Synthesis, characterization and in-silico assessment of novel thiazolidinone derivatives for cyclin-dependent kinases-2 inhibitors

Article abstract of DOI:10.1016/j.molstruc.2020.129311

This work aims to synthesize thiazolidinone derivatives using a rapid, safe and solvent-free grinding assisted approach. The newly synthesized compounds have been targeted due to their bioactivity in wide pharmaceutical uses, especially, as anti-prolifera

Full text of DOI:10.1016/j.molstruc.2020.129311

Journal Pre-proof
Synthesis, characterization and in-silico assessment of novel
thiazolidinone derivatives for cyclin-dependent kinases-2 Inhibitors
Jasim Ali Abdullah , Bilal J M Aldahham , Muwafaq Ayesh Rabeea ,
Fatmah Ali Asmary , Hassna Mohammed Alhajri , Md Ataul Islam
PII:
S0022-2860(20)31629-X
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.129311
MOLSTR 129311
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Accepted date:
9 August 2020
17 September 2020
19 September 2020
Please cite this article as: Jasim Ali Abdullah , Bilal J M Aldahham , Muwafaq Ayesh Rabeea ,
Fatmah Ali Asmary , Hassna Mohammed Alhajri , Md Ataul Islam , Synthesis, characterization and
in-silico assessment of novel thiazolidinone derivatives for cyclin-dependent kinases-2 Inhibitors, Jour-
nal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.129311
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HIGHLIGHTS
 Grinding as a rapid, safe, and solvent-free method for the synthesis of thiazolidinone
derivatives.
 New thiazolidinone derivatives target the Cyclin Dependent Kinase 2
 From the MD simulation, the two synthesized molecules are retained inside the CDK2
receptor cavity.

Synthesis, characterization and in-silico assessment of novel thiazolidinone derivatives for
cyclin-dependent kinases-2 Inhibitors
Jasim Ali Abdullah1, Bilal J M Aldahham2, Muwafaq Ayesh Rabeea 2*, Fatmah Ali Asmary3,
Hassna Mohammed Alhajri3, Md Ataul Islam4,5,6
1Department of Chemistry, College of Education for Pure and Science, University of Mosul, Mosul, Iraq
2Department of Applied Chemistry, College of Applied Sciences-Hit, University Of Anbar, Hit, 31007
Anbar, Iraq
3Chemistry Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi
Arabia
4Division of Pharmacy and Optometry, School of Health Sciences, Faculty of Biology, Medicine and
Health, University of Manchester, Oxford Road, Manchester M13 9PL, United Kingdom
5School of Health Sciences, University of Kwazulu-Natal, Westville Campus, Durban, South Africa
6Department of Chemical Pathology, Faculty of Health Sciences, University of Pretoria and National
Health Laboratory Service Tshwane Academic Division, Pretoria, South Africa.
Email: * muw88@uoanbar.edu.iq
Abstract
This work aims to synthesize thiazolidinone derivatives using a rapid, safe and solvent-free
grinding assisted approach. The newly synthesized compounds have been targeted due to their
bioactivity in wide pharmaceutical uses, especially, as anti-proliferative agents. Two
thiazolidinone derivatives having a diverse set of crucial functional groups were diagnosed with
1
13
melting point (m.p), FT-IR, H-NMR, C-NMR, GC-Mass and CHNS. High outputs (98% and
96%) of thiazolidinone derivatives were offered by the grinding method and the final product
was produced over short reaction times (5 min). This simple strategy would potentially be
promising for the preparation of thiazolidinone derivatives in abundance. Followed by synthesis
both of our molecules were considered for molecular docking study against the Cyclin-
Dependent Kinase 2 (CDK2) protein. A number of potential binding interactions in terms of
hydrogen and hydrophobic bonds were observed with the ligand-binding amino acid of CDK2

protein. The behavior of both molecules inside the CDK2 was explored through all-atoms
molecular dynamics (MD) simulations. Several parameters calculated from the MD simulation
were revealed that both molecules retained inside the receptor cavity of CDK2. Hence, green
synthesized both compounds were found to show strong potentiality against the CDK2 and may
have significant interests in the field of pharmaceutical chemistry.
Keywords: CDK2; Grinding; Thiazolidinone; Molecular docking; Pharmaceutical chemistry.
1. Introduction
Heterocyclic compounds are the aromatic and/or cyclic molecules consisting of several
heteroatoms in the rings included nitrogen, sulfur, oxygen, etc. [1]. The heterocyclic compounds
are pharmaceutically important but still a lot of information about it yet to solve [2,3]. Most of
the plant extracts are comprising of heterocyclic rings as heterocycles form the core structures
for mono and polysaccharides and the DNA sequence bases that make up the genetic code [4–6].
A number of crucial pharmaceuticals are made up of heterocyclic molecules[7,8]. Chemical and
physical properties contribute to improved bioavailability and versatility relative to heterocyclic
aromatic hydrocarbons[9–11]. The solubility in water and polarity of heterocyclic compounds is
dependent on the replacement of a single carbon atom with oxygen, nitrogen, or sulphur [1,12].
Thiazolidinones are one of the most important heterocyclic compounds whose derivatives,
having a carbonyl group in fourth place, are an essential part of numerous synthetic
pharmaceuticals with a wide variety of biological activities [13,14] including anticancer,
antihyperlipidemic, antiparasitic and monoamine oxidase inhibitor [15–19]. Microwave and
ultrasonic irradiation were used to synthesize a variety of heterocyclic compounds, resulting in
the acceleration of chemical reactions without the use of organic solvents [20–23].

Cyclin-dependent kinases-2 (CDK2) is a type of kinase group (CKDs) of the protein responsible
of control the cell-cycle progression by activation of the phosphorylating proteins in
serine/threonine as the site of binding, involved in cell division [24]. CDK2 is approved in
literature, has a critical role in tumor development in several types of cancer [25–29], and the
targeting of CDK2 by inhibiting it is a high efficacy strategy of predicting the activity of anti-
tumor agents. Thiazolidinone based derivatives showed vigorous anticancer activity with
infinitesimal concentrations (nanomolar range) [30–32].
A number of CDK2 inhibitors included Flavopiridol[33], Roscovitine[34], Dinaciclib[35],
SNS032[36], AT7519[37] and Milciclib[38] are entered in the different stages of clinical
trials[39]. It is important to note that Flavopirido, Roscovitine, AT7519 and Milciclib are in
clinical trial stage II. The Dinaciclib and SNS032 are in clinical trials III and I respectively. A
two-dimensional representation of the above CDK2 inhibitors are given in Table S1
(Supplementary data). All the above inhibitors show biological efficacy in a number of diseases
included tumorigenesis.
Many studies were focused on the preparation of some heterocyclic compounds using the
grinding method [40–45], however, as far as we know, there is no mention of the synthesis of
thiazolidinone derivatives from Schiff bases by the use of grinding throughout the entire
production process. Hence, this work deems the new attempt to synthesize two important
pharmaceutical thiazolidinone derivatives by grinding-assisted. Continuously, this protocol
would offer a great incentive to make it easy to synthesize a wide range of new thiazolidinone
derivatives. After successful synthesis two compounds were used for in-silico assessment
included molecular docking and molecular dynamics (MD) simulation studies. Therefore, the
prediction of CDK2 successful inhibitors among the family of thialozidinone derivatives, the

promising anti-cancer group, and seeking the bioactivity of newly synthesized compounds is the
aim of this work.
2. Materials and Methods
2.1. Grinding assisted synthesis
The 4-amino antipyrine (0.01 mole) has been used to synthesize the Schiff bases (Sch-I and Sch-
II) by grinding. It was ground once together with 3-hydroxybenzaldehyde (0.01 mole) and again
with 4-nitrobenzaldehyde (0.01 mole) by porcelain mortar for 5-10 min respectively. Following a
few minutes of grinding, the blend becomes a wet, homogenized substance with a change of
colour. During the grinding process, 15 mL of diethyl ether was added. Finally, the precipitant
output was washed, dried, and recrystallized with absolute ethanol [46].
To synthesis compound 1(Comp1), a mixture of Sch-I (0.001 moles) and thioglycolic acid (0.001
mole) has been ground in a mortar with a pestle made of porcelain for 5 min (Scheme 1). After 2
minutes of grinding, the blend is an organic kneading with a change of colour during the
machining process. The output was eventually filtered, dried, and recrystallized by T.H.F. [47].
Thioglycolic acid (0.001 mole) was mixed with Sch-II (0.001 mole) for synthesis compound 2
(Comp2) under the same conditions as those described above.

Scheme 1. Steps synthesis of Comp1 and Comp2
2.2. Characterization of synthesis product
All organic compounds and solvents were obtained from Sigma-Aldrich and all analyses have
been carried out at the University of Rostock, Germany. Stuat melting apparatus (SM30) has
been used to determine the melting point. Bruker AC 250, Bruker ARX 300 and Bruker ARX
500 have been used to record 1H-NMR and 13C-NMR spectra. All 1HNMR and 13C-NMR spectra
provided in this analysis were obtained in either CDCl3 or DMSO-d6. Both chemical changes
are performed in ppm. TMS has been used as an internal source. A Bruker FT-IR
Spectrophotometer (Tensor 27 Germany) and a biotech Engineering FT-IR-600 (UK., using KBr
discs, Alpha Bruker/ATR Diamond) was used to perform infrared spectra.
Mass spectrometry (MS) was investigated using AMD MS40, Varian MAT CH7, MAT 731 (EI,
70ev), intecta AMD 402 (EI, 70ev, and CI), Finnigan MAT 95 (CI, 200ev). Varian MAT 311,
Intecta AMD 402 was used to record High-Resolution Mass Spectrometry (HRMS). To
Elemental Analysis, Thermoquest Flash EA 1112 (LECO CHNS-932) has been used. Thin-layer
chromatography (TLC) has been performed in the presence of iodine as an aspect of the spot.

Characterization of Sch-I (Figures S1-S4 in supplementary file); Yield %: 96. White
0
crystals. m.p: 280-282 C. Rf: 0.6. FT-IR (KBr), ν (cm-1): 1622 (C=O), 1605 (CH=N), 1580
(C=C), 3156 (O-H). 1H-NMR (25 °C, CDCl3): 2.12 (s, C-CH3), 3.13 (s, N-CH3), 9.48 (s, CH=N),
13
9.46 (s.br, OH), 7.13-7.51 (m, 9H, Ar-H). C-NMR (25 °C, CDCl3): 9.97 (C-*CH3), 35.33 (N-
*CH3), 105.23-136.73 (CH3-*C=*C-N), 153.21 (CH2, C-OH), 157.7 (*CH.CH=N), 116-147 (Ar-
C), 160.33 (C=O). Mass, m/z (%): 308 (16), 307 (75), 199 (23), 188 (25), 171 (19), 121 (33), 89
(15), 77 (17), 56 (100), mol. mass (307.132). CHNS: (C18H17N3O2) 70.130 (Ctheo), 70.230 (Cprac),
5.580 (Htheo), 5.680 (Hprac), 13.670 (Ntheo), 13.880 (Nprac).
Characterization of Sch-II (Figures S5-S8 in supplementary file); Yield %: 98. Orange.
0
1
m.p: 260-261 C. Rf: 0.7. FT-IR (KBr), ν (cm-1): 1641 (C=O), 1596 (CH=N), 1572 (C=C). H-
NMR (25 °C, CDCl3): 2.43 (s, C-CH3), 3.15 (s, N-CH3), 9.52 (s, CH=N), 7.21-8.43 (m, 10H, Ar-
H). 13C-NMR (25 °C, CDCl3): 10.12 (C-*CH3), 34.87 (N-*CH3), 109.83-135.97 (CH3-*C=*C-N),
159.8 (*CH.CH=N), 122-152 (Ar-C), 161.47 (C=O). Mass, m/z (%): 337 (6), 336 (34), 188 (20),
121 (33), 91 (22), 78 (14), 44 (13), mol. mass (336.122). CHNS: (C18H16N4O3) 64.280 (Ctheo),
64.470 (Cprac), 4.790 (Htheo), 4.810 (Hprac), 16.660 (Ntheo), 16.750 (Nprac).
Characterization of Comp 1 (Figures S9-S12 in supplementary file); Yield %: 96. Pale
0
Yellow. m.p: 170-172 C. Rf: 0.55. FT-IR (KBr), ν (cm-1): 1687, 1642 (C=O), 1597 (C=C), 670
1
(C-S), 3156 (O-H). H-NMR (25 °C, CDCl3): 2.01 (s, C-CH3), 3.04 (s, N-CH3), 2.85-4.08 (q,
13
2H, CH2), 9.46 (s.br, OH), 6.12 (s,C-H), 6.85-7.55 (m, 9H, Ar-H). C-NMR (25 °C, CDCl3):
10.80 (C-*CH3), 35.26 (N-*CH3), 107.18-137.22 (CH3-*C=*C-N), 31.65, 152.34 (CH2, C-OH),
61.56 (*CH.CH=N), 115-151 (Ar-C), 167.45 (C=O AP), 170.22 (C=O Lactam). Mass, m/z (%):
382 (16), 381 (79), 348 (13), 306 (31), 246 (16), 215 (54), 188 (100), 137 (13), 121 (65), 56 (77),

mol. mass (381.115). CHNS: (C20H19N3O3S) 62.97 (Ctheo), 62.829 (Cprac), 5.02 (Htheo), 5.294
(Hprac), 11.02 (Ntheo), 11.211 (Nprac), 8.41 (Stheo), 8.443 (Sprac).
Characterization of Comp 2 (Figures S13-S16 in supplementary file); Yield %: 98. Green.
0
m.p: 79-80 C. Rf: 0.53. FT-IR (KBr), ν (cm-1): 1725, 1642 (C=O), 1596 (C=C), 699 (C-S). 1H-
NMR (25 °C, CDCl3): 2.44 (s, C-CH3), 3.16 (s, N-CH3), 2.76-3.93 (q, 2H, CH2), 6.03 (s, C-H),
13
7.35-8.51 (m, 9H, Ar-H). C-NMR (25 °C, CDCl3): 10.01 (C-*CH3), 35.79 (N-*CH3), 105.22-
135.11 (CH3-*C=*C-N), 33.73 (CH2, C-OH), 64.17 (*CH.CH=N), 119-148 (Ar-C), 161.35 (C=O
AP), 170.17 (C=O Lactam). Mass, m/z (%): 411 (24), 410 (69), 372 (15), 371 (73), 330 (19), 329
(29), 238 (41), 203 (15), 189 (79), 188 (55), mol. mass (410.105). CHNS: (C20H18N4O4S) 58.530
(Ctheo), 58.481 (Cprac), 4.42 (Htheo), 4.416 (Hprac), 13.65 (Ntheo), 13.643 (Nprac), 7.81 (Stheo), 7.821
(Sprac).
2.3. Molecular docking
Molecular docking is one of the crucial in-silico approaches to predict the preferred binding
orientation of the small molecule inside the receptor cavity of the macromolecule. Synthesized
two molecules (Comp1 and Comp2) were docked in the CDK2 protein molecule using the
AutoDock vina (ADV) [24]. The crystal structure of the CDK2 was collected from the Research
Collaboratory for Structural Bioinformatics-Protein Data Bank (RCSB-PDB) with PDB ID:
3FZ1[25]. The resolution and R-value of the selected CDK2 protein are 1.90Å and 0.210
respectively. The protein consisted of a single chain having a total of 298 amino residues. Prior
to molecular docking the CDK2 crystal structure was prepared using the AutoDock tools (ADT)
[50]. The crystal water molecules were deleted. The polar hydrogens and charges were added.
The protein atoms were assigned the AD4 (Autodock 4) type and saved as pdbqt format. The
grid coordinates were set as 1.448, 25.918 and 9.073 along X-, Y- and Z-axes respectively. The

grid box size was set to 40 x 40 x 40 along the X-, Y- and Z-axes respectively. Both compounds
were also prepared using ADT. The hydrogens and charges were added. The number of torsions
was set and default docking parameters were considered. Both molecules were saved as pdbqt
file format for the molecular docking input in ADV. After successful docking, the binding
energy was recorded and binding interactions were analysed using the Protein-
Ligand Interaction Profiler (PLIP) [51] online server.
2.4. Molecular dynamics simulation
Molecular dynamics (MD) simulation is an excellent approach to explore the dynamic structural
information on biomacromolecules. Moreover, it is also crucial to analyze the energetic
information about protein and ligand interactions. The structural behavior of novel thiazolidinone
compounds complex with CDK2 protein in dynamic states was explored through an all-atoms
MD simulation for a 100ns period. Individual docked complex of Comp1 and Comp2 with
CDK2 were considered. The simulation was performed in the Amber18 [52] software package
installed at the Computational Shared Facility (CSF3), University of Manchester, UK. Each
complex was immersed in truncated octahedron of TIP3P [53] water prior to the simulation. To
neutralize the system a required number of Na+ and Cl- were added and maintained the ionic
strength of 0.1 M to mimic the physiological pH. The protein topology was generated through
the ff14SB force field [54]. The Nvidia V100-SXM2-16GB Graphic Processing Unit using the
PMEMD.CUDA [55] module was used to execute the simulation. In the simulation, the
temperature of 300K was maintained through the Langevin thermostat with a collision frequency
of 2 ps-1, at 1 atm using a Monte Carlo barostat with volume exchange attempts every 100 fs.
Moreover, a 2-fs integration step was retained. The SHAKE [56] algorithm was used to constrain
the hydrogens involved in covalent bond. For the short-range nonbonded interaction the cut-off

was set to 8Å and the long-range electrostatics were preserved through the particle mesh Ewald
method. A total of 10ns equilibration was performed consisting of rounds of NVT and NPT. A
number of parameters included root-mean-square deviation (RMSD) of CDK2 backbone and
ligands, root-mean-square fluctuation (RMSF) and radius of gyration (RoG) were explored using
CPPTRAJ[57] over full trajectory, taking configuration every 2 ps.
2.5. Binding free energy calculation through MM-GBSA approach
The molecular mechanics-generalized born surface area (MM-GBSA) approach to calculate the
binding free energy (∆Gbind) of any small molecule is considered to be more accurate and widely
accepted in the scientific community. This approach quantitively measures the binding potency
between the protein and small molecules. The ∆Gbind of both molecules was calculated from the
post-processed ensemble of structures obtained from the MD simulation trajectories. The ∆Gbind
calculation using the MM-GBSA approach was carried out through the following expressions.
(1)
(2)
(3)
(4)
(5)
The total binding energy (∆Gbind) is obtained (equation 1) by the difference of free energy
between complex (∆Gcom) and summation of the receptor (∆Grec) and ligand (∆Glig). Further, the
∆Gbind is consisting of two terms, enthalpy (∆H) and entropy (T∆S). The enthalpy term is
calculated using the GBSA, and entropy is obtained from the normal mode analysis (NAM) and
interaction entropy (IE) approaches. The ∆H can be represented by molecular mechanical energy

(∆EMM) and solvation free energy (∆Esol). The ∆EMM is the summation of intra-molecular (∆Eint),
electrostatic (∆Eele) and the van der Waals interaction (∆Evdw) energies. Further, the free energy
of solvation (∆Gsol) can be expressed by the summation of polar (∆Gpol) and non-polar (∆Gnpol
)
energies. The modified generalized Born (GB)[58] is used to calculate the ∆Gpol and ∆Gnpol
obtained from the LCPO algorithm[59] which is based on SASA.
3. Result and Discussion
The use of organic solvents in different chemical reactions is harmful both to the environment
and to human health. As a result, numerous methods have emerged in the synthesis of organic
materials. Green chemistry depends on the absence of chemical solvents, and use of simple
procedures, the shortening of time reactions and excellent yields. Here, grinding-assisted
reactions between 4-amino antipyrine and substituted benzaldehydes were used to synthesize a 4-
(3-hydroxy benzylidene amino) antipyrine (Sch-I) and 4-(4-nitro benzylidene amino) antipyrine
(Sch-II) at room temperature. The preparation of the Schiff bases compounds taking 5 min for
Sch-I with a yield of 96%, which contains an OH group. Sch-II containing NO2 group is
obtained through 10 min with a yield of 98%. The variation of colour, melting point and TLC of
the raw material after the synthesis process is the initial signs of a completed chemical reaction.
For the synthesis of Schiff bases and thiazolidinone derivatives, two precursors with different
functional groups of withdrawing group (NO2) and donating group (OH) were used. The use of
the grinding strategy did not involve catalysing by adding mineral acid (H+) during the
preparation of the Schiff bases, making the nucleophilic attack easier. The NO2 group led to
creating a huge positive charge on the carbon atom of the carbonyl group, which facilitates
interaction with the nucleophile and shows higher product compared to the OH group. The Sch-I
and Sch-II synthesis mechanism (Scheme 2) indicates that the electron pair of amine group

attacks (nucleophilic attack) carbon of the aldehyde carbonyl group[60]. This leads to formation
intermediate compounds (4-((hydroxy(3-hydroxyphenyl)methyl)amino)-1,5-dimethyl-2-phenyl-
1,2-dihydro-3H-pyrazol-3-one and 4-((hydroxy(4-nitrophenyl)methyl)amino)-1,5-dimethyl-2-
phenyl-1,2-dihydro-3H-pyrazol-3-one) which then lose the water molecule and become stable
amine compounds.
Scheme 2 mechanism of Schiff base (Sch-I and Sch-II) preparation
The FT-IR spectra for Sch-I and Sch-II show peaks (1605-1596 cm-1) associated with the
azomethine group. Other peaks (1622-1641 cm-1) and (1580-1572 cm-1) corresponded to the
carbonyl and aromatic groups respectively. Sch-I was distinguished by a peak at (3165 cm-1) due
to the (OH) group. 1H-NMR spectra of Sch-I and Sch-II show a chemical shift in 9.48-9.51 ppm
assigned to (CH = N) while Sch-I reveals a singlet peak at 9.46 ppm associated with Ar-OH. 13C-
NMR spectra appear major peaks at 157.7 ppm and 159.8 ppm related to carbon in CH3-C = N.
Sch-I shows a peak at 153.21 associated with CH2-C-OH [61]. The Sch-I and Sch-II mass
spectra have been registered, and the m/z value obtained of the new compounds matched the

theoretical molecular weight of the compounds. The purities of Sch-I and Sch-II were evaluated
by elementary analysis.
The synthetic route for the target thiazolidine derivatives (comp 1 and comp 2) has been
carried out as illustrated in scheme 1. Sch-I has been reacted with thioglycolic acid by grinding
for 5 min without solvent. Comp 2 was synthesized under the same method conditions by the
reaction of Sch-II with thioglycolic acid. Scheme 3 is a proposed mechanism for the synthesis of
thiazolidine derivatives. Describing the synthesis mechanisms of Comp 1 and Comp 2, a
nucleophilic attack by the electron pair of the sulphur atom in thioglycolic acid attacked the
carbon atom at the Schiff's bases. Continuously, the electron pair on the nitrogen of Schiff's
bases triggers a nucleophilic attack on the carbon atom of the carboxyl group[60,62].
Scheme 3. A possible mechanism to prepare Comp1 and Comp2

The chemical structure of the synthesized derivatives thiazolidine (Comp 1 and Comp 2) was
1
13
interpreted by H-NMR, C-NMR, GC-Mass and CHNS spectra. In FT-IR spectra, the focus
was on the major peak at 1166 cm-1 related to the C-S-N group which appeared in comp1 and
comp2 compared to the compounds of their Schiff bases (Sch-I and Sch-II) [61].
1
The H-NMR spectra of comp1 and comp2 have illustrated all the protons of synthesized
compounds. There is a chemical shift at 9.46 ppm due to proton of OH in comp1 that is not
found in comp2 because OH is replaced by NO2. While the chemical shifts at 9.48 -9.52 ppm(s)
for proton in Schiff bases have been disappeared because of the formation of the thiazolidine
group (CH=N). There is another indicator of the formation of thiazolidinone compounds from
13
the corresponding Schiff bases, depending on the C-NMR spectra. The presence of a new shift
at 60-64 ppm assigned to C-S (newly formed) of comp1 and comp2, as well as a shift at 170 ppm
indicating the formation of a new cyclic moiety in molecules.
GC-Mass spectra of synthesized compounds were recorded. All molecular mass indicates the
possible breakdown in the chemical composition of the compound is prepared. The final result
appears a molecular ion peak at m/z =188 assigned to the (N-N-C = O) group. The description of
CHNS data has been shown to be compatible with the basic elements of our compounds.
3.1. Molecular docking
Molecular docking of both compounds (Comp1 and Comp2) was uncovered that both fit well
into the coupling site and show great communications with the critical amino corrosive build-
ups. To approve the docking conventions of the binding site of CDK2, the molecular docking
experiment was performed in the ADV tool and the binding interactions were visualized in the
PLIP online server. The binding energy was found to be -8.7 and -8.1 Kcal/mol for Comp1 and
Comp2 respectively. The binding interaction profile of Comp1 and Comp2 is given in Figure 1.

Figure 1. Binding interactions profile of Comp1 and Comp2
From binding interactions analysis, it can be seen that the amino acid residues, Ile10, Val18,
Ala31, Phe80, Phe82, Gln131, Leu134, and Ala144 were found to be interacted with Comp1
through hydrophobic interactions. Moreover, a salt bridge was also found between Comp1 and
Asp86. Comp2 was found to establish a hydrogen bond with Glu12. In addition to the above a
number of amino residues included Val18, Ala31, Lys33, Phe80 and Leu134 were interacted
with Comp2 via hydrophobic interactions. The binding mode of both compounds was checked in
a three-dimensional surface view and it is given in Figure 2. It can be seen that both compounds
perfectly fitted almost the same position of CDK2 receptor.

Figure 2. Binding mode of Comp1 and Comp2 in the CDK2
A set of six standard CDK2 inhibitors (Flavopiridol, Roscovitine, Dinaciclib, SNS032, AT7519
and Milciclib)[39] were collected and docked in the CDK2 to explore the binding energy and
binding interactions. The two-dimensional structure along with inhibitory activity and binding
energy of all inhibitors are given in Table S1 (Supplementary file). The binding energy of
Flavopiridol, Roscovitine, Dinaciclib, SNS032, AT7519 and Milciclib was found to be -8.30, -
7.90, -8.50, -7.80, -8.20 and -9.90 Kcal/mol respectively. Hence, it is clear that Comp1 (-8.7
Kcal/mol) possessed a higher binding affinity towards CDK2 in comparison to all standard
CDK2 inhibitors except Milciclib. Comp2 was found to show higher affinity compared to
Roscovitin and SNS032. The binding energy of other CDK2 inhibitors except Milciclib was
found to be comparable with Comp2. The binding interactions of all standard CDK2 inhibitors
were assessed and it is given in Figure S17 (Supplementary data). It is important to note that a
number of ligand-interacting amino acids were found common to the standard CDK2 inhibitors
and, Comp1 and Comp2. Therefore, above analyses clearly indicated that both synthesized

molecules (Comp1 and Comp2) might be potential as of already available standard CDK2
inhibitors.
3.2. Pharmacokinetic analysis
The pharmacokinetic and physicochemical properties are important to explore the potentiality of
the newly synthesized compounds. The SwissADME, an online server was used to calculate the
pharmacokinetic and physicochemical characteristics of both Comp1 and Comp2 and these are
given in Table 1. The relative molecular mass of Comp1 and Comp2 was found to be 381.45 and
410.45 g/mol, respectively. Oral activeness is explained by the value of TPAS. The newly
synthesized compounds were found to have 92.77 and 118.36 for Comp1 and Comp2
respectively, which indicates that they are orally active. The criteria of drug-likeness are derived
from Lipinski’s rule of five, Ghosh’s and Veber’s rule. Both compounds were found to follow
the above drug-likeness rules perfectly. Both molecules are moderately soluble in nature. The
absorption in the gastrointestinal (GI) tract was found to be high for both molecules. Low
synthetic accessibility value was clearly indicated that both molecules not difficult to synthesis.
Hence, the above data undoubtedly favours the potentiality of the molecules.
Table 1. pharmacokinetic and physicochemical properties of Comp1 and Comp2
Parameters
Comp1
Comp2
Formula
aMW
C20H19N3O3S
381.45 g/mol
C20H18N4O4S
410.45 g/mol
bNHA
cNAHA
dNRB
eMR
27
17
3
110.39
92.77
29
17
4
117.19
118.36
fTPSA (Å2)
gLogS
hSC
-4.54
Moderately soluble
High
-4.73
Moderately soluble
High
iGI
jBBB
No
No

kvROF
lvGhose
mvVeber
nBS
0
0
0
0.55
3.74
2.62
0
0
0
0.55
3.87
2.37
oSA
LogP
aMolecular weight; No. of heavy atoms; No.of aromatic heavy atoms; No. of rotatable bonds;
b
c
d
eMolar refractivity; Topological polar surface area; Solubility; Solubility class; Gastrointestinal
absorption; jBlood Brain Barrier Penetration; kViolation of Lipinski’s rule of five; lViolation of Ghose
rule; mViolation of Veber rule; nBioavailability Score; oSynthetic accessibility.
f
g
h
i
3.3. Molecular dynamics simulation
In the drug discovery research, a combined approach of molecular docking and MD simulation
studies became a pivotal method among the scientific community in academia and industry. In
this regard, both Comp1 and Comp2 were docked in the CDK2 and subsequently considered for
100ns MD simulation. After successful completion of the simulation, a number of parameters
included RMSD, RMSF, RoG and binding free energy were calculated and these are given in
Table 2.
The RMSD of each frame of CDK2 backbone bound with Comp1 and Comp2 was calculated
and it is given in Figure 3. It can be seen that initially the RMSD value of CDK2 backbone
bound with both Comp1 and Comp2 was gradually increased. In the case of CDK2- Comp2
complex, the system was achieved stability after about 10ns. On the other hand, the backbone of
CDK2 bound with Comp1 was found to have fluctuation initially but after about 40ns it was
achieved perfect stability till the end of the simulation. The average RMSD value also can give
an idea about the stability of the protein backbone during the simulation. The average RMSD of
CDK2 backbone bound with Comp1 and Comp2 was found to be 2.100 and 1.965Å respectively.
Hence, low average RMSD and consistent variation of backbone undoubtedly explained the
stability of protein-ligand complexes.

Figure 3. RMSD of CDK2 backbone bound with Comp1 and Comp2
The RMSD of both Comp1 and Comp2 was obtained and it is given in Figure 4. It was observed
that ligand RMSD was varied from 0 to 3.100Å. The RMSD of Comp1 was found to be
consistent throughout the simulation except for a small fluctuation around 45ns of simulation
time. Comp 2 was shown very little fluctuation and remained intact during the simulation. No
significant fluctuation was observed. The difference between the maximum and average RMSD
can explain the deviation of the molecules from its mean position. In the case of Comp1 and
Comp2 the difference between the maximum and average RMSD value was found to be 1.924
and 1.613Å respectively. The above data clearly suggested that both molecules remained inside
the pocket without any higher conformational changes.

Figure 4. RMSD of Comp1 and Comp2 during MD simulation
Table 2. RMSD, RMSF, RoG and binding free energy of Comp1 and Comp2
Maximum
2.601
Minimum
0.000
Average
2.100
1.965
1.176
1.265
0.842
0.901
19.907
19.781
Comp1
Comp2
Comp1
Comp2
Comp1
Comp2
Comp1
Comp2
Protein backbone
Ligand
2.592
3.100
2.878
4.633
3.742
20.186
20.161
0.000
0.000
0.000
0.371
0.386
19.602
19.492
RMSD
(Å)
RMSF (Å)
RoG (Å)
Ele.
-29.035
51.736
vwd.
-43.242
-46.135
∆Gbind
-35.094
-32.796
Binding energy (Kcal/mol)
Comp1
Comp2
Ele. = Electrostatic; vwd: van der Waals
RMSF of individual amino residue is an important parameter to explore the stability of protein-
ligand complexes. This parameter tells about the fluctuation of each amino acid which dealings
the average divergence of each amino acid from the reference position. The RMSF of the
individual amino residue of CDK2 bound with Comp1 and Comp2 was calculated from the MD
simulation trajectory and it is given in Figure 5. It is important to note that the variation of
RMSF was found to be more or less similar for both Comp1 and Comp2. The highest RMSF

value was found for Gly146 and His147 when bound with Comp1 and Comp2 respectively. The
above observation might be due to lack of binding interaction with a ligand or the presence of
both amino residues in the flexible region of the CDK2. The average RMSF was found to be
0.842 and 0.901 in the case of CDK2 bound with Comp1 and Comp2 respectively.
Figure 5. RMSF of CDK2 amino acid residues bound with Comp1 and Comp2
The compactness of the system during the MD simulation can be assessed through RoG. RoG of
both complexes was calculated and it is given in Figure 6. Consistent variation of RoG indicates
the stably folding of protein during the MD simulation. The high fluctuation of the RoG
parameters described the unfolding the protein over the time of the simulation. In the current
study, extremely less variation of CDK2 was observed for both cases of Comp1 and Comp2. The
difference between the highest and lowest RoG of CDK2 was found to be 0.584 and 0.669Å
when bound with Comp1 and Comp2 respectively. The above low value and consistent plot of
RoG clearly explained that both the complexes were compact during the simulation.

Figure 6. Radius of gyration of CDK2 bound with Comp1 and Comp2
In order to explore the binding affinity of both Comp1 and Comp2 towards the CDK2, the
binding free energy was calculated through MM-GBSA approach. The binding free energy
obtained from the MM-GBSA is widely accepted and explored by the scientific community and
also thought to be more authentic in contrast to molecular docking based binding energy [63–
65]. The ∆Gbind of both Comp1 and Comp2 was calculated and it is given in Table 2. From Table
2, it can be seen that ∆Gbind of Comp1 and Comp2 was found to be -35.094 and -32.796
Kcal/mol respectively. High negative ∆Gbind explains more affection towards the receptor cavity.
Both molecules were found to have high negative ∆Gbind which clearly suggested that Comp1
and Comp2 might be potent CDK2 inhibitors.
4. Conclusion
The grinding technique has been used in this work to reduce the energy consumption and
chemical additives and, more importantly, the reduction of environmental impacts relative to
prepare thiazolidinone compounds. The grinding-mediated synthesis for two thiazolidinone
1
derivatives exhibits high yields. Characteristics of the final product including H-NMR, 13C-

NMR, GC-Mass, HRMS, CHNS indicate the configuration of the required compounds. Further,
in-silico analyses of both molecules in the CDK2 protein was assessed. The molecular docking
study was explained that both molecules potential enough to form a number of binding
interactions with the catalytic amino residues of CDK2. Best docked pose of each molecule was
further considered for 100ns MD simulations study to explore the dynamic behaviour of Comp1
and Comp2. The CDK2 backbone RMSD, residue RMSF, ligand RMSD and RoG obtained from
MD simulation trajectories were clearly indicated that both molecules capable to retained inside
the CDK2 receptor cavity. The binding affinity of Comp1 and Comp2 was checked by
calculating the binding free energy from the ensemble of MD simulation trajectory. High
negative binding energy suggested that both molecules possessed strong affections towards the
CDK2. Therefore, it can be calculated that synthesized molecules might be crucial
pharmaceutical chemical agents subjected to further experimental validation.
Acknowledgment
The authors extend their appreciation to the Deanship of Scientific Research at King Saud
University for funding this work through research group No (RG-1441-430).
Conflicts of interest
Authors declare that there is no competing interest
CREDIT AUTHOR STATEMENT
Term Author/s
Term Author/s
Jasim Ali Abdullah, Muwafaq Ayesh Rabeea
Bilal J M Aldahham, Jasim Ali Abdullah
Md Ataul Islam, Bilal J M Aldahham, Hassna Mohammed
Alhajri
Conceptualization
Methodology
Software
Validation
Formal analysis
Fatmah Ali Asmary, Bilal J M Aldahham, Md Ataul Islam
Fatmah Ali Asmary, Jasim Ali Abdullah

Investigation
Bilal J M Aldahham, Muwafaq Ayesh Rabeea, Hassna
Mohammed Alhajri
Writing - Original Draft
Md Ataul Islam, Bilal J M Aldahham, Muwafaq Ayesh Rabeea
Writing - Review & Editing Md Ataul Islam, Muwafaq Ayesh Rabeea
Project administration
Fatmah Ali Asmary, Jasim Ali Abdullah
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