WO2010077295A1 - Substituted tricyclic heterocycles and uses to treat tumors and proliferative disorders - Google Patents

Abstract

The present disclosure relates, in part, to substituted tricyclic heterocyclic compounds and to methods of using substituted tricyclic heterocyclic compounds to treat tumors and proliferative disorders and to induce apoptosis.

Description

Substituted Tricyclic Heterocvcles and Uses to Treat Tumors and Proliferative Disorders

Related Applications

This application claims the benefit of the filing date of U.S. provisional patent application 61/201,277, filed December 9, 2008, and entitled "Substituted Tricyclic Heterocycles and Uses to Treat Tumors and Proliferative Disorders." The entire teachings of the referenced provisional patent application are expressly incorporated herein by reference.

Field of the Invention

The present disclosure relates to substituted tricyclic heterocyclic compounds and uses thereof.

Background of the Invention Tumors (benign, pre-malignant, or malignant) and unwanted mammalian cell proliferation are a major cause of morbidity and mortality. Malignant tumors (cancer) cause about 13% of all deaths. According to the American Cancer Society, 7.6 million people died from cancer in the world during 2007.

Treatment of tumors and unwanted mammalian cell proliferation can be approached by several modes of therapy, including surgery, radiation, drug therapy (e.g., chemotherapy), or a combination of any of these treatments. Chemotherapy is an indispensable form of treatment for tumors and unwanted mammalian cell proliferation. However, many chemotherapeutic agents produce unwanted side effects. A need exists to identify novel chemotherapeutic agents.

Summary of the Invention

The present disclosure is based, in part, on the finding that compound of Formula I, a substituted tricyclic heterocyclic compound, induces apoptosis to MDA cells derived from a transformed breast cancer cell line. The disclosure thus provides substituted tricyclic heterocyclic compounds as compositions of matter. The disclosure also encompasses methods of using the conjugates in the treatment of tumors and unwanted mammalian cell proliferation and to induce apoptosis.

According to one aspect of the disclosure, a compound having a structure:

(Formula I) or a salt thereof is provided.

According to another aspect of the disclosure, a pharmaceutical composition is provided. The pharmaceutical composition comprises the compound of Formula I and a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise an agent other than the compound of Formula I. In some embodiments, the agent is an antitumor and/or an antiproliferative agent. Examples of antitumor agents and antiproliferative agents are listed below.

According to another aspect of the disclosure, a method for treating a subject having a tumor is provided. The method comprises administering to the subject an effective amount of a pharmaceutical composition of the compound of Formula I to treat the tumor. The tumor may be a solid tumor or a non-solid tumor. In some preferred embodiments, the tumor is breast cancer.

According to another aspect of the disclosure, a method for treating a subject having an unwanted mammalian cell proliferation is provided. The method comprises administering to the subject an effective amount of a pharmaceutical composition of the compound of Formula I to treat the unwanted mammalian cell proliferation.

According to yet another aspect of the disclosure, a method for inducing apoptosis in a cell is provided. The method comprises contacting the cell with an effective amount of compound of Formula I to induce apoptosis.

According to another aspect of the disclosure, the use of the compound of Formula I:

or a salt thereof in the manufacture of a medicament for treating a tumor in a subject is provided. The medicament is administered in an effective amount to treat the tumor. The tumor may be a solid tumor or a non-solid tumor. In some preferred embodiments, the tumor is breast cancer. According to another aspect of the disclosure, the use of the compound of Formula I:

or a salt thereof in the manufacture of a medicament for treating an unwanted mammalian cell proliferation is provided tumor is provided. The medicament is administered in an effective amount to treat the unwanted mammalian cell proliferation.

These and other aspects of the disclosure, as well as various advantages and utilities, will be more apparent with reference to the Detailed Description. Each aspect of the invention can encompass various embodiments, as will be understood.

Brief Description of the Drawings

Figure 1. Annexin V expression induced by 24 hours treatment at indicated concentrations of compound of Formula I (AT-4). Figure 2. Cell cycle arrest induced by 24 hours treatment at indicated concentrations of compound of Formula I (AT-4).

Figure 3. Annexin V expression induced by 48 hours treatment at indicated concentrations of compound of Formula I (AT-4).

Figure 4. Cell cycle arrest induced by 48 hours treatment at indicated concentrations of compound of Formula I (AT-4).

Figure 5. Annexin V expression induced by 72 hours treatment at indicated concentrations of compound of Formula I (AT-4).

Figure 6. Cell cycle arrest induced by 24 hours treatment at indicated concentrations of compound of Formula I (AT-4). Figure 7. Data showing the selectivity of compound of Formula I (AT-4) at indicated concentrations.

Figure 8. Graph showing the effect of compound of Formula I (AT-4) at indicated concentrations on highly mitotic cells (MDA) vs. poorly mitotic cells (MCF- 12). Figure 9. Photographs showing immunofluorescence staining of cells to visualize chromosomes and spindles at indicated times after treatment with compound of Formula I (AT-4). Cells were stained with PI, DAPI to visualize the chromosomes and α-tubulin antibodies to visualize the spindles.

Figure 10. Photograph of immunoblot showing inhibition of cyclin Bl after indicated times of exposure to compound of Formula I (AT-4). Cyclin Bl was completely inhibited in 48 hours and 72 hours exposure times. Lanes: (1) Control; (2) 24hrs exposure time with 100 μM compound # AT-4; (3) 48hrs exposure time with 100 μM compound # AT-4; (4) 72hrs exposure time with 100 μM compound # AT-4;

Detailed Description of the Invention The disclosure describes substituted tricyclic heterocyclic compounds and methods of using substituted tricyclic heterocyclic compounds (e.g., compound of Formula I) in the treatment of tumors and unwanted mammalian cell proliferation. The disclosure also describes methods of inducing apoptosis in cells using substituted tricyclic heterocyclic compounds (e.g., compound of Formula I). The disclosure provides compositions of matter. In one aspect of the disclosure, the composition of matter is compound of Formula I. The disclosure also provides pharmaceutical compositions comprising compound of Formula I. The pharmaceutical composition comprises the compound of Formula I in a pharmaceutically acceptable carrier or diluent. The term "pharmaceutically acceptable carrier" as used herein refers to compounds suitable for use in contact with recipient subjects, preferably mammals, and more preferably humans, and having a toxicity, irritation, or allergic response commensurate with a reasonable benefit/risk ratio, and effective for their intended use. In some embodiments, the pharmaceutically acceptable carrier is an aqueous solution (e.g., saline).

The pharmaceutical compositions also can contain other components useful in formulating pharmaceutical preparations for administration to subjects, preferably humans, including surfactants, solvents, preservatives, diluents, buffering agents and the like, all of which are standard in the pharmaceutical arts. Suitable surfactants for use include non-ionic agents, such as long-chain fatty acids and their water-insoluble derivatives. These include fatty amines such as lauryl acetyl and stearyl amine, glyceryl esters such as the naturally occurring mono-, di- and triglycerides, and fatty acid esters of fatty amines, such as propylene glycol, polyethylene glycol, sorbitan, sucrose and cholesterol. Also useful are compounds that have polyoxyethylene groups added through an ether linkage with an amine group. Compounds that are also useful include the polyoxyethylene sorbitan fatty acid esters and polyoxyethylene glycerol and steroidal esters. Some of the preferred surfactants are Cremophor^® EL and Cremophor^® EL-P, which are polyoxyethylated castor oil surfactants.

It is contemplated that other surfactants may be used to solubilize the compositions described herein. For example, it is contemplated that polysorbate 80, polysorbate 20, sodium laurate, sodium oleate, and sorbitan monooleate may be useful in certain embodiments of the present invention. Anionic surfactants may also be useful. Examples of these include, but are not limited to, sodium cholate, sodium lauryl sulfate, sodium deoxycholate, sodium laurate, sodium oleate, and potassium laurate.

In certain embodiments, dehydrated ethanol may be used as a solvent for the compositions described herein. In other embodiments, glycols such as propylene glycol or polyethylene glycol may be used. Simple complex polyols may also be suitable solvents. Moreover, the use of non-dehydrated amines may also be suitable. It is recognized that the determination of a solvent and its proper concentration to fully solubilize the compound of Formula I compositions is within the scope of a skilled artisan, and would not require undue experimentation.

Suitable buffering agents include: acetic acid and a salt (1-2% W/V); citric acid and a salt (1-3% W/V); and phosphoric acid and a salt (0.8-2% W/V).

Suitable preservatives include antimicrobial agents, such as, benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9% W/V); parabens (0.01 -0.25% W/V) and thimerosal (0.004-0.02% W/V) and/or suitable antiantioxidants, such as, ascorbic acid, ascorbyl pamitate, BHA, BHT, hypophosphorous acid, monothioglycerol, potassium metabisulfite, propyl gallate, sodium formaldehyde sulfoxylate, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, sulfur dioxide, tocopherol and/or tocopherols excipient. In some embodiments, the compound(s) described herein (e.g., compound of

Formula I) is/are provided in the form of a pharmaceutically acceptable salt. By "pharmaceutically acceptable salt" is meant those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1 (1977). The salts may be prepared during the final isolation and purification of the compounds of the disclosure or separately. The salts may be prepared by reacting a free base function with a suitable acid to form the salt (acid addition salts) or by reacting a carboxylic acid-containing moiety with a suitable base (base addition salts). Examples of suitable bases include hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or organic primary, secondary, or tertiary amine.

Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorolsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate

(isothionate), lactate, maleate, methanesulfonate, nicotinate, 2-Naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate. Examples of acids that can be employed to form pharmaceutically acceptable acid addition salts include inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and organic acids, such as oxalic acid, maleic acid, succinic acid, and citric acid.

Representative pharmaceutically acceptable basic addition salts include, but are not limited to, cations based on alkali metals or alkaline earth metals, such as lithium, sodium, potassium, calcium, magnesium, and aluminum, and the like, and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethyl-ammonium, methylamine, dimethylamine, trimethylamine ethylamine, diethylamine, triethylamine, and the like. Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.

In one aspect, the pharmaceutical compositions comprise a compound of Formula I and one or more therapeutic agents. In some preferred embodiments, the therapeutic agent is one or more an antitumor agent(s) and/or an antiproliferative agent. Examples of antitumor agents that may be used include, but are not limited to: actimomycin D, actinomycin D, AD 32/Valrubicin, Adrenocortical suppressant, Adrenocorticosteroids/antagonists, adriamycin, AG3340, AG3433, alkylating agents such as melphalan and cyclophosphamide, Alkyl sulfonates, 5-Azacitidine, 5- azacytidine, Alfa 2b, Aminoglutethimide, Amsacrine (m-AMSA), Anthracenedione, Antiandrogens, Antibiotics, Antiestrogen, Antimetabolites, Antimitotic drugs, Asparaginase, AraC, Azacitidine, azathioprine, bacteriochlorophyll-a, Batimastat, BAY 12-9566, BB2516/Marmistat, BCH-4556, benzoporphyrin derivatives, Biological response modifiers, Bleomycin, BMS-182751 /oral platinum, busulfan, Busulfan, bromodeozyuridine, 5-bromodeozyuridine, 2-CdA, Caelyx/liposomal doxorubicin, Campto/Levamisole, Camptosar/Irinotecan, Camptothecin, Carboplatin, carmustaine and poliferposan, Carmustine (BCNU), CDP 845, CDK4 and CDK2 inhibitors, Chlorambucil, chloroethylnitrosoureas cisplatin, CI-994, Cisplatin (cis-DDP), 2- chlorodeoxyadenosine, cladribine, CP-358 (774)/EGFR, CP-609 (754)/RAS oncogene inhibitor, CS-682, 9-AC, Cyclopax/oral paclitaxel, Cyclophosphamide, cytosine arabinoside, cytarabine, Cytarabine HCI, Cytokines, D2163, D4809/Dexifosamide, Dacarbazine, Dactinomycin, daunomycin, Daunorubicin HCI, DepoCyt, desmethylmisonidazole, 2'-deoxycoformycin, dexamethasone, diethylstilbestrol ethynyl estradiol, Differentiation Agents, docetaxel, 2,2'-difluorodeoxycytidine,2'- difluorodeoxycytidine, docetaxel etoposide, Doxil/liposomal doxorubicin, doxorubicin, Doxorubicin HCI, DX8951f, E7070, EO9, Edatrexate, Eniluracil/776C85/5FU enhancer, Enzymes, Epipodophylotoxins, Ergamisol/Levamisole, erythrohydroxynonyladenine (EHNA), estramustine, Estramustine phosphate sodium, Estrogens, Erthropoietin, etanidazole, Ethylenimine, Etoposide (V 16-213), Evacet/liposomal doxorubicin, famesyl transferase inhibitor, Folic Acid analogs, FK 317, Floxuridine, Fludara/Fludarabine, fludarabine phosphate, fluorodeoxyuridine, 5-Fluorouracil (5-FU), Flutamide, fluoxymesterone, fragyline, Furtulon/Doxifluridine, Gallium Nitrite, gemcitabine, G- CSF, Gemzar/Gemcitabine, Glamolec, GM-CSF, hydroxyurea, hematoporphyrin derivatives, Hexamethylmelamine (HMM), HMR 1275/Flavopiridol, hormone analogs, Hormones and antagonists, Hycamtin/Topotecan, hydroxyprogesterone acetate, hydroxyprogesterone caproate, Hydroxyurea (hydroxycarbamide), Idarubicin, Inhibitors, Ifes/Mesnex/Ifosamide, Ifosfamide, 5-iododeoxyuridine, Incel/VX-710, Iodine seeds, interferon-alpha, interferon-β, interfon-γ, Interferon Alfa-2a, Interleukin-2, IL-2, irinotecan, ISI641, L-asparaginase, L-Buthiamine Sulfoxide,leuprolide, Lemonal DP 2202, Leuprolide acetate (LHRH-releasing factor analogue), Leustatin/Cladribine, Lomustine (CCNU), LU 79553/Bis-Naphtalimide, LU 103793/Dolastain,

LY264618/Lometexol, Mechlorethamine HCI (nitrogen mustard), medroxyprogesterone acetate, Megestrol, megestrol acetate mitotane, Meglamine GLA, melphalan, 6- Mercaptopurine, Mesna, Metastron/strontium derivative, Metaret/Suramin, metronidazole, Methotrexate (MTX), Methyl glyoxal bis-guanylhydrazone (MGBG), Methylhydrazine derivatives, Methylmelamine, misonidazole, Mitoguazone (methyl- GAG), Mitomycin C, mithramycin, Mitotane (o.p'-DDD), mitoxantrone, Mitoxantrone HCI, MMI270, MMP, MTA/LY231514, naphthalocyanine, naphthalocyanines, nicotinamide, nimorazole, Npe6, Nitrogen mustards, Nitrosourceas, N-methylhydrazine, N-methylhydrazine (MIH), Nonsteroidal antiandrogens, Novantrone/Mitroxantrone, ODN 698, Octreotide, Oral Taxoid, paclitaxel, Paraplatin/Carboplatin, PARP inhibitors, Paxex/Paclitaxel, Pentostatin, PDl 83805, Pharmarubicin/Epirubicin, pheoboride-a, photofrin®, Photosensitizers, phthalocyanine, Picibanil/OK-432, pimonidazole, pimonidazole etanidazole, PKC412, Plantinol/cisplatin, Platinium coordination complexes, Plicamycin, poliferposan, Prednisone, prednisone and equivalents, procarbazine, Procarbazine HCI, Progestins, Purine analogs, Pyrimidine analogs, Radiosensitizers, RAS famesyl transferase inhibitor, retinoic acid derivatives, rubidomycin, RB 6145, RSU 1069, SR4233, Semustine (methyl-CCNU), Semustine Streptozocin, SPU-077/Cisplatin, Substituted urea, TA 2516/Marmistat, tamoxifen, Tamoxifen citrate, Taxane Analog, Taxanes, taxol, Taxol/Paclitaxel, Taxoids, Taxotere, Taxotere/Docetaxel, prodrug of guanine arabinoside, Temodal/Temozolomide, teniposide, Teniposide (VM-26), testosterone propionate, Thioguanine, Thiophosphoramide, 6-Thioguanine, Thiotepa, tin etioporphyrin (SnET2), Thriethylenemelamine, TNP-470, triethylene thiophosphoramide, Tiasofuran, tin etioporphyrin, Topotecan, Triazines, Triethylene, trimetrexate, Tumodex/Ralitrexed, Type I Topoisomerase, UFT(Tegafur/Uracil), valrubicin, Valspodar/PSC833,

Vepeside/Etoposide, vinblastine, vinblastine (VLB), Vinblastine sulfate, Vinca alkaloids, vincristine, Vincristine sulfate, vinorelbine, VX-853, Vumon/Teniposide, ZDOlOl, Xeload/Capecitabine, Yewtaxan/Placlitaxel, YM 116, ZD 0473/Anormed, ZD 1839, ZD 9331, or zinc phthalocyanine.

Other examples of antitumor agents that may be used are listed in Table 1.

Table 1

The compound of Formula I may be administered alone or in conjunction with one or more therapies known or believed to be useful for treating a tumor. Such therapies include, for example, tumor medicaments, radiation and surgical procedures. As used herein, a "tumor medicament" refers to an agent which is administered to a subject for the purpose of treating a tumor. Various types of medicaments for the treatment of tumors are described herein. For the purpose of this specification, tumor medicaments are classified as chemotherapeutic agents, immunotherapeutic agents, tumor vaccines, hormone therapy, and biological response modifiers.

Tumor medicaments function in a variety of ways. Some tumor medicaments work by targeting physiological mechanisms that are specific to tumor cells. Examples include the targeting of specific genes and their gene products (i.e., proteins primarily) which are mutated in tumors. Such genes include but are not limited to oncogenes (e.g., Ras, Her2, bcl-2), tumor suppressor genes (e.g., EGF, p53, Rb), and cell cycle targets (e.g., CDK4, p21, telomerase). Tumor medicaments can alternately target signal transduction pathways and molecular mechanisms which are altered in tumor cells. Targeting of tumor cells via the epitopes expressed on their cell surface is accomplished through the use of monoclonal antibodies. This latter type of tumor medicament is generally referred to herein as immunotherapy.

Other tumor medicaments target cells other than tumor cells. For example, some medicaments prime the immune system to attack tumor cells (i.e., tumor vaccines). Still other medicaments, called angiogenesis inhibitors, function by attacking the blood supply of solid tumors. Since the most malignant tumors are able to metastasize (i.e., exit the primary tumor site and seed a distal tissue, thereby forming a secondary tumor), medicaments that impede this metastasis are also useful in the treatment of tumor. Angiogenic mediators include basic FGF, VEGF, angiopoietins, angiostatin, endostatin, TNF-α, TNP-470, thrombospondin-1, platelet factor 4, CAI, and certain members of the integrin family of proteins. One category of this type of medicament is a metalloproteinase inhibitor, which inhibits the enzymes used by the tumor cells to exit the primary tumor site and extravasate into another tissue.

Immunotherapeutic agents are medicaments which derive from antibodies or antibody fragments which specifically bind or recognize a tumor antigen. As used herein a tumor antigen is broadly defined as an antigen expressed by a tumor cell. Preferably, the antigen is expressed at the cell surface of the tumor cell. Even more preferably, the antigen is one which is not expressed by normal cells, or at least not expressed to the same level as in tumor cells. Antibody-based immunotherapies may function by binding to the cell surface of a tumor cell and thereby stimulate the endogenous immune system to attack the tumor cell. Another way in which antibody-based therapy functions is as a delivery system for the specific targeting of toxic substances to tumor cells. Antibodies are usually conjugated to toxins such as ricin (e.g., from castor beans), calicheamicin and maytansinoids, to radioactive isotopes such as Iodine-131 and Yttrium-90, to chemotherapeutic agents (as described herein), or to biological response modifiers. In this way, the toxic substances can be concentrated in the region of the tumor and non- specific toxicity to normal cells can be minimized. In addition to the use of antibodies which are specific for tumor antigens, antibodies which bind to vasculature, such as those which bind to endothelial cells, are also useful. This is because generally solid tumors are dependent upon newly formed blood vessels to survive, and thus most tumors are capable of recruiting and stimulating the growth of new blood vessels. As a result, one strategy of many tumor medicaments is to attack the blood vessels feeding a tumor and/or the connective tissues (or stroma) supporting such blood vessels. Other types of chemotherapeutic agents which can be used according to the disclosure include Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorambucil, Cytarabine HCl, Dactinomycin, Daunorubicin HCl, Estramustine phosphate sodium, Etoposide (VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factor analogue), Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard), Mercaptopurine, Mesna, Mitotane (o.p'-DDD), Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA), Azacitidine, Erythropoietin, Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (methyl- GAG; methyl glyoxal bis-guanylhydrazone; MGBG), Pentostatin (2'deoxycoformycin), Semustine (methyl-CCNU), Teniposide (VM-26) and Vindesine sulfate.

Tumor vaccines are medicaments which are intended to stimulate an endogenous immune response against tumor cells. Currently produced vaccines predominantly activate the humoral immune system (i.e., the antibody dependent immune response). Other vaccines currently in development are focused on activating the cell-mediated immune system including cytotoxic T lymphocytes which are capable of killing tumor cells. Tumor vaccines generally enhance the presentation of tumor antigens to both antigen presenting cells (e.g., macrophages and dendritic cells) and/or to other immune cells such as T cells, B cells, and NK cells. In some instances, tumor vaccines may be used along with adjuvants, such as those described above.

Tumor antigens, such as those present in tumor vaccines or those used to prepare tumor immunotherapies, can be prepared from crude tumor cell extracts, as described in Cohen PA et al. (1994) Cancer Res 54:1055-8, or by partially purifying the antigens, using recombinant technology, or de novo synthesis of known antigens. Tumor antigens can be used in the form of immunogenic portions of a particular antigen or in some instances a whole cell or a tumor mass can be used as the antigen. Such antigens can be isolated or prepared recombinantly or by any other means known in the art. Other vaccines take the form of dendritic cells which have been exposed to tumor antigens in vitro, have processed the antigens and are able to express the tumor antigens at their cell surface in the context of MHC molecules for effective antigen presentation to other immune system cells. Dendritic cells form the link between the innate and the acquired immune system by presenting antigens and through their expression of pattern recognition receptors which detect microbial molecules like LPS in their local environment.

Examples of antiproloiferative agents that may be used include, but are not limited to: 5-fluorouracil (5-FU), daunomycin, doxorubicin, mitomycin, paclitaxel, rapamycin, (sirolimus), dexamethasone, 2-methoxyestradiol, and Piritrexim isethionate The present disclosure also provides methods of treating a tumor or an unwanted mammalian cell proliferation in a subject, comprising administering to the subject an effective amount of a pharmaceutical compound comprising a compound of Formula I. Preferably, the methods are employed to treat the tumor or the unwanted mammalian cell proliferation in a subject, such as a mammal.

Methods of the disclosure also are readily adaptable for use in assay systems, e.g., assaying a tumor or unwanted mammalian cell proliferation and properties thereof, as well as identifying compounds that affect tumors or unwanted mammalian cell proliferation. As used herein, a subject includes a mammal, such as a human, non-human primate, cow, rabbit, horse, pig, sheep, goat, dog, cat, or rodent such a rat, mouse or a rabbit. In some embodiments, the subject is a human.

The products and methods of the disclosure are useful for treating certain tumors and/or unwanted mammalian cell proliferation. Tumors treatable by the compounds of the disclosure include, for example, benign and malignant solid tumors and benign and malignant non-solid tumors. Examples of solid tumors include but are not limited to: biliary tract cancer, brain cancer (including glioblastomas and medulloblastomas), breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, intraepithelial neoplasms, including Bowen's disease and Paget's disease, liver cancer, lung cancer, lymphomas, including Hodgkin's disease and lymphocytic lymphomas, neuroblastomas, oral cancer, including squamous cell carcinoma, ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells, pancreatic cancer, prostate cancer, rectal cancer, renal cancer including adenocarcinoma and Wilms tumor, sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma, skin cancer, including melanoma, Kaposi's sarcoma, basocellular cancer and squamous cell cancer, testicular cancer, including germinal tumors (seminomas, and non-seminomas such as teratomas and choriocarcinomas), stromal tumors and germ cell tumors, and thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma.

Examples of non-solid tumors include but are not limited to hematological neoplasms. A hematologic neoplasm includes, for example, lymphoid disorders, myeloid disorders, and AIDS associated leukemias.

Lymphoid disorders include but are not limited to acute lymphocytic leukemia and chronic lymphoproliferative disorders (e.g., lymphomas, myelomas, and chronic lymphoid leukemias). Lymphomas include Hodgkin's disease and non-Hodgkin's lymphoma. Chronic lymphoid leukemias include T cell chronic lymphoid leukemias and B cell chronic lymphoid leukemias.

Myeloid disorders include chronic myeloid disorders such as for instance, chronic myeloproliferative disorders, myelodysplastic syndrome and acute myeloid leukemia. Chronic myeloproliferative disorders include but are not limited to angiogenic myeloid metaplasia, essential thrombocythemia, chronic myelogenous leukemia, polycythemia vera, and atypical myeloproliferative disorders. Atypical myeloproliferative disorders include, for example, atypical Chronic Myelogenous Leukemia (CML), chronic neutrophilic leukemia, mast cell disease, and chronic eosinophilic leukemia.

In some preferred embodiments, the tumor is breast cancer.

Conditions of unwanted mammalian cell proliferation and treatable as described in this disclosure include blood vessel proliferative disorders, fibrotic disorders, arteriosclerotic disorders, and dermatological disorders.

Blood vessel proliferative disorders refer to angiogenic and vasculogenic disorders generally resulting in abnormal proliferation of blood vessels. The formation and spreading of blood vessels, or vasculogenesis and angiogenesis, respectively, play important roles in a variety of physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration. They also play a pivotal role in cancer development. Other examples of blood vessel proliferative disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage and ocular diseases such as diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness and neovascular glaucoma.

Another example of abnormal vasculogenesis (called neovascularization) is that associated with solid tumors. It is now established that unrestricted growth of tumors is dependent upon angiogenesis and that induction of angiogenesis by liberation of angiogenic factors can be an important step in carcinogenesis. For example, basic fibroblast growth factor (bFGF) is liberated by several cancer cells and plays a crucial role in cancer angiogenesis. The demonstration that certain animal tumors regress when angiogenesis is inhibited has provided the most compelling evidence for the role of angiogenesis in tumor growth. Other cancers that are associated with neovascularization include hemangioendotheliomas, hemangiomas and Kaposi's sarcoma.

Proliferation of endothelial and vascular smooth muscle cells is the main feature of neovascularization. The compounds, pharmaceutical compositions and methods of the disclosure are useful in inhibiting such proliferation, and therefore in inhibiting or arresting altogether the progression of the angiogenic condition which depends in whole or in part upon such neovascularization. The compounds, pharmaceutical compositions and methods of the disclosure are particularly useful when the condition has an additional element of endothelial or vascular smooth muscle cell proliferation that is not necessarily associated with neovascularization. For example, psoriasis may additionally involve endothelial cell proliferation that is independent of the endothelial cell proliferation associated with neovascularization. Likewise, a solid tumor which requires neovascularization for continued growth may also be a tumor of endothelial or vascular smooth muscle cells. In this case, growth of the tumor cells themselves, as well as the neovascularization, is inhibited by the compounds described herein.

The compounds, pharmaceutical compositions and methods of the disclosure are also useful for the treatment of fibrotic disorders such as fibrosis and other medical complications of fibrosis which result in whole or in part from the proliferation of fibroblasts. Medical conditions involving fibrosis (other than atherosclerosis, discussed below) include undesirable tissue adhesion resulting from surgery or injury.

Other cell proliferative disorders which can be treated by means of the compounds, pharmaceutical compositions and methods of the disclosure include arteriosclerotic conditions. Arteriosclerosis is a term used to describe a thickening and hardening of the arterial wall. An arteriosclerotic condition as used herein means classical atherosclerosis, accelerated atherosclerosis, atherosclerotic lesions and any other arteriosclerotic conditions characterized by undesirable endothelial and/or vascular smooth muscle cell proliferation, including vascular complications of diabetes.

Proliferation of vascular smooth muscle cells is a main pathological feature in classical atherosclerosis. It is believed that liberation of growth factors from endothelial cells stimulates the proliferation of sub-intimal smooth muscle which, in turn, reduces the caliber and finally obstructs the artery. The compounds, pharmaceutical compositions and methods of the disclosure are useful in inhibiting such proliferation, and therefore in delaying the onset of, inhibiting the progression of, or even halting the progression of such proliferation and the associated atherosclerotic condition.

Proliferation of vascular smooth muscle cells produces accelerated atherosclerosis, which is the main reason for failure of heart transplants that are not rejected. This proliferation is also believed to be mediated by growth factors, and can ultimately result in obstruction of the coronary arteries. The compounds, pharmaceutical compositions and methods of the disclosure useful in inhibiting such obstruction and reducing the risk of, or even preventing, such failures.

Vascular injury can also result in endothelial and vascular smooth muscle cell proliferation. The injury can be caused by any number of traumatic events or interventions, including vascular surgery and balloon angioplasty. Restenosis is the main complication of successful balloon angioplasty of the coronary arteries. It is believed to be caused by the release of growth factors as a result of mechanical injury to the endothelial cells lining the coronary arteries. Thus, by inhibiting unwanted endothelial and smooth muscle cell proliferation, the compounds described herein can be used to delay, or even avoid, the onset of restenosis.

Other atherosclerotic conditions which can be treated or prevented by means described in the present disclosure include diseases of the arterial walls that involve proliferation of endothelial and/or vascular smooth muscle cells, such as complications of diabetes, diabetic glomerulosclerosis and diabetic retinopathy.

In addition to the particular disorders enumerated above, the disclosure is also useful in treating or preventing dermatological diseases including keloids, hypertrophic scars, seborrheic dermatosis, psoriasis, papilloma virus infection (e.g., producing verruca vulgaris, verruca plantaris, verruca plan, condylomata, etc.), eczema and epithelial precancerous lesions such as actinic keratosis.

The compounds of the disclosure are administered in effective amounts. An effective amount is a dosage of the therapeutic agent sufficient to provide a medically desirable result. An effective amount means that amount necessary to delay the onset of, inhibit the progression of or halt altogether the onset or progression of the particular condition or disease being treated. In the treatment of tumors or unwanted mammalian cell proliferation, for example, in general, an effective amount will be that amount necessary to inhibit tumor growth or progression of the tumor or the unwanted mammalian cell proliferation, reduce the load of the tumor or the unwanted mammalian cell proliferation, or reduce one or more signs or symptoms of the tumor or unwanted mammalian cell proliferation.

When administered to a subject, effective amounts will depend, of course, on the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size and weight, concurrent treatment, frequency of treatment, and the mode of administration. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. In some embodiments, it is preferred to use a maximum dose, that is, the highest safe dose according to sound medical judgment.

An effective amount typically will vary from about 0.001 mg/kg to about 1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 0.1 mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about 250 mg/kg, from about 10.0 mg/kg to about 150 mg/kg in one or more dose administrations daily, for one or several days (depending of course of the mode of administration and the factors discussed above). Other suitable dose ranges include 1 mg to 10000 mg per day, 100 mg to 10000 mg per day, 500 mg to 10000 mg per day, and 500 mg to 1000 mg per day. In some particular embodiments, the amount is less than 10,000 mg per day with a range of 750 mg to 9000 mg per day. Actual dosage levels of active ingredients in the pharmaceutical compositions of the disclosure can be varied to obtain an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration. The selected dosage level depends upon the activity of the particular compound, the route of administration, the severity of the condition being treated, the condition, and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effort and to gradually increase the dosage until the desired effect is achieved.

The dosage regimen can be determined, for example, by following the response to the treatment in terms of clinical signs. Examples of such clinical signs are well known in the art, and they include for example the pulse, blood pressure, temperature, and respiratory rate. Harrison's Principles of Internal Medicine, 15th Ed., Fauci AS et al., eds., McGraw-Hill, New York, 2001.

The compounds and pharmaceutical compositions of the disclosure can be administered to a subject by any suitable route. For example, the compositions can be administered orally (including bucally and sublingually), nasally, rectally, parenterally, intracisternally, intra vaginal Iy, intraperitoneally, topically and transdermally (as by powders, ointments, or drops). The term "parenteral" administration as used herein refers to modes of administration other than through the gastrointestinal tract, which include intravenous, intramuscular, intraperitoneal, intrasternal, intramammary, intraocular, retrobulbar, intrapulmonary, intrathecal, subcutaneous and intraarticular injection and infusion. Surgical implantation also is contemplated, including, for example, embedding a composition of the disclosure in the body such as, for example, in a tissue, in the abdominal cavity, under the splenic capsule, brain, or in the cornea.

Compounds of the present disclosure also can be administered in the form of liposomes. As is known in the art, liposomes generally are derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any nontoxic, physiologically acceptable, and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present disclosure, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N. Y. (1976), p. 33, et seq. Dosage forms for topical administration of a compound of this disclosure include powders, sprays, ointments, and inhalants as described herein. The active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers, or propellants which may be required. Ophthalmic formulations, eye ointments, powders, and solutions also are contemplated as being within the scope of this disclosure.

Pharmaceutical compositions of the disclosure for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water ethanol, polyols (such as, glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such, as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions also can contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It also may be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of the drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This result can be accomplished by the use of a liquid suspension of crystalline or amorphous materials with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug from is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such a polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations also are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

The injectable formulations can be sterilized, for example, by filtration through a bacterial- or viral-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use. The disclosure provides methods for oral administration of a pharmaceutical composition of the disclosure. Oral solid dosage forms are described generally in Remington's Pharmaceutical Sciences, 18th Ed., 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosage forms for oral administration include capsules, tablets, pills, powders, troches or lozenges, cachets, pellets, and granules. Also, liposomal or proteinoid encapsulation can be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation may include liposomes that are derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). In general, the formulation includes a compound of the disclosure and inert ingredients which protect against degradation in the stomach and which permit release of the biologically active material in the intestine.

In such solid dosage forms, the active compound is mixed with, or chemically modified to include, a least one inert, pharmaceutically acceptable excipient or carrier. The excipient or carrier preferably permits (a) inhibition of proteolysis, and (b) uptake into the blood stream from the stomach or intestine. In one embodiment, the excipient or carrier increases uptake of the compound, overall stability of the compound and/or circulation time of the compound in the body. Excipients and carriers include, for example, sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, cellulose, modified dextrans, mannitol, and silicic acid, as well as inorganic salts such as calcium triphosphate, magnesium carbonate and sodium chloride, and commercially available diluents such as FAST-FLO^®, EMDEX^®, STA-RX 1500^®, EMCOMPRESS^® and AVICEL^®, (b) binders such as, for example, methylcellulose ethylcellulose, hydroxypropyhnethyl cellulose, carboxymethylcellulose, gums (e.g., alginates, acacia), gelatin, polyvinylpyrrolidone, and sucrose, (c) humectants, such as glycerol, (d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, starch including the commercial disintegrant based on starch, EXPLOTAB^®, sodium starch glycolate, AMBERLITE^®, sodium carboxymethylcellulose, ultramylopectin, gelatin, orange peel, carboxymethyl cellulose, natural sponge, bentonite, insoluble cationic exchange resins, and powdered gums such as agar, karaya or tragacanth; (e) solution retarding agents such a paraffin, (f) absorption accelerators, such as quaternary ammonium compounds and fatty acids including oleic acid, linoleic acid, and linolenic acid (g) wetting agents, such as, for example, cetyl alcohol and glycerol monosterate, anionic detergent surfactants including sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate, cationic detergents, such as benzalkonium chloride or benzethonium chloride, nonionic detergents including lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65, and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose; (h) absorbents, such as kaolin and bentonite clay, (i) lubricants, such as talc, calcium sterate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils, waxes, CARBOWAX^® 4000, CARBOWAX^® 6000, magnesium lauryl sulfate, and mixtures thereof; 0) glidants that improve the flow properties of the drug during formulation and aid rearrangement during compression that include starch, talc, pyrogenic silica, and hydrated silicoaluminate. In the case of capsules, tablets, and pills, the dosage form also can comprise buffering agents.

Solid compositions of a similar type also can be employed as fillers in soft and hard-filled gelatin capsules, using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They optionally can contain opacifying agents and also can be of a composition that they release the active ingredients(s) only, or preferentially, in a part of the intestinal tract, optionally, in a delayed manner. Exemplary materials include polymers having pH sensitive solubility, such as the materials available as EUDRAGIT^® Examples of embedding compositions which can be used include polymeric substances and waxes.

The active compounds also can be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol ethyl carbonate ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydroflirfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions also can include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, coloring, flavoring, and perfuming agents. Oral compositions can be formulated and further contain an edible product, such as a beverage.

Suspensions, in addition to the active compounds, can contain suspending agents such as, for example ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar, tragacanth, and mixtures thereof.

Also contemplated herein is pulmonary delivery of the compounds of the disclosure. The compound is delivered to the lungs of a mammal while inhaling, thereby promoting the traversal of the lung epithelial lining to the blood stream. See, Adjei et al., Pharmaceutical Research 7:565-569 (1990); Adjei et al., International Journal of Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquet et al., Journal of Cardiovascular Pharmacology 13 (suppl.5): s.143-146 (1989)(endothelin-l); Hubbard et al., Annals of Internal Medicine 3:206-212 (1989)(αl -antitrypsin); Smith et al., J. Clin. Invest. 84:1145-1146 (1989) (αl -proteinase); Oswein et al., "Aerosolization of Proteins," Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, 1990 (recombinant human growth hormone); Debs et al., The Journal of Immunology 140:3482-3488 (1988) (interferon-γ and tumor necrosis factor α) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor).

Contemplated for use in the practice of this disclosure are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including, but not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

Some specific examples of commercially available devices suitable for the practice of the disclosure are the ULTRA VENT^® nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, MO; the ACORN II^® nebulizer, manufactured by Marquest Medical Products, Englewood, CO.; the VENTOL^® metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.; and the SPINHALER^® powder inhaler, manufactured by Fisons Corp., Bedford, MA.

All such devices require the use of formulations suitable for the dispensing of a compound of the disclosure. Typically, each formulation is specific to the type of device employed and can involve the use of an appropriate propellant material, in addition to diluents, adjuvants, and/or carriers useful in therapy.

The composition is prepared in particulate form, preferably with an average particle size of less than 10 μm, and most preferably 0.5 to 5 μm, for most effective delivery to the distal lung. Carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include lipids, such as DPPC, DOPE, DSPC and DOPC, natural or synthetic surfactants, polyethylene glycol (even apart from its use in derivatizing the inhibitor itself), dextrans, such as cyclodextran, bile salts, and other related enhancers, cellulose and cellulose derivatives, and amino acids.

Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, typically comprise a compound of the disclosure dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation also can include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation also can contain a surfactant to reduce or prevent surface-induced aggregation of the inhibitor composition caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device generally comprise a finely divided powder containing the inhibitor compound suspended in a propellant with the aid of a surfactant. The propellant can be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofiuoromethane, dichlorodifiuoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid also can be useful as a surfactant.

Formulations for dispensing from a powder inhaler device comprise a finely divided dry powder containing the inhibitor and also can include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol, in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation. Nasal delivery of the compounds and composition of the disclosure also is contemplated. Nasal delivery allows the passage of the compound or composition to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. Delivery via transport across other mucous membranes also is contemplated.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of the disclosure with suitable nonirritating excipients or carriers, such as cocoa butter, polyethylene glycol, or suppository wax, which are solid at room temperature, but liquid at body temperature, and therefore melt in the rectum or vaginal cavity and release the active compound.

In order to facilitate delivery of compounds across cell and/or nuclear membranes, compositions of relatively high hybrophobicity are preferred. Compounds can be modified in a manner which increases hydrophobicity, or the compounds can be encapsulated in hydrophobic carriers or solutions which result in increased hydrophobicity.

Generally dosage levels of about 0.1 to about 1000 mg, about 0.5 to about 500 mg, about 1 to about 250 mg, about 1.5 to about 100, and preferably of about 5 to about 20 mg of active compound per kilogram of body weight per day are administered orally or intravenously. If desired, the effective daily dose can be divided into multiple doses for purposes of administration, e.g., two to four separate doses per day.

The present disclosure also provides methods of inducing apoptosis in a cell, comprising administering to the subject an effective amount of a compound of Formula I. Apoptosis is a form of programmed cell death in multicellular organisms. Defective apoptotic processes have been implicated in an extensive variety of diseases. For example, excessive apoptosis causes hypotrophy, such as in ischemic damage, whereas an insufficient or defective apoptosis results in uncontrolled cell proliferation, such as cancer.

Without intending to be bound by any particular mechanism or theory, it is believed that compound of Formula I induces apoptosis by arresting the cellular population on early G2+M phase in the cell cycle checkpoint. It is believed that compound of Formula I might be exerting its effect by acting as a blocking agent at the G2 checkpoint.

The disclosure is exemplified by the following Example.

Examples Example 1.

Compound of Formula I is synthesized from 1,2 phenylenediamine (available from Sigma-Aldrich (St Louis, MO)).

Example 2.

Introduction: Cell cycle checkpoints are important regulators of eukaryotic cell growth. Because loss of Gl checkpoint often occurs in malignant cells, synthetic, small molecule, modulators of the G2/M checkpoint are of particular interest. Several clinically important anticancer compounds, such as vinca alkaloids and taxanes,act as prominent inhibitors of G2/M transition , have complex chemical structure that restrict chemical modification. Compound of formula I (AT-4) is a small synthetic molecule and novel derivative and leading compound. Materials and Methods: Highly invasive (MDA-MB-231) breast cancer cell line was cultured in triplicate. Cells were seeded in 60 mm dishes in RPMI media, containing 10% fetal bovine serum, at 37°C under 5% CO₂. Cells were cultured up to 60% confluence.

Results: Flow cytometry analysis revealed that treatment of MDA-MB-231 cells with lOOμM increased the percentage of cells in G2/M phase from 19.94% to 52.93%. western blotting showed that there is undetectable level of onco protein cyclin Bl, after 48 hrs and 72 hrs exposure time. Immuonfluorescene microscopy showed that compound AT-4 induces premature chromosomal condensation and mitotic catastrophe. The selectivity of compound AT-4 was examined by using non proliferating normal breast cell line without any detectable cytotoxic effect (see Figures 1-10).

Example 3.

In Vivo Models - Inhibition of Tumor Growth

The ability of a compound to inhibit unregulated growth of human tumor cells in vivo is evaluated by implanting tumor cells (e.g., cells from a breast cancer cell line) into the hindflank of athymic mice or NOD/scid mice, administering a test compound (e.g., compound of Formula I) and then quantifying any change in tumor size.

Tumor cells, such as MDA cells derived from a transformed breast cancer cell line are implanted (e.g., 10⁶ cells) subcutaneously into the hindflank of athymic mice (Charles River) and allowed to grow for a few to several days (e.g., 6-10 days). After a measurable tumor is established (as determined by baseline caliper measurement, for example), the animal is administered a dose of the test compound. The test compound may be administered in a pharmaceutically acceptable carrier (e.g., in 5% saline) daily for a period of 10-30 days. Tumor size is measured at intervals (e.g., every three or five days) and the degree of inhibition is determined by comparing drug-treated animals to, for example, vehicle-treated animals (controls). Variations of this method are intended to include variation in the duration of treatment, frequency of treatment and/or various modes of administration (e.g., oral, intramuscular, intraperitoneal, intravenous, or subcutaneous injection or infusion as the route of administration and/or the administration of the test compound either alone or in a combination therapy).

Initial experiments using wide-ranging doses of the test compound are performed to obtain preliminary toxicity and pharmacokinetic information to help determine a dose range(s) to be administered. Differing doses of the test compound (e.g., compound of Formula I) will be utilized based on pharmacokinetic information obtained from toxicity studies. For example, the maximal tolerated dose (MTD) will be calculated using up/down toxicity studies that will be used as the upper limit of dose for treatment. Blood samples for plasma concentrations of the test compound (e.g., compound of Formula I) are collected from mice at various time points/stages to perform pharmacokinetic studies. Examples of time points include: before (pre-dose), during, and after the completion of the administration test compound (e.g., compound of Formula I). Blood or serum samples will be obtained, for example on days 0, 5, 10, and 15 of treatment. Blood or serum chemistry +/- liver and/or kidney function analysis will be performed. Serum concentrations of test compound (e.g., compound of Formula I) will be determined by methods known to those of ordinary skill in the art such as, for example, high performance liquid chromatography (HPLC). Pharmacokinetic parameters are computed according to methods known to those of ordinary skill in the art.

Efficacy outcomes are evaluated based on tumor size and response rate (complete response and partial response), duration of response, progression-free survival, and changes in other biochemical parameters. Further response will be assessed by harvesting the tumor at euthanasia and performing immunohistochemistry and western blot analysis of the proteins known to be involved in cancer cell survival mechanisms such as signal transduction (e.g., Her2, PBkinase), angiogenesis (e.g., HIF-I), and metastasis (AR, MMP2). Each dose and control will be repeated three or more times to confirm results. Immunohistochemistry results may be assessed qualitatively based on staining intensity graded on a scale of 1 to 5.

Statistical analysis may be performed to compare the average tumor size/volume over time between the different doses of the test compound (e.g., compound of Formula I) and the vehicle-treated animals (controls).

This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the above description or illustrated in the drawings. The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing", "involving", and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

We claim:

Claims

Claims

1. A compound having a structure:

(Formula I) or a salt thereof.

2. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition of claim 2 further comprising an agent other than the compound of claim 1.

4. The pharmaceutical composition of claim 3, wherein the agent is an antitumor and/or an antiproliferative agent.

5. A method for treating a subject having a tumor comprising: administering to the subject an effective amount of a pharmaceutical composition of claim 1 to treat the tumor.

6. The method of claim 5, wherein the tumor is a solid tumor.

7. The method of claim 5, wherein the tumor is a non-solid tumor.

8. The method of claim 5, wherein the tumor is breast cancer.

9. A method for treating a subject having an unwanted mammalian cell proliferation other than a tumor comprising: administering to the subject an effective amount of a pharmaceutical composition of claim 1 to treat the unwanted mammalian cell proliferation.

10. A method of inducing apoptosis comprising contacting a cell with a compound of Formula I in an amount effective to induce apoptosis.

11. The method of claim 10, wherein the cell is a tumor cell.

12. The method of claim 10, wherein the apoptosis is induced in vivo.

13. The method of claim 10, wherein the apoptosis is induced in vitro.

14. The use of the compound of Formula I:

or a salt thereof in the manufacture of a medicament for treating a tumor in a subject.

15. The use according to claim 14, wherein the tumor is a solid tumor.

16. The use according to claim 14, wherein the tumor is a non-solid tumor.

17. The use according to claim 14, wherein the tumor is breast cancer.

18. The use of the compound of Formula I:

or a salt thereof in the manufacture of a medicament for treating an unwanted mammalian cell proliferation in a subject.