Title of Invention	NOVEL STEROIDAL ESTERS OF 17-OXIMINO-5-ANDROSTEN-3BETA-OL
Abstract	The present invention relates to steroidal esters of 17-oximino -5-androsten-3-ol,of compound of general formula A wherein R is selected form a group consisting of arylalkyl, aryl, substituted aryl. The ester derivatives are synthesized starting from Dehydroandrosterone acetate. The compounds were tested for their antiproliferative activity and 5α-reductase inhibitory activity in comparison to Finasteride. Decreased androgen level have been found in serum of animal treated with newly synthesized compounds. These compounds have also shown better cytotoxicity in comparison to reference drug Finasteride. Thus such compounds can be useful in treatment of androgen dependent disorder of prostate alone or by synergistic effect they can decrease the size of prostate due to their antiproliferative activity.

Title of Invention

NOVEL STEROIDAL ESTERS OF 17-OXIMINO-5-ANDROSTEN-3BETA-OL

Abstract

The present invention relates to steroidal esters of 17-oximino -5-androsten-3-ol,of compound of general formula A wherein R is selected form a group consisting of arylalkyl, aryl, substituted aryl. The ester derivatives are synthesized starting from Dehydroandrosterone acetate. The compounds were tested for their antiproliferative activity and 5α-reductase inhibitory activity in comparison to Finasteride. Decreased androgen level have been found in serum of animal treated with newly synthesized compounds. These compounds have also shown better cytotoxicity in comparison to reference drug Finasteride. Thus such compounds can be useful in treatment of androgen dependent disorder of prostate alone or by synergistic effect they can decrease the size of prostate due to their antiproliferative activity.

Full Text	Novel steroidal esters of 17-oximino-5-androsten-3β-ol Field of the invention The present invention relates to novel steroidal esters of 17-oximino-5-androsten-3ßol The present invention particularly relates to steroidal esters of compound of formula A wherein R is selected form a group consisting of alkyl, aryl, substituted aryl Formula A (Formula Removed) The compounds are useful as 5α-reductase inhibitor and androgen dependent disorders for the treatment of benign prostatic hyperplasia Background of the invention Hyperplasia is a general medical term referring to excess cell proliferation and benign prostatic hyperplasia is non-malignant enlargement of the prostate gland caused by increase in the number of stromal and epithelial cells resulting in obstruction of the proximal urethera, thus causes urinary flow disturbances [Roehrborn, C G BJU Int 2006, 97, 7,Emberton, M BJU Int 2006, 97, 12 and Fitzpatnck, J M BJU Int 2006, 97, 3 ,] It is leading disorder of the elderly male population and prevalence increases with age affecting around 80% by the age of 80 years Nearly all men will develop microscopic BPH (Benign Prostatic Hyperplasia) by the age of 90 [Berry, S J et al, Br J Urol, 1995, 5, 85] Number of different causes for the overgrowth includes age, late activation of cell growth, abnormal increase in the clonal expansion of transit cell and change in ratios of androgen-estrogen level, which promote growth of prostatic tissue [Theomher, M et al Prostate,1996, 28, 392 and Jenkins,E P et al J Clin Invest ,1992, 89, 293 ] But the most widely accepted hypothesis is the dihydrotestosterone hypothesis which postulates that BPH occurs following an age related changes in the prostate androgen metabolism which favours accumulation of dihydrotestosterone (Formula Removed) FIGURE 1 Dihydrotestosterone (DHT), the most potent circulating androgen hormone, is produced by NADPH dependent stereoselective reduction of testosterone (T), under catalysis of the enzyme steroid 5α-reductase (5AR) [Hitoshi, T et al Chem Pharm Bull 2000, 48, 552] Within prostate dihydrotestosterone binds to cytosol androgen receptor protein (AR) and DHT-AR complex enters the nucleus, interacts with DNA binding sites where it stimulates the RNA synthesis (Figure 1)[ Liao, S Int Rev Cytol 1975, 41, 87] The concentration of dihydrotestosterone is 2 5 fold higher than in normal prostate Therefore, inhibition of androgen action by 5α-reductase is a logical treatment of 5α-reductase activity disorder l e benign prostatic hyperplasia (Figure Removed) (FIGURE 2) Androgen suppression causes reduction in prostatic volume which is believed to decrease the static component of bladder outlet obstruction resulting from benign prostatic hyperplasia (Figure 2) [Reid, P et al J Clin Invest New Drugs 1999, 17, 271] With the discovery of two 5a-reductase isozymes, their physiological and pharmacological roles in BPH, a significant research has been carried out to synthesize and evaluate nonsteroidal [Marisa, C et al Chem Pharm Bull 2001, 49, 525 , Frye, J Med Chem,1994, 37, 2352] and steroidal compounds [Leny, MA et al J Steroid Biochem Mol Biol,1994, 48, 197 , Toshiaki, K et al, Chem Pharm Bull, 1996,44 ,115] as competitive [4-azasteroids, 6-azasteroids ] or non-competitive [3-carboxysteroids ] inhibitors of 5a-reductase Of these, only Finasteride (3) (MK-906) [Foley, C L et al Drugs of Today 2004, 40, 213 ], and Dutastende (4) [Susumu, T , et al Chem Pharm Bull, 2000, 4#,1689] are being used clinically in the treatment of benign prostatic hyperplasia (Formula Removed) It has been reported that various steroidal compounds possess high affinity for 5a-reductase inhibitors on account of their ability to form a coordinate bond with iron heame [Hartmann, R W et al, J J Med Chem 2000, 43, 4266] On other hand, progesterone esters (5,6) synthesised by Mexico laboratory exhibited high antiandrogemc activity [Cabeza, M et al, Chem Pharm Bull (Tokyo) 1999, 47, 1232 and Cabeza, M et al Chem Pharm Bull (Tokyo) 2001, 49, 1081 On long term usage of Finasteride and Dutastende the most common reported side effect include decreased libido, decreased ejaculate amount and erectile dysfunction Whereas progesterone esters have been found to possess good antiandrogemc activity but are still under clinical investigation Based on these observations, it was envisaged to synthesize 17-oximmo-5- androsten-3β-yl esters (10-16) Benign prostatic hyperplasia is also described by abnormal increase in the number of cells in prostate which may result not only from increased cell proliferation but also from decreased level in programmed cell death (apoptosis) These analogues were evaluated for 5α-reductase inhibitory activity and antiproliferative activity These compounds may be useful in androgen dependent benign prostatic hyperplasia, by acting not only at the molecular level for the underlying cause of the disease but also decreasing the growth of prostate by killing cells Objects of the invention The mam object of the present invention is to provide novel steroidal esters of 17-oximino-5-androstene-3β-ol Another object of the invention is to provide a process for preparation of novel steroidal esters of 17-oximino-5-androstene-3β-ol One more object of the present invention is to provide the compounds useful for the treatment of benign prostatic hyperplasia Still another object of the invention is to provide the compounds having antiproliferative activity with a >90% growth inhibition of DU-145 cell Summary of the invention The present invention relates to steroidal esters derivative of 17-oximino -5-androsten-3-ol The compounds have been synthesized starting from Dehydroandrosterone acetate Newly synthesized compounds were tested for their antiproliferative activity and 5α-reductase inhibitory activity in comparison to Finasteride Decreased androgen level has been found in serum of animal treated with newly synthesized compounds These compounds have also shown better cytotoxicity in comparison to reference drug Finasteride Thus such compounds can be useful in treatment of androgen dependent disorder of prostate alone or by synergistic effect they can decrease the size of prostate due to their antiproliferative activity Brief Description of the drawings Figure 5 Log dose-response relationship with regard to cytotoxicity of the compounds on the number of living cells (DU-145) relative to the control Each point represent a mean±SEM of 3 independent experiments Linear regressed line was drawn to calculate the IC50 ANOVA followed by Tukey's was applied Data significantly different from the reference drug (p Figure 6 Effect of compounds on serum level of testosterone Results are mean±SEM of five experiments p Figure 7 Toxicity of the compounds to mouse macrophages (Balb C) Cell viability was determined based on the MTT assay Each point represent a mean value and SEM of 3 independent experiments p Detailed description of the invention Accordingly the present invention provides a compound of general formula A, pharmaceutical^ acceptable salt thereof, NOH (Table Removed) wherein R is selected form a group consisting of arylalkyl, aryl, substituted aryl In an embodiment of the invention wherein the aryalkyl group may be selected from a group consisting of benzyl, phenoxymethyl, or substituted arylalkyl such as 4- chlorobenzyl, 4-mtrobenzyl, 4-methylbenzyl, 4-aminobenzyl, 4- chlorophenxoymethyl, 4-nitrophenoxymethyl, 4-methylphenoxymethyl,4- aminophenoxymethyl In another embodiment of the invention wherein the aryl group may be selected form a group consisting of (1) 4-chlorophenyl (10), (n) 4-mtrophenyl(ll), (in) 4-methylphenyl (β-tolyl)(12) , (IV) phenyl (13), (v) 4-aminophenyl (14), (vi) 4-hydroxyphenyl ( 15), (v11) phenoxymethyl (16) In yet another embodiment of the invention wherein the pharmaceutically acceptable salt may be selected form a group consisting of hydrochloride, sulphates, phosphates acetates, propionates, and sodium , potassium and calcium salts of the phenolic hydroxyl groups containing substrates In still another embodiment of the invention wherein the representative compounds of formula A comprising, (l) 17-oximino-5-androsten-3ß-yl 4-chlorobenzoate (10) (n) 17-oximino-5-androsten-3ß-yl 4-nitrobenzoate (11) (in) 17-oximino-5-androsten-3β-yl 4-methylbenzoate (12) (iv) 17-oximino-5-androsten-3β-yl benzoate (13) (v) 17-oximino-5-androsten-3β-yl 4-aminobenzoate (14) (vi) 17-oximino-5-androsten-3β-yl 4-hydroxybenzoate (15) (vn) 17-oximino-5-androsten-3β-yl phenoxyacetate (16) In a further embodiment of the invention wherein the compounds are useful as 5α-reductase inhibitor, androgen dependent disorders In another embodiment of the invention wherein the compounds showed antiproliferative activity with a >90% growth inhibition of DU-145 cell at a concentration of 5 0µg/ml In another embodiment of the invention wherein the compounds showed increase of serum level of testosterone in rats as compared to control at a dose of 40mg/kg In another embodiment of the mvention wherem the compounds are having LC50 in the range of 19 5 to 89 4 µm In another embodiment of the invention wherein the compounds are non-toxic at a concentration ranging between 0 01 ng/ml to 5 0 µg/ml Accordingly, the present invention provides a process for preparation of novel steroidal esters of 17-oximino-5-androstene-3β-ol of formula A, wherein the process steps comprising (I) reacting 17-oximino-5-androsten-3ß-ol with an organic acid and dicyclohexylcarbodnmide (DCC) in anhydrous dichloromethane for a period ranging between 24-48 hrs at a temperature ranging between 20 to 25°C (n) filtering the precipitated dicyclohexylurea (dcu) and removing the solvent from the filtrate under vacuum and the residue was crystallized to obtain the desired product Accordingly, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula A or a pharmaceutically acceptable salt thereof optionally along with a pharmaceutically acceptable carrier, percipient, diluent In the proposed research project novel steroidal esters of 17-oximino 5-androsten-3β-ol were synthesized in two steps, starting from dehydroepiandrosterone acetate,which was prepared from the diosgemn [Marker, R E et al, JAmer Chem Soc 1939, 61 March, J Advanced Organic Chemistry, John Wiley and Sons New York, 2001, p 395 and Lemeiux, U et al Can J Chem 1955, 33, 1701] The oxime moiety at C-17 of the steroid nucleus was incorporated as proposed in Figure 3 17-Oxo-5-androsten-3p~yl acetate (7) was converted to its oxime (8) by refluxing in ethanol with hydroxylamine hydrochloride and sodium acetate In infrared spectrum stretching due to NOH, C=0 (ester) and C-0 (ester) were found at 3400, 1740 and 1245 cm1, respectively In NMR spectrum signals were recorded at 5 2 3 (s, 3H, CH3COO), 4 60 (m, 1H, 3α-H), 5 4 (br, 1H, 6-vinyhc) and 8 43 ppm (1H, NO//) he oxime 8 was hydrolysed and estenfied as given in Figure 4 NOH FIGURE 4 (Figure Removed) 17-Oximino-5-androsten-3β-yl acetate (8) was hydrolyzed with methanohc potassium hydroxide to get 17-oximino-5-androsten-3β-ol (9) In 1H-NMR spectrum signals for 3a and 6-vinyhc protons appeared at 8 4 54 and 5 5 22, respectively IR spectrum showed disappearance of the stretching due to acetate carbonyl at 1740cm1 17-Oximino-5-androsten-3β-ol (9) was reacted with respective acid and dicyclohexylcarbodiimide (DCC) in anhydrous dichloromethane The reaction mixture was stirred for 48 hr at room temperature and completion of the reaction was monitored by TLC The precipitated dicyclohexylurea (DCU) was filtered, solvent removed under vacuum and the residue was crystallized from ethyl acetate petroleum ether (60 80) to obtain the desired product Following examples are given by way of illustration and should not construed to limit the scope of the invention EXAMPLE 1 17-OXIMINO-5-ANDROSTEN-3P-YL 4-CHLOROBENZOATE (10) To the stirred solution of 17-oximino-5-androsten-3β-ol (9) (0 5 g, 1 6 mmol) and dicyclohexylcarbodnmide (DCC) (0 34 g, 16 mmol) in anhydrous dichloromethane (30 0 ml), 4-chlorobenzoic acid (0 26 g, 1 6 mmol) was added The reaction mixture was stirred for 48 hr at room temperature (20-25°) and completion of the reaction was monitored by TLC The precipitated dicyclohexylurea (DCU) was filtered and the solvent removed under vacuum The resulting residue was crystallized from ethyl acetate petroleum ether (60 80) to give 17-oximino-5-androsten-3β-yl 4-chlorobenzoate (10) (0 30 g, 60 0%) mp 167-170°C (Formula Removed) Anal mp 167-170° C IR(KBr) 3310, 2930, 1740, 1650 and 1235 cm' 1H-NMR (CDC13) 5 1 03 (s, 3H, I8-CH3), 1 04 (s, 3H \9-CH3), 2 17 (1H, NO#), 4 15 (m, 1H, 3α-H;, 5 40 (br, 1H, 6-vinyhc), 7 50 (d, 2H, 3-CH and 5-CH aromatic) and 7 97 ppm (d, 2H, 2-CHand 6-CH aromatic) Calcd for C26H32NO3CI N, 3 17 Found N, 3 25 EXAMPLE 2 17-OXIMINO-5-ANDROSTEN-3P-YL 4-NITROBENZOATE (11) To the stirred solution of 17-oximino-5-androsten-3β-ol (9) (0 5 g, 16 mmol) and dicyclohexylcarbodnmide (DCC) (0 34 g, 1 6 mmol) in anhydrous dichloromethane (30 0 ml), ;?-mtrobenzoic acid (0 27 g, 1 6 mmol) was added The reaction mixture was stirred for 48 hr at room temperature (20-25°) and completion of the reaction was monitored by TLC The precipitated dicyclohexylurea (DCU) was filtered and the solvent removed under vacuum The resulting residue was crystallized from ethyl acetate petroleum ether (60 80) to give 17-oximino-5-androsten-3β-yl 4-mtrobenzoate (11) (0 34 g, 68 0%) mp 185-188°C (Formula Removed) Anal mpl85-188°C IR(KBr) 3300, 2930, 1700, 1650 and 1234 cm ' 'H-NMR (CDC13) 6 0 89 (s, 3H, 18-C/fc), 1 03 (s, 3H \9-CH3), 3 85 (m, 1H, 3a-H), 5 4 (br, 1H, 6-vinyhc), 6 65 (d, 2H, 3-C#and 5-CH aromatic), 7 87 (d, 2H, 2-C#and 6-CH aromatic) Calcd for C26H32N2O5 N, 6 19 Found N, 5 64 EXAMPLE 3 17-OXIMINO-5-ANDROSTEN-3β-YL 4-METHYLBENZOATE (12) 4-Methylbenzoic acid (β-toluic acid) (0 22 g, 1 6 mmol) (0 16 g, 16 mmol) was added to the stirred solution of 17-oximino-5-androsten-3β-ol (9) (0 5 g, 16 mmol) and dicyclohexylcarbodnmide (DCC) (0 34 g, 16 mmol) in anhydrous dichloromethane (30 0 ml) The reaction mixture was stirred for 48 hr at room temperature (20-25°) and completion of the reaction was monitored by TLC The precipitated dicyclohexylurea (DCU) was filtered, solvent removed under vacuum and the residue was crystallized from ethyl acetate petroleum ether (60 80) to obtain 12 (0 29 g, 58 0%) mp 135-139°C (Table Removed) Anal mp 135-139° C IR(KBr) 3310, 2930, 1700, 1650 and 1235 cm ' 1H-NMR (CDC13) 5 0 89 (s, 3H, I8-CH3), 1 03 (s, 3H 19-C/fc), 2 41 (1H, NO//), 4 08(m, 1H, 3α-H;, 6 25 (br,lH, 6-vinyhc), 7 20 (d, 2H, 3-CH and 5-CH aromatic) and 7 43 ppm (d, 2H, 2-CZ/ and 6-CH aromatic) Calcd for C27H35NO3 N, 3 32 Found N, 3 77 EXAMPLE 4 17-OXIMINO-5-ANDROSTEN-3P-YL BENZOATE (13 ) 17-Oximino-5-androsten-3β-ol (9) (0 5 g, 16 mmol) and dicyclohexylcarbodnmide (DCC) (0 34 g, 16 mmol) was dissolved in anhydrous dichloromethane (30 0 ml) and stirred To this was added benzoic acid (0 2 g, 16 mmol) The reaction mixture was stirred for 48 hr at room temperature (20-25°) and completion of the reaction was monitored by TLC The precipitated material was filtered, solvent removed under reduced pressure to give residue which was crystallized from ethyl acetate petroleum ether (60 80) to get 17-oximino-5-androsten-3β-yl benzoate (13 ) (0 32 g, 64 0%) mp 165-170°C (Formula Removed) Anal mp 165-170°C IR(KBr) 3300, 2930, 1700, 1650 and 1235 cm ' 'H-NMR (CDC13) 8 0 89 (s, 3H, 18-C/fc), 1 1 (s, 3H 19-Ci/j), 4 07 (m, 1H, 3a-H), 5 40 (br,lH,6-vinyhc) and 6 84-7 28 (m, 5H, aromatic) Calcd for C26H33NO3 N, 3 44 Found N, 3 74 EXAMPLE 5 17-OXIMINO-5-ANDROSTEN-3β-YL 4-AMINOBENZOATE (14) /7-Aminobenzoic acid (0 22 g, 1 6 mmol) was added to the stirred solution of 17-oximino-5-androsten-3β-ol (9) (0 5 g, 1 6 mmol) and dicyclohexylcarbodnmide (DCC) (0 34 g, 16 mmol) in anhydrous dichloromethane (30 0 ml) The reaction mixture was stirred for 48 hr at room temperature (20-25°) and completion of the reaction was monitored by TLC The precipitated dicyclohexylurea (DCU) was filtered, solvent removed under vacuum and the residue was crystallized from ethyl acetate petroleum ether (60 80) to obtain 17-oximino-5-androsten-3β-yl 4-aminobenzoate (14 ) (0 28 g, 56 0%) mp 165-168°C (Formula Removed) Anal mp 165-168°C IR(KBr) 3330, 2930, 1700, 1630 and 1240 cm ' 'H-NMR (CDCb) 5 0 93 (s, 3H, I8-C//3), 1 05 (s, 3H \9-CH3), 2 17 (1H, NOtf), 4 08 (m, 1H, 3a-H), 6 20 (br, 1H, 6-vinyhc), 7 22 (d, 2H, 3-CH and 5-CH aromatic) and 8 26 ppm (d, 2U,2-CH and 6-CH aromatic) Calcd for C26H34N2O3 N, 6 63 Found N, 6 79 EXAMPLE 6 17-OXIMINO-5-ANDROSTEN-3β-YL 4-HYDROXYBENZOATE (15) 17-Oximino-5-androsten-3β-ol (9) (0 5 g, 1 6 mmol) was taken in anhydrous dichlorormethane (30 0 ml) Dicyclohexylcarbodnmide (DCC) (0 34 g, 16 mmol) was added followed by /7-hydroxybenzoic acid (0 23 g, 1 6 mmol) Reaction mixture was stirred at room temperature for 24-48 hr and completion of the reaction was monitored by TLC The reaction mixture was filtered to remove precipitated dicyclohexylurea (DCU) Excess of solvent was removed under vacuum and the product was crystallized from ethyl acetate petroleum ether (60 80) to yield 17-oximino-5-androsten-3(3-yl 4-hydroxybenzoate (15 ) (0 31g, 62 0%) mp 177-182° C (Formula Removed) Anal mp 177-182° C IR(KBr) 3310, 2930, 1710, 1680 and 1235 cm ' 1H-NMR (CDCI3) δ 0 89 (s, 3H, 18-CH5), 1 05 (s, 3H 19-CH3), 2 17 (IH, NO//), 4 18(m, IH, 3α-H;, 6 35 (br, IH 6-vinylic) 6 83 (d, 2H, 3-CH and 5-CH aromatic) and 7 46 ppm (d, 2H, 2-CH and 6-CH aromatic) Calcd for C26H33NO4 N, 3 31 Found N, 3 32 EXAMPLE 7 17-OXIMINO-5-ANDROSTEN-3β-YL PHENOXYACETATE (16) To the stirred solution of 17-Oximino-5-androsten-3β-ol ( 9) (0 5 g, 1 6 mmol) and dicyclohexylcarbodnmide (DCC) (0 342 g, 1 6 mmol) in anhydrous dichloromethane (30 0 ml), phenoxyacetic acid (0 24 g, 1 6 mmol) was added The reaction mixture was stirred for 48 hr at room temperature (20-25°) and completion of the reaction was monitored by TLC The precipitated dicyclohexylurea (DCU) was filtered and the solvent removed under vacuum The resulting residue was crystallized from ethyl acetate petroleum ether (60 80) to give 17-oximino-5-androsten-3β-yl phenoxyacetate (16 ) (0 27 g, 54 0%) mp 118-122°C (Formula Removed) Anal mpl 18-122° C IR(KBr) 3200, 2940, 1750, 1650 and 1220 cm ' 1H-NMR (CDC13) δ 0 88 (s, 3H, I8-CH3), 1 02 (s, 3H 19-CH3), 4 60 (s, 2H, OCH2), 4 7 (m, 1H, 3α-H), 5 40 (br, 1H, 6-vinyhc) and 7 29(m, 5H, aromatic) Calcd for C27H35NO4 N, 3 20 Found N, 3 60 Biological Evaluation The synthesized steroidal derivatives have been evaluated for in vitro antiproliferative activity using DU-145 cell lines and in vivo studies 1 e 5α-Reductase Inhibitory activity [George, F W et al Endocrinology, 1989,125, 2434] 1) In vitro antiproliferative activity against human prostate cancer cell line (DU-145) Compounds were screened for antiproliferative activity on human prostate cell line DU-145 at National Centre of Human Genome Studies and Research Panjab University, Chandigarh All the compounds (10 to 16), Finasteride and starting material (9) were tested at five different concentrations 0 01, 0 5, 1 0, 2 0, and 5 0 µg/ml Finasteride was the positive control in this growth inhibitory assay Growth inhibitory activity assay (MTT assay) We investigated the effect of newly synthesized compounds on cell growth inhibition using MTT assay This assay quantifies the viable cells by observing the reduction of tetrazohum salt, MTT to formazan crystals by the live cells Based on the absorbance of the cell sample after the test is carried out, cell viable can be measured [Loveland, B E , Jones, T G , Mackay I R „ Vaillant, F , Wang, Z X , Hertzog, P J Bwchem International 1992, 27, 501 and Wright, S A , Thomas, L N , Douglas, R C , Lazier, C B , Rittmaster, R S J Chn Invest 1996, 98, 2558] Cells were cultured at a density of 5 x 103 cells / well in 96 well plates at 37°C in 5 0% CO2 atmosphere and were allowed to attach for 24 hr The cells were treated in triplicate with graded concentration of sample and reference drug Finasteride at 37°C for 48 hr A 20 µl aliquot of MTT solution was added directly to all the appropriate wells Following 4 hr of incubation at 37µl, the media was removed and formazan crystals, which results from the reduction of MTT by active cell were dissolved in 100µl DMSO and vigorously mixed to dissolve the reacted dye The absorbance of each well was read on Ehsa plate reader (Merck) at 570 nm The spectrometer was calibrated to zero absorbance using culture medium without cells The relative cell viability (%) related to control well containing cell culture medium without drug was calculated by [A] test/[A] control x 100 % Growth Inhibition^ [OD] control - [OD] test/ [OD] control X 100 Results* All the newly synthesized compounds were tested for antiproliferative activity using DU-145 cell line The results are expressed in terms of percentage growth inhibition (Table 1) and percentage viable cells (Figure 5) Esters of 17-oximino-5-androsten3ß-ol (10-16) , synthesized in our lab revealed a great reduction in the level of cellular cytotoxicity as compared to Finasteride (3) Examination of data of these analogues 10 to 16 demonstrated a great tolerance of phenyl ring and its parα-substituted analogues Unsubstituted analogue 13 and parα- substituted (electron donating group) compounds 12, 14 and 15 were found to be the most active compounds with >90% growth inhibition at concentration of 5 0 ug/ml relative to Finasteride (78% at 5 0 µg/ml) (Table 1) On the other hand analogues 10, 11 with electron withdrawing substituent decreased the growth of DU-145 cancer cell lines from 81-84%(relatively less), but still better than Finasteride (78%) Parent Compound 9 has not shown any significant inhibitory effect on cell proliferation Table 1 Antiproliferative activity of Finasteride and Compounds 9 to 16 (Table Removed) N, = no significant growth inhibition Number of ester derivatives bearing oxime group (NOH) at C-17, connected directly to the steroidal D ring were synthesized and screened in vitro (Table 1 and Figure 5) Examination of data of these analogues 10 to 16 demonstrated a great tolerance of phenyl ring and its parα-substituted analogues This is in agreement with the previous reports that have pointed out the importance of aromatic and β-substituted aromatic esters Unsubstituted analogue 13 and compounds 12, 14 and 15 with an electron donating moiety at para position have been found to be more potent than the reference Finasteride Compounds 12, 14 and 15 were found to be the most active with >90% growth inhibition at concentration of 5 0 µg/ml relative to Finasteride (78% at 5 0 µg/ml) On the other hand analogues 10, 11 with electron withdrawing substituents demonstrated relatively less activity than above compounds but still better than Finasteride Parent Compound 9 has not shown significant antiproliferative activity b) In vivo (effect on steroid androgen level) Treatment of animals Animals (Sprague Dawley male rats weighing 150-200 g) were divided into 3 groups, vehicle (control), Finasteride (standard), treated (test sample) and each group consist of 5 animals Sprague Dawley rats were treated intraperitoneally with Finasteride 40 mg/kg and equimolar dose of compounds After 6 hr of treatment, blood was withdrawn by cardiac puncture under diethyl ether anesthesia and serum was separated from cells by centnfugation Plasma testosterone values were obtained by ELSIA plate reader at 550 nm and are given in ng/ml Ehsa The aliquots of 50ul of each of standards, control and unknown (serum samples) were added to Testosterone Antibody coated wells 100µl of HRP-testosterone conjugate was added to all the wells and the plates were shaken gently on a shaker for proper mixing of the reagents Following 4 hr of incubation at 37°C, the incubation mixture was removed The wells were washed with phosphate buffer for 5-6 times (200µl each time), followed by addition of 100ul of TMB / H2O2 substrate in each of the wells The plates were further incubated at 37°C for 20 minutes At the end of incubation reaction was stopped by using 100ul of 0 5 M H2SO4 as stopping reagent The absorbance of each well was read on Ehsa plate reader at 450 nm [Rassaie, M J , Kuman, G L , Pandey, P K , Gupta, N , Kochupillai, N , Grover, P K Steroids 1992, 57, 288 and Hosoda, H , Yoshida H, Sakai, Y , Miyam, S , Nambara, T Chem Pharm Bull 1980, 28, 3035] Results The effect of compounds (10 to 16) and Finasteride on the serum concentration of testosterone are reported in Table 2 and Figure 6 Serum 'T' level were increased in Finasteride treated rats (1 26±0 02 ng/ml compared with 0 742±0 07 ng/ml control rats) It is apparent from Table 2 and Figure 6 that almost all the ester derivatives of 17-oximino-5-androsten-3ß-ol were able to increase the serum testosterone level as compared to control except compound 15 The 6 hr post dose serum T levels were 1 05±0 08 ng/ml for the compound 13 with unsubstituted phenyl group Ester derivatives 12 and 14 with electron releasing group at β-position of phenyl ring has been found to have increased activity, while presence of electron withdrawing moiety at this position causes the loss of activity (10, 11) Significant increase in level of serum testosterone level has been found in the compound 16 Table 2 Serum levels of Testosterone (Table Removed) Intact male rats (Sprague Dawley, 200-250 g) were used in the designed study in which various compounds were compared for in vivo 5α-reductase inhibitory potency, as judged by the their ability to attenuate the conversion of testosterone into dihydrotestosterone (DHT) ELISA for testosterone were found to be suitable for determination in serum of rate since the cross reactive DHT levels were extra low in male The procedure measure T equally well, and method met all the requirement of precision, accuracy, sensitivity and selectivity c) In vitro cytotoxicity using mouse macrophages (acute toxicity) In vitro cytotoxicity test using DU-145 cells in the preliminary evaluation of anticancer drug enable us to select most potent compound, but cytotoxic agents however frequently exhibit-unspecific toxicity Nevertheless the ability to selectively kill the target cell remains a highly desirable property of potential new therapeutic cytotoxic agents In this study, we have demonstrated the applicability of Red dye uptake (MTT) assay using mouse macrophages (Balb C) for in vitro toxicity testing of newly synthesized compounds Preparation of test material All steroids were dissolved in ethanol and diluted to appropriate concentration 0 01, 0 5, 1 0, 2 0, 5 0 µg/ml from the two stock solution of 1 mg/ml and 0 0014 µg/ml Stocks were maintained at room temperature Cell Viability Assay Isolation of mouse macrophages Albino mice (laca strain) weighing 20-25 g of either sex were sacrificed by cervical dislocation and small cut was given on peritoneal skin with a help of scissors Approximately 1 ml of Phosphate buffer saline preincubated at 37°C was injected into cavity The abdomen was lavaged for 10 mm and fluid along with macrophages were taken out using a syringe into polypropylene tube The phosphate solution was spun at 800 rpm for 5-10 mm and cells were resuspended in 1 ml RPMI media Cell number was counted using haemocytometer Final cell count =Total cell count x 1000 x dilution factor 0 1x5 0 1 = chamber depth, 5 Number of squares (each square is 1 mm) MTT assay Cells (mouse macrophages) were plated at a density of 5 x 103 cells / well in 96 plate at 37°C in 5% CO2 Cells were exposed in graded concentration of compounds at designated various concentration Each concentration was tested in triplicate wells After 48 hr fresh MTT 20ul (1 mg/ml) was added directly to all the wells and culture was incubated for 4 hr at 37°C During this incubation, MTT was converted into a water insoluble formazan complex by metabolic activity of viable cells Formazan crystal were taken and dissolved in l00µl of DMSO, which give light pink colour The absorbance of each well was read on Elisa plate reader (Merck) at 570 nm The spectrometer was calibrated to zero absorbance using culture medium without cells The relative cell viability (%) related to control well containing cell culture medium without drug was calculated by [A] test / [A] control x 100 % Cell Viability = [A] test/ [A] controi X 100 [A] test= absorbance test sample [A] control= absorbance control sample Results: LC50 Concentration of the compound in µg/ml (µm), lethal to 50% population of cells Red dye uptake (MTT) assay, demonstrated the toxicity of the compounds towards normal cell by using mouse macrophages The assay quantifies the viable cells, after 24 hr incubation of cells with five different concentrations Results in Table 3 and Figure 7 demonstrated a direct and proportional relation between cell number and concentration The results obtained from MTT assay were statistically significant (P Table 3 % Viable Cells of derivative (10 to 16 ) (Table Removed) Table 4 Acute Toxicity of the compounds (Table Removed) In vitro cytotoxicity test using DU-145 cells in the preliminary evaluation of anticancer drug enable us to select most potent compound, but cytotoxic agents however frequently exhibit-unspecific toxicity Nevertheless the ability to selectively kill the target cell remains a highly desirable property of potential new therapeutic cytotoxic agents In this study, we have demonstrated the applicability of Red dye uptake (MTT) assay using mouse macrophages (Balb C) for in vitro toxicity testing of newly synthesized compounds The assay quantifies the viable cells, after 24 hr incubation of cells with five different concentrations Figure 7 and Table 3 demonstrated a direct and proportional relation between cell number and concentration Data from this study Table 4 clearly indicated that compounds 13 and 16 with high LCso values were non toxic to mouse macrophages Acute toxicity of the compounds 10-12,14 and 15 were comparable to finasteride We have synthesized seven new steroidal esters derivative of 17-oximino -5-androsten-3-ol, starting from Dehydroandrosterone acetate Newly synthesized compounds were tested for their antiproliferative activity and 5α-reductase inhibitory activity in comparison to Finasteride Decreased androgen level have been found in serum of animal treated with newly synthesized compounds These compounds have also shown better cytotoxicity in comparison to reference drug Finasteride Thus such compounds can be useful in treatment of androgen dependent disorder of prostate alone or by synergistic effect they can decrease the size of prostate due to their antiproliferative activity We claim: 1. A novel steroidal esters of 17-oximino-5-androsten-3β-ol of general formula A, pharmaceutically acceptable salts thereof; (Formula Removed) General formula A wherein R is selected form a group consisting of arylalkyl, aryl, substituted aryl. 2. A compound as claimed in claim 1 wherein the arylalkyl group is selected from a group consisting of benzyl, phenoxymethyl or substituted arylalkyl such as 4-chlorobenzyl, 4-nitrobenzyl, 4-methylbenzyl, 4-aminobenzyl, 4-chlorophenxoymethyl, 4-nitrophenoxymethyl, 4-methylphenoxymethyl,4-aminophenoxymethy 1. 3. A compound as claimed in claim 1 wherein the aryl group is selected form a group consisting of (i) 4-chlorophenyl (10); (ii) 4-nitrophenyl(ll); (iii) 4-methylphenyl (p-tolyl)(12) ; (iv) phenyl (13); (v) 4-aminophenyl (14); (vi) 4-hydroxyphenyl ( 15); (vii) phenoxymethyl (16). 4. A compound as claimed in claim 1 wherein the pharmaceutically acceptable salt is selected form a group consisting of hydrochloride, sulphates, phosphates acetates, propionates; and sodium , potassium and calcium salts of the phenolic hydroxyl groups containing substrates . A compound as claimed in claim 1 wherein the representative compounds of formula A as claimed in claim 1 comprising; (i) 17-oximino-5-androsten-3β-yl 4-chlorobenzoate (10) (ii) 17-oximino-5-androsten-3β-yl 4-nitrobenzoate (11) (iii) 17-oximino-5-androsten-3β-yl 4-methylbenzoate (12) (iv) 17-oximino-5-androsten-3β-yl benzoate (13) (v) 17-oximino-5-androsten-3β-yl 4-aminobenzoate (14) (vi) 17-oximino-5-androsten-3β-yl 4-hydroxybenzoate (15) (vii) 17-oximino-5-androsten-3β-yl phenoxyacetate (16) 5. A compound as claimed in claim 1 wherein the compounds are useful as 5a-reductase inhibitor, androgen dependent disorders. 6. A compound as claimed in claim 1 wherein the compounds showed antiprolifirative activity with a >90% growth inhibition of DU-145 cell at a concentration of 5.0µg/ml. 7. A compound as claimed in claim 1 wherein the compounds showed increase of serum level of testosterone as compared to control at a dose of 40mg/kg. 8. A compound as claimed in claim 1 wherein the compounds having LC50 in the range of 19.5 to 89.4 µm. 9. A compound as claimed in claim 1 wherein the compounds are non-toxic at a concentration ranging between 0.0lµg/ml to 5.0µg/ml 10. A process for preparation of novel steroidal esters of 17-oximino-5-androstene-3β-ol of formula a as claimed in claim 1, wherein the process comprising : (I) reacting 17-oximino-5-androsten-3β-ol with an organic acid and dicyclohexylcarbodiimide (DCC) in anhydrous dichloromethane for a period ranging between 24-48 hrs at a temperature ranging between 20 to 25°C, (ii) filtering the precipitated dicyclohexylurea (DCU) and removing the solvent from the filtrate under vacuum and the residue was crystallized to obtain the desired product. 11. A pharmaceutical composition comprising: a therapeutically effective amount of a compound of claim lor a pharmaceutically acceptable salt thereof optionally along with a pharmaceutically acceptable carrier, excipient, diluent. 12. A novel steroidal esters of 17-oximino-5-androstene-3β-ol of formula A, process for preparation and a pharmaceutical composition thereof substantially as herein described with reference to the examples and drawings accompanying the specification.

Full Text

Novel steroidal esters of 17-oximino-5-androsten-3β-ol
Field of the invention
The present invention relates to novel steroidal esters of 17-oximino-5-androsten-3ßol The present invention particularly relates to steroidal esters of compound of formula A wherein R is selected form a group consisting of alkyl, aryl, substituted aryl
Formula A (Formula Removed)
The compounds are useful as 5α-reductase inhibitor and androgen dependent
disorders for the treatment of benign prostatic hyperplasia Background of the invention
Hyperplasia is a general medical term referring to excess cell proliferation and benign prostatic hyperplasia is non-malignant enlargement of the prostate gland caused by increase in the number of stromal and epithelial cells resulting in obstruction of the proximal urethera, thus causes urinary flow disturbances [Roehrborn, C G BJU Int 2006, 97, 7,Emberton, M BJU Int 2006, 97, 12 and Fitzpatnck, J M BJU Int 2006, 97, 3 ,] It is leading disorder of the elderly male population and prevalence increases with age affecting around 80% by the age of 80 years Nearly all men will develop microscopic BPH (Benign Prostatic Hyperplasia) by the age of 90 [Berry, S J et al, Br J Urol, 1995, 5, 85]
Number of different causes for the overgrowth includes age, late activation of cell growth, abnormal increase in the clonal expansion of transit cell and change in ratios of androgen-estrogen level, which promote growth of prostatic tissue [Theomher, M et al Prostate,1996, 28, 392 and Jenkins,E P et al J Clin Invest ,1992, 89, 293 ] But the most widely accepted hypothesis is the dihydrotestosterone hypothesis which postulates that
BPH occurs following an age related changes in the prostate androgen metabolism which favours accumulation of dihydrotestosterone
(Formula Removed)
FIGURE 1
Dihydrotestosterone (DHT), the most potent circulating androgen hormone, is produced by NADPH dependent stereoselective reduction of testosterone (T), under catalysis of the enzyme steroid 5α-reductase (5AR) [Hitoshi, T et al Chem Pharm Bull 2000, 48, 552] Within prostate dihydrotestosterone binds to cytosol androgen receptor protein (AR) and DHT-AR complex enters the nucleus, interacts with DNA binding sites where it stimulates the RNA synthesis (Figure 1)[ Liao, S Int Rev Cytol 1975, 41, 87] The concentration of dihydrotestosterone is 2 5 fold higher than in normal prostate
Therefore, inhibition of androgen action by 5α-reductase is a logical treatment of 5α-reductase activity disorder l e benign prostatic hyperplasia

(Figure Removed)
(FIGURE 2)
Androgen suppression causes reduction in prostatic volume which is believed to decrease the static component of bladder outlet obstruction resulting from benign prostatic hyperplasia (Figure 2) [Reid, P et al J Clin Invest New Drugs 1999, 17, 271] With the discovery of two 5a-reductase isozymes, their physiological and pharmacological roles in BPH, a significant research has been carried out to synthesize and evaluate nonsteroidal [Marisa, C et al Chem Pharm Bull 2001, 49, 525 , Frye, J Med Chem,1994, 37, 2352] and steroidal compounds [Leny, MA et al J Steroid Biochem Mol Biol,1994, 48, 197 , Toshiaki, K et al, Chem Pharm Bull, 1996,44 ,115] as competitive [4-azasteroids, 6-azasteroids ] or non-competitive [3-carboxysteroids ] inhibitors of 5a-reductase Of these, only Finasteride (3) (MK-906) [Foley, C L et al Drugs of Today 2004, 40, 213 ], and Dutastende (4) [Susumu, T , et al Chem Pharm Bull, 2000, 4#,1689] are being used clinically in the treatment of benign prostatic hyperplasia
(Formula Removed)

It has been reported that various steroidal compounds possess high affinity for 5a-reductase inhibitors on account of their ability to form a coordinate bond with iron heame [Hartmann, R W et al, J J Med Chem 2000, 43, 4266] On other hand, progesterone esters (5,6) synthesised by Mexico laboratory exhibited high antiandrogemc activity [Cabeza, M et al, Chem Pharm Bull (Tokyo) 1999, 47, 1232 and Cabeza, M et al Chem Pharm Bull (Tokyo) 2001, 49, 1081

On long term usage of Finasteride and Dutastende the most common reported side effect include decreased libido, decreased ejaculate amount and erectile dysfunction Whereas progesterone esters have been found to possess good antiandrogemc activity but are still under clinical investigation
Based on these observations, it was envisaged to synthesize 17-oximmo-5-
androsten-3β-yl esters (10-16) Benign prostatic hyperplasia is also described by
abnormal increase in the number of cells in prostate which may result not only from
increased cell proliferation but also from decreased level in programmed cell death
(apoptosis) These analogues were evaluated for 5α-reductase inhibitory activity and
antiproliferative activity These compounds may be useful in androgen dependent
benign prostatic hyperplasia, by acting not only at the molecular level for the
underlying cause of the disease but also decreasing the growth of prostate by killing cells
Objects of the invention
The mam object of the present invention is to provide novel steroidal esters of 17-oximino-5-androstene-3β-ol
Another object of the invention is to provide a process for preparation of novel steroidal esters of 17-oximino-5-androstene-3β-ol
One more object of the present invention is to provide the compounds useful for the treatment of benign prostatic hyperplasia
Still another object of the invention is to provide the compounds having antiproliferative activity with a >90% growth inhibition of DU-145 cell
Summary of the invention
The present invention relates to steroidal esters derivative of 17-oximino -5-androsten-3-ol The compounds have been synthesized starting from Dehydroandrosterone acetate Newly synthesized compounds were tested for their antiproliferative activity and 5α-reductase inhibitory activity in comparison to Finasteride Decreased androgen level has been found in serum of animal treated with newly synthesized compounds These compounds have also shown better cytotoxicity in comparison to reference drug Finasteride Thus such compounds can be useful in treatment of androgen dependent disorder of prostate alone or by synergistic effect they can decrease the size of prostate due to their antiproliferative activity

Brief Description of the drawings
Figure 5 Log dose-response relationship with regard to cytotoxicity of the compounds on the number of living cells (DU-145) relative to the control Each point represent a mean±SEM of 3 independent experiments Linear regressed line was drawn to calculate the IC50 ANOVA followed by Tukey's was applied Data significantly different from the reference drug (p Figure 6 Effect of compounds on serum level of testosterone Results are
mean±SEM of five experiments *p Figure 7 Toxicity of the compounds to mouse macrophages (Balb C) Cell
viability was determined based on the MTT assay Each point represent a mean value and SEM of 3 independent experiments *p Detailed description of the invention
Accordingly the present invention provides a compound of general formula A, pharmaceutical^ acceptable salt thereof,
NOH (Table Removed)
wherein R is selected form a group consisting of arylalkyl, aryl, substituted aryl
In an embodiment of the invention wherein the aryalkyl group may be selected from a
group consisting of benzyl, phenoxymethyl, or substituted arylalkyl such as 4-
chlorobenzyl, 4-mtrobenzyl, 4-methylbenzyl, 4-aminobenzyl, 4-
chlorophenxoymethyl, 4-nitrophenoxymethyl, 4-methylphenoxymethyl,4-
aminophenoxymethyl
In another embodiment of the invention wherein the aryl group may be selected form a group consisting of (1) 4-chlorophenyl (10), (n) 4-mtrophenyl(ll), (in) 4-methylphenyl (β-tolyl)(12) , (IV) phenyl (13), (v) 4-aminophenyl (14), (vi) 4-hydroxyphenyl ( 15), (v11) phenoxymethyl (16)
In yet another embodiment of the invention wherein the pharmaceutically acceptable salt may be selected form a group consisting of hydrochloride, sulphates, phosphates acetates, propionates, and sodium , potassium and calcium salts of the phenolic hydroxyl groups containing substrates
In still another embodiment of the invention wherein the representative compounds of formula A comprising,
(l) 17-oximino-5-androsten-3ß-yl 4-chlorobenzoate (10)
(n) 17-oximino-5-androsten-3ß-yl 4-nitrobenzoate (11)
(in) 17-oximino-5-androsten-3β-yl 4-methylbenzoate (12)
(iv) 17-oximino-5-androsten-3β-yl benzoate (13)
(v) 17-oximino-5-androsten-3β-yl 4-aminobenzoate (14)
(vi) 17-oximino-5-androsten-3β-yl 4-hydroxybenzoate (15)
(vn) 17-oximino-5-androsten-3β-yl phenoxyacetate (16)
In a further embodiment of the invention wherein the compounds are useful as 5α-reductase inhibitor, androgen dependent disorders
In another embodiment of the invention wherein the compounds showed antiproliferative activity with a >90% growth inhibition of DU-145 cell at a concentration of 5 0µg/ml
In another embodiment of the invention wherein the compounds showed increase of serum level of testosterone in rats as compared to control at a dose of 40mg/kg
In another embodiment of the mvention wherem the compounds are having LC50 in the range of 19 5 to 89 4 µm
In another embodiment of the invention wherein the compounds are non-toxic at a concentration ranging between 0 01 ng/ml to 5 0 µg/ml
Accordingly, the present invention provides a process for preparation of novel steroidal esters
of 17-oximino-5-androstene-3β-ol of formula A, wherein the process steps comprising
(I) reacting 17-oximino-5-androsten-3ß-ol with an organic acid and
dicyclohexylcarbodnmide (DCC) in anhydrous dichloromethane for a period ranging
between 24-48 hrs at a temperature ranging between 20 to 25°C
(n) filtering the precipitated dicyclohexylurea (dcu) and removing the solvent from
the filtrate under vacuum and the residue was crystallized to obtain the desired
product
Accordingly, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula A or a pharmaceutically acceptable salt thereof optionally along with a pharmaceutically acceptable carrier, percipient, diluent
In the proposed research project novel steroidal esters of 17-oximino 5-androsten-3β-ol were synthesized in two steps, starting from dehydroepiandrosterone acetate,which was prepared from the diosgemn [Marker, R E et al, JAmer Chem Soc 1939, 61 March, J Advanced Organic Chemistry, John Wiley and Sons New York, 2001, p 395 and Lemeiux, U et al Can J Chem 1955, 33, 1701] The oxime moiety at C-17 of the steroid nucleus was incorporated as proposed in Figure 3 17-Oxo-5-androsten-3p~yl acetate (7) was converted to its oxime (8) by refluxing in ethanol with hydroxylamine hydrochloride and sodium acetate In infrared spectrum stretching due to NOH, C=0 (ester) and C-0 (ester) were found at 3400, 1740 and 1245 cm1, respectively In NMR spectrum signals were recorded at 5 2 3 (s, 3H, CH3COO), 4 60 (m, 1H, 3α-H), 5 4 (br, 1H, 6-vinyhc) and 8 43 ppm (1H, NO//)
he oxime 8 was hydrolysed and estenfied as given in Figure 4
NOH

FIGURE 4 (Figure Removed)
17-Oximino-5-androsten-3β-yl acetate (8) was hydrolyzed with methanohc potassium hydroxide to get 17-oximino-5-androsten-3β-ol (9) In 1H-NMR spectrum signals for 3a and 6-vinyhc protons appeared at 8 4 54 and 5 5 22, respectively IR spectrum showed disappearance of the stretching due to acetate carbonyl at 1740cm1 17-Oximino-5-androsten-3β-ol (9) was reacted with respective acid and dicyclohexylcarbodiimide (DCC) in anhydrous dichloromethane The reaction mixture was stirred for 48 hr at room temperature and completion of the reaction was monitored by TLC The precipitated dicyclohexylurea (DCU) was filtered, solvent
removed under vacuum and the residue was crystallized from ethyl acetate petroleum ether (60 80) to obtain the desired product
Following examples are given by way of illustration and should not construed to limit the
scope of the invention EXAMPLE 1
17-OXIMINO-5-ANDROSTEN-3P-YL 4-CHLOROBENZOATE (10)
To the stirred solution of 17-oximino-5-androsten-3β-ol (9) (0 5 g, 1 6 mmol) and dicyclohexylcarbodnmide (DCC) (0 34 g, 16 mmol) in anhydrous dichloromethane (30 0 ml), 4-chlorobenzoic acid (0 26 g, 1 6 mmol) was added The reaction mixture was stirred for 48 hr at room temperature (20-25°) and completion of the reaction was monitored by TLC The precipitated dicyclohexylurea (DCU) was filtered and the solvent removed under vacuum The resulting residue was crystallized from ethyl acetate petroleum ether (60 80) to give 17-oximino-5-androsten-3β-yl 4-chlorobenzoate (10) (0 30 g, 60 0%) mp 167-170°C
(Formula Removed)
Anal
mp 167-170° C
IR(KBr) 3310, 2930, 1740, 1650 and 1235 cm'
1H-NMR (CDC13) 5 1 03 (s, 3H, I8-CH3), 1 04 (s, 3H \9-CH3), 2 17 (1H, NO#),
4 15 (m, 1H, 3α-H;, 5 40 (br, 1H, 6-vinyhc), 7 50 (d, 2H, 3-CH and 5-CH aromatic)
and 7 97 ppm (d, 2H, 2-CHand 6-CH aromatic)
Calcd for C26H32NO3CI N, 3 17 Found N, 3 25
EXAMPLE 2
17-OXIMINO-5-ANDROSTEN-3P-YL 4-NITROBENZOATE (11)
To the stirred solution of 17-oximino-5-androsten-3β-ol (9) (0 5 g, 16 mmol) and dicyclohexylcarbodnmide (DCC) (0 34 g, 1 6 mmol) in anhydrous dichloromethane (30 0 ml), ;?-mtrobenzoic acid (0 27 g, 1 6 mmol) was added The reaction mixture was stirred for 48 hr at room temperature (20-25°) and completion of the reaction was monitored by TLC The precipitated dicyclohexylurea (DCU) was filtered and the solvent removed under vacuum The resulting residue was crystallized from ethyl acetate petroleum ether (60 80) to give 17-oximino-5-androsten-3β-yl 4-mtrobenzoate (11) (0 34 g, 68 0%) mp 185-188°C
(Formula Removed)
Anal
mpl85-188°C
IR(KBr) 3300, 2930, 1700, 1650 and 1234 cm '
'H-NMR (CDC13) 6 0 89 (s, 3H, 18-C/fc), 1 03 (s, 3H \9-CH3), 3 85 (m, 1H, 3a-H),
5 4 (br, 1H, 6-vinyhc), 6 65 (d, 2H, 3-C#and 5-CH aromatic), 7 87 (d, 2H, 2-C#and
6-CH aromatic)
Calcd for C26H32N2O5 N, 6 19 Found N, 5 64
EXAMPLE 3
17-OXIMINO-5-ANDROSTEN-3β-YL 4-METHYLBENZOATE (12)
4-Methylbenzoic acid (β-toluic acid) (0 22 g, 1 6 mmol) (0 16 g, 16 mmol) was added to the stirred solution of 17-oximino-5-androsten-3β-ol (9) (0 5 g, 16 mmol) and dicyclohexylcarbodnmide (DCC) (0 34 g, 16 mmol) in anhydrous dichloromethane (30 0 ml) The reaction mixture was stirred for 48 hr at room temperature (20-25°) and completion of the reaction was monitored by TLC The precipitated dicyclohexylurea (DCU) was filtered, solvent removed under vacuum and the residue was crystallized from ethyl acetate petroleum ether (60 80) to obtain 12 (0 29 g, 58 0%) mp 135-139°C
(Table Removed)
Anal
mp 135-139° C
IR(KBr) 3310, 2930, 1700, 1650 and 1235 cm '
1H-NMR (CDC13) 5 0 89 (s, 3H, I8-CH3), 1 03 (s, 3H 19-C/fc), 2 41 (1H, NO//),
4 08(m, 1H, 3α-H;, 6 25 (br,lH, 6-vinyhc), 7 20 (d, 2H, 3-CH and 5-CH aromatic)
and 7 43 ppm (d, 2H, 2-CZ/ and 6-CH aromatic)
Calcd for C27H35NO3 N, 3 32 Found N, 3 77
EXAMPLE 4
17-OXIMINO-5-ANDROSTEN-3P-YL BENZOATE (13 )
17-Oximino-5-androsten-3β-ol (9) (0 5 g, 16 mmol) and dicyclohexylcarbodnmide (DCC) (0 34 g, 16 mmol) was dissolved in anhydrous dichloromethane (30 0 ml) and stirred To this was added benzoic acid (0 2 g, 16 mmol) The reaction mixture was stirred for 48 hr at room temperature (20-25°) and completion of the reaction was monitored by TLC The precipitated material was filtered, solvent removed under reduced pressure to give residue which was crystallized from ethyl acetate petroleum ether (60 80) to get 17-oximino-5-androsten-3β-yl benzoate (13 ) (0 32 g, 64 0%) mp 165-170°C
(Formula Removed)
Anal
mp 165-170°C
IR(KBr) 3300, 2930, 1700, 1650 and 1235 cm '
'H-NMR (CDC13) 8 0 89 (s, 3H, 18-C/fc), 1 1 (s, 3H 19-Ci/j), 4 07 (m, 1H, 3a-H),
5 40 (br,lH,6-vinyhc) and 6 84-7 28 (m, 5H, aromatic)
Calcd for C26H33NO3 N, 3 44 Found N, 3 74
EXAMPLE 5
17-OXIMINO-5-ANDROSTEN-3β-YL 4-AMINOBENZOATE (14)
/7-Aminobenzoic acid (0 22 g, 1 6 mmol) was added to the stirred solution of 17-oximino-5-androsten-3β-ol (9) (0 5 g, 1 6 mmol) and dicyclohexylcarbodnmide (DCC) (0 34 g, 16 mmol) in anhydrous dichloromethane (30 0 ml) The reaction mixture was stirred for 48 hr at room temperature (20-25°) and completion of the reaction was monitored by TLC The precipitated dicyclohexylurea (DCU) was filtered, solvent removed under vacuum and the residue was crystallized from ethyl acetate petroleum ether (60 80) to obtain 17-oximino-5-androsten-3β-yl 4-aminobenzoate (14 ) (0 28 g, 56 0%) mp 165-168°C
(Formula Removed)
Anal
mp 165-168°C
IR(KBr) 3330, 2930, 1700, 1630 and 1240 cm '
'H-NMR (CDCb) 5 0 93 (s, 3H, I8-C//3), 1 05 (s, 3H \9-CH3), 2 17 (1H, NOtf), 4 08 (m, 1H, 3a-H), 6 20 (br, 1H, 6-vinyhc), 7 22 (d, 2H, 3-CH and 5-CH aromatic) and 8 26 ppm (d, 2U,2-CH and 6-CH aromatic) Calcd for C26H34N2O3 N, 6 63 Found N, 6 79
EXAMPLE 6
17-OXIMINO-5-ANDROSTEN-3β-YL 4-HYDROXYBENZOATE (15)
17-Oximino-5-androsten-3β-ol (9) (0 5 g, 1 6 mmol) was taken in anhydrous dichlorormethane (30 0 ml) Dicyclohexylcarbodnmide (DCC) (0 34 g, 16 mmol) was added followed by /7-hydroxybenzoic acid (0 23 g, 1 6 mmol) Reaction mixture was stirred at room temperature for 24-48 hr and completion of the reaction was monitored by TLC The reaction mixture was filtered to remove precipitated dicyclohexylurea (DCU) Excess of solvent was removed under vacuum and the product was crystallized from ethyl acetate petroleum ether (60 80) to yield 17-oximino-5-androsten-3(3-yl 4-hydroxybenzoate (15 ) (0 31g, 62 0%) mp 177-182° C
(Formula Removed)
Anal
mp 177-182° C
IR(KBr) 3310, 2930, 1710, 1680 and 1235 cm '
1H-NMR (CDCI3) δ 0 89 (s, 3H, 18-CH5), 1 05 (s, 3H 19-CH3), 2 17 (IH, NO//),
4 18(m, IH, 3α-H;, 6 35 (br, IH 6-vinylic) 6 83 (d, 2H, 3-CH and 5-CH aromatic)
and 7 46 ppm (d, 2H, 2-CH and 6-CH aromatic)
Calcd for C26H33NO4 N, 3 31 Found N, 3 32
EXAMPLE 7
17-OXIMINO-5-ANDROSTEN-3β-YL PHENOXYACETATE (16)
To the stirred solution of 17-Oximino-5-androsten-3β-ol ( 9) (0 5 g, 1 6 mmol) and dicyclohexylcarbodnmide (DCC) (0 342 g, 1 6 mmol) in anhydrous dichloromethane (30 0 ml), phenoxyacetic acid (0 24 g, 1 6 mmol) was added The reaction mixture was stirred for 48 hr at room temperature (20-25°) and completion of the reaction was monitored by TLC The precipitated dicyclohexylurea (DCU) was filtered and the solvent removed under vacuum The resulting residue was crystallized from ethyl acetate petroleum ether (60 80) to give 17-oximino-5-androsten-3β-yl phenoxyacetate (16 ) (0 27 g, 54 0%) mp 118-122°C
(Formula Removed)
Anal
mpl 18-122° C
IR(KBr) 3200, 2940, 1750, 1650 and 1220 cm '
1H-NMR (CDC13) δ 0 88 (s, 3H, I8-CH3), 1 02 (s, 3H 19-CH3), 4 60 (s, 2H, OCH2),
4 7 (m, 1H, 3α-H), 5 40 (br, 1H, 6-vinyhc) and 7 29(m, 5H, aromatic)
Calcd for C27H35NO4 N, 3 20 Found N, 3 60
Biological Evaluation
The synthesized steroidal derivatives have been evaluated for in vitro antiproliferative
activity using DU-145 cell lines and in vivo studies 1 e 5α-Reductase Inhibitory activity [George, F W et al Endocrinology, 1989,125, 2434]
1) In vitro antiproliferative activity against human prostate cancer cell line (DU-145)
Compounds were screened for antiproliferative activity on human prostate cell
line DU-145 at National Centre of Human Genome Studies and Research Panjab
University, Chandigarh All the compounds (10 to 16), Finasteride and starting material (9) were tested at five different concentrations 0 01, 0 5, 1 0, 2 0, and 5 0 µg/ml Finasteride was the positive control in this growth inhibitory assay
Growth inhibitory activity assay (MTT assay)
We investigated the effect of newly synthesized compounds on cell growth inhibition using MTT assay This assay quantifies the viable cells by observing the reduction of tetrazohum salt, MTT to formazan crystals by the live cells Based on the absorbance of the cell sample after the test is carried out, cell viable can be measured [Loveland, B E , Jones, T G , Mackay I R „ Vaillant, F , Wang, Z X , Hertzog, P J Bwchem International 1992, 27, 501 and Wright, S A , Thomas, L N , Douglas, R C , Lazier, C B , Rittmaster, R S J Chn Invest 1996, 98, 2558] Cells were cultured at a density of 5 x 103 cells / well in 96 well plates at 37°C in 5 0% CO2 atmosphere and were allowed to attach for 24 hr The cells were treated in triplicate with graded concentration of sample and reference drug Finasteride at 37°C for 48 hr A 20 µl aliquot of MTT solution was added directly to all the appropriate wells Following 4 hr of incubation at 37µl, the media was removed and formazan crystals, which results
from the reduction of MTT by active cell were dissolved in 100µl DMSO and vigorously mixed to dissolve the reacted dye The absorbance of each well was read on Ehsa plate reader (Merck) at 570 nm The spectrometer was calibrated to zero absorbance using culture medium without cells The relative cell viability (%) related to control well containing cell culture medium without drug was calculated by [A] test/[A] control x 100
% Growth Inhibition^ [OD] control - [OD] test/ [OD] control X 100
Results*
All the newly synthesized compounds were tested for antiproliferative
activity using DU-145 cell line The results are expressed in terms of percentage growth inhibition (Table 1) and percentage viable cells (Figure 5)
Esters of 17-oximino-5-androsten3ß-ol (10-16) , synthesized in our lab revealed a great reduction in the level of cellular cytotoxicity as compared to Finasteride (3) Examination of data of these analogues 10 to 16 demonstrated a great tolerance of phenyl ring and its parα-substituted analogues Unsubstituted analogue 13 and parα-
substituted (electron donating group) compounds 12, 14 and 15 were found to be the most active compounds with >90% growth inhibition at concentration of 5 0 ug/ml relative to Finasteride (78% at 5 0 µg/ml) (Table 1) On the other hand analogues 10, 11 with electron withdrawing substituent decreased the growth of DU-145 cancer cell lines from 81-84%(relatively less), but still better than Finasteride (78%) Parent Compound 9 has not shown any significant inhibitory effect on cell proliferation
Table 1 Antiproliferative activity of Finasteride and Compounds 9 to 16

(Table Removed)
N, = no significant growth inhibition
Number of ester derivatives bearing oxime group (NOH) at C-17, connected directly to the steroidal D ring were synthesized and screened in vitro (Table 1 and Figure 5) Examination of data of these analogues 10 to 16 demonstrated a great tolerance of phenyl ring and its parα-substituted analogues This is in agreement with the previous reports that have pointed out the importance of aromatic and β-substituted aromatic esters Unsubstituted analogue 13 and compounds 12, 14 and 15 with an electron donating moiety at para position have been found to be more potent than the reference Finasteride Compounds 12, 14 and 15 were found to be the most active with >90% growth inhibition at concentration of 5 0 µg/ml relative to Finasteride (78% at 5 0 µg/ml) On the other hand analogues 10, 11 with electron withdrawing substituents
demonstrated relatively less activity than above compounds but still better than Finasteride Parent Compound 9 has not shown significant antiproliferative activity
b) In vivo (effect on steroid androgen level)
Treatment of animals
Animals (Sprague Dawley male rats weighing 150-200 g) were divided into 3 groups, vehicle (control), Finasteride (standard), treated (test sample) and each group consist of 5 animals Sprague Dawley rats were treated intraperitoneally with Finasteride 40 mg/kg and equimolar dose of compounds After 6 hr of treatment, blood was withdrawn by cardiac puncture under diethyl ether anesthesia and serum was separated from cells by centnfugation Plasma testosterone values were obtained by ELSIA plate reader at 550 nm and are given in ng/ml Ehsa
The aliquots of 50ul of each of standards, control and unknown (serum samples) were added to Testosterone Antibody coated wells 100µl of HRP-testosterone conjugate was added to all the wells and the plates were shaken gently on a shaker for proper mixing of the reagents Following 4 hr of incubation at 37°C, the incubation mixture was removed The wells were washed with phosphate buffer for 5-6 times (200µl each time), followed by addition of 100ul of TMB / H2O2 substrate in each of the wells The plates were further incubated at 37°C for 20 minutes At the end of incubation reaction was stopped by using 100ul of 0 5 M H2SO4 as stopping reagent The absorbance of each well was read on Ehsa plate reader at 450 nm [Rassaie, M J , Kuman, G L , Pandey, P K , Gupta, N , Kochupillai, N , Grover, P K Steroids 1992, 57, 288 and Hosoda, H , Yoshida H, Sakai, Y , Miyam, S , Nambara, T Chem Pharm Bull 1980, 28, 3035] Results
The effect of compounds (10 to 16) and Finasteride on the serum concentration of testosterone are reported in Table 2 and Figure 6
Serum 'T' level were increased in Finasteride treated rats (1 26±0 02 ng/ml compared with 0 742±0 07 ng/ml control rats) It is apparent from Table 2 and Figure 6 that almost all the ester derivatives of 17-oximino-5-androsten-3ß-ol were able to increase the serum testosterone level as compared to control except compound 15 The 6 hr post dose serum T levels were 1 05±0 08 ng/ml for the compound 13 with
unsubstituted phenyl group Ester derivatives 12 and 14 with electron releasing group at β-position of phenyl ring has been found to have increased activity, while presence of electron withdrawing moiety at this position causes the loss of activity (10, 11) Significant increase in level of serum testosterone level has been found in the compound 16
Table 2 Serum levels of Testosterone (Table Removed)

Intact male rats (Sprague Dawley, 200-250 g) were used in the designed study in which various compounds were compared for in vivo 5α-reductase inhibitory potency, as judged by the their ability to attenuate the conversion of testosterone into dihydrotestosterone (DHT) ELISA for testosterone were found to be suitable for determination in serum of rate since the cross reactive DHT levels were extra low in male The procedure measure T equally well, and method met all the requirement of precision, accuracy, sensitivity and selectivity
c) In vitro cytotoxicity using mouse macrophages (acute toxicity)
In vitro cytotoxicity test using DU-145 cells in the preliminary evaluation of anticancer drug enable us to select most potent compound, but cytotoxic agents however frequently exhibit-unspecific toxicity Nevertheless the ability to selectively kill the target cell remains a highly desirable property of potential new therapeutic cytotoxic agents In this study, we have demonstrated the applicability of Red dye uptake (MTT) assay using mouse macrophages (Balb C) for in vitro toxicity testing of newly synthesized compounds
Preparation of test material
All steroids were dissolved in ethanol and diluted to appropriate concentration
0 01, 0 5, 1 0, 2 0, 5 0 µg/ml from the two stock solution of 1 mg/ml and 0 0014 µg/ml Stocks were maintained at room temperature
Cell Viability Assay
Isolation of mouse macrophages
Albino mice (laca strain) weighing 20-25 g of either sex were sacrificed by
cervical dislocation and small cut was given on peritoneal skin with a help of scissors Approximately 1 ml of Phosphate buffer saline preincubated at 37°C was injected into cavity The abdomen was lavaged for 10 mm and fluid along with macrophages were taken out using a syringe into polypropylene tube The phosphate solution was spun at 800 rpm for 5-10 mm and cells were resuspended in 1 ml RPMI media Cell number was counted using haemocytometer
Final cell count =Total cell count x 1000 x dilution factor
0 1x5
0 1 = chamber depth, 5 Number of squares (each square is 1 mm)
MTT assay
Cells (mouse macrophages) were plated at a density of 5 x 103 cells / well in 96 plate at 37°C in 5% CO2 Cells were exposed in graded concentration of compounds at designated various concentration Each concentration was tested in triplicate wells After 48 hr fresh MTT 20ul (1 mg/ml) was added directly to all the wells and culture was incubated for 4 hr at 37°C During this incubation, MTT was converted into a water insoluble formazan complex by metabolic activity of viable cells Formazan crystal were taken and dissolved in l00µl of DMSO, which give light pink colour The absorbance of each well was read on Elisa plate reader (Merck) at 570 nm The spectrometer was calibrated to zero absorbance using culture medium without cells The relative cell viability (%) related to control well containing cell culture medium without drug was calculated by [A] test / [A] control x 100 % Cell Viability = [A] test/ [A] controi X 100 [A] test= absorbance test sample [A] control= absorbance control sample
Results: LC50 Concentration of the compound in µg/ml (µm), lethal to 50%
population of cells
Red dye uptake (MTT) assay, demonstrated the toxicity of the compounds towards normal cell by using mouse macrophages The assay quantifies the viable cells, after 24 hr incubation of cells with five different concentrations Results in Table 3 and Figure 7 demonstrated a direct and proportional relation between cell number and concentration
The results obtained from MTT assay were statistically significant (P Table 3 % Viable Cells of derivative (10 to 16 ) (Table Removed)

Table 4 Acute Toxicity of the compounds (Table Removed)
In vitro cytotoxicity test using DU-145 cells in the preliminary evaluation of anticancer drug enable us to select most potent compound, but cytotoxic agents however frequently exhibit-unspecific toxicity Nevertheless the ability to selectively kill the target cell remains a highly desirable property of potential new therapeutic cytotoxic agents In this study, we have demonstrated the applicability of Red dye uptake (MTT) assay using mouse macrophages (Balb C) for in vitro toxicity testing of newly synthesized compounds The assay quantifies the viable cells, after 24 hr incubation of cells with five different concentrations Figure 7 and Table 3 demonstrated a direct and proportional relation between cell number and concentration
Data from this study Table 4 clearly indicated that compounds 13 and 16 with high LCso values were non toxic to mouse macrophages Acute toxicity of the compounds 10-12,14 and 15 were comparable to finasteride
We have synthesized seven new steroidal esters derivative of 17-oximino -5-androsten-3-ol, starting from Dehydroandrosterone acetate Newly synthesized compounds were tested for their antiproliferative activity and 5α-reductase inhibitory activity in comparison to Finasteride Decreased androgen level have been found in serum of animal treated with newly synthesized compounds These compounds have also shown better cytotoxicity in comparison to reference drug Finasteride Thus such compounds can be useful in treatment of androgen dependent disorder of prostate alone or by synergistic effect they can decrease the size of prostate due to their antiproliferative activity

We claim:
1. A novel steroidal esters of 17-oximino-5-androsten-3β-ol of general formula A,
pharmaceutically acceptable salts thereof;
(Formula Removed)
General formula A wherein R is selected form a group consisting of arylalkyl, aryl, substituted aryl.
2. A compound as claimed in claim 1 wherein the arylalkyl group is selected from a group consisting of benzyl, phenoxymethyl or substituted arylalkyl such as 4-chlorobenzyl, 4-nitrobenzyl, 4-methylbenzyl, 4-aminobenzyl, 4-chlorophenxoymethyl, 4-nitrophenoxymethyl, 4-methylphenoxymethyl,4-aminophenoxymethy 1.
3. A compound as claimed in claim 1 wherein the aryl group is selected form a group consisting of (i) 4-chlorophenyl (10); (ii) 4-nitrophenyl(ll); (iii) 4-methylphenyl (p-tolyl)(12) ; (iv) phenyl (13); (v) 4-aminophenyl (14); (vi) 4-hydroxyphenyl ( 15); (vii) phenoxymethyl (16).
4. A compound as claimed in claim 1 wherein the pharmaceutically acceptable salt is selected form a group consisting of hydrochloride, sulphates, phosphates acetates, propionates; and sodium , potassium and calcium salts of the phenolic hydroxyl groups containing substrates .
A compound as claimed in claim 1 wherein the representative compounds of formula A as claimed in claim 1 comprising;
(i) 17-oximino-5-androsten-3β-yl 4-chlorobenzoate (10) (ii) 17-oximino-5-androsten-3β-yl 4-nitrobenzoate (11)
(iii) 17-oximino-5-androsten-3β-yl 4-methylbenzoate (12)
(iv) 17-oximino-5-androsten-3β-yl benzoate (13)
(v) 17-oximino-5-androsten-3β-yl 4-aminobenzoate (14)
(vi) 17-oximino-5-androsten-3β-yl 4-hydroxybenzoate (15)
(vii) 17-oximino-5-androsten-3β-yl phenoxyacetate (16)
5. A compound as claimed in claim 1 wherein the compounds are useful as 5a-reductase inhibitor, androgen dependent disorders.
6. A compound as claimed in claim 1 wherein the compounds showed antiprolifirative activity with a >90% growth inhibition of DU-145 cell at a concentration of 5.0µg/ml.
7. A compound as claimed in claim 1 wherein the compounds showed increase of serum level of testosterone as compared to control at a dose of 40mg/kg.
8. A compound as claimed in claim 1 wherein the compounds having LC50 in the range of 19.5 to 89.4 µm.
9. A compound as claimed in claim 1 wherein the compounds are non-toxic at a concentration ranging between 0.0lµg/ml to 5.0µg/ml
10. A process for preparation of novel steroidal esters of 17-oximino-5-androstene-3β-ol of formula a as claimed in claim 1, wherein the process comprising :
(I) reacting 17-oximino-5-androsten-3β-ol with an organic acid and
dicyclohexylcarbodiimide (DCC) in anhydrous dichloromethane for a period ranging
between 24-48 hrs at a temperature ranging between 20 to 25°C,
(ii) filtering the precipitated dicyclohexylurea (DCU) and removing the solvent from
the filtrate under vacuum and the residue was crystallized to obtain the desired
product.
11. A pharmaceutical composition comprising: a therapeutically effective amount of a
compound of claim lor a pharmaceutically acceptable salt thereof optionally along
with a pharmaceutically acceptable carrier, excipient, diluent.
12. A novel steroidal esters of 17-oximino-5-androstene-3β-ol of formula A, process for preparation and a pharmaceutical composition thereof substantially as herein described with reference to the examples and drawings accompanying the specification.

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Patent Number

268287

Indian Patent Application Number

1805/DEL/2007

PG Journal Number

35/2015

Publication Date

28-Aug-2015

Grant Date

25-Aug-2015

Date of Filing

24-Aug-2007

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110 001,INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	TILAK RAJ BHARDWAJ	UNIVERSITY INSTITUTE OF PLCENT SCIENCES(UIPS),PUNJAB UNIV.,CHANDIGARH-160014
2	MANOK KUMAR	UNIVERSITY INSTITUTE OF PLCENT SCIENCES (UIPS),PUNJAB UNIV.,CHANDIGARH-160014
3	NEELIMA DHINGRA	UNIVERSITY INSTITUTE OF PLCENT SCIENCES(UIPS),PUNJAB UNIV.,CHANDIGARH-160014
4	NEERAJ MEHTA	UNIVERSITY INSTITUTE OF PLCENT SCIENCES (UIPS),PUNJAB UNIV.,CHANDIGARH-160014

PCT International Classification Number

N/A

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA