Design, synthesis, DFT, docking studies and ADME prediction of some new coumarinyl linked pyrazolylthiazoles: Potential standalone or adjuvant antimicrobial agents

Article abstract of DOI:10.1371/journal.pone.0196016

The control of antimicrobial resistance (AMR) seems to have come to a dead end. The major consequences of the use and abuse of antibacterial drugs are the development of resistant strains due to genetic mutability of both pathogenic and nonpathogenic microorganisms. We, herein, report the synthesis, characterization and biological activities of coumarin-thiazole-pyrazole (CTP) molecular hybrids with an effort to explore and overcome the increasing antimicrobial resistance. The compounds were characterized by analyzing their IR, Mass, ¹H and¹³C NMR spectral data and elemental analysis. The in vitro antimicrobial activity of the synthesized compounds was investigated against various pathogenic strains; the results obtained were further explained with the help of DFT and molecular orbital calculations. Compound 1b and 1f displayed good antimicrobial activity and synergistic effects when used with kanamycin and amphotericin B. Furthermore, in vitro cytotoxicity of compounds 1b and 1f were studied against HeLa cells (cervical cancer cell) and Hek-293 cells. The results of molecular docking study were used to better rationalize the action and prediction of the binding modes of these compounds.

Full text of DOI:10.1371/journal.pone.0196016

RESEARCH ARTICLE
Design, synthesis, DFT, docking studies and
ADME prediction of some new coumarinyl
linked pyrazolylthiazoles: Potential standalone
or adjuvant antimicrobial agents
Sunil Kumar1, Vikram Saini2, Indresh K. Maurya3, Jayant Sindhu4, Mukesh Kumari5,
Ramesh Kataria1☯*, Vinod Kumar6☯¤
*
1 Department of Chemistry & Centre of Advance Studies in Chemistry, Panjab University, Chandigarhs,
India, 2 Department of Biotechnology, AIIMS- New Delhi, Delhi, India, 3 Department of Microbial
Biotechnology, Panjab University, Chandigarh, India, 4 K.M. Government College, Narwana, India,
5 Department of Chemistry, Kurukshetra University, Kurukshetra, India, 6 Department of Chemistry, M. M.
University, Mullana-Ambala, India
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
☯ These authors contributed equally to this work.
¤ Current address: Department of Chemistry, Markanda National College, Shahbad (M), India.
* rkataria@pu.ac.in (RK); vinodbatan@gmail.com (VK)
OPEN ACCESS
Abstract
Citation: Kumar S, Saini V, Maurya IK, Sindhu J,
Kumari M, Kataria R, et al. (2018) Design,
The control of antimicrobial resistance (AMR) seems to have come to a dead end. The
major consequences of the use and abuse of antibacterial drugs are the development of
resistant strains due to genetic mutability of both pathogenic and nonpathogenic microor-
ganisms. We, herein, report the synthesis, characterization and biological activities of cou-
marin-thiazole-pyrazole (CTP) molecular hybrids with an effort to explore and overcome the
increasing antimicrobial resistance. The compounds were characterized by analyzing their
IR, Mass, 1H and13C NMR spectral data and elemental analysis. The in vitro antimicrobial
activity of the synthesized compounds was investigated against various pathogenic strains;
the results obtained were further explained with the help of DFT and molecular orbital calcu-
lations. Compound 1b and 1f displayed good antimicrobial activity and synergistic effects
when used with kanamycin and amphotericin B. Furthermore, in vitro cytotoxicity of com-
pounds 1b and 1f were studied against HeLa cells (cervical cancer cell) and Hek-293 cells.
The results of molecular docking study were used to better rationalize the action and predic-
tion of the binding modes of these compounds.
synthesis, DFT, docking studies and ADME
prediction of some new coumarinyl linked
pyrazolylthiazoles: Potential standalone or adjuvant
antimicrobial agents. PLoS ONE 13(4): e0196016.
https://doi.org/10.1371/journal.pone.0196016
Editor: Mohammad Shahid, Aligarh Muslim
University, INDIA
Received: December 29, 2017
Accepted: April 4, 2018
Published: April 19, 2018
Copyright: © 2018 Kumar et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Introduction
Funding: This work was financially supported by
the University Grant Commission in the form of
UGC-BSR research start up grant (F.30- 86/2014
(BSR)), New Delhi, India. There was no additional
external funding received for this study. The
funders had no role in study design, data collection
Over the past few decades, globally antibiotic repository has become less effective due to both
antibiotic overuse and an increase in antimicrobial drug resistance. Even common bacterial
(e.g. Staphylococcus aureus, Staphylococcus enteric, Escherichia coli) and fungal (e.g. Candida
albicans, Cryptococcus neoformans) pathogens are developing resistance against widely pre-
scribed antibacterial and antifungal drugs like amphotericin B (Amp B), fluconazole (FLC),
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
and analysis, decision to publish, or preparation of
the manuscript.
chloroamphenicol and penicillin [1–3].This is particularly dangerous considering that the
major bacterial as well as fungal pathogens such as Candida sp. account for ~75% of all infec-
tions and represent the 4th leading cause of nosocomial infections [4]. Thus microbial drug
resistance has emerged as a major public health challenge across the world. Consequently,
strategies are needed to develop new therapeutic compounds that could either act as new
drugs or could complement the existing drug therapy more effective by acting as an adjuvant
[5–7].
Competing interests: The authors have declared
that no competing interests exist.
In recent past, azoles have garnered considerable attention especially in the design and syn-
thesis of compounds with significant biological activities [8–11]. In particular, there are vari-
ous drugs incorporating thiazole nucleus (Fig 1) which are being used as the antimicrobial
(sulfathiazole), antiinflammatory (fanetizole) and antifungal (abafungin) agents [12–17]. Simi-
larly, compounds containing pyrazole ring have been found to exhibit a variety of biological
properties and many of them such as celecoxib, pyrazofurin etc. (Fig 1) are used clinically
besides using for agricultural pest management [18–22]. Especially, 3,5-dimethyl-4-aryldiaze-
nylpyrazole derivatives have been reported to exhibit cytotoxic [23], antioxidant [24], analgesic
[25] and significant antimicrobial potential [26]. On the other side, compounds containing
coumarin moiety (Fig 1) have also been proved as a potent antimicrobial, antitumor, anticoag-
ulant, antiviral, and anti-inflammatory agents [23,24,27–31]. It has been widely accepted that
structural properties of thiazole, pyrazole and coumarin derivatives and their stability under in
vivo conditions are the primary factors for their superior pharmacological activities [32–34].
Fig 1. Some thiazole, pyrazole, 4-aryldiazenylpyrazole and coumarin containing biological active heterocycles and target compounds.
https://doi.org/10.1371/journal.pone.0196016.g001
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
A drug discovery program involves the planning of novel chemical entities using molecular
hybridization approach where two active pharmacophoric units of known drugs were con-
nected through a covalent bond in a single matrix [35–37]. The selection of the active units in
the dual drug is usually established on their observed (or anticipated) synergistic or additive
pharmacological activities. In prospect of the above discussed facts and in continuation of our
research towards the development of novel molecular hybrids as potential biologically active
agents [38–42], we have decided to build new molecular hybrids based on coumarin-thiazole-
pyrazole (CTP).
Experimental
Chemistry
General. Melting points were determined by open capillary method and are uncorrected.
Infrared spectrum was obtained neat on a Thermo Scientific Fischer spectrometer. Multinu-
clear NMR (1H, 13C) spectra were recorded on a Bruker avance at 300 MHz. Chemical shifts
are reported in parts per million (ppm). Tetramethylsilane served as an internal standard in
13C and 1H NMR (δ 0.00 ppm). CHNS analyses were obtained on a Perkin Elmer Model 2400
CHNS elemental analyzer and Mass spectroscopy data of synthesized compound was recorded
on waters QQ-TOF micro Mass Spectrometer. All the chemicals used for the synthesis of tar-
get compounds have been purchased from Sigma Aldrich and were used as received. All the
compounds gave C, H and N analysis within 0.5 of the theoretical values. Scanned Spectra
for all compounds are shown in S3–S19 Figs.
General procedure for the synthesis of 4-(coumarin-3-yl)-2-((3,5-dimethyl-4-phenyldia-
zenyl)pyrazol-1-yl)thiazoles (1a-1g). An ethanolic solution (20 mL) of 3-bromoacetylcou-
marin (7, 0.52 g, 2.00 mmol), sodium carbonate (0.11 g, 1.00 mmol) and 3,5-dimethyl-4-(p-
chlorophenyl)diazenylpyrazole-1-thiocarboxamide (5a) (0.59 g, 2.00 mmol) was refluxed on a
water bath for 6 h. On completion of the reaction as observed using TLC (20:80, Ethylacetate:
Hexane (v/v)), the solvent was evaporated. The solid thus obtained was dissolved in chloro-
form (25 mL) and washed with water (50 mL). The organic layer was separated and the excess
solvent was distilled off. The solid thus obtained was crystallized with ethanol to afford 1a in
82% yield. The protocol was further extended using differently substituted amines and couma-
rin which resulted into the formation of compounds 1b-1g in good to excellent yield.
Spectral data
4-(Coumarin-3-yl)-2-(3,5-dimethyl-4-(4-chlorophenyl)diazenyl)pyrazol-1-yl)thiazole (1a).
Rf: 0.6; mp 214–216˚C; Yield: 82%; IR (cm-1): 1719 (C = O), 1605 (C = N); 1H NMR (300
MHz, CDCl3, δ ppm): 8.39 (s, 1H, coumarin 4-H), 8.06 (s, 1H, thiazole 5-H), 7.70 (d, 2H,
J = 8.4 Hz, p-chlorophenyl 2, 6-H), 7.65 (s, 1H, coumarin 5-H), 7.60–7.52 (m, 1H, coumarin
7-H), 7.45 (d, 2H, J = 8.4 Hz, p-chlorophenyl 3,5-H), 7.41–7.33 (m, 2H, coumarin 6,8-H), 2.86
(s, 3H), 2.31 (s, 3H); 13C NMR (75 MHz, CDCl3, δ ppm): 164.33 (C = O), 159.22 (C2-thiazole),
154.38 (C8’-coumarin), 149.26 (C4-thiazole), 147.14 (C4-coumarin), 141.59 (C-Ar), 136.64
(C-Ar), 133.14 (C-Ar), 129.23 (C-Ar), 128.82 (C-Ar), 125.07 (C-Ar), 123.35 (C-Ar), 120.87
(C-Ar), 119.40 (C-Ar), 118.67 (C-Ar), 116.81 (C-Ar), 110.77 (C-Ar), 23.88 (5-CH3 pyrazole),
14.70 (3-CH3 pyrazole); MS (m/z) Found: 463.1, 461.0 (3:1) [M+H]+; Calcd.: 461.92;Anal.
Calcd. for C23H16ClN5O2S: C, 59.80; H, 3.49; N, 15.16. Found: C, 59.85; H, 3.50; N, 15.46.
4-(6-Chlorocoumarin-3-yl)-2-(3,5-dimethyl-4-(4-bromophenyl)diazenyl)pyrazol-1-yl)
thiazole (1b). Rf: 0.6; mp 220–222˚C;Yield: 88%; IR (cm-1): 1719 (C = O), 1602 (C = N); 1H
NMR (300 MHz, CDCl3, δ): 8.30 (s, 1H, coumarin 4-H), 8.08 (s, 1H, thiazole 5-H), 7.65–7.61
(m, 5H, coumarin 5-H, Ph2,6,3,5-H), 7.49 (d, 1H, J = 8.7 Hz, coumarin 8-H), 7.31 (d, 1H,
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
J = 8.7 Hz, coumarin 7-H), 2.74 (s, 3H, 5-CH3), 2.20 (s, 3H, 3-CH3); 13C NMR (75 MHz,
CDCl3, δ ppm): 167.47 (C = O), 159.23 (C2-thiazole), 154.49 (C8’-coumarin), 149.56 (C4-thia-
zole), 147.15 (C4-coumarin), 143.40 (C-Ar), 137.33 (C-Ar), 133.24 (C-Ar), 128.86 (C-Ar),
125.07 (C-Ar), 124.26 (C-Ar), 124.16 (C-Ar), 122.74 (C-Ar), 119.53 (C-Ar), 118.64 (C-Ar),
116.89 (C-Ar), 115.04 (C-Ar), 112.54 (C-Ar), 31.16 (5-CH3 pyrazole), 25.30 (3-CH3 pyrazole);
MS (m/z) Found: 542.1, 540.1; Calcd.: 540.82; Anal. Calcd. for C23H15BrClN5O2S: C, 51.08; H,
2.80; N, 12.95. Found: C, 51.00; H, 2.82; N, 13.14.
4-(Coumarin-3-yl)-2-(3,5-dimethyl-4-(4-methylphenyl)diazenylpyrazol-1-yl)thiazole
(1c). Rf: 0.6; mp 240–241˚C; Yield: 80%; IR (cm-1): 1713 (C = O), 1601 (C = N); 1H NMR (300
MHz, CDCl3, δ): 8.32 (s, 1H, coumarin 4-H), 8.02 (s, 1H, thiazole 5-H), 7.87 (d, 2H, J = 8.7 Hz,
Ph 2,6-H), 7.71 (d, 1H, J = 8.4 Hz, coumarin 5-H), 7.49 (d, 2H, J = 8.4 Hz, Ph 3,5-H), 7.62 (t,
1H, J = 7.5 Hz, coumarin 7-H), 7.43–7.40 (m, 2H, coumarin 6,8-H), 2.74 (s, 3H, 5-CH3), 2.20
(s, 3H, 3-CH3), 2.05 (s, 3H, p-CH3); 13C NMR (75 MHz, CDCl3, δ ppm): 162.73 (C = O),
157.90 (C2-thiazole), 147.90 (C8’-coumarin), 140.92 (C4-thiazole), 140.62 (C4-coumarin),
139.09 (C-Ar), 134.47 (C-Ar), 133.02 (C-Ar), 132.82 (C-Ar), 130.27 (C-Ar), 128.85 (C-Ar),
128.44 (C-Ar), 128.39 (C-Ar), 128.35 (C-Ar), 126.95 (C-Ar), 126.92 (C-Ar), 125.36 (C-Ar),
123.28 (C-Ar), 118.07 (C-Ar), 117.99 (C-Ar), 113.57 (C-Ar), 35.10 (5-CH3 pyrazole), 29.61
(3-CH3 pyrazole), 14.60 (Ph-CH3); MS (m/z) found: 441.3; Calcd. 441.50; Anal. Calcd. For
C24H19N5O2S: C, 65.29; H, 4.34; N, 15.86; Found C, 65.69; H, 4.26; N, 15.85.
4-(6-Chlorocoumarin-3-yl)-2-(3,5-dimethyl-4-(4-methylphenyl)diazenyl)pyrazol-1-yl)
thiazole (1d). Rf: 0.6; mp 255–256˚C; Yield: 83%; IR (cm-1): 1720 (C = O), 1601 (C = N); 1H
NMR (300 MHz, CDCl3, δ): 8.39(s, 1H, coumarin 4-H), 8.00(s, 1H, thiazole 5-H), 7.86 (d, 2H,
J = 8.4 Hz, Ph2,6-H), 7.66 (s, 1H, coumarin 5-H), 7.63 (d, 2H, J = 8.1 Hz, Ph3,5-H), 7.53 (d,
1H, J = 8.4 Hz, coumarin 7-H), 7.35 (d, 1H, J = 8.4 Hz, coumarin 8-H), 2.69 (s, 3H, 5-CH3),
2.19 (s, 3H, 3-CH3), 1.98 (s, 3H, p-CH3);13C NMR (75 MHz, CDCl3, δ ppm): 164.37 (C = O),
152.86 (C2-thiazole), 151.56 (C8’-coumarin), 144.30 (C4-thiazole), 143.71 (C4-coumarin),
139.30 (C-Ar), 138.86 (C-Ar), 131.69 (C-Ar), 131.60 (C-Ar), 129.69 (C-Ar), 128.69 (C-Ar),
128.41 (C-Ar), 126.37 (C-Ar), 126.20 (C-Ar), 120.61 (C-Ar), 120.24 (C-Ar), 119.41 (C-Ar),
116.44 (C-Ar), 113.44 (C-Ar), 20.94 (CH3-Ph), 24.37 (5-CH3 pyrazole), 13.86 (3-CH3 pyra-
zole); MS (m/z) Found: 477.6, 475.8; Calcd. 475.95; Anal. Calcd. for C24H18ClN5O2S: C, 60.56;
H, 3.81; N, 14.71; Found C, 60.55; H, 3.71; N, 14.63.
4-(Coumarin-3-yl)-2-(3,5-dimethyl-4-(4-bromophenyl)diazenyl)pyrazol-1-yl)thiazole
(1e). Rf: 0.6; mp 210–212˚C; Yield: 88%; IR (cm-1): 1717 (C = O), 1600 (C = N); 1H NMR (300
MHz, CDCl3, δ): 8.30 (s, 1H, coumarin 4-H), 8.08 (s, 1H, thiazole 5-H), 7.82 (d, 1H, J = 8.1 Hz,
coumarin 5-H), 7.71 (d, 2H, J = 8.4 Hz, Ph 3,5-H), 7.65–7.61 (m, 1H, coumarin 7-H), 7.47 (d,
2H, J = 8.4 Hz, Ph2,6-H), 7.28–7.24 (m, 2H, coumarin 6,8-H), 2.72 (s, 3H, 5-CH3), 2.32 (s, 3H,
3-CH3);13C NMR (75 MHz, CDCl3, δ ppm): 164.81 (C = O), 159.22 (C2-thiazole), 154.38 (C8’-
coumarin), 149.26 (C4-thiazole), 147.14 (C4-coumarin), 141.59 (C-Ar), 136.64 (C-Ar), 133.14
(C-Ar), 129.23 (C-Ar), 128.82 (C-Ar), 125.07 (C-Ar), 123.35 (C-Ar), 121.87 (C-Ar), 119.40
(C-Ar), 118.67 (C-Ar), 116.81 (C-Ar), 110.77 (C-Ar), 25.08 (5-CH3 pyrazole), 14.71 (3-CH3
pyrazole); MS (m/z) found: 506.1, 504.1 (3:1); Calcd.: 506.37; Anal. Calcd. for C23H16BrN5O2S:
C, 54.55; H, 3.18; N, 13.83. Found: C, 54.48; H, 3.12; N, 13.82.
4-(6-Chlorocoumarin-3-yl)-2-(3,5-dimethyl-4-(4-chlorophenyl)diazenyl)pyrazol-1-yl)
thiazole (1f). Rf: 0.6; mp 235–236˚C;Yield: 85%; IR (cm-1): 1719 (C = O), 1605 (C = N); 1H
NMR (300 MHz, CDCl3, δ): 8.28 (s, 1H, coumarin 4-H), 8.07 (s, 1H,thiazole 5-H), 7.79 (d, 1H,
J = 2.4 Hz, coumarin 5-H), 7.65–7.61 (m, 5H, Ph 4-H and coumarin 6-H), 7.28 (d, 1H, J = 8.1
Hz, coumarin 8-H), 2.84 (s, 3H, 5-CH3), 2.21 (s, 3H, 3-CH3); 13C NMR (75 MHz, CDCl3,
δ ppm 167.44 (C = O), 159.23 (C2-thiazole), 150.22 (C8’-coumarin), 147.40 (C4-thiazole),
142.56 (C4-coumarin), 139.21 (C-Ar), 133.23 (C-Ar), 128.88 (C-Ar), 125.10 (C-Ar), 124.85
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
(C-Ar), 124.80 (C-Ar), 122.43 (C-Ar), 119.34 (C-Ar), 118.64 (C-Ar), 116.81 (C-Ar), 113.49
(C-Ar), 110.39 (C-Ar), 25.16 (5-CH3 pyrazole), 15.54 (3-CH3 pyrazole); MS (m/z) found:
497.2, 495.2 (3:1); Calcd.: 496.37; Anal. Calcd. for C23H15Cl2N5O2S: C, 55.65; H, 3.05; N, 14.11;
Found: C, 55.64; H, 3.10; N, 14.17.
4-(Coumarin-3-yl)-2-(3,5-dimethyl-4-phenyldiazenyl)pyrazol-1-yl)thiazole (1g). Rf: 0.6;
mp 205–206˚C; Yield: 82%; IR (cm-1): 1715 (C = O), 1603 (C = N); 1H NMR (300 MHz,
CDCl3, δ): 8.34 (s, 1H, coumarin 4-H), 8.08 (s, 1H, thiazole 5-H), 7.82–7.78 (m, 2H, Ph 2,6-H),
7.78 (d, 1H, J = 8.4 Hz, coumarin 5-H), 7.65–7.63 (m, 1H, coumarin 7-H), 7.62–7.57 (m, 4H,
Ph 3,4,5-H, coumarin 6-H), 7.33 (d, 1H, J = 8.7 Hz, coumarin 8-H), 2.79 (s, 3H, 5-CH3), 2.24
(s, 3H, 3-CH3); 13C NMR (75 MHz, CDCl3, δ ppm): 167.79 (C = O), 159.35 (C8’-coumarin),
154.35 (C2-thiazole), 148.40 (C4-coumarin), 146.95 (C4-thiazole), 142.99 (C-Ar), 138.95
(C-Ar), 135.97 (C-Ar), 132.95 (C-Ar), 128.78 (C-Ar), 124.99 (C-Ar), 123.43 (C-Ar), 122.59
(C-Ar), 120.05 (C-Ar), 119.79 (C-Ar), 118.76 (C-Ar), 116.75 (C-Ar), 110.52 (C-Ar), 24.83
(5-CH3 pyrazole), 15.22 (3-CH3 pyrazole); MS (m/z) found: 427.3;Calcd.: 427.28; Anal. Calcd.
forC23H17N5O2S: C, 64.62; H, 4.01; N, 16.38; Found C, 64.55; H, 4.05; N, 16.45.
Antimicrobial assay
The microbial strains (bacterial and fungal) used in the study were obtained from the Institute
of Microbial Technology (MTCC-IMTECH), Chandigarh and National Collection of Patho-
genic Fungi (NCPF), Post-Graduate Institute of Medical Education and Research (PGIMER),
Chandigarh. The bacterial strains Escherichia coli MTCC 2961, Staphylococcus aureus MTCC
3160, Enterococcus faecalis MTCC 439, Vibrio cholera MTCC 3906, Streptococcus pyogenes
MTCC 442 were cultured in Mullar Hinton broth (MHB, HiMedia, India). The fungal strains
Candida albicans NCPF 400034, Candida glabrata MTCC3019, Candida krusei NCPF 44002,
Candida parapsilosis NCPF 450002, Candida keyfer, NPCPF 410004 and Candida tropicalis
NPCPF420007 were cultured in yeast extract-peptone-dextrose (YEPD broth, HiMedia, India)
and RPMI 1640 media (HiMedia, India). For agar plates, 2.5% (w/v) bacteriological agar
(HiMedia, India) was added to the medium. The strains were stored with 15% glycerol at
-80˚C as frozen stocks. The cells were freshly revived on respective agar plates from the stock
before each experiment [43,44].
Antibiotic susceptibility testing
Antibacterial activity. All the bacterial strains (E. coli, S. aureus, E. faecalis, V. cholerae, S.
pyogenes) were grown overnight and were diluted in Mueller-Hinton broth (MHB) to a cell
density of 105 CFU/mL. 100 μL of this culture and compounds 1a-1g (250–0.112 μM) dis-
solved in DMSO, were added into the 96-well flat bottomed microtiter plate (Genexy-HiMe-
dia, India). The plate was incubated at 37˚C without shaking for 24 h. The visual and optical
density was measured using microplate reader at 600 nm (Thermo, Model 680). The minimum
inhibitory concentration (MIC) was defined as the concentration of the antimicrobial agent
that inhibit >99% growth. kanamycin (KAN), a known antibacterial drug is used as a positive
control [45].
Antifungal activity. The antifungal activities of compounds 1a-1g against fungal species
(C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. keyfer, C. tropicalis) were performed
according to the Clinical and Laboratory Standards Institute (CLSI, formerly NCCL) in
RPMI-1640 medium by broth micro-dilution methods [46]. The concentrations of com-
pounds 1a-1g ranged between 250 μM and 0.097 μM. The 96 well flat bottom microtiter plates
were incubated without shaking at 30˚C for 48 h. The visual and optical density was measured
using microplate reader at 492 nm (Thermo, Model 680) to study the inhibition in fungal
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
growth. The MIC was defined as the lowest concentration of antifungal drug which resulted in
>99% inhibition of growth compared to that of the untreated control. amphotericin B, a
known antifungal drug is used as a positive control.
Time kill assay. S. aureus exponential phase cells (~1 x 105 CFU/mL) was inoculated in
MHB medium containing compound 1f (at 15.67 μM conc.). The S. aureus cells were incu-
bated (37˚C; 200 rpm) and 100 μl aliquots were removed at pre-determined time points (0, 4,
8, 12, 16, 20 and 24 h). The aliquots were serially diluted (10 fold) in saline and plated on the
MHA (Muller Hinton Agar) plates. The numbers of colonies were counted after incubating
the plates at 37˚C for 24 h. Similarly C. keyfer exponential phase cells (~1 x 104 CFU/mL) were
inoculated in RPMI 1640 medium containing compound 1f (62.25 μM). The tubes were incu-
bated (30˚C; 200 rpm), and 100 μL aliquots were removed at pre-determined time points (0, 4,
8, 12, 16, and 24 h). The aliquots were serially diluted (10 fold) in saline water and plated on
YEPD agar plates. The numbers of colonies were counted after incubating the plates at 30˚C
for 48 h [44].
Checkerboard assay of compound 1b and 1f. The interaction of compound 1b and 1f
with well-known antimicrobials kanamycin, and amphotericin B and fluconazole were evalu-
ated by the checkerboard method as described previously and represented as the fractional
inhibitory concentration (FIC) index (FICI) i.e. sum of the FIC for each compounds. FIC
value of the most effective combination was used in calculating the FICI. FICI = FIC of X
+ FIC of Y = CXcomb/ MICXalone+ CXcomb/ MICYalone, Where, MICXalone and MICYalone are the
MICs of drug X and Y when acting alone and CXcomb and CYcomb are concentrations of drugs
X and Y at the isoeffective combinations, respectively. The interaction was described as syner-
gistic when the FICI was ꢀ 0.5, and when > 4.0, it was interpreted as antagonist and any value
in between as in different [45].
Scanning electron microscopy (SEM) of 1f treated S. aureus and C. keyfer cells. The cell
suspensions of S. aureus and C. keyfer in exponential phase were prepared in MHB and RPMI
1640 medium (pH 7), respectively. Compound 1f (15.67μM) was added to the S. aureus cells
(~1 x 105 CFU/mL), whereas 1f (62.25 μM) was added to the C. keyfer (~1 x 104 CFU/mL) and
incubated at 37˚C (S. aureus for 12 h) and 30˚C (C. keyfer for 12 h). After treatment the sam-
ples for SEM study were prepared as described previously and observed under the SEM
(JEOL-6100F, JEOL, Japan) [47].
Mammalian cell toxicity. The mammalian toxicity of compounds 1a-1g against Hek-293
(normal human embryonic kidney cells) and HeLa (human cervical cancer cells) by MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) protocol [48]. Briefly, the cells
(5 × 104 /well) were cultured in RPMI 1640 medium supplemented with 10% Fetal Bovin
Serum (FBS) in 96-well microtiter plate at 37˚C for overnight. The next day compounds 1a-1g
at MIC conc. were added to the cells in separate wells and incubated at 37˚C for 18 h. The cells
were further incubated at 37˚C for 3 h to 4 h in 20 μL of MTT solution (5 mg/mL) in phos-
phate buffer saline (PBS). Further, after removing supernatant (120 μL), Dimethyl sulfoxide
(DMSO; 100 μL) was added, and the suspension was mixed to dissolve the formazan crystals.
The percent viability of cells was calculated by the ratio of O.D570 of treated cells to the O.D570
of untreated cells. Untreated cells and novobiocin (aminocoumarin antibiotic) were taken as
negative and positive control, respectively.
Computational methods
DFT studies. Khon-Sham’s DFT method that undergo the gradient-corrected hybrid den-
sity functional B3LYP were used for all theoretical calculations [49]. The Becke’s three parame-
ters functional of Lee et al [50], is a combination of non-local exchange potential with the non-
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
local correlation function. A full geometry optimization was performed for all structures,
using this function [50] and the 6–31+g(d,p) bases set as implemented by Gaussian 03 package
[51]. All geometries were visualized using Avogadro 1.2 software package.
Molecular docking. The protein-ligand crystal structure has been optimized to its lower
energy conformation using protein preparation wizard where all the water molecules were
removed during preprocess and missing side chains of residues have been added using prime.
Keeping in mind the appropriate ionization states for the acidic as well as basic amino acids,
hydrogen atoms were added to the protein structure corresponding to the physiological pH
7.0. Finally energy minimization with root-mean-square deviation (RMSD) value of 0.30 Å
was carried out using optimized potentials for liquid simulations (OPLS-2005) force field after
assigning charge and protonation state. Ligand preparation was done by using the Schrodinger
LigPrep utility (Schrodinger, LLC, USA) which generates a low energy 3d structures.
The active site of protein was defined by a bounding box (grid) that was centered on the
native ligand in the crystal complex. Extra-precision glide docking (Glide XP) which docks
ligands flexibly was used to rank the docking poses and to gauze the binding affinity of these
ligands toward the protein.
ADME prediction. ADME properties were predicted for synthesized compounds 1a–1g
using computational methods. In this study, we have calculated QpLogPo/w, QP LogHERG,
QPPCaco, QpLogBB, QPPMDCK, QPlogKp, QpLogHSA, Percentage Human oral absorption
and Lipinski’s rule of five using QikProp.
Results and discussion
Chemistry
In view of our aim of building coumarin-thiazole-pyrazole (CTP) based molecular hybrids the
coumarin analog i.e., 6-substituted-3-acetylcoumarins (6) was synthesised from a reaction of
substituted salicylaldehydes with ethyl acetoacetate in the presence of piperidine as catalyst.
6-substituted-3-acetylcoumarins (6) thus formed was subjected to bromination using Br2/
CHCl3 to yield 6-substituted-3-bromoacetylcoumarins [52] (7a-b) (Fig 2). 3,5-Dimethyl-
4-aryldiazenylpyrazole-1-thiocarboxamides (5a-d) required for the condensation with
6-substituted-3-bromoacetylcoumarins (7a-b) were prepared by the reaction of thiosemicar-
bazide with 4a-d according to the previously reported procedures (Fig 2) [53,54]. The com-
pounds 4a-d were prepared by coupling of diazotized substituted aniline (2) with acetyl
acetone (3) at 0–5˚C (Fig 2).
In order to achieve the synthesis of 4-(coumarin-3-yl)-2-(3,5-dimethyl-4-aryldiazenylpyra-
zol-1-yl)thiazoles(1a-1g) exclusively, the condensation of 3,5-dimethyl-4-hydrazoarylpyra-
zole-1-thiocarboxamides (5) with 6-substituted-3-bromoacetylcoumarins (7a-b) was carried
out in refluxing ethanol under alkaline condition (Fig 2). The structure of all the synthesized
compounds were established by analyzing using IR, NMR (1H, 13C) and mass.
The 1H NMR spectra of compounds (1a-1g) revealed two singlets in the range δ 8.39–8.29
and 8.00–8.10 ppm corresponds to coumarin 4-H and thiazole 5-H, respectively, thereby con-
firming the formation of 1. Two Singlets in the range δ 2.19–2.32 and 2.69–2.86 ppm appeared
due to 3-CH3 and 5-CH3 group attached at pyrazole ring. It has been well established that
5-CH3 is more deshielded compared to 3-CH3 due to a lone pair of electrons present on the
nitrogen atom of the thiazole nucleus and therefore appeared at about δ 2.80ppm [41,55].
Moreover, the appearance of other signals in the NMR spectra corresponding to aromatic
protons and carbons furhter establish the structure of the synthesized compounds and com-
plete assignment of 1H NMR along with other spectral data has been given in experimental
section.
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
Fig 2. Synthesis of 4-(coumarin-3-yl)-2-(3,5-dimethyl-4-hydrazoarylpyrazol-1-yl)thiazoles (1a-1g).
https://doi.org/10.1371/journal.pone.0196016.g002
Biological evaluation
Antimicrobial activity. The antimicrobial activity of compounds (1a-1g) was evaluated
in vitro according to Clinical and Laboratory Standard Institute (CLSI) guidelines using five
clinical bacterial strains (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Vibrio
cholera and Streptococcus pyogens) and six fungal strains (Candida albicans, Candida krusei,
Candida glabrata, Candida keyfer, Candida tropicalis, Candida parapsilosis). The antimicrobial
activity of synthesized compounds (1a-1g) ranged from 250 μM to 15.67 μM and results have
been summarized in Tables 1 and 2. The activities and efficacies of the compounds were com-
pared with kanamycin, a routinely used standard antibiotic, and amphotericin B, a well-estab-
lished antifungal drug [56–58]. Interestingly, the compounds 1b and 1f have been found to be
the most potent antimicrobial agents among all tested compounds. When used as a standalone
antibacterial agent, the MIC of 1b was either comparable (S. aureus) or at least 50% reduced
(S. pyogens) compared to KAN. In case of 1f, the MIC is 50% less compared to that of KAN
when tested against S. aureus, E. faecalis and S. pyogens.
Though MIC was much higher in case of antifungal screening, the compounds 1b and 1f as
standalone found to be more active in comparison to other candidates when Amp B was used
as reference drug. The results of antimicrobial activity study revealed that incorporation of hal-
ogen atoms (Cl and Br) on coumarinyl and phenyl moieties linked to pyrazolylthiazoles may
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
Table 1. Minimum inhibitory concentration (MIC) of the compounds against five bacterial strains.
Compound
Minimum inhibitory concentration valuesà (μM)
S. aureus
>250
31.25
>250
>250
>250
15.67
>250
31.25
E. coli
>250
>250
>250
>250
>250
62.50
>250
03.90
E. faecalis
>250
250
S. pyogens
V. cholerae
>250
250
1a
1b
>250
31.25
>250
>250
>250
31.25
>250
62.50
1c
>250
>250
>250
31.25
>250
62.50
>250
>250
>250
>250
>250
62.50
1d
1e
1f
1g
KAN#
ÃMIC values of the compounds were calculated as CFU plating method as described in materials and methods
#KAN, a frequently used antibacterial drug, was used as positive control for this study.
https://doi.org/10.1371/journal.pone.0196016.t001
be responsible for higher potential of 1b and 1f. Generally, substitution of hydrogen by halo-
gen in coumarin derivatives compounds increases the lipophilicity and thus increases the rate
of cell penetration, which is an important feature of antimicrobial drug efficiency [59]. Substi-
tution of chlorine at position-6 of coumarin ring in 1b and 1f significantly increased activity
against the bacterial and fungal strains. The results demonstrated that the antimicrobial activ-
ity of the investigated compounds was influenced by the physicochemical properties of the
type and position of substituent on the aromatic ring.
Furthermore, it is noteworthy here that S. aureus and candida sp. are responsible for major
opportunistic infections in clinical settings. There are growing concerns about the ability of S.
aureus to acquire resistance, even towards the newest antibacterial available in the market [60].
Similarly, C. keyfer infections mostly occur in patients with hematologic malignancies or carci-
nomas who are already undergoing highly cytotoxic chemotherapy [61]. KAN has been used
as a standard drug in infections with S. aureus [62]. Likewise, Amp B is a one of the primary
standard drugs used in case of candida infections [61,63]. Emergence of resistance against
these front line drugs has been a major public health problem. An important strategy to reduce
the emergence of drug resistant strains is by reducing the effective amount of drug required to
kill the infectious bacterial or fungal agent [64,65]. Therefore, combinatorial therapies or use
of adjuvant is encouraged or used to modulate the antimicrobial activity and mitigate the pos-
sibility of development of drug resistance [46].
Table 2. Minimum inhibitory concentration (MIC) of compounds against six fungal strains.
Compound
Minimum inhibitory concentration valuesà (μM conc.)
C. albicans
>250
62.50
>250
>250
>250
62.50
>250
0.78
C.glabrata
>250
250
C.keyferkeyfer
>250
C. krusei
>250
125
C. tropicalis
>250
125
C. parapsilosis
>250
1a
1b
62.50
62.50
1c
>250
>250
>250
250
>250
>250
>250
>250
62.50
>250
0.78
>250
>250
>250
125
>250
1d
>250
>250
1e
>250
>250
1f
62.50
62.50
1g
Amp B#
>250
0.78
>250
>250
0.78
>250
12.50
0.78
#Amp B, was used as positive control for this study
ÃMIC values of the compounds were calculated as CFU plating as described in materials and methods
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
Table 3. Synergistic effect of compounds 1b and 1f with antibacterial and antifungal drugs against S. aureus and C.keyfer.
Antibacterial/ Antifungal drug (μM) Compd. (μM) Conc. (μM) of the drug in combination with 1b or 1f Fraction inhibitory conc. index (FICI)a
Effect
4.0 (KAN)
31.25 (1b)
15.67 (1f)
62.50 (1b)
62.50 (1b)
62.50 (1f)
62.50 (1f)
0.5 (KAN)+7.81 (1b)
0.5 (KAN)+3.91 (1f)
3.12 (Amp B)+7.81 (1b)
0.08 (FLC)+15.62 (1b)
1.56 (Amp B)+6.25 (1f)
0.05 (FLC)+15.67 (1f)
0.500
0.374
0.364
0.450
0.240
0.375
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
4.0 (KAN)
12.50 (Amp B)
0.40 (FLC)
12.50 (Amp B)
0.40 (FLC)
aThe fraction inhibitory concentration index (FICI) value is defined as the combination of two active agents can exert inhibitory effects that are more than the sum of
their effect alone.
https://doi.org/10.1371/journal.pone.0196016.t003
Synergistic effect. As discussed above, we further evaluated whether the compounds 1b
or 1f can act as an adjunct to potentiate a reduction in MIC of KAN and Amp B against clinical
strains S. aureus and C. Keyfer, respectively. A checkerboard dilution assay has been used to
determine the fraction inhibitory concentration index (FICI) for the compounds 1b and 1f,
when used in combination with KAN and Amp B against S. aureus and C. Keyfer, respectively.
As evident from Table 3, we observed an FICI of ꢀ 0.5 in all drug combinations reported here
indicating a synergistic interaction between the compounds and known drugs. There was a
substantial 8-fold reduction in the MIC of KAN against S. aureus with compounds 1b, and
also with 1f. Likewise, in case of Amp B, there is a 5-fold reduction in MIC with compound 1b
and ~8 fold reduction with compound 1f against fungal pathogen C. keyfer.
Cytotoxic evaluation. Evaluation of cytotoxicity using HeLa cells (cervical cancer cell)
and Hek-293 cells (normal embryonic kidney cells) indicated that the compounds 1b and 1f
have comparable cytotoxicity (> 80% survival) at MIC concentrations (Fig 3) and negligible
cytotoxicity (> 90% survival) at the concentrations used in combination. Their cytotoxicity
was comparable with Novobiocin, a coumarin derivative used as a positive control [48]. Most
potent compounds emerging out of our screen 1b and 1f have comparable cytotoxicity at their
respective MIC concentrations.
Fig 3. Evaluation of cytotoxicity. Cell toxicity of compounds 1a-1g in term of viability of Hek-293and HeLa cells. Data represent
results from three independent experiments. Novobiocin was used as a positive control.
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
Fig 4. Time kill assay. Impact on growth of (A) S. aureus by compound 1f at sub inhibitory conc; (B) C. keyfer by compound 1f at sub
inhibitory conc.
https://doi.org/10.1371/journal.pone.0196016.g004
Since compounds 1b and 1f are active as an adjuvant at much lower concentrations (5–8
time less than their MIC values), we believe that these compounds can act as an ideal adjuvant
to increase the efficacy of KAN, Amp B or possibly another drugs too. Our experiments with
another antifungal agent fluconazole showed that usage of 1b and 1f as adjunct reduced the
MIC ~5-fold with 1b and 8-fold with 1f against C. keyfer substantiating the broad spectrum
effects of these derivatives as adjunct. Compound 1f consistently demonstrated the most
potent activity as a standalone as well as in combination to other drugs as an adjunct. So we
evaluated this compound for further analysis including a time kill kinetic assay.
Time kill kinetic. Time kill kinetic is an important assay to find out the efficacy and kill-
ing rate of antimicrobial compounds [66]. In the time kill assay, the Colony Forming Units
(CFUs) of the S. aureus and C. keyfer were rapidly reduced after treatment with the 1f (MIC
conc. 15.67 μM) and 1f (MIC conc. 62.50 μM), respectively. Maximum killing of S. aureus and
C. keyfer cells with 1f were observed after 24 h in comparison to untreated control. Samples
withdrawn at the indicated time points were evaluated for colony forming units (CFUs) at
MHA (Muller Hinton Agar) and YPEDA (Yeast Extract Peptone Dextrose Agar) medium. At
synergy concentrations of treatment with 1f (0.5 μM of KAN and 07.81 μM of 1f) against S.
aureus and (0.097 μM of Amp B and 31.25 μM of 1f) against C. keyfer showed killing after 12 h
(S. aureus) and 8 h (C. keyfer), respectively (Fig 4).
S. aureus and C. keyfer cells lysis by electron microscopy. The cell surface morphological
effects of the compound 1f on the S. aureus and C. keyfer were also investigated by Scanning
Electron Microscopy (SEM). A change in both S. aureus and C. Keyfer cell morphology was
observed in treated with compound 1f at MIC conc. (at 15.67 μM for S.aureus and 62.50 μM
for C. keyfer). Increased roughness and cell wall damage was observed within 12 h of incuba-
tion (Fig 5). The untreated S. aureus and C. keyfer cells serve as control [67].
As a result, compounds 1b and 1f in our screen displayed significant broad spectrum anti-
microbial activity against clinical strains of bacteria and fungi without any significant mamma-
lian cell cytotoxicity. Comparing the antibacterial activity of coumarin derivatives, 1b showed
the similar level of activity with MIC 31.25 μM and 1f showed an excellent level of activity with
MIC 15.67 μM when compared with standard drug, Kanamycin. Among the synthesized com-
pounds, the 1f showed the best activity against S. aureus (MIC 15.67 μM). The MIC value of
Amp B against C. keyfer is 12.50 μM. More promisingly, when used as an adjunct to standard
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
Fig 5. SEM images. (A) S. aureus, (A1) Untreated cells (40 K, 8.2 mm), (A2) Treated cells with 1f (40 K, 8.1mm), (B) C. keyfer,
(B1) Untreated cells (5 K, 5 mm), (B2) Treated cells (8.5 K, 2 mm). Arrows showed the damage of S. aureus and C. keyfer cells
treated with compound 1f.
https://doi.org/10.1371/journal.pone.0196016.g005
antimicrobial drugs, compounds 1b and 1f significantly enhanced the efficacy of these drugs
KAN, Amp B and FLC by significantly reducing the MIC values of these drugs.
DFT studies: Electrostatic results
The electron donating and receiving ability of a molecule can be determined by considering
HOMO and LUMO energy of that molecule. Higher the energy of HOMO higher will be its
electron donating ability. A full geometry optimization was performed for all compounds (1a-
1g), using Khon-Sham’s DFT method that undergo the gradient-corrected hybrid density
functional B3LYP method and the 6–31+g(d,p) bases set [49]. It has been found that the
energy of HOMO was comparable for all compounds. However, the LUMO orbital energies
were affected by the presence of electron donating or withdrawing or both groups on the struc-
ture. According to Mabkhot et al. [68], HOMO-LUMO energy gap (E gap) is an established
parameter to measure the extent of the intramolecular charge transfer and was used in phar-
maceutical studies. In this study, the compounds exhibited different antimicrobial activity, this
may be due to the difference in LUMO energy levels. Table 4 summarized the theoretical elec-
tronic parameters for investigating the coumarin-thiazole-pyrazole (CTP) based molecular
hybrids.
Correlation of biological assay with electrostatic results. The electrostatic results pre-
sented in Table 4 were plotted against the experimental biological activity of synthesized com-
pounds (Fig 6). The biological activity for excellent to low biological active compounds has
been correlated with energy of their LUMO. The compound 1b and 1f showed illustrious
LUMO energy of -2.647 eV and -2.648 eV respectively and also exhibited higher antibacterial
& antifungal activity than the other compounds. It was observed that compounds with more
stabilized LUMO orbitals have magnificent biological activity (Fig 6).
Table 4. Theoretical results of compounds 1a-1g.
Compound
Dipole moment (D)
HOMO-1 (eV)
-6.528
HOMO (eV)
-6.114
LUMO (eV)
-2.464
LUMO+1 (eV)
-2.435
E
gap (eV)
1a
1b
1c
1d
1e
1f
6.558
4.376
4.108
1.990
6.554
4.390
4.730
-3.65
-6.585
-6.175
-2.647
-2.531
-3.528
-3.567
-3.416
-3.629
-3.536
-3.659
-6.299
-5.933
-2.366
-2.213
-6.530
-5.997
-2.581
-2.268
-6.533
-6.107
-2.478
-2.435
-6.581
-6.184
-2.648
-2.517
1g
-6.368
-6.046
-2.387
-2.301
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
Fig 6. Correlation of biological activity with LUMO energy.
https://doi.org/10.1371/journal.pone.0196016.g006
The effect of LUMO energy on biological activity may be due to the presence of electron
withdrawing bromo and chloro groups in compound 1b and 1f. Presence of chloro group sta-
bilized LUMO to a higher extent than bromo which was evidenced by the comparison of MIC
of compounds 1b and 1f. Halogen substitution on coumarin ring stabilized LUMO energy
more than on phenyl ring. Relating LUMO energy with biological activity was not the only fac-
tor responsible for biological activity. There was a strong demand to look into other aspects
like the placement of these molecular orbitals on the molecule (Fig 6). Such investigations
revealed that location of LUMO along with their energy together influenced the biological
activity. The location of LUMO was particularly affected by the substitution of halogen on cou-
marin ring which was clearly observed in the correlation of LUMO of compounds 1a, 1b and
1f (Fig 6). It has been revealed from electrostatic results that biological activity of coumarin-
thiazole-pyrazole (CTP) hybrids was due to substitution of coumarinyl ring with electron
withdrawing groups which consequently lowered its LUMO energy. Therefore, a theoretically
suggestion about different biological activity of coumarin-thiazole-pyrazole (CTP) hybrids was
made based on the localization and energy of LUMO orbital.
Global reactivity descriptors. The chemical reactivity indices like chemical hardness (η),
electronegativity (χ), electronic chemical potential (μ), and electrophilicity Index (ω), were
also calculated for all the synthesized molecules. The stability and reactivity of a chemical sys-
tem is represented by chemical hardness which is given by η = (ELUMO- EHOMO)/2. This
descriptor is used as a measures of resistance to change in the electron distribution or charge
in a molecule [69]. Electronegativity is given by expression χ = -(EHOMO+ ELUMO) /2, which is
defined as the power of an atom in a molecule to attract electrons towards it [70]. The negative
term of electronegativity of a molecule is defined as Chemical potential [71] which is deter-
mined using equation μ = (EHOMO+ELUMO)/2. Electrophilicity index (ω), is a measures of the
propensity or capacity of a species to accept electrons was introduced by Parr and is calculated
using the electronic chemical potential and chemical hardness and given as ω = μ2/2η[72]. The
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
Table 5. Global reactivity descriptors for compounds 1a-1g.
Compound
E
H (eV)
EL(eV)
-2.464
-2.647
-2.366
-2.581
-2.478
-2.648
-2.387
μ (eV)
-4.289
-4.411
-4.149
-4.289
-4.292
-4.416
-4.216
χ (eV)
4.289
4.411
4.149
4.289
4.292
4.416
4.216
η (eV)
1.85
1.76
1.78
1.70
1.81
1.76
1.82
S (eV-1)
0.2739
0.2834
0.2803
0.2927
0.2755
0.2828
0.2732
ω (eV)
5.03
5.51
4.82
5.38
5.07
5.51
4.85
1a
1b
1c
1d
1e
1f
-6.114
-6.175
-5.933
-5.997
-6.107
-6.184
-6.046
1g
https://doi.org/10.1371/journal.pone.0196016.t005
calculated values of chemical reactivity indices for compounds 1a-1f are summarized in
Table 5. The low chemical hardness (1.76 eV) with high negative value of chemical potential
(-4.41 eV) indicates that compound 1a and 1f are comparatively soft molecule with high polar-
izability than other compounds of this series. The high electronegativity (4.41 eV) and electro-
philicity (5.51 eV) of 1a and 1f indicates its high power to withdrawn electrons and hence to
act as an electrophile.
Docking studies
Molecular docking is the most useful technique to explore the possible binding mode between
ligand and protein complex. The re-docking of native ligand into the crystal complex is a mea-
sure of accuracy of a molecular docking. The comparison between lowest energy pose and the
experimentally determined binding mode can be used to as a tool for determining the preci-
sion of binding mode. First of all the validation of the docking protocol was done by re-dock-
ing the native ligand into the active site of DNA gyrase B (PDB ID: 5L3J) with a root-mean-
square deviation (RMSD) of <1Å. This lower value of RMSD represents the accuracy and reli-
ability of the docking procedure in reproducing the experimentally observed results (Fig 7).
In the present manuscript, an attempt has been made to study the interactions of novel cou-
marin-thiazole-pyrazole (CTP) hybrids with DNA gyrase B. For this purpose molecular dock-
ing studies of all the synthesized compounds 1a-1g were performed in the binding pocket of
DNA gyrase B (PDB ID:5L3J). Four basic parameters-Glide score, Glide energy, H-bonds and
non-bonded interactions (van der Waals and Coulombic) were used to define the binding
affinity of ligands 1a-1g for DNA gyrase B. Glide score with more negative value represents
minimum energy for the formation of complex between ligand and receptor (Glide energy).
The docked poses for compounds 1b, 1g and KAN in the active site of DNA gyrase B are
shown in Figs 8–10 (Table 6). The most active compounds showed substantial binding affini-
ties towards the DNA gyrase B structures (Glide energy range -69.60 kcalmol-1 to -84.78 kcal-
mol-1) and the energy ranges are more than that of reference compounds KAN and CPF
(Table 6). It has been revealed from binding studies that all the synthesized compounds 1a-1g
fit snugly making various close contacts with the residues lining the active site of DNA gyrase.
In the binding of molecules 1a-1g to DNA gyrase B, the contribution made by van der waals
and columbic interactions are more prevalent over the electrostatic interactions. Extensive van
der Waals and columbic interactions have been observed with residues Asn 46, Ile 78, Met 95,
Val 120, Val 43, Val 167, Val 71, Thr 165, Ala 47, Glu 50, Gly 75, Asp 73, Gly 77, Arg 76, Pro
79, Asp 105, Ala 90 and Ile 94 lining the active site of DNA gyrase B. Despite these π–π and π-
cationic interaction are also present in case of all compounds with Phe 104 and Lys 103 residue
respectively. Docked conformations of all compounds (1a-1g) with DNA gyrase B are shown
in S1 Fig and S2 Fig. The binding pattern observed for these coumarin-thiazole-pyrazole
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
Fig 7. Native ligand (grey) overlap with its best docked pose (green) in a re-docking protocol.
https://doi.org/10.1371/journal.pone.0196016.g007
(CTP) based hybrids toward active site of DNA gyrase B opens up a new platform structure-
based design efforts.
Pharmacokinetic effect of synthesized compounds 1a-1g. Pharmacodynamics results
indicated that compounds (1a-1g) may prove good lead as antibacterial drugs by targeting
DNA gyrase B. For drug formulation pharmacokinetic properties are likewise a significant
prospect and should be brought into thoughtfulness. The pharmacokinetic parameters of all
synthesised hybrids have been studied along with standard drug KAN and CPF using QikProp
[73]. The major parameters studied for different synthesised compounds (1a-1g) were seems
to be acceptable and were incorporated in Table 7. The % oral drug absorption predicted for
all the test compounds 1a-1g was highly satisfactory with a high percentage (>80%) of Human
Oral Absorption as compared to the reference, indicating their possibilities in oral drug for-
mulation for the treatment of S. aureus infection.
The bowel-blood barrier was predicted using Caco-2 cell permeability (QPPCaco) as model
along with Lipinski’s rule of 5 [74] all the test compounds and showed very good values com-
pared to the standard Kanamycin and ciprofloxacin. Further, the prediction for human serum
albumin binding using QPlogKhsa, shows that the values for all the inhibitors lie within the
expected range (-1.5 to 1.5). Likewise, the brain/blood partition coefficient (QPlogBB) and
IC50 value of HERG K+ channel blockage (QPlogHERG) for all compounds shows satisfactory
values as compared with reference compounds (Table 7).
Fig 8. 2D and 3D docking pose showing interaction for compound 1b in the binding site of DNA gyrase B crystal structure (PDB ID:5L3J).
https://doi.org/10.1371/journal.pone.0196016.g008
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
Fig 9. 2D and 3D docking pose showing interaction for compound 1f in the binding site of DNA gyrase B crystal structure (PDB ID:5L3J).
https://doi.org/10.1371/journal.pone.0196016.g009
Conclusion
In conclusion, some new coumarinyl linked pyrazolylthiazoles have been synthesized and
explored for their potential as standalone or adjuvant antimicrobial agents against pathogenic
strains. Compounds 1b and 1f displayed significant antimicrobial activity without expressing any
significant mammalian cell cytotoxic effect. Compound 1b showed the similar level of antibacte-
rial activity with MIC 31.25 μM and 1f found best agent with MIC 15.67 μM particularly against
S. aureus when compared with standard drug, kanamycin. Interestingly, as an adjuvant to stan-
dard antimicrobial drugs, compounds 1b and 1f significantly enhanced the efficacy of the stan-
dard drugs KAN, Amp B and FLC by significantly reducing the MIC values of these drugs.
The results demonstrated that the antimicrobial activity of the investigated compounds was
influenced by the physicochemical properties of the type and position of substituent on the aro-
matic ring. The results based on electron microscopic investigations of cell morphology and cel-
lular structural integrity suggest that the antimicrobial activity of these compounds may in part
be attributed to initial cell wall damage, leading to action on the intracellular components of
cells, ultimately leading to cell death. The result of DFT studiy of compounds were correlated
with the observed antimicrobial activities which indicate that coumarin moiety plays an impor-
tant role in antimicrobial activities. Moreover, the molecular docking studies further helped in
supporting the experimental results and the ADME properties predicted further strengthen the
path towards oral drug formulation. Therefore, it is concluded that the compounds 1b and 1f
Fig 10. 2D and 3D docking pose showing interaction for standard drug KAN in the binding site of DNA gyrase B crystal structure (PDB ID:5L3J).
https://doi.org/10.1371/journal.pone.0196016.g010
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
Table 6. Binding data of lead compounds based on MMGB-SA binding energy study.
Entry GScore δG_Bind δG_Bind δG Bind δG_Bind Lipo δG_Bind vdW
Key Protein ligands interaction
(Kcal/mol) coulomb (Kcal/ Hbond (Kcal/
mol)
(Kcal/mol)
(Kcal/mol)
mol)
1a
1b
1c
1d
1e
1f
-5.58
-4.71
-5.61
-4.81
-5.6
-84.78
-81.17
-83.32
-69.60
-84.09
-73.32
-83.22
-5.23
-0.33
-45.12
-58.59
Phe 104, Asn 46, Ile 78, Met 95, Val 120, Val 43, Val 167, Val
71, Thr 165, Ala 47, Glu 50, Gly 75, Asp 73, Gly 77, Arg 76, Lys
103, Pro 79, Asp 105, Ala 90, Ile 94.
-4.92
-4.85
-5.55
-5.33
-5.02
-5.04
-0.17
-0.33
-0.19
-0.33
-0.19
-0.33
-46.15
-44.42
-37.32
-45.16
-40.08
-43.65
-58.55
-58.49
-56.07
-58.76
-56.71
-57.87
Asp 105, Pro 79, Ile 94, Ile 78, Asn 46, Val 120, Val 167, Val 43,
ALA 47, Asp 73, Thr 165, Gly 77, Glu 50, Arg 76, Arg 136, His
55, Lys 103.
Phe 104, Asn 46, Ile 78, Met 95, Val 120, Val 43, Val 167, Val
71, Thr 165, Ala 47, Glu 50, Gly 75, Asp 73, Gly 77, Arg 76, Lys
103, Pro 79, Asp 105, Ala 90, Ile 94.
Phe 104, Asn 46, Ile 78, Met 95, Val 120, Val 43, Val 167, Val
71, Thr 165, Ala 47, Glu 50, Gly 75, Asp 73, Gly 77, Arg 76, Lys
103, Pro 79, Asp 105, Ala 90, Ile 94, His 55, Arg 136.
Phe 104, Asn 46, Ile 78, Met 95, Val 120, Val 43, Val 167, Val
71, Thr 165, Ala 47, Glu 50, Gly 75, Asp 73, Gly 77, Arg 76, Lys
103, Pro 79, Asp 105, Ala 90, Ile 94.
-4.74
-5.69
Phe 104, Asn 46, Ile 78, Thr 165, Ala 47, Glu 50, Gly 75, Asp 73,
Gly 77, Arg 76, Lys 103, Pro 79, Asp 105, Ala 90, Ile 94, His 55,
Arg 136
1g
Phe 104, Ile 78, Asn 46, Met 95, Val 120, Val 43, Val 167, Val
71, Thr 165, Ala 47, Glu 50, Gly 75, Asp 73, Arg 76, Gly 77, Lys
103, Pro 79, Asp 105, Ala 90, Ile 94.
KAN -4.74
CPF -5.11
-27.14
-45.97
-28.36
-1.83
-3.72
-0.88
-15.85
-31.81
-23.81
-31.63
Asp 105, Phe 104, Lys 103, Ala 53, His 55, Glu 50, Pro 79, Ile
78, Gly 77, Arg 76, Arg 136.
Phe 104, Asn 46, Ile 78, Thr 165, Ala 47, Glu 50, Gly 75, Asp 73,
Gly 77, Arg 76, Lys 103, Pro 79, Asp 105, His 55, Arg 136, Ile
94.
https://doi.org/10.1371/journal.pone.0196016.t006
Table 7. Predicted ADME properties of synthesized compounds 1a-1g and standard drug KAN and CPF.
Compounds
QP
logPo/wa
QP
logHERGb
QPPCacoc
QP
logBBd
QPPMDCKe
QPlogKpf QPlogKhsag Percent Human Oral Lipinski’s rule
Absorptionh
of five
Acceptable
range
(-2.0to6.5)
> -5.0
<25 poor > 500
great
(-3 to
1.2)
<25 poor > 500
great
(-8.0 to
-0.1)
(-1.5 to 1.5)
> 80% High, > 25%
low
< 5
1a
1b
4.96
5.55
4.78
5.28
5.03
5.47
4.45
-6.58
0.27
-7.25
-7.16
-7.24
-7.15
-7.29
-7.14
-7.38
-6.19
-3.13
1234.4
1234.5
1234.3
1187.6
1187.6
1234.5
1187.6
0.031
-0.36
-0.19
-0.54
-0.41
-0.37
-0.20
-0.54
-2.93
-0.67
2402.5
6376.3
973.0
2307.2
2481.5
5930.3
934.7
0.009
8.25
-1.59
-1.76
-1.62
-1.82
-1.62
-1.76
-1.45
-11.20
-6.54
0.59
0.74
0.63
0.76
0.62
0.72
0.46
-1.27
0
100
88.874
100
0
2
0
1
2
1
0
2
0
1c
1d
100
1e
85.565
100
1f
1g
100
KAN
CPF
0
12.6
48.315
aPredicted octanol/water partition co-efficient logp.
b Predicted IC50 values to block HERG K+channels.
c Predicted Caco-2 cell permeability.
d Predicted brain/blood partition coefficient.
e Predicted apparent MDCK cell permeability.
f Predicted skin permeability.
g Predicted binding to human serum albumin.
h Predicted oral absorption of drug in percentage term.
https://doi.org/10.1371/journal.pone.0196016.t007
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
may act as lead compounds for the development of potent antibacterial and antifungal agents
and as adjuvant they have potential to unlock the microbial resistance to antibiotics.
Supporting information
S1 Fig. 2D docking pose showing interaction for compound 1a-1g in the binding site of
DNA gyrase B crystal structure (PDB ID:5L3J).
(TIF)
S2 Fig. 3D docking pose showing interaction for compound 1a-1g in the binding site of
DNA gyrase B crystal structure (PDB ID:5L3J).
(TIF)
S3 Fig. 1H NMR spectrum of 4-(coumarin-3-yl)-2-(3,5-dimethyl-4-(4-chlorophenyl) diaze-
nyl)pyrazol-1-yl)thiazole (1a).
(TIF)
S4 Fig. 1H NMR spectrum of 4-(6-chlorocoumarin-3-yl)-2-(3,5-dimethyl-4-(4-bromophe-
nyl)diazenyl)pyrazol-1-yl)thiazole (1b).
(TIF)
S5 Fig. 1H NMR spectrum of 4-(6-chlorocoumarin-3-yl)-2-(3,5-dimethyl-4-(4-methylphe-
nyl)diazenyl)pyrazol-1-yl)thiazole (1d).
(TIF)
S6 Fig. 1H NMR spectrum of 4-(coumarin-3-yl)-2-(3,5-dimethyl-4-(4-bromophenyl) diaze-
nyl)pyrazol-1-yl)thiazole (1e).
(TIF)
S7 Fig. 1H NMR spectrum of 4-(6-chlorocoumarin-3-yl)-2-(3,5-dimethyl-4-(4-chlorophe-
nyl)diazenyl)pyrazol-1-yl)thiazole (1f).
(TIF)
S8 Fig. 13C NMR spectrum of 4-(Coumarin-3-yl)-2-(3,5-dimethyl-4-(4-chlorophenyl) dia-
zenyl)pyrazol-1-yl)thiazole (1a).
(TIF)
S9 Fig. 13C NMR spectrum of 4-(6-Chlorocoumarin-3-yl)-2-(3,5-dimethyl-4-(4-bromophe-
nyl)diazenyl)pyrazol-1-yl)thiazole (1b).
(TIF)
S10 Fig. 13C NMR spectrum of 4-(Coumarin-3-yl)-2-(3,5-dimethyl-4-(4-methylphenyl)
diazenylpyrazol-1-yl)thiazole (1c).
(TIF)
S11 Fig. 13C NMR spectrum of 4-(Coumarin-3-yl)-2-(3,5-dimethyl-4-(4-bromophenyl) dia-
zenyl)pyrazol-1-yl)thiazole (1e).
(TIF)
S12 Fig. 13C NMR spectrum of 4-(6-Chlorocoumarin-3-yl)-2-(3,5-dimethyl-4-(4-chloro-
phenyl)diazenyl)pyrazol-1-yl)thiazole (1f).
(TIF)
S13 Fig. 13C NMR spectrum of 4-(Coumarin-3-yl)-2-(3,5-dimethyl-4-phenyldiazenyl) pyr-
azol-1-yl)thiazole (1g).
(TIF)
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CTP hybrids: Potential standalone or adjuvant antimicrobial agents
S14 Fig. IR spectrum of 4-(coumarin-3-yl)-2-(3,5-dimethyl-4-(4-chlorophenyl) diazenyl)
pyrazol-1-yl)thiazole (1a).
(TIF)
S15 Fig. IR spectrum of 4-(Coumarin-3-yl)-2-(3,5-dimethyl-4-(4-methylphenyl) diazenyl-
pyrazol-1-yl)thiazole (1c).
(TIF)
S16 Fig. IR spectrum of 4-(coumarin-3-yl)-2-(3,5-dimethyl-4-(4-bromophenyl) diazenyl)
pyrazol-1-yl)thiazole (1e).
(TIF)
S17 Fig. Mass spectrum for 4-(6-chlorocoumarin-3-yl)-2-(3,5-dimethyl-4-(4-bromophe-
nyl)diazenyl) pyrazol-1-yl)thiazole (1b).
(TIF)
S18 Fig. Mass spectrum for 4-(coumarin-3-yl)-2-(3,5-dimethyl-4-(4-bromophenyl)diaze-
nyl)pyrazol-1-yl)thiazole (1e).
(TIF)
S19 Fig. Mass spectrum for 4-(6-chlorocoumarin-3-yl)-2-(3,5-dimethyl-4-(4-chlorophenyl)
diazenyl)pyrazol-1-yl)thiazole (1f).
(TIF)
Acknowledgments
Dr. R. Kataria and Dr. I.K. Maurya gratefully acknowledge the SAIF, Punjab university for
spectral data and Central University Bhatinda for providing computational studies.
Author Contributions
Conceptualization: Sunil Kumar, Jayant Sindhu, Ramesh Kataria, Vinod Kumar.
Data curation: Sunil Kumar, Indresh K. Maurya.
Formal analysis: Sunil Kumar, Indresh K. Maurya, Mukesh Kumari.
Investigation: Sunil Kumar, Indresh K. Maurya, Mukesh Kumari.
Methodology: Sunil Kumar, Indresh K. Maurya, Jayant Sindhu.
Supervision: Vikram Saini, Ramesh Kataria, Vinod Kumar.
Validation: Vikram Saini, Indresh K. Maurya, Vinod Kumar.
Writing – original draft: Sunil Kumar, Vikram Saini, Jayant Sindhu, Ramesh Kataria, Vinod
Kumar.
Writing – review & editing: Sunil Kumar, Vikram Saini, Jayant Sindhu, Ramesh Kataria,
Vinod Kumar.
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