Molecular structures, Hirshfeld analysis and biological investigations of isatin based thiosemicarbazones

Journal of Molecular Structure 1198 (2019) 126904 Contents lists available at ScienceDirect Journal of Molecular Structure journal homepage: http://www.elsevier.com/locate/molstruc Molecular structures, Hirshfeld analysis and biological investigations of isatin based thiosemicarbazones Sivaraj Saranya a, 1, Jebiti Haribabu b, c, 1, Vishnunarayanan Namboothiri Vadakkedathu Palakkeezhillam b, Peter Jerome b, Kannayiram Gomathi a, Kodagala Kameswara Rao d, Velakaturi Hari Hara Surendra Babu d, Ramasamy Karvembu b, *, Dasararaju Gayathri e, ** a Department of Biotechnology, Dr. MGR Educational and Research Institute University, Maduravoyal, Chennai 600095, India Department of Chemistry, National Institute of Technology, Tiruchirappalli, 620015, India c Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, 278-8510 Japan d Department of Physics, Sri Venkateswara Arts College, Tirupati, 517501, India e Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai, 600025, India b a r t i c l e i n f o a b s t r a c t Article history: Received 29 April 2019 Received in revised form 26 July 2019 Accepted 5 August 2019 Available online 8 August 2019 The synthesized isatin thiosemicarbazone compounds (1e10) were well characterized by elemental analysis, and UVeVisible, FT-IR and NMR spectroscopic methods. The crystal structure of two of the compounds (6 and 9) was confirmed by single crystal X-ray crystallography. Hirshfeld surface analysis was performed to analyze the intermolecular interactions. The compounds were evaluated for their in vitro antioxidant and cytotoxicity against MCF-7 (breast) cancer cell line through DPPH and MTT assays, respectively. The results clearly indicated that compounds 5, 6, 7 and 8 showed significant antioxidant property and compound 7 exhibited greater cytotoxicity than the other compounds. Antiinflammatory activity of the compounds was determined by in vitro PLA2 inhibition assay and in silico molecular docking study, which showed promising results for all the compounds. © 2019 Elsevier B.V. All rights reserved. Keywords: Thiosemicarbazones Antioxidant Cytotoxicity Anti-inflammatory Phospholipase A2 Molecular docking 1. Introduction Thiosemicarbazide when condensed with aldehyde or ketone with potential donor site(s) yields Schiff base termed as thiosemicarbazone. Thiosemicarbazone compounds and their metal complexes have been studied over the last 50 years for their tremendous biological applications. The presence of sulfur atom and its ability to bind with the metals in the biological system is believed to be the major reason for their biological activities [1] such as anticancer [2], antitumor, antifungal [3], antibacterial [4], antimalarial, antiviral [5] and anti-HIV [6]. Thiosemicarbazone (TSC) derivatives such as marboran, * Corresponding author. ** Corresponding author. E-mail addresses: kar@nitt.edu (R. Karvembu), (D. Gayathri). 1 Both the authors contributed equally to this work. https://doi.org/10.1016/j.molstruc.2019.126904 0022-2860/© 2019 Elsevier B.V. All rights reserved. gayathri@unom.ac.in amithiozone, cutisone, ambazone and anisaldehyde thiosemicarbazone were proved to possess antituberculosis or antitumor effect (Fig. 1) [7]. Recent studies on the antitumor property of thiosemicarbazone derivative DP44 mT (di-2-pyridylketone-4,4dimethyl-3-thiosemicarbazone) have proved that the compound can actively bind iron in a tight chelate complex and deplete tumors as cancer cells need more iron than normal body cells to sustain their abnormally rapid growth [8]. In addition, cytotoxicity effect of a series of cyclohexyl thiosemicarbazones was reported against HER-2 over expressed SKBr-3 cells [9]. Thiosemicarbazone derivatives were reported to possess anticancer activity against various cell lines, cholangiocarcinoma (HuCCA-1), liver carcinoma (HepG2), lung carcinoma (A549) and acute lymphoblastic carcinoma (MOLT-3) [10]. More importantly, N-heterocyclic TSCs showed broad range of activities, which are believed to be at least partially due to their RNR inhibition property [11]. To date, several TSC compounds, namely, 3-amino-2-pyridinecarboxaldehyde TSC (triapine) [12e14], di-2-pyridylketone-4-cyclohexyl-4-methyl-3- 2 S. Saranya et al. / Journal of Molecular Structure 1198 (2019) 126904 Fig. 1. Medicinally important thiosemicarbazone derivatives. thiosemicarbazone (DpC) [15] and (E)-N0 -(6,7-dihydroquinolin8(5H)-ylidene)-4-(pyridine2-yl)piperazine-1-carbothiohydrazide (COTI-2) [16] are undergoing phase I and II clinical trials against various types of cancer. Our previous studies on few thiosemicarbazone derivatives proved their inhibition potential against secretory phospholipase A2 enzyme [17], one of the major enzymes in lipid mediators. Role of lipid mediators in inflammation and various types of cancer emphasises the potential of phospholipases as key enzymes in cancer progression. Isatin and its derivatives are one of the most important and broadly occurring structural units in several natural compounds and drug intermediates. They have shown a wide range of biological properties such as anticonvulsant, anti-inflammatory, antimicrobial, antidepressant, antiviral, antiHIV, and anticancer [18e24]. In continuation of our quest to explore the biological and pharmaceutical potential of thiosemicarbazones, we report here the synthesis, structure and functional characterization of a series of thiosemicarbazone derivatives. Antioxidant and cytotoxicity effect of the compounds were studied in vitro. Anti-inflammatory potential of the compounds was revealed through in vitro and in silico phospholipase A2 enzyme inhibition studies. 2. Experimental 2.1. Materials and methods All the chemicals were purchased from Sigma Aldrich/Merck and used as received. Solvents were purified by distillation and retained under inert atmosphere. Melting points were determined on Lab India instrument and are uncorrected. Elemental analyses were performed using a Vario EL III CHNS analyzer. FT-IR spectra were recorded in the range of 400e4000 cm 1 (as KBr pellets) using a PerkinElmer Frontier FT-IR spectrometer. Electronic spectra were recorded in the range of 250e800 nm using a PG Instruments T90þ UVeVisible spectrophotometer in DMF solution. NMR spectra were recorded in CDCl3 or DMSO‑d6 by using TMS as an internal standard on a Bruker 500/400 MHz spectrometer. (Z)-2-(2oxoindolin-3-ylidene)hydrazinecarbothioamide (1), (Z)-2-(1-allyl2-oxoindolin-3-ylidene)hydrazinecarbo-thioamide (7) and (Z)-2(1-benzyl-2-oxoindolin-3-ylidene)hydrazinecarbothioamide (10) were synthesized by using a reported procedure [25e27]. 2.2. Synthesis of the isatin thiosemicarbazone derivatives (1e10) Thiosemicarbazide (0.911 g, 1 mmol) was dissolved in ethanol (20 mL) and added to an ethanolic solution (20 mL) of appropriate (un)substituted isatin (1 mmol). The reaction mixture was refluxed for 6 h after the addition of a few drops of acetic acid. The yellow/ orange colored solid was formed, which was collected by filtration, washed with ethanol or petroleum ether and dried in vacuo. The product was recrystallized from DMF/CHCl3 mixture (1:3) to get crystals of 6 (red color) and 9 (pale yellow color) suitable for X-ray analysis. 2.2.1. (Z)-2-(5-bromo-2-oxoindolin-3-ylidene) hydrazinecarbothioamide (2) 5-Bromoisatin (0.224 g, 1 mmol) was used. Yield: 90%. Pale yellow solid. m.p.: 169  C. Anal. Calc. C9H7BrN4OS (%): C, 36.13; H, 2.36; N, 18.73; S, 10.72. Found: C, 36.20; H, 2.29; N, 18.81; S, 10.81. UVeVis (DMF): lmax, nm 257, 343. FT-IR (KBr): ʋ, cm 1 3406, 3373, S. Saranya et al. / Journal of Molecular Structure 1198 (2019) 126904 3231 (NeH), 1670 (C]O), 1563 (C]N), 1271 (C]S). 1H NMR (500 MHz, DMSO‑d6): d ppm 12.30 (s, 1H, N]NH), 11.29 (s, 1H, NH), 9.11 (s, 1H, NH2), 8.82 (s, 1H, NH2), 7.89 (s, 1H, H4), 7.51 (d, J ¼ 7.7 Hz, 1H, H6), 6.89 (d, J ¼ 7.9 Hz, 1H, H7). 13C NMR (126 MHz, DMSO‑d6): d ppm 179.1 (C9), 162.7 (C1), 141.6 (C2), 131.4 (C8), 131.0 (C5), 127.1 3 (C6), 122.2 (C4), 121.2 (C3), 112.7 (C7). 2.2.2. (Z)-2-(5-chloro-2-oxoindolin-3-ylidene) hydrazinecarbothioamide (3) 5-Chloroisatin (0.180 g, 1 mmol) was used. Yield: 82%. Pale Scheme 1. List of the synthesized isatin based thiosemicarbazone compounds. 4 S. Saranya et al. / Journal of Molecular Structure 1198 (2019) 126904 yellow solid. m.p.: 190  C. Anal. Calc. C9H7ClN4OS (%): C, 42.44; H, 2.77; N, 22.00; S, 12.59. Found: C, 42.36; H, 2.85; N, 22.09; S, 12.47. UVeVis (DMF): lmax, nm 262, 340. FT-IR (KBr): ʋ, cm 1 3429, 3361, 3249 (NeH), 1676 (C]O), 1570 (C]N), 1279 (C]S). 1H NMR (500 MHz, DMSO‑d6): d ppm 12.31 (s, 1H, N]NH), 11.29 (s, 1H, NH), 9.12 (s, 1H, NH2), 8.81 (s, 1H, NH2), 7.75 (s, 1H, H4), 7.37 (dd, J ¼ 8.5, 1.4 Hz, 1H, H6), 6.93 (d, J ¼ 8.3 Hz, 1H, H7). 13C NMR (126 MHz, DMSO‑d6): d ppm 179.2 (C9), 162.8 (C1), 141.4 (C2), 131.2 (C8), 130.9 (C5), 127.0 (C6), 122.4 (C4), 121.1 (C3), 112.9 (C7). 2.2.3. (Z)-2-(5-fluoro-2-oxoindolin-3-ylidene) hydrazinecarbothioamide (4) 5-Fluoroisatin (0.165 g, 1 mmol) was used. Yield: 77%. Pale yellow solid. m.p.: 175  C. Anal. Calc. C9H7FN4OS (%): C, 45.37; H, 2.96; N, 23.52; S, 13.46. Found: C, 45.22; H, 2.90; N, 23.65; S, 13.57. UVeVis (DMF): lmax, nm 259, 334. FT-IR (KBr): ʋ, cm 1 3411, 3380, 3237 (NeH), 1671 (C]O), 1559 (C]N), 1270 (C]S). 1H NMR (500 MHz, DMSO‑d6): d ppm 12.37 (s, 1H, N]NH), 11.19 (s, 1H, NH), 9.11 (s, 1H, NH2), 8.75 (s, NH2), 7.50 (dd, J ¼ 7.9, 1.8 Hz, 1H, H4), 7.19 (td, J ¼ 20.0, 2.0 Hz, 1H, H6), 6.92 (dd, J ¼ 8.5, 4.0 Hz, 1H, H7). 13C NMR (126 MHz, DMSO‑d6): d ppm 179.2 (C9), 163.1 (C1), 159.6 (C2), 157.7 (C5), 131.8 (C8), 121.9 (C3), 117.9 (C6), 112.5 (C7), 108.3 (C4). 2.2.4. (Z)-2-(5-nitro-2-oxoindolin-3-ylidene) hydrazinecarbothioamide (5) 5-Nitroisatin (0.192 g, 1 mmol) was used. Yield: 86%. Pale yellow solid. m.p.: 181  C. Anal. Calc. C9H7N4O3S (%): C, 40.75; H, 2.66; N, 26.40; S, 12.09. Found: C, 40.83; H, 2.59; N, 26.47; S, 12.15. UVeVis (DMF): lmax, nm 260, 339. FT-IR (KBr): ʋ, cm 1 3418, 3354, 3230 (NeH), 1670 (C]O), 1567 (C]N), 1275 (C]S). 1H NMR (500 MHz, DMSO‑d6): d ppm 12.21 (s, 1H, N]NH), 11.78 (s, 1H, NH), 9.19 (s, 1H, NH2), 9.02 (s, 1H, NH2), 8.58 (s, 1H, H4), 8.24 (dd, J ¼ 8.6, 2.0 Hz, 1H, H6), 7.09 (d, J ¼ 8.7 Hz, 1H, H7). 13C NMR (126 MHz, DMSO‑d6): Table 2 Selected geometric parameters (Å,  ). 6 S1eC10/C9 O1eC1 O2eC6 O2eC9 N1eC1 N1eC4 N2eN3 N2eC2 N3eC10/C9 N4eC10/C9 C2eN2eN3 N2eN3eC10/C9 O1eC1eN1 N3eC10/C9eS1 N4eC10/C9eS1 N4eC10/C9eN3 O1eC1eC2eN2 N1eC1eC2eN2 N2eN3eC10/C9eS1 N2eN3eC10/C9eN4 N3eN2eC2eC1 N3eN2eC2eC3 C2eN2eN3eC10/C9 C4eN1eC1eO1 1.683 (2) 1.228 (2) 1.367 (2) 1.430 (3) 1.353 (3) 1.414 (3) 1.355 (2) 1.287 (2) 1.357 (3) 1.312 (3) 116.3 (2) 120.0 (2) 127.0 (2) 118.0 (2) 124.8 (2) 117.3 (2) 0.3 (4) 178.8 (2) 178.8 (2) 1.1 (3) 2.5 (3) 179.6 (2) 177.1 (2) 178.0 (2) 9 (A) (B) 1.684 (5) 1.233 (6) 1.684 (5) 1.226 (6) 1.363 (7) 1.420 (7) 1.360 (6) 1.286 (7) 1.358 (7) 1.314 (7) 116.1 (4) 120.8 (4) 125.6 (5) 117.5 (4) 125.3 (4) 117.2 (4) 1.1 (1) 179.0 (5) 178.3 (4) 1.8 (8) 2.0 (8) 179.6 (5) 179.4 (5) 178.8 (5) 1.366 (7) 1.414 (7) 1.362 (6) 1.285 (7) 1.361 (6) 1.320 (7) 116.6 (4) 119.8 (4) 126.4(5) 118.1 (3) 124.7 (4) 117.2 (4) 1.9 (1) 178.1 (5) 176.3 (4) 3.0 (8) 0.2 (8) 178.2 (5) 179.1 (5) 179.4 (5) d ppm 179.2 (C9), 163.3 (C1), 147.8 (C2), 143.2 (C5), 130.4 (C8), 127.3 (C3), 121.4 (C6), 116.9 (C7), 111.6 (C4). 2.2.5. (Z)-2-(5-methoxy-2-oxoindolin-3-ylidene) hydrazinecarbothioamide (6) 5-Methoxyisatin (0.177 g, 1 mmol) was used. Yield: 79%. Red Table 1 Crystal data and structure refinement parameters for 6 and 9. 6 9 CCDC number Chemical formula Radiation type, Wavelength Mr Crystal system, space group Temperature (K) Unit cell dimensions (Å,  ) 1567446 C10H10N4O2S MoKa, 0.71073 250.28 Monoclinic, P21/n 110 a ¼ 6.4991 (2) b ¼ 10.520 (3) c ¼ 16.104 (4) b ¼ 92.675 (4) V (Å3) Z, Dx (Mg m 3) Absorption coefficient (m) (mm F(000) Color, Crystal size (mm) 1099.8 (5) 4, 1.512 0.29 520 Red, 0.57  0.12  0.12 27.4, 2.3 3652, 3652, 3157 8/8 0 / 13 0 / 20 TWINABS-2012/1 (Bruker, 2012) 0.636, 0.745 Full matrix least-squares on F2 2652, 156 0.648 H-atom parameters constrained 0.039, 0.092 1.09 0.28, 0.27 1567447 C14H18N4OS CuKa, 1.54178 290.38 Triclinic, P-1 110 a ¼ 5.1361 (3) b ¼ 12.4153 (7) c ¼ 23.7159 (1) a ¼ 78.121 (3) b ¼ 89.286 (3) g ¼ 84.078 (4) 1471.9 (2) 4, 1.310 1.97 616 Pale yellow, 0.25  0.2  0.18 55.0, 1.9 20074, 20074, 16572 5/5 13 / 13 24 / 25 1 ) qmax, qmin ( ) No. of measured, independent and observed [I > 2s(I)] reflections h k l Absorption correction Tmin, Tmax Refinement method No. of reflections, parameters sin q/lmax (Å 1) H-atom treatment R [I > 2s(I)] and wR Goodness of fit on F2 (S) Drmax, Drmin (e Å 3) 0.568, 0.752 20074, 364 0.531 0.056, 0.157 1.05 0.34, 0.32 S. Saranya et al. / Journal of Molecular Structure 1198 (2019) 126904 5 Fig. 2. (a) Thermal ellipsoidal plot of 6 at 30% probability. (b) Molecular packing viewed down a axis showing N3eH3/O1 and N4eH4A/N2 intramolecular interactions, and N4eH4A/S1, C5eH5/S1, N4eH4B/O1 and C8eH8/O2 intermolecular interactions. (c) N1eH1/S1 intermolecular interaction running along [101]. solid. m.p.: 197  C. Anal. Calc. C10H10N4O2S (%): C, 47.99; H, 4.03; N, 22.39; S, 12.81. Found: C, 47.85; H, 4.14; N, 22.27; S, 12.90. UVeVis (DMF): lmax, nm 261, 335. FT-IR (KBr): ʋ, cm 1 3431, 3371, 3227 (NeH), 1676 (C]O), 1570 (C]N), 1279 (C]S). 1H NMR (400 MHz, DMSO‑d6): d ppm 12.44 (s, 1H, N]NH), 11.01 (s, 1H, NH), 9.05 (s, 1H, NH2), 8.72 (s, 1H, NH2), 7.32 (d, J ¼ 2.6 Hz, 1H, H4), 6.92 (dd, J ¼ 8.5, 2.6 Hz, 1H, H6), 6.83 (d, J ¼ 8.5 Hz, 1H, H7), 3.75 (s, 3H, H10). 13C NMR (100 MHz, DMSO‑d6): d ppm 179.1 (C9), 163.2 (C1), 155.7 (C5), 136.4 (C2), 132.7 (C8), 121.2 (C3), 117.9 (C6), 112.2 (C7), 106.6 (C4), 56.0 (C10). 2.2.6. (Z)-2-(2-oxo-1-(prop-2-yn-1-yl)indolin-3-ylidene) hydrazinecarbothioamide (8) N-Propargylisatin (0.185 g, 1 mmol) was used. Yield: 75%. Pale yellow solid. m.p.: 188  C. Anal. Calc. C12H10N4OS (%): C, 55.80; H, 3.90; N, 21.69; S, 12.41. Found: C, 55.68; H, 4.03; N, 21.56; S, 12.50. UVeVis (DMF): lmax, nm 259, 332. FT-IR (KBr): ʋ, cm 1 3424, 3215 (NeH), 1679 (C]O), 1571 (C]N), 1273 (C]S). 1H NMR (500 MHz, DMSO‑d6): d ppm 12.27 (s, 1H, N]NH), 9.11 (s, 1H, NH2), 8.76 (s, 1H, NH2), 7.74 (d, J ¼ 7.0 Hz, 1H, H4), 7.48 (td, J ¼ 7.9, 1.1 Hz, 1H, H6), 7.21 (dd, J ¼ 13.3, 7.7 Hz, 2H, H7), 4.62 (d, J ¼ 2.4 Hz, 2H, H10), 3.33 (t, J ¼ 2.4 Hz, 1H, H12). 13C NMR (125 MHz, DMSO‑d6): d ppm 179.1 (C9), 160.3 (C1), 142.1 (C2), 131.6 (C5), 131.0 (C8), 123.79 (C6), 121.3 (C4), 119.8 (C3), 110.8 (C7), 77.8 (C12), 75.3 (C11), 29.0 (C10). 2.2.7. (Z)-2-(2-oxo-1-pentylindolin-3-ylidene) hydrazinecarbothioamide (9) 5-Pentylisatin (0.217 g, 1 mmol) was used. Yield: 68%. Pale yellow solid. m.p.: 208  C. Anal. Calc. C14H18N4OS (%): C, 57.91; H, 6.25; N, 19.29; S, 11.04. Found: C, 57.82; H, 6.14; N, 19.36; S, 11.16. UVeVis (DMF): lmax, nm 258, 340. FT-IR (KBr): ʋ, cm 1 3417, 3229 (NeH), 1670 (C]O), 1575 (C]N), 1270 (C]S). 1H NMR (500 MHz, CDCl3): d ppm 12.91 (s, 1H, N]NH), 7.57 (d, J ¼ 7.5 Hz, 1H, H4), 7.53 (s, 1H, NH2), 7.39 (t, J ¼ 7.7 Hz, 1H, H6), 7.11 (t, J ¼ 7.6 Hz, 1H, H5), 6.89 (d, J ¼ 7.9 Hz, 1H, H7), 6.60 (s, NH2), 3.74 (t, J ¼ 7.2 Hz, 2H, H10), 1.71 (dd, J ¼ 14.1, 7.1 Hz, 2H, H11), 1.36 (d, J ¼ 3.7 Hz, 4H, H12, H13), 0.91 (t, J ¼ 6.5 Hz, 3H, H14). 13C NMR (125 MHz, CDCl3): d ppm 180.0 (C9), 161.0 (C1), 143.5 (C2), 132.3 (C6), 131.6 (C8), 123.1 (C5), 120.9 (C4), 119.4 (C3), 109.5 (C7), 39.9 (C10), 29.0 (C12), 27.2 (C11), 22.2 (C13), 13.9 (C14). 2.3. X-ray crystallography and Hirshfeld surface analysis Three dimensional crystal structure of compounds 6 and 9 was 6 S. Saranya et al. / Journal of Molecular Structure 1198 (2019) 126904 2.4. Antioxidant assay Antioxidant capability of the compounds was investigated by DPPH method [32] with minor modifications and compared with positive control, ascorbic acid. The DPPH solution (10 mM) was incubated with different concentrations (10, 25, 50, 75, 100, 250 and 500 mg/mL) of the synthesized compounds for 30 min in dark at room temperature. The mixture was analyzed for the absorbance at 517 nm by UVeVis spectrophotometer. Lower the absorbance, higher the free radical scavenging capability. Percentage scavenging was calculated using the formula, % Scavenging ¼ [(Abscontrol-Abssample)/Abscontrol]  100. 2.5. Cytotoxicity MTT [3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed to evaluate the cytotoxic effect of the compounds [33]. The breast cancer cell line MCF-7 was procured from National Center for Cell Sciences (NCCS), Pune and maintained in Dulbecco's Minimal Medium (DMEM) supplemented with 10% FBS in a 5% CO2 incubator at 37  C. When the cells reach confluence, 1  105/well was seeded in a 96 well plate and incubated. After 24 h, different concentrations (25, 50, 100, 250, 500 mg/mL) of the compounds were added to each well and incubated for 72 h. After the incubation time, the cells were washed with phosphate buffer saline (pH 7.4); 10 mL of 0.5% MTT solution was added to each well and incubated for 4 h in 100 mL of solubilization solution (40% DMF in 2% glacial acetic acid dissolved in 16% SDS). The absorbance was read at 570 nm using plate reader. Cell viability percentage was calculated based on the formula, Cell viability % ¼ (Abssample/ Abscontrol)  100. 2.6. Secretory phospholipase A2 inhibition study Secretory phospholipase A2 (PLA2) inhibition assay has been performed using Cayman Chemical sPLA2 assay kit [17]. Absorbance at 414 nm was measured for 15 min at a time scale of every minute, and absorbance difference was calculated using the formula, DA414 ¼ [DA414 (time 2) - DA414 (time 1)]/(time 2 - time 1). 2.7. Molecular docking Fig. 3. (a) Thermal ellipsoidal plot of 9 at 30% probability. (b) Molecular packing viewed down a axis showing intra- and inter-molecular hydrogen bonding interactions. determined by X-ray diffraction studies. Data collection was performed using MoKa radiation for 6 and CuKa radiation for 9 with BRUKER APEX-II CCD diffractometer [28]. SAINT [28] program was used for cell refinement and data reduction. SHELXT [29] and SHELXL [29] programs were used for structure determination (by direct methods) and refinement (with least squares refinement procedure), respectively. PLATON [30] was used to represent thermal ellipsoidal plots, and inter- and intra-molecular hydrogen bonds. Hirshfeld surface analysis has been carried out for molecules 6 and 9 using Crystal-Explorer v.17.5 [31]. The compounds were subjected to molecular docking with sPLA2 as target enzyme to reveal the binding mode of the compounds at the active site of sPLA2. Schrodinger-Maestro [34] was used to perform induced fit molecular docking. Two dimensional coordinates of the thiosemicarbazone derivatives were generated using ChemSketch [35], which were further converted to 3D coordinates and energy minimized using Ligprep module in €dinger-Maestro. Three dimensional coordinates of human Schro non-pancreatic sPLA2 were downloaded from RCSB Protein Data Bank (PDB) [36] and energy minimized using protein preparation wizard. Induced fit molecular docking studies resulted in various binding modes of the compounds at the active site and the best mode of binding was selected based on the docking energy, score and active site interactions. Hydrogen bonding and hydrophobic interactions were identified by PLIP server [37]. 3. Results and discussion 3.1. Synthesis For this study, a series of isatin thiosemicarbazone compounds was obtained from (un)substituted isatin and thiosemicarbazide in 7 S. Saranya et al. / Journal of Molecular Structure 1198 (2019) 126904 Table 3 Intra- and inter-molecular hydrogen bonds. Donor Hydrogen$$$Acceptor 6 N3eH3/O1 N4eH4A$$$N2 N1eH1/S1 ( 1/2þx,1/2-y,-1/2þz) N4eH4A$$$S1 (1/2-x,1/2þy,3/2-z) N4eH4B/O1 (1/2-x,1/2þy,3/2-z) C5eH5/S1 (1/2-x,1/2þy,3/2-z) C8eH8/O2 (1/2-x,-1/2þy,1/2-z) 9 N3eH3/O1 N3BeH3B/O1B N4eH4A$$$N2 N4eH4B/N2B N4eH4B/S1 (3-x,2-y,1-z) N4BeH4B/S1B (2-x,1-y,2-z) C7eH7/O1B ( 1þx,y,z) C7BeH7B/O1 (1þx,-1þy,z) D H (Å) H $$$A (Å) D$$$A (Å) D H$$$A ( ) 0.88 0.88 0.88 0.88 0.88 0.95 0.95 2.06 2.28 2.79 2.63 2.12 2.83 2.52 2.757(2) 2.636(3) 3.322(2) 3.450(2) 2.960(2) 3.715(2) 3.421(3) 135 104 120 155 158 156 159 0.88 0.88 0.88 0.88 0.88 0.88 0.95 0.95 2.00 2.04 2.30 2.29 2.52 2.53 2.52 2.58 2.714(5) 2.738(5) 2.655(6) 2.643(6) 3.389(4) 3.396(5) 3.348(6) 3.450(5) 137 135 104 104 172 168 145 152 Fig. 4. Hirshfeld surface for 6 mapped with dnorm and shape index. Fig. 5. Hirshfeld surface for 9 mapped with dnorm and shape index. the presence of glacial acetic acid (Scheme 1). These compounds were obtained in red/pale yellow solid form, which were insoluble in most of the organic solvents except acetone, DMF and DMSO, and partially soluble in chloroform, dichloromethane and methanol. The compounds were air and light stable. All the thiosemicarbazone derivatives were satisfactorily characterized by elemental analyses and various spectroscopic (UVeVisible, FT-IR and NMR) tools. The molecular structure of compounds 6 and 9 was determined by single crystal X-ray diffraction method. 3.2. Spectroscopy UVeVisible spectra of the thiosemicarbazone compounds in DMF revealed mainly two strong absorption bands at 257e261 and 332e343 nm (Fig. S1) which were assigned to intra-ligand charge transfer p/p* and n/p*, respectively [38]. FT-IR spectra (Figs. S2eS8) exhibited a broad and sharp bands around 32273431 cm 1, which were due to the stretching of the thioamide, isatin and terminal NeH groups [39]. The carbonyl (C]O) 8 S. Saranya et al. / Journal of Molecular Structure 1198 (2019) 126904 Fig. 6. Electronic potential surface for molecules (a) 6 and (b) 9. Fig. 7a. Two-dimensional fingerprint of molecule 6 showing complete and individual contribution of the interactions within the compound contributing to the total Hirshfeld surface area. stretching frequency of the compounds was observed at 16701679 cm 1. The stretching frequencies of imine (C]N) and thiocarbonyl (C]S) were detected at 1559e1579 and 1270-1279 cm 1, respectively [40]. NMR spectra of the thiosemicarbazone derivatives were recorded in CDCl3 or DMSO‑d6. The thioamide NH and terminal NH2 S. Saranya et al. / Journal of Molecular Structure 1198 (2019) 126904 9 Fig. 7b. Two-dimensional fingerprint of molecule 9 showing complete and individual contribution of the interactions within the compound contributing to the total Hirshfeld surface area. protons were appeared at 12.91e12.21 and 9.11e6.60 ppm, respectively [41]. The isatin NH (2e6) proton resonance was observed around 11.78e11.01 ppm as singlet [39]. The resonances due to aromatic ring protons were shown at 8.58e6.83 ppm (Figs. S9eS16). A sharp singlet appeared at 3.75 ppm in the 1H NMR spectrum of 6 was assigned to methoxy protons. In the spectrum of compound 8, the signals due to propargylic group protons were observed at 4.62e3.33 ppm [42]. The signals at 3.74e0.91 ppm in the spectrum of 9 corresponded to pentyl protons. 13C NMR spectra (Figs. S17eS21) of the compounds exhibited resonances due to thiocarbonyl (C]S), carbonyl (C]O) and imine (C]N) carbons in the regions 180.0e177.9, 163.3e159.2 and 159.6e141.1 ppm, respectively [43]. The signals due to propargylic carbons were seen at 77.8, 75.3 and 29.0 ppm. The resonance for the methoxy carbon (6) was shown at 56.0 ppm. The pentyl carbons in 9 were observed at 39.9, 29.0, 27.2, 22.2 and 13.9 ppm in its 13C NMR spectrum. 3.3. Three dimensional structural studies Three dimensional crystal structures of two compounds (6 and 9) are reported here. Data collection and structure refinement details are summarized in Table 1. Compound 6 crystallized in monoclinic and 9 in triclinic system with P21/n and P-1 space groups, respectively. Compound 9 crystallized with two independent molecules in the asymmetric unit cell. Structures were determined to a final R value of 3.88 and 5.64%, respectively for 6 10 S. Saranya et al. / Journal of Molecular Structure 1198 (2019) 126904 and 9. Crystallographic information files have been deposited in Cambridge structure database with CCDC numbers 1567446 and 1567447 for 6 and 9, respectively. Bond lengths and bond angles were in the allowed range and are comparable with the similar structures [27,39,41]. Selected bond lengths, bond angles and torsion angles are listed in Table 2. In the X-ray diffraction data of 6, for component 1, wR2(int) was 0.1030 and 0.0695, respectively, before and after correction [44]. The ratio of minimum to maximum transmission was found to be 0.87 and l/2 correction factor was not present. For component 2, wR2(int) was 0.1169 and 0.0362, respectively, before and after correction. The ratio of minimum to maximum transmission and l/ 2 correction factor were not present. Final HKLF 4 output contains 9495 reflections with Rint ¼ 0.0320 (5096 with I > 3s(I), Rint ¼ 0.0254). For 9, for component 1, wR2(int), before and after correction, was found to be 0.0954 and 0.0614, respectively. The ratio of minimum to maximum transmission was 0.75. For component 2, wR2(int) was found to be 0.0970 and 0.0602, respectively, before and after correction. The ratio of minimum to maximum transmission was not present. Final HKLF 4 output contains 20295 reflections with Rint ¼ 0.0744 (14635 with I > 3s(I), Rint ¼ 0.0705). The l/2 correction factor was not present in both the components. Molecular structure of 6 and 9 was stabilized by N3eH3/O1 and N4eH4A$$$N2 intramolecular interactions which generated S(6) and S(5) motifs, respectively. Crystal packing in 6 was strongly stabilized by intermolecular hydrogen bonding networks formed by NeH/S, CeH/S, NeH/O and CeH/O interactions. N4eH4A$$$S1 and C5eH5/S1 interactions generated C(4) and C(8) chains, respectively, running along b axis wherein S1 acts as a bifurcated acceptor. N1eH1/S1 interaction generated C(8) chain along ac plane. C8eH8/O2 and N4eH4B/O1 interactions generated C(5) and C(8) chains along b axis, respectively. N4eH4/S1 and C7eH7/O1 intermolecular interactions in 9 produced centrosymmetric dimer of R22(8) ring and C(7) chain along b axis, respectively. The thermal ellipsoidal plots and molecular packing are provided in Figs. 2 and 3. Intra- and inter-molecular hydrogen bonding parameters are listed in Table 3. intermolecular interactions using the combination of di (x axis) and de (y axis) which are the closest internal and external distances (in Å) from certain given points on the predicted Hirshfeld surface. Figs. 3 and 4 show the Hirshfeld surface of compounds 6 and 9, which was mapped over dnorm ( 0.03 to 1.3 Å) and shape-index ( 1.0 to 1.0 Å). The vivid red spots in Fig. 4 represent the normalized N/H, O/H and S/H distances corresponding to NeH/N, NeH/O and CeH/S interactions, respectively for molecule 6. Red spots in Fig. 5 represent the N/H, O/H and S/H distances which correspond to NeH/N, CeH/O and NeH/S interactions as established in the analysis of the crystal structure. In the shapeindex surfaces, the blue and red regions represent the hydrogen donor and acceptor groups respectively. Electronic potential surfaces were mapped (Fig. 6) using TONTO [45,46] which was incorporated into the Crystal Explorer software. The figure shows negative potential around the oxygen atoms as light-red clouds and positive potential around hydrogen atoms as light-blue clouds. The contribution made by each type of covalent interactions to the Hirshfeld surface was quantified by 2D fingerprint plots. The intermolecular interactions involved in the structure appeared as distinct spikes in the fingerprint plot and the proportions of each interaction have been mentioned in Fig. 7. The 2D fingerprint plot of 6 (Fig. 7a) showed that the major contribution was due to H/H contacts (33.2%), representing the van der Waals interactions, followed by S/H, O/H, C/H, C/C and N/H interactions contributing 18.9, 17.7, 8.7, 6.9 and 4.7%, respectively. The remaining 9.9% of the interactions were contributed by S/N, O/O, O/N, C/O and N/C. Similarly, the 2D fingerprint maps of 9 (Fig. 7b) revealed that H/H contacts made the major contribution of about 54.3% followed by S/H, C/H, O/H, N/H and C/C contacts being 13.1, 10.7, 6.9, 4.9 and 3.3%, respectively. The S/H and O/H contacts are represented by sharp peaks. The other interactions like S/O, S/N, S/C, C/O, N/N and N/C contributed 6.8% of the remaining interactions. The 2D maps of both the molecules showed that more regions were occupied by the H/H interactions, making the most significant contribution to the Hirshfeld surface. 3.5. DPPH assay 3.4. Hirshfeld surface analysis Hirshfeld surface analysis enables the visualization of intermolecular interactions and also predicts the percentage contributions of various intermolecular contacts in the crystal structures of molecules. The generated 2D fingerprint plots showcase the Free radical scavenging activity of the compounds was carried out in the presence of DPPH (1,1-diphenyl-2-picrylhydrazyl) using ascorbic acid as a positive control [47]. Antioxidant potential of the compounds to scavenge free radicals was measured at 517 nm [48]. The compounds showed dose dependent antioxidant property Fig. 8. Antioxidant activity of ascorbic acid (AA) and the thiosemicarbazone compounds (1e10) at different concentrations using DPPH assay. Each value represents a mean ± SD (n ¼ 3). S. Saranya et al. / Journal of Molecular Structure 1198 (2019) 126904 (Fig. 8). Among the compounds, 1, 5 to 8 and 10 showed good scavenging effect towards DPPH with IC50 of ~25, 10, 10, 10, 25 and 50 mg/mL, respectively. Compounds 2, 3, 4 and 9 showed to have the least radical scavenging property. From the above results, it was evident that compounds 5, 6 and 7 have higher antioxidant potential at the concentration of 10 mg/mL. 3.6. Cytotoxicity Cytotoxic effect of the compounds on breast cancer (MCF-7) cell line was evaluated using MTT assay. The results revealed that the compounds were toxic for the breast cancer cell line in dose dependent manner (Fig. 9). Results indicated that MCF-7 cell line was sensitive towards compound 7 (IC50 ¼ 100 mg/mL). Compounds 1, 3 and 10 showed 50% inhibition at 250 mg/mL concentration. Of all the compounds, 7 showed better cytotoxic effect. 11 inflammation or altering the microenvironment thereby leading to cell growth, survival, migration and invasion [49]. PLA is further classified into PLA1 and PLA2 enzymes as they cleave fatty acid ester bonds at sn-1 and sn-2 positions of glycerol moieties of phospholipids, respectively, to release fatty acid and lysophospholipid. PLA2, irrespective of cytosolic or secretory, plays a key role in inflammation related disorders and also in cancer. Secretory PLA2 (sPLA2) plays a major role in eicosanoid production and in the formation of arachidonic acid cascade which further results in the synthesis of leukotrienes and prostaglandins. In vitro sPLA2 inhibition study has been performed to screen the inhibition potential of istain based thiosemicarbazones against sPLA2 by using the protocol reported in our earlier work [17]. sPLA2 enzyme inhibition activity was calculated based on the absorbance measurement at 414 nm and the percentage inhibition of sPLA2 is shown in Fig. 10. 50% inhibition of sPLA2 was observed at a concentration of ~200 mg/ mL for all the compounds. 3.7. PLA2 inhibition study 3.8. In silico molecular docking Phospholipids are the major structural components of cell membrane which can be broken down to lipid mediators by phospholipases through hydrolysis. This pathway is activated by various factors such as extracellular signals, growth factors and lipids. Based on the hydrolytic cleavage site, phospholipases are classified into three major types, phospholipase A (PLA), phospholipase C (PLC) and phospholipase D (PLD). PLA indirectly or directly influences cancer cells and tumour growth by inducing Results of induced fit molecular docking studies revealed several hydrogen bonds and hydrophobic interactions at the sPLA2 active site [50]. Active site interactions for 1 to 10 are represented using PLIP as shown in Fig. 11. Docking energy and glide score were comparable with the co-crystal ligand (1-benzyl-5-methoxy-2methyl-1h-indol-3-yl)-acetic acid (Table 4). His 47 and Asp 48 are the most important residues at the active site of PLA2 enzyme. Fig. 9. Percentage of MCF-7 cell viability after 72 h of treatment with the thiosemicarbazone derivatives (1e10). Fig. 10. PLA2 inhibition activity of the thiosemicarbazone compounds (1e10). 12 S. Saranya et al. / Journal of Molecular Structure 1198 (2019) 126904 Fig. 11. Interactions of the thiosemicarbazone derivatives at the active site of PLA2. Hydrogen bonds and hydrophobic interactions are represented by dashed and dotted lines, respectively. Ligand binding at the active site and interactions with these residues may inhibit the binding of the substrate and thereby inhibits the progression of down pathway. Clearly, all the compounds used in the present study were observed to interact with these residues in addition to other hydrogen bonding and hydrophobic interactions. 4. Conclusions Ten isatin based thiosemicarbazone compounds were synthesized and characterized by analytical and various spectroscopic methods. Three dimensional crystal structure of compounds 6 and 9 was confirmed by X-ray crystallography and refined to good R factors. Molecular structure of 6 and 9 was stabilized by NeH/N S. Saranya et al. / Journal of Molecular Structure 1198 (2019) 126904 13 Fig. 11. (continued). Table 4 Docking energy and score. Compound Co-crystal ligand 1 2 3 4 5 6 7 8 9 10 Energy (kcal/mol) 50.522 41.968 45.013 45.534 43.578 43.439 47.656 46.600 48.281 45.404 48.438 Score 7.551 5.604 6.269 6.473 6.098 5.005 5.655 5.706 5.778 7.051 5.607 and NeH/O intramolecular interactions which are notable in these derivatives. Crystal packing is stabilized by several hydrogen bonding interactions. Hirshfeld surface and two dimensional fingerprint analyses revealed several interactions. Antioxidant potential of the thiosemicarbazone derivatives was analyzed by in vitro DPPH free radical scavenging assay. Even though all the compounds showed potential scavenging effect, compounds 5, 6 and 7 showed more than 50% scavenging effect at 10 mg/mL concentration. Cytotoxicity of the thiosemicarbazone derivatives was evaluated with MCF-7 cell line and the results revealed that compound 7 showed more than 50% inhibition at 100 mg/mL concentration. Anti-inflammatory activity of the thiosemicarbazones was proved by in vitro PLA2 assay (~50% inhibition at ~200 mg/mL concentration). In silico molecular docking studies of the compounds at the active site of PLA2 revealed the binding potential of the compounds as evident from several hydrogen bonding and hydrophobic interactions. Isatin based thiosemicarbazone derivatives being potential compounds with wide range of promising biological properties may be explored further for the treatment of several diseases. 14 S. Saranya et al. / Journal of Molecular Structure 1198 (2019) 126904 Acknowledgements J. H. thanks the University Grants Commission (F1-17.1/2012-13/ RGNF-2012-13-ST-AND-18716), Government of India for the financial support. DG thanks DST and UGC for the financial support to the department. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.molstruc.2019.126904. References [1] J. Haribabu, G. Sabapathi, M.M. 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