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WO2003011222A2 - Thrombin inhibitors - Google Patents

Thrombin inhibitors Download PDF

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Publication number
WO2003011222A2
WO2003011222A2 PCT/US2002/024219 US0224219W WO03011222A2 WO 2003011222 A2 WO2003011222 A2 WO 2003011222A2 US 0224219 W US0224219 W US 0224219W WO 03011222 A2 WO03011222 A2 WO 03011222A2
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WO
WIPO (PCT)
Prior art keywords
benzyl
mmol
chloro
tricyclo
pentaene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2002/024219
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French (fr)
Other versions
WO2003011222A3 (en
Inventor
Philippe G. Nantermet
Harold G. Selnick
James C. Barrow
Christina L. Newton
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck and Co Inc
Original Assignee
Merck and Co Inc
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Filing date
Publication date
Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Priority to AU2002322802A priority Critical patent/AU2002322802A1/en
Publication of WO2003011222A2 publication Critical patent/WO2003011222A2/en
Anticipated expiration legal-status Critical
Publication of WO2003011222A3 publication Critical patent/WO2003011222A3/en
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/06—Peri-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/06—Peri-condensed systems

Definitions

  • Thrombin is a serine protease present in blood plasma in the form of a precursor, prothrombin. Thrombin plays a central role in the mechanism of blood coagulation by converting the solution plasma protein, fibrinogen, into insoluble fibrin.
  • European Publication 363 284 describes analogs of peptidase substrates in which the nitrogen atom of the scissile amide group of the substrate peptide has been replaced by hydrogen or a substituted carbonyl moiety.
  • Australian Publication 86245677 also describes peptidase inhibitors having an activated electrophilic ketone moiety such as fluoromethylene ketone or a- keto carboxyl derivatives.
  • the invention includes compounds for inhibiting loss of blood platelets, inhibiting formation of blood platelet aggregates, inhibiting formation of fibrin, inhibiting thrombus formation, and inhibiting embolus formation in a mammal, comprising a compound of the invention in a pharmaceutically acceptable carrier.
  • These compounds may optionally include anticoagulants, antiplatelet agents, and thrombolytic agents.
  • the compounds can be added to blood, blood products, or mammalian organs in order to effect the desired inhibitions.
  • the invention also includes a compound for preventing or treating unstable angina, refractory angina, myocardial infarction, transient ischemic attacks, atrial fibrillation, thrombotic stroke, embolic stroke, deep vein thrombosis, disseminated intravascular coagulation, ocular build up of fibrin, and reocclusion or restenosis of recanalized vessels, in a mammal, comprising a compound of the invention in a pharmaceutically acceptable carrier.
  • These compounds may optionally include anticoagulants, antiplatelet agents, and thrombolytic agents.
  • the invention also includes a method for reducing the thrombogenicity of a surface in a mammal by attaching to the surface, either covalently or noncovalently, a compound of the invention.
  • Compounds of the invention are useful as thrombin inhibitors and have therapeutic value in for example, preventing coronary artery disease.
  • the invention includes compounds having the following structure:
  • R 1 is aryl, C ⁇ -4 alkyl, or C 3-7 cycloalkyl;
  • R >2 is hydrogen, C 1-4 alkyl, C 3-7 cycloalkyl, halogen, or CN;
  • R 3 is hydrogen, C 1-4 alkyl, C 3- cycloalkyl, halogen or CN;
  • R 4 is hydrogen, C 1-4 alkyl or -ZCOOH, wherein Z is C 1-4 alkyl;
  • A is selected from the group consisting of a) — (CHz X 1 — where X 1 is O or CH 2j
  • Y 1 is O, S, SO, SO 2 or NR 5
  • X 2 is O or CH 2
  • Y 2 is O, S, SO, SO 2 or NR 5
  • X 3 is O or CH 2
  • X is O or CH 2 , provided that when X is O, n and m are independently 1, 2 or 3, and that when ⁇ is CH 2 , n is 1, 2 or 3, and m is 2 or 3, or e) I— (CH 2 ) n -NR 5 -C(O)-(CH 2 ) m -X 5
  • X is O or CH 2 , provided that when X is O, n and m are independently 1, 2 or 3, and when X is CH 2 , n is 1, 2 or 3 and m is 2 or 3;
  • R 5 is hydrogen, C ⁇ -4 alkyl or -ZOOH, where Z is C 1-4 alkyl.
  • R is hydrogen or halogen and R 3 is hydrogen or halogen.
  • R 2 is hydrogen or CI and R 3 is hydrogen or
  • R 1 is phenyl or cyclopropyl.
  • R 4 is hydrogen, methyl, ethyl, or - CH 2 COOH.
  • R 5 is hydrogen, methyl or ethyl.
  • A is selected from the group consisting of -(CH 2 ) 4 -O-, -(CH 2 ) 3 -O-, -(CH 2 ) 5 -O-, -(CH 2 ) 6 -O-,
  • the compounds of the present invention may have chiral centers and occur as racemates, racemic mixtures and as individual diastereomers, or enantiomers with all isomeric forms being included in the present invention.
  • the compounds of the present invention may also have polymorphic crystalline forms, with all polymorphic crystalline forms being included in the present invention.
  • alkyl is intended to include both branched- and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms (Me is methyl, Et is ethyl, Pr is propyl, Bu is butyl); "alkoxy” represents a linear or branched alkyl group of indicated number of carbon atoms attached through an oxygen bridge; "halogen”, as used herein, means fluoro, chloro, bromo and iodo; and "counterion” is used to represent a small, single negatively-charged species, such as chloride, bromide, hydroxide, acetate, trifluoroacetate, perchlorate, nitrate, benzoate, maleate, sulfate, tartrate, hemitartrate, benzene sulfonate, and the like.
  • cycloC3_7alkyl is intended to include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, and the like.
  • aryl as used herein except where noted, represents a stable 6- to 10-membered mono- or bicyclic ring system such as phenyl, or naphthyl.
  • the aryl ring can be unsubstituted or substituted with one or more of C1 -.4 lower alkyl; hydroxy; alkoxy; halogen; amino.
  • the pharmaceutically-acceptable salts of the compounds of Formula I include the conventional non-toxic salts such as those derived from inorganic acids, e.g. hydrochloric, hydrobromoic, sulfuric, sulfamic, phosphoric, nitric and the like, or the quaternary ammonium salts which are formed, e.g., from inorganic or organic acids or bases.
  • acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pamoate, pectinate, persulfate, 3- ⁇ henylpropionate, picrate, pivalate, propionate, succinate, sulfate,
  • Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth.
  • the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides
  • dialkyl sulfates like dimethyl, diethyl, dibutyl
  • diamyl sulfates long chain halides
  • 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 cerebro vascular disease. Those experienced in this field are readily aware of the circumstances requiring anticoagulant therapy.
  • patient used herein is taken to mean mammals such as primates, including humans, sheep, horses, cattle, pigs, dogs, cats, rats, and mice.
  • Thrombin inhibition is useful not only in the anticoagulant therapy of individuals having thrombotic conditions, but is useful whenever inhibition of blood coagulation is required such as to prevent coagulation of stored whole blood and to prevent coagulation in other biological samples for testing or storage.
  • the thrombin inhibitors can be added to or contacted with any medium containing or suspected of containing thrombin and in which it is desired that blood coagulation be inhibited, e.g., when contacting the mammal's blood with material selected from the group consisting of vascular grafts, stents, orthopedic prosthesis, cardiac prosthesis, and extracorporeal circulation systems.
  • Compounds of the invention are useful for treating or preventing venous thromboembolism (e.g.
  • obstruction or occlusion of a vein by a detached thrombus obstruction or occlusion of a lung artery by a detached thrombus
  • cardiogenic thromboembolism e.g. obstruction or occlusion of the heart by a detached thrombus
  • arterial thrombosis e.g. formation of a thrombus within an artery that may cause infarction of tissue supplied by the artery
  • atherosclerosis e.g. arteriosclerosis characterized by irregularly distributed lipid deposits
  • Examples of venous thromboembolism which may be treated or prevented with compounds of the invention include obstruction of a vein, obstruction of a lung artery (pulmonary embolism), deep vein thrombosis, thrombosis associated with cancer and cancer chemotherapy, thrombosis inherited with thrombophilic diseases such as Protein C deficiency, Protein S deficiency, antithrombin HI deficiency, and Factor V Leiden, and thrombosis resulting from acquired thrombophilic disorders such as systemic lupus erythematosus (inflammatory connective tissue disease). Also with regard to venous thromboembolism, compounds of the invention are useful for maintaining patency of indwelling catheters.
  • cardiogenic thromboembolism examples include thromboembolic stroke (detached thrombus causing neurological affliction related to impaired cerebral blood supply), cardiogenic thromboembolism associated with atrial fibrillation (rapid, irregular twitching of upper heart chamber muscular fibrils), cardiogenic thromboembolism associated with prosthetic heart valves such as mechanical heart valves, and cardiogenic thromboembolism associated with heart disease.
  • arterial thrombosis examples include unstable angina (severe constrictive pain in chest of coronary origin), myocardial infarction (heart muscle cell death resulting from insufficient blood supply), ischemic heart disease (local anemia due to obstruction (such as by arterial narrowing) of blood supply), reocclusion during or after percutaneous transluminal coronary angioplasty, restenosis after percutaneous transluminal coronary angioplasty, occlusion of coronary artery bypass grafts, and occlusive cerebrovascular disease.
  • compounds of the invention are useful for maintaining patency in arteriovenous cannulas.
  • atherosclerosis examples include arteriosclerosis.
  • devices that come into contact with blood include vascular grafts, stents, orthopedic prosthesis, cardiac prosthesis, and extracorporeal circulation systems
  • the thrombin inhibitors of the invention can be administered in such oral forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixers, tinctures, suspensions, syrups, and emulsions. Likewise, they may be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non- toxic amount of the compound desired can be employed as an anti-aggregation agent. For treating ocular build up of fibrin, the compounds may be administered intraocularly or topically as well as orally or parenterally.
  • the thrombin inhibitors can be administered in the form of a depot injection or implant preparation which may be formulated in such a manner as to permit a sustained release of the active ingredient.
  • the active ingredient can be compressed into pellets or small cylinders and implanted subcutaneously or intramuscularly as depot injections or implants.
  • Implants may employ inert materials such as biodegradable polymers or synthetic silicones, for example, Silastic, silicone rubber or other polymers manufactured by the Dow-Corning Corporation.
  • the thrombin inhibitors can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
  • the thrombin inhibitors may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.
  • the thrombin inhibitors may also be coupled with soluble polymers as targetable drug carriers.
  • Such polymers can include polyvinlypyrrolidone, pyran copolymer, polyhydroxy-propyl-methacrylamide-phenol, polyhydroxyethyl- aspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues.
  • the thrombin inhibitors may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels.
  • biodegradable polymers useful in achieving controlled release of a drug
  • a drug for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels.
  • the dosage regimen utilizing the thrombin inhibitors is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed.
  • An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.
  • Oral dosages of the thrombin inhibitors when used for the indicated effects, will range between about 0.01 mg per kg of body weight per day (mg/kg/day) to about 30 mg/kg/day, preferably 0.025-7.5 mg/kg/day, more preferably 0.1-2.5 mg/kg/day, and most preferably 0.1-0.5 mg/kg/day (unless specificed otherwise, amounts of active ingredients are on free base basis).
  • an 80 kg patient would receive between about 0.8 mg/day and 2.4 g/day, preferably 2-600 mg/day, more preferably 8-200 mg/day, and most preferably 8-40 mg/kg/day.
  • a suitably prepared medicament for once a day administration would thus contain between 0.8 mg and 2.4 g, preferably between 2 mg and 600 mg, more preferably between 8 mg and 200 mg, and most preferably 8 mg and 40 mg, e.g., 8 mg, 10 mg, 20 mg and 40 mg.
  • the thrombin inhibitors may be administered in divided doses of two, three, or four times daily.
  • a suitably prepared medicament would contain between 0.4 mg and 4 g, preferably between 1 mg and 300 mg, more preferably between 4 mg and 100 mg, and most preferably 4 mg and 20 mg, e.g., 4 mg, 5 mg, 10 mg and 20 mg.
  • the patient would receive the active ingredient in quantities sufficient to deliver between 0.025-7.5 mg/kg/day, preferably 0.1-2.5 mg/kg/day, and more preferably 0.1-0.5 mg/kg/day.
  • Such quantities may be administered in a number of suitable ways, e.g. large volumes of low concentrations of active ingredient during one extended period of time or several times a day, low volumes of high concentrations of active ingredient during a short period of time, e.g. once a day.
  • a conventional intravenous formulation may be prepared which contains a concentration of active ingredient of between about 0.01-1.0 mg/ml, e.g.
  • 0.1 mg/ml, 0.3 mg/ml, and 0.6 mg/ml and administered in amounts per day of between 0.01 ml/kg patient weight and 10.0 ml/kg patient weight, e.g. 0.1 ml/kg, 0.2 ml/kg, 0.5 ml/kg.
  • an 80 kg patient receiving 8 ml twice a day of an intravenous formulation having a concentration of active ingredient of 0.5 mg/ml, receives 8 mg of active ingredient per day.
  • Glucuronic acid, L-lactic acid, acetic acid, citric acid or any pharmaceutically acceptable acid/conjugate base with reasonable buffering capacity in the pH range acceptable for intravenous administration may be used as buffers. Consideration should be given to the solubility of the drug in choosing an The choice of appropriate buffer and pH of a formulation, depending on solubility of the drug to be administered, is readily made by a person having ordinary skill in the art.
  • the compounds can also be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will, or course, be continuous rather than intermittent throughout the dosage regime.
  • thrombin inhibitors are typically administered as active ingredients in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier” materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixers, syrups and the like, and consistent with convention pharmaceutical practices.
  • carrier suitable pharmaceutical diluents, excipients or carriers
  • the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid form, the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, distintegrating agents and coloring agents can also be incorporated into the mixture.
  • suitable binders, lubricants, distintegrating agents and coloring agents can also be incorporated into the mixture.
  • Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn-sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Disintegrators include, without limitation, starch methyl cellulose, agar, bentonite, xanthan gum and the like.
  • the compounds claimed in this invention can be prepared according to the following general procedures.
  • scheme 1 ethyl oxalyl chloride is converted in four steps to the bromopyrazinone 1.
  • Addition of an -(azidomethyl)-alkylamine hydrochloride to bromopyrazinone 1, followed by chlorination of the pyrazinone and reduction of the azide to the corresponding amine gives access to aminomethyl derivatives of type 2.
  • Alkylation of aminomethyl derivatives of type 2 with tertbutylbromoacetate followed by hydrolysis with aqueous LiOH leads to acids of type 3.
  • Acylation of aminomethyl derivatives of type 2 with allyl chloroformate followed by hydrolysis with aqueous LiOH leads to acids of type 4.
  • phenols of type 5 can be converted to bromides of type 6 by alkylation with dibromide intermediates.
  • Alkylation of aminomethyl derivatives of type 2 with bromides of type 6 leads to Boc-amino ester derivatives of type 7. Removal of the Boc group, followed by hydrolysis of the ester and cyclization under EDC / HO At conditions provides macrocycles of type 8.
  • Scheme 3 illustrates an alternative preparation of ethyl bromide derivative 9 from phenol 5, via alkylation with ethyl bromoacetate, reduction and bromination of the resulting primary alcohol.
  • Alkylation of 2-mercaptoethanol with bromide 9 followed by bromination affords bromides of type 10.
  • Alkylation of aminomethyl derivatives of type 2 with bromides of type 10 leads to Boc-amino ester derivatives of type 11. Removal of the Boc group, followed by hydrolysis of the ester and cyclization under EDC / HOAt conditions provides macrocycles of type 12.
  • Scheme 4 illustrates the preparation of macrocycle 15 with an amide type linker.
  • Alylation of alkylglycine ethyl ester with bromide 9 followed by ester hydrolysis leads to acid 13.
  • EDC/HOAt coupling of acid 13 to aminomethyl derivative 2 provides intermediate 14 which can be cyclized to 15 following the same procedure as described in the previous schemes.
  • Scheme 5 illustrates an alternative mode of macrocyclization.
  • Amine 16 can be prepared by akylation of bromide 9.
  • EDC/HOAt coupling of acid 3 to benzylaminel derivative 16 provides intermediate 17 which can be cyclized to 18 after tertbutyl removal.
  • Scheme 6 illustrates an alternative mode of coupling between the pyrazinone derivative and the phenolic derivative.
  • Aldehyde 19 is obtained from phenol 5 using the following three steps sequence: alkylation with a bromoester, reduction of the ester and oxidation to the corresponding aldehyde.
  • Reductive amination with aminomethyl derivative 2 provides the intermediates of type 20.
  • this alternative mode of linker introduction was found to be more efficient than the original bromide alkylation procedure utilized in schemes 2 and 3. Macrocyclization of intermediate 20 to macrocycle 21 is accomplished using the same sequence as described earlier.
  • Scheme 7 illustrates the preparation of aldehyde 22 and its attachment to the pyrazinone core via reductive amination as described in scheme 6, followed by macrocyclization to 24, as described before.
  • Scheme 8 illustrates the preparation of macrocycles of type 28 which present an amino type linker.
  • Alkylation of a primary amine of type 25 with a bromoester followed by Boc protection and conversion of the ester to the corresponding aldehyde leads to aldehyde derivatives of type 26.
  • Reductive amination with aminomethyl derivative 2 and macrocyclization provides macrocycles of type 28.
  • Scheme 9 illustrates yet another mode of macrocyclization which utilizes Grubbs type olefin metathesis (Grubbs, R. H., Chang, S. Tetrahedron, 1998, 54, 4413-4450).
  • Allyl derivative 29 is prepared from phenol 5 and allyl bromide.
  • Coupling of acid 4 and amine 29 under EDC HOAt conditions leads to bis-olefin derivative 30 which is converted to an E/Z mixture of macrocycles of type 31 via Grubbs olefin metathesis.
  • Hydrogenation on Pd/C provides derivatives of type 32.
  • Scheme 10 illustrates further modifications of the previously described macrocycles.
  • Dehalogenation with hydrogen provides derivatives of type 33 and 34.
  • Reductive alkylation leads to amino derivatives of type 35.
  • a carboxymethyl group can be attached to afford structures of type 36.
  • Step B preparation of (S)-[3-(l-Aminomethyl-2-phenyl-ethylamino)-6-chloro-2-oxo- 2H-pyrazin-l-yl] -acetic acid ethyl ester
  • reaction mixture is concentrated in vacuo and purified by flash chromatography (silica gel, 20% diethyl ether in hexane to 45%) to give (S)-[3-(l-Azidomethyl-2-phenyl-ethylamino)-2-oxo- 2H-6-chloro-pyrazin-l-yl] -acetic acid ethyl ester (5.6 g) as a thick syrup.
  • Step A preparation of [3-(l-(S)-Benzyl-2- ⁇ 4-[2-(tert-butoxycarbonylamino-methyl)- 4-chloro-phenoxy] -butylamino ⁇ -ethylamino)-6-chloro-2-oxo-2H-pyrazin- 1 -yl] -acetic acid ethyl ester
  • reaction mixture is diluted with EtOAc, washed with water and aqueous LiCl (x3), dried on sodium sulfate, concentrated in vacuo and purified by flash chromatography (silica gel, 10% EtOAc in hexane to 20%) to give [2-(4-Bromo- butoxy)-5-chloro-benzyl]-carbamic acid tert-butyl ester (1.13 g).
  • Step B cyclization to 19-(S)-Benzyl-8,24-dichloro-12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,ll)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione dihydrochloride
  • the dihydrochloride salt is obtained by treatment of a solution of the free base in CH 2 C1 2 with 2N HCl in Et O.
  • the crude material is purified by reverse phase preparative HPLC (5% to 95% CH 3 CN in water containing 0.1 % TFA, C18 PRO YMC 20x150 mm) to provide after extraction and hydrochloride preparation under standard conditions (S)-19-Benzyl-8- chloro-12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,ll)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione dihydrochloride.
  • EXAMPLE 8 (S)-18-Benzyl-8,23-dichloro-12-oxa-l,4,16,19,21-pentaaza tricyclo[18.3.1.0(6,11)] tetracosa-6(l 1),7,9,20, 22-pentaene-3,24-dione
  • (S)-18-Benzyl-8,23-dichloro-12-oxa-l,4,16,19,21-pentaaza tricyclo[18.3.1.0(6,ll)] tetracosa-6(ll),7,9,20, 22-pentaene-3,24-dione is prepared from [2-(3-Bromo-propoxy)-5-chloro-benzyl]-carbamic acid tert-butyl ester (prepared from (5-Chloro-2-hydroxy-benzyl)-carbamic acid tert-butyl ester and 1,3- dibromopropane) and (S)-[3-(l-Aminomethyl-2-phenyl-ethylamino)-6-chloro-2-oxo- 2H-pyrazin-l-yl] -acetic acid ethyl ester, using a similar procedure as described in example 5.
  • [21.3.1.0(6,ll)]-heptacosa-6(ll),7,9,23, 25-pentaene-3,27-dione is prepared from [2- (6-BiOmo-hexoxy)-5-chloro-benzyl]-carbamic acid tert-butyl ester (prepared from (5- Chloro-2-hydroxy-benzyl)-carbamic acid tert-butyl ester and 1,6-dibromohexane) and (S)-[3-(l-Aminomethyl-2-phenyl-ethylamino)-6-chloro-2-oxo-2H-pyrazin-l-yl]-acetic acid ethyl ester, using a similar procedure as described in example 5.
  • EXAMPLE 12 (S)-19-Benzyl-17-Methyl-8,24-dichloro-12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,11)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione bis TFA salt
  • EXAMPLE 16 (S)-20-Cyclopropylmethyl-18-Methyl-8,25-dichloro-12-oxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6, 11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione bis TFA salt
  • EXAMPLE 17 (S)-20-Cyclopropylmethyl-8 ,25-dichloro- 12, 15-dioxa- 1 ,4, 18 ,21 ,23-pentaaza tricyclo[20.3.1.0(6,11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione
  • Step A Preparation of ⁇ 5-Chloro-2-[2-(2-hydroxy-ethoxy)-ethoxy]-benzyl ⁇ -carbamic acid tert-butyl ester
  • reaction mixture is concentrated in vacuo, diluted with CH 2 C1 2 , washed with water, concentrated in vacuo and purified by flash chromatography (silica gel, 20% EtOAc in hexane to 70%) to give ⁇ 5-Chloro-2-[2-(2-hydroxy-ethoxy)-ethoxy]-benzyl ⁇ -carbamic acid tert-butyl ester (0.6 g) as a colorless oil.
  • Step B Preparation of ⁇ 3-[2-(2- ⁇ 2-[2-(tert-Butoxycarbonylamino-methyl)-4-chloro- phenoxy] -ethoxy ⁇ -ethylamino)- 1 -cyclopropylmethyl-ethylamino] -6-chloro-2-oxo-2H- pyrazin-1-yl ⁇ -acetic acid ethyl ester
  • reaction mixture is allowed to warm to room temperature, is diluted with CH 2 C1 2 , washed with water, dried on sodium sulfate, and concentrated in vacuo to give ⁇ 5-Chloro-2-[2-(2- oxo-ethoxy)-ethoxy] -benzyl ⁇ -carbamic acid tert-butyl ester which is selectiveely used in the next step without further purification.
  • reaction mixture is stirred at room temperature for 50 min, diluted with CH C1 2 , washed with aqueous sodium bicarbonate, dried on sodium sulfate, concentrated in vacuo, and purified by flash chromatography (silica gel, 1% MeOH containibg 10% NH OH in CH 2 C1 2 to 5%) to give ⁇ 3-[2-(2- ⁇ 2-[2-(tert-Butoxycarbonylarmno-methyl)- 4-chloro-phenoxy] -ethoxy ⁇ -ethylamino)- 1 -cyclopropylmethyl-ethylamino] -6-chloro- 2-oxo-2H-pyrazin-l-yl ⁇ -acetic acid ethyl ester (193 mg).
  • Step C cyclization to (S)-20-Cyclopropylmethyl-8,25-dichloro-12,15-dioxa- 1,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene- 3,26-dione
  • EXAMPLE 18 (S)-20-Benzyl-8,25-dichloro-12,15-dioxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione
  • EXAMPLE 19 15-Ethyl- 20(S)-Benzyl-8,25-dichloro-12-oxa-l,4,15,18,21,23-hexaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,16,26-trione bis TFA salt
  • reaction mixture is diluted with EtOAc, washed with water and aqueous LiCl (x3), dried on sodium sulfate, concentrated in vacuo and purified by flash chromatography (silica gel, 30% EtOAc in hexane to 50%) to give (2- ⁇ 4-[(2-Allyloxy-5-chloro-benzylcarbamoyl)-methyl]-5-chloro-3-oxo- 3,4-dihydro-pyrazin-2-ylamino ⁇ -3-phenyl-propyl)-carbamic acid allyl ester (100 mg) as a white foam.
  • MS ES+ M+l 600.3.
  • EXAMPLE 21 (S)-21-Benzyl-8,26-dichloro-12,17-dioxa-l,4,19,22,24-pentaaza tricyclo- [21.3.1.0(6,11)] heptacosa-6(ll),7,9, 23, 25-pentaene-3,18,27-trione TFA salt and (S)-21-Benzyl-8-chloro-12,17-dioxa-l,4,19,22,24-pentaaza tricyclo-[21.3.1.0(6,ll)] heptacosa-6(ll),7,9,23, 25-pentaene-3,18,27-trione TFA salt
  • EXAMPLE 26 (S)-20-Benzyl- 18-methyl-25-chloro- 12-oxa- 1 ,4, 18 ,21 ,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride
  • (S)-20-Benzyl-18-methyl-25-chloro-12-oxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride is prepared from (S)-20-Benzyl-25-chloro-12-oxa -1,4,18,21,23- pentaaza tricyclo[20.3.1.0(6, 11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride and formaldehyde using a similar procedure as described in example 12.
  • (S)-20-Benzyl-18-carboxymethyl-25-chloro-12-oxa-l,4,18,21,23- pentaaza tricyclo[20.3.1.0(6, 11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride is prepared from (S)-20-Benzyl-25-chloro-12-oxa -1,4,18,21,23- pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride and tert-butyl bromoacetate using a similar procedure as described in example 2 followed by tert-butyl removal in TFA.
  • EXAMPLE 30 (S)-20-Benzyl-18-methyl-25-chloro-12,15-dioxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione .2
  • tert-Butyl 2-(aminomethyl benzylcarbamate hemisulfate To a 3 L, 3 neck flask fitted with a thermocouple, a condenser and nitrogen inlet was added 3 g (23 mmol) of cobaltous chloride, then 1200 mL of THF followed by 59 g (254 mmol) of tert-butyl 2-cyanobenzylcarbamate and 600 mL of ice-water. To the light pink solution at 15°C was added 26 g (684 mmol) of sodium borohydride in portions as follows. The initial 3 g of sodium borohydride resulted in a vigorous hydrogen gas evolution and formation of a black suspension.
  • the batch temperature reached 35°C within 2 hr, and was maintained at this temperature with a heating mantle. Additional sodium borohydride and cobaltous chloride were added as needed to drive the reaction to completion. Typically, 2 x 7.5 g of additional sodium borohydride and 2 x 1 g portions of cobaltous chloride were added at 12 hour intervals. Once complete, the layers were allowed to settle and the clear upper THF layer was decanted from the black aqueous layer. The aqueous layer was washed with 750 mL fresh THF, the two THF layers combined and filtered through a pad of celite.
  • the orange-yellow filtrate was concentrated to about 300 mL in vacuo, resulting in water layer with the product as an oily lower layer.
  • the mixture was extracted with 2 x 250 mL ethyl acetate and the combined extracts reacted with 24 g (200 mmol) of solid sodium hydrogensulfate. A solid formed immediately, and the slurry was stirred for 30 min, filtered and washed with 2 x 100 mL ethyl acetate to give 62 g of a white powder.
  • the powder was slurried in 175 mL water, cooled to 0°C, filtered, washed with 2 x 40 mL cold water and the solid dried in a vacuum oven at 55°C for 24 hr to give 46 g (64% yield from tert-butyl 2-cyanobenzylcarbamate) of tert-butyl 2- (aminomethyl)benzylcarbamate hemisulfate salt as a white powder.
  • Step B Preparation of [2-(tert-Butoxycarbonylamino-methyl)-benzyl]-(4-hydroxy- butyl)-carbamic acid dimethyl-ethyl ester
  • tert-Butyl 2-(aminomethyl)benzylcarbamate hemisulfate (1 g, 3.5 mmol) is partitioned betwwen aqueous NaHCO 3 and CH 2 C1 2 to obtain the free base from the organic layer after back extraction with CH 2 C1 2 .
  • a solution of tert-Butyl 2-(aminomethyl) benzylcarbamate free base (3.5 mmol) in DMF (10 ml) is added ethyl 4-bromo- butyrate (602 ul, 4.2 mmol) and the reaction mixture is stirred at 70 °C for 18 h.
  • reaction mixture is allowed to cool down to room temperature, dilute with EtOAc, washed with water, aqueous NaHCO 3 and aqueous LiCl, dried on Na 2 SO , concentrated in vacuo and purified by flash chromatography (silica gel, 1% MeOH containibg 10% NH 4 OH in CH 2 C1 2 to 10%) to give 4-[2-(tert-Butoxycarbonylamino- methyl)-benzylamino] -butyric acid ethyl ester (912 mg) contaminated with the bis- alkylation product.
  • reaction mixture is concentrated in vacuo and purified by flash chromatography (silica gel, 5% EtOAc in hexane to 40%) to give 4- ⁇ tert-Butoxycarbonyl-[2-(tert-butoxycarbonylamino- methyl)-benzyl] -amino ⁇ -butyric acid ethyl ester (669 mg) as a colorless oil.
  • Step C Reductive alkylation and cyclization toward (S)-20-Benzyl-25-chloro- 1,4,13,18,21,23-hexaaza tricyclo[20.3.1.0(6, 11)] hexacosa-6(ll),7,9,22, 24-pentaene- 3,26-dione tris TFA salt
  • Step A Preparation of tert-Butyl [2-(aminomethyl)phenyl] acetate
  • 2-Bromomethylphenylacetic acid To a solution of 97.0 g (0.646 mol) o-tolylacetic acid in 1.75 L carbon tetrachloride was added 115.0 g (0.646 mol) N-bromosuccinimide and 3.4 g (0.021 mol) 2,2 -azobisisobutyronitrile. The mixture was heated at reflux under a nitrogen atmosphere for 4 h. After the mixture was cooled to 0-5 °C for 30 min, the solids were removed by filtration and washed with a small portion of carbon tetrachloride.
  • Step B Preparation of (2- ⁇ 2-[tert-Butoxycarbonyl-(3-hydroxy-propyl)-amino]-ethyl ⁇ - benzyl)-carbamic acid tert-butyl ester
  • reaction mixture is concentrated in and purified by flash chromatography (silica gel, 30% EtOAc in hexane to 65%) to give (2- ⁇ 2-[tert- Butoxycarbonyl-(3-hydroxy-propyl)-amino]-ethyl ⁇ -benzyl)-carbamic acid tert-butyl ester (439 mg) as a thick oil.
  • Step C reductive alkylation and cyclization toward (S)-20-Benzyl-25-chloro- 1,4,14,18,21,23-hexaaza tricyclo[20.3.1.0(6,11)] hexacosa-6(ll),7,9,22, 24-pentaene- 3,26-dione tris hydrochloride
  • Typical tablet cores suitable for administration of thrombin inhibitors are comprised of, but not limited to, the following amounts of standard ingredients:
  • Mannitol, microcrystallme cellulose and magnesium stearate may be substituted with alternative pharmaceutically acceptable excipients.
  • the thrombin inhibitors can also be co-administered with suitable antiplatelet agents, including, but not limited to, fibrinogen receptor antagonists (e.g. to treat or prevent unstable angina or to prevent reocclusion after angioplasty and restenosis), anticoagulants such as aspirin, thrombolytic agents such as plasminogen activators or streptokinase to achieve synergistic effects in the treatment of various vascular pathologies, or lipid lowering agents including antihypercholesterolemics (e.g. HMG CoA reductase inhibitors such as lovastatin, HMG CoA synthase inhibitors, etc.) to treat or prevent atherosclerosis.
  • fibrinogen receptor antagonists e.g. to treat or prevent unstable angina or to prevent reocclusion after angioplasty and restenosis
  • anticoagulants such as aspirin
  • thrombolytic agents such as plasminogen activators or streptokinase
  • thrombin inhibitors enhance the efficiency of tissue plasminogen activator-mediated thrombolytic reperfusion.
  • Thrombin inhibitors may be administered first following thrombus formation, and tissue plasminogen activator or other plasminogen activator is administered thereafter.
  • Typical doses of thrombin inhibitors of the invention in combination with other suitable anti-platelet agents, anticoagulation agents, or thrombolytic agents may be the same as those doses of thrombin inhibitors administered without coadministration of additional anti-platelet agents, anticoagulation agents, or thrombolytic agents, or may be substantially less that those doses of thrombin inhibitors administered without coadministration of additional anti-platelet agents, anticoagulation agents, or thrombolytic agents, depending on a patient's therapeutic needs.
  • Trypsin assays also contained 1 mM CaCl2- In assays wherein rates of hydrolysis of a p-nitroanilide (pna) substrate were determined, a Thermomax 96-well plate reader was used was used to measure (at 405 nm) the time dependent appearance of p-nitroaniline.
  • p-Nitroanilide substrate concentration was determined from measurements of absorbance at 342 nm using an extinction coefficient of 8270 cm' ⁇ M" 1.
  • Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration ⁇ 0.1 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. Initial velocities of product formation in the absence (V 0 ) or presence of inhibitor (N were measured.
  • the equilibrium constant (Ki) for dissociation of the inhibitor from the enzyme can be obtained from the dependence of N 0 /Ni on [I] shown in the following equation.
  • the activities shown by this assay indicate that the compounds of the invention are therapeutically useful for treating various conditions in patients suffering from unstable angina, refractory angina, myocardial infarction, transient ischemic attacks, atrial fibrillation, thrombotic stroke, embolic stroke, deep vein thrombosis, disseminated intravascular coagulation, and reocclusion or restenosis of recanalized vessels.
  • Active I is compound (S)-19-Benzyl-8,24-dichloro-12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,11)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione dihydrochloride .
  • All of the active compound, cellulose, and a portion of the corn starch are mixed and granulated to 10% corn starch paste.
  • the resulting granulation is sieved, dried and blended with the remainder of the corn starch and the magnesium stearate.
  • the resulting granulation is then compressed into tablets containing 25.0, 50.0, and 100.0 mg, respectively, of active ingredient per tablet.
  • compositions of compound (S)-19-Benzyl-8,24-dichloro- 12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,11)] pentacosa-6(ll),7,9,21, 23- pentaene-3,25-dione dihydrochloride (Active I) tablets are shown below:
  • Active I, mannitol and microcrystallme cellulose were sieved through mesh screens of specified size (generally 250 to 750 ⁇ m) and combined in a suitable blender. The mixture was subsequently blended (typically 15 to 30 min) until the drug was uniformly distributed in the resulting dry powder blend. Magnesium stearate was screened and added to the blender, after which a precompression tablet blend was achieved upon additional mixing (typically 2 to 10 min). The precompression tablet blend was then compacted under an applied force, typically ranging from 0.5 to 2.5 metric tons, sufficient to yield tablets of suitable physical strength with acceptable disintegration times (specifications will vary with the size and potency of the compressed tablet). In the case of the 2, 10 and 50 mg potencies, the tablets were dedusted and film-coated with an aqueous dispersion of water-soluble polymers and pigment.
  • a dry powder blend is compacted under modest forces and remilled to afford granules of specified particle size.
  • the granules are then mixed with magnesium stearate and tabletted as stated above.
  • compositions A-C are as follows:
  • Narious other buffer acids such as L-lactic acid, acetic acid, citric acid or any pharmaceutically acceptable acid/conjugate base with reasonable buffering capacity in the pH range acceptable for intravenous administration may be substituted for glucuronic acid.

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Abstract

Compounds of the invention are useful in inhibiting thrombin and associated thrombotic occlusions having the following structure: e.g. .

Description

TITLE OF THE INVENTION
THROMBi INHIBITORS
BACKGROUND OF THE INVENTION Thrombin is a serine protease present in blood plasma in the form of a precursor, prothrombin. Thrombin plays a central role in the mechanism of blood coagulation by converting the solution plasma protein, fibrinogen, into insoluble fibrin.
Edwards et al., J. Amer. Chem. Soc, (1992) vol. 114, pp. 1854-63, describes peptidyl a-ketobenzoxazoles which are reversible inhibitors of the serine proteases human leukocyte elastase and porcine pancreatic elastase.
European Publication 363 284 describes analogs of peptidase substrates in which the nitrogen atom of the scissile amide group of the substrate peptide has been replaced by hydrogen or a substituted carbonyl moiety. Australian Publication 86245677 also describes peptidase inhibitors having an activated electrophilic ketone moiety such as fluoromethylene ketone or a- keto carboxyl derivatives.
R. J. Brown et al, J. Med. Chem., Vol. 37, pages 1259-1261 (1994) describes orally active, non-peptidic inhibitors of human leukocyte elastase which contain trifluoromethylketone and pyridinone moieties.
H. Mack et al., J. Enzyme Inhibition, Vol. 9, pages 73-86 (1995) describes rigid amidino-phenylalanine thrombin inhibitors which contain a pyridinone moiety as a central core structure.
SUMMARY OF THE INVENTION
The invention includes compounds for inhibiting loss of blood platelets, inhibiting formation of blood platelet aggregates, inhibiting formation of fibrin, inhibiting thrombus formation, and inhibiting embolus formation in a mammal, comprising a compound of the invention in a pharmaceutically acceptable carrier. These compounds may optionally include anticoagulants, antiplatelet agents, and thrombolytic agents. The compounds can be added to blood, blood products, or mammalian organs in order to effect the desired inhibitions.
The invention also includes a compound for preventing or treating unstable angina, refractory angina, myocardial infarction, transient ischemic attacks, atrial fibrillation, thrombotic stroke, embolic stroke, deep vein thrombosis, disseminated intravascular coagulation, ocular build up of fibrin, and reocclusion or restenosis of recanalized vessels, in a mammal, comprising a compound of the invention in a pharmaceutically acceptable carrier. These compounds may optionally include anticoagulants, antiplatelet agents, and thrombolytic agents.
The invention also includes a method for reducing the thrombogenicity of a surface in a mammal by attaching to the surface, either covalently or noncovalently, a compound of the invention.
DETAILED DESCRIPTION OF THE INVENTION AND
PREFERRED EMBODIMENTS
Compounds of the invention are useful as thrombin inhibitors and have therapeutic value in for example, preventing coronary artery disease. The invention includes compounds having the following structure:
Figure imgf000004_0001
and pharmaceutically acceptable salts thereof, wherein R1 is aryl, C ι-4 alkyl, or C 3-7 cycloalkyl; R >2 is hydrogen, C 1-4 alkyl, C 3-7 cycloalkyl, halogen, or CN; R3 is hydrogen, C 1-4 alkyl, C 3- cycloalkyl, halogen or CN; R4is hydrogen, C 1-4 alkyl or -ZCOOH, wherein Z is C 1-4 alkyl; A is selected from the group consisting of a) — (CHz X1— where X1 is O or CH2j
b) ^— (CH2)2.3-Y1-(CH2)2.3-X2-^
where Y1 is O, S, SO, SO2 or NR5, and X2 is O or CH2;
c) - CCOMCB^W-Y^CBULS-X3—
where Y2 is O, S, SO, SO2 or NR5, and X3 is O or CH2,
d) \— (CH2)n-C(O)-NR5-(CH2)m-X4 f
where X is O or CH2, provided that when X is O, n and m are independently 1, 2 or 3, and that when Λ is CH2, n is 1, 2 or 3, and m is 2 or 3, or e) I— (CH2)n-NR5-C(O)-(CH2)m-X5
where X is O or CH2, provided that when X is O, n and m are independently 1, 2 or 3, and when X is CH2, n is 1, 2 or 3 and m is 2 or 3;
R5 is hydrogen, C ι-4 alkyl or -ZOOH, where Z is C 1-4 alkyl. I Inn aa ccllaassss ooff ccoonmpounds of the invention, R is hydrogen or halogen and R3 is hydrogen or halogen.
In a subclass of this class, R2 is hydrogen or CI and R3 is hydrogen or
CL
In a group of this subclass, R1 is phenyl or cyclopropyl. In a subgroup of this group, R4 is hydrogen, methyl, ethyl, or - CH2COOH.
In a family of this subgroup, R5 is hydrogen, methyl or ethyl.
In a subfamily of this family, A is selected from the group consisting of -(CH2)4-O-, -(CH2)3-O-, -(CH2)5-O-, -(CH2)6-O-,
-(CH2)2-O-(CH2)2-O-, -(CH2)C(O)N(CH2CH3)(CH2)2-O-,
-C(O)-O-CH2CHCHCH2-O-, -C(O)-O-(CH2)4-O-,
-(CH2)2S(CH2)2-O-, -C(O)CH2N(CH3)(CH2)2-O-,
-(CH2)4NHCH2-, and -(CH2)3NH(CH2)2-
Specific examples of compounds of the invention include
Figure imgf000006_0001
Figure imgf000006_0002
Figure imgf000007_0001
Figure imgf000007_0002
Figure imgf000008_0001
Figure imgf000008_0002
Figure imgf000009_0001
Figure imgf000009_0002
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000011_0002
The compounds of the present invention, may have chiral centers and occur as racemates, racemic mixtures and as individual diastereomers, or enantiomers with all isomeric forms being included in the present invention. The compounds of the present invention may also have polymorphic crystalline forms, with all polymorphic crystalline forms being included in the present invention.
When any variable occurs more than one time in any constituent or in formula I, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
Some abbreviations that may appear in this application are as follows: ABBREVIATIONS
Designation
AcOH acetic acid
(Boc)2O di-t-butyl dicarbonate
DAST diethylaminosulfurtrifluoride
DBU l,8-diazabicyclo[5.4.0]undec-7-ene
DCE 1 ,2-dichloroethane
DMAP dimethylaminopyridine
DMF dimethylformamide
DPPA diphenylphosphoryl azide
EDC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
EtOAc ethyl acetate
Et2O diethyl ether
HCl hydrochloric acid
HOAc acetic acid
HOAT 1 -hydroxy-7-azabenzotriazole
IPrOH 2-propanol
KOH potassium hydroxide
LAH lithium aluminum hydride
MCPBA m-chloroperoxybenzoic acid
MeOH methanol
MgSO4 magnesium sulfate n-BuLi n-butyllithium
N3PO(Ph)2 diphenyl phosphoryl azide
NaBH4 sodium borohydride
NaHCOs sodium hydrogen carbonate
NaN3 sodium azide
Na2SO4 sodium sulfate
Na2S2O3 sodium thiosulfate
NCS N-chlorosuccinimide
NH4C1 ammonium chloride
P(Ph)3 triphenyl phosphine
Pd-C palladium on activated carbon catalyst
Pd(PPh)3 tetrakis triphenylphosphine palladium
PhCH3 toluene POBr3 phosphorous oxybromide
TEA triethylamine
Tf2O trifluoromethane sulfonic anhydride
THF tetrahydrofuran SiO2 silicon oxide
Zn(CN)2 zinc cyanide
As used herein except where noted, "alkyl" is intended to include both branched- and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms (Me is methyl, Et is ethyl, Pr is propyl, Bu is butyl); "alkoxy" represents a linear or branched alkyl group of indicated number of carbon atoms attached through an oxygen bridge; "halogen", as used herein, means fluoro, chloro, bromo and iodo; and "counterion" is used to represent a small, single negatively-charged species, such as chloride, bromide, hydroxide, acetate, trifluoroacetate, perchlorate, nitrate, benzoate, maleate, sulfate, tartrate, hemitartrate, benzene sulfonate, and the like.
The term "cycloC3_7alkyl" is intended to include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, and the like.
The term "aryl" as used herein except where noted, represents a stable 6- to 10-membered mono- or bicyclic ring system such as phenyl, or naphthyl. The aryl ring can be unsubstituted or substituted with one or more of C1 -.4 lower alkyl; hydroxy; alkoxy; halogen; amino.
In this specification methyl substituents may be represented by
\— CH3 or | — ς . For example, the structures
HN^ CH3 HN^^ ^ and ^
have equivalent meanings.
The pharmaceutically-acceptable salts of the compounds of Formula I (in the form of water- or oil-soluble or dispersible products) include the conventional non-toxic salts such as those derived from inorganic acids, e.g. hydrochloric, hydrobromoic, sulfuric, sulfamic, phosphoric, nitric and the like, or the quaternary ammonium salts which are formed, e.g., from inorganic or organic acids or bases. Examples of acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pamoate, pectinate, persulfate, 3-ρhenylpropionate, picrate, pivalate, propionate, succinate, sulfate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others.
Thrombin Inhibitors - Therapeutic Uses- Method of Using Anticoagulant therapy is indicated for the treatment and prevention of a variety of thrombotic conditions, particularly coronary artery and cerebro vascular disease. Those experienced in this field are readily aware of the circumstances requiring anticoagulant therapy. The term "patient" used herein is taken to mean mammals such as primates, including humans, sheep, horses, cattle, pigs, dogs, cats, rats, and mice.
Thrombin inhibition is useful not only in the anticoagulant therapy of individuals having thrombotic conditions, but is useful whenever inhibition of blood coagulation is required such as to prevent coagulation of stored whole blood and to prevent coagulation in other biological samples for testing or storage. Thus, the thrombin inhibitors can be added to or contacted with any medium containing or suspected of containing thrombin and in which it is desired that blood coagulation be inhibited, e.g., when contacting the mammal's blood with material selected from the group consisting of vascular grafts, stents, orthopedic prosthesis, cardiac prosthesis, and extracorporeal circulation systems. Compounds of the invention are useful for treating or preventing venous thromboembolism (e.g. obstruction or occlusion of a vein by a detached thrombus; obstruction or occlusion of a lung artery by a detached thrombus), cardiogenic thromboembolism (e.g. obstruction or occlusion of the heart by a detached thrombus), arterial thrombosis (e.g. formation of a thrombus within an artery that may cause infarction of tissue supplied by the artery), atherosclerosis (e.g. arteriosclerosis characterized by irregularly distributed lipid deposits) in mammals, and for lowering the propensity of devices that come into contact with blood to clot blood. Examples of venous thromboembolism which may be treated or prevented with compounds of the invention include obstruction of a vein, obstruction of a lung artery (pulmonary embolism), deep vein thrombosis, thrombosis associated with cancer and cancer chemotherapy, thrombosis inherited with thrombophilic diseases such as Protein C deficiency, Protein S deficiency, antithrombin HI deficiency, and Factor V Leiden, and thrombosis resulting from acquired thrombophilic disorders such as systemic lupus erythematosus (inflammatory connective tissue disease). Also with regard to venous thromboembolism, compounds of the invention are useful for maintaining patency of indwelling catheters.
Examples of cardiogenic thromboembolism which may be treated or prevented with compounds of the invention include thromboembolic stroke (detached thrombus causing neurological affliction related to impaired cerebral blood supply), cardiogenic thromboembolism associated with atrial fibrillation (rapid, irregular twitching of upper heart chamber muscular fibrils), cardiogenic thromboembolism associated with prosthetic heart valves such as mechanical heart valves, and cardiogenic thromboembolism associated with heart disease.
Examples of arterial thrombosis include unstable angina (severe constrictive pain in chest of coronary origin), myocardial infarction (heart muscle cell death resulting from insufficient blood supply), ischemic heart disease (local anemia due to obstruction (such as by arterial narrowing) of blood supply), reocclusion during or after percutaneous transluminal coronary angioplasty, restenosis after percutaneous transluminal coronary angioplasty, occlusion of coronary artery bypass grafts, and occlusive cerebrovascular disease. Also with regard to arterial thrombosis, compounds of the invention are useful for maintaining patency in arteriovenous cannulas. Examples of atherosclerosis include arteriosclerosis. Examples of devices that come into contact with blood include vascular grafts, stents, orthopedic prosthesis, cardiac prosthesis, and extracorporeal circulation systems
The thrombin inhibitors of the invention can be administered in such oral forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixers, tinctures, suspensions, syrups, and emulsions. Likewise, they may be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non- toxic amount of the compound desired can be employed as an anti-aggregation agent. For treating ocular build up of fibrin, the compounds may be administered intraocularly or topically as well as orally or parenterally.
The thrombin inhibitors can be administered in the form of a depot injection or implant preparation which may be formulated in such a manner as to permit a sustained release of the active ingredient. The active ingredient can be compressed into pellets or small cylinders and implanted subcutaneously or intramuscularly as depot injections or implants. Implants may employ inert materials such as biodegradable polymers or synthetic silicones, for example, Silastic, silicone rubber or other polymers manufactured by the Dow-Corning Corporation. The thrombin inhibitors can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. The thrombin inhibitors may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The thrombin inhibitors may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinlypyrrolidone, pyran copolymer, polyhydroxy-propyl-methacrylamide-phenol, polyhydroxyethyl- aspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the thrombin inhibitors may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels. The dosage regimen utilizing the thrombin inhibitors is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.
Oral dosages of the thrombin inhibitors, when used for the indicated effects, will range between about 0.01 mg per kg of body weight per day (mg/kg/day) to about 30 mg/kg/day, preferably 0.025-7.5 mg/kg/day, more preferably 0.1-2.5 mg/kg/day, and most preferably 0.1-0.5 mg/kg/day (unless specificed otherwise, amounts of active ingredients are on free base basis). For example, an 80 kg patient would receive between about 0.8 mg/day and 2.4 g/day, preferably 2-600 mg/day, more preferably 8-200 mg/day, and most preferably 8-40 mg/kg/day. A suitably prepared medicament for once a day administration would thus contain between 0.8 mg and 2.4 g, preferably between 2 mg and 600 mg, more preferably between 8 mg and 200 mg, and most preferably 8 mg and 40 mg, e.g., 8 mg, 10 mg, 20 mg and 40 mg. Advantageously, the thrombin inhibitors may be administered in divided doses of two, three, or four times daily. For administration twice a day, a suitably prepared medicament would contain between 0.4 mg and 4 g, preferably between 1 mg and 300 mg, more preferably between 4 mg and 100 mg, and most preferably 4 mg and 20 mg, e.g., 4 mg, 5 mg, 10 mg and 20 mg.
Intravenously, the patient would receive the active ingredient in quantities sufficient to deliver between 0.025-7.5 mg/kg/day, preferably 0.1-2.5 mg/kg/day, and more preferably 0.1-0.5 mg/kg/day. Such quantities may be administered in a number of suitable ways, e.g. large volumes of low concentrations of active ingredient during one extended period of time or several times a day, low volumes of high concentrations of active ingredient during a short period of time, e.g. once a day. Typically, a conventional intravenous formulation may be prepared which contains a concentration of active ingredient of between about 0.01-1.0 mg/ml, e.g. 0.1 mg/ml, 0.3 mg/ml, and 0.6 mg/ml, and administered in amounts per day of between 0.01 ml/kg patient weight and 10.0 ml/kg patient weight, e.g. 0.1 ml/kg, 0.2 ml/kg, 0.5 ml/kg. In one example, an 80 kg patient, receiving 8 ml twice a day of an intravenous formulation having a concentration of active ingredient of 0.5 mg/ml, receives 8 mg of active ingredient per day. Glucuronic acid, L-lactic acid, acetic acid, citric acid or any pharmaceutically acceptable acid/conjugate base with reasonable buffering capacity in the pH range acceptable for intravenous administration may be used as buffers. Consideration should be given to the solubility of the drug in choosing an The choice of appropriate buffer and pH of a formulation, depending on solubility of the drug to be administered, is readily made by a person having ordinary skill in the art.
The compounds can also be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, or course, be continuous rather than intermittent throughout the dosage regime.
The thrombin inhibitors are typically administered as active ingredients in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixers, syrups and the like, and consistent with convention pharmaceutical practices.
For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid form, the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, distintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn-sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch methyl cellulose, agar, bentonite, xanthan gum and the like.
General Preparations
The compounds claimed in this invention can be prepared according to the following general procedures. In scheme 1, ethyl oxalyl chloride is converted in four steps to the bromopyrazinone 1. Addition of an -(azidomethyl)-alkylamine hydrochloride to bromopyrazinone 1, followed by chlorination of the pyrazinone and reduction of the azide to the corresponding amine gives access to aminomethyl derivatives of type 2. Alkylation of aminomethyl derivatives of type 2 with tertbutylbromoacetate followed by hydrolysis with aqueous LiOH leads to acids of type 3. Acylation of aminomethyl derivatives of type 2 with allyl chloroformate followed by hydrolysis with aqueous LiOH leads to acids of type 4.
Scheme 1
Figure imgf000019_0001
Figure imgf000020_0001
In scheme 2, phenols of type 5 can be converted to bromides of type 6 by alkylation with dibromide intermediates. Alkylation of aminomethyl derivatives of type 2 with bromides of type 6 leads to Boc-amino ester derivatives of type 7. Removal of the Boc group, followed by hydrolysis of the ester and cyclization under EDC / HO At conditions provides macrocycles of type 8.
Scheme 2
Figure imgf000021_0001
Figure imgf000022_0001
Scheme 3 illustrates an alternative preparation of ethyl bromide derivative 9 from phenol 5, via alkylation with ethyl bromoacetate, reduction and bromination of the resulting primary alcohol. Alkylation of 2-mercaptoethanol with bromide 9 followed by bromination affords bromides of type 10. Alkylation of aminomethyl derivatives of type 2 with bromides of type 10 leads to Boc-amino ester derivatives of type 11. Removal of the Boc group, followed by hydrolysis of the ester and cyclization under EDC / HOAt conditions provides macrocycles of type 12. Scheme 3
Figure imgf000023_0001
Figure imgf000023_0002
Scheme 4 illustrates the preparation of macrocycle 15 with an amide type linker. Alylation of alkylglycine ethyl ester with bromide 9 followed by ester hydrolysis leads to acid 13. EDC/HOAt coupling of acid 13 to aminomethyl derivative 2 provides intermediate 14 which can be cyclized to 15 following the same procedure as described in the previous schemes.
Scheme 4
Figure imgf000024_0001
Figure imgf000024_0002
EDC, HOAt
Figure imgf000024_0003
Figure imgf000025_0001
Scheme 5 illustrates an alternative mode of macrocyclization. Amine 16 can be prepared by akylation of bromide 9. EDC/HOAt coupling of acid 3 to benzylaminel derivative 16 provides intermediate 17 which can be cyclized to 18 after tertbutyl removal.
Scheme 5
.Br .NHR!
O" O'
LF^NHg
NHBoc NH2
2. HCI(g) 16
X X
Figure imgf000026_0001
Figure imgf000026_0002
17
Figure imgf000026_0003
Scheme 6 illustrates an alternative mode of coupling between the pyrazinone derivative and the phenolic derivative. Aldehyde 19 is obtained from phenol 5 using the following three steps sequence: alkylation with a bromoester, reduction of the ester and oxidation to the corresponding aldehyde. Reductive amination with aminomethyl derivative 2 provides the intermediates of type 20. In some examples, this alternative mode of linker introduction was found to be more efficient than the original bromide alkylation procedure utilized in schemes 2 and 3. Macrocyclization of intermediate 20 to macrocycle 21 is accomplished using the same sequence as described earlier.
Scheme 6
Figure imgf000027_0001
NaBH(OAc)3
Figure imgf000027_0002
Figure imgf000028_0001
Scheme 7 illustrates the preparation of aldehyde 22 and its attachment to the pyrazinone core via reductive amination as described in scheme 6, followed by macrocyclization to 24, as described before.
Scheme 7
Figure imgf000029_0001
NaBH(OAc)3
Figure imgf000029_0002
Scheme 8 illustrates the preparation of macrocycles of type 28 which present an amino type linker. Alkylation of a primary amine of type 25 with a bromoester followed by Boc protection and conversion of the ester to the corresponding aldehyde leads to aldehyde derivatives of type 26. Reductive amination with aminomethyl derivative 2 and macrocyclization provides macrocycles of type 28.
Scheme 8
Figure imgf000030_0001
NaBH(OAc)3
Figure imgf000030_0002
Figure imgf000031_0001
Scheme 9 illustrates yet another mode of macrocyclization which utilizes Grubbs type olefin metathesis (Grubbs, R. H., Chang, S. Tetrahedron, 1998, 54, 4413-4450). Allyl derivative 29 is prepared from phenol 5 and allyl bromide. Coupling of acid 4 and amine 29 under EDC HOAt conditions leads to bis-olefin derivative 30 which is converted to an E/Z mixture of macrocycles of type 31 via Grubbs olefin metathesis. Hydrogenation on Pd/C provides derivatives of type 32.
Scheme 9
O C02H
Figure imgf000032_0001
Figure imgf000032_0002
Figure imgf000033_0001
Grubbs olefin metathesis
H2 Pd/C
Figure imgf000033_0002
Scheme 10 illustrates further modifications of the previously described macrocycles. Dehalogenation with hydrogen provides derivatives of type 33 and 34. Reductive alkylation leads to amino derivatives of type 35. A carboxymethyl group can be attached to afford structures of type 36.
Scheme 10
Figure imgf000034_0001
Unless otherwise stated, all NMR determinations were made using 400 MHz field strength.
EXAMPLE 1 (S)-[3-(l-Armnomethyl-2-phenyl-ethylamino)-6-chloro-2-oxo-2H-pyrazin-l-yl]-acetic acid ethyl ester Step A: Preparation of ethyl 3-bromopyrazin(lH)-2-one-l-acetate
Figure imgf000035_0001
To a suspension of ethyl glycine»HCl (38.4 g, 275 mmol ) in 1,2- dichloroethane (360 mL) was added triethylamine (77.0 mL, 550 mmol) at room temperature. After stirring for 30 minutes the heterogenous mixture was cooled to 0 °C and ethyl oxalyl chloride (30.3 mL, 275 mol) was added dropwise over the course of 1 h. Upon completion of the addition, the cooling bath was removed and the reaction was stirred at room temperature overnight. The reaction was diluted with water (250 mL) and the layers separated. The aqueous layer was backwashed with 2 portions of dichloromethane (250 mL). The combined organic layers were washed with water (250 mL), followed by brine (250 mL), dried over MgSO4 and concentrated to give ethyl N-(ethyl carboxymethyl)oxamate as an oil that was taken directly onto the next step.
To a solution of ethyl N-(ethyl carboxymethyl)oxamate (84.0 g, 414 mmol) in 2-propanol (500 mL) was added aminoacetaldehyde dimethyl acetal (45.7 g, 435 mmol) in one portion. After stirring overnight at room temperature, the reaction mixture was concentrated to a thick orange oil. This thick slurry was diluted with 2- propanol (300 mL) and the solid was broken up with a spatula. Filtration afforded a solid which was further rinsed with an additional portion of 2-propanol. Removal of residual 2-propanol was accomplished via high vacuum to afford N-(ethyl carboxymethyl_-N'-(2,2-dimethoxyethyl)oxamide as a light orange solid. (89.8 g): IH NMR (CDC13) 57.82 (br s, IH), 7.50 (br s, IH), 4.41 (t, IH, 5.3 Hz), 4.24 (q, 2H, 7.1 Hz), 4.09 (d, 2H, 5.9 Hz), 3.47 (dd, 2H, 5.3, 6.2 Hz), 3.40 (s, 6H), 1.30 (t, 3 H, 7.1 Hz).
A solution of the N-(ethyl carboxymethyl_-N'-(2,2- dimethoxyethyl)oxamide (89.8 g, 343 mmol), acetic acid (400 mL), and cone. HCl (2 mL) was heated to reflux. After 1 h the black reaction was concentrated to a thick oil (high vacuum employed to ensure complete removal of AcOH) which was diluted with EtOH (150 mL) and MeOH (150 mL). Scraping the thick black oil with a spatula induced precipitation of the product. The MeOH was removed via rotary evaporation and the remaining slurry was filtered and rinsed with EtOH (200 mL) to deliver a tan solid. Recrystallization from refluxing EtOH (300 mL) afforded ethyl 3- hydroxypyrazin(lH)-2-one-l-acetate as an off-white powder: IH NMR (CD3OD) 5 6.50 (d, IH, 5.9 Hz), 6.36 (d, IH, 5.9 Hz), 4.58 (s, 2H), 4.23 (q, 2H, 7.1 Hz), 1.28 (t, 3 H, 7.1 Hz). Further crude ethyl 3-hydroxypyrazin(lH)-2-one-l-acetate could be obtained upon concentration of the mother liquor.
A solution of the ethyl 3-hydroxypyrazin(lH)-2-one-l-acetate (25.0 g, 126 mmol) 2X3 and phosphorous oxybromide (37.9 g, 132 mmol) in 1,2- dichloroethane (250 mL) was heated to reflux. After 8 h the reaction mixture was treated with sat. aq. Na2CO3 (250 mL) and stirred for lh. The mixture was diluted with water (100 mL) and dichloromethane (100 mL), the layers were separated and the aqueous layer was backwashed with EtOAc (3 x 200mL). The combined organics were dried (MgSO4), and concentrated to give an oil which was stored on a high vacuum line overnite to afford ethyl 3-bromopyrazin(lH)-2-one-l-acetate as a brown solid: 1H NMR (CDC13) δ 7.17 (d, IH, 4.2 Hz), 7.07 (d, IH, 4.2 Hz), 4.65 (s, 2H), 4.27 (q, 2H, 7.2 Hz), 1.31 (t, 3 H, 7.2 Hz).
Step B: preparation of (S)-[3-(l-Aminomethyl-2-phenyl-ethylamino)-6-chloro-2-oxo- 2H-pyrazin-l-yl] -acetic acid ethyl ester
Figure imgf000036_0001
To a solution of ethyl ethyl 3-bromopyrazin(lH)-2-one-l-acetate (8.75 g, 33.5 mmol) in EtOH (60 ml) is added (S)-α-(Azidomethyl)-2-phenethylamine hydrochloride (7.5 g, 35.3 mmol, prepared from (S)-(-)-2-(tertbutoxycarbonylamino)- 3 -phenyl- 1-propanol following the procedure of Horwell et al (J. Med. Chem. 1991, 34, 404-414) and Boc removal under standard HCl(g) conditions) and triethylamine (10.8 ml, 77.6 mmol). The pressure flask is flushed with argon, sealed and the reaction mixture is stirred at 110 °C for 3 days. The reaction mixture is concentrated in vacuo and purified by flash chromatography (silica gel, 20% EtOAc in hexane to 40%) to give (S)-[3-(l-Azidomethyl-2-phenyl-ethylamino)-2-oxo-2H-pyrazin-l-yl]- acetic acid ethyl ester (5.87 g) as a brown syrup. 1H NMR (CDC13, 400 MHz): δ 7.36- 7.20 (m, 5 H); 6.85 (d, J = 4.4 Hz, 1 H); 6.41 (d, J = 4.4 Hz, 1 H); 6.20 (bd, J = 8 Hz, 1 H); 4.58 (A of AB, d, J = 17.5 Hz, 1 H)); ); 4.52 (B of AB, d, J = 17.5 Hz, 1 H)); 4.43-4.33 (m, 1 H); 4.25 (q, J = 7.3 Hz, 2 H); 3.52 (A of ABX, dd, J = 12.5, 5 Hz, 1 H); 3.40 (B of ABX, dd, J = 12.5, 5 Hz, 1 H); 3.00 (A of ABX, dd, J = 14, 6.7 Hz, 1 H); 2.92 (B of ABX, dd, J = 14, 7.9 Hz, 1 H); 1.29 (t, J = 7.3 Hz, 3 H).
To a solution of (S)-[3-(l-Azidomethyl-2-phenyl-ethylamino)-2-oxo- 2H-pyrazin-l-yl]-acetic acid ethyl ester (5.8 g, 16.3 mmol) in dichloroethane (90 ml) was added N-chlorosuccinimide (2.17 g, 16.3 mmol) and the reaction mixture was stirred at 85 °C for 1 h 30 and at room temperature for 18 h. The reaction mixture is concentrated in vacuo and purified by flash chromatography (silica gel, 20% diethyl ether in hexane to 45%) to give (S)-[3-(l-Azidomethyl-2-phenyl-ethylamino)-2-oxo- 2H-6-chloro-pyrazin-l-yl] -acetic acid ethyl ester (5.6 g) as a thick syrup. IH NMR (CDC13, 400 MHz): δ 7.38-7.20 (m, 5 H); 6.85 (s, 1 H); 6.10 (bd, J = 8.8 Hz, 1 H); 4.92 (A of AB, d, J = 17.7 Hz, 1 H)); ); 4.86 (B of AB, d, J = 17.7 Hz, 1 H)); 4.40- 4.30 (m, 1 H); 4.25 (q, J = 7 Hz, 2 H); 3.52 (A of ABX, dd, J = 12.4, 4.6 Hz, 1 H); 3.40 (B of ABX, dd, J = 12.4, 4.6 Hz, 1 H); 3.00 (A of ABX, dd, J = 13.5, 6.4 Hz, 1 H); 2.92 (B of ABX, dd, J = 13.5, 7.4 Hz, 1 H); 1.29 (t, J = 7 Hz, 3 H).
To a solution of (S)-[3-(l-Azidomethyl-2-phenyl-ethylamino)-2-oxo- 2H-6-chloro-pyrazin-l-yl] -acetic acid ethyl ester (5.67 g, 14.5 mmol) in MeOH (80 ml) and THF (40 ml) is added SnCl2 (4.1 g, 21.8 mmol) and the reaction mixture is stirred at room temperature for 18 h. The reaction mixture is concentrated in vacuo, azeothroped with CH C12, and purified by flash chromatography (silica gel, 6%
MeOH containing 10% NH4OH in CH2C12 to 10%) to give (S)-[3-(l-Aminomethyl-2- phenyl-ethylamino)-6-chloro-2-oxo-2H-pyrazin-l-yl]-acetic acid ethyl ester (4.19 g) as a beige solid. 1H NMR (CDC13, 400 MHz): δ 7.35-7.15 (m, 5 H); 6.92 (s, 1 H); 6.17 (bd, J = 8.5 Hz, 1 H); 4.88 (s, 2 H)); ); 4.35-4.15(m, 3 H); 3.00-2.70 (m, 4 H); 1.32 (t, J = 6.9 Hz, 3 H). rotation: [ ]d = -82.6 ° (c = 0.42, MeOH).
EXAMPLE 2 (S)-(3- { 1 - [(tert-Butoxycarbonylmethyl-amino)-methyl]-2-phenyl-ethylamino } -6- chloro-2-oxo-2H-pyrazin-l-yl)-acetic acid
Figure imgf000038_0001
To a solution of (S)-[3-(l-Aminomethyl-2-phenyl-ethylamino)-6- chloro-2-oxo-2H-pyrazin-l-yl]-acetic acid ethyl ester (242 mg, 0.66 mmol) in DMF (5 ml) is added a small amount of 4A molecular sieves, potassium carbonate (92 mg, 0.66 mmol) and tert-butyl bromoacetate (78 ul, 0.53 mmol), and the reaction mixture is stirred at room temperature for 3.5 h. Potassium carbonate (20 mg, 0.13 mmol) and tert-butyl bromoacetate (20 ul, 0.13 mmol) were added and the reaction mixture is stirred at room temperature for 18 h. The reaction mixture was filtered, concentrated in vacuo and purified by flash chromatography (silica gel, 20% EtOAc in hexane) to give (S)-(3-{ l-[(tert-Butoxycarbonylmethyl-amino)-methyl]-2-phenyl-ethylamino}-6- chloro-2-oxo-2H-pyrazin-l-yl)-acetic acid ethyl ester (316 mg) as a yellow oil. 1H NMR (CDC13, 400 MHz): δ 7.30-7.18 (m, 5 H); 6.93 (s, 1 H); 6.23 (bd, J = 8.3 Hz, 1 H); 4.88 (s, 2 H); 4.30-4.20 (m, 1 H); 4.12 (q, J = 7.1 Hz, 2 H); 3.33 (A of AB, d, J = 17.2 Hz, 1 H)); ); 3.22 (B of AB, d, J = 17.2 Hz, 1 H); 2.99 (A of ABX, dd, J = 13.6, 6 Hz, 1 H); 2.86 (B of ABX, dd, J = 13.6, 7.3 Hz, 1 H); 2.73 (d, J = 5.7 Hz, 2 H); 1.43 (s, 9 H); 1.26 (t, J = 7.1 Hz, 3 H). MS ES+ M+l= 479.3.
To a solution of (S)-(3-{l-[(tert-Butoxycarbonylmethyl-amino)- methyl]-2-phenyl-ethylamino}-6-chloro-2-oxo-2H-pyrazin-l-yl)-acetic acid ethyl ester (316 mg, 0.66 mmol) in THF (4 ml) is added 1 N LiOH (720 ul, 0.72 mmol) and the reaction mixture is stirred at room temperature for 18 h. Additional 1 N LiOH
(100 ul, 0.1 mmol) is added and the reaction mixture is stirred at room temperature for 1 h. To the reaction mixture is added 1 N HCl (820 ul, 0.82 mmol) and the reaction mixture is concentrated in vacuo to give (S)-(3-{ l-[(tert-Butoxycarbonylmethyl- armno)-methyl]-2-phenyl-ethylamino}-6-chloro-2-oxo-2H-pyrazin-l-yl)-acetic acid (300 mg) as a yellow solid. MS ES+ M+l= 451.3, M+Na = 473, M+Li = 457.3.
EXAMPLE 3 (S)- {3-[l-(Allyloxycarbonylamino-methyl)-2-phenyl-ethylamino]-6-chloro-2-oxo- 2H-pyrazin-l-yl}-acetic acid
Figure imgf000039_0001
To a solution of (S)-[3-(l-Aminomethyl-2-phenyl-ethylamino)-6- chloro-2-oxo-2H-pyrazin-l-yl] -acetic acid ethyl ester (80 mg, 0.22 mmol) in CH2C12 (2 ml) is added triethylamine (46 ul, 0.33 mmol) and allyl chloroformate (28 ul, 0.26 mmol). The reaction mixture is stirred at room temperature for 30 min, concentrated in vacuo and purified by flash chromatography (silica gel, 25% EtOAc in hexane to 50%) to give (S)- {3-[l-(Allyloxycarbonylamino-methyl)-2-phenyl-ethylamino]-6- chloro-2-oxo-2H-pyrazin-l-yl}-acetic acid ethyl ester (88 mg) as an oil. MS ES+ M+l= 449.2, M+Na = 473.2. To a solution of (S)- {3-[l-(Allyloxycarbonylamino-methyl)-2-phenyl- ethylamino]-6-chloro-2-oxo-2H-pyrazin-l-yl} -acetic acid ethyl ester (83 mg, 0.18 mmol) in MeOH (1.5 ml) is added 1 N LiOH (277 ul, 0.28 mmol) and the reaction mixture is stirred at room temperature for 2h20. To the reaction mixture is added 1 N LiOH (277 ul, 0.28 mmol) and the reaction mixture is stirred at room temperature for 6 h. To the reaction mixture is added 1 N HCl (560 ul, 0.56 mmol) and the reaction mixture is concentrated in vacuo to give (S)- {3-[l-(Allyloxycarbonylamino-methyl)- 2-phenyl-ethylamino]-6-chloro-2-oxo-2H-pyrazin-l-yl}-acetic acid which contains 3 eq LiCl. MS ES+ M+l= 421, M+Na = 444.2, M+Li = 427. 1H NMR (CD3OD, 400 MHz): δ 7.28-7.20 (m, 4 H); 7.20-7.10 (m, 1 H); 6.86 (s, 1 H); 5.96-5.82 (m, 1 H); 5.25 (d, J = 17.7 Hz, 1 H); 5.14 (d, J = 11 Hz, 1 H); 4.84 (s, 2 H); 4.50 (d, J = 5.5 Hz, 2 H); 4.40-4.28 (m, 1 H); 3.44-3.16 (m, 2 H); 2.96-2.80 (m, 2 H).
EXAMPLE 4: (S)-[3-(l-Aminomethyl-2-cyclopropyl-ethylamino)-6-chloro-2-oxo-2H-pyrazin-l-yl]- acetic acid ethyl ester
Figure imgf000040_0001
was prepared from (3-Bromo-2-oxo-2H-pyrazin-l-yl)-acetic acid ethyl ester and (S)- α-(Azidomethyl)-2-cyclopropylethylamine hydrochloride [prepared from L- cyclopropylalanine via reduction according to the procedure of A. Meyers et al (J. Org. Chem. 1993, 58, 3568), Boc protection under standard Boc2O conditions, azide substitution according to the procedure of Horwell et al (J. Med. Chem. 1991, 34, 404-414), and Boc removal under standard HCl(g) conditions], using a similar procedure as described in Example 1.
1H NMR (CDCI3, 300 MHz): δ 8.34 (bs, 2 H); 6.95 (s, 1 H); 6.84 (d, J = 6.7 Hz, 1 H); 4.98 (A of AB, d, J = 17.1 Hz, 1 H)); ); 4.80 (B of AB, d, J = 17.1 Hz, 1 H)); 4.30- 4.10 (m, 1 H); 4.24 (q, J = 7 Hz, 2 H); 3.45-3.35 (m, 1 H); 3.30-3.10 (m, 1 H); 1.95- 1.75 (m, 1 H); 1.65-1.45 (m, 1 H); 1.29 (t, J = 7 Hz, 3 H); 0.90-0.70 (m, 1 H); 0.60- 0.40 (m, 1 H); 0.20-0.05 (m, 1 H). MS ES+ M+l= 329.1
EXAMPLE 5:
(S)-19-Benzyl-8,24-dichloro-12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,11)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione dihydrochloride
Figure imgf000040_0002
Step A: preparation of [3-(l-(S)-Benzyl-2-{4-[2-(tert-butoxycarbonylamino-methyl)- 4-chloro-phenoxy] -butylamino } -ethylamino)-6-chloro-2-oxo-2H-pyrazin- 1 -yl] -acetic acid ethyl ester
Figure imgf000041_0001
To solution of (5-Chloro-2-hydroxy-benzyl)-carbamic acid tert-butyl ester (1.04 g, 4.03 mmol, prepared according to patent US 5,792,779 issued June 8, 1998) in DMF (10 ml) is added cesium carbonate (920 mg, 2.82 mmol) and 1,4- dibromobutane (2.41 ml, 20.2 mmol) and the reaction mixture is stirred at room temperature for 29 h. The reaction mixture is diluted with EtOAc, washed with water and aqueous LiCl (x3), dried on sodium sulfate, concentrated in vacuo and purified by flash chromatography (silica gel, 10% EtOAc in hexane to 20%) to give [2-(4-Bromo- butoxy)-5-chloro-benzyl]-carbamic acid tert-butyl ester (1.13 g). 1H NMR (CDC13, 400 MHz): δ 7.23 (d, J = 3 Hz, 1 H); 7.18 (dd, J = 8.6, 3 Hz, 1 H); 6.75 (d, J = 8.6 Hz, 1 H); 4.9 (bs, 1 H); 4.28 (bd, J = 5.6 Hz, 2 H); 4.0 (t, J = 5.6 Hz, 2 H); 3.49 (t, J = 6.1 Hz, 2 H); 2.12-1.92 (m, 4 H); 1.42 (s, 9 H).
To a solution of [2-(4-Bromo-butoxy)-5-chloro-benzyl]-carbamic acid tert-butyl ester (431 mg, 1.09 mmol) in DMF (10 ml) is added triethylamine (230 ul, 1.64 mmol) and (S)-[3-(l-Aminomethyl-2-phenyl-ethylamino)-6-chloro-2-oxo-2H- pyrazin-1-yl] -acetic acid ethyl ester (400 mg, 1.09 mmol) and the reaction mixture is stirred at 65 °C for 18 h. After cooling to room temperature, the reaction mixture is diluted with EtOAc, washed with water and aqueous LiCl (x3), dried on sodium sulfate, concentrated in vacuo and purified by flash chromatography (silica gel, 2% MeOH containing 10% NH4OH in CH2C12 to 4%) to give [3-(l-(S)-Benzyl-2-{4-[2- (tert-butoxycarbonylamino-methyl)-4-chloro-phenoxy] -butylamino } -ethylamino)-6- chloro-2-oxo-2H-pyrazin-l-yl] -acetic acid ethyl ester, (416 mg) as a foam. 1H NMR (CDCI3, 400 MHz): δ 7.30-7.10 (m, 7 H); 6.92 (s, 1 H); 6.73 (d, J = 8.7 Hz, 1 H); 6.21 (bd, J = 7.8 Hz, 1 H); 5.01 (bs, 1 H); 4.91(A of AB, d, J = 16.9 Hz, 1 H)); ); 4.84 (B of AB, d, J = 16.9 Hz, 1 H)); 4.38-4.20 (m, 5 H); 3.94 (t, J = 6.3 Hz, 2 H); 2.99 (A of ABX, dd, J = 13.1, 5.6 Hz, 1 H); 3.40 (B of ABX, dd, J = 13.1, 6.9 Hz, 1 H); 2.78- 2.67 (m, 2 H); 2.67-2.58 (m, 2 H); 1.86-1.76 (m, 2 H); 1.64-1.56 (m, 2 H); 1.44 (s, 9 H); 1.30 (t, J = 7.5 Hz, 3 H).
Step B: cyclization to 19-(S)-Benzyl-8,24-dichloro-12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,ll)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione dihydrochloride
Figure imgf000042_0001
Through solution of give [3-(l-(S)-Benzyl-2-{4-[2-(tert- butoxycarbonylamino-methyl)-4-chloro-phenoxy]-butylamino}-ethylamino)-6-chloro- 2-oxo-2H-pyrazin-l-yl]-acetic acid ethyl ester (416 mg, 0.61 mmol) in CH2C12 (50 ml) cooled to 0 °C is bubbled HCl (g) for 10 min. The flask is sealed and stirred at room temperature for 2 h. After argon is bubbled through for 5 min, the solution is concentrated in vacuo. To a solution of the residue in THF (16 ml) is added IN LiOH (3.1 ml, 3.1 mmol) and the reaction mixture is stirred at room temperature for 4 h. To the reaction mixture is added 1 N HCl (3.1 ml, 3.1 mmol), the reaction mixture is concentrated in vacuo, and azeothroped with THF then MeOH. . To a solution of the residue in DMF (225 ml) is added triethyl amine (428 ul, 3.07 mmol), l-hydroxy-7- azabenzotriazole (100 mg, 0.74 mmol) and l-ethyl-3-(3 dimethylaminopropyl) carbodiimide (153 mg, 0.8 mmol) and the reaction mixture is stirred at 50 °C for 16 h. The reaction mixture is concentrated to ca. 1/5 in vacuo, diluted with EtOAc and n- BuOH, washed with water. The aqueous layer is backextracted with n-BuOH. The combined organic layer is washed aqueous LiCl (x3), dried on sodium sulfate, concentrated in vacuo and purified by flash chromatography (silica gel, 4% MeOH containibg 10% NH OHin CH2C12 to 6%) to give 19-(S)-Benzyl-8,24-dichloro-12- oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,11)] pentacosa-6(ll),7,9,21, 23- pentaene-3,25-dione (125 mg) as a white solid. The dihydrochloride salt is obtained by treatment of a solution of the free base in CH2C12 with 2N HCl in Et O. 1H NMR (d6 DMSO, 400 MHz): δ 8.78 (bs, 2 H); 7.82 (bs, 1 H); 7.68 (d, J = 8.5 Hz, 1 H); 7.40-7.15 (m, 7 H); 7.00 (d, J = 7.9 Hz, 1 H); 6.92 (s, 1 H); 4.85 (bd, J = 15 Hz, 1 H); 4.60 (bs, 2 H); 4.40 (bd, J = 15 Hz, 1 H); 4.05-3.95 (m, 1 H); 3.95-3.60 (m, H count obliterated by residual water); 3.42-3.28 (m, 1 H); 3.05-3.15 (m, 1 H); 3.15-2.80 (m, 3 H); 2.80-2.65 (m, 1 H); 1.80-1.55 (m, 2 H); 1.55-1.45 (m, 1 H); 1.45-1.30 (m, 1 H).
MS ES exact mass calculated for C26H29Cl2N5O3: 530.1720, found: 530.1737. CHN calculated for C26H29Cl2N5O3 + 0.4 H2O + 2 HCl: G51.14, H:5.25, N:11.47; found: C:51.13, H:5.34, N.11.13. Rotation: [α]d = -15.4 ° (c = 0.21, MeOH).
EXAMPLE 6:
(S)-19-Benzyl-8-chloro-12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,11)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione and (S)-19-Benzyl-12-oxa- 1,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,ll)] pentacosa-6(ll),7,9,21, 23-pentaene- 3,25-dione
Figure imgf000043_0001
To a suspension of 19-(S)-Benzyl-8,24-dichloro-12-oxa-l, 4,17,20,22- pentaaza tricyclo[19.3.1.0(6,ll)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione (83 mg, 0.16 mmol) is added IN HCl (160 ul, 0.16 mmol). To the resulting solution is added catalytic 10% Pd/C and the reaction mixture is hydogeneted under 1 atm H2. Additional amounts of catalyst are added over 2 days until ca. 85% of the starting material is consumed. The reaction mixture is filtered on cellite and concentrated in vacuo. The crude material is purified by reverse phase preparative HPLC (5% to 95% CH3CN in water containing 0.1 % TFA, C18 PRO YMC 20x150 mm) to provide after extraction and hydrochloride preparation under standard conditions (S)-19-Benzyl-8- chloro-12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,ll)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione dihydrochloride.
1H NMR (CD3OD, 400 MHz): δ 7.40-7.15 (m, 7 H); 6.95 (d, J = 8.3 Hz, 1 H); 6.80 (s, 2 H); 4.90-4.60 (m, H count obliterated by residual water); 4.25 (d, J = 15.6 Hz, 1 H); 4.08-3.85 (m, 3 H); 3.18-2.90 (m, 4 H); 1.95-1.53 (m, 4 H). MS ES exact mass calculated for C26H30C1N5O3: 496.2110, found: 496.2134.
CHN calculated for C26H30C1N5O3 + 0.1 H2O + 2 HCl: C:54.72, H:5.69, N: 12.27; found: C:54.83, H:5.69, N: 11.89. Rotation: [α]d = -24 ° (c = 0.13, MeOH).
and (S)-19-Benzyl-12-oxa:l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,11)] pentacosa- 6(11),7,9,21, 23-pentaene-3,25-dione bis TFA salt after concentration. 1H NMR (CD3OD, 400 MHz): δ 7.38-7.16 (m, 7 H); 6.93 (d, J = 7.7 Hz, 1 H); 6.89 (t, J = 7.4 Hz, 1 H); 6.83 (d, J = 4.8 Hz, 1 H); 6.74 (d, J = 4.8 Hz, 1 H); 4.87(A of AB, d, J = 17.7 Hz, 1 H)); ); 4.77 (B of AB, d, J = 17.7 Hz, 1 H)); 4.72-4.60 (m, 2 H); ); 4.18 (d, J = 15.5 Hz, 1 H); 4.06 (d, J = 13.9 Hz, 1 H); 4.03-3.96 (m, 1 H); 3.96-3.86 (m, 1 H); 3.14-3.07 (m, 2 H); ); 3.03 (A of ABX, dd, J = 13.9, 7.7 Hz, 1 H); 2.94 (B of ABX, dd, J = 13.9, 7.4 Hz, 1 H); 1.88-1.53 (m, 4 H). MS ES+ M+l= 462.3.
EXAMPLE 7:
(S)-17-Benzyl-8,22-dichloro-12-oxa-l,4,15,18,20-pentaaza tricyclo[17.3.1.0(6,11)] tricosa-6(ll),7,9,19, 21-pentaene-3,23-dione bis TFA salt
Figure imgf000045_0001
(S)- 17-Benzyl-8,22-dichloro- 12-oxa- 1 ,4, 15 , 18 ,20-pentaaza tricyclo[17.3.1.0(6,11)] tricosa-6(ll),7,9,19, 21-pentaene-3,23-dione bis TFA salt is prepared from [2-(2-Bromo-ethoxy)-5-chloro-benzyl]-carbamic acid tert-butyl ester (prepared from (5-Chloro-2-hydroxy-benzyl)-carbamic acid tert-butyl ester and 1,2- dibromoethane) and (S)-[3-(l-Aminomethyl-2-phenyl-ethylamino)-6-chloro-2-oxo- 2H-pyrazin-l-yl] -acetic acid ethyl ester, using a similar procedure as described in example 5.
1H NMR (CD3OD, 400 MHz): δ 7.36 (d, J = 3 Hz, 1 H); 7.32-7.16 (m, 6 H); 6.98 (d, J = 9 Hz, 1 H); 6.92 (s, 1 H); 4.92-4.78 (m, 1 H); 4.81(A of AB, d, J = 15.7 Hz, 1 H)); 4.68 (B of AB, d, J = 15.7 Hz, 1 H); 4.38-4.30 (m, 1 H); 4.36 (A of AB, d, J = 14.2 Hz, 1 H)); 4.26-4.18 (m, 1 H); 4.13 (B of AB, d, J = 14.2 Hz, 1 H); 3.45-3.25 (m, 4 H); 3.08-2.92 (m, 2 H).
MS ES exact mass calculated for C24H25Cl2N5O3: 502.1407, found: 502.1414. Rotation: [α]d = -11 ° (c = 0.42, MeOH).
EXAMPLE 8: (S)-18-Benzyl-8,23-dichloro-12-oxa-l,4,16,19,21-pentaaza tricyclo[18.3.1.0(6,11)] tetracosa-6(l 1),7,9,20, 22-pentaene-3,24-dione
Figure imgf000046_0001
(S)-18-Benzyl-8,23-dichloro-12-oxa-l,4,16,19,21-pentaaza tricyclo[18.3.1.0(6,ll)] tetracosa-6(ll),7,9,20, 22-pentaene-3,24-dione is prepared from [2-(3-Bromo-propoxy)-5-chloro-benzyl]-carbamic acid tert-butyl ester (prepared from (5-Chloro-2-hydroxy-benzyl)-carbamic acid tert-butyl ester and 1,3- dibromopropane) and (S)-[3-(l-Aminomethyl-2-phenyl-ethylamino)-6-chloro-2-oxo- 2H-pyrazin-l-yl] -acetic acid ethyl ester, using a similar procedure as described in example 5.
1H NMR (CD3OD + CDC13, 300 MHz): δ 7.36-7.16 (m, 7 H); 6.96 (s, 1 H); 6.82 (d, J = 9 Hz, 1 H); 5.04 (A of AB, d, J = 16.4 Hz, 1 H); 4.52 (B of AB, d, J = 16.4 Hz, 1 H); 4.38-4.30 (m, 1 H); 4.33 (A of AB, d, J = 13.7 Hz, 1 H); 4.25 (B of AB, d, J = 13.7 Hz, 1 H); 4.10-3.98 (m, 1 H); 3.98-3.85 (m, 1 H); 3.05-2.70 (m, 3 H); 2.70-2.52 (m, 1 H); 1.85-1.55 (m, 1 H).
MS ES exact mass calculated for C25H27Cl2N5O3: 516.1564, found: 516.1524 CHN calculated for C25H27Cl2N5O3 + 0.45 H2O: C:57.24, H:5.36, N: 13.35; found: C:57.20, H:5.12, N:12.99.
EXAMPLE 9: (S)-20-Benzyl-8,25-dichloro-12-oxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,11)] hexacosa-6(l 1),7,9,22, 24-pentaene-3,26-dione
Figure imgf000047_0001
(S)-20-Benzyl-8,25-dichloro-12-oxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione is prepared from [2-(5-Bromo-pentoxy)-5-chloiO-benzyl]-carbamic acid tert-butyl ester (prepared from (5-Chloro-2-hydroxy-benzyl)-carbamic acid tert-butyl ester and 1,5- dibromopentane) and (S)-[3-(l-Aminomethyl-2-phenyl-ethylamino)-6-chloro-2-oxo- 2H-pyrazin-l-yl] -acetic acid ethyl ester, using a similar procedure as described in example 5.
1H NMR (CD3OD + CDC13, 400 MHz): δ 7.36-7.15 (m, 7 H); 6.87 (s, 1 H); ); 6.78 (d, J = 9 Hz, 1 H); 5.23 (A of AB, d, J = 17 Hz, 1 H); 4.60-4.50 (m, 1 H); 4.41(A of AB, d, J = 14 Hz, 1 H); 4.31 (B of AB, d, J = 17 Hz, 1 H); ); 4.18 (B of AB, d, J = 14 Hz, 1 H); 3.98-3.82 (m, 2 H); 3.03-2.73 (m, 5 H); 2.61-2.52 (m, 1 H); 1.80-1.36 (m, 6 H). MS ES+ M+l= 544.3
EXAMPLE 10:
(S)-20-Benzyl-8-chloro-12-oxa-l,4,18,21,23-pentaaza-tricyclo-[20.3.1.0(6,ll)]- hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride and (S)-20-Benzyl- 12-oxa-l,4,18,21,23-pentaaza-tricyclo-[20.3.1.0(6,ll)]-hexacosa-6(ll),7,9,22,24- pentaene-3,26-dione bis TFA salt.
Figure imgf000048_0001
(S)-20-Benzyl-8-chloro-12-oxa-l,4,18,21,23-pentaaza-tricyclo- [20.3.1.0(6,ll)]-hexacosa-6(ll),7 ,9,22, 24-pentaene-3,26-dione dihydrochloride and (S)-20-Benzyl-12-oxa-l,4,18,21,23-pentaaza-tricyclo-[20.3.1.0(6,ll)]-hexacosa- 6(1 l),7,9,22,24-pentaene-3,26-dione bis TFA salt are prepared from (S)-20-Benzyl- 8,25-dichloro-12-oxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,11)] hexacosa- 6(11),7 ,9,22, 24-pentaene-3,26-dione using a similar procedure as described in example 6: (S)-20-Benzyl-8-chloro-12-oxa-l,4,18,21,23-pentaaza-tricyclo-[20.3.1.0(6,11)]- hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride:
1H NMR (CD3OD + CDC13, 400 MHz): δ 7.37-7.20 (m, 7 H); 6.92 (d, J = 8.9 Hz, 1 H); 6.91 (d, J = 5.3 Hz, 1 H); 6.78 (d, J = 5.3 Hz, 1 H); 4.96-4.64 (m, 3 H); 4.29 (d, J = 16.4 Hz, 1 H); 4.06-3.93 (m, 2 H); 3.93-3.81 (m, 1 H); 3.45-3.25 (m, 2 H); 3.18- 2.95 (m, 4 H); 1.85-1.38 (m, 6 H). MS ES+ M+1= 510.3
(S)-20-Benzyl-12-oxa-l,4,18,21,23-pentaaza-tricyclo-[20.3.1.0(6,ll)]-hexacosa- 6(ll),7,9,22,24-pentaene-3,26-dione bis TFA salt:
1H NMR (CD3OD + CDC13, 400 MHz): δ 7.30-7.16 (m, 7 H); 6.92 (d, J = 8.4 Hz, 1 H); 6.90 (t, J = 7 Hz, 1 H); 6.83 (d, J = 4.8 Hz, 1 H); 6.71 (d, J = 4.8 Hz, 1 H); 4.90 (d, J = 16 Hz, 1 H); 4.72-4.58 (m, 2 H); 4.15-4.08 (m, 1 H); 4.14 (d, J = 16 Hz, 1 H);
4.01-3.92 (m, 1 H); 3.92-3.82 (m, 1 H); 3.40-3.20 (m, 2 H); 3.15-2.84 (m, 4 H); 1.80-
1.36 (m, 6 H).
MS ES+ M+l= 510.3 EXAMPLE 11: (S)-21-Benzyl-8,26-dichloro-12-oxa-l,4,19,22,24-pentaaza tricyclo-[21.3.1.0(6,11)] heptacosa-6(l 1),7,9,23, 25-pentaene-3,27-dione
Figure imgf000049_0001
(S)-21-Benzyl-8,26-dichloro-12-oxa-l,4,19,22,24-pentaaza-tricyclo-
[21.3.1.0(6,ll)]-heptacosa-6(ll),7,9,23, 25-pentaene-3,27-dione is prepared from [2- (6-BiOmo-hexoxy)-5-chloro-benzyl]-carbamic acid tert-butyl ester (prepared from (5- Chloro-2-hydroxy-benzyl)-carbamic acid tert-butyl ester and 1,6-dibromohexane) and (S)-[3-(l-Aminomethyl-2-phenyl-ethylamino)-6-chloro-2-oxo-2H-pyrazin-l-yl]-acetic acid ethyl ester, using a similar procedure as described in example 5.
1H NMR (CD3OD + CDC13, 400 MHz): δ 7.34-7.14 (m, 7 H); 6.89 (s, 1 H); ); 6.79 (d, J = 8.6 Hz, 1 H); 5.18 (A of AB, d, J = 16.3 Hz, 1 H); 4.50(A of AB, d, J = 14.7 Hz, 1 H); 4.49-4.37 (m, 1 H); 4.39 (B of AB, d, J = 16.3 Hz, 1 H); ); 4.13 (B of AB, d, J = 14.7 Hz, 1 H); 4.00-3.84 (m, 2 H); 3.02-2.91 (m, IH); 2.86-2.72 (m, 3 H); 2.72-2.60 (m, 1 H); 2.60-2.50 (m, 1 H); 1.84-1.58 (m, 2 H); 1.58-1.18 (m, 6 H).
MS ES exact mass calculated for C28H33Cl2N5O3: 558.2033, found: 558.2072. Rotation: [α]d = -41.3 ° (c = 0.15, MeOH).
EXAMPLE 12: (S)-19-Benzyl-17-Methyl-8,24-dichloro-12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,11)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione bis TFA salt
Figure imgf000050_0001
To l9-(S)-Benzyl-8,24-dichloro-12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,11)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione bis TFA salt (10 mg, 0.013 mmol) is added formaldehyde (0.5 ml of a solution of 50 ul of 37% aqueous formaldehyde in 5 ml 2:1 dichloroethane:MeOH, 0.065 mmol) and NaBH(OAc)3 (5.6 mg, 0.026 mmol) and the reaction mixture is allowed to stand at room temperature for 3 h. The solvent is blown down under a flow of nitrogen and the crude mixture is purified by reverse phase preparative HPLC (5% to 95% CH3CN in water containing 0.1 % TFA, C18 PRO YMC 20x150 mm) to provide after concentration (S)- 19-Benzyl- 17-Methyl-8 ,24-dichloro- 12-oxa- 1 ,4, 17 ,20,22-pentaaza tricyclo[19.3.1.0(6,ll)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione bis TFA salt. MS ES exact mass calculated for C27H31Cl2N5O3: 544.1877, found: 544.1904. CHN calculated for C27H31Cl2N5O3 + 0.25 H2O + 1.55 TFA: G49.82, H:4.59, N:9.65; found: C:49.82, H:4.57, N:9.74.
EXAMPLE 13: (S)-19-Benzyl-17-Ethyl-8,24-dichloro-12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,11)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione bis TFA salt
Figure imgf000051_0001
(S)- 19-Benzyl- 17-Ethyl-8 ,24-dichloro- 12-oxa- 1 ,4, 17 ,20,22-pentaaza tricyclo[19.3.1.0(6,ll)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione bis TFA salt is prepared from 19-(S)-Benzyl-8,24-dichloro-12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,ll)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione bis TFA salt and acetaldehyde using a similar procedure as described in example 12. MS ES exact mass calculated for C28H33Cl2N5O3: 558.2033, found: 558.2033.
Example 14:
(S)-20-Benzyl-18-Methyl-8,25-dichloro-12-oxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,11)] hexacosa-6(l 1),7,9,22, 24-ρentaene-3,26-dione dihydrochloride
Figure imgf000051_0002
(S)-20-Benzyl- 18-Methyl-8 ,25-dichloro- 12-oxa- 1 ,4, 18 ,21 ,23-pentaaza tricyclo[20.3.1.0(6,11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride is prepared from (S)-20-Benzyl-8,25-dichloro-12-oxa-l,4,18,21,23- pentaaza tricyclo[20.3.1.0(6, 11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione and formaldehyde using a similar procedure as described in example 12. MS ES+ M+1= 558.3.
EXAMPLE 15:
(S)-20-Cyclopropylmethyl-8,25-dichloro-12-oxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione
Figure imgf000052_0001
(S)-20-Cyclopropylmethyl-8,25-dichloro-12-oxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione is prepared from [2-(5-Bromo-pentoxy)-5-chloro-benzyl]-carbamic acid tert-butyl ester (prepared from (5-Chloro-2-hydroxy-benzyl)-carbamic acid tert-butyl ester and 1,5- dibromopentane) and (S)-[3-(l-Aminomethyl-2-cyclopropyl-ethylamino)-6-chloro-2- oxo-2H-pyrazin-l-yl] -acetic acid ethyl ester (example 4), using a similar procedure as described in example 5.
1H NMR (CDC13, 300 MHz): δ 7.34 (s, 1 H); 7.18 (d, J = 9 Hz, 1 H); 6.92 (s, 1 H); 6.83 (bs, 1 H); 6.76 (d, J = 9 Hz, 1 H); 6.47 (bd, J = 6 Hz, 1 H); 5.20 (d, J = 16.1 Hz, 1 H); 4.48-4.20 (m, 4 H); 4.02-3.80 (m, 2 H); 3.15-2.78 (m, 3 H); 2.78-2.60 (m, 1 H); 1.90-1.05 (m, 8 H); 0.77-0.60 (m, 1 H); 0.47 (d, J = 7.6 Hz, 2 H); 0.18-0.02 (m, 2 H). MS ES+ M+l= 508.2
EXAMPLE 16: (S)-20-Cyclopropylmethyl-18-Methyl-8,25-dichloro-12-oxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6, 11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione bis TFA salt
Figure imgf000053_0001
(S)-20-Cyclopropylmethyl- 18-Methyl-8 ,25-dichloro- 12-oxa- 1,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,11)] hexacosa-6(ll),7,9,22, 24-pentaene- 3,26-dione bis TFA salt is prepared from (S)-20-Cyclopropylmethyl-8,25-dichloro-12- oxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24- pentaene-3,26-dione and formaldehyde using a similar procedure as described in example 12.
1H NMR (CDC13, 400 MHz): δ 7.40 (s, 1 H); 7.30 (bs, 1 H); 7.18 (d, J = 9 Hz, 1 H); 6.80 (s, 1 H); 6.77 (d, J = 9 Hz, 1 H); 6.63 (bd, J = 6 Hz, 1 H); 5.39 (d, J = 16.1 Hz, 1 H); 4.88-4.72 (m, 1 H); 4.45-4.35 (m, 1 H); 4.25-4.15 (m, 1 H); 4.05-3.95 (m, 1 H); 3.85-3.75 (m, 1 H);3.75-3.60 (m, 1 H); 3.15-3.05 (m, 1 H);2.90-2.65 (m, 3 H); 2.83 (s, 3 H); 1.95-1.25 (m, 8 H); 1.75-1.60 (m, 1 H); 0.50 (d, J = 7.6 Hz, 2 H); 0.18-0.02 (m, 2 H).
MS ES exact mass calculated for C25H33Cl2N5O3: 522.2033, found: 522.2028. CHN calculated for C25H33Cl2N5O3 + 1.85 TFA: C:47.00, H:4.79, N:9.55; found: C:47.12, H:4.72, N:9.46. Rotation: [α]d = -57.1 ° (c = 0.35, MeOH).
EXAMPLE 17: (S)-20-Cyclopropylmethyl-8 ,25-dichloro- 12, 15-dioxa- 1 ,4, 18 ,21 ,23-pentaaza tricyclo[20.3.1.0(6,11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione
Step A: Preparation of {5-Chloro-2-[2-(2-hydroxy-ethoxy)-ethoxy]-benzyl}-carbamic acid tert-butyl ester
Figure imgf000054_0002
To a solution of (5-Chloro-2-hydroxy-benzyl)-carbamic acid tert-butyl ester (2.01 g, 7.8 mmol) in THF (10 ml) is added triphenylphosphine (2.66 g, 10.16 mmol), allyl hydroxyethyl ether (1.25ml, 11.7 mmol) and diethyl azodicarboxylate (1.6 ml, 10.16 mmol) dropwise, and the reaction mixture is stirred at room temperature for 18 h. The reaction mixture is concentrated in vacuo and purified by flash chromatography (silica gel, 10% EtOAc in hexane to 40%) to give [2-(2-
Allyloxy-ethoxy)-5-chloro-benzyl]-carbamic acid tert-butyl ester as a colorless oil. 1H NMR (CDC13, 400MHz): δ 7.25 (d, J = 0.5 Hz, 1 H); 7.18 (dd, J = 5.4, 0.5 Hz, 1 H); 6.78 (d, J = 5.4 Hz, 1 H); 6.02-5.88 (m, 1 H); 5.48 (bs, 1 H); 5.38-5.18 (m, 2 H); 4.25 (d, J = 5.8 Hz, 2 H); 4.20-4.07 (m, 4 H); 3.85-3.75 (m, 2 H);1.45 (s, 9H). Through a solution of [2-(2-Allyloxy-ethoxy)-5-chloro-benzyl]- carbamic acid tert-butyl ester (0.75 g, 3.1 mmol) in CH2C12 (20 ml) and MeOH (5 ml) cooled to -78 °C is bubbled ozone until the solution remained blue. Ozone is then bubbled for an extra 5 min. The solution is purged with oxygen, allowed to stir at -78 °C for 30 min and carefully quenched with NaBFL (80 mg, 2.1 mmol) and the reaction mixture is allowed to gradually warm up to room temperature over 18 h. The reaction mixture is concentrated in vacuo, diluted with CH2C12, washed with water, concentrated in vacuo and purified by flash chromatography (silica gel, 20% EtOAc in hexane to 70%) to give {5-Chloro-2-[2-(2-hydroxy-ethoxy)-ethoxy]-benzyl}-carbamic acid tert-butyl ester (0.6 g) as a colorless oil.
1H NMR (CDC13, 400MHz): δ 7.20 (d, J = 9 Hz, 1 H); 7.15 (s, 0.5 H); 6.80 (d, J = 9 Hz, 1 H); 6.30 (bt, 0.5 H); 5.50 (bt, 0.5 H); 4.28 (d, J = 5.7 Hz, 1 H); 4.18 (d, J = 5.7 Hz, 1 H); 3.90-3.80 (m, 2 H); 3.90-3.72 (m, 2 H); 3.72-3.55 (m, 2 H); 1.43 (s, 9H).
Step B: Preparation of {3-[2-(2-{2-[2-(tert-Butoxycarbonylamino-methyl)-4-chloro- phenoxy] -ethoxy } -ethylamino)- 1 -cyclopropylmethyl-ethylamino] -6-chloro-2-oxo-2H- pyrazin-1-yl} -acetic acid ethyl ester
Figure imgf000055_0001
To solution of oxalyl chloride (0.35 ml of a 2 M solution in CH C12, 0.70 mmol) in CH2C12 (3 ml) cooled to -78 °C is added DMSO (0.10 ml, 1.38 mmol) and the reaction mixture is stirred at -78 °C for 5 min. The resulting solution is cannulated to a solution of {5-Chloro-2-[2-(2-hydroxy-ethoxy)-ethoxy]-benzyl}- carbamic acid tert-butyl ester (0.22 g, 0.64 mmol) in CH2C12 (4 ml) cooled to -78 °C and the reaction mixture is stirred at -78 °C for 15 min. Triethylamine (0.44 ml, 3.19 mmol) is added and the reaction mixture is stirred at -78 °C for 30 min. The reaction mixture is allowed to warm to room temperature, is diluted with CH2C12, washed with water, dried on sodium sulfate, and concentrated in vacuo to give {5-Chloro-2-[2-(2- oxo-ethoxy)-ethoxy] -benzyl }-carbamic acid tert-butyl ester which is imediately used in the next step without further purification. 1H NMR (CDCI3, 400MHz): δ 9.75 (s, 1 H); 7.20 (d, J = 9 Hz, 1 H); 6.80 (d, J = 9 Hz, 1 H); 5.28 (bt, 1 H); 4.30 (d, J = 5.7 Hz, 2 H); 4.28 (s, 2 H); 4.22-4.18 (m, 2 H); 3.95- 3.90 (m, 2 H); 1.43 (s, 9H).
To a solution of give {5-Chloro-2-[2-(2-oxo-ethoxy)-ethoxy]-benzyl}- carbamic acid tert-butyl ester (255 mg, 0.63 mmol) in dichloroethane (5 ml) is added (S)-[3-(l-Aminomethyl-2-cyclopropyl-ethylamino)-6-chloro-2-oxo-2H-pyrazin-l-yl]- acetic acid ethyl ester (198 mg, 0.60 mmol) and NaBH(OAc)3 (153 mg, 0.73 mmol). The reaction mixture is stirred at room temperature for 50 min, diluted with CH C12, washed with aqueous sodium bicarbonate, dried on sodium sulfate, concentrated in vacuo, and purified by flash chromatography (silica gel, 1% MeOH containibg 10% NH OH in CH2C12 to 5%) to give {3-[2-(2-{2-[2-(tert-Butoxycarbonylarmno-methyl)- 4-chloro-phenoxy] -ethoxy } -ethylamino)- 1 -cyclopropylmethyl-ethylamino] -6-chloro- 2-oxo-2H-pyrazin-l-yl} -acetic acid ethyl ester (193 mg).
1H NMR (CDC13, 400MHz): δ 7.25 (bs, 1 H); 7.18 (dd, J = 7.9, 2.6 Hz, 1 H); 6.91 (s, 1 H); 6.78 (d, J = 7.9 Hz, 1 H); 6.20 (bd, J = 7.8 Hz, 1 H); 5.52 (bt, 1 H); 4.88 (s, 2 H); 4.32-4.22 (m, 3 H); 4.20-4.07 (m, 4 H); 3.81 (bt, J = 5.2 Hz, 2 H); 3.70-3.60 (m, 2 H); 2.90-2.80 (m, 4 H); 1.55-1.35 (m, 2 H); 1.43 (s, 9H); 1.30 (t, J = 6.5 Hz, 3 H); 0.73-0.62 (m, 1 H); 0.43 (d, J = 8.3 Hz, 2H); 0.12-0.02 (m, 2 H). MS ES+ M+l= 656.5
Step C: cyclization to (S)-20-Cyclopropylmethyl-8,25-dichloro-12,15-dioxa- 1,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene- 3,26-dione
Figure imgf000056_0001
The deprotection / cyclization sequence to provide (S)-20- Cyclopropylmethyl-8,25-dichloro- 12, 15-dioxa- 1 ,4, 18 ,21 ,23-pentaaza tricyclo[20.3.1.0(6, 11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione is performed using a similar procedure as described in example 5, step B.
1H NMR (CDC13, 300MHz): δ 7.71 (bt, 1 H); 7.51 (d, J = 2.9 Hz, 1 H); 7.16 (dd, J = 8.9, 2.9 Hz, 1 H); 6.87 (s, 1 H); 6.73 (d, J = 8.9 Hz, 1 H); 6.12 (bd, J = 9.3 Hz, 1 H); 5.36-5.24 (m, 2 H); 4.48-4.22 (m, 3 H); 4.20-4.10 (m, 1 H); 4.05-3.95 (m, 1 H); 3.83- 3.65 (m, 2 H); 3.60-3.52 (m, 2 H); 3.05-2.90 (m, 2 H); 2.78-2.62 (m, 2 H); 1.70-1.52 (m, 1 H); 1.45-1.32 (m, 1 H); 0.95-0.65 (m, 2 H); 0.52-0.42 (m, 1 H); 0.13-0.05 (m, 2 H). MS ES+ M+1= 510.4
EXAMPLE 18: (S)-20-Benzyl-8,25-dichloro-12,15-dioxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione
Figure imgf000057_0001
(S)-20-Benzyl-8,25-dichloro-12,15-dioxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione is prepared from {5-Chloro-2-[2-(2-chloro-ethoxy)-ethoxy]-benzyl}-carbamic acid tert-butyl ester (prepared from (5-Chloro-2-hydroxy-benzyl)-carbamic acid tert-butyl ester and 2,2'- dichloroethyl ether) and (S)-[3-(l-Aminomethyl-2-phenyl-ethylamino)-6-chloro-2- oxo-2H-pyrazin-l-yl] -acetic acid ethyl ester, using a similar procedure as described in example 5. 1H NMR (CDC13, 400 MHz): δ 7.39 (d, J = 3 Hz, 1 H); 7.32-7.14 (m, 6 H); 7.01 (bt, J = 6.1 Hz, 1 H); 6.89 (s, 1 H); 6.74 (d, J = 8.5 Hz, 1 H); 6.15 (d, J = 9.3 Hz, 1 H); 5.18 (A of AB, d, J = 16.3 Hz, 1 H); 4.57-4.46 (m, 1 H); 4.39 (B of AB, d, J = 16.3 Hz, 1 H); 4.40-4.37 (m, 2 H); 4.16-4.08 (m, 1 H); 4.02-3.93 (m, 1 H); 3.80-3.72 (m, 1 H); 3.72-3.64 (m, 1 H); 3.61-3.49 (m, 2 H); 2.98-2.79 (m, 4 H); 2.73-2.63 (m, 2 H). MS ES exact mass calculated for C26H29Cl2N5O4: 546.1669, found: 546.1680.
EXAMPLE 19: 15-Ethyl- 20(S)-Benzyl-8,25-dichloro-12-oxa-l,4,15,18,21,23-hexaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,16,26-trione bis TFA salt
Figure imgf000058_0001
15-Ethyl- 20(S)-Benzyl-8,25-dichloro-12-oxa-l,4,15,18,21,23-hexaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,16,26-trione bis TFA salt was prepared according to the following sequence: (S)-(3-{ l-[(tert- Butoxyc-ιrbonylmethyl-amino)-methyl]-2-phenyl-ethylamino}-6-chloro-2-oxo-2H- pyrazin-l-yl)-acetic acid (example 2) is coupled to [2-(2-Aminomethyl-4-chloro- phenoxy)-ethyl]-ethyl-amine bis hydrochloride (prepared from [2-(2-Bromo-ethoxy)- 5-chloro-benzyl]-carbamic acid tert-butyl ester, example 7, using ethylamine to displace the terminal bromide, followed by Boc removal under standard HCl (g) conditions), using standard EDC / HOAt conditions. The tert-butyl ester is converted to the corresponding carboxylic acid using TFA and final cyclization is performed under EDC / HOAt conditions using a similar procedure as described in example 5, step B. MS ES+ M+1= 587.3. EXAMPLE 20: (S)-21-Benzyl-8,26-dichloro-12,17-dioxa-l,4,19,22,24-pentaaza tricyclo- [21.3.1.0(6,11)] heptacosa-6(ll),7,9,14(Z),23, 25-hexaene-3,18,27-trione TFA salt and (S)-21-Benzyl-8,26-dichloro-12,17-dioxa-l,4,19,22,24-pentaaza tricyclo- [21.3.1.0(6,11)] heptacosa-6(ll),7,9,14(E),23, 25-hexaene-3,18,27-trione TFA salt
Figure imgf000059_0001
To a solution of (S)- {3-[l-(Allyloxycarbonylamino-methyl)-2-phenyl- ethylamino]-6-chloro-2-oxo-2H-pyrazin-l-yl} -acetic acid (0.18 mmol, example 3) in DMF (2 ml) is added 2-AUyloxy-5-chloiO-benzylamine hydrochloride (56 mg, 0.24 mmol, prepared from 5-Chloro-2-hydroxy-benzyl)-carbamic acid tert-butyl ester and allyl bromide, followed by Boc removal under HCl (g) conditions), triethylamine (38 ul, 0.28 mmol), l-hydroxy-7-azabenzotriazole (30 mg, 0.22 mmol) and l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (42.5 mg, 0.22 mmol) and the reaction mixture is stirred at room temperature for 18 h. The reaction mixture is diluted with EtOAc, washed with water and aqueous LiCl (x3), dried on sodium sulfate, concentrated in vacuo and purified by flash chromatography (silica gel, 30% EtOAc in hexane to 50%) to give (2-{4-[(2-Allyloxy-5-chloro-benzylcarbamoyl)-methyl]-5-chloro-3-oxo- 3,4-dihydro-pyrazin-2-ylamino}-3-phenyl-propyl)-carbamic acid allyl ester (100 mg) as a white foam. MS ES+ M+l= 600.3. IH NMR (CDC13, 300 MHz): δ 7.34-7.14 (m, 7 H); 6.94 (s, 1 H); 6.76 (d, J = 8.4 Hz, 1 H); 6.40 (bt, J = 5.3 Hz, 1 H); 6.24 (d, J = 7.9 Hz, 1 H); 6.08-5.80 (m, 4 H); 5.1240 (bt, J = 5.6 Hz, 1 H); 4.84(A of AB, d, J = 15.4 Hz, 1 H)); ); 4.75 (B of AB, d, J = 15.4 Hz, 1 H)); 4.58-4.46 (m, 4 H); 4.48-4.41 (m, 2 H); 4.36-4.23 (m, 1 H); 3.56-3.14 (m, 1 H); 3.51-3.17 (m, 1 H); 2.97 (A of ABX, dd, J = 14.3, 6.7 Hz, 1 H); 2.85 (B of ABX, dd, J = 14.3, 7 Hz, 1 H).
To a solution of (2-{4-[(2-Allyloxy-5-chloro-benzylcarbamoyl)- methyl]-5-chloro-3-oxo-3,4-dihydro-pyrazin-2-ylamino}-3-phenyl-propyl)-carbamic acid allyl ester (25 mg, 0.04 mmol) in CH2C12 (20 ml, degassed with argon) is added bis(tricyclohexylphosphine)benzylidine ruthenium (IV) dichloride (3.4 mg, 0.004 mmol) and the reaction mixture is stirred at room temperature for 6h30. Bis(tricyclohexylphosphine)benzylidine ruthenium (IV) dichloride (3 mg) is added and the reaction mixture is stirred at room temperature for 16 h. The reaction mixture is concentrated in vacuo and purified by flash chromatography (silica gel, 30% EtOAc in hexane to 75%) to give after further purification by preparative HPLC (5% to 95% CH3CN in water containing 0.1 % TFA, C18 PRO YMC 20x150 mm):
(S)-21-Benzyl-8,26-dichloro-12,17-dioxa-l,4,19,22,24-pentaazatricyclo-
[21.3.1.0(6,11)] heρtacosa-6(ll),7,9,14(Z),23, 25-hexaene-3,18,27-trione TFA salt (1.8 mg). 1H NMR (CD3OD + CDC13, 400 MHz): δ 7.32-7.12 (m, 7 H); 6.89 (d, J = 7.8 Hz, 1 H); 6.85 (s, 1 H); 5.80 (dt, J = 11, 5.2 Hz, 1 H); 5.64 (dt, J = 11, 6.7 Hz, 1 H); ); 4.96(A of AB, d, J = 16.3 Hz, 1 H); 4.69-4.52 (m, 3 H); 4.46 (B of AB, d, J = 16.3 Hz, 1 H); 4.29-4.20 (m, 1 H); 4.16-4.07 (m, 1 H); 3.50-3.41 (m, 1 H); 3.21-3.12 (m, 1 H); 2.96 (A of ABX, dd, J = 14, 7.4 Hz, 1 H); 2.79 (B of ABX, dd, J = 14, 7 Hz, 1 H). MS ES+ M+l= 572.3.
and (S)-21-Benzyl-8,26-dichloro-12,17-dioxa-l,4,19,22,24-pentaazatricyclo- [21.3.1.0(6,11)] heptacosa-6(ll),7,9,14(E),23, 25-hexaene-3,18,27-trione TFA salt (9.3 mg). 1H NMR (CD3OD + CDC13, 400 MHz): δ 7.83 (bt, J = 5.2 Hz, 1 H); 7.36- 7.10 (m, 7 H); 6.83 (s, 1 H); 6.82 (d, J = 9.2 Hz, 1 H); 5.89 (A of ABX, dt, J = 16, 4 Hz, 1 H); 5.80 (B of ABX, dt, J = 16, 4.5 Hz, 1 H); 5.16(A of AB, d, J = 16 Hz, 1 H); 4.56-4.29 (m, 6 H); 4.29 (B of AB, d, J = 16 Hz, 1 H); 3.60 (A of ABX, dd, J = 14, 3.6 Hz, 1 H); 3.11 (B of ABX, dd, J = 14, 8.4 Hz, 1 H); 2.99-2.82 (m, 2 H). MS ES+ M+l= 572.3.
EXAMPLE 21: (S)-21-Benzyl-8,26-dichloro-12,17-dioxa-l,4,19,22,24-pentaaza tricyclo- [21.3.1.0(6,11)] heptacosa-6(ll),7,9, 23, 25-pentaene-3,18,27-trione TFA salt and (S)-21-Benzyl-8-chloro-12,17-dioxa-l,4,19,22,24-pentaaza tricyclo-[21.3.1.0(6,ll)] heptacosa-6(ll),7,9,23, 25-pentaene-3,18,27-trione TFA salt
Figure imgf000061_0001
(S)-21-Benzyl-8,26-dichloro-12,17-dioxa-l,4,19,22,24-pentaaza tricyclo-[21.3.1.0(6,ll)] heptacosa-6(ll),7,9, 23, 25-pentaene-3,18,27-trione TFA salt and (S)-21-Benzyl-8-chloro-12,17-dioxa-l,4,19,22,24-pentaaza tricyclo- [21.3.1.0(6,11)] heptacosa-6(ll),7,9,23, 25-pentaene-3,18,27-trione TFA salt are prepared from (S)-21-Benzyl-8,26-dichloro-12,17-dioxa-l,4,19,22,24-pentaaza tricyclo-[21.3.1.0(6,11)] heptacosa-6(ll),7,9,14(Z),23, 25-hexaene-3,18,27-trione using a similar procedure as described in example 6:
(S)-21-Benzyl-8,26-dichloro-12,17-dioxa-l,4,19,22,24-pentaaza-tricyclo- [21.3.1.0(6,11)] heptacosa-6(ll),7,9, 23, 25-pentaene-3,18,27-trione TFA salt. 1H NMR (CD3OD + CDC13, 400 MHz): δ 7.56 (bt, J = 5.4 Hz, 1 H); 7.34-7.16 (m, 7 H); 6.87 (s, 1 H); 6.83 (d, J = 8.5 Hz, 1 H); 5.13(A of AB, d, J = 16.3 Hz, 1 H); 4.53-4.45 (m, 1 H); 4.45-4.21 (m, 2H); 4.36 (B of AB, d, J = 16.3 Hz, 1 H); 4.21-4.01 (m, 2H);4.01-3.89 (m, 2H); 3.54 (A of ABX, dd, J = 14.4, 3.4 Hz, 1 H); 3.12 (B of ABX, dd, J = 14.4, 8.9 Hz, 1 H); 2.94 (A of ABX, dd, J = 14.8, 7.8 Hz, 1 H); 2.86 (B of ABX, dd, J = 14.8, 7 Hz, 1 H); 1.86-1.60 (m, 4 H). MS ES+ M+l= 574.3. (S)-21-Benzyl-8-chloro-12,17-dioxa-l,4,19,22,24-pentaaza tricyclo-[21.3.1.0(6,ll)] heptacosa-6(ll),7,9,23, 25-pentaene-3,18,27-trione TFA salt. 1H NMR (CD3OD + CDCI3, 400 MHz): δ 7.99 (bs, 1 H); 7.35-7.15 (m, 7 H); 6.84 (d, J = 8.8 Hz, 1 H); 6.74 (A of AB, d, J = 5.3 Hz, 1 H); 6.68 (B of AB, d, J = 5.3 Hz, 1 H); 4.92(A of AB, d, J = 16 Hz, 1 H); 4.60-4.50 (m, 1 H); 4.34-4.24 (m, 1 H); 4.20-3.88 (m, 6 H); 3.76- 3.46 (m, 1 H); 3.24-3.14 (m, 1 H); 3.06-2.88 (m, 2 H); 1.88-1.64 (m, 4 H). MS ES+ M+l= 540.3.
EXAMPLE 22: [2-(2-Bromo-ethoxy)-benzyl]-carbamic acid tert-butyl ester
Figure imgf000062_0001
2-methoxybenzylamine (16 ml, 122 mmol) was slowly added to 62% aqueous HBr and the resulting solution was stirred at 120 °C for 16h. The reaction mixture was allowed to cool to room temperature, diluted with EtOH, concentrated in vacuo, and azeothroped with EtOH a few times. To a solution of the residue in DMF (160 ml) is added N-methylmorpholine (40.37 ml, 370 mmol) and ditertbutyldicarbonate (40.12 g, 183 mmol) and the reaction mixture is stirred at room temperature for 18 h. The reaction mixture is diluted with EtOAc, filtered and the filtrate is concentrated in vacuo. The residue is diluted with EtOAc, washed with 10% aqueous KHSO4, water and aqueous NaHCO3, dried on Na2SO4, concentrated in vacuo and purified by flash chromatography (silica gel, 5% EtOAc in hexane to 40%) to give (2-Hydroxy-benzyl)- carbamic acid tert-butyl ester (23.38 g). 1H NMR (CDC13, 300 MHz): δ 8.92 (bs, 1 H); 7.21 (td, J =8, 1.5 Hz, 1 H); 7.06 (dd, J =8, 1.5 Hz, 1 H); 6.94 (dd, J =8, 1.1 Hz, 1 H); 6.82 (td, J =8, 1.1 Hz, 1 H); 5.24 (bs, 1 H); 4.22 (d, J = 7 Hz, 2 H); 1.42 (s, 9 H).
To a solution of (2-Hydroxy-benzyl)-carbamic acid tert-butyl ester (300 mg, 1.34 mmol) in DMF (3 ml) is added cesium carbonate (328 mg, 1 mmol) and ethyl bromoacetate (194 ul, 1.75 mmol) and the reaction mixture is stirred at room temperature for 18 h. The reaction mixture is diluted with water, extracted with EtOAc / Et2O, washed with aqueous LiCl, dried on Na2SO4 and concentrated in vacuo. To a solution of the residue in Et2O (5 ml) cooled to 0 °C is added LiAlH (1.6 ml of 1 M solution in THF, 1.6 mmol) and the reaction mixture is stirred at 0 °C for 5 min. The reaction mixture is quenched with EtOAc, MeOH. Partitioning between EtOAc and Rochelle's salt solution followed by drying on Na2SO , concentration in vacuo and purification by flash chromatography (silica gel, 30% EtOAc in hexane to 70%) provides [2-(2-Hydroxy-ethoxy)-benzyl]-carbamic acid tert-butyl ester (245 mg). 1H NMR (CDC13, 400 MHz): δ 7.30-7.22 (m, 1 H); 7.18 (d, J = 7.6 Hz, 1 H); 6.92 (t, J = 7.6 Hz, 1 H); 6.86 (d, J = 7.6 Hz, 1 H); 4.91 (bs, 1 H); 4.37 d, J = 6 Hz, 2 H); 4.11-4.03 (m, 2 H); 3.97-3.88 (m, 2 H); 3.66 (bt, 1 H); 1.42 (s, 9 H).
To a solution of [2-(2-Hydroxy-ethoxy)-benzyl]-carbamic acid tert- butyl ester (240 mg, 0.9 mmol) in CH2C12 (5 ml) is added carbon tetrabromide (328 mg, 0.98 mmol), triphenyl phosphine (259 mg, 0.98 mmol) and the reaction mixture is stirred at room temperature for 2 h. Concentration in vacuo and purification by flash chromatography (silica gel, 5% EtOAc in hexane to 30%) provides [2-(2-Bromo- ethoxy)-benzyl]-carbamic acid tert-butyl ester (200 mg) as a thick colorless oil. 1H NMR (CDC13, 400 MHz): δ 7.33-7.20 (m, 2 H); 6.96 (t, J = 7.2 Hz, 1 H); 6.82 (d, J = 8 Hz, 1 H); 5.22 (bs, 1 H); 4.39-4.26 (m, 4 H); 3.70 (t, J = 5.8 Hz, 2 H); 1.42 (s, 9 H).
EXAMPLE 23: (S)-20-Benzyl-25-chloro-12-oxa-15-thio-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione
Figure imgf000063_0001
Figure imgf000063_0002
To a solution of [2-(2-Bromo-ethoxy)-benzyl]-carbamic acid tert-butyl ester (235 mg, 0.71 mmol) in DMF (2 ml) is added potassium carbonate (148 mg, 1.07 mmol) and 2-mercaptoethanol (60 ul, 0.85 mmol), and the reaction mixture is stirred at room temperature for 8 h. The reaction mixture is diluted with water, extracted with EtOAc / Et O, washed with aqueous LiCl, dried on Na2SO , concentrated in vacuo and purified by flash chromatography (silica gel, 25% EtOAc in hexane to 75%) to give {2-[2-(2-Hydroxy-ethylsulfanyl)-ethoxy]-benzyl}-carbamic acid tert-butyl ester (210 mg). MS ES+ M+Na= 350.0.
To a solution of {2-[2-(2-Hydroxy-ethylsulfanyl)-ethoxy]-benzyl}- carbamic acid tert-butyl ester (210 mg, 0.64 mmol) in CH2C12 (5 ml) is added carbon tetrabromide (234 mg, 0.71 mmol), triphenyl phosphine (185 mg, 0.71 mmol) and the reaction mixture is stirred at room temperature for 4 h. Concentration in vacuo and purification by flash chromatography (silica gel, 5% EtOAc in hexane to 40%) provides {2-[2-(2-Bromo-ethylsulfanyl)-ethoxy]-benzyl}-carbamic acid tert-butyl ester. 1H NMR (CDC13, 300 MHz): δ 7.33-7.18 (m, 2 H); 6.95 (t, J = 7.2 Hz, 1 H); 6.83 (d, J = 8.2 Hz, 1 H); 5.24 (bs, 1 H); 4.32 (d, J = 6 Hz, 1 H); 4.18 (t, J = 6.2 Hz, 2 H); 3.52 (t, J = 8.6 Hz, 2 H); 3.09-2.92 (m, 4 H); 1.42 (s, 9 H).
(S)-20-Benzyl-25-chloro- 12-oxa- 15-thio- 1 ,4, 18 ,21 ,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione is prepared from {2-[2-(2-Bromo-ethylsulfanyl)-ethoxy]-benzyl}-carbamic acid tert-butyl ester and (S)- [3 -( 1 - Aminomethyl-2-phenyl-ethylamino)-6-chloro-2-oxo-2H-pyrazin- 1 -yl] - acetic acid ethyl ester, using a similar procedure as described in example 5. 1H NMR (CDC13, 300 MHz): δ 7.37-7.16 (m, 7 H); 6.94 (s, 1 H); 6.92 (t, J = 7.2 Hz, 1 H); 6.80 (d, J = 8.2 Hz, 1 H); 6.60 (bt, J = 5.8 Hz, 1 H); 6.25 (d, J = 8.4 Hz, 1 H); 5.16 (A of AB, d, J = 15.5 Hz, 1 H); 4.50-4.28 (m, 4 H); 4.12 (t, J = 6 Hz, 2 H); 3.04-2.66 (m, 10 H). MS ES+ M+1= 528.1.
EXAMPLE 24:
(S)-20-Benzyl-15-methyl-25-chloro-12-oxa -l,4,15,18,21,23-hexaaza tricyclo[20.3.1.0(6,11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,17,26-trione dihydrochloride
Figure imgf000065_0001
Figure imgf000065_0002
(S)-20-Benzyl-15-methyl-25-chloro-12-oxa -l,4,15,18,21,23-hexaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,17,26-trione dihydrochloride is prepared from the coupling of ({2-[2-(tert-Butoxycarbonylamino- methyl)-phenoxy] -ethyl }-methyl-amino)-acetic acid (prepared from [2-(2-Bromo- ethoxy)-benzyl]-carbamic acid tert-butyl ester by alkylation with sarcosine ethyl ester and subsequent hydrolysis of the ethyl ester to the corresponding carboxylic acid) and (S )- [3 -( 1 - Aminomethyl-2-phenyl-ethylamino)-6-chloro-2-oxo-2H-pyrazin- 1 -yl] -acetic acid ethyl ester under standard EDC / HOAt conditions followed by cyclization using a similar procedure as described in example 5.
1H NMR for the corresponding free base (CDC13, 300 MHz): δ 7.36-7.10 (m, 7 H); 6.92 (t, J = 7.4 Hz, 1 H); 6.90 (s, 1 H); 6.80 (d, J = 8.2 Hz, 1 H); ); 6.47 (bt, 1 H); 6.10 (d, J = 8.2 Hz, 1 H); 5.16 (A of AB, d, J = 16.3 Hz, 1 H); 4.60-4.48 (m, 1 H); 4.48- 4.24 (m, 2 H); 4.40 (B of AB, d, J = 16.3 Hz, 1 H); 4.08-3.89 (m, 2 H); 3.66-3.53 (m, 1 H); 3.48-3.34 (m, 1 H);3.12 (s, 2 H); 3.09-3.94 (m, 1 H);2.90-2.66 (m, 3 H);3.37 (s, 3 H).
MS ES exact mass calculated for C27H31ClN6O4: 539.2168, found: 539.2164. CHN calculated for C27H31ClN6O4 + 0.35 H2O + 1.9 HCl: C:53.78, H:5.78, N.13.25; found: C:53.76, H:5.75, N: 13.26. Rotation: [ ]d = -81 ° (c = 0.17, MeOH). EXAMPLE 25: (S)-20-Benzyl-25-chloro-12-oxa -l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride
Figure imgf000066_0001
(S)-20-Benzyl-25-chloro-12-oxa -1,4,18,21,23-ρentaaza tricyclo[20.3.1.0(6, 11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride is prepared from [2- (5-Bromo-pentyloxy)-benzyl]-carbamic acid tert-butyl ester (prepared from (2- Hydroxy-benzyl)-carbamic acid tert-butyl ester and 1,5-dibromopentane) and (S)-[3- (l-Aminomethyl-2-phenyl-ethylamino)-6-chloro-2-oxo-2H-pyrazin-l-yl] -acetic acid ethyl ester, using a similar procedure as described in example 5.
1H NMR (d6 DMSO, 400 MHz): δ 9.12 (bs, 1 H); 8.32 (bs, 1 H);7.81-7.74 (m, 1 H); 7.58 (d, J = 8.9Hz, 1 H); 7.33-7.16 (m, 6 H); 6.96 (s, 1 H); 6.92 (d, J = 7.9 Hz, 1 H); 6.86 (t, J = 6.9 Hz, 1 H); 4.96 (A of AB, d, J = 15.9 Hz, 1 H); 4.65-4.49 (m, 2 H); 4.31 (B of AB, d, J = 15.9 Hz, 1 H); 3.95-3.75 (m, 3 H); 3.35-3.20 (m, 1 H);3.15-2.90 (m, 4 H); 2.85 (dd, J = 13.7, 6.5 Hz, 1 H); 1.75-4.20 (m, 6 H). MS ES+ M+1= 510.68.
EXAMPLE 26: (S)-20-Benzyl- 18-methyl-25-chloro- 12-oxa- 1 ,4, 18 ,21 ,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride
Figure imgf000067_0001
(S)-20-Benzyl-18-methyl-25-chloro-12-oxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride is prepared from (S)-20-Benzyl-25-chloro-12-oxa -1,4,18,21,23- pentaaza tricyclo[20.3.1.0(6, 11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride and formaldehyde using a similar procedure as described in example 12.
1H NMR (d6 DMSO, 300 MHz), fee base: δ 8.04-7.94 (m, 1 H); 7.32-7.10 (m, 6 H); 7.00-6.85 (m, 2 H); ); 6.96 (s, 1 H); 6.82 (d, J = 7.2 Hz, 1 H); 4.88 (A of AB, d, J = 16.1 Hz, 1 H); 4.77 (dd, J = 13.2, 8 Hz, 1 H); 4.37 (B of AB, d, J = 16.1 Hz, 1 H); 4.26-4.10 (m, 1 H); 3.87-3.70 (m, 3 H); 3.01 (dd, J = 13.2, 6.4 Hz, 1 H); 2.84-2.62 (m, 2 H); 2.42-2.24 (m, 2 H); 2.20-2.04 (m, 1 H); 2.07 (s, 3 H); 1.64-1.46 (m, 2 H); 1.46- 1.18 (m, 4 H).
MS ES exact mass calculated for C28H34ClN5O3: 524.2423, found: 524.2453. CHN calculated for C28H34ClN5O3 + 0.3 H2O + 2 HCl: C:55.83, H:6.12, N:11.63; found: C:55.84, H:6.13, N:11.37. Rotation: [α]d = -58 ° (c = 0.32, MeOH).
EXAMPLE 27: (S)-20-Benzyl-18-ethyl-25-chloro-12-oxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione bis TFA salt
Figure imgf000068_0001
(S)-20-Benzyl-18-ethyl-25-chloro-12-oxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-ρentaene-3,26-dione dihydrochloride is prepared from (S)-20-Benzyl-25-chloro- 12-oxa -1,4,18,21,23- pentaaza tricyclo[20.3.1.0(6,11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride and acetaldehyde using a similar procedure as described in example 12. MS ES+ M+l= 538.67.
EXAMPLE 28:
(S)-20-Benzyl-18-carboxymethyl-25-chloro-12-oxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride
Figure imgf000068_0002
(S)-20-Benzyl-18-carboxymethyl-25-chloro-12-oxa-l,4,18,21,23- pentaaza tricyclo[20.3.1.0(6, 11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride is prepared from (S)-20-Benzyl-25-chloro-12-oxa -1,4,18,21,23- pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride and tert-butyl bromoacetate using a similar procedure as described in example 2 followed by tert-butyl removal in TFA. MS ES exact mass calculated for C29H34ClN5O5: 568.2321, found: 568.2325. CHN calculated for C29H34ClN5O5 + 0.85 H2O + 0.4 Et2O + 2 HCl: C:53.58, H.6.13, N: 10.21; found: C:53.67, H:5.75, N:9.87. Rotation: [ ]d = -45.8 ° (c = 0.23, MeOH).
EXAMPLE 29:
(S)-20-Benzyl-25-chloro-12,15-dioxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,11)] hexacosa-6(l 1),7,9,22, 24-pentaene-3,26-dione
Figure imgf000069_0001
(S)-20-Benzyl-25-chloro-12,15-dioxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6, 11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione is prepared from {2-[2-(2-Bromo-ethoxy)-ethoxy]-benzyl}-carbamic acid tert-butyl ester (prepared (2-Hydroxy-benzyl)-carbamic acid tert-butyl ester and 2,2'-dibromoethyl ether) and (S)-[3-(l-Aminomethyl-2-phenyl-ethylamino)-6-chloro-2-oxo-2H-pyrazin- 1-yl] -acetic acid ethyl ester, using a similar procedure as described in example 5.
1H NMR (d6 DMSO, 400 MHz): δ 7.99-7.89 (m, 1 H); 7.32-7.11 (m, 7 H); 6.98 (d, J = 8.4 Hz, 1 H); 6.95 (s, 1 H); 6.90 (t, J = 7.7 Hz, 1 H); 4.82 (A of AB, d, J = 16.9 Hz, 1 H); 4.66 (dd, J = 14, 8.7 Hz, 1 H); 4.36 (B of AB, d, J = 16.9 Hz, 1 H); 4.26-4.13 (m, 1 H); 4.02-3.88 (m, 2 H); 2.94 (dd, J = 14, 6.3 Hz, 1 H); 2.78-2.36 (m, 6 H). MS ES+ M+1= 512.59.
EXAMPLE 30: (S)-20-Benzyl-18-methyl-25-chloro-12,15-dioxa-l,4,18,21,23-pentaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione .2
Figure imgf000070_0001
(S)-20-Benzyl-18-methyl-25-chloro-12,15-dioxa-l,4,18,21,23- pentaaza tricyclo[20.3.1.0(6, 11)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione dihydrochloride is prepared from (S)-20-Benzyl-25-chloro-12,15-dioxa-l,4,18,21,23- pentaaza tricyclo[20.3.1.0(6,11)] hexacosa-6(l 1),7,9,22, 24-pentaene-3,26-dione and formaldehyde using a similar procedure as described in example 12. MS ES exact mass calculated for C27H32ClN5O : 524.2423, found: 524.2418. Rotation: [ ]d = -67.6 ° (c = 0.13, MeOH).
EXAMPLE 31:
(S)-20-Benzyl-25-chloro-l,4,13,18,21,23-hexaaza tricyclo[20.3.1.0(6,11)] hexacosa- 6(11),7,9,22, 24-pentaene-3,26-dione tris TFA salt
Figure imgf000070_0002
Step A: Preparation of tert-Butyl 2-(aminomethyl)benzylcarbamate
Figure imgf000071_0001
2-(Azidomethyl)benzonitrile:
To a solution of 90 g (459 mmol) 2-cyanobenzylbromide in 600 mL THF was added in one portion a solution of 36 g (553 mmol) sodium azide in 100 mL water. The two phase mixture was stirred at 23°C for 18 hr. The THF layer was separated from the lower water layer and used in the next step without further purification.
tert-Butyl 2-cyanobenzylcarbamate: To the THF layer from the previous step was diluted to a volume of 1.6
L, divided into two equal portions and each hydrogenated at 45psi in a Parr pressure bottle containing 6 g of 5% palladium on carbon (50% water by weight). A 5-10°C exotherm was observed within 30 min and shaking continued a total of 1.5 hr. The individual batches were filtered through celite, washed 2x with 100 mL fresh THF and the filtrates combined into a single portion. To the amine mixture without concentration (Caution: attempts to concentrate the solution resulted in a large exotherm and the batch turned black) was added 87.5 mL (381 mmol) of di-tert-butyl dicarbonate neat. After 2 hr the THF was removed in vacuo and flushed with 250 mL of 15% ethyl acetate in hexane. The semi-solid was slurried in 250 mL of 15% ethyl acetate and filtered. The filtrate was concentrated in vacuo, diluted with 10% ethyl acetate in hexane (175 mL), cooled to 0°C and filtered to give 60 g (56% yield from 2-cyanobenzyl bromide) of tert-butyl 2-cyanobenzylcarbamate as a gray solid.
tert-Butyl 2-(aminomethyl benzylcarbamate hemisulfate: To a 3 L, 3 neck flask fitted with a thermocouple, a condenser and nitrogen inlet was added 3 g (23 mmol) of cobaltous chloride, then 1200 mL of THF followed by 59 g (254 mmol) of tert-butyl 2-cyanobenzylcarbamate and 600 mL of ice-water. To the light pink solution at 15°C was added 26 g (684 mmol) of sodium borohydride in portions as follows. The initial 3 g of sodium borohydride resulted in a vigorous hydrogen gas evolution and formation of a black suspension. The batch temperature reached 35°C within 2 hr, and was maintained at this temperature with a heating mantle. Additional sodium borohydride and cobaltous chloride were added as needed to drive the reaction to completion. Typically, 2 x 7.5 g of additional sodium borohydride and 2 x 1 g portions of cobaltous chloride were added at 12 hour intervals. Once complete, the layers were allowed to settle and the clear upper THF layer was decanted from the black aqueous layer. The aqueous layer was washed with 750 mL fresh THF, the two THF layers combined and filtered through a pad of celite. The orange-yellow filtrate was concentrated to about 300 mL in vacuo, resulting in water layer with the product as an oily lower layer. The mixture was extracted with 2 x 250 mL ethyl acetate and the combined extracts reacted with 24 g (200 mmol) of solid sodium hydrogensulfate. A solid formed immediately, and the slurry was stirred for 30 min, filtered and washed with 2 x 100 mL ethyl acetate to give 62 g of a white powder. The powder was slurried in 175 mL water, cooled to 0°C, filtered, washed with 2 x 40 mL cold water and the solid dried in a vacuum oven at 55°C for 24 hr to give 46 g (64% yield from tert-butyl 2-cyanobenzylcarbamate) of tert-butyl 2- (aminomethyl)benzylcarbamate hemisulfate salt as a white powder.
Step B: Preparation of [2-(tert-Butoxycarbonylamino-methyl)-benzyl]-(4-hydroxy- butyl)-carbamic acid dimethyl-ethyl ester
Figure imgf000072_0001
.H2S04
tert-Butyl 2-(aminomethyl)benzylcarbamate hemisulfate (1 g, 3.5 mmol) is partitioned betwwen aqueous NaHCO3 and CH2C12 to obtain the free base from the organic layer after back extraction with CH2C12. To a solution of tert-Butyl 2-(aminomethyl) benzylcarbamate free base (3.5 mmol) in DMF (10 ml) is added ethyl 4-bromo- butyrate (602 ul, 4.2 mmol) and the reaction mixture is stirred at 70 °C for 18 h. The reaction mixture is allowed to cool down to room temperature, dilute with EtOAc, washed with water, aqueous NaHCO3 and aqueous LiCl, dried on Na2SO , concentrated in vacuo and purified by flash chromatography (silica gel, 1% MeOH containibg 10% NH4OH in CH2C12 to 10%) to give 4-[2-(tert-Butoxycarbonylamino- methyl)-benzylamino] -butyric acid ethyl ester (912 mg) contaminated with the bis- alkylation product.
To a solution of 4-[2-(tert-Butoxycarbonylamino-methyl)- benzylamino] -butyric acid ethyl ester (900 mg, 2.57 mmol, contaminated with the bis- alkylation product) in CH2C12 (20 ml) is added ditertbutyldicarbonate (504 mg, 2.31 mmol) and the reaction mixture is stirred at room temperature for 18 h. a little more ditertbutyldicarbonate is added to reach reaction completion. The reaction mixture is concentrated in vacuo and purified by flash chromatography (silica gel, 5% EtOAc in hexane to 40%) to give 4-{tert-Butoxycarbonyl-[2-(tert-butoxycarbonylamino- methyl)-benzyl] -amino} -butyric acid ethyl ester (669 mg) as a colorless oil. 1H NMR (CDC13, 400MHz): δ 7.38-7.29 (m, 1 H); 7.29-7.19 (m, 2 H); 7.19-7.11 (m, 1 H); 4.52 (s, 2 H); 4.31 (d, J = 5.4 Hz, 2 H); 4.11 (q, J = 6.7 Hz, 2 H); 3.20 (bt, 2 H); 2.26 (t, J = 7.8 Hz, 2 H); 1.86-1.74 (m, 2 H); 1.46 (s, 18 H), 1.24 (t, J = 6.7 Hz, 3 H). To a solution of 4-{tert-Butoxycarbonyl-[2-(tert-butoxycarbonylamino- methyl)-benzyl]-amino}-butyric acid ethyl ester (369 mg, 0.82 mmol) in Et2O (10 ml) cooled to 0 °C is added dropwise LiAlELt (820 ul of a 1 M solution in Et2O, 0.82 mmol) and the reaction mixture is stirred at 0 °C for 30 min. The reaction mixture is quenched with EtOAc, water (32 ul), IN NaOH (93 ul) and water (32 ul). The precipitate is filtered after further dilution with Et2O. The filtrate is concentrated in vacuo and purified by flash chromatography (silica gel, 25% EtOAc in hexane to 50%) to give [2-(tert-Butoxycarbonylamino-methyl)-benzyl]-(4-hydroxy-butyl)- carbamic acid dimethyl-ethyl ester (295 mg) as a colorless thick oil. 1H NMR (CDCI3, 400MHz): δ 7.34-7.16 (m, 4 H); 4.51 (s, 2 H); 4.31 (d, J = 5.8 Hz, 2 H); 3.61 (bt, J = 6.6 Hz, 2 H); 3.19 (bs, 2 H); 1.64-1.45 (m, 4 H); 1.44 (s, 18 H).
Step C: Reductive alkylation and cyclization toward (S)-20-Benzyl-25-chloro- 1,4,13,18,21,23-hexaaza tricyclo[20.3.1.0(6, 11)] hexacosa-6(ll),7,9,22, 24-pentaene- 3,26-dione tris TFA salt
(S)-20-Benzyl-25-chloro-l,4,13,18,21,23-hexaaza tricyclo[20.3.1.0(6,11)] hexacosa- 6(11),7,9,22, 24-ρentaene-3,26-dione tris TFA salt is prepared from [2-(tert- Butoxycarbonylamino-methyl)-benzyl]-(4-hyαioxy-butyl)-carbamic acid dimethyl- ethyl ester and (S)-[3-(l-Aminomethyl-2-phenyl-ethylamino)-6-chloro-2-oxo-2H- pyrazin- 1-yl] -acetic acid ethyl ester using a similar procedure as described in example
17 steps B and C.
1H NMR (CD3OD, 400MHz): δ 7.52-7.46 (m, 2 H); 7.44-7.34 (m, 2 H); 7.30-7.14 (m,
5 H); 6.89 (s, 1 H); 4.95 (A of AB, d, J = 16.7 Hz, 1 H); 4.84 (B of AB, d, J = 16.7
Hz, 1 H); 4.73-4.60 (m, 2 H); 4.47 (A of AB, d, J = 14 Hz, 1 H); 4.30 (B of AB, bd, J
= 15.2 Hz, 1 H); 4.20 (B of AB, d, J = 14 Hz, 1 H); 3.06-2.76 (m, 6 H); 1.66-1.49 (m,
3 H); 1.45-1.32 (m, 1 H).
MS ES exact mass calculated for C27H33ClN6O2: 509.2426, found: 509.2431.
CHN calculated for C27H33ClN6O2 + 1.05 H2O + 2.15 TFA: C:48.63, H:4.86,
N: 10.87; found: C:48.63, H:4.82, N.11.00.
Rotation: [ ]d = -80.7 ° (c = 0.15, MeOH).
EXAMPLE 32: (S)-20-Benzyl-25-chloro-l,4,14,18,21,23-hexaazatricyclo[20.3.1.0(6,ll)] hexacosa- 6(11),7,9,22, 24-pentaene-3,26-dione tris hydrochloride
Figure imgf000074_0001
Step A: Preparation of tert-Butyl [2-(aminomethyl)phenyl] acetate
2-Bromomethylphenylacetic acid: To a solution of 97.0 g (0.646 mol) o-tolylacetic acid in 1.75 L carbon tetrachloride was added 115.0 g (0.646 mol) N-bromosuccinimide and 3.4 g (0.021 mol) 2,2 -azobisisobutyronitrile. The mixture was heated at reflux under a nitrogen atmosphere for 4 h. After the mixture was cooled to 0-5 °C for 30 min, the solids were removed by filtration and washed with a small portion of carbon tetrachloride. This solid was triturated with water (0.8 L), collected on a filter, and washed with 500 mL of water to give 86.0g of 2-bromomethylphenylacetic acid. The filtrate was concentrated to a volume of 150 ml, and the resulting slurry cooled to 0-5 °C for 30 min. A second batch of product (10.0 g) was obtained as above to give a total of 96.0 g (65% yield).
2-Bromomethylphenylacetic acid t-butyl ester: To a solution of 80 g (0.349 mol) 2-bromomethylphenylacetic acid in
700 mL 1,4-dioxane in a 2 L heavy- walled flask was added 84 mL (1.571 mol) concentrated sulfinic acid at ambient temperature. The reaction mixture was chilled to -15 oC, and 580 mL isobutylene was condensed directly into the reaction vessel. The sealed pressure flask was shaken mechanically at room temperature for 4 h (the pressure inside the flask rises to ca. 20 psi during this step). The mixture was carefully quenched by slowly pouring it into a 0-5oC stirred mixture of 1.2 L tert- butyl methyl ether and 336 g (4.0 mol) solid sodium bicarbonate before slow dilution with 1.2 L ice-water. The separated organic phase was washed with 0.8 L brine, dried with sodium sulfate, filtered concentrated in vacuo to give an oil, which was used without further purification.
2-Azidomethylphenylacetic acid t-butyl ester:
To a solution of crude 2-bromomethylphenylacetic acid t-butyl ester (0.349 mol) in 600 mL DMF was added 34.1 g (0.524 mol) sodium azide and the mixture stirred at 65 °C for 3 h. After cooling to ambient temperature, the mixture was diluted with 1.2 L ethyl acetate. The organic layer was washed with water (3 x 800 mL), dried over sodium sulfate, filtered and concentrated in vacuo to give 78.0 g (90% from 2-bromophenylacetic acid) as a yellow oil. This material was used without further purification.
tert-Butyl r2-(aminomethyl)phenyl1 acetate oxalate salt:
To a solution of 78.0 g (0.312 mol) 2-azidomethylphenylacetic acid t-butyl ester in 1.36 L THF was added 7.8 g (50% water wet) 5 % Pd on C and the mixture shaken mechanically in a 2 L heavy-walled flask under H at 45 psi for 2 h. The catalyst was removed by filtration through a bed of celite, rinsing with 250 mL THF. To the filtrate was added a solution of 31.25 g (0.347 mol) oxalic acid in 500 mL methyl tert-butyl ether, and the resultant suspension stirred at room temperature for 30 min. The solid was collected on a filter and washed with 300 mL methyl tert- butyl ether (the filtration was very slow, requiring about 3 hours). Drying under reduced pressure at 60 °C for 18 h gave 42.4 g (39% overall from 2- bromomethylphenylacetic acid) tert-butyl [2-(aminomethyl)phenyl] acetate oxalate salt as a white powder. The product is unstable as the free base, and will cyclize to the amide over several hours at room temperature.
Step B: Preparation of (2-{2-[tert-Butoxycarbonyl-(3-hydroxy-propyl)-amino]-ethyl}- benzyl)-carbamic acid tert-butyl ester
Figure imgf000076_0001
To a solution of tert-butyl [2-(aminomethyl)phenyl] acetate oxalate salt (942 mg, 3.03 mmol) in CH2C12 (30 ml) is added triethylamine (1.05 ml, 7.57 mmol) and ditertbutyldicarbonate (763 mg, 3.33 mmol) and the reaction mixture is stirred at room temperature for 2 h. The reaction mixture is diluted with CH2C12, washed with aqueous NaHCO3, dried on Na2SO4 and concentrated in vacuo to give [2-(tert- Butoxycarbonylamino-methyl)-phenyl]-acetic acid tert-butyl ester as an oil which is used in the next step without further purification. To a solution of [2-(tert-Butoxycarbonylamino-methyl)-phenyl]-acetic acid tert-butyl ester (3.03 mmol) in Et2O (30 ml) cooled to 0 °C is added dropwise LiAlEU. (3.03 ml of a 1 M solution in Et2O, 3.03) and the reaction mixture is stirred at 0 °C for 1 h. Further LiAlFύ is added (0.5 ml after 1 h and 0.5 ml after 1 h30).The reaction mixture is quenched with EtOAc, water (152 ul), IN NaOH (456 ul) and water (152 ul). The precipitate is filtered after further dilution with EtOAc. The filtrate is concentrated in vacuo to give [2-(2-Hydroxy-ethyl)-benzyl]-carbamic acid tert-butyl ester (729 mg) as an oil which is used in the next step without further purification. 1H NMR (CDC13, 400MHz): δ 7.32-7.18 (m, 4 H); 5.03 (bs, 1 H); 4.37 (d, J = 5.7 Hz, 2 H); 3.87 (dt, 6, 6 Hz, 2 H); 2.95 (t, J = 6.8 Hz, 2 H); 1.72 (bt, 1 H); 1.45 (s, 9 H).
To a solution of [2-(2-Hydroxy-ethyl)-benzyl] -carbamic acid tert-butyl ester (720 mg, 2.87 mmol) in CH2C12 (20 ml) is added carbon tetrabromide (1.05 g, 3.15 mmol) and triphenylphosphine (827 mg, 3.15 mmol), and the reaction mixture is stirred at room temperature for 3 h. The reaction mixture is concentrated in vacuo and purified by flash chromatography (silica gel, 10% Et2O in hexane to 20%) to give [2- (2-Bromo-ethyl)-benzyl] -carbamic acid tert-butyl ester (480 mg) as a white solid. 1H NMR (CDC13, 400MHz): δ 7.32-7.19 (m, 4 H); 4.72 (bs, 1 H); 4.36 (d, J = 5.5 Hz, 2 H); 3.55 (t, J = 8.3 Hz, 2 H); 3.22 (t, J = 8.3 Hz, 2 H); 1.45 (s, 9 H).
To a solution of [2-(2-Bromo-ethyl)-benzyl]-carbamic acid tert-butyl ester (477 mg, 1.52 mmol) in DMF (5 ml) is added 3-amino-l-propanol (1.16 ml, 15.18 mmol) and the reaction mixture is stirred at room temperature for 1 h and at 50 °C for 2 h. The reaction mixture is diluted with EtOAc, washed with aqueous
NaHCO3, aqueous LiCl, dried on Na2SO4, concentrated in vacuo and purified by flash chromatography (silica gel, 50% EtOAc in hexane followed by 6% MeOH containing 10% NHtOH in CH2C12 to 20%) to give {2-[2-(3-Hydroxy-propylamino)-ethyl]- benzyl} -carbamic acid tert-butyl ester (340 mg) as white solid. 1H NMR (CDC13, 400MHz): δ 7.32-7.16 (m, 4 H); 5.20 (bs, 1 H); 4.33 (d, J = 4.9 Hz, 2 H); 3.78 (t, J = 5.2 Hz, 2 H); 2.95-2.80 (m, 6 H); 1.75-1.65 (m, 2 H); 1.45 (s, 9 H).
To a solution of { 2- [2-(3-Hydroxy-propylamino)-ethyl] -benzyl }- carbamic acid tert-butyl ester (322 mg, 1.04 mmol) in CH2C12 (5 ml) is added ditertbutyldicarbonate (239 mg, 1.1 mmol) and the reaction mixture is stirred at room temperature for 2 h 30. The reaction mixture is concentrated in and purified by flash chromatography (silica gel, 30% EtOAc in hexane to 65%) to give (2-{2-[tert- Butoxycarbonyl-(3-hydroxy-propyl)-amino]-ethyl}-benzyl)-carbamic acid tert-butyl ester (439 mg) as a thick oil. 1H NMR (CDC13, 400MHz): δ 7.32-7.08 (m, 4 H); 5.33 (bs, 0.2 H); 4.72 (bs, 0.8 H); 4.46 (d, J = 5.9 Hz, 2 H); 3.75 (bt, 0.8 H); 3.70-3.48 (m, 2 H); 3.42-3.24 (m, 4 H); 2.95-2.82 (m, 2 H); 2.21 (bs, 0.2 H); 1.80-1.60 (m, 2 H); 1.45 (s, 18 H). Step C: reductive alkylation and cyclization toward (S)-20-Benzyl-25-chloro- 1,4,14,18,21,23-hexaaza tricyclo[20.3.1.0(6,11)] hexacosa-6(ll),7,9,22, 24-pentaene- 3,26-dione tris hydrochloride
Figure imgf000078_0001
Figure imgf000078_0002
(S)-20-Benzyl-25-chloro-l,4,14,18,21,23-hexaaza tricyclo[20.3.1.0(6,ll)] hexacosa-6(ll),7,9,22, 24-pentaene-3,26-dione tris hydrochloride is prepared from (2-{2-[tert-Butoxycarbonyl-(3-hydroxy-propyl)- amino]-ethyl}-benzyl)-carbamic acid tert-butyl ester and (S)-[3-(l-Aminomethyl-2- phenyl-ethylamino)-6-chloro-2-oxo-2H-ρyrazin-l-yl]-acetic acid ethyl ester using a similar procedure as described in example 17 steps B and C.
1H NMR (CD3OD, 400MHz): δ 7.55 (d, J = 7.3 Hz, 1 H); 7.34-7.16 (m, 8 H); 6.93 (s, 1 H); 5.08 (A of AB, d, J = 16.6 Hz, 1 H); 4.70-4.50 (m, 3 H); 4.26 (B of AB, d, J = 15 Hz, 1 H); 3.45-3.75 (m, 12 H); 2.20-1.95 (m, 2 H). MS ES exact mass calculated for C27H33ClN6O2: 509.2426, found: 509.2425. CHN calculated for C27H33ClN6O2 + 1.45 H2O + 0.65 EtOAc + 3 HCl: C:50.65, H:6.33, N: 11.98; found: Q50.67, H:6.02, N: 11.95. Rotation: [ ]d = -75.5 ° (c = 0.2, MeOH). Typical tablet cores suitable for administration of thrombin inhibitors are comprised of, but not limited to, the following amounts of standard ingredients:
Excipient General Range Preferred Range Most Preferred Range (%) (%) (%) mannitol 10-90 25-75 30-60 microcrystallme 10-90 25-75 30-60 cellulose magnesium stearate 0.1-5.0 0.1-2.5 0.5-1.;
Mannitol, microcrystallme cellulose and magnesium stearate may be substituted with alternative pharmaceutically acceptable excipients.
The thrombin inhibitors can also be co-administered with suitable antiplatelet agents, including, but not limited to, fibrinogen receptor antagonists (e.g. to treat or prevent unstable angina or to prevent reocclusion after angioplasty and restenosis), anticoagulants such as aspirin, thrombolytic agents such as plasminogen activators or streptokinase to achieve synergistic effects in the treatment of various vascular pathologies, or lipid lowering agents including antihypercholesterolemics (e.g. HMG CoA reductase inhibitors such as lovastatin, HMG CoA synthase inhibitors, etc.) to treat or prevent atherosclerosis. For example, patients suffering from coronary artery disease, and patients subjected to angioplasty procedures, would benefit from coadministration of fibrinogen receptor antagonists and thrombin inhibitors. Also, thrombin inhibitors enhance the efficiency of tissue plasminogen activator-mediated thrombolytic reperfusion. Thrombin inhibitors may be administered first following thrombus formation, and tissue plasminogen activator or other plasminogen activator is administered thereafter.
Typical doses of thrombin inhibitors of the invention in combination with other suitable anti-platelet agents, anticoagulation agents, or thrombolytic agents may be the same as those doses of thrombin inhibitors administered without coadministration of additional anti-platelet agents, anticoagulation agents, or thrombolytic agents, or may be substantially less that those doses of thrombin inhibitors administered without coadministration of additional anti-platelet agents, anticoagulation agents, or thrombolytic agents, depending on a patient's therapeutic needs. In Vitro Assay For Determining Proteinase inhibition
Assays of human -thrombin and human trypsin were performed by the methods substantially as described in Thrombosis Research, Issue No. 70, page 173 (1993) by S.D. Lewis et al.
The assays were carried out at 25°C in 0.05 M TRIS buffer pH 7.4, 0.15 M NaCl, 0.1% PEG. Trypsin assays also contained 1 mM CaCl2- In assays wherein rates of hydrolysis of a p-nitroanilide (pna) substrate were determined, a Thermomax 96-well plate reader was used was used to measure (at 405 nm) the time dependent appearance of p-nitroaniline. sar-PR-pna was used to assay human - thrombin (Km=125 μM) and bovine trypsin (Km=125 μM). p-Nitroanilide substrate concentration was determined from measurements of absorbance at 342 nm using an extinction coefficient of 8270 cm' ^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 μM) was determined from the increase in fluorescence at 500 nm
(excitation at 400 nm) associated with production of 7-amino-4-trifluoromethyl coumarin. Concentrations of stock solutions of Z-GPR-afc were determined from measurements of absorbance at 380 nm of the 7-amino-4-trifluoromethyl coumarin produced upon complete hydrolysis of an aliquot of the stock solution by thrombin.
Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration < 0.1 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. Initial velocities of product formation in the absence (V0) or presence of inhibitor (N were measured. Assuming competitive inhibition, and that unity is negligible compared Km/[S], [I]/e, and [I]/e (where [S], [I], and e respectively represent the total concentrations, of substrate, inhibitor and enzyme), the equilibrium constant (Ki) for dissociation of the inhibitor from the enzyme can be obtained from the dependence of N0/Ni on [I] shown in the following equation.
Figure imgf000080_0001
The activities shown by this assay indicate that the compounds of the invention are therapeutically useful for treating various conditions in patients suffering from unstable angina, refractory angina, myocardial infarction, transient ischemic attacks, atrial fibrillation, thrombotic stroke, embolic stroke, deep vein thrombosis, disseminated intravascular coagulation, and reocclusion or restenosis of recanalized vessels.
EXAMPLE 33
Tablet Preparation
Tablets containing 25.0, 50.0, and 100.0 mg., respectively, of the following active compounds are prepared as illustrated below (compositions A-C). Active I is compound (S)-19-Benzyl-8,24-dichloro-12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,11)] pentacosa-6(ll),7,9,21, 23-pentaene-3,25-dione dihydrochloride .
Amount-(mg)
Component A B C
Active I 25 50 100
Microcrystallme cellulose 37.25 100 200
Modified food corn starch 37.25 4.25 8.5
Magnesium stearate 0.5 0.75 1.5
All of the active compound, cellulose, and a portion of the corn starch are mixed and granulated to 10% corn starch paste. The resulting granulation is sieved, dried and blended with the remainder of the corn starch and the magnesium stearate. The resulting granulation is then compressed into tablets containing 25.0, 50.0, and 100.0 mg, respectively, of active ingredient per tablet.
EXAMPLE 34
Tablet Preparation
Exemplary compositions of compound (S)-19-Benzyl-8,24-dichloro- 12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,11)] pentacosa-6(ll),7,9,21, 23- pentaene-3,25-dione dihydrochloride (Active I) tablets are shown below:
Component 0.25 mg 2 mg 10 mg 50 mg
Active I 0.500% 1.000% 5.000% 14.29% mannitol 49.50% 49.25% 47.25% 42.61% microcrystalline cellulose 49.50% 49.25% 47.25% 42.61% magnesium stearate 0.500% 0.500% 0.500% 0.500% 2, 10 and 50 mg tablets were film-coated with an aqueous dispersion of hydroxypropyl cellulose, hydroxypropyl methylcellulose and titanium dioxide, providing a nominal weight gain of 2.4%.
Tablet preparation via direct compression
Active I, mannitol and microcrystallme cellulose were sieved through mesh screens of specified size (generally 250 to 750 μm) and combined in a suitable blender. The mixture was subsequently blended (typically 15 to 30 min) until the drug was uniformly distributed in the resulting dry powder blend. Magnesium stearate was screened and added to the blender, after which a precompression tablet blend was achieved upon additional mixing (typically 2 to 10 min). The precompression tablet blend was then compacted under an applied force, typically ranging from 0.5 to 2.5 metric tons, sufficient to yield tablets of suitable physical strength with acceptable disintegration times (specifications will vary with the size and potency of the compressed tablet). In the case of the 2, 10 and 50 mg potencies, the tablets were dedusted and film-coated with an aqueous dispersion of water-soluble polymers and pigment.
Tablet preparation via dry granulation
Alternatively, a dry powder blend is compacted under modest forces and remilled to afford granules of specified particle size. The granules are then mixed with magnesium stearate and tabletted as stated above.
EXAMPLE 35
Intravenous Formulations
Intravenous formulations of compound (S)-19-Benzyl-8,24-dichloro- 12-oxa-l,4,17,20,22-pentaaza tricyclo[19.3.1.0(6,ll)] pentacosa-6(ll),7,9,21, 23- pentaene-3,25-dione dihydrochloride (Active I) were prepared according to general intravenous formulation procedures. Component Estimated range
Active I 0.12 - 0.61 mg
D-glucuronic acid* 0.5 - 5 mg
Mannitol NF 50-53 mg
1 N Sodium Hydroxide q.s. pH 3.9 - 4.1
Water for injection q.s. 1.0 mL
Exemplary compositions A-C are as follows:
Component A B C
Active I 0.61 mg* 0.30** 0.15***
D-glucuronic acid* 1.94 mg 1.94 mg 1.94 mg
Mannitol NF 51.2 mg 51.2 mg 51.2 mg
1 N Sodium Hydroxide q.s. pH 4.0 q.s. pH 4.0 q.s. pH 4.0
Water for injection q.s. 1.0 mL q.s. 1.0 mL q.s. 1.0 mL
* 0.50 mg free base ** 0.25 mg free base *** 0.12 mg free base
Narious other buffer acids, such as L-lactic acid, acetic acid, citric acid or any pharmaceutically acceptable acid/conjugate base with reasonable buffering capacity in the pH range acceptable for intravenous administration may be substituted for glucuronic acid.

Claims

WHAT IS CLAIMED IS:
1. A compound of the general formula:
Figure imgf000084_0001
or a pharmaceutically acceptable salt thereof, wherein R1 is aryl, C 1-4 alkyl, or C 3- cycloalkyl;
R )2 ; is hydrogen, C 1-4 alkyl, C 3- cycloalkyl, halogen, or CN; R3 is hydrogen, C 1- alkyl, C 3- cycloalkyl, halogen or CN;
R4 is hydrogen, C 1- alkyl or -ZCOOH, wherein Z is C 1-4 alkyl; A is selected from the group consisting of
a) .. (CH^g-X1— where X1 is O or CH2;
b) [-(CH2)2-3-Y1-(CH2)2.3-X2-^
where Y1 is O, S, SO, SO2 or NR5, and X2 is O or CH2>
c) ξ— C(O)-(CH2)1.3-Y2-(CH2)1.3-X3—
where Y2 is O, S, SO, SO2 or NR5, and X3 is O or CH2,
d) \— (CH2)n-C(O)-NR5-(CH2)m-X4 1
where 1 is O or CH2, provided that when X is O, n and m are independently 1, 2 or 3, and that when X is CH2, n is 1, 2 or 3, and mis 2 or 3, or e) \— (CH2)n-NR5-C(O)-(CH2)m-X5—
where X is O or CH2, provided that when X is O, n and m are independently 1, 2 or 3, and when X is CH2, n is 1, 2 or 3 and mis 2 or 3;
R5 is hydrogen, C 1-4 alkyl or -ZOOH, where Z is C 1-4 alkyl.
2. A compound of Claim 1 , or pharmaceutically acceptable salt thereof, wherein R is hydrogen or halogen and R is hydrogen or halogen.
3. A compound of Claim 2, or pharmaceutically acceptable salt thereof, wherein R is hydrogen or CI and R is hydrogen or CI.
4. A compound of Claim 3, or pharmaceutically acceptable salt thereof, wherein R1 is phenyl or cyclopropyl.
5. A compound of Claim 4, or pharmaceutically acceptable salt thereof, wherein R4 is hydrogen, methyl, ethyl, or -CH2COOH.
6. A compound of Claim 5, or pharmaceutically acceptable salt thereof, wherein R5 is hydrogen, methyl or ethyl.
7. A compound of Claim 6, or pharmaceutically acceptable salt thereof, wherein A is selected from the group consisting of
-(CH2)4-O-, -(CH2)3-O-, -(CH2)5-O-, -(CH2)6-O-,
-(CH2)2-O-(CH2)2-O-, -(CH2)C(O)N(CH2CH3XCH2)2-O-,
-C(O)-O-CH2CHCHCH2-O-, -C(O)-O-(CH2)4-O-,
-(CH2)2S(CH2)2-O-, -C(O)CH2N(CH3)(CH2)2-O-,
-(CH2)4NHCH2-, and -(CH2)3NH(CH2) .
8. A compound of Claim 7, or pharmaceutically acceptable salt thereof, selected from the group consisting of
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000087_0002
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000089_0002
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000091_0002
9. A composition for inhibiting thrombus formation in blood comprising a compound of Claim 1 and a pharmaceutically acceptable carrier.
10. A method for inhibiting thrombin in blood comprising adding to the blood a composition of Claim 9.
11. A method for inhibiting formation of blood platelet aggregates in blood comprising adding to the blood a composition of Claim 9.
12. A method for inhibiting thrombus formation in blood comprising adding to the blood a composition of Claim 9.
13. The use of a compound of Claim 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting thrombin, inhibiting thrombus formation, treating thrombus formation, or preventing thrombus formation in a mammal.
14. A method for treating or preventing venous thromboembolism and pulmonary embolism in a mammal comprising administering to the mammal a composition of Claim 9.
15. A method for treating or preventing deep vein thrombosis in a mammal comprising administering to the mammal a composition of Claim 9.
16. A method for treating or preventing thromboembolic stroke in humans and other mammals comprising administering to the mammal a composition of Claim 9.
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