Synthesis and anticancer activities of novel 3,5-disubstituted-1,2,4-oxadiazoles

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2739–2741 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl Synthesis and anticancer activities of novel 3,5-disubstituted-1,2,4-oxadiazoles Dalip Kumar a,*, Gautam Patel a, Emmanuel O. Johnson b, Kavita Shah b,* a b Chemistry Group, Birla Institute of Technology and Science, Pilani 333 031, India Department of Chemistry and Purdue Cancer Center, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA a r t i c l e i n f o Article history: Received 11 February 2009 Revised 17 March 2009 Accepted 25 March 2009 Available online 5 April 2009 Keywords: 1,2,4-Oxadiazole Anticancer agent Selective cytotoxicity a b s t r a c t A series of 3,5-disubstituted-1,2,4-oxadiazoles were synthesized and evaluated for their in vitro anti-proliferative activities against various cancer cell lines. Formation of 1,2,4-oxadiazole ring was accomplished by the reaction of amidoxime with carboxylic acids. The in vitro cytotoxic effects of 3,5-disubstituted1,2,4-oxadiazoles have been demonstrated across a wide array of tumor cell types and a few compounds exhibited specificity towards pancreatic (3f, 3h, 3j, and 3k) and prostate (3n) cancer cells. Among the prepared 3,5-disubstituted-1,2,4-oxadiazoles, compound 3n is the most selective (>450-fold) and compound 3p is the most cytotoxic (10 nM) against prostate cancer cell lines. Ó 2009 Elsevier Ltd. All rights reserved. Recently, there has been wide interest in compounds containing the 1,2,4-oxadiazole scaffold because of their unique chemical structure and broad spectrum of biological properties including tyrosine kinase inhibition,1 muscarinic agonism,2 histamine H3 antagonism,3 anti-inflammation,4 antitumor,5 and monoamine oxidase inhibition.6 The 1,2,4-oxadiazoles are also widely used as heterocyclic amide or ester bioisosters7 and in the design of dipeptidomimetics as peptide building blocks.8,9 Cancer is a fatal disease that has posed serious threat to human health. Many classes of anticancer drugs have been developed. However, most drugs cause undesirable side effects due to lack of tumor specificity and multidrug resistance. Several factors aid in cancer cell survival. These include variation in metabolism, absorption and delivery of drug to target tissues, and tumor location. For example, chemotherapy agents such as paclitaxel, vincristine, and doxorubicin have lost their efficacy due to the development of drug resistance from prolonged exposure. Compounds such as epothilone and discodermolide exhibit activities against multi-drug resistant cancer cells and have been evaluated in clinical trials. Recently, ixabepilone, an epothilone derivative was approved for breast cancer in the US. Several other epothilone derivatives are in advanced clinical trials. Discodermolide, however, proved toxic and has been dropped.10 Therefore, the development of novel and effective anticancer agents remains a major challenge. In this letter, we report the synthesis of novel 3,5-disub- * Corresponding authors. Tel.: +91 1596 245073 279; fax: +91 1596 244183 (D.K.); tel.: +1 765 496 9470 (K.S.). E-mail addresses: dalipk@bits-pilani.ac.in (D. Kumar), shah23@purdue.edu (K. Shah). 0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2009.03.158 stituted-1,2,4-oxadiazoles and measure their anticancer activities in various cancer cell lines. The 3,5-disubstituted-1,2,4-oxadiazoles 3 were synthesized according to Scheme 1. The reaction of appropriate benzonitrile 1 with hydroxylamine hydrochloride under refluxing condition afforded amidoxime 211a which was coupled with the corresponding carboxylic acid using DCC in DMF to give 3,5-disubstituted1,2,4-oxadiazoles 3 in moderate yield.11b All synthesized compounds were characterized by NMR and mass spectroscopy.12 A library of 14 compounds was tested in vitro for their anticancer potential in various human cancer cell lines: prostate (PC3, DU145 and LnCaP), breast (MCF7 and MDA231), colon (HCT116), and pancreas (PaCa2).13 All compounds decreased cell viability as determined by colorimetric MTT assay, with IC50 values ranging from nanomolar to greater than 1 mM (Table 1). More importantly, a few compounds were highly potent and exhibited specificity towards one cell type relative to others. The activities of various 3,5-disubstituted-1,2,4-oxadiazoles revealed 3f, 3g, 3n, and 3p as potent cytotoxic agents against various cancer cell lines (IC50 ranging from 10 nM to 860 nM). All of these compounds possess common 1,2,4-oxadiazole nucleus in which substitution at C-3 and C-5 positions play important roles in potency and selectivity of 3,5-disubstituted-1,2,4-oxadiazoles 3. Compounds 3a and 3b, with C-3 phenyl and C-5 4-fluorophenyl/ pyridin-4-yl groups, displayed weak inhibitory activity against tested cancer cell lines. Introduction of 30 -cyclopentyloxy and 40 -methoxy moieties at C-3 aryl ring enhances the activity (3e, 3h in comparison to 3a, 3b, respectively). However, a larger group like benzyloxy at para position of C-3 aryl ring (3c) is detrimental to the activity. Replace- 2740 D. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2739–2741 N R1 CN R1 a R2 OH NH2 a N O b 1 2 O N R3 R1 H or OH Decreases potency R2 R2 N R3 R1 N 3 c 3k d d 3a: R1 = R2 = H, R3 = 4-FC6H4 3b: R1 = R2 = H, R3 = pyridin-4-yl 3c: R1 = OCH3, R2 = OBn, R3 = pyridin-4-yl 3d: R1 = cyclopentyloxy, R2 = OCH3, R3 = C6H5 3e: R1 = cyclopentyloxy, R2 = OCH3, R3 = 4-FC6H4 3f: R1 = cyclopentyloxy, R2 = OCH3, R3 = 4-ClC6H4 3g: R1 = cyclopentyloxy, R2 = OCH3, R3 = 4-HOC6H4 3h: R1 = cyclopentyloxy, R2 = OCH3, R3 = pyridin-4-yl 3i: R1 = cyclopentyloxy, R2 = OCH3, R3 = pyridin-3-yl 3j: R1 = cyclopentyloxy, R2 = OCH3, R3 = (indol-3-yl)methyl 3k: R1 = cyclopentyloxy, R2 = OCH3, R3 = CH2COCH3 3l: R1 = OH, R2 = OCH3, R3 = 4-FC6H4 3m: R1 = cyclopentyloxy, R2 = OCH3, R3 = N-Boc-pyrrolidin-2-yl 3n: R1 = cyclopentyloxy, R2 = OCH3, R3 = pyrrolidin-2-yl 3o: R1 = cyclopentyloxy, R2 = OCH3, R3 = N-Boc-piperidin-4-yl 3p: R1 = cyclopentyloxy, R2 = OCH3, R3 = piperidin-4-yl b Provides potency and specificity R2 H or OBn Decreases potency Piperidin-4-yl C6H5(Not Potent) (Highly potent and specific for several cancer cell lines) Pyrrolidin-2-yl (Highly potent and specific for LnCaP) 3d 3p 3e 3n 3f R3 3k 4-FC6H4 (MCF7 and PaCa2) CH2COCH3 (PaCa2) 3g 3j 3i 3h (Indol-3-yl)methyl (PaCa2) Pyridin-3-yl (PaCa2) 4-ClC6H4 (Highly potent and specific for PaCa2) 4-HOC6H4 (Highly potent for Several cancer cell lines) Pyridin-4-yl (PaCa2) Figure 1. SAR of 1,2,4-oxadiazoles (a) Substitution at R3 provides both specificity and potency. (b) In this panel, R1 = OC5H9 and R2 = OCH3. Phenyl or Pyrindin-3-yl substitution at R3 position decreases potency. CH2COCH3, pyrindin-4-yl or (Indol-3yl)methyl at R3 position confer specificity for PaCa2 cells (IC50 3.0–9.56 lM). Substituents at R3 shown in bold confer high potency. While compounds containing 4-HOC6H5 and piperidin-4-yl substituents at R3 position are highly cytotoxic in multiple cancer cell lines, both 4-ClC6H5 and pyrrolidin-2-yl are highly potent and specific for PaCa2 and LnCaP cells, respectively. Scheme 1. Synthesis of 1,2,4-oxadiazoles. Reagents and conditions: (a) EtOH, H2O, NH2OH.HCl, NaOH, reflux, 18 h; (b) DMF, DCC, aromatic/heteroaromatic/aliphatic carboxylic acid, 0 °C to 30 °C, 3 h then 110 °C 12 h; (c) ethylacetoacetate, reflux; (d) trifluoroacetic acid, DCM. ment of 4-fluorophenyl in 3a with 4-chlorophenyl or 4-hydroxyphenyl group yielded compounds 3f and 3g, respectively, with significant improved activities against different cancer cell lines. Compounds 3f with 4-chlorophenyl, and 3h with pyridin-4-yl groups show selectivity against pancreatic cancer cell line with IC50 values of 0.74 lM and 9.33 lM, respectively. Significant loss in activity was observed upon changing the position of nitrogen atom in the C-5 pyridyl ring of compound 3h to obtain the isomer 3i. When C-5 aryl ring is replaced with an aliphatic ketone moiety as represented by 3k, improved cytotoxicity was observed. Interestingly, compound 3k also exhibited selective cytotoxicity against pancreatic cancer cell line (3.01 lM). Replacement of C-5 phenyl group of 3d with pyrrolidin-2-yl yielded compound 3n, which exhibited high potency as well as selectivity against prostate cancer cell line (LnCaP, 43 nM). Substitution with piperidin-4-yl group in 3d at C-5 position led to the compound 3p with significant improved activity against most cancer cell lines and 6-fold selectivity against LnCaP (10 nM), a prostate cancer cell line. The compound 3j with an (indol-3-yl)methyl group at the C-5 position was also selectively cytotoxic towards pancreatic Table 1 Cytotoxicity profile of 3,5-disubstituted-1,2,4-oxadiazoles against selected human cancer cell lines, IC50 (lM) 2 N O1 R3 R1 N 4 R2 1 Compd R 3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3n 3p H H OCH3 OC5H9 OC5H9 OC5H9 OC5H9 OC5H9 OC5H9 OC5H9 OC5H9 OH OC5H9 OC5H9 R 2 H H OBn OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 R 3 4-FC6H4 Pyridin-4-yl Pyridin-4-yl C6H5 4-FC6H4 4-ClC6H4 4-HOC6H4 Pyridin-4-yl Pyridin-3-yl (Indol-3-yl)methyl CH2COCH3 4-FC6H4 Pyrrolidin-2-yl Piperidin-4-yl 3 PC3 DU145 LnCaP MCF7 MDA231 HCT 116 PaCa2 >103 >103 >103 >103 >103 >103 162.3 ± 0.08 >103 >103 >103 >103 >103 >103 >103 >103 656.6 ± 0.1 >103 >103 20.65 ± 0.1 122.5 ± 0.1 0.78 ± 0.08 >103 >103 >103 302.1 ± 0.1 >103 242 ± 0.1 0.057 ± 0.07 23.7 ± 0.5 710 ± 0.5 >103 >103 87.93 ± 0.5 20.8 ± 0.4 0.86 ± 0.05 >103 >103 >103 28.34 ± 0.4 >103 0.043 ± 0.04 0.010 ± 0.02 >103 >103 >103 >103 3.88 ± 0.3 174.2 ± 0.1 0.39 ± 0.3 >103 >103 >103 174.2 ± 0.1 >103 >103 3.88 ± 0.3 >103 >103 >103 >103 55.47 ± 0.1 395.5 ± 0.1 0.31 ± 0.05 240.1 ± 0.1 >103 >103 53.3 ± 0.1 >103 212.2 ± 0.3 0.37 ± 0.08 >103 > 103 >103 >103 209.7 ± 0.6 >103 0.27 ± 0.05 >103 >103 >103 184.8 ± 0.3 >103 184.8 ± 0.1 1.54 ± 0.03 >103 497 ± 0.09 >103 >103 6.09 ± 0.08 0.74 ± 0.02 0.48 ± 0.08 9.33 ± 0.06 >103 9.56 ± 0.08 3.01 ± 0.03 350.7 ± 0.09 19.49 ± 0.08 0.54 ± 0.03 These experiments were conducted in triplicates at three independent times. IC50 values were obtained using a dose response curve by nonlinear regression using a curve fitting program, GraphPad Prism 5.0. Bold values show IC50 of less than 1 lM. OC5H9 = Cyclopentyloxy. D. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2739–2741 (9.56 lM) cancer cell line. Removal of 30 -cyclopentyl group in compound 3e led to the compound 3l having 30 -hydroxy-40 -methoxyphenyl group at C-3 position, which results in complete loss of activity. Aryl/Heteroaryl substituents with a polar functionality at C-5 and 30 -cyclopentyloxy-40 -methoxyphenyl moiety at C-3 position of 1,2,4-oxadiazole core is very important for the activity and selectivity. The observed structure–activity relationship is illustrated in Figure 1. So far, molecular targets responsible for the observed cytotoxicity of this new series of 3,5-disubstituted1,2,4-oxadiazoles 3 have not been identified, and a reasonable explanation of the substitutions described above is not yet possible. Among synthesized 3,5-disubstituted-1,2,4-oxadiazoles, compounds 3f, 3g, 3n, and 3p were most potent and selective in the in vitro assay. In summary, our SAR study shows that the 3,5-disubstituted1,2,4-oxadiazoles decrease cell viability in various cancer cell lines with IC50 values ranging from 10 nM to >1 mM. While 3n was highly specific and potent for LnCaP cells, a few others (3f, 3h, 3j, and 3k) exhibited specificity towards pancreatic cell line. The substituents at C-3 and C-5 positions of 1,2,4-oxadiazole ring were shown to be vital for potency, suggesting specific interactions of these groups with biological targets. Extensive exploration of structure–activity relationship of this novel 1,2,4-oxadiazole scaffold and its biological target studies are underway. Acknowledgment The authors wish to thank the University Grants Commission, New Delhi (Project F. No. 32-216/2006) for financial support. References and notes 1. Vu, C. B.; Corpuz, E. G.; Merry, T. J.; Pradeepan, S. G.; Bartlett, C.; Bohacek, R. S.; Botfield, M. C.; Eyermann, C. J.; lynch, B. A.; MacNeil, I. A.; Ram, M. K.; Van Schravendijk, M. R.; Violette, S.; Sawyer, T. K. J. Med. Chem. 1999, 42, 4088. 2. Orlek, B. S.; Blaney, F. E.; Brown, F.; Clark, M. S.; Hadley, M. S.; Hatcher, J.; Riley, G. J.; Rosenberg, H. E.; Wadsworth, H. J.; Wyman, P. J. Med. Chem. 1991, 34, 2726. 3. Clitherow, J. W.; Beswick, P.; Irving, W. J.; Scopes, D. I. C.; Barnes, J. C.; Clapham, J.; Brown, J. D.; Evans, D. J.; Hayes, A. G. Bioorg. Med. Chem. Lett. 1996, 6, 833. 4. Nicolaides, D. N.; Fylaktakidou, K. C.; Litinas, K. E.; Hadjipavlou-Litina, D. Eur. J. Med. Chem. 1998, 33, 715. 5. (a) Matsumoto, J.; Takahashi, T.; Agata, M.; Toyofuku, H.; Sasada, N. Jpn. J. Pharmacol. 1994, 65, 51; (b) Zhang, H. Z.; Kasibhatla, S.; Kuemmerle, J.; Kemnitzer, W.; Ollis-Mason, K.; Qiu, L.; Crogan-Grundy, C.; Tseng, B.; Drewe, J.; Cai, S. X. J. Med. Chem. 2005, 48, 5215. 6. Chimirri, A.; Grasso, S.; Montforte, A.-M.; Rao, A.; Zappala, M. Farmaco 1996, 51, 125. 7. Luthman, K.; Borg, S.; Hacksell, U. Methods Mol. Med. 1999, 23, 1. 8. Borg, S.; Vollinga, R. C.; Labarre, M.; Payza, K.; Terenius, L.; Luthman, K. J. Med. Chem. 1999, 42, 4331. 9. Borg, S.; Estenne-Bouhtou, G.; Luthman, K.; Csoregh, I.; Hesselink, W.; Hacksell, U. J. Org. Chem. 1995, 60, 3112. 10. Shaw, S. J. Mini-Rev. Med. Chem. 2008, 8, 276. 11. (a) Synthesis of amidoxime 2: To a mixture of appropriate benzonitrile (10 mmol) and hydroxyl-amine hydrochloride (20 mmol) in 50 mL of ethanol was added dropwise aqueous solution of sodium hydroxide (20 mmol, 10 mL) while maintaining temperature at 0 °C. The resulting mixture was allowed to reflux with stiring for 18 h. Ethanol was distilled off under reduced pressure and the crude product was taken into 50 mL of water. The pH (2) of the solution was adjusted with 1N HCl and the aqueous phase was washed with ethylacetate (2  25 mL). Upon cooling (0 °C) and neutralization with sodium carbonate gave off white precipitate which was filtered, washed and air dried at 60 °C to afford pure amidoxime 2.; b Synthesis of 3,5-disubstituted-1,2,4oxadiazole 3: A solution of appropriate carboxylic acid (0.8 mmol) in dry DMF (1 mL) was cooled to 0 °C and added dicyclohexylcarbodiimide (1.2 mmol) 2741 under nitrogen atmosphere and stirred the reaction mixture at the same temperature for 1 h. To this reaction mixture appropriate benzamidoxime 2 (0.8 mmol) was added and stirred at 0 °C for 0.5 h. The reaction mixture was slowly brought to 30 °C, and stirring continued for another 3 h. Gradually temperature was raised to 110 °C, and further stirred for 10 h. The reaction mixture was cooled to 25 °C and poured into ice-cold water (25 mL). Upon addition of ethylacetate (25 mL) and stirring for 10 min, crystals of dicyclohexylurea separated out and removed by filtration. Separated aqueous phase was extracted with ethylacetate (2  20 mL) and the combined organic phase was washed with brine, dried over anhydrous sodium sulfate. Ethylacetate was distilled off and the residue thus obtained was purified by flash column chromatography using ethylacetate–hexane (0–25%) as eluent to afford pure oxadiazole 3. 12. Data for selected compounds 3a: 1H NMR (CDCl3, 400 MHz): dH = 8.26–8.23 (m, 2H), 8.18–8.16 (m, 2H), 7.54–7.50 (m, 3H), 7.27–7.23 (m, 2H). Compound 3b: 1 H NMR (CDCl3, 400 MHz): dH = 8.89 (dd, 2H, J = 4.48, 1.64 Hz), 8.19–8.17 (m, 2H), 8.07 (dd, 2H, J = 4.44, 1.68 Hz), 7.56–7.52 (m, 3H). Compound 3c: 1H NMR (CDCl3, 400 MHz): dH = 8.88 (d, 2H, J = 5.0 Hz), 8.06 (dd, 2H, J = 4.56, 1.56 Hz), 7.72 (dd, 1H, J = 8.36, 2.0 Hz), 7.67 (d, 1H, J = 1.96 Hz), 7.46 (d, 2H, J = 8.76 Hz), 7.41–7.32 (m, 3H), 7.00 (d, 1H, J = 8.4 Hz), 5.25 (s, 2H), 4.00 (s, 3H). Compound 3d: 1H NMR (CDCl3, 400 MHz): dH = 8.22 (dd, 2H, J = 5.20, 0.9 Hz), 7.76 (dd, 1H, J = 6.24, 1.44 Hz), 7.67 (d, 1H, J = 1.44 Hz), 7.61–7.54 (m, 3H), 6.97 (d, 1H, J = 6.3 Hz), 4.94–4.91 (m, 1H), 3.92 (s, 3H), 2.17–1.84 (m, 6H), 1.67–1.62 (m, 2H). Calcd m/z for C20H20N2O3: 336.1, found: 337.2 (M+H)+, 359.3 (M+Na). Compound 3e: 1H NMR (CDCl3, 400 MHz): dH = 8.25–8.21 (m, 2H), 7.74 (dd, 1H, J = 8.36, 2.04 Hz), 7.66 (d, 1H, J = 1.92 Hz), 7.27–7.22 (m, 2H), 6.97 (d, 1H, J = 8.4 Hz), 4.93–4.90 (m, 1H), 3.92 (s, 3H), 2.04–1.84 (m, 6H), 1.67–1.62 (m, 2H). Calcd m/z for C20H19FN2O3: 354.1, found: 355.2 (M+H)+, 377.2 (M+Na). Compound 3f: 1H NMR (CDCl3, 400 MHz): dH = 8.16 (d, 2H, J = 8.64 Hz), 7.74 (dd, 1H, J = 8.40, 1.92 Hz), 7.56 (d, 1H, J = 1.92 Hz), 7.53 (d, 2H, J = 8.64 Hz), 6.97 (d, 1H, J = 8.40 Hz), 4.93–4.90 (m, 1H), 3.92 (s, 3H), 2.06–1.84 (m, 6H), 1.67– 1.60 (m, 2H). Compound 3g: 1H NMR (CDCl3, 400 MHz): dH = 8.11 (d, 2H, J = 8.80 Hz), 7.74 (dd, 1H, J = 8.28, 1.92 Hz), 7.66 (d, 1H, J = 1.92 Hz), 7.01–6.95 (m, 3H), 6.50 (s, 1H (OH)), 4.93–4.88 (m, 1H), 3.91 (s, 3H), 2.04–1.86 (m, 6H), 1.65–1.56 (m, 2H). Compound 3h: 1H NMR (CDCl3, 400 MHz): dH = 8.88 (dd, 2H, J = 4.48, 1.68 Hz), 8.06 (dd, 2H, J = 4.08, 1.68 Hz), 7.76 (dd, 1H, J = 8.40, 2.04 Hz), 7.66 (d, 1H, J = 1.96 Hz), 6.98 (d, 1H, J = 8.40 Hz), 4.94–4.90 (m, 1H), 3.93 (s, 3H), 2.06–1.85 (m, 6H), 1.68–1.61 (m, 2H). Calculate m/z for C19H19N3O3: 337.1, found: 338.2 (M + H)+, 360.2 (M+Na). Compound 3i: 1H NMR (CDCl3, 400 MHz): dH = 9.45 (dd, 1H, J = 2.08, 0.56 Hz), 8.84 (dd, 1H, J = 4.88, 1.68 Hz), 8.48 (dt, 1H, J = 8.04, 2.0 Hz), 7.76 (dd, 1H, J = 8.4, 2.0 Hz), 7.67 (d, 1H, J = 1.92 Hz), 7.52 (ddd, 1H, J = 8, 4.8, 0.6 Hz), 6.98 (d, 1H, J = 8.44 Hz), 4.93–4.92 (m, 1H), 3.93 (s, 3H), 2.04–1.85 (m, 6H), 1.71–1.62 (m, 2H), Calcd m/z for C19H19N3O3: 337.1, found: 338.2 (M+H)+, 360.2 (M+Na). Compound 3j: 1H NMR (CDCl3, 400 MHz): dH = 8.22 (s, 1H), 7.70 (d, 1H, J = 8.84 Hz), 7.64 (dd, 1H, J = 8.32, 1.92 Hz), 7.57 (d, 1H, J = 1.96 Hz), 7.37 (d, 1H, J = 8.08 Hz), 7.24–7.13 (m, 3H), 6.91 (d, 1H, J = 8.44 Hz), 4.87–4.84 (m, 1H), 4.43 (s, 2H), 3.88 (s, 3H), 2.04–1.80 (m, 6H), 1.67–1.59 (m, 2H). Calcd m/z for C23H23N3O3: 389.2, found: 390.3 (M+H)+, 412.3 (M+Na). Compound 3k: 1H NMR (CDCl3, 400 MHz): dH = 7.65 (dd, 1H, J = 8.40, 2.04 Hz), 7.57 (d, 1H, J = 1.96 Hz), 6.94 (d, 1H, J = 8.44 Hz), 4.89–4.86 (m, 1H), 4.1 (s, 2H), 3.91 (s, 3H), 2.35 (s, 3H), 2.17–1.80 (m, 6H), 1.64–1.58(m, 2H). Calcd m/z for C17H20N2O4: 316.1, found: 317.3 (M+H)+, 339.3 (M+Na). Compound 3l: 1H NMR (CDCl3, 400 MHz): dH = 8.24–8.20 (m, 2H), 7.73–7.69 (m, 2H), 7.23 (d, 2H, J = 4.6 Hz), 6.96 (d, 1H, J = 8.20 Hz), 5.72 (s, 1H (OH)), 3.97 (s, 3H). Compound 3n: 1H NMR (CDCl3, 400 MHz): dH = 7.66 (dd, 1H, J = 8.36, 2.0 Hz), 7.57 (d, 1H, J = 1.96 Hz), 6.93 (d, 1H, J = 8.44 Hz), 4.89–4.87 (m, 1H), 4.56–4.52 (m, 1H), 3.90 (s, 3H), 3.24–3.20 (m, 1H), 3.12–3.08 (m, 1H), 2.33–2.29 (m, 1H), 2.17–1.80 (m, 8H), 1.64–1.58(m, 2H). Calcd m/z for C18H23N3O3: 329.2, found: 330.3 (M+H)+, 352.3 (M+Na). Compound 3o: 1H NMR (CDCl3, 200 MHz): dH = 7.63 (dd, 1H, J = 8.00, 2.00 Hz), 7.55 (d, 1H, J = 2.00 Hz), 6.91(d, 1H, J = 8.00 Hz), 4.89–4.78 (m, 1H), 4.16–4.09 (m, 2H), 3.89 (s, 3H), 3.19–2.87 (m, 2H), 2.14–1.85 (m, 10H), 1.70– 1.55 (m, 3H), 1.46 (s, 9H). Compound 3p: 1H NMR (CDCl3, 200 MHz): dH = 7.63 (dd, 1H, J = 8.00, 2.00 Hz), 7.54 (d, 1H, J = 2.00 Hz), 6.92 (d, 1H, J = 8.00 Hz), 4.86–4.78 (m, 1H), 3.88 (s, 3H), 3.40–2.87 (m, 9H), 2.16–1.61 (m, 8H), Calcd m/z for C19H25N3O3: 343.2, found: 344.2 (M+H)+. 13. Cells were cultured in RPMI-1640 media supplemented with 10% heatinactivated foetal bovine serum and 1% penicillin/streptomycin. For MTT assay, cells were seeded in 96 well plates at a density of 4.0  103 cells per well for 12 h. Cells were incubated with various concentrations of the compounds ranging from 10 nM–1 mM. After 24 h, MTT (3-(4,5dimethyldiazol-2-yl)-2,5-diphenyltetrazoliumbromide) was added to the final concentration of 0.5 mg/ml and incubated for 30 min. The cells were washed twice with PBS and lysed in dimethylsulfoxide, and the absorbance was measured at 570 nm.