Design, synthesis, biological evaluation, and comparative Cox1 and Cox2 docking of p-substituted benzylidenamino phenyl esters of ibuprofenic and mefenamic acids

Article abstract of DOI:10.1016/j.bmc.2011.12.030

Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently associated with gastric mucosal and renal adverse reactions, related to inhibition of cyclooxygenase1 (Cox1) in tissues where prostaglandins exert physiological effects. This led us to develop a set of ibuprofenic acid and mefenamic acid esters, namely: 4-((4-substituted benzylidene)amino)phenyl 2-(4-isobutylphenyl) propanoate and 4-((4-substituted benzylidene)amino)phenyl 2-((2,4- dimethylphenyl)amino)benzoate analogs, which were synthesized by condensation of the corresponding acids with Schiff's bases [4-(4-substituted benzylideneamino)phenols] involving dicyclohexyl carbodiimmide (DCC) as mild dehydrating agent. The main objective is to reduce the GIT toxicity associated with acute and chronic NSAIDs use. Anti-inflammatory, analgesic as well as ulcerogenic activities of the prepared esters were evaluated in vivo and compared with that of ibuprofen as reference standard in all screenings, involving the carrageenan induced paw oedema model and hot plate method. Most of the synthesized esters showed remarkable analgesic and anti-inflammatory activities. Interestingly, all of the compounds were found to be non-ulcerogenic under the tested conditions. This evidence have suggested that modification of the carboxyl function of representative NSAIDs results in retained or enhanced anti-inflammatory and analgesic activities with reduced ulcerogenic potential. Additionally, a comparative AutoDock study into Cox 1 and Cox2 has been done involving both of rigid and flexible docking for potential selectivity of our compounds within different Cox enzymes and to find out the binding orientation of these novel esters into their binding site. Some of the newly prepared aforementioned compounds showed considerable more Cox2 over Cox1 binding affinities by flexible docking better than rigid one.

Full text of DOI:10.1016/j.bmc.2011.12.030

Bioorganic & Medicinal Chemistry 20 (2012) 1259–1270
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, biological evaluation, and comparative Cox1 and Cox2
docking of p-substituted benzylidenamino phenyl esters of ibuprofenic
and mefenamic acids
Gehan H. Hegazy a, Hamed I. Ali b,
⇑
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Helwan University, Helwan, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently associated with gastric mucosal and renal
adverse reactions, related to inhibition of cyclooxygenase1 (Cox1) in tissues where prostaglandins exert
physiological effects. This led us to develop a set of ibuprofenic acid and mefenamic acid esters, namely:
4-((4-substituted benzylidene)amino)phenyl 2-(4-isobutylphenyl)propanoate and 4-((4-substituted ben-
zylidene)amino)phenyl 2-((2,4-dimethylphenyl)amino)benzoate analogs, which were synthesized by
condensation of the corresponding acids with Schiff’s bases [4-(4-substituted benzylideneamino)phe-
nols] involving dicyclohexyl carbodiimmide (DCC) as mild dehydrating agent. The main objective is to
reduce the GIT toxicity associated with acute and chronic NSAIDs use. Anti-inflammatory, analgesic as
well as ulcerogenic activities of the prepared esters were evaluated in vivo and compared with that of
ibuprofen as reference standard in all screenings, involving the carrageenan induced paw oedema model
and hot plate method. Most of the synthesized esters showed remarkable analgesic and anti-inflamma-
tory activities. Interestingly, all of the compounds were found to be non-ulcerogenic under the tested
conditions. This evidence have suggested that modification of the carboxyl function of representative
NSAIDs results in retained or enhanced anti-inflammatory and analgesic activities with reduced ulcero-
genic potential. Additionally, a comparative AutoDock study into Cox 1 and Cox2 has been done involving
both of rigid and flexible docking for potential selectivity of our compounds within different Cox enzymes
and to find out the binding orientation of these novel esters into their binding site. Some of the newly
prepared aforementioned compounds showed considerable more Cox2 over Cox1 binding affinities by
flexible docking better than rigid one.
Received 3 October 2011
Revised 14 December 2011
Accepted 14 December 2011
Available online 22 December 2011
Keywords:
NSAIDs
Molecular docking
Ibuprofen
Mefenamic acid
Cox
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
In recent years, however, serious cardiovascular effects of some
selective Cox2 inhibitors emerged from clinical studies and phar-
Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the
most widely used drugs worldwide and represent a mainstay in
the therapy of acute and chronic pain. However, their use is
frequently associated with a broad spectrum of adverse effects,
which are related to inhibiting prostaglandin synthesis in tissues
where PGs are responsible for physiological homeostasis.1 A lot
of strategies have emerged to improve the therapeutic efficacy
and tolerability of NSAIDs via defining novel targets in the complex
picture of the inflammatory process or modifying classical NSAIDs
by adding chemical moieties that release gastroprotective media-
tors. These trials include development of highly selective Cox2
inhibitors, referred to as ‘coxibs’, were rapidly introduced in the
market and gained an impressive success.2,3
macosurveillance, forcing the drug companies to withdraw from
the market rofecoxib and, soon afterwards, valdecoxib.4,5 Follow-
ing the withdrawal of rofecoxib, which has been considered the
most serious disaster after thalidomide, another strategy involved
the development of dual inhibitors of Cox and 5-lypooxygenase
(5-LOX) by blocking the formation of both prostaglandins and leu-
cotrienes but do not affect lipoxin formation. Such combined inhi-
bition avoids some of the disadvantages of selective Cox2
inhibitors and spares the gastrointestinal mucosa.6 An alternative
strategy to limit the risk of GI damage induced by NSAIDs is to en-
hance the protective mechanisms of the gastric mucosa. This can
be pursued by two gaseous mediators; nitric oxide (NO) and
hydrogen sulfide (H2S) which exert protective effects on gastric
mucosa. The inhibitory effects of NO on nonsteroidal anti-inflam-
matory drugs-induced leukocyte adherence have been exploited
in the development of NO-releasing nonsteroidal anti-inflamma-
tory drugs known as Cox-inhibiting NO donors (CINODs). This class
⇑
Corresponding author. Tel.: +20 101785626, +966560011297; fax:
+9665270000x4225.
E-mail address: hamed_ali37@yahoo.com (H.I. Ali).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.030

1260
G. H. Hegazy, H. I. Ali / Bioorg. Med. Chem. 20 (2012) 1259–1270
of anti-inflammatory agents reduces systemic blood pressure and
might have enhanced cardiovascular safety than coxibs, while
causing less gastrointestinal damage than its parent drug.7 H2S-
releasing NSAID derivatives have been recently developed based
on the observed ability of this gaseous mediator to cause vasodila-
tation and to prevent leukocyte adherence. In preclinical settings,
H2S-releasing NSAIDs produce less gastric damage as compared
to the parent drugs8.
liability. In fact, many of the designed compounds were found to
possess much significant analgesic and anti-inflammatory profile
with significant reduction in potential for ulcerogenic toxicities.
Additionally, comparative docking study was carried out by differ-
ential binding of the synthesized compounds into the active sites
of Cox1 and Cox2. Where, a series of computations were performed
for the prediction of mode of their binding affinities using flexible
AutoDock 4.2.
Another way to avoid GIT complications of NSAIDs involves
synthesis of the ester form of the corresponding irritant parent
compounds. Where, phenylcarbamoylmethyl ester of ibuprofen,
naproxen, and N-acetylanthranilic acid were recently synthesized
and tested for their anti-inflammatory activity. Further the ulcero-
genic liability and PGE2 inhibitory properties for the most active
compounds were determined. Results showed that all the tested
compounds exhibited promising anti-inflammatory activity, com-
pared to ibuprofen and naproxen, with marked decreases in the
ulcerogenic side effects. Moreover, esterification of both ibuprofen
and naproxen derivatives led to increases in the anti-inflammatory
activity, compared to the parent drugs, and this was enhanced in
the case of the 4-methoxyphenylcarbamoyl methyl ester and the
phenylcarbamoylmethyl ester of ibuprofen and naproxen, respec-
tively.9 Ester and amide prodrugs of ibuprofen and naproxen were
synthesized and evaluated for anti-inflammatory activity and gas-
trointestinal toxicity. All prodrugs, except the glycine amide, were
significantly less irritating to the gastric mucosa and exhibited sig-
nificantly better activity (p <0.01) than the parent compounds.10 In
addition esterification increases Cox2 selectivity in esters rather
than the acids.11
Gastroprotective agents are commonly co-prescribed with NSA-
IDs to protect against these side effects such as misoprostol, Cox2
specific and selective NSAIDs, and probably proton pump inhibi-
tors significantly reduce the risk of symptomatic ulcers.12 In spite
of the unprecedented advances in drug discovery, developing a
safe, effective and economical therapy for treating inflammatory
conditions still presents a major challenge. Various Computer
Aided Drug Design (CADD) approaches were introduced to develop
Cyclooxygenase based anti-inflammatory and anticancer drugs.13
Flexible docking was carried out for structurally diverse Cox2
inhibitors. The obtained docking score was correlated with the bio-
logical activities. The detailed analysis of the resulted Cox2–ligand
complexes may improve our knowledge in understanding the
binding interactions in detail. Moreover, a discovery of a phenothi-
azine derivative as a new cyclooxygenase-2 lead compound was
done through 3-D database searching and combinatorial chemistry
to serve as a lead compound for a potentially novel series of anti-
inflammatory compounds.14 Pathway reconstruction and kinetic
modeling are two approaches of computational systems biology
were applied involving classical biochemistry, genomics, proteo-
mics and metabolomics. These models quantitatively describe
the changes in dynamics and regulations of the pathways caused
by the following NSAIDs: aspirin, celecoxib, diclofenac, naproxen,
indomethacin, and ibuprofen.15
2. Results and discussion
2.1. Chemistry
Synthetic approaches based upon chemical modification of
NSAIDs have been taken with the aim of improving safety profile
and in turn therapeutic window of the NSAIDs in this study,
namely ibuprofen and mefenamic acid. Where several studies have
described the derivatization of the carboxylate function23–25 of
NSAIDs with less acidic analogs which resulted in an increased
anti-inflammatory activity with reduced ulcerogenicity. The requi-
site starting intermediate Schiff’s bases, namely 4-(4-substituted
benzylidene-amino)phenols (2a–d), which were used as precur-
sors for synthesis of 4-(4-substituted benzylideneamino)phenyl
2-(4-isobutylphenyl)propanoate (3a–d) and 4-(4-substituted ben-
zylideneamino)phenyl 2-(2,4-dimethylphenylamino) benzoate (
4a–d), were synthesized by treatment of equimole of p-amino phe-
nol (1) and p-substituted aromatic aldehyde in ethanol under re-
flux for 6–8 h as shown in Scheme 1 to afford yellow needles
recrystallized from ethanol in 70–85% yields. On the other hand,
The prodrugs, 4-(4-substituted benzylideneamino)phenyl 2-(4-iso-
butylphenyl)propanoate (3a–d) and 4-(4-substituted benzylide-
neamino) phenyl 2-(2,4-dimethylphenylamino)benzoate (4a–d),
were prepared by condensation reaction of the corresponding
anti-inflammatory acid (ibuprofen or mefenamic acid) with the
intermediate Schiff’s bases, 4-(4-substituted benzylideneami-
no)phenols (2a–d) in THF/dichloromethan at room temperature
overnight, involving dicyclohexyl carbodiimmide (DCC) as mild
dehydrating agent to avoid the hydrolysis of Schiff’s base, where
the products were obtained as yellow needles in 52–84% yields.
IR, 1H NMR spectra, and elemental analyses, were used for deter-
mination and identification of the newly assigned structures. The
structures of the ibuprofen esters 3a–d were confirmed in particu-
lar by the presence of an equivalent proton resonance of ylidenea-
mino moiety (–CH@N) as a singlet signal at dH 8.36–8.62 in 1H NMR
spectra. Also the mefenamic acid esters 4a–d showed that charac-
teristic singlet signal at dH 8.37–8.57. It is implying that ylideneami-
no proton (–CH@N) is the most electron-deficient nucleophile in
the lowest magnetic field. The 4-(4-substituted benzylideneami-
no)phenyl 2-(4-isobutylphenyl)propanoate (3a–d) and 4-(4-substi-
tuted benzylideneamino)phenyl 2-(2,4-dimethylphenylamino)
benzoate (4a–d) were differentiated from the intermediate Schiff’s
base, 4-(4-substituted benzylideneamino)phenols (2a–d) by the
absence of the singlet signal of the aromatic C–OH proton in their
1H NMR spectra which is obviously shown in the starting precur-
sors 2a–d and disappeared on esterification with the appearance
of additional aromatic protons as multiplets at dH 6.72–8.02 and
6.88–8.02 in ibuprofen esters 3a–d and mefenamic acid esters
4a–d, respectively. Moreover, mefenamic acid esters 4a–d exhibit
singlet NH proton, exchangeable with D2O at dH 5.46–5.50.
Schiff’s bases have a wide range of biological activities such as
antimalarial,16 anticancer,17 antibacterial,18 antifungal,19 antitu-
bercular,20 anti-inflammatory, antimicrobial,21 and antiviral,22
etc. They also serve as a back bone for the synthesis of various het-
erocyclic compounds.
The above circumstances led us to seek a convenient synthetic
route for replacing the carboxylic acid group of ibuprofenic and
mefenamic acids with less acidic p-substituted benzylidenamino
phenyl esters. In our attempt to accentuate potency and reduce
GI toxicities associated with the parent ibuprofenic and mefenamic
acids due to their free –COOH group. These compounds were
investigated for their biological activities including in vitro acute
anti-inflammatory effect, analgesic activity, as well as ulcerogenic
2.2. Biological evaluation
In the pharmacological study, we have investigated anti-inflam-
matory and analgesic activities as well as the acute ulcerogenicity
of both p-substituted benzylidenamino phenyl esters of ibuprofe-

G. H. Hegazy, H. I. Ali / Bioorg. Med. Chem. 20 (2012) 1259–1270
1261
Scheme 1. General method for the preparation of 4-(4-substituted benzylideneamino)phenols (2a–d), 4-(4-substituted benzylideneamino)phenyl 2-(4-isobutylphenyl)pro-
panoate (3a–d), and 4-(4-substituted benzylideneamino)phenyl 2-(2,4-dimethylphenylamino) benzoate (4a–d). Reagent and conditions: (a) Ar-CHO, AcOH, EtOH, reflux, 6–
8 h; (b) ibuprofen, Schiff0s base, THF/dichloromethan, dicyclohexyl carbodiimmide (DCC), rt, overnight; (c) mefenamic acid, Schiff’s base, THF/dichloromethan, dicyclohexyl
carbodiimmide (DCC), rt, overnight.
nic acid (3a–d) and p-substituted benzylidenamino phenyl esters
Table 1
of mefenamic acid (4a–d), utilizing the carrageenan-induced rat
Acute anti-inflammatory effect of the tested ester derivatives (3a–d and 4a–d) in
anti-inflammatory testing, hot plate analgesia testing, and influ-
ence on the gastric irritation, respectively. Ibuprofen, one of the
parent compounds, was used as a reference standard control, and
the control group animals were given saline with few drops of car-
boxymethyl cellulose (CMC) 0.5% (0.3 ml/100 g rat).
comparison to ibuprofen involving carrageenan-induced paw oedema technique
Group
Oedema volume (% inhibition)
1 h
2 h
3 h
74.63 7.13*
4 h
78.14 7.10*
Control
3a
3b
3c
3d
4a
4b
4c
55.76 7.84*
53.96 7.03*
29.85 6.15*
71.69 3.65*
36.54 2.63*
35.18 4.13*
36.90 6.59*
41.64 3.74*
31.99 5.25*
30.03 4.11*
68.60 7.14*
56.17 6.43*
44.40 7.51*
96.14 6.89*
40.59 1.94*
49.44 7.06*
41.11 5.53*
45.60 4.38*
38.47 5.78*
36.18 5.04*
61.25 6.05*
50.32 8.91*
103.75 5.62*
44.72 3.46*
57.03 7.30*
43.78 6.38*
53.43 5.58*
46.45 6.54*
41.84 6.04*
64.68 7.10*
53.78 8.86*
107.35 6.81*
49.30 3.96*
62.11 7.80*
52.47 8.43*
63.45 5.09*
51.70 7.03*
43.96 6.06*
2.2.1. Acute anti-inflammatory effect (carrageenan-induced
paw oedema)
Carrageenan-induced paw oedema bioassay in rats was in-
volved as a suitable experimental animal model for evaluating an
anti-inflammatory effect of the newly synthesized compounds:
3a–d and 4a–d. Results were expressed as mean S.E. Difference
between vehicle control and treatment groups were statistically
tested using repeated measures one way ANOVA, followed by least
significant test for multiple comparisons. Methods of statistical
analysis were done according to Armitage et al.32
4d
Ibuprofen
Values represent the mean S.E. of six rats for each group.
Control group animals were given saline with few drops of carboxymethyl cellulose
0.5%.
*
Statistically significant (P <0.05) from the control normal inflamed group at the
To demonstrate the validity of the carrageenan-induced paw
oedema test, rats were administered ibuprofen orally as a positive
control 100 mg/kg bwt, then compared to rats which were admin-
istered various synthesized compounds 3a–d and 4a–d 100 mg/kg
bwt. Both of reference and the test compounds were orally admin-
istered 1 h before carrageenan administration. Obviously, as cited
in Table 1 and Figure 1, all the involved esters of ibuprofenic and
mefenamic acids at 100 mg/kg bwt, decreased the paw volume sig-
nificantly (p <0.05) after all times of its administration at 1, 2, 3,
and 4 h (3a–d, and 4a–d). Where 4-(4-nitro benzylideneami-
no)phenyl 2-(4-isobutylphenyl)propanoate (3b) inhibited paw oe-
dema significantly after all times of its administration especially
after 1 h, where it decreased the inflamed paw volume significantly
(p <0.05) to 29.85% more than that of ibuprofen (30.03%). This indi-
cates that this p-nitro ester derivative of ibuprofen has potential
anti-inflammatory activity slightly higher than its corresponding
acid analog.
corresponding time, using one way ANOVA.
tuted
benzylideneamino)phenyl
2-(2,4-dimethylphenylami-
no)benzoate derivatives (4a–d) significantly reduce the inflamed
paw volume especially compounds 4-(4-nitro benzylideneami-
no)phenyl 2-(2,4-dimethylphenylamino)benzoate (4b), and 4-
(4-bromo benzylideneamino)phenyl 2-(2,4-dimethylphenylami
no)benzoate (4d), where the latter one revealed the highest reduc-
tion of paw volume at all. It is clear to compare all compounds 3a–
d and 4a–d used in this study with ibuprofen as reference standard
as shown in Figure 1, where all of them except compounds 3a and
3c showed significant reduction of paw oedema after carrageenan
injection.
Our entitled compounds can be compared by involving cumula-
tive manner for their % inhibitions of oedema volume (anti-inflam-
matory effects) at all times of 1, 2, 3, and 4 h. Where ibuprofen
exhibited cumulative % inhibitions of 152.01. Whereas, the other
derivatives revealing the closest activities to ibuprofen depending
on their cumulative manner in descending anti-inflammatory or-
der are 4d, 3d, 4b, and 3b being of 168.61, 171.5, 174.26, and
178.35, respectively, as cited in Table 1.
To a better extent, 4-(4-bromo benzylideneamino)phenyl 2-(4-
isobutylphenyl)propanoate (3d), significantly (p <0.05) decreased
the paw volume at 1, 2, 3 and 4 h after carrageenan administration
more than compound 3b at 2, 3 and 4 h being of 40.59%, 44.72%,
and 49.30%, respectively. On the other hand, many of 4-(4-substi-

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G. H. Hegazy, H. I. Ali / Bioorg. Med. Chem. 20 (2012) 1259–1270
Table 2
Analgesic effect of the tested ester derivatives (3a–d and 4a–d) in comparison to
ibuprofen using hot plate method
Group
Basal
Time after induction (s)
0.5 h 1 h
14.3 0.99
1.5 h
16.8 2.12
Control
12.54 1.12
10.52 0.75
9.24 1.29
12.64 2.08
11.74 1.27
11.42 2.10
11.18 1.40
9.60 1.23
11.84 1.67
13.56 1.42
11.16 1.66
20.18 1.03*
23.52 4.82*
22.22 1.93*
19.62 1.08*
21.68 4.19*
18.04 2.57
23.16 2.67*
20.72 2.87*
17.84 3.57
3a
3b
3c
3d
35.14 2.92*
34.62 3.13*
32.14 2.29*
33.44 2.36*
25.08 3.32*
25.98 1.27*
24.40 2.99*
21.22 4.36
16.10 0.94
28.1 3.85*
24.98 0.76*
23.0 1.01*
26.12 2.30*
18.46 0.82
20.82 0.59
15.96 1.63
19.38 2.37
19.0 3.20
4a
4b
4c
4d
Ibuprofen
Values represent the mean S.E. of five mice for each group.
Control group animals were given saline with few drops of carboxymethyl cellulose
0.5%.
Statistically significant (P <0.05) from the control normal group at the corre-
Figure 1. Acute anti-inflammatory effect of the tested ester derivatives (3a–d, and
4a–d) on carrageenan-induced oedema, in comparison with 100 mg/kg bwt,
ibuprofen at 1hr before initiation of inflammation with carrageenan. Oedema was
measured at 1, 2, and 3 h after carrageenan injection. Data shown are means
(n = 6) S.E., p <0.05.
*
sponding time, using one way ANOVA.
This results suggest that p-substituted benzylidenamino phenyl
esters of ibuprofenic and mefenamic acids produces an anti-oede-
matous effect during the first phase, similarly to ibuprofen. The
anti-oedematous effect of low-dose these esters had a prompt on-
set (1 h). This phenomenon may partly be due to the high systemic
bioavailability of p-substituted benzylidenamino phenyl esters fol-
lowing oral dosing, due to their higher and efficient absorptivity.
2.2.2. Analgesic activity
The analgesic activity of the synthesized derivatives was evalu-
ated by applying hot plate test,33,34 using 10 groups each of five
rats, each were given vehicle and/or the different compounds,
where ibuprofen was used as a standard reference. Results were
expressed as mean S.E. Difference between vehicle control and
treatment groups were tested using one way ANOVA followed by
the least significant difference (L.S.D.).
In the classical hot plate test, mice react by licking their paws
and/or jumping. However, tests relying on the unilateral applica-
tion of thermal radiant heat (UHP) to the plantar side of the hind
paw have become popular in recent years since unilateral changes
in nociceptive sensitivity can be detected. Ibuprofen increases
latencies; hence the last group received ibuprofen (250 mg/kg
bwt) 30 min prior to testing. Latency to lick a hind paw or jumping
was recorded sequentially before and at 0.5, 1.0, and 1.5 h post
treatment.
The results revealed that all tested compounds (3a–d and 4a–d)
at the doses of (250 mg/kg bwt) produced significant anti-nocicep-
tion in the hot plate test. According to Table 2, almost all these es-
ter derivatives showed significant analgesic activity higher than
that obtained by ibuprofen at all time post administration. Where
compounds 3a–d exhibited the most potent analgesic effects,
where they increased latency time significantly (p <0.05) after all
times of their administration. Where the most pronounced com-
pounds of analgesic activities are compounds 3b and 4c at 0.5 h,
compounds 3a and 3b at 1.0 h, and compounds 3a and 3d at
1.5 h post administration as cited in Table 2.
Thus, it can be concluded that, compounds 3a–d have signifi-
cant analgesic activity and are the most potent analogs as illus-
trated in Figure 2. Substances that produce strong inhibitory
effects in the hot plate test can inhibit centrally induced pain
and they act as strong analgesics.35,36
Figure 2. The analgesic effect of compounds (3a–d, and 4a–d; 250 mg/Kg bwt)
involving hot plate method, in comparison with ibuprofen. Latency was measured
at 0.5, 1.0, and 1.5 h after oral drug administration. Data shown are means
(n = 5) S.E., p <0.05.
2.2.3. Ulcerogenic liability
Being many of the compounds under investigation having pro-
nounced anti-inflammatory and analgesic activity in comparison to
ibuprofen, therefore the ulcerogenic liability for all of them was
evaluated in albino rats following the reported method.37,38 From
the data obtained (Table 3), it has been surprisingly observed that
all the synthesized p-substituted benzylidenamino phenyl esters of
ibuprofenic and mefenamic acids exhibited no ulcerogenic effect in
all of the experimental animals, compared to that of the standard
ibuprofen (ulcer index of 4.03 0.79). Therefore, the potential
medicinal value of these compounds as anti-inflammatory and
analgesic agents is that they have highly better safety margin on
gastric mucosa than ibuprofen.
This promising ulcer protective properties of the designed and
synthesized esters is greatly support our main objective to avoid
ibuprofen and mefenamic acid limitation of gastric injuries caused
by their free carboxylic group. Therefore, this successful results
support the present work to develop these novel series of ibupro-
fen and mefenamic acid esters to mask their drawbacks.
2.3. Molecular docking study
The observed activities of the aforementioned derivatives in the
hot plate test, therefore suggests that they have strong analgesic
activities.
Molecular docking is a frequently used tool in computer-aided
structure-based rational drug design. It evaluates how small mole-

G. H. Hegazy, H. I. Ali / Bioorg. Med. Chem. 20 (2012) 1259–1270
1263
Table 3
2.3.1. Validation of the accuracy and performance of AutoDock
As cited in literature,41 if the RMSD (root mean square devia-
tion) of the best docked conformation of the native ligand is
ꢀ2.0 Å from the experimental one, the used scoring function is suc-
cessful. Therefore the validation of the docking accuracy was inves-
tigated by docking of the native co-crystallized ligands to inspect
how closely the best docked conformation resembles the bound li-
gand in the biological method.42,43 The obtained success rates of
AutoDock was highly excellent. Where the co-crystallized ligands
of Cox1 and Cox2, namely ibuprofen and S58, respectively seem
exactly superimposed on the native bound ones as shown in Fig-
ure 3. The RMSD of the docked ibuprofen into Cox1 were 0.91
and 2.51 Å, and that of the docked S58 into Cox2 were 0.40 and
0.34 Å by rigid and flexible docking for each receptor, respectively.
Moreover, flexible docking involving AutoDock 4.2 seems to be
more accurate, being of smaller RMSD values, to be of more resem-
blance to the biological co-crystallization.
Ulcerogenic effect of compounds (3a–d, and 4a–d) on the gastric mucosa of six rat
groups indicating number and severity after compounds after 5 h of injection
Group
Ulcer index
No. of ulcer
Severity of ulcer
Control
3a
3b
3c
3d
4a
4b
4c
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4d
Ibuprofen
0
0
4.03 0.79*
9.5 1.78*
Statistical analysis was carried out using Kruskal–Wallis non-parametric one way
ANOVA.
Values represent the mean S.E. of six rats for each group.
*
Statistically significant from the control normal P <0.05.
2.3.2. AutoDock binding affinities of the synthesized
compounds into Cox1 and Cox2 enzymes
The binding affinity was evaluated by the binding free energies
(DGb, kcal/mol), inhibition constants (Ki), hydrogen bonding, and
RMSD values in comparison to the native co-crystallized ligands.
All of these data into the target macromolecule are represented
in Tables 4–7.
cule (substrate, inhibitor, drug or drug candidate) and the target
macromolecule (receptor, enzyme or nucleic acid) fit together. This
can be useful for developing better drug candidates and also for the
understanding the nature of the binding. Herein in silico compara-
tive Cox1 and Cox2 docking study aims to rationalize the obtained
biological data and to explain the possible interactions of the
tested derivatives into the crystal structure of Cox1 and Cox2 en-
zymes. The main interactions of the native co-crystallized ligands
namely, ibuprofen and S58 with Cox1 and Cox2, respectively, were
investigated. The carboxylate group of ibuprofen exhibited three
hydrogen bond with the key amino acids of Cox1: Tyr 355 (p-OH
group) and Arg120 (guanidine –NH2 group). Also other residues
in the binding pocket can be involved in such interactions include
Val349, Met522, Ile523, Gly526, and Ala527 as shown in Figure 3.
Whereas, the main amino acids involved in Cox2 interaction with
the native ligand S58 are His90, Arg120, Val349, Tyr355, Arg53,
Phe518, Gly526, and Ala527. Where His90, Arg120, and Arg513
form four hydrogen bonds with native ligand S58.
The compounds which commonly revealed the highest binding
affinities, that is, lowest binding free energies, and the hydrogen
bond interactions into Cox1 and Cox2 include; to a higher extent;
compounds: 4-(4-nitro benzylideneamino)phenyl 2-(4-isobutyl-
phenyl)propanoate (3b), 4-(4-bromobenzylideneamino) phenyl 2-
(2,4-dimethylphenylamino)benzoate (4d), compounds: 4-(4-nitro
benzylideneamino)phenyl 2-(2,4-dimethylphenylamino)benzoate
(4b), 4-(4-methoxy benzylideneamino)phenyl 2-(4-isobutyl-
phenyl)propanoate (3a), and 4-(4-bromo benzylideneami-
no)phenyl 2-(4-isobutylphenyl)propanoate (3d).
These derivatives exhibited up to three hydrogen bonds be-
tween their carboxylate or imide group with the key amino acids
of Cox1: His90–NH, Arg120–NH2, Tyr 355–OH, Tyr385–OH, and
Arg513–NH2, within highly acceptable RMSD being 1.90–4.72 Å
as cited in Tables 4–7. Compound 4-(4-nitro benzylideneami-
no)phenyl 2-(4-isobutylphenyl)propanoate (3b) on applying its
flexible docking into Cox1 macromolecule, it revealed the highest
binding affinity elucidated by its lowest binding free energies
Computer simulated automated docking study was performed
using the widely distributed molecular grid-based docking pro-
gram. AutoDock3.0539 for docking of flexible ligand within rigid
protein and AutoDock4.240 for docking of flexible ligand within
flexible protein, where flexibility of the target protein is taken into
account in the later type and ignored in the former one.
being, (
D
Gb: ꢁ14.32 kcal/mol) as cited in Table 5. This predicts
its promising anti-inflammatory activity and higher selectivity to-
wards Cox1 more than Cox2 receptor. Moreover, 3b exhibited one
and three hydrogen bonds with Tyr355 and Arg120 by flexible and
rigid docking within Cox1 receptor sites, respectively (Tables 4 and
5). Figure 4 illustrates differential binding affinities of 3b and 4b
AutoDock scans the active site for low energy binding models
and for orientations of the probe molecule, using a modified genet-
ic algorism that employs a local search (GALS) and pre-computed
grids for the evaluation of the interaction energy. Both of the target
Cox1 (PDB code: 1EQG) and Cox2 (PDB code: 1CX2) proteins were
handled by using Accelyrs Discovery Studio visualize v2.5 software
[Accelrys Inc., San Diego, CA (2005)] and the representative amino
acids of the ligand-binding site were selected within 5 Å neighbor-
hood surrounding the embedded ligand; ibuprofen. The results of
10 randomly seeded runs were analyzed for each of the docked
inhibitors. The docked inhibitors were assigned to a cluster if the
atomic coordinates of the docked inhibitors exhibited a root mean
square deviation (RMSD) of less than 0.5 Å difference from each
other (RMSD-tolerance of 0.5 Å). The analysis was carried out for
the top 10 docking clusters. The clusters were ranked from the
averaged lowest energy obtained for members of the cluster to
the highest. Each of the clusters that exhibited significant negative
interaction energies were examined by Accelyrs Discovery Studio
visualize modeling program to determine their binding
orientations.
analogs into Cox1 target site, where, compound 3b
(DGb:
ꢁ10.07 kcal/mol) exhibited three hydrogen bonds between its –
COO moiety and Arg120 within RMSD: 4.72 Å, whereas, compound
4b (
D
Gb: ꢁ10.35 kcal/mol) exhibited two hydrogen bonds between
its NO2 moiety and Tyr385 within RMSD: 4.05 Å as cited in Table 4.
To investigate the potential Cox2 inhibitory activity, com-
pounds 4c (
D
Gb: ꢁ12.76 kcal/mol) and 4d ( Gb: ꢁ13.07 kcal/
D
mol) exhibited selective binding affinities towards Cox2 more
than Cox1, as cited in Table 7. This high binding affinity was sup-
ported more by their close superimposing onto the native co-
crystallized ligand (S58) being their RMSD 1.90 and 2.68 Å,
respectively, providing better RMSD values than other derivatives.
These aforementioned docking results of 4c and 4d indicate that
these compounds are expected to be a reasonable candidate for
Cox2 inhibition.

1264
G. H. Hegazy, H. I. Ali / Bioorg. Med. Chem. 20 (2012) 1259–1270
Figure 3. Differential validation of AutoDock 3.05 (in A, C) and AutoDock 4.2 (in B, D) programs by docking of the native ligands of Cox1 and Cox2 into their binding sites. The
native co-crystallized ibuprofen and S58 are shown in yellow sticks, while the docked ligands are shown in balls and sticks, colored by element. The hydrogen bonds are
shown as blue and green dotted lines, respectively. The docked ligands seem exactly superimposed on the native ones.
Table 4
The rigid docking results (AutoDock 3.05), regarding the binding free energies
DGb) and inhibition constants (Ki) of compounds 3a–d, and 4a–d docked into ovine Cox1(1EQG), the
distances and angles of hydrogen bonds between compounds and amino acids involved in Cox1, and RMSD from the co-crystallized ibuprofen
a
b
Compd
3a
D
Gb (kcal/mol)
Ki
Hydrogen bonds between atoms of compounds and amino acids of ovine Cox1
RMSDc (Å)
4.31
Atom of compd
Amino acid
Distance (Å)
Angle (°)
ꢁ9.40
1.28E-07
4.23E-08
p-C@O
p-COO
p-COO
p-C@O
p-C@O
p-COO
p-COO
p-C@O
p-C@O
d
HN (HE) of Arg120
HN (HE) of Arg120
HN (HH21) of Arg120
HN (HE) of Arg120
HN (HH21) of Arg120
HN (HE) of Arg120
HN (HE) of Arg120
HN (HH21) of Arg120
HN (HE) of Arg120
2.37
2.43
2.20
2.43
1.80
2.18
1.71
1.68
2.34
155.2
147.7
156.0
127.7
142.3
164.0
151.1
170.5
111.7
3b
ꢁ10.07
4.72
3c
3d
ꢁ9.06
ꢁ9.37
2.28E-07
1.36E-07
3.93
4.71
4a
4b
ꢁ8.44
6.46E-07
2.57E-08
4.60
4.05
ꢁ10.35
p-N@O
p-N-O
C@N
p-HO of Tyr385
p-HO of Tyr385
1.85
1.94
1.82
2.10
2.35
2.10
2.20
1.75
1.60
1.91
2.20
135.1
143.2
147.6
136.7
144.5
152.4
155.9
162.0
170.3
109.3
109.6
4c
ꢁ9.21
ꢁ9.65
1.79E-07
8.38E-08
HN (HE) of Arg120
HN (HH21) of Arg120
HN (HE) of Arg120
HN (HH21) of Arg120
HN (HH22) of Arg83
HN (HE) of Arg120
HN (HH21) of Arg120
p-HO of Tyr355
7.96
7.22
C@N
C@N
C@N
H-N
4d
Ibuprofen
ꢁ8.79
3.08E-14
p-C@O
p-COO
p-COO
p-COOH
0.91
p-OH of Tyr35
a
b
c
Binding free energy.
Inhibition constant.
Root mean square deviation.
No hydrogen bond detected.
d

G. H. Hegazy, H. I. Ali / Bioorg. Med. Chem. 20 (2012) 1259–1270
1265
Table 5
The flexible docking results (AutoDock 4.2), regarding the binding free energies (DGb) and inhibition constants (Ki) of compounds 3a–d, and 4a–d docked into ovine Cox1(1EQG),
the distances and angles of hydrogen bonds between compounds and amino acids involved in Cox1, and RMSD from the co-crystallized ibuprofen
RMSDc (Å)
a
b
Compd
D
Gb (kcal/mol)
Ki
Hydrogen bonds between atoms of compounds and amino acids of Cox1
Atom of compd
Amino acid
Distance (Å)
Angle (°)
3a
3b
3c
3d
4a
ꢁ11.78
ꢁ14.32
ꢁ9.46
2.32 nM
32.0 pM
116.5 nM
159.9 pM
11.83 nM
p-C@O
p-COO
d
p-C@O
p-C@O
p-MeO
C@N
C@O
C@O
p-COO
p-COOH
p-HO of Tyr355
p-HO of Tyr355
2.32
2.02
155.0
153.8
1.77
3.92
3.77
3.12
4.39
ꢁ13.36
ꢁ10.81
HN (HH12) of Arg120
HN (HE) of Arg120
p-HO of Tyr355
2.04
1.94
2.01
2.07
2.08
2.10
1.99
2.49
131.3
135.8
144.6
123.7
170.7
135.4
173.2
169.8
4b
4c
4d
Ibuprofen
ꢁ9.71
76.7 nM
393.5 nM
16.81 nM
6.83 nM
p-HO of Tyr355
5.77
5.59
5.58
2.51
ꢁ8.74
HN (HH22) of Arg120
HN (HE) of Arg120
p-HO of Tyr355
ꢁ10.61
ꢁ11.14
p-OH of Ser530
a
b
c
Binding free energy.
Inhibition constant.
Root mean square deviation.
No hydrogen bond detected.
d
Table 6
The rigid docking results (AutoDock 3.05), regarding the binding free energies (
distances and angles of hydrogen bonds between compounds and amino acids involved in Cox2, and RMSD from the co-crystallized S58 ligand
D
Gb) and inhibition constants (Ki) of compounds 3a–d, and 4a–d docked into Cox2 (1CX2), the
RMSDc (Å)
a
b
Compd
D
Gb (kcal/mol)
Ki
Hydrogen bonds between atoms of compounds and amino acids of Cox2
Atom of compd
Amino acid
Distance (Å)
Angle (°)
3a
3b
ꢁ10.53
ꢁ11.63
1.93E-08
2.96E-09
p-C@O
p-COO
p-C@O
p-COO
p-C@O
p-C@O
–C@N
HN
HN (HE21) of Gln192
HN of His90
HN (HH11) of Arg513
HN of His90
p-HO of Tyr355
HN of Ile517
HN (HE22) of Gln192
HN of Asp515
HN of Asp515
HN of His90
HN (HH11) of Arg120
HN of His90
2.02
1.91
1.71
2.44
2.20
1.86
2.47
2.28
2.42
1.86
2.26
2.50
129.7
136.5
130.9
148.3
128.0
149.7
157.6
150.3
124.9
143.6
120.4
113.2
6.84
2.78
3c
3d
4a
4b
4c
ꢁ10.51
ꢁ10.17
ꢁ10.23
ꢁ9.00
1.98E-08
3.50E-08
3.18E-08
2.54E-07
1.25E-08
2.19E-09
3.55E-09
1.32
5.94
10.10
10.37
7.51
1.65
0.40
ꢁ10.78
ꢁ11.81
ꢁ11.53
p-COO
–C@N
C–F
4d
S58d
S@O
a
b
c
Binding free energy.
Inhibition constant.
Root mean square deviation.
1-Phenylsulfonamido-3-trifluoromethyl-5-(p-bromophenyl) pyrazole.
d
Table 7
The flexible docking results (AutoDock 4.2), regarding the binding free energies (DGb) and inhibition constants (Ki) of compounds docked into Cox2 (1CX2), the distances and
angles of hydrogen bonds between compounds and amino acids involved in Cox2, and RMSD from the co-crystallized S58 ligand
a
b
Compd
D
Gb (kcal/mol)
Ki
Hydrogen bonds between atoms of compounds and amino acids of Cox2
RMSDc (Å)
Atom of compd
Amino acid
Distance (Å)
Angle (°)
3a
3b
ꢁ12.01
ꢁ10.94
1.57 nM
9.56 nM
C@O
C@O
C@N
C@O
C@O
C@O
COO
C@N
d
p-HO of Tyr355
p-HO of Tyr355
2.03
2.01
2.40
1.80
2.08
2.10
2.01
2.02
136.8
152.3
113.4
141.9
134.2
134.7
139.7
166.5
3.77
4.32
HN(HH2) of Arg120
HN (HH11) of Arg513
HN (HE) of Arg120
HN (HH2) of Arg120
p-HO of Tyr355
3c
3d
ꢁ11.02
ꢁ10.43
8.82 nM
22.46 nM
5.29
7.07
4a
4b
4c
ꢁ6.64
13.50 mM
3.10 nM
446.8 pM
261.0 pM
115.1 nM
HN (HH1) of Arg120
3.07
3.07
1.90
2.68
0.34
ꢁ11.61
ꢁ12.76
ꢁ13.07
ꢁ9.47
C@N
C@N
S–NH
HN (HE2) of His90
p-HO of Tyr355
O@C of Gln192
1.85
2.31
2.00
138.9
177.8
109.2
4d
S58e
a
b
c
Binding free energy.
Inhibition constant.
Root mean square deviation.
No hydrogen bond detected.
1-Phenylsulfonamido-3-trifluoromethyl-5-(p-bromophenyl)pyrazole.
d
e
Figure 5 illustrates data represented docking of compound 4d
Gb: ꢁ13.07 kcal/mol) which is almost superimposed on the
native ligand (RMSD: 2.68 Å), exhibiting one hydrogen bond be-
tween C@N and p-OH of Tyr355. Relatively, compound 3d ( Gb:
(D
D

1266
G. H. Hegazy, H. I. Ali / Bioorg. Med. Chem. 20 (2012) 1259–1270
3. Conclusion
Oral dosage forms of ibuprofen and mefenamic acid, though
popular, suffer from the limitation of gastric injuries caused by
their free carboxylic group. Therefore, in this study we considered
it interesting to modify NSAIDS structure in such a way that it
would lead to molecules with greatly reduced acidic character by
developing a series of ibuprofen and mefenamic acid esters to
mask the free carboxylic groups. These prodrugs were synthesized
by conventional method of esterification by replacing the carbox-
ylic acid group of ibuprofenic and mefenamic acids with less acidic
p-substituted benzylidenamino phenyl esters. Where the interme-
diate ⁄⁄Schiff’s bases [4-(4-substituted benzylideneamino) phe-
nols; 2a–d] were synthesized by reaction of p-amino phenol and
p-substituted aromatic aldehyde, then subjected to condensation
reaction with ibuprofen or mefenamic acid involving dicyclohexyl
carbodiimmide (DCC) as mild dehydrating agent to afford the pro-
drugs; 4-(4-substituted benzylideneamino)phenyl 2-(4-isobutyl-
Figure 4. Relative docking mode of compounds 3b; ball and stick, colored by
element, and 4b; yellow sticks involving rigid docking into Cox1. They exhibit three
and two hydrogen bonds shown as dashed lines with Arg120 and Tyr385,
respectively. The binding site of the Cox1 is shown with labeled amino acids and
native co-crystallized ibuprofen is shown as cyan stick.
phenyl)propanoate
(3a–d)
and
4-(4-substituted
benzylideneamino)phenyl 2-(2,4-dimethylphenylamino) benzoate
(4a–d). The structures of the newly synthesized compounds were
elucidated by microanalytical and spectral (IR, 1H NMR, mass) data.
In addition to design and synthesis, this work covered testing of
eight drug candidates for their analgesic, anti-inflammatory poten-
cies, in addition to ulcerogenic effects. The anti-inflammatory
activity of the synthesized compounds was evaluated by carra-
geenan induced rat paw oedema model, where significant
(p <0.05) reduction of rat paw oedema was observed by most of
the test compounds at 1–3 h compared to control group (given sal-
ine with few drops of CMC).
Moreover, esterification of ibuprofen led to pronounced in-
crease of its analgesic activity, which was estimated by increasing
the latency time, means strongly inhibited the peripheral pain re-
sponse in the mice compared to the control animals. In addition,
most of the compounds were found to be more active in compari-
son with parent drugs, indicating that esterification of these NSA-
IDs improved their analgesic activity.
Being almost all of these compounds exhibit significant anti-
inflammatory and analgesic activities, they were subjected to ulc-
erogenicity potential test at (100 mg/kg) of 12 times the therapeu-
Figure 5. The comparative binding affinities of compounds 4d; ball and stick,
colored by element, and 3d; sticks, colored by element, involving flexible docking
into Cox2. Compound 4d (
onto the S58 native ligand; yellow sticks, and it exhibited one hydrogen bond with
Tyr355. Compound 3d exhibited lower binding affinity (
DGb: –13.07 kcal/mol; RMSD: 2.68 Å) is superimposed
D
Gb: ꢁ10.43 kcal/mol,
RMSD: 7.07 Å), and it exhibited three hydrogen bond with Arg120 and Tyr355. Cox2
is shown as solid backbone ribbon for protein and its amino acids are shown as
labeled lines, and the binding site is shown with solid surface.
tic doses.
A
thorough examination of the results of
histopathological studies indicated absence of the disruption of
gastric epithelial morphology and absence of ulcers/erosion in test
group animals compared to reference standard, ibuprofen, and
control group animals. These results are attributed to the acquired
acid protective design which reduced their acidic characters of the
synthesized esters compared to the parent acids.
From close inspection of the aforementioned results of in vivo
ulcerogenicity studies, we can conclude that the esterification of
carboxylate moiety of ibuprofenic and mefenamic acids may lead
to the development of novel, useful anti-inflammatory, analgesic,
and cytoprotective pharmacomolecules, with potentially impor-
tant therapeutic applications.
ꢁ10.43 kcal/mol) is docked deviated from the binding site (RMSD:
7.07 Å), bound to Arg120 and Tyr355 amino acids by its C@O moi-
ety. This different binding mode of these compounds may explain
their different biological anti-inflammatory activities of com-
pounds 3d and 4d after 1 and 2 h, where 4d (31.99 5.25%, and
38.47 5.78%) revealed higher anti-inflammatory effect than com-
pound 3d (36.54 2.63% and 40.59 1.94%) as shown in Table 1
and Figure 1.
It is clearly noticed that compounds 4d, 4c, 3a, 4b, and 3c,
respectively revealed more selective Cox2 binding over Cox1 by
flexible docking way as cited in Table 7, whereas, compound 3b
exhibited more selective Cox1 fitting.
Moreover, we have successfully carried out comparative rigid
and flexible docking for eight compounds. The obtained binding free
energies predicted by AutoDock 3.05 and 4.2 were highly correlated
to the biological activities, especially by flexible docking, where a lot
of compounds of pronounced biological activities revealed lowest
binding free energies with smallest RMSD and reasonable number
of hydrogen bonds into the binding site. The results are encouraging
to improve our knowledge in understanding the binding interac-
tions in detail. Where, flexible docking into the cyclooxygenase
receptor revealed more selective Cox2 binding affinities of com-
pounds 3a, 3c, 4b, 4c and 4d over Cox1 one, whereas, compounds
3b and 3d exhibited more selective Cox1 fitting.
In the analysis of docking results we can easily find an overall
excellent correlation between the biological results (anti-inflam-
matory and analgesic activities) and docking studies. Where com-
pounds 3b, 3d, 4b, and 4d which has the highest acute anti-
inflammatory effect (Table 1 and Fig. 1), have also the lowest bind-
ing free energies in both of rigid and flexible docking into Cox1 and
Cox2 with the nearest RMSD values to that of ibuprofen and S58 as
illustrated in Tables 4–7 of the docking results. Additionally, com-
pounds 3a–d revealed the best analgesic activities (Table 2 and
Fig. 2), especially after 0.5 and 1.0 h. These results are directly cor-
related with compounds 3a, 3b, and 3d which have the lowest
binding free energies as cited in Table 5.

G. H. Hegazy, H. I. Ali / Bioorg. Med. Chem. 20 (2012) 1259–1270
1267
dC 20.11 (CH3), 23.31 (isobut. 2,2-CH3), 29.55 (isobut. CH), 44.0
(isobut. CH2), 48.6 (CH), 55.03 (OCH3), 121.06 (C1, C20, C30, C50
and C60), 124.10 (C2, C3, C5 and C6), 137.50 (C200, C300, C500 and
C600), 138.60 (C4 and C100), 141.2 (C10 and C40), 143.4 (C400), 153.0
(C@N), 171.9 (C@O); Anal. Calcd for C27H29NO3: C, 78.04; H, 7.03;
N, 3.37. Found: C, 77.87; H, 6.84; N, 3.00.
4. Experimental
4.1. Chemistry
Melting points were obtained on a Yanagimoto micro melting
point apparatus and are uncorrected. Microanalyses were mea-
sured by Yanaco CHN Corder MT-5 apparatus. IR spectra were re-
corded on a JASCO FT/IR-200 spectrophotometer as Nujol mulls.
1H NMR spectra and 1H and 13C NMR spectra were obtained using
a Varian VXR 300 MHz and 75 MHz spectrophotometer, respec-
tively, and chemical shift values were expressed in d values
(ppm) relative to tetramethylsilane (TMS) as internal standard.
Coupling constants are given in Hz. Coupling constants are given
in Hz. All NH and OH protons were exchangeable with D2O. Micro-
analysis was carried out at microanalytical center, Faculty of Sci-
ence, Cairo University, and the mass spectra were recorded on
GCMC-QP 1000 EX Shimadzo Gas Chromatography MS spectrome-
ter, Japan E.I.70 ev. All reagents were of commercial quality and
were used without further purification. Organic solvents were
dried in the presence of an appropriate drying agent and were
stored over suitable molecular sieves. Reaction progress was mon-
itored by analytical thin layer chromatography (TLC) on pre-coated
glass plates silica gel 60F254-plate-Merck) and the products were
visualized by UV light. Ibuprofen and mefenamic acid were pur-
chased from Sigma Chemical Co. (St. Louis, MO, USA) in the form
of racemic mixtures.
4.1.2.2. 4-(4-Nitro benzylideneamino)phenyl 2-(4-isobutyl-
phenyl)propanoate (3b).
(from MeOH); IR (
max/cmꢁ1): 1620 (C@O); 1H NMR [CDCl3]: d
1.06 (6H, d, J = 6.1 Hz, 4-CH2–CH–(CH3)2), 1.43 (3H, d, J = 6.0 Hz,
-CH–CH3), 2.43 (1H, m, 4-CH2–CH–(CH3)2), 2.45 (2H, d,
J = 6.0 Hz, 4-CH2–CH–(CH3)2), 3.90 (1H, m, -CH–CH3), 7.06–7.25
Yield, (3.61 g, 84%); mp 172 °C
m
a
a
(8H, m, Ar-H), 8.02 (2H, dd, J2,3 = 9.0 Hz, J3,5 = 3.0 Hz, Ar m-H),
8.28 (2H, dd, J2,3 = 9.0 Hz, J2,6 = 3.0 Hz, Ar o-H), 8.56 (1H, s, CH@N);
13C NMR [CDCl3]: dC 21.3 (CH3), 23.7 (isobut. 2,2-CH3), 29.4 (isobut.
CH), 44.0 (isobut. CH2), 44.7 (CH), 125.9 (C1, C20, C30, C50 and C60),
128.6 (C2, C3, C5 and C6), 137.8 (C200, C300, C500 and C600), 139.5 (C4
and C100), 142.0 (C10 and C40), 146.4 (C400), 153.2 (C@N), 174.3
(C@O); Anal. Calcd for C26H26N2O4: C, 72.54; H, 6.09; N, 6.51.
Found: C, 72.57; H, 6.29; N, 6.85. MS, m/z (%): (M+1) at 431.
4.1.2.3. 4-[4-(Dimethylamino)benzylideneamino]phenyl 2-(4-
isobutylphenyl)propanoate (3c).
260 °C (from MeOH); IR (
max/cmꢁ1): 1680 (C@O); 1H NMR
[DMSO-d6]: d 0.81 (6H, d, J = 6.1 Hz, 4-CH2–CH–(CH3)2), 1.24 (3H,
d, J = 6.1 Hz, -CH–CH3), 1.67 (1H, m, 4-CH2–CH–(CH3)2), 2.48
(2H, d, J = 6.1 Hz, 4-CH2–CH–(CH3)2), 2.97 (6H, s, NCH3)2), 3.36
Yield, (2.96 g, 69%); mp
m
a
4.1.1. General procedure for the preparation of the intermediate
Schiff’s bases: 4-(4-substituted benzylideneamino)phenol (2a–
d)26–31
(1H, m, a-CH–CH3), 6.72–7.69 (12H, m, Ar-H), 8.36 (1H, s, CH@N);
13C NMR [CDCl3]: dC 21.0 (CH3), 23.4 (isobut. 2,2-CH3), 28.89 (iso-
but. CH), 32.66 (p-N(CH3)2), 43.67 (isobut. CH2), 44.35 (CH),
125.58 (C20, C30, C50 and C60), 125.99 (C1), 128.26 (C2, C3, C5 and
C6), 128.34 (C200, C300, C500 and C600), 137.6 (C4 and C100), 139.2(C10
and C40), 145.5 (C400), 152.8 (C@N), 173.0 (C@O); Anal. Calcd for
Each reactant of p-aminophenol (1, 10.9 g; 0.1 mol) and p-
substituted aromatic aldehyde (0.1 mol), was dissolved in a mini-
mum amount of absolute ethanol (20 ml), then mixed together
and followed by addition of 0.2 ml of glacial acetic acid. The reac-
tion mixture was refluxed for 6–8 h. After completion of the reac-
tion (monitored by TLC), the resulting clear solution was
concentrated in vacuo. The obtained yellow residue was treated
with ice water and the precipitate powdery crystals were filtered
off, washed well with water, dried and recrystallized from ethanol
to afford the corresponding product as yellow needles in 70–85%
yields.
C28H32N2O2: C, 78.47; H, 7.53; N, 6.54. Found: C, 78.31; H, 7.42;
N, 6.88. MS, m/z (%): (M+1) at 429.
4.1.2.4. 4-(4-Bromo benzylideneamino)phenyl 2-(4-isobutyl-
phenyl)propanoate (3d).
(from MeOH); IR (
max/cmꢁ1): 1650 (C@O); 1H NMR [DMSO-d6]:
d 0.82 (6H, d, J = 8.7 Hz, 4-CH2–CH–(CH3)2), 1.22 (3H, d, J = 8.7 Hz,
-CH–CH3), 2.40 (1H, m, 4-CH2–CH–(CH3)2), 2.50 (2H, d,
J = 8.7 Hz, 4-CH2–CH–(CH3)2), 3.16 (1H, m, -CH–CH3), 7.14–
Yield, (3.81 g, 82%); mp 180 °C
m
a
4.1.2. General procedure for the preparation of 4-(4-substituted
benzylideneamino)phenyl 2-(4-isobutylphenyl)propanoate
(3a–d) and 4-(4-substituted benzylideneamino)phenyl 2-(2,4-
dimethylphenyl amino) benzoate (4a–d)
a
7.81(12H, m, Ar-H), 8.62 (1H, s, CH@N); 13C NMR [CDCl3]: dC 21.2
(CH3), 23.6 (isobut. 2,2-CH3), 29.94 (isobut. CH), 43.9 (isobut.
CH2), 44.55 (CH), 125.8 (C1, C20, C30, C50 and C60), 126.14 (C2, C3,
C5 and C6), 128.6 (C200, C300, C500 and C600), 130.9 (C400), 137.8 (C4
and C100), 139.1(C10 and C40), 153.1 (C@N), 173.8 (C@O); Anal. Calcd
for C26H26BrNO2: C, 67.24; H, 5.64; N, 3.02. Found: C, 67.34; H,
5.89; N, 3.40.
N,N0-Dicyclohexyl carbodiimmide (DCC) (2.48 g, 12 mmol) was
added to the corresponding acid (ibuprofen, 2.06 g; 10 mmol) or
(mefenamic acid, 2.41 g; 10 mmol) in 10 ml dichloromethane/
THF (1:1) on cold. The resulting suspension was vigorously stirred
for 15 min at room temperature. Subsequently, was added a solu-
tion of the appropriate Schiff’s base (10 mmol) in 10 ml dichloro-
methane/THF (1:1). The mixture was left stirring overnight at
room temperature. The mixture was then filtered and evaporated
in vacuo. The crude residue was obtained and crystallized from
methanol to afford the corresponding product as yellow needles
in 52–84% yields.
4.1.2.5. 4-(4-Methoxy benzylideneamino)phenyl 2-(2,4-dimeth-
ylphenylamino)benzoate (4a).
Yield, (2.52 g, 56%); mp
165 °C (from MeOH); IR (
m
max/cmꢁ1): 3500 (NH), 1658 (C@O); 1H
NMR [CDCl3]: d 2.16 (3H, s, p-CH3), 2.31 (3H, s, o-CH3), 3.87 (3H,
s, p-OCH3), 5.47 (1H, br s, NH, exchangeable with D2O), 6.91–7.83
(15H, m, Ar-H), 8.38 (1H, s, CH@N); 13C NMR [CDCl3]: dC 19.8
(2000-CH3), 24.4 (4000-CH3), 55.2 (OCH3), 114.8 (C1), 117.8 (C300 and
C500), 118.5 (C3, and C6000), 121.6 (C5), 124.3 (C30and C50), 124.4
(C20 and C60), 125.1 (C100), 126.8 (C5000), 127.0 (C2), 128.5 (C2000),
128.6 (C4000), 129.4 (C3000), 130.8 (C200 and C600), 137.4 (C4), 138.8
(C1000), 143.4 (C6 and C10), 144.3 (C40), 153.4 (C@N), 157.3 (C400),
169.8 (C@O); Anal. calcd for C29H26N2O3: C, 77.31; H, 5.82; N,
6.22. Found: C, 77.20; H, 6.0; N, 6.55. MS, m/z (%): (M) at 450.
4.1.2.1. 4-(4-Methoxy benzylideneamino)phenyl 2-(4-isobutyl-
phenyl)propanoate (3a).
(from MeOH); IR (
max/cmꢁ1): 1656 (C@O); 1H NMR [CDCl3]: d
1.50 (6H, d, J = 7.2 Hz, 4-CH2–CH–(CH3)2, 1.83 (3H, d, J = 7.2 Hz,
-CH–CH3), 2.15 (1H, m, 4-CH2–CH–(CH3)2, 2.30 (2H, d, J = 7.2 Hz,
4-CH2–CH–(CH3)2, 3.45 (3H, s, p-OCH3), 3.84 (1H, m, -CH–CH3,
6.90–7.25 (12H, m, Ar-H), 8.37 (1H, s, CH@N); 13C NMR [CDCl3]:
Yield, (3.28 g, 79%); mp 210 °C
m
a
a

1268
G. H. Hegazy, H. I. Ali / Bioorg. Med. Chem. 20 (2012) 1259–1270
4.1.2.6. 4-(4-Nitro benzylideneamino)phenyl 2-(2,4-dimethyl-
phenylamino)benzoate (4b). Yield, (2.42 g, 52%); mp 190 °C
(from MeOH); IR (
max/cmꢁ1): 3550 (NH), 1657 (C@O); 1H NMR
4.2.1.3. Analysis of data.
The results are expressed as stan-
dard error of the mean S.E.M. Differences in mean values be-
tween vehicle control and treatment groups were analyzed by a
one-way analysis of variance (ANOVA). Statistical significance
was assessed as p <0.05. Methods of statistical analysis were done
according to Armitage et al.32
m
[CDCl3]: d 2.16 (3H, s, p-CH3), 2.31 (3H, s, o-CH3), 4.30 (1H, br s,
NH, exchangeable with D2O), 6.88–7.26 (11H, m, Ar-H), 8.02 (2H,
dd, J2,3 = 8.9 Hz, J3,5 = 3.0 Hz, Ar m-H), 8.28 (2H, dd, J2,3 = 8.9 Hz,
J2,6 = 3.0 Hz, Ar o-H), 8.57 (1H, s, CH@N); 13C NMR [CDCl3]: dC 13C
NMR [CDCl3]: dC 19.41 (2000-CH3), 24.03 (4000-CH3), 114.92 (C1),
117.48 (C300 and C500), 118.22 (C3, and C6’00), 121.51 (C5), 124.11
(C30and C50), 124.73 (C20 and C60), 126.68 (C2, C100 and C5000),
127.78 (C2000, C3000 and C4000), 130.4 (C200 and C600), 137.02 (C4),
138.34 (C1000), 140.73 (C6), 141.87 (C10), 143.03 (C40), 148.5 (C400),
153.2 (C@N), 169.55 (C@O); Anal. Calcd for C28H23N3O4: C, 72.24;
H, 4.98; N, 9.03. Found: C, 72.18; H, 5.08; N, 9.30.
4.2.2. Acute anti-inflammatory effect (carrageenan-induced
paw oedema test)
Anti-inflammatory activity against carrageenan-induced rat
paw oedema was assayed in adult male Wistar CF rats weighing
180–220 g according to the method of Winter et al.,44 with slight
modifications. The tested compounds (3a–d and 4a–d; 100 mg/kg
bwt), were orally administered into eight groups of six rats, 1 h be-
fore injection of 0.1 ml of a 1% suspension of carrageenan lambda
saline into the subcutaneous tissue of the right hind paw. The left
hind paw was injected in the same way with 0.05 ml of a saline
solution. Rats were fasted 24 h before the experiment and water
(1.5 ml per 100 g body weight) was orally administered twice (2
and 4 h) before injections. One hour before induction of oedema
saline with few drops of CMC 0.5% (0.3 ml/100 g rat) was adminis-
tered orally to a group of animals and served as control. Rats were
kept in the same experimental conditions. Carrageenan caused vis-
ible redness and pronounced swelling that was well developed by
4 h and persisted for more than 48 h. The volume of both hind
paws of control and treated animals were measured immediately
before and after carrageenan and test compounds injections at se-
lected times (1, 2, 3 and 4 h) using a planimeter.45,46 The inhibition
percentage of the inflammatory reaction was determined for each
animal by comparison with controls, and calculated by the
formula:
4.1.2.7. 4-(4-(Dimethylamino)benzylideneamino)phenyl 2-(2,4-
dimethylphenylamino)benzoate (4c).
Yield, (3.85 g, 83%);
mp 155 °C (from MeOH); IR
(
m
max/cmꢁ1): 3500 (NH), 1650
(C@O); 1H NMR [DMSO-d6]: d 2.20 (3H, s, p-CH3), 2.40 (3H, s, o-
CH3), 2.98 (6H, s, NCH3)2), 6.72–7.93 (15H, m, Ar-H), 8.37 (1H, s,
CH@N), 9.32 (1H, br s, NH, exchangeable with D2O); 13C NMR
[CDCl3]: dC 19.66 (2000-CH3), 23.90 (4000-CH3), 32.20 (p-N(CH3)2),
113.84 (C1), 115.72 (C300 and C500), 119.50 (C3, and C6000), 119.71
(C5), 124.36 (C30and C50), 124.64 (C20 and C60), 124.93 (C2, C100
and C5000), 126.20 (C2000, C3000 and C4000), 131.02 (C200 and C600),
137.00 (C4), 138.61 (C1000), 143.30 (C10 and C6), 145.90 (C40 an-
dC400), 153.26 (C@N), 167.86 (C@O); Anal. Calcd for C30H29N3O2:
C, 77.73; H, 6.31; N, 9.06. Found: C, 77.49; H, 6.43; N, 9.36.
4.1.2.8. 4-(4-Bromo benzylideneamino)phenyl 2-(2,4-dimethyl-
phenylamino)benzoate (4d).
Yield, (3.90 g, 78%); mp 160 °C
max/cmꢁ1): 3540 (NH), 1640 (C@O); 1H NMR
Ið%Þ ¼ 100 ꢂ ð1 ꢁ dt=dcÞ
(from MeOH); IR (
m
[CDCl3]: d 2.16 (3H, s, p-CH3), 2.31 (3H, s, o-CH3), 4.29 (1H, br s,
NH, exchangeable with D2O), 6.91–7.37 (15H, m, Ar-H), 8.41 (1H,
s, CH@N); 13C NMR [CDCl3]: dC 13C NMR [CDCl3]: dC 19.45 (2000-
CH3), 24.11 (4000-CH3), 114.47 (C1), 117.52 (C300 and C500), 118.20
(C3, and C6000), 121.64 (C5), 124.13 (C30and C50), 124.80 (C20 and
C60), 126.75 (C2, C100 and C5000), 128.19 (C2000, C3000 and C4000), 130.8
(C200 and C600), 137.09 (C4 and C1000), 138.47 (C6 and C10), 143.08
(C40 and C400), 153.18 (C@N), 169.61 (C@O); Anal. Calcd for
Where dt is the difference in paw volume in the drug-treated
group and dc the difference in paw volume in the control group.
The reference standard drug; ibuprofen 100 mg/kg bwt; was
administered to a group of rats that served as a positive control.
Statistically significant from the control normal inflamed group
at the corresponding time: P <0.05. Statistical analysis was carried
out using repeated measures one way ANOVA followed by least
significant test for multiple comparisons.47
C28H23BrN2O2: C, 67.34; H, 4.64; N, 5.61. Found: C, 67.11; H,
4.56; N, 5.98.
4.2.3. Hot plate analgesia testing
The Central analgesia of the eight synthesized derivatives was
evaluated by applying hot plate test33,34 Mice weighing 20–25 g
were divided into 10 groups (n = 5). The control group animals
were given saline with few drops of carboxymethyl cellulose
(CMC) 0.5% (0.3 ml/100 g rat). The positive control group animals
were given ibuprofen (250 mg/kg bwt) as a standard reference
analgesic. Mice were introduced to electronically controlled hot-
plate surface adjusted to 55 0.1 °C (7280- Hot-plate module,
Ugo Basile, Comerio, Italy). A cut-off time of 45 s was selected to
avoid tissue damage. The latency (licking or jumping) of nocicep-
tive responses was recorded at 0, 0.5, 1 and 1.5 h after oral admin-
istration of the eight test compounds (250 mg/kg bwt). Time
required for mice to lick paw/jump was recorded using built-in
digital timer and designated as withdrawal latency (WDL).
4.2. Biological screening
4.2.1. Materials and methods
4.2.1.1. Animals.
Adult rats of both sexes weighing 150–
200 g and adult mice weighing 20–25 g were used in this study.
They were housed and bred under standardized conditions for
light, temperature, ventilation and free access to feeds (mouse
cubes) and water receiving standard rat chow and libitum. Animals
were randomly assigned to different experimental groups, each
kept in a separate cage. Laboratory investigations on the animals
were carried out in accordance with the ethical guidelines stipu-
lated by ethical committee of the National Research Center and
in accordance with the recommendations for the proper care and
use of laboratory animals (NIH publication No. 85-23, revised
1985) with the internationally accepted principles for laboratory
animal use and care.
4.2.4. Ulcerogenic liability
The experiments were performed on albino rats of Wistar strain
of either sex, following the previously reported standard meth-
od.37,38 Rats weighing 120–140 g maintained at 25 2 °C, 50 5%
relative humidity and 12 h light/dark cycle, were divided into ten
groups of six animals each. Pregnant female rats were excluded.
The animals were fasted 18 h before drug administration. Ibupro-
fen (reference standard) and the tested compounds were sus-
4.2.1.2. Drugs and chemicals.
Carrageenan Iambda Sigma–
Aldrich chemical company (USA), ibuprofen Khahira Pharmaceuti-
cal and Chemical Company (Cairo, Egypt) and 7280- Hot-plate
module, Ugo Basile, Comerio, Italy was used in analgesia testing.

G. H. Hegazy, H. I. Ali / Bioorg. Med. Chem. 20 (2012) 1259–1270
1269
pended in 1% aqueous carboxymethyl cellulose (CMC) solution,
and were administered orally once daily for three consecutive days
to fasted rats in doses which exhibit high activity being (100 mg/
kg). The control group animals were given saline with few drops
of carboxymethyl cellulose (CMC). On the fourth day the animals
were sacrificed by cervical dislocation and the stomachs were
quickly and carefully dissected out. A longitudinal incision along
the greater curvature was made with fine scissor, then stomach
was inverted over the index finger and the presence or the absence
of gastric irritation was examined using a hand held microscope for
the presence of gastric irritation. Ulcers were scored according to
the arbitrary scale used by Singh et al.48 Where 0 = no lesion,
0.5 = hyperaemia, 1 = one or two slight lesions, 3 = very severe le-
sions and 4 = mucosa full of lesions. Ulcer index was calculated
as mean ulcer scores (Tan et al.),49 where the presence of a single
or multiple lesions (erosion, ulcer or perforation) is considered to
be positive.50
rotatable bonds must be found. Flexible residues are amino acids
in the binding site region of the protein that are able to alter their
position via conformational change upon ligand binding. They are
found by comparison of different crystallized structures or by
molecular dynamic simulations. According to the literature, the
flexible residues are Arg120 and Tyr355 in our system. Rotatable
bonds are used by the program to generate rotational isomers of
amino acids and to present enzyme structures with those
conformers.
4.3.1.2. Calculation of affinity maps by using a 3D-grid embrac-
ing the protein and ligand.
A part of ADT, the AutoGrid cal-
culates the energy of the non-covalent interactions between the
protein and probe atoms that are located in the different grid
points of a lattice that defines the area of interest (i.e., the area
of the macromolecule where the possibility of ligand binding is
studied). AutoGrid builds as many files as the number of probe
atoms used. There are about 30 different types of grid maps. Each
one shows the interaction energies for a particular atom type, such
as aliphatic carbons, aromatic carbons, hydrogen bonding oxygens,
etc. The grid itself is a box with determined dimensions that is lo-
cated at the site on the surface of the protein where we expect the
interaction with the ligand. In other words, the grid is our field of
study. The created three-dimensional grids of 60 ꢂ 60 ꢂ 60 Å size
(x,y,z) with a spacing of 0.375 Å centered at 26.64, 32.60, and
200.23 Å that encompassed the active site where the co-crystal-
lized ligand; ibuprofen; was embedded, was used to guide the
docked inhibitors within Cox1 receptor. Whereas, S58 native li-
gand of Cox2 receptor centered at 23.95, 21.58, and 15.44 Å,
respectively. In this part of modeling, we need to determine the
area where the ligand interacts with the enzyme on its surface, size
of that area and particular types of atoms participating in the inter-
action of both a ligand and an enzyme. The first two parameters are
determined by size and position of the grid box; the third param-
eter is given by map type. Once those parameters were set in one
file, AutoGrid calculates grid parameter files for each type of atom
within a given area.
4.3. Molecular docking
Using Auto Dock Tools (ADT), you will perform a docking study
of anti-inflammatory compounds, a known inhibitor of the cyclo-
oxygenase enzyme (Cox) to calculate the position of docked ligand
and flexible residues moved in the process of interaction. Your task
is to compare the energies of the interaction in different conforma-
tions and determine the best fit.
4.3.1. Procedure
AutoDock tool (ADT) consists of two main programs: AutoGrid
pre-calculates these grids. AutoDock performs the docking of the
ligand to a set of grids describing the target protein; and Working
with ADT includes major three steps:
4.3.1.1. Preparation of target protein and ligand files. 4.3.1.1.1.
Preparation of the protein. Two different Cox target enzymes were
investigated. These include ovine Cox-1 complexed with ibuprofen
(PDB code: 1EQG) and Cox2 complexed with a selective inhibitor,
SC-558 (PDB code: 1CX2). Those were retrieved from the Protein
Data Bank, http://www.rcsb.org/pdb/home/home.do. For each
docking target, crucial amino acids of the active site were identi-
fied using data in PDBsum, http://www.ebi.ac.uk/pdbsum/. In this
step we need to place back all hydrogens for ADT calculations.
The other required action is to remove water molecules from the
surface of the protein. This is necessary because the extra water
molecules will mask the protein surface from the ligand.
4.3.1.1.2. Preparation of the ligand. The ligands are originally
drawn with a widely used chemical structure drawing software.
The three-dimensional structures of the aforementioned com-
pounds were constructed using Chem3D Ultra 8.0 software
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
tion, USA (2003)] to obtain standard 3D structures (pdb format).,
then they were energetically minimized by using MOPAC with
100 iterations and minimum RMS gradient of 0.10. It is recom-
mended to confirm whether all hydrogen atoms are in the file be-
fore working with ADT. After opening the ligand, it can be
visualized and ADT now automatically computes Gasteiger charges
(empirical atomic partial charges) and distinguishes between
hybridization state and type of each atom. As a part of preparation,
the program determines rotatable bonds of the ligand to be able to
generate different conformers for the docking.
4.3.1.3. Defining the docking parameters and running the
docking simulation.
When the preparation of the input files
(ligand and protein) and the calculation of the affinity maps are
properly performed, AutoDock will carry out the docking automat-
ically using the newest docking algorithm (Lamarckian Genetic
Algorithm).
4.3.1.3.1. Preparation of the docking parameter file (.dpf). Once
all files are ready, we need to specify for the program what partic-
ular ligand, protein, flexible protein and maps we want to work
with and also what algorithm we want to use, how many iterations
are required and so on. That information is kept usually in docking
parameter file.
4.3.1.3.2. Running Autodock 4 and viewing the docking result-
s. As a result of AutoDock calculations we obtain the output file
with in our case ten conformers of the protein–ligand complex
with flexible residues and the ligand located within the binding
pocket. Each structure are scored and ranked by the program by
the calculated interaction energy.
Acknowledgments
The authors wish to offer their deep gratitude to Assoc. Profes-
sor Mohamed Raafat, Professor of Pharmacology and Toxicology,
Department of Pharmacology and Toxicology, Faculty of Pharmacy,
Umm al Qura University, Saudi Arabia, and to Assoc. Professor
Manal M. Anwar; National Research Centre, Cairo, Egypt, for their
helpful discussion and valuable help in the Pharmacology part of
this manuscript.
4.3.1.1.3. Preparation of the flexible residue file. A unique prop-
erty of the program is its ability to take into account the flexibility
not only of the ligand but also the enzyme during docking process.
It means that ADT is able to model not only how the ligand docks
to the protein but also the position of flexible residues. In order to
use this advantage, the flexible residues must be chosen and the

1270
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24. Duflos, M.; Nourrisson, M. R.; Brelet, J.; Courant, J.; Baut, J. Le.; Grimaud, G. N.;
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