KR20080018908A - Combination therapy comprising a diaryl urea compound and a pi3, akt kinase or mtor inhibitors (rapamycins) for cancer treatment - Google Patents

Abstract

The present invention relates to pharmaceutical compositions and combinations for treating cancer, comprising a diaryl urea compound e.g. 4 {4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy}-pyridine-2-carboxylic acid methylamide and an PI3K/AKT signaling pathway inhibitor. The PI3K/AKI signaling pathway inhibitor comprises PI3 inhibitors {like celecoxilo, viridins, wortmannins}, AKT kinase inhibitors {like perifosine, triciribine} and mTOR inhibitors {like the rapamycins temsirolimus and evorolimus}.

Description

COMBINATION THERAPY COMPRISING A DIARYL UREA COMPOUND AND A PI3, AKT KINASE OR MTOR INHIBITORS (RAPAMYCINS) FOR CANCER TREATMENT

For example the diaryl urea compounds described in US 20050038080, such as 4 {4- [3- (4-chloro-3-trifluoromethylphenyl) -ureido] -3-fluorophenoxy} -pyridine-2- Carboxylic acid methylamides are potent anticancer and antiangiogenic agents with various activities, including inhibitory activity against VEGFR, PDGFR, raf, p38 and / or flt-3 kinase signaling molecules. The RAS / RAF / MEK / ERK pathway is involved in cell proliferation, differentiation and modification and is involved in many cancers. The PI3K / AKT signaling pathway is another important physiological pathway in cells. It mediates extracellular stimuli including growth factors, cytokines, cell-cell adhesion and cell-extracellular matrices (Vivanco and Sawyers, Nat Rev Cancer, 2: 489-501, 2002, Downward, Curr Opin Cell Biol, 10 : 262-267, 1998). The AKT pathway appears to be active in various types of human cancers (Nicholson and Anderson, Cell Signal, 14: 381-395, 2002).

The present invention provides drug combinations, compositions and methods for treating cell proliferative disorders (eg cancer), inflammation, immunomodulatory disorders, and conditions associated with abnormal or undesirable angiogenesis. Drug combinations include a compound of Formula I and one or more second compounds that are inhibitors of the PI3K / AKT signaling pathway. The method may include, for example, administering a diaryl urea compound and signaling pathway inhibitors, pharmaceutically acceptable salts thereof, derivatives thereof, and the like, as described below.

Phosphatidylinositol-3-kinase (PI3K) and AKT (protein kinase B) signaling pathways regulate a variety of biological processes, including cell survival, cell proliferation, cell growth, and cell motility. Aberrations in PI3K-AKT signaling cause many pathologies of disease and conditions, including cell proliferative disorders (eg cancer), inflammatory and immunomodulatory disorders.

Many growth and survival factors activate PI3K population members to specifically convert one lipid signaling molecule, PIP2, to another, PI (3,4,5) P3. Phosphorylated products supplement the internal plasma membrane with Akt population members to stimulate their protein kinase activity. To date, a number of Akt effectors have been identified that are involved in several biological processes. For example, Akt kinase mediates cell survival through phosphorylation and inactivation of apoptotic organ components. PI3K / AKT signaling pathways include all members or components that participate in a signal transduction cascade. These include, for example, PI3-kinase, Akt-kinase, FKBP12, mTOR (mammalian target of rapamycin; also FRAP, RAFT1 or RAPT1), RAPTOR (regulatory related protein in the case of mTOR), TSC (nodular sclerosis complex) tuberous sclerosis complex)), PTEN (phosphatase and tensine homologues), and downstream effectors thereof, but are not limited thereto. The combinations of the present invention can be used to treat and / or prevent all conditions and / or diseases associated with any of the aforementioned activities.

Inhibitors of the PI3K / AKT signaling pathway are compounds that inhibit one or more members of the aforementioned signaling pathways. Such compounds may be called route inhibitors, but the present invention encompasses the use of these inhibitors to treat any of the diseases or conditions mentioned above, regardless of the mechanism of action or how the therapeutic effect is achieved. Indeed, such a compound may have one or more targets and the initial activity recognized for the compound is recognized to be not the activity it has in vivo or thereby achieve its therapeutic effect when administered to a subject. do. Thus, describing a compound as a pathway or protein target (eg, Akt or mTOR) inhibitor indicates that the compound has this activity, but when used as a therapeutic or prophylactic agent, the compound is said to have that activity. It is not limited in any way.

Examples of AKT population members include Akt1, Akt2 (typically overexpressed in tumors; Bellacosa et al., Int . J. Cancer , 64: 280-285, 1995) and Akt3.

Examples of PI3K population members include p110-alpha, p110-beta, p110-delta and p110-gamma (catalytic).

Examples of PI3K / AKT signaling pathway inhibitors include

FTY720 (eg, Lee et al., Carcinogenesis , 25 (12): 2397-2405, 2004);

UCN-01 (eg, Amornphimoltham et al., Clin Cancer Res ., 10 (12 Pt 1): 4029-37, 2004)

These include, but are not limited to these.

Examples of phosphatidylinositol-3-kinase (PI3-kinase) inhibitors include

Celecoxib and its analogs such as OSU-03012 and OSU-03013 (eg, Zhu et al., Cancer Res ., 64 (12): 4309-18, 2004);

3-deoxy-D-myo-inositol analogues such as PX-316 (see, eg, US Application 20040192770; Meuillet et al., Oncol . Res. , 14: 513-27, 2004);

2'-substituted 3'-deoxy-phosphatidyl-myo-inositol analogues (eg, Tabellini et al., Br. J. Haematol ., 126 (4): 574-82, 2004);

Fused heteroaryl derivatives (US Pat. No. 6,608,056);

3- (imidazo [1,2-a] pyridin-3-yl) derivatives (eg, US Pat. Nos. 6,403,588 and 6,653,320);

Ly294002 (eg, Vlahos, et al., J. Biol. Chem, ., 269 (7): 5241-5248, 1994);

Quinazolin-4-one derivatives such as IC486068 (eg, US Application No. 20020161014; Geng et al., Cancer Res ., 64: 4893-99, 2004);

3- (hetero) aryloxy substituted benzo (b) thiophene derivatives (eg WO 04 108715; also WO 04 108713);

PX-866 (acetic acid (1S, 4E, 10R, 11R, 13S, 14R)-[4-diallylaminomethylene-6-hydroxy-1-methoxymethyl-10.13-dimethyl-3,7,17-trioxo -1,3,4,7,10,11,12,13,14,15,16,17-octadecahydro-2-oxa-cyclopenta [a] phenanthrene-11-yl ester) (eg Viridine, including semisynthetic birridines such as, Ihle et al., Mol . Cancer Ther ., 3 (7): 763-72, 2004; US Application No. 20020037276; And

Wortmannin and its derivatives (eg, US Pat. Nos. 5,504,103; 5,480,906; 5,468,773; 5,441,947; 5,378,725; 3,668,222)

Are included, but are not limited to these.

Examples of Akt-kinase (also known as protein kinase B) inhibitors include

Akt-1-1 (inhibits Akt1) (Barnett et al., Biochem . J. , 385 (Pt . 2): 399-408, 2005);

Akt-1-1,2 (inhibiting Ak1 and 2) (Barnett et al., Biochem . J. , 385 (Pt . 2): 399-408, 2005);

API-59CJ-Ome (eg, Jin et al., Br. J. Cancer ., 91: 1808-12, 2004);

1-H-imidazo [4,5-c] pyridinyl compounds (eg, WO05011700);

Indole-3-carbinol and derivatives thereof (eg, US Pat. No. 6,656,963; Sarkar and Li, J Nutr ., 134 (12 Suppl): 3493S-3498S, 2004);

Perifosine (eg, inhibits Akt membrane localization; Dasmahapatra et al., Clin . Cancer Res ., 10 (15): 5242-52, 2004);

Phosphatidylinositol ether lipid analogs (eg, Gills and Dennis, Expert. Opin . Investig . Drugs , 13: 787-97, 2004);

Triciribin (TCN or API-2 or NCI Distinguished Name: NSC 154020; Yang et al., Cancer Res ., 64: 4394-9, 2004)

These include, but are not limited to these.

Examples of mTOR inhibitors include

FKBP12 enhancer;

CCI-779 (Temsirolimus), RAD001 (Everolimus; WO9409010), TAFA93 and AP23573; Rapalogs such as those described, for example, in WO 98/02441 and WO 01/14387, for example AP23573, AP23464, AP23675 or AP23841; 40- (2-hydroxyethyl) rapamycin, 40- [3-hydroxy (hydroxymethyl) methylpropanoate] -rapamycin (also called CC1779), 40-epi- (tetrazolyl) -rapa Mycin (also called ABT578), 32-deoxolarapamycin, 16-pentynyloxy-32 (S) -dihydrorapamycin, and other derivatives described in WO 05005434; USP 5,258,389, WO 94/090101, WO 92/05179, USP 5,118,677, USP 5,118,678, USP 5,100,883, USP 5,151,413, USP 5,120,842, WO 93/111130, WO 94/02136, WO 94/02485, WO 95/14023, WO 94 Rapamycin and derivatives thereof, including the derivatives described in / 02136, WO 95/16691 (eg SAR 943), EP 509795, WO 96/41807, WO 96/41807 and USP 5,256,790;

Phosphorus-containing rapamycin derivatives (eg WO 05016252); And

4H-1-benzopyran-4-one derivatives (eg, US Provisional Application No. 60 / 528,340)

Are included, but are not limited to these.

Examples of preclinical or clinical compounds include AP23573, AP23841, CCI-779 and RAD001.

Examples of phosphatidylinositol-3-kinase (PI3-kinase) inhibitors of interest are wortmannin and its derivatives or analogs, and pharmaceutically acceptable salts of wortmannin and its derivatives or analogs. Thus, the process of the present invention provides a process for preparing a protein of formula (W), which is a PI3-kinase inhibitor of formula (W), a derivative or analogue of a compound of formula (W), a pharmaceutically acceptable salt of a compound of formula (W), and a derivative of a compound of formula (W), or Use of pharmaceutically acceptable salts of analogs includes:

References herein to derivatives and analogues of wortmannin or compounds of Formula (W) include US Pat. No. 5,504,103; 5,480,906; 5,468,773; 5,441,947; 5,378,725; It is intended to include derivatives and analogues identified in US Pat. No. 3,668,222. Suitable derivatives and analogs of the compounds of formula W include the following compounds:

a) a compound of formula W1

Wherein R is H (11-desacetoxywatermannin) or acetoxy and R 'is C ₁ -C ₆ alkyl;

b) Δ9,11-dehydrodesacetoxy watermannin compound of formula W2

Wherein R 'is C ₁ -C ₆ alkyl;

c) 17 (α-dihydro-watermannin compound of formula W3

Wherein R is H or acetoxy, R 'is C ₁ -C ₆ alkyl, and R "is H, C ₁ -C ₆ alkyl, -C (O) OH or -C (O) OC ₁ -C ₆ alkyl;

d) open A-substituted or esters of the wortmannin compound of formula W4

Wherein R ₁ is H, methyl or ethyl and R ₂ is H or methyl; or

e) 11- and 17-substituted derivatives of wortmannin of formula W5

Wherein R ₄ is ═O or —O (CO) R ₆ , R ₃ is ═O, —OH or —O (CO) R ₆ , and R ₆ are each independently phenyl, C ₁ -C ₆ alkyl Or substituted C ₁ -C ₆ alkyl, where R ₄ is ═O or —OH, R ₃ is not ═O.

{4- [3- (4-Chloro-3-trifluoromethylphenyl) -ureido] -3-fluorophenoxy} -pyridine-2-carboxylic acid methylamide, pharmaceutically acceptable salts, polymorphs thereof, Compounds having the structure of formula (I) corresponding to solvates, hydrates, metabolites and prodrugs are referred to collectively herein as "compounds of formula (I)". Formula I is as follows:

When the word compound, salt, and the like are used in plural forms, this is also interpreted to mean a single compound, salt, and the like.

The term does not denote a C _{1 -6} alkyl is in a different way, or cyclic, linear, or single or multiple branching (branching) which can be branched to, a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms it means. Such groups include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclobutyl and the like. The invention also relates to useful forms of the compounds described herein, such as pharmaceutically acceptable salts and metabolites. The present invention also relates to prodrugs of the compounds of formula (I). The term “pharmaceutically acceptable salts” refers to relatively nontoxic inorganic or organic acid addition salts of the compounds of the invention (see, eg, SM Berge, et al ., “Pharmaceutical Salts,” J. Pharm . Sci . 1977, 66, 1-19). Pharmaceutically acceptable salts include reacting the main compound serving as a base with an inorganic or organic acid, such as hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, camphorsulfonic acid, oxalic acid, maleic acid, succinic acid and citric acid. Included are those obtained by forming salts. Pharmaceutically acceptable salts also include those in which the main compound acts as an acid and reacts with the appropriate base to form, for example, sodium, potassium, calcium, magnesium, ammonium and choline salts. Those skilled in the art will also recognize that acid addition salts of the claimed compounds can be prepared by reaction of the compound with an appropriate inorganic or organic acid by any of a number of known methods. Instead, alkali and alkaline earth metal salts are prepared by reacting a compound of the present invention with an appropriate base by a number of known methods.

Representative salts of the compounds of the present invention include, for example, conventional non-toxic and quaternary ammonium salts formed from inorganic or organic acids or bases by methods well known in the art. For example, such acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorlate, camphorsulfonate, cinnamate, cyclopentane Propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, itaconate, lactate, maleate, mandelate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfo Sites, tartrate, thiocyanate, tosylate, include trifluoromethane sulfonate and undecanoate.

Base salts include alkali metal salts such as potassium and sodium salts, alkaline earth metal salts such as calcium and magnesium salts, and ammonium salts with organic bases such as dicyclohexylamine and N-methyl-D-glucamine. In addition, basic nitrogen-containing groups include methyl, ethyl, propyl, and lower alkyl halides such as butyl chloride, bromide and iodide; Dialkyl sulfates such as dimethyl, diethyl and dibutyl sulfate; And long chain halides such as diamyl sulfate, decyl, lauryl, myristyl, and stearyl chloride, bromide and iodide, aryl or aralkyl halides such as benzyl and phenethyl bromide, and other monosubstituted aralkyls Quaternized with materials such as halides or polysubstituted aralkyl halides.

Solvates for the purposes of the present invention are in the form of compounds in which the solvent molecules form complexes in the solid state, including but not limited to, for example, ethanol and methanol. Hydrates are a particular form of solvate when the solvent molecule is water.

Certain pharmacologically active agents can be further modified by unstable functional groups that degrade after administration in vivo to supply the parent active agent and the pharmacologically inactive derivatizing group. These derivatives, commonly referred to as prodrugs, for example, change the physicochemical properties of the active agent, target the active agent to specific tissues, change the pharmacodynamic and pharmacokinetic properties of the active agent, and Can be used to reduce. Prodrugs of the present invention are suitable for the preparation of suitable compounds of the invention that are pharmaceutically acceptable esters with good tolerability, such as, for example, alkyl esters including methyl, ethyl, propyl, isopropyl, butyl, isobutyl or pentyl esters. It includes a ster. Although methyl esters are preferred, additional esters may be used, such as phenyl-C ₁ -C ₅ alkyl.

Methods that can be used to synthesize other prodrugs are described in the following documents on this subject, which are incorporated herein by reference for the description of these synthesis methods:

Metabolites of the compounds of the invention include oxidized derivatives of compounds of formula (I) in which one or more nitrogens are substituted by hydroxy groups, wherein the nitrogen atoms of the pyridine groups are in oxide form (1 in the art) -Oxo-pyridine), derivatives with hydroxy substituents (referred to as 1-hydroxy-pyridine in the art).

General manufacturing method

The compounds of the invention can be prepared using known chemical reactions and the procedures described, for example, in the following known international application WO 2005/009961.

Compounds of formula (I) have already been specified to have a variety of activities including inhibiting the Raf / MEK / ERK pathway, raf kinase, p38 kinase, VEGFR kinase, PDGFR kinase. Their activity and their use in treating various diseases and conditions are described, for example, in WO 2005/009961, which is hereby incorporated by reference in its entirety.

Indication

Drug combinations of the invention can be used to treat any disease or condition associated with or mediated by cellular pathways modulated by the compounds that make up the combination. These pathways include, but are not limited to, signaling pathways including, for example, VEGFR, VEGFR2, Raf / Mek / Erk, Akt / PI3K, MTOR, PTEN, and the like (see also above). Drug combinations are used to treat diseases associated with or mediated by mutations in one of a number of genes present in these pathways, including cancer-associated mutations in PTEN, ras, Raf, Akt, PI3K, and the like. Can be useful.

As mentioned above, although compounds may be known as specific inhibitors, the present invention encompasses any improving or therapeutic effect regardless of the mechanism of action or how it is achieved.

Drug combinations are antiproliferative; Antitumor; Antiangiogenicity; Inhibition of proliferation of endothelial or tumor cells; Anti-neoplastic; Immunosuppressive; Immunomodulatory; One or more of activities including apoptosis-promoting and the like.

Conditions or diseases that can be treated according to the invention include proliferative disorders (eg cancer), inflammatory disorders, immune-modulatory disorders, allergies, autoimmune diseases (eg rheumatoid arthritis or multiple sclerosis), Abnormal or excessive angiogenesis.

having one or more mutations in raf, VEGFR-2, VEGFR-3, PDGFR-beta, Flt-3, ras, PTEN, Akt, PI3K, mTOR, as well as all members upstream or downstream of the signaling pathways they are part of Any tumor or cancer can be treated, including but not limited to cancer. The tumor or cancer can be treated with the drug combination of the present invention regardless of the mechanism by which it is responsible. Any organ, including (but not limited to) colon, pancreas, breast, prostate, bone, liver, kidney, lung, testicle, skin, pancreas, stomach, prostate, ovary, uterus, head and neck, blood cells, lymph, etc. Even cancer can be treated.

Cancers that can be treated according to the invention include, in particular, brain tumors, breast cancers, sarcoma of bones (e.g., osteosarcoma and Ewings sarcoma), bronchial precancerous conditions, endometrial cancer, glioblastoma, hematoma, hepatocellular carcinoma, Hodgkin's disease, renal neoplasm, leukemia, leiomyosarcoma, liposarcoma, lymphoma, Lhermitte-Duclose disease, malignant glioma, melanoma, malignant melanoma, metastatic cancer, multiple myeloma, myelization Live, myeloid leukocyte syndrome, non-small cell lung cancer, pancreatic cancer, prostate cancer, renal cell carcinoma (e.g., advanced, progressive therapeutic resistance), rhabdomyosarcoma, soft tissue sarcoma, squamous cell carcinoma of the skin, loss of PTEN function, activated Cancers associated with Akt (eg, tumors with PTEN-free tumors and ras mutations) are included, but are not limited to these.

Examples of breast cancers include, but are not limited to, invasive ductal cancer, invasive lobular cancer, ductal epithelial cancer and lobular epithelial cancer.

Examples of cancers of the respiratory tract include, but are not limited to, small cell lung cancer, non-small cell lung cancer, bronchial adenoma and pleural blastoma.

Examples of brain cancers include, but are not limited to, brainstem and hypothalamic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, epithelial cell tumor, and neuroectodermal and pineal tumors.

Tumors of the male reproductive organs include, but are not limited to, prostate and testicular cancer. Tumors of female reproductive organs include, but are not limited to, endometrial, oral, ovarian, vaginal and vulvar cancers, as well as sarcomas of the uterus.

Tumors of the digestive system include, but are not limited to, cancers of the anus, colon, colorectal, esophagus, gallbladder, stomach, pancreas, rectum, small intestine, and salivary glands.

Tumors of the urinary tract include, but are not limited to, cancers of the bladder, penis, kidney, renal pelvis, ureters and urethra.

Eye cancers include, but are not limited to intraocular melanoma and retinoblastoma.

Examples of liver cancers include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinoma with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct cancer), and mixed hepatocellular cholangiocarcinoma.

Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.

Head and neck cancers include, but are not limited to, cancers of the larynx, hypopharyngeal, nasopharynx, and / or oropharyngeal, and cancers of the labia and oral cavity.

Lymphomas include, but are not limited to, AIDS-related lymphomas, non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, Hodgkin's disease, and lymphomas of the central nervous system.

Sarcomas include, but are not limited to, soft tissue sarcomas, osteosarcomas, malignant fibrous histiocytoma, lymphosarcomas, and rhabdomyosarcomas.

Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hematopoietic leukemia.

In addition to inhibiting the proliferation of tumor cells, the drug combinations of the present invention can also cause tumor regression, eg, a decrease in the size of a tumor or a range of cancers in the body.

Preferred are for the treatment of melanoma, kidney cancer, hepatocellular cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, colorectal cancer, breast cancer or pancreatic cancer.

Angiogenesis-related conditions and disorders can also be treated with the drug combinations of the present invention. Inappropriate and ectopic expression of angiogenesis can be detrimental to an organism. Many pathological conditions are associated with the growth of blood vessels in vitro. These include, for example, diabetic retinopathy, angiogenic glaucoma, psoriasis, posterior capsular fibrosis, angiofibrosis, inflammation, restenosis, and the like. In addition, increased blood supply associated with cancer and neoplastic tissues promotes growth resulting in rapid tumor expansion and metastasis. Moreover, the growth of new blood vessels in tumors provides an escape pathway for renegade cells to promote metastasis and thus the spread of cancer.

Systems useful for modulating angiogenesis include, for example, angiogenesis of tumor implants (eg, US Pat. Nos. 5,192,744; 6,024,688), chicken chorioallantoic membrane (CAM) tests (eg, Taylor and Folkman, Nature, 297: 307-312, 1982; Eliceiri et al., J. Cell Biol., 140, 1255-1263, 1998), bovine capillary endothelial cell assay (BCE) cell test (e.g. See, for example, US Pat. No. 6,024,688; Polverini, PJ et al., Methods Enzymol., 198: 440-450, 1991), migration assays and HUVECs (human umbilical cord vascular endothelial cells). Growth inhibition testing (eg, US Pat. No. 6,060449). In addition, systems useful for modulating lymphangiogenesis include, for example, rabbit ear models (eg, Szuba et al., FASEB J., 16 (14): 1985-7, 2002).

Modulation of angiogenesis can be measured by any suitable method. For example, the degree of tissue vascular distribution is generally measured by assessing the number and density of blood vessels present in a given sample. For example, microvascular density (MVD) can be used to count the number of endothelial clusters in high magnification microscopic fields, or to grow or markers specific for microvascular endothelial, such as CD31 (also platelet-endothelial adhesion molecules or PECAM). It can be evaluated by detecting other markers of anchored blood vessels. CD31 antibodies are described, for example, in Penfold et al., Br. J. Oral and Maxill. Surg., 34: 37-41; US Pat. No. 6,017,949; Dellas et al., Gyn. Oncol., 67:27 -33, 1997, etc.) can be used in conventional immunohistochemical methods for immunostaining tissue sections. Other markers for angiogenesis include, for example, Vezf1 (eg, Xiang et al., Dev. Bio., 206: 123-141, 1999), angiopoietin, Tie-1, and Tie- 2 (eg, Sato, et al., Nature, 376: 70-74, 1995).

Drug combinations of the present invention may also be used for inflammatory conditions, coronary restenosis, tumor-associated angiogenesis, atherosclerosis, autoimmune diseases, inflammation, certain kidney diseases associated with proliferation of glomerular or glomerular interstitial cells, and retinal vascular proliferation. A wide range of diseases such as eye diseases, psoriasis, cirrhosis, diabetes, atherosclerosis, restenosis, vascular graft restenosis, stent-stenosis, angiogenesis, eye disease, pulmonary fibrosis, obstructive bronchiolitis, glomerulonephritis, rheumatoid arthritis Has a wide range of therapeutic activities to treat or prevent the progression of the class of diseases.

The invention also provides methods of treating, preventing, or modulating one or more of the following conditions in humans and / or other mammals: diabetic retinopathy, ischemic retinal-vein occlusion, premature infants Retinopathy, including retinopathy and age-related macular degeneration; Bullous disorders associated with subepithelial blister formation, including rheumatoid arthritis, psoriasis, or bullous pseudophobic, polymorphic erythema or herpes dermatitis, rheumatic fever, bone resorption, postmenopausal osteoporosis, sepsis, gram negative sepsis, septic shock , Endotoxin shock, toxic shock syndrome, systemic inflammatory reaction syndrome, inflammatory bowel disease (Crohn's disease and ulcerative colitis), Jarisch-Herxheimer reaction, asthma, adult respiratory distress syndrome, acute pulmonary fibrosis Disease, pulmonary sarcoidosis, allergic respiratory disease, silicosis, coal mine pneumoconiosis, alveolar damage, liver failure, liver disease during acute inflammation, severe alcoholic hepatitis, malaria (Plasmodium falciparum malaria and brain malaria) , Non-insulin-dependent diabetes mellitus (NIDDM), congestive heart failure, heart disease damage, atherosclerosis, Alzheimer's disease, acute encephalitis, brain damage , Multiple sclerosis (myelination and multiple dendritic cell loss in multiple sclerosis), advanced cancer, lymphoid tumor, pancreatitis, infection, inflammation and wound healing damaged from cancer, myelodysplastic syndrome, systemic lupus erythematosus, biliary sclerosis, intestinal necrosis, Toxicity by the administration of radiation damage / monoclonal antibodies, graft-versus-host reaction (ischemic reperfusion injury and allograft rejection of kidney, liver, heart and skin), pulmonary allograft rejection (occlusive bronchitis), or total hip replacement Complications due to, and tuberculosis, Helicobacter pylori infection during peptic ulcer disease, Chaga's disease due to Trypanosoma cruzi infection, E. coli. Effects of Shiga-like Toxins from E. coli Infections, Effects of Enterotoxin A Due to Staphylococcus Infections, Meningococcal Infections, and Borrelia burgdorferi, Trefo Infectious diseases selected from infections of Treponema pallidum, cytomegalovirus, influenza virus, Tyler's encephalomyelitis virus and human immunodeficiency virus (HIV), papilloma, blastoglioma, Kaposi's sarcoma , Melanoma, lung cancer, ovarian cancer, prostate cancer, squamous cell cancer, astrocytoma, head cancer, neck cancer, bladder cancer, breast cancer, colorectal cancer, thyroid cancer, pancreatic cancer, stomach cancer, hepatocellular carcinoma, leukemia, lymphoma, Hodgkin's disease, Burkitt's disease (Burkitt's disease), arthritis, rheumatoid arthritis, diabetic retinopathy, angiogenesis, restenosis, stent-stenosis, vascular restenosis, pulmonary fibrosis, liver guard, atherosclerosis Sclerosis, glomerulonephritis, diabetic nephropathy, thrombotic microangiopathy syndrome, graft rejection, psoriasis, diabetes, wound healing, inflammatory and neurodegenerative disorders, hyperimmune disorders, hemangiomas, myocardial angiogenesis, coronary and Cerebral angiogenesis, ischemia, corneal disease, skin redness, angiogenic glaucoma, macular degeneration retinopathy of premature infants, wound healing, ulcer Helicobacter related disease, fractures, endometriosis, diabetic condition, febrile fever, thyroid hyperplasia, asthma, or Burns, trauma, chronic lung disease, stroke, polyps, cysts, synovitis, edema caused by chronic and allergic inflammation, ovarian hyperstimulation syndrome, lung and brain edema, keloid, fibrosis, sclerosis, carpal tunnel syndrome, Adult respiratory distress syndrome, ascites, ocular disease, cardiovascular disease, Crow-Fukase disease, Crohn's disease, glomerulonephritis, osteoarthritis, multiple sclerosis, transplant rejection, Lyme disease me disease, sepsis, von Hippel Lindau disease, swelling, Paget's disease, polycystic kidney disease, sarcoidosis, thyroiditis, oligopoly syndrome, Osler-Weber-Rangdu disease Rendu disease, chronic obstructive pulmonary disease, irradiation, hypoxia, preeclampsia, functional uterine bleeding, endometriosis, infection by herpes simplex, ischemic retinopathy, corneal angiogenesis, herpes zoster ), Human immunodeficiency virus, parapoxvirus, protozoa, toxoplasmosis, spondyloarthritis, ankylosing spondylitis, Morbus Bechterew, eg, avian influenza, including serotype H5N1, and tumors- Associated exudation and edema.

The present invention relates to at least one agent that is a compound of Formula (I) and a PI3K / AKT signaling pathway inhibitor (e.g., a derivative or analog of rapamycin or rapamycin, or a derivative or analog of wortmannin or wortmannin). Provided are methods of treating the aforementioned diseases and / or conditions (including those mentioned in any of the cited documents), including administering an effective amount of two compounds. An "effective amount" is an amount of a compound useful to achieve a desired result, eg, to treat a disease or condition.

The invention also relates to a method of inhibiting angiogenesis in a system comprising cells, comprising administering to the system an effective amount of a combination of the compounds described herein. Systems comprising cells include tumors in patients, isolated organs, tissues or in vivo systems such as cells, in vitro test systems (CAM, BCE, etc.), animal models (eg, in vivo subcutaneous, cancer models), A host in need of treatment (eg, a host suffering from a disease with angiogenic components, such as cancer; a host suffering from restenosis), and the like.

In addition, the drug combination may be administered to modulate one or more of the following processes: cell growth (eg proliferation), tumor cell growth (eg differentiation, cell growth and / or proliferation). Tumor regression, endothelial cell growth (eg, including differentiation, cell survival and / or proliferation), angiogenesis (angiogenesis), angiogenesis and / or hematopoiesis (eg, proliferation, T-cells) Occurrences, etc.).

Compounds or drug combinations of the present invention may be, for example, oral, extraoral, intestinal, intravenous, intraperitoneal, topical, transdermal (eg using any standard patch), intraocular, intranasal, topical, non-oral And may be administered in any form by any effective route including, for example, aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, rectal, intravaginal, intraarterial, intratracheal, and the like. They may be administered alone or in combination with any ingredient (s) active or inactive. They may be administered at any effective dose, eg, from about 0.1 to about 200 mg / kg total weight.

Combinations of the invention can be administered at any time and in any effective form. For example, the compounds may be administered simultaneously, eg, in a single composition or unit dosage form (eg, pills or liquids containing two compositions), or they may be administered simultaneously in separate compositions (eg, One drug may be administered intravenously, and the other may be administered orally or intramuscularly). The drugs may also be administered sequentially at different time points. Agents may be conventionally formulated to achieve the desired release rate over an extended time period, eg, over 12-hours, 24-hours. This can be accomplished by using agents with suitable metabolic half-lives and / or derivatives thereof, or by using controlled release formulations.

Drug combinations can act synergistically, for example, the co-action of drugs can cause the combinational effect to be greater than the algebraic sum of their respective effects. Thus, a reduced amount of drug may be administered to reduce, for example, toxicity or other harmful or unwanted effects, and / or may be used in larger amounts using the same amount as used when the drug is administered alone. Efficacy may be achieved, eg, may have stronger antiproliferative and pre-apoptotic action.

The compound or drug combination of the present invention may be further combined with any other suitable additive or pharmaceutically acceptable carrier. Such additives include all materials already mentioned, as well as Remington: The Science and Practice of Pharmacy (Gennaro and Gennaro, eds, 20th edition, Lippincott Williams & Wilkins, 2000); Theory and Practice of Industrial Pharmacy (Lachman et al., Eds., 3rd edition, Lippincott Williams & Wilkins, 1986); All commonly used ingredients such as those described in Encyclopedia of Pharmaceutical Technology (Swarbrick and Boylan, eds., 2nd edition, Marcel Dekker, 2002) are included. These are referred to herein as "pharmaceutically acceptable carriers" which indicate that they can be safely administered to a subject for therapeutic purposes in combination with an active drug.

In addition, the compounds or drug combinations of the present invention may be administered in conjunction with other active agents or therapies (eg, irradiation) used to treat the above mentioned diseases and / or conditions.

The present invention provides a combination of one or more compounds of formula (I), and one or more second compounds, for example, PI3K / AKT signaling pathway inhibitors useful for treating a disease or disorder. For the purposes of the present invention, "combination" includes:

A single composition or dosage form containing at least one compound of formula (I) and at least one second compound which is a PI3K / AKT signaling pathway inhibitor;

Combination packs containing one or more compounds of formula (I) and one or more second compounds which are PI3K / AKT signaling pathway inhibitors, administered simultaneously or sequentially;

In unit dosage form or in independent unit dosage form, with or without instructions for administering one or more compounds of formula I and one or more second compounds that are PI3K / AKT signaling pathway inhibitors, packaged separately from one another, simultaneously or sequentially Kit comprising a; And

Separate one or more compounds of formula (I) and one or more second compounds that are PI3K / AKT signaling pathway inhibitors that cooperate to achieve a therapeutic effect, eg, the prevention or treatment of the same disease when administered simultaneously or sequentially Independent dosage form.

The dosage of each agent of the combination may be selected with reference to the type of disease and / or disease condition and / or others to provide the desired therapeutic activity. For example, the active agent in the combination can be administered in the presence of a fixed combination. By "fixed combination" is meant herein a pharmaceutical form in which the components are present in a fixed ratio that provides the desired efficacy. These amounts can be routinely determined for individual patients, where appropriate dosages are selected using various parameters (e.g., type of cancer, age of the patient, disease state, patient health, weight, etc.) or the amount is relatively Can be standard.

The combination may comprise an effective amount of one or more compounds of Formula I and one or more second compounds that are PI3K / AKT signaling pathway inhibitors that achieve greater therapeutic efficacy compared to the use of either compound alone. . The combination may produce tumor regression, develop disease stability, prevent or reduce metastasis, or have no therapeutic effect when the agent is used alone or have enhanced effects when the combination is administered. It may be useful to provide other therapeutic endpoints that are observed.

The relative ratios of each compound in the combination can be selected based on their respective mechanism of action and disease biology. For example, activating mutations in the B-RAF gene are observed in at least 60% of human melanoma, and compositions for the treatment of melanoma advantageously comprise one or more compounds wherein the compound of Formula I is a PI3K / AKT signaling pathway inhibitor. It can be included in more powerful amounts. In comparison, where cancer is associated with mutations in the PI3K / AKT signaling pathway (eg, ovarian and breast cancer), agents whose activity is active in the signaling pathway are more potent than Ref / MEK / ERK pathway inhibitors. May exist. The relative ratios of each compound can vary widely, and the present invention includes combinations for cancer treatment that can routinely adjust the amounts of the compounds of Formula I and the second active agent to be present in higher amounts. .

The release of one or more agents of the combination may also be adjusted to provide the desired therapeutic activity, when present in a single dosage form, combination pack, kit, or in separate independent dosage forms, as the case may be.

Test Methods

The activity of the combinations of the present invention can be measured according to effective in vitro or in vivo methods.

Kinase activity

Kinase activity can be routinely measured using conventional test methods. Kinase tests generally include kinase enzymes, substrates, buffers, and components of the detection system. Representative kinase tests involve the production of phosphorylated end-products (eg, phosphoproteins when peptide substrates are used) in response to protein kinases and peptide substrates and ATP such as ³² P-ATP. The resulting end-product can be detected by any suitable method. If radioactive ATP is used, radiolabeled phosphoproteins are separated from unreacted gamma- ³² P-ATP using affinity membrane or gel electrophoresis, visualized using autoradiography or detected with scintillation counters. can do. Non-radioactive methods may be used. This method may utilize an antibody that recognizes a phosphorylated substrate, eg, an anti-phosphotyrosine antibody. For example, the kinase enzyme can be incubated with the substrate in the presence of ATP and kinase buffer under conditions effective for the enzyme to phosphorylate the substrate. The reaction mixture can be separated electrophoretically, for example, and phosphorylation of the substrate can then be measured by western blotting using, for example, anti-phosphotyrosine antibodies. The antibody may be labeled with a detectable label, for example, an enzyme such as HRP, avidin or biotin, chemiluminescent reagents, and the like. Other methods may use an ELISA regime, affinity membrane separation, fluorescence polarization, luminescence, and the like.

An alternative to the radioactive regime is time-resolved fluorescence resonance energy transfer (TR-FRET). This method follows a standard kinase reaction that phosphorylates a substrate, such as biotinylated poly (GluTyr), with a protein kinase in the presence of ATP. Subsequently, the end-product can be detected by europium chelate phosphospecific antibody (anti-phosphotyrosine or phosphoserine / threonine), and streptavidin-APC binding a biotinylated substrate. These two components are spatially contacted together upon binding, and energy transfer from the phosphospecific antibody to the receptor (SA-APC) results in fluorescent readout in a homogeneous regime.

Raf Of MEK Of ERK  activation

c-Raf kinase assays can be performed using (phosphorylated) c-Raf enzyme activated by Lck kinase. Lck-activated c-Raf (Lck / c-Raf) binds GST-c-Raf (amino acids 302 to amino acids 648) and Lck (full-length) in Sf9 insect cells under the control of the polyhedrin promoter. Produced by co-infection with expressing baculovirus. Both baculoviruses are used at a multiplicity of infection of 2.5 and cells are harvested 48 hours after infection.

The MEK-1 protein was also infected by a baculovirus expressing the GST-MEK-1 (full-length) fusion protein in Sf9 insect cells and also infected with duplicate infections of 5 and harvested cells 48 hours after infection. Produced. Similar purification procedures are used for GST-c-Raf 302-648 and GST-MEK-1.

The transfected cells are suspended at 100 mg wet cell biomass per ml in buffer containing 10 mM sodium phosphate, 140 mM sodium chloride pH 7.3, 0.5% Triton X-100 and protease inhibitor cocktail. Cells are disrupted with a polytron homogenizer and centrifuged at 30,000 g for 30 minutes. 30,000 g supernatant is applied onto the GSH-Sepharose phase. The resin is washed with a buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl and 0.01% Triton X-100. GST-tagged proteins are eluted with a solution containing 100 mM glutathione, 50 mM Tris, pH 8.0, 150 mM NaCl and 0.01% Triton X-100. Purified protein is dialyzed into a buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl and 20% glycerol.

Test compounds are serially diluted using 3-fold dilutions in stock solutions of 50 μM to 20 nM in DMSO (for example, the final concentration in the test may range from 1 μM to 0.4 nM). The c-Raf biochemical test is performed by radioactive filtermat assay in 96-well Costar polypropylene plates (Costar 3365). The plate is loaded with 75 μl of solution containing 50 mM HEPES pH 7.5, 70 mM NaCl, 80 ng of Lck / c-Raf and 1 μg MEK-1. Then, 2 μl of series diluted individual compounds are added to the reaction before adding ATP. The reaction is initiated by a 25 μl ATP solution containing 5 μM ATP and 0.3 μCi [33P] -ATP. The plate is sealed and incubated at 32 ° C. for 1 hour. The reaction is quenched by adding 50 μl of 4% phosphoric acid and collected on P30 filtermat using Wallac Tomtec Harvester. The filter mat is washed with primary 1% phosphoric acid and secondary with deionized H ₂ O. The filter is dried in microwave and immersed in scintillation fluid and read on a Wallac 1205 Betaplate Counter (Wallac 1205 Betaplate Counter; Wallac Inc., Atlanta, GA, USA). The results are expressed in% inhibition.

% Inhibition = [100- (Tib / Ti)] × 100

From here,

Tib = (count per minute in the presence of inhibitor)-(background dose)

Ti = (Count per minute without inhibitor)-(Background)

Raf activity can also be monitored by its ability to provide a phospho-ERK by initiating a cascade (ie raf / MEK / ERK) that induces ERK phosphorylation. Bio-Plex phospho-ERK1 / 2 immunoassay tests can be performed as follows:

96-well phospho-ERK (pERK) immunoassay using a laser flow cytometry platform was established to measure the inhibition of basic pERK in cell lines. MDA-MB-231 cells are plated at 50,000 cells per well in 96-well microtiter plates in complete growth medium. For the effect of test compounds on basic pERK1 / 2 inhibition, the day after plating, MDA-MB-231 cells were transferred to DMEM containing 0.1% BSA and at a final concentration of 3 mM to 12 mM in 0.1% DMSO. Incubate with 1: 3 diluted test compounds. Cells were incubated with test compounds for 2 hours, washed and lysed in Bio-Flex Whole Cell Lysis Buffer A. Samples are diluted with Buffer B 1: 1 (v / v) and transferred directly to test plates or frozen at −80 ° C. until processed. 50 ml of diluted MDA-MB-231 cell lysate is incubated overnight on a shaker with about 2000 5 micron bio-plex beads conjugated with anti-ERK1 / 2 antibody at room temperature. The next day, biotinylated phospho-ERK1 / 2 sandwich immunoassay is performed, beads are washed three times in each culture, and then 50 ml of PE-strepavidin is used as a developer. Relative hemoglobin units of pERK1 / 2 are detected by counting 25 beads by high sensitivity bio-plex flow cells (probes). IC50 is calculated by adopting the cell untreated (bead only) as the background amount to the untreated cells to the maximum.

Phosphatidyl Inositol  3- Kinase  activation

PKI3 activity can be routinely measured using, eg, commercially available kits (eg, Perkin-Elmer, FlashPlate Platform) (Frew et al., Anticancer Res ., 14 (6B): 2425- 8, 1994). See also the literature described for PKI3 inhibitors.

Akt activity

AKT can be isolated from insect cells expressing His-tagged AKT1 (aa 136-480) as described in WO 05011700. Expressing cells are lysed with polytron in 25 mM HEPES, 100 mM NaCl, 20 mM imidazole, pH 7.5 (5 ml lysis buffer / g of cells). Cell debris is removed by centrifugation at 28,000 × g for 30 minutes. The supernatant is filtered through a 4.5 micron filter and then loaded onto a nickel-chelated column pre-equilibrated with lysis buffer. The column is washed with 5 column volumes (CV) lysis buffer, followed by 5 CV of 20% buffer B (where buffer B is 25 mM HEPES, 100 mM NaCl, 300 mM imidazole; pH 7). ). His-tagged AKT1 (aa 136-480) is eluted with a 20-100% linear gradient of Buffer B over 10 CVs. The His-tagged AKTI (136-480) elution fractions are pooled and diluted 3-fold with Buffer C, where Buffer C is 25 mM HEPES, pH 7. The sample is then chromatographed on a Q-Sepharose HP column pre-equilibrated with Buffer C. The column is washed with 5 CV buffer and then eluted stepwise with 5 CV 10% D, 5 CV 20% D, 5CV 30% D, 5 CV 50% D and 5 CV 100% D, where buffer D is 25 mM HEPES, 1000 mM NaCl; pH 7.5.

His-tagged AKT1 (aa 136-480) containing fractions were collected and concentrated in a 10-kDa molecular weight cutoff concentrator. His-tagged AKT1 (aa 136-480) was treated with 25 mM HEPES, 200 mM NaCl, 1 mM DTT; Chromatography on a Superdex 75 gelfiltration column pre-equilibrated to pH 7.5. His-tagged AKT1 (aa 136-480) fractions are examined using SDS-PAGE and mass spectrometry. Proteins are collected, concentrated and stored at 80 ° C.

His-tagged AKT2 (aa 138-481) and His-tagged AKT3 (aa 135-479) can be isolated and purified in a similar manner.

AKT Enzyme Assay Compounds can be tested for AKT protein serine kinase inhibitory activity in substrate phosphorylation assays. This assay tests the ability of small molecule organic compounds to inhibit serine phosphorylation of peptide substrates. Substrate phosphorylation assays use the catalytic region of AKT 1, 2 or 3. AKT 17 2 and 3 are also available from Upstate USA, Inc. (Upstate USA, Inc.). This method measures the ability of an isolated enzyme to facilitate the transfer of gamma-phosphate from the ATP onto the serine-72 residue of the biotinylated synthetic peptide (biotin-ahx-ARKRERAYSFGHHA-amide). Substrate phosphorylation can be detected by the following method described in WO 05011700.

Measurement tests are performed on 384 well U-bottom white plates. 10 nM activated AKT enzyme in 50% DMSO in 50 mM MOPS, pH 7.5, 20 mM MgCl ₂ , 4 μM ATP, 8 μM peptide, 0.04 μCi [g- ³³ P] ATP / well, 1 mM CHAPS, 2 mM DTT And incubated for 40 minutes at room temperature in a test volume containing 1 μl of test compound. The reaction was carried out with 50 μl SPA beads mixture (Dulbecco's PBS without Mg2 + and Ca2 +, 0.1% Triton X-100, 5 mM EDTA, 50 μM ATP, 2.5 mg / ml streptavidin-coated SPA beads Stop by adding). The plate was sealed and the beads were allowed to settle overnight, then the plates were counted on a Packard Topcount Microplate Scintillation Counter (Packard Instrument Co., Meriden, CT).

Data on dose response can be shown as Control% calculated by the data reduction formula 100 * (U1-C2) / (C1-C2) versus the concentration of the compound, where U is unknown Value, C1 is the average control value obtained for DIVISO and C2 is the average control value obtained for 0,1 M EDTA. The data correspond to the curve described by y = ((Vmax * x) K + x)), where Vmax is the upper asymptote and K is IC50.

Cell proliferation

Examples of cell proliferation tests are described in the Examples below. However, proliferation testing can be performed by any suitable method. For example, breast cancer cell proliferation can be performed as follows. Other types of cells can be replaced for the MDA-MB-231 cell line.

Human breast cancer cells (MDA MB-231, NCI) were cultured in standard growth medium (DMEM) supplemented with 10% heat-inactivated FBS at 37 ° C. under 5% CO ₂ (vol / vol) in a humidified incubator do. Cells are plated at a density of 3000 cells per well in 90 μl growth medium in 96 well plates. To determine T _Oh CTG values, after 24 hours of plating, 100 μl of CellTiter-Glo light emitting reagent (Promega) is added to each well and incubated for 30 minutes at room temperature. Luminescence is recorded on a Wallac Victor II device. CellTiter-Glo reagents cause cell lysis and the generation of luminescent signals that are proportional to the amount of ATP present that is also directly proportional to the number of cells present.

Test compounds are dissolved in 100% DMSO to prepare 10 mM stock solutions. The stock solution is further diluted 1: 400 in the growth medium to obtain a working stock solution of 25 μM test compound in 0.25% DMSO. Test compounds are serially diluted in growth medium containing 0.25% DMSO to maintain a constant DMSO concentration for all wells. 60 μl of diluted test compound is added to each culture well to obtain a final volume of 180 μl. Cells in the presence and absence of individual test compounds are incubated for 72 hours, at which time ATP dependent luminescence is measured as described above to obtain a T _72h value. Optionally, the IC ₅₀ value can be determined by a least squares analysis program using% inhibition relative to compound concentration.

% Inhibition = [1- (T _{72h test} -T _0h ) / (T _{72h ctrl} -T _0h )] × 100

From here,

T _{72h test} = ATP dependent luminescence at 72 hours in the presence of test compound

T _{72h ctrl} = ATP dependent luminescence in 72 hours in the absence of test compound

T _0h = ATP dependent luminescence at time zero.

Angiogenesis

One useful model for testing angiogenesis is that endothelial cells are enriched into the matrix when a host animal is injected subcutaneously with a reconstituted basement membrane matrix, such as Matrigek, supplemented with growth factors (eg, FGF-I). Based on the observation that new blood vessels form over a period of several days (see, eg, Passaniti et al., Lab. Invest ., 67: 519-528, 1992). Samples of extracts at various time points are taken to examine the angiogenesis temporarily in detail to, for example, migrate into the matrix of endothelial cells, involve endothelial cells in angiogenic pathways, cell extension and formation of pocket-shaped spaces, and red blood cells. Genes involved in all stages of angiogenesis, including the settlement of functional capillaries containing linked linear structures containing can be identified. In order to stabilize the growth factor and / or slow its release from the matrix, the growth factor may be bound to heparin or another stabilizer. The matrix can also be periodically infused with growth factors to enhance and prolong the angiogenic process.

Other systems useful for testing angiogenesis include, for example, angiogenesis of tumor implants (eg, US Pat. No. 5,192,744; 6,024,688), chorionic villus (CAM) testing of chickens (eg, Taylor and Folkman) , Nature, 297: 307-312, 1982; Eliceiri et al., J. Cell Biol., 140, 1255-1263, 1998), bovine capillary endothelial (BCE) cell testing (eg, US Pat. No. 6,024,688) Polverini, PJ et al., Methods Enzymol., 198: 440-450, 1991), migration tests and HUVEC (Human Umbilical Vessel Endothelial Cell) growth inhibition tests (e.g., US Pat. No. 6,060449). do.

The present invention provides one or more of the following features:

A method of treating the aforementioned diseases and / or conditions, comprising administering an effective amount of a compound of Formula I and a second compound that is a PI3K / AKT signaling pathway inhibitor.

A method of modulating (eg, inhibiting) one or more of the aforementioned activities, comprising administering an effective amount of a compound of Formula I and a second compound that is a PI3K / AKT signaling pathway inhibitor.

A composition comprising a compound of Formula I and a second compound that is a PI3K / AKT signaling pathway inhibitor.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. Accordingly, certain preferred embodiments are to be construed merely as illustrative and not in any way limiting the remainder of the description. The entire technical details of all patents and documents cited above and below are hereby incorporated by reference in their entirety.

Claims (16)

A combination comprising a compound of formula (I), a pharmaceutically acceptable salt, polymorph, solvate, hydrate, metabolite or prodrug form thereof, and one or more second compounds that are PI3K / AKT signaling pathway inhibitors: [Formula I]

The method of claim 1, wherein the second compound is celecoxib, OSU-03012, OSU-03013, PX-316, 2'-substituted 3'-deoxy-phosphatidyl- myo-inositol derivative, 3- (already Dazo [1,2-a] pyridin-3-yl) derivatives, Ly294002, IC486068, 3- (hetero) aryloxy substituted benzo (b) thiophene derivatives, PX-866, perifosine, trisiribin, FKBP12 enhancer, A combination that is a phosphatidylinositol ether lipid analog, wortmannin or rapamycin or a derivative thereof, or a pharmaceutically acceptable salt thereof. The method according to claim 1, wherein the second compound is a wortmannin compound of formula W, a derivative or analogue of wortmannin compound of formula W, a pharmaceutically acceptable salt of wortmannin compound of formula W, Or a pharmaceutically acceptable salt of a derivative or analog of the Wartmannin compound of Formula W: [Formula W]

The combination according to claim 3, wherein the derivative or analogue of the compound of formula W is selected from the following compounds a) to e): a) a compound of formula W1 [Formula W1]

Wherein R is H (11-desacetoxywatermannin) or acetoxy and R 'is C ₁ -C ₆ alkyl; b) Δ9,11-dehydrodesacetoxy watermannin compound of formula W2 [Formula W2]

(Wherein R 'is C ₁ -C ₆ alkyl); c) 17 (α-dihydro-watermannin compound of formula W3 [Formula W3]

Wherein R is H or acetoxy, R 'is C ₁ -C ₆ alkyl, and R "is H, C ₁ -C ₆ alkyl, -C (O) OH or -C (O) OC _1- C ₆ alkyl); d) open A-substituted or esters of the wortmannin compound of formula W4 [Formula W4]

(Wherein R ₁ is H, methyl or ethyl and R ₂ is H or methyl); or e) 11- and 17-substituted derivatives of wortmannin of formula W5 [Formula W5]

Wherein R ₄ is ═O or —O (CO) R ₆ , R ₃ is ═O, —OH or —O (CO) R ₆ , and R ₆ are each independently phenyl, C ₁ -C ₆ Alkyl or substituted C ₁ -C ₆ alkyl, wherein if R ₄ is ═O or —OH, R ₃ is not ═O. The combination of claim 1, wherein said second compound is an Akt-kinase inhibitor, The method of claim 1, wherein the second compound is Akt-1-1, Akt-1-1,2, API-59CJ-Ome, 1-H-imidazo [4,5-c] pyridinyl derivative, indole- Combination of 3-carbinol and its derivatives, perifosine, phosphatidylinositol ether lipid analogs, trisiribin, or pharmaceutically acceptable salts thereof. The combination of claim 1, wherein said second compound is an mTOR inhibitor. The method of claim 1, wherein the second compound is rapamycin, temsirolimus, everolimus, AP23573, AP23675, AP23464, AP23841, 40- (2-hydroxyethyl) rapamycin, 40- [3-hydroxy ( Hydroxymethyl) methylpropanoate] -rapamycin, 40-epi- (tetrazolyl) -rapamycin, 32-deoxorapapamycin, or 16-pentynyloxy-32 (S) -dihydrorapamycin, SAR 943, or a pharmaceutically acceptable salt thereof. The combination of claim 1 comprising a compound of formula (I) and wortmannin. The combination of claim 1 comprising a compound of formula I and rapamycin. The combination according to any one of claims 1 to 10, wherein the amount of active ingredient of the combination is a synergistic amount. The combination according to any one of claims 1 to 11 for the treatment of cancer. The combination of claim 12, wherein the cancer is melanoma, hepatocellular cancer, renal cell cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, colorectal cancer, breast cancer or pancreatic cancer. Administering an effective amount of a compound of formula (I), a pharmaceutically acceptable salt, polymorph, solvate, hydrate, metabolite or prodrug thereof, and a second compound that is a PI3K / AKT signaling pathway inhibitor , How to treat cancer in a subject in need of treatment: [Formula I]

12. A method of making a combination of any one of claims 1 to 11 for treating cancer. The method of claim 15, wherein the cancer is melanoma, hepatocellular cancer, renal cell cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, colorectal cancer, breast cancer or pancreatic cancer.