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

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,

Synthesis and Anti-inflammatory Activity Evaluation of Novel

Triazolyl-isatin 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,

inflammatory potential of the synthesized compounds Z-1-[3-(1H-1, 2,

4-Triazol-1-yl)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 mostpotent of all the synthesized derivatives Introduction of 1, 2, 4-triazole ring and electron-donating methoxy group on the phenylhydrazone moiety increased the anti-inflammatoryactivity four fold.

Keywords:Isatin, 1, 2, 4-triazole, anti-inflammatory activity, ICAM-1, triazolylisatins

Table of abbreviations:

ICAM-1 Intercellular Adhesion Molecule-1

MTD Maximal tolerable dose

SAR Structure-activity relationship

VCAM-1 Vascular cell adhesion molecule-1

HUVEC’s Human umbilical vein endothelial cells

MTT 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 syntheticversatility 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 thebrain, peripheral tissues, and body fluids of humans and animals [7] Isatin was first

synthetically obtained as an oxidation product of indigo in the early 19th century byErdman and Laurent [8]

The synthetic interest in the chemistry of isatin and its derivatives stemmed fromits easy synthetic accessibility and exhibition of broad spectrum biological effects,including antibacterial, antifungal, anticonvulsant, antiviral, anticancer, antioxidant, anti-inflammatory and antiproliferative activities [9-16] Isatin derivatives, such ashydrazones, Schiff’s and Mannich bases are of great medicinal value owing to theirnumerous chemotherapeutic properties [17-19]

The 1, 2, 4-triazole moiety is present in a wide variety of therapeuticallyinteresting drugs, such as ribavarin [20], triazolam [21], fluconazole [22] andvoriconazole [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 interestingbiological activities have been described [29-31]

During an inflammatory cascade, various inflammatory mediators, includingcytokines 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 vascularendothelium [32] The increased levels of expression of cell adhesion molecules on theendothelial cells alter the adhesive property of the vasculature, leading to indiscriminateinfiltration of the leukocytes across the blood vessels, and thus causing inflammation Apromising approach for the therapeutic intervention of inflammatory disorders is bypharmacological 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 isatinderivatives, we have synthesised a series of novel isatin derivatives by connecting theisatin core moiety with triazole moiety using various alkyl chain linkers Also, theSchiff’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 bothelectron donating and electron withdrawing substituents on the phenyl ring of the aminefor the ICAM-1 expression inhibition studies

The synthesized triazolylisatins were then screened for their inhibition of TNF-αinduced expression of ICAM-1 in HUVEC’s The structure activity relationship (SAR) ofthe 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,

General: Analytical TLCs were performed on Merck silica gel 60 F254 plates All flashchromatographic separations were performed on 100-200 mesh silica gel The IR spectrawere recorded on a Perkin-Elmer 2000 FT-IR spectrometer The 1H NMR and 13C NMRspectra were recorded on a Bruker AC-300 Avance spectrometer at 300 MHz and 75.5MHz, respectively using TMS as internal standard Chemical shifts are reported on 

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 bis-

hydroxyethyldisulfide (HEDS) doped with sodium acetate as matrix in the Laboratory of

Dr Carl-Erik Olsen at the University of Copenhagen (Denmark) Melting points wererecorded 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 weredistilled over P2O5 and K2CO3, respectively prior to use The compounds 1 & 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) Thefetal 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, dibromoethane/1, 3-dibromopropane (0.03 mol) was added dropwise and the resultantmixture was refluxed for 5 h On completion of the reaction, the reaction mixture was

2-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.

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-d 6): 3.67 (t,

2H, J = 6.6 Hz, C-1'H),  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-d 6): 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]+calcdfor 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-d 6 ): 3.75 (t, 2H, J = 6.6 Hz, C-3'H),  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-d 6):  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 (C-

3) 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, triazole (2 mmol) in acetonitrile (10 mL) was added dropwise and the reaction mixturewas stirred and refluxed for 5 h On completion, the reaction mixture was filtered andsolvent evaporated under reduced pressure The residue thus obtained was purified by

4-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.

DMSO-d 6):  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

-1; 1H NMR (300 MHz, DMSO-d 6):  2.11- 2.15 (m, 2H, 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-d 6):  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 (C-

5''), 158.29 (C-3), 183.28 (C-2) HRMS-FAB: m/z [M + Na+] calcd for C13H12N4O2Na:279.2696; found: 279.2694

(C-2) 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 temperaturefor 24 h, the completion of the reaction was checked by TLC Upon completion, thesolvent was evaporated under reduced pressure and the product was recrystallized from

ethanol (99.5 %) yielding 14-21 in 68-72 % yields.

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 cm

-1; 1H NMR (300 MHz, DMSO-d 6 ): 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, 6H), 7.98 (d, 1H, J = 7.5 Hz, 4H), 8.33 (s, 1H, 3''H),  8.96 (s, 1H,

C-5''H), 13.51 (br s, 1H, OH); 13C NMR (75.5 MHz, DMSO-d 6):  27.05 2'), 36.42 3'), 46.93 (C-1'), 109.02 (C-7), 115.33 (C-6), 122.66 (C-5), 126.89 (C-4), 131.98 (C-8),

(C-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; 1H NMR (300 MHz, DMSO-d 6 ): 2.10-2.13 (m, 2H, 2'H), 3.71 (t, 2H, J = 6.9 Hz, 2'H), 4.26 (t, 2H, J = 6.9 Hz, C-3'H), 7.05 (d, 1H, J = 5.1 Hz, ArH), 7.58 (dd, 1H, J = 2.1

C-Hz each ArH), 8.05 (d, 1H, J = 2.1 C-Hz, ArH), 8.28 (s, 1H, C-3''H), δ 8.90 (s, 1H, C-5''H),

13.80 (br s, 1H, OH); 13C NMR (75.5 MHz, DMSO-d 6):  26.92 (C-2'), 36.61 (C-1'),46.91 (C-3'), 111.15 (C-7), 114.06 (C-6), 116.94 (C-5), 128.84 (C-4), 134.17 (C-8),

C-161.09 (C-2) HRMS-FAB: m/z [M + H+] calcd for C19H18N6O2: 363.1564; found:363.1564

4H), 7.97 (s, 1H, C-3''H), 8.51 (s, 1H, C-5''H), 12.69 (s, 1H, -NH); 13C NMR (75.5 MHz,

DMSO-d 6):  27.58 (C-2'), 36.37 (C-1'), 46.21 (C-3'), 55.32 (–OCH3), 111.04 (C-7),114.24 (C-6), 114.73 (C-3''' & C-5'''), 115.55 (C-2''' & C-6'''), 120.33 (C-5), 122.99 (C-4),123.91 (C-8), 129.68 (C-3), 135.79 (C-1'''), 138.47 (C-9), 144.15 (C-3''), 151.43 (C-5''),

155.65 (C-4'''), 160.80 (C-2) HRMS-FAB: m/z [M + Na+] calcd for C20H19BrN6O2Na:477.0645; found: 477.0644

m/z [M + H+] calcd for C19H13F5N6O: 437.1144; found: 437.1143

Z-1-[3-(1H-1, 2, (perfluorophenyl)hydrazono]indolin-2-one (21)

4-Triazol-1-yl)propyl]-5-bromo-3-[2-Orange solid Yield: 68 % MP: 178-180 °C IR (KBr): 3449, 1680, 1577, 1524, 1444 cm

-1; 1H NMR (300 MHz, DMSO-d 6): 2.14-2.18 (m, 2H, C-2'H),  3.78 (br s, 2H, C-1'H), 

4.26 (br s, 2H, C-3'H),  7.14 (d, 1H, J = 8.1 Hz, ArH), 7.47-7.55 (m, 2H, ArH), 7.95 (s,

1H, C-3''H), 8.47 (s, 1H, C-5''H), 12.42 (br s, 1H,-NH); 13C NMR (75.5 MHz, DMSO-d6):27.31 (C-2'), 36.59 (C-1'), 46.12 (C-3'), 111.71 (C-7), 114.70 (C-6), 121.25 (C-5), 121.67(C-4), 130.22 (C-1'''), 131.93 (C-9), 140.09 (C-3), 141.09 (C-4'''), 144.10 (C-2''', C-3''', C-

5''' and C-6'''), 151.42 (C-3'' & C-5''), 160.79 (C-2) HRMS-FAB: m/z [M + H+] calcd for

C19H12BrF5N6O: 515.0249; found: 515.0227

Cell culture experiments:

Primary endothelial cells were isolated from human umbilical cord using mildtrypsinization as described earlier [36]

Cell viability assay:

The cytotoxicity of these compounds was determined by colorimetric MTT assay asdescribed earlier [35] All experiments were performed at least 3 times in triplicate wells

Cell-ELISA for measurement of ICAM-1:

Cell-ELISA was used for measuring the expression of ICAM-1 on the surface ofendothelial cells [36] The results are expressed in terms of IC50 values (the concentration