Synthesis and anti-inflammatory activity of benzophenone analogues

Article abstract of DOI:10.1016/j.bioorg.2004.04.003

A series of substituted benzophenone analogues has been synthesized and evaluated as orally active anti-inflammatory agents with reduced side effects. The anti-inflammatory and ulcerogenic activities of the compounds were compared with naproxen, indometha

Full text of DOI:10.1016/j.bioorg.2004.04.003

Bioorganic Chemistry 32 (2004) 211–222
www.elsevier.com/locate/bioorg
Synthesis and anti-inXammatory activity
of benzophenone analogues
Shaukath A. Khanum,a Sheena Shashikanth,a,¤
and A.V. Deepakb
a Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
b Applied Botany and Biotechnology, University of Mysore, Manasagangotri, Mysore 570 006, India
Received 27 October 2003
Available online 13 May 2004
Abstract
A series of substituted benzophenone analogues has been synthesized and evaluated as
orally active anti-inXammatory agents with reduced side eVects. The anti-inXammatory and
ulcerogenic activities of the compounds were compared with naproxen, indomethacin, and
phenylbutazone. In carrageenan-induced foot pad edema assay, benzophenone analogues
showed an interesting anti-inXammatory activity. In the air-pouch test, some of the analogues
reduced the total number of leukocytes of the exudate, which indicates inhibition of prosta-
glandin production. Side eVects of the compounds were examined on gastric mucosa, in the
liver and stomach. None of the compounds showed signiWcant side eVects compared with
nonsteroidal anti-inXammatory drugs such as indomethacin and naproxen.
 2004 Elsevier Inc. All rights reserved.
Keywords: Benzphenone analogues; Anti-inXammatory activity.
1. Introduction
InXammatory responses are thought to be mediated in part by the prostaglandins
(PGs) derived from arachidonic acid by the action of prostaglandin H synthase,
which is also referred to as cyclooxygenase (COX) [1,2]. Recent studies have shown
that COX exists in two isoforms COX-1 and COX-2. Both COX are constitutively
¤ Corresponding author.
E-mail address: skanth1@rediffmail.com (S. Shashikanth).
0045-2068/$ - see front matter  2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2004.04.003

212
S.A. Khanum et al. / Bioorganic Chemistry 32 (2004) 211–222
expressed in most tissues, but COX-2, in contrast to COX-1, is the mitogen induc-
ible isoform. The inducing stimuli for COX-2 include pro-inXammatory cytokines
and growth factors, implying a role for COX-2 in both inXammation and control of
cell growth [3–5]. COX isoforms are almost identical in structure but have impor-
tant diVerences in substrate and inhibitor selectivity and in their intracellular
locations [6]. Protective PGs, which preserve the integrity of the stomach lining
and maintain normal renal function in a compromised kidney, are synthesized by
COX-1. In addition to the induction of COX-2 in inXammatory lesions, it is present
constitutively in the brain and spinal cord, where it may be involved in nerve trans-
mission, particularly those for pain and fever. COX is the principal target of nonste-
roidal anti-inXammatory drugs (NSAIDs) and metabolites of the COX pathway are
widely accepted as mediators of the inXammatory response. NSAIDs block the for-
mation of PGs and have anti-inXammatory, analgesic, and antipyretic activity [7].
The discovery of COX-2 has made possible the design of drugs that reduce inXam-
mation without removing the protective PGs in the stomach and kidney made by
COX-1.
Recently, benzophenone analogues were identiWed as potent anti-inXammatory
agents [7,8]. Welstead et al. [9] and Branacaccio et al. [10] have reported the anti-
inXammatory activity of benzoylphenylacetic acid. In addition Vigorita et al. [11]
have identiWed polyaromatic triXuoroacetamides as anti-inXammatory agents. Based
on these observations we synthesized hydroxybenzophenones (2a–c, Scheme 1), aroyl
aryloxyacetic acid (4a–c, Scheme 1), and 2-(2-aroyl aryloxy)-N-phenyl acetamide
analogues (6a–c and 7a–c, Scheme 1), and evaluated them for their anti-inXamma-
tory activity and side eVects.
2. Experimental synthesis
2.1. General
Chemicals were purchased from Aldrich Chemical TLC was performed on alu-
minium-backed silica plated with visualization by UV-light. Melting points were
determined on a Thomas Hoover capillary melting point apparatus with a digital
thermometer. IR spectra were recorded in Nujol on FT-IR Shimadzu 8300 spectro-
1
meter and H NMR spectra were recorded on a Bruker 300 MHz spectrometer in
CDCl3. Chemical shifts were recorded in parts per million downWeld from tetrameth-
ylsilane. Mass spectra were obtained with a VG70-70H mass spectrometer and
elemental analysis results are within 0.4% of the calculated value.
2.1.1. (2-Hydroxy-5-methylphenyl)-(3-chlorophenyl)methanone (2a)
A solution of anhydrous aluminum chloride (3.2g, 0.02mol) in dry nitrobenzene
(25mL) was added to 4-methyl phenyl chloro benzoate (1a, 5 g, 0.02 mol) dissolved in
nitrobenzene (10 mL). The mixture was protected from moisture by a calcium chlo-
ride guard tube and reXuxed with stirring for 30 min. At the end of this period the
solution was cooled and treated with acidic ice-cold water. The nitrobenzene was

S.A. Khanum et al. / Bioorganic Chemistry 32 (2004) 211–222
213
Scheme 1. (a) Anhydrous AlCl3, (b) K2CO3, ClCH2CO2Et, Acetone, (c) NaOH, EtOH, H2O, (d) BF3 ether-
ate, NH2CH2C6H5, Dry benzene, (e) BF3 etherate, H2NC6H4O(CH2)2NEt2, Dry benzene, (f) BF3 etherate,
NH2C6H5, Dry benzene. Note. Bn D
.
removed by steam distillation. The residual solid was crushed into powder, extracted
with 10% sodium hydroxide (3 £50 mL), and the basic aqueous solution was neutral-
ized with 10% hydrochloric acid. The product was extracted into ether and the ether
layer washed well with a saturated sodium chloride solution. Evaporation of the
ether after drying over anhydrous sodium sulphate followed by recrystallization
from alcohol gave (2-hydroxy-5-methyl phenyl) chloro phenyl methanone 2a in 85%
yield. Compounds 2b and 2c were synthesized by analogous procedures using 1b and
1c, respectively, and obtained in 70 and 75% yield, respectively.
1
2a: mp 71–73 °C; IR (Nujol): 1673 (CñO), 3550–3640 cm¡1 (OH); H NMR
(CDCl3): 2.2 (s, 3H, CH3), 7.0–7.65 (m, 7H, Ar-H), 12.15 (bs, 1H, OH); MS (EI) m/z:
246 (M+, 88); Anal. Calcd for C14H11ClO2 (246.5): C, 68.15; H, 4.46; Cl, 14.40. Found:
C, 68.17; H, 4.44; Cl, 14.42%.
1
2b: mp 78–81 °C; IR (Nujol): 1650 (CñO), 3510–3610 cm¡1 (OH); H NMR
(CDCl3): 2.3 (s, 3H, CH3), 6.8–7.75 (m, 7H, Ar-H), 12.0 (bs, 1H, OH); MS (EI) m/z:
291 (M+, 85); Anal. Calcd for C14H11BrO2 (291): C, 57.73; H, 3.78; Br, 27.49. Found:
C, 57.71; H, 3.79; Br, 27.46%.

214
S.A. Khanum et al. / Bioorganic Chemistry 32 (2004) 211–222
1
2c: mp 75–78°C; IR (Nujol): 1668 (CñO), 3540–3650cm¡1 (OH); H NMR
(CDCl3): 2.32 (s, 3H, CH3), 3.75 (s, 3H, OCH3) 7.1–7.75 (m, 7H, Ar-H), 12.0 (bs, 1H,
OH); MS (EI) m/z: 242 (M+, 83); Anal. Calcd for C15H14O3 (242): C, 73.38; H, 5.78.
Found: C, 73.35; H, 5.76%.
2.1.2. Ethyl [2-(3-chlorobenzoyl)-4-methylphenoxy]acetate (3a)
A mixture of 2a (5 g, 0.02 mol) and ethyl chloroacetate (2.4 g, 0.02 mol) in dry ace-
tone (60mL) and anhydrous potassium carbonate (2.8g, 0.02mol) was reXuxed for
8h. Subsequently, the reaction mixture was cooled and the solvent removed under
reduced pressure. The residual mass was triturated with ice water to remove potas-
sium carbonate and extracted with ether (3£ 50 mL). The ether layer was washed
with 10% sodium hydroxide solution (3£ 30mL), followed by water (3 £30 mL), and
then dried over anhydrous sodium sulphate and evaporated to dryness to yield crude
solid. Recrystallization with ethanol gave 3a in 80% yield. Compounds 3b and 3c
were synthesized analogously starting with 2b and 2c, respectively. Compounds 3b
and 3c were obtained in 71 and 72% yield, respectively.
1
3a: mp 60–62 °C; IR (Nujol): 1670 (CñO), 1735cm¡1 (ester, CñO); H NMR
(CDCl3): 1.2 (t, J D7 Hz, 3H, CH3 of ester), 2.3 (s, 3H, CH3), 4.2 (q, J D6 Hz, 2H, CH2
of ester), 4.45 (s, 2H, OCH2), 7.2–7.6 (m, 7H, Ar-H); MS (EI) m/z: 332.5 (M+, 62);
Anal. Calcd for C18H17ClO4 (332.5): C, 64.96; H, 5.11; Cl, 10.67. Found: C, 64.94; H,
5.07; Cl, 10.64%.
1
3b: mp 65–67 °C; IR (Nujol): 1665 (CñO), 1730cm¡1 (ester, CñO); H NMR
(CDCl3): 1.21 (t, J D 7 Hz, 3H, CH3 of ester), 2.3 (s, 3H, CH3), 4.22 (q, J D6 Hz, 2H,
CH2 of ester), 4.46 (s, 2H, OCH2), 7.2–7.6 (m, 7H, Ar-H); MS (EI) m/z: 377 (M+, 61);
Anal. Calcd for C18H17BrO4 (377): C, 57.29; H, 4.50; Br, 21.22. Found: C, 57.26; H,
4.53; Br, 21.25%.
1
3c: mp 58–60 °C; IR (Nujol): 1660 (CñO), 1730 cm¡1 (ester, CñO); H NMR
(CDCl3): 1.2 (t, J D7 Hz, 3H, CH3 of ester), 2.25 (s, 3H, CH3), 3.8 (s, 3H, OCH3), 4.2
(q, JD6Hz, 2H, CH2 of ester), 4.42 (s, 2H, OCH2), 7.0–7.6 (m, 7H, Ar-H); MS (EI) m/z:
328 (M+, 59); Anal. Calcd for C19H20O5 (328): C, 69.51; H, 6.09. Found: C, 69.49; H,
6.05%.
2.1.3. 2-[(2-(3-Chlorobenzoyl)-4-methylphenoxy)ethanoic acid (4a)
Compound 3a (2.0g, 6.0mmol) was dissolved in ethanol (10mL) and treated with
a solution of sodium hydroxide (0.6 g, 15mmol) in water (10 mL). The mixture was
heated under reXuxed for 3 h, cooled and acidiWed with 1 N hydrochloric acid. The
oily precipitate was extracted with dichloromethane (3£ 30 mL) and the solution
washed with water (3£ 25), dried and evaporated to give an oil. Crystallization from
hexane aVorded 4a as a white solid in 75% yield. Compounds 4b and 4c were synthe-
sized analogously starting with 3b and 3c, respectively. Compounds 4b and 4c were
obtained in 74 and 70% yield, respectively.
4a: mp 120–22°C; IR (Nujol): 3400–3500 (acid OH), 1730 (acid CñO), 1675cm¡1
1
(CñO); H NMR (CDCl3): 2.3 (s, 3H, CH3), 4.46 (s, 2H, OCH2), 7.2–7.7 (m, 7H,
Ar-H), 9.5 (s, 1H, COOH); MS (EI) m/z: 304.5 (M+, 60); Anal. Calcd for C16H13ClO4
(304.5): C, 63.06; H, 4.30; Cl, 11.63. Found: C, 63.04; H, 4.26; Cl, 11.60%.

S.A. Khanum et al. / Bioorganic Chemistry 32 (2004) 211–222
215
4b: mp 125–27°C; IR (Nujol): 3410–3510 (acid OH), 1735 (acid CñO), 1670cm¡1
1
(CñO); H NMR (CDCl3): 2.3 (s, 3H, CH3), 4.45 (s, 2H, OCH2), 7.1–7.6 (m, 7H,
Ar-H), 9.4 (s, 1H, COOH); MS (EI) m/z: 349 (M+, 58); Anal. Calcd for C16H13BrO4
(349): C, 55.01; H, 3.72; Br, 22.92. Found: C, 55.04; H, 3.75; Br, 22.94%.
4c: mp 130–32 °C; IR (Nujol): 3470–3575 (acid OH), 1738 (acid CñO), 1660cm¡1
(CñO); 1H NMR (CDCl3): 2.25 (s, 3H, CH3), 3.8 (s, 3H, OCH3), 4.42 (s, 2H, OCH2),
7.0–7.6 (m, 7H, Ar-H), 9.1 (s, 1H, COOH); MS (EI) m/z: 300 (M+, 55); Anal. Calcd for
C17H16O5 (300): C, 68.0; H, 5.33. Found: C, 68.03; H, 5.35%.
2.1.4. N-benzyl-2-[2-(3-chloro-benzoyl)-4-methyl-phenoxy]-acetamide (5a)
A mixture of 4a (1.0 g, 3.2 mmol), benzyl amine (0.43g, 4.0 mmol) and boron triXu-
oride etherate (0.83 g, 6.0 mmol) in dry benzene (15mL) was reXuxed for 6 h. The
reXuxing solvent was dried by circulation over anhydrous sodium sulphate in a Soxh-
let’s apparatus. The reaction mixture was washed with 10% aqueous sodium hydrox-
ide (3 £20 mL), 10% hydrochloric acid (3£ 20 mL) and then with water (3 £ 25 mL).
After being dried with anhydrous sodium sulphate, the organic layer was concen-
trated under reduced pressure. The amide was puriWed by crystallization from etha-
nol. Further puriWcation was carried out by chromatography on silica gel using
hexane and ethanol (7:2) as eluent to give 5a as white solid in 70% yield. Compounds
5b and 5c were synthesized by analogous procedures using 4b and 4c, respectively,
and obtained in 72 and 75% yield, respectively.
1
5a: mp 116–18°C; IR (Nujol): 1705 (amide CñO), 1665cm¡1 (CñO); H NMR
(CDCl3): 2.25 (s, 3 H, CH3), 4.0 (s, 2H, NCH2), 4.4 (s, 2H, OCH2), 4.8 (bs, 1H,
CONH), 7.1–7.7 (bm, 12H, Ar-H); MS (EI) m/z: 394 (M+ + 1, 08); Anal. Calcd for
C23H20ClNO3 (393): C, 70.13; H, 5.08; Cl, 9.02. Found: C, 70.15; H, 5.10; Cl, 9.05%.
1
5b: mp 124–26 °C; IR (Nujol): 1710 (CñO of amide), 1670cm¡1 (CñO); H
NMR (CDCl3): 2.3 (s, 3H, CH3), 4.2 (s, 2H, NCH2), 4.45 (s, 2H, OCH2), 4.9 (bs, 1H,
CONH), 7.2–7.8 (bm, 12H, Ar-H); MS (EI) m/z: 440 (M+ + 1, 07); Anal. Calcd for
C23H20BrNO3 (439): C, 62.87; H, 4.55; Br, 18.22; N, 3.18. Found: C, 62.88; H, 4.52; Br,
18.25; N, 3.15%.
1
5c: mp109–111 °C; IR (Nujol): 1700 (CñO of amide), 1660 cm¡1 (CñO); H
NMR (CDCl3): 2.22 (s, 3H, CH3), 3.75 (s, 3H, OCH3), 3.95 (s, 2H, NCH2), 4.38 (s, 2H,
OCH2), 4.75 (bs, 1H, CONH), 7.0–7.9 (bm, 12H, Ar-H); MS (EI): m/z 390 (M+ + 1,
08); Anal. Calcd for C24H23NO4 (389): C, 74.03; H, 5.91; N, 3.59. Found: C, 74.05; H,
5.93; N, 3.56%.
2.1.5. 2-[2-(3-Chloro-benzoyl)-4-methyl-phenoxy]-N-[4-(2-diethylamino-ethoxy)-phen-
yl]-acetamide (6a)
A mixture of 4a (1.0 g, 3.2 mmol), 4-(2-diethylamino ethoxy) phenylamine (3.17 g,
4.0mmol) and boron triXuoride etherate (0.83 g, 6.0 mmol) in dry benzene (15 mL)
was reXuxed for 6 h. The reXuxing solvent was dried by circulation over anhydrous
sodium sulphate in a Soxhlet’s apparatus. The reaction mixture was washed with
10% aqueous sodium hydroxide (3£ 20 mL), 10% hydrochloric acid (3£ 20mL) and
then with water (3£ 25 mL). After being dried with anhydrous sodium sulphate, the
organic layer was concentrated under reduced pressure. The amide was puriWed by

216
S.A. Khanum et al. / Bioorganic Chemistry 32 (2004) 211–222
crystallization from ethanol. Further puriWcation was carried out by chromatogra-
phy on silica gel using hexane and ethanol (7:2) as eluent to give 6a in 70% yield.
Compounds 6b and 6c were synthesized by analogous procedures using 4b and 4c,
respectively, and obtained in 71 and 73% yield, respectively.
1
6a: mp 142–44°C; IR (Nujol): 1710 (amide CñO), 1670cm¡1 (CñO); H NMR
(CDCl3): 1.02 (t, 6H, 2CH3), 2.2 (s, 3H, Ar-CH3), 2.71 (q, 4H, 2CH2 of ethyl), 2.92 (t,
2H, N-CH2), 4.05 (t, 2H, CH2), 4.45 (s, 2H, OCH2), 7.0–7.9 (bm, 11H, Ar-H), 9.6 (bs,
1H, CONH); MS (EI): m/z 495 (M++1, 10); Anal. Calcd for C28H31ClN2O4 (494): C,
67.94; H, 6.47; Cl, 7.17; N, 5.66. Found: C, 67.96; H, 6.45; Cl, 7.19; N, 5.64%.
1
6b: mp 155–56 °C; IR (Nujol): 1715 (CñO of amide), 1675cm¡1 (CñO); H
NMR (CDCl3): 1.1 (t, 6H, 2CH3), 2.25 (s, 3H, Ar-CH3), 2.75 (q, 4H, 2CH2 of ethyl),
3.05 (t, 2H, N-CH2), 4.1 (t, 2H, CH2), 4.5 (s, 2H, OCH2), 7.1–8.0 (bm, 11H, Ar-H), 9.7
(bs, 1H, CONH); MS (EI): m/z 540 (M+ + 1, 09); Anal. Calcd for C28H31BrN2O4
(539): C, 62.33; H, 5.75; Br, 14.84; N, 5.19. Found: C, 62.35; H, 5.35; Br, 14.85; N,
5.17%.
1
6c: mp 134–36 °C; IR (Nujol): 1708 (CñO of amide), 1665 cm¡1 (CñO); H
NMR (CDCl3): 1.0 (t, 6H, 2CH3), 2.2 (s, 3H, Ar-CH3), 2.7 (q, 4H, 2CH2 of ethyl), 2.91
(t, 2H, N-CH2), 3.78 (s, 3H, OCH3), 4.0 (t, 2H, CH2), 4.4 (s, 2H, OCH2), 6.95–7.9 (bm,
11H, Ar-H), 9.55 (bs, 1H, CONH); MS (EI): m/z 391 (M+ + 1, 08); Anal. Calcd for
C28H34N2O5 (390): C, 71.02; H, 6.93; N, 5.71. Found: C, 71.04; H, 6.95; N, 5.73%.
2.1.6. 2-[2-(3-Chloro-benzoyl)-4-methyl-phenoxy]-N-phenyl-acetamide (7a)
A mixture of 4a (1.0 g, 3.2mmol), aniline (0.37 g, 4.0mmol) and boron triXuoride
etherate (0.83 g, 6.0 mmol) in dry benzene (15 mL) was reXuxed for 6 h. The reXuxing
solvent was dried by circulation over anhydrous sodium sulphate in a Soxhlet’s appa-
ratus. The reaction mixture was washed with 10% aqueous sodium hydroxide
(3£ 20 mL), 10% hydrochloric acid (3£ 20 mL) and then with water (3 £ 25 mL).
After being dried with anhydrous sodium sulphate, the organic layer was concen-
trated under reduced pressure. The amide was puriWed by crystallization from etha-
nol. Further puriWcation was carried out by chromatography on silica gel using
hexane and ethanol (7:2) as eluent to give 7a in 75% yield. Compounds 7b and 7c
were synthesized by analogous procedures using 4b and 4c, respectively, and
obtained in 72 and 73% yield, respectively.
1
7a: mp 125–27°C; IR (Nujol): 1710 (amide CñO), 1655cm¡1 (CñO); H NMR
(CDCl3): 2.22 (s, 3H, Ar-CH3), 4.43 (s, 2H, OCH2), 7.1–7.8 (bm, 12H, Ar-H), 9.6 (bs,
1H, CONH); MS (EI): m/z 380 (M+ + 1, 11); Anal. Calcd for C22H18ClNO3 (379): C,
69.56; H, 4.74; Cl, 9.35; N, 3.68. Found: C, 69.54; H, 4.76; Cl, 9.33; N, 3.66%.
1
7b: mp 135–37 °C; IR (Nujol): 1705 (CñO of amide), 1675cm¡1 (CñO); H
NMR (CDCl3): 2.3 (s, 3H, Ar-CH3), 4.45 (s, 2H, OCH2), 7.2–7.9 (bm, 12H, Ar-H), 9.7
(bs, 1H, CONH); MS (EI): m/z 425 (M+ + 1, 10); Anal. Calcd for C22H18BrNO3 (424):
C, 62.26; H, 4.24; Br, 18.86; N, 3.30. Found: C, 62.28; H, 4.22; Br, 18.89; N, 3.33%.
1
7c: mp 118–20 °C; IR (Nujol): 1705 (CñO of amide), 1675 cm¡1 (CñO); H
NMR (CDCl3): 2.2 (s, 3H, Ar-CH3), 4.41 (s, 2H, OCH2), 7.0–7.75 (bm, 12H, Ar-H),
9.5 (bs, 1H, CONH); MS (EI): m/z 376 (M+ + 1, 08); Anal. Calcd for C23H21NO4
(375): C, 73.60; H, 5.60; N, 3.73. Found: C, 73.63; H, 5.62; N, 3.71%.

S.A. Khanum et al. / Bioorganic Chemistry 32 (2004) 211–222
217
3. Biological evaluation of compounds
The animals were housed in groups of six and acclimatized to room conditions
for at least 2 days before the experiments, with food and water at libitum. The food
was withdrawn on the day before the experiment, but free access to water was
allowed. The compounds (100 mg/kg) and the reference NSAIDs, phenylbutazone
(100 mg/kg), indomethacin (10 mg/kg), and naproxen (30 mg/kg), were suspended in
0.5% carboxymethylcellulose (CMC) and administered orally by animal feeding
needle. The control groups received appropriate volumes of the vehicle (0.5% CMC,
oral) only.
3.1. Anti-inXammatory activity–carrageenan paw edema test (CPE)[12,13]
One hour after oral administration of the compounds, the thickness of right
hind paw was measured by a peacock dial thickness gauge and 0.01 mL 2% carra-
geenan was injected subcutaneously into the plantar surface of the right hind paw.
After 2 h, the volume of the edema was measured again and the anti-edematic
eVects of the drugs were estimated in terms of percent inhibition using following
equation:
Anti-inXammatory activity (%)D [(m ¡mЈ)/m] £ 100,
where m and mЈ indicate the diVerence in thickness between the Wrst and second mea-
surements of hind paws in control and test groups, respectively.
3.2. Air-pouch test [14–16]
The carrageenan powder was dissolved in saline to a concentration of 10 mg/
mL. The solution was sterilized and homogenized by placing it in an oven at 90 °C
for about 1 h. It was then maintained at 37 °C. Air pouches were formed by subcu-
taneous (sc) injection of 1 mL of air for 3 days. On the third day, after the initial
injection of air, carrageenan solution (1 mL) was injected into the air pouch to
induce inXammation. Compounds were administered orally 1 h before injection of
the carrageenan into the pouch. After 4 h, mice were killed by ether exposure and
pouches washed thoroughly with 3 mL of phosphate buVer solution (PBS) contain-
ing 50 ꢀ/mL heparin. Lavage Xuids were centrifuged at 2000 rpm for 15 min at 4 °C
and the pellet was resuspended in 1 mL of PBS-heparin. The total number of poly-
morphonuclear leukocytes (PMNL) inWltration was measured using a Coulter
Counter.
3.3. Histopathological examination
Mice were sacriWced 4 h after the paw edema experiments and their liver, stom-
achs, and kidneys were removed and put into 10% formalin solution. The sections
taken from these specimens were stained with hematoxilen eosine and examined
under the light microscope.

218
S.A. Khanum et al. / Bioorganic Chemistry 32 (2004) 211–222
4. Results and discussion
Compounds 2a–c were synthesized by a Fries rearrangement [17,18] of 1a–c
(Scheme 1). Condensation of 2a–c with ethyl chloroacetate in the presence of anhy-
drous potassium carbonate in dry acetone gave ethyl (2-aroylaryloxy)acetates [19,20]
3a–c. Alkaline hydrolysis of 3a–c gave 2-aroyl aryloxyacetic acid 4a-c. Condensation
of 4a–c with benzyl amine, 4-(2-diethylamino ethoxy) phenylamine or aniline in
presence of boron triXuoride etherate and dry benzene gave 2-(2-aroyl aryloxy) acet-
amide analogues [21] 5a–c, 6a–c, and 7a–c, respectively, in excellent yields.
The pharmacological results (Table 1) indicate that some of the compounds pos-
sess anti-inXammatory properties. In the CPE assay, compounds 2a, 2b, 4a, 4b, 6a, 7a,
and 7b showed anti-inXammatory activity. The most eVective compounds were 2a, 4a
and 7a, which have a chloro group at the meta position. Compounds 2b,4b, and 7b,
which have a bromo group at the ortho position, showed promising activity whereas
compounds 2c, 4c, and 7c showed only weak activity. The acetamide analogues 5a–c
also showed weak activity compared with phenylbutazone.
In addition to the synthesis and identiWcation of new anti-inXammatory com-
pounds, it is desirable to search for a new series of compounds with low ulceration
potential. Our results show that the newly synthesized compounds reduce leukocytes,
which suggests inhibition of PG synthesis. COX-1, is constitutively expressed in most
Table 1
Anti-inXammatory activity of benzophenone compounds
Compounds (100 mg/kg)
CPE (% inhibition SE)a
PMNL (105/cm3) (n D 6, after 4 h)b
Control
2a
2b
2c
4a
4b
4c
5a
5b
5c
6a
6b
121.50 (4.06)
30.15 (0.77)
32.41 (3.14)
69.42 (0.96)
31.75 (0.75)
56.26 (0.37)
n.t
95.25 (0.80)
104.76 (1.56)
n.t
69.50 (9.55c)
60.60 (8.54c)
35.51 (1.21d)
67.32 (9.26c)
51.42 (3.97c)
n.a
22.44 (3.08d)
15.45 (1.67d)
n.a
51.41 (3.98c)
45.18 (9.89c)
n.a
56.25 (0.38)
50.00 (1.32)
n.t
6c
7a
7b
7c
60.62 (8.55c)
51.41 (3.98c)
42.50 (3.82c)
67.50 (1.70c)
53.27 (1.83c)
32.10 (1.38c)
32.43 (3.15)
56.25 (0.38)
46.30 (0.68)
50.20 (1.30)
53.27 (1.71)
68.80 (1.80)
Naproxen (30 mg/kg)
Phenylbutazone (100 mg/kg)
Indomethacin (10 mg/kg)
PMNL, polymorphonuclear leucocytes; n.a.: no activity; n.t: not tested.
a Results are expressed as their mean values (n D 6).
b Mean SEM (n D 6).
c p 0 0.01.
d p 0 0.05.

S.A. Khanum et al. / Bioorganic Chemistry 32 (2004) 211–222
219
tissues and appears to be relevant for tissue homeostatic functions of PGs, whereas
COX-2 is an inducible isozyme and plays a role in many inXammatory reactions [5].
The enzymes are the primary targets of aspirin and other NSAIDs and thus are of
major interest in pharmacology, pharmacogenetics, and epidemiology. NSAIDs have
several potential pharmacologic eVects. However, their anti-inXammatory action
depends primarily on their ability to inhibit the COX enzymes [22]. This results in the
decreased production of pro-inXammatory PGs. Macrophages is known to produce
PGE2 via the COX-2 dependent pathway in response to pro-inXammatory cytokines.
Furthermore, rat peritoneal macrophages have been recently found to have the
capacity to metabolize exogenous arachidonic acid to thromboxane via COX-1 and
to PGE2 via COX-2 [23]. This observation suggested that there is a preferential,
phase-speciWc correlation between the two COX isoforms and the downstream
respective terminal PG synthases.
Previous work has shown that the inXammatory eVect of carrageenan is due to an
inXux of predominantly neutrophilic leukocytes (predominantly PMNL) from blood
circulation into the cavity. In addition, leukocyte migration is induced locally in the
inXammatory process and leukocytes also intensify inXammation by releasing several
inXammatory mediators. The method is used for measuring the edema induced by
injection of carrageenan into the pouches in mice back. Air pouches were highly
reactive to inXammatory stimulus. The enhanced inXammatory reactions in the sites
correlated with formation of lining tissue, the type of cells, and/or the reactivity
of newly formed blood cells [14,15,24,25]. Mean leukocyte numbers per milliliter of
exudates for each drug compared with control values obtained from similar group of
animals receiving vehicle alone and the degree of inXammatory response pro-
duced in the air-pouch cavity was assessed by measuring total cell number of the
exudates. Compounds 2a, 2b, 4a, 4b, 6a, 6b, and 7a–c reduced total number of leuko-
cytes of the exudates. The reduction of leukocytes suggests inhibition of PG produc-
tion. Compounds 2a, 2b, 4a, and 7a were inhibited PMNL production compared with
control and reference compounds [4- to 4.5-fold and 1- to 1.4-fold greater, respectively
(Table 1)].
The ulcerogenic potential of anti-inXammatory compounds can be demonstrated
in animal models using positive (naproxen, idomethacin) and negative (phenylbuta-
zone) controls. Upon microscopic examination, lesions seen in the stomach, kidney,
and liver tissues are graded according to their severity. The grade of the scale is
designed as (+) for mild (++) for moderate and (+++) for severe changes. Stomachs
of the mice are thoroughly sectioned and both corpus and antrum are evaluated. Sur-
face epithelium and the lamina propria of the gastric mucosa are all examined. Acute
gastritis may exist in an earlier or milder nonerosive form with merely mucosal con-
gestion, edema, and histological evidence of inXammation. These earlier changes are
known to be transient and completely reversible within few days, but the develop-
ment of erosions and hemorrhages is more serious and related to an increased risk of
major upper gastrointestinal bleeding. In our study, the animals were sacriWced 4h
after ingestion of the drugs. None of the sections displayed the morphology of the
ulceration, but the nonerosive form of acute gastritis was observed in various degrees
of severity with compounds 5a–c and 6b (Table 2). Upon microscopic examination of

220
S.A. Khanum et al. / Bioorganic Chemistry 32 (2004) 211–222
Table 2
Histopathological examination results benzophenone compounds
Compounds
(100 mg/kg)
p.o.
Kidney
Edema
Stomach
Gastritis
Liver
Infected cell
(MNL)
Fatty
change
Acute hepatitis/
spotty necrosis
Cholestasis
2a
2b
2c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
¡
¡
¡
¡
¡
¡
+
¡
++
¡
¡
++
+
¡
¡
¡
+
++
¡
++
+
¡
¡
++
¡
¡
¡
¡
¡
+
¡
¡
+
¡
¡
¡
¡
¡
¡
¡
¡
¡
++
¡
¡
¡
¡
¡
¡
¡
¡
++
¡
¡
+
+
¡
++
++
¡
+
¡
¡
+
+
¡
¡
¡
+
+
¡
++
¡
++
++
¡
++
¡
¡
¡
¡
+
¡
+
++
++
++
¡
¡
¡
¡
++
¡
¡
Indomethacin
(10 mg/kg)
Naproxen
(30 mg/kg)
+++
++
+
+
+
+
+++
¡
¡, no; +, mild; ++, moderate; +++, severe side eVects.
the kidney sections, the morphologic mononuclear cell inWltrations were characteristic
for tubulointerstitial nephritis. The presence of some scattered eosinophil leukocytes
also provided evidence for the drug eVect. Focal areas displaying variable but gener-
ally mild degree of tubular regeneration were also present. Acute pyelonephritis
should be considered in the diVerential diagnosis of the tubulointerstitial nephritis,
but none of our samples showed interstitial suppurative (inXammation with poly-
morphonuclear leukocytes) inXammation with microabscesses. Therefore, the renal
morphologic Wndings reXect the eVects of the compounds. Liver tissues were also
examined thoroughly and the integrity of the basic structure, degree of lobular and
portal inXammation, and the presence or absence of necrosis, fatty change or chole-
stasis were evaluated. NSAIDs such as phenylbutazone [26] and naproxen [27] are
known to cause acute or chronic hepatitis, conXuent or spotty necrosis, cholestatic
hepatitis and/or fatty change. This shows the importance of examining the liver tis-
sues to better assess the safety of NSAIDs. In compounds 2c, 4c, 5b, 5c, 6b, and 6c
(moderate, ++) showed macro and microvesicular fatty change and several scattered
mild (+) focal spotty necrosis. Multiple foci of spotty necrosis (moderate ++) was
seen with compounds 2c and 4b. Cholestatic hepatitis was observed for compounds
5c, 2c, and 6c (Table 1).
In conclusion, a new series of benzophenone analogues showing anti-inXamma-
tory activities was synthesized. Compounds with a chloro group at the meta position

S.A. Khanum et al. / Bioorganic Chemistry 32 (2004) 211–222
221
(i.e., 2a, 4a, and 7a) showed signiWcant anti-inXammatory proWle with low gastric
ulceration incidence as compared with similar toxic proWles for reference NSAIDs in
the liver.
Acknowledgments
The authors express their sincere gratitude to the University of Mysore, Mysore
for providing laboratory facilities. One of the authors (SAK) is indebted to the Uni-
versity Grant Commission for the award of a teacher’s fellowship. We are also thank-
ful to the Principal of Farooqia College of Pharmacy, Mysore, for providing
laboratory facilities to carry out the pharmacological studies.
References
[1] W.L. Smith, L.J. Marnett, Biochim. Biophys. Acta 1083 (1991) 1–17.
[2] J.R. Vane, Y.S. Bakhle, R.M. Annu, Rev. Pharmacol. Toxicol. 38 (1998) 97–120.
[3] W.L. Smith, D.L. DeWitt, Adv. Immunol. 62 (1996) 167–215.
[4] W.L. Smith, R.M. Garavito, D.L. DeWitt, J. Biol. Chem. 271 (1996) 33157–33160.
[5] M.M. Taketo, J. Natl. Cancer Inst. 90 (1998) 1529–1536.
[6] I.M. Morita, M.K. Schindler, J.C. Regier, T. Otto, D.L. Hori, D.L. DeWitt, W.L. Smith, J. Biol. Chem.
270 (1995) 10902–10908.
[7] (a) A. Palomer, J. Pascual, M. Cabre, L. Borras, G. Gonzalez, M. Aparici, A. Carabaza, F. Cabre, M.L.
Garcia, D. Mauleon, Bioorg. Med. Chem. Lett. 12 (2002) 533–537;
(b) A. Palomer, J.J. Perez, S. Navea, O. Llorens, J. Pascual, M.L. Garcia, D.M. Mauleon, J. Med.
Chem. 43 (2000) 2280–2284.
[8] E.R. Ottosen, M.D. Sorensen, F. Bjorkling, T. Skak-Nielsen, M.S. Fjording, H. Aaes, L. Binderup, J.
Med. Chem. 46 (2003) 5651–5662.
[9] W.J. Welstead, H.W. Moran, H.F. StauVer, L.B. Turnbull, L.F. Sancilio, J. Med. Chem. 46 (1979)
1074–1079.
[10] G. Branacaccio, A. Larizza, G. Lettieri, J. Med. Chem. 24 (1981) 998–1000.
[11] M.G. Vigorita, T. Previtera, A. Trovato, M.T. Monforte, R. Barbera, G. Bisignano, Farmaco 44 (1989)
173–184.
[12] T. Komeshima, T. Osowa, T. Nishitoba, Y. Jinno, T. Kiriu, Chem. Pharm. Bull. 40 (1992) 351–356.
[13] C.A. Winter, E.A. Risley, G.W. Nuss, Proc. Soc. Exp. Biol. Med. 111 (1962) 544–547.
[14] A.D. Sedgwick, A. R Moore, A.R. Al-Duaij, J.C.W. Edwards, D.A. Willoughby, J. Exp. Pathol. 66
(1985) 445–453.
[15] Y.M. Sin, M.K. Wong, Ann. Rheum. Dis. 51 (1992) 112–116.
[16] M. Perretti, J.G. Harris, R.J. Flower, J. Pharmacol. 112 (1994) 801–808.
[17] R. Martin, Org. Prep. Proced. Int. 24 (1992) 373–435.
[18] I. Yoichiro, M. Tomohide, O. Nariyuki, Y. Jiro, Chem. Abstr. 134 (2000) 147375.
[19] M. Hird, W.J. Goodby, N. Gough, K.J. Toyne, J. Mater. Chem. 11 (2001) 2732–2742.
[20] S.A. Khanum, S. Shashikanth, B.S. Sudha, Heteroatom Chem. 15 (2004) 37–42.
[21] J. Tani, T. Oine, I. Inoue, Synthesis (1975) 714–715.
[22] J.R. Vane, Nat. New Biol. 231 (1971) 232–235.
[23] H.H. Matsumoto, M. Naraha, I. Murakami, K. Kudo, A. Yamaki, U.S. Oh-ishi, Biochem. Biophys.
Res. Commun. 230 (1997) 110–115.
[24] J.C.W. Edwards, A.D. Sedgwick, D.A. Willoughby, J. Pathol. Pharmacol. 134 (1981) 147–153.
[25] A.D. Sedgwick, Y.M. Sin, A.R. Mackay, A. Al-Duaij, D.A. Willoughby, J. Pharm. Pharmacol. 36
(1984) 171–174.

222
S.A. Khanum et al. / Bioorganic Chemistry 32 (2004) 211–222
[26] M.D. Mullican, M.W. Wilson, D.T. Connor, C.R. Kostlan, D.J. Schrier, R.D. Dyer, J. Med. Chem. 36
(1993) 1090–1099.
[27] D.H. Boschelli, D.T. Connor, D.A. Bornemeier, R.D. Dyer, J.A. Kennedy, P.J. Kuipers, G.C.
Okonkwo, D.J. Schrier, C.D. Wright, J. Med. Chem. 36 (1993) 1802–1810.