Steroids 84 (2014) 64–69
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Benzylidine pregnenolones and their oximes as potential anticancer agents: Synthesis and biological evaluation Abid H. Banday a,b,⇑, S.M.M. Akram a,c, Shameem A. Shameem a a
Department of Chemistry, Islamia College of Science and Commerce, Srinagar 190009, India Department of Chemistry and Biochemistry, University of Arizona, Tucson 85721, USA c Department of Chemistry, University of Kashmir, Srinagar 190002, India b
a r t i c l e
i n f o
Article history: Received 8 November 2013 Received in revised form 5 March 2014 Accepted 15 March 2014 Available online 1 April 2014 Keywords: Benzylidine pregnenolone Aldol condensation Nucleophillic addition Cytoxicity Dehydration
a b s t r a c t The present study reveals the anticancer activity of benzylidine pregnenolones and their oxime derivatives. The synthesis of the analogs of both series is very simple and involves aldol condensation in the first step followed by nucleophillic addition of hydroxylamine across carbonyl in the second step. Quantitative yields of more than 80% are obtained in both the steps. All the compounds were tested for their cytotoxic activities against a panel of six human cancer cell lines. Amongst all the compounds of both the series screened for their cytotoxic activity, compound 3e, 3f and 4e are very potent especially against HCT-15 and MCF-7 cancer cell lines. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Over the past twenty years, medicinal chemistry has gained enormous popularity for its role in drug discovery. A number of natural products, their semi synthetic analogs and small molecules have been discovered and evaluated for their role in the treatment and cure of various fatal diseases such as cancer, diabetes, microbial infections and cardiovascular diseases etc. [1,2]. However because of the drug resistance and drug tolerance problems, there is always scope for the design and development of new and modified analogs as more efficient drug candidates. This has been done through the rational design and synthesis of receptor based lead molecules which still remains an open area. Natural products have extensively been used as starting tools for the design and synthesis of lead therapeutic scaffolds. The use of natural products for curing human ailments dates back to 1550 BC, since natural products have been used in medicine especially for the treatment of cancer and related diseases. A number of plants have been used as folklore medicinal agents. The modern drug discovery program has made a great success as a number of lead compounds have been developed from the traditionally used medicinal plants. A number of plant
⇑ Corresponding author at: Department of Chemistry, Islamia College of Science and Commerce, Srinagar 190009, India. Fax: +91 194 2429014. E-mail address: [email protected] (A.H. Banday). http://dx.doi.org/10.1016/j.steroids.2014.03.010 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.
based anticancer compounds such as vinblastine (Velban), vincristine (Oncovin), vinorelbine (Navelbine), etoposide (VP-16), teniposide (VM-26), Taxol (paclitaxel), and most recently Taxotere (docetaxel), topotecan (Hycamtin), and irinotecan (Camptosar) have been approved for use as anticancer drugs by the US FDA [3]. Steroids as natural products have been extensively studied as their biological and clinical importance is now well validated. Steroids as well as their derivatives have been found to have the potential to be developed as drugs for the treatment of a large number of diseases including cardiovascular [4], autoimmune diseases [5], brain tumours, breast cancer, prostate cancer, osteoarthritis, etc. [6]. The promise of using steroids for development of lead molecules lies in their regulation of a variety of biological processes and being a fundamental class of signaling molecules [7]. We have been interested in the area of steroid based medicinal chemistry for the past few years and we have successfully evaluated various steroid based congeners as potent pharmacological agents showing activities such as cytotoxic, antimicrobial, antioxidant etc. [8]. This is particularly true of pregnenolone which happens to be a precursor of most other steroids and has been extensively studied for various biological activities. It has been found that the molecule holds a great promise against brain disorders, memory loss, schizophrenia, insulin related disorders and cancer [9]. Thus in continuation of our program, we herein report the anticancer activities of two series of our compounds I,e benzylidine pregnenolones and their oxime derivatives. We are reporting
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the primary in vitro results as the mechanistic studies are in progress and will be communicated in due course of time.
Table 1 Nature of group ‘‘R’’ in the compounds 3a–3h. Entry
2. Experimental
Nature of R
Entry
3a
3e
3b
3f
3c
3g
3d
3h
Nature of R
F
2.1. General methods Melting points were recorded on Buchi Melting point apparatus D-545; IR spectra (KBr discs) were recorded on Bruker Vector 22 instrument. NMR spectra were recorded on Bruker DPX200 instrument in CDCl3 with TMS as internal standard for protons and solvent signals as internal standard for carbon spectra. Chemical shift values are mentioned in d (ppm) and coupling constants are given in Hz. Mass spectra were recorded on EIMS (Shimadzu) and ESI-esquire 3000 Bruker Daltonics instrument. The progress of all reactions was monitored by TLC on 2 5 cm pre-coated silica gel 60 F254 plates of thickness of 0.25 mm (Merck). The chromatograms were visualized under UV 254–366 nm and iodine.
F
OMe
MeO
Table 2 Nature of group ‘‘R’’ in the compounds 4a–4g.
2.2. Chemical synthesis 2.2.1. General procedure for the synthesis of Benzylidine pregnenolones (3) To a solution of pregnenolone 1 (0.316 g, 1 mmol, 1 eq.) in ethanol (10 ml) was added a conc. aq. solution of KOH (2 eq.). Then aldehyde 2 (1.2 eq.) was charged into the reaction mixture to get the corresponding benzylidine derivative 3 (Scheme 1). After completion as revealed by thin layer chromatography (TLC) in an average span of around 1 h, the reaction mixture was precipitated using water because of the limited solubility. The precipitate was filtered, dried and monitored through TLC for the purity. Thin layer chromatography revealed just a single spot which proved the presence of a single product. For further purification, the product was recrystallized from EtOAc:Hexane to give product as solid white powder. It is to be mentioned that when non-aromatic aldehydes were used, the product was formed in a very minor quantity and that too not stable enough at ambient conditions. Thus the study was restricted to the use of aromatic aldehydes only (Tables 1 and 2). 2.2.2. General procedure for the synthesis of Benzylidine pregnenolone oximes (4) To an ethanolic solution of compound 3d (0.418 g, 1 mmol) was added hydroxylamine hydrochloride (0.139 g, 2 mmol) and the reaction was stirred for 2 h at room temperature. A precipitate of the oxime was obtained as a single spot as revealed by the TLC. The reaction mixture was worked up by first evaporating the ethanol and then extracting the solid with EtOAc: H2O (3 25 ml). Quantitative yields of 90–93% were obtained for all the compounds.The spectral data of various compounds from both the series is given as under. 2.2.3.1. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl)-3-phenylprop-2-en-1-one (3a). White powder O
2
R
EtOH/ KOH
1
Nature of R
Entry
4a
F
4c
NO2
4f
F
4g
OMe
MeO 4d
Cl
(86%). M.p.: 128–131 °C; IR (KBr) cm 1: 3425, 2938, 1804, 1637,1403, 1041, 689; 1H NMR (CDCl3): d 0.63 (s, 3H), 1.00 (s, 3H),1.61–1.90 (m, 6H), 2.20–2.38 (m, 3H), 2.82 (t, J = 8.80, 1H); 3.51 (m, 1H); 6.78 (s, J = 16.00, 1H), 7.39 (m, 3H), 7.55 (m, 3H); 13C NMR (500 MHz, CDCl3): d 13.33, 19.26, 21.07, 22.67, 24.62, 31.13, 31.78, 31.99, 37.22, 41.81, 45.11, 48.61, 48.78, 48.95, 49.12, 49.29, 50.04, 57.14, 61.97, 71.22, 121.24, 126.69, 128.29, 128.90, 130.41, 134.62, 140.85, 141.96, 201.32; ESI-MS: 405 (M+H); Anal. Calcd. for C28H36O2: C, 83.12; H, 8.97; Found C, 83.37; H, 8.83. 2.2.3.2. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl)-3-o-tolylprop-2-en-1-one (3b). Yellow powder (89%). M.p.: 208–210 °C; IR (KBr) cm 1: 3405, 2941, 1774, 1674, 1403, 1041, 757; 1H NMR (CDCl3): d 0.62 (s, 3H), 1.00 (s, 3H), 1.63–1.90 (m, 6H), 2.22–2.35 (m, 3H), 2.38 (s, 3H), 2.82 (t, J = 8.43, 1H); 3.52 (m, 1H), 5.32 (s, 1H), 6.69 (d, J = 15.78, 1H), 7.32 (m, 3H), 7.57 (d, J = 6.83, 1H), 7.82 (d, J = 15.78,1H); 13C NMR (500 MHz, CDCl3): d 13.54, 19.46, 19.95, 21.21, 2.84, 24.77, R
HON
NH2OH.HCl EtOH
HO
Nature of R
4e
4b
O OHC
HO
Entry
HO
3 Scheme 1. Synthesis of D-ring substituted benzylidine pregnenolones and their oximes.
4
R
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31.67, 31.92, 32.09, 36.61, 37.27, 37.42, 39.32,42.32, 45.00, 50.13, 57.25, 62.17, 71.70, 71.76, 121.48, 121.72, 126.29, 126.37, 128.01, 129.10, 130.08, 130.93, 133.81, 138.27, 139.06, 140.53, 140.86, 200.51; ESI-MS: 441 (M+Na); Anal. Calcd. for C29H38O2: C, 83.21; H, 9.15; Found C, 83.32; H, 8.83.
2.2.3.3. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl)-3-m-tolylprop-2-en-1-one (3c). Solid white powder (85%). M.p.: 211–213 °C; IR (KBr) cm 1: 3415, 2934, 1804, 1773, 1638, 1403, 1042, 779; 1H NMR (CDCl3): d 0.63 (s, 3H), 1.00 (s, 3H), 1.61–1.90 (m, 6H), 2.20–2.34 (m, 3H), 2.44 (s, 3H), 2.86 (t, J = 8.73,1 H); 3.54 (m, 1H), 5.36 (s, 1H), 6.75 (d, J = 15.93, 1H), 7.27 (m, 4H), 7.51 (d, J = 15.93, 1H); 13C NMR (500 MHz, CDCl3): d 12.36, 19.38, 20.13, 20.48, 21.78, 24.10, 30.62, 30.86, 31.03, 35.54, 36.27, 38.14, 43.26, 43.96, 49.11, 56.21, 60.99, 71.73, 120.44, 124.94, 127.29, 128.65, 131.18, 139.63, 140.54, 198.42; ESI-MS: 441 (M+Na); Anal. Calcd. for C29H38O2: C, 83.21; H, 9.15; Found C, 83.03; H, 9.33.
2.2.3.4. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl)-3-p-tolylprop-2-en-1-one (3d). Solid yellowish powder (78%). M.p.: 243–245 °C; IR (KBr) cm 1: 3397, 2941, 1804, 1773, 1637, 1403, 1216, 1039, 758; 1H NMR (CDCl3): d 0.63 (s, 3 H), 1.00 (s, 3H), 1.61–1.90 (m, 6H), 2.20–2.38 (m, 3H), 2.45 (s, 3H), 2.85 (t, J = 8.43, 1H); 3.52 (m, 1H), 5.36 (s, 1H), 6.74 (d, J = 15.98, 1H), 7.22 (d, J = 7.82, 2H), 7.48 (d, J = 7.82, 2H), 7.54 (d, J = 15.98, 1H); 13C NMR (500 MHz, CDCl3): d 12.42, 18.38, 20.13, 20.48, 21.78, 23.70, 30.62, 30.86, 31.03, 35.54, 36.27, 38.14, 41.27, 43.96, 49.11, 56.21, 60.99, 70.73, 120.44, 124.94, 127.29, 128.65, 131.08, 139.73, 140.55, 199.42; ESI-MS: 441 (M+Na); Anal. Calcd. for C29H38O2: C, 83.21; H, 9.15; Found C, 83.31; H, 8.95.
2.2.3.5. (2E)-3-(3-fluorophenyl)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13, 14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl) prop-2-en-1-one (3e). Solid yellowish powder (84%). M.p.: 228–231 °C; IR (KBr) cm 1: 3407, 2940, 1773, 1678, 1508, 1232, 1040, 756; 1H NMR (CDCl3): d 0.62 (s, 3H), 1.00 (s, 3H), 1.61–1.90 (m, 6H), 2.20–2.32 (m, 3H), 2.84 (t, J = 8.92, 1H); 3.52 (m, 1H), 5.36 (s, 1H), 6.70 (d, J = 15.93, 1H), 7.08 (m, 2H), 7.53 (m, 3H); 13C NMR (500 MHz, CDCl3): d 13.06, 19.28, 20.13, 20.48, 21.78, 25.10, 30.62, 30.86, 31.03, 35.54, 36.27, 38.14, 43.26, 43.96, 49.11, 56.21, 60.99, 71.73, 122.44, 124.94, 126.49, 129.02, 132.18, 139.63, 140.53, 196.92; ESI-MS: 445 (M+Na); Anal. Calcd. for C28H35F O2: C, 79.58; H, 8.35, F, 4.50; Found C, 79.62; H, 8.52, F, 4.32.
2.2.3.6. (2E)-3-(4-fluorophenyl)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13, 14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl) prop-2-en-1-one (3f). Solid White powder (80%). M.p.:196–198 °C; IR (KBr) cm 1: 3417, 2946, 1772, 1678, 1508, 1232, 1045, 755; 1H NMR (CDCl3): d 0.62 (s, 3H), 1.00 (s, 3H), 1.61–1.90 (m, 6H), 2.20–2.33 (m, 3H), 2.83 (t, J = 8.61,1 H), 3.63 (m, 1H), 5.37 (s, 1H), 6.75 (d, J = 15.84, 1H), 7.08 (m, 2H), 7.24–7.32 (m, 2H), 7.50 (d, J = 15.84, 1H); 13C NMR (500 MHz, CDCl3): d 12.22, 19.26, 20.43, 20.48, 21.78, 23.15, 30.62, 30.86, 31.03, 35.54, 36.27, 38.14, 43.26, 43.96, 49.11, 56.21, 60.99, 71.73, 120.52, 124.94, 127.29, 128.65, 131.18, 139.63, 140.54, 196.12; ESI-MS: 445 (M+Na); Anal. Calcd. for C28H35F O2: C, 79.58; H, 8.35, F, 4.50; Found C, 79.42; H, 8.52, F, 4.67.
2.2.3.7. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl)-3-(4-methoxyphenyl)prop-2-en-1-one (3g). Solidwhite powder (87%). M.p.: 200–203 °C; IR (KBr) cm 1: 3415, 2926, 2853, 1803, 1708, 1637, 1508, 1255, 1036, 762; 1H NMR (CDCl3): d 0.60 (s, 3H), 1.00 (s, 3H), 1.65–1.90 (m, 6H), 2.20–2.36 (m, 3H), 2.83 (t, J = 8.89, 1H), 3.53 (m, 1H), 3.87 (s, 3H), 5.36 (s, 1H), 6.65 (d, J = 15.92, 1H), 6.91 (d, J = 8.72, 2H), 7.51 (d, J = 8.72, 2H), 7.53 (d, J = 15.84, 1H); 13C NMR (500 MHz, CDCl3): d 12.13, 18.31, 21.11, 20.48, 21.78, 24.10, 30.62, 30.81, 31.23, 34.52, 36.27, 38.14, 43.26, 43.96, 49.22, 56.21, 60.99, 71.73, 120.44, 124.94, 127.29, 128.65, 133.16, 139.63, 140.54, 196.54; ESI-MS: 457 (M+Na); Anal. Calcd. for C29H38 O3: C, 80.14; H, 8.81; Found C, 80.32; H, 8.67. 2.2.3.8. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl)-3-(2-methoxyphenyl)prop-2-en-1-one (3h). Grey powder (76%). M.p.: 225–227 °C; IR (KBr) cm 1: 3425, 2940,1728, 1636, 1598, 1296, 1024, 753; 1H NMR (CDCl3): d 0.63 (s,3H), 1.00 (s, 3H), 1.65–1.90 (m, 6H), 2.20–2.32 (m, 3H), 2.89 (t, J = 8.85, 1H), 3.52 (m, 1H), 3.91 (s, 3H), 5.37 (s, 1H), 6.94 (d, J = 16.16, 1H), 6.98 (m, 2H), 7.36 (m, 1H), 7.55 (d, J = 6.37, 1H), 7.88 (d, J = 16.16, 1H); 13C NMR (500 MHz, CDCl3): d 12.34, 18.34, 20.13, 20.48, 21.78, 25.10, 30.62, 30.86, 31.03, 35.54, 36.27, 38.14, 43.26, 43.96, 49.11, 56.21, 60.99, 71.73, 118.30, 124.94, 127.29, 128.65, 131.18, 139.63, 140.54, 195.23; ESI-MS: 457 (M+Na); Anal. Calcd. for C29H38O3: C, 80.14; H, 8.81; Found C, 80.02; H, 8.97. 2.2.3.9. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a ]phenanthren-17-yl)-3-p-methylphenylprop-2-en-1-imine (4a). Grey powder (90%). M.p.: 235–240 °C; IR (KBr) cm 1: 3525, 3385, 2948, 1814, 1617, 1512, 1423, 1051, 609; 1H NMR (CDCl3): d 0.78 (s, 3H), 1.200 (s, 3H), 1.54–2.0 (m, 6H), 2.41–2.48 (m, 3H), 2.42 (t, J = 8.80, 1H); 3.21 (m, 1H); 5.98 (s, J = 16.00, 1H), 6.89 (m, 3H), 7.05 (m, 3H), 8.68 (s, 1H); 13C NMR (500 MHz, CDCl3): d 14.23, 21.06, 22.07, 24.02, 31.03, 31.48, 32.09, 36.52, 42.11, 45.21, 48.01, 48.75, 49.32, 49.79, 50.84, 57.44, 61.07, 73.12, 123.26, 127.69, 128.89, 131.41, 135.72, 142.15, 158.6, 201.12; ESI-MS: 420 (M+H); Anal. Calcd. for C28H37NO2: C, 80.15; H, 8.89; N, 3.34; O, 7.63; Found C, 80.10; H, 8.71; N, 3.04; O, 7.52. 2.2.3.10. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[ a ]phenanthren-17-yl)-3-(3-fluorophenyl)-prop-2-en-1-imine (4b). White powder (91%). M.p.: 240–245 °C; IR (KBr) cm 1: 3407, 3312, 2940, 1773, 1618, 1518, 1232, 1040, 781; 1H NMR (CDCl3): d 0.66 (s, 3H), 1.00 (s, 3H), 1.81–2.00 (m, 6H), 2.40–2.62 (m, 3H), 2.94 (t, J = 8.92, 1H); 4.12 (m, 1H), 5.36 (s, 1H), 6.90 (d, J = 15.93, 1H), 7.18 (m, 2H), 7.73 (m, 3H) 9.36 (s, 1H); 13C NMR (500 MHz, CDCl3): d 12.16, 19.18, 20.43, 22.78, 25.90, 30.82, 30.96, 31.13, 35.74, 36.87, 38.24, 43.56, 44.96, 49.71, 56.81, 60.99, 72.73, 122.14, 124.14, 126.79, 129.12, 132.98, 159.73, 196.92; ESI-MS: 438 (M+H); Anal. Calcd. for C28H36FNO2: C, 76.85; H, 8.29; N, 3.02; O, 7.31 F, 4.34; Found: C, 76.76; H, 8.21; N, 2.92; O, 7.11 F, 4.14. 2.2.3.11. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[ a ]phenanthren-17-yl)-3-(4-methoxyphenyl)prop-2-en-1-imine (4c). Solid powder (90%). M.p.: 250–255 °C; IR (KBr) cm 1: 3405, 3322, 2916, 2853, 1803, 1637, 1521, 1275, 1036, 769; 1H NMR (CDCl3): d 0.68 (s, 3H), 1.2(s, 3H), 1.7–1.90 (m, 6H), 2.3–2.56 (m, 3H), 2.93 (t, J = 8.89, 1H), 3.13 (m, 1H), 4.17 (s, 3H), 5.46 (s, 1H), 6.55 (d,
A.H. Banday et al. / Steroids 84 (2014) 64–69
J = 15.92, 1H), 6.81 (d, J = 8.72, 2H), 7.61 (d, J = 8.72, 2H), 7.83 (d, J = 15.84, 1H), 9.36 (s, 1H); 13C NMR (500 MHz, CDCl3): d 12.23, 19.31, 21.21, 21.78, 24.23, 31.51, 32.13, 34.42, 36.17, 38.24, 43.56, 44.96, 49.12, 56.31, 61.89, 75.83, 121.54, 124.74, 127.19, 128.45, 132.46, 139.63, 160.14, 199.94; ESI-MS: 450 (M+H); Anal. Calcd. for C29H39NO3: C, 77.47; H, 8.74; N, 3.12; O, 10.68 Found: C, 77.38; H, 8.64; N, 3.02; O, 10.56. 2.2.3.12. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17-yl)-3-(4-chlorophenyl)-prop-2-en-1-imine (4d). Grey powder (91%). M.p.: 240–250 °C; IR (KBr) cm 1: 3425, 3300, 2917, 1718, 1611, 1513, 1296, 1024, 763; 1H NMR (CDCl3): d 0.61 (s, 3H), 1.03 (s, 3H), 1.65–1.92 (m, 6H), 2.22–2.54 (m, 3H), 2.76 (t, J = 9.12, 1H), 3.62 (m, 1H), 5.45 (s, 1H), 6.74 (d, J = 15.31, 1H), 6.96–7.21 (m, 4H), 7.54 (d, J = 15.31, 1H), 9.96 (s, 1H), 13C NMR (500 MHz, CDCl3): d 13.13, 19.58, 20.23, 24.04, 25.21, 30.92, 31.66, 35.04, 36.17, 38.44, 43.16, 45.76, 49.21, 57.28, 61.29, 75.03, 121.64, 124.04, 127.19, 129.45, 130.98, 142.13, 160.04, 199.83; ESI-MS: 455 (M+H); Anal. Calcd. for C28H36ClNO2: C, 74.07; H, 7.99; N, 3.08; O, 7.05; Cl, 7.81; Found C, 74.01; H, 7.89; N, 3.01; O, 6.99; Cl, 7.78. 2.2.3.13. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17-yl)-3-p-nitrophenylprop-2-en-1-imine (4e). White powder (91%). M.p.: 255–260 °C; IR (KBr) cm 1: 3410, 3315, 2941, 1764, 1651, 1323, 1061, 797; 1H NMR (CDCl3): d 0.71 (s, 3H), 1.09 (s, 3H), 1.93–2.0 (m, 6H), 2.26–2.39 (m, 3H), 2.31 (s, 3H), 3.12 (t, J = 8.43, 1H); 3.82 (m, 1H), 6.12 (s, 1H), 7.01 (d, J = 15.78, 1H), 7.72 (m, 3H), 7.89 (d, J = 6.83, 1H), 8.12 (d, J = 15.78, 1H), 9.31 (s, 1H), 13C NMR (500 MHz, CDCl3): d 13.94, 19.96, 20.15, 21.11, 22.14, 24.87, 31.07, 32.19, 36.71, 37.37, 37.82, 39.82,42.12, 45.18,51.23, 57.75, 63.27, 71.30, 71.86, 121.82, 126.57, 128.11, 129.18, 130.58, 131.93, 134.81, 138.87, 139.76, 140.53, 160.3, 200.51; ESI-MS: 465 (M+H); Anal. Calcd. for C28H36N2O4: C, 72.39; H,7.81; N, 6.03; O, 13.77; Found: C, 72.09; H, 7.68; N, 5.83; O, 13.57. 2.2.3.14. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17-yl)-3-(4-fluorophenyl)-prop-2-en-1-imine (4f). White powder (92%). M.p.:243–250 °C; IR (KBr) cm 1: 3417, 3315, 2946, 1774,1641, 1518, 1232, 1045, 759; 1H NMR (CDCl3): d 0.67 (s, 3H), 1.09 (s, 3H), 1.71–1.99 (m, 6H), 2.23–2.53 (m, 3H), 2.83 (t, J = 8.61, 1H), 3.73 (m, 1H), 5.57 (s, 1H), 6.65 (d, J = 15.84, 1H), 7.18 (m, 2H), 7.14–7.22 (m, 2H), 7.52 (d, J = 15.84, 1H) 8.86 (s, 1H); 13C NMR (500 MHz, CDCl3): d 12.13, 19.36, 20.23, 20.58, 21.68, 23.45, 30.42, 31.86, 32.03, 35.34, 36.17, 38.24, 43.16, 44.96, 49.21, 56.01, 61.99, 70.73, 121.52, 125.04, 127.19, 128.35, 132.08, 139.53, 158.14, 196.82; ESI-MS: 438 (M+H); Anal. Calcd. for C28H36FNO2: C, 76.85; H, 8.29; N, 3.02; O, 7.31 F, 4.34; Found: C, 76.74; H, 8.22; N, 2.91; O, 7.12 F, 4.14. 2.2.3.15. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17-yl)-3-(2-methoxyphenyl)prop-2-en-1-imine (4g). Grey powder (93%). M.p.: 255–260 °C; IR (KBr) cm 1: 3485, 3330, 2950, 1718, 1623, 1528, 1246, 1014, 763; 1H NMR (CDCl3): d 0.70 (s, 3H), 1.70 (s, 3H), 1.9–2.10 (m, 6H), 2.60–2.72 (m, 3H), 2.99 (t, J = 8.85,1H), 3.52 (m, 1H), 3.98 (s, 3H), 5.57 (s, 1H), 6.84 (d, J = 16.16, 1H), 7.01 (m, 2H), 7.46 (m, 1H), 7.59 (d, J = 6.37, 1H), 7.98 (d, J = 16.16, 1H), 10.06 (s, 1H),; 13C NMR (500 MHz, CDCl3): d 13.34, 19.14, 20.38, 21.78, 25.11, 30.67, 30.88, 31.03, 35.44, 36.27, 38.44, 43.56, 44.76, 49.01, 55.29, 61.89, 71.13, 118.75, 124.44, 127.09, 128.15, 130.38, 140.93, 160.34, 199.93; ESI-MS:
67
450 (M+H); Anal. Calcd. for C29H39NO3: C, 77.47; H, 8.74; N, 3.12; O, 10.68; Found: C, 77.38; H, 8.64; N, 3.02; O, 10.56. 2.3. Cell culture and bio-assays The human cancer cell lines used for the test were HT-29, HCT15 (Colon), SF-295 (CNS), HOP-62, A-549 (Lung) and MCF-7 (Breast). All these cancer cell lines were obtained from National cancer institute (NCI), biological testing branch, Federick Research and Development Centre, USA. Cellular viability in the presence and absence of experimental agents was determined using the standard Sulforhodamine B assay. Briefly, cells in their log phase of growth were harvested, counted and seeded (104 cells/well in 100 lL medium) in 96-well microtitre plates. After 24 h of incubation at 37° and 5% CO2 to allow cell attachment, cultures were treated with varying concentrations (0.1–100 lM) of test samples made with 1:10 serial dilutions. Four replicate wells were set up for each experimental condition. Test samples were left in contact with the cells for 48 h under same conditions. Thereafter, cells were fixed with 50% chilled trichloroacetic acid (TCA) and kept at 4 °C for 1 h, washed and air dried. Cells were stained with Sulforhodamine B dye. The adsorbed dye was dissolved in Tris-Buffer and plates were gently shaken for 10 min on a mechanical shaker. The optical density (OD) was recorded on ELISA reader at 540 nm. The cell growth was calculated by subtracting mean OD value of respective blank from the mean OD value of experimental set. Percent growth in presence of test material was calculated considering the growth in absence of any test material as 100% and in turn percent growth inhibition in presence of test material was calculated. Finally the IC50 values (Table 3) were calculated using Microsoft Office Excel. The different steroidal derivatives (test material) were dissolved in a mixture of DMSO:Water (1:1) and then introduced into the medium containing the cancer cell lines. 2.4. Results and discussion 2.4.1. Benzylidines Steroidal benzylidines and their oximes Benzylidines of natural products are considered as resourceful molecules as for as their pharmacological activities are concerned. In reference to steroidal benzylidines, we in our earlier communications [8] have shown their therapeutic potential. As we are interested in thorough pharmacological studies of various steroidal analogs, we examined the anticancer potential of such analogs and the results are presented in this manuscript. Though the importance of different chalcone based natural product analogs is now well validated, only few efforts have been reported for their efficient synthesis and biological evaluation as far as steroids are concerned [10]. Modifications at the D-ring of steroids are essentially important and as per the accepted trend as such alterations result in effective receptor binding or the increased bioavailability. Though there are reports for the synthesis of other such analogs [11], the same is not true for the benzylidine derivatives at the D-ring. Taking inspiration from the number of reported biological activities associated with structurally related analogs, we, in continuation of our efforts towards the synthesis of novel D-ring semisynthetic analogs [8], herein report an efficient and simple synthesis of D-ring benzylidine derivatives of 20-keto pregnenanes and their oximes as effective cytotoxic agents. The preparation the benzylidine analogs (3a–3h) and the oxime derivatives (4a–4g) involves the following synthetic approach. The in vitro cytotoxicity studies of various bezylidine pregnenolones and their oxime derivatives revealed that these derivatives are cell specific as these were found to be active mostly against the HCT-15 and MCF-7 cell lines. Further the oxime derivatives of steroids were more potent than the benzylidines. This may be attributed to the more hydrophilicity and thus more bioavalibilty of the
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Table 3 IC50 values (lM) of benzylidine pregnenolones and their oxime derivatives against a panel of human cancer cell lines. Entry
HT-29
HCT-15
SF-295
HOP-62
3a
5.25
2.12
1.43
2.46
3b
2.44
2.43
3.79
1.51
3.42
1.63
3c
1.53
1.46
8.86
4.48
1.67
1.84
3d
6.25
5.11
3.65
8.11
4.84
3e
3.34
1.02
10.69
1.74
0.79
3f
4.55
0.81
1.67
3.57
1.56
1.00
5.44
6.81
2.17
1.91
11.0
A-545 11.0
ND
MCF-7
R
2.46
F
3g
11.8
23.4
3h
2.37
2.64
5.44
6.81
2.17
1.91
4a
2.44
3.53
3.79
1.5.
1.42
1.90
4b
2.43
1.46
8.86
4.18
1.97
1.34
4c
6.25
5.11
3.65
8.11
4.84
4d
3.44
1.62
2.00
3.69
10.64
10.79
4e
2.35
0.31
1.67
3.57
3.56
0.60
F OMe
MeO
F
4f 4g
11.8 2.37
1.4 0.65
50.6 5.44
ND
50.0
48.1
0.81
7.17
OMe Cl NO2
0.93
F
1.91
MeO ND = not determined. Cell lines: HT-29, HCT-15 (Colon), SF-295 (CNS), HOP-62 (Lung), A-549 (Lung) and MCF-7 (Breast).
1.2 1 0.8 IC50
oxime derivatives. As compounds 3e, 3f and 4e were found to be more active than other analogs, it can be assumed that electron withdrawing groups have an effect over the activity. It is worth to mention that although the oximes formed may be a mixture of syn and anti isomers, however only one of the two was obtained keeping the steric constraints into consideration.
0.6
HCT
0.4
2.4.2. Cell culture and bio-assays The following table gives the cancer cell inhibitory data obtained after treating different cancer cell lines with test doses of the different steroidal benzylidine and oxime derivatives and the values are reported in terms of IC50. It is clear from the IC50 values, that the compounds 3e, 3f and 4e showed significant cytotoxic activity especially against HCT-15 and MCF-7 cancer cell lines. Fig. 1 represents the standard error in the IC50 values of the most potent compounds against HCT and MCF cell lines. It is evident from the IC50 data that even the position of substituent on the aromatic ring influences the relative cytotoxicity which can be attributed to their differences in either the bioavailability or the protein binding properties. As already discussed
MCF
0.2 0 3e
3f 4e Acve Compounds
Fig. 1. Standard error values (% error) of the most active compounds against HCT15 and MCF-7.
it can be assumed that the strong electron withdrawing groups such as flouro- and nitro- increase the in vitro cytotoxicity. Overall the cytotoxicity of these compounds is cancer cell specific as the IC50 values make it evident.
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3. Conclusion A series of novel benzylidine and benzylidine oxime derivatives of pregnenolones were synthesized and screened for anticancer activity against a panel of six human cancer cell lines. From the data it was found that all the compounds are having promising anticancer activity and the compound 4e was found to be the most active in this study.
[7] [8]
References [1] Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years. J Nat Prod 2007;70:461–77. [2] Cutler SJ, Cutler HG. Biologically active natural products: pharmaceuticals. CRC Press; 2000. p. 5–23. [3] Cragg GM, Kingston DGI, Newman DJ, editors. Anticancer agents from natural products. Boca Raton: CRC Press; 2005. [4] Dubey RK, Oparil S, Imthurn B, Jackson EK. Sex hormones and hypertension. Cardiovasc Res 2002;53:688–708. [5] Latham KA, Zamora A, Drought H, Subramanian S, Matejuk A, Offner H, et al. Estradiol treatment redirects the isotype of the autoantibody response and prevents the development of autoimmune arthritis. J Immunol 2003;171(11):5820–7. [6] (a) Sheridan PJ, Blum K, Trachtenberg M. Steroid receptors and disease. New York: Marcel Dekker; 1988. p. 289–564;
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(b) Moudgil VK. Steroid receptors in health and disease. New York/ London: Plenum Press; 1987. (a) O’Malley BW. Hormones and signalling. San Diego: Academic Press; 1998; (b) Parker MG. Steroid hormone action. Oxford: IRL Press; 1993. (a) Banday AH, Mir BP, Lone IH, Suri KA, Kumar HMS. Studies on novel D-ring substituted steroidal pyrazolines as potential anticancer agents. Steroids 2010;75(12):805–9; (b) Banday AH, Shameem SA, Gupta BD, Kumar HMS. D-ring substituted 1,2,3triazolyl 20-keto pregnenanes as potential anticancer agents: Synthesis and biological evaluation. Steroids 2010;75(12):801–4; (c) Banday AH, Zargar MI, Ganai BA. Synthesis and antimicrobial studies of Chalconyl pregnenolones. Steroids 2011;76:1358–62; (d) Banday AH, Singh S, Alam MS, Reddy DM, Gupta BD, Kumar HMS. Synthesis of novel steroidal D-ring substituted isoxazoline derivatives of 17oxoandrostanes. Steroids 2008;73:370–4. (a) Morrow AL. Recent developments in the significance and the therapeutic relevance of neuroactive steroids. Pharmacology and therapeutics 2007;116(1):1–6; (b) Choudhary MI, Alam MS, Atta-ur-Rahman, Yousuf S, Wud YC, Lind AS, et al. Pregnenolone derivatives as potential anticancer agents. Steroids 2011;76:1554–9. Saxena HO, Faridi U, Kumar JK, Luqman S, Darokar MP, Shankar K, et al. Synthesis of chalcone derivatives on steroidal framework and their anticancer activities. Steroids 2007;72:892–900. Warjeet S, Laitonjam TS, Rajkumar BS. Synthesis of some A and D-ring fused steroidal pyrazoles, isoxazoles and pyrimidines. Steroids 2002:67203–9.
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Benzylidine pregnenolones and their oximes as potential anticancer agents: Synthesis and biological evaluation Abid H. Banday a,b,⇑, S.M.M. Akram a,c, Shameem A. Shameem a a
Department of Chemistry, Islamia College of Science and Commerce, Srinagar 190009, India Department of Chemistry and Biochemistry, University of Arizona, Tucson 85721, USA c Department of Chemistry, University of Kashmir, Srinagar 190002, India b
a r t i c l e
i n f o
Article history: Received 8 November 2013 Received in revised form 5 March 2014 Accepted 15 March 2014 Available online 1 April 2014 Keywords: Benzylidine pregnenolone Aldol condensation Nucleophillic addition Cytoxicity Dehydration
a b s t r a c t The present study reveals the anticancer activity of benzylidine pregnenolones and their oxime derivatives. The synthesis of the analogs of both series is very simple and involves aldol condensation in the first step followed by nucleophillic addition of hydroxylamine across carbonyl in the second step. Quantitative yields of more than 80% are obtained in both the steps. All the compounds were tested for their cytotoxic activities against a panel of six human cancer cell lines. Amongst all the compounds of both the series screened for their cytotoxic activity, compound 3e, 3f and 4e are very potent especially against HCT-15 and MCF-7 cancer cell lines. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Over the past twenty years, medicinal chemistry has gained enormous popularity for its role in drug discovery. A number of natural products, their semi synthetic analogs and small molecules have been discovered and evaluated for their role in the treatment and cure of various fatal diseases such as cancer, diabetes, microbial infections and cardiovascular diseases etc. [1,2]. However because of the drug resistance and drug tolerance problems, there is always scope for the design and development of new and modified analogs as more efficient drug candidates. This has been done through the rational design and synthesis of receptor based lead molecules which still remains an open area. Natural products have extensively been used as starting tools for the design and synthesis of lead therapeutic scaffolds. The use of natural products for curing human ailments dates back to 1550 BC, since natural products have been used in medicine especially for the treatment of cancer and related diseases. A number of plants have been used as folklore medicinal agents. The modern drug discovery program has made a great success as a number of lead compounds have been developed from the traditionally used medicinal plants. A number of plant
⇑ Corresponding author at: Department of Chemistry, Islamia College of Science and Commerce, Srinagar 190009, India. Fax: +91 194 2429014. E-mail address: [email protected] (A.H. Banday). http://dx.doi.org/10.1016/j.steroids.2014.03.010 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.
based anticancer compounds such as vinblastine (Velban), vincristine (Oncovin), vinorelbine (Navelbine), etoposide (VP-16), teniposide (VM-26), Taxol (paclitaxel), and most recently Taxotere (docetaxel), topotecan (Hycamtin), and irinotecan (Camptosar) have been approved for use as anticancer drugs by the US FDA [3]. Steroids as natural products have been extensively studied as their biological and clinical importance is now well validated. Steroids as well as their derivatives have been found to have the potential to be developed as drugs for the treatment of a large number of diseases including cardiovascular [4], autoimmune diseases [5], brain tumours, breast cancer, prostate cancer, osteoarthritis, etc. [6]. The promise of using steroids for development of lead molecules lies in their regulation of a variety of biological processes and being a fundamental class of signaling molecules [7]. We have been interested in the area of steroid based medicinal chemistry for the past few years and we have successfully evaluated various steroid based congeners as potent pharmacological agents showing activities such as cytotoxic, antimicrobial, antioxidant etc. [8]. This is particularly true of pregnenolone which happens to be a precursor of most other steroids and has been extensively studied for various biological activities. It has been found that the molecule holds a great promise against brain disorders, memory loss, schizophrenia, insulin related disorders and cancer [9]. Thus in continuation of our program, we herein report the anticancer activities of two series of our compounds I,e benzylidine pregnenolones and their oxime derivatives. We are reporting
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the primary in vitro results as the mechanistic studies are in progress and will be communicated in due course of time.
Table 1 Nature of group ‘‘R’’ in the compounds 3a–3h. Entry
2. Experimental
Nature of R
Entry
3a
3e
3b
3f
3c
3g
3d
3h
Nature of R
F
2.1. General methods Melting points were recorded on Buchi Melting point apparatus D-545; IR spectra (KBr discs) were recorded on Bruker Vector 22 instrument. NMR spectra were recorded on Bruker DPX200 instrument in CDCl3 with TMS as internal standard for protons and solvent signals as internal standard for carbon spectra. Chemical shift values are mentioned in d (ppm) and coupling constants are given in Hz. Mass spectra were recorded on EIMS (Shimadzu) and ESI-esquire 3000 Bruker Daltonics instrument. The progress of all reactions was monitored by TLC on 2 5 cm pre-coated silica gel 60 F254 plates of thickness of 0.25 mm (Merck). The chromatograms were visualized under UV 254–366 nm and iodine.
F
OMe
MeO
Table 2 Nature of group ‘‘R’’ in the compounds 4a–4g.
2.2. Chemical synthesis 2.2.1. General procedure for the synthesis of Benzylidine pregnenolones (3) To a solution of pregnenolone 1 (0.316 g, 1 mmol, 1 eq.) in ethanol (10 ml) was added a conc. aq. solution of KOH (2 eq.). Then aldehyde 2 (1.2 eq.) was charged into the reaction mixture to get the corresponding benzylidine derivative 3 (Scheme 1). After completion as revealed by thin layer chromatography (TLC) in an average span of around 1 h, the reaction mixture was precipitated using water because of the limited solubility. The precipitate was filtered, dried and monitored through TLC for the purity. Thin layer chromatography revealed just a single spot which proved the presence of a single product. For further purification, the product was recrystallized from EtOAc:Hexane to give product as solid white powder. It is to be mentioned that when non-aromatic aldehydes were used, the product was formed in a very minor quantity and that too not stable enough at ambient conditions. Thus the study was restricted to the use of aromatic aldehydes only (Tables 1 and 2). 2.2.2. General procedure for the synthesis of Benzylidine pregnenolone oximes (4) To an ethanolic solution of compound 3d (0.418 g, 1 mmol) was added hydroxylamine hydrochloride (0.139 g, 2 mmol) and the reaction was stirred for 2 h at room temperature. A precipitate of the oxime was obtained as a single spot as revealed by the TLC. The reaction mixture was worked up by first evaporating the ethanol and then extracting the solid with EtOAc: H2O (3 25 ml). Quantitative yields of 90–93% were obtained for all the compounds.The spectral data of various compounds from both the series is given as under. 2.2.3.1. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl)-3-phenylprop-2-en-1-one (3a). White powder O
2
R
EtOH/ KOH
1
Nature of R
Entry
4a
F
4c
NO2
4f
F
4g
OMe
MeO 4d
Cl
(86%). M.p.: 128–131 °C; IR (KBr) cm 1: 3425, 2938, 1804, 1637,1403, 1041, 689; 1H NMR (CDCl3): d 0.63 (s, 3H), 1.00 (s, 3H),1.61–1.90 (m, 6H), 2.20–2.38 (m, 3H), 2.82 (t, J = 8.80, 1H); 3.51 (m, 1H); 6.78 (s, J = 16.00, 1H), 7.39 (m, 3H), 7.55 (m, 3H); 13C NMR (500 MHz, CDCl3): d 13.33, 19.26, 21.07, 22.67, 24.62, 31.13, 31.78, 31.99, 37.22, 41.81, 45.11, 48.61, 48.78, 48.95, 49.12, 49.29, 50.04, 57.14, 61.97, 71.22, 121.24, 126.69, 128.29, 128.90, 130.41, 134.62, 140.85, 141.96, 201.32; ESI-MS: 405 (M+H); Anal. Calcd. for C28H36O2: C, 83.12; H, 8.97; Found C, 83.37; H, 8.83. 2.2.3.2. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl)-3-o-tolylprop-2-en-1-one (3b). Yellow powder (89%). M.p.: 208–210 °C; IR (KBr) cm 1: 3405, 2941, 1774, 1674, 1403, 1041, 757; 1H NMR (CDCl3): d 0.62 (s, 3H), 1.00 (s, 3H), 1.63–1.90 (m, 6H), 2.22–2.35 (m, 3H), 2.38 (s, 3H), 2.82 (t, J = 8.43, 1H); 3.52 (m, 1H), 5.32 (s, 1H), 6.69 (d, J = 15.78, 1H), 7.32 (m, 3H), 7.57 (d, J = 6.83, 1H), 7.82 (d, J = 15.78,1H); 13C NMR (500 MHz, CDCl3): d 13.54, 19.46, 19.95, 21.21, 2.84, 24.77, R
HON
NH2OH.HCl EtOH
HO
Nature of R
4e
4b
O OHC
HO
Entry
HO
3 Scheme 1. Synthesis of D-ring substituted benzylidine pregnenolones and their oximes.
4
R
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31.67, 31.92, 32.09, 36.61, 37.27, 37.42, 39.32,42.32, 45.00, 50.13, 57.25, 62.17, 71.70, 71.76, 121.48, 121.72, 126.29, 126.37, 128.01, 129.10, 130.08, 130.93, 133.81, 138.27, 139.06, 140.53, 140.86, 200.51; ESI-MS: 441 (M+Na); Anal. Calcd. for C29H38O2: C, 83.21; H, 9.15; Found C, 83.32; H, 8.83.
2.2.3.3. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl)-3-m-tolylprop-2-en-1-one (3c). Solid white powder (85%). M.p.: 211–213 °C; IR (KBr) cm 1: 3415, 2934, 1804, 1773, 1638, 1403, 1042, 779; 1H NMR (CDCl3): d 0.63 (s, 3H), 1.00 (s, 3H), 1.61–1.90 (m, 6H), 2.20–2.34 (m, 3H), 2.44 (s, 3H), 2.86 (t, J = 8.73,1 H); 3.54 (m, 1H), 5.36 (s, 1H), 6.75 (d, J = 15.93, 1H), 7.27 (m, 4H), 7.51 (d, J = 15.93, 1H); 13C NMR (500 MHz, CDCl3): d 12.36, 19.38, 20.13, 20.48, 21.78, 24.10, 30.62, 30.86, 31.03, 35.54, 36.27, 38.14, 43.26, 43.96, 49.11, 56.21, 60.99, 71.73, 120.44, 124.94, 127.29, 128.65, 131.18, 139.63, 140.54, 198.42; ESI-MS: 441 (M+Na); Anal. Calcd. for C29H38O2: C, 83.21; H, 9.15; Found C, 83.03; H, 9.33.
2.2.3.4. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl)-3-p-tolylprop-2-en-1-one (3d). Solid yellowish powder (78%). M.p.: 243–245 °C; IR (KBr) cm 1: 3397, 2941, 1804, 1773, 1637, 1403, 1216, 1039, 758; 1H NMR (CDCl3): d 0.63 (s, 3 H), 1.00 (s, 3H), 1.61–1.90 (m, 6H), 2.20–2.38 (m, 3H), 2.45 (s, 3H), 2.85 (t, J = 8.43, 1H); 3.52 (m, 1H), 5.36 (s, 1H), 6.74 (d, J = 15.98, 1H), 7.22 (d, J = 7.82, 2H), 7.48 (d, J = 7.82, 2H), 7.54 (d, J = 15.98, 1H); 13C NMR (500 MHz, CDCl3): d 12.42, 18.38, 20.13, 20.48, 21.78, 23.70, 30.62, 30.86, 31.03, 35.54, 36.27, 38.14, 41.27, 43.96, 49.11, 56.21, 60.99, 70.73, 120.44, 124.94, 127.29, 128.65, 131.08, 139.73, 140.55, 199.42; ESI-MS: 441 (M+Na); Anal. Calcd. for C29H38O2: C, 83.21; H, 9.15; Found C, 83.31; H, 8.95.
2.2.3.5. (2E)-3-(3-fluorophenyl)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13, 14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl) prop-2-en-1-one (3e). Solid yellowish powder (84%). M.p.: 228–231 °C; IR (KBr) cm 1: 3407, 2940, 1773, 1678, 1508, 1232, 1040, 756; 1H NMR (CDCl3): d 0.62 (s, 3H), 1.00 (s, 3H), 1.61–1.90 (m, 6H), 2.20–2.32 (m, 3H), 2.84 (t, J = 8.92, 1H); 3.52 (m, 1H), 5.36 (s, 1H), 6.70 (d, J = 15.93, 1H), 7.08 (m, 2H), 7.53 (m, 3H); 13C NMR (500 MHz, CDCl3): d 13.06, 19.28, 20.13, 20.48, 21.78, 25.10, 30.62, 30.86, 31.03, 35.54, 36.27, 38.14, 43.26, 43.96, 49.11, 56.21, 60.99, 71.73, 122.44, 124.94, 126.49, 129.02, 132.18, 139.63, 140.53, 196.92; ESI-MS: 445 (M+Na); Anal. Calcd. for C28H35F O2: C, 79.58; H, 8.35, F, 4.50; Found C, 79.62; H, 8.52, F, 4.32.
2.2.3.6. (2E)-3-(4-fluorophenyl)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13, 14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl) prop-2-en-1-one (3f). Solid White powder (80%). M.p.:196–198 °C; IR (KBr) cm 1: 3417, 2946, 1772, 1678, 1508, 1232, 1045, 755; 1H NMR (CDCl3): d 0.62 (s, 3H), 1.00 (s, 3H), 1.61–1.90 (m, 6H), 2.20–2.33 (m, 3H), 2.83 (t, J = 8.61,1 H), 3.63 (m, 1H), 5.37 (s, 1H), 6.75 (d, J = 15.84, 1H), 7.08 (m, 2H), 7.24–7.32 (m, 2H), 7.50 (d, J = 15.84, 1H); 13C NMR (500 MHz, CDCl3): d 12.22, 19.26, 20.43, 20.48, 21.78, 23.15, 30.62, 30.86, 31.03, 35.54, 36.27, 38.14, 43.26, 43.96, 49.11, 56.21, 60.99, 71.73, 120.52, 124.94, 127.29, 128.65, 131.18, 139.63, 140.54, 196.12; ESI-MS: 445 (M+Na); Anal. Calcd. for C28H35F O2: C, 79.58; H, 8.35, F, 4.50; Found C, 79.42; H, 8.52, F, 4.67.
2.2.3.7. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl)-3-(4-methoxyphenyl)prop-2-en-1-one (3g). Solidwhite powder (87%). M.p.: 200–203 °C; IR (KBr) cm 1: 3415, 2926, 2853, 1803, 1708, 1637, 1508, 1255, 1036, 762; 1H NMR (CDCl3): d 0.60 (s, 3H), 1.00 (s, 3H), 1.65–1.90 (m, 6H), 2.20–2.36 (m, 3H), 2.83 (t, J = 8.89, 1H), 3.53 (m, 1H), 3.87 (s, 3H), 5.36 (s, 1H), 6.65 (d, J = 15.92, 1H), 6.91 (d, J = 8.72, 2H), 7.51 (d, J = 8.72, 2H), 7.53 (d, J = 15.84, 1H); 13C NMR (500 MHz, CDCl3): d 12.13, 18.31, 21.11, 20.48, 21.78, 24.10, 30.62, 30.81, 31.23, 34.52, 36.27, 38.14, 43.26, 43.96, 49.22, 56.21, 60.99, 71.73, 120.44, 124.94, 127.29, 128.65, 133.16, 139.63, 140.54, 196.54; ESI-MS: 457 (M+Na); Anal. Calcd. for C29H38 O3: C, 80.14; H, 8.81; Found C, 80.32; H, 8.67. 2.2.3.8. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17(b)-yl)-3-(2-methoxyphenyl)prop-2-en-1-one (3h). Grey powder (76%). M.p.: 225–227 °C; IR (KBr) cm 1: 3425, 2940,1728, 1636, 1598, 1296, 1024, 753; 1H NMR (CDCl3): d 0.63 (s,3H), 1.00 (s, 3H), 1.65–1.90 (m, 6H), 2.20–2.32 (m, 3H), 2.89 (t, J = 8.85, 1H), 3.52 (m, 1H), 3.91 (s, 3H), 5.37 (s, 1H), 6.94 (d, J = 16.16, 1H), 6.98 (m, 2H), 7.36 (m, 1H), 7.55 (d, J = 6.37, 1H), 7.88 (d, J = 16.16, 1H); 13C NMR (500 MHz, CDCl3): d 12.34, 18.34, 20.13, 20.48, 21.78, 25.10, 30.62, 30.86, 31.03, 35.54, 36.27, 38.14, 43.26, 43.96, 49.11, 56.21, 60.99, 71.73, 118.30, 124.94, 127.29, 128.65, 131.18, 139.63, 140.54, 195.23; ESI-MS: 457 (M+Na); Anal. Calcd. for C29H38O3: C, 80.14; H, 8.81; Found C, 80.02; H, 8.97. 2.2.3.9. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a ]phenanthren-17-yl)-3-p-methylphenylprop-2-en-1-imine (4a). Grey powder (90%). M.p.: 235–240 °C; IR (KBr) cm 1: 3525, 3385, 2948, 1814, 1617, 1512, 1423, 1051, 609; 1H NMR (CDCl3): d 0.78 (s, 3H), 1.200 (s, 3H), 1.54–2.0 (m, 6H), 2.41–2.48 (m, 3H), 2.42 (t, J = 8.80, 1H); 3.21 (m, 1H); 5.98 (s, J = 16.00, 1H), 6.89 (m, 3H), 7.05 (m, 3H), 8.68 (s, 1H); 13C NMR (500 MHz, CDCl3): d 14.23, 21.06, 22.07, 24.02, 31.03, 31.48, 32.09, 36.52, 42.11, 45.21, 48.01, 48.75, 49.32, 49.79, 50.84, 57.44, 61.07, 73.12, 123.26, 127.69, 128.89, 131.41, 135.72, 142.15, 158.6, 201.12; ESI-MS: 420 (M+H); Anal. Calcd. for C28H37NO2: C, 80.15; H, 8.89; N, 3.34; O, 7.63; Found C, 80.10; H, 8.71; N, 3.04; O, 7.52. 2.2.3.10. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[ a ]phenanthren-17-yl)-3-(3-fluorophenyl)-prop-2-en-1-imine (4b). White powder (91%). M.p.: 240–245 °C; IR (KBr) cm 1: 3407, 3312, 2940, 1773, 1618, 1518, 1232, 1040, 781; 1H NMR (CDCl3): d 0.66 (s, 3H), 1.00 (s, 3H), 1.81–2.00 (m, 6H), 2.40–2.62 (m, 3H), 2.94 (t, J = 8.92, 1H); 4.12 (m, 1H), 5.36 (s, 1H), 6.90 (d, J = 15.93, 1H), 7.18 (m, 2H), 7.73 (m, 3H) 9.36 (s, 1H); 13C NMR (500 MHz, CDCl3): d 12.16, 19.18, 20.43, 22.78, 25.90, 30.82, 30.96, 31.13, 35.74, 36.87, 38.24, 43.56, 44.96, 49.71, 56.81, 60.99, 72.73, 122.14, 124.14, 126.79, 129.12, 132.98, 159.73, 196.92; ESI-MS: 438 (M+H); Anal. Calcd. for C28H36FNO2: C, 76.85; H, 8.29; N, 3.02; O, 7.31 F, 4.34; Found: C, 76.76; H, 8.21; N, 2.92; O, 7.11 F, 4.14. 2.2.3.11. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[ a ]phenanthren-17-yl)-3-(4-methoxyphenyl)prop-2-en-1-imine (4c). Solid powder (90%). M.p.: 250–255 °C; IR (KBr) cm 1: 3405, 3322, 2916, 2853, 1803, 1637, 1521, 1275, 1036, 769; 1H NMR (CDCl3): d 0.68 (s, 3H), 1.2(s, 3H), 1.7–1.90 (m, 6H), 2.3–2.56 (m, 3H), 2.93 (t, J = 8.89, 1H), 3.13 (m, 1H), 4.17 (s, 3H), 5.46 (s, 1H), 6.55 (d,
A.H. Banday et al. / Steroids 84 (2014) 64–69
J = 15.92, 1H), 6.81 (d, J = 8.72, 2H), 7.61 (d, J = 8.72, 2H), 7.83 (d, J = 15.84, 1H), 9.36 (s, 1H); 13C NMR (500 MHz, CDCl3): d 12.23, 19.31, 21.21, 21.78, 24.23, 31.51, 32.13, 34.42, 36.17, 38.24, 43.56, 44.96, 49.12, 56.31, 61.89, 75.83, 121.54, 124.74, 127.19, 128.45, 132.46, 139.63, 160.14, 199.94; ESI-MS: 450 (M+H); Anal. Calcd. for C29H39NO3: C, 77.47; H, 8.74; N, 3.12; O, 10.68 Found: C, 77.38; H, 8.64; N, 3.02; O, 10.56. 2.2.3.12. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17-yl)-3-(4-chlorophenyl)-prop-2-en-1-imine (4d). Grey powder (91%). M.p.: 240–250 °C; IR (KBr) cm 1: 3425, 3300, 2917, 1718, 1611, 1513, 1296, 1024, 763; 1H NMR (CDCl3): d 0.61 (s, 3H), 1.03 (s, 3H), 1.65–1.92 (m, 6H), 2.22–2.54 (m, 3H), 2.76 (t, J = 9.12, 1H), 3.62 (m, 1H), 5.45 (s, 1H), 6.74 (d, J = 15.31, 1H), 6.96–7.21 (m, 4H), 7.54 (d, J = 15.31, 1H), 9.96 (s, 1H), 13C NMR (500 MHz, CDCl3): d 13.13, 19.58, 20.23, 24.04, 25.21, 30.92, 31.66, 35.04, 36.17, 38.44, 43.16, 45.76, 49.21, 57.28, 61.29, 75.03, 121.64, 124.04, 127.19, 129.45, 130.98, 142.13, 160.04, 199.83; ESI-MS: 455 (M+H); Anal. Calcd. for C28H36ClNO2: C, 74.07; H, 7.99; N, 3.08; O, 7.05; Cl, 7.81; Found C, 74.01; H, 7.89; N, 3.01; O, 6.99; Cl, 7.78. 2.2.3.13. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17-yl)-3-p-nitrophenylprop-2-en-1-imine (4e). White powder (91%). M.p.: 255–260 °C; IR (KBr) cm 1: 3410, 3315, 2941, 1764, 1651, 1323, 1061, 797; 1H NMR (CDCl3): d 0.71 (s, 3H), 1.09 (s, 3H), 1.93–2.0 (m, 6H), 2.26–2.39 (m, 3H), 2.31 (s, 3H), 3.12 (t, J = 8.43, 1H); 3.82 (m, 1H), 6.12 (s, 1H), 7.01 (d, J = 15.78, 1H), 7.72 (m, 3H), 7.89 (d, J = 6.83, 1H), 8.12 (d, J = 15.78, 1H), 9.31 (s, 1H), 13C NMR (500 MHz, CDCl3): d 13.94, 19.96, 20.15, 21.11, 22.14, 24.87, 31.07, 32.19, 36.71, 37.37, 37.82, 39.82,42.12, 45.18,51.23, 57.75, 63.27, 71.30, 71.86, 121.82, 126.57, 128.11, 129.18, 130.58, 131.93, 134.81, 138.87, 139.76, 140.53, 160.3, 200.51; ESI-MS: 465 (M+H); Anal. Calcd. for C28H36N2O4: C, 72.39; H,7.81; N, 6.03; O, 13.77; Found: C, 72.09; H, 7.68; N, 5.83; O, 13.57. 2.2.3.14. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17-yl)-3-(4-fluorophenyl)-prop-2-en-1-imine (4f). White powder (92%). M.p.:243–250 °C; IR (KBr) cm 1: 3417, 3315, 2946, 1774,1641, 1518, 1232, 1045, 759; 1H NMR (CDCl3): d 0.67 (s, 3H), 1.09 (s, 3H), 1.71–1.99 (m, 6H), 2.23–2.53 (m, 3H), 2.83 (t, J = 8.61, 1H), 3.73 (m, 1H), 5.57 (s, 1H), 6.65 (d, J = 15.84, 1H), 7.18 (m, 2H), 7.14–7.22 (m, 2H), 7.52 (d, J = 15.84, 1H) 8.86 (s, 1H); 13C NMR (500 MHz, CDCl3): d 12.13, 19.36, 20.23, 20.58, 21.68, 23.45, 30.42, 31.86, 32.03, 35.34, 36.17, 38.24, 43.16, 44.96, 49.21, 56.01, 61.99, 70.73, 121.52, 125.04, 127.19, 128.35, 132.08, 139.53, 158.14, 196.82; ESI-MS: 438 (M+H); Anal. Calcd. for C28H36FNO2: C, 76.85; H, 8.29; N, 3.02; O, 7.31 F, 4.34; Found: C, 76.74; H, 8.22; N, 2.91; O, 7.12 F, 4.14. 2.2.3.15. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17-yl)-3-(2-methoxyphenyl)prop-2-en-1-imine (4g). Grey powder (93%). M.p.: 255–260 °C; IR (KBr) cm 1: 3485, 3330, 2950, 1718, 1623, 1528, 1246, 1014, 763; 1H NMR (CDCl3): d 0.70 (s, 3H), 1.70 (s, 3H), 1.9–2.10 (m, 6H), 2.60–2.72 (m, 3H), 2.99 (t, J = 8.85,1H), 3.52 (m, 1H), 3.98 (s, 3H), 5.57 (s, 1H), 6.84 (d, J = 16.16, 1H), 7.01 (m, 2H), 7.46 (m, 1H), 7.59 (d, J = 6.37, 1H), 7.98 (d, J = 16.16, 1H), 10.06 (s, 1H),; 13C NMR (500 MHz, CDCl3): d 13.34, 19.14, 20.38, 21.78, 25.11, 30.67, 30.88, 31.03, 35.44, 36.27, 38.44, 43.56, 44.76, 49.01, 55.29, 61.89, 71.13, 118.75, 124.44, 127.09, 128.15, 130.38, 140.93, 160.34, 199.93; ESI-MS:
67
450 (M+H); Anal. Calcd. for C29H39NO3: C, 77.47; H, 8.74; N, 3.12; O, 10.68; Found: C, 77.38; H, 8.64; N, 3.02; O, 10.56. 2.3. Cell culture and bio-assays The human cancer cell lines used for the test were HT-29, HCT15 (Colon), SF-295 (CNS), HOP-62, A-549 (Lung) and MCF-7 (Breast). All these cancer cell lines were obtained from National cancer institute (NCI), biological testing branch, Federick Research and Development Centre, USA. Cellular viability in the presence and absence of experimental agents was determined using the standard Sulforhodamine B assay. Briefly, cells in their log phase of growth were harvested, counted and seeded (104 cells/well in 100 lL medium) in 96-well microtitre plates. After 24 h of incubation at 37° and 5% CO2 to allow cell attachment, cultures were treated with varying concentrations (0.1–100 lM) of test samples made with 1:10 serial dilutions. Four replicate wells were set up for each experimental condition. Test samples were left in contact with the cells for 48 h under same conditions. Thereafter, cells were fixed with 50% chilled trichloroacetic acid (TCA) and kept at 4 °C for 1 h, washed and air dried. Cells were stained with Sulforhodamine B dye. The adsorbed dye was dissolved in Tris-Buffer and plates were gently shaken for 10 min on a mechanical shaker. The optical density (OD) was recorded on ELISA reader at 540 nm. The cell growth was calculated by subtracting mean OD value of respective blank from the mean OD value of experimental set. Percent growth in presence of test material was calculated considering the growth in absence of any test material as 100% and in turn percent growth inhibition in presence of test material was calculated. Finally the IC50 values (Table 3) were calculated using Microsoft Office Excel. The different steroidal derivatives (test material) were dissolved in a mixture of DMSO:Water (1:1) and then introduced into the medium containing the cancer cell lines. 2.4. Results and discussion 2.4.1. Benzylidines Steroidal benzylidines and their oximes Benzylidines of natural products are considered as resourceful molecules as for as their pharmacological activities are concerned. In reference to steroidal benzylidines, we in our earlier communications [8] have shown their therapeutic potential. As we are interested in thorough pharmacological studies of various steroidal analogs, we examined the anticancer potential of such analogs and the results are presented in this manuscript. Though the importance of different chalcone based natural product analogs is now well validated, only few efforts have been reported for their efficient synthesis and biological evaluation as far as steroids are concerned [10]. Modifications at the D-ring of steroids are essentially important and as per the accepted trend as such alterations result in effective receptor binding or the increased bioavailability. Though there are reports for the synthesis of other such analogs [11], the same is not true for the benzylidine derivatives at the D-ring. Taking inspiration from the number of reported biological activities associated with structurally related analogs, we, in continuation of our efforts towards the synthesis of novel D-ring semisynthetic analogs [8], herein report an efficient and simple synthesis of D-ring benzylidine derivatives of 20-keto pregnenanes and their oximes as effective cytotoxic agents. The preparation the benzylidine analogs (3a–3h) and the oxime derivatives (4a–4g) involves the following synthetic approach. The in vitro cytotoxicity studies of various bezylidine pregnenolones and their oxime derivatives revealed that these derivatives are cell specific as these were found to be active mostly against the HCT-15 and MCF-7 cell lines. Further the oxime derivatives of steroids were more potent than the benzylidines. This may be attributed to the more hydrophilicity and thus more bioavalibilty of the
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A.H. Banday et al. / Steroids 84 (2014) 64–69
Table 3 IC50 values (lM) of benzylidine pregnenolones and their oxime derivatives against a panel of human cancer cell lines. Entry
HT-29
HCT-15
SF-295
HOP-62
3a
5.25
2.12
1.43
2.46
3b
2.44
2.43
3.79
1.51
3.42
1.63
3c
1.53
1.46
8.86
4.48
1.67
1.84
3d
6.25
5.11
3.65
8.11
4.84
3e
3.34
1.02
10.69
1.74
0.79
3f
4.55
0.81
1.67
3.57
1.56
1.00
5.44
6.81
2.17
1.91
11.0
A-545 11.0
ND
MCF-7
R
2.46
F
3g
11.8
23.4
3h
2.37
2.64
5.44
6.81
2.17
1.91
4a
2.44
3.53
3.79
1.5.
1.42
1.90
4b
2.43
1.46
8.86
4.18
1.97
1.34
4c
6.25
5.11
3.65
8.11
4.84
4d
3.44
1.62
2.00
3.69
10.64
10.79
4e
2.35
0.31
1.67
3.57
3.56
0.60
F OMe
MeO
F
4f 4g
11.8 2.37
1.4 0.65
50.6 5.44
ND
50.0
48.1
0.81
7.17
OMe Cl NO2
0.93
F
1.91
MeO ND = not determined. Cell lines: HT-29, HCT-15 (Colon), SF-295 (CNS), HOP-62 (Lung), A-549 (Lung) and MCF-7 (Breast).
1.2 1 0.8 IC50
oxime derivatives. As compounds 3e, 3f and 4e were found to be more active than other analogs, it can be assumed that electron withdrawing groups have an effect over the activity. It is worth to mention that although the oximes formed may be a mixture of syn and anti isomers, however only one of the two was obtained keeping the steric constraints into consideration.
0.6
HCT
0.4
2.4.2. Cell culture and bio-assays The following table gives the cancer cell inhibitory data obtained after treating different cancer cell lines with test doses of the different steroidal benzylidine and oxime derivatives and the values are reported in terms of IC50. It is clear from the IC50 values, that the compounds 3e, 3f and 4e showed significant cytotoxic activity especially against HCT-15 and MCF-7 cancer cell lines. Fig. 1 represents the standard error in the IC50 values of the most potent compounds against HCT and MCF cell lines. It is evident from the IC50 data that even the position of substituent on the aromatic ring influences the relative cytotoxicity which can be attributed to their differences in either the bioavailability or the protein binding properties. As already discussed
MCF
0.2 0 3e
3f 4e Acve Compounds
Fig. 1. Standard error values (% error) of the most active compounds against HCT15 and MCF-7.
it can be assumed that the strong electron withdrawing groups such as flouro- and nitro- increase the in vitro cytotoxicity. Overall the cytotoxicity of these compounds is cancer cell specific as the IC50 values make it evident.
A.H. Banday et al. / Steroids 84 (2014) 64–69
3. Conclusion A series of novel benzylidine and benzylidine oxime derivatives of pregnenolones were synthesized and screened for anticancer activity against a panel of six human cancer cell lines. From the data it was found that all the compounds are having promising anticancer activity and the compound 4e was found to be the most active in this study.
[7] [8]
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