Synthesis of some newer analogues of substituted dibenzoyl phenol as potent anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2004.08.014

The newly synthesized compounds dibenzoyl phenols 4a-f using microwave irradiation were screened for their anti-inflammatory activity and compared with standard drugs. Benzoylation of hydroxybenzophenones 1a-f affords substituted benzoyl phenyl benzoates

Full text of DOI:10.1016/j.bmcl.2004.08.014

Bioorganic & MedicinalChemistry Letters 14 (2004) 5351–5355
Synthesis of some newer analogues of substituted dibenzoyl
phenol as potent anti-inflammatory agents
b
Shaukath Ara. Khanum,a Venu T. D,a Sheena Shashikantha, and Aiysha Firdouse
*
aDepartment of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
bDepartment of Biochemistry, Farooqia Dental College, Mysore 570 001, India
Received 23 April2004; revised 6 August 2004; accepted 7 August 2004
Available online 9 September 2004
Abstract—Benzoylation of hydroxybenzophenones 1a–f affords substituted benzoylphenylbenzoates 3a–f, which on Fries rear-
rangement using microwave irradiation led to a facile synthesis of solely dibenzoyl phenols 4a–f in excellent yield. The newly syn-
thesized compounds were screened for their anti-inflammatory activity and were compared with standard drugs. Out of the
compounds studied, the compound 4e showed more potent activity than the standard drugs at all doses tested.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
The competence of benzophenone analogues as chemo-
therapeutic agent especially as anti-inflammatory is well
recognized.1,2 Some of these analogues were synthesized
by severalscientists of the world and have been reported
as potent anti-inflammatory agents.10–12 Recently Otto-
sen et al. have reported synthesis and structural activity
relationship of benzophenones as novel class of p38
MAP kinase inhibitors with high anti-inflammatory
activity.13 Our search for new molecules with anti-
inflammatory activity14 encouraged us to synthesize
some newer more potent analogues of hydroxybenzo-
phenones by modifying the aromatic moiety with the
introduction of second benzoylgroup at 2- and 6-posi-
tions. We have focused our interest on the synthesis
and biological evaluation of substituted dibenzoyl phen-
ols for a rational study of the structural activity
relationships.
Nonsteroidalanti-inflammatory drugs (NSAIDs) are
therapeutic agents usefulin the treatment of inflamma-
tion, pain and pyresis.1,2 Inflammatory responses are
considered to be mediated in part by the prostaglandins
(PGs). PGs are produced by the action of cyclooxyge-
nase (COX) enzyme, which is also referred as prosta-
glandin H synthase on arachidonic acid.3,4 Recent
studies have shown that COX exists in two isoforms
COX-1 and COX-2. Both COX are constitutively ex-
pressed in most tissues, but COX-2, in contrast to
COX-1, is the mitogen inducible isoform. The inducing
stimuli for COX-2 include pro-inflammatory cytokines
and growth factors, implying a role for COX-2 in both
5–7
inflammation and controlof cellgrowth.
COX iso-
forms are almost identical in structure but have impor-
tant differences in substrate and inhibitor selectivity
and in their intracellular locations.8 NSAIDs block the
formation of PGs and have anti-inflammatory, analgesic
and anti-pyretic activities.1,2 The discovery of COX-29
isoform has made possible the design of drugs that re-
duce inflammation without removing the protective
PGs in the stomach and kidney made by COX-1. In
addition, its discovery has opened the possibility of
developing COX-2 selective inhibitors to act as an effec-
tive NSAIDs without the gastrotoxic effect.6
2. Chemistry
The synthetic sequence is outlined in Scheme 1. Benzoyl-
ation of substituted hydroxybenzophenones 1a–f with
respective benzoylcholrides
2a–d affords substituted
benzoylphenylbenzoate 3a–f.15 Compounds 3a–f on
thoroughly mixing with an equal amount of montmor-
rillonite k 10 clay in the solid state using vortex mixer
and on subjecting to microwave irradiation for 10–
13min afforded substituted dibenzoylphenosl 4a–f in
excellent yield compared to one pot conventional meth-
od.16 The newly synthesized compounds 3a–f17 and
Keywords: Synthesis; Dibenzoylphenosl; Anti-inflammatory activity.
*
Corresponding author. Fax: +91 0821 2518358; e-mail: shashi56_
2000@yahoo.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.014

5352
S. A. Khanum et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5351–5355
R3
R4
O
OH
COCl
C
R5
R
R2
R1
R2
O
NaOH
+
R
R2
R1
R3
R1
3a-f
1a-f
2a-d
a: R=R3=R4=H, R1=R5=CH3, R2=4-CH3COPh
b: R=R3=R4=R5=H, R1=CH3, R2=COPh
c: R=CH3, R1= 2-CH3COPh, R2=R3=R4=R5=H
d: R=R5=CH3, R1=COPh, R2=R3=R4=H
e: R=R3=R5=H, R1=CH3, R2=3-ClCOPh, R4=Cl
f: R=R4=R5=H, R1=R3=CH3, R2=2-CH3COPh
a: R=H, R1=CH3, R2=4-CH3COPh
b: R=H, R1=CH3, R2=COPh
c: R=CH3, R1= 2-CH3COPh, R2=H
d: R=CH3, R1=COPh, R2=H
e: R=H, R1=CH3, R2=3-ClCOPh
f: R=H, R1=CH3, R2=2-CH3COPh
a: R1=R2=H, R3=CH3
b: R1=R2=R3=H
c: R1=R3=H, R2=Cl
d: R1=CH3, R2=R3=H
OH
O
C
R2
R
R3
R4
k 10 clay
Microwave
R1
4a-f
a: R1=R4=CH3, R=4-CH3COPh, R2=R3=H
b: R2=R3=R4=H, R1=CH3, R=COPh
c: R=CH3, R1= 2-CH3COPh, R2=R3=R4=H
d: R=R4=CH3, R1=COPh, R2=R3=H
e: R2=R4=H, R1=CH3, R=-3-ClCOPh, R3=Cl
f: R3=R4=H, R1=R2=CH3, R=2-CH3COPh
Scheme 1.
1
4a–f18 were characterized by IR, H NMR and mass
spectrophotometer.
examined to find incidence of hyperaemia, shedding of
epithelium, petechial, frank haemorrhages and erosion
or discrete ulceration with or without perforation. The
presence of any of these criteria was considered to be
an evidence of ulcerogenic activity.20
3. Pharmacological evaluation
3.1. Anti-inflammatory activity
3.3. Acute toxicity study
Albino rats were used to perform paw oedema inhibition
test adopting winter et al.19 method. Groups of five rats
(body weight 125–160g), were given a dose of a test
compound. After 30min, 0.2mL of 1% carrageenan sus-
pension in 0.9% sodium chloride solution was injected
subcutaneously, into planter aponeurosis of the hind
paw and the paw volume was measured by a water plet-
hysmometer socreland then measured again after a time
span of 3h. The mean increase of paw volume at each
time intervalwas compared with that of controlgroup
(five rats treated with carrageenan, but not with test
compounds) at the same time intervals. The percentage
inhibition values were calculated using the formula:
Nearly 50% lethal dose (ALD50) of the compounds was
determined in albino mice (body weight 25–30g). The
test compounds were injected intraperitoneally at differ-
ent dose levels in groups of 10 animals. After 24h of
drug administration, percent mortality in each group
was observed from the data obtained ALD50 was calcu-
lated by adopting Smith21 method.
3.4. Cyclooxygenase activity
The in vitro test on microsomalfraction of mucosal
preparation of rabbit distalcooln was carried out in
order to search out the plausible mechanism of the com-
pounds. By adopting Calderano et al.22 procedure the
preparation was carried out. About 2–3g of stripped,
colonic mucosa was minced and homogenized in 3 vols
of Tris buffer 0.1, pH8.0 and the homogenized was cen-
trifuged. The precipitate as suspended in Tris buffer 0.1
M, pH8.0 and recentrifuged. For enzyme assay cyclo-
oxygenase activity, the microsomal pellet was used
immediately. By measuring the rate of conversion of
arachidonic acid to PGE2, cyclooxygenase activity was
assayed. About 50mL of microsomalfractions were
incubated with test agents for 10min at 37ꢁC in 30lL
Tris–HCl, pH8.0 containing 2mM reduced glutathione,
% anti-inflammatory activity ¼ 1 À Gt=Gc  100
where Gt and Gc represent tested and controls groups,
respectively.
3.2. Ulcerogenic activity
Groups of 10 rats (body weight 200–230g), fasted for
24h, were treated with an oraldose of test compound,
except control group. All animals were sacrificed 5h
after the completion of dosing. With the aid of a micro-
scope, the stomach and small intestine of the rats were

Table 1. Anti-inflammatory, ulcerogenic, cyclooxygenase and toxicity data of compounds 4a–f
Compd
Dose (mg/kg po)
Anti-inflammatory
activity % oedema inhibition
relative to control
Dose
(mg/kg po)
Ulcerogenic activity
Cyclooxygenase activity
assay inhibitory action of
some selected compound %
inhibition 10lM
ED50
(mg/kg po)
ALD50
(mg/kg po)
% Of animalwith
hyperaemia
% Of animal
with ulcer
4a
20
40
80
20
40
80
20
40
80
20
40
80
20
40
80
20
40
80
20
40
80
22.2
45.4
77.1
13.7
22.2
45.5
14.1
29.5
55.3
20.1
29.1
62.8
30.3
48.2
94.1
16.6
33.2
64.1
30.4
35.5
59.6
100
200
400
100
200
400
100
200
400
100
200
400
100
200
400
100
200
400
100
200
400
50
70
100
20
40
60
40
60
100
70
90
100
30
60
90
50
70
90
30
60
90
20
30
40
40
50
80
10
20
40
10
20
40
10
20
12
05
10
15
80
90
90
89
60
90
87
NI
20
40
NI
70
99
—
60.2
77.5
78.3
62.5
51.2
77.2
98.3
—
>1000
>1000
>1000
>1000
>1000
>1000
—
4b
4c
4d
4e
4f
Aspirin
Phenyl20
Butazone
31.3
100
30
30
40
80
35.5
57.2
200
400
60
90
Control20
—
30
—
—
NI
—
—
40
80
60
90
NI = no inhibition.

5354
S. A. Khanum et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5351–5355
5mM L-trytophan, 1lM haematin. The substrate 20lM
arachidonic acid with tracer amount of [1À C] arachi-
other compounds 4b and 4e, which do not inhibit the
cyclooxygenase activity, seems to act through some
other mechanism rather than inhibiting prostaglandin
synthesis. It appears that compounds 4b and 4e inhibits
the Phospholipase A2 (PLA2) enzyme. The PLA2 class
of enzymes catalyze hydrolysis of the 2-acyl ester of 3-
Sn phosphoglycerides to yield arachidonic acid, which
is responsible for the production of pro-inflammatory
lipid mediators such as PGs.23–25
14
donic acid [approximately 200 (··) cpm] then added
and the reaction proceeded for 5min at 37ꢁC. The reac-
tion was stopped by addition of 0.2mL of ether/metha-
nol/citric acid 0.2M (30:4:1), which was precooled at
À25ꢁC PGE2, was extracted twice into the same mix-
ture. The solvent was evaporated under nitrogen stream
and radiolabelled arachidonic acid was separated and
from this radiolabelled PGE3 were separated by RP-
HBLC with 2nmol unlabelled PGE2 as an interval
standard. PG chromatographic profile was obtained by
isocratic elution with 150mM H3PO4 in water, pH3.5,
containing 30% acetonitrile, a flow rate of 1mL/min
monitoring the UV absorption at 214nm. Radioactivity
that co-eluted with authentic PGE2 was quantified by
liquid scintillation spectrometry. Test samples were
compared to paired controlincubations. The percentage
of inhibition was calculated as follows.
4.3. ALD50 studies
The toxicity study of these compounds indicates their
good safety margin.
5. Conclusion
From the results of the biological activities, it appears
that introduction of benzoylgroup in 1a–f is fruitful
as it was found that, 4a–f showed good anti-inflamma-
tory with reduced ulcer production activity. Moreover
introduction of second benzoylgroup using microwave
oven is more beneficialwith respect to yiedl compared
to one pot conventionalmethod.
½ðcpm control À cpm test=ðcpm controlÞÞ Â 100ꢀ
4. Results and discussion
The characteristic feature of the title compounds is the
presence of two keto groups at the same phenylring.
All the dibenzoyl phenols 4a–f have shown good anti-
inflammatory activity in the range 22.2–48.2% at a dose
of 40mg/kg po. Among 4a–f, the compounds 4e with
two chloro groups at the meta-position in benzoylmoi-
ety, elicited maximum inhibition of oedema (48.2%).
Compound 4a with three methylgroups, one at the
para-position in the phenylring and the other two at
para-position in benzoylmoiety, exhibited more activity
(45.4%) compared to 4f (33.2%) in which the two methyl
groups in benzoylmoiety are at ortho-position. Whereas
compound 4b having only one methyl group at phenyl
ring exhibited least activity (22.2%) at a dose of 40mg/
kg po. On the contrary compounds 4c (29.5%) and 4d
(29.1%) in which benzoylgroups are at second and
fourth position have shown less activity compared to
compounds 4a,e and 4f, in which benzoylgroups are
at second and sixth position. Based on the above results,
title compounds have been tested at three graded doses
(20, 40 and 80mg/kg po) and compared with standard
drugs aspirin and phenylbutazone. The comparison re-
sults with standard drugs are listed in Table 1.
Acknowledgements
The authors express their sincere gratitude to the Uni-
versity of Mysore, Mysore for providing laboratory
facilities. One of the authors (S.A.K.) is indebted to
the University Grant Commission for the award of a
teacherÕs fellowship.
References and notes
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The title compound 4a–f exhibited low degree of ulcer
production activity (10–50%) at 200mg/kg po. Among
4a–f, compound 4e with two chloro groups at the
meta-position in benzoyl moiety, exhibited lesser ulcero-
genic activity (20%) compared to standard drugs, aspirin
and phenylbutazone.
4.2. Cyclooxygenase assay activity
Compounds 4a,c,d and 4f showed good cyclooxygenase
activity indicating that these compounds reduces inflam-
matory response by inhibition of prostaglandins. The
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2.5 (3s, 9H, 3CH3), 6.9–7.65 (m, 10H, Ar–H), 12.0 (br s,
1H, OH); MS: m/z (64%) M+ 344. Anal. Calcd for
C23H20O3: C, 80.21; H, 5.85. Found: C, 80.20; H, 5.86%.
4b: Yield 80%, mp 159–161ꢁC [lit.: 160];16 IR (Nujol):
1655 and 1620 (C@O), 3400–3500cmÀ1 (OH); 1H NMR
(CDCl3): d 2.3 (s, 3H, CH3), 6.8–7.6 (m, 12H, Ar–H);
12.02 (br s, 1H, OH); MS: m/z (63%) M+ 316. Anal. Calcd
for C21H16O3: C, 79.73; H, 5.10. Found: C, 79.74; H,
5.11%.
4c: Yield 81%, mp 91–93ꢁC; IR (Nujol): 1645 and 1615
1
16. Mandal, S. K.; Nag, K. J. Chem. Soc., Dalton Trans. 1983,
2429.
(C@O), 3360–3470cmÀ1 (OH); H NMR (CDCl3): d 2.3–
2.41 (2s, 6H, 2CH3), 6.8–7.6 (m, 11H, Ar–H), 11.4 (br s,
1H, OH); MS: m/z (63.5%) M+ 330. Anal. Calcd for
C22H18O3: C, 79.98; H, 5.49. Found: C, 79.99; H, 5.47%.
4d: Yield 80%, mp 96–98ꢁC; IR (Nujol): 1640 and
1612cmÀ1 (C@O), 3350–3460cmÀ1 (OH); 1H NMR
(CDCl3): d 2.3–2.42 (2s, 6H, 2CH3), 6.75–7.7 (m, 11H,
Ar–H), 11.2 (br s, 1H, OH); MS: m/z (62.5%) M+ 330.
Anal. Calcd for C22H18O3: C, 79.98; H, 5.49. Found: C,
79.95; H, 5.46%.
17. 3a: Yield 78%, mp 97–99ꢁC; IR (Nujol): 1750 (ester,
C@O); 1668cmÀ1 (C@O), 1H NMR (CDCl3): d 2.3–2.5
(3s, 9H, 3CH3), 6.8–7.6 (m, 11H, Ar–H); MS: m/z (75%)
M+ 344. Anal. Calcd for C23H20O3: C, 80.21; H, 5.85.
Found: C, 80.23; H, 5.83%.
3b: Yield 75%, mp 89–91ꢁC; IR (Nujol): 1760 (ester,
C@O); 1670cmÀ1 (C@O), 1H NMR (CDCl3): d 2.2 (s, 3H,
CH3), 6.9–7.7 (m, 13H, Ar–H); MS: m/z (76%) M+ 316.
Anal. Calcd for C21H16O3: C, 79.73; H, 5.10. Found: C,
79.75; H, 5.12%.
3c: Yield 78%, mp 55–57ꢁC; IR (Nujol): 1740 (ester,
C@O); 1645cmÀ1 (C@O), 1H NMR (CDCl3): d 2.3–2.4
(2s, 6H, 2CH3), 6.7–7.5 (m, 12H, Ar–H); MS: m/z (75.5%)
M+ 330. Anal. Calcd for C22H18O3: C, 79.98; H, 5.49.
Found: C, 79.97; H, 5.48%.
3d: Yield 79%, mp 50–52ꢁC; IR (Nujol): 1745 (ester,
C@O); 1640cmÀ1 (C@O), 1H NMR (CDCl3): d 2.2–2.3
(2s, 6H, 2CH3), 6.75–7.7 (m, 12H, Ar–H); MS: m/z (75%)
M+ 330. Anal. Calcd for C22H18O3: C, 79.98; H, 5.49.
Found: C, 79.96; H, 5.47%.
3e: Yield 77%, mp 93–95ꢁC; IR (Nujol): 1755 (ester,
C@O); 1670cmÀ1 (C@O), 1H NMR (CDCl3): d 2.3 (s, 3H,
CH3), 6.75–7.7 (m, 11H, Ar–H); MS: m/z (77%) M+ 385.
Anal. Calcd for C21H14Cl2O3: C, 65.47; H, 3.66; Cl, 18.41.
Found: C, 65.45; H, 3.65; Cl, 18.40%.
4e: Yield 77%, mp 148–150ꢁC; IR (Nujol): 1660 and
1620cmÀ1 (C@O), 3420–3525cmÀ1 (OH); 1H NMR
(CDCl3): d 2.3 (s, 3H, CH3), 6.75–7.7 (m, 10H, Ar–H),
12.2 (br s, 1H, OH); MS: m/z (62%) M+ 385. Anal. Calcd
for C21H14Cl2O3: C, 65.47; H, 3.66; Cl, 18.41. Found: C,
65.48; H, 3.67; Cl, 18.43%.
4f: Yield 78%, mp 155–57ꢁC; IR (Nujol): 1652 and 1620
1
(C@O), 3415–3518cmÀ1 (OH); H NMR (CDCl3): d 2.1–
2.3 (3s, 9H, 3CH3), 6.92–7.7 (m, 10H, Ar–H), 12.1 (br s,
1H, OH); MS: m/z (64.5%) M+ 344. Anal. Calcd for
C23H20O3: C, 80.21; H, 5.85. Found: C, 80.23; H, 5.87%.
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3f: Yield 79%, mp 96–98ꢁC; IR (Nujol): 1735 (ester,
C@O); 1655cmÀ1 (C@O), 1H NMR (CDCl3): d 2.3–2.5
(3s, 9H, 3CH3), 6.92–7.7 (m, 11H, Ar–H); MS: m/z (74%)
M+ 344. Anal. Calcd for C23H20O3: C, 80.21; H, 5.85.
Found: C, 80.22; H, 5.84%.
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L.; Snyder, D. W.; Hartley, L. W.; Carlson, D. G.;
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18. 4a: Yield 78%, mp 145–147ꢁC; IR (Nujol): 1650 and 1615
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(C@O), 3405–3505cmÀ1 (OH); 1H NMR (CDCl3): d 2.32–