyst was removed by filtration through a pad of Celite with EtOH. The solvents were removed at reduced pressure to give the title compound: Η NMR (CD3OD) δ
8.36 (d, IH, 5.0 Hz), 7.73 (d,lH, 8.6 Hz), 7.54 (dd, IH, 8.4, 5.0 Hz), 4.80 (q, 2H, 8.3
Hz), 4.33 (s, 2 H).
Figure imgf000052_0001
O
Step C
3-(2,2,2-Trifluoroethoxy)-2-pyridylmethyl 3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide. To a stiπed solution of 83 mg (0.23 mmol) of 3-(2,2-difluoro-2-(2- pyridyl-N-oxide)ethylamino)-6-chloropyrazin(lH)-2-one-l-acetic acid and 77 mg (0.28 mmol) 2-aminomethyl-3-(2,2,2-trifluoroethoxy)pyridine dihydrochloride in 1.5 mL of DMF was added 35 mg (0.25 mmol) of EDC, 50 mg (0.253 mmol) of HOAT and 0.50 mL N-methylmorpholine. After stirring overnight, the volatiles were removed en vacuo. The residue was diluted with water, filtered, and rinsed with water to afford a solid. This cmde material was filtered through a short plug of SiO2 with 100% EtOAc to 10:90 MeOH:EtOAc to give a white solid. This material was suspended in MeOH and treated with cone. HCI until the solution became homogeneous. Concentration afforded the title compound as a white solid: Η NMR (CD3OD) δ 8.46 (m, 2H), 8.36 (d, IH, 8.8 Hz), 8.01 (dd, IH, 6.0, 8.5 Hz), 7.79 (m, IH), 7.66 (m, 2H), 6.85 (s, IH), 4.97 (q, 2H, 8.2 Hz), 4.94 (s, 2H), 4.73 (s, 2H), 4.59 (t, 2H, 13.5 Hz).
EXAMPLE 7
Figure imgf000053_0001
Preparation of 2,5-Difluoro-l-phenylmethyl 3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide hydrochloride. A stiπed solution of 200 mg (0.556 mmol) of 3-(2,2-difluoro-2-(2- pyridyl-N-oxide)ethylamino)-6-chloropyrazin(lH)-2-one-l -acetic acid and 66.2 uL (0.566 mmol) 2,5-difluorobenzylamine in 2 mL of DMF was added 321 mg (0.617 mmol) of benzotriazol- 1 -yloxytripπolidinophosphonium hexafluorophosphate (PyBOP) and 0.267 mL (1.67 mmol) N,N-diisopropylethylamine. After stirring for 2 h, add 10 mL water and stir for 10 min. Filter off white solid and dry under vacuum at 40°C for 1 h. This material was suspended in EtOAc and treated with ~1 mL HCl/EtOAc (0.1576g/mL). The solvent was removed in vacuo to give the product as a white solid: Η NMR (DMSO) δ 8.82 (t, IH, 5.7 Hz), 8.35 (d, IH, 6.6 Hz), 7.62-7.54 (m, 2H), 7.39 (t, IH, 7.8), 7.27-7.22 (m, IH), 7.18-7.09 (m, 2H), 6.80 (s, IH), 4.75 (s, 2H), 4.46 (dt, 2H, 6.6, 13.3Hz), 4.31 (d, 2H, 5.6 Hz).
EXAMPLE 8
Figure imgf000054_0001
Preparation of 2-Fluorobenzyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6- cyanopyrazin-2-one- 1 -acetamide hydrochloride.
Prepared in the same manner as above by coupling 3-(2,2-difluoro-2- (2-pyridyl-N-oxide)ethylamino)-6-cyanopyrazin(lH)-2-one-l -acetic acid with 2- fluoro-benzylamine. 'H NMR (CD3OD) δ 8.54 (t, IH, 3.7 Hz), 7.83-7.79 (m, IH), 7.74-7.70 (m, 2H), 7.40-7.25 (m, 2H), 7.26 (s, IH), 7.15-7.04 (m, 2H), 4.74 (s, 2H), 4.64 (t, 2H, 12.9 Hz), 4.47 (s, 2H).
EXAMPLE 9
Figure imgf000054_0002
O
Phenethyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-cyanopyrazin-2-one-l- acetamide trifluoroacetate.
Prepared in the same manner as above by coupling 3-(2,2-difluoro-2-
(2-pyridyl-N-oxide)ethylamino)-6-cyanopyrazin( 1 H)-2-one- 1 -acetic acid with pheethyl amine. Further purification by preparative HPLC afforded the product. Η NMR (DMSO) δ 8.57 (t, IH, 6.6 Hz), 8.41 (t, 2H, 5.5 Hz), 8.36 (d, IH, 6.4 Hz), 7.63-
7.55 (m, 2H), 7.45 (s, IH), 7.40 (t, IH, 7.8 Hz), 7.31-7.27 (m, 2H), 7.22-7.17 (m, 3H),
4.60-4.51 (m, 4H), 3.33-3.28 (m, 2H), 2.72 (t, 2H, 7.5 Hz).
EXAMPLE 10 Preparation of 3-(Methylthio)-2-pyridylmethyl-3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one-l -acetamide
Figure imgf000055_0001
O Step A
2-Cyano-3-methylthiopyridine
A stiπed solution of 1.00 g (8.19 mmol) of 2-cyano-3-fluoropyridine and 0.63 lg (9.01 mmol) of sodium thiomethoxide in 8 mL of DMF was stiπed at room temperature for 1 h. The reaction mixture was diluted with water (80 mL) and stiπed for 5 min. The resulting solid was filtered and dried on a high vacuum line to give the product as an off-white solid: 'H NMR (CDC13) δ 8.46 (d,lH, 4.6 Hz), 7.66 (d, IH, 8.3Hz), 7.44 (dd, IH, 4.6, 8.3Hz), 2.58 (s, 3H).
Step B
2- Aminomethyl-3 -methylthiopyridine dihydrochloride
A stiπed solution of 659 mg (4.39 mmol) of 2-cyano-3- methylthiopyridine in 25 mL of methanol and 5 mL of 6M aq. HCI was hydrogenated over 659 mg of 10% palladium on carbon at 55 psi for 5 h. The catalyst was removed by filtration and the solvents concentrated at reduced pressure. The resulting material was diluted with methanol and concentrated (2x) to give 30! as an off- white solid: IH NMR (CD3OD): δ 2.58 (s, 3 H), 4.28 (s, 2 H), 7.43 (m, 1 H), 7.86 (dd, J = 1.3 and 8.1 Hz, 1 H), 8.43 (dd, J = 1.3 and 4.8 Hz, 1 H).
Step C
3-(Methylthio)-2-pyridylmethyl-3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6- chloropyrazin-2-one- 1 -acetamide
The title compound was prepared according to the general pyBOP coupling procedure and was isolated as a colorless solid: Η NMR (d6-DMSO, 400 MHz): δ 8.69 (m, 1 H), 8.36-8.32 (br m, 2 H), 7.71 (d, J = 8 Hz, 1 H), 7.60 (d, J = 6 Hz, 1 H), 7.55-7.51 (br m, 2 H), 7.41-7.32 (br m, 2 H), 6.78 (s, 1 H), 4.79 (s, 2 H), 4.47^.41 (br m, 4 H), 2.50 (s, 3 H). HRMS (FAB) M+H: 497.0960.
EXAMPLE 11
Figure imgf000056_0001
O
Preparation of 3-(Methylsulfoxy)-2-pyridylmethyl 3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide
To a mixture of 3-(methylthio)-2-pyridylmethyl-3-(2,2-difluoro-2-(2- pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one-l -acetamide (120 mg, 0.241 mmol) in carbon tetrachloride (14 mL) at room temperature was added (-)-[(8,8- dichlorocamphoryl)-sulfonyl]oxaziridine (75.4 mg, 0.253 mmol). The mixture was degassed and purged with argon six times at 25 °C and was then placed in a pre- heated 40 °C oil bath. After 10 min., the mixture was heated to 80 °C under argon and stiπed for 24 h. The reaction mixture was allowed to cool to room temperature and was filtered. The solid residue was washed with diethyl ether and dried in vacuo. Separation of unreacted sulfide and the two enantiomers was performed on a Chiralcel OD column by isocratic elution with 7:3 ethanol:hexanes/0.1% trifluoroacetic acid to collect as colorless solids unreacted sulfide, followed by the faster moving enantiomer and finally the slower moving enantiomer 446:
Faster moving enantiomer : 'H NMR (CD3OD, 400 MHz): δ 8.71 (m, 1 H), 8.43-8.37 (br m 2 H), 7.71-7.64 (m, 2 H), 7.60-7.55 (m, 2 H), 6.72 (m, 1 H), 4.87 (s, 2 H), 4.69-4.52 (4 H), 2.87 (s, 3 H). HRMS (FAB) M+H: 513.0902.
Slower moving enantiomer : 'H NMR (CD3OD, 400 MHz): δ 8.71 (m, 1 H), 8.42- 8.38 (br m, 2 H), 7.71-7.66 (m, 2 H), 7.59-7.54 (m, 2 H), 6.71 (m, 1 H), 4.87 (s, 2 H), 4.65^1.53 (br m, 4 H), 2.86 (s, 3 H). HRMS (FAB) M+H: 513.0929.
EXAMPLE 12
Figure imgf000057_0001
O
Preparation of 3-(2-methyl-2-trifluoroacetamidopropanethio)-2- pyridylmethyl 3-(2,2- difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one-l-acetamide
Step A 2-Amino-2-methylpropanethiol Hydrochloride
A mixture of 2,4,4-trimethyl-2-oxazoline (12.76 mL, 100 mmol) and phosphoms pentasulfide (26.67 g, 60 mmol) in toluene (100 mL) was stiπed at reflux for 16 h. The mixture was cooled, 20% aqueous sodium hydroxide (72 mL) was added and the mixture stiπed until the remaining solids were in suspension. This mixture was filtered through a glass frit and the organic component of the filtrate was washed with water (2x) and dried (Na^O . The resulting solution was extracted with 2.5M HCI (50 mL) and the red aqueous solution was heated to reflux. After 64 h the resulting lime green solution was cooled and evaporated in vacuo, azeotroping with isopropanol. The residue was recrystallized from isopropanol and collected by filtration, washing with cold isopropanol and ether to give the title compound as a colorless crystalline solid: IH NMR (d6 DMSO) δ 1.27 (s, 6 H), 2.73 (s, 2 H).
Step B 2-Methyl-2-trifluroacetamidopropanethiol
Trifluoroacetic anhydride (0.50 mL, 3.53 mmol) was added dropwise to a stiπed mixture of 2-amino-2-methylpropanethiol hydrochloride (500 mg, 3.53 mmol) and triethylamine (0.98 mL, 7.06 mmol) in methylene chloride (25 mL) at 0 C and the resulting mixture was warmed to room temperature. After 16 h the reaction was washed with 10% sodium hydrogen carbonate solution and brine, dried (Na^O and evaporated in vacuo to give the title compound as a colorless oil: IH NMR (CDC13) δ 1.45 (s, 6 H), 2.92 (d, J = 9.2 Hz, 2 H), 6.23 (br s, 1 H).
Step C 2-Cyano-3-(2-methyl-2-trifluoroacetamidopropanethio)-pyridine
A solution of 2-cyano-3-fluoropyridine (0.20 g, 1.64 mmol), 2-methyl- 2-trifluroacetamidopropanethiol (330 mg, 1.64 mmol) and sodium hydrogen carbonate (0.138 g, 1.64 mmol) in methanol (16 mL) was stiπed at room temperature for 64 h. The reaction mixture was evaporated in vacuo and the residue was partitioned between ethyl acetate and brine , dried (Na2SO4) and evaporated in vacuo. The residue was purified by column chromatography on silica (eluting with chloroform) to give the title compound: 'H NMR (CDC13) δ 'H NMR (CDC13) δ 1.55 (s, 6 H), 3.56 (s, 2 H), 6.17 (br s, 1 H), 7.44 (dd, 4.6, 8.2 Hz, 1 H), 7.88 (dd, J = 1.5 and 8.2 Hz, 1 H), 8.46 (dd, J = 1.5 and 4.6 Hz, 1 H).
Step D 2-Aminomethyl-3-(2-methyl-2-trifluoroacetamidopropanethio)-pyridine
A mixture of 2-cyano-3-(2-methyl-2-trifluoroacetamidopropanethio)- pyridine (86 mg, 0.28 mmol) and 10% palladium on carbon (150 mg) in methanol (10 mL) and 6M aq. HCI (2 mL) was shaken in a Pan apparatus under hydrogen (60 psi) for 3 h. More catalyst (50 mg) was added and after a further 16 h, the catalyst was removed by filtration and the solvents concentrated in vacuo. The residue was purified by column chromatography on silica (eluting with 95:5 ammonia saturated chloroform/methanol) to give the title compound: IH NMR (CDC13): δ 1.49 (s, 6 H), 1.83 (br s, 2 H), 3.36 (s, 2 H), 4.14 (s, 2 H), 6.17 (br s, 1 H), 7.14 (dd, 4.6, 7.8 Hz, 1 H), 7.25 (br s, 1 H), 7.71 (dd, J = 1.5 and 7.8 Hz, 1 H), 8.40 (dd, J = 1.5 and 4.6 Hz, 1 H).
Step D
3-(2-methyl-2-trifluoroacetamidopropanethio)-2- pyridylmethyl 3-(2,2-difluoro-2-(2- pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide The title compound was prepared according to the general pyBOP coupling procedure and was isolated as a colorless solid: 'H NMR (CD3OD, 300 MHz): δ 8.36 (m, 2 H), 7.91-7.88 (m, 1 H), 7.69 (m, 1 H), 7.56-7.54 (m, 2 H), 7.30- 7.27 (m, 1 H), 6.70 (s, 1 H), 4.91 (s, 2 H), 4.67 (s, 2 H), 4.56 (t, J - 12 Hz, 2 H), 3.31 (s, 2 H), 1.46 (s, 6 H). HRMS (FAB) M+H: 650.1374.
EXAMPLE 13
Figure imgf000060_0001
3-(2-methyl-2-aminopropanethio)-2- pyridylmethyl 3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one-l -acetamide
To a solution of 3-(2-methyl-2-trifluoroacetamidopropanethio)-2- pyridylmethyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2- one-1 -acetamide (22 mg, 0.034 mmol) in methanol (1.2 mL) and water (1 mL) at room temperature was added potassium carbonate (14.1 mg, 0.102 mmol). The mixture was heated to 60 °C for 3 h. Potassium carbonate (14.1 mg, 0.102 mmol) was again added, and the reaction was stiπed for 3 h. A third portion of potassium carbonate (14.1 mg, 0.102 mmol) was added, and the reaction was stiπed for 5 h. The methanol was removed in vacuo, and the remaining aqueous solution was extracted into chloroform several times. The organic layer was dried over sodium sulfate and concentrated in vacuo to afford the title compound as the free base. Η NMR
(CD3OD, 400 MHz): δ 8.70-8.65 (m, 2 H), 8.45 (m, 1 H), 7.96-7.93 (m, 1 H), 7.78- 7.76 (m, 1 H), 7.65-7.62 (m, 2 H), 6.82 (s, 1 H), 4.98 (s, 2 H), 4.84 (s, 2 H), 4.59 (t, J = 16 Hz, 2 H), 3.57 (s, 2 H), 1.49 (s, 6 H). LCMS (ES) M+H: 554.1.
EXAMPLE 14
Figure imgf000061_0001
2-(Methylthio)benzyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6- chloropyrazin-2-one- 1 -acetamide
The title compound was prepared according to the general pyBOP coupling procedure and was isolated as a colorless solid: Η NMR (CD3OD, 300 MHz): δ 8.37 (m, 1 H), 7.71-7.69 (m, 1 H), 7.56-7.54 (br m, 2 H), 7.30-7.28 (br m, 3 H), 7.15 (m, 1 H), 6.70 (s, 1 H), 4.86 (s, 2 H), 4.57 (t, J = 15 Hz, 2 H), 4.46 (s, 2 H), 2.48 (s, 3 H). HRMS (FAB) M+H: 496.1034.
EXAMPLE 15 Preparation of 2-(Methylsulfonyl)benzyl 3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide
Figure imgf000061_0002
Step A N-Boc-2-(methylsulfonyl)-benzylamine
To a solution of 2-(methylthio)-benzyl amine (160 mg, 1.04 mmol) in dichloromethane (3 mL) was added di-tert-butyl dicarbonate (273 mg, 1.25 mmol) and the reaction mixture was stiπed at room 25 °C for 2 h. Concentration in vacuo provided cmde N-Boc-2-(methylthio)-benzylamine which was used as is in the next step.
To a stiπed solution of N-Boc-2-(methylthio)-benzylamine (1.04 mmol) in dichloromethane (5 mL) was added m-chloroperoxybenzoic acid (540 mg, 1.56 mmol) by portions. After 2 h, the reaction mixture was diluted with dichloromethane (5 mL) and m-chloroperoxybenzoic acid (290 mg) was added and the reaction mixture stiπed for 18 h. The reaction mixture was diluted with methanol (2 mL), m-chloroperoxybenzoic acid (180 mg) was added and the reaction mixture stiπed for 18 h. The reaction mixture was concentrated in vacuo, diluted with diethyl ether, washed with saturated aqueous sodium bicarbonate several times and brine, dried over sodium sulfate, concentrated in vacuo, and the cmde product was purified by flash chromatography (silica gel, hexane to 50% ethyl acetate in hexane) to provide the title compound: Η NMR (CDC13, 400 MHz): δ 8.00 (d, 1 H); 7.70-7.60 (m, 2 H); 7.48 (t, 1 H); 5.48 (b s, 1 H); 4.62 (d, 2 H); 3.15 (s, 3 H); 1.42 (s, 9 H). MS (ES) M+Na: 308.1.
Step B
2-(Methylsulfonyl)-benzylamine hydrochloride Through a solution of N-Boc-2-(methylsulfonyl)-benzylamine (225 mg) in dichloromethane (20 ml) was bubbled HCI (g) for 10 min. The flask is sealed and stiπed for 18 hrs. Argon was bubbled through the reaction mixture which was then concentrated in vacuo to provide the title compound as a white solid. Η NMR
(CD3OD, 300 MHz): δ 8.25 (dd, 1 H); 8.90-8.65 (m, 3 H); 4.50 (s, 2 H); 3.25 (s, 3 H). Step C
2-(Methylsulfonyl)benzyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6- chloropyrazin-2-one- 1 -acetamide
The title compound was prepared according to the general pyBOP coupling procedure and was isolated as a colorless solid: 'H NMR (d6-DMSO, 400 MHz): δ 8.93 (m, 1 H), 8.36-8.34 (m, 1 H), 7.92 (m, 1 H), 7.71 (m, 1 H), 7.61-7.54
(br m, 3 H), 7.40-7.37 (m, 1 H), 6.80 (s, 1 H), 4.79 (s, 2 H), 4.72 (s, 2 H), 4.47 (br m,
2 H), 3.28 (s, 3 H). HRMS (FAB) M+H: 528.0912.
EXAMPLE 16
Figure imgf000063_0001
Benzyl-3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one-l- acetamide.
White solid: TLC: Silica Gf (160/10/1 of CH2Cl2/MeOH/Conc. NH4OH) Rf = 0.22 single, clean spot. HPLC: 100% at 215nm and 254nm.; MS: M+H = 449.9 (ES);
Η NMR (d6-DMSO) δ8.79 (br t, IH), 8.36 (d, IH, 6.4Hz), 7.62-7.54 (m, 3H), 7.42- 7.24 (m, 6H), 6.80 (s, IH), 4.74 (s, 2H), 4.44 (td, 2H, 13.2, 6.4 Hz), 4.30 (d, 2H, 5.5Hz).
EXAMPLE 17
Step A
2-Cyano-3-(2-hydroxy-2-methylpropanethio)pyridine
A stiπed solution of 2.44 g (20.0 mmol) of 2-cyano-3-fluoropyridine, 2.34 g (22.0 mmol) of 2-hydroxy-2-methylpropanethiol, and 1.68 g (20.0 mmol) of sodium hydrogencarbonate in 50 ml of methanol was heated to reflux for 3 hours.
The reaction was cooled to room temperature and partitioned between water (200 mL) and methylene chloride (3 x 150 mL). The organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure. This residue was chromatographed on SiO2 using 3:97 methanol-chloroform, yielding a white solid: Η
NMR (CDC13): δ 1.39 (s, 6 H), 3.18 (s, 2 H), 7.42 (dd, 1 H), 7.94 (dd, 1 H), 8.49 (dd,
I H).
Step B 2-Aminomethyl-3-(2-hydroxy-2-methylpropanethio)pyridine A stiπed solution of 1.9 g (9.1 mmol) of 2-cyano-3-(2-hydroxy-2- methylpropanethio)pyridine in 50 mL of methanol and 10 mL of aqueous 6M HCI was hydrogenated over 3.8 g of 10% palladium on carbon at 55 psi for 72 hours. The catalyst was removed by filtration and the solvents concentrated at reduced pressure. The residue was chromatographed on SiO2 using 5:95 methanol-chloroform saturated with ammonia yielding a yellow oil: Η NMR (CDC13): δ 1.32 (s, 6 H), 3.07 (s, 2 H), 4.18 (s, 2 H), 7.17 (dd, 1 H), 7.78 (dd, 1 H), 8.39 (dd, 1 H).
2-tert-butoxycarbonylaminomethyl-3-(2-hydroxy-2-methylpropanethio)pyridine A stiπed solution of 1.51 g (5.3 mmol) 2-Aminomethyl-3-(2-hydroxy-
2-methylpropanethio)pyridine dihydrochoride, 1.3 g (6.0 mmol), di-tert-butyl- dicarbonate, and 1.5 g (18.0 mmol) sodium hydrogencarbonate in 150 mL of tetrahydrofuran and 50 mL water was heated to 50 °C for 2 hours. The reaction was partitioned between water and methylene chloride. The organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure. This residue was chromatographed on SiO2 using 70:30 ethyl acetate-hexanes, yielding a clear oil: 'H NMR (CDC13): δ 1.33 (s, 6 H), 1.48 (s, 9 H), 3.08 (s, 2 H), 4.58 (d, 2 H), 6.05 (bs, 1 H), 7.16 (dd, 1 H), 7.75 (dd, 1 H), 8.37 (dd, 1 H).
Step D
2-tert-butoxycarbonylaminomethyl-3-(2-hydroxy-2-methylpropanesulfonyl)pyridine
To a stiπed solution of 224 mg (0.7 mmol) 2-tert- butoxycarbonylaminomethyl-3-(2-hydroxy-2-methylpropanethio)pyridine and 10 mg sodium tungstate dihydrate in 1 mL water and 1 mL 1 ,2 dichloroethane at 60 °C was added 0.17 mL (1.5 mmol) 30% H2O2. After 1 hour an additional 0.17 mL 30% H2O2 was added and the reaction stiπed vigorously at 75 °C overnight. The reaction was quenched with excess sodium sulfite solution and partitioned between water and methylene chloride. The organic extracts were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by chromatography on SiO2 using 0:100 to 5:95 methanol-chloroform yielding a clear oil: Η NMR (CDC13): δ 1.41 (s, 9 H), 1.44 (s, 6 H), 3.73 (s, 2 H), 4.74 (d, 2 H), 5.83 (bt, 1 H), 7.42 (dd, 1 H), 8.31 (dd, 1 H), 8.78 (dd, 1 H).
Step E 2-Aminomethyl-3-(2-hydroxy-2-methylpropanesulfonyl)pyridine A solution of 121 mg (0.35 mmol) 2-tert-butoxycarbonylaminomethyl- 3-(2-hydroxy-2-methylpropanesulfonyl)pyridine,10 mL 4M HCl/dioxane and 5 mL methanol was stiπed at room temperature for 2 hours. The reaction was evaporated to dryness. The residue was treated with triethylamine and purified by chromatography on SiO2 using 0:100 to 5:95 methanol-chloroform yielding a yellow oil: 'H NMR (CDC13): δ 1.44 (s, 6 H), 3.56 (s, 2 H), 4.41 (s, 2 H), 7.43 (dd, 1 H), 8.32 (dd, 1 H), 8.79 (dd, 1 H).
Step F 3-(2-hydroxy-2-methylpropanesulfonyl)-2-pyridylmethyl 3-(2,2-difluoro-2-(2- pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide
A solution of 54 mg (0.10 mmol) 3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin(lH)-2-one-l -acetic acid, 24 mg (0.10 mmol) 2- aminomethyl-3-(2-hydroxy-2-methylpropanesulfonyl)pyridine, 29 mg (0.15 mmol) EDC, 20 mg (0.15 mmol) HOBT, and 101 mg (1.0 mmol) NMM in 2 mL DMF was stiπed overnight at room temperature. This solution was partitioned between water and methylene chloride. The organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure. This residue was purified by chromatography on SiO2 using 10:90 methanol-chloroform. The free-base was taken up in methanol and treated with excess IM HCI in ether to generate the bis-HCl salt. This was precipitated from solution with ether, filtered and dried yielding a white solid: 'H NMR (CD3OD): δ 1.41 (s, 6 H), 3.64 (s, 2 H), 4.60 (t, J = 13.4 Hz, 2 H), 4.97 (s, 4 H), 6.86 (s, 1 H), 7.67 (m, 3 H), 7.81 (m, 1 H), 8.47 (m, 2 H), 8.83 (dd, J = 5 Hz, 1.7 Hz, 1 H); HRMS (FAB) calcd C23H25N6O6SClF2 (M+l) 587.1286, found 587.1272.
EXAMPLE 18 Preparation of 2-Cyano-5-methoxybenzyl 3-[2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino]-6-chloropyrazin-2-one-l -acetamide
Figure imgf000065_0001
Step A: 2-Cyano-5-methoxybenzyl alcohol
To a solution of ethyl 2-cyano-5-methoxybenzoate (5 g, 24 mmol) in THF (100 mL) at room temperature was added a solution of LiBH4 in THF (2M, 12 mL, 24 mmol). After 19 h, there was about 60% conversion of starting material. An additional quantity of LiBH4 in THF (2M, 12 mL, 24 mmol) was added and stirring continued for an additional 15 h. The solvent was removed under reduced pressure then water was carefully added to the residue. Excess hydride was destroyed by careful proportionwise addition of a saturated aqueous solution of NH4C1. Once complete, the resulting mixture was extracted with ether and then with ethyl acetate. The combined extracts were dried over NajSO, and the solvents then removed in vacuo. The residue was purified by column chromatography eluting with 2: 1 :2 hexane / chloroform / ethyl acetate. The title compound was isolated as a white solid: 'H NMR (CDC13) δ 7.58 (d, J = 8.5 Hz, 1 H), 7.15 (d, J = 2.7 Hz, 1 H), 6.86 (dd, J = 2.7, 8.5 Hz, 1 H), 4.89 (d, J = 6.0 Hz, 2 H), 3.88 (s, 1 H), 2.01 (t, J = 6.0 Hz, 1 H).
Step B: 2-Cyano-5-methoxybenzyl azide
A solution of 2-cyano-5-methoxybenzyl alcohol (1.8 g, 11 mmol) in THF (50 mL) was cooled to 0°C and then treated sequentially with diphenylphosphoryl azide (2.8 mL, 13 mmol) and DBU 1.9 mL, 13 mmol), the former added in one portion and the latter dropwise. The reaction mixture was allowed to warm gradually to room temperature and to then stir there overnight. The solvent was removed and the residue partitioned between ethyl acetate and water. The organic phase was washed with dilute citric acid, then brine and dried
Figure imgf000066_0001
The solvent was removed in vacuo and the residue purified by column chromatography eluting with 4:1:1 hexane / chloroform / ethyl acetate to give the product as a yellow oil: Η NMR (CDC13) δ 7.62 (d, J = 8.6 Hz, 1 H), 7.02 (d, J = 2.6 Hz, 1 H), 6.92 (dd, J = 2.6, 8.6 Hz, 1 H), 4.59 (s, 2 H), 3.89 (s, 3 H).
Step C: 2-Cyano-5-methoxybenzylamine
A mixture of 2-cyano-5-methoxybenzyl azide (1 g, 5.3 mmol) and 10% palladium on carbon (500 mg) in ethyl acetate (30 ml) was stiπed at room temperature under an atmosphere of hydrogen for 1 h. The catalyst was removed by filtration through a bed of Celite and the filtrate concentrated to yield the product as an orange oil: 1H NMR (CDCI3) δ 7.56 (d, J - 8.5 Hz, 1 H), 7.07 (d, J = 2.5 Hz, 1 H), 6.82 (dd, J = 2.5, 8.5 Hz, 1 H), 4.05 (br s, 2 H), 3.87 (s, 3 H).
Step D:
2-Cyano-5-methoxybenzyl 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino"|-6- chloropyrazin-2-one- 1 -acetamide
The title compound was prepared from 2-cyano-5-methoxy- benzylamine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin- 2-one-l -acetic acid according to the general EDC mediated coupling procedure. When conversion of the starting materials was complete, the DMF was removed under reduced pressure. The resulting residue was then stiπed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water. The product was allowed to dry and then it was further purified by preparative HPLC and characterized as the trifluoroacetate salt: lK NMR (CD3OD) δ 8.86 (m, IH), 8.37 (d,
J = 6.2 Hz, 1 H), 7.68 (m, 1 H), 7.63 (d, J = 8.6 Hz, 1 H), 7.50 - 7.60 (m, 2 H), 7.03 (d, J = 2.6 Hz, 1 H), 6.95 (dd, J = 2.6, 8.6 Hz, 1 H), 6.71 (s, 1 H), 4.87 (s, 2 H), 4.56 (m, 4 H), 3.86 (s, 3 H).
EXAMPLE 19 Preparation of 2,2-Difluoro-2-(2-pyridyl)ethyl 3-r2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino~l-6-chloropyrazin-2-one- 1 -acetamide
Figure imgf000067_0001
The title compound was prepared from 2,2-difluoro-2-(2- pyridyl)ethylamine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6- chloropyrazin-2-one-l -acetic acid according to the general EDC mediated coupling procedure. When conversion of the starting materials was complete, the DMF was removed under reduced pressure. The resulting residue was then stiπed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water. The product was allowed to dry and then it was further purified by preparative HPLC and characterized as the trifluoroacetate salt: *H NMR (CD3OD) δ 8.64 (d, J = 4.8 Hz, 1
H), 8.38 (d, J = 5.5 Hz, 1 H), 7.98 (m, 1 H), 7.70 - 7.74 (m, 2 H), 7.51 - 7.60 (m, 3 H), 6.69 (s, 1 H), 4.78 (s, 2 H), 4.56 (t, J = 13.2 Hz, 2 H), 4.09 (t, J = 14.1 Hz, 2 H).
EXAMPLE 20 Preparation of 3-Fluorophenethyl 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]- 6-chloropyrazin-2-one- 1 -acetamide
Figure imgf000068_0001
The title compound was prepared from 3-fluorophenethyl-amine and 3- [2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin-2-one-l-acetic acid according to the general EDC mediated coupling procedure. When conversion of the starting materials was complete, the DMF was removed under reduced pressure. The resulting residue was then stiπed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration, repeated washing with water and subsequent drying.: ^H NMR (CDCI3) δ 8.26 (d, J = 6.6 Hz, 1 H), 7.63 (m, 1 H), 7.20 - 7.40 (m, 4 H), 6.85 - 6.93 (m, 2 H), 6.89 (s, 1 H), 6.39 (br t, 1 H), 5.80 (br t, 1 H), 4.72 (s, 2 H), 4.64 (td, J = 7.1, 14.1 Hz, 2 H), 3.52 (dd, J = 6.4, 11.8 Hz, 2 H), 2.82 (t, J = 6.8 Hz, 2 H). EXAMPLE 21
Figure imgf000069_0001
Preparation of 3-Trifluoromethoxybenzyl 3-[2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino]-6-chloropyrazin-2-one- 1 -acetamide
The title compound was prepared from 3-trifluoromethoxy- benzylamine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin- 2-one-l -acetic acid according to the general EDC mediated coupling procedure. When conversion of the starting materials was complete, the DMF was removed under reduced pressure. The resulting residue was then stiπed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stiπing was continued until the product solidified whereupon it was collected via filtration, repeated washing with water and subsequent drying.: !H NMR (CDC13) δ 8.22 (d, J = 6.6 Hz, 1 H), 7.62 (dd, J = 2.1, 7.8 Hz, 1 H),
7.00 - 7.38 (m, 6 H), 6.90 (s, 1 H), 6.47 (br t, 1 H), 6.27 (br t, 1 H), 4.84 (s, 2 H), 4.63 (td, J = 6.8, 14.1 Hz, 2 H), 4.49 (m, 2 H).
EXAMPLE 22
Preparation of 2,2-Difluorophenethyl 3-[2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino1-6-chloropyrazin-2-one- 1 -acetamide
Figure imgf000069_0002
The title compound was prepared from 2,2-difluorophenethyl-amine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin-2-one-l-acetic acid according to the modified general EDC mediated coupling procedure. When conversion of the starting materials was complete, the DMF was removed under reduced pressure. The resulting residue was then stiπed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water. The product was allowed to dry and then it was added to a 1 :1 mixture of EtOAc and MeCN. Addition of a 1 M solution of HCI in ether gave the hydrochloride which was then collected via filtration.: !H NMR (CDC13) δ 8.27 (d, J = 6.2 Hz, 1 H), 7.64 (dd, J
= 2.2, 7.9 Hz, 1 H), 7.27 - 7.45 (m, 7 H), 6.88 (s, 1 H), ), 6.55 (br t, 1 H), 6.45 (br t, 1 H), 4.79 (s, 2 H), 4.64 (td, J = 7.0, 14.1 Hz, 2 H), 3.92 (td, J = 6.2, 14.3 Hz, 2 H).
EXAMPLE 23
Preparation of 2-(2,2,2-Trifluoroethoxy)-5-chlorobenzyl 3-[2,2-difluoro-2-(2-pyridyl- N-oxide)ethylamino]-6-chloropyrazin-2-one- 1 -acetamide
Figure imgf000070_0001
Step A:
2-(2,2,2-Trifluoroethoxy)-5-chlorobenzaldehyde
To a solution of trifluoromethanesulfonyl chloride (25 g, 148 mmol) in methylene chloride (200 mL) at -78°C was added sequentially and in a dropwise manner, trifluoroethanol (10 mL, 137 mmol) and triethylamine (21 mL, 151 mmol). The reaction mixture was left to stir for 1.5 hr following which it was allowed to warm to 0°C and stiπed there for 1 hr. The reaction mixture was diluted with ether and stiπed for an additional 0.5 hr following which the triethylamine hydrochloride thus precipitated was filtered off. The filtrate was concentrated though not to dryness (product volatility) and diluted once more with ether. Following filtration to remove any precipitated solids, the filtrate was concentrated almost to dryness and the resulting material used directly.
Using a small volume of DMF, the trifluorethoxy triflate thus prepared was added to a mixture of 5-chlorosalicylaldehyde (5 g, 32 mmol) and cesium carbonate (21 g, 64 mmol) in DMF (50 mL) at room temperature. After stiπing at room temperature overnight the solids were filtered off and the filtrate concentrated under reduced pressure. The residue was diluted with ether and washed well with water. Drying (Na^O and concentration yielded the title compound as an oil: H NMR (CDC13) δ 10.42 (s, 1 H), 7.84 (dd, J = 2.6 Hz, 1 H), 7.54 (m, 1 H), 6.93 (d, J = 8.8 Hz, 1 H), 4.48 (qd, J = 1.5, 7.8 Hz, 2 H).
Step B: 2-(2,2,2-Trifluoroethoxy)-5-chlorobenzyl alcohol
Sodium borohydride (200 mg, 5.3 mmol) was added to a solution of 2- (2,2,2-trifluoroethoxy)-5-chlorobenzaldehyde (2 g, 8.4 mmol) in ethanol at room temperature. After stirring for 1 hr, the solvent was removed under reduced pressure. Aqueous ammonium chloride was added to the residue to destroy excess hydride following which the mixture was extracted with ethyl acetate. The organic extracts were washed with brine and then dried over sodium sulfate. Concentration yielded the title compound as an oil: Η NMR (CDC13) δ 7.41 (d, J = 2.4 Hz, 1 H), 7.25 (m, 1 H), 6.77 (d, J = 8.8 Hz, 1 H), 4.73 (d, J = 4.6 Hz, 2 H), 4.40 (m, 2 H).
Step C: 2-(2,2,2-Trifluoroethoxy)-5-chlorobenzyl azide To a solution of 2-(2,2,2-trifluoroethoxy)-5-chlorobenzyl alcohol (1.9 g, 7.9 mmol) in THF (20 mL) at 0°C was added diphenyl phosphoryl azide (2.2 mL, 10.2 mmol) followed by DBU (1.5 mL, 10.0 mmol). The reaction mixture was allowed to warm to room temperature overnight following which the solvent was removed under reduced pressure. The residue was partitioned between ether and water and the organic layer dried over sodium sulfate. Filtration, concentration and purification by flash chromatography (19:1 hexane / ether) gave the azide as a white solid: Η NMR (CDC13) δ 7.31 (m, 2 H), 6.82 (d, J - 8.8 Hz, 1 H), 4.40 (m, 4 H).
Step D: 2-(2,2,2-Trifluoroethoxy)-5-chlorobenzylamine
To a solution of 2-(2,2,2-trifluoroethoxy)-5-chlorobenzyl azide (1.76 g, 6.63 mmol) in THF (20 mL) was added triphenylphosphine (2 g, 7.63 mmol). After stirring at room temperature for 2 h, water (0.5 mL) was added and the reaction mixture then heated at 65 °C for 2 h. After cooling back to room temperature the solvents were removed under reduced pressure. Following azeotroping with benzene, the residue was dissolved in ether and treated with an excess of cold IM HCI in ether. The hydrochloride salt of the title compound was filtered off and washed well with ether and dichloromethane. Drying gave a white powder.: H NMR (CD3OD) δ 7.46 (m, 2 H), 7.18 (d, J = 8.4 Hz, 1 H), 4.70 (q, J = 8.4 Hz, 2 H), 4.14 (s, 2 H).
Step E:
2-(2,2,2-Trifluoroethoxy)-5-chlorobenzyl 3-[2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino]-6-chloropyrazin-2-one- 1 -acetamide The title compound was prepared from 2-(2,2,2-trifluoroethoxy)-5- chlorobenzylamine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6- chloropyrazin-2-one-l -acetic acid according to the general EDC mediated coupling procedure. When conversion of the starting materials was complete, the DMF was removed under reduced pressure. The resulting residue was then stiπed while water (which had been rendered mildly basic by the addition of 10%> by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water. The product was allowed to dry and then it was added to a 1 :1 mixture of EtOAc and MeCN. Addition of a 1 M solution of HCI in ether gave the hydrochloride which was then collected via filtration.: *H NMR (CD3OD) δ 8.60 (br t, 1 H), 8.36 (d, J = 5.9
Hz, 1 H), 7.68 (m, 1 H), 7.52 - 7.58 (m, 2 H), 7.24 - 7.29 (m, 2 H), 7.02 (d, J = 8.4 Hz, 1 H), 6.71 (s, 1 H), 4.86 (s, 2 H), 4.56 (m, 4 H), 4.39 (m, 2 H).
EXAMPLE 24 Preparation of 2-(2,2,2-Trifluoroethoxy)benzyl 3-[2,2-difluoro-2-(2-pyridyl-N-oxide) ethylamino] -6-chloropyrazin-2-one- 1 -acetamide
Figure imgf000073_0001
O
Step A: 2-(2,2,2-Trifluoroethoxy)benzylamine
A solution of 2-(2,2,2-trifluoroethoxy)-5-chlorobenzylamine hydrochloride (650 mg) in ethanol (200 mL) containing 20% Pd(OH)2/C (1 g) was hydrogenated at 50 psi for 4 h. The catalyst was removed via filtration through Celite and the filtrate then evaporated to dryness to give the title compound as a white powder: Η NMR (CD3OD) δ 7.47 (m, 2 H), 7.19 (m, 2 H), 4.69 (q, J = 8.4 Hz, 2 H), 4.16 (s, 2 H).
Step B:
2-(2,2,2-Trifluoroethoxy)benzyl 3-[2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino]-6-chloropyrazin-2-one- 1 -acetamide
The title compound was prepared from 2-(2,2,2-trifluoroethoxy)-5- chlorobenzylamine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6- chloropyrazin-2-one-l -acetic acid according to the general EDC mediated coupling procedure. When conversion of the starting materials was complete, the DMF was removed under reduced pressure. The resulting residue was then stiπed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water. The product was allowed to dry and then it was added to a 1 :1 mixture of EtOAc and MeCN. Addition of a 1 M solution of HCI in ether gave the hydrochloride which was then collected via filtration.: !H NMR (CDC13) δ 8.25 (d, J = 6.4 Hz, 1 H), 7.61 (dd, J = 1.8, 7.9 Hz, 1 H), 7.27 - 7.36 (m, 4 H), 7.03 (t, J = 7.5 Hz, 1 H), 6.87 (s, 1 H), 6.83 (d, J = 8.2 Hz, 1 H), 6.38 (br t, 1 H), 6.25 (br t, 1 H), 4.77 (s, 2 H), 4.63 (qd, J = 7.0, 13.9 Hz, 2 H), 4.49 (d, J = 6.0 Hz, 2 H), 4.40 (q, J = 8.1 Hz, 2 H).
General Scheme
Figure imgf000075_0001
i) NCS, DCE, reflux ii) MCPBA, Na2CO3 iii) KOH
Figure imgf000075_0002
EXAMPLE 25
Figure imgf000075_0003
Preparation of 2-(2,2,2-Trifluoroethoxy)-5-chlorobenzyl 3-[2-(2-pyridyl-N- oxide)ethylamino]-6-chloropyrazin-2-one-l -acetamide
Step A: Ethyl 3-[2-(2-pyridyl)ethylamino1pyrazin-2-one-l -acetate
A solution of 2-aminoethylpyridine (3.14 mL, 28.5 mmol), ethyl 3- bromo-2 -pyrazinone- 1 -acetate (4.96 g, 19.0 mmol) and triethylamine (2.65 mL, 19.0 mmol) in 3:1 toluene / ethanol was heated in a sealed tube at 120 °C overnight. The reaction mixture was cooled to room temperature and the solids filtered off. The filtrate was concentrated to dryness, combined with the solids and the mixture partitioned between 1 : 1 ethyl acetate / ether and saturated sodium bicarbonate. The aqueous phase was extracted with dichloromethane. The combined organic extracts were dried over sodium sulfate and concentrated to give a yellow brown solid. Purification by flash chromatography (19:1 to 9:1 chloroform / methanol) gave the title compound as a cream colored powder: ^H NMR (CDCI3) δ 8.56 (d, J = 4.6 Hz,
1 H), 7.60 (td, J - 1.5, 7.7 Hz, 1 H), 7.18 (d, J = 7.7 Hz, 1 H), 7.14 (m, 1 H), 6.88 (d, J = 4.8 Hz, 1 H), 6.61 (br t, IH), 6.36 (d, J = 4.6 Hz, 1 H), 4.54 (s, 2 H), 4.25 (q, J = 7.2 Hz, 2 H), 3.84 (q, J = 6.6 Hz, 2 H), 3.12 (t, J = 6.6 Hz, 2 H), 1.29 (t, J = 7.2 Hz, 3 H).
Step B:
Ethyl 3-[2-(2-pyridyl)ethylamino]-6-chloropyrazin-2-one- 1 -acetate
A solution of ethyl 3-[2-(2-pyridyl)ethylamino]pyrazin-2-one-l-acetate
(1.4 g, 4.63 mmol) and N-chlorosuccinimide (0.6 g, 4.49 mmol) in dichloroethane was heated at 70 °C overnight. The reaction mixture was cooled to room temperature and concentrated. The residue was purified by chromatography (1 :4 hexane / EtOAc then EtOAc then 98:2 CH2CI2 / MeOH). The product was found to be contaminated by succinimide. This was removed by dissolving the product in ethyl acetate and washing with water and aqueous sodium bicarbonate. Drying (sodium sulfate) and concentration of the organic phase gave the product as a white solid: ^H NMR (CDCI3) δ 8.56 (d, J = 4.9 Hz, 1 H), 7.61 (t, J = 7.8 Hz, 1 H), 7.17 (d, J = 7.8 Hz, 1
H), 7.15 (m, 1 H), 6.96 (s, 1 H), 6.60 (br t, IH), 4.88 (s, 2 H), 4.25 (q, J = 7.2 Hz, 2 H), 3.83 (q, J = 6.3 Hz, 2 H), 3.12 (t, J = 6.6 Hz, 2 H), 1.30 (t, J = 7.2 Hz, 3 H).
Step C: Ethyl 3 - [2-(2-pyridyl-N-oxide)ethylamino] -6-chloropyrazin-2-one- 1 -acetate m-Chloroperbenzoic acid (70-75%, 1.2 g, ~4.85 mmol) was added to a mixture of ethyl 3-[2-(2-pyridyl)ethylamino]-6-chloropyrazin-2-one-l-acetate (1.4 g, 4.2 mmol) in dichloroethane (30 mL) and 10% sodium carbonate (8 mL) at room temperature. After stirring for 24 h, the reaction mixture was diluted with EtOAc and washed with cold 10% sodium carbonate and then saturated sodium bicarbonate.
Drying (sodium sulfate) and concentration gave the N-oxide as a powder: ^H NMR (CDCI3) δ 8.29 (d, J = 5.3 Hz, 1 H), 7.18 - 7.26 (m, 3 H), 6.93 (s, 1 H), 6.83 (br t,
IH), 4.88 (s, 2 H), 4.25 (q, J = 7.2 Hz, 2 H), 3.84 (q, J = 6.3 Hz, 2 H), 3.12 (t, J = 6.6 Hz, 2 H), 1.30 (t, J = 7.2 Hz, 3 H).
Step D:
3-[2-(2-Pyridyl-N-oxide)ethylamino1-6-chloropyrazin-2-one-l -acetic acid
A suspension of ethyl 3-[2-(2-pyridyl-N-oxide)ethylamino]-6- chloropyrazin-2-one-l -acetate (608 mg, 1.8 mmol) in 1 :1 THF / methanol was treated with 1 M KOH (3 mL) whereupon a homogenous solution was obtained. The reaction mixture was stiπed overnight then the methanol and THF was rotavaped off. 1 M HCI (3 mL) was added and the mixture evaporated to dryness and the resulting solid used directly: !H NMR (CD3OD) δ 8.39 (d, J = 6.4 Hz, 1 H), 7.98 (m, 1 H), 7.44 - 7.60 (m, 3 H), 6.90 (s, 1 H), 4.90 (s, 2 H), 3.83 (t, J = 6.4 Hz, 2 H), 3.32 (t, J = 6.4 Hz, 2 H?, obscured by solvent peak).
Step E:
2-(2,2,2-Trifluoroethoxy)-5-chlorobenzyl 3-[2-(2-pyridyl-N-oxide)ethylamino]-6- chloropyrazin-2-one- 1 -acetamide The title compound was prepared from 2-(2,2,2-trifluoroethoxy)-5- chlorobenzylamine and 3-[2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin-2-one- 1 -acetic acid according to the general EDC mediated coupling procedure. When conversion of the starting materials was complete, the DMF was removed under reduced pressure. The resulting residue was then stiπed while water (which had been rendered mildly basic by the addition of 10%> by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water. The product was allowed to dry and then it was added to a 1 : 1 mixture of EtOAc and MeCN. Addition of a 1 M solution of HCI in ether gave the hydrochloride which was then collected via filtration.: *H NMR (CD3OD) δ 8.59 (d, J = 6.4 Hz, 1 H), 7.63 -
7.85 (m, 3 H), 7.27 - 7.30 (m, 2 H), 7.05 (d, J = 8.6 Hz, 1 H), 7.03 (s, 1 H), 4.97 (s, 2 H), 4.61 (q, J = 8.4 Hz, 2 H), 4.44 (s, 2 H), 3.88 (t, J = 6.8 Hz, 2 H), 3.42 (t, J = 6.8 Hz, 2 H).
EXAMPLE 26
Preparation of 2-Methyl-5-chlorobenzyl 3-[2-(2-pyridyl-N-oxide)ethylamino]-6- chloropyrazin-2-one- 1 -acetamide
Figure imgf000078_0001
Step A: 2-Methyl-5-chlorobenzylamine
A solution of 2-methyl-5-chlorobenzonitrile (2 g, 13.2 mmol), in THF (30 mL) was cooled to -15 °C and treated with 1 M lithium aluminum hydride in THF (13.4 mL). The reaction mixture was allowed to warm gradually to 0 °C over 2 h and was then stiπed at room tenperature for 1.5 h. It was then cooled to 0 °C and quenched sequentially with EtOAc (1 mL), water (0,5 mL), 15% NaOH (0.5 mL), and water (1.5 mL). After stirring for 1 h, the solids were removed via filtration. The filtrate was concentrated and the resulting residue purified by flash chromatography (98:2 methylene chloride / methanol) to give the product as an oil: ^H NMR (CDCI3) δ 7.33 (d, J - 2.0 Hz, 1 H), 7.08 (m, 2 H), , 3.83 (s, 2 H), , 2.28 (s, 3 H), 1.42 (br s, 2 H).
Step B: 2-Methyl-5-chlorobenzyl 3-[2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin-2- one-1 -acetamide
The title compound was prepared from 2-methyl-5-chlorobenzylamine and 3-[2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin-2-one- 1 -acetic acid according to the general EDC mediated coupling procedure. When conversion of the starting materials was complete, the DMF was removed under reduced pressure. The resulting residue was then stiπed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water. The product was allowed to dry and then it was added to a 1 :1 mixture of EtOAc and MeCN. Addition of a 1 M solution of HCI in ether gave the hydrochloride which was then collected via filtration.:
!H NMR (CD3OD) δ 8.60 (d, J = 6.4 Hz, 1 H), 7.65 - 7.88 (m, 3 H), 7.27 (s, 1 H),
7.17 (s, 2 H), 7.04 (s, 1 H), 4.95 (s, 2 H), 4.41 (s, 2 H), 3.88 (t, J = 6.8 Hz, 2 H), 3.43 (t, J = 6.8 Hz, 2 H), 2.31 (s, 3 H).
EXAMPLE 27 Preparation of 2-Methyl-5-chlorobenzyl 3-[2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino]-6-chloropyrazin-2-one-l -acetamide
Figure imgf000080_0001
The title compound was prepared from 2-methyl-5-chloro-benzylamine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin-2-one-l-acetic acid according to the general EDC mediated coupling procedure. When conversion of the starting materials was complete, the DMF was removed under reduced pressure. The resulting residue was then stiπed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration, repeated washing with water and subsequent drying.: ^H NMR (CDCI3) δ
8.22 (d, J = 6.6 Hz, 1 H), 7.62 (d, J = 7.8 Hz, 1 H), 7.11 - 7.37 (m, 5 H), 6.91 (s, 1 H), 6.47 (br t, 1 H), 6.09 (br t, 1 H), 4.84 (s, 2 H), 4.62 (td, J = 6.8, 14.1 Hz, 2 H), 4.44 (d, J = 4.8 Hz, 2 H), 2.26 (s, 3 H).
EXAMPLE 28 Preparation of 2-Trifluoromethoxybenzyl 3-[2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino]-6-chloropyrazin-2-one- 1 -acetamide
Figure imgf000080_0002
The title compound was prepared from 2-trifluoromethoxy- benzylamine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin- 2-one-l -acetic acid according to the general EDC mediated coupling procedure. When conversion of the starting materials was complete, the DMF was removed under reduced pressure. The resulting residue was then stiπed while water (which had been rendered mildly basic by the addition of 10%> by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water. The product was allowed to dry and then it was added to a 1 : 1 mixture of EtOAc and MeCN. Addition of a 1 M solution of HCI in ether gave the hydrochloride which was then collected via filtration.: lR NMR (CD3OD) δ 8.56 (d, J = 5.4 Hz, 1 H), 7.75 -
7.95 (m, 3 H), 7.29 - 7.47 (m, 4 H), 7.03 (s, 1 H), 4.94 (s, 2 H), 4.67 (t, J = 13.9 Hz, 2 H), 4.50 (s, 2 H).
EXAMPLE 29 Preparation of 2,5-Dichlorobenzyl 3-[2-(2-pyridyl-N-oxide)ethylamino]-6- chloropyrazin-2-one- 1 -acetamide
Figure imgf000081_0001
The title compound was prepared from 2,5-dichlorobenzyl-amine and 3-[2-(2-pyridyl-N-oxide)ethylamino]-6-chloropyrazin-2-one-l-acetic acid according to the general EDC mediated coupling procedure. When conversion of the starting materials was complete, the DMF was removed under reduced pressure. The resulting residue was then stiπed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stiπing was continued until the product solidified whereupon it was collected via filtration and repeated washing with water. The product was allowed to dry and then it was added to a 1 :1 mixture of EtOAc and MeCN. Addition of a 1 M solution of
HCI in ether gave the hydrochloride which was then collected via filtration.: ^H NMR (CD3OD) δ 8.63 (d, J = 6.6 Hz, 1 H), 7.80 - 7.93 (m, 2 H), 7.69 (m, 1 H), 7.41
(m, 2 H), 7.32 (m, 1 H), 7.04 (s, 1 H), 4.98 (s, 2 H), 4.49 (s, 2 H), 3.89 (t, J = 6.8 Hz, 2 H), 3.44 (t, J = 6.8 Hz, 2 H).
EXAMPLE 30 Preparation of 2-(2,2,2-Trifluoroethoxy)-5-chlorobenzyl 3-[2,2-difluoro-2-(2-pyridyl- N-oxide)ethylamino]pyrazin-2-one- 1 -acetamide
Figure imgf000082_0001
Step A: 3-[2,2-Difluoro-2-(2-pyridyl-N-oxide)ethylamino]pyrazin-2-one-l-acetic acid
A suspension of ethyl 3-[2,2-Difluoro-2-(2-pyridyl-N-oxide)- ethylamino]pyrazin-2-one-l -acetate (85 mg, 0.24 mmol) in 1 :1 THF / methanol (4 mL) was treated with 1 M KOH (0.3 mL) whereupon a homogenous solution was obtained. The reaction mixture was stiπed overnight then the methanol and THF was rotavaped off. 1 M HCI (0.3 mL) was added and the mixture evaporated to dryness and the resulting solid used directly.
Step B: 2-(2,2,2-Trifluoroethoxy)-5-chlorobenzyl 3-[2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino]pyrazin-2-one- 1 -acetamide
The title compound was prepared from 2-(2,2,2-trifluoroethoxy)-5- chlorobenzylamine and 3-[2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino]pyrazin-2- one-1 -acetic acid according to the general EDC mediated coupling procedure. When conversion of the starting materials was complete, the DMF was removed under reduced pressure. The resulting residue was then stiπed while water (which had been rendered mildly basic by the addition of 10% by volume of saturated sodium bicarbonate) was added. Stirring was continued until the product solidified whereupon it was collected via filtration and repeated washing with water. The product was allowed to dry and then it was added to a 1 : 1 mixture of EtOAc and
MeCN. Addition of a 1 M solution of HCI in ether gave the hydrochloride which was then collected via filtration.: lYl NMR (CD OD) δ 8.52 (m, 1 H), 7.92 (m, 1 H), 7.74
(m, 2 H), 7.31 (m, 2 H), 7.07 (d, J = 5.7 Hz, 1 H), 7.03 (d, J = 8.8 Hz, 1 H), 6.83 (d, J = 5.7 Hz, 1 H), 4.72 (s, 2 H), 4.60 (q, J = 8.4 Hz, 2 H), 4.43 (s, 2 H). EXAMPLE 31
Figure imgf000083_0001
Figure imgf000083_0002
3-Fluorobenzyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2- one-1 -acetamide. To a stiπed solution of 90 mg (0.125 mmol, 50% by weight) of 3- (2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin(lH)-2-one-l -acetic acid and 22 μL (0.19 mmol) 3-fluorobenzylamine in 1 mL of DMF was added 36 mg (0.19 mmol) of EDC, 20 mg (0.15 mmol) of HOAT and 0.52 mL (0.375 mmol) triethylamine. After stirring for 3d, the volatiles were removed en vacuo. The residue was diluted with sat. aq. NaHCO3, filtered, and rinsed with water to afford a gummy solid. This material was triturated with CH2C12 (2 mL) to give a white solid, which was suspended in MeOH and treated with ~1 mL of 2.6M HCI in EtOAc. Concentration afforded the title compound as a solid: Η NMR (CD3OD) δ 8.60 (d, IH, 5.4 Hz), 7.98 (d, IH, 6.6 Hz), 7.82 (m, 2H), 7.35 (ddd, IH, 7.2 Hz), 7.14 (d, IH, 7.6 Hz), 7.07 (s, IH), 7.05 (m, IH), 7.00 (ddd, IH, 2.2, 8.5, 8.5 Hz), 4.96 (s, 2H), 4.71 (t, 2H, 13.9 Hz). 4.45 (s, 2H).
Figure imgf000084_0001
Figure imgf000084_0002
3-Chlorobenzyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2- one-1 -acetamide. To a stiπed solution of 90 mg (0.125 mmol, 50%> by weight) of 3- (2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin(lH)-2-one-l -acetic acid and 23 μL (0.19 mmol) 3-chlorobenzylamine in 1 mL of DMF was added 36 mg (0.19 mmol) of EDC, 20 mg (0.15 mmol) of HOAT and 0.52 mL (0.375 mmol) triethylamine. After stirring for 3d, the volatiles were removed en vacuo. The residue was diluted with sat. aq. NaHCO3, filtered, and rinsed with water to afford a gummy solid. This material was triturated with CH2C12 (2 mL) to give a white solid, which was suspended in MeOH and treated with ~1 mL of 2.6M HCI in EtOAc. Concentration afforded the title compound as a solid: Η NMR (CD3OD) δ 8.61 (d, IH, 5.9 Hz), 7.97 (dd, IH, 1.6, 7.6 Hz), 7.86-7.79 (m, 2H), 7.34-7.24 (m, 4H), 7.07 (s, IH), 4.96 (s, 2H), 4.71 (t, 2H, 14.0 Hz). 4.43 (s, 2H).
Figure imgf000085_0001
Figure imgf000085_0002
3-Chlorobenzyl 3-(2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one- 1 - acetamide. To a stiπed solution of 60 mg (0.133 mmol, 72% by weight) of 3-(2-(2- pyridyl-N-oxide)ethylamino)-6-chloropyrazin(lH)-2-one-l -acetic acid and 25 μL (0.20 mmol) 3-chlorobenzylamine in 1 mL of DMF was added 38 mg (0.20 mmol) of EDC, 20 mg (0.15 mmol) of HOAT and 0.60 mL (0.532 mmol) N-methylmorpholine. After stirring for Id, the volatiles were removed en vacuo. The residue was diluted with sat. aq. NaHCO3, filtered, and rinsed with water to afford a solid. This material was purified by flash chromatography with 2-10% MeOH:CH2Cl2 (with 0.5% NH4OH) to give a solid, which was suspended in MeOH and treated with ~1 mL of 2.6M HCI in EtOAc. Concentration afforded the title compound as a white solid: 'H NMR (CD3OD) δ 8.59 (d, IH, 6.5 Hz), 7.85-7.76 (m, 2H), 7.66 (ddd, IH, 1.8, 7.0, 7.0 Hz), 7.35-7.25 (m, 4H), 7.05 (s, IH), 4.97 (s, 2H), 4.44 (s, 2H), 3.89 (t, 2H, 6.8 Hz). 3.43 (t, 2H, 6.8 Hz).
EXAMPLE 32
Preparation of 3-Chloro-2-pyridylmethyl 3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide
Figure imgf000086_0001
Figure imgf000086_0002
3-Chloro-2-(hydroxymethyl)pyridine
A stiπed solution of 3.31 g (24.0 mmol) of 3-chloro-2-cyanopyridine (Sakamoto et al., Chem. Pharm. Bull. 33(2) 565-571 (1985)) and 50 mL of cone. HCI were heated at 110 °C for 24 h. Concentration and trituration with acetonitrile afforded the carboxylic acid as an off white solid. To a 0 °C suspension of 3.0 g (ca. 18.9 mmol) of this cmde material in 100 mL of THF, was added 25 mL (25.0 mmol) of borane (IM in THF). After 2 d at room temperature, cone. HCI was carefully added until gas evolution subsided. After 20 min of additional stirring the reaction was concentrated, diluted with aq. NaOH and extracted with CH2C12 (4x). The combined organic extracts were dried were dried (MgSO4) and concentrated to a yellow oil. The resultant cmde residue was chromatographed on SiO2 using 60:40 EtOAc-hexanes to afford the title compound: 'H NMR (CDC13) δ 8.49 (d, IH, 4.6 Hz), 7.69 (dd, IH, 1.2, 8.0 Hz), 7.23 (dd, IH, 4.8, 7.9 Hz), 4.80 (d, 2H, 4.6 Hz), 4.33 (t, IH, 4.6 Hz).
Figure imgf000086_0003
Figure imgf000086_0004
►2HCI 2-Aminomethyl-3-chloropyridine dihydrochloride
To a 0 °C solution of 738 mg (5.16 mmol) of 3-chloro-2- (hydroxymethyl)pyridine in 10 mL of toluene, was added 2.13 g (7.74 mmol) of diphenylphosphoryl azide followed by 1.18 g (7.74 mmol) of DBU. After stirrig at room temperature overnight, the reaction was partitioned between EtOAc and sat. aq. NaHCO3. The organic layer was separated and the aqueous phase was washed with EtOAc (3x). The combined organic extracts were dried (MgSO4) and concentrated to a dark oil. This resultant cmde residue was chromatographed on SiO2 using 10:90 EtOAc-hexanes to afford the title compound contaminated with an aromatic impurity. Triphenylphosphine (1.63 g, 6.20 mmol) was added to a solution of the cmde azide in THF : H2O (10 : 0.5 mL). After stirring overnight, the reaction was concentrated. This residue was diluted with EtOAc, and HCI (2.6M in EtOAc) was added dropwise to precipitate out the amine hydrochloride. Stirring was continued for 20 min, after which time the mixture was filtered and rinsed with EtOAc to afford the title compound as a white solid: Η NMR (CD3OD) δ 8.57 (d, IH, 4.7 Hz), 7.94 (d, IH, 8.1 Hz), 7.44 (dd, IH, 4.7, 8.0 Hz), 4.40 (s, 2 Hz).
Figure imgf000087_0001
3-Chloro-2-pyridylmethyl 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6- chloropyrazin-2-one- 1 -acetamide
A stiπed solution of 459 mg (1.28 mmol) of 3-(2,2-difluoro-2-(2- pyridyl-N-oxide)ethylamino)-6-chloropyrazin(lH)-2-one-l -acetic acid and 409 mg (1.91 mmol) 2-aminomethyl-3-chloropyridine dihydrochloride in 10 mL of DMF was added 364 mg (1.9 mmol) of EDC, 258 mg (1.9 mmol) of HOAT and 1.0 mL N- methylmorpholine. After stiπing overnight, the volatiles were removed en vacuo. The residue was diluted with water, filtered, and rinsed with water to afford a solid. This material was suspended in MeOH and treated with cone. HCI until the solution became homogeneous. Concentration afforded the product as a white solid: 'H NMR (CD3OD) δ 8.77 (dd, IH, 1.1, 5.1 Hz), 8.65 (dd, IH, 1.1, 8.3 Hz), 8.56 (br d, IH, 6.3 Hz), 7.98 (dd, IH, 5.7, 8.3 Hz), 7.92 (dd, IH, 2.2, 7.6 Hz), 7.82-7.75 (m, 2H), 6.99 (s, IH), 4.99 (s, 2H), 4.81 (s, 2H), 4.65 (t, 2H, 13.9 Hz).
Example 33
Preparation of 3-(Methylsulfonyl)-2-pyridylmethyl-3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate
Figure imgf000088_0001
Step A N-Phthalimido-3-(methylthio)pyridyl-2-methylamine
To a solution of 3-(methylthio)pyridyl-2-methylamine dihydrochloride (Example 10, Step B, 227 mg, 1.00 mmol) and sodium carbonate (318 mg, 3.00 mmol) in water (8.5 mL) was added N-carbethoxyphthalimide (241 mg, 1.10 mmol). The mixture was stiπed at room temperature for 1 h and was then filtered. The solid residue was washed with water and dried on a high vacuum line to yield the title compound as a white solid:
Η NMR (CDC13, 300 MHz): δ 8.19 (dd, J= 4.8, 1.4 Hz, 1 H), 7.91 - 7.87 (m, 2 H), 7.76 - 7.71 (m, 2 H), 7.54 (dd, J= 7.8, 1.4 Hz, 1 H), 7.13 (dd, J= 7.8, 4.8 Hz, 1 H), 5.05 (s, 2 H), 2.53 (s, 3 H).
Step B N-Phthalimido-3-(methylsulfoxy)pyridyl-2-methylamine
N-Phthalimido-3-(methylthio)pyridyl-2-methylamine was oxidized according to the procedure of Example 11. The cmde product was purified by silica gel chromatography (gradient elution with 60% EtOAc-hexanes to 100% EtOAc to 5% MeOH-CHCl3) to afford the title compound as a mixture of enantiomers which were inseparable by chiral HPLC:
'H NMR (CDClj, 300 MHz): δ 8.56 (dd, J= 4.8, 1.5 Hz, 1 H), 8.34 (dd, J= 7.8, 1.5 Hz, 1 H), 7.91 - 7.88 (m, 2 H), 7.77 - 7.74 (m, 2 H), 7.44 (dd, J= 7.8, 4.7 Hz, 1 H), 5.17 (d, J = 15.9 Hz, 1 H), 4.86 (d, J= 15.9 Hz, 1 H), 2.96 (s, 3 H).
Step C N-Phthalimido-3-(methylsulfonyl)pyridyl-2-methylamine The title compound was prepared according to a procedure which was modified from that described in Giam, C. S.; Kikukawa, K.; Trujillo, D. A.; Org. Prep. Proced. Int. 1981, 13 (2), 137-140 as follows: A mixture of sodium tungstate dihydrate (14 mg, 0.042 mmol), water (4 mL), and acetic acid (1 drop) was heated to reflux for 10 min. After cooling to room temperature, 25 μL of the resulting solution was added to 3-(methylsulfoxy)-2-(phthalimidomethyl)pyridine (59mg, 0.196 mmol) in 1,2-dichloroethane (250 μL). Hydrogen peroxide (50% in water, 12 μL, 0.398 mmol) was added to the mixture, which was then heated to reflux for 2.5 h. The DCE was removed in vacuo, and the resulting residue was taken up in water. Ammonium hydroxide and sodium bisulfite were added in minimal amounts to destroy residual hydrogen peroxide. The aqueous mixture was extracted with CHC13 (x3) and the organic layers were combined and dried over
Figure imgf000089_0001
The solvent was removed in vacuo to afford the title compound as a white, flaky solid:
*H NMR (CDC13, 300 MHz): δ 8.65 (dd, J= 4.5, 1.5 Hz, 1 H), 8.32 (dd, J= 7.8, 1.5 Hz, 1 H), 7.90 (dd, J= 5.4, 3.0 Hz, 2 H), 7.76 (dd, J= 5.4, 3.0 Hz, 2 H), 7.40 (dd, J= 7.8, 4.8 Hz, 1 H), 5.41 (s, 2 H), 3.37 (s, 3 H). MS (FAB) M+H: 317.2.
Step D 2-Aminomethyl-3-(methylsulfonyl)pyridine
To a mixture of N-phthalimido-3-(methylsulfonyl)pyridyl-2- methylamine (50 mg, 0.16 mmol) in ethanol (1 mL) at room temperature was added hydrazine hydrate (7.7 μL, 0.16 mmol). The mixture was heated to reflux for 1 h and the reaction was then quenched with 12 N hydrochloric acid (12 μL, 0.16 mmol). The ethanol was removed in vacuo, and the solid residue was taken up in 1 M hydrochloric acid and heated to 50 °C for 10 min. The mixture was cooled to room temperature and was filtered and washed with 1 M hydrochloric acid. The aqueous filtrate was then washed with two volumes of CH2C12 and concentrated in vacuo to half the volume. Triethylamine was added to pH 8, and the solution was concentrated in vacuo to dryness. The residue was purified by silica gel chromatography (isocratic elution with 8:1 :1 ethyl acetate: ammonium hydroxide:methanol) to afford the title compound as a pink oil:
'H NMR (CD3OD, 300 MHz): δ 8.84 - 8.23 (m, 1 H), 8.37 - 8.35 (m, 1 H), 7.58 - 7.55 (m, 1 H), 4.32 (s, 2 H), 3.25 (s, 3 H), 4.87. MS (FAB) M+H: 187.0.
Step E
3-(Methylsulfonyl)-2-pyridylmethyl-3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate
To a mixture of 3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6- chloropyrazin-2-one-l -carboxylic acid (42.2 mg, 0.117 mmol), 2-(aminomethyl)-3- (methylsulfonyl)pyridine (21.7 mg, 0.117 mmol), and HOAT (8.1 mg, 0.059 mmol) in DMF (1 mL) was added EDC (33.7 mg, 0.176 mmol). The mixture was stiπed at room temperature for 18 h. The cmde reaction mixture was purified directly by reverse phase HPLC (gradient elution with 95:5 water/0.1% trifluoroacetic acid:acetonitrile/0.1% trifluoroacetic acid to 5:95 water/0.1% trifluoroacetic acid:acetonitrile/0.1% trifluoroacetic acid) to afford the title compound as a white powder:
Η NMR (CD3OD, 300 MHz): δ 8.80 (dd, J= 4.8, 1.5 Hz, 1 H), 8.37 - 8.33 (br m, 2 H), 7.68 - 7.67 (m, 1 H), 7.58 - 7.53 (br m, 3 H), 6.68 (s, 1 H), 4.91 (s, 2 H), 4.86 (s, 2 H), 4.55 (t, J= 13.1 Hz, 2 H), 3.30 (s, 3 H). HRMS (FAB) M+H: 529.0880.
Example 34 Preparation of 2-(Ethylthio)benzyl-3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)- 6-chloropyrazin-2-one- 1 -acetamide
Figure imgf000090_0001
Step A Methyl-2-(ethylthio)benzoate
The title compound was prepared according to the procedure described in Abe, H.; Fujii, H.; Masunari, C; Itani, J.; Kashino, S. Chem. Pharm. Bull. 1997, 45 (5), 778-785 and was isolated as a solid:
Η NMR (CDC13, 300 MHz): δ 7.96 (d, J= 7.8, 1.5 Hz, 1 H), 7.47 - 7.41 (m, 1 H), 7.33 - 7.30 (m, 1 H), 7.18 - 7.12 (m, 1 H), 3.92 (s, 3 H), 2.96 (q, J= 7.2 Hz, 2 H), 1.39 (t, J= 7.2 Hz, 3 H).
Step B
2-(Ethylthio)benzyl alcohol
A solution of methyl-2-(ethylthio)benzoate (5.0 g, 25.5 mmol) in dry THF (67 mL) was added to a stiπed solution of LAH (17.5 mL of a 1.0 M solution in THF) in an additional 50 mL dry THF at 0 °C under Ar. The reaction was allowed to warm to room temperature while stirring overnight. The resulting clear solution was diluted with EtOAc (140 mL). The mixture was poured into water (300 mL) and the resulting precipitate was filtered off. The filtrate was extracted with ether (3x200 mL) and the combined organic extracts were dried over MgSO4, filtered, concentrated in vacuo and dried overnight on a high vacuum line to yield the title compound as a pale yellow oil:
'H NMR (CDC13, 300 MHz): δ 7.40 - 7.18 (br m, 4 H), 4.78 (d, J= 6.3 Hz, 2 H), 2.96 (q, J= 7.5 Hz, 2 H), 2.22 (t, J= 6.2 Hz, 1 H), 1.34 - 1.29 (m, 3 H). HRMS (ES) M+NH0: 186.0942.
Step C
2-(Azidomethyl)-ethylthiobenzene
To a stiπed solution of 2-(ethylthio)benzyl alcohol (3.98 g, 23.6 mmol) in THF (48 mL) at 0 °C under Ar was added DPPA (6.1 mL, 28.4 mmol) followed by DBU (4.0 mL, 26.0 mmol). The reaction was allowed to warm to room temperature and stiπed for 2 days. The solution was concentrated to an oil, which was taken up in EtOAc, and washed successively with 10% aqueous citric acid, saturated aqueous NaHCO3 solution and brine. The organic layer was dried over Na2SO4 and concentrated to an oil which was purified by silica gel chromatography (gradient elution with 100% hexanes to 5%> EtOAc-hexanes). Concentration of the product fractions at reduced pressure afforded a pale yellow oil which was dried on a high vacuum line overnight to afford the title compound:
Η NMR (CDClj, 300 MHz): δ 7.42 - 7.19 (m, 4 H), 4.52 (s, 2 H), 2.95 (q, J= 7.2 Hz,
2 H), 1.32 (t, J= 7.2 Hz, 3 H). HRMS (ES) M+NH40 211.1006.
Step D
2-(Aminomethyl)-ethylthiobenzene hydrochloride
To a solution of 2-(azidomethyl)-ethylthiobenzene (3.57 g, 18.4 mmol) in THF (393 mL) was added triphenylphosphine (9.69 g, 36.9 mmol) and water (0.77 mL). The mixture was stiπed overnight at room temperature. An additional 1.0 mL water was added and the mixture was heated to 60°C. After 4 h at 60°C, an additional 2.0 mL water was added and the mixture was heated to reflux overnight. The solution was concentrated to a pale yellow oil which was taken up in CH2C12. HCI gas was bubbled through the solution for approximately 10 min. The resulting precipitate was collected by filtration and dried on a high vacuum line to give the title compound as a white fluffy solid:
'H NMR (CD3OD): δ 7.56 - 7.54 (m, 1 H), 7.45 - 7.43 (m, 2 H), 7.34 - 7.32 (m, 1 H), 4.28 (m, 2 H), 3.04 - 2.98 (m, 2 H), 1.33 - 1.29 (m, 3 H). MS (ES) M+H: 168.5
Step E
2-(Ethylthio)benzyl-3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6- chloropyrazin-2-one- 1 -acetamide
The title compound was prepared according to the EDC coupling procedure which was described in Example 33, Step E (with one equivalent Et3N added to the reaction mixture), and the free base was isolated as an off-white solid: 'H NMR (CD3OD): δ 8.36 (d, J= 6.4 Hz, 1 H), 7.70 - 7.67 (m, 1 H), 7.57 - 7.54 (m, 2 H), 7.39 (d, J= 7.6 Hz, 1 H), 7.30 (d, J= 7.6 Hz, 1 H), 7.26 - 7.23 (m, 1 H), 7.22 - 7.16 (m, 1 H), 6.69 (s, 1 H), 4.85 (m, 2 H), 4.55 (t, J= 13.2 Hz, 2 H), 4.50 (s, 2 H), 2.94 (q, J= 7.2 Hz, 2 H), 1.28 (t, J= 7.2 Hz, 3 H). HRMS (ES) M+H: 510.1159.
Example 35 Preparation of (+)- and (-)-2-(Ethylsulfoxy)benzyl-3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate
Figure imgf000093_0001
Step A 2-(Ethylthio)benzyltrifluoroacetamide
To a solution of 2-(aminomethyl)-ethylthiobenzene hydrochloride (500 mg, 2.46 mmol) and pyridine (0.50 mL, 6.15 mmol) in dry CH2C12 (21 mL) at 0°C under Ar was added rapidly dropwise trifluoroacetic anhydride (0.45 mL, 3.2 mmol). The mixture was allowed to warm to room temperature while stirring overnight. The solution was diluted with CH2C12 (50 mL) and washed with 10% aqueous citric acid (100 mL). The aqueous layer was extracted with an additional 50 mL CH2C12. The combined organic extracts were washed with aqueous saturated NaHCO3 (50 mL) and brine (50 mL). The organic layer was dried over MgSO4 and concentrated at reduced pressure. Silica gel chromatography (isocratic elution with 20% EtOAc/hexanes) afforded the title compound as a white solid:
'H NMR (CDC13, 300 MHz): δ 7.40 - 7.18 (m, 4 H), 6.85 (br s, 1 H), 4.63 (d, J= 6.0 Hz, 2 H), 2.98 (q, J= 7.2 Hz, 2 H), 1.33 (t, J= 7.2 Hz, 3 H).
Step B
(+)- and (-)-2-(Ethylsulfoxy)benzyltrifluoroacetamide
To a stiπed solution of 2-(ethylthio)benzyltrifluoroacetamide (300 mg, 1.16 mmol) in CH2C12 (20 mL) at room temperature was added in portions MCPBA (75%, 280 mg, 1.22 mmol). The reaction was monitored by thin layer chromatography (70% EtOAc-hexanes eluent) and when complete was diluted to a volume of 50 mL with CH2C12 and was washed with aqueous saturated NaHCO3 (2x30 mL). The aqueous washes were further extracted with CH2C12 (20 mL). The combined organic extracts were dried over MgSO4 and concentrated at reduced pressure. Silica gel chromatography (70%EtOAc-hexanes) afforded a racemic mixture of the sulfoxide enantiomers as a colorless oil which was dried on a high vacuum line.
Η NMR (CDC13): δ 8.1 (br s, 1 H), 7.57 - 7.44 (m, 4 H), 4.76 - 4.66 (m, 2 H), 3.02 - 2.88 (m, 2 H), 1.28 (t, J= 7.6 Hz, 3 H). HRMS (ES) M+H: 280.0593. The racemic 2-(ethylsulfoxy)benzyltrifluoroacetamide was resolved into pure enantiomers via chiral HPLC under the following conditions: Chiralpak AS column eluting with an 85:5:10 mixture of 0.2% diethylamine- hexane:isopropanol:methanol. The enantiomers were isolated as colorless oils and carried on directly to the deprotection step (this example, Step C).
Step C
(+)- and (-)-2-(Ethylsulfoxy)benzylamine
A degassed mixture of the faster-moving enantiomer of 2- (ethylsulfoxy)benzyltrifluoroacetamide (110 mg, 0.395 mmol), potassium carbonate (164 mg, 1.19 mmol) and water (0.9 mL) in methanol (13.2 mL) was heated at 65 °C overnight. Silica gel was added to the cmde mixture and the solvent was removed at reduced pressure. The residual powder was loaded onto a pre-packed silica gel column and the product was eluted with 9:0.9:0.1 CHCl3-MeOH-NH4OH. The product fractions were combined, concentrated at reduced pressure and dried on a high vacuum line to afford the title compound as a pale yellow oil: 'H NMR (CDC13): δ 7.96 (dd, J= 7.2, 1.6 Hz, 1 H), 7.52 - 7.40 (m, 3 H), 4.03 (d, J = 14.0 Hz, 1 H), 3.90 (d, J= 14.0 Hz, 1 H), 3.11 - 3.02 (m, 1 H), 2.89 - 2.80 (m, 1 H), 1.30 - 1.26 (m, 3 H). MS (ES) M+H: 184.4 The slower-moving enantiomer of 2-
(ethylsulfoxy)benzyltrifluoroacetamide was deprotected in similar fashion to afford the title compound as a pale orange oil (sample contains significant amount of water by 'H NMR): 'H NMR (CDC13): δ 7.78 - 7.76 (m, 1 H), 7.52 - 7.46 (m, 3 H), 4.18 - 4.08 (m, 2 H), 3.08 - 2.92 (m, 2 H), 1.32 - 1.26 (m, 3 H). MS (ES) M+H: 184.5
Step E
(+)- and (-)- 2-(Ethylsulfoxy)benzyl-3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate The enantiomers of the title compound were prepared separately according to the general EDC coupling procedure described in Example 33, Step E and were isolated as yellow solids:
Enantiomer A (from the amine prepared from the faster-moving isomer of 2-
(ethylsulfoxy)benzyltrifluoroacetamide): 'H NMR (CDC13): δ 8.36 - 8.31 (m, 1 H), 7.72 - 7.70 (m, 1 H), 7.67 - 7.64 (m, 1 H), 7.52 - 7.36 (br m, 5 H), 6.99 - 6.95 (m, 1 H), 6.85 (s, 1 H), 6.44 - 6.34 (m, 1 H), 4.84 - 4.78 (m, 2 H), 4.74 - 4.54 (br m, 4 H), 2.93 (apparent q, J= 7.2 Hz, 2 H), 1.25 (t, J= 6.8 Hz, 3 H). MS (ES) M+H: 526.6. Enantiomer B (from the amine prepared from the slower-moving isomer of 2- (ethylsulfoxy)benzyltrifluoroacetamide): 'H NMR (CDC13): δ 8.40 - 8.38 (m, 1 H), 7.74 - 7.68 (m, 2 H), 7.52 - 7.44 (br m, 5 H), 7.05 (t, J = 5.2 Hz, 1 H), 6.84 (s, 1 H), 6.49 (m, 1 H), 4.87 - 4.75 (m, 2 H), 4.68 - 4.53 (br m, 4 H), 2.99 - 2.92 (m, 2 H), 1.25 (t, J= 7.6 Hz, 3 H). HRMS (ES) M+H: 526.1095.
Example 36 Preparation of 2-(Ethylsulfonyl)benzyl-3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate
Figure imgf000095_0001
Step A
2-(Ethylsulfonyl)benzyltrifluoroacetamide
To a stiπed solution of 2-(ethylthio)benzyltrifluoroacetamide (Example 35, Step A, 100 mg, 0.38 mmol) in CH2C12 (20 mL) was added in portions MCPBA (15%, 180 mg, 0.80 mmol). The mixnire was stiπed at room temperature for 1 h and was then diluted with CH2C12 and washed with saturated aqueous NaHCO3 solution. The aqueous layer was further extracted with CH2C12 and the combined organic extracts were dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel chromatography (isocratic elution with 35% EtOAc-hexanes) to afford the title compound as a colorless oil which was dried on a high vacuum line: IH NMR (CDC13): δ 8.00 (d, J= 7.6 Hz, 1 H), 7.69 - 7.60 (m, 2 H), 7.58 - 7.52 (m, 2 H), 4.76 (d, J= 6.8 Hz, 2 H), 3.19 (q, J= 7.6 Hz, 2 H), 1.34 (t, J= 7.6 Hz). HRMS (ES) M+H: 296.0565.
Step B 2-(Ethylsulfonyl)benzylamine hydrochloride To a stiπed solution of 2-(ethylsulfonyl)benzyltrifluoroacetamide (88 mg, 0.30 mmol) in methanol (11 mL) and water (0.74 mL) was added potassium carbonate (124 mg, 0.90 mmol). The mixture was heated to 65 °C and stiπed under Ar overnight. The mixture was then concentrated to a colorless oil which was taken up in EtOAc and washed with a small amount of brine. The aqueous layer was extracted twice with CHC13 and was then saturated with sodium chloride and further extracted with THF. The combined organic extracts were dried over MgSO4 and concentrated at reduced pressure. The residue was treated with 2 equivalents of 1.0 M HCI in ether. The resulting precipitate was collected by filtration and was dried on a high vacuum line to give the title compound as an off-white solid: IH NMR (CD3OD): δ 8.09 (d, J= 7.8 Hz, 1 H), 7.84 - 7.70 (br m, 3 H), 4.46 (s, 2 H), 3.38 - 3.30 (m, 2 H), 1.28 (t, J= 7.2 Hz, 3 H). HRMS (ES) M+H: 200.0746.
Step C
2-(Ethylsulfonyl)benzyl-3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6- chloropyrazin-2-one-l -acetamide trifluoroacetate
The title compound was prepared according to the general EDC coupling procedure described in Example 33, Step E (with one equivalent Et3N added to the reaction mixture) and was isolated as an off-white solid: Η NMR (CDC13): δ 8.39 (t, J= 4.0 Hz, 1 H), 7.96 (d, J= 8.4 Hz, 1 H), 7.69 - 7.61 (m, 3 H), 7.59 - 7.42 (m, 3 H), 6.83 (t, J= 6.4 Hz, 1 H), 6.80 (s, 1 H), 6.34 (m, 1 H), 4.73 (s, 2 H), 4.69 (d, J= 6.4 Hz, 2 H), 4.62 (td, J= 13.6, 6.0 Hz, 2 H), 3.18 (q, J= 7.6 Hz, 2 H), 1.31 (t, J= 7.6 Hz, 3 H). HRMS (ES) M+H: 542.1049
Example 37 Preparation of 2-(2,2,2-Trifluoroethylthio)benzyl-3-(2,2-difluoro-2-(2 -pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate
Figure imgf000096_0001
Step A
Methyl-2-(2,2,2-trifluoroethylthio)benzoate
Methylthiosalicylate (5.0 mL, 36 mmol) was added dropwise to a stiπed suspension of NaH (60%> oil dispersion, 1.43 g, 36 mmol) in dry DMF (30 mL) at room temperature. The mixture was stiπed in the dark under Ar for 30 min and was then cooled to 0 °C and treated with a solution of 1,1,1 -trifluoro-2-iodoethane
(3.0 mL, 30 mmol) in DMF (10 mL) via cannula. The reaction flask was covered with foil and was allowed to warm to room temperature while stirring overnight.
Water was poured into the mixture which was then extracted with ether (3x150 mL). The combined ether extracts were washed with saturated aqueous NajCO., and brine, dried over MgSO4 filtered and concentrated in vacuo. Silica gel chromatography
(isocratic elution with 10% EtOAc-hexanes) afforded the title compound as a pale yellow oil:
Η NMR (CDC13): δ 7.94 (dd, J= 6.4, 1.2 Hz, 1 H), 7.51 - 7.45 (m, 2 H), 7.31 - 7.27 (m, 1 H), 3.94 (s, 3 H), 3.58 (q, J= 9.6 Hz, 2 H).
Step B 2-(2,2,2-Trifluoroethylthio)benzyl alcohol
The title compound was prepared from methyl-2- (trifluoroethylthio)benzoate according to the procedure of Example 34, Step B and was isolated as a pale yellow oil:
Η NMR (CDC13): δ 7.56 (dd, J= 6.0 Hz, 1.6 Hz, 1 H), 7.46 (dd, J= 5.6 Hz, 2.0 Hz, 1 H), 7.37 - 7.29 (m, 2 H), 4.87 (d, J= 6.4 Hz, 2 H), 3.49 - 3.42 (m, 2 H), 2.02 (t, J =
6.0 Hz, 1 H).
Step C 2-Azidomethyl-(2,2,2-trifluoroethylthio)benzene
The title compound was prepared from 2-(2,2,2- trifluoroethylthio)benzyl alcohol according to the procedure of Example 34, Step C and was isolated as a pale yellow oil:
'H NMR (CDC13): δ 7.63 - 7.59 (m, 1 H), 7.41 - 7.32 (m, 3 H), 4.61 (s, 2 H), 3.44 (q, J= 9.6 Hz, 2 H).
Step D 2-Aminomethyl-(2,2,2-trifluoroethylthio)benzene hydrochloride The title compound was prepared from 2-azidomethyl-(2,2,2- trifluoroethylthio)benzene according to the procedure of Example 34, Step D, and was isolated as a white solid:
'H NMR (CDC13 with 1 drop DMSO-d6): δ 7.81 (d, J= 7.2 Hz, 1 H), 7.64 (d, J= 8.0 Hz, 1 H), 7.42 - 7.37 (m, 2 H), 4.37 (br s, 2 H), 3.45 (q, J= 9.6 Hz, 2 H). MS (ES) M+H: 222.5.
Step E
2-(2,2,2-Trifluoroethylthio)benzyl-3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)- 6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate
The title compound was prepared according to the general EDC coupling procedure described in Example 33, Step E (with one equivalent Et3N added to the reaction mixture) and was isolated as a white solid:
'H NMR (CDC13): δ 8.28 (d, J= 6.0 Hz, 1 H), 7.64 (dd, J= 5.2, 2.4 Hz, 1 H), 7.54 - 7.51 (m, 1 H), 7.39 - 7.26 (br m, 5 H), 6.86 (s, 1 H), 6.44 (t, J= 6.0 Hz, 1 H), 6.33 (br s, 1 H), 4.80 (s, 2 H), 4.65 - 4.54 (m, 4 H), 3.43 (q, J= 9.6 Hz, 2 H). HRMS (ES)
M+H: 564.0889.
Example 38
Preparation of (+)-2-(2,2,2-Trifluoroethylsulfoxy)benzyl-3-(2,2-difluoro-2-(2-pyridyl- N-oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate
Figure imgf000098_0001
O
To a solution of 2-(2,2,2-trifluoroethylthio)benzyl-3-(2,2-difluoro-2- (2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate (29 mg, 0.052 mmol) in methanol and CH2C12 was added MCPBA (75%, 12 mg, 0.052 mmol). The mixture was stiπed at room temperature for 1 h. The solvents were removed at reduced pressure and the residue was purified by reverse phase HPLC (gradient elution with 95:5 water/0.1%) trifluoroacetic acid:acetonitrile/0.1% trifluoroacetic acid to 5:95 water/0.1% trifluoroacetic acid:acetonitrile/0.1% trifluoroacetic acid) to afford the title compound as a yellow solid which was dried on a high vacuum line:
Η NMR (DMSO-d6): δ 8.92 (t, J= 6.0 Hz, 1 H), 8.35 (d, J= 6.8 Hz, 1 H), 7.95 - 7.92 (m, 1 H), 7.61 - 7.54 (br m, 3 H), 7.44 - 7.20 (m, 2 H), 6.80 (s, 1 H), 4.73 (s, 2 H), 4.50 - 4.32 (br m, 3 H), 4.20 - 4.02 (br m, 3 H). HRMS (ES) M+H: 580.0878.
Example 39 Preparation of 2-(2,2,2-Trifluoroethylsulfonyl)benzyl-3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate
Figure imgf000099_0001
Step A N-Boc-2-(2,2,2-Trifluoroethylthio)benzylamine
To a stiπed solution of 2-aminomethyl-(2,2,2- trifluoroethylthio)benzene hydrochloride (500 mg, 1.94 mmol) in CH2C12(20 mL) were added successively (BOC)2O (465 mg, 2.13 mmol), DMAP (23.7 mg, 0.194 mmol) and Et3N (0.27 mL, 1.94 mmol). The reaction mixture was stiπed overnight at room temperature and was then diluted with EtOAc and washed successively with 10%) aqueous citric acid, brine, and water. The organic layer was dried over MgSO4 and concentrated at reduced pressure. Silica gel chromatography (isocratic elution with 20% EtOAc-hexanes) afforded the title compound as a colorless oil: Η NMR (CDC13): δ 7.54 - 7.52 (m, 1 H), 7.39 (d, J= 7.6 Hz, 1 H), 7.34 - 7.26 (m, 2 H), 4.90 (br s, 1 H), 4.51 (d, J= 5.6 Hz, 2 H), 3.46 - 3.38 (m, 2 H), 1.45 (s, 9 H).
Step B N-Boc-2-(2,2,2-Trifluoroethylsulfonyl)benzylamine
The title compound was prepared from N-Boc-2-(2,2,2- trifluoroethylthio)benzylamine according to the procedure described in Example 36, Step A and was isolated as a white solid: 'H NMR (CDC13, 300 MHz): δ 8.05 (d, J= 8.1 Hz, 1 H), 7.72 - 7.64 (m, 2 H), 7.55 - 7.49 (m, 1 H), 5.37 (br s, 1 H), 4.61 (d, J= 6.6 Hz, 2 H), 4.16 - 4.06 (m, 2 H), 1.43 (s, 9 H).
Step C
2-(2,2,2-Trifluoroethylsulfonyl)benzylamine hydrochloride
To a stiπed solution of N-Boc-2-(2,2,2- trifluoroethylsulfonyl)benzylamine (480 mg) in dioxane (4.0 mL) at room temperature was added excess 4.0 M HCI in dioxane. The mixture was stiπed until no starting material remained by TLC and was then concentrated in vacuo to afford the title compound as an off-white solid:
Η NMR (CD3OD, 300 MHz): δ 8.18 (d, J= 7.8 Hz, 1 H), 7.89 - 7.86 (m, 1 H), 7.81 - 7.72 (m, 2 H), 4.65 (q, J= 9.3 Hz, 2 H), 4.49 (s, 2 H).
Step D
2-(2,2,2-Trifluoroethylsulfonyl)benzyl-3-(2,2-difluoro-2-(2-ρyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate
The title compound was prepared according to the general EDC coupling procedure described in Example 33, Step E (with one equivalent Et3N added to the reaction mixture) and was isolated as an off-white solid: Η NMR (CDC13): δ 8.31 (d, J= 6.4 Hz, 1 H), 8.03 (d, J= 8.0 Hz, 1 H), 7.70 - 7.63 (m, 3 H), 7.56 - 7.52 (m, 1 H), 7.41 - 7.31 (m, 2 H), 6.85 (s, 1 H), 6.78 (m, 1 H), 6.39 (m, 1 H), 4.75 (s, 2 H), 4.72 (d, J= 6.4 Hz, 2 H), 4.62 (td, J= 13.6, 6.4 Hz, 2 H), 4.06 (q, J= 9.0 Hz, 2 H). HRMS (FAB) M+Na: 618.0610.
Example 40 Preparation of 3-(ethylthio)-2-pyridylmethyl-3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide dihydrochloride
Figure imgf000100_0001
Step A
3-Bromopyridine- 1 -oxide
To a solution of 3-bromopyridine (1.52 mL, 15.8 mmol) in CH2C12 (35 mL) was added a solution of sodium bicarbonate (3.61 g, 42.9 mmol) in H2O (43 mL).
The biphasic mixture was cooled to 0°C and treated with MCPBA (7.28 g, 31.6 mmol) in portions over 5 min. The mixture was stiπed under N2 overnight and was then transfeπed to a separatory funnel and the aqueous layer was determined to be basic (pH 8-9). The mixture was diluted with CHC13 and the layers were separated. The aqueous layer was further extracted with CHC13 (x3). The combined organic extracts were dried over MgSO4 and concentrated in vacuo to afford a yellow oil.
Silica gel chromatography (isocratic elution with 5% methanol-CHCl3) afforded the title compound as a yellow oil:
'H NMR (CDC13): δ 8.38 (s, 1 H), 8.17 - 8.15 (m, 1 H), 7.43 - 7.41 (m, 1 H), 7.19 - 7.15 (m, 1 H).
Step B
3 -Bromo-2-cyanopyridine
The preparation of the title compound has been described in the literature: Sakamoto, T.; Kaneda, S.; Nishimura, S.; Yamanaka,H. Chem. Pharm. Bull. 1985, 33 (2), 565-571.
Step C 2-Cyano-3-(ethylthio)pyridine To a stiπed suspension of sodium ethanethiolate (600 mg, 5.75 mmol) in dry THF (25 mL) was added 3-bromo-2-cyanopyridine (700 mg, 3.83 mmol). The reaction mixture was heated to reflux under N2 for 3 h. The solvent was removed in vacuo and the resulting residue was taken up in CH2C12 (35 mL). The solution was filtered to remove precipitated sodium bromide and the filtrate was concentrated at reduced pressure to afford the title compound as a yellow oil:
Η NMR (CDC13, 300 MHz): δ 8.49 (dd, J= 3.3, 1.2 Hz, 1 H), 7.76 (d, J= 1.2 Hz, 1 H), 7.45 - 7.41 (m, 1 H), 3.06 (q, J= 7.2 Hz, 2 H), 1.40 - 1.32 (m, 3 H).
Step D 3 -(ethylthio)pyridine-2-methylamine dihydrochloride A mixture of 2-cyano-3-(ethylthio)pyridine (613 mg, 3.73 mmol) and concentrated HCI (0.93 mL, 11.2 mmol) in degassed ethanol (18 mL) was hydrogenated over 10%> palladium on carbon (184 mg) at 60 psi overnight. Additional 10%> palladium on carbon (180 mg) and cone. HCI (1 mL) were added to the reaction and hydrogenation was resumed at 61 psi overnight. The reaction mixture was filtered through Celite and washed with methanol. The filtrate was concentrated at reduced pressure to an orange solid which was taken up in methanol (12 mL). 10% Palladium on carbon (300 mg) and concentrated HCI (2.4 mL of a 6 M aqueous solution) were added and the mixture was hydrogenated at 57 psi for 3 days. The reaction mixture was again filtered through Celite, and the filter cake was washed with ethanol. The product was concentrated to a yellow-orange oil, which was taken up in methanol and treated with 1.0 M HCI in ether. The solvent was removed in vacuo to afford the title compound as an orange solid: 'H NMR (CD3OD): δ 8.48 (m, 1 H), 7.97 (dd, J= 8.0 Hz, 1.2 Hz, 1 H), 7.46 (dd, J= 8.0, 1.2 Hz, 1 H), 4.34 (s, 2 H), 3.08 (q, J= 7.2 Hz, 2 H), 1.34 (t, J= 7.2 Hz, 3 H). MS (ES) M+H: 169.5
Step E 3-(ethylthio)-2-pyridylmethyl-3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6- chloropyrazin-2-one- 1 -acetamide dihydrochloride
The title compound was prepared according to the general EDC coupling procedure as described in Example 33, Step E (with two equivalents Et3N added to the reaction mixture). When the reaction was complete, the product was precipitated from the reaction mixture by addition of water. The solid residue was treated with 1.0 M HCl-ether and was collected by filtration to give the title compound as an off-white solid:
Η NMR (CD3OD, 300 MHz): δ 8.56 - 8.52 (m, 2 H), 8.44 (d, J= 5.1 Hz, 1 H), 7.95 - 7.90 (m, 1 H), 7.78 - 7.75 (m, 1 H), 7.65 - 7.62 (m, 2 H), 6.81 (s, 1 H), 5.25 (s, 2 H), 4.68 (s, 2 H), 4.58 (t, J= 13.5 Hz, 2 H), 3.25 (q, J= 7.2 Hz, 2 H), 1.41 (t, J= 7.2 Hz, 3 H). HRMS (ES) M+H: 511.1162.
Example 41 Preparation of (+)-, (-)- and (±)3-(ethylsulfoxy)-2-pyridylmethyl-3-(2,2-difluoro-2-(2- pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide
Figure imgf000103_0001
Step A
N-Phthalimido-3-(ethylthio)pyridyl-2-methylamine The title compound was prepared from 3-(ethylthio)pyridyl-2- methylamine dihydrochloride (Example 40, Step D) according to the procedure of Example 33, Step A and was isolated as a purple solid:
Η NMR (CDCI3, 300 MHz): δ 8.24 - 8.22 (m, 1 H), 7.91 - 7.87 (m, 2 H), 7.76 - 7.71 (m, 2 H), 7.63 (dd, 7 = 7.8, 1.5 Hz, 1 H), 7.13 - 7.09 (m, 1 H), 5.11 (s, 2 H), 3.00 (q, J = 7.2 Hz, 2 H), 1.38 (t, J= 7.2 Hz, 3 H). MS (ES) M+H: 299.6
Step B (± )-N-Phthalimido-3-(ethylsulfoxy)pyridyl-2-methylamine
The title compound was prepared from N-phthalimido-3- (ethylthio)pyridyl-2-methylamine according to the procedure of Example 35, Step B and was isolated after silica gel chromatography (isocratic elution with 70% EtOAc- hexanes) as a white solid:
Η NMR (CDCI3): δ 8.54 (dd, J= 4.8, 1.6 Hz, 1 H), 8.26 (dd, J= 7.6 Hz, 1.6 Hz, 1 H), 7.91 - 7.88 (m, 2 H), 7.78 - 7.74 (m, 2 H), 7.43 - 7.40 (m, 1 H), 5.16 (d, JAB = 16.0 Hz, 1 H), 4.85 (d, JAB = 16.0 Hz, 1 H), 3.25 - 3.16 (m, 1 H), 3.00 - 2.91 (m, 1 H), 1.35 (t, 7= 7.2 Hz, 3 H).
Step C
(± )-3-(Ethylsulfoxy)pyridyl-2-methylamine N-phthalimido-3-(ethylsulfoxy)pyridyl-2-methylamine was deprotected according to the procedure of Example 33, Step D. The cmde product was purified by silica gel chromatography (isocratic elution with 9:0.9:0.1 CHCl3:methanol:NH4OH). The column fractions containing the product were concentrated at reduced pressure and the residue was dried on a high vacuum line to afford the title compound as a colorless oil: !H NMR (CDC13, 300 MHz): δ 8.67 - 8.65 (m, 1 H), 8.28 - 8.25 (m, 1 H), 7.46 - 7.42 (m, 1 H), 4.11 (d, JAB = 14.7 Hz, 1 H), 3.92 (d, JAB = 14.7 Hz, 1 H), 3.15 - 3.10 (br m, 1 H), 2.87 - 2.76 (br m, 1 H), 1.30 - 1.25 (m, 3 H). MS (ES) M+H: 185.5
Step D
(+)-, (-)- and (+)3-(Ethylsulfoxy)-2-pyridylmethyl-3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide
The racemic title compound was prepared according to the general EDC coupling procedure as described in Example 33, Step E (with one equivalent Et3N added to the reaction mixture) and was isolated as a yellow oil (trifluoroacetate salt):
Η NMR (CDC13): δ 8.73 - 8.70 (m, 1 H), 8.47 - 8.45 (m, 2 H), 8.06 (t, J= 7.2 Hz, 1 H), 7.78 - 7.63 (m, 2 H), 7.54 - 7.47 (m, 2 H), 6.83 (s, 1 H), 6.42 (br s, 1 H), 4.90 (br s, 2 H), 4.68 - 4.39 (br m, 4 H), 3.17 - 3.08 (br m, 1 H), 2.94 - 2.81 (br m, 1 H), 1.34 (t, J= 7.2 Hz, 3 H). MS (ES) M+H = 527.7
The racemate was resolved into pure enantiomers under the following conditions:
Chiralcel OD column eluting with an 80:10:10 mixture of 0.1% diethylamine- hexanes:methanol:ethanol for 60 min, then a 30:30:40 mixture of 0.1 %> diethylamine- hexanes:methanol:ethanol until the isomers eluted separately. Removal of the solvents at reduced pressure afforded the enantiomers as oils (free bases): Faster-moving enantiomer: 'H NMR (CDC13): δ 9.40 (br s, 1 H), 8.62 (dd, J= 5.2, 1.6 Hz, 1 H), 8.29 - 8.27 (m, 2 H), 7.63 (dd, J= 8.0, 2.4 Hz, 1 H), 7.52 - 7.48 (m, 1 H), 7.38 - 7.30 (br m, 2 H), 6.90 (s, 1 H), 6.39 (t, J= 7.2 Hz, 1 H), 4.89 (s, 2 H), 4.69 - 4.49 (br m, 4 H), 3.02 - 2.95 (br m, 1 H), 2.80 - 2.71 (m, 1 H), 1.36 (t, J= 7.6 Hz, 3 H). HRMS (ES) M+H: 527.1057.
Slower-moving enantiomer: Η NMR (CDC13): δ 9.40 (br s, 1 H), 8.62 (dd, J= 4.8, 1.2 Hz, 1 H), 8.29 - 8.27 (m, 2 H), 7.63 (dd, J= 8.0, 2.0 Hz, 1 H), 7.52 - 7.48 (m, 1 H), 7.38 - 7.30 (br m, 2 H), 6.90 (s, 1 H), 6.39 (t, J= 6.8 Hz, 1 H), 4.89 (s, 2 H), 4.69 - 4.49 (br m, 4 H), 3.02 - 2.95 (br m, 1 H), 2.80 - 2.71 (m, 1 H), 1.34 (t, J= 7.6 Hz, 3 H). MS (ES) M+H: 527.7
Example 42 Preparation of 3-(ethylsulfonyl)-2-pyridylmethyl-3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate
Figure imgf000105_0001
Step A
N-Phthalimido-3-(Ethylsulfonyl)pyridyl-2-methylamine The title compound was prepared according to a procedure which was modified from that described in Giam, C. S.; Kikukawa, K.; Trujillo, D. A.; Org. Prep. Proced. Int. 1981, 13 (2), 137-140 as follows: A solution of N-phthalimido-3- (ethylsulfoxy)pyridyl-2-methylamine (Example 41, Step B, 126 mg, 0.40 mmol) in a minimal amount of DCE was treated with 54 μL of a mixture of sodium tungstate (14 mg, 0.042 mmol) and acetic acid (1 drop) in water (4 mL). The mixture was heated to 65 °C and treated with 50% aqueous hydrogen peroxide (24 μL at the rate of 1 drop per 15 s). The mixture was refluxed for 24 h, then treated with an additional 27 μL of the tungstate mixture and refluxed for another 24 h. An additional 54 μL of the tungstate mixture was added and reflux was continued for 24 h. The solvent was removed in vacuo and the residue was taken up in water. The solution was treated with ammonium hydroxide and a small amount of sodium bisulfite to destroy residual hydrogen peroxide, and was then extracted with CHC13 (x2). The combined organic extracts were dried over Na2SO4 and concentrated at reduced pressure. The residue was purified by silica gel chromatography (70%o EtOAc-hexanes) to give the title compound as a white foamy solid:
Η NMR (CDC13, 300 MHz): δ 8.64 (dd, J= 4.8, 1.8 Hz, 1 H), 8.27 (dd, J= 8.1, 1.8 Hz, 1 H), 7.92 - 7.86 (m, 2 H), 7.79 - 7.73 (m, 2 H), 7.41 - 7.37 (m, 1 H), 5.40 (s, 2 H), 3.48 (q, J= 7.2 Hz, 2 H), 1.40 (t, J= 7.2 Hz, 3 H).
Step B
3-(Ethylsulfonyl)pyridyl-2-methylamine dihydrochloride
A mixture of N-phthalimido-3-(ethylsulfonyl)pyridyl-2-methylamine
(40 mg, 0.12 mmol) and hydrazine hydrate (6.0 μL, 0.12 mmol) in ethanol (2.5 mL) was heated to reflux overnight. The mixture was cooled to room temperature and treated with 12 M HCI (10 μL). The solvent was removed in vacuo and the residue was taken up in 1 M HCI and heated to 50 °C for 10 min. The solution was filtered and the residue rinsed with 1 M HCI. The filtrate was washed with CH2C12 (x2) and the aqueous layer was concentrated at reduced pressure. The residue was azeotroped with toluene to afford the title compound as a yellow oil (contaminated with phthalimide by-product):
Η NMR (CDC13 with 1 drop DMSO-d6, 300 MHz): δ 8.87 (d, J= 5.1 Hz, 1 H), 8.32 (d, J= 7.8 Hz, 1 H), 7.63 - 7.57 (m, 1 H), 4.66 - 4.64 (m, 2 H), 3.42 (q, J= 7.5 Hz, 2 H), 1.32 (t, J= 7.5 Hz, 3 H). MS (ES) M+H = 201.5. Phthalimide by-product appears in the Η NMR at δ 8.20 - 8.17 (m, 2 H) and 7.82 - 7.79 (m, 2 H).
Step D
3-(Ethylsulfonyl)-2-pyridylmethyl-3-(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)- 6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate
The title compound was prepared according to the procedure of Example 33, Step E (with two equivalents Et3N added to the reaction mixture) and was isolated as a pale yellow oil:
Η NMR (CDC13): δ 8.79 (dd, J= 5.2, 1.6 Hz, 1 H), 8.55 (d, J= 6.0 Hz, 1 H), 8.39 (dd, J= 8.0, 1.6 Hz, 1 H), 7.78 - 7.74 (m, 2 H), 7.63 - 7.52 (m, 3 H), 6.83 (s, 1 H), 6.46 (br s, 1 H), 4.97 - 4.96 (m, 2 H), 4.88 (s, 2 H), 4.62 (t, J= 13.6 Hz, 2 H), 3.39 (q, J= 7.6 Hz, 2 H), 1.32 (t, J= 7.6 Hz, 3 H). MS (ES) M+H: 543.7.
Example 43 Preparation of 3-(2,2,2-Trifluoroethylthio)-2-pyridylmethyl-3-(2,2-difluoro-2-(2- pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide dihydrochloride
Figure imgf000106_0001
O
Step A 2-Cyano-3-(2,2,2-trifluoroethylthio)pyridine
A suspension of sodium hydride (60% oil dispersion, 180 mg, 4.52 mmol) in dry THF (17 mL) under N2 was treated with trifluoroethanethiol (0.40 mL, 4.52 mmol) and the mixture was heated to 50 °C. When gas evolution ceased (ca. 1 h), 3-bromo-2-cyanopyridine was added and the mixture was heated to reflux for 4 h. The solvent was removed in vacuo and the residue was taken up in CH2C12 and filtered to remove precipitated sodium bromide. The filtrate was concentrated at reduced pressure to afford the title compound as an orange oil:
'H NMR (CDC13): δ 8.67 (dd, J= 4.8, 1.6 Hz, 1 H), 8.01 (dd, J- 8.4, 1.6 Hz, 1 H),
7.53 - 7.50 (m, 1 H), 3.58 (q, J= 9.2 Hz, 2 H).
Step B 3-(2,2,2-Trifluoroethylthio)pyridyl-2-methylamine dihydrochloride
A mixture of 2-cyano-3-(2,2,2-trifluoroethylthio)pyridine (300 mg,
1.37 mmol) and 6 M HCI (1.2 mL) in degassed methanol (6.0 mL) was hydrogenated over 10%) palladium on carbon (146 mg) at 55 psi for 24 h, then at 60 psi for 24 h.
The reaction mixture was filtered through Celite and the filter cake was washed thoroughly with ethanol. Removal of the solvents at reduced pressure afforded the title compound as an orange oil:
'H NMR (CD3OD): δ 8.60 (dd, J= 4.8, 1.2 Hz, 1 H), 8.13 (dd, J= 7.6, 1.2 Hz, 1 H),
7.48 - 7.45 (m, 1 H), 4.48 (s, 2 H), 3.81 (q, J= 10 Hz, 2 H). HRMS (ES) M+H:
223.0522.
Step C
3-(2,2,2-Trifluoroethylthio)-2-ρyridylmethyl-3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide dihydrochloride
The title compound was prepared according to the general EDC coupling procedure described in Example 33, Step E (with two equivalents Et3N added to the reaction mixture). After the described purification by reverse phase HPLC and removal of the solvents at reduced pressure, the dihydrochloride salt was prepared by treatment of the residue with 1 M HCl-ether and was isolated by filtration as an off-white solid: Η NMR (CD3OD): δ 8.75 (d, J= 8.0 Hz, 1 H), 8.68 (dd, J= 5.6, 1.2 Hz, 1 H), 8.42 (d, J= 5.6 Hz, 1 H), 7.97 - 7.93 (br m, 1 H), 7.76 - 7.73 (m, 1 H), 7.65 - 7.58 (br m, 2 H), 6.79 (s, 1 H), 4.96 (s, 2 H), 4.79 (s, 2 H), 4.57 (t, J= 13.4 Hz, 2 H), 4.07 (q, J = 10 Hz, 2 H). HRMS (ES) M+H: 565.0814.
Example 44 Preparation of (+)-3-(2,2,2-Trifluoroethylsulfoxy)-2-pyridylmethyl-3-(2,2-difluoro-2- (2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate
Figure imgf000108_0001
Step A
N-Boc-3-(2,2,2-trifluoroethylthio)pyridyl-2-methylamine
The title compound was prepared from 3-(2,2,2- trifluoroethylthio)pyridyl-2-methylamine dihydrochloride (Example 43, Step B) according to the procedure described in Example 39, Step A and was purified by silica gel chromatography (isocratic elution with 30%> EtOAc-hexanes) to afford a pale yellow oil:
Η NMR (CDC13, 300 MHz): δ 8.51 (dd, J= 4.8, 1.5 Hz, 1 H), 7.84 (d, J= 7.8 Hz, 1
H), 7.24 - 7.20 (m, 1 H), 6.00 (br s, 1 H), 4.64 (d, J= 4.5 Hz, 2 H), 3.41 (q, J= 9.6 Hz, 2 H), 1.46 (s, 9 H).
Step B
(±)-N-Boc-3-(2,2,2-trifluoroethylsulfoxy)pyridyl-2-methylamine
The title compound was prepared from N-Boc-3-(2,2,2- trifluoroethylthio)pyridyl-2-methylamine according to the procedure of Example 35,
Step B and was isolated as a white solid:
]H NMR (CDC13): δ 8.73 (dd, J= 4.8, 1.6 Hz, 1 H), 8.38 (dd, J= 8.0, 1.6 Hz, 1 H),
7.54 - 7.51 (m, 1 H), 5.58 (br s, 1 H), 4.49 - 4.37 (br m, 2 H), 3.93 (br m, 1 H), 3.55 -
3.45 (br m , l H), 1.43 (s, 9 H).
Step C
(±)-3-(2,2,2-trifluoroethylsulfoxy)pyridyl-2-methylamine dihydrochloride
The title compound was prepared from (±)-N-Boc-3-(2,2,2- trifluoroethylsulfoxy)pyridyl-2-methylamine according to the procedure of Example 39, Step C and was isolated as a yellow oil: Η NMR (CD3OD): δ 8.84 (dd, J= 4.8, 1.6 Hz, 1 H), 8.42 (dd, J= 8.0, 1.6 Hz, 1 H), 7.72 (dd, J= 8.0, 4.8 Hz, 1 H), 4.57 - 4.41 (br m, 2 H), 4.12 - 4.07 (br m, 2 H). MS (ES) M+H: 239.5.
Ste D
(±)-3-(2,2,2-Trifluoroethylsulfoxy)-2-pyridylmethyl-3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate
The title compound was prepared according to the general EDC coupling procedure of Example 33, Step E (with two equivalents Et3N added to the reaction mixture) and was isolated as a yellow oil:
'H NMR (CDC13, 300 MHz): δ 8.73 (d, J= 4.8 Hz, 1 H), 8.55 - 8.50 (m, 2 H), 7.81
(br m, 1 H), 7.76 - 7.71 (br m, 2 H), 7.70 - 7.52 (m, 2 H), 7.35 (br m, 1 H), 6.82 (s, 1
H), 4.82 (s, 2 H), 4.65 - 4.46 (m, 4 H), 4.12 - 4.04 (br m, 1 H), 3.63 - 3.51 (br m, 1
H). MS (ES) M+H: 581.7.
Example 45 Preparation of 3-(2,2,2-Trifluoroethylsulfonyl)-2-pyridyl-N-oxide-methyl-3-(2,2- difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one-l -acetamide trifluoroacetate
Figure imgf000109_0001
Step A N-Boc-3-(2,2,2-trifluoroethylsulfonyl)pyridyl-N-oxide-2-methylamine The title compound was prepared from N-Boc-3-(2,2,2- trifluoroethylthio)pyridyl-2-methylamine (Example 44, Step A) according to the procedure of Example36, Step A (using excess MCPBA) and was isolated as a white solid: 'H NMR (CDC13): δ 8.46 (d, J= 6.8 Hz, 1 H), 7.98 (d, J= 8.4 Hz, 1 H), 7.52 - 7.45
(m, 1 H), 6.43 (br s, 1 H), 5.02- 4.94 (br m, 4 H), 1.39 (s, 9 H). MS (ES) M+H: 371.6. Step B
3-(2,2,2-trifluoroethylsulfonyl)pyridyl-N-oxide-2-methylamine dihydrochloride
The title compound was prepared from N-Boc-3-(2,2,2- trifluoroethylsulfonyl)pyridyl-N-oxide-2-methylamine according to the procedure of Example 39, Step C and was isolated as a yellow oil:
■H NMR (CD3OD, 300 MHz): δ 8.71 (d, J= 6.6 Hz, 1 H), 8.13 (d, J= 8.4 Hz, 1 H), 7.83 (dd, J- 8.1, 6.6 Hz, 1 H), 4.72 (s, 2 H), 3.31 - 3.24 (m, 2 H, overlaps with methanol residual peak). MS (ES) M+H: 271.5.
Step C
3-(2,2,2-Trifluoroethylsulfonyl)-2-pyridyl-N-oxide-methyl-3-(2,2-difluoro-2-(2- pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate
The title compound was prepared according to the general EDC coupling procedure described in Example 33, Step E (with two equivalents Et3N added to the reaction mixture) and was isolated as a yellow oil: •H NMR (DMSO-d6): δ 8.94 (m, 1 H), 8.71 (d, J= 6.0 Hz, 1 H), 8.34 (d, J= 6.0 Hz, 1 H), 7.87 (d, J= 7.2 Hz, 1 H), 7.74 - 7.70 (m, 1 H), 7.59 - 7.50 (m, 3 H), 7.40 - 7.36 (br m, 1 H), 6.75 (s, 1 H), 5.24 (q, J= 10.4 Hz, 2 H), 4.89 (m, 2 H), 4.74 (s, 2 H), 4.40 (m, 2 H). MS (ES) M+H: 613.6.
Example 46 Preparation of (+)- and (-)-2-(Methylsulfoxy)benzyl-3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate
Figure imgf000110_0001
Step A 2-(Methylthio)benzyltrifluoroacetamide
The cmde title compound was prepared from commercially available 2-(methylthio)benzylamine according to the procedure of Example 35, Step A and was purified by silica gel chromatography (isocratic elution with 40%> EtOAc- hexanes) to afford a white solid: IH NMR ( CDC13, 300 MHz): d 7.37 - 7.15 (br m, 4 H), 6.77 (br s, 1 H), 4.61 (d, J=
6.0 Hz, 2 H), 2.51 (s, 3 H).
Step B
(+)- and (-)-2-(Methylsulfoxy)benzyltrifluoroacetamide
2-(Methylthio)benzyltrifluoroacetamide was oxidized according to the procedure of Example 35, Step B. The initial cmde mixture of enantiomers was separated from the 2-(methylsulfonyl)benzyltrifluoroacetamide by-product by silica gel chromatography (gradient elution with 100% EtOAc to 10% MeOH-CHC13). The sulfoxide enantiomers were separated on a Chiralpak AD column under the following conditions: 95:5 to 85:15 hexanes/0.1%> diethylamine:ethanol over 60 min, followed by 80: hexanes/0.1%> diethylamine: ethanol for 1 min, followed by 65:35 hexanes/0.1%) diethylamine:ethanol until the separated isomers eluted. The fractions were concentrated to yield the two isomers as white solids: Faster moving enantiomer: 'H NMR (300 MHz, CDC13): δ 7.90 (br s, 1 H), 7.62 - 7.59 (m, 1 H), 7.55 - 7.46 (br m, 3 H), 4.83 - 4.70 (br m, 2 H), 2.87 (s, 3 H). Slower moving enantiomer: 'H NMR (300 MHz, CDC13): δ 7.89 (br s, 1 H), 7.62 - 7.60 (m, 1 H), 7.59 - 7.46 (br m, 3 H), 4.84 - 4.70 (br m, 2 H), 2.87 (s, 3 H).
Step C
(+)- and (-)-2-(Methylsulfoxy)benzylamine
Potassium carbonate (120 mg, 0.871 mmol) was added to a degassed solution of the faster moving enantiomer of 2-(methylsulfoxy) benzyltrifluoroacetamide in methanol (9.7 mL) and water (0.65 mL) and the mixture was heated at 65 °C overnight under Ar atmosphere. Additional potassium carbonate (100 mg) was added and heating was resumed at 70 °C overnight. Silica gel was added to the cmde mixture and the solvent was removed at reduced pressure. The residual powder was loaded onto a pre-packed silica gel column and the product was eluted with 9:0.9:0.1 CHC13-MeOH-NH4OH. The product fractions were combined, concentrated at reduced pressure and dried on a high vacuum line to afford the title compound as a pale yellow oil: Η NMR (CDC13): δ 8.05 (dd, J= 7.6, 1.2 Hz, 1 H), 7.54 - 7.50 (m, 1 H), 7.48 - 7.44 (m, 1 H), 7.38 (d, J= 7.6 Hz, 1 H), 4.03 - 3.93 (m, 2 H), 2.84 (s, 3 H). The slower moving enantiomer of 2- (methylsulfoxy)benzyltrifluoroacetamide was deprotected in the same manner to afford the title compound as a pale yellow oil:
'H NMR (300 MHz, CDC13): δ 8.05 (dd, J= 7.5, 1.5 Hz, 1 H), 7.54 - 7.50 (m, 1 H), 7.49 _ 7.44 (m, l H), 7.39 - 7.36 (m, 1 H), 4.04 - 3.92 (m, 2 H), 2.84 (s, 3 H).
Step D
(+)- and (-)-2-(Methylsulfoxy)benzyl-3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one- 1 -acetamide trifluoroacetate The faster-moving enantiomer (on a Chiralpak AD column eluting with 30:70 0.1%>TFA-hexanes:ethanol) of the title compound was prepared according to the general EDC coupling procedure described in Example 33, Step E using the pure enantiomer of 2-(methylsulfoxy)benzylamine which came from the faster- moving isomer of 2-(methylsulfoxy)benzyltrifluoroacetamide. The cmde coupling product was purified by reverse phase HPLC (gradient elution from 95:5 water/0.1 % trifluoroacetic acid:acetonitrile/0.1%) trifluoroacetic acid to 5:95 water/0.1%) trifluoroacetic acid:acetonitrile/0.1% trifluoroacetic acid) and the fractions containing product were concentrated and dried on a high vacuum line to afford one pure enantiomer as a white solid: Η NMR (300 MHz, DMSO-d6): δ 8.88 (t, J= 5.6 Hz, 1 H), 8.36 (d, J= 6.0 Hz, 1 H), 7.89- 7.86 (m, 1 H), 7.62 - 7.36 (br m, 7 H), 6.80 (s, 1 H), 4.73 (s, 2 H), 4.52 - 4.29 (br m, 4 H), 2.71 (s, 3 H). MS (ES) M+H: 512.1.
The slower-moving enantiomer of the title compound was prepared and purified in similar fashion and was isolated as a white solid: Η NMR (300 MHz, DMSO-d6): δ 8.88 (t, J= 5.6 Hz, 1 H), 8.36 (d, J= 6.3 Hz, 1 H), 7.89- 7.86 (m, 1 H), 7.62 - 7.50 (br m, 5 H), 7.41 - 7.36 (br m, 2 H), 6.80 (s, 1 H), 4.73 (s, 2 H), 4.52 - 4.29 (br m, 4 H), 2.71 (s, 3 H). MS (ES) M+H: 512.1.
I l l EXAMPLE 47 Preparation of 6-chloro-3-{[2,2-difluoro-2-(l-oxidopyridin-2-yl)ethyl]amino}-l-(2- {[(lS)-2-hydroxy-l-phenylethyl]amino}-2-oxoethyl)-2-oxo-l,2-dihydropyrazin-4-ium chloride
Cl¬
Figure imgf000113_0001
The title compound was prepared from (S) phenylglycinol and acid 16 according to the procedure in Example 1, step R. Η NMR (400 mHz, CD3OD) δ 8.45 (dd, IH, J=2.1 and 7.13 Hz); 7.79 (m, IH); 7.65 (m, 2H); 7.33-7.25 (m, 5H); 6.82 (s, IH); 5.0 (m, 3H); 4.59 (t, 2H, J=13.5 Hz); 3.78 (dd, IH, J=5.2 and 11.3 Hz); 3.72 (dd, IH, J=7.5 and 11.3 Hz) Mass Spectmm (electrospray) M+H=480.1
EXAMPLE 48 Preparation of 6-chloro-l-(2-{[(lR)-l-(3-chlorophenyl)ethyl]amino}-2-oxoethyl)-3- {[2,2-difluoro-2-(l-oxidopyridin-2-yl)ethyl]amino}-2-oxo-l,2-dihydropyrazin-4-ium chloride
cr
Figure imgf000113_0002
The title compound was prepared from (lR)l-(3- chlorophenyl)ethylamine (Pickard, Simeon T.; Smith, Howard E. J Am. Chem. Soc. 1990, 112, 5741-7.) phenylglycinol and acid 16 according to the procedure in Example 1, step R. Η NMR Η NMR (400 mHz, CD3OD) δ 8.54 (dd, IH, J=1.6 and 6.95 Hz); 7.90 (m, IH); 7.75 (m, 2H); 7.3 (m, 4H); 6.98 (s, IH); 4.9 (m, 3H); 4.65 (t, 2H, J=13.8 Hz); 1.47, (d, 3H, J=7.5 Hz). Mass Spectmm (electrospray) M+H=498.1 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 Prefeπed Range Most Prefeπed 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.
The thrombin inhibitors can also be co-administered with suitable antiplatelet 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, 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. 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 NaCI, 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 α- thrombin (Km=125 μM) and bovine trypsin (Km=125 μM). p-Nitroanilide substrate concentration was determined from measurements of absorbance at 342 nm using an extinction coefficient of 8270 cm~lM~0
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 (Km=27 μM) was determined from the increase in fluorescence at 500 nm
(excitation at 400 nm) associated with production of 7-amino-4-trifluoromethyl coumarin. Concentrations of stock solutions of Z-GPR-afc were determined from measurements of absorbance at 380 nm of the 7-amino-4-trifluoromethyl coumarin produced upon complete hydrolysis of an aliquot of the stock solution by thrombin.
Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration < 0.1 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. Initial velocities of product formation in the absence (V0) or presence of inhibitor (Vi) were measured. Assuming competitive inhibition, and that unity is negligible compared Km/[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 = 1 + [I]/Kj 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 49 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 3-Fluoro-2-pyridylmethyl 3-(2,2-difluoro-2-(2-pyridyl-N- oxide)ethylamino)-6-chloropyrazin-2-one-l -acetamide (Example 1).
Amount-(mg)
Component A B C Active I 25 50 100 Microcrystalline cellulose 37.25 100 200 Modified food com 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 com starch are mixed and granulated to 10% com starch paste. The resulting granulation is sieved, dried and blended with the remainder of the com 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 50 Tablet Preparation
Exemplary compositions of compound 3-Fluoro-2-pyridylmethyl 3- (2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one-l-acetamide (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 dmg 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 51
Intravenous Formulations Intravenous formulations of compound 3-Fluoro-2-pyridylmethyl 3-
(2,2-difluoro-2-(2-pyridyl-N-oxide)ethylamino)-6-chloropyrazin-2-one-l-acetamide (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 NF 50-53 mg
1 N 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
Active I 0.61 mg* 0.30** 0.15***
D-glucuronic acid* 1.94 mg 1.94 mg 1.94 mg
Mannitol NF 51.2 mg 51.2 mg 51.2 mg
1 N 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:
Figure imgf000119_0001
wherein A is
Figure imgf000119_0002
n = 0-l:
R is hydrogen,
C M alkyl unsubstituted or substituted with halogen, OR9, N(R9)2, COOR9;
CON(R9)2 aryl, or a heterocyclic ring, wherein R9 is independently hydrogen or C M alkyl;
R2 IS hydrogen, C M alkyl, CF3, halogen, cyano, or cyclo C 3.7 alkyl; R3, R7, R8, and R13 are independently chosen from hydrogen, halogen, C ,.4 alkyl;
R14 and R15 are independently chosen from hydrogen, C ,.2 alkyl, and C ,.2 alkyl substituted with OR16 or COOR16, wherein R16 is hydrogen or C alkyl; and
R4, R5 and R6 are independently chosen from hydrogen, halogen, hydroxy,
C ,.4 alkyl,
C ,_4 alkoxy, cyano, CF3O,
CHF2O,
CF3CH2O,
SR10,
SOR10, SO2R10,
OR11,
SR",
NHR"
wherein
R10 is C M alkyl unsubstituted or substituted with C(CH3)2NH2, C(CH3)2OH, C(CH3)2NHCOCF3, or CF3, and R" is phenyl unsubstituted or substituted with one or more of C A alkyl, C M alkoxy, halogen, hydroxy, COOH, CONH2, CH2OH, or CO2R12, wherein R12 is C ,.4 alkyl.
2. A compound of Claim 1, or pharmaceutically acceptable salt thereof, wherein A is
Figure imgf000121_0001
3. A compound of Claim 2, or pharmaceutically acceptable salt
7 4 5 thereof, wherein R is Cl, CH3 or CN; R and R are independently selected from the group consisting of hydrogen, C alkyl, SMe, SOMe, SO2Me, CN, OCH2CF3, OCH3
SCH2C(CH3)2NH2, OCF3, SCH2CH3, SOCH2CH3, SO2CH2CH3, SCH2CF3, SOCH2CF3, SO2CH2CF3, and halogen, R7 is hydrogen or fluoro; and R13 is hydrogen or fluoro.
4. A compound of Claim 3, or pharmaceutically acceptable salt
4 5 thereof, wherein R and R are independently selected from the group consisting of hydrogen, CH3, Cl, F, SMe, SOMe, SO2Me, CN, OCH2CF3, OCH3 SCH2C(CH3)2NH2,
OCF3 , SCH2CH3, SOCH2CH3, SO2CH2CH3, SCH2CF3, SOCH2CF3, and SO2CH2CF3.
5. A compound of Claim 4, or pharmaceutically acceptable salt thereof, wherein. A is selected from the group consisting of
Figure imgf000122_0001
Figure imgf000122_0002
Figure imgf000122_0003
Figure imgf000122_0004
Figure imgf000122_0005
6. A compound of Claim 4, or pharmaceutically acceptable salt thereof, wherein. A is selected from the group consisting of
Figure imgf000123_0001
Figure imgf000123_0002
Figure imgf000123_0003
7. A compound of Claim 5, or pharmaceutically acceptable salt thereof, selected from the group consisting of
Figure imgf000124_0001
Figure imgf000124_0002
Figure imgf000124_0003
Figure imgf000124_0004
Figure imgf000124_0005
Figure imgf000125_0001
Figure imgf000125_0002
Figure imgf000125_0003
Figure imgf000125_0004
Figure imgf000125_0005
Figure imgf000126_0001
Figure imgf000126_0002
O
Figure imgf000126_0003
Figure imgf000126_0004
O
Figure imgf000127_0001
Figure imgf000127_0002
O
Figure imgf000127_0003
O
Figure imgf000127_0004
Figure imgf000128_0001
Figure imgf000128_0002
Figure imgf000128_0003
O
Figure imgf000128_0004
Figure imgf000128_0005
O
Figure imgf000129_0001
o
Figure imgf000129_0002
Figure imgf000129_0003
O
Figure imgf000129_0004
O
Figure imgf000129_0005
O
Figure imgf000130_0001
Figure imgf000130_0002
8. A compound of Claim 6, or pharmaceutically acceptable salt thereof, selected from the group consisting of
Figure imgf000130_0003
Figure imgf000130_0004
Figure imgf000131_0001
Figure imgf000131_0002
Figure imgf000131_0003
Figure imgf000131_0004
Figure imgf000131_0005
Figure imgf000132_0001
O
Figure imgf000132_0002
O
Figure imgf000132_0003
O
Figure imgf000132_0004
O
Figure imgf000133_0001
O
Figure imgf000133_0002
O
Figure imgf000133_0003
O
Figure imgf000133_0004
O
Figure imgf000133_0005
O , and
Figure imgf000134_0001
9. A compound of Claim 7, or pharmaceutically acceptable salt thereof, selected from the group consisting of
Figure imgf000134_0002
10. A compound of Claim 8, or pharmaceutically acceptable salt thereof, selected from the group consisting of
Figure imgf000134_0003
Figure imgf000135_0001
11. A composition for inhibiting thrombus formation in blood comprising a compound of Claim 1 and a pharmaceutically acceptable caπier.
12. A method for inhibiting thrombin in blood comprising adding to the blood a composition of Claim 11.
13. A method for inhibiting formation of blood platelet aggregates in blood comprising adding to the blood a composition of Claim 11.
14. A method for inhibiting thrombus formation in blood comprising adding to the blood a composition of Claim 11.
15. 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.
16. A method for treating or preventing venous thromboembolism and pulmonary embolism in a mammal comprising administering to the mammal a composition of Claim 11.
17. N method for treating or preventing deep vein thrombosis in a mammal comprising administering to the mammal a composition of Claim 11.
18. N method for treating or preventing thromboembolic stroke in humans and other mammals comprising administering to the mammal a composition of Claim 11.
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6515011B2 (en) 2000-12-18 2003-02-04 Merck & Co., Inc. Thrombin inhibitors
US6528503B2 (en) 2000-12-18 2003-03-04 Merck & Co., Inc. Thrombin inhibitors
US7144899B2 (en) 2001-02-09 2006-12-05 Merck & Co., Inc. Thrombin inhibitors
US7262211B2 (en) 2001-12-04 2007-08-28 Dendreon Corporation Aromatic heterocyclic non-covalent inhibitors of urokinase and blood vessel formation
DE102008041214A1 (en) 2008-08-13 2010-02-18 Bayer Cropscience Ag New N-substituted azinylalkyl-azincarboxamide compounds useful e.g. in plant protection, for combating animal pests, arthropods, nematodes and plant diseases, and for protecting industrial materials against attack and destruction by insect
US7666868B2 (en) 2005-03-15 2010-02-23 4Sc Ag N-sulphonylpyrroles and their use as histone deacetylase inhibitors
EP2253612A1 (en) 2005-04-14 2010-11-24 Novartis AG Organic compounds
US7842820B2 (en) 2004-03-11 2010-11-30 4Sc Ag Sulfonylpyrroles
US8188138B2 (en) 2005-09-21 2012-05-29 4Sc Ag Sulphonylpyrrole hydrochloride salts as histone deacetylases inhibitors
US8232297B2 (en) 2005-09-21 2012-07-31 4Sc Ag Sulphonylpyrroles as inhibitors of HDACs novel sulphonylpyrroles
CN103497149A (en) * 2013-10-08 2014-01-08 南京复兴生物科技有限公司 Preparation method of 5-aminomethyl pyridine derivative

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7119094B1 (en) * 2000-11-20 2006-10-10 Warner-Lambert Company Substituted polycyclic aryl and heteroarpyl pyrazinones useful for selective inhibition of the coagulation cascade
US6610701B2 (en) * 2001-02-09 2003-08-26 Merck & Co., Inc. Thrombin inhibitors
DE102004022897A1 (en) * 2004-05-10 2005-12-08 Bayer Cropscience Ag Azinyl-imidazoazines

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997040024A1 (en) * 1996-04-23 1997-10-30 Merck & Co., Inc. Pyrazinone thrombin inhibitors
WO1998042342A1 (en) * 1997-03-24 1998-10-01 Merck & Co., Inc. Thrombin inhibitors
WO1999011267A1 (en) * 1997-09-05 1999-03-11 Merck & Co., Inc. Pyrazinone thrombin inhibitors

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3775686D1 (en) 1986-09-26 1992-02-13 Ciba Geigy Ag AMINOPYRAZINONE AND AMINOTRIAZINONE.
TW201303B (en) 1990-07-05 1993-03-01 Hoffmann La Roche
GB9207145D0 (en) 1991-04-18 1992-05-13 Ici Plc Heterocyclic amides
JP2599665B2 (en) 1991-05-01 1997-04-09 大塚製薬株式会社 Pyrazine derivatives
DE4127404A1 (en) 1991-08-19 1993-02-25 Thomae Gmbh Dr K CYCLIC IMINODERIVATES, MEDICAMENTS CONTAINING SUCH COMPOUNDS AND METHOD FOR THE PRODUCTION THEREOF
TW223629B (en) 1992-03-06 1994-05-11 Hoffmann La Roche
US5672582A (en) 1993-04-30 1997-09-30 Merck & Co., Inc. Thrombin inhibitors
WO1994025051A1 (en) 1993-04-30 1994-11-10 Merck & Co., Inc. Thrombin inhibitors
US5510369A (en) 1994-07-22 1996-04-23 Merck & Co., Inc. Pyrrolidine thrombin inhibitors
JPH11503161A (en) 1995-04-04 1999-03-23 メルク エンド カンパニー インコーポレーテッド Thrombin inhibitor
US5629324A (en) 1995-04-10 1997-05-13 Merck & Co., Inc. Thrombin inhibitors
CA2224437A1 (en) 1995-06-27 1997-01-16 Merck & Co., Inc. Pyridinone-thrombin inhibitors
US5668289A (en) 1996-06-24 1997-09-16 Merck & Co., Inc. Pyridinone thrombin inhibitors
US5981546A (en) 1996-08-29 1999-11-09 Merck & Co., Inc. Integrin antagonists
JP2001508796A (en) 1997-01-22 2001-07-03 メルク エンド カンパニー インコーポレーテッド Thrombin inhibitors
US5866573A (en) 1997-04-21 1999-02-02 Merck & Co., Inc. Pyrazinone thrombin inhibitors
WO1999059591A1 (en) 1998-05-19 1999-11-25 Merck & Co., Inc. Pyrazinone thrombin inhibitors
ES2240100T3 (en) 1999-05-19 2005-10-16 Pharmacia Corporation ARIL- AND HETEROARIL-PIRAZINONAS SUBSTITUTED POLICICLICAS FOR THE SELECTIVE INHIBITION OF THE COAGULATION WATERFALL.

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997040024A1 (en) * 1996-04-23 1997-10-30 Merck & Co., Inc. Pyrazinone thrombin inhibitors
WO1998042342A1 (en) * 1997-03-24 1998-10-01 Merck & Co., Inc. Thrombin inhibitors
WO1999011267A1 (en) * 1997-09-05 1999-03-11 Merck & Co., Inc. Pyrazinone thrombin inhibitors

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6528503B2 (en) 2000-12-18 2003-03-04 Merck & Co., Inc. Thrombin inhibitors
US6515011B2 (en) 2000-12-18 2003-02-04 Merck & Co., Inc. Thrombin inhibitors
US7144899B2 (en) 2001-02-09 2006-12-05 Merck & Co., Inc. Thrombin inhibitors
US7262211B2 (en) 2001-12-04 2007-08-28 Dendreon Corporation Aromatic heterocyclic non-covalent inhibitors of urokinase and blood vessel formation
US8399505B2 (en) 2004-03-11 2013-03-19 4Sc Ag Sulfonylpyrroles
US8969401B2 (en) 2004-03-11 2015-03-03 4Sc Ag Sulfonylpyrroles
US7842820B2 (en) 2004-03-11 2010-11-30 4Sc Ag Sulfonylpyrroles
US8067615B2 (en) 2004-03-11 2011-11-29 4Sc Ag Sulphonylpyrroles
US7666868B2 (en) 2005-03-15 2010-02-23 4Sc Ag N-sulphonylpyrroles and their use as histone deacetylase inhibitors
US8815855B2 (en) 2005-03-15 2014-08-26 4Sc Ag N-sulphonylpyrroles and their use as histone deacetylase inhibitors
EP2253612A1 (en) 2005-04-14 2010-11-24 Novartis AG Organic compounds
US8232297B2 (en) 2005-09-21 2012-07-31 4Sc Ag Sulphonylpyrroles as inhibitors of HDACs novel sulphonylpyrroles
US8188138B2 (en) 2005-09-21 2012-05-29 4Sc Ag Sulphonylpyrrole hydrochloride salts as histone deacetylases inhibitors
DE102008041214A1 (en) 2008-08-13 2010-02-18 Bayer Cropscience Ag New N-substituted azinylalkyl-azincarboxamide compounds useful e.g. in plant protection, for combating animal pests, arthropods, nematodes and plant diseases, and for protecting industrial materials against attack and destruction by insect
CN103497149A (en) * 2013-10-08 2014-01-08 南京复兴生物科技有限公司 Preparation method of 5-aminomethyl pyridine derivative

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JP2003514905A (en) 2003-04-22

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