Synthesis and pharmacological evaluation of pyrazolo[4,3-c]cinnoline derivatives as potential anti-inflammatory and antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2012.08.045

A series of pyrazolo[4,3-c]cinnoline derivatives was synthesized, characterized and evaluated for anti-inflammatory and antibacterial activity. Test compounds that exhibited good anti-inflammatory activity were further screened for their ulcerogenic and l

Full text of DOI:10.1016/j.ejmech.2012.08.045

European Journal of Medicinal Chemistry 57 (2012) 176e184
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and pharmacological evaluation of pyrazolo[4,3-c]cinnoline derivatives
as potential anti-inflammatory and antibacterial agents
,
a
b
a
Rajiv Kumar Tonk a, Sandhya Bawa a , Gita Chawla , Girdhar Singh Deora , Suresh Kumar ,
*
Vandana Rathore b, Naveen Mulakayala b, Azad Rajaram c, Arunasree M. Kalle c, Obaid Afzal a
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi 110 062, India
b Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, India
c Department of Animal Sciences, School of life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrazolo[4,3-c]cinnoline derivatives was synthesized, characterized and evaluated for anti-
inflammatory and antibacterial activity. Test compounds that exhibited good anti-inflammatory
activity were further screened for their ulcerogenic and lipid peroxidation activity. Compounds 4d and
4l showed promising anti-inflammatory activity with reduced ulcerogenic and lipid peroxidation activity
when compared to naproxen. Docking results of these two compounds with COX-2 (PDB ID: 1CX2) also
exhibited a strong binding profile. Among the test derivatives, compound 4i displayed significant anti-
bacterial property against gram-negative (Escherichia coli and Pseudomonas aeruginosa) and gram-posi-
tive (Staphylococcus aureus) bacteria. However, compound 4b emerged as the best dual anti-
inflammatoryeantibacterial agent in the present study.
Received 8 June 2012
Received in revised form
14 August 2012
Accepted 30 August 2012
Available online 7 September 2012
Keywords:
Pyrazolo[4,3-c]cinnoline
Anti-inflammatory agent
COX-2 inhibitors
Ó 2012 Elsevier Masson SAS. All rights reserved.
Ulcerogenic activity
Docking simulations
Antibacterial agent
1. Introduction
may promote bacterial growth [4]. Other reports disclosed that
NSAIDs may enhance the progression of bacterial infection [5,6].
Non-steroidal anti-inflammatory drugs (NSAIDs) are used to
treat inflammation because of their immanent capability of inhib-
iting cyclooxygenases (COXs), the key enzymes involved in the
biosynthesis of prostaglandin from arachidonic acid. However, the
chronic use of NSAIDs may elicit marked gastrointestinal (GI) irri-
tation and ulceration due to their unwanted inhibition of the COX-1
enzyme and the desired blockade of the COX-2 enzyme. The
recognition of COX-2 as a potential target has influenced the
development of drugs that do not cause GI disorders but retain their
clinical efficacy as anti-inflammatory agents [1]. However, all the
selective COX-2 inhibitors are associated with an increased risk of
cardiovascular events. Some recent studies suggest that, among the
NSAIDs, naproxen is safe in terms of cardiovascular toxicity [2,3].
Markedly, inflammation and infection are not equal, even in the
case where infection is the primary cause of the inflammation.
Furthermore, the inflammatory response elicited by an invading
organism can result in host damage, increase the availability of
nutrients and mediate access to host tissues. In addition, inflam-
mation may cause accumulation of fluid in the injured area, which
Hence, a dual anti-inflammatoryeantibacterial agent with an
improved safety profile is required for improved therapeutic
benefits and better patient compliance.
Pyridazine-containing compounds have been reported to exhibit
anti-inflammatory [7e10] and antimicrobial activity [11]. Cinnoline,
a benzfused pyridazine, has been utilized for the development of
potent anti-inflammatory [12,13] and antibacterial agents [14], such
as cinnopentazon and cinoxacin, respectively. Dual effects have also
been reported as a property of cinnoline derivatives [15]. Further-
more, pyrazole-containing compounds have also been documented
as anti-inflammatoryeantibacterial agents [16e18]. These findings
prompted us to construct a new molecular framework that contains
both the cinnoline and pyrazole ring systems in the same matrix
with the hope of developing a compound that possesses both
anti-inflammatory and antibacterial activity.
2. Experimental section
2.1. Chemistry
Melting points, which are uncorrected, were determined using
open capillary tubes on an electrical melting point apparatus.
* Corresponding author. Tel.: þ91 11 26059688x5644.
E-mail addresses: drsbawa@rediffmail.com, sbawa@jamiahamdard.ac.in (S. Bawa).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.045

R.K. Tonk et al. / European Journal of Medicinal Chemistry 57 (2012) 176e184
177
Table 1
Required reagents and chemicals were purchased from
E
Merck (India) Ltd., S. D. Fine (India) and Qualigens (India). Silica gel
60e120 mesh LR (25049 K05) was used for column chromatog-
raphy. IR (KBr) spectra were recorded on a Shimadzu spectrometer
(vmax in cmꢀ1). 1H NMR and 13C NMR spectra were recorded in
DMSO/CDCl3 on a Bruker 300 MHz and 75 MHz spectrometer,
respectively, using tetramethylsilane (TMS) as the internal refer-
Physicochemical constants of the synthesized compounds (4aem).
Compd. R
no.
X
Molecular
formula
m.p., ꢁC Yielda, Rf.b
%
4a
4b
4c
4d
4e
4f
4g
4h
4i
Phenyle
Phenyle
Phenyle
p-methylphenyle
o,p-dichlorophenyle
o,p-dichlorophenyle
p-chlorophenyle
p-fluorophenyle
p-nitrophenyle
p-aminophenyle
e
C17H10ClFN4O 212e213 61
0.44
0.50
0.46
0.52
0.61
0.56
0.58
0.60
0.58
0.62
0.68
0.64
0.56
CH2 C18H12ClFN4O 185e187 66
OCH2 C18H12ClFN4O2 159 52
e
C
C
18H12ClFN4O 197e198 69
17H8Cl3FN4O 226e228 65
ence (chemical shift was measured in
d ppm). Mass spectra
e
(DART-MS) were measured on a JEOL-AccuTOF JMS-T100LS mass
spectrometer with a DART (Direct Analysis in Real Time) source.
The progress of the reaction and the purity of the synthesized
compounds were verified on ascending thin layer chromatography
(TLC) plates coated with silica gel G (Merck). An iodine chamber
and UV lamp were used for the visualization of the TLC spots.
OCH2 C18H10Cl3FN4O2 150e152 58
e
e
e
e
C
C
17H8Cl2FN4O 202
17H9ClF2N4O 167e169 57
62
17H11ClFN5O 180e182 49
19H14ClFN4O3 206 65
18H12ClFN4O2 221e222 60
68
64
C17H9ClFN5O3 239
4j
4k
4l
C
C
C
m,p-dimethoxyphenyle e
p-methoxyphenyle
1-naphthalyle
e
4m
CH2 C22H14ClFN4O 165
a
2.1.1. Ethyl-2-[2-(3-chloro-4-fluorophenyl)hydrazinylidene]-3-
oxobutanoate (2)
After recovery from hexane:AcOEt (4:1).
Toluene:ethylacetate:formic acid (6:3.5:0.5).
b
Compound 2 was synthesized from 3-chloro-4-fluoroaniline (1)
according to the procedure reported in the literature [19,20].
Pale yellow; m.p. 88 ꢁC; IR (KBr) cmꢀ1: 3448 (NeH), 1742 (C]O),
1690 (C]O), 1526 (C]C), 1198 (CeF), 744 (CeCl). 1H NMR
2.1.4. (7-chloro-8-fluoro-3-methyl-1H-pyrazolo[4,3-c]cinnolin-
1-yl)(phenyl)methanone (4a)
(300 MHz, CDCl3):
d
(ppm) 1.28e1.31 (t, 3H, CH3 ester, J ¼ 12 Hz),
Pale yellow. IR (KBr) cmꢀ1: 1650 (C]O),1619 (C]N),1534 (C]C),
1506 (N]N), 1249 (CeF), 752 (CeCl). 1H NMR (300 MHz, CDCl3):
2.56 (s, 3H, COCH3), 4.36e4.39 (q, 2H, CH2 ester, J ¼ 9 Hz), 7.42e7.55
(m, 3H, CHeAr), 14.2 (s, 1H, NH).
d
(ppm) 2.27 (s, 3H, CH3), 7.14e7.20 (m, 5H, HPhenyl), 7.22 (s, 1H, C5e
Cinnoline), 7.49 (s, 1H, C8eHCinnoline). 13C NMR (75 MHz, CDCl3):
d (ppm) 12.2 (CH3),115.3,117.1,126.3,128.8,129.2,131.3,135.4,138.7,
H
2.1.2. 3-acetyl-7-chloro-6-fluorocinnolin-4(1H)-one (3)
Powdered anhydrous AlCl3 (5 g, 37 mmol) was added to a solu-
tion of compound 2 (4 g, 13 mmol) in chlorobenzene (30 ml); the
reaction mixture was kept cool during this addition. The reaction
mixture was then refluxed for 16 h under anhydrous conditions. It
was then cooled and dilute HCl (10% w/w, 200 ml) was added. The
resulting mixture was then heated on a water bath and the excess
chlorobenzene was removed by steam distillation. The separated
solid was filtered, washed with 5% NaOH solution and then with
water. The air dried product was again washed with benzene:e-
thanol (1:1) and re-crystallized with acetic acid:methanol (4:6) to
obtain a pale yellow colored crystalline product.
139.5, 151.5, 159.4, 169.2 (C]O). Anal. Calcd for C17H10ClFN4O: C,
59.92; H, 2.96; N, 16.44. Found: C, 60.10; H, 2.95; N, 16.40. DART-MS
m/z: 340.12 (Mþ), 342.12 (Mþ þ 2).
2.1.5. 1-(7-chloro-8-fluoro-3-methyl-1H-pyrazolo[4,3-c]
cinnolin-1-yl)-2-phenylethanone (4b)
Paleyellow. IR (KBr) cmꢀ1: 1659 (C]O),1622 (C]N),1528 (C]C),
1511 (N]N), 1226 (CeF), 744 (CeCl). 1H NMR (300 MHz, CDCl3):
d
(ppm) 2.35 (s, 3H, CH3), 4.27 (s, 2H, CH2), 7.18e7.25 (m, 5H, HPhenyl),
7.29 (s, 1H, C5eHCinnoline), 7.51 (s, 1H, C8eHCinnoline). 13C NMR
(75 MHz, CDCl3): (ppm) 12.9 (CH3), 39.6, 115.4, 117.2, 126.7, 128.2,
d
Pale yellow; m.p.: 192 ꢁC; IR (KBr) cmꢀ1: 3436 (NeH), 2925,
2840 (CeH), 1712 (C]O), 1659 (C]O), 1522 (C]C), 1215 (CeF), 746
129.5, 131.1, 135.1, 138.6, 139.7, 151.3, 159.6, 168.8 (C]O). Anal. Calcd
for C18H12ClFN4O: C, 60.94; H, 3.41; N,15.79. Found: C, 61.14; H, 3.43;
N, 15.72. DART-MS m/z: 354.14 (Mþ), 356.14 (Mþ þ 2).
(CeCl). 1H NMR (300 MHz, CDCl3)
d
: 2.61 (s, 3H, CH3), 7.29 (s, 1H,
C5eHCinnoline), 7.54 (s, 1H, C8eHCinnoline), 13.8 (s, 1H, NH, D2O-
Exchangeable). 13C NMR (75 MHz, CDCl3)
: 24.0 (CH3), 115.8, 117.1,
d
2.1.6. 1-(7-chloro-8-fluoro-3-methyl-1H-pyrazolo[4,3-c]
cinnolin-1-yl)-2-phenoxyethanone (4c)
126.3,129.3,129.6,151.7,159.5,176.8 (C]O),194.6 (C]O). DART-MS
m/z: 240.18 (Mþ), 242.18 (Mþ þ 2). Anal. Calcd for C10H6ClFN2O2: C,
49.92, H, 2.51, N, 11.64; Found: C, 49.79, H, 2.52, N, 11.60.
Fine yellow. IR (KBr) cmꢀ1: 1664 (C]O), 1604 (C]N), 1530
(C]C), 1494 (N]N), 1219 (CeF), 1046 (CeOeC), 750 (CeCl). 1H
NMR (300 MHz, CDCl3):
CH2), 6.94e7.10 (m, 5H, HPhenyl), 7.33 (s, 1H, C5eHCinnoline), 7.54 (s,
1H, C8eHCinnoline). 13C NMR (75 MHz, CDCl3):
(ppm) 12.4 (CH3),
61.6, 114.8, 116.9, 127.0, 127.8, 129.6, 130.8, 131.0, 135.3, 137.9,
139.4, 151.7, 159.4, 168.3 (C]O). Anal. Calcd for C18H12ClFN4O2: C,
58.31; H, 3.26; N, 15.11. Found: C, 58.42; H, 3.25; N, 15.13. DART-
MS m/z: 370.26 (Mþ), 372.26 (Mþ þ 2).
d (ppm) 2.37 (s, 3H, CH3), 4.67 (s, 2H,
2.1.3. General procedure for the synthesis of compounds (4aem)
Equimolar quantities of compound 3 (0.5 g, 2 mmol) and
aromatic acid hydrazide (benzoic acid hydrazide, phenyl acetic acid
hydrazide, phenoxy acetic acid hydrazide, p-methyl benzoic acid
hydrazide, o,p-dichloro benzoic acid hydrazide, o,p-dichloro phe-
noxyacetic acid hydrazide, p-chloro benzoic acid hydrazide, p-flu-
oro benzoic acid hydrazide, p-nitro benzoic acid hydrazide, p-
amino benzoic acid hydrazide, m,p-dimethoxy benzoic acid
hydrazide, p-methoxy benzoic acid hydrazide, 1-naphthalyl acetic
acid hydrazide) were mixed on a magnetic stirrer with continuous
stirring for 0.5 h, in anhydrous 1,4-dioxane (25 ml) containing
0.3 ml of concentrated hydrochloric acid. The resulting mixture was
boiled under reflux for 12e18 h. After completion of reaction
(monitored by TLC) the reaction mixture was allowed to cool,
concentrated under reduced pressure and poured in to ice-cold
water with constant stirring. The product obtained was filtered,
dried and then purified through column chromatography (Hexane/
AcOEt 4:1) to afford solid product. The molecular weight and
percentage yield of final compounds are presented in Table 1.
d
2.1.7. (7-chloro-8-fluoro-3-methyl-1H-pyrazolo[4,3-c]cinnolin-
1-yl)(4-methylphenyl)methanone (4d)
Greenish yellow. IR (KBr) cmꢀ1: 1652 (C]O), 1624 (C]N),
1533 (C]C), 1509 (N]N), 1238 (CeF), 743 (CeCl). 1H NMR
(300 MHz, CDCl3):
d (ppm) 2.38 (s, 3H, CH3), 3.49 (s, 3H, CH3), 7.32
(s, 1H, C5eHCinnoline), 7.51 (s, 1H, C8eHCinnoline), 7.74e7.77 (d, 2H,
J ¼ 7.2 Hz, C3,5eHPhenyl), 8.58e8.61 (d, 2H, J ¼ 7.5 Hz, C2,6eHPhenyl).
13C NMR (75 MHz, CDCl3):
d (ppm) 12.6 (CH3), 24.4, 115.3,
117.5, 126.1, 129.4, 129.6, 131.3, 135.7, 136.0, 139.3, 151.4, 159.2,
168.2 (C]O). Anal. Calcd for C18H12ClFN4O: C, 60.94; H, 3.41; N,
15.79. Found: C, 61.15; H, 3.42; N, 15.76. DART-MS m/z: 354.10
(Mþ), 356.10 (Mþ þ 2).

178
R.K. Tonk et al. / European Journal of Medicinal Chemistry 57 (2012) 176e184
2.1.8. (7-chloro-8-fluoro-3-methyl-1H-pyrazolo[4,3-c]cinnolin-
1-yl)(2,4-dichlorophenyl)methanone (4e)
d (ppm) 2.36 (s, 3H, CH3), 4.62 (s, 2H, NH2), 7.19e7.21 (d, 2H,
J ¼ 6.3 Hz, C3,5eHPhenyl), 7.39 (s, 1H, C5eHCinnoline), 7.53 (s, 1H, C8e
Pale yellow. IR (KBr) cmꢀ1: 1650 (C]O), 1612 (C]N), 1537
H
Cinnoline), 7.94e7.97 (d, 2H, J ¼ 7.8 Hz, C2,6eHPhenyl). 13C NMR
(C]C), 1504 (N]N), 1230 (CeF), 742 (CeCl). 1H NMR (300 MHz,
(75 MHz, CDCl3): d (ppm) 12.2 (CH3),115.8,116.9,126.5,128.8,129.3,
CDCl3):
J ¼ 6.3 Hz), 7.16e7.24 (m, 2H, HPhenyl), 7.38 (s, 1H, C5eHCinnoline),
7.59 (1H, C8eHCinnoline). 13C NMR (75 MHz, CDCl3):
(ppm) 12.1
(CH3), 115.2, 117.4, 126.5, 128.6, 129.2, 129.5, 130.8, 135.2, 136.5,
139.4, 151.7, 159.3, 168.8 (C]O). Anal. Calcd for C17H8Cl3FN4O: C,
49.85; H, 1.97; N, 13.68. Found: C, 49.92; H, 1.98; N, 13.71. DART-
MS m/z: 409.16 (Mþ), 411.16 (Mþ þ 2), 415.16 (Mþ þ 6).
d
(ppm) 2.34 (s, 3H, CH3), 6.85e6.87 (d, 1H, HPhenyl
,
129.7, 131.5, 139.7, 149.8, 151.6, 159.0, 167.9 (C]O). Anal. Calcd for
C
17H11ClFN5O: C, 57.39; H, 3.12; N, 19.69. Found: C, 57.51; H, 3.13; N,
d
19.64. DART-MS m/z: 355.24 (Mþ), 357.24 (Mþ þ 2).
2.1.14. (7-chloro-8-fluoro-3-methyl-1H-pyrazolo[4,3-c]cinnolin-
1-yl)(3,4-dimethoxyphenyl)methanone (4k)
Pale yellow. IR (KBr) cmꢀ1: 1647 (C]O),1611 (C]N),1538 (C]C),
1497 (N]N), 1229 (CeF), 741 (CeCl). 1H NMR (300 MHz, CDCl3):
2.1.9. 1-(7-chloro-8-fluoro-3-methyl-1H-pyrazolo[4,3-c]
cinnolin-1-yl)-2-(2,4-dichlorophenoxy)ethanone (4f)
d
(ppm) 2.39 (s, 3H, CH3), 3.78 (s, 6H, 2 ꢂ OCH3), 7.15e7.17 (d, 1H,
Phenyl, J ¼ 6), 7.41 (s, 1H, C5eHCinnoline), 7.59 (s, 1H, C8eHCinnoline),
H
Pale yellow. IR (KBr) cmꢀ1: 1666 (C]O), 1604 (C]N), 1539
7.63e7.68 (m, 2H, HPhenyl). 13C NMR (75 MHz, CDCl3):
d
(ppm) 12.7
(C]C), 1498 (N]N), 1226 (CeF), 1058 (CeOeC), 755 (CeCl). 1H
(CH3), 57.5, 115.4, 117.2, 117.6, 126.5, 128.4, 129.2, 130.3, 131.2, 134.8,
139.3, 152.6, 158.9, 168.2 (C]O). Anal. Calcd for C19H14ClFN4O3: C,
56.94; H, 3.52; N, 13.98. Found: C, 57.14; H, 3.51; N, 14.02. DART-MS
m/z: 400.28 (Mþ), 402.28 (Mþ þ 2).
NMR (300 MHz, CDCl3):
d
(ppm) 2.37 (s, 3H, CH3), 5.24 (s, 2H,
7.19e7.26 (m, 2H,
Phenyl), 7.40 (s, 1H, C5eHCinnoline), 7.58 (s, 1H, C8eHCinnoline). 13C
CH2), 6.88e6.90 (d, 1H, HPhenyl, J ¼ 6.6 Hz),
d
H
NMR (75 MHz, CDCl3):
d (ppm) 12.6 (CH3), 61.5, 114.9, 116.9, 127.1,
127.6, 129.5, 129.7, 130.8, 135.6, 136.2, 139.1, 151.9, 159.3, 168.7
(C]O). Anal. Calcd for C18H10Cl3FN4O2: C, 49.17; H, 2.29; N, 12.74.
Found: C, 49.33; H, 2.31; N, 12.69. DART-MS m/z: 439.17 (Mþ),
441.16 (Mþ þ 2), 445.15 (Mþ þ 6).
2.1.15. (7-chloro-8-fluoro-3-methyl-1H-pyrazolo[4,3-c]cinnolin-
1-yl)(4-methoxyphenyl)methanone (4l)
Fineyellow. IR (KBr)cmꢀ1: 1644 (C]O),1623 (C]N),1536 (C]C),
1492 (N]N), 1226 (CeF), 752 (CeCl). 1H NMR (300 MHz, CDCl3):
d
(ppm) 2.33 (s, 3H, CH3), 3.69 (s, 3H, OCH3), 6.96e7.04 (m, 2H, C3,5e
Phenyl), 7.44 (s, 1H, C5eHCinnoline), 7.64 (s, 1H, C8eHCinnoline), 7.85e
7.91 (m, 2H, C2,6eHPhenyl). 13C NMR (75 MHz, CDCl3):
(ppm) 12.3
2.1.10. (7-chloro-8-fluoro-3-methyl-1H-pyrazolo[4,3-c]cinnolin-
1-yl)(4-chlorophenyl)methanone (4g)
H
d
Pale Yellow. IR (KBr) cmꢀ1: 1661 (C]O),1620 (C]N),1538 (C]C),
(CH3), 58.3, 114.7, 117.6, 118.2, 126.2, 128.4, 129.5, 129.8, 132.0, 133.6,
138.5, 153.2, 159.6, 167.4 (C]O). Anal. Calcd for C18H12ClFN4O2: C,
58.31; H, 3.26; N,15.11. Found: C, 58.49;H, 3.27; N,15.14. DART-MSm/
z: 370.25 (Mþ), 372.25 (Mþ þ 2).
1511 (N]N), 1235 (CeF), 744 (CeCl). 1H NMR (300 MHz, CDCl3):
d
(ppm) 2.37 (s, 3H, CH3), 7.38 (s,1H, C5eHCinnoline), 7.45e7.47 (d, 2H,
J ¼ 6.9 Hz, C3,5eHPhenyl), 7.52 (s, 1H, C8eHCinnoline), 7.82e7.84 (d, 2H,
J ¼ 7.8 Hz, C2,6eHPhenyl). 13C NMR (75 MHz, CDCl3):
d (ppm) 12.8
(CH3), 115.2, 118.1, 126.9, 128.6, 129.4, 129.7, 129.9, 131.7, 133.2, 137.1,
139.3, 152.3, 159.7, 169.4 (C]O). Anal. Calcd for C17H9Cl2FN4O: C,
54.42; H, 2.42; N, 14.93. Found: C, 54.59; H, 2.44; N, 14.90. DART-MS
m/z: 375.06 (Mþ), 377.06 (Mþ þ 2), 379.06 (Mþ þ 4).
2.1.16. 1-(7-chloro-8-fluoro-3-methyl-1H-pyrazolo[4,3-c]cinnolin-
1-yl)-2-(naphthalen-1-yl)ethanone (4m)
Brownish yellow. IR (KBr) cmꢀ1: 1658 (C]O), 1620 (C]N), 1536
(C]C), 1492 (N]N), 1218 (CeF), 746 (CeCl). 1H NMR (300 MHz,
CDCl3):
7H, HNaphthalyl), 7.47 (s, 1H, C5eHCinnoline), 7.68 (s, 1H, C8eHCinnoline).
13C NMR (75 MHz, CDCl3):
(ppm) 12.5 (CH3), 44.6, 112.8, 115.5,
d (ppm) 2.39 (s, 3H, CH3), 4.06 (s, 2H, CH2), 7.14e7.22 (m,
2.1.11. (7-chloro-8-fluoro-3-methyl-1H-pyrazolo[4,3-c]cinnolin-
1-yl)(4-fluorophenyl)methanone (4h)
d
Greenish yellow. IR (KBr) cmꢀ1: 1650 (C]O), 1615 (C]N), 1532
117.4, 121.3, 121.7,123.3,124.2,124.8,126.1,129.5,130.8,134.6,136.8,
138.2, 152.0, 159.4, 168.5 (C]O). Anal. Calcd for C22H14ClFN4O: C,
65.27; H, 3.49; N, 13.84. Found: C, 65.44; H, 3.47; N, 13.81. DART-MS
m/z: 404.31 (Mþ), 406.31 (Mþ þ 2).
(C]C), 1510 (N]N), 1222 (CeF), 752 (CeCl). 1H NMR (300 MHz,
CDCl3):
d (ppm) 2.37 (s, 3H, CH3), 7.34 (s, 1H, C5eHCinnoline), 7.51 (s,
1H, C8eHCinnoline), 7.66e7.68 (d, 2H, J ¼ 7.2 Hz, C3,5eHPhenyl), 7.90e
7.93 (d, 2H, J ¼ 9.3 Hz, C2,6eHPhenyl). 13C NMR (75 MHz, CDCl3):
d
(ppm) 12.8 (CH3),115.2,117.4,126.2,129.6,130.7,131.3,132.1,135.3,
2.2. Pharmacology
139.2, 151.0, 159.4, 168.1 (C]O). Anal. Calcd for C17H9ClF2N4O: C,
56.92; H, 2.53; N, 15.62. Found: C, 57.11; H, 2.55; N, 15.57. DART-MS
m/z: 358.22 (Mþ), 360.22 (Mþ þ 2).
2.2.1. Animals
Adult Wistar albino rats (150e200 g) were used to measure
anti-inflammatory, ulcerogenic and lipid peroxidation activity. The
animals were organized randomly in groups of six and each animal
was distinctly marked within its group. The animals were housed
properly and provided food and water ad libitum except during the
experiments.
2.1.12. (7-chloro-8-fluoro-3-methyl-1H-pyrazolo[4,3-c]cinnolin-
1-yl) (4-nitrophenyl)methanone (4i)
Paleyellow. IR (KBr) cmꢀ1: 1649 (C]O),1625 (C]N),1532 (C]C),
1506 (N]N), 1218 (CeF), 740 (CeCl). 1H NMR (300 MHz, CDCl3):
d
(ppm) 2.30 (s, 3H, CH3), 7.31e7.33 (m, 2H, J ¼ 6.9 Hz, C2,6eHPhenyl),
Standard drug and test compounds were administered p.o. as
a suspension in a 0.5% w/v solution of carboxymethyl cellulose
(CMC). The procedure and treatment schedule used for the animal
experiments were approved by the Institutional Animal Ethics
Committee (IAEC).
7.47 (s,1H, C5eHCinnoline), 7.61 (s,1H, C8eHCinnoline), 7.86e7.88 (d, 2H,
J ¼ 7.8 Hz, C3,5eHPhenyl). 13C NMR (75 MHz, CDCl3):
d (ppm) 12.4
(CH3), 115.4, 116.7, 126.0, 129.1, 129.5, 131.4, 135.3, 139.4, 142.6, 151.3,
159.4,168.3(C]O). Anal. CalcdforC17H9ClFN5O3:C, 52.93;H, 2.35;N,
18.16. Found: C, 53.13; H, 2.33; N, 18.20. DART-MS m/z: 385.29 (Mþ),
387.29 (Mþ þ 2).
2.2.2. Anti-inflammatory activity
Anti-inflammatory activity was determined by the carrageenan-
induced rat paw edema method [21]. Group I, which served as the
control, received only 0.5% w/v carboxymethyl cellulose (CMC).
Group II and the other groups received naproxen and the test
2.1.13. (4-aminophenyl)(7-chloro-8-fluoro-3-methyl-1H-pyrazolo
[4,3-c]cinnolin-1-yl)methanone (4j)
Pale yellow. IR (KBr) cmꢀ1: 1654 (C]O),1618 (C]N),1527 (C]C),
1502 (N]N), 1221 (CeF), 749 (CeCl). 1H NMR (300 MHz, CDCl3):
compounds, respectively, at a dose level of 45 mg kgꢀ1
.

R.K. Tonk et al. / European Journal of Medicinal Chemistry 57 (2012) 176e184
179
The hind paw edema was induced in each rat by the sub-planter
injection of 0.1 ml of 1% carrageenan solution in saline 1 h after the
administration of the test compounds and standard drug. The
volume of the paw edema (ml) was determined by a digital Ple-
thysmometer (Panlab LE7500) before and after 3 and 4 h the
carrageenan injection. The percent edema inhibition was calculated
according to the following formula:
10 min at 4000 rpm. The absorbance at 532 nm of the supernatant
organic layer was measured on a UV spectrophotometer and the
results were expressed as nmol MDA/100 mg tissue.
2.2.5. Acute toxicity study
The oral acute toxicity and approximate lethal dose (ALD50) of
the promising compounds were determined in rat using a method
previously described [24,25]. Six animals were kept in each group.
The compounds were administered orally in doses of 200, 300, 400
and 500 mg/kg. The toxic symptoms and mortality rates in each
group were recorded 24 h after drug administration.
Percent edema inhibition ¼ fVc ꢀ Vt=Vcg ꢂ 100
where Vc is the mean increase in paw volume in the absence of test
compound (control) and Vt is the mean increase of paw volume
after treatment with the test compound and standard drug. Results
are presented as mean ꢃ S.E.M. (standard error of mean) of six rats
in Table 2.
2.2.6. Assays for cyclooxygenase-2
Recombinant human COX-2 has been expressed in insect cell
expression system. These enzymes have been purified by employ-
ing conventional chromatographic techniques. Enzymatic activities
of COX-2 were measured according to the method of Copeland et al.
(1994) [26], with slight modifications using a chromogenic assay
based on the oxidation of N,N,N,N,-tetramethyl-p-phenylenedi-
amine (TMPD) during the reduction of PGG2 to PGH2 (Egan et al.,
Pagels et al., 1983) [27]. Briefly, the assay mixture contained Trise
HCl buffer (100 mM, pH 8.0), hematin (15 mM), EDTA (3 mM),
enzyme (100 mg COX-2) and the test compound. The mixture was
pre-incubated at 25 ꢁC for 1 min and then the reaction was initiated
by the addition of arachidonic acid and TMPD, in total volume of
1 ml. The enzyme activity was determined by estimating the
velocity of TMPD oxidation for the first 25 s of the reaction by
following the increase in absorbance at 603 nm. A low rate of non-
enzymatic oxidation observed in the absence of COX-2 was sub-
tracted from the experimental value while calculating the percent
inhibition.
2.2.3. Acute ulcerogenicity study
Acute ulcerogenicity was determined according to the Cioli
et al’s. method [22]. Animals were treated orally at 0 and 12 h with
two equal doses of either naproxen or the test compounds
(45 mg kgꢀ1/dose) except the control group, which received only
0.5% CMC. After the drug treatment, the rats were fed a normal diet
for 17 h and then sacrificed. The stomach was removed and opened
along the greater curvature. It was examined with the aid of
a microscope with a 4ꢂ magnifying lens. The mucosal damage in
each stomach was assessed according to the following scoring
system.
[Half (0.5) for redness; One (1.0) for spot ulcers; One and half
(1.5) for hemorrhagic streaks; Two (2.0) for ulcers more than 3 but
less than 5; Three (3.0) for more than five ulcers. The mean score of
each treated group minus the mean score of control group was
regarded as severity index of gastric mucosal damage.]
2.2.7. Antibacterial activity
2.2.4. Lipid peroxidation study
All the test compounds were evaluated in vitro against Escher-
ichia coli (NCTC 10418), Staphylococcus aureus (NCTC 65710) and
Pseudomonas aeruginosa (NCTC 10662) using the cup plate method
[28] with nutrient agar as the culture medium. A suspension of the
bacterial spore for lawning was prepared with Tween 80 (0.01%) in
normal saline. Liquid agar medium was poured (50 ml) into each
Petri dish (15 cm diameter). The solid agar medium was inoculated
with the bacterial suspension and the plates were then dried in an
incubator at 37 ꢁC for 1 h. Wells were made by an agar punch on
these seeded agar plates and solutions of the test compounds in
DMSO were added into each well, which were labeled previously.
The amount of lipid peroxidation was determined according to
the Ohkawa et al’s. method [23]. After screening for ulcerogenic
activity, the gastric mucosa was scraped with two glass slides,
weighed (100 mg) and homogenized in 1.8 ml of a 1.15% ice-cold
potassium chloride (KCl) solution. The homogenate was supple-
mented with 0.2 ml of 8.1% sodium dodecyl sulphate (SDS),1.5 ml of
acetate buffer (pH 3.5) and 1.5 ml of 0.8% thiobarbituric acid (TBA).
The mixture was then heated at 95 ꢁC for 60 min. After cooling, the
reactants were extracted with 5 ml of a mixture of n-butanol and
pyridine (15:1 v/v), shaken vigorously for 1 min and centrifuged for
Table 2
Anti-inflammatory activity of the synthesized compounds.
Compd. number
Paw volume (mL) ꢃ SEM
% inhibition ꢃ SEM
0 h
3 h
4 h
3 h
4 h
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
Naproxen
Control
0.54 ꢃ 0.042
0.55 ꢃ 0.039
0.55 ꢃ 0.038
0.56 ꢃ 0.046
0.54 ꢃ 0.027
0.56 ꢃ 0.039
0.54 ꢃ 0.036
0.52 ꢃ 0.038
0.56 ꢃ 0.046
0.53 ꢃ 0.042
0.53 ꢃ 0.027
0.54 ꢃ 0.028
0.52 ꢃ 0.034
0.54 ꢃ 0.15
1.08 ꢃ 0.057
1.03 ꢃ 0.035
1.32 ꢃ 0.064
0.94 ꢃ 0.044
1.39 ꢃ 0.075
1.64 ꢃ 0.055
1.42 ꢃ 0.053
0.98 ꢃ 0.052
1.63 ꢃ 0.082
1.10 ꢃ 0.053
1.06 ꢃ 0.078
0.84 ꢃ 0.028
1.63 ꢃ 0.053
0.84 ꢃ 0.018
1.95 ꢃ 0.015
1.12 ꢃ 0.058
1.07 ꢃ 0.053
1.38 ꢃ 0.055
0.97 ꢃ 0.057
1.49 ꢃ 0.055
1.79 ꢃ 0.049
1.55 ꢃ 0.039
1.03 ꢃ 0.052
1.77 ꢃ 0.068
1.15 ꢃ 0.047
1.09 ꢃ 0.059
0.86 ꢃ 0.036
1.78 ꢃ 0.044
0.85 ꢃ 0.020
2.17 ꢃ 0.018
61.31 ꢃ 2.47
65.37 ꢃ 2.16
44.58 ꢃ 3.54
72.34 ꢃ 3.51
39.30 ꢃ 3.53
22.64 ꢃ 1.61
37.19 ꢃ 2.55
67.36 ꢃ 2.03
24.01 ꢃ 3.45
59.20 ꢃ 2.32
62.49 ꢃ 3.72
78.35 ꢃ 2.02
20.51 ꢃ 2.93
78.84 ꢃ 1.39
e
64.40 ꢃ 2.55a
67.94 ꢃ 1.95a
48.65 ꢃ 4.70a
74.67 ꢃ 1.56
41.18 ꢃ 2.46a
24.02 ꢃ 4.14a
37.95 ꢃ 3.31a
68.46 ꢃ 2.50
25.47 ꢃ 2.54a
61.52 ꢃ 2.75a
66.25 ꢃ 2.18a
80.01 ꢃ 2.41
22.13 ꢃ 2.07a
81.23 ꢃ 1.19
e
0.55 ꢃ 0.006
Data were given in mean ꢃ SEM and analyzed by ANOVA followed by Dunnett’s multiple comparison test, (n ¼ 6).
a
P < 0.01 compared to standard drug.

180
R.K. Tonk et al. / European Journal of Medicinal Chemistry 57 (2012) 176e184
Table 3
3. Results and discussion
The ulcerogenic activity, lipid peroxidation and toxicity of test compounds.
3.1. Chemistry
Compound
Ulcerogenic activity
nmols of MDA
content ꢃ SEMb/
100 mg tissue
Toxicity
(mg/kg)
S.I.a ꢃ SEMb
Syntheses of the target compounds were carried out according
to the sequence of reactions outlined in Scheme 1. The key inter-
mediate, 3-acetyl-7-chloro-6-fluorocinnolin-4(1H)-one 3, was
synthesized in excellent yield through the cyclization of ethyl-2-[2-
Control
Naproxen
0.0
3.29 ꢃ 0.19
e
1.75 ꢃ 0.35
0.82 ꢃ 0.39
0.75 ꢃ 0.28*
0.25 ꢃ 0.11**
0.66 ꢃ 0.16*
0.99 ꢃ 0.30
0.41 ꢃ 0.15**
0.16 ꢃ 0.10**
7.83 ꢃ 0.39
e
4a
4b
4d
4h
4j
5.92 ꢃ 0.21**
5.84 ꢃ 0.30**
4.48 ꢃ 0.20**
5.27 ꢃ 0.13**
6.81 ꢃ 0.23
ND
ND
>300
ND
ND
ND
>300
(3-chloro-4-fluorophenyl)hydrazinylidene]-3-oxobutanoate
2 in
the presence of anhydrous AlCl3 and chlorobenzene. Literature
revealed that reactions of aryl hydrazines with 1,3-dicabonyl
compounds in acidic conditions preferentially resulted in one
regioisomer [30,31]. The structures of the newly synthesized
compounds were identified using IR, 1H NMR, 13C NMR and mass
spectral data. Elemental analysis of these synthesized compounds
was also in complete agreement with the proposed structures. The
IR spectrum of 3 showed bands at 3436 cmꢀ1, which indicates NeH
stretching, and at 1712 cmꢀ1 and 1659 cmꢀ1, indicating two
carbonyl groups. The absence of the triplet for eCH3 and the quartet
for eCH2 of eCH2CH3 in the 1H NMR spectrum of 3, which was
observed in the 1H NMR spectrum of 2, confirms the cyclization.
Compound 3 served as a source of 1,3-diketone and its subsequent
condensation with acid hydrazides, in a polar aprotic solvent (1,4-
dioxane) containing a catalytic amount of concentrated HCl, fur-
nished the final compounds 4aem. After the pyrazole formation,
4k
4l
4.63 ꢃ 0.18**
4.03 ꢃ 0.32**
ND denotes not determined.
a
Severity index (S.I.): mean score of each treated group minus the mean score of
the control group.
b
Relative to standard and data were analyzed by ANOVA followed by Dunnett’s
multiple comparison test for n ¼ 6; **P < 0.01, *P < 0.05.
DMSO was used as control. This procedure was repeated for each
bacterial strain and the plates were then incubated at 37 ꢁC for 18e
24 h. Preliminary screening of the test compounds was carried out
at a concentration of 100
mg/ml by measuring the diameter in
millimeters of the zone of growth inhibition formed around each
well. The minimum inhibitory concentration (MIC) was then
determined. This was the lowest concentration at which there was
no visible growth of the microorganism when using two-fold
the characteristic singlet of the cinnolineeNH proton at
disappeared and the singlet of the methyl group shifted upfield to
2.33, as observed in the 1H NMR spectrum of compound 4l. Its 13
NMR spectrum indicated the absence of signals for the carbonyl
groups at 176.8 and
194.6, which were seen in the 13C spectrum
of 3, and showed signals at 12.3 and at 167.4, which represent
d 13.8
dilutions (50, 25, 12.5, 6.25, 3.12
mg/ml) for those compounds
d
C
which showed a good antibacterial profile in the preliminary
screening. The antibacterial activity of each compound (4aem) was
compared against standard ciprofloxacin and the results are
summarized in Table 5.
d
d
d
d
the methyl group at C3 and the carbonyl group at C1, respectively. It
was also supported by the mass spectrum of 4l, which showed
a molecular ion peak at m/z 370.25.
2.3. Docking simulations
Furthermore, an appropriate 1D Nuclear Overhausher Effect
(NOE) experiment of 4l was also carried out for further structural
confirmation of the synthesized compounds. The NOE difference
spectrum of 4l showed that, when 4-methoxy substituted phenyl
H-2 was used for irradiation, H-9 (at the cinnoline ring) achieves
more enhancement than the eCH3 group (at the pyrazole ring),
which is only possible if compound 4l has structure I, in which the
4-methoxy substituted phenyl and methyl groups are in the 1 and 3
positions to each other, as described in Fig. 2.
Docking studies were performed using the Glide module in the
Schrodinger 9.1 program. The crystal structure of COX-2 (1CX2)
was obtained from the protein data bank and prepared with the
protein preparation wizard module in Schrodinger 9.1 [29]. The
binding site was generated by keeping the co-crystallized ligand at
the center of a rectangular box drawn in the receptor. Defining this
binding site is known as receptor grid generation. A 20 Å grid space
was defined for the co-crystallized ligand using the glide grid
module of the software. The LigPrep module was used to produce
low-energy conformers and to correct the chirality of all the
ligands.
3.2. Pharmacology
Ligands were kept flexible by producing the ring conforma-
tions and by penalizing nonpolar amide bond conformations,
whereas the receptor was kept rigid throughout the docking
studies. All other parameters of the Glide module were main-
tained at their default values. The lowest energy conformation
was selected and the ligand interactions (hydrogen bonding and
hydrophobic interaction) with the active sites of COX-2 were
determined.
3.2.1. Anti-inflammatory activity
The carrageenan-induced rat paw edema bioassay was used for
the evaluation of the anti-inflammatory activity of the test
compounds 4aem; the results are summarized in Table 2. Most of
the test compounds showed appreciable inhibition of the edema size
incomparisonwithnaproxen. Thecompounds(7-chloro-8-fluoro-3-
methyl-1H-pyrazolo[4,3-c]cinnolin-1-yl)(4-methylphenyl) meth-
anone (4d) and (7-chloro-8-fluoro-3-methyl-1H-pyrazolo[4,3-c]
cinnolin-1-yl)(4-methoxyphenyl)methanone (4l) showed excellent
protection against inflammation (74.67 and 80.01% inhibition,
respectively), whereas compounds 4f, 4i and 4m were found to have
the least inhibitory effect. Compounds 4a, 4b, 4h, 4j and 4k also
showed good systemic anti-inflammatory activity with a percent
inhibition of 64.40, 67.94, 68.46, 61.52 and 66.25%, respectively, in
comparison to naproxen, which has a percent inhibition of 81.23%.
The rest of the compounds exhibited weak to moderate anti-
inflammatory activity. Introduction of methylene (eCH2e) as
a spacer group between the substituted phenyl ring and the carbonyl
carbon (]C]O) increases the anti-inflammatory activity of the
Table 4
COX-2 inhibition data for selective compounds.
Compound
COX-2 inhibition
COX-2
(at 10
m
M conc.), %
IC50 (mM)
4b
4d
4h
4k
4l
5.82
39.15
14.88
17.79
49.83
ND
12.8
ND
ND
10.0
ND denotes not determined.

R.K. Tonk et al. / European Journal of Medicinal Chemistry 57 (2012) 176e184
181
Table 5
Antibacterial activity of test compounds.
Compound
Diameter of zone of inhibition (mm): Meana ꢃ S.D.b
MICc
(mg/mL)
E. coli (NCTC 10418)
S. aureus (NCTC 65710)
P. aeruginosa (NCTC 10662)
E. coli
S. aureus
P. aeruginosa
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
12.17 ꢃ 0.99
13.28 ꢃ 1.04
11.31 ꢃ 0.39
9.23 ꢃ 1.18
15.34 ꢃ 0.90
10.09 ꢃ 1.05
9.57 ꢃ 1.26
11.49 ꢃ 0.61
16.35 ꢃ 0.85
6.05 ꢃ 0.76
9.84 ꢃ 0.71
9.23 ꢃ 0.59
6.26 ꢃ 0.48
25.32 ꢃ 0.52
00
10.13 ꢃ 0.35
13.52 ꢃ 0.75
11.15 ꢃ 0.43
8.11 ꢃ 0.78
12.06 ꢃ 0.62
7.41 ꢃ 1.13
8.18 ꢃ 0.37
10.22 ꢃ 0.64
13.33 ꢃ 0.87
e
11.06 ꢃ 0.63
12.25 ꢃ 0.73
10.33 ꢃ 0.53
8.36 ꢃ 0.38
14.05 ꢃ 0.53
8.32 ꢃ 0.98
9.1 ꢃ 0.44
10.94 ꢃ 0.48
14.18 ꢃ 0.59
e
25
25
25
Nt
12.5
Nt
50
25
50
Nt
25
Nt
Nt
50
25
Nt
Nt
Nt
Nt
3.12
e
50
25
50
Nt
12.5
Nt
Nt
Nt
25
12.5
Nt
Nt
Nt
Nt
3.12
e
50
12.5
Nt
Nt
Nt
Nt
3.12
e
8.27 ꢃ 1.31
9.14 ꢃ 0.95
e
9.29 ꢃ 0.89
7.16 ꢃ 0.51
5.11 ꢃ 0.87
22.65 ꢃ 0.61
00
4m
Ciprofloxacin
DMSO
23.57 ꢃ 0.58
00
Nt denotes not tested.
a
Mean value of measured diameters of zones of inhibition at 100
S.D. denotes the standard deviation.
MIC denotes minimum inhibitory concentration.
mg/ml.
b
c
compound, as in the case of 4b. On the other hand, a considerable
decrease in activity was observed in compounds containing the eOe
CH2e spacer group, as in the case of 4f. Furthermore, it was found
that the compounds with an electron donating substituent on the
benzoyl ring showed a higher anti-inflammatory activity than those
compounds with a benzoyl ring substituent that has an electron
withdrawing group.
3.2.3. Lipid peroxidation
The lipid peroxidation profile of the selected compounds was
also determined in order to validate the ulcerogenic activity results,
as shown in Table 3. It was observed that 4d and 4l, which
exhibited the least ulcerogenic effect, also showed marked reduc-
tion in lipid peroxidation. It is noteworthy that naproxen, the
reference standard, exhibited the highest lipid peroxidation,
7.83 ꢃ 0.39 nmol MDA/100 mg tissue, whereas the control group
showed a lipid peroxidation of 3.29 ꢃ 0.19 nmol MDA/100 mg
tissue under the same experimental conditions. These results
further suggest that the protective effect of the tested compounds
might be related to the inhibition of lipid peroxidation in the
gastric mucosal wall. It is important to mention here that a cinno-
line derivative [32] was recently isolated by Chen et al. from an
extract of Cichorium endivia L. and has been found to have antiox-
idant properties.
3.2.2. Acute ulcerogenicity study
The test compounds that exhibited notable anti-inflammatory
profiles were further evaluated for their ulcerogenic potential.
The results, which are shown in Table 3, revealed that all the tested
compounds showed a superior GI safety profile (S.I. values 0.16e
0.99) in Wistar rats compared to the standard drug naproxen (S.I.
value 1.75) at an oral dose of 90 mg/day. Compounds 4d and 4l
showed
a
severity index of 0.25
ꢃ
0.11 and 0.16
ꢃ
0.10,
respectively, which is less than one-sixth (1/6) the value obtained
with the standard drug. These findings further support the state-
ment that 4d and 4l may be considered safer in terms of gastric
ulcerogenicity than the conventional drug naproxen.
3.2.4. Acute toxicity study
The active compounds 4d and 4l were further evaluated to
determine their acute oral toxicity. The results (Table 3) indicated
Scheme 1. Synthesis of the intermediate and target compounds: (a) 1. NaNO2/HCl, 2. Ethyl acetoacetate/sodium acetate, 84%. (b) 1. AlCl3, Cl-benzene 2. Dil. HCl, Dil. NaOH, reflux,
16 h, 76%. (c) Anhydrous 1,4-dioxane, Conc. HCl, 12e18 h, 105 ꢁC.

182
R.K. Tonk et al. / European Journal of Medicinal Chemistry 57 (2012) 176e184
Fig. 3. COX-2 inhibition data for selected compounds.
517, Tyr 348, Leu 531, Leu 359, Trp 387, Tyr 385 and Leu 384.
Compound 4l (IC50:10 M) showed hydrogen bond acceptor
m
interactions with His 90, Tyr 355 and Leu 359 (Fig. 5), in addition to
hydrophobic interactions with Val 523, Ala 527, Val 349, Phe 518, Ile
517 and Leu 352. Moreover both the compounds have methyl group
(eCH3 in 4d and eOCH3 in 4l) at the para position of phenyl ring,
which is well occupied in the active site volume and could be
responsible for their higher potency.
Fig. 1. Structures of phenylbutazone, cinnopentazone, and reference ligand (SC-558).
that the tested compounds were found to be non-toxic at the
normal dose and were well tolerated by the experimental animals
up to 300 mg kgꢀ1. In addition, no mortality was observed 24 h
post-administration, even at the highest dose of 500 mg/kg body
weight, which suggests a wide margin of safety.
3.2.5. COX-2 inhibition
Five synthesized compounds that demonstrated the best in vivo
results were evaluated for their anti-inflammatory activity by
measuring their in vitro biochemical COX-2 inhibitory activity. The
results of the COX-2 inhibition are given in Table 4 and Fig. 3.
Compound 4d (IC50:12.8
mM) and compound 4l (IC50:10 mM)
showed promising COX-2 inhibition.
Docking studies of the compounds were performed on the Glide
[33] module of Schrodinger 9.0 to rationalize their anti-
inflammatory results.
A well-reported three-dimensional co-
crystal structure of COX-2 (PDB ID: 1CX2) with a selective inhib-
itor, SC-558 (Fig. 1), was used as the template for the docking
studies. Figs. 4 and 5 show the binding interactions of most potent
compounds 4d and 4l, respectively, to the active site of COX- 2;
these are similar to those observed with SC-558. Compound 4d
(IC50:12.8
mM) displayed hydrogen bond interactions with Tyr 355
and Leu 359 (Fig. 4) and hydrophobic interactions with Phe 381, Ile
Fig. 4. 3D view from a molecular modeling study, showing minimum energy structure
of the complex of 4d docked in Cox-2 (PDB ID: 1CX2).
Fig. 2. Diagnostic NOE in compound 4l.

R.K. Tonk et al. / European Journal of Medicinal Chemistry 57 (2012) 176e184
183
E. coli and P. aeruginosa than S. aureus. Cinnoxacin, a cinnoline
derivative exerts its antibacterial action through the inhibition of
the bacterial DNA-gyrase enzyme [34]. Therefore, the antibacterial
action of the designed compounds might be due to their ability to
inhibit DNA-gyrase.
4. Conclusion
A
new series of pyrazolo[4,3-c]cinnoline derivatives were
synthesized with the objective of developing new anti-
a
inflammatoryeantibacterial agent with minimum gastric irrita-
tion and a good safety margin. Compounds 4d and 4l were found to
be potent anti-inflammatory agents with a percent inhibition after
4 h of 74.67 ꢃ 1.56 and 80.01 ꢃ 2.41, respectively. Compound 4l
displayed stronger binding interactions with the active site of the
human COX-2 enzyme than compound 4d. However, both of these
derivatives did not show specific antibacterial activity against all of
the tested strains. Compounds 4e and 4i were found to be the most
active antibacterial agents in the present study. Moreover, 4b
emerged as a potential dual anti-inflammatoryeantibacterial agent
with low ulcerogenic effect.
Therefore, it can be safely concluded that compound 4b would
constituteausefulmodelforfurtherinvestigationinthedevelopment
of a new class of dual non-acidic anti-inflammatoryeantibacterial
agents.
Acknowledgments
The authors are thankful to Department of Pharmaceutical
Chemistry for providing laboratory facilities and IIT, New Delhi for
recording the 1H NMR and 13C NMR spectra. The authors are also
thankful to Mr. B.R. Sikri for providing gift sample of standard drug
and Dr. Qadir, Head, IGIB, New Delhi for providing bacterial strains.
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m
m
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