Design and synthesis of pyrazolone-based compounds as potent blockers of SARS-CoV-2 viral entry into the host cells

Journal of Molecular Structure 1241 (2021) 130665

Contents lists available at ScienceDirect

Journal of Molecular Structure

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

Design and synthesis of pyrazolone-based compounds as potent

blockers of SARS-CoV-2 viral entry into the host cells

Vincent A. Obakachi^a, Narva Deshwar Kushwaha^a, Babita Kushwaha^a,

Mavela Cleopus Mahlalela^a, Suraj Raosaheb Shinde^a, Idowu Kehinde^b,

Rajshekhar Karpoormath^a,∗

^aDepartment of Pharmaceutical Chemistry, College of Health Sciences, University of KwaZulu-Natal, Durban 4000, South Africa

^bSchool of Laboratory Medicine and Medical Sciences, College of Health Science, University of KwaZulu-Natal, Durban 4000, South Africa

a r t i c l e i n f o

a b s t r a c t

Article history:

SARS-CoV-2 are enveloped positive-stranded RNA viruses that replicate in the cytoplasm. It relies on the

fusion of their envelope with the host cell membrane to deliver their nucleocapsid into the host cell.

The spike glycoprotein (S) mediates virus entry into cells via the human Angiotensin-converting enzyme

2 (hACE2) protein located on many cell types and tissues’ outer surface. This study, therefore, aimed to

design and synthesize novel pyrazolone-based compounds as potential inhibitors that would interrupt the

interaction between the viral spike protein and the host cell receptor to prevent SARS-CoV 2 entrance

into the cell. A series of pyrazolone compounds as potential SARS-CoV-2 inhibitors were designed and

synthesized. Employing computational techniques, the inhibitory potentials of the designed compounds

against both spike protein and hACE2 were evaluated. Results of the binding free energy from the in-silico

analysis, showed that three compounds (7i, 7k and 8f) and six compounds (7b, 7h, 7k, 8d, 8g, and 8h)

showed higher and better binding high aﬃnity to SARS-CoV-2 Sgp and hACE-2, respectively compared

to the standard drugs cefoperazone (CFZ) and MLN-4760. Furthermore, the outcome of the structural

analysis of the two proteins upon binding of the inhibitors showed that the two proteins (SARS-CoV-2

Sgp and hACE-2) were stable, and the structural integrity of the proteins was not compromised. This

study suggests pyrazolone-based compounds might be potent blockers of the viral entry into the host

cells.

Received 14 March 2021

Revised 26 April 2021

Accepted 9 May 2021

Available online 13 May 2021

Keywords:

Sars-CoV-2

Spike glycoprotein

Human angiotensin-converting enzyme 2

Pyrazolone

Computational techniques

Introduction

much more contagious than MERS-CoV or SARS-CoV [5]. The virus

emerged in December 2019 and, as of June 29, 2020, 6 months af-

Novel coronavirus disease (COVID-19) is the current and most

deadly pandemic the world has faced in more than a century. Ac-

cording to the John Hopkins COVID-19 dashboard, the coronavirus,

also known as SARS-CoV-2, has resulted in more than 90.2 mil-

lion infections and more than 1.9 million deaths worldwide as of

January 2021 [1]. Genomic studies have established that SARS-CoV-

2 belongs to the beta coronavirus family, including SARS-CoV and

MERS-CoV, associated with previous outbreaks of relatively smaller

scale [2–4]. Severe acute respiratory syndrome coronavirus 2 is the

seventh known coronavirus to infect humans; four of these coron-

aviruses (HKU1, 229E, NL63 and OC43) only cause slight symptoms

of the common cold. The viruses can cause severe symptoms and

even death, with SARS-CoV-2 having the highest fatality rate and

ter the ﬁrst outbreak of pandemics, the global count is rapidly ap-

proaching 10 million known cases and 500 000 deaths. Due to its

broad clinical scope and high transmissibility, which is responsi-

ble for the high death rate, the eradication of SARS-CoV-2, as was

achieved with SARS-CoV in 2003, does not seem to be a realistic

target in the short term [6]. Since the emergence of the outbreak,

many studies and clinical trials launched on SARS-CoV-2 world-

wide have started yielding positive results, with vaccines’ availabil-

ity in some parts of Europe and the USA after evidence from ran-

domized clinical trials has shown potential therapy improves pa-

tients’ recovery.

SARS-CoV-2 is a single-stranded RNA-enveloped virus [7]. An

RNA-based metagenomic next-generation sequencing method has

been applied to characterize its entire genome, 29,881 bp in length

(GenBank no. MN908947), encoding 9860 amino acids [8]. Gene

fragments show structural and non-structural proteins. The spike

∗

Corresponding author.

ꢀ

[S]-envelope [E]-membrane [M]-nucleocapsid [N])-poly(T)-3 genes

E-mail address: Karpoormath@ukzn.ac.za (R. Karpoormath).

https://doi.org/10.1016/j.molstruc.2021.130665

V.A. Obakachi, N.D. Kushwaha, B. Kushwaha et al.

Journal of Molecular Structure 1241 (2021) 130665

encode structural proteins, in contrast, to nonstructural proteins,

such as 3-chymotrypsin-like protease, papain-like protease, and

RNA-dependent RNA polymerase, are encoded by the ORF region

[9].

SARS-CoV-2 spike protein human angiotensin-converting enzyme

2 (hACE2) employing in silico methods.

2. Results and discussion

Many glycosylated S proteins cover the surface of SARS-CoV-2

and bind to the host cell receptor angiotensin-converting enzyme

2 (ACE2), mediating viral cell entry [10]. When the S protein binds

to the receptor, Transmembrane (TM) protease serine 2 (TMPRSS2),

a type 2 TM serine protease located on the host cell membrane,

promotes the virus to enter into the cell by activating the S pro-

tein [1 1 ]. Once the virus gains entrance to the cell, the viral RNA

is released, polyproteins are translated from the RNA genome, and

replication and transcription of the viral RNA genome occur via

protein cleavage and assembly of the replicase–transcriptase com-

plex. Viral RNA is replicated, and structural proteins are synthe-

sized, assembled, and packaged in the host cell, after which viral

particles are released [12]. The virion envelope of coronaviruses

is typically associated with three viral proteins that include the

membrane protein (M), the envelope protein (E), and the spike

protein (S). Compared to the three proteins, the spike protein (S) is

one of SARS-CoV-II’s key biological characteristics and several other

viruses because it plays a crucial role in penetrating host cells and

initiating infection [13,14]. The S proteins of coronaviruses can be

divided into two important functional subunits: The N-terminal S1

subunit, which forms the S protein’s spherical head, and the C-

terminal S2 region that includes the stalk of the protein, which is

directly embedded into the viral envelope. Upon interaction with a

potential host cell, the S1 subunit will recognize and bind to the

host cell receptors. In contrast, the S2 subunit, which is the most

conserved component of the S protein, will fuse the virus’s enve-

lope with the host cell membrane [15].

2.1. Chemistry

The synthesis of the novel pyrazolone derivatives was achieved

through eﬃcient and versatile synthetic routes. The Vilsmeier–

Haack reaction of the commercially available starting material

2-(phenyl/substituted phenyl)-5-methyl-2,4-dihydro-3H-pyrazol-3-

one (1a-b) lead to chloroformylation to give required 5-chloro-1-

(phenyl/substituted phenyl)-3-methyl-1H-pyrazole-4-carbaldehyde

(2a–b). Through a nucleophilic substitution reaction, ethoxylation

occurs by replacing chloride on the pyrazole ring of compound

(2) to yield 1-(phenyl/substituted phenyl)-5-ethoxy-3-methyl-

1H-pyrazole-4-carbaldehyde (3a,b). By simple aldol condensation

reaction with acetone in the presence of

a base compound

(3a,b) transformed to compound (E)-4-(1-(phenyl/substituted

phenyl)-5-ethoxy-3-methyl-1H-pyrazol-4-yl)but-3-en-2-one

(4a,b). Furthermore, the compound

formation with thiosemicarbazide to afford (E)-2-((E)-4-(1-

(phenyl/substituted phenyl)-5-ethoxy-3-methyl-1H-pyrazol-4-

yl)but-3-en-2-ylidene)hydrazine-1-carbothioamide (5a,b),which

4 underwent Schiff base

were condensed with various commercially available substituted

2-bromo-1-phenylethanones (6b–o). to yield the corresponding

ﬁnal compounds (7b–o and 8a–h; Scheme 1). The anticipated

structures of the intermediate and ﬁnal compounds were con-

ﬁrmed with mass and ¹H NMR spectral analysis and were further

substantiated by IR and ¹³C NMR, which is summarized in the

Supporting Information.

The ¹H NMR spectrum of compound 5a,b exhibited the pres-

ence of distinctive singlet, broad singlet and multiple signals at

around δ 10.12, 8.15 and 7.66 ppm attributed to the N-H, NH₂and

aromatic protons, respectively. The three protons’ singlet signals

at 2.31 and 2.11 authenticate methyl groups’ presence on pyrazole

ring and straight-chain. The quartet of two protons and triplet of

three protons at 4.01 and 1.21 aﬃrm an ethoxy group’s presence.

Also, the appearance of most informative doublet signals

around δ 6.65–6.62 ppm (J =16.77 Hz) and 6.82–6.79 (J =16.77 Hz)

conﬁrms the presence of oleﬁnic protons.

The ¹H NMR spectrum (400 and 600 MHz, DMSO-d₆, CDCl₃) of

the ﬁnal compounds (7b–o, 8a–h) displayed some distinctive sin-

glet signals at around δ 11.07−10.12 ppm for N−H proton, multi-

plet at δ 7.68–7.45 for aromatic protons attached to the 1st posi-

tion of pyrazole ring, singlet signals of 3H at around δ 2.41–2.30

and 2.30–2.05 ppm attributed to the methyl group at 3rd position

of pyrazole ring and straight-chain (N=C-CH₃), respectively. Addi-

tionally, the most informative singlet signal resonated around δ

8.09–7.31 ppm, attributed to the aromatic proton at H-5 of the thi-

azole ring, thus indicating its formation through the cycloconden-

sation process. Whereas most characteristic doublet signals around

δ 6.86–6.68 ppm (J = 16.91–16.00 Hz, Ar-HC=CH-) and δ 6.74–

6.63 ppm (J = 16.84–16.01 Hz, Ar-HC=CH-) indicates the presence

of oleﬁnic protons. This observation was found to be in line with

previously reported works of a similar type of compound [25].

Furthermore, the unique quartet signal of two protons and triple

signal of three protons resonating around δ 4.00–3.99 and 1.36–

1.23 ppm indicated the presence of ethoxy (OCH₂CH₃) group in the

ﬁnal compounds. The various signals appearing as either doublets

or multiplets around δ 8.09–6.96 ppm accounted for aromatic pro-

tons. These ﬁndings were further corroborated from their respec-

tive ¹³C NMR spectra of the title compounds. The characteristic sig-

nals resonating at around δ 170.10–158.80 and 149.65–138.10 ppm

were assigned to carbons C-2 and C-5 of the thiazole ring. The

characteristic carbon signals appearing around δ 71.46–68.70 and

The host cell receptor called Angiotensin-converting enzyme 2

(ACE2) is a protein located on many cell types and tissues’ outer

surface, including the lungs, heart, blood vessels, kidneys, liver

and gastrointestinal tract. It is present in epithelial cells, which

line-speciﬁc tissues and create protective barriers. It is an enzyme

that generates small proteins by cutting up the larger protein an-

giotensinogen, which then regulates functions in the cell. It is the

primary host cell target with which the S protein of SARS-CoV and

SARS-CoV-II associates. The SARS-CoV-II virus interacting with the

spike-like protein on its surface binds to ACE2 – like a key in-

serted into a lock – before entry and cell infection. Hence, ACE2

acts as a cellular doorway – a receptor – for the virus that causes

COVID- 19. The receptor-binding domain (RBD) of the S1 subunit of

these viruses binds to the outer surface of the claw-like structure

of ACE2 [16–18].

According to literature studies, researchers have found that

compounds containing a pyrazole and its related analogues have

received signiﬁcant attention in chemical, medicinal, and pharma-

ceutical research as a structural scaffold found in various drugs.

[19] As shown in Fig. 1, a new pyrazolone compound, edaravone,

also known as MCI-186, has been developed as a medical drug

for brain ischemia and has also been reported useful for myocar-

dial ischemia [20–22]. Compound (A) has been suggested by Anup

Masih and coworkers as a potential lead for therapeutic applica-

tion to control the inﬂammatory response in SARS-CoV-II infec-

tion [23]. Compound (B) 3,5-dioxopyrazolidine has been reported

as a SARS-CoV 3CLpro inhibitor [24]. Ramajayam et al., 2010 iden-

tiﬁed, from high throughput screening, some pyrazolines, partic-

ularly those displaying a 1,3,5-triaryl substitution pattern (C), to

show activities against SARS-CoV 3CLpro, CoV-229E 3CLpro, CVB3

3Cpro, EV71 3Cpro, and RV14 3Cpro [19].

This reported antiviral pyrazole activity has motivated this

study to design and investigate the novel pyrazolone deriva-

tives’ inhibitory potentials synthesized experimentally against the

2

V.A. Obakachi, N.D. Kushwaha, B. Kushwaha et al.

Journal of Molecular Structure 1241 (2021) 130665

Scheme 1. Reagents and conditions; (i) (a) POCl₃, DMF, 0 °C - rt, 1 h, (b)100 °C, 3 h; (ii), CsCO₃, EtOH, Cu(OAc)₂, 80 °C, 15 h; (iii) Acetone, NaOH, rt, 4 h; (iv) Thiosemicar-

bozide, CH₃OH, AcOH, reﬂux, 5 h; (v) methanol, reﬂux, 5 h.

Table 1

Physical properties of the synthesized compounds.

Comp’d Name

R¹

R²

Molecular Formula

₁₇H₂₁N₅OS

Molecular Weight (g/mol)

% Yield

m. p. ( °C.)

Exact Weight Calculated

Exact Weight Found

5a

5b

7b

7c

7d

7e

7f

H

Cl

–

C

343.45

377.89

522.46

461.55

459.56

443.56

489.59

477.55

512.45

538.46

503.62

488.56

473.59

478.01

457.59

503.62

478.01

556.91

496.00

512.45

512.00

523.01

492.04

538.06

87

76

78

72

89

73

76

87

83

74

76

75

77

79

63

68

69

71

81

74

66

64

73

68

150–153 °C

152–155 °C

177–180 °C

124–127 °C

176–179 °C

154–157 °C

132–135 °C

175–179 °C

187–190 °C

178–181 °C

182–185 °C

166–169 °C

194–197 °C

135–137 °C

125–128 °C

190–193 °C

195–198 °C

198–201 °C

195–198 °C

187–190 °C

199–202 °C

197–200 °C

200–203 °C

203–206 °C

343.15

377.11

521.09

461.17

459.17

443.18

489.18

477.16

511.10

537.08

503.2

344

378

522

462

460

444

490

478

512

538

505

489

474

477

458

504

480

556

496

512

523

492

538

–

C₁₇H₂₀ClN₅OS

C₂₅H₂₄BrN₅OS

C₂₅H₂₄FN₅OS

C₂₅H₂₅N₅O₂S

C₂₅H₂₅N₅OS

Br

F

OH

H

2,6-OH, OCH₃

C₂₆H₂₇N₅O₃S

C₂₅H₂₄FN₅O₂S

C₂₅H₂₃Cl₂N₅OS

C₂₅H₂₄BrN₅O₂S

C₂₇H₂₉N₅O₃S

C₂₅H₂₄N₆O₃S

C₂₆H₂₇N₅O₂S

C₂₅H₂₄ClN₅OS

C₂₆H₂₇N₅OS

7 g

7h

7i

2,5-OH, F

2Cl

2,5-OH, Br

7j

2,6–OCH₃

7k

7l

NO₂

488.16

473.19

477.14

457.19

503.2

OCH₃

7m

7n

7o

8a

8b

8c

8d

8e

8f

Cl

CH₃

3,4–OCH₃

C₂₇H₂₉N₅O₃S

C₂₅H₂₄ClN₅OS

C₂₅H₂₃BrClN₅OS

C₂₅H₂₃ClFN₅OS

C₂₅H₂₃Cl₂N₅OS

C₂₅H₂₃ClFN₅O₂S

C₂₅H₂₃ClN₆O₃S

C₂₆H₂₆ClN₅OS

C₂₇H₂₈ClN₅O₃S

H

477.4

Br

555.04

495.13

511.10

511.12

522.12

491.15

537.16

F

Cl

2,5-OH, OCH₃

NO₂

8 g

8h

CH₃

3,4–OCH₃

21.52–11.90 ppm indicate methylene and the three different methyl

groups in the title compounds, while the various aromatic carbons

resonated around δ 140.78–94.33 ppm. The ﬁnal compounds’ phys-

ical properties are given in Table 1.

However, out of all the synthesized compounds, compounds 7i

and 8f exhibited better and higher (higher than CFZ) binding ener-

gies of -34.726 Kcal/mol and -32.956 Kcal/mol, respectively. Table

S2 (Supplementary materials) showed the binding free energies of

all the best-docked compounds. Compound 7k exhibited a rela-

tively close binding energy of -25.898 Kcal/mol. From Table 2, the

result showed the binding free energy of the best six molecules

and the standard inhibitor, MLN-4760 toward hACE-2, with com-

pounds 8h, 8g and 7k exhibiting the highest binding energies

of -49.320 Kcal/mol, -48.229 Kcal/mol, and -46.008 Kcal/mol,

respectively.

2.2. Thermodynamic Binding free energy of the synthesized

compounds and the standard drugs

Several studies have employed the molecular mechan-

ics/generalized born surface area (MMGBSA) computational

technique to estimate the binding free energies (ꢀG_bind) between

a ligand and a protein/enzyme [26,27]. In the study, after 100 ns

molecular dynamic simulations, the best-docked ligands’ bind-

ing energies and the reference drugs for the two proteins were

estimated (Table 2). Table S1 in the supplementary materials

showed the docking results for all the compounds. According to

the study’s outcome, CFZ, a standard inhibitor of the spike protein

(SAR-CoV-2 Sgp), showed binding energy of -32.816 Kcal/mol.

Compounds 7b, 7 h, and 8d with average binding energies of

-43.116 kcal/mol, -42.036 kcal/mol and -41.733 kcal/mol, respec-

tively, might also be potential inhibitors of the human ACE-2 re-

ceptor. These binding energy values might suggest that 7i and 8f

could inhibit the activities of SAR-CoV-2 spike protein, and com-

pounds, 7b, 7 h, 7k, 8d, 8 g and 8 h might be potent and promis-

3

V.A. Obakachi, N.D. Kushwaha, B. Kushwaha et al.

Journal of Molecular Structure 1241 (2021) 130665

Fig. 1. Pyrazolone moieties as drugs or enzyme inhibitors.

ing inhibitors of hACE-2, thereby inhibiting the entry of SAR-CoV-2

into the host cells.

in the interaction plots. As shown in Fig. 2 (SAR-CoV-2 S_gpbound

systems), 7i and 8f have the highest number of interactions of 15

and 19, respectively. Therefore, their higher number of interactions

with the amino acid at the binding site of SAR-CoV-2 Sgp justiﬁed

their higher binding free energies reported in this study. The stan-

dard drug, CFZ, and compound 7k had total interactions of 12 and

13, respectively. From this study, the interaction plots showed that

the three pyrazolone compounds had π-anion interactions residues

Glu152, Phe10, Asp110, and Trp104, with compound 7k having a

π-Sulfur with Cys148 residue. The interactions reported in com-

pounds 7i and 8f together with the estimated binding energy sug-

2.3. Protein-ligand interaction with SAR-CoV-2 s_gpand hACE-2

To better understand the interaction better and further between

the compounds and the amino residues at the two proteins’ bind-

ing pockets, plots of ligand-protein interactions were plotted for

each compound against the proteins. Studies from Ndagi et al. and

Idowu et al. have reportedly used ligand-protein interaction plots

to examine the molecular interactions between amino residues at

the binding sites of a protein and bound ligands [28,29]. Fig. 2

presented the interaction plots for the SAR-CoV-2 S_gpand hACE-

2, respectively. Interactions such as hydrogen bond, π-sigma, π-

anion, π-Sulfur, alkyl, π-alkyl, amide-π stacked interaction, π-π

T-shaped interactions and Van der Waals (vdW) overlaps observed

gest they might be promising inhibitors of SAR-CoV-2 S_gp

.

Fig. 3a and b show the ligand-receptor interaction plots for the

standard drugs and the six compounds with the best binding ener-

gies toward hACE-2. The result showed that the compounds inter-

acted with essential amino residues at the binding pocket of hACE-

4

V.A. Obakachi, N.D. Kushwaha, B. Kushwaha et al.

Journal of Molecular Structure 1241 (2021) 130665

Table 2

Average Thermodynamic Binding Free Energy Proﬁles for the Synthesized compounds and standard drugs towards SAR-CoV-2 S_gp

and hACE-2.

Energy Components (kcal/mol)

Complex

ꢀ E_vdW

ꢀE_elec

ꢀG_gas

ꢀG_solv

ꢀG_bind

SAR-CoV-2 S_gp

CFZ

-45.828

-20.002

-34.080

-47.124

5.481

3.629

10.437

3.565

-27.566

-70.271

9.760

7.274

9.366

-73.394 10.076

-90.273 7.63

38.577 5.720

-32.816 6.070

7i

55.547 10.525

-1.579 26.365

-28.813 9.856

-34.726 8.387

-25.898 9.878

-32.956 3.091

7k

26.189

10.061

-24.319 35.263

-3.942 10.495

8f

43.181

hACE2

MLN-4760

-60.100

-58.982

-59.362

4.673

6.227

4.122

-341.381

-398.646

21.212

20.363

-461.482 23.262

-457.628 25.322

447.923 21.420

414.511 20.383

425.164 7.467

441.322 11.135

428.832 15.841

449.407 10.815

-43.558 4.132

-43.116 6.717

-42.036 5.542

-46.008 6.552

-41.733 5.821

-48.229 5.191

7b

7h

7k

8d

8 g

8h

-407.838 8.108

-427.345 12.415

-467.200

10.210

-59.985 5.699

-57.574 5.421

-487.330 15.695

-470.566 19.570

-497.637 12.474

-412.991

-433.521

-423.331

15.436

11.806

14.438

-64.115

-64.828

3.524

4.295

-488.159

17.120

438.839

12.930

-49.320

6.217

ꢀEele electrostatic energy, ꢀEvdW van der Waals energy, ꢀGbind total binding free energy, ꢀGsol solvation free energy, ꢀEgas

gas-phase free energy

Fig. 2. Interaction types and receptor (SAR-CoV-2 S_gp)-ligand interactions plots of (a) 7i, (b) CFZ, (c) 8f, and (d) 7k.

2 and revealed similar interactions between the synthesized com-

plots, the results showed that pyrazolone-based compounds could

be promising inhibitors of hACE-2 and eventually inhibiting the

formation of receptor-binding domain-hACE-2 (RBD-ACE-2) com-

plex required for SARS-CoV-2 entry.

pounds and the standard drug. The synthesized compounds had an

average of 20 interactions with the amino residues at the binding

pocket of the hACE-2 receptor, which is relatively equal to the 21

interactions observed in MLN-4760. With 7 h having the highest

number of interactions 24 and 7k having the lowest total num-

ber of interactions of 18. Similar to the standard drug, in addi-

tion to the essential hydrogen and Van Der Waal interactions, all

the compounds (except 8 g) had either π-cation, π-anion, or π-

Sulfur interactions with the amino residues. From the interaction

2.4. Structural dynamics of bound SARS-CoV-2 s_gpand hACE-2

After 100 ns of molecular dynamic (MD) simulation, the root

mean square deviation (RMSD), the radius of gyration (RoG)

and Root mean square ﬂuctuation (RMSF) of the unbound (apo-

enzyme) and ligand-bound trajectories were monitored and calcu-

5

V.A. Obakachi, N.D. Kushwaha, B. Kushwaha et al.

Journal of Molecular Structure 1241 (2021) 130665

Fig. 3. (a):Interaction types and receptor (hACE-2)-ligand interactions plots of a) MLN-4760 b) 7b c) 7k and d) 7 h. (b):Interaction types and receptor (hACE-2)-ligand

interactions plots of a) 8 h, b) 8 g and c) 8d

lated to evaluate. The RMSD measured the system’s convergence

and drift from a reference structural and indirectly the structural

stability, and a lowered RMSD values could indicate a more sta-

ble system [30]. From the SARS-CoV-2 S_gpsystems, all three syn-

thesized compounds achieved convergence in less than 10 ns MD

simulation, compared to the CFZ systems, which achieved conver-

gence at approximately 15 ns MD simulation. Signifying their sys-

tems reached structural stability and maintained stable conforma-

tions earlier than the unbound enzyme (Fig. 4). The result also re-

vealed that the binding of the three synthesized compounds (7i,

7k and 8f) with lower RMSD values stabilizes the enzyme more

than the standard drug with a relatively higher RMSD value. Un-

like the SARS-CoV-2 S_gpsystems, the RMSD plots for hACE-2 sys-

tems showed that the systems achieved convergence at 100 ns and

reached structural stability, as evidence by the lower RMSD values

for all the systems (Fig. 4).

six bound ligands are slightly higher than the RoG plot for the apo-

enzyme. The binding of compound 7k lowers the RoG plots slightly

below the apo-enzyme, making it more stable and compacted than

the other compounds and the standard.

RMSF is a measure of the effect of the binding of the ligand

on the behavior of the active site residues of a protein. Higher and

fewer ﬂexible movements are indicated by high and low RMSF val-

ues, respectively. For the SASR-CoV-2 S_gp, as shown in (Fig. 5b), the

standard drug and the apo-enzyme showed the highest ﬂuctuation

values indicating more ﬂexibility in their residues movement. This

result agrees with the high RoG and RMSD plots reported for the

apo-enzyme and the standard drug. Lower RMSF values and ﬂexi-

bility were observed in the three synthesized compounds. For the

hACE-2 systems, similar ﬂuctuations and ﬂexibilities were observed

in all the systems, with higher ﬂuctuations at residues 110–155,

330- 340 and 405–420, respectively.

The binding of the ligands (the six synthesized compounds

and the standard inhibitor) slightly raised the RMSD plots for the

bound system above the unbound system (apo-enzyme). However,

the plots showed that the binding of the compounds to the two

proteins did not affect the proteins’ stability and did not alter the

protein structural integrity.

This study also investigated the number of hydrogen bonds (H-

bonds) formed before and after ligands bind to the two proteins.

H-bonds play vital role in biological system, maintenance of the

protein structural integrity, and facilitate protein-ligand binding

[29]. Fig. 6 (A,B) and 7A shows H-bonds number formed over the

time during the 100 ns simulations of the hACE2 and SARS-CoV-2

Sp respectively. For the hACE2 system, more H-bonds was formed

in the Apo-enzyme, MLN-4760, compounds 7b, 7 h, 8d, and 8 g.

While the lowest H-bonds formed were observed in compounds

7k and 8 h. In the SARS-CoV-2 Sp systems, higher H-bonds were

formed in the Apo-system, CFZ, 7i, 7k than the compound 8f. Re-

duction in H-bonds observed in compounds 7k and 8 h (in the

hACE2), and 8f (in SARS-CoV-2 Sp systems) might lead to struc-

tural imbalances and conformational changes that could eventually

affect drug binding [31, 32].

In addition to the RMSD, the RoG plots of the two proteins’ sys-

tems were evaluated (Figs. 5a). RoG assesses the structural com-

pactness of the proteins upon binding of ligands. The lower the

RoG value, the more stable the system. The result of the RoG plots

for SARS-CoV-2 S_gpsystems correlate with the RMSD plot, show-

ing that the three ligands’ binding enhances the structural com-

pactness of the protein and stabilizes the system more than the

apo-enzyme and the standard drug. The RoG plots for the hACE-2

also correlate with the RMSD plot. The RoG values for ﬁve of the

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V.A. Obakachi, N.D. Kushwaha, B. Kushwaha et al.

Journal of Molecular Structure 1241 (2021) 130665

Fig. 3. Continued

Fig. 4. Comparative (a). RMSD (b). RoG, and (c). RMSF plots of alpha C atoms of the SARS-CoV-2 S_gpsystems calculated throughout 100 ns MD simulations.

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V.A. Obakachi, N.D. Kushwaha, B. Kushwaha et al.

Journal of Molecular Structure 1241 (2021) 130665

Fig. 5. (a) Comparative (a). RMSD b). RoG, and (c). RMSF plots of alpha C atoms of the hACE-2 systems calculated throughout 100 ns MD simulations. (b): Comparative (a).

RMSD (b). RoG, and (c). RMSF plots of alpha C atoms of the hACE-2 systems calculated throughout 100 ns MD simulations.

Fig. 5. Continued

Solvent accessible surface area (SASA) investigates the effect of

the binding of the ligand to a protein, it quantiﬁes the enzyme

exposure to solvent molecules [33]. In the hACE2 system (Fig. 6

C,D), the binding of compounds 8d, 8 g, the standard drug (MLN-

4760), 7b and 7 h increase solvent accessibility compare to the

Apo-enzyme, indicating the binding of the ligand enhanced the

accessibility of hACE2 protein to solvent environment. However,

the upward increase in solvent accessibility by the compounds

showed that hACE2 structural integrity is not altered [27]. Un-

like the hACE2 systems, the bind of the compounds 7i, 7k and 8f

signiﬁcantly lowers the exposure SARS-CoV-2 Sp to solvent envi-

ronment. However, the binding of the standard inhibitor, CFZ, in-

creases the SASA value similar to the Apo-enzyme. Fig. 7.

might lower the bioavailability of the eight selected phytochemical

compounds.

2.6. Assessing the drug-likeness of 8 selected phytochemical

compounds from the synthesized compounds

As shown in Table 3, the eight selected phytochemical com-

pounds (7b,7 h, 7i, 7k 8d, 8f, 8 g and 8 h) from the synthesized

compounds are poorly soluble in water compared to the two stan-

dard drugs (MLN-4760 and CFZ) which are soluble in water. And

might lower the bioavailability of the eight selected phytochemical

compounds.

One of the two conventional drugs passes the drug-likeness test

(MLN-4760), but only ﬁve of the phytochemical compounds (7i, 7k,

8f, 8 g and 8 h) pass the test. These showed that the ﬁve phyto-

chemical compounds have good drug properties.

2.5. Assessing the drug-likeness of 8 selected phytochemical

compounds from the 24 synthesized compounds

Lipinski’s rule is a set of ﬁve rules used to assess a compound’s

drug-likeness with pharmacological or biological activities [34].

The rule says the number of hydrogen bond donors should not be

more than 5, hydrogen bond acceptors not more than 10, molecu-

lar mass less than 500 daltons and partition coeﬃcient (logP) not

higher than 5. From this study, the result revealed one of the con-

As shown in Table 3, the eight selected phytochemical com-

pounds (7b,7 h, 7i, 7k 8d, 8f, 8 g and 8 h) from the synthesized

compounds are poorly soluble in water compared to the two stan-

dard drugs (MLN-4760and CFZ) which are soluble in water. And

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Journal of Molecular Structure 1241 (2021) 130665

Fig. 6. Comparative of H-bond and SASA plots of alpha C atoms of the hACE-2 systems calculated throughout 100 ns MD simulations.

Fig. 7. Comparative of (a). H-bond and (b). SASA plots of alpha C atoms of the SARS-CoV-2 systems calculated throughout 100 ns MD simulations.

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Journal of Molecular Structure 1241 (2021) 130665

ventional drugs (MLN) and two of the phytochemical compounds

from the synthesized compounds (7k and 8 g) were predicted to

pass the rules. It indicates that the two phytochemical compounds

from the synthesized compounds possess the same chemical and

physical properties as MLN. CFZ, the conventional drug and six of

the synthesized compounds failed a maximum of two rules.

All the eight phytochemical compounds from the synthesized

compounds were predicted to have low absorption from the GIT

(7b,7 h, 7i, 7k 8d, 8f, 8 g and 8 h). However, they could still even-

tually attain the required concentration needed for therapeutic ef-

fects, just as in the conventional drug, CFZ. The drug bioavailabil-

ity is a measurement of the degree of absorption and fraction of a

given amount of unchanged drug that goes to the systemic circula-

tion [35]. Compared with the two conventional drugs, CFZ has the

lowest bioavailability scores of 0.11, followed by the 7b, 7 h and

8d with 0.17. Slightly higher bioavailability scores of 0.55 were pre-

dicted for MLN-4760and the other phytochemical compounds from

the synthesized compounds.

3. Conclusion

Inhibiting the interaction of SARS-CoV-2 S_gpand hACE-2 is

signiﬁcant for the prevention of SARS-CoV-2 infections. Sequel

to this fact, this study has examined and evaluated pyrazolone-

based compounds’ possibilities as potential inhibitors of both

SARS-CoV-2 S_gpand hACE-2 to inhibit the virus entry into the

cells. This study employed synthetic chemistry methods to design

pyrazolone-based compounds and computational chemistry tech-

niques to identify these compounds’ potential inhibitory activity

against the spike proteins and hACE-2 receptor. From this study,

24 novel pyrazolone-based compounds were designed and synthe-

sized. According to the in-silico analysis, the results of the bind-

ing free energy showed that three compounds (7i, 7k and 8f) and

six compounds (7b, 7 h, 7k, 8d, 8 g, and 8 h) showed higher and

better binding high aﬃnity to SARS-CoV-2 S_gpand hACE-2_,respec-

tively compared to the standard drugs. Furthermore, the outcome

of the structural analysis of the two proteins upon binding of the

inhibitors showed that the two proteins were stable, and the struc-

tural integrity of the proteins was not compromised. The study

further revealed that the lead compounds possess similar phar-

macokinetic and physicochemical properties to the standard drugs.

Therefore, this study suggests pyrazolone-based compounds might

be potent inhibitors of the virus entry into the host cells.

4. Experimental section

4.1. Chemistry

4.1.1. General consideration

All the ﬁne chemicals, reagents and solvents were purchased

from Sigma Aldrich and Merck and were used without further pu-

riﬁcation unless otherwise stated. The progress of the reactions

and the purity of the compounds was monitored by thin-layer

chromatography (TLC) on pre-coated silica gel plates procured

from E. Merck and Co. (Darmstadt, Germany) visualized by a UV

lamp (254 or 365 nm). Puriﬁcation was performed by using combi-

ﬂash (CombiFlash® NextGen 300+) column chromatography. The

synthesized compounds’ melting points have been determined and

uncorrected using a digital Stuart SMP10 melting point Apparatus.

The Fourier transform infrared (FTIR) spectra were recorded in the

spectral range of 400–4000 cm⁻¹on a Bruker Alpha FT-IR spec-

trometer using the ATR technique. The NMR spectra (¹H and ¹³C)

were recorded using CDCl₃and DMSO-d₆on Bruker AVANCE III

400 and 600 MHz spectrometers. Chemical shifts were determined

relative to internal standard TMS at δ 0.0 parts per million (ppm),

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V.A. Obakachi, N.D. Kushwaha, B. Kushwaha et al.

Journal of Molecular Structure 1241 (2021) 130665

and the coupling constants were reported in Hertz. The multiplici-

ties of the NMR resonances were abbreviated as s (singlet), d (dou-

blet), dd (doublet of doublet), (t (triplet), q (quartet), m (multiplet)

and brs (broad singlet). Mass spectrometry (MS) was performed

using a Bruker ESI-QTOF mass spectrometer in positive and neg-

ative ion mode.

reaction, excess acetone was removed under reduced pressure and

acidify with 6 N HCl, then extracted with DCM. The organic layer

was dried over anhydrous sodium sulfate and concentrated under

reduced pressure to obtain the title compounds (4a,b).

(E)-4-(5-ethoxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)but-3-en-2-

one (4a): Brown Solid, Yield: 91%; ¹H NMR (400 MHz, CDCl₃25 °C)

δ 7.67–7.65 (m, 2H, ArH), 7.44 (t, J =7.80 Hz, 2H, ArH), 7.40 (s,

1H, Ar-HC=CH-), 7.33–7.29 (m, 1H, ArH), 6.56 (d, J =16.53 Hz, 1H,

Ar-HC=CH-), 3.98 (q, J =7.03 Hz, 2H, CH₂), 2.39 (s, 3H, CH₃), 2.33

(s, 3H, CH₃), 1.27 (t, J =7.08 Hz, 3H, CH₃); ¹³C NMR (100 MHz,

CDCl₃25 °C) δ 198.3, 148.9, 139.0, 133.0, 129.2, 127.3, 124.3, 122.5,

104.2, 71.3 (CH₂), 27.6 (CH₃), 15.3 (CH₃), 14.4 (CH₃)

4.1.2. General procedure for the synthesis of intermediate (2a,b)

The Vilsmeier-Haack reagent was freshly prepared by the care-

ful addition of POCl₃(7 eq.) in dimethylformamide (3 eq.) at

0 °C with constant stirring. To the reaction mixture maintain-

ing 0 °C was added 2-(Phenyl/substituted phenyl)-5-methyl-2,4-

dihydro-3H-pyrazol-3-one (1a-b) (1 Eq.) and stirred initially for

30 min and later slowly brought to rt and stirred for 1 h. Finally,

the reaction temperature was raised to 100 °C and allowed to stir

for 3 hrs. After cooling to rt, the reaction mixture was poured into

a mixture of crushed ice and water, the precipitate formed sep-

arated by ﬁltration and washed with ice-cold water. The residue

obtained was ﬁltered, dried, and recrystallized from ethanol to af-

ford the title compounds (2a,b).

(E)-4-(1-(3-chlorophenyl)-5-ethoxy-3-methyl-1H-pyrazol-4-yl)but-

3-en-2-one (4b): Brown Solid, Yield: 74%; ¹H NMR (400 MHz,

CDCl₃25 °C) δ 7.66 (d, J =8.28 Hz, 2H, ArH), 7.44 (s, 1H, ArH), 7.40

(s, 1H, Ar-HC=CH-), 7.33–7.29 (m, 1H, ArH), 6.56 (d, J =16.53 Hz,

1H, Ar-HC=CH-), 3.98 (q, J =7.03 Hz, 2H, CH₂), 2.39 (s, 3H, CH₃),

2.33 (s, 3H, CH₃), 1.27 (t, J =J=7.08 Hz, 3H, CH₃); ¹³C NMR

(100 MHz, CDCl₃25 °C) δ 198.3, 153.0, 148.9, 139.0, 133.0, 129.2,

127.3, 124.3, 122.5, 122.3, 104.2, 71.3 (CH₂), 27.6 (CH₃), 15.3 (CH₃),

14.4 (CH₃)

5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (2a): Off

white, Yield: 75%; ¹H NMR (400 MHz, CDCL₃25 °C) δ 9.98 (s, 1H,

-CHO), 7.567.52 (m, 4H), 7.50–7.47 (m, 1H), 2.55 (s, 3H, CH₃); ¹³C

NMR (100 MHz, CDCL₃25 °C) δ 183.9, 151.9, 137.0, 133.5, 129.4,

129.3, 125.3, 117.5, 13.9 (CH₃)

4.1.5. General procedure for the synthesis of compounds (5a-b)

To a constantly stirred solution of (E)-4-(1-(phenyl/substituted

phenyl)-5-ethoxy-3-methyl-1H-pyrazol-4-yl)but-3-en-2-one (4a-b)

(1.0 eq.) and thiosemicarbazide (1.1eq.) in anhydrous methanol was

added a catalytic amount of glacial acetic acid (0.1 ml) and reﬂuxed

for 4–5 h.. After completing the reaction (monitored via TLC), the

reaction mass was cooled to rt and concentrated under reduced

pressure to give a semi-solid residue. The residue was triturated

with 50% diethyl ether and pentane and ﬁltered under suction to

afford a pure yellow crystalline solid title compound (5a-b).

(E)-2-((E)-4-(5-ethoxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)but-3-

en-2-ylidene)hydrazine-1-carbothioamide (5a) Yield: 87%; mp;150–

153 °C; FTIR (ATR, Vmax, cm-1): 3373.89 (N-H Str. of NH₂), 3262.82

(N-H Str. of NH), 3.177.48 (Ar C-H Str.), 2973.89 (C-H Str. of CH₃),

1597.91 (C = N Str), 1087.70 (C = S); ¹H NMR (400 MHz, DMSO-d₆

25 °C) δ 10.12 (brs, 1H, NH), 8.15 (brs, 1H, NH₂), 7.81 (brs, 1H,

NH₂), 7.65 (d, J = 7.92 Hz, 2H, ArH), 7.49 (t, J = 8.70 Hz, 2H,

ArH), 7.34 (t, J = 8.10 Hz, 1H, ArH), 6.80 (d, J = 16.77 Hz, 1H,

Ar-HC=CH-), 6.63 (d, J = 16.77 Hz, 1H, Ar-HC=CH-), 4.00 (q,

J = 7.03 Hz, 2H, CH₂), 2.30 (s, 3H, CH₃), 2.11 (s, 3H, CH₃), 1.20 (t,

J = 7.76 Hz, 3H, CH₃); MS m/z 344.00 (M+H)⁺.

5-chloro-1-(3-chlorophenyl)-3-methyl-1H-pyrazole-4-carbaldehyde

(2b): White Solid, Yield: 70%; ¹H NMR (400 MHz, CDCL₃25 °C)

δ 9.96 (s, 1H, -CHO), 7.59 (s, 1H), 7.47–7.43 (m, 3H), 2.52 (s, 3H,

CH₃); ¹³C NMR (100 MHz, CDCL₃25 °C) δ 183.8, 152.2, 138.0,

135.1, 133.6, 130.3, 129.4, 125.5, 123.2, 117.8, 13.9 (CH₃)

4.1.3. General procedure for the synthesis of intermediate (3a,b)

To the solution of 5-chloro-1-(phenyl/substituted phenyl)-3-

methyl-1H-pyrazole-4-carbaldehyde (2a,b) (1 eq.) in ethanol was

added cesium carbonate (1.5 eq.) followed by copper acetate (0.25

eq.) and reﬂuxed for 15 hrs. The reaction was monitored with

TLC (30:70, ethyl acetate; hexane); after the reaction completion,

the reaction mixture was cooled to rt, then diluted with ethyl ac-

etate and washed with brine solution. The organic layer was dried

over anhydrous sodium sulfate, ﬁltered, and concentrated under

reduced pressure to get the crude product. The solid crude product

was puriﬁed by column chromatography using 100–200 mesh sil-

ica gel. The desired fractions were collected and concentrated un-

der reduced pressure to afford title products (3a,b).

(E)-2-((E)-4-(1-(3-chlorophenyl)-5-ethoxy-3-methyl-1H-pyrazol-4-

yl)but-3-en-2-ylidene)hydrazine-1-carbothioamide (5b) Yield: 76%;

mp;152–155 °C; FTIR (ATR, Vmax, cm-1): 366.52 (N-H Str. of NH₂),

3258.40 (N-H Str. of NH), 3169.77 (Ar C-H Str.), 2971.39 (C-H Str.

of CH₃), 1640.27 (C = N Str), 1158.23 (C = S); ¹H NMR (400 MHz,

DMSO-d₆25 °C) δ 10.12 (brs, 1H), 8.15 (brs, 1H, NH₂), 7.77 (brs,

1H, NH₂), 7.65 (d, J = 7.90 Hz, 1H), 7.50 (t, J = 7.52 Hz, 2H), 7.34

(d, J = 7.39 Hz, 1H), 6.80 (d, J = 16.30 Hz, 1H Ar-HC=CH-), 6.63

(d, J = 16.98 Hz, 1H, Ar-HC=CH-), 4.01 (q, J = 6.92 Hz, 2H, CH₂),

2.31 (s, 3H, CH₃), 2.11 (s, 3H, CH₃), 1.21 (t, J = 7.16 Hz, 3H, CH₃);

m/z 378.00 (M+H)⁺.

5-ethoxy-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde

(3a):

Brown Solid, Yield: 68%; .); ¹H NMR (400 MHz, CDCl₃25 °C) δ

9.90 (s, 1H, -CHO), 7.64 (d, J = 7.79 Hz, 2H), 7.44 (t, J = 7.91 Hz,

2H), 7.33 (t, J = 7.79 Hz, 1H), 4.41 (q, J = 7.04 Hz, 2H), 2.48 (s,

3H, CH₃), 1.33 (t, J = 7.04 Hz, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃

25 °C) δ 183.1, 155.6, 151.3, 137.5, 129.1, 127.7, 123.1, 108.1, 72.6

(CH₂), 15.3 (CH₃), 14.0 (CH₃)

1-(3-chlorophenyl)-5-ethoxy-3-methyl-1H-pyrazole-4-

carbaldehyde (3b): Brown Solid, Yield: 71%; .); ¹H NMR (400 MHz,

CDCl₃25 °C) δ 9.91 (s, 1H, -CHO), 7.91 (s, 1H), 7.64 (d, J = 7.79 Hz,

1H), 7.44 (d, J = 7.91 Hz, 1H), 7.33 (t, J = 7.79 Hz, 1H), 4.40 (q,

J = 7.04 Hz, 2H, CH₂), 2.48 (s, 3H, CH₃), 1.33 (t, J = 7.04 Hz, 3H,

CH₃); ¹³C NMR (100 MHz, CDCl₃25 °C) δ 183.1, 155.6, 151.3, 147.5,

137.5, 129.1, 127.7, 123.1, 115.6, 108.1, 72.6 (CH₂), 15.3 (CH₃), 14.0

(CH₃)

4.1.6. General procedure for the synthesis of ﬁnal compounds (7b-o

and 8a-h)

To

a stirred solution of (E)-2-((E)-4-(1-(phenyl/substituted

phenyl)-5-ethoxy-3-methyl-1H-pyrazol-4-yl)but-3-en-2-

ylidene)hydrazine-1-carbothioamide (5a,b) (1.0 eq.) in anhydrous

methanol, was added an appropriate 2-bromo-1-(substituted

phenyl)-ethanones (6b-o,1.1 eq.), and the reaction mixture was

reﬂuxed for 4–7 h until TLC showed full consumption of starting

materials. The reaction mass was cooled to rt and concentrated

to remove excess methanol. The obtained reaction mass was

diluted with DCM and washed with saturated NaHCO₃solution.

4.1.4. General procedure for the synthesis of compounds (4a,b)

Compound 1-(phenyl/substituted phenyl)-5-ethoxy-3-methyl-

1H-pyrazole-4-carbaldehyde (3a,b) (1.0 eq.) was dissolved in ace-

tone to which sodium hydroxide solution (7.57 eq.) was slowly

added with continues stirring for 2–3 h. The reaction was moni-

tored with TLC (30:70, ethyl acetate; hexane). After completing the

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V.A. Obakachi, N.D. Kushwaha, B. Kushwaha et al.

Journal of Molecular Structure 1241 (2021) 130665

The organic layer obtained was dried over anhydrous Na₂SO₄

and evaporated under reduced pressure to give a solid residue

washed with diethyl ether and pentane to afford pure solid title

compounds (7b–o and 8a–h).

135 °C; FTIR (ATR, vmax, cm-1): 3052.77 (Ar-H Str.), 2978.74 (C-H

Str. of CH₃), 1600.49 (C = N Str.); ¹H NMR (400 MHz, DMSO-d₆

25 °C) δ 11.27 (brs, 1H, NH), 9.63 (s, 1H, OH), 7.67 (d, J=7.87 Hz,

2H, ArH), 7.57 (d, J=3.13 Hz, 2H, ArH), 7.51 (t, J=7.86 Hz, 2H,

ArH), 7.35 (q, J=4.92 Hz, 2H, ArH), 6.81 (d, J=8.96 Hz, 1H, ArH),

6.77 (d, J=16.80 Hz, 1H, 1H, Ar-HC=CH-), 6.66 (d, J=16.65 Hz,

1H, 1H, Ar-HC=CH-), 4.00 (q, J=7.01 Hz, 2H, CH₂), 3.72 (s, 3H,

OCH₃), 2.32 (s, 3H, CH₃), 2.17 (s, 3H, CH₃), 1.25 (t, J=7.03 Hz,

3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆25 °C) δ 168.3 (C-2 of

thiazole), 152.0, 150.6, 149.7, 149.2 (C-5 of thiazole), 146.9, 137.9,

129.2, 126.7–126.6 (d, J = 13.51 Hz, 1C), 124.9, 121.6, 121.4, 118.7,

117.5, 115.5, 111.0, 104.7, 104.4, 70.6(CH₂), 5₊5.4(OCH₃), 14.9(CH₃),

4.1.6.1. 4-((1E,3E)-3-(2-(4-(4-bromophenyl) cyclopenta-1,3-dien-

1-yl) hydrazono) but-1-en-1-yl)-5-ethoxy-3-methyl-1-phenyl-1H-

pyrazole (7b); Yellowish Solid, Yield: 78%, mp: 177–180 °C; FTIR

(ATR, vmax, cm-1): 3071.06 (Ar-H Str.), 2975.74 (CH Str. of CH₃),

1606.33 (C = N Str.); ¹H NMR (400 MHz, DMSO-d₆25 °C) δ

11.14 (brs, 1H, NH), 7.82 - 7.80 (m, 2H, ArH), 7.68 - 7.65 (m, 2H,

ArH), 7.60 -7.57 (m, 2H, ArH), 7.53 -7.48 (m, 2H, ArH), 7.36 (d,

J = 8.64 Hz, 1H, ArH), 6.73 (d, J =16.83 Hz, 1H, Ar-HC=CH-), 6.64

(d, J =16.83 Hz, 1H, Ar-HC=CH-), 4.00 (q, J = 7.04 Hz, 2H, CH₂),

2.31 (s,3H, CH₃), 2.14 (s, 3H, CH₃), 1.24 (t, J = 7.05 Hz, 3H, CH₃);

¹³C NMR (100 MHz, DMSO-d6 25 °C) δ 159.9(C-2 of thiazole), 151.5,

149.8, 147.8, 138.2(C-5 of thiazole), 134.6, 132.9, 131.4, 129.1, 127.4,

126.4, 121.8, 121.3, 121.1, 120.4, 112.4, 111.7, 68.5 (CH₂), 14.6 (CH₃),

13.4 (CH₃), 13.0 (CH₃). MS m/z 522.00 (M+H)⁺.

13.8(CH₃), 12.0(CH₃). MS m/z 490.00 (M+H)

4.1.6.6.

.

2-(2-(2-((2E,3E)-4-(5-ethoxy-3-methyl-1-phenyl-

1H-pyrazol-4-yl)but-3-en-2-ylidene)hydrazinyl)thiazol-4-yl)-4-

ﬂuorophenol (7 g); Brownish Solid, Yield:87%, mp:175–179 °C;

FTIR (ATR, vmax, cm-1): 3070.26 (Ar-H Str.), 2982.37 (C-H Str. of

CH₃), 1604.59 (C = N Str.); ¹H NMR (400 MHz, DMSO-d₆25 °C) δ

11.21 (brs, 1H), 9.67 (s, 1H, OH), 7.67–7.63 (m, 3H, ArH), 7.52–7.49

(m, 3H, ArH), 7.35 (t, J = 7.43 Hz, 1H, ArH), 7.01- 6.69 (m, 1H, ArH),

6.90 – 6.86 (m, 1H, ArH), 6.77 (d, J = 16.52 Hz, 1H, Ar-HC=CH-),

6.65 (d, J = 16.43 Hz, 1H, Ar-HC=CH-), 4.00 (q, J = 7.00 Hz, 2H,

CH₂), 2.31 (s, 3H, CH₃), 2.16 (s, 3H, CH₃), 1.24 (t, J = 6.96 Hz,

3H, CH₃); ¹³C NMR (100 MHz, DMSO-d6 25 °C) δ 158.8 (C-2 of

thiazole), 154.5, 151.4, 151.2, 150.6, 147.7, 147.0 (C-5 of thiazole),

138.1, 134.4, 129.2–129.1 (d, J = 19.40 Hz, 1c), 128.8, 126.4, 122.2,

120.8 (d, J = 8.00 Hz, 1c), 117.2–117.1 (d, J = 7.67 Hz, 1c), 115.2

4.1.6.2.

5-ethoxy-4-((1E,3E)-3-(2-(4-(4-ﬂuorophenyl)

cyclopenta-1,3-dien-1-yl) hydrazono) but-1-en-1-yl)-3-methyl-1-

phenyl-1H-pyrazole (7c); Brownish Solid,Yield:72%,mp:124–127 °C;

FTIR (ATR, vmax, cm-1): 3238.79 (N-H Str.), 3112.04 (Ar-H Str.),

2978.96 (CH Str. of CH₃), 1549.10 (C = N Str.); ¹H NMR (400 MHz,

DMSO-d₆40 °C) δ 11.07 (brs, 1H, NH), 789–785 (m, 2H, ArH), 7.66–

7.64 (m, 2H, ArH), 7.51–7.47 (m, 2H, ArH), 7.33 (t, J = 7.66 Hz, 1H,

ArH), 7.22–7.18 (m, 3H, ArH), 6.68 (d, J =16.91 Hz, 1H, Ar-HC=CH-),

6.63 (d, J =16.66 Hz, 1H, Ar-HC=CH-), 3.98 (q, J = 7.13 Hz, 2H,

CH₂), 2.29 (s, 3H, CH₃), 2.12 (s, 3H, CH₃), 1.23 (t, J = 7.24 Hz,

3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆25 °C) δ 169.5 (C-2 of

thiazole), 160.3, 150.5, 149.4 (C-5 of thiazole), 148.6, 146.8, 137.9,

131.4, 129.2, 127.4 (d, J = 8.08 Hz, 1C), 127.1, 126.6, 121.6, 120.5,

-115.1 (d, J = 23.54 Hz, 1c), 114.7, 113.9, 113.7, 112.5, 94.5, 68.7

+

(CH₂), 14.6 (CH₃), 13.3 (CH₃), 12.9 (CH₃). MS m/z 478.00 (M+H)

.

4.1.6.7.

4-(3,4-dichlorophenyl)-2-(2-((2E,3E)-4-(5-

ethoxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)but-3-en-2-

15.3 (d, J = 21.56 Hz, 1C), 104.8, 103.4, 70.5 (CH₂), 14.9 (CH₃), 13.8

ylidene)hydrazinyl)thiazole (7 h); Brownish solid, Yield:83%,

mp:187–190 °C; FTIR (ATR, vmax, cm-1): 3054.80 (Ar-H Str.),

2980.41 (C-H Str. of CH₃), 1612.58 (C = N Str.): ¹H NMR (400 MHz,

DMSO-d₆25 °C) δ 11.15 (brs, 1H, NH), 8.10–8.09 (d, J = 2.01 Hz,

1H, ArH), 7.85–7.83 (m, 1H, ArH), 7.67–7.65 (m, 3H, ArH), 7.52–7.48

(m, 3H, ArH), 7.34 (t, J = 7.36 Hz, 1H, ArH), 6.74 (d, J = 16.75 Hz,

1H, Ar-HC=CH-), 6.64 (d, J = 16.75 Hz, 1H, Ar-HC=CH-), 3.99 (q,

J = 7.12 Hz, 2H, CH₂), 2.31 (s, 3H, CH₃), 2.14 (s, 3H, CH₃), 1.24 (t,

J = 7.02 Hz, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆25 °C) δ 160.1

(C-2 of thiazole), 151.6, 148.4, 147.8, 138.1 (C-5 of thiazole), 134.5,

134.2, 131.6, 130.6, 130.3, 128.9, 127.5, 127.1, 126.3, 125.3, 122.8,

+

(CH₃), 11.9 (CH₃). MS m/z 462.00 (M+H)

.

4.1.6.3.

2-(2-(2-((2E,3E)-4-(5-ethoxy-3-methyl-1-phenyl-1H-

pyrazol-4-yl) but-3-en-2-ylidene) hydrazinyl) thiazol-4-yl) phenol

(7d); Brownish Solid, Yield:89%, mp:176–179 °C; FTIR (ATR, vmax,

cm-1): 3070.84 (Ar-H Str.), 2974.54 (CH Str. of CH₃), 1599.88

(C = N Str.); ¹H NMR (400 MHz, DMSO-d₆25 °C) δ 11.44 (brs,

1H, NH), 11.24 (brs, 1H, OH), 7.80 (d, J=7.94 Hz, 1H, ArH), 7.67 (t,

J=8.22 Hz, 2H, ArH), 7.53–7.58 (m, 2H, ArH), 7.36 (t, J=7.33 Hz,

2H, ArH), 7.17–7.13 (m, 1H, ArH), 6.88–6.82 (m, 2H, ArH), 6.77 (d,

J=16.67 Hz, 1H, Ar-HC=CH-), 6.66 (d, J=16.71 Hz, 1H, Ar-HC=CH-),

4.02 (q, J=7.04 Hz, 1H, CH₂), 2.33 (s, 3H, CH₃), 2.17 (s, 3H, CH₃),

1.26 (t, J=7.06 Hz, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆25 °C)

δ 168.5 (C-2 of thiazole), 155, 150.6, 149.3(C-5 of thiazole), 146.9,

137.9, 129.2, 129.1, 128.9, 126.7, 121.8, 121.7, 121.6, 121.2, 119.0,

118.6, 116.9, 116.6, 104.7, 103.6, 70₊.5(CH₂), 14.9 (CH₃), 13.8 (CH₃),

122.1 -122.0 (d, J = 11.63 Hz, 1C), 121.8, 113.0, 112.5, 94.3, 68.6

+

(CH₂), 14.6 (CH₃), 13.3 (CH₃), 12.9 (CH₃). MS m/z 512.00 (M+H)

.

4.1.6.8.

4-bromo-2-(2-(2-((2E,3E)-4-(5-ethoxy-3-methyl-1-

phenyl-1H-pyrazol-4-yl)but-3-en-2-ylidene)hydrazinyl)thiazol-4-

yl)phenol (7i); Brownish solid, Yield:74%, mp: 178–181 °C; FTIR

(ATR, vmax, cm-1): 3051.84 (Ar-H Str.), 2977.06 (C-H Str. of CH₃),

1602.93 (C = N Str.): ¹H NMR (400 MHz, DMSO-d₆25 °C) δ 11.32

(brs, 1H, NH), 10.26 (s, 1H, OH) 8.04 (d, J=2.41 Hz, 1H, ArH), 7.67

(d, J=7.72 Hz, 2H, ArH), 7.52–7.49 (m, 3H, ArH), 7.35 (t, J=7.40 Hz,

1H, ArH), 7.29 (q, J=3.70 Hz, 1H, ArH), 6.88 (d, J=8.66 Hz, 1H,

ArH), 6.78 (d, J=16.58 Hz, 1H, Ar-HC=CH-), 6.66 (d, J=16.67 Hz,

1H, Ar-HC=CH-), 4.01 (q, J=7.01 Hz, 2H, CH₂), 2.32 (s, 3H, CH₃),

2.17 (s, 3H, CH₃), 1.25 (t, J=7.02 Hz, 3H, CH₃); ¹³C NMR (100 MHz,

DMSO-d6 25 °C) δ 168.7 (C-2 of thiazole), 154.8, 147.4(C-5 of

thiazole), 138.3.9, 131.6, 129.9, 129.7, 127.2, 127.0, 122.1, 121.9,

12.0 (CH₃). MS m/z 460.00 (M+H)

.

4.1.6.4. 2-(2-((2E,3E)-4-(5-ethoxy-3-methyl-1-phenyl-1H-pyrazol-

4-yl)but-3-en-2 ylidene)hydrazinyl)-4-phenylthiazole (7e); Light

Brownish Solid, Yield:73% mp:154–157 °C; FTIR (ATR, vmax, cm-1):

3070.84 (Ar-H Str.), 2977.30 (CH Str. of CH₃), 1614.72 (C = N Str.);

¹H NMR (400 MHz, DMSO-d₆25 °C) δ 11.11 (brs, 1H, NH), 7.86 (d,

J=7.30 Hz, 2H, ArH), 7.67 (d, J=7.68 Hz, 2H, ArH), 7.51 (t, J=7.92 Hz,

2H, ArH), 7.40 (t, J=7.62 Hz, 2H, ArH), 7.35 (t, J=7.34 Hz, 1H, ArH),

7.30 (t, J=7.52 Hz, 2H, ArH), 6.73 (d, J=16.69 Hz, 1H, Ar-HC=CH-),

6.65 (d, J=16.68 Hz, 1H, Ar-HC=CH-)), 4.00 (q, J=7.03 Hz, 2H, CH₂),

2.32 (s, 3H, CH₃), 2.15 (s, 3H, CH₃), 1.25 (t, J=7.01 Hz, 3H, CH₃);

¹³C NMR (100 MHz, DMSO-d6 25 °C) δ 169.2 (C-2 of thiazole),

159.8, 147.8, 147.0 (C-5 of thiazole), 137.9, 134.6, 133.7, 129.3, 129.1,

128.6 (d, J = 6.86 Hz, 1C), 128.1, 126.8, 126.4, 125.6 (d, J =7.84 Hz,

119.3, 110.7, 106.5, 105.2, 71.0 (CH₂), 15.4 (CH₃), 14.3 (CH₃), 12.5

+

(CH₃). MS m/z 538.00 (M+H)

4.1.6.9.

.

4-(2,6-dimethoxyphenyl)-2-(2-((2E,3E)-4-(5-

ethoxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)but-3-en-2-

1C), 121.9, 121.7, 121.4, 112.3, 110.9, 104.8, 68.₊5 (CH₂), 14.7 (CH₃₎

,

ylidene)hydrazinyl)thiazole (7j); Yellowish Solid, Yield:76%,

mp:182–185 °C; FTIR (ATR, vmax, cm-1): 3252.00 (N-H Str.),

3162.33 (Ar-H Str.), 2977.32 (C-H Str. of CH₃), 1591.75 (C = N

Str); ¹H NMR (400 MHz, DMSO-d₆25 °C) δ 10.15 (brs, 1H, NH)

7.66 (d, J=7.83 Hz, 2H, ArH), 7.51 (t, J=7.79 Hz, 2H, ArH), 7.41

13.0 (CH₃), 12.1 (CH₃). MS m/z 444.00 (M+H)

.

4.1.6.5.

2-(2-(2-((2E,3E)-4-(5-ethoxy-3-methyl-1-phenyl-

1H-pyrazol-4-yl)but-3-en-2-ylidene)hydrazinyl)thiazol-4-yl)-3-

methoxyphenol (7f); Light Brownish Solid,Yield:76%,mp:132–

12

V.A. Obakachi, N.D. Kushwaha, B. Kushwaha et al.

Journal of Molecular Structure 1241 (2021) 130665

CH₃); ¹³C NMR (100 MHz, CDCl₃) d 169.0 (C-2 of thiazole), 151.3 (d,

J=24.27 Hz, 1C), 148.1, 147.6(C-5 of thiazole), 138.4, 137.5, 132.2,

129.27 (d, J=23.33 Hz, 1 C), 126.86 (d, J=12.07 Hz, 1 C), 125.8,

(t, J=8.32 Hz, 1H, ArH), 7.36 (t, J=7.28, 1H, ArH), 7.16 (s, 1H,

ArH), 7.04 (s, 1H, ArH), 6.93 (d, J=17.79 Hz, 1H, ArH), 6.81 (d,

J=16.59 Hz, 1H, Ar-HC=CH-), 6.70 (d, J=16.43 Hz, 1H, Ar-HC=CH-),

4.02 (q, J=6.99 Hz, 2H, CH₂), 3.78 (s, 6H, OCH₃), 2.33 (s, 3H, CH₃),

2.24 (s, 3H, CH₃), 1.24 (t, J=6.98 Hz, 3H, CH₃); ¹³C NMR (100 MHz,

DMSO-d6 25 °C) δ 158.6 (C-2 of thiazole), 151.5, 147.6, 138.2 (C-5

of thiazole), 132.1, 131.9, 129.7, 127.4, 127.4, 125.6, 122.4, 122.2,

122.2, 121.1, 105.4, 102.9, 70.85 (CH₂), 21.31 (CH₃), 15.33 (CH₃),

+

14.37 (CH₃), 11.01 (CH₃). MS m/z 458.00 (M+H)

4.1.6.14.

.

4-(3,4-dimethoxyphenyl)-2-(2-((2E,3E)-4-(5-

ethoxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)but-3-en-2-

105.1, 104.8, 71.3(CH₂), 56.5(OCH₃), 15.4 (CH₃), 14.4 (CH₃), 13.3

+

ylidene)hydrazinyl)thiazole (7o); Brownish Solid, Yield: 68%,

mp: 190–193 °C; FTIR (ATR, vmax, cm-1): 3060.57 (Ar-H Str.),

2902.04 (C-H Str. of CH₃), 1613.29 (C = N Str.); ¹H NMR (400 MHz,

DMSO-d₆25 °C) δ 10.26 (s, 1H, NH), 7.67 (d, J=7.80 Hz, 2H, ArH),

7.51 (t, J=7.80 Hz, 2H, ArH), 7.38 (m, 3H, ArH), 7.19 (s, 1H, ArH),

6.99 (d, J=8.37 Hz, 1H, ArH), 6.77 (d, J=16.66 Hz, 1H, Ar-HC=CH-),

6.67 (d, J=16.66 Hz, 1H, Ar-HC=CH-), 4.01 (q, J=6.99 Hz, 2H, CH₂),

3.79 (d, J=11.77 Hz, 6H, OCH₃), 2.32 (s, 3H, CH₃), 2.17 (s, 3H, CH₃),

1.25 (t, J=7.02 Hz, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d6 25 °C)

δ 159.5 (C-2 of thiazole), 151.2 (d, J = 8.80 Hz, 1C), 150.8, 148.8(d,

J = 4.37 Hz, 1C), 148.0, 138.2 (C-5 of thiazole), 138.1, 134.3, 129.0,

126.7, 126.3, 121.7, 121.6, 118.0, 112.4, 111.7, 109.2, 109.0, 94.6, 68.7

(CH₃). MS m/z 504.00 (M+H)

4.1.6.10.

.

2-(2-((2E,3E)-4-(5-ethoxy-3-methyl-1-phenyl-

1H-pyrazol-4-yl)but-3-en-2-ylidene)hydrazinyl)-4-(4-

nitrophenyl)thiazole (7k); Brownish Solid, Yield: 75%, mp:166–

169 °C;FTIR (ATR, vmax, cm-1): 3069.55 (Ar-H Str.), 2981.67 (C-H

Str. of CH₃), 1596.47 (C = N Str); ¹H NMR (400 MHz, DMSO-d₆

25 °C) δ 11.29 (brs, 1H, NH), 8.27 (d, J = 8.51 Hz, 2H, ArH), 8.11

(d, J = 8.69 Hz, 2H), 7.69–7.66 (m, 3H, ArH), 7.50 (t, J = 7.59 Hz,

2H, ArH), 7.34 (t, 7.32 Hz, 1H, ArH), 6.73 (d, J = 16.60 Hz, 1H,

Ar-HC=CH-), 6.64 (d, J = 16.60 Hz, 1H, Ar-HC=CH-), 3.99 (q,

J = 7.01 Hz, 2H, CH₂), 2.31 (s, 3H, CH₃), 2.15 (s, 3H, CH₃), 1.24

(t, J = 6.94 Hz, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d6 25 °C) δ

170.1 (C-2 of thiazole), 156.4, 151.0, 149.6(C-5 of thiazole), 147.4,

146.6, 141.2 (d, J = 11.1 Hz, 1C), 138.4, 129.6 (d, J = 4.4 Hz, 1C),

127.2 (d, J = 12.2 Hz 1C), 126.7 (d, J =4.5 Hz, 1C), 124.5 (d,

(CH₂), 55.3 (d, J = 24.2 Hz, 1C, OCH₃), 14.6 (CH₃), 13.3 (CH₃), 13.1

+

(CH₃). MS m/z 504.00 (M+H)

.

4.1.6.15.

2-(2-((2E,3E)-4-(1-(3-chlorophenyl)-5-ethoxy-

3-methyl-1H-pyrazol-4-yl)but-3-en-2-ylidene)hydrazinyl)-4-

J = 16.0 Hz, 1C), 122.1, 121.8, 121.5, 105.2, 71.0 (CH₂), 15.4 (CH₃),

phenylthiazole (8a); Brownish Solid, Yield: 69%, mp: 195–198 °C;

FTIR (ATR, vmax, cm-1): 3087.50 (Ar-H Str.), 2981.46 (C-H Str. of

CH₃), 1618.49 (C = N Str.); ¹H NMR (400 MHz, CDCl₃) d 11.31 (brs,

1H, NH), 7.74 (s, 1H, ArH), 7.70 (d, J=7.09 Hz, 2H, ArH), 7.60 (d,

J=8.16 Hz, 1H, ArH), 7.43 (t, J=7.26 Hz, 2H, ArH), 7.37 (m, 2H, ArH),

7.32 (d, J=8.17 Hz, 1H, ArH) 7.24 (s, 1H, ArH), 6.79 (d, J=16.75 Hz,

1H, Ar-HC=CH-), 6.71 (d, J=16.42 Hz, 1H, Ar-HC=CH-),), 4.00 (q,

J=7.04 Hz, 2H, CH₂), 2.38 (s, 3H, CH₃), 2.28 (s, 3H, CH₃), 1.30 (t,

J=7.06 Hz, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d6 25 °C) δ 169.1

(C-2 of thiazole), 150.7, 147.6 (C-5 of thiazole), 139.9, 134.5, 133.5,

131.1, 129.2, 128.5, 127.5, 126.3, 125.5, 124.6, 120.9, 120.5, 119.8,

+

14.3 (CH₃), 12.4 (CH₃). MS m/z 489.00 (M+H)

.

4.1.6.11.

2-(2-((2E,3E)-4-(5-ethoxy-3-methyl-1-phenyl-

1H-pyrazol-4-yl)but-3-en-2-ylidene)hydrazinyl)-4-(4-

methoxyphenyl)thiazole (7l); Light Brownish Solid, Yield:77%

mp:194–197 °C; FTIR (ATR, vmax, cm-1): 3117.31 (N-H Str.),

3041.74 (Ar-H Str.), 2978.63 (C-H Str. of CH₃), 1612.09 (C = N Str.);

¹H NMR (400 MHz, CDCl₃25 °C) δ 11.29 (brs, 1H, NH), 7.70–7.66

(m, 4H, ArH), 7.46–7.42 (m, 2H, ArH), 7.33–7.29 (m, 1H, ArH), 7.00–

6.69 (m, 2H, ArH), 6.86 (d, J = 16.84 Hz, 1H, Ar-HC=CH-), 6.74 (d,

J = 16.84 Hz, 1H, Ar-HC=CH-), 6.58 (s, 2H), 3.98 (q, J = 7.12 Hz,

2H, CH₂), 3.84 (s, 3H, OCH₃), 2.42 (s, 3H, CH₃), 2.33 (s, 3H, CH₃),

1.29 (t, J = 7.05 Hz, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃25 °C) δ

169.0 (C-2 of thiazole), 160.9, 155.9, 151.8, 147.9 (C-5 of thiazole),

142.6, 138.2, 129.2, 127.3, 127.1, 125.4, 124.8, 122.5, 122.3, 121.4,

114.9, 105.2, 71.1 (CH₂), 55.5 ₊(OCH₃), 15.3 (CH₃), 14.5 (CH₃), 13.3

119.6, 105.2, 103.9, 70.8 (CH₂), 14.9 (CH₃), 13.8 (CH₃), 11.9 (CH₃).

+

MS m/z 480.00 (M+H)

.

4.1.6.16.

4-(4-bromophenyl)-2-(2-((2E,3E)-4-(1-(3-

chlorophenyl)-5-ethoxy-3-methyl-1H-pyrazol-4-yl)but-3-en-2-

ylidene)hydrazinyl)thiazole (8b); Brownish Solid, Yield: 71%,

mp: 198–201 °C; FTIR (ATR, vmax, cm-1): 3116.74 (N-H Str.),

2984.47 (Ar-H Str.), 2924.74 (C-H Str. of CH₃), 1616.80 (C = N

Str.); ¹H NMR (400 MHz, DMSO) d 11.40 (brs, 1H, NH), 7.81 (d,

J=8.36 Hz, 2H, ArH), 7.73 (s, 1H, ArH), 7.68 (d, J=7.22 Hz, 1H,

ArH), 7.59 (d, J=8.38 Hz, 2H, ArH), 7.53 (d, J=8.14 Hz, 1H, ArH),

7.40 (d, J=7.95 Hz, 1H, ArH), 7.35 (d, J=4.91 Hz, 1H, ArH), 6.72 (d,

J=16.93 Hz, 1H, Ar-HC=CH-), 6.64 (d, J=16.78 Hz, 1H, Ar-HC=CH-),

4.05 (q, J=7.02 Hz, 2H, CH₂), 2.31 (s, 3H, CH₃), 2.14 (s, 3H, CH₃),

1.27 (t, J=6.95 Hz, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆25 °C)

δ 169.1 (C-2 of thiazole), 150.6, 148.8, 147.6 (C-5 of thiazole), 139.0,

133.8, 133.4, 131.5, 131.0, 129.1, 127.5, 126.3, 124.5, 123.4, 120.9,

120.5, 120.4, 119.6, 115.1, 105.2, 104.8, 70₊.8 (CH₂), 14.9 (CH₃), 13.8

(CH₃). MS m/z 474.00 (M+H)

.

4.1.6.12.

4-(4-chlorophenyl)-2-(2-((2E,3E)-4-(5-

ethoxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)but-3-en-2-

ylidene)hydrazinyl)thiazole (7 m); Light Brownish Solid, Yield:

79%, mp: 135–137 °C; FTIR (ATR, vmax, cm-1): 3234.58 (N-H Str.),

3112.25 (Ar-H Str.), 2977.59 (C-H Str. of CH₃), 1597.50 (C = N Str);

¹H NMR (400 MHz, DMSO-d₆25 °C) δ 10.12 (brs, 1H, NH), 7.85

(d, J = 8.28 Hz, 2H, ArH), 7.65 (d, J = 7.36 Hz, 2H, ArH), 7.50

(t, J = 7.89 Hz, 2H, ArH), 7.44 (d, J = 8.65 Hz, 2H, ArH), 7.34 (t,

J = 7.13 Hz, 1H, ArH), 7.28 (s, 1H, ArH), 6.69 (d, J = 16.67 Hz,

1H, Ar-HC=CH-), 6.64 (d, J = 16.67 Hz, 1H, Ar-HC=CH-), 3.98 (q,

J = 7.04 Hz, 2H, CH₂), 2.30 (s, 3H, CH₃), 2.12 (s, 3H, CH₃), 1.23

(t, J = 7.13 Hz, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆25 °C) δ

169.9 (C-2 of thiazole), 150.7, 149.4, 148.9, 147.2(C-5 of thiazole),

138.0, 133.8, 132.0, 129.4, 128.8, 127.4, 127.0, 121.9, 120.7, 105.0,

(CH₃), 11.9 (CH₃). MS m/z 556.00 (M+H)

.

4.1.6.17. 2-(2-((2E,3E)-4-(1-(3-chlorophenyl)-5-ethoxy-3-

methyl-1H-pyrazol-4-yl)but-3-en-2-ylidene)hydrazinyl)-4-(4-

ﬂuorophenyl)thiazole (8c); Brownish solid, Yield: 81%, mp:

195–198 °C; FTIR (ATR, vmax, cm-1): 3221.52 (N-H Str.), 3094.49

(Ar-H Str.), 2982.67 (C-H Str. of CH₃), 1618.01 (C = N Str.); ¹H

NMR (400 MHz, DMSO-d₆25 °C) δ 7.90 (m, J=3.55 Hz, 2H,

ArH), 7.73 (t, J=1.93 Hz, 1H, ArH), 7.68 (m, J=1.82 Hz, 1H, ArH),

7.59 (q, J=3.44 Hz, 1H, ArH), 7.41 (q, J=3.03 Hz, 1H, ArH), 7.26

(t, J=4.01 Hz, 2H, ArH), 7.22 (d, J=8.87 Hz, 2H, ArH), 6.73 (d,

J=16.60 Hz, 1H, Ar-HC=CH-), 6.65 (d, J=16.70 Hz, 1H, Ar-HC=CH-

),), 4.05 (q, J=7.02 Hz, 2H, CH₂), 2.32 (s, 3H, CH₃), 2.14 (s, 3H,

CH₃), 1.28 (t, J=7.01 Hz, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d6

25 °C) δ 169.2 (C-2 of thiazole), 162.8, 160.3, 150.7, 147.6 (C-5

104.6, 70.7 (CH₂), 30.8 (CH₃), 15.1 (CH₃), 13.9 (CH₃), 12.1 (CH₃).

+

MS m/z 477.00 (M+H)

.

4.1.6.13. 2-(2-((2E,3E)-4-(5-ethoxy-3-methyl-1-phenyl-1H-pyrazol-

4-yl)but-3-en-2-ylidene)hydrazinyl)-4-(p-tolyl)thiazole (7n); Brown-

ish Solid, Yield:63% mp:125–128 °C; FTIR (ATR, vmax, cm-1):

3306.52 (N-H Str.), 3115.66 (Ar-H Str.), 2977.50 (C-H Str. of CH₃),

1592.79 (C = N Str.) ¹H NMR (400 MHz, CDCl₃, 25 °C), δ 8.71

(s, 1H, NH), 7.69 (q, J=5.63 Hz, 4H, ArH), 7.44 (m, J=3.96 Hz, 2H,

ArH), 7.30 (m, J=3.99 Hz, 1H, ArH), 7.19 (d, J=7.92 Hz, 2H, ArH),

6.82 (s, 1H, ArH), 6.79 (d, J=16.72 Hz, 1H, Ar-HC=CH-), 6.64 (d,

J=16.72 Hz, 1H, Ar-HC=CH-), 3.99 (q, J=7.06 Hz, 2H, CH₂), 2.41 (s,

3H, CH₃), 2.36 (s, 3H, CH₃), 2.05 (s, 3H, CH₃), 1.30 (t, J=7.07 Hz, 3H,

13

V.A. Obakachi, N.D. Kushwaha, B. Kushwaha et al.

Journal of Molecular Structure 1241 (2021) 130665

of thiazole), 139.0, 133.5, 131.2, 130.9 (d,

J

=

10.96 Hz, 1C),

4.1.6.22.

2-(2-((2E,3E)-4-(1-(3-chlorophenyl)-5-ethoxy-3-

129.2, 128.3, 127.5 (d, J = 8.13 Hz, 1C), 126.3, 124.8, 124.5, 123.4,

120.7 (d, J = 38.48 Hz, 1C), 119.7 (d, J = 24.30 Hz, 1C), 115.4 (d,

methyl-1H-pyrazol-4-yl)but-3-en-2-ylidene)hydrazinyl)-4-(3,4-

dimethoxyphenyl)thiazole (8 h); Brownish Solid, Yield: 68%, mp:

203–206 °C; FTIR (ATR, vmax, cm-1): 3246.20 (N-H Str.), 3077.26

(Ar-H Str.), 2901.07 (C-H Str. of CH₃), 1608.77 (C = N Str.); ¹H

NMR (400 MHz, DMSO-d₆25 °C) δ 11.13 (s, 1H, NH), 7.67 (d, J

=7.80 Hz, 2H, ArH), 7.51 (t, J =7.80 Hz, 2H, ArH), 7.38 (m, 3H,

ArH), 7.19 (s, 1H, ArH), 6.99 (d, J =8.37 Hz, 1H, ArH), 6.77 (d, J

=16.66 Hz, 1H, Ar-HC=CH-), 6.67 (d, J =16.66 Hz, 1H, Ar-HC=CH-),

4.01 (q, J =6.99 Hz, 2H, CH₂), 3.79 (d, J =11.77 Hz, 6H, OCH₃), 2.32

(s, 3H, CH₃), 2.17 (s, 3H, CH₃), 1.25 (t, J =7.02 Hz, 3H, CH₃); ¹³C

NMR (100 MHz, DMSO-d6 25 °C) δ 162.4 (C-2 of thiazole), 159.5,

151.2 (d, J = 8.80 Hz, 1C), 150.8, 148.8(d, J = 4.37 Hz, 1C), 148.0,

138.2(C-5 of thiazole), 138.1, 134.3, 129.0, 126.7, 126.3, 121.7, 121.6,

J = 21.37 Hz, 1C), 115.1, 105.2, 103.6, 70.8 (CH₂), 14.9 (CH₃), 13.8

+

(CH₃), 11.9 (CH₃). MS m/z 496.00 (M+H)

.

4.1.6.18.

4-(4-chlorophenyl)-2-(2-((2E,3E)-4-(1-(3-

chlorophenyl)-5-ethoxy-3-methyl-1H-pyrazol-4-yl)but-3-en-2-

ylidene)hydrazinyl)thiazole (8d); Light Brownish Solid, Yield: 74%

mp: 187–190 °C; FTIR (ATR, vmax, cm-1): 3084.66 (Ar-H Str.),

2982.50 (C-H Str. of CH₃), 1616.31 (C = N Str.); ¹H NMR (400 MHz,

DMSO-d₆25 °C) δ 11.29 (brs, 1H, NH), 7.89 (d, J=8.52 Hz, 2H, ArH),

7.75 (t, J=1.91 Hz, 1H, ArH), 7.60 (s, 1H, ArH), 7.55 (t, J=8.13 Hz,

1H, ArH), 7.47 (d, J=8.64 Hz, 2H, ArH), 7.42 (d, J=7.12 Hz, 1H, ArH),

7.38 (s, 1H, ArH), 6.74 (d, J=16.91 Hz, 1H, Ar-HC=CH-), 6.66 (d,

J=16.68 Hz, 1H, Ar-HC=CH-), 4.29 (s, 2H, CH₂), 2.34 (s, 3H, CH₃),

2.17 (s, 3H, CH₃), 1.30 (t, J=7.01 Hz, 3H, CH₃); ¹³C NMR (100 MHz,

DMSO-d6 25 °C) δ 169.5 (C-2 of thiazole), 150.9, 149.2, 147.9 (C-5

of thiazole), 139.2, 133.7, 133.6, 132.7, 132.1, 131.2, 128.8, 127.4,

118.0, 112.4, 111.7, 109.2, 109.0, 68.7 (CH₂), 55.3 (d, J = 24.2 Hz, 1C,

+

OCH₃), 14.6 (CH₃), 13.3 (CH₃), 13.1 (CH₃). MS m/z 538.00 (M+H)

.

4.2. Molecular docking and simulation

126.5, 121.6, 121.3, 121.1, 120.8, 120.1, 119.8, 105.4, 104.9, 71.0

+

(CH₂), 15.1 (CH₃), 13.9 (CH₃), 12.1(CH₃). MS m/z 512.00 (M+H)

4.1.6.19.

methyl-1H-pyrazol-4-yl)but-3-en-2-ylidene)hydrazinyl)thiazol-4-

.

4.2.1. Proteins (COVID-19 s_gpand hACE2) acquisition and preparation

2-(2-(2-((2E,3E)-4-(1-(3-chlorophenyl)-5-ethoxy-3-

The X-ray crystal structures of the SARS-CoV-2 S_gp(PDB code:

6LZG) and hACE2 (PDB code: 1R4l) were obtained from the RSCB

Protein Data Bank [36,37]. The two proteins’ structures were then

prepared on the UCSF Chimera software package [38]. The struc-

tures of the proteins were prepared by removing water molecules,

nonstandard naming, protein residue connectivity. The missing

atoms of sidechains and protein backbone were added to the pro-

tein structure before the molecular docking. The standard drugs

cefoperazone (CFZ) and MLN-4760 were accessed from PubChem

[39]. The 3-D structures of the pyrazolone compounds and the

standard drugs were prepared on the Avogadro software package

[40].

yl)-4-ﬂuorophenol (8e); Brownish Solid, Yield: 66%, mp: 199–

202 °C; FTIR (ATR, vmax, cm-1): 3073.91 (Ar-H Str.), 2895.89 (C-H

Str. of CH₃), 1600.42 (C = N Str.); ¹H NMR (400 MHz, DMSO-d₆

25 °C) δ 11.26 (brs, 1H), 10.12 (s, 1H, OH), 7.72 (s,1 H, ArH), 7.67

(d, J=1.96 Hz, 1H, ArH), 7.63 (q, J=4.26 Hz, 2H, ArH), 7.56 (s, 1H),

7.52 (d, J=8.14 Hz, 1H, ArH), 7.48 (d, J=4.35 Hz, 1H, ArH), 7.40

(d, J=7.30 Hz, 1H, ArH), 6.99 (q, J=5.56 Hz, 1H, ArH), 6.89 (t,

J=4.43 Hz, 1H, ArH), 6.76 (d, J=16.63 Hz, 1H, Ar-HC=CH-), 6.64 (d,

J=16.68 Hz, 1H, Ar-HC=CH-), 4.04 (q, J=6.99 Hz, 2H, CH₂), 2.31

(s, 3H, CH₃), 2.15 (s, 3H, CH₃), 1.26 (t, J=7.00 Hz, 3H, CH₃); ¹³C

NMR (100 MHz, DMSO-d6 25 °C) δ 168.2 (C-2 of thiazole), 154.2,

151.4, 147.7 (C-5 of thiazole), 139.0, 138.5, 133.5, 131.0, 127.1, 124.6,

4.2.2. Molecular docking

123.4, 120.9, 119.6, 115.0, 112.8, 105.6, 105.1, 70.8 (CH₂), 15.0 (CH₃),

+

Autodock available on Chimera was used for molecular dock-

ing [41] with default docking parameters. Before docking, Gasteiger

charges were added to the molecules, and the non-polar hydrogen

atoms were merged into carbon atoms. The molecules were then

docked into the proteins’ binding pocket, COVID-19 S_gpand hACE2,

13.8 (CH₃), 12.0 (CH₃). MS m/z 512.00 (M+H)

4.1.6.20.

.

2-(2-((2E,3E)-4-(1-(3-chlorophenyl)-5-ethoxy-3-

methyl-1H-pyrazol-4-yl)but-3-en-2-ylidene)hydrazinyl)-4-(4-

nitrophenyl)thiazole (8f); Brownish Solid, Yield: 64%, mp: 197–

200 °C; FTIR (ATR, vmax, cm-1): 3087.80 (Ar-H Str.), 2978.29 (C-H

Str. of CH₃), 1620.73 (C = N Str.); ¹H NMR (400 MHz, DMSO-d₆

25 °C) δ 11.66 (brs, 1H, NH), 8.27 (d, J=8.91 Hz, 2H, ArH), 8.11

(d, J=8.86 Hz, 2H, ArH), 7.72 (t, J=1.87 Hz, 1H, ArH), 7.68–7.66

(m, 2H, ArH), 7.54 (t, J=8.16 Hz, 1H, ArH), 7.40 (d, J=6.87 Hz, 1H,

ArH), 6.73 (d, J=16.76 Hz, 1H, Ar-HC=CH-), 6.64 (d, J=16.69 Hz,

1H, Ar-HC=CH-), 4.05 (q, J=7.00 Hz, 2H, CH₂), 2.31 (s, 3H, CH₃),

2.14 (s, 3H, CH₃), 1.27 (t, J=7.00 Hz, 3H, CH₃); ¹³C NMR (100 MHz,

DMSO-d6 25 °C) δ 169.8 (C-2 of thiazole), 150.9, 149.3, 148.7, 147.9

(C-5 of thiazole), 146.3, 140.9, 139.2, 133.7, 131.2, 127.5, 126.5,

˚

by deﬁning the grid box with a spacing of 1 A each and size and

(64 × 108 × 46) and (78 × 102 × 90) pointing in x, y and z di-

rections, respectively. A duplicate docking and of the The standard

drug systems and all molecules with the two best docking scores

(for duplication) were then subjected to molecular dynamics sim-

ulations.

4.2.3. Molecular dynamic (MD) simulations

The MD simulation was performed as described by Idowu et al.

[29]. The simulations were performed using the GPU version pro-

vided with the AMBER package (AMBER 18), in which the FF18SB

variant of the AMBER force ﬁeld (Nair and Miners, 2014) was used

to describe the systems.

124.3, 121.1, 120.9, 119.8, 109.0, 105.3, 71.0 (CH₂), 15.1 (CH₃), 14.0

+

(CH₃), 12.1(CH₃). MS m/z 523.00 (M+H)

4.1.6.21.

.

2-(2-((2E,3E)-4-(1-(3-chlorophenyl)-5-ethoxy-3-

methyl-1H-pyrazol-4-yl)but-3-en-2-ylidene)hydrazinyl)-4-(p-

tolyl)thiazole (8 g); Brownish Solid, Yield: 73%, mp: 200–203 °C;

FTIR (ATR, vmax, cm-1): 3088.16 (Ar-H Str.), 2980.80 (C-H Str. of

CH₃), 1615.82 (C = N Str.); ¹H NMR (400 MHz, CDCl₃, 25 °C), δ

11.10 (brs, 1H, NH), 7.76 (s, 1H, ArH), 7.61 (d, J=7.85 Hz, 3H, ArH),

7.56 (d, J=8.07 Hz, 3H, ArH), 7.35 (t, J=8.07 Hz, 1H, ArH), 7.23 (s,

3H, ArH), 6.76 (d, J=16.00 Hz, 1H, Ar-HC=CH-), 6.67 (d, J=16.01 Hz,

1H, Ar-HC=CH-), 4.01 (q, J=7.03 Hz, 2H, CH₂), 2.39 (s, 3H, CH₃),

2.36 (s, 3H, CH₃), 2.30 (s, 3H, CH₃), 1.31 (t, J=7.06 Hz, 3H, CH₃);

¹³C NMR (100 MHz, CDCl_3,25 °C), δ 169.0 (C-2 of thiazole), 151.3

(d, J=24.27 Hz, 1C), 148.1, 147.6 (C-5 of thiazole), 138.4, 137.5,

132.2, 129.2 (d, J=23.33 Hz, 1 C), 126.8 (d, J=12.07 Hz, 1 C), 125.8,

122.2, 121.1, 105.4, 102.9, 70.8 (CH₂), 21.3₊(CH₃), 15.3 (CH₃), 14.3

ANTECHAMBER was used to generate atomic partial charges for

the ligand by utilizing the Restrained Electrostatic Potential (RESP)

and the General Amber Force Field (GAFF) procedures. The Leap

module of AMBER 18 allowed for the addition of hydrogen atoms

and Cl⁻and Na⁺counter ions to COVID-19 S_gpand hACE2, respec-

tively, to neutralize all systems. The systems were then suspended

implicitly within an orthorhombic box of TIP3P water molecules

˚

such that all atoms were within 8 A of any box edge [42].

An initial minimization of 2000 steps was carried out with

an applied restraint potential of 500 kcal/mol for both solutes.

They were performed for 1000 steps using the steepest descent

method followed by 1000 steps of conJugate gradients. An addi-

tional full minimization of 1000 steps was further carried out us-

ing the conJugate gradient algorithm without restraint. A gradual

(CH₃), 11.01 (CH₃). MS m/z 492.00 (M+H)

.

14

V.A. Obakachi, N.D. Kushwaha, B. Kushwaha et al.

Journal of Molecular Structure 1241 (2021) 130665

heating MD simulation from 0 K to 300 K was executed for 50 ps,

such that the systems maintained a ﬁxed number of atoms and

ﬁxed volume. The systems’ solutes were imposed with a poten-

tial harmonic restraint of 10 kcal/mol and collision frequency of

1.0 ps. Following heating, an equilibration estimating 500 ps of

each system was conducted; the operating temperature was kept

constant at 300 K. Additional features such as several atoms and

pressure were also held constant, mimicking an isobaric-isothermal

ensemble. The system’s pressure was maintained at 1 bar using the

Berendsen barostat (Gonnet, 2007; Basconi and Shirts, 2013).

The total time for the MD simulations conducted was 100 ns.

In each simulation, the SHAKE algorithm was employed to con-

strict hydrogen atoms’ bonds [43]. The step size of each simulation

was 2fs, and an SPFP precision model was used. The simulations

coincided with the isobaric-isothermal ensemble (NPT), with ran-

domized seeding, the constant pressure of 1 bar maintained by the

Berendsen barostat a pressure-coupling constant of 2 ps, a temper-

ature of 300 K and Langevin thermostat with a collision frequency

of 1.0 ps [42–44].

Credit author statement

Vincent A. Obakachi and Narva Deshwar Kushwaha synthe-

sized the compounds and drafted the manuscript. Babita Kush-

waha contributed to the data analysis and experimental writ-

ing. Idowu Kehinde performed in-silico analysis and assisted with

manuscript drafting. Mavela Cleopus Mahlalela and Suraj Raosaheb

Shinde contributed to the data analysis and manuscript drafting.

Rajshekhar Karpoormath designed the experiments and supervised

the project.

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

Vincent A. Obakachi: Conceptualization, Investigation, Method-

ology, Writing - original draft. Narva Deshwar Kushwaha: Con-

4.2.4. Post-Dynamic analysis

ceptualization, Investigation, Methodology, Writing

- original

Analysis of root mean square deviation (RMSD), root means

square ﬂuctuation (RMSF) Solvent accessible surface area (SASA),

Hydrogen bond (Hbond) and radius of gyration (RoG) was done us-

ing the CPPTRAJ module employed in the AMBER 18 suit. All aver-

age raw data plots were generated using the Origin data analysis

software [45].

draft. Babita Kushwaha: Visualization, Investigation, Data cu-

ration. Mavela Cleopus Mahlalela: Writing - review & editing.

Suraj Raosaheb Shinde: Writing - review & editing. Idowu Ke-

hinde: Writing - original draft, Software. Rajshekhar Karpoor-

math: Supervision, Project administration.

Acknowledgments

4.2.5. Binding free energy calculations

To estimate and compare the systems’ binding aﬃnity, the free

binding energy was calculated using the Molecular Mechanics/GB

Surface Area method (MM/GBSA) (Ylilauri and Pentikäinen, 2013).

Binding free energy was averaged over 100,000 snapshots ex-

tracted from the 100 ns trajectory. The free binding energy (ꢀG)

computed by this method for each molecular species (complex, lig-

and, and receptor) can be represented as:

The authors are thankful to the Discipline of Pharmaceutical

Sciences, College of Health Sciences, University of KwaZulu-Natal

(UKZN), Durban, South Africa, for providing all the necessary facil-

ities. R.K. gratefully acknowledges National Research Foundation-

South Africa (NRF-SA) for funding this project (Grant Nos. 103728

and 1 12079). The authors would also like to acknowledge fellow

UKZN colleagues Dr. Vuyisa Mzozoyana (NMR spectroscopy).

ꢀG_bind= G_complex− G_receptor− G

1

_ligand( )

Supplementary materials

Supplementary material associated with this article can be

found, in the online version, at doi:10.1016/j.molstruc.2021.130665.

ꢀG_bind= E_gas+ G − TS

sol

2

( )

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Design and synthesis of pyrazolone-based compounds as potent blockers of SARS-CoV-2 viral entry into the host cells

DOI: 10.1016/j.molstruc.2021.130665

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