Synthesis and Biological Study of Novel Indole-3 ... - Ingenta Connect

Send Orders of Reprints at [email protected] Letters in Drug Design & Discovery, 2013, 10, 19-26

19

Synthesis and Biological Study of Novel Indole-3-Imine-2-on Derivatives as Src Kinase and Glutathione S-Transferase Inhibitors Z. Kılıç Kurta, D. Aydına, Y. G. görb, B. S. görb and S. Olgen*,a a

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ankara, 06100, Tandogan, Ankara, Turkey; bChemistry Group, Faculty of Enginerring, Atılım University, 06836, ncek, Ankara, Turkey Abstract: The aim of this study is to design and synthesize novel dual inhibitors of Src protein tyrosine kinase (PTK) and Glutathione S-transferases (GSTs), as a potential drug lead with therapeutic efficacy on cancer and immune disorders. The biological activity profiling of small molecule inhibitors via miniaturized biochemical techniques compatible with medium throughput screening and focused screening methodologies were performed. To determining the effects of small molecule inhibitors on Src kinase and Phase II detoxification enzyme GST isozymes in liver homogenates used to verify their roles in drug resistance mechanism for cancer chemotherapeutics. In this study, 14 indole-3-imine-2-on and N-benzyl indole-3-imine-2-on derivatives were synthesized for dual activities against Src and GST. The chemical structures and purities of compounds were verified by IR, 1H NMR, MASS spectroscopy, and elemental analysis. The compounds 2, 3 and 9 are found slightly active against both enzyme Src and GST.

Keywords: Cancer, Src tyrosine kinase; Isatine; Glutathion-S-transferase; Drug resistance; Synthesis. INTRODUCTION Src family kinases (SFKs), as members of nonreceptor tyrosine kinases (Blk, Brk, Fgr, Frc, Fyn, Hck, Lck, Lyn, Src, Srm, and Yes) control the signaling networks regulating metabolism, proliferation, differentiation and migration [1]. Among these proteins Src (c-Src) is the first discovered and studied non-receptor protein tyrosine kinases (PTKs), and also the protypical member of SFKs [2]. Despite the structural resemblance and highly conserved kinase domain of SFK enzymes, the studies shown the diversity in their tissue distribution: Src, Yes, Fyn, and Lyn are widely expressed throughout the organism, whereas Hck, Fgr, Lck and Blk are limited to lymphoid and myeloid tissues [3]. The SFKs, as being the largest family of PTKs, and especially the Src itself are essential part of cellular function including cytoskeletal alterations, differentiation, proliferation, cellcycle progression, adhesion, and migration in both normal and transformed cells [2, 4]. They are known to act as an integral part of signaling from receptors on cell surface to the relevant targets in nucleus: the recruitment of the relevant surface receptors upon growth factor, cytokine, integrin or immunologic stimulation activates the receptor associated tyrosine kinases that interact with downstream components of signaling [3, 5-8]. Being involved in various mechanisms in cellular functioning, deregulation of SFK signaling were found explicit in the development and progress of multiple human cancer types in tissues including breast, gastrointestinal system, lung, ovary, pancreas, prostate, and skin [6, 9-12]. Therefore SFK members became universal molecular targets and the efforts to discover novel small molecules against cancer. With an emphasis on combination therapies with standard chemotherapeutic agents, the dual *Address correspondence to this author at the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, 06100, Tandogan/Ankara, Turkey; Tel: +90 (312) 2033073; Fax: +90 (312) 2131081; E-mail: [email protected] 17-;/13 $58.00+.00

function of SFK inhibitors may occur by increasing the sensitivity of tumors to established chemotherapeutic agents and by preventing tumors to metastasize [12]. In the recent years, some novel substituted indolin-2-ones were also discovered as potent non-receptor tyrosine Src kinase inhibitors [13, 14]. Compounds I and II Fig. (1) inhibited Src with IC50 values of 10 nM and 70 nM, which were about 6-8 folds more potent than those to VEGFR. Studies on indole scaffold bearing inhibitor research revealed that compound III [15] has ability to inhibit Src with IC50 of 1.34 M Fig. (1). Another indole derivative compound IV [16] was found promising inhibitor for Src, with IC50 of 4.69 M Fig. (1). Screening for inhibitors of cell migration yielded several noteworthy small molecules, including indole derivative the Rho-kinase inhibitor Rockout Fig. (1), which inhibited disassembly of stress fibers in the human melanoma cell line M2 [17]. The drug resistance of tissues is also found associated with the Glutathione S-transferases (GSTs), a family of phase II detoxification system enzymes [18-20]. Many endogenous or xenobiotic lipophilic substances are eliminated from the cells by the sequence of oxidation, conjugation to an anionic group (glutathione, glucuronate or sulfate) and transport across the plasma membrane into the extracellular space [21]. GSTs are capable of recognize diverse chemical structures, catalyze their conjugation to glutathione, and hence reduce cytotoxic reactivity of those chemicals. These enzymes are generally considered to be detoxification of both endogenous and xenobiotic compounds; however, GSTs can also lead to the formation of more reactive intermediates and may cause the deregulated signaling which may predispose malignant transformation [22]. Besides catalyzing the inactivation of various electrophile-producing anticancer agents via conjugation to the glutathione, GSTs are promising negative regulator for oxidative stress and drug-induced cell apoptosis through the © 2013 Bentham Science Publishers

20 Letters in Drug Design & Discovery, 2013, Vol. 10, No. 1

Kurt et al.

HN O S O

O O N H

Cl

HN

HN

NH2

O S O

N

N H

O

N

N

PP2

N

O N

OCH2COOH Cl

O .HCl HN

N N

N H III

N

OH II

I

N N

IV

F

rockout

Cl COCHCH2CH3 CH3 Ethacrynic acid

Fig. (1). Potent Src kinase inhibitors.

interaction with specific signaling kinases. The major roles of glutathione (GSH) and glutathione S-transferases (GSTs) in the detoxification of xenobiotics predicts their important role in drug resistance [23]. As such, both GSH and GSTs have been manipulated as targets in the design of novel chemotherapeutic drugs. The discovery that GSTs have additional roles in the cell as regulatory molecules in the mitogen-activated protein kinase pathways together with the more recent discovery of GSH as a regulatory posttranslational modification lend further weight to their already important roles in the anticancer drug resistance response. These findings highlight the importance of these targets in the creation of future novel anticancer drugs. On the other hand, the multidrug resistance-associated proteins (MRPs) have potential role in clinical multidrug resistance. MRPs transfer the glutathione conjugates out of cells to prevent the accumulation of anionic conjugates and GSSG in the cytoplasm, and therefore, have critical and an essential role in defense against oxidative stres [24-26]. In addition, inhibition of Src tyrosine kinase has been shown to enhance the cytotoxicity of several anticancer drugs and restore sensitivity in drug-resistant cells [27]. As it known, GSTs have been implicated in the development of resistance towards chemotherapeutic agents. On the other hand, GSTs have emerged as a therapeutic target because spesific isozymes are overexpressed in a wide variety of tumors and may play a role in the etiology of other diseases, including neurodegenerative diseases, multiple sclerosis, and asthma [28]. Ethacrynic acid was reported as inhibitor of GST and might be used to overcome acquired resistance to alkylating agents Fig. (1), [29]. In this context, for effective therapeutic efficacy it seems critical to have dual targeting capacity of inhibitors aimed at a specific enzyme such as Src responsible in disease progress and those responsible in developing drug resistance in that target tissue [30]. Based on above information about the Src kinase, GST and drug resistance relationship from literature, indole derivatives were tested to generate effective dual inhibitors as Src kinase and GSTs.

MATERIALS AND METHOD Chemistry Anhydrous magnesium sulphate, sodium sulphate, hexane, ethyl acetate, anhydrous dimethylformamide, sodium dihydrogen phosphate, potassium hydrogen phosphate, sodium chloride, dimethylsulfoxide, sulfuric acid, mercaptoethanol, potassium chloride, EDTA (from Merck, Darmstadt, Germany); deutero dimethylsulfoxide, 4chloroaniline, 3-chloro-4-fluoroaniline (from Acros Organics, Geel, Belgium); methanol, hydrochloric acid, dichloromethane, anhydrous ethanol, isatin, 4-chloroisatin, sodium hydride, benzyl bromide, tween 20, 1-chloro-2,4dinitrobenzene, glutathione (from Sigma-Aldrich, St. Louis, MO, USA); Src kinase (Invitrogen, NY, USA) and ProFluor Src-family kinase assay kit, 8 plate (Promega, Madison, USA) were purchased. Melting points were measured with a capillary melting point apparatus (Electrothermal 9100, Essex, UK) and uncorrected. The Nuclear Magnetic Resonance (1H NMR) spectra were recorded on Varian Mercury 400 NMR spectrometer for 400 MHz (Varian Inc., Palo Alto, CA, USA). The chemicals shift values were expressed in parts per million (ppm) relative to tetramethylsilane as an internal standard and signals were reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet). Mass spectra were recorded on a Waters ZQ Micromass LC-MS spectrometer (Waters Corporation, Milford, MA, USA) Electrosprey Ionization (ESI) method. Infrared (IR) spectra were measured on Jasco FT/IR-420 (Jasco, Tokyo, Japan). Elemental analysis was taken on a Leco-932 CHNS-O analyzer (St. Joseph, MI, USA). Molecular Devices Spectra MAX 190 (from Molecular Devices Corporation, Sunnyvale, CA, USA) was used to measure of absorbance of phoshorylation reaction. Analytical TLC was carried out on Merck 0.2 mm precoated silica gel (60 F-254) aluminium sheets (from Merck, Darmstadt, Germany), with visualization by irradiation with a UV lamp. The flash column chromatography was accomplished on silica gel 60

Indole-3-imine-2-on Derivatives as Src and GST Inhibitors

(230-400 mesh, Merck, Darmstadt, Germany). The doseresponse curves of the compounds and non-linear regression analysis were performed using GraphPad Prism 4.0 (GraphPad Software for Windows, San Diego, California, USA). Synthesis of N-benzyl isatin and N-benzyl-5-chloro isatin Isatin and 5-chloro isatin (0.03 mol) was dissolved in DMF (6 ml) and the solution was cooled to 0oC. NaH (0.09 mol) was added and the mixture stirred for 15 min at 0oC and then 45 min at room temperature. Benzyl bromide (0.07 mol) was added dropwise to the rection mixture and stirred for 72 h at room temperature. It was diluted with water and extracted with EtOAc (3x50 ml). The combined organic phase was dried over anhydrous Na2SO4. Evaporation of the solvent gave crude compounds, which were purified by column chromatography using hexane: ethylacetate (9.5:0.5) and following EtOH crystallization. Synthesis of Compounds 1-14 A reaction mixture of isatin, 5-chloro isatin, N-benzyl isatin, N-benzyl-5-chloro isatin (1 equiv.), and corresponding amines (1 equiv.), were boiled for 0.5 h and then stirred at room temperature for 24 h. After cooling, the precipitates were collected and crystallization with ethanol gave pure compounds as either a mixture of both E and Z or only one isomer. The data for minor isomers are not reported. N-Benzyl isatin M.p.: 123ºC, yield: 57.0%, 1H NMR, , ppm (DMSO-d6): 4.90 (s, 2H, -CH2), 6.97 (d, J = 8.4 Hz, 1H, H4), 7.12 (t, 1H, H4’), 7.29 (d, 1H, H6), 7.35 (t, 2H, H3’,5’), 7.42 (d, 2H, H2’,6’), 7.61-7.64 (m, 2H, H5, H7); ESI-MS: m/z, 238.29 [M + 1]. N-Benzyl-5-chloro isatin M.p.: 135ºC, yield: 66.0%, 1H NMR, , ppm (DMSO-d6): 4.91 (s, 2H, -CH2), 6.96 (d, J = 8 Hz, 1H, H4), 7.28 (m, 1H, H4’), 7.34 (t, 2H, H3’,5’), 7.42 (d, 2H, H2’,6’), 7.61-7.64 (m, 2H, H6, H7); ESI-MS: m/z, 272 [M + 1], 274.27 [M + 3]. 3-[(4-Chlorophenyl)imino]-1,3-dihydro-2H-indol-2-on (1) M.p.: 269ºC, yield: 90.3%, IR (KBr): enolic OH (cm-1): 3266.34, C=O (cm-1): 1733.69, C=N (cm-1): 1608.82; 1 H NMR, , ppm (DMSO-d6): major (E) isomer 6.44 (d, 1H, H4), 6.78 (t, 1H, H5), 6.90 (d, 1H, H7), 7.05 (d, 2H, H2’,6’), 7.36 (t, 1H, H6), 7.53 (d, 2H, H3’,5’), 11.03 (s, 1H, NH); ESIMS: m/z, 257.23 [M + 1], 259.24 [M + 3]; Anal. for C14H9ClN2O.0.2H2O: Calc. C: 64.60, H: 3.64, N: 10.76. Found C: 64.67, H: 3.52, N: 11.00. [31]. 3-[(3-Chloro-4-flurophenyl)imino]-1,3-dihydro-2H-indol2-on (2) M.p.: 234ºC, yield: 65.8%, IR (KBr): enolic OH (cm-1): 3142.44, C=O (cm-1): 1724.53, C=N (cm-1): 1610.27; 1 H NMR, , ppm, (DMSO-d6): major (Z) isomer 6.46 (d, 1H, H4), 6.80 (t, 1H, H5), 6.91 (d, 1H, H7), 7.03-7.40 (m, 2H, H2’,6 ), 7.37 (d, 1H, H6’ ), 7.53 (t, 1H, H5’), 11.05 (s, 1H, NH); ESI-MS: m/z, 275.24 [M + 1], 277.24 [M + 3]; Anal. for C14H8ClFN2O.0.3H2O: Calc. C: 60.04, H: 3.09, N: 10.00. Found C: 59.80, H: 2.83, N: 10.05.

Letters in Drug Design & Discovery, 2013, Vol. 10, No. 1

21

5-Chloro-3-[(4-chlorophenyl)imino]-1,3-dihydro-2H-indol2-on (3) M.p.: 282ºC, yield: 87.0%, IR (KBr): enolic OH (cm-1): 3240.79, C=O (cm-1): 1750.08, C=N (cm-1): 1605.93; 1 H NMR, , ppm, (DMSO-d6): major (E) isomer 6.33 (s, 1H, H4), 6.93 (d, 1H, H7), 7.07 (d, 2H, H2’,6’), 7.43 (d, 1H, H6 ), 7.57 (d, 2H, H3’,5’), 11.13 (s, 1H, NH); ESI-MS: m/z, 291.26 [M + 1], 293.19 [M + 3]; Anal. for C14H8Cl2N2O: Calc. C: 57.76, H: 2.77, N: 9.62. Found C: 57.56, H: 2.58, N: 9.84. [32]. 5-Chloro-3-[(4-fluorophenyl)imino]-1,3-dihydro-2H-indol2-on (4) M.p.: 240ºC, yield: 78.8%, IR (KBr): enolic OH (cm-1): 3241, C=O (cm-1): 1738, C=N (cm-1): 1611; 1H NMR, , ppm, (DMSO-d6): A mixture of the E and Z isomers (3:1 E:Z), major (E) isomer 6.31 (d, J = 2 Hz, 1H, H4), 6.93 (d, J = 8.4 Hz, 1H, H7), 7.01-7.10 (m, 2H, H2',6'), 7.36 (t, 2H, J = 7.6 Hz, H3',5'), 7.42 (dd, J = 1.6, 2.4, 8.4 Hz, 1H, H6), 11.15 (s, 1H, NH), minor (Z) isomer 6.88 (d, J = 8.4 Hz, 1H, H7), 7.14-7.17 (m, 4H, H2',3',5',6'), 7.49 (dd, J = 2, 2.4, 8.4 Hz, 1H, H6), 7.56 (d, J = 2 Hz, 1H, H4), 11.15 (s, 1H, NH); ESI-MS: m/z, 275 [M + 1], 277 [M + 2]; Anal. for C14H8ClFN2: Calc. C: 61.22, H: 2.94, N: 10.20. Found C: 61.32, H: 2.81, N: 10.26. [33]. 5-Chloro-3-[(3-chloro-4-fluorophenyl)imino]-1,3-dihydro2H-indol-2-on (5) M.p.: 230ºC, yield: 65.3%, IR (KBr): enolic OH (cm-1): 3219.58, C=O (cm-1): 1749.12, C=N (cm-1): 1604.97; 1 H NMR, , ppm, (DMSO-d6): major (E) isomer 6.36 (s, 1H, H4), 6.94 (d, 1H, H7), 7.07-7.40 (m, 3H, H2’,6’,6 ), 7.44 (dd, 1H, H5’ ), 11.13 (s, 1H, NH); ESI-MS: m/z, 309.27 [M + 1], 311.27 [M + 3]; Anal. for C14H7Cl2FN2O: Calc. C: 54.40, H: 2.28, N: 9.06. Found C: 54.18, H: 2.35, N: 9.10. 5-Chloro-3-[(2,5-dichlorophenyl)imino]-1,3-dihydro-2Hindol-2-on (6) M.p.: 228ºC, yield: 56.0%, IR (KBr): enolic OH (cm-1): 3183, C=O (cm-1): 1712, C=N (cm-1): 1616; 1H NMR, , ppm, (DMSO-d6): A mixture of the E and Z isomers (1:1 E:Z), major (E) isomer 6.21 (d, J = 2 Hz, 1H, H4), 6.97 (d, J = 8.4 Hz, 1H, H7), 7.18 (dd, J = 2.4, 8.4 Hz, 1H, H4'), 7.23 (d, J = 2.4 Hz, 1H, H6'), 7.50 (dd, J = 2.4, 8.8 Hz, 1H, H6), 7.69 (d, J = 8.4 Hz, 1H, H3'), 11.20 (s, 1H, NH), minor (Z) isomer 6.93 (d, J = 8 Hz, 1H, H7), 7.27 (d, J = 2.4 Hz, 1H, H6'), 7.40 (dd, J = 2.4, 8.4 Hz, 1H, H4'), 7.56 (dd, J = 2, 2.4, 8 Hz, 1H, H6), 7.68 (d, J = 2 Hz, 1H, H4), 7.69 (d, J = 8.4 Hz, 1H, H3'), 11.20 (s, 1H, NH); ESI-MS: m/z, 325.26 [M +], 327.00 [M + 2]; Anal. for C14H7Cl3N2O: Calc. C: 51.65, H: 2.17, N: 8.60. Found C: 51.38, H: 2.21, N: 8.59. 5-Chloro-3-[(4-thiomethylphenyl)imino]-1,3-dihydro-2Hindol-2-on (7) M.p.: 269ºC; yield: 90.3%, IR (KBr): enolic OH (cm-1): 3241, C=O (cm-1): 1749, C=N (cm-1): 1605; 1H NMR, , ppm, (DMSO-d6): A mixture of the E and Z isomers (3:1 E:Z), major (E) isomer 2.53 (s, 3H, -CH3), 6.48 (d, J = 2.4 Hz, 1H, H4), 6.92 (d, J = 8.4 Hz, 1H, H7), 7.02 (d, J = 8 Hz, 2H, H2',6'), 7.40 (d, J = 8 Hz, 2H, H3',5'), 7.42 (dd, J = 1.6, 2.4, 8.4 Hz, 1H, H6), 11.10 (s, 1H, NH), minor (Z) isomer 2.53


(s, 3H, -CH3), 6.87 (d, J = 8.8 Hz, 1H, H7), 7.11 (d, J = 8.8 Hz, 2H, H2',6'), 7.22 (d, J = 8.4 Hz, 2H, H3',5'), 7.48 (dd, J = 2, 2.4, 8 Hz, 1H, H6), 7.56 (d, J = 2 Hz, 1H, H4), 11.10 (s, 1H, NH); ESI-MS: m/z, 303.30 [M +], 305.00 [M + 2]; Anal. for C15H11ClN2OS: Calc. C: 59.50, H: 3.66, N: 9.25. Found C: 59.70, H: 3.58, N: 9.29. N-Benzyl-3-[(4-chlorophenyl)imino]-1,3-dihydro-2H-indol2-on (8) M.p.: 202ºC, yield: 75.4%, IR (KBr): C=O (cm-1): 1738.51, C=N (cm-1): 1609.79; 1H NMR, , ppm, (DMSOd6): major (E) isomer 4.88 (s, 2H, -CH2), 6.50 (d, 1H, H4), 6.84 (t, 1H, H5), 7.01-7.49 (m, 9H, H2’,6’, H2’’,6’’, H3’’,5’’, H4’’, H6, H7), 7.54 (d, 2H, H3’,5’); ESI-MS: m/z, 347.41 [M + 1], 349.42 [M + 3]; Anal. for C21H15ClN2O: Calc. C: 72.73, H: 4.36, N: 8.08. Found C: 72.53, H: 4.20, N: 8.20. [34]. N-Benzyl-3-[(3-chloro-4-fluorophenyl)imino]-1,3-dihydro2H-indol-2-on (9) M.p.: 166ºC, yield: 79.4%, IR (KBr): C=O (cm-1): 1740.92, C=N (cm-1): 1606.90; 1H NMR, , ppm, (DMSOd6): major (E) isomer 4.99 (s, 2H, -CH2), 6.53 (d, 1H, H4), 6.86 (t, 1H, H5), 7.03 (d, 2H, H2’’,6’’), 7.08-7.43 (m, 6H, H2’,6,7, H3’’,5’’, H4’’), 7.55 (t, 1H, H5’), 7.67 (d, 1H, H6’); ESIMS: m/z, 365.41 [M + 1], 367.51 [M + 3]; Anal. for C21H14ClFN2O: Calc. C: 69.14, H: 3.87, N: 7.68. Found C: 68.78, H: 3.75, N: 7.72. N-Benzyl-5-chloro-3-[(4-chlorophenyl)imino]-1,3-dihydro2H-indol-2-on (10) M.p.: 164ºC, yield: 59.5%, IR (KBr): C=O (cm-1): 1724.53, C=N (cm-1): 1602.07; 1H NMR, , ppm, (DMSOd6): major (E) isomer 4.99 (s, 2H, -CH2), 6.39 (s, 1H, H4) 7.05 (d, 2H, H2’’,6’’), 7.14 (m, 3H, H3’’,5’’, H4’’), 7.38 (m, 4H, H6, H7, H2’,6’), 7.59 (d, 2H, H3’,5’ ); ESI-MS: m/z, 381.30 [M + 1], 383.45 [M + 3]; Anal. for C21H14Cl2N2O.0.2H2O: Calc. C: 65.54, H: 3.77, N: 7.28. Found C: 65.42, H: 3.73, N: 7.28. N-Benzyl-5-chloro-3-[(4-fluorophenyl)imino]-1,3-dihydro2H-indol-2-on (11) M.p.: 143ºC, yield: 22.3%, IR (KBr): C=O (cm-1): 1740, C=N (cm-1): 1606; 1H NMR, , ppm, (DMSO-d6): A mixture of the E and Z isomers (3:1 E:Z), major (E) isomer 5.00 (s, 2H,-CH2), 6.39 (d, J = 2 Hz, 1H, H4), 7.05 (d, J = 8.8 Hz, 1H, H7), 7.11-7.15 (m, 2H, H2',6'), 7.31 (t, 2H, J = 8 Hz, H3',5'), 7.35-7.42 (m, 5H, -CH2Ph), 7.47 (dd, J = 2, 2.8, 8 Hz, 1H, H6), minor (Z) isomer 4.86 (s, 2H, -CH2), 6.99 (d, J = 8.8 Hz, 1H, H7), 7.19-7.22 (m, 4H, H2',3',5',6'), 7.27-7.40 (m, 5H, -CH2Ph), 7.53 (dd, J = 2, 2.4, 8 Hz, 1H, H6), 7.66 (d, J = 2.4 Hz, 1H, H4); ESI-MS: m/z, 365.43 [M + 1], 367.00 [M + 3]; Anal. for C21H14ClFN2O: Calc. C: 69.14, H: 3.87, N: 7.68. Found C: 69.27, H: 3.76, N: 7.68. N-Benzyl-5-chloro-3-[(3-chloro-4-fluorophenyl)imino]-1,3dihydro-2H-indol-2-on (12) M.p.: 120ºC, yield: 21.2%, IR (KBr): C=O (cm-1): 1712.96, C=N (cm-1): 1612.68; 1H NMR, , ppm, (DMSOd6): major (Z) isomer 5.00 (s, 2H, -CH2), 6.43 (s, 1H, H4), 7.06 (d, 2H, H2’’,6’’), 7.13-7.67 (m, 8H, H2’,6’, H6, H7, H5’, H3’’,5’’, H4’’); ESI-MS: m/z, 399.77 [M + 1], 402.43 [M + 3]; Anal. for C21H13Cl2FN2O.0.3C6H12 : Calc. C: 64.51, H: 3.94, N: 6.59. Found C: 64.61, H: 4.02, N: 6.77.

Kurt et al.

N-Benzyl-5-chloro-3-[(2,5-dichlorophenyl)imino]-1,3dihydro-2H-indol-2-on (13) M.p.: 102ºC, yield: 16.5%, IR (KBr): C=O (cm-1): 1720, C=N (cm-1): 1611; 1H NMR, , ppm, (DMSO-d6): A mixture of the E and Z isomers (1:1 E:Z), major (E) isomer 5.01 (s, 2H, -CH2), 6.29 (d, J = 2 Hz, 1H, H4), 7.10 (d, J =8.4 Hz, 1H, H7), 7.22 (dd, J = 2, 2.4, 8 Hz, 1H, H4'), 7.28-7.42 (m, 6H, H6', -CH2Ph), 7.53 (d, J = 8.4 Hz, 1H, H3') 7.54 (J = 2, 2.4, 8 Hz, 1H, H6), minor (Z) isomer 4.87 (s, 2H, -CH2), 7.04 (d, J = 8.8 Hz, 1H, H7), 7.28-7.42 (m, 7H, H4',6', -CH2Ph), 7.60 (dd, J = 2, 2.4, 8 Hz, 1H, H6), 7.71 (d, J = 8.8 Hz, 1H, H3'), 7.76 (d, J = 2 Hz, 1H, H4); ESI-MS: m/z, 415.00 [M+], 417.00 [M + 2]. Anal. for C21H13Cl3N2O: Calc. C: 60.67, H: 3.15, N: 6.74. Found C: 60.69, H: 3.37, N: 6.92. N-Benzyl-5-chloro-3-[(4-thiomethylphenyl)imino]-1,3dihydro-2H-indol-2-on (14) M.p.: 148°C, yield 74.5%, IR (KBr): C=O (cm-1): 1729, C=N (cm-1): 1601; 1H NMR, , ppm, (DMSO-d6): A mixture of the E and Z isomers (3:1 E:Z), major (E) isomer 3.34 (s, 3H, -CH3) 5.00 (s, 2H, -CH2), 6.55 (d, J = 2 Hz, 1H, H4), 7.04 (d, J = 8.8 Hz, 1H, H7), 7.07 (d, J = 8 Hz, 2H, H2',6'), 7.27-7.43 (m, 7H, H3',5', -CH2Ph), 7.46 (dd, J = 2, 2.4, 8 Hz, 1H, H6), minor (Z) isomer 3.34 (s, 3H, -CH3), 4.86 (s, 2H, CH2), 6.99 (d, J = 8.4 Hz, 1H, H7), 7.19 (d, J = 8.4 Hz, 2H, H2',6'), 7.24-7.43 (m, 7H, H3',5', -CH2Ph), 7.51 (dd, J = 2, 2.4, 8 Hz, 1H, H6), 7.65 (d, J = 2.4 Hz, 1H, H4); ESI-MS: m/z, 392.00 [M + 1], 395.00 [M + 3]. Anal. for C22H17ClN2OS: Calc. C: 67.25, H: 4.36, N: 7.13. Found C: 66.94, H: 4.16, N: 7.16. Biology Assay for Glutathione-S-Transferase Activity All compounds were dissolved in DMSO (Dimethylsulfoxide) to form the stock solutions with the final concentration of 15-25 mM. The serial dilutions were prepared from these stocks to yield 0.3-5 μM in relevant reaction buffer. The method of Habig to measure the kinetic change in substrate (GSH) utilization by Glutathione Stransferase were slightly modified and adopted for microscale applications [35, 36]. Briefly, the total GST activity was measured in a 100 mM potassium phosphate buffer at pH 6.5 with 2.4 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 3.2 mM GSH. In the assay, the compounds to be tested or DMSO (vehicle control), CDNB and reduced GSH were transferred to microplate in reaction buffer (100 mM potassium phosphate buffer at pH 6.5) and incubated at 22oC for 5 minutes. Upon addition of enzyme source (bovine liver cytosol) microplate transferred to spectrophotometer and GSH-CDNB conjugate formation was monitored as an increase in the absorbance at 340 nm for 240 second. Initial rates of enzymatic reactions were determined as nanomoles of the conjugation product of GSH and reported as nmole/minute/mg protein, where the total protein content was determined by the Lowry method [37]. In the analysis of compounds the kinetic measurement performed but the calculations were made with respect to the vehicle control. All measurements were performed at 340 nm, in 96 well microplate, using Spectramax M2e.


Letters in Drug Design & Discovery, 2013, Vol. 10, No. 1 R2 O

4

R1

R1

O N 2 1

6 7

2'

3'

1' 6'

3'

5'

6'

R5

O

N

EtOH, reflux

R3 4' R 4

1'

R5

4'

BrCH2Ph

N

R4

O R1

2'

3

5 NaH, DMF

R2

R3

H2N

23

2'' 3''

1''

5'

O N H

6''

R1= H, N-benzyl isatin

8-14

R1= Cl, N-benzyl 5-chloro isatin R1= H, Cl EtOH, reflux R2 R2

R3

H2N

2' R1

R4

4 5

R5

N 2 N1 H

3'

4'

3

6 7

1'

R3

6' O

R4

8 9 10 11 12 13 14

4'' 5''

R1=H, R2=H, R3=H, R4= Cl, R5=H R1=H, R2=H, R3=Cl, R4=F, R5=H R1= Cl, R2=H, R3=H, R4=Cl, R5=H R1=Cl, R2=H, R3=H, R4=F, R5=H R1=Cl, R2=H, R3=Cl, R4=F, R5=H R1=Cl, R2=Cl, R3=H, R4=H, R5=Cl R1=Cl, R2=H, R3=H, R4=SCH3, R5=H

5' R 5

1-7 1 2 3 4 5 6 7

R1=H, R2=H, R3=H, R4= Cl, R5=H R1=H, R2=H, R3=Cl, R4=F, R5=H R1= Cl, R2=H, R3=H, R4=Cl, R5=H R1=Cl, R2=H, R3=H, R4=F, R5=H R1=Cl, R2=H, R3=Cl, R4=F, R5=H R1=Cl, R2=Cl, R3=H, R4=H, R5=Cl R1=Cl, R2=H, R3=H, R4=SCH3, R5=H

Scheme 1. Synthesis of indole-3-imine-2-on (1-7) and N-benzyl indole-3-imine-2-on (8-14) derivatives.

Assay for Src Tyrosine Kinase Activity: In vitro Kinase Assay The activity measurement were performed using (ProFluor Src-Family Kinase Assay protocol Promega) with some modifications of the manufacturer’s protocol [12]. The pure enzyme Src (Invitrogen) were used at certain activities that yield 20% of the maximum fluorescent signal and the molecules to be tested (compounds to be inhibitors) were used in final concentrations bearing 0.5% DMSO. A titration assay was performed for each kinase to determine the amount of the enzyme that results in approximately 80% phosphorylation as suggested by the manufacturer. The compounds were dissolved in DMSO and tested at 1, 10 and 100 M concentrations. Briefly, the molecules were mixed with a reaction buffer that included a specific substrate for Src-family kinases (R110), a control substrate and the kinase. The reaction was initiated by the addition of ATP. After incubating the 96-well reaction plate at 22°C for 1 h, protease solution was added to each well and the plate was incubated for 1 h. The fluorercence of the liberated R110 was read at an excitation wavelength of 485 nm and emission wavelength of 530 nm. The strength of fluorescence is inversely correlated with the kinase activity. The Src kinase activity is measured as the difference between the total activity of no vehicle (DMSO) and the activity of enzyme in the presence of DMSO. % inhibition value of compounds was determined as the mean of triplicate measurements. The IC50 values were determined by nonlinear regression analysis, the four parameter logistic

equation (Sigmoidal dose response, GRAPHPAD PRISM version 4.0 for Windows; GraphPad Software, San Diego, CA, USA). RESULTS AND DISCUSSION Indole-3-imine-2-on (1-7) and N-benzyl-indole-3-imine2-on (8-14) derivatives were synthesized from isatin, 5chloro isatin, N-benzyl isatin and N-benzyl 5-chloro isatin (Scheme 1) [38]. N-benzyl isatin and N-benzyl 5-chloro isatin were obtained with substitution of isatin or 5-chloro isatin and benzyl bromide in the presence of NaH in DMF [39, 40]. The final compounds were obtained as either Z or E isomers and mixture of Z/E isomers, depending on the characteristics of the substitutions at the C-3 position. The configuration of some compounds was determined using NOE analysis, which show a neat correlation between H 4 and H2’ and also a weak correlation between H4 and H3’ for major E isomers. In addition, the signal for H4 of the Eisomer was considerably shifted upfield relative to the H 4 signal of the parent compound and the minor Z-isomers. These assignments were also confirmed using 1H NMR spectral data. The signal from H4 of the E-isomer was markedly shifted relative to the H4 signal of the parent compound. In contrast, the chemical shifts of H4 and H6 of the Z-isomer showed little difference in chemical shift to the parent compound. The mass spectra of compounds were taken with ESI methods and the mass values of compounds were monitored as [M + 1] and [M+]. The expected isotop


(a)

Kurt et al.

(b)

Fig. (2). GST Inhibition of Compound 2 with IC50 of 48.35 M at 346 u/l activity of GST (a), and Src Kinase Inhibition with IC50 of 131.1 M at 320X10-7 u/l activity of Src kinase (b).

(a)

(b)

Fig. (3). GST Inhibition of Compound 3 with IC50 of 21.61 M at 346 u/l activity of GST (a), and Src Kinase Inhibition with IC50 of 131.6 M at 320X10-7 u/l activity of Src kinase (b).

(a) (b) Fig. (4). GST Inhibition of Compound 9 with IC50 of 42.00 M at 346 u/l activity of GST (a), and Src Kinase Inhibition with IC50 of 142.5 M at 320X10-7 u/l activity of Src kinase (b).

peaks of Cl atoms were detected as a ratio of 3:1 and numbers of peaks were also detected depending on the number of atoms. In the IR spectra, all compounds showed stretching values of amide C=O in the range of 1712-1750

cm-1, enolic O-H 3219-3266 cm-1 and C=N stretching of imine values were found as 1601-1616 cm-1. Element compositions were measured within 0.4% of the calculated values.


The compounds were analyzed for their inhibitory activity toward Src kinase and GST to identify those with dual activity against biological targets. Among the compounds analyzed for Src inhibitory activity, except compound 7 and 14, all the compounds resulted in dose response curves with IC50s in the range of 20-200 M. The compounds 1, 8, 9 and 10 showed only the reasonable Src inhibition with IC50s of 151.9, 106, 142.5 and 197.3 M, respectively; comparison with known Src inhibitor PP2 (IC50= 5 nM, Fig. (1). However, for those the dose response curves against GST showed the maximum inhibition less than or around 50% and not enough to conclude that they are good GST inhibitiors at the active range of compounds for Src inhibition. Although compound 8 showed most potent inhibition against Src, it did not inhibit GST at all. This result may indicate that compound 8 does not play a role as the resistant drug. The compounds 2, 3 and 9 are found slightly active against both enzyme Src and GST Figs. (2, 3, and 4). The IC50 values for the compounds 2, 3 and 9 against Src were determined as 131.1, 131.6 and 142.5 M, respectively. The GST inhibition of these compounds were yielded the IC50s of 48.35, 21.61 and 42.0 M for compounds 2, 3 and 9, respectively; comparison with known GST inhibitor ethacrynic acid (IC50= 3.3 M, Fig. (1) [29]. The compounds 7 and 14 are found complete inactive in our assays. The compounds 4, 5 and 6 showed less than 50% of the inhibitory activity and are found inactive within the concentration range used in assays. If these compounds show any inhibition, they may further be tested using the wide range of high doses, since their dose-response curves show some sigmoid curve and reveals a potential inhibitor pattern with varying concentrations. However, only those that may result in sufficient response profile at high dose titration assay should further be analyzed against Src. As a result, none of compounds can be considered as good inhibitor for dual activity. Nevertheless, it was noteworthy to test indole derivatives to find new Src kinase and GST dual inhibitors to solve drug resistance problem of Src inhibitors for several cancer types especially, breast, prostate and liver. CONFLICT OF INTEREST

Letters in Drug Design & Discovery, 2013, Vol. 10, No. 1

[6] [7]

[8] [9] [10] [11] [12] [13]

[14] [15]

[16]

[17]

[18]

[19]

[20]

[21]

The authors have declared no conflict of interest. [22]

ACKNOWLEDGEMENT This project is received a funding support from Ankara University (BAP 09B3336002) Scientific Research Funds. REFERENCES [1] [2] [3] [4] [5]

Biscardi, J. S.; Tice, D. A.; Parsons, S. J. c-Src, receptor tyrosine kinases, and human cancer. Adv. Cancer Res., 1999, 76, 61-119. Martin, G. S. The road to Src. Oncogene, 2004, 23, 7910-7917. Lowell, C. A.; Soriano, P. Knockouts of Src-family kinases: stiff bones, wimpy T cells, and bad memories. Genes Dev., 1996, 10, 1845-1857. Bjorge, J. D.; Jakymiw, A.; Fujita, D. J. Selected glimpses into the activation and function of Src kinase. Oncogene, 2000, 19, 56205635. Tice, D. A.; Biscardi, J. S.; Nickles, A. L.; Parsons, S. J. Mechanism of biological synergy between cellular Src and

[23]

[24]

[25]

[26]

25

epidermal growth factor receptor. Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 1415-1420. Warmuth, M.; Damoiseaux, R.; Liu, Y.; Fabbro, D.; Gray, N. SRC family kinases: potential targets for the treatment of human cancer and leukemia. Curr. Pharm. Des., 2003, 9, 2043-2059. DeMali, K. A.; Balciunaite, E.; Kazlauskas, A. J. Integrins enhance platelet-derived growth factor (PDGF)-dependent responses by altering the signal relay enzymes that are recruited to the PDGF beta receptor. Biol. Chem., 1999, 274, 19551-19558. Courtneidge, S. A. Isolation of novel Src substrates. Biochem. Soc. Trans., 2003, 31, 25-28. Alvarez, R. H.; Kantarjian, H. M.; Cortes, J. E. The role of Src in solid and hematologic malignancies: development of newgeneration Src inhibitors. Cancer, 2006, 107, 1918-1929. Belsches-Jablonski, A. P.; Demory, M. L.; Parsons, J. T. The src pathway as a therapeutic strategy. Drug Discovery Today: Therapeutic Strategies, 2005, 2, 313-321. Frame, M. C. Src in cancer: deregulation and consequences for cell behaviour. Biochim. Biophys. Acta, 2002, 1602, 114-130. Ingley, E. Src family kinases: Regulation of their activities, levels and identification of new pathways. Biochim. Biophys. Acta, 2008, 1784, 56-65. Guan, H. P.; Laird, D.; Blake, R. A.; Tang, C.; Liang, C. Design and synthesis of aminopropyl tetrahydroindole-based indolin-2ones as selective and potent inhibitors of Src and Yes tyrosine kinase. Bioorg. Med. Chem. Lett., 2004, 14, 187-190. Olgen, S.; Akaho, E.; Nebiolu, D. Synthesis and docking-based studies of 3-substituted indolin-2-on and thione derivatives as tyrosine kinase inhibitors. IL Farmaco, 2005, 60, 497-506. Olgen, S.; Akaho, E.; Nebioglu, D. J. Evaluations of indole esters as inhibitors of p60c-Src receptor tyrosine kinase and investigation of enzyme inhibition using receptor docking studies. Enzym. Inhib. Med. Chem., 2003, 8, 485-490. Kılıç, Z.; Igör, Y. G.; Ölgen, S. Synthesis and pp60c-Src tyrosine kinase inhibitory activity of novel indole-3-imine derivatives and their amine congeners substituted at N1 and C5. Arch. Pharm., 2009, 342, 333-343. Tolliday, N.; Clemons, P. A.; Ferraiolo, P.; Koehler, A. N.; Lewis, T. A.; Li, X.; Schreiber, S. L.; Gerhard, D. S.; Eliasof, S. Small molecules, big players: the national cancer institute's initiative for chemical genetics. Cancer Res., 2006, 66, 8935-8942. Juronen, E.; Tasa, G.; Uusküla, M.; Pooga, M.; Mikelsaar, A. V. Purification, characterization and tissue distribution of human class theta glutathione S-transferase T1-1. Biochem. Mol. Biol. Int., 1996, 39, 21-29. Hayes, J. D.; Pulford, D. J. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol., 1995, 30, 445-600. Gusson, F.; Carletti, M.; Albo, A. G.; Dacasto, M.; Nebbia, C. Comparison of hydrolytic and conjugative biotransformation pathways in horse, cattle, pig, broiler chick, rabbit and rat liver subcellullar fractions. Vet. Res. Commun., 2006, 30, 271-283. Homolya, L.; Váradi, A.; Sarkadi, B. Multidrug resistanceassociated proteins: Export pumps for conjugates with glutathione, glucuronate or sulfate. Biofactors, 2008, 17, 103-114. Di Pietro, G.; Magno, L.; Rios-Santos, F. Glutathione Stransferases: an overview in cancer research. Expert. Opin. Drug Metab. Toxicol., 2010, 6, 153-170. Findlay, V. J.; Townsend, D. M.; Tew, K. D. Glutathione and glutathione s-transferases in drug resistance. Cancer drug resistance, Edited by Beverly A. Teicher, Humana Press, NJ, USA 2006. Baez, S.; Segura-Aguilar, J.; Widersten, M.; Johansson, A. S.; Mannervik, B. Glutathione transferases catalyse the detoxication of oxidized metabolites (o-quinones) of catecholamines and may serve as an antioxidant system preventing degenerative cellular processes. Biochem. J., 1997, 324, 25-28. Scotlandi, K.; Remondini, D.; Castellani, G.; Manara, M. C.; Nardi, F.; Cantiani, L.; Francesconi, M.; Mercuri, M.; Caccuri, A. M.; Serra, M.; Knutila, S.; Picci, P. Overcoming resistance to conventional drugs in Ewing sarcoma and identification of molecular predictors of outcome. J. Clin. Oncol., 2009, 27, 22092216. Hayeshi, R.; Chinyanga, F.; Chengedza, S.; Mukanganyama, S. Inhibition of human glutathione transferases by multidrug


[27] [28] [29] [30] [31] [32] [33]

[34]

Kurt et al.

resistance chemomodulators in vitro. J. Enzyme Inhib. Med. Chem., 2006, 21, 581-587. Chen, Y., Taylor, C. C. Src Inhibition Enhances Paclitaxel Cytotoxicity in Ovarian Cancer Cells by Caspase-9-independent Activation of Caspase-3. Mol. Cancer Ther., 2005, 4, 217-224. Townsend, D. M.; Tew, K. D. The role of glutathione-S-transferase in anti-cancer drug resistance. Oncogene, 2003, 22, 7369-7375. Tew, K. D.; Bomber, A. M.; Hoffman, S. J. Ethacrynic acid and priprostat as enhancers of cyctotoxicity in drug resistant and sensitive cell lines. Cancer Res. 1988, 48, 3622-3625. Noolvi, M. N.; Patel, H. M. small molecule tyrosine kinase inhibitors: the new dawn for cancer therapy. Lett. Drug. Des. Discov. 2012, 9, 84-125. Azizian, J.; Mohammadi, M. K.; Firuzi, O.; Razzaghi, N.; Miri, R. Synthesis, biological activity and docking study of some new isatin schiff base derivatives. Med. Chem. Res., 2011, 21, 3730-3740. Rajopadhye, M.; Popp, F. D. Synthesis and antileukemic activity of spiro[indoline-3,2'-thiazolidine]-2,4'-diones. J. Het. Chem., 1987, 24, 1637-1642. Varma, R. S.; Khan, I. A. Potential biologically active agents. Part XV. Synthesis of 5-chloro-3-arylimino-, 1-methyl-5-chloro-3arylimino- and 1-morpholino-(piperidino) methyl-5-chloro-3arylimino-2-indolinones. Indian J. Chem. Section B: Organic Chemistry Including Medicinal Chemistry, 1978, 16B, 1113-1114. Shi, Y. H.; Wang, Z.; Shi, Y.; Deng, W. P. Facile and highly diastereoselective synthesis of 3-aminooxindoles via AgOAc-

Received: June 03, 2012

[35] [36]

[37] [38] [39]

[40]

catalyzed vinylogous Mannich reaction. Tetrahedron, 2012, 68, 3649-3653. Habig, W. H.; Jakoby, W. B. Assays for differentiation of glutathione S-transferases. Methods Enzymol., 1981, 77, 398-405. Depeille, P.; Pierre Cuq, P.; Mary, S.; Passagne, I.; Evrard, A.; Cupissol, D; Vian, L. Glutathione S-transferase M1 and multidrug resistance protein 1 act in synergy to protect melanoma cells from Vincristine Effects. Mol. Pharmacol., 2004, 65, 897-905. Agrez, M.; Garg, M.; Dorahy, D.; Ackland, S. Synergistic antitumor effect of cisplatin when combined with an anti-src kinase integrin-based peptide. J. Cancer Ther., 2011, 2, 295-301. Sridhar, S. K.; Ramesh, A. Synthesis and pharmacological activities of hydrazones, schiff and mannich bases of isatin derivatives. Biol. Pharm. Bull., 2001, 24, 1149-1152. Wee, X. K.; Yeo, W. K.; Zhang, B.; Tan, V. B. C.; Lim, K. M.; Tay, T. E.; Go, M. L. Synthesis and evaluation of functionalized isoindigos as antiproliferative agents. Bioorg. Med. Chem., 2009, 17, 7562-7571. Konkel, M. J.; Lagu, B.; Boteju, L. W.; Jimenez, H.; Noble, S.; Walker, M. W.; Chendrasena, G.; Blackburn, T. P.; Nikam, S. S.; Wright, J. L.; Kornberg, B. E.; Gregory, T.; Pugsley, T. A.; Akunne, H.; Zoski, K.; Wise, L. D. 3-Arylimino-2-indolones are potent and selective galanin GAL3 receptor antagonists. J. Med. Chem., 2006, 49, 3757-3758.

Revised: September 22, 2012

Accepted: October 08, 2012