CA2245811A1 - Pyridinone thrombin inhibitors - Google Patents

Abstract

Compounds of the invention are useful in inhibiting thrombin and associated thrombotic occlusions and have structure (I), for example (II).

Description

W O 97/30708 PCTrUS971U2408 . TITLE~ OF THE INVENTION
PYRIDINONE THROMBlN ~HIBITORS

BA(~KGROUND 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 mech~nism 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 or~-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analogs of peptidase substrates in which the nitrogen atom of the scissile amide group of the substrate peptide has been replaced by hydrogen or a substituted carbonyl moiety.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 hllm~n leukocyte elastase which contain trifluoromethylketone and pyridinone moieties.
H. Mack et al., J. Enzyme lnhibition, Vol. 9, pages 73-86 (1995~ describes rigid amidino-phenyl~l~nine thrombin inhibitors which contain a pyridinone moiety as a central core structure.

SUMMARY OF THE INVENTION
The invention includes a composition for inhibiting loss of " 30 blood platelets, inhibiting formation of blood platelet aggregates,inhibiting formation of fibrin, inhibiting thrombus formation, and inhibiting embolus formation in a m~mm~l, comprising a compound of the invention in a pharmaceutically acceptable carrier. These compositions may optionally include anticoagulants, antiplatelet agents, -and thrombolytic agents. The compositions can be added to blood, blood f products, or m:~mm~ n organs in order to effect the desired inhibitions.
The invention also includes a composition for preventing or treating unstable angina, refractory ~n~in~, myocardial infarction, S transient ischemic attacks, atrial ~lbrillation, thrombotic stroke, embolicstroke, deep vein thrombosis, dissemin~ted intravascular coagulation, ocular build up of fibrin, and reocclusion or restenosis of rec~n~li7ed vessels, in a m~mms~l, comprising a compound of the invention in a pharmaceutically acceptable carrier. These compositions may optionally include anticoagulants, antiplatelet agents, and thrombolytic agents.
The invention also includes a method for reducing the thrombogenicity of a surface in a m?~mm~l by attaching to the surface, either covalently or noncovalently, a compound of the invention.
The invention also includes the use of a compound of the invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting th~ombus formation, preventing thrombus formation, inhibiting thrombin, inhibiting formation of fibrin, and inhibiting formation of blood platelet aggregates, in a m~mm~l 2{) Some abbreviations that may appear in this application are as follows.

ABBREVlLATIONS

Designation Protecting Group BOC (Boc) t-butyloxycarbonyl CBZ (Cbz) benzyloxycarbonyl(carbobenzoxy) TBS (TBDMS) t-butyl-dimethylsilyl Activating Group l~BT(HOBT or HOBt) l-hydroxybenzotriazole hydrate W O 97/30708 PCTrUS97/02408 Designation Coupling Rea~ent BOP reagent benzotriazol-l-yloxytris-(dimethylamino)phosphonium hexafluorophosphate BOP-Cl bis(2-oxo-3-oxazolidinyl)phosphinic chloride EDC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride Other (BOC)20 (130C20) di-t-butyl dicarbonate n-Bu4N+F- tetrabutyl amrnonium fluoride nBuLi (n-Buli) n-butyllithillm DMF dimethylform~mide Et3N (TEA) triethylamine EtOAc ethyl acetate TFA trifluoroacetic acid DMAP dimethylaminopyridine DME dimethoxyethane NMM N-methylmorpholine DPPA diphenylphosphoryl azide THF tetrahydrofuran DIPEA diisopropyl~mine Amino Acid Ile Isoleucine Phe Phenyl~l~nint Pro Proline " Ala ~l~nine Val Valine W O 97/30708 PCT~US97/02408 DETAILED DESCRIPTION OF THE INVENTrION ANlD
PREFERRED EMBODIMENTS
Compounds of the invention are useful as thrombin inhibitors and have therapeutic value in for exarnple, preventing coronary 5 artery disease, and have the following structure:
R3~ R30 R4 ' N ~N~ N

wherein wis 1 0 hydrogen, Rl-, RlOC(O)-, RlC(O)-, RlS02-, RlNHS02-, (R 1 )2NSO2-, R 1 (CH2)nNHC(O)-, (R 1 )2CH(CH2)nNHC(O)-, or wherein n is 0-4;
Rl is R2(CH2)r-, where r is 0-4, (R2)(0R2)CH(CH2)p-, where p is 1-4, (R2)2CH(CH2)r-, where r is 0-4 and each R2 can be the same or different, and wherein (R2)2 can also form a ring with CH
represented by C3 7 cycloalkyl, C7_l2 bicylic alkyl, C:~10-16 tricylic aLkyl, or a 5- to 7- membered mono- or bicyclic W O 97/30708 PCT~US97/~2408 heterocyclic ring which can be saturated or Im~:ltllrated, and which contains from one to three heteroatoms selected from the group consisting of N, O and S, R20(CH2)p-, wherein p is 1-4, or R2(COOR3)(CH2)r-, where r is 1-4;

R2 and R14 are independently selected from -phenyl, unsubstituted or substituted with one or more of Cl 4 aLkyl, Cl 4 aLkoxy, halogen, hydroxy, COOH, or CONH2, 1 0 naphthyl, biphenyl, a ~- to 7- membered mono- or a 9- to 10-membered bicyclic heterocyclic ring which can be saturated or lm~ lrated, and which contains from one to four heteroatoms selected from the ~5 group consisting of N, O and ~, C 1-4 aLkyl, C3 7 cycloalkyl, C7 12 bicyclic aLkyl, or C10 16 tricyclic aLkyl, R3 and R3 are independently Cl 4aLkyl, C3 7 cycloalkyl, or trifluoromethyl;
2~
R4 is hydrogen, Cl 4 aLkyl, Cl 4aLkoxy, halogen, -OCH2CF3, -COOH, -OH, -COOR6, where R6 is Cl 4aLkyl, W O 97f30708 PCTnUS97/02408 -CoNR7R8, where R7 and R8 are independently hydrogenorCl 4aLkyl, -(CH2) 1-40H, -CH2NHC(O)CH3, -CH2NHC(O)CF3, -CH2NHS02CH3, -S02NH2, -(CH2)1 4So2NR7R8 -(CH2)1 4S02R6, a 5- to 7- membered mono- or a 9- to 1 O-membered bicyclic heterocyclic ring which can be saturated or unsaturated, and which contains from one to four heteroatoms selected from the group consisting of N, O and S, -XCH2C02H, -XCH2C02CH3, -XCH2C02(CH2)1 3CH3, -X(CHR9)1 3C(O~NRlOR
wherein R9 is H or Cl 4 alkyl, 2~ R 1 0 and R l 1 are independently hydrogen, C3 7 cycloaLkyl, aryl, heteroaryl, 2~ heterocycloaLkyl, Cl 4 aLkyl unsubstituted or substituted with one or more of hydroxy, COOH, amino, diaLkylamino, aryl, heteroaryl, or heterocycloaLkyl, or R 10 and R 1 1 are joined to form a four to seven CA 022458il 1998-08-10 W O 97/30708 PCTrUS97/02408 membered cycloaLkyl ring unsubstituted or substituted with hydroxy, amino or aryl, or -XCH2R 1 4, wherein X is 0, S or CH2;

R5 is hydrogen, halogen, Cl 4aLkyl, C1 4 alkoxy, CN, or C~02NH2, and ph~rm~ceutically acceptable salts thereof.
In one class of compounds and ph~ ceutically acceptable salts thereof, W is Rl or RlS02.
In a subclass of this class of compounds and pharmaceutically acceptable salts thereof, Rl is R2(CH2)r, or (R2)2CH(CH2)r, phenyl-CH2S02, or diphenyl-CHS02.
In a group of this subclass of compounds and pharmaceutically acceptable salts thereof, R 1 is phenyl-CH2S02, or diphenyl-CHS02-In a subgroup of this group of compounds and pharmaceutically accepta~le salts thereof, R3 is C 1-4 alkyl.
In a family of this subgroup of compounds and pharmaceutically acceptable salts thereof, R3 is methyl and R3 is hydrogen.
In a subfamily of this family of compounds and pharmaceutically acceptable salts thereof, R4 is hydrogen, chlorine, OH, W O 97r30708 PCT/US97/02408 OCH2C(0)NH2 OCH2C(O)NHCH2CH3, or OCH2C~O)NH(CHCH2CH2); amd R5 is chlorine.
Specific embodiments of this subfamily include (note that the methyl group is conventionally indicated as a single bond attached to a ring~:
1~

~,S~ N J~'~ N

~ s'NJ~N~

Cl W O 97/30708 PCTrUS97/02408 o '13 '~S'N~N/'~¢J

HJ~ NH
Cl --N ~N/~JI--N~ ~

Cl The compounds of the present invention, may have chiral 10 centers and occur as racemates, racemic mixtures and as individual diastereomers, or enantiomers with all isomeric forms being included in the present invention. A racemate or racemic mixture does not imply a 50:50 mixture of stereoisomers.

W 097/30708 PCTnUS97/02408 When any variable occurs more than one time in any constituent or in formula I, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such 5 combinations result in stable compounds.
As used herein except where noted, "aLkyl" is intended to include both branched- and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms (Me is methyl, Et is ethyl, Pr is propyl, 13u is butyl); "aLkoxy" represents a linear or branched 10 alkyl group of indicated number of carbon atoms attached through an oxygen bridge; "Halo", as used herein, means lluoro, 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, 1~ hemitartrate, benzene sulfonate, and the like.
The term "C3 7cycloa~cyl" is intended to include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, and the like.
The term "C7 12 bicyclic aLkyl" is intended to include 20 bicyclo[2.2. 1 ]heptyl (norbornyl) , bicyclo[2.2.2]octyl, 1,1 ,3-trime~yl-bicyclo[2.2.1]heptyl ~bornyl~, 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 unsubstih~te-l or substituted with one or 25 more of C1-4 lower aLkyl; hydroxy; aLkoxy; halogen; amino.
The term "heterocycler' or "heterocyclic ring", as used herein except where noted, represents a stable 5- to 7-membered mono- or bicyclic or stable 7- to 10-membered bicyclic heterocyclic ring system any ring of which may be saturated or unsaturated, and which consists of 30 carbon atoms and from one to four heteroatoms selected from ~e group consisting of N, O and ~, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring.

CA 0224~811 1998-08-10 W O 97/30708 PCT~US97/02408 - Especially useful are rings cont~ining one oxygen or sulfur, one to four nitrogen atoms, or one oxygen or sulfur combined with one or two nitrogen atoms. I~he heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure.
Examples of such heterocyclic groups include piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, 10 morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, quirluclidinyl, isothiazolidinyl, indolyl, quinolinyl, isocluinolinyl, benzimidazolyl, thiadiazoyl, benzopyranyl, benzothiazolyl, benzoxazolyl, furyl, tetrahydrofuryl, tetrahydropyranyl, tetrazole, thienyl, benzothienyl, th~amorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, and 15 oxadiazolyl. Morpholino is the same as morpholinyl.
The ph~ ceutically-acceptable salts of the compounds of Formula I (in the form of water- or oil-soluble or dispersible products) include the conventional non-toxic salts or the quaternary ammonium salts which are formed, e.g., from inorganic or organic acids or bases.
20 Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium salts, aL~ali metal salts such 30 as sodium and potassium salts, ~lk~linP 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-cont~ining groups may be quaternized with such agents as lower allcyl halides, such as W O 97130708 PCT~US97/02408 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, araL~cyl 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 10 readily aware of the circumstances requiring anticoagulant therapy. The term "patient" used herein is taken to mean m~mm~l~ such as primates, including hllm~n~, 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 15 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 cont~inin~ or suspected of cont~ining thrombin and in which it is desired that blood coagulation be 20 inhibited, e.g. when contacting the mz~mm~l's blood with material selected from the group consisting of vascular grafts, stents, olthopedic prosthesis, cardiac prosthesis, and extracorporeal circulation systems The thrombin inhibitors of the invention can be ~ ini~tered in such oral forms as tablets, capsules (each of which includes sustained 2~ release or timed release formulations), pills, powders, granules, elixers, tinctures, suspensions, syrups, and emulsions. Likewise, they may be ~lmini.~tered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or i~ scular form, all using folms well known to those o~ ordinary skill in the pharmaceutical arts. An effective but non-toxic 30 amount of the compound desired can be employed as an anti-aggregation agent. For treating ocular build up of fibrin, the compounds may be :~flmini~tered intraocularly or topically as well as orally or parenterally.
The thrombin inhibitors can be ~lmini~tered in the form of a depot injection or implant preparation which may be formulated in such a CA 0224~811 1998-08-lO
W O 97/30708 PCTrUS97/02408 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, ~ilastic, silicone rubber or other polymers manufactured by the Dow-Corning Corporation.
The thrombin inhibitors can also be a-lmini~tered in the form of liposome delivery systems, such as small unilamellar vesicles, large lmil~mellar vesicles and multi~amellar vesicles. Liposomes can be 10 formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
The thrombin inhibitors may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The thrombin inhibitors may also be coupled 15 with soluble polymers as targetable drug carriers. Such polymers can include polyvinlypyrrolidone, pyran copolymer, polyhydroxy-propyl-methacrylamide-phenol, polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues.
Furthermore, the thrombin inhibitors may be coupled to a class of 20 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 lltili7in~ 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 ~lmini~tration; the renal and hepatic f~nction of the patient; and the particular compound or salt thereof 30 employed. An ordinarily skilled physician or veterinarian can readily determ~ne and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.
Oral dosages of the thrombin inhibitors, when used for the indicated effects, will range between about 0.1 mg per kg of body weight W O 97130708 PCTrUS97/02408 per day (mgfkg/day) to about 100 mg/lcg/day and preferably 1.0-100 mglkg/day and most preferably 1-20 mg/kg/day. A suitably prepared medicament for once a day ~flmini.~tration would thus contain between 9 mg and 9 g, preferably between 90 mg and 9 g, and most preferably between 90 mg and 1.8 g, e.g. lOOmg, 500 mg and 1 g. Advantageously, the thrombin inhibitors may be ~flminictered in divided doses of two, three, or four times daily. Por such multiple ~lm;ni~tration, individual medicament strengths can be, for example, 25 mg, 33 mg or 50 mg, .
Intravenously, the most preferred doses will range from about 0.01 to 10 about 10 mg/kg/minute during a constant rate infusion. Furtherrnore, they can be ~lmini~tered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transde~nal skin patches well known to those of ordinary skill in that art.
To be ~lmini~tered in the forrn of a transdermal delivery system, the 1~ dosage ~lmini~tration will, or course, be continuous rather than intermittent throughout the dosage regime.
The thrombin inhibitors are typically ~dmini~tered as active ingredients in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier"
20 materials) suitably selected with respect to the intended form of ~tlrnini~tration, that is, oral tablets, capsules, elixers, syrups and the like,and consistent with convention pharmaceutical practices.
For instance, for oral ~llmini~tration in the form of a tablet or capsule, ~e active drug component can be combined with an oral, non-25 toxic, pharmaceutically acceptable, inert carrier such as lactose, starch,sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, m~nnitol, sorbitol and the like; for oral a-lmini~tration in liquid form, the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as 30 ethanol~ glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, distintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn-sweeteners, natural and synthetic gums such as acacia, tragacanth or W O 97/30708 PCTrUS97102408 sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodilLm stearate, m;~gn~sium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limi~tion, 5 starch methyl cellulose, agar, bentonite, x~nth~n gum and the like.
The thrombin inhibitors can also be co-~-lministered with suitable anti-coagulation agents, including, but not limited to, fibrinogen receptor antagonists, heparin, aspirin, or warfarin, or thrombolytic agents such as pl~sminogen activators or streptokinase to achieve synergistic 10 effects in the treatment of various ascular pathologies. For example, thrombin inhibitors enhance the efficiency of tissue pl~sminogen activator-mediated thrombolytic reperfusion. Thrombin inhibitors may be ~tlministered first following thrombus formation, and tissue pl~sminogen activator or other pl~minogen activator is ~lministered 1 5 thereafter.

METHODS OF MAKlNG

The following synthetic methods can be used to prepare the 20 compounds of the present invention:

As exemplified by Example 1 (Scheme 1).
Starting 6-methyl-2-hydroxy-pyridine-3-carboxylic acid is reacted with diphenylphosphoryl azide (DPPA) and benzyl alcohol in 2S Step A to afford the protected aminopyridine. This is aLkylated in Step B
with t-butylbromoacetate and the CBZ group is removed in Step C by hydrogenation over a catalyst. The resulting amine is then reacted with the a~roL~liate reagent, in this case benzylsulfonyl chloride, in Step D
with pyridine as an acid scavenger and the t-butyl ester is then removed 30 under acidic conditions in Step E. The acid is then coupled in Step F
with an amine, such as 2,S-dichlorobenzylamine, to afford the final product.

W O 9713Q708 PCTrUS97/02408 DPPA, BrCH2CO2tBu HO2&~ Et3N, BnOH CbzNH~ NaH, THF

HO N StepA HO N Step B
CbzN H H2N
~ H2, Pd(OH)2/C ~
O~NJ~ EtOH, H20 O~N~\
~~'1< Step C ~~~1<

l~h H
S - N~ BnSO2CI, Pyridine N Step D
~ ~<
Step E ¦ HCI, EtOAc ~2 ~3~ DCC, amin, CHzC12 o ~ O &l Amide couplings, e.g., Step F in Scheme 1, to form the S compounds of this invention can be performed by the carbodiimide method with reagents such as dicyclohexylcarbodiimide, or l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide. Other methods of forming the amide or peptide bond include, but are not limited to the synthetic routes via an acid chloride, azide, mixed anhydride or activated ester.

W O 97/30708 PCT~US97/02408 Typically, solution phase amide couplings are performed, but solid-phase synthesis by classical Merrifield techniques may be employed instead.
The addition and removal of one or more protecting groups is also typical practice.
S Modifications of the method will allow different W, R3, R4 and R5 groups contemplated by the scope of the broad claim below to be present by the use of an a~l~ro~iate reagent or ~ iately substituted starting material in the indicated synthetic step. For example the starting pyridine in Step A, can have as the 6-substituent, ethyl, isopropyl, cyclopropyl, trifluoromethyl, and the like, to achieve the different operable values of R3. Likewise, different W groups can be present by the use of an appropriate aLkylating, carbonylating, sulfonylating, ure~l~ting agent, and the like, in Step D. Use of, for example, an aLkyl halide, aLkoxycarbonyl halide, acyl halide, alkylsulfonyl halide, or alkyl 1~ isocyanate will yield the corresponding values of W where W is Rl, RlOCO, R1CO, R1S02, or (R1)m(CH2)nNCO. Likewise, an appropriate choice of the amine in Step F will allow the different operable values of R4 and R5 to be achieved. Obvious variations and modifications of the method to produce similar and obvious variants 2() thereo~, will be apparent to one skilled in the art.
A method for preparing compounds which c~-nt~in, or are derivatives of, 2-hydroxybenzylamine as an end group is illustrated in Scheme 2 and is exemplified by Example 2.
4-Chlorosalicaldehyde is condensed with hydroxylamine hydrochloride in ethanolic aqueous sodium carbonate solution. The oxime is reduced by hydrogenation over a catalyst such as rhodium and the amine is protected as its BOC derivative under standard conditions.
The phenol is aLkylated in Step D with an acetate equivalent such as ethylbromoacetate and the resulting ester is hydrolysed with lithium 3~) hydroxide. The product carboxylic acid is coupled to an amine such as ethylamine in Step F, and the E~OC group is removed by strong acid. The amine is coupled to the product of Scheme 1, Step E to give the final product.

WO 97/30708 PCTrUS97/02408 Cl Cl NH20H.HCI ~ H2, Rh/C
ll INa2CO37 EtOH/H20 ll I EtOH, H2SO4 OHC~ Step A ~ Step B
OH Ho~N OH

J~ Step C ~¢~ Cs2CO37 D'I~F Step D

Cl Cl Step E f~
CNH O~CO2H BOCNH O~,CO2Et Step F I EDC, EtNH2.HCI
HOBT, NMM, DMF
Cl Cl TFA, CH2cl2 Ih CH2CI2 Step G ~ Step H
CNH O~CONHEt NH2 O~CONHEt FnSO2NH ~ Cl O~N~ H

O ~0 CONHEt CA 0224~811 1998-08-10 W O 97130708 PCT~US97/02408 Modifications of this method will allow different R4 and R5 groups contemplated by the scope of the broad claim below to be present by ~e use of an appropriate reagent or appropriately substituted starting material in the indicated synthetic step. For example, appropriate choice of the amine in Step F will allow different values of R7 and R8 to be achieved. Obvious variations and modifications of the method to produce similar and obvious variants thereof, will be apparent to one skilled in the art.
A method for preparing compounds which are derivatives 10 of 3-amino-4-trifluoromethyl-2-pyridinone is illustrated in Scheme 3.
In Step A, 1,1,1, trifluoro-2,4-pentanedione is condensed with nitroacetarnide in ethanolic diethylamine to give 2-hydroxy-6-methyl-3-nitro-4-trifluoromethylpyridine. This is aLkylated in Step B with an acetate equivalent such as t-butyl bromoacetate in THF using sodium 15 hydride as a base and the nitro group is reduced in Step C by hydrogenation using a catalyst such as palladium on carbon. The t-butyl group is removed in Step D by a strong acid such as HCl and the resulting carboxylic acid is coupled to ethyl-2-aminomethyl-4-chlorophenoxyacetamide in Step E to give the final product.
2{) Modifications of the method will allow different R3, R4 and R5 groups contemplated by the scope of the broad claim below to be present by the use of an appropriate reagent or ~ropliately substituted starting material in the indicated synthetic step. For example use of the appropriate dione in Step A will allow the different 25 operable values of R3 to be achieved. Similarly an appropriate choice of the amine in Step E will allow the different operable values of R4 and R5 to be achieved. Obvious variations and modifications of the method to produce similar and obvious variants thereof, will be apparent to one skilled in the art.

W O 97130708 PCTrUS97/Q2408 CF3 NO2cH2cONH2 CF3 BrCH2CO2tBu 0~ Et2N, EtOH 02N~ NaH, THF
O step A HO NJ\ step B

02N~ H2, Pd/C H2N~ HCI, EtOAc O N O N step D
~0~< stepC ~O

H2N~ EDC, HOBT H2N~ Cl O step E ~N ~

CONHEt The following examples are illustrative of the invention as 5 contemplated by the inventors and should not be construed as being limits ~n the scope or spirit of the instant invention.

~ =

W O 97/30708 PCT~US97/02408 Preparation of 3-Benzylsulfonylamino-6-methyl-1-(methylene-carboxamidomethyl-2~5-dichlorophenyl)-2-pyridinone ~ N~~ N

Cl Step A: 3-Benzyloxycarbonylamino-6-methyl-2-pyridinone DPPA (35.6 ml, 165 mmol) was added to a stirred solution of 2-hydroxy-6-methylpyridine-3-carboxylic acid (22.97 g, 165 mmol) and triethyl~mine (23.0 ml, 165 mmol) in dry dioxane (300 ml) and the resulting solution was heated to reflux. After 16 h more triethyl~mine (23.0 ml, 165 mmol) and benzyl alcohol (17.1 ml, 165 mmol) were added and the solution was refluxed for a further 24 h. The reaction was concentrated in vacuo to remove most of the volatiles. The residue was partitioned between methylene chloride (500 ml) and brine (500 ml), acidified to pH 1 with 1 M HCl (165 ml). The aqueous layer was extracted methylene chloride (two times) and the combined organic layers were washed with sodium hydrogen carbonate solution and brine, dried (Na2SO4) and evaporated in vacuo to a brown solid. This was recrystallized from methanol, to give the title compound as a tan solid:
lH NMR (300 MHz, CDC13) ~ 2.29 (s, 3H, CH3), 5.20 (s, 2H, PhCH2), 6.06 (d, J=7.6 Hz, pyridinone-5-H), 7.32-7.43 (m, SH, Ph), 7.67 ~br s, lH, CbzNH), 8.03 (br d, pyridinone-4-H).

Step B: 3-Benzyloxycarbonylamino-6-methyl-1-(t-butyl-methylenecarboxy~-2-pyridinone Sodium hydride (5.3 g, 0.22 mol) was added to a stirred slurry of 3-benzyloxycarbonylamino-6-methyl-2-pyridinone (53.2 g, 0.20 mol) at 0~C. t-Butylbromoacetate ~45 ml, 0.27 mol) was added to the resulting solution and a precipitate rapidly forms. The reaction was ..

W O 97130708 PCTrUS97/02408 warmed to rt over lh and after 2h ~e solvent was evaporated in vacllo and the residue was partitioned between 1: 1 water/brine (200 ml) and 6: 1 THF/methylene chloride (700 ml). The organic layer was dried (Na2SO4) and evaporated in vacuo to a solid which was triturated with S hexane to give the title compound as a crystalline solid:
lH NMR (400 Mz, CDC13) o 1.47 (s, 9H), 2.25 (s, 3H), 4.75 (s, 2 H), 5.19 (s, 2H), 6.Q9 (d, J=7.8 Hz), 7.30-7.40 (m, SH), 7.75 (br s, lH), 7.94 (br d, lH).
~0 Ste~ C: 3-Amino-6-methyl-1-(t-butyl-methylenecarboxy)-2-pyridinone A mixture of 3-benzyloxycarbonyla~nino-6-methyl-1-(t-butyl-methylenecarboxy)-2-pyridinone (5.59 g, 15.0 mmol) and Pearlman's catalyst (0.56 g) in 4: 1 ethanol/water (200 ml) was shaken in a 15 Parr apparatus under H2 (S0 psi) for 2 h. The reaction ~ni~lur~ was filtered through celite and evaporated in vacuo, azeotroping with ethanol to give the title compound as a solid:
lH NMR (400 Mz, CDCl3) o 1.46 (s, 9H, t-Bu), 2.18 (s, 3H, Me), 4.02 (br s, 2H, NH2~, 4.74 (s, 2H, CH2), S.90 (d, J=7.3Hz, 1 H, pyridinone H-20 S), 6.47 (d, J=7.3 Hz, lH, pyrid~none H-4).

Step D: 3-Benzylsulfonylamino-6-methyl- 1 -(t-butyl-met~ylenecarboxy)-2-pyridinone Benzylsulfonyl chloride (3.15 g, 16.5 mmol) was added to a 25 stirred solution of 3-amino-6-me~yl-1-(t-butyl-methylenecarboxy)-2-pyridinone (3.55 g, 14.9 mmol) in pyridine (30 ml) at 0~C and as the resulting solution was st~lTed a thick precipitate formed. After lh the reaction mixture was evaporated in vacuo to a paste which was partitioned between methylene chloride and 10% potassium hydrogen 3~) sulfate solution. The organic layer was dried (Na2S04) and evaporated in vacuo to give the title compound as a pale pink solid:
1H NMR (400 Mz, CDC13) ~ 1.51 (s, 9H, t-Bu), 2.26 (s, 3H, Me), 4.31 (s, 2H, PhCH2), 4.75 (s, 2H, NCH2), 6.01 (d, J=7.7Hz, lH, pyridinone H-5), 7.22-7.34 (m, 7H, rem~inin~ H).

W O 97/30708 PCTrUS97/02408 Step E: 3-Benzylsulfonylamino-6-methyl-1-methylenecarboxy-2-pyridinone HCl gas was bubbled through a stirred suspension of 3-benzylsulfonylamino-6-methyl- 1 -(t-butyl-methylenecarboxy)-2-pyridinone (5.70 g, 14.52 mmol) in ethyl acetate (60 ml) at 0~C lmtil a solution had formed which was saturated with HCl. After 1.5 h at RT a thick suspension had formed. The mixture was degassed with nitrogen and filtered to give the title compound as a solid:
lH NMR (400 Mz, CD30D) ~i 2.32 (s, 3H, Me), 4.43 (s, 2H, PhCH2), 4.89 (s, 2H, NCH2)~ 6.14 (d, J=7.7Hz, lH, pyridinone H-5), 7.28-7.33 (m, 6H, rem~ininf~ H).

Step F: 3-Benzylsulfonylamino-6-methyl-1-(N-2,5-Dichloro benzyl -acetamido)-2-pyridinone EDC Hydrochloride (48 mg, 0.25 mmol) was added to a stirred mi~ul~ of 3-benzylsulfonylamino-6-methyl-1-methylenecarboxy-2-pyridinone (70 mg, 0.21 mmol), HOBT (34 mg, 0.25 mrnol), 2,5-dichlorobenzylamine (44 mg, 0.25 mmol) and triethykqmine (0.070 ml, 0.50 mmol) in DMF (2.5 ml) and the mixture was stirred for 16 h. The reaction was diluted with ethyl acetate and was washed with brine, dried (Na2S04) and evaporated in vacuo to a solid. The crude product was purified by flash column chromatography on silica (50%
ethylacetate/hexanes), to give the title compound as a crystalline solid:
lH NMR (400 Mz, CDC13) ~i 2.43 (s, 3H, Me), 4.30 (s, 2H), 4.49 (d, J=6.1Hz, 2 H, CH2N), 4.68 (s, 2H), 6.05 (d, J=7.7Hz, lH), 7.16 (dt, =2.4 and 8.9 Hz, 1 H), 7.24-7.40 (m, 8H); MS (FAB) 494 (M+l)+-~XAMPLE II

Preparation of 3-Benzylsulfonylamino-6-methyl-1-[Ethyl-(2-methylenecarboxamidomethyl -4-chlorophenoxy)-acetamido] -2-S pyridinone ~ HJ~N

Step A: 4-Chlorosalicaldehyde Oxime A solution of hydroxylarnine hydrochloride (16.7 g, 0.24 mol) and sodium carbonate (12.7 g, 0.12 mol) in water (120 ml) was added to a stirred solution of 4-chlorosalicaldehyde (25.0 g, 0.16 mol) in ethanol (160 ml) and the resulting solution was heated to reflux. After 1 h the reaction was cooled, water (320 rnl) was added and the resulting crystalline precipitate was isolated by filtration. A second crop was similarly collected and the combined solids were dried to give the title 15 compound:
H NMR (400 Mz, CD(~13) ~ 6.92 (d, J=8.8Hz, 1 ~), 7.15 (d, J=2.6Hz, lH), 7.23 (dd, J=2.6 and 8.8 Hz, lH), 7.26 (s, lH), 8.16 (s, lH), 9.71 (s, lH).

20 Step B: 2-Hydroxy-5-Chlorobenzylamine A llli~tUlC~ of 4-chlorosalicaldehyde oxime (10 g, 58.3 mmol) and 5% Rh/C (2.0 g) in ethanol (100 ml) cont~ining concentrated sulfuric acid (10 ml) was shaken in a Parr apparatus under H2 (60 psi) for 24 h. Water (100 ml) was added and ~e mixture was filtered through 25 celite. The filtrate was concentrated until the product had crystallized out of solution. The solid was collected by filtration and the filtrate was further concentrated, ~ lin~; water to give a second crop which was combined with the first to give after drying the title compound.

..

CA 022458il 1998-08-10 W O 97/30708 PCT~US97/02408 - 2~ -lH NMR (400 Mz, CD30D) ~ 4.07 (s, 2 H), 6.88 (d, J=8.6Hz, 1 H), 7.25 (dd, J=2.6 and 8.6Hz, lH), 7.31 (d, J=2.6Hz, 1 H).

Step C: N-t-Butoxycarbonyl-2-Hydroxy-5-Chlorobenzylamine A mixture of 2-hydroxy-5-chlorobenzyl~min~ (1.22 g, 4.77 mmol assuming the bisulfate salt), (BOC)20 (1.56 g, 7.16 mmol) and N-methylmorpholine (1.05 ml, 9.54 mmol) in DMF (10 ml) was stirred for 5 h at r.t. The reaction was partitioned between water and ethyl acetate and the organic layer was washed with 5% KHS04 solution (2 times), sodium hydrogen carbonate solution and brine, dried (Na2S04) and evaporated in vacuo to a solid. The crude product was recrystallized from ethyl acetate/hexanes (1:5, 12 ml) to give the title compound:
lH NMR (400 Mz, CDC13) ~i 1.44 (s, 9 H, t-Bu), 4.17 (d, J=6.8Hz, 2H, CH2), 5.22 (br t, lH, NH), 6.87 (d, J=8.6Hz, lH, H-3), 7.03 (d, J =
2.6Hz, lH, H-6), 7.15 (dd, J=2.6 and 8.6 Hz, lH, H-4).

Step D: Ethyl-(2-t-Butoxycarbonylaminomethyl-4-Chlorophen-oxy)-Acetate A mixture of N-t-butoxycarbonyl-2-hydroxy-5-chloro-20 benzyl~mine (730 mg, 2.83 mmol), Cs2C03 (923 mg, 2.83 mmol) andethylbromoacetate (0.314 ml, 2.83 mmol) on DMF (5 ml) was stirred for 2 h. The crude reaction mixture was partitioned between ethyl acetate and water and the organic layer was washed with brine, dried (Na2S04) and evaporated in vacuo to an oil which was used for the next step.
Step E: 2-t-Butoxycarbonylaminomethyl-4-Chlorophenoxyacetic Acid The product from Step D was suspended in 1: 1: 1 methanol/THF/ water (9 ml) and lithium hydroxide hydrate (126 mg, 3.0 30 mmol) was added. After 16 h the volatiles were removed in vacuo and the solution was diluted with water and was washed with ethyl acetate, ~ adding sufficient brine to disperse the emulsion. The aqueous layer was acidified with 5% KHS04 solution and was extracted with methylene W O 97130708 PCT~US97/02408 chloride which was then dried (Na2S04) and evaporated in vacllo to give the title compound as a solid:
lH NMR (400 Mz, CDC13) ~ 1.44 (s, 9H, t-Bu), 4.35 (br s, 2H, NCH2), 4.62 (s, 2H, OCH2), 5.04 (br s, 1 H, NH), 6.74 (d, J=7.9Hz, lH, H-3), 7.20 (d, J=2.6Hz, lH, H-6), 7.24 (d obscured, lH, H-4).

Step F: Ethyl-(2-t-Butoxycarbonylaminomethyl-4-Chlorophen-oxy)-Acetamide EDC Hydrochloride (249 mg, 1.3 mmol) was added to a stîrred mixture of 2-t-butoxycarbonylaminomethyl-4-chloro-phenoxyacetic acid (316 mg, 1.0 mmol), HOBT (I76 mg, 1.3 mmol), ethylamine hydrochloride (106 mg, 1.3 mrnol) and N-methylmorpholine (0.396 ml, 3.6 mmol) in DMF (4 ml) and the mixture was stirred for 16 h.
The reaction was partitioned between ethylacetate and 5% KHSO4 solution and the organic layer was washed with 5% KHSO4 solution, water, NaHCO3 solution and brine, dried (Na2~04) and evaporated in vacuo to a solid (333 mg) which was used for the next step.

Step G: Ethyl-(2-Arninomethyl-4-Chlorophenoxy)-Acetamide l~thyl-(2-t-butoxycarbonylaminomethyl4-chlorophenoxy)-acetamide from Step F was dissolved in 2:1 methylene chloride/TFA (3 ml) and after 15 min the solvent was evaporated in vacuo. The residue was dissolved in water and the solution was washed with methylene chloride (twice). The aqueous layer was then basified with saturated 2~ sodium carbonate solution and NaCl was added to saturation. The mixture was extracted with ethyl acetate, and the organic layer was dried (Na2S04) a~d evaporated in l~acuo to give the title compound as a crs~stalline solid:
lH NMR ~300 MHz, CDC13) ~ 1.12 (t, J=7.3 Hz, 3H, Me), 1.54 (s, 9H, t-Bu), 3.31 (quintet, J=7.3 Hz, 2H, CH2Me), 3.90 (s, 2H, NcH2)~ 4.58 (s, 2H7 OCH2), 6.80 (d, J=8.3 Hz, IH, H-3), 7.19-7.23 (m, 2H, H-4, H-6), 8.0 I (br s, 1 H, CONEI).

W O 97/30708 PCTrUS97/02408 Step H: 3-Benzylsulfonylamino-6-methyl-1-[Ethyl-(2-Methyl-enecarboxamidomethyl-4-Chlorophenoxy)-Acetamide DCC (103 mg, 0.50 mrnol) was added to a stirred solution of ethyl-(2-aminomethyl-4-chlorophenoxy)-acetamide (137 mg, 0.57 mmol) and 3-benzylsulfonylamino-6-methyl-1-methylenecarboxy-2-pyridinone (168 mg, 0Ø50 mmol) in methylene chloride (3 ml). After 3 h the resulting thick mixture was filtered through celite, washing the pad with a large volume of methylene chloride since the product is sparingly soluble, and ~e filtrate was evaporated in vacuo to a solid. The crude product was purified by flash colurnn chromatography on silica (eluting with 3:1 hexanes/acetone followed by a methanol/chloroform gradient, 2-4% methanol) to give the title compound as a crystalline solid, m.p. 210-213"C:
lH NMR (400 Mz, DMSO) ~ 0.98 (t, J=7.2Hz, 3H, Me), 2.25 (s, 3H, Me), 3.11 (quintet, J-7.2Hz, 2H, CH2Me), 4.40 (d, J=5.5Hz, 2H, C~2NH~, 4.49 (s, 2H), 4.50 (s, 2H), 4.79 (s, 2H), 6.09 (d, J=7.7Hz, lH), 6.94 (d, J=8.6Hz, 1 H), 7.12 (d, J=7.5Hz, lH), 7.26-7.33 (m, 7H), 8.02 (br t, lH), 8.57 (s, 1 H), 8.75 (br s, lH); MS (PAB) 561 (M+l)+.

2Q Using the procedure of Example 2, the following eompounds were prepared:

W O 97~30708 PCTAUS97/02408 EXAM[PLE 3 3-Benzylsulfonylamino-6-methyl- 1 -[cyclopropyl-(2-Me~ylene-carboxamidomethyl -4-Chlorophenoxy)-Acetamidol -2-Pyridinone H
~~

Cl DCC (103 mg, 0.50 m~nol) was added to a stirred solution of cyclopropyl-(2-aminomethyl-4-chlorophenoxy)-acetamide (127 mg, 0.50 mrnol,) and 3-benzylsul~onylamino-6-methyl-1-me~ylenecarboxy-2-pyridinone ~168 mg, 0.50 mmol) in methylene chloride (2 ml). After 16 10 h the resulting thick mixtllre was ~iltered through celite, washing through with 1:1 methanol/chloroform and ~e filtrate was evaporated in vacuo to a solid. The crude product was purified by flash column chromatography on silica (eluting with 3:1 hexanes/acetone followed by a m~fh~nol/chloroform gradient, 2-4% methanol) to give the title 1~ compound as a crystalline solid, m.p. > 200~C:
lH NMR (400 Mz, CD30D) ~ 0.50-0.65 (m, 4H, CH2CH2), 2.29 (s, 3H, Me), 2.72 (m, lH, CH), 4.41 (s, 2H), 4.48 (s, 2H), 4.49 (s, 2H), 4.81 (s, 2H), 6.15 (d, J=7.7Hz, lH), 6.90 (d, J=8.8Hz, lH), 7.24-7.35 (m, 8H);
MS (FAB) 573 (M+ l )+.

W O 97/30708 PCTrUS97/02408 3 -Benzylsulfonylamino-6-methyl- 1 -(2-Methylenecarboxamidomethyl-4-Chlorophenoxyacetamido) -2-Pyr~dinone ~S~NJ~ N/\~ ~

Cl DCC (63 mg, 0.307 mmol) was added to a stirred solution of 2-aminomethyl-4-chlorophenoxyacetamide (101 mg, 0.307 mmol,) and 3 -benzylsulfonylamino-6-methyl- 1 -methylenecarboxy-2-pyridinone (103 mg, 0.307 mmol) in methylene chloride (2 ml). After 66 h the resulting thick mixture was filtered through celite, washing through with 1:1 methanollmethylene chloride and the filtrate was evaporated in vacuo to a solid. The crude product was purified by flash column chromatography on silica (eluting with 3:1 hexanes/acetone followed by a methanoVchloroform gradient, 2-5% methanol) to give the title compound as a crystalline solid, m.p. > 200~C:
lHNMR(300MHz,DMSO) o2.48(s,3H,Me),4.36(d,J=5.9Hz,2 H, C~2NH), 4.46 (s, 2H), 4.50 (s, 2H), 4.77 (s, 2H), 6.08 (d, J=7.6Hz, lH), 6.91 (d, J=8.6Hz, lH), 7.12 (d, J=7.6Hz, lH), 7.24-7.33 (m, 7H), 7.45 ~br s, 2H, NH2), 8.57 (s, lH, SO2NH), 8.71 (br t, lH, CONH); MS
~FAB3 533 (M+l)+.

In addition, the following examples were prepared:

W O 97/30708 PCT~US97/OZ408 3 -Benzylsulfonylamino-6-methyl- 1 -(2-~ethylenecarboxamidomethyl-4-Chlorophenoxy)-2-Pyridinone ~ ~2 ~ ~

Cl EDC Hydrochloride (236 mg, 1.23 mmol) was added to a stirred mixture of 3-benzylsul~onylamino-6-methyl-1-methylenecarboxy-2-pyridinone (415 mg, 1.23 mmol), HOBT (167 mg, 1.23 mmol), 2-hydroxy-5-chlorobenzyl~mine (195 mg, the product of Step B, Example B~ and NMM (0.136 ml, 1.23 mmol) in DMF (1.2 ml) and the mixture was stirred for 3 h. The reaction was diluted with ethyl acetate and was washed with water, dried (Na2S04) and evaporated in vacuo to a solid.
The crude product was purified by flash column chromatography on silica (ethylacetate/hexanes/2% acetic acid gradient, 50-80% ethyl 1~ acetate), to give t~e title compound as a crystalline solid, m.p. > 200~C:
lH NMR (400 Mz, CDC13) ~ 2.42 (s, 3H, Me), 4.22 (s, 2H), 4.26 (d, J=6.4Hz, 2 H, CH2NH), 4.44 (s, 2H), 6.08 (d, J=7.7Hz, lH), 6.72 (d, J=8.6Hz, lH), 7.01 -7.15 (m, 7H), 7.43 (d, J=7.7Hz, lH), 8.01 (br t, lH, CONH), MS (FAB) 476 (M+l)+.

W O 97130708 PCTrUS97/02408 3-Benzylsulfonylamino-6-methyl- 1 -[2,2,2-Trifluoroethyl-(2-Methyl-enecarboxamidomethyl -4-Chloro)-phenoxyl -2-Pyridinone 3~ 0 ~ ~ ,~CF3 Cl DCC (25 mg, 0.12 mmol) was added to a stirred solution of 2-(2,2,2-trifluoroethyl)-5-chlorobenzylamine (29 mg, 0.12 mmol,) and 3-benzylsulfonylamino-6-methyl-1-methylenecarboxy-2-pyridinone (Gl mg, 0.18 mmol) in methylene chloride (0.7 ml). After 16 h the resulting 10 ~ick mix1~lre was filtered through celite, w~hin~ with methylene chloride and the filtrate was evaporated in vacuo to a solid. The crude product was purified by flash column chromatography on silica (eluting with 3:1 hexanes/acetone followed by 50% ethyl acetate/hexanes) to give a crystalline solid which was triturated with methylene chloride to give 15 the title compound as a crystalline solid, m.p. > 200~C:
lH NMR (300 MHz, DMSO) o 2.26 (s, 3H, Me), 4.28 (d, J=5.6Hz, 2H, CH2NH), 4.51 (s, 2H), 4.75-4.83 (rn, 4 H), 6.09 (d, J='7.6Hz, lH), 7.01-7.31 ~m, 9 H), 8.56 (br s, lH, SO2NH), 8.70 (br t, lH, CONH); MS
(FAB) 598 (M+ l )+.
2{~

W O 97/30708 PCTrUS97/02408 3-Benzylsulfonylamino-6-methyl- 1 -(Methylenecarboxamidomethyl-3 -Chloropheny)-2-Pyridinone O H

Cl DCC (43 mg, 0.21 mmol) was added to a stirred solution of 3-chlorobenzyl~mine (0.025 ml, 0.21 mmol,) and 3-benzylsulfonylamino-6-methyl-1-methylenecarboxy-2-pyridinone (70 mg, 0.21 mmol) in m~thylene chloride (1.0 ml). After 16 h the resulting thick mixture was filtered through celite, washing with methylene chloride and the ~lltrate was evaporated in vacuo to a solid. The crude product was puri~led by flash column chromatography on silica (eluting with 3: 1 hexanes/acetone followed by an ethyl acetate/hexanes gradient, 60-100% ethyl acetate) to give a crystalline solid which was triturated with methanol to give the title eompound as a solid, m.p. 176-178~C:
lH NMR (400 Mz, CDC13, 1 drop CD30D) ~ 2.41 (s, 3H, Me), 4.31 (s, 2H), 4.41 (s, 2H), 4.67 (s, 2H), 6.08 (d, ~=7.6 Hz, lH), 7.15-7.34 (m, 10H); MS (FAB) 460 ~M+1)+.

~N V~T~O ASSAY FOR DETERMlN~G PROTEINASE ~HIBITION
Assays of human ~-thrombin and hl-m~n trypsin were per~ormed by the methods subst~n~i~lly as described in Thrombosis Research, Issue No. 70, page 173 (1993) by S.D. Lewis et al.
The assays were carried out at 25~C in 0.05 M TRIS buffer pH 7.4, 0.15 M NaCl, 0.1% PEG. Trypsin assays also contained 1 mM
CaC12. In assays wherein rates of hydrolysis of a p-nitroanilide (pna) substrate were deterlnined, a Thermomax 96-well plate reader was used CA 0224~811 1998-08-10 W O 97/30708 PCTrUS97/024~8 was used to measure (at 405 nm) the time dependent appearance of p-nitroaniline. sar-PR-pna was used to assay hllm~n o~-thrombin (Km=125 ~M) and bovine trypsin (Km=125 ,uM). p-Nitroanilide substrate concentration was determined from measurements of absorbance at 342 S nm using an extinction coefficient of 8270 cm- 1 M- 1 In certain studies with potent inhibitors (Ki < 10 nM) where the degree of inhibition of thrombin was high, a more sensitive activity assay was employed. In this assay the rate of thrombin catalyzed hydrolysis of the fluorogenic substrate Z-GPR-afc (Km=27 ,uM) was determined from the increase in fluorescence at S00 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 lS 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 cont~inin~ enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were 20 determined in control experiments. Initial velocities of product formation in the absence (VO) or presence of inhibitor (Vi) were measured.
Assllmin~ competitive inhibition, and that unity is negligible compared Km/[S], [Il/e, and [Il/e (where [S], [I], and e respectively represent the total concentrations, of substrate, inhibitor and enzyme), the equilibrium 25 constant (Ki) for dissociation of the inhibitor from the enzyme can be obtained from the dependence of Vo/Vi on [I] shown in ec uation 1.

Vo/Vi = 1 + [I]/Ki (1) 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 ~ngin:~? refractory ~ngin~, myocardial infarction, transient ischemic attacks, atrial fibrillation, thrombotic stroke, embolic stroke, deep vein thrombosis, dissemin~ted W O 97/30708 PCT~US97/02408 intravascular coagulation, and reocclusion or restenosis of rec~n~li7ed vessels. The inhibitory activity of each of the following compounds against human thrombin, represented by Ki, is less than 15 nM. These are selective compounds, as evidenced by their inhibitory activity against S human trypsin (represented by Ki), which is at least 1800 nM.

NH J~' H--OH
OC H2CON H Et CA 0224~811 1998-08-10 W O 97/30708 PCT~USg7/02408 EXAM:PL~ 9 In Vivo Studies To Measure Thrombotic Occlusions Applicants have conducted in vivo studies of the compounds 5 claimed herein using the following rat ferric chloride assay subst~nti~lly as described in Thrombosis Kesearch, No. 60, page 269(1990) by Kurtz et al.
In the assay used to determine in vivo activity of the thrombin inhibitors or the invention, Male Sprague-Dawley rats (body weights 200-350 grams) were anesthetized with dial-urethane solution (0.1 ml/100 gm body weight i.p.), and a lateral tail vein was c~nn~ ted with a 23 gauge needle connected to a 12 inch length of PE50 tubing.
The tubing was attached to a 3-way valve by a tubing adapter. Saline (control) or test comppund, as appropriate, was ~minictered via the tail 15 vein catheter. A tracheostomy was performed with a 0.75 inch length of PE205 tubing. The right carotid artery was exposed and a 1.3 mm diameter Doppler flow probe was placed on the vessel. Body temperature was m~int~ined at 37~C using a heat lamp.
Rats (8-10/group) were randomized to continuous 20 intravenous infusions of saline or test compound ~lmini~tered via the tail vein at a rate of 0.028 ml/min. Treatment infusions were initiated 60 min before the placement of a 3 mm square piece of Wh~tm~n No. 1 filter paper saturated with 35% FeCl3 onto ~e exposed carotid artery distal to the flow probe. Treatment infusions were continued for an additional 90 25 mimltes after the application of FeC13 (total infusion duration 150 mimltes) if thrombotic occlusions did not occur, or were termin:~ted 30 min~ s after thrombotic occlusion of the vessel. Time to occlusion was defined as the time from application of FeCl3 to thrombotic occlusion of the vessel. At the termination of the study (90 minutes after application 30 of FeC13 in ~nim~l~ which did not occlude~ or at 30 minutes after thrombotic occlusion), 3 ml blood samples were drawn by cardiac puncture into 0.3 ml of 3.8% sodium citrate.
The results show that the compounds of the invention are effective in preventing thrombotic occlusions.

W O 97130708 PCT~US97/02408 Tablet Preparation Tablets cont~ining 25.0, 50.0, and 100.0 mg., respectively, of the following active compounds are prepared as illustrated below:

3-Benzylsulfonylamino-6-methyl- 1 -(methylenecar~oxamidomethyl-2,5-dichlorophenyl)-2-pyridinone, 3-Benzylsulfonylamino-6-methyl- 1 -[Ethyl-(2-methylene-carboxamidomethyl4-chlorophenoxy)-acetamido]-2-pyrid}none, 3-Benzylsulfonylamino-6-methyl- 1 -[cyclopropyl-(2-Methylene-carboxamidomethyl-4-Chlorophenoxy)-Acetamido] -2-Pyridinone, 3-Benzylsulfonylamino-6-methyl- 1 -(2-Methylenecarboxamidomethyl-4-Chlorophenoxyacetamido)-2-Pyridinone TABLE FOR DOSES CONTA~G FROM
25-lOOMG OF THE ACTIVE COMPOUND

Amount-mg Active Compound 25.0 50.0 100.0 Microcrystalline cellulose 37.25 100.0 200.0 Modified food corn starch 37.25 4.25 8.5 Magnesium stearate 0.50 0.75 1.5 All of the active compound, cellulose, and a portion of the com starch are mixed and granulated to 10% corn starch paste. The resulting granulation is sieved, dried and blended with the remainder of CA 02245sil 1998-08-10 W O 97/30708 PCTrUS97/02408 the corn starch and the m ~n~,siu m stearate. The resulting gr~mll~tion is then compressed into tablets cont~ining 25.0,50.0, and 100.0 mg, respectively, of active ingredient per tablet.

Claims (16)

WHAT IS CLAIMED IS:

1. A compound having the formula:

wherein W is hydrogen, R1-, R1OC(O)-, R1C(O)-, R1SO2-, R1NHSO2-, (R1)2NSO2-, R1(CH2)nNHC(O)-, (R1)2CH(CH2)nNHC(O)-, or wherein n is 0-4;

R1 is R2(CH2)r-, where r is 0-4, (R2)(OR2)CH(CH2)p-, where p is 1-4, (R2)2CH(CH2)r-, where r is 0-4 and each R2 can be the same or different, and wherein (R2)2 can also form a ring with CH
represented by C3-7 cycloalkyl, C7-12 bicylic alkyl, C10-16 tricylic alkyl, or a 5- to 7- membered mono- or bicyclic heterocyclic ring which can be saturated or unsaturated, and which contains from one to three heteroatoms selected from the group consisting of N, O and S, R2O(CH2)p-, wherein p is 1-4, or R2(COOR3)(CH2)r-, where r is 1-4;

R2 and R14 are independently selected from -phenyl, unsubstituted or substituted with one or more of C1-4 alkyl, C1-4 alkoxy, halogen, hydroxy, COOH, or CONH2, naphthyl, biphenyl, a 5- to 7- membered mono- or a 9- to 10-membered bicyclic heterocyclic ring which can be saturated or unsaturated, and which contains from one to four heteroatoms selected from the group consisting of N, O and S, C1-4 alkyl, C3-7 cycloalkyl, C7-12 bicyclic alkyl, or C10-16 tricyclic alkyl;

R3 and R3' are independently C1-4 alkyl, C3-7 cycloalkyl, or trifluoromethyl;

R4 is hydrogen, C1-4 alkyl, C1-4 alkoxy, halogen, -OCH2CF3, -COOH, -OH, -COOR6, where R6 is C1-4alkyl, -CONR7R8, where R7 and R8 are independently hydrogen or C1-4alkyl, -(CH2)1-4OH, -CH2NHC(O)CH3, -CH2NHC(O)CF3, -CH2NHSO2CH3, -SO2NH2, -(CH2)1-4SO2NR7R8, -(CH2)1-4SO2R6, a 5- to 7- membered mono- or a 9- to 10-membered bicyclic heterocyclic ring which can be saturated or unsaturated, and which contains from one to four heteroatoms selected from the group consisting of N, O and S, -XCH2CO2H, -XCH2CO2CH3, -XCH2CO2(CH2)1-3CH3, -X(CHR9)1-3C(O)NR10R11, wherein R9 is H or C1-4 alkyl, R10 and R11 are independently hydrogen, C3-7 cycloalkyl, aryl, heteroaryl, heterocycloalkyl, C1-4 alkyl unsubstituted or substituted with one or more of hydroxy, COOH, amino, dialkylamino, aryl, heteroaryl, or heterocycloalkyl, or R10 and R11 are joined to form a four to seven membered cycloalkyl ring unsubstituted or substituted with hydroxy, amino or aryl, or -XCH2R14, wherein X is O, S or CH2;

R5 is hydrogen, halogen, C1-4 alkyl, C1-4 alkoxy, CN, or CO2NH2, and pharmaceutically acceptable salts thereof.

2. A compound of Claim 1, and pharmaceutically acceptable salts thereof, wherein W is R1 or R1SO2.

3. A compound of Claim 2, and pharmaceutically acceptable salts thereof, wherein R1 is R2(CH2)r, or (R2)2CH(CH2)r, phenyl-CH2SO2, or diphenyl-CHSO2.

4. A compound of Claim 3, and pharmaceutically acceptable salts thereof, wherein R1 is phenyl-CH2SO2, or diphenyl-CHSO2.

5. A compound of Claim 4, and pharmaceutically acceptable salts thereof, wherein R3 is C1-4 alkyl.

6. A compound of Claim 5, and pharmaceutically acceptable salts thereof, wherein R3 is methyl and R3 is hydrogen.

7. A compound of claim 6, and pharmaceutically acceptable salts thereof, wherein R4 is hydrogen, chlorine, OH, OCH2C(O)NH2 OCH2C(O)NHCH2CH3, or OCH2C(O)NH(CHCH2CH2); and R5 is chlorine.

8. The compound of Claim 7, and pharmaceutically acceptable salts thereof, selected from:

9. A composition for inhibiting thrombin in blood comprising a compound of Claim 1 and a pharmaceutically acceptable carrier.

10. A composition for inhibiting formation of blood platelet aggregates in blood comprising a compound of Claim 1 and a pharmaceutically acceptable carrier.

11. A composition for inhibiting thrombus formation in blood comprising a compound of Claim 1 and a pharmaceutically acceptable carrier.

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

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

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

15. A method for inhibiting thrombus formation in blood comprising adding to the blood a compound of Claim 1 with a fibrinogen recerptor antagonist.

16. The use of a compound of Claim 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting thrombus formation, preventing thrombus formation, inhibiting thrombin, inhibiting formation of fibrin, and inhibiting formation of blood platelet aggregates, in a mammal