Synthesis of new nicotinic acid derivatives and their evaluation as analgesic and anti-inflammatory agents

Article abstract of DOI:10.1248/cpb.c13-00261

A series of 2-substituted phenyl derivatives of nicotinic acid 4a-l were synthesized and evaluated for their analgesic and anti-inflammatory activities. Compounds including 2-bromophenyl substituent, 4a, c, and d, proved to display distinctive analgesic and anti-inflammatory activities in comparison to mefenamic acid as a reference drug. Compound 4c could be identified as the most biologically active member within this study with an interesting dual anti-inflammatory analgesic profile. Effect of the compounds 4a-l on the serum level of certain inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 was also determined.

Full text of DOI:10.1248/cpb.c13-00261

September 2013
Regular Article
Chem. Pharm. Bull. 61(9) 933–940 (2013)
933
Synthesis of New Nicotinic Acid Derivatives and Their Evaluation as
Analgesic and Anti-inflammatory Agents
,a
Nadia Abdalla Khalil,a Eman Mohamed Ahmed,* Khaled Omar Mohamed,a and
Sawsan Abo-Bakr Zaitoneb
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University; Cairo 11562, Egypt: and
b Department of Pharmacology & Toxicology, Faculty of Pharmacy, Suez Canal University; Ismailia 41522, Egypt.
Received April 4, 2013; accepted June 17, 2013
A series of 2-substituted phenyl derivatives of nicotinic acid 4a–l were synthesized and evaluated for
their analgesic and anti-inflammatory activities. Compounds including 2-bromophenyl substituent, 4a, c,
and d, proved to display distinctive analgesic and anti-inflammatory activities in comparison to mefenamic
acid as a reference drug. Compound 4c could be identified as the most biologically active member within
this study with an interesting dual anti-inflammatory analgesic profile. Effect of the compounds 4a–l on the
serum level of certain inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6
was also determined.
Key words nicotinic acid; fenamate; analgesic; anti-inflammatory
Osteoarthritis (OA) is the most common cause of chronic modification of classic NSAIDs have been taken with the aim
joint pain. Degeneration of joint cartilage is the central feature of improving their safety profile. Pyridine derivatives form
in OA, but the disease is associated with concomitant changes an important class of heterocyclic compounds that have at-
in synovium and subchondral bone metabolism, causing in- tracted the attention of many scientists. The pyridine nucleus
flammation of the synovial membrane in the involved joints.1) is present in niflumic acid and flunixin, which are a traditional
One of the factors that stimulate OA, results from the failure NSAIDs belonging to the class of fenamates (Fig. 1). Unlike
of chondrocytes that lie within the joint to synthesize a good- most other NSAIDs, the fenamates appear also to compete
quality matrix, and to maintain a balance between synthesis with PGs for binding at the PG receptor site and, thus, po-
and degradation of the extracellular matrix. Thus, synovial tentially antagonize the physiopathological effects of PGs that
inflammation and local concentration of pro-inflammatory have already been formed.12) However, fenamates are still
mediators seems to be directly involved in the generation of exhibit most of the GI adverse effects induced by NSAIDs.
pain in OA joints.2)
GI damage from NSAIDs may be generally attributed to
Recent studies pointed out the key roles of inflammatory the direct contact of the carboxylic acid (–COOH) moiety
cytokines, such as tumor necrosis factor (TNF)-α and inter- of NSAIDs with GI mucosal cells (topical effect) as well as
leukin (IL)-6, in the etiology and pathogenesis of rheumatoid decreased tissue PG production in tissues thus reducing the
arthritis (RA) and osteoarthritis.3,4) Thus, therapies suppress- cytoprotective effect of PGs in maintaining GI health.13)
ing these inflammatory cytokines have been focused on, as an
effective treatment of these inflammatory diseases. In addi-
tion, myeloperoxidase (MPO) plays an important role in infec-
tion and inflammation through converting hydrogen peroxide
and chloride to hypochlorous acid and water.5) The excessive
MPO activity, and production of reactive oxygen species
(ROS), especially HOCl, are implicated in many inflammatory
processes, including atherosclerosis, cancer, and Alzheimer’s
disease.6,7) MPO is released in the extracellular medium by
highly stimulated and dying neutrophils in pathological condi-
tions of acute and chronic inflammation. Under these condi-
tions, MPO is able to exert oxidant activity on neighbouring
cells and tissues.8)
The medications most commonly used to treat OA are
symptom-modifying drugs, such as nonsteroidal anti-inflam-
matory drugs (NSAIDs), which inhibit prostaglandin (PG)
formation through inhibition of both cyclooxygenase (COX)-1
and COX-2 enzymes. However, the chronic use of currently
available NSAIDs may elicit appreciable gastrointestinal (GI)
toxicity leading to ulceration, bleeding and nephrotoxicity,
which causes some patients to abandon NSAID therapy.9–11)
On this basis, synthetic approaches based upon chemical
Fig. 1. Fenamate Lead Compounds and General Structure of the Syn-
The authors declare no conflict of interest.
thesized Compounds
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: dr_eman2001@hotmail.com

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Vol. 61, No. 9
Several studies have described the derivatization of the accounts for formation of E-isomers exclusively, in DMSO-
carboxylic function with amide or N-acylarylhydrazone moi- d6 solution, whereas Z-isomer can be stabilized in less polar
eties.14–17) N-Acylhydrazones have been widely described as solvents by an intramolecular hydrogen bond.20) In the present
potent anti-inflammatory, antinociceptive, and anti-platelet study, the spectral data were recorded in DMSO-d6, and the
compounds, due to their ability to mimic the bis-allylic moiety signal corresponding to the imine hydrogen was observed in
of unsaturated fatty acids and amides, such as arachidonic the range of 8.19–8.93ppm.
1
acid.14)
Analytical and spectral data (IR, H-NMR, 13C-NMR, mass
In light of the above facts, this work comprises the syn- spectra) of the synthesized compounds were in full agreement
thesis and evaluation of analgesic and anti-inflammatory ac- with the proposed structures.
tivities of new series of N-acylarylhydrazone derivatives of 2-
Analgesic Activity In this study, the new compounds
arylaminonicotinic acids and 2-aryloxynicotinic acids (Fig. 1). were tested for analgesic activity using the writhing test in
mice21) and compared to mefenamic acid, as a reference stan-
dard at the same dose level (25mg/kg, per os (p.o.)). Writh-
Results and Discussion
Chemistry The synthetic route to the target compounds ing was induced by intraperitoneal (i.p.) injection of freshly
4a–l is outlined in Chart 1. Briefly, the 2-substituted nicotinic prepared acetic acid solution (1%, 10mL/kg) in mice. The
acids 1a–c, obtained according to a procedure previously de- number of writhes (constriction of abdomen, turning of trunk,
scribed,18) were esterified to give the corresponding ethyl nico- extension of hind limbs) due to acetic acid was expressed
1
tinate derivatives 2a–c. The appearance of signals in H-NMR as a nociceptive response. Vehicle treated control mice were
spectra, at δ 1.28–1.35ppm and 4.25–4.40ppm assignable to given acetic acid solution (1%) and writhing response was
CH3 and CH2 protons respectively of –COOCH2CH3 con- noted during the period of 15min. The results showed that the
firms the formation of ester 2. The latter compounds were tested compounds exhibited analgesic activity ranging from
also confirmed by their IR spectra, which revealed a band at 22.2 1.2% to 73.7 6.2%. Compounds 4a, c, and d showed
1735–1750 cm−1 indicating ester carbonyl function.
significant reduction in the writhing response (61.7 4.8 to
Reaction of 2a–c with hydrazine hydrate in absolute etha- 73.7 6.2). Compound 4c showed analgesic effect similar to
nol, afforded the corresponding hydrazides 3a–c, which are that exhibited by mefenamic acid (72.4 4.6%). The com-
key intermediates for the production of the title compounds pounds that displayed the highest activity in the writhing test
4a–l. IR spectra of 3a–c, showed bands due to carbonyl amide include 2-bromo substituent in the phenyl ring. Replacement
function in the range of 1639–1697cm−1. The target hydrazone of the 2-bromo substituent by carboxamide moiety results in
compounds 4a–l were prepared in good yields by the conden- significant reduction in activity (compounds 4e–h). It was
sation of the hydrazides 3 with different substituted aromatic also obvious that, among the compounds bearing carboxamide
aldehydes in refluxing ethanol. The compounds containing substituent in the 2-position of the phenyl ring (compounds
arylidene–hydrazide structure may exist as E/Z geometri- 4e–l), higher analgesic activity was observed with the aryloxy
cal isomers about –C=N– double bond and cis/trans amide analogues (X=O), (compounds 4i–l) (Table 1).
conformers.19) According to literature, the presence of single
Anti-inflammatory Activity Anti-inflammatory activity
downfield resonating (8.43–8.99ppm) imine hydrogen signal was determined by using carrageenan induced rat paw edema
Reagents and conditions: a: Abs. C2H5OH, conc. H2SO4, 8h, b: N2H4·H2O, C2H5OH, 3h, c: ArCHO, C2H5OH, 4h.
Chart 1

September 2013
935
Table 1. Analgesic, Anti-inflammatory and Ulcerogenic Potential of the Tested Compounds
Compound
Vehicle
Analgesic activity (% protection)
Anti-inflammatory activity (% inhibition) Ulcerogenic potential (Severity index)
0
0
0
Mefenamic acid
72.4 4.6*
61.7 4.8*
54.8 4.2*,#
73.7 6.2*
70.5 4.8*
25.4 2.1*,#
28.2 1.1*,#
22.2 1.2*,#
55.2 4.3*,#
24.8 2.3*,#
52.3 0.42*,#
42.1 0.38*,#
52.3 0.42*,#
59.3 4.21*
52.9 3.8*,#
39.2 2.5*,#
62.2 5.4*
56.3 4.2*
15.5 0.8*,#
22.3 1.2*,#
23.1 0.62*,#
32.1 1.8*,#
12.0 0.9*,#
41.0 3.4*,#
31.2 0.16*,#
22.1 0.18*,#
1.0 0.06*
4a
4b
4c
4d
4e
4f
0.82 0.05*,#
0.72 0.05*,#
1.3 0.09*,#
0.86 0.05*
0.5 0.02*,#
0.3 0.01*,#
0.41 0.02*,#
0.69 0.05*,#
0.2 0.01*,#
0.64 0.03*,#
0.18 0.01*,#
0.22 0.01*,#
4g
4h
4i
4j
4k
4l
The vehicle group was pretreated with 1% carboxymethylcellulose solution. All compounds were tested in a dose equals 25mg/kg (p.o.) 30min before testing. Data are
presented as mean S.E.M. and analyzed using one-way ANOVA followed by Tukey’s test for multiple comparisons. *p<0.05 compared to the vehicle group and # p<0.05
compared to mefenamic acid group.
Table 2. Serum Level of Tumor Necrosis Factor-α (TNF-α) and Interleukin 6 (IL-6) in Mice Pretreated with the Mefenamic Acid or the Tested Com-
pounds in Carrageenan-Induced Paw Edema Test
Compound
Vehicle
TNF-α (mg/mL)
IL-6 (mg/mL)
5.24 0.42
20.4 1.6
Mefenamic acid
3.04 0.27*
3.32 0.28*
3.55 0.32*
2.67 0.21*
3.12 0.27*
4.38 0.42*,#
4.23 0.36*,#
4.78 0.45,#
3.42 0.31*,#
4.54 0.43,#
3.72 0.31*,#
5.12 0.47,#
4.98 0.45,#
8.2 0.54*
10.7 0.87*,#
14.7 0.12*,#
8.3 0.65*
9.6 0.76*
16.5 1.22,#
16.8 1.31,#
17.7 1.42,#
12.2 1.16*,#
17.1 0.12,#
4a
4b
4c
4d
4e
4f
4g
4h
4i
,#
4j
13.5 0.11*
21.34 0.24,#
22.34 0.21,#
4k
4l
All compounds were tested in a dose equals 25mg/kg (p.o.) 30min before testing. Data are represented as mean S.E.M. and analyzed using one-way ANOVA followed by
Tukey’s test for multiple comparisons. *p<0.05 compared to the vehicle group and # p<0.05 compared to mefenamic acid group.
model.22) Carrageenan (1% w/v) was used to produce paw verity index than mefenamic acid. The results of ulcerogenic-
edema. Edema is presented as percentage increase in right ity studies revealed that derivatization of the carboxylic acid
hind paw, in comparison to the uninjected left hind paw. Per- group slightly improved the safety margin of the all final new
centage change in paw volume was calculated and expressed compounds with the exception of 4c. Thus, the ulcerogenic
as the amount of inflammation. Compounds 4a, c, and d effects of the compounds 4a–l may be attributed to the non-
significantly induced strong anti-inflammatory effect that was selective suppression of cyclooxygenases COX-1 and COX-2
comparable to that exhibited by mefenamic acid. These results isoforms, thereby preventing the biosynthesis of PGs that have
were in agreement with those shown in the analgesic activity a role in homeostasis and gastroprotection.
test (Table 1).
Immunohistochemistry for Myeloperoxidase Enzyme
Furthermore, mefenamic acid as well as the tested com- MPO is released in the extracellular medium by highly stimu-
pounds 4a–d, have shown significant suppression of serum lated and dying neutrophils in pathological conditions of acute
IL-6 and TNF-α levels compared to the control group (Table and chronic inflammation. Under these conditions, MPO is
2).
able to exert oxidant activity on neighbouring cells and tis-
Acute Ulcerogenicity Studies In current study, test- sues. Compounds that possess antioxidant activity have the
ing acute ulcerogenic potential revealed that mefenamic acid ability to inhibit MPO level, and thus suppress oxidative dam-
showed an ulcer severity index of 1.0 0.06. All tested com- age and may consequently, prevent inflammatory conditions.23)
pounds except 4c showed lower mean severity index than that
Histopathological examination revealed that the mucosa of
observed by mefenamic acid, indicating lower ulcerogenic mefenamic acid group as well as groups treated with com-
effect (Table 1). Although compound 4c revealed promising pounds 4a–e, h, and j showed higher staining for myeloper-
analgesic and anti-inflammatory results, it displayed higher se- oxidase enzyme compared to the vehicle group (Figs. 2A,B).

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Vol. 61, No. 9
Fig. 2. (A) Photomicrographs Showing the Immunohistochemical Staining for Myeloperoxidase Enzyme (MPO) in the Gastric Mucosa of Normal
Mice and Mice Treated with Some of the Screened Compounds (×200) and (B) Relative MPO Expression in Different Groups Compared to the Ve-
hicle Group Determined by Measuring the Optical Density for MPO Immunopositive Cells in Each Group
Data are expressed as mean S.E.M. and analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test at p<0.05. *p<0.05 compared to the vehicle group
and #p<0.05 compared to mefanamic acid group.
sion of ulcerogenicity, compared to the free carboxylic acid
Various 2-substitutedphenyl derivatives of nicotinic acid analogues.
Conclusion
4a–l structurally related to niflumic acid were synthesized and
In light of these results, one can say that the most promis-
screened for their potential analgesic, anti-inflammatory and ing results as the anti-inflammatory and analgesic agents were
ulcerogenic potentials. Promising analgesic and anti-inflam- observed with the nicotinic acid derivatives 4a, c, and d, con-
matory activities compared to the reference drug, mefenamic taining 2-bromophenyl substituent in their structure.
acid, was observed with the compounds 4a, c, and d. It has
been also obvious that the latter compounds significantly re-
duced serum TNF-α and IL-6 levels.
Experimental
Chemistry All chemicals and reagents were obtained
The tested compounds 4a–l were also screened for ulcero- from Aldrich (Sigma-Aldrich, St. Louis, MO, U.S.A.), and
genic adverse effect. The results revealed that all compounds were used without further purification. Reactions were moni-
except 4c showed lower ulcerogenic potential than the refer- tored by TLC, performed on silica gel glass plates containing
ence drug.
60 GF-254, and visualization on TLC was achieved by UV
From close inspection of the results of in vivo experiments light or iodine indicator. IR spectra were determined on Shi-
1
in this study and our previous investigation in this field,18) it madzu IR 435 spectrophotometer (KBr, cm−1). H-NMR spec-
is obvious that derivatization of carboxylic acid functionality tra were carried out using a Mercury 300-BB 300MHz using
with N-acyl hydrazone in compounds 4a, c, and d resulted in tetramethylsilane (TMS) as internal standard. 13C-NMR spec-
significant increase in both analgesic and anti-inflammatory tra were carried out using a Mercury 300-BB 75MHz using
activities, without affecting the ulcerogenic potential com- TMS as internal standard. Chemical shifts (δ) are recorded
pared to the corresponding analogue with free carboxylic acid in ppm on δ scale, Micro Analytical Center, Cairo University,
grouping. On the other hand, derivatization of the carboxylic Egypt. Mass spectra were recorded on Shimadzu Qp-2010
acid function suppress the analgesic and anti-inflammatory plus Spectrometer, Micro Analytical Center, Cairo Univer-
properties of compounds 4e–4h with concomitant suppres- sity, Egypt. Elemental analyses were carried out at the Micro

September 2013
937
Analytical Center, Cairo University, Egypt. Melting points δ (ppm): 6.46–6.60 (m, 3H, 2H Ar, H5, pyr.), 7.68–7.71 (d,
were determined with Stuart apparatus and are uncorrected. 2H, 1H Ar, H4, pyr.), 8.40 (s, 1H, NH, D2O exchangeable),
Progress of the reactions was monitored using TLC sheets 8.28–8.31 (m, 2H, 1H Ar, H6, pyr.), 10.54 (s, 3H, NH, NH2,
precoated with UV fluorescent silica gel Merck 60F 254 using D2O exchangeable); MS (EI) m/z (% rel. Int.): 308 (M+2,
acetone–benzene (1 : 9) and were visualized using UV lamp.
The starting materials 2-(2-bromophenylamino)nicotinic H, 3.61; N, 18.24. Found: C, 46.88; H, 3.55; N, 18.46.
4.64), 306 (M+, 4.91); Anal. Calcd for C12H11BrN4O: C, 46.93;
acid 1a, 2-(2-carbamoylphenylamino)nicotinic acid 1b and
2-[(3-Hydrazinecarbonyl)pyridine-2-ylamino]benzamide
2-(2-carbamoylphenoxy)nicotinic acid 1c were prepared as re- (3b): Yield 60%; mp 219–220°C; IR (KBr) cm−1: 3444–3302
ported.18) Other chemicals were obtained from Aldrich, Fluka, (NH, NH2), 3078 (C–H aromatic), 1697, 1647 (2C=O);
or Merck Chemicals.
1H-NMR (DMSO-d6, 300MHz) δ (ppm): 4.93 (s, 2H, NH2,
General Procedure for the Preparation of 2a–c To a D2O exchangeable), 6.65–6.70 (m, 1H, Ar), 7.45–7.84 (m, 4H,
solution of an appropriate 1a–c (10mmol) in absolute ethanol 3H Ar, H5, pyr.), 8.22 (d, 1H, H4, pyr.), 8.76 (d, 1H, H6, pyr.),
(30mL), conc H2SO4 (1mL) was added, and the mixture was 9.28 (s, 1H, NH, D2O exchangeable), 12.23 (s, 3H, NH, NH2,
heated under reflux for 8h. After cooling, the reaction mixture D2O exchangeable); 13C-NMR (DMSO-d6 ppm) δ: 106.48,
was concentrated under reduced pressure, an aqueous sodium 116.15, 121.00, 125.63, 126.15, 126.70, 127.20, 134.53, 139.21,
carbonate solution (20mL, 10%, w/v) was added, and the ester 140.96, 148.69 (aromatic C’s), 160.32, 166.20 (2C=O); MS (EI)
was extracted with chloroform (2×15mL). The combined ex- m/z (% rel. Int.): 272 (M+H, 52.78), 271 (M+, 57.41); Anal.
tract was dried (Na2SO4), filtered, concentrated under reduced Calcd for C13H13N5O2: C, 57.56; H, 4.83; N, 25.82. Found: C,
pressure, and the resulting ester was collected.
57.36; H, 4.68; N, 25.95.
Ethyl 2-(2-Bromophenylamino)nicotinate (2a): Yield 68%;
2-[3-(Hydrazinecarbonyl)pyridin-2-yloxy]benzamide
(3c):
mp 41–42°C; IR (KBr) cm−1: 3429 (NH), 3055 (C–H aro- Yield 65%; mp 129–130°C; IR (KBr) cm−1: 3410–3201 (NH,
matic), 2981, 2830 (C–H aliphatic), 1740 (C=O); 1H-NMR NH2), 3124 (C–H aromatic), 1689, 1639 (2C=O); 1H-NMR
(DMSO-d6, 300MHz) δ (ppm): 1.32 (t, 3H, J=7.2Hz, CH3), (DMSO-d6, 300MHz) δ (ppm): 4.61 (s, 2H, NH2, D2O ex-
4.33 (q, 2H, J=7.2Hz, CH2), 5.21 (s, 1H, NH, D2O exchange- changeable), 6.45–6.50 (m, 3H, 2H Ar, H5, pyr.), 6.66–6.68
able), 6.78–7.66 (m, 5H, 4H Ar and H5, pyr.), 8.25 (d, 1H, (m, 2H, 1H Ar, H4, pyr.), 8.28–8.32 (m, 2H, 1H Ar, H6, pyr.),
J=6.2Hz, H4, pyr.), 8.66 (d, 1H, J=5.4Hz, H6, pyr.); MS (elec- 10.51 (s, 3H, NH, NH2, D2O exchangeable); MS (EI) m/z (%
tron ionization (EI)) m/z (% rel. Int.): 322 (M+2, 1.74), 320 rel. Int.): 273 (M+H, 23.74), 272 (M+, 35.02); Anal. Calcd for
(M+, 1.88); Anal. Calcd for C14H13BrN2O2: C, 52.36; H, 4.08; C13H12N4O3: C, 57.35; H, 4.44; N, 20.58. Found: C, 57.24; H,
N, 8.72. Found: C, 52.11; H, 3.98; N, 8.45.
4.15; N, 20.35.
Ethyl 2-(2-Carbamoylphenylamino)nicotinate (2b): Yield
General Procedure for the Synthesis of 4a–l An equi-
60%; mp 104–105°C; IR (KBr) cm−1: 3500–3200 (NH, NH2), molar mixture of an appropriate hydrazide 3a–c and the corre-
3124 (C–H aromatic), 2985, 2850 (C–H aliphatic), 1645, 1735 sponding aldehyde (1.5mmol) each in absolute ethanol (10mL)
1
(2C=O); H-NMR (DMSO-d6, 300MHz) δ (ppm): 1.35 (t, 3H, was heated under reflux for 4h in the presence of hydrochloric
J=7.5Hz, CH3), 4.40 (q, 2H, J=7.5Hz, CH2), 7.04–7.09 (m, acid (2 drops). The resulting precipitate was filtered while hot,
1H, Ar), 7.52–7.55 (m, 1H, Ar), 7.70 (d, 1H, Ar), 7.88–7.94 (m, washed with water, and crystallized from ethanol to give 4a–i.
2H, 1H Ar, H5, pyr.), 8.25 (d, 1H, J=6.0Hz, H4, pyr.), 8.88 (d,
N′-(2-Bromobenzylidene)-2-(2-bromophenylamino)-
1H, J=5.1Hz, H6, pyr.), 11.44 (s, 2H, NH2, D2O exchangeable); nicotinohydrazide (4a): Yield 80%; mp 289–290°C; IR (KBr)
MS (EI) m/z (% rel. Int.): 286 (M+H, 0.16), 285 (M+, 0.47), cm−1: 3437–3228 (NH), 1666 (C=O); 1H-NMR (DMSO-d6,
241 (C14H13N2O2, 64.46); Anal. Calcd for C15H15N3O3: C, 63.15; 300MHz) δ (ppm): 6.57 (t, 1H, J=7.2Hz, Ar), 7.36–7.54 (m,
H, 5.30; N, 14.73. Found: C, 63.23; H, 5.05; N, 14.43.
5H, Ar), 7.68–7.99 (m, 3H, 2H Ar, H5, pyr.), 8.16 (d, 1H,
Ethyl 2-(2-Carbamoylphenoxy)nicotinate (2c): Yield 55%; J=5.1Hz, H4, pyr.), 8.44 (d, 1H, J=5.4Hz, H6, pyr.), 8.59 (s,
mp 45–46°C; IR (KBr) cm−1: 3429–3380 (NH, NH2), 3065 1H, =CH), 12.78, 13.05 (2s, 1H, NH, D2O exchangeable, cis
(C–H aromatic), 2950, 2845 (C–H aliphatic), 1640, 1750 and trans conformers); MS (EI) m/z (% rel. Int.): 474 (M+2,
(2C=O); 1H-NMR (DMSO-d6, 300MHz) δ (ppm): 1.28 (t, 14.09); Anal. Calcd for C19H14Br2N4O: C, 48.13; H, 2.98; N,
3H, J=7.2Hz, CH3), 4.25 (q, 2H, J=7.2Hz, CH2), 6.81–6.87 11.82. Found: C, 48.22; H, 2.68; N, 11.98.
(m, 1H, Ar), 7.33–7.41 (m, 1H, Ar), 7.52–7.58 (m, 1H, Ar),
2-(2-Bromophenylamino)-N′-(2-chlorobenzylidene)-
7.83–7.85 (m, 2H, 1H Ar, H5, pyr.), 8.23 (d, 1H, J=7.8Hz, H4, nicotinohydrazide (4b): Yield 62%; mp 283–284°C; IR (KBr)
pyr.), 8.56 (d, 1H, J=4.8Hz, H6, pyr.), 10.55 (s, 2H, NH2, D2O cm−1: 3313–3201 (NH), 1670 (C=O); 1H-NMR (DMSO-d6,
exchangeable); MS (EI) m/z (% rel. Int.): 285 (M−H, 0.22); 300MHz) δ (ppm): 6.56 (t, 1H, J=6.9Hz, Ar), 7.40–7.59 (m,
Anal. Calcd for C15H14N2O4: C, 62.93; H, 4.93; N, 9.79. Found: 5H, Ar), 7.76–8.01 (m, 3H, 2H Ar, H5, pyr.), 8.16 (dd, 1H,
C, 62.78; H, 5.11; N, 9.44.
J=6.3, 1.8Hz, H4, pyr.), 8.43 (dd, 1H, J=5.1, 1.8Hz, H6, pyr.),
General Procedure for the Preparation of 3a–c A mix- 8.67 (s, 1H, =CH), 12.70, 13.06 (2s, 1H, NH, D2O exchange-
ture of the appropriate 2a–c (1mmol) and hydrazine hydrate able, cis and trans conformers); 13C-NMR (DMSO-d6 ppm) δ:
(80%, 5mmol) in ethanol, was heated under reflux for 3h. The 106.79, 119.46, 127.12, 127.53, 127.68, 128.16, 129.86, 130.0,
reaction mixture was concentrated under reduced pressure 130.12, 131.42, 131.52, 133.09, 133.13, 140.32, 143.95, 145.00,
and left overnight. The resulting crystalline solid product was 158.09, 160.31 (aromatic C’s and =CH), 162.00 (C=O); MS
filtered then washed with ethanol to give pure acid hydrazides (EI) m/z (% rel. Int.): 429 (M+H, 2.16); Anal. Calcd for
3a–c.
C19H14BrClN4O: C, 53.11; H, 3.28; N, 13.04. Found: C, 53.27;
2-(2-Bromophenylamino)nicotinohydrazide (3a): Yield 74%; H, 3.43; N, 13.18.
mp 199–200°C; IR (KBr) cm−1: 3433–3201 (NH, NH2), 3124
2-(2-Bromophenylamino)-N′-(4-chlorobenzylidene)-
(C–H aromatic), 1689 (C=O); H-NMR (DMSO-d6, 300MHz) nicotinohydrazide (4c): Yield 70%; mp 185–186°C; IR (KBr)
1

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Vol. 61, No. 9
cm−1: 3444–3309 (NH), 1680 (C=O); 1H-NMR (DMSO-d6, 139.21, 140.96, 148.81, 150.16, 159.25, 159.34 (aromatic C’s and
300MHz) δ (ppm): 7.58 (d, 2H, J=8.4Hz, Ar), 7.64–7.81 (m, =CH), 160.22, 160.31 (2C=O); EI-MS (% rel. abundance): 403
5H, 4H Ar, H5, pyr.), 7.97 (d, 2H, J=8.4Hz, Ar), 8.20 (d, 1H, (M+H, 15.07), 402 (M+, 13.88); Anal. Calcd for C22H22N6O2:
H4, pyr.), 8.45 (d, 1H, H6, pyr.), 8.69 (s, 1H, =CH), 10.40 (s, C, 65.66; H, 5.51; N, 20.88. Found: C, 65.48; H, 5.80; N, 20.65.
1H, NH, D2O exchangeable); MS (EI) m/z (% rel. Int.): 432
2-{3-[2-(2-Bromobenzylidene)hydrazinecarbonyl]pyridin-2-
(M+4, 32.60), 430 (M+2, 29.67), 428 (M+, 32.60); Anal. Calcd yloxy}benzamide (4i): Yield 70%; mp 230–231°C; IR (KBr)
for C19H14BrClN4O: C, 53.11; H, 3.28; N, 13.04. Found: C, cm−1: 3435–3220 (NH, NH2), 1700, 1666 (2C=O); H-NMR
53.42; H, 3.34; N, 13.16.
1
(DMSO-d6, 300MHz) δ (ppm): 6.56 (t, 1H, J=6.9Hz, Ar),
2-(2-Bromophenylamino)-N′-[4-(dimethylamino)- 7.34–7.50 (m, 5H, Ar), 7.68–7.70 (m, 3H, 2H Ar, H5, pyr.),
benzylidene]nicotinohydrazide (4d): Yield 66%; mp 253–255°C; 7.81 (brs, 2H, NH2, D2O exchangeable), 7.99 (dd, 1H, J=5.7,
IR (KBr) cm−1: 3441–3201 (NH), 1665 (C=O); 1H-NMR 2.1Hz, H4, pyr.), 8.44 (dd, 1H, J=5.4, 1.8Hz, H6, pyr.), 8.61
(DMSO-d6, 300MHz) δ (ppm): 2.98 (s, 6H, 2CH3), 6.74 (d, (s, 1H, =CH), 12.72, 13.07 (2s, 1H, NH, D2O exchangeable,
2H, J=8.7Hz, Ar), 6.90–7.55 (m, 5H, 4H Ar, H5, pyr.), 7.62 (d, cis and trans conformers); MS (EI) m/z (% rel. Int.): 438 (M+,
2H, J=8.7Hz, Ar), 8.14 (d, 1H, H4, pyr.), 8.43 (d, 1H, H6, pyr.), 1.51); Anal. Calcd for C20H15BrN4O3: C, 54.69; H, 3.44; N,
8.49 (s, 1H, =CH); Anal. Calcd for C21H20BrN5O: C, 57.54; H, 12.75. Found: C, 54.55; H, 3.57; N, 12.68.
4.60; N, 15.98. Found: C, 57.34; H, 4.45; N, 16.08.
2-{3-[2-(2-Chlorobenzylidene)hydrazinecarbonyl]pyridin-2-
2-{3-[2-(2-Bromobenzylidene)hydrazinecarbonyl]pyridin-2- yloxy}benzamide (4j): Yield 75%; mp 276–277°C; IR (KBr)
1
ylamino}benzamide (4e): Yield: 66%; mp 250–251°C; IR cm−1: 3420–3194 (NH, NH2), 1678, 1670 (2C=O); H-NMR
(KBr) cm−1: 3433–3228 (NH, NH2), 1690, 1670 (2C=O); (DMSO-d6, 300MHz) δ (ppm): 6.57 (t, 1H, J=6.9Hz, Ar),
1H-NMR (DMSO-d6, 300MHz) δ (ppm): 4.05 (s, 2H, NH2, 7.43–7.55 (m, 5H, Ar), 7.75–7.78 (m, 3H, 2H Ar, H5, pyr.), 7.81
D2O exchangeable), 7.45–7.67 (m, 5H, Ar), 7.76–7.80 (m, 4H, (s, 2H, NH2, D2O exchangeable), 8.01 (dd, 1H, J=6.9, 2.4Hz,
3H Ar, H5, pyr.), 8.16 (d, 1H, J=6.9Hz, H4, pyr.), 8.34 (d, H4, pyr.), 8.44 (dd, 1H, J=5.1, 2.4Hz, H6, pyr.), 8.65 (s, 1H,
1H, H6, pyr.), 8.92 (s, 1H, =CH), 11.78 (s, 1H, NH, D2O ex- =CH), 12.76, 13.09 (2s, 1H, NH, D2O exchangeable, cis and
changeable), 11.98, 12.35 (2s, 1H, NH, D2O exchangeable, cis trans conformers); Anal. Calcd for C20H15ClN4O3: C, 60.84; H,
and trans conformers); EI-MS (% rel. abundance): 439 (M+ 3.83; N, 14.19. Found: C, 60.77; H, 3.52; N, 14.45.
2, 40.72), 437 (M+, 33.51); Anal. Calcd for C20H16BrN5O2: C,
2-{3-[2-(4-Chlorobenzylidene)hydrazinecarbonyl]pyridin-2-
54.81; H, 3.68; N, 15.98. Found: C, 54.69; H, 3.46; N, 15.78.
yloxy}benzamide (4k): Yield 68%; mp 181–182°C; IR (KBr)
2-{3-[2-(2-Chlorobenzylidene)hydrazinecarbonyl]pyridin-2- cm−1: 3429–3360 (NH, NH2), 1674, 1651 (2C=O); H-NMR
ylamino}benzamide (4f): Yield: 75%; mp 260–261°C; IR (DMSO-d6, 300MHz) δ (ppm): 7.56 (d, 2H, J=8.7Hz, Ar),
(KBr) cm−1: 3441–3201 (NH, NH2), 1693, 1670 (2C=O); 7.66–7.72 (m, 5H, 4H Ar, H5, pyr.), 7.88 (d, 2H, J=8.7Hz,
1H-NMR (DMSO-d6, 300MHz) δ (ppm): 4.05 (s, 2H, NH2, Ar), 8.22 (d, 1H, H4, pyr.), 8.50 (d, 1H, H6, pyr.), 8.71 (s, 1H,
D2O exchangeable), 6.68 (t, 1H, J=7.2Hz, Ar), 7.35–7.93 (m, =CH); MS (EI) m/z (% rel. int.): 396 (M+2, 23.37), 394 (M+,
8H, 7H Ar, H5, pyr.), 8.14 (d, 1H, J=6.3Hz, H4, pyr.), 8.23 (d, 19.29); Anal. Calcd for C20H15ClN4O3: C, 60.84; H, 3.83; N,
1H, H6, pyr.), 8.58 (s, 1H, =CH), 11.81 (s, 1H, D2O exchange- 14.19. Found: C, 60.79; H, 4.00; N, 14.32.
1
able), 12.37, 13.31 (2s, 1H, NH, D2O exchangeable, cis and
2-{3-[2-(4-Dimethylamino)benzylidene)hydrazinecarbonyl)-
trans conformers); EI-MS (% rel. abundance): 393 (M+, 1.50); pyridin-2-yloxy)benzamide (4l): Yield 70%; mp 264–265°C;
Anal. Calcd for C20H16ClN5O2: C, 60.99; H, 4.09; N, 17.78. IR (KBr) cm−1: 3440–3325 (NH, NH2), 1674, 1651 (2C=O);
Found: C, 61.23; H, 4.18; N, 17.95.
1H-NMR (DMSO-d6, 300MHz) δ (ppm): 2.97 (s, 6H, 2CH3),
2-{3-[2-(4-Chlorobenzylidene)hydrazinecarbonyl]pyridin-2- 6.54 (t, 1H, J=7.2Hz, Ar), 6.72–6.76 (m, 4H, Ar), 7.55–7.59
ylamino]benzamide (4g): Yield: 60%; mp 258–259°C; IR (m, 4H, 3H Ar, H5, pyr.), 7.77 (d, 1H, J=6.0Hz, H4, pyr.), 8.19
(KBr) cm−1: 3440–3250 (NH, NH2), 1700, 1662 (2C=O); (s, 1H, =CH), 8.43 (d, 1H, J=5.1Hz, H6, pyr.), 12.64, 12.76
1H-NMR (DMSO-d6, 300MHz) δ (ppm): 4.03 (s, 2H, NH2, (2s, 1H, NH, D2O exchangeable, cis and trans conformers);
D2O exchangeable), 6.68 (t, 1H, J=7.8Hz, Ar), 7.48–7.86 (m, 13C-NMR (DMSO-d6 ppm) δ: 40.33 (2CH3), 106.70, 111.60,
8H, 7H Ar, H5, pyr.), 8.14 (d, 1H, J=6.3Hz, H4, pyr.), 8.23 (d, 111.69, 119.85, 119.90, 121.38, 121.44, 128.57, 139.60, 144.28,
1H, J=5.4Hz, H6, pyr.), 8.75 (s, 1H, =CH), 11.69 (s, 1H, NH, 144.36, 148.96, 151.35, 151.55, 159.35, 159.40 (aromatic C’s
D2O exchangeable), 12.36, 13.32 (2s, 1H, NH, D2O exchange- and =CH), 162.08, 162.12 (2C=O); MS (EI) m/z (% rel. Int.):
able, cis and trans conformers); EI-MS (% rel. abundance): 401 (M-2, 1.51); Anal. Calcd for C22H21N5O3: C, 65.50; H,
397 (M+4, 40.84), 395 (M+2, 43.98), 394 (M+H, 28.80); 5.25; N, 17.36. Found: C, 65.78; H, 5.33; N, 17.18.
Anal. Calcd for C20H16ClN5O2: C, 60.99; H, 4.09; N, 17.78.
Pharmacological Activity. Animals The investigations
Found: C, 60.87; H, 4.27; N, 17.98.
were carried out using healthy male Swiss albino mice with
2-{3-[2-(4-Dimethylaminobenzylidene)hydrazinecarbonyl]- a weight of 20–25g. Mice were supplied by the Modern Vet-
pyridin-2-ylamino}benzamide (4h): Yield: 76%; mp 254–255°C; erinary Office for Laboratory Animals (Cairo, Egypt). All ani-
IR (KBr) cm−1: 3302–3217 (NH, NH2), 1697, 1667 (2C= mals were naive to drug treatment and experimentation at the
O); 1H-NMR (DMSO-d6, 300MHz) δ (ppm): 2.96 (s, 6H, beginning of the study. Mice were allowed to acclimatize for
2CH3), 3.98 (s, 2H, NH2, D2O exchangeable), 6.68 (t, 1H, 1–2 weeks prior to use, with standard pellet diet and water ad
J=7.8Hz, Ar), 7.48–7.86 (m, 8H, 7H, Ar, H5, pyr.), 8.14 (d, 1H, libitum. The animals were randomly assigned to experimental
J=6.3Hz, H4, pyr.), 8.21 (d, 1H, J=5.7Hz, H6, pyr.), 8.75 (s, groups, 6 mice each. All experiments were conducted between
1H, =CH), 11.33 (s, 1H, NH, D2O exchangeable), 12.38, 13.32 10:00a.m. and 16:00p.m. to eliminate circadian influence on
(2s, 1H, NH, D2O exchangeable, cis and trans conformers); animal behaviour. All experimental protocols were approved
13C-NMR (DMSO-d6 ppm) δ: 40.34 (2CH3), 106.47, 116.15, by the Institutional Animal Care and Use Committee at the
121.00, 121.10, 125.84, 126.15, 126.20, 127.15, 127.20, 134.53, Faculty of Pharmacy, Suez Canal University.

September 2013
939
Preparation of the Tested Compounds and the Standard separated, collected in clean tubes and stored at −80°C until
Drug Test compounds were given orally to test animals after being used for enzyme-linked immunosorbent assay (ELISA)
suspending in a 1% sodium carboxymethylcellulose (CMC) assays. ELISA kits for IL-6 (Glory Science Co., Ltd., Del Rio,
aqueous solution. The control group animals received the TX, U.S.A.) was used for determination of tissue levels of this
same experimental handling as those of the test groups except marker. The assays were carried out following the instructions
that the drug treatment was replaced with appropriate volumes of the manufacturer using an automated ELISA reader (Eu-
of the dosing vehicle. Mefenamic acid (25mg/kg) was kindly rope S.A., Belgium).
provided by Al-Qahira Pharmaceutical Company (Cairo,
Egypt). It was prepared in 1% CMC and used as a reference mined according to Cioli et al.24) Ulcerogenic activity was
drug.
evaluated after p.o. administration of the test compound or
Acute Ulcerogenesis Acute ulcerogenesis test was deter-
Analgesic Activity Analgesic activity was carried out by mefenamic acid at the dose of 50mg/kg. Control mice were
acetic acid induced writhing method.21) Aqueous acetic acid treated orally with the vehicle (suspension of 1% CMC). Food
solution (0.1mL, 1%, i.p.) was used to induce writhing. Mice but not water was removed 24h before administration of the
were kept individually in the test cage, before acetic acid test compounds. After the drug treatment, mice were fed
injection and habituated for 30min. Screening of analgesic normal diet for 17h. Mice were anesthetized with a mixture
activity was performed after p.o. administration of test drugs of ketamine (50mg/kg, i.p.)/xylazine (10mg/kg, i.p.), then sac-
at the dose of 25mg/kg. All compounds were dissolved in 1% rificed by decapitation. The stomach was removed and opened
CMC solution. One group was kept for the control experiment along the greater curvature, washed with distilled water and
and received p.o. administration of 1% CMC. Mefenamic acid cleaned gently by dipping in saline. The gastric mucosa of
was used as a reference drug. After 30min of drug admin- the mice was examined by means of a magnifying glass. For
istration, acetic acid solution (0.1mL, 1%, i.p.) was given to each stomach, the severity of mucosal damage was assessed
each mouse.
by measuring severity index i.e., severity of drug to cause mu-
Stretching movements consisting of arching of the back, cosal damage, which is the difference between the mean score
elongation of body and extension of hind limbs were counted of each treated group and the mean score of the control group.
for 5–15min after acetic acid injection. The analgesic activity
was expressed as follows:
Immunohistochemistry25) Three different sections were
cut on positively charged slides and fixed in a 65°C oven for
1h. The slides were placed in a coplin jar filled with 60mL
of triology (Cell Marque®, CA, U.S.A.) working solution and
the jar is securely positioned in an autoclave. The autoclave
%analgesic activity={(n−n′)/n}×100
where n=mean number of writhes of control group and was kept at a temperature of 120°C for 15min. The pressure
n′=mean number of writhes of test group.
was released, the coplin jar was removed and left to cool for
Anti-inflammatory Activity Anti-inflammatory activity 30min. Sections were washed and immersed in Tris-buffered
test was performed following the method of Winter et al.22) saline to adjust the pH. This was repeated between each step
Carrageenan (Sigma, St. Louis, Missouri, U.S.A.) was freshly of the immunohistochemical procedure. Quenching endo-
prepared as suspension (0.05mL, 1% w/v solution in 0.9% genous peroxidase activity was performed by immersing
saline). Carrageenan solution was injected into the subplantar slides in 3% hydrogen peroxide for 10min.
tissue of the right hind paw of each mouse. Control animals
were injected with saline into that of the left hind paw.
Excess serum was drained from each slide without washing,
and 2–3 drops of the primary antibodies against myeloperoxi-
Animals were divided in groups of six each. One group dase (Thermo Fischer Scientific®, Fremont, CA, U.S.A.) were
was kept as control and the animals of other groups were pre- added. The slides were incubated in the humidity chamber
treated with the test drugs suspended in 1% CMC and given for 1h. Henceforward, biotinylated secondary antibody was
orally 30min prior carrageenan injection. The paw volume applied to each slide for 20min, followed by another 20min
was measured before and after 4h of carrageenan injection. incubation with the enzyme conjugate. Three drops of 3,3′-di-
The difference in thickness between the right and left hind aminobenzidine (DAB) chromogen were added to each slide
paw was measured with a pair of dial thickness gauge calli- for 2min. DAB was rinsed and the slides were counterstain-
pers (Ozaki Co., Tokyo, Japan). Mean values of treated groups ing with Mayer’s hematoxylin. Cover slipping was performed
were compared to those of control group and analysed using as the final steps before slides being examined using a light
statistical methods. The percentage inhibition of inflammation microscope (Olympus CX21, Japan). The count of the opti-
was calculated by applying the following formula:
cal density was carried out by means of a computer assessed
image analysis system “Image J 1.45F” (National Institute of
Health, U.S.A.) on five randomly selected regularly spaced
sections (4×). The average optical density in each slide was
used to calculate the relative optical density compared to the
anti-inflammatory activity (% inhibition)
=(Vc −Vt ) /Vc ×100
where Vc=edema volume in control group, Vt=edema volume vehicle group.
in groups treated with the test compounds.
Statistical Analysis Data was collected, tabulated and
Furthermore, the mice were anesthetized with a mixture expressed as mean S.E.M. Statistical differences between
of ketamine (50mg/kg, i.p.)/xylazine (10mg/kg, i.p.), then the treatments and the control were evaluated using one-way
sacrificed by decapitation. Thereafter, a midline incision was ANOVA followed by Tukey’s test for multiple comparisons.
made, and blood samples were withdrawn from the heart. All statistical analyses were carried out using The Statistical
Thirty minutes after collection, blood samples were processed Package for Social Sciences, version 17 (SPSS Inc., Chicago,
by centrifugation at 2000×g for 15min. Serum samples were IL, U.S.A.). p<0.05 was considered to be statistically signifi-

940
Vol. 61, No. 9
Rofecoxib Osteoarthritis Endoscopy Multinational Study Group,
Arthritis Rheum., 43, 370–377 (2000).
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14) Duarte C. D., Tributino J. L., Lacerda D. I., Martins M. V.,
Alexandre-Moreira M. S., Dutra F., Bechara E. J., De-Paula F. S.,
Goulart M. O., Ferreira J., Calixto J. B., Nunes M. P., Bertho A. L.,
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Med. Chem., 8, 2243–2248 (2000).
Acknowledgment Authors wish to acknowledge Al-
Qahira Pharmaceutical Company for the generous gift of
mefenamic acid.
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