Synthesis and anti-inflammatory activity evaluation of novel triazolyl-isatin hybrids

Article abstract of DOI:10.3109/14756366.2016.1151015

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)

Full text of DOI:10.3109/14756366.2016.1151015

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and anti-inflammatory activity
evaluation of novel triazolyl-isatin hybrids
Pramod K. Sharma, Sakshi Balwani, Divya Mathur, Shashwat Malhotra,
Brajendra K. Singh, Ashok K. Prasad, Christophe Len, Erik V. Van der Eycken,
Balaram Ghosh, Nigel G. J. Richards & Virinder S. Parmar
To cite this article: Pramod K. Sharma, Sakshi Balwani, Divya Mathur, Shashwat Malhotra,
Brajendra K. Singh, Ashok K. Prasad, Christophe Len, Erik V. Van der Eycken, Balaram Ghosh,
Nigel G. J. Richards & Virinder S. Parmar (2016): Synthesis and anti-inflammatory activity
evaluation of novel triazolyl-isatin hybrids, Journal of Enzyme Inhibition and Medicinal
Chemistry, DOI: 10.3109/14756366.2016.1151015
To link to this article: http://dx.doi.org/10.3109/14756366.2016.1151015
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Date: 12 May 2016, At: 02:42

http://www.tandfonline.com/ienz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–7
2016 Taylor & Francis. DOI: 10.3109/14756366.2016.1151015
!
RESEARCH ARTICLE
Synthesis and anti-inflammatory activity evaluation of novel
triazolyl-isatin hybrids
Pramod K. Sharma1,2, Sakshi Balwani3, Divya Mathur1,4, Shashwat Malhotra1, Brajendra K. Singh1, Ashok K. Prasad1,
Christophe Len5,6, Erik V. Van der Eycken7, Balaram Ghosh3, Nigel G. J. Richards8,9, and Virinder S. Parmar1,5,8
2
1Department of Chemistry, Bioorganic Laboratory, University of Delhi, Delhi, India, Chemical Research Laboratory, Wockhardt Research Centre,
3
4
Aurangabad, Maharashtra, India, Immunogenetics Laboratory, CSIR-Institute of Genomics and Integrative Biology, Delhi, India, Department of
5
Chemistry, Daulat Ram College, University of Delhi, Delhi, India, Sorbonne Universites, Universite de Technologie de Compiegne (UTC), Ecole
´
´
`
6
Superieure de Chimie Organique et Minerale (ESCOM), Compiegne Cedex, France, Department of Chemistry, University of Hull, Hull, UK,
´
´
`
7Department of Chemistry, Laboratory for Organic & Microwave-Assisted Chemistry (LOMAC), University of Leuven (KU Leuven), Leuven, Belgium,
9
8Department of Chemistry and Chemical Biology, Indiana University-Purdue University (IUPUI), Indianapolis, IN, USA, and School of Chemistry,
College of Physical Sciences & Engineering, Cardiff University, Cardiff, UK
Abstract
Keywords
New isatin-triazole based hybrids have been synthesized and evaluated for their inhibitory
activity of TNF-a 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 anti-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 ¼ 20 mM and 89% ICAM-1 inhibition with MTD at
200 mM was found to be the most potent of all the synthesized derivatives. Introduction of
1,2,4-triazole ring and electron-donating methoxy group on the phenylhydrazone moiety
resulted in four-fold increase of the anti-inflammatory activity.
1,2,4-Triazole, anti-inflammatory activity,
ICAM-1, isatin, triazolylisatins
History
Received 12 December 2015
Accepted 28 January 2016
Published online 4 May 2016
Introduction
bases are of great medicinal value owing to their numerous
chemotherapeutic properties17–19
.
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
Isatis1, Calanthe discolor LINDL2 and Couroupita guianensis
Aubl3. Various substituted isatins have been isolated from plants,
e.g. melosatin alkaloids from Melochia tomentosa, a Caribbean
tumorigenic plant4; 6-(30-methylbuten-20-yl)isatin from fungi,
Streptomyces albus5 and 5-(30-methylbuten-20-yl)isatin from
Chaetomium globosum6. It is also known that isatins act as
endogenous biological regulators, found in the brain, peripheral
tissues, and body fluids of humans and animals7. Isatin was Erst
synthetically obtained as an oxidation product of indigo in the
early 19th century by Erdman and Laurent8.
The 1,2,4-triazole moiety is present in a wide variety of
therapeutically interesting drugs, such as ribavarin20, triazolam21,
fluconazole22 and voriconazole23. Anticancer24, antitubercular25,
analgesic26, antimicrobial27 and anti-inflammatory activities of
1,2,4-triazole derivatives have also been reported28. Recently,
some 1,2,4-triazole containing isatin derivatives possessing
interesting biological activities have been described29–31
.
During an inflammatory cascade, various inflammatory
mediators, including cytokines, such as TNF-a, IL-1b 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 endothelium32. 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
cells33. 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-kB and abrogate TNF-a-induced
expression of E-selectin, VCAM-1 and ICAM-1 on human
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 anti-
bacterial, antifungal, anticonvulsant, antiviral, anticancer, anti-
oxidant, anti-inflammatory and antiproliferative activities9–16
.
Isatin derivatives, such as hydrazones, Schiff’s and Mannich
Address for correspondence: Prof. Virinder S. Parmar, Department of
Chemistry, Bioorganic Laboratory, University of Delhi, Delhi – 110 007,
India. E-mail: virparmar@gmail.com
umbilical vein endothelial cells (HUVEC’s)34–36
.

2
P. K. Sharma et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
In drug discovery, the development of hybrid molecules 1-(2-Bromoethyl)indoline-2,3-dione (3)
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 synthesized a series of novel isatin
derivatives by connecting the isatin core moiety with triazole
Orange solid. Yield: 65%; m.p.: 128–130 ꢀC (literature m.p.: 131–
132 ꢀC)37.
1-(3-Bromopropyl)indoline-2,3-dione (4)
moiety using various alkyl chain linkers. Also, the Schiff’s bases Orange solid. Yield: 60%; m.p.: 182–184 ꢀC (literature m.p.: 187-
and oximes were prepared using substituted hydrazines and 188 ꢀC)37. IR (KBr): 3454, 2956, 1738, 1614, 1468 cmꢁ1 1H
;
hydroxylamine. Both p-methoxyphenyl hydrazine and pentafluor- NMR (300 MHz, DMSO-d6): d 2.13–2.16 (m, 2H, C-20H), 3.60 (t,
ophenyl hydrazine were used as amines for the condensation step, 2H, J ¼ 3.0 Hz, C-10H), 3.78 (t, 2H, J ¼ 3.0 Hz, C-30H), 7.09–7.12
as we wanted to evaluate the effects of both electron donating and (m, 1H, ArH), 7.17–7.20 (m, 1H, ArH), 7.54-7.55 (m, 1H, ArH),
electron withdrawing substituents on the phenyl ring of the amine 7.64-7.66 (m, 1H, ArH); 13C NMR (75.5 MHz, DMSO-d6):
for the ICAM-1 expression inhibition studies.
d 30.04 (C-30), 31.83 (C-20), 58.24 (C-10), 110.48 (C-7), 117.71
The synthesized triazolylisatins were then screened for their (C-4), 123.13 (C-5), 124.44 (C-6), 138.03 (C-8), 150.50 (C-9),
inhibition of TNF-a induced expression of ICAM-1 in HUVEC’s. 158.32 (C-2), 183.28 (C-3). HRMS-FAB: m/z [M + Na+] calcd for
The structure activity relationship (SAR) of the synthesized C11H10BrNO2Na: 289.1102; found: 289.1100.
compounds has also been well established to search for potential
lead compounds as anti-inflammatory drugs. Our findings 5-Bromo-1-(2-bromoethyl)indoline-2,3-dione (5)
revealed that Z-1-[3-(1H-1,2,4-triazol-1-yl)propyl]-5-bromo-3-
Orange solid. Yield: 62%; m.p.: 180–182 ꢀC (literature m.p.: 180-
[2-(4-methoxyphenyl)hydrazono]indolin-2-one (19) with an IC50
184 ꢀC)37. IR (KBr): 3458, 1738, 1602, 1470, 1437 cmꢁ1 1H
;
value of 20 mM and 89% inhibition in HUVEC’s is the most potent
ICAM-1 expression inhibitor.
NMR (300 MHz, DMSO-d6): d 3.67 (t, 2H, J ¼ 6.6 Hz, C-10H), d
4.10 (t, 2H, J ¼ 6.6 Hz, C-20H), 7.30 (d, 1H, J ¼ 8.4 Hz, C-6H),
7.66 (s, 1H, C-4H), 7.85 (d, 1H, J ¼ 7.2 Hz, ArH); 13C NMR
(75.5 MHz, DMSO-d6): d 28.95 (C-20), 41.28 (C-10), 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.
Materials and methods
General
Analytical TLCs were performed on Merck silica gel 60 F254
plates. All flash chromatographic separations were performed on
100-200 mesh silica gel. The IR spectra were recorded on a
1
Perkin-Elmer 2000 FT-IR spectrometer (Waltham, MA). The H
5-Bromo-1-(3-bromopropyl)indoline-2,3-dione (6)
NMR and 13C NMR spectra were recorded on a Bruker AC-300
Avance spectrometer (Billerica, MA) at 300 and 75.5 MHz, Orange solid. Yield: 60%; m.p.: 155–158 ꢀC (literature m.p.:
respectively, using TMS as internal standard. Chemical shifts are 155 ꢀC)38. 1H NMR (300 MHz, DMSO-d6): d 3.75 (t, 2H,
reported on d scale and coupling constants (J) are in Hz. The J ¼ 6.6 Hz, C-30H), d 2.16–2.73 (m, 2H, C-20H), 7.1 (d, 1H,
HRMS determinations were made in FAB positive mode on a J ¼ 8.4 Hz, C-6H), 7.68 (s, 1H, C-4H), 7.83 (d, 1H, J ¼ 8.4 Hz, C-
13
JEOL JMS-AX505W high-resolution mass spectrometer using 7H); C NMR (75.5 MHz, DMSO-d6): d 30.17 (C-30), 31.70 (C-
bis-hydroxyethyldisulfide (HEDS) doped with sodium acetate as 20), 40.34 (C-10), 112.62 (C-7), 114.80 (C-6), 119.52 (C-5),
matrix in the Laboratory of Dr. Carl-Erik Olsen at the University 126.60 (C-4), 139.60 (C-8), 149.38 (C-9), 157.93 (C-2), 182.01
of Copenhagen (Denmark). Melting points were recorded in a (C-3). HRMS-FAB: m/z [M + Na+] calcd for C10H7Br2NO2Na:
sulfuric acid bath and are uncorrected.
367.8892; found: 367.8889.
Materials
General method for the preparation of compounds 7–10
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 1 and 2, the
endothelial cell growth factor (ECGF), M199 medium, l-glutam-
ine, methylthiazolydiphenyl-tetrazolium bromide (MTT), trypsin,
o-phenylenediamine and goat anti-mouse IgG–HRP conjugate
were procured from Sigma Chemical Co. (St. Louis, MO). The
fetal calf serum (FCS) was procured from Biological Industries
(Kibbutz Beit Haemek, Israel).
To a solution of compounds 3–6 (2 mmol) and anhydrous
potassium carbonate (2 mmol) in acetonitrile (10 mL), 1,2,4-
triazole (2 mmol) in acetonitrile (10 mL) was added dropwise and
the reaction mixture was stirred and refluxed for 5 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)
Methods
Orange solid. Yield: 65%; m.p.: 105–108 ꢀC. IR (KBr):
General method for the preparation of compounds 3–6
3116, 1739, 1612, 1510, 1469 cmꢁ1
;
1H NMR (300 MHz,
DMSO-d6): d 4.04 (t, 2H, J ¼ 5.4 Hz, C-10H), 4.45 (t, 2H,
J ¼ 5.7 Hz, C-20H), 6.77 (d, 1H, J ¼ 8.1 Hz, C-7H), 7.07 (t, 1H,
J ¼ 7.5 Hz, C-5), 7.50-7.56 (m, 2H, C-4H and C-6H) 7.90 (s, 1H,
C-300), 8.50 (s, 1H, C-500); 13C NMR (75.5 MHz, DMSO-d6):
d 46.37 (C-10 and C-20), 110.0 (C-7), 117.35 (C-4), 123.27 (C-
5), 124.52 (C-6), 138.12 (C-8), 144.77 (C-300), 150.36 (C-9),
151.68 (C-500), 158.18 (C-3), 183.03 (C-2). HRMS-FAB:
m/z [M + Na+] calcd for C12H10N4O2Na: 265.0696; found:
265.0696.
To a solution of compound 1/2 (2 mmol) and anhydrous
potassium carbonate (2 mmol) in acetonitrile (20 mL), 1,2-
dibromoethane/1,3-dibromopropane (0.03 mol) was added drop-
wise and the resultant mixture was refluxed for 5 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.

DOI: 10.3109/14756366.2016.1151015
Synthesis and anti-inflammatory activity evaluation of novel triazolyl-isatin hybrids
3
1-[3-(1H-1,2,4-Triazol-1-yl)propyl]-indoline-2,3-dione (8)
Z-1-[3-(1H-1,2,4-Triazol-1-yl)propyl]-3-(hydroxyimino)indolin-2-
one (15)
Orange solid. Yield: 68%; m.p.: 103–106 ꢀC. IR (KBr): 3446,
3100, 1724, 1605, 1468 cmꢁ1; 1H NMR (300 MHz, DMSO-d6): d Orange solid. Yield: 68%; m.p.: 239–241 ꢀC. IR (KBr): 3449,
2.11–2.15 (m, 2H, C-20H), 3.65-3.70 (m, 2H, C-30H), 4.24–4.29 1681, 1611, 1581, 1540 cmꢁ1; 1H NMR (300 MHz, DMSO-d6): d
(m, 2H, C-10 H), 7.09–7.15 (m, 2H, C-4H, C-6H), 7.52 (d, 1H, 2.14-2.19 (m, 2H, C-20H), 3.75 (t, 2H, J ¼ 6.6 Hz, C-10H), 4.30 (t,
J ¼ 7.2 Hz, C-7H), 7.64 (t, 1H, J ¼ 7.8 Hz, C-5H), 8.44 (s, 1H, C- 2H, J ¼ 6.9 Hz, C-30H), 7.07–7.13 (m, 2H, C-5H and C-7H) 7.43
300), 8.50 (s, 1H, C-500); 13C NMR (75.5 MHz, DMSO-d6): d 27.06 (t, 1H, J ¼ 7.8 Hz, C- 6H), 7.98 (d, 1H, J ¼ 7.5 Hz, C-4H), 8.33 (s,
(C-20), 36.83 (C-10), 46.13 (C-30), 110.49 (C-7), 117.69 (C-5), 1H, C-300H), d 8.96 (s, 1H, C-500H), 13.51 (br s, 1H, OH); 13C
123.14 (C-6), 124.41 (C-4), 138.01 (C-8), 144.10 (C-300), 150.38 NMR (75.5 MHz, DMSO-d6): d 27.05 (C-20), 36.42 (C-30), 46.93
(C-9), 151.46 (C-500), 158.29 (C-3), 183.28 (C-2). HRMS-FAB: (C-10), 109.02 (C-7), 115.33 (C-6), 122.66 (C-5), 126.89 (C-4),
m/z [M + Na+] calcd for C13H12N4O2Na: 279.2696; found: 131.98 (C-8), 142.70 (C-3) 143.39 (C-9 and C-300), 149.11 (C-500),
279.2694.
163.19 (C-2). HRMS-FAB: m/z [M + Na+] calcd for
C13H13N5O2Na: 294.9896; found: 294.9894.
1-[2-(1H-1,2,4-Triazol-1-yl)ethyl]-5-bromoindoline-2,3-dione (9)
Z-1-[2-(1H-1,2,4-Triazol-1-yl)ethyl]-5-bromo-3-(hydroxyimi-
no)indolin-2-one (16)
Orange solid. Yield: 70%; m.p.: 104–106 ꢀC. IR (KBr): 3449,
3117, 1739, 1611, 1440 cmꢁ1; 1H NMR (300 MHz, DMSO-d6): d
4.06 (t, 2H, J ¼ 5.4 Hz, C-10H), 4.46 (t, 2H, J ¼ 4.5 Hz, C-20H), Orange solid. Yield: 72%; m.p.: 251–252 ꢀC. IR (KBr): 3433,
6.75 (d, 1H, J ¼ 8.2 Hz, C-7H), 7.55–7.70 (m, 1H, C-6H), 7.70 (s, 3042, 1722, 1607, 1437 cmꢁ1; 1H NMR (300 MHz, DMSO-d6): d
1H, C-4H), 7.93 (s, 1H, C-300H), 8.51 (s, 1H, C-500H); 13C NMR 4.10 (t, 2H, J ¼ 5.4 Hz, C-20H), 4.50 (t, 2H, J ¼ 5.1 Hz, C-10H),
(75.5 MHz, DMSO-d6): d 46.45 (C-20 and C-10), 112.45 (C-7), 6.85 (t, 1H, J ¼ 7.2 Hz, C-7H), 7.50-7.52 (m, 1H, C-6H), 8.05 (s,
119.12 (C-5), 121.44 (C-6), 128.23 (C-4), 138.72 (C-8), 144.11 1H, C-4H), 8.08 (s, 1H, C-300H) and 8.73 (s, 1H, C-500H), 13.84
13
(C-300), 148.39 (C-9), 151.47 (C-500), 160.32 (C-3), 183.07 (C-2). (brs, 1H, OH); C NMR (75.5 MHz, DMSO-d6): d 46.73 (C-20
HRMS-FAB: m/z [M + Na+] calcd for C12H9BrN4O2Na: and C-10), 110.67 (C-7), 114.02 (C-6), 116.71 (C-4), 128.77
342.9801; found: 342.9801.
(C-5), 138.02 (C-8), 141.77 (C-3), 142.29 (C-300), 144.16 (C-9),
150.31 (C-500), 162.60 (C-2). HRMS-FAB: m/z [M + H+] calcd for
C12H10BrN5O2: 336.0091; found: 336.0085.
1-[3-(1H-1,2,4-Triazol-1-yl)propyl]-5-bromoindoline-2,3-dione
(10)
Z-1-[3-(1H-1,2,4-Triazol-1-yl)propyl]-5-bromo-3-(hydroxyimi-
no)indolin-2-one (17)
Orange solid. Yield: 75%; m.p.: 102–105 ꢀC. IR (KBr): 3451,
2928, 1739, 1607, 1509 cmꢁ1; H NMR (300 MHz, DMSO-d6):
1
Orange solid. Yield: 70%; m.p.: 234–236 ꢀC. IR (KBr): 3487,
2969, 1698, 1604, 1436 cmꢁ1; 1H NMR (300 MHz, DMSO-d6): d
2.10–2.13 (m, 2H, C-20H), 3.71 (t, 2H, J ¼ 6.9 Hz, C-20H), 4.26 (t,
2H, J ¼ 6.9 Hz, C-30H), 7.05 (d, 1H, J ¼ 5.1 Hz, ArH), 7.58 (dd,
1H, J ¼ 2.1 Hz each ArH), 8.05 (d, 1H, J ¼ 2.1 Hz, ArH), 8.28 (s,
1H, C-300H), d 8.90 (s, 1H, C-500H), 13.80 (brs, 1H, OH); 13C
NMR (75.5 MHz, DMSO-d6): d 26.92 (C-20), 36.61 (C-10), 46.91
(C-30), 111.15 (C-7), 114.06 (C-6), 116.94 (C-5), 128.84 (C-4),
134.17 (C-8), 141.91 (C-3), 142.56 (C-300), 143.33 (C-9), 148.99
(C-500), 162.74 (C-2). HRMS-FAB: m/z [M + Na+] calcd for
C13H12BrN5O2Na: 372.0067; found: 372.0055.
d 2.10 (brs, 2H, C-20), 3.65 (brs, 2H, C-30H), 4.24 (brs, 2H, C-
10H), 7.12 (d, 1H, J ¼ 8.4 Hz, C-7H), 7.52 (d, 1H, J ¼ 7.5 Hz,
C-6H), 7.79 (s, 1H, C-4H), 7.92 (s, 1H, C-300H), 8.47 (s, 1H,
13
C-500); C NMR (75.5 MHz, DMSO-d6): d 27.08 (C-20), 36.97
(C-10), 45.89 (C-30), 110.52 (C-7), 112.70 (C-6), 119.46 (C-5),
126.16 (C-4), 139.71 (C-8), 144.11 (C-300), 150.39 (C-9),
151.47 (C-500), 158.32 (C-3), 182.07 (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
Z-1-[2-(1H-1,2,4-Triazol-1-yl)ethyl]-3-[2-(4-methoxyphenyl)hy-
drazono]indolin-2-one (18)
Equimolar quantities of compounds 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.
Orange solid. Yield: 70%; m.p.: 154–157 ꢀC. IR (KBr): 3448,
1
2928, 1671, 1612, 1558, 1514, 1467 cmꢁ1; H NMR (300 MHz,
DMSO-d6): d 3.75 (s, 3H, –OCH3), 4.19 (brs, 2H, C-10H), d 4.52
(brs, 2H, C-20H), 6.86 (d, 2H, J ¼ 7.8 Hz, C-2000H and C-6000H),
6.96 (d, 2H, J ¼ 8.7 Hz, C-3000H and C-5000H), 7.06–7.09 (m, 1H,
ArH), 7.20–7.39 (m, 2H, ArH), 7.53–7.55 (m, 1H, ArH), 7.90 (s,
1H, C-300H), 8.43 (s, 1H, C-500H), 12.59 (s, 1H, -NH); 13C NMR
(75.5 MHz, DMSO-d6): d 46.60 (C-10 and C-20), 55.30 (–OCH3),
114.77 (C-3000 and C-5000), 115.58 (C-2000 and C-6000), 117.99 (C-7),
120.51 (C-6), 122.21 (C-5), 125.10 (C-4), 127.67 (C-3), 135.98
(C-1000), 139.50 (C-9), 144.42 (C-300), 151.54 (C-500), 155.68 (C-
4000), 161.09 (C-2). HRMS-FAB: m/z [M + H+] calcd for
C19H18N6O2: 363.1564; found: 363.1564.
Z-1-[2-(1H-1, 2, 4-Triazol-1-yl)ethyl]-3-(hydroxyimino)indolin-2-
one (14)
Orange solid. Yield: 70%; m.p.: 228–230 ꢀC. IR (KBr): 3437,
1
1715, 1611, 1518, 1465 cmꢁ1; H NMR (300 MHz, DMSO-d6):
d 4.17 (t, 2H, J ¼ 5.7 Hz, C-10H), 4.54 (t, 2H, J ¼ 5.7 Hz, C-
20H), 6.90 (d, 1H, J ¼ 7.8 Hz, C-4H), 7.12 (t, 1H, J ¼ 7.5 Hz, C-
6H), 7.41 (t, 1H, J ¼ 7.5 Hz, C-5H), 7.97 (s, 1H, C-300H), 8.02
(d, 1H, J ¼ 7.2 Hz, C-7H), 8.53 (s, 1H, C-500H), 13.49 (d, 1H,
J ¼ 3.6 Hz, OH); 13C NMR (75.5 MHz, DMSO-d6): d 46.42 (C-
10 & C-20), 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-300),
151.55 (C-500), 163.03 (C-2). HRMS-FAB: m/z [M + Na+] calcd
for C12H11N5O2Na: 280.0805; found: 280.0815.
Z-1-[2-(1H-1,2,4-Triazol-1-yl)propyl]-3-[2-(4-methoxyphenyl)hy-
drazono]indolin-2-one (19)
Orange solid. Yield: 69%; m.p.: 195–198 ꢀC. IR (KBr): 2926,
1664, 1592, 1557, 1466 cmꢁ1; 1H NMR (300 MHz, DMSO-d6): d
2.14–2.18 (m, 2H, C-20H), 3.75 (s, 3H, –OCH3), 3.82 (brs, 2H,
C-10H), 4.24 (brs, 2H, C-30H), 6.95 (d, 2H, J ¼ 9.0 Hz, C-2000H and

4
P. K. Sharma et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
C-6000H), 7.08 (d, 2H, J ¼ 8.1 Hz, C-3000H and C-5000H), 7.38–7.44 121.67 (C-4), 130.22 (C-1000), 131.93 (C-9), 140.09 (C-3), 141.09
(m, 2H, C-6 and C-7H), 7.67 (s, 1H, C-4H), 7.97 (s, 1H, C-300H), (C-4000), 144.10 (C-2000, C-3000, C-5000 and C-6000), 151.42 (C-300 and
8.51 (s, 1H, C-500H), 12.69 (s, 1H, –NH); 13C NMR (75.5 MHz, C-500), 160.79 (C-2). HRMS-FAB: m/z [M + H+] calcd for
DMSO-d6): d 27.58 (C-20), 36.37 (C-10), 46.21 (C-30), 55.32 C19H12BrF5N6O: 515.0249; found: 515.0227.
(–OCH3), 111.04 (C-7), 114.24 (C-6), 114.73 (C-3000 and C-5000),
115.55 (C-2000 and C-6000), 120.33 (C-5), 122.99 (C-4), 123.91 (C- Cell culture experiments
8), 129.68 (C-3), 135.79 (C-1000), 138.47 (C-9), 144.15 (C-300),
Primary endothelial cells were isolated from human umbilical
151.43 (C-500), 155.65 (C-4000), 160.80 (C-2). HRMS-FAB: m/z
cord using mild trypsinization as described earlier36.
[M + Na+] calcd for C20H19BrN6O2Na: 477.0645; found:
477.0644.
Cell viability assay
The cytotoxicity of these compounds was determined by colori-
Z-1-[3-(1H-1,2,4-Triazol-1-yl)propyl]-3-[2-(perfluorophenyl)hy-
metric MTT assay as described earlier35. All experiments were
drazono]indolin-2-one (20)
performed at least three times in triplicate wells.
Orange solid. Yield: 68%; m.p.: 132–133 ꢀC. IR (KBr): 3436,
1
2924, 1668, 1609, 1526, 1500, 1467 cmꢁ1; H NMR (300 MHz,
Cell-ELISA for measurement of ICAM-1
DMSO-d6): d 2.17–2.22 (m, 2H, C-20H), d 3.81 (t, 2H, J ¼ 6.6 Hz,
Cell-ELISA was used for measuring the expression of ICAM-1 on
C-10H), d 4.28 (t, 2H, J ¼ 6.9 Hz, C-30H), d 7.11–7.19 (m, 2H,
the surface of endothelial cells36. The results are expressed in
ArH), 7.37–7.40 (m, 1H, ArH), 7.48-7.51 (m, 1H, ArH), 7.97 (s,
terms of IC50 values (the concentration of the inhibitor that gives
1H, C-300H), 8.50 (s, 1H, C-500H) 12.44 (s, 1H, -NH); 13C NMR
50% inhibition of the ICAM-1 expression) for each derivative.
(75.5 MHz, DMSO-d6): 27.42 (C-20), 36.47 (C-10), 46.19 (C-30),
The ICAM-1 assay was validated by using the reference
109.69 (C-7), 119.18 (C-6), 119.59 (C-5), 122.84 (C-4), 129.92
compound ‘‘piperine’’, which exhibits an IC50 value of 100 mM
for ICAM-1 expression inhibition on HUVECs.
(C-3), 141.14 (C-3000 and C-5000), 144.12 (C-2000 and C-6000), 151.44
(C-300 and C-500), 161.19 (C-2). HRMS-FAB: m/z [M + H+] calcd
for C19H13F5N6O: 437.1144; found: 437.1143.
Results and discussion
Chemistry
Z-1-[3-(1H-1,2,4-Triazol-1-yl)propyl]-5-bromo-3-[2-(perfluoro-
phenyl)hydrazono]indolin-2-one (21)
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 1,2-dibromoethane/1,3-dibro-
mopropane (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 N-alkylation of 1,2,4-
triazole could afford a mixture of 1-substituted and 4-substituted
Orange solid. Yield: 68%; m.p.: 178–180 ꢀC. IR (KBr): 3449,
1680, 1577, 1524, 1444 cmꢁ1; 1H NMR (300 MHz, DMSO-d6): d
2.14-2.18 (m, 2H, C-20H), d 3.78 (brs, 2H, C-10H), d 4.26 (brs,
2H, C-30H), d 7.14 (d, 1H, J ¼ 8.1 Hz, ArH), 7.47–7.55 (m, 2H,
ArH), 7.95 (s, 1H, C-300H), 8.47 (s, 1H, C-500H), 12.42 (brs, 1H,-
13
NH); C NMR (75.5 MHz, DMSO-d6): 27.31 (C-20), 36.59
(C-10), 46.12 (C-30), 111.71 (C-7), 114.70 (C-6), 121.25 (C-5),
Scheme 1. Synthesis of triazolylisatin derivatives.

DOI: 10.3109/14756366.2016.1151015
Synthesis and anti-inflammatory activity evaluation of novel triazolyl-isatin hybrids
5
products. The NMR spectra of compounds 7–21 showed protons using various spectroscopic techniques, viz. 1H NMR, 13C
H-300 between d 7.90 and 8.44 ppm, and the H-500 between d 8.43 NMR, IR, HRMS, etc (see Supplementary material).
and 8.96 ppm instead of a single signal for two equivalent protons,
thus confirming the formation of 1-substituted triazolyl product Anti-inflammatory activity evaluation of compounds 3–10
(Figure 1, see Supplementary material). Finally, treatment of and 14–21
compounds 7–10 with hydroxylamine (11) and substituted
Compounds 3–10 and 14–21 were screened for their
phenylhydrazines (12–13) in ethanol afforded the compounds
anti-inflammatory activities with respect to the TNF-a induced
14–21 in 68–72% yields (Scheme 1). The 3-substituted indolin-2-
expression of ICAM-1 inhibition in human endothelial cells. The
one may exist as either the Z- or E-isomer and there are several
results summarized in Table 1 revealed that the synthesized
1
factors that can determine the configuration39. In our case, the H
compounds were potent in inhibiting the ICAM-1 expression.
NMR spectra of the synthesized compounds 14–21 indicated the
Interestingly, Z-1-[3-(1H-1,2,4-triazol-1-yl)propyl]-5-bromo-3-[2-
formation of a single stereoisomer. The downfield shift of the
(4-methoxyphenyl)hydrazono]indolin-2-one (19) showed max-
proton peak of the OH group in compounds 14–17 between d
imum inhibition of ICAM-1 expression – 89% with an IC50
13.49 and13.84 ppm, and the NH proton peak in compounds 18–
value of 20 mM at a maximal tolerable dose of 200 mM and was
21 appearing between d 12.42 and 12.69 ppm indicates an
found to be the most active compound among the series of 16
intramolecularly hydrogen-bonded proton (see Supplementary
synthesized triazolyisatins investigated for anti-inflammatory
material). The intramolecular hydrogen bonding between NH of
activities in the present study.
the hydrazone moiety and the carbonyl group of the indolinone
Also, Z-1-[3-(1H-1,2,4-triazol-1-yl)propyl]-5-bromo-3(hydro-
unit leading to the formation of the pseudo six-membered ring
xyimino)indolin-2-one (17) showed 77% inhibition at maximal
indicated the formation of Z-hydrazones (Figure 2).
tolerable dose of 150 mM with an IC50 value of 30 mM (Table 1,
Similarly, isatin oximes also exist as Z isomers due to
Scheme 1).
hydrogen bonding between hydroxyl proton and carbonyl
group. The FTIR spectra also confirmed the Z-configured
Structure activity relationship
structure. The frequencies of ꢀ(NH) and ꢀ(C¼O) vibrations
were found to be lower than usual values due to the We examined the effect of following structural modifications in
intramolecular hydrogen bonding (see Supplementary material). isatin scaffold on their ICAM-1 expression inhibitory activity as
The analysis of the 13C NMR spectra of compounds 14–21 shown in Table 1.
also showed C-2 carbonyl chemical shifts between d 160.79
and 163.19 ppm, thus confirming the Z stereochemical con- Effect of the bromine at the C-5 position in the benzenoid ring of
figuration (see Supplementary material). The structures of all isatin
the synthesized compounds were unambiguously established
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 1 and 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 mM as compared to its more potent
bromo derivative 9 with an IC50 value of 55 mM. Similarly, oxime
derivative 14 exhibited an IC50 value of 90 mM in comparison to
bromo-substituted oxime 16 having an IC50 value of 50 mM. 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 activity14.
However, it was also observed that in case of compound 21
1H NMR
δ 8.47-8.51 ppm
1H NMR
δ 7.90-8.44 ppm
bearing bromo group at C-5 and pentafluorophenyl 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
Figure 1. Formation of N-1 substituted 1,2,3-triazolyl derivatives 7–10.
Figure 2. Z-configured synthesized oximes 14–17 and hydrazones 18–21.

6
P. K. Sharma et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
Table 1. ICAM-1 inhibitory activity of compounds 3–10 and 14–21.
activity when compared to isatin-3-oxime derivatives 14 and 17
with IC50 values of 90 and 30 mM, respectively. It is worth
mentioning that compounds 20 and 21 with electron withdrawing
pentafluoro phenyl group showed poor activity (IC50
value ¼ 80 mM) 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.
% ICAM-1 IC50 (mM) standard
Compound Maximum tolerable
No.
inhibition
at MTD
error of
mean (SEM)
dose (MTD), mM
3
4
5
6
7
8
9
10
14
15
16
17
18
19
20
21
Piperine
150
150
150
150
150
150
150
150
150
150
150
150
200
200
80
41
52
60
62
55
67
61
71
66
73
68
77
70
89
45
43
90
ꢂ150
135
75
70
140
100
3
3
3
1
2
2
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
55 2.382
52
90
48
50
30
53
20
80
2
1
3
1
2
3
1
3
2
1
position of isatin increases its pharmacological activity40–42
.
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 effect29–31. In
the 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 3 versus 7, 4 versus 8, 5 versus 9, and 6 versus 10
that the ICAM-1 expression inhibitory activity increases with the
addition of triazole ring (Table 1 and Figure 3). Thus, the IC50
value of compound 10 was 52 mM as opposed to that of 6 that
does not have the triazole ring and has the IC50 value of 70 mM.
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.
80
250
80
100
Conclusions
In the current study, 12 novel and four known triazolylisatin
hybrids have been synthesized and their anti-inflammatory
activities have been investigated with respect to their inhibition
of TNF-a 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 mM with a maximal tolerable dose of
200 mM 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 the
targeted compounds. All these results yield valuable information
for further optimization of structure-based drug design towards
ICAM-1 expression inhibitors.
Figure 3. Effect of functional groups on ICAM-1 inhibitory activity of
triazolylisatin derivatives.
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.
Effect of value of ‘n’ in the linker
Declaration of interest
On comparing the IC50 value of compound 3 with that of 4 (ꢂ150
and 135 mM, respectively), 7 with 8 (140 and 100 mM, respect-
ively) and 14 with 15 (90 and 48 mM, respectively), which differ
only in the value of ‘n’, we observed that increasing the chain
length of the linker increases the inhibition of TNF-a induced
expression of ICAM-I on endothelial cells (Table 1 and Figure 3).
The authors acknowledge the financial support from the
University of Delhi, University Grants Commission (UGC, New
Delhi) and the Department of Biotechnology (DBT, New Delhi).
B.G. thanks the Council of Scientific & Industrial Research
(CSIR, Govt. Of India) for the financial support (Project:
BSC0116).
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 electron-donating methoxy
group on phenyl ring at C-3 position is the most promising among
the tested compounds. We compared the parent isatin derivatives
7 and 10 having IC50 values of 140 and 52 mM, respectively, with
the corresponding C-3 substituted compounds. Isatin-3-hydra-
zones 18 and 19 bearing electron donating methoxy group had
IC50 values of 53 and 20 mM, respectively, and showed improved
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Supplementary material available online
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for further explanation of supplemental data and underlying research materials.