WO2010135568A1 - Benzoxazépines en tant qu'inhibiteurs de mtor et leur utilisation pour traiter le cancer - Google Patents

Benzoxazépines en tant qu'inhibiteurs de mtor et leur utilisation pour traiter le cancer Download PDF

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Publication number
WO2010135568A1
WO2010135568A1 PCT/US2010/035639 US2010035639W WO2010135568A1 WO 2010135568 A1 WO2010135568 A1 WO 2010135568A1 US 2010035639 W US2010035639 W US 2010035639W WO 2010135568 A1 WO2010135568 A1 WO 2010135568A1
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Prior art keywords
tetrahydro
methyl
mmol
benzoxazepin
benzoxazepine
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PCT/US2010/035639
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English (en)
Inventor
Neel Kumar Anand
Tae-Gon Baik
Charles M. Blazey
Joerg Bussenius
Jeffry Kimo Curtis
Steven Charles Defina
Larisa Dubenko
Jason R. Harris
Eileen Jackson-Ugueto
Anagha Joshi
Angie Inyoung Kim
Jean-Claire Limun Manalo
Csaba J. Peto
Kenneth D. Rice
Tsze H. Tsang
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Exelixis, Inc.
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Publication of WO2010135568A1 publication Critical patent/WO2010135568A1/fr

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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
    • C07D413/14—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
    • 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
    • C07D413/02—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
    • 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
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed 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
  • mTOR 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 mT0RC2 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 Engl J 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.
  • breast cancer Nagata, Lan et al., Cancer Cell 2004, 6(2), 117-27; Pandolfi N Engl J Med 2004, 351(22), 2337-8; Nahta, Yu et al. Nat Clin Pract Oncol 2006, 3(5), 269-280
  • MCL antle cell lymph
  • 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.
  • 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.
  • 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. ScL, 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-naphthalenesulf
  • 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 ternary 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, JV-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.
  • 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.
  • 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.
  • “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. 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.
  • Embodiments of the Invention 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.
  • the invention comprises compounds of structural formula
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 emulsifiers, 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
  • 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. [0048] 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.
  • 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 th Edition) and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).
  • 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.
  • 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)CF 3 and may exist in part or in whole as the -C(OH 2 )CF 3 form. Regardless of whether 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. Although 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 )CF 3 form and that the ratio of the two forms may vary depending on the compound and the conditions in which it exists. [0054] 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. 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. [0056] 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 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-specific 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.
  • Reagent Preparation 1 4-chloro-6-(phenylmethoxy)-quinazoline.
  • 4-chloro-6-(phenylmethoxy)-quinazoline Prepared according to the method described in (J. Med. Chem. 2002, 45(17), 3772-3793) using 2-amino-5- benzyloxybenzoic acid methyl ester (J. Org. Chem. 2001, 66(8), 2784-2788).
  • Reagent Preparation 2 4-chloro-5-[(4-fluorophenyl)methyl]-6-methylpyrimidine [0061]
  • STEP 1 A solution of methyl 2-[(4-fluorophenyl)methyl]-3-oxobutanoate (90.0 g, 0.41 mol), thiourea (50.0 g, 0.66 mol), and l,8-diazabicyclo[5.4.0]undec-7-ene (80.0 mL, 0.52 mol) in acetonitrile (250 mL) was heated to reflux and the reaction mixture was stirred for 2 hours. After cooling to room temperature the reaction mixture was concentrated.
  • the resulting viscous oil was dissolved in 2M aqueous sodium hydroxide (400 mL) and washed with ethyl acetate (500 mL). The organic layer was separated and extracted with 2M aqueous sodium hydroxide (2x 100 mL). The aqueous layers were combined and cooled to O 0 C then the pH was adjusted to 5 by the slow addition of concentrated hydrochloric acid. The precipitated product was collected by filtration, washed with water and dried in vacuo to give 5-[(4-fluorophenyl)methyl]-6- methyl-2-thioxo-2,3-dihydropyrimidin-4(lH)-one (75 g, 72%).
  • STEP 1 To a cooled (0 0 C) solution of 4,4-dimethylcyclohexanone (21 g, 0.17 mol) and dimethyl carbonate (45 g, 0.50 mol) in THF (400 mL) was added NaH (60% wt/wt in mineral oil, 17 g, 0.43 mol) portionwise over 30 minutes. The resulting slurry was allowed to stir at ambient temperature for 30 minutes followed by two hours at reflux. The reaction mixture was cooled (0 0 C) and MeOH (30 mL) was added dropwise over 20 minutes. The resulting slurry was partitioned between 10% aqueous citric acid and ethyl acetate.
  • STEP 2 A solution of methyl 2-hydroxy-5,5-dimethylcyclohex-l- enecarboxylate (10.0 g, 54 mmol) and ammonium acetate (10 g, 130 mmol) in ethanol (50 niL) was heated to reflux for 2 hours. The reaction was concentrated to one third original volume, and then diluted with ethyl acetate (100 rnL). The organic solution was washed with water (100 mL) and brine (50 rnL) and then dried over anhydrous sodium sulfate.
  • STEP 3 2-amino-5,5-dimethylcyclohex-l-enecarboxylate (7.42 g, 40 mmol) was dissolved in N,N-dimethylformamide dimethylacetal (50 mL) and heated to 110 0 C for 18 hours. The resulting solution was cooled to room temperature and concentrated to provide methyl 2-((dimethylamino)methyleneamino)-5,5-dimethylcyclohex-l- enecarboxylate (9.5 g, 98% yield) as an oil.
  • STEP 5 To 6,6-dimethyl-5,6,7,8-tetrahydroquinazolin-4(3H)-one (6.41 g, 36 mmol) in chloroform (10 mL) added phosphorus oxy chloride (10 mL) and refluxed for 2 hours. The mixture was concentrated to an oil, then diluted with ethyl acetate (80 mL) and washed with saturated sodium carbonate (50 mL) and brine (25 mL).
  • the reaction mixture was concentrated and partitioned between ethyl acetate (100 mL) and water (50 mL). The organic layer was separated, washed with water (2x 50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel gradient chromatography 100% ethyl acetate to 10% of (10% ammonium hydroxide in methanol) in ethyl acetate to give 2-amino-N-(2-amino-2-methylpropyl)-5-bromopyridine-3- sulfonamide (0.50 g, 84%).
  • STEP 1 A mixture of 6-bromo- 1 ,3-dihydro-2H-imidazo[4,5-6]pyridine-2-one (400 mg, 1.7 mmol, WO 2004035549) and azetidine (450 mg, 7.9 mmol) in ethanol (2 mL) was heated at 100 0 C with microwave irradiation for 30 min. Concentration and column chromatography on silica (95:5 dichloromethane/methanol) gave 2-azetidin-l-yl- 6-bromo-lH-imidazo[4,5-6]pyridine (135 mg, 31% yield) as a yellow solid. MS (EI) for C 9 H 9 BrN 4 : 254 (MH + ).
  • STEP 2 A suspension of 2-azetidin-l-yl-6-bromo-lH-imidazo[4,5- ⁇ ]pyridine (135 mg 0.53 mmol) in dimethylformamide (5 mL) was cooled to O 0 C, sodium hydride (60% dispersion in mineral oil, 22 mg, 0.53 mmol) was added, and the mixture was stirred at O 0 C for 30 min. (2-(chloromethoxy)ethyl)trimethylsilane (techn. 90%, 99 mg, 0.53 mmol) was added, and the mixture was stirred for 1 h while warming to room temperature.
  • Reagent Preparation 7 N-(5-bromo-2-isopropoxypyridin-3-yl)methanesulfonamide
  • STEP 1 A solution of S-bromo ⁇ -chloro-S-nitropyridine (480 mg, 2.02 mmol) and l,8-diazabicyclo[5.4.0]undec-7-ene (1.5 niL, 10 mmol) in isopropanol (6 mL) was heated to 50 0 C for 3.5 h and then cooled to rt. I N HCl was added, and the resulting aqueous mixture was extracted twice with ethyl acetate.
  • STEP 2 To a solution of 5-bromo-2-isopropoxy-3-nitropyridine (125 mg, 0.48 mmol) in acetic acid (2.5 mL) at 60 0 C was added iron powder (268 mg, 4.8 mmol), and the mixture was stirred for 25 min. After cooling to rt, ethyl acetate was added, and the solids were removed by filtration through celite. The organic filtrate was washed with water followed by saturated aqueous sodium bicarbonate.
  • STEP 3 A solution of 5-bromo-2-isopropoxypyridin-3-amine (118 mg, 0.51 mmol) and diisopropylethylamine (195 uL, 1.12 mmol) in dichloromethane (2 mL) was cooled to 0 0 C and methanesulfonyl chloride (79 uL, 1.02 mmol) was added. The mixture was allowed to warm to rt over 15 h, and was then diluted with dichloromethane. The organic mixture was washed with 10% aqueous citric acid followed by water. The organic phase was dried over magnesium sulfate, filtered, and concentrated in vacuo.
  • step 1 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.
  • N-(2-(benzyloxy)-5-bromopyridin-3-yl)methanesulfonamide Prepared according to the methods described in reagent preparation 7 using benzyl alcohol in step 1.
  • 1 H NMR 400 MHz, CDCl 3 ) ⁇ 8.00 (d, IH), 7.91 (d, IH), 7.44-7.34 (m, 5H), 6.71 (br s, IH), 5.40 (s, 2H), 2.99 (s, 3H); MS (EI) for Ci 3 Hi 3 BrN 2 O 3 S: 357, 359 (Br isotopes, MH + ).
  • N-(2-(azetidin- 1 -yl)-5-bromopyridin-3-yl)methanesulfonamide Prepared according to the methods described in reagent preparation 7 using azetidine in step 1.
  • N-(5-bromo-2-morpholinopyridin-3-yl)methanesulfonamide Prepared according to the methods described in reagent preparation 7 using morpholine in step 1.
  • Reagent Preparation 8 tert-Butyl 3-(bis(tert-butoxycarbonyl)amino)-6-bromo-lH- indazole-1-carboxylate [0096] To a cooled (0 0 C) solution of 6-bromo-lH-indazol-3 -amine (0.30 g, 1.4 mmol), DIPEA (2.5 mL, 14 mmol) and di tert-butyl dicarbonate (1.5 g, 7.0 mmol) in THF (15 mL) was added DMAP (0.09 g, 0.70 mmol). The reaction mixture was then stirred at ambient temperature for three hours.
  • Step 1 Trifluoroethylamine hydrochloride salt (275 mg, 2.03 mmol) was suspended in THF (3 mL) followed by addition of DIPEA (1.7 mL, 10.2 mmol) and the resulting solution was cooled to O 0 C. Thiophosgene (156 uL, 2.03 mmol) was added to the solution by syringe and the mixture was allowed to stir 15 min.
  • Step 2 5-Bromo-N-(2,2,2-trifluoroethyl)-lH-benzo[d]imidazol-2-amine as obtained above was taken into THF (15 mL) followed by addition of DIPEA (0.5 mL, 2.9 mmol) and isobutyl chloroformate (0.3 mL, 2.3 mmol) and the resulting mixture was allowed to stir at room temperature for one hour. The solution was then concentrated and taken into ethyl acetate then washed twice with 10% aqueous citric acid then brine, dried over anhydrous sodium sulfate, filtered and concentrated.
  • DIPEA 0.5 mL, 2.9 mmol
  • isobutyl chloroformate 0.3 mL, 2.3 mmol
  • Step 1 5-Bromo-2-chloropyridin-3-amine (55.3 mg, 0.27 mmol) was taken into dichloromethane (1 mL) followed by addition of DIPEA (50 uL, 0.29 mmol) and propionyl chloride (25 uL) and the mixture was stirred at room temperature for 30 min. at which point additional DIPEA (50 uL) and propionyl chloride (20 uL) were added. After one hour the mixture was concentrated and the residue was partitioned with ethyl acetate and 10% aqueous citric acid.
  • Step 2 N-(5-bromo-2-chloropyridin-3-yl)propionamide (29.7 mg, 0.11 mmol), phosphorous pentasulfide (66 mg, 0.15 mmol) and sodium carbonate (16 mg) were taken into THF (2 mL) and the mixture was stirred for 12h. at room temperature. Additional phosphorous pentasulfide (88 mg) and sodium carbonate (20 mg) were then added and stirring was continued 12 h. Aqueous sodium hydroxide (2M, 1 mL) was then added to the reaction mixture and stirred 2 h. at room temperature then partitioned with ethyl ether and water.
  • Step 1 6-bromo-N-ethylthiazolo [5,4-6] pyridin-2-amine
  • Step 1 5-Bromo-2-chloropyridin-3-amine (27 mg, 0.13 mmol) was taken into dimethylacetamide (1 mL) followed by addition of ethyl isothiocyanate (100 uL) and the mixture was heated to 9O 0 C over 24 h at which point an additional aliquot of ethyl isothiocyanate (100 uL) was added and the mixture was heated at 12O 0 C an additional 24 h.
  • Reagent Preparation 12 4-chloro-6,6-dimethyl-2-(methylthio)-5,6,7,8- tetrahydroquinazoline
  • STEP 1 To a freshly prepared solution of sodium metal (0.72 g, 30.0 mmol) in dry ethanol (50 mL) was added thiourea (1.60 g, 21.0 mmol) and methyl 5,5-dimethyl- 2-oxocyclohexanecarboxylate (3.00 g, 16.0 mmol), and the mixture was refluxed for 17 h. The reaction was concentrated, water (40 mL) was added and the pH was adjusted to 6.
  • STEP 2 A mixture of 2-mercapto-6,6-dimethyl-5,6,7,8-tetrahydroquinazolin- 4-ol (2.82 g, 13.4 mmol), potassium carbonate (9.27 g, 67.0 mmol), and iodomethane (1.90 g, 13.4 mmol) in dimethylformamide (30 mL) was stirred at room temperature for 23 h. Ethyl acetate (300 mL) was added, and the organic layer was washed with water (100 mL), 5% aqueous lithum chloride (2 x 100 mL), and brine (100 mL), dried over sodium sulfate, filtered and concentrated.
  • STEP 3 A solution of 6,6-dimethyl-2-(methylthio)-5,6,7,8-tetrahydro- quinazolin-4-ol (0.33 g, 1.47 mmol) in phosphorus oxychloride (10 mL) was stirred at 60 0 C for 2 h. The mixture was concentrated, ethyl acetate (10 mL) and saturated sodium bicarbonate (10 mL) were added, and the biphasic mixture was stirred for 45 min. More ethyl acetate (50 mL) was added and the layers were separated.
  • STEP 1 Commercially-available 5-bromo-2-hydroxybenzaldehyde (4.0 g, 10 mmol) and 2-aminoethanol were combined in T ⁇ F/MeO ⁇ (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 tert-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.
  • Reagent Preparation 14 isobutyl 6-bromo-2-methyl-lH-imidazo[4,5-6]pyridine-l- carboxylate [00107]
  • STEP 1 To a solution of 6-bromo-2-methyl-l/f-imidazo[4,5-6]pyridine (3.40 g, 16.0 mmol) and diisopropylethylamine (6.5 mL, 65 mmol) in ⁇ /, ⁇ /-dimethylformamide (20 mL) cooled in an ice bath was added dropwise isobutyl chloro formate (2.51 mL, 19.2 mmol) and the mixture was warmed to room temperature.
  • STEP 2 A solution of 1,1-dimethylethyl 7-bromo-2,3-dihydro-l,4- benzoxazepine-4(5H)-carboxylate (30.0 g, 91.4 mmol) and triisopropyl borate (22.4 g, 119 mmol) in THF (300 mL) was cooled to -78 0 C, and a 2.5 M solution of n-butyllithium in hexanes (47.6 mL, 119 mmol) was added dropwise over 40 min at this temperature.
  • 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- IH- benzimidazole-1-carboxylate (11.3 g, 36 mmol), (4- ⁇ [(l,l-dimethylethyl)oxy]carbonyl ⁇ - 2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)boronic acid (example 5, step I)(11.7 g, 40 mmol), dichloro [1,1 - ⁇ (diphenylphosphino] 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 -dimethylethyl 7-(l- ⁇ [(1,1- 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 2 To a solution of 1,1-dimethylethyl 7- ⁇ 4- [(methyloxy)carbonyl]phenyl ⁇ -2,3-dihydro- l,4-benzoxazepine-4(5H)-carboxylate (1.90 g, 4.96 mmol) in dry methanol (10 mL) was added drop wise 4 N hydrogen chloride in dioxane (10 mL) at room temperature. The reaction mixture was warmed to 55 0 C for 60 min, at which time it was cooled to room temperature.
  • STEP 3 A mixture of methyl 4-(2,3,4,5-tetrahydro-l,4-benzoxazepin-7- yl)benzoate hydrochloride (0.20 g, 0.64 mmol), 4-chloro-3-[(4-fluorophenyl)methyl]-2- methylpyridine (0.15 g, 0.64 mmol) (reagent preparation 2) and potassium carbonate (0.26 g, 1.90 mmol) in dimethylformamide (5.0 mL) was stirred at 140 0 C for 16 hours. The reaction mixture was cooled to room temperature and diluted with water (25 mL). The aqueous layer was extracted with ethyl acetate (2x 100 mL).
  • Example 3 7- [3,4-bis(methyloxy)phenyl] -4- ⁇ 5- [(4-fluor ophenyl)methyl] -6- methylpyrimidin-4-yl ⁇ -2,3,4,5-tetrahydro-l,4-benzoxazepine
  • STEP 1 1,1 -dimethylethyl 7-bromo-2,3-dihydro- 1 ,4-benzoxazepine-4(5H)- carboxylate (reagent preparation 13) (500 mg, 1.52 mmol) and 3,4- dimethoxyphenylboronic acid (305 mg, 1.68 mmol) were taken into a mixture of 1,2- dimethoxyethane (4 mL) and water (0.4 mL) followed by addition of potassium carbonate (842 mg, 6.1 mmol) and [l,r-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (112 mg, 0.15
  • STEP 2 1,1 -dimethylethyl 7-[3,4-bis(methyloxy)phenyl]-2,3-dihydro-l,4- benzoxazepine-4(5H)-carboxylate (508 mg, 1.32 mmol) was dissolved in methanol (5 mL) followed by addition of 4M hydrogen chloride in dioxane (1.2 mL). The resulting solution was heated to reflux then allowed to slowly cool to room temperature over 1 h.
  • STEP 3 To a mixture of 7-[3,4-bis-(methyloxy)phenyl]-2,3,4,5-tetrahydro- 1 ,4-benzoxazepine hydrochloride salt (150 mg, 0.47 mmol) and 4-chloro-5-(4- fluorobenzyl)-6-methylpyrimidine (re ⁇ ust prep.o ⁇ noo ⁇ ) (100 mg, 0.42 mmol) in 1- methyl-2-pyrrolidinone (3 mL) was added DIPEA (0.3 mL, 1.69 mmol.) then heated at 120C for 4 h in a microwave reactor. On cooling to room temperature the mixture was partitioned with ethyl acetate and water.
  • STEP 1 To a slurry of 1 , 1 -dimethylethyl 7-bromo-2,3-dihydro- 1 ,4- benzoxazepine-4(5H)-carboxylate (reagent preparation 13) (5.0 g, 17 mmol) in methanol (50 mL) was added anhydrous hydrogen chloride (20 mL, 4N in dioxane, 80 mmol) and the mixture was heated (50 0 C). After 1.5 h the reaction mixture was concentrated to 10 mL and diluted with ethyl ether (100 mL).
  • STEP 2 To a solution of 7-bromo-2,3,4,5-tetrahydro-l,4-benzoxazepine hydrochloride (1.0 g, 3.8 mmol) and DIPEA (3.3 mL, 19 mmol) in NMP (15 mL) was added 4-chloro-5-(4-fluorobenzyl)-6-methylpyrimidine (reagent preparation 2) (0.90 g, 3.8 mmol).
  • the resulting mixture was heated (99 0 C) for 24 h and then partitioned between ethyl acetate (10 mL) and water (10 mL). The aqueous layer was washed with ethyl acetate (2 x 10 mL) and the combined organic layers were washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated.
  • Example 5 4-(6,6-dimethyl-5,6,7,8-tetrahydroquinazolin-4-yl)-7-(2- ethyl [ 1 ,3] thiazolo [5,4- b ⁇ pyridin-6-yl)-2,3,4,5-tetrahydro- 1 ,4-benzoxazepine [00151]
  • STEP 1 A solution of 1 , 1 -dimethylethyl 7-bromo-2,3-dihydro- 1 ,4- benzoxazepine-4(5H)-carboxylate (reagent preparation 13) (30.0 g, 91.4 mmol) and triisopropyl borate (22.4 g, 119 mmol) in THF (300 mL) was cooled to -78 0 C, and a 2.5M solution of n-butyllithium in hexanes (47.6 mL, 119 mmol) was added dropwise over 40 min at this
  • reaction mixture was stirred at -78 0 C for an additional 30 min, then quenched by dropwise addition of 2N 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 To a slurry of 2,3,4,5-tetrahydro-l,4-benzoxazepin-7-ylboronic acid hydrochloride salt (5.7 g, 25 mmol) (example 8, step 1) and 4-chloro-6,6-dimethyl- 5,6,7,8-tetrahydroquinazoline (reagent preparation 3) (3.0 g, 15 mmol) in dioxane (75 mL) and H 2 O (75 mL) was added DIPEA (17 mL, 100 mmol) and the resulting mixture was heated (90 0 C). After 72 hours the solution was concentrated and partitioned between 2M aqueous sodium hydroxide and ethyl ether.
  • reagent preparation 3 4-chloro-6,6-dimethyl- 5,6,7,8-tetrahydroquinazoline
  • Step 4 5-bromo-2-ethylbenzo[d]thiazole (reagent preparation 10) (17 mg, 0.07 mmol) and [4-(6,6-dimethyl-5,6,7,8-tetrahydroquinazolin-4-yl)-2,3,4,5-tetrahydro- 1 ,4-benzoxazepin-7-yl]boronic acid (25.9 mg, 0.07 mmol) were taken into dioxane (0.5 mL) and water (0.1 mL) followed by addition of diisopropylethylamine (0.05 mL, 0.28 mmol.) and [l,r-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (3.7 mg).
  • the mixture was heated at 95 0 C in a sealed vessel for 18 h then cooled to room temperature.
  • the mixture was diluted with ethyl acetate and dried over sodium sulfate then filtered through a plug of silica gel using ethyl acetate as eluent.
  • N-(2-(azetidin-l-yl)-5-bromopyridin-3- yl)methanesulfonamide (reagent preparation 7) in step 4.
  • STEP 2 tert-Butyl 7-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-2,3- dihydrobenzoxazepine-4(5H)-carboxylate (500 mg, 2.33 mmol), 4-bromo-2- methoxyaniline (296 mg, 1.47 mmol), potassium carbonate (737 mg, 5.34 mmol) and [l,r-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (97.5 mg, 0.13 mmol) were heated at HO 0 C in DME (7 mL) and water (1 mL) over 48 h.
  • STEP 4 2-(methyloxy)-4-(2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)aniline hydrochloride salt (150 mg, 0.55 mmol), 4-chloro-5-(4-fluorobenzyl)-6-methylpyrimidine (reagent preparation 2) (119 mg, 0.5 mmol) in l-methyl-2-pyrrolidinone (3 mL) was added DIPEA (0.35 mL, 2.01 mmol.) then heated at 12O 0 C for 5 h in a microwave reactor. On cooling to room temperature the mixture was partitioned with ethyl acetate and water.
  • STEP 2 A mixture of 1 , 1 -dimethylethyl 7-(2-methyl- 1 - ⁇ [(2-methy lpropyl)- oxy]carbonyl ⁇ -lH-imidazo[4,5- ⁇ ]pyridine-6-yl)-2,3-dihydro-l,4-benzoxazepine- 4(5H)carboxylate (1.1 g, 2.3 mmol) in methanol (6 ml) and 4N hydrochloric acid in dioxane (12 ml) was stirred at room temperature for 1 h and then concentrated.
  • STEP 3 To a solution of 2-methylpropyl 2-methyl-6-(2,3,4,5-tetrahydro-l,4- benzoxazepin-7-yl)-lH-imidazo[4,5- ⁇ ]pyridine-l-carboxylate (0.065 g, 0.14 mmol) and DIPEA (0.2 niL, 1.1 mmol) in NMP (1 mL) was added 4-chloro-6-(trifluoromethyl)- 5,6,7,8-tetrahydroquinazoline (reagent preparation 3) (0.037 g, 0.16 mmol) and the resulting mixture was was heated (120 0 C) for twelve 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. [00179] 4-(2,6-dimethylpyrimidin-4-yl)-7-(2-methyl-lH-imidazo[4,5- ⁇ ]pyridin-6-yl)- 2,3,4,5-tetrahydro-l,4-benzoxazepine. Prepared as acetate salt according to the method of example 7 by using 4-chloro-2,6-dimethylpyrimidine in step 3.
  • Example 8 4- ⁇ 5- [(4-fluorophenyl)methyl] -6-methylpyrimidin-4-yl ⁇ -7-(2-methyl- 1 ,3- thiazol-5-yl)-2,3,4,5-tetrahydro-l,4-benzoxazepine
  • STEP 1 A solution of 1,1-dimethylethyl 7-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)-2,3-dihydro-l,4-benzoxazepine-4(5H)-carboxylate (example 6, step 1) (3.0 g, 8.00 mmol) in dichloromethane (90 mL) and trifluoroacetic acid (10 mL) was heated to reflux for 1 h, and then cooled to room temperature.
  • STEP 2 A mixture of 7-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-2,3- dihydro-l,4-benzoxazepine trifluoroacetate salt (2.9 g, 8.00 mmol, 4-chloro-5-[(4- fluorophenyl)methyl]-6-methylpyrimidine U ⁇ 3 ⁇ on ⁇ p;q>;sX3u ⁇ vi 2 s (1.9 g, 8.00 mmol) and N,N-diisopropylethylamine (7.0 mL, 40.0 mmol) in N-methyl-2-pyrrolidone (10 mL) was reacted in a microwave apparatus (250 W) for 2 h at 150 0 C.
  • a microwave apparatus 250 W
  • the reaction mixture was stirred at 100 0 C for 20 h, and then cooled to room temperature.
  • the reaction mixture was concentrated, and then partitioned between ethyl acetate (100 mL) and water (50 mL). The layers were separated and the organic layer was washed with brine, dried over magnesium sulfate then filtered and concentrated.
  • STEP 3 A suspension of -(2,3,4, 5-tetrahydro-l,4-benzoxazepin-7- yl)benzoate hydrochloride (1.30 g, 4.14 mmol), 4-chloro-5-[(4-fluorophenyl)methyl]-6- methylpyrimidine (reagent preparation 2) (0.98 g, 4.14 mmol), and potassium carbonate (1.71 g, 12.4 mmol) in DMF (20 mL) was heated to 130 0 C for 18 h. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (40 mL), and then washed with water (50 mL) and brine (20 mL).
  • STEP 5 To a solution of 4-(4- ⁇ 5-[(4-fluorophenyl)methyl]-6- methylpyrimidin-4-yl ⁇ -2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl)benzoic acid (0.50 g, 1.07 mmol) and DMF (20 ⁇ L) in chloroform (15 mL) was added drop wise oxalyl chloride (0.35 mL, 4.0 mmol).
  • STEP 6 STEP 6: To a solution of 4-(4- ⁇ 5-[(4-fiuorophenyl)methyl]-6- methylpyrimidin-4-yl ⁇ -2,3,4,5-tetrahydro-l ,4-benzoxazepin-7-yl)benzoyl chloride (0.06g, 0.12 mmol) and 2-fluoroethanamine hydrochloride (0.05 g, 0.50 mmol) in tetrahydrofuran (8 mL) at O 0 C was added triethylamine (0.25 mL, 2.00 mmol) and the reaction mixture was stirred for 2 hours at room temperature.
  • the reaction mixture was concentrated and the residue was partitioned between water (10 mL) and ethyl acetate (30 mL). The organic layer was separated, washed with saturated aqueous sodium hydrogencarbonate (10 mL) and brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The resulting residue was purified by gradient flash chromatography (10% to 100% ethyl acetate in hexane).
  • STEP 1 A suspension of 5-bromo-3-nitropyridin-2-amine (4.84 g, 22.2 mmol) , (4- ⁇ [(l,l-dimethylethyl)oxy]carbonyl ⁇ -2,3,4,5-tetrahydro-l,4-benzoxazepin-7- yl)boronic acid (6.51 g, 22.2 mmol) (example 5, step 1), dichloro[l,l-bis(diphenyl)- phosphino]ferrocenepalladium (II) dichloromethane adduct (1.60 g, 10 mol %) in dioxane (75 mL) and water (15 mL) was degassed with nitrogen, and then cesium carbonate (14.46 g, 44.4 mmol) was added.
  • STEP 2 A mixture of 1,1-dimethylethyl 7-(6-amino-5-nitropyridin-3-yl)-2,3- dihydro-l,4-benzoxazepine-4(5H)-carboxylate (3.5 g, 9.1 mmol) in methanol (75 mL) and 4N hydrogen chloride in dioxane (11 mL) was stirred at 50 0 C for 1.5 h and then concentrated.
  • STEP 3 A solution of 3-nitro-5-(2,3,4,5-tetrahydro-l,4-benzoxazepin-7- yl)pyridin-2-amine dihydrochloride (540 mg, 1.50 mmol), 4-chloro-6,6-dimethyl-5, 6,7,8- tetrahydroquinazoline (270 mg, 1.37 mmol, reagent preparation 3), and diisopropylethylamine (970 mg, 7.49 mmol) in N-methylpyrrolidinone (3 mL) was stirred at 120 0 C for 18 h.
  • step 3 or 5 Using analogous synthetic techniques and substituting with alternative starting reagents in step 3 or 5 the following compounds of the invention were prepared. Alternative starting materials were obtained commercially unless otherwise indicated. [00210] 7-(2-pyridin-2-yl-l/f-benzimidazol-6-yl)-4-quinolin-4-yl-2,3,4,5-tetrahydro- 1,4-benzoxazepine. Synthesized according to the method of example 10 using picolinic acid in step 5.
  • reaction mixture was stirred at 90 0 C for 1 hour. It was cooled to room temperature and partitioned between water (50 ml) and ethyl acetate (120 mL). The organic layer was separated, washed with water (50 mL) and brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated.
  • STEP 2 A solution of 4-[4-(6,6-dimethyl-5,6,7,8-tetrahydroquinazolin-4-yl)- 2,3,4,5-tetrahydro-l,4-benzoxazepin-7-yl]-2-nitroaniline (0. 36 g, 0.80 mmol) in a mixture of tetrahydrofuran (80 mL) and methanol (20 mL) in the presence of 5% palladium on carbon (50% water) was hydrogenated at atmospheric pressure for 16 hours. The mixture was filtered through a pad of Celite and concentrated.
  • STEP 4 A solution of methyl ⁇ 6-[4-(6,6-dimethyl-5,6,7,8- tetrahydroquinazolin-4-yl)-2,3 ,4,5-tetrahydro- 1 ,4-benzoxazepin-7-yl]- lH-benzimidazol- 2-yl ⁇ carbamate (0.10 g, 0.20 mmol) and 2M aqueous sodium hydroxide (8.0 mL, 16.0 mmol) in methanol (10 mL) was heated to reflux for 4 hours. After cooling to room temperature it was concentrated and partitioned between ethyl acetate (100 mL) and brine (10 mL).
  • 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 mTORc2 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 vzVo 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-BPl 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 En Vision plate reader. [00222] 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 0.40 ⁇ M or less.
  • the Compound of the Invention has an mTOR-inhibitory activity of about 0.10 ⁇ M or less. In another embodiment the Compound of the Invention has an mTOR-inhibitory activity of about 0.20 ⁇ 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 15-29, 42-47, 51, 53-56, 63, 64, and 71 have an IC50 in this assay of less than or equal to 20 nM.
  • Compounds 7-14, 41, 58-60, 65 have an IC50 in this assay of greater than 20 nM but less than or equal to 50 nM.
  • Compounds 1-6, 40, 57, 61-62, 67-70 have an IC50 in this assay of greater than 50 nM but less than or equal to 100 nM.
  • Compounds 30-39, 48-50, 52, and 66 have an IC50 in this assay of greater than 100 nM but less than or equal to 690 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% cholamidopropyldimethylammoniopropanesulfonate (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 VO 4 , 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, 0.
  • the immune complexes (equivalent to IxIO 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) 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 1.5 ⁇ M or less. In another embodiment, the Compound of the Invention tested in this assay in MCF-7 cells had an inhibitory activity of 1.0 ⁇ M or less. In another embodiment, the Compound of the Invention tested in this assay in MCF-7 cells had an inhibitory activity of 0.5 ⁇ M or less. In one embodiment, the Compound of the Invention tested in this assay in MCF-7 cells had an inhibitory activity of 0.25 ⁇ M or less. In one embodiment, the Compound of the Invention tested in this assay in MCF-7 cells had an inhibitory activity of 0.2 ⁇ M or less. In one embodiment, the Compound of the Invention tested in this assay in MCF-7 cells had an inhibitory activity of 0.1 ⁇ M or less. Biological Example 4-10
  • mice 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. On day 0, 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.
  • DMEM Cellgro
  • Fetal Bovine Serum Cellgro
  • Penicillin-Streptomycin Penicillin-Streptomycin
  • non-essential amino acids at 37 0 C in a humidified 5% CO 2 atmosphere.
  • cells
  • 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 (Mediatech) supplemented with 10% 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 3x10 6 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.
  • 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 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.
  • DMEM Mediatech
  • Fetal Bovine Serum Hyclone
  • Penicillin- Streptomycin and non-essential amino acids at 37 0 C in a humidified 5% CO 2 atmosphere.
  • cells are harvested by trypsinization and 3x10 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 non-essential amino acids at 37 0 C in a humidified 5% CO 2 atmosphere.
  • DMEM Mediatech
  • Fetal Bovine Serum Hyclone
  • Penicillin- Streptomycin and non-essential amino acids
  • 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 A549 human lung carcinoma model
  • 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 model
  • 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% CO 2 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 model [00237] WM-266-4 human melanoma cells are cultured in vitro in DMEM (Mediatech) supplemented with 10% 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 3x10 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).

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Abstract

La présente invention concerne des inhibiteurs de mTOR et leurs sels ou solvates pharmaceutiquement acceptables, ainsi que des procédés pour les utiliser. Ces inhibiteurs répondent d'une manière générale à la formule développée suivante : dans laquelle R1 et R2 sont chacun tels que définis dans la description, et ses sels pharmaceutiquement acceptables.
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WO2012071519A1 (fr) * 2010-11-24 2012-05-31 Exelixis, Inc. Benzoxazépines en tant qu'inhibiteurs de pi3k/mtor et procédés de leurs utilisation et fabrication
WO2012071501A1 (fr) * 2010-11-24 2012-05-31 Exelixis, Inc. Benzoxazépines en tant qu'inhibiteurs de pi3k/mtor et procédés de leurs utilisation et fabrication
WO2012074869A1 (fr) * 2010-11-24 2012-06-07 Exelixis, Inc. Benzoxazépines en tant qu'inhibiteurs de pi3k/mtor, et leurs procédés d'utilisation et de fabrication
WO2012068106A3 (fr) * 2010-11-15 2012-08-16 Exelixis, Inc. Benxazépines en tant qu'inhibiteurs de pi3k/mtor et leurs procédés d'utilisation et de fabrication
WO2012068096A3 (fr) * 2010-11-15 2012-11-08 Exelixis, Inc. Benzoxazépines en tant qu'inhibiteurs de pi3k/mtor et leurs méthodes d'utilisation et de fabrication
CN103361082A (zh) * 2012-04-03 2013-10-23 Jsr株式会社 液晶配向剂、液晶配向膜、液晶显示元件、聚合物及化合物
JP2013540782A (ja) * 2010-10-26 2013-11-07 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング キナゾリン誘導体

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WO2012068106A3 (fr) * 2010-11-15 2012-08-16 Exelixis, Inc. Benxazépines en tant qu'inhibiteurs de pi3k/mtor et leurs procédés d'utilisation et de fabrication
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CN103459384A (zh) * 2010-11-24 2013-12-18 埃克塞里艾克西斯公司 作为PI3K/mTOR抑制剂的苯并氧氮杂环庚三烯以及它们使用和制造方法
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