Synthesis of novel thiophene fused pyrazoline-thiocyanatoethanone derivative: Spectral, DFT, pharmacological, docking and in vitro antibacterial studies

Article abstract of DOI:10.1016/j.molstruc.2020.129487

Novel thiophene fused pyrazoline-thiocyanatoethanone compound was synthesized with good yield. The synthesized compound molecular structure was authenticated by SCXRD. The spectral studies of the compound were recorded through NMR, FTIR and UV to ascertai

Full text of DOI:10.1016/j.molstruc.2020.129487

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Synthesis of novel thiophene fused pyrazoline-thiocyanatoethanone
derivative: Spectral, DFT, pharmacological, docking and in vitro
antibacterial studies
K. Saranya , S. Murugavel
PII:
S0022-2860(20)31802-0
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.129487
MOLSTR 129487
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Journal of Molecular Structure
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Accepted date:
2 September 2020
18 October 2020
Please cite this article as: K. Saranya , S. Murugavel , Synthesis of novel thiophene fused pyrazoline-
thiocyanatoethanone derivative: Spectral, DFT, pharmacological, docking and in vitro antibacterial
studies, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.129487
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Highlights
 Novel Thiophene fused pyrazoline-thiocyanatoethanone was synthesized and
characterized by various experimental and DFT methods.
 The inhibition activities of syntheised compound was evaluated against various
bacterial strains via in vitro studies (MIC)
 The MEP investigation was revealed that negative charge spreads around thiocyanate,
carbonyl groups and positive charge intensely appears over pyrazole region
 The in silica pharmacological analysis of TKSCN pointed out well drug likeness
nature.
 Molecular docking studies figured out the binding interactions of synthesized
molecule.
1

Synthesis of novel thiophene fused pyrazoline-thiocyanatoethanone derivative: Spectral,
DFT, pharmacological, docking and in vitro antibacterial studies
K. Saranyaa*, S. Murugavelb
a Department of Physics, Government College of Engineering, Salem-636 011, Tamilnadu, India.
b
Department of Physics, Thanthai Periyar Government Institute of Technology, Vellore-632
002, Tamilnadu, India.
*Corresponding author. Tel.: +919952818941. Email address: saranya.gce11@gmail.com
(K.Saranya)
Abstract
Novel thiophene fused pyrazoline-thiocyanatoethanone compound was synthesized with good
yield. The synthesized compound molecular structure was authenticated by SCXRD. The
spectral studies of the compound were recorded through NMR, FTIR and UV to ascertain
structural parameters. The synthesized structure geometrical characteristics were optimized by
DFT calculations. The computed structural characteristics of a molecule were will agreed with
experimental studies. The compound molecular orbits were investigated through HOMO-LUMO
analysis. The MEP analysis revealed that negative potential spreads around thiocyanate and
carbonyl moieties. However, the positive potential appeared intensely covered around pyrazole
moiety. The pharmacological analyses of compound were performed through in silica approach
to realize drug properties. The molecular docking studies were executed against antibacterial
target of Thymidylate kinase for synthesized compound. The docking results emphasized good
binding behavior over the standard drug. The antibacterial behavior of the compound was
screened against bacterial strains through MIC studies. The synthesized compound in vitro
results exhibited notable antibacterial activity.
Keywords: Pyrazole; Thiophene; Thiocyanate; XRD; DFT; Molecular docking; Antibacterial
pathogens
1. Introduction
2

Bacterial strains are more familiar with human life and create leading threat along with other
diseases to affect human health [1]. The heterocyclic molecules occupy a significant space in
antibacterial activities in new therapeutic drug derivatives [2]. Pyrazole moiety is fused with any
compound it can able to alter the charge distribution and also significantly influences the
geometric properties [3]. Pyrazole fused molecules are strongly placed in various medical
therapeutics like antibacterial [4], anticancer [5], antifungal [6], analgesic [7], anti-inflammatory
[8] and etc. Thiophene incorporated heterocyclic molecules are important in medicinal field
because of its superior therapeutic activities [9]. Thiophene ring attached compound actively
visible in many drugs due to its bioactive characteristics [10]. Thiophene molecule is behaves
like a structure brick in drug compound and subsequently enhance the compound
pharmacokinetic characteristics [11]. An influential biological property of heterocyclic
molecules (pyrazole, thiophene) attracts many researchers to utilize these types of molecule for
new drug derivatives [12]. The thiocyanate (SCN) is a naturally available compound in human
saliva and it acts an inhibiting agent against bacteria [13]. SCN is always found in mammals
extracellular fluids with various concentrations and it defenses against external microbial
intrusion [14]. Our previous study [15] pyrazoline-thiocyanate fused with phenyl moieties
synthesis scheme and antibacterial activities were discussed. The present study is to investigate
hybridization of thiophene moiety with pyrazoline-thiocyanate to explore the insight molecular
characteristics and antibacterial activities. The novel thiophene fused pyrazoline-
thiocyanatoethanone compound is synthesized and its molecular structure established through
1
FTIR, H & 13C NMR and UV studies. SCXRD technique is used to reveals the structural
geometries and obtained results are compared via numerical studies (DFT). The synthesized
molecule antibacterial potentials are assessed by in vitro method (broth dilution).
2. Experimental & Numerical Methods
2.1 Synthesis procedure of TKSCN
The synthesis scheme was established based on our previous study [15] as shown in Figure 1.
The high purity chemicals were used for synthesis and Thin-layer chromatography (TLC) was
executed on aluminum foils layered via silica gel absorbent (F254) and pictured through UV-
chamber. The Bruker spectrometer was utilized to record chemical shifts via 1H &13C NMR
spectra with corresponding ppm and J values. A mixture of pyrazoline-thiophene [16] and
3

potassium thiocyanate of 1.0 mmol each were refluxed through water (5mL) about 10 minutes.
The growth of reaction was supervised through TLC and yielded chemical blend was extracted
through dichloromethane. The anhydrous sodium sulphate was used to dry produced mixture
through condensed pressure. The deposited compound was purified through chromatography by
pet. ether: ethyl acetate (93:7) and the afforded pure eluent as thiophene fused pyrazoline-
thiocyanatoethanone with 94% yield. The compound attained was 1-(3-Phenyl-5-(thiophen-2-
yl)-4,5-dihydro-1H-pyrazol-1-yl)-2-thiocyanatoethanone (TKSCN).
The compound (TKSCN) was prepared through pyrazoline with thiophene yielded 94% of white
crystal: M.P = 125-127 °C, 13C NMR (75 MHz, CDCl3) δ 162.68, 156.13, 142.35, 139.88,
1
133.58, 131.11, 129.83, 128.78, 128.65, 126.81, 125.42, 111.99, 60.35, 42.90, 36.93. H NMR
(300 MHz, CDCl3) δ 7.79 – 7.76 (m, 2H), 7.51 – 7.45 (m, 3H), 7.23 (d, J = 12.0 Hz, 1H), 7.07
(d, J = 3.0 Hz, 1H), 6.96 – 6.94 (m, 1H), 5.91 (dd, J = 11.3, 4.2 Hz, 1H), 4.41 (d, J = 15.0 Hz,
1H), 4.35 (d, J = 15.0 Hz, 1H), 3.82 (dd, J = 17.9, 11.3 Hz, 1H), 3.45 (dd, J = 17.9, 4.2 Hz, 1H).
Fig. 1. Synthesis of TKSCN
2.2 SC-XRD
The crystallographic intensity data of TKSCN were acquired through CCD detector via graphite-
monochromated (λ = 0.71073 Å) MoKα radiation in Bruker single crystal X-ray diffractometer
(SC-XRD - AXS Kappa Apex II). The SADABS [17] program was executed to perform
4

empirical corrections (m-scan). The software modules (SHELXS-97 & SHELXL-97) were
obtained from the WinGX package [18] was utilized to solve and refine the TKSCN crystal
structure. The carbon atom confined H atoms were acquired geometrically through isotropic
displacement fixed as 1.2 or 1.5 times of U values for carbon atoms. The geometrical parameters
and crystal packing results of TKSCN were accomplished via software modules (PLATON [19],
ORTEP [20] & MERCURY [21]). The crystal structure refinement information of TKSCN is
described in Table S1.
2.3 Spectral instruments
The synthesized molecule 13C & 1H NMR spectra, UV spectra and FT-IR were recorded through
Bruker 300 MHz spectrometer, Shimadzu UV-1800 and IRAffinity-1 respectively. UV spectra
and FT-IR were captured in the range of 200-800 nm and 400-4000 cm-1 respectively.
2.4 Antibacterial test
The synthesized compound TKSCN was evaluated against five bacterial pathogens of two gram-
negative (E.coli & S.aureus) and three gram-positive (S.pyogenes, S.typhi & P.aeruginosa)
through in vitro method to identify the antibacterial potentials. The broth dilution technique
(BDT) was employed to assess the Minimum inhibitory concentration value (MIC) against each
pathogen [22]. The five different concentrations of compound were 50, 25, 12.5, 6.25 and 3.125
µg/ml prepared by two-fold dilution technique [23]. The five bacterial solutions (1ml) were
added to each concentration tubes and maintained under 37°C (incubation temperature) about 24
hours. The microbe’s visible growths of all test tubes were observed to identify the MIC values
of bacterial pathogens.
2.5 Computational details
TKSCN molecular structure was optimized using DFT-B3LYP/6-311++G(d,p) method. The
DFT calculations were carried out by Gaussian 09 [24] and visualized with Gauss View
program. The vibrational frequencies, UV spectra, and NMR spectra were acquired through DFT
files. The synthesized molecule PED assignments, HOMO-LUMO, MEP and Mulliken charges
were calculated through DFT studies. The pharmacological activity of a molecule depends on its
crystal structure, geometric parameters and intermolecular interactions [25]. The TKSCN
5

structure pharmacological parameters were computed by PreADMET [26] and Molinspiration
[27] online server. The molecular docking investigation of synthesized compound (TKSCN) was
performed against Thymidylate Kinase (PDB: 4QGG) by AutoDock software [28]. Thymidylate
Kinase enzyme is bacterial biosynthesized DNA inhibitors used to develop antibacterial
derivatives. The receptor-ligand interactions are evaluated based on docking score and H-bond
binding interactions.
3. Results and Discussion
3.1 Structural description (XRD)
The synthesized TKSCN crystal structure was attained by SCXRD is displayed in Figure 2. The
TKSCN molecule CIF file information’s are available as CCDC NO: 1899126. In TKSCN,
thiocyanatoethanone, benzene and thiophene rings fragments are fused at pyrazole ring through
N2, C3 and C1 positions. The pyrazole moiety adopts C-envelope formation and C1 (flap atom)
diverging through mean plane via other 4 atoms (N1-N2, C2-C3) by 0.0076(3) Å. Pyrazole
moiety ring (C1-C3, N1-N2) conformation of TKSCN is authenticated by puckering parameters
of q2 = 0.121(3) Å & φ2= 79.1(1)° [29].
Fig. 2. TKSCN ellipsoid plot (30% level)
6

The compound TKSCN dihedral angle between pyrazole ring (C1-C3, N1-N2) and benzene ring
(C4-C9) is 12.4(2)° and appeared as coplanar. The thiophene and benzene rings are formed
nearly orthogonal to each other at an angle of 89.0(2)°. Thiophene ring and pyrazole ring
included angle of 76.7(2)° and also thiophene ring attached with pyrazole ring at equatorial
position. The thiocyanate fragment (S1-C12-N3) bond angle 178.9 (4)° of TKSCN is seen as
almost linear. The molecule TKSCN geometrical parameters are shown in Table S2 and these
parameters are well agreed with other pyrazole reported structures [30].
TKSCN molecular conformation is stabilized through π-π interaction with Cg3-Cg2=3.652(1)Å
[Centroids represents Cg3 (pyrazole ring N1/N2/C1-C3) and Cg2 (thiophene ring S2/C13-C16)].
The supramolecular chains of TKSCN structure are formed and linked along molecules by C16-
H16…O1i (i: 1/2+x, 1/2-y, 1/2+z) H-bonds and two C-H…π interactions through C11-
H11A…Cg1ii (ii: -1/2+x, 1/2-y, -1/2+z) and C5-H5…Cg2i (i: 1/2+x, 1/2-y, 1/2+z) [Table 1.
Cg1: centroid of benzene (C4-C9) ring] in 3-D crystal packing. These supramolecular chains are
stacked along b axis without any further interactions are shown in Figure 3.
Fig. 3. Supramolecular assembly of TKSCN
Table 1 H-bonding geometry of TKSCN (Å,°).
D–H...A
D–H
d(H...A)
d(D...A)
<(DHA)
7

C16-H16…Oi
0.93
0.93
0.93
2.50
2.91
2.95
3.390(4)
3.624(4)
3.726(3)
160
133
141
C11-H11A…Cg1ii
C5-H5…Cg2i
Cg1- benzene (C4–C9) and Cg2 - thiophene (S2/C13-C16) rings centroids,
Symmetry codes : (i) 1/2+x, 1/2-y, 1/2+z & (ii) -1/2+x, 1/2-y, -1/2+z
3.2 IR spectrum
FT-IR spectrum is revealing the various characteristic peaks and corresponding chemical bands
of synthesized compound TKSCN. The experimental and numerical characteristic peaks are
assigned with its PED values described in Table S3 and spectrums are picturized in Figure S1.
The compound TKSCN C-H stretching bands in asymmetric and symmetric were appeared at
2993 cm-1, 2908 cm-1experimentally and 2936, 2940 cm-1 in DFT [31]. The vibrational peaks
(IR) at 833 cm-1 943 cm-1 & numerically at 850 cm-1 were recognized as C-H out of plane
,
bending. The magnitude peaks at 1435 cm-1 (IR) & 1473 cm-1 (DFT) were identified as aromatic
C-H bends [32]. The vibrational peaks at 3317 cm-1, 3641 cm-1 were realized as O-H vibrations
experimentally [33]. The characteristic peak at 1411 cm-1(IR) & 1473 cm-1(DFT) were assigned
to C=C stretching of phenyl and thiophene rings [34]. The C-C stretching was viewed at 1435
cm-1 (IR) & 1452 cm-1 calculated numerically for phenyl ring. The wave peak at 1751 cm-1 was
evidenced carbonyl (C=O) bond (Exp) & 1670 cm-1 (DFT) with PED of 86% as lied in reported
region [35]. The cyano (C≡N) bond was visualized at 2113 cm-1 (Exp) & 2161 cm-1(DFT) with
PED of 94% for SCN moiety [36]. The frequency peak of 1311 cm-1 (IR) & 1396 cm-1(DFT)
were authenticated pyrazole ring carbon (C-N). C-S frequency seemed at 740 cm-1 (IR) & 790,
834 cm-1(DFT) for SCN group carbon (C12) and also at 578 cm-1 for thiophene carbon (C14)
[37].
3.3 NMR and UV studies
13
1H & C NMR spectra of TKSCN molecule was recorded in between δ 3.45 – δ 7.79
and δ 36.93 - δ 162.68 respectively are shown in Figure S2 and Table S4 in supplementary. The
CH2 and CH protons of pyrazole ring were noticed at δ 5.98, δ 3.49 and δ 3.42 (Exp) and δ 3.63,
8

δ 3.06 (DFT) [38]. The thiophene hydrogen’s were exhibited at δ 6.96-6.94 (Exp) as multiplets
and δ 6.99 (DFT) [39]. The CH2 protons lies between SCN and Carbonyl moieties were recorded
at δ 4.41 and δ 4.35 and phenyl protons were resonated at δ 7.22, δ 7.45 [40].
The carbonyl (C=O) group carbon atom (C11) was found at δ 162.68 (Exp) and δ 163.23
(DFT) as lies between reported values of δ 159- δ 165.37 [41]. The SCN carbon atom (C12) was
observed at δ111.99 (Exp) and δ 117.56 (DFT) [42]. The thiophene carbon (C13-C16) atoms
were exhibited at δ 125.72, δ 131.11 and δ 133.58 (Exp) and δ 127.23, δ 131.58, 136.48 (DFT)
[43]. The phenyl carbon (C4-C9) atoms were observed at δ 125.72, δ 129.83, δ 131.11, δ 133.58
(Exp) and δ 130.88, δ 132.89, δ 134.04, δ 135.25(DFT).
UV-Spectrum of synthesized molecule TKSCN was seen in between 200 - 400 nm and
200 - 400 nm through experimental and theoretical respectively as reported in Table S5. The UV
spectrum peak value of 331 nm (Exp) evidenced the phenyl ring [44] and numerically achieved
at 301.70 nm with energy of 4.11 eV yields HOMO-LUMO influence of 96.75%. The second
peak recorded at 252.5 nm (Exp) confirmed the pyrazole moiety in synthesized molecule [45].
Theoretical spectrum at 278.8 nm with energy of 4.45 eV yielded strength of 0.0121 of HOMO
to LUMO contribution of 95.62%. The wave peak of 246.5 nm was authorized thiocyanate
fragment of TKSCN [46]. The SCN peak calculated theoretically at 273.4 nm of energy level
4.53 eV with HOMO-LUMO+1 influence of 94.95%.
3.4 Antibacterial analysis
The compound TKSCN bacterial activity assessed against two gram-negative and three gram-
positive pathogens through MIC scores are shown Figure 4.
9

Fig. 4. MIC scores of TKSCN and Ciprofloxacin (µg/ml)
The compound TKSCN exhibited equally potent with ciprofloxacin of MIC values 3.125 µg/ml
& 6.25 µg/ml against S. typhi & S.pyogenes respectively. In specific, TKSCN yields superior
inhibition activity against S. aureus (3.125 µg/ml) compared to standard drug (6.25 µg/ml). The
inhibition activity of compound against E.coli & P.aeruginosa recorded less potential related
with ciprofloxacin. The TKSCN molecule has better antibacterial activity against Gram +Ve
strains compared with Gram –Ve starins. Comparison with our previous studies [15], the
thiophene fused pyrazoline-thiocyanatoethanone yielded better inhibition activity against S. typi,
S. aureus and S. pyogenes. TKSCN molecule revealed good antibacterial nature against bacterial
strains.
3.5 Computational studies
The synthesized molecule structure (TKSCN) was optimized through DFT and compared with
XRD obtained geometrical characteristics described in Table S4. The HOMO-LUMO, MEP and
charge distributions (MPA & NPA) were calculated by DFT analysis.
3.5.1 MPA & NPA analyses
The atomic charges of synthesized molecule play key role in quantum analysis. The molecule
pairs (Donor & Acceptor) charge distribution sketched inner molecule transport behavior. The
synthesized molecule charge distributions obtained via MPA (Mulliken population analysis) and
10

NPA (Natural population analysis) methods [47, 48] by DFT calculations are described in Figure
S3 & Table S6. The H-atoms of TKSCN were seen as +Ve in both analysis (MPA & NPA). The
carbonyl fragment O1 (oxygen atom) was calculated as –Ve charge in both population studies
The S1 atom of SCN fragment yielded +Ve (MPA) and –Ve (NPA) as comparable with earlier
reports [49]. The nitrogen (N2) atom of pyrazole moiety marked as -Ve in MPA & NPA studies.
The carbon (C13) atom showed –Ve charge due to pyrazole and thiophene ring fusion. The
nitrogen (N3) atom of SCN showed –Ve charge in both analyses. N2 and C10 atoms charge
magnitude values of -0.24 and -1.43 respectively were confirmed pyrazole and carbonyl groups.
3.5.2 HOMO and LUMO analysis
It delivers a qualitative assessment of excitation ability and electron transport features of the
molecule. The HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied
Molecular Orbital) are acted as electron acceptor and electron donor respectively. The molecular
chemical reactivity described via energy gap of HOMO-LUMO and small energy gap yields less
kinetic stability. HOMO-LUMO mapped the intermolecular charge movement between electron
donor and acceptor groups and in Table S7 and Figure 5. The HOMO π nature was visualized
around thiophene, SCN moieties and LUMO appeared over C=O, phenyl ring. The electron
donor nature was seen around thiocyanatoethanone and thiophene ring in HOMO. The LUMO
was localized around carbonyl, phenyl ring and pyrazole ring capitulate electron acceptor. The
LUMO was localized around carbonyl, phenyl ring and pyrazole ring yielded as electron
acceptor. The energy gap values of HOMO & LUMO were calculated as -6.58 eV and -2.10 eV.
The HOMO-LUMO energy transition values marked that electron transfer appears from
thiophene to phenyl and carbonyl via pyrazole ring.
11

Fig. 5. HOMO-LUMO of TKSCN
3.5.3 MEP Analysis
The hydrogen binding potentials are described via Molecular Electrostatic Potential (MEP) plot
with the aid of electron density and electrophilic, nucleophilic effects of molecule. Electrophilic
and nucleophilic characteristics were plotted through DFT solved geometries. The MEP surface
visualized through different color shades (most negative – red; most positive – blue; zero –
green) as picturized in Figure 6. The molecule attractive and repulsive regions of proton
represented as negative and positive values. The negative potential looked around C=O, SCN
fragments and negative potential covered around pyrazole and thiophene rings.
12

Fig. 6. MEP plot of TKSCN
3.5.4 Pharmacological investigation
The physicochemical characteristics of synthesized molecule are assessed via Lipinski's rule
[50]. The molecular properties of synthesized molecule was calculated through in silico
(Molinspriation) online module and shown in Table S8. The molecular weight (327.43) of
synthesized molecule evidenced that good solubility. The molecular weight of TKSCN
calculated as 327.43 and it indicates that good solubility nature. TPSA value (56.47 Å) of
TKSCN molecule was less than 140 Å indicated that good physiochemical properties [51]. The
molinspiration assessment of TKSCN molecule possessed good drug-likeness characteristics
compared with ciprofloxacin. The pharmacokinetic compartments (Absorption, Distribution,
Metabolism & Excretion - ADME) of synthesized molecule evaluated via PreADMET module
and showed in Table S9. HIA magnitude of TKSCN as 99.2 % evidenced as excellent intestinal
absorption. The CaCo2 value (29.07 nm/sec) of TKSCN was marked as higher than standard
drug. The synthesized molecule TKSCN produced higher MDCK value of 98.76 nm/s was
evidenced that good transportation characteristics. The Plasma protein binding (PPB) calculated
as 95.68% proved that strong binding energy with plasma proteins. The toxicology prediction of
TKSCN estimated theoretically and described in Table S10. The toxicity of synthesized
molecule TKSCN was reported as non-Carcinogenin nature in both Mouse and Rat models [52].
The pharmacological and toxicity results of TKSCN proved that suitable drug properties.
3.5.5 Molecular docking analysis
The docking analyses of new ligands are essential to explore insight molecular details during
interactions with target microbial enzymes [53]. The ligand TKSCN was placed in TMK enzyme
(4QGG) and visualized nine H-bond interactions are emphasized in Figure 7.
13

Fig. 7(a) TKSCN – 4QGG complex
Fig. 7(b) Docking interactions of TKSCN
The N3 atom (nitrogen) of SCN fragment formed quadfurcated H-bond binding interaction with
SER13, LYS 15 and GLY14 (amino acids). N3 atom was interacted with SER13 as bifurcated
type binding of NG and N atoms stabilized at 2.35 Å and 2.44 Å respectively. Also, N3 binding
with LYS15 and GLY14 stabilized at 2.29 Å and 2.51 Å. The atom O1 (C=O) docking
conformation visualized with ARG92 as bifurcated type and N1 atom of pyrazole ring interacted
with ARG92. The least stabilized distance recorded as 1.92 Å for O1 atom with LYS15. Our
earlier study [15] the docking results reported as six H-bond conformation (Fluorophenyl), five
H-bond conformation (Chlorophenyl) for pyrazoline-thiocyanatoethanone. The docking
predication of TKSCN recorded as superior binding potential compared with formerly
synthesized compounds and ciprofloxacin.
4. Conclusions
The synthesized compound molecular structure was characterized by spectral investigations (IR,
NMR & UV). The 3-D geometrical characteristics of TKSCN were validated by SCXRD and
compared with theoretical model (DFT). HOMO-LUMO analysis sketched the molecular charge
transfer and 4.48 eV (energy gap) specified that chemically stable and more reactive. Thiophene
and thiocyanate moieties were picturized as electron donor nature. MEP analysis visualized the
charge strength of synthesized molecule. In silico pharmacological, drug-likeness studies
sketched that TKSCN holds better score than ciprofloxacin. The docking analysis exhibited nine
14

binding interactions of molecule with TMK protein and interestingly N3 atom stabilized with
quadfurcated H-bonds. The MIC scores of synthesized molecule evidenced that significant
antibacterial role against Gram +Ve strains. The computational and in vitro studies of TKSCN
were identified as potent antibacterial molecule.
Conflict of interest: All the authors mentioned in the manuscript declared that no conflict of
interest themselves.
DECLARATION
a. We wish to inform you that there are no known conflicts of interest associated with this
publication.
b. No funding was received for this work
c. We confirm that the manuscript has been read and approved by all named authors.
d. We confirm that the order of authors listed in the manuscript has been approved by all named
authors.
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GRAPHICAL ABSTRACT
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