Design, synthesis and biological evaluation of novel 5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazole-3-thiols as an anticancer agent

Journal of Molecular Structure 1231 (2021) 130000

Contents lists available at ScienceDirect

Journal of Molecular Structure

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

Design, synthesis and biological evaluation of novel

5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazole-3-thiols as an

anticancer agent

Krupa R. Patel^a, Jpan G. Brahmbhatt^b, Pranav A. Pandya^a, Drashti G. Daraji^a,

Hitesh D. Patel^a,∗, Rakesh M. Rawal^b, Sujit K. Baran^c

^aDepartment of Chemistry, School of Sciences, Gujarat University, Ahmedabad-380009, Gujarat, India

^bDepartment of Life Science, School of Sciences, Gujarat University, Ahmedabad-380009, Gujarat, India

^cDepartment of Chemistry, The M. S. University of Baroda, Vadodara-390002, Gujarat, India

a r t i c l e i n f o

a b s t r a c t

Article history:

Cellular tumor antigen p53 is signiﬁcant for cancer prevention and its mutation is most documented ge-

nomic change in human cancers. Thus, restoration of p53 function by interruption of the p53-MDM2

interaction opens up a prospect for a nongenotoxic anticancer therapeutic strategy. A novel series of

molecules comprising 1,2,4-triazole-3-thiol scaffold were successfully discovered by structure-based de-

signing approach. In silico modules predicted that 5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazole-3-thiol

derivatives have draggability and ability to mimic critical binding residues of p53. All target compounds

were assayed for their in vitro antiproliferative activity against A549, U87 and HL60 cell lines. Twelve out

of sixteen compounds exhibited good in vitro inhibitory activity in micromolar range. Especially, com-

pound 6h possessed acute antitumor activity with IC₅₀values 3.854, 4.151 and 17.522 μM against three

tested cell lines. It represents as a promising lead for further optimization and a template for develop-

ment of novel antitumor agents.

Received 27 October 2020

Revised 13 December 2020

Accepted 18 January 2021

Available online 28 January 2021

Keywords:

Structure-based design

1,2,4-triazole-3-thiol

Single crystal X-ray diffraction

Molecular docking and dynamics

In silico ADME

In vitro antiproliferative activity

1. Introduction

stress, the protein level of p53 is elevated and leads to the tran-

scription of multiple downstream target genes implicated to cell

Cancer is the rapid creation of abnormal cells that grow beyond

their usual boundaries, which can then invade adjoining parts of

the body and spread to other organs. According to World Health

Organization (WHO), cancer is the second leading cause of mor-

bidity and mortality, and is responsible for an estimated 9.6 mil-

lion deaths in 2018 worldwide [1]. Despite the use of conventional

chemotherapy and newly approved targeted therapies, cancer still

represents an unachieved medical need [2-4]. An ideal therapy

would be non-genotoxic and the one, which control the instinc-

tive mechanism of cancer cell to annihilate tumor growth, such as

activating a dormant apoptosis pathway.

cycle arrest, apoptosis, DNA repair and senescence [7]. As per Inter-

national Agency for Research on Cancer (IARC) TP53 database, TP53

gene encoding p53 protein is deleted or mutated at DNA binding

domain in approximately half of the human cancers, which facil-

itates carcinogenesis [8]. In the other half of cancer tissues, p53

expresses wild-type status but its tumor suppressor function is ef-

fectively impaired by overexpression or ampliﬁcation of oncopro-

tein MDM2 (HDM2 in humans). MDM2 inhibits the antiprolifera-

tive transcription activity of p53 by multiple mechanisms: 1) di-

rect binding to the N-terminal transactivation domain, 2) ubiqui-

tination via E3 ligase activity to trigger proteasomal degradation,

and 3) promoting the export of p53 from the nucleus to the cyto-

plasm [9].

The tumor suppressor p53 protein is a transcriptional factor

plays a momentous role in maintaining genetic integrity, so named

as “guardian of genome” [5]. p53 protein is very unstable in nor-

mal unstressed cell and its low cellular concentration is sustained

by negative regulatory protein Murine Double Minute 2 (MDM2)

through continuous degradation [6]. In response to various cellular

The co-crystal structure of MDM2 bound to a short peptide of

p53 clearly disclose the presence of a deep hydrophobic cleft on

the surface of the MDM2 molecule on which the p53 protein binds

as a helix. Steric complementarity between the MDM2 and the hy-

drophobic face of the p53 relies on three amino acid residues from

the α-helix of p53 peptide: Phe19, Trp23 and Leu26 [10]. These

amino acid residues have extended conformations and pack closely

∗

Corresponding Author: Tel. No. 07926300969.

E-mail address: drhiteshpatel1@gmail.com (H.D. Patel).

https://doi.org/10.1016/j.molstruc.2021.130000

K.R. Patel, J.G. Brahmbhatt, P.A. Pandya et al.

Journal of Molecular Structure 1231 (2021) 130000

.

Scheme 1. Synthesis of compound 6a-6e. Reagents and conditions: (i) NH₂-NH₂H₂O (Hydrazine monohydrate), Ethanol, 25°C; (ii) pyridine, 25°C; (iii) NaOH/MeOH (Methyl

Alcohol), reﬂux; (iv) TEA (Triethyl Amine), THF (Tetrahydrofuran), reﬂux.

Scheme 2. Synthesis of compound 6f-6p. Reagents and conditions: (i) CAC (Chloroacetyl chloride), TEA, MDC (Methylene dichloride), 25°C; (ii) TEA, THF, reﬂux.

to each other, making sequential and immense van der Waals

interactions with the surface of MDM2. Protein-Protein Interac-

tions (PPIs) usually involves large and ﬂat surfaces with lack of

deep and well-deﬁned binding pockets. However, p53-MDM2 com-

plex has well-structured, deep and druggable hydrophobic binding

site. Reactivation of p53 tumor suppressive function by interrupt-

ing p53-MDM2 protein-protein interaction with small molecule in-

hibitors is explored as most promising, non-genotoxic and attrac-

tive therapeutic strategy for the treatment of a broad spectrum

of cancers [1 1 -13]. In last few decades, numerous novel classes

of small molecule inhibitors i.e imidazolines (nutlins) [14], ben-

zodiazepines [15], spiro-oxindoles [16], piperazines [1 7 ], pyrroli-

done [18], aminothiophene [19], tetrazoles [20], etc. have been

shown to antagonise the MDM2-p53 interaction. Among them,

some highly potent MDM2 inhibitors RG7112 [21], RG7388 [22],

SAR405838 [23], AMG 232 [24], MK-8242 [25], CGM-097 [26],

DS-3032b [27] and HDM201 [28] have been advanced in clinical

trials.

2. Result and discussion

2.1. Design

A widely accepted three-ﬁnger pharmacophore model for de-

signing of p53-MDM2 inhibitors was taken into consideration,

since it was based on strategy to mimic three crucial hydropho-

bic pockets: Phe19, Trp23 and Leu26 [32]. Five membered rings

were evaluated for core heterocycles as they are more ﬂexible

by pseudo-rotational mobility compared to six membered rings.

Among them triazole was selected as heterocyclic core, being

abundantly found in marketed drugs and isostere of imidazole (a

potent core of nutlins). For further designing we have retained

pharmacophoric features of previously reported potent p53-MDM2

inhibitors: 1) two cis-phenyl groups to ensure the hydrophobic

interactions, and 2) the functional groups (mostly halogens [33])

that are capable of forming hydrogen-bonding interactions. Thou-

sands of enumerated structures incorporating above mention re-

quirements, were generated with the help of Marvin sketch. Opti-

mization was carried out by Computer-Aided Drug Design (CADD)

software Schrödinger. An approach for optimization involves: 1)

Molecular docking simulation with Simple Precision (SP) followed

by Extra Precision (XP), and 2) In silico ADME prediction. The se-

lection process was based on their docking scores, scaffold diver-

sity, synthesizability, pharmacokinetic proﬁle and most importantly

their docking poses. In selected potential hits, triazole residue oc-

cupied the central part and served as a hub providing appropriate

substitution vectors to access the three hydrophobic sub-pockets.

Heterocyclic compounds possess nitrogen as a hetero atom are

extensively found in therapeutic agents. Triazoles and their fused

heterocyclic derivatives has become the most fortunate structures

in the ﬁeld of medicine due to wide spectrum of biological ac-

tivities, low toxicity, good pharmacokinetic and pharmacodynamic

proﬁles [29]. Out of the two isomeric forms, 1,2,4-triazoles are in-

vestigated to be more important isomer pharmacologically [30].

Triazole heterocycles which incorporate sulphur in a form of mer-

capto and thione substitution shows more potency compared to

parent derivatives [31]. 1,2,4-triazole ring is also part of mar-

keted anticancer drugs i.e. Vorozole, Letrozole, Anastrozole used

for treatment of breast cancer. But, 1,2,4-triazole-3-thiol derivatives

have not been reported for their anticancer potential especially as

MDM2 antagonist.

2.2. Chemistry

A general synthetic pathway for designed 5-(4-chlorophenyl)-

Herein, we report design and synthesis of a novel series of

1,2,4-triazole-3-thiol derivatives and ﬁnd promising lead com-

pounds with high anticancer properties.

4-phenyl-4H-1,2,4-triazole-3-thiol derivatives (6a-6p) was en-

capsulated in scheme

1 and 2. Scaffold synthesis was ini-

tiated by reaction of isothiocyanatobenzene with slight ex-

2

K.R. Patel, J.G. Brahmbhatt, P.A. Pandya et al.

Journal of Molecular Structure 1231 (2021) 130000

Figure 1. Design strategy of the target compounds and schematic binding mode representation of 1,2,4-triazole-3-thiol scaffold with key p53 residues.

cess hydrazine hydrate to avoid dimerization of product N-

phenylhydrazinecarbothioamide (1). Synthetic procedure for scaf-

fold (3) from compound (1) was optimized by using various

combinations of solvent & base, and fetch up with highly eﬃ-

cient two-step process. In which, compound (1) undergoes re-

action with 4-chlorobenzoyl chloride in pyridine to give 2-(4-

chlorobenzoyl)-N-phenylhydrazinecarbothioamide intermediate (2).

Then compound (2) undergoes intramolecular condensation in re-

ﬂuxing methanolic NaOH (Sodium hydroxide) to give desired scaf-

fold 5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazole-3-thiol (3). The

scaffold (3) was identiﬁed by LCMS (Liquid Chromatography–Mass

Spectrometry) analysis and the crude was used in the next step

without further puriﬁcation.

Derivatization of scaffold to achieve both sets of products (6a-

6e) and (6f-6p), follows identical synthetic mechanism, which is

nucleophilic SN2 substitution reaction between thiol (-SH) and

methyl chloride derivatives (-CH₂Cl). As nucleophilic SN2 substi-

tution reactions were not favourable in polar protic solvents like

3

K.R. Patel, J.G. Brahmbhatt, P.A. Pandya et al.

Journal of Molecular Structure 1231 (2021) 130000

methanol & ethanol, we had performed model reaction in polar

aprotic solvent conditions like dichloromethane, tetrahydrofuran,

acetone and N,N-dimethylformamide. Model reaction optimization

was also performed in terms of different basic condition by us-

ing K₂CO₃(Potassium carbonate), Cs₂CO₃(Cesium carbonate), DBU

(1,8-Diazabicyclo[5.4.0]undec-7-ene) and triethylamine along with

temperature factor. After some optimization, we came up with

highly eﬃcient and versatile reaction condition in tetrahydrofuran

and triethylamine under reﬂuxing condition for 5-8 hours. Out of

two possible tautomeric forms of scaffold (3), the substitution re-

action took place on sulfur instead of nitrogen atom due to the

stronger nucleophilic activity of sulfur atom.

higher potencies than 3-(benzylthio)-5-(4-chlorophenyl)-4-phenyl-

4H-1,2,4-triazole derivatives (6a-6e) against all three tested cell

lines. The introduction of electron donating as well as electron

withdrawing substituents on para position of phenyl ring (6b-

6e) decreases potency of primarily hit (6a) except compound

6e. Furthermore, phenyl substitution at 2, 3 and 4 position of

compound 6f leads into dramatically increases and decreases

of antiproliferative activities. In spite of the difference in chlo-

rine position in compounds 6g, 6h and 6i, they presented acute

anti-cancer potencies. However, in case of ﬂuorine ortho position

(6j) expressed strong potency, meta position offered complete

inactivity and interestingly para position expressed good potency

only against U87 cells. Besides that, comparatively larger atom

of halogens bromine at para position appeared with quite good

anti tumor potency. Presence of ring activating methoxy group

(6d and 6o) triggers a potential reduction in parent unsubstituted

derivatives (6a and 6f). In contrast, strong ring deactivating nitro

group insertion resulted into one of the highly potent analogous

(6e) and least potent analogous (6n). Moreover, carboxylic group

substitution at para position reported moderate antiproliferative

activity.

Commercially available benzyl chloride derivatives (5a-5e) were

used to synthesize designed analogous (6a-6e) (see scheme-1).

Aromatic side chains (5f-5p) were synthesized from appropriate

amines (4f-4p) as per previously reported method [34], which was

further condensed with scaffold (3) to give targeted compounds

(6f-6p) (see scheme-2).

All the targeted molecules were characterized by ¹H NMR,

¹³C NMR and ESI-MS (Electrospray Ionization Mass Spectrometry)

analysis. The mass spectrum of 6a revealed that observed molec-

ular ion peaks were in agreement with the molecular weight of

respective compounds. ¹H NMR of compound 6a in DMSO-d6 at

400 MHz revealed that δ value 4.41 ppm corresponding to -CH₂- of

the molecule. Aromatic protons of three phenyl rings shows peaks

in deshielded region at 10.66 (s, 2H), 7.95 – 7.93 (m, 2H), 7.62 (d,

J = 4.6 Hz, 2H), 7.54 – 7.51 (m, 2H), 7.46 – 7.42 (m, 2H), 7.35 –

7.33 (m, 2H) and 7.30 (dt, J = 5.8, 1.4 Hz, 2H) δ value. Protons

of one of the phenyl rings are highly deshielded due to 4-chloro

substitution and attachment with 1,2,4-triazol. ¹³C NMR spectral of

6a also in good agreement with the structure assigned. In which,

only aliphatic carbon present at 36.15 δ ppm and C3 and C5 of

1,2,4-triazol ring appears at 164.80 and 153.43 δ ppm, respectively.

Compound 6a was recrystallized from methanol by vapor diffusion

and a high-resolution X-ray crystal structure was obtained that es-

tablished the absolute conformation (ﬁgure 2). [CCDC No. 2015884]

2.4. Docking

To elucidate the binding interaction between designed 1,2,4-

triazole-3-thiol derivatives and p53-MDM2 binding pocket, we

have conducted molecular docking studies utilizing Glide pro-

gramme of Schrödinger package [36]. The X-ray crystal struc-

ture of the MDM2 oncoprotein bound to the p53 tumor suppres-

sor transactivation domain (PDB ID:1YCR) was used as a recep-

tor for docking stimulation. The structure revealed that MDM2-

p53 interaction is mediated by a distinct hydrophobic surface

pocket in MDM2 and three key hydrophobic residues (Phe19,

Trp23, and Leu26) in p53 [10]. On the basis of the target analy-

sis, we presented a hypothetical binding model for 1,2,4-triazole-

3-thiol-MDM2 complex. Our docking model demonstrated that, 3-

(benzylthio)-5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazole deriva-

tives (6a-6e) own ‘U’ shape in its lowest energy conformation.

Where, both phenyl and 4-chloro phenyl substituents of triazole

lies perpendicular to each other and accommodated in Leu26

and Trp23 hydrophobic pockets, respectively (Figure 1). In addi-

tion, sulphur atom with its side chain were exposed to the sol-

vent. In contrast, 2-((5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazol-

3-yl)thio)-N-phenylacetamide derivatives (6f-6p) display ‘T’ shaped

stable conformation. Here, potent core 1,2,4-triazole remain into

Trp23 pocket whereas both perpendicular 4-chloro phenyl and

phenyl rings were directed towards hydrophobic Leu26 pocket and

hydrophilic solvent environment, respectively. A tail portion of ‘T’,

which is long polar side chain ending with phenyl substituents

were accounted into relatively larger hydrophobic pocket Phe19

(Figure 1). Numerous interrelationships between designed 1,2,4-

triazole-3-thiol derivatives and MDM2 protein are listed in tabu-

lar form (Table 2). Moreover, docking score, XP Gscore, glide score

and glide energy data of targeted molecules are available in sup-

plementary information.

2.3. In vitro antiproliferative activities and structure activity

relationships

The synthesized molecules 6a-6p were tested for their ef-

fect on the growth and viability of cultured cancer cells using

the standard MTT method [35]. The in vitro antiproliferative ac-

tivities of the 5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazole-3-thiol

derivatives were examined against three human cancer cell lines,

namely A549 (p53 wild type), U87 (p53 wild type) and HL60 (p53

null). The resulting cytostatic potency in terms of IC₅₀(the concen-

tration causing 50% inhibition of the tumor cell proliferation) val-

ues are summarized in table 1. In general, these compounds dis-

played moderate to high in vitro antitumor activity against lung

cancer A549 cell line, brain cancer U87 cell line and leukemia HL60

cell line in comparison to standard antitumor drug Cisplatin. Com-

pound 6h exhibited acute activity, with IC₅₀values 3.854, 4.151

and 17.522 μM against three tested cell lines A549, U87 and HL60,

respectively. In addition to that, compound 6g and 6i displayed

strong in vitro antitumor eﬃcacy against both wild type p53 cell

lines and null p53 cell line. Compound 6a, 6e, 6f and 6j also ex-

pressed better antitumor activity. Whereas, compound 6b, 6c, 6m,

6o and 6p were found to have moderate anti-cancer activity. Un-

fortunately, compound 6k and 6n presented poor antiproliferative

activity. Comprehensive result of cellular proliferation (MTT) assay

disclosed that synthesized derivatives show lower values of IC₅₀

against wild-type p53 cell lines compared to null p53 cell line.

The inhibitory activity comparison of these (6a-6p) synthesised

virtual hits validates the presumed binding model at many extend.

From these assessments of in silico binding mode and actual in

vitro inhibitory activities, we can justify potency of almost every

synthesized derivatives i.e. 1) 6a-6e derivatives were less potent

than 6f-6p because of inability to approach Phe19 pocket; 2) out of

6a-6e, only 6e expressed good potential due to addition π-cation

interaction between NO₂and Phe55; and 3) anti tumor potential of

6f-6p derivatives mostly depend upon 1,2,4-triazole position inside

Trp23 pocket and its interaction with alpha-helix of MDM2, deriva-

tives (6n and 6k) who lost this interaction reported as inactive

In terms of structure

activity relationships, 2-((5-

(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)-N-

phenylacetamide derivatives (6f-6p) exhibited comparatively

4

K.R. Patel, J.G. Brahmbhatt, P.A. Pandya et al.

Journal of Molecular Structure 1231 (2021) 130000

Table 1

Structure and in vitro antiproliferative activity of synthesized compounds

IC₅₀(μM)^a

Compound code

Product

A549

U87

HL60

(wild-type p53)

(null p53)

6a

15.813

11.205

20.423

6b

62.894

70.639

86.294

6c

37.030

43.865

63.610

82.331

23.696

6d

>100

6e

10.487

11.882

6f

10.140

14.830

17.341

6g

5.729

13.026

24.492

6h

3.854

4.151

17.522

(continued on next page)

5

K.R. Patel, J.G. Brahmbhatt, P.A. Pandya et al.

Journal of Molecular Structure 1231 (2021) 130000

Table 1 (continued)

Compound code

Product

IC₅₀(μM)^a

A549

U87

HL60

(wild-type p53)

(null p53)

6i

5.561

15.120

>100

8.802

6.620

>100

19.664

28.410

>100

6j

6k

6l

6m

6n

>100

20.696

>100

34.715

27.902

>100

50.739

n.a.

6o

70.690

87.691

>100

6p

51.520

33.801

71.099

37.296

88.440

23.633

Cis-platin^b

-

a

Each compound was tested in triplicate and the data are presented as the mean value

b

Standard antitumor drug

n.a. = not active

6

K.R. Patel, J.G. Brahmbhatt, P.A. Pandya et al.

Journal of Molecular Structure 1231 (2021) 130000

Figure 2. X-ray structure of compound 6a.

Table 2

Binding interactions of docked compounds with amino acid present in binding pocket

H-bond π- π interaction

Bond

Halogen bond

π-cation

Salt bridge

Comp. code

Atom of ligand

Amino

acid

Amino

Bond

Amino

Bond

length^a

Amino

Bond

length^a

Amino

Bond

length^a

acid

length^a

acid

6a

6b

6c

6d

6e

-

4-Cl Ph

-

PHE 55

5.27

5.16

5.10

-

GLN 59

2.67

-

4-Cl Ph

-

PHE 55

GLN 59

2.48

-

4-Cl Ph

-

PHE 55

GLN 59

1.79

LYS 24

5.08

-

N of NO₂

1,2,4-triazole

O of C=O

1,2,4-triazole

O of C=O

1,2,4-triazole

O of C=O

1,2,4-triazole

O of C=O

1,2,4-triazole

O of C=O

4-Cl Ph

-

PHE 55

4.18

-

6f

6g

6h

6i

-

PHE 55

5.21

-

LYS 24

1.85

-

PHE 55

5.19

-

LYS 24

1.85

-

PHE 55

5.21

-

LYS 24

1.86

-

PHE 55

5.19

-

LYS 24

1.85

2.69

2.03

-

6j

GLN 59

PHE 55

5.09

-

GLN 59

-

6k

6l

-

PHE 55

4.90

-

LYS 24

4.68

-

H of -NH

1,2,4-triazole

O of C=O

1,2,4-triazole

O of C=O

4-Cl Ph

GLU 28

1.63

-

PHE 55

5.21

-

LYS 24

1.86

-

6m

6n

-

PHE 55

5.21

-

LYS 24

1.83

-

PHE 55

5.26

5.20

-

4-NO₂Ph

N of NO₂

1,2,4-triazole

O of C=O

O of COOH

-

PHE 55

-

GLU 52

3.82

6o

-

PHE 55

5.22

-

LYS 24

GLN 59

1.84

2.06

-

6p

-

a

˚

Bond length in A.

anti-cancer agents. The 2D (2-dimensional) and 3D (3-dimensional)

view of ligand interaction diagram of potential hits 6g, 6h and 6i

in the active site of MDM2 are illustrated in Figure 3.

study due to its intrinsically dynamic nature. Thus, Molecular Dy-

namic (MD) simulations were performed at 50 ns using Desmond

to analyse stability and dynamic aspects of p53-MDM2 protein

complex and its molecular interactions with potent derivatives (6g,

6h and 6i). The overall statistical data for compound 6g, 6h and

6i after the analysis of the MD trajectories are explained with

ﬁgure 4, 5 and 6, respectively. Here, (A) the plot shows Root Mean

Square Deviation (RMSD) values of a protein (left Y-axis) and Lig-

2.5. Molecular dynamics

The Cognizance of protein-ligand binding mode is pivotal for

biomedical research, but the process had always been arduous to

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K.R. Patel, J.G. Brahmbhatt, P.A. Pandya et al.

Journal of Molecular Structure 1231 (2021) 130000

Figure 3. (A) 2D and 3D docked representation of compound 6g at the binding site of MDM2 protein. (B) 2D and 3D docked representation of compound 6h at the binding

site of MDM2 protein. (C) 2D and 3D docked representation of compound 6i at the binding site of MDM2 protein. (PDB ID:1YCR)

and (right Y-axis) to examine their stability over the course of sim-

ulation period; (B) the bar chart summarized protein-ligand inter-

actions over the course of the trajectory, where contacts are cat-

egorized into four types: hydrogen bonds, hydrophobic, ionic and

water bridges; and (C) a timeline representation of the contacts,

where top panel illustrates the total number of speciﬁc contacts

throughout the simulation period and the bottom panel portrays

particular residue interactions with the ligand in each trajectory

frame. Some residues make more than one speciﬁc contact with

the ligand, which is represented by a darker shade of orange.

8

K.R. Patel, J.G. Brahmbhatt, P.A. Pandya et al.

Journal of Molecular Structure 1231 (2021) 130000

Figure 4. Dynamics of compound 6g bound to p53-MDM2 complex. (A) RMSD of protein-ligand; (B) Protein-ligand contacts; (C) A timeline representation of the interactions

and contacts.

Figure 5. Dynamics of compound 6h bound to p53-MDM2 complex. (A) RMSD of protein-ligand; (B) Protein-ligand contacts; (C) A timeline representation of the interactions

and contacts.

Monitoring the RMSD values of three novel inhibitors conﬁrmed

the stability of ligands with respect to the protein and its bind-

ing pocket. Protein-ligand RMSD plots of 6g and 6h did not show

signiﬁcant ﬂuctuation, whereas protein-ligand RMSD plots of 6i

shows small amount of ﬂuctuation but that fall into perfectly ac-

ceptable order. Protein-ligand interaction charts for all three lig-

ands validates the vital contribution of the key docking interac-

tion for strong activity, which is with Phe55 amino acid residue

of MDM2 (chain A). Interaction fraction value corresponding to

Phe55 is almost 1.0 and above in all three cases, which means

this interaction retains for around 100% of simulation time by mak-

ing multiple contacts of same subtype with the ligand. Potent lig-

9

K.R. Patel, J.G. Brahmbhatt, P.A. Pandya et al.

Journal of Molecular Structure 1231 (2021) 130000

Figure 6. Dynamics of compound 6i bound to p53-MDM2 complex. (A) RMSD of protein-ligand; (B) Protein-ligand contacts; (C) A timeline representation of the interactions

and contacts.

Table 3

Predicted ADME parameters for synthesized compounds

Comp. code

M.W.^a

CNS^b

logPo/w^c

QPlogS^d

QPPCaco^e

QPlogBB^f

QPPMDCK^g

QPlogKp^h

QPlogKhsaⁱ

PHOA^j

ROF^k

6a

6b

6c

6d

6e

6f

377.89

412.336

395.881

407.917

422.888

420.915

455.361

438.906

499.812

465.913

450.942

464.925

1

5.867

6.41

-6.176

-6.745

-6.453

-7.359

-7.247

-7.43

3472.651

3480.942

3447.187

3146.355

392.829

0.226

0.37

7017.362

10000

-0.737

-0.897

-0.863

-0.665

-2.464

-1.202

-1.345

-1.35

1.137

1.244

1.177

1.282

1.179

1.027

0.923

0.931

1.088

1.072

1.061

0.903

1.168

0.772

0.821

0.594

100

1

0

1

0

1

6.19

0.318

0.046

-0.905

-0.47

10000

100

1

6.468

5.608

5.666

5.551

5.643

6.179

5.875

5.903

5.492

6.229

4.503

5.253

4.904

5946.671

658.825

2053.35

5694.351

7531.652

9377.904

3288.645

4032.756

5242.868

5318.32

351.678

3417.717

50.682

100

-1

0

93.256

100

1166.548

1667.234

1739.463

1951.231

1093.221

1243.664

1516.035

1147.169

216.53

6g

6h

6i

0

-6.348

-6.523

-7.746

-7.802

-7.745

-6.509

-8.311

-6.004

-5.857

-7.177

-0.043

0.011

-0.014

-0.408

-0.324

-0.103

-0.319

-1.101

-0.183

-1.694

100

1

100

0

-1.002

-1.375

-1.288

-1.412

-1.384

-3.062

-1.308

-3.252

100

6j

0

100

6k

6l

0

100

0

100

6m

6n

6o

6p

0

100

-2

0

95.114

100

1783.161

30.296

-2

82.171

Descriptor standard range:

a

M.W = Molecular weight (<500);

b

c

d

e

f

CNS = Central Nervous System activity [-2(inactive) to +2(active)];

logPo/w = Octanol-water partition co-eﬃcient (<5);

QPLogS = Aqueous solubility (-6.5 to 0.5)

QPPCaco = Caco-2 cell permeability (<25 poor,>500 great)

QPLogBB = Blood/Brain partition co-eﬃcient (-3.0 to 1.2);

QPPMDCK = MDCK cell permeability (<25 poor,>500 great);

QPlogKp = Skin permeability (-8.0 to -1.0);

g

h

i

QPlogKhsa = Human serum albumin binding (-1.5 to 1.5)

j

PHOA = Percentage of Human Oral Absorption (>80%-high, <25%-poor)

ROF = Rule of Five (<4).

k

ands reported to form various additional contacts with amino acid

residues of both MDM2 (chain A) and p53 (chain B) during MD

simulations, which were not observed in their molecular docking

poses. During the period of 50 ns simulations, all three potent

ligands interacted consistently with p53-MDM2 complex by 0-9

contacts of different kinds i.e hydrogen bonds, hydrophobic con-

tacts and water bridging bonds. Detailed graphical representation

of protein-ligand contacts with respect to interaction fraction and

simulation time are elucidated in ﬁgure 4, 5 and 6.

2.6. In silico ADME proﬁle

In conventional drug design process ADME tools were applied

at the terminal stage of the drug development pipeline, whereas in

10

K.R. Patel, J.G. Brahmbhatt, P.A. Pandya et al.

Journal of Molecular Structure 1231 (2021) 130000

modern process in silico ADME properties are predicted at an early

phase, to remove molecules with poor ADME properties which

leads to remarkable saving of cost and time in drug develop-

ment. In silico ADME models recognize structure-property relation-

ships and predict ADME (Absorption, Distribution, Metabolism, Ex-

cretion) properties from structure of compound. ADME properties

of titled compounds (6a-6p) were predicted by QikProp module

of Schrödinger and their pharmacophoric properties with accept-

able limits were represented in table 3. Among all the predicted

44 different physical and pharmaceutical relevant properties, we

have selected properties which known to inﬂuence metabolism,

cell permeation and bioavailability i.e. Molecular weight, Cen-

tral Nervous System activity (CNS), Octanol-water partition co-

eﬃcient (logPo/w), Aqueous solubility (QPLogS), Caco-2 cell per-

meability (QPPCaco), Blood/Brain partition co-eﬃcient (QPLogBB),

MDCK cell permeability (QPPMDCK), Skin permeability (QPlogKp),

Human serum albumin binding (QPlogKhsa), Percentage of human

oral absorption and Lipinski’s rule of 5.

cokinetic properties for this class of inhibitors, and the results will

be reported in future publications.

4. Experimental Section

4.1. Material and apparatus

Unless otherwise stipulated, all reactions were carried out in

standard commercially available glassware using standard syn-

thetic chemistry methods. Air-sensitive and moisture-sensitive re-

actions were performed under an atmosphere of nitrogen in oven

dried glassware. Solvents and chemicals used in the entire re-

action were purchased from commercial sources (Sigma Aldrich,

Merck, TIC, Spectrochem, Astron, Renkem, and Finar chemical com-

pany) and used without further puriﬁcation. Proton nuclear mag-

netic resonance (1H NMR) & Carbon nuclear magnetic resonance

(13C NMR) experiment were performed on Bruker Avance III HD

400MHz NMR equipped with a 5mm PABBO-19F Z Grd probe and

Topspin 3.2 software using DMSO-d6 as a solvent. Chemical shifts

(δ) were presented in terms of parts per million (ppm), relative

to residual solvent peaks as an internal standard set to δ 2.50

(1H NMR), 39.52 (13C NMR) ppm for DMSO-d6. Coupling constant

(J) were reported in Hertz. Splitting patterns of signal were doc-

umented as s = singlet, d = doublet, t = triplet, q = quartet,

m = multiplate, dd = double doublet, dt = doublet of triplets,

dq = doublet of quartets, ddd = doublet of doublet of doublets,

dtd = doublet of triplet of doublets. Mass spectroscopy analy-

sis was performed on water’s SQ Detector 2 with ESI ionization

source on both positive as well as negative 30 & 10 CV. Sam-

ple preparation was done in methanol with 500ppm of which

2μL was injected in mass spectrometry via water’s autosampler.

10mM ammonium acetate: acetonitrile (60:40) was used as mo-

bile phase with 0.3 ml/min ﬂow rate. Analytical thin-layer chro-

matography (TLC) was performed on Pre-coated aluminum sheets

(Silica gel 60 F₂₅₄, Merck Germany) to examine reaction progres-

sion. TLC Visualization was carried out by BTL UV (Ultraviolet) cab-

inet at 254 nm & 365 nm wavelength. Speciﬁc functional group on

TLC was detected by exposing to iodine vapor and spraying vari-

ous reagents (Ninhydrin, Potassium permanganate) on it. Melting

points were noted on optimelt (MPA100) automated melting point

system which was uncorrected. Elemental analysis was performed

on vario MICRO cube, elementar CHNS (Carbon, Hydrogen, Nitro-

gen, Sulphur) analyser serial no.: 15084053. In silico experiments

were carried out computationally on Linux-based operating system

Centos 6.5. Molecular docking study was performed using Glide

module and ADME prediction was performed by QikProp mod-

ule of Maestro 11.4.011, Schrödinger, LLC, New York, 2015. While

Molecular dynamics was conducted using Desmond, module of

Schrödinger, LLC, New York, 2020.

The human oral absorption percentage explain bioavailability

of drug when administrated orally. All analysed compounds were

noted in acceptable values with range of 82 to 100%, where com-

pound 6a-6d, 6f-6m and 6o showed 100% oral absorption. All

tested compounds were reported within the permissible range of

absorption descriptors such as Caco-2 cell permeability (QPPCaco),

Skin permeability (QPlogKp) and MDCK cell permeability (QPP-

MDCK). Out of two distribution factors, all synthesized compounds

were found to be in acceptable range for Blood/Brain partition

co-eﬃcient (QPLogBB), whereas in case of Central Nervous Sys-

tem activity (CNS) only 6n and 6p did not fall into satisfactory

range. Human serum albumin binding (QPlogKhsa) reported to in-

ﬂuence ADME properties of drug molecule, all synthesized com-

pound falls into acceptable range of this descriptor. Some com-

pounds (6a, 6c, 6g, 6h, 6l, 6n, 6o) possessed permissible range of

the aqueous solubility parameter (QPlogS). The Lipinsky rule of ﬁve

evaluate probability of a molecule to be developed as an orally

active drug candidate based on properties like Molecular weight

(<500), Donor HB (<5), Acceptor HB (<10), logPo/w (<5), Vol-

ume (500.0 – 2000.0). In the present study, compound 6n and

6p reported zero violation of the rule, however remaining com-

pounds exhibited violation in Octanol-water partition co-eﬃcient

(logPo/w).

3. Conclusion

A combination of good understanding of the target and its bind-

ing pocket, structure-based design approach, architecture of po-

tent inhibitors and medicinal chemistry inputs resulted into an

interesting addition to the small-molecule inhibitors that can in-

hibit p53-MDM2 interaction. We have demonstrated the utility of

docking and other in silico modules in the optimization of series

of inhibitors for MDM2-p53 interaction based on 1,2,4-triazole-3-

thiol scaffold, and synthesized novel series of 5-(4-chlorophenyl)-

4-phenyl-4H-1,2,4-triazole-3-thiol derivatives to probe their capac-

ity of inhibiting the interaction. All synthesized compounds are

in general druggable by virtue of in silico ADME proﬁle and the

Pﬁzer rules. Among these derivatives, 6g, 6h and 6i exhibited the

acute in vitro antiproliferative activities against three human cancer

cell lines in comparison with Cisplatin. Moreover, binding mode

of potential hits were veriﬁed by molecular docking and dynam-

ics, but precise molecular mechanism of the p53-MDM2 upregu-

lation by potent molecules entails additional investigation. In con-

clusion, potency of 1,2,4-triazole-3-thiols warrant them as a useful

lead compound for the development of more potent and selective

p53-MDM2 interaction inhibitors. Further optimization is still un-

der investigation to address the eﬃcacy, selectivity and pharma-

4.2. Synthetic procedures and analytical data

4.2.1. Preparation of N-phenylhydrazinecarbothioamide (1)

Synthesis was carried out in oven-dried ﬂat bottom ﬂask

equipped with a Teﬂon-coated magnetic stirring bar and cooling

bath. It was charged with isothiocyanatobenzene (1.0 mmol) and

absolute alcohol (20 mL) as a solvent. Let the mixture for stir-

ring in cooling bath until temperature reaches in-between 0-5°C

on thermometer. Latterly, hydrazine hydrate (1.2 mmol) was added

into cooling mixture in dropwise manner via syringe. Within few

minutes shiny precipitates were started appearing. Let the mixture

for starring up to 1 hour at ambient temperature until conﬁrma-

tion of completion by TLC Ethyl acetate: Hexane (70:30). Later, re-

action mixture was quenched by adding it into ice water. The mix-

ture was ﬁltered and washed subsequently with ice water (2 × 20

11

K.R. Patel, J.G. Brahmbhatt, P.A. Pandya et al.

Journal of Molecular Structure 1231 (2021) 130000

mL). Filter cake was dried in oven at 48°C temp and recrystalized

in hot ethanol to get titled compound.

4.2.4.2. 3-((4-chlorobenzyl)thio)-5-(4-chlorophenyl)-4-phenyl-4H-

1,2,4-triazole (6b). A white crystalline solid (Yield: 92%). m.p.:

204.5°C. MS(ESI) m/z 412.08 [M]+; 414.28 [M+2]+. ¹H NMR

(400 MHz, DMSO-d₆) δ (ppm) 8.18 (d, J = 8.4 Hz, 2H), 7.67 (d,

J = 8.4 Hz, 2H), 7.57 – 7.51 (m, 3H), 7.44 (d, J = 8.3 Hz, 2H), 7.35

(td, J = 5.5, 4.8, 2.2 Hz, 4H), 4.55 (s, 2H). ¹³C NMR (101 MHz,

DMSO-d₆) δ (ppm) 153.49, 151.58, 136.35, 134.61, 133.52, 132.03,

130.87, 130.13, 129.96, 129.58, 128.72, 128.35, 127.55, 125.41, 35.13.

Anal calcd for: C₂₁H₁₅Cl₂N₃S: C, 61.17; H, 3.67; N, 10.19; S, 7.78 %

Found C, 61.19; H, 3.65; N, 10.18; S, 7.77 %.

4.2.2. Preparation of

5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazole-3-thiol (3)

An oven-dried ﬂat bottom ﬂask furnished with a Teﬂon-coated

magnetic stirring bar, rubber septa and nitrogen balloon was

charged with N-phenylhydrazinecarbothioamide (1.0 mmol) and

pyridine (10 mL) which served as a catalyst and solvent. Then, 4-

chlorobenzoyl chloride (1.0 mmol) was added dropwise via syringe

in stirring mixture, and the resulting solution was stirred at am-

bient temperature for 6 hours. Afterward pour that solution in ice

cold water, ﬁlter off precipitates and let them dry at room temp.

Dried precipitates were taken in ﬂat bottom ﬂask with methanolic

NaOH solution (15 mL, 60% solution) and reﬂux for 30 hours. Re-

action progression was monitored by TLC in Ethyl acetate: Hexane

(50:50), quenched with ice cold water after completion. The solu-

tion was acidiﬁed by 50% hydrochloric acid, ﬁltered and washed

subsequently with ice water (3 × 20 mL). Precipitates of 5-(4-

chlorophenyl)-4-phenyl-4H-1,2,4-triazole-3-thiol was dried in oven

at 48°C temp and promoted without further puriﬁcation.

4.2.4.3. 3-(4-chlorophenyl)-5-((4-ﬂuorobenzyl)thio)-4-phenyl-4H-

1,2,4-triazole (6c).

A white crystalline solid (Yield: 88%). m.p.:

169.4°C. MS(ESI) m/z 396.03 [M+1]+. ¹H NMR (400 MHz, DMSO-

d₆) δ (ppm) 10.66 (s, 1H), 7.96 – 7.92 (m, 1H), 7.63 – 7.60 (m,

1H), 7.54 – 7.51 (m, 2H), 7.43 (dq, J = 8.3, 2.9 Hz, 3H), 7.35 –

7.30 (m, 3H), 7.18 – 7.09 (m, 2H), 4.41 (s, 2H). ¹³C NMR (101

MHz, DMSO-d₆) δ (ppm) 164.80, 162.66, 160.24, 153.46, 151.70,

136.77, 134.61, 133.55, 133.38, 133.35, 131.14, 131.08, 131.00,

130.10, 129.96, 129.58, 129.48, 129.36, 129.09, 128.71, 128.67,

127.55, 127.26, 125.42, 117.07, 115.30, 115.09, 35.17. Anal calcd for:

C₂₁H₁₅ClFN₃S: C, 63.71; H, 3.82; N, 10.61; S, 8.10 % Found C, 63.72;

H, 3.81; N, 10.63; S, 8.10 %.

4.2.3. Preparation of 2-chloro-N-phenylacetamide derivatives (5f-5p)

Assembly for synthesis includes oven-dried ﬂat bottom ﬂask fa-

cilitated with a Teﬂon-coated magnetic stirring bar, rubber septa,

nitrogen balloon and cooling bath. An appropriate primary amine

(1.0 mmol) was added drop wise to a mixture of triethylamine (1.2

mmol) in dichloromethane (4.5 mL) under nitrogen atmosphere at

0°C. Chloroacetyl chloride (1.2 mmol) was added drop wise at 0°C,

then the reaction mixture was stirred at ambient temperature for

20 h. Consumption of starting material was monitored by TLC in

Ethyl acetate: Hexane (80:20), and ninhydrin spraying reagent. The

reaction mixture was concentrated under reduced pressure and the

residue washed with ice water (3 × 20 mL) and ﬁltered. Precipi-

tates of desired compound were dried in oven at 48°C temp and

was carried on without further puriﬁcation [34].

4.2.4.4. 3-(4-chlorophenyl)-5-((4-methoxybenzyl)thio)-4-phenyl-4H-

1,2,4-triazole (6d). A white crystalline solid (Yield: 79%). m.p.:

176.9°C. MS(ESI) m/z 408.37 [M+1]+. ¹H NMR (400 MHz, DMSO-

d₆) δ (ppm) 10.66 (s, 1H), 7.97 – 7.91 (m, 2H), 7.65 – 7.60 (m,

2H), 7.54 – 7.50 (m, 2H), 7.45 – 7.42 (m, 1H), 7.36 – 7.33 (m,

1H), 7.32 – 7.30 (m, 1H), 7.28 – 7.26 (m, 1H), 6.88 – 6.83 (m,

2H), 4.36 (s, 2H), 3.72 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆)

δ (ppm) 164.80, 158.63, 153.37, 151.88, 138.50, 136.76, 134.58,

133.60, 131.13, 130.26, 130.07, 129.93, 129.58, 129.49, 129.36,

129.10, 128.71, 128.68, 128.59, 127.56, 127.27, 125.47, 117.07, 113.82,

55.06, 35.76. Anal calcd for: C₂₂H₁₈ClN₃OS: C, 64.78; H, 4.45; N,

10.30; S, 7.86 % Found C, 64.77; H, 4.45; N, 10.32; S, 7.85 %.

4.2.4. Preparation of

4.2.4.5. 3-(4-chlorophenyl)-5-((4-nitrobenzyl)thio)-4-phenyl-4H-1,2,4-

triazole (6e). A white crystalline solid (Yield: 94%). m.p.: 180.4°C.

MS(ESI) m/z 423.01 [M+1]+. ¹H NMR (400 MHz, DMSO-d₆) δ

(ppm) 8.28 – 8.13 (m, 2H), 7.74 – 7.63 (m, 2H), 7.60 – 7.50 (m,

3H), 7.46 – 7.41 (m, 2H), 7.34 (dd, J = 7.5, 5.0 Hz, 4H), 4.54 (d,

J = 4.6 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm) 153.59,

151.31, 146.65, 145.61, 134.64, 133.45, 130.26, 130.18, 130.00,

129.59, 128.72, 127.56, 125.35, 123.49, 34.95. Anal calcd for:

C₂₁H₁₅ClN₄O₂S: C, 59.64; H, 3.58; N, 13.25; S, 7.58 % Found C,

59.64; H, 3.58; N, 13.25; S, 7.58 %.

3-(benzylthio)-5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazole

derivatives (6a-6e)

Synthetic procedure was performed in oven-dried ﬂat bot-

tom ﬂask enabled with

a Teﬂon-coated magnetic stirring bar,

rubber septa and nitrogen balloon. It was charged with 5-(4-

chlorophenyl)-4-phenyl-4H-1,2,4-triazole-3-thiol (1.0 mmol) in 20

mL tetrahydrofuran followed by dropwise addition of triethyl

amine (1.0 mmol) in stirring condition. After few minutes suitable

(chloromethyl)benzene derivative (1.0 mmol) was added and re-

ﬂux for 5-8 hours. Reaction completion was concluded by TLC in

Ethyl acetate: Hexane (40:60). Reaction mixture was quenched by

pouring it into cold water. Crude product was ﬁltered and washed

with ice water (3 × 20 mL). Precipitates were dried in oven at 48°C

temp to remove bound water molecules. Puriﬁcation was achieved

by crystallization in hot methanol, results in characteristic shaped

pure crystalline product.

4.2.5. Preparation of 2-((5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-

triazol-3-yl)thio)-N-phenylacetamide derivatives

(6f-6p)

Synthetic procedure was performed in oven-dried ﬂat bot-

tom ﬂask enabled with

a Teﬂon-coated magnetic stirring bar,

rubber septa and nitrogen balloon. It was charged with 5-

(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazole-3-thiol (1.0 mmol) in

20 mL tetrahydrofuran followed by dropwise addition of tri-

ethyl amine (1.0 mmol) in stirring condition. After few minutes

suitable 2-chloro-N-phenylacetamide derivative (1.0 mmol) was

added and reﬂux for 5-8 hours. Reaction completion was con-

cluded by TLC in Ethyl acetate: Hexane (40:60). Reaction mixture

was quenched by pouring it into cold water. Crude product was

ﬁltered and washed with ice water (3 × 20 mL). Precipitates were

dried in oven at 48°C temp to remove bound water molecules. Pu-

riﬁcation was achieved by crystallization in hot methanol, results

in characteristic shaped pure crystalline product.

4.2.4.1. 3-(benzylthio)-5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazole

(6a). A white crystalline solid (Yield: 89%). m.p.: 196.5°C. MS(ESI)

m/z 377.97 [M]+; 379.33 [M+2]+. ¹H NMR (400 MHz, DMSO-d₆)

δ (ppm) 10.66 (s, 2H), 7.95 – 7.93 (m, 2H), 7.62 (d, J = 4.6 Hz, 2H),

7.54 – 7.51 (m, 2H), 7.46 – 7.42 (m, 2H), 7.35 – 7.33 (m, 2H), 7.30

(dt, J = 5.8, 1.4 Hz, 2H), 4.41 (s, 2H). ¹³C NMR (101 MHz, DMSO-

d₆) δ (ppm) 164.80, 153.43, 151.75, 136.93, 136.76, 134.60, 133.56,

131.13, 130.09, 129.94, 129.58, 129.49, 129.36, 129.10, 128.99,

128.71, 128.68, 128.44, 127.56, 127.47, 127.27, 125.45, 121.95, 117.07,

36.15. Anal calcd for: C₂₁H₁₆ClN₃S: C, 66.75; H, 4.27; N, 11.12; S,

8.49 % Found C, 66.73; H, 4.29; N, 11.14; S, 8.48 %.

12

K.R. Patel, J.G. Brahmbhatt, P.A. Pandya et al.

Journal of Molecular Structure 1231 (2021) 130000

4.2.5.1. 2-((5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)-N-

phenylacetamide (6f). A white crystalline solid (Yield: 91%). m.p.:

194.3°C. MS(ESI) m/z 421.36 [M+1]+. ¹H NMR (400 MHz, DMSO-

d₆) δ (ppm) 10.39 (s, 1H), 7.61 – 7.54 (m, 5H), 7.48 – 7.41 (m, 4H),

7.33 (dd, J = 19.2, 8.2 Hz, 4H), 7.06 (t, J = 7.3 Hz, 1H), 4.22 (s, 2H).

¹³C NMR (101 MHz, DMSO-d₆) δ (ppm) 165.40, 153.43, 151.89,

138.76, 134.61, 133.49, 130.23, 130.07, 129.60, 128.80, 128.73,

127.54, 125.37, 123.49, 119.03, 36.81. Anal calcd for: C₂₂H₁₇ClN₄OS:

C, 62.78; H, 4.07; N, 13.31; S, 7.62 % Found C, 62.77; H, 4.04; N,

13.31; S, 7.61 %.

7.32 (m, 3H), 7.29 (dt, J = 8.2, 1.4 Hz, 1H), 6.91 (td, J = 8.5, 2.6 Hz,

1H), 4.22 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm) 165.85,

163.29, 160.89, 153.46, 151.81, 140.51, 140.40, 134.63, 133.46,

130.53, 130.43, 130.24, 130.08, 129.59, 128.73, 127.53, 125.35,

114.82, 114.80, 110.07, 109.86, 105.97, 105.71, 36.72. Anal calcd for:

C₂₂H₁₆ClFN₄OS: C, 60.20; H, 3.67; N, 12.77; S, 7.31 % Found C,

60.21; H, 3.65; N, 12.79; S, 7.31 %.

4.2.5.7. 2-((5-(4-chlorophenyl)-4-phenyl-4 H-1,2,4-triazol-3-yl)thio)-

N-(4-ﬂuorophenyl)acetamide (6l). A white crystalline solid (Yield:

86%). m.p.: 243.9°C. MS(ESI) m/z 438.97 [M]+; 440.45 [M+2]+. ¹H

NMR (400 MHz, DMSO-d₆) δ (ppm) 10.42 (s, 1H), 7.58 (dtt, J = 9.3,

6.4, 3.3 Hz, 5H), 7.47 – 7.41 (m, 4H), 7.36 (d, J = 8.7 Hz, 2H), 7.16

(t, J = 8.9 Hz, 2H), 4.19 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ

(ppm) 165.34, 159.26, 156.87, 153.44, 151.85, 135.18, 135.15, 134.62,

133.48, 130.23, 130.07, 129.84, 129.59, 128.73, 128.55, 127.79,

127.54, 125.37, 120.87, 120.79, 115.49, 115.27, 36.70. Anal calcd for:

C₂₂H₁₆ClFN₄OS: C, 60.20; H, 3.67; N, 12.77; S, 7.31 % Found C,

60.21; H, 3.65; N, 12.76; S, 7.36 %.

4.2.5.2. N-(2-chlorophenyl)-2-((5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-

triazol-3-yl)thio)acetamide (6g). A white crystalline solid (Yield:

93%). m.p.: 201.6°C. MS(ESI) m/z 455.33 [M]+; 457.31 [M+2]. ¹H

NMR (400 MHz, DMSO-d₆) δ (ppm) 10.58 (s, 1H), 7.79 (t, J = 2.0

Hz, 1H), 7.64 – 7.52 (m, 3H), 7.44 (dq, J = 8.9, 2.1 Hz, 5H), 7.39

– 7.30 (m, 3H), 7.19 – 7.08 (m, 1H), 4.21 (s, 2H). ¹³C NMR (101

MHz, DMSO-d₆) δ (ppm) 165.88, 153.46, 151.79, 140.18, 134.63,

133.45, 133.10, 130.54, 130.24, 130.08, 129.59, 128.73, 127.52,

125.34, 123.22, 118.51, 117.46, 36.70. Anal calcd for: C₂₂H₁₇ClN₄OS:

C, 58.03; H, 3.54; N, 12.30; S, 7.04 % Found C, 58.00; H, 3.55; N,

12.30; S, 7.02 %.

4.2.5.8. N-(4-bromophenyl)-2-((5-(4-chlorophenyl)-4-phenyl-4

H-

1,2,4-triazol-3-yl)thio)acetamide (6m). A white crystalline solid

(Yield: 88%). m.p.: 215.7°C. MS(ESI) m/z 499.29 [M]+; 501.27

[M+2]+. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 10.52 (s, 1H),

7.60 – 7.54 (m, 5H), 7.53 – 7.48 (m, 2H), 7.47 – 7.42 (m, 4H),

7.38 – 7.33 (m, 2H), 4.21 (s, 2H).¹³C NMR (101 MHz, DMSO-d₆) δ

(ppm) 165.63, 153.45, 151.81, 138.13, 134.62, 133.46, 131.63, 130.24,

130.07, 129.59, 128.73, 127.53, 125.36, 120.97, 115.08, 36.76. Anal

calcd for: C₂₂H₁₆BrClN₄OS: C, 52.87; H, 3.23; N, 11.21; S, 6.42 %

Found C, 52.86; H, 3.22; N, 11.21; S, 6.40 %.

4.2.5.3. N-(3-chlorophenyl)-2-((5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-

triazol-3-yl)thio)acetamide (6h). A white crystalline solid (Yield:

90%). m.p.: 201.5°C. MS(ESI) m/z 455.35 [M]+; 457.31 [M+2]+.¹H

NMR (400 MHz, DMSO-d₆) δ (ppm) 10.58 (s, 1H), 7.79 (t, J = 2.1

Hz, 1H), 7.65 – 7.51 (m, 3H), 7.44 (ddd, J = 8.6, 4.6, 2.2 Hz,

5H), 7.40

– 7.30 (m, 3H), 7.19 – 7.06 (m, 1H), 4.22 (s, 2H).

¹³C NMR (101 MHz, DMSO-d₆) δ (ppm) 165.88, 153.46, 151.80,

140.18, 134.64, 133.45, 133.11, 130.53, 130.24, 130.08, 129.59,

128.73, 127.52, 125.34, 123.22, 118.51, 117.46, 36.71. Anal calcd for:

C₂₂H₁₆Cl₂N₄OS: C, 58.03; H, 3.54; N, 12.30; S, 7.04 % Found C,

58.01; H, 3.54; N, 12.31; S, 7.02 %.

4.2.5.9. 2-((5-(4-chlorophenyl)-4-phenyl-4 H-1,2,4-triazol-3-yl)thio)-

N-(4-nitrophenyl)acetamide (6n).

A pale yellow crystalline solid

(Yield: 90%). m.p.: 252.5°C. MS(ESI) m/z 466.36 [M+1]+. ¹H NMR

(400 MHz, DMSO-d₆) δ (ppm) 11.00 (s, 1H), 8.25 (d, J = 9.2 Hz,

2H), 7.90 – 7.80 (m, 2H), 7.57 (dt, J = 6.2, 3.2 Hz, 3H), 7.50 –

7.41 (m, 4H), 7.35 (d, J = 8.6 Hz, 2H), 4.27 (s, 2H). ¹³C NMR (101

MHz, DMSO-d₆) δ (ppm) 166.56, 144.86, 142.34, 133.43, 130.27,

130.09, 129.60, 128.74, 127.52, 125.08, 118.80, 36.75. Anal calcd

for: C₂₂H₁₆ClN₅O₃S: C, 56.71; H, 3.46; N, 15.03; S, 6.88 % Found C,

56.71; H, 3.45; N, 15.05; S, 6.87 %.

4.2.5.4. N-(4-chlorophenyl)-2-((5-(4-chlorophenyl)-4-phenyl-4

H-

1,2,4-triazol-3-yl)thio)acetamide (6i). A white crystalline solid

(Yield: 88%). m.p.: 208.7°C. MS(ESI) m/z 455.00 [M]+; 457.32

[M+2]+. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 10.54 (s, 1H),

7.68 – 7.53 (m, 5H), 7.45 (td, J = 6.3, 3.0 Hz, 4H), 7.41 – 7.33 (m,

4H), 4.22 (s, 2H). 1H NMR (400 MHz, DMSO-d6, D₂O) δ (ppm)

7.68 – 7.53 (m, 5H), 7.45 (td, J = 6.3, 3.0 Hz, 4H), 7.41 – 7.33

(m, 4H), 4.22 (s, 2H).¹³C NMR (101 MHz, DMSO-d₆) δ (ppm)

165.61, 153.46, 151.82, 137.72, 134.62, 133.46, 130.24, 130.08,

129.60, 128.73, 127.54, 127.04, 125.36, 120.60, 36.73. Anal calcd

for: C₂₂H₁₆Cl₂N₄OS: C, 58.03; H, 3.54; N, 12.30; S, 7.04 % Found C,

58.00; H, 3.53; N, 12.29; S, 7.03 %.

4.2.5.10. 2-((5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)-

N-(4-methoxyphenyl)acetamide (6o).

A grey solid (Yield: 87%).

m.p.: 258.4°C. MS(ESI) m/z 451.38 [M+1]+. ¹H NMR (400 MHz,

DMSO-d₆) δ (ppm) 10.22 (d, J = 2.5 Hz, 1H), 7.63 – 7.53 (m, 3H),

7.50 – 7.41 (m, 6H), 7.36 (d, J = 8.7 Hz, 2H), 6.95 – 6.85 (m, 2H),

4.17 (s, 2H), 3.72 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm)

164.85, 155.33, 153.42, 151.92, 134.61, 133.50, 131.90, 130.22,

130.06, 129.84, 129.59, 128.73, 128.56, 127.80, 127.55, 125.39,

120.60, 116.83, 113.88, 55.11, 36.75. Anal calcd for: C₂₃H₁₉ClN₄O₂S:

C, 61.26; H, 4.25; N, 12.42; S, 7.11 % Found C, 61.25; H, 4.27; N,

12.42; S, 7.09 %.

4.2.5.5. 2-((5-(4-chlorophenyl)-4-phenyl-4 H-1,2,4-triazol-3-yl)thio)-

N-(2-ﬂuorophenyl)acetamide (6j). A white crystalline solid (Yield:

92%). m.p.: 211.5°C. MS(ESI) m/z 438.97 [M]+; 440.45 [M+2]+.

¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 10.20 (s, 1H), 7.92 (ddt,

J = 7.8, 5.9, 3.9 Hz, 1H), 7.63 – 7.52 (m, 3H), 7.51 – 7.42 (m,

4H), 7.40 – 7.34 (m, 2H), 7.32 – 7.24 (m, 1H), 7.16 (dq, J = 6.5,

3.6, 3.0 Hz, 2H), 4.26 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆)

δ (ppm) 166.08, 154.46, 153.45, 152.02, 151.85, 134.63, 133.50,

130.23, 130.06, 129.60, 128.74, 127.55, 125.92, 125.81, 125.37,

125.31, 124.41, 124.38, 123.55, 115.57, 115.38, 36.43. Anal calcd for:

C₂₂H₁₆ClFN₄OS: C, 60.20; H, 3.67; N, 12.77; S, 7.31 % Found C,

60.21; H, 3.67; N, 12.76; S, 7.35 %.

4.2.5.11. 4-(2-((5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazol-3-

yl)thio)acetamido)benzoic acid (6p). A brown solid (Yield: 81%).

m.p.: 231.4°C. MS(ESI) m/z 465.36 [M+1]+. ¹H NMR (400 MHz,

DMSO-d₆) δ (ppm) 12.80 (s, 1H), 10.71 (s, 1H), 7.92 – 7.89 (m,

2H), 7.69 (d, J = 8.7 Hz, 2H), 7.59 – 7.55 (m, 3H), 7.44 (dq, J = 9.1,

2.2 Hz, 4H), 7.37 – 7.34 (m, 2H), 4.25 (s, 2H). ¹³C NMR (101 MHz,

DMSO-d₆) δ (ppm) 36.81,118.36, 125.34, 127.54, 128.73, 129.49,

129.60, 130.08, 130.25, 130.46, 131.12, 133.46, 134.63, 142.77,

151.81, 153.46, 166.02, 166.84. Anal calcd for: C₂₃H₁₇ClN₄O₃S: C,

59.42; H, 3.69; N, 12.05; S, 6.90 % Found C, 59.4; H, 3.70; N, 12.01;

S, 6.98 %.

4.2.5.6. 2-((5-(4-chlorophenyl)-4-phenyl-4 H-1,2,4-triazol-3-yl)thio)-

N-(3-ﬂuorophenyl)acetamide (6k).

A off white crystalline solid

(Yield: 89%). m.p.: 209.6°C. MS(ESI) m/z 438.98 [M]+; 440.37

[M+2]+. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 10.61 (s, 1H), 7.57

(dtd, J = 7.3, 4.5, 2.4 Hz, 4H), 7.44 (dq, J = 9.2, 2.1 Hz, 4H), 7.40 –

13

K.R. Patel, J.G. Brahmbhatt, P.A. Pandya et al.

Journal of Molecular Structure 1231 (2021) 130000

4.3. MTT assay

0.787 and comprises residues earlier reported in literature i.e.

Phe19, Trp23 and Leu26.

4.3.1. Cell culture and source of lines

Receptor Grid Generation tool was used to generate a grid box

at the centroid of the active site for docking. The grid was created

by picking one of the site points followed by applying a van der

Waals radii scaling factor of 1.00 with a partial charge cut-off of

less than 0.25e.

The adenocarcinomic human alveolar basal epithelial cell line

of lung cancer A549 (p53 wild type), Human primary glioblastoma

cell line of brain cancer U87 (p53 wild type) and acute promyelo-

cytic leukemia cell line of peripheral blood HL60 (p53 null) were

procured from NCCS (National Centre for Cell Science, Pune, Maha-

rashtra, India). Cell lines A549, U87 and HL60 were grown at 37°C

in 5% CO₂, maintained in F-12K medium, Eagle’s Minimum Essen-

tial Medium (EMEM) and RPMI1640 medium, respectively. All cell

lines were routinely tested for Mycoplasma and Epstein-Barr Virus.

4.4.2. Ligand preparation

The 3D structures of ligand were generated in the SDF (Stan-

dard Database Format) using MarvinSketch (version 5.5.1.0). Lig-

ands were generated for docking by OPLS3e force ﬁeld in LigPrep

module of maestro, Schrödinger. A number of structures from each

input structure was produced with stereo chemical, ionization, tau-

tomeric variations, as well as energy minimization and optimized

for their geometry, desalted and corrected for their chiralities. The

ionization and tautomeric states were generated at physiological

4.3.2. In vitro antiproliferative assay

The MTT [3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium

bromide] method was used to evaluate the antiproliferative ac-

tivities of the synthesized 5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-

triazole-3-thiol derivatives. Targeted cell lines A549, U87 and HL60

were seeded into 96-well plates (104 cells per well). After cultur-

ing in a humidiﬁed atmosphere with 5% CO₂at 37°C for 24 h, test

compounds were added onto triplicate wells with different con-

centrations (0.01, 0.1, 1, 10, 100 and 1000 μM). Cisplatin was used

as the positive control and untreated cells as blank. Subsequently

incubated for 24h, 10 μL of MTT (M P Biochemicals, USA) solu-

tion (5 mg/mL) was added to each well and after the samples

were shaken for 1 min, the plate was incubated further for 4 h

at 37°C in dark condition. The purple coloured formazan crystals

were dissolved in 100.00 μL of DMSO. The absorbance (OD- Op-

tical Density) was recorded at 570 nm using Epoch^TMMicroplate

Spectrophotometer (BioTek Instruments, VT, USA). Wells containing

plain media without cells as were used as blanks. The concentra-

tion of the compounds that inhibited cell growth by 50% (IC₅₀) was

calculated by comparing the optical density reading with the con-

trol in GraphPad software [35].

pH value 7.0

2.0 using Epik.

4.4.3. Molecular docking

Glide module of the Schrödinger program was applied for scor-

ing process of ligands with standard settings in Extra Precision

(XP) mode [36]. Glide used hierarchical series of ﬁlters to search

for possible locations of the ligand in the active site region of the

receptor, by keeping ligands ﬂexible and receptor rigid except that

active site. Docking scores were calculated and ligands were ranked

on the basis of Gscore (XP). An empirical formula of XPGlide score

is as follows:

GScore = a vdW + b Coul + Lipo + Hbond + Metal + BuryP +

RotB + Site

Where, vdW = van der Waals energy; Coul = coulomb energy;

Lipo = lipophilic contact term; Hbond = the hydrogen-bonding

term; Metal = metal binding term; BuryP = penalty for buried

polar groups; RotB = penalty for freezing rotatable bonds and

Site = polar interactions at the active site. The coeﬃcients of vdW

and Coul are: a = 0.065, b = 0.130.

4.4. Docking

4.5. Molecular Dynamics

Docking is a molecular modelling technique that is used to pre-

dict how macromolecules (protein) interacts with small molecules

(ligand) in three-dimensional space. Docking procedures includes

numerous steps as follow:

Static structure-based approaches determine the effect of com-

pounds on the stability of receptor in terms of binding energy.

However, at the physiological domain protein and ligand are dy-

namic in nature; hence, scrutinizing their motions at the molecu-

lar level is indispensable to understand key physicochemical phe-

nomena. Molecular Dynamics simulations were performed using

Desmond module of Schrödinger 2020 [42]. MD simulations were

executed for 50 ns using Isothermal-isobaric (NPT) ensemble class,

where temperature and pressure were reserved at 300 K and

1.01325 bar pressure employing Nose-Hoover temperature coupling

and isotropic scaling. The molecular systems were solvated with

water molecules in orthorhombic box via simple point charge (SPC)

method. Neutralization of systems were achieved by adding min-

imum amount of sodium and chloride ions, to balance the net

charge of solvated system and to mimic the osmotic effect of wa-

ter. Equilibration of the systems and MD simulations were carried

out using the default protocol provided in Desmond. Conforma-

tional variations in ligand structures and dynamic behaviour of the

protein were analysed by calculating the Root Mean Square Devia-

tion and potential energy. Input and output ﬁles of 3-D structures

and trajectories were visually examined using the Maestro graphi-

cal interface.

4.4.1. Receptor selection and preparation

The X-ray crystal structure of MDM2 bound to the transacti-

˚

vation domain of p53 (PDB ID: 1YCR, Resolution: 2.6 A) was re-

trieved from the protein data bank [37]. Schrödinger has assem-

bled Protein Preparation Wizard in Maestro to ensure chemical cor-

rectness and optimization in protein suitable for modelling calcu-

lations [38]. In this step, bond orders were assigned, all hydrogen’s

in the structure were added, disulphide bonds were created, ad-

justed zero order bond on the metal and deleting waters that were

beyond 5 A° distance to het groups. Ionization predication and tau-

tomeric states of the het group at pH (Potential of Hydrogen) 7

was achieved by het states options. Prime was used for adding all

missing side chain present in the crystal structure of protein [39].

Hydrogen bonds equivalent to pH 7.0 were added as it is an ap-

propriate ionization state for both the acidic and basic amino acid

residues. The steric clashes among the residues due to addition of

hydrogen atoms was relieved by energy minimization using OPLS-

˚

2005 force ﬁeld till RMSD value of 0.30 A [40].

The active site (binding pockets) of MDM2-p53 complex was

investigated and characterized by SiteMap module of Schrödinger

[41]. A SiteMap calculates the overall site score and generate con-

tour maps, producing hydrophobic and hydrophilic maps. The only

active site generated and utilized for grid generation had site score

4.6. ADME

Clinical phase drug development failure due to undesirable ab-

sorption, distribution, metabolism and excretion (ADME) properties

14

K.R. Patel, J.G. Brahmbhatt, P.A. Pandya et al.

Journal of Molecular Structure 1231 (2021) 130000

of some new drug candidates has resulted in a paradigm shift to

identify such problems early in the drug discovery process. Thus,

in silico ADME prediction has been explored as a tool to opti-

mize selection of the most suitable drug candidates for develop-

ment. In silico ADME parameters of the compound (6a-6p) were

calculated using QikProp, module of Schrödinger software which

was designed by Professor William L. Jorgensen [43]. QikProp pre-

dicts an immense variety of pharmaceutically relevant properties

i.e. octanol/water and water/gas log Ps, aqueous solubility (Log

S), brain and blood partition co-eﬃcient (log BB), overall CNS ac-

tivity, Caco-2 and MDCK cell permeabilities, log Khsa for human

serum albumin binding, percent human oral absorption, log IC₅₀

for HERG K+-channel blockage, etc. It also evaluates the accept-

ability of analogous based on Lipinski’s rule of ﬁve such as log P,

H-bond donors, H-bond acceptors, number of rotatable bonds and

molecular weight [44] which is essential to ensure drug-like phar-

macokinetic proﬁle. The compounds prepared by LigPrep module

were utilized for the calculation of ADME parameters. QikProp was

run in normal processing mode with default options.

Hitesh D. Patel) is thankful to GUJCOST for the ﬁnancial support

(MRP: No. GUJCOST/MRP/2017-18/2323).

Supplementary materials

Supplementary material associated with this article can be

found, in the online version, at doi:10.1016/j.molstruc.2021.130000.

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NaOH= Sodium hydroxide; K₂CO₃

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=

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Jpan G. Brahmbhatt: Investigation (In vitro), Validation (In

vitro)

Pranav A. Pandya: Formal analysis

Drashti G. Daraji: Resources

Hitesh D. Patel: Supervision, Writing - Review & Editing

Rakesh M. Rawal: Supervision

Sujit K. Baran: Investigation (XRD), Validation (XRD)

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.

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[21] ClinicalTrials.gov identiﬁers for RG7112: NCT00559533, NCT00623870,

NCT01677780, NCT01164033, NCT01605526, NCT01143740, NCT02407080

and NCT01635296.

Acknowledgment

The authors are thankful to the Department of Chemistry and

Department of Life Science, Gujarat University, Ahmedabad, Gu-

jarat, India, for providing the necessary laboratory facilities. UGC-

Info net and INFLIBNET Gujarat University are acknowledged for

providing the e-sources. We are grateful to DST-Purse program at

faculty of science, The M. S. University of Baroda, Gujarat, India,

for single crystal X-ray diffraction studies. We would also like to

thank Schrödinger team for assistance in computational study. One

of the authors (Krupa R. Patel) is thankful to DST-INSPIRE Fellow-

ship (IF160734) for the ﬁnancial support. Corresponding author (Dr.

[22] ClinicalTrials.gov identiﬁers for RG7388: NCT02407080, NCT02828930,

NCT01901172, NCT01462175, NCT02633059, NCT02545283, NCT02624986,

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16

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Design, synthesis and biological evaluation of novel 5-(4-chlorophenyl)-4-phenyl-4H-1,2,4-triazole-3-thiols as an anticancer agent

DOI: 10.1016/j.molstruc.2021.130000

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