WO2020076728A1 - SUBSTITUTED OXAZINOPTERIDINONES AS INHIBITORS OF mTOR - Google Patents

SUBSTITUTED OXAZINOPTERIDINONES AS INHIBITORS OF mTOR Download PDF

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Publication number
WO2020076728A1
WO2020076728A1 PCT/US2019/055045 US2019055045W WO2020076728A1 WO 2020076728 A1 WO2020076728 A1 WO 2020076728A1 US 2019055045 W US2019055045 W US 2019055045W WO 2020076728 A1 WO2020076728 A1 WO 2020076728A1
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Prior art keywords
compound
formula
kinase
pharmaceutically acceptable
compounds
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PCT/US2019/055045
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French (fr)
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Sean Murphy
Matthew Marx
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Takeda Pharmaceutical Company Limited
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Publication of WO2020076728A1 publication Critical patent/WO2020076728A1/en

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    • 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
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/14—Ortho-condensed systems

Definitions

  • This invention relates to substituted oxazinopteridinones which are selective inhibitors of mammalian target of rapamycin (mTOR), to pharmaceutical compositions which contain them, and to the use of the substituted oxazinopteridinones to treat diseases, disorders, and conditions associated with mTOR, including immunological disorders, cancer, and neurodegenerative diseases.
  • mTOR mammalian target of rapamycin
  • Mammalian target of rapamycin is a serine/threonine kinase and has been identified as a regulator of protein synthesis and of cell growth and proliferation.
  • mTOR has been shown to regulate the response of tumor cells to nutrients and growth factors and to regulate the ability of tumors to promote angiogenesis.
  • inhibitors of mTOR activity are actively being studied as potential anti-proliferative agents. Inhibitors of mTOR are currently approved for immunosuppression and cancer treatment.
  • Inhibition of mTOR function by small molecules results in a loss of transmission of upstream activating signals (i.e., from growth factor receptors) to downstream effectors of cell growth.
  • Rapamycin an inhibitor of mTOR, inhibits proliferation or growth of cells derived from a range of tissue types, including smooth muscle and T-cells, as well as cells derived from a diverse range of tumor types including rhabdomyosarcoma, neuroblastoma, glioblastoma and
  • rapamycin and its derivatives have shown the ability to potentiate the cytotoxicity of a number of common cancer chemotherapies including cisplatin, camptothecin and doxorubicin.
  • mTOR functions in two distinct complexes (mTORCl and mTORC2). Rapamycin primarily inhibits the mTORCl complex while largely sparing mTORC2 activity. Thus, one strategy is to identify compounds that are capable of inhibiting mTORCl - and mTORC2-mediated activity in the cell.
  • mTOR Complexl-S6Kl integrates various extrinsic signals that regulate cell growth and metabolism.
  • rapamycin provide a link between mTOR
  • WO 2011/079114 and WO 2011/079118 describe various pteridinones which are inhibitors of polo-like kinase (PLK); and published patent application WO 2012/148548 describes various N- substituted oxazinopteridines and oxazinopteridinones which are inhibitors of certain isoforms of phosphoinositide 3-kinase (PI3K).
  • PLK polo-like kinase
  • PI3K phosphoinositide 3-kinase
  • This invention provides substituted oxazinopteridinones and pharmaceutically acceptable salts thereof, which are selective inhibitors of mammalian target of rapamycin (mTOR).
  • mTOR mammalian target of rapamycin
  • This invention also provides pharmaceutical compositions which contain the substituted oxazinopteridinones, and provides for their use to treat diseases, disorders, and conditions associated with mTOR, including immunological disorders, cancer, and neurodegenerative diseases.
  • One aspect of the invention provides a compound of Formula 1,
  • Formula 1 depicts the compound fV)-5-(4-hydroxy-
  • Another aspect of the invention provides a compound of the formula,
  • a further aspect of the invention provides a compound of the formula
  • An additional aspect of the invention provides a pharmaceutical composition which includes a compound of Formula 1 or a pharmaceutically acceptable salt thereof, or any one of the compounds or pharmaceutically acceptable salts defined in the preceding paragraphs; and a pharmaceutically acceptable excipient.
  • Another aspect of the invention provides a compound of Formula 1 or a
  • a further aspect of the invention provides a use of a compound of Formula 1 or a pharmaceutically acceptable salt thereof, or any one of the compounds or pharmaceutically acceptable salts defined in the preceding paragraphs, for the manufacture of a medicament for the treatment of a disease, disorder or condition associated with mTOR.
  • An additional aspect of the invention provides a method of treating a disease, disorder or condition associated with mTOR, the method comprising administering to the subject an effective amount of a compound of Formula 1 or a pharmaceutically acceptable salt thereof, or any one of the compounds or pharmaceutically acceptable salts defined in the preceding paragraphs.
  • Another aspect of the invention provides a method of treating a disease, disorder or condition in a subject, the method comprising administering to the subject an effective amount of a compound of Formula 1 or a pharmaceutically acceptable salt thereof, or any one of the compounds or pharmaceutically acceptable salts defined in the preceding paragraphs, wherein the disease, disorder or condition is selected from immunological disorders, cancer, and
  • a further aspect of the invention provides an effective amount of a compound of Formula 1 or a pharmaceutically acceptable salt thereof, or any one of the compounds or pharmaceutically acceptable salts defined in the preceding paragraphs; and at least one additional pharmacologically active agent.
  • the drawing shows the dose response of a human PC3 xenograft mouse model following treatment with the compound prepared in Example 3 or with compounds A, B or C, which correspond to Examples 17, 26, and 33, respectively, in published patent application WO 2011/025889A1.
  • Condition associated with mTOR relate to a disease, disorder or condition in a subject for which inhibition of mTOR may provide a therapeutic or prophylactic benefit.
  • Selective mTOR inhibitor means a compound that inhibits mTOR at a concentration that is at least ten times less than it inhibits either PLK or PI3K (alpha, beta, delta or gamma isoforms).
  • Optesite enantiomer refers to a molecule that is a non-superimposable mirror image of a reference molecule, which may be obtained by inverting all of the stereogenic centers of the reference molecule. For example, if the reference molecule has S absolute stereochemical configuration, then the opposite enantiomer has R absolute stereochemical configuration.
  • the opposite enantiomer has R,R stereochemical configuration, and so on.
  • Stereoisomer and“stereoisomers” of a compound with given stereochemical configuration refer to the opposite enantiomer of the compound and to any diastereoisomers, including geometrical isomers ( Z!E) of the compound.
  • Z!E geometrical isomers
  • stereoisomer refers to any one of the possible stereochemical configurations of the compound.
  • “Substantially pure stereoisomer” and variants thereof refer to a sample containing a compound having a specific stereochemical configuration and which comprises at least about 95% of the sample, based on the total amount of all stereoisomers of the compound present in the sample.
  • “Pure stereoisomer” and variants thereof refer to a sample containing a compound having a specific stereochemical configuration and which comprises at least about 99.5% of the sample, based on the total amount of all stereoisomers of the compound present in the sample.
  • Subject refers to a mammal, including a human.
  • “Pharmaceutically acceptable” substances refer to those substances which are suitable for administration to subjects.
  • Treating refers to reversing, alleviating, inhibiting the progress of, or preventing a disease, disorder or condition to which such term applies, or to reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of such disease, disorder or condition.
  • Treatment refers to the act of“treating,” as defined immediately above.
  • “Drug,”“drug substance,”“active pharmaceutical ingredient,” and the like refer to a compound (e.g., compounds of Formula 1, Formula 2, and Formula 3) that may be used for treating a subject in need of treatment.
  • Effective amount of a drug refers to the quantity of the drug that may be used for treating a subject and may depend on the weight and age of the subject and the route of administration, among other things.
  • Excipient refers to any diluent or vehicle for a drug.
  • “Pharmaceutical composition” refers to the combination of one or more drug substances and one or more excipients.
  • “Drug product,”“pharmaceutical dosage form,”“dosage form,”“final dosage form” and the like refer to a pharmaceutical composition suitable for treating a subject in need of treatment and generally may be in the form of tablets, capsules, sachets containing powder or granules, liquid solutions or suspensions, patches, films, and the like.
  • this disclosure concerns a compound of Formula 1 and its pharmaceutically acceptable salts.
  • This disclosure also concerns materials and methods for preparing the compound of Formula 1, pharmaceutical compositions which contain it, and the use of the compound of Formula 1 and its pharmaceutically acceptable salts (optionally in
  • the compound of Formula 1 includes compounds specifically named above and in the examples, and may exist as salts, complexes, solvates, hydrates, and liquid crystals. Likewise, compounds of Formula 1 that are salts may exist as complexes, solvates, hydrates, and liquid crystals. [0038] The compound of Formula 1 may form pharmaceutically acceptable complexes, salts, solvates and hydrates. These salts include acid addition salts (including di-acids) and base salts.
  • Pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, and phosphorous acids, as well nontoxic salts derived from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl -substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.
  • inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, and phosphorous acids
  • organic acids such as aliphatic mono- and dicarboxylic acids, phenyl -substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, alipha
  • Such salts include acetate, adipate, aspartate, benzoate, besylate, bicarbonate, carbonate, bisulfate, sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate, hydrogen phosphate, dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate
  • Pharmaceutically acceptable base salts include salts derived from bases, including metal cations, such as an alkali or alkaline earth metal cation, as well as amines.
  • suitable metal cations include sodium, potassium, magnesium, calcium, zinc, and aluminum.
  • suitable amines include arginine, NfT -dibenzylethylenediamine, chloroprocaine, choline, diethylamine, diethanolamine, dicyclohexylamine, ethylenediamine, glycine, lysine, N- methylglucamine, olamine, 2-amino-2-hydroxymethyl-propane-l,3-diol, and procaine.
  • salts may be prepared using various methods. For example, a compound of Formula 1 may be reacted with an appropriate acid or base to give the desired salt. Alternatively, a precursor of the compound of Formula 1 may be reacted with an acid or base to remove an acid- or base-labile protecting group or to open a lactone or lactam group of the precursor. Additionally, a salt of the compound of Formula 1 may be converted to another salt (or free form) through treatment with an appropriate acid or base or through contact with an ion exchange resin. Following reaction, the salt may be isolated by filtration if it precipitates from solution, or by evaporation to recover the salt. The degree of ionization of the salt may vary from completely ionized to almost non-ionized.
  • the compound of Formula 1 may exist in a continuum of solid states ranging from fully amorphous to fully crystalline.
  • amorphous refers to a state in which the material lacks long range order at the molecular level and, depending upon temperature, may exhibit the physical properties of a solid or a liquid. Typically such materials do not give distinctive X-ray diffraction patterns and, while exhibiting the properties of a solid, are more formally described as a liquid.
  • a change from solid to liquid properties occurs which is characterized by a change of state, typically second order (“glass transition”).
  • crystalline refers to a solid phase in which the material has a regular ordered internal structure at the molecular level and gives a distinctive X-ray diffraction pattern with defined peaks. Such materials when heated sufficiently will also exhibit the properties of a liquid, but the change from solid to liquid is characterized by a phase change, typically first order (“melting point”).
  • the compound of Formula 1 may also exist in unsolvated and solvated forms.
  • solvate describes a molecular complex comprising the compound and one or more
  • solvent molecules e.g., ethanol
  • hydrate is a solvate in which the solvent is water.
  • Pharmaceutically acceptable solvates include those in which the solvent may be isotopically substituted (e.g., D2O, aceton e-de, DMSO-r/r,).
  • a currently accepted classification system for solvates and hydrates of organic compounds is one that distinguishes between isolated site, channel, and metal-ion coordinated solvates and hydrates. See, e.g., K. R. Morris (H. G. Brittain ed.) Polymorphism in
  • Isolated site solvates and hydrates are ones in which the solvent (e.g., water) molecules are isolated from direct contact with each other by intervening molecules of the organic compound.
  • the solvent molecules lie in lattice channels where they are next to other solvent molecules.
  • metal-ion coordinated solvates the solvent molecules are bonded to the metal ion.
  • the complex When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and in hygroscopic compounds, the water or solvent content will depend on humidity and drying conditions. In such cases, non-stoichiometry will typically be observed.
  • the compound of Formula 1 may also exist as multi-component complexes (other than salts and solvates) in which the compound (drug) and at least one other component are present in stoichiometric or non-stoichiometric amounts.
  • Complexes of this type include clathrates (drug- host inclusion complexes) and co-crystals. The latter are typically defined as crystalline complexes of neutral molecular constituents which are bound together through non-covalent interactions, but could also be a complex of a neutral molecule with a salt.
  • Co-crystals may be prepared by melt crystallization, by recrystallization from solvents, or by physically grinding the components together. See, e.g., O. Almarsson and M. J. Zaworotko, Chem. Commun. (2004) 17: 1889-1896.
  • the compound of Formula 1 may exist in a mesomorphic state (mesophase or liquid crystal).
  • the mesomorphic state lies between the true crystalline state and the true liquid state (either melt or solution).
  • Mesomorphism arising as the result of a change in temperature is described as“thermotropic” and mesomorphism resulting from the addition of a second component, such as water or another solvent, is described as “lyotropic.”
  • Compounds that have the potential to form lyotropic mesophases are described as “amphiphilic” and include molecules which possess a polar ionic moiety
  • the compound of Formula 1 may exist as polymorphs, stereoisomers, tautomers, or some combination thereof, may be isotopically-labeled, may result from the administration of a prodrug, or form a metabolite following administration.
  • Prodrugs refer to compounds having little or no pharmacological activity that can, when metabolized in vivo , undergo conversion to compounds having desired pharmacological activity. Prodrugs may be prepared by replacing appropriate functionalities present in
  • prodrugs include ester, ether or amide derivatives of compounds of Formula 1 having carboxylic acid, hydroxy, or amino functional groups, respectively.
  • prodrugs see e.g., T. Higuchi and V. Stella “Pro-drugs as Novel Delivery Systems,” ACS Symposium Series 14 (1975) and E. B. Roche ed., Bioreversible Carriers in Drug Design (1987).
  • Methodabolites refer to compounds formed in vivo upon administration of
  • pharmacologically active compounds examples include hydroxymethyl, hydroxy, secondary amino, primary amino, phenol, and carboxylic acid derivatives of compounds of Formula 1 having methyl, alkoxy, tertiary amino, secondary amino, phenyl, and amide groups, respectively.
  • the compound of Formula 1 may exist as a particular stereoisomer.
  • Stereoisomers are configuration isomers resulting, for example, from the presence of one or more stereogenic centers, double bonds, cyclic groups, or some combination of these structural features. Such stereoisomers may also result from acid addition or base salts in which the counter-ion is optically active, for example, when the counter-ion is D-lactate or L-lysine. Stereoisomers may be pure, substantially pure, or mixtures.
  • the compound of Formula 1 may exhibit more than one type of stereoisomerism.
  • the stereogenic carbon (position 6a) of the tricyclic core shown in Formula 1 has S stereochemical configuration.
  • the compound of Formula 1 may exist as cis or trans stereoisomers based on the relative positions of substituents attached to the cyclohexyl moiety.
  • the compound of Formula 1 includes geometric isomers (,S)-c/.
  • the cis stereoisomer has the name S)-5-((U,4f?)-4-hydroxy-4-methylcyclohexyl)-2-(li7-indol-4-yl)-6a-methyl- 6a, 7,9, 10-tetrahydro-[ 1 ,4]oxazino[3,4-/?]pteridin-6(5//)-one
  • the trans stereoisomer has the name S)-5-((lr,4,S)-4-hydroxy-4-methylcyclohexyl)-2-(li7-indol-4-yl)-6a-methyl-6a,7,9,l0- tetrahydro-[ 1 ,4]oxazino[3,4-/?]pteridin-6(5H)-one.
  • Compounds of Formula 1 may exist as tautomers, which are isomers resulting from tautomerization.
  • tautomeric isomerism includes, for example, imine-enamine, keto- enol, oxime-nitroso, and amide-imidic acid tautomerism.
  • Geometrical (cis/trans) isomers may be separated by conventional techniques such as chromatography and fractional crystallization.
  • Conventional techniques for preparing or isolating a compound having a specific stereochemical configuration include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).
  • HPLC high pressure liquid chromatography
  • the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound contains an acidic or basic moiety, an acid or base such as tartaric acid or 1- phenylethylamine.
  • the resulting diastereomeric mixture may be separated by chromatography, fractional crystallization, etc., and the appropriate diastereoisomer converted to the compound having the requisite stereochemical configuration.
  • chromatography fractional crystallization, etc.
  • diastereoisomer converted to the compound having the requisite stereochemical configuration.
  • the compound of Formula 1 may possess isotopic variations, in which at least one atom is replaced by an atom having the same atomic number, but an atomic mass different from the atomic mass usually found in nature. Isotopes suitable for inclusion in compound of
  • Formula 1 include, for example, isotopes of hydrogen, such as 2 H and 3 H; isotopes of carbon, such as u C, 13 C and 14 C; isotopes of nitrogen, such as 13 N and 15 N; and isotopes of oxygen, such as 15 0, 17 0 and 18 0.
  • ETse of isotopic variations e.g., deuterium, 2 H
  • radioactive isotope e.g., tritium, 3 H, or 14 C
  • substitution with positron emitting isotopes, such as U C, 15 0, and 13 N, may be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
  • Isotopically-labeled compounds may be prepared by processes analogous to those described elsewhere in the disclosure using an appropriate isotopically-labeled reagent in place of a non-labeled reagent.
  • the compound of Formula 1 may be prepared using the techniques described below. Some of the schemes and examples may omit details of common reactions, including oxidations, reductions, and so on, separation techniques (extraction, evaporation, precipitation,
  • reaction intermediates may be used in subsequent steps without isolation or purification (i.e., in situ).
  • certain compounds can be prepared using protecting groups, which prevent undesirable chemical reaction at otherwise reactive sites.
  • Protecting groups may also be used to enhance solubility or otherwise modify physical properties of a compound.
  • protecting group strategies a description of materials and methods for installing and removing protecting groups, and a compilation of useful protecting groups for common functional groups, including amines, carboxylic acids, alcohols, ketones, aldehydes, and so on, see T. W. Greene and P. G. Wuts, Protecting Groups in Organic Chemistry (1999) and P. Kocienski, Protective Groups (2000).
  • the chemical transformations described throughout the specification may be carried out using substantially stoichiometric amounts of reactants, though certain reactions may benefit from using an excess of one or more of the reactants. Additionally, many of the reactions disclosed throughout the specification may be carried out at about room temperature (RT) and ambient pressure, but depending on reaction kinetics, yields, and so on, some reactions may be run at elevated pressures or employ higher temperatures (e.g., reflux conditions) or lower temperatures (e.g., -78°C to 0°C). Any reference in the disclosure and claims to a stoichiometric range, a temperature range, a pH range, etc., whether or not expressly using the word“range,” also includes the indicated endpoints.
  • the one or more solvents may be polar protic solvents (including water), polar aprotic solvents, non-polar solvents, or some combination.
  • Representative solvents include saturated aliphatic hydrocarbons (e.g., «-pentane, «-hexane, «-heptane, «-octane, cyclohexane,
  • methylcyclohexane methylcyclohexane
  • aromatic hydrocarbons e.g., benzene, toluene, xylenes
  • halogenated hydrocarbons e.g., methylene chloride, chloroform, carbon tetrachloride
  • aliphatic alcohols e.g., methanol, ethanol, propan- l-ol, propan-2-ol, butan-l-ol, 2-methyl-propan- l-ol, butan-2-ol, 2- methyl-propan-2-ol, pentan-l-ol, 3-methyl-butan-l-ol, hexan-l-ol, 2-m ethoxy-ethanol, 2-ethoxy- ethanol, 2-butoxy-ethanol, 2-(2-methoxy-ethoxy)-ethanol, 2-(2-ethoxy-ethoxy)-ethanol, 2-(2- butoxy-ethoxy)-ethanol); ethers (e.g.
  • sulfur-containing solvents e.g., carbon disulfide, dimethyl sulfoxide, tetrahydro-thiophene-l, l,- dioxide
  • phosphorus-containing solvents e.g., hexamethylphosphoric triamide
  • the compounds of Formula 1, which includes compounds named above, and their pharmaceutically acceptable complexes, salts, solvates and hydrates, should be assessed for their biopharmaceutical properties, such as solubility and solution stability across pH, permeability, and the like, to select an appropriate dosage form and route of administration.
  • Compounds that are intended for pharmaceutical use may be administered as crystalline or amorphous products, and may be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, spray drying, evaporative drying, microwave drying, or radio frequency drying.
  • the compound of Formula 1 may be administered alone or in combination with one another or with one or more pharmacologically active compounds which are different than the compound of Formula 1.
  • one or more of these compounds are administered as a pharmaceutical composition (a formulation) in association with one or more pharmaceutically acceptable excipients.
  • the choice of excipients depends on the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form, among other things.
  • Useful pharmaceutical compositions and methods for their preparation may be found, for example, in A. R. Gennaro (ed.), Remington: The Science and Practice of Pharmacy (20th ed., 2000).
  • the compound of Formula 1 may be administered orally.
  • Oral administration may involve swallowing in which case the compound enters the bloodstream via the gastrointestinal tract.
  • oral administration may involve mucosal administration (e.g., buccal, sublingual, supralingual administration) such that the compound enters the bloodstream through the oral mucosa.
  • Formulations suitable for oral administration include solid, semi-solid and liquid systems such as tablets; soft or hard capsules containing multi- or nano-particulates, liquids, or powders; lozenges which may be liquid-filled; chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal or mucoadhesive patches.
  • Liquid formulations include suspensions, solutions, syrups and elixirs.
  • Such formulations may be employed as fillers in soft or hard capsules (made, e.g., from gelatin or hydroxypropylmethylcellulose) and typically comprise a carrier (e.g., water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil) and one or more emulsifying agents, suspending agents or both.
  • a carrier e.g., water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil
  • emulsifying agents e.g., ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil
  • Liquid formulations may also be prepared by the reconstitution of a solid (e.g., from a sachet).
  • the compound of Formula 1 may also be used in fast-dissolving, fast-disintegrating dosage forms such as those described in Liang and Chen, Expert Opinion in Therapeutic Patents (2001) 1 l(6):98l-986.
  • the active pharmaceutical ingredient may comprise from about 1 wt% to about 80 wt% of the dosage form or more typically from about 5 wt% to about 60 wt% of the dosage form.
  • tablets may include one or more disintegrants, binders, diluents, surfactants, glidants, lubricants, anti-oxidants, colorants, flavoring agents, preservatives, and taste-masking agents.
  • disintegrants examples include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, Ci- 6 alkyl-substituted hydroxypropylcellulose, starch, pregelatinized starch, and sodium alginate.
  • the disintegrant will comprise from about 1 wt% to about 25 wt% or from about 5 wt% to about 20 wt% of the dosage form.
  • Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropylcellulose and hydroxypropylmethylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous), mannitol, xylitol, dextrose, sucrose, sorbitol,
  • microcrystalline cellulose starch and dibasic calcium phosphate dihydrate.
  • Tablets may also include surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc.
  • surface active agents such as sodium lauryl sulfate and polysorbate 80
  • glidants such as silicon dioxide and talc.
  • surface active agents may comprise from about 0.2 wt% to about 5 wt% of the tablet, and glidants may comprise from about 0.2 wt% to about 1 wt% of the tablet.
  • Tablets may also contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulfate.
  • Lubricants may comprise from about 0.25 wt% to about 10 wt% or from about 0.5 wt% to about 3 wt% of the tablet.
  • Tablet blends may be compressed directly or by roller compaction to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or extruded before tableting. If desired, prior to blending one or more of the components may be sized by screening or milling or both.
  • the final dosage form may comprise one or more layers and may be coated, uncoated, or encapsulated.
  • Exemplary tablets may contain up to about 80 wt% of API, from about 10 wt% to about 90 wt% of binder, from about 0 wt% to about 85 wt% of diluent, from about 2 wt% to about 10 wt% of disintegrant, and from about 0.25 wt% to about 10 wt% of lubricant.
  • Consumable oral films for human or veterinary use are pliable water-soluble or water- swellable thin film dosage forms which may be rapidly dissolving or mucoadhesive.
  • a typical film includes one or more film-forming polymers, binders, solvents, humectants, plasticizers, stabilizers or emulsifiers, viscosity-modifying agents, and solvents.
  • film ingredients may include anti-oxidants, colorants, flavorants and flavor enhancers, preservatives, salivary stimulating agents, cooling agents, co-solvents (including oils), emollients, bulking agents, anti-foaming agents, surfactants, and taste-masking agents.
  • Some components of the formulation may perform more than one function.
  • the amount of API in the film may depend on its solubility. If water soluble, the API would typically comprise from about 1 wt% to about 80 wt% of the non-solvent components (solutes) in the film or from about 20 wt% to about 50 wt% of the solutes in the film. A less soluble API may comprise a greater proportion of the composition, typically up to about 88 wt% of the non-solvent components in the film.
  • the film-forming polymer may be selected from natural polysaccharides, proteins, or synthetic hydrocolloids and typically comprises from about 0.01 wt% to about 99 wt% or from about 30 wt% to about 80 wt% of the film.
  • Film dosage forms are typically prepared by evaporative drying of thin aqueous films coated onto a peelable backing support or paper, which may carried out in a drying oven or tunnel (e.g., in a combined coating-drying apparatus), in lyophilization equipment, or in a vacuum oven.
  • Useful solid formulations for oral administration may include immediate release formulations and modified release formulations.
  • Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.
  • suitable modified release formulations see US Patent No. 6, 106,864.
  • Other useful release technologies such as high energy dispersions and osmotic and coated particles, see Verma et al, Pharmaceutical Technology On-line (2001) 25(2): 1-14.
  • the compound of Formula 1 may also be administered directly into the blood stream, muscle, or an internal organ of the subject.
  • Suitable techniques for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, and subcutaneous administration.
  • Suitable devices for parenteral administration include needle injectors, including microneedle injectors, needle-free injectors, and infusion devices.
  • Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (e.g., pH of from about 3 to about 9).
  • excipients such as salts, carbohydrates and buffering agents (e.g., pH of from about 3 to about 9).
  • the compound of Formula 1 may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
  • a suitable vehicle such as sterile, pyrogen-free water.
  • the preparation of parenteral formulations under sterile conditions may be readily accomplished using standard pharmaceutical techniques.
  • solubility of compounds which are used in the preparation of parenteral solutions may be increased through appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.
  • Formulations for parenteral administration may be formulated to be immediate or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release.
  • the compound of Formula 1 may be formulated as a suspension, a solid, a semi-solid, or a thixotropic liquid for administration as an implanted depot providing modified release of the active compound.
  • examples of such formulations include drug-coated stents and semi-solids and suspensions comprising drug-loaded poly(DL-lactic-coglycolic)acid (PGLA) microspheres.
  • the compound of Formula 1 may also be administered topically, intradermally, or transdermally to the skin or mucosa.
  • Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages and microemulsions.
  • Liposomes may also be used.
  • Typical carriers may include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol.
  • Topical formulations may also include penetration enhancers. See, e.g., Finnin and Morgan, J. Pharm. Sci.
  • Topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis and microneedle or needle-free (e.g. PowderjectTM and BiojectTM) injection.
  • Formulations for topical administration may be formulated to be immediate or modified release as described above.
  • the compound of Formula 1 may also be administered intranasally or by inhalation, typically in the form of a dry powder, an aerosol spray, or nasal drops.
  • An inhaler may be used to administer the dry powder, which comprises the API alone, a powder blend of the API and a diluent, such as lactose, or a mixed component particle that includes the API and a phospholipid, such as phosphatidylcholine.
  • the powder may include a bioadhesive agent, e.g., chitosan or cyclodextrin.
  • a pressurized container, pump, sprayer, atomizer, or nebulizer may be used to generate the aerosol spray from a solution or suspension comprising the API, one or more agents for dispersing, solubilizing, or extending the release of the API (e.g., EtOH with or without water), one or more solvents (e.g., l,l,l,2-tetrafluoroethane or l,l,l,2,3,3,3-heptafluoropropane) which serve as a propellant, and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.
  • An atomizer using electrohydrodynamics may be used to produce a fine mist.
  • the drug product Prior to use in a dry powder or suspension formulation, the drug product is usually comminuted to a particle size suitable for delivery by inhalation (typically 90% of the particles, based on volume, having a largest dimension less than 5 microns). This may be achieved by any appropriate size reduction method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing, high pressure homogenization, or spray drying.
  • Capsules, blisters and cartridges made, for example, from gelatin or
  • hydroxypropylmethyl cellulose for use in an inhaler or insufflator may be formulated to contain a powder mixture of the active compound, a suitable powder base such as lactose or starch, and a performance modifier such as L-leucine, mannitol, or magnesium stearate.
  • the lactose may be anhydrous or monohydrated.
  • Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose, and trehalose.
  • a suitable solution formulation for use in an atomizer using electrohydrodynamics to produce a fine mist may contain from about 1 pg to about 20 mg of the API per actuation and the actuation volume may vary from about 1 pL to about 100 pL.
  • a typical formulation may comprise one or more compounds of Formula 1, propylene glycol, sterile water, EtOH, and NaCl.
  • Alternative solvents, which may be used instead of propylene glycol, include glycerol and polyethylene glycol.
  • Formulations for inhaled administration, intranasal administration, or both may be formulated to be immediate or modified release using, for example, PGLA. Suitable flavors, such as menthol and levomenthol, or sweeteners, such as saccharin or sodium saccharin, may be added to formulations intended for inhaled/intranasal administration.
  • the dosage unit is determined by means of a valve that delivers a metered amount.
  • Units are typically arranged to administer a metered dose or“puff’ containing from about 10 pg to about 1000 pg of the API.
  • the overall daily dose will typically range from about 100 pg to about 10 mg which may be administered in a single dose or, more usually, as divided doses throughout the day.
  • the active compounds may be administered rectally or vaginally, e.g., in the form of a suppository, pessary, or enema. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate. Formulations for rectal or vaginal administration may be formulated to be immediate or modified release as described above.
  • the compound of Formula 1 may also be administered directly to the eye or ear, typically in the form of drops of a micronized suspension or solution in isotonic, pH-adjusted, sterile saline.
  • Other formulations suitable for ocular and aural administration include ointments, gels, biodegradable implants (e.g. absorbable gel sponges, collagen), non-biodegradable implants (e.g. silicone), wafers, lenses, and particulate or vesicular systems, such as niosomes or liposomes.
  • the formulation may include one or more polymers and a preservative, such as benzalkonium chloride. Typical polymers include crossed-linked polyacrylic acid,
  • formulations may also be delivered by iontophoresis.
  • Formulations for ocular or aural administration may be formulated to be immediate or modified release as described above.
  • the compound of Formula 1 may be combined with soluble macromolecular entities, including cyclodextrin and its derivatives and polyethylene glycol-containing polymers.
  • soluble macromolecular entities including cyclodextrin and its derivatives and polyethylene glycol-containing polymers.
  • API- cyclodextrin complexes are generally useful for most dosage forms and routes of administration. Both inclusion and non-inclusion complexes may be used.
  • the cyclodextrin may be used as an auxiliary additive, i.e. as a carrier, diluent, or solubilizer.
  • auxiliary additive i.e. as a carrier, diluent, or solubilizer.
  • Alpha-, beta- and gamma-cyclodextrins are commonly used for these purposes. See, e.g., WO 91/11172, WO 94/02518, and WO 98/55148.
  • the compound of Formula 1 including compounds specifically named above, and their pharmaceutically active complexes, salts, solvates and hydrates, may be combined with each other or with one or more other active pharmaceutically active compounds to treat various diseases, conditions and disorders.
  • the active compounds may be combined in a single dosage form as described above or may be provided in the form of a kit which is suitable for coadministration of the compositions.
  • the kit comprises (1) two or more different pharmaceutical compositions, at least one of which contains a compound of Formula 1; and (2) a device for separately retaining the two pharmaceutical compositions, such as a divided bottle or a divided foil packet.
  • An example of such a kit is the familiar blister pack used for the packaging of tablets or capsules.
  • the kit is suitable for administering different types of dosage forms (e.g., oral and parenteral) or for administering different pharmaceutical compositions at separate dosing intervals, or for titrating the different pharmaceutical compositions against one another.
  • the kit typically comprises directions for
  • administration and may be provided with a memory aid.
  • the total daily dose of the claimed and disclosed compounds is typically in the range of about 0.1 mg to about 3000 mg depending on the route of administration.
  • oral administration may require a total daily dose of from about 1 mg to about 3000 mg
  • an intravenous dose may only require a total daily dose of from about 0.1 mg to about 300 mg.
  • the total daily dose may be administered in single or divided doses and, at the physician’s discretion, may fall outside of the typical ranges given above.
  • these dosages are based on an average human subject having a mass of about 60 kg to about 70 kg, the physician will be able to determine the appropriate dose for a patient (e.g., an infant) whose mass falls outside of this weight range.
  • the compound of Formula 1 may be used to treat diseases, disorders, and conditions for which inhibition of mTOR is indicated.
  • diseases, disorders, and conditions generally relate to any unhealthy or abnormal state in a subject for which the inhibition of mTOR provides a therapeutic benefit.
  • diseases, disorders, and conditions include neurodegenerative diseases, cancer, diseases involving the immune system, inflammation, autoimmune diseases, and fibrotic disorders.
  • diseases, disorders, and conditions associated with mTOR they include more specific conditions mentioned in diagnostic manuals and other materials.
  • the treatment of cancer includes treatment of all neoplasia, regardless of their histopathological appearance.
  • the compound of Formula 1 may be used to treat cancer of blood, including leukemia (e.g., acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia), and cancer of the skin, including melanoma, basal cell carcinoma, and squamous cell carcinoma.
  • leukemia e.g., acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia
  • cancer of the skin including melanoma, basal cell carcinoma, and squamous cell carcinoma.
  • the compound of Formula 1 may also be used to treat cancer of the bone, liver, and lung (e.g., small-cell lung tumor, non small-cell lung cancer, and bronchioalveolar cancer); cancer of the brain, breast, prostate, larynx, gall bladder, pancreas, rectum, bile duct, parathyroid, thyroid, adrenal gland, neural tissue, bladder, spleen, head and neck, including cancer of the jaw, mouth, and nose; and cancer of the colon, stomach, testes, esophagus, uterus, cervix, vulva, bronchi, bile duct, bladder, kidney, ovary, and pancreas.
  • small-cell lung tumor e.g., non small-cell lung cancer, and bronchioalveolar cancer
  • the compound of Formula 1 may be used to treat multiple myeloma, lymphomas, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, osteosarcoma, Ewing’s sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, islet cell tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuromas, intestinal
  • ganglioneuromas hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm’s tumor, seminoma, leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, myelodysplastic syndrome, mycosis fungoides, rhabdomyosarcoma, astrocytoma, non-Hodgkin’s lymphoma, osteogenic and other sarcoma, malignant hypercalcemia,
  • polycythermia vera polycythermia vera, adenocarcinoma, glioblastoma multiforma, glioma, lymphomas, epidermoid carcinomas, and other carcinomas and sarcomas.
  • the compound of Formula 1 may also be used to treat benign tumors including hemangiomas, hepatocellular adenoma, cavernous haemangioma, focal nodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, nodular regenerative hyperplasia, trachomas, pyogenic granulomas, and the like, and hamartoma conditions such as Koz-Jeghers Syndrome (PJS), Cowden disease, Bannayan-Riley-Ruvalcaba Syndrome (BRRS), Proteus syndrome, Lhermitte-Duclos disease and Tuberous Sclerosis (TSC).
  • benign tumors including hemangiomas, hepatocellular adenoma, cavernous haemangioma, focal nod
  • the compound of Formula 1 may be used to treat abnormal cell proliferation due to insults to body tissue during surgery, repetitive motion disorders, including carpal tunnel syndrome. These insults may arise as a result of a variety of surgical procedures such as joint or bowel surgery, and include keloid scarring. [0096] The compound of Formula 1 may be used to prevent restenosis, i.e., undesirable proliferation of normal cells in the blood vessel in response to the introduction of stents in the treatment of cardiovascular disease.
  • the compound of Formula 1 may also be used to treat proliferative responses associated with organ transplantation, which may contribute to potential organ rejection or associated complications. Such proliferative responses may occur during transplantation of the heart, lung, liver, kidney, and other body organs or organ systems.
  • the compound of Formula 1 may be used to treat abnormal angiogenesis, including the abnormal angiogenesis accompanying rheumatoid arthritis, ischemic-reperfusion related brain edema and injury, cortical ischemia, ovarian hyperplasia and hypervascularity, polycystic ovary syndrome, endometriosis, psoriasis, diabetic retinopathy, and other ocular angiogenic diseases such as retinopathy of prematurity (retrolental fibroplastic), macular degeneration (e.g., wet and dry age-related macular degeneration), corneal graft rejection, neovascular glaucoma, Oster Webber syndrome, retinal/choroidal neovascularization and corneal neovascularization, Best’s disease, myopia, optic pits, Stargart’s diseases, Pagets disease, vein occlusion, artery occlusion, sickle cell anemia, sarcoid, s
  • abnormal angiogenesis
  • the compound of Formula 1 may also be used to treat neurodegenerative diseases, disorders, and conditions, including Huntington Disease, Alzheimer Disease, Niemann-Pick disease, Parkinson’s disease, prion-mediated disease, spinocerebellar ataxia, progressive multifocal encephalopathy, amyotrophic lateral sclerosis, axonal neuropathies, muscular dystrophies, and age-related nervous system degeneration.
  • neurodegenerative diseases, disorders, and conditions including Huntington Disease, Alzheimer Disease, Niemann-Pick disease, Parkinson’s disease, prion-mediated disease, spinocerebellar ataxia, progressive multifocal encephalopathy, amyotrophic lateral sclerosis, axonal neuropathies, muscular dystrophies, and age-related nervous system degeneration.
  • Formula 1 may be used to treat Huntington’s disease.
  • the compound of Formula 1 may be used to treat inflammation.
  • inflammation examples include treatment of acute pancreatitis, chronic pancreatitis, asthma, allergies, chronic obstructive pulmonary disease, and adult respiratory distress syndrome, and chronic inflammatory diseases associated with uncontrolled angiogenesis, inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis, psoriasis, sarcoidosis, rheumatoid arthritis, and multisystem granulomatous disorder.
  • the compound of Formula 1 may be used to treat autoimmune disorders, including glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves’ disease, autoimmune gastritis, diabetes, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, atopic dermatitis, chronic active hepatitis, myasthenia gravis, multiple sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn’s disease, psoriasis, graft vs. host disease, multiple sclerosis, and Sjogren’s syndrome.
  • autoimmune disorders including glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves’ disease, autoimmune gastritis, diabetes, autoimmune hemolytic anemia, autoimmune neutropenia,
  • the compound of Formula 1 may be used to treat cardiovascular and metabolic diseases, disorders and conditions, including obesity, diabetes, insulin resistance, metabolic syndrome, and hyperlipidemia.
  • the compound of Formula 1 may also be used to treat fibrotic disorders, which include primary fibrotic disorders, fibrosis associated with autoimmune and inflammatory diseases, disorders, and conditions, respiratory fibrotic disorders, and secondary fibrosis.
  • Primary fibrotic disorders include local and systemic variants of scleroderma; mediastinal, retroperitoneal, and nephrogenic systemic fibrosis; as well as endomyocardial fibrosis, pericarditis, pleuritis, and primary myocardial fibrosis.
  • Fibrotic disorders include fibrosis associated with autoimmune and inflammatory diseases, disorders, and conditions, such as rheumatoid arthritis, systemic lupus erythematosus, primary and secondary Sjogren syndrome, inflammatory bowel disease, including Crohn disease and ulcerative colitis; spondarthropathy, including ankylosing spondylitis; psoriatic arthritis, enteropathic arthritis, and reactive arthritis; inflammatory muscle disease including polymositis, dermatomyositis, and inclusion body myopathies, whether primary or accompanying malignant disease; conjunctivis, ulceris, keratitis, keratoconjunctivitis, scleritis, and uveitis; retinopathies, including retinal detachment; myasthenia gravis, atopic dermatitis, eczema, and alopecia areata; familial Mediterranean fever; acute, intercritical, chronic recurring, polyarticular, and tophaceous forms of
  • pyrophosphate tissue deposition disease both acute and chronic variants; primary and secondary osteoarthritis; adhesive capsulitis; psoriasis; juvenile arthropathies including systemic-onset, polyarticular, pauci-articular and enthesopathic variants, and childhood-onset variants of rheumatoid arthritis and systemic lupus erythematosus; multiple sclerosis, neuromyelitis optica, and other demyelinating conditions of the central or peripheral nervous systems; thyroiditis, including Hashimoto disease and Grave’s ophthalmopathy; autoimmune endocrine glandular dysfunction, including Addison disease; automuune bone marrow and hematologic disorders including immune and idiopathic forms of thrombocytopenic purpura and hemolytic anemia, aplastic anemia; psoriasis, including psoriasis vulgaris, guttate psoriasis, psoriasis affecting the scalp or nails, and palmo
  • blistering skin and mucosal disease including pemphigus vulgaris, bullous pemphigoid, Stevens- Johnson syndrome, and cutaneous drug reactions; primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis; allergic reactions, including reactions to drugs, pollens, animal dander, arthropod or snake bites, and insect feces; celiac disease; sarcoidosis; lichen planus; Behcets disease; and idiopathic hearing or visual loss.
  • Fibrotic respiratory disorders refers to respiratory fibrosis, including idiopathic pulmonary fibrosis (IPF); interstitial lung diseases, including diffuse parenchymal lung diseases, idiopathic interstitial pneumonias, granulomatous lung disorders (e.g., sarcoidosis); and other forms of interstitial lung disease (ILD) including lymphangioleiomyomatosis (LAM), pulmonary Langerhans’ cell histiocytosis/histiocytosis X, and eosinophilic pneumonia; interstitial pneumonias (IPs), which include nonspecific interstitial pneumonia, desquamative interstitial pneumonia, respiratory bronchiolitis-associated interstitial lung disease, acute interstitial pneumonia, cryptogenic organizing pneumonia, lymphocytic interstitial pneumonia, and bronchitis obliterans syndrome (BOS).
  • IPF idiopathic pulmonary fibrosis
  • interstitial lung diseases including diffuse parenchymal lung diseases, idiopathic inter
  • Other fibrotic disorders include secondary fibrosis, which includes fibrosis of the liver following drug or alcohol exposure, non-alcoholic steatohepatosis, and metals exposure, including exposure to iron and copper; viral hepatitis, including Hepatitis A, B, C, D or E or other hepatotrophic viruses or bacteria alone or in combination; myocardial and pericardial fibrosis following myocardial infarction, coronary artery bypass, coronary stenting, and other forms of cardiac surgery; fibrosis from other forms of cardiac damage; myelofibrosis; kidney fibrosis, including fibrosis complicating diabetes, drug or heavy metal-related renal injury, or
  • fibrotic disorders include non-malignant forms of excessive or abnormal tissue proliferation or expansion including polycystic kidney disease, neurofibromatosis, uterine fibroids, colonic polyps, meningioma, tuberous sclerosis, and familial and sporadic forms of lymphangioleiomyomatosis; sebaceous cysts; fibrocystic breast disease; nodules of the thyroid gland; granulomas; arteriovenous malformations; syndesmophyte and osteophyte formation; and disseminated idiopathic skeletal hyperostosis.
  • Other fibrotic disorders include fibrosis or other connective tissue proliferation or expansion following the implantation of temporary or permanent therapeutic devices including artificial joints, ocular lenses, cardiac valves, pacemakers, and drug-eluting or other implantable drug-delivery systems.
  • the compound of Formula 1 which include compounds specifically named above and their pharmaceutically acceptable complexes, salts, solvates and hydrates, may be administered simultaneously, sequentially or separately in combination with one or more compounds or therapies for treating neurodegenerative diseases, cancer, diseases involving the immune system, inflammation, autoimmune diseases, fibrotic disorders, and others.
  • Such combinations may offer significant therapeutic advantages, including fewer side effects, improved ability to treat underserved patient populations, or synergistic activity.
  • the compound of Formula 1 may be combined with one or more nonsteroidal anti-inflamatory drugs (NSAIDs), analgesics, corticosteroids, biological response modifiers, and protein-A immunoadsorption therapy.
  • NSAIDs nonsteroidal anti-inflamatory drugs
  • analgesics corticosteroids
  • corticosteroids biological response modifiers
  • protein-A immunoadsorption therapy Alternatively or additionally, the compound of Formula 1 may be combined with one or more disease modifying antirheumatic drugs
  • NSAIDs include apazone, aspirin, celecoxib, diclofenac (with and without misoprostol), diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate sodium, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, choline and magnesium salicylates, salsalate, and sulindac.
  • Representative analgesics include acetaminophen and morphine sulfate, as well as codeine, hydrocodone, oxycodone, propoxyphene, and tramadol, all with or without acetaminophen.
  • Representative corticosteroids include betamethasone, cortisone acetate, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, and prednisone.
  • Representative biological response modifiers include TNF-a inhibitors, such as adalimumab, etanercept, and infliximab; selective B-cell inhibitors, such as rituximab; IL-l inhibitors, such as anakinra, and selective costimulation modulators, such as abatacept.
  • TNF-a inhibitors such as adalimumab, etanercept, and infliximab
  • selective B-cell inhibitors such as rituximab
  • IL-l inhibitors such as anakinra
  • selective costimulation modulators such as abatacept.
  • Representative DMARDs include auranofm (oral gold), azathioprine, chlorambucil, cyclophosamide, cyclosporine, gold sodium thiomalate (injectable gold), hydroxychloroquine, leflunomide, methotrexate, minocycline, myophenolate mofetil, penicillamine, and sulfasalazine.
  • Representative osteoporosis agents include bisphosphonates, such as alendronate, ibandronate, risedronate, and zoledronic acid; selective estrogen receptor modulators, such as droloxifene, lasofoxifene, and raloxifene; hormones, such as calcitonin, estrogens, and parathyroid hormone; and immunosuppressant agents such as azathioprine, cyclosporine, and rapamycin.
  • bisphosphonates such as alendronate, ibandronate, risedronate, and zoledronic acid
  • selective estrogen receptor modulators such as droloxifene, lasofoxifene, and raloxifene
  • hormones such as calcitonin, estrogens, and parathyroid hormone
  • immunosuppressant agents such as azathioprine, cyclosporine, and rapamycin.
  • Particularly useful combinations include a compound of Formula 1 and methotrexate; a compound of Formula 1 and one or more biological response modifiers, such as lefluonomide, etanercept, adalimumab, and infliximab; or a compound of Formula 1, methotrexate, and one or more biological response modifiers, such as lefluonomide, etanercept, adalimumab, and infliximab.
  • biological response modifiers such as lefluonomide, etanercept, adalimumab, and infliximab.
  • the compound of Formula 1 may be combined with one or more cardiovascular agents such as calcium channel blockers, statins, fibrates, beta-blockers, ACE inhibitors, and platelet aggregation inhibitors.
  • cardiovascular agents such as calcium channel blockers, statins, fibrates, beta-blockers, ACE inhibitors, and platelet aggregation inhibitors.
  • the compound of Formula 1 may also be combined with one or more compounds or therapies for treating cancer (i.e., anti-cancer agent).
  • cancer i.e., anti-cancer agent
  • chemotherapeutic agents i.e., cytotoxic or antineoplastic agents
  • alkylating agents antibiotics, antimetabolic agents, plant-derived agents, and topoisomerase inhibitors
  • molecularly targeted drugs include both small molecules and biologies.
  • Representative alkylating agents include bischloroethylamines (nitrogen mustards, e.g., chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, and uracil mustard); aziridines (e.g., thiotepa); alkyl alkone sulfonates (e.g., busulfan); nitrosoureas (e.g., carmustine, lomustine, and streptozocin); nonclassical alkylating agents (e.g., altretamine, dacarbazine, and procarbazine); and platinum compounds (e.g., carboplatin, cisplatin, nedaplatin, oxaliplatin, satraplatin, and triplatin tetranitrate).
  • nitrogen mustards e.g., chlorambucil, cyclophosphamide, ifosfamide,
  • antibiotic agents include anthracyclines (e.g., aclarubicin, amrubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, pirarubicin, valrubicin, and zorubicin);
  • anthracyclines e.g., aclarubicin, amrubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, pirarubicin, valrubicin, and zorubicin
  • anthracenediones e.g., mitoxantrone and pixantrone
  • streptomyces e.g., actinomycin, bleomycin, dactinomycin, mitomycin C, and plicamycin
  • Representative antimetabolic agents include dihydrofolate reductase inhibitors (e.g., aminopterin, methotrexate, and pemetrexed); hymidylate synthase inhibitors (e.g., raltitrexed and pemetrexed); folinic acid (e.g., leucovorin); adenosine deaminase inhibitors (e.g., pentostatin); halogenated/ribonucleotide reductase inhibitors (e.g., cladribine, clofarabine, and fludarabine); thiopurines (e.g, thioguanine and mercaptopurine); thymidylate synthase inhibitors (e.g., fluorouracil, capecitabine, tegafur, carmofur, and floxuridine); DNA polymerase inhibitors (e.g., cytarabine); ribonucle
  • Representative plant-derived agents include vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinzolidine, and vinorelbine), podophyllotoxins (e.g., etoposide and teniposide), and taxanes (e.g., docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel).
  • vinca alkaloids e.g., vincristine, vinblastine, vindesine, vinzolidine, and vinorelbine
  • podophyllotoxins e.g., etoposide and teniposide
  • taxanes e.g., docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel.
  • Representative type I topoisomerase inhibitors include camptothecins, such as belotecan, irinotecan, rubitecan, and topotecan.
  • Representative type II topoisomerase inhibitors include amsacrine, etoposide, etoposide phosphate, and teniposide, which are derivatives of
  • Molecularly targeted therapies include biologic agents such as cytokines and other immune-regulating agents.
  • useful cytokines include interleukin-2 (IL-2, aldesleukin), interleukin 4 (IL-4), interleukin 12 (IL-12), and interferon, which Dincludes more than 23 related subtypes.
  • Other cytokines include granulocyte colony stimulating factor (CSF) (filgrastim) and granulocyte macrophage CSF (sargramostim).
  • immuno-modulating agents include bacillus Calmette- Guerin, levamisole, and octreotide; monoclonal antibodies against tumor antigens, such as trastruzumab and rituximab; and cancer vaccines, which induce an immune response to tumors.
  • EGF epiderma
  • Specific molecularly targeted drugs include selective estrogen receptor modulators, such as tamoxifen, toremifene, fulvestrant, and raloxifene; antiandrogens, such as bicalutamide, nilutamide, megestrol, and flutamide; and aromatase inhibitors, such as exemestane, anastrozole, and letrozole.
  • selective estrogen receptor modulators such as tamoxifen, toremifene, fulvestrant, and raloxifene
  • antiandrogens such as bicalutamide, nilutamide, megestrol, and flutamide
  • aromatase inhibitors such as exemestane, anastrozole, and letrozole.
  • agents which inhibit signal transduction include agents which inhibit signal transduction, such as imatinib, dasatinib, nilotinib, trastuzumab, gefitinib, erlotinib, cetuximab, lapatinib, panitumumab, and temsirolimus; agents that induce apoptosis, such as bortezomib; agents that block angiogensis, such as bevacizumab, sorafenib, and sunitinib; agents that help the immune system destroy cancel cells, such as rituximab and alemtuzumab; and monoclonal antibodies which deliver toxic molecules to cancer cells, such as gemtuzumab ozogamicin, tositumomab, l3 ll-tositumoab, and ibritumomab tiuxetan.
  • agents which inhibit signal transduction such as imatinib, dasatin
  • Bioactivity may be determined using a variety of methods, including in vitro and in vivo methods.
  • the inhibitory activity of five (5) compounds is evaluated using a lO-point dose-response analysis (0.510 to 10,000 nM) against a set of six (6) kinases via the SelectScreenTM Kinase Profiling Service by Invitrogen, a Life Technologies company (Madison, WI, ETSA), which includes the Adapta® ETniversal Kinase Assay (four (4) phospholipid kinases) and the Z’-LYTE® biochemical assay (FRAP (mTOR) and PLK2 kinase). All enzymes are tested using an adenosine 5 '-triphosphate (ATP) concentration equal to the Michaelis constant (K m ) apparent.
  • ATP adenosine 5 '-triphosphate
  • the Adapta® Universal Kinase Assay is a homogenous, fluorescent-based immunoassay for the detection of adenosine 5’ -diphosphate (ADP) using a time-resolved (TR)-FRET signal.
  • the assay can be divided into 2 phases: a kinase reaction phase and an ADP detection phase. In the kinase reaction phase, all components required for the kinase reaction are added to the well, and the reaction is allowed to incubate for 60 minutes.
  • a detection solution consisting of a europium (EU)-labeled anti-ADP antibody, an Alexa Fluor® 647-labeled ADP tracer, and ethylenediaminetetraacetic acid (EDTA) (to stop the kinase reaction) are added to the assay well.
  • ADP formed by the kinase reaction (in the absence of an inhibitor) displaces the Alexa Fluor 647-labeled ADP tracer from the antibody, resulting in a decrease in the TR-FRET signal.
  • ADP formation is determined by calculating the emission ratio from the assay well. The emission ratio is calculated by dividing the intensity of the tracer (acceptor) emission by the intensity of the Eu (donor) emission at 615 nm, as shown in Eqn 1 :
  • the detection mix consists of EDTA (30 mM), Eu-anti-ADP antibody (30 nM), and ADP tracer.
  • the test compounds are screened in 1% DMSO (final) in the well. The detailed protocol used for testing each of the four (4) recombinant human phospholipid kinases is described below.
  • Adapta® Assay Controls are made for each individual kinase and are located on the same plate as the kinase.
  • the maximum Emission Ratio is established by the 0% Conversion Control (100% Inhibition Control), which contains no ATP in the kinase reaction and therefore exhibits no kinase activity.
  • ATP is added to these wells. ATP addition is required for the 0% conversion controls wells because the ADP antibody binds ATP with low affinity. The ATP in wells with maximum kinase inhibition will displace the ADP tracer slightly, though much less efficiently than ADP.
  • the 100% Conversion Control wells contain ADP instead of ATP and are designed to allow for the calculation of percent ATP conversion.
  • the 0% Conversion and 100% Conversion Controls allow one to estimate the percent ATP conversion achieved in a specific reaction well. Control wells do not include any kinase inhibitors.
  • the minimum Emission Ratio in a screen is established by the 0% Inhibition Control, which contains active kinase. This control is designed to produce ⁇ 40% ATP conversion in the Kinase Reaction. (The range of ATP conversion allowed is different for each kinase and set in the linear region.).
  • a known inhibitor control standard curve, 10 point titration, is run for each individual kinase on the same plate as the kinase to ensure the kinase is inhibited within an expected IC50 range previously determined.
  • Adapta® assays are run in the linear range determined for each kinase. Full ATP/ADP standard curves are run during validation to define this range. In addition, ATP/ADP standard curves are used to calculate the percent ATP conversion of each sample. SelectScreen® Kinase Profiling Service uses XLfit from IDBS. The ATP/ADP standard curve is fit to model number 205 (sigmoidal dose-response model), shown here as Eqn 2:
  • PIK3 C A/PIK3 R 1 (pl 10 alpha/p85 alpha)
  • Adapta® assay The 2X PIK3 C A/PIK3 R 1 (pl 10 alpha/p85 alpha)/PIP2:PS reaction mixture is prepared in 50 mM pH 7.5 HEPES buffer containing 100 mM NaCl, 0.03% CHAPS, 3 mM MgCk, and 1 mM EGTA.
  • the final 10 pL kinase reaction consists of 0.25 - 2 ng PIK3CA/PIK3R1 (pl 10 alpha/p85 alpha) enzyme and 50 pM PIP2:PS in 32.5 mM HEPES pH 7.5, 50 mM NaCl, 0.015% CHAPS, 1.5 mM MgCk, 0.5 mM EGTA. After the 1 hour kinase reaction incubation, 5 pL of detection mix is added.
  • PIK3CB/PIK3R1 (pl 10 beta/p85 alpha)
  • Adapta® assay The 2X PIK3CB/PIK3R1 (pl 10 beta/p85 alpha) / PIP2:PS mixture is prepared in 50 mM pH 7.5 HEPES buffer containing 100 mM NaCl, 0.03% CHAPS, 3 mM MgCk, and 1 mM EGTA.
  • the final 10 pL kinase reaction consists of 6.25 - 60 ng PIK3CB/PIK3R1 (pl 10 beta/p85 alpha) and 50 pM PIP2:PS in 32.5 mM pH 7.5 HEPES buffer containing 50 mM NaCl, 0.015% CHAPS, 1.5 mM MgCk, and 0.5 mM EGTA. After the 1 hour kinase reaction incubation, 5 pL of detection mix is added.
  • PIK3CD/PIK3R1 (pl 10 delta/p85 alpha)
  • Adapta® assay The 2X PIK3 CD/PIK3 R 1 (pl 10 delta/p85 alpha) / PIP2:PS mixture is prepared in 50 mM pH 7.5 HEPES buffer containing 100 mM NaCl, 0.03% CHAPS, 3 mM MgCk, and 1 mM EGTA.
  • the final 10 pL kinase reaction consists of 0.2 - 3 ng PIK3 CD/PIK3 R 1 (pl 10 delta/p85 alpha) and 50 pM PIP2:PS in 32.5 mM pH 7.5 HEPES buffer containing 50 mM NaCl, 0.015% CHAPS, 1.5 mM MgCk, and 0.5 mM EGTA. After the 1 hour kinase reaction incubation, 5 pL of detection mix is added.
  • PIK3CG (pl 10 gamma)
  • Adapta® assay The 2X PIK3CG (pl 10 gamma) / PIP2:PS mixture is prepared in 50 mM pH 7.5 HEPES buffer containing 3 mM MgCb and 1 mM EGTA.
  • the final 10 pL kinase reaction consists of 6.25 - 70 ng PIK3CG (pl 10 gamma) and 50 mM PIP2:PS in 32.5 mM pH 7.5 HEPES buffer containing 1.5 mM MgCh and 0.5 mM EGTA. After the 1 hour kinase reaction incubation, 5 pL of detection mix is added.
  • the lipid substrates are prepared by creating lipid vesicles.
  • these vesicles include a carrier lipid, such as phosphatidylserine (PS).
  • PS phosphatidylserine
  • PIP2:PS refers to large unilamellar vesicles (LETVs) containing five mole percent L-a- phosphatidylinositol-4,5-bisphosphate (PIP2) and ninety -five percent phosphatidylserine.
  • the concentration listed refers only to the PIP2 substrate, not the PS carrier lipid.
  • the Z’-LYTE® biochemical assay for mTOR (FRAP) and PLK2 protein kinases are conducted by Invitrogen/Life Technologies and uses a fluorescence-based, coupled-enzyme format that is based on the differential sensitivity of phosphorylated and nonphosphorylated peptides to proteolytic cleavage.
  • the peptide substrate is labeled with 2 fluorophores - 1 at each end, a donor (coumarin) and an acceptor (fluorescein) - that make up a fluorescence resonance energy transfer (FRET) pair.
  • the kinase transfers the g-phosphate of ATP to a single tyrosine, serine, or threonine residue in a synthetic FRET peptide.
  • a site-specific protease recognizes and cleaves nonphosphorylated FRET peptides.
  • FRET peptides suppresses cleavage by the development reagent. Cleavage disrupts FRET between the donor and acceptor fluorophores on the FRET -peptide, whereas uncleaved, phosphorylated FRET peptides maintain FRET.
  • a ratiometric method which calculates the emission ratio of donor emission (coumarin at 445 nm) to acceptor emission (fluorescein at 520 nm) after excitation of the donor fluorophore at 400 nm, was used to quantitate the reaction progress. The extent of phosphorylation of the FRET peptide can be calculated from the emission ratio according to Eqn 3:
  • the emission ratio remains low if the FRET peptide is phosphorylated (i.e., no kinase inhibition) and is high if the FRET peptide is nonphosphorylated (i.e., kinase inhibition).
  • the test compounds are screened in 1% DMSO (final) in the well. The detailed protocol used for testing recombinant human mTOR (FRAP) and PLK2 protein kinases are described below. [0136] Z' -LYTE® Assay Controls are made for each individual kinase and are located on the same plate as the kinase.
  • the maximum Emission Ratio is established by the 0% Phosphorylation Control (100% Inhibition Control), which contains no ATP and therefore exhibits no kinase activity. This control yields 100% cleaved peptide in the development reaction.
  • the 100% Phosphorylation Control which consists of a synthetically phosphorylated peptide of the same sequence as the peptide substrate, is designed to allow for the calculation of percent
  • the 0% Phosphorylation and 100% Phosphorylation Controls allow one to calculate the percent Phosphorylation achieved in a specific reaction well. Control wells do not include any kinase inhibitors.
  • the minimum Emission Ratio in a screen is established by the 0% Inhibition Control, which contains active kinase. This control is designed to produce a 10-50%
  • Interference is established by comparing the Test Compound Control wells that do not contain ATP versus the 0% Phosphorylation Control (which does not contain the Test Compound).
  • the expected value for a non-interfering compound should be 100%. Any value outside of 90% to 110% is flagged.
  • the Test Compound Fluorescence Interference is determined by comparing the Test Compound Control wells that do not contain the Kinase/Peptide Mixture (zero peptide control) versus the 0% Inhibition Control.
  • the expected value for a non-fluorescence compound should be 0%. Any value > 20% is flagged.
  • SelectScreen® Kinase Profiling Service uses XLfit from IDBS.
  • the dose response curve is curve fit to model number 205 (sigmoidal dose-response model) as described above for Eqn 2. If the bottom of the curve does not fit between -20% & 20% inhibition, it is set to 0% inhibition. If the top of the curve does not fit between 70% and 130% inhibition, it is set to 100% inhibition.
  • FRAP1 (mTOR) Z'-LYTE® Assay The 2X FRAP1 (mTOR) / Ser/Thr 11 mixture is prepared in 50 mM pH 7.5 HEPES buffer containing 0.01% BRIJ-35, 10 mM MnCh, 1 mM EGTA, 2 mM DTT, and 0.02% NaN3.
  • the final 10 pL kinase reaction consists of 11.1 - 56 ng FRAPl (mTOR) enzyme and 2 pM Ser/Thr 11 in 50 mM pH 7.5 HEPES buffer containing 0.01% BRIJ-35, 5 mM MgCh, 5 mM MnCh, 1 mM EGTA, 1 mM DTT, and 0.01% NaN3.
  • 5 pL of a 1 : 16 dilution of development reagent B is added.
  • PLK2 Z'-LYTE® Assay The 2X PLK2 / Ser/Thr 16 mixture is prepared in 50 mM pH 7.5 HEPES buffer containing 0.01% BRIJ-35, 10 mM MgCb, and 1 mM EGTA. The final 10 pL kinase reaction consists of 9.59 - 160 ng PLK2 and 2 pM Ser/Thr 16 in 50 mM pH 7.5 HEPES buffer containing 0.01% BRIJ-35, 10 mM MgCh, and 1 mM EGTA. After 1 hour kinase reaction incubation, 5 pL of a 1 : 16 dilution of development reagent B is added.
  • a pharmacokinetic analysis is conducted in male Sprague-Dawley rats following intravenous and oral administration at 0.3 mg/kg (Doseiv) and 5 mg/kg (Dosepo), respectively, of the test compounds. Animals are fasted overnight and food is returned approximately 4 hours post dose.
  • the dose administration intravenously is formulated in 20% beta-cyclodextrin in 0.05M MSA pH 3 at 0.15 mg/mL and is delivered at a 2 mL/kg dose volume.
  • the dose administered orally is formulated as a suspension in 0.5% methylcellulose at 1 mg/mL and delivered at 5 mL/kg dose volume.
  • Blood samples are collected into tubes with K 2 EDTA at 0, 0.033, 0.25, 0.5, 1, 3, 8, 24 and 48 hours post intravenous administration and 0, 0.25, 0.5, 1, 3, 6, 8, 24 and 48 hours post oral dose, and are stored on wet ice until processed to plasma by centrifugation (3500 rpm at 5°C) within 30 minutes of collection. Resulting plasma samples are stored at -80°C until bioanalysis.
  • Compound levels are quantified from plasma with an LC/MS bioanalytical method following extraction with 75% acetonitrile/25% methanol/0.2% formic acid mixture, under gradient conditions.
  • Pharmacokinetic parameters of Cmax, Tmax, Half-life, AETC, CL and Vss are determined using non-compartmental analysis using Watson software.
  • Permeability of a given test compound is determined using bidirectional transport studies in LLC-PK1 cells over-transfected to express MDR1.
  • the LLC-PK1-MDR1 cells are seeded at 2.25 x 10 6 cells/mL into Coming HTS Transwell-96 well permeable support plates (0.4 pm pore size, polycarbonate membrane). Cells are seeded in two plates: plate 1 in the A-B (apical to basolateral) direction and plate 2 in the B-A direction. Fresh media (50 pL) is added to each well and the plates are incubated at 5% CCb/95% air/95% humidity for 7 days.
  • the culture medium is prepared using Invitrogen 199 medium (500 mL), FBS (50 mL), geneticin (5 mL) and colchicine 1.5 mg/mL (50 pL). The medium is replaced from the cell culture at minimum once before initiating the assay.
  • Bidirectional transport studies are performed at 37°C in an atmosphere of 5% CO2 in air and 95% humidity.
  • the confluent cell monolayers on Transwell inserts are equilibrated for 30 - 60 minutes with assay medium (Invitrogen 199 medium [500 mL], 10 mM HEPES [0.5 mL] and BSA [5 g]).
  • assay medium Invitrogen 199 medium [500 mL], 10 mM HEPES [0.5 mL] and BSA [5 g].
  • 250 pL blank assay medium is added to plate 1 (A-B direction), and 250 pL dosing solution is added to plate 2 (B-A direction).
  • Dosing solution contains 1 or 10 mM of a test compound.
  • Atenolol a cell monolayer integrity marker
  • Samples are quantified by liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) using a Shimazu LC-ABSciex API4000 QTrap mass spectrometer.
  • Mobile phase A consists of 0.04% formic acid in HPLC grade water and mobile phase B is 0.04% formic acid in acetonitrile.
  • Eight calibration standards are used to construct the standard curve, ranging from 2.29 nM to 5000 nM.
  • MDA-MB-361 and PC3 tumor cell lines are maintained in accordance with the supplier (American Type Culture Collection, Rockville, MD). Cells are seeded in 96-well tissue culture microplates at 5,000-20,000 cells per well and cultured for 24 hours prior to addition of test compounds or DMSO (dimethylsulfoxide) vehicle. After 72 hours of treatment, viable cells are detected using CellTiter-Glo® Luminescent Cell Viability (Promega, Madison, WI).
  • Luminescence is measured on a PHERAstar FS microplate reader (BMG Labtech, Cary, NC). To generate concentration-response curves, cells are treated in duplicate with a range of serial compound dilutions (final DMSO concentration is 0.5%). The percentage of viable cells per well is determined by correcting for background and normalizing against DMSO-treated cells. EC so values for inhibition of cell viability are calculated using XLfit4 Microsoft Excel curve-fitting software.
  • Athymic nude mice are inoculated subcutaneously in the right flank with suspensions of the human prostate cancer cell line PC3.
  • tumors in implanted mice reach the target size of 100-150 mm 3
  • animals are randomized into treatment groups.
  • Treatment and control groups consist of 5 mice each.
  • Test compounds are administered daily by oral gavage in 0.5% carboxymethylcellulose (CMC) at 5 pL per gram body weight. Tumor volumes are measured using digital calipers.
  • CMC carboxymethylcellulose
  • Antitumor efficacy is expressed as mean relative growth of treated versus control tumors (%T/C). This is calculated using the formula: [(T-To)/(C-Co)] x 100, in which C and T are mean control and compound-treated tumor volume and Co and To are initial mean control and compound- treated tumor volume, respectively.
  • regression percentage was calculated using the formula: [l-(T/To)] x 100, in which T and To are treated and initial tumor volume, respectively.
  • Complete regression (CR) was defined as tumor shrinkage to below measurable size ( ⁇ 13.5 mm 3 ) for 3 consecutive measurements
  • partial regression (PR) was defined as reduction to ⁇ 50% of initial tumor volume and >13.5 mm 3 for 3 consecutive measurements.
  • Reverse phase chromatography is typically carried out on a column (e.g., Phenomenex GeminiTM 5m, C18, 30 mm x 150 mm; AxiaTM, 5m, 30 mm x 75 mm) under acidic conditions (“acid mode”) eluting with CFECN and water mobile phases containing 0.035% and 0.05% trifluoroacetic acid (TFA), respectively, or under basic conditions (“basic mode”) eluting with water and 20/80 (v/v) water/acetonitrile mobile phases, both containing 10 mM NH4HCO3.
  • acid mode acidic conditions
  • basic mode basic conditions
  • Preparative TLC is typically carried out on silica gel 60 F254 plates.
  • PREPARATION 1 4-amino- l-methylcy cl ohexan-l-ol
  • PREPARATION 2 /tvV-butyl (( 1.v,4.v)-4-hydroxy-4-methyl cyclohexyl (carbamate or 67S _ l-methyl-4-(ter/-butoxycarbonylamino)cy cl ohexan-l-ol
  • PREPARATION 3 /ert-butyl ((lr,4r)-4-hydroxy-4-methylcyclohexyl)carbamate or trans- 1 -methyl-4-(/er/-butoxycarbonylamino)cyclohexan- 1 -ol
  • PREPARATION 4 ( 1.v,4.v)-4-amino- 1 -methylcyclohexan- 1 -ol or cA-4-amino-l- methylcyclohexan-l-ol
  • PREPARATION 5 (lr,4r)-4-amino-l-methylcyclohexan-l-ol or /ra//.s-4-amino- l - methylcyclohexan-l-ol
  • PREPARATION 6 methyl f V)-4-(2-chl oro-5 -fl uoropy ri i di n-4-y 1 )-3 - methylmorpholine-3-carboxylate
  • PREPARATION 7 methyl (ri)-4-(5-fluoro-2-(l7/-indol-4-yl)pyrimidin-4-yl)-3- methylmorpholine-3-carboxylate
  • the reaction mixture was stirred at 90°C for 18 hours and then cooled to 23°C.
  • the contents of the flask were filtered through CeliteTM and the CeliteTM was rinsed with EtOAc, which was combined with the filtrate.
  • the filtrate was further diluted with EtOAc, washed with saturated aqueous NaHC03, saturated aqueous NH 4Q , and brine, and then dried over MgS0 4.
  • the product-containing mixture was filtered and the solvent removed in vacuo.
  • the residue was treated with diethyl ether (200 mL) and concentrated in vacuo.
  • the diethyl ether wash was repeated three times and concentrated in vacuo to give the title compound as a brown solid (119 g, 100%).
  • PREPARATION 8 (ri)-4-(5-fluoro-2-(l77-indol-4-yl)pyrimidin-4-yl)-3- methylmorpholine-3 -carboxylic acid
  • the aqueous layer was brought to pH 4 with 1 M HC1 (750 mL).
  • the resulting solid was isolated by filtration, washed with water, and dried on a Buchner funnel to give Crop-l of the title compound as a brown solid (7.0 g).
  • the aqueous acidic filtrate still contained product, which was subsequently extracted with EtOAc (3 x 200 mL).
  • the organic extract was dried with MgS0 4 , filtered, concentrated and dried in vacuo to give Crop-2 as a yellow solid (12.5 g). During the first precipitation, a black sludge formed around the stir bar.
  • PREPARATION 9 (ri)-4-(5-fluoro-2-(l77-indol-4-yl)pyrimidin-4-yl)-/V-(4-hydroxy-4- methylcyclohexyl)-3-methylmorpholine-3 -carboxamide
  • the reaction mixture was stirred at 23°C for 5 days, then partitioned between EtOAc (1.5 mL) and saturated NLLCl (0.75 mL). The aqueous and organic layers were separated. The aqueous phase was washed with EtOAc (2 x 1.5 mL) and the organic extracts were combined. The crude product was extracted from the organic phase with 1N HC1 aq (6 x 0.5 mL). The aqueous extracts were combined and basified to a pH of about 5-6 with a slow addition of solid NaHC03 (0.30 g). The product was extracted from the aqueous phase with EtOAc (5 x 0.5 mL).
  • PREPARATION 10 (ri)-4-(5-fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-A-((n,4i?)-4- hydroxy-4-methylcyclohexyl)-3-methylmorpholine-3 -carboxamide or (S)-cis- 1 -methyl-4-(4-(5- fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-3-methylmorpholin-3-carbonylamino)-cyclohexan-l-ol
  • PREPARATION 11 (ri)-4-(5-fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-A-((lr,4ri)-4- hydroxy-4-methylcyclohexyl)-3-methylmorpholine-3 -carboxamide or (S)-trans- l-methyl-4-(4-(5- fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-3-methylmorpholin-3-carbonylamino)-cyclohexan-l-ol
  • the reaction mixture was stirred at 23°C for 23 hours, partitioned between EtOAc (3.0 mL) and saturated aqueous NLLCl (1.5 mL), and the layers were separated.
  • the aqueous phase was washed with EtOAc (2 x 3.0 mL) and the organic extracts were combined.
  • the crude product was extracted from the organic phase with 1N HC1 aq (5 x 1.0 mL).
  • the product was extracted from the aqueous phase with EtOAc (5 x 1.0 mL).
  • the organic extracts were combined, washed with saturated NaHC03 (1.0 mL) and brine (1.0 mL), dried over MgS0 4 , filtered, rinsed with EtOAc and dried in vacuo to provide the crude product as an orange oil.
  • the crude product was combined with the organic extract from Preparation 9.
  • the combined extracts were dissolved in DCM ( ⁇ l mL) and purified by medium pressure chromatography (Moritex Purif-PackTM NH, 60 mM, size 20 column) eluting with a gradient of 0-5% MeOH in DCM.
  • PREPARATION 12 (ri)-4-(5-fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-A-((n,4i?)-4- hydroxy-4-methylcyclohexyl)-3-methylmorpholine-3 -carboxamide or (S)-cis- 1 -methyl-4-(4-(5- fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-3-methylmorpholin-3-carbonylamino)-cyclohexan-l-ol
  • the reaction mixture was stirred at 23°C for 22 hours and then partitioned between EtOAc (7 mL) and saturated aqueous NLLCl (3.5 mL). The aqueous and organic layers were separated. The aqueous phase was washed with EtOAc (2 x 7 mL) and the organic extracts were combined. The crude product was extracted from the organic phase with 1N HC1 aq (6 x 2 mL). The aqueous extracts were combined and basified to pH 8 with a slow addition of solid NaHC03 (1.02 g). The product was extracted from the aqueous phase with EtOAc (5 x 2 mL).
  • PREPARATION 13 (ri)-4-(5-fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-A-((lr,4ri)-4- hydroxy-4-methylcyclohexyl)-3-methylmorpholine-3 -carboxamide or (S)-trans- l-methyl-4-(4-(5- fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-3-methylmorpholin-3-carbonylamino)-cyclohexan-l-ol
  • the reaction mixture was stirred at 23°C for 22 hours and then partitioned between EtOAc (4 mL) and saturated aqueous NLLCl (2 mL). The aqueous and organic layers were separated. The aqueous phase was washed with EtOAc (2 x 4 mL) and the organic extracts were combined. The crude product was extracted from the organic phase with 1N HC1 aq (6 x 1.5 mL). The aqueous extracts were combined and basified to pH 7with a slow addition of solid NaHC03 (0.76 g). The product was extracted from the aqueous phase with EtOAc (5 x 1.5 mL).
  • EXAMPLE 1 S)-5-((H,4f?)-4-hydroxy-4-methylcyclohexyl)-2-(li7-indol-4-yl)-6a- methyl-6a,7,9, l0-tetrahydro-[l,4]oxazino[3,4-/z]pteridin-6(5i7)-one or (S)-cis- 1 -m ethyl -4-(2-( 1 H- indol-4-yl)-6a-methyl-6(5F/)-oxo-6a,7,9,l0-tetrahydro-[l,4]oxazino[3,4-/i]pteridin-5-yl)- cyclohexan-l-ol
  • the resulting crude material was reconstituted in DMSO and filtered through a hydrophilic PTFE 0.45 pm filter (Millipore Millex-LCRTM). The filter was rinsed with DMSO. The combined rinse solution and filtrate was purified by preparative mass-triggered LCMS, eluting with a gradient of 15-40%
  • EXAMPLE 2 S)-5-((lr,4ri)-4-hydroxy-4-methylcyclohexyl)-2-(li7-indol-4-yl)-6a- methyl-6a,7,9, l0-tetrahydiO-[l,4]oxazino[3,4-/z]pteridin-6(5i7)-one or (S)-/rans- ⁇ -methyl-4-(2- (li7-indol-4-yl)-6a-methyl-6(5F/)-oxo-6a,7,9, l0-tetrahydro-[l,4]oxazino[3,4-/i]pteridin-5-yl)- cyclohexan-l-ol
  • EXAMPLE 3 fV)-5-(( l .s,4A > )-4-hydroxy-4-methylcyclohexyl)-2-( l //-indol-4-yl)-6a- methyl-6a,7,9, l0-tetrahydro-[l,4]oxazino[3,4-/2]pteridin-6(5F/)-one or (S)-cis- 1 -m ethyl -4-(2-( 1 H- indol-4-yl)-6a-methyl-6(5F/)-oxo-6a,7,9, l0-tetrahydro-[l,4]oxazino[3,4-/i]pteridin-5-yl)- cyclohexan-l-ol
  • the TFA salt was dissolved in MeOH (5.0 mL) and the solution was passed through a 500 mg VariPureTM IPE cartridge (HCO3MP) to remove the TFA.
  • the cartridge was rinsed with MeOH (3 x 2 mL).
  • the combined rinse solution and filtrate was concentrated via rotary evaporation.
  • the concentrate was dried in vacuo to give a tan solid.
  • the tan solid was re-suspended in EtOH (3 mL), heated at reflux for 5 minutes, and cooled to 23 °C.
  • the solids were filtered, rinsed with EtOH (3 x 1 mL), and dried in vacuo to give an off-white solid.
  • the filtrate contained a significant amount of product, the filtrate was dried in vacuo and the residue was re-suspended in EtOH (0.5 mL), heated at reflux for 5 minutes, and then cooled to 23°C. The mixture was filtered. The solids were rinsed with EtOH (3 x 1 mL), combined with the first crop of solids, and dried in vacuo to give the title compound as an off-white solid (71.3 mg, 32.3%).
  • EXAMPLE 4 S)-5-((lr,4ri)-4-hydroxy-4-methylcyclohexyl)-2-(l77-indol-4-yl)-6a- methyl-6a,7,9, l0-tetrahydro-[l,4]oxazino[3,4-/2]pteridin-6(57/)-one or (S)-/rans- ⁇ -methyl-4-(2- (l77-indol-4-yl)-6a-methyl-6(57/)-oxo-6a,7,9, l0-tetrahydro-[l,4]oxazino[3,4-/i]pteridin-5-yl)- cyclohexan-l-ol
  • the TFA salt was dissolved in MeOH (5.0 mL) and passed through a 500 mg VariPureTM IPE cartridge (HCO3MP) to remove the TFA.
  • the cartridge was rinsed with MeOH (3 x 2 mL).
  • the combined rinse solution and filtrate was concentrated via rotary evaporation and dried in vacuo to give the product as an off-white solid.
  • the off-white solid was re-suspended in EtOH (3 mL), heated at reflux for 5 minutes, and cooled to 23°C.
  • the solids were filtered, rinsed with EtOH (3 x 1 mL), and dried in vacuo to give the title compound as an off-white solid (106.7 mg, 50.1%).
  • Table 1 lists enzyme inhibition data and Table 2 lists rat pharmacokinetics and cell permeability for compounds shown in Examples 3 and 4 and for comparator compounds A, B, C, and D, which are described below. Each compound was tested in accordance with the assays for in vitro kinase inhibition, rat pharmacokinetics and cell permeability described in the Biological Activity section, above. In Table 1 smaller ICso corresponds to greater potency. In Table 2 larger %F and Papp A-B correspond to greater bioavailability and cell permeability, respectively, and smaller efflux ratio (ER) corresponds to lower cell efflux. Generally, an ER greater than or equal to 2 indicates compound efflux is occurring.
  • Tables 3 and 4 compare antitumor activity of the compounds in cell lines and a xenograft mouse model.
  • Table 3 lists cell viability of carcinoma cell lines PC3 and MDA-MB- 361 following treatment with compounds of Examples 3 or 4 or with comparator compounds A, B, C or D. Each compound was tested in accordance with the assay for cell viability described in the Biological Activity section, above. In Table 3, smaller ECso corresponds to greater efficacy.
  • Table 4 lists the mean relative growth of treated tumors versus untreated (control) tumors (%T/C) for the compound of Example 3 and comparator compounds A, B, and C. Each compound was tested in accordance with the xenograft study procedure described in the Biological Activity section, above. In Table 4, better efficacy is indicated by lower values of %T/C or by larger regression percentage.
  • the drawing shows the dose response of a human PC3 xenograft mouse model following treatment with the compound prepared in Example 3 or with comparator compounds A, B or C.
  • mice treated with the compound of Example 3 exhibit slower tumor growth than mice treated with comparator compounds A, B or C.
  • mice treated with the compound of Example 3 exhibited tumor shrinkage (regression).
  • comparator compounds A, B, C, and D correspond to Examples 17 (S-stereoisomer), 26, 33, and 45 (S-stereoisomer), respectively, described in international patent application PCT/US2010/046839 (published as WO 2011/025889A1).
  • COMPOUND A (,S)-l-(4-(5-cyclopropyl-6a-methyl-6-oxo-5,6,6a,7,9,l0-hexahydro- [l,4]oxazino[3,4-/z]pteridin-2-yl)phenyl)-3-methylurea
  • COMPOUND B (L')-I -methyl -3 -(4-(6a-methyl-6-oxo-5-(tetrahydro2//-pyran-4-yl)- 5,6,6a,7,9, l0-hexahydro-[l,4]oxazino[3,4-/z]pteridin-2-yl)phenyl)urea
  • COMPOUND C (L')- I -cyclopropyl-3-(4-(6a-methyl-6-oxo5-(tetrahydro-2//-pyran-4- yl)-5,6,6a,7,9, l0-hexahydro-[l,4]oxazino[3,4-/2]pteridin-2-yl)phenyl)urea
  • COMPOUND D (S)- 1 -cy d opropy 1 -3 -(4- [5 -( 1 , 1 -di oxi dotetrahy dro-2//-thi opy ran-4-y 1 )- 6a-methyl-6-oxo-5,6,6a,7,9,l0-hexahydro[l,4]oxazino[3,4-/2]pteridin-2-yl]phenyl)urea

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Abstract

Disclosed is a compound of Formula 1, and pharmaceutically acceptable salts thereof. This disclosure also relates to materials and methods for preparing the compound of Formula 1, to pharmaceutical compositions which contain it, and to its use for treating diseases, disorders, and conditions associated with mTOR.

Description

SUBSTITUTED OXAZINOPTERIDINONES AS INHIBITORS OF mTOR
FIELD OF THE INVENTION
[0001] This invention relates to substituted oxazinopteridinones which are selective inhibitors of mammalian target of rapamycin (mTOR), to pharmaceutical compositions which contain them, and to the use of the substituted oxazinopteridinones to treat diseases, disorders, and conditions associated with mTOR, including immunological disorders, cancer, and neurodegenerative diseases.
BACKGROUND OF THE INVENTION
[0002] Mammalian target of rapamycin (mTOR) is a serine/threonine kinase and has been identified as a regulator of protein synthesis and of cell growth and proliferation. In addition, mTOR has been shown to regulate the response of tumor cells to nutrients and growth factors and to regulate the ability of tumors to promote angiogenesis. Thus, inhibitors of mTOR activity are actively being studied as potential anti-proliferative agents. Inhibitors of mTOR are currently approved for immunosuppression and cancer treatment.
[0003] Inhibition of mTOR function by small molecules results in a loss of transmission of upstream activating signals (i.e., from growth factor receptors) to downstream effectors of cell growth. Rapamycin, an inhibitor of mTOR, inhibits proliferation or growth of cells derived from a range of tissue types, including smooth muscle and T-cells, as well as cells derived from a diverse range of tumor types including rhabdomyosarcoma, neuroblastoma, glioblastoma and
medulloblastoma, small cell lung cancer, osteosarcoma, pancreatic carcinoma and breast and prostate carcinoma. Moreover, rapamycin and its derivatives have shown the ability to potentiate the cytotoxicity of a number of common cancer chemotherapies including cisplatin, camptothecin and doxorubicin.
[0004] It has been shown that mTOR functions in two distinct complexes (mTORCl and mTORC2). Rapamycin primarily inhibits the mTORCl complex while largely sparing mTORC2 activity. Thus, one strategy is to identify compounds that are capable of inhibiting mTORCl - and mTORC2-mediated activity in the cell.
[0005] In addition, mTOR Complexl-S6Kl integrates various extrinsic signals that regulate cell growth and metabolism. Experiments with rapamycin provide a link between mTOR
Complexl-S6Kl and adipogenesis. Researchers have also demonstrated that S6K1 -deficient mice are protected from diet and age induced obesity. [0006] Published international patent application WO 2011/025889 describes various hexahydrooxazinopterines which are inhibitors of mTOR; published patent applications
WO 2011/079114 and WO 2011/079118 describe various pteridinones which are inhibitors of polo-like kinase (PLK); and published patent application WO 2012/148548 describes various N- substituted oxazinopteridines and oxazinopteridinones which are inhibitors of certain isoforms of phosphoinositide 3-kinase (PI3K).
SUMMARY OF THE INVENTION
[0007] This invention provides substituted oxazinopteridinones and pharmaceutically acceptable salts thereof, which are selective inhibitors of mammalian target of rapamycin (mTOR). This invention also provides pharmaceutical compositions which contain the substituted oxazinopteridinones, and provides for their use to treat diseases, disorders, and conditions associated with mTOR, including immunological disorders, cancer, and neurodegenerative diseases.
[0008] One aspect of the invention provides a compound of Formula 1,
Figure imgf000003_0001
or a pharmaceutically acceptable salt thereof. Formula 1 depicts the compound fV)-5-(4-hydroxy-
4-methylcyclohexyl)-2-(liT-indol-4-yl)-6a-methyl-6a,7,9,l0-tetrahydro-[l,4]oxazino[3,4-
/z]pteridin-6(5F/)-one.
[0009] Another aspect of the invention provides a compound of the formula,
Figure imgf000003_0002
or a pharmaceutically acceptable salt thereof.
[0010] A further aspect of the invention provides a compound of the formula,
Figure imgf000004_0001
or a pharmaceutically acceptable salt thereof.
[0011] An additional aspect of the invention provides a pharmaceutical composition which includes a compound of Formula 1 or a pharmaceutically acceptable salt thereof, or any one of the compounds or pharmaceutically acceptable salts defined in the preceding paragraphs; and a pharmaceutically acceptable excipient.
[0012] Another aspect of the invention provides a compound of Formula 1 or a
pharmaceutically acceptable salt thereof, or any one of the compounds or pharmaceutically acceptable salts defined in the preceding paragraphs, for use in the treatment of a disease, disorder or condition associated with mTOR.
[0013] A further aspect of the invention provides a use of a compound of Formula 1 or a pharmaceutically acceptable salt thereof, or any one of the compounds or pharmaceutically acceptable salts defined in the preceding paragraphs, for the manufacture of a medicament for the treatment of a disease, disorder or condition associated with mTOR.
[0014] An additional aspect of the invention provides a method of treating a disease, disorder or condition associated with mTOR, the method comprising administering to the subject an effective amount of a compound of Formula 1 or a pharmaceutically acceptable salt thereof, or any one of the compounds or pharmaceutically acceptable salts defined in the preceding paragraphs.
[0015] Another aspect of the invention provides a method of treating a disease, disorder or condition in a subject, the method comprising administering to the subject an effective amount of a compound of Formula 1 or a pharmaceutically acceptable salt thereof, or any one of the compounds or pharmaceutically acceptable salts defined in the preceding paragraphs, wherein the disease, disorder or condition is selected from immunological disorders, cancer, and
neurodegenerative diseases.
[0016] A further aspect of the invention provides an effective amount of a compound of Formula 1 or a pharmaceutically acceptable salt thereof, or any one of the compounds or pharmaceutically acceptable salts defined in the preceding paragraphs; and at least one additional pharmacologically active agent. BRIEF DESCRIPTION OF THE DRAWING
[0017] The drawing shows the dose response of a human PC3 xenograft mouse model following treatment with the compound prepared in Example 3 or with compounds A, B or C, which correspond to Examples 17, 26, and 33, respectively, in published patent application WO 2011/025889A1.
DETAILED DESCRIPTION OF THE INVENTION
[0018] ETnless otherwise indicated, this disclosure uses definitions provided below.
[0019] “About” or“approximately,” when used in connection with a measurable numerical variable, refers to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value or within ±10 percent of the indicated value, whichever is greater.
[0020] “ Condition associated with mTOR” and similar phrases relate to a disease, disorder or condition in a subject for which inhibition of mTOR may provide a therapeutic or prophylactic benefit.
[0021] “ Selective mTOR inhibitor” means a compound that inhibits mTOR at a concentration that is at least ten times less than it inhibits either PLK or PI3K (alpha, beta, delta or gamma isoforms).
[0022] “Opposite enantiomer” refers to a molecule that is a non-superimposable mirror image of a reference molecule, which may be obtained by inverting all of the stereogenic centers of the reference molecule. For example, if the reference molecule has S absolute stereochemical configuration, then the opposite enantiomer has R absolute stereochemical configuration.
Likewise, if the reference molecule has S,S absolute stereochemical configuration, then the opposite enantiomer has R,R stereochemical configuration, and so on.
[0023] “ Stereoisomer” and“stereoisomers” of a compound with given stereochemical configuration refer to the opposite enantiomer of the compound and to any diastereoisomers, including geometrical isomers ( Z!E) of the compound. For example, if a compound has S,R,Z stereochemical configuration, its stereoisomers would include its opposite enantiomer having R,S,Z configuration, and its diastereomers having S,S,Z configuration, R,R,Z configuration, S,R,E configuration, R,S,E configuration, S,S,E configuration, and R,R,E configuration. If the stereochemical configuration of a compound is not specified, then“stereoisomer” refers to any one of the possible stereochemical configurations of the compound. [0024] “Substantially pure stereoisomer” and variants thereof refer to a sample containing a compound having a specific stereochemical configuration and which comprises at least about 95% of the sample, based on the total amount of all stereoisomers of the compound present in the sample.
[0025] “Pure stereoisomer” and variants thereof refer to a sample containing a compound having a specific stereochemical configuration and which comprises at least about 99.5% of the sample, based on the total amount of all stereoisomers of the compound present in the sample.
[0026] “Subject” refers to a mammal, including a human.
[0027] “Pharmaceutically acceptable” substances refer to those substances which are suitable for administration to subjects.
[0028] “Treating” refers to reversing, alleviating, inhibiting the progress of, or preventing a disease, disorder or condition to which such term applies, or to reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of such disease, disorder or condition.
[0029] “ Treatment” refers to the act of“treating,” as defined immediately above.
[0030] “Drug,”“drug substance,”“active pharmaceutical ingredient,” and the like, refer to a compound (e.g., compounds of Formula 1, Formula 2, and Formula 3) that may be used for treating a subject in need of treatment.
[0031] “Effective amount” of a drug,“therapeutically effective amount” of a drug, and the like, refer to the quantity of the drug that may be used for treating a subject and may depend on the weight and age of the subject and the route of administration, among other things.
[0032] “Excipient” refers to any diluent or vehicle for a drug.
[0033] “Pharmaceutical composition” refers to the combination of one or more drug substances and one or more excipients.
[0034] “Drug product,”“pharmaceutical dosage form,”“dosage form,”“final dosage form” and the like, refer to a pharmaceutical composition suitable for treating a subject in need of treatment and generally may be in the form of tablets, capsules, sachets containing powder or granules, liquid solutions or suspensions, patches, films, and the like.
[0035] The following abbreviations may be used in the specification: Ac (acetyl); ACN (acetonitrile); AIBN (azo-bis-isobutyronitrile); API (active pharmaceutical ingredient); aq (aqueous); AETC (area under the [concentration versus time] curve); BINAP (2,2'- bis(diphenylphosphino)- 1,1 '-binaphthyl); Boc (ter/-butoxy carbonyl); Cbz (carbobenzyloxy); dba (dibenzylideneacetone); CD (cyclodextrin); DCC (l,3-dicyclohexylcarbodiimide); DCE (1,1- dichloroethane); DCM (dichloromethane); DIPEA (N,N-d\ i sopropyl ethyl a i ne, Hiinig’s Base); DMA (Af,A-di methyl acetami de); DMAP (4-dimethylaminopyridine); DME (1,2- dimethoxyethane); DMF (Af,A-dirnethylformamide); DMSO (dimethylsulfoxide); dppf (1, 1 '- bis(diphenylphosphino)ferrocene); DTT (dithiothreitol); ECso (effective concentration at half maximal response); EDA ethoxylated dodecyl alcohol, Brj®35); EDC (A-(3- di methyl ami nopropyl )-Ap -ethyl carbodii mi de); EDTA (ethylenediaminetetraacetic acid); ee (enantiomeric excess); eq (equivalents); Eqn (equation); Et (ethyl); Et3N (triethyl-amine); EtOAc (ethyl acetate); EtOH (ethanol); %F (bioavailability, i.e., fraction (percent) of dose reaching systemic circulation unchanged); ELATU (2-(3A/-[l,2,3]triazolo[4,5-£]pyridin-3-yl)-l,l,3,3- tetramethyluronium hexafluorophosphate(V)); HEPES (4-(2-hydroxyethyl)piperazine-l- ethanesulfonic acid); AcOH (acetic acid); HOBt ( l //-benzo[z/][ 1 ,2,3]triazol- 1 -ol); ICso
(concentration at 50% inhibition); IPA (isopropanol); IP Ac (isopropyl acetate); IPE
(isopropylether); LDA (lithium diisopropylamide); LiHMDS (lithium bis(trimethylsilyl)amide); mCPBA (zzz-chloroperoxybenzoic acid); Me (methyl); MeOH (methanol); MSA (methanesulfonic acid); MTBE (methyl Zf/V-butyl ether); mp (melting point); NaOZ-Bu (sodium tertiary butoxide); NMM (N-methylmorpholine); NMP (A-methyl-pyrrolidone); OTf (triflate); PE (petroleum ether); Ph (phenyl); pECso (-logio(EC5o), where ECso is given in molar (M) units); pICso (-logio(ICso), where ICso is given in molar (M) units); Pr (propyl); c-Pr (cyclopropyl), z-Pr (isopropyl); PTFE (polytetrafluoroethylene); RT (room temperature, approximately 20°C to 25°C); TCEP (tr/s(2- carboxyethyl)phosphine); TFA (trifluoroacetic acid); TFAA (2,2,2-trifluoroacetic anhydride); THF (tetrahydrofuran); TMS (trimethylsilyl); and Tris buffer (2-amino-2-hydroxymethyl- propane-l,3-diol buffer).
[0036] As described, below, this disclosure concerns a compound of Formula 1 and its pharmaceutically acceptable salts. This disclosure also concerns materials and methods for preparing the compound of Formula 1, pharmaceutical compositions which contain it, and the use of the compound of Formula 1 and its pharmaceutically acceptable salts (optionally in
combination with other pharmacologically active agents) for treating diseases, disorders or conditions associated with mTOR, including immunological disorders, cancer, and
neurodegenerative diseases.
[0037] The compound of Formula 1 includes compounds specifically named above and in the examples, and may exist as salts, complexes, solvates, hydrates, and liquid crystals. Likewise, compounds of Formula 1 that are salts may exist as complexes, solvates, hydrates, and liquid crystals. [0038] The compound of Formula 1 may form pharmaceutically acceptable complexes, salts, solvates and hydrates. These salts include acid addition salts (including di-acids) and base salts. Pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, and phosphorous acids, as well nontoxic salts derived from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl -substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Such salts include acetate, adipate, aspartate, benzoate, besylate, bicarbonate, carbonate, bisulfate, sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate, hydrogen phosphate, dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts.
[0039] Pharmaceutically acceptable base salts include salts derived from bases, including metal cations, such as an alkali or alkaline earth metal cation, as well as amines. Examples of suitable metal cations include sodium, potassium, magnesium, calcium, zinc, and aluminum. Examples of suitable amines include arginine, NfT -dibenzylethylenediamine, chloroprocaine, choline, diethylamine, diethanolamine, dicyclohexylamine, ethylenediamine, glycine, lysine, N- methylglucamine, olamine, 2-amino-2-hydroxymethyl-propane-l,3-diol, and procaine. For a discussion of useful acid addition and base salts, see S. M. Berge et ak, ./. Pharm. Sci. (1977) 66: 1-19; see also Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (2002).
[0040] Pharmaceutically acceptable salts may be prepared using various methods. For example, a compound of Formula 1 may be reacted with an appropriate acid or base to give the desired salt. Alternatively, a precursor of the compound of Formula 1 may be reacted with an acid or base to remove an acid- or base-labile protecting group or to open a lactone or lactam group of the precursor. Additionally, a salt of the compound of Formula 1 may be converted to another salt (or free form) through treatment with an appropriate acid or base or through contact with an ion exchange resin. Following reaction, the salt may be isolated by filtration if it precipitates from solution, or by evaporation to recover the salt. The degree of ionization of the salt may vary from completely ionized to almost non-ionized. [0041] The compound of Formula 1 may exist in a continuum of solid states ranging from fully amorphous to fully crystalline. The term“amorphous” refers to a state in which the material lacks long range order at the molecular level and, depending upon temperature, may exhibit the physical properties of a solid or a liquid. Typically such materials do not give distinctive X-ray diffraction patterns and, while exhibiting the properties of a solid, are more formally described as a liquid. Upon heating, a change from solid to liquid properties occurs which is characterized by a change of state, typically second order (“glass transition”). The term“crystalline” refers to a solid phase in which the material has a regular ordered internal structure at the molecular level and gives a distinctive X-ray diffraction pattern with defined peaks. Such materials when heated sufficiently will also exhibit the properties of a liquid, but the change from solid to liquid is characterized by a phase change, typically first order (“melting point”).
[0042] The compound of Formula 1 may also exist in unsolvated and solvated forms. The term “solvate” describes a molecular complex comprising the compound and one or more
pharmaceutically acceptable solvent molecules (e.g., ethanol). The term“hydrate” is a solvate in which the solvent is water. Pharmaceutically acceptable solvates include those in which the solvent may be isotopically substituted (e.g., D2O, aceton e-de, DMSO-r/r,).
[0043] A currently accepted classification system for solvates and hydrates of organic compounds is one that distinguishes between isolated site, channel, and metal-ion coordinated solvates and hydrates. See, e.g., K. R. Morris (H. G. Brittain ed.) Polymorphism in
Pharmaceutical Solids (1995). Isolated site solvates and hydrates are ones in which the solvent (e.g., water) molecules are isolated from direct contact with each other by intervening molecules of the organic compound. In channel solvates, the solvent molecules lie in lattice channels where they are next to other solvent molecules. In metal-ion coordinated solvates, the solvent molecules are bonded to the metal ion.
[0044] When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and in hygroscopic compounds, the water or solvent content will depend on humidity and drying conditions. In such cases, non-stoichiometry will typically be observed.
[0045] The compound of Formula 1 may also exist as multi-component complexes (other than salts and solvates) in which the compound (drug) and at least one other component are present in stoichiometric or non-stoichiometric amounts. Complexes of this type include clathrates (drug- host inclusion complexes) and co-crystals. The latter are typically defined as crystalline complexes of neutral molecular constituents which are bound together through non-covalent interactions, but could also be a complex of a neutral molecule with a salt. Co-crystals may be prepared by melt crystallization, by recrystallization from solvents, or by physically grinding the components together. See, e.g., O. Almarsson and M. J. Zaworotko, Chem. Commun. (2004) 17: 1889-1896. For a general review of multi-component complexes, see J. K. Haleblian, J.
Pharm. Sci. (1975) 64(8): 1269-88.
[0046] When subjected to suitable conditions, the compound of Formula 1 may exist in a mesomorphic state (mesophase or liquid crystal). The mesomorphic state lies between the true crystalline state and the true liquid state (either melt or solution). Mesomorphism arising as the result of a change in temperature is described as“thermotropic” and mesomorphism resulting from the addition of a second component, such as water or another solvent, is described as “lyotropic.” Compounds that have the potential to form lyotropic mesophases are described as “amphiphilic” and include molecules which possess a polar ionic moiety
(e.g., -COO Na+, -COO K+, -S03~Na+) or polar non-ionic moiety (such as -NTS[+(CH3)3). See, e.g., N. H. Hartshome and A. Stuart, Crystals and the Polarizing Microscope (4th ed, 1970).
[0047] The compound of Formula 1 may exist as polymorphs, stereoisomers, tautomers, or some combination thereof, may be isotopically-labeled, may result from the administration of a prodrug, or form a metabolite following administration.
[0048] “Prodrugs” refer to compounds having little or no pharmacological activity that can, when metabolized in vivo , undergo conversion to compounds having desired pharmacological activity. Prodrugs may be prepared by replacing appropriate functionalities present in
pharmacologically active compounds with“pro-moieties” as described, for example, in
H. Bundgaar, Design of Prodrugs (1985). Examples of prodrugs include ester, ether or amide derivatives of compounds of Formula 1 having carboxylic acid, hydroxy, or amino functional groups, respectively. For further discussions of prodrugs, see e.g., T. Higuchi and V. Stella “Pro-drugs as Novel Delivery Systems,” ACS Symposium Series 14 (1975) and E. B. Roche ed., Bioreversible Carriers in Drug Design (1987).
[0049] “Metabolites” refer to compounds formed in vivo upon administration of
pharmacologically active compounds. Examples include hydroxymethyl, hydroxy, secondary amino, primary amino, phenol, and carboxylic acid derivatives of compounds of Formula 1 having methyl, alkoxy, tertiary amino, secondary amino, phenyl, and amide groups, respectively.
[0050] The compound of Formula 1 may exist as a particular stereoisomer. Stereoisomers are configuration isomers resulting, for example, from the presence of one or more stereogenic centers, double bonds, cyclic groups, or some combination of these structural features. Such stereoisomers may also result from acid addition or base salts in which the counter-ion is optically active, for example, when the counter-ion is D-lactate or L-lysine. Stereoisomers may be pure, substantially pure, or mixtures.
[0051] The compound of Formula 1 may exhibit more than one type of stereoisomerism. For example, the stereogenic carbon (position 6a) of the tricyclic core shown in Formula 1 has S stereochemical configuration. In addition, the compound of Formula 1 may exist as cis or trans stereoisomers based on the relative positions of substituents attached to the cyclohexyl moiety. Thus the compound of Formula 1 includes geometric isomers (,S)-c/. '-l-methyl-4-(2-(liT-indol-4- yl)-6a-methyl-6(5F/)-oxo-6a,7,9,l0-tetrahydro-[l,4]oxazino[3,4-/z]pteridin-5-yl)-cyclohexan-l-ol (i.e., the“cis stereoisomer”), which is represented by the formula
Figure imgf000011_0001
and (S)-trans-l -methyl-4-(2-(li7-indol-4-yl)-6a-methyl-6(5F/)-oxo-6a, 7,9,10-tetrahydro- [l,4]oxazino[3,4-/z]pteridin-5-yl)-cyclohexan-l-ol (i.e., the“trans stereoisomer”), which is represented by the formula
Figure imgf000011_0002
Using the naming function in ChemDraw® Professional (version 15.0.0.106), the cis stereoisomer has the name S)-5-((U,4f?)-4-hydroxy-4-methylcyclohexyl)-2-(li7-indol-4-yl)-6a-methyl- 6a, 7,9, 10-tetrahydro-[ 1 ,4]oxazino[3,4-/?]pteridin-6(5//)-one, and the trans stereoisomer has the name S)-5-((lr,4,S)-4-hydroxy-4-methylcyclohexyl)-2-(li7-indol-4-yl)-6a-methyl-6a,7,9,l0- tetrahydro-[ 1 ,4]oxazino[3,4-/?]pteridin-6(5H)-one.
[0052] Compounds of Formula 1 may exist as tautomers, which are isomers resulting from tautomerization. Generally, tautomeric isomerism includes, for example, imine-enamine, keto- enol, oxime-nitroso, and amide-imidic acid tautomerism.
[0053] Geometrical (cis/trans) isomers may be separated by conventional techniques such as chromatography and fractional crystallization. [0054] Conventional techniques for preparing or isolating a compound having a specific stereochemical configuration include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound contains an acidic or basic moiety, an acid or base such as tartaric acid or 1- phenylethylamine. The resulting diastereomeric mixture may be separated by chromatography, fractional crystallization, etc., and the appropriate diastereoisomer converted to the compound having the requisite stereochemical configuration. For a further discussion of techniques for separating stereoisomers, see E. L. Eliel and S. H. Wilen, Stereochemistry of Organic Compounds (1994).
[0055] The compound of Formula 1 may possess isotopic variations, in which at least one atom is replaced by an atom having the same atomic number, but an atomic mass different from the atomic mass usually found in nature. Isotopes suitable for inclusion in compound of
Formula 1 include, for example, isotopes of hydrogen, such as 2H and 3H; isotopes of carbon, such asuC, 13C and 14C; isotopes of nitrogen, such as13N and 15N; and isotopes of oxygen, such as 150, 170 and 180. ETse of isotopic variations (e.g., deuterium, 2H) may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements. Additionally, certain isotopic variations of the disclosed
compounds may incorporate a radioactive isotope (e.g., tritium, 3H, or 14C), which may be useful in drug and/or substrate tissue distribution studies. Substitution with positron emitting isotopes, such as UC, 150, and 13N, may be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds may be prepared by processes analogous to those described elsewhere in the disclosure using an appropriate isotopically-labeled reagent in place of a non-labeled reagent.
[0056] The compound of Formula 1 may be prepared using the techniques described below. Some of the schemes and examples may omit details of common reactions, including oxidations, reductions, and so on, separation techniques (extraction, evaporation, precipitation,
chromatography, filtration, trituration, crystallization, and the like), and analytical procedures, which are known to persons of ordinary skill in the art of organic chemistry. The details of such reactions and techniques can be found in a number of treatises, including Richard Larock, Comprehensive Organic Transformations (1999), and the multi -volume series edited by Michael B. Smith and others, Compendium of Organic Synthetic Methods (1974 et seq.). Starting materials and reagents may be obtained from commercial sources or may be prepared using literature methods. Some of the reaction schemes may omit minor products resulting from chemical transformations (e.g., an alcohol from the hydrolysis of an ester, CO2 from the decarboxylation of a di-acid, etc.). In addition, in some instances, reaction intermediates may be used in subsequent steps without isolation or purification (i.e., in situ).
[0057] In some of the reaction schemes and examples below, certain compounds can be prepared using protecting groups, which prevent undesirable chemical reaction at otherwise reactive sites. Protecting groups may also be used to enhance solubility or otherwise modify physical properties of a compound. For a discussion of protecting group strategies, a description of materials and methods for installing and removing protecting groups, and a compilation of useful protecting groups for common functional groups, including amines, carboxylic acids, alcohols, ketones, aldehydes, and so on, see T. W. Greene and P. G. Wuts, Protecting Groups in Organic Chemistry (1999) and P. Kocienski, Protective Groups (2000).
[0058] Generally, the chemical transformations described throughout the specification may be carried out using substantially stoichiometric amounts of reactants, though certain reactions may benefit from using an excess of one or more of the reactants. Additionally, many of the reactions disclosed throughout the specification may be carried out at about room temperature (RT) and ambient pressure, but depending on reaction kinetics, yields, and so on, some reactions may be run at elevated pressures or employ higher temperatures (e.g., reflux conditions) or lower temperatures (e.g., -78°C to 0°C). Any reference in the disclosure and claims to a stoichiometric range, a temperature range, a pH range, etc., whether or not expressly using the word“range,” also includes the indicated endpoints.
[0059] Many of the chemical transformations may also employ one or more compatible solvents, which may influence the reaction rate and yield. Depending on the nature of the reactants, the one or more solvents may be polar protic solvents (including water), polar aprotic solvents, non-polar solvents, or some combination. Representative solvents include saturated aliphatic hydrocarbons (e.g., «-pentane, «-hexane, «-heptane, «-octane, cyclohexane,
methylcyclohexane); aromatic hydrocarbons (e.g., benzene, toluene, xylenes); halogenated hydrocarbons (e.g., methylene chloride, chloroform, carbon tetrachloride); aliphatic alcohols (e.g., methanol, ethanol, propan- l-ol, propan-2-ol, butan-l-ol, 2-methyl-propan- l-ol, butan-2-ol, 2- methyl-propan-2-ol, pentan-l-ol, 3-methyl-butan-l-ol, hexan-l-ol, 2-m ethoxy-ethanol, 2-ethoxy- ethanol, 2-butoxy-ethanol, 2-(2-methoxy-ethoxy)-ethanol, 2-(2-ethoxy-ethoxy)-ethanol, 2-(2- butoxy-ethoxy)-ethanol); ethers (e.g., diethyl ether, di-isopropyl ether, dibutyl ether, 1,2- dimethoxy-ethane, l,2-di ethoxy-ethane, l-methoxy-2-(2-methoxy-ethoxy)-ethane, 1 -ethoxy -2-(2- ethoxy-ethoxy)-ethane, tetrahydrofuran, l,4-dioxane); ketones (e.g., acetone, methyl ethyl ketone); esters (methyl acetate, ethyl acetate); nitrogen-containing solvents (e.g., formamide, /V,/V- dimethylformamide, acetonitrile, A'- ethyl -pyrrol idone, pyridine, quinoline, nitrobenzene);
sulfur-containing solvents (e.g., carbon disulfide, dimethyl sulfoxide, tetrahydro-thiophene-l, l,- dioxide); and phosphorus-containing solvents (e.g., hexamethylphosphoric triamide).
[0060] The compounds of Formula 1, which includes compounds named above, and their pharmaceutically acceptable complexes, salts, solvates and hydrates, should be assessed for their biopharmaceutical properties, such as solubility and solution stability across pH, permeability, and the like, to select an appropriate dosage form and route of administration. Compounds that are intended for pharmaceutical use may be administered as crystalline or amorphous products, and may be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, spray drying, evaporative drying, microwave drying, or radio frequency drying.
[0061] The compound of Formula 1 may be administered alone or in combination with one another or with one or more pharmacologically active compounds which are different than the compound of Formula 1. Generally, one or more of these compounds are administered as a pharmaceutical composition (a formulation) in association with one or more pharmaceutically acceptable excipients. The choice of excipients depends on the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form, among other things. Useful pharmaceutical compositions and methods for their preparation may be found, for example, in A. R. Gennaro (ed.), Remington: The Science and Practice of Pharmacy (20th ed., 2000).
[0062] The compound of Formula 1 may be administered orally. Oral administration may involve swallowing in which case the compound enters the bloodstream via the gastrointestinal tract. Alternatively or additionally, oral administration may involve mucosal administration (e.g., buccal, sublingual, supralingual administration) such that the compound enters the bloodstream through the oral mucosa.
[0063] Formulations suitable for oral administration include solid, semi-solid and liquid systems such as tablets; soft or hard capsules containing multi- or nano-particulates, liquids, or powders; lozenges which may be liquid-filled; chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal or mucoadhesive patches. Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules (made, e.g., from gelatin or hydroxypropylmethylcellulose) and typically comprise a carrier (e.g., water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil) and one or more emulsifying agents, suspending agents or both. Liquid formulations may also be prepared by the reconstitution of a solid (e.g., from a sachet).
[0064] The compound of Formula 1 may also be used in fast-dissolving, fast-disintegrating dosage forms such as those described in Liang and Chen, Expert Opinion in Therapeutic Patents (2001) 1 l(6):98l-986.
[0065] For tablet dosage forms, depending on dose, the active pharmaceutical ingredient (API) may comprise from about 1 wt% to about 80 wt% of the dosage form or more typically from about 5 wt% to about 60 wt% of the dosage form. In addition to the API, tablets may include one or more disintegrants, binders, diluents, surfactants, glidants, lubricants, anti-oxidants, colorants, flavoring agents, preservatives, and taste-masking agents. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, Ci-6 alkyl-substituted hydroxypropylcellulose, starch, pregelatinized starch, and sodium alginate. Generally, the disintegrant will comprise from about 1 wt% to about 25 wt% or from about 5 wt% to about 20 wt% of the dosage form.
[0066] Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropylcellulose and hydroxypropylmethylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous), mannitol, xylitol, dextrose, sucrose, sorbitol,
microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.
[0067] Tablets may also include surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. When present, surface active agents may comprise from about 0.2 wt% to about 5 wt% of the tablet, and glidants may comprise from about 0.2 wt% to about 1 wt% of the tablet.
[0068] Tablets may also contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulfate. Lubricants may comprise from about 0.25 wt% to about 10 wt% or from about 0.5 wt% to about 3 wt% of the tablet.
[0069] Tablet blends may be compressed directly or by roller compaction to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or extruded before tableting. If desired, prior to blending one or more of the components may be sized by screening or milling or both. The final dosage form may comprise one or more layers and may be coated, uncoated, or encapsulated. Exemplary tablets may contain up to about 80 wt% of API, from about 10 wt% to about 90 wt% of binder, from about 0 wt% to about 85 wt% of diluent, from about 2 wt% to about 10 wt% of disintegrant, and from about 0.25 wt% to about 10 wt% of lubricant. For a discussion of blending, granulation, milling, screening, tableting, coating, as well as a description of alternative techniques for preparing drug products, see A. R. Gennaro (ed.), Remington: The Science and Practice of Pharmacy (20th ed., 2000); H. A. Lieberman et al. (ed.), Pharmaceutical Dosage Forms: Tablets, Vol. 1-3 (2d ed., 1990); and D. K. Parikh & C. K. Parikh, Handbook of Pharmaceutical Granulation Technology, Vol. 81 (1997).
[0070] Consumable oral films for human or veterinary use are pliable water-soluble or water- swellable thin film dosage forms which may be rapidly dissolving or mucoadhesive. In addition to the API, a typical film includes one or more film-forming polymers, binders, solvents, humectants, plasticizers, stabilizers or emulsifiers, viscosity-modifying agents, and solvents.
Other film ingredients may include anti-oxidants, colorants, flavorants and flavor enhancers, preservatives, salivary stimulating agents, cooling agents, co-solvents (including oils), emollients, bulking agents, anti-foaming agents, surfactants, and taste-masking agents. Some components of the formulation may perform more than one function.
[0071] In addition to dosing requirements, the amount of API in the film may depend on its solubility. If water soluble, the API would typically comprise from about 1 wt% to about 80 wt% of the non-solvent components (solutes) in the film or from about 20 wt% to about 50 wt% of the solutes in the film. A less soluble API may comprise a greater proportion of the composition, typically up to about 88 wt% of the non-solvent components in the film.
[0072] The film-forming polymer may be selected from natural polysaccharides, proteins, or synthetic hydrocolloids and typically comprises from about 0.01 wt% to about 99 wt% or from about 30 wt% to about 80 wt% of the film.
[0073] Film dosage forms are typically prepared by evaporative drying of thin aqueous films coated onto a peelable backing support or paper, which may carried out in a drying oven or tunnel (e.g., in a combined coating-drying apparatus), in lyophilization equipment, or in a vacuum oven.
[0074] Useful solid formulations for oral administration may include immediate release formulations and modified release formulations. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release. For a general description of suitable modified release formulations, see US Patent No. 6, 106,864. For details of other useful release technologies, such as high energy dispersions and osmotic and coated particles, see Verma et al, Pharmaceutical Technology On-line (2001) 25(2): 1-14.
[0075] The compound of Formula 1 may also be administered directly into the blood stream, muscle, or an internal organ of the subject. Suitable techniques for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, and subcutaneous administration. Suitable devices for parenteral administration include needle injectors, including microneedle injectors, needle-free injectors, and infusion devices.
[0076] Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (e.g., pH of from about 3 to about 9). For some applications, however, the compound of Formula 1 may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions (e.g., by lyophilization) may be readily accomplished using standard pharmaceutical techniques.
[0077] The solubility of compounds which are used in the preparation of parenteral solutions may be increased through appropriate formulation techniques, such as the incorporation of solubility-enhancing agents. Formulations for parenteral administration may be formulated to be immediate or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release. Thus, the compound of Formula 1 may be formulated as a suspension, a solid, a semi-solid, or a thixotropic liquid for administration as an implanted depot providing modified release of the active compound. Examples of such formulations include drug-coated stents and semi-solids and suspensions comprising drug-loaded poly(DL-lactic-coglycolic)acid (PGLA) microspheres.
[0078] The compound of Formula 1 may also be administered topically, intradermally, or transdermally to the skin or mucosa. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages and microemulsions. Liposomes may also be used. Typical carriers may include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Topical formulations may also include penetration enhancers. See, e.g., Finnin and Morgan, J. Pharm. Sci. 88(l0):955-958 (1999). [0079] Other means of topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis and microneedle or needle-free (e.g. Powderject™ and Bioject™) injection. Formulations for topical administration may be formulated to be immediate or modified release as described above.
[0080] The compound of Formula 1 may also be administered intranasally or by inhalation, typically in the form of a dry powder, an aerosol spray, or nasal drops. An inhaler may be used to administer the dry powder, which comprises the API alone, a powder blend of the API and a diluent, such as lactose, or a mixed component particle that includes the API and a phospholipid, such as phosphatidylcholine. For intranasal use, the powder may include a bioadhesive agent, e.g., chitosan or cyclodextrin. A pressurized container, pump, sprayer, atomizer, or nebulizer, may be used to generate the aerosol spray from a solution or suspension comprising the API, one or more agents for dispersing, solubilizing, or extending the release of the API (e.g., EtOH with or without water), one or more solvents (e.g., l,l,l,2-tetrafluoroethane or l,l,l,2,3,3,3-heptafluoropropane) which serve as a propellant, and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid. An atomizer using electrohydrodynamics may be used to produce a fine mist.
[0081] Prior to use in a dry powder or suspension formulation, the drug product is usually comminuted to a particle size suitable for delivery by inhalation (typically 90% of the particles, based on volume, having a largest dimension less than 5 microns). This may be achieved by any appropriate size reduction method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing, high pressure homogenization, or spray drying.
[0082] Capsules, blisters and cartridges (made, for example, from gelatin or
hydroxypropylmethyl cellulose) for use in an inhaler or insufflator may be formulated to contain a powder mixture of the active compound, a suitable powder base such as lactose or starch, and a performance modifier such as L-leucine, mannitol, or magnesium stearate. The lactose may be anhydrous or monohydrated. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose, and trehalose.
[0083] A suitable solution formulation for use in an atomizer using electrohydrodynamics to produce a fine mist may contain from about 1 pg to about 20 mg of the API per actuation and the actuation volume may vary from about 1 pL to about 100 pL. A typical formulation may comprise one or more compounds of Formula 1, propylene glycol, sterile water, EtOH, and NaCl. Alternative solvents, which may be used instead of propylene glycol, include glycerol and polyethylene glycol. [0084] Formulations for inhaled administration, intranasal administration, or both, may be formulated to be immediate or modified release using, for example, PGLA. Suitable flavors, such as menthol and levomenthol, or sweeteners, such as saccharin or sodium saccharin, may be added to formulations intended for inhaled/intranasal administration.
[0085] In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve that delivers a metered amount. Units are typically arranged to administer a metered dose or“puff’ containing from about 10 pg to about 1000 pg of the API. The overall daily dose will typically range from about 100 pg to about 10 mg which may be administered in a single dose or, more usually, as divided doses throughout the day.
[0086] The active compounds may be administered rectally or vaginally, e.g., in the form of a suppository, pessary, or enema. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate. Formulations for rectal or vaginal administration may be formulated to be immediate or modified release as described above.
[0087] The compound of Formula 1 may also be administered directly to the eye or ear, typically in the form of drops of a micronized suspension or solution in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ocular and aural administration include ointments, gels, biodegradable implants (e.g. absorbable gel sponges, collagen), non-biodegradable implants (e.g. silicone), wafers, lenses, and particulate or vesicular systems, such as niosomes or liposomes. The formulation may include one or more polymers and a preservative, such as benzalkonium chloride. Typical polymers include crossed-linked polyacrylic acid,
polyvinylalcohol, hyaluronic acid, cellulosic polymers (e.g., hydroxypropylmethylcellulose, hydroxyethylcellulose, methyl cellulose), and heteropolysaccharide polymers (e.g., gelan gum). Such formulations may also be delivered by iontophoresis. Formulations for ocular or aural administration may be formulated to be immediate or modified release as described above.
[0088] To improve their solubility, dissolution rate, taste-masking, bioavailability, or stability, the compound of Formula 1 may be combined with soluble macromolecular entities, including cyclodextrin and its derivatives and polyethylene glycol-containing polymers. For example, API- cyclodextrin complexes are generally useful for most dosage forms and routes of administration. Both inclusion and non-inclusion complexes may be used. As an alternative to direct
complexation with the API, the cyclodextrin may be used as an auxiliary additive, i.e. as a carrier, diluent, or solubilizer. Alpha-, beta- and gamma-cyclodextrins are commonly used for these purposes. See, e.g., WO 91/11172, WO 94/02518, and WO 98/55148. [0089] As noted above, the compound of Formula 1, including compounds specifically named above, and their pharmaceutically active complexes, salts, solvates and hydrates, may be combined with each other or with one or more other active pharmaceutically active compounds to treat various diseases, conditions and disorders. In such cases, the active compounds may be combined in a single dosage form as described above or may be provided in the form of a kit which is suitable for coadministration of the compositions. The kit comprises (1) two or more different pharmaceutical compositions, at least one of which contains a compound of Formula 1; and (2) a device for separately retaining the two pharmaceutical compositions, such as a divided bottle or a divided foil packet. An example of such a kit is the familiar blister pack used for the packaging of tablets or capsules. The kit is suitable for administering different types of dosage forms (e.g., oral and parenteral) or for administering different pharmaceutical compositions at separate dosing intervals, or for titrating the different pharmaceutical compositions against one another. To assist with patient compliance, the kit typically comprises directions for
administration and may be provided with a memory aid.
[0090] For administration to human patients, the total daily dose of the claimed and disclosed compounds is typically in the range of about 0.1 mg to about 3000 mg depending on the route of administration. For example, oral administration may require a total daily dose of from about 1 mg to about 3000 mg, while an intravenous dose may only require a total daily dose of from about 0.1 mg to about 300 mg. The total daily dose may be administered in single or divided doses and, at the physician’s discretion, may fall outside of the typical ranges given above.
Although these dosages are based on an average human subject having a mass of about 60 kg to about 70 kg, the physician will be able to determine the appropriate dose for a patient (e.g., an infant) whose mass falls outside of this weight range.
[0091] As noted above, the compound of Formula 1 may be used to treat diseases, disorders, and conditions for which inhibition of mTOR is indicated. Such diseases, disorders, and conditions generally relate to any unhealthy or abnormal state in a subject for which the inhibition of mTOR provides a therapeutic benefit. Such diseases, disorders, and conditions include neurodegenerative diseases, cancer, diseases involving the immune system, inflammation, autoimmune diseases, and fibrotic disorders. When general terms are used to describe diseases, disorders, and conditions associated with mTOR, they include more specific conditions mentioned in diagnostic manuals and other materials.
[0092] For example, the treatment of cancer includes treatment of all neoplasia, regardless of their histopathological appearance. Thus, the compound of Formula 1 may be used to treat cancer of blood, including leukemia (e.g., acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia), and cancer of the skin, including melanoma, basal cell carcinoma, and squamous cell carcinoma. The compound of Formula 1 may also be used to treat cancer of the bone, liver, and lung (e.g., small-cell lung tumor, non small-cell lung cancer, and bronchioalveolar cancer); cancer of the brain, breast, prostate, larynx, gall bladder, pancreas, rectum, bile duct, parathyroid, thyroid, adrenal gland, neural tissue, bladder, spleen, head and neck, including cancer of the jaw, mouth, and nose; and cancer of the colon, stomach, testes, esophagus, uterus, cervix, vulva, bronchi, bile duct, bladder, kidney, ovary, and pancreas.
[0093] In addition, the compound of Formula 1 may be used to treat multiple myeloma, lymphomas, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, osteosarcoma, Ewing’s sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, islet cell tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuromas, intestinal
ganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm’s tumor, seminoma, leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, myelodysplastic syndrome, mycosis fungoides, rhabdomyosarcoma, astrocytoma, non-Hodgkin’s lymphoma, osteogenic and other sarcoma, malignant hypercalcemia,
polycythermia vera, adenocarcinoma, glioblastoma multiforma, glioma, lymphomas, epidermoid carcinomas, and other carcinomas and sarcomas.
[0094] The compound of Formula 1 may also be used to treat benign tumors including hemangiomas, hepatocellular adenoma, cavernous haemangioma, focal nodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, nodular regenerative hyperplasia, trachomas, pyogenic granulomas, and the like, and hamartoma conditions such as Peutz-Jeghers Syndrome (PJS), Cowden disease, Bannayan-Riley-Ruvalcaba Syndrome (BRRS), Proteus syndrome, Lhermitte-Duclos disease and Tuberous Sclerosis (TSC).
[0095] The compound of Formula 1 may be used to treat abnormal cell proliferation due to insults to body tissue during surgery, repetitive motion disorders, including carpal tunnel syndrome. These insults may arise as a result of a variety of surgical procedures such as joint or bowel surgery, and include keloid scarring. [0096] The compound of Formula 1 may be used to prevent restenosis, i.e., undesirable proliferation of normal cells in the blood vessel in response to the introduction of stents in the treatment of cardiovascular disease.
[0097] The compound of Formula 1 may also be used to treat proliferative responses associated with organ transplantation, which may contribute to potential organ rejection or associated complications. Such proliferative responses may occur during transplantation of the heart, lung, liver, kidney, and other body organs or organ systems.
[0098] In addition, the compound of Formula 1 may be used to treat abnormal angiogenesis, including the abnormal angiogenesis accompanying rheumatoid arthritis, ischemic-reperfusion related brain edema and injury, cortical ischemia, ovarian hyperplasia and hypervascularity, polycystic ovary syndrome, endometriosis, psoriasis, diabetic retinopathy, and other ocular angiogenic diseases such as retinopathy of prematurity (retrolental fibroplastic), macular degeneration (e.g., wet and dry age-related macular degeneration), corneal graft rejection, neovascular glaucoma, Oster Webber syndrome, retinal/choroidal neovascularization and corneal neovascularization, Best’s disease, myopia, optic pits, Stargart’s diseases, Pagets disease, vein occlusion, artery occlusion, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum carotid abstructive diseases, chronic uveitis/vitritis, mycobacterial infections, Lyme’s disease, systemic lupus erythematosis, retinopathy of prematurity, Eales disease, diabetic retinopathy, Behcets disease, infections causing retinitis or chroiditis, presumed ocular histoplasmosis, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post- laser complications, diseases associated with rubesis (neovascularization of the iris), diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, Mooren’s ulcer, Terrien’s marginal degeneration, marginal keratolysis, polyarteritis, Wegener sarcoidosis, scleritis, periphigoid radial keratotomy, neovascular glaucoma and retrolental fibroplasia, mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, epilepsy, seizures, polyglutamine diseases, traumatic brain injury, ischemic and hemorrhaging stroke, cerebral ischemias or neurodegenerative disease, including apoptosis-driven neurodegenerative disease, caused by traumatic injury, acute hypoxia, ischemia or glutamate neurotoxicity. [0099] The compound of Formula 1 may also be used to treat neurodegenerative diseases, disorders, and conditions, including Huntington Disease, Alzheimer Disease, Niemann-Pick disease, Parkinson’s disease, prion-mediated disease, spinocerebellar ataxia, progressive multifocal encephalopathy, amyotrophic lateral sclerosis, axonal neuropathies, muscular dystrophies, and age-related nervous system degeneration. In particular, the compound of
Formula 1 may be used to treat Huntington’s disease.
[0100] The compound of Formula 1 may be used to treat inflammation. Examples include treatment of acute pancreatitis, chronic pancreatitis, asthma, allergies, chronic obstructive pulmonary disease, and adult respiratory distress syndrome, and chronic inflammatory diseases associated with uncontrolled angiogenesis, inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis, psoriasis, sarcoidosis, rheumatoid arthritis, and multisystem granulomatous disorder.
[0101] In addition, the compound of Formula 1 may be used to treat autoimmune disorders, including glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves’ disease, autoimmune gastritis, diabetes, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, atopic dermatitis, chronic active hepatitis, myasthenia gravis, multiple sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn’s disease, psoriasis, graft vs. host disease, multiple sclerosis, and Sjogren’s syndrome.
[0102] The compound of Formula 1 may be used to treat cardiovascular and metabolic diseases, disorders and conditions, including obesity, diabetes, insulin resistance, metabolic syndrome, and hyperlipidemia.
[0103] The compound of Formula 1 may also be used to treat fibrotic disorders, which include primary fibrotic disorders, fibrosis associated with autoimmune and inflammatory diseases, disorders, and conditions, respiratory fibrotic disorders, and secondary fibrosis. Primary fibrotic disorders include local and systemic variants of scleroderma; mediastinal, retroperitoneal, and nephrogenic systemic fibrosis; as well as endomyocardial fibrosis, pericarditis, pleuritis, and primary myocardial fibrosis.
[0104] Fibrotic disorders include fibrosis associated with autoimmune and inflammatory diseases, disorders, and conditions, such as rheumatoid arthritis, systemic lupus erythematosus, primary and secondary Sjogren syndrome, inflammatory bowel disease, including Crohn disease and ulcerative colitis; spondarthropathy, including ankylosing spondylitis; psoriatic arthritis, enteropathic arthritis, and reactive arthritis; inflammatory muscle disease including polymositis, dermatomyositis, and inclusion body myopathies, whether primary or accompanying malignant disease; conjunctivis, iritis, keratitis, keratoconjunctivitis, scleritis, and uveitis; retinopathies, including retinal detachment; myasthenia gravis, atopic dermatitis, eczema, and alopecia areata; familial Mediterranean fever; acute, intercritical, chronic recurring, polyarticular, and tophaceous forms of gout; vasculitis, including Wegener’s granulomatosis, giant cell arteritis, polymyalgia rheumatic, polyarteritis nodosa, and Churg-Strauss syndrome; polychondritis; calcium
pyrophosphate tissue deposition disease, both acute and chronic variants; primary and secondary osteoarthritis; adhesive capsulitis; psoriasis; juvenile arthropathies including systemic-onset, polyarticular, pauci-articular and enthesopathic variants, and childhood-onset variants of rheumatoid arthritis and systemic lupus erythematosus; multiple sclerosis, neuromyelitis optica, and other demyelinating conditions of the central or peripheral nervous systems; thyroiditis, including Hashimoto disease and Grave’s ophthalmopathy; autoimmune endocrine glandular dysfunction, including Addison disease; automuune bone marrow and hematologic disorders including immune and idiopathic forms of thrombocytopenic purpura and hemolytic anemia, aplastic anemia; psoriasis, including psoriasis vulgaris, guttate psoriasis, psoriasis affecting the scalp or nails, and palmo-plantar psoriasis; cervical and lumbar spondylitis or spondylosis;
blistering skin and mucosal disease including pemphigus vulgaris, bullous pemphigoid, Stevens- Johnson syndrome, and cutaneous drug reactions; primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis; allergic reactions, including reactions to drugs, pollens, animal dander, arthropod or snake bites, and insect feces; celiac disease; sarcoidosis; lichen planus; Behcets disease; and idiopathic hearing or visual loss.
[0105] Fibrotic respiratory disorders refers to respiratory fibrosis, including idiopathic pulmonary fibrosis (IPF); interstitial lung diseases, including diffuse parenchymal lung diseases, idiopathic interstitial pneumonias, granulomatous lung disorders (e.g., sarcoidosis); and other forms of interstitial lung disease (ILD) including lymphangioleiomyomatosis (LAM), pulmonary Langerhans’ cell histiocytosis/histiocytosis X, and eosinophilic pneumonia; interstitial pneumonias (IPs), which include nonspecific interstitial pneumonia, desquamative interstitial pneumonia, respiratory bronchiolitis-associated interstitial lung disease, acute interstitial pneumonia, cryptogenic organizing pneumonia, lymphocytic interstitial pneumonia, and bronchitis obliterans syndrome (BOS).
[0106] Other fibrotic disorders include secondary fibrosis, which includes fibrosis of the liver following drug or alcohol exposure, non-alcoholic steatohepatosis, and metals exposure, including exposure to iron and copper; viral hepatitis, including Hepatitis A, B, C, D or E or other hepatotrophic viruses or bacteria alone or in combination; myocardial and pericardial fibrosis following myocardial infarction, coronary artery bypass, coronary stenting, and other forms of cardiac surgery; fibrosis from other forms of cardiac damage; myelofibrosis; kidney fibrosis, including fibrosis complicating diabetes, drug or heavy metal-related renal injury, or
glomerulonephritis; keloid and other forms of excessive scar formation; fibrosis complicating tissue damage from other forms of traumatic, inflammatory, or degenerative disease.
[0107] Other fibrotic disorders include non-malignant forms of excessive or abnormal tissue proliferation or expansion including polycystic kidney disease, neurofibromatosis, uterine fibroids, colonic polyps, meningioma, tuberous sclerosis, and familial and sporadic forms of lymphangioleiomyomatosis; sebaceous cysts; fibrocystic breast disease; nodules of the thyroid gland; granulomas; arteriovenous malformations; syndesmophyte and osteophyte formation; and disseminated idiopathic skeletal hyperostosis. Other fibrotic disorders include fibrosis or other connective tissue proliferation or expansion following the implantation of temporary or permanent therapeutic devices including artificial joints, ocular lenses, cardiac valves, pacemakers, and drug-eluting or other implantable drug-delivery systems.
[0108] The compound of Formula 1 may be combined with one or more other
pharmacologically active compounds or therapies for treating the diseases, disorders, and conditions described above. The compound of Formula 1, which include compounds specifically named above and their pharmaceutically acceptable complexes, salts, solvates and hydrates, may be administered simultaneously, sequentially or separately in combination with one or more compounds or therapies for treating neurodegenerative diseases, cancer, diseases involving the immune system, inflammation, autoimmune diseases, fibrotic disorders, and others. Such combinations may offer significant therapeutic advantages, including fewer side effects, improved ability to treat underserved patient populations, or synergistic activity.
[0109] For example, the compound of Formula 1 may be combined with one or more nonsteroidal anti-inflamatory drugs (NSAIDs), analgesics, corticosteroids, biological response modifiers, and protein-A immunoadsorption therapy. Alternatively or additionally, the compound of Formula 1 may be combined with one or more disease modifying antirheumatic drugs
(DMARDs) or osteoporosis agents.
[0110] Representative NSAIDs include apazone, aspirin, celecoxib, diclofenac (with and without misoprostol), diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate sodium, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, choline and magnesium salicylates, salsalate, and sulindac. Representative analgesics include acetaminophen and morphine sulfate, as well as codeine, hydrocodone, oxycodone, propoxyphene, and tramadol, all with or without acetaminophen. Representative corticosteroids include betamethasone, cortisone acetate, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, and prednisone.
Representative biological response modifiers include TNF-a inhibitors, such as adalimumab, etanercept, and infliximab; selective B-cell inhibitors, such as rituximab; IL-l inhibitors, such as anakinra, and selective costimulation modulators, such as abatacept.
[0111] Representative DMARDs include auranofm (oral gold), azathioprine, chlorambucil, cyclophosamide, cyclosporine, gold sodium thiomalate (injectable gold), hydroxychloroquine, leflunomide, methotrexate, minocycline, myophenolate mofetil, penicillamine, and sulfasalazine. Representative osteoporosis agents include bisphosphonates, such as alendronate, ibandronate, risedronate, and zoledronic acid; selective estrogen receptor modulators, such as droloxifene, lasofoxifene, and raloxifene; hormones, such as calcitonin, estrogens, and parathyroid hormone; and immunosuppressant agents such as azathioprine, cyclosporine, and rapamycin.
[0112] Particularly useful combinations include a compound of Formula 1 and methotrexate; a compound of Formula 1 and one or more biological response modifiers, such as lefluonomide, etanercept, adalimumab, and infliximab; or a compound of Formula 1, methotrexate, and one or more biological response modifiers, such as lefluonomide, etanercept, adalimumab, and infliximab.
[0113] For the treatment of thrombis and restensosis, the compound of Formula 1 may be combined with one or more cardiovascular agents such as calcium channel blockers, statins, fibrates, beta-blockers, ACE inhibitors, and platelet aggregation inhibitors.
[0114] The compound of Formula 1 may also be combined with one or more compounds or therapies for treating cancer (i.e., anti-cancer agent). These include chemotherapeutic agents (i.e., cytotoxic or antineoplastic agents) such as alkylating agents, antibiotics, antimetabolic agents, plant-derived agents, and topoisomerase inhibitors, as well as molecularly targeted drugs which block the growth and spread of cancer by interfering with specific molecules involved in tumor growth and progression. Molecularly targeted drugs include both small molecules and biologies.
[0115] Representative alkylating agents include bischloroethylamines (nitrogen mustards, e.g., chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, and uracil mustard); aziridines (e.g., thiotepa); alkyl alkone sulfonates (e.g., busulfan); nitrosoureas (e.g., carmustine, lomustine, and streptozocin); nonclassical alkylating agents (e.g., altretamine, dacarbazine, and procarbazine); and platinum compounds (e.g., carboplatin, cisplatin, nedaplatin, oxaliplatin, satraplatin, and triplatin tetranitrate). [0116] Representative antibiotic agents include anthracyclines (e.g., aclarubicin, amrubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, pirarubicin, valrubicin, and zorubicin);
anthracenediones (e.g., mitoxantrone and pixantrone); and streptomyces (e.g., actinomycin, bleomycin, dactinomycin, mitomycin C, and plicamycin).
[0117] Representative antimetabolic agents include dihydrofolate reductase inhibitors (e.g., aminopterin, methotrexate, and pemetrexed); hymidylate synthase inhibitors (e.g., raltitrexed and pemetrexed); folinic acid (e.g., leucovorin); adenosine deaminase inhibitors (e.g., pentostatin); halogenated/ribonucleotide reductase inhibitors (e.g., cladribine, clofarabine, and fludarabine); thiopurines (e.g, thioguanine and mercaptopurine); thymidylate synthase inhibitors (e.g., fluorouracil, capecitabine, tegafur, carmofur, and floxuridine); DNA polymerase inhibitors (e.g., cytarabine); ribonucleotide reductase inhibitors (e.g., gemcitabine); hypomethylating agent (e.g., azacitidine and decitabine); and ribonucleotide reductase inhibitor (e.g., hydroxyurea); and an asparagine depleter (e.g., asparaginase)
[0118] Representative plant-derived agents include vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinzolidine, and vinorelbine), podophyllotoxins (e.g., etoposide and teniposide), and taxanes (e.g., docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel).
[0119] Representative type I topoisomerase inhibitors include camptothecins, such as belotecan, irinotecan, rubitecan, and topotecan. Representative type II topoisomerase inhibitors include amsacrine, etoposide, etoposide phosphate, and teniposide, which are derivatives of
epipodophyllotoxins.
[0120] Molecularly targeted therapies include biologic agents such as cytokines and other immune-regulating agents. Useful cytokines include interleukin-2 (IL-2, aldesleukin), interleukin 4 (IL-4), interleukin 12 (IL-12), and interferon, which Dincludes more than 23 related subtypes. Other cytokines include granulocyte colony stimulating factor (CSF) (filgrastim) and granulocyte macrophage CSF (sargramostim). Other immuno-modulating agents include bacillus Calmette- Guerin, levamisole, and octreotide; monoclonal antibodies against tumor antigens, such as trastruzumab and rituximab; and cancer vaccines, which induce an immune response to tumors.
[0121] In addition, molecularly targeted drugs that interfere with specific molecules involved in tumor growth and progression include inhibitors of epidermal growth factor (EGF), transforming growth factor-alpha (TGFa), TGFp, heregulin, insulin-like growth factor (IGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), colony stimulating factor (CSF), erythropoietin (EPO), interleukin-2 (IL-2), nerve growth factor (NGF), platelet-derived growth factor (PDGF), hetaptocyte growth factor (HGF), vascular endothelial growth factor (VEGF), angiopoietin, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), HER4, insulin-like growth factor 1 receptor (IGF1R), IGF2R, fibroblast growth factor 1 receptor (FGF1R), FGF2R, FGF3R, FGF4R, vascular endothelial growth factor receptor (VEGFR), tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 2 (Tie-2), platelet-derived growth factor receptor (PDGFR), Abl, Bcr-Abl, Raf, FMS-like tyrosine kinase 3 (FLT3), c-Kit, Src, protein kinase c (PKC), tropomyosin receptor kinase (Trk), Ret, mammalian target of rapamycin (mTOR), Aurora kinase, polo-like kinase (PLK), mitogen activated protein kinase (MAPK), mesenchymal-epithelial transition factor (c-MET), cyclin-dependant kinase (CDK), Akt, extracellular signal-regulated kinases (ERK), poly(ADP) ribose polymerase (PARP), and the like.
[0122] Specific molecularly targeted drugs include selective estrogen receptor modulators, such as tamoxifen, toremifene, fulvestrant, and raloxifene; antiandrogens, such as bicalutamide, nilutamide, megestrol, and flutamide; and aromatase inhibitors, such as exemestane, anastrozole, and letrozole. Other specific molecularly targeted drugs include agents which inhibit signal transduction, such as imatinib, dasatinib, nilotinib, trastuzumab, gefitinib, erlotinib, cetuximab, lapatinib, panitumumab, and temsirolimus; agents that induce apoptosis, such as bortezomib; agents that block angiogensis, such as bevacizumab, sorafenib, and sunitinib; agents that help the immune system destroy cancel cells, such as rituximab and alemtuzumab; and monoclonal antibodies which deliver toxic molecules to cancer cells, such as gemtuzumab ozogamicin, tositumomab, l3 ll-tositumoab, and ibritumomab tiuxetan.
[0123] BIOLOGICAL ACTIVITY
[0124] Biological activity may be determined using a variety of methods, including in vitro and in vivo methods.
[0125] A. In Vitro Kinase Inhibition
[0126] The inhibitory activity of five (5) compounds (Example compound 3 and comparator compounds A, B, C, and D) is evaluated using a lO-point dose-response analysis (0.510 to 10,000 nM) against a set of six (6) kinases via the SelectScreen™ Kinase Profiling Service by Invitrogen, a Life Technologies company (Madison, WI, ETSA), which includes the Adapta® ETniversal Kinase Assay (four (4) phospholipid kinases) and the Z’-LYTE® biochemical assay (FRAP (mTOR) and PLK2 kinase). All enzymes are tested using an adenosine 5 '-triphosphate (ATP) concentration equal to the Michaelis constant (Km) apparent.
[0127] The Adapta® Universal Kinase Assay is a homogenous, fluorescent-based immunoassay for the detection of adenosine 5’ -diphosphate (ADP) using a time-resolved (TR)-FRET signal. The assay can be divided into 2 phases: a kinase reaction phase and an ADP detection phase. In the kinase reaction phase, all components required for the kinase reaction are added to the well, and the reaction is allowed to incubate for 60 minutes. After this incubation, a detection solution consisting of a europium (EU)-labeled anti-ADP antibody, an Alexa Fluor® 647-labeled ADP tracer, and ethylenediaminetetraacetic acid (EDTA) (to stop the kinase reaction) are added to the assay well. ADP formed by the kinase reaction (in the absence of an inhibitor) displaces the Alexa Fluor 647-labeled ADP tracer from the antibody, resulting in a decrease in the TR-FRET signal.
In the presence of an inhibitor, the amount of ADP formed by the kinase reaction is reduced, and the resulting intact antibody -tracer interaction results in a high TR-FRET signal. ADP formation is determined by calculating the emission ratio from the assay well. The emission ratio is calculated by dividing the intensity of the tracer (acceptor) emission by the intensity of the Eu (donor) emission at 615 nm, as shown in Eqn 1 :
Emission Ratio =
[Alexa Fluor® 647 emission (665 nM)]/[europium emission (615 nM)] Eqn 1
The detection mix consists of EDTA (30 mM), Eu-anti-ADP antibody (30 nM), and ADP tracer. The test compounds are screened in 1% DMSO (final) in the well. The detailed protocol used for testing each of the four (4) recombinant human phospholipid kinases is described below.
[0128] Adapta® Assay Controls are made for each individual kinase and are located on the same plate as the kinase. The maximum Emission Ratio is established by the 0% Conversion Control (100% Inhibition Control), which contains no ATP in the kinase reaction and therefore exhibits no kinase activity. After addition of the detection mix containing EDTA, ATP is added to these wells. ATP addition is required for the 0% conversion controls wells because the ADP antibody binds ATP with low affinity. The ATP in wells with maximum kinase inhibition will displace the ADP tracer slightly, though much less efficiently than ADP. The 100% Conversion Control wells contain ADP instead of ATP and are designed to allow for the calculation of percent ATP conversion. The 0% Conversion and 100% Conversion Controls allow one to estimate the percent ATP conversion achieved in a specific reaction well. Control wells do not include any kinase inhibitors. The minimum Emission Ratio in a screen is established by the 0% Inhibition Control, which contains active kinase. This control is designed to produce < 40% ATP conversion in the Kinase Reaction. (The range of ATP conversion allowed is different for each kinase and set in the linear region.). A known inhibitor control standard curve, 10 point titration, is run for each individual kinase on the same plate as the kinase to ensure the kinase is inhibited within an expected IC50 range previously determined.
[0129] Adapta® assays are run in the linear range determined for each kinase. Full ATP/ADP standard curves are run during validation to define this range. In addition, ATP/ADP standard curves are used to calculate the percent ATP conversion of each sample. SelectScreen® Kinase Profiling Service uses XLfit from IDBS. The ATP/ADP standard curve is fit to model number 205 (sigmoidal dose-response model), shown here as Eqn 2:
Percent Inhibition =
Bottom + (Top - Bottom)/(l + (ICso/[compound concentration])11) Eqn 2 where Bottom and Top are the plateau percent inhibition values at zero and saturating inhibitor concentration, respectively, IC50 is the concentration that gives 50% inhibition, and n is the Hill slope value. The dose response curve is also curve fit to model number 205 (Eqn 2). If the bottom of the curve does not fit between -20% & 20% inhibition, it is set to 0% inhibition. If the top of the curve does not fit between 70% and 130% inhibition, it is set to 100% inhibition.
[0130] PIK3 C A/PIK3 R 1 (pl 10 alpha/p85 alpha) Adapta® assay: The 2X PIK3 C A/PIK3 R 1 (pl 10 alpha/p85 alpha)/PIP2:PS reaction mixture is prepared in 50 mM pH 7.5 HEPES buffer containing 100 mM NaCl, 0.03% CHAPS, 3 mM MgCk, and 1 mM EGTA. The final 10 pL kinase reaction consists of 0.25 - 2 ng PIK3CA/PIK3R1 (pl 10 alpha/p85 alpha) enzyme and 50 pM PIP2:PS in 32.5 mM HEPES pH 7.5, 50 mM NaCl, 0.015% CHAPS, 1.5 mM MgCk, 0.5 mM EGTA. After the 1 hour kinase reaction incubation, 5 pL of detection mix is added.
[0131] PIK3CB/PIK3R1 (pl 10 beta/p85 alpha) Adapta® assay: The 2X PIK3CB/PIK3R1 (pl 10 beta/p85 alpha) / PIP2:PS mixture is prepared in 50 mM pH 7.5 HEPES buffer containing 100 mM NaCl, 0.03% CHAPS, 3 mM MgCk, and 1 mM EGTA. The final 10 pL kinase reaction consists of 6.25 - 60 ng PIK3CB/PIK3R1 (pl 10 beta/p85 alpha) and 50 pM PIP2:PS in 32.5 mM pH 7.5 HEPES buffer containing 50 mM NaCl, 0.015% CHAPS, 1.5 mM MgCk, and 0.5 mM EGTA. After the 1 hour kinase reaction incubation, 5 pL of detection mix is added.
[0132] PIK3CD/PIK3R1 (pl 10 delta/p85 alpha) Adapta® assay: The 2X PIK3 CD/PIK3 R 1 (pl 10 delta/p85 alpha) / PIP2:PS mixture is prepared in 50 mM pH 7.5 HEPES buffer containing 100 mM NaCl, 0.03% CHAPS, 3 mM MgCk, and 1 mM EGTA. The final 10 pL kinase reaction consists of 0.2 - 3 ng PIK3 CD/PIK3 R 1 (pl 10 delta/p85 alpha) and 50 pM PIP2:PS in 32.5 mM pH 7.5 HEPES buffer containing 50 mM NaCl, 0.015% CHAPS, 1.5 mM MgCk, and 0.5 mM EGTA. After the 1 hour kinase reaction incubation, 5 pL of detection mix is added. [0133] PIK3CG (pl 10 gamma) Adapta® assay: The 2X PIK3CG (pl 10 gamma) / PIP2:PS mixture is prepared in 50 mM pH 7.5 HEPES buffer containing 3 mM MgCb and 1 mM EGTA. The final 10 pL kinase reaction consists of 6.25 - 70 ng PIK3CG (pl 10 gamma) and 50 mM PIP2:PS in 32.5 mM pH 7.5 HEPES buffer containing 1.5 mM MgCh and 0.5 mM EGTA. After the 1 hour kinase reaction incubation, 5 pL of detection mix is added.
[0134] Note the lipid substrates are prepared by creating lipid vesicles. In some cases, these vesicles include a carrier lipid, such as phosphatidylserine (PS). In the above assay conditions, “PIP2:PS” refers to large unilamellar vesicles (LETVs) containing five mole percent L-a- phosphatidylinositol-4,5-bisphosphate (PIP2) and ninety -five percent phosphatidylserine. The concentration listed refers only to the PIP2 substrate, not the PS carrier lipid.
[0135] The Z’-LYTE® biochemical assay for mTOR (FRAP) and PLK2 protein kinases are conducted by Invitrogen/Life Technologies and uses a fluorescence-based, coupled-enzyme format that is based on the differential sensitivity of phosphorylated and nonphosphorylated peptides to proteolytic cleavage. The peptide substrate is labeled with 2 fluorophores - 1 at each end, a donor (coumarin) and an acceptor (fluorescein) - that make up a fluorescence resonance energy transfer (FRET) pair. In the primary reaction, the kinase transfers the g-phosphate of ATP to a single tyrosine, serine, or threonine residue in a synthetic FRET peptide. In the secondary reaction, a site-specific protease recognizes and cleaves nonphosphorylated FRET peptides.
Phosphorylation of FRET peptides suppresses cleavage by the development reagent. Cleavage disrupts FRET between the donor and acceptor fluorophores on the FRET -peptide, whereas uncleaved, phosphorylated FRET peptides maintain FRET. A ratiometric method, which calculates the emission ratio of donor emission (coumarin at 445 nm) to acceptor emission (fluorescein at 520 nm) after excitation of the donor fluorophore at 400 nm, was used to quantitate the reaction progress. The extent of phosphorylation of the FRET peptide can be calculated from the emission ratio according to Eqn 3:
Emission Ratio =
[coumarin emission (445 nM)]/[fluorescein emission (520 nM)] Eqn 3
The emission ratio remains low if the FRET peptide is phosphorylated (i.e., no kinase inhibition) and is high if the FRET peptide is nonphosphorylated (i.e., kinase inhibition). The test compounds are screened in 1% DMSO (final) in the well. The detailed protocol used for testing recombinant human mTOR (FRAP) and PLK2 protein kinases are described below. [0136] Z' -LYTE® Assay Controls are made for each individual kinase and are located on the same plate as the kinase. The maximum Emission Ratio is established by the 0% Phosphorylation Control (100% Inhibition Control), which contains no ATP and therefore exhibits no kinase activity. This control yields 100% cleaved peptide in the development reaction. The 100% Phosphorylation Control, which consists of a synthetically phosphorylated peptide of the same sequence as the peptide substrate, is designed to allow for the calculation of percent
phosphorylation. This control yields a very low percentage of cleaved peptide in the development reaction. The 0% Phosphorylation and 100% Phosphorylation Controls allow one to calculate the percent Phosphorylation achieved in a specific reaction well. Control wells do not include any kinase inhibitors. The minimum Emission Ratio in a screen is established by the 0% Inhibition Control, which contains active kinase. This control is designed to produce a 10-50%
phosphorylated peptide in the kinase reaction. A known inhibitor control standard curve, 10-point titration, is run for each individual kinase on the same plate as the kinase to ensure the kinase is inhibited within an expected ICso range previously determined. Additionally, controls are prepared for each concentration of Test Compound assayed. The Development Reaction
Interference is established by comparing the Test Compound Control wells that do not contain ATP versus the 0% Phosphorylation Control (which does not contain the Test Compound). The expected value for a non-interfering compound should be 100%. Any value outside of 90% to 110% is flagged. The Test Compound Fluorescence Interference is determined by comparing the Test Compound Control wells that do not contain the Kinase/Peptide Mixture (zero peptide control) versus the 0% Inhibition Control. The expected value for a non-fluorescence compound should be 0%. Any value > 20% is flagged.
[0137] For analysis of Z’-LYTE® data, SelectScreen® Kinase Profiling Service uses XLfit from IDBS. The dose response curve is curve fit to model number 205 (sigmoidal dose-response model) as described above for Eqn 2. If the bottom of the curve does not fit between -20% & 20% inhibition, it is set to 0% inhibition. If the top of the curve does not fit between 70% and 130% inhibition, it is set to 100% inhibition.
[0138] FRAP1 (mTOR) Z'-LYTE® Assay: The 2X FRAP1 (mTOR) / Ser/Thr 11 mixture is prepared in 50 mM pH 7.5 HEPES buffer containing 0.01% BRIJ-35, 10 mM MnCh, 1 mM EGTA, 2 mM DTT, and 0.02% NaN3. The final 10 pL kinase reaction consists of 11.1 - 56 ng FRAPl (mTOR) enzyme and 2 pM Ser/Thr 11 in 50 mM pH 7.5 HEPES buffer containing 0.01% BRIJ-35, 5 mM MgCh, 5 mM MnCh, 1 mM EGTA, 1 mM DTT, and 0.01% NaN3. After 1 hour kinase reaction incubation, 5 pL of a 1 : 16 dilution of development reagent B is added. [0139] PLK2 Z'-LYTE® Assay: The 2X PLK2 / Ser/Thr 16 mixture is prepared in 50 mM pH 7.5 HEPES buffer containing 0.01% BRIJ-35, 10 mM MgCb, and 1 mM EGTA. The final 10 pL kinase reaction consists of 9.59 - 160 ng PLK2 and 2 pM Ser/Thr 16 in 50 mM pH 7.5 HEPES buffer containing 0.01% BRIJ-35, 10 mM MgCh, and 1 mM EGTA. After 1 hour kinase reaction incubation, 5 pL of a 1 : 16 dilution of development reagent B is added.
[0140] B. Rat Pharmacokinetics
[0141] A pharmacokinetic analysis is conducted in male Sprague-Dawley rats following intravenous and oral administration at 0.3 mg/kg (Doseiv) and 5 mg/kg (Dosepo), respectively, of the test compounds. Animals are fasted overnight and food is returned approximately 4 hours post dose. The dose administration intravenously is formulated in 20% beta-cyclodextrin in 0.05M MSA pH 3 at 0.15 mg/mL and is delivered at a 2 mL/kg dose volume. The dose administered orally is formulated as a suspension in 0.5% methylcellulose at 1 mg/mL and delivered at 5 mL/kg dose volume.
[0142] Blood samples are collected into tubes with K2 EDTA at 0, 0.033, 0.25, 0.5, 1, 3, 8, 24 and 48 hours post intravenous administration and 0, 0.25, 0.5, 1, 3, 6, 8, 24 and 48 hours post oral dose, and are stored on wet ice until processed to plasma by centrifugation (3500 rpm at 5°C) within 30 minutes of collection. Resulting plasma samples are stored at -80°C until bioanalysis.
[0143] Compound levels are quantified from plasma with an LC/MS bioanalytical method following extraction with 75% acetonitrile/25% methanol/0.2% formic acid mixture, under gradient conditions. Pharmacokinetic parameters of Cmax, Tmax, Half-life, AETC, CL and Vss are determined using non-compartmental analysis using Watson software. Bioavailability (%F) is calculated from the expression %F = (AUCPO/AUCIV) x (Doseiv/Dosepo) x 100, where AUCPO and AUCiv are the areas under the concentration-versus-time curves following oral dosing and intravenous dosing, respectively.
[0144] C. Cell Permeability
[0145] Permeability of a given test compound is determined using bidirectional transport studies in LLC-PK1 cells over-transfected to express MDR1. The LLC-PK1-MDR1 cells are seeded at 2.25 x 106 cells/mL into Coming HTS Transwell-96 well permeable support plates (0.4 pm pore size, polycarbonate membrane). Cells are seeded in two plates: plate 1 in the A-B (apical to basolateral) direction and plate 2 in the B-A direction. Fresh media (50 pL) is added to each well and the plates are incubated at 5% CCb/95% air/95% humidity for 7 days. The culture medium is prepared using Invitrogen 199 medium (500 mL), FBS (50 mL), geneticin (5 mL) and colchicine 1.5 mg/mL (50 pL). The medium is replaced from the cell culture at minimum once before initiating the assay.
[0146] Bidirectional transport studies are performed at 37°C in an atmosphere of 5% CO2 in air and 95% humidity. The confluent cell monolayers on Transwell inserts are equilibrated for 30 - 60 minutes with assay medium (Invitrogen 199 medium [500 mL], 10 mM HEPES [0.5 mL] and BSA [5 g]). Following equilibration, 250 pL blank assay medium is added to plate 1 (A-B direction), and 250 pL dosing solution is added to plate 2 (B-A direction). Dosing solution contains 1 or 10 mM of a test compound. Atenolol (a cell monolayer integrity marker) at 10 mM is used as a positive control.
[0147] All culture media, control and test compound solutions are pre-warmed at 37°C prior to addition to the cell culture plates. Following addition of control and test compounds, the cell culture plates are incubated for 2 hours. All control and test compounds are tested in triplicate. Following incubation, 50 pL samples are collected from the top and bottom plates (apical and basolateral, respectively). The samples are mixed with 100 pL cold acetonitrile using a plate shaker for 10 minutes and are centrifuged for 10 minutes at 3000 rpm, 4°C. The supernatants are mixed with the internal standard (IS; 0.2mM buspirone and taurocholic acid) and concentrations of the test compounds are determined using LC-MS/MS.
[0148] Samples are quantified by liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) using a Shimazu LC-ABSciex API4000 QTrap mass spectrometer. Mobile phase A consists of 0.04% formic acid in HPLC grade water and mobile phase B is 0.04% formic acid in acetonitrile. Eight calibration standards are used to construct the standard curve, ranging from 2.29 nM to 5000 nM.
[0149] Permeability A-B and B-A, Recovery %, and ER (efflux ratio) are calculated based on the following equations:
Figure imgf000034_0001
AxtxlO)
(ConcBLx0.25 + ConcAPxl0x0.075)
Recoveiy%A-B =
Dosing Concx0.075
(ConcAPxO.075x10000)
Papp B-A (nm/sec) = (AxtxlO)
(ConcAPx0.075 + ConcBLxl0x0.25)
Recoveiy% B-A =
Dosing Concx0.25 app B-A
app A-B
[0150] In the above equations,
P app A-B apparent permeability A-B
Papp B-A apparent permeability B-A
ConcBL basolateral well concentration
ConcAP apical well concentration
Dosing cone starting concentration
A well surface area (cm2), A = 0.143 cm2
t incubation time (seconds), t = 7200 seconds
[0151] D. Cell Viability Assay
[0152] MDA-MB-361 and PC3 tumor cell lines are maintained in accordance with the supplier (American Type Culture Collection, Rockville, MD). Cells are seeded in 96-well tissue culture microplates at 5,000-20,000 cells per well and cultured for 24 hours prior to addition of test compounds or DMSO (dimethylsulfoxide) vehicle. After 72 hours of treatment, viable cells are detected using CellTiter-Glo® Luminescent Cell Viability (Promega, Madison, WI).
Luminescence is measured on a PHERAstar FS microplate reader (BMG Labtech, Cary, NC). To generate concentration-response curves, cells are treated in duplicate with a range of serial compound dilutions (final DMSO concentration is 0.5%). The percentage of viable cells per well is determined by correcting for background and normalizing against DMSO-treated cells. EC so values for inhibition of cell viability are calculated using XLfit4 Microsoft Excel curve-fitting software.
[0153] E. PC3 Xenograft Study
[0154] Athymic nude mice are inoculated subcutaneously in the right flank with suspensions of the human prostate cancer cell line PC3. When tumors in implanted mice reach the target size of 100-150 mm3, animals are randomized into treatment groups. Treatment and control groups consist of 5 mice each. Tumor volume is measured twice weekly and is calculated using the formula: tumor volume (mm3) = (a c b2)/2, in which“a” is the largest diameter and“b” is smallest diameter of the measured tumor. Animal health is closely monitored and distressed animals are immediately euthanized.
[0155] Test compounds are administered daily by oral gavage in 0.5% carboxymethylcellulose (CMC) at 5 pL per gram body weight. Tumor volumes are measured using digital calipers.
Antitumor efficacy is expressed as mean relative growth of treated versus control tumors (%T/C). This is calculated using the formula: [(T-To)/(C-Co)] x 100, in which C and T are mean control and compound-treated tumor volume and Co and To are initial mean control and compound- treated tumor volume, respectively. For tumor shrinkage, regression percentage was calculated using the formula: [l-(T/To)] x 100, in which T and To are treated and initial tumor volume, respectively. Complete regression (CR) was defined as tumor shrinkage to below measurable size (<13.5 mm3) for 3 consecutive measurements, and partial regression (PR) was defined as reduction to <50% of initial tumor volume and >13.5 mm3 for 3 consecutive measurements.
EXAMPLES
[0156] The following examples are intended to be illustrative and non-limiting, and represent specific embodiments of the present invention.
[0157] 'H Nuclear magnetic resonance (NMR) spectra were obtained for many of the compounds in the following examples. Characteristic chemical shifts (d) are given in parts-per- million downfield from tetramethylsilane using conventional abbreviations for designation of major peaks, including s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad). The following abbreviations are used for common solvents: CDCb (deuterochloroform), DMSO- ck (deuterodimethylsulfoxide), CD3OD (deuteromethanol), CD3CN (deuteroacetonitrile), and THF-i¾ (deuterotetrahydrofuran). The mass spectra (m/z for [M+H]+) were recorded using either electrospray ionization (ESI-MS) or atmospheric pressure chemical ionization (APCI-MS) mass spectrometry.
[0158] Where indicated, products of certain preparations and examples are purified by mass- triggered HPLC (Pump: Waters™ 2525; MS: ZQ™; Software: MassLynx™), flash
chromatography or preparative thin layer chromatography (TLC). Reverse phase chromatography is typically carried out on a column (e.g., Phenomenex Gemini™ 5m, C18, 30 mm x 150 mm; Axia™, 5m, 30 mm x 75 mm) under acidic conditions (“acid mode”) eluting with CFECN and water mobile phases containing 0.035% and 0.05% trifluoroacetic acid (TFA), respectively, or under basic conditions (“basic mode”) eluting with water and 20/80 (v/v) water/acetonitrile mobile phases, both containing 10 mM NH4HCO3. Preparative TLC is typically carried out on silica gel 60 F254 plates. After isolation by chromatography, the solvent is removed and the product is obtained by drying in a centrifugal evaporator (e.g., GeneVac™), rotary evaporator, evacuated flask, etc. Reactions in an inert (e.g., nitrogen) or reactive (e.g., Eh) atmosphere are typically carried out at a pressure of about 1 atmosphere (14.7 psi).
[0159] PREPARATION 1 : 4-amino- l-methylcy cl ohexan-l-ol
Figure imgf000037_0001
[0160] STEP A: /er/-butyl /V-(4-hydroxy-4-methyl-cyclohexyl)carbamate
Figure imgf000037_0004
[0161] A solution of /er/-butyl /V-(4-oxocyclohexyl)carbamate (50.00 g, 234.44 mmol, 1.00 eq) in THF(500 mL) was added drop wise to a solution of CEEMgBr (3 M, 234.44 mL, 3.00 eq) in THF (1000 mL) at -20°C. When addition was completed, the mixture was allowed to warm to 20°C and stirred for another 17 hours. The reaction mixture was then quenched by aqueous NH4CI (300 mL) with cooling in an ice-bath. The mixture was evaporated under reduced pressure to remove most of the THF, and the residue was extracted with ethyl acetate (3 x 300 mL). The combined organic layers were washed with brine (2 x 300 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography on silica gel (Petroleum ether/Ethyl acetate=l0/l to 1/2) to give the title compound as a white solid (4.46 g, 8.30%). ¾ NMR (400 MHz, CDCh) 4.30 (br, 1H, NH), 3.40
(br, 1H, OH), 1.80-1.78 (m, 2H), 1.67-1.64 (m, 2H), 1.52-1.45 (m, 13H), 1.23 (s, 3H), 1.08 (m, 1H).
[0162] STEP B: 4-amino- l-methylcy cl ohexan-l-ol
Figure imgf000037_0002
[0163] To a vial charged with /cvV-butyl /V-(4-hydroxy-4-methyl-cyclohexyl)carbamate (128.3 mg, 0.559 mmol) in ethyl acetate (2.0 mL) was added hydrogen chloride, 4.0 M solution in dioxane (1.049 mL, 4.20 mmol) at 23°C. The mixture was stirred at 23°C for 1 hour to give a suspension. The resulting solid was filtered, rinsed with EtOAc (2 x 1 mL) and hexanes (2 x
1 mL), and dried in vacuo to provide the HC1 salt of the title compound as a light yellow solid
(59.5 mg, 64.2%). ESI-MS m/z [M+H]+ 130.2.
[0164] PREPARATION 2: /tvV-butyl (( 1.v,4.v)-4-hydroxy-4-methyl cyclohexyl (carbamate or 67S_ l-methyl-4-(ter/-butoxycarbonylamino)cy cl ohexan-l-ol
Figure imgf000037_0003
[0165] PREPARATION 3: /ert-butyl ((lr,4r)-4-hydroxy-4-methylcyclohexyl)carbamate or trans- 1 -methyl-4-(/er/-butoxycarbonylamino)cyclohexan- 1 -ol
Figure imgf000038_0002
[0166] To a 200 mL flask charged with /V-4-Boc-aminocyclohexanone (2.0 g, 9.38 mmol) in THF (40 mL) was added, dropwise, methylmagnesium chloride (3M solution in THF, 9.38 mL,
28.1 mmol) at -78°C. The addition of the Grignard reagent was carried out under a nitrogen atmosphere over a 1 hour period using a syringe pump. The mixture was stirred at -78°C for 3 hours and then warmed slowly to 23°C. The reaction mixture was stirred for an additional 11 hours at 23 °C. Next, aqueous NLLCl (20 mL) was added at 0°C to quench the reaction, and the mixture was stirred for an additional 15 minutes. The resulting mixture was concentrated via rotary evaporation and partitioned between DCM (80 mL) and water (40 mL) with the aid of solid citric acid (4.0 g). The organic phase was washed with saturated aqueous NaHCCb (40 mL), water (40 mL), and brine (40 mL), dried over Na2S04, filtered, rinsed with DCM, and concentrated via rotary evaporation. The crude material was dissolved in toluene (2 mL) and purified by medium pressure chromatography (Single Step™ 80 g silica gel column) eluting with a gradient of EtOAc (20-80%) in heptane. The early eluting fractions were combined, concentrated via rotary evaporation, and dried in vacuo to provide the cis stereoisomer, tert- butyl (( 1.v,4.v)-4-hydroxy-4- methylcyclohexyl)carbamate, as a white solid (252.8 mg, 11.76%). 1H MR (400 MHz, CDCb) d ppm 1.23 (s, 3 H), 1.44 (s, 9 H), 1.46 - 1.52 (m, 4 H), 1.60 - 1.69 (m, 2 H), 1.76 - 1.85 (m, 2 H),
3.31 - 3.49 (m, 1 H), 4.33 - 4.54 (m, 1 H). ESI-MS m/z [M+H]+ 156.3, 174.3, 230.3.
[0167] The late eluting fractions were combined, concentrated via rotary evaporation, and dried in vacuo to provide the trans stereoisomer, tert- butyl ((lr,4r)-4-hydroxy-4- methylcyclohexyl)carbamate, as a white solid (136.9 mg, 6.37%). 1H MR (400 MHz, CDCb) d ppm 1.25 (s, 3 H), 1.32 - 1.40 (m, 2 H), 1.45 (s, 9 H), 1.50 - 1.58 (m, 2 H), 1.60 - 1.68 (m, 2 H),
1.87 - 2.01 (m, 2 H), 3.46 - 3.64 (m, 1 H), 4.36 - 4.56 (m, 1 H). ESI-MS m/z [M+H]+ 156.3, 174.3, 230.3.
[0168] PREPARATION 4: ( 1.v,4.v)-4-amino- 1 -methylcyclohexan- 1 -ol or cA-4-amino-l- methylcyclohexan-l-ol
Figure imgf000038_0001
[0169] To a vial charged with /er/-butyl (( 1.v,4.v)-4-hydroxy-4-methyl cyclohexyl (carbamate (249.7 mg, 1.089 mmol) in EtOAc (4.0 mL) was added hydrogen chloride (4.0 M solution in dioxane, 2.042 mL, 8.17 mmol) at 23°C. The mixture was stirred at 23°C for 1.5 hours to give a suspension. The mother liquor was decanted, and the resulting solid was suspended in EtOAc (2.0 mL). The mother liquor was decanted again, and the process was repeated with EtOAc (2.0 mL) and heptane (2 x 2.0 mL). The resulting solid was dried in vacuo to give an HC1 salt of the title compound as a white solid (141 mg, 78%).
[0170] PREPARATION 5: (lr,4r)-4-amino-l-methylcyclohexan-l-ol or /ra//.s-4-amino- l - methylcyclohexan-l-ol
Figure imgf000039_0001
[0171] To a vial charged with /er/-butyl ((lr,4r)-4-hydroxy-4-methylcyclohexyl)carbamate (135.7 mg, 0.592 mmol) in EtOAc (2.0 mL) was added hydrogen chloride (4.0 M solution in dioxane, 1.110 mL, 4.44 mmol) at 23°C. The mixture was stirred at 23°C for 1.5 hours to give a suspension. The resulting solid was filtered, rinsed with EtOAc (2 x 1 mL) and hexanes (2 x 1 mL), and dried in vacuo to give an HC1 salt of the title compound as a white solid (78 mg, 80%).
[0172] PREPARATION 6: methyl f V)-4-(2-chl oro-5 -fl uoropy ri i di n-4-y 1 )-3 - methylmorpholine-3-carboxylate
Figure imgf000039_0002
[0173] A 3 -neck, round bottom, reaction flask equipped with a mechanical stirrer, an addition funnel, and an N2 inlet was charged with fV)-3 -(methoxycarbonyl )-3 -methyl morpholin-4-ium (7,7-dimethyl-2-oxobicyclo[2.2. l]heptan-l-yl)methanesulfonate (148.7 g, 380 mmol), 2,4- dichloro-5-fluoropyrimidine (57.7 g, 345 mmol) and DMSO (691 mL) to give a white suspension. Next, potassium fluoride (26.1 g, 449 mmol) was added at 23°C followed by NN- diisopropylethylamine (150 ml, 863 mmol) from the addition funnel over a 10 minute period. The mixture was stirred at 23 °C for 3 days under nitrogen to give a yellow-orange suspension. The reaction mixture cooled on ice to +l2°C and diluted with saturated aqueous NLLCl (1.4 L), which was added dropwise over a 2-hour period. A gum formed on the stir bar. The gum was taken up in EtOAc, washed with brine and combined with the extracts from the aqueous phase. The aqueous phase was extracted with EtOAc (750 mL), washed with brine (750 mL), dried over MgS04, filtered, rinsed with EtOAc and concentrated in vacuo. The residue was taken up in diethyl ether (150 mL) and concentrated in vacuo. This was repeated three times to provide the title compound (93.6 g, 94%) as a yellow-orange solid. ¾ NMK (400 MHz, DMSO-dis) d ppm 1.55 (s, 3 H), 3.51 - 3.59 (m, 1 H), 3.61 (s, 3 H), 3.68 - 3.81 (m, 3 H), 3.90 - 4.02 (m, 2 H), 8.38 (d, 7=6.06 Hz, 1 H). ESI-MS m/z [M + H]+ 390.1.
[0174] PREPARATION 7: methyl (ri)-4-(5-fluoro-2-(l7/-indol-4-yl)pyrimidin-4-yl)-3- methylmorpholine-3-carboxylate
Figure imgf000040_0001
[0175] A 2 L round-bottomed flask equipped with a reflux condenser and a magnetic stir bar was charged with (k)-methyl 4-(2-chloro-5-fluoropyrimidin-4-yl)-3-methylmorpholine-3- carboxylate (93 g, 321 mmol), 4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-l7/-indole (82 g, 337 mmol), PdCl2(dppf) (7.05 g, 9.63 mmol), and potassium phosphate (76 g, 803 mmol) in dioxane (535 mL) and water (535 mL). The reaction mixture was stirred at 90°C for 18 hours and then cooled to 23°C. The contents of the flask were filtered through Celite™ and the Celite™ was rinsed with EtOAc, which was combined with the filtrate. The filtrate was further diluted with EtOAc, washed with saturated aqueous NaHC03, saturated aqueous NH4Q, and brine, and then dried over MgS04. The product-containing mixture was filtered and the solvent removed in vacuo. The residue was treated with diethyl ether (200 mL) and concentrated in vacuo. The diethyl ether wash was repeated three times and concentrated in vacuo to give the title compound as a brown solid (119 g, 100%). ¾ NMR (400 MHz, DMSO-7e) d ppm 1.70 (s, 3 H), 3.51 (s, 3 H), 3.54 - 3.63 (m, 1 H), 3.70 - 3.76 (m, 1 H), 3.78 - 3.88 (m, 2 H), 3.91 - 4.02 (m, 2 H), 7.19 (t, .7=7.71 Hz, 1 H), 7.37 (d, 7=2.02 Hz, 1 H), 7.44 (t, 7=2.78 Hz, 1 H), 7.54 (d, 7=7.83 Hz, 1 H),
7.86 (d, 7=7.07 Hz, 1 H), 8.60 (d, 7=5.81 Hz, 1 H), 11.28 (br s, 1 H). ESI-MS m/z [M + H]+
371.3.
[0176] PREPARATION 8: (ri)-4-(5-fluoro-2-(l77-indol-4-yl)pyrimidin-4-yl)-3- methylmorpholine-3 -carboxylic acid
Figure imgf000041_0001
[0177] A 2 L round-bottomed flask equipped with a magnetic stir bar was charged with (S)- methyl 4-(5-fluoro-2-(l77-indol-4-yl)pyrimidin-4-yl)-3-methylmorpholine-3-carboxylate (119 g, 321 mmol) and aqueous Li OH (803 mL, 1606 mmol) in dioxane (800 mL). The reaction mixture was stirred at l00°C for 18 hours, then concentrated in vacuo to remove dioxane, diluted with water (500 mL), and extracted with iPrOAc (3 x 250 mL). The aqueous layer was brought to pH 4 with 1 M HC1 (750 mL). The resulting solid was isolated by filtration, washed with water, and dried on a Buchner funnel to give Crop-l of the title compound as a brown solid (7.0 g). The aqueous acidic filtrate still contained product, which was subsequently extracted with EtOAc (3 x 200 mL). The organic extract was dried with MgS04, filtered, concentrated and dried in vacuo to give Crop-2 as a yellow solid (12.5 g). During the first precipitation, a black sludge formed around the stir bar. This material was re-suspended in 1N LiOH aq (500 mL) and neutralized to pH 4 a second time with 1N HC1 aq to give Crop-3 as a brown solid, which was isolated by filtration (87.6 g). The aqueous acidic filtrate was extracted with EtOAc to give additional product that was combined with Crop-2. All crops were combined and dried in a vacuum oven at 50°C to provide the title compound. ¾ NMR (400 MHz, DMSO-Tis) d ppm 1.66 (s, 3 H), 3.49 -
3.61 (m, 1 H), 3.69 (d, .7=11.12 Hz, 1 H), 3.77 - 3.88 (m, 2 H), 3.88 - 4.00 (m, 2 H), 7.15 (t, .7=7.71 Hz, 1 H), 7.36 (br s, 1 H), 7.42 (t, .7=2.65 Hz, 1 H), 7.52 (d, 7=8.08 Hz, 1 H), 7.95 (d, 7=7.33 Hz, 1 H), 8.57 (d, 7=6.06 Hz, 1 H), 11.24 (br s, 1 H), 12.67 (br s, 1 H). ESI-MS: m/z 357.3
[M+H]+.
[0178] PREPARATION 9: (ri)-4-(5-fluoro-2-(l77-indol-4-yl)pyrimidin-4-yl)-/V-(4-hydroxy-4- methylcyclohexyl)-3-methylmorpholine-3 -carboxamide
Figure imgf000041_0002
[0179] To a solution of (ri)-4-(5-fluoro-2-(li7-indol-4-yl)pyrimidin-4-yl)-3-methylmorpholine- 3-carboxylic acid (47 mg, 0.132 mmol), 4-amino- l-methylcy cl ohexanol hydrochloride (32.8 mg, 0.198 mmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (37.9 mg, 0.198 mmol), and HOBt (20.20 mg, 0.132 mmol) in DMF (0.35 mL) was added /V,/V- diisopropylethylamine (0.069 mL, 0.396 mmol) at 23°C. The reaction mixture was stirred at 23°C for 5 days, then partitioned between EtOAc (1.5 mL) and saturated NLLCl (0.75 mL). The aqueous and organic layers were separated. The aqueous phase was washed with EtOAc (2 x 1.5 mL) and the organic extracts were combined. The crude product was extracted from the organic phase with 1N HC1 aq (6 x 0.5 mL). The aqueous extracts were combined and basified to a pH of about 5-6 with a slow addition of solid NaHC03 (0.30 g). The product was extracted from the aqueous phase with EtOAc (5 x 0.5 mL). The organic extracts were combined, washed with saturated aqueous NaHC03 (0.5 mL) and brine (0.5 mL), dried over MgS04, filtered, rinsed with EtOAc, and dried in vacuo to provide the title compound as an orange oil (50 mg, 81%). ESI-MS m/z [M+H]+ 468.4.
[0180] PREPARATION 10: (ri)-4-(5-fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-A-((n,4i?)-4- hydroxy-4-methylcyclohexyl)-3-methylmorpholine-3 -carboxamide or (S)-cis- 1 -methyl-4-(4-(5- fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-3-methylmorpholin-3-carbonylamino)-cyclohexan-l-ol
Figure imgf000042_0001
[0181] PREPARATION 11 : (ri)-4-(5-fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-A-((lr,4ri)-4- hydroxy-4-methylcyclohexyl)-3-methylmorpholine-3 -carboxamide or (S)-trans- l-methyl-4-(4-(5- fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-3-methylmorpholin-3-carbonylamino)-cyclohexan-l-ol
Figure imgf000042_0002
[0182] To a solution of (ri)-4-(5-fluoro-2-(l77-indol-4-yl)pyrimidin-4-yl)-3-methylmorpholine- 3-carboxylic acid (81 mg, 0.227 mmol), 4-amino- l-methylcy cl ohexanol hydrochloride (56.5 mg, 0.341 mmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (65.4 mg, 0.341 mmol), and HOBt (34.8 mg, 0.227 mmol) in DMF (0.4 mL) was added /V,/V- diisopropylethylamine (0.119 mL, 0.682 mmol) at 23°C. The reaction mixture was stirred at 23°C for 23 hours, partitioned between EtOAc (3.0 mL) and saturated aqueous NLLCl (1.5 mL), and the layers were separated. The aqueous phase was washed with EtOAc (2 x 3.0 mL) and the organic extracts were combined. The crude product was extracted from the organic phase with 1N HC1 aq (5 x 1.0 mL). The aqueous extracts were combined and basified to pH=6-7 with a slow addition of solid NaHC03 (0.42 g). The product was extracted from the aqueous phase with EtOAc (5 x 1.0 mL). The organic extracts were combined, washed with saturated NaHC03 (1.0 mL) and brine (1.0 mL), dried over MgS04, filtered, rinsed with EtOAc and dried in vacuo to provide the crude product as an orange oil. The crude product was combined with the organic extract from Preparation 9. The combined extracts were dissolved in DCM (~l mL) and purified by medium pressure chromatography (Moritex Purif-Pack™ NH, 60 mM, size 20 column) eluting with a gradient of 0-5% MeOH in DCM. The early fractions were combined and dried in vacuo to give the cis stereoisomer, (ri)-4-(5-fluoro-2-(l77-indol-4-yl)pyrimidin-4-yl)-/V-((n,47?)-4- hydroxy-4-methylcyclohexyl)-3-methylmorpholine-3 -carboxamide as a light yellow foam (71.8 mg, 67.6%). ¾ NMR (400 MHz, DMSO-i¾) d ppm 0.97 (s, 3 H), 1.00 - 1.05 (m, 1 H), 1.09 - 1.25 (m, 3 H), 1.29 - 1.39 (m, 1 H), 1.41 - 1.47 (m, 1 H), 1.55 - 1.62 (m, 1 H), 1.64 (s, 3 H), 1.67 -
I .71 (m, 1 H), 3.40 - 3.53 (m, 2 H), 3.57 (d, .7=11.12 Hz, 1 H), 3.79 (d, .7=11.37 Hz, 1 H), 3.87 (s,
1 H), 3.89 - 4.04 (m, 3 H), 7.12 (t, =7.83 Hz, 1 H), 7.34 - 7.42 (m, 2 H), 7.46 (d, =8.34 Hz, 1 H), 7.49 (dt, =8.08, 0.88 Hz, 1 H), 8.01 (dd, =7.58, 1.01 Hz, 1 H), 8.52 (d, =6.32 Hz, 1 H),
I I .21 (br s, 1 H). ESI-MS m/z [M+H]+ 468.4.
[0183] The later fractions were combined and dried in vacuo to provide the trans stereoisomer, (ri)-4-(5-fluoro-2-(l77-indol-4-yl)pyrimidin-4-yl)-/V-((lr,4ri)-4-hydroxy-4-methylcyclohexyl)-3- methylmorpholine-3 -carboxamide as a light yellow foam (31.9 mg, 30.0%). ¾ NMR (400 MHz, DMSO-£¾) d ppm 0.95 (s, 3 H), 0.98 - 1.04 (m, 2 H), 1.10 - 1.16 (m, 1 H), 1.22 - 1.42 (m, 4 H), 1.50 - 1.55 (m, 1 H), 1.65 (s, 3 H), 3.43 - 3.55 (m, 2 H), 3.58 (d, .7=11.37 Hz, 1 H), 3.78 (d,
.7=11.12 Hz, 1 H), 3.84 - 4.01 (m, 3 H), 4.08 (s, 1 H), 7.13 (t, .7=7.83 Hz, 1 H), 7.31 - 7.38 (m, 2 H), 7.39 - 7.42 (m, 1 H), 7.50 (dt, =7.96, 0.95 Hz, 1 H), 7.98 (dd, =7.45, 0.88 Hz, 1 H), 8.54 (d, J=6.06 Hz, 1 H), 11.22 (br s, 1 H). ESI-MS m/z [M+H]+ 468.4. [0184] PREPARATION 12: (ri)-4-(5-fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-A-((n,4i?)-4- hydroxy-4-methylcyclohexyl)-3-methylmorpholine-3 -carboxamide or (S)-cis- 1 -methyl-4-(4-(5- fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-3-methylmorpholin-3-carbonylamino)-cyclohexan-l-ol
Figure imgf000044_0001
[0185] To a solution of (ri)-4-(5-fluoro-2-(li7-indol-4-yl)pyrimidin-4-yl)-3-methylmorpholine- 3-carboxylic acid (197 mg, 0.553 mmol), (lr,4r)-4-amino-l-methylcyclohexanol hydrochloride (137 mg, 0.829 mmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (159 mg, 0.829 mmol), and HOBt (85 mg, 0.553 mmol) in DMF (1.0 mL) was added /V,/V- diisopropylethylamine (0.289 mL, 1.658 mmol) at 23°C. The reaction mixture was stirred at 23°C for 22 hours and then partitioned between EtOAc (7 mL) and saturated aqueous NLLCl (3.5 mL). The aqueous and organic layers were separated. The aqueous phase was washed with EtOAc (2 x 7 mL) and the organic extracts were combined. The crude product was extracted from the organic phase with 1N HC1 aq (6 x 2 mL). The aqueous extracts were combined and basified to pH 8 with a slow addition of solid NaHC03 (1.02 g). The product was extracted from the aqueous phase with EtOAc (5 x 2 mL). The organic extracts were combined, washed with saturated aqueous NaHC03 (2 mL) and brine (2 mL), dried over MgS04, filtered, rinsed with EtOAc, and dried in vacuo to give the title compound as an orange oil (234.5 mg, 91%). ESI-MS m/z [M+H]+ 468.5.
[0186] PREPARATION 13: (ri)-4-(5-fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-A-((lr,4ri)-4- hydroxy-4-methylcyclohexyl)-3-methylmorpholine-3 -carboxamide or (S)-trans- l-methyl-4-(4-(5- fluoro-2-(liT-indol-4-yl)pyrimidin-4-yl)-3-methylmorpholin-3-carbonylamino)-cyclohexan-l-ol
Figure imgf000044_0002
[0187] To a solution of (ri)-4-(5-fluoro-2-(li7-indol-4-yl)pyrimidin-4-yl)-3-methylmorpholine- 3-carboxylic acid (109 mg, 0.306 mmol), (lr,4r)-4-amino-l-methylcyclohexanol hydrochloride (76 mg, 0.459 mmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (88 mg, 0.459 mmol), and HOBt (46.8 mg, 0.306 mmol) in DMF (0.55 mL) was added /V,/V- diisopropylethylamine (0.160 mL, 0.918 mmol) at 23°C. The reaction mixture was stirred at 23°C for 22 hours and then partitioned between EtOAc (4 mL) and saturated aqueous NLLCl (2 mL). The aqueous and organic layers were separated. The aqueous phase was washed with EtOAc (2 x 4 mL) and the organic extracts were combined. The crude product was extracted from the organic phase with 1N HC1 aq (6 x 1.5 mL). The aqueous extracts were combined and basified to pH 7with a slow addition of solid NaHC03 (0.76 g). The product was extracted from the aqueous phase with EtOAc (5 x 1.5 mL). The organic extracts were combined, washed with saturated aqueous NaHC03 (1.5 mL) and brine (1.5 mL), dried over MgS04, filtered, rinsed with EtOAc, and dried in vacuo to give the title compound as an orange oil (143 mg, 100%). ESI-MS m/z
[M+H]+ 468.4.
[0188] EXAMPLE 1 : S)-5-((H,4f?)-4-hydroxy-4-methylcyclohexyl)-2-(li7-indol-4-yl)-6a- methyl-6a,7,9, l0-tetrahydro-[l,4]oxazino[3,4-/z]pteridin-6(5i7)-one or (S)-cis- 1 -m ethyl -4-(2-( 1 H- indol-4-yl)-6a-methyl-6(5F/)-oxo-6a,7,9,l0-tetrahydro-[l,4]oxazino[3,4-/i]pteridin-5-yl)- cyclohexan-l-ol
Figure imgf000045_0001
[0189] To a solution of (ri)-4-(5-fluoro-2-(li7-indol-4-yl)pyrimidin-4-yl)-/V-((n,4f?)-4-hydroxy- 4-methylcyclohexyl)-3-methylmorpholine-3 -carboxamide (67.3 mg, 0.144 mmol, Preparation 10) in DMA (1.5 mL) was added potassium /ert-butoxide (1.0 M in THF) (0.432 mL, 0.432 mmol) at 40°C in one portion. The reaction mixture was stirred at 40°C for 30 minutes and then cooled to 23°C. Acetic acid (0.021 mL, 0.360 mmol) was added to quench the reaction. The resulting crude material was reconstituted in DMSO and filtered through a hydrophilic PTFE 0.45 pm filter (Millipore Millex-LCR™). The filter was rinsed with DMSO. The combined rinse solution and filtrate was purified by preparative mass-triggered LCMS, eluting with a gradient of 15-40%
ACN in water (0.05% TFA). The fractions were collected and combined, and the solvent was removed via rotary evaporation. The resulting mixture was lyophilized to give a TFA salt of the title compound as a yellow solid (32.7 mg, 40.5%). ¾ NMR (400 MHz, DMSO- is) d ppm 1.16 (s, 3 H), 1.33 (s, 3 H), 1.36 - 1.59 (m, 4 H), 1.61 - 1.70 (m, 2 H), 2.55 - 2.67 (m, 2 H), 3.30 - 3.39 (m, 1 H), 3.59 - 3.68 (m, 1 H), 3.70 (d, J=l 1.87 Hz, 1 H), 3.99 (d, J=l 1.37 Hz, 1 H), 4.08 - 4.19 (m, 2 H), 4.49 - 4.57 (m, 1 H), 7.21 (t, =7.83 Hz, 1 H), 7.33 (dd, J=3.03, 1.26 Hz, 1 H), 7.48 (t, J=2.65 Hz, 1 H), 7.56 (d, =8.08 Hz, 1 H), 8.03 (d, =7.33 Hz, 1 H), 8.55 (s, 1 H), 11.33 (br s, 1 H). ESI-MS m/z [M+H]+ 448.4.
[0190] EXAMPLE 2: S)-5-((lr,4ri)-4-hydroxy-4-methylcyclohexyl)-2-(li7-indol-4-yl)-6a- methyl-6a,7,9, l0-tetrahydiO-[l,4]oxazino[3,4-/z]pteridin-6(5i7)-one or (S)-/rans- \ -methyl-4-(2- (li7-indol-4-yl)-6a-methyl-6(5F/)-oxo-6a,7,9, l0-tetrahydro-[l,4]oxazino[3,4-/i]pteridin-5-yl)- cyclohexan-l-ol
Figure imgf000046_0001
[0191] To a solution of (ri)-4-(5-fluoro-2-(li7-indol-4-yl)pyrimidin-4-yl)-/V-((lr,4X)-4-hydroxy- 4-methylcyclohexyl)-3-methylmorpholine-3 -carboxamide (30.4 mg, 0.065 mmol, Preparation 11) in DMA (0.75 mL) was added potassium /er/-butoxide (1.0 M in THF) (0.195 mL, 0.195 mmol) at 40°C in one portion. The reaction mixture was stirred at 40°C for 30 minutes and then cooled to 23°C. Acetic acid (9.31 pL, 0.163 mmol) was added to quench the reaction. The resulting crude material was reconstituted in DMSO and filtered through a hydrophilic PTFE 0.45 pm filter (Millipore Millex-LCR™). The filter was rinsed with DMSO. The combined rinse solution and filtrate was purified by preparative mass-triggered LCMS, eluting with a gradient of 15-40%
ACN in water (0.05% TFA). The fractions collected and combined, and the solvent was removed via rotary evaporation. The resulting mixture was lyophilized to give a TFA salt of the title compound as a yellow solid (20.5 mg, 56.1%). ¾ NMR (400 MHz, DMSO- is) d ppm 1.24 (s, 3 H), 1.31 (s, 3 H), 1.49 - 1.72 (m, 6 H), 2.30 - 2.43 (m, 1 H), 2.52 - 2.62 (m, 1 H), 3.29 - 3.38 (m, 1 H), 3.59 - 3.70 (m, 2 H), 3.97 (d, =l l .62 Hz, 1 H), 4.08 - 4.22 (m, 3 H), 7.21 (t, =7.7l Hz, 1 H), 7.32 - 7.35 (m, 1 H), 7.48 (t, =2.78 Hz, 1 H), 7.55 (d, J=8.08 Hz, 1 H), 8.01 - 8.05 (m, 1 H), 8.54 (s, 1 H), 11.32 (br s, 1 H). ESI-MS m/z [M+H]+ 448.4.
[0192] EXAMPLE 3 : fV)-5-(( l .s,4A>)-4-hydroxy-4-methylcyclohexyl)-2-( l //-indol-4-yl)-6a- methyl-6a,7,9, l0-tetrahydro-[l,4]oxazino[3,4-/2]pteridin-6(5F/)-one or (S)-cis- 1 -m ethyl -4-(2-( 1 H- indol-4-yl)-6a-methyl-6(5F/)-oxo-6a,7,9, l0-tetrahydro-[l,4]oxazino[3,4-/i]pteridin-5-yl)- cyclohexan-l-ol
Figure imgf000047_0001
[0193] To a solution of (ri)-4-(5-fluoro-2-(li7-indol-4-yl)pyrimidin-4-yl)-/V-((n,4f?)-4-hydroxy- 4-methylcyclohexyl)-3-methylmorpholine-3 -carboxamide (230.3 mg, 0.493 mmol, Preparation 12) in DMA (2.0 mL) was added potassium /ert-butoxide (1.0 M in THF, 1.478 mL, 1.478 mmol) at 40°C in one portion. The reaction mixture was stirred at 40°C for 30 minutes and then cooled to 23°C. Acetic acid (0.070 mL, 1.231 mmol) was added to quench the reaction. The resulting crude material was reconstituted in DMSO (1 mL) and filtered through a hydrophilic PTFE 0.45 pm filter (Millipore Millex-LCR™). The filter was rinsed with DMSO (2 x 0.5 mL). The combined rinse solution and filtrate was purified by preparative mass-triggered LCMS, eluting with a gradient of 15-40% ACN in water (0.05% TFA). The fractions were collected and combined, and the solvent was removed via rotary evaporation. The resulting mixture was lyophilized to provide a TFA salt of the title compound as a yellow-orange solid. The TFA salt was dissolved in MeOH (5.0 mL) and the solution was passed through a 500 mg VariPure™ IPE cartridge (HCO3MP) to remove the TFA. The cartridge was rinsed with MeOH (3 x 2 mL). The combined rinse solution and filtrate was concentrated via rotary evaporation. The concentrate was dried in vacuo to give a tan solid. The tan solid was re-suspended in EtOH (3 mL), heated at reflux for 5 minutes, and cooled to 23 °C. The solids were filtered, rinsed with EtOH (3 x 1 mL), and dried in vacuo to give an off-white solid. Since the EtOH filtrate contained a significant amount of product, the filtrate was dried in vacuo and the residue was re-suspended in EtOH (0.5 mL), heated at reflux for 5 minutes, and then cooled to 23°C. The mixture was filtered. The solids were rinsed with EtOH (3 x 1 mL), combined with the first crop of solids, and dried in vacuo to give the title compound as an off-white solid (71.3 mg, 32.3%). 'H NMR (400 MHz, DMSO-r/r,) d ppm 1.16 (s, 3 H), 1.28 (s, 3 H), 1.31 - 1.39 (m, 1 H), 1.40 - 1.59 (m, 3 H), 1.60 - 1.72 (m, 2 H), 2.52 - 2.68 (m, 2 H), 3.25 - 3.33 (m, 1 H), 3.60 - 3.66 (m, 1 H), 3.68 (d, J=\ 1.37 Hz, 1 H), 3.99 (d, =l l.37 Hz, 1 H), 4.05 - 4.15 (m, 2 H), 4.33 (s, 1 H), 4.48 - 4.61 (m, 1 H), 7.18 (t, 7=7.71 Hz,
1 H), 7.36 - 7.40 (m, 1 H), 7.44 (t, =2.78 Hz, 1 H), 7.51 (dt, J=8.02, 0.92 Hz, 1 H), 8.09 (dd, =7.58, 1.01 Hz, 1 H), 8.61 (s, 1 H), 11.25 (br s, 1 H). ESI-MS m/z [M+H]+ 448.3. Melting point, 290.0 - 291.2°C (decomposed). [0194] EXAMPLE 4: S)-5-((lr,4ri)-4-hydroxy-4-methylcyclohexyl)-2-(l77-indol-4-yl)-6a- methyl-6a,7,9, l0-tetrahydro-[l,4]oxazino[3,4-/2]pteridin-6(57/)-one or (S)-/rans- \ -methyl-4-(2- (l77-indol-4-yl)-6a-methyl-6(57/)-oxo-6a,7,9, l0-tetrahydro-[l,4]oxazino[3,4-/i]pteridin-5-yl)- cyclohexan-l-ol
Figure imgf000048_0001
[0195] To a solution of (ri)-4-(5-fluoro-2-(l77-indol-4-yl)pyrimidin-4-yl)-/V-((lr,4ri)-4-hydroxy- 4-methylcyclohexyl)-3-methylmorpholine-3 -carboxamide (222.6 mg, 0.476 mmol, Preparation 13) in DMA (2.0 mL) was added potassium /er/-butoxide (1.0 M in THF, 1.428 mL, 1.428 mmol) at 40°C in one portion. The reaction mixture was stirred at 40°C for 30 minutes and then cooled to 23°C. Acetic acid (0.068 mL, 1.190 mmol) was added to quench the reaction. The resulting crude material was reconstituted in DMSO (1 mL) and filtered through a hydrophilic PTFE 0.45 pm filter (Millipore Millex-LCR™). The filter was rinsed with DMSO (2 x 0.5 mL). The combined rinse solution and filtrate was purified by preparative mass-triggered LCMS, eluting with a gradient of 15-40% ACN in water (0.05% TFA). The fractions were collected and combined, and the solvent was removed via rotary evaporation. The resulting mixture was lyophilized to give a TFA salt of the title compound as a yellow solid. The TFA salt was dissolved in MeOH (5.0 mL) and passed through a 500 mg VariPure™ IPE cartridge (HCO3MP) to remove the TFA. The cartridge was rinsed with MeOH (3 x 2 mL). The combined rinse solution and filtrate was concentrated via rotary evaporation and dried in vacuo to give the product as an off-white solid. The off-white solid was re-suspended in EtOH (3 mL), heated at reflux for 5 minutes, and cooled to 23°C. The solids were filtered, rinsed with EtOH (3 x 1 mL), and dried in vacuo to give the title compound as an off-white solid (106.7 mg, 50.1%). 'H NMR (400 MHz, DMSO-£¾) d ppm 1.24 (s, 3 H), 1.26 (s, 3 H), 1.47 - 1.75 (m, 6 H), 2.30 - 2.40 (m, 1 H), 2.52 - 2.60 (m, 1 H), 3.24 - 3.33 (m, 1 H), 3.59 - 3.69 (m, 2 H), 3.97 (d, .7=11.37 Hz, 1 H), 4.04 - 4.14 (m, 2 H), 4.19 (t, .7=12.13 Hz, 1 H), 4.43 (s, 1 H), 7.18 (t, =7.83 Hz, 1 H), 7.37 - 7.41 (m, 1 H), 7.42 - 7.46 (m, 1 H), 7.51 (dt, =8.02, 0.92 Hz, 1 H), 8.09 (dd, =7.58, 1.01 Hz, 1 H), 8.56 (s, 1 H), 11.25 (br s, 1 H). ESI-MS m/z [M+H]+ 448.3. Melting point, 278.6 - 280.8°C (decomposed). [0196] Table 1 lists enzyme inhibition data and Table 2 lists rat pharmacokinetics and cell permeability for compounds shown in Examples 3 and 4 and for comparator compounds A, B, C, and D, which are described below. Each compound was tested in accordance with the assays for in vitro kinase inhibition, rat pharmacokinetics and cell permeability described in the Biological Activity section, above. In Table 1 smaller ICso corresponds to greater potency. In Table 2 larger %F and Papp A-B correspond to greater bioavailability and cell permeability, respectively, and smaller efflux ratio (ER) corresponds to lower cell efflux. Generally, an ER greater than or equal to 2 indicates compound efflux is occurring.
TABLE 1. Enzyme Inhibition, ICso (nM)
Figure imgf000049_0001
TABLE 2. Rat Pharmacokinetics and Cell Permeability
Figure imgf000049_0002
[0197] Tables 3 and 4 compare antitumor activity of the compounds in cell lines and a xenograft mouse model. Table 3 lists cell viability of carcinoma cell lines PC3 and MDA-MB- 361 following treatment with compounds of Examples 3 or 4 or with comparator compounds A, B, C or D. Each compound was tested in accordance with the assay for cell viability described in the Biological Activity section, above. In Table 3, smaller ECso corresponds to greater efficacy. Table 4 lists the mean relative growth of treated tumors versus untreated (control) tumors (%T/C) for the compound of Example 3 and comparator compounds A, B, and C. Each compound was tested in accordance with the xenograft study procedure described in the Biological Activity section, above. In Table 4, better efficacy is indicated by lower values of %T/C or by larger regression percentage.
TABLE 3. Cell Viability in PC3 and MDA-MB-361 Cell Lines
Figure imgf000050_0001
TABLE 4. Mean relative growth of treated tumors versus untreated (control) tumors (%T/C) or regression percentage in human PC3 xenograft mouse model
Figure imgf000050_0002
[0198] The drawing shows the dose response of a human PC3 xenograft mouse model following treatment with the compound prepared in Example 3 or with comparator compounds A, B or C. At a given dose, mice treated with the compound of Example 3 exhibit slower tumor growth than mice treated with comparator compounds A, B or C. At a dose of 10 mg/Kg, mice treated with the compound of Example 3 exhibited tumor shrinkage (regression).
[0199] In the above assays, comparator compounds A, B, C, and D correspond to Examples 17 (S-stereoisomer), 26, 33, and 45 (S-stereoisomer), respectively, described in international patent application PCT/US2010/046839 (published as WO 2011/025889A1). [0200] COMPOUND A: (,S)-l-(4-(5-cyclopropyl-6a-methyl-6-oxo-5,6,6a,7,9,l0-hexahydro- [l,4]oxazino[3,4-/z]pteridin-2-yl)phenyl)-3-methylurea
Figure imgf000051_0001
[0201] COMPOUND B: (L')-I -methyl -3 -(4-(6a-methyl-6-oxo-5-(tetrahydro2//-pyran-4-yl)- 5,6,6a,7,9, l0-hexahydro-[l,4]oxazino[3,4-/z]pteridin-2-yl)phenyl)urea
Figure imgf000051_0002
[0202] COMPOUND C: (L')- I -cyclopropyl-3-(4-(6a-methyl-6-oxo5-(tetrahydro-2//-pyran-4- yl)-5,6,6a,7,9, l0-hexahydro-[l,4]oxazino[3,4-/2]pteridin-2-yl)phenyl)urea
Figure imgf000051_0003
[0203] COMPOUND D: (S)- 1 -cy d opropy 1 -3 -(4- [5 -( 1 , 1 -di oxi dotetrahy dro-2//-thi opy ran-4-y 1 )- 6a-methyl-6-oxo-5,6,6a,7,9,l0-hexahydro[l,4]oxazino[3,4-/2]pteridin-2-yl]phenyl)urea
Figure imgf000051_0004

Claims

WHAT IS CLAIMED IS:
1. A compound of F ormul a 1 ,
Figure imgf000052_0001
or a pharmaceutically acceptable salt thereof.
2. The compound according to claim 1, which is represented by the formula,
Figure imgf000052_0002
or a pharmaceutically acceptable salt thereof.
3. The compound according to claim 1, which is represented by the formula,
Figure imgf000052_0003
or a pharmaceutically acceptable salt thereof.
4. A pharmaceutical composition comprising:
a compound or pharmaceutically acceptable salt as defined in any one of claims 1 to 3; and
a pharmaceutically acceptable excipient.
5. A compound or pharmaceutically acceptable salt as defined in any one of claims 1 to 3 for use as a medicament.
6. A compound or pharmaceutically acceptable salt as defined in any one of claims 1 to 3 for use in the treatment of a disease, disorder or condition selected from immunological disorders, cancer, and neurodegenerative diseases.
7. A method of treating a disease, disorder or condition in a subject, the method comprising administering to the subject a compound or pharmaceutically acceptable salt as defined in any one of claims 1 to 3, wherein the disease, disorder or condition is selected from immunological disorders, cancer, and neurodegenerative diseases.
8. A combination comprising a compound or pharmaceutically acceptable salt as defined in any one of claims 1 to 3, and at least one additional pharmacologically active agent.
9. The combination according to claim 8, wherein the additional pharmacologically active agent is an anti-cancer agent.
PCT/US2019/055045 2018-10-08 2019-10-07 SUBSTITUTED OXAZINOPTERIDINONES AS INHIBITORS OF mTOR WO2020076728A1 (en)

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