Design, synthesis and anti-inflammatory activity of structurally simple anthranilic acid congeners devoid of ulcerogenic side effects

Article abstract of DOI:10.1248/cpb.c12-00516

Simple, three classes of new anthranilic acid derivatives were aimed at, synthesized and tested for their toxicity, anti-inflammatory, analgesic, antipyretic activity. Also, their potential protective role against ulcerative colitis in rats was performed.

Full text of DOI:10.1248/cpb.c12-00516

1290
Regular Article
Chem. Pharm. Bull. 60(10) 1290–1300 (2012)
Vol. 60, No. 10
Design, Synthesis and Anti-inflammatory Activity of Structurally Simple
Anthranilic Acid Congeners Devoid of Ulcerogenic Side Effects
,a
Amal Abdel Haleem Mohamed Eissa,* Gamal Abd El-Hakeem Soliman,b and
Moayad Hussein Khataibehb
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University; Kasr El-Aini Street, POB 11562,
b
Cairo, Egypt: and Pharmacology Department, Faculty of Pharmacy; Salman Bin Abdulaziz University; Al-Kharj,
Saudi Arabia. Received June 11, 2012; accepted July 16, 2012; advance publication released online August 2, 2012
Simple, three classes of new anthranilic acid derivatives were aimed at, synthesized and tested for their
toxicity, anti-inflammatory, analgesic, antipyretic activity. Also, their potential protective role against ul-
cerative colitis in rats was performed. Furthermore, their effect on liver and kidney functions was detected
through measurement of the serum level of alanine transaminase (ALT), aspartate aminotransferase (AST),
urea, creatinine and other parameters. Compounds 4, 5, 6b, 6c, 7c and 7e showed significant anti-inflamma-
tory activity. From those 6b and 7e best improved the inflammatory indices even producing better reduction
in the intensity of lesion score, ulcer area and wet weight/length ratio and showed good analgesic activity.
Fortunately, none of the tested compounds showed any hepatotoxicity or nephrotoxicity. None of the tested
compounds showed any antipyretic activity. Conclusively, presence of a phenyl ring in the substituent added
is a must, since any alteration in its nature led to decrease in activity. Also, the presence of an extra halogen
in addition to the one already embedded in the main structure was detrimental to activity.
Key words anthranilic acid derivative; anti-inflammatory activity; ulcerative colitis; hepatotoxicity evalua-
tion; nephrotoxicity evaluation
Non steroidal anti-inflammatory drugs (NSAID) continue to and fecal blood loss. Over the disease course, these symptoms
be one of the more widely used groups of therapeutic agents gave way to more serious complications such as hemorrhage,
and useful tools in treatment of acute and chronic inflam- obstruction, perforation or cancer.15) NSAIDs could induce
mation states, pain and fever. The therapeutic anti-inflam- forms of acute kidney and liver injury or malfunction16) which
matory action of NSAID is produced by inhibition of cyclo- is directly related to the reduction of prostaglandin synthe-
oxygenase-2 (COX-2), while unwanted side effects arise from sis induced by NSAID. The release of prostaglandin E2 and
inhibition of cyclooxygenase-1 (COX-1) activity. These two prostacyclin was increased by underlying glomerular disease,
distinct isoforms of COXs were a constitutive form COX-1 renal insufficiency and the vasoconstrictors angiotensin II and
and an inducible form COX-2.1) The COX-1 known as a house- norepinephrin (NE).17,18) COX-2 inhibitors might have several
keeping enzyme and expressed in resting cells of most tissues adverse effects on the cardiovascular system including an
involved in protection of gastric mucosa, platelet aggregation increase in cardiovascular events and exacerbation of heart
and renal blood flow. On the other hand, the COX-2 was failure. Recent work suggested that COX-2 is equally impor-
rapidly induced in response to a variety of pro-inflammatory tant to human renal function and significant nephrotoxicity
stimuli such as tumor necrosis factor, interleukins and growth of selective COX-2 inhibitors was reported in salt-depleted
factors. It played an important role in pain, oncogenesis, and patients.19,20) Renal side effects of nonselective cyclooxygenase
various acute and chronic inflammatory symptoms.2–7)
inhibitors included hyperkalemia, edema, interstitial nephritis,
NSAIDs inhibited COX-1 and COX-2 with a varying degree minimal change glomerular lesions and transient impairment
of selectivity.8) Despite the known benefits of NSAID; chronic of glomerular perfusion.21) Congestive heart failure, cirrhosis
use of these agents might elicit appreciable gastrointestinal and intrinsic renal disease such as the nephrotic syndrome
ulceration, bleeding and other complications.9) The incidence were other well-established risk factors for nephrotoxicity due
of clinically significant gastro-intestinal side effects due to to non selective COX inhibitors.22)
NSAIDs was over 30% which led some patients to abandon
N-Aryl anthranilic acid derivatives are a valued class of
their NSAIDs therapy.10,11) Since the early 1990s, it was re- compounds that has been used for their pain killing, anti-
ported that cyclooxygenases (COXs) were responsible for the pyretic and anti-inflammatory properties. Amongst these are
production of prostaglandins H2,12,13) which was a precursor mefenamic acid (MA), flufenamic acid (Fig. 1) and others.
for the biosynthesis of prostaglandins, thromboxanes and pros- The main side effects of mefenamic acid included GIT dis-
tacyclins.14)
turbances, peptic ulceration and gastric bleeding. These
Inflammatory bowl diseases (IBDs) are chronic relapsing gastroenteropathies were generally believed to be the result
conditions with a high morbidity and remain largely incurable. of direct contact effect which could be attributed to the com-
Ulcerative colitis (UC) is characterized by chronic mucosal bination of local irritation produced by the free carboxylic
inflammation of the large intestine and the rectum and is lim- acid group present in its structure and/or the local blockage of
ited to the first two layers of the intestinal lining; the mucosa prostaglandin biosynthesis in the GIT.23) Literature survey re-
and submucosa. This inflammatory process led to the devel- vealed the many efforts to synthesize prodrugs or derivatives
opment of ulcerations, resulting in diarrhea, abdominal pain of many NSAIDs24,25) via temporary masking the carboxylic
group through ester or amide formation to give some relief to
The authors declare no conflict of interest.
the patient from GI irritation.25–27)
*To whom correspondence should be addressed. e-mail: phd_amalabdelhaleem@hotmail.com
© 2012 The Pharmaceutical Society of Japan

October 2012
1291
methyl ester (CDE; 4) with hydrazine hydrate. This chlorodi-
methyl ester33) was obtained in turn through the reaction of
the corresponding 2-(2-carboxyphenylamino)-4-chlorobenzoic
acid (3) with methanol in presence of thionyl chloride. This
chlorodiacid (3) was formed from reaction of anthranilic acid
with 2,4-dichloro benzoic acid under Ullmann reaction con-
ditions.34) The chlorodihydrazide congener (CDH; 5) was the
building block for the preparation of the forthcoming deriva-
tives (Chart 1).
Fig. 1. Drugs with Anthranilic Acid Moiety
Condensation of CDH (5) with various aldehydes; afforded
the corresponding chloro-hydrazones (6a–h) while reaction
Also, the use of several drugs to treat inflammatory condi- of 5 with different acetophenones gave the disubstituted
tions especially in case of patients with problems associated chloro-hydrazones (7a–g). On the other hand, reaction of
with liver or kidney function was always a challenge.28)
some chosen isothiocyanates with CDH (5) produced the rela-
In spite of this class of compounds, not being extensively tive chloro-thiosemicarbazide derivatives (8a–c) (Chart 2).
studied; several anthranilic acid derivatives were synthesized
and evaluated for their antipyretic, analgesic, anti-inflamma-
tory and cytotoxic29) (I and II) and hypotensive30) (III and IV)
efficacy (Fig. 2).
Results and Discussion
Pharmacological Activities. Acute Toxicity Tests The
tested new compounds were characterized by a low degree of
In continuation of previous work; some diphenylamines toxicity. Oral administration of the new compounds in doses
derived from anthranilic acid were synthesized, evaluated and up to 200mg/kg failed to kill any mouse within 24h of obser-
found active as anti-human immunodeficiency virus (HIV)31) vation. The calculated LD50 of the tested new derivatives in
and anticancer,31,32) respectively (compounds V, VI and VII) mice was found to be above 360mg/kg. LD50 of compounds
(Fig. 3).
6f, 6h, 7c and 8a was 370mg/kg. LD50 of compounds 5, 6c, 7g
Focusing on modifying the carboxylic acid function with and 8b was 380mg/kg. LD50 of compounds 4, 6a, 6e, 7a, 7b,
the aim of improving the pharmaceutical profile in mind; 7d, 7e, and 8c was 400mg/kg. Also, LD50 for compounds 6b,
and since one drug out of three appeared to be a halogenated 6d and 7f was 420mg/kg. Accordingly, the new synthesized
derivative for example chloro which increased lipophilicity, compounds are considered safe since substances possessing
activity and decreased metabolism and discovering new activ- LD50 higher than 50mg/kg are non toxic.35)
ities for this type of compounds; new series of chlor-N-arylan-
Anti-inflammatory Activity Anti-inflammatory activ-
thranilic acid derivatives were synthesized through for- ity of the tested compounds was measured against acute
mation of the corresponding chloro-diester (CDE-4) and paw edema induced by carrageenan (Table 1). Carrageenan-
chloro-dihydrazide (CDH-5) analogues. This was followed by induced inflammation in the rat paw represents a classical
reaction of this chloro dihydrazide (CDH-5) with chosen alde- model of acute inflammation that was used for evaluation of
hydes, acetophenones and isothiocyanates to yield 6a–h, 7a–g anti-inflammatory activity of drugs. Sub plantar injection of
and 8a–c, respectively.
1.0% carrageenan induced edema in the foot pad of rat hind
Chemistry The designed compounds were synthe- paw.36) The data shown in Table 1 are the mean changes in
sized following the general procedure as depicted in paw volume at 3h after carrageenan administration and the
Charts 1 and 2. The key starting compound 4-chloro-2-(2- results were presented as percentage reduction in paw swell-
(hydrazinecarbonyl)phenylamino)benzohydrazide (CDH; 5) ing.
was prepared via the reaction of its precursor; the chlorodi-
The starting compound CDE (4) and the new intermediate
Fig. 2. Examples of Some Anthranilic Acid Derivatives
Fig. 3. Examples of Some Diphenylamines Derived from Anthranilic Acid

1292
Vol. 60, No. 10
Chart 1
Chart 2
CDH (5) showed almost equal good anti-inflammatory ac- number was increased to two (6e) and three methoxy groups
tivity affecting 38.46 and 35.90% reduction in paw swelling (6f) there was progressive decrease in the anti-inflammatory
respectively; they both show significant difference from both activity to 20 and 10% respectively. Presence of halogen as
the control and mefenamic acid. This might throw the light on p-chloro (6a) dropped the anti-inflammatory activity to 4.10%.
lipophilicity/hydrophilicity of both compounds. The CDE is Replacement of the phenyl moiety with a heterocyclic furan
more lipophilic while the CDH is more hydrophilic.
ring (6f) also decreased the activity to 7.18% only. The above
Concerning the first group of compounds (6a–h); the hy- results strengthened the assumptions that the size, number in
drazones; all the compounds of this series bore phenyl ring addition to the nature of the substituent—whether lipophilic
variously substituted except 6h which had a heterocyclic ring or hydrophilic, electron withdrawing or donating—is of im-
namely furan. The most active was 6b (48.72% reduction in portance in this group of derivatives to presence and intensity
paw swelling) featuring a hydroxyl group attached to the phe- of the activity. Also, there is significant difference between
nyl ring. When two methyl groups were added to this hydroxy compounds 6b, 6c and 6d and the control.
(6c); the activity decreased to 29.74% which might be attrib-
The second batch of derivatives is what could be presumed
uted to the increase in bulkiness or size and the orientation as disubstituted hydrazones; the story was different. Here
of the final compound affected by the hydroxy and the two two parameters could affect the activity; the alkyl side chain
methyls. Putting a p-methoxy group (6d); the activity was and the substituted phenyl portion. The alkyl side chain was
almost as 6c (28% reduction in rat paw swelling) but when the restricted to either methyl or ethyl groups. The phenyl, on

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1293
Table 1. Anti-inflammatory Activity of the New Compounds (40mg/kg) Table 2. Analgesic Efficacy of the New Compounds (40mg/kg) Using
against Carrageenan-Induced Paw Edema in Rats (n=6)
the Hot-Plate Test in Mice (n=6)
Mean changes in paw volume
(mL) 3h after carrageenan
administration
Hot plate reaction time
Increase in pain
threshold (%)
Reduction of paw
swelling (%)
Groups
Groups
(s)
Control (DMSO)
5.85 0.46‡
9.06 0.75*
6.94 0.49‡
6.82 0.48‡
6.25 0.55‡
7.20 0.51
6.54 0.45‡
6.23 0.47‡
6.12 0.49‡
6.15 0.42‡
6.15 0.57‡
6.22 0.51‡
6.27 0.46‡
6.62 0.53‡
6.21 0.39‡
7.21 0.60
6.43 0.52‡
6.27 0.47‡
6.28 0.47‡
6.31 0.48‡
6.55 0.38‡
00.00
54.87
18.63
16.58
06.83
23.07
11.79
06.49
04.61
05.12
05.12
06.32
07.17
13.16
06.15
23.24
09.91
07.17
07.35
07.86
11.96
Control (DMSO)
1.95 0.13‡
0.79 0.05*
1.27 0.10*,‡
1.25 0.10*,‡
1.87 0.14‡
1.20 0.11*,‡
1.37 0.13*,‡
1.40 0.13*,‡
1.56 0.14‡
1.76 0.14‡
1.81 0.15‡
1.63 0.13‡
1.80 0.14‡
1.41 0.13*,‡
1.88 0.15‡
1.16 0.13*,‡
1.38 0.14*,‡
1.86 0.16‡
1.86 0.14‡
1.81 0.15‡
1.36 0.12*,‡
00.00
59.49
38.46
35.90
04.10
48.72
29.74
28.20
20.00
09.74
07.18
16.41
07.69
29.69
03.59
40.51
29.23
04.61
04.61
07.18
30.25
Mefenamic acid
Mefenamic acid
4
4
5
5
6a
6b
6c
6d
6e
6f
6a
6b
6c
6d
6e
6f
6h
7a
7b
7c
7d
7e
7f
6h
7a
7b
7c
7d
7e
7f
7g
8a
8b
8c
7g
8a
8b
8c
The results were expressed as means S.E.M. *Significantly different versus con-
trol group at p≤0.05. ‡ Significantly different versus mefenamic acid group at p≤0.05.
The results were expressed as means S.E.M. *Significantly different versus con-
trol group at p≤0.05. ‡ Significantly different versus mefenamic acid group at p≤0.05.
activity to 4.61% and this confirmed the assumption that aro-
the other hand was variably substituted or unsubstituted with maticity rather than alkyl chains were favored since by hydro-
electron donating or withdrawing groups of variable size and genation of the phenyl ring; the activity apparently decreased
nature. Both parameters collectively affected the activity and when the cyclohexyl was substituted by an ethyl further
reaching a sensitive balance between hydrophilicity and li- decrease took place.
pophilicity; the steric effect exemplified by the size and nature
Analgesic Activity The analgesic activity of the new
of the substituents had an impact on activity as well. The most compounds was tested in mice using two different techniques;
active was that bearing two methyl groups; one on phenyl the hot plate method and the acetic acid-induced writhing
and the other as side chain (7e), causing 40.51% reduction in reaction. The hot plate test in mice is an efficient analge-
paw swelling. The activity decreased to 29.69% when p-tolyl sic model for the screening of centrally acting compounds
was exchanged by p-hydroxyphenyl retaining the methyl as against acute noxious thermal stimulation37) while the acetic
a side chain (7c) or 29.23% for 7f with unsubstituted phenyl acid-induced writhing test in mice is an efficient analgesic
and an ethyl side chain. These above mentioned three deriv- model for the screening of analgesic compounds against vis-
atives showed significant difference from the control. When ceral inflammatory pain.38) The reference drug, mefenamic
one methyl from the side chain of 7f was removed to afford acid produced 54.87% increase in pain threshold in hot plate
7a; the anti-inflammatory activity decreased further to 16.41% test model and 62.78% as percentage inhibition of induced ab-
reduction in paw swelling. As in the first type (6a–h); the dominal contractions in acetic acid model (Tables 2, 3).
appearance of a halogen- here a bromo- even in the presence
Concerning the hot plate test; all tested new compounds
of the methyl side chain (7b); led to reduction in activity to showed varying degrees of activity compared to reference me-
7.69%. In this group of derivatives the hydroxy decreased the fenamic acid but not as significant (Table 2). CDE and CDH
anti-inflammatory activity comparatively 7e versus 7c and 7f (4 and 5) showed about 19 and 17% increase in pain threshold
versus 7g and the least active was the one featuring a methoxy relative to the control. It could be observed that the most po-
group with methyl side chain (7d, 4.61%). It was apparent tent anti-inflammatory compounds proved to be effective anal-
from the results of the second type that also lipophilicity, size gesic agents where the hydrazone derivatives 6b and 6c were
and nature of the substituents embedded and consequently the most active in the first type of compounds showing 23.07
orientation of the whole compound affected the anti-inflam- and 11.79% inhibition respectively; 7e and 7c in the second
matory activity.
(23.24 and 13.16%, respectively) and 8c in the third (11.96%).
The third group was of substituted thiosemicarbazides
The results of analgesic activity by the acetic acid induced
where only the phenyl substituted derivative (8c) affected abdominal writhing method (Table 3) differed slightly. Still
30.25% reduction in paw swelling compared to the control and compound 6b was the most active (22.27%) in the first group
had significant difference versus control, after which came the followed by 6f (13%) indicating the importance of the size
cyclohexyl (8b) derivative whose activity dropped severely and nature of the substituent. The most active in second group
to 7.18%. The ethyl substitution (8a) further decreased the was also 7e as the rest showed very weak activity. In the third

1294
Vol. 60, No. 10
group, 8b and 8c (10.50, 10.25%, respectively) were active they did not increase the severity of the lesions.
while the least was 8a (4.55%).
Effect on Liver and Kidney Function The non toxic
These results might give an indication that these new com- nature of the new compounds in acute toxicity study was well
pounds may be acting centrally rather than against visceral supported by the biochemical data following 5-d treatment pe-
inflammatory pain.
riod in rats by oral dosing of the new synthesized compounds
Antipyretic Activity The antipyretic activity of the to rats in a dose of 40mg/kg. Compounds 6d, 6e, 7b, 7g and
newly synthesized compounds was screened in male
Sprague-Dawley rats by using yeast-induced hyperpyrexia
Table 3. Antinociceptive Effect of the New Compounds (40mg/kg)
Using Acetic Acid-Induced Abdominal Writhing Test in Mice (n=6)
method. Rectal temperature was recorded after subcutaneous
injection of an aqueous suspension of brewer’s yeast.39) Un-
Number of writhing
(mean S.E.M.)
Groups
Inhibition (%)
fortunately, all tested compounds hadn’t shown any significant
effect on pyrexia induced by yeast in rats, while mefenamic
acid (reference drug) reversed yeast induced fever (Table 4).
Effect on Ulcerative Colitis The model of acetic acid
induced colitis shares many of the histologic features of UC
in human beings including mucosal edema and submucosal
ulceration.40)
The start (4) and key intermediate (5) compounds improved
inflammatory indices not as mefenamic acid but definitely
better than the control colitis rats (Table 5).
Same pattern for activity was observed with few additions.
6b was the best from the first set of derivatives (6a–h); it
showed better reduction in intensity of lesion score, ulcer
area, ulcer index and wet weight/length ratio compared to the
control group. 6c came next followed by 6d. The other com-
pounds 6a, 6e, 6f and 6h were the least but still better than
the control.
The second set of compounds (7a–g); 7e was best produc-
ing reduction in the lesion score intensity, ulcer area and wet
weight/length ratio compared to the control group. This was
followed by 7c, 7f, then 7a, 7d, 7b and 7g respectively.
Third set was better than the control and lower than me-
fenamic acid with 8c showing highest and 8a lowest (Table 5).
The compounds could be described as non-ulcer inducers as
Control (DMSO)
31.60 1.97‡
11.76 0.87*
27.57 1.85‡
27.80 1.60‡
29.87 1.88‡
24.56 1.57*,‡
29.37 1.77‡
28.28 1.65‡
30.12 1.32‡
27.49 1.78‡
30.16 1.92‡
30.63 1.37‡
30.55 1.31‡
29.41 1.18‡
29.50 1.37‡
25.49 1.24*,‡
29.38 1.26‡
30.17 1.15‡
30.16 1.81‡
28.28 1.75‡
28.36 1.25‡
00.00
62.78
12.75
12.02
05.47
22.27
07.05
10.50
04.68
13.00
04.55
03.06
03.32
06.93
06.64
19.33
07.02
04.52
04.55
10.50
10.25
Mefenamic acid
4
5
6a
6b
6c
6d
6e
6f
6h
7a
7b
7c
7d
7e
7f
7g
8a
8b
8c
The results were expressed as means S.E.M. *Significantly different versus con-
trol group at p≤0.05. ‡ Significantly different versus mefenamic acid group at p≤0.05.
Table 4. Antipyretic Activity of the New Compounds (40mg/kg) in Hyperthermic Rats (n=6)
Mean rectal temperature (°C) after
Groups
1h
2h
3h
4h
Control (DMSO)
39.6 0.53‡
37.5 0.65*
39.1 0.74
39.2 0.59
39.0 0.77
39.0 0.73
39.5 0.78
39.1 0.50
38.6 0.59
39.1 0.41
39.7 0.74
39.1 0.85
38.5 0.38
39.0 0.87
39.6 0.74
39.2 0.73
39.4 0.50
38.7 0.57
39.5 0.58
39.3 0.60
39.2 0.59
39.0 0.54‡
37.2 0.41*
39.1 0.60‡
38.4 0.92
38.9 0.96
38.9 0.76
38.8 0.51
38.4 0.49
39.1 0.50‡
38.5 0.64
39.0 0.89
38.5 0.37
39.2 0.58‡
38.8 0.66
39.2 0.45‡
38.9 0.55
39.1 0.61
38.7 0.60
39.0 0.75
38.4 0.55
38.6 0.45
38.6 0.52‡
37.3 0.35*
38.5 0.53
38.4 0.36
38.5 0.61
38.2 0.50
38.1 0.36
38.5 0.75
38.5 0.32‡
38.3 0.37
38.6 0.44
38.7 0.49
38.5 0.44
38.2 0.37
38.4 0.69
38.5 0.74
38.1 0.62
38.4 0.50
38.3 0.58
38.5 0.83
38.1 0.50
39.1 0.37
37.9 0.58
38.5 0.64
38.7 0.38
38.7 0.50
38.9 0.51
38.6 0.64
38.8 0.47
38.5 0.85
38.9 0.58
38.6 0.83
38.8 0.47
38.4 0.85
38.5 0.61
38.4 0.70
38.3 0.64
38.8 0.73
38.7 0.60
38.8 0.70
38.6 0.55
38.7 0.40
Mefenamic acid
4
5
6a
6b
6c
6d
6e
6f
6h
7a
7b
7c
7d
7e
7f
7g
8a
8b
8c
The results were expressed as means S.E.M. *Significantly different versus control group at p≤0.05. ‡ Significantly different versus mefenamic acid group at p≤0.05.

October 2012
1295
Table 5. Effect of Mefenamic Acid (25mg/kg) and the New Compounds
(40mg/kg) on the Macroscopic Parameters of Ulcerative Colitis Induced
by Acetic Acid in Rats (n=6)
levels of ALT, AST, total bilirubin, total proteins and albumin
(Table 6).
Urea and creatinine are the most sensitive biochemical
markers employed in the diagnosis of renal damage. In kidney
damage, there will be retention of urea and creatinine in the
blood,41) therefore marked increase in serum urea and creatin-
ine are indications of functional damage to the kidney.42) None
of the tested compounds showed significant change in the
mean values of urea and creatinine in sera of rats following
five days of administration at 40mg/kg dose when compared
with the control rats. According to these indicators, all the
new compounds are therefore, not nephrotoxic in rats. It is
worthy to mention that most of the novel compounds were non
toxic especially the most active ones (6b, 6c, 7c and 7e) while
the rest were slightly toxic.
Groups
Lesion score (0–5) Ulcer area (cm2) Wet W/L (g/cm)
Sham
0.0 0.0
0.0 0.0
0.33 0.02
Control colitis
4.8 0.24‡
2.7 0.18*
3.5 0.25*,‡
3.6 0.25*,‡
4.5 0.25‡
3.1 0.20*
3.7 0.27*,‡
3.8 0.21*,‡
4.5 0.28‡
4.2 0.25‡
4.3 0.27‡
4.3 0.26‡
4.4 0.25‡
3.9 0.22*,‡
4.4 0.27‡
3.2 0.21*
4.0 0.25*,‡
4.5 0.28‡
4.5 0.26‡
4.3 0.24‡
4.0 0.26*,‡
5.5 0.37‡
2.9 0.19*
3.8 0.29*,‡
3.9 0.27*,‡
4.9 0.28‡
3.3 0.25*
4.0 0.33*,‡
4.1 0.26*,‡
5.1 0.36‡
4.8 0.30‡
4.7 0.29‡
4.8 0.30‡
5.0 0.43‡
4.1 0.27*,‡
4.9 0.31‡
3.5 0.26*
4.2 0.28*,‡
5.0 0.35‡
5.3 0.32‡
5.0 0.36‡
4.2 0.27*,‡
0.95 0.05‡
0.52 0.03*
0.67 0.04*,‡
0.69 0.04*,‡
0.87 0.04‡
0.61 0.03*
0.69 0.04*,‡
0.74 0.05*,‡
0.88 0.04‡
0.83 0.05‡
0.83 0.05‡
0.83 0.05‡
0.86 0.04‡
0.73 0.05*,‡
0.85 0.06‡
0.62 0.04*
0.76 0.05*,‡
0.87 0.06‡
0.90 0.05‡
0.85 0.06‡
0.75 0.06*,‡
Mefenamic acid
4
5
6a
6b
6c
6d
6e
6f
6h
7a
7b
7c
7d
7e
7f
Conclusion
In summary, three series of chloro-containing anthranilic
acid derivatives were synthesized. Their anti-inflammatory,
analgesic, antipyretic activities were evaluated. Effect on
ulcerative colitis activities in addition to their effect on liver
and kidney function was performed. Most of the compounds
had moderate anti-inflammatory, analgesic activities with the
exception of compounds 6b, 6c, 7c, 7e and 8c which showed
significantly promising good activity. Most of the compounds
affected positively UC improving the inflammatory indexes.
Fortunately, most of the new derivatives showed no damaging
effect on the liver tissue and were not nephrotoxic. These re-
sults established the idea that in the first type of derivatives;
the most active compounds were those bearing hydroxy moi-
7g
8a
8b
8c
The results were expressed as means S.E.M. *Significantly different versus con-
trol colitis group at p≤0.05. ‡ Significantly different versus mefenamic acid group at
p≤0.05.
8c showed some partial damaging effect on the liver tissue ety (6b) or hydroxy and methyl groups (6c) in the phenyl sub-
indicated by significant elevation (p<0.05) in serum alanine stituent. Also in the second group; the activity of the hydroxy
transaminase (ALT) and aspartate transaminase (AST) activ- phenyl substituted compound (7c) was apparent as well as the
ities although showed insignificant change in total protein, tolyl (7e) and both had methyl group as the second substituent.
total bilirubin and albumin levels. Fortunate enough, the rest The third group had a sulphur atom incorporated in its struc-
of the new compounds had not any significant effect on the ture which changed the nature of the resulting derivatives,
Table 6. Effect of Oral Administration of the New Compounds (40mg/kg) for 5d on Liver Function Parameters in Rats (n=6)
Groups
ALT (U/L)
AST (U/L)
Total bilirubin (mg/dL)
Total protein (g/dL)
Albumin (g/dL)
Control (DMSO)
68.8 3.28
74.3 3.66
66.6 2.78
62.3 2.94
64.8 2.88
71.2 3.16
81.5 3.64*
82.2 4.16*
66.5 2.75
70.3 3.23
74.9 3.11
85.9 4.25*
72.4 3.10
75.4 3.57
69.3 2.73
78.6 3.34
82.5 4.14*
70.5 3.38
74.3 3.23
85.8 4.50*
141.4 6.62
152.8 6.57
140.9 5.34
148.5 6.23
154.4 6.84
146.9 5.34
171.8 7.56*
175.0 8.74*
132.4 5.42
162.6 8.34
136.1 6.62
172.3 8.11*
142.0 6.33
156.6 6.84
160.4 6.54
150.7 6.87
174.5 8.36*
152.4 5.77
164.8 6.45
174.5 8.98*
1.71 0.07
1.86 0.08
1.80 0.07
1.66 0.08
1.80 0.06
1.66 0.08
1.83 0.11
1.75 0.17
1.83 0.07
1.74 0.07
1.86 0.12
1.80 0.08
1.84 0.13
1.84 0.12
1.66 0.10
1.90 0.08
1.74 0.17
1.82 0.05
1.66 0.09
1.82 0.11
8.4 0.25
8.4 0.32
8.5 0.33
8.5 0.25
8.7 0.33
8.3 0.32
8.6 0.26
8.2 0.38
8.8 0.27
8.4 0.35
8.8 0.33
8.7 0.30
8.1 0.40
8.1 0.27
8.3 0.32
8.0 0.39
8.7 0.29
8.4 0.34
8.3 0.27
7.9 0.30
3.5 0.19
3.3 0.18
3.6 0.20
3.5 0.12
3.4 0.20
3.4 0.12
3.4 0.14
3.1 0.12
3.5 0.15
3.6 0.17
3.1 0.11
3.7 0.17
3.2 0.15
3.9 0.11
3.2 0.12
3.2 0.16
3.3 0.16
3.3 0.22
3.2 0.20
3.2 0.11
4
5
6a
6b
6c
6d
6e
6f
6h
7a
7b
7c
7d
7e
7f
7g
8a
8b
8c
The results were expressed as means S.E.M. *Significantly different versus control group at p≤0.05.

1296
Vol. 60, No. 10
hence the simplicity of the substituents added. The most active under reflux with stirring for 25h and concentrated under vac-
was the phenyl substituted (8c). It was obvious from the dif- uum. Enough ethanol was added to affect complete precipita-
ferent tests results that the presence of phenyl ring is impor- tion, precipitate filtered and dried to yield 0.77g (77% yield).
tant for any activity. Any deviation caused drop like phenyl The product was recrystallized from DMF–H2O to give faint
saturation, switching to heterocyclic ring or aliphatic chains beige light powder.
1
even addition of halogens to this phenyl affected the activity
mp 262–263°C; H-NMR (DMSO-d6) δ: 4.48 (4H, s, 2NH2
negatively. Also, a certain balance between lipophilicity and exch. D2O), 6.86–7.02 (2H, m), 7.19 (1H, s), 7.31–7.42 (2H, m),
hydrophilicity was essential for the compounds to show activ- 7.48–7.51 (2H, m), 9.69 (1H, s, CONH exch. D2O), 9.75 (1H,
ity. Hence, the various activities tested gave a clue of the trend s, CONH exch. D2O), 10.38 (1H, s, NH exch. D2O). IR (KBr)
how these simple compounds act especially for future work.
cm−1: 3350, 3300, 1660, 1640, 1615, 1580, 1560, 1510, 770.
From comprehensive examination of data, it was interesting Anal. Calcd for C14H14ClN5O2 (319.76): C, 52.59; H, 4.41; N,
to note that minor alteration in the molecular configuration 21.90. Found: C, 52.74; H, 4.26; N, 22.00.
of the investigated compounds exemplified by variation in
General Procedure for 4-Chloro-N′-(substituted)-2-
the size and nature of the substituents added; might have a {2-[2(substituted)hydrazinecarbonyl]phenylamino}benzo-
pronounced effect on the different pharmacological screening hydrazide 6a–h 4-Chloro-2-[2-(hydrazinecarbonyl)phenyl-
techniques applied.
amino]benzohydrazide 5 (0.60g; 1.88mmol) and the appro-
priate aldehyde (3.80mmol) in 30mL absolute ethanol were
heated under reflux for 20–30h with stirring in presence of
Experimental
Chemistry Unless otherwise noted, all materials were 1mL glacial acetic acid. Precipitate formed was filtered, left
obtained from commercial suppliers and used without further to dry then crystallized from the suitable solvent.
purification. TLC was monitored on FLUKA silica gel TLC
aluminium cards (0.2mm thickness) with fluorescent indicator lidene)hydrazinecarbonyl]phenylamino}benzohydrazide
4-Chloro-N′-(4-chlorobenzylidene)-2-{2-[2-(4-chlorobenzy-
6a:
254nm using chloroform–ethanol (9:1) as eluent and visual- mp 165–166°C; yield: 98%. Crystallization solvent: N,N-
ized by UV lamp. Melting points were performed on Schorpp dimethylformamide (DMF)–ethanol. Yellowish white pow-
1
Geratetechnik Schmelznunktbestimmer SMP II melting point der. H-NMR (DMSO-d6) δ: 7.07–7.16 (2H, m), 7.31 (1H, s),
apparatus and were uncorrected. Elemental microanalyses 7.51–7.55 (6H, m), 7.75–7.76 (6H, m), 8.40 (2H, s, 2N=CH),
were performed at the Microanalytical Center, Cairo Univer- 10.37 (1H, s, NH exch. D2O), 12.04 (2H, s, 2 CONH exch.
sity and the Regional Center for Mycology and Biotechnology, D2O). IR (KBr) cm−1: 3225, 2900, 1670, 1590, 1580, 750. MS,
Al-Azhar University. NMR spectra were recorded on Varian m/z: 565.15 [M+] (Calcd for C28H20Cl3N5O2: 564.87). Anal.
mercury 300BB at 300MHz, Gemini-200 at 200MHz and Jeol Calcd for C28H20Cl3N5O2: C, 59.54; H, 3.57; N, 12.39. Found:
FX 90 Q 90MHz Fourier Transform NMR spectrometer for C, 59.64; H, 3.69; N, 12.32.
1H-NMR, and at 75.45MHz for 13C-NMR; using tetrameth-
ylsilane (TMS) as internal reference. Chemical shift values benzylidene)hydrazinecarbonyl]phenylamino}benzohydrazide
4-Chloro-N′-(4-hydroxybenzylidene)-2-{2-[2(4-hydroxy-
were given in ppm. Mass spectra were performed on Fennigan 6b: mp 206–207°C; yield: 88%. Crystallization solvent: etha-
1
MAT, SSQ 7000 mass spectrophotometer at 70eV. IR spectra nol–H2O. Creamy powder. H-NMR (DMSO-d6) δ: 6.61–6.64
were recorded on Bruker FT-IR spectrophotometer as potas- (2H, m), 6.87–7.11 (5H, m), 7.25–7.45 (6H, m), 7.58–7.62 (2H,
sium bromide disc.
m), 8.27 (2H, s, 2N=CH), 9.97 (2H, s (br) 2OH exch. D2O),
2-(2-Carboxyphenylamino)-4-chlorobenzoic Acid 3 The 10.35 (1H, s, NH exch. D2O), 11.72 (2H, s, 2CONH D2O
compound was prepared according to the literature exch.). IR (KBr) cm−1: 3464, 3334, 2916, 2849, 1644, 1570,
procedure34) (mp 286–287°C as reported).
1500, 755. Anal. Calcd for C28H22ClN5O4 (527.97): C, 63.69; H,
Methyl-4-chloro-2-[2-(methoxycarbonyl)phenylamino]- 4.19; N, 13.26. Found: C, 63.75; H, 4.45; N, 13.51.
benzoate 433) To a cold solution of 2-(2-carboxyphenyl-
4-Chloro-N′-(4-hydroxy-3,5-dimethylbenzylidene)-2-{2-
amino)-4-chlorobenzoic acid 3 (5.00g; 17.15mmol) in 200mL [2(4-hydroxy-3,5-dimethylbenzylidene)hydrazinecarbonyl]-
methanol, 15mL thionyl chloride was added drop wise with phenylamino}benzohydrazide 6c: mp 202–203°C; yield:
stirring. Stirring was continued for another 20min at room 98.5%. Crystallization solvent: ethanol–H2O. Beige powder.
temperature. The reaction mixture was then heated under re- 1H-NMR (DMSO-d6) δ: 3.43 (12 H, s, 4CH3), 6.98–7.01 (6H,
flux conditions with stirring for 4h. The resulting solution was m), 7.28 (1H, s), 7.46–7.48 (2H, m), 7.67–7.70 (2H, m), 8.29
concentrated under vacuum and left to cool. The precipitate (2H, s, 2N=CH), 8.91 (2H, s, 2 OH exch. D2O), 10.32 (1H, s,
formed was collected, left to dry to give 4.85g (88%) and then NH exch. D2O), 11.83 (2H, s, 2 CONH exch. D2O). IR (KBr)
crystallized from ethanol to give cottony white solid.
mp 151–152°C; H-NMR (DMSO-d6) δ: 3.85 (6H, s, 2CH3), Calcd for C32H30ClN5O4 (584.08): C, 65.81; H, 5.18; N, 11.99.
cm−1: 3300, 3264, 2910, 2845, 1640, 1584, 1509, 756. Anal.
1
6.98–7.12 (2H, m), 7.39 (1H, s), 7.40–7.57 (2H, m), 7.91–7.95 Found: C, 65.75; H, 5.34; N, 12.11.
(2H, m), 10.80 (1H, s, NH exch. D2O). IR (KBr) cm−1: 3275,
2975, 1700, 1580, 1520, 750. Anal. Calcd for C16H14ClNO4 benzylidene)hydrazinecarbonyl]phenylamino}benzohydrazide
4-Chloro-N′-(4-methoxybenzylidene)-2-{2-[2-(4-methoxy-
(319.75): C, 60.10; H, 4.41; N, 4.38. Found: C, 60.30; H, 4.00; 6d: mp 159–160°C; yield: 99%. Crystallization solvent:
1
N, 4.40.
DMF–H2O. Yellow powder. H-NMR (DMSO-d6) δ: 3.81 (6H,
4-Chloro-2-[2-(hydrazinecarbonyl)phenylamino]benzo- s, 2OCH3), 6.99–7.03 (6H, m), 7.26 (1H, s), 7.46–7.48 (2H,
hydrazide Methyl-4-chloro-2-(2-(methoxycarbonyl)- m), 7.66–7.69 (6H, m), 8.32 (2H, s, 2N=CH), 10.29 (1H, s,
5
phenylamino)benzoate 4 (1.00g; 3.13mmol) was heated in NH exch. D2O), 11.79 (2H, s, 2 CONH exch. D2O). IR (KBr)
40mL butanol till solution then hydrazine hydrate 99% was cm−1: 3200, 2900, 1640, 1600, 1550, 750. Anal. Calcd for
added (0.35g; 6.99mmol). The reaction mixture was heated C30H26ClN5O4 (556.03): C, 64.81; H, 4.71; N, 12.59. Found: C,

October 2012
1297
1
64.87; H, 4.58; N, 12.89.
Yellow powder. H-NMR (DMSO-d6) δ: 2.31 (s, 6H, 2CH3),
4-Chloro-N′-(3,4-dimethoxybenzylidene)-2-{2-[2(3,4- 6.92–7.28 (m, 4H, aromatic), 7.41 (7H, s, (br)), 7.62–7.71 (4H,
dimethoxybenzylidene)hydrazinecarbonyl]phenylamino}- m), 9.90 (1H, s (br) NH exch. D2O), 10.92 (2H, s, 2CONH
benzohydrazide 6e: mp 164–165°C; yield: 97%. Crystallization exch. D2O). IR (KBr) cm−1: 3250, 2950, 2850, 1660, 1580,
solvent: DMF–ethanol–H2O. Pale beige powder. 1H-NMR 1510, 760. MS, m/z: 524.25 [M+] (Calcd for C30H26ClN5O2:
(DMSO-d6) δ: 3.82 (12H, s, 4OCH3), 6.99–7.02 (4H, m), 524.03). Anal. Calcd for C30H26ClN5O2: C, 68.76; H, 5.00; N,
7.16–7.20 (2H, m), 7.28 (1H, s), 7.38 (2H, s (br)), 7.48 (2H, s 13.36. Found: C, 68.64; H, 5.29; N, 13.25.
(br)), 7.71–7.73 (2H, m), 8.37 (2H, s, 2N=CH), 10.38 (1H, s,
N′-[1-(4-Bromophenyl)ethylidene]-2-{2-[2-(1-(4-
NH exch. D2O), 11.96 (2H, s, 2CONH exch. D2O). IR (KBr) bromophenyl)ethylidene)hydrazinecarbonyl]phenyl-
cm−1: 3227, 3158, 2910, 2840, 1652, 1576, 1500, 754. MS, m/z: amino}-4-chlorobenzohydrazide 7b: mp 145–146°C; yield:
616.35 [M+] (Calcd for C32H30ClN5O6: 616.08). Anal. Calcd for 98%. Crystallization solvent: DMF–H2O. Faint yellow powder.
C32H30ClN5O6: C, 62.39; H, 4.91; N, 11.37. Found: C, 62.45; H, 1H-NMR (DMSO-d6) δ: 2.49 (6H, s, 2CH3), 6.89–7.17 (3H, m),
4.70; N, 11.01.
7.42–7.44 (4H, m), 7.62–7.68 (8H, m), 9.90 (1H, s, NH exch.
4-Chloro-N′-(2,4,6-trimethoxybenzylidene)-2-{2- D2O), 10.95 (2H, s, 2 CONH exch. D2O). IR (KBr) cm−1: 3300,
[2(2,4,6-trimethoxybenzylidene)hydrazinecarbonyl]- 3264, 2916, 2849, 1662, 1571, 1509, 756, 600. Anal. Calcd for
phenylamino}benzohydrazide 6f: mp 130–132°C; yield: C30H24Br2ClN5O2 (682.01): C, 52.83; H, 3.55; N, 10.27. Found:
92%. Crystallization solvent: ethanol. Light brown powder. C, 53.07; H, 3.79; N, 10.20.
1H-NMR (DMSO-d6) δ: 3.8–3.86 (18H, d, 6OCH3), 6.17–6.3
4-Chloro-N′-[1-(4-hydroxyphenyl)ethylidene]-2-{2-[2-
(4H, m), 6.97–7.08 (2H, m), 7.16 (1H, s), 7.44 (2H, s (br)), (1-(4-hydroxyphenyl)ethylidene)hydrazinecarbonyl]phenyl-
7.65–7.71 (2H, m), 8.54 (2H, s, 2N=CH), 10.21 (1H, s, NH amino}benzohydrazide 7c: mp 148–151°C; yield: 83%. Crys-
exch. D2O), 11.56 (2H, s, 2CONH exch. D2O). 13C-NMR tallization solvent: ethanol. Dark yellow powder. 1H-NMR
(DMSO-d6) δ: 55.54 (OCH3), 55.92 (OCH3), 90.63–134.00 (DMSO-d6) δ: 2.24 (6H, s, 2CH3), 6.74–6.83 (4H, m),
(aromatic Cs), 144.00 (CH=N), 159.89 (C–O), 163.30 (C=O). 6.94–7.05 (4H, m), 7.21 (1H, s), 7.42–7.47 (4H, m), 7.57–7.62
IR (KBr) cm−1: 3253, 3159, 2916, 2843, 1643, 1601, 1570, 757. (2H, m), 9.81 (1H, s, NH exch. D2O), 10.36 (2H, s, 2OH exch.
MS, m/z: 676.35 [M+] (Calcd for C34H34ClN5O8: 676.13). Anal. D2O), 10.78 (2H, s, 2 CONH exch. D2O). IR (KBr) cm−1:
Calcd for C34H34ClN5O8: C, 60.39; H, 5.07; N, 10.36. Found: 3250 br, 2900, 2850, 1650, 1580, 1510, 750. MS, m/z: 555.50
C, 60.55; H, 5.20; N, 10.36.
[M+] (Calcd for C30H26ClN5O4: 556.03). Anal. Calcd for
4-Chloro-N′-[4-(trif luromethyl)benzylidene]-2-{2- C30H26ClN5O4: C, 64.81; H, 4.71; N, 12.59. Found: C, 64.65; H,
[2(4-trifluromethylbenzylidene)hydrazinecarbonyl]phenyl- 4.90; N, 12.50.
amino}benzohydrazide 6g: mp 142–143°C; yield: 92%.
Crystallization solvent: ethanol–H2O. Buff powder. H-NMR (4-methoxyphenylethylidene)hydrazinecarbonyl]phenylamino}-
4-Chloro-N′-[1-(4-methoxyphenyl)ethylidene]-2-{2-[2-(1-
1
(DMSO-d6) δ: 6.98–7.02 (2H, m), 7.36 (1H, s), 7.45–7.79 (6H, benzohydrazide 7d: mp 154–155°C; yield: 91%. Crystallization
1
m, aromatic), 7.91–7.96 (6H, m), 8.41 (2H, s, 2N=CH), 10.76 solvent: ethanol. Dark yellow powder. H-NMR (DMSO-d6) δ:
(1H, s, NH exch. D2O), 12.05 (2H, s, 2CONH exch. D2O). IR 2.34 (6H, s, 2CH3), 3.88 (6H, s, 2 OCH3), 6.99–7.16 (7H, m),
(KBr) cm−1: 3248 br, 2924, 2850, 1651, 1578, 1508, 752. Anal. 7.26 (1H, s), 7.34–7.55 (3H, m), 7.74–7.83 (4H, m), 9.98 (1H, s,
Calcd for C30H20ClF6N5O2 (631.97): C, 57.02; H, 3.19; N, 11.08. NH exch. D2O), 10.89 (2H, s, 2 CONH exch. D2O). IR (KBr)
Found: C, 57.24; H, 3.16; N, 11.27.
cm−1: 3200, 2950, 2850, 1660, 1600, 1580, 760. MS, m/z:
4-Chloro-N′-(furan-2-ylmethylene)-2-{2-[2(furan-2- 584.25 [M+] (Calcd for C32H30ClN5O4: 584.08). Anal. Calcd for
ylmethylene)hydrazinecarbonyl]phenylamino}benzohydrazide C32H30ClN5O4: C, 65.81; H, 5.81; N, 11.99. Found: C, 66.09; H,
6h: mp 179–180°C; yield: 95.5%. Crystallization solvent: 5.02; N, 12.09.
ethanol–H2O. Orange yellow powder. 1H-NMR (DMSO-d6)
4-Chloro-N′-(1-p-tolylethylidene)-2-{2-[2-(1-p-tolylethyli-
δ: 6.64 (2H, d, 2H-3 furan, J=1.90Hz), 6.94–7.12 (4H, m, 2H dene)hydrazinecarbonyl]phenylamino}benzohydrazide 7e: mp
and 2H-4 furan), 7.24 (s, 1H), 7.46 (2H, m), 7.65–7.68 (2H, m), 220–222°C; yield: 95%. Crystallization solvent: DMF–etha-
7.85 (2H, d, 2H-5 furan, J=2.10Hz), 8.28 (2H, s, 2N=CH), nol–H2O. Dark buff powder. 1H-NMR (DMSO-d6) δ: 2.28 (6H,
10.21 (1H, s, NH exch. D2O), 11.87 (2H, s, 2 CONH exch. s, 2 C-CH3), 3.42 (6H, s, 2 CH3), 7.16–7.23 (7H, m), 7.40–7.44
D2O). IR (KBr) cm−1: 3252, 2915, 2847, 1650, 1570, 1509, 751. (2H, m), 7.61–7.64 (6H, m), 9.60 (1H, s, NH exch. D2O), 10.86
MS, m/z: 475.25 [M+] (Calcd for C24H18ClN5O4: 475.89). Anal. (2H, s, 2 CONH exch. D2O). IR (KBr) cm−1: 3300, 3200,
Calcd for C24H18ClN5O4 (475.89): C, 60.57; H, 3.81; N, 14.72. 2900, 1650, 1580, 1510, 755. MS, m/z: 552.25 [M+] (Calcd
Found: C, 60.68; H, 3.96; N, 14.43.
General Procedure for 4-Chloro-N′-[1-(4-un/substi- 69.62; H, 5.48; N, 12.69. Found: C, 69.83; H, 5.56; N, 12.65.
tutedphenyl)ethylidene]-2-{2-[2-(1-(4-un/substitutedphenyl- 4-Chloro-N′-(1-phenylpropylidene)-2-{2-[2-(1-phenyl-
for C32H30ClN5O2: 552.08). Anal. Calcd for C32H30ClN5O2: C,
ethylidene)hydrazinecarbonyl]phenylamino}benzohydra- propylidene)hydrazinecarbonyl]phenylamino}benzohydrazide
zide 7a–g The chosen acetophenone (4.38mmol), 4-chloro- 7f: mp 109–111°C; yield: 88%. Crystallization solvent: eth-
benzohydrazide intermediate 5 (0.70g; 2.19mmol), glacial anol–H2O. Creamy white powder. 1H-NMR (DMSO-d6) δ:
acetic acid (2mL) and absolute ethanol (30mL) were refluxed 1.01–1.17 (6H, t, 2CH2CH3, J=7.10Hz), 2.79–3.00 (4H, m, 2
with stirring for 25h. The reaction mixture was concentrated CH2CH3, overlapped), 6.95–7.04 (3H, m), 7.13–7.18 (2H, m),
and poured over ice-water. The solid obtained was collected, 7.39–7.42 (6H, m), 7.58–7.66 (6H, m), 9.89 (1H, s, NH exch.
dried and crystallized from the appropriate solvent.
D2O), 11.02 (2H, s, 2 CONH exch. D2O). IR (KBr) cm−1: 3250,
4-Chloro-N′-(1-phenylethylidene)-2-{2-[2-(1-phenylethyli- 2950, 1660, 1580, 1510, 760. MS, m/z: 552.25 [M+] (Calcd for
dene)hydrazinecarbonyl]phenylamino}benzohydrazide 7a: mp C32H30ClN5O2: 552.08). Anal. Calcd for C32H30ClN5O2 (552.08):
137–138°C; yield: 98%. Crystallization solvent: ethanol. C, 69.62; H, 5.48; N, 12.69. Found: C, 69.39; H, 5.70; N, 12.75.

1298
Vol. 60, No. 10
4-Chloro-N′-[1-(4-hydroxyphenyl)propylidene]-2-{2-
Pharmacological Activities. Animals Swiss albino
[2-(1-(4-hydroxyphenyl)propylidene)hydrazinecarbonyl]- mice of both sex (27–30g body weight (b.wt)) and male
phenylamino}benzohydrazide 7g: mp 100–102°C; yield: 81%. Sprague-Dawley rats (200–220g b.wt) were obtained from the
Crystallization solvent: ethanol. Yellow powder. 1H-NMR Animal Center of National Research Center, Cairo. Animals
(DMSO-d6) δ: 0.99–1.15 (6H, t, 2CH2CH3, J=7.38Hz), were maintained under standard conditions of temperature
2.97–3.05 (4H, m, 2CH2CH3), 6.81–6.90 (4H, d), 7.19 (1H, s), (23 1.0°C), humidity (55 10%), and 12h light/12h dark cycle
7.37–7.47 (4H, m), 7.57 (2H, s (br)), 7.81–7.90 (4H, d), 9.90 and fed with a standard pellet diet with water ad libitum. All
(1H, s (br) NH exch. D2O), 10.40 (2H, s, 2OH exch. D2O), the rats were housed in standard polypropylene cages with
10.90 (2H, s, 2CONH exch. D2O). IR (KBr) cm−1: 3200 br, wire mesh top. Animals were allowed to adapt to the labora-
2900, 1655, 1570, 1510, 750. MS, m/z: 583.25 [M+−1] (Calcd tory environment for one week before experimentation. The
for C32H30ClN5O4: 584.08). Anal. Calcd for C32H30ClN5O4: C, experimental tests on animals have been performed in accor-
65.81; H, 5.18; N, 11.99. Found: C, 65.62; H, 5.53; N, 11.69.
dance with the Institutional Ethical Committee approval.
General Procedure for 2-{4-Chloro-2-[2-(2-(substituted-
Preparation of the Tested Chemical Compounds All
carbamothioyl)hydrazine carbonyl)phenylamino]benzoyl}- the tested chemicals and the reference drug (mefenamic acid)
N-substitutedhydrazinecarbthioamide 8a–c The 4-chloro- were dissolved in 2% v/v dimethylsulfoxide (DMSO) before
benzohydrazide derivative 5 (0.60g; 1.88mmol) and the oral administration to the experimental animals.
chosen isothiocyanate (3.75mmol) in 25mL absolute ethanol
Acute Toxicity (LD50) Test The oral median lethal doses
were heated under reflux with stirring for more than 30h. (LD50) of the tested compounds (4–8) were determined in
The reaction solution obtained was concentrated and poured Swiss albino mice as described by Lorke.43) In a preliminary
over ice- water to precipitate the desired compounds. The test, mice of either sex in groups of three, received one of 10,
precipitate was filtered, left to dry and crystallized from the 100, or 1000mg/kg of the tested compounds. Animals were
appropriate solvent.
observed for 24h for signs of toxicity and mortality. From the
2-{4-Chloro-2-[2-(2-(ethylcarbamothioyl)hydrazine- results of the preliminary test, doses of 200, 400, 600, 800
carbonyl)phenylamino]benzoyl}-N-ethylhydrazine- and 1000mg/kg of the tested compounds were administered
carbthioamide 8a: mp 134–136°C; yield: %. Crystalliza- to fresh groups, each of 5 mice. Control animals received the
tion solvent: ethanol–H2O. Fluffy grey powder. 1H-NMR solvent and kept under the same conditions without any treat-
(DMSO-d6) δ: 1.03–1.29 (2H, m, 2CH2CH3), 3.87–3.88 (6H, ment. Signs of toxicity and number of deaths per dose during
m, 2CH2CH3), 6.98–7.15 (2H, m), 7.40 (1H, s), 7.56–7.57 (2H, 24h were recorded and the LD50 was calculated as the geo-
m), 7.63–7.96 (2H, m), 9.19 (2H, s (br) 2NH–C2H5 exch. D2O), metric mean of the minimum dose that resulted in 100% mor-
10.30 (2H, s (br) 2NHC=S exch. D2O), 10.50 (1H, s, NH exch. tality and the maximum dose that caused no lethality at all.
D2O), 10.76 (2H, s, 2 CONH exch. D2O). IR (KBr) cm−1: 3294
br, 2920, 2851, 1701, 1639, 1562, 1512, 1215, 748. Anal. Calcd 40mg/kg that’s equal to /10 LD50 of most of the tested com-
for C20H24ClN7O2S2 (494.04): C, 48.62; H, 4.89; N, 19.85. pounds (4, 6a, 6e, 7a, 7b, 7d, 7e and 8c) were selected to be
Doses In this investigation, an experimental dose of
1
Found: C, 48.84; H, 4.98; N, 20.12.
given orally to rats. The reference drug; mefenamic acid was
2-{4-Chloro-2-[2-(2-(cyclohexylcarbamothioyl)hydrazine- given orally at a dose of 25mg/kg. The doses were calculated
carbonyl)phenylamino]benzoyl}-N-cyclohexylhydrazine- by converting the therapeutic dose used in humans to rat's
carbothioamide 8b: mp 165–167°C; yield: 90%. Crystalli- dose according to the Table of Paget and Barnes.44) Acetic acid
zation solvent: DMF–ethanol–H2O. White fluffy powder. was diluted in saline to be 4% and infused into the colon of
1H-NMR (DMSO-d6) δ: 1.05–1.24 (10H, m), 1.56–1.79 (10H, rats through a rubber catheter at the dose of 5mL/kg.
m), 4.08 (2H, m, 2H-1′ cyclohexyl), 6.96–7.07 (2H, m), 7.25
Anti-inflammatory Activity The anti-inflammatory ac-
(1H, s), 7.41–7.50 (2H, m), 7.82–7.86 (2H, m), 9.19 (2H, s, tivity was evaluated in male Sprague-Dawley rats using a
2NH-cyclohexyl exch. D2O), 10.23 (3H, s (br) 2NHC=S carrageenan-induced paw edema test.36) The rats were divided
and 1H, NH exch. D2O), 10.49 (2H, s, 2 CONH exch. D2O). into 21 groups (n=6). Rats of the 1st (normal control) and 2nd
13C-NMR (DMSO-d6) δ: 24.67 (C-3′ and C-5′), 25.08 (C-4′), (standard) groups were treated orally with the vehicle and me-
31.75 (C-2′ and C-6′), 52.67 (C-1′), 115.50–144.71 (aromatic fenamic acid (25mg/kg), respectively. Animals of the 3rd–21st
Cs), 162.21 (C=O), 166.95 (C=S). IR (KBr) cm−1: 3264 br, groups were orally given the tested compounds (4–8), respec-
2930, 2853, 1661, 1577, 1536, 1509, 1216, 770. MS, m/z: 601.90 tively in a dose of 40mg/kg. Acute inflammation was induced
[M+] (Calcd for C28H36ClN7O2S2: 602.23). Anal. Calcd for by the subplantar injection of 0.1mL of 1% carrageenan in
C28H36ClN7O2S2: C, 55.85; H, 6.03; N, 16.28. Found: C, 55.88; normal saline in the right hind paw of all rats after 30min of
H, 6.01; N, 16.11.
administration of the tested compounds. The volume of the
2-{4-Chloro-2-[2-(2-(phenylcarbamothioyl)hydrazine- injected paws was measured in mL using a plethysmometer
carbonyl)phenylamino]benzoyl}-N-phenylhydrazinecarbothio- immediately before and 3h following carrageenan injection.
amide 8c: mp 154–156°C; yield: 90%. Crystallization sol- The mean change in the volume of injected paw with respect
vent: ethanol–H2O. Off-white powder. 1H-NMR (DMSO-d6) to the initial paw volume was calculated and the percentage
δ: 7.02–7.19 (3H, m), 7.33–7.37 (8H, m), 7.48–7.50 (4H, m), inhibition of paw edema with respect to control group was
7.94–7.96 (2H, m), 9.83 (2H, s (br) 2NH-phenyl D2O exch.), estimated using the formula:
10.58 (2H, s (br) 2NHC=S exch. D2O), 10.75 (1H, s, NH
% inhibition of paw edema =(1Vt /Vc )×100
exch. D2O), 11.07 (2H, s, 2CONH exch. D2O). IR (KBr) cm−1:
3254 br, 1652, 1625, 1591, 1558, 1220, 748. Anal. Calcd for Where: Vt and Vc are the mean change in paw volume of
C28H24ClN7O2S2 (590.13): C, 56.99; H, 4.09; N, 16.61. Found: treated and control rats, respectively.
C, 57.00; H, 3.80; N, 16.59.
Analgesic Activity Analgesic activity of the newly

October 2012
1299
synthesized compounds was carried out in Swiss albino mice inflammation. These inflammatory indices were significantly
(27–30g b.wt) by using two different models.
improved by oral dosing of mefenamic acid.
Induction of Ulcerative Colitis All animals were fasted
Hot Plate Method The hot plate test45) was used with few
modifications. Mice (25–30g) were divided into 21 groups, overnight, with access to water ad libitum, and then anesthe-
each containing six animals. Mice of the 1st (normal control) tized with ether inhalation before induction of the ulcerative
and 2nd (reference) groups were treated orally with the vehicle colitis. A polyethylene catheter with 2mm diameter was in-
and mefenamic acid (25mg/kg), respectively. Animals of the serted into the lumen of the colon via the anus to a distance
3rd–21st groups were orally given the tested compounds (4–8), of 8cm.47) A solution of 2mL (4%, v/v) acetic acid in saline
respectively in a dose of 40mg/kg. After one h of medication, was slowly infused into the colon through the catheter. The
mice were placed individually on a hot plate maintained at rats were then maintained in a supine Trendelenburg position
55 1°C. The time taken by the animals to lick the fore or hind for 30s to prevent early leakage of the acetic acid.48) In sham
paw or jump out of the place was taken as the reaction time.
group, equivolume of normal saline was infused into the colon
Acetic Acid-Induced Writhing Test The writhing test instead of acetic acid. The acetic acid was then aspirated
was carried out as described by Collier et al.46) Mice were and 2mL of phosphate buffer solution (pH=7) was instilled
assigned into 21 groups, each containing 6 animals and med- into the rectum of each rat.49) The catheter was left in place
icated as those in the hot plate method. Writhing was induced for a few seconds then gently removed. Two days after the
by an intraperitoneal injection of a 0.6% acetic acid solution induction of colitis, each animal was sacrificed using ether
(0.25mL/animal) after 30min of medication with the tested anesthesia and laparatomy was performed. Colonic segments
compounds. Immediately after administration of acetic acid, (8cm in length and 3cm proximal to the anus) were excised,
each animal was isolated in an individual cage to be observed opened along its mesenteric border, washed with saline, and
for 15min. The number of writhing per animal was recorded were used for macroscopic scoring.
and expressed as the percentage of protection.
Assessment of Colonic Lesions The colon specimens
Antipyretic Activity The antipyretic activity of the were weighted and wet weight/length ratio was calculated for
newly synthesized compounds was screened in male all the rats. The specimens were examined under a dissecting
Sprague-Dawley rats by using yeast-induced hyperpyrexia microscope and the mucosal lesions were quantified by the
method.39) After measuring rectal temperature of each rat by scoring system (0–5) given by Millar et al.,48) after some mod-
introducing 1.5cm of digital thermometer into rectum, py- ifications.
rexia was induced by subcutaneous injection of 1mL of 15%
The Lesion Scores were: 0=no damage, 1=local edema and
brewer’s yeast suspension in saline solution. After 18h of inflammation without ulcers; 2=one ulcer without inflam-
yeast injection, rats which showed a rise in temperature of at mation; 3=one to two ulcers with inflammation and lesion
least 0.6°C were taken for the study. One hundred thirty two diameter <1cm; 4=more than two ulcers with lesion diameter
hyperthermic rats were divided into 21 equal groups. Rats 1–2cm; 5=severe ulceration with lesion diameter >2cm.
of the 1st (hyperthermic control) and 2nd (reference) groups
Ulcer Area was measured using plane glass square. Each
were treated orally with the vehicle (5mL/kg) and mefenamic cell on the glass square was 1mm2 in area and the number of
acid (25mg/kg), respectively. Animals of the 3rd–21st groups cells was counted and the ulcer area was determined for each
were orally given the tested compounds (4–8), respectively colon.
in a dose of 40mg/kg. Rectal temperature of each rat was
Blood Sampling for Liver and Kidney Functions One
then recorded at 1h interval after administration for 4h. The hundred twenty six male Sprague-Dawley rats were randomly
antipyretic efficacy was decided on the basis of the difference divided into 20 equal groups. Rats of the 1st group were given
in the mean temperature between the control and the tested the solvent (DMSO) in a dose of 5mL/kg and left as normal
compounds.
control. Rats of the 2nd–20th groups were administered the
Effect on Ulcerative Colitis Male Sprague-Dawley rats test compounds 4–8, respectively in a dose of 40mg/kg b.wt.
were divided into 22 experimental groups, each of 6 animals. All medications were administered orally via the aid of an
Rats of groups 1 and 2 (Sham and Control colitis groups, orogastric cannula for 5d. The animals were observed for
respectively) were given the solvent only (2%, v/v, DMSO) signs of abnormalities throughout the experiment. At the end
in a dose of 5mL/kg. Group 3 (reference group) was given of the experimental period, blood samples (2mL) were col-
mefenamic acid in a dose of 25mg/kg. Rats of groups 4–22 lected from the retro-orbital venous plexus of each rat (under
received the test compounds 4–8, respectively in a dose of ether anesthesia) and kept for 30min at 4°C. Serum was sep-
40mg/kg. All medications were administered orally, once arated by centrifugation at 3000rpm for 10min and used for
daily via the aid of an orogastric cannula for 5 successive days the biochemical estimations.
and the last dose was administered 2h before colitis induction.
Biochemical Estimation of Liver and Kidney Functions
In rats of sham group, no abnormal changes were observed Liver functions were evaluated by measuring the serum activ-
suggesting that handling procedure had no interference with ity of ALT and AST.50) Serum levels of total bilirubin,51) total
the experimental outputs. Macroscopic damage parameters proteins,52) and albumin53) were also assayed. Serum concen-
of the colon of control colitis rats, 2d after rectal infusion of trations of urea54) and creatinine55) were determined colormet-
acetic acid revealed dark brown lesions, mucosal hyperemia, rically as measures of kidney functions.
edema, erosion, and ulceration. Control colitis rats showed
lesion score, ulcer area and values of 4.8 0.24 and 5.5
Statistical Analysis Statistical analysis was performed
using SPSS 10.0 statistical software. Differences among
0.37cm2 respectively (Table 5). The inflammatory changes of groups were examined using parametric one-way analysis
the intestinal tract were associated with a significant increase of variance (ANOVA) with Tukey HSD as post hoc test.
of wet weight/length of the colon specimens as an indicator of Non-parametric data were analyzed by Kruskal–Wallis

1300
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