Synthesis of benzoyl phenyl benzoates as effective inhibitors for phospholipase A2 and hyaluronidase enzymes

Article abstract of DOI:10.1016/j.bmcl.2005.06.012

Benzoylation of (hydroxy phenyl) phenyl methanone 2a-g to benzoyl phenyl benzoates 4a-g, a benzophenone analogue, was achieved in good yield. All the newly synthesized compounds were evaluated for their phospholipase A₂ [E.C. 3.1.1.4] and hyaluronidase [E.C. 3.2.1.35] enzyme inhibitory activity in snake venom as source and their structure-activity relationship with respect to different groups is reported for the first time. The in vitro PLA₂ enzyme inhibitory activity and in vivo anti-inflammatory activity studies of benzoyl phenyl benzoates are illustrated.

Full text of DOI:10.1016/j.bmcl.2005.06.012

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4100–4104
Synthesis of benzoyl phenyl benzoates as effective inhibitors
for phospholipase A2 and hyaluronidase enzymes
Shaukath Ara Khanum,a Satish Kumar Murari,b Bannikuppe Sannanaik Vishwanthb
and Sheena Shashikantha,*
aDepartment of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
bDepartment of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
Received 8 March 2005; revised 28 May 2005; accepted 3 June 2005
Available online 1 July 2005
Abstract—Benzoylation of (hydroxy phenyl) phenyl methanone 2a–g to benzoyl phenyl benzoates 4a–g, a benzophenone analogue,
was achieved in good yield. All the newly synthesized compounds were evaluated for their phospholipase A2 [E.C. 3.1.1.4] and hyal-
uronidase [E.C. 3.2.1.35] enzyme inhibitory activity in snake venom as source and their structure–activity relationship with respect
to different groups is reported for the first time. The in vitro PLA2 enzyme inhibitory activity and in vivo anti-inflammatory activity
studies of benzoyl phenyl benzoates are illustrated.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
The competence of benzophenone analogues as chemo-
therapeutic agents, especially as anti-inflammatory com-
pounds is well recognized.8–13 Recently, synthesis and
structure–activity relationship of benzophenones as nov-
el class of p38 MAP kinase inhibitors with high anti-in-
Phospholipase A2 (PLA2) is a promising family of dis-
tinct enzymes that exhibits different substrate specifici-
ties, cofactor requirements, subcellular localization and
cellular functions.1 The PLA2 class of enzymes catalyzes
hydrolysis of the 2-acyl ester of 3-Sn phosphoglycerides
to yield arachidonic acid (metabolized to eicosanoids by
cyclooxygenase and lipoxygenase) and lysophospholip-
id, which is a rate limiting step of the production of
pro-inflammatory lipid mediators such as prostaglan-
dins, leukotrienes, lipoxins, and platelet activating fac-
tor.2–5 In many inflammatory diseases, high levels of
PLA2 enzymes are identified and are believed to be
responsible for part of the inflammatory reactions.
Injection of purified PLA2 enzyme from synovial fluid
and from snake venom into animal joints confirmed
the development of an acute inflammatory response
with edema, swelling of synovial cells, and hyperpla-
sia.6,7 Clinical results with cyclooxygenase and lipoxyge-
nase inhibitions demonstrate that inhibition of PLA2
enzyme results in reduction of both lipid mediators,
indicating that these PLA2 inhibitors can be used as
anti-inflammatory drugs.
flammatory activity has been reported.14 Moreover,
15
Katsuichi et al.
have reported benzophenone ana-
logues as PLA2 inhibitors. In view of these observations
and due to our ongoing work on microwave tech-
nique,16,17 this investigation is directed towards the syn-
thesis and evaluation of benzophenone analogues as
anti-inflammatory agents.
The enzyme hyaluronidase and its substrate hyaluronan
are known to be involved in fundamental, physiological
and pathological, processes such as embryological devel-
opment, migration, adhesion, proliferation and differen-
tiation of cells, immune surveillance, inflammation,
wound healing, angiogenesis, tumorigenesis, virulency,
and venom.18 Hyaluronidases are present in virtually
all snake venoms.19 In snake venoms, the hyaluronidases
are generally referred to as spreading factors.20 The
spreading property of enzyme is presumed to be the
critical event in the spreading of toxins from the site of
injection to systemic circulation.21 This process is accom-
plished by the degradation of hyaluronan and eventual
loss of integrity in extracellular matrix of soft connective
tissue surrounding the blood vessels. This leads to the
easy diffusion of other toxic components of venoms.
Keywords: Phospholipase A2 inhibitor; Hyaluronidase inhibitor; Ben-
zoyl phenyl benzoates.
*
Corresponding author. Tel.: +91 082 125 47279; fax: +91 082 125
18358; e-mail: skanth1@rediffmail.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.012

S. A. Khanum et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4100–4104
4101
Distortion in the integrity of the extracellular matrix of
local tissue(s) due to degradation of hyaluronan with
an eventual dissemination of target specific toxins is pre-
sumed to be the critical event in the enzyme-mediated
spreading process.18 The infective nematodes release
hyaluronidase during the gastrointestinal invasive stage
and are thought to have a role in tissue degradation
and mucosal invasion, as well as in the pathogenesis of
the associated enteritis.21 Certain anti-inflammatory
drugs that are claimed as possessing hyaluronidase inhib-
itory activity include salicylates indomethacin, sodium
cromoglycate, and sodium auro-thiomalate.22 These
drugs may exert a portion of their inflammatory activity
by preventing the generation of small Hyaluronic acid
(HA) fragments, though the fragments of HA are potent
inducers of inflammatory cytokine release.23 Other drugs
that are used to suppress allergic reactions, such as diso-
dium cromoglycate, flavonoids, tannins, curcumins, gly-
cyrrhizin, cinnamic acid derivatives, and tranilast may
function as hyaluronidase inhibitors.24,25 Synthetic
inhibitors prevent venom induced systemic coagulopathy
and local tissue damage.26 No information is so far avail-
able on the active site residues and mechanism of hyalu-
ronan degradation by snake venom hyaluronidases.18
Inhibition of hyaluronidase(s) contributes toward the
better management of snakebites. It is likely that the
cocktail of the inhibitors are of great value as first aid
agents and further, incorporation would complement
Scheme 1.

4102
S. A. Khanum et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4100–4104
and enhance the efficiency of antivenom therapy as sys-
temically administered antivenoms have minimal protec-
tive effects on local tissue damage.19,20 This study
emphasizes the need for screening of compounds and a
thorough characterization of locally acting enzymes/tox-
ins and suggests for identification/designing of novel
inhibitor(s).
and counted in a Hewlett Packard liquid Scintillation
Analyzer TRI CARB 2100 TR.
3.2. Determination of edema inducing activity
Groups of six mice (22–24 g) were injected in the right
footpads of hind limbs with 3 mM dose of benzoyl phen-
yl benzoates in 20 ll saline. The left footpads received
20 ll of saline, which served as control. After 45 min,
the mice were sacrificed by cervical dislocation and both
legs were cut 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
leg · 100/weight of the normal leg Minimum edema dose
is defined as micrograms of protein causing an edema
ration. of 120%. The time-course of the edema-inducing
activity was obtained by injecting a fixed dose of protein
into mice footpads and killing them at regular time
periods. Edema ratio was calculated as defined.30
2. Chemistry
The synthetic sequence is outlined in Scheme 1. Com-
pounds 1a–g on thoroughly mixing with an equal
amount of montmorrillonite k 10 clay in the solid
state using vortex mixer and on subjecting to micro-
wave irradiation for 10–13 min afforded substituted
(hydroxy phenyl)phenyl methanone 2a–g in excellent
yield.16,17 Benzoylation of 2a–g with the respective
benzoyl chlorides 3a–d affords substituted benzoyl
phenyl benzoate 4a–g.13 The compounds 2a–g27 and
1
4a–g28 were characterized by IR, H NMR, and mass
3.3. Hyaluronidase assay
spectroscopies.
Hyaluronidase activity was assayed by estimating the
amount of N-acetylglucosamine released.31 Snake ven-
om 100 lg each was separately incubated with 50 lg of
hyaluronic acid in 300 ll 0.2 M sodium acetate buffer,
pH 5.0 containing 0.15 M NaCl and 0.88 mM
4-(2-methylbenzoyl)-2-methylphenyl 4-methylbenzoate
derivatives at 37 ꢁC for 2.5 h. The change in absorbance
was monitored at 585 nm. The results are shown in
Table 2.
3. Pharmacological evaluation
3.1. PLA2 activity
This was assayed with [14C]oleate-labeled autoclaved
Escherichia coli as the substrate.29 The reaction mixture,
350 ll contained 100 mM Tris–HCl, pH 8.0, 5 mM
Ca2+, and 3.15 · 109 autoclaved E. coli cells (corre-
sponding to 10,000 cpm and 60 nmol of lipid phospho-
rous). 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 benzoyl phenyl benzoates. Adding labeled
E. coli substrate started the reaction. The reaction was
terminated by adding 100 ll 2.0 M HCl and 100 ll of
fatty acid free BSA (100 mg/ml). The tubes were vor-
tex-mixed and centrifuged at 20,000g for 5 min. An ali-
quot (140 ll) of the supernatant containing released
[14C]oleic acid was mixed with scintillation cocktail
4. Results and discussion
As shown in Table 1, among compounds 4a–g, f and g
demonstrated strong in vitro and in vivo PLA2 enzyme
inhibition. Compounds 4c and b inhibited the in vitro
PLA2 activity of Naja melanoleuca venom and Tri-
meresurus flavoviridis venom isoenzymes, respectively.
The edema ratio at 1 lg PLA2 enzyme concentration
dropped below 120% in the presence of 4a–g at
300 lM concentration with all used PLA2 isoenzymes.
Compounds 4f and g were more effective compared to
Table 1. Neutralizing edema inducing activity and IC50 values in lM of compounds 4a–g on PLA2 enzyme activity
Phospholipase A2
4a
4b
4c
4d
4e
4f
4g
Ursolic acid
2.9
Naja melanoleuca venom
73
(ND)
87
60
(ND)
94
57
(103 6)
96
98
(102 5)
105
132
(ND)
112
53
(104 1)
40
35
(101 7)
31
101
2.3
6
3
1
2
Echis carinatus venom
Trimeresurus flavoviridis venom
Synovial fluida
(ND)
75
(ND)
69
(ND)
108
(ND)
139
(ND)
89
(103 4)
46
(101 3)
53
101
2.5
(ND)
56
(101 6)
(105 2)
46
(109 2)
(ND)
123
(ND)
(ND)
86
(106 3)
(ND)
156
(ND)
(105 3)
32
(102 1)
(109 3)
34.9
(103 4)
102
2.5
101
Edema ratio (given in parentheses) = weight of edematous leg · 100/weight of normal leg.
ND = not determined.
The PLA2 enzyme (1 lg): compounds 4a–g/ursolic acid (300 lM) mixture was preincubated at 37 ꢁC for 1 h prior to injection into the mice foot pads.
Values of edema ratio are expressed as mean SD (n = 4), P values < 0.05 were considered significant when compared to the control by StudentÕs
t test.
a Amount of venom taken for each assay was 1.5 lg (600 ng).

S. A. Khanum et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4100–4104
Table 2. Effect of compounds 4a–g on hyaluronidase enzyme activity
4103
Hyaluronidase enzyme source
Percent inhibition of compounds (inhibitors)
4a
4b
4c
4d
4e
4f
4g
Naja naja naja
42.4
36.7
36.9
48.9
13.9
NI
69.3
NI
58.7
46.9
NI
NI
85.3
90.6
71.6
76.8
89.3
79.2
87.8
92.5
Naja melanoleuca
Vipera russelli russelli
Trimeresurus elegans
16.9
26.2
33.1
28.2
NI
46
76.8
42.4
NI = No inhibition.
Values are presented as mean of four independent experiments. Heparin was used as known inhibitor in the same concentration as compounds 4a–g
and it showed 100% inhibition of hyaluronidase enzyme in all mentioned snake venoms as source.
4c and e in both in vivo anti-inflammatory activity and
in vitro PLA2 enzyme inhibition.
S.K.M. thank the Department of Science and Technol-
ogy, Government of India for supporting this research
and for Senior Research Fellowship.
Compounds 4a–g alone did not cause edema when
injected into mice footpads. Furthermore, it may be seen
from Table 1 that compounds 4f and g with a benzoyl
group at the ortho position carrying the electron-with-
drawing chloro group show higher inhibitory activity
than compounds 4a–e. In conclusion, it may be said that
4f and g in vitro inhibitory activity correlates with its in
vivo inhibition of edema induced by PLA2 enzyme iso-
forms. Similar trend has been observed in 4b and c with
T. flavoviridis venom PLA2 and Naja melanoleuca ven-
om PLA2. Synovial fluid PLA2 inhibitory activity was
higher with 4b than compounds 4a, c, and d. Our current
efforts are directed toward improving the anti-inflamma-
tory properties. We propose that 4f and g are potential
anti-inflammatory compounds.
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As shown in Table 2, compounds 4a, f, and g demon-
strate inhibition toward Elapidae, Viperidae, and Crotal-
idae families of snake venom hyaluronidases and among
them 4f and g showed strong inhibition in the snake ven-
om used as source for hyaluronidase enzyme. Com-
pounds 4c–e, showed no inhibition to N. melanoleuca,
Vipera russelii russelii and Naja naja naja, respectively.
Further 4f and g were found to show good inhibition
of hyaluronidases in all species of snake venoms. So far
no information is available on the active site residues
and mechanism of hyaluronan degradation by snake
venom hyaluronidases. However X-ray crystallography
studies indicate the primary catalytic residues as Asn
349, His 399 and Tyr 408. The hyaluronidase enzyme
binds compounds 4f and g in which ortho-substituted
benzoyl group carries the electron-withdrawing chloro
group. Therefore a higher inhibition was obtained with
4f and g than with the corresponding compounds
4a–e. Our current efforts are directed toward identifying
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muscle necrosis from naja and viper bites.
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Calcd for C14H11ClO2: C, 68.15; H, 4.46; Cl, 14.40.
Found: C, 68.17; H, 4.44; Cl, 14.42.
28. Compound 4a:13 Yield 78%, mp 53–55 ꢁC; IR (Nujol) m
(cmÀ1): 1750 (ester, C@O); 1665 (C@O), 1H NMR
(CDCl3) d: 2.32–2.51 (3s, 9H, 3CH3), 6.85–7.6 (m, 11H,
Ar-H); EI-MS: m/z (75%) M+ 344. Anal. Calcd for
C23H20O3: C, 80.21; H, 5.85. Found: C, 80.22; H, 5.86.
Compound 4b:13 Yield 75%, mp 89–91 ꢁC; IR (Nujol) m
(cmÀ1): 1760 (ester, C@O); 1670 (C@O), 1H NMR
(CDCl3) d: 2.2 (s, 3H, CH3), 6.9–7.7 (m, 13H, Ar-H);
EI-MS: m/z (76%) M+ 316. Anal. Calcd for C21H16O3: C,
79.73; H, 5.10. Found: C, 79.75; H, 5.12.
26. Anai, K.; Sugiki, M.; Yoshida, E.; Maruyama, M.
Toxicon 2002, 40, 63.
27. Compound 2a: Yield 92%, mp 120–122 ꢁC; IR (Nujol) m
(cmÀ1): 1640 (C@O), 3320–3450 (OH); 1H NMR (CDCl3)
d: 2.3 (s, 6H, 2CH3), 6.8–7.5 (m, 7H, Ar-H), 9.7 (br s, 1H,
OH); EI-MS: m/z (85%) M+ 226. Anal. Calcd for
C15H14O2: C, 79.64; H, 6.19. Found: C, 79.62; H, 6.20.
Compound 2b: Yield 91%, mp 81–83 ꢁC; IR (Nujol) m
(cmÀ1): 1670 (C@O), 3545–3649 (OH); 1H NMR (CDCl3)
d: 2.3 (s, 3H, CH3), 6.85–7.75 (m, 8H, Ar-H), 12.05 (br s,
1H, OH); EI-MS: m/z (87%) M+ 212. Anal. Calcd for
C14H12O2: C, 79.24; H, 5.66. Found: C, 79.26; H, 5.64.
Compound 2c: Yield 92%, mp 75–77 ꢁC; IR (Nujol) m
(cmÀ1): 1665 (C@O), 3510–3641 (OH); 1H NMR (CDCl3)
d: 2.3–2.35 (2s, 6H, 2CH3), 6.9–7.7 (m, 7H, Ar-H), 12.1 (br
s, 1H, OH); EI-MS: m/z (83%) M+ 226. Anal. Calcd for
C15H14O2: C, 79.64; H, 6.19. Found: C, 79.63; H, 6.16.
Compound 2d: Yield 90%, mp 71–73 ꢁC; IR (Nujol) m
(cmÀ1): 1665 (C@O), 3515–3645 (OH); 1H NMR (CDCl3)
d: 2.25–2.3 (2s, 6H, 2CH3), 6.9–7.7 (m, 7H, Ar-H), 12.05
(br s, 1H, OH); EI-MS: m/z (83%) M+ 226. Anal. Calcd for
C15H14O2: C, 79.64; H, 6.19. Found: C, 79.62; H, 6.18.
Compound 2e: Yield 86%, mp 154–156 ꢁC; IR (Nujol) m
(cmÀ1): 1645 (C@O), 3300–3410 (OH); 1H NMR (CDCl3)
d: 2.3 (s, 3H, CH3), 6.9–7.65 (m, 6H, Ar-H), 10.1 (br s, 1H,
OH); EI-MS: m/z (85%) M+ 281. Anal. Calcd for
C14H10Cl2O2 (281): C, 59.78; H, 3.55; Cl, 25.26. Found:
C, 59.79; H, 3.58; Cl, 25.28.
Compound 4c:13 Yield 78%, mp 97–99 ꢁC; IR (Nujol) m
(cmÀ1): 1750 (ester, C@O); 1668 (C@O), 1H NMR
(CDCl3) d: 2.3–2.5 (3s, 9H, 3CH3), 6.8–7.6 (m, 11H, Ar-
H); EI-MS: m/z (75%) M+ 344. Anal. Calcd for C23H20O3:
C, 80.21; H, 5.85. Found: C, 80.23; H, 5.83.
Compound 4d:13 Yield 79%, mp 96–98 ꢁC; IR (Nujol) m
(cmÀ1): 1735 (ester, C@O); 1655 (C@O), 1H NMR
(CDCl3) d: 2.3–2.5 (3s, 9H, 3CH3), 6.92–7.7 (m, 11H,
Ar-H); EI-MS: m/z (74%) M+ 344. Anal. Calcd for
C23H20O3: C, 80.21; H, 5.85. Found: C, 80.22; H, 5.84.
Compound 4e: Yield 77%, mp 56–57 ꢁC; IR (Nujol) m
(cmÀ1): 1737 (ester, C@O); 1658 (C@O), 1H NMR
(CDCl3) d: 2.3 (s, 3H, CH3), 6.9–7.65 (m, 11H, Ar-H);
EI-MS: m/z (73.5%) M+ 385. Anal. Calcd for C21H14
Cl2O3: C, 65.47; H, 3.66; Cl, 18.41. Found: C, 65.46; H,
3.65; Cl, 18.42.
Compound 4f: Yield 77%, mp 90–92 ꢁC; IR (Nujol) m
(cmÀ1): 1740 (ester, C@O); 1660 (C@O), 1H NMR
(CDCl3) d: 6.9–7.65 (m, 11H, Ar-H); EI-MS: m/z (74%)
M+ 405.5. Anal. Calcd for C20H11Cl3O3: C, 59.22; H, 2.73;
Cl, 26.22. Found: C, 59.23; H, 2.74; Cl, 26.23.
Compound 4g:13 Yield 77%, mp 93–95 ꢁC; IR (Nujol) m
(cmÀ1): 1755 (ester, C@O); 1670 (C@O), 1H NMR
(CDCl3) d: 2.3 (s, 3H, CH3), 6.75–7.7 (m, 11H, Ar-H);
EI-MS: m/z (77%) M+ 385. Anal. Calcd for C21H14 Cl2O3:
C, 65.47; H, 3.66; Cl, 18.41. Found: C, 65.45; H, 3.65; Cl,
18.40%.
Compound 2f: Yield 85%, mp 78–80 ꢁC; IR (Nujol) m
(cmÀ1): 1648 (C@O), 3305–3410 (OH); 1H NMR (CDCl3)
d: 6.9–7.65 (m, 6H, Ar-H), 11.9 (br s, 1H, OH); EI-MS:
m/z (84%) M+ 301.5. Anal. Calcd for C13H7Cl3O2: C,
51.78; H, 2.34; Cl, 35.27. Found: C, 51.77; H, 2.33; Cl,
35.26. Compound 2g: Yield 92%, mp 71–73 ꢁC; IR (Nujol)
m(cmÀ1): 1673 (C@O), 3550–3640 (OH); 1H NMR
(CDCl3) d: 2.2 (s, 3H, CH3), 7.0–7.65 (m, 7H, Ar-H),
12.15 (br s, 1H, OH); EI-MS: m/z (88%) M+ 246.5. Anal.
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