PREPARATION THEREOF

FIELD OF INVENTION

[0001] The present invention relates to 16a-heteroayl-l -yl pregnenolone acetate and a process for its preparation. The present invention particularly relates to compounds of 16cc-(lH-benzimidazol- l -yl)-pregnenolone acetate (1) and 16ct-(lH-imidazol- l -yl)- pregnenolone acetate (2). The present invention also relates to a process for the preparation of said compounds froml6-dehydropregnenolone acetate (16-DPA). More particularly the present invention relates to the anticancerous activity of the said compounds (1 and 2).

BACKGROUND OF THE INVENTION

[0002] Benzimidazole, imidazole and their derivatives are very important pharmacophores in medicinal chemistry showing a wide range of biological activities like antibacterial, antifungal, anticancer, antiviral, antituberculosis and antiinflammatory and antiageing agents.

[0003] Synthesis of several modified steroids comprising aza heterocyclic rings either fused or linked to the steroid framework have received considerable attention in the recent years due to their wide range of biological activities. These steroidal heterocycles are well known as inhibitors of enzymes such as 5 -reductase, 1 7 -Iyase and aromatase. Steroids comprising heterocyclic rings such as pyridine, imidazole, benzimidazole and diazine ring linked to 17-position show anticancer activity. Even 16-substituted steroids either by heterocycles or nonheterocyclic compounds have shown wide range of pharmacological activities. For example many \ 6E- arylidenosteroids are reported which show in vitro activity against many types of tumor cells. Even, 16-substituted steroids with imidazole heterocycle such as 16/5- imidazolyl androsterone derivatives are known which are synthesized using 17-oxo-5- androsten-3 ?-ol as the starting material and these compounds are found to exhibit moderate cytotoxic effect in sixty cancer cell lines derived from nine cancer types.

[0004] Conjugated addition of benzimidazole/imidazole to a, ?-unsaturated carbonyl compounds are known using Lewis acid catalyzed addition, base catalyzed addition, high pressure promoted addition, iodine catalyzed addition, and acid catalyzed addition reactions. Unfortunately, many of these processes suffer from limitations, such as the use of expensive catalyst, expensive reagents, harsh conditions, relatively long reaction times, high catalyst loading, tedious work-up procedures and use of hazardous organic solvents. Hence, the development of a sustainable environment as an alternative procedure for Michael addition reaction avoiding the use of harmful solvents and expensive catalysts remains a challenging task to organic chemists. On the other hand, microwave-assisted organic synthesis (MAOS) which is recognized as a "green" technology has been applied as a very efficient way to accelerate the course of many organic reactions, producing high yields, higher selectivity, and lower quantities of side products.

[0005] Reference may be made to G A Potter, S E Barrie, M Jarman & M G Rowlands J. Med. Chem. 1995, 38, 2463 wherein, steroidal compounds having 17-(3- pyridyl) substituent together with 16, 17-double bond were synthesized which showed potent inhibition of human testicular 17«-hydroxylase-C i 7 2o-lyase. [0006] Another reference may be made to V D Handrattu, T S Vasaitis, V C O Njar, L K Gediya, R Kataria, P Chopra, D Newman, Jr.R Farquhar, Z Guo, Y Qi & A M H Brodie J. Med. Chem. 2005, 48, 2972 wherein, the syntheses and biological activities of novel 17-benzoazoles and 17-diazines were reported. The 17-benzimidazoIe steroidal compounds were shown to exhibit potent in vitro activities for CYP 17 inhibition, AR antagonism and inhibition of prostate cancer cell growth.

[0007] Still another reference may be made to R D Bruno, TS Vasaitis, L K Gediya, P Purnshottamachar, A M Godhole, Z Ates-Alagoz, A M H Brodie & V C O Njar Steroids 2011, 76 1268 wherein it was found that 17-benzimidazole steroidal compound VN/ 124-1 was a potent inhibitor of human prostate cancer tumor growth and was remarkably more effective than castration or compound abiraterone that is currently undergoing phase III clinical trials in prostate cancer patients. [0008] Still another reference may be made to R Bansal & S Guleria Steroids 2008, 73, 1391 wherein the synthesis of 16-substituted steroidal compounds such as 16E-[3- methoxy-4-(2-aminoethoxy)-benzylidene]androstene derivatives and their cytotoxicity studies were reported.

[0009] Still another reference may be made to R Bansal, S Guleria, S Thota, S L Bodhankar, MR Patwardhan, C Zimmer, R W Hartmann & A L Harvey Steroids 2012, 77, 621 wherein, syntheses of 16 ?-imidazolyl-17-keto steroids and its 16 ?-imidazolyl- 17/?-hydroxyl derivatives were reported which exhibited moderate cytotoxic effects in sixty cancer cell lines derived from nine cancers types.

[0010] Still another reference may be made to J P Morrison, C A Stein, D S Casebier, J Morrison, C Stein and D Casebier WO/2011/017534 _^ wherein, compounds, pharmaceutical compositions and medicaments comprising such compounds, and methods of using such compounds that decrease androgen biosynthesis, decrease androgen receptor signaling and decrease androgen receptor sensitivity are reported.

[0011] Still another reference may be made to R Bansal, S Guleria, G Narang and H R Wolfgang WO/2007/031833, wherein a novel series of imidazolyl substituted steroidal and indan-l -one derivatives are reported.

[0012] Still another reference may be made to A Brodie and V C O Njar US5994335, wherein 17-azolyl steroids are reported to be useful as androgen synthesis inhibitors.

OBJECTIVES OF THE INVENTION

[0013] The main object of the present invention is to provide 16a-heteroayl- l -yl pregnenolone acetate and a process for preparation of the compounds. [0014] Another object of the present invention is to provide compounds 16a-( lH- benzimidazol-l -yl)-pregnenolone acetate (1) and 16ec-(lH-imidazol-l -yl)- pregnenolone acetate (2).

[0015] Another object of the present invention is to provide a process for the preparation of 16a-(lH-benzimidazol-l -yl)-pregnenolone acetate (1) and 16a-(lH- imidazol-l -yl)-pregnenolone acetate (2) from 16-dehydropregnenolone acetate ( 16- DPA) without using solvent and catalyst under microwave irradiation.

[0016] Still another objective of the present invention is to evaluate the in vitro cytotoxic activities of compound 1 and 2 against cervical HeLa cancer cell line, prostate DU 205 cancer cell line and breast cancer MCF-7 cell line in comparison to the drug doxorubicin.

SUMMARY OF INVENTION

[0017] Accordingly the present invention provides a compound of formula A,

[0018] In an embodiment of the present invention compound, wherein structural formula of representative compound comprising are:

1 ( C ₃₀ H ₃₈ N ₂ O ₃ ) 2 (C ₂₆ H ₃₆ N ₂ 0 ₃ ) [0019] In another embodiment of the present invention a compound wherein the formula is represented by the group of the following compounds:- a) 16a-(lH-benzimidazol-l -yl)-pregnenolone acetate (1)

b) 16a-(lH-imidazol-l -yl)-pregnenolone acetate (2)

[0020] In another embodiment of the present invention a compound wherein 16a- (lH-benzimidazol-l -yl)-pregnenolone acetate (1) and 16a-(lH-imidazol-l-yl)- pregnenolone acetate (2) exhibited in vitro cytotoxic activity against cervical HeLa cancer cell line, prostate DU 205 cancer cell line and breast cancer MCF-7 cell line.

[0021] In another embodiment of the present invention a compound wherein cytotoxic activities of compound 1 with ICso= 8.3317 against HeLa cancer cell line, IC ₅ o= 12.0192 against prostate DU 205 cancer cell line and IC ₅₀ = 8.2855 against breast cancer MCF-7 cell line.

[0022] In yet another embodiment of the present invention a compound wherein cytotoxic activities of compound 2 with IC ₅₀ = 48.2584 against HeLa cancer cell line, IC ₅₀ = 37.8069 against prostate DU 205 cancer cell line and IC ₅₀ = 20.0288 against breast cancer MCF-7 cell line.

[0023] In yet another embodiment of the present invention a process for the preparation of compound of formula ( I ) wherein the said process comprises of following steps:- a) mixing a, ?-unsaturated ketone 16-DPA with compounds selected from benzimidazole or imidazole in mortar,

b) irradiating the mixture obtained in step a) in a microwave reactor at a frequency of 2450 MHz for a time period ranging between 10 to 30 minutes,

c) purifying the crude product obtained in step b) by silica gel column chromatography using EtOAc-hexane taken in the ratio of 1 :4. [0024] In another embodiment of the present invention a process wherein the compounds are prepared by solvent free and catalyst-free method by the Michael addition of benzimidazole to 16-DPA under microwave irradiation.

[0025] In another embodiment of the present invention a process, wherein the yield of the compound of formula A is as high as 47% to 86%.

BRIEF DESCRIPTION OF DRAWINGS & FIGURES

[0026] Figure 1 shows the synthesis of compounds 16a-(lH-benzimidazol-l -yl)- pregnenolone acetate (1) and 16a-(lH-imidazol-l -yl)-pregnenolone acetate (2) from 16-DPA. [0027] Figure 2 shows the key NOESY correlations and relative stereochemistry of compounds 1 and 2. From this correlation the trarcs-orientation of the substituents in 16 and 17 positions was confirmed.

DETAILED DESCRIPTION OF THE INVENTION

1 ( C ₃₀ H38N ₂ O ₃ ) 2 (C2BH3 ₆ N ₂ 0 ₃ )

[0028] The invented compounds l 6a-( l H-benzimidazol- l -yl)-pregnenolone acetate (1) and 16a-( l H-imidazol- l -yl)-pregnenolone acetate (2) are of pregnenolone acetate. One benzimidazole group and imidazole group are substituted at 16-position in compounds 1 and 2, respectively. These compounds were characterized by Ή NMR, l ³ C NMR and mass spectroscopy. These substituents at 16 position are a-oriented which are /ra -orientated with respect to the acetyl group at C- l 7 position. This /ram- orientation is proved by the NOESY spectra of both the compounds 1 and 2. Compound 1 is a yellow solid, (m.p.: 1 1 8 °C) and compound 2 is a brown solid (m.p.: 131 °C). Both the compounds 1 and 2 show in vitro cytotoxic activity against cervical HeLa cancer cell line, prostate DU 205 cancer cell line and breast cancer MCF-7 cell line.

[0029] Accordingly, the present invention provides a process for the preparation of 16«-( lH-benzimidazol-l -yl)-pregnenolone acetate (1) and 16oc-(lH-imidazol-l -yl)- pregnenolone acetate (2) by the solvent-free and catalyst-free Michael addition of nitrogen containing heterocycles benzimidazole and imidazole to 16- dehydropregnenolone acetate under microwave irradiation. The present invention also provides biological evaluation of compounds 1 and 2 as cytotoxic agent. [0030] The reaction of equimolar amount of 16-DPA (1) and benzimidazole in protic solvents ethanol and isopropanol under thermal heating conditions could not provide the product 1 (Figure 1 ). Similar results were obtained using nonprotic solvents acetonitrile and dimethylformamide under reflux condition (8 hours). Interestingly, when the reaction was performed in a solvent-free condition, under thermal heating ( 140 °C) for 8 hours, the product 1 was obtained in satisfactory yield (40%). It was noteworthy to observe that finely ground equimolar amounts of 1 and benzimidazole under microwave irradiation (720 Watt) for 10 minutes afforded 1 in 69% yield. The formation of 16et-substituted N-heterocycles having trans orientation with respect to the acetyl group at C- 17 position of the steroid moiety was determined by comparing the optical rotational data of these hetero steroids with the reported 16-substituted steroidal heterocycles by Gould et al. (D Gould, E L Shapiro, L E Finckenor, F Gruen & E B Hershberg J. Am. Chem. Soc. 1956, 78, 3158). The a orientation of benzimidazole at 16-position and the β orientation of acetyl group at 17-position was then confirmed by NOESY spectrum of compound 1. In the NOESY spectrum of compound 1, the presence of correlation of CH ₃ -18 protons with H- 16 and CH3- I 8 protons with H-21 protons and the absence of correlation between CH3- I 8 protons with H- 1 7 also indicated that H- 16 and H- 17 were β and a oriented respectively in compound 1 (Figure 2). Similarly, the reaction of 16-DPA with imidazole under microwave irradiation (720 Watt) for 10 minutes afforded Michael adduct 2 in 86% yield under microwave irradiation (Figure 1 ).

16-DPA

Figure 1 [0031] In vitro cytotoxic activity studies of 16a-( lH-benzimidazol-l -yl)- pregnenolone acetate (1):

[0032] These cell lines were procured from ATCC in 2009. Catalog numbers HeLa (ATCC®CCL-2TM)-15th passage cells used for the present experiment. DU 145 (ATCC® HTB-81™)-12 the passage cells used for the present experiment. MCF7 (ATCC® HTB-22™)-l 5 the passage cells used for the present experiment.

[0033] Cellular viability in the presence of test compounds was determined by MTT- micro cultured tetrazolium assay. The cells seeded to flat bottom 96 ( l OOOcells/Ι ΟΟμΙ) well plates and cultured in the medium containing 1 0% serum and al lowed to attach and recover for 24 hours in a humid chamber containing 5% C0 ₂ MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; sigma catalog No M2128) was dissolved in PBS at 5 mg/mL, filtered to sterilize and remove a small amount of insoluble residue present in MTT. Then different concentrations of the compound were added to the cells. After 48 hours, stock MTT solution ( 10 μΐ) was added to the culture plate. Cells were again kept in C0 ₂ incubator for 2 hours. After incubation, 100 μΙ of DMSO was added and mixed. The absorbance was read at 562 nm in a plate reader. The results were represented as percentage of cytotoxicity/viability. All the experiments were carried out in triplicates. From the percentage of cytotoxicity the 1C- 50 value was calculated (S Myadaraboina, M Alia, V Saddanapu, V R Boenaa & A Addlagatta. Eur. J. Med. Chem. 2010, 45, 5208).

[0034] As showed in Table 1 , compound 1 displayed good cytotoxicity to the tested cancer cell lines. Compound 1 had IC ₅₀ values of 8.3317, 12.0192 and 8.2855 μΜ against HeLa cell line, DU 145 cell line and MCF-7 cancer cell lines, respectively which is almost comparable to the drug doxorubicin. Compound 2 exhibited moderate activity against the tested cell lines. It had IC ₅₀ values of 48.2584, 37.8069 and 20.0288 μΜ against HeLa cell line, DU 145 cell line and MCF-7 cancer cell lines, respectively.

Table 1. IC50 values of compound 1 &2 determined by MTT assay

[0035] The steroidal conjugated enone 16-DPA could be converted into corresponding Michael adduct 1 and 2 with benzimidazole and imidazole, respectively in high yield under microwave irradiation. The steroidal ketone 1 has shown cytotoxic activity similar to drug doxorubicin against I leLa cancer cell line, prostate DU 205 cancer cell line and breast cancer MCF-7 cell line.

EXAMPLES

[0036] The following examples are given by illustrations and should not construe the scope of the invention.

Example-I

[0037] Preparation of 16ct-( 1 H-benzimidazol- 1 -yl)-pregnenolone acetate (1): α, Jnsaturated ketone 16-DPA (356 mg, 1 .0 mmol) and benzimidazole (1 18 mg, 1 .0 mmol) were mixed intimately in a mortar and irradiated in a microwave reactor (720 Watt, 2450 MHz) for 10 minutes. On completion of the reaction (by TLC), the crude product was purified by silica gel column chromatography using EtOAc-hexane (1 :4) as the eluent to afford steroidal Michael adduct 1. (Yield 69%, 327 mg). The crude product can be purified by neutral alumina also using EtOAc-hexane (1 :4) as the eluent to afford steroidal Michael adducts 1. (Yield 67%, 318 mg). Yellow solid, m.p.: 1 17 °C;

R _f = 0.3 (EtOAc/Hexane = 1 :4); [a] _D = -6.6 (c, 0.5, CHC1 ₃ ); IR (CHC1 ₃ , cm- ¹ ): 2927, 2856, 1732, 1709, 1655, 1361, 1250, 1033, 762; Ή NMR (CDC1 ₃ , 300 MHz): δ 8.10 (s, 1H), 7.79 (dd, 1H, J = 6.8 & 3.5 Hz), 7.50 (dd, 1H, J = 8.2 & 2.0 Hz), 7.33-7.23 (m, 2H), 5.59-5.53 (m, 1H), 5.41-5.38 (m, 1H), 4.66-4.60 (m, 1H), 3.06 (d, 1H, J = 8.5 Hz), 2.09 (s, 3H), 2.05 (s, 3H), 1.07 (s, 3H), 2.36-0.89 (m, 17H), 0.81 (s, 3H); ¹³ C NMR (CDC1 ₃ , 75 MHz): δ 206.2, 170.6, 144.0, 142.1 , 139.9, 132.7, 122.9, 122.2, 121.7, 120.5, 1 10.5, 73.7, 70.6, 58.4, 56.6, 54.6, 49.7, 45.2, 38.6, 38.0, 36.9, 36.6, 32.0, 31 .6, 27.7, 21 .4, 20.7, 19.3, 18.4, 13.9; MS (EI, m/z) = 474 (M ⁺ ), 414 (M ⁺ -60). Anal, calcd. for C30H38 2O3 : C, 75.92; H, 8.07; N, 5.90. Found: C, 76.22; H, 8.18; N, 6.03.

[0038] Preparation of 3p-acetoxy- 16a-( l H-imidazol-l -yl)-17p-acetyl-androst-5-ene

16 DPA (356 mg, 1 .0 mmol) and imidazole (68 mg, 1.0 mmol) were mixed intimately in a mortar and irradiated in a microwave reactor (720 Watt) for 10 minutes. On completion of the reaction (by TLC), the crude product was purified by silica gel column chromatography using EtOAc-hexane ( 1 :4) as the eluent to afford steroidal Michael adduct 2. Brown solid, m.p.: 130 °C. Yield 86% (365 mg); R _r : 0.4 (EtOAc/Hexane = 3:7); [a] _D = -8.4 (c 0.5, CHCI ₃ ); IR (CHCI ₃ ) cm ^" ' : v 2943, 2854, 1731 , 1705, 1649, 1 364, 1247, 1034, 754; Ή NMR (CDC1 ₃ , 300 MHz): δ 7.55 (s, 1 H), 7.03 (s, 1 H), 6.91 (s, 1 H), 5.40-5.37 (m, 1 H), 5.30-5.22 (m, 1 H), 4.67-4.57 (m, 1 H), 2.80 (d, 1 H, J = 7.8 Hz), 2.10 (s, 3H), 2.04 (s, 3H), 1 .04 (s, 3H), 0.73 (s, 3H), 2.40-0.89 (m, 1 7H); ^{l 3} C NMR (CDC1 ₃ , 75 MHz): δ 206.0, 170.6, 141 .4, 139.8 (2C), 121 .7 (2C), 73.7, 72.8, 56.1 , 55.8, 49.5, 45.2, 38.0, 36.6, 33.6, 31.9, 31.5, 29.7, 29.4, 27.7, 22.7, 21.4, 20.6, 19.3, 14.1 ; MS (EI) m/z = 364 (M ⁺ -60), 296 [(M ⁺ -60)-imidazole]. Anal, calcd. for C ₂₆ H36N ₂ 0 ₃ : C, 73.55; H, 8.55; N, 6.60; Found: C, 73.69; H, 8.51 ; N, 6.80. Example-II

[0039] Preparation of 16a-(lH-benzimidazol-l -yl -pregnenolone acetate (1): a, ?-Unsaturated ketone 16-DPA (2.136 g, 6.0 mmol) and benzimidazole (708 mg, 6.0 mmol) were mixed intimately in a mortar and irradiated in a microwave reactor (720 Watt, 2450 MHz) for 10 minutes. On completion of the reaction (by TLC), the crude product was purified by silica gel column chromatography using EtOAc-hexane (1 :4) as the eluent to afford steroidal Michael adduct 1. Yellow solid, m.p.: 1 19 °C. Yield 68% (1.93 g).

[0040] Preparation of 3P-acetoxy-16a-(l H-irnidazol-l -yl)-17p-acetyl-androst-5-ene £2)1

16 DPA (2.136 g, 6.0 mmol) and imidazole (408 mg, 6.0 mmol) were mixed intimately in a mortar and irradiated in a microwave reactor (720 Watt) for 10 minutes. On completion of the reaction (by TLC), the crude product was purified by silica gel column chromatography using EtOAc-hexane ( 1 :4) as the eluent to afford steroidal Michael adduct 2. Brown solid, m.p.: 13 1 °C. Yield 85% (2.16 g).

Example-Ill

[0041] Preparation of 16a-( l H-benzimidazol-l -yl)-pregnenolone acetate (1 ): a, ?-Unsaturated ketone 16-DPA (4.272 g, 12.0 mmol) and benzimidazole ( 1 .42 g, 12.0 mmol) were mixed intimately in a mortar and irradiated in a microwave reactor (720 Watt, 2450 MHz) for 10 minutes. On completion of the reaction (by TLC), the crude product was purified by silica gel column chromatography using EtOAc-hexane ( 1 :4) as the eluent to afford steroidal Michael adduct 1. Yellow solid, m.p.: 1 18 °C. Yield 69% (3.92 g).

[0042] Preparation of 3 β-acetoxy- 16 -( 1 H-imidazol- 1 -yl)- 1 7P-acetyl-androst-5-ene 16 DPA (4.272 g, 12.0 mmol) and imidazole (816 mg, 12.0 mmol) were mixed intimately in a mortar and irradiated in a microwave reactor (720 Watt, 2450 MHz) for 10 minutes. On completion of the reaction (by TLC), the crude product was purified by silica gel column chromatography using EtOAc-hexane (1 :4) as the eiuent to afford steroidal Michael adduct 2. Brown solid, m.p.: 130 °C. Yield 83% (4.22 g).

Example-IV

[0043] Preparation of 16a-( l H-benzimidazol-l -yr)-pregnenolone acetate (1): a, ?-Unsaturated ketone 16-DPA (356 mg, 1.0 mmol) and benzimidazole (1 18 mg, 1.0 mmol) were mixed intimately in a mortar and irradiated in a microwave reactor (540 Watt, 2450 MHz) for 10 minutes. On completion of the reaction (by TLC), the crude product was purified by silica gel column chromatography using EtOAc-hexane ( 1 :4) as the eiuent to afford steroidal Michael adduct 1. (Yield 47%, 223 mg). Yellow solid, m.p.: 1 17 °C.

[0044] Preparation of 3p-acetoxy-16 -(l H-imidazol-l -yl)-17P-acetyl-androst-5-ene £2):

16 DPA (356 mg, 1 .0 mmol) and imidazole (68 mg, 1 .0 mmol) were mixed intimately in a mortar and irradiated in a microwave reactor (540 Watt, 2450 MHz) for 10 minutes. On completion of the reaction (by TLC), the crude product was purified by silica gel column chromatography using EtOAc-hexane ( 1 :4) as the eiuent to afford steroidal Michael adduct 2. Brown solid, m.p.: 1 30 °C. Yield 62% (263 mg).

Example-V

[0045] Preparation of 16a-( l H-benzimidazol- l -yl)-pregnenolone acetate (1): a,/?-Unsaturated ketone 16-DPA (356 mg, 1.0 mmol) and benzimidazole ( 1 1 8 mg, 1 .0 mmol) were mixed intimately in a mortar and irradiated in a microwave reactor (540 Watt, 2450 MHz) for 20 minutes. On completion of the reaction (by TLC), the crude product was purified by silica gel column chromatography using EtOAc-hexane ( 1 :4) as the eiuent to afford steroidal Michael adduct 1. (Yield 52%, 247 mg). Yellow solid, m.p.: 1 1 7 °C. [0046] Preparation of 3 β-acetoxy- 16a-( 1 H-imidazol- 1 -vD- 17p-acetyl-androst-5-ene £211

16 DPA (356 mg, 1.0 mmol) and imidazole (68 mg, 1.0 mmol) were mixed intimately in a mortar and irradiated in a microwave reactor (540 Watt, 2450 MHz) for 20 minutes. On completion of the reaction (by TLC), the crude product was purified by silica gel column chromatography using EtOAc-hexane (1 :4) as the eluent to afford steroidal Michael adduct 2. Brown solid, m.p.: 130 °C. Yield 66% (280 mg).

Example- VI

[0047] Preparation of 16a-(lH-benzimidazol-l-yl)-pregnenolone acetate (1): α, Jnsaturated ketone 16-DPA (356 mg, 1.0 mmol) and benzimidazole (1 18 mg, 1.0 mmol) were mixed intimately in a mortar and irradiated in a microwave reactor (720 Watt, 2450 MHz) for 20 minutes. On completion of the reaction (by TLC), the crude product was purified by silica gel column chromatography using EtOAc-hexane (1 :4) as the eluent to afford steroidal Michael adduct 1. (Yield 69%, 327 mg). Yellow solid, m.p.: 1 16 °C.

[0048] Preparation of 3 β-acetoxy- 16a-( 1 H-imidazol- 1 -yl)- 17P-acetyl-androst-5-ene

16 DPA (356 mg, 1 .0 mmol) and imidazole (68 mg, 1 .0 mmol) were mixed intimately in a mortar and irradiated in a microwave reactor (720 Watt, 2450 MHz) for 20 minutes. On completion of the reaction (by TLC), the crude product was purified by silica gel column chromatography using EtOAc-hexane ( 1 :4) as the eluent to afford steroidal Michael adduct 2. Brown solid, m.p.: 131 °C. Yield 86% (365 mg).

Example-VII

10049] Preparation of 16a-( l H-benzimidazol- l -yl)-pregnenolone acetate (1): α,/3-Unsaturated ketone 16-DPA (356 mg, 1.0 mmol) and benzimidazole ( 1 1 8 mg, 1 .0 mmol) were mixed intimately in a mortar and irradiated in a microwave reactor (900 Watt, 2450 MHz) for 10 minutes. On completion of the reaction (by TLC), the crude product was purified by silica gel column chromatography using EtOAc-hexane (1 :4) as the eluent to afford steroidal Michael adduct 1. (Yield 65%, 308 mg). Yellow solid, m.p.: 1 16 °C.

[0050] Preparation of 3p-acetoxy- 16a-( 1 H-imidazol- 1 -yl)- 17P-acetyl-androst-5-ene (21:

16 DPA (356 mg, 1.0 mmol) and imidazole (68 mg, 1.0 mmol) were mixed intimately in a mortar and irradiated in a microwave reactor (900 Watt, 2450 MHz) for 10 minutes. On completion of the reaction (by TLC), the crude product was purified by silica gel column chromatography using EtOAc-hexane ( 1 :4) as the eluent to afford steroidal Michael adduct 2. Brown solid, m.p.: 132 °C. Yield: 79% (335 mg).

ADVANTAGES OF THE PRESENT INVENTION [0051] The main advantages of the present invention are:

1 . The compound 16a-( l H-benzimidazol- l -yl)-pregnenolone acetate (1) exhibited cytotoxic activity against cervical HeLa cancer cell line, prostate DU 205 cancer cell line and breast cancer MCF-7 cell line similar to drug doxorubicin.

2. The compound 16<¾-( 1 H-imidazol- l -yl)-pregnenolone acetate (2) exhibited cytotoxic activity against cervical HeLa cancer cell l ine, prostate DU 205 cancer cell line and breast cancer MCF-7 cell line.

3. Incorporation of a benzimidazole group and imidazole group at 16-position of steroidal D-ring via solvent and catalyst free Michael addition reaction of benzimidazole and imidazole with commercially available 16-DPA molecule under microwave irradiation.

4. Incorporation of a benzimidazole/imidazole group at 16-position affords 16a- substituted heterosteroid which has /ram-orientation with respect to 17-acetyl group.