Synthesis and biological evaluation of new heteroaryl carboxylic acid derivatives as anti-inflammatory-analgesic agents

Article abstract of DOI:10.1248/cpb.c12-00949

A series of nicotinic acid derivatives structurally related to niflumic acid and certain pyridazine-containing compounds have been synthesized and characterized by analytical and spectral data. All compounds were screened for their potential analgesic and

Full text of DOI:10.1248/cpb.c12-00949

222
Regular Article
Chem. Pharm. Bull. 61(2) 222–228 (2013)
Vol. 61, No. 2
Synthesis and Biological Evaluation of New Heteroaryl Carboxylic Acid
Derivatives as Anti-inflammatory-Analgesic Agents
,b
Khaled Abouzid Mohamed Abouzid,a Nadia Abdalla Khalil,b Eman Mohamed Ahmed,* and
Sawsan Abo-Bakr Zaitonec
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University; Cairo 11566, Egypt:
b Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University; Cairo 11562, Egypt: and
c Department of Pharmacology & Toxicology, Faculty of Pharmacy, Suez Canal University; Ismailia 41522, Egypt.
Received October 28, 2012; accepted November 20, 2012
A series of nicotinic acid derivatives structurally related to niflumic acid and certain pyridazine-con-
taining compounds have been synthesized and characterized by analytical and spectral data. All compounds
were screened for their potential analgesic and anti-inflammatory activities. The compounds which displayed
analgesic and anti-inflammatory activities were tested for ulcerogenicity and screened for in vivo inhibition
of certain inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and
cyclooxygenase-2 (COX-2). Compounds 1c, 2a, 2b, and 5a have shown potent analgesic and anti-inflammatory
activities.
Key words nicotinic acid; niflumic acid; pyridazine; analgesic; anti-inflammatory
Rheumatoid arthritis (RA) is an immune-based chronic activities.13–16) Niflumic acid and flunixin are two traditional
inflammatory synovitis presenting with pain, stiffness and NSAIDs belonging to the class of fenamates enclosing pyri-
swelling of the affected joints. RA results in secondary bone dine nucleus in their structure (Fig. 1). They bind at the pros-
and cartilage destruction causing joint deformity. Current taglandin receptor site thus, potentially antagonizing the phys-
therapies include conventional non-steroidal anti-inflammatory iopathological effects of the already produced prostaglandins.
agents (NSAIDs), which inhibit prostaglandin (PG) forma- However, fenamates are still exhibited most of the GI adverse
tion through inhibition of both cyclooxygenase-1 (COX-1) and effects induced by NSAIDs.16)
COX-2 enzymes.1,2) PGs are important lipid mediators that are
Furthermore, a lot of 3(2H)-pyridazinone derivatives have
produced at elevated levels in inflamed tissues including rheu- been reported as analgesic and anti-inflammatory agents with-
matoid synovium.3,4) However, long term NSAID treatment is out gastrointestinal side effect.14,17,18) In the same direction,
often limited due to gastrointestinal (GI) ulcerogenicity that we planned to design new pyridazinone scaffolds as potential
may result from the suppression of physiological PG produc- anti-inflammatory agents (Fig. 2). This is agreement with in
tion in these tissues.5) Certain studies have revealed key roles our experience in the pyridazinone field.19)
for inflammatory cytokines, such as tumor necrosis factor-al-
Considering the above results and as a part of our ongoing
pha (TNF-α), interleukin-1b (IL-1b) and IL-6, in pathogenesis program to design analgesic and anti-inflammatory agents,
of rheumatoid arthritis (RA).6,7) IL-1, TNFα, and PGE2 over- herein, we describe the synthesis, and biological evaluation of
production play potential pathogenic roles in the establishment certain 2-arylamino, heteroarylamino or aryloxynicotinic acid
of rheumatoid synovitis, in the formation of pannus tissue and derivatives structurally related to niflumic acid. Keeping the
in the process of joint destruction.8) Thus, therapies suppress- 2-aminonicotinic acid framework, and either modifying the
ing these inflammatory cytokines have been focused on as aryl substituents on the benzene ring or replacing the aryl ring
effective treatment for RA.
by a heterocycle. We also aimed at developing certain pyr-
Selective COX-2 inhibitors demonstrated the ability to idazine and pyridazinone derivatives (Fig. 2). The analgesic
block PG production9) and acute tissue inflammation10) in vivo and anti-inflammatory activities were investigated for the title
at dosages that do not affect stomach PG production, sug- compounds utilizing the writhing test and the carrageenan-
gesting that COX-2 inhibitors may provide a safer therapeutic induced paw edema test (CPE test), respectively. The com-
alternative to NSAIDs. Nevertheless, the voluntary withdrawal pounds with analgesic and anti-inflammatory potentials were
of rofecoxib worldwide in 2004 due to an increased risk of also tested for the irritative and ulcerogenic action on gastric
cardiovascular events,11) raised serious concerns about safety mucosa. The serum cytokine level has been analysed as well,
of selective COX-2 inhibitors.12) Therefore, there exists an for the compounds that revealed significant anti-inflammatory
unmet medical need for ‘Safe NSAIDs’ that do not cause ad-
verse effects.
Synthetic approaches based upon chemical modification of
NSAIDs have been taken with the aim of improving safety
profile and in turn, therapeutic window of these NSAIDs. It
has been reported in the literature that certain compounds
bearing pyridine, pyridazine and pyridazinone hetero-
cycles, possess significant analgesic and anti-inflammatory
The authors declare no conflict of interest.
Fig. 1. Selected Fenamate Derivatives
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: dr_eman2001@hotmail.com

February 2013
223
Fig. 2. General Structures of the Synthesized Compounds
activity in the rat paw edema model.
Results and Discussion
Chemistry In Chart 1, new 2-substituted-nicotinic de-
rivatives 1a–e were synthesized through nucleophilic displace-
ment reaction of the commercially available 2-chloronicotinic
acid with various aromatic and heterocyclic primary amino
compounds following procedure reported for analogous
compounds.15) On the other hand, reaction of 2-chloro-
nicotinic acid with either salicylamide or anthranilamide
resulted in the formation of 2-(2-carbamoylphenoxy)- and
2-(2-carbamoylphenylamino)nicotinic acids 2a,b, respectively.
New benzoic acid derivatives 3a,b, 4a,b, 5a,b, and 6a,b
were synthesized according to Chart 2. Initially, nucleophilic
displacement reaction of commercial 3,6-dichloropyridazine
with the appropriate aminobenzoic acid in isopropanol af-
forded the appropriate 3-(6-chloropyridazine amino)benzoic
acids 3a,b according to previously reported procedure.19) The
latter were reacted with thiourea to give the isothiouronium
intermediates, which upon alkaline hydrolysis yielded the
corresponding thiones 4a,b following procedure reported for
analogous compounds.20) Hydrolysis of the chloropyridazine
derivatives 3a,b was carried out upon heating in acetic acid to
afford the corresponding 3(2H)-pyridazinone derivatives 5a,b.
Furthermore, 3-(6-ethoxypyridazinamino)benzoic acids 6a,b
were synthesized by heating 3a,b in absolute ethanol in the
Reagents and conditions: a) isopropanol/K2CO3/reflux 4h.
Chart 1
presence of sodium ethoxide. Analytical and spectral data (IR, anti-inflammatory activities. Furthermore, the thiazoline and
1H-NMR, mass spectra) of the synthesized compounds were in benzimidazole conjugates (1d,e, respectively) showed no an-
full agreement with the proposed structures.
algesic and anti-inflammatory potentials. It was also obvious
Analgesic Activity In current study, the new compounds that replacement oxygen atom in by nitrogen in the nicotinic
were tested for analgesic activity using the writhing test in acid derivatives 2a,b has only slight effect on the analgesic
mice21) and compared to mefenamic acid, as a standard drug activity. On the other hand, the pyridazine derivatives 3a, 4a,
at the same dose level (25mg/kg, per os (p.o.)). Writhing was and 4b were almost devoid of activity. However, the pyrid-
induced by intraperitoneal (i.p.) injection of freshly prepared azinone analogues 5a,b showed significant protection. The
acetic acid solution (1%, 10mL/kg, i.p.) in mice. The number compound 5a displayed the most potent analgesic activity
of writhes (constriction of abdomen, turning of trunk, exten- with % protection 78.2 6.8 (Table 1). Compounds 6a,b with
sion of hind limbs) due to acetic acid was expressed as a noci- 6-ethoxy substituent in the pyridazine ring showed lower an-
ceptive response. Vehicle treated control mice were given 1% algesic activity compared to their hydroxyl analogues, with no
acetic acid and writhing response was noted for 15min. The significant change in anti-inflammatory activity.
results showed that the tested compounds exhibited analgesic
Anti-inflammatory Activity Anti-inflammatory activity
activity ranging from 10.2 1.2% to 78.2 6.8%. Compounds was determined by using carrageenan induced rat paw edema
1c, 2a, 2b, 5a, and 6a showed significant reduction in the model.22) Carrageenan (1% w/v) was used to produce paw
writhing response (72.1 6.5 to 78.2 6.8) superior to that edema. Edema is presented as percentage increase in right
exhibited by mefenamic acid (70.3 2.5%). Among the com- hind paw, in comparison to the uninjected left hind paw. Per-
pounds 1a–e, the 2-chloropyridin-3-yl derivative 1c, exhibited centage change in paw volume was calculated and expressed
the highest analgesic activity. Replacement of the 2-chloro- as the amount of inflammation. Compounds 1c, 2a,b, 3b, 5a,
pyridine moiety with phenyl ring with halogen substituents and 6a significantly induced strong anti-inflammatory activity
in 2 and/or 4 position resulted in reduction of analgesic and that was slightly superior to that exhibited by mefenamic acid.

224
Vol. 61, No. 2
Reagents and conditions: a) isopropanol, K2CO3, reflux 4h; b) H2NCSNH2, abs. C2H5OH, reflux; c) acetic acid, reflux, 5h; d) abs. C2H5OH, CH3ONa reflux, 0.5h.
Chart 2
Table 1. Analgesic, Anti-inflammatory and Ulcerogenic Potential of the
Tested Compounds
Table 2. Serum Level of TNF-α, IL-6 and COX-2 in Mice Pretreated
with the Mefenamic Acid or the Tested Compounds in Carrageenan-
Induced Paw Edema Test
Anti-inflammatory Ulcerogenic
Analgesic activity
(% protection)
Compound
activity (% in-
hibition)
potential
(Severity index)
Compound
TNF-α (ng/mL)
IL-6 (ng/mL)
COX-2 (ng/mL)
Vehicle
Mefenamic acid
5.61 0.42
3.23 0.27*
4.23 0.39*
4.12 0.37*
3.25 0.21*
—
22.1 1.8
12.6 0.91
18.3 0.15#
19.3 1.4#
9.2 0.61*
—
43.2 3.5
39.2 2.8
45.2 3.5
42.6 3.9
38.5 3.1
—
Vehicle
0
0
0
Mefenamic acid
70.3 4.5*
46.2 3.3*,#
53.2 4.3*,#
74.2 5.3*
0
54.5 3.45*
18.3 1.5*,#
24.2 1.8*,#
64.5 4.9*
—
1.1 0.03*
0.4 0.02*,#
0.5 0.03*,#
0.9 0.04*
—
1a
1b
1c
1d
1e
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
1a
1b
1c
1d
1e
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
—
—
—
0
—
—
2.45 0.18*
2.67 0.15*
—
3.56 0.27*
—
14.3 0.56*
10.1 0.82*
—
16.4 0.43*
—
35.3 2.9*
41.1 4.3
—
41.3 3.7
—
72.1 6.5*
73.4 7.2*
0
59.4 5.2*
0
10.2 1.2*,#
78.2 6.8*
47.2 4.2*,#
72.4 4.8*
56.4 5.3*,#
61.3 5.6*
56.2 5.7*,#
—
55.4 4.3*
—
0.9 0.03*
1.0 0.08*
—
0.7 0.02*,#
—
0.1 0.01#
0.7 0.02*,#
0.2 0.01*,#
1.3 0.09*
0.8 0.06*
—
—
—
0
2.69 0.16*
4.62 0.42
2.94 0.18*
3.54 0.23*
15.4 0.43*
20.3 0.21#
9.3 0.61*
17.2 0.22#
35.1 2.8*
42.3 2.6
38.3 2.8
42.5 3.2
56.4 4.3*
23.3 2.1*,#
57.4 4.7*,#
43.2 4.1*
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.
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.
compounds 1c, 2b and 5b, they did not suppress serum COX-2
level. This may be attributed to inhibition of enzyme activity
These results were in agreement with the results shown in the with no influence on enzyme expression (Table 2).
analgesic activity test (Table 1).
Acute Ulcerogenicity Studies In current study, test-
Additionally, mefenamic acid as well as the tested com- ing acute ulcerogenic potential revealed that mefenamic acid
pounds 1c, 2a,b, 3b, 5a, and 6a have shown suppression of showed an ulcer severity index of 1.1 0.03. All tested com-
serum IL-6 as well as TNF-α levels compared to the control pounds except 5c showed lower mean severity index than that
group. However, only compounds 2a and 5a significantly observed by mefenamic acid, indicating lower ulcerogenic
reduced serum COX-2 level compared to the vehicle group. effect (Table 1).
In spite of the potent anti-inflammatory activity observed by
Histopathological examination revealed that mefenamic

February 2013
225
Fig. 3. Total Ulcerogenic Score of the Tested Compounds
*p<0.05 compared to the vehicle group. # p<0.05 compared to mefenamic acid group.
acid-treated mice showed a marked change in the architecture
of the mucosal cells, and erosion of the surface epithelia ex-
tending, in some cases to the muscularis mucosa. Compound
6a showed non-significant increase in the mean total ulcero-
genic score compared to mefenamic acid. In addition, the
ulcerogenic effect of the compounds 1c, 6b, 2a, and 2b was
comparable to that of the reference drug. On the other hand,
the tested compounds 1a,b, 3b, and 5a produced a lesser de-
gree of erosion and inflammatory cells infiltration, whereas
compound 5b showed fairly normal mucosal structure (Figs.
3, 4).
Conclusion
Various pyridazine derivatives and nicotinic acid deriva-
tives structurally related to niflumic acid were synthesized and
screened for their potential analgesic, anti-inflammatory and
ulcerogenic potentials. The results of the analgesic and anti-
inflammatory activities of the synthesized compounds showed
moderate enhancement in activity with the compounds 1a,b,
5b, and 6b. In acetic-acid-induced abdominal constriction test,
compounds 1c, 2a,b, 5a, and 6a displayed the most potent
analgesic activity. Moreover, these compounds in addition to
compound 3b exhibited the most prominent and consistent
Fig. 4. Histopathological Examination of the Gastric Mucosa of Some
of the Tested Compounds
Experimental
Chemistry All chemicals and reagents were obtained
anti-inflammatory activity which was superior to mefenamic from Aldrich (Sigma-Aldrich, St. Louis, MO, U.S.A.), and
acid. In contrast, compounds 1d,e, 3a, 4a, andb were almost were used without further purification. Reactions were moni-
devoid of activity. It has been also observed that compounds tored by TLC, performed on silica gel glass plates containing
1c, 2a,b, 3b, 5a, and 6a significantly reduced the level of 60 GF-254, and visualization on TLC was achieved by UV
TNF-α and IL-6 in the serum. However, only the compounds, light or iodine indicator. IR spectra were determined on Shi-
1
2a and 5a markedly reduced the level of COX-2 as well.
madzu IR 435 spectrophotometer (KBr, cm−1). H-NMR spec-
The compounds that displayed significant analgesic and tra were carried out using a Mercury 300-BB 300MHz using
anti-inflammatory activities were also screened for ulcero- tetramethylsilane (TMS) as internal standard. Chemical shifts
genic adverse effect at 50mg/kg dose level. After microscopic (δ) are recorded in ppm on δ scale, Micro analytical Center,
elimination, it was obvious that all compounds which showed Cairo University, Egypt. 13C-NMR spectra were carried out
higher potency than mefenamic acid (except 6a) in both anal- using a Mercury 300-BB 75MHz using TMS as internal
gesic and anti-inflammatory screens, showed lower ulcerogen- standard. Chemical shifts (δ) are recorded in ppm on δ scale,
ic potential than the reference drug. No significant ulceration Micro analytical Center, Cairo University, Egypt. Mass spec-
risk was observed in the compound 5b.
tra were recorded on Shimadzu Qp-2010 plus spectrometer,
In light of these results, one can say that the most promis- Micro analytical Center, Cairo University, Egypt. Elemental
ing results as the anti-inflammatory and analgesic agents was analyses were carried out at the Micro analytical Center, Cairo
observed with the nicotinic acid derivatives 1c, 2a, andb as University, Egypt. Melting points were determined with Stuart
well as the pyridazinone derivative 5a.
apparatus and are uncorrected. Progress of the reactions was

226
Vol. 61, No. 2
monitored using TLC sheets precoated with UV fluorescent mp 164–165°C; IR (KBr) cm−1: 3398–2360 (O–H and NH2),
1
silica gel Merck 60F 254 using acetone–benzene (1:9) and 1716, 1681 (2C=O); H-NMR (DMSO-d6, 300MHz) δ (ppm):
were visualized using UV lamp.
6.82–6.88 (m, 1H, Ar-H), 7.36–7.50 (m, 1H, Ar-H), 7.52–7.55
The starting material 2-chloronicotinic acid and other (m, 1H, Ar-H), 7.82–7.90 (m, 1H, H5, pyr.), 8.22 (dd, 1H, J=
chemicals were obtained from Aldrich, Fluka, or Merck 5.4, 1.8Hz, H4, pyr.), 8.40 (s, 2H, NH2, D2O exchangeable),
Chemicals.
8.54 (dd, 1H, J=4.5, 1.8Hz, H6, pyr.), 13.79 (br, 1H, O–H,
+
·
General Procedure for 1a–e, 2a,b A mixture of 2-chlo- D2O exchangeable); MS (EI) m/z (% rel. int.): 258 (M , 0.17).
ronicotinic acid (0.157g, 0.01mol), anhydrous K2CO3 (2.76g, Anal. Calcd for C13H10N2O4 (258.23): C, 60.47; H, 3.90; N,
0.02mol) and an appropriate substituted amino compound 10.85; Found: C, 60.33; H, 4.05; N, 10.78.
(0.01mol) in isopropanol (30mL) was heated under reflux for
2-(2-Carbamoylphenylamino)nicotinic Acid (2b): Yield
4h. The reaction mixture was concentrated under reduced 80%; mp 150–151°C; IR (KBr) cm−1: 3429–2400 (O–H and
1
pressure to half its volume then cooled. The separated solid NH2), 1716, 1680 (2C=O); H-NMR (DMSO-d6, 300MHz) δ
was filtered, dried and crystallized from isopropanol.
(ppm): 7.51–7.55 (m, 3H, 2Ar-H, H5, pyr.), 8.13–8.28 (m, 2H,
2-[(2-Bromophenyl)amino]nicotinic Acid (1a): Yield 75%; Ar-H and H4, pyr.), 8.53–8.55 (m, 2H, Ar-H, H6, pyr.), 13.70
mp 187–188°C; IR (KBr) cm−1: 3200–2200 (O–H and NH), (br, 1H, O–H, D2O exchangeable); MS (EI) m/z (% rel. int.):
3097 (C–H aromatic), 1720 (C=O); 1H-NMR (DMSO-d6, 256 (M−H, 67.86), 213 (15.48). Anal. Calcd for C13H11N3O3
300MHz) δ (ppm): 5.54 (s, 1H, NH, D2O exchangeable), (257.24): C, 60.70; H, 4.31; N, 16.33; Found: C, 60.53; H, 4.25;
7.51–7.55 (m, 5H, 4H arom. and H5, pyr.), 8.23 (dd, 1H, J=6.0, N, 16.57.
1.8Hz, H4, pyr.), 8.55 (dd, 1H, J= 4.8, 1.8Hz, H6, pyr.), 13.75
General Procedure for 3a,b A mixture of 3,6-dichlo-
(br, 1H, OH, D2O exchangeable); MS (electron ionization (EI)) ropyridazine (1.49g, 0.01mol), anhydrous K2CO3 (2.76g,
m/z (% rel. int.): 294 (M+2, 0.20), 293 (M+H), 171 (9.30), 0.02mol) and the appropriate substituted amino benzoic acid
157 (100), 122 (5.78), 78 (23.63). Anal. Calcd for C12H9BrN2O2 (1.37g, 0.01mol) in isopropanol (30mL) was heated under
(293.12): C, 49.17; H, 3.09; N, 9.56; Found: C, 49.34 H, 3.25; reflux for 4h. The reaction mixture was concentrated under
N, 9.23.
reduced pressure to half its volume then cooled. The separated
2-[(2,4-Dichlorophenyl)amino]nicotinic Acid (1b): Yield solid was filtered, dried and crystallized from isopropanol.
70%; mp 51–52°C; IR (KBr) cm−1: 3417–2580 (O–H and NH),
3 - [ ( 6 - C h l o r o p y r i d a z i n - 3 - y l ) a m i n o ] b e n z o i c A c i d ( 3a): Yield
3190 (C–H aromatic), 1700 (C=O); 1H-NMR (DMSO-d6, 80%; mp 189–190°C; IR (KBr) cm−1: 3298–2553 (O–H and
1
300MHz) δ (ppm): 5.52 (s, 1H, NH, D2O exchangeable), 6.78 NH), 1685 (C=O); H-NMR (DMSO-d6, 300MHz) δ (ppm):
(d, 1H, Ar-H), 7.08 (d, 1H, Ar-H), 7.26 (s, 1H, Ar-H), 7.51–7.55 6.43 (d, 1H, Ar-H), 6.77–6.81 (m, 2H, Ar-H), 7.06 (d, 1H,
(m, 1H, H5, pyr.), 8.22 (d, 1H, H4, pyr.), 8.55 (d, 1H, H6, pyr.); Ar-H), 7.96 (d, 1H, pyridazine H-4), 8.37 (d, 1H, pyridazine
+
·
MS (EI) m/z (% rel. int.): 286 (M+4, 58.74), 282 (M , 3.72). H-5), 9.80 (s, 1H, NH, D2O exchangeable); MS (EI) m/z (% rel.
+
·
Anal. Calcd for C12H8Cl2N2O2 (283.11): C, 50.91; H, 2.85; N, int.): 251 (M+2, 10.51), 249 (M , 32.00), 248 (M−H, 100).
9.89; Found: C, 50.86; H, 2.98; N, 10.08. Anal. Calcd for C11H8ClN3O2 (249.65): C, 52.92; H, 3.23; N,
2-[(2-Chloropyridin-3-yl)amino]nicotinic Acid (1c): Yield 16.83; Found: C, 52.75; H, 3.45; N, 16.66.
65%; mp 174–175°C; IR (KBr) cm−1: 3452–2360 (O–H and
4 - [ ( 6 - C h l o r o p y r i d a z i n - 3 - y l ) a m i n o ] b e n z o i c A c i d ( 3b): Yield
1
NH), 1720 (C=O); H-NMR (DMSO-d6, 300MHz) δ (ppm): 70%; mp 244–245°C; IR (KBr) cm−1: 3294–2549 (O–H and
1
5.55 (s, 1H, NH, D2O exchangeable), 7.10 (d, 1H, H4, pyr.), NH), 1666 (C=O); H-NMR (DMSO-d6, 300MHz) δ (ppm):
7.51–7.57 (m, 2H, Ar-H), 8.22 (d, 1H, H4, pyr.), 8.53–8.56 (m, 7.27 (d, 2H, Ar-H), 7.64 (d, 2H, Ar-H), 7.81 (d, 1H, pyridazine
2H, H6, pyr.), 13.79 (br, 1H, OH, D2O exchangeable); MS (EI) H-4), 7.92 (d, 1H, pyridazine H-5), 9.91 (s, 1H, NH, D2O ex-
m/z (% rel. int.): 249 (M, 0.19), 251 (M+2, 0.18). Anal. Calcd changeable); MS (EI) m/z (% rel. int.): 251 (M+2, 1.11), 249
+
·
for C11H8ClN3O2 (249.65): C, 52.92; H, 3.23; N, 16.83; Found: (M , 0.92), 248 (M–H, 0.92). Anal. Calcd for C11H8ClN3O2
C, 52.68; H, 3.42; N, 16.98.
(249.65): C, 52.92; H, 3.23; N, 16.83; Found: C, 52.65; H, 3.18;
2-[(4,5-Dihydrothiazol-2-yl)amino]nicotinic Acid (1d): Yield N, 16.78.
50%; mp 110–111°C; IR (KBr) cm−1: 3400–2353 (O–H and
General Procedure for 4a,b A mixture of an appropri-
NH), 1643 (C=O); H-NMR (DMSO-d6, 300MHz) δ (ppm): ate 3a,b (0.5g, 0.002mol) and thiourea (0.23g, 0.003mol) in
1
3.45 (t, 2H, J=7.5Hz, CH2), 3.86 (t, 2H, J=7.5Hz, CH2), 5.55 absolute ethanol (20mL) was heated under reflux for 3h. The
(s, 1H, NH, D2O exchangeable), 7.33–7.37 (m, 1H, H5, pyr.), reaction mixture was allowed to cool to room temperature and
7.89 (d, 1H, H4, pyr.), 8.31 (d, 1H, H6, pyr.); MS (EI) m/z (% the excess solvent was removed under diminished pressure.
+
·
rel. int.): 223 (M , 3.86), 224 (M+H, 7.26). Anal. Calcd for The crude isothiouronium salt was combined with sodium
C9H9N3O2S (223.25): C, 48.42; H, 4.06; N, 18.82; Found: C, hydroxide solution (10%, 20mL) and the mixture was heated
48.16; H, 4.25; N, 18.79.
under reflux for 2h. After cooling, the mixture was acidified
2-[(1H-Benzo[d]imidazol-2-yl)amino]nicotinic Acid (1e): with glacial acetic acid, the precipitated orange solid product
Yield 65%; mp 189–190°C; IR (KBr) cm−1: 3290–2588 (O–H was filtered, washed with water and crystallized from ethanol.
and NH), 1681 (C=O); 1H-NMR (DMSO-d6, 300MHz) δ
3-[(6-Sulphanylpyridazin-3-yl)amino]benzoic Acid (4a):
(ppm): 7.07–7.10 (m, 2H, Ar-H), 7.25–7.28 (m, 2H, Ar-H), Yield 50%; mp 149–150°C; IR (KBr) cm−1: 3444–2650 (O–H
7.39–7.43 (m, 1H, H5, pyr), 8.24 (s, 2H, 2NH, D2O exchange- and NH), 1681 (C=O), ; 1H-NMR (DMSO-d6, 300MHz) δ
able), 8.01 (dd, 1H, J=6.0, 1.8Hz, H4, pyr.), 8.37 (dd, 1H, J= (ppm): 7.05–7.91 (m, 4H, Ar-H), 7.99 (d, 1H, pyridazine H-4),
+
·
4.8, 1.8Hz, H6, pyr.); MS (EI) m/z (% rel. int.): 254 (M , 8.10 (d, 1H, pyridazine H-5), 8.98 (s, 1H, NH or SH, D2O ex-
0.92). Anal. Calcd for C13H10N4O2 (254.24): C, 61.41; H, 3.96; changeable), 9.55 (s, 1H, NH or SH, D2O exchangeable); MS
+
·
N, 22.04; Found: C, 61.25; H, 3.77; N, 21.96.
(EI) m/z (% rel. int.): 247 (M , 20.97), 246 (M−H, 20.14).
2-(2-Carbamoylphenoxy)nicotinic Acid (2a): Yield 85%; Anal. Calcd for C11H9N3O2S (247.27): C, 53.43; H, 3.67; N,

February 2013
227
16.99; Found: C, 53.15; H, 3.50; N, 17,35.
Pharmacological Activity. Animals The investigations
4-[(6-Sulphanylpyridazin-3-yl)amino]benzoic Acid (4b): were carried out using healthy male Swiss albino mice with
Yield 55%; mp 269–270°C; IR (KBr) cm−1: 3500–2538 (O–H a weight of 20–25g. Mice were supplied by the Modern Vet-
and NH), 1701 (C=O); 1H-NMR (DMSO-d6, 300MHz) δ erinary Office for Laboratory Animals (Cairo, Egypt). All ani-
(ppm): 7.07 (d, 2H, J=9.3Hz, Ar-H), 7.44 (d, 2H, J=9.3Hz, mals were naive to drug treatment and experimentation at the
Ar-H), 7.60 (d, 1H, J=9Hz, pyridazine H-4), 7.87 (d, 1H, beginning of the study. Mice were allowed to acclimatize for
J=9Hz, pyridazine H-5), 9.45 (s, 1H, NH or SH, D2O ex- 1–2 weeks prior to use with standard pellet diet and water ad
changeable), 9.78 (s, 1H, NH or SH, D2O exchangeable); MS libitum. The animals were randomly assigned to experimental
+
·
(EI) m/z (% rel. int.): 247 (M , 77.89), 246 (M−H, 100). Anal. groups, 6 mice each. All experiments were conducted between
Calcd for C11H9N3O2S (247.27): C, 53.43; H, 3.67; N, 16.99; 10:00 a.m. and 16:00 p.m. to eliminate circadian influence on
Found: C, 53.58; H, 3.43; N, 17.05.
animal behaviour. All experimental protocols were approved
General Procedure for 5a,b An appropriate 3a,b (0.25g, by the Institutional Animal Care and Use Committee at the
0.001mol) in glacial acetic acid (25mL) was heated under re- Faculty of Pharmacy, Suez Canal University.
flux for 5h. The reaction mixture was concentrated to half its
Preparation of the Tested Compounds and the Standard
volume under reduced pressure then cooled. The precipitated Drug Test compounds were given orally to test animals after
crystalline solid was filtered, washed with water, and recrys- suspending in a 1% sodium carboxymethylcellulose (CMC)
tallized from ethanol to give 5a,b.
aqueous solution. The control group animals received the
3-[(6-Oxo-1,6-dihydropyridazin-3-yl)amino]benzoic
Acid same experimental handling as those of the test groups except
(5a): Yield 60%; mp 273–274°C; IR (KBr) cm−1: 3336–2507 that the drug treatment was replaced with appropriate volumes
(O–H and NH), 1693, 1674 (2C=O); 1H-NMR (DMSO-d6, of the dosing vehicle. Mefenamic acid (25mg/kg) was kindly
300MHz) δ (ppm): 6.86 (d, 1H, J=9.3Hz, pyridazine-H4), provided by Al-Qahira Pharmaceutical Company (Cairo,
7.20 (d, 1H, Ar-H), 7.34–7.40 (m, 1H, Ar-H), 7.45 (d, 1H, Egypt). It was prepared in 1% CMC and used as a reference
Ar-H), 7.74 (d, 1H, J=9.3Hz, pyridazine-H5), 8.15 (s, 1H, drug.
Ar-H), 9.13 (s, 1H, NH, D2O exchangeable), 12.10 (s, 1H,
Analgesic Activity Analgesic activity was carried out
+
OH, D2O exchangeable); MS (EI) m/z (% rel. int.): 231 (M , by acetic acid induced writhing method.21) A 1% aqueous
·
89.85); 230 (100). Anal. Calcd for C11H9N3O3 (231.21): C, 57.14; acetic acid solution (i.p. injection; 0.1mL) was used to induce
H, 3.92; N, 18.17; Found: C, 57.23; H, 3.88; N, 18.10.
4-[(6-Oxo-1,6-dihydropyridazin-3-yl)amino]benzoic
writhing. Mice were kept individually in the test cage, before
Acid acetic acid injection and habituated for 30min. Screening of
(5b): Yield 55%; mp 329–330°C; IR (KBr) cm−1: 3414–2500 analgesic activity was performed after p.o. administration of
(O–H and NH), 1685, 1670 (2C=O); 1H-NMR (DMSO-d6, test drugs at the dose of 25mg/kg. All compounds were dis-
300MHz) δ (ppm): 6.88 (d, 1H, pyridazine-H4), 7.25 (d, 1H, solved in 1% CMC solution. One group was kept for the con-
pyridazine-H5), 7.77 (d, 2H, Ar-H), 7.85 (d, 2H, Ar-H), 9.33 trol experiment and received p.o. administration of 1% CMC.
(s, 1H, NH, D2O exchangeable), 12.14 (brs, 1H, OH, D2O Mefenamic acid was used as a reference drug. After 30min
+
·
exchangeable); MS (EI) m/z (% rel. int.): 231 (M , 11.74). of drug administration, 0.1mL of 1% acetic acid solution was
Anal. Calcd for C11H9N3O3 (231.21): C, 57.14; H, 3.92; N, 18.17; given to each mouse intraperitoneally.
Found: C, 57.05; H, 4.15; N, 18.35.
Stretching movements consisting of arching of the
General Procedure for 6a,b An appropriate 3a,b (0.25g, back, elongation of body and extension of hind limbs
0.001mol) in absolute ethanol (25mL) containing metallic were counted for 5–15min of acetic acid injection. The
sodium (0.46g, 0.02mol) was heated under reflux for 0.5h. analgesic activity was expressed as follows: % analgesic
The reaction mixture was concentrated under reduced pres- activity={(n−n′)/n}×100 where n=mean number of writhes of
sure, poured into ice cold water then acidified by dilute hy- control group and n′=mean number of writhes of test group.
drochloric acid. The precipitated crystalline solid was filtered,
washed with water, and recrystallized from ethanol.
Anti-inflammatory Activity Anti-inflammatory activity
test was performed following the method of Winter et al.22)
3-[(6-Ethoxypyridazin-3-yl)amino]benzoic Acid (6a): Yield Carrageenan (Sigma, St. Louis, MO, U.S.A.) was freshly pre-
50%; mp 214–215°C; IR (KBr) cm−1: 3417–2546 (O–H and pared as suspension (0.05mL, 1% w/v solution in 0.9% saline).
1
NH), 1666 (C=O); H-NMR (DMSO-d6, 300MHz) δ (ppm): Carrageenan solution was injected into the subplantar tissue
1.05 (t, 3H, J= 6.9Hz, CH3), 3.42 (q, 2H, J=6.9Hz, CH2), 7.23 of the right hind paw of each mouse. Control animals were
(t, 1H, Ar-H), 7.35 (d, 1H, pyridazine H-4), 7.52–7.56 (m, 2H, injected with saline into that of the left hind paw.
Ar-H), 7.85 (d, 1H, pyridazine H-5), 8.17 (s, 1H, Ar-H), 9.90
(s, 1H, NH, D2O exchangeable); MS (EI) m/z (% rel. int.): 259 group was kept as control and the animals of other groups
Animals were divided in groups of six in each group. One
+
·
(M , 0.35). Anal. Calcd for C13H13N3O3 (259.26): C, 60.22; H, were pre-treated with the test drugs suspended in 1% CMC
5.05; N, 16.21; Found: C, 60.45; H, 5.32; N, 16.05.
given orally 30min before carrageenan injection. The paw
4-[(6-Ethoxypyridazin-3-yl)amino]benzoic Acid (6b): Yield volume was measured before and after 4h of carrageenan
45%; mp 255–256°C; IR (KBr) cm−1: 3417–2546 (O–H and injection. The difference in thickness between the right and
1
NH), 1666 (C=O); H-NMR (DMSO-d6, 300MHz) δ (ppm): left hind paw was measured with a pair of dial thickness
1.15 (t, 3H, CH3), 3.55 (q, 2H, CH2), 7.29 (d, 1H, pyridazine gauge callipers (Ozaki Co., Tokyo, Japan). Mean values of
H-4), 7.81 (d, 1H, pyridazine H-5), 7.84 (d, 2H, Ar-H), 7.92 treated groups were compared with those of control group and
(d, 2H, Ar-H), 9.93 (s, 1H, NH, D2O exchangeable); MS (EI) analysed using statistical methods. The percentage inhibition
+
·
m/z (% rel. int.): 259 (M , 74.39), 258 (100). Anal. Calcd for of inflammation was calculated by applying the following
C13H13N3O3 (259.26): C, 60.22; H, 5.05; N, 16.21; Found: C, formula:
60.38; H, 5.11; N, 16.48.

228
Vol. 61, No. 2
anti-inflammatory activity (% inhibition)= (Vc Vt ) /Vc ×100
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Acknowledgements Authors wish to acknowledge Al-
Qahira Pharmaceutical Company for the generous gift of
mefenamic acid.