Design, synthesis and evaluation of mutual prodrug of 4-biphenylacetic acid and quercetin tetramethyl ether (BPA-QTME) as gastrosparing NSAID

Article abstract of DOI:10.1016/j.ejmech.2010.02.047

A novel mutual prodrug consisting of 4-biphenylacetic acid (BPA) and quercetin tetramethyl ether (QTME) has been synthesized as a gastrosparing NSAID, devoid of ulcerogenic side effects. The physicochemical properties, including aqueous solubility, partition coefficient, chemical stability and enzymatic hydrolysis of synthesized derivative have been studied to assess its prodrug potential. Its antiinflammatory, antiulcer and analgesic activities were also evaluated. The results indicated that BPA-QTME derivative is chemically stable, biolabile and possesses optimum lipophilicity. The synthesized compound also exhibited retention of antiinflammatory activity with reduced ulcerogenicity. Based on these observations, the therapeutic potential of this mutual prodrug is discussed.

Full text of DOI:10.1016/j.ejmech.2010.02.047

European Journal of Medicinal Chemistry 45 (2010) 2591e2596
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and evaluation of mutual prodrug of 4-biphenylacetic acid
and quercetin tetramethyl ether (BPAeQTME) as gastrosparing NSAID
*
Mamta Madhukar, Shruti Sawraj, Pritam Dev Sharma
University Institute of Pharmaceutical Sciences, Panjab University, Sector-14, Chandigarh 160014, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel mutual prodrug consisting of 4-biphenylacetic acid (BPA) and quercetin tetramethyl ether
(QTME) has been synthesized as a gastrosparing NSAID, devoid of ulcerogenic side effects. The physi-
cochemical properties, including aqueous solubility, partition coefficient, chemical stability and enzy-
matic hydrolysis of synthesized derivative have been studied to assess its prodrug potential. Its
antiinflammatory, antiulcer and analgesic activities were also evaluated. The results indicated that
BPAeQTME derivative is chemically stable, biolabile and possesses optimum lipophilicity. The synthe-
sized compound also exhibited retention of antiinflammatory activity with reduced ulcerogenicity. Based
on these observations, the therapeutic potential of this mutual prodrug is discussed.
Received 18 February 2009
Received in revised form
17 February 2010
Accepted 19 February 2010
Available online 25 February 2010
Keywords:
4-Biphenylacetic acid
Mutual prodrug
Reactive oxygen species
Cyclooxygenase
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
ulcer exacerbation in high risk patients, delayed GI ulcer healing,
kidney toxicity, as well as, cardiovascular side effects [11]. These
Nonsteroidal antiinflammatory drugs (NSAIDs) belong to one of
the most commonly used therapeutically group of agents world-
wide for the treatment of pain, fever and inflammation. However,
usefulness of these agents is limited due to higher incidence of
gastrointestinal (GI) damage including gastric ulceration, perfora-
tion and their associated complications. These side effects are
related to the intrinsic mechanism responsible for their desired
activity [1,2]. NSAIDs exert their therapeutic effects by inhibiting
the activity of the enzyme cyclooxygenase (COX), resulting in the
prevention of prostaglandins synthesis [3]. Now, it is known that
COX exists in two isoforms, namely COX-1 and COX-2 [4,5]. COX-1 is
constitutive and provides cytoprotection in the GI tract, whereas
COX-2 is inducible and mediates inflammation. The mucosal
integrity in normal GI tract is primarily maintained by the prosta-
glandins that are derived from COX-1 and therefore, inhibition of
COX-1 rather than COX-2 by NSAIDs is responsible for their
ulcerogenic side effect [6e8]. Based on these observations, it has
been suggested that selective COX-2 inhibitors may act as safer
NSAIDs, devoid of their ulcerogenic side effects and a number of
such selective COX-2 inhibitors have been developed and intro-
duced in the market for clinical use [9,10]. However, long term use
of these agents has shown some potential limitation, including
observations indicated that safety of these agents is questionable
on their long term use and some of these agents have been with-
drawn from the market [12]. Thus, the initial enthusiasm of
developing selective COX-2 inhibitors has faded away and need for
design and development of safer NSAIDs still remains.
During recent years, it has been well established that generation
of reactive oxygen species (ROS) plays a significant role in the
formation of gastric mucosal lesions associated with NSAIDs
therapy [13,14]. Based on these observations, it has been suggested
that coadministration of antioxidants and NSAIDs in pharmaceu-
tical dosage forms may possibly decrease the risk of NSAIDs
induced GI ulcerogenicity [15,16]. However, there are potential
advantages in giving such agents with complementary pharmaco-
logical activities in the form of a single chemical entity. Such agents
are named as mutual prodrugs or codrugs that are designed with
improved physicochemical properties and release the parent drugs
at the site of action [17e19]. We have reported the synthesis of
a number of mutual prodrugs of 4-biphenylacetic acid (BPA) and
different phytophenolics having single hydroxyl groups [20]. In the
present study, quercetin, a naturally occurring polyphenolic flavo-
noid was selected to conjugate with BPA to obtain BPA-Antioxidant
mutual prodrug. Quercetin is well known for antiulcerogenic
activity due to its antioxidant properties [21,22]. Although, linking
of quercetin with BPA in 1:1 ratio is difficult due to the presence of
a number of hydroxyl groups, we have been able to conjugate this
agent in the form of its derivative, quercetin tetramethyl ether
* Corresponding author. Tel.: þ91 1722534117; fax: þ91 1722541142.
E-mail address: pritamdevsharma@hotmail.com (P.D. Sharma).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.047

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M. Madhukar et al. / European Journal of Medicinal Chemistry 45 (2010) 2591e2596
(QTME) with BPA. In this paper, design, synthesis and evaluation of
BPAeQTME mutual prodrug as safer NSAID are reported.
accordingly. To assess the lipophilicity, the Log P of BPAeQTME (9)
was determined in n-octonol and buffer layers and calculated
by correlating the absorbance with the concentration in standard
plot. The obtained values of Log P and solubility at pH 7.4 were
2. Chemistry
found to be 3.95 and 14.5 mg/ml respectively, indicating that the
synthesized derivative 9 meets the requirement for gastrointes-
tinal absorption.
To conjugate polyphenolic flavonoid, quercetin (1) with carboxyl
group containing NSAIDs in 1:1 ratio, an alternative strategy was
developed. For this purpose, rutin (2), the glycoside of 1 was treated
with dimethyl sulfate (3) in dry acetone in the presence of potas-
sium carbonate. The methylated glycoside (4) was obtained as
semisolid, which was not purified and subjected to hydrolysis by
refluxing in ethanolic sulfuric acid to obtain the quercetin deriva-
tive, quercetin tetramethyl ether (QTME, 5). The free hydroxyl group
generated at 3-position in this derivative was used as a synthetic
handle for conjugation with the BPA (6).
BPA (6) was treated with thionyl chloride (7) by stirring at room
temperature and after removing excess of thionyl chloride, biphe-
nylacetyl chloride (8) was obtained as yellow amorphous powder in
84% yield. Quercetin derivative, QTME (5) was treated with 8, trie-
thylamine and 4-dimethylaminopyridine using dichloromethane
as solvent and the desired derivative, BPAeQTME (9) was obtained
as light yellow solid. Sequence of various steps involved in the
reaction is shown in Scheme 1.
3.2. Chemical stability
A prodrug should be chemically stable so that it can be formu-
lated in an appropriate pharmaceutical dosage form with optimum
half life. At the same time, it should be biolabile to regenerate the
parent drug molecule(s) to exhibit therapeutic activity. Therefore,
the BPAeQTME (9) was assayed in vitro to evaluate its chemical
stability [27]. The kinetics of chemical hydrolysis was studied at
37 ꢀC using buffer solutions of pH 2 and 7.4. The reactivity to
chemical hydrolysis was evaluated by pseudo first order rate
constants obtained from slopes of semi logarithmic plots of 9
concentrations against time. The rate constants at pH 7.4 and 2
were found to be 0.0180 and 0.0066 hꢁ1 respectively, indicating the
stability in buffer solutions. The derivative showed considerable
chemical stability at pH 2 (t½ > 100 h) which implies that it passed
unhydrolysed through the stomach after oral administration. At pH
7.4, the compound showed enough stability (t½ > 38 h) to be
absorbed intact from the intestine.
3. Results and discussion
3.1. Solubility and partition coefficient
3.3. Enzymatic hydrolysis
Lipophilicity is an important factor controlling the interaction of
drugs with biological membranes. It is generally accepted that
good absorption of an orally administered drug could be obtained
when apparent coefficient partition (Log P) value is more than 2
In 80% human plasma (human plasma containing 20%, 0.02 m
phosphate buffer, pH 7.4), BPAeQTME ester bond was found to be
cleaved, forming the parent molecules, BPA (6) and QTME (5). The
degradation process was found to correlated with pseudo first
order kinetics for several half lives. The rate of hydrolysis of 9 was
found to be 2.09 hꢁ1 and half life (t½) being 19.8 min respectively,
indicating that 9 is readily hydrolyzed in plasma to release the
parent drug molecules. The rapid rate of hydrolysis observed in
and the aqueous solubility is more than 10 mg/ml [23]. The evalu-
ation of solubility and apparent partition coefficient (Log P) of 9
was performed by saturation shake flask method [24e26]. For
the quantification of 6 and 9, standard plots were constructed
using UV spectrophotometer and quantification was carried out
OCH3
OCH3
OH
OH
1) (CH ) SO (3)
3 2
4
2) K CO
3) Reflux
H3CO
O
2
3
HO
O
O
O-Rutinose
O-Rutinose
OCH
3O
OH
(4)
(2)
HOOC H2C
(6)
1) SOCl (7)
2
2) Room Temperature
1) Ethanolic
2) H SO (2%)
2
4
3) Reflux
ClOC H2C
OCH3
OCH3
OCH3
(8)
OCH3
H3CO
O
H3CO
O
O-C-CH2
O
OH
OCH
3O
1) TEA, DMAP, DCM
o
OCH
3O
2) -10
C
(9)
(5)
Scheme 1. Sequence of steps involved in the synthesis of BPAeQTME (9) mutual prodrug.

M. Madhukar et al. / European Journal of Medicinal Chemistry 45 (2010) 2591e2596
2593
plasma and more stability in the absence of plasma under similar
conditions in buffers (pH 2 and 7.4) is important and implies that
enzymatic reactivity of 9 is independent of their intrinsic ester
reactivity.
observations, this agent was also evaluated for various pharmaco-
logical activities.
3.5. Pharmacological evaluation
3.4. Biochemical evaluation
BPA and BPAeQTME mutual prodrug were evaluated for their
antiinflammatory activity by carrageenan induced paw edema
methods [28]. In this method, carrageenan (1% w/v) was used to
produce paw edema in control group. BPA (15 mg/kg, 70 mmol,
p.o.) significantly decreased the carrageenan induced increase in
paw volume as compared to control. BPAeQTME at equivalent dose
(39 mg/kg, 70 mmol) significantly inhibited carrageenan induced
inflammation and showed antiinflammatory activity comparable to
parent drug, BPA (Table 2).
These agents were studied for acute gastric damage evaluation
[29]. For this purpose, BPA (54 mg/kg, 254 mmol, p.o.) was adminis-
tered to produce a significant increase in ulcer index (6.2 ꢂ 0.41) as
compared to control group (0.2 ꢂ 0.12). Equivalent dose of
BPAeQTME 9 (140 mg/ml, 254 mmol, p.o.) showed significantly
reduced gastric damage (ulcer index 0.45 ꢂ 0.27). BPA þ QTME
physical mixture in equivalent doses (54 mg/kg, 254 mmol) þ
(86 mg/kg, 254 mmol, p.o.) also showed reduction in gastric damage
(ulcer index 3.4 ꢂ 0.57) but the inhibition of gastric damage by
BPAeQTME 9 was to a greater extend which may be due to combined
effect of improved physicochemical properties of the conjugate,
resulting in improved absorption and antioxidant activity of the
released QTME (Table 2).
For the evaluation of analgesic activity, the abdominal writhing
assay method was followed [30]. In this method, vehicle treated
control mice were given 1% acetic acid and exhibited writhing
response (86 ꢂ 3.1). BPA (9.9 mg/kg, 46 mmol, p.o.) significantly
reversed the incidents of writhing (68.6 ꢂ 3.8). BPAeQTME prodrug
at equivalent dose (25.5 mg/kg, 46 mmol, p.o.) also significantly
reduced the writhing response (62.4 ꢂ 4.0). The BPAeQTME 9
showed analgesic activity comparable to the parent drug (Table 2).
The results listed in Table 2 showed that BPAeQTME 9 lack
gastrointestinal ulcerogenic side effects with retention of antiin-
flammatory and analgesic activity.
The effects of BPA (6), BPAeQTME (9) and BPA þ QTME physical
mixture were studied on various peripheral markers of oxidative
stress including lipid peroxidation (MDA levels), myeloperoxidase
activity (MPO levels), superoxide dismutase activity (SOD), catalase
activity and shown in Table 1.
MDA levels were found to be significantly increased (P < 0.01) in
BPA treated group (22.57 ꢂ 2.34) in comparison to control group
(4.61 ꢂ1.28), indicating gastric damage. However, BPAeQTME
derivative 9 showed MDA level (6.42 ꢂ 0.55) close to the control
group (4.61 ꢂ1.28) and significantly reduced as compared with the
BPA þ QTME physical mixture (13.36 ꢂ 1.92) and BPA (22.57 ꢂ 2.34).
These results suggested that BPAeQTME may exhibit protective
effect against gastric damage as compared to BPA þ QTME physical
mixturewhich may be due tothe inhibition of lipid peroxidation and
cell damage (Table 1).
Similarly, myeloperoxidase activity (%MPO) was significantly
increased in BPA treated group (238.47 ꢂ 19.39) as compared to
control. BPAeQTME (9) significantly reduced myeloperoxidase
activity (118.93 ꢂ 5.55) as compared to BPA þ QTME physical
mixture treated group (227.33 ꢂ13.32), which is close to control
group (100 ꢂ 0). These results indicated lesser neutrophil adher-
ence in BPAeQTME mutual prodrug treated group, which may
result in reduction of ulcerogenic side effects (Table 1).
SOD is an endogenous antioxidant enzyme that scavenges
ꢃ
superoxide radical (Oꢁ2 ) by catalyzing its dismutation to H2O2 and
ꢃ
O2. Decreased level of SOD indicates increased generation of O2ꢁ
and increased gastric mucosal injury. SOD activity was significantly
decreased in BPA treated group (26.23 ꢂ 6.31) as compared to the
control group (325.40 ꢂ 69.0). BPAeQTME treated group showed
significantly increased levels of SOD (223.46 ꢂ 24) as compared
with BPA þ QTME physical mixture treated group (60.41 ꢂ13.28)
(Table 1).
Catalase is endogenous antioxidant enzyme that scavenges
hydroxyl radical (ꢃOH) by converting H2O2 to water. Decreased level
of catalase indicates increased gastric damage. Catalase activity was
significantly decreased in BPA treated group (3.52 ꢂ 0.33) as
compared to the control group (28.38 ꢂ 2.85). BPAeQTME treated
group showed maintenance levels of catalase (22.41 ꢂ1.68) close to
control as compared with BPA þ QTME physical mixture treated
group (8.69 ꢂ 0.30).
4. Conclusion
In the present study, BPAeQTME (9) mutual prodrug has been
designed, synthesized and evaluated as safer NSAID in which it has
been possible to conjugate BPA (6) and QTME (5) in 1:1 ratio. The
derivative has been found to be chemically stable and biolabile. It
exhibited retention in antiinflammatory with significant reduced
ulcerogenicity as compared to the BPA þ QTME physical mixture.
This may be due to improved physicochemical properties required
for enhanced bioavailability. On the basis of these observations,
it can be concluded that there is advantage of giving BPA and
QTME in the form of a single molecule i.e. BPAeQTME mutual
prodrug.
The results of studies on lipid peroxidation (MDA levels), mye-
loperoxidase activity (MPO levels), superoxide dismutase activity
(SOD) and catalase activity suggested that BPAeQTME conjugate 9
exhibited significant activity as compared to BPA þ QTME physical
mixture. Therefore, this conjugate may exhibit antiinflammatory
activity without/or reduced gastric damage. On the basis of these
Table 1
Effect of BPA, BPAeQTME and BPA þ QTME (physical mixture) on superoxide dismutase activity, catalase activity, lipid peroxidation (MDA levels) and myeloperoxidase activity.
Treatment
SOD activity (U/mg protein)
Catalase activity (U/mg protein)
nmoles MDA/mg protein
%MPO activity
Control
325.40 ꢂ 69.00
223.46 ꢂ 24.01a
60.41 ꢂ 13.26c
26.23 ꢂ 6.31**
28.38 ꢂ 2.85
22.41 ꢂ 1.68a
8.69 ꢂ 0.30a
3.52 ꢂ 0.33*
4.61 ꢂ 1.28
6.42 ꢂ 0.55b
13.36 ꢂ 1.92c
22.57 ꢂ 2.34**
100 ꢂ 0
BPAeQTME
BPA þ QTME
BPA
118.93 ꢂ 5.55b
227.33 ꢂ 13.32
238.47 ꢂ 19.39**
Data are expressed as mean ꢂ SE of five experiments; (*) p < 0.0001 extremely significant as compared to control; (**) p < 0.01 very significant as compared to control;
ap < 0.0001 extremely significant as compared to BPA; bp < 0.001 very significant as compared to BPA; cp < 0.05 significant as compared to BPA.

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M. Madhukar et al. / European Journal of Medicinal Chemistry 45 (2010) 2591e2596
Table 2
Antiinflammatory, analgesic and ulcerogenic activities of BPA, BPAeQTME mutual prodrug; and BPA þ QTME (physical mixture).
Treatment
% Increase in paw edema
%Inhibition in writhing
Ulcer index
2 h
3 h
4 h
5 h
Control
BPAeQTME
BPA
BPA þ QTME
68.8 ꢂ 2.06
34.2 ꢂ 1.03a
26.9 ꢂ 0.81*
e
81.1 ꢂ 2.43
37.5 ꢂ 0.83a
26.5 ꢂ 0.80*
e
78.4 ꢂ 2.35
31.6 ꢂ 0.95a
25.2 ꢂ 0.76*
e
65.2 ꢂ 1.96
33.1 ꢂ 0.99c
29.1 ꢂ 0.87*
e
e
0.2 ꢂ 0.12
0.45 ꢂ 0.27a
6.2 ꢂ 0.41*
3.4 ꢂ 0.57b
62.4 ꢂ 4.0
68.6 ꢂ 3.8
62.6 ꢂ 2.4
Data are expressed as mean ꢂ SE of five experiments; e not determined; (*) p < 0.0001 extremely significant as compared to control; ap < 0.0001 extremely significant as
compared to BPA; bp < 0.001 very significant as compared to BPA; cp < 0.05 significant as compared to BPA.
5. Experimental protocols
5.1.2. Biphenylacetyl chloride (8)
BPA (6) (2.12 g, 0.01 mmol) was added to thionyl chloride (7)
(1.44 g, 0.96 ml, 0.012 mol) and stirred at room temperature for
18 h. The excess of thionyl chloride was removed under reduced
pressure to give biphenylacetyl chloride (8) as yellow amorphous
solid (2.057 g, 84%), mp 49 ꢀC, Rf 0.72 (toluene), IR (KBr): 2940 (Ar
CeH st), 2810 (aliphatic CeH st), 1690 (carboxylic C]O st), 1480 (Ar
5.1. Chemistry
Melting points (mp) were determined on a Veego melting point
apparatus and are uncorrected. For TLC, glass plates coated with
silica gel (E. Merck) were used. The TLC plates were activated at
110 ꢀC for 30 min and visualized by exposure to iodine vapors. Glass
columns of appropriate sizes were used. Silica gel (60e120 mesh,
BDH) was used as adsorbent. UV(max) spectra were recorded on
Perkin Elmer Lambda 15 UV/VIS spectrometer using ethanol as
solvent. IR spectra were recorded on Perkin Elmer 882 spectrom-
eter using potassium bromide pellets. 1HNMR and 13CNMR spectra
were recorded with Bruker AC 300F, 300 MHz spectrometer using
CDCl3 or DMSO-d6 as solvents and tetramethylsilane as internal
standard. Mass spectra were obtained with Vg-11-250J 70s mass
spectrometer at 70 ev using electron ionization (EI) sources. The
synthetic reactions were monitored by TLC. The identity of all new
C]C st), 1255 (CeO st) cmꢁ1, 1HNMR (CDCl3)
d: 3.75 (2H, s, Ar-CH2),
7.37 (4H, ABq, J ¼ 9.02 Hz, p-substituted Ar-H overalapping 1H, m,
Ar-H), 7.30e7.45 (4H, m, Ar-H).
5.1.3. BPAeQTME mutual prodrug (9)
QTME (1.74 g, 0.005 mol) was dissolved in dichloromethane
(20 ml) containing triethylamine (5 drops) and 4-dimethylamino-
pyridine (1 pinch). The reaction mixture was cooled to ꢁ10 ꢀC and
biphenylacetyl chloride (7) (2.057 g, 0.01 mol) dissolved in
dichloromethane (30 ml) was added dropwise over a period of 1 h.
The reaction mixture was stirred overnight and the solvent was
removed under reduced pressure. The solid product obtained was
chromatographed on silica gel column using dichloromethane:ethyl
acetate (99:1) as eluent and the solvent was removed under reduced
pressure to obtain BPAeQTME (9); yield (0.7 g, 25.4%), mp
118e120 ꢀC, Rf 0.85(dichloromethane), UV (lmax) nm: EtOH: 332,
compounds was confirmed by 1HNMR, 13CNMR, IR data, UV (lmax
)
data and mass spectrometer; homogeneity was confirmed by TLC.
Solutions were routinely dried over anhydrous sodium sulfate prior
to evaporation. Rutin trihydrate was purchased from Lancaster.
Nitroblue tetrazolium, ethylenediaminetetracetic acid (EDTA), thi-
obarbituric acid, trichloroacetic acid were obtained from SD Fine
Chem. p-Nitrosodimethyl aniline was purchased from LOBA chem.
Hydrogen peroxide was purchased from Qualigens fine Chem. Pvt.
Ltd. All other reagents and solvents were of AR grade.
1%
265; E : 300, IR (KBr): 3123 (Ar CeH st), 2358 (CeOeCH3 st and
1cm
CeH st), 1604 (Ar C]O st), 1510 (Ar C]C st), 1463 (CeH bend),
1310.5, 1251.1 (C]CeOeC ether st), 1159.3, 1086 (CeOeC st)cmꢁ1
.
1HNMR(CDCl3)
d
: 3.65, 3.77 (12H, overlapping s, 4 ꢄ OCH3), 4.0 (2H,
s, Ar-CH2), 6.36 (1H, d, J ¼ 2.0 Hz, C-6-H), 6.51 (1H, d, J ¼ 2.0 Hz, C-8-
H), 6.96 (2H, ABq, J ¼ 9 Hz, C-50-H, C-60-H), 7.22 (1H, d, J ¼ 2.0 Hz,
C-20-H), 7.35 (1H, m, Ar-H), 7.51 (4H, ABq, J ¼ 9.02 Hz, p-substituted
5.1.1. Quercetin 5,7,30, 40-tetramethyl ether (QTME, 5)
To a fine suspension of rutin trihydrate (2) (2 g, 0.003 mol) in
dry acetone (200 ml), anhydrous potassium carbonate (8 g,
0.056 mol) and dimethyl sulfate (3) (8 ml, 0.059 mol) were added
and the reaction mixture was refluxed for 60 h. The solution was
filtered and insoluble potassium salts were washed with acetone.
The washings were combined with the filtrate and solvent was
removed under reduced pressure to obtain methylated glycoside
(4) as semisolid residue.
Ar-H overlapping 4H, m, Ar-H), 13CNMR (CDCl3)
d: 171.6 (C-4), 168.4
(ester C]O), 164.4 (C-7), 161.4 (C-5), 159.7 (C-9), 149.2 (C-2), 140.7
(C-40), 140.2 (C-30), 135.0 (C-3), 132.4, 130.0, 128.7, 127.2, 126.9 (12C,
Ar C of biphenyl), 122.3 (C-10), 121.9 (C-60), 111.7 (C-20), 111.4 (C-50),
108.0 (C-10), 96.3 (C-6), 93.0 (C-8), 56.17 (4 ꢄ OCH3), 40.7 (CH2), MS:
m/z 552 (Mþ), 358 (Mþ ꢁ C14H11O, 100%), 343 (Mþ ꢁ C14H11OeCH3),
328 (Mþ ꢁ C14H11Oe2 ꢄ CH3), 314, 300 and 167.
The product was refluxed with ethanolic sulfuric acid (2%, 50 ml)
for 2 h. The solvent was removed under reduced pressure and the
residue obtained was recrystallized from ethanol to give QTME (5)
(0.8 g, 85%), mp 195e197 ꢀC, Rf 0.3 (dichloromethane:ethyl acetate:
9.5:0.5), UV (lmax) nm: EtOH: 360, 250.8; AlCl3: 419.0, 258.6;
AlCl3 þ HCl: 219.0, 255.4, IR (KBr): 3338 (Ar OeH st), 2975 (Ar CeH
st), 2861 (CeOeCH3 st and CeH st), 1616 (Ar C]O st), 1518 (Ar C]C
st), 1461 (CeH bend), 1375, 1220 (C]CeOeC ether st), 1156.4, 1086
5.2. Physicochemical properties
5.2.1. Solubility studies
Solutions of BPA were prepared in methanol. The lmax was
determined by scanning solution containing 20e50 mg/ml of BPA
between 200 and 400 nm using UVevisible spectrophotometer.
The lmax of BPA was found to be 253 nm. Similarly, lmax of
BPAeQTME was determined and found to be 332 nm.
(CeOeC st) cmꢁ1 1HNMR (CDCl3)
, d: 3.4, 3.6 (12H, overlapping s,
4 ꢄ OCH3), 6.36 (1H, d, J ¼ 2.02 Hz, C-6-H), 6.56 (1H, d, J ¼ 2.02 Hz,
The standard plot for BPA was constructed in methanol. The
stock solution containing 1 mg/ml of BPA was diluted to obtain
solutions of concentration in the range of 2e10 mg/ml. The spec-
C-8-H), 7.29 (2H, ABq, J ¼ 9 Hz, C-50-H, C-60-H), 7.3 (1H, s, C-3-OH),
7.79 (1H, d, J ¼ 2.0 Hz, C-20-H), 13CNMR (CDCl3)
d
: 171.6 (C-4), 164.3
(C-9), 160.5 (C-7), 156.6 (C-2), 150.3 (C-5), 146.6 (C-40), 142.0 (C-30),
137.5 (C-3), 123.7 (C-10), 120.6 (C-60), 110.9 (C-20), 110.4 (C-50),
106.1 (C-10), 96.2 (C-6), 95.6 (C-8), 56.3, 55.9 (2C), 55.6 (4 ꢄ OCH3).
MS: m/z 358 (Mþ, 100%), 343 (Mþ ꢁ CH3), 328 (Mþ ꢁ 2 ꢄ CH3), 312,
and 297.
trophotometeric absorbances were recorded at 253 nm using
methanol as blank. A highly linear calibration curve was obtained
1%
with slope as 0.0979 and E
979. Similarly, calibration plot of
1cm
1%
BPAeQTME was established in chloroform at 332 nm and E
BPAeQTME was found to be 300.
for
1cm

M. Madhukar et al. / European Journal of Medicinal Chemistry 45 (2010) 2591e2596
2595
The solubility studies of BPA and BPAeQTME were carried out in
phosphate buffer (pH 7.4). Excess amount of each compound was
added to 10 ml of buffer and shaken for 24 h at 25 ꢂ 2 ꢀC using
water bath shaker. Solutions were filtered and analyzed spectro-
photometrically for determining the amount of compounds.
Animals were treated with BPA (54 mg/kg, p.o.) and equimolar doses
of BPAeQTME and BPA þ QTME (physical mixture). Animals
were sacrificed 4 h after the treatment. The stomach was removed,
opened along greater curvature, washed with saline and
observed for ulcers. The ulcers were scored as: 0 ¼ no observable
damage; 1 ¼ punctiform lesion <1 mm, 3 ¼ filiform lesion <5 mm,
4 ¼ punctiform lesion >1 mm or filiform lesion >5 mm.
5.2.2. Partition coefficient determination
Partition coefficients of BPA and BPAeQTME were determined in
octonol/phosphate buffer (pH 7.4) system using shake flask method.
Saturated solutions of the compounds were prepared in n-octonol
(10 ml) and equal volumes of phosphate buffer were added to the
solutions in conical flasks. The sealed flasks were kept for shaking in
a water bath shaker maintained at 25 ꢂ 20 ꢀC for 24 h and then
allowed to stand for 30 min for the phases to fully separate. There-
after, the respective phases were analyzed spectrophotometrically.
5.3.4. Analgesic activity
Analgesic activity was determined by using abdominal writhing
assay. Mice were divided into 4 groups; i) vehicle (control); ii) BPA
(standard); iii) BPAeQTME; and iv) BPA þ QTME (physical mixture).
The animals were food deprived overnight prior to the experiments.
The samples were suspended in carboxymethylcellulose (0.5%, CMC),
and administered orally (0.1 ml/10 g of body weight) at 1 h before the
administration of freshly prepared acetic acid solution (1%, 10 ml/kg,
i.p.) intraperitoneal. The number of writhes (constriction of abdomen,
turning of trunk and extension of hind limbs) for each animal was
recorded during 20 min period, beginning 3 min after the adminis-
tration of acetic acid. The average number of writhes in each group of
drug treated mice was compared with that of control group and
degree of analgesia was expressed as % inhibition as follows:-
5.2.3. Kinetics of hydrolysis in aqueous solution
The rate of chemical hydrolysis of BPAeQTME was determined
in isotonic phosphate buffer (pH 7.4) and HCl buffer (pH 2) at 37 ꢀC.
An appropriate amount of BPAeQTME codrug was dissolved in
preheated buffer, and solution was placed in a thermostatically
controlled water bath at 37 ꢀC. At appropriate time intervals,
samples were taken and analyzed for the appearance of BPA at
253 nm spectrophotometrically. Pseudo first order half time (t½)
for the hydrolysis of BPAeQTME was calculated from the slope of
the linear portion of the plotted logarithm of BPA concentration vs
time.
ꢀ
ꢁ
Nt
Nc
% Inhibition ¼ 1 ꢁ
ꢄ 100
Where, Nc ¼ number of writhes in control, and
Nt ¼ number of writhes in drug treated mice.
5.4. Biochemistry
5.2.4. Kinetics of hydrolysis in human plasma
Plasma from human was obtained by centrifugation of blood
samples containing 0.3% citric acid at 3000 g for 15e20 min.
Human plasma fractions (4 ml) were diluted with 1 ml of isotonic
phosphate buffer (pH 7.4) to give a final volume of 5 ml (80%
plasma). Incubation was performed at 37 ꢂ 0.5 ꢀC using shaking
5.4.1. Antioxidant activity (in vivo)
Preparation of tissue homogenate: The glandular parts of
excised stomachs from ulcer studies were homogenized in ice cold
phosphate buffer (pH 7.4) with a Potter-Elvehjerr glass homoge-
nizer for 30 s. The homogenate was centrifuged at 800 g for 10 min.
The supernatant was again centrifuged at 12,000 g for 15 min and
the obtained post mitochondrial fraction (PMF) was used for
following estimations.
water bath. The reactions were initiated by adding 100 ml of stock
solution of (1 mg/ml) to 5 ml of preheated plasma. At appropriate
time intervals, samples were taken and diluted with phosphate
buffer and analyzed spectrophotometrically at 253 nm for the
appearance of BPA. The value of rate constants (K) and half lives
(t½) for the hydrolysis of BPAeQTME was calculated from the linear
portion of the plotted logarithm of BPA concentration vs time.
5.4.1.1. Catalase assay. To 0.05 ml of PMF, freshly prepared
hydrogen peroxide (3 ml, 30 mM in 50 mM phosphate buffer, pH 7)
was added and change in absorbance was measured at 240 nm for
2 min at 30 s intervals. Catalase activity was estimated using the
following equation:
5.3. Pharmacology
5.3.1. Animals
2:3
A1
A2
Catalase ¼
log
Sprague-Dawley (sd) rats (weighing 150e200 g) of both sex and
LACCA mice (male, 25e35 g) procured from central animal house,
Panjab University, Chandigarh, India were used. The animals were
housed in plastic cages (five rats/cage) under standard laboratory
conditions and maintained on rat chow and water.
D
t
A1 ¼ initial absorbance, and
A2 ¼ final absorbance, when
Dt ¼ 1 min.
Catalase activity was expressed as units/mg protein. One unit
catalase is defined as the amount of enzyme required for decom-
posing 1 m
mol of H2O2/min at 25 ꢀC at pH 7.0.
5.3.2. Antiinflammatory activity
Antiinflammatory activity was assessed by the carrageenan
induced rat paw edema model. Rats were divided into 3 groups; i)
vehicle (control); ii) BPA (standard); and iii) BPAeQTME. The rats
were fasted for 12 h prior to test. The test compounds were sus-
pended in carboxymethylcellulose (0.5%, CMC) and administered
orally. Control animals were given the corresponding amount of
vehicle (0.5%, CMC). The compounds were administered on molar
equivalent basis of BPA.
5.4.1.2. Superoxide dismutase (SOD) activity. To 0.1 ml of PMF, 2 ml
of nitroblue tetrazolium (96 mM, freshly prepared in 50 mM
Na2CO3 solution containing 0.1 mM EDTA) and 0.5 ml of hydrox-
ylamine hydrochloride (20 mM) were added and the change in
absorbance was measured at 560 nm for 2 min at 30 s intervals.
SOD activity was calculated from the following equation:
ꢀ
ꢁ
Anonꢁenz: ꢁ Aenz:
SOD Activity ¼
ꢄ 100
Aenz:
5.3.3. Antiulcer activity
For the study of antiulcer activity of BPAeQTME, rats were fasted
overnight and divided into 4 groups; i) vehicle (control); ii) BPA
(standard); iii) BPAeQTME; and iv) BPA þ QTME (physical mixture).
Anon-enz. e Absorbance of control (without homogenate)
Aenz. e Absorbance of test (with homogenate).

2596
M. Madhukar et al. / European Journal of Medicinal Chemistry 45 (2010) 2591e2596
5.4.1.3. Myeloperoxidase (MPO) activity. The stomach homogenate
(prepared in 0.01 M phosphate buffer) was centrifuged at
10,000 rpm at 10 ꢀC for 10 min. Supernatant was collected and
the levels of significance in antioxidant studies. P values <0.05 were
considered statistically significant.
mixed with o-phenylenediamine (660 mg/ml in phosphate buffer)
Acknowledgment
and hydrogen peroxide (300 mM) was added to initiate the
reaction. Absorbance was recorded at 492 nm at intervals of 30 s for
5 min. Change in optical density per min was calculated and results
were expressed as % MPO activity, considering 100% MPO activity in
control group.
The financial support from University Grant Commission (UGC)
and the research facilities provided by University Institute of
Pharmaceutical Sciences, Panjab University, India are gratefully
acknowledged.
5.4.1.4. Lipid peroxidation (LPO) activity. To 0.5 ml of PMF (phos-
phate buffer, pH 7.4), 0.5 ml of tris hydrochloric acid buffer (0.1 M,
pH 7.4) was added. The mixture was incubated at 37 ꢂ 2 ꢀC for 2 h.
To this mixture, ice cold trichloroacetic acid (1 ml, 10%) was added.
The turbid solution was centrifuged at 1000 rpm for 10 min. To 1 ml
of the supernatant obtained, thiobarbituric acid (1 ml, 0.67% w/v)
was added. The mixture was boiled for exactly 10 min and cooled.
To this solution, 1 ml distilled water was added and absorbance was
recorded at 532 nm.
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