Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/655—Azo (—N=N—), diazo (=N2), azoxy (>N—O—N< or N(=O)—N<), azido (—N3) or diazoamino (—N=N—N<) compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/193—Colony stimulating factors [CSF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/21—Interferons [IFN]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D253/00—Heterocyclic compounds containing six-membered rings having three nitrogen atoms as the only ring hetero atoms, not provided for by group C07D251/00
  • C07D253/02—Heterocyclic compounds containing six-membered rings having three nitrogen atoms as the only ring hetero atoms, not provided for by group C07D251/00 not condensed with other rings
  • C07D253/04—1,2,3-Triazines
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D517/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having selenium, tellurium, or halogen atoms as ring hetero atoms
  • C07D517/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having selenium, tellurium, or halogen atoms as ring hetero atoms in which the condensed system contains three hetero rings
  • C07D517/14—Ortho-condensed systems

Definitions

• the present invention relates to Selenophene compounds and Selenophene triazene compounds, their geometrical isomeric forms, stereoisomers, configurational isomers, polymorphs, hydrates, solvates and pharmaceutically acceptable salts thereof.
• the present invention further relates to a process for the preparation of the above said Selenophene compounds and Selenophene triazene compounds and their pharmaceutically acceptable compositions.
• a goal of the present invention is to provide Selenopheno triazene compounds as active ingredient(s) either alone or in combination with other pharmaceutically acceptable drugs for the prevention and treatment of cancer and other vascular diseases.
• the compounds of the present invention are also potent angiogenesis inhibitors and can be effectively used for pharmaceutical compositions that prevent angiogenesis related diseases including cancer and other vascular diseases.
• novel compounds and compositions are useful for the prevention, control and treatment of metastatic malignant melanoma and for carcinomas of the solid tumors, and all other cancers including but not limited to lymphomas, sarcomas and gliomas either alone or in combination with other pharmaceutically acceptable drugs or excipients.
• Melanoma a malignant neoplasm
• melanin a malignant neoplasm
• the cutaneous form is characterized by a proliferation of cells at the dermal epidermal junction which soon invades adjacent tissues.
• the cells vary in amount and pigmentation of cytoplasm; the nuclei are relatively large and frequently playful in shape, with prominent acidophilic nucleoli; the mitotic figures tend to be numerous.
• Melanomas frequently metastasize widely; regional lymph nodes, skin, liver, lungs, and brain are likely to be involved.
• UV radiation represents a definitive risk factor for skin cancer, especially when exposure occurs in combination with certain underlying genetic traits, such as red hair and fair skin. Pigmentation of the skin results from the synthesis of melanin in the pigment-producing cells, the melanocytes, followed by distribution and transport of pigment granules to neighboring keratinocytes. It is commonly believed that melanin is crucial for absorption of free radicals that have been generated within the cytoplasm by UV and acts as a direct shield from UV and visible light radiation.
• UV-induced pigmentation requires induction of ⁇ -melanocyte-stimulating hormone ( ⁇ -MSH) secretion by keratinocytes.
• ⁇ -MSH and other bioactive peptides are cleavage products of Pro-Opiomelanocortin (POMC).
• POMC Pro-Opiomelanocortin
• the p53 tumor suppressor gene is one of the most frequent targets for genetic alterations in cancer.
• the p53 is a transcriptional regulator of the POMC gene, which translates to proteins that cause the melanocytes to produce melanin, which wards off skin cancer by absorbing UV radiation. Direct mutational inactivation of p53 is observed in close to half of all human tumors.
• DTIC dacarbazine
• DTIC is the only agent used to treat metastatic malignant melanoma.
• dacarbazine is also indicated for Hodgkin's lymphoma as a secondary line therapy when used in combination with other effective drugs.
• DTIC is 5- (3,3-dimethyl-l-trizeno)-imidazole-4-carboxamide with the following structural formula:
• dacarbazine requires bioactivation in vivo by the liver.
• One of the methyl groups of the dimethyltriazeno functionality is activated by liver microsomal enzymes and, in particular, by the Cytochrome P450, to oxidation, resulting in a hydroxymethyl group.
• the oxidative mono-demethylation of the dimethyitriazeno functionality affords mono-methyltriazene.
• MTIC monomethyltriazene metabolite, 3-methyl-(triazen-l- yl)-imidazole-4-carboxamide
• AIC 5-amino-imidazole-4- carboxamide
• AIC 5-amino-imidazole-4- carboxamide
• the Cytochrome P450 enzymes play only a minor role in the metabolism of MTIC.
• Temozolomide is also a similar imidazotetrazine alkylator that methylates DNA at nucleophilic site. Temozolomide as a bicyclic compound is orally bioavailable, more lipophilic, and spontaneously converted to MTIC, and also seems to generate less nausea. The O 6 -methylguanine adducts causes a mismatch during DNA replication and the addition of a thymidine, instead of cytosine, to the newly formed DNA strand. Because of the excellent CNS biodistribution, temozolomide has been useful as a radiosensitizer in both primary brain tumors and CNS metastases. Temozolomide improves quality of life when used with radiation in patients with brain metastases.
• Temozolomide has activity against sarcoma.
• the analogous bicyclic Temozolomide derivative having Selenium may be useful in sarcoma radiosensitization for primary control as well as for the treatment of metastases.
• Temozolomide is a radiosensitizer that is well tolerated and has modest side effects.
• the combination of Temozolomide and Irinotecan is more than additive against some cancers.
• the author(s) Patel, VJ. et al., Clin. Cancer Res., 2000, 6, 4154-4157 report that their experience confirms a high response rate in relapsed Ewing's sarcoma and DSRCT that is possibly even higher than that reported in the literature.
• Temozolomide plus Irinotecan combination is less immune suppressive than standard cyclophosphamide-containing regimens. This might be especially important in Ewing's sarcoma since the author(s) De Angulo, G. et al., J Pediatr. Hematol. Oncol., 2007, 29, 48-52 have shown that lymphocyte recovery (i.e., absolute lymphocyte count >500 on day 15 after the first cycle of chemotherapy) is associated with significantly higher survival in Ewing's sarcoma. Temozolomide or dacarbazine has also been combined with other drugs including Gemcitabine and Doxorubicin liposomes.
• DTIC DTIC is extensively degraded. Besides unchanged DTIC, AIC is a major metabolite of DTIC excreted in the urine. Although the exact mechanism of action of DTIC is not known, three hypotheses have been offered:
• the biochemical mechanism of action of the resulting MTIC reactive species whose cytotoxicity involved in generation of methyl carbonium ion in vivo is thought to be primarily due to alkylation of DNA.
• Alkylation occurs mainly at the O 6 and N 7 positions of guanine.
• DTIC prior to its metabolism to the monomethyltriazene, is oxidized initially to monohydroxymethyl and finally to an aldehyde.
• the monomethyltriazene in its aldehyde form prior to oxidative monodemethylation, is cyclized to the cyclic compound (as shown in Scheme 1) which interferes with the double helix DNA structure and blocks replication of the cancer cells.
• the imidazole ring system of the dacarbazine is hydrophilic in nature. Therefore, there is a need in the art for possibly effective binding to the melanin such that the cytotoxic functionality of the molecule is one hundred percent effective. Thus, the present inventors have aimed to provide novel compounds with increased lipophilicity thereby providing more target specificity.
• Selenophene which has a five-membered aromatic heterocyclic ring system, with Selenium in the ring is lipophilic in nature and may have effective binding by increased avidity to the melanin; as a result, one would be able to get the same therapeutic effectiveness at a significantly lower dose, hence minimizing the toxicity. This would in turn afford high specificity with a larger window of the Therapeutic Index (TI).
• a larger therapeutic index is preferred. This is because; one would like to start the therapeutic regimen with a very high Maximum Tolerated Dose (MTD) such that the cancer cells would be hit hard in the first chemotherapy itself. Otherwise, the surviving cancer cells would repair the DNA damage and subsequently metastasize to the other organs. In addition, the cancer cells that survived from the first treatment would become resistant to the second chemotherapy again, if needed. And besides, due to weakness of the immune system from the first chemotherapy, a suboptimal dose would be given in the second treatment that would contribute to toxicity.
• MTD Maximum Tolerated Dose
• the novel triazeno selenophene analogs have several additional advantages inherently built in within the structure over dacarbazine for increased activity. Therefore, in order for the dose regimen to be effective, possibly high melanin binding moieties such as selenophene system could offer a therapeutic treatment having all the three biochemical mechanisms of action superior to DTIC (Dacarbazine) with a possible positive outcome leading to complete responses.
• the present invention aims to fulfill this unmet medical need of selectively binding to the targeted melanoma cells and sparing the normal cells thereby increasing the target to non-target cell ratio and further providing other related advantages as described herein.
• Angiogenesis or neovascularization, is the process of generating new blood vessels derived as extensions from the existing vasculature. Angiogenesis plays an important role in the growth and spread of cancer. New blood vessels "feed" the cancer cells with oxygen and nutrients, allowing these cells to grow, invade nearby tissue, spread to other parts of the body, and form new colonies of cancer cells. Therefore, anti-angiogenesis or inhibition of angiogenesis has been considered as a potential therapeutic strategy for controlling tumor growth and metastatic spread of cancer cells.
• the present invention provides novel Selenophene and selenopheno triazene compounds of general formulae (I) and (II), respectively.
• the present invention also provides selenopheno triazene compounds of the general formula (I):
• R is selected from H, CH 3 and CH 2 OH.
• N NN(CH 3 )CH 2 OH, CONH 2 , CONHR 4 , CONR 4 R 5 , CONHNH 2 , CONHNHR 4 ,
• CONHNR 4 R 5 COOCH 3 , COOCH 2 CH 3 , COOH, COSH, CN, C ⁇ CH, SO 2 NH 2 ,
• R 4 and R 5 are independently selected from H, CH 3 , Ci-Cio alkyl, alkenyl, alkylol, alkoxy, alkylamine, etc.; alternatively, any two of R 1 , R 2 and R 3 can be joined together to form alicyclic, aromatic, heterocyclic systems comprising cyclohexyl, cyclopentyl, phenyl and pyridyl.
• the present invention provides selenopheno triazene compounds of the general formula (II).
• X, Y and Z are independently selected from C and Se such that the resulting five membered aromatic heterocyclic moieties involving the bicyclic system are un- substituted and substituted Selenophenes. such that the double bond is either in between X and Y or in between Y and Z;
• R 8 and R 9 are independently selected from H, CH 3 , Ci-C 1 O alkyl, alkenyl, alkylol, alkoxy, alkylamine, etc.; alternatively R 6 and, R 7 can be joined together to form alicyclic, aromatic, heterocyclic systems comprising cyclohexyl, cyclopentyl, phenyl and pyridyl.
• the present invention provides pharmaceutically acceptable salts of the compounds of the general formulae (I) and (II), for example organic or inorganic salts.
• the invention provides the optical enantiomers or diastereomers of the optically active compounds of general formulae (I) and (II).
• the invention provides pharmaceutical compositions comprising: a) at least one selenopheno triazene compound selected from the above general formula (I) and derivatives thereof; b) at least one selenopheno triazene compound selected from the above general formula (II) and derivatives thereof; or c) a mixture of at least one selenopheno triazene compound selected from the above general formula (I) and at least one selenopheno triazene compound selected from the above general formula (II).
• the pharmaceutical compositions including a compound selected from the above general formula (I) and/or a compound selected from the above general formula (II) optionally further include at least one pharmaceutically acceptable excipient, carrier, or diluent.
• the pharmaceutical compositions may optionally include, in addition to a compound selected from the above general formula (I) and/or a compound selected from the above general formula (II), one or more pharmaceutical drugs.
• the present invention provides a method of binding selenopheno triazene compound(s) to melanin or killing a cancer cell for a therapeutic purpose. The method includes the step of administering to a subject in need thereof a therapeutically effective amount of the said compound(s) of general formula (I) or (II) or their pharmaceutical composition(s).
• the invented compound(s) are useful in the management of metastatic malignant melanoma and for carcinomas of solid tumors and all other cancers.
• the invention further provides a method of inhibiting angiogenesis and for ameliorating the angiogenesis modulators which include but not limited to VEGF, PDGF, TGF-beta and FGF in warm blooded animals in need thereof comprising administering a therapeutically effective amount of selenophene and/or selenopheno triazene compounds of general formulae (I) and (II), respectively, or their pharmaceutical composition(s).
• angiogenesis modulators include but not limited to VEGF, PDGF, TGF-beta and FGF in warm blooded animals in need thereof comprising administering a therapeutically effective amount of selenophene and/or selenopheno triazene compounds of general formulae (I) and (II), respectively, or their pharmaceutical composition(s).
• the present invention provides a method for remodeling of the vasculatures by modifying the proliferation, migration, invasion of endothelial cells and vascular smooth muscle cells in warm blooded animals in need thereof.
• the present invention further provides a method for inhibiting metastatic tumor growth and its spread in warm blooded animals in need thereof.
• FIG. 1 Bar diagrams show percent increase in leaked Lactate dehydrogenase (LDH) from B 16 FO mouse melanoma cells (A), and in A375 human melanoma cells (B) , treated with various concentrations of DTIC and Compound 1 as indicated in the diagrams. Each bar indicates the percent increase in leaked LDH with respect to the vehicle control cultures (0.5% DMSO), calculated form a mean of quadruplicate wells.
• LDH Lactate dehydrogenase
• Figure 2 Bar diagram represents percentage of growth inhibition of Hs.531.sk normal human skin cells by compound 1 and DTIC at 25 ⁇ g/ml and 50 ⁇ g/ml.
• Figure 3 Panel A represents images showing inhibition of B 16F0 colony formation in presence of Compound 1 and DTIC in vitro. B 16F0 cells were treated with 0.1% DMSO (1), or 1, 5, 10, 20 ⁇ g/ml of Compound 1 (2-5, respectively) or 100 ⁇ g/ml of DTIC (6). The average number of colonies and the average size of the colonies are presented in panels B and C, respectively.
• Figure 4 Photomicrographs show invaded B 16F0 cells in vehicle control (A), 100 ⁇ g/ml of DTIC (B), and 20 ⁇ g/ml of Compound 1 (C) treated cultures. Bar diagram represents the average number ⁇ SD of invaded cells in respective cultures counted from 20 independent fields observed at 20 x objective. * indicates significance (p ⁇ 0.05) vs. vehicle control (Student T-test).
• Figure 5 Pictures show inhibitory effect of Compound 1 on human endothelial cell migration. Microphotographs illustrate the migration of HUVECs in the presence of either 100 ⁇ g/ml of DTIC (B) or Compound 1 at 5 ⁇ g/ml (C) or Compound 1 at 10 ⁇ g/ml (D), respectively.
• Panel A represents cellular migration in 0.1% DMSO treated vehicle control wells.
• the bar graph shows the average number of migrated cells under different culture conditions as indicated under each bar. Each bar represents mean of migrated cells calculated from at least twenty fields under 2OX objective.
• FIG. 6 Pictures show inhibition of capillary-like tube formation by Compound 1.
• Human umbilical vein endothelial cells (HUVECs) were laid on Cultrex coated plates in presence of either DTIC (100 ⁇ g/ml) or Compound 1 (5 and 10 ⁇ g/ml) (C, D) and allowed to form endothelial capillary tubes for 16 h at 37 0 C.
• Picture panels A-D represent, capillary-like tube formation in 0.1% DMSO treated vehicle control wells, DTIC treated culture and 5 and 10 ⁇ g/ml of Compound 1 treated cultures, respectively.
• the bars (A to D) in the bar graph represent the average number of branching points under different culture conditions, respectively as indicated in the picture panels.
• composition of the present invention means and includes compounds of formula (I) or (II) along with atleast one pharmaceutical carrier/excipient.
• pharmaceutically acceptable is meant the excipient(s), carrier(s), diluent(s), and/or salt(s) compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
• melanoma accounts for 1-3% of all malignant tumors and is increasing in incidence by 6-7% every year. In patients with advanced disease, 5-year life expectancy- is less than 10%, with a median survival of 6- 8.5 months.
• Melanoma metastases affect skin, lymph nodes, lung, liver, brain, bone, and sometimes other organs such as the pancreas. Different therapeutic approaches for metastatic melanoma have been evaluated, including chemotherapy and biological therapies, both as single treatments and in combination.
• DTIC dacarbazine
• dacarbazine can result in overexpression of angiogenic factors such as interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) (Lev DC et al., MoI Cancer Ther; 2:753-763, 2003; Lev DC et al., J. Clin. Oncol. 22: 2092- 2100, 2004).
• IL-8 interleukin-8
• VEGF vascular endothelial growth factor
• the present invention utilizes selenophene as a back bone in place of imidazole in DTIC to significantly increase its activity for possible cure in the early diagnosis and to significantly increase efficacy in the treatment of late stage malignancy.
• the selenophene analog (Compound 1) showed 31.2% inhibition in the tumor growth compared to 17% inhibition showed by DTIC in mouse melanoma xenograft model of C57B6J mice (Figure 7).
• the compound 1 showed significantly lesser cytotoxic effect in normal human skin epithelial cells ( Figure 2). This observation suggests that Compound 1 is able to inhibit efficiently the • melanoma tumor cell growth without or minimally affecting the normal human cells.
• the novel analog (Compound 1) is significantly better than the marketed drug DTIC, in terms of efficacy and safety.
• Compound 1 exhibited anti-angiogenic properties in endothelial capillary formation assay, whereas, DTIC could not inhibit the endothelial capillary formation in vitro ( Figure 6).
• Compound 1 inhibited endothelial cell migration, which suggests that this novel analog also might help in inhibiting tissue remodeling during the angiogenesis process required for tumor growth.
• Compound 1 also significantly inhibited the invasion of malignant melanoma tumor cells ( Figure 4), whereas, DTIC failed to inhibit the tumor cell invasion.
• invention provides selenopheno triazene compounds represented by the following general formula (I) and (II):
• R is selected from H, CH 3 and CH 2 OH;
• X, Y and Z are independently selected from C and Se such that the resulting bicyclic systems involving five membered aromatic heterocyclic moieties of un-substituted and substituted Selenophenes; such that the double bond is either in between X and Y or in between Y and Z.
• Alkyl groups in general formulae (I) and (II) encompass, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl ., isobutyl, tertiary butyl, pentyl, hexyl, heptyl, octyl and other Ci to C 30 alkyl groups; alkenyl groups in general refer to ethene, propene, butene, pentene and higher alkenyl groups, including C 6 to C 3 0 alkenyl groups with one or more double bonds; alkylol groups in general refer to hydroxymethyl, hydroxyethyl, hydroxypropyl and other hydroxyalkyl groups; alkoxy groups in general refer to methoxy, ethoxy, propoxy, butoxy, pentoxy and other higher alkoxy groups, include C 6 to C 30 carbon atoms; alkylamine groups refer to aminomethyl, aminoethyl, aminopropyl and other
• any two of R , R and R in general formula (I) can be joined together to form alicyclic, aromatic, heterocyclic systems including but not limited to cyclopentyl, cyclohexyl, phenyl and pyridyl.
• R 6 and R 7 in general formula (II) can be joined together to form alicyclic, aromatic, or heterocyclic systems including but not limited to cyclopentyl, cyclohexyl, phenyl and pyridyl.
• the present invention relates to novel selenopheno triazene compounds of the general formulae (I) and (II).
• the compounds of the general formulae (I) and (II) are disclosed together because of their metabolic relationship.
• a compound of general formula (I) upon in vivo activation by liver microsomal enzymes (cytochrome P450) followed by oxidative demethylation affords its monomethyltriazene derivative.
• analogous compound of general formula (II) upon in vivo hydrolysis affords similar monomethyltriazene derivative. Therefore, due to their metabolic relationship in vivo, the compounds of general formulae (I) and (II) constitute a single invention.
• the compounds of general formulae (I) and (II) are disclosed separately for convenience.
• the present invention relates to the geometrical isomeric forms, stereoisomers, configurational isomers, polymorphs, hydrates, solvates and pharmaceutically acceptable salts thereof of general formulae (I) and (II).
• the invention provides the synthesis of following selenopheno triazene compounds of general formulae (I) or (II), the preparation of which is described in examples 1-16: 1. 4-[(dimethylamino)diazenyl]-5-methylselenophene-2-carboxamide (compound 1).
• the invention provides the synthesis of preferred compounds as well as their intermediates obtained during the process of synthesis, as described in examples 1- 16.
• pharmaceutically acceptable acid(s) and base addition salts of compounds of general formulae (I) and (II) comprise wide variety of organic and inorganic acids and bases and include but not limited to the physiologically acceptable salts which are often used in pharmaceutical industry. Such salts are also part of this invention.
• Typical inorganic acids used to form such salts include hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, hypophosphoric and the like.
• Salts derived from organic acids, such as aliphatic mono and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkanoic and hydroxyalandioic acids, aromatic acids, aliphatic and aromatic sulfonic acids may also be used.
• Such pharmaceutically acceptable salts thus include acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, cinnamate, citrate, formate, fumarate, glycollate, heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate, oxalate, phthalate, terephthalate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite, bisulfate, sulfonate
• the pharmaceutically acceptable salts generally have enhanced aqueous solubility characteristics compared to the compound's native form from which they are derived, and thus are often more amenable to formulation as liquids or emulsions, and can have enhanced bioavailability.
• the invention further provides a process for the preparation of the above said selenophene compounds and analogs of general formulae (I) and (II) and their pharmaceutically acceptable compositions.
• Reagents' & conditions (i) HNO 3 , Ac 2 O, room temperature (rt) (ii) Fe, HCl, MeOH, reflux, 1 h (iii) HCl, NaNO 2 , K 2 CO 3 /dimethylamine, O 0 C (iv) Aq. NH 3 , rt, 40 h.
• Nitration of methyl S-methylselenophene-l-carboxylate provided methyl 5-methyl-4- nitroselenophene-2-carboxylate.
• the nitro functionality is reduced to amines using suitable reducing agents, for example, iron powder or any other nitro reducing agents in good yield.
• Diazotization of methyl 4-amino-5-methylselenophene-2-carboxylate with sodium nitrite followed by treatment with potassium carbonate/dimethylamine provides methyl 4-[(dimethylamino)diazenyl]-5-methylselenophene-2-carboxylate.
• Treatment of the ester with ammonia gave the required 4-[(dimethylamino)diazenyl]-5- methylselenophene-2-carboxamide (compound 1).
• Reagents & conditions (i) SeO 2 , AcOH, reflux, 8 h (ii) AgNO 3 , NaOH, rt, 1 h (iii) HNO 3 , H 2 SO 4 , O°C-rt, 2 h (iv) MeOH, SOCl 2 , reflux, 2 h (v) Fe, HCl, MeOH, reflux, 3 h (vi) HCl, NaNO 2 , K 2 CO 3 /dimethylamine, O 0 C, 2 h (vii) Aq. NH 3 , rt, 24 h.
• Oxidation of methyl 5-methylselenophene-2-carboxylate with selenium dioxide provided methyl 5-formylselenophene-2-carboxylate, which on further oxidation with silver nitrate gave selenophene-2,5-dicarboxylic acid.
• Nitration of selenophene-2,5-dicarboxylic acid and esterfication provided methyl 5-(methoxycarbonyl)-3-nitroselenophene-2-carboxylate in good yield.
• the nitro functionality is reduced to amines using suitable reducing agents, for example, iron powder or any other nitro reducing agents in good yield.
• Reagents & conditions (i) POCl 3 , DMF, O 0 C, NH 2 OFLHCl, 145-155 0 C, 30 min (ii) Na 2 Se, DMF, ClCH 2 COOEt, NaOMe, MeOH, 60-70 0 C, 5 h (iii) HCl, NaNO 2 , K 2 CO 3 /dimethylamine, O 0 C, 2 h (iv) Aq. NH 3 , PEG-400, it, 48 h.
• the 3-chloro-3-substituted prop-2-enenitrile is prepared starting from the corresponding ketone with DMF-phosphorous oxychloride followed by hydroxylamine hydrochloride.
• the obtained products are reacted with sodium selenide, ethyl chloroacetate in presence of a base to provide 3 -substituted ethyl 3-amino-selenophene-2-carboxylates in good yields.
• Diazotization of the amino compounds with sodium nitrite followed by treatment with potassium carbonate/dimethylamine provides triazine compounds.
• the 3-chlorocyclohex-l-enecarbonitrile is prepared starting from the cyclohexanone with DMF-phosphorous oxychloride and hydroxyl amine hydrochloride, which is reacted with sodium selenide to provide ethyl 3-amino-4,5,6,7-tetrahydrobenzo[2,l-d]selenophene-2- carboxylate in good yield.
• Reagents & conditions (i) Na 2 Se, DMF, NaOMe, MeOH, 60-70 0 C, 5 h (ii) HCl, NaNO 2 , K 2 CO 3 /dimethylamine, O 0 C, 1 h (iii) Aq. NH 3 , rt, 16 h.
• the ethyl 3-aminoselenopheno[2,3-b]pyridine-2-carboxylate is prepared starting from the 2-chloropyridine, sodium selenide and ethyl chloroacetate, which on diazotization with sodium nitrite followed by treatment with dimethylamine provides ethyl 3- [(dimethylamino)diazenyl]selenopheno[2,3-b]pyridine-2-carboxylate. Treatment of the ester with ammonia gave the required 3-[(dimethylamino)diazenyl]selenopheno[2,3- b]pyridine-2-carboxamide (compound 6).
• Reagents & conditions (i) MeOH, NaOH, rt, 16 h.
• Reagents & conditions (i) MeOH, NaOH, rt, 2 h.
• Reagents & conditions (i) Na 2 Se, DMF, ClCH 2 CN, NaOMe, MeOH, 60-70 0 C, 5 h (ii) HBF 4 , NaNO 2 , K 2 C0 3/ dimethylamine, O 0 C, 2 h.
• the 3-amino-5-(tert-butyl)selenophene-2-carbonitrile is prepared starting from the 3- chloro-4,4-dimethylpent-2-enenitrile, sodium selenide and chloroacetonitrile, which on diazotization with sodium nitrite followed by treatment with dimethylamine gave the required 3-[(dimethylamino)diazenyl]-5-(tert-butyl)selenophene-2-carbonitrile
• Reagents & conditions (i) Na 2 Se, DMF, ClCH 2 CN, NaOMe, MeOH, 60-70 0 C, 5 h (ii) aq. NaOH, EtOH, reflux, 45 min (iii) NaNO 2 , H 2 SO 4 , O 0 C, 1 h, rt, 1 h (iv) CH 3 I, K 2 CO 3 , acetone, rt, 16 h.
• Reagents & conditions (i) Na 2 Se, DMF, ClCH 2 CN, NaOMe, MeOH, 6O 0 C, 3 h (ii) aq. NaOH, EtOH, reflux, 1 h (iii) NaNO 2 , H 2 SO 4 , O 0 C, 2 h (iv) CH 3 I, K 2 CO 3 , acetone, PEG- 400, rt, 16 h.
• the 3-amino-4,5,6,7-tetrahydrobenzo[l,2-b]selenophene-2-carbonitrile is prepared starting from 2-chlorocyclohex-l-enecarbonitrile, sodium selenide and chloroacetonitrile, which on treatment with aqueous sodium hydroxide gave the corresponding amide in good yield.
• Diazotization of the amine with sodium nitrite followed by treatment with iodomethane in presence of a base gave the required 3-methyl-6,7,8,9- tetrahydrobenzo[l,2-b] l,2,3-triazino-[4,5-d]-selenophen-4-one (compound 14).
• X C Reagents & conditions: (i) Na 2 Se, DMF, NaOMe, MeOH, 60-70 0 C, 3 h (ii) aq. NaOH, EtOH, reflux, 45 min (iii) NaNO 2 , H 2 SO 4 , O 0 C, 2 h (iv) CH 3 I, K 2 CO 3 , acetone, PEG-400, rt, 16 h.
• 3-amino-selenophenocarbonitriles are prepared starting from the 2- chloropyridine-3-carbonitrile or 2-chlorobenzonitrile, sodium selenide and chloroacetonitrile, which on treatment with aqueous sodium hydroxide gave the corresponding amides in good yield.
• the invention provides pharmaceutical or veterinary compositions (hereinafter, referred to as a pharmaceutical composition) comprising selenopheno triazene analogs in combination with a pharmaceutically acceptable excipient(s) or carrier(s) or diluent(s).
• a pharmaceutical composition comprising selenopheno triazene analogs in combination with a pharmaceutically acceptable excipient(s) or carrier(s) or diluent(s).
• the invention provides pharmaceutical or veterinary compositions comprising selenopheno triazene analogs in combination with a pharmaceutically acceptable excipient(s)/carrier(s)/diluent(s), further optionally comprising one or more pharmaceutically acceptable anti-cancer drugs.
• the invention provides pharmaceutical or veterinary compositions comprising selenopheno triazene analogs in combination with a pharmaceutically acceptable excipient(s)/carrier(s)/diluent(s), further optionally comprising one or more pharmaceutically acceptable anti-angiogenic drugs.
• the invention provides pharmaceutical or veterinary compositions (hereinafter, referred to as a pharmaceutical composition) comprising selenopheno triazene analogs that contains a melanin targeted analog as described above, in combination with a pharmaceutically acceptable excipient(s)/carrier(s)/diluent(s).
• a pharmaceutical composition comprising selenopheno triazene analogs that contains a melanin targeted analog as described above, in combination with a pharmaceutically acceptable excipient(s)/carrier(s)/diluent(s).
• the invention provides novel compositions for use as pharmaceutical or veterinary compositions comprising selenopheno triazene analogs in combination with a pharmaceutically acceptable excipient(s)/carrier(s)/diluent(s), further optionally comprising one or more pharmaceutically acceptable drugs.
• the pharmaceutically acceptable drugs can be one or more drugs selected from the list comprising anti-cancer drugs, anti-melanoma drugs, anti-metastatic melanoma drugs, anti-angiogenesis drugs, anti-inflammatory drugs, anti-obese drugs, anti-diabetic drugs, anti-metabolic disease drugs, biologic response modifying agents and other chemotherapy drugs.
• compositions of the methods described herein may comprise additional agents, such as adjuvants or antineoplastic agents.
• Antineoplastic agents include but are not limited to, RNAi reagents, tumor cells and antibodies.
• the antineoplastic agent is selected from but not limited to:
• 5-fluorouracil or a pharmaceutically acceptable composition of 5-fluorouracil including but not limited to Adrucil®, Carac®, Efudex® and Fluoroplex®;
• 6-mercatopurine or a pharmaceutically acceptable 6-mercatopurine composition including but not limited to Purinethol®;
• Actinomycin, aminoglutethimide or a pharmaceutically acceptable aminoglutethimide composition including but not limited to Cytadren®;
• Anastrozole or a pharmaceutically acceptable Anastrozole composition including but not limited to Arimidex®; bevacizumab or a pharmaceutically acceptable bevacizumab composition including but not limited to Avastin®;
• Docetaxel or a pharmaceutically acceptable Docetaxel composition including but not limited to Taxotere®;
• Doxorubicin or a pharmaceutically acceptable Doxorubicin composition including but not limited to Adriamycin®;
• Epirubicin or a pharmaceutically acceptable Epirubicin composition including but not limited to Ellence® and Pharmorubicin®);
• Etoposide or a pharmaceutically acceptable Etoposide composition including but not limited to Eposin®, Etopophos®, Vepesid®, and VP- 16®;
• Exemestane or a pharmaceutically acceptable Exemestane composition including but not limited to Aromasin®;
• Fluoxymesterone or a pharmaceutically acceptable Fluoxymesterone composition including but not limited to Halotestin®;
• Letrozole or a pharmaceutically acceptable Letrozole composition including but not limited to Femara®;
• Megestrol or Megestrol acetate or a pharmaceutically acceptable Megestrol acetate composition including but not limited to Megace®;
• Mitomycin or a pharmaceutically acceptable Mitomycin composition including but not limited to Mutamycin®;
• Mitoxantrone or a pharmaceutically acceptable Mitoxantrone composition including but not limited to Novantrone®;
• Paclitaxel or a pharmaceutically acceptable Paclitaxel composition including but not limited to Taxol®;
• Pamidronate or a pharmaceutically acceptable Pamidronate composition including but not limited to Aredia®; Prednisone; tamoxifen or a pharmaceutically acceptable tamoxifen composition including but not limited to Nolvadex®, Istubal®, Tamofen®, Tamone®, Tamoplex®, and Valodex®;
• Trastuzumab or a pharmaceutically acceptable Trastuzumab composition including but not limited to Herceptin®;
• Vinblastine or a pharmaceutically acceptable Vinblastine composition including but not limited to Velbe®;
• Vincristine or a pharmaceutically acceptable Vincristine composition including but not limited to Oncovin®; or
• Vinorelbine or a pharmaceutically acceptable Vinorelbine composition including but not limited to Navelbine®.
• compositions of the present invention include one or more pharmaceutical drugs selected from but not limited to angiostatin, endostatin, thalidomide, osteopontin, maspin, canstatin, proliferin related protein, restin and other related molecules.
• composition(s) comprise immunomodulatory agents, such as cytokines.
• Cytokines include but not limited to IFN- ⁇ , interleukins (IL-I, IL-2, IL-4, IL- 9, IL-I l, IL-12), monoclonal antibodies, interferons (interferon- ⁇ ), various types of colony stimulating factors (CSF, GM-CSF, G-CSF), TNF- ⁇ receptor blocker drugs and the like.
• the composition(s) further comprise an immunomodulatory drug, such as cyclophosphamide.
• the composition(s) comprise adjuvants.
• the compound(s) or composition(s) of the present invention are useful for treating cancer with one or more therapies including but not limited to anti- angiogenesis therapy, chemotherapy, cytokine therapy, radiotherapy, gene therapy, hormonal therapy, surgery, biological therapy or a combination thereof.
• the compound(s) or composition(s) of the present invention are useful for treating cancer wherein a cancer cell can originate from any part of the body, and not limited to any organ of human body such as brain, lung, adrenal glands, pituitary gland, breast, prostate, pancreas, ovaries, gastrointestinal tract, kidneys, liver, spleen, testicles, cervix, upper, lower, or middle esophagus either primary, secondary or tertiary tumors of all types.
• the compound(s) or composition(s) of the present invention are useful for the amelioration of potentially useful apoptosis markers including but not limited to cleaved cytokeratin-18 (c-CK18), cleaved caspase-3 (c-cas-3), cleaved lamin A (c-lam- A), phosphorylated histone H2AX (TH2AX), cleaved poly(ADP ribose) polymerase (c- PARP), and translocation of apoptosis-inducing factor (AIF), Bcl-2-associated X protein (Bax), Claudin-18 (CLD-18), cytokeratin-18 (CK 18), Apo 2.7 and Apo-2 Ligand/(TNF)- related apoptosis-inducing ligand.
• apoptosis markers including but not limited to cleaved cytokeratin-18 (c-CK18), cleaved caspase-3 (c-cas-3), cle
• novel compounds and compositions of the present invention are also potent angiogenesis inhibitors and can be effectively used alone or as a pharmaceutical composition(s) to prevent, treat and cure diseases including but not limited to cancer, cancer-related, cancer-associated, angiogenesis related and other vascular diseases.
• the present invention relates to the use of novel selenopheno triazene analogs of general formulae (I) and (II), their geometrical isomeric forms, stereoisomers, configurational isomers, polymorphs, hydrates, solvates and pharmaceutically acceptable salts thereof for metastatic malignant melanoma and for carcinomas of the solid tumors, and all other cancers including but not limited to lymphomas, sarcomas, leukemias and gliomas either alone or in combination with other approved chemotherapeutic drugs.
• the invention further provides a method of inhibiting angiogenesis and for ameliorating the angiogenesis modulators which include but not limited to VEGF, PDGF, TGF-beta and FGF in warm blooded animals in need thereof comprising administering a therapeutically effective amount of selenopheno triazene analogs of general formula (I) or formula (II) or their pharmaceutical composition(s).
• the present invention provides a method for remodeling of the vasculatures by modifying the proliferation, migration, invasion of endothelial cells and vascular smooth muscle cells in warm blooded animals in need thereof.
• the invention provides a method of treating a mammal having, or at risk of, a vascular indication associated with TGF-beta deficiency.
• Another preferred embodiment is an agent that increases the level of TGF-beta which is capable of binding to the TGF-beta receptors.
• compositions of the present invention may be in any form which allows for the composition to be administered to a subject.
• the composition may be in the form of a solid, liquid or gas (aerosol).
• routes of administration include, without limitation, topical, parenteral, sublingual, intraperitoneal (IP), intravenous (IV), oral (PO), intramuscular (IM), intracutaneous (IC), intradermal (ID), intrauterine and intrarectal.
• IP intraperitoneal
• IM intravenous
• IC intracutaneous
• ID intradermal
• intrauterine and intrarectal intrarectal.
• parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
• Pharmaceutical compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a subject.
• compositions that will be administered take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of triazene analog in topical form may hold a plurality of dosage units and using nanoparticles of different sizes in an emulsion to a warm blooded animal, in need thereof.
• compositions of the present invention may be delivered in any form of drug delivery system including but not limited to Liposome- based, Polymeric surfactant-based, Biodegradable block copolymers, Microencapsulation and Nanoparticles.
• Nanoparticles drug delivery can comprise Polymer-lipid hybrid nanoparticle system, Carbon nanotubes, Liquid filled nanoparticles, Liposomes Encapsulating Chitosan Nanoparticles, Organically Modified Silica Nanoparticles, Fluorocarbon nanoparticles and Dendrimer nanotechnology.
• the pharmaceutical compositions of the present invention can be useful for the preparation of medicaments for use in warm blooded animals in need thereof. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition(s) will depend on a variety of factors. Relevant factors include, without limitation, the type of subject (e.g., human), the particular form of the active ingredient, the manner of administration and the composition(s) employed.
• the pharmaceutical composition(s) of the present invention may include a compound of general formula (I) or (II) as described herein, in admixture with one or more carriers.
• the carrier(s) may be particulate, so that the compositions are, for example, in tablet or powder form.
• the carrier(s) may be liquid, with the compositions being, for example oral syrup or injectable liquid.
• the carrier(s) may be gaseous, so as to provide an aerosol composition useful in e.g., inhalatory administration.
• the invention provides the administration of the inventive compounds in any suitable form.
• the composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
• a solid composition for oral administration can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form.
• a solid composition will typically contain one or more inert diluents or edible carriers.
• binders such as carboxy methyl cellulose, ethyl cellulose, microcrystalline cellulose, or gelatin
• excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, primogel, corn starch and the like
• lubricants such as magnesium stearate or sterotex
• glidants such as colloidal silicon dioxide
• sweetening agents such as sucrose or saccharin, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent.
• the composition can be formulated as a capsule, e.g., a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or fatty oil.
• a liquid carrier such as polyethylene glycol or fatty oil.
• the composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension.
• the liquid may be for oral administration or for delivery by injection, as two examples.
• preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
• a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
• the liquid pharmaceutical composition of the invention may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylene diamine tetra acetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride
• fixed oils such as synthetic mono or diglycerides which may
• parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• Physiological saline is a preferred adjuvant.
• An injectable pharmaceutical composition comprising present inventive compounds of the general formula (I) or (II) are preferably sterile.
• the composition may include various materials which modify the physical form of a solid or liquid dosage unit.
• the composition may include various materials which modify the physical form of a solid or liquid dosage unit.
• the composition may include materials that form a coating shell around the active ingredients.
• the materials which form the coating shell are typically inert, and may be selected from, for example sugar, shellac, and other enteric coating agents.
• the active ingredients may be encased in a gelatin capsule.
• the composition in solid or liquid form may include an agent which binds to the active melanin targeted analog component(s) or derivatives thereof and thereby assists in the delivery of the active components.
• Suitable agents which may act in this capacity comprise a monocyclic, bicyclic, tricyclic, polycyclic aromatic, heterocyclic hydrophobic ring system as backbone moieties.
• the pharmaceutical composition of the present invention may consist of gaseous dosage units, e.g., it may be in the form of an aerosol.
• aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system which dispenses the active ingredients.
• Aerosols of compounds of the invention may be delivered in any suitable form such as monophasic, biphasic or triphasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, sub-containers, spacers and the like, which together may form a kit. Preferred aerosols may be determined by one skilled in the art, without undue experimentation.
• the invention concerns a method for treatment wherein an effective amount of a compound or composition(s) of the present invention is used to treat diseases of cells having melanoma and other cancers. These cells are typically mammalian cells.
• Methods of administering effective amounts of the analogs or derivatives are well known in the art and include the administration of inhalation, oral or parenteral forms.
• dosage forms includes, but are not limited to, parenteral solutions, tablets, capsules, sustained release implants and transdermal delivery systems; or inhalation dosage systems employing dry powder inhalers or pressurized multi-dose inhalation devices.
• the dosage amount and frequency are selected to create an effective level of the agent without harmful effects.
• a liquid composition intended for either parenteral or oral administration should contain a therapeutically effective amount of the inventive compounds of the general formula (I) or (II). Typically, this amount is at least 0.01% of the composition. When intended for oral administration, this amount may be varied to be between 0.01 and 70% of the weight of the composition.
• Preferred oral compositions contain between 4% and about 50% of the active triazene compound.
• Preferred compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01% and 1% by weight of inventive compound.
• the pharmaceutical composition of the present invention may be intended for any suitable administration such as topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.
• the base may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.
• Thickening agents may be present in a pharmaceutical composition for topical administration.
• the composition may include a transdermal patch or ionophoresis device.
• Topical formulations may contain a concentration of the inventive compound from about 0.1 to about 10% w/v (weight per unit volume).
• composition of the present invention may be intended for rectal administration, in the form, e.g., of a suppository which will melt in the rectum and release the drug.
• the composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient.
• bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
• the invention employs a method of administration wherein a composition intended to be administered by injection can be prepared by combining the selenopheno triazene analog or derivative thereof with water so as to form a solution.
• a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
• Surfactants are compounds that non-covalently interact with the triazene analog ,or derivative so as to facilitate dissolution or homogeneous suspension of the triazene analog or derivative in the aqueous delivery system.
• the inventive selenopheno triazene analogs of general formulae (I) and (II) are used in the treatment of melanoma, Hodgkin's lymphoma, carcinomas, sarcomas, gliomas, and other cancers in warm blooded animals in need thereof.
• the present invention relates to the method of treating metastatic malignant melanoma and for carcinomas of the solid tumors, and all other cancers of solid, liquid or lymphatic origin including but not limited to lymphomas, sarcomas and gliomas comprising administering to a warm blooded animal in need thereof a therapeutically effective dose of atleast one compound selected from the novel selenophene compounds of general formulae (I) and (H), their geometrical isomeric forms, stereoisomers, configurational isomers, polymorphs, hydrates, solvates and pharmaceutically acceptable salts thereof.
• the present invention relates to the method of treating metastatic malignant melanoma and for carcinomas of the solid tumors, and all other cancers including but not limited to lymphomas, sarcomas and gliomas comprising administering to a warm blooded animal in need thereof a therapeutically effective dose of composition comprising atleast one compound selected from the novel selenophene compounds of general formulae (I) and (II), their geometrical isomeric forms, stereoisomers, configurational isomers, polymorphs, hydrates, solvates and pharmaceutically acceptable salts thereof in combination with pharmaceutically acceptable excipient(s)/diluent(s)/carrier(s), further optionally containing one or more drug(s).
• composition comprising atleast one compound selected from the novel selenophene compounds of general formulae (I) and (II), their geometrical isomeric forms, stereoisomers, configurational isomers, polymorphs, hydrates, solvates and pharmaceutically acceptable salts thereof in combination
• Methyl 5-methyl-4-nitroselenophene-2-carboxylate To an ice cold (0-10 0 C) solution of methyl S-methylselenophene ⁇ -carboxylate (5.4 g, 26.6 mmol) in acetic anhydride (15 mL) was added an ice cold mixture of nitric acid (5.5 mL, 61.1 mmol, 70%) and acetic anhydride (10 mL) for 10 min. The reaction mixture was slowly allowed to rt for 1 h and stirred at room temperature (rt) for 16 h. The mixture was poured into ice cold water and stirred for 10 min.
• Step b
• Methyl 4-amino-5-methylselenophene-2-carboxylate To a solution of methyl 5-methyl- 4-nitroselenophene-2-carboxylate (2.3 g, 9.28 mmol) in a mixture of water (5 mL) and methanol (40 mL) was added cone, hydrochloric acid (1.0 mL). To the above solution was added iron powder (2.6 g, 46.4 mmol) followed by ammonium chloride (2.5 g, 46.4 mmol) at rt. The reaction mixture was refluxed for 1 h and was then allowed to cool to rt. The solution was filtered and basified with saturated sodium bicarbonate solution.
• the reaction mixture was added to the solution of potassium carbonate (2.9 g, 20.9 mmol) and dimethylamine (2.23 mL, 40%, 19.8 mmol) in water (15.6 mL) at O 0 C.
• the mixture was stirred at 0-1O 0 C for 1 h and poured into ice cooled water.
• the solution was extracted with chloroform (3 x 100 mL) and the combined chloroform layer was washed with water, brine and dried over sodium sulfate. The solution was filtered and evaporated the solvent.
• Methyl 5 -form ylselenophene-2-carboxy late To a solution of methyl 5- methylselenophene-2-carboxylate (5.0 g, 24.6 mmol) in acetic acid (30 mL) was added selenium dioxide (10.84 g, 98.4 mmol) at rt and the mixture was refluxed for 8 h. The cooled reaction mixture was poured into ice cooled water and stirred for 15 min. The solution was extracted with chloroform (3 x 100 mL) and the combined organic layer was washed with water, brine and dried over sodium sulfate. The solution was filtered and evaporated the solvent.
• Step b
• Methyl 5-(methoxycarbonvD-3-nitroselenophene-2-carboxylate To a solution of 3- nitroselenophene-2,5-dicarboxylic acid (10 g, 37.73 mmol; mixture of 2 compounds) in methanol (100 mL) was added thionyl chloride (10.9 mL, 150.92 mmol) drop wise under stirring at ice cold temperature. The reaction mixture was refluxed for 2 h and attained to rt. The mixture was poured into ice cooled water and stirred for 15 min. The solution was extracted with chloroform (3 * 200 mL) and the combined organic layer was washed with water, dil.
• Methyl 3-amino-5-(methoxycarbonyl)selenophene-2-carboxylate To a solution of methyl 5-(methoxycarbonyl)-3-nitroselenophene-2-carboxyIate (8.0 g, 27.3 mmol) in a mixture of water (20 mL) and methanol (150 mL) was added cone, hydrochloric acid (2.75 mL, 27.3 mmol). To the above solution was added iron powder (7.64 g, 136.5 mmol) followed by ammonium chloride (7.3 g, 136.5 mmol) at rt. The reaction mixture was refluxed for 3 h and was then allowed to cool to rt.
• Step f
• Methyl 3-f(dimethylamino)diazenyl1-5-(methoxycarbonyl)selenophene-2-carboxylate To a solution of methyl 3-amino-5-(methoxycarbonyl)selenophene-2-carboxylate (0.6 g, 2.28 mmol) and cone. HCl (1.0 mL, 9.12 mmol) in water (5.7 mL) was added sodium nitrite (173 mg, 2.51 mmol) in portions for 5 min at O 0 C.
• the reaction mixture was added to the solution of potassium carbonate (1.1 g, 8.66 mmol) and dimethylamine (1.0 mL, 40%, 8.21 mmol) in water (6.8 mL) at O 0 C.
• the mixture was stirred at 0-5 0 C for 2 h and poured into ice cold water.
• the solution was extracted with chloroform (3 x 100 mL) and the combined chloroform layer was washed with water, brine and dried over sodium sulfate. The solution was filtered and evaporated the solvent.
• Step g
• Ethyl 3-amino-5-phenylselenophene-2-carboxylate To a suspension of sodium selenide (1.31 g, 10.4 mmol) in DMF (10.5 mL) was added a solution of 3-chloro-3- ⁇ henylprop-2- enenitrile (1.7 g, 10.4 mmol) in DMF (4 mL) at room temperature (rt) for 5 min and stirred the mixture at 60-70 0 C for 2 h. Then ethyl chloroacetate (1.1 mL, 10.4 mmol) was added dropwise to the reaction mixture and again stirred at 60-70 0 C for 2 h.
• Ethyl 3-[(dimethylamino ' )diazenv ⁇ -5-phenylselenophene-2-carboxylate To a solution of ethyl 3-amino-5-phenylselenophene-2-carboxylate (0.6 g, 2.03 mmol) and cone. HCl (0.79 mL, 8.12 mmol) in water (4.7 mL) was added sodium nitrite (150 mg, 2.23 mmol) in portions for 5 min at O 0 C.
• the reaction mixture was added to the solution of potassium carbonate (1.06 g, 7.7 mmol) and dimethylamine (0.82 mL, 40%, 7.3 mmol) in water (5.7 mL) at O 0 C.
• the mixture was stirred at 0-5 0 C for 2 h and poured into ice cold water.
• the solution was extracted with chloroform (3 x 100 mL) and the combined layer was washed with water, brine and dried over sodium sulfate. The solution was filtered and evaporated the solvent.
• Step b
• Ethyl 3-amino-5-(tert-butyl ' )selenophene-2-carboxylate To a suspension of sodium selenide (4.4 g, 34.84 mmol, prepared from 2.78 g of selenium as described above) in DMF (35 mL) was added a solution of 3-chloro-4,4-dimethylpent-2-enenitrile (5 g, 34.84 mmol) in DMF (10 mL) at rt for 5 min and stirred the mixture at 60-70 0 C for 2 h.
• Ethyl 3-r(dimethylamino)diazenyl]-5-(tert-butv ⁇ selenophene-2-carboxylate To a solution of ethyl 3-amino-5-(tert-butyl)selenophene-2-carboxylate (6 g, 21.8 mmol) and cone. HCl (8.38 mL, 87.2 mmol) in water (50 mL) was added sodium nitrite (1.65 g, 23.9 mmol) in portions for 5 min at O 0 C.
• the reaction mixture was added to the solution of potassium carbonate (11.43 g, 82.8 mmol) and dimethylamine (9 mL, 40%, 78.48 mmol) in water (62 mL) at O 0 C.
• the mixture was stirred at 0-5 0 C for 2 h and poured into ice cold water.
• the solution was extracted with chloroform (3 * 200 mL) and the combined organic layer was washed with water, brine and dried over sodium sulfate. The solution was filtered and evaporated the solvent.
• Step b
• the reaction mixture was stirred at 6O 0 C for 3 h. Then, a solution of sodium methoxide (2.0 g, 37.3 mmol) in methanol (37 mL) was added dropwise and stirring was continued for 2 h at the same temperature. The mixture was allowed to rt and poured into ice cold water and stirred for 15 min. The solution was extracted with chloroform (3 x 200 mL) and the combined chloroform layer was washed with water, brine and dried over sodium sulfate. The solution was filtered and evaporated the solvent.
• Zl-d]selenophene-2- carboxylate To a solution of ethyl and methyl esters of 3-amino-4,5,6,7- tetrahydrobenzo[2,l-d]selenophene-2-carboxylate (400 mg, 1.54 mmol), cone. HCl (0.6 mL, 6.17 mmol) in water (3.5 mL) and acetone (3 mL) was added sodium nitrite (120 mg, 1.69 mmol) in portions for 5 min at O 0 C.
• the reaction mixture was added to the solution of potassium carbonate (800 mg, 5.85 mmol) and dimethylamine (0.62 mL, 40%, 5.54 mmol) in water (4.3 mL) at O 0 C.
• the mixture was stirred at 0-5 0 C for 1 h and poured into ice cold water.
• the solution was extracted with chloroform (3 x 200 mL) and the combined organic layer was washed with water, brine and dried over sodium sulfate. The solution was filtered and evaporated the solvent.
• Ethyl 3-aminoselenophenor2.3-blpyridine-2-carboxylate To a suspension of sodium selenide (0.9 g, 7.2 mmol, prepared from 0.75 g of selenium as described above) in DMF (7 mL) was added a solution of 2-chloropyridine-3-carbonitrile (1 g, 7.2 mmol) in DMF (3 mL) at rt for 5 min and stirred the mixture at 60-70 0 C for 2 h. Then ethyl chloroacetate (0.78 mL, 7.22 mmol) was added dropwise to the reaction mixture and again stirred at 60-70 0 C for 2 h.
• Step b
• Ethyl 3-[(dimethylamino)diazenyllselenopheno[2,3-b1pyridine-2-carboxylate A suspension of ethyl 3-aminoselenopheno[2,3-b]pyridine-2-carboxylate (1 g, 3.7 mmol) and aqueous fluoroboric acid (5.8 mL, 29.6 mmol, 45%) in water (40 mL) was heated to dissolve the compound. The mixture was again cooled to O 0 C and added sodium nitrite (300 mg, 4.4 mmol) in portions for 5 min at O 0 C.
• the reaction mixture was added to the solution of potassium carbonate (3.8 g, 28 mmol) and dimethylamine (3 mL, 40%, 26.8 mmol) in water (60 mL) at O 0 C.
• the mixture was stirred at 0-5 0 C for 1 h and poured into ice cold water.
• the solution was extracted with chloroform (3 * 200 mL) and the combined organic layer was washed with water, brine and dried over sodium sulfate. The solution was filtered and evaporated the solvent.
• Step b
• 3-Amino-5-phenylselenophene-2-carboxamide To a suspension of 3-amino-5- phenylselenophene-2-carbonitrile (4.0 g) in aqueous sodium hydroxide solution (120 mL, 10%) was added ethanol (50 mL) and the mixture refluxed for 45 min. The mixture was allowed to rt and the crystals separated were filtered off, washed with cold water and dried to give the product as golden yellow color solid (2.8 g, 67%), mp 184-186 0 C.
• 6-Phenyl-3H-selenophenor3,2-c11,2,3-triazin-4-one To an ice cold solution (O 0 C) of 3- amino-5-phenylselenophene-2-carboxamide (0.27 g, 1.01 mmol) in concentrated sulfuric acid (7 mL) was added a cold (O 0 C) solution of sodium nitrite (77 mg, 1.11 mmol) in concentrated sulfuric acid (2.5 mL) for 10 min (while adding, the temperature should keep between -5-O 0 C). After addition, the mixture was stirred at O 0 C for 1 h and at rt for 1 h.
• 3-Methyl-6-phenylselenopheno[3,2-dl 1 ,2,3-triazin-4-one To a solution of 6-phenyl-3H- selenopheno[3,2-c]l,2,3-triazin-4-one (0.6 g, 2.16 mmol) in acetone (25 mL) was added sequentially potassium carbonate (0.59 g, 4.33 mmol), iodomethane (0.16 mL, 2.6 mmol) and potassium iodide (catalytic) at rt and the mixture was stirred at rt for 16 h. The solution was filtered and the solids were washed with acetone.
• Step b
• 3-Amino-5-(tert-butyl)selenophene-2-carboxamide To a suspension of 3-amino-5-(tert- butyl)selenophene-2-carbonitrile (5.0 g) in aqueous sodium hydroxide solution (80 mL, 10%) was added ethanol (50 mL) and the mixture refluxed for 1 h. Ethanol was distilled off under vacuum (appr. 25 mL) and the mixture was allowed to cool to 5-1O 0 C. The separated crystals were filtered off, washed with cold water and dried to give the product as a off-white color solid (4.5 g, 84%), mp 160-162 0 C.
• 6-(tert-Butyl)-3-methylselenophenor3.2-d]l,2,3-triazin-4-one To a solution of 6-(tert- butyl)-3H-selenopheno[3,2-d]l,2,3-triazin-4-one (2.0 g, 7.78 mmol) in acetone (70 mL) was added sequentially potassium carbonate (2.14 g, 15.56 mmol), iodomethane (0.58 mL, 9.3 mmol) and potassium iodide (catalytic) at rt and the mixture was stirred at rt for 16 h. The solution was filtered and the solids were washed with acetone.
• Step b
• the reaction mixture was poured into crushed ice slowly with stirring for 15 min and stirred at the same temperature for 15 min.
• the solution was extracted with ethyl acetate (3 x 100 mL) and the combined EtOAc layer was washed with water, brine and dried over sodium sulfate.
• the solution was filtered and evaporated the solvent to give the product as a pale brown color solid (450 mg, 31%), mp 150-152 0 C.
• Step b
• 3-Aminoselenopheno[2.3-b1pyridine-2-carboxamide To a suspension of 3- aminoselenopheno [2,3-b]pyridine-2-carbonitrile (1.0 g) in aqueous sodium hydroxide solution (20 mL, 10%) was added ethanol (20 mL) and the mixture refluxed for 45 min. Ethanol was distilled off under vacuum (appr. 25 mL) and the mixture was allowed to cool to 5-1O 0 C. The separated crystals and the solution was poured into ice cooled water and stirred for 15 min. The solid, was filtered off, washed with cold water and dried to give the product as a pale yellow color solid (0.53 g, 50%), mp 256-26O 0 C.
• the reaction mixture was poured into crushed ice slowly with stirring for 15 min and stirred at the same temperature for 15 min.
• the solution was extracted with ethyl acetate (3 * 100 mL) and the combined organic layer was washed with water, brine and dried over sodium sulfate.
• the solution was filtered and evaporated the solvent to give the product as a reddish brown color solid (350 mg, 67%), mp 184-186 0 C (decomposed).
• Step b
• 3-Aminobenzo[b]selenophene-2-carboxamide To a suspension of 3- aminobenzo[b]selenophene-2-carbonitrile (5.0 g) in aqueous sodium hydroxide solution (100 mL, 10%) was added ethanol (60 mL) and the mixture refluxed for 1 h. Ethanol was distilled off under vacuum (appr. 25 mL) and the mixture was allowed to cool to 5-1O 0 C. The separated crystals were filtered off, washed with cold water and dried to give the product as a off-white color solid (3 g, 60%), mp 180-182 0 C.
• MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] incorporation based cell proliferation assay was performed using standard procedure.
• MTT based cell proliferation assays indicate that among the test compounds (compounds 1 to 16), 4-[(dimethylamino)diazenyl]-5-methylselenophene-2-carboxamide (Compound 1 ) exhibited the best efficacy in inhibiting melanoma tumor cell growth in vitro (Table 1). In comparison with the marketed standard drug, dacarbazine, Compound 1 has shown 5 times and 8 times more, potent inhibition in mouse melanoma B 16 cells and in human melanoma A375 cells, respectively.
• Anti-tumor growth potential of Compound 1 and DTIC were evaluated in various human tumor cells such as A549 lung tumor cells, DU 145 prostate tumor cells, HT29 colon cancer cells and MCF-7 (ER + ) breast tumor cells in vitro, by using MTT based cell proliferation assay as described earlier (example 17).
• the results (mean OD ⁇ SD) obtained from quadruplicate wells were used in calculation to determine the inhibition of cell proliferation (50% of inhibitory concentration, IC50) of the test compounds (Table
• Table 2 Comparative anti-tumor growth potential of Compound 1 in some other human cancer cells of different tissue origin
• Cytotoxicity potential of DTIC and the Compound 1 was evaluated by measuring the leaked lactate dehydrogenase (LDH) into the tumor cells culture supernatant (LDH Cytotoxicity Detection Kit plus , Roche Applied Sciences, Germany).
• LDH lactate dehydrogenase
• the leaked LDH is directly proportional to the cell damage done by the cytotoxic compounds. Briefly, equal number of human malignant melanoma A375 cells or mouse melanoma cells B 16 FO were treated with test compounds at various concentrations and incubated for 48 h. Vehicle control culture wells received only a maximum of 0.5% DMSO. The cell free culture supernatants were mixed with catalyst and dye solution and allowed to incubate for 15 min at room temperature.
• a bar diagram depicts the loss of cell viability of B 16 FO and A375 cells as indicated by percent increase of leaked LDH in DTIC and compound 1 treated versus drug concentration as depicted in Figure IA and IB, respectively.
• compound 1 and DTIC exhibited 2.33% and 34.58% growth inhibition of HS.531.sk normal human skin epithelial cells; and at 50 ⁇ g/ml concentration, these two anti-melanoma drugs showed 23.83% and 43.46% growth inhibitions in the normal skin epithelial cells, respectively.
• Inhibitory efficacy in clone formation of the Compound 1 and DTIC was tested by following the procedure described earlier with some modifications . Briefly, B 16F0 cells were harvested and seeded into 6-well plates (100 cells/ml). The cells were allowed to grow for 4 days and thereafter, the cells were incubated with DMEM containing either 0.1% DMSO or 100 ⁇ g/ml DTIC or different concentrations (1, 5, 10 or 20 ⁇ g/ml) of Compound 1 for further 8 days. Fresh medium containing test agents was replaced at every 24h. Finally, the wells were washed three times with PBS and fixed in methanol for 15 min. The cells were stained with Giemsa stain and observed under microscope.
• Figure 3 shows inhibition of B 16 colony growth in DTIC and Compound 1 treated wells.
• Compound 1 exhibited significant inhibition in Bl 6 tumor cell colony growth compared with DTIC.
• DTIC showed only 35.8% inhibition, in contrast, 1 1%, 35%, 54% and 94% reductions in number of colonies were achieved by 1, 5, 10 or 20 ⁇ g/ml of Compound 1, respectively.
• Compound 1 inhibits invasion of B 16F0 mouse melanoma cells:
• the inhibitory effects of DTIC and compound 1 on invasive ability of B 16F0 were tested in cell invasion assay performed with by using Matrigel (BME - Cultrex®, R&D Systems, USA) coated cell culture inserts (Becton Dickinson, USA) with 8 ⁇ m-pore membrane. Equal number (fifty thousands) of B 16F0 cells were applied in each insert well and allowed to attach for 2h at 37 0 C and in presence of 5% CO 2 . Thereafter, the cellular invasion through the matrigel layer was performed in presence or absence of test compounds. Either 100 ⁇ g/ml of DTIC or 20 ⁇ g/ml of Compound 1 was applied in the lower chamber of the invasion assembly. 0.1% DMSO was applied in the vehicle control culture chambers.
• Compound 1 inhibits migration of human endothelial cells:
• FALCONTM Cell Culture inserts (Becton Dickinson, USA) with 8 ⁇ m-pores in their PET membrane was coated with 0.1 mg/ml of collagen.
• Human umbilical vein endothelial cells (HUVEC) were added to the cell culture inserts (Becton Dickinson) at a density of 5*10 4 cells/insert. Cells were allowed to migrate through the insert for 18h in presence of different concentrations of either DTIC or Compound 1.
• the control culture containing migration assembly received only 0.1% DMSO.
• Compound 1 inhibits endothelial capillary tube formation in vitro:
• this compound was tested for its anti-melanoma efficacy in A375 human melanoma xenograft model of nu/nu BALB/c nude mice.
• the animals of 6-8 weeks age (body weight 18-22 g) were used in this study.
• Animals study protocols were approved by Institutional Ethics Committee (LAEC). All the studies were performed in compliance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines and OECD guidelines. Animals were allowed free access to sterile standard rodent diet and provided with charcoal filtered and UV exposed water ad libitum. The animals were housed in a sterile room and were located individually in ventilated cages. The room was maintained at a controlled temperature (24-26°C), humidity (45-70%), and 12h/12h of light/dark cycle.
• melanoma tumor To induce the melanoma tumor, sub-confluent A375 human melanoma cells were harvested by brief trypsinization and 1x10 6 cells were injected subcutaneously in 0.2 ml phosphate-buffered saline. Drug treatment was started after development of palpable tumors (6-7 days after implantation of the cells). At this point the tumor take rate was 100%.
• Compound 1 was prepared in phosphate-buffered saline containing 10% DMSO (v/v); and 25 mg/kg of compound 1 was administered daily through intra-peritoneal route. Vehicle treated control animals received only 10% DMSO in phosphate buffered saline. After twenty one days of treatment, the animals were sacrificed by CO 2 inhalation and tumors growth was measured by the following formula (Friedman HS et al., MoI Cancer Ther 2002; 1:943-948).
• Compound 1 is able to inhibit 46.52% melanoma tumor growth in human melanoma xenograft model of nude mice. This observation substantiates its anti-melanoma efficacy and strengthens our finding that compound 1 can used ,as a potential therapeutic agent to treat human malignant melanoma.

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