Newer N-substituted anthranilic acid derivatives as potent anti-inflammatory agents

Article abstract of DOI:10.1016/S0223-5234(02)01340-5

The new 5-bromo-N-[2′-amino(1″-acetyl-5″substitutedaryl-2″- pyrazolin-3″-yl)-1′,3′,4′-oxadiazol-5′- ylmethyl]anthranilic acids 7a-7e and N-[2′-amino-(1″-acetyl-5″-substiutedaryl-2″- pyrazolin-3″-yl)-1′,3′,4′-thiadiazol-5′- ylmethyl]anthranilic acids 6′a-6′c have been synthesised from 5-bromo-N-(2′-aminosubstituedbenzylideneacetyl-1′,3′, 4′-oxadiazol-5′-ylmethyl)anthranilic acids 6a-6e and N-(2′-aminosubstitutedbenzylideneacetyl-1′,3′,4′- thiadiazol-5′-ylmethyl)anthranilic acids 5′a-5′e, respectively. All these compounds have been screened in vivo for their anti-inflammatory and acute toxicity. Compounds 7b and 6′b were found to be potent member of this series, which showed 50.66 and 47.56%, respectively, inflammation inhibitory activity at a dose of 50 mg kg^-1 p.o., while standard drug, phenylbutazone, exhibited 45.52% anti-inflammatory activity at the same dose. However, 5-bromo-N-{2′-amino-[1″-acety1-5″-(para-methoxyphenyl)- 2″-pyrazolin-3″-yl]-1′,3′,4′-oxidiazol- 5′-ylmethyl}anthranilic acid (7b) was found to be the most active and less ulcerogcnic compound than the standard drag mid rest of the compounds of this series. The structures of these compounds have been established by IR, ¹H-NMR spectroscopic data and elemental analyses.

Full text of DOI:10.1016/S0223-5234(02)01340-5

Eur. J. Med. Chem. 37 (2002) 689–697
www.elsevier.com/locate/ejmech
Short communication
Newer N-substituted anthranilic acid derivatives as potent
anti-inflammatory agents
Shalabh Sharma 1, Virendra Kishor Srivastava, Ashok Kumar *
Medicinal Chemistry Di6ision, Department of Pharmacology, Lala Lajpat Rai Memorial Medical College, Meerut, U.P. 250 004, India
Received 12 July 2001; accepted 23 January 2002
Abstract
The new 5-bromo-N-[2%-amino(1¦-acetyl-5¦substitutedaryl-2¦-pyrazolin-3¦-yl)-1%,3%,4%-oxadiazol-5%-ylmethyl]anthranilic acids 7a–
7e and N-[2%-amino-(1¦-acetyl-5¦-substiutedaryl-2¦-pyrazolin-3¦-yl)-1%,3%,4%-thiadiazol-5%-ylmethyl]anthranilic acids 6%a–6%c have
been synthesised from 5-bromo-N-(2%-aminosubstituedbenzylideneacetyl-1%,3%,4%-oxadiazol-5%-ylmethyl)anthranilic acids 6a–6e and
N-(2%-aminosubstitutedbenzylideneacetyl-1%,3%,4%-thiadiazol-5%-ylmethyl)anthranilic acids 5%a–5%e, respectively. All these compounds
have been screened in vivo for their anti-inflammatory and acute toxicity. Compounds 7b and 6%b were found to be potent member
of this series, which showed 50.66 and 47.56%, respectively, inflammation inhibitory activity at a dose of 50 mg kg−1 p.o., while
standard drug, phenylbutazone, exhibited 45.52% anti-inflammatory activity at the same dose. However, 5-bromo-N-{2%-amino-
[1¦-acety1-5¦-(para-methoxyphenyl)-2¦-pyrazolin-3¦-yl]-1%,3%,4%-oxidiazol-5%-ylmethyl}anthranilic acid (7b) was found to be the
most active and less ulcerogcnic compound than the standard drag mid rest of the compounds of this series. The structures of
1
´
these compounds have been established by IR, H-NMR spectroscopic data and elemental analyses. © 2002 Editions scientifiques
et me´dicales Elsevier SAS. All rights reserved.
Keywords: N-substituted anthranilic acid-oxadiazolyl-pyrazolines; Thiadiazolyl-pyrazolines; Synthesis; Albino rats; Acute toxicity; Anti-inflamma-
tory activity; Ulcerogenic activity
anti-inflammatory activity [2,3]. Furthermore, looking
into the structure activity relationship of various
NSAIDs, it is obvious that pyrazoline [4–6], oxadiazole
[7,8] and thiadiazole [9,10] have also been found to
possess strong anti-inflammatory activity. Hence, it is
not irrelevant to speculate that incorporating these
pyrazoline, oxadiazole, thiadiazole rings into the 2-posi-
tion of anthranilic acid may enhance the anti-inflamma-
tory activity of such compounds. Therefore, it was
thought worthwhile to synthesise some new N-substi-
tuted anthranilic acid derivatives by incorporating oxa-
diazolyl, thiadiazolyl and pyrazolinyl moieites with the
hope to get better anti-inflammatory molecules.
1. Introduction
Mefenamic acid and meclofenamates [1], both N-
phenylanthranilic acid derivatives, have been used as
anti-inflammatory agents in therapy. A considerable
amount of work has been done on the structural varia-
tion of this subclass of drugs broadly known as Non-
Steroidal anti-inflammatory Drugs (NSAIDs). It has
been observed that best known NSAIDs are acidic in
nature. In view of this, our attention has been directed
to the variation at 2-position of anthranilic acid by
incorporating different acidic functional five membered
heterocyclic rings with a view to synthesise new analo-
gies with improved anti-inflammatory effects. Recent
literature shows that substitutions at 2-position of an-
thranilic acid (2-amino benzoic acid) by different substi-
tutedaryl or heteroaryl moieties markedly modulate the
2. Chemistry
The synthetic routes of compounds are outlined in
Figs. 1 and 2. In the first synthetic route, 5-bromo
antharnilic acid was reacted with ethyl chloroacetate to
give compound 2 i.e. 5-bromo-N-ethylacetoanthranilic
* Correspondence and reprints
E-mail address: rajput ak@indiatimes.com (A. Kumar).
–
1 This paper is a part of the Ph.D. thesis of Shalabh Sharma.
´
0223-5234/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0223-5234(02)01340-5

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S. Sharma et al. / European Journal of Medicinal Chemistry 37 (2002) 689–697
acid, which was further reacted with semicarbazide
hydrochloride to yield the compound 3. The compound
3 i.e. 5-bromo-N-(semicarbazido carbonyl methyl)-
anthranilic acid on dehydration with conc. H2SO4 af-
nilic acid, which further condensed with proper aro-
matic aldehydes yielding 5-bromo-N-(2%-aminosubstitut-
edbenzylidene
acetyl-1%,3%,4%-oxadiazol-5%-ylmethyl)-
anthranilic acids (6a–6e), which on cyclization with 99%
hydrazine hydrate in the presence of few drops of glacial
acetic acid gave final compounds 7a–7e i.e. 5-bromo-N-
[2%-amino-(1¦-acetyl-5¦-substitutedaryl-2¦-pyrazolin-3¦-
yl)-1%,3%,4%-oxadiazol-5%-ylmethyl]anthranilic acids.
forded
5-Bromo-N-(2%-amino-1%,3%,4%-oxadiazol-5%-yl-
methyl)anthranilic acid. This compound further reacted
with acetylchloride to give compound 5 i.e. 5-bromo-N-
(2%-aminoacetyl-1%,3%,4%-oxadiazol-5%-ylmethyl)anthra-
Fig. 1.

S. Sharma et al. / European Journal of Medicinal Chemistry 37 (2002) 689–697
691
activity. These two compounds showed better anti-infl-
ammatory activity at all three tested doses than the
reference drug. However, compound 7b exhibited maxi-
mum percent anti-inflammatory activity.
3.3. Ulcerogenic acti6ity
The two most active compounds 7b, 6%b and reference
drug, phenylbutazone, were screened for their ulcero-
genic activity. The UD50 of compounds 7b, 6%b and
phenylbutazone are 170.52, 156.2 and 66.6 mg kg−1
i.p., respectively.
Fig. 2. The bar diagram showing mean percent anti-inflammatory
activity of the most potent compounds 7b, 6%b and reference drug,
Phenylbutazone, at the three graded doses.
4. Discussion
In the second synthetic route, anthranilic acid reacted
with ethyl chloroacetate to yield compound N-ethylace-
toanthranilic acid (1%), which on condensation with
thiosemicarbazide yielded the compound 2%. This com-
pound was dehydrated with conc. H2SO4 to give com-
The characteristic feature of route-first and route-
second is that 5-bromoanthranilic acid and anthranilic
acid were substituted at N-position with five membered
ring structure oxadiazolyl and thiadiazolyl, respectively.
Furthermore, these compounds were converted in their
corresponding substituted benzylidene derivatives,
which were finally cyclized into pyrazoline derivatives.
All these new compounds (6a–6e; 7a–7e; 5%a–5%e;
and 6%a–6%e) were tested in vivo in order to evaluate
their pharmacological activity. The pharmacological
data are given in Table 3. These compounds were
screened for their anti-inflammatory profile tested at 50
mg kg−1 p.o, exhibited substantive anti-inflammatory
property. It was observed that compounds, 6a and 5%a,
having phenyl group as a substituent showed the least
percent inhibition of oedema i.e. 32.57 and 30.2%,
respectively, while the compounds, 6b and 5%h, substi-
tuted with para-methoxyphenyl group exhibited the
maximum anti-inflammatory activity, 40.39 and
39.72%, respectively. On the other hand, compounds,
6c and 5%c, having meta-methoxy-para-hydroxyphenyl
group as a substituent showed interesting anti-inflam-
matory activity.
pound
3%
i.e.
N-(2%-amino-1%,3%,4%-thiadiazol-5%-
ylmethyl)anthranilic acid. Compound 3% was further
acetylated with acetyl chloride to afford N-(2%-arni-
noacetyl-1%,3%,4%-thiadiazol-5%-ylmethyl)anthranilic acid
(4%). This acetylated product was reacted with proper
aromatic aldehydes giving N-(2%-aminosubstitutedben-
zylideneacetyl-1%,3%,4%-thiadiazol-5%-ylmethyl)anthranilic
acids (5%a–5%e). Finally, compounds 5%a–5%e were cy-
clised with 99% hydrazine hydrate in the presence of
few drops of glacial acetic acid to give N-[2%-amino-(1¦-
acetyl-5¦-substitutedaryl-2¦-pyrazolin-3¦-yl)-1%,3%,4%-thia-
diazol-5%-ylmethyl]anthranilic acids (6%a–6%e).
3. Results
3.1. Acute toxicity study
All the compounds of this series showed ALD50\
800 mg kg−1 p.o., with maximum in compound 7b and
6%b (1600 mg kg−1 p.o). All the results are depicted in
Table 3.
It is clear from the results obtained, that cyclisation
of substituted benylidene derivatives, 6a–6e and 5%a–
5%e, into their corresponding pyrazoline derivatives, 7a–
7e and 6%a–6%e, enhanced the anti-inflammatory activity
and compounds, 6a–6e and 7a–7e, having oxadiazolyl
moiety generally showed better anti-inflammatory
property in comparison to the compounds, 5%a–5%e and
6%a–6%c, contained thiadiazolyl moiety.
3.2. Anti inflammatory acti6ity
Random screening of compounds [(6a–6e; (7a–7e);
(5%a–5%e) and (6%a–6%e)] and reference drug, phenylbuta-
zone, was performed at 50 mg kg−1 p.o. The two
compounds 7b and 6%b were found to possess the most
potent anti-inflammatory activity (50.66% and 47.56
and, respectively) at 50 mg kg−1 p.o. in comparison
with reference drug, phenylbutazone, which showed
45.52% of inhibition of oedema at same dose. Further-
more, the two test compounds 7b, 6%b and phenylbuta-
zone were subjected to screening at three graded doses
of 25, 50 and 100 mg kg−1 p.o. for anti-inflammatory
Compounds 7b and 6%b (50.66 and 47.53%, respec-
tively) exhibited more potent anti-inflammatory activity
than that of Phenylbutazone (45.52%). However, 5-
bromo-N-{2%-amino-[1¦-acetyl-5¦-(para-methoxyphenyl)-
2¦-pyrazolin-3¦-yl]-1%,3%,4%-oxadiazol-5%-ylmethyl]
an-
thranilic acid (7b) was found to be the most active
compound of the present series, which has oxadiazolyl
moiety. On considering their potentialities compounds,
7b, 6%b and reference drug, Phenylbutazone, were

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S. Sharma et al. / European Journal of Medicinal Chemistry 37 (2002) 689–697
further tested at three graded doses i.e. 25, 50 and 100
mg kg−1, p.o., and these two compounds, 7b and 6%b,
showed higher anti-inflammatory activity than the refe-
rence drug at all three tested doses. Furthermore, these
two compounds and reference drug have been tested for
ulcerogenic liability, and these compounds, 7b and 6%b,
exhibited less ulcerogenie potentiality as compared with
reference drug, Phenylbutazone (UD50 of 7b=170.52
mg kg−1 i.p.; UD50 of 6%b=156.2 mg kg−1 i.p.; UD50
of phenylbutazone=66.6 mg kg−1 per i.p.). Moreover
compounds 7b and 6%b have shown ALD50\1600 mg
kg−1 p.o.
5.1.1. 5-Bromoanthranilic acid (1)
It was prepared by following the method of Wheeler
et al. [15], bromine (0.8 mol) in acetic acid (20 mL) was
added to the solution of anthranilic acid (0.4 mol) in
absolute acetic acid. The reaction mixture was kept
overnight. Then, 200–250 mL coldwater was added
into it. The solid product was filtered off, washed with
water and dried. It was recrystallised with ethanol-wa-
ter m.p., 220 °C, yield 55%, while Wheeler et al.,
reported m.p. 210 °C and yield was 60%.
5.1.2. 5-Bromo-N-ethylacetoanthranilic acid (2)
Hence, it may be concluded:
(a) Substituted benzylidene derivatives exhibit mild to
moderate the anti-inflammatory profile.
(b) Cyclization of these substituted benzylidene deriva-
tives into pyrazoline derivatives enhance the inflam-
mation inhibiting property.
(c) Presence of para-methoxyphenyl or meta-methoxy-
para-hydroxyphenyl as a substituent elicit a re-
markable increase in anti-inflammatory activity.
However, para-methoxy isomer showed better anti-
inflammatory activity than meta-methoxy isomer.
(d) Compounds having oxadiazolyl moiety generally
showed better anti-inflammatory property in com-
parison to thiadiazolyl. Similar results have also
been reported by Mazzone et al. [14].
A mixture of 5-bromoanthranilic acid (1) (0.1 mol),
ethyl chloroacetate (0.1 mol) and anhydrous K2CO3
(5.0 g) in acetone (80 mL) was refluxed for about 15 h.
The excess of solvent was distilled off under reduced
pressure and the resulting solid mass poured into ice
water, filtered and the separated solid recrystallised
from ethanol–water to give compound 2, Compound 2:
m.p. 125 °C, yield 70%, C11H12O4NBr [Calc. (Found):
C, 43.71 (43.82); H, 3.97 (3.60); N, 4.64 (4.79)]%. IR
(KBr) w in cm−1: 3480 (OꢀH); 3125 (NꢀH); 3030 (CꢀH
aromatic); 2925 (CH2); 1710 (CꢁO); 1590 (C···C of
aromatic ling), 660 (CꢀBr). 1H-NMR (CDC13). l in
ppm: 7.60–7.40 (m, 3H, ArꢀH), 1.25 (t, 3H, J=7 Hz,
ꢀCOOCH2CH3) 4.20 (q, 2H, J=7 Hz, ꢀCOOCH2CH3),
4.50 (s, 2H, NꢀCH2), 5.8 (s, 1H, NH, exchangeable
with D2O), 11.2 (s, 1H, ꢀCOOH, exchangeable with
D2O).
(e) Presence of electronegative atom, Br, may play a
pivotal role in the modulation of anti-inflammatory
activity.
5.1.3. 5-Bromo-N-(semicarbazido carbonyl
methyl)anthranilic acid (3)
5-Bromo-N-ethylaceto-anthranilic acid (2) (0.02 mol)
and semicarbazide hydrochloride (0.02) in methanol (50
mL) in the presence of anhydrous NaOH (5.0 g), was
refluxed for 10 h. The excess of solvent removed under
reduced pressure and the viscous mass recrystallised
from ethanol–water to afford compound 3. Compound
3: m.p. 140 °C. yield 62%, C10H11O4N4Br [Calc.
(Found): C, 36.25 (36.10); H, 3.32 (3.56); N, 16.92
(16.63)]%. IR (KBr) w in cm−1: 3475 (OꢀH); 3130
(NꢀH); 3020 (CꢀH aromatic); 2920 (CH2); 1715 (CꢁO);
5. Experimental protocols
5.1. Chemistry
Melting points of newly synthesised compounds were
taken in open capillaries with help of thermonic melting
point apparatus and are uncorrected. The purity of the
compounds was checked by thin layer chromatography
on silica gel-G, eluent was a mixture of methanol–ben-
zene in 2:8 proportion and spots were located by
iodine. The structure of these compounds was eluci-
1
1575 (C···C of aromatic ring), 665 (CꢀBr). H-NMR
(CDCl3) l in ppm: 7.62–7.45 (m, 3H, ArꢀH), 8.40 (m,
4H, NHNHCONH2, exchangeable with D2O), 4.70 (s,
2H, NꢀCH2). 5.70 (s, 1H, NH, exchangeable with
D2O), 11.31 (s, 1H, ꢀCOOH, exchangeable with D2O).
1
dated by IR, H-NMR and elemental analyses. The IR
(KBr) spectra were recorded on FTIR Paragon 500
(Perkin–Elmer), w max in cm−1. The H-NMR spectra
1
were recorded in CDCl3 or DMSO-d6 on Brucker-300
FT instrument, chemical shift (l) in ppm. Tetramethyl-
silane (TMS) was used as internal reference standard.
The carbon, hydrogen and nitrogen analyses were per-
formed on Heracus, Carlo Erba-1108 analyser at Cen-
tral Drug Research Institute (CDRI) at Lucknow (UP),
India. The carbon, hydrogen and nitrogen analyses
were found within 90.4% of the theoretical value.
5.1.4. 5-Bromo-N-(2%-amino-1%,3%,4%-oxadiazol-5%-
ylmethyl)anthranilic acid (4)
A mixture of compound 3 (0.05 mol) and conc.
H2SO4 (20 mL) was kept overnight at room tempera-
ture., poured into ice cold water, neutralised with liquid
ammonia and filtered. The product obtained was
recrystallised from methanol–water. Compound 4: m.p.
150 °C, yield, 50%, C10H9O3N4Br [Calc. (Found): C,

S. Sharma et al. / European Journal of Medicinal Chemistry 37 (2002) 689–697
693
38.33 (38.59); H, 2.88 (2.60); N, 17.89 (17.71)]%. IR
(KBr) w in cm−1: 3485 (OꢀH): 3140 (NꢀH), 3330
(NH2); 3025 (CꢀH aromatic); 2920 (CH2); 1720(CꢁO);
1585 (C···C of aromatic ring), 1600 (CꢁN), 1215 (CꢀN),
5NꢀCH2), 5.70 (s, 1H, NH, exchangeable with D2O),
11.20 (s, 1H, ꢀCOOH, exchangeable with D2O), 2.30 (s,
3H, ꢀCOCH3).
1
1040 (NꢀN), 655 (CꢀBr); H-NMR (CDC13) l in ppm;
5.1.6. 5-Bromo-N-(2%-aminosubstitutedbenzylidene-
acetyl-1%,3%,4%-oxadiazol-5%-ylmethyl)anthranilic acids
(6a–6e)
7.65–7.40 (m, 3H, ArꢀH), 6.25 (bs, 2H, NH2, ex-
changeable with D2O). 4.75 (s, 2H, NꢀCH2), 5.95 (s,
1H, NH, exchangeable with D2O), 11.25 (s, 1H,
ꢀCOOH, exchangeable with D2O).
A solution of compound 5 (0.01 mol) in absolute
ethanol (60 mL) with proper aromatic aldehydes (0.01
mol) in the presence of 2% NaOH was refluxed for 10
h, while progress and completion of the reaction was
monitored by TLC. The reaction mixture was distilled
off, cooled and then poured into ice–water and filtered.
The solid thus obtained was recrystallised from the
appropriate solvent giving compound 6. By this proce-
dure, compounds 6a–6e were obtained starting from
benzaldehyde, 4-methoxybenzaldehyde, 4-hydroxy-3-
methoxybenzaldehyde, 4-dimeihylaminobenzaldehyde
and 4-hydroxybenzaldehyde, respectively. Their physi-
cal and analytical data are given in Table 1. Compound
6b: m.p. 120 °C, yield 68%, C20H17O5N4Br [% Calc.
(Found): C, 50.74 (50.40); H, 3.59 (3.88); N, 11.84
(11.65)]%. IR (KBr) w in cm−1: 3485 (OꢀH); 3155
(NꢀH); 3060 (CꢀH aromatic); 2920 (CH2), 1710(CꢁO);
1590 (C···C of aromatic ring), 1600 (CꢁN), 1200 (CꢀN),
1124 (CꢀOꢀC), 1040 (NꢀN), 550 (CꢀBr). 1H-NMR
(CDC13) l in ppm: 7.65–7.20 (m, 7H, ArꢀH), 8.SO (bs,
5.1.5. 5-Bromo-N-(2%-aminoacetyl-l%,3%,4%-oxadiazol-5%-
ylmethyl)anthranilic acid (5)
To a solution of compound 4 (0.01 mol) in dry
benzene (50 mL), acetyl chloride (0.01 mol) was added
drop by drop at 0–5 °C temperature. The reaction
mixture was stirred for 2 h, refluxed for 6 h and then
distilled off and poured onto crushed ice. The solid thus
obtained was recrystallised from methanol–water and
purity of product was checked by TLC. Compound 5;
m.p. 133 °C, yield 70%, C12H11O4N4Br [Calc. (Found);
C, 40.56 (40.39); H, 3.09 (3.27); N, 15.77 (15.50)]%. IR
(KBr) w in cm−1: 3475 (OꢀH), 3150 (NꢀH); 3030 (CꢀH
aromatic); 2925 (CH2); 2840 (CꢀH of COCH3), 1710
(CꢁO); 1580 (C···C of aromatic ring), 1565 (CꢁN), 1210
1
(CꢀN), 1040 (NꢀN), 650 (CꢀBr); H-NMR (CDC13) l
in ppm; 7.60–7.40 (m, 3H, ArꢀH), 8.35 (bs, 1H,
NHCO, exchangeable with D2O), 4.60 (s, 2H,
Table 1
Physical and analytical data of compounds 6a–6e and 7a–7e
a
C, H, N were found within 90.4% of theoretical value.

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S. Sharma et al. / European Journal of Medicinal Chemistry 37 (2002) 689–697
1H, NHCO, exchangeable with D2O), 4.50 (s, 2H,
NꢀCH2), 5.60 (s, 1H, NH, exchangeable with D2O),
11.40 (s, 1H, ꢀCOOH, exchangeable with D2O), 3.47 (s,
3H, ArꢀOCH3), 6.85 (d, 1H, ꢀCOCH), 8,20 (d, 1H,
ꢁCHꢀAr).
mol) and thiosemicarbazide (0.02) in methanol (50 mL)
was refluxed for 10 h. The solvent was removed under
reduced pressure and the viscous mass poured over
ice–water, filtered and recrystallised from methanol–
water to afford compound 2%. Compound 2%: m.p.
128 °C, yield 75%, C10H12O3N4S [Calc. (Found): C,
44.78 (44.52); H, 4.48 (4.10); N, 22.90 (20.70)]%. IR
(KBr) w in cm−1: 3475 (OꢀH); 3135 (NꢀH); 3060 (CꢀH
aromatic); 2930 (CH2); 1700 (CꢁO), 1560 (C···C of
5.1.7. 5-Bromo-N-[2%-amino-(1¦-acetyl-5¦-substitute-
daryI-2%-pyrazolin-3¦-yl)-1%,3%,4%-oxadiazol-5%-ylmethyl]-
anthranilic acids (7a–7e)
1
To a solution of the proper compound 6 (0.02 mol)
in absolute ethanol (50 mL), 99% hydrazine hydrate
(0.04 mol) was added drop by drop with constant
stirring in the presence of few drops of glacial acetic
acid. The reaction mixture was refluxed for 12 h, dis-
tilled off and cooled. The separated solid was filtered,
washed with pet.ether and recrystallised from the ap-
propriate solvent to give compound 7, By this proce-
dure compounds 7a–7e were obtained starting from
6a–6e, respectively. The physical and analytical data
of compounds 7a–7e are given in Table 1. Compound
7b: m.p. 192 °C, yield 65%, C22H21O5N6Br [Calc.
(Found): C, 49.90 (49.58); H, 3.97 (4.21); N, 15.88
(16.15)]%. IR (KBr) w in cm−1: 3480 (OꢀH); 3160
(NꢀH); 3065 (CꢀH aromatic); 2923 (CH2), 2850 (CꢀH
of COCH3), 1690 (CꢁO); 1550 (C···C of aromatic
ring), 1580 (CꢁN), 1220 (CꢀN), 1115 (CꢀOꢀC), 1030
(NꢀN), 555 (CꢀBr). 1H-NMR (CDC13) l in ppm:
7.90–7.25 (m, 7H, ArꢀH), 6,15 (bs, 1H, NH, ex-
changeable with D2O), 4.55 (s, 2H, NꢀCH2), 5.65 (s,
1H, NH, exchangeable with D2O), 11.15 (s, 1H,
ꢀCOOH, exchangeable with D2O), 3.45 (s, 3H,
ArꢀOCH3), 5.25 (d, 2H, CH2), 6.95 (t, 1H, ꢀCHꢀAr)
2.35 (s, 3H, COCH3).
aromatic ring), H-NMR (CDCl3) l in ppm: 7.60–7.45
(m, 4H, ArꢀH), 8.15 (m, 4H, NHNHCSNH2, ex-
changeable with D2O), 4.55 (s, 2H, NꢀCH2), 5.82 (s,
1H, NH, exchangeable with D2O), 12.45 (s, 1H,
ꢀCOOH, exchangeable with D2O).
5.1.10. N-(2%-amino-1%,3%,4%-thiadiazol-5%-ylmethyl)-
anthranilic acid (3%)
A mixture of compound 2% (0.05 mol) and cone,
H2SO4 (20 mL) was kept overnight at room tempera-
ture, then poured into cold water, neutralised with
liquid ammonia and filtered. The product thus obtained
was recrystallised from ethanol–water. Compound 3%;
m.p. 137 °C, yield 62%, C10H10O2N4S [Calc. (Found):
C, 48.0 (48.39); H, 4.0 (3.88); N, 22.4 (22.12)]%. IR
(KBr) w in cm−1: 3475 (OꢀH); 3175 (NꢀH), 3350
(NH2); 3065 (CꢀH aromatic); 2930 (CH2); 1720(CꢁO);
1585 (C···C of aromatic ring), 1595 (CꢁN), 1210 (CꢀN),
1045 (NꢀN), 730 (CꢀSꢀC); 1H-NMR (CDC13) l in
ppm; 7.62–7.35 (m, 4H, ArꢀH), 6.35 (bs, 2H, NH2,
exchangeable with D2O), 4.60 (s, 2H, NꢀCH2), 5.80 (s,
1H, NH, exchangeable with D2O), 12.35 (s, 1H,
ꢀCOOH, exchangeable with D2O).
5.1.8. N-ethylacetoanthranilic acid (1%)
A mixture of anthranilic acid (0.1 mol), ethyl
chloroacetate (0.1 mol) and anhydrous K2CO3 (5.0 g) in
acetone (80 mL) was refluxed for about 15 h. The
excess of solvent was distilled of under reduced pres-
sure and the resulting solid mass poured into ice water,
filtered and the separated solid recrystallised from
methanol–water to give compound 1%. Compound 1%:
m.p. 115 °C, yield 60%, C11H13O4N [Calc. (Found): C,
59.19 (59.37); H, 5.83 (5.58); N, 6.28 (6.12)]%. IR
(KBr) w in cm−1; 3500 (OꢀH); 3150 (NꢀH); 3040 (CꢀH
aromatic); 2930 (CH2); 1720 (CꢁO); 1595 (C···C of
aromatic ring), 1H-NMR (CDC13) l in ppm: 7.50–
7.25 (m, 4H, ArꢀH), 1.35 (t, 3H, J=7 Hz,
5.1.11. N-(2%-aminoacetyl-1%,3%,4%-thiadiazol-5%-
ylmethyl)anthranilic acid (4%)
To a solution of compound 3% (0.01 mol) in dry
benzene (50 mL), acetyl chloride (0.01 mol) was added
drop by drop at 0–5 °C temperature with constant
stirring. The reaction mixture was further stirred for 2
h at room temperature and refluxed for 6 h, then
distilled off. The resulting mixture was poured onto
crushed ice. The solid thus obtained was recrystallised
from methanol–water to afford pure compound 4%.
Compound 4%: m.p. 160 °C, yield 65%, C12H12O3N4S
[Calc. (Found): C, 49.32 (49.13); H, 4.11 (4.30); N,
19.18 (19.42)]%. IR (KBr) w in cm−1: 3480 (OꢀH); 3180
(NꢀH); 3070 (CH2); 2935 (CꢀH aliphatic); 2840 (CꢀH
of COCH3), 1715 (CꢁO); 1565 (C···C of aromatic ring),
1590 (CꢁN), 1180 (CꢀN), 1040 (NꢀN), 730 (CꢀSꢀC);
1H-NMR (CDC13) l in pprn: 7.50–7.30 (m, 4H,
ArꢀH), 8.20 (bs, 1H, NHCO, exchangeable with D2O),
4.55 (s, 2H, NꢀCH2), 5,85 (s, 1H, NH, exchangeable
with D2O), 12.22 (s, 1H, ꢀCOOH, exchangeable with
D2O), 2.35 (s, 3H, COCH3).
ꢀCOOCH2CH3),
4.25
(q,
2H,
J=7
Hz,
ꢀCOOCH2CH3), 4.65 (s, 2H, NꢀCH2), 5.90 (s, 1H,
NH, exchangeable with D2O), 12.40 (s, 1H, ꢀCOOH,
exchangeable with D2O).
5.1.9. N-(thiosemicarbazido carbonyl methyl)anthranilic
acid (2%)
A mixture of N-ethylacetoanthranilic acid (1%) (0.02

S. Sharma et al. / European Journal of Medicinal Chemistry 37 (2002) 689–697
695
Table 2
Physical and analytical data of compounds 5%a–5%e and 6%a–6%e
a
C, H, N were found within 90.4% of theoretical value.
5.1.12. N-(2%-aminosubstitutedbenzylideneacetyl-1%,3%,4%-
thiadiazol-5%-ylmethyl)anthranilic acids (5%a–5%e)
5.1.13. N-[2%-amino(1¦-acetyl-5¦-substitutedaryl-2¦-
pyrazolin-3¦-yl)-1%,3%,4%-thiadiazol-5%-ylmethyl]-
anthranilic acids (6%a–6%e)
A solution of compound 4% (0.01 mol) in methanol (60
mL) was refluxed with proper aromatic aldehyde (0.01
mol) in the presence of 2% NaOH for 10 h, while
progress and completion of the reaction was monitored
by TLC. The excess of solvent was removed through
distillation, the separated solid was poured onto crushed
ice, filtered. The product thus obtained was recrys-
tallised from the appropriate solvent giving compound
5%, By this procedure compounds 5%a–5%e were synthe-
sized starting from methoxybenzaldchyde, 4-dimethy-
To a solution of the proper compound 5% (0.02 mol)
in absolute ethanol (50 mL), 99% hydrazine hydrate
(0.04 mol) was added drop by drop with constant
stirring in the presence of glacial acetic acid. The reac-
tion mixture was refluxed for 12 h, then distilled in
vacuum and cooled. The separated solid was filtered,
washed with pet.ether and recrystallised from the ap-
propriate solvent to give compound 6%. By this proce-
dure, compounds 6%a–6%e were obtained starting from
5%a–5%e, respectively. The physical and analytical data
of compounds 6%a–6%e are depicted in Table 2. Com-
pound 6%b: m.p. 145 °C, yield 72%, [Calc. (Found): C,
56.65 (56.50); H, 4.72 (4.96); N, 18.0.3 (18.27)]%. IR
(KBr) w in cm−1: 3500 (OꢀH); 3180 (NꢀH); 3020 (CꢀH
aromatic); 2900 (CH2), 2860 (CꢀH of COCH3),
1715(CꢁO); 1555 (C···C of aromatic ring), 1600 (CꢁN),
735 (CꢀSꢀC), 1035 (NꢀN); 1H-NMR (CDCl3) l in ppm:
7.65–7.10 (m, 8H, ArꢀH), 6.20 (bs, 1H, NH, exchange-
able with D2O), 4.60 (s, 2H, NꢀCH2), 5.95 (s, 1H, NH,
exchangeable with D2O), 12.15 (s, 1H, ꢀCOOH,
exchangeable with D2O), 3.45 (s, 3H, ArꢀOCH3,), 2.40
(s, 3H, COCH3), 5.20 (d, 2H, CH2), 6.70 (t, 1H,
ꢁCHꢀAr).
laminobenaldehyde
and
4-hydroxybenzaldehyde,
respectively. The physical and analytical data of com-
pounds 5%a–5%e are given in Table 2. Compound 5%b:
m.p. 125 °C, yield 60%, C20H18O4N4S [Calc. (Found):
C, 58.54 (58.30); H, 4.39 (4.59); N, 13.66 (13.80)]%. IR
(KBr) w in cm−1: 3490 (OꢀH); 3180 (NꢀH); 3025 (CꢀH
aromatic); 2910 (CH2), 1700 (CꢁO); 1570 (C···C of
aromatic ring), 1605 (CꢁN), 1190 (CꢀN), 720 (CꢀSꢀC),
1035 (NꢀN), 1H-NMR (CDCl3) l in ppm; 7.70–7.15 (m,
8H, ArꢀH), 8.55 (bs, 1H, NHCO, exchangeable with
D2O), 4.75 (s, 2H, NꢀCH2), 5.90 (s, 1H, NH exchange-
able with D2O), 12.10 (s, 1H, ꢀCOOH, exchangeable
with D2O), 6.55 (d, 1H, ꢀCOCH), 8.15 (d, 1H,
ꢁCHꢀAr) 3.40 (s, 3H, ArꢀOCH3).

696
S. Sharma et al. / European Journal of Medicinal Chemistry 37 (2002) 689–697
5.2. Pharmacology
mate Lethal Dose (ALD50) was calculated by the
method of Smith [11].
The compounds [6a–6e; 7a–7e; 5%a–5%e and 6%a–6%c]
were evaluated for their anti-inflammatory activity and
acute toxicity. Phenylbutaone, a potent anti-inflamma-
tory compound, was used as reference drug for com-
parison. The experiments were performed on albino
rats of the Charles Foster strain of either sex of 70–95
days weighing 80–140 g, and pregnancy was excluded
in females. The animals were maintained at the follow-
ing conditions 2592 °C, 5095% relative humidity, 12
h light/dark cycle. Food and water were freely available
up to the time of experiments. Acute toxicity was tested
in albino mice (20–25 g) maintained at above men-
tioned conditions. The test compounds were dissolved
in propylene glycol.
5.2.2. Anti-inflammatory acti6ity
This activity was performed by the following the
procedure of Winter et al. [12] on groups of six animals
each. A freshly prepared suspension of carrageenan (1.0
in 0.9% saline; 0.05 mL) was injected under the planter
aponeurosis of right hind paw of the rat. One group
was kept as control and treated with propylene glycol.
The animals of standard drug and drug treated groups
were pretreated with standard drug and test compounds
given orally 1 h before the carrageenan injection, res-
pectively. The volume of foot was measured before 1
and 3 h after carrageenan treatment, with help of
pleythysmometer. The percent anti-inflammatory
activity was calculated according to formula given
below:
5.2.1. Acute toxicity study
The test compounds were administered orally at dif-
ferent dose levels in separate groups of animals. After
24 h of drug administration percent mortality in each
group was observed. From the data obtained, Approxi-
% anti-inflammatory activity=(1−Vt/Vc)×100
where Vt and Vc are the volumes of oedema in drug
treated and the control groups, respectively.
Table 3
Pharmacological data of compounds 6a–6e, 7a–7e, 5%a–5%e and 6%a–6%e
Compound
number
Acute toxicity (ALD50 mg kg−1
p.o.)
Anti-inflammatory activity
Ulcerogenic activity (UD50 mg kg−1 i.p.)
mg kg−1 p.o.
% Inhibition of
oedema
6a
6b
6c
6d
6e
7a
7b
\800
\800
\800
\800
\800
\800
\1600
50
50
50
50
50
50
25
50
100
50
50
50
50
50
50
50
50
50
25
50
100
50
50
50
25
50
100
32.57 a
40.39
–
–
–
–
–
b
38.7
24.78 b
28.23 a
40.2 a
29.75 b
50.66 c
70.18 b
44.7 a
170.52
7c
7d
7e
5%a
5%b
5%c
5%d
5%e
6%a
6%b
\800
\800
\800
\800
\800
\800
\800
\800
\800
\1600
–
–
–
32.92 b
36.3 b
30.2
39.72 b
35.92 a
21.52 a
29.35 a
39.71 b
27.52 a
47.56 b
67.20 b
41.72 a
36.8 b
–
–
–
–
156.2
6%c
6%d
6%e
\800
\800
\800
–
–
38.75 c
26.56 a
45.52 c
64.70 b
Phenylbutazone \280
66.6
a PB0.05.
b PB0.01.
c PB0.001.

S. Sharma et al. / European Journal of Medicinal Chemistry 37 (2002) 689–697
697
5.2.3. Ulcerogenic acti6ity
The Pharmacological Basis of Therapeutics, McGraw Hill, New
York, 1996, pp. 638–657.
This activity was done according to the method of
Verma et al. [13]. In this method, adult albino rats,
fasted 24 h prior to the administration of drugs, were
divided into groups of ten animals each. Water was
allowed ad libitum to the animals. The test compounds
and standard drugs were given intraperitoneally and the
animals sacrificed 8 h after drugs treatment. The stom-
ach, duodenum and jejunum were removed and exam-
ined with a hand lens for any evidence of (a) shedding
of epithelium (b) petechial and frank haemorrhage and
(c) erosion or discrete ulceration with or without perfo-
ration. The presence of any one of these criteria was
considered to be an evidence of ulcerogenic activity.
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Acknowledgements
[11] Q.E. Smith, Medicinal Chemistry, vol. 1, Butterworth, London,
1960.
[12] C.A. Winter, E.A. Fisley, G.W. Nuss, Proc. Soc. Exp. Biol. New
York 111 (1962) 544.
We are thankful to CDRI, Lucknow, for elemental
and spectral analyses of newly synthesised compounds.
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