Title of Invention

COMPOUNDS OF BIS - BROMO BENZOIC ACID HYDRAZONE AND PROCESS OR PREPARATION THEREOF,

Abstract

Steroidal derivatives of immense potential as drug molecules have been prepared on the basis tigogenin-sapogenin from the plant Yucca gloriosa cultivated in Georgia Preparation methods to get Bis-m-bromobenzoic acid hydrazone of 5 a-androstane-3, 17-dione and Bis-m-nitrobenzoic acid hydrazone of 5 a-androstane-3, 17-dione are given. The steroids Bis-m-bromobenzoic acid hydrazone of 5 a-androstane-3, 17-dione and Bis-m-nitrobenzoic acid hydrazone of 5 a-androstane-3, 17-dione have shown promising anti TB, anticancer and anti HIV activities in vitro.

Full Text

FORM 2 The Patent Act 1970, (39 of 1970) & The Patent rule 2003 COMPLETE SPECIFICATION (See Section 10 and Rule 13) 1. TITLE OF THE INVENTION Medicinal applications of compounds synthesized on the basis of steroidal Sapogenin-tigogenin from the plant Yucca gloriosa cultivated in Georgia 2.APPLICANT (S) (a) NAME: (b) NATIONALITY: (c) ADDRESS: Shelar Ashok Ranganath Indian Dr.Ashok Shelar (Ph.D) Plot No.8,Unit No.1 Ambai Defence Colony, Kolhapur-416008 Maharashtra State(lndia) 2. COMPLETE SPECIFICATION DESCRIPTION The following specification particularly describes the invention and the manner in which it is to be performed. FIELD OF INVENTION In recent years chemical research in the steroid field has gone hand in hand with chemical investigation to develop a wide variety of steroid derivatives,not found in nature,which have specific physiological action and medical application. Small variations in the structure of steroid molecules frequently results in wide variations in the physiological activity and help in search of new drugs with enhanced potency, broader applicability,lower toxicity and fewer undesirable side effects. Steroid therapy is becoming increasingly important in modern medicine,and runs the gamut from preventing abortion to arresting certain cancers,from controlling pregnancy to treating arthritis,and from correcting hormonal abnormality to to treating dermatitis.Dexamethasone,a fluorine containing steroid,is used in treating inflammation,the acetylinic derivative of 19-norethisterone exerts control over the menstrual cycle and used as oral contraceptive,and the triketone predinisone finds general application in the field of cortisone therapy. Steroids include a wide variety of natural products containing the cyclo pentano perhydrophenanthrene ring system present in cholesterol. STATEMENT OF INVENTION Tuberculosis The significant increase in the incidence and morbidity from tuberculosis since the start of the 1990 s prompted the World Health Organization t o regard the disease as a worldwide danger. One of the factors leading to the increased incidence is the development of resistance in the Mycobacterium tuberculosis. One in every 20 new cases of TB worldwide is now resistant to two or more drugs. Half a million new cases of MDR-TB and 40,000 new cases of XDR-TB are emerging each year across the globe. 110,000 people with MDR-TB die every year from the disease as per the data collected between 2002-2006 on TB patients in 81 countries. There fore the search for new effective antituberculosis compounds has become urgent. Cancer Cancer chemotherapy uses compounds that can differentiate to some degree between normal tissue cells and cancer cells. The decision to use a certain anti neoplastic drug depends on type and location of tumor. Therefore it is imperative to keep searching for new compounds. HIV HTV infection in humans is now a pandemic. As of January 2006,the joint United Nations Programme on HIV/AIDS (UNAIDS) and the World Health Organization ( WHO ) estimate that AIDS has killed more than 25 million people since it was first recognized on December 1,1981 making it one of the most destructive pandemics in recorded history in 2005 alone, AIDS claimed an estimated 2.4-3.3 million lives. About 0.6% of worlds living population is infected with HIV. Antiretroviral reduces both mortality and morbidity of HIV infection, but access to antiretroviral medication is not available in all countries. 4 PRIOR ART OF THE INVENTION A number of steroidal compounds with NH2, N-alkyl, N-alkyloxy, N,N-dialkyl etc. substituents in the C-17 position that exhibit a broad spectrum of biological activity have been synthesized based on tigogenin. Synthesis of 5 a-androstan-3 B, 17-B-diols were reported as potential anticancer compounds. Novel steroidal isonicotin hydrazones and thiosemicarbazones were reported as potential anti T.B. agents SOME COPYRIGHT COMPOUNDS (A) Formula: C20H31 N3 S CA Index Name: Androst-2-en-17-one(aminothioxomethyl)hydrazone Registry No. 487039-91-8 Copyright 2007 American Chemical Society (B) Formula: C26 H36 03 S CA Index Name: Androst-2-en-17-ol, 4-methylbenzenesulphonate Registry No. 913816-27-0 Copyright 2007 American Chemical Society (C)Formula: C19H30O CA Index Name: Androst-2-en-17-ol Registry No. 6699-64-5 Copyright 2007 ACS (C) Formula: C19 H33 NO CA Index Name: 5 a-androstane-3-ol,17-amino-, Registry No. 32911-76-5 Copyright 2007 ACS REFERENCES (1) Camoutis C.,Trafalis D.Int.New Drugs 2003 21 47. (2) Amiranashvili L.,Merlani M.,Menshova N.,Suvorov N.,Bull.Georg. Acad.Sci.1998 158(2)273 (3) Merlani M.I.,Kemertelidze E.P.,Papadopoulos K.,Menshova N.I., Bioorg. Khim.2004 30 552 [Engl.transl.Russ.J.Bioorg.Chem.2004 30 000]. 6 DESCRIPTION OF INVENTION IN DETAIL The Institute of Pharmaceutical Chemistry of the Georgia proposed the steroidal sapogenin tigogenin as starting material for synthesizing hormonal preparations of the 5 a-series. Tigogenin is isolated froin the plant Yucca gloriosa, which is cultivated in Georgia [1]. We developed a synthetic scheme for acetate eoiandrosterone based on tigogenin (1) that involves conversion of 1 to pregnenolone acetate (2), of 2 to epiandrosterone acetate. For conversion of 1 to 2, we chose oxidative dehydration using TiC14 as catalyst. The yield of 2 from 1 was 69.5% [2]. Compound 2 was converted to epiandrosterone acetate using the Schmidt-Thome method [3], according to which pregnenolone acetate oxime (3) underwent Beckmann rearrangement by POCl3 in pyridine. Acid hydrolysis of intermediate 17-acetylamino derivative 4 gave epiandrosterone acetate (5) in 65% yield [4]. 3p-Acetoxy-5a-pregn-16-en-20-One (2). A mixture of 1 (50 g, 120.0 mmol), (CH3CO)20 (150 mL), and C5H5N (10 mL) was boiled for 1 h, cooled to 100°C, stirred, treated with TiCl4 (2.5 g, 13.16 mmol) in (CH3CO)20 (2.5 mL), boiled an additional 2 h, cooled to 40°C, treated gradually with CH3COONa (10 g) dissolved in water (25 mL), stirred 20 min, cooled to room temperature, poured into CH3COCH3 (220 mL) and CH3COOH (220 mL), oxidized by addition of Cr03 (15 g) in water (7.5 mL) at 15-18°C, stirred an additional hour, treated with isopropanol (7.5 mL), gradually heated to distill off acetone and reach a temperature of 115-117°C, boiled for 1.5 h, cooled to room temperature, and treated with water (425 mL). The resulting precipitate was filtered off, washed with water, and recrystaUized from methanol:acetone (3:1) to afford 2 (29 g, 69.5%), mp 158-162°C, lit. mp 158-162°C [5]. A mixture of 2 (2.5 g, 6.97 mmo)), NH2OH-HC} 7 (0.55 g, 7.91 mmol), and dry C5H5N (12 mL) was heated at 65-68°C for 2 h, cooled to room temperature, treated with water (45 mL), and stirred for 30 min. The resulting precipitate was filtered off and washed with water to afford 3 (2.5 g, 98.07%), mp 196-198°C, lit. mp 195.5-98.5°C [4]. 3P-Acetoxy-5oc-androstan-17-one (5). A mixture of 3 (1 g, 2.67 mmol), dry C5H5N (3.2 mL), and dry CH3COCH3 (3.2 mL) at 18-20°C was treated with POC13 (1.2 mL), stirred for 30 min, cooled to -5°Cα, treated with dilute HC1 (1:1 with water, 28 mL), stirred for 30 min, and treated with water until neutral. The resulting precipitate was filtered off and washed with water to afford crude product (0.83 g) that was chromatographed over a column of silica gel (L 100-160) with elution by low-boiling petroleum ether:ether (20:1) to afford 5 (0.58 g, 65%), mp 111-113°C, lit. mp 111-13°C [4]. 3p-hydroxy- -androstan-17-one (6). A mixture of 5 (1 g, 3.00mmol), NaOH 0.12g (3.44 mmol) in methanol was refluxed for 10 min, cooled to room temperature and treated with water. The resulting precipitate was filtered off and washed with water to afford crude product 6 (0.82 g, 95%) 5a-androstan-3,17-dione (7). To the mixture of 6 (5g, 17.2 mmol) and 75 ml acetone at room temperature 1.5 ml of Jones reagent (CrO3, H2SO4, H2O) was added by drops. After the reaction was completed, NaOH was added, liquid phase was separated and then 90 ml water was added. The resulting precipitate was filtered off to afford product 7 (4.72 g, 94%). M.p. 134-137°C. s AcO AcO 6 7 9 References 1.E. P. Kemertelidze and T. A. Pkheidze, Khim-Farm. Zh., 6, 44 (1972). 2.L. K. Kavtaradze, R. I. Dabrundashvili, N. I. Men'shova, N. A. Korzinkina, and E. P. Kemertelidze, Soobshch. Akad. Nauk Gruz. SSR, 132, No. 3, 537 (1988). 3.J. Schmidt-Thome, Chem, Ber., 88, 895 (1955). 4.N. I. Men'shova, N. A. Korzinkina, E. P. Kemertelidze, N. Sh. Nadaraia, M. G. Davitishvili, L. I. Lishcheta, and V. S. Grosheva, Sb. Nauchn. Tr. VNIKhFi im. S. Ordzhonikidze, 10, 83 (1982). 5a-androstan-3,17-dione (7 ) + m-bromobenzoic acid hydrazide gives compound Formula(I) Formula (I) Bis-m-bromobenzoic acid hydrazone of 5a-androstane-3,17 - dione PREPARATION OF NEW STEROIDAL DERIVATIVES Bis-m-brombenzoic acid hydrazone of 5a-androstane-3,17-dione. A mixture of 5a- androstane-3,17-dione (1 g, 3.46 mmol), m-brombenzoic acid hydrazide (1.49 g, 6.93 mmol) and acetic acid (1 ml) in ethanol (10 ml) was refluxed for 2 h and cooled to room temperature. The precipitated solid was filtered, washed with water, and recrystallized from ethanol to give desired hydrazone; yield 93%; mp 165-167°C Structural Formula (I) IR (KBr, cm"1): 3475 (NH), 1700 (NHC=0), 1643 (C=N), 1550 (aromatic ring), 'H NMR (500MHZ, CDC13), 8 : 0.83 (3H, s, C18-H3), 0.90 (3H, s, 19-CH3), 7.64-7.89 (10H, m, aromatic protons), 8.17 (1H, br s, NH), 8.31 (1H, br s, NH) 13C NMR(500MHz, CDCI3), 8 :11,11(CH3), 16.95(CH3), 122.91-150.1 l(aromatic ring) 161.21 (C=N), 162,22 (C=N), 171.22(C=0) Bis-m-nitrobenzoic acid hydrazone of 5a-androstane-3,17-dione. A mixture of 5a- androstane-3,17-dione (1 g, 3.46 mmol), m-nitrobenzoic acid hydrazide (1.25 g, 6.93 mmol) and acetic acid (1 ml) in ethanol (10 ml) was refluxed for 2 h and cooled to room temperature. The precipitated solid was filtered, washed with water, and recrystallized from ethanol to give desired hydrazone; yield 90%; mp 202.-205°C Structural Formula (II) IR (KBr, cm-1): 3484 (NH), 1700 (NHC=0), 1639 (C=N), 1528 (aromatic ring), 'H NMR (500MHz, CDC13), 8 : 0.83 (3H, s, C18-H3), 0.90 (3H, s, 19-CH3), 7.64-7.89 (10H, m, aromatic protons), 8.17 (1H, br s, NH), 8.31 (1H, br s, NH) 13C NMR(500MHz, CDC13), 8 :11,23(CH3), 17.26 CH3), 122.91-147.1 l(aromatic ring), 161.21 (C=N), 162,22 (C=N), 176.22(C=0) BIOLOGICAL ACTIVITY PROFILE FOR THE COMPOUNDS: Following Methods have been employed for screening for biological activities. Antimicrobial activities were determined by the Microbroth Dilution Method ( NCCLS ) Anti Tubercular activities were determined against the H 37 Rv strain of M.tuberculosis Cyto toxic effects was evaluated using the human HeLa cell line srocured from the National Center For Cell Science,Pune Anti retro viral tests for evaluation of anti HIV activity was performed by the Method of Viral Load by RT-PCR The details of procedure are given in the following pages Anti T.B. tests M.tuberculosis maintained in the laboratory.Middle brook 7H9 medium was prepared in bottles and starilised by autoclaving.ADC growthsupplement containing bovine albumin fraction V,dextrose and catalase was added to each bottle with sterile precautions. A sterile suspension(10 mg/ml) of each compound to be tested was prepared using appropriate solvents under sterile conditions.Each compound was tested at a concentration say of 5 micro grams/ml the required amount was added to each bottle of middle brook medium.M.tuberculosis culture suspension was added at a concentration of 10 raise to 5 organisms per ml and the media were incubated at 37 degree centigrade in a Humidified chamber for at least 4 weeks. Controls of antimycobacterial agents i.e. Ciprofloxacin 5 micro grams/ml,streptomycin 7.5 micro grams/ml and pyrazinamide 7.5s mg/ml were set up with each batch. A medium control and growth control tubes also were includeEach bottle was examined periodically to observe for growth or contamination if any at the end of four weeks a smear was prepared from each bottle showing turbidity stained with Z-N stain to confirm the presence of M.tuberculosis.Those media with the presence of organisms were considered as resistant to the compound and those without any bacteria were deemed as sensitive. CONCLUSIONS: The compounds of Formula (I) and Formula (II) have shown excellent activity against H37Rv strain of M.Tuberculosis.The lab results were further confirmed by field tests on actual samples from patients. Anti Cancer Tests In vitro evaluation of cytotoxic effects on human tumour cell lines: Cell culture :The HeLa cell line(procured from National Centre For Cell Science Pune University,India)was maintained in the labs by serial sub cultures. The cell line was maintained as mono layers in 75 cm square culture flasks in Eagles minimum essential medium supplemented with 10% fetal calf serum 2mg of gentamicin, 100 Unit/ml of penicillin and 100 micro gram/ml of streptomycin. The cells were grown in humidified 37 degree incubator with a regulated supply of 5% carbon dioxide. Medium was changed every 5-6 days. On the day of the experiment cells were washed with phosphate buffered saline and were detached from the surface of the flask by addition of 0.25% trypsin in EDTA buffer.The cells were then plated at a density of 5X10 to the power of 3 cells per well in 96 well micro plates containing 250 micro litre of cell culture medium per well and allowed to adhere over night. Stock solutions(10 micro gram per ml) of the compounds to be tested were freshly prepared just before use under sterile conditions.Each compound was tested in two concentration 10 and 100 micro grams per ml.Requisite amount of the compound was added to each well and were allowed to act on tumour cells for a period of 72 hours. A cell control was also set up with each compound for comparison. At the end of the incubation period, each well was studied under 40X power inverted microscope and analyzed for cytotoxic effects of each concentration of the compound on tumour cell lines. CONCLUSIONS: The compounds of Formula (I) and Formula (IT) have shown excellent anti cancer activity.The Photographs are records of the cell growth inhibition. WE CLAIM 1. A process for the preparation of Bis-m-bromo benzoic acid hydrazone of 5 a-androstane-3,17-dione of structural Formua (T) and its salt/pharmaceutically acceptable form thereof of the Formula (1) 2.A process of claim 1, wherein a mixture of 5 a-androstane-3, 17-dione ( 3.46 mmol ),m-bromobenzoic acid hydrazide ( 6.93 mmol), and acetic acid (1 ml) in ethanol (10 ml) was refluxed for 2 h and cooled to get hydrazone of Formula (X). 3.A process for the preparation of Bis-m-nitrobenzoic acid hydrazone of 5 a-androstane-3,17-dione of Formula (II)and its salt/pharmaceutically acceptable form thereof of structural Formula (II) 4.A process of claim 3, wherein a mixture of 5 a-androstane-3, 17-dione ( 3.46 mmol), m-nitrobenzoic acid hydrazide ( 6.93 mmol) and acetic acid (1 ml) in ethanol (10 ml) was refiuxed for 2 h and cooled to get the hydrazone of structural Formula (II). 5.A method for inhibiting the growth of pathogenic microorganisms ( ex.bacteria, M.tuberculum etc.) wherein the chemotherapeutic agent is a compound of Formula (I) or Formula (II), its salts or the pharmaceutically acceptable forms. 6.A method for inhibiting the growth/replication of any form of cancer (ex. Tumor etc) wherein the chemotherapeutic agent is a compound of Formula (I) or Formula (II), its salts or the pharmaceutically acceptable forms. 7.A method for inhibiting the growth/replication ofviruses(ex. HIV etc ) wherein the chemotherapeutic agent is a compound of Formula (I) or Formula (II), its salts or the pharmaceutically acceptable forms. 8.A method for use as CNS stimulant (ex.psychotropic agent) wherein The chemotherapeutic agent is a compound of Formula ( I ) or Formula ( II ), its salts or pharmaceutically acceptable forms.

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