Steroids 79 (2014) 19–27

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Epoxide group relationship with cytotoxicity in withanolide derivatives from Withania somnifera Pallavi Joshi a, Laxminarain Misra a,⇑, Amreen A. Siddique a, Monica Srivastava a, Shiv Kumar b, Mahendra P. Darokar b a b

Chemical Sciences Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, India Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, India

a r t i c l e

i n f o

Article history: Received 1 July 2013 Received in revised form 17 October 2013 Accepted 23 October 2013 Available online 1 November 2013 Keywords: Withania somnifera Withanolides Epoxide Amination Reduction Cytotoxicity

a b s t r a c t Withania somnifera is one of the highly reputed medicinal plants of India. Its steroidal constituents exist in the form of two major substitution patterns, viz. withaferin A (1) and withanone (5). Withaferin A with oxidation at carbons 4, 5, and 6 is considered as an active type, especially as anticancer, whereas the withanones with oxidation at carbons 5, 6, and 7 rarely show any activity. We prepared a series of derivatives with modifications at carbons 5, 6, and 7 in ring B of these withanolides to study the role of the epoxide group towards the cytotoxic property of these bioactive steroids. We have converted withanolides into the respective thiiranes, amino alcohols and alcohols by selective reactions at the epoxide ring and were evaluated for in vitro anticancer activity against four cancer cell lines to study the structure activity relationships. The transformations of the epoxide group in withanolides of the withaferin A type showed moderate reduction in their cytotoxicity whereas the almost inactive withanones have shown some improvements in their alcohol derivatives. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Withania somnifera L. Dunal (Solanaceae), popularly known as Ashwagandha, is one of the most popular medicinal plants of India and is highly valued for its medicinal and nutraceutical properties [1,2]. It is an annual herb growing in dry and arid soil as a wild. Its popularity as health promoting and therapeutic medicinal plant in traditional systems of medicine in India such as Ayurveda, Siddha and Unani, has been very well documented [3,4]. Most of the therapeutic properties viz., antioxidant, anti-tumour [5], adaptogenic, anti-stress, anti-convulsant, immuno-modulatory, neurological effects [6–8], selective COX-2 inhibition, apoptosis, etc. [5,9–11], have a direct relationship with these withanolides. Our continuing interest in the chemical composition of various parts of this plant has yielded a variety of new and known chemical structures belonging to withasteroids [12–13]. Recently we suggested that the epoxide functionality in ring B of withanolides, which are also known as withasteroids, has a positive relationship with its anticancer activity [12]. In continuation of these investigations, we have further isolated several withasteroids from the leaves of W. somnifera and applied simple reactions to convert

⇑ Corresponding author. Address: CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India. Tel.: +91 522 2717529; fax: +91 522 2342666. E-mail address: [email protected] (L. Misra). 0039-128X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.steroids.2013.10.008

the epoxide of withanolides into thiiranes, amino alcohols and alcohols and tested their cytotoxicity. The present paper deals with the results of these transformations vis-a-vis their structure activity relationship. 2. Experimental 2.1. General experimental procedure The experimental procedure of instrumentation has been described in our previous paper [14]. 2.2. Extraction and isolation of withanolides from W. somnifera leaves One of the strains of W. somnifera rich in withaferin A types of steroids is under cultivation in our experimental farm. The fresh leaves (6.0 kg) were ground in liquid nitrogen and soaked with MeOH: H2O = 2:3 (3  8.0 L). Methanol was distilled off and the aqueous portion of the extract was extracted with CHCl3 (3  5.0 L), using a separatory funnel. The chloroform extract (50 g) was chromatographed on a silica gel column with n-hexane as mobile phase and then elution was carried out with n-hexane, ethyl acetate and methanol with solvent gradients [14,15]. The polarity was increased by sequentially adding ethyl acetate in nhexane (5–100% ethyl acetate in n-hexane) followed by 2–10% methanol in ethyl acetate. The obtained fractions, as detailed earlier

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[14], were further subjected to successive flash chromatography. Fractions 11 (12.50 g) and 12 (11.40 g) were subjected to flash chromatography using silica gel (230–400 mesh size). Elution was carried out with solvent system (A) CHCl3:C6H6 = 2:1 and (B) CHCl3:MeOH = 1:5. The polarity was increased sequentially by adding 2–12% solvent B in A, yielding pure withaferin A (1, 2.70 g). Other fractions after repetitive flash chromatography and preparative TLC [14–16] yielded withaferin A (1, 3.20 g), 27-deoxywithaferin A (2, 0.25 g), 17-hydoxywithaferin A (3, 0.18 g), 17-hydroxy-27-deoxy withaferin A (4, 0.15 g), and withanone (5, 0.25 g). Similarly, the fresh leaves of second strain of a W. somnifera, rich in withanone (5) type of steroids, after extraction and exhaustive purification as described above for the first strain, yielded withaferin A (1, 0.65 g), withanone (5, 2.50 g), withanolide A (7, 0.45 g), 27-hydroxywithanone (6, 0.15 g), and 27-deoxywithaferin A (2, 0.09 g).

2.3.3. 4b,17a,27-trihydroxy-1-oxowitha-2,24-dienolide-5a,6athiirane (3a) 17-Hydoxywithaferin A (3, 0.030 g) gave 3a (0.020 g) with 68% yield: ½a20 D = +71 (c 0.3, MeOH); IR (KBr, t): 3640 (OH), 1710 (dlactone), 1690 (ACH@CH–CHO), 660 (C–S); 1H NMR (300 MHz, CDCl3, d): 6.58 (dd, J = 10.0, 3.5 Hz, H-3); 6.10 (d, J = 10.0 Hz, H2); 4.60 (two d, J = 12.0 Hz, H-27); 4.35 (dt, J = 13.0, 5.5, 3.5 Hz, H-22); 3.68 (d, J = 3.5 Hz, H-4); 2.95 (dd, J = 3.0, 6.0 Hz, H-6); 2.46 (d, J = 8.0 Hz, H-23); 1.98 (s, H-28); 1.44 (s, H-19); 1.04 (d, J = 7.0 Hz, H-21); 0.70 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 201.9 (C-1), 130.7 (C-2), 143.0 (C-3), 68.0 (C-4), 57.7 (C-5), 55.4 (C-6), 36.4 (C-7), 29.0 (C-8), 43.2 (C-9), 42.0 (C-10), 20.6 (C-11), 38.6 (C-12), 47.2 (C-13), 52.0 (C-14), 23.1 (C-15), 36.0 (C-16), 84.5 (C-17), 18.0 (C-18), 15.9 (C-19), 47.1 (C-20), 13.4 (C-21), 78.2 (C-22), 30.2 (C-23), 151.9 (C-24), 125.9 (C-25), 166.2 (C-26), 56.4 (C-27), 19.5 (C-28); HRESI MS (m/z): 502.2382 (calculated for C28H38O6S 502.2384), ESI MS (m/z): 525 [M+Na]+.

2.3. General procedure for preparation of thiiranes from withanolide [17,18]

2.3.4. 4b,17a-dihydroxy-1-oxowitha-2,24-dienolide-5a,6a-thiirane (4a) 17-Hydroxy-27-deoxy withaferin A (4, 0.030 g) gave 4a (0.022 g) with 72% yield: ½a20 D = +88 (c 0.3, MeOH); IR (KBr, t): 3640 (OH), 1710 (d-lactone), 1690 (ACH@CH–CHO), 660 (C–S); 1 H NMR (300 MHz, CDCl3, d): 6.61 (dd, J = 10.0, 3.5 Hz, H-3); 5.98 (d, J = 10.0 Hz, H-2); 4.50 (m, H-22); 3.69 (d, J = 3.5 Hz, H-4); 2.98 (dd, J = 3.0, 6.0 Hz, H-6); 2.20 (d, J = 8 Hz, H-23); 1.93 (s, H-28); 1.88 (s, H-27); 1.44 (s, H-19); 1.05 (d, J = 7.0 Hz, H-21); 0.80 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 201.8 (C-1), 129.7 (C-2), 143.1 (C-3), 67.2 (C-4), 57.9 (C-5), 56.4 (C-6), 36.7 (C-7), 29.0 (C-8), 43.6 (C-9), 42.1 (C-10), 20.9 (C-11), 38.7 (C-12), 48.5 (C13), 46.1 (C-14), 23.5 (C-15), 36.1 (C-16), 84.6 (C-17), 10.5 (C-18), 15.0 (C-19), 43.0 (C-20), 12.3 (C-21), 78.7 (C-22), 33.0 (C-23), 150.5 (C-24), 124.0 (C-25), 166.9 (C-26), 14.5 (C-27), 20.5 (C-28); HRESI MS (m/z): 486.2430 (calculated for C28H38O5S 486.2434), ESI MS (m/z): 509 [M+Na]+.

To a solution of withanolides (1–4) in THF, ammonium thiocyanate and cyanuric chloride were added in molar ratio 2:6:1 and the reaction mixture were stirred under reflux for 15 h. After the completion of the reaction, as indicated by TLC, the reaction mixture was diluted with water (5 mL) and extracted with dichloromethane three times. The combined organic phases were dried over anhydrous Na2SO4 and concentrated under vacuum to provide the products 1a–4a as white masses. The crystallized derivatives did not melt below 250 °C. 2.3.1. 4b, 27-dihydroxy-1-oxowitha-2,24-dienolide-5a,6a-thiirane (1a) Withaferin A (1, 0.050 g) gave 1a (0.038 g) with 76% yield: ½a20 D = +25 (c 0.3, MeOH); IR (KBr, t): 3640 (OH), 1710 (d-lactone), 1690 (ACH@CH–CHO), 660 (C–S); 1H NMR (300 MHz, CDCl3, d): 6.50 (dd, J = 10.0, 3.5 Hz, H-3); 5.99 (d, J = 10.0 Hz, H-2); 4.61 (two d, J = 12.0 Hz, H-27); 4.35 (dt, J = 13.0, 5.5, 3.5 Hz, H-22); 3.74 (d, J = 3.5 Hz, H-4); 2.95 (dd, J = 3.0, 6.0 Hz, H-6); 2.53 (d, J = 8.0 Hz, H-23); 2.06 (s, H-28); 1.71 (s, H-19); 0.86 (d, J = 7.0 Hz, H-21); 0.70 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 201.9 (C-1), 125.4 (C-2), 143.1 (C-3), 68.3 (C-4), 57.8 (C-5), 55.4 (C-6), 36.5 (C-7), 29.0 (C-8), 43.5 (C-9), 41.8 (C-10), 20.8 (C-11), 38.6 (C-12), 47.1 (C-13), 55.5 (C-14), 23.6 (C-15), 26.8 (C-16), 51.5 (C-17), 11.2 (C-18), 16.3 (C-19), 38.1 (C-20), 12.6 (C-21), 78.1 (C-22), 30.2 (C-23), 153.2 (C-24), 128.5 (C-25), 166.0 (C-26), 56.1 (C-27), 19.5 (C-28); HRESI MS (m/z): 486.2430 (calculated for C28H38O5S 486.2434), ESI MS (m/z): 509 [M+Na]+. 2.3.2. 4b-hydroxy-1-oxowitha-2,24-dienolide-5a,6a-thiirane (2a) 27-Deoxywithaferin A (2, 0.030 g) gave 2a (0.021 g) with 70% yield: ½a20 D = +105 (c 0.3, MeOH); IR (KBr, t): 3640 (OH), 1710 (dlactone), 1690 (ACH@CH–CHO), 660 (C–S); 1H NMR (300 MHz, CDCl3, d): 6.60 (dd, J = 10.0, 3.5 Hz, H-3); 5.99 (d, J = 10.0 Hz, H2); 4.33 (m, H-22); 3.69 (d, J = 3.5 Hz, H-4); 2.96 (dd, J = 3.0, 6.0 Hz, H-6); 2.42 (d, J = 8.0 Hz, H-23); 1.88 (s, H-28); 1.88 (s, H27); 1.44 (s, H-19); 1.04 (d, J = 7.0 Hz, H-21); 0.71 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 201.8 (C-1), 128.4 (C-2), 143.2(C-3), 67.1 (C-4), 57.9 (C-5), 56.5 (C-6), 36.8 (C-7), 29.0 (C-8), 43.5 (C9), 42.1 (C-10), 20.8 (C-11), 38.6 (C-12), 47.0 (C-13), 55.5 (C-14), 23.5 (C-15), 26.7 (C-16), 51.4 (C-17), 11.2 (C-18), 15.0 (C-19), 38.2 (C-20), 12.7 (C-21), 78.1 (C-22), 32.0 (C-23), 151.0 (C-24), 122.0 (C-25), 166.0 (C-26), 15.0 (C-27), 20.4 (C-28); HRESI MS (m/z): 470.2488 (calculated for C28H38O4S 470.2485), ESI MS (m/z): 493 [M+Na]+.

2.4. General procedure for preparation of amino alcohols using benzyl amine, water and withanolide [19] In a round-bottomed flask, equipped with a magnetic stirrer, 0.1 mmol of the respective withanolide (1–4) was suspended in 1 mL distilled water. Benzyl amine was added to it (molar ratio 1:1) and the reaction mixture was vigorously stirred at room temperature for 40 h. The progress of the reaction was monitored by TLC. On completion of the reaction, 2 mL of water was added and the organic matter was extracted with chloroform (3  5 mL) and dried over anhydrous Na2SO4. The solvent was removed under reduced pressure to give the amino alcohols (1b–4b). 2.4.1. 6a-benzylamino-4b,5b,27-trihydroxy-1-oxowitha-2,24dienolide (1b) Withaferin A (1, 0.040 g) gave 1b (0.032 g) with 80% yield: ½a20 D = +49 (c 0.1, MeOH); IR (KBr, t): 3640 (OH), 3050 (Ar-H), 1710 (d-lactone), 1690 (ACH@CH–CHO), 1600 (C@C in C6H5 str), 1200 (C–N), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 7.32 (m; C6H5 protons); 6.52 (dd, J = 10.0, 3.5 Hz, H-3); 5.98 (d, J = 10.0 Hz, H-2); 5.08 (–NH–CH2); 4.60 (two d, J = 12.0 Hz, H-27); 4.35 (dt, J = 13.0, 5.5, 3.5 Hz, H-22); 3.19 (dd, J = 12.5, 5.0 Hz, H-6); 3.91 (d, J = 3.5 Hz, H-4); 2.45 (d, J = 8.0 Hz, H-23); 2.04 (s, H-28); 1.38 (s, H-19); 0.98 (d, J = 7.0 Hz, H-21); 0.70 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 201.8 (C-1), 132.2 (C-2), 144.1 (C-3), 70.5 (C4), 79.2 (C-5), 52.3 (C-6), 28.8 (C-7), 29.0 (C-8), 43.8 (C-9), 41.9 (C-10), 20.9 (C-11), 38.5 (C-12), 46.8 (C-13), 55.1 (C-14), 23.8 (C15), 26.5 (C-16), 51.4 (C-17), 11.5 (C-18), 15.9 (C-19), 38.3 (C-20), 12.8 (C-21), 77.8 (C-22), 29.9 (C-23), 154.0 (C-24), 125.4 (C-25), 165.8 (C-26), 56.5 (C-27), 19.6 (C-28), 54.2 (-NH-CH2-), 140.6,

P. Joshi et al. / Steroids 79 (2014) 19–27

128.2, 128.3, 126.4, 126.3, 126.8 (–C6H5); HRESI MS (m/z): 577.3399 (calculated for C35H47O6N 577.3398), ESI MS (m/z): 600 [M+Na]+.

2.4.2. 6a-benzylamino-4b,5b-dihydroxy-1-oxowitha-2,24-dienolide (2b) 27-Deoxywithaferin A (2, 0.015 g) gave 2b (0.012 g) with 77% yield: ½a20 D = +110 (c 0.1, MeOH); IR (KBr, t): 3640 (OH), 3050 (Ar–H), 1710 (d-lactone), 1690 (ACH@CH–CHO), 1600 (C@C in C6H5 str), 1200 (C–N), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 7.32 (m; C6H5 protons); 6.50 (dd, J = 10.0, 3.5 Hz, H-3); 5.99 (d, J = 10.0 Hz, H-2); 4.40 (m, H-22); 3.81 (d, J = 3.5 Hz, H-4); 3.19 (dd, J = 12.5, 5.0 Hz, H-6); 2.41 (d, J = 8.0 Hz, H-23); 1.93 (s, H28); 1.88 (s, H-27); 1.40 (s, H-19); 1.05 (d, J = 7.0 Hz, H-21); 0.70 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 202.0 (C-1), 132.0 (C-2), 144.2 (C-3), 70.8 (C-4), 79.1 (C-5), 52.2 (C-6), 28.8 (C-7), 29.0 (C8), 43.7 (C-9), 42.5 (C-10), 20.8 (C-11), 38.2 (C-12), 46.9 (C-13), 55.2 (C-14), 23.7 (C-15), 26.8 (C-16), 51.3 (C-17), 11.2 (C-18), 15.0 (C-19), 38.1 (C-20), 12.7 (C-21), 78.3 (C-22), 32.2 (C-23), 151.1 (C-24), 150.0 (C-25), 165.9 (C-26), 15.0 (C-27), 20.4 (C-28), 54.2 (–NH–CH2–), 140.6, 128.2, 128.3, 126.4, 126.3, 126.8 (–C6H5); HRESI MS (m/z): 561.3447 (calculated for C35H47O5N 561.3449), ESI MS (m/z): 584 [M+Na]+.

2.4.3. 6a-benzylamino-4b,5b,17a,27-tetrahydroxy-1-oxowitha-2,24dienolide (3b) 17-Hydoxywithaferin A (3, 0.015 g) gave 3b (0.011 g) with 73% yield: ½a20 D = +90 (c 0.1, MeOH); IR (KBr, t): 3640 (OH), 3050 (Ar–H), 1710 (d-lactone), 1690 (ACH@CH–CHO), 1600 (C@C), 1200 (C–N), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 7.32 (m; C6H5 protons); 6.60 (dd, J = 10.0, 3.5 Hz, H-3); 5.92 (d, J = 10.0 Hz, H-2); 4.58 (two d, J = 12.0 Hz, H-27); 4.39 (dt, J = 13.0, 5.5, 3.5 Hz, H-22); 4.30 (d, J = 3.5 Hz, H-4); 3.10 (dd, J = 12.5, 5.0 Hz, H-6); 2.40 (d, J = 8.0 Hz, H-23); 2.00 (s, H-28); 1.42 (s, H-19); 1.05 (d, J = 7.0 Hz, H-21); 0.74 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 202.2 (C-1), 132.2 (C-2), 144.2 (C-3), 70.9 (C-4), 79.1 (C-5), 52.2 (C-6), 29.1 (C-7), 28.8 (C-8), 43.5 (C-9), 42.2 (C-10), 20.5 (C-11), 38.7 (C-12), 46.0 (C-13), 51.1 (C-14), 23.0 (C-15), 35.8 (C-16), 85.0 (C-17), 18.5 (C-18), 16.2 (C-19), 42.1 (C-20), 13.4 (C-21), 78.1 (C-22), 30.5 (C-23), 152.0 (C-24), 126.0 (C-25), 166.2 (C-26), 56.4 (C-27), 19.8 (C-28), 54.2 (–NH–CH2–), 140.6, 128.2, 128.3, 126.4, 126.3, 126.8 (–C6H5); HRESI MS (m/z): 593.3345 (calculated for C35H47O7N 593.3347), ESI MS (m/z): 616 [M+Na]+.

2.4.4. 6a-benzylamino-4b,5b,17a-trihydroxy-1-oxowitha-2,24dienolide (4b) 17-Hydroxy-27-deoxy withaferin A (4, 0.015 g) gave 4b (0.011 g) with 70% yield: ½a20 D = +92 (c 0.1, MeOH); IR (KBr, t): 3640 (OH), 3050 (Ar-H), 1710 (d-lactone), 1690 (ACH@CH–CHO), 1600 (C@C), 1200 (C–N), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 7.32 (m; C6H5 protons); 6.89 (dd, J = 10.0, 3.5 Hz, H-3); 6.19 (d, J = 10.0 Hz, H-2); 4.50 (m, H-22); 3.84 (d, J = 3.5 Hz, H-4); 2.89 (dd, J = 12.5, 5.0 Hz, H-6); 2.40 (d, J = 8.0 Hz, H-23); 1.93 (s, H28); 1.88 (s, H-27); 1.40 (s, H-19); 1.05 (d, J = 7.0 Hz, H-21); 0.80 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 202.2 (C-1), 131.9 (C-2), 144.9 (C-3), 70.8 (C-4), 79.0 (C-5), 50.9 (C-6), 28.9 (C-7), 29.2 (C8), 43.7 (C-9), 42.2 (C-10), 20.8 (C-11), 38.0 (C-12), 48.8 (C-13), 46.2 (C-14), 23.2 (C-15), 36.1 (C-16), 84.9 (C-17), 10.5 (C-18), 15.4 (C-19), 43.0 (C-20), 12.4 (C-21), 78.7 (C-22), 32.9 (C-23), 150.4 (C-24), 124.6 (C-25), 166.9 (C-26), 14.5 (C-27), 20.5 (C-28), 54.2 (–NH–CH2–), 140.6, 128.2, 128.3, 126.4, 126.3, 126.8 (–C6H5); HRESI MS (m/z): 577.3396 (calculated for C35H47O6N 577.3398), ESI MS (m/z): 600 [M+Na]+.

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2.5. General procedure for preparation of amino alcohols using n-butylamine, water, lithiumperchlorate and withanolides [20] The respective withanolide (0.1 mmol 1–4) was stirred in a solution of lithium perchlorate (0.2 mmol) in 1 mL water. n-Butylamine (0.2 mmol) was added and the mixture was refluxed for 16 h. On completion of the reaction, as indicated by TLC, the resultant mixture was extracted with ethyl acetate. The organic layer was separated and dried over anhydrous Na2SO4. The residue was a mixture of two products which were separated and purified by preparative TLC using chloroform to give the final products 1c–4c and 1d–4d. 2.5.1. 6a-n-butylamino-4b,5b,27-trihydroxy-1-oxowitha-2,24dienolide (1c) Withaferin A (1, 0.050 g) gave 1c (0.018 g) with 36% yield: ½a20 D = +140 (c 0.1, MeOH); IR (KBr, t): 3640 (OH), 1710 (d-lactone), 1690 (ACH@CH–CHO), 1185 (C–N), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 6.61 (dd, J = 10.0, 3.5 Hz, H-3); 5.89 (d, J = 10.0 Hz, H-2); 4.58 (two d, J = 12.0 Hz, H-27); 4.37 (dt, J = 13.0, 5.5, 3.5 Hz, H-22); 3.69 (d, J = 3.5 Hz, H-4); 3.12 (dd, J = 12.5, 5.0 Hz, H-6); 2.52 (d, J = 8.0 Hz, H-23); 2.11 (s, H-28); 1.31 (s, H19); 0.95 (d, J = 7.0 Hz, H-21); 0.76 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 202.0 (C-1), 131.7 (C-2), 144.0 (C-3), 70.5 (C-4), 78.5 (C-5), 51.2 (C-6), 28.1 (C-7), 28.9 (C-8), 43.7 (C-9), 41.8 (C-10), 20.9 (C-11), 38.6 (C-12), 46.9 (C-13), 55.2 (C-14), 23.9 (C-15), 26.6 (C-16), 51.5 (C-17), 11.6 (C-18), 15.9 (C-19), 38.2 (C-20), 12.8 (C-21), 77.7 (C-22), 29.8 (C-23), 154.2 (C-24), 125.5 (C-25), 165.9 (C-26), 56.6 (C-27), 19.6 (C-28), 44.8, 35.9, 19.8, 13.7 (–NH–R); HRESI MS (m/z): 543.3551 (calculated for C32H49O6N 543.3554), ESI MS (m/z): 566 [M+Na]+. 2.5.2. 5a-n-butylamino-4b,6b,27-trihydroxy-1-oxowitha-2,24dienolide (1d) Withaferin A (1, 0.05 g) gave 1d (0.020 g) with 40% yield: ½a20 D = +103 (c 0.1, MeOH); IR (KBr, t): 3640 (OH), 1710 (d-lactone), 1690 (ACH@CH–CHO), 1185 (C–N), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 6.59 (dd, J = 10.0, 3.5 Hz, H-3); 5.88 (d, J = 10.0 Hz, H-2); 4.50 (two d, J = 12.0 Hz, H-27); 4.38 (dt, J = 13.0, 5.5, 3.5 Hz, H-22); 3.92 (d, J = 3.5 Hz, H-4); 3.60 (t, J = 2.5 Hz, H6); 2.52 (d, J = 8.0 Hz, H-23); 2.11 (s, H-28); 1.30 (s, H-19); 1.01 (d, J = 7.0 Hz, H-21); 0.77 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 202.1 (C-1), 132.5 (C-2), 144.8 (C-3), 70.1 (C-4), 53.4 (C-5), 77.6 (C-6), 29.0 (C-7), 29.1 (C-8), 43.7 (C-9), 41.8 (C-10), 20.8 (C-11), 38.6 (C-12), 46.8 (C-13), 55.1 (C-14), 23.8 (C-15), 26.5 (C-16), 51.5 (C-17), 13.7 (C-18), 15.9 (C-19), 38.3 (C-20), 12.8 (C-21), 77.8 (C-22), 29.9 (C-23), 154.0 (C-24), 125.4 (C-25), 165.8 (C-26), 56.5 (C-27), 19.6 (C-28), 19.6 (C-28), 44.8, 35.9, 19.8, 13.7 (–NH–R); HRESI MS (m/z): 543.3557 (calculated for C32H49O6N 543.3554), ESI MS (m/z): 566 [M+Na]+. 2.5.3. 6a-n-butylamino-4b,5b-dihydroxy-1-oxowitha-2,24-dienolide (2c) 27-Deoxywithaferin A (2, 0.030 g) gave 2c (0.010 g) with 33% yield: ½a20 D = +150 (c 0.1, MeOH); IR (KBr, t): 3640 (OH), 1710 (dlactone), 1690 (ACH@CH–CHO), 1185 (C–N), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 6.88 (dd, J = 10.0, 3.5 Hz, H-3); 6.00 (d, J = 10.0 Hz, H-2); 4.38 (m, H-22); 3.81 (d, J = 3.5 Hz, H-4); 2.91 (dd, J = 12.5, 5.0 Hz, H-6); 2.50 (d, J = 8.0 Hz, H-23); 1.93 (s, H28); 1.88 (s, H-27); 1.40 (s, H-19); 1.06 (d, J = 7.0 Hz, H-21); 0.80 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 202.2 (C-1), 132.4 (C-2), 145.0 (C-3), 70.8 (C-4), 78.9 (C-5), 52.0 (C-6), 28.8 (C-7), 29.0 (C-8), 43.7 (C-9), 42.1 (C-10), 20.8 (C-11), 38.0 (C-12), 48.9 (C-13), 46.1 (C-14), 23.2 (C-15), 36.6 (C-16), 85.1 (C-17), 11.6 (C-18), 16.0 (C-19), 43.1 (C-20), 12.4 (C-21), 78.7 (C-22), 32.9 (C23), 150.5 (C-24), 125.5 (C-25), 166.8 (C-26), 14.6 (C-27), 120.6

22

P. Joshi et al. / Steroids 79 (2014) 19–27

(C-28), 44.8, 35.9, 19.8, 13.7 (–NH–R); HRESI MS (m/z): 527.3604 (calculated for C32H49O5N 527.3605), ESI MS (m/z): 550 [M+Na]+. 2.5.4. 5a-n-butylamino-4b,6b-dihydroxy-1-oxowitha-2,24-dienolide (2d) 27-Deoxywithaferin A (2, 0.030 g) gave 2d (0.011 g) with 37% yield: ½a20 D = +142 (c 0.1, MeOH); IR (KBr, t): 3640 (OH), 1710 (dlactone), 1690 (ACH@CH–CHO), 1185 (C–N), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 6.87 (dd, J = 10.0, 3.5 Hz, H-3); 6.00 (d, J = 10.0 Hz, H-2); 3.80 (d, J = 3.5 Hz, H-4); 3.50 (t, J = 2.5 Hz, H-6); 2.45 (d, J = 8.0 Hz, H-23); 2.00 (s, H-28); 1.88 (s, H-27); 1.37 (s, H-19); 1.18 (d, J = 7.0 Hz, H-21); 0.82 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 202.0 (C-1), 132.1 (C-2), 144.5 (C-3), 70.3 (C4), 53.5 (C-5), 78.0 (C-6), 29.3 (C-7), 28.9 (C-8), 43.2 (C-9), 41.5 (C-10), 20.6 (C-11), 38.7 (C-12), 48.4 (C-13), 55.1 (C-14), 23.8 (C15), 26.5 (C-16), 51.5 (C-17), 13.7 (C-18), 15.9 (C-19), 38.3 (C-20), 12.8 (C-21), 78.7 (C-22), 32.8 (C-23), 154.5 (C-24), 125.4 (C-25), 166.9 (C-26), 14.7 (C-27), 120.4 (C-28), 44.8, 35.9, 19.8, 13.7 (–NH–R); HRESI MS (m/z): 527.3603 (calculated for C32H49O5N 527.3605), ESI MS (m/z): 550 [M+Na]+. 2.5.5. 6a-n-butylamino-4b,5b,17a,27-tetrahydroxy-1-oxowitha-2,24dienolide (3c) 17-Hydoxywithaferin A (3, 0.030 g) gave 3c (0.012 g) with 40% yield: ½a20 D = +110 (c 0.1, MeOH); IR (KBr, t): 3640 (OH), 1710 (dlactone), 1690 (ACH@CH–CHO), 1185 (C–N), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 6.90 (dd, J = 10.0, 3.5 Hz, H-3); 6.10 (d, J = 10.0 Hz, H-2); 4.55 (two d, J = 12.0 Hz, H-27); 4.39 (dt, J = 13.0, 5.5, 3.5 Hz, H-22); 3.82 (d, J = 3.5 Hz, H-4); 2.90 (dd, J = 12.5, 5.0 Hz, H-6); 2.81 (t, NH–CH2–); 2.50 (d, J = 8.0 Hz, H-23); 1.93 (s, H-28); 1.40 (s, H-19); 1.06 (d, J = 7.0 Hz, H-21); 0.80 (s, H-18). 13 C NMR (75 MHz, CDCl3, d): 202.2 (C-1), 132.4 (C-2), 145.0 (C-3), 70.8 (C-4), 78.9 (C-5), 52.0 (C-6), 28.8 (C-7), 29.0 (C-8), 43.7 (C9), 42.1 (C-10), 20.8 (C-11), 38.0 (C-12), 48.9 (C-13), 46.1 (C-14), 23.2 (C-15), 36.6 (C-16), 85.1 (C-17), 11.6 (C-18), 16.0 (C-19), 43.1 (C-20), 12.4 (C-21), 78.7 (C-22), 32.9 (C-23), 150.5 (C-24), 125.5 (C-25), 166.8 (C-26), 14.6 (C-27), 120.6 (C-28), 44.8, 35.9, 19.8, 13.7 (–NH–R); HRESI MS (m/z): 559.7456 (calculated for C32H49O7N 559.7459), ESI MS (m/z): 582 [M+Na]+. 2.5.6. 5a-n-butylamino-4b,6b,17a,27-tetrahydroxy-1-oxowitha-2,24dienolide (3d) 17-Hydoxywithaferin A (3, 0.030 g) gave 3d (0.012 g) with 40% yield: ½a20 D = +104 (c 0.1, MeOH); IR (KBr, t): 3640 (OH), 1710 (dlactone), 1690 (ACH@CH–CHO), 1185 (C–N), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 6.87 (dd, J = 10.0, 3.5 Hz, H-3); 6.08 (d, J = 10.0 Hz, H-2); 4.50 (two d, J = 12.0 Hz, H-27); 4.38 (dt, J = 13.0, 5.5, 3.5 Hz, H-22); 3.80 (d, J = 3.5 Hz, H-4); 3.50 (t, J = 2.5 Hz, H6); 2.91 (t, NH–CH2–); 2.45 (d, J = 8.0 Hz, H-23); 2.01 (s, H-28); 1.37 (s, H-19); 1.18 (d, J = 7.0 Hz, H-21); 0.82 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 202.0 (C-1), 132.1 (C-2), 144.5 (C-3), 70.3 (C4), 53.5 (C-5), 78.0 (C-6), 29.3 (C-7), 28.9 (C-8), 43.2 (C-9), 41.5 (C-10), 20.6 (C-11), 38.7 (C-12), 48.4 (C-13), 55.1 (C-14), 23.8 (C15), 26.5 (C-16), 51.5 (C-17), 13.7 (C-18), 15.9 (C-19), 38.3 (C-20), 12.8 (C-21), 78.7 (C-22), 32.8 (C-23), 154.5 (C-24), 125.4 (C-25), 166.9 (C-26), 14.7 (C-27), 120.4 (C-28), 44.8, 35.9, 19.8, 13.7 (–NH–R); HRESI MS (m/z): 559.7461 (calculated for C32H49O7N 559.7459), ESI MS (m/z): 582 [M+Na]+. 2.5.7. 6a-n-butylamino-4b,5b,17a-trihydroxy-1-oxowitha-2,24dienolide (4c) 17-Hydroxy-27-deoxy withaferin A (4, 0.030 g) gave 4c (0.010 g) with 33% yield: ½a20 D = +115 (c 0.1, MeOH); IR (KBr, t): 3640 (OH), 1710 (d-lactone), 1690 (ACH@CH–CHO), 1185 (C–N), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 6.88 (dd, J = 10.0, 3.5 Hz, H-3); 6.00 (d, J = 10.0 Hz, H-2); 4.38 (m, H-22); 3.81 (d,

J = 3.5 Hz, H-4); 2.91 (dd, J = 12.5, 5.0 Hz, H-6); 2.50 (d, J = 8.0 Hz, H-23); 1.93 (s, H-28); 1.88 (s, H-27); 1.40 (s, H-19); 1.06 (d, J = 7.0 Hz, H-21); 0.80 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 202.2 (C-1), 132.4 (C-2), 145.0 (C-3), 70.8 (C-4), 78.9 (C-5), 52.0 (C-6), 28.8 (C-7), 29.0 (C-8), 43.7 (C-9), 42.1 (C-10), 20.8 (C-11), 38.0 (C-12), 48.9 (C-13), 46.1 (C-14), 23.2 (C-15), 36.2.6 (C-16), 85.1 (C-17), 11.6 (C-18), 16.0 (C-19), 43.1 (C-20), 12.4 (C-21), 78.7 (C-22), 32.9 (C-23), 150.5 (C-24), 125.5 (C-25), 166.8 (C-26), 14.6 (C-27), 120.6 (C-28), 44.8, 35.9, 19.8, 13.7 (–NH–R); HRESI MS (m/z): 543.3556 (calculated for C32H49O6N 543.3554), ESI MS (m/z): 566 [M+Na]+. 2.5.8. 5a-n-butylamino-4b,6b,17a-trihydroxy-1-oxowitha-2,24dienolide (4d) 17-Hydroxy-27-deoxy withaferin A (4, 0.030 g) gave 4d (0.012 g) with 40% yield: ½a20 D = +105 (c 0.1, MeOH); IR (KBr, t): 3640 (OH), 1710 (d-lactone), 1690 (ACH@CH–CHO), 1185 (C–N), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 6.87 (dd, J = 10.0, 3.5 Hz, H-3); 6.00 (d, J = 10.0 Hz, H-2); 4.39 (m, H-22); 3.80 (d, J = 3.5 Hz, H-4); 3.50 (t, J = 2.5 Hz, H-6); 2.45 (d, J = 8.0 Hz, H-23); 2.02 (s, H-28); 1.88 (s, H-27); 1.37 (s, H-19); 1.18 (d, J = 7.0 Hz, H-21); 0.82 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 202.0 (C-1), 132.1 (C-2), 144.5 (C-3), 70.3 (C-4), 53.5 (C-5), 78.0 (C-6), 29.3 (C-7), 28.9 (C-8), 43.2 (C-9), 41.5 (C-10), 20.6 (C-11), 38.7 (C-12), 48.4 (C-13), 55.1 (C-14), 23.8 (C-15), 26.5 (C-16), 51.5 (C-17), 13.7 (C-18), 15.9 (C-19), 38.3 (C-20), 12.8 (C-21), 78.7 (C-22), 32.8 (C-23), 154.5 (C-24), 125.4 (C-25), 166.9 (C-26), 14.7 (C-27), 120.4 (C-28), 44.8, 35.9, 19.8, 13.7 (–NH–R); HRESI MS (m/z): 543.3551 (calculated for C32H49O6N 543.3554), ESI MS (m/z): 566 [M+Na]+. 2.6. General procedure for reductive opening of epoxide rings in withanolides with polymethylhydrosiloxane [21] To a solution of withanolide (1–7) in 3.5 mL chloroform, a 2-fold excess of polymethylhydrosiloxane was added in a round bottomed flask under cooling with ice. Iodine (3.00 mg, 0.011 mmol) was added and mixed well. The reaction was then stirred at room temperature for 90 min. On completion of reaction, it was washed with dilute HCl, thiosulfate solution and water. After extraction with chloroform, the organic layer was dried over anhydrous Na2SO4 to give the product (1e–7e). Products (1e–7e, 10 mg each individually), 2,2-dimethoxypropane (1.0 ml) and catalytic amount of p-TsOH in CHCl3 were stirred for 40 min at room temperature, then MeOH (1.0 ml) was added and stirred overnight. After addition of CHCl3 (5.0 ml), the organic layer was washed with saturated aqueous NaHCO3 (2.0 ml) and dried over anhydrous Na2SO4. The solvent was distilled off in vacuo to afford the respective acetonides (40% yield) [22]. 2.6.1. 4b,5b,27-trihydroxy-1-oxowitha-2,24-dienolide (1e) Withaferin A (1, 0.040 g) gave 1e (0.029 g) with 74% yield: ½a20 D = +210 (c 0.2, MeOH); IR (KBr, t): 36450 (OH), 1710 (d-lactone), 1700 (ACH@CH–CHO), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 6.46 (dd, J = 10.0, 3.5 Hz, H-3); 5.99 (d, J = 10.0 Hz, H2); 4.60 (two d, J = 12.0 Hz, H-27); 4.40 (dt, J = 13.0, 5.5, 3.5 Hz, H-22); 4.14 (d, J = 3.5 Hz, H-4); 2.35 (s, H-6); 2.45 (d, J = 8.0 Hz, H-23); 2.03 (s, H-28); d 1.39 (s, H-19); 1.03 (d, J = 7.0 Hz, H-21); 0.72 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 200.3 (C-1), 127.5 (C2), 142.9 (C-3), 66.4 (C-4), 76.6 (C-5), 39.1 (C-6), 28.0 (C-7), 29.8 (C-8), 43.2 (C-9), 43.0 (C-10), 22.9 (C-11), 38.6 (C-12), 46.3 (C13), 55.1 (C-14), 23.9 (C-15), 27.2 (C-16), 51.7 (C-17), 13.3 (C-18), 14.1 (C-19), 38.3 (C-20), 12.8 (C-21), 78.5 (C-22), 30.3 (C-23), 125.7 (C-24), 153.0 (C-25), 166.5 (C-26), 56.8 (C-27), 20.7 (C-28);

P. Joshi et al. / Steroids 79 (2014) 19–27

HRESI MS (m/z): 472.2815 (calculated for C28H40O6 472.2819), ESI MS (m/z): 495 [M+Na]+. 2.6.2. 4b,5b-dihydroxy-1-oxowitha-2,24-dienolide (2e) 27-Deoxywithaferin A (2, 0.015 g) gave 2e (0.011 g) with 73% yield: ½a20 D = +224 (c 0.1, MeOH); IR (KBr, t): 36450 (OH), 1710 (d-lactone), 1700 (ACH@CH–CHO), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 6.46 (dd, J = 10.0, 3.5 Hz, H-3); 5.99 (d, J = 10.0 Hz, H-2); 4.35 (m, H-22); 4.15 (d, J = 3.5 Hz, H-4); 3.09 (s, H-6); 2.41 (d, J = 8.0 Hz, H-23); 1.93 (s, H-28); 1.88 (s, H-27); 1.50 (s, H-19); 1.05 (d, J = 7.0 Hz, H-21); 0.75 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 201.8 (C-1), 131.5 (C-2), 144.4 (C-3), 70.5 (C-4), 78.5 (C-5), 27.8 (C-6), 29.3 (C-7), 29.0 (C-8), 43.5 (C-9), 43.0 (C10), 20.5 (C-11), 38.6 (C-12), 47.1 (C-13), 55.4 (C-14), 23.7 (C-15), 26.7 (C-16), 51.4 (C-17), 11.2 (C-18), 15.4 (C-19), 38.2 (C-20), 12.6 (C-21), 78.4 (C-22), 32.2 (C-23), 151.0 (C-24), 149.0 (C-25), 166.8 (C-26), 14.9 (C-27), 20.2 (C-28); HRESI MS (m/z): 456.2868 (calculated for C28H40O5 456.2870), ESI MS (m/z): 479 [M+Na]+. 2.6.3. 4b,5b,17a,27-tetrahydroxy-1-oxowitha-2,24-dienolide (3e) 17-Hydoxywithaferin A (3, 0.015 g) gave 3e (0.012 g) with 80% yield: ½a20 D = +209 (c 0.1, MeOH); IR (KBr, t): 36450 (OH), 1710 (dlactone), 1700 (ACH@CH–CHO), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 6.50 (dd, J = 10.0, 3.5 Hz, H-3); 5.90 (d, J = 10.0 Hz, H2); 4.55 (two d, J = 12.0 Hz, H-27); 4.39 (dt, J = 13.0, 5.5, 3.5 Hz, H-22); 4.20 (d, J = 3.5 Hz, H-4); 2.10 (s, H-6); 2.40 (d, J = 8.0 Hz, H-23); 1.99 (s, H-28); 1.50 (s, H-19); 1.04 (d, J = 7.0 Hz, H-21); 0.79 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 202.0 (C-1), 131.2 (C-2), 144.5 (C-3), 70.6 (C-4), 78.6 (C-5), 27.9 (C-6), 29.1 (C-7), 29.2 (C-8), 43.4 (C-9), 42.2 (C-10), 20.5 (C-11), 38.6 (C-12), 45.9 (C-13), 50.1 (C-14), 22.9 (C-15), 35.7 (C-16), 84.6 (C-17), 18.5 (C18), 16.2 (C-19), 42.0 (C-20), 13.4 (C-21), 78.2 (C-22), 30.5 (C-23), 152.1 (C-24), 125.9 (C-25), 166.3 (C-26), 56.2 (C-27), 19.8 (C-28); HRESI MS (m/z): 488.2770 (calculated for C28H40O7 488.2768), ESI MS (m/z): 511 [M+Na]+. 2.6.4. 4b,5b,17a-trihydroxy-1-oxowitha-2,24-dienolide (4e) 17-Hydroxy-27-deoxy withaferin A (4, 0.015 g) gave 4e (0.011 g) with 73% yield: ½a20 D = +214 (c 0.1, MeOH); IR (KBr, ˆ): 36450 (OH), 1710 (d-lactone), 1700 (ACH@CH–CHO), 1140 (ether); 1 H NMR (300 MHz, CDCl3, d): 6.90 (dd, J = 10.0, 3.5 Hz, H-3); 6.24 (d, J = 10.0 Hz, H-2); 4.60 (m, H-22); 3.42 (d, J = 3.5 Hz, H-4); 2.03 (s, H-6); 2.40 (d, J = 8.0 Hz, H-23); 1.94 (s, H-28); 1.88 (s, H-27); 1.50 (s, H-19); 1.05 (d, J = 7.0 Hz, H-21); 0.82 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 202.2 (C-1), 131.8 (C-2), 144.5 (C-3), 70.7 (C4), 78.7 (C-5), 27.9 (C-6), 28.9 (C-7), 29.1 (C-8), 43.7 (C-9), 42.2 (C-10), 20.5 (C-11), 38.6 (C-12), 48.7 (C-13), 46.0 (C-14), 23.1 (C15), 36.3 (C-16), 85.0 (C-17), 10.5 (C-18), 15.4 (C-19), 43.0 (C-20), 12.4 (C-21), 78.8 (C-22), 33.0 (C-23), 150.4 (C-24), 125.0 (C-25), 166.9 (C-26), 14.9 (C-27), 20.6 (C-28); HRESI MS (m/z): 472.2817 (calculated for C28H40O6 472.2819), ESI MS (m/z): 495 [M+Na]+. 2.6.5. 5a,6a,17a-trihydroxy-1-oxowitha-2,24-dienolide (5e) Withanone (5, 0.022 g) gave 5e (0.018 g) with 80% yield: ½a20 D = +163 (c 0.1, MeOH); IR (KBr, t): 36450 (OH), 1710 (d-lactone), 1700 (ACH@CH–CHO), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 6.60 (dt, J = 10.0, 3.5 Hz, H-3); 5.85 (d, J = 10.0 Hz, H-2); 4.60 (m, H-22); 2.8, 2.46 (d, H-4); 3.87 (dd, J = 12.0, 5.0 Hz, H-6); 2.50 (d, J = 8.0 Hz, H-23); 1.94 (s, H-28); 1.89 (s, H-27); 1.50 (s, H-19); 1.05 (d, J = 7.0 Hz, H-21); 0.82 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 203.1 (C-1), 128.6 (C-2), 143.8 (C-3), 36.2 (C4), 73.5 (C-5), 70.4 (C-6), 30.5 (C-7), 35.1 (C-8), 36.1 (C-9), 39.2 (C-10), 21.6 (C-11), 32.4 (C-12), 48.7 (C-13), 45.7 (C-14), 23.0 (C15), 36.0 (C-16), 84.6 (C-17), 18.6 (C-18), 16.0 (C-19), 43.0 (C-20),

23

12.4 (C-21), 78.7 (C-22), 32.6 (C-23), 151.0 (C-24), 125.5 (C-25), 167.4 (C-26), 14.9 (C-27), 20.6 (C-28); HRESI MS (m/z): 472.2818 (calculated for C28H40O6 472.2819), ESI MS (m/z): 495 [M+Na]+. 2.6.6. 5a,6a,17a,27-tetrahydroxy-1-oxowitha-2,24-dienolide (6e) 27-Hydroxywithanone (6, 0.015 g) gave 6e (0.012 g) with 77% yield: ½a20 D = +170 (c 0.1, MeOH); IR (KBr, t): 36450 (OH), 1710 (d-lactone), 1700 (ACH@CH–CHO), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 6.78 (dt, J = 10.0, 3.5 Hz, H-3); 5.90 (d, J = 10.0 Hz, H-2); 4.58 (m, H-22); 4.35 (d, H-27); 3.83 (dd, J = 12.0, 5.0 Hz, H-6); 2.90 (d, H-4); 2.50 (d, J = 8.0 Hz, H-23); 2.05 (s, H-28); 1.40 (s, H-19); 1.05 (d, J = 7.0 Hz, H-21); 0.78 (s, H-18). 13 C NMR (75 MHz, CDCl3, d): 202.0 (C-1), 130.1 (C-2), 144.1 (C-3), 129.5 (C-4), 74.2 (C-5), 70.5 (C-6), 30.4 (C-7), 35.1 (C-8), 36.0 (C9), 43.2 (C-10), 22.1 (C-11), 38.7 (C-12), 48.7 (C-13), 51.1 (C-14), 23.1 (C-15), 36.1 (C-16), 84.5 (C-17), 18.6 (C-18), 16.0 (C-19), 43.0 (C-20), 12.4 (C-21), 78.5 (C-22), 33.0 (C-23), 152.1 (C-24), 125.9 (C-25), 166.5 (C-26), 56.4 (C-27), 20.6 (C-28); HRESI MS (m/z): 488.2770 (calculated for C28H40O7 488.2769), ESI MS (m/z): 511 [M+Na]+. 2.6.7. 5a,6a,20b-trihydroxy-1-oxowitha-2,24-dienolide (7e) Withanolide A (7, 0.015 g) gave 7e (0.012 g) with 77% yield: ½a20 D = +175 (c 0.1, MeOH); IR (KBr, t): 36450 (OH), 1710 (dlactone), 1700 (ACH@CH–CHO), 1140 (ether); 1H NMR (300 MHz, CDCl3, d): 6.80 (dt, J = 10.0, 3.5 Hz, H-3); 5.99 (d, J = 10.0 Hz, H-2); 4.26 (m, H-22); 3.82 (dd, J = 12.0, 5.0 Hz, H-6); 2.90 (d, H-4); 2.50 (d, J = 8.0 Hz, H-23); 1.93 (s, H-28, H-27); 1.20 (s, H-19); 1.05 (d, J = 7.0 Hz, H-21); 0.90 (s, H-18). 13C NMR (75 MHz, CDCl3, d): 202.0 (C-1), 130.0 (C-2), 144.0 (C-3), 29.4 (C-4), 74.1 (C-5), 70.4 (C-6), 30.5 (C-7), 35.1 (C-8), 36.1 (C-9), 43.2 (C-10), 21.8 (C-11), 32.5 (C-12), 48.7 (C-13), 46.0 (C-14), 23.1 (C-15), 36.3 (C-16), 72.1 (C-17), 18.6 (C-18), 16.8 (C-19), 81.2 (C-20), 72.3 (C-21), 79.9 (C-22), 33.8 (C-23), 150.5 (C-24), 125.9 (C-25), 166.9 (C-26), 14.9 (C-27), 20.6 (C-28); HRESI MS (m/z): 472.2815 (calculated for C28H40O6 472.2819), ESI MS (m/z): 495 [M+Na]+. 2.7. Anticancer activity test Compounds were evaluated using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for in vitro cytotoxicity against four human cancer cell lines WRL68 (liver cancer cells), CaCO-2 (colon cancer cells), PC-3 (prostate cancer cells) and MCF7 (hormone dependent breast cancer cells); results are listed in Table 1. Doxorubicin was used as reference compound and in vitro tests were performed using the method of Woerdenbag et al. [23]. 1–2  104 cells/well were incubated in the 5% CO2 incubator for 24 h to enable them to adhere properly to the 96-well polystyrene microplate. The test compounds were dissolved in DMSO and added in concentrations of 100, 10, 01, 0.1, and 0.01 lg/ml medium. After incubation for 6 h, the media were replaced with fresh media and the cells were incubated for another 48 h in the CO2 incubator at 37 °C. The concentration of DMSO used in our experiments never exceeded 1.25%, which was found to be non-toxic to cells. Then, 10 lL MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromide] was added, and plates were incubated at 37 °C for 4 h. Calibrated micropipette (Eppendorf) was used to dispense the accurate volume. 100 lL of DMSO was added to all wells and mixed thoroughly to dissolve the dark blue crystals. Proper dissolution of crystals was ensured by keeping them for few minutes at room temperature, the plates were read on a spectrofluorometer FLUOstar Omega at 570 nm. Plates were normally read within 1 h after adding the DMSO. The experiment was done in triplicate and the inhibitory concentration (IC) values were calculated as follows:

24

P. Joshi et al. / Steroids 79 (2014) 19–27

Table 1 Anticancer activity along cancer cell lines. Compound

1 2 3 4 5 6 7 1a 2a 3a 4a 1b 2b 3b 4b 1c 1d 2c 2d 3c 3d 4c 4d 1e 2e 3e 4e 5e 6e 7e Doxorubicin

CACO-2

PC-3

WRL-68

MCF-7

IC50

IC90

IC50

IC90

IC50

IC90

IC50

IC90

5.2 6.0 6.4 6.6 >100 >100 78.0 >100 >100 100 >100 100 100 >100 >100 100 100 100 100 >100 >100 100 100 46.0 48.0 55.0 57.0 >100 >100 >100 4.8

12.8 16.4 17.4 17.5 >100 >100 >100 >100 >100 100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 85.0 88.0 90.0 95.0 >100 >100 >100 >100

5.4 6.2 6.8 6.7 >100 >100 68.0 77.0 84.0 90.0 89.0 100 100 100 100 100 100 100 100 100 100 98.0 100 15.5 17.0 20.0 22.0 79.0 85.0 72.0 5.0

12.2 16.6 16.9 17.0 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 80.0 80.0 85.0 90.0 >100 >100 >100 >100

4.0 5.1 4.9 4.8 >100 >100 >100 77.0 86.0 88.0 87.0 85.0 88.0 90.0 89.0 100 100 98.0 100 100 100 99.0 100 18.0 19.0 21.0 25.0 91.0 95.0 90.0 0.9

9.0 10.5 9.2 9.5 >100 >100 >100 >100 >100 >100 >100 100 100 100 100 >100 >100 >100 >100 >100 >100 100 100 90.0 95.0 100 >100 99.0 >100 >100 9.4

3.8 4.6 4.8 4.9 >100 >100 84.0 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 59.0 64.0 70.0 72.0 >100 >100 >100 5.3

10.4 12.6 12.9 13.0 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100

WRL-68 = Liver cancer cell lines, CaCO2 = colon cancer, MCF-7 = breast cancer, PC-3 = prostate cancer. Doxorubicin Sigma D-1515 is the standard used.

% inhibition ¼ ð1  OD at 570 nm of sample well=OD at 570

3.1. Conversion of epoxide into thiirane

nm of control wellÞ  100: IC50 is the concentration lg/mL required for 50% inhibition of cell growth as compared to that of untreated control.

3. Results and discussion Withanolides are C28 ergostane based steroidal skeleton, having 2-en-1-one, 24-ene and 22,26-olide functional groups, are common structural features in both withaferin A and withanone types of molecules. However, they vary in the oxidation pattern at C-4, 5, 6 and 7 [24,25]. The a,b-unsaturated ketone in ring A, the epoxide in ring B and the conjugated lactone in ring E are prominent electrophilic centers in these steroids, showing the possibility of biological intervention [26,27]. The importance of boat conformations in the opening of epoxides fused to cyclohexanes has already been emphasized [28]. There has been an explicit discussion of the importance of the C-27 hydroxy group, a,b-unsaturated 1-ketone and 5b,6b-epoxide moiety in withanolides over the past few decades [9,29–30], however the analogs of withanolides other than withaferin A and withanone have rarely been studied. In order to comprehend the role of epoxide in the structure activity relationship in both withanone and withaferin A steroidal nucleus, we performed a series of reactions which are discussed herewith. In the present work, 23 derivatives of natural withanolides, viz. withaferin A (1), 27-deoxywithaferin A (2), 17-hydoxywithaferin A (3), 17-hydroxy-27-deoxywithaferin A (4), withanone (5), 27hydroxywithanone (6) and withanolide A (7) have been prepared. The important inference drawn after these derivatizations and subsequent biological activity testing is that epoxide ring is often indispensable in withanolides for their cytotoxicity because it mostly decreased in analogs which have no epoxide functional group.

Thiiranes find broad application in pharmaceutical and polymer industries. In an attempt to check the variations in the cytotoxicity of withanolide analogs, several thiirane derivatives were prepared. The 5b,6b-epoxide induces a strained A and B ring fusion in the steroidal structure as already established in cholestane skeleton [31]. A series of sulfurating agents, specifically at epoxide, were attempted. Thiocyanate gave the best results under the reactions using two different catalysts as depicted in Scheme 1. In an attempt, to convert oxiranes to thiiranes using ammonium thiocyanate and SbCl3 as catalyst, only low yields of 5,6a-thiiranes were obtained [17]. However, with ammonium thiocyanate and cyanuric chloride [18] the reaction showed improved yields. On treatment with thiocyanate, the chiral epoxide converted into the corresponding thiirane with inversion in stereochemistry by ring cleavage in anti-manner [32]. The MS, NMR and IR data of the

H

21 18 12

O

19

1

11

10

3

5

4

6

14

H

22 26

O

O

O

O

R1 O

13

8

R

R

25

R1

17

9

2

20

27

24

23

16 15

(i) (ii)

7

O OH 1 R= OH, R1= H 2 R= R1= H 3 R= R1= OH 4 R= H, R1= OH

S OH 1a R= OH, R1= H 2a R= R1= H 3a R= R1= OH 4a R= H, R1= OH

Scheme 1. (i) Ammonium thiocyanate, SbCl3, acetonitrile, 3 h; (ii) Ammonium thiocyanate, cyanuric chloride, THF, overnight.

P. Joshi et al. / Steroids 79 (2014) 19–27

products prepared by this route also showed matching signals with 1a–4a, discussed as above. The structure of 1a was confirmed by the NMR data, which showed an upfield shift for the H-6 proton from d 3.24 in 1 to d 2.95 in its 1H NMR spectrum and from d 60.2 to d 55.5 in its 13C NMR spectrum, suggesting the conversion of the oxirane to a thiirane, in agreement with the MS data. The inversion of stereochemistry at C-5,6 from b-epoxide in 1 to a-thiirane in 1a, was evident by the change of the coupling constant of H-6a (broad singlet in 1) to H-6b (double doublet, J = 3.0, 6.0 Hz in 1a). Similarly, 2a showed in its 1H NMR spectrum a double doublet (J = 3.0, 6.0 Hz) at d 2.95 for H-6 and in its 13C NMR spectrum at d 55.5 for C-6. Also, 3a gave H-6 in 1H NMR spectrum at d 2.96 and 4a at d 2.98 with similar coupling constants and in 13C NMR spectrum, the signals for C-6 were obtained at d 55.0 in 3a and at d 56.4 in 4a. However, none of the above mentioned thiirane conversions was successful in case of withanolides 5–7 having 6a,7a-epoxide moiety. Most probably, the stereochemistry at 6,7 positions in these withanolides restricts its reactivity with the thiocyanate nucleophile, as has previously been observed [12]. Another method of oxirane to thiirane conversion by ammonium thiocyanate and polyethylene glycol [33] failed in case of withanolides with 6a,7a-epoxide. 3.2. Aminolysis of epoxide In another set of conversions, the epoxide group was subjected to aminolysis to obtain medicinally important amino alcohols and check their cytotoxic properties. In order to improve the yield of amino alcohols of withanolides, two different approaches were made under mild conditions using water as the reaction medium. In the first approach, the aminolysis of epoxide was carried out using benzyl amine in aqueous condition without any catalyst [19]. The reaction lasted for 40 h to furnish the amino alcohol in good yield (75–85%). Thus, a 5-hydroxy-6-amino substitution was generated from the 5b,6b-epoxide group. Under these conditions, only one product was formed in a highly stereoselective manner. The unsymmetrical 5b,6b-epoxide shows regioselective addition of the nucleophile to the lesser substituted carbon. During the epoxide ring opening, the hydroxyl group at C-4 may have obstructed the approach of bulky benzyl group and in turn the favored 6-amino-5-hydroxy product was formed [20]. Product 1b, thus obtained from 1, showed H-6 as double doublet at d 3.19 (J = 12.5, 5.0 Hz) in its 1H NMR spectrum and a doublet at d 52.3 in 13C NMR spectrum for C-6. The change in the coupling constant of 1 at H-6 (broad singlet) to a double doublet [34] in 1b, indicated that the a-substitution of C-N at C-6 was followed by the formation of the expected b-hydroxide at C-5. The presence of hydroxyl at C-5 was supported by the signal of quaternary carbon at d 79.2 in its 13C NMR. The remaining 1H and 13C NMR spectral data were similar to the reactant, except the additional signals for benzyl moiety (Experimental) in 1b, suggesting the sole reaction of the amine at the epoxide group. Compound 2b also showed the phenyl signal as multiplet at d 7.32 and H-6 proton at d 3.19 (J = 12.5, 5.0 Hz) in its 1H NMR spectrum and C-6 at d 52.2 in the 13C NMR spectrum, as stated in case of 1a. Similarly the amino alcohols 3b and 4b showed H-6 as double doublet at d 3.10 (J = 12.5, 5.0 Hz) and 2.89 (J = 12.5, 5.0 Hz), respectively in their 1H NMR spectra and at d 52.2 and 50.9 in the 13C NMR spectra, respectively. Since the aminolysis of epoxide with aliphatic saturated amines viz. n-butylamine, under same reaction conditions gave products in poor yields (10–20%) for most of the withanolides, a catalyst (lithiumperchlorate) was added (Scheme 2(ii)). The Li+ ion assisted aminolysis proceeded through an A-1 type mechanism [20]. It afforded two regioisomers 1c–4c and 1d–4d for each of the wit-

25

hanolides. As anticipated, the 1H NMR spectrum of 1c and 1d differed only at the H-6 position; a double doublet (J = 12.5, 5.0 Hz) at d 3.10 for 1c and a triplet (J = 2.5 Hz) [34] at d 3.60 for 1d indicated the 4b-OH and 5a-NH substitution in 1c and 4a-NH and 5b-OH substitution in 1d, respectively. Compounds 2c and 2d showed a similar pattern in the 1H NMR spectrum as in case of 1c and 1d for these isomers by the presence of double doublets at d 2.91 (J = 12.5, 5.0 Hz) and a triplet at d 3.50 (J = 2.5 Hz), respectively, for H-6. The 13C NMR values also differed from 1 for C-6 giving signals at d 51.9 and d 77.9, respectively. Similar observations were made in the 1H NMR and 13C NMR spectral data of 3c and 3d as well as 4c and 4d (Experimental). However, the aminolysis reactions were ineffective on 6a,7aepoxide of withanone skeleton. The reaction was tried on withanolides 5–7 under various conditions but the expected reaction did not occur. The probable reason for this could be the strong interaction of the –NH nucleophile with the angular methyl at C-10 position in these withanolides which, in turn, hinders the di-axial substitution at the epoxide moiety [12]. 3.3. Reaction with PMHS The reaction of withanolides with polymethylhydrosiloxane, an inexpensive and non-toxic reducing reagent, in the presence of iodine as catalyst [21,35], yielded 4,5-diols for reactants 1–4 and 5,6-diols for reactants 5–7. The products obtained in case of 5b,6b-epoxides 1–4 were the respective 4b-5b-diols (Scheme 3). The position of the diol in 1e was confirmed by the disappearance of epoxide signal of 1 at C-6 in its 1H and 13C NMR spectra. The product 1e showed in its 1H NMR spectrum H-4 as a doublet at d 4.14 (J = 3.5 Hz) and the C-4 and C-5 signals appeared in the 13C NMR and DEPT 135 spectra at d 66.4 as doublet and at d 76.6 as singlet, respectively. The formation of a 4,5-diol was evident by the coupling of H-4 with H-3 (J = 3.5 Hz), but not with further partners. The stereochemistry of the product was confirmed by the formation of its acetonide having additional singlets at d 1.65 and 1.85 for (CH3)2C in its 1H NMR spectrum. Therefore, we assume that the two hydroxyl groups are syn to each other and the hydroxyl at C-5 is b-oriented. Compounds 2e, 3e and 4e also showed diol formation at C-4,5 as the 1H NMR and 13C NMR signals for H-4 and C-4, respectively, also shifted downfield as described in case of 1e. The stereochemistry of diols were also found as 4b,5b since they also formed the respective acetonides. Withanone (5), on reaction with polymethylhydrosiloxane resulted in reductive opening of the epoxide ring and yielded 5a,6adiols (Scheme 4). The stereochemistry of the diols was established by the acetonide formation in compounds 5e–7e. The structure of compound 5e was again confirmed as 5a,6a-diol since it showed a downfield shift for H-6 proton in its 1H NMR spectrum at d 3.87 as double doublet [36] and disappearance of the epoxide signals at d 3.05 (d, H-6) and d 3.20 (d, H-7) in the 1H NMR spectrum clearly indicating the 1,2-diol at C-5 and C-6. Its 13C NMR and DEPT 135 spectra also supported the reaction, as the C-6 signal moved downfield from d 57.1 in 5 to d 70.4 as doublet in 5e and C-7 carbon signal showed an upfield shift from d 56.3 in 5 to d 30.5 as triplet in 5e. Similarly, the compounds 6e and 7e also showed similar changes in the spectral data at C-5, C-6 and C-7 as explained for 5e thereby supporting the formation of 5a,6a-diol in 6e and 7e, as well. 3.4. Cytotoxicity test 3.4.1. Derivatives of withaferin A type of withanolides There were appreciable variations in the cytotoxic properties of derivatives of withaferin A (1). The thiirane derivatives of 1 and congeners showed moderate reduction in the activities against the prostate (IC50 = 77.0 lg/mL for 1a) and liver cell lines

26

P. Joshi et al. / Steroids 79 (2014) 19–27

R R

H

H

O R1

O

O O

(i)

O

O

R1

(ii) OH O

OH

R2

OH 1b R= OH, R1= H, R2= NHCH2C6H5 2b R= R1= H, R2= NHCH2C6H5 3b R= R1= OH, R2= NHCH2C6H5 4b R= H, R1= OH, R2= NHCH2C6H5 1c R= OH, R1= H, R2= NH(CH2)3CH3 2c R= R1= H, R2= NH(CH2)3CH3 3c R= R1= OH, R2= NH(CH2)3CH3 4c R= H, R1= OH, R2= NH(CH2)3CH3

1 R= OH, R1= H 2 R= R1= H 3 R= R1= OH 4 R= H, R1= OH

R H O

O

R1

O

R2 OH

1d R= OH, R1= H, R2= NH(CH2)3CH3 2d R= R1= H, R2= NH(CH2)3CH3 3d R= R1= OH, R2= NH(CH2)3CH3 4d R= H, R1= OH, R2= NH(CH2)3CH3

OH

Scheme 2. (i) Benzyl amine, water, 48 h; (ii) Butyl amine, lithium perchlorate, water, 16 h.

R

R

H

H O

O

O

R1

O

R1

O

O (i) O

OH

OH

OH

1 R= OH, R 1= H 2 R= R1= H 3 R= R1= OH 4 R= H, R 1= OH

1e R= OH, R 1= H 2e R= R1= H 3e R= R1= OH 4e R= H, R 1= OH

Scheme 3. (i) Polymethylhydrosiloxane, iodine, 1.5 h.

R

R R2

R2 H

O

O

R1

O

H

O

O

R1

O (i)

OH 2=

O R 1=

OH 5 R= R H, 6 R= R1= OH, R 2= H 1= H, R 2= OH 7 R= R

atom results in an appreciable decrease in the cytotoxicity of derivatives of 1. The epoxide ring opening and amino alcohol formation again resulted in the decrease of activity. The activity was drastically reduced after butyl amination in derivatives 1c and 1d (IC50 > 100.0 lg/mL), however, a moderate activity, specifically against the liver cell lines, was observed after benzyl amination in 1b (IC50 = 85.0 lg/mL). Thus, aromatic amino derivatives have shown better scope to enhance its cytotoxicity against liver cell lines. Reduction to diol using polymethylhydrosiloxane gave positive results against all 4 cell lines. The derivative 1e was active, in particular, against prostate (IC50 = 15.5 lg/mL) and liver cell lines (IC50 = 18.0 lg/mL), at lower micro molar concentrations (IC90 = 80.0 lg/mL). Similarly, withanolides 2, 3 and 4 showed IC50 values as 6.0, 6.4 and 6.6 lg/mL against colon cell lines, 6.2, 6.8, 6.7 lg/mL against prostate cell lines, 5.1, 4.9, 4.8 lg/mL against liver cell lines and 4.6, 4.8, 4.9 lg/mL against breast cell lines, respectively. However, a similar trend with moderate reduction in activity in thiirane, aromatic amino derivatives and PMHS reduction products (2a–2d, 3a–3d, 4a–4d) was observed for the derivatives of 2, 3 and 4 as described in case of 1 (see Table 1).

OH OH 5e R= R2= H, R 1= OH 6e R= R1= OH, R 2= H 7e R= R1= H, R 2= OH

Scheme 4. (i) Polymethylhydrosiloxane, iodine, 1.5 h.

(IC50 = 77.0 lg/mL for 1a), as compared to withaferin A (IC50 = 5.4 lg/mL and 4.0 lg/mL for the prostate and liver cell lines, respectively). The activity against the breast and colon cell lines, however, disappeared in the thiirane derivatives 1a (IC50 > 100.0 lg/mL). Thus, replacement of oxygen atom in epoxide with sulfur

3.4.2. Derivatives of withanone type of withanolides Withanone (5) and 27-hydroxywithanone (6) showed negligible activity against any cancer cell line (IC50 values >100.0 lg/ mL) whereas withanolide A (7) showed IC50 = 78.0, 68.0, >100.0, 84.0 lg/mL against colon, prostate, liver and breast cell lines, respectively. The withanone type of withanolides did not react successfully for thiiration and amination reactions. The only successful reaction which showed moderate cytotoxicity for these derivatives happened to be the PMHS reduction. Compound 5e showed moderate activity against prostate (IC50 = 79.0 lg/mL) and liver (IC50 = 91.0 lg/mL) cell lines which is higher than the activity of withanone (IC50 > 100.0 lg/mL against all 4 cell lines). Similarly, the derivatives 6e and 7e showed moderate activity against the prostate (IC50 = 85.0 lg/mL for 6e and 72.0 lg/mL for 7e) and liver (IC50 = 95.0 lg/mL for 6e and 90.0 lg/mL for 7e) cell lines but exhibited negligible cytotoxicity against the breast and colon cell lines.

P. Joshi et al. / Steroids 79 (2014) 19–27

4. Conclusion The present work has demonstrated the application of various reactions on withanolides to achieve selective reactivity at the epoxide moiety. Conversion of epoxides to thiiranes was easily achieved by using thiocyanate with cyanuric chloride as catalyst. The epoxide ring opening by aminolysis was performed in aqueous medium using different aliphatic and aromatic amines under catalyzed and non-catalyzed conditions. Reductive opening of epoxide ring was done using polymethylhydrosiloxane (PMHS) as the reducing agent and molecular iodine as a catalyst. The yields obtained were significant and PMHS even reacted with the withanone nucleus, which is otherwise less reactive. The biological activity of all 23 derivatives was tested in vitro against 4 cancer cell lines and the results obtained varied significantly for different derivatives. The anticancer activity of withasteroid derivatives showed significant reduction in biological activity. Interestingly, a moderate activity was also observed for the withanone nucleus which is, otherwise, generally less active. Our observation is fully supported by a recent bioinformatics paper on withasteroids, which also concludes that withaferin A nucleus is comparatively more active than withanone. Withaferin A binds strongly to the target, thus acting as an efficient anticancer agent. The authors have concluded that the withanone nucleus, on the other hand, shows weak binding to the targets thereby showing a lesser activity towards cancer cell lines [37].

Acknowledgement We thank Director, CSIR-CIMAP, Lucknow for the facilities and encouragements. PJ is thankful to UGC for a Junior Research Fellowship.

References [1] Anonymous. The Wealth of India PID, vol. 8. New Delhi: CSIR; 1976 [37–38]. [2] Sangwan RS, Chaurasiya ND, Misra LN, Lal P, Uniyal GC, Sharma R, et al. Phytochemical variability in commercial herbal products and preparations. Curr Sci 2004;86:461–5. [3] Tuli R, Sangwan RS, Kumar S, Bhattacharya S, Misra LN, et al. Ashwagandha (Withania somnifera): a model Indian medicinal plant. New Delhi: CSIR; 2009. [4] Misra LN. Traditional phytomedicinal systems, scientific validations and current popularity as nutraceuticals. Int J Trad Natl Med 2013;2:27–75. [5] Jayaprakasam B, Nair MG. Cyclooxygenase-2 enzyme inhibitory withanolides from Withania somnifera leaves. Tetrahedron 2003;59:841–9. [6] Singh N, Nath R, Lata A, Singh SP, Kohli RP, Bhargava KP. Withania somnifera (Ashwagandha), a rejuvenating herbal drug which enhances survival during stress (an adaptogen). Int J Crude Drug Res 1982;20:29–35. [7] Budhiraja RD, Krishan P, Sudhir S. Biological activity of withanolides. J Sci Ind Res 2000;59:904–11. [8] Furmanowa M, Gajdzis KD, Ruszkowska J, Czarnocki Z, Obidoska G, Sadowska A, et al. In vitro propagation of Withania somnifera and isolation of withanolides with immunosuppressive activity. Planta Med 2001;67:146–9. [9] Glotter E. Withanolides and related ergostane-type steroids. Nat Prod Rep 1991;8:415–40. [10] Chen LX, He H, Qiu F. Natural withanolides: an overview. Nat Prod Rep 2011;28:705–40. [11] Salvador JA, Carvalho JF, Neves MA, Silvestre SM, Leitão AJ, Silva MM, et al. Anticancer steroids: linking natural and semi-synthetic compounds. Nat Prod Rep 2013;30:324–74.

27

[12] Misra LN, Lal P, Chaurasia ND, Sangwan RS, Sinha S, Tuli R. Selective reactivity of 2-mercaptoethanol with 5,6-epoxide in steroids from Withania somnifera. Steroids 2008;73:245–51. [13] Sabir F, Sangwan RS, Singh J, Misra LN, Pathak N, Sangwan NS. Biotransformation of withanolides from cell suspension cultures of Withania somnifera (Dunal). Biotechnol Lett 2011;5:127–34. [14] Misra LN, Lal P, Sangwan RS, Sangwan NS, Tuli R. Unusually sulfated and oxygenated steroids from Withania somnifera leaves. Phytochemistry 2005;66:2702–7. [15] Sangwan RS, Chaurasiya ND, Misra LN, Lal P, Uniyal GC, Sangwan NS. An improved process for isolation of withaferin A from plant materials and products therefrom. U.S.Patent 7, 108, 870 B2, 2006. [16] Mirjalili MH, Moyano E, Bonfill M, Cusido RM, Palazon J. Steroidal lactones from Withania somnifera, an ancient plant for novel medicine. Molecules 2009;14:2373–93. [17] Baltork M, Khosropour AR. Antimony trichloride; a mild, efficient and convenient catalyst for conversion of oxiranes to thiiranes under nonaqueous conditions. Indian J Chem 1999;38B:605–6. [18] Bandgar BP, Joshi NS, Kamble VT. 2,4,6-Trichloro-1,3,5-triazine catalyzed synthesis of thiiranes from oxiranes under solvent-free and mild conditions. Tetraherdon Lett 2006;47:4775–7. [19] Azizi N, Saidi MR. Highly chemoselective addition of amines to epoxides in water. Org Lett 2005;7:3649–51. [20] Chini M, Crotti P, Macchia F. Metal salts as new catalysts for mild and efficient aminolysis of oxiranes. Tetrahedron Lett 1990;31:4661–4. [21] Yadav JS, Reddy BVS, Shanker S, Swamy T. The reductive etherification of carbonyl compounds using polymethylhydrosiloxane activated by molecular iodine. Tetrahedron Lett 2010;51:46–8. [22] Watanabe B, Nakagawa Y, Ogura T, Miyagawa H. Stereoselective synthesis of (22R)- and (22S)-castasterone/ponasterone A hybrid compounds and evaluation of their molting hormone activity. Steroids 2004;69:483–93. [23] Woerdenbag HJ, Moskal TA, Pras N, Malingre TM, Farouk S, El-Feraly H, et al. Cytotoxicity of artemisinin-related endoperoxides to ehrlich ascites tumorcells. J Nat Prod 1993;56:849–56. [24] Ray AB, Gupta M. Withasteroids a growing group of naturally occurring steroidal lactones. In: Herz W, Kirby GW, Moore RE, Steglich W, Tamm C, Herz W, Kirby GW, Moore RE, Steglich W, Tamm C, editors. Progress in the chemistry of organic natural products, vol. 63. New York: Springer; 1994. p. 1–106. [25] Dhalla NS, Sastry MS, Malhotra CL. Chemical studies on the leaves of Withania somnifera. J Pharm Sci 1961;50:876–7. [26] Fuska J, Fuskova A, Rosazza JP, Nicholas AW. Novel cytotoxic and antitumour agents. IV. Withaferin A. relation of its structure to the in vivo cytotoxic effects on P-388 cells. Neoplasma 1984;31:31–6. [27] Misra LN, Mishra P, Pandey A, Sangwan RS, Sangwan NS, Tuli R. Withanolides from Withania somnifera roots. Phytochemistry 2008;69:1000–4. [28] Lavie D, Greenfield S, Glotter E. Constituents of Withania somnifera Dun. Part VI. The stereochemistry of Withaferin A. J Chem Soc (C) 1965:1753–6. [29] Jayaprakasham B, Zhang Y, Seeram NP, Nair MG. Growth inhibition of human tumor cell lines by withanolides from Withania somnifera leaves. Life Sci 2003;74:125–32. [30] Machin RP, Veleiro AS, Nicotra VE, Oberti JC, Padron JM. Antiproliferative activity of withanolides against human breast cancer cell lines. J Nat Prod 2010;73:966–8. [31] Lavie D, Glotter E, Shavo Y. Constituents of Withania somnifera Dun. The structure of Withaferin A. J Chem Soc 1965:7517. [32] Wang LS, Hollis TK. Demonstration of a phosphazirconocene as a catalyst for the ring opening of epoxides with TMSCl. Org Lett 2003;13:2543–5. [33] Das B, Reddy VS, Krishnaih M. An efficient catalyst-free synthesis of thiiranes from oxiranes using polyethylene glycol as the reaction medium. Tetrahedron Lett 2006;47:8471–3. [34] Bonetto GM, Gil RR, Oberti JC. Novel withanolides from Jaborosa sativa. J Nat Prod 1995;58:705–11. [35] Patel JP, Li AH, Donga H, Korlipara VL, Mulvihill MJ. Polymethylhydrosiloxane (PMHS)/trifluoroacetic acid (TFA): a novel system for reductive amination reactions. Tetrahedron Lett 2009;50:5975–7. [36] Cirigliano AM, Veleiro AS, Bonetto GM, Oberti JC, Burton G. Spiranoid withanolides from Jaborosa runcinata and Jaborosa araucana. J Nat Prod 1996;59:717–21. [37] Vaishnavi K, Saxena N, Shah N, Singh R, Manjunath K, Uthayakumar M, et al. Differential activities of the two closely related withanolides, withaferin A and withanone: bioinformatics and experimental evidences. PLoS ONE 2012;7:e44419.

Epoxide group relationship with cytotoxicity in withanolide derivatives from Withania somnifera.

Withania somnifera is one of the highly reputed medicinal plants of India. Its steroidal constituents exist in the form of two major substitution patt...
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