WO2010138490A1 - Benzoxazepines as inhibitors of mtor and methods of their use and manufacture - Google Patents

Benzoxazepines as inhibitors of mtor and methods of their use and manufacture Download PDF

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Publication number
WO2010138490A1
WO2010138490A1 PCT/US2010/036038 US2010036038W WO2010138490A1 WO 2010138490 A1 WO2010138490 A1 WO 2010138490A1 US 2010036038 W US2010036038 W US 2010036038W WO 2010138490 A1 WO2010138490 A1 WO 2010138490A1
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Prior art keywords
methyl
carbonyl
benzimidazol
tetrahydro
benzoxazepme
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PCT/US2010/036038
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French (fr)
Inventor
Naing Aay
Charles M. Blazey
Owen Joseph Bowles
Joerg Bussenius
Jeffry Kimo Curtis
Steven Charles Defina
Larisa Dubenko
Jason R. Harris
Eileen E. Jackson-Ugueto
Angie Inyoung Kim
Jean-Claire Limun Manalo
Michael Pack
Csaba J. Peto
Kenneth D. Rice
Tsze H. Tsang
Longcheng Wang
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Exelixis, Inc.
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Publication of WO2010138490A1 publication Critical patent/WO2010138490A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D451/00—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof
    • C07D451/02—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D451/00—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof
    • C07D451/02—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof
    • C07D451/04—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof with hetero atoms directly attached in position 3 of the 8-azabicyclo [3.2.1] octane or in position 7 of the 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D451/00—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof
    • C07D451/02—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof
    • C07D451/04—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof with hetero atoms directly attached in position 3 of the 8-azabicyclo [3.2.1] octane or in position 7 of the 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring system
    • C07D451/06—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D453/00—Heterocyclic compounds containing quinuclidine or iso-quinuclidine ring systems, e.g. quinine alkaloids
    • C07D453/06—Heterocyclic compounds containing quinuclidine or iso-quinuclidine ring systems, e.g. quinine alkaloids containing isoquinuclidine ring systems
    • 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
    • C07D487/08—Bridged systems

Definitions

  • This invention relates to the field of protein kinases and inhibitors thereof.
  • the invention relates to inhibitors of mammalian target of rapamycin (mTOR) signaling pathways, and methods of their use.
  • mTOR mammalian target of rapamycin
  • Background of the Invention [0002]
  • the mammalian target of rapamycin, mTOR is a protein kinase that integrates both extracellular and intracellular signals of cellular growth, proliferation, and survival. Extracellular mitogenic growth factor signaling from cell surface receptors and intracellular pathways that convey hypoxic stress, energy and nutrient status all converge at mTOR.
  • mTOR exists in two distinct complexes: mTOR complex 1 (mTORCl) and mTOR complex 2 (mT0RC2).
  • mTORCl is a key mediator of transcription and cell growth (via its substrates p70S6 kinase and 4E-BP1) and promotes cell survival via the serum and glucocorticoid-activated kinase SGK, whereas mTORC2 promotes activation of the pro-survival kinase AKT.
  • mTOR signaling is frequently dysregulated in cancer and other diseases (Bjornsti and Houghton Rev Cancer 2004, 4(5), 335- 48; Houghton and Huang Microbiol Immunol 2004, 279, 339-59; Inoki, Corradetti et al. Nat Genet 2005, 37(1), 19-24).
  • mTOR is a member of the PIKK (PI3K-related Kinase) family of atypical kinases which includes ATM, ATR, and DNAPK, and its catalytic domain is homologous to that of PI3K.
  • Dyregulation of PI3K signaling is a common function of tumor cells.
  • mTOR inhibition may be considered as a strategy in many of the tumor types in which PI3K signaling is implicated such as those discussed below.
  • Inhibitors of mTOR may be useful in treating a number of cancers, including the following: breast cancer (Nagata, Lan et al., Cancer Cell 2004, 6(2), 117-27; Pandolfi N EnglJ Med 2004, 351(22), 2337-8; Nahta, Yu et al. Nat Clin Pract Oncol 2006, 3(5), 269-280); antle cell lymphoma (MCL) (Dal Col, Zancai et al. Blood 2008, 111(10), 5142-51); renal cell carcinoma (Thomas, Tran et al. Nat Med 2006, 12(1), 122-7; Atkins, Hidalgo et al.
  • Neoplasia 2006, 8(5), 394-401 ovarian cancer
  • ovarian cancer Shayesteh, Lu et al. Nat Genet, 1999, 21(1), 99-102; (Lee, Choi et al. Gynecol Oncol 2005, 97(1) 26-34); endometrial tumors (Obata, Morland et al. Cancer Res 1998, 58(10), 2095-7; Lu, Wu et al. Clin Cancer Res 2008, 14(9), 2543-50); non small cell lung carcinoma (NSCLC) (Tang, He et al. Lung Cancer 2006, 51(2), 181-91; Marsit, Zheng et al.
  • NSCLC non small cell lung carcinoma
  • a first aspect of the invention provides a compound of Table 1, optionally as a pharmaceutically acceptable salt thereof.
  • the invention is directed to a pharmaceutical composition which comprises 1) a compound of Table 1 or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt or solvate thereof and 2) a pharmaceutically acceptable carrier, excipient, or diluent.
  • a third aspect of the invention is a method of inhibiting the in vivo activity of mTOR, the method comprising administering to a patient an effective mTOR-inhibiting amount of a compound of Table 1 or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt or solvate thereof or pharmaceutical composition thereof.
  • the Invention provides a method for treating a disease, disorder, or syndrome which method comprises administering to a patient a therapeutically effective amount of a compound of Table 1 or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising a therapeutically effective amount of a compound of Table 1 or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.
  • a pharmaceutically acceptable carrier excipient, or diluent.
  • administering and variants thereof (e.g., “administering” a compound) in reference to a compound of the invention means introducing the compound or a prodrug of the compound into the system of the animal in need of treatment.
  • a compound of the invention or prodrug thereof is provided in combination with one or more other active agents (e.g., surgery, radiation, and chemotherapy, etc.)
  • “administration” and its variants are each understood to include concurrent and sequential introduction of the compound or prodrug thereof and other agents.
  • Yield for each of the reactions described herein is expressed as a percentage of the theoretical yield.
  • Methodabolite refers to the break-down or end product of a compound or its salt produced by metabolism or biotransformation in the animal or human body; for example, biotransformation to a more polar molecule such as by oxidation, reduction, or hydrolysis, or to a conjugate (see Goodman and Gilman, "The Pharmacological Basis of Therapeutics” 8.sup.th Ed., Pergamon Press, Gilman et al. (eds), 1990 for a discussion of biotransformation).
  • the metabolite of a compound of the invention or its salt may be the biologically active form of the compound in the body.
  • a prodrug may be used such that the biologically active form, a metabolite, is released in vivo.
  • a biologically active metabolite is discovered serendipitously, that is, no prodrug design per se was undertaken.
  • An assay for activity of a metabolite of a compound of the present invention is known to one of skill in the art in light of the present disclosure.
  • "Patient" for the purposes of the present invention includes humans and other animals, particularly mammals, and other organisms. Thus the methods are applicable to both human therapy and veterinary applications. In a specific embodiment the patient is a mammal, and in a more specific embodiment the patient is human.
  • a "pharmaceutically acceptable salt” of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. It is understood that the pharmaceutically acceptable salts are non-toxic. Additional information on suitable pharmaceutically acceptable salts can be found in Remington 's Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, PA, 1985, which is incorporated herein by reference or S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977;66: 1-19 both of which are incorporated herein by reference.
  • Examples of pharmaceutically acceptable acid addition salts include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, 3-(4-hydroxybenzoyl)benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 2-
  • Examples of a pharmaceutically acceptable base addition salts include those formed when an acidic proton present in the parent compound is replaced by a metal ion, such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Specific salts are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins.
  • organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, tromethamine, N-methylglucamine, polyamine resins, and the like.
  • Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.”
  • Platinum(s)," and “platin-containing agent(s)” include, for example, cisplatin, carboplatin, and oxaliplatin.
  • Prodrug refers to compounds that are transformed (typically rapidly) in vivo to yield the parent compound of the above formulae, for example, by hydrolysis in blood.
  • Common examples include, but are not limited to, ester and amide forms of a compound having an active form bearing a carboxylic acid moiety.
  • Examples of pharmaceutically acceptable esters of the compounds of this invention include, but are not limited to, alkyl esters (for example with between about one and about six carbons) the alkyl group is a straight or branched chain. Acceptable esters also include cycloalkyl esters and arylalkyl esters such as, but not limited to benzyl.
  • Examples of pharmaceutically acceptable amides of the compounds of this invention include, but are not limited to, primary amides, and secondary and tertiary alkyl amides (for example with between about one and about six carbons).
  • Amides and esters of the compounds of the present invention may be prepared according to conventional methods. A thorough discussion of prodrugs is provided in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," VoI 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference for all purposes.
  • “Therapeutically effective amount” is an amount of a compound of the invention, that when administered to a patient, ameliorates a symptom of the disease.
  • the amount of a compound of the invention which constitutes a “therapeutically effective amount” will vary depending on the compound, the disease state and its severity, the age of the patient to be treated, and the like. The therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their knowledge and to this disclosure.
  • “Treating” or “treatment” of a disease, disorder, or syndrome includes (i) preventing the disease, disorder, or syndrome from occurring in a human, i.e.
  • Embodiments of the Invention [0022] Another embodiment provides a pharmaceutical composition which comprises 1) a compound, as a single stereoisomer or mixture of isomers thereof, selected from Table 1 , optionally as a pharmaceutically acceptable salt thereof, and 2) a pharmaceutically acceptable carrier, excipient, and/or diluent thereof.
  • Another embodiment is a method of treating disease, disorder, or syndrome where the disease is associated with uncontrolled, abnormal, and/or unwanted cellular activities effected directly or indirectly by mTOR which method comprises administering to a human in need thereof a therapeutically effective amount of a Compound selected from Table 1 , optionally as a pharmaceutically acceptable salt or pharmaceutical composition thereof.
  • the disease is cancer.
  • Embodiment (A) Another embodiment is directed to a method of treating a disease, disorder, or syndrome which method comprises administering to a patient a therapeutically effective amount of a Compound selected from Table 1, optionally as a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a therapeutically effective amount of a Compound selected from Table 1 , and a pharmaceutically acceptable carrier, excipient, or diluent.
  • the disease is cancer.
  • the cancer is breast cancer, mantle cell lymphoma, renal cell carcinoma, acute myelogenous leukemia, chronic myelogenous leukemia, NPM/ALK-transformed anaplastic large cell lymphoma, diffuse large B cell lymphoma, rhabdomyosarcoma, ovarian cancer, endometrial cancer, cervical cancer, non small cell lung carcinoma, small cell lung carcinoma, adenocarcinoma, colon cancer, rectal cancer, gastric carcinoma, hepatocellular carcinoma, melanoma, pancreatic cancer, prostate carcinoma, thyroid carcinoma, anaplastic large cell lymphoma, hemangioma, glioblastoma, or head and neck cancer.
  • R 1 and R 2 are as defined in one of the compounds in Table 1, below, and pharmaceutically acceptable salts thereof.
  • the invention comprises a compound in Table 1, and pharmaceutically acceptable salts thereof.
  • the invention provides pharmaceutical compositions comprising an inhibitor of mTOR according to the invention and a pharmaceutically acceptable carrier, excipient, or diluent.
  • administration is by the oral route.
  • Administration of the compounds of the invention, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration or agents for serving similar utilities.
  • administration can be, for example, orally, nasally, parenterally (intravenous, intramuscular, or subcutaneous), topically, transdermally, intravaginally, intravesically, intracistemally, or rectally, in the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, or aerosols, or the like, specifically in unit dosage forms suitable for simple administration of precise dosages.
  • the compositions will include a conventional pharmaceutical carrier or excipient and a compound of the invention as the/an active agent, and, in addition, may include carriers and adjuvants, etc.
  • Adjuvants include preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • a pharmaceutical composition of the invention may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, antioxidants, and the like, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylalted hydroxytoluene, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, antioxidants, and the like, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylalted hydroxytoluene, etc.
  • formulation depends on various factors such as the mode of drug administration (e.g., for oral administration, formulations in the form of tablets, pills or capsules) and the bioavailability of the drug substance.
  • pharmaceutical formulations have been developed especially for drugs that show poor bioavailability based upon the principle that bioavailability can be increased by increasing the surface area i.e., decreasing particle size.
  • U.S. Pat. No. 4,107,288 describes a pharmaceutical formulation having particles in the size range from 10 to 1,000 nm in which the active material is supported on a crosslinked matrix of macromolecules.
  • compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • aqueous and nonaqueous carriers, diluents, solvents or vehicles examples include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • a coating such as lecithin
  • surfactants One specific route of administration is oral, using a convenient daily dosage regimen that can be adjusted according to the degree of severity of the disease-state to be treated.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or
  • fillers or extenders as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid
  • binders as for example, cellulose derivatives, starch, alignates, gelatin, polyvinylpyrrolidone, sucrose, and gum acacia
  • humectants as for example, glycerol
  • disintegrating agents as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, croscarmellose sodium, complex silicates, and sodium carbonate
  • solution retarders as for example paraffin
  • absorption accelerators as for example,
  • Solid dosage forms as described above can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain pacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedded compositions that can be used are polymeric substances and waxes. The active compounds can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients. [0038] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs.
  • Such dosage forms are prepared, for example, by dissolving, dispersing, etc., a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like; solubilizing agents and emulsif ⁇ ers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide; oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan; or mixtures of these substances, and the like, to thereby form a
  • Suspensions in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • suspending agents as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • compositions for rectal administrations are, for example, suppositories that can be prepared by mixing the compounds of the present invention with for example suitable non- irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt while in a suitable body cavity and release the active component therein.
  • suitable non- irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt while in a suitable body cavity and release the active component therein.
  • Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays, and inhalants.
  • the active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required.
  • Ophthalmic formulations, eye ointments, powders, and solutions are also contemplated as being within the scope of this invention.
  • Compressed gases may be used to disperse a compound of this invention in aerosol form.
  • Inert gases suitable for this purpose are nitrogen, carbon dioxide, etc.
  • the pharmaceutically acceptable compositions will contain about 1% to about 99% by weight of a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, and 99% to 1 % by weight of a suitable pharmaceutical excipient.
  • the composition will be between about 5% and about 75% by weight of a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, with the rest being suitable pharmaceutical excipients.
  • composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease-state in accordance with the teachings of this invention.
  • the compounds of the invention are administered in a therapeutically effective amount which will vary depending upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of the compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular disease-states, and the host undergoing therapy.
  • the compounds of the present invention can be administered to a patient at dosage levels in the range of about 0.1 to about 1,000 mg per day. For a normal human adult having a body weight of about 70 kilograms, a dosage in the range of about 0.01 to about 100 mg per kilogram of body weight per day is an example. The specific dosage used, however, can vary.
  • the dosage can depend on a number of factors including the requirements of the patient, the severity of the condition being treated, and the pharmacological activity of the compound being used.
  • the determination of optimum dosages for a particular patient is well known to one of ordinary skill in the art.
  • Compounds of this invention can be made by the synthetic procedures described below.
  • the starting materials and reagents used in preparing these compounds are either available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wis.), or Bachem (Torrance, Calif.), or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and Sons, 4 Edition) and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).
  • the reactions described herein take place at atmospheric pressure and over a temperature range from about -78 0 C to about 150 0 C, more specifically from about 0 0 C. to about 125 0 C and more specifically at about room (or ambient) temperature, e.g., about 20 0 C. Unless otherwise stated (as in the case of an hydrogenation), all reactions are performed under an atmosphere of nitrogen.
  • Prodrugs can be prepared by techniques known to one skilled in the art. These techniques generally modify appropriate functional groups in a given compound. These modified functional groups regenerate original functional groups by routine manipulation or in vivo. Amides and esters of the compounds of the present invention may be prepared according to conventional methods. A thorough discussion of prodrugs is provided in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," VoI 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference for all purposes.
  • the compounds of the invention may have asymmetric carbon atoms or quaternized nitrogen atoms in their structure.
  • Compounds of the Invention that may be prepared through the syntheses described herein may exist as single stereoisomers, racemates, and as mixtures of enantiomers and diastereomers.
  • the compounds may also exist as geometric isomers. All such single stereoisomers, racemates and mixtures thereof, and geometric isomers are intended to be within the scope of this invention.
  • Some of the compounds of the invention contain an active ketone -C(O)CF3 and may exist in part or in whole as the -C(OH 2 )CF 3 form.
  • the compound is drawn as the -C(O)CF 3 or -C(OH 2 )CF 3 form, both are included within the scope of the Invention.
  • an individual compound may be drawn as the -C(O)CF 3 form, one of ordinary skill in the art would understand that the compound may exist in part or in whole as the -C(OH 2 )CFs form and that the ratio of the two forms may vary depending on the compound and the conditions in which it exists.
  • Some of the compounds of the invention may exist as tautomers.
  • R 1 can be 5-oxo-lH-l,2,4-triazol-3-yl, depicted structurally below:
  • Both 5-oxo-lH-l,2,4-triazol-3-yl and the above structure 1 include, and are equivalent to, 3-hydroxy-4H-l,2,4-triazol-5-yl and its structure 2:
  • the present invention also includes N-oxide derivatives and protected derivatives of compounds of the Invention.
  • compounds of the Invention when compounds of the Invention contain an oxidizable nitrogen atom, the nitrogen atom can be converted to an N-oxide by methods well known in the art.
  • compounds of the Invention contain groups such as hydroxy, carboxy, thiol or any group containing a nitrogen atom(s), these groups can be protected with a suitable "protecting group” or "protective group”.
  • a comprehensive list of suitable protective groups can be found in T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, Inc.
  • Enantiomers may be resolved by methods known to one of ordinary skill in the art, for example by: formation of diastereoisomeric salts or complexes which may be separated, for example, by crystallization; via formation of diastereoisomeric derivatives which may be separated, for example, by crystallization, selective reaction of one enantiomer with an enantiomer-specif ⁇ c reagent, for example enzymatic oxidation or reduction, followed by separation of the modified and unmodified enantiomers; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support, such as silica with a bound chiral ligand or in the presence of a chiral solvent.
  • enantiomer may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents or by converting on enantiomer to the other by asymmetric transformation.
  • enantiomer enriched in a particular enantiomer, the major component enantiomer may be further enriched (with concomitant loss in yield) by recrystallization.
  • the compounds of the present invention can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like.
  • pharmaceutically acceptable solvents such as water, ethanol, and the like.
  • the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.
  • STEP 1 To a solution of tert-butyl 2-oxopiperidine- 1 -carboxylate (0.22 g, 1.08 mmol) in tetrahydrofuran (5 mL) at -78 0 C was added slowly over 15 minutes 4-chloro-3- fluorophenylmagnesium bromide (0.5 M in tetrahydrofuran) (2.60 mL, 1.66 mmol) and the mixture was allowed to warm to 25 °C over 30 minutes. The reaction mixture was quenched by the addition of 1.0 N hydrochloric acid (1.0 mL) and partitioned with diethyl ether (50 mL).
  • STEP 2 A solution of tert-butyl 5-oxo-5-(4-chloro-3-fluorophenyl)pentylcarbamate (0.25 g, 0.76 mmol) was stirred in trifluoroacetic acid (1.5 mL) for 1 hour then concentrated. The residue was partitioned between dichloromethane (40 mL) and 2 M aqueous sodium hydroxide (20 mL). The organic layer was separated and washed with brine (20 mL), dried over anhydrous magnesium sulfate filtered and concentrated to provide 5-amino-l-(4-chloro-3- fluorophenyl)pentan-l-one.
  • STEP 1 A suspension of potassium tert-butoxidc (1.25 g, 11.1 mmol) and methyltriphenylphosphonium bromide (3.86 g, 1.1 mmol) in tetrahydrofuran (100 mL) was stirred at 40 0 C for 30 minutes. The mixture was then cooled to room temperature and a solution of tert-butyl 4-oxo-2-phenylpiperidine-l-carboxylate (2.35 g, 8.5 mmol) in tetrahydrofuran (30 mL) was added slowly. The reaction mixture was stirred at 40 0 C for 24 hours.
  • STEP 1 To a solution of tert-butyl (4-hydroxymethyl)piperidine-l-carboxylate (0.52 g, 2.40 mmol, (J. Labelled Compounds and Radiopharmaceuticals 1999, 42, 1289-1300) in dichloromethane (20 mL) was added Dess-Martin-periodinane (1.13 g, 2.66 mmol), and the mixture was stirred at room temperature for 2 h. A 10% aqueous solution of sodium thiosulfate (20 mL) was added followed by saturated sodium bicarbonate (20 mL), and the biphasic mixture was stirred at room temperature for 45 min.
  • STEP 2 To a solution of DAST (1.16 g, 7.20 mmol) in dichloromethane (30 mL) was added a solution of tert-butyl 4-formylpiperidine-l-carboxylate (0.51 g, 2.40 mmol) in dichloromethane (5 mL) at 0 0 C. The reaction mixture was warmed to room temperature and stirred for 18 h.
  • STEP 1 To a solution of DAST (1.83 g, 11.35 mmol) in dichloromethane (30 mL) was added 4-acetylpyridine (1.00 g, 8.25 mmol) at 0 0 C. The reaction mixture was warmed to room temperature and stirred for 2 d. More DAST (0.61 g, 3.78 mmol) was added and stirring was continued for 1 d.
  • STEP 2 The mixture was dissolved in methanol (10 mL) and 1 M hydrochloric acid (10 mL) then catalytic platinum oxide was added and the resulting suspension was hydrogenated in a Parr apparatus at 40 psi for 3 d. Filtration through celite and concentration of the filtrate gave a complex mixture containing 20% of the desired 4-(l,l-difluoroethyl)piperidine as the hydrochloride salt which was used without further purification.
  • STEP 1 To a solution of tert-butyl 4-hydroxypiperidine- 1 -carboxylate (4.0 g, 20.0 mmol) and triethylamine (4.0 g, 40 mmol) in dichloromethane (50 mL) was added methanesulfonyl chloride (2.8 g, 24.4 mmol) at 0 0 C. The solution was stirred at 0 0 C for 10 min, then at room temperature for 2 h. The reaction mixture was partitioned between 10% citric acid and ethyl acetate.
  • STEP 3 A solution of tert-butyl 4-(methylthio)piperidine-l -carboxylate (63 mg, 0.27 mmol) in methanol (1 mL) and 4 N hydrogen chloride in dioxane (4 mL) was refluxed for 2 min and then concentrated and dried to provide 4-(methylthio)piperidine hydrochloride as a colorless oil.
  • Step 1 Trimethyl(trifluoromethyl)silane (0.32 g, 2.25 mmol) was added to a mixture of tert-butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (0.50 g, 2.2 mmol), cesium carbonate (1.1 g, 3.4 mmol) in N,N-dimethylformamide (5 mL) at O 0 C. The resulting mixture was warmed to room temperature and stirred for two hours.
  • the mixture was diluted with ethyl acetate (80 mL), washed with water (3 x 50 mL) then brine (50 mL), dried over sodium sulfate, filtered, and concentrated. The residue was taken into methanol (20 mL) and potassium carbonate (0.62 g, 4.5 mmol) was added then stirred at room temperature for 18 hours. The mixture was diluted with ethyl acetate (150 mL) then filtered and concentrated.
  • Step 2 tert-Butyl 3-(enJo)-hydroxy-3-(trifluoromethyl)-8-azabicyclo[3.2.1]octane-8- carboxylate 1 (0.36 g, 1.2 mmol) was taken into acetonitrile (2 mL) and 4 M hydrogen chloride in 1 ,4-dioxane (2 mL) then stirred at 70 0 C for 15 minutes. The reaction mixture was concentrated and dried to give 3-(trifluoromethyl)-8-azabicyclo[3.2.1]octan-3-(e « ⁇ fo)-ol hydrochloride (0.28g, 100% yield). MS (EI) for C 8 H 12 F 3 NO: 196 (MH + ).
  • Step 1 Methylmagnesium bromide (3 M solution in ether, 2.7 mmol) was added to a solution of tert-butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (0.50 g, 2.2 mmol), in tetrahydrofuran (20 mL) at 0 0 C and the resulting mixture was stirred one hour. The reaction mixture was quenched with saturated aqueous ammonium chloride solution (20 mL) then partitioned with ethyl acetate (80 mL). The organic portion was separated, washed with water, then brine, dried over sodium sulfate, filtered and concentrated.
  • Step 2 tert-Butyl 3-(endo)-hydroxy-3-methyl-8-azabicyclo[3.2.1]octane-8- carboxylate (0.22 g, 1.2 mmol) was taken into acetonitrile (1 mL), and 4 M hydrogen chloride in 1,4-dioxane (1 mL) then stirred at 70 0 C for 15 minutes. The reaction mixture was concentrated and dried to give 3-methyl-8-azabicyclo[3.2. l]octan-3-(endo)-ol hydrochloride salt (0.16 g, 100% yield). MS (EI) for C 8 H 12 F 3 NO: 142 (MH + ).
  • Step 1 Dimethylaminosulfur trifluoride (81 mg, 0.61 mmol) was added to a solution of tert-butyl 3-(e/? ⁇ io)-(hydroxymethyl)-8-azabicyclo[3.2.1 ]octane-8-carboxylate (50 mg, 0.21 mmol) (reagent preparation 18, step 2) in dichloromethane (2 mL) at 0 0 C, and the resulting mixture was stirred one hour.
  • Step 2 A mixture of tert-butyl 3-fluoro-3-(e « ⁇ io)-methyl-8-azabicyclo[3.2.1 Joctane- 8-carboxylate (0.22 g, 1.2 mmol), acetonitrile (1 mL) and 4 M hydrogen chloride in 1,4-dioxane (1 mL) was stirred at 70 0 C for 15 minutes. The reaction mixture was concentrated and dried to give 3-fluoro-3-(en ⁇ io)-methyl-8-azabicyclo[3.2.1]octane hydrochloride salt (20 mg, 100% yield). MS (EI) for C 8 Hi 4 FN: 144 (MH + ).
  • Step 1 Potassium tert-butoxide (0.62 g, 5.5 mmol) was added to a suspension of methyltriphenylphosphonium bromide (1.98 g, 5.5 mmol) in tetrahydrofuran (20 mL) and the resulting mixture was stirred at room temperature for one hour. A solution of tert-butyl 3-oxo-8- azabicyclo[3.2.1]octane-8-carboxylate (0.50 g, 2.2 mmol) in tetrahydrofuran (5 mL) was then added and the resulting mixture was stirred at 35 0 C for two hours.
  • Step 2 Borane (I M solution in tetrahydrofuran, 1.79 mL) was added to a solution of tert-butyl 3-methylene-8-azabicyclo[3.2.1]octane-8-carboxylate (0.20 g, 0.87 mmol) in tetrahydrofuran (20 mL) at 0 0 C. The reaction mixture was slowly warmed to room temperature and stirred for 18 hours. It was then cooled to 0 0 C, followed by sequential addition of 2 M sodium hydroxide solution (1 mL) and hydrogen peroxide solution (30% in water, 0.46 mL). The mixture was warmed to room temperature and stirred for 1.5 hours.
  • 2 M sodium hydroxide solution (1 mL
  • hydrogen peroxide solution 30% in water, 0.46 mL
  • reaction mixture was quenched with saturated sodium bicarbonate solution (10 mL), diluted with water (20 mL) and partitioned with ethyl acetate (20 mL). The organic portion was separated and washed twice with saturated sodium bisulfite solution (20 mL), water then brine, dried over sodium sulfate, filtered and concentrated.
  • Step 3 Methanesulfonyl chloride (154 mg, 1.35 mmol) was added to a mixture of tert-butyl 3-(e «6? ⁇ )-(hydroxymethyl)-8-azabicyclo[3.2.1]octane-8-carboxylate (325 mg, 1.4 mmol), triethylamine (136 mg, 1.4 mmol), and l,4-diazabicyclo[2.2.2]octane (31 mg, 0.28 mmol) in toluene (10 mL) at 0 0 C. The resulting mixture was stirred at 0 0 C for 15 minutes, and at room temperature for another 15 minutes.
  • Step 4 A mixture of tert-butyl 3-((ercdo)-methylsulfonyloxy)methyl)-8- azabicyclo[3.2.1]octane-8-carboxylate (330 mg, 1.0 mmol), triethylamine (136 mg, 1.4 mmol), and tetrabutylammonium fluoride hexahydrate (489 mg, 1.3 mmol) in tetrahydrofuran (10 mL) was stirred at 60 0 C for 18 hours.
  • Step 5 A mixture of tert-butyl 3-(endo)-(fluoromethyl)-8-azabicyclo[3.2.1]octane-8- carboxylate (50 mg, 0.21 mmol), acetonitrile (1 niL), and 4 M hydrogen chloride in 1,4-dioxane (1 mL) was stirred at 70 0 C for 15 minutes. The reaction mixture was concentrated and dried to give 3-(endo)-(fluoromethyl)-8-azabicyclo[3.2.1]octane hydrochloride salt (37 mg, 100% yield). MS (EI) for C 8 Hi 5 FN: 144 (MH + ).
  • STEP 1 To a solution of ter?-butyl 4-oxopiperidine-l-carboxylate (0.6 g, 3.0 mmol) and cesium carbonate (1.1 g, 3.3 mmol) in dimethylformamide (10 mL) was added dropwise trimethyl(trifluoromethyl)silane (2 mL, 13.5 mmol) at 0 0 C. The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with diethyl ether (100 ml) washed with water (50 mL) and brine (50 mL).
  • Step 2 tert-Butyl 4-cyano-4-methylpiperidine- 1 -carboxylate as obtained in step 1 (100 mg, 0.21 mmol), acetonitrile (1 mL), and 4 M hydrogen chloride in 1,4-dioxane (1 mL) were combined and stirred at 70 0 C for 15 minutes. The reaction mixture was concentrated and dried to give 4-methylpiperidine-4-carbonitrile hydrochloride salt (56 mg) contaminated with 4- methylpiperidine-4-carboxamide hydrochloride salt. MS (EI) for C7H 12 N 2 (4-methylpiperidine- 4-carbonitrile): 125 (MH + ). Reagent Preparation 14
  • STEP 1 Sodium borohydride (178 mg, 4.7 mmol) was added to a solution of tert-butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (0.50 g, 2.2 mmol) in ethanol (10 mL), and the resulting mixture was stirred at room temperature for one hour. The mixture was quenched with saturated ammonium chloride solution (30 mL), and extracted with ethyl acetate (3x 20 mL).
  • step 2 tert-Butyl 3-hydroxy-8-azabicyclo[3.2.1 ]octane-8-carboxylate as obtained in step 1 (100 mg, 2.0 mmol), acetonitrile (2 mL) and 4 M hydrogen chloride in 1,4-dioxane (2 mL) were combined and stirred at 70 0 C for 15 minutes. The reaction mixture was concentrated and dried to give 8-azabicyclo[3.2.1]octan-3-ol hydrochloride salt (71 mg, 100% yield). MS (EI) for C 7 H 13 NO: 128 (MH + ). Reagent Preparation 15
  • STEP 1 A mixture of tert-butyl 3-methylene-8-azabicyclo[3.2. l]octane-8- carboxylate (0.10 g, 0.44 mmol) (reagent preparation 18), 10% palladium on charcoal (10 mg) and ethanol (15 mL) was hydrogenated in a Parr apparatus at 40 psi for 18 hours. The mixture was filtered and concentrated then dried to give tert-butyl 3-(endo)-methyl-8- azabicyclo[3.2.1]octane-8-carboxylate (96 mg, 95% yield); GC-MS (EI) for Ci 3 H 23 NO 2 : 225
  • STEP 2 A mixture of tert-butyl 3-(endo)-methyl-8-azabicyclo[3.2.1]octane-8- carboxylate (96 mg, 0.43 mmol), acetonitrile (1 mL), and 4 M hydrogen chloride in 1,4-dioxane (1 mL) was stirred at 70 0 C for 15 minutes. The reaction mixture was concentrated and dried to give 3-(endo)-methyl-8-azabicyclo[3.2.1]octane hydrochloride salt (68 mg, 100% yield). MS (EI) for C 8 Hi 5 N: 126 (MH + ).
  • STEP 5 A suspention of ( ⁇ )-(2R,4R)-tert-buty ⁇ 4-acetoxy-2-(4- fluorophenyl)piperidine- 1 -carboxylate (150 mg, 0.40 mmol) and potassium carbonate (1.0 g) in methanol: water 10:1 (11 mL) was stirred for 1 hour then diluted with ethyl acetate (40 mL) and washed with water (25 mL) and brine (25 mL).
  • STEP 1 Potassium tert-butoxide (0.62 g, 5.5 mmol) was added to a suspension of methyltriphenylphosphonium bromide (1.98 g, 5.5 mmol) in tetrahydrofuran (20 mL) and the resulting mixture was stirred at room temperature for one hour. A solution of tert-butyl 3-oxo-8- azabicyclo[3.2.1]octane-8-carboxylate (0.50 g, 2.2 mmol) in tetrahydrofuran (5 mL) was then added and the resulting mixture was stirred at 35 0 C for two hours.
  • STEP 2 To a solution of tert-butyl 3-methylene-8-azabicyclo[3.2.1]octane- carboxylate (0.9 g, 4.0 mmol) (reagent preparation 18, step 1) in acetone (16 mL) and water (4 mL) was added osmium tetroxide (0.25 mL of a 4% aqueous solution, 0.04 mmol) and
  • STEP 3 A solution of tert-butyl 3-(hydroxy)-3-(en Jo)-(hydroxymethyl)-8- azabicyclo[3.2.1]octane-carboxylate (50 mg, 0.20 mmol) in dichloromethane (1 mL) and trifluoroacetic acid (1 mL) was stirred at room temperature for 1 h and then concentrated and dried to give 3-(en ⁇ io)-(hydroxymethyl)-8-azabicyclo[3.2.1]octan-3-ol as the trifluoroacetate salt, which was used without further purification.
  • STEP 1 Benzyl 2-(4-fluorophenyl)-4-oxo-3,4-dihydropyridine-l(2H)-carboxylate was prepared according to the method in (Tetrahedron Lett., 1986, 27, 4549-4552) using 4-methoxypyridine (29.8 mL, 290 mmol), benzyl chloroformate (50.0 mL, 350 mmol) and 4-fluorophenyl magnesium bromide (0.8 M solution in T ⁇ F), (450 mL, 0.36 mmol), to yield (81 g, 86% yield) of the title compound.
  • STEP 2 Benzyl 2-(4-fluorophenyl)-4-oxopiperidine- 1 -carboxylate was prepared according to the method described in (J. Am. Chem. Soc, 2001, 66, 2181-2182) using benzyl 2-(4-fluorophenyl)-4-oxo-3,4-dihydropyridine-l(2H)-carboxylate (16.5 g, 50.7 mmol) and zinc dust (9.8 g, 150 mmol) to afford (16.0 g, 96% yield) of the title compound.
  • STEP 1 To a solution of 6-bromo-2-methyl- lH-imidazo[4,5-6]pyridme (3.8 g, 17.5 mmol) in dimethylformamide (20 mL) at 0 0 C was added 60% sodium hydride in mineral oil (1.05 g, 26.2 mmol) and the reaction mixture was stirred for 30 minutes, followed by the addition of a solution of triphenylmethyl chloride (5.4 g, 19.4 mmol) in dimethylformamide (10 mL).
  • STEP 1 Commercially-available 5-bromo-2-hydroxybenzaldehyde (4.0 g, 10 mmol) and 2-aminoethanol were combined in THF/MeOH (100 mL, 10:1) and sodium borohydride (0.76 g, 2.0 mmol) was added with stirring. The resulting reaction mixture was stirred at 40 0 C for 4 h, concentrated on a rotary evaporator then diluted with EtOAc (50 mL) and saturated NaHCO 3 (30 mL). To this suspension was added di-tert-buty ⁇ dicarbonate (2.83 g, 13 mmol). The mixture was stirred at rt overnight.
  • STEP 2 ter ⁇ -Butyl-5-bromo-2-hydroxybenzyl(2-hydroxyethyl)carbamate (3.46 g, 10 mmol) and triphenylphosphine (3.96 g, 15 mmol) were combined in DCM (100 mL) and diisopropyl azodicarboxylate (3.03 g, 15 mmol) was added. The resulting reaction mixture was stirred at rt for 12 h. The reaction mixture was washed with water, dried, filtered, and concentrated on a rotary evaporator.
  • STEP 2 To a solution of tert-butyl 4-(oxoethyl)piperidine-l-carboxylate as obtained in step 1 in dichloromethane (50 mL) was added DAST (1.2 g, 7.8 mmol) at 0 0 C. The reaction mixture was warmed to room temperature and stirred for 17 h. A 5% aqueous solution of sodium bicarbonate was added and the layers were separated. The organic layer was washed with saturated sodium bicarbonate, and brine, dried over sodium sulfate, filtered and concentrated to provide tert-butyl 4-(2,2-difiuoroethyl)piperidine-l-carboxylate that was used directly without further purification.
  • STEP 3 tert-Butyl 4-(2,2-difluoroethyl)piperidine-l-carboxylate as obtained in step 2 was dissolved in a minimum of trifiuoroacetic acid and the resulting solution was stirred at room temperature for 2 h. The solution was then concentrated to give 4-(2,2- difluoroethyl)piperidine as the trifluoroacetate salt. MS (EI) for C 7 Hi 3 F 2 N: 150 (MH + ).
  • reaction mixture was stirred at -78 0 C for an additional 30 min, then quenched by dropwise addition of 2 N hydrochloric acid (80 ml), and allowed to warm up to room temperature.
  • Ethyl acetate (100 mL) and water (100 mL) were added, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (100 mL).
  • the combined organic layers were washed with water, dried over sodium sulfate, and concentrated. Hexane (200 mL) was added to the residue and the mixture was stirred overnight.
  • STEP 3 A suspension of 1,1 -dimethylethyl 6-bromo-2-methyl-lH-benzimidazole-l- carboxylate (11.3 g, 36 mmol), (4- ⁇ [(l,l-dimethylethyl)oxy]carbonyl ⁇ -2,3,4,5-tetrahydro-l,4- benzoxazepin-7-yl)boronic acid (11.7 g, 40 mmol), dichloro[l,l-bis(diphenyl- phosphino]ferrocenepalladium (II) dichloromethane adduct (3.0 g, 10 mol %) in dioxane (115 mL) and water (28.5 mL) was degassed with nitrogen, and then diisopropylethylamine (18.6 g, 144 mmol) was added.
  • STEP 4 A solution of 1 , 1 -dimethyl 7-(l- ⁇ [(l,l-dimethylethyl)oxy]carbonyl ⁇ -2- methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepine-4(5H)-carboxylate (13.1 g, 27 mmol) in a mixture of methanol (20 mL) and 4 N hydrogen chloride in dioxane (30 mL) was refluxed for 15 min. After cooling to room temperature ethyl ether (100 mL) was added, and the reaction mixture was concentrated.
  • step 6 prop-2-en-l-yl 7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4- benzoxazepine-4(5H)-carboxylate as obtained in step 5 was taken into T ⁇ F (100 mL) followed by addition of pyridine (2.5 mL, 30 mml) and di-tert-butyl dicarbonate (4.9 g, 22.4 mmol) and the mixture was allowed to stir at room temperature over 12 h. The resulting solution was concentrated and the residue partitioned with ethyl acetate and 10% aqueous citric acid.
  • STEP 7 7-[l-[(l,l-dimethylethoxy)carbonyl]-2-methyl-lH-benzimidazol-6-yl]-2,3- dihydro- 1 ,4-benzoxazepine-4(5H)-carboxylic acid 2-propenyl ester (110 mg, 0.27 mmol) was taken into T ⁇ F (1 mL) followed by addition of sodium triacetoxyborohydride (254 mg, 1.2 mmol) then tetrakis(triphenylphosphine)palladium (0) (6.1 mg, 0.005 mmol) and the mixture was stirred for 1 h at room temperature.
  • STEP 10 1,1-dimethylethyl 6-(4- ⁇ [2-(4-chlorophenyl)piperidin- l-yl]carbonyl ⁇ - 2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)-2-methyl-lH-benzimidazole-l-carboxylate as obtained in step 9 was taken into methanol (1 mL) followed by addition of 4 N hydrogen chloride in dioxane solution (1 mL) and the resulting solution was allow to stir for 2 h at room temperature.
  • step 2 1 , 1 -dimethylethyl 7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4- benzoxazepine-4(5H)-carboxylate as prepared in step 1 (9.6 g) was taken into dichloromethane (100 mL) followed by sequential addition of DIPEA (4.3 mL, 24.9 mmol) and isobutyl chloroformate (2.7 mL, 20.8 mmol). The mixture was stirred for 1 h at room temperature then partitioned with 0.5 N aqueous hydrochloric acid .
  • step 2 1 , 1 -dimethylethyl 7-(2-methyl-l-[ ⁇ (2-methylpropyl)oxy]carbonyl ⁇ -lH- benzimidazol-6-yl-2,3-dihydro-l,4-benzoxazepine-4(5H)-carboxylate as obtained in step 2 (10.3 g) was taken into 1 : 1 TFA: dichloromethane (100 mL) and the resulting solution was stirred for 1 h at room temperature then concentrated. The residue was treated with saturated aqueous sodium bicarbonate (100 mL) and the aqueous mixture was treated with portion wise solid sodium bicarbonate until p ⁇ 8.5.
  • Step 4 Phosgene (20 W% in toluene, 9.5 mL) was diluted into dichloromethane (40 mL) and the resulting solution cooled to 0 0 C.
  • 2-Methylpropyl 2-methyl-6-(2,3,4,5-tetramethyl- l,4-benzoxazepin-7-yl)-lH-benzimidazole-l-carboxylate as obtained in step 3 (6.8 g, 17.9 mmol)) was taken into dichloromethane (30 mL) followed by addition of DIPEA (7.8 mL, 44.8 mmol) and the resulting solution was slowly added to the cooled phosgene solution over 5 minutes by addition funnel.
  • STEP 1 To a solution of 1,1-dimethylethyl 6-[4-(chlorocarbonyl)-2,3,4,5-tetrahydro- l,4-benzoxazepin-7-yl)-lH-benzimidazole-l-carboxylate (example 1, step 8) (5.5mg, 16 ⁇ mol) and decahydroisoquinoline (6.7 mg, 48 ⁇ mol) in anhydrous DCM (2 mL) was added PL-DIPAM (85mg, 3.27 mmol/g loading, 315 ⁇ mol, Polymer Labs) and the reaction mixture was shaken overnight at room temperature.
  • PL-DIPAM 85mg, 3.27 mmol/g loading, 315 ⁇ mol, Polymer Labs
  • the resulting mixture was drained into PL-PETA (55mg, 2.7 mmol/g loading, 175 ⁇ mol, Polymer Labs) and PL-MIA, (35 mg, 2.65 mmol/g loading, 104 ⁇ mol, Polymer Labs).
  • the reaction mixture was shaken overnight at room temperature, drained and the resin was washed with 3.0 mL of methanol.
  • the combined methanol solution was transferred to a 2 dram vial and concentrated under reduced pressure.
  • the resulting oil was dissolved in methanol (2 mL) followed by the addition of 4 N anhydrous hydrogen chloride in dioxane (0.5 mL, Aldrich). The mixture was shaken at room temperature for an additional 18 hours.
  • STEP 2 7-bromo-2,3,4,5-tetrahydro-l,4-benzoxazepine hydrochloride (3.0 g, 11.34 mmol) was suspended in dichloromethane (30 mL) followed by addition of DIPEA (3 mL, 34 mmol) and pyridine (4 mL, 49 mmol) and the resulting partially heterogeneous mixture was added portionwise over 5 minutes to a 0 0 C cooled solution of phosgene (20 W% in toluene, 15 mL, 28 mmol) in dichloromethane (15 mL). The resulting mixture was then allowed to slowly warm to room temperature over 30 minutes then concentrated.
  • DIPEA 3 mL, 34 mmol
  • pyridine 4 mL, 49 mmol
  • step 3 7-bromo-2,3-dihydro-l,4-benzoxazepine-4(5H)-carbonyl chloride as obtained in step 2 was taken into dichloromethane (35 mL) followed by portionwise addition of 4-methylpiperidine (3.5 mL, 28.4 mmol) over 5 minutes. The resulting mixture was stirred an additional 5 minutes then concentrated.
  • STEP 4 7-bromo-4-[(4-methylpiperidin-l-yl)carbonyl]-2,3,4,5-tetrahydro-l,4- benzoxazepine as obtained in step 3 (3.91 g, 11.07 mmol) was azeotroped twice from warm heptane then taken into anhydrous THF and cooled to -78 0 C under nitrogen. Triisopropyl borate (3.3 mL, 14.4 mmol) was added by syringe followed by dropwise addition of n-butyllithium (2.5M in hexane, 5.8 mL, 14.4 mmol) over 30 minutes.
  • the mixture was allowed to stir an additional 30 minutes at -78 0 C then quenched by careful addition of 2M aqueous hydrochloric acid (10 mL) and warmed to room temperature. The mixture was stirred for 1 h at room temperature then concentrated to remove THF. The resulting aqueous mixture was then diluted with additional water and basified to pH greater than 12 by addition of 50% aqueous sodium hydroxide. The aqueous mixture was extracted once with ethyl ether then acidified to pH 1 by addition of concentrated aqueous hydrochloric acid.
  • STEP 5 To a mixture of 6-bromo-lH-benzimidazole (0.09 g, 0.46 mmol), ⁇ 4-[(4- methylpiperidin-l-yl)carbonyl]-2,3,4,5-tetrahydro-l,4-benzoxazepine -7-yl ⁇ boronic acid (0.15 g, 0.47 mmol) and potassium carbonate (0.32 g, 2.3 mmol) in DMA (5.0 mL) and water (0.5 mL) was added dichloro[l,l-bis(diphenylphosphino]ferrocenepalladium (II) dichloromethane adduct (0.03 g, 0.05 mmol).
  • STEP 1 A mixture of N-(5-bromothiazol-2-yl)acetamide (1.00 g, 4.52 mmol), (4- ⁇ [(1 , 1 -dimethylethyl)oxy]carbonyl ⁇ -2,3,4, 5-tetrahydro- 1 ,4-benzoxazepin-7-yl)boronic acid (example 1, step 2) (1.54 g, 5.43 mmol ), [l,r-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane (0.33 g, 0.40 mmol), potassium carbonate (2.50 g, 18.1 mmol) in 1,4-dioxane (20 mL) and water (2 mL) was degassed with nitrogen for 5 minutes and then stirred at 93 0 C for 18 hours.
  • reaction mixture was cooled to room temperature, diluted with ethyl acetate (80 mL), filtered over celite. The filtrate was washed with brine (2 x 50 mL), dried over sodium sulfate, filtered, concentrated.
  • STEP 2 A mixture 1,1-dimethylethyl 7-[2-(acetylamino)-l,3-thiazol-5-yl]-2,3- dihydro- 1 ,4-benzoxazepine-4(5H)-carboxylate (0.29g, 0.75 mmol), in methanol (2 mL) and 4 M hydrogen chloride in 1,4-dioxane (2 mL) was stirred at 7O 0 C for 15 minutes.
  • STEP 3 4-Methylpiperidine-l-carbonyl chloride (reagent preparation 37) (103 mg, 0.64 mmol) was added to a mixture of N-[5-(2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)-l,3- thiazol-2-yl]acetamide hydrochloride, (173 mg, 0.53 mmol) and potassium carbonate (374 mg, 2.7 mmol) in 7V,N-dimethylformamide (2 mL). The resulting mixture was stirred at room temperature for 18 hours, then methanol (2 mL) was added and concentrated.
  • STEP 4 A mixture of N-(5- ⁇ 4-[(4-methylpiperidin-l-yl)carbonyl]-2,3,4,5- tetrahydro-l,4-benzoxazepin-7-yl ⁇ -l,3-thiazol-2-yl)acetamide (50 mg, 0.12 mmol) in 1.5 mL of 6 M hydrochloric acid was stirred at 9O 0 C for two hours.
  • step 3 Using analogous synthetic techniques and substituting with alternative starting reagents in step 3 the following compounds of the invention were prepared. Alternative starting materials were obtained commercially unless otherwise indicated. [00221 ] 5 -(4- ⁇ [4-(trifluoromethyl)piperidin- 1 -yl] carbonyl ⁇ -2,3 ,4,5 -tetrahydro- 1 ,4- benzoxazepin-7-yl)-l,3-thiazol-2-amine. Synthesized according to the method of example 5 using 4-(trifluoromethyl)piperidine-l -carbonyl chloride (reagent preparation 19) in step 3.
  • STEP 1 1,1-Dimethylethyl 7-bromo-2,3-dihydro-l,4-benzoxazepine-4(5H)- carboxylate (reagent preparation 21) (5.0 g, 20.1 mmol), bis(pinacolato)diboron (5.6 g, 22.1 mmol), potassium acetate (5.9 g, 60.2 mmol) and [1,1 '- bis(diphenylphosphino)ferrocene]dichloropalladium(II) (440 mg, 0.62 mmol) were heated in DMSO (5 mL) solution at 80 0 C for 1.5 h.
  • DMSO 5 mL
  • step 4 7-(4,4,5,5-Tetramethyl-l,3,2-dioxaboralan-2-yl)-2,3-dihydro-l,4- benzoxazepine-4(5H)-carbonyl chloride (3.0 g) as obtained in step 3 was taken into dichloromethane (50 mL) followed by addition of DIPEA (40 mmol, 7 mL) then 4-trifluoromethylpiperidine hydrochloride salt (1.55 g, 8.2 mmol). The mixture was allowed to stir 30 minutes at room temperature then concentrated. The residue was partitioned with ethyl acetate and 5% aqueous citric acid.
  • step 5 Using analogous synthetic techniques and substituting with alternative starting reagents in step 5 then conducting protecting group removal as required according to literature techniques appropriate for a given protecting group (see for example: Greene and Wuts,
  • STEP 1 (4- ⁇ [(l,l-dimethylethyl)oxycarbonyl ⁇ -2,3,4,5-tetrahydro-l,4-benzoxazepin- 7-yl)boronic acid (example 1, step 2) (2.22 g, 7.57 mmol) and 4-bromo-2-nitroaniline (1.56 g, 7.20 mmol) were taken into dioxane (20 mL), water (4 mL) and DIPEA (5.4 mL, 31 mmol) followed by addition of dichloro[l,l- ⁇ (diphenylphosphino]ferrocenepalladium (II) dichloromethane adduct (322 mg, 0.39 mmol) and the mixture was heated to 95 0 C for 2 h.
  • STEP 2 1,1-dimethylethyl 7-(4-amino-3-nitrophenyl)-2,3-dihydro-l,4- benzoxazepine-4(5H)-carboxylate (6.0 g, 15.6 mmol) was taken into warm methanol (50 mL) followed by addition of 4M hydrogen chloride in dioxane (50 mL) in portions and the warm solution was allowed to slowly cool to room temperature over Ih.
  • STEP 4 Prop-2-en-l-yl 7-(4-amino-3-mtrophenyl)-2,3-dihydro-l,4-benzoxazepine- 4(5H)-carboxylate (4.2 g, 11.2 mmol) was taken into glacial acetic acid (25 mL) and the solution was warmed to 45 0 C. Tin (II) chloride (8.51 g, 44.8 mmol) was added in portions over 5 minutes and the mixture was allowed to stir at this temperature for 6h. The mixture was then cooled to room temperature and diluted with MTBE (100 mL).
  • STEP 7 N,N'-di-BOC prop-2-en-l-yl 7-(2- ⁇ [(methyloxy)carbonyl]amino ⁇ -lH- benzimidazol-5-yl)-2,3-dihydro-l,4-benzoxazepine-4(5H)-carboxylate (3.3 g, 5.3 mmol) was taken into T ⁇ F (30 mL) followed by addition of sodium triacetoxyborohydride (5.6 g, 26.5 mmol) and palladium tetrakis-triphenylphosphine (612 mg) and the mixture was stirred for 30 minutes at room temperature.
  • sodium triacetoxyborohydride 5.6 g, 26.5 mmol
  • palladium tetrakis-triphenylphosphine 612 mg
  • STEP 9 A mixture of NN'-di-BOC methyl ⁇ 5-[4-(chlorocarbonyl)-2,3,4,5- tetrahydro-l,4-benzoxazepin-7-yl]-lH-benzimidazol-2-yl ⁇ carbamate (140 mg, 0.23 mmol), 3-methyl-8-azabicyclo[3.2.1]octan-3-(endo)-ol hydrochloride (reagent preparation 9) (40 mg, 0.23mmol), and diisoproylethylamine (101 mg, 0.78 mmol), in dichloromethane (2 mL) was stirred at room temperature for one hour.
  • STEP 10 A solution of NN'-di-BOC methyl (6- ⁇ 4-[(3-(en ⁇ fo)-hydroxy-3-methyl-8- azabicyclo[3.2.1 ]oct-8-yl)carbonyl]-2,3 ,4,5-tetrahydro- 1 ,4-benzoxazepin-7-yl ⁇ -IH- benzimidazol-2-yl)carbamate, (20 mg, 0.028 mmol) in trifluoroacetic acid (0.2 mL) and dichloroethane (1.8 mL) was stirred at room temperature for one hour.
  • STEP 1 7-Bromo-2,3-dihydro-l,4-benzoxazepine-4(5H)-carbonyl chloride (373 mg, 1.28 mmol) (example 4, step 2) was taken into dichloromethane (10 mL) followed by addition of DIPEA (0.66 mL, 3.84 mmol) and 4-(difluoromethyl)piperidine trifluoroacetate salt (reagent preparation 4) (320 mg, 1.28 mmol) and the mixture was allowed to stir at room temperature for 1.5 h. The mixture was then quenched by addition of 5% aqueous citric acid and the aqueous portion extracted twice with dichloromethane.
  • DIPEA 0.66 mL, 3.84 mmol
  • reagent preparation 4 4-(difluoromethyl)piperidine trifluoroacetate salt
  • STEP 2 7-Bromo-4- ⁇ [4-(difluoromethyl)piperidin-l-yl]carbonyl ⁇ -2,3,4,5- tetrahydro-l,4-benzoxazepine (356 mg, 0.91 mmol) and 2-mtro-4-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)aniline (240 mg, 0.91 mmol) were taken into dioxane (10 mL) and water (1 mL) followed by addition of dichloro[ 1,1 -bis(diphenylphosphino]ferrocenepalladium (II) dichloromethane adduct (74 mg, 0.09 mmol) and cesium carbonate (1.5 g, 4.55 mmol).
  • the mixture was then heated to 9O 0 C over 12h then cooled to room temperature and diluted with ethyl acetate.
  • the aqueous phase was extracted once with with ethyl acetate and the combined organic layers were washed with brine, dried over sodium sulfate then filtered and concentrated.
  • STEP 3 4-(4- ⁇ [4-(Difluoromethyl)piperidin-l-yl]carbonyl ⁇ -2,3,4,5-tetrahydro-l,4- benzoxazepin-7-yl)-2-nitroaniline (350 mg, 0.78 mmol) and 10% palladium on carbon (400 mg) were suspended in methanol (10 mL) and the mixture was hydrogenated at 35 psi using a Parr apparatus for 3.5 h.
  • STEP 4 4-(4- ⁇ [4-(Difluoromethyl)piperidin-l-yl]carbonyl ⁇ -2,3,4,5-tetrahydro-l,4- benzoxazepin-7-yl)benzene- 1 ,2-diamine (140 mg, 0.34 mmol) was taken into acetic acid (10 mL) followed by addition of l,3-bis(methoxycarbonyl)-2-methyl-2-thiopseudourea (90 mg, 0.44 mmol) and the mixture was heated at 80 0 C for 2 h. On cooling to room temperature the mixture was diluted with ethyl acetate and partitioned with saturated aqueous sodium bicarbonate.
  • STEP 2 2,3,4,5-Tetrahydro-l,4-benzoxazepin-7-ylboronic acid hydrochloride salt (1.0 g, 5.18 mmol) was taken into 1 :1 aqueous THF (30 mL) followed by addition of sodium bicarbonate (2.17 g, 25.9 mmol) then 2-(4-fluorophenyl)-4-oxopiperidine-l-carbonyl chloride (reagent preparation 19) (1.32 g, 5.18 mmol) as a THF solution and the mixture was stirred at room temperature 12 h. The mixture was then partitioned with ethyl acetate and water.
  • STEP 3 (4- ⁇ [2-(3-fluorophenyl)-4-oxopiperidin-l-yl]carbonyl ⁇ -2,3,4,5-tetrahydro- 1 ,4-benzoxazepin-7-yl)boronic acid (100 mg, 0.24 mmol) and isobutyl 5-(4-bromophenyl)-lH- imidazole-1-carboxylate (78 mg, 0.24 mmol) were taken into dioxane (9 mL) and water (1 mL) followed by addition of DIPEA (0.21 mL, 1.2 mmol) and dichloro[l,l-bis(diphenyl- phosphinojferrocenepalladium (II) dichloromethane adduct (20 mg) then the mixture was warmed to 60 0 C for 12 h.
  • DIPEA 0.21 mL, 1.2 mmol
  • STEP 1 7-(4,4,5,5-Tetramethyl-l,3,2-dioxaboralan-2-yl)-2,3-dihydro-l,4- benzoxazepine-4(5H)-carbonyl chloride (example 6, step 3) (3.0 g) was taken into dichloromethane (50 mL) followed by addition of DIPEA (40 mmol, 7 mL) then 4-trifiuoromethylpiperidine hydrochloride salt (1.55 g, 8.2 mmol). The mixture was allowed to stir 30 minutes at room temperature then concentrated. The residue was partitioned with ethyl acetate and 5% aqueous citric acid.
  • STEP 2 To a solution of 7-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-4- ⁇ [4- (trifluoromethyl)piperidin-l-yl]carbonyl ⁇ -2,3,4,5-tetrahydro-l,4-benzoxazepine (1.27 g, 2.81 mmol) and methyl 4-bromobenzoate (0.604 g, 2.81 mmol) in dimethoxyethane (10 mL) and water (1.25 mL) was added potassium carbonate (1.55 g, 11.2 mmol).
  • STEP 3 To a solution of methyl 4-(4- ⁇ [trifluoromethyl)piperidin-l-yl]carbonyl ⁇ - 2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)benzoate (0.525 g, 1.14 mmol) in methanol (15 mL) was added a solution of 1 M sodium hydroxide (3 mL). The resulting solution was heated at 60 0 C for 22 hours. After cooling to room temperature some of the methanol was evaporated, water was added to the resulting mixture, and the pH adjusted to 3 with IN aqueous hydrochloric acid.
  • STEP 4 A solution of ethyl 4-(4- ⁇ [4-(trifluoromethy)piperidin-l-yl]carbonyl ⁇ - 2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)benzoic acid (0.150 g, 0.334 mmol), 4-fluorobenzene- 1,2-diamine (0.422 g, 0.334 mmol), HATU (0.133 g, 0.351 mmol), and diisopropylethylamine (0.130 g, 1.00 mmol) in dimethylformamide (4 mL) was stirred at room temperature for 18 hours.
  • reaction mixture was diluted with ethyl acetate (100 mL), washed with saturated sodium bicarbonate (100 mL) and brine (50 mL), dried over sodium sulfate. Filtration and concentration afforded a crude brown oil that was purified by silica gel chromatography (99: 1 dichloromethane/methanol) to provide N-(2-amino-4-fluorophenyl)-4-(4- ⁇ [4-
  • Compounds of this invention have been tested using the assay described in Biological Example 1 and have been determined to be mTORcl inhibitors. As such compounds of Formula I are useful for treating diseases, particularly cancer in which mTOR activity contributes to the pathology and/or symptomatology of the disease.
  • Suitable in vitro assays for measuring mTORcl and mT0Rc2 activity and the inhibition thereof by compounds, as well as cell-based assays for measurement of in vitro efficacy in treatment of cancer are known in the art and examples are described below.
  • Suitable in vivo models for cancer are known to those of ordinary skill in the art and examples are disclosed in below. Following the examples disclosed herein, as well as that disclosed in the art, a person of ordinary skill in the art can determine the mTOR- inhibitory activity of a compound of this invention.
  • the assay conditions were as follows; 0.2 nM mTORCl, 10 ⁇ M ATP and 50 nM NHis-tagged 4E-BP1 in 20 mM Hepes, pH 7.2, 1 mM DTT, 50 mM NaCl, 10 mM MnCl 2 , 0.02 mg/mL BSA, 0.01% CHAPS, 50 mM ⁇ -glycerophosphate. Following an incubation of 120 minutes at ambient temperature, 20 ⁇ L of the reaction volume was transferred to a Ni-Chelate-coated 384-well plate. The binding step of the 4E-BP1 protein proceeded for 60 minutes, followed by washing 4 times each with 50 ⁇ L of Tris-buffered saline solution (TBS).
  • TBS Tris-buffered saline solution
  • Anti-phospho-4E-BP 1 rabbit-IgG (20 ⁇ L, 1 :5000) in 5% BSA-TBST (0.2% Tween-20 in TBS) was added and further incubated for 60 minutes. Incubation with a secondary HRP -tagged anti-IgG was similarly performed after washing off the primary antibody (4 washes of 50 ⁇ L). Following the final wash step with TBST, 20 ⁇ L of SuperSignal ELISA Femto (Pierce Biotechnology) was added and the luminescence measured using an EnVision plate reader.
  • the Compound of the Invention has an mTOR-inhibitory activity of about 0.4 ⁇ M or less. In another embodiment, the Compound of the Invention has an mTOR-inhibitory activity of about 0.3 ⁇ M or less. In another embodiment, the Compound of the Invention has an mTOR-inhibitory activity of about 0.2 ⁇ M or less. In another embodiment, the Compound of the Invention has an mTOR- inhibitory activity of about 0.1 ⁇ M or less. In another embodiment, the Compound of the Invention has an mTOR-inhibitory activity of about 0.05 ⁇ M or less. In another embodiment, the Compound of the Invention has an mTOR-inhibitory activity of about 0.02 ⁇ M or less. In another embodiment, the Compound of the Invention has an mTOR-inhibitory activity of about 0.01 ⁇ M or less.
  • Compounds 15, 46, 47, and 53-86 have an IC 50 in this assay of less than or equal to 20 nM.
  • Compounds 13, 14, and 19-45 have an IC50 in this assay of greater than 20 nM but less than or equal to 100 nM.
  • Compounds 1-12 and 16-18 have an IC50 in this assay of greater than 100 nM but less than or equal to 352 nM.
  • HeLa (ATCC) cells are grown in suspension culture and lysed in ice-cold lysis buffer containing 40 mM HEPES pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM sodium pyrophosphate, 10 mM ⁇ -glycerophosphate, 10 mM NaF, 10 mM NaN 3 , one tablet of protease inhibitors (Complete-Mini, EDTA-free, Roche), 0.3% cholamidopropyl- dimethylammoniopropanesulfonate (CHAPS), 1 mM AEBSF, 0.5 mM benzamidine HCl, 20 ⁇ g/mL heparin, and 1.5 mM Na 3 VO 4 .
  • HEPES pH 7.5 120 mM NaCl
  • 1 mM EDTA 10 mM sodium pyrophosphate
  • 10 mM ⁇ -glycerophosphate 10 mM NaF
  • the mTORC2 complex is immunoprecipitated with anti-RICTOR antibody for 2 h.
  • the immune complexes are immobilized on Protein A sepharose (GE Healthcare, 17-5280-01), washed sequentially 3 times with wash buffer (40 mM HEPES pH 7.5, 120 mM NaCl, 10 mM ⁇ -glycerophosphate, 0.3% CHAPS, 1 mM AEBSF, 20 ⁇ g/mL heparin, 1.5 mM Na 3 VOz I , and Complete-Mini, EDTA-free) and resuspended in kinase buffer (40 mM HEPES, pH 7.5, 120 mM NaCl, 0.3% CHAPS, 20 ⁇ g/mL heparin, 4 mM MgCl 2 , 4 mM MnCl 2 , 10% Glycerol, and 10 mM DTT).
  • wash buffer 40 mM HEPES pH 7.5, 120 mM NaCl,
  • the immune complexes (equivalent to I xIO 7 cells) are pre-incubated at 37 0 C with a test compound or 0.6% DMSO for 5 min, and then subjected to a kinase reaction for 8 min in a final volume of 33 ⁇ L (including 5 ⁇ L bed volume) containing kinase buffer, 50 ⁇ M ATP, and 0.75 ⁇ g full length dephosphorylated AKTl .
  • Kinase reactions are terminated by addition of 11 ⁇ L 4x SDS sample buffer containing 20% ⁇ -mercaptoethanol and resolved in a 10% Tris Glycine gels. The gels are transferred onto PVDF membrane at 50 V for 20 h at 4 0 C.
  • the membranes are blocked in 5% non-fat milk in TBST for 1 h and incubated overnight at 4 0 C with 1/1000 dilution of rabbit anti-pAKT (S473) (Cell Signaling Technology, 4060) in 3% BSA/TBST.
  • the membranes are washed 3 times in TBST and incubated for 1 h with a 1/10000 dilution of secondary goat anti-rabbit HRP antibody (Cell Signaling Technology, 2125) in 5% non-fat milk/TBST.
  • the signal is detected using Amersham ECL-plus.
  • the scanned data are analyzed using ImageQuant software.
  • IC50 for the test compound is determined relative to DMSO treated sample using XLfit4 software.
  • MCF-7 cells (ATCC) or PC-3 cells were seeded at 24000 cells per well in 96-well plates (Corning, 3904) in DMEM (Cellgro) containing 10% FBS (Cellgro), 1% NEAA (Cellgro) and 1% penicillin-streptomycin (Cellgro). Cells were incubated at 37°C, 5% CO2 for 48 h, and the growth medium was replaced with serum-free DMEM or in medium containing 0.4% BSA. Serial dilutions of the test compound in 0.3% DMSO (vehicle) were added to the cells and incubated for 3h.
  • Anti-pS6 (S240/244) antibody (Cell Signaling Technology, 2215) and anti-total-S6 antibody (R&D systems, MAB5436) were diluted 1 :400 in Odyssey blocking buffer, and 50 ⁇ L of the antibody solution containing both antibodies was added to one plate to detect pS6 and total S6. After incubation overnight at 4°C, plates were washed 4 times with 200 ⁇ L TBS containing 0.1% Tween20 (Bio-Rad, catalog # 170-6351) (TBST).
  • Goat anti-rabbit and Goat anti-mouse secondary antibody (Li-Cor Biosciences, catalog # 926-32221 and 926- 32210) conjugated to IRDye were diluted 1 :400 in Odyssey blocking buffer containing 0.1% Tween20. 50 ⁇ L of antibody solution containing both antibodies was added to each well and incubated for Ih at RT. Plates were washed 3 times with 200 ⁇ L TBST and 2 times with 200 ⁇ L TBS. Fluorescence was read on an Odyssey plate reader. IC50 values were determined based on the ratio of pS6 to total S6 signal for compound treated wells, normalized to the DMSO-treated control wells.
  • the Compound of the Invention tested in this assay in MCF-7 cells had an inhibitory activity of 0.5 ⁇ M or less. In another embodiment, the Compound of the Invention tested in this assay in MCF-7 cells had an inhibitory activity of 0.25 ⁇ M or less, the Compound of the Invention tested in this assay in MCF-7 cells had an inhibitory activity of 0.2 ⁇ M or less, the Compound of the Invention tested in this assay in MCF-7 cells had an inhibitory activity of 0.1 ⁇ M or less.
  • the Compound of the Invention tested in this assay in PC-3 cells had an inhibitory activity of 2.0 ⁇ M or less. In another embodiment, the Compound of the Invention tested in this assay in PC-3 cells had an inhibitory activity of 1.0 ⁇ M or less. In another embodiment, the Compound of the Invention tested in this assay in PC-3 cells had an inhibitory activity of 0.5 ⁇ M or less. In another embodiment, the Compound of the Invention tested in this assay in PC-3 cells had an inhibitory activity of 0.25 ⁇ M or less. In another embodiment, the Compound of the Invention tested in this assay in PC-3 cells had an inhibitory activity of 0.2 ⁇ M or less. In another embodiment, the Compound of the Invention tested in this assay in PC-3 cells had an inhibitory activity of 0.1 ⁇ M or less.
  • Biological Example 5-11 Pharmacodynamic xenograft tumor models Female and male athymic nude mice (NCr) 5-8 weeks of age and weighing approximately 20-25 g are used in the following models. Prior to initiation of a study, the animals are allowed to acclimate for a minimum of 48 h. During these studies, animals are provided food and water ad libitum and housed in a room conditioned at 70-75 0 F and 60% relative humidity. A 12 h light and 12 h dark cycle is maintained with automatic timers. All animals are examined daily for compound-induced or tumor-related deaths. MCF-7 Breast adenocarcinoma model
  • MCF7 human mammary adenocarcinoma cells are cultured in vitro in DMEM (Cellgro) supplemented with 10% Fetal Bovine Serum (Cellgro), Penicillin-Streptomycin and non-essential amino acids at 37 0 C in a humidified 5% CO 2 atmosphere.
  • DMEM Cellgro
  • Fetal Bovine Serum Cellgro
  • Penicillin-Streptomycin Cellgro
  • non-essential amino acids 37 0 C in a humidified 5% CO 2 atmosphere.
  • cells are harvested by trypsinization, and 5 x 10 6 cells in 100 ⁇ L of a solution made of 50% cold Hanks balanced salt solution with 50% growth factor reduced matrigel (Becton Dickinson) implanted subcutaneously into the hindflank of female nude mice.
  • a transponder is implanted into each mouse for identification and data tracking, and animals are monitored daily for clinical symptoms and survival.
  • Tumors are established in female athymic nude mice and staged when the average tumor weight reached 100-200 mg.
  • a Compound of the Invention is orally administered as a solution/fine suspension in water (with 1 : 1 molar ratio of 1 N HCL) once-daily (qd) or twice-daily (bid) at 10, 25, 50 and 100 mg/kg for 14 days. During the dosing period of 14-19 days, tumor weights are determined twice-weekly and body weights are recorded daily.
  • Colo-205 human colorectal carcinoma cells are cultured in vitro in DMEM
  • Tumors are established in female athymic nude mice and staged when the average tumor weight reached 100-200 mg.
  • a Compound of the Invention is orally administered as a solution/fine suspension in water (with 1 : 1 molar ratio of 1 N HCL) once-daily (qd) or twice-daily (bid) at 10, 25, 50 and 100 mg/kg for 14 days. During the dosing period of 14 days, tumor weights are determined twice-weekly and body weights are recorded daily.
  • PC-3 prostate adenocarcinoma model [00272] PC-3 human prostate adenocarcinoma cells are cultured in vitro in DMEM (Mediatech) supplemented with 20% Fetal Bovine Serum (Hyclone), Penicillin-Streptomycin and non-essential amino acids at 37 0 C in a humidified 5% CO 2 atmosphere. On day 0, cells are harvested by trypsinization and 3xlO 6 cells (passage 10-14, >95% viability) in 0.1 mL of ice-cold Hank's balanced salt solution are implanted subcutaneously into the hindflank of 5-8 week old male nude mice. A transponder is implanted in each mouse for identification, and animals are monitored daily for clinical symptoms and survival.
  • Tumors are established in male athymic nude mice and staged when the average tumor weight reached 100-200 mg.
  • a Compound of the Invention is orally administered as a solution/fine suspension in water (with 1 : 1 molar ratio of 1 N HCl) once-daily (qd) or twice-daily (bid) at 10, 25, 50, or 100-mg/kg for 19 days.
  • qd once-daily
  • bid twice-daily
  • U-87 MG human glioblastoma cells are cultured in vitro in DMEM (Mediatech) supplemented with 10% Fetal Bovine Serum (Hyclone), Penicillin-Streptomycin and nonessential amino acids at 37 0 C in a humidified 5% CO 2 atmosphere.
  • DMEM Mediatech
  • Fetal Bovine Serum Hyclone
  • Penicillin-Streptomycin nonessential amino acids at 37 0 C in a humidified 5% CO 2 atmosphere.
  • cells are harvested by trypsinization and 2xlO 6 cells (passage 5, 96% viability) in 0.1 mL of ice-cold Hank's balanced salt solution are implanted intradermally into the hindflank of 5-8 week old female nude mice.
  • a transponder is implanted in each mouse for identification, and animals are monitored daily for clinical symptoms and survival. Body weights are recorded daily.
  • A549 human lung carcinoma model [00275] A549 human lung carcinoma cells are cultured in vitro in DMEM (Mediatech) supplemented with 10% Fetal Bovine Serum (Hyclone), Penicillin-Streptomycin and nonessential amino acids at 37 0 C in a humidified 5% CO 2 atmosphere. On day 0, cells are harvested by trypsinization and 1OxIO 6 cells (passage 12, 99% viability) in 0.1 mL of ice-cold Hank's balanced salt solution are implanted intradermally into the hindflank of 5-8 week old female nude mice. A transponder is implanted in each mouse for identification, and animals are monitored daily for clinical symptoms and survival. Body weights are recorded daily.
  • A2058 human melanoma cells are cultured in vitro in DMEM (Mediatech) supplemented with 10% Fetal Bovine Serum (Hyclone), Penicillin-Streptomycin and nonessential amino acids at 37 0 C in a humidified, 5% CO2 atmosphere.
  • DMEM Mediatech
  • Fetal Bovine Serum Hyclone
  • Penicillin-Streptomycin nonessential amino acids
  • cells are harvested by trypsinization and 3xlO 6 cells (passage 3, 95% viability) in 0.1 mL ice-cold Hank's balanced salt solution are implanted intradermally in the hind-flank of 5-8 week old female athymic nude mice.
  • a transponder is implanted in each mouse for identification, and animals are monitored daily for clinical symptoms and survival. Body weights are recorded daily.
  • WM-266-4 human melanoma cells are cultured in vitro in DMEM (Mediatech) supplemented with 10% Fetal Bovine Serum (Hyclone), Penicillin-Streptomycin and nonessential amino acids at 37 0 C in a humidified, 5% CO 2 atmosphere.
  • DMEM Mediatech
  • Fetal Bovine Serum Hyclone
  • Penicillin-Streptomycin nonessential amino acids
  • cells are harvested by trypsinization and 3xlO 6 cells (passage 5, 99% viability) in 0.1 mL ice-cold Hank's balanced salt solution are implanted intradermally in the hind-flank of 5-8 week old female athymic nude mice.
  • a transponder is implanted in each mouse for identification, and animals are monitored daily for clinical symptoms and survival. Body weights are recorded daily.
  • TGI Percent inhibition of tumor growth
  • tumor size is calculated individually for each tumor to obtain a mean ⁇ SEM value for each experimental group.
  • Statistical significance is determined using the 2-tailed Student's t-test (significance defined as P ⁇ 0.05).

Abstract

The invention is directed to inhibitors of mTOR and pharmaceutically acceptable salts or solvates thereof, as well as methods of using them. The inhibitors are generally of structural formula wherein the combination of R1 and R2 are as defined herein, and pharmaceutically acceptable salts thereof.

Description

BENZOXAZEPINES AS INHIBITORS OF mTOR AND METHODS OF THEIR USE
AND MANUFACTURE
BACKGROUND OF THE INVENTION
Field of the Invention [0001] This invention relates to the field of protein kinases and inhibitors thereof. In particular, the invention relates to inhibitors of mammalian target of rapamycin (mTOR) signaling pathways, and methods of their use. Background of the Invention [0002] The mammalian target of rapamycin, mTOR, is a protein kinase that integrates both extracellular and intracellular signals of cellular growth, proliferation, and survival. Extracellular mitogenic growth factor signaling from cell surface receptors and intracellular pathways that convey hypoxic stress, energy and nutrient status all converge at mTOR. mTOR exists in two distinct complexes: mTOR complex 1 (mTORCl) and mTOR complex 2 (mT0RC2). mTORCl is a key mediator of transcription and cell growth (via its substrates p70S6 kinase and 4E-BP1) and promotes cell survival via the serum and glucocorticoid-activated kinase SGK, whereas mTORC2 promotes activation of the pro-survival kinase AKT. Given its central role in cellular growth, proliferation and survival, it is perhaps not surprising that mTOR signaling is frequently dysregulated in cancer and other diseases (Bjornsti and Houghton Rev Cancer 2004, 4(5), 335- 48; Houghton and Huang Microbiol Immunol 2004, 279, 339-59; Inoki, Corradetti et al. Nat Genet 2005, 37(1), 19-24).
[0003] mTOR is a member of the PIKK (PI3K-related Kinase) family of atypical kinases which includes ATM, ATR, and DNAPK, and its catalytic domain is homologous to that of PI3K. Dyregulation of PI3K signaling is a common function of tumor cells. In general, mTOR inhibition may be considered as a strategy in many of the tumor types in which PI3K signaling is implicated such as those discussed below.
[0004] Inhibitors of mTOR may be useful in treating a number of cancers, including the following: breast cancer (Nagata, Lan et al., Cancer Cell 2004, 6(2), 117-27; Pandolfi N EnglJ Med 2004, 351(22), 2337-8; Nahta, Yu et al. Nat Clin Pract Oncol 2006, 3(5), 269-280); antle cell lymphoma (MCL) (Dal Col, Zancai et al. Blood 2008, 111(10), 5142-51); renal cell carcinoma (Thomas, Tran et al. Nat Med 2006, 12(1), 122-7; Atkins, Hidalgo et al. J Clin Oncol 2004, 22(5), 909-18; Motzer, Hudes et al. J Clin Oncol 2007, 25(25), 3958-64); acute myelogenous leukemia (AML) (Sujobert, Bardet et al.Blood 2005, 106(3), 1063-6; Billottet, Grandage et al. Oncogene 2006, 25(50), 6648-6659; Tamburini, EHe et al. Blood 2007, 110(3), 1025-8); chronic myelogenous leukemia (CML) (Skorski, Bellacosa et al. Embo J 1997, 16(20), 6151-61; Bai, Ouyang et al. Blood 2000, 96(13), 4319-27; Hickey and Cotter Biol Chem 2006, 281(5), 2441-50); diffuse large B cell lymphoma (DLBCL) (Uddin, Hussain et al. Blood 2006, 108(13), 4178-86); several subtypes of sarcoma (Hernando, Charytonowicz et al. Nat Med 2007, 13(6), 748-53; Wan and Helman Oncologist 2007, 12(8), 1007-18); rhabdomyosarcoma (Cao, Yu et al. Cancer Res 2008, 68(19), 8039-8048; Wan, Shen et al. Neoplasia 2006, 8(5), 394-401); ovarian cancer (Shayesteh, Lu et al. Nat Genet, 1999, 21(1), 99-102; (Lee, Choi et al. Gynecol Oncol 2005, 97(1) 26-34); endometrial tumors (Obata, Morland et al. Cancer Res 1998, 58(10), 2095-7; Lu, Wu et al. Clin Cancer Res 2008, 14(9), 2543-50); non small cell lung carcinoma (NSCLC) (Tang, He et al. Lung Cancer 2006, 51(2), 181-91; Marsit, Zheng et al. Hum Pathol 2005, 36(7), 768-76); small cell, squamous, large cell and adenocarcinoma (Massion, Taflan et al. Am JRespir Crit Care Med 2004, 170(10), 1088-94); lung tumors in general (Kokubo,
Gemma et al. Br J Cancer 2005, 92(9), 1711-9; Pao, Wang et al. Pub Library of Science Med 2005, 2(1), el 7); colorectal tumors (Velho, Oliveira et al. Eur J Cancer 2005, 41(11), 1649-54; Foukas, Claret et al. Nature, 2006, 441(7091), 366-370), particularly those that display microsatellite instability (Goel, Arnold et al. Cancer Res 2004, 64(9), 3014-21; Nassif, Lobo et al. Oncogene 2004, 23(2), 617-28), KRAS-mutated colorectal tumors (Bos Cancer Res 1989. 49(17), 4682-9; Fearon ^rø N Y Acad Sd 1995, 768, 101-10); gastric carcinomas (Byun, Cho et al. Int J Cancer 2003, 104(3), 318-27); hepatocellular tumors (Lee, Soung et al. Oncogene 2005, 24(8), 1477-80); liver tumors (Hu, Huang et al. Cancer 2003, 97(8), 1929-40; Wan, Jiang et al. Cancer Res Clin Oncol 2003, 129(2), 100-6); primary melanomas and associated increased tumor thickness (Guldberg, thor Straten et al. Cancer Res 1997, 57(17), 3660-3; Tsao, Zhang et al. Cancer Res 2000, 60(7), 1800-4; Whiteman, Zhou et al. Int J Cancer2002, 99(1), 63-7; Goel, Lazar et al. J Invest Dermatol 126(1), 2006, 154-60); pancreatic tumors (Asano, Yao et al. Oncogene 2004, 23(53), 8571-80); prostate carcinoma (Cairns, Okami et al. Cancer Res 1997, 57(22), 4997-5000; Gray, Stewart et al. Br J Cancer 1998, 78(10), 1296-300; Wang, Parsons et al. Clin Cancer Res 1998, 4(3), 811-5; Whang, Wu et al. Proc Natl Acad Sd USA 1998, 95(9), 5246-50; Majumder and Sellers Oncogene 2005, 24(50) 7465-74; Wang, Garcia et al. Proc Natl Acad Sci USA 2006, 103(5), 1480-5; (Lu, Ren et al. Int J Oncol 2006, 28(1), 245-51; Mulholland, Dedhar et al. Oncogene 25(3), 2006, 329-37; Xin, Teitell et al. Proc Natl Acad Sci USA 72006, 03(20), 7789-94; Mikhailova, Wang et al. Adv Exp Med Biol 2008, 617, 397-405; Wang, Mikhailova et al. Oncogene 2008, 27(56), 7106-7117); thyroid carcinoma, particularly in the anaplastic subtype (Garcia-Rostan, Costa et al. Cancer Res 2005, 65(22), 10199-207); follicular thyroid carcinoma (Wu, Mambo et al. J Clin Endocrinol Metab 2005, 90(8), 4688-93); anaplastic large cell lymphoma (ALCL); hamaratomas, angiomyelolipomas, TSC-associated and sporadic lymphangioleiomyomatosis: Cowden's disease (multiple hamaratoma syndrome) (Bissler, McCormack et al. N EnglJ Med 2008, 358(2), 140-151); sclerosing hemangioma (Randa M. S. Amin Pathology International 2008, 58(1), 38-44); Peutz-Jeghers syndrome (PJS); head and neck cancer (Gupta, McKenna et al. Clin Cancer Res 2002, 8(3), 885-892); neurofibromatosis (F erner Eur J Hum Genet 2006, 15(2), 131-138; Sabatini Nat Rev Cancer 2006, 6(9), 729-734; Johannessen, Johnson et al. Current Biology 2008, 18(1), 56-62); macular degeneration; macular edema; myeloid leukemia; systemic lupus; and autoimmune lymphoproliferative syndrome (ALPS).
SUMMARY OF THE INVENTION
[0005] The following only summarizes certain aspects of the invention and is not intended to be limiting in nature. These aspects and other aspects and embodiments are described more fully below. All references cited in this specification are hereby incorporated by reference in their entirety. In the event of a discrepancy between the express disclosure of this specification and the references incorporated by reference, the express disclosure of this specification shall control.
[0006] In view of the important role of mTOR in biological processes and disease states, the inventors realized that inhibitors of this protein kinase, including dual inhibitors mTORCl are desirable. Compounds of the Invention are potent and specific inhibitors of mTORCl .
[0007] A first aspect of the invention provides a compound of Table 1, optionally as a pharmaceutically acceptable salt thereof.
[0008] In a second aspect, the invention is directed to a pharmaceutical composition which comprises 1) a compound of Table 1 or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt or solvate thereof and 2) a pharmaceutically acceptable carrier, excipient, or diluent.
[0009] In a third aspect of the invention is a method of inhibiting the in vivo activity of mTOR, the method comprising administering to a patient an effective mTOR-inhibiting amount of a compound of Table 1 or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt or solvate thereof or pharmaceutical composition thereof.
[0010] In a fourth aspect, the Invention provides a method for treating a disease, disorder, or syndrome which method comprises administering to a patient a therapeutically effective amount of a compound of Table 1 or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising a therapeutically effective amount of a compound of Table 1 or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, excipient, or diluent. DETAILED DESCRIPTION OF THE INVENTION Abbreviations and Definitions The following abbreviations and terms have the indicated meanings throughout: Abbreviation Meaning
Figure imgf000006_0001
Figure imgf000007_0001
[0012] "Administration" and variants thereof (e.g., "administering" a compound) in reference to a compound of the invention means introducing the compound or a prodrug of the compound into the system of the animal in need of treatment. When a compound of the invention or prodrug thereof is provided in combination with one or more other active agents (e.g., surgery, radiation, and chemotherapy, etc.), "administration" and its variants are each understood to include concurrent and sequential introduction of the compound or prodrug thereof and other agents.
[0013] "Yield" for each of the reactions described herein is expressed as a percentage of the theoretical yield.
[0014] "Metabolite" refers to the break-down or end product of a compound or its salt produced by metabolism or biotransformation in the animal or human body; for example, biotransformation to a more polar molecule such as by oxidation, reduction, or hydrolysis, or to a conjugate (see Goodman and Gilman, "The Pharmacological Basis of Therapeutics" 8.sup.th Ed., Pergamon Press, Gilman et al. (eds), 1990 for a discussion of biotransformation). As used herein, the metabolite of a compound of the invention or its salt may be the biologically active form of the compound in the body. In one example, a prodrug may be used such that the biologically active form, a metabolite, is released in vivo. In another example, a biologically active metabolite is discovered serendipitously, that is, no prodrug design per se was undertaken. An assay for activity of a metabolite of a compound of the present invention is known to one of skill in the art in light of the present disclosure. [0015] "Patient" for the purposes of the present invention includes humans and other animals, particularly mammals, and other organisms. Thus the methods are applicable to both human therapy and veterinary applications. In a specific embodiment the patient is a mammal, and in a more specific embodiment the patient is human.
[0016] A "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. It is understood that the pharmaceutically acceptable salts are non-toxic. Additional information on suitable pharmaceutically acceptable salts can be found in Remington 's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, which is incorporated herein by reference or S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977;66: 1-19 both of which are incorporated herein by reference. [0017] Examples of pharmaceutically acceptable acid addition salts include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, 3-(4-hydroxybenzoyl)benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4'-methylenebis-(3- hydroxy-2-ene- 1 -carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, p-toluenesulfonic acid, and salicylic acid and the like. [0018] Examples of a pharmaceutically acceptable base addition salts include those formed when an acidic proton present in the parent compound is replaced by a metal ion, such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Specific salts are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins. Examples of organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, tromethamine, N-methylglucamine, polyamine resins, and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine."Platin(s)," and "platin-containing agent(s)" include, for example, cisplatin, carboplatin, and oxaliplatin.
[0019] "Prodrug" refers to compounds that are transformed (typically rapidly) in vivo to yield the parent compound of the above formulae, for example, by hydrolysis in blood. Common examples include, but are not limited to, ester and amide forms of a compound having an active form bearing a carboxylic acid moiety. Examples of pharmaceutically acceptable esters of the compounds of this invention include, but are not limited to, alkyl esters (for example with between about one and about six carbons) the alkyl group is a straight or branched chain. Acceptable esters also include cycloalkyl esters and arylalkyl esters such as, but not limited to benzyl. Examples of pharmaceutically acceptable amides of the compounds of this invention include, but are not limited to, primary amides, and secondary and tertiary alkyl amides (for example with between about one and about six carbons). Amides and esters of the compounds of the present invention may be prepared according to conventional methods. A thorough discussion of prodrugs is provided in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," VoI 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference for all purposes.
[0020] "Therapeutically effective amount" is an amount of a compound of the invention, that when administered to a patient, ameliorates a symptom of the disease. The amount of a compound of the invention which constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the age of the patient to be treated, and the like. The therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their knowledge and to this disclosure. [0021] "Treating" or "treatment" of a disease, disorder, or syndrome, as used herein, includes (i) preventing the disease, disorder, or syndrome from occurring in a human, i.e. causing the clinical symptoms of the disease, disorder, or syndrome not to develop in an animal that may be exposed to or predisposed to the disease, disorder, or syndrome but does not yet experience or display symptoms of the disease, disorder, or syndrome; (ii) inhibiting the disease, disorder, or syndrome, i.e., arresting its development; and (iii) relieving the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by one of ordinary skill in the art.
Embodiments of the Invention [0022] Another embodiment provides a pharmaceutical composition which comprises 1) a compound, as a single stereoisomer or mixture of isomers thereof, selected from Table 1 , optionally as a pharmaceutically acceptable salt thereof, and 2) a pharmaceutically acceptable carrier, excipient, and/or diluent thereof.
[0023] Another embodiment is a method of treating disease, disorder, or syndrome where the disease is associated with uncontrolled, abnormal, and/or unwanted cellular activities effected directly or indirectly by mTOR which method comprises administering to a human in need thereof a therapeutically effective amount of a Compound selected from Table 1 , optionally as a pharmaceutically acceptable salt or pharmaceutical composition thereof. In another embodiment the disease is cancer. [0024] Embodiment (A): Another embodiment is directed to a method of treating a disease, disorder, or syndrome which method comprises administering to a patient a therapeutically effective amount of a Compound selected from Table 1, optionally as a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a therapeutically effective amount of a Compound selected from Table 1 , and a pharmaceutically acceptable carrier, excipient, or diluent. In another embodiment the disease is cancer. [0025] In another embodiment of any of the embodiments of Embodiment (A), the cancer is breast cancer, mantle cell lymphoma, renal cell carcinoma, acute myelogenous leukemia, chronic myelogenous leukemia, NPM/ALK-transformed anaplastic large cell lymphoma, diffuse large B cell lymphoma, rhabdomyosarcoma, ovarian cancer, endometrial cancer, cervical cancer, non small cell lung carcinoma, small cell lung carcinoma, adenocarcinoma, colon cancer, rectal cancer, gastric carcinoma, hepatocellular carcinoma, melanoma, pancreatic cancer, prostate carcinoma, thyroid carcinoma, anaplastic large cell lymphoma, hemangioma, glioblastoma, or head and neck cancer.
Representative Compounds [0026] Compoudns of the Invention are depicted below. In each instance the embodiment includes both the recited compounds, as well as a single stereoisomer or mixture of stereoisomers thereof, as well as a pharmaceutically acceptable salt thereof. Compounds of the invention are named according to systematic application of the nomenclature rules agreed upon by the International Union of Pure and Applied Chemistry (IUPAC), International Union of Biochemistry and Molecular Biology (IUBMB), and the Chemical Abstracts Service (CAS) Specifically, names in Table 1 were generated using ACD/Labs naming software 8.00 release, product version 8.08 or later. [0027] In one embodiment, the invention comprises compounds of structural formula
Figure imgf000011_0001
wherein the combination of R1 and R2 are as defined in one of the compounds in Table 1, below, and pharmaceutically acceptable salts thereof.
[0028] In another embodiment, the invention comprises a compound in Table 1, and pharmaceutically acceptable salts thereof.
Table 1
Figure imgf000011_0002
1 -
-yl] c arbonyl } -
} -
,4,5-
Figure imgf000012_0001
} - } -
pyπdm-2 -
- 7 - (2-
{4-
Figure imgf000013_0001
- } -
- 7 -(2 -
IH-
Figure imgf000014_0001
1 - {[7-(2- 1 ,4-
3 ,4 , 5 -
amine } -
-
-
Figure imgf000015_0001
-
- 7 -(2 - 5-
Figure imgf000016_0001
,4 , 5 -
-ol
-
-yl] carbonyl } -
Figure imgf000017_0001
-7-
-
1 -
- 1 -
Figure imgf000018_0001
-(endo)- XH-
IH- XH-
-
,3-thiazol-
Figure imgf000019_0001
Figure imgf000020_0001
General Administration
[0029] In one aspect, the invention provides pharmaceutical compositions comprising an inhibitor of mTOR according to the invention and a pharmaceutically acceptable carrier, excipient, or diluent. In certain other specific embodiments, administration is by the oral route. Administration of the compounds of the invention, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration or agents for serving similar utilities. Thus, administration can be, for example, orally, nasally, parenterally (intravenous, intramuscular, or subcutaneous), topically, transdermally, intravaginally, intravesically, intracistemally, or rectally, in the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, or aerosols, or the like, specifically in unit dosage forms suitable for simple administration of precise dosages. [0030] The compositions will include a conventional pharmaceutical carrier or excipient and a compound of the invention as the/an active agent, and, in addition, may include carriers and adjuvants, etc.
[0031] Adjuvants include preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. [0032] If desired, a pharmaceutical composition of the invention may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, antioxidants, and the like, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylalted hydroxytoluene, etc.
[0033] The choice of formulation depends on various factors such as the mode of drug administration (e.g., for oral administration, formulations in the form of tablets, pills or capsules) and the bioavailability of the drug substance. Recently, pharmaceutical formulations have been developed especially for drugs that show poor bioavailability based upon the principle that bioavailability can be increased by increasing the surface area i.e., decreasing particle size. For example, U.S. Pat. No. 4,107,288 describes a pharmaceutical formulation having particles in the size range from 10 to 1,000 nm in which the active material is supported on a crosslinked matrix of macromolecules. U.S. Pat. No. 5,145,684 describes the production of a pharmaceutical formulation in which the drug substance is pulverized to nanoparticles (average particle size of 400 nm) in the presence of a surface modifier and then dispersed in a liquid medium to give a pharmaceutical formulation that exhibits remarkably high bioavailability. [0034] Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. [0035] One specific route of administration is oral, using a convenient daily dosage regimen that can be adjusted according to the degree of severity of the disease-state to be treated. [0036] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, cellulose derivatives, starch, alignates, gelatin, polyvinylpyrrolidone, sucrose, and gum acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, croscarmellose sodium, complex silicates, and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, magnesium stearate and the like (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.
[0037] Solid dosage forms as described above can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain pacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedded compositions that can be used are polymeric substances and waxes. The active compounds can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients. [0038] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Such dosage forms are prepared, for example, by dissolving, dispersing, etc., a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like; solubilizing agents and emulsifϊers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide; oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan; or mixtures of these substances, and the like, to thereby form a solution or suspension. [0039] Suspensions, in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
[0040] Compositions for rectal administrations are, for example, suppositories that can be prepared by mixing the compounds of the present invention with for example suitable non- irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt while in a suitable body cavity and release the active component therein.
[0041] Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays, and inhalants. The active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required. Ophthalmic formulations, eye ointments, powders, and solutions are also contemplated as being within the scope of this invention.
[0042] Compressed gases may be used to disperse a compound of this invention in aerosol form. Inert gases suitable for this purpose are nitrogen, carbon dioxide, etc. [0043] Generally, depending on the intended mode of administration, the pharmaceutically acceptable compositions will contain about 1% to about 99% by weight of a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, and 99% to 1 % by weight of a suitable pharmaceutical excipient. In one example, the composition will be between about 5% and about 75% by weight of a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, with the rest being suitable pharmaceutical excipients.
[0044] Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Company, Easton, Pa., 1990). The composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease-state in accordance with the teachings of this invention.
[0045] The compounds of the invention, or their pharmaceutically acceptable salts or solvates, are administered in a therapeutically effective amount which will vary depending upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of the compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular disease-states, and the host undergoing therapy. The compounds of the present invention can be administered to a patient at dosage levels in the range of about 0.1 to about 1,000 mg per day. For a normal human adult having a body weight of about 70 kilograms, a dosage in the range of about 0.01 to about 100 mg per kilogram of body weight per day is an example. The specific dosage used, however, can vary. For example, the dosage can depend on a number of factors including the requirements of the patient, the severity of the condition being treated, and the pharmacological activity of the compound being used. The determination of optimum dosages for a particular patient is well known to one of ordinary skill in the art.
[0046] If formulated as a fixed dose, such combination products employ the compounds of this invention within the dosage range described above and the other pharmaceutically active agent(s) within its approved dosage range. Compounds of the instant invention may alternatively be used sequentially with known pharmaceutically acceptable agent(s) when a combination formulation is inappropriate.
General Synthesis
[0047] Compounds of this invention can be made by the synthetic procedures described below. The starting materials and reagents used in preparing these compounds are either available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wis.), or Bachem (Torrance, Calif.), or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and Sons, 4 Edition) and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). These schemes are merely illustrative of some methods by which the compounds of this invention can be synthesized, and various modifications to these schemes can be made and will be suggested to one skilled in the art having referred to this disclosure. The starting materials and the intermediates of the reaction may be isolated and purified if desired using conventional techniques, including but not limited to filtration, distillation, crystallization, chromatography and the like. Such materials may be characterized using conventional means, including physical constants and spectral data.
[0048] Unless specified to the contrary, the reactions described herein take place at atmospheric pressure and over a temperature range from about -78 0C to about 1500C, more specifically from about 00C. to about 125 0C and more specifically at about room (or ambient) temperature, e.g., about 200C. Unless otherwise stated (as in the case of an hydrogenation), all reactions are performed under an atmosphere of nitrogen.
[0049] Prodrugs can be prepared by techniques known to one skilled in the art. These techniques generally modify appropriate functional groups in a given compound. These modified functional groups regenerate original functional groups by routine manipulation or in vivo. Amides and esters of the compounds of the present invention may be prepared according to conventional methods. A thorough discussion of prodrugs is provided in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," VoI 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference for all purposes.
[0050] The compounds of the invention, or their pharmaceutically acceptable salts, may have asymmetric carbon atoms or quaternized nitrogen atoms in their structure. Compounds of the Invention that may be prepared through the syntheses described herein may exist as single stereoisomers, racemates, and as mixtures of enantiomers and diastereomers. The compounds may also exist as geometric isomers. All such single stereoisomers, racemates and mixtures thereof, and geometric isomers are intended to be within the scope of this invention. [0051] Some of the compounds of the invention contain an active ketone -C(O)CF3 and may exist in part or in whole as the -C(OH2)CF3 form. Regardless of whether the compound is drawn as the -C(O)CF3 or -C(OH2)CF3 form, both are included within the scope of the Invention. Although an individual compound may be drawn as the -C(O)CF3 form, one of ordinary skill in the art would understand that the compound may exist in part or in whole as the -C(OH2)CFs form and that the ratio of the two forms may vary depending on the compound and the conditions in which it exists. [0052] Some of the compounds of the invention may exist as tautomers. For example, where a ketone or aldehyde is present, the molecule may exist in the enol form; where an amide is present, the molecule may exist as the imidic acid; and where an enamine is present, the molecule may exist as an imine. All such tautomers are within the scope of the invention. Further, for example, in this application R1 can be 5-oxo-lH-l,2,4-triazol-3-yl, depicted structurally below:
Figure imgf000025_0001
100.
Both 5-oxo-lH-l,2,4-triazol-3-yl and the above structure 1 include, and are equivalent to, 3-hydroxy-4H-l,2,4-triazol-5-yl and its structure 2:
Figure imgf000025_0002
200.
Regardless of which structure or which terminology is used, each tautomer is included within the scope of the Invention. [0053] The present invention also includes N-oxide derivatives and protected derivatives of compounds of the Invention. For example, when compounds of the Invention contain an oxidizable nitrogen atom, the nitrogen atom can be converted to an N-oxide by methods well known in the art. When compounds of the Invention contain groups such as hydroxy, carboxy, thiol or any group containing a nitrogen atom(s), these groups can be protected with a suitable "protecting group" or "protective group". A comprehensive list of suitable protective groups can be found in T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, Inc. 1991, the disclosure of which is incorporated herein by reference in its entirety. The protected derivatives of compounds of the Invention can be prepared by methods well known in the art. [0054] Methods for the preparation and/or separation and isolation of single stereoisomers from racemic mixtures or non-racemic mixtures of stereoisomers are well known in the art. For example, optically active (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. Enantiomers (R- and S-isomers) may be resolved by methods known to one of ordinary skill in the art, for example by: formation of diastereoisomeric salts or complexes which may be separated, for example, by crystallization; via formation of diastereoisomeric derivatives which may be separated, for example, by crystallization, selective reaction of one enantiomer with an enantiomer-specifϊc reagent, for example enzymatic oxidation or reduction, followed by separation of the modified and unmodified enantiomers; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support, such as silica with a bound chiral ligand or in the presence of a chiral solvent. It will be appreciated that where a desired enantiomer is converted into another chemical entity by one of the separation procedures described above, a further step may be required to liberate the desired enantiomeric form. Alternatively, specific enantiomer may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents or by converting on enantiomer to the other by asymmetric transformation. For a mixture of enantiomers, enriched in a particular enantiomer, the major component enantiomer may be further enriched (with concomitant loss in yield) by recrystallization. [0055] In addition, the compounds of the present invention can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.
[0056] The chemistry for the preparation of the compounds of this invention is known to those skilled in the art. In fact, there may be more than one process to prepare the compounds of the invention. The following examples illustrate but do not limit the invention. All references cited herein are incorporated by reference in their entirety.
Synthetic Examples
Reagent Preparation 1 2-(4-chloro-3-fluorophenyl)piperidine
[0057] STEP 1 : To a solution of tert-butyl 2-oxopiperidine- 1 -carboxylate (0.22 g, 1.08 mmol) in tetrahydrofuran (5 mL) at -78 0C was added slowly over 15 minutes 4-chloro-3- fluorophenylmagnesium bromide (0.5 M in tetrahydrofuran) (2.60 mL, 1.66 mmol) and the mixture was allowed to warm to 25 °C over 30 minutes. The reaction mixture was quenched by the addition of 1.0 N hydrochloric acid (1.0 mL) and partitioned with diethyl ether (50 mL). The organic layer was separated and washed with brine (25 mL), dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (diethyl ether/hexanes 1 :4) to give tert-butyl 5-oxo-5-(4-chloro3-fluorophenyl)pentylcarbamate (0.25 g, 70% yield). 1H NMR (400 MHz, CDCl3): 7.73 (dd, IH), 7.69 (m, IH), 7.50 (dd, IH), 4.59 (brs, IH), 3.18 (m, 2H) 2.97 (t, 2H), 1.77 (m, 2H), 1.57 (m, 2H), 1.44 (s, 9H). MS (EI) for Ci6H2IFClNO3: 330 (MH+).
[0058] STEP 2: A solution of tert-butyl 5-oxo-5-(4-chloro-3-fluorophenyl)pentylcarbamate (0.25 g, 0.76 mmol) was stirred in trifluoroacetic acid (1.5 mL) for 1 hour then concentrated. The residue was partitioned between dichloromethane (40 mL) and 2 M aqueous sodium hydroxide (20 mL). The organic layer was separated and washed with brine (20 mL), dried over anhydrous magnesium sulfate filtered and concentrated to provide 5-amino-l-(4-chloro-3- fluorophenyl)pentan-l-one. To a solution of the crude product in a mixture of methanol (50 mL) and water (0.5 mL) was added sodium borohydride (43 mg, 1.14 mmol) and stirred for 16 hours at room temperature. The reaction mixture was quenched by the addition of IM aqueous hydrochloric acid (1.0 mL) and concentrated. The residue was partitioned between dichloromethane (100 mL) and saturated aqueous sodium hydrogencarbonate (20 mL), washed with brine (20 mL), filtered and concentrated to give 2-(4-chloro-3-fluorophenyl)piperidine (0.14 g, 85% yield) as an oil. [0059] Using analogous techniques and substituting alternative reagents in step 1 the following reagents were prepared. All starting materials were commercially obtained unless otherwise stated.
[0060] 2-(4-chlorophenyl)piperidine. Synthesized according to the method of reagent preparation 1 using 4-chlorophenylmagnesium bromide in step 1. MS (EI) for CHHHCIN: 196 (MH+).
[0061] 2-(4-(trifluoromethoxy)phenyl)piperidine. Synthesized according to the method of reagent preparation 1 using 4-trifluoromethoxyphenylmagnesium bromide in step 1. MS (EI) for Ci2Hi4F3NO: 246 (MH+). [0062] 2-(3-fluoro-4-methoxyphenyl)piperidine. Synthesized according to the method of reagent preparation 1 using 3-fluoro-4-methoxyphenylmagnesium bromide in step 1. MS (EI) for Ci2Hi6FNO: 210 (MH+).
[0063] 2-(2-fluorophenyl)piperidine. Synthesized according to the method of reagent preparation 1 using 2-fluorophenylmagnesium bromide in step 1. MS (EI) for C11H14FN: 180 (MH+). [0064] 2-(3,4-dichlorophenyl)piperidine. Synthesized according to the method of reagent preparation 1 using 3,4-dichlorophenylmagnesium bromide in step 1. MS (EI) for CHHI3CI2N: 230 (MH+). Reagent Preparation 2 (±)-(2Jff,45)-2-pheny]piperidin-4-ylmethanol
Figure imgf000028_0001
[0065] STEP 1 : A suspension of potassium tert-butoxidc (1.25 g, 11.1 mmol) and methyltriphenylphosphonium bromide (3.86 g, 1.1 mmol) in tetrahydrofuran (100 mL) was stirred at 40 0C for 30 minutes. The mixture was then cooled to room temperature and a solution of tert-butyl 4-oxo-2-phenylpiperidine-l-carboxylate (2.35 g, 8.5 mmol) in tetrahydrofuran (30 mL) was added slowly. The reaction mixture was stirred at 40 0C for 24 hours. The mixture was cooled to room temperature and quenched by the addition of water and diluted with ethyl acetate (250 mL), The organic layer was separated then washed with water, 10% aqueous citric acid and brine, dried over anhydrous sodium sulfate, filtered and concentrated. Column chromatography on silica gel (hexane:ethyl acetate 95:5 to 9:1) provided tert-butyl 4-methylene-2- phenylpiperidine-1-carboxylate (2.24 g, 96%). 1H NMR (400 MHz, CDCl3): 7.31 (m, 4H), 7.21 (m, IH), 5.48 (br d, IH), 4.84 (dd, 2H), 4.07 (br dd, IH), 2.85 (br, t, IH), 2.78 (dtr, IH), 2.64 (dd, IH), 2.28 (dtr, IH), 2.20 (br d, IH), 1.42 (s, 9H). GC/MS (EI) for CnH23NO2: 273 (M+). [0066] STEP 2: To solution of tert-butyl 4-methylene-2-phenylpiperidine-l-carboxylate (2.20 g, 8.04 mmol) in tetrahydrofuran (50 mL) at 0 0C was added borane-tetrahydrofuran complex (IM solution in in tetrahydrofuran) (12.1 mL, 12.1 mmol) and the reaction mixture was stirred at 0 0C for 1 hour. The reaction mixture was allowed to warm to room temperature then stirred for an additional 2 hours. It was cooled to 0 0C and 2M aqueous sodium hydroxide (8.0 mL, 16.0 mmol) was added slowly followed by the slow addition of 30% aqueous hydrogen peroxide (5.5 mL, 48.4 mmol). The mixture was stirred for another hour then diluted with water (100 mL) and partitioned with ethyl acetate (250 mL). The organic layer was separated and washed with 2M aqueous sodium thiosulfate (100 mL), brine, dried over anhydrous sodium sulfate, filtered and concentrated. Column chromatography in silica gel (chlorofornrmethanol 9:1 to 4:1) provided tert-butyl 4-(hydroxymethyl)-2-phenylpiperidine-l-carboxylate (1.86 g, 79%). 1H NMR (400 MHz, CDCl3): 7.30 (m, 2H), 7.20 (m, 3H), 4.86 (dd, IH), 4.04 (m, IH), 3.62 (m, 0.5H), 3.44 (m, 3H), 3..24 (m, IH), 2.12 (m, 0.5H), 1.93(m, IH), 1.64 (m, 2H), 1.42 (m, IH), 1.26 (s, 9H). GC/MS (EI) for Ci7H25NO3: 235 (M-tBu+). [0067] STEP 3 : To a solution of tert-butyl 4-(hydroxymethyl)-2-phenylpiperidine- 1 - carboxylate (0.29 g, 1.00 mmol) in dichloromethane (50 mL) was added trifluoroacetic acid (10 mL) and the reaction mixture was heated to reflux. After cooling to room temperature the solvent was evaporated. The residue was twice taken into 50% ethyl acetate in toluene then concentrated (2x100 mL) and the resulting solid then dried to give (±)-(2R,4S)-2- phenylpiperidin-4-ylmethanol as the trifluoroacetic acid salt (0.26 g, quantitative). MS (EI) for Ci2Hi7NO: 192 (MH+).
Reagent Preparation 3 2-(trifluoromethyl)piperidine [0068] A mixture of 2-(trifluoromethyl)pyridine (0.38 g, 2.60 mmol) and platinum oxide (0.04 g, 0.18 mmol) in acetic acid (15 mL) and concentrated hydrochloric acid (2 mL) was hydrogenated in a Parr apparatus at 40 psi for 3 d. Filtration through celite and concentration of the filtrate provided 2-(trifluoromethyl)piperidine as hydrochloride salt which was used without further purification. 1H NMR (400 MHz, methanol-d4): 4.18 (m, IH), 3.50 (m, IH), 3.15 (m, IH), 2.16 (m, IH), 1.99 (m, 2H), 1.71 (m, 3H).
[0069] Using analogous synthetic techniques and substituting with alternative starting reagents the following reagents were prepared. Alternative starting materials were obtained commercially unless otherwise indicated. [0070] 4-cyclopropylpiperidine. Prepared as hydrochloride salt according to reagent preparation 3 by using 4-cyclopropylpyridine. MS (EI) for C8Hi5N: 125 (M+).
Reagent Preparation 4 4-(difluoromethy])piperidine
[0071] STEP 1 : To a solution of tert-butyl (4-hydroxymethyl)piperidine-l-carboxylate (0.52 g, 2.40 mmol, (J. Labelled Compounds and Radiopharmaceuticals 1999, 42, 1289-1300) in dichloromethane (20 mL) was added Dess-Martin-periodinane (1.13 g, 2.66 mmol), and the mixture was stirred at room temperature for 2 h. A 10% aqueous solution of sodium thiosulfate (20 mL) was added followed by saturated sodium bicarbonate (20 mL), and the biphasic mixture was stirred at room temperature for 45 min. The layers were separated and the aqueous layer was extracted with dichloromethane (2 x). The combined organic layers were washed with saturated sodium bicarbonate, brine, dried over sodium sulfate then filtered and concentrated to afford tert-butyl 4-formylpiperidine-l-carboxylate. 1H NMR (400 MHz, CDCl3): 9.67 (s, IH), 3.99 (m, 2H), 2.93 (m, 2H), 2.42 (m, IH), 1.89 (m, 2H), 1.55 (m, 2H), 1.46 (s, 9H). [0072] STEP 2: To a solution of DAST (1.16 g, 7.20 mmol) in dichloromethane (30 mL) was added a solution of tert-butyl 4-formylpiperidine-l-carboxylate (0.51 g, 2.40 mmol) in dichloromethane (5 mL) at 0 0C. The reaction mixture was warmed to room temperature and stirred for 18 h. A 5% aqueous solution of sodium bicarbonate was added, the layers were separated, the organic layer was washed with saturated sodium bicarbonate, and brine, dried over sodium sulfate, filtered and concentrated to provide tert-butyl 4-(difiuoromethyl)piperidine-l- carboxylate. 1H NMR (400 MHz, CDCl3): 5.59 (m, IH), 4.20 (m, 2H), 2.69 (m, 2H), 1.91 (m, IH), 1.74 (m, 2H), 1.46 (s, 9H), 1.34 (m, 2H). [0073] STEP 3: A solution of tert-butyl 4-(difluoromethyl)piperidine- 1 -carboxylate in trifluoroacetic acid was stirred at room temperature for 1 h then concentrated and dried to give 4-(difluoromethyl)piperidine as the trifluoroacetate salt. 1H NMR (400 MHz, CDCl3): 5.67 (m, IH), 3.55 (m, 2H), 2.96 (m, 2H), 2.04 (m, 3H), 1.80 (m, 2H). Reagent Preparation 5
4-(fluoromethyl)piperidine
[0074] A solution of tert-butyl 4-(fluoromethyl)piperidine-l -carboxylate (J. Labelled Compounds and Radiopharmaceuticals 1999, 42, 1289-1300) in trifluoroacetic acid was stirred at room temperature for 1 h and then concentrated and dried to give 4-(fluoromethyl)-piperidine as the trifluoroacetate salt. 1H NMR (400 MHz, CDCl3): 4.33 (dd, 2H), 3.49 (m, 2H), 2.92 (m, 2H), 2.07 (m, IH), 1.97 (m, 2H), 1.64 (m, 2H).
Reagent Preparation 6
4-(l,l-difluoroethyl)piperidine
[0075] STEP 1 : To a solution of DAST (1.83 g, 11.35 mmol) in dichloromethane (30 mL) was added 4-acetylpyridine (1.00 g, 8.25 mmol) at 0 0C. The reaction mixture was warmed to room temperature and stirred for 2 d. More DAST (0.61 g, 3.78 mmol) was added and stirring was continued for 1 d. A 5% aqueous solution of sodium bicarbonate was added, the layers were separated and the organic layer was washed with saturated sodium bicarbonate, and brine then dried over sodium sulfate, filtered and concentrated to provide a 5: 1 mixture of 4-(l ,1- difluoroethyl)pyridine and 4-acetylpyridine.
[0076] STEP 2: The mixture was dissolved in methanol (10 mL) and 1 M hydrochloric acid (10 mL) then catalytic platinum oxide was added and the resulting suspension was hydrogenated in a Parr apparatus at 40 psi for 3 d. Filtration through celite and concentration of the filtrate gave a complex mixture containing 20% of the desired 4-(l,l-difluoroethyl)piperidine as the hydrochloride salt which was used without further purification.
Reagent Preparation 7 4-(methylthio)piperidine
[0077] STEP 1 : To a solution of tert-butyl 4-hydroxypiperidine- 1 -carboxylate (4.0 g, 20.0 mmol) and triethylamine (4.0 g, 40 mmol) in dichloromethane (50 mL) was added methanesulfonyl chloride (2.8 g, 24.4 mmol) at 0 0C. The solution was stirred at 0 0C for 10 min, then at room temperature for 2 h. The reaction mixture was partitioned between 10% citric acid and ethyl acetate. The organic layer was washed with sodium bicarbonate, and brine, dried over sodium sulfate, filtered and concentrated to give tert-butyl 4- (methylsulfonyloxy)piperidine- 1 -carboxylate (6.4 g, quantitative yield). MS (EI) for CnH2INO5S: 279 (M+). [0078] STEP 2: A solution of tert-butyl 4-(methylsulfonyloxy)piperidine- 1 -carboxylate (2.0 g, 7.2 mmol) and sodium thiomethoxide (1.0 g, 14.4 mmol) in methanol (30 mL) was refluxed for 15 h and then concentrated. The residue was partitioned between water and ethyl acetate. The aqueous layer was extracted twice with ethyl acetate and the combined organic extracts washed with brine, dried over sodium sulfate, filtered and concentrated. Column chromatography on silica (3% ethyl acetate in hexanes) afforded tert-butyl 4- (methylthio)piperidine- 1 -carboxylate (0.98 g, 58% yield) as a colorless oil. MS (EI) for CnH21NO2S: 231 (M+). [0079] STEP 3: A solution of tert-butyl 4-(methylthio)piperidine-l -carboxylate (63 mg, 0.27 mmol) in methanol (1 mL) and 4 N hydrogen chloride in dioxane (4 mL) was refluxed for 2 min and then concentrated and dried to provide 4-(methylthio)piperidine hydrochloride as a colorless oil.
Reagent Preparation 8 3-(trifluoromethyl)-8-azabicyclo [3.2.1] octan-3-(endo)-ol [0080] Step 1 : Trimethyl(trifluoromethyl)silane (0.32 g, 2.25 mmol) was added to a mixture of tert-butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (0.50 g, 2.2 mmol), cesium carbonate (1.1 g, 3.4 mmol) in N,N-dimethylformamide (5 mL) at O0C. The resulting mixture was warmed to room temperature and stirred for two hours. The mixture was diluted with ethyl acetate (80 mL), washed with water (3 x 50 mL) then brine (50 mL), dried over sodium sulfate, filtered, and concentrated. The residue was taken into methanol (20 mL) and potassium carbonate (0.62 g, 4.5 mmol) was added then stirred at room temperature for 18 hours. The mixture was diluted with ethyl acetate (150 mL) then filtered and concentrated. The residue was purified by silica gel chromatography (10% to 25% ethyl acetate in hexanes gradient) to give tert-butyl 3-(endo)-hydroxy-3-(trifluoromethyl)-8-azabicyclo[3.2.1]octane-8-carboxylate (0.36 g, 55% yield), GC-MS (EI) for Ci3H20F3NO3: 295 (M+).
[0081] Step 2: tert-Butyl 3-(enJo)-hydroxy-3-(trifluoromethyl)-8-azabicyclo[3.2.1]octane-8- carboxylate 1 (0.36 g, 1.2 mmol) was taken into acetonitrile (2 mL) and 4 M hydrogen chloride in 1 ,4-dioxane (2 mL) then stirred at 700C for 15 minutes. The reaction mixture was concentrated and dried to give 3-(trifluoromethyl)-8-azabicyclo[3.2.1]octan-3-(e«ύfo)-ol hydrochloride (0.28g, 100% yield). MS (EI) for C8H12F3NO: 196 (MH+).
Reagent Preparation 9
3-methyl-8-azabicyclo [3.2.1 ] octan-3-(endo)-o\
[0082] Step 1 : Methylmagnesium bromide (3 M solution in ether, 2.7 mmol) was added to a solution of tert-butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (0.50 g, 2.2 mmol), in tetrahydrofuran (20 mL) at 00C and the resulting mixture was stirred one hour. The reaction mixture was quenched with saturated aqueous ammonium chloride solution (20 mL) then partitioned with ethyl acetate (80 mL). The organic portion was separated, washed with water, then brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (5% to 35% ethyl acetate in hexanes gradient) to give tert-butyl 3-(endo)-hydroxy-3-methyl-8-azabicyclo[3.2.1]octane-8-carboxylate (0.22 g, 41% yield), GC- MS (EI) for Ci3H23NO3: 241 (M+).
[0083] Step 2: tert-Butyl 3-(endo)-hydroxy-3-methyl-8-azabicyclo[3.2.1]octane-8- carboxylate (0.22 g, 1.2 mmol) was taken into acetonitrile (1 mL), and 4 M hydrogen chloride in 1,4-dioxane (1 mL) then stirred at 700C for 15 minutes. The reaction mixture was concentrated and dried to give 3-methyl-8-azabicyclo[3.2. l]octan-3-(endo)-ol hydrochloride salt (0.16 g, 100% yield). MS (EI) for C8H12F3NO: 142 (MH+).
Reagent Preparation 10
3-fluoro-3-(e«rfo)-methyl-8-azabicyclo [3.2.1] octane [0084] Step 1 : Dimethylaminosulfur trifluoride (81 mg, 0.61 mmol) was added to a solution of tert-butyl 3-(e/?<io)-(hydroxymethyl)-8-azabicyclo[3.2.1 ]octane-8-carboxylate (50 mg, 0.21 mmol) (reagent preparation 18, step 2) in dichloromethane (2 mL) at 00C, and the resulting mixture was stirred one hour. The reaction mixture was quenched with saturated aqueous sodium bicarbonate solution (10 mL) then partitioned with dichloromethane (20 mL). The organic portion was separated, washed with water, then brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (5% to 35% ethyl acetate in hexanes gradient) to give tert-butyl 3-fluoro-3-(en<io)-methyl-8- azabicyclo[3.2.1]octane-8-carboxylate (28 mg, 56% yield), GC-MS (EI) for Ci3H22FNO2: 243 (M+). [0085] Step 2 : A mixture of tert-butyl 3-fluoro-3-(e«<io)-methyl-8-azabicyclo[3.2.1 Joctane- 8-carboxylate (0.22 g, 1.2 mmol), acetonitrile (1 mL) and 4 M hydrogen chloride in 1,4-dioxane (1 mL) was stirred at 700C for 15 minutes. The reaction mixture was concentrated and dried to give 3-fluoro-3-(en<io)-methyl-8-azabicyclo[3.2.1]octane hydrochloride salt (20 mg, 100% yield). MS (EI) for C8Hi4FN: 144 (MH+).
Reagent Preparation 11 3-(en</o)-(fluoromethyl)-8-azabicyc]o[3.2.1]octane
[0086] Step 1 : Potassium tert-butoxide (0.62 g, 5.5 mmol) was added to a suspension of methyltriphenylphosphonium bromide (1.98 g, 5.5 mmol) in tetrahydrofuran (20 mL) and the resulting mixture was stirred at room temperature for one hour. A solution of tert-butyl 3-oxo-8- azabicyclo[3.2.1]octane-8-carboxylate (0.50 g, 2.2 mmol) in tetrahydrofuran (5 mL) was then added and the resulting mixture was stirred at 350C for two hours. The mixture was cooled, diluted with hexane (100 niL), filtered, and the filtrate was washed with water then brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (20% ethyl acetate in hexanes) to give tert-butyl 3-methylene-8- azabicyclo[3.2.1]octane-8-carboxylate (0.45g, 91% yield). GC-MS (EI) for Ci3H2iNO2: 223 (M+).
[0087] Step 2: Borane (I M solution in tetrahydrofuran, 1.79 mL) was added to a solution of tert-butyl 3-methylene-8-azabicyclo[3.2.1]octane-8-carboxylate (0.20 g, 0.87 mmol) in tetrahydrofuran (20 mL) at 00C. The reaction mixture was slowly warmed to room temperature and stirred for 18 hours. It was then cooled to 00C, followed by sequential addition of 2 M sodium hydroxide solution (1 mL) and hydrogen peroxide solution (30% in water, 0.46 mL). The mixture was warmed to room temperature and stirred for 1.5 hours. The reaction mixture was quenched with saturated sodium bicarbonate solution (10 mL), diluted with water (20 mL) and partitioned with ethyl acetate (20 mL). The organic portion was separated and washed twice with saturated sodium bisulfite solution (20 mL), water then brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (20% to 90% ethyl acetate hexanes gradient) to give tert-butyl 3-(e«ύfo)-(hydroxymethyl)-8- azabicyclo[3.2.1]octane-8-carboxylate (0.19 g, 88% yield), GC-MS (EI) for Ci3H23NO3: 241 (M+). [0088] Step 3: Methanesulfonyl chloride (154 mg, 1.35 mmol) was added to a mixture of tert-butyl 3-(e«6?ø)-(hydroxymethyl)-8-azabicyclo[3.2.1]octane-8-carboxylate (325 mg, 1.4 mmol), triethylamine (136 mg, 1.4 mmol), and l,4-diazabicyclo[2.2.2]octane (31 mg, 0.28 mmol) in toluene (10 mL) at 00C. The resulting mixture was stirred at 00C for 15 minutes, and at room temperature for another 15 minutes. The reaction mixture was quenched with a cold mixture of water and ethyl acetate. The organic portion was separated, washed with water, then brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (5% to 25% ethyl acetate in hexanes gradient) to give tert-butyl 3-((endo- methylsulfonyloxy)methyl)-8-azabicyclo[3.2.1]octane-8-carboxylate (330 mg, 77% yield), GC- MS (EI) for Ci4H25NO5S: 319 (M+). [0089] Step 4: A mixture of tert-butyl 3-((ercdo)-methylsulfonyloxy)methyl)-8- azabicyclo[3.2.1]octane-8-carboxylate (330 mg, 1.0 mmol), triethylamine (136 mg, 1.4 mmol), and tetrabutylammonium fluoride hexahydrate (489 mg, 1.3 mmol) in tetrahydrofuran (10 mL) was stirred at 600C for 18 hours. The reaction mixture was cooled, concentrated and the residue purified by silica gel chromatography (5% to 15% ethyl acetate in hexanes gradient) to give tert- butyl 3-(endo)-(fluoromethyl)-8-azabicyclo[3.2.1]octane-8-carboxylate (120 mg, 36% yield), GC-MS (EI) for Ci3H22FNO2: 243 (M+). [0090] Step 5: A mixture of tert-butyl 3-(endo)-(fluoromethyl)-8-azabicyclo[3.2.1]octane-8- carboxylate (50 mg, 0.21 mmol), acetonitrile (1 niL), and 4 M hydrogen chloride in 1,4-dioxane (1 mL) was stirred at 700C for 15 minutes. The reaction mixture was concentrated and dried to give 3-(endo)-(fluoromethyl)-8-azabicyclo[3.2.1]octane hydrochloride salt (37 mg, 100% yield). MS (EI) for C8Hi5FN: 144 (MH+).
Reagent Preparation 12 4-(trifluoromethyl)piperidin-4-ol
[0091] STEP 1 : To a solution of ter?-butyl 4-oxopiperidine-l-carboxylate (0.6 g, 3.0 mmol) and cesium carbonate (1.1 g, 3.3 mmol) in dimethylformamide (10 mL) was added dropwise trimethyl(trifluoromethyl)silane (2 mL, 13.5 mmol) at 0 0C. The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with diethyl ether (100 ml) washed with water (50 mL) and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to afford tert-butyl 4-(trifluoromethyl)-4- (trimethylsilyloxy)piperidine-l-carboxylate (0.512 g, 50% yield) as an orange residue that was used without further purification. MS (EI) for Ci4H26F3NO3Si: 341 (MH+). [0092] STEP 2: To a solution of tert-butyl 4-(trifluoromethyl)-4- (trimethylsilyloxy)piperidine-l-carboxylate (0.512 g, 1.50 mmol), in methanol (10 mL) was potassium carbonate (0.25 g, 1.81 mmol). The resulting mixture was stirred at room temperature for 12 hours. Filtration and concentration provided an orange residue that was purified by silica gel chromatography (97:3 dichloromethane:methanol) to give tert-butyl 4-hydroxy-4-(trifluoromethyl)piperidine-l-carboxylate (0.07 g, 14% yield). MS (EI) for CHHI8F3NO3 : 269 (MH+).
[0093] STEP 3: To a solution of tert-butyl 4-hydroxy-4-(trifiuoromethyl)piperidine- 1 - carboxylate (0.07 g, 0.26 mmol) in dichloromethane (1 mL) was added trifluoroacetic acid (1 mL). The resulting mixture was stirred at room temperature for 30 minutes. Concentration and drying afforded 4-(trifluoromethyl)piperidin-4-ol (0.044 g, 100%). MS (EI) for C6Hi0F3NO: 269 (MH+).
Reagent Preparation 13 4-methylpiperidine-4-carbonitrile [0094] STEP 1 : Trifluoroacetic acid anhydride (75 uL, 0.82 mmol) was added to a mixture of tert-butyl 4-carbamoyl-4-methylpiperidine-l -carboxylate (100 mg, 0.41 mmol) and pyridine (118 uL, 1.6 mmol) in tetrahydrofuran (2 mL), and the resulting mixture was stirred at room temperature for one hour. The mixture was concentrated then taken into ethyl acetate (20 mL) and partitioned with 0.5 M hydrochloric acid. The organic layer was washed with water then brine, dried over sodium sulfate, filtered, and concentrated to provide a 1 : 1 mixture of tert-butyl 4-cyano-4-methylpiperidine-l-carboxylate and tert-butyl 4-carbamoyl-4-methylpiperidine-l- carboxylate (100 mg) that was carried forward without further purification. GC-MS (EI) for C12H20N2O2 (tert-butyl 4-cyano-4-methylpiperidine-l-carboxylate): 224 (M+). [0095] STEP 2: tert-Butyl 4-cyano-4-methylpiperidine- 1 -carboxylate as obtained in step 1 (100 mg, 0.21 mmol), acetonitrile (1 mL), and 4 M hydrogen chloride in 1,4-dioxane (1 mL) were combined and stirred at 700C for 15 minutes. The reaction mixture was concentrated and dried to give 4-methylpiperidine-4-carbonitrile hydrochloride salt (56 mg) contaminated with 4- methylpiperidine-4-carboxamide hydrochloride salt. MS (EI) for C7H12N2 (4-methylpiperidine- 4-carbonitrile): 125 (MH+). Reagent Preparation 14
8-azabicyclo [3.2.1 ] octan-3-ol
[0096] STEP 1 : Sodium borohydride (178 mg, 4.7 mmol) was added to a solution of tert-butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (0.50 g, 2.2 mmol) in ethanol (10 mL), and the resulting mixture was stirred at room temperature for one hour. The mixture was quenched with saturated ammonium chloride solution (30 mL), and extracted with ethyl acetate (3x 20 mL). The combined extract was washed with water then brine, dried over sodium sulfate, filtered and concentrated to give tert-butyl 3-hydroxy-8-azabicyclo[3.2.1]octane-8-carboxylate (463 mg, 92% yield) as a mixture of endo and exo stereoisomers. GC-MS (EI) for C12H21NO3: 227 (M+). [0097] STEP 2: tert-Butyl 3-hydroxy-8-azabicyclo[3.2.1 ]octane-8-carboxylate as obtained in step 1 (100 mg, 2.0 mmol), acetonitrile (2 mL) and 4 M hydrogen chloride in 1,4-dioxane (2 mL) were combined and stirred at 700C for 15 minutes. The reaction mixture was concentrated and dried to give 8-azabicyclo[3.2.1]octan-3-ol hydrochloride salt (71 mg, 100% yield). MS (EI) for C7H13NO: 128 (MH+). Reagent Preparation 15
3-(e/fύ?ø)-niethyl-8-azabicyclo [3.2.1] octane
[0098] STEP 1 : A mixture of tert-butyl 3-methylene-8-azabicyclo[3.2. l]octane-8- carboxylate (0.10 g, 0.44 mmol) (reagent preparation 18), 10% palladium on charcoal (10 mg) and ethanol (15 mL) was hydrogenated in a Parr apparatus at 40 psi for 18 hours. The mixture was filtered and concentrated then dried to give tert-butyl 3-(endo)-methyl-8- azabicyclo[3.2.1]octane-8-carboxylate (96 mg, 95% yield); GC-MS (EI) for Ci3H23NO2: 225
(M+).
[0099] STEP 2: A mixture of tert-butyl 3-(endo)-methyl-8-azabicyclo[3.2.1]octane-8- carboxylate (96 mg, 0.43 mmol), acetonitrile (1 mL), and 4 M hydrogen chloride in 1,4-dioxane (1 mL) was stirred at 700C for 15 minutes. The reaction mixture was concentrated and dried to give 3-(endo)-methyl-8-azabicyclo[3.2.1]octane hydrochloride salt (68 mg, 100% yield). MS (EI) for C8Hi5N: 126 (MH+).
Reagent Preparation 16 (±)-(2/?,4R)-2-(4-fluorophenyl)piperidin-4-ol [00100] STEP 1 : Benzyl 2-(4-fluorophenyl)-4-oxo-3,4-dihydropyridine-l(2H)-carboxylate was prepared according to the method in (Tetrahedron Lett., 1986, 27, 4549-4552) using 4-methoxypyridine (29.8 mL, 290 mmol), benzyl chloroformate (50.0 mL, 350 mmol) and 4-fluorophenyl magnesium bromide (0.8 M solution in TΗF), (450 mL, 0.36 mmol), to yield (81 g, 86% yield) of the title compound. MS (EI) for Ci9Hi6FNO3: 326 (MH+). [00101] STEP 2: A mixture of benzyl 2-(4-fluorophenyl)-4-oxo-3,4-dihydropyridine- 1(2H)- carboxylate (1.00 g, 3.07 mmol) (reagent preparation 20) and 5% Pd on carbon (0.1 g) in acetic acid:methanol 1 : 10 (20 mL) was hydrogenated at 45 psi using a Parr apparatus for 16 hours. The catalyst was removed by filtering through Celite, and the filtrate concentrated to give (±)-(25,4i?)-2-(4-fluorophenyl)piperidin-4-ol as an oil. The material was taken into chloroform (100 mL) and di-tert-butyl dicarbonate (0.74 g, 3.4 mmol) was added, followed by the dropwise addition of diisopropylethylamine (1.5 g, 12 mmol). The reaction was warmed to reflux for 10 minutes, then allowed to cool to 25°C over 30 minutes. The organic solution was washed with 0.1M aqueous hydrochloric acid (45 mL), water (50 mL) and saturated sodium bicarbonate (50 mL), then dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate:hexanes, 1 : 1) to give (±)-(2S,4R)- tert-butyl 2-(4-fluorophenyl)-4-hydroxypiperidine-l-carboxylate (0.59 g, 65% yield). 1H NMR (400 MHz, d6-DMSO): 7.25 (m, 2H), 7.10 (m, 2H), 4.96 (t, IH), 4.46 (d, IH), 3.90 (m, IH), 3.77 (m, IH), 3.23 (dt, IH), 2.06 (m, IH), 1.95 (m, IH) 1.73 (m, IH), 1.45 (m, IH), 1.29 (s, 9H). [00102] STEP 3 : To (±)-(2,S',4i?)-tert-butyl 2-(4-fiuorophenyl)-4-hydroxypiperidine- 1 - carboxylate (0.55 g, 1.90 mmol) in tetrahydrofuran (20 mL) was added methanesulfonyl chloride (0.158 mL, 2.05 mmol), followed by dropwise addition of diisopropylethylamine (0.50 g, 3.9 mmol) and Λ/,N-dimethylpyridin-4-amine (10 mg). After 30 minutes the reaction was diluted with ethyl acetate (50 mL) and washed with 0.1 M hydrochloric acid (25 mL) then saturated sodium bicarbonate (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (ethyl acetate :hexanes, 1 :4) to give
(±)-(2S,4R)-tert-butyl 2-(4-fluorophenyl)-4-(methylsulfonyloxy)piperidine- 1 -carboxylate (0.62 g, 88% yield). 1H NMR (400 MHz, CDCl3): 7.19 (dd, IH), 7.05 (t, 2H), 5.38 (d, IH), 5.14 (m, IH), 4.14 (m, IH), 3.25 (m, IH), 2.68 (m, IH), 2.59 (s, 3H), 2.21 9M, IH), 1.93 (m, 2H), 1.42 (s, 9H). [00103] STEP 4: A solution of (±)-(2S,4R)-tert-butyl 2-(4-fluorophenyl)-4- (methylsulfonyloxy)piperidine-l-carboxylate (0.30 g, 0.80 mmol) and sodium acetate (0.33 g, 4.0 mmol) in dimethylsulfoxide (15 mL) was heated to 90 0C for 2.5 hours. After cooling, the reaction mixture was diluted with ethyl acetate (40 mL), and washed with water (25 mL) and brine (25 mL). The organic layer was dried over anhydrous sodium sulfate then filtered and concetrated. The residue was purified by silica gel column chromatography (ethyl acetate :hexanes 1 :10) to give (±)-(2R,4R)-tert-butyl 4-acetoxy-2-(4-fluorophenyl)piperidine- 1 - carboxylate (150 mg, 49% yield). 1H NMR (400 MHz, d6-DMSO): 7.24 (m, 4H), 5.14 (br s, IH), 4.63 (m, IH), 4.00 (br d, IH), 2.72 (m, IH), 2.56 (br d, IH), 1.88 (s, 3H), 1.84 (br d IH), 1.78 (m, IH), 1.44 (m, IH), 1.39 (s, 9H).
[00104] STEP 5 : A suspention of (±)-(2R,4R)-tert-buty\ 4-acetoxy-2-(4- fluorophenyl)piperidine- 1 -carboxylate (150 mg, 0.40 mmol) and potassium carbonate (1.0 g) in methanol: water 10:1 (11 mL) was stirred for 1 hour then diluted with ethyl acetate (40 mL) and washed with water (25 mL) and brine (25 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concetrated to give {±)-(2R,4R)-tert-bvXy\ 2-(4-fluorophenyl)-4- hydroxypiperidine-1 -carboxylate (117 mg, 99% yield). 1H NMR (400 MHz, d6-DMSO): 7.17 (m, 4H), 5.34 (br d, IH), 4.73 (d, IH), 4.34 (br d, IH), 3.41 (m, IH), 2.67 (m, IH), 2.42 (br d, IH), 1.57 (m, IH), 1.38 (s, 9H). [00105] STEP 6: To (±)-(2R,4R)-tert-butyl 2-(4-fluorophenyl)-4-hydroxypiperidine-l- carboxylate (0.10 g, 0.34 mmol) in dichloromethane (10 mL) added trifluoroacetic acid: dichloromethane 1 :4 (5 mL) and the mixture was stirred at 25 °C for 30 minutes. The solution was concentrated and dried to give title compound (±)-(2i?,4i?)-2-(4-fluorophenyl)piperidin-4-ol (105 mg, 99% yield) as the trifluoracetic acid salt. 1H NMR (400 MHz, d6-DMSO): 7.56 (m, 2H), 7.31 (m, 2H), 4.53 (t, IH), 4.12 (br s, IH), 3.32 (q, IH), 3.20 (d, IH), 2.10 (t, IH), 1.85 (br d, 2H), 1.79 (dd, IH). MS (EI) for CHHI4FNO: 196 (MH+).
Reagent Preparation 17
3-(eM</ø)-(hydroxymethyl)-8-azabicyclo[3.2.1]octan-3-ol
[00106] STEP 1 : Potassium tert-butoxide (0.62 g, 5.5 mmol) was added to a suspension of methyltriphenylphosphonium bromide (1.98 g, 5.5 mmol) in tetrahydrofuran (20 mL) and the resulting mixture was stirred at room temperature for one hour. A solution of tert-butyl 3-oxo-8- azabicyclo[3.2.1]octane-8-carboxylate (0.50 g, 2.2 mmol) in tetrahydrofuran (5 mL) was then added and the resulting mixture was stirred at 350C for two hours. The mixture was cooled, diluted with hexane (100 mL), filtered, and the filtrate was washed with water then brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (20% ethyl acetate in hexanes) to give tert-butyl 3-methylene-8- azabicyclo[3.2.1]octane-8-carboxylate (0.45g, 91% yield). GC-MS (EI) for Ci3H2iNO2: 223 (M+).
[00107] STEP 2: To a solution of tert-butyl 3-methylene-8-azabicyclo[3.2.1]octane- carboxylate (0.9 g, 4.0 mmol) ( reagent preparation 18, step 1) in acetone (16 mL) and water (4 mL) was added osmium tetroxide (0.25 mL of a 4% aqueous solution, 0.04 mmol) and
N-methylmorpholine N-oxide (1.4 g, 12.0 mmol). The reaction mixture was stirred at room temperature for 15 h, concentrated, and the residue was partitioned between 20% citric acid and ethyl acetate. The organic layer was washed twice with brine, dried over sodium sulfate, filtered and concentrated to give tert-butyl 3-(hydroxy)-3-(e«<io)-(hydroxymethyl)-8- azabicyclo[3.2.1]octane-carboxylate (1.0 g, quantitative yield). MS (EI) for Ci3H23NO4: 257 (M+).
[00108] STEP 3 : A solution of tert-butyl 3-(hydroxy)-3-(en Jo)-(hydroxymethyl)-8- azabicyclo[3.2.1]octane-carboxylate (50 mg, 0.20 mmol) in dichloromethane (1 mL) and trifluoroacetic acid (1 mL) was stirred at room temperature for 1 h and then concentrated and dried to give 3-(en<io)-(hydroxymethyl)-8-azabicyclo[3.2.1]octan-3-ol as the trifluoroacetate salt, which was used without further purification.
Reagent Preparation 18
3-(4-bromophenyl)-l-(tetrahydro-2H-pyran-2-yl)-lH-pyrazole [00109] STEP 1 : To a heated (80 0C) solution of 3-(4-bromophenyl)-lH-pyrazole (1.0 g, 4.5 mmol) and trifluoroacetic acid (0.02 mL, 0.23 mmol) in toluene (5 mL) was added 3,4-dihydro- 2H-pyran (0.43 mL, 4.7 mol) over 1 hour. The reaction mixture was stirred for an additional hour and was then concentrated and dried to provide 3-(4-bromophenyl)-l-(tetrahydro-2H- pyran-2-yl)-lH-pyrazole (1.3 g, 94% yield). MS (EI) for Ci4H15BrN2O: 308 (MH+).
Reagent Preparation 19 2-(4-fluorophenyl)-4-oxopiperidine-l-carbonyl chloride
Figure imgf000038_0001
[00110] STEP 1 : Benzyl 2-(4-fluorophenyl)-4-oxo-3,4-dihydropyridine-l(2H)-carboxylate was prepared according to the method in (Tetrahedron Lett., 1986, 27, 4549-4552) using 4-methoxypyridine (29.8 mL, 290 mmol), benzyl chloroformate (50.0 mL, 350 mmol) and 4-fluorophenyl magnesium bromide (0.8 M solution in TΗF), (450 mL, 0.36 mmol), to yield (81 g, 86% yield) of the title compound. MS (EI) for Ci9Hi6FNO3: 326 (MH+). [00111] STEP 2: Benzyl 2-(4-fluorophenyl)-4-oxopiperidine- 1 -carboxylate was prepared according to the method described in (J. Am. Chem. Soc, 2001, 66, 2181-2182) using benzyl 2-(4-fluorophenyl)-4-oxo-3,4-dihydropyridine-l(2H)-carboxylate (16.5 g, 50.7 mmol) and zinc dust (9.8 g, 150 mmol) to afford (16.0 g, 96% yield) of the title compound. 1H NMR (400 MHz, CDCl3): 7.39-7.32 (m, 5H), 7.21 (m, 2H), 7.00 (t, 2H), 5.82 (br s, IH), 5.21 (dd, 2H), 4.28 (br s, IH), 3.15 (m, IH), 2.92 (m, IH), 2.88 (dd, IH), 2.54 (m, IH), 2.37 (m, IH). MS (EI) for Ci9Hi8FNO3: 328 (MH+).
[00112] STEP 3: A solution of benzyl 2-(4-fluorophenyl)-4-oxopiperidine- 1 -carboxylate (4.75 g, 14.50 mmol) in a mixture of ethyl acetate and tetrahydrofuran (1 : 1, 100 mL) was hydrogenated in the presence of 10% Pd/C at atmospheric pressure over 12h. The catalyst was filtered off and the filtrate was concentrated. The residue was dissolved in ethyl acetate (250 mL) and washed twice with saturated aqueous bicarbonate (100 mL), brine, then dried over anhydrous sodium sulfate, filtered and concentrated. The residue was dried to give 2-(4- fiuorophenyl)piperidin-4-one (2.8 g, quantitative). MS (EI) for CHHI2FNO: 194 (MH+). [00113] STEP 4: To a slurry of 2-(4-fluorophenyl)piperidin-4-one hydrochloride (2.8 g,
12.19 mmol) and N,N-diisopropylethylamine (6.4 mL, 36.57 mmol) in tetrahydrofuran (30 mL) at O0C was added phosgene (20% solution in toluene, 6.7 mL, 13.41 mmol) in tetrahydrofuran (30 mL) and the reaction mixture was stirred for 1 hour. It was quenched with water (2.0 mL) and the reaction mixture was concentrated. The residue was partitioned between water (100 mL) and ethyl acetate (250 mL). The organic layer was separated, washed with water (2x 100 mL) and brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The resulting crude was purified by gradient flash chromatography (5% to 20% ethyl acetate in hexane) to give 2-(4-fluorophenyl)-4-oxopiperidine- 1 -carbonyl chloride (2.85 g, 91%). GC-MS (EI) for CI2HI IC1FNO2: 255 (M+). [00114] Using analogous techniques and substituting alternative reagents in step 4 the following reagents were prepared. All starting materials were commercially obtained unless otherwise stated.
[00115] 4-(trifluoromethyl)piperidine-l -carbonyl chloride. Synthesized according to reagent preparation 19 by using 4-(trifluoromethyl)piperidine in step 4. GC-MS (EI) for C7HgClF3NO: 215 (M+).
[00116] 3-(enύ?o)-hydroxy-3-(trifluoromethyl)-8-azabicyclo[3.2.1]octane-8-carbonyl chloride. Synthesized according to the method of reagent preparation 19 using 3-(trifluoromethyl)-8- azabicyclo[3.2.1]octan-3-ol hydrochloride salt (reagent preparation 8) in step 4. GC-MS (EI) for C9HIiClF3NO2: 257 (M+). Reagent Preparation 20 6-bromo-2-methyl-l-(triphenylmethyl)-lH-iniidazo[4,5-/>]pyridine.
[00117] STEP 1 : To a solution of 6-bromo-2-methyl- lH-imidazo[4,5-6]pyridme (3.8 g, 17.5 mmol) in dimethylformamide (20 mL) at 0 0C was added 60% sodium hydride in mineral oil (1.05 g, 26.2 mmol) and the reaction mixture was stirred for 30 minutes, followed by the addition of a solution of triphenylmethyl chloride (5.4 g, 19.4 mmol) in dimethylformamide (10 mL). The reaction mixture was stirred at room temperature for 16 hours then it was quenched by the careful addition of water and diluted with ethyl acetate (450 mL), washed with 10% aqueous citric acid (2 x 100 mL), brine (250 mL), dried over anhydrous sodium sulfate and concentrated. Column chromatography (hexane ethyl acetate 9:1 to 4: 1) provided 6-bromo-2 -methyl- 1-
(triphenylmethyl)-lH-imidazo[4,5-έ]pyridine (0.51 g, 6%). 1H NMR (400 MHz, CDCl3): 7.99 (d, IH), 7.96 (d, IH), 7.33-7.23 (m, 15H), 2.00 (s, 3H). MS (EI) for C26H20BrN3: 454 (MH+).
Reagent Preparation 21 1,1-dimethylethyl 7-bromo-2,3-dihydro-l,4-benzoxazepine-4(5H)-carboxylate
Figure imgf000040_0001
[00118] STEP 1 : Commercially-available 5-bromo-2-hydroxybenzaldehyde (4.0 g, 10 mmol) and 2-aminoethanol were combined in THF/MeOH (100 mL, 10:1) and sodium borohydride (0.76 g, 2.0 mmol) was added with stirring. The resulting reaction mixture was stirred at 40 0C for 4 h, concentrated on a rotary evaporator then diluted with EtOAc (50 mL) and saturated NaHCO3 (30 mL). To this suspension was added di-tert-buty\ dicarbonate (2.83 g, 13 mmol). The mixture was stirred at rt overnight. The organic layer was washed with water, dried over anhydrous magnesium sulfate, filtered, and concentrated on a rotary evaporator. Hexane was subsequently added to the crude reaction product which resulted in the formation of a white solid. This slurry was filtered to obtain ter£-butyl-5-bromo-2-hydroxybenzyl(2- hydroxyethyl)carbamate (6.8 g, 98 %) as a white solid. MS (EI) for Ci4H20BrNO4, found 346 (MH+).
[00119] STEP 2: terϊ-Butyl-5-bromo-2-hydroxybenzyl(2-hydroxyethyl)carbamate (3.46 g, 10 mmol) and triphenylphosphine (3.96 g, 15 mmol) were combined in DCM (100 mL) and diisopropyl azodicarboxylate (3.03 g, 15 mmol) was added. The resulting reaction mixture was stirred at rt for 12 h. The reaction mixture was washed with water, dried, filtered, and concentrated on a rotary evaporator. The resulting crude product was purified via silica gel chromatography eluting with 8:2 hexane/ethyl acetate to give the desired product (1.74 g, 53 %) as a white solid. MS (EI) for Ci4Hi8BrNO3, found 328 (MH+). Reagent Preparation 22 4-(2,2-difluoroethyl)piperidine
[00120] STEP 1 : To a solution of ter£-butyl 4-(2-hydroxyethyl)piperidine-l-carboxylate (0.6 g, 2.6 mmol) in dichloromethane (30 mL) was added Dess-Martin-periodinane (1.2 g, 2.8 mmol), and the mixture was stirred at room temperature for 90 min. A 10% aqueous solution of sodium thiosulfate (15 mL) was added followed by saturated sodium bicarbonate (15 mL), and the biphasic mixture was stirred at room temperature for 1 h. The layers were separated, the aqueous layer was extracted twice with dichloromethane. The combined organic layers were washed with saturated sodium bicarbonate, and brine, dried over sodium sulfate,fϊltered and concentrated to afford tert-butyl 4-(oxoethyl)piperidine-l-carboxylate that was used directly without further purification
[00121] STEP 2: To a solution of tert-butyl 4-(oxoethyl)piperidine-l-carboxylate as obtained in step 1 in dichloromethane (50 mL) was added DAST (1.2 g, 7.8 mmol) at 0 0C. The reaction mixture was warmed to room temperature and stirred for 17 h. A 5% aqueous solution of sodium bicarbonate was added and the layers were separated. The organic layer was washed with saturated sodium bicarbonate, and brine, dried over sodium sulfate, filtered and concentrated to provide tert-butyl 4-(2,2-difiuoroethyl)piperidine-l-carboxylate that was used directly without further purification. [00122] STEP 3: tert-Butyl 4-(2,2-difluoroethyl)piperidine-l-carboxylate as obtained in step 2 was dissolved in a minimum of trifiuoroacetic acid and the resulting solution was stirred at room temperature for 2 h. The solution was then concentrated to give 4-(2,2- difluoroethyl)piperidine as the trifluoroacetate salt. MS (EI) for C7Hi3F2N: 150 (MH+).
Synthetic Example 1 4-{[2-(4-chlorophenyl)piperidin-l-yl]carbonyl}-7-(2-methyl-lH-benzimidazol-6-yl)-2,3,4,5- tetrahydro-l,4-benzoxazepine
[00123] STEP 1 : To 5-bromo-2-methylbenzimidazole (38 g, 180 mmol) in TΗF (400 mL) was added di-tert-butyl dicarbonate (39 g, 189 mmol). The reaction mixture was stirred at room temperature for 24 h and then concentrated. Ethyl acetate (400 mL) was added to the residue, and the solution was washed with 10% aqueous citric acid (2 x 100 mL), water (100 mL), and brine (100 mL), dried over sodium sulfate, and concentrated. Column chromatography on silica (gradient 20-30% ethyl acetate in hexane) provided 1,1 -dimethyl 6-bromo-2-methyl-lH- benzimidazole-1-carboxylate (27 g, 48% yield) as a beige solid. MS (EI) for Ci3Hi5BrN2O2: 312 (MH+) [00124] STEP 2: A solution of 1,1-dimethylethyl 7-bromo-2,3-dihydro-l,4-benzoxazepine- 4(5H)-carboxylate (reagent preparation 21) (30.0 g, 91.4 mmol) and triisopropyl borate (22.4 g, 119 mmol) in THF (300 mL) was cooled to -78 0C, and a 2.5M solution of n-butyllithium in hexanes (47.6 mL, 119 mmol) was added dropwise over 40 min at this temperature. The reaction mixture was stirred at -78 0C for an additional 30 min, then quenched by dropwise addition of 2 N hydrochloric acid (80 ml), and allowed to warm up to room temperature. Ethyl acetate (100 mL) and water (100 mL) were added, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate (100 mL). The combined organic layers were washed with water, dried over sodium sulfate, and concentrated. Hexane (200 mL) was added to the residue and the mixture was stirred overnight. The precipitate was filtered, washed several times with hexane, and dried to give (4-{[(l,l-dimethylethyl)oxy]carbonyl}-2,3,4,5-tetrahydro- 1 ,4-benzoxazepin-7-yl)boronic acid (23.4g, 87%) as a colorless solid. MS (EI) for Ci4H20BNO5: 294 (MH+).
[00125] STEP 3: A suspension of 1,1 -dimethylethyl 6-bromo-2-methyl-lH-benzimidazole-l- carboxylate (11.3 g, 36 mmol), (4-{[(l,l-dimethylethyl)oxy]carbonyl}-2,3,4,5-tetrahydro-l,4- benzoxazepin-7-yl)boronic acid (11.7 g, 40 mmol), dichloro[l,l-bis(diphenyl- phosphino]ferrocenepalladium (II) dichloromethane adduct (3.0 g, 10 mol %) in dioxane (115 mL) and water (28.5 mL) was degassed with nitrogen, and then diisopropylethylamine (18.6 g, 144 mmol) was added. The reaction mixture was stirred at 90 0C for 220 min, cooled to room temperature, and concentrated. Column chromatography on silica of the residue (gradient 25- 30% ethyl acetate in hexane) afforded 1,1-dimethyl 7-(l-{[(l,l-dimethylethyl)oxy]carbonyl}-2- methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepine-4(5H)-carboxylate (13.2 g, 76% yield) as an amorphous solid. MS (EI) for C27H33N3O5: 480(MH+).
[00126] STEP 4: A solution of 1 , 1 -dimethyl 7-(l-{[(l,l-dimethylethyl)oxy]carbonyl}-2- methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepine-4(5H)-carboxylate (13.1 g, 27 mmol) in a mixture of methanol (20 mL) and 4 N hydrogen chloride in dioxane (30 mL) was refluxed for 15 min. After cooling to room temperature ethyl ether (100 mL) was added, and the reaction mixture was concentrated. Another portion of ethyl ether (100 mL) was added, the precipitate was filtered off, washed several times with ethyl ether, and dried to give 7-(2-methyl- lH-benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine dihydrochloride (8.9 g, 93% yield) as a light beige solid. 1HNMR (400 MHz, CD3OD): 7.93(s, IH), 7.86-7.67(m, 4H), 7.28(s, IH), 4.54(s, 2H), 4.33-4.23(m, 2H), 3.65-3.54(m, 2H), 2.91(s, 3H); MS (EI) for CnH17N3O: 280 (MH+).
[00127] STEP 5: 7-(2-methyl-lH-benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine dihydrochloride (6.95 g, 19.73 mmol) was suspended in dichloromethane (10OmL) and cooled to 0 0C. To the resulting mixture was added DIPEA (19 mL, 109 mmol) followed by allyl chloroformate (4.6 mL, 43.4 mmol) and stirring was continued at 0 0C for 30 minutes then warmed to room temperature. The reaction mixture was then charged with additional DIPEA (3.4 mL) and allyl chloroformate (1 mL) then stirred an additional 30 minutes at room temperature. The resulting solution was then concentrated and the residue azeotroped once from methanol (100 mL). The residue was then taken back into methanol (100 mL) followed by portionwise addition of 2 M aqueous sodium hydroxide (20 mL) and the mixture was allowed to stir for 1 h at room temperature. The solution was then concentrated and the residue partitioned with chloroform and dilute brine. The organic phase was then dried over anhydrous sodium sulfate, filtered and concentrated to give prop-2-en-l-yl 7-(2-methyl-lH-benzimidazol-6-yl)-2,3- dihydro- 1 ,4-benzoxazepine-4(5H)-carboxylate as an amorphous residue which was carried forward directly into step 6.
[00128] STEP 6: prop-2-en-l-yl 7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4- benzoxazepine-4(5H)-carboxylate as obtained in step 5 was taken into TΗF (100 mL) followed by addition of pyridine (2.5 mL, 30 mml) and di-tert-butyl dicarbonate (4.9 g, 22.4 mmol) and the mixture was allowed to stir at room temperature over 12 h. The resulting solution was concentrated and the residue partitioned with ethyl acetate and 10% aqueous citric acid. The organic phase was washed twice with additional 10% aqueous citric acid then brine, dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography using 3:2 to 4:1 ethyl acetate in hexanes as eluent to afford 7-[l-[(l,l- dimethylethoxy)carbonyl]-2-methyl-lH-benzimidazol-6-yl]-2,3-dihydro-l,4-benzoxazepine- 4(5H)-carboxylic acid 2-propenyl ester (10.2 g) as a pale yellow amorphous residue. MS (EI) for C26H29N3O5: 465 (MH+).
[00129] STEP 7: 7-[l-[(l,l-dimethylethoxy)carbonyl]-2-methyl-lH-benzimidazol-6-yl]-2,3- dihydro- 1 ,4-benzoxazepine-4(5H)-carboxylic acid 2-propenyl ester (110 mg, 0.27 mmol) was taken into TΗF (1 mL) followed by addition of sodium triacetoxyborohydride (254 mg, 1.2 mmol) then tetrakis(triphenylphosphine)palladium (0) (6.1 mg, 0.005 mmol) and the mixture was stirred for 1 h at room temperature. The mixture was diluted with chloroform and partitioned with dilute aqueous sodium bicarbonate. The aqueous phase was extracted twice with chloroform and the combined organic layers were dried over anhydrous sodium sulfate then filtered and concentrated to give crude 1,1-dimethylethyl 2-methyl-6-(2,3,4,5-tetrahydro-l,4- benzoxazepin-7-yl)-lH-benzimidazole-l-carboxylate (109.5 mg) as an amorphous residue. MS (EI) for C22H25N3O3: 380 (MH+).
[00130] STEP 8: Phosgene (20 W% in toluene) (190 uL, 0.38 mmol) was added by syringe to a 0 0C cooled solution of pyridine (100 uL, 1.2 mmol) in chloroform (3 mL) followed by addition of 1 , 1 -dimethylethyl 2-methyl-6-(2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)-lH- benzimidazole-1-carboxylate as obtained in step 7 as a solution in chloroform (1 mL). The mixture was stirred for 15 minutes at 0 0C then partitioned with 10% aqueous citric acid. The organic phase was dried over anhydrous sodium sulfate then filtered and concentrated. The residue was purified by silica gel chromatography to give 1,1-dimethylethyl 6- [4- (chlorocarbonyl)-2,3 ,4,5-tetrahydro- 1 ,4-benzoxazepin-7-yl]- lH-benzimidazole- 1 -carboxylate (62.6 mg, 59% yield) as a yellow amorphous residue. MS (EI) for C23H24ClN3O4: 442 (MH+). [00131] STEP 9: 1,1-dimethylethyl 6-[4-(chlorocarbonyl)-2,3,4,5-tetrahydro-l,4- benzoxazepin-7-yl]-lH-benzimidazole-l -carboxylate (40 mg, 0.09 mmol), 2-(4- chlorophenyl)piperidine (reagent preparation 1) and DIPEA (78 uL, 0.45 mmol) in TΗF (2 mL) was heated at 60 0C over 12 h. The resulting solution was concentrated and the residue purified by silica gel chromatography using 20-50% ethyl acetate in hexanes as eluent to provide
. , 1 -dimethylethyl 6-(4- { [2-(4-chlorophenyl)piperidin- 1 -yljcarbonyl} -2,3 ,4,5 -tetrahydro- 1,4- benzoxazepin-7-yl)-2-methyl-lH-benzimidazole-l -carboxylate (38 mg, 70% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3): 8.14 (d, 0.4H), 7.94 (dd, 0.6H), 7.82 (d, 0.6H), 7.70 (dd, 0.4H), 7.57 -7.50 (m, 3H), 7.16 (dd, IH), 4.79 (s, IH), 4.71 (br s, IH), 4.20-4.13 (m, 3H), 4.03 (m, IH), 2.86 (s, 3H), 1.73 (s, 9H).
[00132] STEP 10: 1,1-dimethylethyl 6-(4-{ [2-(4-chlorophenyl)piperidin- l-yl]carbonyl}- 2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)-2-methyl-lH-benzimidazole-l-carboxylate as obtained in step 9 was taken into methanol (1 mL) followed by addition of 4 N hydrogen chloride in dioxane solution (1 mL) and the resulting solution was allow to stir for 2 h at room temperature. The solution was then concentrated and the residue purified by preparative reverse phase ΗPLC to give 4-{[2-(4-chlorophenyl)piperidin-l-yl]carbonyl}-7-(2-methyl-lH- benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine (17 mg, 55 % yield) as a colorless solid. 1H NMR (400 MHz, de-DMSO): 7.68 (s, 0.5H), 7.54-7.43 (m, 3.5H), 7.30-7.21 (m, 5H),
7.02 (d, IH), 4.59-4.48 (m, 4H), 4.21-4.09 (m, 3H), 3.64 (s, 3H), 3.05 (m, IH), 1.94 (m, IH), 1.78 (m, IH), 1.63-1.43 (br m, 5H). MS (EI) for C29H29ClN4O2: 502 (MH+).
[00133] Using analogous synthetic techniques and substituting with alternative starting reagents in step 9 the following compounds of the invention were prepared. Alternative starting materials were obtained commercially unless otherwise indicated. [00134] 7-(2-methyl-lH-benzimidazol-6-yl)-4-[(4-methylpiperidin-l-yl)carbonyl]-2,3,4,5- tetrahydro- 1,4-benzoxazepine. Synthesized according to the method of example 1 using 4- methylpiperidine in step 9. MS (EI) for C24H28N4O2: 406 (MH+).
[00135] 7-(2-methyl-lH-benzimidazol-6-yl)-4-[(2-methylpiperidin-l-yl)carbonyl]-2,3,4,5- tetrahydro- 1,4-benzoxazepine. Prepared according to the method of example 1 by using racemic 2-methylpiperidine in step 9. 1H NMR (400 MHz, methanol-d4): 7.64 (s, IH), 7.52 (d, 2H), 7.47 (dd, IH), 7.42 (dd, IH), 7.04 (d, IH), 4.48 (m, 2H), 4.20 (m, 2H), 3.95 (m, IH), 3.67 (m, 2H), 3.35 (m, IH), 3.12 (m, IH), 2.58 (s, 3H), 1.79-49 (m, 6H), 1.19 (d, 3H); MS (EI) for C24H28N4O2: 405 (MH+).
[00136] 7-(2-methyl-lH-benzimidazol-6-yl)-4-{[4-(trifluoromethyl)piperidin-l-yl]carbonyl}- 2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using 4-(trifluoromethyl)piperidine in step 9. 1H NMR (400 MHz, methanol-dt): 7.66 (s, IH), 7.53 (m, 2H), 7.47 (m, 2H), 7.04 (d, IH), 4.51 (s, 2H), 4.21 (m, 2H), 3.77 (m, 2H), 3.70 (m, 2H), 2.87 (m, 2H), 2.61 (s, 3H), 2.39 (m, IH), 1.86 (m, 2H), 1.58 (m, 2H); MS (EI) for C24H25F3N4O2: 459 (MH+). [00137] 4-[(4-fluoropiperidin-l-yl)carbonyl]-7-(2-methyl-lH-benzimidazol-6-yl)-2,3,4,5- tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using 4- fiuoropiperidine in step 9. 1H NMR (400 MHz, methanol-d4): 7.64 (s, IH), 7.53 (d, IH), 7.51 (s, IH), 7.47 (dd, IH), 7.43 (dd, 1H),7.O4 (d, IH) 4.52 (s, 2H), 4.21 (m, 2H), 3.70 (m, 2H), 3.41 (m, 2H), 3.24 (m, 2H), 2.58 (s, 3H), 1.93 (m, 3H), 1.81 (m, 2H); MS (EI) for C23H25FN4O2: 409 (MH+). [00138] l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl}piperidine-4-carbonitrile. Prepared according to the method of example 1 by using piperidine-4-carbonitrile in step 9. 1H NMR (400 MHz, methanol^): 7.67 (s, IH), 7.55 (d, IH), 7.52 (d, IH), 7.47 (m, 2H), 7.04 (d, IH), 4.52 (s, 2H), 4.21 (m, 2H), 3.70 (m, 2H), 3.47 (m, 2H), 3.15 (m, 2H), 2.99 (m, IH), 2.61 (s, 3H), 1.97 (m, 2H), 1.83 (m, 2H); MS (EI) for C24H25N5O2: 416 (MH+).
[00139] 3-(l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl}piperidin-4-yl)propan-l-ol. Prepared according to the method of example 1 by using 3-(piperidin-4-yl)propan-l-ol in step 9. 1H NMR (400 MHz, methanol-d4): 7.65 (s, IH), 7.52 (m, 2H), 7.47 (dd, IH), 7.43 (dd, IH), 7.04 (d, IH), 4.49 (s, 2H), 4.20 (m, 2H), 3.69 (m, 4H), 3.54 (t, 2H), 2.82 (t, 2H), 2.58 (s, 3H), 1.73 (m, 2H), 1.56 (m, 2H), 1.47 (m, IH), 1.34 (m, 2H), 1.23 (m, 2H); MS (EI) for C26H32N4O3: 449 (MH+).
[00140] 4-[(4-ethylpiperidin-l-yl)carbonyl]-7-(2-methyl-lH-benzimidazol-6-yl)-2,3,4,5- tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using 4- ethylpiperidine in step 9. 1H NMR (400 MHz, methanol-d4): 7.64 (s, IH), 7.52 (m, 2H), 7.47 (dd, IH), 7.43 (d, IH), 7.04 (d, IH), 4.49 (s, 2H), 4.20 (m, 2H), 3.69 (m, 4H), 2.82 (m, 2H), 2.59 (s, 3H), 1.72 (m, 21H), 1.32 (m, 3H), 1.20 (m, 2H), 0.92 (t, 3H); MS (EI) for C25H30N4O2: 419 (MH+).
[00141] (±)-4-{[(25,45)-2,4-dimethylpiperidin-l-yl]carbonyl}-7-(2-methyl-lH-benzimidazol- 6-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using racemic 2,4-dimethylpiperidine in step 9 and purification of the title compound by preparatory reverse phase HPLC (Phenomenex Luna 150x3 Omm, 0.1% aqueous ammonium acetate-acetonitrile 20-90%, 15min, retention time 8.7 min). 1H NMR (400 MHz, methanol-d4): 7.68 (s, IH), 7.56 (d, IH), 7.52-7.46 (m, 3H), 7.05 (d, IH), 4.46 (m, 2H), 4.21 (m, 2H), 4.07 (m, IH), 3.65 (m, 2H), 3.47 (m, IH), 3.12 (m, IH), 2.63 (s, 3H), 1.85 (m, IH), 1.64 (m, IH), 1.45 (m, IH), 1.37 (m, IH), 1.21 (d, 3H), 1.16 (m, IH), 0.93 (d, 3H); MS (EI) for C25H30N4O2: 419 (MH+).
[00142] (±)-4- { [(2,S,4i?)-2,4-dimethylpiperidin- 1 -yl]carbonyl} -7-(2-methyl- lH-benzimidazol- 6-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using racemic 2,4-dimethylpiperidine in step 9 and purification of the title compound by preparatory reverse phase ΗPLC (Phenomenex Luna 150x30mm, 0.1% aqueous ammonium acetate-acetonitrile 20-90%, 15min, retention time 9.5 min). 1H NMR (400 MHz, methanol-d4): 7.66 (m, IH), 7.53 (m, 2H), 7.47 (dd, IH), 7.44 (dd, IH), 7.05 (d, IH), 4.70 (d, IH), 4.60 (d, IH), 4.19 (m, IH), 4.11 (m, IH), 3.94 (m, IH), 3.79 (m, IH), 3.27 (m, IH), 2.96 (m, IH), 2.69 (m, IH), 2.59 (s, 3H), 1.65 (m, 2H)1.51 (m, IH), 1.27 (m, IH), 1.02 (m, IH), 0.95 (t, 3H), 0.94 (t, 3H); MS (EI) for C25H30N4O2: 419 (MH+)
[00143] 7-(2-methyl-lH-benzimidazol-6-yl)-4-{[4-(methylthio)piperidin-l-yl]carbonyl}- 2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using 4-(methylthio)piperidine (synthesized according to reagent preparation 7) in step 9. 1H NMR (400 MHz, DMSO-de): 7.66 (m, IH), 7.54 (m, 2H), 7.48 (m, IH), 7.40 (m, IH), 7.01 (m, IH), 4.44 (s, 2H), 4.18 (m, 2H), 3.59 (m, 2H), 3.51 (m, 2H), 2.80 (m, 3H), 2.54 (m, 3H), 2.05 (m, 3H), 1.92 (m, 3H), 1.45 (m, 2H); MS (EI) for C24H28N4O2S: 437 (MH+). [00144] l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl}-4-[3-(trifluoromethyl)phenyl]piperidin-4-ol. Prepared according to the method of example 1 by using 4-(3-(trifluoromethyl)phenyl)piperidin-4-ol in step 9. 1H NMR (400 MHz, methanol-d4): 7.85 (s, IH), 7.71 (d, IH), 7.64 (s, IH), 7.56-7.40 (m, 6H), 7.05 (d, IH), 4.55 (s, 2H), 4.23 (m, 2H), 3.74 (m, 2H), 3.64 (m, 2H), 3.39 (m, 2H), 2.57 (s, 3H), 2.12 (m, 2H), 1.70 (m, 2H); MS (EI) for C30H29F3N4O3: 551 (MH+).
[00145] 7-(2-methyl-lH-benzimidazol-6-yl)-4-{[2-(trifluoromethyl)piperidin-l-yl]carbonyl}- 2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using racemic 2-(trifluoromethyl)piperidine (synthesized according to reagent preparation 3) in step 9. 1H NMR (400 MHz, methanol-d4): 7.68 (s, IH), 7.55 (m, IH), 7.49 (m, 3H), 7.05 (d, IH), 4.64 (m, IH), 4.51 (m, 2H), 4.31 (m, IH), 4.10 (m, IH), 3.70 (m, 2H), 3.60 (m, IH), 2.62 (s, 3H), 2.01 (m, IH), 1.85 (m, IH), 1.71 (m, 2H), 1.48 (m, IH); MS (EI) for C24H25N4O2: 459 (MH+). [00146] 7-(2-methyl-lH-benzimidazol-6-yl)-4-[(4-propylpiperidin-l-yl)carbonyl]-2,3,4,5- tetrahydro-l,4-benzoxazepine. Prepared as the acetate salt according to the method of example 1 by using 4-propylpiperidine in step 9. 1H NMR (400 MHz, methanol-d4): 7.63 (m, IH), 7.53- 7.39 (m, 4H), 7.02 (d, IH), 4.46 (s, 2H), 4.18 (m, 2H), 3.66 (m, 4H), 2.79 (m, 2H), 2.58 (s, 3H), 1.98 (s, 3H), 1.68 (m, 2H), 1.44 (m, IH), 1.33 (m, 2H), 1.28-1.12 (m, 4H), 0.89 (t, 3H); MS (EI) for C26H32N4O2: 433 (MH+). [00147] 4-(2-azabicyclo[2.2.2]oct-2-ylcarbonyl)-7-(2-methyl-lH-benzimidazol-6-yl)-2,3,4,5- tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using 2- azabicyclo[2.2.2]octane in step 9. 1H NMR (400 MHz, DMSO-d6): 12.30 (br. s, IH), 7.62 (br. s, IH), 7.53 (d, IH), 7.50 (m, IH), 7.46 (dd, IH), 7.36 (dd, IH), 7.00 (d, IH), 4.42 (s, 2H), 4.18 (m, 2H), 3.56 (m, 2H), 1.91 (s, 3H), 1.89 (m, IH), 1.81 (m, IH), 1.57 (m, 6H); MS (EI) for C25H28N4O2: 417 (MH+).
[00148] 4-[(4,4-dimethylpiperidin-l-yl)carbonyl]-7-(2-methyl-lH-benzimidazol-6-yl)-2,3,4,5- tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using 4,4- dimethylpiperidine in step 9. 1H NMR (400 MHz, methanol-d4): 7.64 (s, IH), 7.52 (m, 2H), 7.47 (dd, IH), 7.43 (dd, IH), 7.04 (d, IH), 4.49 (s, 2H), 4.20 (m, 2H), 3.67 (m, 2H), 3.27 (m, 4H), 2.58 (s, 3H), 1.41 (m, 4H), 0.99 (s, 6H); MS (EI) for C25H30N4O2: 419 (MH+).
[00149] 4-(8-azabicyclo[3.2.1]oct-8-ylcarbonyl)-7-(2-methyl-lH-benzimidazol-6-yl)-2,3,4,5- tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 using 8- azabicyclo[3.2.1]octane step 9. 1H NMR (400 MHz, methanol-d4): 7.63 (s, IH), 7.53-7.39 (m, 4H), 7.03 (d, IH), 4.59 (s, 2H), 4.21 (t, 2H), 4.06 (br s, 2H), 3.75 (t, 2H), 2.57 (s, 3H), 1.92-1.49 (m, 10H); MS (EI) for C25H28N4O2: 417 (MH+).
[00150] 4- {[4-(difluoromethyl)piperidin-l-yl]carbonyl}-7-(2 -methyl- lH-benzimidazol-6-yl)- 2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using 4-(difluoromethyl)piperidine (synthesized according to reagent preparation 4) in step 9. 1H NMR (400 MHz, methanol-d4): 7.68 (s, IH), 7.56 (d, IH), 7.49 (m, 3H), 7.04 (d, IH), 4.51(s, 2H), 4.20 (m, 2H), 3.75 (m, 2H), 3.70 (m, 2H), 2.84 (m, 2H), 2.63 (s, 3H), 1.99 ( (m, 2H), 1.75 (m, 2H), 1.47 (m, 2H); MS (EI) for C24H26F2N4O2: 441 (MH+).
[00151] N-methyl-7-(2-methyl- lH-benzimidazol-6-yl)-N-[(15)- 1 -phenylethyl]-2,3-dihydro- l,4-benzoxazepine-4(5H)-carboxamide. Prepared according to the method of example 1 by using (,S)-N-methyl-l-phenylethanamine in step 9. 1H ΝMR (400 MHz, methanol-d4): 7.50 (d, IH), 7.41 (d, IH), 7.37 (dd, IH), 7.26 (dd, IH), 7.23 (d, IH), 7.18 (m, 2H), 7.11 (m, 3H), 6.96 (d, IH), 5.01 (q, IH), 4.41 (m, 2H), 4.15 (m, 2H), 3.61 (m, 2H), 2.55 (s, 3H), 2.49 (s, 3H), 1.44 (d, 3H); MS (EI) for C27H28N4O2: 441 (MH+).
[00152] 4- {[4-(fluoromethyl)piperidin-l-yl]carbonyl}-7-(2 -methyl- lH-benzimidazol-6-yl)- 2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using 4-(fiuoromethyl)piperidine (synthesized according to reagent preparation 5) in step 9. 1H NMR (400 MHz, methanol-d4): 7.71 (s, IH), 7.60 (d, IH), 7.55 (dd, IH), 7.52 (d, IH), 7.48 (dd, IH), 7.05 (d, IH), 4.50 (s, 2H), 4.35 (d, IH), 4.23 (d, IH), 4.20 (m, 2H), 3.72 (m, 4H), 2.85 (m, 2H), 2.68 (s, 3H), 1.88 (m, IH), 1.72 (m, 2H), 1.36 (m, 2H); MS (EI) for C24H27FN4O2: 423 (MH+). [00153] 4- { [4-( 1 , 1 -difluoroethyl)piperidin- 1 -yl] carbonyl} -7-(2 -methyl- lH-benzimidazol-6- yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using 4-(l,l-difluoroethyl)piperidine (synthesized according to reagent preparation 6) in step 9. 1H NMR (400 MHz, methanol-d4): 7.67 (s, IH), 7.53 (m, 2H), 7.47 (m, 2H), 7.04 (d, IH), 4.50 (s, 2H), 4.21 (m, 2H), 3.77 (m, 2H), 3.69 (m, 2H), 2.83 (m, 2H), 2.61 (s, 3H), 1.80 (m, 2H), 1.55 (t, 3H), 1.48 (m, 2H); MS (EI) for C25H28F2N4O2: 455 (MH+). [00154] 7-(2-methyl- 1 H-benzimidazol-6-yl)-4- { [4-( 1 -methylethyl)piperidin- 1 -yl]carbonyl} - 2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using 4-isopropylpiperidine in step 9. 1H NMR (400 MHz, DMSO-d6): 12.23 (br. s, IH), 7.72-7.42 (m, 4H), 7.35 (m, IH), 7.01 (d, IH), 4.42 (s, 2H), 4.17 (m, 2H), 3.59 (m, 4H), 2.67 (m, 2H), 1.66 (m, 2H), 1.42 (m, IH), 1.17 (m, 3H), 0.85 (d, 6H); MS (EI) for C26H32N4O2: 433 (MH+). [00155] 8-{[7-(2-Methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl}-3-(trifluoromethyl)-8-azabicyclo[3.2.1]octan-3-(endo)-ol. Prepared according to the method of example 1 by using 3-(trifluoromethyl)-8-azabicyclo[3.2.1]octan-3-(endo)-ol hydrochloride (reagent preparation 8) in step 9. (400 MHz, methanol-d4): 7.63 (br, IH), 7.50 (d, IH), 7.47 to 7.44 (m, 2H), 7.41 (dd, IH), 7.03 (d, IH), 4.57 (s, 2H), 4.21 (m, 2H), 4.15 (br, 2H), 3.75 (m, 2H), 2.21 (s, 3H), 2.21 to 2.11 (m, 4H), 1.91 to 1.86 (m, 2H), 1.78 (d, 2H); MS (EI) for C26H27F3N4O3: 501 (MH+).
[00156] 3-Methyl-8-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin- 4(5H)-yl]carbonyl}-8-azabicyclo[3.2.1]octan-3-(endo)-ol. Prepared according to the method of example 1 by using 3-methyl-8-azabicyclo[3.2.1]octan-3-(endo)-ol hydrochloride (reagent preparation 9) in step 9. (400 MHz, methanol-d4): 7.54 (d, IH), 7.41 (m, IH), 7.38 (m, IH), 7.35 (d, IH), 7.32 (dd, IH), 6.93 (d, IH), 4.47 (s, 2H), 4.11 (m, 2H), 3.96 (m, 2H), 3.64 (m, 2H), 2.48 (s, 3H), 2.07 (q, 2H), 2.07 (dd, 2H), 1.73 to 1.68 (m, 2H), 1.65 (d, 2H), 1.06 (s, 3H); MS (EI) for C26H30N4O3: 447 (MH+). [00157] 4-[(3-Fluoro-3-(CTύfo)-methyl-8-azabicyclo[3.2.1]oct-8-yl)carbonyl]-7-(2-methyl-lH- benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using 3-fluoro-3-(endo)-methyl-8-azabicyclo[3.2.1]octane hydrochloride (reagent preparation 10) in step 9. (400 MHz, methanol-d4): 7.64 (br, IH), 7.53 to 7.49 (m, 2H), 7.48 (dd, 2H), 7.43 (dd, IH), 7.04 (d, IH), 4.61 (s, 2H), 4.22 (m, 2H), 4.13 (br, 2H), 3.77 (m, 2H), 2.57 (s, 3H), 2.40 (dd, IH), 2.30 (dd, IH), 1.96 to 1.87 (m, 4H), 1.77 to 1.70 (m, 2H), 1.56 to 1.49 (d, 3H); MS (EI) for C26H29FN4O2: 449 (MH+). [00158] (±)-[(2R,41S)-l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin- 4(5H)-yl]carbonyl}-2-phenylpiperidin-4-yl]methanol. Prepared according to the method of example 1 by using (±)-(2i?,4S)-2-phenylpiperidin-4-yl)methanol (reagent preparation 2) in step 9. 1H NMR (400 MHz, d6-DMSO): 12.20 (br s, IH), 7.65 (s, IH), 7.55 (d, IH), 7.50 (m, IH), 7.47 (d, IH), 7.43 (dd, IH), 7.33 (m, IH), 7.11 (dd, 2H), 7.07-7.00 (m, 3H), 5.18 (t, IH), 4.48 (s, 2H), 4.08 (m, 2H), 3.98 (m, 2H), 3.84 (m, 2H), 3.70 (m, 2H), 2.51 (s, 3H), 1.84 (m, 2H), 1.71 (m, IH), 1.58 (m, 2H). MS (EI) C30H32N4O3: 497 (MH+).
[00159] 7-(2-methyl-lH-benzimidazol-6-yl)-4-[(l-phenyl-3,4-dihydroisoquinolin-2(lH)- yl)carbonyl]-2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using 1 -phenyl- 1,2, 3, 4-tetrahydroisoquino line in step 9. 1H NMR (400 MHz, d3-Me0H): 7.58 (d, IH), 7.49 (m, 2H), 7.32 (dd, IH), 7.24 (s, IH), 7.21 (m, 3H), 7.12 (m, 3H), 7.06 (d, IH), 7.01 (m, 3H), 6.82 (d, IH), 6.03 (s, IH), 4.54 (dd, 2H), 4.25 (m, 2H), 3.70 (m, 3H), 3.30 (m, IH), 3.00 (m, IH), 2.85 (2m, IH), 2.59 (s, 3H). MS (EI) C33H30N4O2: 515 (MH+). [00160] 4-{[3-(en<io)-(Fluoromethyl)-8-azabicyclo[3.2.1]oct-8-yl]carbonyl}-7-(2-methyl-lH- benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 1 by using 3-(endo)-(fluoromethyl)-8-azabicyclo[3.2.1]octane hydrochloride (reagent preparation 11) in step 9. (400 MHz, methanol-d4): 7.64 (br, IH), 7.54 to 7.49 (m, 2H), 7.45 (dd, 2H), 7.42 (d, IH), 7.05 (d, IH), 4.60 (s, 2H), 4.37 (d,d, 2H), 4.22 (m, 2H), 4.08 (br, 2H), 3.77 (m, 2H), 2.58 (s, 3H), 2.27 to 2.17 (m, 3H), 1.96 to 1.87 (m, IH), 1.67 to 1.58 (m, 2H), 1.56 to 1.49 (m, 2H); MS (EI) for C26H29FN4O2: 449 (MH+).
Synthetic Example 2 4-(3-fluorophenyl)- 1- { [7-(2-methyl- lH-benzimidazol-6-yl)-2,3-dihydro- 1,4-benzoxazepin-
4(5H)-yl] carbonyl}piperidin-4-ol [00161] STEP 1 : 7-(2-methyl-lH-benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine dihydrobromide (prepared as in step 5 example 1) (9.16 g, 20.76 mmol) was suspended in dichloromethane (10OmL) followed by addition of DIPEA (12.6 mL, 72.7 mmol) and pyridine (1.7 mL, 20.8 mmol). Di-tert-butyl dicarbonate (10.0 g, 45.7 mmol) and the solution was stirred for 12 h at room temperature. The mixture was concentrated and the residue partitioned with ethyl acetate and 10% aqueous citric acid. The organic phase was washed twice with additional 10% aqueous citric acid then brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue of 1,1-dimethyl 7-(l-{[(l,l-dimethylethyl)oxy]carbonyl}-2-methyl- lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepine-4(5H)-carboxylate was taken into methanol (100 mL) followed by addition of sodium hydroxide (1.0 g, 25 mmol) in water (10 mL). The mixture was stirred for 1 h at room temperature then concentrated. The residue was partitioned with ethyl acetate and 1 : 1 brine: 10% aqueous citric acid. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give 1 , 1 -dimethylethyl 7-(2- methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepine-4(5H)-carboxylate (9.6 g) as an amorphous residue. MS (EI) for C22H25N3O3: 380(MH+).
[00162] STEP 2: 1 , 1 -dimethylethyl 7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4- benzoxazepine-4(5H)-carboxylate as prepared in step 1 (9.6 g) was taken into dichloromethane (100 mL) followed by sequential addition of DIPEA (4.3 mL, 24.9 mmol) and isobutyl chloroformate (2.7 mL, 20.8 mmol). The mixture was stirred for 1 h at room temperature then partitioned with 0.5 N aqueous hydrochloric acid . The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to afford 1 , 1 -dimethylethyl 7-(2- methyl- 1 -[ {(2-methylpropyl)oxy]carbonyl} -lH-benzimidazol-6-yl-2,3-dihydro-l ,4- benzoxazepine-4(5Η)-carboxylate (10.3 g) as an amorphous residue. MS (EI) for C27H33N3O5:
480(MH+).
[00163] STEP 3: 1 , 1 -dimethylethyl 7-(2-methyl-l-[{(2-methylpropyl)oxy]carbonyl}-lH- benzimidazol-6-yl-2,3-dihydro-l,4-benzoxazepine-4(5H)-carboxylate as obtained in step 2 (10.3 g) was taken into 1 : 1 TFA: dichloromethane (100 mL) and the resulting solution was stirred for 1 h at room temperature then concentrated. The residue was treated with saturated aqueous sodium bicarbonate (100 mL) and the aqueous mixture was treated with portion wise solid sodium bicarbonate until pΗ 8.5. The aqueous mixture was then saturated with sodium chloride and partitioned with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated then dried in vacuo. The residue was taken into 1 :1 ethyl acetate:ethyl ether then washed with dilute aqueous sodium bicarbonate, water then brine and dried over anhydrous sodium sulfate. Silica gel was added to the mixture and stirred for 5 minutes then filtered through a celite bed. The filtrate was concentrated to provide 2-methylpropyl 2-methyl- 6-(2,3,4,5-tetramethyl-l,4-benzoxazepin-7-yl)-lH-benzimidazole-l-carboxylate (6.8 g, 86% overall yield).
[00164] Step 4: Phosgene (20 W% in toluene, 9.5 mL) was diluted into dichloromethane (40 mL) and the resulting solution cooled to 0 0C. 2-Methylpropyl 2-methyl-6-(2,3,4,5-tetramethyl- l,4-benzoxazepin-7-yl)-lH-benzimidazole-l-carboxylate as obtained in step 3 (6.8 g, 17.9 mmol)) was taken into dichloromethane (30 mL) followed by addition of DIPEA (7.8 mL, 44.8 mmol) and the resulting solution was slowly added to the cooled phosgene solution over 5 minutes by addition funnel. The mixture was stirred an additional 5 minutes at 0 0C then allowed to warm to room temperature and concentrated. The residue was partitioned with ethyl acetate and 10% aqueous citric acid then washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The resulting residue was purified by silica gel chromatography using 50% ethyl acetate in hexanes as eluent to give 2-methylpropyl 6-[4- (chlorocarbonyl)-2,3 ,4,5-tetrahydro- 1 ,4-benzoxazepin-7-yl)- lH-benzimidazole- 1 -carboxylate (3.73 g) as an amorphous solid.
[00165] STEP 5: 2-methylpropyl 6-[4-(chlorocarbonyl)-2,3,4,5-tetrahydro-l,4-benzoxazepin- 7-yl)-lH-benzimidazole-l -carboxylate (100 mg, 0.09 mmol) and 4-(3-fluorophenyl)piperidin-4- ol (prepared according to the method given in WO 2005118587) (88 mg, 0.45 mmol) were taken into dichloromethane (3 mL) and the mixture was stirred for 1 h at room temperature. The reaction mixture was treated with 5% aqueous citric acid and the aqueous phase extracted twice with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate then filtered and concentrated. The residue was chromatographed on silica gel using 3% methanol in dichloromethane as eluent. Product fractions were pooled and concentrated and the residue taken into methanol solution followed by addition of solid potassium carbonate. The mixture was stirred for 12 h at room temperature then diluted with saturated aqueous ammonium chloride. The aqueous mixture was extracted with ethyl acetate and the combined organic layers dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by preparative reverse phase ΗPLC to give 4-(3 -fluorophenyl)- 1 - { [7-(2 -methyl- lH-benzimidazol-6- yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)-yl]carbonyl}piperidin-4-ol (10 mg, 22% yield). 1H NMR (400 MHz, methanol-d4): 7.64 (m, IH), 7.54 (m, IH), 7.51 (d, IH), 7.47 (dd, IH), 7.42 (dd, IH), 7.33-7.22 (m, 3H), 7.05 (d, IH), 6.94 (m, IH), 4.54 (s, 2H), 4.23 (m, 2H), 3.73 (m, 2H), 3.62 (m, 2H), 3.36 (m, 2H), 2.58 (s, 3H), 2.08 (m, 2H), 1.69 (m, 2H). [00166] Using analogous synthetic techniques and substituting with alternative starting reagents in steps 2 and/or 5 the following compounds of the invention were prepared. Alternative starting materials were obtained commercially unless otherwise indicated. [00167] l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl}-4-(trifluoromethyl)piperidin-4-ol. Prepared according to the method of example 2 by using methyl chloroformate in step 2 and 4-(trifluoromethyl)piperidin-4-ol (reagent preparation 12) in step 5. 1H NMR (400 MHz, DMSO-de): 12.30 (br. s, IH), 7.72-7.42 (m, 4H), 7.35 (m, IH), 7.00 (d, IH), 6.04 (br. s, IH), 4.46 (s, 2H), 4.18 (m, 2H), 3.62 (m, 2H), 3.49 (m, 2H), 3.02 (m, 2H), 1.66 (m, 4H); MS (EI) for C24H25F3N4O3: 475 (MH+). [00168] 8-{[7-(2-Methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl}-8-azabicyclo[3.2.1]octan-3-one. Prepared according to the method of example 2 using 8-azabicyclo[3.2.1]octan-3-one in step 5. 1H NMR (400 MHz, DMSO-d6): 7.63 (br, IH), 7.57 (d, IH), 7.50-7.46 (m, 2H), 7.36 (dd, IH), 7.00 (d, IH), 4.63 (s, 2H), 4.23 (br, 2H), 3.40 (br, 2H), 2.81 (dd, 4H), 2.45 (s, 3H), 2.19 (dd, 2H), 1.90 (m, 2H), 1.54 (m, 2H); MS (EI) for C25H26N4O3: 431(MH+). [00169] 4-Methyl-l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin- 4(5H)-yl]carbonyl}piperidine-4-carbonitrile. Prepared according to the method of example 2 by using 4-methylpiperidine-4-carbonitrile hydrochloride (reagent preparation 13) in step 5. 1H NMR (400 MHz, methanol-d4): 7.64 (br, IH), 7.53 to 7.50 (m, 2H), 7.45 (dd, IH), 7.43 (dd, IH), 7.02(d, IH), 4.50 (s, 2H), 4.19 (m, 2H), 3.71 to 3.64 (m, 4H), 3.07 (t, 2H), 2.58 (s, 3H), 1.92 to 1.86 (m, 2H), 1.66 to 1.57 (m, 2H), 1.40 (s, 3H); MS (EI) for C25H27N5O2: 430 (MH+). [00170] l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl}-4-phenylpiperidin-4-ol. Prepared according to the method of example 2 by using methyl chloro formate in step 2 and 4-phenylpiperidin-4-ol in step 5. 1H NMR (400 MHz, methanol-d4): 7.87 (m, IH), 7.80 (m, IH), 7.75 (d, IH), 7.59 (m, IH), 7.54-7.45 (m, 3H), 7.31 (m, 2H), 7.21 (m, IH), 7.09 (d, IH), 4.57 (s, 2H), 4.26 (m, 2H), 3.76 (m, 2H), 3.59 (m, 2H), 3.36 (m, 2H), 2.86 (s, 3H), 2.09 (m, 2H), 1.70 (m, 2H); MS (EI) for C29H30N4O3: 483 (MH+). [00171] 8-{[7-(2-Methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl}-8-azabicyclo[3.2.1]octan-3-(exo)-ol. Prepared according to the method of example 2 by using 8-azabicyclo[3.2.1]octan-3-ol hydrochloride (reagent preparation 14) in step 5 then purification by reverse phase preparative ΗPLC (acetonitrile-aqueous ammonium acetate mobile phase) to give exo-isomer, tr= 9.82 minutes. 1H NMR (400 MHz, methanol-d4): 7.64 (br, IH),
7.52 to 7.41 (m, 4H), 7.02 (d, IH), 4.60 (s, 2H), 4.23 (m, 2H), 4.10 (br, 2H), 4.02 (m, IH), 3.76 (m, 2H), 2.57 (s, 3H), 1.94 to 1.85 (m, 4H), 1.72 to 1.64 (m, 4H); MS (EI) for C25H28N4O3: 433 (MH+).
[00172] 8-{[7-(2-Methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl}-8-azabicyclo[3.2.1]octan-3-ol. Prepared according to the method of example 2 by using 8-azabicyclo[3.2.1]octan-3-ol hydrochloride (reagent preparation 14) in step 5 then purification by reverse phase preparative ΗPLC (acetonitrile-aqueous ammonium acetate mobile phase) to give exo-isomer, tr= 10.35 minutes. 1H NMR (400 MHz, methanol-d4): 7.64 (br, IH),
7.53 to 7.40 (m, 4H), 7.03 (d, IH), 4.58 (s, 2H), 4.21 (m, 2H), 4.10 to 4.02 (m, 3H), 3.74 (m, 2H), 2.58 (s, 3H), 2.21 to 2.12 (m, 4H), 1.88 to 1.76 (m, 4H); MS (EI) for C25H28WO3: 433 (MH+).
[00173] 7-(2-methyl-lH-benzimidazol-6-yl)-4-{[2-(4-methylphenyl)piperidin-l-yl]carbonyl}- 2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 2 by using methyl chloroformate in step 2 and racemic 2-p-tolylpiperidine in step 5. 1H NMR (400 MHz, d6-DMSO): 7.82 (s, IH), 7.76 (d,lH), 7.60 (dd, IH), 7.54 (dd, IH), 7.43 (m, IH), 7.16 - 7.02 (m, 5H), 4.63 (t, IH), 4.51 (m, 2H), 4.19 (m, 2H), 3.75-3.40 (m, 5H), 396 (m, IH), 276 (s, 3H), 2.22 (s, 3H), 2.07-2.01 (m, IH), 1.78 (m, IH), 1.60-1.36 (m, 4H); MS (EI) for C3iH33N3O2: 480 (MH+). [00174] 4-{[4-(2,2-difluoroethyl)piperidin-l-yl]carbonyl}-7-(2-methyl-lH-benzimidazol-6- yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 2 by using methyl chloroformate in step 2 and 4-(2,2-difluoroethyl)piperidine (reagent preparation 22) in step 5. 1H NMR (400 MHz, methanol-d4): 7.65 (s, IH), 7.55-7.41 (m, 4H), 7.04 (m, IH), 5.97 (m, IH), 4.50 (s, 2H), 4.19 (m, 2H), 3.69 (m, 4H), 2.84 (m, 2H), 2.58 (s, 3H), 1.87-1.71 (m, 5H), 1.33 (m, 2H); MS (EI) for C25H28F2N4O2: 455 (MH+).
[00175] 4-(7-azabicyclo[2.2.1 ]hept-7-ylcarbonyl)-7-(2-methyl-lH-benzimidazol-6-yl)- 2,3,4,5-tetrahydro-l,4-benzoxazepine. Synthesized according to the method of example 2 using methyl chloroformate in step 2 and azabicyclo[2.2.1]heptane in step 5. 1H NMR (400 MHz, DMSO-de): 12.24 (bs, IH), 7.40-7.72 (m, 4H), 7.34 (d, IH), 6.99 (d, IH), 4.56 (s, 2H), 4.14- 4.22 (m, 2H), 3.94 (s, 2H), 3.67-3.77 (m, 2H), 3.34 (s, 3H), 1.66-1.76 (m, 4H), 1.30-1.41 (m, 4H); MS (EI) for C24H26N4O2: 403 (MH+).
[00176] 4-[(4-cyclopropylpiperidin-l-yl)carbonyl]-7-(2 -methyl- lH-benzimidazol-6-yl)- 2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 2 by using methyl chloroformate in step 2 and 4-cyclopropylpiperidine (reagent preparation 3) in step 5. 1H NMR (400 MHz, methanol-d4): 7.87 (s, IH), 7.78 (m, 2H), 7.56 (d, IH), 7.51 (dd, IH), 7.08 (d, IH), 4.51 (s, 2H), 4.23 (m, 2H), 3.68 (m, 4H), 2.86 (s, 3H), 2.76 (m, 2H), 1.75 (m, 2H), 1.37 (m, 2H), 0.69 (m, IH), 0.56 (m, IH), 0.40 (m, 2H), 0.10 (m, 2H); MS (EI) for C26H30N4O2: 431 (MH+). [00177] 7-(2-methyl-lH-benzimidazol-6-yl)-4-({2-[4-(methyloxy)phenyl]piperidin-l- yl}carbonyl)-2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 2 by using methyl chloroformate in step 2 and racemic 2-(4- methoxyphenyl)piperidinein step 5. 1H NMR (400 MHz, d6-DMSO): 7.83 (s, IH), 7.75 (d, IH), 7.55 (dd, 2H), 7.44 (s, 1H),7.16 (d, 2H), 7.05 (d, IH), 6.70 (d, 2H), 4.64 (t, IH), 4.51 (m, 2H), 4.25-4.14 (m, 2H), 3.67 (m, 6H), 22.96 (t,lH), 2.75 (s, 3H), 2.03 (d, IH), 1.77 (t, IH), 1.53-1.40 (m, 4H); MS (EI) for C30H32N4O3: 497 (MH+).
[00178] 4-{[2-(4-chloro-3-fluorophenyl)piperidin-l-yl]carbonyl}-7-(2-methyl-lH- benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine. Synthesized according to the method of example 2 using methyl chloroformate in step 2 and 2-(4-chloro-3-fluorophenyl)piperidine (reagent preparation 1) in step 5. 1H NMR (400 MHz, CDCl3): 8.95 (br s, IH), 7.80 (br s, 0.5H), 7.70 (br d, 0.5H), 7.48 (m, IH), 7.40 (m, 0.5H), 7.32 (m, 2H), 7.0 (m, 1.5H), 7.10 (d, IH), 6.99 (m, 2H), 4.63 (m, IH), 4.52 (m, 2H), 4.20 (m, IH), 4.10 (m, 2H), 3.81 (m, IH), 3.70 (m, IH), 3.32 (m, IH), 3.14 (m, IH), 2.52 (s, 3H), 1.90 (m, 3H), 1.72 (m, 2H). MS (EI) for C29H28ClFN4O2: 520 (MH+). [00179] 4-[(3-(enJo)-methyl-8-azabicyclo[3.2.1]oct-8-yl)carbonyl]-7-(2-methyl-lH- benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared according to the method of example 2 by using 3-(en<io)-methyl-8-azabicyclo[3.2.1]octane hydrochloride (reagent preparation 15) in step 5. 1H NMR (400 MHz, methanol-d4): 7.63 (br, IH), 7.52 to 7.49 (m, 2H), 7.46 (dd, IH), 7.42 (dd, IH), 7.03 (d, IH), 4.58 (s, 2H), 4.22 (m, 2H), 4.06 (br, 2H), 3.75 (m, 2H), 2.57 (s, 3H), 1.94 to 1.04 (m, 2H), 1.71 to 1.58 (m, 4H), 1.44 (t, 2H), 0.93 (d, 3H); MS (EI) for C26H30N4O2: 431(MH+).
[00180] 7-(2-methyl-lH-benzimidazol-6-yl)-4-[(2-{4-[(trifluoromethyl)oxy]phenyl}piperidin- l-yl)carbonyl]-2,3,4,5-tetrahydro-l,4-benzoxazepine. Synthesized according to the method of example 2 using methyl chloro formate in step 2 and 2-(4-(trifluoromethoxy)phenyl)piperidine (reagent preparation 1) in step 5. MS (EI) for C30H29F3N4O3: 551 (MH+). [00181] 4-( {2- [3 -fluoro-4-(methyloxy)phenyl]piperidin- 1 -yl} carbonyl)-7-(2-methyl- IH- benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine. Synthesized according to the method of example 2 using methyl chloro formate in step 2 and 2-(3-fluoro-4-methoxyphenyl)piperidine (reagent preparation 1) in step 5. 1H NMR (400 MHz, CDCl3): 9.20 (br s, IH), 7.64 (brs, IH), 7.50 (m, IH), 7.46 (d, IH), 7.38 (m, 0.5H), 7.31 (m 1.5H), 7.28 (s, 2H), 7.12-6.78 (m, 2H), 4.78 (m, IH), 4.48 (m, IH), 4.58 (m, IH), 4.22 (m, 2H), 3.86 (m 2H), 3.83 (s, 3H), 3.62 (m, IH), 3.18 (m IH), 2.52 (s, 3H), 1.88 (m IH), 1.62 (m, 3H), 1.06 (m, 2H). MS (EI) for C30H3iFN4O3: 515 (MH+). [00182] 4-{[2-(2-fluorophenyl)piperidin-l-yl]carbonyl}-7-(2-methyl-lH-benzimidazol-6-yl)- 2,3,4,5-tetrahydro-l,4-benzoxazepine. Synthesized according to the method of example 2 using methyl chloro formate in step 2 and 2-(2-fluorophenyl)piperidine (reagent preparation 1) in step 5. 1H NMR (400 MHz, d6-DMSO): 12.22 (br s, IH), 7.53 (br s, IH), 7.47 (dd, IH), 7.44 (br m, IH), 7.32 (br d, IH), 7.10 (dd, IH), 7.07 (m, IH), 6.99 (d, IH), 6.96 (dd, IH), 6.82 (t, IH), 4.60 (dd, 2H), 4.42 (m, IH), 4.08 (m, 2H), 3.72 (m, IH), 3.58 (2t, IH), 3.30 (m, 2H), 2.88 (m, IH), 2.49 (s, 3H), 1.78-1.52 (m, 5H), 1.44 (m, IH). MS (EI) for C29H29FN4O2: 485 (MH+). [00183] 4- { [2-(3 ,4-dichlorophenyl)piperidin- 1 -yljcarbonyl} -7-(2 -methyl- lH-benzimidazol-6- yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine. Synthesized according to the method of example 2 using methyl chloro formate in step 2 and 2-(3,4-dichlorophenyl)piperidine (reagent preparation 1) in step 5. 1H NMR (400 MHz, d6-DMSO): 12.22 (br s, IH), 7.68 (r s, 0.5H), 7.54 (br s, IH), 7.49 (br s, 2.H), 7.47 (d, 0.5H), 7.43-7.39 (m, 3H), 7.29 (br d, IH), 7.17 (dd, IH), 7.02 (d, IH), 4.56 (dd, 2H), 4.42 (m, IH), 4.19 (m, IH), 4.12 (m, IH), 3.66 (m, 2H), 3.10 (m, 2H), 2.49 (s, 3H), 1.96 (m, 2H), 1.93 (m, 2H), 1.90 (m, 2H). MS (EI) for C29H28Cl2N4O2: 536 (MH+). [00184] 4-(4-chlorophenyl> 1 - { [7-(2-methyl- lH-benzimidazol-6-yl)-2,3 -dihydro- 1,4- benzoxazepin-4(5H)-yl]carbonyl}piperidin-4-ol. Prepared as the free base as described in example 2 using methyl chloro formate in step 2 and 4-(4-chlorophenyl)piperidin-4-ol in step 5. 1H NMR (400 MHz, d6-DMSO): 12.24 (br s, IH), 7.77-7.33 (m, 9H), 7.02 (d, IH), 5.16 (s, IH), 4.48 (s, 2H), 4.19 (br s, 2H), 3.62 (br s, 2H), 3.46 (d, 2H), 3.17 (t, 2H), 2.50 (s, 3H), 1.93 (m, 2H), 1.55 (d, 2H); MS (EI) for C29H29ClN4O3: 518 (MH+). [00185] 4-(4-bromophenyl)-l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4- benzoxazepin-4(5H)-yl]carbonyl}piperidin-4-ol. Prepared as described in example 2 using 4-(4- bromophenyl)piperidin-4-ol in step 5. 1H NMR (400 MHz, methanol-d4): 7.64 (s, IH), 7.55- 7.35 (m, 8H), 7.05 (d, IH), 4.53 (s, 2H), 4.22 (t, 2H), 3.72 (t, 2H), 3.60 (d, 2H), 3.32 (m, 2H), 2.60 (s, 3H), 2.04 (m, 2H), 1.67 (d, 2H); MS (EI) for C29H29BrN4O3: 562 (MH+). [00186] 8-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl} -3-[3-(trifluoromethyl)phenyl]-8-azabicyclo[3.2.1 ]octan-3-(en<io)-ol. Synthesized according to the method of example 2 using 3-(3-(trifluoromethyl)phenyl)-8- azabicyclo[3.2.1]octan-3-(em/o)-ol (Ηuaxue Shiji 2002, 24(3), 165-166) in step 5. 1H NMR (400 MHz, DMSO-d6): δ 7.81 (bs, IH), 7.62 (s, IH), 7.38-7.59 (m, 6H), 7.28 (t, IH), 7.05 (d, IH), 4.63 (s, 2H), 4.13-4.28 (m, 4H), 3.80-3.84 (m, 2H), 2.59 (s, 3H), 2.29-2.43 (m, 4H), 1.84- 1.96 (m, 4H); MS (EI) for C32H3IF3N4O3: 577 (MH+).
[00187] 4-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl}piperazin-2-one. Prepared according to the method of example 2 by using piperazin- 2-one in step 5. 1H NMR (400 MHz, ds-DMSO): 7.99 (s, IH), 7.71-7.36 (m, 5H), 6.70 (d, IH), 4.47 (s, 2H), 4.19 (s, 2H), 3.74 (s, 2H), 3.62 (s, 2H), 3.23 (s, s2H), 3.03 (s, 2H), 2.49 (s, 3H). MS (EI) for C22H23N5O3: 406 (MH+).
[00188] l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl}-4-[3-(methyloxy)phenyl]piperidin-4-ol. Prepared as described in example 2 using 4-(3-methoxyphenyl)piperidin-4-ol (synthesized according to the method in WO 2005118587) in step 5. 1H NMR (400 MHz, d6-DMSO): 11.87 (br s, IH), 7.85 (s, IH), 7.78 (d, IH), 7.62-7.52 (m, 4H), 7.42 (t, 2H), 7.31(d, IH), 7.01 (d, IH), 5.32 (s, IH), 4.48 (s, 2H), 4.20 (t, 2H), 3.75 (s, 3H), 3.64 (t, 2H), 3.48 (d, 2H), 3.19 (t, 2H), 2.00 (t, 2H), 1.58 (d, 2H); MS (EI) for C30H32N4O4: 513 (MH+). [00189] l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl}-4-(3-methylphenyl)piperidin-4-ol. Prepared as described in example 2 using 4-m- tolylpiperidin-4-ol (synthesized according to the method in WO 2005118587) in step 5. 1H NMR (400 MHz, methanol-d4): 7.64 (s, IH), 7.54-7.40 (m, 4H), 7.31 (s, IH), 7.25 (d, IH), 7.17 (t, IH), 7.06-7.01 (m, 2H), 4.53 (s, 2H), 4.22 (t, 2H), 3.72 (t, 2H), 3.60 (d, 2H), 3.36 (t, 2H), 2.57 (s, 3H), 2.31 (s, 3H), 2.09 (t, 2H), 1.70 (d, 2H); MS (EI) for C30H32N4O3: 497 (MH+). [00190] 4-(2-fluorophenyl)-l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4- benzoxazepin-4(5H)-yl]carbonyl}piperidin-4-ol. Prepared according to the method of example 2 by using 4-(2-fluorophenyl)piperidin-4-ol in step 5. 1H NMR (400 MHz, methanol-dzt): 7.63 (m, 2H), 7.53 (m, IH), 7.50-7.40 (m, 3H), 7.26 (m, IH), 7.15 (t, IH), 7.05 (d, IH), 7.01 (m, IH), 4.54 (s, 2H), 4.22 (m, 2H), 3.72 (m, 2H), 3.61 (m, 2H), 3.38 (m, 2H), 2.57 (s, 3H), 2.39 (m, 2H), 1.68 (m, 2H); MS (EI) for C29H29FN4O3: 501 (MH+).
[00191] 4-[4-chloro-3-(trifluoromethyl)phenyl]-l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3- dihydro-l,4-benzoxazepin-4(5H)-yl]carbonyl}piperidin-4-ol. Prepared as described in example 2 using 4-(4-chloro-3-(trifluoromethyl)phenyl)piperidin-4-ol in step 5. 1H NMR (400 MHz, methanol-d4): 7.94 (s, IH), 7.67-7.62 (m, 2H), 7.55-7.40 (m, 5H), 7.05 (d, IH), 4.55 (s, 2H), 4.22 (t, 2H), 3.72 (t, 2H), 3.63 (d, 2H), 3.40-3.30 (m, 2H), 2.58 (s, 3H), 2.09 (t, 2H), 1.67 (d, 2H); MS (EI) for C30H28ClF3N4O3: 586 (MH+).
[00192] (l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl}piperidm-4-yl)methanol. Prepared as described in example 2 using piperidin-4- ylmethanol in step 5. 1H NMR (400 MHz, methanol-d4): 7.64 (s, IH), 7.54-7.41 (m, 4H), 7.03 (d, IH), 4.49 (s, 2H), 4.20 (t, 2H), 3.76-3.66 (m, 4H), 3.43 (d, 2H), 2.83 (t, 2H), 2.58 (s, 3H), 1.77-1.61 (m, 3H), 1.27 (q, 2H); MS (EI) for C24H28N4O3: 421 (MH+). [00193] 4-(3-chlorophenyl)-l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4- benzoxazepin-4(5H)-yl]carbonyl}piperidin-4-ol. Prepared according to the method of example 2 by using 4-(3-chlorophenyl)piperidin-4-ol (synthesized according to the method in WO
2005118587) in step 5. 1H NMR (400 MHz, DMSO-de): 12.26 (br. s, IH), 7.70-7.26 (m, 9H), 7.01 (d, IH), 4.48 (s, 2H), 4.19 (m, 2H), 3.63 (m, 2H), 3.47 (m, 2H), 3.17 (m, 2H), 2.49 (s, 3H), 1.96 (m, 2H), 1.56 (m, 2H); MS (EI) for C29H29ClN4O3: 517 (MH+). [00194] 3-(e«ύ?o)-(hydroxymethyl)-8-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4- benzoxazepin-4(5H)-yl]carbonyl}-8-azabicyclo[3.2.1]octan-3-ol. Prepared according to the method of example 2 by using 3-(endo)-(hydroxymethyl)-8-azabicyclo[3.2.1]octan-3-ol (reagent preparation 17) in step 5. 1H NMR (400 MHz, DMSO-de): 12.05 (br. s, IH), 7.66 (s, IH), 7.49 (m, 3H), 7.37 (m, IH), 7.01 (d, IH), 4.54 (s, 2H), 4.19 (m, 2H), 3.94 (m, 2H), 3.68 (m, 2H), 3.30 (s, 2H), 2.49 (s, 3H), 1.83 (m, 4H), 1.73 (m, 2H), 1.52 (m, 2H); MS (EI) for C26H30N4O4: 463 (MH+).
[00195] (±)-(2i?,4i?)-2-(4-fluorophenyl)-l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro- l,4-benzoxazepin-4(5H)-yl]carbonyl}piperidm-4-ol. Prepared according to the method of example 2 by using racemic (2i?,4i?)-2-(4-fiuorophenyl)piperidin-4-ol (reagent preparation 16) in step 5. 1H NMR (400 MHz, d6-DMSO): 12.20 (d,lH), 7.64 (s, IH), 7.51 - 7.20 (m,7H), 7.06 (t, 2H), 6.98 (dd, IH), 4.84 (1, IH), 4.71 (dd, IH), 4.47 (m, 2H), 4.21 - 4.10 (m, 4H), 2.86 (t, IH), 2.31 - 2.25 (m, IH), 1.72 ( m, IH), 1.63 (m, IH), 1.41 (d, IH) ). MS (EI) for C29H29FN4O3: 501 (MH+).
Synthetic Example 3
7-(2-Methyl-lH-benzimidazol-6-yl)-4-(octahydroquinolin-l(2H)-ylcarbonyl)-2,3,4,5- tetrahydro-l,4-benzoxazepine
[00196] STEP 1 : To a solution of 1,1-dimethylethyl 6-[4-(chlorocarbonyl)-2,3,4,5-tetrahydro- l,4-benzoxazepin-7-yl)-lH-benzimidazole-l-carboxylate (example 1, step 8) (5.5mg, 16 μmol) and decahydroisoquinoline (6.7 mg, 48 μmol) in anhydrous DCM (2 mL) was added PL-DIPAM (85mg, 3.27 mmol/g loading, 315 μmol, Polymer Labs) and the reaction mixture was shaken overnight at room temperature. The resulting mixture was drained into PL-PETA (55mg, 2.7 mmol/g loading, 175 μmol, Polymer Labs) and PL-MIA, (35 mg, 2.65 mmol/g loading, 104 μmol, Polymer Labs). The reaction mixture was shaken overnight at room temperature, drained and the resin was washed with 3.0 mL of methanol. The combined methanol solution was transferred to a 2 dram vial and concentrated under reduced pressure. The resulting oil was dissolved in methanol (2 mL) followed by the addition of 4 N anhydrous hydrogen chloride in dioxane (0.5 mL, Aldrich). The mixture was shaken at room temperature for an additional 18 hours. The resulting solution was concentrated under reduced pressure to give 7-(2 -methyl- IH- benzimidazol-6-yl)-4-(octahydroquinolin-l(2H)-ylcarbonyl)-2,3,4,5-tetrahydro-l,4- benzoxazepine. MS (EI) for C27Η32N4O2: 445.6 (MH+). The compound was analyzed by LC- MS (Mux) and demonstrated purity requirement was measured above 80% AUC based on UV absorbance.
[00197] Using the above automated synthesis technique and substituting with alternative starting amines the following compounds of the invention were prepared. Alternative starting materials were all obtained commercially. All compounds of the invention demonstrated purity requirement was measured above 80% AUC based on UV absorbance.
[00198] 7-(2-methyl-lH-benzimidazol-6-yl)-4-({4-[2-(methyloxy)phenyl]piperazin-l- yl}carbonyl)-2,3,4,5-tetrahydro-l,4-benzoxazepine. MS (EI) for C29H3IN5O3: 498.6 (MH+). [00199] 4-[(2-ethylpiperidin-l-yl)carbonyl]-7-(2-methyl-lH-benzimidazol-6-yl)-2,3,4,5- tetrahydro-l,4-benzoxazepine. MS (EI) for C25H30N4O2: 419.5 (MH+). [00200] 4- {[4-(2,3-dimethylphenyl)piperazin-l-yl]carbonyl}-7-(2 -methyl- lH-benzimidazol- 6-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine. MS (EI) for C30H33N5O2: 496.6 (MH+). [00201] 4-({4-[2-(ethyloxy)phenyl]piperazin-l-yl}carbonyl)-7-(2 -methyl- lH-benzimidazol-6- yl)-2,3,4,5-tetrahydro-l,4-benzoxazepme. MS (EI) for C30H33N5O3: 512.6 (MH+). [00202] l-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl}-4-phenylpiperidine-4-carbonitrile. MS (EI) for C30H29N5O2: 492.6 (MH+). [00203] N-ethyl-7-(2-methyl-lH-benzimidazol-6-yl)-N-(pyridin-4-ylmethyl)-2,3-dihydro-l,4- benzoxazepine-4(5H)-carboxamide. MS (EI) for C26H27N5O2: 442.5 (MH+). [00204] 7-(2-methyl- lH-benzimidazol-6-yl)-4-( {4-[3-(trifluoromethyl)phenyl]piperazin- 1 - yl}carbonyl)-2,3,4,5-tetrahydro-l,4-benzoxazepine. MS (EI) for C29H28F3N5O2: 536.6 (MH+). [00205] 4-(4-{[7-(2-methyl-lH-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl]carbonyl}piperazin-l-yl)phenol. MS (EI) for C28H29N5O3: 484.6 (MH+).
Synthetic Example 4
7-(lH-benzimidazol-6-yl)-4-[(4-methylpiperidin-l-yl)carbonyl]-2,3,4,5-tetrahydro-l,4- benzoxazepine [00206] STEP l : l,l-dimethylethyl 7-bromo-2,3-dihydro-l,4-benzoxazepine-4(5H)- carboxylate (reagent preparation 21) (10 g, 30.5 mmol) was taken into hot ethanol (10 mL) followed by addition of 4M hydrogen chloride in dioxane solution (2.1 eq, 16 mL) and the resulting solution was allowed to slowly cool to ambient temperature over one hour. An excess of ethyl ether was then added and the resulting slurry was filtered. The filter cake was washed with ethyl ether and dried to give 7-bromo-2,3,4,5-tetrahydro-l,4-benzoxazepine hydrochloride (7.9 g, 98% yield) as a colorless crystalline solid. MS (EI) for C9Hi0NOBr: 229 (MH+). [00207] STEP 2: 7-bromo-2,3,4,5-tetrahydro-l,4-benzoxazepine hydrochloride (3.0 g, 11.34 mmol) was suspended in dichloromethane (30 mL) followed by addition of DIPEA (3 mL, 34 mmol) and pyridine (4 mL, 49 mmol) and the resulting partially heterogeneous mixture was added portionwise over 5 minutes to a 0 0C cooled solution of phosgene (20 W% in toluene, 15 mL, 28 mmol) in dichloromethane (15 mL). The resulting mixture was then allowed to slowly warm to room temperature over 30 minutes then concentrated. The residue was partitioned with ethyl acetate and water and the organic phase washed twice with IM aqueous hydrochloric acid then brine, dried over anhydrous magnesium sulfate, filtered and concentrated to give 7-bromo- 2,3-dihydro-l,4-benzoxazepine-4(5H)-carbonyl chloride (3.35 g) as a pale yellow oil. MS (EI) for Ci0H9BrClNO2: 292 (MH+).]
[00208] STEP 3: 7-bromo-2,3-dihydro-l,4-benzoxazepine-4(5H)-carbonyl chloride as obtained in step 2 was taken into dichloromethane (35 mL) followed by portionwise addition of 4-methylpiperidine (3.5 mL, 28.4 mmol) over 5 minutes. The resulting mixture was stirred an additional 5 minutes then concentrated. The residue was partitioned with ethyl acetate and water and the organic phase washed with 1 M aqueous hydrochloric acid then brine, dried over anhydrous magnesium sulfate, filtered and concentrated to give 7-bromo-4-[(4-methylpiperidin- l-yl)carbonyl]-2,3,4,5-tetrahydro-l,4-benzoxazepine (3.91 g) as a clear oil. MS (EI) for Ci6H2IBrN2O2: 292 (MH+). [00209] STEP 4: 7-bromo-4-[(4-methylpiperidin-l-yl)carbonyl]-2,3,4,5-tetrahydro-l,4- benzoxazepine as obtained in step 3 (3.91 g, 11.07 mmol) was azeotroped twice from warm heptane then taken into anhydrous THF and cooled to -78 0C under nitrogen. Triisopropyl borate (3.3 mL, 14.4 mmol) was added by syringe followed by dropwise addition of n-butyllithium (2.5M in hexane, 5.8 mL, 14.4 mmol) over 30 minutes. The mixture was allowed to stir an additional 30 minutes at -78 0C then quenched by careful addition of 2M aqueous hydrochloric acid (10 mL) and warmed to room temperature. The mixture was stirred for 1 h at room temperature then concentrated to remove THF. The resulting aqueous mixture was then diluted with additional water and basified to pH greater than 12 by addition of 50% aqueous sodium hydroxide. The aqueous mixture was extracted once with ethyl ether then acidified to pH 1 by addition of concentrated aqueous hydrochloric acid. The acidic mixture was extracted once with ethyl acetate then washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to afford {4-[(4-methylpiperidm-l-yl)carbonyl]-2,3,4,5-tetrahydro-l,4- benzoxazepine -7-yl} boronic acid (2.86 g, 81% yield). MS (EI) for Ci6H23BrN2O4: 319 (MH+). [00210] STEP 5: To a mixture of 6-bromo-lH-benzimidazole (0.09 g, 0.46 mmol), {4-[(4- methylpiperidin-l-yl)carbonyl]-2,3,4,5-tetrahydro-l,4-benzoxazepine -7-yl} boronic acid (0.15 g, 0.47 mmol) and potassium carbonate (0.32 g, 2.3 mmol) in DMA (5.0 mL) and water (0.5 mL) was added dichloro[l,l-bis(diphenylphosphino]ferrocenepalladium (II) dichloromethane adduct (0.03 g, 0.05 mmol). The reaction mixture was stirred at 90 0C for 12 hours and then partitioned between ethyl acetate (10 mL) and water (10 mL). The aqueous layer was extracted with ethyl acetate (2x 10 mL) and the combined organic layers were dried of anhydrous magnesium sulfate, filtered and concentrated. The resulting brown residue was dissolved in acetonitrile and purified by preparative reverse phase ΗPLC to afford 7-(lH-benzimidazol-6-yl)-4-[(4-methylpiperidin-l- yl)carbonyl]-2,3,4,5-tetrahydro-l,4-benzoxazepine (7.7 mg, 4% yield) as a white solid. 1H NMR (400 MHz, DMSO-de): δ 12.54 (bs, IH), 8.24 (s, IH), 7.65 (bs, IH), 7.54 (s, IH), 7.43-7.51 (m, 4H), 7.02 (d, IH), 4.43 (s, 2H), 4.12-4.20 (m, 2H), 3.50-3.60 (m, 4H), 2.71 (t, 2H), 1.58 (d, 2H), 1.44-1.54, (m; IH), 1.12 (q, 2H), 0.92 (d, 3H); MS (EI) for C23H26N4O2: 391 (MH+). [00211] Using analogous synthetic techniques and substituting with alternative starting reagents in step 5 then conducting protecting group removal as required according to literature techniques appropriate for a given protecting group (see for example: Greene and Wuts,
Protective Groups in Organic Synthetic, Wiley-Interscience) the following compounds of the invention were prepared. Alternative starting materials were obtained commercially unless otherwise indicated. [00212] 4-[(4-methylpiperidin-l-yl)carbonyl]-7-(2-methyl-l,3-thiazol-5-yl)-2,3,4,5- tetrahydro-l,4-benzoxazepine. Synthesized according to the method of example 4 using 5- bromo-2-methylthiazole in step 5. 1H NMR (400 MHz, CDCl3): 7.71 (s, IH), 7.34 (m, 2H), 7.02 (d, IH), 4.31 (s, 2H), 4.71 (t, 2H), 3.67 (m, 2H), 2.76 (m, 5H), 1.65 (d, 2H), 1.21 (m, 3H), 0.98 (d, 3H); MS (EI) for C20H25N3O2S: 372 (MH+).
[00213] 5-{4-[(4-methylpiperidin-l-yl)carbonyl]-2,3,4,5-tetrahydro-l,4-benzoxazepin-7- yl}pyridin-2-amine. Synthesized according to the method of example 4 using 5-bromopyridin-2- amine in step 5. 1H NMR (400 MHz, d6-DMSO): 8.24 (dd, IH), 8.21 (s, 2H), 8.04 (br, 2H), 7.54 (d, IH), 7.47 (dd, IH), 7.05 (d, IH), 7.02 (d, IH), 4.42 (s, 2H), 4.19 (br s, 2H), 3.58 (br s, 2H), 3.50 (d, 2H), 2.69 (tr, 2H), 1.56 (d, 2H), 1.49 (m, IH), 1.08 (q, 2H), 0.90 (d, 3H). MS (EI) for C2IH26N4O2: 367 (MH+). [00214] 4- {4-[(4-methylpiperidin- 1 -yl)carbonyl]-2,3 ,4,5-tetrahydro- 1 ,4-benzoxazepin-7-yl} - N-[(3i?)-pyrrolidin-3-yl]benzamide. Synthesized according to the method of example 4 using (R)-tert-butyl 3-(4-bromobenzamido)pyrrolidine-l-carboxylate in step 5 followed by BOC deprotection. 1H NMR (400 MHz, d6-DMSO): 8.44 (d, IH), 7.93 (d, 2H), 7.71 (d, 2H), 7.60 (d, IH), 7.55 (dd, IH), 7.03 (d, IH), 4.44 (s, 2H), 4.39-4.31 (m, IH), 4.22-4.16 (m, 2H), 3.61-3.48 (m, 4H), 3.01-2.91 (m, 2H), 2.82-2.64 (m, 4H), 2.04-1.94 (m, IH), 1.75-1.65 (m, IH), 1.61-1.45 (m, 3H), 1.17-1.05 (m, 2H), 0.91 (d, 3H). MS (EI) for C27H34N4O3: 463(MH+). [00215] N-methyl-6- {4-[(4-methylpiperidin- 1 -yl)carbonyl]-2,3,4,5-tetrahydro- 1 ,4- benzoxazepin-7-yl}pyridine-3-carboxamide. Synthesized according to the method of example 4 using 6-bromo-Ν-methylnicotinamide in step 5. 1H NMR (400 mHz, DSMO-d6): 9.03 (s, IH), 8.63-8.69 (m, IH), 8.24 (dd, IH), 8.06 (s, IH), 8.02 (d, IH), 7.96 (d, IH), 7.04 (d, IH), 4.46 (s, 2H), 4.20-4.25 (m, 2H), 3.57-3.62 (m, 2H), 3.50 (d, 2H), 2.83 (d, 3H), 2.68 (t, 2H), 1.42-1.61 (m, 3H), 1.04-1.18 (m, 2H), 0.91 (d, 3H); MS (EI) for C23H28N4O3: 409 (MH+).
Synthetic Example 5
5-{4-[(4-methylpiperidin-l-yl)carbonyl]-2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl}-l,3- thiazol-2-amine
[00216] STEP 1 : A mixture of N-(5-bromothiazol-2-yl)acetamide (1.00 g, 4.52 mmol), (4- {[(1 , 1 -dimethylethyl)oxy]carbonyl} -2,3,4, 5-tetrahydro- 1 ,4-benzoxazepin-7-yl)boronic acid (example 1, step 2) (1.54 g, 5.43 mmol ), [l,r-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane (0.33 g, 0.40 mmol), potassium carbonate (2.50 g, 18.1 mmol) in 1,4-dioxane (20 mL) and water (2 mL) was degassed with nitrogen for 5 minutes and then stirred at 930C for 18 hours. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (80 mL), filtered over celite. The filtrate was washed with brine (2 x 50 mL), dried over sodium sulfate, filtered, concentrated. The residue was purified by flash chromatography (20% to 80% ethyl acetate- hexane gradient) to give 1 , 1 -dimethylethyl 7-[2-(acetylamino)-l,3-thiazol-5-yl]-2,3-dihydro-l,4- benzoxazepine-4(5H)-carboxylate (0.99 g, 2.54 mmol, 56.2% yield); MS (EI) for Ci9H23N3O4S: 390 (MH+).
[00217] STEP 2: A mixture 1,1-dimethylethyl 7-[2-(acetylamino)-l,3-thiazol-5-yl]-2,3- dihydro- 1 ,4-benzoxazepine-4(5H)-carboxylate (0.29g, 0.75 mmol), in methanol (2 mL) and 4 M hydrogen chloride in 1,4-dioxane (2 mL) was stirred at 7O0C for 15 minutes. The reaction mixture was cooled and concentrated, and dried in vacuum to give the de-protected product N-[5-(2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)-l,3-thiazol-2-yl]acetamide hydrochloride (0.24 g, 0.24 mmol, 99% yield); MS (EI) for Ci4Hi5N3O2S: 290 (MH+). [00218] STEP 3: 4-Methylpiperidine-l-carbonyl chloride (reagent preparation 37) (103 mg, 0.64 mmol) was added to a mixture of N-[5-(2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)-l,3- thiazol-2-yl]acetamide hydrochloride, (173 mg, 0.53 mmol) and potassium carbonate (374 mg, 2.7 mmol) in 7V,N-dimethylformamide (2 mL). The resulting mixture was stirred at room temperature for 18 hours, then methanol (2 mL) was added and concentrated. The residue was diluted with ethyl ether (40 mL), a solid was collected by filtration, and washed with ether and water to give N-(5-{4-[(4-methylpiperidin-l-yl)carbonyl]-2,3,4,5-tetrahydro-l,4-benzoxazepin- 7-yl}-l,3-thiazol-2-yl)acetamide (140 mg, 0.34 mol, 63% yield), 1H NMR (400 MHz, DMSO- d6): 7.58 (s, IH), 7.35 to 7.32 (m, 2H), 6.91 (d, IH), 4.36 (s , 2H), 4.12 (br , 2H), 3.54 to 3.47 (m, 4H), 2.71 to 2.65 (m, 2H), 2.00 (s, 3H), 1.60 to 1.43 (m, 3H), 1.16 to 1.067 (m, 2H), 0.92 (d, 3H); MS (EI) for C2iH26N4O3S: 415 (MH+). [00219] STEP 4: A mixture of N-(5-{4-[(4-methylpiperidin-l-yl)carbonyl]-2,3,4,5- tetrahydro-l,4-benzoxazepin-7-yl}-l,3-thiazol-2-yl)acetamide (50 mg, 0.12 mmol) in 1.5 mL of 6 M hydrochloric acid was stirred at 9O0C for two hours. The reaction mixture was cooled, neutralized with 50% aqueous sodium hydroxide, diluted with methanol (0.5 mL) and purified by preparative reverse phase HPLC to give 5-{4-[(4-methylpiperidin-l-yl)carbonyl]-2,3,4,5- tetrahydro-l,4-benzoxazepin-7-yl}-l,3-thiazol-2-amine (14.2 mg, 32% yield); 1H ΝMR (400 MHz, CDCl3): 7.25 to 7.21 (m, 2H), , 7.23 (s, IH), 6.99 (d, IH), 4.35 (s , 2H), 4.15 (m , 2H), 3.70 to 3.63 (m, 4H), 2.76 (t, 2H), 1.66 (d, 2H), 1.56 (br, IH), 1.25 to 1.14 (m, 2H), 0.98 (d, 3H); MS (EI) for Ci9H24N4O2S: 373 (MH+). [00220] Using analogous synthetic techniques and substituting with alternative starting reagents in step 3 the following compounds of the invention were prepared. Alternative starting materials were obtained commercially unless otherwise indicated. [00221 ] 5 -(4- { [4-(trifluoromethyl)piperidin- 1 -yl] carbonyl} -2,3 ,4,5 -tetrahydro- 1 ,4- benzoxazepin-7-yl)-l,3-thiazol-2-amine. Synthesized according to the method of example 5 using 4-(trifluoromethyl)piperidine-l -carbonyl chloride (reagent preparation 19) in step 3. 1H NMR (400 MHz, DMSO-de): 7.26 (m, 2H), 7.22 (dd, IH), 7.09 (s, 2H), 6.89 (d, IH), 4.36 (s, 2H), 4.13 (m, 2H), 3.58 (m, 3H), 2.75 (m, 2H), 1.77 (m, 2H), 1.45 (m, 2H); MS (EI) for Ci9H2IF3N4O2S: 427 (MH+).
Synthetic Example 6
5-(4-{[4-(trifluoromethyl)piperidin-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4-benzoxazepin-7- yl)pyridin-2-amine
[00222] STEP 1 : 1,1-Dimethylethyl 7-bromo-2,3-dihydro-l,4-benzoxazepine-4(5H)- carboxylate (reagent preparation 21) (5.0 g, 20.1 mmol), bis(pinacolato)diboron (5.6 g, 22.1 mmol), potassium acetate (5.9 g, 60.2 mmol) and [1,1 '- bis(diphenylphosphino)ferrocene]dichloropalladium(II) (440 mg, 0.62 mmol) were heated in DMSO (5 mL) solution at 80 0C for 1.5 h. The mixture was then cooled to room temperature and diluted with an excess of ethyl acetate and filtered through a bed of celite. The filtrate was partitioned with IM aqueous hydrochloric acid and the organic phase washed with brine and dried over anhydrous sodium sulfate. The mixture was filtered and concentrated and the residue purified by silica chromatography using 4: 1 hexanes:ethyl acetate as eluent to give tert-butyl 7-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzoxazepine-4(5)-carboxylate (7.6g, 100%). 1H NMR (400 MHz, CDCl3): 7.77 (s, 0.4H), 7.67 (s, IH), 7.65 (s, 0.6H), 7.04- 6.98 (m, IH), 4.54 (s, 0.7H), 4.43 (s, 1.3H), 4.09-4.01 (m, 2H), 3.79 (dd, 2H), 1.40 (br s, 9H), 1.26 (s, 12H). MS (EI) for C20H30BNO5: 376 (MH+). [00223] STEP 2: To a solution of 1 , 1 -dimethylethyl 7-(4,4,5,5-tetramethyl-l,3,2- dioxaboralan-2-yl)-2,3-dihydro-l,4-benzoxazepine benzoxazepin-4(5H)-carboxylate (3.Og, 8.00 mmol) in dichloromethane (90 mL) was added trifluoroacetic acid (10 mL) and the reaction mixture was heated to reflux. After cooling to room temperature the solvent was evaporated and the residue was taken into ethyl acetate (250 mL). The solution was partitioned with saturated aqueous sodium bicarbonate (200 mL) and the organic layer was separated. It was washed again with saturated aqueous bicarbonate (150 mL) then brine. The combined aqueous phase was extracted once with ethyl acetate (200 mL). The combined organic phases were then washed with brine, dried over anhydrous anhydrous sodium sulfate, filtered and concentrated to give 7-(4,4,5,5-tetramethyl-l,3,2-dioxaboralan-2-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine (2.1 g, 96%). MS (EI) for C23H25N3O3: 276 (MH+). [00224] STEP 3: 7-(4,4,5,5-tetramethyl-l,3,2-dioxaboralan-2-yl)-2,3,4,5-tetrahydro-l,4- benzoxazepine (2.2 g, 8.0 mmol) was taken into dichloromethane (30 mL) followed by addition of DIPEA (3.2 mL, 17.1 mmol) and the resulting solution was added dropwise to a 0 0C cooled solution of phosgene (20W% in toluene, 4.2 mL, 8.0 mmol) in dichloromethane (25 mL). The mixture was then allowed to warm to room temperature over 30 minutes and concentrated. The residue was partitioned with dilute aqueous hydrochloric acid and ethyl acetate. The organic phase was washed with brine, dried over anhydrous sodium sulfate then filtered and concentrated to give 7-(4,4,5,5-tetramethyl-l,3,2-dioxaboralan-2-yl)-2,3-dihydro-l,4-benzoxazepine-4(5H)- carbonyl chloride (3.0 g) as a pale yellow amorphous residue. MS (EI) for C16Η21BNO4CI: 356 (M+H20+). [00225] STEP 4: 7-(4,4,5,5-Tetramethyl-l,3,2-dioxaboralan-2-yl)-2,3-dihydro-l,4- benzoxazepine-4(5H)-carbonyl chloride (3.0 g) as obtained in step 3 was taken into dichloromethane (50 mL) followed by addition of DIPEA (40 mmol, 7 mL) then 4-trifluoromethylpiperidine hydrochloride salt (1.55 g, 8.2 mmol). The mixture was allowed to stir 30 minutes at room temperature then concentrated. The residue was partitioned with ethyl acetate and 5% aqueous citric acid. The organic phase was washed with brine, dried over anhydrous sodium sulfate then filtered and concentrated to give 7-(4,4,5,5-tetramethyl-l,3,2- dioxaboralan-2-yl)-4-{[4-(trifluoromethyl)piperidin-l-yl]carbonyl}2,3,4,5-tetrahydro-l,4- benzoxazepine. MS (EI) for C22H30BF3N2O4Cl: 455 (MH+). [00226] STEP 5: 7-(4,4,5,5-Tetramethyl-l,3,2-dioxaboralan-2-yl)-4-{[4- (trifluoromethyl)piperidin-l-yl]carbonyl}2,3,4,5-tetrahydro-l,4-benzoxazepine (100 mg, 0.27 mmol), 5-bromo-2-aminopyridine (50.8 mg, 0.30 mmol), potassium carbonate (149 mg, 1.1 mmol) and [l,r-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (19.7 mg, 0.03 mmol) were taken into dimethylacetamide (5 mL) and water (0.5 mL) and the resulting mixture was heated at 950C over 12h. The mixture was then partitioned with ethyl acetate and water and the organic solution extracted with water (3x), then brine, dried over sodium sulfate, filtered and concentrated. The residue was then taken into a minimum of acetonitrile and purified by preparative reverse phase HPLC to afford 5-(4-{[4-(trifluoromethyl)piperidin-l-yl]carbonyl}- 2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)pyridin-2-amine (38.5 mg, 35%) as an amorphous solid after lyophillization of pure eluent fractions. 1H NMR (400 MHz, de-DMSO): 8.22 (dd, IH), 8.19 (d, IH), 8.02 (br s, 2H), 7.54 (d, IH), 7.45 (dd, IH), 7.03 (d, IH), 7.00 (d, IH), 4.42 (s, 2H), 4.18 (m, 2H), 3.60 (m, 4H), 2.74 (t, 2H), 1.74 (d, 2H), 1.46 (m, 2H). MS (EI) for C2IH23F3N4O2: 421 (MH+).
[00227] Using analogous synthetic techniques and substituting with alternative starting reagents in step 5 then conducting protecting group removal as required according to literature techniques appropriate for a given protecting group (see for example: Greene and Wuts,
Protective Groups in Organic Synthetic, Wiley-Interscience) the following compounds of the invention were prepared. Alternative starting materials were obtained commercially unless otherwise indicated. [00228] 7-[4-(lH-pyrazol-5-yl)phenyl]-4-{[4-(trifluoromethyl)piperidin-l-yl]carbonyl}- 2,3,4,5-tetrahydro-l,4-benzoxazepine. Synthesized according to the method of example 6 using 3-(4-bromophenyl)-l-(tetrahydro-2H-pyran-2-yl)-lH-pyrazole (reagent preparation 18) in step 5 followed by THP group deprotection. 1H NMR (400 MHz, d6-DMSO): 12.91 (s, IH), 7.93-7.49 (m, 7H), 7.02 (d, IH), 6.77 (s, IH), 4.46 (s, 2H), 4.23-4.17 (m, 2H), 3.67-3.54 (m, 4H), 2.78 (t, 2H), 1.81-1.73 (m, 2H), 1.53-1.39 (m, 2H). MS (EI) for C25H25F3N4O2: 471(MH+). [00229] N-[(3i?)-pyrrolidin-3-yl]-4-(4-{[4-(trifluoromethyl)piperidin-l-yl]carbonyl}-2,3,4,5- tetrahydro-l,4-benzoxazepin-7-yl)benzamide. Synthesized according to the method of example 6 using (R)-tert-butyl 3-(4-bromobenzamido)pyrrolidine-l-carboxylate in step 5 followed by BOC group deprotection. 1H ΝMR (400 MHz, d6-DMSO): 9.11-8.88 (m, 2H), 8.71 (d, IH), 7.96 (d, 2H), 7.75 (d, 2H), 7.64 (d, IH), 7.56 (dd, IH), 7.03 (d, IH), 4.59-4.44 (m, 3H), 4.25- 4.16 (m, 2H), 3.66-3.55 (m, 5H), 3.33-3.13 (m, 4H), 2.81-2.70 (m, 2H), 2.26-2.15 (m, IH), 2.09- 1.98 (m, IH), 1.82-1.71 (m, 2H), 1.52-1.38 (m, 2H). MS (EI) for C27H31F3N4O3S: 517(MH+). [00230] 7-(lH-benzimidazol-6-yl)-4-{[4-(trifluoromethyl)piperidin-l-yl]carbonyl}-2,3,4,5- tetrahydro-l,4-benzoxazepine. Synthesized according to the method of example 6 using 5- bromo-lΗ-benzimidazole in step 5. 1H NMR (400 MHz, CDCl3): 8.49-8.43 (m, IH), 8.24-8.00 (m, IH), 7.94-7.80 (m, IH), 7.60-7.34 (m, 3H), 7.13-7.05 (m, IH), 4.48-4.42 (m, 2H), 4.24-4.18 (m, 2H), 3.86-3.69 (m, 4H), 2.85-2.4 (m, 2H), 2.34-2.12 (m, 2H), 1.95-1.84 (m, 2H), 1.81-1.54 (m, 2H). MS (EI) for C23H23F3N4O3: 445.2 (MH+).
[00231] N-methyl-5-(4-{[4-(trifluoromethyl)piperidin-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4- benzoxazepin-7-yl)pyridine-2-carboxamide. Synthesized according to the method of example 6 usmg ό-bromo-N-methylnicotmamide in step S. 1H ΝMR (400 mHz, DSMO-d6): 8.90 (d, IH), 8.75-8.84 (m, IH), 8.21 (dd, IH), 8.06 (d, IH), 7.71 (s, IH), 7.63 (dt, IH), 7.06 (d, IH), 4.49 (s, 2H), 4.20-4.25 (m, 2H), 3.57-3.65 (m, 4H), 2.83 (d, 3H), 2.77 (t, 2H), 1.71-1.81 (m, 2H), 1.38- 1.52 (m, 2H), 0.91 (d, 3H); MS (EI) for C23H25F3N4O3: 463 (MH+).
Synthetic Example 7 Methyl (6-{4-[(3-(enrfo)-hydroxy-3-methyl-8-azabicyclo[3.2.1]oct-8-yl)carbonyl]-2,3,4,5- tetrahydro-l,4-benzoxazepin-7-yl}-lH-benzimidazol-2-yl)carbamate
[00232] STEP 1 : (4-{[(l,l-dimethylethyl)oxycarbonyl}-2,3,4,5-tetrahydro-l,4-benzoxazepin- 7-yl)boronic acid (example 1, step 2) (2.22 g, 7.57 mmol) and 4-bromo-2-nitroaniline (1.56 g, 7.20 mmol) were taken into dioxane (20 mL), water (4 mL) and DIPEA (5.4 mL, 31 mmol) followed by addition of dichloro[l,l-δώ(diphenylphosphino]ferrocenepalladium (II) dichloromethane adduct (322 mg, 0.39 mmol) and the mixture was heated to 95 0C for 2 h. The mixture was cooled to room temperature and partitioned with ethyl acetate and 10% aqueous citric acid. The biphasic mixture was filtered through a celite pad and the organic filtrate was washed once with 0.5 M aqueous hydrochloric acid, brine then dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography to give 1,1-dimethylethyl 7-(4-amino-3-nitrophenyl)-2,3-dihydro-l,4-benzoxazepine-4(5H)-carboxylate (2.01 g, 69% yield) as a yellow crystalline solid.
[00233] STEP 2: 1,1-dimethylethyl 7-(4-amino-3-nitrophenyl)-2,3-dihydro-l,4- benzoxazepine-4(5H)-carboxylate (6.0 g, 15.6 mmol) was taken into warm methanol (50 mL) followed by addition of 4M hydrogen chloride in dioxane (50 mL) in portions and the warm solution was allowed to slowly cool to room temperature over Ih. The mixture was diluted with ethyl ether (100 mL) and the yellow solid was collected by filtration and dried to give 2-nitro-4- (2,3,4, 5-tetrahydro-l,4-benzoxazepin-7-yl)aniline hydrochloride salt (3.61 g, 72% yield) as a yellow solid. 1H NMR (400 MHz, D2O): [00234] STEP 3: 2-nitro-4-(2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)aniline hydrochloride salt (3.61 g, 11.2 mmol) and DIPEA (5.2 mL, 30 mmol) were taken into dichloromethane (50 mL) followed by dropwise addition of allyl chloroformate (1.23 mL, 11.2 mmol) over 5 minutes. The mixture was allowed to stir 30 minutes at room temperature then concentrated. The residue was partitioned with ethyl acetate and 10% aqueous citric acid and the organic solution washed with brine then dried over anhydrous sodium sulfate, filtered and concentrated to give prop-2-en- 1-yl 7-(4-amino-3-nitrophenyl)-2,3-dihydro-l,4-benzoxazepine-4(5H)-carboxylate (4.2 g, 100% yield) as a red amorphous residue. MS (EI) for C19Η19N3O5: 370 (MH+). [00235] STEP 4: Prop-2-en-l-yl 7-(4-amino-3-mtrophenyl)-2,3-dihydro-l,4-benzoxazepine- 4(5H)-carboxylate (4.2 g, 11.2 mmol) was taken into glacial acetic acid (25 mL) and the solution was warmed to 45 0C. Tin (II) chloride (8.51 g, 44.8 mmol) was added in portions over 5 minutes and the mixture was allowed to stir at this temperature for 6h. The mixture was then cooled to room temperature and diluted with MTBE (100 mL). 50% aqueous sodium hydroxide was then added in small portions with stirring until complete precipitation of tin salts occured. Anhydrous sodium sulfate was then added in portions until the precipitated salts formed a fine granular solid and the mixture was filtered. The filter cake was washed with additional MTBE and the combined organic filtrate was concentrated. The residue was partitioned with ethyl acetate and saturated aqueous sodium bicarbonate. 50% aqueous sodium hydroxide was added to the biphasic mixture in portions until the aqueous pΗ was 9-10. The layers were then separated and the organic solution washed with brine then dried over anhydrous sodium sulfate, filtered and concentrated to provide prop-2-en-l-yl 7-(3,4-diaminophenyl)-2,3-dihydro-l ,4- benzoxazepine-4(5H)-carboxylate (2.7 g, 71% yield) as a yellow solid. MS (EI) for C19Η21N3O3: 340 (MH+).
[00236] STEP 5: Prop-2-en-l-yl 7-(3, 4-diaminophenyl)-2,3-dihydro-l,4-benzoxazepine- 4(5H)-carboxylate (2.7 g, 7.96 mmol) was taken into glacial acetic acid (15 mL) followed by addition of l,3-(dimethoxycarbonyl)-2-methyl-2-thiopseudourea (1.81 g, 8.8 mmol) and the mixture was heated to 80 0C for 30 minutes then concentrated to a thick residue. The residue was treated with saturated aqueous sodium bicarbonate and the aqueous mixture basifϊed with portionwise addition of solid sodium bicarbonate with pH 8-9. The aqueous mixture was then partitioned with hexanes then filtered. The filter cake was washed with water then hexanes and dried to give prop-2-en-l-yl 7-(2-{[(methyloxy)carbonyl]amino}-lH-benzimidazoi-5-yl)-2,3- dihydro-l,4-benzoxazepine-4(5H)-carboxylate (3.46 g, 100% yield) as a pale yellow solid. [00237] STEP 6: Prop-2-en-l-yl 7-(2-{[(methyloxy)carbonyl]amino}-lH-beiizimidazol-5- yl)-2,3-dihydro-l,4-benzoxazepine-4(5H)-carboxylate (3.46 g, 7.96 mmol) was suspended in TΗF (75 mL) followed by addition of di-tert-butyl dicarbonate (4.3 g, 19.9 mmol) and pyridine (2 mL, 23.9 mmol). The mixture was stirred at room temperature Ih then warmed to reflux for an additional hour. The solution was then cooled to room temperature and concentrated. The residue was partitioned with ethyl acetate and 10% aqueous citric acid and the organic solution washed with brine then dried over anhydrous sodium sulfate, filtered and concentrated. The residue was chromatographed on silica gel (ethyl acetate :hexanes 1 :1) to provide N,N'-di-BOC prop-2-en- 1 -yl 7-(2- { [(methyloxy)carbonyl]amino} - lH-benzimidazol-5-yl)-2,3 -dihydro-1 ,4- benzoxazepine-4(5H)-carboxylate (3.3 g, 67% yield) as an impure amorphous residue. MS (EI) 6Dr C32H38N4O9: 624 (MH+).
[00238] STEP 7: N,N'-di-BOC prop-2-en-l-yl 7-(2-{[(methyloxy)carbonyl]amino}-lH- benzimidazol-5-yl)-2,3-dihydro-l,4-benzoxazepine-4(5H)-carboxylate (3.3 g, 5.3 mmol) was taken into TΗF (30 mL) followed by addition of sodium triacetoxyborohydride (5.6 g, 26.5 mmol) and palladium tetrakis-triphenylphosphine (612 mg) and the mixture was stirred for 30 minutes at room temperature. The mixture was concentrated and the residue partitioned with chloroform and saturated aqueous sodium bicarbonate. The biphasic mixture was saturated with solid sodium chloride and the aqueous phase extracted twice with chloroform. The combined organic solution was then dried over anhydrous sodium sulfate, filtered and concentrated. The residue was chromatographed on silica gel (100% ethyl acetate to 10% methanol in chloroform) to afford N,N'-di-BOC methyl [5-(2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)-lH-benzimidazol- 2-yl] carbamate (830 mg, 29% yield) as an amorphous residue. MS (EI) for
Figure imgf000066_0001
540 (MH+). [00239] STEP 8: To a solution of triphosgene (137 mg, 0.56 mmol) in THF (5 mL) was added a solution of N,N'-di-BOC methyl [5-(2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yi)-lH- benzimidazol-2-yl]carbamate (830 mg, 1.54 mmol), DIPEA (0.4 mL, 2.3 mmol) and pyridine (15 uL, 0.15 mmol) in THF (10 mL) in a dropwise manner over 5 minutes. The mixture was stirred an addition 10 minutes then concentrated. The residue was partitioned with ethyl acetate and 10% aqueous citric acid and the organic solution washed with brine then dried over anhydrous sodium sulfate, filtered and concentrated. The residue was chromatographed on silica gel (ethyl acetate:hexanes 2:3) to provide N,N'-di-BOC methyl {5-[4-(chlorocarbonyl)-2, 3,4,5- tetrahydro-l,4-benzoxazepin-7-yl]-lH-benzimidazol-2-yl}carbamate (384 mg, 41 % yield). MS (EI) for C29H33ClN4O8: 602 (MH+). [00240] STEP 9: A mixture of NN'-di-BOC methyl {5-[4-(chlorocarbonyl)-2,3,4,5- tetrahydro-l,4-benzoxazepin-7-yl]-lH-benzimidazol-2-yl}carbamate (140 mg, 0.23 mmol), 3-methyl-8-azabicyclo[3.2.1]octan-3-(endo)-ol hydrochloride (reagent preparation 9) (40 mg, 0.23mmol), and diisoproylethylamine (101 mg, 0.78 mmol), in dichloromethane (2 mL) was stirred at room temperature for one hour. The mixture was concentrated and purified directly by silica gel chromatography (0-15% methanol-dichlomethane) to give N,N'-di-BOC methyl (6-{4- [(3-(e«do)-hydroxy-3-methyl-8-azabicyclo[3.2.1 ]oct-8-yl)carbonyl]-2,3,4,5-tetrahydro- 1 ,4- benzoxazepin-7-yl}-lH-benzimidazol-2-yl)carbamate, (96 mg, 58% yield), MS (EI) for C37H47N5O9: 706 (MH+). [00241] STEP 10: A solution of NN'-di-BOC methyl (6-{4-[(3-(en<fo)-hydroxy-3-methyl-8- azabicyclo[3.2.1 ]oct-8-yl)carbonyl]-2,3 ,4,5-tetrahydro- 1 ,4-benzoxazepin-7-yl} -IH- benzimidazol-2-yl)carbamate, (20 mg, 0.028 mmol) in trifluoroacetic acid (0.2 mL) and dichloroethane (1.8 mL) was stirred at room temperature for one hour. The reaction mixture was concentrated and purified by preparative reverse phase ΗPLC to give methyl (6-{4-[(3-(en<io)- hydroxy-3-methyl-8-azabicyclo[3.2.1]oct-8-yl)carbonyl]-2,3,4,5-tetrahydro-l,4-benzoxazepin-7- yl}-lH-benzimidazol-2-yl)carbamate (3.3 mg, 23% yield), 1H ΝMR (400 MHz, methanol-d4): 7.61 (d, IH), 7.47 (m, 2H), 7.45 (d, IH), 7.34 (dd, IH), 7.02 (d, IH), 4.57 (s, 2H), 4.20 (m, 2H), 4.06 (m, 2H), 3.85 (s, 3H), 3.75 (m, 2H), 2.17 (q, 2H), 1.98 to 1.93 (m, 2H), 1.84 to 1.78 (m, 2H), 1.75 (d, 2H), 1.16 (s, 3H); MS (EI) for C27H31N5O5: 506 (MH+), and 8-{[7-(2-amino-lH- benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5H)-yl]carbonyl}-3-methyl-8- azabicyclo[3.2.1]octan-3-(e«io)-ol, 1H NMR (400 MHz, methanol-d4): 7.66-7.41 (m, 3H), 7.34 (dd, IH), 7.29(d, IH), 7.02 (d, IH), 4.56 (s, 2H), 4.20 (m, 2H), 4.05 (m, 2H), 3.74 (m, 2H), 2.16 (q, 2H), 1.98 to 1.93 (m, 2H), 1.82 to 1.78 (m, 2H), 1.75 (d, 2H), 1.16 (s, 3H); MS (EI) for C25H29N5O3: 448 (MH+).
Synthetic Example 8 6-(4-{[4-(difluoromethyl)piperidin-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4-benzoxazepin-7- yl)-lH-benzimidazol-2-amine
[00242] STEP 1 : 7-Bromo-2,3-dihydro-l,4-benzoxazepine-4(5H)-carbonyl chloride (373 mg, 1.28 mmol) (example 4, step 2) was taken into dichloromethane (10 mL) followed by addition of DIPEA (0.66 mL, 3.84 mmol) and 4-(difluoromethyl)piperidine trifluoroacetate salt (reagent preparation 4) (320 mg, 1.28 mmol) and the mixture was allowed to stir at room temperature for 1.5 h. The mixture was then quenched by addition of 5% aqueous citric acid and the aqueous portion extracted twice with dichloromethane. The combined organic layers were dried over sodium sulfate, filtered and concentrated. The residue was then purified by silica gel chromatography using 30% to 50% ethyl acetate in hexanes to afford 7-bromo-4-{[4- (difluoromethyl)piperidin-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4-benzoxazepine (356 mg, 71%). 1H NMR (400 MHz, CDCl3): 7.37 (d, IH), 7.30 (dd, IH), 6.90 (d, IH), 5.62 (d tr, CHF2, IH), 4.32 (s, 2H), 4.15-4.11 (m, 2H), 3.74 (d, 2H), 3.67 (m, 2H), 2.78 (tr, 2H), 2.01-1.90 (m, IH), 1.79 (d, 2H), 1.47 (m, 2H). [00243] STEP 2: 7-Bromo-4-{[4-(difluoromethyl)piperidin-l-yl]carbonyl}-2,3,4,5- tetrahydro-l,4-benzoxazepine (356 mg, 0.91 mmol) and 2-mtro-4-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)aniline (240 mg, 0.91 mmol) were taken into dioxane (10 mL) and water (1 mL) followed by addition of dichloro[ 1,1 -bis(diphenylphosphino]ferrocenepalladium (II) dichloromethane adduct (74 mg, 0.09 mmol) and cesium carbonate (1.5 g, 4.55 mmol). The mixture was then heated to 9O0C over 12h then cooled to room temperature and diluted with ethyl acetate. The aqueous phase was extracted once with with ethyl acetate and the combined organic layers were washed with brine, dried over sodium sulfate then filtered and concentrated. The residue was purified by silica gel chromatography using 10% ethyl acetate in hexanes to 100% ethyl acetate as eluent to give 4-(4-{[4-(difluoromethyl)piperidin-l-yl]carbonyl}-2,3,4,5- tetrahydro-l,4-benzoxazepin-7-yl)-2-nitroaniline (350 mg, 86%). 1H NMR (400 MHz, CDCl3): 8.30 (d, IH), 7.59 (dd, IH), 7.40-7.38 (m, 2H), 7.08 (d, IH), 6.89 (d, IH), 6.13 (br s, 2H), 5.64 (d tr, CHF2, IH), 4.44 (s, 2H), 4.19 (m, 2H), 3.78 (d, 2H), 3.71 (m, 2H), 2.80 (tr, 2H), 2.02-1.93 (m, IH), 1.80 (d, 2H), 1.50 (m, 2H).
[00244] STEP 3: 4-(4-{[4-(Difluoromethyl)piperidin-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4- benzoxazepin-7-yl)-2-nitroaniline (350 mg, 0.78 mmol) and 10% palladium on carbon (400 mg) were suspended in methanol (10 mL) and the mixture was hydrogenated at 35 psi using a Parr apparatus for 3.5 h. The mixture was then filtered through a bed of celite and the filtrate concentrated to afford 4-(4-{[4-(difluoromethyl)piperidin-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4- benzoxazepin-7-yl)benzene- 1 ,2-diamine which was carried forward without further purification. 1H NMR (400 MHz, CD3OD): 7.37 (d, IH), 7.34 (dd, IH), 6.96-6.94 (m, 2H), 6.84 (dd, IH), 6.74 (d, IH), 5.71 (d tr, CHF2, IH), 4.45 (s, 2H), 4.16 (m, 2H), 3.74 (d, 2H), 3.65 (m, 2H), 2.82 (tr, 2H), 2.02-1.95 (m, IH), 1.74 (d, 2H), 1.45 (m, 2H).
[00245] STEP 4: 4-(4-{[4-(Difluoromethyl)piperidin-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4- benzoxazepin-7-yl)benzene- 1 ,2-diamine (140 mg, 0.34 mmol) was taken into acetic acid (10 mL) followed by addition of l,3-bis(methoxycarbonyl)-2-methyl-2-thiopseudourea (90 mg, 0.44 mmol) and the mixture was heated at 800C for 2 h. On cooling to room temperature the mixture was diluted with ethyl acetate and partitioned with saturated aqueous sodium bicarbonate. The organic phase was then dried over sodium sulfate, filtered and concentrated. The residue was triturated with methanol and washed with water then dried in vacuo to give methyl [6-(4-{[4- (difluoromethyl)piperidin- 1 -yljcarbonyl} -2,3 ,4,5 -tetrahydro- 1 ,4-benzoxazepin-7-yl)- 1 H- benzimidazol-2-yl]carbamate (97 mg, 57%). 1H NMR (400 MHz, DMSO-d6): 11.72 (br. s, IH), 7.58 (s, IH), 7.48 (s, IH), 7.43 (d, 2H), 7.31 (d, IH), 7.00 (d, IH), 5.93 (m, IH), 4.43 (s, 2H), 4.17 (m, 2H), 3.76 (s, 3H), 3.60 (m, 4H), 2.75 (m, 2H), 1.99 (m, IH), 1.65 (m, 2H), 1.36 (m, 2H); MS (EI) for C25H27F2N5O4: 500 (MH+). [00246] STEP 5: Methyl [6-(4-{[4-(difluoromethyl)piperidin-l-yl]carbonyl}-2,3,4,5- tetrahydro- l,4-benzoxazepin-7-yl)-lH-benzimidazol-2-yl]carbamate (70 mg, 0.14 mmol) was suspended in methanol (10 mL) followed by addition of 2M aqueous potassium hydroxide (10 mL) and the mixture was stirred at 650C over 96 h. On cooling to room temperature the mixture was neutralized to pH 7-8 by addition of IM aqueous hydrochloric acid then concentrated to remove methanol. The aqueous residue was extracted with ethyl acetate (6x) and the combined organic layers dried over sodium sulfate then filtered and concentrated. The residue was taken into a minimum of aqueous acetonitrile and purified by preparative reverse phase HPLC to give 6-(4- {[4-(difluoromethyl)piperidin- 1 -yljcarbonyl} -2,3 ,4,5-tetrahydro- 1 ,4-benzoxazepin-7-yl)- lH-benzimidazol-2-amine (12 mg) as an amorphous solid after lyophillization of the combined pure fractions. 1H NMR (400 MHz, methanol-d4): 7.43 (m, 3H), 7.28 (m, 2H), 7.01 (m, IH), 5.71 (m, IH), 4.48 (m, 2H), 4.19 (m, 2H), 3.72 (m, 4H), 2.83 (m, 2H), 1.99 (m, IH), 1.94 (s, 3H), 1.74 (m, 2H), 1.46 (m, 2H); MS (EI) for C23H25F2N5O2: 442 (MH+).
Synthetic Example 9
2-(4-fluorophenyl)-l-({7-[4-(lH-imidazol-5-yl)phenyl]-2,3-dihydro-l,4-benzoxazepin-4(5H)- yl} carbonyl)piperidin-4-one [00247] STEP 1 : (4-{[(l,l-dimethylethyl)oxycarbonyl}-2,3,4,5-tetrahydro-l,4-benzoxazepin- 7-yl)boronic acid (Example 1, step 2) (1.07 g, 3.64 mmol) was dissolved into 4M hydrogen chloride in dioxane and the resulting solution was allowed to stir at room temperature for 1.3 h. The heterogeneous mixture was then diluted with ethyl ether (100 mL) and the solid collected by filtration to give 2,3,4,5-tetrahydro-l,4-benzoxazepin-7-ylboronic acid hydrochloride salt (791 mg, 95%). 1H NMR (400 MHz, D2O): 7.79 (dd, IH), 7.74 (d, IH), 7.21 (d, IH), 4.47 (s, 2H), 4.36 (m, 2H), 3.69 (m, 2H).
[00248] STEP 2: 2,3,4,5-Tetrahydro-l,4-benzoxazepin-7-ylboronic acid hydrochloride salt (1.0 g, 5.18 mmol) was taken into 1 :1 aqueous THF (30 mL) followed by addition of sodium bicarbonate (2.17 g, 25.9 mmol) then 2-(4-fluorophenyl)-4-oxopiperidine-l-carbonyl chloride (reagent preparation 19) (1.32 g, 5.18 mmol) as a THF solution and the mixture was stirred at room temperature 12 h. The mixture was then partitioned with ethyl acetate and water. The organic phase was washed with 0.5M aqueous hydrochloric acid then brine, dried over magnesium sulfate, filtered and concentrated. The residue was the triturated with hexanes and dried in vacuo to afford (4-{[2-(3-fluorophenyl)-4-oxopiperidin-l-yl]carbonyl}-2, 3,4,5- tetrahydro-l,4-benzoxazepin-7-yl)boronic acid (1.75 g, 82%) as a pale yellow solid.
[00249] STEP 3: (4-{[2-(3-fluorophenyl)-4-oxopiperidin-l-yl]carbonyl}-2,3,4,5-tetrahydro- 1 ,4-benzoxazepin-7-yl)boronic acid (100 mg, 0.24 mmol) and isobutyl 5-(4-bromophenyl)-lH- imidazole-1-carboxylate (78 mg, 0.24 mmol) were taken into dioxane (9 mL) and water (1 mL) followed by addition of DIPEA (0.21 mL, 1.2 mmol) and dichloro[l,l-bis(diphenyl- phosphinojferrocenepalladium (II) dichloromethane adduct (20 mg) then the mixture was warmed to 60 0C for 12 h. The mixture was then cooled to room temperature and concentrated. The residue was chromatographed on silica gel using 5% to 55% ethyl acetate in hexanes. Concentration of the product containing fractions afforded 20 mg of residue which was taken into a minimum of aqueous acetonitrile and further purified by preparative reverse phase ΗPLC to provide 2-(4-fluorophenyl)-l-({7-[4-(lH-imidazol-5-yl)phenyl]-2,3-dihydro-l,4- benzoxazepin-4(5H)-yl}carbonyl)piperidin-4-one (4.3 mg) as an amorphous solid after lyophillization of the combined pure fractions. 1H NMR (400 MHz, de-DMSO): 12.22 (s, IH), 7.48 (d, 2H), 7.72-7.58 (m, 5H), 7.51 (d, IH), 7.31 (q, IH), 7.13-7.00 (m, 4H), 5.18 (t, IH), 4.58 (s, 2H), 4.22 (m, 2H), 3.68 (m, 2H), 2.85 (m, 2H), 2.60-2.54 (m, IH), 2.45 (m, IH), 2.37-2.30 (m, 2H), 1.75 (s, IH). MS (EI) for C30H27FN4O3: 511 (MH+).
[00250] Using analogous synthetic techniques and substituting with alternative starting reagents in steps 2 and/or 3 then conducting protecting group removal as required according to literature techniques appropriate for a given protecting group (see for example: Greene and Wuts, Protective Groups in Organic Synthetic, Wiley-Interscience) the following compounds of the invention were prepared.
[00251] 8-{[7-(2-methyl-lH-imidazo[4,5-b]pyridin-6-yl)-2,3-dihydro-l,4-benzoxazepin- 4(5H)-yl]carbonyl}-3-(trifluoromethyl)-8-azabicyclo[3.2. l]octan-3-ol. Synthesized according to the method of example 9 using 3-(endo)-hydroxy-3-(trifluoromethyl)-8-azabicyclo[3.2.1]octane- 8-carbonyl chloride (reagent preparation 19) in step 2 and 6-bromo-2-methyl-l-trityl-lH- imidazo[4,5-b]pyridine (reagent preparation 20) in step 3. 1H NMR (400 MHz, d6-DMSO): 8.75 (s, IH), 8.33 (s, IH), 7.64 (s, IH), 7.61 (d, IH), 7.06 (d, IH), 5.84 (br s, IH), 4.54 (s, 2H), 4.25 (br s, 2H), 4.05 (br s, 2H), 3.69 (br s, 2H), 2.05-2.01 (m, 4H), 1.77-1.72 (m, 4H). MS (EI) for C25H26F3N5O3: 503 (MH+). Synthetic Example 10
7-[4-(5-fluoro-lH-benzimidazol-2-yl)phenyl]-4-{[4-(trifluoromethyl)piperidin-l- yl]carbonyl}-2,3,4,5-tetrahydro-l,4-benzoxazepine
[00252] STEP 1 : 7-(4,4,5,5-Tetramethyl-l,3,2-dioxaboralan-2-yl)-2,3-dihydro-l,4- benzoxazepine-4(5H)-carbonyl chloride (example 6, step 3) (3.0 g) was taken into dichloromethane (50 mL) followed by addition of DIPEA (40 mmol, 7 mL) then 4-trifiuoromethylpiperidine hydrochloride salt (1.55 g, 8.2 mmol). The mixture was allowed to stir 30 minutes at room temperature then concentrated. The residue was partitioned with ethyl acetate and 5% aqueous citric acid. The organic phase was washed with brine, dried over anhydrous sodium sulfate then filtered and concentrated to give 7-(4,4,5,5-tetramethyl-l,3,2- dioxaboralan-2-yl)-4-{[4-(trifluoromethyl)piperidin-l-yl]carbonyl}2,3,4,5-tetrahydro-l,4- benzoxazepine. MS (EI) for C22H30BF3N2O4Cl: 455 (MH+).
[00253] STEP 2: To a solution of 7-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-4-{[4- (trifluoromethyl)piperidin-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4-benzoxazepine (1.27 g, 2.81 mmol) and methyl 4-bromobenzoate (0.604 g, 2.81 mmol) in dimethoxyethane (10 mL) and water (1.25 mL) was added potassium carbonate (1.55 g, 11.2 mmol). The solution was sparged with N2(g) for ten minutes before the addition of [1,1'- bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.229 g, 0.281 mmol). The resulting suspension was heated at 90 0C for 2 hours in a sealed tube vessel. On cooling to room temperature the mixture was diluted with ethyl acetate (100 mL) then washed with saturated aqueous sodium bicarbonate (50 mL). The organic layer was washed with brine (100 mL) and then dried over anhydrous sodium sulfate. Filtration and concentration afforded a crude brown oil that was purified by silica gel chromatography (7:3 hexanes/ethyl acetate) to provide methyl 4-(4-{[trifluoromethyl)piperidin-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4-benzoxazepin-7- yl)benzoate (0.525 g, 40.7% yield) as a light tan solid. 1H NMR (400 MHz, methanol-d4): 8.07 (d, 2H), 7.71 (d, 2H), 7.58 (s, IH), 7.53 (d, IH), 7.06 (d, IH), 4.51 (s, 2H), 4.23 (t, 2H), 3.92 (s, 3H), 3.79-3.68 (m, 4H), 2.86 (t, 2H), 2.38 (m, IH), 1.86 (d, 2H), 1.57 (m, 2H); MS (EI) for C24H25F3N2O4: 463 (MH+). [00254] STEP 3: To a solution of methyl 4-(4-{[trifluoromethyl)piperidin-l-yl]carbonyl}- 2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)benzoate (0.525 g, 1.14 mmol) in methanol (15 mL) was added a solution of 1 M sodium hydroxide (3 mL). The resulting solution was heated at 60 0C for 22 hours. After cooling to room temperature some of the methanol was evaporated, water was added to the resulting mixture, and the pH adjusted to 3 with IN aqueous hydrochloric acid. The precipitate obtained was filtered, washed with water, and dried to afford 4-(4-{[4- (trifluoromethy)piperidin-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)benzoic acid (0.274 g, 53.7% yield) as a white solid. 1H NMR (400 MHz, methanol-d4): 8.06 (d, 2H), 7.69 (d, 2H), 7.57 (s, IH), 7.53 (d, IH), 7.06 (d, IH), 4.51 (s, 2H), 4.23 (t, 2H), 3.79-3.68 (m, 4H), 2.86 (t, 2H), 2.38 (m, IH), 1.86 (d, 2H), 1.57 (m, 2H); MS (EI) for C23H23F3N2O4: 449 (MH+). [00255] STEP 4: A solution of ethyl 4-(4-{[4-(trifluoromethy)piperidin-l-yl]carbonyl}- 2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)benzoic acid (0.150 g, 0.334 mmol), 4-fluorobenzene- 1,2-diamine (0.422 g, 0.334 mmol), HATU (0.133 g, 0.351 mmol), and diisopropylethylamine (0.130 g, 1.00 mmol) in dimethylformamide (4 mL) was stirred at room temperature for 18 hours. The reaction mixture was diluted with ethyl acetate (100 mL), washed with saturated sodium bicarbonate (100 mL) and brine (50 mL), dried over sodium sulfate. Filtration and concentration afforded a crude brown oil that was purified by silica gel chromatography (99: 1 dichloromethane/methanol) to provide N-(2-amino-4-fluorophenyl)-4-(4- { [4-
(trifluoromethyl)piperidin- 1 -yl]carbonyl} -2,3 ,4,5-tetrahydro- 1 ,4-benzoxazepin-7-yl)benzamide (0.186 g, 100% yield) as a golden oil. 1H ΝMR (400 MHz, methanol-d4): 8.04 (d, 2H), 7.75 (d, 2H), 7.60-7.53 (m, 2H), 7.14 (m, IH), 7.07 (d, IH), 6.60 (d, IH), 6.44 (m, IH), 4.53 (s, 2H), 4.23 (t, 2H), 3.79-3.68 (m, 4H), 2.86 (t, 2H), 2.38 (m, IH), 1.86 (d, 2H), 1.57 (m, 2H); MS (EI) for C29H28F4N4O3: 557 (MH+).
[00256] STEP 5: A solution ofN-(2-amino-4-fluorophenyl)-4-(4-{[4- (trifluoromethyl)piperidin- 1 -yl]carbonyl} -2,3 ,4,5-tetrahydro- 1 ,4-benzoxazepin-7-yl)benzamide (0.186 g, 0.334 mmol) in glacial acetic acid (2 mL) was heated for 1 hour at 100 0C. After cooling to room temperature the reaction mixture was concentrated and then diluted with ethyl acetate (100 mL) and washed with saturated aqueous sodium bicarbonate (100 mL). The organic layer was dried over anhydrous sodium sulfate. Filtration and concentration afforded a crude brown oil that was purified by preparative reverse phase HPLC (0.1% aqueous ammonium acetate-acetonitrile) providing 7-[4-(5-fluoro-lH-benzimidazol-2-yl)phenyl]-4- {[4- (trifluoromethyl)piperidin-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4-benzoxazepine (0.022 g, 12% yield) as a white solid. 1H ΝMR (400 MHz, methanol-d4): 8.13 (d, 2H), 7.79 (d, 2H), 7.61-7.53 (m, 3H), 7.30 (d, IH), 7.09-7.02 (m, 2H), 4.53 (s, 2H), 4.23 (t, 2H), 3.79-3.68 (m, 4H), 2.87 (t, 2H), 2.39 (m, IH), 1.87 (d, 2H), 1.59 (m, 2H); MS (EI) for C29H26F4N4O2: 539 (MH+). [00257] Using analogous synthetic techniques and substituting with alternative starting reagents in step 1 the following compounds of the invention were prepared. Alternative starting materials were obtained commercially unless otherwise indicated.
[00258] 7-[4-(5-fluoro- lH-benzimidazol-2-yl)phenyl]-4- { [4-(fluoromethyl)piperidin- 1 - yl]carbonyl}-2,3,4,5-tetrahydro-l,4-benzoxazepine. Synthesized according to the method of example 10 using 4-(fluoromethy)piperidine (reagent preparation 5) in step 2. 1H NMR (400 mHz, DSMO-de): 10.23 (bs, IH), 8.04 (d, IH), 6.93-7.76 (m, 8H), 4.15-4.36 (m, 4H), 3.68-3.84 (m, 4H), 2.83 (t, 2H), 1.48-2.00 (m, 6H), 1.18-1.46 (m, 2H); MS (EI) for C29H28F2N4O2: 503 (MH+).
Biological Examples
[00259] Compounds of this invention have been tested using the assay described in Biological Example 1 and have been determined to be mTORcl inhibitors. As such compounds of Formula I are useful for treating diseases, particularly cancer in which mTOR activity contributes to the pathology and/or symptomatology of the disease. Suitable in vitro assays for measuring mTORcl and mT0Rc2 activity and the inhibition thereof by compounds, as well as cell-based assays for measurement of in vitro efficacy in treatment of cancer, are known in the art and examples are described below. Suitable in vivo models for cancer are known to those of ordinary skill in the art and examples are disclosed in below. Following the examples disclosed herein, as well as that disclosed in the art, a person of ordinary skill in the art can determine the mTOR- inhibitory activity of a compound of this invention.
Biological Example 1 mTOR/GbL/Raptor fmTORCl) ELISA Assay
[00260] The measurement of mTORCl enzyme activity was performed in an ELISA assay format following the phosphorylation of 4E-BP1 protein. All experiments were performed in the 384-well format. Generally, 0.5 μL DMSO containing varying concentrations of the test compound was mixed with 15 μL enzyme solution. Kinase reactions were initiated with the addition of 15 μL of substrates-containing solution. The assay conditions were as follows; 0.2 nM mTORCl, 10 μM ATP and 50 nM NHis-tagged 4E-BP1 in 20 mM Hepes, pH 7.2, 1 mM DTT, 50 mM NaCl, 10 mM MnCl2, 0.02 mg/mL BSA, 0.01% CHAPS, 50 mM β-glycerophosphate. Following an incubation of 120 minutes at ambient temperature, 20 μL of the reaction volume was transferred to a Ni-Chelate-coated 384-well plate. The binding step of the 4E-BP1 protein proceeded for 60 minutes, followed by washing 4 times each with 50 μL of Tris-buffered saline solution (TBS). Anti-phospho-4E-BP 1 rabbit-IgG (20 μL, 1 :5000) in 5% BSA-TBST (0.2% Tween-20 in TBS) was added and further incubated for 60 minutes. Incubation with a secondary HRP -tagged anti-IgG was similarly performed after washing off the primary antibody (4 washes of 50 μL). Following the final wash step with TBST, 20 μL of SuperSignal ELISA Femto (Pierce Biotechnology) was added and the luminescence measured using an EnVision plate reader.
[00261] All Compounds in Table 1 were tested in the above assay. In one embodiment, the Compound of the Invention has an mTOR-inhibitory activity of about 0.4 μM or less. In another embodiment, the Compound of the Invention has an mTOR-inhibitory activity of about 0.3 μM or less. In another embodiment, the Compound of the Invention has an mTOR-inhibitory activity of about 0.2 μM or less. In another embodiment, the Compound of the Invention has an mTOR- inhibitory activity of about 0.1 μM or less. In another embodiment, the Compound of the Invention has an mTOR-inhibitory activity of about 0.05 μM or less. In another embodiment, the Compound of the Invention has an mTOR-inhibitory activity of about 0.02 μM or less. In another embodiment, the Compound of the Invention has an mTOR-inhibitory activity of about 0.01 μM or less.
[00262] As numbered in Table 1, Compounds 15, 46, 47, and 53-86 have an IC50 in this assay of less than or equal to 20 nM. As numbered in Table 1, Compounds 13, 14, and 19-45 have an IC50 in this assay of greater than 20 nM but less than or equal to 100 nM. As numbered in Table 1, Compounds 1-12 and 16-18 have an IC50 in this assay of greater than 100 nM but less than or equal to 352 nM.
Biological Example 2
Immune-Complex mTORC2 Kinase (mT0RC2 IP -Kinase) Assay
[00263] HeLa (ATCC) cells are grown in suspension culture and lysed in ice-cold lysis buffer containing 40 mM HEPES pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM sodium pyrophosphate, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM NaN3, one tablet of protease inhibitors (Complete-Mini, EDTA-free, Roche), 0.3% cholamidopropyl- dimethylammoniopropanesulfonate (CHAPS), 1 mM AEBSF, 0.5 mM benzamidine HCl, 20 μg/mL heparin, and 1.5 mM Na3VO4. The mTORC2 complex is immunoprecipitated with anti-RICTOR antibody for 2 h. The immune complexes are immobilized on Protein A sepharose (GE Healthcare, 17-5280-01), washed sequentially 3 times with wash buffer (40 mM HEPES pH 7.5, 120 mM NaCl, 10 mM β-glycerophosphate, 0.3% CHAPS, 1 mM AEBSF, 20 μg/mL heparin, 1.5 mM Na3VOzI, and Complete-Mini, EDTA-free) and resuspended in kinase buffer (40 mM HEPES, pH 7.5, 120 mM NaCl, 0.3% CHAPS, 20 μg/mL heparin, 4 mM MgCl2, 4 mM MnCl2, 10% Glycerol, and 10 mM DTT). The immune complexes (equivalent to I xIO7 cells) are pre-incubated at 37 0C with a test compound or 0.6% DMSO for 5 min, and then subjected to a kinase reaction for 8 min in a final volume of 33 μL (including 5 μL bed volume) containing kinase buffer, 50 μM ATP, and 0.75 μg full length dephosphorylated AKTl . Kinase reactions are terminated by addition of 11 μL 4x SDS sample buffer containing 20% β-mercaptoethanol and resolved in a 10% Tris Glycine gels. The gels are transferred onto PVDF membrane at 50 V for 20 h at 4 0C. The membranes are blocked in 5% non-fat milk in TBST for 1 h and incubated overnight at 4 0C with 1/1000 dilution of rabbit anti-pAKT (S473) (Cell Signaling Technology, 4060) in 3% BSA/TBST. The membranes are washed 3 times in TBST and incubated for 1 h with a 1/10000 dilution of secondary goat anti-rabbit HRP antibody (Cell Signaling Technology, 2125) in 5% non-fat milk/TBST. The signal is detected using Amersham ECL-plus. The scanned data are analyzed using ImageQuant software. IC50 for the test compound is determined relative to DMSO treated sample using XLfit4 software.
Biological Example 3 t>S6 (S240/244) ELISA Assay
[00264] MCF-7 cells (ATCC) or PC-3 cells were seeded at 24000 cells per well in 96-well plates (Corning, 3904) in DMEM (Cellgro) containing 10% FBS (Cellgro), 1% NEAA (Cellgro) and 1% penicillin-streptomycin (Cellgro). Cells were incubated at 37°C, 5% CO2 for 48 h, and the growth medium was replaced with serum-free DMEM or in medium containing 0.4% BSA. Serial dilutions of the test compound in 0.3% DMSO (vehicle) were added to the cells and incubated for 3h. To fix the cells, medium was removed and lOOμL/well of 4% formaldehyde (Sigma Aldrich, F8775) in TBS (20 mM Tris, 500 mM NaCl) was added to each well at RT for 30 min. Cells were washed 4 times with 200μL TBS containing 0.1% Triton X-100 (Sigma, catalog # T9284). Plates were blocked with lOOμL Odyssey blocking buffer (Li-Cor Biosciences, 927-40000) for Ih at RT. Anti-pS6 (S240/244) antibody (Cell Signaling Technology, 2215) and anti-total-S6 antibody (R&D systems, MAB5436) were diluted 1 :400 in Odyssey blocking buffer, and 50μL of the antibody solution containing both antibodies was added to one plate to detect pS6 and total S6. After incubation overnight at 4°C, plates were washed 4 times with 200μL TBS containing 0.1% Tween20 (Bio-Rad, catalog # 170-6351) (TBST). Goat anti-rabbit and Goat anti-mouse secondary antibody (Li-Cor Biosciences, catalog # 926-32221 and 926- 32210) conjugated to IRDye were diluted 1 :400 in Odyssey blocking buffer containing 0.1% Tween20. 50μL of antibody solution containing both antibodies was added to each well and incubated for Ih at RT. Plates were washed 3 times with 200μL TBST and 2 times with 200μL TBS. Fluorescence was read on an Odyssey plate reader. IC50 values were determined based on the ratio of pS6 to total S6 signal for compound treated wells, normalized to the DMSO-treated control wells.
[00265] In one embodiment, the Compound of the Invention tested in this assay in MCF-7 cells had an inhibitory activity of 0.5 μM or less. In another embodiment, the Compound of the Invention tested in this assay in MCF-7 cells had an inhibitory activity of 0.25 μM or less, the Compound of the Invention tested in this assay in MCF-7 cells had an inhibitory activity of 0.2 μM or less, the Compound of the Invention tested in this assay in MCF-7 cells had an inhibitory activity of 0.1 μM or less.
[00266] In one embodiment, the Compound of the Invention tested in this assay in PC-3 cells had an inhibitory activity of 2.0 μM or less. In another embodiment, the Compound of the Invention tested in this assay in PC-3 cells had an inhibitory activity of 1.0 μM or less. In another embodiment, the Compound of the Invention tested in this assay in PC-3 cells had an inhibitory activity of 0.5 μM or less. In another embodiment, the Compound of the Invention tested in this assay in PC-3 cells had an inhibitory activity of 0.25 μM or less. In another embodiment, the Compound of the Invention tested in this assay in PC-3 cells had an inhibitory activity of 0.2 μM or less. In another embodiment, the Compound of the Invention tested in this assay in PC-3 cells had an inhibitory activity of 0.1 μM or less.
Biological Example 5-11 Pharmacodynamic xenograft tumor models [00267] Female and male athymic nude mice (NCr) 5-8 weeks of age and weighing approximately 20-25 g are used in the following models. Prior to initiation of a study, the animals are allowed to acclimate for a minimum of 48 h. During these studies, animals are provided food and water ad libitum and housed in a room conditioned at 70-750F and 60% relative humidity. A 12 h light and 12 h dark cycle is maintained with automatic timers. All animals are examined daily for compound-induced or tumor-related deaths. MCF-7 Breast adenocarcinoma model
[00268] MCF7 human mammary adenocarcinoma cells are cultured in vitro in DMEM (Cellgro) supplemented with 10% Fetal Bovine Serum (Cellgro), Penicillin-Streptomycin and non-essential amino acids at 37 0C in a humidified 5% CO2 atmosphere. On day 0, cells are harvested by trypsinization, and 5 x 106 cells in 100 μL of a solution made of 50% cold Hanks balanced salt solution with 50% growth factor reduced matrigel (Becton Dickinson) implanted subcutaneously into the hindflank of female nude mice. A transponder is implanted into each mouse for identification and data tracking, and animals are monitored daily for clinical symptoms and survival. [00269] Tumors are established in female athymic nude mice and staged when the average tumor weight reached 100-200 mg. A Compound of the Invention is orally administered as a solution/fine suspension in water (with 1 : 1 molar ratio of 1 N HCL) once-daily (qd) or twice-daily (bid) at 10, 25, 50 and 100 mg/kg for 14 days. During the dosing period of 14-19 days, tumor weights are determined twice-weekly and body weights are recorded daily.
Colo-205 colon model [00270] Colo-205 human colorectal carcinoma cells are cultured in vitro in DMEM
(Mediatech) supplemented with 10% Fetal Bovine Serum (Hyclone), Penicillin-Streptomycin and non-essential amino acids at 37 0C in a humidified, 5% CO2 atmosphere. On day 0, cells are harvested by trypsinization, and 3xlO6 cells (passage 10-15, >95% viability) in 0.1 mL ice-cold Hank's balanced salt solution are implanted intradermally in the hind-flank of 5-8 week old female athymic nude mice. A transponder is implanted in each mouse for identification, and animals are monitored daily for clinical symptoms and survival.
[00271] Tumors are established in female athymic nude mice and staged when the average tumor weight reached 100-200 mg. A Compound of the Invention is orally administered as a solution/fine suspension in water (with 1 : 1 molar ratio of 1 N HCL) once-daily (qd) or twice-daily (bid) at 10, 25, 50 and 100 mg/kg for 14 days. During the dosing period of 14 days, tumor weights are determined twice-weekly and body weights are recorded daily.
PC-3 prostate adenocarcinoma model [00272] PC-3 human prostate adenocarcinoma cells are cultured in vitro in DMEM (Mediatech) supplemented with 20% Fetal Bovine Serum (Hyclone), Penicillin-Streptomycin and non-essential amino acids at 37 0C in a humidified 5% CO2 atmosphere. On day 0, cells are harvested by trypsinization and 3xlO6 cells (passage 10-14, >95% viability) in 0.1 mL of ice-cold Hank's balanced salt solution are implanted subcutaneously into the hindflank of 5-8 week old male nude mice. A transponder is implanted in each mouse for identification, and animals are monitored daily for clinical symptoms and survival.
[00273] Tumors are established in male athymic nude mice and staged when the average tumor weight reached 100-200 mg. A Compound of the Invention is orally administered as a solution/fine suspension in water (with 1 : 1 molar ratio of 1 N HCl) once-daily (qd) or twice-daily (bid) at 10, 25, 50, or 100-mg/kg for 19 days. During the dosing period of 14-19 days, tumor weights are determined twice-weekly and body weights are recorded daily.
U-87 MG human glioblastoma model
[00274] U-87 MG human glioblastoma cells are cultured in vitro in DMEM (Mediatech) supplemented with 10% Fetal Bovine Serum (Hyclone), Penicillin-Streptomycin and nonessential amino acids at 37 0C in a humidified 5% CO2 atmosphere. On day 0, cells are harvested by trypsinization and 2xlO6 cells (passage 5, 96% viability) in 0.1 mL of ice-cold Hank's balanced salt solution are implanted intradermally into the hindflank of 5-8 week old female nude mice. A transponder is implanted in each mouse for identification, and animals are monitored daily for clinical symptoms and survival. Body weights are recorded daily.
A549 human lung carcinoma model [00275] A549 human lung carcinoma cells are cultured in vitro in DMEM (Mediatech) supplemented with 10% Fetal Bovine Serum (Hyclone), Penicillin-Streptomycin and nonessential amino acids at 37 0C in a humidified 5% CO2 atmosphere. On day 0, cells are harvested by trypsinization and 1OxIO6 cells (passage 12, 99% viability) in 0.1 mL of ice-cold Hank's balanced salt solution are implanted intradermally into the hindflank of 5-8 week old female nude mice. A transponder is implanted in each mouse for identification, and animals are monitored daily for clinical symptoms and survival. Body weights are recorded daily.
A2058 human melanoma model
[00276] A2058 human melanoma cells are cultured in vitro in DMEM (Mediatech) supplemented with 10% Fetal Bovine Serum (Hyclone), Penicillin-Streptomycin and nonessential amino acids at 37 0C in a humidified, 5% CO2 atmosphere. On day 0, cells are harvested by trypsinization and 3xlO6 cells (passage 3, 95% viability) in 0.1 mL ice-cold Hank's balanced salt solution are implanted intradermally in the hind-flank of 5-8 week old female athymic nude mice. A transponder is implanted in each mouse for identification, and animals are monitored daily for clinical symptoms and survival. Body weights are recorded daily.
WM-266-4 human melanoma model
[00277] WM-266-4 human melanoma cells are cultured in vitro in DMEM (Mediatech) supplemented with 10% Fetal Bovine Serum (Hyclone), Penicillin-Streptomycin and nonessential amino acids at 37 0C in a humidified, 5% CO2 atmosphere. On day 0, cells are harvested by trypsinization and 3xlO6 cells (passage 5, 99% viability) in 0.1 mL ice-cold Hank's balanced salt solution are implanted intradermally in the hind-flank of 5-8 week old female athymic nude mice. A transponder is implanted in each mouse for identification, and animals are monitored daily for clinical symptoms and survival. Body weights are recorded daily. [00278] Tumor weight (TW) in the above models is determined by measuring perpendicular diameters with a caliper, using the following formula: tumor weight (mg) = [tumor volume = length (mm) x width2 (mm2)]/2
These data were recorded and plotted on a tumor weight vs. days post-implantation line graph and presented graphically as an indication of tumor growth rates. Percent inhibition of tumor growth (TGI) is determined with the following formula:
Figure imgf000078_0001
where Xo = average TW of all tumors on group day Xf = TW of treated group on Day f Yf = TW of vehicle control group on Day f
If tumors regress below their starting sizes, then the percent tumor regression is determined with the following formula:
Figure imgf000078_0002
Tumor size is calculated individually for each tumor to obtain a mean ± SEM value for each experimental group. Statistical significance is determined using the 2-tailed Student's t-test (significance defined as P<0.05).
[00279] The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity and understanding. The invention has been described with reference to various specific embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention. It will be obvious to one of skill in the art that changes and modifications may be practiced within the scope of the appended claims. Therefore, it is to be understood that the above description is intended to be illustrative and not restrictive. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the following appended claims, along with the full scope of equivalents to which such claims are entitled. All patents, patent applications and publications cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual patent, patent application or publication were so individually denoted.

Claims

What is claimed is:
1. A compound of structural formula
Figure imgf000080_0001
when the combination of R1 and R2 are as defined in one of the following compounds:
4-{4-[(4-methylpipeπdin-l-yl)carboπyl]-2,3,4,5-tetrahydro-l,4-beπzoxazepin-7-yl}-Λ^-[(3Λ)-pyrrolidm-3- yl]benzamide
Λr-methyl-6-{4-[(4-methylpipeπdin-l-yl)carbonyl]-2,3,4,5-tetrahydro-l,4-benzoxazepm-7-yl}pyπdme-3- carboxamide
4-[(4-methylpiperidm-l-yl)carbonyl]-7-(2 -methyl- 1, 3 -thiazol-5-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepme
4- { [4-(2,3 -dimethylphenyl)piperazm- 1 -yl] carbonyl} -7-(2 -methyl- 177-benzimidazol-6-yl)-2,3 ,4,5-tetrahydro- 1 ,4-benzoxazepme
4-[(2-ethylpipeπdm-l-yl)carbonyl]-7-(2 -methyl- li/-benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepme
4-({4-[2-(ethyloxy )phenyl]piperazm-l-yl}carbonyl)-7-(2 -methyl- l//-benzimidazol-6-yl)-2, 3,4, 5-tetrahydro- 1 ,4-benzoxazepme
3-(endo)-(hydroxymethyl)-8-{[7-(2 -methyl- 177-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepm-4(5//)- yljcarbonyl} -8-azabicyclo[3.2.1 ]octan-3-ol
7-(2 -methyl- li/-benzimidazol-6-yl)-4-({4-[3-(tπfluoromethyl)phenyl]piperazin-l-yl}carbonyl)-2,3,4, 5- tetrahydro- 1 ,4-benzoxazepme
7-(2-methyl-l//-benzimidazol-6-yl)-4-({4-[2-(methyloxy)phenyl]piperazm-l-yl}carbonyl)-2,3,4,5-tetrahydro- 1 ,4-benzoxazepme
ΛT-ethyl-7-(2 -methyl- li/-benzimidazol-6-yl)-iV-(pyridm-4-ylmethyl)-2,3-dihydro-l,4-benzoxazepme -4(5//)- carboxamide
Λ?-methyl-5-(4-{[4-(tπfluoromethyl)pipeπdm-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4-benzoxazepm-7- yl)pyndme-2-carboxamide
4- { [2-(2-fluorophenyl)pipeπdin-l -yl]carbonyl} -7-(2 -methyl- 177-benzimidazol-6-yl)-2,3 ,4,5-tetrahydro- 1 ,4- benzoxazepme
Λf-methyl-7-(2-methyl-l/f-benzimidazol-6-yl)-Λr-[(15)-l-phenylethyl]-2,3-dihydro-l,4-benzoxazepme-4(5i/)- carboxamide
4-(4-{[7-(2-methyl-l//-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepm-4(5//)-yl]carbonyl}piperazin-l- yl)phenol
7-(2-methyl-li/-benzimidazol-6-yl)-4-{[4-(tπfluoromethyl)pipeπdm-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4- benzoxazepme
7-(2 -methyl- li/-benzimidazol-6-yl)-4-(octahydroqumohn-l(2i/)-ylcarbonyl)-2, 3, 4, 5 -tetrahydro- 1,4- benzoxazepme
Λf-[(3Λ)-pyrrolidin-3-yl]-4-(4-{[4-(tπfluoromethyl)pipeπdm-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4- benzoxazepm-7-yl)benzamide
7-(2-methyl-li/-benzimidazol-6-yl)-4-{[2-(tπfluoromethyl)pipeπdm-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4- benzoxazepme
(±)-(2/J,4iJ)-2-(4-fluorophenyl)- 1 - { [7-(2 -methyl- l//-benzimidazol-6-yl)-2,3 -dihydro- 1 ,4-benzoxazepin-4(5//)- yl] carbonyl } pipeπdm-4-ol
5-{4-[(4-methylpipeπdin-l-yl)carbonyl]-2,3,4,5-tetrahydro-l,4-benzoxazepm-7-yl}pyπdm-2-amme
1 - { [7-(2-methyl- l//-benzimidazol-6-yl)-2, 3 -dihydro- 1 ,4-benzoxazepm-4(5i/)-yl] carbonyl} -4-phenylpipeπdme- 4-carbonitnle
4- { [7-(2-methyl- l//-benzimidazol-6-yl)-2, 3 -dihydro- 1 ,4-benzoxazepm-4(5ij)-yl] carbonyl}piperazm-2-one
4-[(4-fluoropiperidm-l-yl)carbonyl]-7-(2 -methyl- l//-benzimidazol-6-yl)-2, 3, 4,5-tetrahydro-l,4-benzoxazepme
7-(l//-benzimidazol-6-yl)-4-[(4-methylpiperidin-l-yl)carbonyl]-2,3,4,5-tetrahydro-l,4-benzoxazepine
7-(2 -methyl- li/-benzimidazol-6-yl)-4-[(2-{4-[(tπfluoromethyl)oxy]phenyl}pipeπdm-l-yl)carbonyl]-2, 3, 4,5- tetrahydro- 1 ,4-benzoxazepme
1 - { [7-(2-methyl- l//-benzirmdazol-6-yl)-2, 3 -dihydro- 1 ,4-benzoxazepm-4(5i/)-yl] carbonyl}pipeπdme-4- carbonitπle
4-(7-azabicyclo[2.2.1]hept-7-ylcarbonyl)-7-(2-methyl-li/-benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4- benzoxazepme 4-(4-bromophenyl)- 1 - {[7-(2 -methyl- 17f-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepm-4(5//)- yl] carbonyl } pipeπdm-4-ol
(l-{[7-(2 -methyl- l//-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepm-4(5//)-yl]carboπyl}pipeπdm-4- yl)methanol
7-[4-(l//-pyrazol-5-yl)phenyl]-4-{[4-(tnfluoromethyl)pipeπdm-l-yl]carbonyl}-2,3,4,5-tetraliydro-l,4- benzoxazepme
8- { [7-(2-methyl- l//-benzirmdazol-6-yl)-2, 3 -dihydro- 1 ,4-benzoxazepm-4(5i/)-yl] carbonyl} -8- azabicyclo[3.2.1]octan-3-(exo)-ol
4-[(4,4-dimethylpipeπdm- 1 -yl) carbonyl] -7-(2-methyl- li/-benzimidazol-6-yl)-2, 3 ,4, 5-tetrahydro- 1 ,A- benzoxazepme
7-(2 -methyl- li/-benzimidazol-6-yl)-4-[(2-methylpiperidm-l-yl)carbonyl] -2,3,4, 5 -tetrahydro- 1,4 -benzoxazepme
7-(2-methyl-li/-benzimidazol-6-yl)-4-[(4-methylpipendm-l-yl)carbonyl]-2,3,4,5-tetrahydro-l,4-benzoxazepme
4-(4-chlorophenyl)- 1 - {[7-(2 -methyl- li/-benzimidazol-6-yl)-2,3-dihydro- 1 ,4-benzoxazepm-4(5//)- yl] carbonyl } pipeπdm-4-ol
4-methyl-l-{[7-(2-methyl-17/-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepin-4(5//)-yl]carbonyl}pipeπdme- 4-carbonitπle
4-[4-chloro-3-(tπfluoromethyl)phenyl]-l-{[7-(2-methyl-l//-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepm- 4 (5H) -yl] c arbonyl } pipeπdm-4 -ol
4-(2-azabicyclo[2 2.2]oct-2-ylcarbonyl)-7-(2-methyl-li/-benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4- benzoxazepme
7-(2-methyl-li/-benzimidazol-6-yl)-4-{[4-(methylthio)pipeπdm-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4- benzoxazepme
7-(2 -methyl- li/-benzimidazol-6-yl)-4-({2-[4-(metliyloxy)phenyl]piperidm-l-yl}carbonyl)-2,3,4,5-tetrahydro- 1 ,4-benzoxazepme
6-(4-{[4-(difluoromethyl)pipeπdm-l-yl]carbonyl}-2,3,4,5-tetrahydro-l,4-benzoxazepm-7-yl)-li/- benzimidazol-2 - amine
7-( l//-benzimidazol-6-yl)-4- {[4-(tπfluoromethyl)piperidm- 1 -yl] carbonyl} -2,3 ,4, 5-tetrahydro- 1 ,4- benzoxazepme
3-(l-{[7-(2-methyl-l//-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepm-4(5//)-yl]carbonyl}piperidm-4- yl)propan- 1 -ol
5-(4-{[4-(tπfluoromethyl)piperidin-l-yl] carbonyl} -2, 3, 4,5-tetrahydro-l,4-benzoxazepin-7-yl)pyridm-2-amme
4- { [2-(3 ,4-dichlorophenyl)pipeπdm- 1 -yl] carbonyl} -7-(2-methyl- l//-benzrmidazol-6-yl)-2, 3 ,4, 5-tetrahydro- 1 ,A- benzoxazepme
4-({2-[3-fluoro-4-(methyloxy)phenyl]pipeπdm-l-yl}carbonyl)-7-(2 -methyl- li/-benzimidazol-6-yl)-2,3,4, 5- tetrahydro- 1 ,4-benzoxazepme
1 - { [7-(2-methyl- l//-benzimidazol-6-yl)-2,3 -dihydro- 1 ,4-benzoxazepin-4(5//)-yl] carbonyl} -4-phenylpipeπdin- 4-ol
7-(2 -methyl- li/-benzimidazol-6-yl)-4-[(4-propylpipendm-l -yl)carbonyl]-2,3,4,5-tetrahydro- 1 ,4-benzoxazeprne
8- { [7-(2-methyl- l//-benzimidazol-6-yl)-2, 3 -dihydro- 1 ,4-benzoxazepm-4(5i/)-yl] carbonyl} -8- azabicyclo[3.2.1]octan-3-(endo)-ol
4-{[4-(2,2-difluoroethyl)pipeπdm-l-yl]carbonyl}-7-(2-methyl-li/-benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4- benzoxazepme
8- { [7-(2-methyl- l//-benzimidazol-6-yl)-2,3 -dihydro- 1 ,4-benzoxazepm-4(5if)-yl] carbonyl} -8- azabicyclo[3.2.1]octan-3-one
4- {[4-(difluoromethyl)pipeπdm-l-yl]carbonyl}-7-(2 -methyl- 177-benzimidazol-6-yl)-2, 3, 4, 5-tetrahydro- 1,4- benzoxazepme
4-[(4-cyclopropylpipeπdin-l-yl)carbonyl]-7-(2-methyl-l//-benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4- benzoxazepme
7-(2 -methyl- li/-benzimidazol-6-yl)-4- {[2-(4-methylphenyl)pipeπdm- 1 -yl] carbonyl} -2,3,4,5-tetrahydro- 1 ,A- benzoxazepme
4-(8-azabicyclo[3.2.1]oct-8-ylcarbonyl)-7-(2-methyl-l//-benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4- benzoxazepme
2-(4-fluorophenyl)-l-({7-[4-(li/-imidazol-5-yl)phenyl]-2,3-dihydro-l,4-benzoxazepm-4(5i/)- yl} carbonyl)pipeπdm-4-one
4-[(4-ethylpipendm-l-yl)carbonyl]-7-(2 -methyl- li/-benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine methyl (6-{4-[(3-(endo)-hydroxy-3-methyl-8-azabicyclo[3.2.1]oct-8-yl)carbonyl]-2,3,4,5-tetrahydro-l,4- benzoxazepm-7-yl}-l//-benzimidazol-2-yl)carbamate
1 - { [7-(2-methyl- l//-benzimidazol-6-yl)-2, 3 -dihydro- 1 ,4-benzoxazepm-4(5i/)-yl] carbonyl} -4-[3- (methyloxy)phenyl]piperidm-4-ol
1 - { [7-(2-methyl- l//-benzimidazol-6-yl)-2, 3 -dihydro- 1 ,4-benzoxazepm-4(5//)-yl] carbonyl} -4-(3- methylphenyl)piperidm-4-ol 7-(2 -methyl- li/-benzimidazol-6-yl)-4- {[4-( 1 -methylethyl)pipeπdm- 1 -yl]carbonyl} -2,3 ,4,5-tetrahydro- 1 ,4- benzoxazepme
7-[4-(5-fluoro-l//-benzimidazol-2-yl)phenyl]-4-{[4-(tπfluoromethyl)pipeπdm-l-yl]carbonyl}-2,3,4,5- tetrahydro- 1 ,4-benzoxazepme
4-{[2-(4-chloroplienyl)pipeπdin-l-yl]carbonyl}-7-(2-methyl-l/f-benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4- benzoxazepme
4- { [4-(fluoromethyl)pipeπdm- 1 -yl] carbonyl} -7-(2-methyl- l//-benzimidazol-6-yl)-2,3 ,4,5-tetrahydro- 1,4- benzoxazepme
(±)-4- { [(25',4_R)-2,4-dimethylpipeπdin- 1 -yl] carbonyl} -7-(2 -methyl- l//-benzimidazol-6-yl)-2,3 ,4,5-tetrahydro- 1 ,4-benzoxazepme
8- { [7-(2-methyl- l//-benzirmdazol-6-yl)-2, 3 -dihydro- 1 ,4-benzoxazepm-4(5i/)-yl] carbonyl} -3 -[3- (tπfluoromethyl)phenyl] -8-azabicyclo[3.2.1 ]octan-3 -(endo)-ol
4- { [3 -(e«(io)-(fluoromethyl)-8-azabicyclo[3.2.1 ]oct- 8 -yl] carbonyl} -7-(2-methyl- l//-benzimidazol-6-yl)- 2,3,4,5-tetrahydro-l,4-benzoxazepme
4-{[4-(l,l-difluoroethyl)piperidm-l-yl]carbonyl}-7-(2-methyl-li/-benzimidazol-6-yl)-2,3,4,5-tetrahydro-l,4- benzoxazepme
8-{[7-(2-ammo-li/-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepm-4(5//)-yl]carbonyl}-3-methyl-8- azabicyclo[3.2.1]octan-3-(endo)-ol
4-{[2-(4-chloro-3-fluorophenyl)pipeπdm-l-yl]carbonyl}-7-(2-methyl-l//-benzimidazol-6-yl)-2,3,4,5- tetrahydro- 1 ,4-benzoxazepme
(±)-4-{[(25,45)-2,4-dimethylpipeπdm-l-yl]carbonyl}-7-(2-methyl-l//-benzimidazol-6-yl)-2,3,4,5-tetrahydro- 1 ,4-benzoxazepme
7-[4-(5-fluoro-l//-benzimidazol-2-yl)phenyl]-4-{[4-(fluoromethyl)pipeπdm-l-yl]carbonyl}-2,3,4,5-tetrahydro- 1 ,4-benzoxazepme
7-(2-methyl-li/-benzimidazol-6-yl)-4-[(l-phenyl-3,4-dihydroisoqumolm-2(l//)-yl)carbonyl]-2,3,4,5- tetrahydro- 1 ,4-benzoxazepme
3-methyl-8-{[7-(2-methyl-l//-benzimidazol-6-yl)-2,3-dihydro-l,4-benzoxazepm-4(5//)-yl]carbonyl}-8- azabicyclo[3.2.1]octan-3-(endo)-ol
4-(3 -fluorophenyl)- 1 - {[7-(2-methyl- l//-benzimidazol-6-yl)-2,3 -dihydro- 1 ,4-benzoxazepm-4(5//)- yl] carbonyl } pipeπdm-4-ol
1 - { [7-(2-methyl- l//-benzirmdazol-6-yl)-2, 3 -dihydro- 1 ,4-benzoxazepm-4(5i/)-yl] carbonyl} -4- (trifluoromethyl)pipendm-4-ol
4-[(3-fluoro-3-(e«ύ?o)-methyl-8-azabicyclo[3.2.1]oct-8-yl)carbonyl]-7-(2 -methyl- l//-benzimidazol-6-yl)- 2,3,4,5-tetrahydro-l,4-benzoxazepme
4-(2-fluorophenyl)- 1 - {[7-(2 -methyl- l//-benzimidazol-6-yl)-2,3 -dihydro- 1 ,4-benzoxazepm-4(5//)- yl] carbonyl } pipeπdm-4-ol
4-(3 -chlorophenyl)- 1 - {[7-(2 -methyl- li/-benzimidazol-6-yl)-2,3-dihydro- 1 ,4-benzoxazepm-4(577)- yl] carbonyl } pipeπdm-4-ol
8-{[7-(2-methyl-l/f-imidazo[4,5-b]pyridm-6-yl)-2,3-dihydro-l,4-benzoxazepm-4(5//)-yl]carbonyl}-3- (trifluoromethyl)-8-azabicyclo[3.2.1]octan-3-(endo)-ol
4-[(3-(endo)-methyl-8-azabicyclo[3 2.1]oct-8-yl)carbonyl]-7-(2-methyl-l//-benzimidazol-6-yl)-2,3,4,5- tetrahydro- 1 ,4-benzoxazepme
5- {4-[(4-methylpipeπdm- 1 -yl)carbonyl]-2,3,4,5-tetrahydro-l ,4-benzoxazepm-7-yl} - 1 ,3-thiazol-2-amme
1 - { [7-(2-methyl- l/f-benzimidazol-6-yl)-2,3 -dihydro- 1 ,4-benzoxazepm-4(5i/)-yl] carbonyl} -4-[3- (trifluoromethyl)phenyl]pipendm-4-ol
8- { [7-(2-methyl- l//-benzimidazol-6-yl)-2, 3 -dihydro- 1 ,4-benzoxazepm-4(5i/)-yl] carbonyl} -3 -(tπfluoromethyl)- 8-azabicyclo[3.2.1]octan-3-(endo)-ol
5-(4- {[4-(tπfluoromethyl)pipeπdm- 1 -yl] carbonyl} -2,3 ,4,5-tetrahydro- 1 ,4-benzoxazepm-7-yl)- 1 ,3 -thiazol-2- amme, and
(±)-[(2R,4S)- 1 - { [7-(2 -methyl- l//-benzimidazol-6-yl)-2,3-dmydro- 1 ,4-benzoxazepm-4(5i/)-yl] carbonyl} -2- phenylpiperidm-4 -yl] methanol optionally as a pharmaceutically acceptable salt thereof.
2. A pharmaceutical composition which comprises a compound, optionally as pharmaceutically acceptable salt thereof, of Claim 1 and a pharmaceutically acceptable carrier, excipient, or diluent.
3. A method for treating a disease, disorder, or syndrome which method comprises administering to a patient a therapeutically effective amount of a compound of Claim 1, optionally as a pharmaceutically acceptable salt thereof, or administering to a patient a pharmaceutical composition comprising a therapeutically effective amount of a compound of Claim 1 , optionally as a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.
4. The method of Claim 3 where the disease is cancer.
5. The method of Claim 4 where the cancer is breast cancer, mantle cell lymphoma, renal cell carcinoma, acute myelogenous leukemia, chronic myelogenous leukemia, NPM/ALK- transformed anaplastic large cell lymphoma, diffuse large B cell lymphoma, rhabdomyosarcoma, ovarian cancer, endometrial cancer, cervical cancer, non small cell lung carcinoma, small cell lung carcinoma, adenocarcinoma, colon cancer, rectal cancer, gastric carcinoma, hepatocellular carcinoma, melanoma, pancreatic cancer, prostate carcinoma, thyroid carcinoma, anaplastic large cell lymphoma, hemangioma, glioblastoma, or head and neck cancer.
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