WO2012154610A1 - Inhibiteurs réactifs de la pi3 kinase et leurs utilisations - Google Patents

Inhibiteurs réactifs de la pi3 kinase et leurs utilisations Download PDF

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Publication number
WO2012154610A1
WO2012154610A1 PCT/US2012/036664 US2012036664W WO2012154610A1 WO 2012154610 A1 WO2012154610 A1 WO 2012154610A1 US 2012036664 W US2012036664 W US 2012036664W WO 2012154610 A1 WO2012154610 A1 WO 2012154610A1
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aryl
ioalkyl
heteroaryl
ioalkynyl
alkyl
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PCT/US2012/036664
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English (en)
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Pingda Ren
Yi Liu
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Intellikine, Llc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • kinases signaling pathways play a central role in numerous biological processes. Defects in various components of signal transduction pathways have been found to account for a vast number of diseases, including numerous forms of cancer, inflammatory disorders, metabolic disorders, vascular and neuronal diseases (Gaestel et al. Current Medicinal Chemistry (2007) 14:2214-2234). In recent years, kinases that are associated with oncogenic signaling pathways have emerged as important drug targets in the treatment of various diseases including many types of cancers.
  • PI 3-kinases Phosphatidylinositol 3-kinases
  • PI3Ks Phosphatidylinositol 3-kinases
  • These lipid kinases phosphorylate the 3-position hydroxyl group of the inositol ring of phosphatidylinositol (Ptdlns), activating signaling cascades associated with such processes as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking.
  • Disruption of these processes involing PI3K leads to many diseases including cancer, allergic contact dermatitis, rheumatoid arthritis, osteoarthritis, inflammatory bowel diseases, chronic obstructive pulmonary disorder, psoriasis, multiple sclerosis, asthma, disorders related to diabetic complications, and inflammatory
  • the PI3K family comprises 15 kinases with distinct substrate specificities, expression patterns, and modes of regulation.
  • the class I PI3Ks (pi 10a, pi 10 ⁇ , pi 10 ⁇ , and pi 10 ⁇ ) are typically activated by tyrosine kinases or G-protein coupled receptors to generate phosphatidylinositol-3,4,5- trisphosphate (PIP 3 ), which engages downstream effectors such as those in the Akt/PDKl pathway, ⁇ , the Tec family kinases, and the Rho family GTPases.
  • the alpha (a) isoform of type I PI3K has been implicated in a variety of human cancers.
  • Angiogenesis has been shown to selectively require the a isoform of PI3K in the control of endothelial cell migration. (Graupera et al, Nature 2008;453;662-6). Mutations in the gene coding for PI3K a or mutations which lead to upregulation of PI3K a are believed to occur in many human cancers such as lung, stomach, endometrial, ovarian, bladder, breast, colon, brain and skin cancers.
  • mutations in the gene coding for PI3K a are point mutations clustered within several hotspots in helical and kinase domains, such as E542K, E545K, and H1047R. Many of these mutations have been shown to be oncogenic gain- of- function mutations. While other PI3K isoforms such as PI3K ⁇ or PI3K ⁇ are expressed primarily in hematopoietic cells, PI3K a, along with PI3K ⁇ , is expressed constitutively. [0006] The delta ( ⁇ ) isoform of class I PI3K has been implicated, in particular, in a number of diseases and biological processes.
  • PI3K ⁇ is expressed primarily in hematopoietic cells including leukocytes such as T-cells, dendritic cells, neutrophils, mast cells, B-cells, and macrophages.
  • PI3K ⁇ is integrally involved in mammalian immune system functions such as T-cell function, B-cell activation, mast cell activation, dendritic cell function, and neutrophil activity. Due to its integral role in immune system function, PI3K ⁇ is also involved in a number of diseases related to undesirable immune response such as allergic reactions, inflammatory diseases, inflammation mediated angiogenesis, rheumatoid arthritis, auto-immune diseases such as lupus, asthma, emphysema and other respiratory diseases.
  • Other class I PI3K involved in immune system function includes PI3K ⁇ , which plays a role in leukocyte signaling and has been implicated in inflammation, rheumatoid arthritis, and autoimmune diseases such as lupus.
  • PI3K ⁇ has been implicated primarily in various types of cancer including PTEN-negative cancer (Edgar et al. Cancer Research (2010) 70(3): 1164-1172), and HER2-overexpressing cancer such as breast cancer and ovarian cancer.
  • kinases particularly protein kinases such as PI3Ks are prime targets for drug development.
  • the present invention addresses this need in the art by providing a new class of kinase inhibitors the bind PI3Ks with high affinity.
  • the invention provides a compound of Formula I or III:
  • one of X 1 and X 2 is N-R'-W'-R ⁇ Z or C-R'-W'-R ⁇ Z, and the other is N or C-E 1 ;
  • X 5 is N or C-E 5 ;
  • X a is NR 31 , S or O;
  • R'and R 5 are independently -C3_gcycloalkyl, -aryl, -heteroaryl, -aralkyl, -heteroaralkyl, or - heterocyclyl, each of which is unsubstituted or substituted by one or more independent R 3 substituents;
  • W 1 is a bond or where one or more CH 2 groups have been replaced by -0-, -NR 7 -, -S(0)o_ 2-,-C(0)-,-C(0)N(R 7 )-, -N(R 7 )C(0)-, -N(R 7 )S(0)-, -N(R 7 )S(0) 2 - -C(0)0- - CH(R 7 )N(C(0)OR 8 )-, -CH(R 7 )N(C(0)R 8 )- -CH(R 7 )N(S0 2 R 8 )-, -CH(R 7 )N(R 8 )-, - CH(R 7 )C(0)N(R 8 )-, -CH(R 7 )N(R 8 )C(0)-, -CH(R 7 )N(R 8 )S(0)-, or -CH(R 7 )N(R 8 )S(0) 2 -;
  • each of R , R , and R is independently H or Ci.ioalkyl , wherein the Ci.ioalkyl is unsubstituted or is substituted with one or more aryl, heteroalkyl, heterocyclyl, or heteroaryl substituent, wherein each of said aryl, heteroalkyl, heterocyclyl, or heteroaryl substituent is unsubstituted or is substituted with one or more halo, -OH, - Ci_i 0 alkyl, -CF 3 , -O-aryl, -OCF 3 , -OCi_i 0 alkyl, -NH 2 , - N(Ci_i 0 alkyl)(Ci_ l oalkyl), - NH(Ci.ioalkyl), - NH( aryl), -NR 34 R 35 , -C(O)(Ci_i 0 alkyl), -C(O)(Ci.i 0 al
  • each of R 7 and R 8 is independently hydrogen, Ci.ioalkyl, C2_ioalkenyl, aryl, heteroaryl, heterocyclyl or C 3 _iocycloalkyl, each of which except for hydrogen is unsubstituted or is substituted by one or more independent R 6 substituents;
  • Z is -W 3 -R 9 , where:
  • W 3 is a bond or Ci.galkyl, where zero, one, two or three CH 2 groups have been replaced by -0-, -
  • R 9 is H, C3_gcycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or heterocyclyl, where R 9 is substituted by one, two, three or four independent R x substituents;
  • each R x is independently selected from -Z'-Z 2 , oxo, N0 2 , halogen, CN, a suitable leaving group, orCi. 6 alkyl optionally substituted with oxo, halogen, N0 2 , or CN, wherein Z 1 is a bond or a Ci_6 saturated or unsaturated, straight or branched,hydrocarbon chain, wherein one or two methylene units of Z 2 are optionally andindependently replaced by -N(R)-, -S-, -0-, -C(O)-, -OC(O)-, -C(0)0-, -SO-, -S0 2 -, - N(R)C(0)-, -C(0)N(R)-, -N(R)S0 2 -, or -S0 2 N(R)-; and Z 2 is hydrogen or Ci_ 6 alkyl optionally substituted with oxo, halogen, N0 2 , or CN.
  • X 1 is N. In other embodiments, X 1 and X 5 are N.
  • E 3 , E 4 and E 6 are hydrogen.
  • the compound has the Formula:
  • the compound has the Formula:
  • the invention further provides a compound of Formula II:
  • X 5 is N or C-E 5 ;
  • X 6 is N
  • X 7 and X 8 are independently N or C; wherein no more than three of X 5 , X 6 , X 7 and X 8 are N;
  • one of X 9 and X 10 is C-R'-W'-R ⁇ Z, and the other is C-E 2 ;
  • X a is NR 31 , O or S;
  • loalkenylheteroalkyl C 2 _ioalkenylheterocyclyl, C 2 _ioalkenyl-C 3 _gcycloalkyl, C 2 _ioalkynyl-C 3 _ gcycloalkyl, C 2 _ioalkynylaryl, C 2 _ioalkynylheteroaryl, C 2 _ioalkynylheteroalkyl, C 2 _
  • R'and R 5 are independently -C 3 _gcycloalkyl, -aryl, -heteroaryl, -aralkyl, -heteroaralkyl, or - heterocyclyl, each of which is unsubstituted or substituted by one or more independent R 3 substituents;
  • W 1 is a bond or where one or more CH 2 groups have been replaced by -0-, -NR 7 -, -S(0)o_ 2 -,-C(0)-,-C(0)N(R 7 )-, -N(R 7 )C(0)-, -N(R 7 )S(0)-, -N(R 7 )S(0) 2 - -C(0)0- - CH(R 7 )N(C(0)OR 8 )-, -CH(R 7 )N(C(0)R 8 )-, -CH(R 7 )N(S0 2 R 8 )-, -CH(R 7 )N(R 8 )-, - CH(R 7 )C(0)N(R 8 )-, -CH(R 7 )N(R 8 )C(0)-, -CH(R 7 )N(R 8 )S(0)-, or -CH(R 7 )N(R 8 )S(0) 2 -;
  • loalkenylheteroalkyl C 2 _ioalkenylheterocyclyl, C 2 _ioalkenyl-C 3 _gcycloalkyl, C 2 _ioalkynyl-C 3 _ gcycloalkyl, C 2 _ioalkynylaryl, C 2 _ioalkynylheteroaryl, C 2 _ioalkynylheteroalkyl, C 2 .
  • each of R , R , and R is independently H or Ci.ioalkyl , wherein the Ci.ioalkyl is unsubstituted or is substituted with one or more aryl, heteroalkyl, heterocyclyl, or heteroaryl substituent, wherein each of said aryl, heteroalkyl, heterocyclyl, or heteroaryl substituent is unsubstituted or is substituted with one or more halo, -OH, - Cuoalkyl, -CF 3 , -O-aryl, -OCF 3 , -OCuoalkyl, -NH 2 , - N(Ci_i 0 alkyl)(Ci_ 10 alkyl), - NH(Ci.ioalkyl), - NH( aryl), -NR 34 R 35 , -C(O)(C 1 .
  • each of R 7 and R 8 is independently hydrogen, Cuoalkyl, C 2 _i 0 alkenyl, aryl, heteroaryl, heterocyclyl or C3_iocycloalkyl, each of which except for hydrogen is unsubstituted or is substituted by one or more independent R 6 substituents;
  • Z is -W 3 -R 9 , wherein: W 3 is a bond or Ci.galkyl, where zero, one, two or three CH 2 groups have been replaced by -0-, -NR 7 -, -S(O) 0 - 2 -,-C(O)-,-C(O)N(R 7 )-, -N(R 7 )C(0)-, - N(R 7 )S(0)-, -N(R 7 )S(0) 2 - -C(0)0- -CH(R 7 )N(C(0)OR 8 )-, -CH(R 7 )N(C(0)R 8 )-, - CH(R 7 )N(S0 2 R 8 )-, -CH(R 7 )N(R 8 )-, -CH(R 7 )C(0)N(R 8 )-, -CH(R 7 )N(R 8 )C(0)N(R 8 )-, -CH(R 7 )N(R
  • X 6 is N. In some embodiments, X 5 is C-E 5 .
  • E 2 , E 3 and E 4 are H.
  • X 7 is N and X 8 is C. In other embodiments, X 7 is C and X 8 is N. [0019] In some embodiments, the compound has the Formula:
  • the compound has the Formula:
  • the present invention provides a composition comprising a pharmaceutically acceptable excipient and one or more compound disclosed herein.
  • the composition is a liquid, solid, semi-solid, gel, or an aerosol form.
  • the present invention provides a method for inhibiting a phosphatidyl inositol-3 kinase (PI3 kinase), comprising: contacting the PI3 kinase with an effective amount of a compound disclosed herein.
  • the method is performed in conditions suitable to promote formation of a covalent bond between the compound and the kinase.
  • the PI3 kinase is PI3 kinase alpha.
  • the step of contacting may further comprise contacting a cell that expresses one or more type I PI3 kinases, including PI3 kinease alpha.
  • the method further comprises administering a second therapeutic agent to the cell.
  • the present invention also provides a method for treating a condition associated with PI3 kinase, comprising administering to a subject in need thereof an effective amount of the compound disclosed herein.
  • the condition associated with PI3 kinase is selected from the group consisting of asthma, emphysema, bronchitis, psoriasis, allergy, anaphylaxis, rheumatoid arthritis, graft versus host disease, lupus erythematosus, psoriasis, restenosis, benign prostatic hypertrophy, diabetes, pancreatitis, proliferative glomerulonephritis, diabetes- induced renal disease, inflammatory bowel disease, atherosclerosis, eczema, scleroderma, diabetes, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, hemangioma, glioma, melanoma
  • the present invention provides compounds capable of inhibiting a kinase protein.
  • the compounds of the invention are capable of covalently reacting with the kinase protein. Such binding is generally expected to lead to an increase in the affinity of the compound for the kinase, which is an advantageous property in many applications, including therapeutic and diagnostic uses.
  • the compounds of the invention may be electrophilic groups capable of reacting with any nucleophilic group present in a kinase protein. Suitable electrophilic groups are described throughout the application, while suitable nucleophilic groups include, for example, cysteine moieties present in kinase proteins. PI3 kinases are known to possess such cysteine moieties, including in the binding domain.
  • Cysteine residues that may be targeted by compounds of the invention include, but are not limited to, residue 862 of PI3Ka (sequence QCKGGLKGALQFNSHTLHQW), residue 838 of PI3Ka (sequence LPYGCLS), residue 841 of ⁇ 3 ⁇ (sequence LPYGCLA), and residue 1119 of ⁇ 3 ⁇ (sequence VIFRCFS).
  • the compounds of the invention are capable of covalently binding to cysteine residue 862 of PI3Ka.
  • the invention provides a conjugate of a compound of the invention with a kinase such as a PI3 kinase.
  • a kinase such as a PI3 kinase.
  • the invention provides a conjugate of a compound of the invention with cysteine residue 862 of PI3Ka.
  • "about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value.
  • the term can mean within an order of magnitude, preferably within 5- fold, and more preferably within 2-fold, of a value.
  • Treatment refers to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated.
  • a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder.
  • the compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
  • neoplastic condition refers to the presence of cells possessing abnormal growth characteristics, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, perturbed oncogenic signaling, and certain characteristic morphological features.
  • tumor cells tumor cells
  • tumors that proliferate by expressing a mutated tyrosine kinase or overexpression of a receptor tyrosine kinase
  • benign and malignant cells of other proliferative diseases in which aberrant tyrosine kinase activation occurs any tumors that proliferate by receptor tyrosine kinases
  • any tumors that proliferate by aberrant serine/threonine kinase activation and (5) benign and malignant cells of other proliferative diseases in which aberrant serine/threonine kinase activation occurs.
  • the term "effective amount” or “therapeutically effective amount” refers to that amount of an inhibitor described herein that is sufficient to effect the intended application including but not limited to disease treatment, as defined below.
  • the therapeutically effective amount may vary depending upon the intended application ⁇ in vitro or in vivo), or the subjectand disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the term also applies to a dose that will induce a particular response in target cells, e.g., reduction of proliferation or downregulation of activity of a target protein.
  • the specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.
  • a "sub-therapeutic amount" of an agent or therapy is an amount less than the effective amount for that agent or therapy, but when combined with an effective or sub-therapeutic amount of another agent or therapy can produce a result desired by the physician, due to, for example, synergy in the resulting efficacious effects, or reduced side effects.
  • a "synergistically effective therapeutic amount" of an agent or therapy is an amount which, when combined with an effective or sub-therapeutic amount of another agent or therapy, produces a greater effect than when either of the two agents are therapies are used alone.
  • a syngergistically effective therapeutic amount of an agent or therapy produces a greater effect when used in combination than the additive effects of each of the two agents or therapies when used alone.
  • agent refers to a biological, pharmaceutical, or chemical compound or other moiety.
  • Non-limiting examples include simple or complex organic or inorganic molecule, a peptide, a protein, an oligonucleotide, an antibody, an antibody derivative, antibody fragment, a vitamin derivative, a carbohydrate, a toxin, or a chemotherapeutic compound.
  • Various compounds can be synthesized, for example, small molecules and oligomers (e.g., oligopeptides and oligonucleotides), and synthetic organic compounds based on various core structures.
  • various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.
  • agonist refers to a compound having the ability to initiate or enhance a biological function of a target protein, whether by inhibiting the activity or expression of the target protein. Accordingly, the term “agonist” is defined in the context of the biological role of the target polypeptide. While preferred agonists herein specifically interact with (e.g., bind to) the target, compounds that initiate or enhance a biological activity of the target polypeptide by interacting with other members of the signal transduction pathway of which the target polypeptide is a member are also specifically included within this definition.
  • antagonists are used interchangeably, and they refer to a compound having the ability to inhibit a biological function of a target protein, whether by inhibiting the activity or expression of the target protein. Accordingly, the terms “antagonist” and “inhibitors” are defined in the context of the biological role of the target protein. While preferred antagonists herein specifically interact with (e.g., bind to) the target, compounds that inhibit a biological activity of the target protein by interacting with other members of the signal transduction pathway of which the target protein is a member are also specifically included within this definition.
  • a preferred biological activity inhibited by an antagonist is associated with the development, growth, or spread of a tumor, or an undesired immune response as manifested in autoimmune disease.
  • mTOR inhibitor that binds to and directly inhibits both mTORCl and mTORC2 kinases refers to an mTOR inhibitor that interacts with and reduces the kinase activity of both mTORCl and mTORC2 complexes.
  • an "anti-cancer agent”, “anti-tumor agent” or “chemotherapeutic agent” refers to any agent useful in the treatment of a neoplastic condition.
  • One class of anti-cancer agents comprises chemotherapeutic agents.
  • “Chemotherapy” means the administration of one or more
  • chemotherapeutic drugs and/or other agents to a cancer patient by various methods, including intravenous, oral, intramuscular, intraperitoneal, intravesical, subcutaneous, transdermal, buccal, or inhalation or in the form of a suppository.
  • cell proliferation refers to a phenomenon by which the cell number has changed as a result of division. This term also encompasses cell growth by which the cell morphology has changed (e.g., increased in size) consistent with a proliferative signal.
  • co-administration encompass administration of two or more agents to an animal so that both agents and/or their metabolites are present in the animal at the same time.
  • Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.
  • Co-administered agents may be in the same formulation.
  • Co-administered agents may also be in different formulations.
  • a "therapeutic effect,” as used herein, encompasses a therapeutic benefit and/or a prophylactic benefit as described above.
  • a prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • salts refers to salts derived from a variety of organic and inorganic counter ions well known in the art.
  • Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids.
  • Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
  • Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like.
  • Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine,
  • the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts.
  • “Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions of the invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Signal transduction is a process during which stimulatory or inhibitory signals are transmitted into and within a cell to elicit an intracellular response.
  • a modulator of a signal transduction pathway refers to a compound which modulates the activity of one or more cellular proteins mapped to the same specific signal transduction pathway.
  • a modulator may augment (agonist) or suppress (antagonist) the activity of a signaling molecule.
  • selective inhibition or “selectively inhibit” as applied to a biologically active agent refers to the agent's ability to selectively reduce the target signaling activity as compared to off- target signaling activity, via direct or interact interaction with the target.
  • Subject refers to an animal, such as a mammal, for example a human.
  • the methods described herein can be useful in both human therapeutics, pre-clinical, and veterinary applications.
  • the subject is a mammal, and in some embodiments, the subject is human.
  • in vivo refers to an event that takes place in a subject's body.
  • an in vitro assay refers to an event that takes places outside of a subject's body.
  • an in vitro assay encompasses any assay run outside of a subject assay.
  • In vitro assays encompass cell-based assays in which cells alive or dead are employed.
  • In vitro assays also encompass a cell-free assay in which no intact cells are employed.
  • PI3K Phosphoinositide 3 -kinase
  • PI phosphatidylinositol
  • connection of compound name moieties are at the rightmost recited moiety. That is, the substituent name starts with a terminal moiety, continues with any linking moieties, and ends with the linking moiety.
  • heteroarylthio C M alkyl has a heteroaryl group connected through a thio sulfur to a C alkyl radical that connects to the chemical species bearing the substituent.
  • a formula such as, for example "-L-C MO alkyl - C 3 _gcycloalkyl” is represented.
  • the terminal group is a C 3 _gcycloalkyl group attached to a linking CMO alkyl moiety which is attached to an element L, which is itself connected to the chemical species bearing the substituent.
  • Alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to ten carbon atoms (e.g., Ci-Cio alkyl).
  • a numerical range such as “1 to 10” refers to each integer in the given range; e.g., "1 to 10 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the present definition also covers the occurrence of the term "alkyl" where no numerical range is designated.
  • Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl isobutyl, tertiary butyl, pentyl, isopentyl, neopentyl, hexyl, septyl, octyl, nonyl, decyl, and the like.
  • the alkyl is attached to the rest of the molecule by a single bond,for example, methyl (Me), ethyl (Et), n-propyl, 1 -methylethyl (z o-propyl), n-butyl, n-pentyl, 1 , 1 -dimethylethyl (i-butyl), 3-methylhexyl, 2-methylhexyl, and the like.
  • an alkyl group is optionally substituted by one or more of substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR a , -SR a , -OC(0)-R a , -N(R a ) 2 , -C(0)R a , -C(0)OR a , -OC(0)N(R a ) 2 , -C(0)N(R a ) 2 , -N(R a )C(0)OR a , -N(R a )C(0)R a , -N(R a )C(0)OR a , -N(R a )C(0)R
  • halo or halogen refers to fluoro, chloro, bromo, or iodo.
  • haloalkyl refers to an alkyl group substituted with one or more halo groups, for example chloromethyl, 2-bromoethyl, 3-iodopropyl, trifluoromethyl, perfluoropropyl, 8-chlorononyl, and the like.
  • Acyl refers to the groups (alkyl)-C(O)-, (aryl)-C(O)-, (heteroaryl)-C(O)-,
  • acyl radical which refers to the total number of chain or ring atoms of the alkyl, aryl, heteroaryl or heterocycloalkyl portion of the acyloxy group plus the carbonyl carbon of acyl, i.e three other ring or chain atoms plus carbonyl. If the R radical is heteroaryl or heterocycloalkyl, the hetero ring or chain atoms contribute to the total number of chain or ring atoms. Unless stated otherwise specifically in the specification, the "R" of an acyloxy group is optionally substituted by one or more substituents which
  • heterocycloalkyl heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.
  • Cycloalkyl refers to a monocyclic or polycyclic radical that contains only carbon and hydrogen, and may be saturated, or partially unsaturated. Cycloalkyl groups include groups having from 3 to 10 ring atoms (i.e., C 2 -Cio cycloalkyl). Whenever it appears herein, a numerical range such as “3 to 10" refers to each integer in the given range; e.g., "3 to 10 carbon atoms” means that the cycloalkyl group may consist of 3 carbon atoms, etc., up to and including 10 carbon atoms. In some embodiments, it is a C3-C8 cycloalkyl radical.
  • cycloalkyl groups include, but are not limited to the following moieties: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloseptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl, and the like.
  • a cycloalkyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR a , -SR a , -OC(0)-R a , -N(R a ) 2 , -C(0)R a , -C(0)OR a , -OC(0)N(R a ) 2 , -C(0)N(R a ) 2 , -N(R a )C(0)OR a , -N(R a )C(0)R a , -N(R a )C(0)OR a , -N(R a )
  • heterocycloalkyl heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.
  • Cycloalkyl- C 3 - 8 cycloalkyl is used to describe an alkyl group, branched or straight chain and containing 1 to 10 carbon atoms, attached to a linking cycloalkyl group which contains 3 to 8 carbons, such as for example, 2-methyl cyclopropyl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • bicycloalkyl refers to a structure consisting of two cycloalkyl moieties, unsubstituted or substituted, that have two or more atoms in common. If the cycloalkyl moieties have exactly two atoms in common they are said to be "fused”. Examples include, but are not limited to, bicyclo[3.1.0]hexyl, perhydronaphthyl, and the like. If the cycloalkyl moieties have more than two atoms in common they are said to be "bridged”. Examples include, but are not limited to, bicyclo[3.2.1]heptyl ("norbornyl"), bicyclo[2.2.2]octyl, and the like.
  • heteroatom or "ring heteroatom” is meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).
  • Heteroalkyl “heteroalkenyl” and “heteroalkynyl” include optionally substituted alkyl, alkenyl and alkynyl radicals and which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof.
  • a numerical range may be given, e.g., C 1 -C4 heteroalkyl which refers to the chain length in total, which in this example is 4 atoms long.
  • a -CH 2 OCH 2 CH 3 radical is referred to as a "C4" heteroalkyl, which includes the heteroatom center in the atom chain length description.
  • a heteroalkyl group may be substituted with one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, -OR a , -SR a , -OC(0)-R a , -N(R a ) 2 , -C(0)R a , -C(0)OR a , -C(0)N(R a ) 2 , -N(R a )C(0)OR a , -N(R a )C(0)OR a , -N(R a )C(0)OR a , -N(R a )C(0)OR a , -N(R a )C(0)R
  • heteroalkylaryl refers to a heteroalkyl group as defined above which is attached to an aryl group, and may be attached at a terminal point or through a branched portion of the heteroalkyl, for example, an benzyloxymethyl moiety. Either portion of the moiety is unsubstituted or substituted.
  • heteroalkylheteroaryl refers likewise to a heteroalkyl group which is attached to a heteroaryl moiety, for example, an ethoxymethylpyridyl group. Either portion of the moiety is unsubstituted or substituted.
  • heteroalkyl-heterocyclyl refers to a heteroalkyl group as defined above, which is attached to a heterocyclic group, for example, 4(3-aminopropyl)-N-piperazinyl. Either portion of the moiety is unsubstituted or substituted.
  • heteroalkyl-C3_gcycloalkyl refers to a heteroalkyl group as defined above, which is attached to a cyclic alkyl containing 3 to 8 carbons, for example, 1 -aminobutyl-4-cyclohexyl. Either portion of the moiety is unsubstituted or substituted.
  • heterocycloalkyl refers to a bicycloalkyl structure, which is unsubstituted or substituted, in which at least one carbon atom is replaced with a heteroatom independently selected from oxygen, nitrogen, and sulfur.
  • heterospiroalkyl refers to a spiroalkyl structure, which is unsubstituted or substituted, in which at least one carbon atom is replaced with a heteroatom independently selected from oxygen, nitrogen, and sulfur.
  • An "alkene” moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond
  • an “alkyne” moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon triple bond.
  • the alkyl moiety, whether saturated or unsaturated, may be branched, straight chain, or cyclic.
  • alkenyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, and having from two to ten carbon atoms (i.e., C2-C10 alkenyl).
  • a numerical range such as “2 to 10” refers to each integer in the given range; e.g., "2 to 10 carbon atoms” means that the alkenyl group may consist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms.
  • an alkenyl comprises two to eight carbon atoms.
  • an alkenyl comprises two to five carbon atoms (e.g., C2-C5 alkenyl).
  • the alkenyl is attached to the rest of the molecule by a single bond, for example, ethenyl (i.e., vinyl), prop-l-enyl (i.e., allyl), but-l-enyl, pent-l-enyl, penta-l,4-dienyl, and the like.
  • an alkenyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR a , -SR a ,
  • each R a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.
  • C2-10 alkenyl-heteroalkyl refers to a group having an alkenyl moiety, containing 2 to 10 carbon atoms and is branched or straight chain, which is attached to a linking heteroalkyl group, such as, for example, allyloxy, and the like. Either portion of the moiety is unsubstituted or substituted.
  • C2-10 alkynyl-heteroalkyl refers to a group having an alkynyl moiety, which is unsubstituted or substituted, containing 2 to 10 carbon atoms and is branched or straight chain, which is attached to a linking heteroalkyl group, such as, for example, 4-but-l-ynoxy, and the like. Either portion of the moiety is unsubstituted or substituted.
  • haloalkenyl refers to an alkenyl group substituted with one or more halo groups.
  • cycloalkenyl refers to a cyclic aliphatic 3 to 8 membered ring structure, optionally substituted with alkyl, hydroxy and halo, having 1 or 2 ethylenic bonds such as methylcyclopropenyl, trifluoromethylcyclopropenyl, cyclopentenyl, cyclohexenyl, 1,4- cyclohexadienyl, and the like.
  • Alkynyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond, having from two to ten carbon atoms (i.e., C2-C10 alkynyl). Whenever it appears herein, a numerical range such as “2 to 10" refers to each integer in the given range; e.g., "2 to 10 carbon atoms” means that the alkynyl group may consist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. In certain
  • an alkynyl comprises two to eight carbon atoms. In other embodiments, an alkynyl has two to five carbon atoms (e.g., C2-C5 alkynyl).
  • the alkynyl is attached to the rest of the molecule by a single bond, for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.
  • an alkynyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,
  • heterocycloalkyl aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR a ,SR a , -OC(0)-R a , -N(R a ) 2 , -C(0)R a , -C(0)OR a , -OC(0)N(R a ) 2 , -C(0)N(R a ) 2 , -N(R a )C(0)OR a , -N(R a )C(0)R a , - N(R a )C(0)N(R a ) 2 ,
  • R a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.
  • C2-10 alkynyl- C3.8 cycloalkyl refers to a group containing an alkynyl group, containing 2 to 10 carbons and branched or straight chain, which is attached to a linking cycloalkyl group containing 3 to 8 carbons, such as, for example 3-prop-3-ynyl- cyclopent-lyl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • haloalkynyl refers to an alkynyl group substituted with one or more independent halo groups.
  • Amino refers to a -N(R a ) 2 radical group, where each R a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, unless stated otherwise specifically in the specification.
  • R a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, unless stated otherwise specifically in the specification.
  • R a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroaryl
  • -N(R a ) 2 is meant to include, but not be limited to, 1 -pyrrolidinyl and 4-morpholinyl. Unless stated otherwise specifically in the specification, an amino group is optionally substituted by one or more substituents which
  • Amide refers to a chemical moiety with formula -C(0)N(R) 2 or -NHC(0)R, where R is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon), each of which moiety may itself be optionally substituted. In some embodiments it is a C1-C4 amido or amide radical, which includes the amide carbonyl in the total number of carbons in the radical.
  • the R 2 of - N(R) 2 of the amide may optionally be taken together with the nitrogen to which it is attached to form a 4-, 5-, 6-, or 7-membered ring.
  • an amido group is optionally substituted independently by one or more of the substituents as described herein for alkyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl.
  • An amide may be an amino acid or a peptide molecule attached to a compound of Formula (I), thereby forming a prodrug. Any amine, hydroxy, or carboxyl side chain on the compounds described herein can be amidified.
  • Aromatic or “aryl” refers to an aromatic radical with six to ten ring atoms (e.g., C6-C10 aromatic or C6-C10 aryl) which has at least one ring having a conjugated pi electron system which is carbocyclic (e.g., phenyl, fluorenyl, and naphthyl).
  • Bivalent radicals formed from substituted benzene derivatives and having the free valences at ring atoms are named as substituted phenylene radicals.
  • Bivalent radicals derived from univalent polycyclic hydrocarbon radicals whose names end in "-yl” by removal of one hydrogen atom from the carbon atom with the free valence are named by adding "-idene” to the name of the corresponding univalent radical, e.g., a naphthyl group with two points of attachment is termed naphthylidene.
  • a numerical range such as “6 to 10” refers to each integer in the given range; e.g., "6 to 10 ring atoms” means that the aryl group may consist of 6 ring atoms, 7 ring atoms, etc., up to and including 10 ring atoms.
  • an aryl moiety is optionally substituted by one or more substituents which are independently: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR a , -SR a , -OC(0)-R a , -N(R a ) 2 , -C(0)R a , -C(0)OR a , -OC(0)N(R a ) 2 , -C(0)N(R a ) 2 , -N(R a )C(0)OR a , -OC(0)N(R a ) 2 , -C(0)N(R a ) 2 , -N(R a )C(0)OR a ,
  • R a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.
  • Heteroaryl or, alternatively, “heteroaromatic” refers to a 5- to 18-membered aromatic radical (e.g., C5-C 13 heteroaryl) that includes one or more ring heteroatoms selected from nitrogen, oxygen and sulfur, and which may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system.
  • a numerical range such as “5 to 18” refers to each integer in the given range; e.g., "5 to 18 ring atoms” means that the heteroaryl group may consist of 5 ring atoms, 6 ring atoms, etc., up to and including 18 ring atoms.
  • Bivalent radicals derived from univalent heteroaryl radicals whose names end in "-yl” by removal of one hydrogen atom from the atom with the free valence are named by adding "-idene” to the name of the corresponding univalent radical, e.g., a pyridyl group with two points of attachment is a pyridylidene.
  • An N-containing "heteroaromatic” or “heteroaryl” moiety refers to an aromatic group in which at least one of the skeletal atoms of the ring is a nitrogen atom.
  • the polycyclic heteroaryl group may be fused or non- fused.
  • the heteroatom(s) in the heteroaryl radical is optionally oxidized.
  • heteroaryl is attached to the rest of the molecule through any atom of the ring(s).
  • heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1 ,3-benzodioxolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[6] [l ,4]dioxepinyl, benzo[b] [l ,4]oxazinyl, 1 ,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzoxazolyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranyl, benzofur
  • a heteraryl moiety is optionally substituted by one or more substituents which are independently: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, -OR a , -SR a , -OC(0)-R a , -N(R a ) 2 , -C(0)R a , -C(0)OR a , -C(0)N(R a ) 2 , -N(R a )C(0)OR a , -N(R a )C(0)OR a , -N(R a )C(0)R a , -N(R a )S(0) t R a (where t is 1 or 2),
  • aryl-alkyl arylalkyl
  • arylalkyl arylalkyl
  • aralkyl a group wherein the alkyl chain can be branched or straight chain forming a linking portion with the terminal aryl, as defined above, of the aryl-alkyl moiety.
  • aryl-alkyl groups include, but are not limited to, optionally substituted benzyl, phenethyl, phenpropyl and phenbutyl such as 4-chlorobenzyl, 2,4- dibromobenzyl, 2-methylbenzyl, 2-(3-fluorophenyl)ethyl, 2-(4-methylphenyl)ethyl, 2-(4- (trifluoromethyl)phenyl) ethyl, 2-(2-methoxyphenyl)ethyl, 2-(3-nitrophenyl)ethyl, 2-(2,4- dichlorophenyl)ethyl, 2-(3,5-dimethoxyphenyl)ethyl, 3-phenylpropyl, 3-(3-chlorophenyl)propyl, 3-(2- methylphenyl)propyl, 3-(4-methoxyphenyl)propyl, 3-(4-(trifluoromethyl)phenyl)propyl
  • Ci.ioalkylaryl refers to an alkyl group, as defined above, containing 1 to 10 carbon atoms, branched or unbranched, wherein the aryl group replaces one hydrogen on the alkyl group, for example, 3-phenylpropyl. Either portion of the moiety is unsubstituted or substituted.
  • C 2 - 10 alkyl monocycloaryl refers to a group containing a terminal alkyl group, branched or straight chain and containing 2 to 10 atoms attached to a linking aryl group which has only one ring, such as for example, 2-phenyl ethyl. Either portion of the moiety is unsubstituted or substituted.
  • C o alkyl bicycloaryl refers to a group containing a terminal alkyl group, branched or straight chain and containing 2 to 10 atoms attached to a linking aryl group which is bicyclic, such as for example, 2-(l-naphthyl)- ethyl. Either portion of the moiety is unsubstituted or substituted.
  • aryl-cycloalkyl and "arylcycloalkyl” are used to describe a group wherein the terminal aryl group is attached to a cycloalkyl group, for example phenylcyclopentyl and the like. Either portion of the moiety is unsubstituted or substituted.
  • heteroaryl-C3_gcycloalkyl and “heteroaryl- C3_gcycloalkyl" are used to describe a group wherein the terminal heteroaryl group is attached to a cycloalkyl group, which contains 3 to 8 carbons, for example pyrid-2-yl-cyclopentyl and the like. Either portion of the moiety is unsubstituted or substituted.
  • heteroaryl- heteroalkyl refers to a group wherein the terminal heteroaryl group is attached to a linking heteroalkyl group, such as for example, pyrid-2-yl methylenoxy, and the like. Either portion of the moiety is unsubstituted or substituted.
  • aryl-alkenyl arylalkenyl
  • arylalkenyl arylalkenyl
  • aralkenyl a group wherein the alkenyl chain can be branched or straight chain forming a linking portion of the aralkenyl moiety with the terminal aryl portion, as defined above, for example styryl (2-phenylvinyl), phenpropenyl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • aryl -C2-ioalkenyl means an arylalkenyl as described above wherein the alkenyl moiety contains 2 to 10 carbon atoms such as for example, styryl (2-phenylvinyl), and the like. Either portion of the moiety is unsubstituted or substituted.
  • C2-ioalkenyl-aryl is used to describe a group wherein the terminal alkenyl group, which contains 2 to 10 carbon atoms and can be branched or straight chain, is attached to the aryl moiety which forms the linking portion of the alkenyl-aryl moiety, such as for example, 3-propenyl- naphth-l-yl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • aryl-alkynyl arylalkynyl
  • arylalkynyl arylalkynyl
  • aralkynyl a group wherein the alkynyl chain can be branched or straight chain forming a linking portion of the aryl-alkynyl moiety with the terminal aryl portion, as defined above, for example 3 -phenyl- 1-propynyl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • aryl- C2-ioalkynyl means an arylalkynyl as described above wherein the alkynyl moiety contains two to ten carbons, such as, for example 3 -phenyl- 1-propynyl, and the like . Either portion of the moiety is unsubstituted or substituted.
  • C2-ioalkynyl- aryl means a group containing an alkynyl moiety attached to an aryl linking group, both as defined above, wherein the alkynyl moiety contains two to ten carbons, such as, for example 3-propynyl-naphth-l -yl. Either portion of the moiety is unsubstituted or substituted.
  • aryl-oxy aryloxy
  • aryloxy aryloxy
  • aroxy a terminal aryl group attached to a linking oxygen atom.
  • Typical aryl-oxy groups include phenoxy, 3,4-dichlorophenoxy, and the like. Either portion of the moiety is unsubstituted or substituted.
  • aryl-oxyalkyl aryloxyalkyl
  • aryloxyalkyl aryloxyalkyl
  • aroxyalkyl a group wherein an alkyl group is substituted with a terminal aryl-oxy group, for example
  • Ci_ioalkoxy-Ci_ioalkyl refers to a group wherein an alkoxy group, containing 1 to 10 carbon atoms and an oxygen atom within the branching or straight chain, is attached to a linking alkyl group, branched or straight chain which contains 1 to 10 carbon atoms, such as, for example methoxypropyl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • Ci_ioalkoxy-C2-ioalkenyl refers to a group wherein an alkoxy group, containing 1 to 10 carbon atoms and an oxygen atom within the branching or straight chain, is attached to a linking alkenyl group, branched or straight chain which contains 1 to 10 carbon atoms, such as, for example 3-methoxybut-2-en-l-yl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • Ci_ioalkoxy-C2-ioalkynyl refers to a group wherein an alkoxy group, containing 1 to 10 carbon atoms and an oxygen atom within the branching or straight chain, is attached to a linking alkynyl group, branched or straight chain which contains 1 to 10 carbon atoms, such as, for example 3-methoxybut-2-in-l -yl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • heterocycloalkenyl refers to a cycloalkenyl structure, which is unsubstituted or substituted in which at least one carbon atom is replaced with a heteroatom selected from oxygen, nitrogen, and sulfur.
  • heteroaryl-oxy used to describe a terminal heteroaryl group, which is unsubstituted or substituted, attached to a linking oxygen atom.
  • Typical heteroaryl-oxy groups include 4,6- dimethoxypyrimidin-2-yloxy and the like.
  • heteroarylalkyl used to describe a group wherein the alkyl chain can be branched or straight chain forming a linking portion of the heteroaralkyl moiety with the terminal heteroaryl portion, as defined above, for example 3-furylmethyl, thenyl, furfuryl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • heteroaryl-Ci.ioalkyl is used to describe a heteroaryl alkyl group as described above where the alkyl group contains 1 to 10 carbon atoms. Either portion of the moiety is unsubstituted or substituted.
  • Ci_ioalkyl-heteroaryl is used to describe a alkyl attached to a hetary group as described above where the alkyl group contains 1 to 10 carbon atoms. Either portion of the moiety is unsubstituted or substituted.
  • heteroarylalkenyl used to describe a heteroarylalkenyl group wherein the alkenyl chain can be branched or straight chain forming a linking portion of the heteroaralkenyl moiety with the terminal heteroaryl portion, as defined above, for example 3-(4-pyridyl)-l -propenyl. Either portion of the moiety is unsubstituted or substituted.
  • heteroaryl- C2-ioalkenyl group is used to describe a group as described above wherein the alkenyl group contains 2 to 10 carbon atoms. Either portion of the moiety is unsubstituted or substituted.
  • C2-ioalkenyl- heteroaryl is used to describe a group containing an alkenyl group, which is branched or straight chain and contains 2 to 10 carbon atoms, and is attached to a linking heteroaryl group, such as, for example 2-styryl-4-pyridyl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • heteroarylalkynyl used to describe a group wherein the alkynyl chain can be branched or straight chain forming a linking portion of the heteroaralkynyl moiety with the heteroaryl portion, as defined above, for example 4-(2-thienyl)-l-butynyl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • heteroaryl- C2-ioalkynyl is used to describe a heteroarylalkynyl group as described above wherein the alkynyl group contains 2 to 10 carbon atoms. Either portion of the moiety is unsubstituted or substituted.
  • C2-ioalkynyl- heteroaryl is used to describe a group containing an alkynyl group which contains 2 to 10 carbon atoms and is branched or straight chain, which is attached to a linking heteroaryl group such as, for example, 4(but-l-ynyl) thien-2-yl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • heterocyclyl refers to a four-, five-, six-, or seven-membered ring containing one, two, three or four heteroaroms independently selected from nitrogen, oxygen and sulfur.
  • the four- membered ring has zero double bonds
  • the five-membered ring has zero to two double bonds
  • the siz- and seven-membered rings have zero to three double bonds.
  • heterocyclyl also includes bicyclic groups in which the heterocyclyl ring is fused to another monocyclic heterocyclyl , or a four- to se-membered aromatic or nonaromatic carbocyclic ring.
  • the heterocyclyl group can be attached to the parent molecular moiety through any carbon atom or nitrogen atom in the group.
  • Heterocycloalkyl refers to a stable 3- to 18-membered non-aromatic ring radical that comprises two to twelve carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. Whenever it appears herein, a numerical range such as “3 to 18" refers to each integer in the given range; e.g., "3 to 18 ring atoms” means that the heterocycloalkyl group may consist of 3 ring atoms, 4 ring atoms, etc., up to and including 18 ring atoms. In some embodiments, it is a C 5 -Cio heterocycloalkyl. In some embodiments, it is a C4-C 10 heterocycloalkyl.
  • the heterocycloalkyl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems.
  • the heteroatoms in the heterocycloalkyl radical may be optionally oxidized.
  • One or more nitrogen atoms, if present, are optionally quaternized.
  • the heterocycloalkyl radical is partially or fully saturated.
  • the heterocycloalkyl may be attached to the rest of the molecule through any atom of the ring(s).
  • heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl,imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1 -o
  • heterocycloalkyl moiety is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, -OR a , SR a , -OC(0)-R a , -N(R a ) 2 , -C(0)R a , -C(0)OR a , -C(0)N(R a ) 2 , -N(R a )C(0)OR a , -N(R a )C(0)OR a , -N(R a )C(0)R a , -N(R a )S(0) t R a (where t is 1 or 2), -
  • Heterocycloalkyl also includes bicyclic ring systems wherein one non-aromatic ring, usually with 3 to 7 ring atoms, contains at least 2 carbon atoms in addition to 1 -3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, as well as combinations comprising at least one of the foregoing heteroatoms; and the other ring, usually with 3 to 7 ring atoms, optionally contains 1-3 heteroatoms independently selected from oxygen, sulfur, and nitrogen and is not aromatic.
  • heterocyclylalkyl refers to the divalent derivative of heterocycloalkyl.
  • Ci.ioalkyl-heterocycyl refers to a group as defined above where the alkyl moiety contains 1 to 10 carbon atoms. Either portion of the moiety is unsubstituted or substituted.
  • heterocyclic group refers to a group containing a terminal heterocyclic group attached to a linking alkyl group which contains 1 to 10 carbons and is branched or straight chain, such as, for example, 4-morpholinyl ethyl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • heterocyclylalkenyl used to describe a group wherein the alkenyl chain can be branched or straight chain forming a linking portion of the heterocyclylalkenyl moiety with the terminal heterocyclyl portion, as defined above, for example 2-morpholinyl-l-propenyl and the like.
  • alkenyl chain can be branched or straight chain forming a linking portion of the heterocyclylalkenyl moiety with the terminal heterocyclyl portion, as defined above, for example 2-morpholinyl-l-propenyl and the like.
  • heterocycloalkenylene refers to the divalent derivative of heterocyclylalkenyl. Either portion of the moiety is unsubstituted or substituted.
  • heterocycyl- C2-10 alkenyl refers to a group as defined above where the alkenyl group contains 2 to 10 carbon atoms and is branched or straight chain, such as, for example, 4-(N- piperazinyl)-but-2-en-l-yl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • heterocyclylalkynyl refers to a group wherein the alkynyl chain can be branched or straight chain forming a linking portion of the heterocyclylalkynyl moiety with the terminal heterocyclyl portion, as defined above, for example 2-pyrrolidinyl-l-butynyl and the like. Either portion of the moiety is unsubstituted or substituted.
  • heterorocycyl- C2-10 alkynyl refers to a group as defined above where the alkynyl group contains 2 to 10 carbon atoms and is branched or straight chain, such as, for example, 4-(N- piperazinyl)-but-2-yn-l-yl, and the like.
  • aryl- heterocycyl refers to a group containing a terminal aryl group attached to a linking heterocyclic group, such as for example, N4-(4-phenyl)- piperazinyl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • heteroaryl- heterocycyl refers to a group containing a terminal heteroaryl group attached to a linking heterocyclic group, such as for example, N4-(4-pyridyl)- piperazinyl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • carboxylalkyl refers to a terminal carboxyl (-COOH) group attached to branched or straight chain alkyl groups as defined above.
  • carboxylalkenyl refers to a terminal carboxyl (-COOH) group attached to branched or straight chain alkenyl groups as defined above.
  • carboxylalkynyl refers to a terminal carboxyl (-COOH) group attached to branched or straight chain alkynyl groups as defined above.
  • carboxylcycloalkyl refers to a terminal carboxyl (-COOH) group attached to a cyclic aliphatic ring structure as defined above.
  • carboxylcycloalkenyl refers to a terminal carboxyl (-COOH) group attached to a cyclic aliphatic ring structure having ethylenic bonds as defined above.
  • cycloalkylalkyl and “cycloalkyl-alkyl” refer to a terminal cycloalkyl group as defined above attached to an alkyl group, for example cyclopropylmethyl, cyclohexylethyl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • cycloalkylalkenyl and “cycloalkyl-alkenyl” refer to a terminal cycloalkyl group as defined above attached to an alkenyl group, for example cyclohexylvinyl, cycloheptylallyl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • cycloalkylalkynyl and “cycloalkyl-alkynyl” refer to a terminal cycloalkyl group as defined above attached to an alkynyl group, for example cyclopropylpropargyl, 4-cyclopentyl-2- butynyl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • cycloalkenylalkyl and “cycloalkenyl-alkyl” refer to a terminal cycloalkenyl group as defined above attached to an alkyl group, for example 2-(cyclopenten-l -yl)ethyl and the like. Either portion of the moiety is unsubstituted or substituted.
  • cycloalkenylalkenyl and “cycloalkenyl-alkenyl” refer to terminal a cycloalkenyl group as defined above attached to an alkenyl group, for example l-(cyclohexen-3-yl)allyl and the like.
  • cycloalkenylalkynyl and “cycloalkenyl-alkynyl” refer to terminal a cycloalkenyl group as defined above attached to an alkynyl group, for example l-(cyclohexen-3-yl)propargyl and the like. Either portion of the moiety is unsubstituted or substituted.
  • alkoxy refers to the group -O-alkyl, including from 1 to 8 carbon atoms of a straight, branched, cyclic configuration and combinations thereof attached to the parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy and the like.
  • Lower alkoxy refers to alkoxy groups containing one to six carbons. In some embodiments, C 1 -C4 alkyl, is an alkyl group which encompasses both straight and branched chain alkyls of from 1 to 4 carbon atoms.
  • haloalkoxy refers to an alkoxy group substituted with one or more halo groups, for example chloromethoxy, trifluoromethoxy, difluoromethoxy, perfluoroisobutoxy, and the like.
  • alkoxyalkoxyalkyl refers to an alkyl group substituted with an alkoxy moiety which is in turn is substituted with a second alkoxy moiety, for example methoxymethoxymethyl, isopropoxymethoxyethyl, and the like. This moiety is substituted with further substituents or not substituted with other substituents.
  • alkylthio includes both branched and straight chain alkyl groups attached to a linking sulfur atom, for example methylthio and the like.
  • alkoxyalkyl refers to an alkyl group substituted with an alkoxy group, for example isopropoxymethyl and the like. Either portion of the moiety is unsubstituted or substituted.
  • alkoxyalkenyl refers to an alkenyl group substituted with an alkoxy group, for example 3-methoxyallyl and the like. Either portion of the moiety is unsubstituted or substituted.
  • alkoxyalkynyl refers to an alkynyl group substituted with an alkoxy group, for example 3-methoxypropargyl and the like. Either portion of the moiety is unsubstituted or substituted.
  • C 2 -ioalkenylC 3 .gCycloalkyl refers to an alkenyl group as defined above substituted with a three to eight membered cycloalkyl group, for example, 4-(cyclopropyl) -2-butenyl and the like. Either portion of the moiety is unsubstituted or substituted.
  • C 2 -ioalkynylC 3 .gCycloalkyl refers to an alkynyl group as defined above substituted with a three to eight membered cycloalkyl group, for example, 4-(cyclopropyl) -2-butynyl and the like. Either portion of the moiety is unsubstituted or substituted.
  • heterocyclyl-Ci.ioalkyl refers to a heterocyclic group as defined above substituted with an alkyl group as defined above having 1 to 10 carbons, for example, 4-(N-methyl)-piperazinyl, and the like. Either portion of the moiety is unsubstituted or substituted.
  • heterocyclyl-C 2 -ioalkenyl refers to a heterocyclic group as defined above, substituted with an alkenyl group as defined above, having 2to 10 carbons, for example, 4-(N-allyl) piperazinyl, and the like. Moieties wherein the heterocyclic group is substituted on a carbon atom with an alkenyl group are also included. Either portion of the moiety is unsubstituted or substituted.
  • heterocyclyl-C 2 -ioalkynyl refers to a heterocyclic group as defined above, substituted with an alkynyl group as defined above, having 2 to 10 carbons, for example, 4-(N- propargyl) piperazinyl, and the like.
  • Moieties wherein the heterocyclic group is substituted on a carbon atom with an alkenyl group are also included. Either portion of the moiety is unsubstituted or substituted.
  • oxo refers to an oxygen that is double bonded to a carbon atom.
  • an "oxo” requires a second bond from the atom to which the oxo is attached. Accordingly, it is understood that oxo cannot be subststituted onto an aryl or heteroaryl ring, unless it forms part of the aromatic system as a tautomer.
  • oligomer refers to a low-molecular weight polymer, whose number average molecular weight is typically less than about 5000 g/mol, and whose degree of polymerization (average number of monomer units per chain) is greater than one and typically equal to or less than about 50.
  • a sulfonamido group is optionally substituted by one or more of the substituents described for alkyl, cycloalkyl, aryl, heteroaryl respectively.
  • Compounds described can contain one or more asymmetric centers and may thus give rise to diastereomers and optical isomers.
  • the present invention includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Compounds may be shown without a definitive stereochemistry at certain positions.
  • the present invention includes all stereoisomers of the disclosed compounds and pharmaceutically acceptable salts thereof. Further, mixtures of
  • stereoisomers as well as isolated specific stereoisomers are also included.
  • the products of such procedures can be a mixture of stereoisomers.
  • the present invention includes all manner of rotamers and conformationally restricted states of an inhibitor of the invention.
  • R', R", R'" and R" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1 -3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.
  • each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present.
  • R' and R" or R" and R'" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring.
  • -NR'R is meant to include, but not be limited to, 1 -pyrrolidinyl, 4 piperazinyl, and 4-morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF 3 and -CH 2 CF 3 ) and acyl (e.g., -C(0)CH 3 , -C(0)CF 3 , -C(0)CH 2 OCH 3 , and the like).
  • haloalkyl e.g., -CF 3 and -CH 2 CF 3
  • acyl e.g., -C(0)CH 3 , -C(0)CF 3 , -C(0)CH 2 OCH 3 , and the like.
  • 0-2 in the context of -S(0) ( o_ 2) - are integers of 0, 1 , and 2.
  • Two of the substituents on adjacent atoms of aryl or heteroaryl ring may optionally form a ring of the formula -T-C(0)-(CRR') q -U-, wherein T and U are independently -NR-, -0-, -CRR'- or a single bond, and q is an integer of from 0 to 3.
  • two of the substituents on adjacent atoms of aryl or heteroaryl ring may optionally be replaced with a substituent of the formula
  • a and B are independently -CRR'-, -0-, -NR-, -S-, -S(O)-, -S(0) 2 -, -S(0) 2 NR'- or a single bond, and r is an integer of from 1 to 4.
  • One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
  • two of the substituents on adjacent atoms of aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR') S - X'-(C"R"') d -, where s and d are independently integers of from 0 to 3, and X' is -0-, -NR'-, -S-, -S(O)- , -S(0) 2 -, or -S(0) 2 NR'-.
  • R, R', R" and R'" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
  • structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by 13 C- or 14 C-enriched carbon are within the scope of this invention.
  • the compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds.
  • the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( 3 H), iodine- 125 ( 125 I) or carbon- 14 ( 14 C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.
  • the present invention provides a method for treating a disease condition associated with a PI3-kinase in a subject.
  • the method typically comprises administering to a subject a therapeutically effective amount of a compound of the invention.
  • the therapeutically effective amount of the subject combination of compounds may vary depending upon the intended application (in vitro or in vivo), or the subjectand disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the term also applies to a dose that will induce a particular response in target cells, e.g., reduction of proliferation or downregulation of activity of a target protein.
  • the specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.
  • the PI3-kinase inhibitor utilized in the subject methods typically exhibits selective inhibition of PI3-kinase a relative to one or more type I phosphatidylinositol 3-kinases (PI3-kinase) including, e.g., PI3-kinase ⁇ , PI3-kinase ⁇ , and PI3-kinase ⁇ .
  • PI3-kinase type I phosphatidylinositol 3-kinases
  • Selective inhibition of PI3-kinase a can be ascertained by an in vitro or an in vivo method. Any assay known in the art may be used, including without limitation, immunoassays, immunoprecipitation, fluorescence or cell-based assays.
  • an in vitro assay is used to determine selective inhibition of PI3-kinase aby an assay which measures the activity of the PBKa protein relative to the activity of another PI3 -kinase such as PI3-kinase ⁇ , PI3-kinase ⁇ , and PI3-kinase ⁇ .
  • a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K may be used to determine an IC50 value for a test compound for PI3 -kinase a and/or any of the other PI3 -kinases.
  • IC50 refers to the half maximal inhibitory concentration of an inhibitorin inhibiting biological or biochemical function. This quantitative measure indicates how much of a particular inhibitor is needed to inhibit a given biological process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half. In other words, it is the half maximal (50%) inhibitory concentration (IC) of a substance (50% IC, or IC50).
  • IC50 refers to the plasma concentration required for obtaining 50%> of a maximum effect in vivo.
  • Determination of IC50 can be made by determining and constructing a dose-response curve and examining the effect of different concentrations of an inhibitor on reversing agonist activity. In vitro assays that are useful in making these determinations are referred to as "in vitro kinase assays.”
  • an in vitro kinase assay includes the use of labeled ATP as phosphodonor, and following the kinase reaction the substrate peptide is captured on an appropriate filter. Unreacted labeled ATP and metabolites are resolved from the radioactive peptide substrate by various techniques, involving trichloroacetic acid precipitation and extensive washing. Addition of several positively charged residues allows capture on phosphocellulose paper followed by washing. Radioactivity incorporated into the substrate peptide is detected by scintillation counting.
  • This assay is relatively simple, reasonably sensitive, and the peptide substrate can be adjusted both in terms of sequence and concentration to meet the assay requirements.
  • a cell-based assay is used to ascertain selective inhibition of PI3-kinase a.
  • an inhibitor can be shown to be selective for PI3-kinase a if it selectively
  • PI3-kinase signal transduction in cells that expressPB-kinase a, preferably in cells that exhibit abnomally high level or activity of PI3-kinase a.
  • PB-kinase a a variety of cells having PI3-kinase a mutations and hence exhibiting such PI3-kinase a abnormalities are known in the art.
  • Non- limiting examples of cell lines harboring such mutations include those that carry point mutations, deletions, substituations, or translation of nucleic acid sequence of the PI3-kinase a gene.
  • cell lines include but are not limited to BT20 (H1047R mutation), MCF-7 (E545K mutation), MDA-MB- 361 (E545K mutation), MDA-MB-453 (HI 047R mutation), T47D (HI 047R mutation), Hec-IA (G1049R mutation) and HCT-116 (H1047R mutation).
  • Other cell lines having mutations in the PI3Ka protein may be used, such as cells harboring mutations in the p85, C2, helical or kinase domains.
  • inhibition of PI3-kinase aactivity can be determined by a reduction in signal transduction of the PI3 -kinase a pathway.
  • a wide variety of readouts can be utilized to establish a reduction of the output of such signaling pathway.
  • Some non-limiting exemplary readouts include (1) a decrease in phosphorylation of Akt at residues, including but not limited to S473 and T308; (2) a decrease in activation of Akt as evidenced by a reduction of phosphorylation of Akt substrates including but not limited to Fox01/03a T24/32, GSK3a/ S21/9, and TSC2 T1462; (3) a decrease in phosphorylation of signaling molecules downstream of PI3-kinase a, including but not limited to ribosomal S6 S240/244, 70S6K T389, and 4EBPl T37/46; (4) inhibition of proliferation of cells including but not limited to normal or neoplastic cells, mouse embryonic fibroblasts, leukemic blast cells, cancer stem cells, and cells that mediate autoimmune reactions; (5) induction of apoptosis of cells or cell cycle arrest; (6) reduction of cell chemotaxis; and (7) an increase in binding of 4EBP1 to e
  • the PI3-kinase a inhibitor selectively inhibits PI3-kinase a relative to one, two or three other type I phosphatidylinositol 3-kinases (PI3-kinases) consisting of PI3-kinase ⁇ , PI3-kinase ⁇ , and PI3-kinase ⁇ .
  • PI3-kinases type I phosphatidylinositol 3-kinases
  • some of the subject inhibitors selectively inhibit PI3-kinase a and PI3-kinase ⁇ as compared to the rest of the type I PI3-kinases.
  • some of the subject inhibitors selectively inhibit PI3-kinase a and PI3-kinase ⁇ as compared to the rest of the type I PI3-kinases. In still yet other embodiments, some of the subject inhibitors selectively inhibit PI3-kinase a and PI3-kinase ⁇ as compared to the rest of the type I PI3- kinases.
  • the subject methods utilizes a PI3-kinase a inhibitor with an IC50 value of about or less than a predetermined value, as ascertained in an in vitro kinase assay.
  • the PI3-kinase a inhibitor inhibits PI3-kinase a with an IC50 value of about 1 nM or less, 2 nM or less, 5 nM or less, 7 nM or less, 10 nM or less, 20 nM or less, 30 nM or less, 40 nM or less, 50 nM or less, 60 nM or less, 70 nM or less, 80 nM or less, 90 nM or less, 100 nM or less, 120 nM or less, 140 nM or less, 150 nM or less, 160 nM or less, 170 nM or less, 180 nM or less, 190 nM or less, 200 nM or less,
  • the PI3-kinase a inhibitor selectively inhibits PI3-kinase a with an IC50 value that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, or 1000 times less than its IC50 value against one, two, or three other type I PB-kinase(s) selected from the group consisting of PI3-kinase ⁇ , PI3-kinase ⁇ , and PI3-kinase ⁇ .
  • the PI3-kinase a inhibitor selectively inhibits PI3-kinase a with an IC50 value that is less than about 1 nM, 2 nM, 5 nM, 7 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 225 nM, 250 nM, 275 nM, 300 nM, 325 nM, 350 nM, 375 nM, 400 nM, 425 nM, 450 nM, 475 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800
  • the PI3-kinase a inhibitor inhibits PI3-kinase a with an IC50 value of about 200 tiM or less as ascertained in an in vitro kinase assay and the IC50 value is at least 5, 10, 15, 20, 25, 50, 100, or 1000 times less than its IC50 value against all other type I PI3-kinases selected from the group consisting of PI3-kinase ⁇ , PI3-kinase ⁇ , and PI3-kinase ⁇ .
  • the PI3-kinase a inhibitor selectively inhibits PI3-kinase a with an IC50 value that is less than about 100 tiM, and said IC50 value is at 5, 10, 15, 20, 25, 50, or 100, 1000 times less than its IC50 value against all other type I PI3-kinases selected from the group consisting of PI3-kinase ⁇ , PI3-kinase ⁇ , and PI3- kinase ⁇ .
  • the PI3-kinase a inhibitor inhibits PI3-kinase a with an IC50 value of about 50 tiM or less as ascertained in an in vitro kinase assay and the IC50 value is at least 5, 10, 15, 20, 25, 50, 100, or 1000 times less than its IC50 value against all other type I PI3-kinases selected from the group consisting of PI3-kinase ⁇ , PI3-kinase ⁇ , and PI3-kinase ⁇ .
  • the PI3-kinase a inhibitor selectively inhibits PI3-kinase a with an IC50 value that is less than about 20 tiM, and said IC50 value is at 5, 10, 15, 20, 25, 50, or 100, 1000 times less than its IC50 value against all other type I PI3-kinases selected from the group consisting of PI3 -kinase ⁇ , PI3 -kinase ⁇ , and PI3 -kinase ⁇ .
  • the PI3-kinase a inhibitor selectively inhibits PI3-kinase a with an IC50 value that is less than about 20 tiM, and said IC50 value is at 5, 10, 15, 20, 25, 50, or 100, 1000 times less than its IC50 value against all other type I PI3-kinases selected from the group consisting of PI3 -kinase ⁇ , PI3 -kinase ⁇ , and PI3 -kinase ⁇ .
  • the PI3-kinase a inhibitor inhibits PI3-kinase a with an IC50 value of about 20 tiM or less as ascertained in an in vitro kinase assay and the IC50 value is at least 100 times less than its IC50 value against all other type I PI3-kinases selected from the group consisting of PI3- kinase ⁇ , PI3-kinase ⁇ , and PI3-kinase ⁇ .
  • the PI3-kinase a inhibitor inhibits PI3-kinase a with an EC50 value of about 10 ⁇ or less, 5 ⁇ or less, 2.5 ⁇ or less, ⁇ M or less, 500nM or less, 100 tiM or less, 75 tiM or less, 50 tiM or less, 25 tiM or less, 10 tiM or less, 5 tiM or less, 1 tiM or less, 500 pM or less, or 100 pM or less as ascertained in an in vitro kinase assay.
  • the PI3-kinase a inhibitor selectively inhibits PI3-kinase a with an EC50 value that is at least 5, 10, 15, 20, 25, 50, 100, or 1000 times less than its EC50 value against one, two or thre other type I PI3-kinases selected from the group consisting of PI3-kinase ⁇ , PI3- kinase ⁇ , and PI3-kinase ⁇ .
  • the PI3-kinase a inhibitor inhibits PI3-kinase a with an EC50 value of about 10 ⁇ or less, 5 ⁇ or less, 2.5 ⁇ or ⁇ , ⁇ ⁇ or less, 500nM or less, 100 nM or less, 75 nM or less, 50 nM or less, 25 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, 500 pM or less, or 100 pM or less as ascertained in an in vitro kinase assay, and such EC50 value is at least 5, 10, 15, 20, 25, 50, or 100, 1000 times less than its EC50 value against one, two or three other type I PI3-kinases selected from the group consisting of PI3-kinase ⁇ , PI3-kinase ⁇ , and PI3-kinase ⁇ .
  • any cells that express PI3-kinase a,mTorCl, mTorC2 and/or Akt can be utilized.
  • specific cell types whose proliferation can be inhibited include fibroblast, cells of skeletal tissue (bone and cartilage), cells of epithelial tissues (e.g. liver, lung, breast, skin, bladder and kidney), cardiac and smooth muscle cells, neural cells (glia and neurones), endocrine cells (adrenal, pituitary, pancreatic islet cells), melanocytes, and many different types of haemopoietic cells (e.g., cells of B-cell or T-cell lineage, and their corresponding stem cells, lymphoblasts).
  • haemopoietic cells e.g., cells of B-cell or T-cell lineage, and their corresponding stem cells, lymphoblasts.
  • cells exhibiting a neoplastic propensity or phenotype are also of interest.
  • diseases involving abnormal functioning of genes include but are not limited to autoimmune diseases, cancer, obesity, hypertension, diabetes, neuronal and/or muscular degenerative diseases, cardiac diseases, endocrine disorders, and any combinations thereof.
  • the subject methods are useful for treating a disease condition associated with PI3-kinase a. Any disease condition that results directly or indirectly from an anbornal activity or expression level of PI3-kinase a can be an intended disease condition.
  • PI3-kinase a A vast diversity of disease conditions associated with PI3-kinase a have been reported. PI3- kinase a has been implicated, for example, in a variety of human cancers. Angiogenesis has been shown to selectively require the a isoform of PI3K in the control of endothelial cell migration.
  • PI3K a Mutations in the gene coding for PI3K a or mutations which lead to upregulation of PI3K a are believed to occur in many human cancers such as lung, stomach, endometrial, ovarian, bladder, breast, colon, brain and skin cancers. Often, mutations in the gene coding for PI3K a are point mutations clustered within several hotspots in helical and kinase domains, such as E542K, E545K, and H1047R. Many of these mutations have been shown to be oncogenic gain-of-function mutations. Because of the high rate of PI3K a mutations, targeting of this pathway provides valuable therapeutic opportunities. While other PI3K isoforms such as PI3K ⁇ or PI3K ⁇ are expressed primarily in hematopoietic cells, PI3K a, along with PI3K ⁇ , is expressed constitutively.
  • Disease conditions associated with PI3-kinase a can also be characterized by abnormally high level of activity and/or expression of downstream messengers of PI3-kinase a.
  • proteins or messengers such as PIP2, PIP3, PDK, Akt, PTEN, PRAS40, GSK-3 , p21, p27 may be present in anbnormal amounts which can be identified by any assays known in the art.
  • the present invention provides for a method comprising: (a) determining the presence in a subject of a mutation in PI3-kinase a that is associated with a disease condition mediated by PI3 -kinase a; and (b) administering to said subject a pharmaceutical composition of the invention.
  • Non- limiting examples of such conditions include but are not limited to Acanthoma,Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblasts leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T- cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Ana
  • Gastrointestinal cancer Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor,
  • Gastrointestinal stromal tumor Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor
  • Lymphangioma Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occul
  • Pheochromocytoma Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lympho
  • the methods of using a PBKa inhibitor described herein are applied to the treatment of heart conditions including atherosclerosis, heart hypertrophy, cardiac myocyte dysfunction, elevated blood pressure and vasoconstriction.
  • the invention also relates to a method of treating diseases related to vasculogenesis or angiogenesis in a mammal that comprises administering to said mammal a therapeutically effective amount of a ⁇ 3 ⁇ inhibitor of the present invention, or any pharmaceutically acceptable salt, ester, prodrug, solvate, hydrate or derivative thereof.
  • said method is for treating a disease selected from the group consisting of tumor angiogenesis, chronic inflammatory disease such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease, skin diseases such as psoriasis, eczema, and scleroderma, diabetes, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, hemangioma, glioma, melanoma, Kaposi's sarcoma and ovarian, breast, lung, pancreatic, prostate, colon and epidermoid cancer.
  • a disease selected from the group consisting of tumor angiogenesis, chronic inflammatory disease such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease, skin diseases such as psoriasis, eczema, and scleroderma
  • diabetes diabetic retinopathy, retinopathy of prematurity
  • age-related macular degeneration hemangio
  • the invention provides for the use of a PI3Ka inhibitor for treating a disease condition associated with PI3-kinase a, including, but not limited to, conditions related to an undesirable, over-active, harmful or deleterious immune response in a mammal, collectively termed "autoimmune disease.”
  • Autoimmune disorders include, but are not limited to, Crohn's disease, ulcerative colitis, psoriasis, psoriatic arthritis, juvenile arthritis and ankylosing spondilitis.
  • Other non- limiting examples of autoimmune disorders include autoimmune diabetes, multiple sclerosis, systemic lupus erythematosus (SLE), rheumatoid spondylitis, gouty arthritis, allergy, autoimmune uveitis, nephrotic syndrome, multisystem autoimmune diseases, autoimmune hearing loss, adult respiratory distress syndrome, shock lung, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis, silicosis
  • Undesirable immune response can also be associated with or result in, e.g., asthma, emphysema, bronchitis, psoriasis, allergy, anaphylaxsis, auto-immune diseases, rhuematoid arthritis, graft versus host disease, transplantation rejection, lung injuries, and lupus erythematosus.
  • the pharmaceutical compositions of the present invention can be used to treat other respiratory diseases including but not limited to diseases affecting the lobes of lung, pleural cavity, bronchial tubes, trachea, upper respiratory tract, or the nerves and muscle for breathing.
  • compositions of the invention can be further used to treat multiorgan failure.
  • the invention also providesmethods of using a PBKa inhibitor for the treatment of liver diseases (including diabetes), pancreatitis or kidney disease (including proliferative),
  • the invention also provides a method of using a ⁇ 3 ⁇ inhibitor for the treatment of sperm motility.
  • the invention further provides a method of using a PI3Ka inhibitor for the treatment of neurological or neurodegenerative diseases including, but not limited to, Alzheimer's disease,
  • the invention further provides a method of using a PI3Ka inhibitor for the prevention of blastocyte implantation in a mammal.
  • the invention also relates to a method of using a PI3Ka inhibitor for treating a disease related to vasculogenesis or angiogenesis in a mammal which can manifest as tumor angiogenesis, chronic inflammatory disease such as rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, skin diseases such as psoriasis, eczema, and scleroderma, diabetes, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, hemangioma, glioma, melanoma, Kaposi's sarcoma and ovarian, breast, lung, pancreatic, prostate, colon and epidermoid cancer.
  • chronic inflammatory disease such as rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, skin diseases such as psoriasis, eczema, and scleroderma
  • diabetes diabetic retinopathy, retinopathy of prem
  • the invention further provides a method of using a PI3Ka inhibitor for the treatment of disorders involving platelet aggregation or platelet adhesion, including but not limited to Bernard- Soulier syndrome, Glanzmann's thrombasthenia, Scott's syndrome, von Willebrand disease,
  • methods of using a PI3Ka inhibitor are provided for treating a disease which is skeletal muscle atrophy, skeletal muscle hypertrophy, leukocyte recruitment in cancer tissue, invasion metastasis, melanoma, Kaposi's sarcoma, acute and chronic bacterial and viral infections, sepsis, glomerulo sclerosis, glomerulo, nephritis, or progressive renal fibrosis.
  • Certain embodiments contemplate a human subject such as a subject that has been diagnosed as having or being at risk for developing or acquiring adisease condition associated with PI3-kinase a.
  • a non-human subject for example a non-human primate such as a macaque, chimpanzee, gorilla, vervet, orangutan, baboon or other non-human primate, including such non- human subjects that can be known to the art as preclinical models, including preclinical models for inflammatory disorders.
  • non-human subject that is a mammal, for example, a mouse, rat, rabbit, pig, sheep, horse, bovine, goat, gerbil, hamster, guinea pig or other mammal.
  • subject or biological source can be a non-mammalian vertebrate, for example, another higher vertebrate, or an avian, amphibian or reptilian species, or another subject or biological source.
  • a transgenic animal is utilized.
  • a transgenic animal is a non-human animal in which one or more of the cells of the animal includes a nucleic acid that is non- endogenous (i.e., heterologous) and is present as an extrachromosomal element in a portion of its cell or stably integrated into its germ line DNA (i.e., in the genomic sequence of most or all of its cells).
  • a nucleic acid that is non- endogenous (i.e., heterologous) and is present as an extrachromosomal element in a portion of its cell or stably integrated into its germ line DNA (i.e., in the genomic sequence of most or all of its cells).
  • the invention provides a compound of Formula I or Formula III:
  • one of X 1 and X 2 is N-R'-W'-R ⁇ Z or C-R'-W'-R ⁇ Z, and the other is N or C-E 1 ;
  • X 5 is N or C-E 5 ;
  • X a is NR 31 , S or O;
  • loalkenylheteroalkyl C 2 _ioalkenylheterocyclyl, C 2 _ioalkenyl-C 3 _gcycloalkyl, C 2 _ioalkynyl-C 3 _ gcycloalkyl, C 2 _ioalkynylaryl, C 2 _ioalkynylheteroaryl, C 2 _ioalkynylheteroalkyl, C 2 _
  • R'and R 5 are independently -C 3 _gcycloalkyl, -aryl, -heteroaryl, -aralkyl, -heteroaralkyl, or - heterocyclyl, each of which is unsubstituted or substituted by one or more independent R 3 substituents;
  • W 1 is a bond or where one or more CH 2 groups have been replaced by -0-, -NR 7 -, -S(0)o_ 2 -,-C(0)-,-C(0)N(R 7 )-, -N(R 7 )C(0)-, -N(R 7 )S(0)-, -N(R 7 )S(0) 2 - -C(0)0- - CH(R 7 )N(C(0)OR 8 )-, -CH(R 7 )N(C(0)R 8 )-, -CH(R 7 )N(S0 2 R 8 )-, -CH(R 7 )N(R 8 )-, - CH(R 7 )C(0)N(R 8 )-, -CH(R 7 )N(R 8 )C(0)-, -CH(R 7 )N(R 8 )S(0)-, or -CH(R 7 )N(R 8 )S(0) 2 -;
  • loalkenylheteroalkyl C 2 _ioalkenylheterocyclyl, C 2 _ioalkenyl-C 3 _gcycloalkyl, C 2 _ioalkynyl-C 3 _ gcycloalkyl, C 2 _ioalkynylaryl, C 2 _ioalkynylheteroaryl, C 2 _ioalkynylheteroalkyl, C 2 _
  • each of R 7 and R 8 is independently hydrogen, Ci.ioalkyl, C 2 _ioalkenyl, aryl, heteroaryl, heterocyclyl or C3_iocycloalkyl, each of which except for hydrogen is unsubstituted or is substituted by one or more independent R 6 substituents;
  • the invention provides a compound of Formula II:
  • X 5 is N or C-E 5 ;
  • X 6 is N;
  • X 7 and X 8 are independently N or C; wherein no more than three of X 5 , X 6 , X 7 and X 8 are N;
  • one of X 9 and X 10 is C-R'-W'-R ⁇ Z, and the other is C-E 2 ;
  • X a is NR 31 , O or S;
  • loalkenylheteroalkyl C 2 _ioalkenylheterocyclyl, C 2 _ioalkenyl-C 3 _gcycloalkyl, C 2 _ioalkynyl-C 3 _ gcycloalkyl, C 2 _ioalkynylaryl, C 2 _ioalkynylheteroaryl, C 2 _ioalkynylheteroalkyl, C 2 .
  • R ⁇ nd R 5 are independently -C 3 _gcycloalkyl, -aryl, -heteroaryl, -aralkyl, -heteroaralkyl, or - heterocyclyl, each of which is unsubstituted or substituted by one or more independent R 3 substituents;
  • W 1 is a bond or Ci_ 6 alkyl, where one or more CH 2 groups have been replaced by -0-, -NR 7 -, -S(O) 0 . 2 -,-C(0)-,-C(0)N(R 7 )-, -N(R 7 )C(0)-, -N(R 7 )S(0)-, -N(R 7 )S(0) 2 - -C(0)0- - CH(R 7 )N(C(0)OR 8 )-, -CH(R 7 )N(C(0)R 8 )-, -CH(R 7 )N(S0 2 R 8 )-, -CH(R 7 )N(R 8 )-, - CH(R 7 )C(0)N(R 8 )-, -CH(R 7 )N(R 8 )C(0)-, -CH(R 7 )N(R 8 )S(0)-, or -CH(R 7 )N(R 8 )S(0) 2 -;
  • each of R , R , and R is independently H or Cuoalkyl , wherein the Ci.ioalkyl is unsubstituted or is substituted with one or more aryl, heteroalkyl, heterocyclyl, or heteroaryl substituent, wherein each of said aryl, heteroalkyl, heterocyclyl, or heteroaryl substituent is unsubstituted or is substituted with one or more halo, -OH, - Cuoalkyl, -CF 3 , -O-aryl, -OCF 3 , -OCuoalkyl, -NH 2 , - N(Ci_i 0 alkyl)(Ci_ l oalkyl), - NH(Ci.ioalkyl), - NH( aryl), -NR 34 R 35 , -C(O)(Ci_i 0 aikyl), -C(O)(Ci.i 0 alkyl-aryl),
  • NR 34 R 35 -NO 2 , -CN, -S(0) o- 2 Cuoalkyl, -S(0) ⁇ 2 aryl, -S0 2 NR 34 R 35 , -S0 2 NR 31 R 32 , Cuoalkyl, C 2 _ l oalkenyl, C 2 _ioalkynyl, aryl-Cuoalkyl, aryl-C 2 _ioalkenyl, aryl-C 2 _ioalkynyl, heteroaryl-Ci.ioalkyl, heteroaryl-C 2 _ioalkenyl, or heteroaryl-C 2 _ioalkynyl, each of which is unsubstituted or is substituted with one or more independent halo, cyano, nitro, -OCi.ioalkyl, Cuoalkyl, C 2 _ioalkenyl, C 2 _ioalkynyl, hal
  • Z is a group capable of reacting with a cysteine residue.
  • X a is O.
  • X 9 is C-R 1 -W 1 -R 5 -Z. In certain embodiments of compounds of Formula I, X 1 is C-R 1 -W 1 -R 5 -Z.
  • Z is -W 3 -R 9 , where: W 3 is a bond or Ci.galkyl, where zero, one, two or three CH 2 groups have been replaced by -0-, -NR 7 -, -S(0)o_ 2 -,- C(0)-,-C(0)N(R 7 )-, -N(R 7 )C(0)-, -N(R 7 )S(0)-, -N(R 7 )S(0) 2 - -C(0)0- -CH(R 7 )N(C(0)OR 8 )-, -CH(R 7 )N(C(0)R 8 )-, -CH(R 7 )N(S0 2 R 8 )-, -CH(R 7 )N(R 8 )-, -CH(R 7 )C(0)N(R 8 )-, - CH(R 7 )N(R 8 )C(0)-, -CH(R 7 )N(R
  • R 1 is an optionally substituted ring selected from phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, a 7-10 membered saturated or partially unsaturated bicyclic carbocyclic ring, a 7-12 membered saturated or partially unsaturated bridged bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 7-10 membered saturated or partially unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered bicyclic aryl ring, a 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1 -4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 1 is an optionally substituted 6-membered saturated heterocyclic ring having one or two heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 1 is a piperazinyl or piperidinyl ring.
  • R 1 is piperdinyl.
  • R 1 is substituted with one or more oxo groups.
  • R 1 is thiomorpholine optionally substituted with one or more oxo groups.
  • R 1 is an optionally substituted 6-membered partially unsaturated heterocyclic ring having one or two heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 1 is tetrahydropyridyl. In some embodiments, R 1 is phenyl. In some embodiments, R 1 is an optionally substituted 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R 1 is pyridyl. In some embodiments, R 1 is an optionally substituted 3-7 membered saturated or partially unsaturated carbocyclic ring. In certain embodiments, R 1 is cyclohexyl. In some embodiments, R 1 is a 7-12 membered saturated or partially unsaturated bridged bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 5 is an optionally substituted ring selected from phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, a 7-10 membered saturated or partially unsaturated bicyclic carbocyclic ring, a 7-12 membered saturated or partially unsaturated bridged bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 7-10 membered saturated or partially unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered bicyclic aryl ring, a 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1 -4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 5 is an optionally substituted 6-membered saturated heterocyclic ring having one or two heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 5 is a piperazinyl or piperidinyl ring.
  • R 5 is piperdinyl.
  • R 5 is substituted with one or more oxo groups.
  • R 5 is thiomorpholine optionally substituted with one or more oxo groups.
  • R 5 is an optionally substituted 6-membered partially unsaturated heterocyclic ring having one or two heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 5 is tetrahydropyridyl. In some embodiments, R 5 is phenyl. In some embodiments, R 5 is an optionally substituted 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R 5 is pyridyl. In some embodiments, R 5 is an optionally substituted 3-7 membered saturated or partially unsaturated carbocyclic ring. In certain embodiments, R 5 is cyclohexyl. In some embodiments, R 5 is a 7-12 membered saturated or partially unsaturated bridged bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Z is -W 3 -R 9 .
  • W 3 is a bond or Ci.galkyl, where zero, one, two or three CH 2 groups have been replaced by -0-, -NR 7 -, -S(O) 0 - 2 -,-C(O)-,-C(O)N(R 7 )-, -N(R 7 )C(0)-, - N(R 7 )S(0)-, -N(R 7 )S(0) 2 - -C(0)0- -CH(R 7 )N(C(0)OR 8 )-, -CH(R 7 )N(C(0)R 8 )-, - CH(R 7 )N(S0 2 R 8 )-, -CH(R 7 )N(R 8 )-, -CH(R 7 )C(0)N(R 8 )-, -CH(R 7 )N(R 8 )C(0)-, -CH(R 7 )N(R 8 )C(0)-,
  • W 3 is a covalent bond. In other embodiments, W 3 is a bivalent Ci_ galkyl group. In certain embodiments, W 3 is -CH 2 -. In some embodiments, W 3 is a covalent bond, - CH 2 , -NH-, -CH 2 NH-, -NHCH 2 -, -NHC(O)-, -NHC(0)CH 2 OC(0)-, -CH 2 NHC(0)-, -NHS0 2 , - NHS0 2 CH 2 -, or -S0 2 NH-.
  • W 3 is a bivalent Ci.galkyl group wherein at least one methylene unit of W 3 is replaced by -C(O)-. In certain embodiments, W 3 is a bivalent Ci.galkyl group wherein at least two methylene units of W 3 are replaced by -C(O)-. In some embodiments, W 3 is -C(0)CH 2 CH 2 C(0)-, -C(0)CH 2 NHC(0)-, -C(0)CH 2 NHC(0)CH 2 CH 2 C(0)-, or -C(0)CH 2 CH 2 CH 2 NHC(0)CH 2 CH 2 C(0)-.
  • W 3 is a bivalent Ci.galkyl group wherein one methylene unit of W 3 is replaced by -S(0) 2 -. In certain embodiments, W 3 is a bivalent Ci.galkyl group wherein at least one methylene unit of W3 is replaced by S(0) 2 - and at least one methylene unit of W 3 is replaced by - C(O)-. In certain embodiments, W 3 is a bivalent Ci.galkyl group wherein at least one methylene unit of W 3 is replaced by S(0) 2 - and at least two methylene units of W 3 are replaced by -C(O)-. In some embodiments, W 3 is -S(0) 2 CH 2 CH 2 NHC(0)CH 2 CH 2 C(0)- or -S(0) 2 CH 2 CH 2 NHC(0)-.
  • W 3 is a bivalent C 2 .galkenyl and zero, one or two additional methylene units of W 3 are independently replaced by -NRC(O)-, -C(0)NR 7 -, -N(R 7 )S0 2 , -S0 2 N(R 7 )-, -S-, -S(O)- , -S0 2 -, -OC(O)-, -C(0)0-, cyclopropylene, -0-, -N(R 7 )-, or -C(O)-, wherein R 7 is H or unsubstituted or substituted
  • W 3 is a bivalent C 2 .galkenyl and at least one methylene unit of W 3 is replaced by -C(O)-, -NR 7 C(0)-, -C(0)NR 7 -, -N(R 7 )S0 2 -, -S0 2 N(R 7 )-, -S-, -S(O)-, -S0 2 -, -OC(O)-, - C(0)0-, and zero, one or two additional methylene units of W 3 are independently replaced by cyclopropylene, -0-, -N(R 7 )-, or -C(O)- wherein R 7 is H or unsubstituted or substituted Ci_ 6 alkyl.
  • W 3 is a bivalent C 2 .galkenyl and at least one methylene unit of W 3 is replaced by -C(O)-, and zero, one or two additional methylene units of W 3 are independently replaced by cyclopropylene, -0-, -N(R 7 )-, or -C(O)- wherein R 7 is H or unsubstituted or substituted Ci_ 6 alkyl.
  • W 3 is a bivalent C 2 _galkynyl and zero, one or two additional methylene units of W 3 are independently replaced by -NRC(O)-, -C(0)NR 7 -, -N(R 7 )S0 2 -, -S0 2 N(R 7 )-, -S-, - S(O)-, -S0 2 -, -OC(O)-, -C(0)0-, cyclopropylene, -0-, -N(R 7 )-, or -C(O)-, wherein R 7 is H or unsubstituted or substituted
  • W 3 is -C ⁇ C-, -C ⁇ CCH 2 N(isopropyl)-, - NHC(0)C ⁇ CCH 2 CH 2 -, -CH 2 -C ⁇ C-CH 2 -, -C ⁇ CCH 2 0-, -CH 2 C(0)C ⁇ C-, -C(0)
  • W 3 is a bivalent C 2 _galkyl wherein one CH2 unit is replaced by cyclopropylene and zero, one, or two additional CH2 units are independently replaced by -NRC(O)-, - C(0)NR 7 -, -N(R 7 )S0 2 , -S0 2 N(R 7 )-, -S-, -S(O)-, -S0 2 -, -OC(O)-, -C(0)0-, cyclopropylene, -0-, - N(R 7 )-, or -C(O)-.
  • W 3 is -NHC(0)-cyclopropylene-S0 2 - and -NHC(0)-cyclopropylene.
  • R 9 is H, C 3 _gcycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or heterocyclyl, where R 9 is substituted by one, two, three or four independent R x substituents.
  • R 9 is H.
  • R 9 is C 2 _ 6 alkenyl. In other embodiments, R 9 is C 2 _ 6 alkynyl.
  • R 9 is C 3 _gcycloalkyl.
  • R 9 is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Each such R 9 is substituted with 1 or more R x groups.
  • R 9 is cyclobutyl or cyclopentyl substituted with 1 or 2 R x groups.
  • R 9 is C 4- gcycloalkenyl.
  • R 9 is cyclobutenyl, cyclop entenyl, or cyclohexenyl. Each such R 9 is substituted with 1 or more R x groups.
  • R 9 is cyclobutenyl or cyclopentenyl substituted with 1 or 2 R x groups.
  • R 9 is heterocyclyl.
  • R 9 is epoxide, oxetane, pyrrolidine, piperidine, or piperazine each of which is substituted with at least one R x group.
  • R 9 is pyrrolidine or piperidine, substitued with 1, 2, 3 or 4 R x groups.
  • R 9 is saturated heterocyclyl. In other embodiments, R 9 is unsaturated heterocyclyl.
  • R 9 is pyrrolidinone, pyrrolidinedione (e.g. succinimide), pyrrole-2,5-dione, or pyrrolone.
  • R 9 is phenyl, additionally substituted by 1, 2, 3 or 4 R x groups.
  • R 9 is a monocyclic heteroaryl group such as a pyridine, pyridazine, pyrimidine, or pyrazine, substituted by 1, 2, 3 or 4 R x groups.
  • R 9 is triazole, imidazole, isoxazole, thiazole, imidazole, pyrazole, pyrrole, thiophene, furan or oxadiazole, substituted by 1 , 2, or 3 R x groups.
  • R 9 is a bicyclic aryl or heteroaryl group, substituted by 1, 2, or 3 R x groups.
  • Each R x is independently selected from -Z'-Z 2 , oxo, NO 2 , halogen, CN, a suitable leaving group, or optionally substituted with oxo, halogen, NO 2 , or CN, wherein Z 1 is a bond or a Ci_ 6 saturated or unsaturated, straight or branched, hydrocarbon chain, wherein one or two methylene units of Z 1 are optionally and independently replaced by -N(R 7 )-, -S-, -0-, -C(O)-, -OC(O)-, -C(0)0-, -SO-, -SO 2 -, -N(R 7 )C(0)-, -C(0)N(R 7 )-, -N(R 7 )S0 2 -, or -S0 2 N(R 7 )-; and Z 2 is hydrogen or Ci_ 6 alkyl optionally substituted with oxo, halogen, NO 2 , or CN.
  • Z 1 is C 2 _ 6 alkenyl wherein one or two methylene units of z e optionally and independently replaced by -N(R 7 )-, -S-, -0-, -C(O)-, -OC(O)-, -C(0)0-, -SO-, - SO 2 -, -N(R 7 )C(0)-, -C(0)N(R 7 )-, -N(R 7 )S0 2 -, or -S0 2 N(R 7 )-; and Z 2 is hydrogen or d_ 6 alkyl optionally substituted with oxo, halogen, NO 2 , or CN.
  • R x can further be a leaving group subject to nucleophilic displacement.
  • groups include chemical moieties reactive with thiol groups such as halogen, alkoxy, sulphonyloxy,
  • R x can be chloro, iodo, bromo, fluoro, acetoxy, methanesulfonyloxy, tosyloxy, triflyloxy, nitro- phenylsulfonyloxy, and bromo-phenylsulfonyloxy.
  • Z is selected from the group consisting of:
  • X 1 is N. In other embodiments of the compound of Formula I, X 1 and X 5 are N.
  • E 3 , E 4 and E 6 are hydrogen.
  • the compound has the Formula:
  • the compound of Formula I has the Formula:
  • X 6 is N.
  • X 5 is C-E 5 .
  • E 2 , E 3 and E 4 are H.
  • X s is C.
  • X 8 is N.
  • the compound of Formula II has the Formula:
  • the compound has the Formula:
  • the compounds of the invention may be synthesized via a reaction scheme represented generally in Schemes A', A" and B'.
  • the synthesis proceeds via coupling a compound of Formula A with a compound of Formula B to yield a compound of Formula C.
  • the coupling step is typically catalyzed by using a palladium catalyst, including but not limited to palladium tetrakis
  • the coupling is generally performed in the presence of a suitable base, a nonlimiting example being sodium carbonate.
  • a suitable solvent for the reaction is aqueous dioxane.
  • a compound of Formula A for use in Scheme A' or A" has a structure of Formula A, wherein Ti is halo including bromo, chloro, fluoro, and iodo, and wherein the remaining substituents are defined for Formulas I and II of compounds of the invention.
  • X is either O or S, and the benzoxazole or benzothiazole moiety can be attached at the4-, 5-, 6- or 7- position.
  • G is hydrogen or R G i, wherein R G i is alkyl, alkenyl, or aryl.
  • B(OG) 2 is taken together to form a 5- or 6- membered cyclic moiety.
  • the compound of Formula B is a compound having a structure of Formula E:
  • G is H or R G i;
  • R G I is alkyl, alkenyl, or aryl.
  • B(OG) 2 is taken together to forma 5- or 6- membered cyclic moiety; and
  • RQ 2 is H, tert- butyl carbamate,or acyl.
  • Scheme C depicts an exemplary scheme for synthesizing a compound of Formula B' or, optionally, Formula B" for use in Reaction Scheme C.
  • M is a heterocyclic moiety such as a benzoxazolyl or benzothiazolyl moiety as described by Formula B.
  • This reaction proceeds via reacting a compound of Formula D with a trialkyl borate or a boronic acid derivative to produce a compound of Formula B'.
  • the trialkyl borate includes but is not limited to triisopropyl borate and the boronic acid derivative includes but is not limited to bis(pinacolato)diboron.
  • the reaction typically is run in the presence of a base, a nonlimiting example being potassium acetate.
  • the reaction may be run in a solvent such as dioxane or tetrahydrofuran.
  • a compound of Formula D for use in Scheme C is a compound wherein T 2 is halo or another leaving group, and M is as defined above.
  • the compound of Formula B' may further be converted to a compound of Formula B"by treatment with an acid such as hydrochloric acid.
  • deprotection of a substituent e.g., removal of Boc protection from an amino substituent
  • a substituent on the benzoxazolyl moiety i.e. Mi of Formula C
  • compositions are typically formulated to provide a therapeutically effective amount of a compound of the present invention as the active ingredient, or a
  • compositions contain pharmaceutically acceptable salt and/or coordination complex thereof, and one or more pharmaceutically acceptable excipients, carriers, including inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants.
  • compositions can be administered alone or in combination with one or more other agents, which are also typically administered in the form of pharmaceutical compositions.
  • the one or more compounds of the invention and other agent(s) may be mixed into a preparation or both components may be formulated into separate preparations to use them in combination separately or at the same time.
  • the concentration of one or more compounds provided in the pharmaceutical compositions of the present invention is less than 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w/w, w/v or v/v.
  • the concentration of one or more compounds of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%
  • the concentration of one or more compounds of the invention is in the range from approximately 0.0001% to approximately 50%, approximately 0.001% to approximately 40 %, approximately 0.01% to approximately 30%, approximately 0.02% to approximately 29%, approximately 0.03% to approximately 28%, approximately 0.04% to approximately 27%, approximately 0.05% to approximately 26%, approximately 0.06% to approximately 25%, approximately 0.07% to approximately 24%, approximately 0.08% to approximately 23%, approximately 0.09% to approximately 22%, approximately 0.1% to approximately 21%,
  • the concentration of one or more compounds of the invention is in the range from approximately 0.001% to approximately 10%, approximately 0.01% to approximately 5%, approximately 0.02% to approximately 4.5%, approximately 0.03% to approximately 4%, approximately 0.04% to approximately 3.5%, approximately 0.05% to approximately 3%, approximately 0.06% to approximately 2.5%, approximately 0.07%> to approximately 2%, approximately 0.08%> to approximately 1.5%, approximately 0.09% to approximately 1%, approximately 0.1% to approximately 0.9% w/w, w/v or v/v.
  • the amount of one or more compounds of the invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009
  • the amount of one or more compounds of the invention is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g
  • the amount of one or more compounds of the invention is in the range of 0.0001-10 g, 0.0005-9 g, 0.001-8 g, 0.005-7 g, 0.01-6 g, 0.05-5 g, 0.1-4 g, 0.5-4 g, or 1-3 g.
  • the compounds according to the invention are effective over a wide dosage range.
  • dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used.
  • An exemplary dosage is 10 to 30 mg per day. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician.
  • a pharmaceutical composition of the invention typically contains an active ingredient (e.g., a compound) of the present invention or a pharmaceutically acceptable salt and/or coordination complex thereof, and one or more pharmaceutically acceptable excipients, carriers, including but not limited to inert solid diluents and fillers, diluents, sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants.
  • compositions for oral administration In some embodiments, the invention provides a pharmaceutical composition for oral administration containing a compound of the invention, and a pharmaceutical excipient suitable for oral administration.
  • the invention provides a solid pharmaceutical composition for oral administration containing: (i) an effective amount of a compound of the invention; optionally (ii) an effective amount of a second agent; and (iii) a pharmaceutical excipient suitable for oral administration.
  • the composition further contains: (iv) an effective amount of a third agent.
  • the pharmaceutical composition may be a liquid pharmaceutical composition suitable for oral consumption.
  • Pharmaceutical compositions of the invention suitable for oral administration can be presented as discrete dosage forms, such as capsules, cachets, or tablets, or liquids or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid, an oil-in- water emulsion, or a water-in-oil liquid emulsion.
  • Such dosage forms can be prepared by any of the methods of pharmacy, but all methods include the step of bringing the active ingredient into association with the carrier, which constitutes one or more necessary ingredients.
  • compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
  • a tablet can be prepared by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free- flowing form such as powder or granules, optionally mixed with an excipient such as, but not limited to, a binder, a lubricant, an inert diluent, and/or a surface active or dispersing agent.
  • Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • This invention further encompasses anhydrous pharmaceutical compositions and dosage forms comprising an active ingredient, since water can facilitate the degradation of some compounds.
  • water may be added (e.g., 5%) in the pharmaceutical arts as a means of simulating long- term storage in order to determine characteristics such as shelf- life or the stability of formulations over time.
  • Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions.
  • Pharmaceutical compositions and dosage forms of the invention which contain lactose can be made anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.
  • An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained.
  • anhydrous compositions may be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits.
  • suitable packaging include, but are not limited to, hermetically sealed foils, plastic or the like, unit dose containers, blister packs, and strip packs.
  • An active ingredient can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
  • the carrier can take a wide variety of forms depending on the form of preparation desired for administration.
  • any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used in the case of oral solid preparations, in some embodiments without employing the use of lactose.
  • suitable carriers include powders, capsules, and tablets, with the solid oral preparations. If desired, tablets can be coated by standard aqueous or nonaqueous techniques.
  • Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixtures thereof.
  • natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrol
  • suitable fillers for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.
  • talc calcium carbonate
  • microcrystalline cellulose e.g., powdere., powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.
  • Disintegrants may be used in the compositions of the invention to provide tablets that disintegrate when exposed to an aqueous environment. Too much of a disintegrant may produce tablets which may disintegrate in the bottle. Too little may be insufficient for disintegration to occur and may thus alter the rate and extent of release of the active ingredient(s) from the dosage form. Thus, a sufficient amount of disintegrant that is neither too little nor too much to detrimentally alter the release of the active ingredient(s) may be used to form the dosage forms of the compounds disclosed herein. The amount of disintegrant used may vary based upon the type of formulation and mode of administration, and may be readily discernible to those of ordinary skill in the art.
  • Disintegrants that can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums or mixtures thereof.
  • Lubricants which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, or mixtures thereof.
  • Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, or mixtures thereof.
  • a lubricant can optionally be added, in an amount of less than about 1 weight percent of the pharmaceutical composition.
  • the active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if so desired, emulsifying and/or suspending agents, together with such diluents as water, ethanol, propylene glycol, glycerin and various combinations thereof.
  • the tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.
  • Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.
  • Surfactant which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. That is, a mixture of hydrophilic surfactants may be employed, a mixture of lipophilic surfactants may be employed, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant may be employed.
  • a suitable hydrophilic surfactant may generally have an HLB value of at least 10, while suitable lipophilic surfactants may generally have an HLB value of or less than about 10.
  • An empirical parameter used to characterize the relative hydrophilicity and hydrophobicity of non-ionic amphiphilic compounds is the hydrophilic-lipophilic balance ("HLB" value).
  • HLB hydrophilic-lipophilic balance
  • Surfactants with lower HLB values are more lipophilic or hydrophobic, and have greater solubility in oils, while surfactants with higher HLB values are more hydrophilic, and have greater solubility in aqueous solutions.
  • Hydrophilic surfactants are generally considered to be those compounds having an HLB value greater than about 10, as well as anionic, cationic, or zwitterionic compounds for which the HLB scale is not generally applicable.
  • lipophilic (i.e., hydrophobic) surfactants are compounds having an HLB value equal to or less than about 10.
  • HLB value of a surfactant is merely a rough guide generally used to enable formulation of industrial, pharmaceutical and cosmetic emulsions.
  • Hydrophilic surfactants may be either ionic or non-ionic. Suitable ionic surfactants include, but are not limited to, alkylammonium salts; fusidic acid salts; fatty acid derivatives of amino acids, oligopeptides, and polypeptides; glyceride derivatives of amino acids, oligopeptides, and
  • polypeptides lecithins and hydrogenated lecithins; lysolecithins and hydrogenated lysolecithins; phospholipids and derivatives thereof; lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acyl lactylates; mono- and di- acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof.
  • ionic surfactants include, by way of example: lecithins, lysolecithin, phospholipids, lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof.
  • Ionic surfactants may be the ionized forms of lecithin, lysolecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine,
  • lysophosphatidylcholine lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidic acid, lysophosphatidylserine, PEG-phosphatidylethanolamine, PVP -phosphatidylethanolamine, lactylic esters of fatty acids, stearoyl-2-lactylate, stearoyl lactylate, succinylated monoglycerides, mono/diacetylated tartaric acid esters of mono/diglycerides, citric acid esters of mono/diglycerides, cholylsarcosine, caproate, caprylate, caprate, laurate, myristate, palmitate, oleate, ricinoleate, linoleate, linolenate, stearate, lauryl sulfate, teracecyl sulfate, docusate, la
  • Hydrophilic non-ionic surfactants may include, but are not limited to, alkylglucosides
  • hydrophilic-non-ionic surfactants include, without limitation, PEG- 10 laurate, PEG- 12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32 dilaurate, PEG- 12 oleate, PEG- 15 oleate, PEG-20 oleate, PEG-20 dioleate, PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG- 15 stearate, PEG-32 distearate, PEG-40 stearate, PEG- 100 stearate, PEG-20 dilaurate, PEG-25 glyceryl trioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30 glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate, PEG-30 glyceryl oleate, PEG-30 glyce
  • Suitable lipophilic surfactants include, by way of example only: fatty alcohols; glycerol fatty acid esters; acetylated glycerol fatty acid esters; lower alcohol fatty acids esters; propylene glycol fatty acid esters; sorbitan fatty acid esters; polyethylene glycol sorbitan fatty acid esters; sterols and sterol derivatives; polyoxyethylated sterols and sterol derivatives; polyethylene glycol alkyl ethers; sugar esters; sugar ethers; lactic acid derivatives of mono- and di-glycerides; hydrophobic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids and sterols; oil-soluble vitamins/vitamin derivatives; and mixtures thereof.
  • preferred lipophilic surfactants include glycerol fatty acid esters, propylene glycol fatty acid esters, and mixtures thereof, or are hydrophobic transesterification products of a polyol with at least one member of the group consisting of vegetable oils, hydrogenated vegetable oils, and triglycerides.
  • the composition may include a solubilizer to ensure good solubilization and/or dissolution of the compound of the present invention and to minimize precipitation of the compound of the present invention. This can be especially important for compositions for non-oral use, e.g., compositions for injection.
  • a solubilizer may also be added to increase the solubility of the hydrophilic drug and/or other components, such as surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion.
  • solubilizers include, but are not limited to, the following: alcohols and polyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediols and isomers thereof, glycerol, pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide, polyethylene glycol, polypropylene glycol, polyvinylalcohol, hydroxypropyl
  • alcohols and polyols such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediols and isomers thereof, glycerol, pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide, polyethylene glycol, polypropylene glycol, polyvinylalcohol,
  • solubilizers may also be used. Examples include, but not limited to, triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, dimethylacetamide, N-methylpyrrolidone, N- hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrins, ethanol, polyethylene glycol 200-100, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide. Particularly preferred solubilizers include sorbitol, glycerol, triacetin, ethyl alcohol, PEG-400, glycofurol and propylene glycol.
  • the amount of solubilizer that can be included is not particularly limited.
  • the amount of a given solubilizer may be limited to a bioacceptable amount, which may be readily determined by one of skill in the art.
  • the solubilizer can be in a weight ratio of 10%, 25%o, 50%), 100%o, or up to about 200%> by weight, based on the combined weight of the drug, and other excipients.
  • solubilizer may also be used, such as 5%>, 2%>, 1%) or even less.
  • the solubilizer may be present in an amount of about 1%> to about 100%, more typically about 5%> to about 25%> by weight.
  • the composition can further include one or more pharmaceutically acceptable additives and excipients.
  • additives and excipients include, without limitation, detackifiers, anti-foaming agents, buffering agents, polymers, antioxidants, preservatives, chelating agents, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.
  • an acid or a base may be incorporated into the composition to facilitate processing, to enhance stability, or for other reasons.
  • pharmaceutically acceptable bases include amino acids, amino acid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide, sodium hydrogen carbonate, aluminum hydroxide, calcium carbonate, magnesium hydroxide, magnesium aluminum silicate, synthetic aluminum silicate, synthetic hydrocalcite, magnesium aluminum hydroxide, diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine, triethylamine, triisopropanolamine, trimethylamine,
  • tris(hydroxymethyl)aminomethane and the like.
  • bases that are salts of a pharmaceutically acceptable acid, such as acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p- toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid, uric acid, and the like.
  • a pharmaceutically acceptable acid such as acetic acid, acrylic acid, adipic acid,
  • Salts of polyprotic acids such as sodium phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate can also be used.
  • the cation can be any convenient and pharmaceutically acceptable cation, such as ammonium, alkali metals, alkaline earth metals, and the like.
  • Example may include, but not limited to, sodium, potassium, lithium, magnesium, calcium and ammonium.
  • Suitable acids are pharmaceutically acceptable organic or inorganic acids.
  • suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, nitric acid, boric acid, phosphoric acid, and the like.
  • suitable organic acids include acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acids, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid, uric acid and the like.
  • compositions for injection In some embodiments, the invention provides a pharmaceutical composition for injection containing a compound of the present invention and a pharmaceutical excipient suitable for injection. Components and amounts of agents in the compositions are as described herein.
  • Aqueous solutions in saline are also conventionally used for injection.
  • Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and the like (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils may also be employed.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, for the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • Sterile injectable solutions are prepared by incorporating the compound of the present invention in the required amount in the appropriate solvent with various other ingredients as enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • certain desirable methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Pharmaceutical compositions for topical (e.g.. transdermal) delivery In some embodiments, the invention provides a pharmaceutical composition for transdermal delivery containing a compound of the present invention and a pharmaceutical excipient suitable for transdermal delivery.
  • compositions of the present invention can be formulated into preparations in solid, semisolid, or liquid forms suitable for local or topical administration, such as gels, water soluble jellies, creams, lotions, suspensions, foams, powders, slurries, ointments, solutions, oils, pastes,
  • suppositories sprays, emulsions, saline solutions, dimethylsulfoxide (DMSO)-based solutions.
  • DMSO dimethylsulfoxide
  • carriers with higher densities are capable of providing an area with a prolonged exposure to the active ingredients.
  • a solution formulation may provide more immediate exposure of the active ingredient to the chosen area.
  • compositions also may comprise suitable solid or gel phase carriers or excipients, which are compounds that allow increased penetration of, or assist in the delivery of, therapeutic molecules across the stratum corneum permeability barrier of the skin. There are many of these penetration- enhancing molecules known to those trained in the art of topical formulation.
  • humectants e.g., urea
  • glycols e.g., propylene glycol
  • alcohols e.g., ethanol
  • fatty acids e.g., oleic acid
  • surfactants e.g., isopropyl myristate and sodium lauryl sulfate
  • pyrrolidones e.g., isopropyl myristate and sodium lauryl sulfate
  • pyrrolidones e.glycerol monolaurate, sulfoxides, terpenes (e.g., menthol)
  • amines amides, alkanes, alkanols, water, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • transdermal delivery devices patches
  • Such transdermal patches may be used to provide continuous or discontinuous infusion of a compound of the present invention in controlled amounts, either with or without another agent.
  • transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
  • compositions for inhalation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable
  • compositions are administered by the oral or nasal respiratory route for local or systemic effect.
  • Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices that deliver the formulation in an appropriate manner. [00292] Other pharmaceutical compositions.
  • compositions may also be prepared from compositions described herein and one or more pharmaceutically acceptable excipients suitable for sublingual, buccal, rectal, intraosseous, intraocular, intranasal, epidural, or intraspinal administration. Preparations for such pharmaceutical compositions are well-known in the art.
  • Administration of the compounds or pharmaceutical composition of the present invention can be effected by any method that enables delivery of the compounds to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, intraarterial, subcutaneous, intramuscular, intravascular, intraperitoneal or infusion), topical (e.g. transdermal application), rectal administration, via local delivery by catheter or stent or through inhalation. Compounds can also abe administered intraadiposally or intrathecally.
  • an effective dosage is in the range of about 0.001 to about 100 mg per kg body weight per day, preferably about 1 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to 7 g/day, preferably about 0.05 to about 2.5 g/day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, e.g. bydividing such larger doses into several small doses for administration throughout the day.
  • a compound of the invention is administered in a single dose.
  • Such administration will be by injection, e.g., intravenous injection, in order to introduce the agent quickly.
  • injection e.g., intravenous injection
  • other routes may be used as appropriate.
  • a single dose of a compound of the invention may also be used for treatment of an acute condition.
  • a compound of the invention is administered in multiple doses. Dosing may be about once, twice, three times, four times, five times, six times, or more than six times per day. Dosing may be about once a month, once every two weeks, once a week, or once every other day. In another embodiment a compound of the invention and another agent are administered together about once per day to about 6 times per day. In another embodiment the administration of a compound of the invention and an agent continues for less than about 7 days. In yet another embodiment the administration continues for more than about 6, 10, 14, 28 days, two months, six months, or one year. In some cases, continuous dosing is achieved and maintained as long as necessary.
  • Administration of the compounds of the invention may continue as long as necessary.
  • a compound of the invention is administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 days.
  • a compound of the invention is administered for less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day.
  • a compound of the invention is administered chronically on an ongoing basis, e.g., for the treatment of chronic effects.
  • An effective amount of a compound of the invention may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including rectal, buccal, intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneous ly, orally, topically, or as an inhalant.
  • compositions of the invention may also be delivered via an impregnated or coated device such as a stent, for example, or an artery-inserted cylindrical polymer.
  • an impregnated or coated device such as a stent, for example, or an artery-inserted cylindrical polymer.
  • a compound of the invention may be administered, for example, by local delivery from the struts of a stent, from a stent graft, from grafts, or from the cover or sheath of a stent.
  • a compound of the invention is admixed with a matrix.
  • a matrix may be a polymeric matrix, and may serve to bond the compound to the stent.
  • Polymeric matrices suitable for such use include, for eample, lactone-based polyesters or copolyesters such as polylactide, polycaprolactonglycolide, polyorthoesters, polyanhydrides, polyaminoacids, polysaccharides, polyphosphazenes, poly (ether-ester) copolymers (e.g. PEO-PLLA);
  • lactone-based polyesters or copolyesters such as polylactide, polycaprolactonglycolide, polyorthoesters, polyanhydrides, polyaminoacids, polysaccharides, polyphosphazenes, poly (ether-ester) copolymers (e.g. PEO-PLLA);
  • polydimethylsiloxane poly(ethylene-vinylacetate), acrylate-based polymers or copolymers (e.g. polyhydroxyethyl methylmethacrylate, polyvinyl pyrrolidinone), fluorinated polymers such as polytetrafluoroethylene and cellulose esters.
  • Suitable matrices may be nondegrading or may degrade with time, releasing the compound or compounds.
  • Compounds of the invention may be applied to the surface of the stent by various methods such as dip/spin coating, spray coating, dip-coating, and/or brush-coating. The compounds may be applied in a solvent and the solvent may be allowed to evaporate, thus forming a layer of compound onto the stent.
  • the compound may be located in the body of the stent or graft, for example in microchannels or micropores. When implanted, the compound diffuses out of the body of the stent to contact the arterial wall.
  • Such stents may be prepared by dipping a stent manufactured to contain such micropores or microchannels into a solution of the compound of the invention in a suitable solvent, followed by evaporation of the solvent. Excess drug on the surface of the stent may be removed via an additional brief solvent wash.
  • compounds of the invention may be covalently linked to a stent or graft. A covalent linker may be used which degrades in vivo, leading to the release of the compound of the invention.
  • bio-labile linkage may be used for such a purpose, such as ester, amide or anhydride linkages.
  • Compounds of the invention may additionally be administered intravascularly from a balloon used during angioplasty. Extravascular administration of the compounds via the pericard or via advential application of formulations of the invention may also be performed to decrease restenosis.
  • the compounds of the invention may be administered in dosages. It is known in the art that due to intersubject variability in compound pharmacokinetics, individualization of dosing regimen is necessary for optimal therapy. Dosing for a compound of the invention may be found by routine experimentation in light of the instant disclosure.
  • the subject pharmaceutical composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution, suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository.
  • the pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages.
  • the pharmaceutical composition will include a conventional pharmaceutical carrier or excipient and a compound according to the invention as an active ingredient. In addition, it may include other medicinal or pharmaceutical agents, carriers, adjuvants, etc.
  • Exemplary parenteral administration forms include solutions or suspensions of active compound in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.
  • the subject is a human in need of treatment for cancer, or a precancerous condition or lesion, wherein the cancer is preferably NSCL, breast, colon or pancreatic cancer.
  • Subjects that can be treated with combination treatments of the present invention, or pharmaceutically acceptable salt, ester, prodrug, solvate, hydrate or derivatives of thetherapeutic agents, according to the methods of this invention include, for example, subjects that have been diagnosed as having psoriasis; restenosis; atherosclerosis; BPH; breast cancer such as a ductal carcinoma in duct tissue in a mammary gland, medullary carcinomas, colloid carcinomas, tubular carcinomas, and inflammatory breast cancer; ovarian cancer, including epithelial ovarian tumors such as adenocarcinoma in the ovary and an adenocarcinoma that has migrated from the ovary into the abdominal cavity; uterine cancer; cervical cancer such as adenocarcinoma in the cer
  • Oral cavity and oropharyngeal cancer such as, hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, and oropharyngeal cancer
  • stomach cancer such as lymphomas, gastric stromal tumors, and carcinoid tumors
  • testicular cancer such as germ cell tumors (GCTs), which include seminomas and nonseminomas, and gonadal stromal tumors, which include Leydig cell tumors and Sertoli cell tumors
  • thymus cancer such as to thymomas, thymic carcinomas, Hodgkin disease, non-Hodgkin lymphomas carcinoids or carcinoid tumors
  • rectal cancer and colon cancer.
  • the invention also relates to a method of treating diabetes in a mammal that comprises administering to said mammal a therapeutically effective amount of a combination treatment of the present invention.
  • the combination treatments described herein may be used to treat acne.
  • arteriosclerosis is a general term describing any hardening of medium or large arteries.
  • Atherosclerosis is a hardening of an artery specifically due to an atheromatous plaque.
  • Glomerulonephritis is a primary or secondary autoimmune renal disease characterized by inflammation of the glomeruli. It may be asymptomatic, or present with hematuria and/or proteinuria. There are many recognized types, divided in acute, subacute or chronic glomerulonephritis. Causes are infectious (bacterial, viral or parasitic pathogens), autoimmune or paraneoplastic.
  • the combination treatments described herein may be used for the treatment of bursitis, lupus, acute disseminated encephalomyelitis (ADEM), addison's disease, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hepatitis, coeliac disease, Crohn's disease, diabetes mellitus (type 1), goodpasture's syndrome, graves' disease, guillain-barre syndrome (GBS), hashimoto's disease, inflammatory bowel disease, lupus erythematosus, myasthenia gravis, opsoclonus myoclonus syndrome (OMS), optic neuritis, ord's thyroiditis,ostheoarthritis, uveoretinitis, pemphigus, polyarthritis, primary biliary cirrhosis, reiter's syndrome, takayasu's arteritis, temporal arteritis, warm autoimmune hemolytic
  • the invention also relates to a method of treating a cardiovascular disease in a mammal that comprises administering to said mammal a therapeutically effective amount of a combination treatment of the present invention.
  • cardiovascular conditions include, but are not limited to, atherosclerosis, restenosis, vascular occlusion, carotid obstructive disease, or ischemic conditions.
  • the present invention provides methods of disrupting the function of a leukocyte or disrupting a function of an osteoclast.
  • the method includes contacting the leukocyte or the osteoclast with a function disrupting amount of a combination treatment of the invention.
  • methods are provided for treating ophthalmic disease by applying one or more of the subject combination treatments to the eye of a subject.
  • Methods are further provided for administering the combination treatments of the present invention via eye drop, intraocular injection, intravitreal injection, topically, or through the use of a drug eluting device, microcapsule, implant, or microfluidic device.
  • combination treatments are administered with a carrier or excipient that increases the intraocular penetrance of the compound such as an oil and water emulsion with colloid particles having an oily core surrounded by an interfacial film.
  • the colloid particles include at least one cationic agent and at least one non- ionic sufactant such as a poloxamer, tyloxapol, a polysorbate, a polyoxyethylene castor oil derivative, a sorbitan ester, or a polyoxyl stearate.
  • the cationic agent is an alkylamine, a tertiary alkyl amine, a quarternary ammonium compound, a cationic lipid, an amino alcohol, a biguanidine salt, a cationic compound or a mixture thereof.
  • the cationic agent is a biguanidine salt such as chlorhexidine, polyaminopropyl biguanidine, phenformin, alkylbiguanidine, or a mixture thereof.
  • the quaternary ammonium compound is a benzalkonium halide, lauralkonium halide, cetrimide, hexadecyltrimethylammonium halide, tetradecyltrimethylammonium halide, dodecyltrimethylammonium halide, cetrimonium halide, benzethonium halide, behenalkonium halide, cetalkonium halide, cetethyldimonium halide, cetylpyridinium halide, benzododecinium halide, chlorallyl methenamine halide, rnyristylalkonium halide, stearalkonium halide or a mixture of two or more thereof.
  • cationic agent is a benzalkonium chloride, lauralkonium chloride, benzododecinium bromide, benzethenium chloride, hexadecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, dodecyltrimethylammonium bromide or a mixture of two or more thereof.
  • the oil phase is mineral oil and light mineral oil, medium chain triglycerides (MCT), coconut oil; hydrogenated oils comprising hydrogenated cottonseed oil, hydrogenated palm oil, hydrogenate castor oil or hydrogenated soybean oil; polyoxyethylene hydrogenated castor oil derivatives comprising poluoxyl-40 hydrogenated castor oil, polyoxyl-60 hydrogenated castor oil or polyoxyl- 100 hydrogenated castor oil.
  • MCT medium chain triglycerides
  • coconut oil hydrogenated oils comprising hydrogenated cottonseed oil, hydrogenated palm oil, hydrogenate castor oil or hydrogenated soybean oil
  • polyoxyethylene hydrogenated castor oil derivatives comprising poluoxyl-40 hydrogenated castor oil, polyoxyl-60 hydrogenated castor oil or polyoxyl- 100 hydrogenated castor oil.
  • the invention further provides methods of modulating a PI3K and/or mTOR kinase activity by contacting the kinase with an effective amount of a compound of the invention. Modulating can be inhibiting or activating kinase activity. In some embodiments, the invention provides methods of inhibiting kinase activity by contacting the kinase with an effective amount of a compound of the invention in solution. In some embodiments, the invention provides methods of inhibiting the kinase activity by contacting a cell, tissue, or organ that expresses the kinase of interest.
  • the invention provides methods of inhibiting kinase activity in subject including but not limited to rodents and mammal (e.g., human) by administering into the subject an effective amount of a composition comprising a compound of the invention.
  • the percentage of inhibiting exceeds 50%, 60%, 70%, 80%, or 90%.
  • the present invention also provides methods for further combination therapies in which, in addition to a compound of the invention, an agent known to modulate other pathways, or other components of the same pathway, or even overlapping sets of target enzymes is used or a
  • such therapy includes but is not limited to the compound with chemotherapeutic agents, therapeutic antibodies, and radiation treatment, to provide, where desired, a synergistic or additive therapeutic effect.
  • compositions can be used in combination with commonly prescribed drugs including but not limited to Enbrel ® , Remicade ® , Humira ® , Avonex ® , and Rebif ® .
  • the subject compounds or pharmaceutical compositions can be administered in combination with commonly prescribed drugs including but not limited to Xolair ® , Advair ® , Singulair ® , and Spiriva ® .
  • the compounds of the invention may be formulated or administered in conjunction with other agents that act to relieve the symptoms of inflammatory conditions such as encephalomyelitis, asthma, and the other diseases described herein.
  • agents include non-steroidal anti-inflammatory drugs (NSAIDs), e.g., acetylsalicylic acid; ibuprofen; naproxen; indomethacin; nabumetone; tolmetin; etc.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • Corticosteroids are used to reduce inflammation and suppress activity of the immune system.
  • the most commonly prescribed drug of this type is Prednisone. Chloroquine (Aralen) or
  • hydroxychloroquine may also be very useful in some individuals with lupus. They are most often prescribed for skin and joint symptoms of lupus. Azathioprine (Imuran) and
  • cyclophosphamide suppress inflammation and tend to suppress the immune system.
  • Other agents e.g., methotrexate and cyclosporin are used to control the symptoms of lupus.
  • Anticoagulants are employed to prevent blood from clotting rapidly. They range from aspirin at very low dose which prevents platelets from sticking, to heparin/coumadin.
  • this invention also relates to methods and pharmaceutical compositions for inhibiting abnormal cell growth in a mammal which comprises an amount of an inhibitor of the present invention, or a pharmaceutically acceptable salt, ester, prodrug, solvate, hydrate or derivative thereof, in combination with an amount of an anti-cancer agent (e.g., a chemotherapeutic agent).
  • an anti-cancer agent e.g., a chemotherapeutic agent.
  • Many chemotherapeutics are presently known in the art and can be used in combination with the compounds of the invention.
  • the chemotherapeutic is selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones, angiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, and anti-androgens.
  • chemotherapeutic agents include cytotoxic agents, and non-peptide small molecules such as Gleevec (Imatinib Mesylate), Velcade (bortezomib), Casodex (bicalutamide), Iressa
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide
  • alkyl sulfonates such as busulfan, improsulfan and piposulfan
  • aziridines such as benzodopa, carboquone, meturedopa, and uredopa
  • ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine
  • nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard
  • nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine
  • antibiotics such as anthracyclins, actinomycins and bleomycins including aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, CasodexTM, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin,
  • antibiotics such as anthracyclins, actinomycins and bleomycins including aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, Casodex
  • marcellomycin mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens such as calusterone, dromostanolone propionate, epitiostan
  • chemotherapeutic cell conditioners are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti- estrogens including for example tamoxifen (NolvadexTM), raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum or platinum analogs and complexes such as cisplatin and carboplatin; anti- microtubule such as diterpenoids, including paclitaxel and docetaxel, or Vinca alkaloids including vinblastine, vincristine, vinflunine,
  • mitomycin C mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase I and II inhibitors including camptothecins (e.g., camptothecin-11), topotecan, irinotecan, and epipodophyllotoxins; topoisomerase inhibitor RFS 2000; epothilone A or B; difluoromethylornithine (DMFO); histone deacetylase inhibitors; compounds which induce cell differentiation processes; gonadorelin agonists; methionine aminopeptidase inhibitors; compounds targeting/decreasing a protein or lipid kinase activity; compounds which target, decrease or inhibit the activity of a protein or lipid phosphatase; anti-androgens; bisphosphonates; biological response modifiers; antiproliferative antibodies; hepara
  • the compounds or pharmaceutical composition of the present invention can be used in combination with commonly prescribed anti-cancer drugs such as Herceptin ® , Avastin ® , Erbitux ® , Rituxan ® , Taxol ® , Arimidex ® , Taxotere ® , and Velcade ® . Further information on compounds which may be used in conjunction with the compounds of the invention is provided below.
  • Proteasome inhibitors include compounds which target, decrease or inhibit the activity of the proteasome.
  • Compounds which target, decrease or inhibit the activity of the proteasome include e.g., Bortezomid (VelcadeTM)and MLN 341.
  • Matrix metalloproteinase inhibitors include, but are not limited to, collagen peptidomimetic and nonpeptidomimetic inhibitors, tetracycline derivatives, e.g., hydroxamate peptidomimetic inhibitor batimastat and its orally bioavailable analogue marimastat (BB-2516), prinomastat (AG3340), metastat (NSC 683551 ) BMS- 279251, BAY 12-9566, TAA211, MMI270B or AAJ996.
  • MMP matrix metalloproteinase inhibitors
  • FMS-like tyrosine kinase inhibitors e.g., compounds targeting, decreasing or inhibiting the activity of FMS-like tyrosine kinase receptors (Flt- 3R); interferon, 1 -b-D-arabinofuransylcytosine (ara-c) and bisulfan; and ALK inhibitors e.g., compounds which target, decrease or inhibit anaplastic lymphoma kinase.
  • FMS-like tyrosine kinase receptors are especially compounds, proteins or antibodies which inhibit members of the Flt-3R receptor kinase family, e.g., PKC412, midostaurin, a staurosporine derivative, SU11248 and MLN518.
  • Hsp90 inhibitors include compounds such as 17-AAG (17-allylaminogeldanamycin, NSC330507), 17-DMAG (17-dimethylaminoethylamino-17-demethoxy-geldanamycin, NSC707545), IPI-504, CNFIOIO, CNF2024, CNFIOIO from Conforma Therapeutics; temozo- lomide
  • TEMODAL® kinesin spindle protein inhibitors, such as SB715992 or SB743921 from
  • MEK inhibitors such as ARRY142886 from Array PioPharma, AZD6244 from AstraZeneca, PD181461 from Pfizer, leucovorin, EDG binders, antileukemia compounds, ribonucleotide reductase inhibitors, S- adenosylmethionine decarboxylase inhibitors, antiproliferative antibodies or other chemotherapeutic compounds.
  • Histone deacetylase inhibitors include compounds which inhibit a histone deacetylase and which possess antiproliferative activity. This includes compounds disclosed in WO 02/22577, especially N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(lH-indol-3-yl)ethyl]- amino]methyl]phenyl]-2E-2-propenamide, N-hydroxy-3-[4-[[[2-(2-methyl-lH-indol-3-yl)-ethyl] amino]methyl]phenyl]-2E-2-propenamide and pharmaceutically acceptable salts thereof. It further especially includes Suberoylanilide hydroxamic acid (SAHA).
  • SAHA Suberoylanilide hydroxamic acid
  • Bisphosphonates for use in combination with the compounds of the invention include, but are not limited to, etridonic, clodronic, tiludronic, pamidronic, alendronic, ibandronic, risedronic and zoledronic acid.
  • Compounds of the invention may also be used in conjunction with compounds targeting or decreasing a protein or lipid kinase activity, a protein or lipid phosphatase activity, or further anti- angiogenic compounds.
  • Such compounds include, but are not limited to, protein tyrosine kinase and/or serine and/or threonine kinase inhibitors or lipid kinase inhibitors, e.g.,: compounds targeting, decreasing or inhibiting the activity of the platelet- derived growth factor-receptors (PDGFR), such as compounds which target, decrease or inhibit the activity of PDGFR, especially compounds which inhibit the PDGF receptor, e.g., a N-phenyl-2-pyrimidine-amine derivative, e.g., imatinib, SU101, SU6668 and GFB-1 11 ; compounds targeting, decreasing or inhibiting the activity of the fibroblast growth factor-receptors (FGFR); compounds targeting, decreasing or inhibiting the activity of
  • compounds targeting, decreasing or inhibiting the activity of the C-kit receptor tyrosine kinases - such as compounds which target, decrease or inhibit the activity of the c-Kit receptor tyrosine kinase family, especially compounds which inhibit the c-Kit receptor, e.g., imatinib; compounds targeting, decreasing or inhibiting the activity of members of the c-Abl family, their gene- fusion products (e.g., BCR-AbI kinase) and mutants, such as compounds which target decrease or inhibit the activity of c-Abl family members and their gene fusion products, e.g., a N-phenyl-2- pyrimidine-amine derivative, e.g., imatinib or nilotinib (AMN107); PD180970; AG957; NSC 680410; PD173955 from ParkeDavis; or dasatinib (BMS-354825); compounds targeting, decreasing
  • a tyrphostin is preferably a low molecular weight (Mr ⁇ 1500) compound, or a pharmaceutically acceptable salt thereof, especially a compound selected from the benzylidenemalomtrile class or the S-arylbenzenemalonirile or bisubstrate quinoline class of compounds, more especially any compound selected from the group consisting of Tyrphostin
  • A23/RG-50810 AG 99; Tyrphostin AG 213; Tyrphostin AG 1748; Tyrphostin AG 490; Tyrphostin B44; Tyrphostin B44 (+) enantiomer; Tyrphostin AG 555; AG 494; Tyrphostin AG 556, AG957 and adaphostin (4- ⁇ [(2,5- dihydroxyphenyl)methyl]amino ⁇ -benzoic acid adamantyl ester; NSC 680410, adaphostin).
  • Compounds of the invention may also be used in combination with compounds targeting, decreasing or inhibiting the activity of the epidermal growth factor family of receptor tyrosine kinases (EGFR, ErbB2, ErbB3, ErbB4 as homo- or heterodimers) and their mutants, such as compounds which target, decrease or inhibit the activity of the epidermal growth factor receptor family are especially compounds, proteins or antibodies which inhibit members of the EGF receptor tyrosine kinase family, e.g., EGF receptor, ErbB2, ErbB3 and ErbB4 or bind to EGF or EGF related ligands, and are in particular those compounds, proteins or monoclonal antibodies generically and specifically disclosed in WO 97/02266, e.g., the compound of ex.
  • EGFR epidermal growth factor family of receptor tyrosine kinases
  • ErbB2, ErbB3, ErbB4 as homo- or heterodimers
  • Non-receptor kinase angiogenesis inhibitors may also be useful in conjunction with the compounds of the present invention.
  • Angiogenesis in general is linked to erbB21EGFR signaling since inhibitors of erbB2 and EGFR have been shown to inhibit angiogenesis, primarily VEGF expression. Accordingly, non-receptor tyrosine kinase inhibitors may be used in combination with the compounds of the present invention.
  • anti-VEGF antibodies which do not recognize VEGFR (the receptor tyrosine kinase), but bind to the ligand; small molecule inhibitors of integrin (alphav beta3) that will inhibit angiogenesis; endostatin and angiostatin (non-RTK) may also prove useful in combination with the disclosed compounds.
  • VEGFR the receptor tyrosine kinase
  • small molecule inhibitors of integrin alphav beta3
  • endostatin and angiostatin non-RTK
  • Compounds which target, decrease or inhibit the activity of a protein or lipid phosphatase include e.g., inhibitors of phosphatase 1, phosphatase 2A, or CDC25, e.g., okadaic acid or a derivative thereof.
  • Compounds which induce cell differentiation processes are e.g., retinoic acid, ⁇ - ⁇ - or ⁇ - tocopherol or a- ⁇ - or ⁇ -tocotrienol.
  • Cyclooxygenase inhibitors include, but are not limited to, e.g., Cox-2 inhibitors, 5-alkyl substituted 2-arylaminophenylacetic acid and derivatives, such as celecoxib (CELEBREX), rofecoxib (VIOXX), etoricoxib, valdecoxib or a 5-alkyl-2- arylaminophenylacetic acid, e.g., 5-methyl-2-(2'-chloro-6'-fluoroanilino)phenyl acetic acid, and lumiracoxib.
  • Cox-2 inhibitors such as celecoxib (CELEBREX), rofecoxib (VIOXX), etoricoxib, valdecoxib or a 5-alkyl-2- arylaminophenylacetic acid, e.g., 5-methyl-2-(2'-chloro-6'-fluoroanilino)phenyl acetic acid, and lumiracoxi
  • Heparanase inhibitors includes compounds which target, decrease or inhibit heparin sulfate degradation, including, but not limited to, PI-88.
  • Biological response modifiers include lymphokines and interferons, e.g., interferon ⁇ .
  • Inhibitors of Ras oncogenic isoforms include H-Ras, K-Ras, N-Ras, and other compounds which target, decrease or inhibit the oncogenic activity of Ras.
  • Farnesyl transferase inhibitors include, but are not limited to, e.g., L-744832, DK8G557 and Rl 15777 (Zarnestra).
  • Telomerase inhibitors include compounds which target, decrease or inhibit the activity of telomerase.
  • Compounds which target, decrease or inhibit the activity of telomerase are especially compounds which inhibit the telomerase receptor, e.g., telomestatin.
  • Methionine aminopeptidase inhibitors are, for example, compounds which target, decrease or inhibit the activity of methionine aminopeptidase.
  • Compounds which target, decrease or inhibit the activity of methionine aminopeptidase are e.g., bengamide or a derivative thereof.
  • Antiproliferative antibodies include, but are not limited to, trastuzumab (HerceptinTM), Trastuzumab-DMl,erbitux, bevacizumab (AvastinTM), rituximab (Rituxan®), PR064553 (anti-CD40) and 2C4 Antibody.
  • antibodies is meant e.g., intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least 2 intact antibodies, and antibodies fragments so long as they exhibit the desired biological activity.
  • compounds of the invention can be used in combination with standard leukemia therapies, especially in combination with therapies used for the treatment of AML.
  • compounds of the invention can be administered in combination with, e.g., farnesyl transferase inhibitors and/or other drugs useful for the treatment of AML, such as Daunorubicin, Adriamycin, Ara-C, VP- 16, Teniposide, Mitoxantrone, Idarubicin, Carboplatinum and PKC412.
  • Antileukemic compound for use in combination with compounds of the invention include, for example, Ara-C, a pyrimidine analog, which is the 2'-alpha-hydroxy ribose (arabinoside) derivative of deoxycytidine. Also included is the purine analog of hypoxanthine, 6-mercaptopurine (6-MP) and fludarabine phosphate.
  • HDAC histone deacetylase
  • SAHA suberoylanilide hydroxamic acid
  • HDAC inhibitors include MS275, SAHA, FK228 (formerly FR901228), Trichostatin A and compounds disclosed in US 6,552,065, in particular, A/-hydroxy-3-[4-[[[2-(2-methyl-l H-indol-3-yl)-ethyl]- amino]methyl]phenyl]-2E-2- propenamide, or a pharmaceutically acceptable salt thereof and A/-hydroxy-3-[4-[(2-hydroxyethyl) ⁇ 2- (l/-/-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide, or a pharmaceutically acceptable salt thereof, e.g., the lactate salt.
  • Somatostatin receptor antagonists include compounds which target, treat or inhibit the somatostatin receptor such as octreotide, and SOM230 (pasireotide).
  • Tumor cell damaging approaches include approaches such as ionizing radiation, e.g., ionizing radiation that occurs as either electromagnetic rays (such as X-rays and gamma rays) or particles (such as alpha and beta particles). Ionizing radiation is provided in, but not limited to, radiation therapy and is known in the art. See Hellman, Principles of Radiation Therapy, Cancer, in Principles and Practice of Oncology, Devita et al., Eds., 4th Edition, Vol. 1, pp. 248-275 (1993).
  • EDG binders includes immunosuppressants that modulate lymphocyte recirculation, such as FTY720.
  • Ribonucleotide reductase inhibitors include pyrimidine or purine nucleoside analogs including, but not limited to, fludarabine and/or cytosine arabinoside (ara-C), 6-thioguanine, 5- fluorouracil, cladribine, 6-mercaptopurine (especially in combination with ara-C against ALL) and/or pentostatin.
  • ara-C cytosine arabinoside
  • 6-thioguanine 5- fluorouracil
  • cladribine cladribine
  • 6-mercaptopurine especially in combination with ara-C against ALL
  • pentostatin especially in combination with ara-C against ALL
  • Ribonucleotide reductase inhibitors are e.g., hydroxyurea or 2-hydroxy-l /-/-isoindole- 1,3-dione derivatives, such as PL-1, PL-2, PL-3, PL-4, PL-5, PL-6, PL-7 or PL-8 mentioned in Nandy et al., Acta Oncologica, Vol. 33, No. 8, pp. 953-961 (1994).
  • S-adenosylmethionine decarboxylase inhibitors include, but are not limited to the compounds disclosed in US 5,461,076.
  • VEGF vascular endothelial growth factor
  • WO 98/35958 e.g., l-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a
  • ANGIOSTATIN described by O'Reilly et al., Cell, Vol. 79, pp. 315-328 (1994); ENDOSTATIN, described by O'Reilly et al., Cell, Vol. 88, pp. 277-285 (1997); anthranilic acid amides; ZD4190; ZD6474; SU5416; SU6668; bevacizumab; or anti- VEGF antibodies or anti-VEGF receptor antibodies, e.g., rhuMAb and RHUFab, VEGF aptamer e.g., Macugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibody, Angiozyme (RPI 4610) and Bevacizumab (AvastinTM).
  • ENDOSTATIN described by O'Reilly et al., Cell, Vol. 88, pp. 277-285 (1997)
  • anthranilic acid amides ZD4190; ZD6474;
  • the compounds of the invention are also useful as co-therapeutic compounds for use in combination with other drug substances such as anti- inflammatory, bronchodilatory or antihistamine drug substances, particularly in the treatment of obstructive or inflammatory airways diseases such as those mentioned hereinbefore, for example as potentiators of therapeutic activity of such drugs or as a means of reducing required dosaging or potential side effects of such drugs.
  • An inhibitor of the invention may be mixed with the other drug substance in a fixed pharmaceutical composition or it may be administered separately, before, simultaneously with or after the other drug substance.
  • the invention includes a combination of an inhibitor of the invention as described with an anti-inflammatory, bronchodilatory, antihistamine or anti-tussive drug substance, said compound of the invention and said drug substance being in the same or different pharmaceutical composition.
  • Suitable anti-inflammatory drugs include steroids, in particular glucocorticosteroids such as budesonide, beclamethasone dipropionate, fluticasone propionate, ciclesonide or mometasone furoate, or steroids described in WO 02/88167, WO 02/12266, WO 02/100879, WO 02/00679 (especially those of Examples 3, 1 1, 14, 17, 19, 26, 34, 37, 39, 51, 60, 67, 72, 73, 90, 99 and 101 ), WO
  • Non-steroidal glucocorticoid receptor agonists such as those described in WO 00/00531, WO 02/10143, WO 03/082280, WO 03/082787, WO 03/104195, WO 04/005229; LTB4 antagonists such LY29311 1, CGS025019C, CP- 195543, SC-53228, BILL 284, ONO 4057, SB 209247 and those described in US 5451700; LTD4 antagonists such as montelukast and zafirlukast; PDE4 inhibitors such cilomilast (Ariflo® GlaxoSmithKline), Roflumilast (Byk Gulden),V-l 1294A (Napp), BAY19-8004 (Bayer), SCH-351591 (Schering- Plough), Arofylline
  • WO 02/22630 WO 02/96462, WO 03/086408, WO 04/ 039762, WO 04/039766, WO 04/045618 and WO 04/046083;
  • A2b antagonists such as those described in WO 02/42298; and beta-2 adrenoceptor agonists such as albuterol (salbutamol), metaproterenol, terbutaline, salmeterol fenoterol, procaterol, and especially, formoterol and pharmaceutically acceptable salts thereof, and compounds (in free or salt or solvate form) of formula I of WO 0075114, which document is incorporated herein by reference, preferably compounds of the Examples thereof, as well as compounds (in free or salt or solvate form) of formula I of WO 04/16601, and also compounds of WO 04/033412.
  • Suitable bronchodilatory drugs include anticholinergic or antimuscarinic compounds, in particular ipratropium bromide, oxitropium bromide, tiotropium salts and CHF 4226 (Chiesi), and glycopyrrolate, but also those described in WO 01/041 18, WO 02/51841, WO 02/53564, WO 03/00840, WO 03/87094, WO 04/05285, WO 02/00652, WO 03/53966, EP 424021, US 5171744, US 3714357, WO 03/33495 and WO 04/018422.
  • Suitable antihistamine drug substances include cetirizine hydrochloride, acetaminophen, clemastine fumarate, promethazine, loratidine, desloratidine, diphenhydramine and fexofenadine hydrochloride, activastine, astemizole, azelastine, ebastine, epinastine, mizolastine and tefenadine as well as those disclosed in WO 03/099807, WO 04/026841 and JP 2004107299.
  • Other useful combinations of compounds of the invention with anti-inflammatory drugs are those with antagonists of chemokine receptors, e.g., CCR-1, CCR-2, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7, CCR-8, CCR-9 and CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, particularly CCR- 5 antagonists such as Schering-Plough antagonists SC-351 125, SCH- 55700 and SCH-D, Takeda antagonists such as TAK-770, and CCR-5 antagonists described in US 6166037 (particularly claims 18 and 19), WO 00/66558 (particularly claim 8), WO 00/66559 (particularly claim 9), WO 04/018425 and WO 04/026873.
  • chemokine receptors e.g., CCR-1, CCR-2, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7, CCR-8, CCR-9 and CCR10
  • Anti-microtubule or anti-mitotic agents include phase specific agents active against the microtubules of tumor cells during M or the mitosis phase of the cell cycle.
  • anti- microtubule agents include, but are not limited to, diterpenoids and vinca alkaloids.
  • Diterpenoids which are derived from natural sources, are phase specific anti-cancer agents that operate at the G2/M phases of the cell cycle. It is believed that the diterpenoids stabilize the ⁇ -tubulin subunit of the microtubules, by binding with this protein. Disassembly of the protein appears then to be inhibited with mitosis being arrested and cell death following.
  • diterpenoids examples include, but are not limited to, paclitaxel and its analog docetaxel.
  • Paclitaxel 5P,20-epoxy-l,2a,4,7P,10P,13a-hexa- hydroxytax-1 l-en-9-one 4, 10-diacetate 2-benzoate 13-ester with (2R,3S)-N-benzoyl-3- phenylisoserine; is a natural diterpene product isolated from the Pacific yew tree Taxus brevifolia and is commercially available as an injectable solution TAXOL®. It is a member of the taxane family of terpenes.
  • Paclitaxel has been approved for clinical use in the treatment of refractory ovarian cancer in the United States and for the treatment of breast cancer. It is a potential candidate for treatment of neoplasms in the skin and head and neck carcinomas. The compound also shows potential for the treatment of polycystic kidney disease, lung cancer and malaria.
  • Treatment of subjects with paclitaxel results in bone marrow suppression (multiple cell lineages, Ignoff, R. J. et. al, Cancer Chemotherapy Pocket Guide, 1998) related to the duration of dosing above a threshold concentration (50 tiM) (Kearns, C. M.
  • Docetaxel (2R,3S)-N-carboxy-3-phenylisoserine, N-tert-butyl ester, 13-ester with 5P-20-epoxy- l,2a,4,7 ,10 ,13a-hexahydroxytax-l-l-en-9-one 4-acetate 2-benzoate, trihydrate; is commercially available as an injectable solution as TAXOTERE®.
  • Docetaxel is indicated for the treatment of breast cancer.
  • Docetaxel is a semisynthetic derivative of paclitaxel q.v., prepared using a natural precursor, 10-deacetyl-baccatin III, extracted from the needle of the European Yew tree.
  • the dose limiting toxicity of docetaxel is neutropenia.
  • BCL-2 inhibitors Other compounds that can regulate apoptosis (e.g., BCL-2 inhibitors) can be used in conjunction.
  • Vinca alkaloids include phase specific anti-neoplastic agents derived from the periwinkle plant. Vinca alkaloids act at the M phase (mitosis) of the cell cycle by binding specifically to tubulin. Consequently, the bound tubulin molecule is unable to polymerize into microtubules. Mitosis is believed to be arrested in metaphase with cell death following. Examples of vinca alkaloids include, but are not limited to, vinblastine, vincristine, and vinorelbine. Vinblastine, vincaleukoblastine sulfate, is commercially available as VELBAN® as an injectable solution.
  • vincaleukoblastine, 22-oxo-, sulfate is commercially available as ONCOVIN® as an injectable solution.
  • Vincristine is indicated for the treatment of acute leukemias and has also found use in treatment regimens for Hodgkin's and non-Hodgkin's malignant lymphomas.
  • Alopecia and neurologic effects are the most common side effect of vincristine and to a lesser extent myelosupression and gastrointestinal mucositis effects occur.
  • Vinorelbine 3',4'-didehydro-4'-deoxy-C- norvincaleukoblastine [R— (R*,R*)-2,3-dihydroxybutanedioate (1 :2)(salt)], commercially available as an injectable solution of vinorelbine tartrate (NAVELBINE®), is a semisynthetic vinca alkaloid. Vinorelbine is indicated as a single agent or in combination with other chemotherapeutic agents, such as cisplatin, in the treatment of various solid tumors, particularly non-small cell lung, advanced breast, and hormone refractory prostate cancers. Myelosuppression is the most common dose limiting side effect of vinorelbine.
  • Platinum coordination complexes include non-phase specific anti-cancer agents, which interact with DNA.
  • the platinum complexes enter tumor cells, undergo, aquation and form intra- and interstrand crosslinks with DNA causing adverse biological effects to the tumor.
  • Examples of platinum coordination complexes include, but are not limited to, cisplatin and carboplatin.
  • Cisplatin, cis-diamminedichloroplatinum is commercially available as PLATINOL® as an injectable solution.
  • Cisplatin is primarily indicated in the treatment of metastatic testicular and ovarian cancer and advanced bladder cancer.
  • the primary dose limiting side effects of cisplatin are nephrotoxicity, which may be controlled by hydration and diuresis, and ototoxicity.
  • Carboplatin platinum, diammine [1,1- cyclobutane-dicarboxylate(2-)-0,0'], is commercially available as PARAPLATIN®) as an injectable solution.
  • Carboplatin is primarily indicated in the first and second line treatment of advanced ovarian carcinoma. Bone marrow suppression is the dose limiting toxicity of carboplatin.
  • Alkylating agents include non-phase anti-cancer specific agents and strong electrophiles. Typically, alkylating agents form covalent linkages, by alkylation, to DNA through nucleophilic moieties of the DNA molecule such as phosphate, amino, sulfhydryl, hydroxyl, carboxyl, and imidazole groups. Such alkylation disrupts nucleic acid function leading to cell death.
  • alkylating agents include, but are not limited to, nitrogen mustards such as cyclophosphamide, melphalan, and chlorambucil; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine; and triazenes such as dacarbazine.
  • Cyclophosphamide 2-[bis(2-chloroethyl)amino]tetrahydro-2H-l,3,2- oxazaphosphorine 2-oxide monohydrate, is commercially available as an injectable solution or tablets as CYTOXAN®. Cyclophosphamide is indicated as a single agent or in combination with other chemotherapeutic agents, in the treatment of malignant lymphomas, multiple myeloma, and leukemias. Alopecia, nausea, vomiting and leukopenia are the most common dose limiting side effects of cyclophosphamide.
  • Melphalan 4-[bis(2-chloroethyl)amino]-L-phenylalanine, is commercially available as an injectable solution or tablets as ALKERAN®. Melphalan is indicated for the palliative treatment of multiple myeloma and non-resectable epithelial carcinoma of the ovary. Bone marrow suppression is the most common dose limiting side effect of melphalan. Chlorambucil, 4-[bis(2- chloroethyl)amino]benzenebutanoic acid, is commercially available as LEUKERAN® tablets.
  • Chlorambucil is indicated for the palliative treatment of chronic lymphatic leukemia, and malignant lymphomas such as lymphosarcoma, giant follicular lymphoma, and Hodgkin's disease. Bone marrow suppression is the most common dose limiting side effect of chlorambucil.
  • Busulfan, 1,4-butanediol dimethanesulfonate, is commercially available as MYLERAN® TABLETS.
  • Busulfan is indicated for the palliative treatment of chronic myelogenous leukemia. Bone marrow suppression is the most common dose limiting side effects of busulfan.
  • Carmustine, l,3-[bis(2-chloroethyl)-l-nitrosourea, is commercially available as single vials of lyophilized material as BiCNU®.
  • Carmustine is indicated for the palliative treatment as a single agent or in combination with other agents for brain tumors, multiple myeloma, Hodgkin's disease, and non-Hodgkin's lymphomas. Delayed myelosuppression is the most common dose limiting side effects of carmustine.
  • dacarbazine, 5-(3,3-dimethyl-l-triazeno)- imidazole-4-carboxamide is commercially available as single vials of material as DTIC-Dome®.
  • dacarbazine is indicated for the treatment of metastatic malignant melanoma and in combination with other agents for the second line treatment of Hodgkin's Disease. Nausea, vomiting, and anorexia are the most common dose limiting side effects of dacarbazine.
  • Antibiotic anti-neoplastics include non-phase specific agents, which bind or intercalate with DNA. Typically, such action results in stable DNA complexes or strand breakage, which disrupts ordinary function of the nucleic acids leading to cell death.
  • antibiotic anti-neoplastic agents include, but are not limited to, actinomycins such as dactinomycin, anthrocyclins such as daunorubicin and doxorubicin; and bleomycins.
  • Dactinomycin also know as Actinomycin D, is commercially available in injectable form as COSMEGEN®. Dactinomycin is indicated for the treatment of Wilm's tumor and rhabdomyosarcoma.
  • Daunorubicin, (8S-cis-)-8-acetyl-10-[(3-amino- 2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]-7,8,9, 10-tetrahydro-6,8, 11 -trihydroxy-1 -methoxy-5, 12 naphthacenedione hydrochloride, is commercially available as a liposomal injectable form as DAUNOXOME® or as an injectable as CERUBIDINE®.
  • Daunorubicin is indicated for remission induction in the treatment of acute nonlymphocytic leukemia and advanced HIV associated Kaposi's sarcoma. Myelosuppression is the most common dose limiting side effect of daunorubicin.
  • Doxorubicin (8S,10S)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]-8- glycoloyl, 7,8,9,10-tetrahydro-6,8,l l-trihydroxy-l-methoxy-5,12 naphthacenedione hydrochloride, is commercially available as an injectable form as RUBEX® or ADRIAMYCiN RDF®.
  • Doxorubicin is primarily indicated for the treatment of acute lymphoblastic leukemia and acute myeloblasts leukemia, but is also a useful component in the treatment of some solid tumors and lymphomas. Myelosuppression is the most common dose limiting side effect of doxorubicin.
  • Bleomycin a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of Streptomyces verticillus, is
  • Bleomycin is indicated as a palliative treatment, as a single agent or in combination with other agents, of squamous cell carcinoma, lymphomas, and testicular carcinomas. Pulmonary and cutaneous toxicities are the most common dose limiting side effects of bleomycin.
  • Topoisomerase II inhibitors include, but are not limited to, epipodophyllotoxins.
  • Epipodophyllotoxins are phase specific anti-neoplastic agents derived from the mandrake plant. Epipodophyllotoxins typically affect cells in the S and G2 phases of the cell cycle by forming a ternary complex with topoisomerase II and DNA causing DNA strand breaks. The strand breaks accumulate and cell death follows. Examples of epipodophyllotoxins include, but are not limited to, etoposide and teniposide. Etoposide, 4'-demethyl-epipodophyllotoxin 9[4,6-0-(R)-ethylidene-P-D- glucopyranoside], is commercially available as an injectable solution or capsules as VePESID® and is commonly known as VP- 16.
  • Etoposide is indicated as a single agent or in combination with other chemotherapy agents in the treatment of testicular and non-small cell lung cancers. Myelosuppression is the most common side effect of etoposide. The incidence of leucopenia tends to be more severe than thrombocytopenia.
  • Teniposide, 4'-demethyl-epipodophyllotoxin 9[4,6-0-(R)-thenylidene-P-D- glucopyranoside] is commercially available as an injectable solution as VUMON® and is commonly known as VM-26.
  • Teniposide is indicated as a single agent or in combination with other chemotherapy agents in the treatment of testicular and non-small cell lung cancers. Myelosuppression is the most common side effect of etoposide. The incidence of leucopenia tends to be more severe than thrombocytopenia.
  • topoisomerase II inhibitors include epirubicin, idarubicin, nemorubicin, mitoxantrone, and losoxantrone.
  • Antimetabolite neoplastic agents include phase specific anti-neoplastic agents that act at S phase (DNA synthesis) of the cell cycle by inhibiting DNA synthesis or by inhibiting purine or pyrimidine base synthesis and thereby limiting DNA synthesis. Consequently, S phase does not proceed and cell death follows.
  • Examples of antimetabolite anti-neoplastic agents include, but are not limited to, fluorouracil, methotrexate, cytarabine, mercaptopurine, thioguanine, and gemcitabine. 5- fluorouracil, 5-fluoro-2,4-(lH,3H) pyrimidinedione, is commercially available as fluorouracil.
  • 5-fluorouracil leads to inhibition of thymidylate synthesis and is also incorporated into both RNA and DNA. The result typically is cell death.
  • 5-fluorouracil is indicated as a single agent or in combination with other chemotherapy agents in the treatment of carcinomas of the breast, colon, rectum, stomach and pancreas. Myelosuppression and mucositis are dose limiting side effects of 5-fluorouracil.
  • Other fluoropyrimidine analogs include 5-fluoro deoxyuridine (floxuridine) and 5- fluorodeoxyuridine monophosphate.
  • Cytarabine 4-amino-l-P-D-arabinofuranosyl-2(lH)- pyrimidinone, is commercially available as CYTOSAR-U® and is commonly known as Ara-C. It is believed that cytarabine exhibits cell phase specificity at S-phase by inhibiting DNA chain elongation by terminal incorporation of cytarabine into the growing DNA chain. Cytarabine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. Other cytidine analogs include 5-azacytidine and 2',2'-difluorodeoxycytidine (gemcitabine).
  • Cytarabine induces leucopenia, thrombocytopenia, and mucositis.
  • Mercaptopurine l,7-dihydro-6H- purine-6-thione monohydrate, is commercially available as PURINETHOL®.
  • Mercaptopurine exhibits cell phase specificity at S-phase by inhibiting DNA synthesis by an as of yet unspecified mechanism.
  • Mercaptopurine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. Myelosuppression and gastrointestinal mucositis are expected side effects of mercaptopurine at high doses.
  • a useful mercaptopurine analog is azathioprine.
  • Thioguanine 2-amino-l,7-dihydro-6H-purine-6-thione, is commercially available as TABLOID®.
  • Thioguanine exhibits cell phase specificity at S-phase by inhibiting DNA synthesis by an as of yet unspecified mechanism.
  • Thioguanine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia.
  • Myelosuppression including leucopenia, thrombocytopenia, and anemia, is the most common dose limiting side effect of thioguanine administration. However, gastrointestinal side effects occur and can be dose limiting.
  • Other purine analogs include pentostatin, erythrohydroxynonyladenine, fludarabine phosphate, and cladribine.
  • Gemcitabine 2'-deoxy-2',2'-difluorocytidine monohydrochloride ( ⁇ -isomer), is commercially available as GEMZA ®.
  • GEMZA 2'-deoxy-2',2'-difluorocytidine monohydrochloride
  • Gemcitabine exhibits cell phase specificity at S-phase and by blocking progression of cells through the Gl/S boundary.
  • Gemcitabine is indicated in combination with cisplatin in the treatment of locally advanced non-small cell lung cancer and alone in the treatment of locally advanced pancreatic cancer.
  • Myelosuppression including leucopenia, thrombocytopenia, and anemia, is the most common dose limiting side effect of gemcitabine administration.
  • Methotrexate N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L- glutamic acid, is commercially available as methotrexate sodium. Methotrexate exhibits cell phase effects specifically at S-phase by inhibiting DNA synthesis, repair and/or replication through the inhibition of dyhydrofolic acid reductase which is required for synthesis of purine nucleotides and thymidylate.
  • Methotrexate is indicated as a single agent or in combination with other chemotherapy agents in the treatment of choriocarcinoma, meningeal leukemia, non-Hodgkin's lymphoma, and carcinomas of the breast, head, neck, ovary and bladder.
  • Myelosuppression (leucopenia,
  • thrombocytopenia, and anemia are expected side effect of methotrexate administration.
  • Topoisomerase I inhibitors include camptothecins such as camptothecin and camptothecin derivatives. Camptothecin cytotoxic activity is believed to be related to its Topoisomerase I inhibitory activity. Examples of camptothecins include, but are not limited to irinotecan and topotecan.
  • Irinotecan is a derivative of camptothecin which binds, along with its active metabolite SN-38, to the topoisomerase I-DNA complex.
  • cytotoxicity occurs as a result of irreparable double strand breaks caused by interaction of the topoisomerase I:DNA:irinotecan or SN-38 ternary complex with replication enzymes.
  • Irinotecan is indicated for treatment of metastatic cancer of the colon or rectum.
  • the dose limiting side effects of irinotecan HC1 are myelosuppression, including neutropenia, and GI effects, including diarrhea.
  • Topotecan HC1 (S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-lH-pyrano[3',4',6,7]- indolizino[l,2-b]quinoline-3,14-(4H,12H)-dione monohydrochloride, is commercially available as the injectable solution HYCAMTIN®.
  • Topotecan is a derivative of camptothecin which binds to the topoisomerase I-DNA complex and prevents religation of singles strand breaks caused by Topoisomerase I in response to torsional strain of the DNA molecule.
  • Topotecan is indicated for second line treatment of metastatic carcinoma of the ovary and small cell lung cancer.
  • the dose limiting side effect of topotecan HC1 is myelosuppression, primarily neutropenia.
  • Hormones and hormonal analogues are useful compounds for treating cancers in which there is a relationship between the hormone(s) and growth and/or lack of growth of the cancer.
  • hormones and hormonal analogues useful in cancer treatment include, but are not limited to, adrenocorticosteroids such as prednisone and prednisolone which are useful in the treatment of malignant lymphoma and acute leukemia in children; aminoglutethimide and other aromatase inhibitors such as aminoglutethimide, roglethimide, pyridoglutethimide, trilostane, testolactone, ketokonazole, vorozole, fadrozole, anastrozole, letrazole, formestane, atamestane and exemestane useful in the treatment of adrenocortical carcinoma and hormone dependent breast carcinoma containing estrogen receptors; progestrins such as megestrol acetate useful in the treatment of hormone dependent breast cancer and end
  • GnRH gonadotropin-releasing hormone
  • LH leutinizing hormone
  • FSH follicle stimulating hormone
  • SH2/SH3 domain blockers are agents that disrupt SH2 or SH3 domain binding in a variety of enzymes or adaptor proteins including, PI3-K p85 subunit, Src family kinases, adaptor molecules (She, Crk, Nek, Grb2) and Ras-GAP.
  • SH2/SH3 domains as targets for anti-cancer drugs are discussed in Smithgall, T. E. (1995), Journal of Pharmacological and Toxicological Methods. 34(3) 125-32.
  • Inhibitors of Serine/Threonine Kinases including MAP kinase cascade blockers which include blockers of Raf kinases (rafk), Mitogen or Extracellular Regulated Kinase (MEKs), and Extracellular Regulated Kinases (ERKs); and Protein kinase C family member blockers including blockers of PKCs (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta).
  • IkB kinase family IKKa, IKKb
  • PKB family kinases akt kinase family members
  • TGF beta receptor kinases TGF beta receptor kinases.
  • Serine/Threonine kinases and inhibitors thereof are described in Yamamoto, T., Taya, S., Kaibuchi, K., (1999), Journal of Biochemistry. 126 (5) 799-803; Brodt, P, Samani, A, and Navab, R. (2000), Biochemical Pharmacology, 60. 1 101 -1 107; Massague, J., Weis-Garcia, F. (1996) Cancer Surveys. 27:41 -64; Philip, P. A., and Harris, A. L. (1995), Cancer Treatment and Research. 78: 3-27, Lackey, K. et al Bioorganic and Medicinal Chemistry Letters, (10), 2000, 223-226; U.S. Pat. No.
  • inhibitors are signal transduction pathway inhibitors such as inhibitors of Ras Oncogene.
  • Such inhibitors include inhibitors of farnesyltransferase, geranyl-geranyl transferase, and CAAX proteases as well as anti-sense oligonucleotides, ribozymes and immunotherapy.
  • Such inhibitors have been shown to block ras activation in cells containing wild type mutant ras, thereby acting as antiproliferation agents.
  • Ras oncogene inhibition is discussed in Scharovsky, O. G., Rozados, V. R., Gervasoni, S. I. Matar, P. (2000), Journal of Biomedical Science. 7(4) 292-8; Ashby, M. N. (1998), Current Opinion in Lipidology. 9 (2) 99-102; and BioChim. Biophys. Acta, (19899) 1423(3):19-30.
  • This invention further relates to a method for using the compounds or pharmaceutical composition in combination with other tumor treatment approaches, including surgery, ionizing radiation, photodynamic therapy, or implants, e.g., with corticosteroids, hormones, or used as radiosensitizers.
  • One such approach may be, for example, radiation therapy in inhibiting abnormal cell growth or treating the hyperproliferative disorder in the mammal.
  • Techniques for administering radiation therapy are known in the art, and these techniques can be used in the combination therapy described herein.
  • the administration of the compound of the invention in this combination therapy can be determined as described herein.
  • Radioactive isotopes e.g., At-211, 1-131, 1-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, and radioactive isotopes of Lu.
  • Suitable radiation sources for use as a cell conditioner of the present invention include both solids and liquids.
  • the radiation source can be a radionuclide, such as 1-125, 1-131, Yb-169, Ir-192 as a solid source, 1-125 as a solid source, or other radionuclides that emit photons, beta particles, gamma radiation, or other therapeutic rays.
  • the radioactive material can also be a fluid made from any solution of radionuclide(s), e.g., a solution of I- 125 or 1-131, or a radioactive fluid can be produced using a slurry of a suitable fluid containing small particles of solid radionuclides, such as Au-198, Y-90.
  • the radionuclide(s) can be embodied in a gel or radioactive micro spheres.
  • the compounds of the present invention can render abnormal cells more sensitive to treatment with radiation for purposes of killing and/or inhibiting the growth of such cells.
  • this invention further relates to a method for sensitizing abnormal cells in a mammal to treatment with radiation which comprises administering to the mammal an amount of an inhibitor of the present invention or pharmaceutically acceptable salt, ester, prodrug, solvate, hydrate or derivative thereof, which amount is effective is sensitizing abnormal cells to treatment with radiation.
  • the amount of the compound, salt, or solvate in this method can be determined according to the means for ascertaining effective amounts of such compounds described herein.
  • Photodynamic therapy includes therapy which uses certain chemicals known as
  • photosensitizing compounds to treat or prevent cancers examples include treatment with compounds, such as e.g., VISUDYNE and porfimer sodium.
  • Angiostatic steroids include compounds which block or inhibit angiogenesis, such as, e.g., anecortave, triamcinolone, hydrocortisone, 11-a-epihydrocotisol, cortexolone, 17a-hydroxyprogesterone, corticosterone, desoxycorticosterone, testosterone, estrone and dexamethasone.
  • Implants containing corticosteroids include compounds, such as e.g., fluocinolone and dexamethasone.
  • Other chemotherapeutic compounds include, but are not limited to, plant alkaloids, hormonal compounds and antagonists; biological response modifiers, preferably lymphokines or interferons; antisense oligonucleotides or oligonucleotide derivatives; shRNA or siRNA; or miscellaneous compounds or compounds with other or unknown mechanism of action.
  • the compounds or pharmaceutical compositions of the present invention can be used in combination with an amount of one or more substances selected from anti-angiogenesis agents, signal transduction inhibitors, and antiproliferative agents.
  • Anti-angiogenesis agents such as MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, and COX-11 (cyclooxygenase 11) inhibitors, can be used in conjunction with an inhibitor of the present invention and pharmaceutical compositions described herein.
  • MMP-2 matrix-metalloproteinase 2
  • MMP-9 matrix-metalloproteinase 9
  • COX-11 cyclooxygenase 11
  • useful COX-II inhibitors include CELEBREXTM (alecoxib), valdecoxib, and rofecoxib.
  • Examples of useful matrix metalloproteinase inhibitors are described in WO 96/33172 (published October 24,1996), WO 96/27583 (published March 7,1996), European Patent Application No. 97304971.1 (filed July 8,1997), European Patent Application No.
  • MMP-2 and MMP-9 inhibitors have little or no activity inhibiting MMP-1, or selectively inhibit MMP-2 and/or AMP-9 relative to the other matrix- metalloproteinases (i.
  • MMP inhibitors useful in the present invention are AG-3340, RO 32-3555, and RS 13-0830.
  • the invention also relates to a method of and to a pharmaceutical composition of treating a cardiovascular disease in a mammal which comprises an amount of an inhibitor of the present invention, or a pharmaceutically acceptable salt, ester, prodrug, solvate, hydrate or derivative thereof, or an isotopically-labeled derivative thereof, and an amount of one or more therapeutic agents use for the treatment of cardiovascular diseases.
  • agents for use in cardiovascular disease applications are anti-thrombotic agents, e.g., prostacyclin and salicylates, thrombolytic agents, e.g., streptokinase, urokinase, tissue plasminogen activator (TP A) and anisoylated plasminogen-streptokinase activator complex (APSAC), anti-platelets agents, e.g., acetyl-salicylic acid (ASA) and clopidrogel, vasodilating agents, e.g., nitrates, calcium channel blocking drugs, anti-proliferative agents, e.g., colchicine and alkylating agents, intercalating agents, growth modulating factors such as interleukins, transformation growth factor-beta and congeners of platelet derived growth factor, monoclonal antibodies directed against growth factors, anti- inflammatory agents, both steroidal and non-steroidal, and other agents that can modulate
  • Antibiotics can also be included in combinations or coatings comprised by the invention. Moreover, a coating can be used to effect therapeutic delivery focally within the vessel wall. By incorporation of the active agent in a swellable polymer, the active agent will be released upon swelling of the polymer.
  • Medicaments which may be administered in conjunction with the compounds described herein include any suitable drugs usefully delivered by inhalation for example, analgesics, e.g., codeine, dihydromorphine, ergotamine, fentanyl or morphine; anginal preparations, e.g., diltiazem; antiallergics, e.g., cromoglycate, ketotifen or nedocromil; anti-infectives, e.g., cephalosporins, penicillins, streptomycin, sulphonamides, tetracyclines or pentamidine; antihistamines, e.g., methapyrilene; anti-inflammatories, e.g., beclomethasone, flunisolide, budesonide, tipredane, triamcinolone acetonide or fluticasone; antitussives, e.g., noscapine;
  • the medicaments may be used in the form of salts (e.g., as alkali metal or amine salts or as acid addition salts) or as esters (e.g., lower alkyl esters) or as solvates (e.g., hydrates) to optimize the activity and/or stability of the medicament.
  • salts e.g., as alkali metal or amine salts or as acid addition salts
  • esters e.g., lower alkyl esters
  • solvates e.g., hydrates
  • exemplary therapeutic agents useful for a combination therapy include but are not limited to agents as described above, radiation therapy, hormone antagonists, hormones and their releasing factors, thyroid and antithyroid drugs, estrogens and progestins, androgens,
  • adrenocorticotropic hormone adrenocortical steroids and their synthetic analogs; inhibitors of the synthesis and actions of adrenocortical hormones, insulin, oral hypoglycemic agents, and the pharmacology of the endocrine pancreas, agents affecting calcification and bone turnover: calcium, phosphate, parathyroid hormone, vitamin D, calcitonin, vitamins such as water-soluble vitamins, vitamin B complex, ascorbic acid, fat-soluble vitamins, vitamins A, K, and E, growth factors, cytokines, chemokines, muscarinic receptor agonists and antagonists; anticholinesterase agents; agents acting at the neuromuscular junction and/or autonomic ganglia; catecholamines,
  • sympathomimetic drugs and adrenergic receptor agonists or antagonists
  • 5-hydroxytryptamine (5-HT, serotonin) receptor agonists and antagonists 5-hydroxytryptamine
  • Therapeutic agents can also include agents for pain and inflammation such as histamine and histamine antagonists, bradykinin and bradykinin antagonists, 5-hydroxytryptamine (serotonin), lipid substances that are generated by biotransformation of the products of the selective hydrolysis of membrane phospholipids, eicosanoids, prostaglandins, thromboxanes, leukotrienes, aspirin, nonsteroidal anti- inflammatory agents, analgesic-antipyretic agents, agents that inhibit the synthesis of prostaglandins and thromboxanes, selective inhibitors of the inducible cyclooxygenase, selective inhibitors of the inducible cyclooxygenase-2, autacoids, paracrine hormones, somatostatin, gastrin, cytokines that mediate interactions involved in humoral and cellular immune responses, lipid-derived autacoids, eicosanoids, ⁇ -adrenergic agonists, i
  • Additional therapeutic agents contemplated herein include diuretics, vasopressin, agents affecting the renal conservation of water, rennin, angiotensin, agents useful in the treatment of myocardial ischemia, anti-hypertensive agents, angiotensin converting enzyme inhibitors, ⁇ - adrenergic receptor antagonists, agents for the treatment of hypercholesterolemia, and agents for the treatment of dyslipidemia.
  • Other therapeutic agents contemplated include drugs used for control of gastric acidity, agents for the treatment of peptic ulcers, agents for the treatment of gastroesophageal reflux disease, prokinetic agents, antiemetics, agents used in irritable bowel syndrome, agents used for diarrhea, agents used for constipation, agents used for inflammatory bowel disease, agents used for biliary disease, agents used for pancreatic disease.
  • Therapeutic agents used to treat protozoan infections drugs used to treat Malaria, Amebiasis, Giardiasis, Trichomoniasis, Trypanosomiasis, and/or Leishmaniasis, and/or drugs used in the chemotherapy of helminthiasis.
  • therapeutic agents include antimicrobial agents, sulfonamides, trimethoprim-sulfamethoxazole quinolones, and agents for urinary tract infections, penicillins, cephalosporins, and other, ⁇ -Lactam antibiotics, an agent comprising an aminoglycoside, protein synthesis inhibitors, drugs used in the chemotherapy of tuberculosis, mycobacterium avium complex disease, and leprosy, antifungal agents, antiviral agents including nonretroviral agents and antiretroviral agents.
  • therapeutic antibodies that can be combined with a subject compound include but are not limited to anti-receptor tyrosine kinase antibodies (cetuximab, panitumumab, trastuzumab), anti CD20 antibodies (rituximab, tositumomab), and other antibodies such as alemtuzumab, bevacizumab, and gemtuzumab.
  • anti-receptor tyrosine kinase antibodies cetuximab, panitumumab, trastuzumab
  • anti CD20 antibodies rituximab, tositumomab
  • other antibodies such as alemtuzumab, bevacizumab, and gemtuzumab.
  • therapeutic agents used for immunomodulation such as immunomodulators, immunosuppressive agents, tolerogens, and immunostimulants are contemplated by the methods herein.
  • therapeutic agents acting on the blood and the blood-forming organs such as hematopoietic agents, growth factors, minerals, and vitamins, anticoagulant, thrombolytic, and antiplatelet drugs.
  • Example 1 Expression and Inhibition Assays of ⁇ 110 ⁇ / ⁇ 85 ⁇ , ⁇ 110 ⁇ / ⁇ 85 ⁇ , ⁇ 110 ⁇ / ⁇ 85 ⁇ , and ⁇ :
  • kits or systems for assaying PI3-K activities are avaiable.
  • the commercially available kits or systems can be used to screen for inhibitors and/or agonists of PI3Ks including but not limited to PI3-Kinase ⁇ , ⁇ , ⁇ , and ⁇ .
  • An exemplary system is PI 3-Kinase (human) HTRFTM Assay from Upstate. The assay can be carried out according to the procedures suggested by the
  • the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K.
  • the kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20ul per well.
  • each well receives 2ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration.
  • approximately 14.5ul of a kinase/PIP2 mixture (diluted in IX reaction buffer) is added per well for a final concentration of 0.25-0.3ug/ml kinase and ⁇ PIP2.
  • the plate is sealed and incubated for 15 minutes at room temperature.
  • ATP diluted in IX reaction buffer
  • ⁇ ATP diluted in IX reaction buffer
  • the plate is sealed and incubated for 1 hour at room temperature.
  • the reaction is stopped by adding 5ul of Stop Solution per well and then 5ul of Detection Mix is added per well.
  • the plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader. Data is analyzed and IC50s are generated using GraphPad Prism 5. For PI3K ⁇ , ⁇ , ⁇ , and ⁇ , the nM conentration of inhibitor to reach IC50 is provided.
  • PI3K a Inhibition of PI3K a at lower concentrations than those for ⁇ , ⁇ , and ⁇ provides evidence of specificity within this group of kinases. Similar assays, and others known in the art, can be used to measure the percent inhibition of other kinases, including but not limited to PI3K class II kinases, phosphoinositide 4 kinases (PI4K), and phosphoinositide 5 kinases (PI5K).
  • PI3K class II kinases phosphoinositide 4 kinases
  • PI5K phosphoinositide 5 kinases
  • the cross-activity or lack thereof of one or more compounds of the present invention against Abl kinase can be measured according to any procedures known in the art or methods disclosed below.
  • the compounds described herein can be assayed in triplicate against recombinant full-length Abl or Abl (T315I) (Upstate) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl 2 , 200 ⁇ ATP (2.5 ⁇ of ⁇ -32 ⁇ - ⁇ ), and 0.5 mg/mL BSA.
  • the optimized Abl peptide substrate EAIYAAPFAKKK is used as phosphoacceptor (200 ⁇ ). Reactions are terminated by spotting onto phosphocellulose sheets, which are washed with 0.5% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • the cross-activity or lack thereof of one or more compounds of the present invention against Hck kinase can be measured according to any procedures known in the art or methods disclosed below.
  • the compounds described herein can be assayed in triplicate against recombinant full-length Hck in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl 2 , 200 ⁇ ATP (2.5 ⁇ of ⁇ -32 ⁇ - ATP), and 0.5 mg/mL BSA.
  • the optimized Src family kinase peptide substrate EIYGEFKKK is used as phosphoacceptor (200 ⁇ ). Reactions are terminated by spotting onto phosphocellulose sheets, which are washed with 0.5% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • the cross-activity or lack thereof of one or more compounds of the present invention against IR receptor kinase can be measured according to any procedures known in the art or methods disclosed below.
  • the compounds described herein can be assayed in triplicate against recombinant insulin receptor kinase domain (Upstate) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl 2 , 10 mM MnCl 2 , 200 ⁇ ATP (2.5 ⁇ of ⁇ -32 ⁇ - ⁇ ), and 0.5 mg/mL BSA.
  • Poly E-Y Sigma; 2 mg/mL is used as a substrate.
  • Reactions are terminated by spotting onto nitrocellulose, which is washed with 1M NaCl/l%> phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • the cross-activity or lack thereof of one or more compounds of the present invention against Src kinase can be measured according to any procedures known in the art or methods disclosed below.
  • the compounds described herein can be assayed in triplicate against recombinant full-length Src or Src (T338I) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl 2 , 200 ⁇ ATP (2.5 ⁇ of ⁇ -32 ⁇ - ⁇ ), and 0.5 mg/mL BSA.
  • the optimized Src family kinase peptide substrate EIYGEFKKK is used as phosphoacceptor (200 ⁇ ). Reactions are terminated by spotting onto phosphocellulose sheets, which are washed with 0.5% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • DNA-PK can be purchased from Promega and assayed using the DNA-PK Assay System (Promega) according to the manufacturer's instructions.
  • the cross-activity or lack thereof of one or more compounds of the present invention against mTor can be measured according to any procedures known in the art or methods disclosed below.
  • the compounds described herein can be tested against recombinant mTOR (Invitrogen) in an assay containing 50 mM HEPES, pH 7.5, ImM EGTA, 10 mM MgCl 2 , 2.5 mM, 0.01% Tween, 10 ⁇ ATP (2.5 ⁇ of ⁇ -32 ⁇ - ⁇ ), and 3 ⁇ g/mL BSA.
  • Rat recombinant PHAS-1/4EBP1 (Calbiochem; 2 mg/mL) is used as a substrate.
  • Reactions are terminated by spotting onto nitrocellulose, which is washed with 1M NaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • kits or systems for assaying mTOR activity are commercially available.
  • Invitrogen's LanthaScreenTM Kinase assay to test the inhibitors of mTOR disclosed herein.
  • This assay is a time resolved FRET platform that measures the phosphorylation of GFP labeled 4EBP1 by mTOR kinase.
  • the kinase reaction is performed in a white 384 well microtitre plate.
  • the total reaction volume is 20ul per well and the reaction buffer composition is 50mM HEPES pH7.5, 0.01% Polysorbate 20, ImM EGTA, lOmM MnCl 2 , and 2mM DTT.
  • each well receives 2ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final
  • Example 8 Expression and Inhibition Assays of Vascular endothelial growth receptor
  • the cross-activity or lack thereof of one or more compounds of the present invention against VEGF receptor can be measured according to any procedures known in the art or methods disclosed below.
  • the compounds described herein can be tested against recombinant KDR receptor kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl 2 , 0.1% BME, 10 ⁇ ATP (2.5 ⁇ of ⁇ -32 ⁇ - ⁇ ), and 3 ⁇ g/mL BSA.
  • Poly E-Y Sigma; 2 mg/mL
  • Reactions are terminated by spotting onto nitrocellulose, which is washed with 1M
  • the cross-activity or lack thereof of one or more compounds of the present invention against EphB4 can be measured according to any procedures known in the art or methods disclosed below.
  • the compounds described herein can be tested against recombinant Ephrin receptor B4 kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl 2 , 0.1% BME, 10 ⁇ ATP (2.5 ⁇ of ⁇ -32 ⁇ - ⁇ ), and 3 ⁇ g/mL BSA.
  • Poly E-Y Sigma; 2 mg/mL is used as a substrate.
  • Reactions are terminated by spotting onto nitrocellulose, which is washed with 1M NaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • the cross-activity or lack thereof of one or more compounds of the present invention against EGFR kinase can be measured according to any procedures known in the art or methods disclosed below.
  • the compounds described herein can be tested against recombinant EGF receptor kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl 2 , 0.1% BME, 10 ⁇ ATP (2.5 ⁇ of ⁇ -32 ⁇ - ⁇ ), and 3 ⁇ g/mL BSA.
  • Poly E-Y Sigma; 2 mg/mL
  • Reactions are terminated by spotting onto nitrocellulose, which is washed with 1M
  • the cross-activity or lack thereof of one or more compounds of the present invention against KIT kinase can be measured according to any procedures known in the art or methods disclosed below.
  • the compounds described herein can be tested against recombinant KIT kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl 2 , lmM DTT, lOmM MnCl 2 , 10 ⁇ ATP (2.5 ⁇ of ⁇ -32 ⁇ - ⁇ ), and 3 ⁇ g/mL BSA.
  • Poly E-Y Sigma; 2 mg/mL is used as a substrate.
  • Reactions are terminated by spotting onto nitrocellulose, which is washed with 1M NaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • the cross-activity or lack thereof of one or more compounds of the present invention against RET kinase can be measured according to any procedures known in the art or methods disclosed below.
  • the compounds described herein can be tested against recombinant RET kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl 2 , 2.5mM DTT,10 ⁇ ATP (2.5 ⁇ of ⁇ -32 ⁇ - ⁇ ), and 3 ⁇ g/mL BSA.
  • the optimized Abl peptide substrate EAIYAAPFAKKK is used as phosphoacceptor (200 ⁇ ). Reactions are terminated by spotting onto phosphocellulose sheets, which are washed with 0.5% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • the cross-activity or lack thereof of one or more compounds of the present invention against PDGFR kinase can be measured according to any procedures known in the art or methods disclosed below.
  • the compounds described herein can be tested against recombinant PDG receptor kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl 2 , 2.5mM DTT, 10 ⁇ ATP (2.5 ⁇ of ⁇ -32 ⁇ - ⁇ ), and 3 ⁇ g/mL BSA.
  • EAIYAAPFAKKK is used as phosphoacceptor (200 ⁇ ). Reactions are terminated by spotting onto phosphocellulose sheets, which are washed with 0.5% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • Example 14 Expression and Inhibition Assays of FMS-related tyrosine kinase 3 (FLT-3)
  • the cross-activity or lack thereof of one or more compounds of the present invention against FLT-3 kinase can be measured according to any procedures known in the art or methods disclosed below.
  • the compounds described herein can be tested against recombinant FLT-3 kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl 2 , 2.5mM DTT,10 ⁇ ATP (2.5 ⁇ of ⁇ -32 ⁇ - ⁇ ), and 3 ⁇ g/mL BSA.
  • EAIYAAPFAKKKis used as phosphoacceptor (200 ⁇ ). Reactions are terminated by spotting onto phosphocellulose sheets, which are washed with 0.5% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • TIE2 TEK receptor tyrosine kinase
  • the cross-activity or lack thereof of one or more compounds of the present invention against TIE2 kinase can be measured according to any procedures known in the art or methods disclosed below.
  • the compounds described herein can be tested against recombinant TIE2 kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl 2 , 2mM DTT, lOmM MnCl 2 , 10 ⁇ ATP (2.5 ⁇ of ⁇ -32 ⁇ - ⁇ ), and 3 ⁇ g/mL BSA.
  • Poly E-Y Sigma; 2 mg/mL is used as a substrate.
  • Reactions are terminated by spotting onto nitrocellulose, which is washed with 1M NaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • the ability of one or more subject compounds to inhibit B cell activon and proliferation is determined according to standard procedures known in the art. For example, an in vitro cellular proliferation assay is established that measures the metabolic activity of live cells. The assay is performed in a 96 well microtiter plate using Alamar Blue reduction. Balb/c splenic B cells are purified over a Ficoll-PaqueTM PLUS gradient followed by magnetic cell separation using a MACS B cell Isolation Kit (Miletenyi). Cells are plated in 90ul at 50,000 cells/well in B Cell Media (RPMI + 10%FBS + Penn/Strep + 50 ⁇ bME + 5mM HEPES).
  • a compound disclosed herein is diluted in B Cell Media and added in a lOul volume. Plates are incubated for 30min at 37C and 5% C0 2 (0.2% DMSO final concentration). A 50ul B cell stimulation cocktail is then added containing either lOug/ml LPS or 5ug/ml F(ab')2 Donkey anti-mouse IgM plus 2ng/ml recombinant mouse IL4 in B Cell Media. Plates are incubated for 72 hours at 37°C and 5% C0 2 . A volume of 15 ⁇ of Alamar Blue reagent is added to each well and plates are incubated for 5 hours at 37C and 5% C0 2 . Alamar Blue fluoresce is read at 560Ex/590Em, and IC50 or EC50 values are calculated using GraphPad Prism 5.
  • an in vitro cellular proliferation assay can be performed to measure the metabolic activity of live cells.
  • the assay is performed in a 96 well microtiter plate using Alamar Blue reduction.
  • Human tumor cell lines are obtained from ATCC (e.g., MCF7, U-87 MG, MDA-MB-468, PC-3), grown to confluency in T75 flasks, trypsinized with 0.25% trypsin, washed one time with Tumor Cell Media (DMEM + 10%FBS), and plated in 90ul at 5,000 cells/well in Tumor Cell Media.
  • a compound disclosed herein is diluted in Tumor Cell Media and added in a lOul volume. Plates are incubated for 72 hours at 37C and 5% CO 2 . A volume of 1 OuL of Alamar Blue reagent is added to each well and plates are incubated for 3 hours at 37C and 5% C0 2 . Alamar Blue fluoresce is read at 560Ex/590Em, and IC50 values are calculated using GraphPad Prism 5. The assay can be used to show that some of the compounds of the present invention are potent inhibitors of tumor cell line proliferation under the conditions tested.
  • This tumor model is established from a tumor biopsy of an ovarian cancer patient. Tumor biopsy is taken from the patient.
  • A2780Tax is a paclitaxel-resistant human ovarian carcinoma model. It is derived from the sensitive parent A2780 line by co-incubation of cells with paclitaxel and verapamil, an MDR-reversal agent. Its resistance mechanism has been shown to be non-MDR related and is attributed to a mutation in the gene encoding the beta-tubulin protein.
  • the compounds described herein can be administered to mice bearing staged tumors on an every 2 days x 5 schedule.
  • HCTl 16/VM46 is an MDR-resistant colon carcinoma developed from the sensitive HCTl 16 parent line. In vivo, grown in nude mice, HCTl 16/VM46 has consistently demonstrated high resistance to paclitaxel.
  • the compounds described herein can be administered to mice bearing staged tumors on an every 2 days x 5 schedule.
  • M5076 is a mouse fibrosarcoma that is inherently refractory to paclitaxel in vivo.
  • the compounds described herein can be administered to mice bearing staged tumors on an every 2 days x 5 schedule.
  • One or more compounds of the invention can be used in combination other therapeutic agents in vivo in the multidrug resistant human colon carcinoma xenografts HCT/VM46 or any other model known in the art including those described herein.
  • the assay can be used to show that one or more compounds of the present invention are potent inhibitors of tumor growth in vivo under the conditions tested.
  • the stability of one or more subject compounds is determined according to standard procedures known in the art. For example, stability of one or more subject compounds is established by an in vitro assay. In particular, an in vitro microsome stability assay is established that measures stability of one or more subject compounds when reacting with mouse, rat or human microsomes from liver. The microsome reaction with compounds is performed in 1.5 mL Eppendorf tube. Each tube contains 0.1 ⁇ of 10.0 mg/ml NADPH; 75 ⁇ of 20.0 mg/ml mouse, rat or human liver microsome; 0.4 ⁇ of 0.2 M phosphate buffer, and 425 ⁇ of ddH 2 0.
  • Negative control (without NADPH) tube contains 75 ⁇ of 20.0 mg/ml mouse, rat or human liver microsome; 0.4 ⁇ of 0.2 M phosphate buffer, and 525 ⁇ of ddH 2 0.
  • the reaction is started by adding 1.0 ⁇ of 10.0 mM tested compound.
  • the reaction tubes are incubated at 37°C. 100 ⁇ sample is collected into new Eppendorf tube containing 300 ⁇ cold Methanol at 0, 5, 10, 15, 30 and 60 minutes of reaction. Samples are centrifuged at 15,000 rpm to remove protein. Supernatant of centrifuged sample is transferred to new tube. Concentration of stable compound after reaction with microsome in the supernatant is measured by Liquid Chromatography/Mass Spectrometry (LC-MS).
  • LC-MS Liquid Chromatography/Mass Spectrometry
  • the stability of one or more subject compounds in plasma is determined according to standard procedures known in the art. See, e.g., Rapid Commun. Mass Spectrom., 10: 1019-1026. The following procedure is an HPLC-MS/MS assay using human plasma; other species including monkey, dog, rat, and mouse are also available. Frozen, heparinized human plasma is thawed in a cold water bath and spun for 10 minutes at 2000 rpm at 4 °C prior to use. A subject compound is added from a 400 ⁇ stock solution to an aliquot of pre-warmed plasma to give a final assay volume of 400 ⁇ (or 800 ⁇ for half-life determination), containing 5 ⁇ test compound and 0.5 % DMSO.
  • Reactions are incubated, with shaking, for 0 minutes and 60 minutes at 37 °C, or for 0, 15, 30, 45 and 60 minutes at 37 C for half life determination. Reactions are stopped by transferring 50 ⁇ of the incubation mixture to 200 ⁇ of ice-cold acetonitrile and mixed by shaking for 5 minutes. The samples are centrifuged at 6000 x g for 15 minutes at 4°C and 120 ⁇ of supernatant removed into clean tubes. The samples are then evaporated to dryness and submitted for analysis by HPLC-MS/MS.
  • one or more control or reference compounds are tested simultaneously with the test compounds: one compound, propoxycaine, with low plasma stability and another compound, propantheline, with intermediate plasma stability.
  • Samples are reconstituted in acetonitrile/methanol/water (1/1/2, v/v/v) and analyzed via (RP)HPLC-MS/MS using selected reaction monitoring (SRM).
  • the HPLC conditions consist of a binary LC pump with autosampler, a mixed-mode, CI 2, 2 x 20 mm column, and a gradient program. Peak areas corresponding to the analytes are recorded by HPLC-MS/MS. The ratio of the parent compound remaining after 60 minutes relative to the amount remaining at time zero, expressed as percent, is reported as plasma stability. In case of half- life determination, the half- life is estimated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) vs. time, assuming first order kinetics.
  • the chemical stability of one or more subject compounds is determined according to standard procedures known in the art. The following details an exemplary procedure for ascertaining chemical stability of a subject compound.
  • the default buffer used for the chemical stability assay is phosphate- buffered saline (PBS) at pH 7.4; other suitable buffers can be used.
  • PBS phosphate- buffered saline
  • a subject compound is added from a 100 ⁇ stock solution to an aliquot of PBS (in duplicate) to give a final assay volume of 400 ⁇ , containing 5 ⁇ test compound and 1% DMSO (for half- life determination a total sample volume of 700 ⁇ . is prepared).
  • Reactions are incubated, with shaking, for 0 minutes and 24 hours at 37°C; for half-life determination samples are incubated for 0, 2, 4, 6, and 24 hours. Reactions are stopped by adding immediately 100 ⁇ . of the incubation mixture to 100 ⁇ . of acetonitrile and vortexing for 5 minutes. The samples are then stored at -20°C until analysis by HPLC-MS/MS. Where desired, a control compound or a reference compound such as chlorambucil (5 ⁇ ) is tested simultaneously with a subject compound of interest, as this compound is largely hydrolyzed over the course of 24 hours. Samples are analyzed via (RP)HPLC-MS/MS using selected reaction monitoring (SRM).
  • SRM selected reaction monitoring
  • the HPLC conditions consist of a binary LC pump with autosampler, a mixed- mode, CI 2, 2 x 20 mm column, and a gradient program. Peak areas corresponding to the analytes are recorded by HPLC- MS/MS. The ratio of the parent compound remaining after 24 hours relative to the amount remaining at time zero, expressed as percent, is reported as chemical stability. In case of half-life determination, the half- life is estimated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) vs. time, assuming first order kinetics.
  • Cells comprising components of the Akt/mTOR pathway including but not limited to L6 myoblasts, B-ALL cells, B-cells, T-cells, leukemia cells, bone marrow cells, pi 90 transduced cells, philladelphia chromosome positive cells (Ph+), and mouse embryonic fibroblasts, are typically grown in cell growth media such as DMEM supplemented with fetal bovine serum and/or antibiotics, and grown to confluency.
  • cell growth media such as DMEM supplemented with fetal bovine serum and/or antibiotics
  • said cells are serum starved overnight and incubated with one or more compounds disclosed herein or about 0.1% DMSO for approximately 1 minute to about 1 hour prior to stimulation with insulin (e.g., 100 nM) for about 1 minutes to about 1 hour.
  • insulin e.g., 100 nM
  • Cells are lysed by scraping into ice cold lysis buffer containing detergents such as sodium dodecyl sulfate and protease inhibitors (e.g., PMSF).
  • the solution After contacting cells with lysis buffer, the solution is briefly sonicated, cleared by centrifugation, resolved by SDS-PAGE, transferred to nitrocellulose or PVDF and immunob lotted using antibodies to phospho- Akt S473, phospho- Akt T308, Akt, and ⁇ -actin (Cell Signaling Technologies).
  • PI3K7 Akt /mTor signaling is measured in blood cells using the phosflow method (Methods Enzymol. 2007;434:131-54).
  • the advantage of this method is that it is by nature a single cell assay so that cellular heterogeneity can be detected rather than population averages. This allows concurrent dinstinction of signaling states in different populations defined by other markers. Phosflow is also highly quantitative.
  • unfractionated splenocytes, or peripheral blood mononuclear cells are stimulated with anti-CD3 to initiate T-cell receptor signaling. The cells are then fixed and stained for surface markers and intracellular phosphoproteins. It is expected that inhibitors disclosed herein inhibit anti-CD3 mediated
  • TCR T cell receptor
  • BCR B cell receptor
  • Fab'2 fragments anti- kappa light chain antibody
  • samples are fixed (e.g., with cold 4% paraformaldehyde) and used for phosflow.
  • Surface staining is used to distinguish T and B cells using antibodies directed to cell surface markers that are known to the art.
  • the level of phosphrylation of kinase substrates such as Akt and S6 are then measured by incubating the fixed cells with labeled antibodies specific to the phosphorylated isoforms of these proteins. The population of cells is then analyzed by flow cytometry.
  • the assay can be used to show that one or more of the compounds of the present invention are potent and selective inhibitors of one or more members of one or more of PI3K, mTOR, and Akt signaling in blood cells under the conditions tested.
  • Murine bone marrow cells freshly transformed with a pi 90 BCR-Abl retrovirus (herein referred to as pi 90 transduced cells) are plated in the presence of various drug combinations in M3630 methylcellulose media for about 7 days with recombinant human IL-7 in about 30% serum, and the number of colonies formed is counted by visual examination under a microscope.
  • human peripheral blood mononuclear cells are obtained from Philadelphia chromosome positive (Ph+) and negative (Ph-) patients upon initial diagnosis or relapse. Live cells are isolated and enriched for CD19+ CD34+ B cell progenitors. After overnight liquid culture, cells are plated in methocult GF+ H4435, Stem Cell Tehcnologies) suplemented with cytokines (IL-3, IL-6, IL-7, G-CSF, GM-CSF, CF, Flt3 ligand, and erythropoietin) and various concentrations of known chemotherapeutic agents in combination with either compounds of the present disclosure. Colonies are counted by microscopy 12-14 days later. This method can be used to test for evidence of additive or synergistic activity.
  • cytokines IL-3, IL-6, IL-7, G-CSF, GM-CSF, CF, Flt3 ligand, and erythropoietin
  • the assay can be used to show that one or more the compounds of the present invention are potent and selective inhibitors of pi 90 transduced cell colony formation under the conditions tested.
  • mice Female recipient mice are lethally irradiated from a ⁇ source in two doses about 4 hr apart, with approximately 5Gy each. About lhr after the second radiation dose, mice are injected i.v. with about lxlO 6 leukemic cells (e.g., Ph+ human or murine cells, or pl90 transduced bone marrow cells). These cells are administered together with a radioprotective dose of about 5x10 6 normal bone marrow cells from 3-5 week old donor mice. Recipients are given antibiotics in the water and monitored daily. Mice who become sick after about 14 days are euthanized and lymphoid organs are harvested for analysis. Kinase inhibitor treatment begins about ten days after leukemic cell injection and continues daily until the mice become sick or a maximum of approximately 35 days post-transplant. Inhibitors are given by oral lavage.
  • leukemic cells e.g., Ph+ human or murine cells, or pl90 transduced bone marrow cells.
  • Peripheral blood cells are collected approximately on day 10 (pre-treatment) and upon euthanization (post treatment), contacted with labled anti-hCD4 antibodies and counted by flow cytometry.
  • This method can be used to demonstrate that the synergistic effect of one or more compounds disclosed herein in combination with known chemotherapeutic agents significantly reduce leukemic blood cell counts as compared to treatment with known chemotherapeutic agents (e.g., Gleevec) alone under the conditions tested.
  • the R2KO mice develop lupus-like disease with anti-nuclear antibodies, glomerulonephritis and proteinurea within about 4-6 months of age.
  • the rapamycin analogue RAD001 available from LC Laboratories
  • This compound has been shown to ameliorate lupus symptoms in the B6.Slelz.Sle3z model (T. Wu et al. J. Clin Invest. 117:2186-2196).
  • mice such as R2KO, BXSB or MLR/lpr are treated at about 2 months old, approximately for about two months.
  • Mice are given doses of: vehicle, RAD001 at about lOmg/kg, or compounds disclosed herein at approximately 1 mg/kg to about 500 mg/kg.
  • Blood and urine samples are obtained at approximately throughout the testing period, and tested for antinuclear antibodies (in dilutions of serum) or protein concentration (in urine). Serum is also tested for anti- ssDNA and anti-dsDNA antibodies by ELISA. Animals are euthanized at day 60 and tissues harvested for measuring spleen weight and kidney disease. Glomerulonephritis is assessed in kidney sections stained with H&E. Other animals are studied for about two months after cessation of treatment, using the same endpoints.
  • This model established in the art can be employed to test that the kinase inhibitors disclosed herein can suppress or delay the onset of lupus symptoms in lupus disease model mice.
  • mice Female recipient mice are lethally irradiated from a ⁇ ray source. About lhr after the radiation dose, mice are injected with about 1x106 leukemic cells from early passage pl90 transduced cultures (e.g., as described in Cancer Genet Cytogenet. 2005 Aug;161(l):51-6) . These cells are administered together with a radioprotective dose of approximately 5x106 normal bone marrow cells from 3-5wk old donor mice. Recipients are given antibiotics in the water and monitored daily. Mice who become sick after about 14 days are euthanized and lymphoid organs harvested for flow cytometry and/or magnetic enrichment.
  • Treatment begins on approximately day 10 and continues daily until mice become sick, or after a maximum of about 35 days post-transplant. Drugs are given by oral gavage (p.o.).
  • chemotherapeutic that is not curative but delays leukemia onset by about one week or less is identified; controls are vehicle-treated or treated with chemotherapeutic agent, previously shown to delay but not cure leukemogenesis in this model (e.g., imatinib at about 70mg/kg twice daily).
  • chemotherapeutic agent previously shown to delay but not cure leukemogenesis in this model (e.g., imatinib at about 70mg/kg twice daily).
  • pi 90 cells that express eGFP are used, and postmortem analysis is limited to enumeration of the percentage of leukemic cells in bone marrow, spleen and lymph node (LN) by flow cytometry.
  • pi 90 cells that express a tailless form of human CD4 are used and the postmortem analysis includes magnetic sorting of hCD4+ cells from spleen followed by immunoblot analysis of key signaling endpoints: p Akt -T308 and S473; pS6 and p4EBP-l .
  • sorted cells are incubated in the presence or absence of kinase inhibitors of the present disclosure inhibitors before lysis.
  • "phosflow" is used to detect p Akt -S473 and pS6-S235/236 in hCD4-gated cells without prior sorting.
  • Samples of peripheral blood are obtained weekly from all mice, starting on day 10 immediately prior to commencing treatment. Plasma is used for measuring drug concentrations, and cells are analyzed for leukemia markers (eGFP or hCD4) and signaling biomarkers as described herein.
  • eGFP eGFP or hCD4
  • Example 28 Rat Developing Type II Collagen Induced Arthritis Assay
  • a collagen induced developing arthritis model is used.
  • Female Lewis rats are given collagen injections at day 0.
  • Bovine type II collagen is prepared as a 4mg/ml solution in 0.01N acetic acid.
  • Equal volumes of collagen and Freund's incomplete adjuvant are emulsified by hand mixing until a bead of the emulsified material holds its form in water.
  • Each rodent receives a 300 ⁇ injection of the mixture at each injection time spread over three subcutaneous sites on the back.
  • Oral compound administration begins on day 0 and continues through day 16 with vehicle (5% NMP, 85% PEG 400, 10% Solutol) or compounds of the present invention in vehicle or control (e.g., methotrexate) at 12 hour intervals daily. Rats are weighed on days 0, 3, 6, 9-17 and caliper measurements of ankles are taken on days 9-17. Final body weights are taken, and then the animals are euthanized on day 17. After euthanization, blood is drawn and hind paws and knees are removed. Blood is further processed for pharmacokinetics experiments as well as an anti-type II collagen antibody ELISA assay. Hind paws are weighed and then, with the knees, preserved in 10%> formalin. The paws and knees are subsequently processed for microscopy. Livers, spleen and thymus are weighed. Sciatic nerves are prepared for histopathology.
  • vehicle or control e.g., methotrexate
  • Knee and ankle joints are fixed for 1-2 days and decalcified for 4-5 days. Ankle joints are cut in half longitudinally, and knees are cut in half along the frontal plane. Joints are processed, embedded, sectioned and stained with toluidine blue. Scoring of the joints is done according to the following criteria:
  • the assay can be used to show, relative to vehicle only control or to methotrexate control, that the compounds of the present invention exhibit a siginificant reduction in arthritis induced ankle diameter increase over time, and reduction of ankle histopathology in at least one or more of the categories of inflammation, pannus, cartilage damage, and bone resporption as described above.
  • the assay can be used to show that one or more compounds of the present invention may be useful for the treatment and reduction of arthritis disease symptoms.
  • mice Female Lewis rats are anesthetized and given collagen injections prepared and administered as described previously on day 0. On day 6, animals are anesthetized and given a second collagen injection. Caliper measurements of normal (pre-disease) right and left ankle joints are performed on day 9. On days 10-11, arthritis typically occurs and rats arerandomized into treatment groups.
  • the assay can be used to show reduction in mean ankle diamter increase over time for selected test compounds under the conditions tested.
  • Isoflurane-anesthetized Lewis rats 200-250 g are implanted with an intrathecal (IT) catheter. After a 6 d recovery period, all animals except those that appeared to have sensory or motor abnormalities (generally fewer than 5% of the total number) are used for experiments.
  • IT administration 10 ⁇ of drug or saline followed by 10 ⁇ of isotonic saline is injected through the catheter.
  • Drug e.g., one or more compounds of the present invention or or vehicle
  • Treatment is generally started on day 8 and is continued daily until day 20.
  • Clinical signs of arthritis generally begin on day 10, and paw swelling is determined every second day by water displacement plethysmometry.
  • the assay can be used to show that one or more compounds of the present invention may be useful for the treatment of one or more of the diseases or conditions described herein.
  • mice In order to study the pharmacokinetics of the compounds of the present invention a set of 4-10 week old mice are grouped according to the following table:
  • Compounds of the present invention are dissolved in an appropriate vehicle (e.g., 5% 1- methyl-2-pyrrolidinone, 85% polyethylene glycol 400, 10% Solutor) and administered orally at 12 hour intervals daily. All animals are euthanized in CO 2 2 hours after the final compound is administered. Blood is collected immediately and kept on ice for plasma isolation. Plasma is isolated by centrifuging at 5000 rpm for 10 minutes. Harvested plasma is frozen for pharmacokinetic detection.
  • an appropriate vehicle e.g., 5% 1- methyl-2-pyrrolidinone, 85% polyethylene glycol 400, 10% Solutor
  • the assay can be used to demonstrate the pharmacokinetic parameters such as absorption, distribution, metabolism, excretion, and toxicity for the compounds of the present invention.
  • the basotest assay is performed using Orpegen Pharma Basotest reagent kit. Heparinized whole blood is pre- incubated with test compound or solvent at 37C for 20min. Blood is then incubated with assay kit stimulation buffer (to prime cells for response) followed by allergen (dust mite extract or grass extract) for 20min. The degranulation process is stopped by incubating the blood samples on ice. The cells are then labeled with anti-IgE-PE to detect basophilic granulocytes, and anti-gp53-FITC to detect gp53 (a glycoprotein expressed on activated basophils). After staining red blood cells are lysed by addition of Lysing Solution. Cells are washed, and analyzed by flow cytometry.
  • Test compounds when evaluated in this assay inhibit allergen induced activation of basophilic granulocytes at sub micromolar range.
  • the assay can be used to demonstrate that under the conditions tested one or more compounds of the present invention are capable of inhbiting allergen induced activation of basophils.
  • Example 33 Use of the compounds of the present invention for inhibition of tumor growth
  • Cell lines of interest (A549, U87, ZR-75-1 and 786-0) are obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells are proliferated and preserved cryogenically at early passage (e.g., passage 3). One aliquot is used for further proliferation to get enough cells for one TGI study (at about passage 9). Animals
  • mice Female athymic nude mice are supplied by Harlan. Mice are received at 4 to 6 weeks of age. All mice are acclimated for about one day to two weeks prior to handling. The mice are housed in microisolator cages and maintained under specific pathogen- free conditions. The mice are fed with irradiated mouse chow and freely available autoclaved water is provided.
  • mice are inoculated subcutaneous ly in the right flank with 0.01 to 0.5 ml of tumor cells (approximately 1.0 x 10 5 to 1.0 x 10 s cells/mouse). Five to 10 days following inoculation, tumors are measured using calipers and tumor weight is calculated, for example using the animal study management software, such as Study Director V.1.6.70 (Study Log). Mice with tumor sizes of about 120 mg are pair-matched into desired groups using Study Director (Day 1). Body weights are recorded when the mice are pair-matched. Tumor volume and bodyweight measurements are taken one to four times weekly and gross observations are made at least once daily.
  • mice On Day 1, compounds of the present invention and reference compounds as well as vehicle control are administered by oral gavage or iv as indicated.
  • mice are sacrificed and their tumors are collected 1-4 hours after the final dose. The tumors are excised and cut into two sections. One third of the tumor is fixed in formalin and embedded in paraffin blocks and the remaining two thirds of tumor is snap frozen and stored at -80°C.
  • TS tumor shrinkage
  • the model can be employed to show whether the compounds of the present invention can inhibit tumor cell growth such as renal carcinomoa cell growth, breast cancer cell growth, lung cancer cell growth, or glioblastoma cell growth under the conditions tested.
  • tumor cell growth such as renal carcinomoa cell growth, breast cancer cell growth, lung cancer cell growth, or glioblastoma cell growth under the conditions tested.
  • Inhibition of angiogenesis in the presence of test compound is evaluated using a tube formation assay, such as by using a tube formation assay kit (e.g., commerically available from Invitrogen).
  • Angiogenic capacity can be measured in vitro using an endothelial cell line, such as human umbilical vein endothelial cells (HUVEC).
  • the assay is conducted according to the kit instructions, in the presence or absence of compound.
  • a gel matrix is applied to a cell culture surface, cells are added to the matrix-covered surface along with growth factors, with some samples also receiving an inhibitor compound, cells are incubated at 37°C and 5% CO 2 long enough for control samples (no compound added) to form tube structures (such as overnight), cells are stained using a cell-permeable dye (e.g., calcein), and cells are visualized to identify the degree of tube formation. Any decrease in tube formation relative to un-inhibited control cells is indicative of angiogenic inhibition. Based on doses tested and the corresponding degree of tube formation inhibition, IC50 values for tube formation are calculated. IC50 values for cell viability can be measured using any number of methods known in the art, such as staining methods that distinguish live from dead cells (e.g., Image-iT DEAD Green viability stain commercially available from Invitrogen.
  • TNP-Ficoll B-cell activation assay is used as described herein.
  • Compounds of the present invention are dissolved in an appropriate vehicle (e.g. 5% l-methyl-2- pyrrolidinone, 85% polyethylene glycol 400, 10% Solutor).
  • Compounds are administered orally approximately lhr before TNP-Ficoll treatment to 4-10 week old mice.
  • an appropriate vehicle e.g. 5% l-methyl-2- pyrrolidinone, 85% polyethylene glycol 400, 10% Solutor
  • TNP- BSA is coated onto a Nunc Maxisorb microtiter plate with ⁇ /well at a concentration of lC ⁇ g/ml in phosphate buffered saline (PBS). The Maxisorb plate is incubated for 1.5 hours at room
  • blocking buffer e.g. 1% BSA in PBS
  • PBS 0.05% Tween-20 wash buffer
  • a 1 :2 dilution of serum from each mouse in blocking buffer is added to each well in the first column (1) of the microtiter plate.
  • the serum in each well of column 1 is then diluted 3-fold in blocking buffer and added to column 2.
  • the serum in each well of column 2 is diluted 3-fold in blocking buffer and added to column 3.
  • the procedure is repeated across the twelve columns of the microtiter plate.
  • the microtiter plate is incubated 1 hr at room temperature.
  • Serum is removed from the plate and the plate is washed three times with wash buffer.
  • 100 ⁇ /well of goat anti-mouse IgG3-HRP diluted 1 :250 in blocking buffer is added to each well and incubated lhr at room temperature.
  • the anti-mouse IgG3-HRP is removed from the microtiter plate and the plate is washed six times with wash buffer.
  • HRP substrate 200 ⁇ ABTS solution + 30% H 2 0 2 + 10ml citrate buffer
  • the amount of anti-TNP IgG3 is determined spectrophotometrically at 405nm.
  • anti-TNP IgM and total anti-TNP Ab are determined using anti-mouse IgM-HRP and anti-mouse Ig-HRP respectively.

Abstract

La présente invention concerne des composés hétérocycliques bicycliques en tant qu'inhibiteurs de PI3 kinase, par exemple de la PI3 kinase alpha, apte à réagir de façon covalente avec la protéine kinase par liaison à un résidu cystéine de la PI3 kinase conduisant à la formation d'un produit conjugué. L'invention concerne également des procédés d'utilisation de tels inhibiteurs de PI3 kinase pour le traitement d'un état ou d'une maladie associés à la PI3 kinase chez un sujet.
PCT/US2012/036664 2011-05-06 2012-05-04 Inhibiteurs réactifs de la pi3 kinase et leurs utilisations WO2012154610A1 (fr)

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US10800782B2 (en) 2016-08-31 2020-10-13 Agios Pharmaceutical, Inc. Inhibitors of cellular metabolic processes
USRE49934E1 (en) 2022-03-08 2024-04-23 Servier Pharmaceuticals Llc Inhibitors of cellular metabolic processes

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014151147A1 (fr) * 2013-03-15 2014-09-25 Intellikine, Llc Combinaison d'inhibiteurs de kinase et ses utilisations
US10800782B2 (en) 2016-08-31 2020-10-13 Agios Pharmaceutical, Inc. Inhibitors of cellular metabolic processes
US11325914B1 (en) 2016-08-31 2022-05-10 Servier Pharmaceuticals Llc Inhibitors of cellular metabolic processes
USRE49934E1 (en) 2022-03-08 2024-04-23 Servier Pharmaceuticals Llc Inhibitors of cellular metabolic processes

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