Synthesis of new ibuprofen derivatives with their in silico and in vitro cyclooxygenase-2 inhibitions

Article abstract of DOI:10.1016/j.bioorg.2013.10.002

Cyclooxygenase-2 (COX-2) is one of the important targets for treatment of inflammation related diseases. In the literature, most of drug candidates are first synthesized and then their COX-2 inhibitory activities are tested by in vitro and in vivo experim

Full text of DOI:10.1016/j.bioorg.2013.10.002

Bioorganic Chemistry 52 (2014) 8–15
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of new ibuprofen derivatives with their in silico and in vitro
cyclooxygenase-2 inhibitions
Cevher Gundogdu-Hizliates a,b, Hakan Alyuruk a, Mustafa Gocmenturk a, Yavuz Ergun a,b, Levent Cavas a,c,
⇑
a
_
Dokuz Eylül University, Graduate School of Natural and Applied Sciences, Department of Chemistry, Kaynaklar Campus, 35160 Izmir, Turkey
b
_
Dokuz Eylül University, Faculty of Science, Department of Chemistry, Division of Organic Chemistry, Kaynaklar Campus, 35160 Izmir, Turkey
c
_
Dokuz Eylül University, Faculty of Science, Department of Chemistry, Division of Biochemistry, Kaynaklar Campus, 35160 Izmir, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 May 2012
Available online 5 November 2013
Cyclooxygenase-2 (COX-2) is one of the important targets for treatment of inflammation related diseases.
In the literature, most of drug candidates are first synthesized and then their COX-2 inhibitory activities
are tested by in vitro and in vivo experiments. However, synthesis of dozens of drug analogues without
any interpretations on their inhibitory activity can result in loss of time and chemicals. Therefore, syn-
thetic drug designs with molecular modeling are of importance to synthesize selective drug candidates
against inflammatory diseases. The synthesis of the novel ibuprofen derivatives through their in silico
and in vitro COX-2 inhibitory activities were investigated in the present study. Starting from ibuprofen,
ibuprofen amide and ibuprofen acyl hydrazone derivatives were synthesized. According to the results
of the in silico molecular docking and in vitro enzyme inhibition studies, the synthesized novel ibuprofen
derivatives have selective COX-2 inhibition, and molecule 3a and 3c were showed higher inhibition com-
pared to ibuprofen. In conclusion, the newly synthesized ibuprofen derivatives can be used in model
in vivo studies.
Keywords:
Cyclooxygenase-2
Ibuprofen
Molecular docking
Molecular modeling
Selective inhibitor
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
[1,4]. COX has two well known isoforms, COX-1 and COX-2
[1,2,5]. When they are aligned (Fig. 1), it is seen that they have
Prostaglandin H synthase (PGHS) catalyses the first step in the
cyclic pathway of eicosanoid metabolism. PGHS have two different
catalytic activities, a cyclooxygenase (COX) and a peroxidase activ-
ity. Radical mediated addition of two molecules of oxygen through
Tyr385 residue to arachidonic acid is catalyzed by COX. The formed
product is called as PGG2. Peroxidase activity of PGHS transforms
PGG2 into PGH2 that is precursor of some important bio-molecules
such as prostaglandins, prostacyclins and thromboxanes [1–3]. The
COX subunit within the PGHS is found on the opposite side of the
heme and it is located at the end of a long narrow hydrophobic
channel. Arg120, Tyr385 and Ser530 are important residues of
the active site of COXs. Acetylsalicylic acid (aspirin) based studies
revealed that Ser530 is not responsible for catalytic activities of
COXs. Acetylation of Ser530 by aspirin prevents arachidonic acid
to reach to the Tyr385 in the active site of COX. Arg120 is of also
great importance for inhibition of COX according to a flurbiprofen
based study [1,4]. Carboxyl group of flurbiprofen interacts with
Arg120, and this interaction also closes the channel of PGHS that
is responsible for entrance of substrate to the COX’s active site
quite similar sequence and structural identity. Although COX-1 is
expressed in many organs, COX-2 is expressed in some tissues
for forming a response against inflammatory stimuli. Since most
of the developed non-steroidal inflammatory drugs (NSAIDs) are
not specific for COXs, they affect both isoforms [1]. Therefore, there
is a great need for development of selective inhibitors for COX-2.
Molecular dockings is a powerful approach to study structure
based drug design. This method provides a big contribution to
the development of novel selective drugs for COX-2. Organic syn-
thesis strategies of novel ibuprofen derivatives were described
and COX-2 inhibitory activities of novel ibuprofen derivatives were
supported with in silico and in vitro molecular docking analysis in
the present study.
2. Results
Molecular docking studies showed that the interaction of the
inhibitor with Arg120 through a strong hydrogen bond, hydropho-
bic interactions between the substrate binding site of COX-2 and
hydrophobic tale of the inhibitor are key factors in designing of
NSAIDs. Docking studies with LeadIT revealed that among synthe-
sized COX-2 inhibitors, molecules 2a, 2b, 2c, 3b and 3d were imi-
tated the arachidonic acid better than classical NSAIDs (Table 1).
⇑
Corresponding author at: Dokuz Eylül University, Faculty of Science, Depart-
ment of Chemistry, Kaynaklar Campus, 35160 Izmir, Turkey. Fax: +90 232 453 41
_
88.
E-mail address: levent.cavas@deu.edu.tr (L. Cavas).
0045-2068/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2013.10.002

C. Gundogdu-Hizliates et al. / Bioorganic Chemistry 52 (2014) 8–15
9
Fig. 1. Alignment of COX-1 (UniProt ID: P23219) and COX-2 (UniProt ID: P35354).
Table 1
Molecular docking results obtained with LeadIT 2.0.2 for newly synthesized potential COX-2 inhibitors and commercial inhibitors on COX-2 crystal structures (PDB ID: 1CVU,
1CX2 and 1PXX).
COX-2
inhibitor
COX-2 structure
PDB ID: 1CVU
HYDE score
PDB ID: 1CX2
HYDE score
PDB ID: 1PXX
HYDE score
Mean
score
H-bonded residues
Arg120
H-bonded residues
Arg513, Tyr355
H-bonded
residues
(kJ molꢀ1
)
(kJ molꢀ1
)
(kJ molꢀ1
)
Arachidonic
acid
ꢀ74
ꢀ50
ꢀ68
Arg120
ꢀ64
Celecoxib
Curcumin
ꢀ39
ꢀ36
Arg120, Val116
Arg120, Tyr385,
Ser530
ꢀ37
ꢀ34
Arg120
Arg120, Arg513,
Asp515
ꢀ42
ꢀ32
Tyr385, Ser530
Arg120, Met522
ꢀ39
ꢀ34
Diclofenac
Etoricoxib
Meloxicam
Naproxen
Rofecoxib
S58
Valdecoxib
Molecule 2a
Molecule 2b
Molecule 2c
Molecule 2d
Molecule 3a
Molecule 3b
Molecule 3c
ꢀ41
ꢀ34
ꢀ27
ꢀ36
ꢀ34
ꢀ32
ꢀ38
ꢀ50
ꢀ54
ꢀ65
ꢀ40
ꢀ44
ꢀ50
ꢀ42
Arg120
Arg120
Arg120, Ser530
Arg120
Arg120
Arg120, Tyr355
Arg120, Tyr355
Arg120
Arg120
Arg120
ꢀ37
ꢀ31
ꢀ19
ꢀ35
ꢀ34
ꢀ25
ꢀ30
ꢀ46
ꢀ52
ꢀ46
ꢀ31
ꢀ34
ꢀ35
ꢀ30
Val523
Arg513
ꢀ32
ꢀ38
ꢀ35
ꢀ40
ꢀ34
ꢀ43
ꢀ36
ꢀ42
ꢀ49
ꢀ49
ꢀ38
ꢀ40
ꢀ45
ꢀ40
Tyr385, Val523
Tyr385
Val523, Ser530
Tyr385, Ser530
Ser530
Tyr385, Ser530
Arg120
Arg120
ꢀ37
ꢀ34
ꢀ27
ꢀ37
ꢀ34
ꢀ33
ꢀ35
ꢀ46
ꢀ52
ꢀ53
ꢀ36
ꢀ39
ꢀ43
ꢀ37
Arg120, Val523
Arg120, Tyr385
Arg120, Tyr385
Gln192, Arg513
Arg120
Arg120, Tyr355
Arg120
Arg120, Arg513
Arg120
Arg120
Arg120
Arg120, Tyr355,
Glu524
Arg120
Arg120, Val523
Tyr385, Ser530
Tyr385, Ser530
Tyr385, Ser530
Tyr385, Ser530
Val523
Arg120
Arg120
Val523
Molecule 3d
ꢀ59
Arg120
ꢀ40
Arg120, Tyr355
ꢀ41
Tyr385, Ser530
ꢀ47
The highest binding affinity was found as ꢀ65 kJ molꢀ1 for mole-
cule 2c and this score was very close to that of arachidonic acid
on 1CVU crystal structure. Among novel ibuprofen derivatives,
higher binding affinities were observed when the inhibitor
interacts with Arg120. On the other hand, the binding affinities
of diclofenac and celecoxib were not very high. As can be seen from
Fig. 2, molecule 2c interacts with active site of COX-2 similar to
arachidonic acid; however, diclofenac was not able to interact with

10
C. Gundogdu-Hizliates et al. / Bioorganic Chemistry 52 (2014) 8–15
3. Discussion and conclusion
Before the synthesis of COX-2 inhibitors in wet lab condi-
tions, the evaluation of molecular interactions between enzyme
and inhibitor is an important step for successful design of selec-
tive COX-2 inhibitors. Also, the structural differences between
the active sites of COX-1 and COX-2 should be investigated
extensively for designing selective COX-2 inhibitors. The major
difference between COX-1 and COX-2 is the larger volume
(about 20%) of the active site channel of COX-2 [1]. Thus, the de-
sign of an inhibitor which is able to bind to the active site of
COX-2 by preventing the entrance of the arachidonic acid is very
important. On the other hand, arachidonic acid binds to the ac-
tive site from Arg120 with its carboxyl oxygens and Tyr385 is
involved in the radicalic transformation of arachidonic acid to
further intermediates. Therefore, both steric hinderance and en-
zyme-inhibitor side chain interactions are of great importance
for successful drug design. Most of the classical NSAIDs are
known to inhibit both COX-1 and COX-2 non-selectively. Since
some NSAIDs have negative side effects on human metabolism
such as cardiovascular and gastrointestinal system problems,
some of them (e.g., rofecoxib, valdecoxib) were withdrawn from
the market [6–8]. Ibuprofen is one of the NSAIDs that inhibit
COXs non-selectively. It was found to interact with Arg120 and
Tyr355 in the active site of COX-1 [9]. Modification of ibuprofen
from its carboxyl group has been studied in the literature [10–
19] and improvements in the selectivity and anti-inflammatory
activity were achieved. Hegazy and Ali [10] have reported re-
duced ulcerogenic potential for NSAIDs that modified from their
carboxyl group in addition to their anti-inflmmatory and analge-
sic acitivities. Also, NSAIDs esterified from their carboxyl group
showed higher binding affinity and good selectivity for COX-2
in molecular docking studies [10]. Raval et al. [11] have observed
good anti-inflammatory and analgesic activities with reduced
ulcerogenic effect for ibuprofen derivatives, synthesized with
cyclization of carboxyl group and bearing thiadiazolo[3,2-
a][1,3,5]triazine-5-thione nucleus. Amides of arylpropionic acids
have been studied in numerous researches, but only few of them
were related with ibuprofen amide derivatives [20,21]. It is well
known that amide derivatives of arylpropionic acids may retain
the activity of the parent acids and decrease their gastrointesti-
nal toxicity [22,23]. In this study, it was aimed at synthesizing
selective ibuprofen analogues that differ in functional group
reactivity and steric hindrance. Also, inhibitory activities of syn-
thesized molecules were estimated by in silico molecular docking
and in vitro enzyme inhibition studies. Among synthesized mol-
ecules, binding affinities of molecules 2a, 2b and 2c were close
to that of arachidonic acid and also higher than commercial
COX-2 inhibitors. This theoretical result indicates the effect of
aliphatic hydrocarbon chain that is fixed to carboxyl group of
ibuprofen by amide bond when compared to other synthesized
molecules. This linear hydrocarbon chain could be able to mimic
the arachidonic acid by its hydrophobic nature and it might pre-
vent the entrance of substrate by closing the active site channel
around Arg120. However, molecule 3a and 3c were found more
effective than ibuprofen and other synthesized ibuprofen deriva-
tives in in vitro analyses. The predicted binding affinity of mole-
cule 3a was closer to reflect the in vitro % inhibition of COX-2. In
addition, the results of LeadIT did not confirm the findings of
AutoDock Vina. Spearman’s rank correlation coefficient
Fig. 2. 3D representation of docked molecules and their interactions on active site
of COX-2 (PDB ID: 1CVU). (a) Arachidonic acid, (b) Diclofenac, and (c) Molecule 2c.
same number of amino acids. 2D representations of enzyme-inhib-
itor interactions showed that arachidonic acid and molecule 2c
were able to interact with active site through their long hydropho-
bic tales (Fig. 3). Binding affinity results obtained from AutoDock
Vina were not parallel to results of LeadIT. According to the results
of AutoDock Vina, the molecules 2d, 2a and 2b had greater mean
binding affinities compared to other inhibitors (Table 2). When
compared to the docking result of ibuprofen, only molecule 3d
has lower binding affinity against COX-2. The in silico binding affin-
ities of synthesized COX-2 inhibitors were also found for COX-1 to
compare their selectivity theoretically. According to the AutoDock
Vina results, the binding affinities of the synthesized ibuprofen
analogues against COX-1 were close to that of COX-2 (Table 3).
However, binding affinity of the ibuprofen against COX-1 was
found higher compared to COX-2.
The in vitro inhibition studies on COX-2 showed that molecule
3a and 3c were the most effective inhibitors among other studied
ibuprofen derivatives at 50 lM of final inhibitor concentration
(Table 4). The inhibition percentage of ibuprofen (positive control)
was 9.7%. Molecule 2a and 3d were the least effective compounds
compared to ibuprofen and their binding energies (or docking
scores) were also low according to the in silico analysis.
(q
= ꢀ0.8) between the results of LeadIT and AutoDock Vina
can confirm this controversial result. It can be explained by
the different theoretical backgrounds of both softwares. Similar
controversial results were also obtained by Maldonado-Rojas
and Olivero-Verbel for AutoDock Vina, GOLD and Surflex-Dock

C. Gundogdu-Hizliates et al. / Bioorganic Chemistry 52 (2014) 8–15
11
Fig. 3. 2D representation of interactions between docked molecules and active site of COX-2. (a) Arachidonic acid, (b) Diclofenac, and (c) Molecule 2c.
[24]. Therefore, the differences observed between results of in
silico and in vitro analyses could be due to the rigid prediction
methods used in molecular docking softwares that do not in-
clude instant conformational changes in the protein or inhibitor,
and close prediction results arising from similarity of functional
groups in the inhibitors [25–39]. Moreover, the controversies be-
tween in vivo and in vitro experiments were also reported in the
literature [40–43]. There are increasing numbers of studies that
use computer aided drug design approaches including molecular
docking to predict interactions of novel inhibitor molecules [44–
59]. Also, theoretical results such as binding affinity are reported
to be well in line with experimental results in the great number
of molecular docking studies [24,53,60,61]. Since Spearman’s
rank correlation coefficient was found as 0.6 between mean
scores (Table 2) and percent inhibition (Table 4), the molecular
docking calculations in our study could be considered as reliable.
In conclusion, the newly synthesized ibuprofen derivatives men-
tioned in the present paper can be used in model in vitro and
in vivo studies.
4. Experimental section
4.1. Molecular docking studies
Crystal structures of COX-1 (PDB ID: 1CQE and 1EQH) and COX-
2 (PDB ID: 1CVU, 1CX2, and 1PXX) were retrieved from Protein
Data Bank [62]. 3D structures of commercial COX-1 and COX-2
inhibitors were obtained from PubMed Compound Database in
SDF file format. 2D structures of novel ibuprofen analogues were
drawn with Accelrys Draw 4.0 and saved as MOL file format. Pre-
pared 2D coordinates of inhibitors were converted to 3D, opti-
mized and explicit hydrogens were removed with CHEMSKETCH
12.01 (freeware) and saved as MOL file format. 3D coordinates in
MOL format were converted to MOL2 file format with YASARA
11.11.2 [63]. PyRx 0.8 was used for energy minimization calcula-
tions and MOL to PDBQT file format conversion [64]. Molecular
docking calculations were comparatively performed with LeadIT
v2.0.2 and AutoDock Vina 1.1.2 [65,66]. LeadIT is a molecular drug
design platform for docking applications. In LeadIT, possible

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C. Gundogdu-Hizliates et al. / Bioorganic Chemistry 52 (2014) 8–15
Table 2
Molecular docking results obtained with AutoDock Vina 1.1.2 for newly synthesized potential COX-2 inhibitors and commercial inhibitors on COX-2 crystal structures (PDB ID:
1CVU, 1CX2 and 1PXX).
COX-2 inhibitor
COX-2 structure
PDB ID: 1CVU
PDB ID: 1CX2
PDB ID: 1PXX
Mean score
Vina score (kcal molꢀ1
)
Vina score (kcal molꢀ1
)
Vina score (kcal molꢀ1
)
Arachidonic acid
Celecoxib
Curcumin
Diclofenac
Etoricoxib
Meloxicam
Naproxen
Rofecoxib
S58
Valdecoxib
Ibuprofen
Molecule 2a
Molecule 2b
Molecule 2c
Molecule 2d
Molecule 3a
Molecule 3b
Molecule 3c
Molecule 3d
ꢀ6.43 0.21
ꢀ5.93 0.06
ꢀ7.23 0.06
ꢀ6.00 0.35
ꢀ6.03 0.11
ꢀ7.30 0.00
ꢀ6.67 0.31
ꢀ6.23 0.23
ꢀ5.90 0.17
ꢀ6.77 0.11
ꢀ6.60 0.00
ꢀ6.57 0.06
ꢀ5.80 0.35
ꢀ5.67 0.15
ꢀ6.87 0.06
ꢀ6.57 0.06
ꢀ6.40 0.10
ꢀ7.53 0.06
ꢀ6.50 0.52
ꢀ6.43 0.15
ꢀ7.13 0.23
ꢀ7.53 0.23
ꢀ5.80 0.00
ꢀ6.60 0.00
ꢀ7.73 0.06
ꢀ6.67 0.23
ꢀ7.47 0.06
ꢀ6.60 0.00
ꢀ7.07 0.32
ꢀ5.93 0.12
ꢀ6.73 0.15
ꢀ6.90 0.00
ꢀ7.07 0.15
ꢀ8.30 0.00
ꢀ7.47 0.06
ꢀ7.57 0.06
ꢀ8.30 0.26
ꢀ7.83 0.15
ꢀ5.87 0.21
0.03 2.02
ꢀ4.20 2.17
ꢀ5.50 0.10
0.60 2.36
ꢀ6.24
ꢀ4.34
ꢀ6.32
ꢀ5.77
ꢀ4.01
ꢀ7.47
ꢀ6.47
ꢀ6.33
ꢀ4.75
ꢀ6.47
ꢀ6.01
ꢀ6.20
ꢀ6.13
ꢀ6.12
ꢀ6.40
ꢀ6.52
ꢀ6.57
ꢀ6.15
ꢀ4.81
ꢀ7.37 0.06
ꢀ6.07 0.06
ꢀ5.30 0.70
ꢀ1.76 2.14
ꢀ5.57 0.49
ꢀ5.50 0.00
ꢀ5.30 0.10
ꢀ5.70 0.20
ꢀ5.63 0.55
ꢀ4.03 2.48
ꢀ5.53 1.16
ꢀ5.73 0.23
ꢀ2.63 4.34
ꢀ0.10 3.29
Table 3
Molecular docking results obtained with AutoDock Vina 1.1.2 for newly synthesized ibuprofen derivatives and commercial inhibitors on COX-1 crystal structures (PDB ID: 1CQE
and 1EQH).
COX-1 inhibitor
COX-1 structure
PDB ID: 1CQE
PDB ID: 1EQH
Mean score (kcal molꢀ1
)
Vina score (kcal molꢀ1
)
Vina score (kcal molꢀ1
)
Arachidonic acid
Aspirin
Celecoxib
Flurbiprofen
Ibuprofen
Indomethacin
Mofezolac
Molecule 2a
Molecule 2b
Molecule 2c
Molecule 2d
Molecule 3a
Molecule 3b
Molecule 3c
Molecule 3d
SC 560
ꢀ7.13 0.06
ꢀ6.70 0.00
ꢀ4.70 0.00
ꢀ9.00 0.00
ꢀ7.80 0.00
ꢀ4.90 0.00
ꢀ5.97 0.23
ꢀ6.23 0.06
ꢀ6.83 0.58
ꢀ7.53 0.06
ꢀ6.63 0.55
ꢀ6.73 0.21
ꢀ5.87 0.81
ꢀ5.43 0.06
ꢀ5.63 0.84
ꢀ5.87 0.06
ꢀ7.53 0.06
ꢀ6.80 0.00
ꢀ3.70 0.00
ꢀ9.67 0.06
ꢀ7.80 0.00
ꢀ4.20 0.00
ꢀ6.40 0.00
ꢀ6.80 0.00
ꢀ5.86 0.12
ꢀ6.00 0.00
ꢀ6.23 0.58
ꢀ6.83 0.06
ꢀ5.40 0.00
ꢀ6.80 0.69
ꢀ4.20 0.10
ꢀ4.76 0.06
ꢀ7.33
ꢀ6.75
ꢀ4.20
ꢀ9.34
ꢀ7.80
ꢀ4.55
ꢀ6.19
ꢀ6.51
ꢀ6.34
ꢀ6.77
ꢀ6.43
ꢀ6.78
ꢀ5.64
ꢀ6.12
ꢀ4.92
ꢀ5.32
[67]. Molecular docking calculations with AutoDock Vina were per-
formed according to Maldonado-Rojas and Olivero-Verbel [24].
Briefly, AutoDock Vina performs calculations by using coordinates
belong to the binding area of native ligand as a template in cubic
geometry. Thus, each dimension of the cubic area was set to 24 Å
and Cartesian coordinates (X, Y and Z) of the center of cubic area
were selected for each crystal structures as follows: 25.277,
22.358 and 49.308 for 1CVU; 25.374, 21.657 and 17.292 for
1CX2; 27.058, 24.431 and 15.437 for 1PXX; 25.3693, 33.9205 and
207.8642 for 1CQE; 27.6342, 33.7218 and 199.0601 for 1EQH,
respectively. Average binding affinities of inhibitors were calcu-
lated after three runs of the software by selecting the value of best
pose in each run. The chiral centers of all the designed compounds
were at S-configuration for in silico analysis.
Table 4
Percent inhibition of COX-2 by the synthesized ibuprofen derivatives. Data is obtained
from in vitro inhibition assay of COX-2 and the study was performed as three
replicates.
Inhibitor
% Inhibition of COX-2
Molecule 2a
Molecule 2b
Molecule 2c
Molecule 2d
Molecule 3a
Molecule 3b
Molecule 3c
Molecule 3d
Ibuprofen
8.3
12.4
11.4
15.6
22.9
12.4
21.5
5.0
9.7
binding regions of inhibitors were selected around amino acid res-
idues which were 6.5 Å away from the binding site of arachidonic
acid in spherical geometry. Binding affinities of inhibitors were cal-
culated with HYDE function in LeadIT. Interactions of inhibitors
with COX-2 were viewed in 2D with PoseView program in LeadIT
4.2. Organic synthesis of novel ibuprofen derivatives
Molecular structures of synthesized ibuprofen amide and ibu-
profen acyl hydrazone derivatives were given in Fig. 4.

C. Gundogdu-Hizliates et al. / Bioorganic Chemistry 52 (2014) 8–15
13
Fig. 4. Molecular structures of novel ibuprofen derivatives as potential COX-2 inhibitors.
Scheme 1. Overall scheme of reagents and reaction conditions: (i) SOCl2, reflux, 2 h; (ii) THF, triethyl amine, amine, 70 °C, 6 h; (iii) HetArCHO, C2H5OH, AcOH, reflux, 4 h.
Molecule 1 is ibuprofen.
4.3. General procedure for the synthesis of ibuprofen amide derivatives
Triethylamine (5 mmoles) and amine derivatives or hydrazide
(5 mmoles) were added into this solution and then the solution
was heated at 70 °C for 6 h. The reaction mixture was filtered
and the solvent was evaporated. The residue was chromatographed
over silica gel with ethyl acetate-hexane (1:1). The solvent was
evaporated under reduced pressure to obtain ibuprofen amide
derivatives (Scheme 1).
A mixture of ibuprofen (1 in Scheme 1) (5 mmoles) and thionyl
chloride (5.5 mmoles) was refluxed on an oil bath for 2 h. The ex-
cess thionyl chloride was removed under reduced pressure to pro-
duce ibuprofen acyl chloride as yellow oil. Then, ibuprofen acyl
chloride (5 mmoles) was dissolved in tetrahydrofuran (20 mL).

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C. Gundogdu-Hizliates et al. / Bioorganic Chemistry 52 (2014) 8–15
4.4. General procedure for the synthesis of ibuprofen acyl hydrazone
derivatives
4.9. N0-(furan-2-ylmethylene)-2-(4-isobutylphenyl)propanehydrazide
(molecule 3a)
Ibuprofen acyl chloride (5 mmoles) was dissolved in tetrahy-
drofuran (20 mL). Triethylamine (5 mmoles) and hydrazine hy-
drate (5 mmoles) were added into this solution and then the
solution was heated at 70 °C for 6 h. The reaction mixture was
poured into the cold water. Then the precipitate was filtered and
washed with water. The crude ibuprofen hydrazide was refluxed
with heteroaryl aldehydes in the presence of glacial acetic acid in
ethanol for 4 h. Then the reaction mixture was cooled and precip-
itate was filtered. The crude product was re-crystallized from eth-
anol yielded ibuprofen acyl hydrazone derivatives (Scheme 1).
Light brown solid (74%); mp 148 °C. This compound was syn-
thesized previously by our group [68], and also Nakka et al. [69].
4.10. 2-(4-isobutylphenyl)-N0-(thiophen-2-
ylmethylene)propanehydrazide (molecule 3b)
Light brown solid (72%). mp: 141–142 °C. IR (KBr):
m 3447 (NH),
2953 (CH), 1667 (C@O) cmꢀ1. 1H NMR (400 MHz, CDCl3): d 0.88 (d,
6H, J = 6.9 Hz, 2xCH3), 1.53 (d, 3H, J = 6.8 Hz, CH3), 1.75–1.94 (m,
1H, CH), 2.38 (d, 2H, J = 7.1 Hz, CH2), 4.66 (q, 1H, J = 7.0 Hz, CH),
6.96 (d, 2H, J = 4.0 Hz, Thiophene-H), 7.06 (d, 2H, J = 7.6 Hz, ArH),
7.14 (d, 1H, J = 3.6 Hz, Thiophene-H), 7.22 (t, 1H, J = 4.6 Hz, Thio-
phene-H), 7.33 (d, 2H, J = 8.0 Hz, ArH), 7.91 (s, 1H, N@CH), 10.20
(s, 1H, NH). Anal. Calcd. for C18H22N2SO: 68.75C; 7.05H; 8.91N;
10.20S. Found: 68.83C; 7.13H; 8.82N; 10.22S.
4.5. N-(2,2-dimethoxyethyl)-2-(4-isobutylphenyl)propanamide
(molecule 2a)
Oily product (50%). IR (KBr):
m 3308 (NH), 2952 (CH), 1651
(C@O), 1131 (CAO) cmꢀ1 1H NMR (400 MHz, CDCl3): d 0.89 (d,
.
4.11. N0-((1H-indol-3-yl)methylene)-2-(4-
isobutylphenyl)propanehydrazide (molecule 3c)
6H, J = 6.4 Hz, 2xCH3), 1.51 (d, 3H, J = 7.2 Hz, CH3), 1.84 (m, 1H,
CH), 2.45 (d, 2H, J = 7.2 Hz, CH2), 3.27 (d, 2H, J = 1.6 Hz, CH2), 3.32
(s, 6H, 2xOCH3), 3.53 (q, 1H, J = 6.8 Hz, CH), 4.27 (t, 1H,
J = 10.8 Hz, CH), 5.57 (s, 1H, NH), 7.11 (d, 2H, J = 8 Hz, ArH), 7.19
White solid (71%). mp: 165–166 °C. IR (KBr):
m 3447 (NH), 3395
(NH), 2975 (CH), 1662 (C@O) cmꢀ1. 1H NMR (400 MHz, d6-DMSO):
d 0.82 (d, 6H, J = 7.1 Hz, 2xCH3), 1.41 (d, 3H, J = 6.9 Hz, CH3), 1.73–
1.78 (m, 1H, CH), 2.36 (d, 2H, J = 7.1 Hz, CH2), 4.74 (q, 1H, J = 7.2 Hz,
CH), 7.03–7.42 (m, 8H, ArH and indole-H), 7.72 (d, 1H, J = 13.2 Hz,
indole-H), 8.18 (s, 1H, N@CH), 11.13 (s, 1H, NH), 11.49 (s, 1H, NH).
Anal. Calcd. for C22H25N3O: 76.05C; 7.25H; 12.09N. Found: 75.91C;
7.33H; 12.14N.
(d, 2H, J = 8.4 Hz, ArH). Anal. Calcd. for
C17H27NO3: 69.59C;
9.28H; 4.77N. Found: 69.45C; 9.34H; 4.81N.
4.6. N-(2,2-dimethoxyethyl)-2-(4-isobutylphenyl)-N-
methylpropanamide (molecule 2b)
Oily product (45%). IR (KBr):
m 2954 (CH), 1649 (C@O), 1124
4.12. N0-((9-hexyl-9H-carbazol-3-yl)methylene)-2-(4-
isobutylphenyl)propanehydrazide (molecule 3d)
(CAO) cmꢀ1 1H NMR (400 MHz, CDCl3): d 0.89 (d, 6H, J = 14 Hz,
.
2xCH3), 1.26 (t, 1H, J = 14 Hz, CH), 1.42 (d, 3H, J = 7.2 Hz, CH3),
1.84 (m, 1H, CH), 2.44 (d, 2H, J = 6.8 Hz, CH2), 2.93 (s, 3H, NCH3),
3.30 (s, 6H, 2xOCH3), 3.35 (d, 2H, J = 4.4 Hz, CH2), 3.85 (q, 1H,
J = 6.8 Hz, CH), 7.08 (d, 2H, J = 8 Hz, ArH), 7.16 (d, 2H, J = 8.4 Hz,
ArH). Anal. Calcd. for C18H29NO3: 70.32C; 9.51H; 4.56N. Found:
70.22C; 9.63H; 4.62N.
Pale yellow solid (62%). mp: 177–178 °C. IR (KBr):
m 3420 (NH),
2982 (CH), 1664 (C@O) cmꢀ1. 1H NMR (400 MHz, d6-DMSO): d 0.75
(t, 3H, J = 6.8 Hz, CH3), 0.81 (d, 6H, J = 6.9 Hz, 2xCH3), 1.16–1.22 (m,
6H, CH2), 1.40 (d, 3H, J = 7.0 Hz, CH3), 1.71–1.87 (m, 3H, CH and
CH2), 2.38 (d, 2H, J = 7.3 Hz, CH2), 4.32 (t, 2H, J = 6.8 Hz, NCH2),
4.72 (q, 1H, J = 6.8 Hz, CH), 7.02–7.59 (m, 7H, ArH), 7.79 (m, 1H,
ArH), 8.09 (s, 1H, N@CH), 8.13–8.39 (m, 3H, ArH), 11.41 (s, 1H,
NH). Anal. Calcd. for C32H39N3O: 79.79C; 8.16H; 8.72N. Found:
80.16C; 8.24H; 8.64N.
4.7. N-(4,4-diethoxybutyl)-2-(4-isobutylphenyl)propanamide
(molecule 2c)
Oily product (48%). IR (KBr):
m 3320 (NH), 2930 (CH), 1650
(C@O), 1128 (CAO) cmꢀ1 1H NMR (400 MHz, CDCl3): d 0.89 (d,
.
4.13. In vitro evaluation of cyclooxygenase-2 enzyme activity
inhibition
3H, J = 6.8 Hz, CH3), 0.91 (d, 3H, J = 6.8 Hz, CH3), 1.16 (t, 3H,
J = 6.8 Hz, CH3),1.20 (t, 3H, J = 5.6 Hz, CH3), 1.47 (d, 3H, J = 6.8 Hz
CH3), 1.47–1.53 (m, 4H, 2x CH2), 1.85 (m, 1H, CH), 2.45 (d, 2H,
J = 7.6 Hz, CH2), 3.20 (m, 2H, CH2), 3.40–3.53 (m, 4H, 2xCH2), 3.6
(m, 1H, CH), 4.12 (t, 1H, J = 7.2 Hz CH), 5.49 (s, 1H, NH), 7.09 (d,
2H, J = 8 Hz, ArH), 7.18 (d, 2H, J = 8 Hz, ArH). Anal. Calcd. for
The inhibition of COX-2 enzyme activity was studied by using
the colorimetric ovine inhibitor screening assay kit (Cayman
Chemical Company, catalogue no 760111). The assay buffer
(0.1 M Tris–HCl, pH 8.0), heme and the (ovine) COX-2 were pre-
pared according to the instructions of the producer and added
into a 96-well plate. The arachidonic acid was prepared at
C21H35NO3: 72.17C; 10.09H; 4.01N. Found: 72.25C; 10.22H; 4.08N.
0.55 mM before its use to achieve 50
tration in the wells. Then, the inhibitors were dissolved in di-
methyl sulfoxide and added into the wells at final
concentration of 50 M. Following the five minutes of incubation
at 25 °C, 20 L of the colorimetric substrate (TMPD) and the ara-
chidonic acid were added into the wells. After a second incuba-
tion for five minutes at 25 °C, the absorbances of the wells
were measured at 590 nm by using a microplate reader (BioTek
Instruments, USA, ELx800). Ibuprofen was used as positive control
and the results are obtained from three independent measure-
ments (n = 3). Inhibiton percentage (% Inhibition) of the COX-2
enzyme activity was calculated as below:
lM final substrate concen-
4.8. N0-(2-(4-isobutylphenyl)propanoyl)isonicotinohydrazide
(molecule 2d)
a
l
Oily product (76%). IR (KBr):
m
.
3445 (NH), 3232 (NH), 2953 (CH),
l
1695 (C@O), 1651 (C@O) cmꢀ1
1H NMR (400 MHz, CDCl3): d 0.90
(d, 6H, J = 6.8 Hz, 2xCH3), 1.50 (d, 3H, J = 6.8 Hz, CH3), 1.81 (m,
1H, CH), 2.41 (d, 2H, J = 7.2 Hz, CH2), 3.76 (q, 1H, J = 6.8 Hz, CH),
7.07 (d, 2H, J = 7.6 Hz, ArH), 7.21 (d, 2H, J = 7.6 Hz, ArH), 7.57 (d,
2H, J = 7.6 Hz, ArH), 8.60 (d, 2H, J = 6.8 Hz, ArH), 9.36 (s, 1H, NH),
10.47 (s, 1H, NH). Anal. Calcd. for C19H23N3O2: 70.13C; 7.12H;
12.91N. Found: 70.02C; 7.20H; 12.95N.

C. Gundogdu-Hizliates et al. / Bioorganic Chemistry 52 (2014) 8–15
15
ðA100% ꢀ ABlÞ ꢀ ðAsample ꢀ ABlÞ
ðA100% ꢀ ABlÞ
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%Inhibition ¼
ꢁ 100
ð1Þ
where A100% is the mean absorbance value of 100% enzyme activity
wells, ABl is the mean absorbance of blank wells, and Asample is the
mean absorbance value of the wells including the inhibitors.
4.14. Statistical analysis
Spearman’s rank correlation coefficients between in silico and
in vitro data were calculated by using Minitab 16.
Acknowledgments
The authors thank Ms. Gloria Kraschinski from BioSolveIT for
providing temporary license of LeadIT. H. Alyuruk is thankful to
Turkish Scientific and Technological Research Council for scholar-
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