Chlorzoxazone esters of some non-steroidal anti-inflammatory (NSAI) carboxylic acids as mutual prodrugs: Design, synthesis, pharmacological investigations and docking studies

Article abstract of DOI:10.1016/j.bmc.2009.03.065

The discovery of the inducible isoform of cyclooxygenase enzyme (COX-2) spurred the search for anti-inflammatory agents devoid of the undesirable effects associated with classical NSAIDs. New chlorzoxazone ester prodrugs (6-8) of some acidic NSAIDs (1-3)

Full text of DOI:10.1016/j.bmc.2009.03.065

Bioorganic & Medicinal Chemistry 17 (2009) 3665–3670
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chlorzoxazone esters of some non-steroidal anti-inflammatory (NSAI)
carboxylic acids as mutual prodrugs: Design, synthesis, pharmacological
investigations and docking studies
*
Ahmed Z. Abdel-Azeem, Atef A. Abdel-Hafez , Gamal S. El-Karamany, Hassan H. Farag
Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery of the inducible isoform of cyclooxygenase enzyme (COX-2) spurred the search for anti-
inflammatory agents devoid of the undesirable effects associated with classical NSAIDs. New chlorzoxa-
zone ester prodrugs (6–8) of some acidic NSAIDs (1–3) were designed, synthesized and evaluated as
mutual prodrugs with the aim of improving the therapeutic potency and retard the adverse effects of gas-
trointestinal origin. The structure of the synthesized mutual ester prodrugs (6–8) were confirmed by IR,
1H NMR, mass spectroscopy (MS) and their purity was ascertained by TLC and elemental analyses. In vitro
chemical stability revealed that the synthesized ester prodrugs (6–8) are chemically stable in hydrochlo-
ric acid buffer pH 1.2 as a non-enzymatic simulated gastric fluid (SGF) and in phosphate buffer pH 7.4 as
non-enzymatic simulated intestinal fluid (SIF). In 80% human plasma, the mutual prodrugs were found to
be susceptible to enzymatic hydrolysis at relatively faster rate (t1/2 ꢀ 37 and 34 min for prodrugs 6 and 7,
respectively). Mutual ester prodrugs (6–8) were evaluated for their anti-inflammatory and muscle relax-
ation activities. Scanning electromicrographs of the stomach showed that the ester prodrugs induced
very little irritancy in the gastric mucosa of rats after oral administration for 4 days. In addition, docking
of the mutual ester prodrugs (6–8) into COX-2 active site was conducted in order to predict the affinity
and orientation of these prodrugs at the enzyme active site.
Received 10 December 2008
Revised 23 March 2009
Accepted 29 March 2009
Available online 8 April 2009
Keywords:
Chlorzoxazone
Non-steroidal anti-inflammatory
Lipophilicity
Muscle relaxant
Ulcerogenicity
Docking
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
synthesis in the GI tract. The indirect effect can be attributed to
combination of an ion trapping mechanism of NSAIDs from the
Limited utilization of a therapeutically significant drug in
clinical practice may be attributed to various shortcomings like
poor organoleptic properties, poor bioavailability, short duration
of action, incomplete absorption or undesirable side effects.1 The
non-steroidal anti-inflammatory drugs (NSAIDs) are among the
most widely prescribed drugs worldwide.2 NSAIDs are widely used
for indications extending from inflammation and pain to cardio-
vascular and genitourinary diseases.3 Despite the intensive
research that has been aimed at the development of NSAIDs, their
clinical usefulness is still restricted by their gastrointestinal side
effects like gastric irritation, ulceration, bleeding, and perforation
and in some cases may develop into life threatening conditions.4
GI mucosal injury produced by NSAIDs is generally believed to be
caused by two different mechanisms.5 The first mechanism
involves a local action composed of a direct contact while the other
has indirect effect on the GI mucosa. The direct contact effect can
be attributed to a combination of a local irritation produced by
acidic group of NSAIDs and local inhibition of prostaglandin
lumen into the mucosa. The second mechanism is based on a gen-
eralized systemic action occurring after absorption. Since the dis-
covery of COX-2, a second subtype of cyclooxygenase, selective
inhibitors or ‘coxibs’ were developed with the idea that this iso-
form was inducible at the site of inflammation. This new class of
non steroidal anti-inflammatory agents was thought to be safer
for ulcerations of the gastrointestinal mucosa observed with non
selective COX-2 inhibitors. Nevertheless, at the end of September
2004, Merck & Co announced the voluntary withdrawal of rofecox-
ib worldwide because of an increased risk of cardiovascular events.
This decision raised serious concerns about safety of selective COX-
2 inhibitors which are actively marketed today, and the ones cur-
rently under development.6,7 The GIT mucosal injury problems
have been overcome by derivatization of carboxylic function of
NSAIDs into ester and amide mutual prodrugs using amino acids
like L-tryptophan, L-histidine, and L-glycine as carriers that have
marked antiinflammatory activity of their own.8 Other analgesic,
antiinflammatory drugs like paracetamol and salicylamide have
also been used as carriers to synthesize mutual prodrugs of NSA-
IDs. Benorylate is a mutual prodrug of aspirin and paracetamol,
linked through ester linkage, which claims to have decreased
* Corresponding author. Tel.: +20 88 230 3006; fax: +20 88 233 2776.
E-mail address: atef@aun.edu.eg (A.A. Abdel-Hafez).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.065

3666
A. Z. Abdel-Azeem et al. / Bioorg. Med. Chem. 17 (2009) 3665–3670
gastric irritancy with synergistic analgesic action.9 Glycine methyl
ester conjugate of ketoprofen,10 histidine methyl ester conjugate of
diclofenac,11 and various conjugates of flurbiprofen with amino
CH2OH
N
H
N
Cl
Cl
HCHO, H O
2
O
O
Reflux
acid like
L
-tryptophan,
L
-histidine, phenylalanine and alanine as
O
O
mutual prodrugs12 were reported to have less ulcerogenicity with
better anti-inflammatory and analgesic action than their parent
drugs. Mutual prodrugs of ibuprofen with paracetamol and salicyl-
amide have been reported with better lipophilicity and reduced
gastric irritancy than the parent drug.13 Naproxen propyphenazone
mutual prodrugs were synthesized with an aim to improve thera-
peutic index through prevention of GI irritation and bleeding.14
Chlorzoxazone [5-chloro-2(3H)-benzoxazolone] is an active
muscle relaxant, while acetaminophen (N-acetyl-p-aminophenol)
exhibits analgesic properties. Owing to their synergistic effects,
these two drugs can be prescribed together.15,16 Using this ratio-
nale, a mutual prodrug of chlorzoxazone and acetaminophen has
been designed, and its synthesis and kinetics have been reported.17
A mutual prodrug consists of two pharmacologically active agents
coupled together so that each acts as a promoiety for the other
agent and vice versa.18 The active moiety selected may have the
same biological action as that of the parent drug and thus might
give synergistic action, or this moiety may have some additional
biological action that is lacking in the parent drug, thus ensuring
some additional benefit. The active moiety may also be a drug that
might help to target the parent drug to a specific site or organ or
cells or may improve site specificity of a drug. The drug may be
used to overcome some side effects of the parent drugs as well.18
Chlorzoxazone is a marketed as a single component muscle relax-
ant or in combination with some NSAIDs. On the basis of the men-
tioned reports, the present work reports on the design and
synthesis of different mutual ester prodrugs of chlorzoxazone 4
and some NSAIDs (1–3) with the objective of minimizing gastroin-
testinal side effects of NSAIDs and improving pharmacokinetic
properties of both chlorzoxazone 4 and NSAIDs (1–3) while main-
taining the useful anti-inflammatory and skeletal muscle relaxa-
tion activities.
5
4
RCOOH
DMAP, DCC
O
CH2O
R
Cl
N
O
O
R=
H3CO
7
6
H
N
8
Scheme 1. Pathway for the synthesis of the target ester prodrugs (6–8).
Lipophilicities of chlorzoxazone 4, hydroxymethyl chlorzoxa-
zone 5, the synthesized prodrugs (6–8) and their parent non-steroi-
dal anti-inflammatory drugs (1–3) were calculated using
PC-software package, which helps computation of the logP on the
basis of the fragment method developed by Leo.20 The results listed
in Table 2 (Supplementary data) revealed that the lipophilicities of
the prodrugs (6–8) increased vividly compared with the parent
drugs (1–3). The lipophilicity increament of the synthesized mutual
prodrugs (6–8) probably enhances their absorption and conse-
quently their bioavailability.
a
COOH
COOH
2.2. Stability studies
H3CO
2
1
The chemical and enzymatic hydrolysis of the synthesized ester
prodrugs (6–8) of the selected NSAIDs were studied at 37 °C in
aqueous buffer solutions of pH 1.2 and 7.4, which are considered
as a non-enzymatic simulated gastric fluid (SGF) and non-enzy-
matic simulated intestinal fluid (SIF), respectively, as well as in
80% human plasma and the reactions were monitored by HPLC.
The results indicate that the synthesized prodrugs (6–8) are suffi-
ciently stable at pH 1.2 for about one day monitoring, so that no
hydrolysis to the free acids might occur in the stomach. Similarly,
the chemical stability of the ester prodrugs (6–8) at pH 7.4 for one
day suggested that they are absorbed almost unchanged from the
intestine. On the other hand, the enzymatic hydrolysis of the ester
prodrugs (6–8) in human plasma demonstrates the susceptibility
of these esters for plasma esterases. The results revealed that the
rate of hydrolysis of ibuprofen and naproxen ester prodrugs 6
and 7 in human plasma are markedly accelerated, t1/2 is 37 and
34 min, respectively, compared with those in aqueous buffers.
The rate of hydrolysis of mepfenamic ester prodrug 8 was difficult
to be determined owing to solubility problems in human plasma.
The chemical stability of these orally designed ester prodrugs (6–
8) at pH values simulating the gastric fluids and their liability to
release the parent non-steroidal anti-inflammatory drugs (1–3)
and the muscle relaxant chlorzoxazone 4 are essential require-
ments for improving the bioavailability.
COOH
H
N
3
2. Results and discussion
2.1. Chemistry
Schematic representation of the reactions for the synthesis of
the mutual ester prodrugs (6–8) is depicted in Scheme 1. The syn-
thesis of the key intermediate, hydroxymethyl chlorzoxazone 5,
necessitate the reflux of chlorzoxazone 4 with formaldehyde solu-
tion (40%) according to the reported procedure.19 Subsequent
esterification of the resulting precursor 5 with the corresponding
non-steroidal anti-inflammatory carboxylic acid drugs (1–3) affor-
ded the mutual ester prodrugs (6–8) in good yields. The structure
of the synthesized prodrugs (6–8) were confirmed by IR, 1H NMR,
mass spectroscopy (MS) and their purity was ascertained by TLC
and elemental analyses.

A. Z. Abdel-Azeem et al. / Bioorg. Med. Chem. 17 (2009) 3665–3670
3667
2.3. Pharmacological evaluation
2.4. Docking studies
Non-steroidal anti-inflammatory drugs (1–3) and their corre-
sponding mutual ester prodrugs (6–8) were evaluated for their
in vivo systemic effect using carrageenan-induced paw edema bio-
assay in rats.21 Table 3 shows the results (% inhibition) of the anti-
inflammatory activity of the tested targets at different time inter-
vals. Ibuprofen 1 and ibuprofen ester prodrug 6 showed maximum
inhibition of inflammation ranging from 16.7% to 50%. On the other
hand, naproxen 2, mefenamic acid 3 and naproxen ester prodrug 7
were slightly active while the minimum inhibitory activity of
inflammation was observed to mefenamic ester prodrug 8. From
the results obtained the mutual ester prodrugs (6–8) exhibited com-
parable anti-inflammatory activity to their parents carboxylic acid
drugs (1–3). Also, muscle relaxant activity of the mutual ester pro-
drugs (6–8) was evaluated in comparison with muscle relaxant drug
chloroxazone 4 using rat sciatic nerve tibialis muscle preparation.22
The percentage inhibition of muscle contraction listed in Table 4
showed that all the tested targets (6–8) revealed muscle relaxation
activity. Moreover, the ester prodrug 6 displayed muscle relaxation
activity comparable to that of the reference drug chlorzoxazone 4.
Gastrointestinal toxicity of ibuprofen-chlorzoxazone mutual
prodrug ester 6 compared with the parent drug ibuprofen 1, the
most active anti-inflammatory drug tested, was examined under
electron microscope.23 Investigation of the stomach specimens of
the treated experimental animals under scanning electron
microscope afforded a highly precise method for examination of
ulcerogenic potential of NSAIDs. Figure 1, represents scanning elec-
tromicrographs for stomach specimens of rats treated with chronic
doses (150 mg/kg) of ibuprofen 1 (Fig. 1A), ibuprofen ester prodrug
6 (Fig. 1B), and the control group (Fig. 1C), which received only the
vehicle. Ibuprofen 1 treated animals were characterized by com-
plete damage of the mucous layer besides ulceration of submuco-
sal cells compared with that of control. The ester prodrug of
ibuprofen 6 treated animals showed damage of the mucosal layer
but to mush less extent than that of ibuprofen 1. From the above
finding, ibuprofen ester mutual produrg 6 proved to have superior
GI safety profile as compared to the reference drug ibuprofen 1.
Docking studies of non-steroidal anti-inflammatory drugs (1–3)
and their mutual prodrug esters with chlorzoxazone (6–8) were
performed by MOE (Molecular Operating Enviroment) using murine
COX-2 co-crystallized with SC-558 (PDB ID: 1CX2) as a template.24
The recent determination of the three-dimensional co-crystal
structure of murine COX-2 complexed with SC-558 has led to the
development of a model for the topography of NSAIDs binding site
in human COX-2. This might enable the prediction of the orienta-
tion and interaction of these drugs (1–3) and their ester prodrugs
(6–8) into COX-2 active site.25 We performed 100 docking itera-
tions for each ligand and the top scoring configuration of each of
the ligand-enzyme complexes was selected on energetic ground.
The output of docking simulation are the scoring function which
reflects the binding free energy dG in kcal/mol (S), value propor-
tional to the sum of Gaussian R1R2 exp (ꢁ0.5d2), where R1 and R2
are the radii of atoms in Angstrom Å and d is the distance between
the pair in Å (ASE) a linear combination of (S, ASE, Econf) where Econf
is an estimated self-energy of the ligand in kcal/mol (E). Docking
simulations revealed that the mutual ester prodrugs (6–8) inhibit
COX-2 enzyme in a quiet similar manner to those of their parent
non-steroidal anti-inflammatory drugs (1–3). These mutual ester
prodrugs (6–8) docked on cyclooxygenase enzyme demonstrates
dGester prodrug ꢀ dGNSAIDs 1 kcal/mol as shown in Table 5 (Supple-
mentary data). Naproxen 2 and ibuprofen ester prodrug 6 showed
the highest dG values of ꢁ12.65 and ꢁ11.69 kcal/mol, respectively.
Figure 2 shows the orientation of these drug and ester prodrug (2
and 6, respectively) into COX-2 enzyme active site. The above men-
tioned observations may provide an explanation for the potent
inhibitory activity of the ester prodrugs.
The introduction of mutual prodrug in human therapy has given
successful results in overcoming undesirable properties like
absorption, non-specificity, poor bioavailability and GIT toxicity.
In vitro and in vivo evaluation of the synthesized mutual ester
prodrugs (6–8) of chlorzoxazone 4 and some non-steroidal anti-
inflammatory drugs (1–3) revealed adequately enhanced lipophil-
icity, chemical stability, reduced ulcerogenic liability compared
Table 3
Anti-inflammatory activity of non-steroidal anti-inflammatory drugs (1–3) and their target esters (6–8)
Compound no.
Anti-inflammatory activity % inhibition of edema (mean) SEMa,*
0.5 h
1 h
2 h
3 h
4 h
5 h
Control
0.00
0.00
0.00
0.00
0.00
0.00
1
2
3
6
7
8
16.7 0.00
9.4 3013
16.7 1.36
16.7 0.00
19.1 0.95
14.3 1.1
16.7 0.00
16.3 3.61
18.3 3.47
16.7 1.36
19.6 2.86
14.8 0.00
45.8 4.17
28.1 3.13
33.3 4.1
33.3 0.00
18.7 2.35
23.4 3.21
62.5 4.17
46.9 3.13
41.7 4.81
45.8 4.17
33.3 3.96
30.5 4.5
50:0 0.00
37.5 0.00
45.8 4.17
50:0 0.00
38.1 4.17
25.7 3.3
41.7 4.81
18.8 3.6
33.3 2.72
45.8 4.17
23.8 0.00
20:0 3.47
a
SME denotes the standard error of the mean.
*
All data are significantly different from control (P < 0.01).
Table 4
Muscle relaxation activity of chlorzoxazone 4 and non-steroidal anti-inflammatory prodrugs (6–8)
Compound no
Muscle relaxation activity % inhibition of muscle contraction SEMa
10 min
20 min
30 min
45 min
60 min
75 min
90 min
120 min
4
6
7
8
18.5 1.2**
2.5 1.04
25.88 0.6**
10.5 0.68**
8.6 0.66**
5.59 1.81**
33.43 1.23**
19.95 1.96**
14.25 0.63**
10.78 0.62**
35.26 1.37**
28.35 5.07**
20.73 0.93**
16.55 1.83**
33.45 2.83**
35.13 2.33**
23.65 3.89**
19.48 1.62**
21.65 1.46**
33.2 1.72**
12.9 1.45**
9.65 1.40**
15.25 0.85**
0.00
27.05 1.68**
8.00 0.51**
4.58 0.92*
18.25 1.3**
0.00
5.89 0.49**
2.78 1.00
0.00
a
SEM denotes the standard error of the mean.
*
Data are significantly different from control (P < 0.05).
All data are significantly different from control (P < 0.01).
**

3668
A. Z. Abdel-Azeem et al. / Bioorg. Med. Chem. 17 (2009) 3665–3670
Figure 2. Docking of (A) naproxen 2 (yellow stick) and (B) ibuprofen ester prodrug
6 (green stick) in the active site of COX-2 enzyme. Hydrogen atoms of the amino
acid residues have been removed to improve clarity.
Thin layer chromatography was carried out on pre-coated Silica
gel 60 F254 plates (0.25 mm thickness, Merck, Darmastat,
Germany) and spots were detected under UV light. Silica gel 60
(60–230 mesh, Merck, Darmastat, Germany) was used for column
chromatography. IR spectra were recorded as KBr discs on a shima-
dzu IR 400-91527 (Shimadzu, Kyoto, Japan) at the Faculty of Phar-
macy, Assiut University, Assiut, Egypt. 1H NMR spectra were
measured on Varian EM-360L, NMR Spectrophotometer (60 MHz)
(Varian, USA) at the Faculty of Pharmacy, Assiut University, Assiut,
Egypt. Chemical shifts were given in d ppm relative to tetrameth-
ylsilane (TMS). Electron impact (EI) Mass Spectra were measured
with JEOL spectrophotometer (JEOL, Tokyo, Japan) at an ionization
voltage of 70 eV at the central laboratory, Assiut University, Assiut,
Egypt and at the Microanalytical center, Cairo University, Cairo,
Egypt. Elemental microanalyses were performed at the Microana-
lytical Center, Faculty of Science, Assiut University, Assiut, Egypt.
All chemicals used were of analytical grade.
Figure 1. Scanning electromicrographs of rat stomach specimen after chronic doses
(4 days) of: (A) ibuoprofen; (B) chlorzoxazone ester with ibuoprofen; (C) control.
with the corresponding parent drugs. The minimized side effects
obtained in the prodrug might be due to inhibition of direct contact
of carboxyl group of the drug to the gastric mucosa, which is
mainly responsible for the damage. It is also due to negligible dis-
solution as well as hydrolysis in acidic buffer (pH 1.2). On the basis
of the results, it is concluded that prodrug approach can be suc-
cessfully applied in attaining the goal of minimized gastrointesti-
nal toxicity without loss of desired anti-inflammatory and
analgesic activities of the drug. As indicated, mutual prodrug
approach offers a very fruitful area of research and an efficient tool
for improving the clinical and therapeutic effectiveness of a drug
that is suffering from some undesirable properties hindering its
clinical usefulness otherwise.
HPLC analysis was carried out a Knauer HPLC system, consisting
of a pump (KNAUER WellChrom Mini-Star K-500 HPLC pump, Ger-
many), a variable wavelength UV detector (KNAUER), a Shimadzu
C-R 6A chromatopac recording integrator, and a 20
loop (KNAUER). The column used was a reversed phase SUPELCO
m particle) connected with a
lL injection
Discovery C18 (250 ꢂ 4.6 mm; 5
l
cartridge guard column. The chromatographic conditions for each
of the studied compounds are summarized in Table 2 (Supplemen-
tary data). Quantification of the eluted compounds was obtained
from the area under the peak measurements in relation to those
of standards chromatographed under the same conditions.
3. Experimental
Melting points were determined on an electrothermal melting
point apparatus (Stuart scientific, England) and are uncorrected.
The anti-inflammatory and muscle relaxation activities were
performed at the Department of Pharmacology, Faculty of

A. Z. Abdel-Azeem et al. / Bioorg. Med. Chem. 17 (2009) 3665–3670
3669
Medicine, Assiut University, Assiut, Egypt. The ulcerogenic poten-
7.4) and incubated at 37 °C. At appropriate time intervals 200
of the plasma reaction were withdrawn and deproteinized by mix-
ing with 300 L of acetonitrile. After centrifugation for 10 min at
104 rpm, 20 L of the clear supernatant was analyzed by HPLC.
lL
tial was detected with a JEOL, JSM-5400LV Scanning Electron
Microscope (Electron Microscope Unit, Assiut University, Assiut,
Egypt).
l
l
Docking studies were carried out at the Department of Pharma-
ceutical Medicinal Chemistry, Faculty of Pharmacy, Assiut Univer-
sity, Assiut, Egypt.
3.4. Biological evaluations
3.4.1. Anti-inflammatory activity
3.1. Chemical synthesis
The anti-inflammatory activity of the synthesized derivatives
(6–8) in comparison with their parent NSAI drugs (1–3) was eval-
uated against carrageenan-induced rat paw edema.21 Adult male
Wister rats weighing (100–130 g) were divided into seven groups,
four animals each. Target compounds were suspended in aqueous
solution of carboxymethyl cellulose (CMC, 0.5% w/v) and adminis-
tered orally at a dose level of 150, 40, 20 mg/kg of NSAI drugs (1–
3), respectively and their equivalent doses of the target derivatives
(6–8). Control animals were similarly treated with aqueous solu-
tion of carboxymethyl cellulose (CMC, 0.5% w/v). After 30 min
0.1 mL of freshly prepared 1% carrageenan solution in normal sal-
ine was injected into the subplantar region of the right hind paw
of each rat. The thickness of paw edema was determined by means
of skin caliper at different time intervals (1/2, 1, 2, 3, 4, and 5 h)
after administration of the test compounds. The difference
between the thicknesses of two paws (right and left) was taken
as a measure of edema. The percent inhibition of edema was calcu-
lated as follows:
3.1.1. Synthesis of 5-chloro-3-(hydroxymethyl)benzo[d]oxazol-
2(3H)-one (5)19
A suspension of chlorzoxazone 4 (1.7 g, 0.01 mol) and 5 mL
formaldehyde solution (40%) in water (100 mL) was refluxed for
three hours. The hot solution was left to cool where crystals are
formed. The separated crystalline product was filtered and dried
to afford hydroxymethyl chloroxazone 5. Physical, IR, and 1H
NMR data are listed in Table 1 (Supplementary data).
3.1.2. General method for the synthesis of the target derivatives
(6–8)
To an ice-cold solution of the appropriate non-steroidal anti-
inflammatory drug (3 mmol) in 30 ml dichloromehane, hydroxy-
methyl chlorzoxazone 5 (3 mmol), dimethylaminopyridine (DMAP,
20 mg), and dicyclohexylcarbodiimde (DCC, 3.3 mmol) were
added. The reaction mixture was stirred at 4 °C for 1 h and kept
overnight at room temperature. The precipitated formed was sep-
arated by filtration and the filtrate was washed with cooled 0.05 N
HCl followed by saturated solution of NaHCO3 and finally with
brine, dried over anhydrous Na2SO4, and evaporated under
reduced pressure. The residue was purified with column chroma-
tography using a mobile phase system of hexane and ethyl acetate
in ratio (9:1) for esters (6 and 7), and ratio (20:1) for ester (8).
Physical, IR, and 1H NMR data are listed in Table 1 (Supplementary
data).
ðVR ꢁ VLÞ control ꢁ ðVR ꢁ VLÞ treated ꢂ 100
% Edema inhibition ¼
ðVR ꢁ VLÞ control
Where VR represents the mean of the right paw displacement vol-
ume, and VL represents the mean of the left paw displacement vol-
ume. The results of the anti-inflammatory evaluation are listed in
Table 3.
3.4.2. Muscle relaxant activity
The muscle relaxant activity of hydroxymethyl chlorzoxazone 5,
and the target esters (6–8) in comparison with chlorzoxazone 4
was evaluated using rat sciatic nerve tibialis muscle preparation.22
Male adult Wister rats (100–130 g), were divided into four groups,
four rats each, were anesthetized by intraperitoneal injection with
50 mg/kg urethane. The anterior tibialis muscle was carefully dis-
sected and exposed so that the blood and nerve supply were intact.
The sciatic nerve which supplies the muscle was exposed in the
gluteal region. The distal tendon of the muscle was tied to silk
threads which were attached to a T2 isotonic transducer and an
amplifier of a two-channel oscillograph MD2 (Bioscience, Kent,
UK). The hind limb was tidily fixed in a horizontal position and
the muscle was adjusted to resting length. The nerve was stimu-
lated using an electronic square wave physiograph stimulator with
pulses of 2 ms duration, 5 V intensity, and 5 Sꢁ1 frequencies. After
the preparation becomes stable, the height of the tibialis muscle
contractions was determined before and after injection, intraperi-
toneally at a dose of 50 mg/kg chlorzoxazone 4 or equivalent dose
of the target ester prodrugs (6–8), at different time intervals of any
of the tested compounds under investigation into the animal. The
percentage change in the height of contraction was determined
as indicator of muscle relaxation activity. The results of the muscle
relaxation evaluation are listed in Table 4.
3.2. Calculation of logP values
The logP values of the NSAIDs (1–3), chloroxazone 4, hydroxy-
methyl chlorxazone 5 and the target derivatives (6–8) were
computed with a routine method called calculated logP (ClogP)
contained in a PC-software package (Mac logP 2.0, BioByte Corp.,
CA, USA). A representation of the molecular structure where hydro-
gens are omitted, or suppressed (SMILES notation), is entered into
the program, which computes the logP based on the fragment
method developed by Leo.20 Results are given in Table 2 (Supple-
mentary data).
3.3. In vitro experiments
3.3.1. Chemical hydrolysis
The hydrolysis of the synthesized derivatives (6–8) was studied
in hydrochloric acid buffer (pH 1.2) and phosphate buffer (pH 7.4),
containing 1% tween 20. The ionic strength (
maintained 0.2 by adding a calculated amount of potassium chlo-
ride. The reactions were initiated by adding 200 L of stock meth-
anolic or acetonitrile solution of the target derivative (3 mg/mL) to
9.8 mL of preheated buffer solutions in screw-capped test tubes.
The solutions were kept in a water bath at constant temperature
l) for each buffer was
l
(37 °C), at appropriate time intervals, 20 lL were withdrawn and
analyzed by HPLC for the residual target derivative.
3.4.3. Ulcerogenicity studies23
Male adult Wister rats (150–200 g) were divided into three
groups, three animals each. Rats were starved but had free access
to water for 12 h prior to the administration of the drug. The first
group was administered a daily oral dose, (150 mg/kg) as a 1 mL
suspension of Ibuprofen 1 in 0.5% solution of carboxymethyl
3.3.2. Enzymatic hydrolysis
The reactions were initiated by adding 50 lL of stock solution of
the target derivatives (6–8) in DMSO (4 mg/mL) to 1.95 mL of pre-
incubated 80% human plasma with isotonic phosphate buffer (pH

3670
A. Z. Abdel-Azeem et al. / Bioorg. Med. Chem. 17 (2009) 3665–3670
cellulose (CMC), for four successive days. In a similar manner, the
second group received equivalent doses of the corresponding ester
6. The third group was administered equivalent amount of vehicle
and considered as the control group. Food was withdrawn from all
groups until 24 hr after the last dose. The rats were then sacrificed;
so that the stomach could be removed, opened along the greater
curvature and cleaned gently by dipping with saline. Randomly
selected specimens were then taken and prepared for scanning in
an electron microscope. Specimens were fixed by soaking in glutar-
aldehyde solution (5% in cacodylate buffer; pH 7.2) for 24 h fol-
lowed three washings each for 20 min with cacodylate buffer.
The specimens were then treated with osmium tetraoxide (1%
solution) for 2 h and washed with cacodylate buffer as shown
above. The specimens were subjected to dehydration by treatment
for 30 min with each of 30%, 50% and 70% ethanolic solution fol-
lowed by 90% ethanol for 1 h and finally in absolute ethanol for
two days. After discharge of the alcohol the specimens were soaked
in amyl acetate solution for two days, dried under reduced pres-
sure, mounted on holders and coated for scanning in electron
microscopy.
(ii) Hydrogen atoms were added to the structure with their stan-
dard geometry. (iii) MOE Alpha Site Finder was used for the active
sites search in the enzyme structure and dummy atoms were
created from the obtained alpha spheres. The obtained ligand–en-
zyme complex model was then used in calculating the energy
parameters using MMFF94x force field energy calculation and
predicting the ligand–enzyme interactions at the active site.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.03.065.
References and notes
1. Brahmankar, D. M.; Jaiswal, S. B. Biopharmaceutics and Pharmacokinetics a
Treatise; Vallabh Prakashan: New Delhi, 1995.
2. Fiorucci, S.; Meli, R.; Bucci, M.; Cirino, G. Biochem. Pharmacol. 2001, 62, 1433.
3. Mishra, A.; Veerasamy, R.; Jain, P. K.; Dixit, V. K.; Agrawal, R. K. Eur. J. Med.
Chem. 2008, 43, 2464.
4. Robert, T. S.; Ronald, J. V. Am. J. Med. 1989, 86, 449.
5. Buckley, M. M. M.; Brogden, R. N. Drugs 1990, 39, 86.
6. Dogne, J. M.; Supuran, C. T.; Praticò, D. J. Med. Chem. 2005, 48, 2251.
7. Dogne, J. M.; Hanson, J.; Supuran, C.; Pratico, D. Curr. Pharm. Des. 2006, 12, 971.
8. Meyers, J.; Braine, E.; Moonka, D. K.; Davis, R. H. Inflammation 1979, 3, 225.
9. Croft, D. N.; Cuddigan, J. H. P.; Sweetland, C. Br. Med. J. 1972, 3, 545.
10. Dhaneshwar, S. S.; Chaturvedi, S. C. Indian Drugs 1994, 31, 374.
11. Dhaneshwar, S. S.; Dhaneshwar, S. R.; Chaturvedi, S. C. Eastern Pharm. 1996, 119.
12. Gairola, N.; Nagpal, D.; Dhaneshwar, S. R.; Dhaneshwar, S. S.; Chaturvedi, S. C.
Indian J. Pharm. Sci. 2005, 67, 369.
13. Bhosale, A. V.; Agarwal, G. P.; Mishra, P. Indian J. Pharm.Sci. 2004, 66, 158.
14. Sheha, M.; Khedr, A.; Elsherief, H. Eur. J. Pharm. Sci. 2002, 17, 121.
15. Elenbaas, J. K. Am. J. Hospital Pharm. 1980, 37, 1313.
16. Stewart, J. T.; Janicki, C. A. Anal. Profile Drug Substances 1987, 16, 119.
17. Vigroux, A.; Bergon, M. Bioorg. Med. Chem. Lett. 1995, 5, 427.
18. Bhosle, D.; Bharambe, S.; Gairola, N.; Dhaneshwar, S. S. Indian J. Pharm. Sci.
2006, 3, 286.
19. Varma, R. S.; Nobles, W. L. J. Pharm. Sci. 1968, 57, 39.
20. Leo, A. J. Chem. Rev. 1993, 63, 1281.
21. Di Rosa, M.; Willoughby, D. A. J. Pharm. Pharmacol. 1971, 23, 297.
22. Ahmed, O. A. Pharmacol. Res. 2000, 41, 163.
23. Omar, F. A.; Mahfouz, N. M.; Rahman, M. A. Eur. J. Med. Chem. 1996, 31, 819.
24. Molecular Operating Environment (MOE), version 2005.06. Chemical Computing
Group, Inc. Montreal, Quebec, Canada, 2005, http://www.chemcomp.com.
25. Garavito, R. M.; Malkowski, M. G.; Witt, D. L. Prostagland. Lipids Mediators 2002,
68, 129.
3.5. Docking studies
Docking studies were performed using ‘Molecular Operating
Environment (MOE) version 2005.06, Chemical Computing Group
Inc., 1010 Sherbrooke Street West, Suite 910, Montreal, H3A 2R7,
Canada. The program operated under ‘Windows XP’ operating sys-
tem installed on an Intel Pentium IV PC with a 2.8 MHz processor
and 512 RAM. Non-steroidal anti-inflammatory drugs (1–3) and
their corresponding ester prodrugs (6–8) were built using the
builder interface of the MOE program and subjected to energy min-
imization tool using the included MOPAC 7.0. The produced model
was subjected to Systematic Conformational Search where all
items were set as default with RMS gradient of 0.01 kcal/mol and
RMS distance of 0.1 A°.
The X-ray crystallographic structure of cyclooxygenase-2
(murine COX-2) complexed with SC-558 (PDB ID: 1CX2) was ob-
tained from the Protein Data Bank.26 The enzyme was prepared
for docking studies where: (i) the ligand molecule with any exist-
ing solvent molecules were removed from the enzyme active site.
26. Pouplana, R.; Lozano, J. J.; Ruiz, J. J. Mol. Graph. Model. 2002, 20, 329.