Synthesis and evaluation of benzophenone oximes derivatized with sydnone as inhibitors of secretory phospholipase A2 with anti-inflammatory activity

Article abstract of DOI:10.1248/cpb.57.16

A series of benzophenone oximes appended with sydnone (3a-h) bearing different substituents on aroyl moiety were synthesized to evaluate in vivo and in vitro for their inhibitory activity against purified phospholipase A ₂ (PLA₂) enz

Full text of DOI:10.1248/cpb.57.16

16
Chem. Pharm. Bull. 57(1) 16—21 (2009)
Vol. 57, No. 1
Synthesis and Evaluation of Benzophenone Oximes Derivatized with
Sydnone as Inhibitors of Secretory Phospholipase A2 with
Anti-inflammatory Activity
Ravindra Ramappa KAMBLE, ,a Sudha Sathyanarayanrao BELGUR,b Ravindranath ALADKATTI,c and
*
a
Imthiyaz Ahmed KHAZI
a Post-Graduate Department of Studies in Chemistry, Karnatak University; Dharwad–580 003, India: b Yuvaraja’s College,
c
University of Mysore; Mysore–570 005, India: and Central Animal Facility, Indian Institute of Science; Bangalore–560
012, India. Received July 9, 2008; accepted October 18, 2008; published online October 28, 2008
A series of benzophenone oximes appended with sydnone (3a—h) bearing different substituents on aroyl
moiety were synthesized to evaluate in vivo and in vitro for their inhibitory activity against purified phospholi-
pase A2 (PLA2) enzymes from snake venom and human inflammatory pleural and ascites fluid. In vivo and in
vitro inhibition studies were carried out against PLA2 with respect to the modification of the pharmacophore
(substituent) to analyze the specificity for PLA2. The substituent at the aroyl ring was responsible for enhancing
the inhibition towards PLA2 enzymes. Most of the newly synthesized compounds inhibit the purified PLA2 en-
zyme, and the inhibition was more in hydrophobic and aromatic substituents and less when no such substituents
were present. The inhibitory effect of the compounds appeared to be due to the direct interaction of compounds
with the enzyme. Inhibition is substrate dependent, and the inhibition competes with the substrate for the same
binding site of the enzyme. The most active interacting compound 3h from in vitro inhibition of PLA2 activity
showed similar potency in the in vivo neutralization of PLA2 induced mouse paw edema and hemolytic activity.
Thus, the in vitro inhibition correlated well with the in vivo inhibition and hence the reported derivatives are
therapeutically important anti-inflammatory drugs.
Key words benzophenone oxime; nonsteroidal anti-inflammatory drug; phospholipase A2; sydnone; synthesis
Phospholipases A2 (PLA2) are a family of key enzymes in tory drugs (NSAIDs) to treat the inflammation is still in
the metabolism of phospholipids possessing various substrate progress. A literature survey revealed the high potency of
specificities, co-factor requirements, sub cellular localization anti-inflammatory and other pharmacological activity by
and cellular functions. All of these enzymes catalyze hydrol- benzophenone oxime analogues. The potent anti-inflamma-
ysis of the 2-acylester of 3-sn phosphoglycerides to yield tory activity of these compounds has been shown to be due to
arachidonic acid (AA) and lysophospholipid, which is a rate the inhibition of cycloxygenase enzyme. Inhibition of cy-
limiting step in the production of pro-inflammatory lipid me- cloxygenase and lipoxygenase also indicated the inhibition of
diators such as prostaglandins, leukotrienes, lipoxins and PLA2 enzyme and hence resulted in the reduction of lipid
platelet activating factor (PAF). The AA is metabolized fur- mediators. Hence, Such PLA2 inhibitors can be used as anti-
ther to eicosanoids by cycloxygenase and lipoxygenase. inflammatory drugs. Benzophenone oximes have structural
These Lipid mediators in turn exert a wide range of potent resemblance with fenoprofen, which belongs to the class of
physiological effects, with excess production linked to dis- aryl acetic acids. Introduction of hydrophobic alkyl groups
eases such as inflammation, allergy, brain injury, cancer de- on the aryloxy moiety of benzophenone oxime assists in
velopment and metastasis, and cardiovascular disorders. binding with PLA2 enzyme resulting in the increase of anti-
More than ten different forms of the PLA2 enzymes have inflammatory activity.12,13) A series of derivatives of 3-(2-
been identified.1) Group I PLA2 is present in pancreatic juice phenylthio)-ethylsydnones have been synthesized which are
and old world snakes, where as II PLA2 are found in mam- more potent than hydrocortisone and phenylbutazone to-
mals and new world snakes.2—5) Increased levels of group II wards adjuvant arthritis.14) 3-[4-[3-(Substituted aryl)-1-oxo-
are found in rheumatoid synovial fluid6) and in plasma after 2-propenyl]-phenyl]-sydnones were condensed with hy-
an inflammatory challenge.7) Injection of purified PLA2 en- drazine to yield 3-[4-[4,5-dihydro-5-(substituted aryl)-1H-
zyme into the animal joints developed an acute inflammatory pyrazol-3-yl]-phenyl]-sydnones and have shown anti-inflam-
response viz., edema, swelling of synovial cells and hyperpla- matory activity in carrageenan induced edema assay in
sia.8) Thus, several findings point to the importance of PLA2 rats.15) Upadhya et al. have reported 3-substituted 4-(4ꢀ-thia-
in inflammatory reactions. Inhibition of such a PLA2 enzyme zolyl)-sydnones which have potent anti-inflammatory proper-
by endogenous inhibitors, xenobiotics and synthetic com- ties.16) It has been revealed that coupling of two or more bio-
pounds is of potential therapeutic relevance in many inflam- dynamic molecules resulted in the enhanced biological activ-
matory diseased states. The existence of different types of ity. Hence, in this study we report the synthesis of eight dif-
PLA2 drew attention to the importance of finding the selec- ferent derivatives of benzophenone oximes appended to syd-
tive and specific inhibitors for the group PLA2 enzyme.
nones along with variable substituents viz., methyl, n-propyl
Extensive research in preliminary studies has been com- and n-butyl groups at para position of aryl moiety and a
mitted to finding novel compounds for the treatment of in- phenyl ring at 4-position of sydnone ring leading to in-
flammation, haemostasis, and bacterial diseases in our labo- creased hydrophobicity and aromaticity respectively. A com-
ratory.9—11) The search for potent nonsteroidal anti-inflamma- parison of PLA2 inhibitory, neutralization of edema inducing
∗ To whom correspondence should be addressed. e-mail: kamchem9@gmail.com
© 2009 Pharmaceutical Society of Japan

January 2009
17
1H-NMR (300 MHz, CDCl3) d: 3.77 (1H, s, CH), 7.10—7.35 (14H, bm, Ar-
H), 9.80 (s, 1H, OH).
and neutralization of indirect hemolytic activity of these de-
rivatives with purified PLA2 from Vipera russelli, Naja naja,
Trimeresurus malabaricus, synovial and ascites fluid was
performed. This study revealed additional information on the
extensive structure–activity relationship (SAR) for hydrocar-
bon chain length and unsaturated aromatic ring on aroyl and
sydnone rings respectively.
N-Nitroso-[4-{(4ꢀ-methyl)-benzoyl)}-phenyl]-phenyl-amino Acetic Acid
2f Compound 2f was obtained as pale yellow solid in 87% yield, mp
133—135 °C; IR (film): 3492 (carboxylic OH), 1742 (carboxylic CꢁO),
1
1609 (CꢁO) cmꢂ1; H-NMR (300 MHz, CDCl3) d: 1.95 (3H, s, CH3), 3.75
(1H, s, CH), 6.95 (2H, d, Ar-H, Jꢁ7.15 Hz), 7.05 (2H, d, Ar-H, Jꢁ7.15 Hz),
7.10 (2H, d, Ar-H, Jꢁ7.0 Hz), 7.19 (2H, d, Ar-H, Jꢁ7.0 Hz), 7.26—7.7 (5H,
m, Ar-H), 9.25 (1H, s, OH).
Experimental
N-Nitroso-[4-{(4ꢀ-n-propyl)-benzoyl)}-phenyl]-phenyl-amino Acetic
Acid 2g Compound 2g was obtained as white solid in 72% yield, mp
165—167 °C; IR (film): 3457 (carboxylic OH), 1750 (carboxylic CꢁO),
Materials The starting compounds 4-aroyl anilines (1a—d) were pre-
pared according to the reported method.17) All the reagents were purchased
from the Merck Chemicals (Merck India) and used without further purifica-
tions.
Methods Melting points were determined using a Thomas–Hoover cap-
illary melting point apparatus and are uncorrected. Infrared (IR) spectra
were as films with KBr plates on JASCO (Japan) FTIR-460 plus spectrome-
ter. Nuclear Magnetic Resonance (1H-NMR) spectra were recorded on a
Bruker Varian–300 MHz FT-NMR spectrometer. J values are given in Hz.
Male Albino mice weighing 20—25 g were obtained from the Central Ani-
mal Facility, Indian Institute of Science, Bangalore.
N-Nitroso-(4-benzoyl-phenyl)-amino Acetic Acid 2a: General Proce-
dure 4-Amino benzophenone (1.97 g, 10 mmol) 1a, was mixed with neu-
tralized monochloro acetic acid (0.94 g, 10 mmol) and refluxed on the sand
bath for 5 h. The solid obtained was cooled, filtered dried, and subjected to
nitrosation at 0—5 °C using sodium nitrite and hydrochloric acid. After
completion of the reaction, the formed pale yellow solid nitroso derivative
was washed thoroughly with water until the washings were neutral. Purifica-
tion of this solid by recrystallization using absolute ethanol gave 2a (2.27 g,
80%) as pale yellow needles; mp 128—130 °C; IR (film): 3471 (carboxylic
OH), 1776 (carboxylic CꢁO), 1642 (CꢁO) cmꢂ1; 1H-NMR (CDCl3) d: 3.75
(2H, s, CH2), 7.10—7.28 (5H, m, Ar-H), 7.35 (2H, d, Ar-H, Jꢁ6.85 Hz),
7.54 (2H, d, Ar-H, Jꢁ6.85 Hz), 9.2 (1H, s, OH).
1
1614 (CꢁO) cmꢂ1; H-NMR (300 MHz, CDCl3) d: 1.04 (3H, t, CH3), 1.54
(2H, m, CH2), 2.44 (2H, t, CH2), 3.80 (1H, s, CH), 6.74 (2H, d, Ar-H,
Jꢁ5.8 Hz), 6.97 (2H, d, Ar-H, Jꢁ5.8 Hz), 7.06 (2H, d, Ar-H, Jꢁ7.0 Hz),
7.12 (2H, d, Ar-H, Jꢁ7.0 Hz), 7.20—7.54 (5H, m, Ar-H), 9.2 (1H, s, OH).
N-Nitroso-[4-{(4ꢀ-n-butyl)-benzoyl)}-phenyl]-phenyl-amino Acetic
Acid 2h Compound 2h was obtained as pale yellow solid in 65% yield, mp
173—175 °C; IR (film): 3400 (carboxylic OH), 1762 (carboxylic CꢁO),
1
1620 (CꢁO) cmꢂ1; H-NMR (300 MHz, CDCl3) d: 1.00 (3H, t, CH3), 1.33
(2H, m, CH2), 1.57 (2H, m, CH2), 2.04 (2H, t, CH2), 3.72 (1H, s, CH), 6.72
(2H, d, Ar-H, Jꢁ6.0 Hz), 6.83 (2H, d, Ar-H, Jꢁ6.0 Hz), 6.95 (2H, d, Ar-H,
Jꢁ6.8 Hz), 7.08 (2H, d, Ar-H, Jꢁ6.8 Hz), 7.14—7.49 (5H, m, Ar-H), 8.95
(1H, s, OH).
3-[4ꢀ-(Hydroxyimino-phenyl-methyl)-phenyl]-sydnone 3a N-Nitroso-
(4-benzoyl-phenyl)-amino acetic acid 2a (2.84 g, 10 mmol) was added to
acetic anhydride (5.0 ml), and the mixture was refluxed for 3 h. After cooling
to 25 °C, the solution was poured into water. The solid obtained was filtered,
dried and further refluxed with hydroxyl amine hydrochloride (0.69 g,
10 mmol) for 2 h in 20 ml of absolute ethanol. The reaction mixture was con-
centrated and the solid obtained was filtered, dried and recrystallized from
ethanol to get colorless needles of 3a in 90% yield; mp 184—186 °C; IR
(film): 3400 (OH), 3100 (sydnone C4–H), 1732 (sydnone CꢁO), 1600
1
(CꢁN) cmꢂ1; H-NMR (300 MHz, CDCl3) d: 6.70 (1H, s, sydnone C–H),
Compounds 2b—h were prepared using a similar sequence of reactions
using different substituents on the aroyl moiety of 4-aminobenzophenone.
For the preparation of compounds 2e—h ethyl-a-chloro-a-phenyl acetate
(1.98 g, 10 mmol) (followed by acid hydrolysis) was used in stead of mono-
chloroacetic acid. The physical and spectral data for 2b—h are listed below.
N-Nitroso-[4-{(4ꢀ-methyl)-benzoyl}-phenyl]-amino Acetic Acid 2b
Compound 2b was obtained as pale yellow solid in 82% yield; mp 125—
126 °C; IR (film): 3450 (carboxylic OH), 1763 (carboxylic CO), 1635
6.75 (2H, d, Ar-H, Jꢁ7.35 Hz), 6.91 (2H, d, Ar-H, Jꢁ6.85 Hz), 7.14 (2H, d,
Ar-H, Jꢁ6.2 Hz), 7.26 (2H, Jꢁ6.2 Hz, d, Ar-H), 8.85 (1H, s, OH).
Compounds 3b—h were prepared using similar cyclization reaction using
acetic anhydride followed by ammoximation using hydroxylamine hy-
drochloride. The stereochemistry of the oximes prepared was not estab-
lished. The physical and spectral data for the same are listed below.
3-[4ꢀ-(Hydroxyimino-p-tolyl-methyl)-phenyl]-sydnone 3b Compound
3b was obtained as pale yellow solid in 61% yield, mp 146—145 °C; IR
(film): 3403 (OH), 3110 (sydnone C4–H), 1728 (sydnone CꢁO), 1613
(CꢁO) cmꢂ1 1H-NMR (CDCl3) d: 2.70 (3H, s, CH3), 3.70 (2H, s, CH2),
;
6.85 (2H, Jꢁ6.5 Hz, d, Ar-H), 7.01 (2H, Jꢁ7.0 Hz, d, Ar-H), 7.10 (2H,
Jꢁ6.0 Hz, d, Ar-H), 7.30 (2H, d, Ar-H, Jꢁ6.0 Hz), 9.0 (1H, s, OH).
1
(CꢁN) cmꢂ1; H-NMR (300 MHz, CDCl3) d: 2.17 (3H, s, CH3), 6.65 (1H,
N-Nitroso-[4-{(4ꢀ-propyl)-benzoyl}-phenyl]-amino Acetic Acid 2c
Compound 2c was obtained as white solid in 65% yield; mp 187—189 °C;
IR (film): 3501 (carboxylic OH), 1751 (carboxylic CꢁO), 1641 (CꢁO)
s, sydnone C–H), 6.90 (2H, d, Jꢁ6.0 Hz, Ar-H), 7.0 (2H, d, Ar-H,
Jꢁ6.0 Hz), 7.15 (2H, d, Ar-H, Jꢁ5.4 Hz), 7.22 (2H, d, Ar-H, Jꢁ5.4 Hz),
8.64 (1H, s, OH).
1
cmꢂ1; H-NMR (300 MHz, CDCl3) d: 1.0 (3H, t, CH3), 1.57 (2H, m, CH2),
3-[4-{Hydroxyimino-(4ꢁ-n-propyl-phenyl)-methyl}-phenyl]-sydnone 3c
Compound 3c was obtained as pale yellow solid in 56% yield, mp 164—
166 °C; IR (film): 3397 (OH), 3106 (sydnone C4–H), 1745 (sydnone CꢁO),
2.40 (2H, t, CH2), 3.78 (s, 2H, CH2), 6.67 (d, Jꢁ5.8 Hz, Ar-H), 6.91 (2H, d,
Ar-H, Jꢁ5.8 Hz), 7.22 (2H, d, Ar-H, Jꢁ6.0 Hz), 7.35 (2H, d, Ar-H,
Jꢁ6.0 Hz), 9.0 (s, 1H, OH).
1
1612 (CꢁN) cmꢂ1; H-NMR (300 MHz, CDCl3) d: 1.00 (3H, t, CH3), 1.39
N-Nitroso-[4-{(4ꢀ-n-butyl)-benzoyl}-phenyl]-amino Acetic Acid 2d
Compound 2d was obtained as pale yellow solid in 60% yield; mp 122—
124 °C; IR (film): 3466 (carboxylic OH), 1760 (carboxylic CꢁO), 1625
(2H, m, CH2), 2.50 (2H, t, CH2), 6.72 (1H, s, sydnone C–H), 7.05 (2H, d,
Ar-H, Jꢁ5.0 Hz), 7.13 (2H, d, Ar-H, Jꢁ5.0 Hz), 7.20 (2H, d, Ar-H,
Jꢁ6.2 Hz), 7.26 (2H, d, Ar-H, Jꢁ6.2 Hz), 8.85 (1H, s, OH).
(CꢁO) cmꢂ1 1H-NMR (300 MHz, CDCl3) d: 0.92 (t, 3H, CH3), 1.34 (m,
;
3-[4-{Hydroxyimino-(4ꢁ-n-butyl-phenyl)-methyl}-phenyl]-sydnone 3d
Compound 3d was obtained as yellow solid in 50% yield, mp 112—114 °C;
IR (film): 3421 (OH), 3096 (sydnone C4–H), 1744 (sydnone CꢁO), 1607
2H, CH2), 1.62 (m, 2H, CH2), 2.55 (t, 2H, CH2), 3.70 (s, 2H, CH2), 6.60
(2H, d, Ar-H, Jꢁ6.2 Hz), 6.84 (2H, d, Ar-H, Jꢁ6.2 Hz), 7.05 (2H, d, Ar-H,
Jꢁ5.4 Hz), 7.14 (2H, d, Ar-H, Jꢁ5.4 Hz), 9.2 (s, 1H, OH).
1
(CꢁN) cmꢂ1; H-NMR (300 MHz, CDCl3) d: 1.04 (3H, t, CH3), 1.42 (2H,
N-Nitroso-[4-(benzoyl)-phenyl]-phenyl-amino Acetic Acid 2e Com-
pound 2e was obtained as white solid in 80% yield; mp 127—130 °C; IR
m, CH2), 1.50 (2H, m, CH2), 2.10 (2H, t, CH2), 6.80 (1H, s, sydnone C–H),
6.98 (2H, d, Ar-H, Jꢁ6.4 Hz), 7.09 (2H, Jꢁ6.4 Hz, d, Ar-H), 7.14 (2H, d,
Ar-H, Jꢁ6.0 Hz), 7.20 (2H, d, Ar-H, Jꢁ6.0 Hz), 8.9 (1H, s, OH).
(film): 3480 (carboxylic OH) 1739 (carboxylic CꢁO), 1615 (CꢁO) cmꢂ1
;
Reagents and conditions: i) ClCHR1COOR2 (4i—j)/heat, ii) NaNO2/HCl, 0—-5 °C, iii) Ac2O/reflux, iv) NH2OH·HCl/reflux.
Chart 1

18
Vol. 57, No. 1
3-[4ꢀ-(Hydroxyimino-phenyl-methyl)-phenyl]-4-phenyl-sydnone 3e
in absolute alcohol to get the final product (3a—h). The
structures of nitroso derivative (2a—h) and the final products
(3a—h) were confirmed by their IR, NMR data which are in
complete agreement.
Compound 3e was obtained as yellow solid in 66% yield, mp 203—205 °C;
1
IR (film): 3401 (OH), 1722 (sydnone CꢁO), 1610 (CꢁN) cmꢂ1; H-NMR
(300 MHz, CDCl3) d: 6.89 (2H, d, Ar-H, Jꢁ6.0 Hz), 7.09 (2H, d, Ar-H,
Jꢁ6.0 Hz), 7.14—7.40 (10H, m, Ar-H), 8.8 (1H, s, OH).
Derivatives were of two series based on the substitution in
the 4th position of sydnone ring. Derivatives with –H in the
4th position of the sydnone 3a—d along with the hydropho-
bic groups on para position of the benzoyl moiety and those
with phenyl ring at 4th position of and hydrophobic sub-
3-[4ꢀ-(Hydroxyimino-p-tolyl-methyl)-phenyl]-4-phenyl-sydnone 3f
Compound 3f was obtained as yellow solid in 58% yield, mp 147—149 °C;
1
IR (film): 3421 (OH), 1701 (sydnone CꢁO), 1608 (CꢁN) cmꢂ1; H-NMR
(300 MHz, CDCl3) d: 2.00 (3H, s, CH3), 6.95 (2H, d, Ar-H, Jꢁ5.3 Hz), 7.02
(2H, d, Ar-H, Jꢁ5.3 Hz), 7.09—7.36 (9H, m, Ar-H), 9.0 (1H, s, OH).
3-[4-{Hydroxyimino-(4ꢁ-n-propyl-phenyl)-methyl}-phenyl]-4-phenyl-
sydnone 3g Compound 3g was obtained as yellow solid in 62% yield, mp stituents on the para position of benzoyl moiety 3e—h. In
221—223 °C; IR (film): 3412 (OH), 1706 (sydnone CꢁO), 1597 (CꢁN)
both series, 3a—d have only the alkyl substituents which are
hydrophobic in nature. Whereas, the compounds 3e—h have
both hydrophobic alkyl groups and phenyl group which adds
up the aromatic character.
cmꢂ1; 1H-NMR (300 MHz, CDCl3) d: 1.14 (3H, t, CH3), 1.40 (2H, m, CH2),
2.42 (2H, t, CH2), 6.87 (2H, d, Ar-H, Jꢁ6.8 Hz), 6.93 (2H, d, Ar-H,
Jꢁ6.8 Hz), 7.00—7.30 (9H, m, Ar-H), 8.67 (1H, s, OH).
3-[4-{Hydroxyimino-(4ꢁ-n-butyl-phenyl)-methyl}-phenyl]-4-phenyl-
sydnone 3h Compound 3h was obtained as yellow solid in 57% yield, mp
160—162 °C; IR (film): 3400 (OH), 1713 (sydnone CꢁO), 1611 (CꢁN)
cmꢂ1; 1H-NMR (300 MHz, CDCl3) d: 0.97 (3H, t, CH3), 1.35 (2H, m, CH2),
1.58 (2H, m, CH2), 2.44 (2H, t, CH2), 6.94 (2H, d, Ar-H, Jꢁ5.6 Hz), 7.0
(2H, d, Ar-H, Jꢁ5.6 Hz), 7.14—7.35 (9H, m, Ar-H), 8.95 (1H, s, OH).
Determination of Edema Inducing Activity All protocols of animal
experiments have been approved by the Institutional Animal Ethics Commit-
tee (IAEC). Groups of six mice (22—24 g) were injected in the right foot-
pads of hind limbs with 3 mM dose of benzophenone analogues in 20 ml
saline. The left footpads received 20 ml of saline, which served as control.
After 45 min mice were sacrificed by cervical dislocation and both legs were
removed at the ankle joint and weighed individually. The increase in weight
due to edema was calculated as the edema ratio, which equals the weight of
edematous legꢃ100/weight of the normal leg. Minimum edema dose is de-
fined as the microgram of protein causing an edema ratio of 120%. Injecting
a fixed dose protein into mice footpads and sacrificing them at regular period
of time obtained time course curve of edema inducing activity. Edema ratio
was calculated as defined.12)
The parent compound benzophenone is known to have
anti-inflammatory properties. The compounds 3a—h were
tested for anti-inflammatory properties by studying their in-
teraction with different PLA2 enzymes. Inflammatory PLA2
from V. russelli, N. naja, T. malabaricus, synovial and ascites
fluid were used to study interaction with benzophenones de-
rivatized with sydnone ring. Figure 1 shows the interaction of
all 8 benzophenone oxime analogues (75 mM concentration)
with different types of enzymes. All of them inhibited PLA2
enzyme activity. These derivatives especially 3e—h have
inhibited all the types of PLA2 enzymes. The compounds
3g—h have shown the inhibition activity almost equal to the
standard Luffariellin B due to the presence of hydrophobic
group on the aroyl moiety and the aromatic phenyl ring at the
4th position of the sydnone moiety. The compounds 3b—d
have shown moderate activity due to the presence of the only
the presence of hydrophobic alkyl groups. The compound 3a
has shown very weak activity due to the absence of both type
of groups viz., hydrophobic and aromatic. The similar trend
is observed also in case of the edema inducing activity.
Determination of PLA2 Activity Assayed with [14C] oleate-labelled au-
toclaved Escherichia coli as the substrate.19) The reaction mixture, 350 ml
contained 100 mM Tris/HCl, pH 8.0, 5 mM Ca2ꢄ and 3.15ꢃ109 autoclaved E.
coli cells (corresponding to 10000 cpm and 60 nmol of lipid phosphorous).
The amount of enzyme protein was chosen such that 10—15% hydrolysis of
substrate was obtained when incubated at 37 °C for 60 min. The reaction
components were mixed in the following order: buffer, calcium, water and
benzophenone analogue. Adding labelled E. coli substrate started the reac-
tion. The reaction was terminated by adding 100 ml 2.0 M HCl and 100 ml of
fatty acid free BSA (100 mg/ml). The tubes were vortex mixed and cen-
trifuged at 20000 mg for 5 min. Aliquot (140 ml) of the supernatant contain-
ing released [14C] oleic acid was mixed with scintillation cocktail and
counted in a Hewlett Packard liquid Scintillation Analyzer TRICARB 2100
TR.
Discussion
Understanding the molecular interaction between the
PLA2 enzyme and agents inhibiting its activity is crucial in
designing the powerful inhibitors. Several endogenous and
exogenous agents such as lipocortin,19) cis-unsaturated fatty
acids,20) gangliosides,21) manoalide,22) cacosponginolide, pet-
rosasponginolide,23) surfactin,24) retinoids,10,25) flavonoids,26)
aristolochic acid,27) natural plant lipids,28) and synthetic
lipids,29) have been shown to inhibit the PLA2 enzyme. From
studies with the different inhibitors, it is generally under-
stood that most PLA2 inhibitors are hydrophobic compounds
containing long hydrocarbon chain with one to several aro-
Neutralization of Indirect Hemolytic Activity Indirect hemolytic ac-
tivity was assayed using the method of Boman and Kaletta.18) Briefly, packed
human erythrocyte egg yolk and phosphate-buffered saline (1 : 1 : 8, v/v)
were mixed. This suspension (1 ml) was incubated with the enzyme (20 mg)
for 10 min at 37 °C. After adding 9 ml of ice-cold phosphate-buffered saline,
the reaction mixture was centrifuged at 4 °C for 10 min at 2000 rpm. The
amount of hemoglobin released in the supernatant due to hemolysis was
measured at 540 nm. The hydrolysis of erythrocyte caused by the addition of
9 ml distilled water taken as 100%. Values are presented as the mean of 4 in- matic rings.19—30) Molecules with a more hydrophobic nature
dependent determinations.
have a better interaction with the PLA2 enzyme and greater
inhibitory activity. The parent compounds benzophenone de-
Results
rivatives have been shown to have anti-inflammatory proper-
The synthetic route of compounds 3a—h is as depicted in ties by selective inhibition of cycloxygenase-2 (COX-2) en-
Chart 1. The starting compounds 4-aroyl aniline (1a—c) was zyme.31,32) The clinical results with COX and lipoxygenase
refluxed with 4i—j in alkaline medium to get the correspon- inhibitors demonstrated that inhibition of the PLA2 enzyme
ding esters of substituted amino acids. The esters of amino results in a reduction of both lipid mediators. As a result,
acids were hydrolyzed with 10% NaOH to get substituted these PLA2 inhibitors can be used as anti-inflammatory drugs
amino acid which was nitrosated at 0—5 °C using NaNO2 in that are as powerful as anti-inflammatory steroidal com-
25% HCl to give the N-nitroso derivative (2a—h). The ni- pounds. Some derivatives of benzophenone oximes exhibited
troso derivative (2a—h) was cyclized to get the sydnone de- potent anti-inflammatory activity but failed to exhibit COX-
rivative on refluxing with acetic anhydride. This was fol- 2, thus suggesting the action by a different mechanism. This
lowed by ammoximation using hydroxylamine hydrochloride observation led to the inference that these compounds might

January 2009
19
Fig. 1. In Vitro Inhibition of PLA2 Enzyme Activity Data for 3-[4-{Hydroxyimino-(aryl)-methyl}-phenyl]-4-(un)-substituted-sydnones 3a—h
Inhibition of PLA2 enzymes by compounds at 75 mM concentration (LB is Luffariellin B, standard). Each compound is preincubated with enzyme for 30 min. The 350 ml reaction
mixture contained 30 ml of substrate (added after preincubation), 35 ml of 1 M Tris–HCl buffer pH 7.5, 5 mM CaCl2 and distilled water, and was incubated for 1 h at 37 °C. Data rep-
resent meanꢆS.E.M. for nꢁ4.
interact with the PLA2 enzyme and exhibit stronger anti-in-
flammatory activity. In this study, several benzophenone de-
rivatives were synthesized with increased hydrophobicity at
the aroyl moiety and 4th position of sydnone ring of the par-
ent compound 3a, and their effect on in vitro PLA2 inhibition
and edema inhibiting activity in a mouse model was exam-
ined. The variable extent to which different PLA2 enzymes
are inhibited may be due to differential binding affinities.
These differential affinities with benzophenones derivatized
with sydnone varied as the hydrophobicity and aromaticity
were increased in the ring (Fig. 1). The percentage inhibition
was increased from 3a to 3h as the chain length of alkyl
group was increased. The inhibition was minimum when no
alkyl substituent was present (e.g., the inhibition of 3a
against V. russeli was 55.6% and that of 3h with n-butyl sub-
stituent was 79.3%).
The inhibition is dose dependent and increases with the in-
crease in hydrophobicity. The increased inhibition is some-
what linear with increased hydrophobicity in the aliphatic
chain length of methyl, n-propyl and n-butyl groups. When
Fig. 2. Dose Dependent Inhibition of PLA2 Enzymes by 3-[4ꢀ-(Hydroxy-
the aromatic phenyl ring is introduced along with the
aliphatic side chains, the inhibition was much stronger and
further enhanced indicating a preference by the enzyme for
hydrophobic molecules along with unsaturated aromatic
rings as better inhibitors (Fig. 2). Similar hydrophobic com-
pounds, which are inhibitors of PLA2, are chemically modi-
imino-phenyl-methyl)-phenyl]-sydnone 3a and 3-[4-{Hydroxyimino-(4ꢅ-n-
butyl-phenyl)-methyl}-phenyl]-4-phenyl-sydnone 3h
The 350 ml reaction mixture containing 2 mg of V. russelli in 100 mM Tris–HCl buffer
pH 7.5, 5 mM CaCl2 and indicated concentrations of 3a and 3h was preincubated for
30 min at 37 °C. The PLA2 activity was initiated by adding substrate (60 nmol E. coli
phospholipid), then continued incubation at 37 °C for 1 h.
fied tetracyclins,33) derivatives of isoxazolines,11,13) indole inflammatory fluids into animal joints resulted in acute in-
analogues,12) and flavonoids,26) all exhibiting stronger PLA2 flammatory response with edema, swelling of synovial cells
inhibition with increased hydrophobicity and unsaturated and hyperplasia. This may be due to their combined effect to
rings in their structure. These studies clearly indicate that the hydrolyze the membrane integrity, as well as the generation
hydrophobicity and aromatic nature is critical for inhibition and metabolism of products such as eicosanoids, which then
of the enzyme.
Injection of purified PLA2 enzyme from snake venom and with the specific PLA2 enzyme-modifying agents, such as p-
function to amplify inflammatory events.34,35) Several studies

20
Vol. 57, No. 1
Fig. 3. In Vivo Edema Inducing Activity Data for 3-[4-{Hydroxyimino-(aryl)-methyl}-phenyl]-4-(un)-substituted-sydnones 3a—h
Percent inhibition of sample concentration of 2.67 mM. Edema ratioꢁweight of edematous legꢃ100/weight of normal leg. Amount of venom taken for each
assay was 1 mg. The PLA2 enzyme (1 mg): benzophenone analogues/Luffariellin B (3 mM) mixture was preincubated at 37 °C for 1 h prior to injection into the
mice footpads. Prolonged preincubation time up to 12 h, at 37 °C did not have any additional effect on edema ratio. Values of edema ratios are expressed as
meanꢆS.D. (nꢁ6), p values ꢇ0.05 were considered significant when compared to the control by student’s t-test. Inhibitory rate (%)ꢁnanomoles of labelled
substrate released as compared to the control.
bromophenacylbromide (p-BPB) resulting in the loss of en-
zyme activity and edema inducing activity, directly indicates
that the edema produced by these PLA2 enzymes is mediated
by the catalytic domain of the enzyme.36) Benzophenones de-
rivatized with sydnones 3a—h with a co-injection of snake
venom PLA2 enzyme activity decreased the edema-inducing
activity in dose dependent manner. The concomitant inhibi-
tion of PLA2 enzyme activity and in vivo edema-inducing
activity by these derivatives suggests a strong correlation
between lipolytic activity and pro-inflammatory activities.
Since the benzophenone derivative binds at the catalytic site
of the enzymes, it is effective in preventing the enzyme from
inducing edema (Figs. 3, 4). In this case also the compounds
with hydrophobic alkyl groups and presence of an aromatic
ring on the 4th position of sydnone ring inhibit the enzyme
thus reducing the edema inducing activity.
Inhibition of the PLA2 enzyme by benzophenone deriva-
tives is reflected in the inhibition of indirect hemolytic activ-
ity (Fig. 5) where it was assayed with crude egg phospho-
Fig. 4. Dose Dependent Neutralization of Edema Inducing Activity of V.
russelli by 3-[4ꢀ-(Hydroxyimino-phenyl-methyl)-phenyl]-sydnone 3a and 3-
[4-{Hydroxyimino-(4ꢅ-n-butyl-phenyl)-methyl}-phenyl]-4-phenyl-sydnone
lipids mixture dispersed in the buffer as micelles, rather than
an intact E. coli membrane. Since the benzophenone deriva-
tive binds at the substrate-binding regions of the enzymes,
enzyme activity is inhibited irrespective of the substrate na-
ture provided for its activity. It has been observed that in-
crease in the dose has increased the inhibition of indirect he-
molytic activity.
3h
The reaction mixture 30 ml contains 5 mg PLA2 enzyme was incubated for 30 min
with increasing concentration of 3a and 3h. Saline (30 ml) injected into the mouse foot-
pad served as control. Data represent ꢆS.E.M. for nꢁ4.
drophobic and aromatic groups on the para position of aroyl
In summary, the above-synthesized compounds are good moiety and 4th position of the sydnone ring which is essen-
inhibitors of the PLA2 enzyme. The inhibition by benzophe- tial for binding at the catalytic domain of the enzyme. As the
none derivatives was further enhanced by substituting hy- in vitro inhibition of the enzyme by these derivatives corre-

January 2009
21
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Acknowledgements The authors (RRK and BSS) are thankful to the
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