Synthesis and Anti-inflammatory Activity Evaluation of Novel Triazolyl-isatin Hybrids

Synthesis and Anti-inflammatory Activity Evaluation of Novel Triazolylisatin Hybrids Pramod K Sharmaa,b, Sakshi Balwanic, Divya Mathura,d, Shashwat Malhotraa, Brajendra K Singha, Ashok K Prasada, Christophe Lene,f, Erik V Van der Eyckeng, Balaram Ghoshc , Nigel G J Richardsh,i and Virinder S Parmara,e,h* a Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi-110 007, India b Chemical Research Laboratory, Wockhardt Research Centre, Aurangabad – 431 210, Maharashtra, India c Immunogenetics Laboratory, CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi – 110 007, India d Department of Chemistry, Daulat Ram College, University of Delhi, Delhi-110 007, India e Sorbonne universités, Université de Technologie de Compiègne (UTC), Ecole Supérieure de Chimie Organique et Minérale (ESCOM), CS 60319, 60203 Compiègne Cedex, France f Department of Chemistry, University of Hull, Hull HU6 7RX, UK g Laboratory for Organic & Microwave-Assisted Chemistry (LOMAC), Department of Chemistry, University of Leuven (KU Leuven), Celestijnenlaan 200F, B-3001 Leuven, Belgium h Department of Chemistry and Chemical Biology, Indiana University-Purdue University (IUPUI), 402 North Blackford Street, Indianapolis, IN 46202-3074, USA i School of Chemistry, College of Physical Sciences & Engineering, Cardiff University, Park Place, Cardiff, CF10 3AT, UK Email: virparmar@gmail.com Abstract: New isatin-triazole based hybrids have been synthesized and evaluated for their inhibitory activity of TNF-α induced expression of Intercellular Adhesion Molecule-1 (ICAM-1) on the surface of human endothelial cells Structure-activity relationship (SAR) studies revealed that the presence of the electron-attracting bromo substituent at position-5 of the isatin moiety played an important role in enhancing the antiinflammatory potential of the synthesized compounds Z-1-[3-(1H-1, 2, 4-Triazol-1yl)propyl]-5-bromo-3-[2-(4-methoxyphenyl)hydrazono]indolin-2-one (19) with an IC50 = 20 µM and 89 % ICAM-1 inhibition with MTD at 200 µM was found to be the most potent of all the synthesized derivatives Introduction of 1, 2, 4-triazole ring and electrondonating methoxy group on the phenylhydrazone moiety increased the anti-inflammatory activity four fold Keywords: Isatin, 1, 2, 4-triazole, anti-inflammatory activity, ICAM-1, triazolylisatins Table of abbreviations: ICAM-1 MTD SAR VCAM-1 HUVEC’s MTT Intercellular Adhesion Molecule-1 Maximal tolerable dose Structure-activity relationship Vascular cell adhesion molecule-1 Human umbilical vein endothelial cells Methylthiazolydiphenyl-tetrazolium bromide Introduction Indoline-2, 3-diones or indole-1H-2, 3-diones, commonly known as isatins have been extensively studied due to their diverse pharmacological properties and synthetic versatility Isatins are a well-known class of natural products found in plants of the genus Isatis [1], Calanthe discolor LINDL [2] and Couroupita guianensis Aubl [3] Various substituted isatins have been isolated from plants, e.g melosatin alkaloids from Melochia tomentosa, a caribbean tumorigenic plant [4]; 6-(3′ -methylbuten-2′ -yl)isatin from fungi, Streptomyces albus [5] and 5-(3′ -methylbuten-2′ -yl)isatin from Chaetomium globosum [6] It is also known that isatins act as endogenous biological regulators, found in the brain, peripheral tissues, and body fluids of humans and animals [7] Isatin was first synthetically obtained as an oxidation product of indigo in the early 19 th century by Erdman and Laurent [8] The synthetic interest in the chemistry of isatin and its derivatives stemmed from its easy synthetic accessibility and exhibition of broad spectrum biological effects, including antibacterial, antifungal, anticonvulsant, antiviral, anticancer, antioxidant, antiinflammatory and antiproliferative activities [9-16] Isatin derivatives, such as hydrazones, Schiff’s and Mannich bases are of great medicinal value owing to their numerous chemotherapeutic properties [17-19] The 1, 2, 4-triazole moiety is present in a wide variety of therapeutically interesting drugs, such as ribavarin [20], triazolam [21], fluconazole [22] and voriconazole [23] Anticancer [24], antitubercular [25], analgesic [26], antimicrobial [27] and anti-inflammatory activities of 1, 2, 4-triazole derivatives have also been reported [28] Recently, some 1, 2, 4-triazole containing isatin derivatives possessing interesting biological activities have been described [29-31] During an inflammatory cascade, various inflammatory mediators, including cytokines such as TNF-α, IL-1β, and bacterial lipopolysaccharides induce the expression of endothelial cell adhesion molecules, viz intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, on the vascular endothelium [32] The increased levels of expression of cell adhesion molecules on the endothelial cells alter the adhesive property of the vasculature, leading to indiscriminate infiltration of the leukocytes across the blood vessels, and thus causing inflammation A promising approach for the therapeutic intervention of inflammatory disorders is by pharmacological inhibition of the CAM expression of endothelial cells [33] In order to develop safer and potent anti-inflammatory agents/drugs, our laboratories have identified a number of small molecules from natural/synthetic sources that efficiently block nuclear accumulation of NF-ĸB and abrogate TNF-α induced expression of E-selectin, VCAM-1 and ICAM-1 on human umbilical vein endothelial cells (HUVEC’s) [34-36] In drug discovery, the development of hybrid molecules through the combination of different pharmacophores leads to compounds with interesting biological profiles Prompted by the anti-inflammatory activities associated with 1, 2, 4-triazole and isatin derivatives, we have synthesised a series of novel isatin derivatives by connecting the isatin core moiety with triazole moiety using various alkyl chain linkers Also, the Schiff’s bases and oximes were prepared using substituted hydrazines and hydroxylamine Both p-methoxyphenyl hydrazine and pentafluorophenyl hydrazine were used as amines for the condensation step, as we wanted to evaluate the effects of both electron donating and electron withdrawing substituents on the phenyl ring of the amine for the ICAM-1 expression inhibition studies The synthesized triazolylisatins were then screened for their inhibition of TNF-α induced expression of ICAM-1 in HUVEC’s The structure activity relationship (SAR) of the synthesized compounds has also been well established to search for potential lead compounds as anti-inflammatory drugs Our findings revealed that Z-1-[3-(1H-1, 2, 4triazol-1-yl)propyl]-5-bromo-3-[2-(4-methoxyphenyl)hydrazono]indolin-2-one (19) with an IC50 value of 20 µM and 89 % inhibition in HUVEC’s was found to be the most potent ICAM-1 expression inhibitor Materials and Methods General: Analytical TLCs were performed on Merck silica gel 60 F 254 plates All flash chromatographic separations were performed on 100-200 mesh silica gel The IR spectra were recorded on a Perkin-Elmer 2000 FT-IR spectrometer The 1H NMR and 13C NMR spectra were recorded on a Bruker AC-300 Avance spectrometer at 300 MHz and 75.5 MHz, respectively using TMS as internal standard Chemical shifts are reported on δ scale and coupling constants (J) are in Hz The HRMS determinations were made in FAB positive mode on a JEOL JMS-AX505W high-resolution mass spectrometer using bishydroxyethyldisulfide (HEDS) doped with sodium acetate as matrix in the Laboratory of Dr Carl-Erik Olsen at the University of Copenhagen (Denmark) Melting points were recorded in a sulfuric acid bath and are uncorrected Materials: Materials were obtained from commercial suppliers and were used without further purification unless otherwise noted Petroleum ether and ethyl acetate were distilled over P2O5 and K2CO3, respectively prior to use The compounds & 2, the endothelial cell growth factor (ECGF), M199 medium, l-glutamine, MTT (methylthiazolydiphenyl-tetrazolium bromide), trypsin, o-phenylenediamine and goat anti-mouse IgG–HRP conjugate were procured from Sigma Chemical Co (USA) The fetal calf serum (FCS) was procured from Biological Industries (Israel) Methods General method for the preparation of compounds 3-6: To a solution of compound 1/2 (2 mmol) and anhydrous potassium carbonate (2 mmol) in acetonitrile (20 mL), 1, 2dibromoethane/1, 3-dibromopropane (0.03 mol) was added dropwise and the resultant mixture was refluxed for h On completion of the reaction, the reaction mixture was filtered and solvent evaporated under reduced pressure The residue was purified by silica gel column chromatography using ethyl acetate/petroleum ether (3:1, v/v) as an eluent to afford the compounds 3-6 in 60-65 % yields 1-(2-Bromoethyl)indoline-2, 3-dione (3) Orange solid Yield: 65 % MP: 128-130 °C (literature MP: 131-132 °C) [37] 1-(3-Bromopropyl)indoline-2, 3-dione (4) Orange solid Yield: 60 % MP: 182-84°C (literature MP: 187-188 °C) [37] IR (KBr): 3454, 2956, 1738, 1614, 1468 cm-1; 1H NMR (300 MHz, DMSO-d6):δ 2.132.16 (m, 2H, C-2'H), 3.60 (t, 2H, J = 3.0 Hz, C-1'H), 3.78 (t, 2H, J = 3.0 Hz, C-3'H), 7.09-7.12 (m, 1H, ArH), 7.17-7.20 (m, 1H, ArH), 7.54-7.55 (m, 1H, ArH), 7.64-7.66 (m, 1H, ArH); 13C NMR (75.5 MHz, DMSO-d6): δ 30.04 (C-3'), 31.83 (C-2'), 58.24 (C-1'), 110.48 (C-7), 117.71 (C-4), 123.13 (C-5), 124.44 (C-6), 138.03 (C-8), 150.50 (C-9), 158.32 (C-2), 183.28 (C-3) HRMS-FAB: m/z [M + Na+] calcd for C11H10BrNO2Na: 289.1102; found: 289.1100 5-Bromo-1-(2-bromoethyl)indoline-2,3-dione (5) Orange solid Yield: 62 % MP: 180-182 °C (literature MP: 180-184 °C) [37] IR (KBr): 3458, 1738, 1602, 1470, 1437 cm -1; 1H NMR (300 MHz, DMSO-d6):δ 3.67 (t, 2H, J = 6.6 Hz, C-1'H), δ 4.10 (t, 2H, J = 6.6 Hz, C-2'H), 7.30 (d, 1H, J = 8.4 Hz, C-6H), 7.66 (s, 1H, C-4H), 7.85 (d, 1H, J = 7.2 Hz, ArH); 13C NMR (75.5 MHz, DMSO-d6): δ 28.95 (C-2'), 41.28 (C-1'), 113.29 (C-7), 115.14 (C-5), 119.17 (C-6), 126.80 (C-4), 139.90 (C-8), 149.12 (C-9), 157.74 (C-2), 181.73 (C-3) HRMS-FAB:m/z [M+Na]+calcd for C10H7Br2NO2Na: 353.8736; found: 353.8726 5-Bromo-1-(3-bromopropyl)indoline-2,3-dione (6) Orange solid Yield: 60 % MP: 155-158 °C (literature MP: 155 °C) [38] 1H NMR (300 MHz, DMSO-d6):δ 3.75 (t, 2H, J = 6.6 Hz, C-3'H), δ 2.16-2.73 (m, 2H, C-2'H), 7.1 (d, 1H, J = 8.4 Hz, C-6H), 7.68 (s, 1H, C-4H), 7.83 (d, 1H, J = 8.4 Hz, C-7H); 13CNMR (75.5 MHz, DMSO-d6): δ 30.17 (C-3'), 31.70 (C-2'), 40.34 (C-1'), 112.62 (C-7), 114.80 (C-6), 119.52 (C-5), 126.60 (C-4), 139.60 (C-8), 149.38 (C-9), 157.93 (C-2), 182.01 (C3) HRMS-FAB: m/z [M + Na+] calcd for C10H7Br2NO2Na: 367.8892; found: 367.8889 General method for the preparation of compounds 7-10: To a solution of compound 3-6 (2 mmol) and anhydrous potassium carbonate (2 mmol) in acetonitrile (10 mL), 1, 2, 4triazole (2 mmol) in acetonitrile (10 mL) was added dropwise and the reaction mixture was stirred and refluxed for h On completion, the reaction mixture was filtered and solvent evaporated under reduced pressure The residue thus obtained was purified by silica gel column chromatography using ethyl acetate/petroleum ether (3:1, v/v) as an eluent to afford the compounds 7-10 in 65-75 % yields 1-[2-(1H-1, 2, 4-Triazol-1-yl)ethyl]-indoline-2,3-dione (7) Orange solid Yield: 65 % MP: 105-108 °C IR (KBr): 3116,1739, 1612, 1510, 1469 cm; H NMR (300 MHz, DMSO-d6): δ 4.04 (t, 2H, J = 5.4 Hz, C-1'H), 4.45 (t, 2H, J = 5.7 1 Hz, C-2'H), 6.77 (d, 1H, J = 8.1 Hz, C-7H), 7.07 (t, 1H, J = 7.5 Hz, C-5), 7.50-7.56 (m, 2H, C-4H and C-6H) 7.90 (s, 1H, C-3''), 8.50 (s, 1H, C-5''); 13 C NMR (75.5 MHz, DMSO-d6): δ 46.37 (C-1' & C-2'), 110.0 (C-7), 117.35 (C-4), 123.27 (C-5), 124.52 (C-6), 138.12 (C-8), 144.77 (C-3''), 150.36 (C-9), 151.68 (C-5''), 158.18 (C-3), 183.03 (C-2) HRMS-FAB: m/z [M + Na+] calcd for C12H10N4O2Na: 265.0696; found: 265.0696 1-[3-(1H-1, 2, 4-Triazol-1-yl)propyl]-indoline-2,3-dione (8) Orange solid Yield: 68 % MP: 103-106 °C IR (KBr): 3446, 3100, 1724, 1605, 1468 cm; 1H NMR (300 MHz, DMSO-d6): δ 2.11- 2.15 (m, 2H, C-2'H), 3.65-3.70 (m, 2H, C- 3'H), 4.24-4.29 (m, 2H, C-1' H), 7.09-7.15 (m, 2H, C-4H, C-6H), 7.52 (d, 1H, J = 7.2 Hz, C-7H), 7.64 (t, 1H, J = 7.8 Hz, C-5H), 8.44 (s, 1H, C-3''), 8.50 (s, 1H, C-5''); 13C NMR (75.5 MHz, DMSO-d6): δ 27.06 (C-2'), 36.83 (C-1'), 46.13 (C-3'), 110.49 (C-7), 117.69 (C-5), 123.14 (C-6), 124.41 (C-4), 138.01 (C-8), 144.10 (C-3''), 150.38 (C-9), 151.46 (C5''), 158.29 (C-3), 183.28 (C-2) HRMS-FAB: m/z [M + Na+] calcd for C13H12N4O2Na: 279.2696; found: 279.2694 1-[2-(1H-1, 2, 4-Triazol-1-yl)ethyl]-5-bromoindoline-2,3-dione (9) Orange solid Yield: 70 % MP: 104-106 °C IR (KBr): 3449, 3117, 1739, 1611, 1440 cm1 ; 1H NMR (300 MHz, DMSO-d6):δ 4.06 (t, 2H, J = 5.4 Hz, C-1'H), 4.46 (t, 2H, J = 4.5 Hz, C-2'H), 6.75 (d, 1H, J = 8.2 Hz, C-7H), 7.55-7.70 (m, 1H, C-6H), 7.70 (s, 1H, C-4H), 7.93 (s, 1H, C-3''H), 8.51 (s, 1H, C-5''H); 13C NMR (75.5 MHz, DMSO-d6): δ 46.45 (C-2' & C-1'), 112.45 (C-7), 119.12 (C-5), 121.44 (C-6), 128.23 (C-4), 138.72 (C-8), 144.11 (C-3''), 148.39 (C-9), 151.47 (C-5''), 160.32 (C-3), 183.07 (C-2) HRMS-FAB: m/z [M + Na+] calcd for C12H9BrN4O2Na: 342.9801; found: 342.9801 1-[3-(1H-1, 2, 4-Triazol-1-yl)propyl]-5-bromoindoline-2,3-dione (10) Orange solid Yield: 75 % MP: 102-105 °C IR (KBr): 3451, 2928, 1739, 1607, 1509 cm- 1 ; H NMR (300 MHz, DMSO-d6):δ 2.10 (br s, 2H, C-2'), 3.65 (br s, 2H, C-3'H), 4.24 (br s, 2H, C-1'H), 7.12 (d, 1H, J = 8.4 Hz, C-7H), 7.52 (d, 1H, J = 7.5 Hz, C-6H), 7.79 (s, 1H, C-4H), 7.92 (s, 1H, C-3''H), 8.47 (s, 1H, C-5''); 13C NMR (75.5 MHz, DMSO-d6): δ 27.08 (C-2'), 36.97 (C-1'), 45.89 (C-3'), 110.52 (C-7), 112.70 (C-6), 119.46 (C-5), 126.16 (C-4), 139.71 (C-8), 144.11 (C-3''), 150.39 (C-9), 151.47 (C-5''), 158.32 (C-3), 182.07 (C2) HRMS-FAB: m/z [M + H+] calcd for C13H12BrN4O2: 335.0138; found: 335.0129 General method for the preparation of compounds 14-21: Equimolar quantities of compound 7-10 (2 mmol) and hydroxylamine (11)/ aryl hydrazine 12-13 (2 mmol) were taken in 20 mL of absolute ethanol The reaction mixture was stirred at room temperature for 24 h, the completion of the reaction was checked by TLC Upon completion, the solvent was evaporated under reduced pressure and the product was recrystallized from ethanol (99.5 %) yielding 14-21 in 68-72 % yields Z-1-[2-(1H-1, 2, 4-Triazol-1-yl)ethyl]-3-(hydroxyimino)indolin-2-one (14) Orange solid Yield: 70 % MP: 228-230 °C IR (KBr): 3437, 1715, 1611, 1518, 1465 cm1 ; 1H NMR (300 MHz, DMSO-d6):δ 4.17 (t, 2H, J = 5.7 Hz, C-1'H), 4.54 (t, 2H, J = 5.7 Hz, C-2'H), 6.90 (d, 1H, J = 7.8 Hz, C-4H), 7.12 (t, 1H, J = 7.5 Hz, C-6H), 7.41 (t, 1H, J = 7.5 Hz, C-5H), 7.97 (s, 1H, C-3''H) 8.02 (d, 1H, J = 7.2 Hz, C-7H), 8.53 (s, 1H, C5''H), 13.49 (d, 1H, J = 3.6 Hz, OH); 13C NMR (75.5 MHz, DMSO-d6): δ 46.42 (C-1' & C-2'), 108.49 (C-7), 115.14 (C-6), 122.58 (C-5), 126.85 (C-4), 131.83 (C-8), 142.70 (C-3) 143.27 (C-9), 144.48 (C-3''), 151.55 (C-5''), 163.03 (C-2) HRMS-FAB: m/z [M + Na+] calcd for C12H11N5O2Na: 280.0805; found: 280.0815 Z-1-[3-(1H-1, 2, 4-Triazol-1-yl)propyl]-3-(hydroxyimino)indolin-2-one (15) Orange solid Yield: 68 % MP: 239-241 °C IR (KBr): 3449,1681, 1611, 1581, 1540 cm1 ; 1H NMR (300 MHz, DMSO-d6):δ 2.14- 2.19 (m, 2H, C-2'H), 3.75 (t, 2H, J = 6.6 Hz, C-1'H), 4.30 (t, 2H, J = 6.9 Hz, C-3'H), 7.07-7.13 (m, 2H, C-5H and C-7H) 7.43 (t, 1H, J = 7.8 Hz, C- 6H), 7.98 (d, 1H, J = 7.5 Hz, C-4H), 8.33 (s, 1H, C-3''H), δ 8.96 (s, 1H, C5''H), 13.51 (br s, 1H, OH); 13C NMR (75.5 MHz, DMSO-d6): δ 27.05 (C-2'), 36.42 (C3'), 46.93 (C-1'), 109.02 (C-7), 115.33 (C-6), 122.66 (C-5), 126.89 (C-4), 131.98 (C-8), 142.70 (C-3) 143.39 (C-9 & C-3''), 149.11 (C-5''), 163.19 (C-2) HRMS-FAB: m/z [M + Na+] calcd for C13H13N5O2Na: 294.9896; found: 294.9894 Z-1-[2-(1H-1, 2, 4-Triazol-1-yl)ethyl]-5-bromo-3-(hydroxyimino)indolin-2-one (16) Orange solid Yield: 72 % MP: 251-252 °C IR (KBr): 3433, 3042, 1722, 1607, 1437 cm1 ; 1H NMR (300 MHz, DMSO-d6):δ 4.10 (t, 2H, J = 5.4 Hz, C-2'H), 4.50 (t, 2H, J = 5.1 Hz, C-1'H), 6.85 (t, 1H, J = 7.2 Hz, C-7H), 7.50-7.52 (m, 1H, C-6H), 8.05 (s, 1H, C-4H), 8.08 (s, 1H, C-3''H) and 8.73 (s, 1H, C-5''H), 13.84 (br s, 1H, OH); 13C NMR (75.5 MHz, DMSO-d6): δ 46.73 (C-2'& C-1'), 110.67 (C-7), 114.02 (C-6), 116.71 (C-4), 128.77 (C-5), 138.02 (C-8), 141.77 (C-3), 142.29 (C-3''), 144.16 (C-9), 150.31 (C-5''), 162.60 (C-2) HRMS-FAB: m/z [M + H+] calcd for C12H10BrN5O2: 336.0091; found: 336.0085 Z-1-[3-(1H-1, 2, 4-Triazol-1-yl)propyl]-5-bromo-3-(hydroxyimino)indolin-2-one (17) Orange solid Yield: 70 % MP: 234-236 °C IR (KBr): 3487, 2969, 1698, 1604, 1436 cm 1 ; H NMR (300 MHz, DMSO-d6):δ 2.10-2.13 (m, 2H, C-2'H), 3.71 (t, 2H, J = 6.9 Hz, C- 10 Results and discussion Chemistry In order to get the desired triazolylisatin derivatives, we first carried out the synthesis of 5-bromoisatin (2) and N-substituted derivatives 3-6 of isatin (1) using 1, 2dibromoethane/1, 3-dibromopropane (Scheme-1) Subsequently, the triazolylisatins 7-10 were synthesized by treating N-bromoalkylisatins 3-6 with 1, 2, 4-triazole and anhydrous potassium carbonate using acetonitrile as solvent in 65-75 % yields (Scheme-1) The Nalkylation of 1, 2, 4-triazole could afford a mixture of 1-substituted and 4-substituted products The NMR spectra of compounds 7-21 showed protons H-3'' between δ 7.908.44 ppm and the H-5'' between δ 8.43-8.96 ppm instead of a single signal for two equivalent protons thus confirming the formation of 1-substituted triazolyl product (Figure 1) Figure 1: Formation of N-1 substituted 1, 2, 3-triazolyl derivatives 7-10 Finally, treatment of compounds 7-10 with hydroxylamine 11 and substituted phenylhydrazines 12-13 in ethanol afforded the compounds 14-21 in 68-72 % yields 14 (Scheme-1) The 3-substituted indolin-2-one may exist as either the Z- or E- isomer and there are several factors that can determine the configuration [39] In our case, the 1H NMR spectra of the synthesized compounds 14-21 indicated the formation of a single stereoisomer The downfield shift of the proton peak of the OH group in compounds 1417 between δ 13.49-13.84 ppm and the NH proton peak in compounds 18-21 appearing between δ 12.42-12.69 ppm indicates an intramolecularly hydrogen-bonded proton The intramolecular hydrogen bonding between NH of the hydrazone moiety and the carbonyl group of the indolinone unit leading to the formation of the pseudo six-membered ring indicated the formation of Z-hydrazones (Figure 2) Figure 2: Z-configured synthesized oximes 14-17 and hydrazones 18-21 Similarily, isatin oximes also exist as Z isomer due to hydrogen bonding between hydroxyl proton and carbonyl group The FTIR spectra also confirmed the Z-configured structure The frequencies of ν(NH) and ν(C=O) vibrations were found to be lower than usual values due to the intramolecular hydrogen bonding The analysis of the 13C NMR spectra of compounds 14-21 also showed C-2 carbonyl chemical shifts between δ 160.7915 163.19 ppm, thus confirming the Z stereochemical configuration The structures of all the synthesized compounds were unambiguously established using various spectroscopic techniques viz 1H NMR, 13C NMR, IR, HRMS, etc Anti-inflammatory activity evaluation of compounds 3-10 and 14-21 Compounds 3-10 and 14-21 were screened for their anti-inflammatory activities with respect to the TNF-α induced expression of ICAM-1 inhibition in human endothelial cells The results summarized in Table revealed that the synthesized compounds were potent in inhibiting the ICAM-1 expression Interestingly, Z-1-[3-(1H-1, 2, 4-triazol-1yl)propyl]-5-bromo-3-[2-(4-methoxyphenyl)hydrazono]indolin-2-one (19) showed maximum inhibition of ICAM-1 expression - 89 % with an IC 50 value of 20 µM at a maximal tolerable dose of 200 µM and was found to be the most active compound among the series of sixteen synthesized triazolyisatins investigated for anti-inflammatory activities in the present study Also, Z-1-[3-(1H-1, 2, 4-triazol-1-yl)propyl]-5-bromo-3(hydroxyimino)indolin-2-one (17) showed 77 % inhibition at maximal tolerable dose of 150 µM with an IC 50 value of 30 µM (Table 1, Scheme 1) 16 Scheme 1: Synthesis of triazolylisatin derivatives Structure activity relationship (SAR) We examined the effect of following structural modifications in isatin scaffold on their ICAM-1 expression inhibitory activity as shown in Table Table 1: ICAM-1 inhibitory activity of compounds 3-10 and 14-21 Compound No Maximum % ICAM-1 inhibition IC50 , (µM) Tolerable at MTD ± standard error of Dose (MTD), mean (SEM) µM 150 41 ≥ 150 ± 150 52 135 ± 150 60 75 ± 150 62 70 ± 17 150 55 140 ± 150 67 100 ± 150 61 55 ± 2.382 10 150 71 52 ± 14 150 66 90 ± 15 150 73 48 ± 16 150 68 50 ± 17 150 77 30 ± 18 200 70 53 ± 19 200 89 20 ± 20 80 45 80 ± 21 80 43 80 ± Piperine 250 90 100 ± (i) Effect of the bromine at the C-5 position in the benzenoid ring of isatin: As evident from the data given in Table 1, introduction of bromine at the C-5 position of the isatin derivatives significantly increases the ICAM-1 expression inhibitory activity (Table & Figure 3) The IC50 value of 1-[2-(1H-1, 2, 4-triazol-1-yl)ethyl]-indoline-2, 3-dione (7) was found to be 140 µM as compared to its more potent bromo derivative with an IC50 value of 55 µM Similarly, oxime derivative 14 exhibited an IC50 value of 90 µM in comparison to bromo substituted oxime 16 having an IC50 value of 50 µM This is most likely due to the increased cell permeability and hydrophobicity of bromo substituted derivatives Our results strengthen the earlier findings that electron withdrawing groups at C-5 position of isatin increases the ICAM-1 inhibitory activity [14] However, it was also observed that in case of compound 21 bearing bromo group at C-5 and pentafluorophenyl 18 group at C-3, there was not much increase in the inhibitory activity as compared to compound 20 bearing hydrogen at C-5 position of isatin It can therefore be inferred that increase in activity due to the presence of bromine substituent in the triazolyl-isatin is being masked by the deactivating effect of electron withdrawing pentaflurophenyl group Thus, simultaneous presence of electron-attracting groups in the isatin scaffold as well as in the phenyl ring at C-3 position could result in an inactive ICAM-1 inhibitor (ii) Effect of value of ‘n’ in the linker: On comparing the IC50 value of compound with that of (≥ 150 µM and 135 µM, respectively), with (140 µM and 100 µM, respectively) and 14 with 15 (90 µM and 48 µM, respectively), which differ only in the value of ‘n’, we observed that increasing the chain length of the linker increases the inhibition of TNF-α induced expression of ICAM-I on endothelial cells (Table & Figure 3) (iii) Effect of modification at C-3 position in the isatin ring: It was observed that derivatization at C-3 position in the isatin ring influences the ICAM-1 inhibitory activity According to the data shown in Table 1, as a general trend, arylhydrazone and oxime derivatives 14-21 of triazolylisatin were found to be more potent when compared to parent triazolyl derivatives 7-10 The results show that compound 19 with electrondonating methoxy group on phenyl ring at C-3 position is the most promising among the tested compounds We compared the parent isatin derivatives and 10 having IC50 values of 140 µM and 52 µM, respectively with the corresponding C-3 substituted compounds Isatin-3-hydrazones 18 and 19 bearing electron donating methoxy group had IC 50 values 19 of 53 µM and 20 µM, respectively, and showed improved activity when compared to isatin-3-oxime derivatives 14 and 17 with IC50 values of 90 µM and 30 µM, respectively It is worth mentioning that compounds 20 and 21 with electron withdrawing pentafluoro phenyl group showed poor activity (IC50 value = 80 µM) as compared to other derivatives Thus, it can be inferred that electron donating group at the C-3 position in the isatin ring leads to a potent ICAM-1 expression inhibitor (iv) Effect of triazole ring: It has been observed that the introduction of aromatic ring bearing electron withdrawing groups through carbon chain linker at N-1 position of isatin increases its pharmacological activity [40-42] 1, 2, 4-Triazole ring is a basic aromatic heterocycle which has been incorporated in the isatin scaffold at various positions to enhance its pharmacological property by synergetic effect [29-31] In our current study, the 1, 2, 4-traizole ring is tethered to the isatin by carbon chain linker It was observed by comparing the IC50 values of compounds vs 7, vs 8, vs 9, and vs 10 that the ICAM-1 expression inhibitory activity increases with the addition of triazole ring (Table1 & Figure 3) Thus, the IC50 value of compound 10 was 52 µM as opposed to that of that does not have the triazole ring and has the IC 50 value of 70 µM This corroborates the fact that hybrid molecules incorporating two or more biologically active moieties in a single structure are comparatively more active than the individual components 20 Figure 3: Effect of functional groups on ICAM-1 inhibitory activity of triazolylisatin derivatives Conclusions In the current study, twelve novel and four known triazolylisatin hybrids have been synthesized and their anti-inflammatory activities have been investigated with respect to their inhibition of TNF-α induced expression of ICAM-1 in HUVEC’s The synthesized compounds exhibited potent ICAM-1 expression inhibition and our findings/evaluations led to the conclusion that compound 19 potently inhibited the ICAM-1 expression by 89 % at an IC50 value of 20 µM with a maximal tolerable dose of 200 µM in HUVEC’s On comparing the ICAM-1 expression inhibition results, structure-activity relationship has also been established with respect to the substituents present on the core isatin moiety of the synthesized compounds which impose the requirement of the desired functional group / substituents in 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µM and 135 µM, respectively), with (140 µM and 100 µM, respectively) and 14 with 15 (90 µM and 48 µM, respectively), which differ only in the value of ‘n’,... group had IC 50 values 19 of 53 µM and 20 µM, respectively, and showed improved activity when compared to isatin-3-oxime derivatives 14 and 17 with IC50 values of 90 µM and 30 µM, respectively... of functional groups on ICAM-1 inhibitory activity of triazolylisatin derivatives Conclusions In the current study, twelve novel and four known triazolylisatin hybrids have been synthesized and