FIELD OF THE INVENTION

The present invention relates to a compound of formula (1) (as described herein), or a pharmaceutically acceptable salt or a derivative thereof. The compound may be obtained by fermentation of a microorganism belonging to marine actinomycetes strain (PM0895117/MTCC 5675). The present invention further relates to processes for the production of the compound of formula (1), to the microorganism belonging to marine actinomycetes strain (PM0895117/MTCC 5675) and to pharmaceutical compositions containing the compound of formula (1) as an active ingredient and use thereof for the treatment of cancer.

BACKGROUND OF THE INVENTION

Cancer has become an increasing public health problem due to its high rates of morbidity and mortality. Despite continuing advances in treatment regimens for cancer, this disease still remains one of the leading causes of death in the world for the reason that the available treatment options are associated with undesirable side effects and limited efficacy. It is the second most common cause of death in developed countries, exceeded only by heart diseases. Cancer accounts for nearly one of every four deaths (Source: Cancer Facts and Figures 2010 of the American Cancer Society). By the year 2020, cancer could kill more than ten million people per year unless action is taken in both the field of prevention and treatment (Biotechnology Advances, 29, 2011, 531-547).

Therefore, there still exists a continuing need to develop newer anticancer agents that may address the drawbacks of the existing anticancer agents.

Cancer therapy currently falls under the following categories including surgery, radiation therapy, chemotherapy, bone marrow transplantation, stem cell transplantation, hormonal therapy, immunotherapy, antiangiogenic therapy, targeted therapy, gene therapy and others.

The marine environment, covering seventy percent of the earth's surface and ninety five percent of its tropical biosphere represents thirty four of the thirty six phyla of life and provides a fascinating variety of biodiversity exceeding that of the terrestrial environment (Life Sciences, 78, 2005, 442^-53). Marine natural product bio prospecting, which is exploration, extraction and screening of biological diversity and indigenous knowledge for commercially valuable genetic and biochemical resources, has yielded a considerable number of drug candidates (Drug Discovery Today, 8 (12), 2003, 536-544). Marine natural products have become fascinating targets for biologists and chemists for discovery of lead compounds for clinical development since the past five decades (Evidence-Based Complementary and Alternative Medicine, 2011, 2011, 1-11).

Many pharmaceutical agents have been discovered by screening natural products from marine organisms and microorganisms. Examples of anticancer agents originating from marine sources include citarabine, bryostatin-1, aplidine, dolastatin 10 and ET-743. (Current Opinion in Pharmacology, 1, 2001, 364—369).

Plicacetin is a nucleoside antibiotic isolated from actinomycete Streptomyces plicatus.

It is a potent inhibitor of protein synthesis and is reposted to inhibit the KB strain of human epidermoid carcinoma cells and increase survival time of mice with leukemia (Journal of American Chemical Society, 94 (9), 1972, 3280-3281). Cytosaminomycin A is a nucleoside anticoccidial antibiotic isolated from the fermentation broth of Streptomyces amakusaensis KO-81 191 (The Journal Of Antibiotics, 47 (7), 1994, 774-781, and 47 (7), 1994, 782-786). In view of this, it would be prudent to explore this area of research to identify and develop new drugs which can be used effectively to treat various types of cancers.

The inventors of the present invention have directed their efforts in carrying out an extensive research in this area which resulted in the present invention.

SUMMARY OF THE INVENTION

The present invention relates to a nucleoside analogue designated herein as compound of formula (1) (as described herein).

The present invention also relates to a purified compound of formula (1), isolated from the fermented broth of the marine actinomycetes strain (PM0895117/MTCC 5675) or one of its variants or mutants.

The invention also relates to all isomeric forms and tautomeric forms of the compound of formula (1), and a pharmaceutically acceptable salt or a derivative thereof.

The compound of formula (1), or an isomer, a tautomer,or a pharmaceutically acceptable salt or a derivative thereof, for use in the treatment of cancer.

The invention further relates to a pharmaceutical composition comprising the compound of formula (1), or an isomer, a tautomer, a pharmaceutically acceptable salt, or a derivative thereof, as an active ingredient in association with at least one pharmaceutically acceptable excipient, carrier or a vehicle. The present invention also relates to processes for the production of the compound of formula (1) and/or its isomers or its tautomers from the marine actinomycetes strain (PM0895117/MTCC 5675).

The present invention also relates to processes for the isolation of the marine actinomycetes strain (PM0895117/MTCC 5675), which on cultivation produces the compound of formula (1), or its isomers or tautomers.

The present invention relates to a method for the treatment of cancer in a subject comprising administering to the subject a therapeutically effective amount of a compound of formula (1), or an isomer or a tautomer or a pharmaceutically acceptable salt or a derivative thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates H NMR spectrum (DMSO-d ₆ ; 500 MHz; Instrument: Bruker) of the compound of formula (1).

Figure 2 illustrates ¹³ C NMR spectrum (DMSO-d ₆ ; 75 MHz; Instrument: Bruker) of the compound of formula (1).

Figure 3 illustrates effect of compound of formula (1) on apoptosis in Panc-1 cancer cells.

DETAILED DESCRIPTION OF THE INVENTION

The compound of formula (1) has the molecular formula C ₂₂ H ₃₄ N ₄ O ₇ S and a molecular weight 498.2. The compound of formula (1) may be characterized by using any one or more of the physico-chemical and spectral methods, such as high performance liquid chromatography (HPLC), mass spectrometry (MS), infra red spectroscopy (IR), and nuclear magnetic resonance (NMR) spectroscopic data as discussed herein below.

The compound of formula (1) is structurally represented as follows:

formula (1) The gram-positive bacteria, which may be used for the production of the compound of formula (1), is a marine actinomycetes strain (PM0895117/MTCC 5675), herein after referred to as culture no. PM0895117, which was isolated from the deep sea sediment collected from the offshore region of Mumbai, Maharashtra, India.

One aspect of the present invention provides processes for the production of the compound of formula (1) from the culture no. PM0895117 comprising the steps of:

(a) cultivating the culture no. PM0895117/MTCC 5675 or one of its variants or mutants under submerged aerobic conditions in a nutrient medium containing one or more sources of carbon and one or more sources of nitrogen and nutrient inorganic salts to produce a culture broth containing the compound of formula (1);

(b) isolating the compound of formula (1) from the culture broth; and

(c) purifying the compound of formula (1).

The step (c) involving purification of the compound of formula (1) is carried out by purification procedures generally used in the related art.

The compound of formula (1) produced according to the process of the present invention is a substantially pure compound. Thus, the compound of formula (1) is an isolated pure compound produced from the culture no. PM0895117.

As used herein, the term "mutant" refers to an organism or cell carrying a mutation, which is an alternative phenotype to the wild-type.

As used herein, the term "variant" refers to an individual organism that is recognizably different from an arbitrary standard type in that species.

The term "mammal" as used herein, refers to a human as well as non-human mammal, including but not limited to, cows, horses, pigs, dogs and cats. The term "mammal" may be used interchangeably with the term "patient" or "subject".

The term "whole broth" may be used interchangeably with the terms "nutrient broth", "culture broth" or "fermented broth".

The term "active ingredient" as used herein, refers to the compound of formula (1) or to an isomer or a tautomer or a derivative or a pharmaceutically acceptable salt thereof.

The term "compound of formula (1)" includes the compound of formula (1) itself and an isomer, a tautomer, or a pharmaceutically acceptable salt thereof.

The term "substantially pure" as used herein, means that the compound of formula (1) or an isomer thereof is sufficiently pure such that further purification would not detectably alter its physical and chemical properties, such as enzymatic and biological activities, of the substance. The compound of formula (1) can be purified substantially by following the methods known to those skilled in the art.

As used herein the term "therapeutically effective amount" in reference to the treatment of cancer (as listed herein) using the compound of formula (1), a pharmaceutically acceptable salt thereof or a derivative thereof, refers to an amount capable of invoking one or more of the following effects in a subject receiving the compound of the present invention: (i) inhibition, to some extent, of tumor growth, including, slowing down and complete growth arrest; (ii) reduction in the number of tumor cells; (iii) reduction in tumor size; (iv) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into peripheral organs; (v) inhibition (i.e., reduction, slowing down or complete stopping) of metastasis; (vi) enhancement of anti-tumor immune response, which may, but does not have to, result in the regression of the tumor; and/or (vii) relief, to some extent, of one or more symptoms associated with the cancer being treated.

The term "pharmaceutically acceptable salt(s)", as used herein, means those salts of compound(s) of the invention that are safe and effective in mammals and that possess the desired biological activity. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, phosphate, acetate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, formate, benzoate, glutamate, methanesulfonate, benzensulfonate, or p-toluenesulfonate salts. Suitable base addition salts include, but are not limited to, calcium, lithium, magnesium, potassium, sodium, or zinc, salts.

Preliminary identification of culture no. PM0895117, from which the compound of formula (1) is produced, was performed by examination of its colony characteristics. Microscopic studies on the strain of isolated culture no. PM0895117 were carried out on modified actinomycetes isolation agar medium. The observations were made after 20-22 days of incubation at 20°C to 30°C, till colonies were observed. Culture no. PM0895117 has been identified as a marine actinomycetes strain.

Culture no. PM0895117 has been deposited with Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Sector 39-A, Chandigarh -160 036, India, a World Intellectual Property Organization (WIPO) recognized International Depository Authority (IDA) and has been given the accession number MTCC 5675.

In addition to the specific microorganism described herein, it should be understood that mutants of PM0895117, such as those produced by the use of chemical or physical mutagens including X-rays, U.V. rays etc. and organisms whose genetic makeup has been modified by molecular biology techniques, may also be cultivated to produce the compound of formula (1).

The screening for suitable mutants and variants which can produce the compound according to the invention, can be confirmed by HPLC, NMR, IR, MS and/or determination of biological activity of the active compounds accumulated in the culture broth, for example by testing the compounds for anticancer activity or by a combination thereof.

The medium and/or nutrient medium used for isolation and cultivation of culture no. PM0895117, which produces the compound of formula (1), preferably contains sources of carbon, nitrogen and nutrient inorganic salts. The carbon sources are, for example, one or more of soluble starch, glucose, sucrose, dextrin, fructose, molasses, glycerol, lactose, or galactose. A preferred carbon source is soluble starch and glucose. The sources of nitrogen are, for example, one or more of soyabean meal, peanut meal, yeast extract, beef extract, peptone, malt extract, corn steep liquor, gelatin, sodium caseinate, L-Asparagine or casamino acids. Preferred nitrogen source is soyabean meal, peptone, sodium caseinate, L-Asparagine, corn steep liquor and yeast extract. The nutrient inorganic salts are, for example, one or more of sodium chloride, potassium chloride, calcium chloride, manganese chloride, magnesium chloride, strontium chloride, cobalt chloride, potassium bromide, sodium fluoride, sodium hydrogen phosphate, potassium hydrogen phosphate, dipotassium hydrogen phosphate, disodium phosphate, calcium carbonate, sodium propionate, sodium bicarbonate, sodium silicate, sodium nitrate, ammonium nitrate, potassium nitrate, sodium sulphate, ammonium sulphate, ammonium heptamolybdate, ferric citrate, copper sulphate, magnesium sulphate, ferrous sulphate, zinc sulphate or boric acid. Sodium chloride, dipotassium hydrogen phosphate, magnesium sulphate, ferrous sulphate, copper sulphate, manganese chloride, zinc sulphate and calcium carbonate are preferred nutrient inorganic salts.

The maintenance of culture no. PM0895117 may be carried out at a temperature ranging from 24°C to 32°C. Typically, culture no. PM0895117 is maintained at 27°C - 29°C. The well-grown cultures may be preserved in the refrigerator at 4°C - 8°C.

Seed culture cultivation of culture no. PM0895117 may be carried out at a temperature ranging from 27°C to 33°C and a pH of about 5.5 to 8.5, for 60-80 hrs at 210- 260 rpm (revolutions per minute). Typically, culture no. PM0895117 seed is cultivated at 29 °C - 31 °C and a pH of about 6.5- 7.5, for 70-74 hrs at 230-250 rpm.

The production of the compound of formula (1) may be carried out by cultivating culture no. PM0895117 by fermentation in shake flasks at a temperature ranging from 27°C to 33°C and a pH of about 5.5 to 8.5, for 80-110 hrs at 200- 250 rpm. Typically, culture no. PM0895117 is cultivated at 29°C-31°C and pH 6.5- 7.5, for 90-100 hrs at 210- 230 rpm.

The production of the compound of formula (1) may be carried out by cultivating culture no. PM0895117 by fermentation in a fermenter at a temperature ranging from 27°C to 33°C and a pH of about 5.5 to 8.5, for 80-110 hrs at 40- 70 rpm and 400- 500 1pm (liters per minute) aeration. Typically, culture no. PM0895117 is cultivated at 28°C-31°C and pH 6.5- 7.5, for 90-100 hrs at 50- 60 rpm and 435- 465 1pm aeration.

The production of the compound of formula (1) can be carried out by cultivating culture no. PM0895117 in a suitable nutrient broth under conditions described herein, preferably under submerged aerobic conditions in shake flasks or fermenters. The progress of fermentation and production of the compound of formula (1) can be detected by high performance liquid chromatography (HPLC) and by measuring the bioactivity of the culture broth by testing against the cancer cell lines.

Fermentation is a process of growing microorganisms for the production of various chemical or pharmaceutical compounds. Microbes are normally incubated under specific conditions in the presence of nutrients. Whole broth is obtained after completing the process of fermentation. The whole broth is subjected to centrifugation which results in formation of cell mass and culture filtrate, which can be processed further by processes, described herein.

The compound of formula (1) present in the culture broth may be isolated using different extraction methods and chromatographic techniques.

Thus, the compound of formula (1) can be recovered from the culture filtrate by extraction with a water immiscible solvent such as petroleum ether, dichloromethane, chloroform, ethyl acetate, diethyl ether or butanol, or by hydrophobic interaction chromatography using polymeric resins such as "Diaion HP-20 ^® " (Mitsubishi Chemical Industries Limited, Japan), "Amberlite XAD ^® " (Rohm and Haas Industries, USA) or adsorption on activated charcoal. These techniques may be used repeatedly, alone or in combination.

The compound of formula (1) can be recovered from the cell mass by extraction with a water miscible solvent such as methanol, acetone, acetonitrile, n-propanol, or iso-propanol or with a water immiscible solvent such as petroleum ether, dichloromethane, chloroform, ethyl acetate or butanol. Alternatively, the whole broth may be extracted with a solvent selected from petroleum ether, dichlorome thane, chloroform, ethyl acetate, methanol, acetone, acetonitrile, n-propanol, iso-propanol, or butanol.

Typically, the compound of formula (1) is extracted from the whole broth using ethyl acetate. Concentration of the extract gives the active crude material.

The compound of formula (1) of the present invention can be recovered from the crude material by fractionation using any of the following techniques: normal phase chromatography (using alumina or silica gel as stationary phase; eluents such as petroleum ether, ethyl acetate, chloroform, dichloromethane, acetone, methanol, or combinations thereof; and if required, additions of amines such as triethylamine); reverse phase chromatography (using reverse phase silica gel such as dimethyloctadecylsilylsilica gel, (RP- 18) or dimethyloctylsilyl silica gel (RP-8) as stationary phase; and eluents such as water, buffers (for example, phosphate, acetate, citrate (pH 2-8)), and organic solvents (for example methanol, acetonitrile, acetone, tetrahydrofuran, or combinations of these solvents)); gel permeation chromatography (using resins such as Sephadex LH-20 ^® (Pharmacia Chemical Industries, Sweden), TSKgel ^® Toyopearl HW (TosoHaas, Tosoh Corporation, Japan) in solvents such as methanol, chloroform, acetone, ethyl acetate, or their combinations); or by counter-current chromatography (using a biphasic eluent system made up of two or more solvents such as water, methanol, ethanol, iso-propanol, n-propanol, tetrahydrofuran, acetone, acetonitrile, dichloromethane, chloroform, ethyl acetate, petroleum ether, benzene, and toluene). These techniques may be used repeatedly, alone or in combination. A typical method is chromatography over normal phase silica gel and reverse phase silica gel (RP-18).

As used herein, the term "isomer" is a general term used for all isomers of the compound of formula (1) or of the derivative thereof that differ only in the orientation of their atoms in space. The term isomer includes mirror image isomers (enantiomers), mixtures of mirror image isomers (racemates, racemic mixtures) and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereoisomers). The compound of the present invention may have asymmetric centers and occur as racemates, racemic mixtures, individual diastereoisomers, or enantiomers, or may exist as geometric isomers, with all isomeric forms of said compound being included in the present invention.

As used herein, the term "tautomer" refers to the coexistence of two (or more) compounds that differ from each other only in the position of one (or more) mobile atoms and in electron distribution, for example, keto-enol tautomers. The compound of formula (1), an isomer or a tautomer thereof can be converted into their pharmaceutically acceptable salts and derivatives, such as esters and ethers, which are all contemplated by the present invention.

The salts can be prepared by standard procedures known to one skilled in the art, for example, salts like sodium and potassium salts, can be prepared by treating the compound of formula (1), or an isomer, a tautomer or a derivative thereof, with a suitable sodium or potassium base, for example sodium hydroxide or potassium hydroxide. Similarly, salts like hydrochloride and sulphate salts, can be prepared by treating the compound of formula (1), or an isomer, a tautomer, and a derivative thereof, with a suitable acid, for example hydrochloric acid or sulphuric acid.

The derivative of the compound of formula (1) is preferably an ester or an ether of the compound of formula (1). The esters and ethers of the compound of formula (1) can be prepared by the methods given in the literature (Advanced Organic Chemistry, 1992, 4 ^th Edition, J. March, John Wiley & Sons) and known by one skilled in the art.

The compound of formula (1) or a pharmaceutically acceptable salt or a derivative thereof has anticancer activity against a wide range of cancer cells.

The compound of formula (1), or an isomer, a tautomer, a pharmaceutically acceptable salt or a derivative thereof, can be administered to a subject in need thereof as a pharmaceutical and in the form of a pharmaceutical composition. The compound of formula (1) or an isomer, a tautomer, a pharmaceutically acceptable salt or a derivative thereof, can be administered to a subject who is diagnosed having cancer.

Accordingly, the present invention also relates to the compound of formula (1), to the use of the compound of formula (1), or an isomer, a tautomer, a pharmaceutically acceptable salt or a derivative thereof for the manufacture of a medicament for the treatment of cancer.

The present invention further relates to a pharmaceutical composition comprising a therapeutically effective amount of the compound of formula (1) or an isomer or a tautomer or a pharmaceutically acceptable salt or a derivative such as an ester or ether thereof, and a pharmaceutically acceptable excipient or a carrier. The pharmaceutical composition is provided for use in the treatment of cancer. The therapeutically effective amount of the compound of formula (1), or its stereoisomer, or its tautomer or its pharmaceutically acceptable salt or its derivative as the active ingredient in the pharmaceutical preparations may range from about 0.01 mg to 1000 mg.

The compounds of the present invention find use in the treatment of cancers. Compounds of present invention are used to reduce, inhibit, or diminish the proliferation of tumor cells, and thereby assist in reducing the size of a tumor. Representative cancers that may be treated by such compounds include but are not limited to leukemia, lung cancer, brain tumors, non-Hodgkin's lymphoma, Hodgkin's disease, liver cancer, kidney cancer, bladder cancer, cancer of urinary tract, breast cancer, head and neck cancer, endometrial cancer, lymphoma, melanoma, cervical cancer, thyroid cancer, gastric cancer, germ cell tumor, cholangiocarcinoma, extracranial cancer, sarcoma, mesothelioma, malignant fibrous histiocytoma of bone, retinoblastoma, esophageal cancer, multiple myeloma, head and neck cancer, pancreatic cancer, ependymoma, neuroblastoma, skin cancer, ovarian cancer, recurrent ovarian cancer, prostate cancer, testicular cancer, colorectal cancer, lymphoproliferative disease, refractory multiple myeloma, resistant multiple myeloma or myeloproliferative disorder.

According to another embodiment of the present invention, the cancer is selected from acute lymphocytic leukemia, acute myeloid leukemia, adult acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, non-small-cell lung cancer, small-cell lung cancer, brain stem glioma, glioblastoma, astrocytoma including cerebellar astrocytoma and cerebral astrocytoma, visual pathway glioma, hypothalamic glioma, supratentorial primitive neuroectodermal, pineal tumors, medulloblastoma, primary central nervous system lymphoma, mantle cell lymphoma, Hodgkin's disease, hepatocellular carcinoma, renal cell carcinoma, Wilms' tumor, bladder cancer, cancer of urinary tract, Ewing's sarcoma family of tumors, osteosarcoma, rhabdomyosarcoma, soft tissue sarcomas, mesothelioma, breast cancer, endometrial cancer, oral cancer, melanoma, cervical cancer, thyroid cancer, gastric cancer, germ cell tumor, cholangiocarcinoma, extracranial cancer, malignant fibrous histiocytoma of bone, retinoblastoma, esophageal cancer, multiple myeloma, pancreatic cancer, ependymoma, neuroblastoma, skin cancer, ovarian cancer, recurrent ovarian cancer, prostate cancer, testicular cancer, colorectal cancer, lymphoproliferative disease, refractory multiple myeloma, resistant multiple myeloma or myeloproliferative disorder, among others.

According to an embodiment, the present invention provides a method for the treatment of cancer by administering to a mammal in need thereof a therapeutically effective amount of a compound of formula (1).

The compound of formula (1) or a pharmaceutically acceptable salt or a derivative thereof can be administered orally, nasally, topically, subcutaneously, intramuscularly, intravenously, or by other modes of administration. The method of administration which is suitable in a specific case depends on the type of cancer to be treated and on the stage of the cancer. Further, the method of administration can be optimized by a medical practitioner using methods known in the art.

As is customary, the dosage range which are suitable in a specific case depend on the type of cancer to be treated and on the state of the respective condition or disease, and can be optimized using methods known in the art. On average, the daily dose of active compound (the compound of formula (1)) in a patient is 0.05 mg to 200 mg per kg, typically 1 mg to 100 mg per kg.

Pharmaceutical compositions which contain compound of formula (1) or an isomer or a tautomer, a pharmaceutically acceptable salt or a derivative thereof, can be prepared by mixing the compound of formula (1) with one or more pharmacologically tolerated auxiliaries and/or excipients such as, wetting agents, solubilisers such as surfactants, vehicles, tonicity agents, fillers, colorants, masking flavors, lubricants, disintegrants, diluents, binders, plasticizers, emulsifiers, ointment bases, emollients, thickening agents, polymers, lipids, oils, cosolvents, complexation agents, or buffer substances, and converting the mixture into a suitable pharmaceutical form such as, for example, tablets, coated tablets, capsules, granules, powders, creams, ointments, gels, syrup, emulsions, suspensions, or solutions suitable for parenteral administration.

Examples of auxiliaries and/or excipients that may be mentioned for use in preparation of pharmaceutical composition are cremophor, poloxamer, benzalkonium chloride, sodium lauryl sulphate, dextrose, glycerin, magnesium stearate, polyethylene glycol, starch, dextrin, lactose, cellulose, carboxymethylcellulose sodium, talc, agar-agar, mineral oil, animal oil, vegtetable oil, organic and mineral waxes, paraffin, gels, propylene glycol, benzyl alcohol, dimethylacetamide, ethanol, polyglycols, Tween 80, solutol HS 15, and water. It is also possible to administer the compound of formula (1) as such, without vehicles or diluents, in a suitable form, for example, in capsules.

In one embodiment of the present invention, the pharmaceutical compositions which contain compound of formula (1) or an isomer or a tautomer, a pharmaceutically acceptable salt or a derivative thereof, may be used in combination with one or more anticancer agents such as asparaginase, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, doxorubicin, epirubicin, etoposide, fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide, leucovorin, lomustine, mechlorethamine, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone, prednisone, procarbazine, streptozocin, tamoxifen, thioguanine, vinblastine, vincristine, vindesine, aminoglutethimide, 5-azacytidine, cladribine, busulfan, diethylstilbestrol, 2',2'-difluorodeoxycytidine, docetaxel, erythro-9-(2-hydroxy-3- nonyl) adenine, ethinyl estradiol, 5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, fludarabine phosphate, fluoxymesterone, flutamide, hydroxyprogesterone caproate, idarubicin, interferon, medroxyprogesterone acetate, megestrol acetate, melphalan, mitotane, paclitaxel, pentostatin, N-phosphonoacetyl-L-aspartate (PALA), plicamycin, teniposide, testosterone propionate, thiotepa, trimethylmelamine, uridine, vinorelbine, alsterpaullone, butyrolactone I, 2-(2-hydroxyethylamino)-6-(3-chloroanilino)-9- isopropylpurine, indirubin-3'-monoxime, kenpaullone, olomoucine, iso-olomoucine, N ⁹ - isopropyl-olomoucine, purvalanol A, roscovitine, (S)-isomer roscovitine and WHI-P180 [4- (3 ' -hydroxyphenyl)amino-6,7-dimethoxyquinazoline] .

The following are provided as illustrative examples of the present invention and do not limit the scope thereof.

EXAMPLES

The following terms/symbol/abbreviations/chemical formulae are employed in the examples:

Example 1

Isolation of culture no. PM0895117 from marine source

a) Composition of the isolation medium:

Modified Actinomycetes Isolation Agar medium: sodium caseinate 0.2%, L- asparagine 0.01%, sodium propionate 0.4%, dipotassium hydrogen phosphate 0.05%, magnesium sulphate 0.01%, ferrous sulphate 0.0001%, glucose 1% and agar 1.5%; demineralized water ; pH 8.1. Composition of the artificial seawater (ASW): sodium chloride 24.6 g/L, potassium chloride 0.67 g/L, calcium chloride 1.36 g/L, magnesium sulphate 6.29 g/L, magnesium chloride 4.66 g/L, sodium bicarbonate 0.18 g/L; demineralized water 1 L.

b) Procedure:

Deep sea sediment sample was collected from the offshore region of Mumbai,

Maharashtra, India, and stored at -20°C throughout the journey to Piramal Healthcare Limited (Formerly NCE Research Division of PiramalLife Sciences Limited), Goregaon, Mumbai, India.

Molecular approach was implemented for the isolation of uncultured marine actinomycetes from the collected sediment sample. For this, genomic DNA was isolated from the sediment samples and amplified for the presence of polyketide synthase (PKS), non- ribosomal peptide synthetases (NRPS) and glycosyl transferase biosynthetic pathways by performing PCR. Culture of interest was then isolated from the sediment sample having Glycosyl transferase pathway.

The sample was stored at -20°C to -22°C and later thawed to room temperature

(25±2°C) for isolation of the microbes. The soil sample (~2 g) was suspended in 25 ml of sterile artificial sea water (ASW) in autoclaved mortar and pestle and crushed thoroughly. 1 ml of this suspension was transferred in to a test tube and vortexed for 30 seconds. Serial dilutions up to 10 ^"5 were prepared in sterile ASW. 100 of 10 ^"5 dilution was surface spread on plate containing modified Actinomycetes Isolation agar medium. The plate was incubated at room temperature (25±2°C) till colonies were observed. After incubation for 20-22 days, the colony which appeared on this medium was streaked on petriplates containing modified actinomycetes isolation agar, as mentioned above. The isolate was purified and was provided culture ID no. PM0895117. The culture no. PM0895117 was thus isolated from amongst the growing microorganisms as single isolate.

Example 2

Purification of culture no. PM0895117

a) Composition of the purification medium:

Modified Actinomycetes Isolation Agar medium: sodium caseinate 0.2%, L- asparagine 0.01%, sodium propionate 0.4%, dipotassium hydrogen phosphate 0.05%, magnesium sulphate 0.01%, ferrous sulphate 0.0001%, glucose 1% and agar 1.5%; demineralized water; pH 8.1. b) Procedure:

The culture no. PM0895117 was streaked on a petriplate containing modified Actinomycetes Isolation Agar. The petriplate was incubated for ten days at 27°C. One of the isolated colonies from the petriplate was transferred to slants containing ISP4 Agar medium (HiMedia, India). The slants were incubated for ten days at 27°C.

Example 3

Maintenance of producer strain - culture no. PM0895117

This particular strain was maintained on slants containing ISP4 media (HiMedia, India). The media was dissolved thoroughly by heating; the resultant solution was distributed in test tubes and sterilized at 121°C for 30 minutes. The test tubes were cooled and allowed to solidify in a slanting position. The agar slants were streaked with well grown culture no. PM0895117, by a wire loop and incubated at 27°C to 29°C until a significant growth was observed. The well-grown cultures were stored in the refrigerator at 4°C to 8°C.

Example 4

Fermentation of the culture no. PM0895117 in shake flasks

a) Composition of the seed medium:

MBA8 medium: calcium carbonate 0.2%, sodium chloride 0.5%, corn steep liquor 0.5%, peptone 0.75%, glucose 1.5% and yeast extract 0.75%; demineralized water to make up the volume up to 100 ml; pH-7.0.

b) Procedure:

50 ml of the seed medium as prepared in Example 4 (a) was distributed in 500 ml capacity Erlenmeyer flasks and autoclaved at 121°C for 30 minutes. The flasks were cooled to room temperature (25±2°C) and each flask was inoculated with a loopful of the well- grown producing strain (culture no. PM895117) on the slant and shaken on a rotary shaker for 72 hrs at 230 rpm to 250 rpm at 30°C (±1°C) to give seed culture.

c) Composition of the production medium:

MBA8 medium: calcium carbonate 0.2%, sodium chloride 0.5%, corn steep liquor 0.5%, peptone 0.75%, glucose 1.5% and yeast extract 0.75%; demineralized water to make up the volume up to 1 L; pH-7.0. d) Procedure:

100 ml of the above production media was distributed in 500 ml capacity Erlenmeyer flasks and autoclaved at 121°C for 30 minutes, cooled to 29°C to 30°C and seeded with 3% (v/v) of the seed culture mentioned in Example 4 (b).

e) Fermentation parameters:

The production flasks were incubated on shaker at 30°C and 220 rpm for 96 hrs. The production flasks were harvested (harvest pH: 7.0 to 8.0) and the whole broth from each media flask was extracted with equal volume of solvent mixture (1 :9 methanol: ethyl acetate). These flasks were kept at room temperature for 4-6 hrs for extraction followed by separation of the supernatant. The supernatant was used for testing for anti cancer activity.

Example 5

Preparation of the seed culture in shake flasks for fermenter batch

a) Composition of the seed medium 274(1):

glucose 15g, corn steep liquor 5g, peptone 7.5g, yeast extract 7.5g, calcium carbonate

2g, sodium chloride 5g, demineralized water 1.0 L, pH 6.5-7.5 (before sterilization).

b) Procedure:

200 ml of the above medium was distributed in 1000 ml Erlenmeyer flasks and autoclaved at 121 °C for 30 minutes. The flasks were cooled to room temperature and each flask was inoculated with a loopful of the well-grown producing strain (PM0895117) on the slant and shaken on a rotary shaker for 70-74 hrs at 230-250 rpm at 29°C -30°C to obtain the seed culture.

Example 6

Cultivation of the culture no. PM0895117 in fermenter

a) Composition of the production medium (135SS):

soluble starch 25g, glucose lOg, yeast extract 2g, soyabean meal 20g, sodium chloride

5g, calcium carbonate 3g, magnesium sulphate lg, trace salt solution 1 ml/L (composition of the trace salt solution: copper sulphate 7g, ferrous sulphate lg, manganese chloride 8g, zinc sulphate 2g and demineralized water 1.0 L), demineralized water 1.0 L, pH 7.0 (before sterilization). b) Procedure:

450 L of the production medium in 1KL fermenter along with 180 ml of Desmophen as an antifoaming agent was sterilized in situ for 30 minutes at 121°C, cooled to 29-30°C and seeded with 8-10 L of the seed culture as obtained in Example 5 (b).

c) Fermentation parameters:

The production fermenter was incubated at 29°C -30°C, agitated at 54 rpm and aeration of 450 1pm for 96 hrs. The harvest pH of the culture broth was 6.5-7.5. The production of the compound of formula (1) in the fermentation broth was determined by HPLC and by testing for anti cancer activity.

Example 7

Isolation and purification of the compound of formula (1)

The harvested whole broth (450 L) from the Example 6 was extracted using ethyl acetate (450 L). The organic layer was separated using disc stack separator (Alfa Laval Sweden), model no LAPX404) and concentrated to obtain the crude extract (600 g).

The crude extract was processed by column chromatography (silica gel, solvent: methanol/chloroform). The compound of formula (1) eluted with 5% methanol to 10% methanol in chloroform, which was concentrated to obtain the semipure compound (130 g). Fractions were monitored by TLC [Silica; Chloroform: Methanol (90: 10); Detection: UV 254 nm; Rf: 0.4]

Further purification was carried out by repeated reversed phase preparative HPLC. Preparative HPLC conditions are as follows:

Column : Tekhroma Tracer C-18 (Teknokroma Spain)

(10μ, 51x250 mm)

Eluent Water (A) : Methanol (B) gradient

Row rate 120 ml/min

Detection (UV) 310 nm and 254 nm; Retention time: 124 min

Time (mins) % A % B

0.01 - 10 98 2

12 - 25 90 10

30 - 50 70 30

60 - 115 50 50

120 - 130 40 60 135 - 145 10 90

150 - 160 98 2

Purity of fractions was checked by analytical HPLC. The eluates containing the compound of formula (1) were pooled and concentrated under reduced pressure to remove the solvent to obtain compound of formula (1) (200 mg). Physical & Spectral properties of the compound of formula (1) are given in Table 1.

Analytical HPLC conditions are as follows:

Column : ASCENTIS CI 8, Sigma- Aldrich (2.7μ, 4.6 x 50 mm)

Solvent system : acetonitrile (A %): 0.01M NH ₄ OAc in water + 0.5% TEA, pH 5 with AcOH (B %) gradient

Row rate 1 ml/min

Detection (UV) 320 nm

Retention time 1.5 min

Table 1 : Physical & Spectral properties of the compound of formula (1)

Appearance Off -White powder

Melting point 131-135°C (decomposes)

Solubility Soluble in methanol, acetonitrile, chloroform,

ethyl acetate and water

MS [HR-ESI(+)MS)] m/z 499.2213 (M+H)

Molecular weight 498.2

Molecular formula C ₂₂ H ₃₄ N ₄ 0 ₇ S

IR (KBr) 3314, 2927, 1653, 1577, 1490, 1316, 1268

and 1039 cm ^"1

UV (λ^) 316 nm

'H NMR δ 8.1 (d, 1H), 7.8 (d, 1H), 7.3 (d, 1H), 6.2 (d,

(500 MHz, DMSO-d ₆ ) 1H), 5.7(d, 1H), 4.8 (d, 1H), 4.7 (bs, 1H), 4.5 (bs, 1H), 3.7 (dd, 1H), 3.6 (dd, 2H), 3.2

(bs, 2H), 2.4 (s, 9H), 2.3 (dd, 1H), 2.0 (m, 1H), 1.9 (dd, 1H), 1.8 (dd, 1H), 1.7 (dd, 1H), 1.5 (dd, 1H), 1.2 (d, 3H) and 1.1 (d, 3H) (bs refers to broad singlet) (as given in Figure 1) l ⁱ C NMR δ 164.27, 163.47, 154.43, 147.03, 145.61,

(75 MHz, DMSO-d ₆ ) 116.53, 96.50, 95.08, 82.51, 76.79, 73.41(2),

70.47, 68.70, 66.19, 42.61(2), 29.52, 26.98, 19.60, 18.90 and 14.56 (as given in Figure 2)

Biological evaluation of the compound of formula (1):

In vitro assays

The assays were designed as described in the reference BMC Cancer, 10, 610, 2010, 1-11, the disclosure of which is incorporated by reference for the teaching of the assay.

Example 8

Monolayer assay

Step 1

Maintenance of the cell lines:

Human tumour cell lines Panc-1 (Pancreatic Cancer), HCT 116 (Colorectal Cancer), ACHN (Renal Cell Carcinoma), Calu-1 (Lung Carcinoma) were grown in Minimal Essential Media with Eagle's Basal Salts (MEM - EBS) obtained from AMIMED (BioConcept - Switzerland). Tumour cell lines H460 (Non-small cell lung cancer) were cultured in RPMI 1640 (AMIMED, BioConept, Switzerland). All tumour cell lines were supplemented with 10% Foetal Bovine Serum (FBS) (GIBCO), 1 % Penicillin/Streptomycin (Sigma) and 1% Anti-Anti (GIBCO) and grown in T-175 tissue culture flasks (Nunc). MCF-IOA non- tumourigenic cell line was cultured in Mammary Epithelial Basal Medium (MEBM) with all standard additions (Lonza, Catalog. No. CC-3150). All cells were grown at 37°C with 5% CO ₂ incubator. Cells were passaged at 80 - 90% confluence. Adherent cells were trypsinised using Trypsin-EDTA (Sigma) and maintained. Step 2

Sample preparation:

Compound of formula (1) was dissolved in DMSO to give a required stock solution of 20 mM. Eight different concentrations of the compound of formula (1) were prepared by serial dilution of the stock solution finally resulting in a 200-fold higher concentration (compared to the test concentration). Compound of formula (1) was tested at the concentrations of 30 μΜ, 10 μΜ, 3 μΜ, 1 μΜ, 0.3 μΜ, 0.1 μΜ, 0.03 μΜ and 0.01 μΜ. Each concentration was evaluated in triplicate.

Step 3

Assay

Method for determination of IC5 ₀ of compound of formula (1):

a) Different cancer/normal cells were seeded at a density of 3000 cells/200 μΕ well, in a tissue culture grade 96 well plate and allowed them to recover for 24 hrs in a humidified 5 ± 0.2% C0 ₂ incubator at 37 ± 0.5 °C.

b) After 24 hrs, 1 μΕ of 200 X (200 times higher than required concentration is denoted as 200 X) compound of formula (1) dissolved in DMSO was added to the above tissue culture plate seeded with cancer/normal cells. The final concentration of DMSO was 0.5% in wells. DMSO was used as a vehicle control.

c) After 72 hrs the plates were removed from CO ₂ incubator and 5 μΕ of CCK-8 (Dojindo Molecular Technologies, Inc. USA, Catalog. No. CK04-20) per well was added. d) The plate was then placed at 37 °C for 2 hrs.

e) The absorbance was recorded at 450 nm.

f) The percent cytotoxicity was calculated using the following formula:

(Reading of Vehicle Control - Reading of Treated cells)

Percent Cytotoxicity = X 100

Reading of Vehicle contr

Results are given in Table 2.

Table 2: IC5 ₀ values of compound of formula (1) in different cell lines at 72 hrs

Sr. No. Human tumor cell lines IC50 in μΜ

1 ACHN 2.23

2 Panc-1 2.69

3 Calu-1 3.72

4 H460 3.8 5 HCT 116 3.5

6 MCF-IOA 14.32

Conclusion: The result revealed that, the compound of formula (1) showed IC5 ₀ in the range of 2.23 μΜ to 3.8 μΜ in different cancer cells; while in normal cells (MCF-IOA) it showed an IC5 ₀ of 14.32 μΜ. This data shows the selectivity of this compound towards highly proliferating cancer cells.

Example 9

Assay

Detection of FITC AnnexinV apoptosis:

The annexinV assay, a classical technique for detecting apoptosis, is the most commonly used method for detecting apoptosis by flow cytometry. One of the earliest features of apoptosis is the translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane, thereby exposing phosphatidylserine to the external environment. AnnexinV binds to phosphatidylserine exposed on the cell surface and identifies cells at an earlier stage of apoptosis.

a) Panc-1 cells [referred to in step 1 of example 8] were seeded at a density of 0.5 x 10 ⁶ cells/2000 μΐ _^ well, in a tissue culture grade 96 well plate and allowed them to recover for 24 hrs in a humidified 5 ± 0.2% C0 ₂ incubator at 37 ± 0.5 °C.

b) After 24 hrs, 5 μΐ _^ of 200 X (200 times higher than required concentration is represented as 200 X) compound of formula (1) dissolved in DMSO [referred to in step 2 of example 8] was added to above tissue culture plate seeded with Panc-1 cells. The final test concentration of compound of formula (1) was 3 μΜ and 5 μΜ. The final concentration of DMSO in wells was 0.5%.

c) After 48 hrs the plates were removed from CO ₂ incubator and stained for FITC AnnexinV using Apoptosis Detection Kit as per the manufacture's instruction. (Catalogue number: 556547, BD biosciences, USA).

d) The processed cells were analyzed in BD flow cytometer for annexin V detection. The population present in Ml and M2 were determined using FACS analysis.

Results: The vehicle control cells showed 2.31 % annexinV positive cells (M2), while cells exposed to compound of formula (1) with 3 μΜ and 5 μΜ concentrations for 48 hrs showed 32.14 % and 59.62 % cells positive for annexinV staining (M2) respectively (Figure 3). Conclusion: This data indicates that, compound of formula (1) induces cell death through apoptosis in Panc-1 cancer cells.

Example 10

Assay

Study of Protein expression by High content screening:

Protein expressions of p53 (Santa cruz biotechnology, Inc. USA, Catelog. No. sc- 65334), phospho C-JUN (Santa cruz biotechnology, Inc. USA, Catelog. No. sc- 101723) and caspase 3 (Santa cruz biotechnology, Inc. USA, Catelog. No. sc-22171) was carried out in Panc-1 cells [referred to in step 1 of example 8] by using Cellomics high content imaging. Briefly, 1 X 10 ⁴ cells were seeded in 96 well tissue culture grade black plate (Nunc brand, Thermofisher Scientific Inc. USA, Catalog. No. 165305) with transparent bottom and allowed them to adhere for 24 hrs. After 24 hrs the media was replaced with fresh media and the cells were treated with 5 μΜ, 3 μΜ and 1 μΜ concentrations of compound of formula (1) [referred to in step 2, of example 8] and incubated for 12 hrs and 24 hrs. After respective time point, cells were fixed with 3.7% formaldehyde (Sigma St. Louis, MO) for 10 minutes at RT, followed by permeabilization with 0.15% Triton X-100 (Sigma St. Louis, MO) for 10 minutes. Then the cells were blocked with 5% Bovine Serum Albumin for 2 hrs. After blocking step specific primary antibody were added for lhr and primary antibodies of different protein were localized by secondary antibody labeled with Dylight 548 (red) (Thermo Scientific, USA). Following Secondary incubation, the nucleus was stained with Hoechst 3342 (blue) (Sigma, USA). Immunofluorescence was determined by scanning the plates on Cellomics Array Scan® VTI HCS Reader (Thermo- Fisher Scientific Inc., Waltham, MA). All the data points were analyzed using the Compartmental Analysis bio- algorithm of Cellomics and quantitative data were expressed as % activation in comparison to the un-treated cells. 1000 cells were counted for each replicate well and the results were presented as an average ± SE.

The results are summarized in the Table 3. Table 3: Effect of compound of formula (1) on p53, pC-JUN and cleaved caspase3 expression in Pane

Conclusion: The results revealed that compound of formula (1) significantly activates p53, phospho-c-JUN and cleaved caspase3 expression levels thereby causing apoptosis in Panc-1 cells.