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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Vatsala et al.

World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 2.786

Volume 3, Issue 8, 1895-1914.

Research Article

ISSN 2278 – 4357

DESIGN, AN EFFICIENT ECOFRIENDLY SYNTHESIS OF SPIROOXINDOLE DERIVATIVES AND THEIR ANTICANCER ACTIVITY SUPPORTED BY MOLECULAR DOCKING STUDIES Yakaiah Erugu1, Bhavanarushi Sangepu1, Kanakaiah Varre1, Rajesh Pamanji2, Yashwanth Bomma2, Venkateswara Rao Janapala2, Vankadari Srinivasarao3, Parthasarathy Tigulla3, Vatsala Rani Jetti1* 1

Fluoroorganic division, Indian Institute of Chemical Technology, Hyderabad-500007, India. 2

Biology division, Indian Institute of Chemical Technology, Hyderabad -500007, India 3

Univeristy college of science, Osmania Univerisity, Hyderabad -500007, India.

Article Received on 20 May 2014, Revised on 24 June 2014, Accepted on 30 July 2014

ABSTRACT An efficient, operationally easy and ecofriendly one-pot three component

synthesis

of

novel

trifluoromethylpyranopyrazole-4-spiro-oxindole

6-amino-5-cyno-3derivatives

was

developed by the domino reaction of 3-trifluoromethyl-5-hydroxy *Correspondence for Author Dr. J Vatsala Rani Fluoroorganic division, Indian Institute of Chemical

pyrazole, isatin and malononitrile in water reflux at 80-85oC for 45 min. The major returns of this protocol are excellent yield, operational cleanness and formation of three new bonds in one operation. The

Technology, Hyderabad-

synthesized compounds were evaluated for in vitro cytotoxicity against

500607, India

cell lines U937 (human histiocytic lymphoma) and B16F10 (mouse mealanocarcinoma) by using MTT assay. IC50 values (µg/mL), which

is the concentration required to inhibit 50% of cell viability by the test compounds after exposure to cells, have been determined. An exploration of the docking studies of tested Spirooxindole derivatives was made to explain the observed variance in their anticancer activity. Keywords: Isatin, 5-Hydroxypyrazole, malononitrile, multicomponent reaction, anticancer activity and molecular docking.

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INTRODUCTION The isatin (1H-indole-2,3-Dione) moiety is responsible for a broad spectrum of biological properties in many synthetically versatile molecules. Among these properties, cytotoxic and antineoplastic activities of this moiety have been found to be interesting.[1-5] Cancer has become the second cause of mortality in the world and the development of effective and specific anticancer agents is urgently needed because of the problems like severe toxicity as well as resistance with the existing drugs. Anticancer agents exert their biological effect usually by targeting various intracellular targets; therefore, current research mainly focuses on therapeutic targets involved in cell proliferation. However, identification of the exact target for particular class of compounds for their anticancer activity is one of the challenges to improve efficacy. Spirooxindole derivatives have been found to exhibit anticancer activity by interacting with different intracellular targets.[6,7] The indigoids are a group of isatins that have lately emerged as a promising scaffold for anticancer activity. Furthermore, indole substituted heterocycles at the 3-position are present as an interesting array of bioactive natural products and pharmaceutical compounds.[8,9] The isatin frame work bearing spiro cyclic quaternary stereo-center at the C3 position has significant advantages this heterocyclic scaffold, appears in a excess of natural alkaloids such as horsifikine, spirotryprostatine A and B, elacomine etc. (Fig.1) and also acts as effective non-peptide inhibitor of the p53-MDM2 interactions.[10]

Isatin incorporating a quaternary carbon center at the C3 position are

omnipresent in nature and exploited as building blocks for alkaloid synthesis as well as development of possible therapeutic agents.[11] Condensed heterocyclic compounds restraining a isatin nucleus or a 4H-pyrano fragment have diverse pharmacological activities.[12] Spirooxindole compounds were evaluated for in vitro cytotoxicity against a panel of five human cancer cell lines including lung (A-549), CNS (SK-N-SH), breast (MCF7), liver (Hep-2) and prostate (DU-145) by using MTT assay.[13] Multi-component reactions (MCRs) and the methods to improve the reactions are of extensive interest in the present day research. Multi-component reactions (MCRs) have emerged as a powerful tool for the construction of novel and complex molecular structures due to their advantages over conventional multi-step synthesis.[14] The major advantages of MCRs include lower costs, shorter reaction times, high atom-economy, energy saving, and the avoidance of time consuming and expensive purification processes. It is established that MCRs are generally much more environmentally friendly, and offer rapid access to large compound libraries with diverse functionalities. As a one-pot reaction, allow rapid access to

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compound molecules particularly in drug discovery.[15] The synthesis of heterocyclic’s bearing spiro-oxindole unit by designing new sequencing MCRs (multi-component reactions) with transformations like cyclization, and construction of new functional groups is a very useful process for developing molecular architectures.[16] The utility of organic synthesis in water has received a great deal of interest due to their relatively non-toxic, ecofriendly and readily available cheap as compared to other conventional organic solvents and also more stability to air properties. The synthetic convenience of carrying out one-pot MCRs

has

additional significance when the reactions are carried out in water.[17] When organic compounds are floating in water their relative insolubility causes them to combine, diminishing the water hydrocarbon interfacial area.[18] In other words the hydrophobic effect of water creates internal pressure and promotes the association of the reactants in the solvent cavity during the activation processes and accelerates the reaction. In this paper we wish to report an efficient and convenient synthesis of trifluoromethylpyranopyrazole substituted spiro-oxindole derivatives via 3-trifluoromethyl-5-hydroxy pyrazole, isatin and malononitrile in water at reflux condition for 45 min to produce good yields of the products.

Fig 1: Some biologically active isatin containing drugs MATERIALS AND METHODS All the substituted pyrazoles (1) were prepared by the known method which involves reaction of the corresponding ethyltrifluoroacetoacetate with phenyl hydrazine in the presence of aq HCl catalyst using EtOH as the solvent. 4-Fluorophenyhydrazine hydrochloride, isatine and ethyltrifluoroacetoacetate (ETFAA) were purchased from M/s Sigma Aldrich (St. Louise,

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USA) and malanonitrile M/s Lancaster (India). Melting points of all the compounds were recorded on Casiae–Siamia (VMP-AM) melting point apparatus and are uncorrected. IR spectra were recorded on Perkine-Elmer FT-IR 240-C spectrophotometer using KBr optics. 1

H NMR and 13C NMR spectra were recorded on Bruker AV 300 MHz and Inova 400 MHz

spectrometer in DMSO-d6 using TMS as internal standard. Electron Spray ionization (ESI) spectra was recorded on QSTARXL hybrid MS/MS system (Applied Biosystems,USA) under electrospray ionization. All the reactions were monitored by thin layer chromatography (TLC) on precoated silica gel 60 F254 (mesh) spots were visualized using UV light.

Scheme1: Synthesis of trifluoromethylpyranopyrazole substituted spiro-oxindole derivatives ( 4a-l ).

Scheme 2: Reaction Mechanism of spiro-oxindole derivatives

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Table 1: The spirooxindole derivatives (4a-l) are synthesized; yields were 90-96%. and results shown in table 1

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EXPERIMENTAL General procedure for the synthesis of spiro-oxindole derivatives (4a-l): The reaction mixture of 3-trifluoromethyl-5-hydroxypyrazole 1 (1.0 mol), isatin 2 (1.0 mol), and malononitrile 3 (1.0 mol) in water reflux at 80-85oC for 45 min. After completion of the reaction the solid product was filtered off, washed with water, dried and recrystallized from ethanol. All compounds were identified by the spectral data. 6'-amino-1'-(4-fluorophenyl)-5-nitro-2-oxo-3'-(trifluoromethyl)-1'H-spiro[indoline-3,4'pyrano[2,3-c]pyrazole]-5'-carbonitrile: (4a) Brown crystalline solid (Yield 94%). m.p 236238 °C 1H NMR (DMSO-d6, 300 MHz): 6.84(s, NH2, 2H), 7.09(d, J = 8.68 Hz, 1H), 7.25(d, J = 8.76 Hz, 2H), 7.80(m, 2H), 8.03(s, 1H), 8.24(d, J = 8.68 Hz, 1H), 11.18(s, NH, 1H); 13C NMR in DMSO-d6 (300MHz): δ 48.6, 59.4, 109.2, 115.8, 116.4, 117.8, 119.2, 122.8, 123.4, 126.2, 128.6, 133.2, 138.6, 144.2, 147.6, 156.2, 160.6, 168.4, 176.2; Vmax (KBr): 3399(NH), 3290, 3181(NH2), 2197(CN), 1703(CO), 1647, 1609, 1586, 1158; MS (m/z): 487 [M+H]. 6'-amino-5-bromo-1'-(4-fluorophenyl)-2-oxo-3'-(trifluoromethyl)-1'H-spiro[indoline-,4'pyrano[2,3-c]pyrazole]-5'-carbonitrile: (4b) Brown crystalline solid (Yield

89%). m.p

268-270 °C 1H NMR (DMSO-d6, 300 MHz): 6.87 (d, J = 8.30z, 2H), 7.14(s, NH2, 2H),7.25(d, J = 8.876 Hz, 2H), 7.68(s, 1H), 7.84(m, 2H), 10.69(s, NH, 1H);

13

C NMR in

DMSO-d6 (300 MHz): δ 49.4, 58.6, 115.2, 115.8, 116.6, 118.8, 123.6, 124.8, 130.2, 138.8, 133.6, 144.2, 139.2, 140.6, 154.2, 160.2, 168.0, 174.8; Vmax (KBr): 3410(NH), 3305, 3196 (NH2), 2190(CN), 1699(CO), 1645, 1525, 1140; MS (m/z): 521 [M+H]. 6'-amino-2-oxo-1'-phenyl-3'-(trifluoromethyl)-1'H-spiro[indoline-3,4'-pyrano[2,3c]pyrazole]-5'-carbonitrile: (4c) Brown crystalline solid (Yield 94 %). m.p 248-250 °C 1H NMR (DMSO-d6, 300 MHz): 6.94(d, J = 7.743 Hz, 2H), 7.01(s, NH2, 2H), 7.25(t, J = 7.54Hz, 1H), 7.44(t, J = 7.35Hz, 1H), 7.55(t, J = 7.743 Hz, 3H), 7.81(d, J = 8.12 Hz, 2H), 10.52(s, NH, 1H);

13

C NMR in DMSO-d6 (300 MHz): δ 48.9, 59.2, 115.8, 117.4, 119.0,

122.8, 123.2, 125.2, 126.6, 127.8, 128.4, 129.2, 137.6, 139.8, 140.2, 153.8, 167.2, 173.8; Vmax (KBr): 3406(NH), 3222, 3183(NH2), 2195(CN), 1706(CO), 1646, 1485, 1145. MS (m/z): 424 [M+H]. 6'-amino-5-bromo-2-oxo-1'-phenyl-3'-(trifluoromethyl)-1'H-spiro[indoline-3,4'pyrano[2,3-c]pyrazole]-5'-carbonitrile: (4d) Brown crystalline solid (Yield 92 %). m.p 234-236 °C

1

H NMR (DMSO-d6, 300 MHz): 6.89 (s, 2H, NH2), 6.97 (s, 1H), 6.94(d, J =

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7.46 Hz, 2H), 7.44(t, J = 7.365 Hz, 1H,), 7.54(t, J = 8.12 Hz, 2H), 7.80(d, J = 7.93 Hz, 2H), 10.52(s, 1H NH);

13

C NMR in DMSO-d6 (300 MHz): δ 46.8, 62.2, 116.8, 119.4, 122.0,

124.8, 125.5, 126.0, 128.8, 129.6, 130.2, 134.6 138.7, 140.3, 140.8, 154.8, 166.2, 174.2; Vmax (KBr): 3402(NH), 3236, 3193(NH2), 2198(CN), 1698(CO), 1649, 1625, 1532, 1150. MS (m/z): 503 [M+H]. 6'-amino-1'-(4-chlorophenyl)-2-oxo-3'-(trifluoromethyl)-1'H-spiro[indoline-3,4'pyrano[2,3-c]pyrazole]-5'-carbonitrile: (4e) Brown crystalline solid (Yield 258-260 °C

1

86 %). m.p

H NMR (DMSO-d6, 300 MHz): 6.88(d, J = 8.12 Hz, 1H), 7.01(s, 2H, NH2),

7.38(d, J = 6.61 Hz,1H), 7.45(d, J = 7.365 Hz, 1H), 7.55(t, J = 7.932 Hz, 2H), 7.62(d, J = 7.46 Hz, 1H), 7.81(d, J = 7.74 Hz, 2H), 10.66(s, 1H, NH); 13C NMR in DMSO-d6 (300 MHz): δ 49.2, 58.6, 115.2, 117.6, 119.2, 120.2, 124.5, 125.3, 127.6, 128.2, 130.2, 131.4, 136.2 139.7, 141.3, 154.8, 168.2, 172.4; Vmax (KBr): 3398(NH), 3296, 3195(NH2), 2203(CN), 1701(CO), 1647, 1609,1586, 1158. MS (m/z): 458 [M+H]. 6'-amino-1'-(4-chlorophenyl)-5-fluoro-2-oxo-3'-(trifluoromethyl)-1'H-spiro[indoline3,4'-pyrano[2,3-c]pyrazole]-5'-carbonitrile: (4f) Brown crystalline solid ( Yield

92 % ).

1

m.p 244-246 °C H NMR (DMSO-d6, 300 MHz): 6.93(d, J=7.36 Hz,1H), 7.13(d, J=7.17 Hz, 1H), 7.25(s, 1H), 7.40(s, 2H, NH2), 7.54(d, J=7.36 Hz,2H), 7.88(d, J=7.36 Hz, 2H), 10.63(s, 1H, NH).; Vmax (KBr): 3404(NH), 3321, 3185(NH2), 2195(CN), 1706(CO), 1643, 1525, 1485, 1145. MS (m/z): 475 [M+H]. 6'-amino-1'-(4-chlorophenyl)-5-nitro-2-oxo-3'-(trifluoromethyl)-1'H-spiro[indoline-3,4'pyrano[2,3-c]pyrazole]-5'-carbonitrile: (4g) Brown crystalline solid (Yield 256-258 °C

1

92 %). m.p

H NMR (DMSO-d6, 300 MHz): 6.87 (d, J= 8.30 Hz, 2H), 7.14(s, NH2,

2H),7.25(d, J= 8.876 Hz, 2H), 7.28(s, 1H), 7.84(m, 2H), 10.69(s, NH, 1H).; Vmax (KBr): 3398(NH), 3320, 3190(NH2), 2203(CN), 1717(CO), 1659, 1626, 1527, 1147. MS (m/z): 503 [M+H]. 6'-amino-5-nitro-2-oxo-1'-phenyl-3'-(trifluoromethyl)-1'H-spiro[indoline-3,4'-pyrano [2,3-c]pyrazole]-5'-carbonitrile: (4h) Brown crystalline solid (Yield °C

1

95 %). m.p 252-254

H NMR(DMSO-d6, 300 MHz): 7.10(t, J=8.67 Hz,1H), 7.16(s,2H, NH2), 7.53(t, J=8.49

Hz, 2H,), 7.84(d, J=8.50 Hz, 2H), 8.04(s, 1H), 8.23(m, 2H)11.26(s, 1H, NH).; Vmax (KBr): 3404(NH), 3321, 3185(NH2), 2195(CN), 1706(CO), 1643, 1525, 1485, 1145. MS (m/z): 469 [M+H].

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6'-amino-5-bromo-1'-(4-chlorophenyl)-2-oxo-3'-(trifluoromethyl)-1'H-spiro[indoline3,4'-pyrano[2,3-c]pyrazole]-5'-carbonitrile: (4i) Brown crystalline solid ( Yield m.p 240–241 °C

1

90 % ).

H NMR (DMSO-d6, 300 MHz): 7.13(d, J=8.68 Hz, 1H), 7.47(d, J=7.36

Hz, 1H), 7.54(s, 2H, NH2), 7.57(s, 1H), 7.85(d, J=7.93 Hz, 2H), 8.25(d, J=7.26 Hz, 2H), 11.38(s, 1H, NH)).; Vmax (KBr): 3404(NH), 3321, 3185(NH2), 2195(CN), 1706(CO), 1643, 1525, 1485, 1145. MS (m/z): 536 [M+H]. 6'-amino-1'-(4-fluorophenyl)-2-oxo-3'-(trifluoromethyl)-1'H-spiro[indoline-3,4'-pyrano [2,3-c]pyrazole]-5'-carbonitrile: (4j) Brown crystalline solid (Yield °C

1

89 %). m.p 236-238

H NMR (DMSO-d6, 300 MHz): 6.56(s, 2H, NH2), 6.86(d, J=8.30 Hz, 1H), 7.23(t,

J=7.17 Hz, 2H), 7.39(d, J=8.36 Hz, 1H), 7.49(d, J=8.87 Hz, 2H), 7.76(d, J=8.06 Hz, 2H), 10.42(s, 1H, NH).; Vmax (KBr): 3401(NH), 3326, 3182(NH2), 2196(CN), 1704(CO), 1645, 1525,1498, 1152. MS (m/z): 442 [M+H]. 6'-amino-5-fluoro-1'-(4-fluorophenyl)-2-oxo-3'-(trifluoromethyl)-1'H-spiro[indoline3,4'-pyrano[2,3-c]pyrazole]-5'-carbonitrile: (4k) Brown crystalline solid (Yield

92 %).

m.p 256-258 °C 1H-NMR-(300MHz)) in DMSO-d6: δ 6.88( s, 2H, NH2), 6.90(d, J=7.43 Hz 1H), 6.93(s, 1H), 6.94(d, J=7.43 Hz, 1H), 6.98 (d, J=7.86 HZ, 2H), 7.23(d, J=7.38 Hz, 2H,), 10.49(s, 1H, NH); Vmax (KBr): 3394(NH), 3324, 3190(NH2), 2201(CN), 1712(CO), 1650, 1628, 1526, 1148. MS (m/z): 460 [M+H]. 6'-amino-5-fluoro-2-oxo-1'-phenyl-3'-(trifluoromethyl)-1'H-spiro[indoline-3,4'-pyrano[ 2,3-c]pyrazole]-5'-carbonitrile: (4l) Brown crystalline solid (Yield 94 %). m.p 234-236 °C 1

H-NMR-(300MHz)) in DMSO-d6: δ 6.94( s, 2H, NH2), 6.98 (s, 1H), 6.90(d, J=7.62 Hz 1H),

6.89(d, J=7.23 Hz 1H), 7.45 (d, J=7.89 Hz, 2H), 7.54 (t, J=8.03 Hz, 3H,), 10.52 (s, 1H, NH). 13

C-NMR in DMSO-d6 (300MHz): δ 48.2, 58.4, 110.2, 113.4, 118.2, 119.0, 121.8, 123.4,

124.6, 128.9, 137.2, 137.5, 139.4, 152.9, 159.2, 168.2. Vmax (KBr): 3397(NH), 3310, 3190(NH2), 2203(CN), 1710(CO), 1652, 1626, 1527, 1148. MS (m/z): 442 [M+H]. PHARMACOLOGICAL SCREENING Cytotoxic Evaluation of Spirooxindole Derivatives on U937& B16F10 Cells. Cell Lines and Cell Culture The cell lines U937 (human histiocytic lymphoma) and B16F10 (mouse mealanocarcinoma) cell lines were obtained from the National Centre for Cellular Sciences (NCCS), Pune, India. Cells were cultured either in RPMI -1640 (U937) or DMEM (B16F10) media, supplemented

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with 10% heat-inactivated fetal bovine serum (FBS), 1mM NaHCO3, 2 mM -glutamine, 100 units/ml penicillin and 100 µg/mL streptomycin. All cell lines were maintained in culture at 37° C in an atmosphere of 5% CO2. Test Concentrations Initially, stock solutions of each test substances were prepared in 100% Dimethyl Sulfoxide (DMSO, Sigma Chemical Co., St. Louis, MO) with a final concentration of 8 mg/ml. Exactly 50µl of stock was diluted to 1 ml in culture medium to obtain experimental stock concentration of 400µg/ml. This solution was further serially diluted with media to generate a dilution series of 10µg to 200µg/ml. Precisely, 100 µl of each test concentration was added to 100 µl of cell suspension (total assay volume of 200 µl efficacy of the derivatives were evaluated with three different set of experiments) and incubated for 24h at 37 °C in 5% CO2. RESULTS AND DISCUSSION The synthesized spirooxindoles are potentially bioactive heterocyclic compounds. Herein we report the results of our investigation the condensation of 3-trifluoromethyl-1H-pyrazole-5-ol (1a-c), isatin (2a-d) and malononitrile (3) was refluxed for 45 min in water (without catalyst) to provide a desire product, the yields were 86-96%. Twelve derivatives of spirooxindole derivatives 4a-l are synthesized and results shown in table 1 and it can be applied to a variety of substrates, thus demonstrating wide scope of this methodology. The compounds were characterized to be novel fluorinated pyranopyrazole substituted spiro-oxindole derivatives, after completion of the reaction the solid product is isolable cleanly by filtration alone. A probable rationale for this catalyst-free reaction is outlined in Scheme1. Initially a cyanoolefin intermediate is formed by the knoevenagel condensation between 2 and 3 followed by Michel addition and cyclization in scheme2. The structures of the desire spirooxindole derivatives 4a-l were confirmed by the spectroscopic studies. In all cases the solid products were separated by filtration. The products were fully characterized by 1H and 13

C NMR, MS and IR spectrum.

BIOLOGICAL SCREENING Cytotoxic Cytotoxicity was measured using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay, according to the method of Mossman [19]. Briefly, the cells (2 x 104) were seeded in each well containing 100 µl of medium in 96 well plates. After overnight incubation at 37 °C in 5% CO2, exactly 100 µl of different test concentrations (10 µg to 200 www.wjpps.com

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µg/ml) were added to the cell suspension, which is equivalent to 2 to 40 µg per 20 0µl of assay volume. The viability of cells was assessed after 24 h, by adding 10 µl of MTT (5 mg/mL) per well and incubated at 37°C for additional three hours. The medium was discarded and the formazan blue, which formed in the cells, was dissolved in 100 µl of DMSO. The intensity of colour formation was measured at 570 nm in a spectrophotometer (Spectra MAX Plus; Molecular Devices; supported by SOFTmax PRO-5.4). The percent inhibition of cell viability was determined with reference to the control values. The data were subjected to linear regression analysis and the regression lines were plotted for the best straight-line fit. The IC50 (inhibition of cell viability) concentrations were calculated using the respective regression equation.[19] Exponentially growing cells were treated with different concentrations of spirooxindole derivatives for 24h and cell growth inhibition was analyzed through MTT assay. a

IC50 is defined as the concentration, which results in a 50% decrease in cell number as

compared with that of the control cultures in the absence of an inhibitor and were calculated using the respective regression analysis. The values represent the mean ± SE of three individual observations. All the synthesized compounds were evaluated for in vitro cytotoxicity against cell lines U937 (human histiocytic lymphoma) and B16F10 (mouse mealanocarcinoma) by using MTT assay. IC50 values (µg/mL), which is the concentration required to inhibit 50% of cell viability by the test compounds after exposure to cells, have been determined results in Table 2, and indicate that most of the compounds displayed anticancer potency against the cell lines tested in the assay. Table-2: In vitro cytotoxicity of Spirooxindole derivatives against human U937 and mouse B16F10 cancer cells by MTT assay. S.No 1 2 3 4 5 6 7 8 9 10

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Compound codes 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j

a

IC50 (µg/ml)

U937c 96.66±2.08 86.78±2.31 108.48±1.72 89.51±2.92 146.54±2.57 100.72±1.11 95.92±1.69 139.69±2.18 116.82±2.71 82.09±2.16

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B16F10d 73.92±0.89 30.18±0.53 40.37±1.01 134.29±2.84 24.66±0.17 34.55±0.43 20.59±0.95 50.56±1.16 34.35±0.29 70.43±0.52

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11 4k 12 4l Doxorubicin Etoposide

127.8±1.92 97.8±2.92 0.07±0.01 6.02±0.79

46.89±0.49 30.463±1.32 5.79±0.13 4.12±0.42

c- Human histolytic lymphoma d- Mouse mealanocarcinoma The activity of the sprirooxidole derivatives was prominent on cell line B16F10 than U937. The potency of the compound 4g was best when nitro group was present in spirooxindole moiety and a chlorine atom in pyrazole moiety. The potency of the compounds 4e, 4f and 4i decreased marginally when nitro group was absent or replaced by other halogen groups in oxindole but chlorine atom still retained in pyrazole moiety. The potency of the compound 4d was very low when chlorine atom was removed from pyrazole moiety. The results further indicate that the activity of the sprio-oxindole analogs depend on substituted pyrazole moiety. MOLECULAR MODELLING STUDIES In addition to the synthetic work, an exploration of the docking studies of tested Spirooxindole derivatives was made to explain the observed variance in their anticancer activity. This predicts the best drug candidate providing an insight into the substitutional and configurational requirements for optimum receptor pit which leads to the development of best pharmacophore activity. Compounds (4a-l) were constructed and energy-minimized with the Hyperchem 7.5 version.[20] The resulting structures were used for the docking study of the ATP binding site. Docking solutions with the ATP binding site were obtained with human topo II-α co-crystallized with phosphoraminophosphonic acid-adenylate ester, an ATP analogue, in the ATPase domain (PDB code 1ZXM, resolution 1.87 Å) was used.[21] The TOPO-II structure contains Mg2+ ions. The interaction of the ligand with the receptor in the modeled complexes is investigated and observed the binding energy function ability of hydrolase protein by different inhibitors. The 3D structure of selected Protein [Human TopoIIa ATPase (1ZXM)] was selected from PDB Bank RCSB[22] with an X-ray resolution in the range of 1.87Å. Among the proteins (1ZXM) was selected based on the literature. The Successful docking has been performed for the selected set of viz (4a-l) inhibitors and their corresponding binding energy values have been produced in the Argus Lab 4.0.1[23] is molecular modeling and docking software. Argus lab was used to visualize the binding conformations of these analogs within the active site of (1ZXM) protein and details are displayed in Fig 2. In the active site region (15Å) of (1ZXM) protein Ala167, Gly166, www.wjpps.com

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Ser148, Ser149, Asn91, Ile141, Thr147, Arg162,Asn163,Gly164, Tyr165, Gly166 amino acids can play important role and are shown in Fig 3. The binding energy values estimated by Argus Lab 4.0.1 was found to have a good correlation with the experimental anti cancer activity. Docking results revealed that all the molecules have comparable binding energy values. Among the Spirooxindole derivatives, compounds 4k, 4b, 4e & 4d showed best binding energy values with best interactions. The experimental inhibition values of these four compounds also supported with docking with respect to standard Doxorubicin in case of U937 cell lines. The compounds 4a, 4g & 4c showed moderate binding energy values, 4j, 4l, 4i & 4f showed low binding energy values which also correlated with experimental biological activity with respect to standard Doxorubicin.The results obtained from the docking studies also supported by the experimental anticancer activity values. The

binding orientations of

database ball cylinder low model with coloured amino acid residues compounds 4a, 4b, 4c, 4f, 4h, 4i, 4k & 4l with crystallographic conformation of active site (PDB ID 1ZXM). Hydrogen bonds are shown in red color dotted lines. These compounds were docked in the active site of protein, with a significant different binding mode. The binding energy values of these compounds (4a-l) of Spirooxindole derivatives were estimated by Argus Lab 4.0.1 and values was shown in Table 3. Table 3: Binding energy and Elapsed time values of Spirooxindole derivatives

Comp 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l

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Argus energy (K cal/mol) -5.8740 -7.7013 -4.6903 -7.10022 -7.6425 -3.4159 -5.5344 -5.91317 -3.6854 -3.9093 -8.06133 -3.7863

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4a active site area

4b active site area

4c active site area

(4a) B. Energy =-5.9131 Kcal/mol (4b) B. Energy =-5.5344 Kcal/mol (4c) B. Energy =-3.9093 Kcal/mol

4f active site area

4h active site area

4i active site area

(4f) B. Energy =-5.8740 Kcal/mol (4h) B. Energy =-3.6854 Kcal/mol (4i) B. Energy =-8.0613 Kcal/mol

4k active site area

4l active site area

(4k) B. Energy =-4.6903 Kcal/mol (4l) B. Energy =-7.1002 Kcal/mol Fig 2: Binding orientations of database ball cylinder low model compounds 4a, 4b, 4c, 4f, 4h, 4i, 4k & 4l with crystallographic conformation of active site (PDB ID 1ZXM). Hydrogen bonds are shown in red colour dotted lines.

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Fig 3: Active site amino acids of crystallographic protein (1ZXM) Calculation of drug-likeness properties: Drug-likeness can be considered as a delicate balance

among

the

molecular

properties

of

a

compound

that

influences

its

pharmacodynamics, pharmacokinetics and ultimately ADME (absorption, distribution, metabolism and excretion) in human body like a drug.[24] These parameters allow as certaining oral absorption, or membrane permeability that occurs when evaluated molecules obey Lipinski’s rule-of-five[25] other parameters that included are number of rotatable bonds, molecular volume, topological polar surface area, and. The above mentioned parameters were calculated for (4a-l) and the results were presented in Table 4. It was observed that all the compounds (4a-l) have optimum logP (