Gallic acid-based indanone derivatives as anticancer agents

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3914–3918 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl Gallic acid-based indanone derivatives as anticancer agents q Hari Om Saxena a, Uzma Faridi b, Suchita Srivastava b, J. K. Kumar b, M. P. Darokar b, Suaib Luqman b, C. S. Chanotiya b, Vinay Krishna b, Arvind S. Negi b,*, S. P. S. Khanuja b a b Rain Forest Research Institute, Jorhat, India Central Institute of Medicinal and Aromatic Plants (CIMAP), PO CIMAP, Kukrail Road, Lucknow 226 015, India a r t i c l e i n f o Article history: Received 28 March 2008 Revised 15 May 2008 Accepted 11 June 2008 Available online 14 June 2008 Keywords: Gallic acid Indanones Anticancer Osmotic fragility HMQC a b s t r a c t Gallic acid-based indanone derivatives have been synthesised. Some of the indanones showed very good anticancer activity in MTT assay. Compounds 10, 11, 12 and 14 possessed potent anticancer activity against various human cancer cell lines. The most potent indanone (10, IC50 = 2.2 lM), against MCF-7, that is, hormone-dependent breast cancer cell line, showed no toxicity to human erythrocytes even at higher concentrations (100 lg/ml, 258 lM). While, indanones 11, 12 and 14 showed toxicities to erythrocytes at higher concentrations. Ó 2008 Elsevier Ltd. All rights reserved. Indanones and related compounds are important bioactive molecules. These compounds have been studied for various biological activities including cancer and Alzheimer’s type of diseases. Indanones are also used as drug intermediates, ligands of olefinic polymerisation catalysts2a,b and discotic liquid crystals.3 Indanocine (1, Fig. 1) and its analogues are being developed to combat drug-resistant malignancies.4 Another indanone analogue Donepezil hydrochloride (2, Fig. 1) has been approved by US-FDA for the treatment of mild to moderate Alzheimer’s disease. This drug acts as an AChE (Acetylcholinesterase) inhibitor.5 Dilemmaone A6 (3, Fig. 1) and some other indanones have been isolated from natural products. Being such a useful moiety, several synthetic strategies have also been developed for their synthesis.7a–j In continuation of our studies on modification of plant phenolics,8a–e we modified gallic acid to an indanone moiety (4, Fig. 1). Gallic acid (5), a plant phenolic acid is present as hydrolysable tannins in almost all woody perennials. The modified gallic acid moiety i.e., a 3,4,5-trimethoxy phenyl unit has been established as an essential structural requirement for several anticancer leads9 like Combretastatin A4, Podophyllotoxin, Colchicine, etc. (Fig. 2). In the present letter, gallic acid-based indanone derivatives have been synthesised and evaluated for their anticancer activity. One of the potent indanone (10) has further been modified to establish its structure and activity relationship (SAR). All the compounds showq See Ref. 1. * Corresponding author. Tel.: +91 522 2717529x327; fax: +91 522 2342666. E-mail address: arvindcimap@rediffmail.com (A.S. Negi). 0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2008.06.039 ing potent anticancer activity were further evaluated for toxicity to human erythrocytes by performing erythrocyte fragility test. The synthetic strategy was as depicted in Scheme 1, gallic acid (5) was taken as the starting material. It was fully methylated at phenolic as well as carboxylic acid positions by refluxing it with dimethyl sulphate in 20% aqueous alkali to get 3,4,5-trimethyl gallic acid methyl ester (6) in 60% yield. The ester 6 underwent Grignard reaction with methylmagnesium iodide to yield the desired substrate 3,4,5-trimethoxyacetophenone (7). The acetophenone 7 and aldehyde 8 were condensed together in 3% aqueous methanolic sodium hydroxide to get a corresponding chalcone10 (9). Similarly, other aldehydes were condensed with 7 to get respective chalcones first and then modified to corresponding indanones (11–14).11,12 All these chalcones were further modified to corresponding indanones by heating with trifluoroacetic acid in a sealed glass tube (Borosil).13a However, indanone 15 was obtained on condensation of 3,4-dimethoxyacetophenone with 8 to get the respective chalcone and further modified to corresponding indanone (15) as described for other indanones (11–14). Chalcones lacking an electron releasing groups in the phenyl ring of benzoyl group did not undergo Nazarov’s cyclisation reaction, due to deactivation by the carbonyl group. Therefore, chalcones synthesised from simple acetophenones could not be transformed into indanones. All the compounds were characterised by spectroscopic means.17 All these indanones were evaluated for in vitro anticancer activity by MTT assay14 (Table 1) against various human cancer 3915 H. O. Saxena et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3914–3918 NH2 O H3CO O H3CO CH3 H3CO . HCl N H3CO OH 2 CH3 1 O OCH3 H3CO NH O H3CO OCH3 R H3C 3 4 Figure 1. Structures of Indanocine (1), Donepezil hydrochloride (2), Dilemmaone A (3) and gallic acid-based indanone (4). OH O H3CO O H3CO OCH3 OCH3 O O OH H3CO Combretastatin A4 O OCH3 OCH3 Podophyllotoxin CH3 NH H3CO H3CO O OCH3 Colchicine OCH3 H3CO OCH3 OCH3 3,4,5-Trimethoxyphenyl unit Figure 2. Structures of some lead molecules possessing 3,4,5-trimethoxyphenyl moiety as a common unit. cell lines i.e., KB403 (oral and mouth cancer cells), WRL68 (liver cancer cells), CaCO2 (colon cancer cells), HepG2 (liver cells) and MCF7 (hormone-dependent breast cancer cells). Taxol (Paclitaxel) and Podophyllotoxin were used as reference compounds. Among all these indanone derivatives compound 14 (IC50 = 0.022 lM) showed the highest level of activity followed by 12 (IC50 = 0.023 lM) against WRL liver cancer cell lines, while indanone 10 (IC50 = 2.2 lM) was found to be most active against MCF-7 hormone-dependent breast cancer cell lines. Indanones 11 and 12 possessed highest anticancer activity against KB-403 oral and mouth cancer cell lines. Indanone 13 was found to be inactive against almost all the cell lines. Rest of the compounds showed moderate to low level of activity against these human cancer cell lines. Compound 10 having trimethoxyphenyl units identical to gallic acid in both the rings possessed potent anticancer activity against MCF-7, HEPG2 and WRL68 human cancer cell lines. To establish structure and activity relationship (SAR) of compound 10, it was further modified by simple derivatisations (Scheme 2).13b,c Compound 10 was refluxed with selenium dioxide in 1,4-dioxane to introduce another keto group at 2-position. On oxidising compound 16 was obtained as 1,2-keto-enol derivative rather than a 1,2-diketo derivative. An unexpected polycyclic derivative 17 (Fig. 3) was also obtained, which was characterised by various spectroscopic means. Compound 10, on refluxing with hydroxylamine hydrochloride in ethanol and pyridine transformed to its oxime in excellent yields (94%). On sodium borohydride reduction in methanol, a corresponding secondary alcohol 19 was formed in quantitative yield, while sodium borohydride reduction in trifluoroacetic acid yielded a 1-deoxy derivative 20 in good yield. But all these derivatives possessed either lower cytotoxicities or were found to be inactive as compared to the parent molecule 10. From this, it was concluded that in indanone 10, all the above modifications are not favourable. Hence, a keto group at 1-position along with no such substitutions at 2-position is desirable for its better activity. The structure proposed for compound 17 has been confirmed by spectroscopic means using IR, NMR experiments like 1H NMR, 13 C NMR, DEPT 135 and HMBC correlation experiments and finally by mass spectrometry. The proton spectra taken on 300 MHz FT NMR in CDCl3 showed six distinct singlets at 3.90, 3.96, 3.98, 4.00, 4.03 and 4.07 ppm for six methoxy groups. Two singlet protons were also observed in the aromatic region at 7.04 and 8.07 ppm. The 13C NMR spectra showed presence of total 21 carbons in 17. 13C coupled with DEPT 135 experiments clearly indicated the presence of six methyls (all oxygenated) and two methines (aromatic) and 13 quaternary (nine oxygenated) carbons. Absence of one of the aromatic proton of ring C (which is otherwise a singlet for two enantiotopic protons) and enolic hydroxyl suggested the possibility of cyclisation. It was further indicated by the downfield shifts of both the aromatic protons and presence of one more quaternary carbon at the loss of one aromatic methine as compared to the 13C spectrum of 16. It was further ascertained by HMBC correlations of compound 17 (Fig. 3). Indanone derivatives 10, 11, 12 and 14 showing potent anticancer activity were also evaluated for erythrocyte osmotic fragility (Fig. 4) to determine their toxicity.15 Among these indanone 10 showing most potent activity against MCF-7 was found to be non-toxic to human erythrocytes even at higher concentrations (100 lg/ml, 258 lM). Indanones 11, 12 and 14 increased the haemolysis of erythrocytes, hence these may be considered toxic at higher concentrations. In conclusion, gallic acid-based indanone derivatives showed potent anticancer activity against hormone-dependent breast cancer, oral and liver cancer cell lines. As one of the potent molecules was found non-toxic to human erythrocytes, this compound may further be optimised to better anticancer leads with no or low toxicities to normal cells. 3916 H. O. Saxena et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3914–3918 HO HO H3CO H3CO i COOH H3CO HO O C CH3 ii H3CO COOCH3 7 H3CO H3CO 6 5 + H3CO OH C CH3 CH3 H3CO H3CO Minor O CH3 O CHO OCH3 H3CO iii + OCH3 OCH3 OCH3 7 8 H3CO OCH3 H3CO H3CO OCH3 OCH3 9 OCH3 iv O H3CO H3CO OCH3 OCH3 10 O OCH3 OCH3 O R1 R2 iv R1 R2 1 2 R= R= 10: 4,5,6-trimethoxy- , 3',4',5'-trimethoxy; 11: 4,5,6-trimethoxy- , H; 12: 4,5,6-trimethoxy- , 2',3',4'-trimethoxy-; 13: 4,5,6-trimethoxy- , 3'-methoxy-; 14: 4,5,6-trimethoxy- , 2',4',6'-trimethoxy-; 15: 5,6-dimethoxy- , 3',4',5'-trimethoxy- . Scheme 1. Reagents and conditions: (i) 20% aq alkali, dimethyl sulphate, refluxed for 3 h, 60%; (ii) CH3I, Mg turnings, THF, 20 min at RT then reflux for 1 h, 42%; (iii) 3% aq methanolic NaOH, RT, overnight 16–18 h, 62–84% respective acetophenones and aldehydes used; (iv) TFA, refluxed in a sealed tube, 3–4 h, 28–52%. Table 1 Cytotoxicitiesa of indanones and their analogues against various human cancer cell lines by MTT assay Compound 10 11 12 13 14 15 16 17 18 19 20 Taxol Podophyllotoxin a Human cancer cell lines KB403 IC50 (lM) WRL68 IC50 (lM) CaCO2 IC50 (lM) HEPG2 IC50 (lM) MCF7 IC50 (lM) 10.3 0.84 1.54 Inactive 12.90 139.6 Inactive Inactive 136.4 192.3 Inactive 0.001 20.5 12.8 Inactive 0.023 Inactive 0.022 195.5 124.3 62.5 223.3 192.3 153.8 0.004 4.8 Inactive Inactive 206 Inactive 226 139.6 Inactive 112.5 49.6 6.4 115.3 0.008 0.002 9.0 188 Inactive Inactive 1.49 111.7 74.6 Inactive 124 102.5 89.7 0.009 4.8 2.20 Inactive Inactive Inactive 211 Inactive Inactive 50 Inactive 243.5 Inactive 0.006 8.5 IC50 P 250 lM was considered as inactive. 3917 H. O. Saxena et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3914–3918 O O H3CO v + H3CO 16 O OCH3 H3CO OCH3 vi H3CO OH OCH3 OCH3 OCH3 17 OCH3 OCH3 OCH3 NOH H3CO H3CO O OCH3 OCH3 H3CO 18 OCH3 OCH3 H3CO OCH3 OCH3 vii OCH3 OCH3 10 OH H3CO H3CO OCH3 OCH3 19 OCH3 OCH3 viii H3CO H3CO OCH3 20 OCH3 OCH3 OCH3 Scheme 2. Reagents and conditions: (v) SeO2, 1,4-Dioxane, refluxed for 3 h, 16: 67%, 17: 14%; (vi) NH2OHHCl, Ethanol, Pyridine, refluxed for 2 h, 94%; (vii) NaBH4–MeOH, 58%; (viii) NaBH4–TFA, 0 °C–RT, 6 h, 72%. Acknowledgment H O The financial support from the CSIR Networking Project (NWP09) is duly acknowledged. H3CO O H3CO OCH3 OCH3 H H3CO Supplementary data OCH3 Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmcl.2008.06.039. Figure 3. HMBC correlations of compound 17. References and notes 10 12 14 11 CONTROL 120 Percent Hemolysis 100 80 60 40 20 0 0.85 0.65 0.4 0.2 0.1 Percent Phosphate Buffer Saline Figure 4. Osmotic haemolysis curve of erythrocytes. 1. CIMAP Communication No. 2008-80J. 2. (a) Schumann, H.; Stenzel, O.; Girgsdies, F. Organometallics 2001, 20, 1743; (b) Herzog, M. N.; Chien, J. C. W.; Rausch, M. D. J. Organomet. Chem. 2002, 654, 29. 3. Sato, Y. Jpn. Kokai Tokkyo Koho 1998, 10298126.; Sato, Y. Chem. Abstr. 1999, 130, 13852. 4. Leoni, L. M.; Hamel, E.; Genini, D.; Shih, H.; Carrera, C. J.; Cottam, H. B.; Carson, D. A. J. Natl. Cancer Inst. 2000, 92, 217. 5. Sugimoto, H. Pure Appl. Chem. 1999, 71, 2031. 6. Beukes, D. R.; Davies-Coleman, M. T.; Kelly-Borges, M.; Harper, M. K.; Faulkner, D. J. J. Nat. Prod. 1998, 61, 699. 7. (a) Pellissier, H. Tetrahedron 2005, 61, 6479; (b) Frontier, A.; Collinson, C. Tetrahedron 2005, 61, 7577; (c) Coyanis, E. M.; Panayides, J. L.; Fernandes, M. A.; de Koning, C. B.; van Ottoerlo, W. A. L. J. Organomet. Chem. 2006, 691, 5222; (d) Clark, W. M.; Tickner-Eldridge, A. M.; Huang, G. K.; Pridgen, L. N.; Olsen, M. A.; Mills, R. J.; Lantos, I.; Baine, N. H. J. Am. Chem. Soc. 1998, 120, 4550; (e) Wang, G.; Zheng, C.; Zhao, G. Tetrahedron: Asymmetry 2006, 17, 2074; (f) Kajiro, H.; Mitamura, S.; Mori, A.; Hiyama, T. Tetrahedron: Asymmetry 1998, 9, 907; (g) Torisawa, Y.; Aki, S.; Minamikawa, J. Bioorg. Med. Chem. Lett. 2007, 17, 453; (h) Cui, D. M.; Zhang, C.; Kawamura, M.; Shimada, S. Tetrahedron Lett. 2004, 45, 1741; (i) Yadav, J. S.; Reddy, G. S. K. K.; Sabitha, G.; Krishna, A. D.; Prasad, A. R.; Rahaman, H. R. R.; Rao, K. V.; Rao, A. B. Tetrahedron: Asymmetry 2007, 18, 717; (j) Sharma, A. K.; Subramani, A. V.; Gorman, C. B. Tetrahedron 2007, 63, 389. and Refs. 2–9 cited in the paper. 8. (a) Negi, A. S.; Darokar, M. P.; Chattopadhyay, S. K.; Garg, A.; Bhattacharya, A. K.; Srivastava, V.; Khanuja, S. P. S. Bioorg. Med. Chem. Lett. 2005, 15, 1243; 3918 9. 10. 11. 12. 13. 14. 15. H. O. Saxena et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3914–3918 (b) Negi, A. S.; Chattopadhyay, S. K.; Srivastava, S.; Bhattacharya, A. K. Synth. Commun. 2005, 35, 15; (c) Srivastava, V.; Negi, A. S.; Kumar, J. K.; Faridi, U.; Sisodia, B. S.; Darokar, M. P.; Luqman, S.; Khanuja, S. P. S. Bioorg. Med. Chem. Lett. 2006, 16, 911; (d) Srivastava, V.; Saxena, H. O.; Shanker, K.; Kumar, J. K.; Luqman, S.; Gupta, M. M.; Khanuja, S. P. S.; Negi, A. S. Bioorg. Med. Chem. Lett. 2006, 16, 4603; (e) Srivastava, V.; Darokar, M. P.; Fatima, A.; Kumar, J. K.; Chowdhury, C.; Dwivedy, G. R.; Shrivastava, K.; Gupta, V.; Chattopadhyay, S. K.; Luqman, S.; Saxena, H. O.; Gupta, M. M.; Negi, A. S.; Khanuja, S. P. S. Bioorg. Med. Chem. 2007, 13, 518. Srivastava, V.; Negi, A. S.; Kumar, J. K.; Gupta, M. M.; Khanuja, S. P. S. Bioorg. Med. Chem. 2005, 13, 5892. Furniss, B. S.; Hannaford, A. J.; Smith, P. W. G.; Tatchel, A. R.. In Book, Vogel’s Text Book of practical organic chemistry; Addison-Wesley Longman, Inc.: Reading, MA, 1998; p 1034. Lawrence, N. J.; Armitage, E. S. M.; Greedy, B.; Cook, D.; Ducki, S.; McGown, A. T. Tetrahedron Lett. 2006, 47, 1637. Saxena, H. O.; Faridi, U.; Kumar, J. K.; Luqman, S.; Daroker, M. P.; Shanker, K.; Chanotiya, C. S.; Gupta, M. M.; Negi, A. S. Steroids 2007, 72, 892. (a) Syntheses: General procedure for preparing indanone derivatives from chalcones: Synthesis of 3-(30 ,40 ,50 -Trimethoxyphenyl)-4,5,6-trimethoxyindan-1-one (10). In a Borosil test tube, chalcone 9 (150 mg, 0.39 mmol) was taken in trifluoroacetic acid (0.5 ml) and the tube was sealed carefully with flame. The reaction mixture was heated at 120 °C for 4 h. The reaction mixture was poured into crushed ice and extracted with ethyl acetate, organic layer was washed with water, dried over anhydrous sodium sulphate and evaporated in vacuo. The crude residue thus obtained was purified through column chromatography on silica gel using ethyl acetate–hexane as eluent. The desired indanone 10 was obtained as a solid. It was recrystallised with chloroform–hexane (1:3) to get 10 as a light brown solid. (b) Synthesis of 3-(30 ,40 ,50 -Trimethoxyphenyl)-4,5,6-trimethoxy-indan (20). Indanone 10 (100 mg, 0.26 mmol) was taken in trifluoroacetic acid (2.5 ml) and the reaction flask was stirred in an ice-bath. After stirring for 5 min sodium borohydride (100 mg, 2.6 mmol) was added in portions with maintaining the bath temperature 0–15 °C for an hour. After that the reaction mixture was stirred at room temperature for 6 h. On completion, 10 ml water was added to reaction mixture and it was extracted with ethyl acetate, organic layer was washed with water, dried over anhydrous sodium sulphate and evaporated in vacuo. The crude residue thus obtained was purified through column chromatography on silica gel using ethyl acetate–hexane as eluent. The desired indan 20 was obtained as oil. (c) 2-Hydroxy, 3-(30 ,40 ,50 -trimethoxyphenyl)-4,5,6-trimethoxy-ind-2-en-1-one (16). Indanone 10 (100 mg, 0.26 mmol) was taken in a round-bottomed flask with 1,4-dioxane (10 ml) and selenium dioxide (290 mg, 2.6 mmol). The reaction mixture was refluxed for 6 h. On completion, reaction mixture was filtered and filtrate was evaporated in vacuo. The crude mass thus obtained was purified through column chromatography on silica gel using ethyl acetate–hexane as eluent. The cyclised product 17 was first obtained followed by the desired derivative 16. In-vitro anticancer activity using MTT assay. In-vitro cytotoxicity testing was performed as per reported method.16 2  103 cells/well were incubated in the 5% CO2 incubator for 24 h to enable them to adhere properly to the 96-well polystyrene microplates (Grenier, Germany). Test compound dissolved in dimethyl sulphoxide (DMSO, Merck, Germany), in at least five concentrations, were added into the wells and left for 4 h. After the incubation, the compound plus media was replaced with fresh media and the cells were incubated for another 48 h in the CO2 incubator at 37 °C. The concentration of DMSO were always kept below 1.25%, which was found to be non-toxic to cells. Then, 10 lL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added to each well and plates were incubated at 37 °C for 4 h. DMSO (100 lL) was added to all wells and mixed thoroughly to dissolve the dark blue crystals. The plates were read on SpectraMax 190 Microplate reader (Molecular Devices Inc. USA) at 570 nm within 1 h of DMSO addition. Determination of osmotic haemolysis of erythrocytes: Blood from healthy human male volunteers (n = 3) with informed consent was collected for experiments using heparin (10 U/ml) as the anti-coagulant. The collected blood was stored at 4 °C and was used for experiments within 4 h of collection.18 Experiments were carried in-vitro by adding heparinised blood to hypotonic solutions of varying concentrations of phosphate buffered saline (0.85% to 0.10%). Phosphate-buffered saline stock (10%) was prepared by dissolving 5 g of sodium chloride, 1.3655 g of disodium hydrogen orthophosphate and 0.243 g of sodium dihydrogen orthophosphate in 100 ml of autoclaved double distilled water. From this stock, working standards of 0.85% to 0.10% were prepared. The tubes were incubated at 37 °C for 60 min with mild shaking and the extent of haemolysis was measured colorimetrically at 540 nm.19 Results are expressed in terms of mean erythrocyte fragility (MEF50), which is the level of haemolysis of the erythrocytes at 50% saline concentrations. Similarly, prior to the experiment, heparinised blood was incubated with effective concentration of 16. 17. 18. 19. 20. 21. indanone derivatives (5–100 lg/ml) at 37 °C for 60 min. The concentrations of indanones were chosen higher than the concentrations at which the compounds showed anticancer activity. Aliquots of saline solutions of decreasing concentration (from 10 to 1 g/L) were prepared as described earlier.19,20 The test compound treated erythrocytes were then transferred to tubes containing decreasing concentrations of saline solutions. After careful mixing, the cell suspensions were left to equilibrate for 30 min and then centrifuged at 3000 rpm for 5 min. The absorbance of supernatants was read at 540 nm, with the standardised against an assay blank (the 10 g/L saline supernatant corresponds to 0% haemolysis). The recorded optical density (OD) of the supernatant reflects the degree of haemolysis of the erythrocytes. The percentage lysis was calculated by dividing the OD of the supernatant obtained from a particular saline concentration by the OD of the standard (1 g/L) representing 100% haemolysis.21 Osmotic fragility curves were constructed by plotting the percentage lysis against the concentration of saline solutions. The MEF50 (mean erythrocyte fragility) value, which is the saline concentration at which 50% of the cells haemolyse at standard pH and temperature, was then obtained from the curve. Woerdenbag, H. J.; Moskal, T. A.; Pras, N.; Malingré, T. M.; Farouk, S.; EI-Feraly, H.; Kampinga, H.; Konings, A. W. T. J. Nat. Prod. 1993, 56, 849. Selected physical data:Compound 10: Yield = 64%; mp = 107–110 °C; IR (KBr, cm1): 2938, 1705, 1591, 1509, 1500, 1129. 1H NMR(CDCl3, 300 MHz) d 2.58– 2.65 (dd, 1H, 2-CH, J = 2.58, 19.29 Hz), 3.13–3.22(dd, 1H, 2-CH, J = 7.98, 19.26 Hz), 3.42 (s, 3H, OCH3), 3.78 (s, 6H, 2 OCH3), 3.81 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 4.50–4.53 (dd, 1H, 3-CH, J = 7.95, 2.49 Hz), 6.31 (s, 2H, aromatic protons), 7.09 (s, 1H, aromatic proton); 13C NMR(CDCl3, 75.46 MHz) d 42.30, 47.48, 56.58, 56.68, 56.68, 60.44, 61.14, 61.14, 100.81, 105.03, 132.65, 137.75, 140.42, 140.42, 144.38, 149.21, 150.89, 153.84, 153.84, 155.45, 205.08. EI Mass GC–MS (CH3CN): 388 [M+], 373, 357, 181. Compound 12: Yield = 71%; mp = 119–123 °C; IR (KBr, cm1): 2936, 2838, 1704, 1599, 1498, 1470, 1101. 1H NMR(CDCl3, 300 MHz) d 2.54–2.61 (dd, 1H, 2-CH, J = 1.99, 19.07 Hz), 3.07–3.16 (dd, 1H, 2-CH, J = 8.03, 19.09 Hz), 3.38 (s, 3H, OCH3), 3.69 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 4.75–4.78 (br d, 1H, -CH, J = 7.95, 2.49 Hz), 6.53–6.58 (br s, 2H, aromatic protons), 7.08 (s, 1H, aromatic proton); EI mass GC–MS (CH3CN): 388 [M+], 357, 373, 358, 342. Compound 13: Yield: 48%; mp = 112–115 °C; 1H NMR(CDCl3, 300 MHz) d 2.58– 2.65 (dd, 1H, 2-CH, J = 2.42, 19.26 Hz), 3.14–3.23(dd, 1H, 2-CH, J = 7.97, 19.27 Hz), 3.39 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 4.54–4.58 (dd, 1H, –CH, J = 2.36 and 7.90 Hz), 6.66–7.23 (m, 8H, aromatic protons); EI mass GC–MS (CH3CN): 328 [M+], 313, 207. Compound 16: Yield = 67%; mp = oil; IR (KBr, cm1): 3444, 2939, 1727, 1594, 1499, 1466, 1126. 1H NMR(CDCl3, 300 MHz) d 3.33 (s, 3H, OCH3), 3.78 (s, 6H, 2 OCH3), 3.84 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 6.65 (s, 2H, aromatic protons), 6.97 (s, 1H, aromatic proton); 13C NMR(CDCl3, 75 MHz) d 42.30, 47.48, 56.58, 56.68, 56.68, 60.44, 61.14, 61.14, 100.81, 105.03, 132.65, 137.75, 140.42, 140.42,144.38, 149.21, 150.89, 153.84, 153.84, 155.45, 205.08. EI Mass GC–MS (CH3CN): 402 [M+], 387, 195. Compound 17: Yield = 16%; mp = 145–148 °C; IR (KBr, cm1): 2933, 1697, 1474, 1384, 1096. 1H NMR(CDCl3, 300 MHz) d 3.90 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 4.03 (s, 3H, OCH3), 4.07 (s, 3H, OCH3), 7.04 (s, 1H, aromatic proton), 8.07 (s, 1H, aromatic proton); 13C NMR(CDCl3, 75 MHz) d 56.55, 56.93, 61.10, 61.37, 61.51, 62.09, 105.96, 106.96, 127.09, 130.38, 133.06, 136.87, 137.10, 141.73, 147.17, 147.52, 149.42, 153.86, 154.26, 156.33, 187.84. EI Mass GC–MS (CH3CN): 400 [M]+. Compound 19: Yield = 58%; mp = 132–136 °C ; IR (KBr, cm1): 3516, 2941, 1593, 1503, 1463, 1419, 1335, 1120. 1H NMR(CDCl3, 300 MHz) d 1.92–2.01 (m, 1H, 2CH), 2.94–2.99 (m, 1H, 2-CH), 3.45 (s, 3H, OCH3), 3.79 (s, 12H, 4 OCH3), 3.90 (s, 3H, OCH3), 4.22–4.27 (m, 1H, 3-CH), 5.16–5.20 (m, 1H, 1-CH), 6.48 (s, 2H, aromatic protons), 6.81 (s, 1H, aromatic proton); 13C NMR(CDCl3, 75.46 MHz) d 46.35, 47.58, 56.45, 56.53, 60.21, 60.42, 60.94, 60.98, 75.35, 103.54, 105.24, 105.88, 130.49, 137.01, 141.54, 142.30, 142.82, 150.36, 153.31, 154.46. EI Mass  GC–MS (CH3CN): 390 [M+], 372, 357. ½a28 589 þ 6:99 (1.04, MeOH). Compound 20: Yield = 72%; mp = 71–74 °C ; 1H NMR(CDCl3, 300 MHz) d 1.99– 2.06 (m, 1H, 2-CH), 2.52–2.59 (m, 1H, 2-CH), 2.85–2.89 (m, 1H, 1-CH), 2.99– 3.06 (m, 1H, 1-CH), 3.51 (s, 3H, OCH3), 3.77 (s, 6H, 2 OCH3), 3.80 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 4.37–4.42 (m, 1H, 3-CH), 6.33 (s, 2H, aromatic proton), 6.63 (s, 1H, aromatic proton); 13C NMR(CDCl3, 75.46 MHz) d 22.66, 29.67, 31.93, 49.43, 56.35, 56.35, 60.15, 60.73, 60.81, 104.01, 105.19, 130.84, 139.95, 142.38, 150.29, 153.93, 153.76. EI Mass GC–MS (CH3CN): 390 [M+], 372, 357. Luqman, S.; Rizvi, S. I. Asia Pacific J. Pharmacol. 2004, 16, 53. Luqman, S.; Obli Prabu, K. V.; Pal, A.; Saikia, D.; Darokar, M. P.; Khanuja, S. P. S. Nat. Prod. Commun. 2006, 1, 481. Luqman, S.; Kumar, N.; Rizvi, S. I. University Allahabad Studies 2004, 3, 29. Dacie, J. V.; Lewis, S. M. Practical Hematology, 1984; Orient Longman, p 152.