European Journal of Medicinal Chemistry 89 (2015) 128e137

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Investigation of podophyllotoxin esters as potential anticancer agents: Synthesis, biological studies and tubulin inhibition properties Mohd Adil Shareef a, 1, Divya Duscharla b, 1, G. Ramasatyaveni b, 1, Neha R. Dhoke b, Amitava Das b, Ramesh Ummanni b, Ajay Kumar Srivastava a, * a b

Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Tarnaka, Hyderabad 500 007, India Center for Chemical Biology, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Tarnaka, Hyderabad 500 007, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 May 2014 Received in revised form 14 October 2014 Accepted 16 October 2014 Available online

A series of fifteen podophyllotoxin derived esters have been synthesized and their anti-cancer properties have been evaluated against A549 (lung cancer), DU-145 (prostate cancer), HepG2 (liver cancer), HeLa (cervical cancer) and MCF-7 (breast cancer) cell lines. Five compounds of the series 8a, 8geh, 8m and 8o showed IC50 values in the range of 0.71e10.94 mM. Among compounds, 8g and 8h showed significant cytotoxicity towards all the types of cancer studied. Cell cycle analysis revealed that the compounds 8a, 8m and 8o inhibit proliferation by cell cycle arrest. Also Hoechst-positive nucleus indicating apoptosis of these cells was observed in presence of 8geh. Further studies revealed that these compounds inhibit tubulin polymerization and leads to the inactivation of AKT/PKB that are known to play an important role in the proliferation of cancer cells. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Podophyllotoxin CA-4 Tubulin polymerization inhibition Anticancer agents

1. Introduction With the alarming increase in the number of cancer cases across the globe, several approaches are being investigated to develop an effective and affordable cure for this deadly disease [1]. Among various approaches, exploiting natural products have been one of the most successful methods to identify novel hits and leads [2]. Podophyllotoxin (1) is a naturally occurring cytotoxic lignan, isolated from podophyllin, a resin produced by species of the genera podophyllum (berberidaceae) [3] and has been exploited enormously to develop new drug candidates [4]. This unique natural product disrupts microtubule formation by inhibiting tubulin polymerization [5] and the three most potent semi-synthetic derivatives of 1, etoposide (2), etophos (3) and teniposide (4) inhibit DNA-topoisomerase II to stop cell proliferation (Fig. 1). These derivatives are used for the treatment of germ-cell malignancies, small-cell lung cancer, non-Hodgkin’s lymphoma, leukaemia, Kaposi's sarcoma, neuroblastoma, and soft tissue sarcoma [6]. With these findings various podophyllotoxin hybrids and conjugates have been developed to investigate the pharmacophoric properties and to identify a better molecule with enhanced bioavailability [7].

* Corresponding author. E-mail address: [email protected] (A.K. Srivastava). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.ejmech.2014.10.050 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

The C-4 position of 1 is one of the major targets for derivatization and several C-4 modified clinical candidates have been identified [8]. SAR reports suggest that C-4 position of 1 can bear bulky groups and thus has huge scope for functionalization [9]. In our efforts to develop new anticancer drugs, we envisioned that C-4 functionalized podophyllotoxin esters with properly functionalized counterpart could be highly effective as anticancer agents with improved activity. Several esters of podophyllotoxin have been reported in literature with functionalized aliphatic chains (5) and (6) with improved bioavailability but with lower cytotoxicity than the parent compound [10,11]. Bathini and coworkers have reported the ester derivatives of epipodophyllotoxin with increased cytotoxic potential [12]. Recently, PAMAM dendrimer linked podophyllotoxin derivatives were studied by Sk et al. to enhance the bioavailability and were found stable against hydrolytic cleavage. These derivatives showed sustained release characteristics with potent cytotoxicity against various cancer cell lines [13]. Carbamate derivatives of 4b-(1,2,3-triazol-1-yl)-podophyllotoxin and 40 demethylepipodophyllotoxin have been reported by Chen et al. and some of these compounds were found more potent than etoposide. These carbamates inhibited microtubule formation and also inhibit DNA-topoisomerase II [14,15]. Herein, we report synthesis and biological studies of a series of podophyllotoxin esters where five molecules showed good cytotoxicity values (IC50) 100

8.83 ± 2.53 26.54 ± 5.5 24.37 ± 12.8 51.16 ± 6.9 48.61 ± 2.02 27.34 ± 8.8 1.47 ± 0.514 1.08 ± 0.374 27.96 ± 12.7 22.91 ± 6.8 27.30 ± 13.8 23.71 ± 9.17 10.94 ± 2.06 23.65 ± 4.56 5.13 ± 1.8 3.82 ± 0.012 5.48 2.27

2.06 ± 1.00 17.58 ± 0.91 17.09 ± 0.66 15.46 ± 1.22 13.19 ± 0.57 13.69 ± 0.144 1.00 ± 0.189 1.12 ± 0.23 15.93 ± 0.050 14.30 ± 0.788 14.10 ± 0.593 11.75 ± 0.496 2.30 ± 1.17 15.91 ± 0.532 2.02 ± 0.680 0.90 ± 0.02 96.26 7.58

1.56 15.27 19.51 17.38 21.03 19.68 1.35 1.23 20.34 17.61 20.10 22.95 1.83 23.33 1.74 2.78 e e

a b c d e f g

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.69 7.2 6.2 6.9 11.6 7.2 0.28 0.28 8.6 5.2 8.9 11.7 0.94 10.4 0.608 0.06

50% inhibitory concentrations and the values are the average of three individual experiments. Lung cancer. Prostate cancer. Liver cancer. Cervical cancer. Breast cancer. Literature values [21].

drug delivery approaches can be developed for the targeted delivery of such molecules.

Table 2 FACS analysis (at 2 mM concentration). Entry

Sub G1

G0/G1

S

G2/M

8a 8m 8o DMSO

40.72 38.88 73.0 0.17

41.81 42.80 22.39 78.96

4.24 5.69 1.68 5.88

13.23 12.63 2.94 15.00

and also compared with that CA-4 showed 69.44% of inhibition (Table 3).

2.2.6. Effect on AKT signalling Akt is a serine/threonine protein kinase, which is downstream to the phosphoinositide 3-kinase. Cancer cells expressing a higher level of Akt, is known to influence a variety of physiological factors, including regulation of cell growth, cell cycle and cell survival. Recent studies have indicated that inhibitors of tubulin polymerisation cause a decrease in the phosphorylation (Ser473), of the Akt protein [24]. A dose dependent decrease in the expression of pAKT was observed with increasing concentrations of the 8a, 8m and 8o from 2 mM to 5 mM (Fig. 6).

3. Conclusions We have studied the cytotoxicity profile of series podophyllotoxin derivatives and observed that some of these derivatives have potential to be taken further for anticancer drug development. The IC50 values from Table 1 showed that the triple bond containing compounds 8geh were found most active thereby reflecting the importance of the alkyne group. Among the compounds containing acrylate counterpart, only 8m and 8o showed good cytotoxicity. Increasing bulk at the benzene ring of acrylate counterpart (8iej) did not improve the activity. It was abundant that the acrylate esters exhibit better activity than benzoate esters of podophyllotoxin. However, nirobenzoate derivative 8a showed good cytotoxicity. The Despite the fact that esters hydrolyse in biological system, new

4. Experimental section 4.1. General information All chemicals, reagents, and solvents for the synthesis of the compounds were of analytical grade, purchased from commercial sources and used without further purification, unless otherwise specified. All the reactions were monitored by thin layer chromatography over silica gel coated TLC plates. The spots on TLC were visualized by warming ceric sulphate (2% CeSO4 in 2 N H2SO4) sprayed plates in hot plate or in an oven at about 100  C. Silica gel (100e200 mesh) was used for column chromatography. IR spectra were recorded on Perkin Elmer 881 or FT IR 820/PC instrument and values are expressed in cm-1. ESMS were recorded using a Micromass LCeMS system. 1H and 13C NMR were recorded on Bruker Advance DPX 500 MHz using TMS as an internal standard.

4.2. Preparation of compounds 8aeo General Procedure: Podophyllotoxin was purified from the resin and was used directly to couple with separately synthesized acids by following typical procedure of esterification: To the solution of acid (1.2 mmols) in anhydrous CH2Cl2 (4 mL) was added EDCI (1.2 mmols) followed by HOBt (1.2 mmols) and podophyllotoxin (1.0 mmol) at 0  C and the reaction mixture was warmed to rt. Stirring was continued for 4e6 h at rt till the complete consumption of podophyllotoxin. Progress of the reaction was monitored by TLC. After complete consumption of 1, reaction mixture was diluted by CH2Cl2 (10 mL) and washed by water (5 mL  3) followed by brine (5 mL). The organic layer was dried on Na2SO4 and solvent was removed in vaccuo. Crude was subject to column chromatography by using silica gel to afford the pure product.

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Fig. 3. Apoptosis assay was performed by using Hoechst 33342 staining of DU-145 cells treated with compound 8g (C) and 8h (D) at 2 mM concentration. DMSO vehicle (A) was used as negative control and doxorubicin (B) at 2 mM was used as positive control. Nuclear fragmentation was observed in compound treated groups as depicted by arrows in the figure. Results depicted are representative image of experiments performed more than three times. Scale bar represents 20 mm.

Fig. 4. A colony Formation Soft Agar assay was performed to analyse the long term effect of podophyllotoxin derivatives on anchorage independent proliferative capacity of DU-145 cells. DMSO was taken as a negative control and doxorubicin was taken as positive control. The inhibitory effect of the compounds was observed. Representative images depict results of experiments performed at least three times. Scale bar represents 20 mm.

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133

4.61e4.54 (m, 2H), 4.30 (t, J ¼ 9.0 Hz, 1H), 3.80 (s, 3H), 3.78 (s, 6H), 3.05e2.96 (m, 2H). 13 C NMR: (75 MHz, CDCl3): d 173.3, 164.9, 152.6, 150.8, 148.4, 147.7, 134.6, 132.5, 130.7, 127.5, 123.7, 109.9, 108.0, 106.8, 101.7, 75.3, 71.2, 60.7, 56.1, 45.5, 43.6, 41.9, 38.6. ESIMS: [MþH]þ ¼ 564. HRMS (ESI): calcd for C29H26NO11 [MþH] ¼ 564.1506; found ¼ 564.1496.

Fig. 5. In vitro tubulin polymerization assay was performed at 5 mM concentration of compounds 8a, 8g, 8h, 8m, 8o and podophyllotoxin, respectively and CA-4 at 10 mM was included as a positive control. The assay was performed in triplicates and the mean percentage inhibition of tubulin polymerization was presented.

Table 3 Tubulin polymerization inhibition assay. S. No.

Compound

% Inhibition of tubulin polymerization at 5 mM

1. 2. 3. 4. 5. 6. 7.

8a 8g 8h 8m 8o Podophyllotoxin Combretastatin A-4

63.96 62.07 53.84 51.22 46.75 66.63 69.44

4.2.1. a-Podophyllotoxin-4-nitrobenzoate, 8a Compound 8a was prepared by coupling podophyllotoxin with 4-nitrobenzoic acid. Yield ¼ 68% (93 mg from 100 mg of 1). IR (KBr): 3340, 2967, 2934, 1616, 1572, 1522, 1463, 1247, 1169, 1129 cm1 1 H NMR: (500 MHz, CDCl3): d 8.35 (d, J ¼ 7.5 Hz, 2H, ArH), 8.24 (d, J ¼ 7.5 Hz, 2H, ArH), 6.84 (s, 1H, ArH), 6.61 (s, 1H, ArH), 6.44 (s, 2H, ArH), 6.13 (pseudo d, J ¼ 8.68 Hz, 1H), 6.02 (d, J ¼ 10.0 Hz, 2H),

4.2.2. a-Podophyllotoxin-2-nitro-4-methoxybenzoate, 8b Compound 8b was prepared by coupling podophyllotoxin with 2-nitro-4-methoxybenzoic acid. Yield ¼ 73% (105 mg from 100 mg of 1). IR (KBr): 3340, 2967, 2934, 1779, 1724, 1615, 1573, 1454, 1244, 1125, 1034 cm1 1 H NMR: (500 MHz, CDCl3): d 7.87 (d, J ¼ 9.0 Hz, 1H, ArH), 7.35 (d, J ¼ 8.5 Hz, 1H, ArH), 7.20 (dd, J ¼ 8.0, 1.2 Hz, 1H, ArH), 6.78 (s, 1H, ArH), 6.53 (s, 1H, ArH), 6.39 (s, 2H, ArH), 6.13 (pseudo d, J ¼ 8.68 Hz, 1H), 5.97 (d, J ¼ 6.0 Hz, 2H), 4.59e4.54 (m, 2H), 4.30 (t, J ¼ 9.0 Hz, 1H), 3.81 (s, 6H), 3.76 (s, 6H), 3.02e2.96 (m, 2H). 13 C NMR: (75 MHz, CDCl3): d 173.4, 165.1, 164.5, 162.2, 157.0, 152.5, 147.5, 145.5, 134.6, 132.1, 127.3, 117.6, 109.7, 109.3, 107.8, 106.7, 101.5, 75.3, 71.2, 60.6, 56.0, 43.7, 41.9, 45.3, 38.0. ESIMS: [MþH]þ ¼ 594. HRMS (ESI): calc for C30H27NNaO12 [MþNa] ¼ 616.1431; Found ¼ 616.1426. 4.2.3. a-Podophyllotoxin-2-chlorobenzoate, 8c Compound 8c was prepared by coupling podophyllotoxin with 2-chlorobenzoic acid. Yield ¼ 88.7% (118 mg from 100 mg of 1). IR (KBr): 3340, 2967, 1483, 1437, 1419, 1240, 1128, 1038 cm1 1 H NMR: (500 MHz, CDCl3): d 7.85 (d, J ¼ 8.3 Hz, 1H, ArH), 7.50e7.49 (m, 2H, ArH), 7.39e7.35 (m, 1H, ArH), 6.89 (s, 1H, ArH), 6.57 (s, 1H, ArH), 6.42 (s, 2H, ArH), 6.17 (pseudo d, J ¼ 8.68 Hz, 1H), 5.98 (d, J ¼ 4.1 Hz, 2H), 4.65e4.63 (m, 1H), 4.50e4.48 (m, 1H), 4.32 (t, J ¼ 10.4 Hz, 1H), 3.76 (s, 3H), 3.74 (s, 6H), 3.02e3.01 (m, 2H). 13 C NMR: (75 MHz, CDCl3): d 173.5, 166.2165.4, 157.0, 152.2, 148.2, 147.5, 136.9, 134.7, 133.1, 132.4, 131.2, 121.7, 126.8, 109.6, 107.8, 101.5, 74.7, 71.3, 60.6, 56.0, 45.5, 43.7,41.9, 38.6. ESIMS: [MþNa]þ ¼ 575. HRMS (ESI): calcd for C29H26ClO9 [MþH] ¼ 553.1266; found ¼ 553.1260.

Fig. 6. DU-145 cells were treated with Compounds 8a, 8m and 8o at 2, 3, 5 mM concentrations, respectively for 48 h and processed to Western blot analysis. In 3 different drug treatments pAKT was observed to be decreasing with the increasing concentration of drug. Representative blots are from experiments that were replicated at least three times.

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4.2.4. (E)-a-Podophyllotoxin-3-(3,4-dimethoxyphenyl)-2-(4methoxyphenyl)-acrylate, 8d Compound 8d was prepared by coupling podophyllotoxin with (E)-2-(3,4-dimethoxyphenyl)-3-(4-methoxyphenyl)acrylic acid. Yield ¼ 68% (116 mg from 100 mg of 1). IR (KBr): 3340, 2966, 2930, 1778, 1701, 1609, 1513, 1483, 1325, 1244, 1171, 1125, 1029 cm1 1 H NMR: (500 MHz, CDCl3): d 7.81 (s, 1H), 7.02 (d, J ¼ 8.0 Hz, 2H, ArH), 6.86e6.83 (m, 2H, ArH), 6.76 (d, J ¼ 8.0 Hz, 2H, ArH), 6.71 (d, J ¼ 9.0 Hz, 2H, ArH), 6.53 (s, 1H, ArH) 6.31 (s, 2H, ArH), 6.01e5.99 (m, 3H), 4.59 (d, J ¼ 4.0 Hz, 1H), 4.44 (t, J ¼ 6.0 Hz, 1H), 4.27 (t, J ¼ 9.0 Hz, 1H), 3.90 (s, 3H), 3.87e3.80 (m, 6H), 3.79 (s, 3H), 3.78 (s, 3H), 3.77 (s, 3H), 2.97e2.93 (m, 1H), 2.88e2.79 (m. 1H). 13 C NMR: (75 MHz, CDCl3): d 173.4, 167.2, 160.4, 153.2, 149.2, 148.2, 146.3, 141.5, 136.7, 133.7, 132.2, 130.2, 127.9, 128.4, 126.8, 121.7, 109.8, 107.8, 106.8, 101.4, 74.9, 70.3, 60.9, 56.4, 44.5, 43.2,41.5, 38.9. ESIMS: [MþNa]þ ¼ 733. HRMS (ESI): calcd for C40H39O12[MþH] ¼ 711.2442; found ¼ 711.2438. 4.2.5. a-Podophyllotoxin-2,3-dimethoxybenzoate, 8e Compound 8e was prepared by coupling podophyllotoxin with 2,3-dimethoxybenzoic acid. Yield ¼ 71% (99 mg from 100 mg of 1). IR (KBr): 3340, 2967, 2933, 2875, 1816, 1577, 1521, 1439, 1384, 1247, 1169, 1129 cm1 1 H NMR (500 MHz, CDCl3): d 8.24 (s, 1H, ArH), 8.22 (d, J ¼ 8.5 Hz, 2H, ArH), 6.84 (s, 1H, ArH), 6.61 (s,1H, ArH), 6.44 (s, 2H, ArH), 6.15 (d, J ¼ 8.0 Hz, 1H, ArH), 6.02 (d, J ¼ 10.0 Hz, 2H), 4.67 (s, 1H), 4.45 (t, J ¼ 7.2 Hz, 1H), 4.34 (t, J ¼ 9.4 Hz, 1H), 3.89 (s, 3H), 3.84 (s, 6H), 3.78 (s, 6H), 3.03e3.00 (m, 2H). 13 C NMR: (75 MHz, CDCl3): d 173.7, 166.5, 163.1, 157.0, 153.5, 152.5, 148.8, 147.6, 134.8, 132.3, 128.5, 123.6, 121.5, 11.9, 110.2, 109.7, 108.0, 107.0, 101.5, 73.8, 71.5, 60.7, 55.9, 45.6, 473.7, 41.9, 38.8. ESIMS: [MþH]þ ¼ 579. HRMS (ESI): calcd for C31H30NaO11 [MþNa] ¼ 601.1686; found ¼ 601.1429. 4.2.6. a-Podophyllotoxin-2-amino-5-nitrobenzoate, 8f Compound 8f was prepared by coupling podophyllotoxin with 5-nitroanthralic acid. Yield ¼ 49% (68 mg from 100 mg of 1). IR (KBr): 3427, 2923, 2853, 1745, 1599, 1459, 1254, 1119, 1030 cm1 1 H NMR: (500 MHz, CDCl3) d 8.74 (d, J ¼ 2.7 Hz, 1H), 8.24e8.19 (m, 1H), 8.06 (bs, 1H), 6.82 (s, 1H), 6.78e6.74 (m, 2H), 6.61 (s, 1H), 6.45 (s, 2H), 6.16 (d, J ¼ 8.3 Hz, 1H), 6.03 (d, J ¼ 8.0 Hz, 2H), 4.67 (bs, 1H), 4.49e4.42 (m, 2H), 3.83 (s, 3H), 3.81 (s, 6H), 3.01e2.96 (m, 2H). 13 C NMR: (75 MHz, CDCl3): d 173.4, 161.9, 157.0, 155.4152.6, 148.4, 147.8, 139.0, 136.8, 127.3, 124.3, 116.7, 109.6, 107.1101.6, 74.1, 71.3, 60.6, 55.9, 48.8, 45.8, 41.9, 38.7, 43.1; ESIMS: [MþH]þ ¼ 579. HRMS (ESI): calcd for C29H27N2O11 ¼ 579.1615; found ¼ 579.1592. 4.2.7. a-Podophyllotoxin-but-2-ynoate, 8g Compound 8g was prepared by coupling podophyllotoxin with 2-butynoic acid. Yield ¼ 61% (70 mg from 100 mg of 1). IR (KBr): 3340, 2967, 2933, 1780, 1707, 1617, 1507, 1246, 1062 cm1. 1 H NMR (500 MHz, CDCl3): d 6.82 (s, 1H), 6.54 (s, 1H), 6.39 (s, 2H), 5.99 (s, 2H), 5.98 (d, J ¼ 8.3 Hz, 1H), 4.60 (d, J ¼ 3.3 Hz, 1H), 4.42 (t, J ¼ 7.2 Hz, 1H), 4.19 (t, J ¼ 9.4 Hz, 1H), 3.81 (s, 3H), 3.77 (s, 6H), 2.91 (s, 2H), 2.03 (s, 3H). 13 C NMR: (75 MHz, CDCl3): d 173.1, 156.7, 153.6, 152.3, 136.8, 134.3, 132.1, 127.1, 109.4, 107.7, 106.8, 101.3, 87.5, 74.6, 70.8, 60.4, 55.8, 45.2, 43.4, 41.7, 38.2, 35.4.

ESIMS: [MþH]þ ¼ 481. HRMS (ESI): calcd for C26H24NaO9 [MþNa] ¼ 503.1318; found ¼ 503.1312. 4.2.8. a-Podophyllotoxin-3-phenylpropiolate, 8h Compound 8h was prepared by coupling podophyllotoxin with 3-phenylpropiolic acid. Yield ¼ 74% (96 mg from 100 mg of 1). IR (KBr): 3340, 2967, 2933, 1780, 1707, 1616, 1578, 1506, 1485, 1274, 1184, 1126, 1036 cm1 1 H NMR (500 MHz, CDCl3): d 7.59 (d, J ¼ 7.5 Hz, 2H, ArH), 7.49 (t, J ¼ 7.5 Hz, 1H, ArH), 7.39 (t, J ¼ 7.5 Hz, 2H, ArH), 6.99 (s,1H, ArH), 6.59 (s, 1H, ArH), 6.42 (s, 2H, ArH), 6.01e5.99 (m, 1H), 6.00 (s, 2H), 4.63 (s, 1H), 4.48e4.45 (m, 1H), 4.28e4.21 (m, 1H), 3.81 (s, 3H), 3.78 (s, 6H), 2.96 (bs, 2H). 13 C NMR (75 MHz, CDCl3): d 172.9, 156.5, 153.9, 152.2, 147.9, 147.2, 136.7, 134.1, 132.5, 130.6, 128.2, 127.0, 118.6, 109.2, 107.6, 106.8, 101.2, 87.8, 79.4, 70.7, 60.2, 55.7, 45.2, 43.2, 38.1. ESIMS: [MþH]þ ¼ 543. HRMS (ESI): calcd for C31H27O9 [MþH] ¼ 543.1655; Found ¼ 543.1639. 4.2.9. (E)-a-Podophyllotoxin-3-(4-(benzyloxy)-3-methoxyphenyl)2-(3,4-dimethoxyphenyl) acrylate, 8i Compound 8i was prepared by coupling podophyllotoxin with (E)-3-(4-(benzyloxy)-3-methoxyphenyl)-2-(3,4-dimethoxyphenyl) acrylic acid. Yield ¼ 70% (137 mg from 100 mg of 1). IR (KBr): 3340, 2966, 2929, 2361, 1702, 1617, 1581, 1515, 1243, 1126, 1027 cm1 1 H NMR: (500 MHz, CDCl3): d 7.77 (s, 1H), 7.39e7.31 (m, 5H, ArH), 7.29 (d, J ¼ 7.0 Hz, 1H, ArH), 6.86 (d, J ¼ 8.1 Hz, 1H, ArH), 6.82 (d, J ¼ 7.3 Hz, 2H, ArH), 6.74 (s, 2H, ArH), 6.50 (d, J ¼ 10.4 Hz, 2H, ArH), 6.30 (s, 2H, ArH), 5.99e5.97 (m, 1H), 5.98 (d, J ¼ 11.9 Hz, 2H), 5.13 (s, 2H), 4.59 (d, J ¼ 4.3 Hz, 1H), 4.44 (t, J ¼ 7.31 Hz, 1H), 4.27 (t, J ¼ 10.1 Hz, 1H), 3.88 (s, 3H), 3.80 (s, 3H), 3.78 (s, 3H), 3.62 (s, 6H), 3.49 (s, 3H), 2.94e2.90 (m, 2H). 13 C NMR: (75 MHz, CDCl3): d 183.6, 168.3, 152.5, 149.6, 148.7, 147.5, 141.6, 136.9, 134.7, 132.4, 136.9, 128.5, 127.9, 122.2, 107.0, 107.8, 101.5, 109.6, 111.4, 113.0, 74.0, 71.6, 70.6, 60.7, 55.8, 45.6.43.7, 38.8. ESIMS: [MþNa]þ ¼ 839. HRMS (ESI): calcd for C47H44NaO13 [MþNa] ¼ 839.2680; found ¼ 839.2674. 4.2.10. (E)-a-Podophyllotoxin 3-(3-(benzyloxy)-4-methoxyphenyl)2-(3,4-dimethoxyphenyl)acrylate, 8j Compound 8j was prepared by coupling podophyllotoxin with 3-(3-(benzyloxy)-4-methoxyphenyl)-2-(3,4-dimethoxyphenyl) acrylic acid. Yield ¼ 58% (114 mg from 100 mg of 1). IR (KBr): 3010, 2941, 2836, 1699, 1588, 1491, 1460, 1239, 1208, 1125, 995 cm1 1 H NMR (500 MHz, CDCl3): d 7.76 (s, 1H), 7.36e7.30 (m, 2H, ArH), 7.29e7.23 (m, 3H, ArH), 6.84 (t, J ¼ 8.4 Hz, 1H, ArH), 6.81 (s, 2H, ArH), 6.78e6.74 (m, 3H, ArH), 6.58 (d, J ¼ 1.8 Hz, 1H, ArH), 6.52 (s, 1H, ArH), 6.29 (s, 2H, ArH), 6.00 (d, J ¼ 8.2 Hz, 1H), 5.99 (d, J ¼ 10.7 Hz, 2H), 4.75 (d, J ¼ 12.3, 24.3 Hz, 2H), 4.69 (d, J ¼ 12.3 Hz, 1H), 4.58 (d, J ¼ 5.1 Hz, 1H), 4.45 (t, J ¼ 8.7 Hz, 1H), 4.26 (t, J ¼ 9.4 Hz, 1H), 3.88 (s, 3H), 3.86 (s, 3H), 3.79 (s, 3H), 3.78 (s, 3H), 3.76 (s, 3H), 3.62 (s 3H), 2.96e2.93 (m, 1H), 2.86e2.78 (m, 1H). 13 C NMR: (75 MHz, CDCl3): d 183.4, 168.7, 153.5, 148.6, 148.3, 147.3, 140.6, 136.9, 134.9, 133.4, 136.2, 128.5, 127.2, 122.1, 107.9, 107.8, 101.2, 108.6, 110.4, 112.0, 75.0, 72.6, 76.6, 60.2 54.8, 44.6, 43.9, 38.5. ESIMS: [MþNa]þ ¼ 839. HRMS (ESI): calcd for C47H44NaO13 [MþNa] ¼ 839.2680; found ¼ 839.2651.

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4.2.11. (E)-a-Podophyllotoxin- 2-(3-methoxyphenyl)-3-(3,4,5trimethoxy-phenyl)acrylate, 8k Compound 8k was prepared by coupling podophyllotoxin with 2-(3-methoxyphenyl)-3-(3,4,5-trimethoxy-phenyl)acrylic acid. Yield ¼ 67% (119 mg from 100 mg of 1). IR (KBr): 3340, 2966, 2935, 1778, 1704, 1617, 1588, 1508, 1419, 1242, 1222, 1125, 1034 cm1 1 H NMR (500 MHz, CDCl3): d 7.81 (s, 1H), 7.72 (s, 2H, ArH), 7.10 (s, 1H, ArH), 7.04e6.99 (m, 4H, ArH), 6.46 (s, 1H, ArH), 6.39 (s, 2H, ArH), 6.03e5.99 (m, 1H), 6.02 (d, J ¼ 4.2 Hz, 2H), 4.59 (d, J ¼ 4.0 Hz, 1H), 4.44 (t, J ¼ 6.2 Hz, 1H), 4.29 (t, J ¼ 9.0 Hz, 1H), 3.90 (s, 3H), 3.86 (s, 6H), 3.79 (s, 3H), 3.78 (s, 3H), 3.77 (s, 3H), 2.97e2.93 (m, 1H), 2.88e2.79 (m. 1H). 13 C NMR (75 MHz, CDCl3): d 173.4, 167.2, 160.4, 153.2, 149.2, 148.2, 146.3, 141.5, 136.7, 133.7, 132.2, 130.2, 127.9, 128.4, 126.8, 121.7, 109.8, 107.8, 106.8, 101.4, 74.9, 70.3, 60.9, 56.4, 44.5, 43.2,41.5, 38.9. ESIMS: [MþNa]þ ¼ 763. HRMS(ESI): calcd for C41H40NaO13 ¼ 763.2367; found ¼ 763.2353. 4.2.12. (E)-a-Podophyllotoxin 2-(3,4-dimethoxyphenyl)-3-(3,4,5trimethoxyphenyl)acrylate, 8l Compound 8l was prepared by coupling podophyllotoxin with 2-(3,4-dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)acrylic acid. Yield ¼ 72% (133 mg from 100 mg of 1). IR (KBr): 3340, 2967, 2931, 1680, 1617, 1578, 1513, 1464, 1326, 1254, 1127, 1027 cm1 1 H NMR (500 MHz, CDCl3): d 7.79 (s, 1H), 7.14 (s, 1H, ArH), 6.96e6.81 (m, 4H, ArH), 6.56 (s, 1H, ArH), 6.39 (s, 2H, ArH), 6.33 (s, 1H, ArH), 5.99 (s, 2H), 4.79 (bs, 1H), 4.44 (t, J ¼ 3.2 Hz, 1H), 4.34e4.26 (m, 1H), 4.16e4.07 (m, 1H), 3.90 (s, 3H), 3.84 (s, 6H), 3.81 (s, 3H), 3.78 (s, 6H), 3.64 (s, 3H), 3.61 (s, 3H), 2.90e2.74 (m, 2H). 13 C NMR: (75 MHz, CDCl3): d 173.5, 168.2, 159.0, 152.2, 149.7, 148.5, 147.3, 141.3, 136.7, 134.6, 132.0, 128.0, 125.5, 126.9, 120.5, 114.0, 110.5, 107.6, 106.8, 101.3, 109.2, 106.6, 73.8, 71.5, 60.5, 55.6, 45.3, 43.5, 40.7; ESIMS: [MþH]þ ¼ 771. HRMS (ESI): calcd for C42H42NaO14 [MþNa] ¼ 793.2472; found ¼ 739.2436. 4.2.13. (E)-a-Podophyllotoxin 3-(4-acetoxy-3-methoxyphenyl)-2(3,4-dimethoxyphenyl)acrylate, 8m Compound 8m was prepared by coupling podophyllotoxin with 3-(4-acetoxy-3-methoxyphenyl)-2-(3,4-dimethoxyphenyl)acrylic acid. Yield ¼ 70% (130 mg from 100 mg of 1) IR (KBr): 3340, 2967, 2926, 1617, 1577, 1247, 1169 cm1 1 H NMR (500 MHz, CDCl3): d 7.91 (s, 1H), 6.90 (s, 1H, ArH), 6.88 (s, 1H, ArH), 6.85e6.84 (m, 2H), 6.82 (s, 1H, ArH), 6.78e6.76 (m, 1H, ArH), 6.73e6.71 (m, 1H, ArH), 6.54 (s, 1H, ArH), 6.39 (s, 2H), 5.98 (d, J ¼ 5.5 Hz, 2H), 5.88 (d, J ¼ 9.1 Hz, 1H), 4.61 (d, J ¼ 4.5 Hz, 1H), 4.38 (t, J ¼ 7.0 Hz, 1H), 4.20 (t, J ¼ 4.0 Hz, 1H), 3.87 (s, 3H), 3.81 (s, 3H), 3.76 (s, 6H), 3.72 (s, 3H), 3.54 (s, 3H), 2.94e2.90 (m, 1H), 2.85e2.80 (m, 1H), 2.28 (s, 3H). 13 C NMR (75 MHz, CDCl3): d 171.1, 168.3, 167.6, 156.5, 153.6, 152.1, 149.6, 146.8, 142.7, 140.0, 137.4, 133.9, 130.0, 128.5, 122.2, 121.2, 117.0, 110.8, 112.6, 109.5, 73.5, 63.6, 60.1, 55.4, 50.9, 52.4, 55.4, 47.0, 45.8, 38.9, 55.7, 55.4, 20.3. ESIMS: [MþH]þ ¼ 769. HRMS (ESI): calcd for C42H41O14 [MþH] ¼ 769.2496; found ¼ 769.3339. 4.2.14. (E)- a-podophyllotoxin 2-(4-methoxyphenyl)-3-(3,4,5trimethoxyphenyl)acrylate, 8n Compound 8n was prepared by coupling podophyllotoxin with 2-(4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)acrylic acid.

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Yield ¼ 69% (123 mg from 100 mg of 1) IR (KBr): 3431, 2922, 2850, 1666, 1611, 1584, 1460, 1189, 1125, 1014 cm1 1 H NMR (500 MHz, CDCl3): d 7.81 (s, 1H), 7.19 (d, J ¼ 7.8 Hz, 2H), 6.84 (d, J ¼ 8.2 Hz, 2H), 6.67 (s, 1H), 6.42 (s, 1H), 6.39e6.21 (m, 4H, ArH), 6.01 (d, J ¼ 4.2 Hz, 1H), 5.98 (d, J ¼ 6.0 Hz, 2H), 4.59 (d, J ¼ 5.0 Hz, 1H), 4.49e4.42 (m, 1H), 4.33e4.26 (m, 1H), 3.89 (s, 3H), 3.78 (s, 6H), 3.77 (s, 6H), 3.66 (s, 6H), 3.05e3.00 (m, 1H), 2.96e2.85 (m, 1H). 13 C NMR (75 MHz, CDCl3): d 173.2, 167.8, 158.8, 156.5, 152.1, 147.6, 147.1, 141.0, 138.6, 134.3, 131.8, 130.5, 129.8, 129.0, 127.8, 127.2, 118.6, 113.3, 109.0, 107.6, 106.5, 101.0, 73.6, 71.1, 60.2, 55.4, 55.4, 54.8, 42.2, 41.5, 38.2. ESIMS: [MþNa]þ ¼ 763. HRMS(ESI): calcd for C41H40NaO13 ¼ 763.2367; found ¼ 763.2352. 4.2.15. (E)- a-podophyllotoxin 2,3-bis(3,4-dimethoxyphenyl) acrylate, 8o Compound 8o was prepared by coupling podophyllotoxin with 2,3-bis(3,4-dimethoxyphenyl)acrylic acid. Yield ¼ 81% (144 mg from 100 mg of 1); IR (KBr): 3418, 2966, 2924, 2256, 1633, 1513, 1463, 1255, 1025, 1000 cm1 1 H NMR (500 MHz, CDCl3): d 7.80 (s, 1H), 6.88 (d, J ¼ 8.0 Hz, 1H, ArH), 6.83e6.80 (m, 4H, ArH), 6.74 (d, J ¼ 8.5 Hz, 1H, ArH), 6.53 (s, 1H, ArH), 6.30 (s, 2H, ArH), 6.02 (d, J ¼ 4.0 Hz, 2H), 5.97 (bs, 1H), 4.59 (d, J ¼ 4.0 Hz, 1H), 4.45 (t, J ¼ 4.0 Hz, 1H), 4.28 (t, J ¼ 9.5 Hz, 1H), 3.89 (s, 3H), 3.86 (s, 3H), 3.81 (s, 3H), 3.78 (s, 3H), 3.63 (s, 6H), 3.48 (s, 3H), 2.96e2.92 (m, 1H), 2.88e2.80 (m, 1H); 13C NMR (75 MHz, CDCl3): d 173.3, 168.2, 152.4, 150.3, 149.1, 148.5, 148.1, 148.0, 147.4, 141.6, 136.7, 134.6, 132.3, 128.6, 126.9, 125.6, 122.1, 112.6, 112.3, 111.2, 110.4, 109.5, 107.6, 107.0, 101.5, 73.9, 71.5, 60.6, 55.8, 55.1, 45.49, 43.6, 38.7; ESIMS (m/z): [MþNa]þ ¼ 763. HRMS(ESI): calcd for C41H40NaO13 ¼ 763.2367; found ¼ 763.2353. 4.3. Biology experiment 4.3.1. Cell culture The various human cancer cell lines including A-549 (non-small lung cancer), Du-145 (prostate cancer), HepG2 (hepatocellular carcinoma), HeLa (cervical cancer) and MCF-7 (breast cancer cell) were maintained in Dulbecco's Minimum Essential Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin antibiotics in a humidified incubator and 5% CO2 atmosphere at 37  C. 4.3.2. MTT assay The in vitro anti-proliferative activity of the synthesized compounds was evaluated against A-549, DU-145, HepG2, HeLa and MCF-7 cancer cell lines using MTT assay. This assay involves the reduction of yellow tetrazolium salt (MTT) by the metabolically active cells and formation of purple formazan crystals. Briefly, the cells were seeded in a 96-well cell culture plate at a density of 5  103 cells/well and incubated for 24 h at 37  C in a humidified 5% CO2 incubator. The compounds were dissolved in DMSO and diluted with culture medium. Cell were treated with the compounds at different concentrations to achieve a final concentration of 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 and 100.0 mM and incubated for 48 h at 37  C in a humidified 5% CO2 incubator. This was followed by addition of 10% v/v of MTT reagent dye (5 mg/mL) per well and incubation for 4 h at 37  C in a humidified atmosphere with 5% CO2. The medium was replaced with DMSO to dissolve the formazan crystals and absorbance was read at 540 nm using a plate-reader (Thermo Scientific,

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USA). The IC50 values were calculated from the chart of percent cell viability against dose of compounds (mM) treated. 4.3.3. Cell cycle analysis To understand the effect of 8a, 8m and 8o on cell cycle, DU-145 cells were treated with above listed compounds for 48 h at 2 mM concentration. Cells were harvested after stimulation, washed with PBS and fixed in 70% ethanol at 20  C for overnight. Ethanol is removed by Centrifugation and pellet were resuspended in PI solution (RNase 0.1 mg/mL, Triton X-100-0.05 %, PI-50 mg/mL) and incubated for 1 h in dark at room temperature. Then the cells were washed with PBS buffer and DNA content was analysed by fluorescence activated cell sorting (FACS Calibur System; BD Bioscience, Erembodegem, Belgium) on an FL-2 fluorescence detector (10000 events were recorded for each condition). Flow cytometry data were analysed using FCS express 4 software (De Novo Software, Los Angeles, CA). 4.3.4. Apoptosis assay The prostate cancer cells, DU-145 were seeded at a density of 3  104 cells/well and cultured in 6 well plates on a cover slip for 24 h at 37  C. The cells were treated with compounds 8g, 8h and doxorubicin at a concentration of 2 mM and incubated for 24 h. Following the treatment the cells were fixed with 4% Paraformaldehyde and stained with Hoechst 33342 (2 mg/mL; Invitogen, USA) for 30 min. The cover slip was inverted and placed on a glass slide and mounted. Apoptotic cells were identified by condensation and fragmentation of nuclei using confocal microscope (Olympus, USA) [25]. 4.3.5. Colony formation assay DU-145 (1.5  104/well) were mixed with medium containing the compound and top agar to obtain the final concentration of 0.35% agar (Bacto Agar: Becton, Dickinson, Sparks, MD) and plated in a 6-well plate containing already solidified 1% base agar and incubated for 9 days under culture conditions to assess the clonogenic potential of cells at low seeding density. The culture medium was changed every 3 days and the formation of cell colonies was monitored by staining with 0.005% crystal violet for 1 h. Excess stain was removed by washing for 5 min in MQ water and colonies were counted using microscope. All experiments were performed in triplicates and representative images were shown in Fig. 4. 4.3.6. Tubulin polymerisation assays The tubulin polymerisation assay was performed by using fluorescence-based tubulin polymerisation assay kit (Cytoskeleton, Inc.) according to the manufacturer's protocol. The reaction mixture contained, 2 mg/ml tubulin, 10 mM fluorescent reporter, and 1 mM GTP in the presence of Podophyllotoxin esters in buffer 1 at 37  C in a final volume of 16 mL. Fluorescence readings were recorded on a multimode reader at 340/410 nm (excitation/emission) every 1 min for up to 1 h. Combretastatin A-4 was used as positive control under similar conditions. The assay was performed in triplicates and the mean percentage inhibition of tubulin polymerization is presented Fig. 5. 4.3.7. Immunoblot analysis After stimulation of DU-145 cells with 8a, 8m and 8o, all cells were harvested in cold PBS buffer and washed 3 times in PBS. Cell pellets were lysed in complete lysis buffer (8 M urea, 2 M thiourea, 4% CHAPS, 4 mM Tris base and 65 mM DTT) containing inhibitors against proteases and phosphatase. The protein concentration was determined by modified Bradford assay. Protein samples were resolved by SDSe10% polyacrylamidegel electrophoresis and transferred onto nitrocellulose membrane, incubated in blocking

buffer containing 3% BSA in TBST and membrane was incubated overnight at 4  C with appropriate primary antibody (Cell Signaling Technology) diluted in blocking buffer (1:1000). Excess and unbound antibody was washed for 15 min in TBST and membrane was incubated with horseradish peroxidase-conjugated anti-rabbit antibody diluted 1:5000 in TBST at room temperature for 1 h after three washes, membrane was developed by chemiluminescence detection system. The representative blots were shown in Fig. 6. Acknowledgements Authors acknowledge Director CSIR-IICT for support and financial assistance by CSIR, Govt. of India, New Delhi under 12th Fiveyear plan projects: ACT (CSC-0301) and (SMiLE CSC-0111). Fellowships provided by CSIR, UGC and ICMR are gratefully acknowledged by MAS and DD (CSIR-JRF), RG (UGC-JRF) and ND (ICMR-JRF). Authors thank Dr. Ahmed Kamal for his generous support by providing podophyllotoxin resin and valuable suggestions during the progress of the project. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.10.050. References [1] a) http://www.who.int/gho/ncd/mortality_morbidity/cancer/en/index.html. b) S. Neidle, D.E. Thurston, Nat. Rev. Cancer 5 (2005) 285. [2] a) D.J. Newman, G.M. Cragg, J. Nat. Prod. 75 (2012) 311; b) M.S. Butler, Nat. Prod. Rep. 25 (2008) 475. [3] H. Xu, M. Lv, X. Tian, Curr. Med. Chem. 16 (2009) 327. [4] A.K. Mukherjee, S. Basu, N. Sarkar, A.C. Ghosh, Curr. Med. Chem. 8 (2001) 1467. [5] Y. Damayanthi, J.W. Lown, Curr. Med. Chem. 5 (1998) 205. [6] F. Cortese, B. Bhattacharyya, J. Wolff, J. Biol. Chem. 252 (1977) 1134. [7] X.-K. Zhu, J. Guan, Y. Tachibana, K.F. Bastow, S.J. Cho, H.-H. Cheng, Y.-C. Cheng, M. Gurwith, K.-H. Lee, J. Med. Chem. 42 (1999) 2441. [8] Y.-Q. Liu, J. Tian, K. Qian, X.-B. Zhao, S.L. Morris-Natschke, L. Yang, X. Nan, X. Tian, K.-H. Lee, Recent progress on C-4-modified podophyllotoxin analogs as potent antitumor agents, Med. Res. Rev. (2014), http://dx.doi.org/10.1002/ med.21319. [9] a) A. Kamal, J.R. Tamboli, M.J. Ramaiah, S.F. Adil, S.N.C.V.L. Pushpavalli, R. Ganesh, P. Sarma, U. Bhadra, M. Pal-Bhadra, Bioorg. Med. Chem. 21 (2013) 6414 (and references cited therein); b) A. Kamal, B.A. Kumar, P. Suresh, S.K. Agrawal, G. Chashoo, S.K. Singh, A.K. Saxena, Bioorg. Med. Chem. 18 (2010) 8493; c) V.K. Singh, R. Kadu, H. Roy, Eur. J. Med. Chem. 74 (2014) 552; d) A. Kamal, N.L. Gayatri, D.R. Reddy, P.S.M.M. Reddy, M. Arifuddin, S.G. Dastidar, A.K. Kondapi, M. Rajkumar, Bioorg. Med. Chem. 13 (2005) 6218; e) A. Kamal, E. Laxman, G.B.R. Khanna, P.S.M.M. Reddy, T. Rehana, M. Arifuddin, K. Neelima, A.K. Kondapi, S.G. Dastidar, Bioorg. Med. Chem. 12 (2004) 4197; f) A. Kamal, B.A. Kumar, M. Arifuddin, S.G. Dastidar, Lett. Drug Des. Discov. 3 (2006) 205.  pez-Pe rez, E. del Olmo, B. de Pascual-Teresa, A. Abad, A.S. Feliciano, [10] J.L. Lo Bioorg. Med. Chem. Lett. 14 (2004) 1283. [11] N.J. Lee, I.C. Jeong, M.Y. Cho, C.W. Jeon, B.C. Yun, Y.O. Kim, S.H. Kim, I. Chung, Eur. Polym. J. 42 (2006) 3352. [12] Y. Bathini, A. Scholte, S. Kelly, Syn. Commun. 29 (1999) 379. [13] U.H. Sk, D. Dixit, E. Sen, Eur. J. Med. Chem. 68 (2013) 47. [14] J.-F. Liu, C.-Y. Sang, X.-H. Xu, L.-L. Zhang, X. Yang, L. Hui, J.-B. Zhang, S.W. Chen, Eur. J. Med. Chem. 64 (2013) 621. [15] C.-Y. Sang, J.-F. Liu, W.-W. Qin, J. Zhao, Lin Hui, Y.-X. Jin, S.-W. Chen, Eur. J. Med. Chem. 70 (2013) 59. [16] M.A. Jordan, L. Wilson, Nat. Rev. Cancer 4 (2004) 253. [17] J. Chen, T. Liu, X. Dong, Y. Hu, Mini Rev. Med. Chem. 9 (2009) 1174. [18] M. Jordan, Curr. Med. Chem. Anti Cancer Agents 2 (2012) 1. [19] a) A. Kamal, P. Suresh, M.J. Ramaiah, A. Mallareddy, B.A. Kumar, P. Raju, J.V. Gopal, S.N.C.V.L. Pushpavalli, A. Lavanya, P. Sarma, M.P. Bhadra, Bioorg. Med. Chem. 19 (2011) 4589; b) Y. Liu, C.-M. Park, X. Xu, Angew. Chem. Int. Ed. 51 (2012) 9372. [20] A. Kamal, M. Arifuddin, B. A. Kumar, S. G. Dastidar, US patent (2007) US20070066837A1. [21] J. Ren, L. Wu, W.Q. Xin, X. Chen, K. Hu, Bioorg. Med. Chem. Lett. 22 (2012) 4778.

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