Journal of Molecular Structure 1264 (2022) 133234

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Journal of Molecular Structure

journal homepage: www.elsevier.com/locate/molstr

Synthesis, characterization, computational, anticancer and

anti-inflammatory studies of pregabalin esters and their complexes
with heavy metals
Ayesha Asbat a, Farooq Saleem a,∗, Saima Najm b, Javed Iqbal a, Muhammad Ali Syed a,
Sarfraz Ahmad c, Sana Hanif a, Muhammad Azeem a
a
Faculty of Pharmacy, The University of Lahore, Lahore, Pakistan
b
Faculty of Pharmacy, Lahore College of Pharmaceutical Sciences, Lahore, Pakistan
c
Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: The current study entails a series of pregabalin ester complexed with zinc and copper; further character-
Received 22 June 2021 ized by spectroscopic techniques and biological assays. Docking analysis revealed three complexes with
Revised 17 February 2022
favorable binding interactions which were further subjected to in-vitro anti-inflammatory and anticancer
Accepted 4 May 2022
activities. The complex-5 displayed most potent anti-inflammatory activity against Luminol-enhanced
Available online 18 May 2022
Chemiluminescence assay with IC50 value of 7.7 ± 0.8 μM as compared to standard Ibuprofen with IC50
Keywords: value of 11.2 ± 1.9 μM; whereas none of them showed significant outcomes against HeLa cell lines for
Pregabalin antiproliferative activity as compared with standard doxorubicin. Computational studies were carried out
Esters using the reported crystal structure of multidrug resistant Alpha2S protein (PDB-ID: 6ND9), neurokinin-
Auto dock 1 receptor (PDB-ID: 2KSA) and dihydrofolate reductase (PDB-ID: 3GHW); indicating the compound 3
Luminol showed significant interactions at all receptors. As the analogues were more active than parent drug in
Antitumor
some respects; therefore, the expansion of the project can be planned to carry out more pharmacological
Anti-inflammatory
screening.
© 2022 Elsevier B.V. All rights reserved.

1. Introduction drowsiness while using it in generalized anxiety disorder. However,

Pregabalin, [3 - (amino methyl hexonic acid)] is used in the
treatment of epilepsy [1]. It was synthesized in 1990 as an an-
ticonvulsant drug [2]. The drug treats convulsions at the dose of
600 mg/day. However, some patients feel dizziness at initial dose
[3]. Numerous scientists have used functional MRI to study brain
systems; most of them have used graph theory analysis. However,
functional techniques can be analyzed by the effects of antiepilep-
tic drugs [4]. The drug was accepted to treat partial seizures in
adults as well as to cure neuropathic pain and diabetic neuropathy
[5]. Pregabalin (PGB) binds with high affinity to the R2-δ subunit
of voltage gated calcium channels in cell membrane. Nitroalkane-
mediated cycloproponation as a central step was used to prepare
some new amino acids [6,7]. PGB lacks plasma protein binding and
so it has no interactions with other anticonvulsants, antidiabetics,
and oral contraceptives. However, it readily infiltrates the blood-
brain barrier [1]. PGB shows adverse effects such as dizziness and
∗
Corresponding author.
E-mail address: farooq.saleem@pharm.uol.edu.pk (F. Saleem).
it would be safe to use these types of medications at night and
avoid them to use during traveling [8].
On the high-pitched side, many drugs possess improved
toxicological and pharmacological properties (anti-inflammatory
and anti-cancer) in the form of metal complexes. Some metal
complexes e.g. silver metal complex was synthesized for anti-
inflammatory activity and the quinolone derivatives along with
their antimony (III) complexes were assessed for their cytotoxicity
against HL-60 and Jurkat human leukemia cell lines [9,10]. How-
ever, some essential trace metals including zinc, copper and iron
enable our body to function normally and their deficiency leads
to abnormal growth, poor immunity and decline oxygen transport
to body [11]. A new platinum (IV) complex, [Pt(Cl)2(Pregabalin)2],
holding the drug PGB was synthesized, characterized by differ-
ent techniques and observed to bind with Human serum albumin
(HSA) and causes strong fluorescence quenching of HAS [11,12].
Besides, metal complexes of PGB derivatives have played a cen-
tral role in improvement of co-ordination chemistry. In this study,
various esters and metal complexes of PGB were synthesized. For
better understanding between ligands and targets, molecular dock-

https://doi.org/10.1016/j.molstruc.2022.133234
0022-2860/© 2022 Elsevier B.V. All rights reserved.
A. Asbat, F. Saleem, S. Najm et al.
Scheme 1. General scheme of synthesis of pregabalin derivatives and their metal complexes (Zn and Cu).
M = ZnCl2, Cu(CH3 COO)2. H2 O 1=pregabalin; 2=formaldehyde; 3=Ligand formaldehyde with PGB; 4 a, b = pregabalin ester with metal complexes (Zn and Cu).
ing studies was planned and assessed their interactions against α 2-
δ −1 subunit (PDB-ID: 6ND9); neurokinin-1 (PDB ID: 2KSA) and di-
hydrofolate reductase (PDB ID: 3GHW). The findings explored the
significant anticancer and anti-inflammatory potential PGB deriva-
tives, making them suitable for advanced pharmacological screen-
ing.
2. Results and discussion
2.1. Chemistry
The target compounds were synthesized as sketched in
Scheme 1. The synthesis of PGB esters (3) were achieved by reac-
tion of compound 1 with 2 (Scheme 1). The synthesis of PGB esters
is the main intermediate for synthesis of our target compounds.
Moreover, zinc chloride and copper acetate form coordinate bonds
with derivatives by using methanol to complete the formation
of required heavy metal complexes (4a and 4b) of PGB [12]. Fi-
nally, the structure elucidation was achieved by FTIR, 1 H NMR and
ESI-MS spectra. In FTIR spectra all compounds showed carbonyl
stretching vibrations in the range of 1668–1670 cm−1 . Initially we
were expecting an open chain structure of coordinate complexes;
however, characterization confirmed the presence of straight chain
structures of amines (see supplementary Figs. S1,S2). FTIR spectra
of PGB ester and its metal complexes proved the straight chain
structure of amines. However, in the 1 H NMR spectra the multi-
ple signals present in the region of δ 0.78–4.69 also represents
straight chain structure of alkyl amines (see sup Figs. S3, S4). Spec-
troscopic analysis of ESI-MS was used to recognize the formation
of PGB derivative and its metal complexes. The HRMS (ESI+) cal-
culated for C9 H19 NO2 [M+H+] is 173.292 Da, found = 174.148 Da
where as; HRMS (ESI+) calculated for compound C9H19NO2 is
[M+H+] = 408.204 Da, and found = 408.2458 Da. This data fur-
ther supported the proposed structure and purity of the com-
pounds was confirmed (sup Fig. S5). The absorption bands at 2953,
2598, 1641, 1386, 1332 and 1162 cm−1 confirmed the comport-
ment of -CH, -OH, -NH2, -CH3,=CH2 and C-O, respectively. How-
ever, 1HNMR data of compound 3 showed a doublet at δ = 0.78
for CH3 functional group and also showed a clear singlet at δ =
2
Journal of Molecular Structure 1264 (2022) 133234
4.69 for NH2 group. All other peaks at 2.24, 2.86 and 4.69 showed
presence of -CH3 group. Resultantly, above mentioned peaks con-
firm the structure of compound 3. 1 HNMR data of compound 4a
showed singlet peak at δ = 3.55 for -CH3 functional group and also
showed a clear singlet at δ = 4.70 which indicated the presence of
–NH2 group and hence this spectroscopic analysis shows the con-
firmation of 4a. The absorption bands at 3266.8, 2963.9, 1669.8,
1546.8, 1485, 1388.4 and 1276.6 cm−1 confirmed the presence of
NH, -CH, -C=O, -NH2, -CH3,=CH2 and C-O, respectively so we have
seen the confirmation of compound 4b via this spectroscopic anal-
ysis.
2.2. Solubility studies
The solubility of newly synthesized metal complexes was eval-
uated. A measured amount of each compound was dissolved in
weighed amount of solvent and clarity of solution was observed.
Water, methanol, ethanol, DMSO and chloroform were used for this
study. For good solubility the threshold value of <0.1/100 ml with
the clear colored solution in a given solvent was considered [13].
The solubility of various ligands and their metal complexes is given
in Table S2.
2.3. Computational studies
Molecular docking studies were used to evaluate the binding
energies, kinetic properties and enzymes interaction for selected
compounds [14]. The three dimensional coordinates of 6ND9, 2KSA
and 3GHW were retrieved from RCSB Protein Data Bank (PDB);
their respective PDB ID’s along with their affinity with α 2-δ−1,
NK1R and DHFR receptors, respectively were tabulated in Table 1.
The molecular properties of respective ligands were deduced from
Auto Dock Tools [15–17] also tabulated in Table 1. Structure-based
sequence configuration of α 2-δ −1, NK1R and DHFR against the se-
lected model was performed. The 3D homology model of these re-
ceptors in homo-sapiens was predicted using Auto Dock Tools1.5.6.
High quality models demands an accurate sequence alignment be-
tween the model and the template protein. Auto Dock generates
10 clusters, the cluster with least binding energy was found to
A. Asbat, F. Saleem, S. Najm et al. Journal of Molecular Structure 1264 (2022) 133234

Table 1
Binding properties of receptor proteins to different ligands.

Molecular Active Receptors Interacting Efficacy Least Binding Inhibition Torsional No. of Interacting residues
Formula compound Protein(PDB energy (kcal/mol) Constant energy hydrogen
ID) (μM) bonds

C9H19NO2 3 α 2-δ −1 6ND9 Anticonvulsant −5.41 125.7 0.89 1 LEU72, ARG73, CYS81,
GLN102, MET103,
CYS104, GLN105,
TYR118,
C8H17NO2 PGB α 2-δ −1 6ND9 Anticonvulsant −5.19 157.4 0.6 1 ARG73, MET103,
CYS104, GLN105,
TYR118
C9H19NO2 3 NK1R 2KSA Anti-Inflammatory −6.41 20.15 0.89 1 LEU5, PRO6, VAL7,
THR173, MET174,
TRP184, PRO185
C13H18O2 IBU NK1R 2KSA Anti-Inflammatory −6.47 31.63 1.19 1 VAL66, THR65,
ARG130, ALA133,
HIS136, LEU138,
ARG141, LEU142,
LYS243
C9H19NO2 3 DHFR 3 ghw Anti-cancer −6.73 62.93 0.89 0 ASP21, LEU22, PHE34,
THR56, SER59, ILE60,
VAL115
C27H29NO11 DOX DHFR 3 ghw Anti-cancer −6.96 7.94 0.89 0 ILE7, VAL8, ILE16,
GLY17, ASP21, LEU22,
GLU30, PHE31, PHE34,
SER59, PRO61, ASN64,
GLY116, GLY117,
TYR121

Fig. 1. (A) Docked structure of PGB shown in orange color; with modelled 6ND9
receptor with interacting residues shown in blue color; blue dashes show hydrogen
bonding calculated by PLP. (B) The 2D interacting diagram of ligand prepared from
protein plus online server. (C) Ligand-receptor interaction calculated by auto dock;
the binding energy of docked complex was found to be −5.19 Kcal/mole implies
more stable structure.
Fig. 2. (A) Docked ligand (3) was shown in orange color with blue colored amino
acid residues (GLN105, and TYR118) in the binding pocket of 6ND9 calculated by
PLP, the binding energy of docked complex was found to be −5.41 Kcal/mol implies
more stable structure. (B) Two dimensional binding interaction of ligand show one
hydrogen bond at receptor site. (C) Docked structure of 3 with modelled 6ND9 re-
ceptor for anticonvulsant activity with interacting residues calculated by Auto Dock.

be more stable for pharmacological assessment. Structural assess-

ments and authentication of protein model were performed by the
programs called Pyrex, Protein Plus and Protein Ligand Interaction
Profiler (PLP) online servers [18–20] binding site prediction tools.
The main interacting residues at 6ND9 receptor protein with stan-
dard antiepileptic drug PGB are ARG73, MET103, CYS104, GLN105,
and TYR118 as shown in Fig. 1; with the best dock binding en-
ergy of −5.19 kcal/mol as cleared from Table 1. Whereas; the main
interacting residues of compound 3 at 6ND9 receptor are LEU72,
ARG73, GLN102, MET103, CYS104, GLN105, TYR118, and CYS81 as
revealed in Fig. 2. Receptor protein binding information of com-
pound 3 is provided in Table 1.
The main interacting residues of 2KSA protein with stan- Fig. 3. (A) Docked structure of standard IBU with modelled NK1R receptor with
interacting residues by Auto Dock for anti-inflammatory activity. (B) The 2D inter-
dard anti-inflammatory drug IBU are VAL66, THR65, ARG130,
acting diagram of ligand prepared from protein plus online server at 2KSA receptor
ALA133, HIS136, LEU138, ARG141, LEU142, and LYS243 as shown protein.
in Fig. 3. The best docked conformation of IBU has binding en-

3
A. Asbat, F. Saleem, S. Najm et al. Journal of Molecular Structure 1264 (2022) 133234

Fig. 4. (A) Compound 3 shown in orange color with interacting residues of NK1R shown in blue color in the binding pocket of 2KSA receptor calculated by online PLP
web server. (B) 2D interacting diagram of 3 showing hydrogen bond in the binding pocket of 2KSA enzyme. (C) Docked structure of 3 with modelled 2KSA enzyme with
interacting residues by Auto Dock for anti-inflammatory activity.

Fig. 5. (A) DOX was shown in orange color with blue colored amino acid residues in the binding pocket of 3GHW; calculated by PLP online server. (B) Docked structure of
DOX with modelled 3GHW receptor with interacting residues calculated by Auto Dock for anticancer potential.

ergy of −6.47 kcal/mol with 1 hydrogen bond. The main interacting

residue of potent PGB compound (3) with 2KSA receptor are LEU5,
PRO6, VAL7, THR173, MET174, TRP184, and PRO185 as shown in
Fig. 4. Binding information of 3 and IBU is provided in Table 1. The
best dock conformation of this ligand has −6.41 Kcal/mol bind-
ing energy with 1 hydrogen bond. The main interacting residues of
3GHW protein with standard anticancer drug DOX are ILE7, VAL8,
ILE16, GLY17, ASP21, LEU22, GLU30, PHE31, PHE34, SER59, PRO61,
ASN64, GLY116, GLY117, and TYR121 as shown in Fig. 5. The best
docked conformation of DOX has binding energy of −6.96 kcal/mol
with 1 hydrogen bond. The main interacting residue of potent PGB
ester (3) with 3GHW receptor are ASP21, LEU22, PHE34, THR56, Fig. 6. (A) Potent ligand 3 was shown in orange color with blue colored amino
SER59, ILE60, and VAL115 as revealed in Fig. 6. The best dock con- acid residues in the binding pocket of 3GHW; blue line show hydrogen bonding
formation of this ligand has −6.73 Kcal/mol binding energy with whereas gray dotted line represents hydrophobic interactions; calculated by PLP on-
no hydrogen bond. Receptor binding information about DOX and 3 line server. (B) Docked structure of 3 with modelled 3GHW receptor with interact-
ing residues in the binding pocket of DHFR calculated by Auto Dock.
is tabulated in Table 1.

3. Biological screening hibited substantial anti-inflammatory potential, with Inhibi-

3.1. Anti-inflammatory activity
Overall many complexes show varied anti-inflammatory activ-
ity. Among them, silver complexes depict better anti-inflammatory
effect [9]. In the current research, the compound (4b) ex-
tion/Stimulation 84.2% and IC50 = 7.7 μM. On the other hand,
other compounds displayed no activity. As it is cleared from Table
S3 and Fig. 7; when synthesized metallic compounds were com-
pared with standard ibuprofen; copper complex of PGB ester (4b)
showed excellent activity, whereas, compounds 3 and 4a were in-
active against oxidative burst assay.

4
A. Asbat, F. Saleem, S. Najm et al.
Fig. 7. Anti-inflammatory potential of PGB derivative and their metal complexes
shown with positive values show significant activity.
Fig. 8. Cytotoxic activity (HeLa cell line) shown by different groups.
3.2. Anticancer (Cytotoxic) activity
Different derivatives and metal complexes of pregabalin pos-
sess anti-cancer activity with their respected growth prolifera-
tion rates. As it is cleared from data in the Table S4 and Fig. 8
that when we compared standard doxorubicin with synthesized
metal complexes such as PGB-LF-Cu ester (4b) showed some In-
hibition/Stimulation = 35.9% whereas; other compounds appeared
to be inactive against Hela cell lines.
4. Statistical analysis
Data was expressed as mean standard deviation (±S.E), where
appropriate one-way ANOVA followed by post-hoc Dunnet’s test
was employed. Values P < 0.5 were considered as significant.
5. Conclusion
We have synthesized esters of pregabalin and their complexes
with heavy metals like Zn and Cu. Few of them possessed signif-
icant anti-inflammatory potential. Furthermore, all controlled pa-
rameters and their efficacies were validated via their characteriza-
tion by different spectroscopic analysis techniques.
Computational studies have also shown a good correlation be-
tween experiments and estimated binding energy. Considering the
increased interest towards the utilization of these metal complexes,
5
Journal of Molecular Structure 1264 (2022) 133234
the study climaxes the metal co-ordination with pregabalin for
high efficacy and increased therapeutic potential. Resultantly, these
compounds will prove themselves more fruitful in future research
era in terms of their therapeutic fitness and less adverse effects.
6. Experimental
6.1. Material and method
Pregabalin (PGB) was gifted by Briell Pharmaceuticals Pvt. Ltd.,
Lahore- Pakistan. The chemicals and solvents used in research were
purchased from Sigma Aldrich (Germany). IR-spectra of conjugate-
metal complexes were recorded by Agilet FTIR spectrophotome-
ter. 1 H Nuclear magnetic resonance (NMR) spectra were recorded
on a Bruker Avance-300 spectrometer (300 MHz) by dissolving in
dimethyl sulfoxide (DMSO). Electrospray Ionization Mass spectra
(ESI-HR-MS, +/-) were measured at Bruker Compass Data Analyzer
4.2. Three dimensional crystal structures of enzymes were obtained
from Protein Data Bank. Likewise, 3D of ligand and metal com-
plexes were drawn and optimized by ACD/Chem Sketch software.
Molecular docking by Auto Dock Tools 1.5.6 and biological activi-
ties were also performed on analogues.
6.2. Synthesis
6.2.1. General procedure for synthesis of compound 3
Pregabalin (2 M) was mixed with formaldehyde (2 M) in 20 ml
ethanol. Few drops of glacial acetic acid were added as catalyst.
The mixture was stirred with magnetic stirrer for 7 to 8 h. The
progress of reaction was monitored by TLC and the final product
was evaporated at room temperature in petri dish placed in desic-
cator [12].
6.2.2. Methyl 3-(amino methyl)−5-methylhexanoate (3)
Yield: 70%; M. P.: 130 °C; IR ʋ (cm−1): 2953(-CH), 2598 (-OH),
1641, 1543 (-NH2), 1386 (-CH3), 1332 (-CH2), 1162, 1101 (C-0). 1H
NMR (300 MHz, D2O) δ (ppm): δ 0.78 (d, J = 6.60 Hz, 3H, CH3),
1.10 (dt, J = 7.69, 7.20 Hz, 1H, CH), 2.24 (d, J = 6.94 Hz, 1H, CH),
2.86 (d, J = 7.40 Hz, 1H, CH), 4.69 (3H, s, NH2); HRMS (ESI+) cal-
culated for C9H19NO2 [M + H+] = 173.292 was found 174.148.
6.2.3. General procedure for synthesis of metal complexes of
pregabalin derivatives (4a, b)
20 ml of methanolic solution of pregabalin derivative having
concentration of 2 mM was mixed with same quantity of methano-
lic solution of ZnCl2 (2 mM) in a round bottom flask with con-
stant stirring. The precipitates were then filtered off, washed with
methanol and dried in desiccator. Same procedure was repeated
with Cu(CH3COO)2. (2 mM) instead of ZnCl2. Moreover, ester Cu
complex was refrigerated for overnight [20,21].
6.2.4. Methyl 3-(amino methyl)−5-methylhexanoate zinc chloride
(4a)
Yield: 70%; M. P.: 220 °C. 1H NMR (300 MHz, CDCl3) δ (ppm):
δ 3.55 (3H, s, CH3), 4.70 (3H, s, NH). 1HNMR data of compound
showed singlet peak at δ = 3.55 for CH3 functional group and also
showed a clear singlet at δ = 4.70 which indicated the presence of
NH group and hence this spectroscopic analysis shows the confir-
mation of FZC/4a.
6.2.5. Methyl 3-(amino methyl)−5-methylhexanoate copper acetate
(4b)
Yield: 90%; M. P.: 230 °C; IR ʋ (cm−1 ): 3266.8 (NH), 2963.9,
2896.1 (-CH), 1669.8 (C=O), 1546.8 (-NH2), 1485, 1459 (-CH3),
1388.4,1332 (-CH2), 1276.6 (C-O). HRMS (ESI+) calculated for
C18H36CuN2O4 [M+H+] = 408.204, found = 408.245.
A. Asbat, F. Saleem, S. Najm et al.
6.3. Solubility studies
Apparent solubility of all the synthesized metals was deter-
mined. A measured amount of synthesized compounds were dis-
solved in weighed amount of solvent and clarity of solution was
observed. Water, methanol, ethanol, DMSO and chloroform were
used for this study. For good solubility the threshold value of
<0.1/100 ml with the clear colored solution in the given solvent
was considered [13].
6.3.1. Molecular docking studies
Docking model was based on 3D crystal structure of α 2-δ −1
subunit (PDB-ID: 6ND9) (https://www.rcsb.org/ structure/6ND9)
for anti-convulsant activity, neurokinin-1 receptor (PDB-ID: 2KSA)
(https://www.rcsb.org/structure/2KSA) for anti-inflammatory activ-
ity and dihydrofolate reductase receptor (PDB-ID: 3GHW) (https://
www.rcsb.org/structure/3GHW) for antitumor activity; were re-
trieved from RCSB Protein Data Bank (PDB). ACD/Chemsketch soft-
ware was used for the 3D structure buildup of various ligands and
save as Mol file; Open Babel-GUI 3.0 program was used to convert
Mol file to PDB file for automated docking by Auto Dock Tools 1.5.6
software [16]. The docking protocol was tested by removing the
co-crystallized inhibitor from the protein and docks it on the same
side. Protein structure was added by polar hydrogens and gestesian
charges during protein preparation by Auto dock Tools graphical
user interface. The cuboid grid box was used for the computation
with a purpose to embrace all the minimized inhibitors spanning
10 A˚ in all three dimensions. The Lamarckian genetic algorithm
(LGA) was used to generate conformations of ligands within the
binding site, with an initial population size of 150 individuals, with
a maximum number of 250,0 0 0 energy evaluations, 150,0 0 0 gen-
erations with a mutation rate of 0.02, and a cross-over rate of 2
points. The rigidity parameters were set for the receptor, keeping
the ligand flexible. Out of the ten docked conformations obtained,
one having the lowest binding energy was selected andthe active
site of enzyme. Root mean square deviation (RMSD) was calculated
and in all cases RMSD value of <2.0 Å was considered significant
in predicting binding orientation of ligand [11].
6.4. Biological screenings
6.4.1. Anti-inflammatory activity
In-vitro anti-inflammatory activity of ligand esters and their
metal complexes were evaluated (7–9). Luminol-enhanced Chemi-
luminescence assay was performed, as described by Helfand et al.
[22]. For this purpose, 25 μl of diluted whole blood HBSS++ (Hanks
Balanced Salt Solution, containing calcium chloride and magne-
sium chloride) was incubated with 25 μl of three varied concen-
trations of compounds (1, 10 and 100 μg/ml), each in triplicate.
Both (HBSS++ and cells having no compounds) were introduced
into control wells. Test was implemented in white half area of 96
well plates and was incubated at 37 °C for 15 min in the ther-
mostat chamber of luminometer. Subsequently, 25 μl of serum op-
sonized zymosan (SOZ) and 25 μl of intracellular reactive oxygen
species detecting probe, luminol were added into all wells individ-
ually except the blank which possessed only HBSS++. The level of
the reactive oxygen species (ROS) was examined in luminometer in
the form of relative light units (RLU) (10, 11). Moreover, standard
drug used for the assay was Ibuprofen with IC50 = 11.2 ± 1.9 μM
[22–24].
6.4.2. Anticancer activity
Standard MTT (3-[4, 5-dimethylthiazole-2-y1]−2, 5-diphenyl-
tetrazolium bromide) 96 well plates colorimetric assay was used
to assess the cytotoxic activity of pregabalin derivatives. To achieve
6
Journal of Molecular Structure 1264 (2022) 133234
this, HeLa cell line was cultured in Minimum Essential Medium Ea-
gle, accompanied with 5% of fetal bovine serum (FBS), 100 IU/ml
of penicillin and 100 μg/ml of streptomycin in 75 cm2 flasks, and
preserved in 5% CO2 incubate at 37 °C. Additionally, exponen-
tially growing cells were harvested, calculated with hemocytome-
ter and diluted with a respective media. Cell culture possessing
the concentration of 6 × 104 cells/ml was prepared and trans-
ferred (100 μl/well) into 96-well plates. Medium was removed af-
ter overnight incubation, and 200 μl of fresh medium was added
with varied concentrations of compounds (1–30 μM). However,
200 μl MTT (0.5 mg/ml) was added in all wells individually after
48 h and then incubated additionally for 4 h. Successively, 100 μl
of DMSO was added to each well separately. The degree of MTT re-
duction to formazan within cells was examined by determining the
absorbance at 570 nm, using a micro plate reader. Finally, the cy-
totoxicity was calculated as concentration causing 50% growth in-
hibition (IC50) for HeLa cell line [25,26].
The percent inhibition was calculated by following formula:
% age inhibition=100-((mean of O.D of test compound-mean
of O.D of negative control)/(mean of O.D of positive control-
mean of O.D of negative control) *100).
Doxorubicin% inhibition at 30μ = 101.2%
Doxorubicin IC50 = 0.9 ± 0.14
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
CRediT authorship contribution statement
Ayesha Asbat: Data curation, Formal analysis, Investigation,
Methodology, Software, Writing – original draft, Funding acqui-
sition, Writing – review & editing. Farooq Saleem: Data cura-
tion, Formal analysis, Methodology, Supervision, Validation, Writ-
ing – review & editing. Saima Najm: Conceptualization, Data
curation, Formal analysis, Investigation, Methodology, Resources,
Software, Supervision, Validation, Visualization, Writing – original
draft, Writing – review & editing. Javed Iqbal: Data curation, Fund-
ing acquisition, Writing – review & editing. Muhammad Ali Syed:
Data curation, Supervision, Writing – review & editing. Sarfraz Ah-
mad: Writing – review & editing. Sana Hanif: Formal analysis, In-
vestigation, Writing – review & editing. Muhammad Azeem: Data
curation, Investigation, Writing – review & editing.
Acknowledgement
The authors are grateful Muhammad Mubbashir Khan (Quality
Control Manager), Briell Pharmaceuticals (Pvt.) Ltd, Lahore-Pakistan
for his skillful assistance in recording FTIR. Thanks are also due
to Quaid-e-Azam University Islamabad for characterization of com-
pounds and International center for Chemical and Biological sci-
ences (Karachi) for ESI-MS and in-vitro (anti-inflammatory and
anti-cancer) studies of synthesized chemical compounds.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2022.133234.
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