Combination therapy for the treatment of cancer

专利摘要:
The present invention relates to a method for the treatment or prevention of neoplastic diseases using protein tyrosine kinase inhibitors in combination with cyclooxygenase inhibitors, in particular cyclooxygenase-2-selective inhibitors.
公开号:KR20040043193A
申请号:KR10-2004-7002183
申请日:2002-08-15
公开日:2004-05-22
发明作者:제이미 마스퍼러；파룰 도시；줄리 셰링톤
申请人:수젠, 인크.；파마시아 코포레이션；
IPC主号:

专利说明:

COMBINATION THERAPY FOR THE TREATMENT OF CANCER
[2] Neoplasms or tumors are abnormal, unregulated and histological proliferation of cell growth. If the neoplasm has destructive growth, invasive and metastatic properties, it is malignant or incurable. Invasion refers to the local spread of neoplasms that often enter the body's circulatory system by invasion or destruction of surrounding tissue, typically through breaks through the base plate that define the tissue's boundaries. Metastasis typically refers to the transmission of tumor cells by lymphatic vessels or blood vessels. Metastasis also refers to the migration of tumor cells by direct expansion through the mesenteric cavity, or subarachnoid or other space. Through the metastasis process, tumor cell migration to other parts of the body allows the neoplasm to settle away from the site of early appearance.
[3] Cancer is currently the second leading cause of death in the United States, with more than 8 million people diagnosed with cancer in the United States. In 1995, cancer reached 23.3% of all deaths in the United States (see US Dept. of Health and Human Services, National Center for Health Statistics, Health United States 1996-97 and Injury Chartbook 117 (1997)). .
[4] Cancer is not currently fully understood at the molecular level. Exposure of cells to certain viruses, certain chemicals or carcinogens such as radiation is known to induce DNA denaturation that inactivates the "inhibitory" gene or activates the "tumor gene". Inhibitor genes are growth regulator genes that can no longer regulate cell growth upon mutation. An oncogene is a normal gene (called a proto-oncogene) that is initially transformed by a mutated or denatured expression environment. Generation of modified genes leads to inadequate cell growth. More than 20 different normal cell genes can be "tumor genes" by genetic degeneration. Modified cells differ from normal cells in many ways, including cell morphology, cell-to-cell interactions, membrane contents, cytoskeletal structure, protein secretion, gene expression and death (modified cells can grow indefinitely).
[5] Cancer is currently primarily treated with one or a combination of three types of therapies: surgery, radiation and chemotherapy. Surgery involves the massive removal of damaged tissue. Surgery is sometimes effective in removing tumors located in certain areas, such as breasts, colons, and skin, but cannot be used to treat tumors located in other areas, such as the spine, or in the treatment of transmitted tumor signs, such as leukemia.
[6] Chemotherapy includes disruption of cell replication or cell metabolism. It is most commonly used for the treatment of breast cancer, lung cancer and testicular cancer.
[7] The adverse effect of systemic chemotherapy on the treatment of neoplastic disease is the most feared for patients receiving cancer treatment. Among these adverse effects, nausea and vomiting are the most common and serious side effects. Other negative side effects include hemocytopenia, infections, cachexia, mucositis in patients receiving high dose chemotherapy and radiation therapy to regenerate bone marrow; Toxins (hair loss); Skin complications (MD: Abeloff et al .; Alopecia and Cutaneous Complications.P. 755-56.In Abeloff, MD, Armitage, JO, Lichter, AS, and Niedehuber, JE (eds) Clinical Oncology.Churchill Livingston, New York, 1992 for cutaneous reactions to chemotherapy agents) such as pruritus, urticaria and angioedema; Neurological complications; Lung and heart complications in patients undergoing radiation or chemotherapy; And reproductive and endocrine gland complications.
[8] Chemotherapy-induced side effects have a significant impact on the quality of life of the patient and can have a very significant effect on the patient as a result of treatment complications.
[9] In addition, negative side effects associated with chemotherapeutic agents generally have major dose-limiting toxicity upon administration of such drugs. For example, mucositis is one of the major dose limiting toxicities for several anticancer agents, including the metabolite cytotoxic drug 5-FU, methotrexate, and anticancer antibiotics such as doxorubicin. Many of these chemotherapy-induced side effects can lead to hospitalization in severe cases, or may require treatment with painkillers to treat pain.
[10] Negative side effects induced by chemotherapeutic agents and radiation therapy are of major importance for the clinical management of cancer patients.
[11] Currently, scientists are looking at the treatment of cancer through the use of antiangiogenic agents. Angiogenesis is believed to be a mechanism through which tumors acquire, grow, and metastasize to other locations in the body. Antiangiogenic agents interfere with this process and destroy or control tumors.
[12] For example, US Pat. No. 5,854,205 describes isolated endostatin proteins, ie inhibitors of endothelial cell proliferation and angiogenesis; US Pat. No. 5,843,925 discloses a method for inhibiting angiogenesis and endothelial cell proliferation using 7- [substituted amino] -9- [substituted glyzalamido] -6-demethyl-6-deoxytetracycline. It is described; US Patent No. 5,861,372 describes the use of an aggregate endothelial inhibitor, angiostatin, used to inhibit angiogenesis; PCT / GB97 / 00650 describes the use of cynoline derivatives used to produce angiogenesis and / or vascular permeability reducing effects; Tai-Ping, D, describes potent antiangiogenic therapies (Trends Phamacol. Sci. 16, No. 2, 57-66, 1995); Rod, H et al. Describe the eradication of spontaneous tumor metastasis by synergistic effects between antiangiogenic integrin alpha v antagonists and antibody-cytokine fusion proteins (Proc. Nat. Acd. Sci. USA., 96 (4), 1591-1596, 1999); Giannis, A., et al. Describe integrin antagonists and other low molecular weight compounds as angiogenesis inhibitors (see New drugs incancer therapy. Angew. Chem. Int. Ed. Engl. 36 (6), 588). -590, 1997); WO 97/41844 describes a method of using a combination of angiostatic compounds for the prevention and / or treatment of angiogenesis in human patients; WO 98/22101 describes a method of using [yrazol-1-yl] benzenesulfonamide as an angiogenesis agent; US Pat. No. 5,792,783 describes the use of 3-heteroaryl-2-indolinone, an antiangiogenic and protein kinase inhibitor, for the treatment of various cancers.
[13] Recently, treatment of colon cancer with cyclooxygenase-2-selective inhibitors, in particular Celecoxib® in combination with ART-2 inhibitors, in particular Herceptin®, It has been reported to be more effective than when one drug is used alone. Thus, there is a need for the discovery of new combinations of chemotherapeutic agents that are more potent than when used alone.
[14] Summary of the Invention
[15] In one aspect, the invention provides a method of treating cancer comprising administering a protein kinase inhibitor of Formula I to a mammal in need of such treatment in combination with a cyclooxygenase inhibitor or a pharmaceutically acceptable salt thereof It is about prevention method.
[16]
[17] Where
[18] R is hydrogen, piperazin-1-ylmethyl, 4-methylpiperazin-1-ylmethyl, piperidin-1-ylmethyl, 2-hydroxymethylpyrrolidin-1-ylmethyl, 2-carboxy Rollidin-1-ylmethyl and pyrrolidin-1-ylmethyl;
[19] R ¹ is hydrogen, halo, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, -C (O) NR ⁸ R ⁹ , -NR ¹³ R ¹⁴ ,-(CO) R ¹⁵ and-(CH ₂ ) _r R ¹⁶ ;
[20] R ² is hydrogen, halo, alkyl, substituted alkyl, trihalomethyl, hydroxy, alkoxy, cyano, -NR ¹³ R ¹⁴ , -NR ¹³ C (O) R ¹⁴ , -C (O) R ¹⁵ , Aryl, heteroaryl, and -S (O) ₂ NR ¹³ R ¹⁴ ;
[21] R ³ is hydrogen, halogen, alkyl, substituted alkyl, trihalomethyl, hydroxy, alkoxy, aryl, heteroaryl, -NR ¹³ R ¹⁴ , -NR ¹³ S (O) ₂ R ¹⁴ , -S (O) ₂ NR ¹³ R ¹⁴ , -NR ¹³ C (O) R ¹⁴ , -NR ¹³ C (O) OR ¹⁴ ,-(CO) R ¹⁵ and -SO ₂ R ¹⁹ ;
[22] R ⁴ is selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, hydroxy, alkoxy and —NR ¹³ R ¹⁴ ;
[23] R ⁵ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, and —C (O) R ¹⁰ ;
[24] R ⁶ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, and —C (O) R ¹⁰ ;
[25] R ⁷ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, -C (O) R ^l7 and -C (O) R ^l0 , provided that R ⁵ is hydrogen and R ⁶ and R ^At least one of ⁷ is —C (O) R ¹⁰ ; or
[26] R ⁶ and R ⁷ combine to form a group selected from the group consisting of — (CH ₂ ) ₄ —, — (CH ₂ ) ₅ — and — (CH ₂ ) ₆ —;
[27] R ⁸ and R ⁹ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl and aryl;
[28] R ¹⁰ is selected from the group consisting of hydroxy, alkoxy, aryloxy, -N (R ^ll ) (alkylene) _n R ^l2 , wherein the alkylene group is optionally substituted with a hydroxy group and -NR ¹³ R ¹⁴ Selected;
[29] R ¹¹ is selected from the group consisting of hydrogen, alkyl and substituted alkyl;
[30] R ¹² is -NR ¹³ R ^14, hydroxy, -C (O) R ^15, aryl, ^{heteroaryl, -N + (O -) R} 13 R 14, -N (OH) R 13 and -NHC (O) R ¹⁸ is selected from the group consisting of (wherein, R ^l8 is alkyl, substituted alkyl, haloalkyl, or aralkyl);
[31] R ¹³ and R ¹⁴ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, lower alkyl substituted with hydroxyalkylamino, cyanoalkyl, cycloalkyl, substituted cycloalkyl, aryl and heteroaryl; or
[32] R ¹³ and R ¹⁴ may combine to form a heterocyclo group;
[33] R ¹⁵ is selected from the group consisting of hydrogen, hydroxy, alkoxy and aryloxy;
[34] R ¹⁶ is selected from the group consisting of hydroxy, -NR ¹³ R ¹⁴ , -C (O) R ¹⁵ and -C (O) NR ¹³ R ¹⁴ ;
[35] R ¹⁷ is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, aryl and heteroaryl;
[36] R ¹⁹ is selected from the group consisting of alkyl, substituted alkyl, aryl, aralkyl, heteroaryl or heteroaralkyl;
[37] n and r are independently 1, 2, 3 or 4.
[38] Preferably, the protein kinase inhibitor of formula I is a cyclooxygenase-2-selective inhibitor selected from the group consisting of (i) a compound of formula II, (ii) a compound of formula III, or a pharmaceutically acceptable salt thereof Used in combination with
[39]
[40] Where
[41] G is selected from the group consisting of O, S and -NR ^a , wherein R ^a is hydrogen or alkyl;
[42] R ^10a is selected from the group consisting of hydrogen and aryl;
[43] R ^lla is selected from the group consisting of carboxyl, alkyl, aralkyl, aminocarbonyl, alkylsulfonylaminocarbonyl and alkoxycarbonyl;
[44] R ^1a is selected from the group consisting of haloalkyl, alkyl, aralkyl, cycloalkyl, and aryl optionally substituted with one or more radicals selected from alkylthio, nitro and alkylsulfonyl;
[45] R ^13a is hydrogen, halo, alkyl, aralkyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, haloalkyl, haloalkoxy, alkylamino, arylamino, aralkylamino, heteroarylamino, Heteroarylalkylamino, nitro, amino, aminosulfonyl, alkylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, aralkylaminosulfonyl, heteroaralkylaminosulfonyl, heterocyclosulfonyl, alkylsulfonyl Independently from the group consisting of hydroxyarylcarbonyl, nitroaryl, optionally substituted aryl, optionally substituted heteroaryl, aralkylcarbonyl, heteroarylcarbonyl, arylcarbonyl, aminocarbonyl and alkylcarbonyl At least one radical selected; or
[46] R ^13a together with ring E form a naphthyl ring.
[47]
[48] Where
[49] A is selected from the group consisting of partially unsaturated or unsaturated heterocyclyl and partially unsaturated or unsaturated carbocyclic rings;
[50] R ^lb is selected from the group consisting of heterocyclyl, cycloalkyl, cycloalkenyl and aryl, which is alkyl, haloalkyl, cyano, carboxyl, alkoxycarbonyl, hydroxyl, hydroxyalkyl, haloalkoxy at substitutable positions Optionally substituted with one or more radicals independently selected from amino, alkylamino, arylamino, nitro, alkoxyalkyl, alkylsulfinyl, halo, alkoxy and alkylthio;
[51] R ^2b is selected from the group consisting of methyl and amino;
[52] R ^3b is hydrogen, halo, alkyl, alkenyl, alkynyl, oxo, cyano, carboxyl, cyanoalkyl, heterocyclyloxy, alkyloxy, alkylthio, alkylcarbonyl, cycloalkyl, aryl, haloalkyl, hetero Cyclyl, cycloalkenyl, aralkyl, heterocyclylalkyl, acyl, alkylthioalkyl, hydroxyalkyl, alkoxycarbonyl, arylcarbonyl, aralkylcarbonyl, aralkenyl, alkoxyalkyl, arylthioalkyl, aryl Oxyalkyl, aralkylthioalkyl, aralkoxyalkyl, alkoxyaralkoxyalkyl, alkoxycarbonylalkyl, aminocarbonyl, aminocarbonylalkyl, alkylaminocarbonyl, N-arylaminocarbonyl, N-alkyl-N-aryl Aminocarbonyl, alkylaminocarbonylalkyl, carboxyalkyl, alkylamino, N-arylamino, N-aralkylamino, N-alkyl-N-aralkylamino, N-alkyl-N-arylamino, aminoalkyl, alkyl Aminoalkyl, N-arylaminoalkyl, N-aralkylaminoal , N-alkyl-N-aralkylaminoalkyl, N-alkyl-N-arylaminoalkyl, aryloxy, aralkoxy, arylthio, aralkylthio, alkylsulfinyl, alkylsulfonyl, aminosulfonyl, alkylaminosul Phonyl, N-arylaminosulfonyl, arylsulfonyl and N-alkyl-N-arylaminosulfonyl.
[53] Preferably, the compound is administered as a pharmaceutical composition comprising at least one such compound and a pharmaceutically acceptable excipient. In particular, the therapeutic agents of the invention may be formulated in separate compositions provided simultaneously or at different times, or may be provided as a single composition.
[54] The method of the present invention is terminal acroma melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma, adenosarcoma, adenosquamous cell carcinoma, astrocytic tumor, bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, capillary carcinoma , Carcinoid, carcinoma, carcinosarcoma, cavernous carcinoma, cholangiocarcinoma, chondsarcoma, choroid plexus / carcinoma, clear cell carcinoma, cystic adenocarcinoma, endodermal sinus tumor, endometrial hyperplasia, stromal endometrial sarcoma, endometrial adenocarcinoma Sarcoma, Epithelial Sarcoma, Ewing's Sarcoma, Fibrous Lamellar Tumor, Local Nodular and Proliferative Tumor, Gastrinoma, Germ Cell Tumor, Glioblastoma, Glucagon, Hemangioblastoma, Hemangioendothelioma, Hemangioma, Liver Adenoma, Hepatic Adenopathy, Hepatocellular Carcinoma, Insulinoma, intraepithelial tumor, interepithelial squamous cell tumor, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, malignant melanoma, malignant melanoma, malignant mesothelioma, medulloblastoma, medulla Carcinoma, melanoma, meningioma, mesothelioma, metastatic carcinoma, mucous epidermoid carcinoma, neuroblastoma, neuroepithelial adenocarcinoma nodular melanoma, oat cell carcinoma, oligodendricular adenocarcinoma, osteosarcoma, pancreatic polypeptide adenocarcinoma, papillary serous Adenocarcinoma, pineal cell tumor, pituitary tumor, plasmacytoma, adenocarcinoma, lung blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous carcinoma, squamous carcinoma Renal including cell carcinoma, submesothelial melanoma, superficially expanded melanoma, undifferentiated carcinoma, uveal melanoma, warty carcinoma, vascular activating peptide (vipoma), highly differentiated carcinoma, and Wilm's tumor It is useful for the treatment or prevention of biological diseases.
[55] The method of the present invention provides one or more advantages. One of the advantages is that the compositions, drugs and therapies of the present invention are administered in combination in lower dosages, i.e., in amounts less than those normally used in the clinical context for each of the individual components administered alone.
[56] Advantages of reducing the dosages of the compounds, compositions, drugs and therapies of the invention administered to a mammal include a reduction in the incidence of adverse effects associated with higher dosages. For example, by reducing the dose of a chemotherapeutic agent such as methotrexate, the frequency and severity of nausea and vomiting will be reduced compared to that observed at higher doses. Similar advantages are contemplated when the protein kinase inhibitors of the invention are used in combination with cyclooxygenase inhibitors, in particular cyclooxygenase-2-selective inhibitors.
[57] By reducing the incidence of adverse effects, improvements in the quality of life of patients undergoing cancer treatment are considered. Additional benefits of reducing the incidence of adverse effects include improved patient acceptance, a reduction in the number of hospitalizations required to treat adverse effects, and a reduction in the administration of analgesics required to treat pain associated with adverse effects.
[58] Another advantage to consider is that when protein kinase inhibitors are administered in combination with cyclooxygenase inhibitors, preferably cyclooxygenase-2-selective inhibitors, tumor suppression is greater than when only one of these drugs is administered alone. Is the point.
[59] The invention further includes a kit comprising a COX-2 inhibitor and a protein kinase inhibitor of Formula (I).
[1] The present invention relates to a method for the treatment or prophylaxis of neoplastic diseases using protein triosine kinase inhibitors in combination with cyclooxygenase inhibitors, in particular cyclooxygenase-2-selective inhibitors.
[60] Justice
[61] Unless defined otherwise, the following terms used in the specification and claims of the present invention have the meanings discussed below.
[62] "Alkyl" means a saturated aliphatic hydrocarbon radical comprising straight and branched chains of 1 to 20 carbon atoms (where a numerical range such as "1-20" is used herein, if it is an alkyl group, this group is 1 It can contain up to 20 carbon atoms, including 2 carbon atoms, 2 carbon atoms, and 3 carbon atoms). Alkyl groups having 1 to 4 carbon atoms are referred to as lower alkyl groups. When the lower alkyl group lacks a substituent, it is referred to as lower alkyl group. More preferably, the alkyl group is medium alkyl having 1 to 10 carbon atoms, for example methyl, ethyl, propyl, 2-propyl, n-butyl, iso-butyl, tert-butyl, pentyl and the like. Most preferably it is lower alkyl having 1 to 4 carbon atoms, for example methyl, ethyl, propyl, 2-propyl, n-butyl, iso-butyl or tert-butyl and the like.
[63] "Substituted alkyl" is halo; Hydroxy; Lower alkoxy; Aryl optionally substituted with one or more, preferably 1, 2 or 3 groups independently of one another consisting of halo, hydroxy, lower alkyl or lower alkoxy groups; Aryloxy optionally substituted with one or more, preferably one, two or three groups, independently of one another, consisting of halo, hydroxy, lower alkyl or lower alkoxy groups; 6 having from 1 to 3 nitrogen atoms in the ring, wherein the carbon in the ring is optionally substituted with one or more, preferably 1, 2 or 3 groups independently of one another consisting of halo, hydroxy, lower alkyl or lower alkoxy groups Circle heteroaryl; One or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, wherein at least one carbon and nitrogen atom in the group is independently of one another, preferably 1, 2, with a halo, hydroxy, lower alkyl or lower alkoxy group Or 5-membered heteroaryl optionally substituted with 3 groups; Having from 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, the carbon and nitrogen (if any) atoms in the group are halo, hydroxy, lower alkyl or lower alkoxy groups, mercapto, (lower alkyl) thio , Arylthio (optionally substituted with one or more, preferably one, two or three groups, independently of one another, halo, hydroxy, lower alkyl or lower alkoxy groups), cyano, acyl, thioacyl, O- Carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido, R ¹⁸ S (O) —, R ¹⁸ S (O) ₂ —, —C (O) OR ¹⁸ , R ¹⁸ C (O) O—, and —NR ¹⁸ R ¹⁹ , wherein R ¹⁸ and R ¹⁹ are hydrogen; lower alkyl; trihalomethyl (C ₃ -C ₆ ) cycloalkyl; lower alkenyl; lower alkynyl; and one independently composed of halo, hydroxy, lower alkyl or lower alkoxy groups Above, preferably independently selected from the group consisting of aryl optionally substituted with 1, 2 or 3 groups), independently of one another, preferably 5, optionally substituted with 1, 2 or 3 groups Or an alkyl group as defined above substituted with one or more, more preferably 1 to 3, even more preferably 1 or 2 substituent (s) independently selected from the group consisting of 6-membered heteroalicyclic groups Means.
[64] Preferably, the alkyl group is hydroxy; At least one heteroatom selected from the group consisting of nitrogen, oxygen and sulfur, and at least one carbon and nitrogen atom (if present) in the group, independently of one another, halo, hydroxy, lower alkyl or lower alkoxy group, Preferably 5- or 6-membered heteroaryl optionally substituted with 1, 2 or 3 groups; One or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, wherein at least one carbon and nitrogen atom in the group is independently of one another, preferably 1, 2, with a halo, hydroxy, lower alkyl or lower alkoxy group Or 5-membered heteroaryl optionally substituted with 3 groups; 6 having from 1 to 3 nitrogen atoms in the ring and optionally substituted with one or more, preferably 1, 2 or 3 groups, wherein the carbon atoms in the group are independently of each other composed of halo, hydroxy, lower alkyl or lower alkoxy groups Circle heteroaryl; Or -NR ¹⁸ R ¹⁹ , wherein R ¹⁸ and R ¹⁹ are independently selected from the group consisting of hydrogen or lower alkyl. Even more preferably, the alkyl group is hydroxy, dimethylamino, ethylamino, diethylamino, dipropylamiro, pyrrolidino, piperidino, morpholino, piperazino, 4-lower alkylpiperazino, Phenyl, imidazolyl, pyridinyl, pyridazinyl, pyrimidinyl, oxazolyl, triazinyl, and the like, substituted with one or two substituents independently of one another.
[65] "Cycloalkyl" means a 3-8 membered monocyclic ring in which all ring atoms are carbon, a 5-membered / 6-membered or 6-membered / 6-membered fused bicyclic ring or polycyclic fused ring in which all ring atoms are carbon ("fusion"); Ring system means that each ring in the system shares an adjacent pair of carbon atoms with a different ring in the system), wherein one or more of the rings may contain one or more double bonds, but any ring is completely conjugated. Does not have a pi-electronic system.
[66] Examples of cycloalkyl include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, adamantane, cycloheptane, cyclohexatriene, and the like.
[67] "Substituted cycloalkyl" is lower alkyl; Trihaloalkyl; Halo; Hydroxy; Lower alkoxy; Aryl optionally substituted with one or more, preferably one or two groups independently of one another consisting of halo, hydroxy, lower alkyl or lower alkoxy groups; Aryloxy optionally substituted with one or more, preferably one or two groups, independently of one another, halo, hydroxy, lower alkyl or lower alkoxy groups; 6-membered hetero atoms having 1 to 3 nitrogen atoms in the ring, wherein the carbon in the ring is optionally substituted with one or more, preferably 1 or 2 groups, independently of one another, with halo, hydroxy, lower alkyl or lower alkoxy groups Aryl; One or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur and at least one, preferably 1 or 2, of the carbon and nitrogen atoms in the group independently of one another in halo, hydroxy, lower alkyl or lower alkoxy groups 5-membered heteroaryl optionally substituted with 2 groups; Having from 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, the carbon and nitrogen (if any) atoms in the group are halo, hydroxy, lower alkyl or lower alkoxy groups, mercapto, (lower alkyl) thio , Arylthio (optionally substituted with one or more, preferably one or two groups, independently of one another, halo, hydroxy, lower alkyl or lower alkoxy groups), cyano, acyl, thioacyl, O-carbamyl , N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido, R ¹⁸ S (O)-, One or more independently selected from the group consisting of R ¹⁸ S (O) _2- , -C (O) OR ¹⁸ , R ¹⁸ C (O) O- and -NR ¹⁸ R ¹⁹ (as defined above); It means a cycloalkyl group as defined above, preferably substituted with one or two substituents.
[68] "Alkenyl" refers to a lower alkyl group, as defined above, consisting of two or more carbon atoms and one or more carbon-carbon double bonds. Representative examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, and the like.
[69] "Alkynyl" refers to a lower alkyl group, as defined above, consisting of two or more carbon atoms and one or more carbon-carbon triple bonds. Representative examples include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-, 2- or 3-butynyl, and the like.
[70] "Aryl" refers to a monocyclic or fused ring polycyclic (ie, ring shares a pair of adjacent carbon atoms) groups having from 1 to 12 carbon atoms with a fully conjugated pi-electron system, all of which are carbon atoms; Refers to. Examples of aryl groups are, but are not limited to, phenyl, naphthalenyl and anthracenyl. Aryl groups may be substituted or unsubstituted. When substituted, the substituents are preferably one or more, more preferably 1, 2 or 3, even more preferably 1 or 2, and lower alkyl, trihaloalkyl, halo, hydroxy, lower alkoxy, mercapto , (Lower alkyl) thio, cyano, acyl, thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, nitro, N -Sulfonamido, S-sulfonamido, R ¹⁸ S (O)-, R ¹⁸ S (O) _2- , -C (O) OR ¹⁸ , R ¹⁸ C (O) O- and -NR ¹⁸ R ¹⁹ Wherein R ¹⁸ and R ¹⁹ are as defined above. Preferably the aryl group is independently selected from halo, lower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono- or di-alkylamino, carboxy or N-sulfonamido Or optionally substituted with two substituents.
[71] "Heteroaryl" means a group of 5 to 12 ring atoms containing 1, 2 or 3 ring heteroatoms selected from N, O or S and the remaining ring atoms are C and also having a fully conjugated pi-electron system It refers to a cyclic or fused ring (ie, rings share an adjacent pair of atoms). Examples of unsubstituted heteroaryl groups are, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine and carbazole. Heteroaryl groups may be substituted or unsubstituted. When substituted, the substituents are preferably one or more, more preferably 1, 2 or 3, even more preferably 1 or 2, and lower alkyl, trihaloalkyl, halo, hydroxy, lower alkoxy, mercapto , (Lower alkyl) thio, cyano, acyl, thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, nitro, N -Sulfonamido, S-sulfonamido, R ¹⁸ S (O)-, R ¹⁸ S (O) _2- , -C (O) OR ¹⁸ , R ¹⁸ C (O) O- and -NR ¹⁸ R ¹⁹ Wherein R ¹⁸ and R ¹⁹ are as defined above. Preferably, the heteroaryl group is independently from halo, lower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono- or di-alkylamino, carboxy or N-sulfonamido Optionally substituted with one or two substituents selected.
[72] “Heteroalicyclic” is a group having from 5 to 9 ring atoms wherein one or two ring atoms are N, O or S (O) _n (where n is an integer from 0 to 2) and the remaining ring atoms are C; It refers to a cyclic or fused ring group. The ring may also have one or more double bonds. However, the ring does not have a fully conjugated pi-electronic system. Examples of unsubstituted heteroalicyclic groups include, but are not limited to, pyrrolidino, piperidino, piperazino, morpholino, thiomorpholino, homopiperazino, and the like. Heteroalicyclic rings may be substituted or unsubstituted. When substituted, the substituents are preferably one or more, more preferably 1, 2 or 3, even more preferably 1 or 2, and lower alkyl, trihaloalkyl, halo, hydroxy, lower alkoxy, mercapto , (Lower alkyl) thio, cyano, acyl, thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, nitro, N -Sulfonamido, S-sulfonamido, R ¹⁸ S (O)-, R ¹⁸ S (O) _2- , -C (O) OR ¹⁸ , R ¹⁸ C (O) O- and -NR ¹⁸ R ¹⁹ Wherein R ¹⁸ and R ¹⁹ are as defined above. Preferably, the heteroaryl group is independently from halo, lower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono- or di-alkylamino, carboxy or N-sulfonamido Optionally substituted with one or two substituents selected.
[73] Preferably, the heteroalicyclic group is selected from halo, lower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono- or di-alkylamino, carboxy or N-sulfonamido Optionally substituted with one or two substituents independently selected.
[74] "Heterocycle" is a heteroatom selected from one or two ring atoms of N, O or S (O) _n , where n is an integer from 0 to 2, and the remaining ring atoms are C, wherein one or two C Atom represents a saturated cyclic radical of 3 to 8 ring atoms, which may be optionally substituted by a carbonyl group. Heterocyclyl rings are optionally substituted lower alkyl, which is substituted with one or two substituents independently selected from carboxy or esters, haloalkyl, cyanoalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino Independent from 1, 2 or 3 substituents selected from, monoalkylamino, dialkylamino, aralkyl, heteroaralkyl, -COR (where R is alkyl) or -COOR (where R is hydrogen or alkyl) May be optionally substituted. More specifically, the term heterocyclyl refers to tetrahydropyranyl, 2,2-dimethyl-1,3-dioxolane, piperidino, N-methylpiperidin-3-yl, piperazino, N- Methylpyrrolidin-3-yl, 3-pyrrolidino, morpholino, thiomorpholino, thiomorpholino-1-oxide, thiomorpholino-1,1-dioxide, 4-ethyloxycarbonylpy Ferrazino, 3-oxopiperazino, 2-imidazolidone, 2-pyrrolidinone, 2-oxohomopiperazino, tetrahydropyrimidin-2-one and derivatives thereof Do not. Preferably, heterocycle groups include, but are not limited to, halo, lower alkyl, lower alkyl substituted with carboxy, ester hydroxy, mono- or di-alkylamino.
[75] "Hydroxy" refers to an -OH group.
[76] "Alkoxy" refers to both -O- (alkyl) and -O- (cycloalkyl) groups. Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like.
[77] "Aryloxy" refers to both an -O-aryl and -O-heteroaryl group as defined herein. Representative examples include, but are not limited to, phenoxy, pyridinyloxy, furanyloxy, thienyloxy, pyrimidinyloxy, pyrazinyloxy, and the like and derivatives thereof.
[78] "Mercapto" refers to the -SH group.
[79] "Alkylthio" refers to both -S-alkyl and -S-cycloalkyl groups. Representative examples include, but are not limited to, methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio, and the like.
[80] "Arylthio" refers to both -S-aryl and -S-heteroaryl groups as defined herein. Representative examples include, but are not limited to, phenylthio, pyridinylthio, furanylthio, thienylthio, pyrimidinylthio, and the like and derivatives thereof.
[81] "Acyl" refers to the group -C (O) -R ", wherein R" is hydrogen; Lower alkyl; Trihalomethyl; Cycloalkyl; Aryl optionally substituted with one or more, preferably 1, 2 or 3 substituents selected from the group consisting of lower alkyl, trihalomethyl, lower alkoxy, halo and -NR ⁹ R ¹⁰ group; Heteroaryl optionally substituted with one or more, preferably one, two or three substituents selected from the group consisting of lower alkyl, trihaloalkyl, lower alkoxy, halo and -NR ⁹ R ¹⁰ group being); And heteroalicyclic optionally substituted with one or more, preferably 1, 2 or 3 substituents selected from the group consisting of lower alkyl, trihaloalkyl, lower alkoxy, halo and -NR ⁹ R ¹⁰ group. Coupled through). Representative acyl groups include, but are not limited to, acetyl, trifluoroacetyl, benzoyl and the like.
[82] "Aldehyde" refers to an acyl group in which R "is hydrogen.
[83] "Tioacyl" refers to the group -C (S) -R ", wherein R" is as defined above.
[84] "Ester" refers to the group -C (O) O-R ", wherein R" is as defined above except that it may not be hydrogen.
[85] An "acetyl" group refers to a -C (O) CH ₃ group.
[86] "Halo" group refers to fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.
[87] A "trihalomethyl" group refers to a -CX ₃ group, where X is a halo group as defined above.
[88] A "trihalomethanesulfonyl" group refers to an X ₃ CS (= 0) ₂ -group where X is as defined above.
[89] "Cyano" refers to the group -C≡N.
[90] "Methylenedioxy" refers to an -OCH ₂ O- group wherein two oxygen atoms are bonded to an adjacent carbon atom.
[91] "Ethylenedioxy" refers to -OCH ₂ CH ₂ O-, wherein two oxygen atoms are bonded to adjacent carbon atoms.
[92] "S-sulfonamido" refers to the group -S (O) ₂ NR ¹⁸ R ¹⁹ , wherein R ¹⁸ and R ¹⁹ are as defined above.
[93] "N-sulfonamido" refers to the group -NR ¹⁸ S (O) ₂ R ¹⁹ , wherein R ¹⁸ and R ¹⁹ are as defined above.
[94] "O-carbamyl" group refers to the group -OC (O) NR ¹⁸ R ¹⁹ , wherein R ¹⁸ and R ¹⁹ are as defined above.
[95] "N-carbamyl" refers to an R ¹⁸ OC (O) NR ¹⁹ -group where R ¹⁸ and R ¹⁹ are as defined above.
[96] "O-thiocarbamyl" refers to the group -OC (S) NR ¹⁸ R ¹⁹ , wherein R ¹⁸ and R ¹⁹ are as defined above.
[97] "N-thiocarbamyl" refers to an R ¹⁸ OC (S) NR ¹⁹ -group where R ¹⁸ and R ¹⁹ are as defined above.
[98] "Amino" refers to the group -NR ¹⁸ R ¹⁹ , wherein R ¹⁸ and R ¹⁹ are both hydrogen.
[99] “C-amido” refers to the group —C (O) NR ¹⁸ R ¹⁹ , wherein R ¹⁸ and R ¹⁹ are as defined above.
[100] “N-amido” refers to an R ¹⁸ C (O) NR ¹⁹ -group where R ¹⁸ and R ¹⁹ are as defined above.
[101] "Nitro" refers to the -NO ₂ group.
[102] "Haloalkyl" is an alkyl as defined above, preferably substituted with one or more identical or different halo atoms, preferably lower alkyl, eg -CH ₂ Cl, -CF ₃ , -CH ₂ CF ₃ , -CH ₂ CCl ₃ And the like.
[103] "Aralkyl" is an alkyl as defined above, preferably substituted with an aryl group as defined above, preferably lower alkyl such as -CH ₂ phenyl,-(CH ₂ ) ₂ phenyl,-(CH ₂ ) ₃ Phenyl, CH ₃ CH (CH ₃ ) CH ₂ phenyl, and the like and derivatives thereof.
[104] A "heteroaralkyl" group is an alkyl as defined above, preferably substituted with a heteroaryl group, preferably lower alkyl, -CH ₂ pyridinyl,-(CH ₂ ) ₂ pyrimidinyl,-(CH ₂ ) ₃ imide. Zolyl and the like and derivatives thereof.
[105] "Monoalkylamino" means a radical -NHR where R is an unsubstituted alkyl or cycloalkyl group as defined above, for example methylamino, (1-methylethyl) amino, cyclohexylamino and the like. do.
[106] "Dialkylamino" refers to a radical -NRR wherein each R is independently an unsubstituted alkyl or cycloalkyl group as defined above, for example dimethylamino, diethylamino, (1-methylethyl)- Ethylamino, cyclohexylmethylamino, cyclopentylmethylamino and the like.
[107] "Cyanoalkyl" means alkyl as defined above, preferably lower alkyl, which is substituted with one or two cyano groups.
[108] “Optional” or “optionally” means that an event or situation described subsequently may occur, but does not necessarily need to occur, and includes and does not occur when the event or situation occurs. For example, "heterocycle group optionally substituted with an alkyl group" means that an alkyl group may be present but not necessarily present, the state where the heterocycle group is substituted with an alkyl group and the heterocyclo group is an alkyl group Includes an unsubstituted state.
[109] The terms "2-indolinone", "indolin-2-one" and "2-oxindole" are used interchangeably herein to refer to a molecule of the formula:
[110]
[111] The term "pyrrole" refers to a molecule of the formula
[112]
[113] The terms "pyrrole substituted 2-indolinone" and "3-pyrrolidenyl-2-indolinone" are used interchangeably herein to refer to compounds having the general formula represented by Formula (I).
[114] Compounds having the same molecular formula but different atomic bond properties or sequence or atomic arrangement in space are referred to as "isomers". Isomers that differ in the arrangement of their atoms in space are called "stereoisomers".
[115] Stereoisomers that are not mirror images of one another are called "diastereomers", and mirror images that are not superimposed on one another are called "enantiomers". When the compound has an asymmetric center, for example when the compound is bound to four different groups, a pair of enantiomers are possible. Enantiomers are characterized by an absolute configuration of asymmetric centers, described by Cahn and Prelog's R- and S-array rules or by the way the molecules rotate the plane of polarization and clockwise or counterclockwise. As directions (ie, (+) or (-)-isomers, respectively). Chiral compounds may exist as individual enantiomers or mixtures thereof. Mixtures containing equal proportions of the enantiomers are referred to as "racemic mixtures".
[116] The compounds of the present invention may possess one or more asymmetric centers, and such compounds may thus be produced as individual (R)-or (S) -stereoisomers or mixtures thereof. For example, when the R ⁶ substituent in the compound of formula (I) is 2-hydroxyethyl, the carbon to which the hydroxy group is attached is an asymmetric center, so that the compound of formula (I) is a (R)-or (S) -isomer May be present as. Unless otherwise indicated, the description or nomenclature of a particular compound in this specification and claims is intended to include both individual enantiomers and mixtures, racemics or the like. Methods for crystallization of stereochemistry and separation of stereoisomers are well known in the art (see Chapter 4 of "Advanced Organic Chemistry", 4th edition J. March, John Wiley and Sons, New York, 1992). ).
[117] Compounds of the present invention may exhibit tautomerization and structural isomerization phenomena. For example, the compounds of formula (I) described herein can adopt an E or Z configuration for a double bond connecting a 2-indolinone moiety to a pyrrole moiety or can be a mixture of E and Z. The present invention includes any tautomeric or structural isomeric forms and mixtures thereof that have the ability to modulate RTK, CTK and / or STK activity and are not limited to any one tautomeric or structural isomeric form.
[118] A "pharmaceutical composition" refers to a mixture of one or more compounds described herein or physiologically / pharmaceutically acceptable salts or prodrugs thereof and other chemical ingredients such as physiologically / pharmaceutically acceptable carriers and excipients. Refer. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
[119] The compounds of the present invention may also act as prodrugs. "Prodrug" refers to a drug that is converted to a parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. For example, prodrugs are bioavailable by oral administration, while parent drugs are not. Prodrugs also have improved solubility in pharmaceutical compositions over parent drugs. Examples of prodrugs are administered as esters ("prodrugs") to facilitate delivery across cell membranes when water solubility is impaired, but then metabolically hydrolyzed once inside the cell to a carboxylic acid, activator, if water solubility is beneficial. There are compounds of the invention that decompose, but are not limited to these.
[120] Further examples of prodrugs may be, but are not limited to, short polypeptides, eg, 2 to 10 amino acid polypeptides, linked to the carboxy group of a compound of the invention via terminal amino groups, wherein the polypeptide is hydrolyzed or metabolized in vivo To release the active molecule. Prodrugs of compounds of formula I are within the scope of this invention.
[121] It is also contemplated that compounds of formula (I) are metabolized by enzymes in the body of an organism such as humans to produce metabolites that can regulate the activity of protein kinases. Such metabolites are within the scope of the present invention.
[122] As used herein, "physiologically / pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not destroy the biological activity and properties of the administered compound.
[123] "Pharmaceutically acceptable excipient" refers to an inert substance added to the pharmaceutical composition to facilitate administration of the compound. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
[124] As used herein, the term “pharmaceutically acceptable salts” refers to salts that retain the biological effects and properties of the parent compound. Such salts include:
[125] (1) the free base of the parent compound and inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid and perchloric acid, or acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid acid addition salts obtained by the reaction of organic acids, such as p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid or malonic acid, preferably hydrochloric acid or (L) -malic acid, such as 5- (5-fluoro L-maleate salt of 2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl) amide; or
[126] (2) Salts formed when acidic protons present in the parent compound are substituted by metal ions such as alkali metal ions, alkaline earth metal ions or coordinated with an organic base. The ions include conventional valences of aluminum, calcium, lithium, magnesium, potassium, sodium and zinc. Preferred organic bases include trimethylamine, diethylamine, N, N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine And quantized tertiary amines and quaternary ammonium cations.
[127] “Methods” include manners, means, techniques and processes known in the chemistry, pharmacy, biology, biochemistry and medicine or readily developed from the manners, means, techniques and processes known by those skilled in the art. It refers to ways, means, techniques and processes for accomplishing a given task, but not limited to these.
[128] "Treat", "treating" and "treatment" refer to all procedures, behaviors, applications, healings, etc., when a mammal, including humans, needs medical help for the purpose of directly or indirectly improving its manifestations. Refer. Particularly for cancer, the term simply means increasing the life expectancy of an individual affected by cancer or reducing one or more symptoms of the disease. This may, among other things, delay the appearance of primary or secondary tumors, slow the development of primary or secondary tumors, reduce the incidence of primary or secondary tumors, and slow or reduce the severity of secondary effects of the disease. And inhibit tumor growth and degenerate the tumor.
[129] The term "prevention" includes the prevention of the expression of clinically apparent neoplasia entirely or in the preclinical phase of neoplasia in an individual at risk. In addition, the definition is intended to include the prevention of onset of malignant cells or the inhibition or conversion of precancerous cells to malignant cells. This includes prophylactic treatment of patients at risk of neoplastic development.
[130] The phrase "healing effective" limits the amount of each drug that will achieve the goal of improving neoplastic disease severity and frequency of neoplastic disease for treatment with each drug itself, while avoiding the side effects typically associated with other therapies. It is intended to be.
[131] "Healing effect" or "healing effective amount" means: 1) reducing the number of cancer cells; 2) reducing tumor size; 3) inhibit (ie, slow to some extent, preferably stop) the invasion of cancer cells into peripheral organs; 3) inhibit (ie, slow to some extent, preferably stop) metastasis of the tumor; 4) inhibiting to some extent tumor growth; 5) alleviating or reducing to some extent one or more of the symptoms associated with the disease; And / or 6) limiting the amount of anticancer agent required to alleviate to some extent one or more of the symptoms of a neoplastic disease, including but not limited to alleviating or reducing the side effects associated with the administration of the anticancer agent.
[132] The phrase “combination therapy” (or “co-therapy”) refers to protein kinase inhibitors and cyclooxygenases as part of a specific therapeutic regimen intended to provide a beneficial effect from the co-action of such therapy. -2 administration of the inhibitor. Beneficial effects of such combinations include, but are not limited to, pharmacokinetic or pharmacokinetic co-actions resulting from the combination of therapeutic agents. Administration of such combinations of therapy is typically administered over a defined period of time (typically minutes, hours, days or weeks, depending on the combination selected). “Combination therapy” is generally intended to include the administration of two or more of these therapies as part of a separate monotherapy regimen that results in a combination of the present invention incidentally and independently. "Combination therapy" refers to the administration of these therapies in a continuous manner, that is, the administration of each therapy at different times, as well as the administration of two or more of these agents or therapies in a substantially simultaneous manner. It is intended to be included. Substantial simultaneous administration can be achieved, for example, by administering to the patient a single capsule having a fixed ratio of each therapy or multiple, single capsules for each therapy. Continuous or substantially simultaneous administration of each therapy may be by any suitable route, including but not limited to oral route, intravenous route, intramuscular route, direct absorption through mucosal tissue. The therapy may be administered by the same route or by different routes. For example, the first therapy of the selected combination may be administered by intravenous injection, while other therapies of the combination may be administered orally. Alternatively, for example, both therapies may be administered orally, or both therapies may be administered by intravenous injection. The order in which the therapies are administered is of little importance. “Combination therapy” also includes other biologically active ingredients (eg, but not limited to, second and other anti-neoplastic drugs) and non-drug therapy (eg, but not limited to surgery or radiation therapy). And in further combinations with the foregoing therapeutic agents. If the combination therapy further comprises radiation therapy, radiation therapy can be performed at any suitable time so long as a beneficial effect is achieved from the co-action of the therapeutic agent and the radiation therapy combination. For example, where appropriate, a beneficial effect can be achieved even when the radiation treatment is temporarily eliminated upon administration of the therapy for days or even weeks.
[133] “Additional therapies” include, for example, drugs that reduce the toxic effects of anticancer drugs (eg, bone resorption inhibitors, cardioprotectants), drugs that prevent or reduce the occurrence of nausea and vomiting associated with chemotherapy, radiotherapy or surgery, or myelosuppression Treating the patient with a drug that reduces or eliminates the side effects associated with the combination therapy of the present invention, including but not limited to drugs that reduce the incidence of infection associated with administration of sex anticancer drugs.
[134] Preferred Embodiment
[135] While a broad definition is disclosed in the above summary of the invention, certain compounds of formulas (I), (II) and (III) are preferred for carrying out the invention.
[136] A. Protein Tyrosine Kinase Inhibitors-Compounds of Formula (I)
[137] (1) Preferred groups of compounds of formula (I) are those in which R ¹ , R ³ and R ⁴ are hydrogen.
[138] (2) Another preferred group of compounds of Formula (I) are those in which R ¹ , R ² and R ⁴ are hydrogen.
[139] (3) Another preferred group of compounds of Formula (I) are those in which R ¹ , R ² and R ³ are hydrogen.
[140] (4) Another preferred group of compounds of Formula (I) are those in which R ² , R ³ and R ⁴ are hydrogen.
[141] (5) Another preferred group of compounds of Formula (I) are those in which R ¹ , R ² , R ³ and R ⁴ are hydrogen.
[142] (6) A further preferred group of compounds of formula I is R ⁵ , R ⁶ or R ⁷ , preferably R ⁵ or R ⁶ , more preferably R ⁶ is —COR ¹⁰ , wherein R ¹⁰ Is —NR ¹¹ (CH ₂ ) _n R ¹² wherein R ¹¹ is hydrogen or lower alkyl, preferably hydrogen or methyl; n is 2, 3 or 4, preferably 2 or 3; R ¹² is -NR ¹³ R ¹⁴ , wherein R ¹³ and R ¹⁴ are independently alkyl, more preferably lower alkyl, or R ¹³ and R ¹⁴ are bonded to-(CH ₂ ) ₄ -,-(CH ₂ ) _{5 -, - (CH 2)} 2 -0- (CH 2) 2 - or _{- (CH 2) 2 N (} CH 3) (CH 2) 2 - may be from, and preferably selected form the group R ^13, and R ¹⁴ is independently hydrogen, methyl, ethyl or combined to form morpholin-4-yl, pyrrolidin-1-yl, piperazin-1-yl or 4-methylpiperazin-1-yl] .
[143] More preferably, R ⁵ or R ⁶ in the above (6) is N- (2-dimethylaminoethyl) aminocarbonyl, N- (2-ethylaminoethyl) -N-methylaminocarbonyl, N- ( 3-dimethylaminopropyl) -aminocarbonyl, N- (2-diethylaminoethyl) aminocarbonyl, N- (3-ethylaminopropyl) aminocarbonyl, N- (3-diethylaminopropyl) aminocarbon Nyl, 3-pyrrolidin-1-yl-propylaminocarbonyl, 3-morpholin-4-ylpropyl-aminocarbonyl, 2-pyrrolidin-1-ylethylaminocarbonyl, 2-morpholine- 4-ylethylaminocarbonyl, 2- (4-methylpiperazin-1-yl) ethylaminocarbonyl, 2- (4-methylpiperazin-1-yl) propylaminocarbonyl, 2- (3,5 -Dimethylpiperazin-1-yl) ethylaminocarbonyl or 2- (3,5-dimethylpiperazin-1-yl) propylaminocarbonyl, even more preferably N- (2-diethyl-aminoethyl) amino Carbonyl or N- (2-ethylaminoethyl) amino-carbonyl.
[144] (7) A further preferred group of compounds of formula I is R ⁵ , R ⁶ or R ⁷ , preferably R ⁵ or R ⁶ , more preferably R ⁶ is -COR ¹⁰ , wherein R ¹⁰ is -NR ¹³ R ¹⁴ wherein R ¹³ is hydrogen; R ¹⁴ is lower alkyl, aryl, heteroaryl, heteroalicyclic or carboxy substituted with alkyl, preferably hydroxy, more preferably methyl, ethyl, propyl or butyl, aryl, heteroalicye substituted with hydroxy Click (eg, piperidine, piperazine, morpholine, etc.), hetero aryl, or carboxy]. Even more preferably R ⁵ or R ⁷ of this group (7) is 2-ethoxycarbonylmethyl-aminocarbonyl, carboxymethylamino-carbonyl, 3-hydroxypropyl-aminocarbonyl, 2-hydroxyethyl Aminocarbonyl, 3-triazin-1-ylpropylamino-carbonyl, triazin-1-ylethylaminocarbonyl, 4-hydroxy-phenylethylaminocarbonyl, 3-imidazol-1-ylpropyl- Aminocarbonyl, pyridin-4-ylmethylaminocarbonyl, 2-pyridin-2-ylethylaminocarbonyl or 2-imidazol-1-ylethylaminocarbonyl.
[145] (8) A further preferred group of compounds of formula (I) is R ⁵ , R ⁶ or R ⁷ , preferably R ⁵ or R ⁶ , more preferably R ⁶ is -COR ¹⁰ , wherein R ¹⁰ is -NR ¹¹ (CH ₂ ) _n R ¹² wherein R ¹¹ is hydrogen or alkyl, preferably hydrogen or methyl; n is 2, 3 or 4, preferably 2 or 3; R ¹² is —R ¹³ R ¹⁴ , wherein R ¹³ and R ¹⁴ combine together to form a heterocycle, preferably a 5, 6 or 7 membered heterocycle containing carbonyl groups and 1 or 2 nitrogen atoms. ]. Preferably, R ⁵ or R ⁶ is 2- (3-ethoxycarbonylmethylpiperazin-1-yl) ethylaminocarbonyl, 2- (3-oxopiperazin-1-yl) ethylaminocarbonyl, 2- (imidazolin-1-yl-2-one) ethylaminocarbonyl, 2- (tetrahydropyrimidin-1-yl-2-one) ethylaminocarbonyl, 2- (2-oxopyrrolidine -1-yl) -ethylaminocarbonyl, 3- (4-methylpiperazin-1-yl) propylaminocarbonyl, 3- (3-ethoxycarbonylmethylpiperazin-1-yl) -propylaminocarbon Nyl, 3- (3-oxopiperazin-1-yl) propyl-aminocarbonyl, 3- (imidazolidin-yl-2-one) propyl-aminocarbonyl, 3- (tetrahydropyrimidine-1 -Yl-2-one) -propylaminocarbonyl, 3- (2-oxopyrrolidin-1-yl) propyl-aminocarbonyl, 2- (2 oxohomopiperidin-1-yl) ethylamino- Carbonyl or 3- (2-oxohomopiperidin-1-yl) propylaminocarbonyl.
[146] (9) A further preferred group of compounds of formula (I) is R ⁵ , R ⁶ or R ⁷ , preferably R ⁵ or R ⁶ , more preferably R ⁶ is —COR ¹⁰ , wherein (a ) R ¹⁰ is —NR ¹¹ (CH ₂ ) _n R ¹² where R ¹¹ is hydrogen or alkyl, preferably hydrogen or methyl; n is 2, 3 or 4, preferably 2 or 3; R ¹² is -NR ¹³ R ¹⁴ , wherein R ¹³ is hydrogen and R ¹⁴ is cyanoalkyl or -NHCOR ^a (R ^a is alkyl), or (b) R ¹⁰ is -NR ¹³ R ¹⁴ [ Wherein R ¹³ and R ¹⁴ combine with each other to form a heterocycle containing no carbonyl group in the ring. Preferably, R ⁵ or R ⁶ is 2- (2-cyanoethylamino) ethylaminocarbonyl, 2- (acetylamino) -ethylaminocarbonyl, morpholinocarbonyl, piperidin-1-yl -Carbonyl, 2-cyanomethylaminoethylaminocarbonyl or piperadin-1-ylcarbonyl.
[147] (10) Another preferred group of compounds of Formula I is that R ⁵ is -COR ¹⁰ , wherein R ¹⁰ is -NR ¹³ R ¹⁴ , wherein R ¹³ is hydrogen and R ¹⁴ is lower alkyl substituted by hydroxy , Lower alkyl, carboxy, or —NR ¹⁸ R ¹⁹ substituted with hydroxyalkylamino, wherein R ¹⁸ and R ¹⁹ are independently hydrogen or lower alkyl, more preferably R ⁵ is 2-[(di Ethylamino) -2-hydroxyethyl] aminocarbonyl, 2- (N-ethyl-N-2-hydroxyethylamino) ethylaminocarbonyl, carboxymethylamino-carbonyl or 2- (2-hydroxyethyl Amino) ethylamino-carbonyl.
[148] (11) A further preferred group of compounds of Formula (I) is that R ⁶ is -COR ¹⁰ , wherein R ¹⁰ is -NR ¹³ R ¹⁴ , wherein R ¹³ is hydrogen and R ¹⁴ is substituted with hydroxy Lower alkyl, lower alkyl substituted with hydroxyalkylamino, carboxy, or —NR ¹⁸ R ¹⁹ , wherein R ¹⁸ and R ¹⁹ are independently hydrogen or lower alkyl, and more preferably R ⁶ is [2] -(Diethylamino) -2-hydroxy] ethylaminocarbonyl, 2- (N-ethyl-N-2-hydroxyethyl-amino) ethylaminocarbonyl, carboxymethylaminocarbonyl or 2- (2- Hydroxyethylamino) ethylamino-carbonyl.
[149] (12) Another more preferred group of the formula (I) and R ⁵ is -COR ^10, in the above, R ¹⁰ is _{^{-NR 11 (CH 2) n R}} 12 [ wherein, R ¹² is -N ⁺ (O ^-) NR ¹³ R ¹⁴ or —N (OH) R ¹³ , wherein R ¹³ and R ¹⁴ are independently selected from the group consisting of hydrogen and lower alkyl], more preferably R ⁵ is 2- (N-hydroxy -N- ethylamino) ethylamino-carbonyl, or ^{2- [N + (O -)} (C 2 H 5) 2] - is an amino-carbonyl ethyl.
[150] (13) Another more preferred group of the formula (I) and R ⁶ is -COR ^10, in the above, R ¹⁰ is _{^{-NR 11 (CH 2) n R}} l2 [ wherein, R ¹² is -N ⁺ (O ^-) NR ^l3 R ^l4 or -N (OH) R ¹³ , wherein R ¹³ and R ¹⁴ are independently selected from the group consisting of hydrogen and lower alkyl, preferably R ⁶ is 2- (N-hydroxy- N-ethylamino) ethylaminocarbonyl or 2- [N ⁺ (O ⁻ ) (C ₂ H ₅ ) ₂ ] ethyl-aminocarbonyl.
[151] (14) In the above preferred groups (6) to (13) wherein R ⁵ is -COR ¹⁰ , a more preferred group of compounds is
[152] R ⁶ is selected from the group consisting of hydrogen and alkyl, preferably hydrogen, methyl, ethyl, isopropyl tert-butyl, isobutyl or n-butyl, more preferably hydrogen or methyl;
[153] R ⁷ is hydrogen, alkyl, aryl, heteroaryl and -C (O) R ^l7 [wherein, R ¹⁷ is hydroxy, alkyl or aryl], more preferably hydrogen, methyl, ethyl, propyl, n-, iso tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more preferably methyl, hydrogen or phenyl.
[154] (15) In the above preferred groups (6) to (13) in which R ⁵ is -COR ¹⁰ , another more preferred group of compounds is that R ⁶ and R ⁷ combine to form-(CH ₂ ) _4- . .
[155] (16) In the above preferred groups (6) to (13) wherein R ⁶ is -COR ¹⁰ , a more preferred group of compounds is
[156] R ⁵ is selected from the group consisting of hydrogen and alkyl, preferably hydrogen, methyl, ethyl, isopropyl tert-butyl, isobutyl or n-butyl, more preferably hydrogen or methyl;
[157] R ⁷ is hydrogen, alkyl, aryl, heteroaryl and -C (O) R ^l7 [wherein, R ¹⁷ is hydroxy, alkyl or aryl], more preferably hydrogen, methyl, ethyl, isopropyl, n-, Iso-, tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more preferably selected from the group consisting of methyl, hydrogen or phenyl.
[158] (17) In the above preferred and more preferred groups (6) to (16), even more preferred groups of compounds are
[159] R ¹ is hydrogen, alkyl, —C (O) NR ⁸ R ⁹ , cycloalkyl or aryl, preferably hydrogen, phenyl, 3,4-dimethoxyphenylaminocarbonyl, 4-methoxy-3-chlorophenyl- Aminocarbonyl, even more preferably hydrogen or methyl, most preferably hydrogen;
[160] R ² is cyano, hydrogen, halo, lower alkoxy, aryl or —S (O) ₂ NR ¹³ R 1 ⁴ where R ¹³ is hydrogen and R ¹⁴ is hydrogen, aryl or alkyl, preferably R ² is hydrogen, chloro, bromo, fluoro, methoxy, ethoxy, phenyl, dimethylamino-sulfonyl, 3-chlorophenyl aminosulfonyl, carboxy, methoxy, aminosulfonyl, aminosulfonyl-methyl, aminosulfonyl-phenyl Phonyl, pyridin-3-yl-aminosulfonyl, dimethylaminosulfonyl, isopropylamino-sulfonyl, more preferably hydrogen, fluoro or bromo;
[161] R ³ is optionally substituted with one or two substituents selected from the group consisting of hydrogen, lower alkoxy, -C (O) R ¹⁵ , -NR ¹³ C (O) R ¹⁴ , aryl, preferably lower alkyl, halo or lower alkoxy Aryl substituted with heteroaryl, and heteroaryl optionally substituted with one or two substituents selected from the group consisting of lower alkyl, halo or lower alkoxy; Preferably hydrogen, methoxy, carboxy, phenyl, pyridin-3-yl, 3,4-dichlorophenyl, 2-methoxy-5-isopropylphenyl, 4-n-butylphenyl, 3-isopropylphenyl, more Preferably selected from the group consisting of hydrogen or phenyl;
[162] R ⁴ is hydrogen.
[163] (18) Another more preferred group of formula (I) is
[164] R ¹ is hydrogen, alkyl, —C (O) NR ⁸ R ⁹ , cycloalkyl or aryl, preferably hydrogen, 3,4-dimethoxyphenylaminocarbonyl, 4-methoxy-3-chlorophenylaminocarbonyl Even more preferably hydrogen or methyl, in particular hydrogen;
[165] R ² is cyano, hydrogen, halo, lower alkoxy, aryl or —S (O) ₂ NR ¹³ R 1 ⁴ where R ¹³ is hydrogen and R ¹⁴ is hydrogen, aryl or alkyl, preferably R ² is hydrogen, chloro, bromo, fluoro, methoxy, ethoxy, phenyl, dimethylamino-sulfonyl, 3-chlorophenyl aminosulfonyl, carboxy, methoxy, aminosulfonyl, aminosulfonyl-methyl, aminosulfonyl-phenyl Phonyl, pyridin-3-yl-aminosulfonyl, dimethylaminosulfonyl, isopropylamino-sulfonyl, more preferably hydrogen, fluoro or bromo;
[166] R ³ is optionally substituted with one or two substituents selected from the group consisting of hydrogen, lower alkoxy, -C (O) R ¹⁵ , -NR ¹³ C (O) R ¹⁴ , aryl, preferably lower alkyl, halo or lower alkoxy Aryl substituted with heteroaryl, and heteroaryl optionally substituted with one or two substituents selected from the group consisting of lower alkyl, halo or lower alkoxy; Preferably hydrogen, methoxy, carboxy, phenyl, pyridin-3-yl, 3,4-dichlorophenyl, 2-methoxy-5-isopropylphenyl, 4-n-butylphenyl, 3-isopropylphenyl, more Preferably selected from the group consisting of hydrogen or phenyl;
[167] R ⁴ is hydrogen.
[168] In this preferred group (18), more preferred groups of compounds are
[169] R ⁵ is —COR ¹⁰ , wherein R ¹⁰ is as defined in the Summary of the Invention, preferably —NR ¹¹ (CH ₂ ) _n R ¹² or -NR ¹³ R as defined in the Summary of the Invention. ¹⁴ ;
[170] R ⁶ is selected from the group consisting of hydrogen and alkyl, preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl, isobutyl or n-butyl, more preferably hydrogen or methyl;
[171] R ⁷ is hydrogen, alkyl, aryl, heteroaryl and —C (O) R ¹⁷ wherein R ¹⁷ is hydroxy, alkyl or aryl, more preferably hydrogen, methyl, ethyl, isopropyl, n-, iso or tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more preferably methyl, hydrogen or phenyl.
[172] In this preferred group (18), more preferred groups of compounds are
[173] R ⁶ is —COR ¹⁰ , wherein R ¹⁰ is as defined in the Summary of the Invention, preferably —NR ¹¹ (CH ₂ ) _n R ¹² or -NR ¹³ R as defined in the Summary of the Invention. ¹⁴ ;
[174] R ⁵ is selected from the group consisting of hydrogen and alkyl, preferably hydrogen, methyl, ethyl, isopropyl, tert-butyl, isobutyl or n-butyl, more preferably hydrogen or methyl;
[175] R ⁷ is hydrogen, alkyl, aryl, heteroaryl and —C (O) R ¹⁷ wherein R ¹⁷ is hydroxy, alkyl or aryl, more preferably hydrogen, methyl, ethyl, isopropyl, n-, iso or tert-butyl, phenyl, benzoyl, acetyl or carboxy, even more preferably methyl, hydrogen or phenyl.
[176] (19) Another more preferred group of formula (I) is
[177] R ¹ and R ⁴ are hydrogen;
[178] R ² is selected from the group consisting of hydrogen, halo, lower alkoxy, —C (O) R ¹⁵ and —S (0) ₂ NR ¹³ R ¹⁴ ;
[179] R ³ is selected from the group consisting of hydrogen, lower alkoxy, —C (O) R ¹⁵ , —S (O) ₂ NR ¹³ R ¹⁴ , aryl and heteroaryl;
[180] R ⁵ is —C (O) R ¹⁰ ;
[181] R ⁶ is selected from the group consisting of hydrogen and lower alkyl;
[182] R ⁷ is selected from the group consisting of hydrogen, lower alkyl and —C (O) R ¹⁷ .
[183] In compounds having a structure as described in (15) above, R ¹⁰ is hydroxy, lower alkoxy and NR ¹¹ (CH ₂ ) _n R ^12, wherein n is 2 or 3; R ¹¹ is selected from the group consisting of hydrogen and lower alkyl; R ¹² is selected from the group consisting of aryl and —NR ¹³ R ¹⁴ ] is another presently preferred embodiment of the present invention.
[184] In compounds having the structure described in the previous two paragraphs, R ¹³ and R ¹⁴ are hydrogen, lower alkyl and-(CH ₂ ) ₄ -,-(CH ₂ ) ₅ -,-(CH ₂ ) ₂ O (CH _{2) 2} - or _{- (CH 2) 2 N (} CH 3) (CH 2) 2 - is independently selected from the group consisting of a presently preferred further embodiment of the invention.
[185] (20) Another presently preferred embodiment of the present invention is
[186] R ¹ is selected from the group consisting of hydrogen, lower alkyl, — (CH ₂ ) _r R ¹⁶ and —C (O) NR ⁸ R ⁹ ;
[187] R ² is selected from the group consisting of hydrogen, halogen, aryl and —S (O) ₂ NR ¹³ R ¹⁴ ;
[188] R ³ is selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, aryl, heteroaryl and -C (O) R ¹⁵ ;
[189] R ⁴ is hydrogen;
[190] R ⁵ is selected from the group consisting of hydrogen and lower alkyl;
[191] R ⁶ is —C (O) R ¹⁰ ;
[192] R ⁷ is selected from the group consisting of hydrogen, lower alkyl and aryl;
[193] R ¹⁶ is selected from the group consisting of hydroxy and -C (O) R ¹⁵ ;
[194] r is 2 or 3;
[195] A presently preferred embodiment of the invention is a compound having the structure described in the preceding paragraph wherein R ³ is optionally substituted with one or more groups selected from the group consisting of lower alkyl, lower alkoxy and halo.
[196] (21) In addition, a presently preferred embodiment of the present invention,
[197] R ¹ is selected from the group consisting of hydrogen, lower alkyl, — (CH ₂ ) _r R ¹⁶ and —C (O) NR ⁸ R ⁹ ;
[198] R ² is selected from the group consisting of hydrogen, halogen, aryl and —S (O) ₂ NR ¹³ R ¹⁴ ;
[199] R ³ is selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, aryl, heteroaryl and -C (O) R ¹⁵ ;
[200] R ⁴ is hydrogen;
[201] R ⁵ is selected from the group consisting of hydrogen and lower alkyl;
[202] R ⁶ is —C (O) R ¹⁰ ;
[203] R ⁷ is selected from the group consisting of hydrogen, lower alkyl and aryl;
[204] R ¹⁶ is selected from the group consisting of hydroxy and -C (O) R ¹⁵ ;
[205] r is 2 or 3;
[206] R ¹⁰ is hydroxy, lower alkoxy, -NR ¹³ R ¹⁴ and -NR ¹¹ (CH ₂ ) _n R ¹² where n is 1, 2 or 3, R ¹¹ is hydrogen, R ¹² is hydroxy, lower Alkoxy, —C (O) R ¹⁵ , heteroaryl and —NR ¹³ R ¹⁴ ].
[207] (22) A further presently preferred embodiment of the invention is-(CH ₂ ) ₄ -,-(CH ₂ ) ₅ -,-(CH ₂ ) wherein R ¹³ and R ¹⁴ are hydrogen, lower alkyl, heteroaryl and combined ₂ O (CH ₂ ) ₂ -or-(CH ₂ ) ₂ N (CH ₃ ) (CH ₂ ) ₂ -is a compound having the structure described in the preceding paragraph independently selected from the group consisting of.
[208] (23) Another presently preferred embodiment of the present invention is
[209] R ¹ is —C (O) NR ⁸ R ⁹ wherein R ⁸ is hydrogen and R ⁹ is aryl optionally substituted with one or more groups selected from the group consisting of halo, hydroxy and lower alkoxy;
[210] R ² is selected from the group consisting of hydrogen, halogen, aryl and —S (O) ₂ NR ¹³ R ¹⁴ ;
[211] R ³ is selected from the group consisting of hydrogen, lower alkyl, lower alkoxy, aryl, heteroaryl and -C (O) R ¹⁵ ;
[212] R ⁴ is hydrogen;
[213] R ⁵ is selected from the group consisting of hydrogen and lower alkyl;
[214] R ⁶ is —C (O) R ¹⁰ ;
[215] R ⁷ is selected from the group consisting of hydrogen, lower alkyl and aryl;
[216] R ¹⁶ is selected from the group consisting of hydroxy and -C (O) R ¹⁵ ;
[217] r is 2 or 3;
[218] (24) Another further presently preferred embodiment of the present invention is
[219] R ¹ is selected from the group consisting of hydrogen and lower alkyl;
[220] R ² is selected from the group consisting of hydrogen, halo, lower alkoxy, aryl, —C (O) R ¹⁵ and —S (O) ₂ NR ¹³ R ¹⁴ ;
[221] R ³ is selected from the group consisting of hydrogen, halo, aryl, heteroaryl and -C (O) R ¹⁵ ;
[222] R ⁴ is hydrogen;
[223] R ⁵ is —C (O) R ¹⁰ ;
[224] R ⁶ and R ⁷ are bonded to form a-(CH ₂ ) ₄ -group.
[225] In compounds having a structure as described in the preceding paragraphs, R ¹⁰ is hydroxy, alkoxy, —NR ¹³ R ¹⁴ and —NH (CH ₂ ) _n NR ¹³ R ¹⁴ , where n is 2 or 3 Selected from the group consisting of are presently preferred embodiments.
[226] In compounds having a structure as described in the previous two paragraphs, R ¹³ and R ¹⁴ are hydrogen, lower alkyl, and-(CH ₂ ) ₄ -,-(CH ₂ ) ₅ -,-(CH ₂ ) in combination Independently selected from the group consisting of ₂ O (CH ₂ ) ₂ -or-(CH ₂ ) ₂ N (CH ₃ ) (CH ₂ ) ₂ -is a presently preferred embodiment of the present invention.
[227] Representative compounds of formula (I) are shown in Table I below.
[228]
[229]
[230]
[231]
[232]
[233]
[234]
[235]
[236]
[237]
[238]
[239]
[240]
[241]
[242]
[243]
[244]
[245]
[246]
[247]
[248]
[249]
[250]
[251]
[252]
[253] The compound number corresponds to the example number of the example portion of the present invention. That is, the synthesis of the compounds of Table I is described in Example 1. The compounds shown in Table I are merely examples and are in no way regarded as limiting the scope of the invention. Additional protein kinase inhibitors that may be used in this method include 3- (3,5-dimethylpyrrole-2-ylmethylidene) -2-indolinone (su 5416), 3- [3,5-dimethyl-4- ( 2-carboxyethyl) pyrrole-2-ylmethylidene] -2-indolinone (su 6668) and 3- [3- (2-carboxyethyl) -5-methylpyrrole-2-ylmethylidene-2-indolinone It includes.
[254] B. Cyclooxygenase-2-Selective Inhibitors-Compounds of Formulas II and III
[255] Non-limiting examples of COX-2 inhibitors that can be used in the present invention are set forth in Tables II and III below.
[256]
[257]
[258]
[259]
[260] Pyrazoles can be prepared by the process described in WO 95/15316. Pyrazoles can be further prepared by the process described in WO 95/15315. Pyrazoles can also be prepared by the process described in WO 96/03385. Thiophene analogs can be prepared by the methods described in WO 95/00501. The preparation of thiophene analogs is also described in WO 94/15932. Oxazoles can be prepared by the process described in WO 95/00501. The preparation of oxazoles is also described in WO 94/27980. Isoxazoles can be prepared by the process described in WO 96/25405. Imidazoles can be prepared by the process described in WO 96/03388. The preparation of imidazoles is also described in WO 96/03387. Cyclopentane cyclooxagenase-2-inhibitors can be prepared by the methods described in US Pat. No. 5,344,991. The preparation of cyclopentane Cox-2 inhibitors is also described in WO 95/00501. Terphenyl compounds can be prepared by the process described in WO 96/16934. Thiazole compounds can be prepared by the process described in WO 96/03392. Pyridine compounds are also described in WO 96/24585.
[261] Celecoxib used in the therapeutic combinations of the present invention may be prepared in the manner disclosed in US Pat. No. 5,466,823.
[262] Valdecoxib used in the therapeutic combinations of the present invention may be prepared in the manner disclosed in US Pat. No. 5,633,272.
[263] Parecoxib used in the therapeutic combinations of the present invention may be prepared in the manner disclosed in US Pat. No. 5,932,598.
[264] Rofecoxib used in the therapeutic combinations of the present invention may be prepared in the manner disclosed in US Pat. No. 5,968,974.
[265] Japan Tobacco JTE-552 used in the therapeutic combination of the present invention can be prepared in the manner disclosed in JP 90/52882.
[266] Lumiracoxib (Cox-189) used in the therapeutic combinations of the present invention may be prepared in the manner disclosed in WO 99/11605.
[267] Ethoxycoxib (MK 663) used in the therapeutic combinations of the present invention can be prepared in the manner disclosed in WO 98/03484.
[268] Bristol Meyers Squibb's BMS 34070 used in the therapeutic combinations of the present invention can be prepared in the manner disclosed in US Pat. No. 6,180,651.
[269] The above references listed in Tables II and III, listing various COX-2 inhibitors suitable for use in the present invention described herein and methods for their preparation, are individually incorporated by reference.
[270] Preferred COX-2 inhibitors that can be used in the present invention include but are not limited to the following compounds.
[271] C1)
[272]
[273] JTE-522, 4- (4-cyclohexyl-2-methyloxazol-5-yl) -2-fluorobenzenesulfonamide,
[274] C2)
[275] 5-chloro-3- (4- (methylsulfonyl) phenyl) -2- (methyl-5-pyridinyl) pyridine,
[276] C3)
[277] 2- (3,5-difluorophenyl) -3-4- (methylsulfonyl) phenyl) -2-cyclopenten-l-one,
[278] C4)
[279]
[280] 4- [5- (4-methylphenyl) -3- (trifluoromethyl) -1H-pyrazol-1-yl] benzenesulfonamide,
[281] C5)
[282]
[283] Rofecoxib, 4- (4- (methylsulfonyl) phenyl] -3-phenyl-2 (5H) -furanone,
[284] C6)
[285]
[286] 4- (5-methyl-3-phenylisoxazol-4-yl) benzenesulfonamide,
[287] C7)
[288] N-[[4- (5-methyl-3-phenylisoxazol-4yl] phenyl] sulfonyl] propanamide,
[289] C8)
[290]
[291] 4- [5- (4-chlorophenyl) -3- (trifluoromethyl) -1H-pyrazol-1-yl] benzenesulfonamide,
[292] C9)
[293] ;
[294] C10)
[295] ;
[296] C11)
[297]
[298] 6-[[5- (4-chlorobenzoyl) -1,4-dimethyl-1H-pyrrol-2-yl] methyl] -3 (2H) -pyridazinone,
[299] C12)
[300]
[301] N- (4-nitro-2-phenoxyphenyl) methanesulfonamide,
[302] C13)
[303]
[304] C14)
[305]
[306] 3- (3,4-difluorophenoxy) -5,5-dimethyl-4- [4- (methylsulfonyl) phenyl] -2 (5H) -furanone,
[307] C15)
[308]
[309] N- [6-[(2,4-difluorophenyl) thio] -2,3-dihydro-1-oxo-1H-inden-5-yl] methanesulfonamide,
[310] C16)
[311]
[312] 3- (4-chlorophenyl) -4- [4- (methylsulfonyl) phenyl] -2 (3H) -oxazolone,
[313] C17)
[314]
[315] 4- [3- (4-fluorophenyl) -2,3-dihydro-2-oxo-4-oxazolyl] benzenesulfonamide,
[316] C18)
[317]
[318] 3- [4- (methylsulfonyl) phenyl] -2-phenyl-2-cyclopenten-l-one,
[319] C19)
[320]
[321] 4- (2-methyl-4-phenyl-5-oxazolyl) benzenesulfonamide,
[322] C20)
[323]
[324] 3- (4-fluorophenyl) -4- [4- (methylsulfonyl) phenyl] -2- (3H) -oxazolone,
[325] C21)
[326]
[327] 5- (4-fluorophenyl) -1- [4- (methylsulfonyl) phenyl] -3- (trifluoromethyl) -1H-pyrazole,
[328] C22)
[329]
[330] 4- [5-phenyl) -3- (trifluoromethyl) -1H-pyrazol-1-yl) benzenesulfonamide,
[331] C23)
[332]
[333] 4- [l-phenyl-3- (trifluoromethyl) -lH-pyrazol-5-yl] benzenesulfonamide,
[334] C24)
[335]
[336] 4- [5- (4-fluorophenyl) -3- (trifluoromethyl) -lH-pyrazol-1yl] benzenesulfonamide,
[337] C25)
[338]
[339] N- [2- (cyclohexyloxy) -4-nitrophenyl] methanesulfonamide,
[340] C26)
[341]
[342] N- [6- (2,4-difluorophenoxy) -2,3-dihydro-1-oxo-1H-inden-5-yl] methanesulfonamide,
[343] C27)
[344]
[345] 3- (4-chlorophenoxy) -4-[(methylsulfonyl) amino] benzenesulfonamide,
[346] C28)
[347]
[348] 3- (4-fluorophenoxy) -4-[(methylsulfonyl) amino] benzenesulfonamide,
[349] C29)
[350]
[351] 3-[(1-methyl-1H-imidazol-2-yl) thio] -4 [(methylsulfonyl) amino] benzenesulfonamide,
[352] C30)
[353]
[354] 5,5-dimethyl-4- [4- (methylsulfonyl) phenyl] -3-phenoxy-2 (5H) -furanone,
[355] C31)
[356]
[357] N- [6-[(4-ethyl-2-thiazolyl) thio] -1,3-dihydro-1-oxo-5-isobenzofuranyl] methanesulfonamide,
[358] C32)
[359]
[360] 3-[(2,4-dichlorophenyl) thio] -4-[(methylsulfonyl) amino] benzenesulfonamide,
[361] C33)
[362]
[363] 1-fluoro-4- [2- [4- (methylsulfonyl) phenyl] cyclopenten-1-yl] benzene,
[364] C34)
[365]
[366] 4- [5- (4-chlorophenyl) -3- (difluoromethyl) -1H-pyrazol-1-yl] benzenesulfonamide,
[367] C35)
[368]
[369] 3- [1- [4- (methylsulfonyl) phenyl] -4- (trifluoromethyl) -1H-imidazol-2-yl] pyridine,
[370] C36)
[371]
[372] 4- [2- (3-pyridinyl) -4- (trifluoromethyl) -1H-imidazol-1-yl] benzenesulfonamide,
[373] C37)
[374]
[375] 4- [5- (hydroxymethyl) -3-phenylisoxazol-4-yl] benzenesulfonamide,
[376] C38)
[377]
[378] 4- [3- (4-chlorophenyl) -2,3-dihydro-2-oxo-4-isoxazolyl] benzene sulfonamide,
[379] C39)
[380]
[381] 4- [5- (difluoromethyl) -3-phenylisoxazol-4-yl] benzenesulfonamide,
[382] C40)
[383]
[384] [1,1 ': 2', 1 "-terphenyl] -4-sulfonamide,
[385] C41)
[386]
[387] 4- (methylsulfonyl) -1,1 ', 2], 1 "-terphenyl,
[388] C42)
[389]
[390] 4- (2-phenyl-3-pyridinyl) benzenesulfonamide,
[391] C43)
[392]
[393] N- (2,3-dihydro-1,1-dioxido-6-phenoxy-1,2-benzisothiazol-5-yl) methanesulfonamide,
[394] C44)
[395]
[396] N- [3- (formylamino) -4-oxo-6-phenoxy-4H-1-benzopyran-7-yl] methanesulfonamide,
[397] C45)
[398] ;
[399] C46)
[400] ;
[401] C47)
[402] ;
[403] C48)
[404] ; And
[405] C49) lumiracoxib (COX-189)
[406]
[407] 2-[(2-chloro-6-fluorophenyl) amino] -5-methylbenzeneacetic acid,
[408] C50) etoricoxib (MK 663)
[409]
[410] 5-chloro-6'-methyl-3- [4- (methylsulfonyl) phenyl] -2,3'-bipyridine,
[411] C51) BMS 34070
[412]
[413] More preferred COX-2 inhibitors that can be used in the present invention are selected from the group consisting of the following compounds.
[414] I)
[415]
[416] JTE-522, 4- (4-cyclohexyl-2-methylisoxazol-5-yl) -2-fluorobenzenesulfonamide,
[417] II)
[418] 5-chloro-3- (4- (methylsulfonyl) phenyl) -2- (methyl-5-pyridinyl) pyridine,
[419] III)
[420] 2- (3,5-difluorophenyl) -3-4- (methylsulfonyl) phenyl) -2-cyclopenten-l-one,
[421] IV)
[422]
[423] 4- [5- (4-methylphenyl) -3- (trifluoromethyl) -1 H-pyrazol-1-yl] -benzenesulfonamide,
[424] V)
[425]
[426] Rofecoxib, 4- (4- (methylsulfonyl) phenyl] -3-phenyl-2 (5H) -furanone,
[427] VI)
[428]
[429] 4- (5-methyl-3-phenylisoxazol-4-yl) benzenesulfonamide,
[430] VII)
[431] N-[[4- (5-methyl-3-phenylisoxazol-4yl] phenyl] sulfonyl] propanamide,
[432] VIII)
[433]
[434] 4- [5- (4-chlorophenyl) -3- (trifluoromethyl) -1H-pyrazol-1yl] benzenesulfonamide, and
[435] IX) Lumiracoxib (COX-189)
[436]
[437] 2-[(2-chloro-6-fluorophenyl) amino] -5-methylbenzeneacetic acid.
[438] Even more preferably, COX-2 inhibitors that may be used in the present invention include, but are not limited to, celecoxib, valdecoxib, parecoxib, rofecoxib, lumiracoxib and Japan Tobacco JTE0522.
[439] Also included in the combinations of the present invention are isomeric forms of the described compounds, precursors and tautomers, and pharmaceutically acceptable salts thereof. Examples of exemplary pharmaceutically acceptable salts include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, Benzoic acid, atlanic acid, mesyl acid, stearic acid, salicylic acid, p-hydrobenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, toluenesulfonic acid, It is prepared from 2-hydroxyethanesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, alginic acid, b-hydrobutyric acid, galactaric acid and galacturonic acid.
[440] Usage
[441] Compounds of the invention are inhibitors of protein kinases (PKs) and cyclooxygenase enzymes, in particular cyclooxygenase-2 enzymes, and are therefore useful for the treatment of cancer.
[442] PKs whose catalytic activity is regulated by the compounds of formula (I) of the invention include protein tyrosine kinases such as receptor tyrosine kinases (RTK), cellular tyrosine kinases (CTK) and serine-threonine kinases (STK). RTK mediated signal transduction is achieved by initiation of extracellular interactions with specific growth factors (ligands) followed by dimerization of receptors and transient stimulation and phosphorylation of intrinsic protein tyrosine kinase activity. This allows the binding site to complex with a wide range of cytoplasmic signaling molecules that are made for intracellular signal transduction molecules and that facilitate appropriate cellular responses (eg, cell division, metabolic effects on extracellular microenvironments, etc.). do. See Schlessinger and Ulrich (1992, Neuron , 9: 303-391).
[443] Tyrosine phosphorylation sites on growth factor receptors have been shown to act as binding sites with high affinity for the SH2 (src homologue) of signaling molecules. Fanl et al., 1992, Cell , 69: 413-423, Songyang et al . , 1994, Mol. Cell. Biol. 14: 2777-2785, Songyang et al. (1993, Cell , 72: 767-778) and Koch et al. (1991, Science 252: 668-678). Several intracellular matrix proteins associated with RTK have been identified. They can be divided into two main groups: 1) substrates with catalytic domains, and (2) substrates without these domains but acting as adapters and associated with catalytically active molecules. See Songyang et al., 1993, Cell 72: 767-778. The specificity of the interaction between the receptor and the SH2 domain of these substrates is determined by the amino acid residues immediately surrounding the phosphorylated tyrosine residues. The difference in binding affinity between the SH2 domain and the amino acid sequence surrounding phosphotyrosine residues on a specific receptor is consistent with the difference observed in the substrate phosphorylation profile. See Songyang et al., 1993, Cell 72: 767-778. This observation suggests that the action of each RTK is determined by its expression pattern and ligand availability as well as the arrangement of downstream signal transduction pathways activated by specific receptors. Thus, phosphorylation provides an important regulatory step to determine the selectivity of signaling pathways complemented by differentiation factor receptors as well as specific growth factor receptors.
[444] STK, which is predominantly cytoplasmic, often affects the cell's internal biochemistry as a down-line response to PTK events. STK has been involved in the signaling process of proliferating cells by initiating DNA synthesis and subsequent mitosis.
[445] Thus, PK signal transduction leads to cell proliferation, differentiation, growth and metabolism, among other reactions. Abnormal cell proliferation may include the development of neoplasms (e.g. carcinomas, sarcomas, gliomas and hemangiomas), diseases (e.g. leukemia, psoriasis, atherosclerosis, arthritis and diabetic retinopathy) and uncontrolled angiogenesis and / or It can cause a wide range of diseases and disorders, including other diseases associated with angiogenesis.
[446] It is not necessary to accurately understand the mechanism by which the compounds of the present invention inhibit PK in order to practice the present invention. However, without being bound by any particular mechanism or theory, it is believed that the compounds of formula (I) will interact with amino acids in the catalytic region of PK. PK has a typical bilobal structure that appears to bind ATP to the valley between two lobes, the region where amino acids are conserved between PKs. Inhibitors of PK appear to be bound by non-covalent interactions such as hydrogen bonding, van der Waals forces and ionic interactions in the same overall region where ATP binds to PK. More specifically, it is believed that the 2-indolinone element of the compound of formula (I) normally binds to the overall space occupied by the adenine ring of ATP. If so, the specificity of a particular molecule for a particular PK may arise as a result of further interactions between the various substituents on the 2-indolinone core and the specific amino acid domain for that particular PK. Thus, different indolinone substituents may contribute to preferential binding to a particular PK. The ability to select compounds that are active at different ATP (or other nucleotide) binding sites makes the compounds of formula (I) useful for targeting any protein having such sites. Thus, the compounds of formula (I) disclosed herein not only exhibit therapeutic effects in vivo through interaction with such proteins, but also have utility in in vitro assays for such proteins.
[447] In addition, the compounds of formula (I) may be used for the treatment of many types of solid tumors, including but not limited to carcinomas, sarcomas (including Kaposi's sarcoma), erythromatoma, glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma Provide a therapeutic approach. Treatment or prevention of non-solid tumor cancers such as leukemia is also contemplated by the present invention. Symptoms include, but are not limited to, brain cancer, bladder cancer, ovarian cancer, gastric cancer, pancreatic cancer, colon cancer, blood cancer, lung cancer, and bone cancer.
[448] Further examples of the types of diseases associated with inappropriate PK activity in which the compounds of the present invention may be useful for prevention, treatment, and research include, but are not limited to, cell proliferative diseases, fibrotic diseases, and metabolic diseases.
[449] Cell proliferative diseases that can be prevented, treated or further studied by the present invention include cancer, vascular proliferative diseases and glomerular interstitial cell proliferative diseases.
[450] Vascular proliferation disease refers to a disease associated with abnormal angiogenesis (angiogenesis) and angiogenesis (diffusion of blood vessels). Angiogenesis and angiogenesis play an important role in a variety of normal physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration, but they also result in the development of cancers that form new capillaries that are needed to keep tumors alive. Even plays a pivotal role. Other examples of vascular proliferative diseases include arthritis (where fresh capillaries invade joints and destroy cartilage) and diabetic retinopathy (where new capillaries in the retina invade the vitreous, causing bleeding and blindness). It is the same eye disease.
[451] Two structurally related RTKs were identified that bind VEGF with high affinity: fms type tyrosine 1 (fit-1) receptor (Shibuya et al., 1990, Oncogene , 5: 519-524 De Vries et al. (1992, Science , 255: 989-991), and the KDR / FLK-1 receptor, also known as VEGF-R2. Vascular endothelial growth factor (VEGF) has been reported as a mitotic substance specific for endothelial cells having endothelial cell growth promoting activity in vitro. Ferrara and Henzel, 1989, Biochem, Biophys. Res. Comm. 161: 851-858; Vaisman et al . , 1990, J. Biol. Chem. , 265: 19461-19566. The information disclosed in US patent applications 08 / 193,829, 08 / 038,596 and 07 / 975,750 strongly suggest that VEGF not only causes endothelial cell proliferation, but also is a major regulator of normal angiogenesis and pathological angiogenesis. Suggest. In general, Klagsburn and Soker et al., Current Biology , 3 (10) 699-702; Houck et al . , 1992, J. Biol. Chem. , 267: 26031-26037.
[452] Normal angiogenesis and angiogenesis play an important role in various physiological processes such as embryonic development, wound healing, period regeneration and female reproductive processes (for example, follicle growth in the corpus luteum during ovulation and placental growth after pregnancy). . Folkman and Singh, 1992, J. Biological Chem. , 267 (16): 10931-34. Uncontrolled angiogenesis and / or angiogenesis is associated with diseases such as diabetes, as well as malignant solid tumors that rely on angiogenesis for growth. Clasburn and Soker (1993, Current Biolgoy , 3 (10): 699-702); Folkman , 1991, J. Natl. Cancer Inst. , 82: 4-6; Weidner et al . , 1991, New Engl. J. Med. , 324: 1-5.
[453] The presumed role of VEGF in the proliferation and migration of endothelial cells during angiogenesis and angiogenesis suggests an important role for KDR / FLK-1 receptors in these processes. Diseases, such as diabetes (Forkman, 198, XI ^th Congress of Thrombosis and Haemostasis (Verstraeta et al., Eds.) Pp. 583-596, Leuven University Press, Leuven), and arthritis, also malignant Tumor growth may be the result of uncontrolled angiogenesis. See, eg, Folkman , 1971, N. Engl. J. Med. , 285: 1182-1186. Receptors to which VEGF specifically binds are important and powerful targets for the regulation and coordination of several serious diseases including angiogenesis and / or angiogenesis and abnormal cell growth caused by these processes. See, eg, Plowman et al., 1994, DN & P , 7 (6): 334-339. More specifically, the highly specific role of the KDR / FLK-1 receptor in new angiogenesis makes it an excellent target for therapeutic approaches to the treatment of cancer and other diseases, including the uncontrolled formation of blood vessels.
[454] Accordingly, the present invention provides compounds capable of controlling and / or tuning tyrosine kinase signal transduction, including KDR / FLK-1 receptor signal transduction, to inhibit or promote angiogenesis and / or angiogenesis, ie ligands such as VEGF. Provided are compounds that, when activated by, inhibit, prevent or interfere with signal transduction by KDR / FLK-1. Although the compounds of the present invention are believed to act on receptors or other elements along tyrosine kinase signaling pathways, they may also act directly on tumor cells that are the result of uncontrolled angiogenesis.
[455] Although the names of human and rat counterparts of the generic "flk-I" receptor are different, they are interchangeable in many respects. The murine receptor Flk-1 and its human counterpart KDR share 93.4% sequence homology within the intracellular domain. Similarly, murine FLK-1 binds to human VEGF with the same affinity as murine VEGF and is thus activated by ligands derived from either species. Milluer et al., 1993, Cell , 72: 835-846; Quinn et al . , 1993, Proc. Natl. Acad. Sci. USA , 90: 7533-7537. FLK-1 also phosphorylates tyrosine after binding to human RTK substrates (eg PLC-γ or p85) when co-expressed in 293 cells (human embryonic kidney fibroblasts).
[456] Thus, models that depend on the FLK-1 receptor can be directly applied to understanding the KDR receptor. For example, the use of murine FLK-1 receptors in methods of identifying compounds that modulate murine signaling pathways has led to the identification of compounds that can be used to modulate human signaling pathways, i.e. compounds that modulate activity associated with KDR receptors. Can be applied directly to Thus, chemical compounds identified as ex vivo inhibitors of KDR / FLK-1 can be identified in a suitable in vivo model. Both in vivo mouse and rat animal models have proven to be of great value in investigating the clinical potential of drugs acting on KDR / FLK-1 induced signal transduction pathways.
[457] Accordingly, the present invention provides compounds that affect, regulate and / or inhibit angiogenesis and / or angiogenesis by affecting the enzyme activity of the KDR / FLK-1 receptor and interfering with signal transduction by KDR / FLK-1. do. Accordingly, the present invention provides therapeutics for the treatment of many types of solid tumors, including but not limited to glioblastoma, melanoma and Kaposi's sarcoma, and ovarian cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, colon cancer and epidermal carcinoma. Provide an approach. In addition, the data suggest that administration of compounds that inhibit KDR / Flk-1 mediated signal transduction pathways can also be used to treat hemangiomas, restenosis and diabetic retinopathy.
[458] The invention also relates to inhibition of angiogenesis and angiogenesis by other receptor-mediated pathways, including pathways comprising flt-1 receptors.
[459] Receptor tyrosine kinase mediated signal transduction is initiated by extracellular interactions with specific growth factors (ligands), followed by dimerization of the receptors, transient promotion of native protein tyrosine kinase activity and autophosphorylation. Thereby binding sites are made for intracellular signal transduction molecules, which result in complex formation with a wide range of cytoplasmic signal molecules that facilitate appropriate cellular responses (eg, metabolic effects on cell division and extracellular microenvironment). . See Schlesinger and Ulrich (1992, Neuron, 9: 1-20).
[460] The close homology of the intracellular regions of KDR / FLK-1 and PDGF-β receptors (50.3% homology) and / or associated flt-1 receptors indicates induction of overlapping signal transduction pathways. For example, for PDGF-β receptors, members of the src family (Twamley et al . , 1993, Proc. Natl. Acad / Sci . USA, 90: 7696-7700), phosphatidylinositol-3'- Kinases (Hu et al . , 1992, Mol. Cell. Biol. , 12: 981-990), phospholipase cγ (Kashishian and Cooper, 1993, Mol. Cell) Biol., 4: 49-51]), ras-GTPase activating protein (Cassician et al. [ EMBO J., 11: 1373-1382]), PTP-ID / syp (Kazlauskas et al. 1993, Proc. Natl. Acad. Sci. USA , 10 90: 6939-6943), Grb2 (Arvidsson et al . , 1994, Mol. Cell. Biol. , 14: 6715-6726 ] And the adapter molecules Shc and Nck (Nishimura et al . (1993, Mol. Cell. Biol. , 13: 6889-6896)) have been shown to bind to regions containing different autophosphorylation sites. In general, Claeson-Welsh, 1994, Prog. Growth Factor Res. , 5: 37-54. Thus, signal transduction pathways activated by KDR / FLK-1 are probably the ras pathway (Rozakis et al. , 1992, Nature , 360: 689-692), PI-3′-kinase, arc-mediated. Pathway and plcγ-mediated pathway. These pathways may each play an important role in the angiogenic and / or angiogenic effects of KDR / FLK-1 in endothelial cells. In conclusion, another aspect of the present invention relates to the use of the organic compounds disclosed herein to modulate angiogenesis and angiogenesis regulated by these pathways.
[461] Conversely, diseases associated with shrinkage, contraction or closure of blood vessels, such as restenosis, are also contemplated and can be treated or prevented by the methods of the invention.
[462] Fibrosis disease refers to abnormal formation of the extracellular matrix. Examples of fibrotic diseases include cirrhosis of the liver and glomerular interstitial cell proliferation. Liver cirrhosis is characterized by an increase in extracellular matrix components resulting in scarring of the liver. The increase in extracellular matrix that produces scarring of the liver can also be due to viral infections such as hepatitis. Fat cells play an important role in cirrhosis. Other fibrotic diseases that are expected include atherosclerosis.
[463] Glomerular interstitial cell proliferative disease means a disease caused by abnormal proliferation of glomerular interstitial cells. Glomerular interstitial proliferative diseases include various human kidney diseases, such as glomerulonephritis, diabetic nephropathy and malignant neurosis, and also diseases such as thrombotic microvascular disorder syndrome, transplant rejection and glomerulopathy. RTK PDGFR has been linked to maintenance of glomerular interstitial cell proliferation. Floege et al., 1993, Kidney International 43: 47S-54S.
[464] Many cancers are cell proliferative diseases, and as mentioned previously, PK is associated with cell proliferative diseases. Thus, it is not surprising that members of the PK family, such as the RTK family, are involved in the development of cancer. Some of these receptors are described, for example, in EGFR (Tuzi et al . , 1991, Br. J. Cancer 63: 227-233, Torp et al., 1992, APMIS 100: 713-719). ), HER2 / neu (Slamon et al. (1989, Science 244: 707-712)) and PDGF-R (Kumabe et al. 1992, Oncogene , 7: 627-633) Is overexpressed in many tumors and / or continuously activated by a self-secreting loop. Indeed, in most common and severe cancers, overexpression of these receptors (Akbasak and Suner-Akbasak et al . , 1992, J. Neurol. Sci. , 111: 119-133), Dickson ( Dickson et al . , 1992, Cancer Treatment Res. 61: 249-273, Corc et al. , 1992, J. Clin. Invest . 90: 1352-1360, and self-secreting loops (Lee). ) And Donoghue's (1992, J. Cell. Biol. , 118: 1057-1070), Cork et al . , Above, such as Akbasak and Sunner-Arkbaksak). . For example, EGFR is associated with squamous cell carcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancer and bladder cancer. HER2 is associated with breast cancer, ovarian cancer, gastric cancer, lung cancer, pancreatic cancer and bladder cancer. PDGFR is associated with gliomas and melanoma, and also lung cancer, ovarian cancer and prostate cancer. RTK c-met is also associated with malignant tumor formation. For example, c-met is associated with colorectal cancer, thyroid cancer, pancreatic cancer, gastric cancer and hepatocellular carcinoma and lymphoma, among other cancers. In addition, c-met is associated with leukemia. Overexpression of the c-met gene has also been detected in patients with Hawkinson's and Burkitts' disease.
[465] In addition to being associated with nutritional support and type II diabetes, IGF-IR is also associated with several types of cancer. For example, IGF-I has been described in human breast cancer carcinoma cells (Arteaga et al. (1989, J. Clin. Invest . 84: 1418-1423)) and small lung cancer cells (Macauley et al. 1990, Cancer Res. , 50: 2511-2517], is involved as a self-distributing growth promoter for several tumor types. In addition, IGF-I appears to be an integral part of normal growth and differentiation of the nervous system and to be a self-distributing promoter of human glycoma. Sandberg-Nordqvist et al . , 1993, Cancer Res. 53: 2475-2478. The importance of IGF-IR and its ligands in cell proliferation is due to the fact that many cell types (fibroblasts, endothelial cells, smooth muscle cells, T-lymphocytes, bone marrow cells, chondrocytes and osteoblasts (stem cells of the bone marrow) in cultures have been identified as IGF-IR. It is supported by the fact that it is stimulated and grown by I. See Goldring and Goldring, 1991, Eukaryotic Gene Expression , 1: 301-326. Baserga and Coppola suggest that IGF-IR plays a pivotal role in the mechanism of transformation and may therefore be a desirable target for therapeutic intervention in a wide range of human malignancies. Waserga, 1995, Cancer Res. , 55: 249-252, Waserga et al. (1994, Cell , 79: 927-930), Coppola et al . , 1994, Mol. Cell. Biol. , 14: 4588-4595.
[466] STK has been associated with many types of cancer, especially breast cancer (Cance et al. , Int. J. Cancer , 54: 571-77 (1993)).
[467] The association between abnormal PK activity and disease is not limited to cancer. For example, RTK may include psoriasis, diabetes, endometriosis, angiogenesis, atheromatous plaque development, Alzheimer's disease, restenosis, von Hippel-Lindau disease, epidermal hyperplasia, neurodegenerative diseases, age and Associated spot degeneration and hemangiomas have been associated. For example, EGFR appears in the treatment of corneas and skin wounds. Defects in insulin-R and IGF-1R appear in type II diabetes. A more complete correlation between specific RTKs and their therapeutic regimens is disclosed in Plowman et al. (1994, DN & P 7: 334-339).
[468] As previously mentioned, CTK (Bolen et al., 1992, including but not limited to RTK as well as src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk) , FASEB J. , 6: 3403-3409) are involved in proliferation and metabolic signal transduction pathways and, therefore, may be expected to be involved in many PTK-mediated diseases of interest in the present invention. For example, the mutated src (v-src) appears to be a tumor protein (pp60 ^v-src ) in chickens. Moreover, its cell homologue, the proto-oncogene pp60 ^c-src , carries oncogenic signals of many receptors. In tumors, pp60 ^c-src is constitutively activated by overexpression of EGFR or HER2 / neu, a characteristic of malignant cells not found in normal cells. On the one hand, mice lacking the expression of c-src exhibit an osteophysogenic phenotype, indicating a possible association in disease associated with the crucial participation of c-src in osteoclast function.
[469] Similarly, Zap70 is involved in T-cell signaling that may be associated with autoimmune disease.
[470] STK is associated with inflammation, autoimmune diseases, immune responses and hyperproliferative diseases such as restenosis, fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis.
[471] PK is also involved in embryo implantation. Accordingly, the compounds of the present invention can provide an effective method for preventing the implantation of such embryos, and are useful as birth control agents. Further diseases that can be treated or prevented using the compounds of the present invention are immune diseases such as autoimmune diseases, AIDS and cardiovascular diseases such as atherosclerosis.
[472] Finally, it is assumed that both RTK and CTK are currently involved in highly immune disease.
[473] Examples of the effects of many exemplary compounds of the invention on various PTKs are shown in Table 2 below. The compounds and data presented should not be construed as limiting the scope of the invention in any way.
[474] B. Cyclooxygenase-2 inhibitors or COX-2 inhibitors or cyclooxygenase-2 inhibitors specifically inhibit cyclooxygenase-2, a class of enzymes, and less inhibitory cyclooxygenase-1 Contains drugs. Preferably, it has a cyclooxygenase-2 IC ₅₀ of less than about 0.2 μM and also selectively inhibits cyclooxygenase-2 at least 50-fold, preferably at least 100-fold over cyclooxygenase-1. Compound. Even more preferably, the compound has a cyclooxygenase-1 IC ₅₀ of at least about 1 μM, more preferably at least 10 μM.
[475] Studies show that prostaglandins synthesized by cyclooxygenase play a critical role in the initiation and promotion of cancer. COX-2 is also overexpressed in neoplastic lesions of the colon, breast, lung, prostate, esophagus, pancreas, intestine, cervix, ovary, bladder and head and neck. COX-2 inhibitors have inhibited tumor growth and metastasis in several ex vivo and animal models.
[476] In addition to the cancer itself, COX-2 is also expressed in the neovascularized vessels within and adjacent to hyperplastic and tumorigenic lesions, indicating that COX-2 plays an important role in angiogenesis. In both mice and rats, COX-2 inhibitors significantly inhibit bFGF-induced angiogenesis. The use of COX-2 inhibitors as chemoprophylactic agents, angiogenesis inhibitors and chemotherapeutic agents is described in Koki et al., Potential utility of COX-2 inhibitors in chemoprevention and chemotherapy. Exp. Opin. Invest. Drugs (1999) 8 (10) pp. 1623-1638. Amplification and / or overexpression of HER-2 / nue (ErbB2) occurs in 20-30% of human breast and ovarian cancers, and also 5-15% of gastric and esophageal cancers, and is associated with a poor prognosis. In addition, we have recently discovered in vitro that COX-2 expression is upregulated in cells overexpressing the HER-2 / neu oncogene (Increased expression of cyclooxygenase-, Subbaramaiah et al., Herein incorporated by reference). 2 in HER-2 / neu-overexpressing breast cancer.Cancer Research (1999)]. In this study, significantly increased levels of PGE ₂ production, COX-2 protein and mRNA were detected in NER-2 / neu transformed breast epithelial cells compared to non-transformed corresponding cell lines. The product of COX-2 activity, ie prostaglandins, promotes proliferation, increases the invasiveness of malignant cells, increases the production of vascular endothelial growth factors, which promote angiogenesis. HER-2 / neu also induces the production of angiogenic factors such as vascular endothelial growth factor.
[477] As a result, the use of a COX-2 inhibitor in combination with an anti HER-2 / neu antibody, such as trastuzumab®, Herceptin, and other therapeutic agents intended to inhibit HER-2 / neu Administration is expected to treat cancer in which HER-2 / neu is overexpressed.
[478] In addition, COX-2 levels are increased in tumors where other oncogenes are amplified and / or overexpressed, including but not limited to c-myc, N-myc, L-myc, K-ras, H-ras, N-ras. It is expected to increase. Products of COX-2 activity promote cell proliferation, inhibit immune supervision, increase the invasiveness of malignant tumor cells, and promote neovascularization. In conclusion, administration of the protein kinase inhibitor in combination with the COX-2 inhibitor of the present invention is useful for preventing or treating cancers in which the oncogene is overexpressed.
[479] Certain COX-2 inhibitors are useful for the treatment of cancer (WO98 / 16227) and reduce angiogenesis induced by various growth factors in several animal models (WO98 / 22101). Anti-angiogenesis is achieved using COX-2 inhibitors in rats implanted with bFGF, vascular endothelial growth factor (VEGF) or carrageenan (a protein with well-known angiogenic properties) (Masferrer et al. (89 ^th Annual Meeting of the American Association for Cancer Reserach, March 1998).
[480] Pharmaceutical Compositions and Administration
[481] The compounds of the present invention or pharmaceutically acceptable salts thereof may be administered to a human patient as they are, or may be administered in a pharmaceutical composition in which the aforementioned materials are mixed with a suitable carrier or excipient. Techniques for the preparation and administration of drugs can be found in "Remington's Pharmacological Sciences", Mack Publishing Co., Easton, PA, latest edition.
[482] As used herein, "administration" refers to a pharmaceutical composition containing a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a compound of formula (I) or a pharmaceutically acceptable salt thereof of the present invention for the prevention of PK-associated diseases or Means delivery into raw vegetables for treatment.
[483] Suitable routes of administration include oral, rectal, topical, transmucosal or visceral administration, intramuscular, subcutaneous, intramedullary, intradermal, direct intraventricular, intravascular, intravitreal, intraperitoneal, intranasal or intraocular injections, but It is not limited. Preferred routes of administration are oral and parenteral.
[484] Alternatively, the compound may often be administered in a topical rather than systemic manner, for example via direct infusion of the compound into a solid or sustained release formulation.
[485] The drug can also be administered in a target drug delivery system, such as liposomes coated with tumor-specific antibodies. Liposomes are targeted to tumors and selectively absorbed.
[486] The pharmaceutical compositions of the present invention may be prepared through processes well known in the art, such as conventional mixing, dissolving, granulating, dragging-making, grinding, emulsifying, encapsulating, capturing or lyophilizing processes.
[487] Pharmaceutical compositions for use according to the invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Can be. Proper formulation will depend on the route of administration chosen.
[488] For injection, the compounds of the present invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution or physiological saline buffer. For transmucosal administration, penetrants suitable for the permeable barrier are used in the formulation. Such penetrants are generally known in the art.
[489] For oral administration, the compounds may be prepared by mixing the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers allow the compounds of the present invention to be formulated in tablets, pills, lozenges, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like for ingestion by the patient. Pharmaceutical formulations for oral use can be prepared by using solid excipients, optionally grinding the resulting mixture, optionally adding other suitable auxiliaries and then processing the mixture of granules to obtain a tablet or dragee core. . Useful excipients are especially for example sugars (eg lactose, sucrose, mannitol or sorbitol), cellulose preparations (eg corn starch, wheat starch, rice starch and sweet potato starch) and other substances such as gelatin, Fillers comprising tragacanth rubber, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinyl pyrrolidone (PVP). If desired, disintegrants such as crosslinked polyvinyl pyrrolidone, agar or alginic acid can be added. Salts such as sodium alginate can also be used.
[490] Dragee cores are provided with suitable coatings. Concentrated sugar solutions can be used for this purpose, which optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. can do. Dyestuffs or pigments may be added to the tablets or dragee coatings to identify and characterize different combinations of dosages of the active compounds.
[491] Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, and also soft sealed capsules made of gelatin and a plasticizer (eg glycerol or sorbitol). Push-fit capsules may contain the active ingredient in admixture with a filler (eg lactose), a binder (eg starch) and / or a lubricant (eg talc or magnesium stearate) and optionally a stabilizer. . In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin or liquid polyethylene glycols. Stabilizers may also be added to these formulations.
[492] Capsules may be packaged in brown glass or plastic bottles to protect the active ingredient from light. Containers containing the active compound capsule formulation must be stored at controlled room temperature (15-30 ° C.).
[493] For administration by inhalation, the compounds according to the invention are conveniently condensed packs or nebulizers and suitable propellants (such as, but not limited to, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetra-fluoroethane or carbon dioxide). Is delivered in the form of an aerosol spray. In the case of a compressed aerosol, the dosage unit can be adjusted by providing a valve to deliver a measurand. For example, capsules and cartridges of gelatin for use in an inhaler or blower may be formulated containing a mixed powder of the compound and a suitable powder base (eg, lactose or starch).
[494] In addition, the compounds may be formulated for parenteral administration, eg by bolus injection or continuous infusion. Injectable formulations may be presented in unit dosage form, such as ampoules or in multi-dose containers, with an added preservative. The compositions may take the form of suspensions, solutions or emulsions in oil or aqueous carriers and may contain preparations such as suspensions, stabilizers and / or dispersants.
[495] Pharmaceutical compositions for parenteral administration include, for example, aqueous solutions in water-soluble forms, including but not limited to salts of the active compounds. In addition, suspensions of the active compounds may be prepared with lipophilic vehicles. Suitable lipophilic vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate and triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain agents which increase the solubility of the compound to enable the preparation of suitable stabilizers and / or highly concentrated solutions.
[496] Alternatively, the active ingredient may be in powder form consisting of a suitable carrier such as sterile pyrogen-free water before use.
[497] The compounds may also be formulated in rectal compositions such as suppositories or delayed enema using, for example, conventional suppository bases such as cocoa butter or other glycerides.
[498] In addition to the formulations described above, the compounds may also be formulated as depot formulations. Such long acting formulations may be administered by implantation (such as subcutaneously or intramuscularly) or by intramuscular injection. Compounds of the invention may be used for such routes of administration using suitable polymeric or hydrophobic materials (such as emulsions with pharmacologically acceptable oils) with ion exchange resins or sparingly soluble derivatives (such as sparingly soluble). Salts, but not limited thereto).
[499] Non-limiting examples of pharmaceutical carriers for the hydrophobic compounds of the present invention are cosolvent systems comprising an aqueous phase, such as benzyl alcohol, nonpolar surfactants, water-miscible organic polymers, and VDP co-solvent systems. VDP is 3% w / v benzyl alcohol, 8% w / v nonpolar surfactant Polysorbate 80, and 65% w / v polyethylene glycol 300 (made up in volume of anhydrous ethanol). The VDP co-solvent system (VDP: D5W) consists of VDP diluted 1: 1 with 5% dextrose in aqueous solution. This co-solvent system dissolves hydrophobic compounds well and itself produces low toxicity upon system administration. Naturally, the ratio of these co-solvent systems can vary considerably without destroying solubility and toxicity properties. Moreover, the identification of co-solvent components can vary, for example low-toxic nonpolar surfactants can be used in place of polysorbate 80, the fraction size of polyethylene glycol can be varied, and other biocompatible polymers can be Glycols such as polyvinyl pyrrolidone may be substituted and other sugars or polysaccharides may be substituted for dextrose.
[500] Alternatively, other delivery systems for hydrophobic pharmaceutical compounds can be used. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. In addition, certain organic solvents, such as dimethylsulfoxide, may also be used, often at the expense of greater toxicity.
[501] In addition, the compounds can be delivered using sustained release systems, such as semipermeable matrices of solid hydrophobic polymers containing the healing agent. Various sustained release materials have been established and are well known by those skilled in the art. Sustained release capsules can release the compounds depending on their chemical properties for weeks to over 100 days. Depending on the chemical properties and biological stability of the therapeutic reagent, additional silver methods for protein stabilization can be used.
[502] The pharmaceutical compositions herein may also include suitable solid or gel carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycol.
[503] Many of the PK tuning compounds of the present invention may be provided as pharmacologically acceptable salts in which the claimed compounds may form negatively or positively charged species. Examples of salts in which the compounds form negatively charged residues include quaternary amines (as defined elsewhere herein), salts such as hydrochlorides, sulfates, carbonates, lactates, tartrates, maleates, maleates, succinates Including but not limited to (the nitrogen atom of the quaternary amine is the nitrogen of selected compounds of the present invention that react with a suitable acid). Salts in which the compounds of the present invention form negatively charged species include a suitable base (e.g., sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium hydroxide (Ca (OH) ₂ ), etc.) and a carboxylic acid group in the compound. Include, but are not limited to, sodium, potassium, calcium and magnesium salts formed by the reaction.
[504] Pharmaceutical compositions suitable for use in the present invention include compositions containing the active ingredient in an amount sufficient to achieve the intended purpose, such as coordination of PK activity or treatment or prevention of PK-related diseases.
[505] More specifically, a therapeutically effective amount means an amount effective to prevent, alleviate or ameliorate the symptoms of a disease or prolong the survival of a patient being treated.
[506] A therapeutically effective amount can be well defined within the capabilities of those skilled in the art, in particular in light of the detailed disclosure provided herein.
[507] For any compound used in the methods of the invention, the therapeutically effective amount or dosage can be assessed initially from cell culture assays. The dosage can then be formulated for use in an animal model to achieve a range of circulating concentrations that includes an IC ₅₀ (i.e., the concentration of test compound that achieves half of the maximum inhibition of PK activity) measured in cell culture. This information can then be used to more accurately determine useful administration to humans.
[508] Toxicity and therapeutic efficacy of the compounds described herein can be measured by measuring standard pharmaceutical procedures in cell culture or experimental animals, such as IC ₅₀ and LD ₅₀ (both disclosed elsewhere herein) for the compound of interest. . Data obtained from these cell culture assays and animal studies can be used to formulate a range of dosage for use in humans. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the physician's individual consideration of the patient's condition. (See Fingl et al., "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1 (1975)).
[509] Dosage and interval may be individually adjusted to provide sufficient plasma levels of active species to maintain kinase tuning effects. This plasma level is referred to as minimum effect concentration (MEC). The MEC will vary for each compound but can be assessed from ex vivo data, such as concentrations necessary to achieve 50-90% inhibition can be identified using the assays described herein. Dosages required to achieve the MEC will depend on the individual characteristics and route of administration. HPLC assays or bioassays can be used to measure plasma concentrations.
[510] Dosage intervals can also be determined using MEC values. The compound may be administered using a regimen that maintains the MEC at the plasma level for a time of 10 to 90%, preferably 30 to 90%, most preferably 50 to 90%.
[511] At present, the therapeutically effective amount of the compound of formula (I) may be approximately 0.25-1500 mg / m 2 / day, preferably about 3 mg / m 2 / day, even more preferably at 50 mg / qm qd at 400 mg / qd.
[512] The therapeutically effective amount of a cyclooxygenase inhibitor is about 0.1 to about 10,000 mg for the treatment of these indications, and the preferred level is about 1.0 to about 1,000 mg.
[513] The amount of active ingredient that can be combined with other anticancer agents to produce a single dosage form will vary depending upon the recipient treated and the particular mode of administration.
[514] In the case of topical administration or selective absorption, the effective local concentration of the drug may not be related to the plasma concentration, and other procedures known in the present invention may be used to define the correct dosage and interval.
[515] Of course, the amount of composition administered may vary depending on the patient being treated, the severity of the pain, the mode of administration, the judgment of the attending physician and the like.
[516] The composition may be present in a pack or dispenser device, such as an FDA approved kit, containing one or more unit dosage forms containing the active ingredient as desired. The pack may, for example, comprise a metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. In addition, the pack or dispenser may be accompanied by a notice relating to the container in a form defined by a governmental agency prescribing the manufacture, use or sale of the pharmaceutical product, which notice may be provided by an organ in the form of a composition of human or veterinary administration. It reflects approval. Such notice may be, for example, a label approved by the US Food and Drug Administration for prescription drugs of the insert, or it may be an approved product insert. Compositions comprising a compound of the invention formulated with a miscible pharmaceutical carrier are also prepared and placed in appropriate containers and labeled for the treatment of the indicated indications. Suitable indications indicated on the label may include tumors, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like.
[517] It is also an aspect of the present invention that the compounds, salts or prodrugs thereof described herein may be combined with other chemotherapeutic agents for the treatment of the diseases and disorders described above. For example, the compounds, salts or prodrugs of the present invention may be combined with alkylating agents such as fluorouracil (5-FU) alone or further in combination with leuboline; Or other pyrimidine analogs (eg, UFT, capecitabine, gemcitabine and cytarabine), alkyl sulfonates such as busulfan (used to treat chronic granulocytic leukemia), improsulfan and pifosulfan; Aziridines such as benzodepa, cabocuone, meturedepa and uredepa; Ethyleneimines and methylmelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethyleneethiophosphoramide and trimethylolmelamine; And nitrogen mustards such as chlorambucil (used to treat chronic lymphocytic leukemia, primary macroglobulinemia and non-Hodgkin's lymphoma), cyclophosphamide (Hodgkin's disease, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, Used in the treatment of lung cancer, Wilm's tumor and rhabdomyosarcoma), esturamustine, ifosfamide, norshambrin, prednisostin and uracil mustard (primary thrombocytopenia, non-Hodgkin's lymphoma, Hodgkin's disease and ovarian cancer) Used for); And triazines such as dacarbazine (used for the treatment of soft tissue sarcoma), but may be combined with alkylating agents.
[518] Compounds, salts or prodrugs of the invention include, but are not limited to, folic acid analogs such as methotrexate (used in the treatment of acute lymphocytic leukemia, choriocarcinoma, myxosarcoma breast cancer, head and neck cancer, and osteosarcoma); And purine analogues known to be used for the treatment of acute granulocytic leukemia, acute lymphocytic leukemia and chronic granulocytic leukemia such as mercaptopurine and thioguanine, to be used in combination with other metabolic chemotherapeutic agents. It may be.
[519] Compounds, salts or prodrugs of the invention also include vinblastine (used in the treatment of breast and testicular cancer), vincristine and vindesine; Epipodopyrrotocins such as etoposide and teniposide (both useful for the treatment of testicular cancer and Kaposi's sarcoma); Antibiotic chemotherapeutic agents such as daunorubicin, doxorubicin, epirubicin, mitomycin (used to treat stomach cancer, cervical cancer, colon cancer, breast cancer, bladder cancer and pancreatic cancer), dactinomycin, temozolomide, flakamycin, bleomycin (Used to treat cancer in the skin, esophagus and fertilizer); And enzyme chemotherapeutic agents such as L-asparaginase, but may be used in combination with natural product chemotherapeutic agents.
[520] In addition to the above, the compounds, salts or prodrugs of the present invention may also include platinum coordination complexes (cisplatin and the like); Substituted ureas such as hydroxyurea; Methylhydrazine derivatives such as procarbazine; Corticosteroids such as mitotans, aminoglutetimides; Hormones and hormonal antagonists such as corticosteroids (eg prednisone), progesterone (eg hydroxyprogesterone caproate); Estrogens such as diethylstilbestol; Antiestrogens such as tamoxifen; Androgens such as testosterone propionate; And aromatase inhibitors such as anastrozole.
[521] Other anti-neoplastic agents known in the art can be used in combination with the combination therapy of the present invention. Examples of such anti-neoplastics and other anti-neoplastics are described in US patent application Ser. No. 60 / 113,786 filed December 23, 1998; And PCT Application Publication No. WO 00/38716, the disclosures of which are incorporated herein by reference.
[522] Finally, the combinations of the compounds of the present invention will be effective in combination with mitosantron for the treatment of leukemias including but not limited to solid tumor cancer or acute myeloid (non-lymphoid) leukemia.
[523] Synthetic Example
[524] The following preparations and examples are provided to enable those skilled in the art to more clearly understand and to practice the present invention. It should not be considered as limiting the scope of the invention, but should only be considered as exemplary and representative.
[525] Synthesis of Protein Kinase Inhibitors of Compounds of Formula (I) of the Invention
[526] General Synthetic Procedures:
[527] The following general methodology can be used to prepare the compounds of the present invention.
[528] Appropriately substituted 2-oxindol (1 equiv), suitably substituted aldehyde (1.2 equiv) and base (0.1 equiv) were mixed in a solvent (1-2 mL / mmimolar 2-oxyndol), and then the mixture was about 2-2 Heat for about 12 hours. After cooling, the precipitate formed is filtered off, washed with cold ethanol or ether and dried in vacuo to give a solid product. If no precipitate is formed, the reaction mixture is concentrated, the residue is triturated with dichloromethane / ether and the resulting solid is collected by filtration and then dried. The product may optionally be further purified by chromatography.
[529] The base can be an organic or inorganic base. If an organic base is used, it is preferably a nitrogen base. Examples of organic nitrogen bases include diisopropylamine, trimethylamine, triethylamine, aniline, pyridine, 1,8-diazabicyclo [5.4.1] undec-7-ene, pyrrolidine and piperidine. It may be, but is not limited to such.
[530] Examples of inorganic bases include, but are not limited to, ammonia, alkali metal or alkaline earth metal hydroxides, phosphates, carbonates, bicarbonates, bicarbonates and amides. Alkali metals include lithium, sodium and potassium, and alkaline earth metals include calcium, magnesium and barium.
[531] In a presently preferred embodiment of the invention, when the solvent is a protic solvent such as water or alcohol, the base is an alkali metal or alkaline earth metal inorganic base, preferably an alkali metal or alkaline earth metal hydroxide.
[532] In accordance with both the general principles of known organic synthesis and the disclosure herein, it will be apparent to those skilled in the art that the most suitable base for the reaction is considered.
[533] The solvent to carry out the reaction may be a protic or aprotic solvent, preferably a protic solvent. A “protic solvent” is a solvent that has hydrogen atom (s) covalently bonded with oxygen or nitrogen atoms that can be “covalent” with the solute through hydrogen bonding by causing the hydrogen atoms to be detectably acidic.
[534] The "aprotic solvent" may be polar or nonpolar, but in no case contains acidic hydrogen and therefore hydrogen cannot bind to the solute. Examples of nonpolar aprotic solvents include, but are not limited to, pentane, hexane, benzene, toluene, methylene chloride and carbon tetrachloride. Examples of polar aprotic solvents are chloroform, tetrahydrofuran, dimethylsulfoxide and dimethylformamide.
[535] In a presently preferred embodiment of the invention, the solvent is a protic solvent, preferably water or an alcohol such as ethanol.
[536] The reaction is carried out at temperatures above room temperature. The temperature is generally about 30 ° C. to about 150 ° C., preferably about 80 ° C. to about 100 ° C., most preferably about 75 ° C. to about 85 ° C., which is approximately the boiling point of ethanol. By “about” is meant that the temperature range is preferably within 10 ° C. of the mentioned temperature, more preferably within 5 ° C. of the mentioned temperature and most preferably within 2 ° C. of the mentioned temperature. Thus, for example, "about 75 ° C" means 75 ° C ± 10 ° C, preferably 75 ° C ± 5 ° C, most preferably 75 ° C ± 2 ° C.
[537] 2-oxindole and aldehydes can be readily synthesized using techniques well known in the chemical art. It will be understood by those skilled in the art that other synthetic routes to form the compounds of the present invention can be used and the following are provided by way of example and not by way of limitation.
[538] Method A: Formylation of Pyrrole
[539] POCl ₃ (1.1 equiv) is added dropwise to dimethylformamide (3 equiv) at −10 ° C., then the appropriate pyrrole dissolved in dimethylformamide is added. After stirring for 2 hours, the reaction mixture is diluted with H ₂ O and basified to pH 11 with 10N KOH. The precipitate formed is collected by filtration, washed with H ₂ O and dried in a vacuum oven to give the desired aldehyde.
[540] Method B: saponification of pyrrolecarboxylic acid ester
[541] The mixture of pyrrolecarboxylic acid ester and KOH (2-4 equivalents) in EtOH is refluxed until completion of reaction is confirmed by thin layer chromatography (TLC). The cooled reaction mixture is acidified to pH 3 with 1N HCl. The precipitate formed is collected by filtration, washed with H ₂ O and dried in a vacuum oven to give the desired pyrrolecarboxylic acid.
[542] Method C: Amidation
[543] To a stirred solution of pyrrolecarboxylic acid dissolved in dimethylformamide (0.3M) 1-ethyl-3- (3-dimethylamino-propyl) carbodiimide (1.2 equiv), 1-hydroxybenzotriazole (1.2 equiv) And triethylamine (2 equiv). Appropriate amine is added (1 equiv) and the reaction is stirred until complete by TLC. Ethyl acetate is then added to the reaction mixture and the solvent is washed with saturated NaHCO ₃ and brine (extra salts used), dried over anhydrous MgSO ₄ and concentrated to give the desired amide.
[544] Method D: Condensation of oxindole and aldehyde containing carboxylic acid substituent
[545] A mixture of oxindole (1 equiv), aldehyde and 1 to 3 equivalents of piperidine (or pyrrolidine) in ethanol (0.4 M) is stirred at 90-100 ° C. until completion of reaction is confirmed by TLC. . The mixture is then concentrated and the residue is acidified with 2N HCl. The precipitate formed is washed with H ₂ O and EtOH and then dried in a vacuum oven to give the product.
[546] Method E: Condensation of oxindoles and aldehydes containing no carboxylic acid substituents
[547] A mixture of oxindole (1 equiv), aldehyde and 1 to 3 equivalents of piperidine (or pyrrolidine) in ethanol (0.4 M) is stirred at 90-100 ° C. until completion of reaction is confirmed by TLC. . The mixture is cooled to room temperature and the solid formed is collected by vacuum filtration, washed with ethanol and dried to give the product. If a precipitate does not form upon cooling of the reaction mixture, the mixture is concentrated and purified by column chromatography.
[548] C: Example of Oxidol Synthesis
[549] The following examples of synthesis of representative auxindol are not considered to limit the scope of the invention in any way. Other routes for the presented auxindol as well as other auxindol used to prepare the compounds of the invention will be apparent to those skilled in the art based on the following disclosure. Such synthesis and auxindol is within the scope and spirit of the present invention.
[550] 5-amino-2-oxindole
[551] 5-Nitro-2-oxindol (6.3 g) is hydrogenated over 10% palladium on carbon in methanol to yield 3.0 g (6.0% yield) of the title compound as a white solid.
[552] 5-bromo-2-oxindole
[553] 2-oxindol (1.3 g) in 20 mL acetonitrile was cooled to -10 ° C, and 2.0 g of N-bromosuccinimide was slowly added under stirring. The reaction was stirred at −10 ° C. for 1 hour and at 0 ° C. for 2 hours. The precipitate was collected, washed with water and dried to give 1.9 g (90% yield) of the title compound.
[554] 4-methyl-2-oxindol
[555] Diethyl oxylate (30 mL) in 20 mL of anhydrous ether was added to 19 g of potassium ethoxide suspended in 50 mL of anhydrous ether with stirring. The mixture was cooled in an ice bath and 20 mL of 3-nitro-o-oxyylene in 20 mL of anhydrous ether was added slowly. The dark dark red mixture was heated at reflux for 0.5 h, concentrated to a dark red solid and treated with 10% sodium hydroxide until most of all solids were dissolved. The dark red mixture was treated with 30% hydrogen peroxide until the red color turned yellow. Alternatively, the mixture was treated with 10% sodium hydroxide and 30% hydrogen peroxide until dark red was no longer present. The solid was filtered off and the filtrate was acidified with 6N hydrochloric acid. The resulting precipitate was collected by vacuum filtration, washed with water and dried under vacuum to yield 9.8 g (45% yield) of 2-methyl-6-nitrophenylacetic acid as an off-white solid. The solid was hydrogenated over 10% palladium on carbon in methanol to yield 9.04 g of the title compound as a white solid.
[556] 7-bromo-5-chloro-2-oxindol
[557] 5-chloro-2-oxindol (16.8 g) and 19.6 g N-bromosuccinimide were suspended in 140 ml of acetonitrile and refluxed for 3 hours. Thin layer chromatography at 2 hours of reflux (silica, ethyl acetate) showed N-bromosuccinimide (Rf 0.8), product (Rf 0.85) and second product (Rf 0.9) (fractions were changed after further reflux time). Not shown). The mixture was cooled to 10 ° C., the precipitate was collected by vacuum filtration, washed with 25 ml of ethanol and suction dried for 20 minutes in a funnel to yield 14.1 g (56% yield) of the wet product. The solid was suspended in 200 ml of denatured ethanol and the slurry was washed under stirring and refluxed for 10 minutes. The mixture was cooled to 10 ° C. in an ice bath. The solid product was collected by vacuum filtration, washed with 25 ml of ethanol and dried at 40 ° C. in vacuo to give 12.7 g (51% yield) of 7-bromo-5-chloro-2-oxindol.
[558] 5-fluoro-2-oxindol
[559] 5-fluoroisatin (8.2 g) was dissolved in 50 ml of hydrazine hydrate and refluxed for 1.0 h. The reaction mixture was then poured into ice water. The precipitate was then filtered off, washed with water and dried in a vacuum oven to afford the title compound.
[560] 5-nitro-2-oxindole
[561] 2-oxindol (6.5 g) was dissolved in 25 ml of concentrated sulfuric acid and the mixture was kept at -10 to -15 [deg.] C. while 2.1 ml of fuming nitric acid was added dropwise. After addition of nitric acid, the reaction mixture was stirred at 0 ° C. for 0.5 h and poured into ice water. The precipitate was collected by filtration, washed with water and crystallized from 50% acetic acid. The crystalline product was then filtered off, washed with water and dried under vacuum to afford 6.3 g (70%) of 5-nitro-2-oxindole.
[562] 5-aminosulfonyl-2-oxindol
[563] To a 100 ml flask filled with 27 ml of chlorosulfonic acid was added 13.3 g of 2-oxindole slowly. The reaction temperature was kept below 30 ° C. during the addition. After addition, the reaction mixture was stirred at room temperature for 1.5 hours, heated to 68 ° C. for 1 hour, cooled and poured into water. The precipitate was washed with water and dried in a vacuum oven to give 11.0 g (50% yield) of 5-chlorosulfonyl-2-oxindol which was used without further purification.
[564] 5-chlorosulfonyl-2-oxindol (2.1 g) was added to 10 ml of ammonium hydroxide in 10 ml of ethanol and stirred overnight at room temperature. The mixture was concentrated and the solid was collected by vacuum filtration to yield 0.4 g (20% yield) of the title compound as off white solid.
[565] 5-isopropylaminosulfonyl-2-oxindol
[566] To a 100 ml flask filled with 27 ml of chlorosulfonic acid was added 13.3 g of 2-oxindole slowly. The reaction temperature was kept below 30 ° C. during the addition. The reaction mixture was stirred at rt for 1.5 h, heated to 68 ° C. for 1 h, cooled and poured into water. The precipitate formed was filtered off, washed with water and dried in a vacuum oven to yield 11.0 g (50%) of 5-chlorosulfonyl-2-oxindol which was used without further purification.
[567] A suspension of 3 g of 5-chlorosulfonyl-2-oxindol, 1.15 g of isopropylamine and 1.2 ml of pyridine in 50 ml of dichloromethane was stirred at room temperature for 4 hours during which time a white solid was formed. The solid was collected by vacuum filtration, the slurry was washed with hot ethanol, cooled, collected by vacuum filtration and dried overnight at 40 ° C. under vacuum to yield 1.5 g of 5-isopropylaminosulfonyl-2-oxindol ( 45%) was obtained.
[568]
[569] 5-phenylaminosulfonyl-2-oxindol
[570] A suspension of 5-chlorosulfonyl-2-oxindol (1.62 g, 7 mmol), aniline (0.782 mL, 8.4 mmol) and pyridine (1 mL) in dichloromethane (20 mL) was stirred at room temperature for 4 hours. The reaction mixture was diluted with ethyl acetate (300 mL) and acidified with 1N hydrochloric acid (16 mL). The organic layer was washed with sodium bicarbonate and brine, dried and concentrated. The residue was washed with ethanol (3 mL) and then chromatographed on silica gel eluting with methanol / dichloromethane 1: 9 to afford 5-phenylaminosulfonyl-2-oxindol.
[571]
[572] 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid pyridin-3-ylamide
[573] A solution of 5-chlorosulfonyl-2-oxindol (3 g) and 3-aminopyridine (1.46 g) in pyridine (15 mL) was stirred overnight, ie at room temperature while a brown solid was present. The solid was filtered, washed with ethanol and dried under vacuum to afford 1.4 g (38%) of 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid pyridin-3-ylamide.
[574]
[575] 5-phenyloxindol
[576] 5-bromo-2-oxindol (5 g, 23.5 mmol) was stirred and dissolved in 110 ml of toluene and 110 ml of ethanol with little heating. Tetrakis (triphenylphosphine) palladium (()) (1.9 g, 1.6 mmol) was added followed by 40 mL (80 mmol) of 2M aqueous sodium carbonate. Benzene boronic acid (3.7 g, 30.6 mmol) was added to this mixture and the mixture was heated in a 100 ° C. oil bath for 12 hours. The reaction was cooled, diluted with ethyl acetate (500 mL) and washed with saturated sodium bicarbonate (200 mL), water (200 mL), 1N HCl (200 mL) and brine (200 mL). The organic layer was dried over magnesium sulfate and concentrated to give a brown solid. Trituration with dichloromethane gave 3.8 g (77%) of 5-phenyl-2-oxindol as a tan solid.
[577]
[578] In a similar manner, the following auxindol can be prepared:
[579] 6- (3,5-dichlorophenyl) -1,3-dihydroindol-2-one
[580]
[581] 6- (4-butylphenyl) -1,3-dihydroindol-2-one
[582]
[583] 6- (5-isopropyl-2-methoxyphenyl) -1,3-dihydroindol-2-one
[584]
[585] 6- (4-ethylphenyl) -1,3-dihydroindol-2-one
[586]
[587] 6- (3-isopropylphenyl) -1,3-dihydroindol-2-one
[588]
[589] 6- (2,4-dimethoxyphenyl) -1,3-dihydroindol-2-one
[590]
[591] 6-pyridin-3-yl-1,3-dihydroindol-2-one
[592]
[593] 2-Oxo-2,3-dihydro-1 H-indole-4-carboxylic acid (3-chloro-4-ethoxyphenyl) -amide
[594] Benzotriazole- at room temperature in a solution of 4-carboxy-2-oxindole (200 mg, 1.13 mmol) and 3-chloro-4-methoxyphenylamine (178 mg, 1.13 mmol) in dimethylformamide (15 mL). 1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP reagent, 997 mg, 2.26 mmol) followed by 4-dimethylaminopyridine (206 mg, 1.69 mmol) was added. The mixture was stirred at rt for 72 h. The reaction was then diluted with ethyl acetate (300 mL) and washed with saturated sodium bicarbonate (100 mL), water, 2N hydrochloric acid (100 mL), water (3 x 200 mL) and brine. Then dried over magnesium sulfate and concentrated. The residue is triturated with ethyl acetate to afford 2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid (3-chloro-4-methoxyphenyl) -amide as a pink solid.
[595]
[596] 4-carboxy-2-oxindole
[597] A solution of trimethylsilyldiazomethane in hexane (2M) was added dropwise to a solution of 2.01 g of 2-chloro-3-carboxy-nitrobenzene in 20 ml of methanol at room temperature until no further gas evolution appeared. Acetic acid was then added to quench with excess trimethylsildiazomethane. The reaction mixture was evaporated in vacuo and the residue was dried in an oven overnight. The 2-chloro-3-methoxycarbonylnitrobenzene obtained was sufficiently pure for the following reaction.
[598] Dimethyl malonate (6.0 mL) was added to an ice-cooled suspension of 2.1 g sodium hydride in 15 mL DMSO. The reaction mixture was stirred at 100 ° C. for 1 hour and then cooled to room temperature. 2-chloro-3-methoxycarbonylnitrobenzene (2.15 g) was added and the mixture was heated to 100 ° C for 1.5 h. The reaction mixture was then cooled to room temperature, poured into ice water, acidified to pH 5 and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated to yield 3.0 g of dimethyl 2-methoxycarbonyl-6-nitrophenyl-malonate.
[599] Dimethyl 2-methoxycarbonyl-6-nitrophenylmalonate (3.0 g) was refluxed in 50 mL of 6N hydrochloric acid overnight. The mixture was concentrated to dryness, 20 ml of ethanol and 1.1 g of tin (II) chloride were added and the mixture was refluxed for 2 hours. The mixture was filtered through celite, concentrated and chromatographed on silica gel using ethyl acetate: hexane: acetic acid as eluent to yield 0.65 g (37%) of 4-carboxy-2-oxindol as a white solid.
[600]
[601] D. Synthesis of pyrrole substituted 2-indolinones
[602] Example 1
[603] 4-Methyl-5- (2-oxo-1,2-dihydroindole-3-ylidenemethyl) -1 H-pyrrole-2-carboxylic acid
[604] 4-methyl-2-pyrrolecarboxylic acid ethyl ester (commercially available) was formylated using Method A to give 5-formyl-4-methyl-2-pyrrolecarboxylic acid ethyl ester (73%). Then hydrolysis using Method B yielded 5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid (58%).
[605] Oxidol (113 mg, 1 mmol) was condensed with 5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid (153 mg) using Method D to give 268 mg (100%) of the title compound as an orange-red solid. ) Was obtained.
[606]
[607] Example 2
[608] 4-Methyl-5- (1-methyl-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -1 H-pyrrole-2-carboxylic acid
[609] Condensation of 1-methyl-1,3-dihydroindol-2-one (146 mg, 1 mmol) with 5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid (153 mg) using Method D To give 250 mg (86%) of the title compound.
[610]
[611] Example 3
[612] 4-Methyl-5- (2-oxo-1,2-dihydroindole-3-ylidenemethyl) -1 H-pyrrole-2-carboxylic acid methyl ester
[613] Auxindol (105 mg, 0.79 mmol) was condensed with 5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid methyl ester (110 mg, 0.67 mmol) using Method E to give 153.2 mg (81%) of the title compound. ) Was obtained.
[614]
[615] Example 4
[616] 5- (5-Chloro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -4-methyl-1 H-pyrrole-2-carboxylic acid ethyl ester
[617] 5-chloro-1,3-dihydroindol-2-one (2.22 g, 13.2 mmol) 5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid ethyl ester (2.43 g) using Method E Condensation with 4.1 g (94%) of the title compound as an orange solid.
[618]
[619] Example 5
[620] 5- (5-Chloro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -4-methyl-1 H-pyrrole-2-carboxylic acid
[621] Ethyl 5- (5-chloro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -4-methyl-1H-pyrrole-2-carboxylic acid in methanol (25 mL) and ethanol (25 mL) A mixture of ester (1.3 g, 4 mmol) and potassium hydroxide was heated at reflux overnight. Insoluble material was removed by filtration and the mixture was neutralized with 6N hydrochloric acid to yield 0.876 g (70%) of the title compound.
[622]
[623] Example 6
[624] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -4-methyl-1H-pyrrole-2-carboxylic acid (3-pyrrolidin-1-yl-propyl )amides
[625] 5-Bromo-1,3-dihydroindol-2-one (0.16 g, 0.76 mmol) to 5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid (3-pyrrolidin-1-yl Condensation with propyl) amide (0.2 g, prepared by Method C) gave 60 mg (17%) of the title compound as an orange solid.
[626]
[627] Example 7
[628] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -4-methyl-1H-pyrrole-2-carboxylic acid (3-diethylamino-propyl) amide
[629] 5-Bromo-1,3-dihydroindol-2-one (0.16 g, 0.75 mmol) was converted to 5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid (3-diethylaminopropyl) amide ( 0.2 g, prepared by Method C), to give 30 mg (8%) of the title compound as an orange solid.
[630]
[631] Example 8
[632] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -1H-pyrrole-2-carboxylic acid (2-diethylaminoethyl) amide
[633] 5-Bromo-1,3-dihydroindol-2-one (212 mg, 1 mmol) was converted to 5-formyl-1H-pyrrole-2-carboxylic acid (2-diethylaminoethyl) amide (methods A and B). And C from ethyl pyrrole-2-carboxylate) to give 162 mg (38%) of the title compound.
[634]
[635] Example 9
[636] 5- (2-Oxo-6-phenyl-1,2-dihydroindole-3-ylidenemethyl) -1 H-pyrrole-2-carboxylic acid (2-diethylaminoethyl) amide
[637] 6-phenyl-1,3-dihydroindol-2-one (209 mg, 1 mmol) was condensed with 5-formyl-1H-pyrrole-2-carboxylic acid (2-diethylaminoethyl) amide to give the title compound 182. MG (42%) was obtained.
[638]
[639] Example 10
[640] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -1H-pyrrole-2-carboxylic acid (2-diethylaminoethyl) amide-methyl-amide
[641] 5-bromo-1,3-dihydroindol-2-one (212 mg, 1 mmol) was condensed with 5-formyl-1H-pyrrole-2-carboxylic acid (2-diethylaminoethyl) methylamide to give the title 246 mg (55%) of compound was obtained.
[642]
[643] Example 11
[644] 5- (2-Oxo-6-phenyl-1,2-dihydroindole-3-ylidenemethyl) -1 H-pyrrole-2-carboxylic acid (2-diethylaminoethyl) methylamide
[645] Condensation of 6-phenyl-1,3-dihydroindol-2-one (209 mg, 1 mmol) with 5-formyl-1H-pyrrole-2-carboxylic acid (2-diethylaminoethyl) methylamide to give the title compound 277 mg (63%) was obtained.
[646]
[647] Example 12
[648] 3-Methyl-5- (2-oxo-6-phenyl-1,2-dihydroindole-3-ylidenemethyl) -1 H-pyrrole-2-carboxylic acid (3-diethylaminopropyl) amide
[649] Auxindol (66.5 mg, 0.5 mmol) was converted to 5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid (3-diethylaminopropyl) amide (3-formyl-3-methyl by methods B and C). Condensation with -1H-pyrrole-2-carboxylic acid ethyl ester to give 39 mg (21%) of the title compound.
[650]
[651] Example 13
[652] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1H-pyrrole-2-carboxylic acid (3-diethylamino-propyl) amide
[653] 5-Bromo-1,3-dihydroindol-2-one (106 mg, 0.5 mmol) was mixed with 5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid (3-diethylaminopropyl) amide. Condensation gave 35 mg (15%) of the title compound.
[654]
[655] Example 14
[656] 3-Methyl-5- (2-oxo-6-phenyl-1,2-dihydroindole-3-ylidenemethyl) -1 H-pyrrole-2-carboxylic acid (3-diethylaminopropyl) amide
[657] Condensation of 6-phenyl-1,3-dihydroindol-2-one (105 mg, 0.5 mmol) with 5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid (3-diethylaminopropyl) amide To give 67.8 mg (30%) of the title compound.
[658]
[659] Example 15
[660] 5- (5-methoxy-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1H-pyrrole-2-carboxylic acid (3-diethylamino-propyl) amide
[661] 5-methoxy-1,3-dihydroindol-2-one (82.5 mg, 0.5 mmol) with 5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid (3-diethylaminopropyl) amide Condensation gave 80 mg (39%) of the title compound.
[662]
[663] Example 16
[664] 5- (6-methoxy-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1H-pyrrole-2-carboxylic acid (3-diethylamino-propyl) amide
[665] 6-methoxy-1,3-dihydroindol-2-one (82.5 mg, 0.5 mmol) with 5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid (3-diethylaminopropyl) amide Condensation gave 63 mg (31%) of the title compound.
[666]
[667] MSm / z 410 [M⁺].
[668] Example 17
[669] 3- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid (2-di Ethylamino-ethyl) amide
[670] 4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid ethyl ester (May, Donald A .; Lash, Timothy D . ; J. Org . Chem. , 1992, 57:18, 4820-4828 ) Was formylated using Method A followed by B to yield 3-formyl-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid.
[671] 5-Bromo-1,3-dihydroindol-2-one (1.43 g, 6.8 mmol) was converted to 3-formyl-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid (2 Condensation with -diethylaminoethyl) amide (1.97 g) gave 2.2 g (67%) of the title compound as a yellow-orange solid.
[672]
[673] Example 18
[674] 3- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid (3-di Ethylamino-propyl) amide
[675] 5-Bromo-1,3-dihydroindol-2-one (20 mg, 0.1 mmol) was added to 3-formyl-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid (3 Condensation with diethylaminopropyl) amide (30 mg) gave 33 mg (46%) of the title compound as an orange solid.
[676]
[677] Example 19
[678] 3- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid (3-py Rollidin-1-ylpropyl) amide
[679] 5-Bromo-1,3-dihydroindol-2-one (80 mg, 0.4 mmol) was added to 3-formyl-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid (3 Condensation with -pyrrolidin-1-ylpropyl) amide (120 mg) gave 43 mg (22%) of the title compound as a tan-orange solid.
[680]
[681] Example 20
[682] 3- (2-Oxo-6-pyridin-3-yl-1,2-dihydroindole-3-ylidenemethyl) -4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid ( 2-diethylaminoethyl) amide
[683] 6-pyridin-3-yl-1,3-dihydroindol-2-one (60 mg, 0.4 mmol) 3-formyl-4,5,6,7-tetrahydro-2H-isoindole-1- Condensation with carboxylic acid (2-diethylaminoethyl) amide (80 mg) gave 50 mg (38%) of the title compound as a reddish solid.
[684]
[685] Example 21
[686] 4-Benzoyl-5- (5-bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1 H-pyrrole-2-carboxylic acid (3-diethylaminopropyl) amides
[687] To a mixture of benzoyl chloride (1 equiv) and aluminum chloride (1 equiv) in dichloroethane was added ethyl 3,5-dimethyl-2-pyrrolecarboxylate (1 equiv) at 0 ° C. The mixture was stirred for 4 h at 80 ° C. The mixture was then extracted with ethyl acetate (EtOAc) and H ₂ O. The combined organic extracts were washed with saturated sodium bicarbonate and brine, dried and concentrated to give 4-benzoyl-3,5-dimethyl-1H-pyrrole-2-carboxylic acid (51%).
[688] Tetrahydrofuran (THF): acetic acid (HOAc): H ₂ O 1: 1: 4-benzoyl-3,5-dimethyl-1H-pyrrole-2-carboxylic acid ethyl ester (4.13 g, 15.2 mmol) in 50 mL A mixture of ceric ammonium nitrate (33 g, 4 equiv) was refluxed overnight. The reaction mixture was then cooled, extracted with EtOAc and basified to pH 9 with sodium carbonate. The organic layer was then washed with brine, dried (MgSO ₄ ), concentrated and column chromatographed to give 4-benzoyl-5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid ethyl as a yellow solid. 3.25 g (75%) of ester were obtained.
[689] Condensation of 5-bromo-1,3-dihydro-indol-2-one with 4-benzoyl-5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid using Method D to yield 4-benzoyl- 5- (5-Bromo-2-oxo-1,2-dihydro-indole-3-ylidenemethyl) -3-methyl-1H-pyrrole-2-carboxylic acid was obtained.
[690] The carboxylic acid was then coupled with N, N-diethyl-1,3-propanediamine using Method C to afford the title compound.
[691]
[692] Example 22
[693] 4-Benzoyl-5- (5-bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1 H-pyrrole-2-carboxylic acid (3-morpholine-4- Monopropyl) amide
[694]
[695] Example 23
[696] 4-benzoyl-3-methyl-5- (2-oxo-1,2-dihydroindole-3-ylidenemethyl) -1H-pyrrole-2-carboxylic acid (3-pyrrolidin-1-ylpropyl) amide
[697]
[698] Example 24
[699] 4-Benzoyl-5- (5-bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1 H-pyrrole-2-carboxylic acid (3-pyrrolidine-4 -Ylpropyl) amide
[700]
[701] Example 25
[702] 4-benzoyl-3-methyl-5- (2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl) -1H-pyrrole-2-carboxylic acid (3-pyrrolidine-1- Monopropyl) amide
[703]
[704] Example 26
[705] 4-benzoyl-5- (6-methoxy-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1H-pyrrole-2-carboxylic acid (3-pyrrolidine-1 -Ylpropyl) amide
[706]
[707] Example 27
[708] 4-benzoyl-5- (5-methoxy-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1H-pyrrole-2-carboxylic acid (3-pyrrolidine-1 -Ylpropyl) amide
[709]
[710] Example 28
[711] 4-Benzoyl-5- (5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1 H-pyrrole-2-carboxylic acid (3-pyrrolidine-1 -Ylpropyl) amide
[712]
[713] Example 29
[714] 4-acetyl-5- (5-bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1H-pyrrole-2-carboxylic acid (3-diethylaminopropyl) amides
[715] 5-Bromo-1,3-dihydro-indol-2-one to 4-acetyl-5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid (3-diethylaminopropyl) amide (method B Condensation with 4-acetyl-5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid ethyl ester by the method of C) to afford the title compound.
[716]
[717] Example 30
[718] 4-Acetyl-5- (5-bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1H-pyrrole-2-carboxylic acid (3-pyrrolidine-1 -Ylpropyl) amide
[719]
[720] Example 31
[721] 4-Acetyl-5- (5-bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1H-pyrrole-2-carboxylic acid (3-morpholine-4- Monopropyl) amide
[722]
[723] Example 32
[724] 4-acetyl-5- (5-bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1H-pyrrole-2-carboxylic acid (3-hydroxypropyl) amide
[725]
[726] MS-EI m / z 445 & 447 [M ⁺ -1] & [M ⁺ +1].
[727] Example 34
[728] 4-Acetyl-5- (5-bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1H-pyrrole-2-carboxylic acid (2-morpholine-4- Monoethyl) amide
[729]
[730] Example 35
[731] 4-Acetyl-5- (5-bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1H-pyrrole-2-carboxylic acid (2-pyrrolidine-1 -Ylethyl) amide
[732]
[733] Example 36
[734] 4-acetyl-5- (5-bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -3-methyl-1H-pyrrole-2-carboxylic acid [2- (4-hydroxy Phenyl) ethyl] amide
[735]
[736] Example 37
[737] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2-isopropyl-4-phenyl-1 H-pyrrole-3-carboxylic acid (3-diethylaminopropyl )amides
[738] A mixture of 2-aminoacetophenone hydrochloride (1 equiv), ethyl isobutyryl acetate (1.2 equiv) and sodium acetate (2,4 equiv) in H ₂ O was stirred at 100 ° C. for 18 hours and then cooled to room temperature I was. The aqueous layer was decanted and the oil dissolved in ethyl acetate. It was then washed with water and brine and dried to give 2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylic acid ethyl ester as reddish brown oil (93%).
[739]
[740] The pyrrole was formylated using Method A to give 5-formyl-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylic acid ethyl ester as a reddish solid.
[741]
[742] The pyrrolecarboxylic acid ester was hydrolyzed using Method B to give 5-formyl-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylic acid as a beige solid (57%).
[743]
[744] 5-Bromo-1,3-dihydroindol-2-one (120 mg, 0.31 mmol) was converted to 5-formyl-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylic acid (3-diethyl Condensation with aminopropyl) amide (prepared by Method C) afforded 120 mg (71%) of the title compound.
[745]
[746] Example 38
[747] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2-isopropyl-4-phenyl-1 H-pyrrole-3-carboxylic acid (3-pyrrolidine- 1-propyl) amide
[748] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2-isopropyl-4-phenyl-1 H-pyrrole-3-carboxylic acid (127 mg, 0.28 mmol) Was condensed with 3-pyrrolidin-1-yl-propylamine (43 mg, 0.336 mmol) to give 140 mg (66%) of the title compound.
[749]
[750] Example 39
[751] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2-isopropyl-4-phenyl-1 H-pyrrole-3-carboxylic acid (2-diethylaminoethyl )amides
[752] 5-Bromo-1,3-dihydroindol-2-one (57 mg, 0.27 mmol) was converted to 5-formyl-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylic acid (2-diethyl Condensation with aminoethyl) amide (120 mg) afforded 78 g (53%) of the title compound as a yellow solid.
[753]
[754] Example 40
[755] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2-isopropyl-4-phenyl-1 H-pyrrole-3-carboxylic acid [3- (4-methyl Piperazin-l-yl) propyl] amide
[756] 5-Bromo-1,3-dihydroindol-2-one (53 mg, 0.25 mmol) was substituted with 5-formyl-2-isopropyl-4-phenyl-lH-pyrrole-3-carboxylic acid [3- (4 Condensation with -methylpiperazin-1-yl) propyl] amide (300 mg) gave 65 mg of the title compound.
[757]
[758] Example 41
[759] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2-isopropyl-4-phenyl-lH-pyrrole-3-carboxylic acid
[760] 5-Bromo-1,3-dihydroindol-2-one (170 mg, 0.8 mmol) 5-formyl-2-isopropyl-4-phenyl-1H-pyrrole-3-carboxylic acid using Method D Condensation with (205 mg) gave 210 mg (58%) of the title compound as a yellow solid.
[761]
[762] Example 42
[763] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidine-1 -Ylethyl) amide
[764] 5-Bromo-1,3-dihydroindol-2-one (44 mg, 0.21 mmol) was converted to 5-formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidine Condensation with -1-ylethyl) amide (70 mg, prepared in the same manner as the isopropyl analogue) afforded 0.03 g (27%) of the title compound as a yellow solid.
[765]
[766] Example 43
[767] 5- [6- (2-methoxyphenyl) -2-oxo-1,2-dihydroindole-3-ylidenemethyl] -2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid (2- Pyrrolidin-l-ylethyl) amide
[768] 6- (2-methoxyphenyl) -1,3-dihydroindol-2-one (50 mg, 0.21 mmol) was added to 5-formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid ( Condensation with 2-pyrrolidin-1-ylethyl) amide (70 mg) gave 0.04 g (35%) of the title compound as an yellow-orange solid.
[769]
[770] Example 44
[771] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2-methyl-4-phenyl-lH-pyrrole-3-carboxylic acid (2-dimethylaminoethyl) amide
[772] 5-Bromo-1,3-dihydroindol-2-one (46 mg, 0.22 mmol) was converted to 5-formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid (2-dimethylaminoethyl Condensation with) amide (65 mg) gave 60 mg (55%) of the title compound as a yellow solid.
[773]
[774] Example 45
[775] 5- [6- (2-methoxyphenyl) -2-oxo-1,2-dihydroindole-3-ylidenemethyl] -2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid (2- Dimethylaminoethyl) amide
[776] 6- (2-methoxyphenyl) -1,3-dihydroindol-2-one (53 mg, 0.22 mmol) was added to 5-formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid ( Condensation with 2-dimethylaminoethyl) amide (65 mg) gave 0.05 g (44%) of the title compound as an orange gum.
[777]
[778] Example 46
[779] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid ethyl ester
[780] 5-Bromo-1,3-dihydroindol-2-one (60 mg, 0.29 mmol) was converted to 5-formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid ethyl ester (75 mg). Condensation with afforded 78 mg (60%) of the title compound as an orange solid.
[781]
[782] Example 47
[783] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid (3-diethylaminopropyl) amides
[784] 5-Bromo-1,3-dihydroindol-2-one (0.47 g, 2.2 mmol) was converted to 5-formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acid (3-diethylamino Condensation with propyl) amide (0.75 g) gave 0.11 g (42%) of the title compound as an orange solid.
[785]
[786] Example 48
[787] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-dimethylamino-ethyl) amide
[788] A mixture of tert-butyl 3-oxobutyrate and sodium nitrite in acetic acid was stirred at room temperature to give tert-butyl-2-hydroxymino-3-oxobutyrate.
[789] Ethyl-3-oxobutyrate (1 equiv), zinc mal (3.8 equiv) and crude tert-butyl-2-hydroxyimino-3-oxobutyrate in acetic acid were stirred at 60 ° C. for 1 hour. The reaction mixture was poured into H ₂ O and the filtrate was collected to give 2-tert-butyloxycarbonyl-3,5-dimethyl-4-ethoxycarbonylpyrrole.
[790] A mixture of 2-tert-butyloxycarbonyl-3,5-dimethyl-4-ethoxycarbonylpyrrole and triethyl orthoformate (1.5 equiv) in trifluoroacetic acid was stirred at 15 ° C. for 1 hour. The reaction was concentrated and the residue was purified to give 2,4-dimethyl-3-ethoxycarbonyl-5-formylpyrrole as a yellow needle.
[791] 2,4-dimethyl-3-ethoxycarbonyl-5-formylpyrrole was hydrolyzed using Method B to yield 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (90 %).
[792]
[793] 5-Bromo-1,3-dihydroindol-2-one (0.17 g, 0.8 mmol) 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2- Condensation with dimethylaminoethyl) amide (0.2 g, prepared by Method C) yielded 0.3 g (83%) of the title compound as a yellow solid.
[794]
[795] Example 49
[796] 2,4-dimethyl-5- (2-oxo-6-phenyl-1,2-dihydroindole-3-ylidenemethyl) -1H-pyrrole-3-carboxylic acid (2-dimethylaminoethyl) amide
[797] 6-phenyl-1,3-dihydroindol-2-one (0.17 g, 0.8 mmol) was added to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-dimethylaminoethyl) amide ( 0.2 g) yielded 0.13 g (36%) of the title compound as a yellow-orange solid.
[798]
[799] Example 50
[800] 5- (5-Chloro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1 H-pyrrole-3-carboxylic acid (2-dimethylamino-ethyl) amide
[801] 5-Chloro-1,3-dihydroindol-2-one (0.1 g, 0.6 mmol) was added to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-dimethylaminoethyl) amide ( 0.15 g) to afford 0.22 g (90%) of the title compound as a yellow solid.
[802]
[803] Example 51
[804] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) amide
[805] 5-Bromo-1,3-dihydroindol-2-one (0.17 g, 0.8 mmol) was converted to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-diethylaminoethyl) Condensation with amide (0.2 g) gave 0.09 g (26%) of the title compound as a yellow solid.
[806]
[807] Example 52
[808] 5- (5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-yl Ethyl) amide
[809] 5-Bromo-1,3-dihydroindol-2-one (0.09 g, 0.4 mmol) was converted to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-pyrrolidine-1 Condensation with -ylethyl) amide (0.1 g) gave 0.14 g (81%) of the title compound as a yellow-orange solid.
[810]
[811] Example 53
[812] 5- (5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (3-imidazol-1-yl- Propyl) amide
[813] 5-Bromo-1,3-dihydroindol-2-one (0.09 g, 0.4 mmol) was added to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (3-imidazol-1- Condensation with propyl) amide (0.1 g) gave 0.1 g (59%) of the title compound as an orange solid.
[814]
[815] Example 54
[816] 5- [6- (2-methoxyphenyl) -2-oxo-1,2-dihydroindole-3-ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-dimethylamino Ethyl) amide
[817] 6- (2-methoxyphenyl) -1,3-dihydroindol-2-one (30 mg, 0.13 mmol) to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2- Condensation with dimethylaminoethyl) amide (30 mg) gave 0.06 g (100%) of the title compound as a yellow-orange solid.
[818]
[819] Example 55
[820] 5- [6- (3-methoxyphenyl) -2-oxo-1,2-dihydroindole-3-ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-dimethylamino Ethyl) amide
[821] 6- (3-methoxyphenyl) -1,3-dihydroindol-2-one (30 mg, 0.13 mmol) in 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2- Condensation with dimethylaminoethyl) amide (30 mg) gave 8 mg (14%) of the title compound as a yellow-orange solid.
[822]
[823] Example 56
[824] 2,4-dimethyl-5- (2-oxo-5-phenyl-1,2-dihydroindol-3-ylidenemethyl) -lH-pyrrole-3-carboxylic acid (2-diethylaminoethyl) amide
[825] 5-phenyl-1,3-dihydroindol-2-one (80 mg, 0.4 mmol) 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-di Condensation with ethylaminoethyl) amide (0.1 g) gave 79 mg (46%) of the title compound.
[826]
[827] Example 57
[828] 2,4-dimethyl-5- (2-oxo-5-phenyl-1,2-dihydroindol-3-ylidenemethyl) -1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-ylethyl )amides
[829] 5-phenyl-1,3-dihydroindol-2-one (0.04 g, 0.2 mmol) to 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidine-1- Condensation with monoethyl) amide (0.04 g) gave the title compound as a yellow-orange solid.
[830]
[831] Example 58
[832] 2,4-dimethyl-5- (2-oxo-5-phenyl-1,2-dihydroindol-3-ylidenemethyl) -1H-pyrrole-3-carboxylic acid (3-imidazol-1-ylpropyl) amides
[833] 5-phenyl-1,3-dihydroindol-2-one (8 mg, 0.04 mmol) to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (3-imidazol-1-yl Condensation with propyl) amide (10 mg) gave 10 mg (59%) of the title compound as an orange solid.
[834]
[835] Example 59
[836] 2,4-dimethyl-5- (2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl) -lH-pyrrole-3-carboxylic acid (2-diethylaminoethyl) amide
[837] 6-phenyl-1,3-dihydroindol-2-one (0.08 g, 0.4 mmol) 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl) amide Condensation with (0.1 g) gave 65 mg (38%) of the title compound as a yellow solid.
[838]
[839] Example 60
[840] 2,4-dimethyl-5- (2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl) -lH-pyrrole-3-carboxylic acid (2-pyrrolidin-1-ylethyl )amides
[841] 6-phenyl-1,3-dihydroindol-2-one (30 mg, 0.15 mmol) was added to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-pyrrolidine-1- Condensation with monoethyl) amide (40 mg) gave 5.9 mg (8.5%) of the title compound as a yellow-orange solid.
[842]
[843] Example 61
[844] 2,4-dimethyl-5- (2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl) -lH-pyrrole-3-carboxylic acid (3-imidazol-1-ylpropyl) amides
[845] 6-phenyl-1,3-dihydroindol-2-one (8 mg, 0.04 mmol) to 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (3-imidazol-1-yl Condensation with propyl) amide (10 mg) gave 7.3 mg (43%) of the title compound as an orange solid.
[846]
[847] Example 62
[848] 5- [6- (3,5-Dichlorophenyl) -2-oxo-1,2-dihydroindole-3-ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-di Ethylaminoethyl) amide
[849] 6- (3,5-Dichlorophenyl) -1,3-dihydroindol-2-one (64 mg, 0.23 mmol) to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2 Condensation with diethylaminoethyl) amide (60 mg) gave 53 mg (44%) of the title compound as a light brown solid.
[850]
[851] Example 63
[852] 2,4-dimethyl-5- (2-oxo-6-pyridin-3-yl-1,2-dihydroindol-3-ylidenemethyl) -lH-pyrrole-3-carboxylic acid (2-diethylaminoethyl )amides
[853] 6-pyridin-3-yl-1,3-dihydroindol-2-one (40 mg, 0.19 mmol) in 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-diethyl Condensation with aminoethyl) amide (50 mg) gave 29 mg (33%) of the title compound as a light orange solid.
[854]
[855] Example 64
[856] 2,4-dimethyl-5- (2-oxo-6-pyridin-3-yl-1,2-dihydroindol-3-ylidenemethyl) -1H-pyrrole-3-carboxylic acid (2-pyrrolidine- 1-ylethyl) amide
[857] 6-pyridin-3-yl-1,3-dihydroindol-2-one (60 mg, 0.28 mmol) was added to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-pyrroli). Condensation with din-1-ylethyl) amide (75 mg) gave 90 mg (71%) of the title compound as a light orange solid.
[858]
[859] Example 65
[860] 2,4-dimethyl-5- (2-oxo-6-pyridin-3-yl-1,2-dihydroindol-3-ylidenemethyl) -1H-pyrrole-3-carboxylic acid (3-dimethylaminopropyl) amides
[861] 6-pyridin-3-yl-1,3-dihydroindol-2-one (42 mg, 0.2 mmol) in 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (3-dimethylamino Condensation with propyl) amide (50 mg) gave 67 mg (75%) of the title compound as a yellow-brown solid.
[862]
[863] Example 66
[864] 2,4-dimethyl-5- (2-oxo-5-phenyl-1,2-dihydroindole-3-ylidenemethyl) -1H-pyrrole-3-carboxylic acid (3-dimethylaminopropyl) amide
[865] 5-phenyl-1,3-dihydroindol-2-one (67 mg, 0.32 mmol) was converted to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (3-dimethylaminopropyl) amide ( 81 mg) gave 40 mg (28%) of the title compound as an orange solid.
[866]
[867] Example 67
[868] 2,4-dimethyl-5- (2-oxo-5-phenyl-1,2-dihydroindole-3-ylidenemethyl) -lH-pyrrole-3-carboxylic acid (3-diethylaminopropyl) amide
[869] 5-phenyl-1,3-dihydroindol-2-one (1.5 g, 7.16 mmol) to 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (3-diethylaminopropyl) amide Condensation with (2 g) gave 1.3 g (40%) of the title compound as a yellow-orange solid.
[870]
[871] MS-EI m / z 470 [M ⁺ ].
[872] Example 68
[873] 2,4-dimethyl-5- (2-oxo-6-phenyl-1,2-dihydroindole-3-ylidenemethyl) -1H-pyrrole-3-carboxylic acid (3-diethylaminopropyl) amide
[874] 6-phenyl-1,3-dihydroindol-2-one (1.5 g, 7.16 mmol) to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (3-diethylaminopropyl) amide Condensation with (2 g) gave 1.9 g (57%) of the title compound as an orange solid.
[875]
[876] Example 69
[877] 3- [4- (3-Diethylaminopropylcarbamoyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -2-oxo-2,3-dihydro-lH-indole-4-carboxylic acid (3-chloro-4-methoxyphenyl) amide
[878] 2-oxo-2,3-dihydro-1H-indole-4-carboxylic acid (3-chloro-4-methoxyphenyl) amide (1 g, 3.16 mmol) was changed to 5-formyl-2,4-dimethyl-1H-. Condensation with pyrrole-3-carboxylic acid (3-diethylaminopropyl) amide (1 g, 3.58 mmol) afforded 1.7 g (85%) of the title compound as a yellow-orange solid.
[879]
[880] Example 70
[881] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (3-diethylamino-propyl) amide
[882] 5-bromo-1,3-dihydroindol-2-one (0.5 g, 2.36 mmol) to 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (3-diethylaminopropyl) Condensation with amide (0.51 g) gave 0.84 g of the title compound as a red-orange solid.
[883]
[884] Example 71
[885] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-diisopropyl-lH-pyrrole-3-carboxylic acid (2-diethylamino-ethyl )amides
[886] 5-Bromo-1,3-dihydroindol-2-one (100 mg, 0.47 mmol) was added to 5-formyl-2,4-diisopropyl-1H-pyrrole-3-carboxylic acid (2-diethylamino Condensation with ethyl) amide (150 mg) gave 0.15 g (62%) of the title compound as a yellow-orange solid.
[887]
[888] Example 72
[889] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-diisopropyl-1H-pyrrole-3-carboxylic acid (3-diethylamino-propyl )amides
[890] 5-Bromo-1,3-dihydroindol-2-one (90 mg, 0.42 mmol) was converted to 5-formyl-2,4-diisopropyl-lH-pyrrole-3-carboxylic acid (3-diethylamino Condensation with propyl) amide (140 mg) gave 54 mg (25%) of the title compound as a red-brown solid.
[891]
[892] Example 73
[893] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-diisopropyl-1H-pyrrole-3-carboxylic acid (3-pyrrolidine-1 -Ylpropyl) amide
[894] 5-Bromo-1,3-dihydroindol-2-one (130 mg, 0.6 mmol) was converted to 5-formyl-2,4-diisopropyl-1H-pyrrole-3-carboxylic acid (3-pyrrolidine Condensation with -1-ylpropyl) amide (150 mg, 0.45 mmol) afforded 36 mg (15%) of the title compound as a tan-orange solid.
[895]
[896] Example 74
[897] 5- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (pyridin-4-ylmethyl) -amide
[898] 5-Bromo-1,3-dihydroindol-2-one (170 mg, 0.8 mmol) was converted to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (pyridin-4-ylmethyl) Condensation with amide (200 mg) gave 14 mg (4%) of the title compound as a yellow solid.
[899]
[900] Example 75
[901] 5- [6- (4-Butylphenyl) -2-oxo-1,2-dihydroindole3-ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidine- 1-ylethyl) amide
[902] 5- [6- (4-butylphenyl)]-1,3-dihydroindol-2-one (50 mg, 0.19 mmol) was converted to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid. Condensation with (2-pyrrolidin-1-ylethyl) amide (50 mg) gave 74 mg (76%) of the title compound as an orange solid.
[903]
[904] Example 76
[905] 5- [6- (5-Isopropyl-2-methoxyphenyl) -2-oxo-1,2-dihydroindole-3-ylidenemethyl] -2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-pyrrolidin-1-ylethyl) amide
[906] 6- (5-isopropyl-2-methoxyphenyl) -1,3-dihydroindol-2-one (50 mg, 0.17 mmol) 5-formyl-2,4-dimethyl-lH-pyrrole-3 Condensation with carboxylic acid (2-pyrrolidin-1-ylethyl) amide (45 mg) gave 67 mg (75%) of the title compound as an orange solid.
[907]
[908] Example 77
[909] 5- [6- (4-ethylphenyl) -2-oxo-1,2-dihydroindole-3-ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidine -1-ylethyl) amide
[910] 6- (4-ethylphenyl) -1,3-dihydroindol-2-one (45 mg, 0.19 mmol) in 5-formyl-2, 4-dimethyl-lH-pyrrole-3-carboxylic acid (2-py Condensation with Ralidin-1-ylethyl) amide (50 mg) gave 60 mg (65%) of the title compound as a yellow-orange solid.
[911]
[912] Example 78
[913] 5- [6- (2,4-Dimethoxyphenyl) -2-oxo-1,2-dihydroindol-3-ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2- Pyrrolidin-1-ylethyl) amide
[914] 6- (2,4-dimethoxyphenyl) -1,3-dihydroindol-2-one (51 mg, 0.19 mmol) was substituted with 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid ( Condensation with 2-pyrrolidin-1-ylethyl) amide (50 mg) gave 30 mg (31%) of the title compound as an orange solid.
[915]
[916] Example 79
[917] 5- [6- (3-Isopropylphenyl) -2-oxo-1,2-dihydroindole-3-ylidenemethyl] -2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-pyrroli Din-1-ylethyl) amide
[918] 6- (3-isopropylphenyl) -1,3-dihydroindol-2-one (48 mg, 0.19 mmol) to 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2- Condensation with pyrrolidin-1-ylethyl) amide (50 mg) gave 59 mg (63%) of the title compound as an orange solid.
[919]
[920] Example 80
[921] 5- [5-Fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-diethylamino- Ethyl) -amide
[922] 5-Fluoro-1,3-dihydroindol-2-one (0.54 g, 3.8 mmol) was converted to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-diethylaminoethyl) Condensation with amide gave 0.83 g (55%) of the title compound as a greenish yellow solid.
[923]
[924] Example 80 (Other Synthesis)
[925] 5- [5-Fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino- Ethyl) -amide
[926] Hydrazine hydrate (55%, 3000 mL) and 5-fluoroisatin (300 g) were heated to 100 ° C. Additional 5-fluoro-isatin (500 g) was added in portions (100 g) with stirring over 120 minutes. The mixture was heated to 110 ° C. and stirred for 4 hours. The mixture was cooled to room temperature and the solids were collected by vacuum filtration to afford crude (2-amino-5-fluoro-phenyl) -acetic acid hydrazide (748 g). Hydrazide was suspended in water (700 mL) and the pH of the mixture was adjusted to less than pH 3 with 12N hydrochloric acid. The mixture was stirred at rt for 12 h. The solid was collected by vacuum filtration and washed twice with water. The product was dried under vacuum to afford 5-fluoro-1,3-dihydro-indol-2-one (600 g, 73% yield) as a brown powder. ¹ H-NMR (dimethylsulfoxide-d ₆ ) δ 3.46 (s, 2H, CH ₂ ), 6.75, 6.95, 7.05 (3 × m, 3H, aromatic), 10.35 (s, 1H, NH). MS m / z 152 [M + l].
[927] 10N hydrochloric acid (3650 mL) was added slowly with vigorous stirring of 3,5-dimethyl-1H-pyrrole-2,4-bicarboxylic acid 2-tert-butyl ester 4-ethylester (2600 g) and ethanol (7800 mL). The temperature was raised from 25 ° C. to 35 ° C. and gas evolution began. The mixture was warmed to 54 ° C. and stirred with additional heating for 1 hour at which temperature reached 67 ° C. The mixture was cooled to 5 ° C. and 32 L of ice and water were added slowly with stirring. The solid was collected by vacuum filtration and washed three times with water. The solid was air dried to constant weight to afford 2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethyl ester (1418 g, 87% yield) as a pinkish solid.
[928]
[929] Dimethylformamide (322 g) and dichloromethane (3700 mL) were cooled to 4 ° C. in an ice bath and phosphorus oxychloride (684 g) was added under stirring. Solid 2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethyl ester (670 g) was added in aliquots over 15 minutes. The maximum temperature reached was 18 ° C. The mixture was heated at reflux for 1 h, cooled to 10 ° C. in 1 ice bath, and rapidly added 1.6 L of ice water with vigorous stirring. The temperature rose to 22 ° C. The mixture was left for 30 minutes and the layers were allowed to separate. The temperature reached a maximum of 40 ° C. The aqueous layer was adjusted to pH 12-13 with 10N potassium hydroxide (3.8L) at a rate such that the temperature reached and remained at 55 ° C during the addition. After the addition was completed, the mixture was cooled to 10 ° C. and stirred for 1 hour. The solid was collected by vacuum filtration and washed four times with water to give 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethyl ester (778 g, 100% yield) as a yellow solid.
[930]
[931] 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethyl ester (806 g), potassium hydroxide (548 g), water (2400 mL) and methanol (300 mL) were refluxed under stirring for 2 hours. Cooled to 8 ° C. The mixture was extracted twice with dichloromethane. The aqueous layer was adjusted to pH 4 with 1000 mL of 10N hydrochloric acid while maintaining the temperature below 15 ° C. Water was added to facilitate stirring. The solid was collected by vacuum filtration, washed three times with water and dried under vacuum at 50 ° C. to give 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (645 g, 935% yield) as a yellow solid. Obtained.
[932]
[933] While adding 1- (3-dimethyl-aminopropyl-3-ethylcarbodiimide hydrochloride (2071 g), hydroxybenzotriazole (1460 g), triethylamine (2016 mL) and diethylethylenediamine (1215 mL) , 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (1204 g) and 6020 mL of dimethylformamide were stirred at room temperature The mixture was stirred at room temperature for 20 hours The mixture was stirred for 3000 mL of water, Dilute with 2000 mL brine and 3000 mL saturated sodium bicarbonate solution and adjust the pH to more than 10 with 10 N sodium hydroxide The mixture is extracted twice with 5000 mL 10% methanol in dichloromethane each, the extracts are combined, and anhydrous sulfuric acid Dry over magnesium and rotary evaporate to dryness The mixture was diluted with 1950 mL toluene and again rotary evaporated to dryness The residue was triturated with 3: 1 hexanes: diethyl ether (4000 mL) The solid was vacuum filtered. Pick up, Washed twice with 400 ml of ethyl acetate and dried for 21 h at 34 ° C. vacuum to yield 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) as a light brown solid. -Amide (819 g, 43% yield) was obtained.
[934]
[935] 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl) -amide (809 g), 5-fluoro-1,3-dihydro-indol-2-one ( 438 g), ethanol (8000 mL) and pyrrolidine (13 mL) were heated at 78 ° C. for 3 hours. The mixture was cooled to rt, the solid was collected by vacuum filtration and washed with ethanol. The solid was stirred with ethanol (5900 mL) at 72 ° C. for 30 minutes. The mixture was cooled to room temperature. The solid was collected by vacuum filtration, washed with ethanol and dried under vacuum at 54 ° C. for 130 hours to give 5- [5-fluoro-2-oxo-1,2-dihydro-indole- (3Z) as an orange solid. -Ilidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) -amide (1013 g, 88% yield) was obtained.
[936]
[937] Example 81
[938] 3- [4- (2-Diethylaminoethylcarbamoyl) -3,5-dimethyl-1H-pyrrole-2-ylmethylene] -2-oxo-2,3-dihydro-1H-indole-6-carboxylic acid
[939] 2-oxo-2,3-dihydro-lH-indole-6-carboxylic acid (80 mg, 0.45 mmol) to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-diethylamino Condensation with ethyl) amide gave 210 mg (92%) of the title compound as a yellow orange solid.
[940]
[941] Example 82
[942] 5- (5-dimethylsulfamoyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3carboxylic acid (2-pyrrolidin-1-yl Ethyl) amide
[943] 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide (90 mg, 0.38 mmol) to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2- Condensation with pyrrolidin-1-ylethyl) amide (100 mg) gave 100 mg (54%) of the title compound as a yellow solid.
[944]
[945] Example 83
[946] 5- [5- (3-Chlorophenylsulfamoyl) -2-oxo-1,2-dihydroindol-3-ylidenemethyl] -2,4-dimethyl-1 H-pyrrole-3-carboxylic acid (2-py Rollidin-1-ylethyl) amide
[947] 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (3-chloro-phenyl) amide (120 mg, 0.38 mmol) was replaced with 5-formyl-2,4-dimethyl-1H-pyrrole- Condensation with 3-carboxylic acid (2-pyrrolidin-1-ylethyl) amide (100 mg) gave 150 mg (69%) of the title compound as a yellow orange solid.
[948]
[949] Example 84
[950] 2,4-dimethyl-5- [2-oxo-5- (pyridin-3-ylsulfamoyl) -1,2-dihydroindol-3-ylidenemethyl] -1 H-pyrrole-3-carboxylic acid (2- Pyrrolidin-1-ylethyl) amide
[951] 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid pyridin-3-ylamide (110 mg, 0.38 mmol) in 5-formyl-2,4-dimethyl-1H-pyrrole-3- Condensation with carboxylic acid (2-pyrrolidin-1-ylethyl) amide (100 mg) gave 150 mg (74%) of the title compound as an orange solid.
[952]
[953] Example 85
[954] 3- [3,5-dimethyl-4- (4-methylpiperazin-1-carbonyl) -lH-pyrrole-2-ylmethylene] -4- (2-hydroxyethyl) -1,3-dihydro Indole-2-one
[955] 4- (2-hydroxyethyl) -1,3-dihydroindol-2-one (71 mg, 0.4 mmol) was added to 3,5-dimethyl-4- (4-methyl-piperazine-1-carbonyl) Condensation with -1H-pyrrole-2-carbaldehyde gave 90 mg (55%) of the title compound as an orange solid.
[956]
[957] Example 86
[958] 3- [3,5-dimethyl-4- (4-methylpiperazin-1-carboyl) -lH-pyrrole-2-ylmethylene] -2-oxo-2,3-dihydro-1H-indole-5 Sulfonic acid phenylamide
[959] 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid phenylamide (110 mg, 0.4 mmol) was added to 3,5-dimethyl-4- (4-methylpiperazin-1-carbonyl)- Condensation with 1H-pyrrole-2-carbaldehyde (100 mg) gave 50 mg (24%) of the title compound as a yellow solid.
[960]
[961] Example 87
[962] 5- (5-Dimethylsulfamoyl-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1 H-pyrrole-3-carboxylic acid (2-diethylaminoethyl) amide
[963] 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide (90 mg, 0.38 mmol) to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2- Condensation with diethylaminoethyl) amide (100 mg) gave 80 mg (43%) of the title compound as a yellow solid.
[964]
[965] Example 88
[966] 5- [5- (3-Chlorophenylsulfamoyl) -2-oxo-1,2-dihydroindole-3-ylidenemethyl] -2,4-dimethyl-1 H-pyrrole-3-carboxylic acid (2-di Ethylaminoethyl) amide
[967] 2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (3-chloro-phenyl) amide (120 mg, 3.8 mmol) was replaced with 5-formyl-2,4-dimethyl-1H-pyrrole- Condensation with 3-carboxylic acid (2-diethylaminoethyl) amide (100 mg) gave 80 mg (37%) of the title compound as a yellow solid.
[968]
[969] Example 95
[970] 3- (2-oxo-5-phenyl-1,2-dihydroindole-3-ylidenemethyl) -4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid ethyl ester
[971]
[972] Example 99
[973] 3- (2-Oxo-5-phenylsulfamoyl-1,2-dihydroindole-3-ylidenemethyl) -4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid ethyl ester
[974]
[975] Example 109
[976] 3- [3- (morpholin-4-carbonyl) -4,5,6,7-tetrahydro-2H-isoindol-1-ylmethylene] -2-oxo-2,3-dihydro-1H- Indole-6-carboxylic acid
[977]
[978] Example 112
[979] 5-bromo-3- [3- (pyrrolidin-1-carbonyl) -4,5,6,7-tetrahydro-2H-isoindol-1-ylmethylene] -1,3 dihydro-indole 2-on
[980]
[981] Example 114
[982] 3- (3-Dimethylcarbamoyl-4,5,6,7-tetrahydro-2H-isoindol-1-ylmethylene) -2-oxo-2,3-dihydro-lH-indole-6-carboxylic acid
[983]
[984] Example 115
[985] 4-Methyl-5- (5-methylsulfamoyl-2-oxo-1,2-dihydro-indol-3-ylidenemethyl) -1 H-pyrrole-3-carboxylic acid
[986]
[987] Example 116
[988] {[4-Methyl-5- (4-methyl-5-methylsulfamoyl-2-oxo-1,2-dihydro-indol-3-ylidenemethyl) -1 H-pyrrole-3-carbonyl] -amino } -Acetic acid ethyl ester
[989] 4-methyl-1H-pyrrole-3-carboxylic acid ethyl ester (DO Cheng, TL Bowman and E. LeGoff; J. Heterocyclic Chem .; 1976; 13; 1145-1147) was formylated using Method A and After hydrolysis using Method B, amidation (method C) yielded [(5-formyl-4-methyl-1H-pyrrole-3-carbonyl) -amino] -acetic acid ethyl ester.
[990] 4-Methyl-5-methylaminosulfonyl-2-oxindol (50 mg, 0.21 mmol) was dissolved in [(5-formyl-4-methyl-1H-pyrrole-3-carbonyl)-in ethanol (2 ml). Condensation with amino] -acetic acid ethyl ester (100 mg, 0.42 mmol) and piperidine (0.1 mL) gave 50 mg (52%) of the title compound.
[991]
[992] Example 117
[993] {[4-Methyl-5- (5-methylsulfamoyl-2-oxo-1,2-dihydro-indol-3-ylidenemethyl) -1 H-pyrrole-3-carbonyl] -amino} -ethyl acetate ester
[994] 5-Methylaminosulfonyl-2-oxindol (0.06 g, 0.22 mmol), [(5-formyl-4-methyl-1H-pyrrole-3-carbonyl) -amino] -acetic acid in ethanol (5 mL) A mixture of ethyl ester (0.075 g, 0.27 mmol) and piperidine (2 drops) was heated in a sealed tube at 90 ° C. for 12 hours. After cooling, the precipitate was collected by vacuum filtration, washed with ethanol, triturated with dichloromethane / ether and dried to afford 0.035 g (36%) of the title compound as a yellowish brown solid.
[995]
[996] Example 118
[997] {[4-Methyl-5- (5-methylsulfamoyl-2-oxo-1,2-dihydro-indol-3-ylidenemethyl) -1 H-pyrrole-3-carbonyl] -amino} -acetic acid
[998] A mixture of [(5-formyl-4-methyl-lH-pyrrole-3-carbonyl) -amino] -acetic acid ethyl ester (0.142 g, 0.59 mmol) and 1N NaOH (1.2 mL) in methanol (10 mL) was prepared. Stir at room temperature for 1 hour. The reaction was concentrated and the residue was condensed with 5-methylaminosulfonyl-2-oxindole (0.13 g, 0.48 mmol) and piperidine (0.12 mL) in ethanol (12 mL) to give 0.11 g (52%) of the title compound. ) Was obtained.
[999]
[1000] Example 120
[1001] 5-Methyl-2- (2-oxo-1,2-dihydro-indole-3-ylidenemethyl) -1 H-pyrrole-3-carboxylic acid
[1002]
[1003] Example 121
[1004] 5-Methyl-2- (2-oxo-1,2-dihydro-indole-3-ylidenemethyl) -1 H-pyrrole-3-carboxylic acid ethyl ester
[1005]
[1006] Example 122
[1007] 2- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -5-methyl-1H-pyrrole-3-carboxylic acid ethyl ester
[1008]
[1009] Example 123
[1010] 2- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -5-methyl-1H-pyrrole-3-carboxylic acid
[1011]
[1012] Example 124
[1013] 2- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -5-methyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-ylethyl) -amides
[1014] 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide in a solution of 2-formyl-5-methyl-1H-pyrrole-3-carboxylic acid (250 mg, 1.63 mmol) in dimethylformamide (3 mL) (376 mg, 1.2 equiv), 1-hydroxybenzotriazole (265 mg, 1.2 equiv), triethylamine (0.45 mL, 2 equiv) and 1- (2-aminoethyl) pyrrolidine (0.23 mL, 1.1 Equivalent)) was added. After stirring overnight at room temperature, the reaction was diluted with saturated sodium bicarbonate and brine (extra salt used) and extracted with 10% methanol in dichloromethane. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated to give 130 mg of 2-formyl-5-methyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-yl-ethyl) -amide. Obtained.
[1015] 5-Bromo-2-oxindole (106 mg, 0.5 mmol), 2-formyl-5-methyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-yl- in ethanol (2 mL) A mixture of ethyl) -amide (125 mg, 1 equiv) and piperidine (0.2 mL) was heated in a sealed tube at 80 ° C. for 1 hour and then cooled. The precipitate formed was collected by vacuum filtration, washed with ethanol and ethyl acetate and dried to give the title compound as an orange solid.
[1016]
[1017] Example 125
[1018] 2- (5-Bromo-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -5-methyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl) -amide
[1019] 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (483) to 2-formyl-5-methyl-1H-pyrrole-3-carboxylic acid (320 mg, 2.1 mmol) in dimethylformamide (3 mL) Mg, 1.2 equiv), 1-hydroxybenzotriazole (340 mg, 1.2 equiv), triethylamine (0.59 mL, 2 equiv) and N, N-diethylethylenediamine (0.32 mL, 1.1 equiv) were added . After stirring overnight at room temperature, the reaction was diluted with saturated sodium bicarbonate and brine (extra salt used) and extracted with 10% methanol in dichloromethane. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated to afford 2-formyl-5-methyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl) -amide.
[1020] 5-Bromo-2-oxindole (106 mg, 0.5 mmol), 2-formyl-5-methyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) -amide in ethanol (2 mL) A mixture of (126 mg, 1 equiv) and piperidine (0.2 mL) was heated in an encapsulation tube at 80 ° C. for 1 hour and then cooled. The precipitate was collected by vacuum filtration, washed with ethanol and ethyl acetate and dried to give the title compound as an orange solid.
[1021]
[1022] Example 126
[1023] 2,4-Dimethyl-5- (2-oxo-1,2-dihydroindole-3-ylidenemethyl) -1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) -amide
[1024] 1,3-dihydro-indol-2-one (266 mg, 2 mmol), 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) in ethanol A mixture of -amide (530 mg, 2 mmol) and piperidine (1 drop) was heated at 90 ° C. for 2 hours. The reaction was cooled to room temperature and the resulting precipitate was collected by vacuum filtration, washed with ethanol and dried to give 422 mg (55%) of the title compound as a light yellow solid.
[1025]
[1026] Example 127
[1027] 5- (5-Chloro-2-oxo-1,2-dihydro-indole-3-ylidenemethyl) -2,4-dimethyl-1 H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl)- amides
[1028] 5-Chloro-1,3-dihydro-indol-2-one (335 mg, 2 mmol), 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-diethyl in ethanol A mixture of amino-ethyl) -amide (530 mg, 2 mmol) and piperidine (1 drop) was heated at 90 ° C. for 2 hours. The reaction was cooled to room temperature and the resulting precipitate was collected by vacuum filtration, washed with ethanol and dried to give 565 mg (68%) of the title compound as an orange solid.
[1029]
[1030] Example 128
[1031] 2,4-Dimethyl-5- (2-oxo-1,2-dihydro-indole-3-ylidenemethyl) -1H-pyrrole-3-carboxylic acid (2-pyrrolidine-1-ethyl) -amide
[1032] Condensation of 1,3-dihydro-indol-2-one with 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-pyrrolidin-1-yl-ethyl) -amide to give the title The compound was obtained.
[1033] MS + ve APCI 379 [M ⁺ + 1]
[1034] Example 129
[1035] 5- (5-Fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl) -2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-pyrrolidine-1- Mono-ethyl) -amide
[1036] 5-Fluoro-1,3-dihydro-indol-2-one to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-pyrrolidin-1-yl-ethyl)- Condensation with an amide gave the title compound.
[1037] MS + ve APCI 397 [M ⁺ + 1]
[1038] Scale-up procedure:
[1039] 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (61 g), 5-fluoro-1,3-dihydro-indol-2-one (79 g), ethanol (300 mL) and blood Rollidine (32 mL) was refluxed for 4.5 h. Acetic acid (24 mL) was added to the mixture and reflux was continued for 30 minutes. The mixture was cooled to rt, the solid was collected by vacuum filtration and washed twice with ethanol. The solid was stirred for 130 min in 40% acetone in water (400 mL) containing 12N hydrochloric acid (6.5 mL). The solid was collected by vacuum filtration and washed twice with 40% acetone in water. The solid was dried under vacuum to yield 5- [5-fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3 as an orange solid. -Carboxylic acid (86 g, 79% yield) was obtained.
[1040]
[1041] 5- [5-Fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (100 g) and dimethylformamide (500 mL) was stirred and benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (221 g), 1- (2-aminoethyl) pyrrolidine (45.6 g) and triethylamine (93 mL) was added. The mixture was stirred at ambient temperature for 2 hours. The solid product was collected by vacuum filtration and washed with ethanol. The solid was slurry-washed by stirring in ethanol (500 mL) at 64 ° C. for 1 hour and cooled to room temperature. The solid was collected by vacuum filtration, washed with ethanol and dried under vacuum to afford 5- [5-fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2, 4-Dimethyl] -1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-yl-ethyl) -amide (101.5 g, 77% yield) was obtained.
[1042]
[1043] Example 130
[1044] 5- (5-Chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl) -2,4-dimethyl-1 H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-yl -Ethyl) -amide
[1045] 5-Chloro-1,3-dihydro-indol-2-one to 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-pyrrolidin-1-yl-ethyl) -amide Condensation with to yield the title compound.
[1046]
[1047] Example 131
[1048] 2,4-Dimethyl-5- (2-oxo-1,2-dihydro-indole-3-ylidenemethyl) -1 H-pyrrole-3-carboxylic acid (2-dimethylaminoethyl) -amide
[1049] 1,3-Dihydro-indol-2-one was condensed with 5-formyl-2,4-dimethyl-lH-pyrrol-3-carboxylic acid (2-dimethylamino-ethyl) amide to afford the title compound.
[1050]
[1051] Example 132
[1052] 5- (5-Fluoro-2-oxo-1,2-dihydro-indole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-dimethylaminoethyl) -amide
[1053] Condensation of 5-fluoro-1,3-dihydro-indol-2-one with 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-dimethylaminoethyl) amide gives the title compound. Obtained.
[1054]
[1055] Example 193
[1056] 5- [5-Fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-ethylamino-ethyl )-amides
[1057] 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (2-ethylamino-ethyl) -amide (99 g), ethanol (400 mL), 5-fluoro-2-oxindol (32 g) And pyrrolidine (1.5 g) was refluxed for 3 hours under stirring. The mixture was cooled to rt and the solid was collected by vacuum filtration. The solid was stirred in ethanol at 60 ° C., cooled to room temperature and collected by vacuum filtration. The product was dried under vacuum to afford 5- [5-fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid ( 2-ethylamino-ethyl) -amide (75 g, 95% yield) was obtained.
[1058]
[1059] Example 195
[1060] 5- [5-Fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethyl-N Oxoamino-ethyl) -amide
[1061] Method A:
[1062] 5- [5-Fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino- Ethyl) -amide (598 mg) and dichloromethane (60 mL) were treated with 3-chloroperbenzoic acid (336 mg) in an ice bath and the mixture was stirred at rt overnight. The solvent was rotary evaporated and the residue suspended in methanol (20 mL). Water (20 mL) containing sodium hydroxide (240 mg) was added and the mixture was stirred for 1 hour. The precipitate was collected by vacuum filtration, washed with 5 ml of water and dried under vacuum to give 5- [5-fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl as an orange solid. ] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethyl-N-oxoamino-ethyl) -amide (510 mg, 82% yield) was obtained.
[1063]
[1064] Method B:
[1065] 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) -amide (10 g) is suspended in dichloromethane (100 mL) and cooled in an ice bath. 3-Chloro-peroxybenzoic acid (13.1 g) is added under stirring and the mixture is allowed to warm to room temperature and then stirred overnight. The mixture was rotary evaporated to dryness and chromatographed on a silica gel column eluting with 20% methanol in dichloromethane. Fractions containing product were combined and rotary evaporated to dryness to 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethyl-N-oxoamino-ethyl) -amide (9 g, 83% Yield).
[1066] 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethyl-N-oxoamino-ethyl) -amide (9 g), 5-fluoro-1,3-dihydro-indole 2-one (9 g, 83% yield) and pyrrolidine (9 g, 83% yield (0.1 g)) were refluxed in ethanol (30 mL) for 4 hours. The mixture was cooled in an ice bath and the precipitate was collected by vacuum filtration and washed with ethanol. The solid was stirred in ethyl acetate (30 mL), collected by vacuum filtration, washed with ethyl acetate and dried under vacuum to give 5- [5-fluoro-2-oxo-1,2-dihydro as an orange solid. -Indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethyl-N-oxoamino-ethyl) -amide (10.3 g, 80% yield) was obtained It was.
[1067]
[1068] Example 190
[1069] 5- [5-Fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2- (pyridine-1 -Yl) ethyl] -amide
[1070] 5- [5-Fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-lH-pyrrole-3-carboxylic acid (120 mg, 0.4 mmol) Was purchased from EDC, HCl (96 mg, 0.5 mmol), anhydrous 1-hydroxy-benztriazole (68 mg, 0.5 mmol) and Aldrich in anhydrous DMF (3 mL) for 2-3 days at room temperature. Shake with one 2- (2-aminoethylpyridine. The reaction mixture was diluted with 1M NaHCO 3 (1.5 mL) followed by 8 mL of water The precipitated crude product was collected by filtration, washed with water, dried and Purification by crystallization or chromatography to give 5- [5-fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-lH-pyrrole-3- Carboxylic acid [2- (pyridin-1-yl) -ethyl] amide was obtained.
[1071] Example 189
[1072] 5- [5-Chloro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2- (pyridine-1- (Ethyl) amide
[1073] 5- [5-Fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid 5- [5-chloro Previous practice except replacing with 2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-lH-pyrrole-3-carboxylic acid (127 mg) Treated as described in the examples to give 5- [5-chloro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-lH-pyrrole-3-carboxylic acid [ 2- (pyridin-1-yl) ethyl] amide was obtained.
[1074] Example 192
[1075] 5- [5-Bromo-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2- (pyridine-1 -Yl) ethyl] amide
[1076] 5- [5-Fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-lH-pyrrole-3-carboxylic acid Except for replacing with mother-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (145 mg) Treatment as described in Example 190 resulted in 5- [5-bromo-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3- Carboxylic acid [2- (pyridin-1-yl) ethyl] amide was obtained.
[1077] Example 191
[1078] 5- [2-Oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2- (pyridin-1-yl) ethyl] amides
[1079] 5- [5-Fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidene-methyl] -2,4-dimethyl-lH-pyrrole-3-carboxylic acid 5- [2- Example 190 described above, except that oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-lH-pyrrole-3-carboxylic acid (113 mg) was substituted. Treat as described to 5- [2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-lH-pyrrole-3-carboxylic acid [2- (pyridine-1- Il) ethyl] amide.
[1080] Example 203
[1081] 5- [5-Cyano-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2- (pyridine-1 -Yl) ethyl] amide
[1082] 5- [5-Fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-lH-pyrrole-3-carboxylic acid 5- [5-sia The above procedure, except that it was replaced by no-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-lH-pyrrole-3-carboxylic acid (123 mg). Treated as described in Example 190 to treat 5- [5-cyano-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3- Carboxylic acid [2- (pyridin-1-yl) ethyl] amide was obtained.
[1083] Examples 142, 186, 187, 188 and 204
[1084] Except for replacing 2- (2-aminoethyl) pyridine with 1- (2-aminoethyl) pyrrolidine (purchased from Aldrich Chemical Company, Inc.) Treatment as described in Examples 190, 189, 191, 192 and 203 gave the desired compound.
[1085] Examples 143-147
[1086] 2- (2-aminoethyl) pyridine is disclosed in U.S. Patent No. 2613212 (1950) issued to 1- (2-aminoethyl) imidazolin-2-one (Rohm & Hass Co.). Prepared by heating dimethyl carbonate in a sealed flask with bis (2-aminoethyl) amine (2 equiv) for 30 min according to the procedure described, eluting the crude product with chloroform-methanol-aqueous ammonia 80: 25: 2. Was treated as described in Examples 190, 189, 191, 192 and 203 above except that the mixture was purified on silica gel) to give the desired compound.
[1087] Examples 148-151 and 184
[1088] 2- (2-aminoethyl) pyridine to 4- (2-aminoethyl) piperazine-1-acetic acid ethyl ester (prepared as follows: piperazine-1-acetic acid ethyl ester (11.22 g) at 0 ° C. ethyl Treatment with iodoacetonitrile (5.0 mL) in the presence of potassium carbonate (6.9 g) in acetate (260 mL) After completion of the addition of iodoacetonitrile (45 min), the reaction mixture was allowed to stand at room temperature for 11 h. The reaction mixture was filtered and the filtrate was evaporated The residue was hydrogenated in ethanol in the presence of cobalt boride (prepared from CoCl 2 and sodium borohydride) at 50 psi for 2 days at room temperature. And chromatographic purification using an elution mixture of chloroform-methanol-aqueous ammonia 80: 25: 2 to give the desired amine (3.306 g) as a pale yellow oil). , 191, 1 Treatment as described in 92 and 203 gave the desired compound.
[1089] Examples 152 to 153
[1090] 2- (2-aminoethyl) pyridine to 2-[(2-aminoethylamino)] acetonitrile (prepared as follows: A solution of iodoacetonitrile (50 mmol) in ethanol (80 mL) was added for 30 minutes. Was added to a solution of ethylene diamine (150 mL) in ethanol (60 mL) at 0 ° C. Stirred continuously at 0 ° C. for an additional 1 hour and then at room temperature for 14 hours 55 mmol of potassium carbonate was added. The mixture was stirred for 30 minutes, filtered and the filtrate was concentrated at room temperature The residue was purified on silica gel using an elution mixture of chloroform-methanol-aqueous ammonia 80: 15: 1.5 and immediately used 2-[( 2-aminoethylamino)]-acetonitrile (3.550 g) was obtained, except that the compound was treated as described in Examples 190, 189, 191, 192 and 203 to obtain the desired compound.
[1091] Examples 154-158
[1092] 2- (2-aminoethyl) pyridine to 1- (3-aminopropyl) -azin-2-one (lithium hydroxide (1 hr), DBU 5 ml, water 2 ml, lithium hydroxide hydrate 420 mg Prepared according to the procedure in Kraft A .: J. Chem. Soc. Perkin Trans. 1, 6, 1999, 705-14, except that hydrolysis of DBU was performed at 145 ° C. in the presence of The crude product was purified on silica using chloroform-methanol-aqueous ammonia 80: 40: 4 as the elution mixture to afford 1- (3-aminopropyl) azin-2-one (4.973 g, 87% yield) Except as replaced by Example 190, 189, 191, 192 and 203 was treated as described to give the desired compound.
[1093] Examples 133-135, 159 and 200
[1094] 2- (2-aminoethyl) pyridine was prepared by heating a mixture of N-acetyl ethylene diamine (ethylene diamine (1.5 equiv) and ethyl acetate at 160 ° C. for 1 hour in an enclosed vessel. In 56% yield by vacuum distillation). Obtain the desired product, N-acetylethylene diamine is also obtained from Aldrich) and treated as described in Examples 190, 189, 191, 192 and 203 to give the desired compound.
[1095] Examples 146-140
[1096] 2- (2-aminoethyl) pyridine was converted to 1- (3-aminopropyl) -tetrahydro-pyrimidin-2-one (Kraft. A: J. Chem. Soc. Perkin Trans. 1, 6, 1999, 705-14, prepared in the same manner as 1- (3-aminopropyl) -azin-2-one: simply, 1,3,4,6,7,8-hexahydro-2H Pyridino [1,2-a] pyrimidine (4.939 g), lithium hydroxide hydrate (918 mg) and 2 ml of water were heated in a sealed container without solvent at 145 ° C. for 1 hour. Example 190, 189, 191, 192 and 203, except that purification on silica column with methanol-aqueous ammonia 80: 40: 4 replaced with pure amine (5.265 g, 94% yield)) Treatment as described yields the desired compound.
[1097] Examples 141, 160-162 and 185
[1098] 2- (2-aminoethyl) pyridine to 1- (2-aminoethyl) -piperazin-2-one (prepared as follows: pure tert-butyldiphenylsilyl chloride (25 mL, 97.7 mmol) in 5 minutes Was added dropwise at room temperature into a solution of DBU (19.5 mL, 130 mmol) and bis (2-aminoethyl) amine (4.32 mL, 40 mmol) in anhydrous dimethyl acetamide (80 mL) while cooling on a water bath. Bromoacetic acid ethyl ester (6.70 mL, 60 mmol) was added while cooling to room temperature The reaction was stirred for 25 minutes and then evaporated in high vacuum The residue was dissolved in methanol (200 mL), KHCO ₃ (10 g) and KF (12 g, 200 mmol) were added and the mixture was stirred for 5 hours at 60 ° C. 10 g of Na ₂ CO ₃ was added, stirred for 10 minutes, cooled and filtered. The residue was extracted with hexane (2 × 250 mL) hexane-fire The crude material was dissolved in ethanol (60 mL), filtered and evaporated The residue was purified on a silica column in chloroform-methanol-aqueous ammonia 80: 40: 4 to give pure amine (4.245 g, 74% yield) Treatment as described in Examples 190, 189, 191, 192, and 203, except for replacement with)), gave the desired compound.
[1099] Examples 163-167
[1100] 2- (2-aminoethyl) pyridine was converted to 3-[(2-aminoethyl) amino] propionitrile (as described in Israel, M. et al ., J. Med Chem . 7, 1964, 710-16). The desired compound was treated as described in Examples 190, 189, 191, 192 and 203 except at room temperature with the substitution of ethylene diamine (150 mmol) and acrylonitrile (50 mmol) in THF. Obtained.
[1101] Example 168
[1102] 5- (5-Fluoro-2-oxo-1,2-dihydro-indole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2- (4-methylpiperazine -1-yl) -ethyl] -amide
[1103] 5- [5-fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid in 20 ml reaction tube (90 mg), BOP reagent (199 mg) was added to the yellow turbid stirred mixture of DMF (0.8 mL) and TEA (0.084 mL). The mixture became clear after 5 minutes. 2- (4-methylpiperazin-1-yl) ethylamine ¹ (51 mg) was added to the clear mixture. The resulting solution was stirred overnight at room temperature. A yellow solid product precipitated out of the reaction system. Thin layer chromatography (10% methanol in methylene chloride) showed that all starting material had been converted to the product. The solid was isolated by vacuum filtration and washed once with ethanol (1 mL). The solid was sonicated for 20 min in diethyl ether (2 mL) and collected by vacuum filtration. After drying in vacuo, 5- (5-fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (4- Methylpiperazin-1-yl-ethyl) -amide (79 mg, 62% yield) was obtained.
[1104]
[1105] Example 169
[1106] 5- (5-Chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl) -2,4-dimethyl-lH-pyrrole-3-carboxylic acid (4-methylpiperazin-1-yl -Ethyl) -amide
[1107] 5- [5-Fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-lH-pyrrole-3-carboxylic acid 5- [5-chloro 2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-lH-pyrrole-3-carboxylic acid (95 mg, 0.3 mmol) except that Following the procedure in Example 168 above 5- (5-chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid ( 4-Methylpiperazin-1-yl-ethyl) -amide (76 mg, 58%) was obtained.
[1108]
[1109] Example 170
[1110] 5- (5-Bromo-2-oxo-1,2-dihydro-indole-3-ylidenemethyl) -2,4-dimethyl-lH-pyrrole-3-carboxylic acid (4-methylpiperazin-1- Mono-ethyl) -amide
[1111] 5- (5-Chloro-2-oxo-1,2-dihydro-indole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid 5- (5-bromo-2 -(Oxo-1,2-dihydro-indole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid except that 5- ( 5-Bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (4-methylpiperazin-1-yl-ethyl ) -Amide (39 mg, 54%) was obtained and obtained from SU011670 (54 mg, 0.15 mmol).
[1112]
[1113] Example 172
[1114] 5- (2-Oxo-1,2-dihydro-indol-3-ylidenemethyl) -2,4-dimethyl-lH-pyrrole-3-carboxylic acid (4-methylpiperazin-1-yl-ethyl)- amides
[1115] 5- (5-Fluoro-2-oxo-1,2-dihydro-indole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid SU014900 5- (2-oxo- 5- (2- according to the procedure described in Example 168 above, except that 1,2-dihydro-indole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid was replaced. Oxo-1,2-dihydro-indol-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (4-methylpiperazin-1-yl-ethyl) -amide (136 mg, 84%) was obtained from (112.8 mg, 0.4 mmol).
[1116]
[1117] Example 171
[1118] 5- [2-Oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2- (3,5-dimethylpiperazine- 1-yl) ethyl) amide
[1119] 5- [2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (112.8 mg, 0.4 mmol) in 20 ml reaction tube ), A yellow cloudy, stirred mixture of DMF (0.5 mL) and triethylamine (0.111 mL) was added BOP reagent (265 mg). The mixture became clear after 5 minutes. 2- (2,6-dimethylpiperazin-1-yl) ethylamine (68.6 mg) (Tapia, L. Alonso-Cires, P. Lopez-Tudanca, R. Mosquera, L. Labeaga, A. Innerarity, A. Orjales, J. Med. Chem. , 1999, 42, 2870-2880) were added to the clear mixture. The resulting solution was stirred overnight at room temperature. Thin layer chromatography (10% methanol in methylene chloride) showed that the starting materials were all converted to the product. The reaction mixture was evaporated to dryness, purified by flash chromatography (CH ₂ Cl ₂ / CH ₃ OH = 20/1 to 15/1), and then recrystallized to give 5- [2-oxo-1,2-di Hydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2- (3,5-dimethylpiperazin-1-yl) ethyl) amide (83 mg, 50 % Yield) was obtained.
[1120]
[1121] Example 173
[1122] 5- [5-Fluoro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2- (3,5 -Dimethylpiperazin-1-yl) ethyl) amide
[1123] The desired compound was obtained following the procedure described in Example 168 above (60 mg, 0.2 mmol).
[1124]
[1125] Example 174
[1126] 5- [5-Chloro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2- (3,5- Dimethylpiperazin-1-yl) ethyl) amide
[1127] The desired compound (31.2 mg, 34%) was prepared in accordance with the procedure for Example 171 above with 5- [5-chloro-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2. Obtained from, 4-dimethyl-1H-pyrrole-3-carboxylic acid (63 mg, 0.2 mmol).
[1128]
[1129] Example 175
[1130] 5- [5-Bromo-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2,4-dimethyl-1H-pyrrole-3-carboxylic acid [2- (3,5 -Dimethylpiperazin-1-yl) ethyl) amide
[1131] The desired compound (40 mg, 40%) was prepared according to the procedure described in Example 171 to 5- [5-bromo-2-oxo-1,2-dihydro-indole- (3Z) -ylidenemethyl] -2. Obtained from, 4-dimethyl-lH-pyrrole-3-carboxylic acid (74 mg, 0.2 mmol).
[1132]
[1133] Example 205
[1134] (3Z) -3-[(3,5-dimethyl-lH-pyrrol-2-yl) -methylidene] -1- [1- (4-methylpiperazinyl) methyl] -1,3-dihydro- 2H-indole-2-one
[1135] N-methylpiperazine (10 g, 100 mmol) was dissolved in methanol (100 mL) with aqueous formaldehyde (10 g of 38% solution, 100 mmol) and 3- (3,5-dimethyl-lH-pyrrol-2-ylmethylidene) To a stirred solution of -1,3-dihydro-indol-2-one (2.38 g, 10 mmol) was added. The solution was heated at 60 ° C. for 1 hour, concentrated to a small volume, the precipitate was filtered off, washed with methanol and dried to give 2.38 g of the title compound (melting point 160 to 164 ° C.).
[1136]
[1137] (3Z) -3-[(3,5-dimethyl-1H-pyrrole-2-yl) -methylidene] -1- [1- (4-methylpiperazinyl) -methyl] -1,3-dihydro -2H-indol-2-one was converted to the dihydrochloride salt.
[1138] Example 206
[1139] (3Z) -3-[(3,5-dimethyl-1H-pyrrole-2-yl) -methylidene] -1- (1-pyrrolidinylmethyl) -1,3-dihydro-2H-indole-2 Synthesis of -one
[1140] Pyrrolidine (450 mg, 6.3 mmol) was dissolved in aqueous formaldehyde (580 mg, 6.0 mmol) in methanol (50 mL) and 3- (3,5-dimethyl-lH-pyrrole-2-ylmethylidene). -1,3-dihydro-indol-2-one (900 mg, 3.8 mmol) was added after 15 minutes, the solution was cooled to 0 ° C., the precipitate was filtered off, washed with water and dried to give 1.08 g of the title compound Was obtained (melting point: 129 to 132 ° C).
[1141]
[1142] Biological Example
[1143] Protein Kinase Inhibitors of Formula (I)
[1144] The following assays can be used to determine the level of activity and effect of a compound of formula (I) on one or more protein kinases (hereafter PK). Similar assays for any PK can be designed according to the same scheme using techniques well known in the art.
[1145] A. Analysis Procedure
[1146] Some of the assays described herein are described in the Enzyme-Linked Immunosorbent Sandwich Assay (ELISA) format (Voller et al., 1980, "Enzyme-Linked Immunosorbent Assay," Manual of Clinical Immunology, 2d ed., Rose and Fredman, Am "). . Soc. Of Microbiology, Washington, D.C., pp. 359-371]. The general procedure is as follows: Compounds of Formula (I) are introduced into cells that naturally or recombinantly express test kinases for a selected period of time after the addition of a ligand known to activate the receptor if the test kinase is a receptor. Cells are lysed and the lysates are transferred to wells of an ELISA plate already coated with a specific antibody that recognizes the substrate of the enzyme-phosphorylation reaction. The non-substrate component of the cell lysate is washed away and an antibody that specifically recognizes phosphotyrosine is used to detect the amount of phosphorylation on the substrate as compared to control cells that are not in contact with the test compound.
[1147] Current preferred protocols for conducting ELISA experiments for specific PKs are presented below. However, these protocols for measuring the activity of compounds against cytosolic tyrosine kinase (CTK) and serine threonine tyrosine kinase (STK), as well as other receptor tyrosine kinases (RTK), are adopted within the knowledge category of those skilled in the art. It is preferable. Other assays described herein determine the amount of DNA produced in response to the activity of a test kinase, which is a general measure of the proliferative response. The general procedure for this analysis is as follows. The compound is added to a cell that naturally or recombinantly expresses the test kinase for a selected period of time after the addition of a ligand known to activate the receptor if the test kinase is a receptor. After incubation at least overnight, a DNA-labeled reagent such as 5-bromodeoxyuridine (BrdU) or H ³ -thymidine is added. The amount of labeled DNA is detected by using an anti-BrdU antibody or by measuring radioactivity and compared to control cells that are not in contact with the test compound.
[1148] GST-FLK-1 Bioassay
[1149] This assay analyzes the tyrosine kinase activity of GST-Flk 1 against poly (glu, tyr) peptides.
[1150] Substances and Reagents:
[1151] 1. Corning 96-well ELISA plate (Corning Catalog No. 5805-96).
[1152] 2. Glue tyr 4: 1, lyophilisate (Sigma Catalog No. P0275).
[1153] 3. Preparation of glu, tyr (pEY) -coated assay plates: 2 μg / well of glu, tyr (pEY) in 100 μl of PBS and coated at 4 ° C. at room temperature or overnight for 2 hours. Secure with. Cover the plate well to prevent evaporation.
[1154] 4. PBS buffer: For 1 L, mix 0.2 g KH ₂ PO ₄ , 1.15 g Na ₂ HPO ₄ , 0.2 g KCl and 8 g NaCl in about 900 mL dH ₂ O. If all reagents are dissolved, adjust the pH to 7.2 with HCl. The total volume is made up to 1 L with dH ₂ O.
[1155] 5. PBST Buffer: Add 1.0 ml of Tween-20 to 1 liter of PBS buffer.
[1156] 6. TBB-blocking buffer: For 1 L, mix 1.21 g TRIS, 8.77 g NaCl, 1 mL TWEEN-20 in about 900 mL dH ₂ O. Adjust the pH to 7.2 using HCl. 10 g of BSA is added and stirred to dissolve. The total volume is made up to 1 L with dH ₂ O. Filtration removes particulate matter.
[1157] 7. 1% BSA in PBS: To prepare a 1 × working solution, 10 g of BSA is added to about 990 ml of PBS buffer and stirred to dissolve. The total volume is made up to 1 L with dH ₂ O. Filtration removes particulate matter.
[1158] 8. 50 mM HEPES at pH 7.5.
[1159] 9. GST-Flk 1cd (SUGEN, Inc.) purified from sf9 recombinant baculovirus transformation.
[1160] 10. 4% DMSO in dH ₂ O.
[1161] 11. 10 mM ATP in dH ₂ O.
[1162] 12. 40 mM MnCl ₂ .
[1163] Is mixed with HEPES (pH 7.5) 10㎖, 5M NaCl 1㎖, 100mM sodium ortho vanadate 40㎕ and dH ₂ O to 5% BSA in dH ₂ O 0.4㎖ 88.56㎖: 13. Kinase Dilution Buffer (KDB).
[1164] 14. NUNC 96-well V bottom polypropylene plate, Applied Scientific Catalog No. AS-72092.
[1165] 15. EDTA: 14.12 g of ethylenediaminetetraacetic acid (EDTA) are mixed in about 70 ml of dH ₂ O. Add 10N NaOH until EDTA is dissolved. Adjust pH to 8.0. The total volume is made up to 100 ml using dH ₂ O.
[1166] ¹⁰ 16. Antibody dilution buffer: 5% BSA 10㎖ are mixed in the PBS buffer and TBST 89.5㎖.
[1167] 17. Anti-phosphotyrosine mononuclear antibody conjugated to horseradish peroxidase (PY99 HRP, Santa Cruz Biotech).
[1168] 18. 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, Moss, Cat.No. ABST).
[1169] 19. 10% SDS.
[1170] step:
[1171] 1. As described in the third step of the material and reagent portions, Corning 96-well ELISA plates are coated with 2 μg polyEY peptide in sterile PBS.
[1172] 2. Turn the plate over to remove the unbound liquid from the wells. Wash once with TBST. Tap on a plate of paper towel to remove excess liquid.
[1173] 3. Add 100 μl of 1% BSA in PBS to each well. Incubate with shaking at room temperature for 1 hour.
[1174] 4. Repeat step 2.
[1175] 5. Wet the wells with 50 mM HEPES pH 7.5 (150 μl / well).
[1176] 6. Dilute the test compound four times with dH ₂ O / 4% DMSO to the desired final assay concentration in a 96-well polypropylene plate.
[1177] 7. Add 25 μl of diluted test compound to the ELISA plate. 25 μl of dH ₂ O / 4% DMSO is placed in the control well.
[1178] 8. Add 25 μL of 40 mM MnCl ₂ to each well as 4 × ATP (2 μM).
[1179] 9. Add 25 μl 0.5 M EDTA to the negative control wells.
[1180] 10. Dilute GST-Flk 1 to 0.005 μg (5 ng) / well using KDB.
[1181] 11. Add 50 μl of diluted enzyme to each well.
[1182] 12. Incubate for 15 minutes at room temperature with shaking.
[1183] 13. Stop the reaction by adding 50 μl 250 mM EDTA (pH 8.0).
[1184] 14. Wash three times with TBST and tap on a plate of paper towel to remove excess liquid.
[1185] 15. Add anti-phosphotyrosine HRP conjugate in antibody dilution buffer at 100 μl / well at a dilution of 1: 5,000. Incubate with shaking at room temperature for 90 minutes.
[1186] 16. Wash as in step 14.
[1187] 17. Add 100 μl of room temperature ABTS solution to each well.
[1188] 18. Incubate with shaking for 10-15 minutes. Remove the bubbles.
[1189] 19. Add 20 μl of 10% SDS to each well to stop the reaction.
[1190] 20. Read the results on a Dynatech MR7000 ELISA reader using a test filter at 410 nM and a reference filter at 630 nM.
[1191] PYK2 Bioassay
[1192] This assay is used to measure the in vitro kinase activity of HA epitope-labeled full length pyk2 (FL.pyk2-HA) in ELISA assays.
[1193] Substances and Reagents:
[1194] 1. Corning 96-well ELISA plate.
[1195] 2. 12CA5 mononuclear anti-HA antibody (Suzen Inc.).
[1196] 3. PBS (Dulbecco's Phosphate-Buffered Saline, Dulbecco's Phosphate-Buffered Saline) (Gibco Catalog No. 450-1300EB).
[1197] 4. TBST buffer: For 1 L, 8.766 g NaCl, 6.057 g TRIS, 1 ml Triton X-100 are mixed in about 900 ml dH ₂ O. Adjust the pH to 7.2 and bring the total volume to 1 l.
[1198] 5. Blocking Buffer: For 1 L, mix 100 g of 10% BSA, 12.1 g of 100 mM TRIS, 58.44 g of 1 M NaCl, 10 ml of 1% TWEEN-20.
[1199] 6. FL.pyk2-HA (Susen Inc.) purified from sf9 cell lysate.
[1200] 7. 4% DMSO in MilliQue H ₂ O.
[1201] 8. 10 mM ATP in dH ₂ O.
[1202] 9. 1 M MnCl ₂ .
[1203] 10. 1 M MgCl ₂ .
[1204] 11. 1M Dithiothreitol (DTT).
[1205] 12. 10 × Kinase Buffered Phosphorylation: 5.0 ml of 1 M HEPES (pH 7.5), 0.2 ml of 1 M MnCl ₂ , 1.0 ml of 1 M MgCl _{2, and} 1.0 ml of 10% Triton X-100 in 2.8 ml of dH ₂ O. Immediately before use, 0.1 ml of 1M DTT is added.
[1206] 13. NUNC 96-well V bottom polypropylene plate.
[1207] 14. 500 mM EDTA in dH ₂ O.
[1208] 15. Antibody Dilution Buffer: For 100 ml, 1 ml of 5% BSA / PBS and 1 ml of 10% Tween-20 in 88 ml of TBS.
[1209] 16. HRP-conjugated anti-Ptyr PY99 (Santa Cruz Biotech, Cat. No. SC-7020).
[1210] 17. ABTS, Moss, Cat. No. ABST-2000.
[1211] 18. 10% SDS.
[1212] step:
[1213] 1. Corning 96-well ELISA plates are coated with 0.5 μg / well anti-HA antibody in PBS. Store overnight at 4 ° C.
[1214] 2. Invert the plate to remove unbound HA antibody from the wells. Wash the plate with dH ₂ O. Tap on a plate of paper towel to remove excess liquid.
[1215] 3. Add 150 μl of blocking buffer to each well. Incubate with shaking at room temperature for 30 minutes.
[1216] 4. Wash the plate 4 times with TBS-T.
[1217] 5. Dilute the lysate in PBS (1.5 μg lysate / 100 μl PBS).
[1218] 6. Add 100 μl of diluted lysate to each well. Shake at room temperature for 1 hour.
[1219] 7. Wash as in step 4.
[1220] 8. Add 50 [mu] l of 2X kinase buffer to an ELISA plate containing captured pyk2-HA.
[1221] 9. Add 25 μL of 400 μM test compound in 4% DMSO to each well. For control wells, only 4% DMSO is used.
[1222] 10. Add 25 μL 0.5 M EDTA to the negative control wells.
[1223] 11. Add 25 μl 20 μM ATP to each well. Incubate with shaking for 10 minutes.
[1224] 12. Stop the reaction by adding 25 μl 500 mM EDTA (pH 8.0) to each well.
[1225] 13. Wash as in step 4.
[1226] 14. Add 100 μl of anti-Ptyr diluted 1: 5,000 conjugated HRP in antibody dilution buffer to each well. Incubate with shaking at room temperature for 1 hour.
[1227] 15. Wash three times with TBST and once with PBS.
[1228] 16. Add 100 μL ABST solution to each well.
[1229] 17. If necessary, add 20 μl of 10% SDS to each well to stop the reaction.
[1230] 18. Read the plate on the ELISA reader using the test filter at 410 nM and the reference filter at 630 nM.
[1231] FGFR1 Bioassay
[1232] This assay is used to measure the ex vivo kinase activity of FGF1-R in ELISA assays.
[1233] Substances and Reagents
[1234] 1.Costar 96-well ELISA plate (Corning Catalog No. 3369).
[1235] 2. Glu-Tyr (Sigma Catalog No. PO275).
[1236] 3. PBS (Gibco Catalog No. 450-1300EB).
[1237] 4. 50 mM HEPES buffer solution.
[1238] 5. Blocking buffer (5% BSA / PBS).
[1239] 6. Purified GST-FGFR1 (Sujen Incorporated)
[1240] 7. Kinase Dilution Buffer: Mix 500 μl 1 M HEPES (Gibco), 20 μl 5% BSA / PBS, 10 μl 100 mM sodium orthovanadate and 50 μl 5M sodium chloride.
[1241] 8. 10 mM ATP.
[1242] 9. ATP / Manganese Chloride Phosphorylation Mixture: Mix 20 μL of ATP, 400 μL of ₁ M manganese chloride and 9.56 mL of dH ₂ O.
[1243] 10. NUNC 96-well V bottom polypropylene plate (Applied Scientific Catalog No. AS-72092).
[1244] 11. 0.5 M EDTA.
[1245] 12. 0.05% TBST: Add 500 μl TWEEN to 1 L of TBS.
[1246] 13. Rabbit multinuclear anti-phosphotyrosine serum (Suzen Inc.).
[1247] 14. Goat anti-rabbit IgG peroxidase conjugate (Biosource, catalog ALI0404).
[1248] 15. ABTS solution.
[1249] 16. ABTS / H ₂ O ₂ solution.
[1250] step:
[1251] 1. Coat a Costa 96 well ELISA plate with 1 μg / well poly (Glu, Tyr) in 100 μl PBS. Store overnight at 4 ° C.
[1252] 2. Wash the coated plate once with PBS.
[1253] 3. Add 150 μl 5% BSA / PBS blocking buffer to each well. Incubate with shaking at room temperature for 1 hour.
[1254] 4. Wash the plate twice with PBS and once with 50 mM HEPES. Tap on a plate of paper towel to remove excess liquid and bubbles.
[1255] 5. Add 25 μL of 0.4 mM test compound or 4% DMSO (control) in 4% DMSO to the plate.
[1256] 6. Dilute purified GST-FGFR1 (5 ng kinase / 50 μl KDB / well) in kinase dilution buffer.
[1257] 7. Add 50 μl of diluted kinase to each well.
[1258] 8. Initiate the kinase reaction by adding 25 μl / well of freshly prepared ATP / Mn ⁺⁺ (0.4 mL of freshly prepared 1M manganese chloride, 40 μl of 10 mM ATP, 9.56 mL of dH ₂ 0).
[1259] 9. This is a fast kinase reaction and should be stopped using 25 μl 0.5 M EDTA in a similar manner to the ATP addition.
[1260] 10. Wash the plate 4 times with fresh TBST.
[1261] 11. Preparation of antibody dilution buffer: Mix 50 ml, 5 ml of 5% BSA, 250 µl 5% milk and 50 µl 100 mM sodium vanadate and make a final volume with 0.05% TBST.
[1262] 12. Add 100 μl / well of anti-phosphotyrosine (1: 0000 dilution in ADB). Incubate with shaking for 1 hour at room temperature.
[1263] 13. Wash as in step 10.
[1264] 14. Add 100 μl / well of biosource goat anti-rabbit IgG peroxidase conjugate (1: 6000 dilution in ADB). Incubate with shaking for 1 hour at room temperature.
[1265] 15. Wash as in step 10 and then with PBS to remove foam and excess TWEEN.
[1266] 16. Add 100 μL of ABTS / H ₂ O ₂ solution to each well.
[1267] 17. Incubate with shaking for 10-20 minutes. Remove the bubbles.
[1268] 18. Analytical Reading on Dynatech MR 7000 ELISA Reader: The filter is tested at 410 nM and the reference filtrate is read at 630 nM.
[1269] EGFR Bioassay
[1270] This assay is used to measure the ex vivo kinase activity of FGF1-R in ELISA assays.
[1271] Substances and Reagents:
[1272] 1. Corning 96-well ELISA plate.
[1273] 2. SUMO1 mononuclear anti-EGFR antibody (Susen Inc.).
[1274] 3. PBS.
[1275] 4. TBST buffer.
[1276] 5. Blocking buffer: For 100 ml, 5.0 g of Carnation Instant Milk® is mixed with 100 ml of PBS.
[1277] 6. A431 cell lysate (Suzen Inc.).
[1278] 7. TBS buffer.
[1279] 8. TBS + 10% DMSO: For 1 L, mix 1.514 g TRIS, 2.192 g NaCl and 25 mL DMSO and bring the total volume to 1 L with dH ₂ O.
[1280] 9. ATP (adenosine-5'-triphosphate, Equine muscle, Sigma Cat. No A-5394), 10 mM solution in dH ₂ O. This reagent should be formulated immediately before use and stored on ice.
[1281] 10. 1.0 mM MnCl ₂ .
[1282] 11. ATP / MnCl ₂ phosphorylation mixture: To make up 10 ml, mix 300 μl of 1 mM ATP, 500 μl MnCl ₂ and 9.2 ml of dH ₂ O. Prepared immediately before use and stored on ice.
[1283] 12. NUNC 96-well V bottom polypropylene plate.
[1284] 13. EDTA.
[1285] 14. Rabbit multinuclear anti-phosphotyrosine serum (Suzen Inc.).
[1286] 15. Goat anti-rabbit IgG peroxidase conjugate (Biosource, catalog ALI0404).
[1287] 16. ABTS.
[1288] 17. 30% hydrogen peroxide.
[1289] 18. ABTS / H ₂ O ₂ .
[1290] 19. 0.2 M HCl.
[1291] step:
[1292] 1. Coat Corning 96-well ELISA plates with 0.5 μg SUMO1 in 100 μl PBS and store at 4 ° C. overnight.
[1293] 2. Remove unbound SUMO1 from the well by inverting the plate to remove the liquid. Wash once with dH ₂ O. Tap on a plate of paper towel to remove excess liquid.
[1294] 3. Add 150 μl of blocking buffer to each well. Incubate with shaking at room temperature for 30 minutes.
[1295] 4. Wash the plate three times with deionized water and then once with TBST. Tap on a plate of paper towel to remove excess liquid and bubbles.
[1296] 5. Dilute the lysate in PBS (7 μg lysate / 100 μl PBS).
[1297] 6. Add 100 μl of diluted lysate to each well. Shake at room temperature for 1 hour.
[1298] 7. Clean the plate as in step 4 above.
[1299] 8. Add 120 μl of TBS to the ELISA plate containing the captured EGFR.
[1300] 9. Dilute test compound to 1:10 in TBS and place in well.
[1301] 10. Add 13.5 μl of diluted test compound to the ELISA plate. To the control wells add 13.5 μL of TBS in 10% DMSO.
[1302] 11. Incubate with shaking at room temperature for 30 minutes.
[1303] 12. Add 15 μl phosphorylation mixture to all wells except the negative control wells. The final well volume should be about 150 μl with 3 μM ATP / 5 mM MnCl ₂ in each well. Incubate with shaking for 5 minutes.
[1304] 13. Stop the reaction by adding 16.5 μl EDTA solution with shaking. Shake for an additional 1 minute.
[1305] 14. Wash four times with deionized water and twice with TBST.
[1306] 15. Add 100 μl / well of anti-phosphotyrosine (1: 3000 dilution in TBST). Incubate with shaking at room temperature for 30-45 minutes.
[1307] 16. Wash as in the fourth step.
[1308] 17. Add 100 μl / well of biosource goat anti-rabbit IgG peroxidase conjugate (1: 2000 dilution in TBST) to each well. Incubate with shaking for 30 minutes at room temperature.
[1309] 18. Wash as in the fourth step.
[1310] 19. Add 100 μL of ABTS / H ₂ O ₂ solution to each well.
[1311] 20. Incubate with shaking for 5-10 minutes. Remove the bubbles.
[1312] 21. If necessary, stop the reaction by adding 100 μl / well of 0.2 M HCl.
[1313] 22. Read the analysis on a Dynatech MR7000 ELISA reader using a test filter at 410 nM and a reference filter at 630 nM.
[1314] PDGFR Bioassay
[1315] This assay is used to measure the ex vivo kinase activity of FGF1-R in ELISA assays.
[1316] Substances and Reagents:
[1317] 1. Corning 96-well ELISA plate.
[1318] 2. 28D4C10 mononuclear anti-EGFR antibody (Susen Inc.).
[1319] 3. PBS.
[1320] 4. TBST buffer.
[1321] 5. Blocking buffer (same as in EGFR bioassay).
[1322] 6. PDGFR-β expressing NIH 3T3 cell lysate (Susen Inc.).
[1323] 7. TBS buffer.
[1324] 8.TBS + 10% DMSO
[1325] 9. ATP.
[1326] 10. MnCl ₂ .
[1327] 11. Kinase buffer phosphorylation mixture: For 10 ml, make 10 ml by mixing 250 μl 1M TRIS, 200 μl 5M NaCl, 100 μl 1M MnCl ₂ and 50 μl 100 mM Triton X-100 in sufficient dH ₂ O.
[1328] 12. NUNC 96-well V bottom polypropylene plate.
[1329] 13. EDTA.
[1330] 14. Rabbit multinuclear anti-phosphotyrosine serum (Suzen Inc.).
[1331] 15. Goat anti-rabbit IgG peroxidase conjugate (Biosource, catalog ALI0404).
[1332] 16. ABTS.
[1333] 17. Hydrogen peroxide, 30% solution.
[1334] 18. ABTS / H ₂ O ₂ .
[1335] 19. 0.2 M HCl.
[1336] step:
[1337] 1. Coat Corning 96-well ELISA plates with 0.5 μg of 28D4C10 in 100 μl of PBS and store at 4 ° C. overnight.
[1338] 2. Remove unbound 28D4C10 from the well by turning the plate upside down to remove the liquid. Wash once with dH ₂ O. Tap on a plate of paper towel to remove excess liquid.
[1339] 3. Add 150 μl of blocking buffer to each well. Incubate with shaking at room temperature for 30 minutes.
[1340] 4. Wash the plate three times with deionized water and then once with TBST. Tap on a plate of paper towel to remove excess liquid and bubbles.
[1341] 5. Dilute lysate in HNTG (10 μg lysate / 100 μl HNTG).
[1342] 6. Add 100 μl of diluted lysate to each well. Shake for 60 minutes at room temperature.
[1343] 7. Clean the plate as described in the fourth step.
[1344] 8. Add 80 μL of TBS to the ELISA plate containing the captured PDGFR.
[1345] 9. Dilute the test compound 1:10 in TBS in a 96-well polypropylene plate.
[1346] 10. Add 10 μl of diluted test compound to the ELISA plate. Add 10 μl TBS + 10% DMSO to control wells. Incubate with shaking at room temperature for 30 minutes.
[1347] 11. Add 10 μL of ATP directly to all wells except the negative control wells. (The final well volume should be about 100 μl with 20 μM ATP in each well.) Incubate for 30 minutes with shaking.
[1348] 12. Stop the reaction by adding 10 [mu] l of EDTA solution to each well.
[1349] 13. Wash four times with deionized water and twice with TBST.
[1350] 14. Add 100 μl / well of anti propostyrosine (1: 3000 dilution in TBST). Incubate with shaking at room temperature for 30-45 minutes.
[1351] 15. Wash as in step 4.
[1352] 16. Add 100 μl / well of biosource goat anti-rabbit IgG peroxidase conjugate (1: 2000 dilution in TBST) to each well. Incubate with shaking for 30 minutes at room temperature.
[1353] 17. Wash as in the fourth step.
[1354] 18. Add 100 μL of ABTS / H ₂ O ₂ solution to each well.
[1355] 19. Incubate with shaking for 10-30 minutes. Remove the bubbles.
[1356] 20. If necessary, stop the reaction by adding 100 μl / well of 0.2 M HCl.
[1357] 21. Read the analysis on a Dynatech MR7000ELISA reader using a test filter at 410 nM and a reference filter at 630 nM.
[1358] Cellular HER-2 Kinase Assay
[1359] This assay is used to measure HER-2 kinase activity in all cells in ELISA form.
[1360] Substances and Reagents:
[1361] 1. DMEM (Gibco Catalog No. 11965-092).
[1362] 2. Bovine placental serum (FBS, Gibco Catalog No. 16000-044), heat inactivated in water bath at 56 ° C. for 30 minutes.
[1363] 3. Trypsin (Gibco catalog 25200-056).
[1364] 4. L-Glutamine (Gibco Catalog No. 25030-081).
[1365] 5. HEPES (Gibco Catalog No. 15630-080).
[1366] 6. Growth medium: Mix 500 ml of DMEM, 55 ml of heat inactivated FBS, 10 ml of HEPES and 5.5 ml of L-glutamine.
[1367] 7. Deficient medium: Mix 500 ml of DMEM, 2.5 ml of heat inactivated FBS, 10 ml of HEPES and 5.5 ml of L-glutamine.
[1368] 8. PBS.
[1369] 9. Flat bottom 96-well tissue culture micro titer plate (coating catalog 25860).
[1370] 10. 15 cm tissue culture dish (Corning Catalog No. 08757148).
[1371] 11. Corning 96-well tissue ELISA plate.
[1372] 12. NUNC 96-well ELISA plate.
[1373] 13. Costa Transfer Cartridge for Transta 96 (Costa Catalog No. 7610).
[1374] 14. SUMO1: mononuclear anti-EGFR antibody (Susen Inc.).
[1375] 15. TBST buffer.
[1376] 16. Blocking Buffer: 5% Carnation Instant Milk® in PBS.
[1377] 17. EGF Ligand: EGF-201, Shinko American. The powder is suspended in 100 μl of 10 mM HCl. Add 100 μl 10 mM NaOH. 800 μl of PBS is added, transferred to an Eppendorf tube and stored at −20 ° C. until use.
[1378] 18. HNTG Lysis Buffer: For stock 5X HNTG, 1 L of the total solution is made by mixing 23.83 g HEPES, 43.83 g NaCl, 500 ml glycerol, 100 ml Triton X-100 and sufficient dH ₂ O. For 1 × HNTG, mix 2 ml HNTG, 100 μl 0.1 M Na ₃ VO ₄ , 250 μl 0.2 M Na ₄ P ₂ O ₇ and 100 μl EDTA.
[1379] 19. EDTA.
[1380] 20. Na ₃ VO ₄ : To prepare the raw material solution, 1.84 g of Na ₃ VO _{4 is} mixed with 90 ml of dH ₂ O. Adjust pH to 10. Boil in the microwave for 1 minute (clear solution). Cool to room temperature. Adjust pH to 10. The heating / cooling cycle is repeated until the pH is maintained at 10.
[1381] 21. 200 mM Na ₄ P ₂ O ₇ .
[1382] 22. Rabbit multinuclear antiserum specific to phosphotyrosine (anti-Ptyr antibody, Suzen Inc.).
[1383] 23. Affinity purified antiserum, goat anti-rabbit antibody, peroxidase-conjugated conjugate (Biosource Catalog ALI0404).
[1384] 24. ABTS solution.
[1385] 25. 30% hydrogen peroxide solution.
[1386] 26. ABTS / H ₂ O ₂ .
[1387] 27. 0.2 M HCl.
[1388] step:
[1389] 1. Corning 96-well ELISA plates are coated with 0.5 μg / well SUMO1 in PBS. Store overnight at 4 ° C.
[1390] 2. On the day of use, remove the coating buffer, wash the plate three times with dH ₂ O and once with TBST buffer. Unless otherwise indicated, all washes of this assay should be performed in this manner.
[1391] 3. Add 100 μl of blocking buffer to each well. Incubate the plate with shaking for 30 minutes at room temperature. Clean the plates just before use.
[1392] 4. Use the EGFr / HER-2 chimera / 3T3-C7 cell line for this assay.
[1393] 5. Select dishes with 80-90% confluence. Cells are harvested by trypsinization and centrifuged at 1000 rpm for 5 minutes at room temperature.
[1394] 6. Resuspend cells in deficient medium and count trypan blue. More than 90% viability is required. Cells in deficient medium are seeded into 96-well microtiter plates at a density of 2,500 cells / well, 90 μl / well. The seeded cells are incubated overnight at 37 ° C. under 5% CO ₂ .
[1395] 7. Begin analysis two days after seeding.
[1396] 8. Dissolve the test compound in 4% DMSO. The sample is then diluted directly on the plate with deficiency-DMEM. Typically this dilution will be 1:10 moving. All wells are then transferred to the cell plate at an additional 1:10 dilution (10 μl sample and medium into 90 μl of deficient medium). The final DMSO concentration should be below 1%. A series of standard dilutions can also be used.
[1397] 9. Incubate at 37 ° C. under 5% CO ₂ for 2 hours.
[1398] 10. EGF ligand was prepared by diluting the raw material EGF (16.5 μM) in warm DMEM to 150 μM.
[1399] 11. Prepare enough fresh HNTG ^* at 100 μl per well and place on ice.
[1400] 12. After 2 hours of incubation with the test compound, the prepared EGF ligand is added to the cells at 50 μl per well for a final concentration of 50 nM. Positive control wells receive the same amount of EGF. Negative controls do not receive EGF. Incubate at 37 ° C. for 10 minutes.
[1401] 13. Remove test compound, EGF and DMEM. The cells are washed once with PBS.
[1402] 14. Transfer HNTG ^* to cells at 100 μl per well. Place on ice for 5 minutes. Blocking buffer, on the other hand, is removed from the ELISA plate and washed.
[1403] 15. Rake the cells from the plate using a micropipette and homogenize the cell material by repeating inhaling and dispensing HNTG ^* in Lice buffer. Lysates are transferred to coated, blocked and washed ELISA plates. Alternatively, lysate is transferred to the plate using a Costa transfer cartridge.
[1404] 16. Incubate with shaking at room temperature for 1 hour.
[1405] 17. Remove lysate and wash. Freshly diluted anti-Ptyr antibody (1: 3000 in TBST) is transferred to ELISA plate at 100 μl / well.
[1406] 18. Incubate for 30 minutes at room temperature with shaking.
[1407] 19. Remove and wash anti-Ptyr antibody. Freshly diluted BIOSOURCE antibody is transferred to an ELISA plate (1: 3000 in TBST, 100 μl / well).
[1408] 20. Incubate for 30 minutes at room temperature with shaking.
[1409] 21. Remove and wash BIOSOURCE antibody FMF. Transfer the freshly prepared ABTS / H ₂ O ₂ solution to the ELISA plate at 100 μl / well.
[1410] 22. Incubate with shaking for 5-10 minutes. Remove the bubbles.
[1411] 23. Stop the reaction by adding 100 μl / well of 0.2 M HCl.
[1412] 24. Read the analysis on a Dynatech MR7000 ELISA reader using a test filter at 410 nM and a reference filter at 630 nM.
[1413] CDK2 / Cyclin A Assay
[1414] This assay is used to measure the ex vivo serine / threonine kinase activity of human cdk2 / cycline in scintillation proximity assay (SPA).
[1415] Substances and Reagents:
[1416] 1. Wallac 96-well polyethylene terephthalate (flexi) plate (wallac catalog 1450-401).
[1417] 2. Amersham Redivue [γ ³³ P] ATP (Amersham Catalog No. AH 9968).
[1418] 3. Amersham streptavidin-coated polyvinyltoluene SPA beads (Amersham catalog RPNQ0007). Beads should be reconstituted at 20 mg / ml in PBS without magnesium or calcium.
[1419] 4. Activated cdk2 / cyclin A enzyme complex purified from Sf9 cells (Suzen Inc.).
[1420] 5. Biotinylated Peptide Substrate (Debtide) Peptide Biotin-X-PKTPKKAKKL is dissolved in dH ₂ O at a concentration of 5 mg / ml.
[1421] 6. Peptide / ATP Mixture: For 10 ml, mix 9.970 ml of dH ₂ O, 0.00125 ml of cold ATP, 0.010 ml of Deptide and 0.010 ml of γ ³³ P ATP. The final concentration per well will be 0.5 μM cold ATP, 0.1 μg depide and 0.2 μCiγ ³³ P ATP.
[1422] 7. Kinase buffer: Mix 10 mL, freshly added just before use, 8.85 mL dH ₂ O, 0.625 mL TRIS (pH 7.4), 0.25 mL 1M MgCl _2, 0.25 mL 10% NP40, 0.025 mL 1M DTT. do.
[1423] 8. 10 mM ATP in dH ₂ O.
[1424] 9. Adjust the pH of 1M TRIS to 7.4 with HCl.
[1425] 10. 1 M MgCl ₂ .
[1426] 11. 1M DTT.
[1427] 12. PBS (Gibco Catalog No. 14190-144).
[1428] 13. 0.5 M EDTA.
[1429] 14. Stop Solution: For 10 ml, mix 9.25 ml of PBS, 0.005 ml of 100 mM ATP, 0.1 ml of 0.5 M EDTA, 0.1 ml of 10% Triton X-100 and 1.25 ml of 20 mg / ml SPA beads.
[1430] step:
[1431] 1. A solution of test compound is prepared at 5 times the desired final assay concentration in 5% DMSO.
[1432] 2. Dilute 5 μl of cdk2 / cyclin A solution twice with 2.1 ml of kinase buffer.
[1433] 3. Add 20 μl of enzyme to each well.
[1434] 4. Add 10 μl 0.5 M EDTA to the negative control wells.
[1435] 5. To start the kinase reaction, add 20 [mu] l of peptide / ATP mixture to each well. Incubate for 1 hour without shaking.
[1436] 6. Add 200 μl of stop solution to each well.
[1437] 7. Last at least 10 minutes.
[1438] 8. Rotate the plate at about 2300 rpm for 3 to 5 minutes.
[1439] 9. Count plates using Trilux or similar reader.
[1440] MET Phosphorylation Exchange Analysis
[1441] This assay is used to determine phosphotyrosine levels for poly (glutamic acid: tyrosine (4: 1)) as a means of identifying agonists / antagonists of met phosphorylation exchange reactions of substrates.
[1442] Substances and Reagents:
[1443] 1. Corning 96-well ELISA Plate, Corning Catalog No. 25805-96.
[1444] 2. Glue tyr 4: 1, Sigma Catalog No. P0275.
[1445] 3. PBS, Gibco Catalog No. 450-1300EB.
[1446] 4. 50mM HEPES.
[1447] 5. Blocking Buffer: 25 g of bovine serum albumin (Sigma Cat. No A-7888) is dissolved in 500 ml of PBS and filtered through a 4 μm filter.
[1448] 6. Purified GST fusion protein (Suzen Inc.) containing MET chibase domain.
[1449] 7. TBST buffer.
[1450] 8. 10% aqueous (MilliQue H ₂ O) DMSO.
[1451] 9. 10 mM aqueous (dH ₂ O) adenosine-5'-triphosphate (Sigma Cat. No A-5394).
[1452] 10. 2X Kinase Dilution Buffer: For 100 ml, 10 ml of 1 M HEPES (pH 7.5), 0.4 ml of 5% BSA / PBS, 0.2 ml of 0.1 M sodium orthovanadate and 1 ml of 5 M sodium chloride in 88.4 ml of dH ₂ O. Mix.
[1453] 11. 4X ATP reaction mixture: For 10 ml, 0.4 ml of 1 M magnesium chloride and 0.02 ml of 0.1 M ATP are mixed in 9.56 ml of dH ₂ O.
[1454] 12. 4 × negative control mixture: For 10 ml, 0.4 ml of ₁ M magnesium chloride is mixed in 9.6 ml of dH ₂ O.
[1455] 13. NUNC 96-well V bottom polypropylene plate, Applied Scientific Catalog No. AS-72092.
[1456] 14. 500 mM EDTA.
[1457] 15. Antibody Dilution Buffer: For 100 ml, mix 10 ml of 5% BSA / PBS, 5% Carnation Instant Milk® in PBS and 0.1 ml of 0.1 M sodium orthovanadate in 88.4 ml of TBST.
[1458] 16. Rabbit Multinuclear Antophosphotyrosine Antibody (Suzen Inc.).
[1459] 17. Goat Anti-Rabbit Horseradish Peroxidase-Conjugated Antibody (Biosource Inc., Biosource Inc.)
[1460] 18. ABTS solution: For 1 liter, 19.21 g citric acid, 35.49 g Na ₂ HPO ₄ and 500 mg ABTS are mixed together with sufficient dH ₂ O to make 1 liter.
[1461] 19. ABTS / H ₂ O ₂ : Mix 15 ml ABST with ₂ μl of H ₂ O ₂ 5 minutes before use.
[1462] 20. 0.2 M HCl.
[1463] step:
[1464] 1. Coat ELISA plate with 2 μg of Poly (Glu-Tyr) in 100 μl of PBS and store at 4 ° C. overnight.
[1465] 2. Block the plate with 150 μl of 5% BSA / PBS for 60 minutes.
[1466] 3. Wash the plate twice with PBS and once with 50 mM HEPES buffer (pH 7.4).
[1467] 4. Add 50 μl of diluted kinase to all wells. (Diluted purified kinase with kinase dilution buffer. Final concentration should be 10 ng / well.)
[1468] 5. Add 25 μL of test compound (in 4% DMSO) or DMSO alone (4% in dH ₂ O).
[1469] 6. Incubate the kinase / compound mixture for 15 minutes.
[1470] 7. Add 25 μL of 40 mM MnCl ₂ to the negative control wells.
[1471] 8. Add 25 [mu] l of ATP / MnCl ₂ mixture to all other wells except negative control. Incubate for 5 minutes.
[1472] 9. Stop the reaction by adding 25 [mu] l of 500 mM EDTA.
[1473] 10. Wash the plate three times with TBST.
[1474] 11. Add 100 [mu] l of rabbit multinuclear anti-Ptyr diluted 1: 10,000 in antibody dilution buffer to each well. Incubate with shaking for 1 hour at room temperature.
[1475] 12. Wash the plate three times with TBST.
[1476] 13. Dilute biosource HRP conjugated anti-rabbit antibody diluted 1: 6,000 in antibody dilution buffer. Add to 100 μl / well and incubate with shaking for 1 hour at room temperature.
[1477] 14. Wash the plate once with PBS.
[1478] 15. Add 100 μL of ABTS / H ₂ O ₂ solution to each well.
[1479] 16. If necessary, stop the development reaction by adding 100 μl / well of 0.2 M HCl.
[1480] 17. Read the analysis on a Dynatech MR7000 ELISA reader using a test filter at 410 nM and a reference filter at 630 nM.
[1481] IGF-1 Phosphorylation Exchange Analysis
[1482] This assay is used to determine phosphotyrosine levels for poly (glutamic acid: tyrosine (4: 1)) as a means of identifying agonists / antagonists of gst-IGF-1 phosphorylation exchange of substrates.
[1483] Substances and Reagents
[1484] 1. Corning 96-well ELISA plate.
[1485] 2. Glu-Tyr (4: 1) (Sigma Catalog No. P 0275).
[1486] 3. PBS (Gibco Catalog No. 450-1300EB).
[1487] 4. 50mM HEPES.
[1488] 5. TBB-Blocking Buffer: For 1 L, mix 100 g BSA, 1.21 g TRIS (pH 7.5), 58.44 g NaCl, 10 ml 1% TWEEN-20.
[1489] 6. Purified GST fusion protein (Suzen Inc.) containing IGF-1 kinase domain.
[1490] 7. TBST Buffer: For 1 L, mix 6.057 g TRIS, 8.766 g NaCl and 0.5 mL TWEEN-20 with sufficient dH ₂ O to make 1 L.
[1491] 8. 4% DMSO in Milli-Q H ₂ O.
[1492] 9. 10 mM ATP in dH ₂ O.
[1493] 10. 2 × Kinase Dilution Buffer: For 100 ml, 10 ml of 1 M HEPES (pH 7.5), 0.4 ml of 5% BSA in dH ₂ O, 0.2 ml of 0.1 M sodium orthovanadate and 1 ml of 5M sodium chloride with sufficient dH ₂ O Mix together to make 100 ml.
[1494] 11. 4X ATP reaction mixture: For 10 ml, mix 0.4 ml of 1 M MnCl ₂ , 0.008 ml of 0.01 M ATP and 9.56 ml of dH ₂ O.
[1495] 12. 4X negative control mixture: For 0.4 ml, mix 0.4 ml of 1M manganese chloride in 9.6 ml of dH ₂ O.
[1496] 13. NUNC 96-well V bottom polypropylene plate.
[1497] 14. 500 mM EDTA in dH ₂ O.
[1498] 15. Antibody Dilution Buffer: For 100 ml, mix 10 ml of 5% BSA in PBS, 5% Carnation Instant Non-Pat Milk® in PBS and 0.1 ml of 0.1 M sodium orthovanadate in 88.4 ml of TBST.
[1499] 16. Rabbit Multinuclear Antophosphotyrosine Antibody (Suzen Inc.).
[1500] 17. Goat anti-rabbit horseradish peroxidase-conjugated antibody (biosource).
[1501] 18. ABTS solution.
[1502] 20. ABTS / H ₂ O ₂ : Mix 15 ml ABST with ₂ μl of H ₂ O ₂ 5 minutes before use.
[1503] 21. 0.2 M HCl in dH ₂ O.
[1504] step:
[1505] 1. Coat ELISA plates with 2.0 μg / well poly (Glu, Tyr) in 100 μl PBS. Store overnight at 4 ° C.
[1506] 2. Wash the coated plate once with PBS.
[1507] 3. Add 100 [mu] l of TBB blocking buffer to each well. Incubate with shaking at room temperature for 1 hour.
[1508] 4. Wash the plate once with PBS, then twice with 50 mM HEPES buffer (pH 7.5).
[1509] 5. Add 25 μl of test compound in 4% DMSO (obtained by diluting the stock solution of 10 mM test compound in 100% DMSO with dH ₂ O) to the plate.
[1510] 6. Add GST-FGF-1 kinase in kinase dilution buffer to all wells.
[1511] 7. Start the kinase reaction by adding 25 μL of 4 × ATP reaction mixture to all test wells and positive control wells. 25 μl of 4 × negative control mixture is added to all negative control wells. Incubate with shaking at room temperature for 10 minutes.
[1512] 8. Add 25 μl 0.5 M EDTA, pH 8.0 to all wells.
[1513] 9. Wash the plate four times with TBST buffer.
[1514] 10. Add rabbit polynuclear anti-phosphotyrosine anti-serum diluted 1: 10,000 in 100 μl of antibody dilution buffer to all wells. Incubate with shaking for 1 hour at room temperature.
[1515] 11. Clean the plate as in step 9.
[1516] 12. Add 100 μl of BioSource Goat Anti-Rabbit HRP diluted 1: 10,000 in antibody dilution buffer. Incubate with shaking for 1 hour at room temperature.
[1517] 13. After washing the plate as in step 9, wash once with PBS to reduce foam and excess TWEEN-20.
[1518] 14. Develop by adding 100 μl / well of ABTS / H ₂ O ₂ solution to each well.
[1519] 15. After about 5 minutes, filter is tested at 410 nM and reference filtrate is read on an ELISA reader at 630 nM.
[1520] BRDU Incorporation Analysis
[1521] The following assays assess the effect of the compound on the activity of ligand-induced DNA synthesis by measuring BrdU incorporation into DNA after using cells engineered to express selected receptors.
[1522] The following materials, reagents and procedures are general for each of the following BrdU incorporation assays. Variations in certain analyzes are noted.
[1523] Substances and Reagents
[1524] 1. The appropriate ligand.
[1525] 2. Appropriate processed cells.
[1526] 3. BrdU labeling reagent: 10 mM in PBS (pH 7.4) (Boehringer Mannheim, Germany).
[1527] 4. FixDenat: Fixing solution (prepared for use) (Möllinger Mannheim, Germany).
[1528] 5. Anti-BrdU-POD: mouse mononuclear antibody conjugated with peroxidase (Möllinger Mannheim, Germany).
[1529] 6. TMB substrate solution: tetramethylbenzidine (TMB, Schöllinger Mannheim, Germany).
[1530] 7. PBS wash solution: 1 × PBS, pH 7.4.
[1531] 8. Albumin, Bovine (BSA), Fraction V Powder (Sigma Chemical Company, USA).
[1532] step:
[1533] 1. Seed cells in 96-well plates at 10% CS in DMEM, 8000 cells / well in 2 mM Gln. Cells are incubated overnight at 37 ° C. in 5% CO ₂ .
[1534] 2. After 24 hours, cells are washed with PBS and then serum-deficient in serum-free medium (0% CS DMEM + 0.1% BSA) for 24 hours.
[1535] 3. On day 3, the appropriate ligand and test compound are added to the cells simultaneously. Negative control wells receive serum-free DMEM with only 0.1% BSA, positive control wells accept ligands but no test compounds. Test compounds are prepared in serum-free DMEM with ligands in 96-well plates and diluted to a series of seven test concentrations.
[1536] 4. After 18 hours of ligand activation, diluted BrdU labeling reagent (1: 100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final concentration = 10 μM) for 1.5 hours.
[1537] 5. After incubation with the labeling reagent, slowly tilt and tap the inverted plate on the paper towel to remove the medium. FixDenat solution is added (50 μl / well) and the plate is incubated for 45 minutes at room temperature on a plate shaker.
[1538] 6. Slowly tilt and tap the inverted plate on the paper towel to remove the FixDenat solution completely. Milk is added as blocking solution (5% dehydrated milk in PBS, 200 μl / well) and plates are incubated for 30 minutes at room temperature on a plate shaker.
[1539] 7. Gently remove the blocking solution and wash the wells once with PBS. Anti-BrdU-POD solution (1: 200 dilution in PBS, 1% BSA) is added (50 μl / well) and the plates incubated for 90 minutes at room temperature on a plate shaker.
[1540] 8. The wells are slowly tilted and rinsed five times with PBS to completely remove the antibody conjugate, and the plates are inverted on a paper towel and patted dry.
[1541] 9. Add TMB substrate solution (100 μl / well) and incubate for 20 minutes at room temperature on a plate shaker until sufficient color development for luminosity detection.
[1542] 10. Measure the absorbance of the sample at 410 nm (in "dual wavelength" mode with filter reading at 490 nm as reference wavelength) on a Dynatech ELISA plate reader.
[1543] EGF-induced BrdU incorporation assay
[1544] Substances and Reagents
[1545] 1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).
[1546] 2. 3T3 / EGFRc7.
[1547] EGF-induced Her-2-induced BrdU incorporation assay
[1548] Substances and Reagents
[1549] 1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).
[1550] 2. 3T3 / EGFr / Her2 / EGFr (EGFr with Her-2 kinase domain).
[1551] EGF-induced Her-4-induced BrdU incorporation assay
[1552] Substances and Reagents
[1553] 1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).
[1554] 2. 3T3 / EGFr / Her4 / EGFr (EGFr with Her-4 kinase domain).
[1555] PDGF-Induced BrdU Incorporation Assay
[1556] Substances and Reagents
[1557] 1. Human PEGF B / B (Möllinger Mammheim, Germany).
[1558] 2. 3T3 / EGFRc7.
[1559] FGF-induced BrdU incorporation assay
[1560] Substances and Reagents
[1561] 1. Human FGF2 / bFGF (Gibco BRL, USA).
[1562] 2. 3T3c7 / EGFr.
[1563] IGF1-induced BrdU incorporation assay
[1564] Substances and Reagents
[1565] 1. Human, recombinant (G511, Promega Corporation, USA).
[1566] 2. 3T3 / IGF1r.
[1567] Insulin-Induced BrdU Incorporation Assay
[1568] Substances and Reagents
[1569] Insulin, crystals, bovine, zinc (13007, Gibco BRL, USA).
[1570] 2. 3T3 / H25.
[1571] HGF-induced BrdU incorporation assay
[1572] Substances and Reagents
[1573] 1. Recombinant human HGF (Catalog No. 249-HG, R & D Systems Inc., USA).
[1574] 2. BxPC-3 cells (ATCC CRL-1687).
[1575] step:
[1576] 1. Seed the cells at 9000 cells / well in RPMI 10% FBS in 96-well plates. Cells are incubated overnight at 37 ° C. in 5% CO ₂ .
[1577] 2. After 24 hours, cells are washed with PBS and then serum-deficient in serum-free medium (RPMI + 0.1% BSA) for 24 hours.
[1578] 3. On day 3, 25 μl of ligand (prepared at 1 μg / ml in RPMI with 0.1% BSA; final HGF concentration is 200 ng / ml) and test compound are added to the cells. Negative control wells receive serum-free RPMI with only 0.1% BSA and positive control wells accept ligand (HGF) but no test compound. Test compounds are prepared in serum-free RPMI with ligands in 96-well plates and diluted to a series of seven test concentrations. Typically, the highest final concentration of the test compound is 100 μM and a 1: 3 dilution is used (ie the concentration range of the final test compound is 0.137 to 100 μM).
[1579] 4. After 18 hours of ligand activation, diluted BrdU labeling reagent (1: 100 in RPMI, 0.1% BSA) is added to each well and cells are incubated with BrdU (final concentration = 10 μM) for 1 hour.
[1580] 5. Same as general procedure.
[1581] 6. Same as general procedure.
[1582] 7. Gently remove the blocking solution and wash the wells once with PBS. Anti-BrdU-POD solution (1: 100 dilution in PBS, 1% BSA) is added (100 μl / well) and plates are incubated for 90 minutes at room temperature on a plate shaker.
[1583] 8. Same as general procedure.
[1584] 9. Same as general procedure.
[1585] 10. Same as general procedure.
[1586] HUV-EC-C Analysis
[1587] This assay is used to measure the activity of compounds against PDGF-R, FGF-R, VEGF, aFGF or Flk-1 / KDR, all of which are naturally expressed by HUV-EC cells.
[1588] 0 days
[1589] 1. Wash and trypsinize HUV-EC-C cells (Human Belly Vein Endothelial Cells, American Type Culture Collection, Catalog 1730 CRL). Wash twice in a tissue culture flask of about 1 ml / 10 cm 2 with Dulbecco's phosphate-buffered saline. Trypsinize with 0.05% trypsin-EDTA in non-enzymatic cell dissociation solution (Sigma Chemical Company, Catalog No. C-1544). 0.05% trypsin is prepared by diluting 0.25% trypsin / 1 mM EDTA (Gibco, catalog 25200-049) in cell dissociation solution. Trypsinize to a tissue culture flask of about 1 ml / 25-30 cm 2 at 37 ° C. for about 5 minutes. After detaching cells from the flask, the same volume of assay medium is added and transferred to a 50 ml sterile centrifuge tube (Fisher Scientific, catalog 05-539-6).
[1590] 2. Add about 35 ml of assay medium into a 50 ml sterile centrifuge tube to wash the cells, centrifuge for 10 min at about 200 × g, aspirate the supernatant and resuspend with 35 ml D-PBS. Wash twice or more with D-PBS and resuspend the cells in about 1 ml assay medium / 15 cm 2 tissue culture flask. Assay medium consists of F12K medium (Gibco BRL, catalog 21127-014) and 0.5% heat-inactivated bovine placental serum. Cells are counted with a Coulter Counter® (Culter Electronics Inc.) and assay medium is added to the cells to obtain a concentration of 0.8-1.0 × 10 ⁵ cells / ml.
[1591] 3. Add cells to a 96-well flat-bottom plate at a concentration of 100 μl / well or 0.8 to 1.0 × 10 ⁵ cells / ml and incubate at 35 ° C. with 5% CO _{2 for} about 24 hours.
[1592] 1 day
[1593] 1. Prepare 2 × test compounds, typically 50 μM to 0 μM, in separate 96-well plates. The same assay medium as mentioned in the second step on day 0 is used. 90 μl / well of test compound is titrated by adding 200 μM (4 × final well concentration) to the upper wells of a specific plate column. Since the raw test compound is typically 20 mM in DMSO, the 200 μM drug concentration contains 2% DMSO.
[1594] The test compound is diluted but the DMSO concentration is kept constant using a diluent to make 2% DMSO in the assay medium as a diluent for titration of the test compound. This dilution is added at 60 μl / well to the remaining wells in the column. 60 μl in 120 μl 200 μM test compound dilution in the top well of the column is taken and mixed with 60 μl in the second well of the column. Take 60 μL from this well, mix with 60 μL in the third well of the column, and continue until 2 × titration is complete. If the wells before the final well are mixed, 60 μl of 120 μl in this well is taken and discarded. 60 μl of DMSO / medium dilution is left in the final well as a non-test compound-containing control. (1) VEGF (manufactured from Peptro Tech Inc., catalog No. 100-200), (2) endothelial cell growth factor (ECGF) (acid fibroblast growth factor or FGF also Known), or (3) nine columns of titrated test compounds sufficient for each 3 × well for human PDGF B / B (1276-956, Schöllinger Mainheim, Germany) and the assay medium adjusted . ECGF becomes a product with sodium heparin.
[1595] 2. Transfer 50 μl / well of test compound dilution to a 96-well assay plate containing 0.8-1.0 × 10 ⁴ cells / 100 μl / well of HUV-EC-C cells from day 0, and add 5% CO ₂ 37 Incubate at C for about 2 hours.
[1596] 3. At 3 ×, add 80 μg / ml VEGF, 20 ng / ml ECGF or media control at 50 μl / well of each test compound to the conditions. For the test compound, the growth factor concentration is four times the desired final concentration. The assay medium from step 0 on day 0 is used to formulate concentrations of growth factors. Incubate at 5% CO _{2 at} 37 ° C. for about 24 hours. Each well will have 50 μl of dilution of the test compound, 50 μl of growth factor or medium and 100 μl of cells, calculated as a total of 200 μl / well. Thus, the 4X concentration of test compound and growth factor will be equal to the total added once to the wells.
[1597] 2 days
[1598] 1. Add ³ H-thymidine (Amersham, Catalog No. TRK-686) to 1 μCi / well (100 μCi / ml solution + 10% heat-inactivated placental serum 10 μl / well) prepared in RPMI medium and Incubate at 5% CO _{2 at} 37 ° C. for about 24 hours. RPMI is obtained from Gibco BRL Catalog No. 11875-051.
[1599] 3 days
[1600] 1. Freeze the plate overnight at -20 ° C.
[1601] 4 days
[1602] The plates are thawed and harvested on a filter mat (Wallac, Catalog No. 1205-401) using a 96-well plate harvester (Tomtech Harvester 96®), and Wallac beta Counts are read on a plate (Wallac Betaplate) liquid scintillation counter.
[1603] Table 2 sets forth the biological test results for some examples of compounds of Formula (I). The results are reported for the IC ₅₀ (μM concentration) of the tested compound resulting in a 50% change in the activity of the target PKT compared to the activity of PTK in the control without the test compound. In particular, the results presented indicate the concentration of test compound required to cause a 50% reduction in the activity of the target PKT. The bioassays that have been or can be used to evaluate compounds are described in detail below.
[1604]
[1605]
[1606]
[1607] In vivo animal models
[1608] Xenograft animal model
[1609] The ability of human tumors to grow as xenografts in mice without thyroid gland provides a useful in vivo model for studying biological responses to the treatment of human tumors. After the first successful case of xenografting human tumors into mice without thyroid gland (Rygaard and Povlsen, 1969, Acta Pathol. Microbial. Scand. 77: 758-760), many Several human tumor cell lines (eg, mammals, lungs, genitourinary glands, sheaths, head and neck, glioblastoma, bone and malignant melanoma) have been transplanted into nude mice and successfully grown. The assay below can be used to determine the activity level, in particular the effect of various compounds of formula (I).
[1610] Three general types of assays are useful for evaluating compounds of formula (I). Cell / catalytic, cell / biological and in vivo. The purpose of the cell / catalytic assay is to determine the effect of compounds of formula (I) on the ability of TK to phosphorylate tyrosine on known substrates in cells. The purpose of the cell / biological analysis is to determine the effect of the compound of formula (I) on the biological response stimulated by TK. The purpose of in vivo analysis is to determine the effect of the compounds of formula (I) in animal models with certain disorders such as cancer.
[1611] Suitable cell lines for subcutaneous xenograft experiments are C6 cells (glioma, ATCC # CCL 107), A375 cells (melanoma, ATCC # CRL 1619), A431 cells (pseudo-epidermal carcinoma, ATCC # CRL 1555), Calu 6 cells ( Lungs, ATCC # HTB 56), PC3 cells (prostate, ATCC # CRL 1435), SKOV3TP5 cells, and NIH 3T3 subfibers genetically engineered to overexpress EGFR, PDGFR, IGF-1R or any other test kinase. do. The following protocol can be used to conduct xenograft experiments.
[1612] Female mice without thymus (BALB / c, nu / nu) are obtained from Simonsen Laboratories (Gilroy, Canada). All animals are kept under clean room conditions. These are often supplied with sterile rodent food and water.
[1613] Cell lines are grown in appropriate medium (eg, MEM, DMEM, Ham's F10, or Ham's F12 plus 5% -10% bovine placental serum (FBS) and 2 mM glutamine (GLN)). All cell culture media, glutamine and bovine placental serum are purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise noted. All cells are grown at 37 ° C. in a humid atmosphere of 90-95% air and 5-10% CO ₂ . All cell lines are usually subcultured twice a week and are negative for mycoplasma as measured by the Mycotect method (Gibco).
[1614] Cells are harvested with or similar to 0.05% Trypsin-EDTA and pelleted at 450 × g for 10 minutes. Pellets are resuspended at specific concentrations in sterile PBS and medium (without PBS) and cells are implanted into the hind limbs of mice (8-10 mice / group, 2-10 × 10 ⁶ cells / animal). Tumor growth is measured over three to six weeks using a venier caliper. Tumor volume is calculated as a result of length × width × height unless otherwise indicated. Calculate the P value using the Student's t-test. Test compounds of formula (I) in 50-100 μl of excipients (DMSO or VPD: D5W) can be delivered by IP infusion at various concentrations generally starting one day after implantation.
[1615] Tumor infiltration model
[1616] The following tumor penetration models have been developed and can be used to assess the therapeutic value and efficacy of the compounds of formula (I) defined to selectively inhibit KDR / FLK-1 receptors.
[1617] step
[1618] Eight weeks of age nude mice (females) (Simonsen Inc.) are used as experimental animals. Transplantation of tumor cells can be performed in a laminar flow hood. For anesthesia, xylazine / ketamine cocktail (100 mg / kg ketamine and 5 mg / kg xylazine) is administered intraperitoneally. An intermediate incision is made so that the abdominal cavity is exposed (about 1.5 cm long) and 10 ⁷ tumor cells are injected into 100 μl volume of medium. Cells are injected into the duodenum of interest or below the colon of the colon. The peritoneum and muscles are sutured with a 6-0 facet suture and the skin is closed using a wound clip. Observe the animals daily.
[1619] analysis
[1620] After 2-6 weeks, according to the overall observation of the animals, mice are sacrificed and local tumor metastases to various organs (lungs, liver, brain, stomach, visa, heart, gold) are incised and analyzed (tumor size). , Penetration, immunochemistry, hybridization measurements in the same region, etc.).
[1621] C-Kit Analysis
[1622] This assay is used to detect the level of c-kit tyrosine phosphorylation.
[1623] MO7E (human acute myeloid leukemia) cells are serum deficient overnight in 0.1% serum. Cells were pretreated with a compound of formula (I) (with serum depletion) prior to ligand stimulation. Cells were stimulated with 250ng / ml rh-SCF for 15 minutes. After stimulation, cells are lysed and immunoprecipitated with anti-c-kit antibody. Western blotting was used to determine phosphotyrosine and protein levels.
[1624] MTT proliferation assay
[1625] MO7E cells are serum deficient and pretreated with compounds of formula I as described in phosphorylation experiments. The cells are placed in ⁴ × 10 ⁵ cells / well in 100 μl RPMI + 10% serum in 96-well dishes. rh-SCF (100 ng / ml) was added and the plates incubated for 48 hours. After 48 hours, 10 μl of 5 mg / ml MTT [3- (4,5-dimeththiazol-2-yl) -2,5-diphenyl tetrazolium bromide) is added and incubated for 4 hours. Acid isopropanol (100 μl of 0.04N HCl in isopropanol) was added and the optical density was measured at a wavelength of 550 nm.
[1626] Apoptosis Assay
[1627] MO7E cells were incubated with rh-GM-SCF (10 ng / ml) and rh-IL-3 (10 ng / ml) with +/- SCF and +/- compounds of formula I in 10% FBS. Samples were analyzed at 24 and 48 hours. To measure activated caspase-3, the samples were washed with PBS and permeabilize with ice cold 70% ethanol. Cells were then stained with PE-conjugated multinuclear rabbit anti-active caspase-3 and analyzed by FACS. To determine the dissected PARP, the samples were lysed and analyzed by western blotting using anti-PARP antibodies.
[1628] Further analysis
[1629] Additional assays that may be used to evaluate compounds of formula I include, but are not limited to, bio-flk-1 assay, EGF receptor-HER2 chimeric receptor assay, bio-src assay, bio-lck assay, and raf of all cells. Analysis to measure phosphorylation function is included. The protocol for each of these analyzes can be found in US patent application Ser. No. 09 / 099,842, which is incorporated herein by reference, in any figure.
[1630] Measurement of cytotoxicity
[1631] The therapeutic compound should be more effective in inhibiting receptor tyrosine kinase activity than exerting a cytotoxic effect. Determination of the potency and cytotoxicity of the compounds can be obtained by measuring the therapeutic index, ie IC ₅₀ / LD ₅₀ . Dosages required to reach 50% inhibition (IC ₅₀ ) can be measured using standard techniques as described herein. Dosages showing 50% toxicity (LD ₅₀ ) may be determined using standard techniques (Mossman's literature "1983, J. Immunol. Methods , 65: 55-63"), or by measuring the amount of LDH J. Immunol. Methods , 64: 313 by Korzeniewski and Callewaert, Decker and Lohmann-Matthes, 1988, J. Immunol. Methods , 115: 61 "], or by measuring the lethal dose in an animal model. Compounds with large therapeutic indices are preferred. The therapeutic index should be greater than 2, preferably at least 10, more preferably at least 50.
[1632] B. 5- (5-Fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl Example of cell assay using amide (Compound (80))
[1633] (Below) 5- (5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid detected in biochemical analysis ( To identify the potency of 2-diethylamino-ethyl) amide (Compound (80)), NIH engineered to overexpress Flk-1 or human PDGFRβ the ability of the compound to inhibit ligand-dependent RTK phosphorylation. Evaluation was made in cell-based assays using -3T3 mouse cells. 5- (5-Fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) amide (Compound (80)) had an IC ₅₀ value of about 0.03 μM, thus inhibiting VEGF-dependent Flk-1 tyrosine phosphorylation. This value is 5- (5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid as measured in biochemical analysis. Similar to a K _i value of 0.009 μM as measured by inhibition of Flk-1 by (2-diethylamino-ethyl) amide (Compound (80)). It is 5- (5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) It shows that the amide (compound 80) easily permeates into the cell. 5- (5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2- Consistent with the indication that diethylamino-ethyl) amide (Compound 80) has significant activity against Flk-1 or human PDGFRβ, it is also PDGF-dependent in cells with an IC ₅₀ value of about 0.03 μM. It has been found to inhibit receptor phosphorylation. 5- (5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -2,4-inhibiting c-kit, a highly related RTK that binds to stem cell factor (SCF) The ability of dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) amide (Compound (80)) was measured using MO7E cells expressing this receptor. In these cells, 5- (5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino -Ethyl) amide (Compound (80)) had an IC ₅₀ value of 0.01-0.1 μM, thus inhibiting SCF-dependent c-kit phosphorylation. This compound also inhibited SCF-dependent c-kit phosphorylation in acute myeloid leukemia (AML) blasts isolated from the blood of the patient's peripheral tissues.
[1634] 5- (5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-inhibits ligand-dependent receptor phosphorylation intracellularly In addition to testing the ability of the carboxylic acid (2-diethylamino-ethyl) amide (Compound 80), its effect on the ligand-dependent proliferative response of the cells was also tested in vitro (see Table 4). In these studies, cells arrested by overnight serum deficiency induce DNA to be synthesized upon addition of the appropriate promoting ligand. As shown in Table 3, 5- (5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2- diethylamino-ethyl) amide (compound 80) are respectively 0.031 and of inhibiting the PDGF- induced proliferation of NIH-3T3 for overexpressing PDEFRβ or PDGFRα with IC ₅₀ value of 0.069μM, 0.007μM and IC ₅₀ SCF-induced proliferation of MO7E cells with values was inhibited.
[1635]
[1636] As shown in Table 3, 5- (5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2- There is an overall integral between the biochemical and cytological activity of these compounds, which supports the conclusion that diethylamino-ethyl) amide (compound (80)) crosses the cell membrane. It can also be concluded that the cellular response is the result of the activity of compound 80 on the intellectual target. In contrast, when tested in the presence of in vitro complete growth medium, 5- (5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole The actual high concentration (> 10 μM) of -3-carboxylic acid (2-diethylamino-ethyl) amide (compound (80)) was required to inhibit the growth of various human tumor cells (see Table 4). This means that the compounds do not directly inhibit the growth of these cells at the concentrations necessary to inhibit ligand-dependent receptor phosphorylation and cell proliferation.
[1637]
[1638] In summary, the results presented in Table 4 show that cells were serially diluted in 5- (5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole. Obtained by incubation for 48 h in complete growth medium in the presence of -3-carboxylic acid (2-diethylamino-ethyl) amide (compound (80)). At the end of the growth period, the relative number of cells was measured. IC ₅₀ values were calculated at concentrations of compounds that inhibited cell growth by 50% compared to untreated cells. The LD ₅₀ value was calculated at the concentration of the compound which reduced the number of cells by 50% compared to the beginning of the experiment.
[1639] 5- (5-Fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) amide A more relative cell-based assay that assesses the anti-angiogenic potential of (Compound 80) is an extracellular cell that uses human navel venous endothelial cells (HUVEC) as a model system for endothelial cell proliferation important for angiogenesis processes. Cleavage analysis. In this assay, cell division responses determined to increase in DNA synthesis are induced in serum-deficient HUVECs following the addition of VEGF or FGF. In these cells, 5- (5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino -Ethyl) amide (Compound (80)) is a VEGF- and FGF-induced cell division reaction in a dose-dependent manner with IC ₅₀ values of 0.004 and 0.7 μM, respectively, when the compound is present through a 48 hour assay. Was suppressed.
[1640] In summary, a series of dilutions 5- (5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (for a 24-hour period) The above-described results were obtained using serum-deficient HUVECs incubated with cell division concentrations of VEGF (100 ng / ml) and FGF (30 ng / ml) in the presence of 2-diethylamino-ethyl) amide (Compound (80)). Obtained. Cell division reactions for the next 24 hours in the presence of ligand and inhibitor were quantified by measurement of DNA synthesis based on incorporation of bromodeoxyuridine into cellular DNA.
[1641] In a separate experiment, Compound 80 inhibited the VEGF-dependent phosphorylation of ERK 1/2 (p42 / 44MAP kinase), the initial downstream target of Flk-1 / KDR, in a dose-dependent manner. The inhibitory activity of compound 80 is also long lasting in this system, with short exposure (2 hours) exposure to the micromolar concentration of the compound followed by 5- (5-fluoro-2-oxo-1,2 from the medium. After removing dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) amide (compound (80)), VEGF-dependent phosphorylation of ERK 1/2 was inhibited during the period.
[1642] VEGF has been recognized as an important survival factor for endothelial cells. 5- (5-Fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) amide Since (Compound (80)) inhibited the VEGF-dependent cell division reaction of HUVEC, the effect of the compound on HUVEC survival was investigated. In these experiments, cleavage of caspase 3 substrate poly-ADP-ribosyl polymerase (PARP) was used as a readout for apoptosis. HUVECs cultured in serum-free conditions for 24 hours showed the actual level of PARP cleavage as detected by the accumulation of 23kDa PARP cleavage fragments. This is largely prevented by adding VEGF to the cell medium, which means that VEGF acts as a survival factor in this assay. 5- (5-Fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) amide (Compound (80)) has been shown to inhibit KDR signaling. Thus, 5- (5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl ) Amide (Compound 80) inhibits VEGF-mediated HUVEC survival in a dose-dependent manner. Thus, these data indicate that Compound 80 induced apoptosis in endothelial cells in culture in the presence of VEGF.
[1643] C. Intracellular Efficacy Studies
[1644] i. Efficacy on Established Tumor Grafts
[1645] In a subcutaneous (SC) transplantation model using human tumor cells transplanted into the hind limb lateral region of mice without thyroid, 5- (5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl Intracellular efficacy of) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) amide (Compound (80)) was studied. After transplantation, tumors were established to a size of 100-550 mm 3 before starting with oral treatment with the compound.
[1646] Daily oral administration of Compound 80 resulting in dose-dependent inhibition of A431 tumor growth upon treatment was started after tumors grew to a size of 400 mm 3. Statistically significant (P <0.05) inhibition of tumor growth was seen at doses of 40 mg / kg / day (74% inhibition) and 80 mg / kg / day (84% inhibition) (see Table 6). In preliminary experiments, the high (160 mg / kg / day) dose of the compound is less potent on established A431 tumors than the 80 mg / kg / day dose. In addition, body weights were reduced in mice treated at 160 mg / kg / day dose of the compound, indicating that high doses are not resistant. Similar results were obtained in experiments that resulted in A431 tumors reaching 100 mm 3 size (see Table 4). In this second experiment, complete degeneration of the tumor was seen in 6 of 8 animals treated at 80 mg / kg / day for 21 days. In these six animals, tumors did not regrow during the 110 day observation period after treatment. In two animals whose tumors regrowed to large size (2000-3000 mm 3), the tumors regressed in response to the second treatment with compound (80). Important for all efficacy experiments, 5- (5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole at 80 mg / kg / day 3-carboxylic acid (2-diethylamino-ethyl) amide (compound (80)) had good resistance even in the case of continuous administration for a period of 100 days or more.
[1647]
[1648] In summary, the results presented in Table 5 were obtained using A431 cells (0.5 × 10 ⁶ cells / mouse) implanted with SC in the lateral region of the hind limb of mice without thyroid. When tumors reach a specified mean volume, 5- (5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2 in a cremofo-based vehicle or vehicle control Daily oral administration of, 4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) amide (Compound 80) was started. Tumors were measured using veneer calipers and tumor volumes were calculated as a result of length × width × height. At the end of the experiment using the Student's t-test, the P value is calculated by comparing the tumor size of the animal treated with Compound (80) (n = 8) to that of the vehicle treated animal (n = 16). It was.
[1649] Colo205 (colon carcinoma), SF763T (glioma) and NCI-H460 (small cell lung carcinoma) grafts were used to measure the efficacy of Compound (80) against established human tumors of different origins (see Table 6). . These experiments consisted of 5- (5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3 administered orally at 80 mg / kg / day. It was carried out using carboxylic acid (2-diethylamino-ethyl) amide (Compound 80), which was a dose with effective and good resistance.
[1650]
[1651] In the experiments described above, once tumors reached the indicated size, Compound (80) was administered once at 80 mg / kg / day in a cremofo-based vehicle daily. Percent inhibition compared to vehicle-treated control was calculated at the end of the experiment. Using a two-tailed Student's t-test, the P value was calculated by comparing the tumor size of the animal treated with the compound to the tumor size of the animal treated with the vehicle.
[1652] 5- (5-Fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl) amide Although (Compound (80)) inhibited the growth of all tumor types shown in Table 7, different results were shown in response to different transplant models. In particular, 5- (5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl When treated with) amide (Compound 80), the growth of NCI-H460 and SF763T tumors was arrested or significantly slowed, while Colo205 tumors such as A431 were regressed.
[1653] SF763T tumors were studied to determine on a molecular basis the differences in response between transplant models. Thus, 5- (5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl SF763T tumors, which are less responsive to treatment with) amide, were assessed at the molecular level using immunohistochemical techniques to determine the effectiveness of treatment with these compounds. These studies were initially conducted in this type of tumor because SF763T tumors are highly conduited with microvessels that express largely the endothelial cell marker CD31 and are suitable for the study of tumor microvessel density (MVD). Immunohistologic evaluation of SF763T tumors showed that tumors from treated animals had reduced MVD compared to vehicle-treated controls, and the anti-angiogenic mechanism of activity for Compound (80) was consistent. The MVD was 24 ± 4.1 in animals treated with Compound 80, compared to 39.3 ± 5.7 in animals treated only with vehicle. As expected from the associated tumor growth arrest, excellent inhibition of tumor cell proliferation was demonstrated in tumors treated with Compound (80). These tumors had a 2/1 mitotic index than in vehicle-treated tumors (data not shown). The effect of Compound 80 on MVD and tumor cell proliferation appears to be that the compound has large anti-angiogenic and anti-tumor effects even under conditions where tumors do not regress.
[1654] In addition, the ability of compound 80 to inhibit PDGFR phosphorylation and subsequent in vivo signaling was evaluated in SF763T tumors expressing high levels of PDGFRβ. Treatment of SF763T tumors with Compound (80) significantly inhibited PDGFRβ tyrosine phosphorylation in established SF763T tumors. Compound 80 also reduced the levels of phosphorylated (activated) phospholipase C gamma (PLC-γ), a fast-acting downstream indicator of PDGFR activity. These data demonstrate that oral administration of Compound (80) shows a direct effect on target in tumor (PDGFR) activity in vivo.
[1655] Based on the evidence that the ability of compound 80 to inhibit VEGE-dependent signaling in HUVECs in vitro (as described below) lasts for a long time (described below), the efficacy of the compound was evaluated when the compound was frequently administered in the Colo205 tumor model. As shown in Table 7, 80 mg / kg / day (91% inhibition) and 40 mg / kg / day (84% inhibition) were effective for daily administration, but not twice for weekly administration. In contrast, high doses of Compound 80 (160 mg / kg) inhibited the growth of established Colo205 tumors (52% inhibition) for twice weekly administration. This suggests that this compound can demonstrate the efficacy of frequent administration at high doses. It should be noted that the dosage regimen may be determined by one of ordinary skill in the art without inappropriate experimentation.
[1656]
[1657] In summary, the results presented in Table 7 were obtained using Colo205 cells (0.5 × 10 ⁶ cells / mouse) with SC implanted in the hind lateral region of mice without thyroid. When tumors reached 400 mm 3, oral administration of Compound 80 was started according to the designated schedule. Tumors were measured using veneer calipers and calculated as a result of length × width × height. At the end of the experiment using the two-tailed student's t-test, the P value was calculated by comparing the tumor size of the animal treated with Compound 80 with that of the vehicle treated with vehicle.
[1658] ii. Efficacy of Compound (80) in Models with Infectious Diseases
[1659] In support of the sustained growth of solid primary tumors, angiogenesis is also an essential factor in supporting the development of the disease transmitted due to metastases from said primary tumors. The effect of Compound (80) on the development of infectious disease was examined in the B16-F1 mouse melanoma lung colonization model. In this model, B16-F1 cells inoculated intravenously through the tail vein of mice without thyroid gland form colonies and tumors in the lungs. As shown in Table 7, oral administration of Compound (80) at 80 mg / kg / day effectively reduced the burden of B16-F1 cells in the lungs as assessed by measurement of total lung weight. These data suggest that Compound (80) can inhibit infectious diseases in vivo.
[1660]
[1661] In summary, the results presented in Table 8 were obtained using mice without thymus inoculated with B16-F1 tumor cells (5 × 10 ⁵ cells / mouse) via the tail vein. For 24 days after tumor cell inoculation, daily treatment was performed with oral administration (n = 10) or vehicle (n = 18) of 80 mg / kg / day. At the end of the treatment period, mice were sacrificed and their lungs removed and weighed. Percent inhibition was calculated by comparing the lung weight of the animal treated with Compound (80) to that of the animal treated with vehicle. P values were calculated using a two-tailed student's t-test.
[1662] II. COX-2 inhibitor
[1663] The Lewis Lung Model, described below, was used to test the ability of COX-2 inhibitors to arrest tumor growth.
[1664] Mice were subcutaneously injected into the left foot (1 × 10 ⁶ tumor cells suspended in 30% Matrigel) and tumor volume was measured using a phlethysmometer twice weekly for 30 to 60 days. Blood was taken twice during the experiment in a 24 hour protocol evaluating plasma concentration and total exposure by AUC analysis. Data is expressed as mean +/− SEM. The Student's and Mann-Whitney tests were used to assess the difference between the means of using the Insta software package. Celecoxib given on a diet at a dosage between 160 and 3200 ppm delayed the growth of these tumors. The inhibitory effect of celecoxib was dose dependent and ranged from 48% to 85% as compared to the control tumor. Analysis of the lung metastasis was carried out in all animals by counting the metastasis under a stereomicroscope and subsequent histological analysis of the lung part. Celecoxib did not affect lung metastasis at lower 160 ppm doses, but surface metastases were reduced by at least 50% when given at doses between 480 and 3200 ppm. In addition, histopathological analysis revealed that celecoxib decreased with dose the size of metastatic lesions in the lungs.
[1665] 2. HT-29 Model:
[1666] Mice were subcutaneously injected in the left foot (1 × 10 ⁶ tumor cells suspended in 30% Matrigel) and tumor volume was measured using a flutemometer twice weekly for 30 to 60 days. Transplantation of human colorectal cancer cells into nude mice forms tumors that will reach 0.6-2 ml between 30-50 days. Blood was taken twice during the experiment in a 24-hour protocol evaluating plasma concentrations and total exposure by AUC analysis. Data is expressed as mean +/− SEM. The Student's and Mann-Whitney tests were used to assess the differences between the means of using the Insta software package.
[1667] A. Mice injected with HT-29 cancer cells were treated with cytotoxic intraperitoneal at a dose of 50 mg / kg on days 5, 7, and 9 in the presence or absence of celecoxib in the diet. The efficacy of both agents was determined by measuring tumor volume. Treatment with celecoxib associated COX-2 inhibitor (SC-58236) reduced tumor volume by 89%. In the same test, indomethacin given in close proximity to the maximum tolerated dose of 2 mg / kg / day in drinking water inhibited tumor formation by 77%. Moreover, COX-2 selected inhibitors completely inhibit the formation of lung metastasis, whereas non-selective NSAID indomethacin was not effective. The results from this study demonstrate that celecoxib administered as a diet to mice suffering from tumors can delay the growth of tumors and metastases when administered alone. Moreover, a positive benefit is observed when celecoxib is administered with a cytotoxic agent such as cyclophosphamide.
[1668] B. In the second trial, mice injected with HT-29 colon cancer cells were treated with celecoxib (10, 40 or 160 ppm) in a diet starting on day 10. Approximate dose-dependent effects were observed (Table 9).
[1669]
[1670] III. In vivo testing using protein kinases in conjunction with cyclooxygenase-2 selective inhibitors to treat cancer
[1671] The ability of protein kinase inhibitors to delay tumor growth with COX-2 selective inhibitors can be tested in the 1483 transplantation model described below.
[1672] The 1483 graft is an animal graft modeling human epithelial cancer showing cyclooxygenase-2 (COX-2) in tumor cells and in the vasculature.
[1673] Human tumor transplantation nude mouse models for head and neck squamous cell carcinoma (1483 cell line) showing tumor cells and COX-2 in the vasculature are similar to human epithelial cancer. Applicants believe that this model represents an excellent epithelial cancer in humans and relates the efficacy of anticancer drugs to humans, including COX-2 inhibitors.
[1674] Substances and Methods:
[1675] Cell culture:
[1676] 1483 Human head and neck squamous cell carcinoma (HNSCC) cells are stored in frozen vials containing 3 × 10 ⁶ cells, 90% fetal placental serum (FBS) and 10% dimethyl sulfoxide (DMSO). Freeze vials are taken and quickly dissolved at 37 ° C. and placed in T-162 cm 2 (Corning) flasks containing D-MEM / F12 medium (GibcoBRL) with 12 mM Hepes buffer, L-glutamine, pyridoxine hydrochloride and 10% FBS. . Cells are grown in incubators with 5% CO ₂ and a temperature of 37 ° C. Medium is changed every other day and cells are passed at 80-90% confluence. For cell passage, the phosphate buffered saline (PBS) flask is washed, aspirated and placed in the incubator 5 minutes after addition of 2 ml trypsin / EDTA (0.25% / 1 mM, GibroBRL). 8 ml of this medium is added to the flask rinse and transferred to a sterile 50 ml centrifuge tube. More than 30 ml of medium is added and mixed, the cells are counted using a hemasidometer, and the cells are plated in T-162 cm ² containing 3 to 4 × 10 ⁶ .
[1677] 1483 animal models
[1678] The medium is changed 24 hours before harvest of 1483 cells before injection into nude mice. Trypsinize 1483 cells as described above in the cell culture section. Count the cells and measure the number of cells. Centrifuge at 1000 rpm for 5 minutes at room temperature. The cell pellets are resuspended and collected together with Hank's buffered saline solution (HBSS, GibcoBRL) in one 50 ml centrifuge tube and centrifuged as before. You can have about 25% more cells that want other cells for injection. If 72 mice are injected and have 100 × 10 ⁶ cells, all cells are prepared for injection into the mice. Inject 1483 cells into 1 × 10 ⁶ cells in 0.03 ml / mouse.
[1679] Cells are injected with 30% Matrigel (Collaborative Biomedical Products) and 70% HBSS. The pelleted pellet is resuspended with cold HBSS 2.1 (70%) followed by 0.9 ml (30%) of thawed cold liquefied Matrigel. Always store this cell preparation on ice before injecting it into the mouse. Nude mouse males, 4-6 weeks of age, are used in these studies (Harlen). After anesthesia with CO ₂ / O ₂ gas, they are injected into the center of the right hind paw using a 0.5 cc tuberculin syringe (Beckerson & Dickerson). For baseline weights to begin the study, mice are weighed for body weight on the day of infusion (day 0). Mice are weighed on day 7, and the right hind paw is measured for foot tumor volume using a flutemometer (Stoelting Co.). A flute thermometer is a machine that measures foot volume by replacing it with water. Left uninjected feet are measured and averaged against background measurements and subtracted from the foot with the right tumor.
[1680] If the tumor is between 100 and 200 μl in size, the animal is placed on the test compound in feed form and the compound feed is continued throughout the study. Some mice provide only protein kinase inhibitors or cyclooxygenase-2 selective inhibitors. Some mice receive both daily protein kinase inhibitors and cyclooxygenase-2 selective inhibitors at appropriate dosages determined based on ex vivo assay results.
[1681] The mice are weighed and measured at days 7, 10, 14, 17, 21, 24 and 28 throughout the study. Compound treatment can begin on day 0 (prevention), or there is a tumor established around 7 days once treatment (treatment). Near 30 days, vehicle (control) mice will have large tumors (about 1.0-1.5 ml) and weight loss will begin, killing the vehicle animals. If tumor suppression is seen in the treatment group and they are in good health, half of the treatment group is anesthetized and half of the treatment group is alive to determine the delay in tumor growth.
[1682] Those skilled in the art will readily understand that the present invention has been adapted to carry out the objects and to obtain the objects and advantages originally present as well as those mentioned herein. The molecular complexes, methods, procedures, treatments, molecules, and specific compounds described herein are representative of the preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that modifications and other uses are included within the spirit of the invention as defined by the claims.
[1683] Those skilled in the art will readily appreciate that substitutions and changes to the inventions disclosed herein may be modified without departing from the scope and spirit of the invention.
[1684] All patents and documents mentioned in the specification are indicative of the level of skill in the art related to the invention. All patents and documents are incorporated herein by reference in the same way as to indicate in particular that each individual document is incorporated by reference.

权利要求:
Claims (50)
[1" claim-type="Currently amended] A method of treating or preventing cancer comprising administering a protein kinase inhibitor of formula (I) to a mammal in need of treatment in combination with a cyclooxygenase inhibitor or a pharmaceutically acceptable salt thereof.
Formula I

Where
R is hydrogen, piperazin-1-ylmethyl, 4-methylpiperazin-1-ylmethyl, piperidin-1-ylmethyl, 2-hydroxymethylpyrrolidin-1-ylmethyl, 2-carboxy Rollidin-1-ylmethyl and pyrrolidin-1-ylmethyl;
R ¹ is hydrogen, halo, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, -C (O) NR ⁸ R ⁹ , -NR ¹³ R ¹⁴ ,-(CO) R ¹⁵ and-(CH ₂ ) _r R ¹⁶ ;
R ² is hydrogen, halo, alkyl, substituted alkyl, trihalomethyl, hydroxy, alkoxy, cyano, -NR ¹³ R ¹⁴ , -NR ¹³ C (O) R ¹⁴ , -C (O) R ¹⁵ , Aryl, heteroaryl, and -S (O) ₂ NR ¹³ R ¹⁴ ;
R ³ is hydrogen, halogen, alkyl, substituted alkyl, trihalomethyl, hydroxy, alkoxy, aryl, heteroaryl, -NR ¹³ R ¹⁴ , -NR ¹³ S (O) ₂ R ¹⁴ , -S (O) ₂ NR ¹³ R ¹⁴ , -NR ¹³ C (O) R ¹⁴ , -NR ¹³ C (O) OR ¹⁴ ,-(CO) R ¹⁵ and -SO ₂ R ¹⁹ ;
R ⁴ is selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, hydroxy, alkoxy and —NR ¹³ R ¹⁴ ;
R ⁵ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, and —C (O) R ¹⁰ ;
R ⁶ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, and —C (O) R ¹⁰ ;
R ⁷ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, -C (O) R ^l7 and -C (O) R ^l0 , provided that R ⁵ is hydrogen and R ⁶ and R ^At least one of ⁷ is —C (O) R ¹⁰ ; or
R ⁶ and R ⁷ combine to form a group selected from the group consisting of — (CH ₂ ) ₄ —, — (CH ₂ ) ₅ — and — (CH ₂ ) ₆ —;
R ⁸ and R ⁹ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl and aryl;
R ¹⁰ is selected from the group consisting of hydroxy, alkoxy, aryloxy, -N (R ^ll ) (alkylene) _n R ^l2 , wherein the alkylene group is optionally substituted with a hydroxy group and -NR ¹³ R ¹⁴ Selected;
R ¹¹ is selected from the group consisting of hydrogen, alkyl and substituted alkyl;
R ¹² is -NR ¹³ R ^14, hydroxy, -C (O) R ^15, aryl, ^{heteroaryl, -N + (O -) R} 13 R 14, -N (OH) R 13 and -NHC (O) R ¹⁸ is selected from the group consisting of (wherein, R ^l8 is alkyl, substituted alkyl, haloalkyl, or aralkyl);
R ¹³ and R ¹⁴ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, lower alkyl substituted with hydroxyalkylamino, cyanoalkyl, cycloalkyl, substituted cycloalkyl, aryl and heteroaryl; or
R ¹³ and R ¹⁴ may combine to form a heterocyclo group;
R ¹⁵ is selected from the group consisting of hydrogen, hydroxy, alkoxy and aryloxy;
R ¹⁶ is selected from the group consisting of hydroxy, -NR ¹³ R ¹⁴ , -C (O) R ¹⁵ and -C (O) NR ¹³ R ¹⁴ ;
R ¹⁷ is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, aryl and heteroaryl;
R ¹⁹ is selected from the group consisting of alkyl, substituted alkyl, aryl, aralkyl, heteroaryl or heteroaralkyl;
n and r are independently 1, 2, 3 or 4.
[2" claim-type="Currently amended] A protein kinase inhibitor of formula (I) in combination with (i) a compound of formula (II), (ii) a cyclooxygenase-2-selective inhibitor selected from the group consisting of a compound of formula (III) or a pharmaceutically acceptable salt thereof A method of treating or preventing cancer comprising administering to a mammal in need thereof a therapeutically effective amount.
Formula I

In Formula I,
R is hydrogen, piperazin-1-ylmethyl, 4-methylpiperazin-1-ylmethyl, piperidin-1-ylmethyl, 2-hydroxymethylpyrrolidin-1-ylmethyl, 2-carboxy Rollidin-1-ylmethyl and pyrrolidin-1-ylmethyl;
R ¹ is hydrogen, halo, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, -C (O) NR ⁸ R ⁹ , -NR ¹³ R ¹⁴ ,-(CO) R ¹⁵ and-(CH ₂ ) _r R ¹⁶ ;
R ² is hydrogen, halo, alkyl, substituted alkyl, trihalomethyl, hydroxy, alkoxy, cyano, -NR ¹³ R ¹⁴ , -NR ¹³ C (O) R ¹⁴ , -C (O) R ¹⁵ , Aryl, heteroaryl, and -S (O) ₂ NR ¹³ R ¹⁴ ;
R ³ is hydrogen, halogen, alkyl, substituted alkyl, trihalomethyl, hydroxy, alkoxy, aryl, heteroaryl, -NR ¹³ R ¹⁴ , -NR ¹³ S (O) ₂ R ¹⁴ , -S (O) ₂ NR ¹³ R ¹⁴ , -NR ¹³ C (O) R ¹⁴ , -NR ¹³ C (O) OR ¹⁴ ,-(CO) R ¹⁵ and -SO ₂ R ¹⁹ ;
R ⁴ is selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, hydroxy, alkoxy and —NR ¹³ R ¹⁴ ;
R ⁵ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, and —C (O) R ¹⁰ ;
R ⁶ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, and —C (O) R ¹⁰ ;
R ⁷ is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, -C (O) R ^l7 and -C (O) R ^l0 , provided that R ⁵ is hydrogen and R ⁶ and R ^At least one of ⁷ is —C (O) R ¹⁰ ; or
R ⁶ and R ⁷ combine to form a group selected from the group consisting of — (CH ₂ ) ₄ —, — (CH ₂ ) ₅ — and — (CH ₂ ) ₆ —;
R ⁸ and R ⁹ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl and aryl;
R ¹⁰ is selected from the group consisting of hydroxy, alkoxy, aryloxy, -N (R ^ll ) (alkylene) _n R ^l2 , wherein the alkylene group is optionally substituted with a hydroxy group and -NR ¹³ R ¹⁴ Selected;
R ¹¹ is selected from the group consisting of hydrogen, alkyl and substituted alkyl;
R ¹² is -NR ¹³ R ^14, hydroxy, -C (O) R ^15, aryl, ^{heteroaryl, -N + (O -) R} 13 R 14, -N (OH) R 13 and -NHC (O) R ¹⁸ is selected from the group consisting of (wherein, R ^l8 is alkyl, substituted alkyl, haloalkyl, or aralkyl);
R ¹³ and R ¹⁴ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, lower alkyl substituted with hydroxyalkylamino, cyanoalkyl, cycloalkyl, substituted cycloalkyl, aryl and heteroaryl; or
R ¹³ and R ¹⁴ may combine to form a heterocyclo group;
R ¹⁵ is selected from the group consisting of hydrogen, hydroxy, alkoxy and aryloxy;
R ¹⁶ is selected from the group consisting of hydroxy, -NR ¹³ R ¹⁴ , -C (O) R ¹⁵ and -C (O) NR ¹³ R ¹⁴ ;
R ¹⁷ is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, aryl and heteroaryl;
R ¹⁹ is selected from the group consisting of alkyl, substituted alkyl, aryl, aralkyl, heteroaryl or heteroaralkyl;
n and r are independently 1, 2, 3 or 4.
Formula II

In Chemical Formula II,
G is selected from the group consisting of O, S and -NR ^a , wherein R ^a is hydrogen or alkyl;
R ^10a is selected from the group consisting of hydrogen and aryl;
R ^lla is selected from the group consisting of carboxyl, alkyl, aralkyl, aminocarbonyl, alkylsulfonylaminocarbonyl and alkoxycarbonyl;
R ^1a is selected from the group consisting of haloalkyl, alkyl, aralkyl, cycloalkyl, and aryl optionally substituted with one or more radicals selected from alkylthio, nitro and alkylsulfonyl;
R ^13a is hydrogen, halo, alkyl, aralkyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, haloalkyl, haloalkoxy, alkylamino, arylamino, aralkylamino, heteroarylamino, Heteroarylalkylamino, nitro, amino, aminosulfonyl, alkylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, aralkylaminosulfonyl, heteroaralkylaminosulfonyl, heterocyclosulfonyl, alkylsulfonyl Independently from the group consisting of hydroxyarylcarbonyl, nitroaryl, optionally substituted aryl, optionally substituted heteroaryl, aralkylcarbonyl, heteroarylcarbonyl, arylcarbonyl, aminocarbonyl and alkylcarbonyl At least one radical selected; or
R ^13a together with ring E form a naphthyl ring.
Formula III

In Chemical Formula III,
A is selected from the group consisting of partially unsaturated or unsaturated heterocyclyl and partially unsaturated or unsaturated carbocyclic rings;
R ^lb is selected from the group consisting of heterocyclyl, cycloalkyl, cycloalkenyl and aryl, which is alkyl, haloalkyl, cyano, carboxyl, alkoxycarbonyl, hydroxyl, hydroxyalkyl, haloalkoxy at substitutable positions Optionally substituted with one or more radicals independently selected from amino, alkylamino, arylamino, nitro, alkoxyalkyl, alkylsulfinyl, halo, alkoxy and alkylthio;
R ^2b is selected from the group consisting of methyl and amino;
R ^3b is hydrogen, halo, alkyl, alkenyl, alkynyl, oxo, cyano, carboxyl, cyanoalkyl, heterocyclyloxy, alkyloxy, alkylthio, alkylcarbonyl, cycloalkyl, aryl, haloalkyl, hetero Cyclyl, cycloalkenyl, aralkyl, heterocyclylalkyl, acyl, alkylthioalkyl, hydroxyalkyl, alkoxycarbonyl, arylcarbonyl, aralkylcarbonyl, aralkenyl, alkoxyalkyl, arylthioalkyl, aryl Oxyalkyl, aralkylthioalkyl, aralkoxyalkyl, alkoxyaralkoxyalkyl, alkoxycarbonylalkyl, aminocarbonyl, aminocarbonylalkyl, alkylaminocarbonyl, N-arylaminocarbonyl, N-alkyl-N-aryl Aminocarbonyl, alkylaminocarbonylalkyl, carboxyalkyl, alkylamino, N-arylamino, N-aralkylamino, N-alkyl-N-aralkylamino, N-alkyl-N-arylamino, aminoalkyl, alkyl Aminoalkyl, N-arylaminoalkyl, N-aralkylaminoal , N-alkyl-N-aralkylaminoalkyl, N-alkyl-N-arylaminoalkyl, aryloxy, aralkoxy, arylthio, aralkylthio, alkylsulfinyl, alkylsulfonyl, aminosulfonyl, alkylaminosul Phonyl, N-arylaminosulfonyl, arylsulfonyl and N-alkyl-N-arylaminosulfonyl.
[3" claim-type="Currently amended] The method of claim 2,
Cyclooxygenase-2-selective inhibitors are compounds of Formula II,
G is selected from the group consisting of oxygen and sulfur;
R ^lla is selected from the group consisting of carboxyl, lower alkyl, lower aralkyl, lower alkoxycarbonyl;
R ^l2a is selected from the group consisting of lower haloalkyl, lower cycloalkyl and phenyl;
R ^13a is H, halo, lower alkyl, lower alkoxy, lower haloalkyl, lower haloalkoxy, lower alkylamino, nitro, amino, aminosulfonyl, lower alkylaminosulfonyl, 5-membered heteroarylalkylaminosulfonyl, 6-membered Heteroarylalkylaminosulfonyl, lower aralkylaminosulfonyl, 5-membered nitrogen-containing heterocyclosulfonyl, 6-membered nitrogen-containing heterocyclosulfonyl, lower alkylsulfonyl, optionally substituted phenyl, lower aralkylcarbonyl and lower At least one radical selected from the group consisting of alkylcarbonyl; Or R ^13a together with ring E forms a naphthyl ring.
[4" claim-type="Currently amended] The method of claim 3, wherein
R ^lla is carboxyl;
R ^l2a is lower haloalkyl;
R ^13a is H, halo, lower alkyl, lower haloalkyl, lower haloalkoxy, lower alkylamino, amino, aminosulfonyl, lower alkylaminosulfonyl, 5-membered heteroarylalkylaminosulfonyl, 6-membered heteroarylalkylaminosul One or more radicals selected from the group consisting of fonyl, lower aralkylaminosulfonyl, lower alkylsulfonyl, six-membered nitrogen-containing heterocyclosulfonyl, optionally substituted phenyl, lower aralkylcarbonyl and lower alkylcarbonyl; Or R ^13a together with ring E form a naphthyl ring,
Method of treating or preventing cancer.
[5" claim-type="Currently amended] The method of claim 4, wherein
R ^12a is fluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluoroethyl, difluoropropyl, dichloroethyl, dichloropropyl, difluoromethyl and trifluoro Romethyl;
R ^1a is hydrido, chloro, fluoro, bromo, iodo, methyl, ethyl, isopropyl, tert-butyl, butyl, isobutyl, pentyl, hexyl, methoxy, ethoxy, isopropyloxy, tert- Butyloxy, trifluoromethyl, difluoromethyl, trifluoromethoxy, amino, N, N-dimethylamino, N, N-diethylamino, N-phenylmethylaminosulfonyl, N-phenylethylaminosulfonyl , N- (2-furylmethyl) aminosulfonyl, nitro, N, N-dimethylaminosulfonyl, aminosulfonyl, N-methylaminosulfonyl, N-ethylsulfonyl, 2,2-dimethylethylaminosulfonyl , N, N-dimethylaminosulfonyl, N- (2-methylpropyl) aminosulfonyl, N-morpholinosulfonyl, methylsulfonyl, benzylcarbonyl, 2,2-dimethylpropylcarbonyl, phenylacetyl and phenyl At least one radical selected from the group consisting of; Or R ^13a together with ring E form a naphthyl ring,
Method of treating or preventing cancer.
[6" claim-type="Currently amended] The method of claim 5, wherein
R ^12a is selected from the group consisting of trifluoromethyl and pentafluoroethyl;
R ^1a is hydrido, chloro, fluoro, bromo, iodo, methyl, ethyl, isopropyl, tert-butyl, methoxy, trifluoromethyl, trifluoromethoxy, N-phenylmethylaminosulfonyl, N-phenylethylaminosulfonyl, N- (2-furylmethyl) aminosulfonyl, N, N-dimethylaminosulfonyl, N-methylaminosulfonyl, N- (2,2-dimethylethyl) aminosulfonyl, At least one radical selected from the group consisting of dimethylaminosulfonyl, 2-methylpropylaminosulfonyl, N-morpholinosulfonyl, methylsulfonyl, benzylcarbonyl and phenyl; Or R ^13a together with ring E forms a naphthyl ring,
Method of treating or preventing cancer.
[7" claim-type="Currently amended] The method of claim 2,
Cyclooxygenase-2-selective inhibitors,
6-chloro-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-chloro-7-methyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
8- (1-methylethyl) -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-chloro-7- (1,1-dimethylethyl) -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-chloro-8- (1-methylethyl) -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
2-trifluoromethyl-3H-naphthopyran-3-carboxylic acid;
7- (l, l-dimethylethyl) -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-bromo-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
8-chloro-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-trifluoromethoxy-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
5,7-dichloro-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
8-phenyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
7,8-dimethyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6,8-bis (dimethylethyl) -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
7- (1-methylethyl) -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
7-phenyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-chloro-7-ethyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-chloro-8-ethyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-chloro-7-phenyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6,7-dichloro-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6,8-dichloro-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
2-trifluoromethyl-3H-naphtho [2,1-b] pyran-3-carboxylic acid;
6-chloro-8-methyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
8-chloro-6-methyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
8-chloro-6-methoxy-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-bromo-8-chloro-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
8-bromo-6-fluoro-2-trifluoromethyl-2H-l-benzopyran-3-carboxylic acid;
8-bromo-6-methyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
8-bromo-5-fluoro-2-trifluoromethyl-2H-l-benzopyran-3-carboxylic acid;
6-chloro-8-fluoro-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-bromo-8-methoxy-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-[[(phenylmethyl) amino] sulfonyl] -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-[(dimethylamino) sulfonyl] -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-[(methylamino) sulfonyl] -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-[(4-morpholino) sulfonyl] -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-[(l, l-dimethylethyl) aminosulfonyl] -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-[(2-methylpropyl) aminosulfonyl] -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-methylsulfonyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
8-chloro-6-[[(phenylmethyl) amino] sulfonyl] -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-phenylacetyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6,8-dibromo-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
8-chloro-5,6-dimethyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6,8-dichloro- (S) -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-benzylsulfonyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-[[N- (2-furylmethyl) amino] sulfonyl] -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-[[N- (2-phenylethyl) amino] sulfonyl] -2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
6-iodo-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid;
7- (l, l-dimethylethyl) -2-pentafluoroethyl-2H-1-benzopyran-3-carboxylic acid; And
6-chloro-2-trifluoromethyl-2H-1-benzothiopyran-3-carboxylic acid; or
Selected from the group consisting of pharmaceutically acceptable salts thereof,
How to treat and prevent cancer.
[8" claim-type="Currently amended] The method of claim 2,
The cyclooxygenase-2-selective inhibitor is a compound selected from the group consisting of the compounds of a) to o), a pharmaceutically acceptable salt thereof, or any combination thereof.
a)
6-nitro-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid
b)
6-chloro-8-methyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid
c)
(S) -6-chloro-7- (1,1-dimethylethyl) -2- (trifluoromethyl-2H-1-benzopyran-3-carboxylic acid
d)
2-Trifluoromethyl-2H-naphtho [2,3-b] pyran-3-carboxylic acid
e)
6-chloro-7- (4-nitrophenoxy) -2- (trifluoromethyl) -2H-1-benzopyran-3-carboxylic acid
f)
((S) -6,8-dichloro-2- (trifluoromethyl) -2H-1-benzopyran-3-carboxylic acid
g)
6-chloro-2- (trifluoromethyl) -4-phenyl-2H-1-benzopyran-3-carboxylic acid
h)
6- (4-hydroxybenzoyl) -2- (trifluoromethyl) -2H-1-benzopyran-3-carboxylic acid
i)
2- (Trifluoromethyl) -6-[(trifluoromethyl) thio] -2H-1-benzothiopyran-3-carboxylic acid
j)
6,8-dichloro-2-trifluoromethyl-2H-1-benzothiopyran-3-carboxylic acid
k)
6- (1,1-dimethylethyl) -2- (trifluoromethyl) -2H-1-benzothiopyran-3-carboxylic acid
l)
6,7-difluoro-1,2-dihydro-2- (trifluoromethyl) -3-quinolinecarboxylic acid
m)
6-Chloro-1,2-dihydro-l-methyl-2- (trifluoromethyl) -3-quinolinecarboxylic acid
n)
6-chloro-2- (trifluoromethyl) -1,2-dihydro [1,8] naphthyridine-3-carboxylic acid
o)
((S) -6-chloro-1,2-dihydro-2- (trifluoromethyl) -3-quinolinecarboxylic acid.
[9" claim-type="Currently amended] The method of claim 2,
The cyclooxygenase-2-selective inhibitor is a compound of formula III selected from the group consisting of the compounds of a) to f), a pharmaceutically acceptable salt thereof, or any combination thereof.
a)
b)
c)
d)
e)
f)
[10" claim-type="Currently amended] The method of claim 3, wherein
A cyclooxygenase-2-selective inhibitor is a compound selected from the group consisting of a) and b), a pharmaceutically acceptable salt thereof, or any combination thereof.
a)
b)
[11" claim-type="Currently amended] The method of claim 2,
A cyclooxygenase-2-selective inhibitor is a compound of formula (1) or a pharmaceutically acceptable salt thereof, the method of treating and preventing cancer.

[12" claim-type="Currently amended] The method of claim 2,
A cyclooxygenase-2-selective inhibitor is a compound of Formula 2 or a pharmaceutically acceptable salt thereof, the method of treating and preventing cancer.

[13" claim-type="Currently amended] The method of claim 1,
Treatment and prevention of cancer, wherein the cyclooxygenase-2-selective inhibitor is 4-[(4-methyl) -sulfonyl) phenol] -3-phenyl-2 (5H) -furanone or a pharmaceutically acceptable salt thereof Way.
[14" claim-type="Currently amended] The method of claim 2,
A cyclooxygenase-2-selective inhibitor is 4-[(5-methyl-3-phenyl) -4-isoxazole] phenylsulfonamide or a pharmaceutically acceptable salt thereof.
[15" claim-type="Currently amended] The method of claim 1,
The cyclooxygenase-2-selective inhibitor is 2- (6-methylpyrid-3-yl) -3- (4-methylsulfonylphenyl) -5-chloropyridine or a pharmaceutically acceptable salt thereof Treatment and prevention methods.
[16" claim-type="Currently amended] The method of claim 2,
The cyclooxygenase-2-selective inhibitor is 4- [5- (4-methylphenyl) -3- (trifluoromethyl) -1H-pyrazol-1-yl] phenylsulfonamide or a pharmaceutically acceptable salt thereof , Methods of treatment and prevention of cancer.
[17" claim-type="Currently amended] The method of claim 2,
A method of treating and preventing cancer wherein the cyclooxygenase-2-selective inhibitor is 4-[(2-methyl-4-cyclohexyl) -5-oxazolyl] phenylsulfonamide or a pharmaceutically acceptable salt thereof.
[18" claim-type="Currently amended] The method of claim 2,
Cyclooxygenase-2-selective inhibitor is 4- [5- (3-fluoro-4-methoxyphenyl) -3- (difluoromethyl) -1H-pyrazol-1-yl] -benzenesulfonamide Or a pharmaceutically acceptable salt thereof.
[19" claim-type="Currently amended] The method of claim 2,
Of the cancer, wherein the cyclooxygenase-2-selective inhibitor is (S) -6,8-dichloro-2- (trifluoromethyl) -2H-1-benzopyran-3-carboxylic acid or a pharmaceutically acceptable salt thereof Treatment and prevention methods.
[20" claim-type="Currently amended] The method of claim 2,
The cyclooxygenase-2-selective inhibitor is a compound or a pharmaceutically acceptable salt thereof, the method of treating and preventing cancer.

Where
X is O or S;
R ^l2b is lower haloalkyl;
R ^3b is selected from the group consisting of hydrido and halo;
R ^{13b ′} is hydrido, halo, lower alkyl, lower haloalkoxy, lower alkoxy, lower aralkylcarbonyl, lower dialkylaminosulfonyl, lower alkylaminosulfonyl, lower aralkylaminosulfonyl, lower heteroaralkyl Aminosulfonyl, 5-membered nitrogen-containing heterocyclosulfonyl, and 6-membered nitrogen-containing heterocyclosulfonyl;
R ^{13b ″} is selected from the group consisting of hydrido, lower alkyl, halo, lower alkoxy and aryl;
R ^{13b ″ ′} is selected from the group consisting of hydrido, halo, lower alkyl, lower alkoxy and aryl.
[21" claim-type="Currently amended] The method of claim 20,
R ^1b is selected from the group consisting of trifluoromethyl and pentafluoroethyl;
R ^13b is selected from the group consisting of hydrido, chloro and fluoro;
R ^{13b ′} is hydrido, chloro, bromo, fluoro, iodo, methyl, tert-butyl, trifluoromethoxy, methoxy, benzylcarbonyl, dimethylaminosulfonyl, isopropylaminosulfonyl, methylamino Sulfonyl, benzylaminosulfonyl, phenylethylaminosulfonyl, methylpropylaminosulfonyl, methylsulfonyl and morpholinosulfonyl;
R ^{13b ″} is selected from the group consisting of hydrido, methyl, ethyl, isopropyl, tert-butyl, chloro, methoxy, diethylamino and phenyl;
R ^{13b ″ ′} is selected from the group consisting of hydrido, chloro, bromo, fluoro, methyl, ethyl, tert-butyl, methoxy and phenyl,
How to treat and prevent cancer.
[22" claim-type="Currently amended] The method of claim 2,
Protein kinase inhibitors are compounds of Formula (I) or pharmaceutically acceptable salts thereof,
R ¹ is hydrogen, halo, alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, -C (O) R ¹⁵ , -NR ¹³ R ¹⁴ ,-(CH ₂ ) _r R ¹⁶ and -C (O) NR ⁸ R ⁹ ;
R ² is hydrogen, halo, alkyl, trihalomethyl, hydroxy, alkoxy, -NR ¹³ R ¹⁴ , -NR ¹³ C (O) R ¹⁴ , -C (O) R ¹⁵ , aryl, heteroaryl and -S (O) ₂ NR ¹³ R ¹⁴ ;
R ³ is hydrogen, halogen, alkyl, trihalomethyl, hydroxy, alkoxy,-(CO) R ¹⁵ , -NR ¹³ R ¹⁴ , aryl, heteroaryl, -NR ¹³ S (O) ₂ R ¹⁴ , -S (O) ₂ NR ¹³ R ¹⁴ , -NR ¹³ C (O) R ^14, and -NR ¹³ C (O) OR ¹⁴ ;
R ⁴ is selected from the group consisting of hydrogen, halogen, alkyl, hydroxy, alkoxy and —NR ¹³ R ¹⁴ ;
R ⁵ is selected from the group consisting of hydrogen, alkyl and -C (O) R ¹⁰ ;
R ⁶ is selected from the group consisting of hydrogen, alkyl and -C (O) R ¹⁰ ;
R ⁷ is selected from the group consisting of hydrogen, alkyl, aryl, 3-carboxypropyl, heteroaryl, -C (O) R ¹⁷ and -C (O) R ¹⁰ ;
R ⁶ and R ⁷ combine to form a group selected from the group consisting of — (CH ₂ ) ₄ —, — (CH ₂ ) ₅ — and — (CH ₂ ) ₆ —;
R ⁸ and R ⁹ are independently selected from the group consisting of hydrogen, alkyl and aryl;
R ¹⁰ is selected from the group consisting of hydroxy, alkoxy, aryloxy, -N (R ^ll ) (CH ₂ ) _n R ^l2 and -NR ¹³ R ¹⁴ ;
R ¹¹ is selected from the group consisting of hydrogen and alkyl;
R ¹² is selected from the group consisting of —NR ¹³ R ¹⁴ , hydroxy, —C (O) R ¹⁵ , aryl and heteroaryl;
R ¹³ and R ¹⁴ are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl and heteroaryl;
R ^l3 and R ^l4 combine to form — (CH ₂ ) ₄ —, — (CH ₂ ) ₅ —, — (CH ₂ ) ₂ O (CH ₂ ) ₂ — and — (CH ₂ ) ₂ N (CH ₃ ) ( CH ₂ ) ₂ -to form a group selected from the group consisting of;
R ¹⁵ is selected from the group consisting of hydrogen, hydroxy, alkoxy and aryloxy;
R ¹⁶ is selected from the group consisting of hydroxy, -C (O) R ¹⁵ , -NR ¹³ R ¹⁴ and -C (O) NR ¹³ R ¹⁴ ;
R ¹⁷ is selected from the group consisting of alkyl, cycloalkyl, aryl and heteroaryl;
wherein n and r are independently 1, 2, 3 or 4;
[23" claim-type="Currently amended] The method of claim 2,
Protein kinase inhibitors are compounds of Formula (I) or pharmaceutically acceptable salts thereof,
R ⁶ is -COR ¹⁰ ,
R ¹⁰ is —NR ¹¹ (CH ₂ ) _n R ¹² ,
R ¹¹ is hydrogen or lower alkyl,
n is 2 or 3,
R ^l2 is -NR ¹³ R ¹⁴ ,
R ¹³ and R ¹⁴ are independently lower alkyl.
[24" claim-type="Currently amended] The method of claim 2,
Protein kinase inhibitors are compounds of Formula (I) or pharmaceutically acceptable salts thereof,
R ⁶ is -COR ¹⁰ ,
R ¹⁰ is —NR ¹¹ (CH ₂ ) _n R ¹² ,
R ¹¹ is hydrogen or lower alkyl,
n is 2 or 3,
R ^l2 is -NR ¹³ R ¹⁴ ,
R ¹³ and R ¹⁴ combine to form — (CH ₂ ) ₄ —, — (CH ₂ ) ₅ —, — (CH ₂ ) ₂ —O— (CH ₂ ) ₂ — or — (CH ₂ ) ₂ N (CH ₃₎ ) (CH ₂ ) ₂ -to form a group selected from the group consisting of.
[25" claim-type="Currently amended] The method of claim 2,
Protein kinase inhibitors are compounds of formula I wherein R ⁶ is N- (2-dimethylamino-ethyl) aminocarbonyl, N- (2-diethyl-aminoethyl) -N-methylaminocarbonyl, N- (3 -Dimethylamino-propyl) -aminocarbonyl, N- (2-diethylaminoethyl) aminocarbonyl, N- (2-ethylaminoethyl) -aminocarbonyl, N- (3-ethylaminopropyl) aminocarbon Neyl or N- (3-diethylamino-propyl) aminocarbonyl. The method of treating and preventing cancer.
[26" claim-type="Currently amended] The method of claim 2,
The protein kinase inhibitor is a compound of Formula I, wherein R ⁶ is N- (2-diethylaminoethyl) aminocarbonyl or N- (2-ethylamino-ethyl) aminocarbonyl.
[27" claim-type="Currently amended] The method of claim 2,
Protein kinase inhibitors are compounds of formula I wherein R ⁶ is 3-pyrrolidin-1-ylpropylaminocarbonyl, 3-morpholin-4-ylpropylamino-carbonyl, 2-pyrrolidin-1-yl Ethylamino-carbonyl, 2-morpholin-4-ylethylaminocarbonyl, 2- (4-methylpiperazin-1-yl) ethyl-aminocarbonyl, 2- (3,5-dimethylpiperazine-1 -Yl) ethyl-aminocarbonyl, 3- (4-methylpiperazin-1-yl) propylamino-carbonyl or 3- (3,5-dimethylpiperazin-1-yl) propylamino-carbonyl, How to treat and prevent cancer.
[28" claim-type="Currently amended] The method of claim 2,
Protein kinase inhibitors are compounds of formula I, wherein R ⁶ is -COR ¹⁰ , R ¹⁰ is -NR ¹³ R ¹⁴ , R ¹³ is hydrogen, and R ¹⁴ is substituted with hydroxy, lower alkyl, aryl, heteroalicye Click, heteroaryl or carboxyin, methods of treating and preventing cancer.
[29" claim-type="Currently amended] The method of claim 2,
Protein kinase inhibitors are compounds of formula I wherein R ⁶ is 2-triazin-1-ylpropylaminocarbonyl, 2-triazin-1-ylethylaminocarbonyl, 3-imidazol-1-ylpropylaminocarbon Neyl, pyridin-4-ylmethyl-aminocarbonyl, 2-pyridin-2-ylethylaminocarbonyl or 2-imidazol-1-yl ethylaminocarbonyl.
[30" claim-type="Currently amended] The method of claim 2,
Protein kinase inhibitors are compounds of Formula I,
R ⁶ is -COR ¹⁰ ,
R ¹⁰ is —NR ¹¹ (CH ₂ ) _n R ¹² ,
R ¹¹ is hydrogen or lower alkyl,
n is 2 or 3,
R ^l2 is -NR ¹³ R ¹⁴ ,
R ¹³ and R ¹⁴ combine together to form a heterocycle.
[31" claim-type="Currently amended] The method of claim 2,
Protein kinase inhibitors are compounds of Formula I,
R ⁶ is -COR ¹⁰ ,
R ¹⁰ is —NR ¹¹ (CH ₂ ) _n R ¹² ,
R ¹¹ is hydrogen or lower alkyl,
n is 2 or 3,
R ^l2 is -NR ¹³ R ¹⁴ ,
R ¹³ and R ¹⁴ combine together to form a 5, 6 or 7 membered heterocycle containing 1 or 2 nitrogen atoms and a carbonyl group in the ring.
[32" claim-type="Currently amended] The method of claim 2,
Protein kinase inhibitors are compounds of Formula I wherein R ⁶ is 2- (3-oxopiperazin-1-yl) ethylaminocarbonyl, 2- (imidazolidin-1-yl-2-one) ethylaminocarbo Nyl, 2- (tetrahydropyrimidin-1-yl-2-one) ethylaminocarbonyl, 2- (2-oxopyrrolidin-1-yl) -ethylaminocarbonyl, 3- (3-oxopipepe Razin-1-yl) propyl-aminocarbonyl, 3- (imidazolidin-1-yl-2-one) propyl-aminocarbonyl, 3- (tetrahydropyrimidin-1-yl-2-one) -Propylaminocarbonyl or 3- (2-oxopyrrolidin-1-yl) propyl-aminocarbonyl.
[33" claim-type="Currently amended] 33. The method according to any one of claims 22 to 32,
Protein kinase inhibitors are compounds of Formula I,
R ⁵ is selected from the group consisting of hydrogen and lower alkyl;
R ⁷ is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, and -C (O) R ¹⁷ , wherein R ¹⁷ is hydroxy, lower alkyl or aryl.
[34" claim-type="Currently amended] The method of claim 33, wherein
R ¹ is hydrogen, lower alkyl, —C (O) NR ⁸ R ⁹ , cycloalkyl or aryl;
R ² is hydrogen, halo, lower alkoxy, cyano, aryl, —SO ₂ R 20 or —S (O) ₂ NR ¹³ R ¹⁴ , wherein R ¹³ is hydrogen and R ¹⁴ is hydrogen, aryl or alkyl ;
R ³ is selected from the group consisting of hydrogen, lower alkoxy, —C (O) R ¹⁵ , —NR ¹³ C (O) R ¹⁴ , aryl and heteroaryl;
The method of treating and preventing cancer, wherein R ⁴ is hydrogen.
[35" claim-type="Currently amended] The method of claim 34, wherein
R ¹ is hydrogen or phenyl;
R ² is hydrogen, chloro, bromo, fluoro, methoxy, ethoxy, phenyl, dimethylaminosulfonyl, cyano, methylsulfonyl, ethylsulfonyl, benzylsulfonyl, 3-chlorophenyl-aminosulfonyl, Carboxy, methoxy, aminosulfonyl, methylaminosulfonyl, phenylaminosulfonyl, pyridin-3-yl-aminosulfonyl, dimethylaminosulfonyl or isopropylamino-sulfonyl;
R ³ is hydrogen, methoxy, carboxy, phenyl, pyridin-3-yl, 3,4-dichlorophenyl, 2-methoxy-5-isopropylphenyl, 4-n-butylphenyl, 3-isopropylphenyl;
The method of treating and preventing cancer, wherein R ⁴ is hydrogen.
[36" claim-type="Currently amended] 36. The method of claim 35 wherein
R ¹ is hydrogen;
R ² is hydrogen, cyano, fluoro, chloro or bromo;
R ³ is phenyl;
The method of treating and preventing cancer, wherein R ⁴ is hydrogen.
[37" claim-type="Currently amended] The method of claim 2,
R ¹ is hydrogen, lower alkyl, —C (O) NR ⁸ R ⁹ , cycloalkyl or aryl;
R ² is hydrogen, halo, lower alkyl, cyano, aryl or —S (O) ₂ NR ¹³ R ¹⁴ wherein R ¹³ is hydrogen and R ¹⁴ is hydrogen, aryl or alkyl;
R ³ is selected from the group consisting of hydrogen, lower alkoxy, —C (O) R ¹⁵ , —NR ¹³ C (O) R ¹⁴ , aryl and heteroaryl;
The method of treating and preventing cancer, wherein R ⁴ is hydrogen.
[38" claim-type="Currently amended] The method of claim 2,
R ¹ is hydrogen or methyl;
R ² is hydrogen, cyano, chloro, fluoro or bromo;
R ³ is selected from the group consisting of hydrogen or phenyl;
The method of treating and preventing cancer, wherein R ⁴ is hydrogen.
[39" claim-type="Currently amended] The method of claim 2,
R ⁶ is -COR ¹⁰ ,
R ¹⁰ is -NR ¹³ R ¹⁴ ,
R ¹³ is hydrogen,
Cancer, wherein R ¹⁴ is lower alkyl substituted with hydroxy, lower alkyl substituted with hydroxyalkylamino, carboxy, or —NR ¹⁸ R ¹⁹ , wherein R ¹⁸ and R ¹⁹ are independently hydrogen or lower alkyl Treatment and prevention methods.
[40" claim-type="Currently amended] The method of claim 2,
R ⁶ is [2- (diethylamino) -2-hydroxy] ethylaminocarbonyl, 2- (N-ethyl-N-2-hydroxyethylamino) -ethylaminocarbonyl, carboxymethylaminocarbonyl or A method of treating and preventing cancer, which is 2-hydroxyethyl-aminocarbonyl.
[41" claim-type="Currently amended] The method of claim 2,
R ⁶ is -COR ¹⁰ ,
R ¹⁰ is —NR ¹¹ (CH ₂ ) _n R ¹² ,
And NR ¹³ R ¹⁴ or ^{-N (OH) R 13, -} R 12 is -N ⁺ (O)
R ¹³ and R ¹⁴ are independently selected from the group consisting of lower alkyl.
[42" claim-type="Currently amended] The method of claim 2,
Treatment and prevention of cancer, wherein R ⁶ is 2- (N-hydroxy-N-ethylamino) ethylaminocarbonyl or 2- [N ⁺ (O ⁻ ) (C ₂ H ₅ ) ₂ ] ethyl-aminocarbonyl Way.
[43" claim-type="Currently amended] 43. The method of claim 41 or 42,
R ⁵ is selected from the group consisting of hydrogen or methyl;
R ⁷ is selected from the group consisting of methyl, hydrogen or phenyl.
[44" claim-type="Currently amended] The method of claim 2,
R ¹ is hydrogen;
R ² is hydrogen, cyano, chloro, fluoro or bromo;
R ³ is hydrogen;
The method of treating and preventing cancer, wherein R ⁴ is hydrogen.
[45" claim-type="Currently amended] The method of claim 2,
Kinase inhibitors,

And their L-maleate salts.
[46" claim-type="Currently amended] The method of claim 2,
A method of treating and preventing cancer wherein the kinase inhibitor is a compound of:

[47" claim-type="Currently amended] Treatment of cancer comprising administering a protein kinase inhibitor of Formula 4 to a mammal in need of such treatment in combination with a cyclooxygenase-2-selective inhibitor of Formula 1 or a pharmaceutically acceptable salt thereof And preventive methods.
Formula 1


[48" claim-type="Currently amended] Treatment of cancer comprising administering a protein kinase inhibitor of Formula 4 to a mammal in need of such treatment in combination with a cyclooxygenase-2-selective inhibitor of Formula 2 or a pharmaceutically acceptable salt thereof And preventive methods.
Formula 2

Formula 4

[49" claim-type="Currently amended] Treatment of cancer comprising administering a protein kinase inhibitor of Formula 3 to a mammal in need of such treatment in combination with a cyclooxygenase-2-selective inhibitor of Formula 1 or a pharmaceutically acceptable salt thereof And preventive methods.
Formula 1

Formula 3

[50" claim-type="Currently amended] Treatment of cancer comprising administering a protein kinase inhibitor of Formula 3 to a mammal in need of such treatment in combination with a cyclooxygenase-2-selective inhibitor of Formula 2 or a pharmaceutically acceptable salt thereof And preventive methods.
Formula 2

Formula 3

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同族专利:

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公开号 | 公开日

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EA008137B1|2007-04-27|

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HRP20040170A2|2004-08-31|

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NZ530792A|2005-09-30|

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TNSN04015A1|2006-06-01|

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EP1427326A2|2004-06-16|

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SK1412004A3|2004-10-05|

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AR038957A1|2005-02-02|

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MA27060A1|2004-12-20|

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HU0500424A2|2007-02-28|

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EP1427326A4|2005-06-08|

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AU2002329744B2|2007-08-30|

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AP200402995A0|2004-03-31|

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KR100656004B1|2007-02-28|

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CO5560544A2|2005-09-30|

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ZA200400849B|2005-05-03|

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GEP20063868B|2006-07-10|

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WO2003015608A2|2003-02-27|

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IL159887D0|2004-06-20|

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CZ2004356A3|2004-12-15|

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TWI230609B|2005-04-11|

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NO20040516L|2004-04-13|

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MXPA04001464A|2005-02-17|

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EP1427326B1|2008-11-19|

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ECSP045021A|2004-04-28|

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JP2005501843A|2005-01-20|

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CN1541098A|2004-10-27|

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PL368921A1|2005-04-04|

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BR0211978A|2004-07-20|

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US20030216410A1|2003-11-20|

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DE60229959D1|2009-01-02|

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EA200400235A1|2004-10-28|

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CA2457745A1|2003-02-27|

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OA12651A|2006-06-15|

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AT414512T|2008-12-15|

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RS13204A|2007-02-05|

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BG108622A|2005-10-31|

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IS7137A|2004-01-30|

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US7320996B2|2008-01-22|

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WO2003015608A3|2003-10-30|

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PE20030332A1|2003-04-05|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2001-08-15|Priority to US31241301P

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2001-08-15|Priority to US60/312,413

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2002-08-15|Application filed by 수젠, 인크., 파마시아 코포레이션

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2002-08-15|Priority to PCT/US2002/025797

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2004-05-22|Publication of KR20040043193A

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2007-02-28|Application granted

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2007-02-28|Publication of KR100656004B1

优先权:

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申请号 | 申请日 | 专利标题

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US31241301P| true| 2001-08-15|2001-08-15|

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US60/312,413|2001-08-15|

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PCT/US2002/025797|WO2003015608A2|2001-08-15|2002-08-15|Combination therapy for the treatment of cancer|