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MEDICINAL CHEMISTRY RESEARCH

Med Chem Res (2013) 22:1604–1617 DOI 10.1007/s00044-012-0150-7

ORIGINAL RESEARCH

Synthesis, characterization, in vitro anticancer activity, and docking of Schiff bases of 4-amino-1,2-naphthoquinone S. Shukla • R. S. Srivastava • S. K. Shrivastava A. Sodhi • Pankaj Kumar



Received: 6 February 2012 / Accepted: 8 June 2012 / Published online: 30 June 2012 Ó Springer Science+Business Media, LLC 2012

Abstract A series of Schiff bases of 4-amino-1,2-naphthoquinone were synthesized, purified, characterized, and evaluated for cytotoxicity against a panel of human cancer cell lines (Hep-G2, MG-63, and MCF-7). The cells were dosed with these Schiff bases at varying concentrations, and cell viability was measured by a 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Significant anticancer activities were observed in vitro for some members of the series and compounds 4-(3,4,5-trimethoxybenzylideneamino)naphthalene-1,2-dione (S10) as well as 4-(4-hydroxy-3-methoxybenzylideneamino)naphthalene-1,2-dione (S13) are active cytotoxic agents against different cancer cell lines with IC50 values in the range of 5.91–9.98 lM. The structures of synthesized compounds were established by spectroscopic (FT-IR, 1H NMR, 13C NMR) and elemental analysis. To study the molecular basis of interaction and affinity of binding of the target molecules, all the compounds were docked into the ATPase domain of Topoisomerase-II (TP-II) by using Schro¨dinger molecular modeling software package. Docking experiments showed a good correlation between their predicted glide scores and the observed IC50 values of synthesized compounds. Structure– activity relationships indicated that presence of electron

S. Shukla  R. S. Srivastava (&)  S. K. Shrivastava Department of Pharmaceutics, I.T., Banaras Hindu University, Varanasi 221005, India e-mail: [email protected]; [email protected] A. Sodhi School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi 221005, India P. Kumar School of Pharmaceutical Science, Siksha ‘O’ Anusandhan University, Bhubaneshwar, Orrisa, India

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donating groups on phenyl ring of Schiff bases enhances the activity but Schiff base with electron withdrawing substituents on phenyl ring shows diminished activity. Keywords Hep-G2  MG-63  MCF-7  Cytotoxicity  Schiff bases  Docking

Introduction According to World Cancer Report from the International Agency for Research on Cancer, cases of cancer doubled globally between 1975 and 2000, will double again by 2020, and will nearly triple by 2030 (Mulcahy, 2008). Therefore various research initiatives are going on worldwide for the treatment of malignancy with the objective to discover some novel potent and effective antineoplastic agents, particularly those interacting with novel biological targets. Mass screening programs of natural products by the National Cancer Institute have identified the quinone as an important pharmacophoric moiety for cytotoxic activity (Driscoll et al., 1974; Liu et al., 2004). Literature survey described a variety of naturally occurring and synthetic 1,2-naphthoquinones receiving a great deal of attention for their anticancer activity. Among them some naturally occurring 1,2-naphthoquinones include rhinacanthone (Thirumurugan et al., 2000; Siripong et al., 2006, 2009; Kongkathip et al., 2003), beta lapachone (Dubin et al., 2001; Lai et al., 1998; Pardee et al., 2002), mansonones (Wang et al., 2004), etc. (Fig. 1a–c) as well as synthetic 1, 2-naphthoquinones include 4-hydroxy-3-methyl-1,2-naphthoquinone thiosemicarbazone (HM-NQTS, Saha et al., 2002), 1,2-naphthoquinone-2-thiosemicarbazone (NQTS, Chen et al., 2004), 4-alkyl/aryl-1,2-naphthoquinones thiosemicarbazones and their copper complex (Afrasiabi et al.,

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Fig. 1 Structures of some potent natural and synthetic anticancer 1,2-naphthoquinone analogues

2004) (Fig. 1d–f). Our research group had also explored and reported a series of 1,2-naphthoquinone derivatives having antiproliferative activity (Shukla et al., 2012). Several 1, 2-napthoquinone derivatives can induce DNA-TOPO IImediated cleavages of DNA, and this effect is crucial for the cytotoxicity of these compounds (Frydman et al., 1997). Schiff bases also gained importance because of the physiological and pharmacological activities associated with them. Compounds containing azomethine group (–C=N–) in the structure are known as Schiff bases, which are usually synthesized by the condensation of primary amines and active carbonyl groups. Various schiff bases are found to have anticancer activity (Hu et al., 2008; Sharma et al., 1998; Pathak et al. 2000; Xu and Xu, 2000). Therefore, these above-mentioned results prompted us to continue our investigation towards synthesis of Schiff bases after introducing amino group in 1,2-naphthoquinone ring system in order to achieve new lead compounds for future development as anticancer agents and docking studies were conducted to get an insight about their binding preferences at the active site of the receptor (ATPase domain of Topoisomerase-II). In this context, new series of 4-(arylideneamino)naphthalene-1,2-dione (S01–S16) and 4-(cyclohexylidene/cyclopentylidine amino)naphthalene-1,2-dione (S17, S18) were synthesized and their anticancer activity was evaluated.

Materials and methods Chemistry The target compounds 4-(arylideneamino)naphthalene-1, 2-dione (S01–S16) and 4-(cyclohexylidene/cyclopentylidine

amino)naphthalene-1,2-dione (S17, S18) were prepared by following the synthetic pathway depicted in Fig. 2. According to previously reported method, the starting material 1,2-naphthoquinone was conveniently prepared by oxidation of 1-amino-2-naphthol hydrochloride, which was obtained by reduction of water soluble dye b naphthol orange (Furniss et al., 1995; Vogel’s Text Book Of Practical Organic Chemistry). The synthesized 1,2-naphthoquinone aminated with sodium azide in acetic acid and gave 4-amino-1,2-naphthoquinone (Fieser and Hartwell, 1935). Then Schiff bases (S01–S16) were prepared by condensing substituted aryl aldehydes with 4-amino-1, 2-naphthoquinone in suitable solvent (methanol/ethanol) in the presence of few drops of glacial acetic acid as catalyst. Compounds (S17, S18) were synthesized by the above method using cyclohexanone/cyclopentanone as starting material. The chemical structures of the synthesized compounds were established by spectroscopic (FT-IR, 1H NMR, 13C NMR) and elemental analysis. Experimental All reactions were monitored by thin layer chromatography (TLC) using silica gel G (Spectrochem Pvt. Ltd., Mumbai). The plates were developed by exposing to iodine chamber. Melting points were determined by BARNSTEAD/ Electrothermal Stuart-SMP10, open capillary melting point apparatus and were uncorrected. IR spectra were recorded on KBr disks using SHIMADZU Infrared-spectrophotometer, FTIR-8400S. The 1H NMR and 13C NMR spectra were measured by JEOL, AL300, FT-NMR spectrophotometer with Operating frequency, 300 MHz at temperature 25 °C using DMSO-d6/CDCl3 as solvent containing TMS as internal standard and reported chemical

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Fig. 2 Scheme for synthesis of 4-amino-1,2-naphthoquinone and its Schiff bases, reagents, and conditions (i) Na2S2O4, stirring at 40–50 °C, SnCl2 in conc. HCl with 100 ml water added, and cooled to 0 °C. (ii) FeCl3 in conc. HCl with crushed ice and added with

stirring. (iii) Sodium azide in water and glacial acetic acid, heated to 40 °C. (iv, a) Aromatic aldehydes, glacial acetic acid, ethanol/ methanol, reflux 6–10 h (iv, b) Cyclic ketones, conc. H2SO4, ethanol/ methanol, reflux 8–10 h

shift in d values (ppm). C, H, N analysis has been performed with Exeter Analytical Inc., USA, CE-440 elemental analyzer.

after 2 h the reaction product was crystallized from water and it formed beautiful deep red needles. M.P. 172–173 °C, yield 80 %, TLC (benzene:chloroform, 2:8) Rf: 0.73. IR (KBr, tmax cm-1): 3367.82 (N–H str.), 1683.91, 1576.54 (C=O str. of carbonyl group), 3115.14 (Aromatic –C–H str.); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 11.46 (s, 2H, NH2), 6.77 (s, 1H, ethylene proton), 7.48–8.02 (m, 4H, Ar–H); 13C NMR (DMSO-d6) d (ppm): 101 (C3, ethylene proton), 127.44– 143.34 (Aromatic C, C5–C10), 174.64, 182.21 (C1–C2, C=O); Anal. Calc. for C10H7NO2: C, 69.36; H, 4.07; N, 8.09. Found: C, 69.41; H, 4.02; N, 8.12 (cf. Fig. 2).

Procedure for preparation of 4-amino-1, 2-naphthoquinone (ANQ) Solution of 0.01 mol of 1,2-naphthoquinone in 15 ml of glacial acetic acid at 40 °C was treated with a solution of 0.017 mol of sodium azide in 5 ml of water. Gas was evolved; the solution became reddish brown in color, and

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General procedure for synthesis of schiff bases [4-(arylideneamino)naphthalene-1,2-dione (S01–S16)]

(Aromatic-C, C5–C10 C10 –C60 ), 156.25 (C=N–), 178.58, 180.04 (C1–C2, C=O) (cf. Fig. 2).

A mixture of equimolar quantities of substituted aryl aldehydes (0.01 mol) and 4-amino-1,2-naphthoquinone (0.01 mol) in ethanol/methanol (20 ml) was refluxed on a water bath for 6–10 h in the presence of few drops of glacial acetic acid as catalyst. The progress of reaction was monitored by TLC at appropriate time interval. After completion of reaction, the solution was cooled, separated solid was filtered and washed with ice-cold water and dried. Finally, the product thus obtained was recrystallized from ethyl acetate and ethanol in different proportions depending on the nature of the compound. Physicochemical data and results of elemental analysis of the synthesized Schiff bases are listed in Table 1. The spectral data of all the compounds are given below.

4-(4-Hydroxybenzylideneamino)naphthalene-1,2-dione (S04)

4-(Benzylideneamino)naphthalene-1,2-dione (S01) TLC (methanol:chloroform, 2:8) Rf: 0.62. IR (KBr, tmax cm-1): 1638.84 (C=N, azomethine), 3159.83 (Aromatic C–H str.), 1701.39, 1688.49 (C=O str. of carbonyl group), 1538.54 (Aromatic C=C str.); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 6.52 (s, 1H, C=CH– group), 7.38–7.95 (m, 9H, Ar–H), 8.34 (s, 1H, –CH=N– group); 13C NMR (DMSO-d6) d (ppm): 124.52–138.72 (Aromatic-C, C5–C10 C10 –C60 ), 139.78 (C=N) 180.43, 181.23 (C1–C2, C=O) (cf. Fig. 2).

TLC (methanol:chloroform, 1:9) Rf: 0.73. IR (KBr, tmax cm-1): 3138.79, 3056.69 (Aromatic C–H str.), 1621.83 (C=N, azomethine), 1703.65, 1683.33 (C=O str. of carbonyl group), 3451.27 (O–H str.); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 6.71 (s, 1H, C=CH– group), 7.12–7.91 (m, 8H, Ar–H), 8.21 (s, 1H, –CH=N– group), 9.61 (s, 1H, –OH); 13C NMR (DMSO-d6) d (ppm): 123.99–140.16 (Aromatic-C, C5–C10 C10 –C60 ), 151.20 (C=N–), 179.14, 182.81 (C1–C2, C=O) (cf. Fig. 2). 4-(4-Methoxybenzylideneamino)naphthalene-1,2-dione (S05) TLC (methanol:chloroform, 1:9) Rf: 0.57. IR (KBr, tmax cm-1): 3085.88 (Aromatic C–H str.), 2935.43 (C–H str., O–CH3 group), 1641.28 (C=N, azomethine), 1705.54, 1678.87 (C=O str. of carbonyl group); 1H NMR (DMSOd6, 300 MHz) d (ppm): 3.81 (s, 3H, OCH3), 6.57 (s, 1H, C=CH– group), 7.19–7.95 (m, 8H, Ar–H), 8.07 (s, 1H, HC=N group); 13C NMR (DMSO-d6) d (ppm): 55.67 (OCH3), 121.19–150.3 (Aromatic-C, C5–C10 C10 –C60 ), 154.90 (C=N–), 177.87, 179.51 (C1–C2, C=O) (cf. Fig. 2).

4-(4-Nitrobenzylideneamino)naphthalene-1,2-dione (S02)

4-(2-Chlorobenzylideneamino)naphthalene-1,2-dione (S06)

TLC methanol:chloroform, 2:8) Rf : 0.67. IR (KBr, tmax cm-1): 1631.83 (C=N, azomethine), 3129.50 (Aromatic C–H str.), 1359.50 (N=O str.) 1707.69, 1679.30 (C=O str. of carbonyl group); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 6.44 (s, 1H, C=CH– group), 7.33–8.07 (m, 8H, Ar–H), 8.53 (s, 1H, –CH=N– group); 13C NMR (DMSOd6) d (ppm): 123.99–140.16 (Aromatic-C, C5–C10 C10 –C60 ), 158.10 (C=N–), 179.14, 182.81 (C1–C2, C=O) (cf. Fig. 2).

TLC methanol:chloroform, 1:9) Rf: 0.61. IR (KBr, tmax cm-1): 3117.98, 3053.67 (Aromatic C–H str.), 1617.22 (C=N, azomethine), 1709.19, 1682.82 (C=O str. of carbonyl group), 1053.17 (C–Cl str.); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 6.62 (s, 1H, C=CH– group), 7.40–7.90 (m, 8H, Ar–H), 8.71 (s, 1H, HC=N– group); 13C NMR (DMSO-d6) d (ppm): 121.85–135.96 (Aromatic-C, C5–C10 C10 –C60 ), 154.85 (C=N–),181.04, 180.58 (C1–C2, C=O) (cf. Fig. 2).

4-(2,4-Dichlorobenzylideneamino)naphthalene-1,2-dione (S03)

4-(3-Bromobenzylideneamino)naphthalene-1,2-dione (S07)

TLC (methanol:chloroform, 1:9) Rf: 0.71. IR (KBr, tmax cm-1): 3115.93, 3033.61 (Aromatic C–H str.), 1634.63 (C=N, azomethine), 1705.25, 1681.66 (C=O str. of carbonyl group), 1107.19, 1046.55 (C–Cl str.); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 6.62 (s, 1H, C=CH– group), 7.26–8.08 (m, 7H, Ar–H), 8.59 (s, 1H, –CH=N– group); 13C NMR (DMSO-d6) d (ppm):122.14–135.06

TLC (methanol:chloroform, 1:9) Rf: 0.63. IR (KBr, tmax cm-1): 3108.54, 3023.18 (Aromatic C–H str.), 1636.47 (C=N, azomethine), 1702.91, 1679.52 (C=O str. of quinone), 771.55 (C–Br str.); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 6.64 (s, 1H, C=CH– group), 7.22–8.37 (m, 8H, Ar–H), 8.81 (s, 1H, HC=N– group); 13C NMR (DMSO-d6) d (ppm): 123.12–142.62 (Aromatic-C, C5–C10 C10 –C60 ), 157.52 (C=N–), 180.27, 181.78 (C1–C2, C=O) (cf. Fig. 2).

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Table 1 Analytical and physicochemical data of the synthesized compounds O

O O

N

O O

O

N

N

C R'

R

R1

Compounds R S01

H

S02

H

S18

S17

S01-S16

Molecular formula M.W.a M.P. (°C)b Yield (%) % Analysis of C H and N found (calc)c C, H, N C17H11NO2

261

154–156

51

78.11 (78.15), 4.21 (4.24), 5.33 (5.36)

C17H10N2O4

306

142–143

65

66.69 (66.67), 3.27 (3.29), 9.12 (9.15)

C17H9Cl2NO2

329

133–134

71

61.82 (61.84), 2.77 (2.75), 4.26 (4.24)

C17H11NO3

277

141–143

67

73.66 (73.64), 3.99 (4.00), 5.07 (5.05)

C18H13NO3

291

171–173

44

74.24 (74.22), 4.48 (4.50), 4.79 (4.81)

C17H10ClNO2

295

158–159

57

69.07 (69.05), 3.45 (3.41), 4.71 (4.74)

C17H10BrNO2

339

183–185

78

60.05 (60.02), 2.93 (2.96), 4.09 (4.12)

C18H13NO2

275

147–149

63

78.50 (78.53), 4.73 (4.76), 5.11 (5.09)

C19H16N2O2

304

127–129

74

74.95 (74.98), 5.33 (5.30), 9.17 (9.20)

C20H17NO5

351

139–141

72

68.39 (68.37), 4.91 (4.88), 4.03 (3.99)

NO2

S03

H Cl

Cl

S04

H OH

S05

H OCH3

S06

H

Cl

S07

H

Br

S08

H CH3

S09

H N

S10

H

OCH3

OCH3

OCH3

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Table 1 continued R1

Compounds R

S11

H

Molecular formula M.W.a M.P. (°C)b Yield (%) % Analysis of C H and N found (calc)c C, H, N OCH3

C19H15NO4

321

167–169

69

71.05 (71.02), 4.68 (4.71), 4.33 (4.36)

C17H10N2O4

306

179–181

41

66.64 (66.67), 3.31 (3.29), 9.12 (9.15)

C18H13NO4

307

147–148

59

70.31 (70.35), 4.24 (4.26), 4.58 (4.56)

C17H11NO3

277

123–125

77

73.67 (73.64), 3.98 (4.00), 5.08 (5.05)

C19H12N2O2

300

189–190

62

76.01 (75.99), 4.06 (4.03), 9.35 (9.33)

C19H13NO2

287

204–205

78

79.39 (79.43), 4.59 (4.56), 4.91 (4.88)

C16H15NO2 C15H13NO2

253 239

152–153 168–170

64 55

75.85 (75.87), 5.94 (5.97) 5.57 (5.53) 75.28 (75.30), 5.51 (5.48), 5.88 (5.85)

OCH3

S12

H

S13

H

O2N

OCH3

OH

S14

H

S15

H

HO

N H

S16

H

S17 S18

– –

a

– –

Molecular weight (M.W.) of the compound

b

Melting point (M.P.) of the compounds at their decomposition

c

Elemental analysis for C, H, and N were within 0.04 % of the theoretical value

4-(4-Methylbenzylideneamino)naphthalene-1,2-dione (S08) TLC (ethylacetate:hexane, 7:3) Rf: 0.54. IR (KBr, tmax cm-1): 2889.61 (C–H str. of CH3 group), 3048.84 (Aromatic C–H str.), 1633.90 (C=N, azomethine), 1701.73, 1669.80 (C=O str. of carbonyl group); 1H NMR (DMSOd6, 300 MHz) d (ppm): 2.88 (s, 3H, –CH3), 6.61(s, 1H, C=CH– group), 7.08–7.89 (m, 8H, Ar–H), 8.18 (s, 1H, HC=N– group); 13C NMR (DMSO-d6) d (ppm): 21.10 (–CH3), 101.03–134.25 (Aromatic-C, C5–C10 C10 –C60 ), 152.10 (C=N–), 179.58, 182.21 (C1–C2, C=O) (cf. Fig. 2). 4-(4-(Dimethylamino)benzylidene amino)naphthalene-1, 2-dione (S09) TLC (ethylacetate:hexane, 7:3) Rf: 0.51. IR (KBr, tmax cm-1): 1338.64 (–C–N stretch), 2881.06 (C–H str. of CH3 group), 3066.35 (Aromatic C–H str.), 1620.26 (C=N,

azomethine), 1721.40, 1681.49 (C=O str. of carbonyl group); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 3.29, 3.31 (d, 6H, CH3), 7.11–7.83 (m, 8H, Ar–H), 8.27 (s, 1H, – HC=N– group); 13C NMR (DMSO-d6) d (ppm): 43.2, 44.19 (2CH3–), 111.57–136.96 (Aromatic-C, C5–C10 C10 – C60 ), 148.05 (C=N–), 179.66, 180.14 (C1–C2, C=O) (cf. Fig. 2). 4-(3,4,5-Trimethoxybenzylideneamino)naphthalene-1, 2-dione (S10) TLC (methanol:chloroform, 1:9) Rf: 0.49. IR (KBr, tmax cm-1): 3184.91, 3124.54, 3089.52 (Aromatic C–H str.), 2924.18, 2854.58, 2804.17 (C–H str., O–CH3 groups), 1632.91 (C=N, azomethine), 1701.27, 1687.47 (C=O str. of carbonyl group); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 3.86 (t, 9H, OCH3), 7.11–7.96 (m, 6H, Ar–H), 8.23 (s, 1H, HC=N–group); 13C NMR (DMSO-d6) d (ppm): 57.14, 58.66, 58.94 (OCH3), 116.36–139.11 (Aromatic-C, C5–C10

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C10 –C60 ), 149.87 (C=N–), 177.82, 182.14 (C1–C2, C=O) (cf. Fig. 2).

C10 –C60 ), 153.97 (C=N–), 176.58, 182.15 (C1–C2, C=O) (cf. Fig. 2).

4-(3,4-Dimethoxybenzylideneamino)naphthalene-1,2-dione (S11)

4-((1H-indol-3-yl)methyleneamino)naphthalene-1,2-dione (S15)

TLC (methanol:chloroform, 1:9) Rf: 0.52. IR (KBr, tmax cm-1): 3071.49 (Aromatic C–H str.), 2873.59, 2804.76 (C–H str., O–CH3 groups), 1638.25 (C=N, azomethine), 1707.48, 1668.13 (C=O str. of carbonyl group);1H NMR (DMSO-d6, 300 MHz) d (ppm): 3.88 (d, 6H, OCH3) 6.91– 7.81 (m, 7H, Ar–H), 8.28 (s, 1H, –HC=N– group); 13C NMR (DMSO-d6) d (ppm): 55.57, 55.81 (OCH3), 113.31– 147.11 (Aromatic-C, C5–C10 C10 –C60 ), 154.11 (C=N–),179.87, 181.51(C1–C2, C=O) (cf. Fig. 2).

TLC (methanol:chloroform, 1:9) Rf: 0.68. IR (KBr, tmax cm-1) 3109.35 (Aromatic C–H str.), 1620.26 (C=N str., azomethine), 1703.40, 1682.25 (C=O str. of carbonyl group), 3290.67 (–N–H str.); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 11.51 (s, 1H, Indole NH), 7.07–7.82 (m, 9H, Ar–H), 8.48 (s, 1H, –HC=N– group);13C NMR (DMSO-d6) d (ppm): 111.58–136.70 (Aromatic-C), 147.11 (C=N–), 179.56, 180.47 (C1–C2, C=O) (cf. Fig. 2).

4-(2-Nitrobenzylideneamino)naphthalene-1,2-dione (S12) TLC (methanol:chloroform, 1:9) Rf: 0.65. IR (KBr, tmax cm-1): 3142.11, 3123.59 (Aromatic C–H str.), 1346.24 (–N=O str.), 1635.69 (C=N, azomethine), 1718.39, 1682.34 (C=O str. of carbonyl group); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 6.92 (s, 1H, C=CH– group), 7.60–8.61 (m, 8H, Ar–H), 8.82 (s, 1H, –HC=N– group); 13C NMR (DMSO-d6) d (ppm): 123.10–140.06 (Aromatic-C, C5–C10 C10 –C60 ), 155.07 (C=N–),178.34, 182.31 (C1–C2, C=O) (cf. Fig. 2). 4-(4-Hydroxy-3-methoxybenzylideneamino)naphthalene1,2-dione (S13) TLC (methanol:chloroform, 1:9) Rf: 0.69. IR (KBr, tmax cm-1): 3059.20, 3032.24 (Aromatic C–H str.), 1638.73 (C=N, azomethine), 1713.25, 1668.13 (C=O str. of carbonyl group), 3484.12 (O–H str.), 2871.90 (C–H str., O–CH3 group); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 10.24 (br, s, 1H, –O–H group), 3.80 (s, 3H, OCH3), 6.96–7.72 (m, 7H, Ar–H), 8.27 (s, 1H, –HC=N– group); 13C NMR (DMSO-d6) d (ppm): 56.62 (OCH3), 127.29–139.72 (Aromatic-C, C5–C10 C10 –C60 ), 147.73 (C=N–), 178.29, 180.17(C1–C2, C=O) (cf. Fig. 2). 4-(2-Hydroxybenzylideneamino)naphthalene-1,2-dione (S14) TLC (methanol:chloroform, 1:9) Rf: 0.71. IR (KBr, tmax cm-1): 3128.23, 3041.81 (Aromatic C–H str.), 1635.84 (C=N, azomethine), 1707.48, 1681.13 (C=O str. of carbonyl group), 3481.23 (O–H str.); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 10.18 (s, 1H, –O–H group), 7.20–7.89 (m, 8H, Ar–H), 8.43 (s, 1H, –HC=N– group); 13C NMR (DMSO-d6) d (ppm): 119.45–136.16 (Aromatic-C, C5–C10

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4-((3-Phenylallylidene)amino)naphthalene-1,2-dione (S16) TLC (methanol:chloroform, 1:9) Rf: 0.51. IR (KBr, tmax cm-1): 3164.19, 3051.49 (Aromatic C–H str.), 2962.76, 2834.59 (C–H str. of allyl group), 1640.12 (C=N str., azomethine), 1711.24, 1683.52 (C=O str. of carbonyl group); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 7.50– 8.29 (m, 9H, Ar–H), 8.61 (s, 1H, –HC=N– group), 6.63– 6.82 (dd, 2H, –CH=CH–); 13C NMR (DMSO-d6): d (ppm): 123.06–137.46 (Aromatic-C), 152.12 (N=CH– group) 174.60, 182.14 (C1–C2, C=O) (cf. Fig. 2). General procedure for synthesis of schiff bases from cyclic ketones [4-(cyclohexylidene/cyclopentylidine amino)naphthalene-1,2-dione (S17–S18)] For the preparation of schiff bases with cyclic ketones, ethanolic solution of 4-amino-1,2-naphthoquinone (0.01 mol) was taken in 100-ml round bottom flask, to this equimolar (0.01 mol) ethanolic solution of respective cyclic ketones were added and few drops of conc. H2SO4 were added as catalyst. The solution was allowed to reflux on a water bath for 8–10 h. The solution was poured on to the crushed ice, neutralized with K2CO3 and kept aside. The precipitate thus separated was collected by filtration. 4-(Cyclohexylideneamino)naphthalene-1,2-dione (S17) TLC (ethylacetate:hexane, 7:3) Rf: 0.62. IR (KBr, tmax cm-1): 3121.23–3038.31 (Aromatic C–H str.), 2932.28– 2848.81 (C–H str. of cylohexyl group), 1619.83 (C=N str., azomethine), 1713.27, 1679.61 (C=O str. of carbonyl group); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 1.80– 2.37 (m, 10H, cyclohexylidene–H), 6.50 (s, 1H, C=CH– proton, C4), 7.40–8.09 (m, 4H, Ar–H); 13C NMR (DMSOd6) d (ppm): 23.57–34.51 (Cyclohexyl-C), 125.94–136.16 (Aromatic-C), 150.65 (C=N group), 181.04, 180.58 (C1–C2, C=O) (cf. Fig. 2).

Med Chem Res (2013) 22:1604–1617

4-(Cyclopentylideneamino)naphthalene-1,2-dione (S18) TLC (ethylacetate:hexane, 7:3) Rf: 0.57 .IR (KBr, tmax cm-1): 3087.14, 3058.54 (Aromatic C–H str.), 2924.17, 2843.88 (C– H str. of cylopentyl group), 1637.73 (C=N str., azomethine), 1702.29, 1677.47 (C=O str. of carbonyl group); 1H NMR (DMSO-d6, 300 MHz) d (ppm), 1.46–1.75 (m, 8H, cyclopentylidene–H), 6.61 (s, 1H, C=CH– proton, C4), 7.40–8.61 (m, 4H, Ar–H); 13C NMR (DMSO-d6) d (ppm): 23.66–31.50 (Cyclopentyl-C), 123.93–134.20 (Aromatic-C), 143.37 (C=N group), 177.60, 179.14 (C1–C2, C=O) (cf. Fig. 2).

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preferences at the active site of the receptor. The computation was carried out using Schro¨dinger 2011 molecular modeling software package. Docking was performed by using the Glide 5.7 integrated with Maestro 9.2 (Schro¨dinger, LLC, 2011) interface on the Linux operating system. Crystal structure selection

Anticancer activity

The starting coordinates of the human Topoisomerase-II ATPase (TP-II)–AMP-PNP complex [PDB:1ZXM] was taken from the Protein Data Bank (www.rcsb.org) and further modified for docking calculations.

Maintenance of cancer cell lines

Preparation of ligands

The cell lines MCF-7 (human breast cancer), Hep-G2 (liver sarcoma), MG-63 (Osteosarcoma), were obtained from National Centre for Cell Sciences Repository, Pune, India. The cells were maintained in Dulbecco’s minimum essential medium (DMEM) with 10 % fetal calf serum and 0.1 % antibiotic solution (complete medium-CM) at 37 °C in humidified air containing 5 % CO2 in the CO2 incubator, Thermo Scientific Heraeus Cytoperm.

A compound library of synthesized Schiff bases were built on Maestro 9.2 build panel and minimized in Schro¨dinger using the parental structure of doxorubicin as a template by Lig Prep 2.5 version v25111 (Schro¨dinger, LLC., USA) which generates a single low energy 3D structure with correct chiralities for each successfully processed input structure based upon the OPLS 2005 molecular mechanics force field.

Preparation of samples for cell line testing

Docking

The compounds S01–S18 were dissolved in DMSO and then diluted with complete medium-CM to obtain different concentrations of the compounds (1, 2.5, 5, 10, 15, 20, 25 lM).

The protein (TP-II complex) was optimized with the ‘‘protein preparation wizard’’ workflow by subjecting a cycle of constrained minimization steps allowing a maxi˚ from mum root mean square deviation (RMSD) of 0.30 A the original structure. For Glide (Schro¨dinger) calculations, TP-II complex was imported to Maestro (Schro¨dinger), the co-crystallized ligands were identified and removed from the structure. The ligands were docked with the binding site using the ‘standard precision’ Glide algorithm in Schrodinger.

Testing of compounds on various cell lines The cells were seeded in a 96-well plate at the density of 20,000 cells per well for MCF-7, 25,000 cells per well for Hep-G2 and 15,000 cells per well for MG-63 and allowed to attach for 24 h. A range of concentrations of the compounds diluted in CM were added to the cells and incubated at 37 °C in a CO2 incubator for 48 h. Triplicate wells were prepared for each individual dose. After the incubation, the cells were visualized using an Olympus microscope. All experiments were carried out in laminar flow hoods, Laminar Flow Ultraclean Air Unit, Microfilt, India. After completion of treatment, MTT solution was added to the monolayers and incubated for 4 h at 37 °C. The MTT reaction was terminated by the addition of 0.04 N HCl in isopropanol. The MTT formazan formed was measured on ELISA plate reader at 540 nm.

Pharmacokinetic predictions The molecules were also subjected to Qikprop analysis to predict the in silico pharmacokinetic properties. The QikProp 3.4 (V34111) program predicts both physically significant descriptors and pharmaceutically relevant properties.

Result and discussion Chemistry

In silico studies Molecular docking studies were carried out on the synthesized compounds to get insight about their binding

IR spectrum of synthesized compounds showed C=O stretching bands in the range of 1,710–1,580 cm-1 that confirm the presence of carbonyl groups in quinone ring system. In 13C NMR

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presence of peaks at 174.64 –182.41 also confirm the presence of two carbonyl groups in 1,2-naphthoquinone ring system. The FT-IR spectra of Schiff bases of 4-amino-1,2-naphthoquinone showed absorption bands at 3,030–3,170 cm-1 for aromatic C–H and at 1,590–1,650 cm-1 for azomethine group (–CH=N–). Formation of Schiff bases also confirmed by the disappearance of N–H stretching (3,130–3,400 cm-1) of 4-amino-1,2-naphthoquinone. 1H NMR also showed a characteristic singlet peak in the range of 8.07–8.82 d ppm which also confirms the presence of azomethine proton (–CH=N–). The appearance of multiplets at d 6.9–8.6 confirms presence of aromatic protons. Moreover, 13C NMR spectra showed peak in the range of 143.37–160.00 ppm which was due to azomethine (–CH=N–) carbon. The multiplets at d 3.8–3.9 ppm indicated the presence of –OCH3 groups of the benzene ring which was also confirmed by presence of peaks in the range of d 55.57–58.94 ppm in 13C NMR spectra of schiff bases having methoxy groups. Resonating position of the azomethine proton is strongly affected by the electro-negativity of the substituent’s group on adjoining benzene ring. It is noteworthy that the downfield chemical shift (d 8.58–8.82 ppm) corresponds to the azomethine proton was observed in compounds having electron withdrawing substituents (Cl, Br, NO2) due to de-shielding of proton while upfield chemical shift (d 8.07– 8.43 ppm) was observed in compounds with electron donating substituents (OCH3, OH) due to shielding of proton.

Anticancer activity The cytotoxicity of compounds S01–S18 was evaluated against human breast adenocarcinoma (MCF-7), liver sarcoma (Hep-G2), and Osteosarcoma (MG-63) cell lines. Doxorubicin was taken as reference compound. The IC50 values derived from in vitro screening studies revealed that most of the compounds possess significant cytotoxicity against MCF-7 and Hep-G2 cancer cell lines (Table 2). However, a lesser activity was observed against MG-63 cancer cell line. Morphological changes in cells were observed on treatment with compounds and microscopic images after treatment with compound S10 are shown in Fig. 3. The results are represented graphically in Figs. 4, 5, 6, and 7. Compounds S09, S10, S11, S13, S17, and S18 showed significant cytotoxicity against all the cancer cells with IC50 values in the range of 5.91–13.31 lM. Compounds S01, S06, S07, S08, S14, and S16 were found to be significant for MCF-7 and Hep-G2 but non-significant for MG-63. Compounds S02 and S03 were found to be least active in the series. Structure–activity relationship (SAR) studies from the results of the antiproliferative activity revealed that

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Med Chem Res (2013) 22:1604–1617 Table 2 Docking score and IC50 of compounds S1–S18 in MCF-7, Hep-G2, and MG 63 cell lines Compounds

Docking score

IC50 (lM) MCF-7

Hep-G2

MG-63 [25

NQ

-4.34

16.92

15.89

ANQ

-5.23

14.8

15.68

24.6

S01 S02

-6.07 -6.16

13.75* 14.74*

11.47* 15.61ns

16.17ns 24.97ns

S03

-5.33

14.66*

15.73ns

24.31ns

S04

-6.82

11.12**

8.92**

S05

-5.80

11.16**

9.03**

S06

-5.92

14.28*

12.10*

17.12ns

S07

-5.27

14.63*

12.19*

17.34ns

S08

-5.90

12.61*

10.43*

15.05ns

S09

-6.71

10.59**

S10

-7.84

S11

-6.66

10.86**

S12

-6.21

14.4*

S13

-7.96

S14

-5.68

13.25*

S15

-6.11

11.33**

10.14*

13.85*

S16 S17

-5.84 -5.35

12.9* 9.3**

10.63* 7.37**

14.84ns 11.52*

S18

-5.45

9.64**

7.77**

11.88*

Dox

-8.06

2.67

1.69

7.65***

8.24**

7.72** 5.91** 9.15** 11.82* 6.37** 11.04*

13.64* 12.71*

12.91* 9.69** 13.31* 17.01ns 9.98** 16.05ns

3.17

The bold values revealed the focus on compounds having more significant activity against all the cell lines IC50 (lM): dose of the compound which caused 50 % reduction of cell survival. Values were calculated from dose–response curves for triplicate dose of each compound Level of significance checked by using one way ANOVA followed by Dunnett’s t test * P \ 0.05, ** P \ 0.01, *** P \ 0.001, ns: nonsignificant

conversion of amino group to azomethine group via schiff base formation enhances the activity. Schiff bases having electron donating substituents on phenyl ring and schiff bases of cyclic ketones were found to be more active than Schiff bases with electron withdrawing substituents. Statistical analysis Statistical analysis was performed using Graph Pad prism software, version 5 (Graph Pad software) by using nonlinear regression analysis, IC50 values were calculated from each set of triplicate wells.

Docking study Molecular docking studies were conducted to explore the interaction between target (ATPase domain of

Med Chem Res (2013) 22:1604–1617

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Fig. 3 Morphological changes in MCF-7 cells incubated for 48 h (a) negative control (b) with 10 lM concentration of compound S10. Morphological changes in Hep-G2 cells incubated for 48 h (c) negative control (d) with 10 lM concentration of compound S10. Morphological changes in MG-63 cells incubated for 48 h (e) negative control (f) with 10 lM concentration of compound S10. The cells were observed under phase contrast microscope 9100

Fig. 4 Effect of concentration (1–25 lM) of compounds S09, S10, S11, S13, and doxorubicin on viability of human breast cancer (MCF-7) cell line

Fig. 5 Effect of concentration (1–25 lM) of compounds S09, S10, S11, S13, and doxorubicin on viability of Osteosarcoma (MG-63) cell line

Topoisomerase-II) and synthesized compounds. Glide calculations were carried out with Impact version v57111 and the G (Glide)-scores are shown in Table 2.

As per glide prediction, compounds S04, S09, S10, S11, S13, were nicely accommodated in the ATPase domain of Topoisomerase-II and showed good interaction with TP-II.

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Fig. 6 Effect of concentration (1–25 lM) of compounds S09, S10, S11, S13, and doxorubicin on viability of liver sarcoma (Hep-G2) cell line

Med Chem Res (2013) 22:1604–1617

These results were matching with wet lab findings. It is evident that the compounds S10 and S13, which were predicted in silico to be the most active, has been observed the most active. As seen in Fig. 8, the carbonyl groups of 1,2-naphthoquinone ring system in S10 as the hydrophilic head is involved in hydrogen bonds formation with Asn 150 and Ser 149 and nitrogen of azomethine linkage of schiff base showed hydrogen bonds interaction with Ser 149. Compound S13 was having strong H bonds interactions with Asn 150, Ser 148, Ser 149, and Gly 164 (Fig. 9). As seen in Fig. 10 the standard drug doxorubicin showed hydrophilic binding interaction with Arg 98, Tyr 186, and Ser 148 and having the glide score -8.06, which is comparable with glide scores of compounds S10 (-7.84) and S13 (-7.96). Some compounds like S17 and S18 did not exhibit good glide scores but found to have significant activity. It is possible that these compounds may act through a different mechanism, and further studies are required to elucidate their mechanism of action. Pharmacokinetic predictions of the best fit molecules

Fig. 7 Comparative IC50 values of compound S09, S10, S11, S13, S17, S18, and doxorubicin against MCF-7, Hep-G2, and MG-63 cell lines

Fig. 8 Binding pose for compound S10 within the ATPase domain of Topoisomerase-II, active site amino acid residues, i.e., Asparagine (ASN) 150 and Serine (SER) 148 bind with oxygen of 1,2-naphthoquinone ring system, Serine (SER) 149 bind with nitrogen of azomethine linkage of Schiff base. Hydrogen bondings are shown as dashed yellow lines (Color figure online)

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Unfavorable ADME and toxicity properties have been identified as a major cause of failure for candidate molecules in drug development. Therefore with the objective of increasing the success rate of compounds reaching development, the in silico ADME prediction studies were performed on QikProp module of the software for ligands having comparable G-scores with doxorubicin (Sengupta et al., 2007). The best-fit ligands were neutralized before being used by QikProp and significant ADME properties were predicted for the best-fit molecules (Ligand Nos. S04, S09, S10, S11, and S13), consisting of principal descriptors and physiochemical properties with analysis of the log P

Med Chem Res (2013) 22:1604–1617

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Fig. 9 Binding pose for compound S13 within the ATPase domain of Topoisomerase-II, i.e., ASN 150 and SER 148 bind with oxygen of carbonyl group of 1,2naphthoquinone ring system, SER 149 bind with nitrogen of azomethine linkage and GLY 164 bind with hydroxyl group of Schiff base

Fig. 10 Binding pose for standard drug doxorubicin which shows binding interaction with Arginine (ARG) 98, Tyrosine (TYR) 186, and Serine (SER) 148 residue at the active site of ATPase domain of Topoisomerase-II

(octanol/water), % human oral absorption, CNS activity, and permeability through Madin-Darby Canine Kidney (MDCK) cells in nm/s, etc. (MDCK cells are considered to be a good mimic for the blood–brain barrier) (cf. Tables 3, 4). All the compounds were found to be non-toxic for CNS, value of permeability through MDCK cells is \500 in all cases so the compounds will not cross blood–brain barrier which is desirable. The predicted drug likeliness of the synthesized compounds follow the Lipinski ‘‘Rule of Five’’, all four parameter values for a compound, i.e., Log P \ 5, H-bond donors \5, H-bond acceptors \10 and molecular weight \500 suggested that the compounds might have good absorption or permeability properties (Moorthy et al., 2009).

Conclusion Schiff bases of 4-amino-1,2-naphthoquinone were synthesized and characterized successfully. All the synthesized compounds gave satisfactory analytical and spectroscopic data, which were in full accordance with their depicted structures. The results of in vitro antiproliferative screening of synthesized compounds revealed that the compounds S04, S05, S09, S10, S11, S13, S15, S17, and S18 showed significant cytotoxicity against all the cancer cell lines. Most of the compounds having significant cytotoxicity are also found to have nice glide scores which reveal the support of wet lab findings with docking studies. On the basis of in silico ADME studies, we concluded that these

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Med Chem Res (2013) 22:1604–1617

Table 3 Pharmacokinetic prediction of the best fit compounds by QikProp 3.4 Comp code

Percentage human oral absorption

QPlogPo/w

QPPMDCK

CNS

#Stars

DonorHB

AccptHB

Rule of five violation

S04

80.22

1.8

107.80

-2

0

1

5.75

0

S09

85.34

2.1

168.43

-2

0

0

6

0

S10

86.62

2.2

179.43

-2

0

0

7.25

0

S11

82.87

1.9

159.27

-2

0

0

6.5

0

S13

84.34

2.0

153.98

-2

0

1

6.5

0

Table 4 QikProp properties and descriptors S. No. 1

Descriptor % Human oral absorption

Description

Recommended range

It predicts human oral absorption on 0–100 % scale. The prediction is based on a quantitative multiple linear regression model. This property usually correlates well with human oral-absorption

[80 % is high \25 % is poor

2

QPlog Po/w

Predicted octanol/water coefficient

-2.0–6.5

3

QPPMDCK

Predicted apparent MDCK cell permeability in nm/s. MDCK cells are considered to be a good mimic for the blood–brain barrier. QikProp predictions are for non-active transport

\25 poor, [500 great

4

CNS

Predictive Central Nervous Activity on a -2 (inactive) to ?2 (active) scale

-2 to ?2

5

#Stars

Number of property or descriptor values that fall outside the 95 % range of similar values for known drugs. A large number of stars suggest that a molecule is less drug-like than molecules with few stars. The following are some of properties and descriptors are included in the determination of #stars: Molecular weight, dipole, QPlogPw, QPlogPo/w, QlogS, solvent accessible surface area (SASA), etc.

0–5

6

DonorHB

Estimated number of hydrogen bonds that would be donated by the solute to water molecules in an aqueous solution. Values are averages taken over a number of configurations, so they can be non-integer

0.0–6.0

7

AccptHB

Estimated number of hydrogen bonds that would be accepted by the solute from water molecules in an aqueous solution. Values are averages taken over a number of configurations, so they can be non-integer

2–20

8

Rule of five

Number of violations of Lipinski’s rule of five. The rules are: mol_MW \ 500, Q Plog Po/w \ 5, donorHB B 5, accptHB B 10. Compounds that satisfy these rules are considered drug-like

Maximum is 4

compounds can be used for second generation of development and needed for further modifications to obtain more effective anticancer compounds. Acknowledgments The authors are grateful to The Head, Department of Chemistry, Faculty of Science, Banaras Hindu University (BHU) Varanasi, India for providing the facilities of 1H NMR and 13C NMR spectroscopy. One of the author, Shubhanjali Shukla likes to thank Jawaharlal Nehru Memorial Fund, New Delhi for providing financial assistance to continue the research.

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