JP2001517623A - Thrombin inhibitors - Google Patents

Abstract

  (57) [Summary] The present invention provides a novel fused lactam compound that inhibits human thrombin. The present invention includes a method for inhibiting thrombin in blood. The invention also includes compositions and methods for treating or preventing a thrombotic disorder.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION [0002] Thrombin is a serine protease present in plasma in the form of its precursor prothrombin. Thrombin plays a central role in the mechanism of blood coagulation by converting fibrinogen, a solution plasma protein, to insoluble fibrin.

[0002] Edwards et al ., J. Amer. Chem. Soc. (1992) vol. 114 , pp. 1854
-63) describe piperidyl a-ketobenzoxazoles, reversible inhibitors of the serine proteases human leukocyte elastase and porcine pancreatic elastase.

[0003] EP 363284 describes analogs of peptidase substrates in which the nitrogen atom of the scissile amide group of the substrate peptide has been replaced by a hydrogen or substituted carbonyl moiety.

[0004] Australian Patent Publication No. 86245677 also describes peptidase inhibitors having an activated electrophilic ketone moiety, such as a fluoromethylene ketone or a-ketocarbonyl derivative.

The thrombin inhibitors described in the above publications have arginine and lysine side chains. These structures show less selectivity for thrombin compared to other trypsin-like enzymes. Some show hypotensive toxicity and hepatotoxicity.

[0006] EP-A-601,459 discloses N- [4-[(aminoiminomethyl) amino] butyl] -1- [N- (2-naphthalenylsulfonyl) -L-phenylalanyl] -L-. Sulfonamide heterocyclic thrombin inhibitors such as prolinamide have been described.

[0007] WO 94/29336 describes compounds useful as thrombin inhibitors.

DISCLOSURE OF THE INVENTION [0008] Disclosed are compounds that inhibit human thrombin and have the following general structure:

[0009]

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Examples include the following compounds:

[0011]

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[0012] The present invention includes unstable angina, refractory angina, myocardial infarction, transient ischemic attack in a mammal, comprising a compound of the present invention in a pharmaceutically acceptable carrier. Atrial fibrillation,
Included are compositions that prevent or treat thrombotic stroke, embolic stroke, deep vein thrombosis, disseminated intravascular coagulation, ocular deposition of fibrin and reocclusion or restenosis of revascularized vessels. The composition can also include anticoagulants, antiplatelet agents, and thrombolytic agents.

The invention comprises inhibiting a loss of platelets in a mammal, comprising inhibiting a platelet aggregate, inhibiting the formation of fibrin, comprising a compound of the invention in a pharmaceutically acceptable carrier; Thrombus formation; including compositions that inhibit embolism formation.
The composition may optionally contain an anticoagulant, an antiplatelet agent and a thrombolytic agent. The desired inhibition can be achieved by adding / administering the composition to blood, blood products or mammalian organs.

The present invention further includes unstable angina, intractable angina, myocardial infarction, transient ischemic attacks, atrial fibrillation, thrombotic stroke, embolic stroke, deep vein thrombosis in mammals. Also included is the use of a compound of the invention in the manufacture of a medicament for the prevention or treatment of disseminated intravascular coagulation, fibrin ocular deposition and reocclusion or restenosis of revascularized vessels.

[0015] The present invention also includes a method of reducing surface thrombogenicity in a mammal by covalently or non-covalently binding a compound of the present invention to a surface.

BEST MODE FOR CARRYING OUT THE INVENTION The compound of the present invention is a compound having the following structure and a pharmaceutically acceptable salt of the compound.

[0017]

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Wherein X is O or H ₂ ; R ¹ and R ² are independently hydrogen, C _1-6 alkyl-, C _2-6 alkenyl, C _2-6 alkynyl, C _3-8 cyclo alkyl _{-, C 3-8} cycloalkyl _{C 1-6} alkyl -, di _{(C 3-8 cycloalkyl) C 1-6} alkyl -, aryl, aryl _{C 1-6} alkyl -, di _{(aryl) C 1-6} Alkyl-; wherein aryl is unsubstituted or -OH,
-NH _{2, C 1-6 alkyl, C 3-8} cycloalkyl or 6-membered carbocyclic ring halogen substituted unsaturated; B is

[0019]

Embedded image Is;

In the above formula, R⁴And R⁵Is independently hydrogen, C_1-4Alkyl, C_2-4Alkenyl, C_2-4Alkynyl, C_1-4Alkoxy, halogen, -COOH, -OH, -COOR⁷(R⁷Is C_1-4Alkyl)), -CONR⁸R⁹(R⁸And R⁹Is independently hydrogen or C_1-4Alkyl
), -OCH₂CO₂H, -OCH₂CO₂CH₃, -OCH₂CO₂(CH₂)_1-3CH₃, -O (CH₂)_1-3C (O) NR¹⁰R¹¹(R¹⁰And R¹¹Is independent
, Hydrogen, C_1-4Alkyl, C_3-7Cycloalkyl or -CH₂CF₃so
Yes),-(CH₂)_1-4OH, -NHC (O) CH₃, -NHC (O) CF₃, -NHSO₂CH₃, -SO₂NH₂  B is selected from the group consisting of

[0021]

Embedded image Is;

In the above formula, R ⁶ and R ¹² may be the same or different, and include hydrogen, C _1-6 alkyl-, C _2-6 alkenyl-, C _2-6 alkynyl-, C _3-8 cycloalkyl -, aryl, aryl _{C 1-6} alkyl -; and aryl in the above is substituted or unsubstituted _-OH, -NH _{2, C 1- 6 alkyl,} in _{C 3-8} cycloalkyl or halogen- Saturated 6
Membered carbocycle.

One class of these compounds is the following compounds and pharmaceutically acceptable salts of the compounds.

[0024]

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Wherein R ¹ and R ² are independently hydrogen, C _1-6 alkyl-, C _3-8 cycloalkyl C _1-6 alkyl-, di (C _3-8 cycloalkyl) C _1-6 alkyl -, aryl C _1-6 alkyl -, di (aryl) C _1-6 alkyl - is selected from the group consisting of; aryl in the above is optionally monosubstituted or disubstituted by or unsubstituted halogen; B Is

[0026]

Embedded image Is;

In the above formula, R ⁴ and R ⁵ are independently selected from the group consisting of hydrogen, halogen or —OCH ₂ C (O) NHR ^11, wherein R ¹¹ is C _1-4 alkyl; Or B is

[0028]

Embedded image Is;

In the above formula, R ⁶ and R ¹² may be the same or different and represent hydrogen or —
CH _3.

One group of this class of compounds is compounds of the formula: and pharmaceutically acceptable salts thereof.

[0031]

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Wherein R ¹ is hydrogen,

[0033]

Embedded image Is;

R ² is hydrogen, CH ₃ , C _1-6 alkyl and aryl C _1-6 alkyl; B is

[0035]

Embedded image It is.

One sub-group of this group of compounds is compounds of the following formulas and pharmaceutically acceptable salts of said compounds.

[0037]

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Wherein R ¹ is hydrogen,

[0039]

Embedded image It is.

Specific examples of this class of compounds are shown in the table below.

[0041]

[Table 1]

[0042]

[Table 2]

Pharmaceutically acceptable salts of the compounds shown above are also included by way of example.

[0044]

[Table 3]

The compounds of the present invention may have chiral centers and may be obtained as racemates, racemic mixtures and as individual diastereomers or enantiomers, all of which are included in the present invention. The compounds of the present invention may also have polymorphic crystal forms, but any polymorphic crystal form is included in the present invention.

When any variable occurs more than one time in any constituent or in formula I,
Its definition for each individual is independent of its definition in all others. further,
Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

Unless otherwise specified, the term “alkyl,” as used herein, unless otherwise indicated, includes both branched and straight-chain saturated aliphatic hydrocarbon groups having 1-8 carbon atoms. (Me is methyl; Et is ethyl; Pr is propyl; Bu is butyl). "Alkenyl" means, unless otherwise indicated.
It includes both branched and straight-chain unsaturated aliphatic hydrocarbon groups having 1 to 8 carbon atoms, and includes, for example, ethenyl and propenyl. The term "cycloalkyl" includes cyclic saturated aliphatic hydrocarbon group having 3 to 8 carbon atoms (e.g., a "C _{3- 8} cycloalkyl", cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, etc. ). The term “C _7-12 bicyclic alkyl” refers to bicyclo [2.2.1] heptyl (norbornyl)
, Bicyclo [2.2.2] octyl, 1,1,3-trimethyl-bicyclo [2.
2.1] heptyl (bornyl) and the like. The term “aryl,” as used herein, unless otherwise specified, refers to a stable 6-10 membered monocyclic or bicyclic ring system such as phenyl or naphthyl. The aryl ring may be unsubstituted or substituted by one or more C _1-4 lower alkyl; hydroxyl; alkoxy; halogen; "Alkoxy" represents an alkyl group of 1 to 8 carbon atoms attached through an oxygen bridge. As used herein, the term "halo" refers to fluorine, chlorine, bromine and iodine. "Counter ion" means chloride ion, bromide ion, hydroxide ion, acetate ion, trifluoroacetate ion, perchlorate ion, nitrate ion, benzoate ion, maleate ion, tartrate ion, hemitartrate ion, benzenesulfone. It is used to represent small, monovalent negatively charged species such as acid ions.

Unless otherwise specified, standard nomenclature used throughout this disclosure describes the terminal portion of the designated side chain first, followed by the adjacent functional moiety toward the point of attachment. I do. For example, an ethyl substituent substituted with "methylcarbonylamino" is:

[0049]

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The pharmaceutically acceptable salts of the compounds of the formula I (in the form of water-soluble or oil-soluble or dispersible pharmaceuticals) include the conventional non-toxic salts formed, for example, from inorganic or organic acids or bases. Salts or quaternary ammonium salts. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorate Sulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride , Hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
Pamoate, pectate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, 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 salts and magnesium salts; salts with organic bases such as dicyclohexylamine salts; N-methyl-D-glucamine And salts with amino acids such as arginine and lysine. Further, lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate and diamyl sulfate; decyl, lauryl, myristyl and stearyl chlorides; Basic-containing nitrogen groups can be quaternized by agents such as long-chain halides such as bromide and iodide; aralkyl halides such as benzyl and phenethyl bromide, and the like.

[0051]Thrombin inhibitors-therapeutic use  Anticoagulant therapy is used to treat and predict various thrombotic conditions, especially coronary and cerebrovascular disease.
Prevention is the adaptation. Those skilled in the art can easily understand the situation where anticoagulant therapy is required.
. As used herein, the term "patient" refers to primates, including humans, sheep, and cormorants.
Mammals, cattle, pigs, dogs, cats, mammals such as rats and mice
And

Thrombin inhibition is required not only in anticoagulant therapy in patients with thrombotic conditions, but also to prevent clotting of stored whole blood and to prevent clotting in other biological specimens for testing or storage. It is useful in any case. Accordingly, a medium containing or expected to contain thrombin, such as a material selected from the group consisting of vascular prostheses, stents, orthopedic prostheses, cardiac prostheses and extracorporeal circulation, and mammalian blood The thrombin inhibitor can be added to or contacted with the medium in which it is desired to inhibit clotting, as in the case of contact with the thrombin inhibitor.

Compounds of the present invention may be used in mammals to treat venous thromboembolism (eg, impaired or obstructed venous blood flow due to detached thrombus; impaired or obstructed pulmonary artery due to detached thrombus), cardiogenic thromboembolism (eg, , Impaired blood flow or obstruction of the heart due to detached thrombus)
Arterial thrombosis (eg, thrombus formation in the artery that can cause infarction of the tissue supplied by the artery), atherosclerosis (eg, atherosclerosis characterized by irregularly distributed lipid deposits) This is useful in the treatment or prevention of curing, and in reducing the propensity of medical devices to coagulate blood in contact with blood.

Examples of venous thromboembolism that can be treated or prevented by the compounds of the present invention include impaired venous blood flow; impaired pulmonary artery blood flow (pulmonary embolism); deep venous thrombosis; cancer and cancer Thrombosis associated with chemotherapy of thrombosis; thrombosis inherent to thrombogenic diseases such as protein C deficiency, protein S deficiency, antithrombin III deficiency and Factor V Leiden; systemic lupus erythematosus Thrombosis caused by acquired thrombogenic disorders such as tissue diseases). Also for venous thromboembolism, the compounds of the invention are useful for maintaining the patency of an indwelling catheter.

Examples of cardiogenic thromboembolism that can be treated or prevented by the compounds of the present invention include thromboembolic stroke (stripped thrombus causing neuropathy associated with impaired blood supply to the brain), atrial fibrosis Cardiogenic thromboembolism associated with movement (rapid irregular spasm of the upper chamber myofibrils), cardiogenic thromboembolism associated with prosthetic heart valves such as prosthetic heart valves, and cardiogenic heart associated with heart disease And thromboembolism.

Examples of arterial thrombosis include unstable angina (severe astringent pain in the chest of coronary origin), myocardial infarction (cardiomyocyte death caused by insufficient blood supply), ischemic heart disease (blood supply Lesions (eg, local anemia due to arterial stenosis), reocclusion during or after percutaneous transluminal coronary angioplasty, restenosis after percutaneous transluminal coronary angioplasty, occlusion and occlusion of coronary artery bypass grafts And cerebrovascular disease. Also for arterial thrombosis, the compounds of the invention are useful for maintaining patency in arteriovenous cannulas.

Examples of atherosclerosis include arteriosclerosis.

Examples of medical devices that come into contact with blood include artificial blood vessels, stents, orthopedic prostheses, cardiac prostheses, and extracorporeal circulation systems. For example, a compound of the present invention can be used to coat a stent to prevent local coagulation that occurs frequently during stent implantation.

The thrombin inhibitors of the present invention include tablets, capsules (each containing a sustained release or sustained release formulation), pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions. And other oral preparations. Similarly, the compound
It can be administered in the form of intravenous (bolus or infusion), intraperitoneal, subcutaneous or intramuscular dosage forms, all of which are known to those skilled in the pharmaceutical arts. As an anticoagulant, an effective but non-toxic amount of the desired compound can be used. For ocular deposition of fibrin, the compounds may be administered intraocularly or topically, orally or parenterally.

These thrombin inhibitors can be administered in the form of a depot injection or implant preparation which can 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 a depot injection or implant. Implants include, for example, Dow-Corning Corp.
Oration) Biodegradable polymers such as manufactured Silastic, silicone rubber or other polymers or inert materials such as synthetic silicones can be used.

The thrombin inhibitor can further be administered in the form of a liposome administration system such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes are
It can be formed from various phospholipids such as cholesterol, stearylamine or phosphatidylcholines.

The thrombin inhibitor can further be administered using a monoclonal antibody as an individual carrier to which the compound molecule is bound. The thrombin inhibitor,
It can also be coupled with a soluble polymer as a targetable pharmaceutical carrier. Such polymers may include polyvinylpyrrolidone, pyran copolymer, polyhydroxy-propyl-methacrylamide-phenol, polyhydroxyethyl-aspartamide-phenol or polyethylene oxide-polylysine substituted with palmitoyl residues. . Further, the thrombin inhibitor includes, for example, polyacetic acid, polyglycolic acid, a copolymer of polyacetic acid and polyglycolic acid, polyε-caprolactone, polyhydroxybutyric acid, polyorthoesters,
The polyacetals, polydihydropyrans, polycyanoacrylates, and hydrogels can be conjugated to a class of biodegradable polymers useful in providing sustained release of drugs, such as cross-linking or amphiphilic block copolymers.

The method of administration using the thrombin inhibitor is based on the type of patient, animal species, age, body weight,
The choice will depend on various factors such as gender and medical condition; severity of the condition to be treated; route of administration; renal and hepatic function of the patient; and the particular compound or salt thereof used. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, treat or arrest the progress of the condition.

The oral dose of the thrombin inhibitor when used to achieve the above effects is about 0.01 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, most preferably 0.1-0.5 mg / kg / day (unless otherwise noted, the active ingredient Amounts are based on free base). For example, for a patient weighing 80 kg, about 0.8
mg / day to 2.4 g / day, preferably 2 to 600 mg / day, more preferably 8 to
200 mg / day, most preferably 8-40 mg / day will be administered. Thus, pharmaceuticals suitably prepared for once daily administration include 0.8 mg to 2.4 g,
Preferably, it will contain from 2 mg to 600 mg, more preferably from 8 mg to 200 mg, most preferably from 8 mg to 40 mg, for example 8 mg, 10 mg
mg, 20 mg and 40 mg. Advantageously, the thrombin inhibitor can be administered in divided doses twice, three or four times daily. For twice-daily administration, suitably prepared pharmaceuticals include 0.4 mg to 4 g, preferably 1 mg to 4 mg.
300 mg, more preferably 4 mg to 100 mg, most preferably 4 mg to 20 mg
It would be expected to contain 4 mg, 5 mg, 10 mg and 20 mg.

For intravenous administration, patients receive 0.025-7.5 mg / kg / day, preferably 0.1 mg / kg / day.
It is contemplated that the active ingredient will be administered in an amount sufficient to administer 1-2.5 mg / kg / day, more preferably 0.1-0.5 mg / kg / day. Such amounts can be adjusted in many suitable ways, e.g. by administering large amounts of the low-concentration active ingredient once a day or several times a day, or by administering the high-concentration active ingredient in a short time once a day. It can be administered by administering it in a low dose. Typically, about 0.01 to 1.0 mg / mL
(Example: 0.1 mg / mL, 0.3 mg / mL and 0.6 mg / mL) A conventional intravenous formulation containing the active ingredient was prepared, and it was added at 0.01 mL / kg to 10 mL / kg.
. 0 mL / kg (eg, 0.1 mL / mg, 0.2 mL / mg and 0.5 mL / mg
mg) per day. As an example, an active ingredient concentration of 0.5
A patient weighing 80 kg receiving 8 mg of the mg / mL intravenous formulation twice a day was
The active ingredient will be administered at 8 mg per day. PH acceptable for intravenous administration
Glucuronic acid, L-lactic acid, acetic acid, citric acid or a pharmaceutically acceptable acid / conjugate base having a reasonable buffer capacity in the range can be used as the buffer. The choice must take into account the solubility of the drug. One of skill in the art can easily select an appropriate buffer and pH for the formulation depending on the solubility of the drug to be administered.

The compounds can also be administered in a nasal formulation by topical use of a suitable nasal vehicle, or by a transdermal route using transdermal patches in a manner known to those skilled in the art. Of course, for administration in the form of a transdermal administration system, the administration will be continuous rather than intermittent throughout the dosage regimen.

The thrombin inhibitor will typically be suitably selected for the intended dosage form, ie, oral tablet, capsule, elixir, syrup, etc., and will be a suitable pharmaceutical diluent compatible with conventional pharmaceutical practice. , Excipient or carrier (collectively referred to herein as "
(Referred to as the "carrier" material) in the form of an active ingredient.

For example, for oral administration in the form of a tablet or capsule, the active drug component can be formulated with lactose, starch, sucrose, glucose, methylcellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Can be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier; for oral administration in a liquid formulation, the oral drug component should be an oral, non-toxic, pharmaceutical such as ethanol, glycerin, water, etc. It can be combined with a chemically acceptable inert carrier. In addition, if desired or necessary, the mixture may comprise a suitable binder,
Lubricants, disintegrants and coloring agents can also be incorporated. Suitable binders include starch; gelatin; natural sugars such as glucose or β-lactose; corn sweeteners; natural and synthetic gums such as acacia, tragacanth or sodium alginate; carboxymethyl cellulose; polyethylene glycol; Lubricants used in these formulations 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.

[0069] Representative tablet cores suitable for administration of the thrombin inhibitor comprise, but are not limited to, the following standard component amounts:

Proposed Composition Range for Excipients in Uncoated Tablet Cores

[0071]

[Table 4] Manitol, microcrystalline cellulose and magnesium stearate can be substituted for another pharmaceutically acceptable excipient.

The thrombin inhibitor may be a fibrinogen receptor antagonist (eg, for treating or preventing unstable angina or for preventing reocclusion after angioplasty and restenosis), aspirin, heparin or Anticoagulants such as warfarin,
It may be administered in combination with a suitable antiplatelet agent such as, but not limited to, a plasminogen activator or a thrombolytic agent such as streptokinase to achieve a synergistic effect in the treatment of various vascular diseases. Or in combination with lipid lowering agents such as antihypercholesterolemia drugs (eg, HMG CoA reductase inhibitors such as lovastatin or simvastatin, HMG CoA synthase inhibitors, etc.) Atherosclerosis can be treated or prevented. For example, a combination of a fibrinogen receptor antagonist and a thrombin inhibitor may be effective for patients suffering from coronary artery disease and undergoing angioplasty. In addition, the thrombin inhibitors enhance the efficacy of tissue plasminogen activator-mediated thrombolytic reperfusion. First, the thrombin inhibitor can be administered after thrombus formation, followed by administration of a tissue plasminogen activator or other plasminogen activator.

A typical dose of a thrombin inhibitor of the present invention in combination with another suitable antiplatelet, anticoagulant or thrombolytic agent is one with another antiplatelet, anticoagulant and thrombolytic agent. Can be the same as the dose of the thrombin inhibitor given without the combination of, or given without the combination with another antiplatelet, anticoagulant and thrombolytic agent The dose of the thrombin inhibitor can be substantially lower, depending on the therapeutic needs of the patient.

The following examples are intended to illustrate the invention intended by the inventor and should not be construed as limiting the scope or spirit of the invention.

The amine coupling used to form the compounds of the present invention typically involves dicyclohexylcarbodiimide or 1-ethyl-3- (3-dimethylaminopropyl)
It is performed by a carbodiimide method using a reagent such as carbodiimide. Other methods of forming amide or peptide bonds include, but are not limited to, synthetic routes via acid chlorides, azides, mixed anhydrides or activated esters. Typically, liquid phase amide coupling is performed, but solid phase synthesis by the classic Merrifield method can be used instead. Addition and elimination of one or more protecting groups is also a representative method.

The compounds of the present invention can be prepared, for example, using the starting materials, intermediates and general procedures described below.

[0077]Production of raw materials  4-formyl-2-methoxy-6-methyl-3-nitropyridine

[0078]

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Step A: Ethyl 6-methyl-3-nitropyridone-4-carboxylate

[0080]

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A slurry of nitroacetamide ammonium salt (70.3 g, 581 mmol) in deionized water (400 mL) was added to 100 g of ethyl 2,4-dioxovalerate (63 g).
3 mmol, 1.09 equivalents) and then piperidinium acetate solution (acetic acid (21 mL)
An aqueous solution (100 mL of water, prepared by adding 36 mL of piperidine) was added. The resulting solution was stirred at 40 ° C. for 16 hours, then cooled in an ice bath. The precipitated product was filtered and washed with 50 mL of cold water to give the title pyridone as a yellow solid.

¹ H NMR (CDCl ₃ ) d: 6.43 (s, 1H), 4.35 (q, J = 7)
Hz, 2H), 2.40 (s, 3H), 1.35 (t, J = 7 Hz, 3H). Step B: ethyl 2-methoxy-6-methyl-3-nitropyridone-4-carboxylate

[0083]

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The pyridone from Step A (6.2 g, 27.4 mmol) in DCM (50 mL)
4.47 g of solid trimethyloxonium tetrafluoroborate (30.
2 mmol) was added and the mixture was stirred at 40 ° C. until the reaction was judged complete by HPLC (typically 24-72 h). The reaction mixture was concentrated to 1/3 volume, loaded onto a silica gel column and eluted with 2: 3 EtOAc / hexane to give the title methoxypyridine as a yellow liquid.

¹ H NMR (CDCl ₃ ) d: 7.2 (s, 1H), 4.35 (q, J = 7H)
z, 2H), 4.05 (s, 3H), 2.55 (s, 3H), 1.35 (t, J
= 7 Hz, 3H). Step C: 4-hydroxymethyl-2-methoxy-6-methyl-3-nitropyridine

[0086]

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The ester from Step B (5.4 g, 22.5 mmol) in DCM (140 mL)
) The solution was brought to -70 ° C, and DIBAL-H (1M hexane solution) was 56.2 m.
L (56.2 mmol) was added via dropping funnel. The resulting solution was stirred for 1 hour and allowed to warm to room temperature over 1 hour. The reaction mixture was quenched by the careful addition of saturated NaK tartrate. Stirring is continued for 30 minutes, then the solid is filtered,
Washed with DCM (100 mL). The filtrate was extracted twice with 50 mL of saturated NaK tartrate and then extracted with brine (25 mL). The yellow solution was concentrated and chromatographed (2: 3 EtOAc / hexane) to give the desired alcohol as a yellow solid.

¹ H NMR (CDCl ₃ ) d: 7.00 (s, 1H), 4.70 (s, 2H)
4.05 (s, 3H), 2.50 (s, 3H), 2.10 (bs, 1H). Step D: 4-formyl-2-methoxy-6-methyl-3-nitropyridine

[0089]

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A solution of oxalyl chloride (2.0 mL, 22 mmol) in DCM (50 mL) was brought to −70 ° C., and DMSO (3.4 mL, 44 mmol) in DCM (1 mL) was added.
0 mL) solution was added via dropping funnel. After 2 minutes, a solution of 3.99 g (20 mmol) of the alcohol from Step C in DCM (20 mL) was added to the reaction mixture. The solution was stirred at −70 ° C. for a further 15 minutes, 14 mL (50 mmol) of Et ₃ N was added, and the temperature was raised to room temperature over 90 minutes. The reaction was stopped by adding 100 mL of water,
The two phases were separated. The aqueous phase was extracted with DCM (100 mL), the combined organic extracts were washed with brine 50 mL, dried over MgSO _4. The yellow solution was concentrated and chromatographed (2: 3 EtOAc / hexane) to give the title aldehyde as a yellow solid.

¹ H NMR (CDCl ₃ ) d: 10.05 (s, 1H), 7.10 (s, 1H)
), 4.70 (s, 2H), 4.05 (s, 3H), 2.60 (s, 3H).

General Procedure for the Synthesis of Intermediate Phosphate Esters

[0093]

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Intermediate 1 (I-1) : Trimethyl 2-benzyl-2-phosphonoacetate (R = PhC
H ₂ -) in DMSO (12 mL) medium slurry NaH400mg (16.7mmol), was added neat trimethyl phosphonoacetate (2.4mL, 14.9mmol). The reaction mixture was stirred for 20 minutes and benzyl bromide 1.4 mL (11.9 mmol)
Was added and stirring was continued for 16 hours. Dilute the homogeneous solution with 25 mL of ether and add 10 mL of water.
Washed 6 times with mL and then with brine. The solution was dried (MgSO ₄ ), concentrated and chromatographed (4: 1 EtOAc / hexane) to give the title compound as a colorless liquid.

¹ H NMR (CDCl ₃ ) d: 7.25 (m, 5H), 3.85 (s, 3H)
, 3.80 (s, 3H), 3.65 (s, 3H), 3.25 (m, 3H). Intermediate 2 (I-2): 2- cyclobutyl-2-phosphonopropionic acid trimethyl _{(R = c-BuCH 2 -} ) in DMSO (12 mL) medium slurry NaH2.0g (80.0mmol),
Undiluted trimethyl phosphonoacetate (12.6 mL, 69.2 mmol) was added. The reaction mixture was stirred for 20 minutes and 7.9 mL of (bromomethyl) cyclobutane (5 mL)
(3.3 mmol) were added and stirring was continued for 16 hours. 100 m of ether solution
Diluted with L, quenched with 0.25N HCl to pH = 7, washed 6 times with 10 mL of water and then with brine. The solution was dried (MgSO ₄ ), concentrated and chromatographed (4: 1 EtOAc / hexane) to give the title compound as a colorless liquid.

¹ H NMR (CDCl ₃ ) d: 3.85 (s, 3H), 3.80 (s, 3H)
3.65 (s, 3H), 2.90 (m, 1H), 2.4-1.6 (m, 9H)
. Intermediate 3 (I-3): 2- cyclopropyl-2-phosphonopropionate trimethyl _{(R = c-PrCH 2 -} ) in DMSO (12 mL) medium slurry NaH400mg (16.7mmol), neat phosphonoacetic acid Trimethyl (2.4 mL, 14.9 mmol) was added. The reaction mixture was stirred for 20 minutes and 1.2 mL of (bromomethyl) cyclopropane (
11.9 mmol) was added and stirring was continued for 16 hours. Add homogeneous solution to 100
Diluted in mL, quenched with 0.25N HCl to pH = 7, washed 6 times with 10 mL of water and then with brine. The solution was dried (MgSO ₄ ), concentrated and chromatographed (4: 1 EtOAc / hexane) to give the title compound as a colorless liquid.

¹ H NMR (CDCl ₃ ) d: 3.85 (s, 3H), 3.80 (s, 3H)
, 3.78 (s, 3H), 3.10 (m, 1H), 2.0 (m, 1H), 1.7
0 (m, 1H), 0.75 (m, 1H), 0.4 (m, 2H), 0.10 (m,
2H). Intermediate 4 (I-4) : Trimethyl 2- (2-fluorobenzyl) -2-phosphonoacetate (R = 2-F-Bn-) Undiluted with a slurry of 400 mg (16.7 mmol) of NaH in DMSO (12 mL) Of trimethyl phosphonoacetate (2.4 mL, 14.9 mmol) was added. The reaction mixture was stirred for 30 minutes and 1.44 mL of 2-fluorobenzyl bromide
(11.9 mmol) was added and stirring continued for 16 hours. Homogeneous solution in ether 10
Diluted with 0 mL, quenched with 0.25 N HCl to pH = 7, washed 6 times with 10 mL of water and then with brine. The solution was dried (MgSO ₄ ), concentrated,
Chromatography (4: 1 EtOAc / hexane) provided the title compound as a colorless liquid.

¹ H NMR (CDCl ₃ ) d: 7.4 to 7.0 (m, 4H), 3.85 (s,
3H), 3.80 (s, 3H), 3.65 (s, 3H), 3.4-3.2 (m,
3H). Intermediate 5 (I-5) : Trimethyl 2- (3-fluorobenzyl) -2-phosphonoacetate (R = 3-F-Bn-) undiluted in a slurry of 400 mg (16.7 mmol) of NaH in DMSO (12 mL) Of trimethyl phosphonoacetate (2.4 mL, 14.9 mmol) was added. The reaction mixture was stirred for 30 minutes and 1.44 mL of 3-fluorobenzyl bromide
(11.9 mmol) was added and stirring continued for 16 hours. Homogeneous solution in ether 10
Diluted with 0 mL, quenched with 0.25 N HCl to pH = 7, washed 6 times with 10 mL of water and then with brine. The solution was dried (MgSO ₄ ), concentrated,
Chromatography (4: 1 EtOAc / hexane) provided the title compound as a colorless liquid.

¹ H NMR (CDCl ₃ ) d: 7.2 (m, 1H), 6.90 (m, 3H),
3.85 (s, 3H), 3.80 (s, 3H), 3.65 (s, 3H), 3.4
~ 3.2 (m, 3H). Intermediate 6 (I-6): 4- (S) - methyl-2-phosphono-hexanoic acid trimethyl _{[R = -CH 2 CH (S} ) - (CH 3) CH 2 CH 3] NaH200mg (8.3mmol) To a slurry of DMSO in DMSO (5 mL) was added neat trimethyl phosphonoacetate (1.1 mL, 6.9 mmol). The reaction mixture was stirred for 20 minutes and 1-iodo-2- (S) -methylbutane 0.66 mL
(5.1 mmol) was added and stirring continued for 16 hours. 20 m of homogeneous solution in ether
L, quenched with 0.25N HCl to pH = 7, washed 6 times with 5 mL of water and then with brine. The solution was dried (MgSO ₄ ), concentrated and chromatographed (4: 1 EtOAc / hexane) to give the title compound as a colorless liquid.

¹ H NMR (CDCl ₃ ) d: 3.85 to 3.70 (6 singlet, 9H),
3.05 (m, 1H), 2.2-1.2 (m, 5H), 0.85 (m, 6H).
Intermediate 7 (I-7) : trimethyl 2- (2,5-difluorobenzyl) -2-phosphonoacetate (R = 2,5-di-F-Bn-) 339 mg (14.1 mmol) of NaH in DMSO (10 mL) To the medium slurry was added neat trimethyl phosphonoacetate (1.95 mL, 9.5 mmol). The reaction mixture was stirred for 20 minutes and 2,5-difluorobenzyl bromide 1.2
4 mL (9.67 mmol) were added and stirring was continued for 16 hours. The homogeneous solution was diluted with 50 mL of ether, quenched with 0.25 N HCl to pH = 7, washed 6 times with 5 mL of water and then with brine. The solution was dried (MgSO ₄ ), concentrated and chromatographed (4: 1 EtOAc / hexane) to give the title compound as a colorless liquid.

¹ H NMR (CDCl ₃ ) d: 6.95 (m, 3H), 3.85 (s, 3H)
, 3.80 (s, 3H), 3.70 (s, 3H), 3.4-3.2 (m, 3H)
. Intermediate 8 (I-8) : Trimethyl 2- (3,5-difluorobenzyl) -2-phosphonoacetate (R = 3,5-di-F-Bn-) NaH 169 mg (7.0 mmol) in DMSO (5 mL) To the medium slurry was added neat trimethyl phosphonoacetate (0.97 mL, 4.75 mmol).
The reaction mixture was stirred for 20 minutes and 1.0 g of 3,5-difluorobenzyl bromide
(4.83 mmol) was added and stirring was continued for 16 hours. Homogeneous solution in ether 30
Diluted with mL, quenched with 0.25N HCl to pH = 7, washed 6 times with 5 mL of water and then with brine. The solution was dried (MgSO ₄ ), concentrated and chromatographed (EtOAc) to give the title compound as a colorless liquid.

¹ H NMR (CDCl ₃ ) d: 6.85 (m, 3H), 3.85 (s, 3H)
, 3.80 (s, 3H), 3.70 (s, 3H), 3.3 to 3.1 (m, 3H)
.

General Procedure for Production of Fusion Lactam Thrombin Inhibitors

[0104]

Embedded image

[0105]

Embedded image

[0106]Example 1  Production of the following compounds

[0107]

Embedded image

Step 1-A:

[0109]

Embedded image

The intermediate phosphate I-1 (1.36 g, 5.0 mmol) in THF (25
The solution was brought to 0 ° C. and 145 mg (4.75 mmol) of NaH was added thereto. After the mixture was stirred for 30 minutes, a solution of 930 mg (4.75 mmol) of 4-formyl-2-methoxy-3-nitropyridine in 15 mL of THF was added dropwise.
The solution was then stirred at 50 ° C. for 3 hours, cooled and evaporated. Residue in EtOA
redissolved in c (100 mL) and quenched with saturated NH ₄ Cl to pH = 7. The organic phase was washed with brine, dried over MgSO _4. The desired olefin was purified by column chromatography (2: 3 EtOAc / hexane)
-Obtained as a mixture of isomers.

¹ H NMR (CDCl ₃ ) d: 7.60 (s, 1H), 7.40 to 7.00 (
m, 6H), 6.60 (two singlet, 2H), 4.00 (two singlet, 6H
), 3.75 (two singlets, 8H), 2.40 (two singlets, 6H). Step 1-B:

[0112]

Embedded image

The nitroolefin 1-1 (1.6 g, 4.75 mmol) in EtOAc (50
(mL) solution, 400 mg of 10% Pd (C) was added. Hydrogen gas was added and the solution was heated at 50 ° C. for 16 hours. The reaction mixture was filtered through celite, and the filtrate was evaporated. Column chromatography (2: 3 EtOAc / hexane) provided the bicyclic lactam as a white solid.

¹ H NMR (CDCl ₃ ) d: 7.45 (bs, 1H), 7.40 to 7.20
(M, 5H), 6.45 (s, 1H), 3.95 (s, 3H), 3.35 (dd)
, 1H), 2.80 (m, 2H), 2.60 (m, 2H), 2.40 (s, 3H)
). Step 1-C:

[0115]

Embedded image

To a solution of methoxypyridine 1-2 (700 mg, 2.48 mmol) in dichloroethane (25 mL) at 23 ° C. was added 8.0 mL (8.0 mmol) of BBr ₃ (1M in DCM). The insoluble gum precipitates within 5 minutes and the reaction is
After stirring for 0 min, the reaction was stopped by adjusting the pH to 8 with saturated NaHCO ₃ .
The mixture was diluted with 100 mL of EtOAc and 10 mL of THF. The aqueous phase was discarded and the organic solution was washed with 10 mL of water and then with 10 mL of brine. Evaporation of the solvent gave a tan solid which was used without further purification.

¹ H NMR (CDCl ₃ ) d: 8.20 (bs, 1H), 7.40 to 7.10
(M, 5H), 5.88 (s, 1H), 3.35 (dd, 1H), 2.80-2
. 50 (m, 4H), 2.25 (s, 3H). Step 1-D:

[0118]

Embedded image

Pyridone 1-3 (630 mg, 2.5 mmol) in DMF (20 mL) at 23 ° C.
) Was added CsCO ₃ 812 mg (2.5 mmol) and bromoacetic acid tert-
0.37 mL (2.5 mmol) of butyl was added. The reaction mixture was stirred for 16 hours before the solvent was removed under reduced pressure. The mixture was diluted with 100 mL of EtOAc and 25 mL of water. The aqueous phase was discarded and the organic solution was washed with 20 mL of brine. The solvent was evaporated and the resulting oil was chromatographed (1: 1 EtOAc / hexane) to give the alkylated pyridone as a white solid.

¹ H NMR (CDCl ₃ ) d: 7.84 (bs, 1H), 7.33 to 7.17
(M, 5H), 5.87 (s, 1H), 4.79 (q, J = 17.2 Hz, 2H
), 3.36 (dd, J = 4.1, 13.5 Hz, 1H), 2.79 (m, 1H
), 2.65 (m, 2H), 2.48 (m, 1H), 2.23 (s, 3H), 1
. 48 (s, 9H). Step 1-E:

[0121]

Embedded image

A solution of the ester 1-4 (310 mg, 0.85 mmol) in DCM (30 mL) was brought to 0 ° C., and 8 mL of trifluoroacetic acid was added thereto. The reaction mixture was brought to room temperature over 5 hours with stirring and the solvent was removed under reduced pressure. The obtained solid is benzene,
Evaporated with EtOAc, then ether. In this way, the desired carboxylic acid was obtained as a white solid.

¹ H NMR (DMSO-d ₆ ) d: 8.92 (bs, 1H), 7.35 to 7.
10 (m, 5H), 6.04 (s, 1H), 4.75 (q, J = 17.2 Hz,
2H), 3.16 (dd, J = 4.2, 13.7 Hz, 1H), 2.79 (m,
1H), 2.65 to 2.40 (m, 3H), 2.1 (s, 3H). Step 1-F:

[0124]

Embedded image

The acid 1-5 (240 mg, 0.90 mmol) and 5-aminomethyl-2-b
237 mg (1.0 mmol) of oc-amino-6-methylpyridine in DMF (5
EDCI (192 mg, 1.0 mmol), HOBT (135 m
g, 1.0 mmol) and 0.22 mL of N-methylmorpholine. The reaction mixture was stirred for 16 hours before the solvent was removed under reduced pressure. The mixture was diluted with 20 mL of EtOAc and 5 mL of water. The aqueous phase was discarded and the organic solution was washed three times with 5 mL of water and then with 10 mL of brine. The solvent was distilled off, and chromatography (9: 1) was performed.
EtOAc / MeOH) to give the desired product as a white solid.

¹ H NMR (CDCl ₃ ) d: 8.47 (bs, 1H), 7.64 (d, J =
9.0 Hz, 1H), 7.43 to 7.19 (m, 7H), 7.05 (bs, 1H)
), 5.95 (s, 1H), 4.75 (s, 2H), 4.37 (s, 2H), 3
. 32 (dd, J = 4.1, 13.5 Hz, 1H), 2.79 to 2.0 (m, 4
H), 2.45 (s, 3H), 2.35 (s, 3H), 1.58 (s, 9H).
Stage 1-G:

[0127]

Embedded image

A solution of Bicyclic Compound 1-6 (410 mg, 0.85 mmol) was dissolved in 12 mL of a 1: 1 mixture of DCM and TFA. The reaction mixture was stirred for 3 hours and then
The solvent was removed under reduced pressure. The resulting solid was azeotroped with benzene, EtOAc and ether. In this manner, the desired compound was obtained as a white solid. Several 3 mg samples of the free base of this compound were each dissolved in MeOH (1 mL) and applied to a Chiralcel OD column (250 × 4.6 mm; A = hexane containing 0.1% diethylamine, B = EtOH, A: B = 15/85; flow rate = 7 mL
/ Min).

¹ H NMR (CD ₃ OD) d: 7.84 (d, J = 9.0 Hz, 1H), 7.
40-7.20 (m, 5H), 6.79 (d, J = 9.0 Hz, 1H), 6.1
8 (s, 1H), 4.80 (s, 2H), 4.27 (s, 2H), 3.16 (d
d, J = 4.1, 13.5 Hz, 1H), 2.85 to 2.44 (m, 4H), 2
. 0 (s, 3H), 2.38 (s, 3H).

Embodiment 2

[0131]

Embedded image

Compound 2 was prepared using the procedure described in Example 1 except that trimethyl 2-cyclobutyl-2-phosphonopropionate (I-2) was used instead of the Emmon's reagent in Step 1-A. -1 was prepared.

¹ H NMR (CD ₃ OD) d: 7.86 (d, J = 9.0 Hz, 1H), 6.
82 (d, J = 9.0 Hz, 1H), 6.20 (s, 1H), 4.80 (s, 2
H), 4.25 (s, 2H), 2.95 (dd, J = 4.1, 13.5 Hz, 1
H), 2.60 (m, 1H), 2.59 (s, 3H), 2.48 (m, 2H),
2.38 (s, 3H), 2.20-1.60 (m, 8H).

Embodiment 3

[0135]

Embedded image

Instead of 2- (3-fluorobenzyl) -2 as the emon reagent in Step 1-A
-Compound 3-1 was prepared using the procedure described in Example 1 except using trimethyl phosphonoacetate (I-5). The free base of this compound is released by the action of ammonia in chloroform and the compound is chromatographed (9: 1
NH ₃ saturated CHCl ₃ / CH ₃ OH) to give the final product.

¹ H NMR (CD ₃ OD) d: 7.40 (d, J = 9.0 Hz, 1H), 7.
35 (m, 2H), 7.00 (m, 2H), 6.42 (d, J = 9.0 Hz, 1
H), 6.15 (s, 1H), 4.80 (s, 2H), 4.25 (s, 2H),
3.25 (dd, J = 4.1, 13.5 Hz, 1H), 3.20 (m, 2H),
2.75 (m, 2H), 2.35 (s, 3H), 2.20 (s, 3H).

Embodiment 4

[0139]

Embedded image

Using the procedure described in Example 1, except that instead of trimethyl 2- (3,5-difluorobenzyl) -2-phosphonoacetate (I-8) as the emon reagent in Step 1-A. Compound (4-1) was produced. By the action of chloroform solution of ammonia to liberate the free base of the compound, chromatographed on the compound (9: 1N H ₃ saturated CHCl ₃ / _{CH 3} OH) performed to give the final product.

¹ H NMR (CD ₃ OD) d: 7.40 (d, J = 9.0 Hz, 1H), 7.
35 (m, 2H), 7.00 (m, 2H), 6.42 (d, J = 9.0 Hz, 1
H), 6.15 (s, 1H), 4.80 (s, 2H), 4.25 (s, 2H),
3.25 (dd, J = 4.1, 13.5 Hz, 1H), 3.20 (m, 2H),
2.75 (m, 2H), 2.35 (s, 3H), 2.20 (s, 3H).

Embodiment 5

[0143]

Embedded image

Using the procedure described in Example 1, except that instead of trimethyl 2- (2,5-difluorobenzyl) -2-phosphonoacetate (I-7) as the emon reagent in step 1-A. Compound (5-1) was produced.

¹ H NMR (CD ₃ OD) d: 7.80 (d, J = 9.0 Hz, 1H), 7.
2-6.9 (m, 3H), 6.82 (d, J = 9.0 Hz, 1H), 6.05 (s
, 1H), 4.80 (s, 2H), 4.35 (s, 2H), 3.25 (dd, J
= 4.1, 13.5 Hz, 1H), 2.8 to 2.6 (m, 4H), 2.52 (s
, 3H), 2.35 (s, 3H).

Embodiment 6

[0147]

Embedded image

The compound was prepared using the procedure described in Example 1 except that trimethyl 4- (S) -methyl-2-phosphonohexanoate (I-6) was used instead as the emon reagent in Step 1-A. (6-1) was produced.

¹ H NMR (CD ₃ OD) d: 7.86 (d, J = 9.0 Hz, 1H), 6.
82 (d, J = 9.0 Hz, 1H), 6.11 (s, 1H), 4.80 (s, 2H)
), 4.25 (s, 2H), 2.99 (m, 1H), 2.64 (m, 2H), 2
. 55 (s, 3H), 2.30 (s, 3H), 1.75 (m, 1H), 1.55
(M, 2H), 1.40 (m, 2H), 1.20 (m, 2H), 0.90 (m,
6H).

Embodiment 7

[0151]

Embedded image

Compound 7-1 was prepared using the procedure described in Example 1 except that commercially available trimethyl phosphonoacetate was used instead as the emon reagent in Step 1-A. The free base of this compound was liberated by the action of ammonia in chloroform and the compound was chromatographed (1: 1 NH ₃ saturated CHCl ₃ / CH ₃ OH) to give the final product.

¹ H NMR (CD ₃ OD) d: 7.40 (d, J = 9.0 Hz, 1H), 6.
40 (d, J = 9.0 Hz, 1H), 6.21 (s, 1H), 4.80 (s, 2
H), 4.25 (s, 2H), 2.85 (t, J = 7 Hz, 1H), 2.60 (
t, J = 7 Hz, 2H), 2.35 (s, 3H), 2.30 (s, 3H).

[0154]An in vitro assay for measuring proteinase inhibition.  Assays for human α-thrombin and human trypsin were performed at 25 ° C. in 0.05 M TRIS buffer (pH 7.4), 0.15 M NaCl, 0.1% PEG. In the trypsin assay, 1 mM CaCl₂Was also contained.

For the measurement of the rate of hydrolysis of p-nitroanilide (pna) substrate, the time-dependent appearance of p-nitroaniline was measured (at 405 nm) using a Thermomax 96-well plate reader. Assays for human α-thrombin (K _m = 125 μM) and human trypsin (K _m = 59 μM) were performed using sar-PR-pna (sarcosine-Pro-Arg-p-nitroanilide).
The p-nitroanilide substrate concentration was determined using an extinction coefficient of 8270 cm ⁻¹ M ⁻¹ .
It was determined from the measured absorbance at 342 nm.

Certain studies with potent inhibitors of high thrombin inhibition (K _i <10 nM) used a more sensitive activity assay. In the assay, the fluorogenic substrate Z-GPR-afc (Cbz-Gly-Pro-Arg-7-amino-
4-trifluoromethyl _coumarin) thrombin-catalyzed hydrolysis rate of _(K m = _27μM), associated with the 7-amino-4-trifluoromethyl coumarin production 500
Determined from the increase in fluorescence at nm (excitation at 400 nm). The concentration of the stock solution of Z-GPR-afc was determined from the measured absorbance at 380 nm of 7-amino-4-trifluoromethylcoumarin produced by complete hydrolysis of an aliquot sample of the stock solution with thrombin.

Assays for activity are performed by diluting a stock stock of substrate 10-fold or more to a final concentration ≦ 0.
. 5K_mAnd a solution containing the enzyme in equilibrium with the enzyme or inhibitor.
Was. The time required to achieve equilibrium between enzyme and inhibitor was determined in control experiments
. Initial rate of product formation in the absence of inhibitor (V₀) Or raw in the presence of inhibitors
Initial rate of product formation (V_i) Was measured. There is competitive inhibition, K_m/ [S], [
I] / e and [I] / e ([S], [I] and e are the substrate and inhibitor, respectively)
And represents the total concentration of the enzyme), assuming that 1 is negligible compared to
Equilibrium constant for dissociation of drug from enzyme (K_i) Is V to the [I] shown in Equation 1. ₀ / V_iFrom the dependence of

[0158]

(Equation 1)

The activity demonstrated by this assay indicates that the compounds of the invention show that the compounds of the present invention are unstable angina, refractory angina, myocardial infarction, transient ischemic attack, atrial fibrillation, thrombotic stroke, embolic Stroke,
It has shown therapeutic utility in treating various conditions in patients suffering from deep vein thrombosis, disseminated intravascular coagulation, and reocclusion or restenosis of revascularized vessels.

[0160]In vivo test to measure thrombotic occlusion  The method described in the report by Kurtz et al. (Kurtz et al.,Thrombosis Research, No.60,
p.269 (1990)), using the rat ferric chloride assay described below.
3- (2-phenylethylamino) -6-methyl-1- (2-amino-6-
Trial of methyl-5-methylenecarboxamidomethylpyridinyl) -2-pyridinone
The thrombin inhibitor of the present inventionin vivoActivity was determined. Dial-urethane solution (0.1 mL per 100 g body weight, intraperitoneal injection)
Anesthetize Plague Dawley rats (weight: 200-350 g) and have a length of 12 a.
Cannulate the lateral tail vein using a 23 gauge needle connected to the
Les intubated. The tubing was attached to the three-way valve by a tubing adapter. Physiological saline (control)
Alternatively, the appropriate test compound was administered via a tail vein catheter. Length 0
. Tracheostomy was performed using a 75 inch PE205 tube. Exposed right carotid artery
And a 1.3 mm diameter Doppler flow probe was attached to the vessel. Use heating lamp
And the body temperature was maintained at 37 ° C.

[0161] Six rats were randomized to continuous intravenous infusion of saline or test compound administered via the tail vein. The test compound was administered at a rate of 10 μg / kg / min. 60 minutes after the start of dosing infusion, ₃ saturated with 35% FeCl 3
mm square Whatman no. One filter paper was attached to the exposed carotid artery distal to the flow probe. When thrombus occlusion did not occur, administration was continued for an additional 90 minutes after administration of FeCl ₃ (total infusion period of 150 minutes), or the infusion was terminated 30 minutes after thrombus occlusion of the blood vessel occurred. The time to occlusion is F
The time from eCl ₃ administration to thrombotic occlusion of the blood vessel was defined as At the end of the study (90 min after FeCl ₃ administration in animals without occlusion or 30 min after thrombotic occlusion), cardiac puncture into 0.3 mL of 3.8% sodium citrate.
3 mL of blood was collected.

[0162]Example 8 Tablet manufacturing  Compound 1-7 ("active compound") was added at 25.0, 50.0 and 100, respectively.
. Tablets containing 0 mg are prepared as described below.

[0163]

[Table 5]

The active compound, cellulose and part of the corn starch are all mixed and granulated to give a 10% corn starch paste. The resulting granules are sieved, dried and mixed with the remaining corn starch and magnesium stearate. Next, the obtained fine granules were tableted, and the active ingredient per tablet was 25.0 mg and 50.0 mg, respectively.
And a tablet containing 100.0 mg.

[0165]Example 9 Tablet manufacturing  A composition example of the compound 1-7 tablet is shown below.

[0166]

[Table 6]

The 2 mg, 10 mg and 50 mg tablets are hydroxypropylcellulose,
The film was coated with an aqueous dispersion of hydroxypropylmethylcellulose and titanium oxide to give an apparent weight gain of 2.4%.

[0168]Tablet production by direct compression  The active ingredient IV, mannitol and microcrystalline cellulose are added to a specified diameter (generally 2 μm).
(50-750 μm) mesh screen and combined with a suitable mixer. Next
The mixture was mixed until the drug was evenly dispersed in the resulting dry powder mixture.
(Usually 15-30 minutes). Sift magnesium stearate and add to mixer
Followed by further mixing (typically 2-10 minutes) to obtain a pre-compressed mixture. Next
In addition, the pre-compressed mixture has an acceptable disintegration time (specifications are
And force to obtain tablets of suitable physical strength (depending on
Compression (usually in the range of 0.5-2.5 metric tons). titer
For 2 mg, 10 mg and 50 mg, the tablets are dedusted and the water-soluble polymer and
The film was coated with an aqueous dispersion of the pigment.

[0169]Tablet manufacturing using dried fine granules  Alternatively, the dry powder mixture can be compressed by applying light force and reground,
Fine particles of a predetermined particle size are obtained. Mix the granules with magnesium stearate and mix as described above.
Tablet.

[0170]Example 10 Intravenous formulation  An intravenous formulation of compound 1-7 was prepared according to a general intravenous formulation method.

[0171]

[Table 7]

Examples of the compositions A to C include the following.

[0173]

[Table 8]

Instead of glucuronic acid, L-lactic acid, acetic acid, citric acid or any other variety such as any pharmaceutically acceptable acid / conjugate base having a reasonable buffering capacity in the pH range suitable for intravenous administration. Buffer acid can be used.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 9/10 A61P 9/10 43/00 43/00 111 111 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ) , MD, RU, TJ, TM), AL, AM, AU, AZ, BA, BB, BG, BR, BY, CA, CN, CU, CZ, EE, GE, HR, HU, ID, IL, IS, JP, KG, KR, KZ, LC, LK, LR, LT, LV, MD, MG, MK, MN, MX, NO, NZ, PL, RO, RU, SG, SI, SK, SL , TJ, TM, TR, TT, UA, US, UZ, VN, YU Williams, Peter Day United States, New Jersey 07065, Lowway, East Lincoln Avenue, 126 F-term (reference) ZA45 ZA54

Claims (32)

[Claims] 1. A compound having the structure: and a pharmaceutically acceptable compound thereof.
Salt. Embedded image Wherein X is O or H₂R¹And R²Is independently hydrogen, C_1-6Alkyl-, C_2-6Alkenyl, C_2-6Alkynyl, C_3-8Cycloalkyl-, C_3-8Cycloalkyl C_1-6Alkyl-, di (C_3-8Cycloalkyl) C_1-6Alkyl-, aryl, aryl C_1-6Alkyl-, di (aryl) C_1-6Alkyl-; wherein aryl is unsubstituted or -OH,
-NH₂, C_1-6Alkyl, C_3-8Substitute with cycloalkyl or halogen
B is an unsaturated 6-membered carbocyclic ring;In the above formula, R⁴And R⁵Is independently hydrogen, C_1-4Alkyl, C_2-4Alkenyl, C_2-4Alkynyl, C_1-4Alkoxy, halogen, -COOH, -OH, -COOR⁷(R⁷Is C_1-4Alkyl)), -CONR⁸R⁹(R⁸And R⁹Is independently hydrogen or C_1-4Alkyl
), -OCH₂CO₂H, -OCH₂CO₂CH₃, -OCH₂CO₂(CH₂)_1-3CH₃, -O (CH₂)_1-3C (O) NR¹⁰R¹¹(R¹⁰And R¹¹Is independent
, Hydrogen, C_1-4Alkyl, C_3-7Cycloalkyl or -CH₂CF₃so
Yes),-(CH₂)_1-4OH, -NHC (O) CH₃, -NHC (O) CF₃, -NHSO₂CH₃, -SO₂NH₂  Or B is selected from the group consisting of:In the above formula, R⁶And R¹²May be the same or different, hydrogen, C_1-6Alkyl-, C_2-6Alkenyl-, C_2-6Alkynyl-, C_3-8Cycloalkyl-, aryl, aryl C_1-6Wherein the aryl is unsubstituted or -OH, -NH₂, C_{1- 6} Alkyl, C_3-8Unsaturated 6 substituted by cycloalkyl or halogen
Membered carbocycle. ] 2. The compound according to claim 1, having the following structure: and a pharmaceutically acceptable salt of said compound. Embedded image Wherein R ¹ and R ² are independently hydrogen, C _1-6 alkyl-, C _3-8 cycloalkyl C _1-6 alkyl-, di (C _3-8 cycloalkyl) C _1-6 alkyl- ArylC _1-6 alkyl-, di (aryl) C _1-6 alkyl-; wherein aryl is unsubstituted or mono- or di-substituted by halogen; Formula 5 Wherein R ⁴ and R ⁵ are independently selected from the group consisting of hydrogen, halogen, or —OCH ₂ C (O) NHR ^11, wherein R ¹¹ is C _1-4 alkyl; Or B is In the above formula, R ⁶ and R ¹² may be the same or different, and represent hydrogen or-
CH _3. ] 3. The compound according to claim 2, having the following structure: and a pharmaceutically acceptable salt of said compound. Embedded image Wherein R ¹ is hydrogen, R ² is hydrogen, CH ₃ , C _1-6 alkyl and aryl C _1-6 alkyl; B is It is. ] 4. The compound according to claim 3, having the structural formula: and a pharmaceutically acceptable salt of said compound. Embedded image Wherein R ¹ is hydrogen, It is. ] 5. The compound according to claim 4, which is selected from the group consisting of the following compounds. Embedded image Embedded image 6. A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier. 7. A method for inhibiting thrombin in blood, comprising the step of adding the composition according to claim 6 to blood. 8. A method for inhibiting platelet aggregate formation in blood, comprising the step of adding the composition according to claim 6 to blood. 9. A method for inhibiting thrombus formation in blood, comprising the step of adding the composition according to claim 6 to blood. 10. A method for inhibiting thrombus formation in blood, comprising a step of adding the compound according to claim 1 to blood together with a fibrinogen receptor antagonist. 11. The compound according to claim 1 or a medicament of said compound for the manufacture of a medicament for inhibiting thrombus formation, preventing thrombus formation, inhibiting thrombin, inhibiting fibrin formation and inhibiting platelet aggregate formation in a mammal. Use of chemically acceptable salts. 12. A method of treating or preventing venous thromboembolism and pulmonary embolism in a mammal, comprising administering the composition of claim 6 to the mammal. 13. A method for treating or preventing deep vein thrombosis in a mammal, comprising administering the composition according to claim 6 to the mammal. 14. A method for treating or preventing cardiogenic thromboembolism in a mammal, comprising administering the composition of claim 6 to the mammal. 15. A method for treating or preventing thromboembolic stroke in humans and other mammals, comprising administering the composition of claim 6 to said mammal. 16. A method of treating or preventing cancer and thrombosis associated with cancer chemotherapy in a mammal, comprising administering the composition of claim 6 to the mammal. 17. A method for treating or preventing unstable angina in a mammal, comprising administering the composition of claim 6 to the mammal. 18. A method for treating or preventing myocardial infarction in a mammal, comprising administering the composition according to claim 6 to the mammal. 19. A method for treating or preventing atrial fibrillation-related cardiogenic thromboembolism in a mammal, comprising administering the composition according to claim 6 to the mammal. 20. A method for treating or preventing prosthetic heart valve-related cardiogenic thromboembolism in a mammal, comprising administering the composition according to claim 6 to the mammal. 21. A method for treating or preventing heart disease-related cardiogenic thromboembolism in a mammal, comprising administering the composition according to claim 6 to the mammal. 22. A method for treating or preventing atherosclerosis in a mammal, comprising administering the composition according to claim 6 to the mammal. 23. Protein C deficiency, protein S deficiency, antithrombin III and factor V, comprising the step of administering the composition of claim 6 to a mammal.
A method for treating or preventing thrombosis in a mammal having a congenital thrombogenic disease such as Leiden. 24. A method for treating or preventing thrombosis in a mammal having an acquired thrombogenic disorder such as systemic lupus erythematosus, comprising administering the composition according to claim 6 to the mammal. 25. A method for treating or preventing ischemic heart disease in a mammal, comprising administering the composition according to claim 6 to the mammal. 26. A method of reducing the tendency of a device in contact with blood to coagulate blood, comprising the step of coating the device with the composition of claim 6. 27. A method for treating or preventing reocclusion in a mammal during or after percutaneous transluminal coronary angioplasty, comprising the step of administering the composition according to claim 6 to the mammal. 28. A method for treating or preventing restenosis in a mammal after percutaneous transluminal coronary angioplasty, comprising administering the composition of claim 6 to the mammal. 29. A method for treating or preventing occlusion of a coronary artery bypass graft in a mammal, comprising administering the composition according to claim 6 to the mammal. 30. A method for treating or preventing obstructive cerebrovascular disease in a mammal, comprising a step of administering the composition according to claim 6 to the mammal. 31. A method for maintaining patency in an arteriovenous cannula intubated in a mammal, comprising administering the composition according to claim 6 to the mammal. 32. A method for maintaining the patency of an indwelling catheter in a mammal, comprising administering the composition according to claim 6 to the mammal.