Synthesis of novel thymol derivatives against MRSA and ESBL producing pathogenic bacteria

Article abstract of DOI:10.1080/14786419.2018.1474465

Twelve substituted aryl-azo-thymol derivatives (4a to 4 l) were synthesized and characterized by several spectral techniques such as, FTIR, UV-vis, proton NMR, Mass spectrometry and elemental analysis. Antimicrobial activities were evaluated by agar-well diffusion method against isolated MRSA, ESBL-producing pathogenic bacteria and antifungal resistant fungi, in vitro. In addition, drug likeness properties of derivatives were assessed through bioinformatic tools such as, PASS prediction, molecular docking and Lipinski rules of five, along with determination of toxic nature and LD₅₀ values. Among 12 derivatives, 4a, 4b, 4c, 4 g, 4i, 4j and 4 k had significant antibacterial and antifungal activities with minimum inhibitory concentration values, 40 to 80 μg/ml. Moreover, the docking scores of derivatives were ?8.27 to ?11.44 kcal/mol, against 4 bacterial targets and ?9.45 to ?12.49 kcal/mol against 2 fungal targets. Thus, from in vitro and in silico studies, thymol derivatives had control of MRSA, ESBL-producing bacteria and antifungal resistant fungi.

Full text of DOI:10.1080/14786419.2018.1474465

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Synthesis of novel thymol derivatives against
MRSA and ESBL producing pathogenic bacteria
Shasank S. Swain, Sudhir K. Paidesetty & Rabindra N. Padhy
To cite this article: Shasank S. Swain, Sudhir K. Paidesetty & Rabindra N. Padhy (2018):
Synthesis of novel thymol derivatives against MRSA and ESBL producing pathogenic bacteria,
Natural Product Research, DOI: 10.1080/14786419.2018.1474465
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Natural Product research, 2018
https://doi.org/10.1080/14786419.2018.1474465
Synthesis of novel thymol derivatives against MRSA and ESBL
producing pathogenic bacteria
Shasank S. Swaina, Sudhir K. Paidesettyb and Rabindra N. Padhya
acentral research laboratory, IMs and sum hospital, siksha ‘o’ anusandhan (deemed to be university),
Bhubaneswar, India; bdepartment of Pharmaceutical chemistry, school of Pharmaceutical sciences, siksha‘o’
anusandhan (deemed to be university), Bhubaneswar, India
ABSTRACT
ARTICLE HISTORY
received 8 January 2018
accepted 5 May 2018
Twelve substituted aryl-azo-thymol derivatives (4a to 4 l) were
synthesized and characterized by several spectral techniques such
as, FTIR, UV-vis, proton NMR, Mass spectrometry and elemental
analysis. Antimicrobial activities were evaluated by agar-well diffusion
method against isolated MRSA, ESBL-producing pathogenic bacteria
and antifungal resistant fungi, in vitro. In addition, drug likeness
properties of derivatives were assessed through bioinformatic tools
such as, PASS prediction, molecular docking and Lipinski rules of five,
along with determination of toxic nature and LD50 values. Among 12
derivatives, 4a, 4b, 4c, 4 g, 4i, 4j and 4 k had significant antibacterial
and antifungal activities with minimum inhibitory concentration
values, 40 to 80 μg/ml. Moreover, the docking scores of derivatives
were −8.27 to −11.44 kcal/mol, against 4 bacterial targets and −9.45
to −12.49 kcal/mol against 2 fungal targets. Thus, from in vitro and in
silico studies, thymol derivatives had control of MRSA, ESBL-producing
bacteria and antifungal resistant fungi.
KEYWORDS
thymol derivative;
antibacterial activities;
antifungal activity; molecular
docking
1. Introduction
Gram-negative (GN) and Gram-positive (GP) pathogenic bacteria cause morbidity and mor-
tality from infections. The first antibiotic penicillin or 6-aminopenicillanic acid (6-APA) could
control infections from those bacteria, and later on penicillin was resisted by target bacteria
(Chambers and Deleo 2009; Canton and Morosini 2011). Moreover, pathogenic bacteria
CONTACT rabindra N. Padhy
rnpadhy54@gmail.com, rabindranathpadhy@soa.ac.in
© 2018 Informa uK limited, trading as taylor & Francis Group

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S. S. SWAIN ET AL.
developed further resistance individually to newly introduced antibiotics (Davies and Davies
2010; Canton and Morosini 2011; Swain and Padhy 2015). Thus, multidrug resistant (MDR)
bacteria must be controlled with an iron-hand, which has been giving impetus for search
for newer antibacterials from conventional and non-conventional sources, apart from chem-
ical modification of antibiotics. Several phytochemicals were reported having antibacterial
and antifungal activities and lend themselves to the preparation of mainstream medicine
(Cowan 1999; Vázquez-Sánchez et al. 2015).
Thymol, 2-isopropyl-5-methylphenol is a natural monocyclic monoterpene phenol avail-
able in several plants such as, Thymus vulgaris and other Thymus sp., Cinnamonum zeylanicum,
Eugenia caryophyllus, etc., had been reported as a potent antibacterial, antifungal and anti-
oxidative agent (Falcone et al. 2005; Bollenbach 2015; Vázquez-Sánchez et al. 2015). However,
phytochemicals could not be used directly for unknown pharmacological/ chemical con-
straint/ lack of drug likeness properties (Brown and Dawson 2015; Hughes et al. 2011; Swain
et al. 2017). Thus, a solution of this problem to improve the antibacterial activity along with
drug likeness property is attempted in the present study. Twelve thymol derivatives (4a-4 l)
were developed through conjugation with several amines for probable control of gruesome
human pathogenic bacteria and fungi, isolated from clinical samples; and those were
recorded as having improvement of several physico-chemical properties, suitable for further
consideration as drugable candidate. Moreover, molecular docking study was carried out
using β-lactamase as the bacterial target and sterol α-demethylase along with glu-
cosamine-6-phosphate synthase as fungal targets for evaluating activity of each derivative,
in silico.
2. Results and discussion
2.1. Chemistry, synthesis and characterization of thymol derivatives
Wave length values of individual aryl-azo-thymol derivatives were observed at a concentra-
tion 10−5 to 10−6 M by UV–visible spectrophotometry using ethanol as solvent. Derivatives
had strong bathochromic shifts, as the λmax values ranged from 344 to 361 nm. Similarly,
comparative HPLC chromatograms were carried out in reverse-phase Grace Alltima HP Amide
analytical column with a solution (v/v) in the ratio, 55:45:: phosphate buffer:acetonitrile; it
was evident that the synthesized derivatives had > 93 % purity, for example the derivative
4e (Figure S1).
Several spectral peaks in structures of 12 synthesized derivatives were confirmed from
FTIR and 1H NMR spectral data: peaks at 3225–3150, 1524–1470 and 1610–1600 cm−1 in FTIR
analysis are the groups of –OH str., –N = N–, and –C = C–aromatic str., respectively. FTIR of
4e confirms that functionalities were characterized (Figure S2). Moreover, the 1H NMR spec-
trum exhibited 4 sets of proton signals characteristic of the expected thymol moiety. With
the synthesized derivatives, two singlet aromatic proton signals were observed at ranges, δ
7.552–7.853 ppm and δ 6.921–7.253 ppm of H-3 and H-6 positions of thymol, respectively.
In addition to the cited protons, gem-dimethyl (isopropyl) and methyl protons of thymol
nucleus also appeared at ranges of δ 3.083–3.283 and 1.306–2.271 ppm as multiples and a
singlet, respectively. The proton NMR spectrum of 4e is elucidated (Figure S3).

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Table 1. antibacterial and antifungal activities of synthesized thymol derivatives monitored by agar-well
diffusion method in vitro.
Inhibition zone size (mm)
Isolated bacterial strains
Isolated fungal strains
Derivatives/RA
S. aureus
E. coli
K. pneumoniae
P. aeruginosa
A. fumigatus
C. albicans
4a
4b
4c
4d
4e
4f
4 g
4 h
4i
4j
4 k
4 l
19.2 0.34
20.0 0.63
21.3 0.76
18.8 0.45
19.4 0.48
16.8 0.62
19.2 0.86
16.8 0.71
19.5 0.18
22.9 0.58
15.2 1.20
18.8 0.82
17.6 0.34
–
19.3 0.73
18.6 0.60
20.8 0.56
19.3 0.76
16.9 0.42
18.0 1.67
19.4 0.28
16.7 0.78
19.6 0.38
19.8 0.54
18.8 0.90
17.0 0.82
16.0 0.42
–
18.3 0.73
21.4 0.58
19.8 0.61
18.4 0.56
16.9 0.42
18.0 1.67
19.4 0.28
19.7 0.78
19.6 0.38
20.4 0.54
19.5 0.90
18.2 0.46
17.6 0.28
–
18.8 0.62
20.6 0.70
21.4 0.47
19.3 0.38
17.8 0.66
19.9 0.81
18.0 0.44
17.9 0.36
20.4 0.42
21.6 0.34
19.3 0.72
17.6 0.56
17.9 0.52
–
21.3 0.62
22.8 0.70
23.2 0.47
19.9 0.38
20.8 0.66
19.9 0.81
22.6 0.44
21.4 0.36
24.4 0.42
25.6 0.34
20.3 0.72
20.6 0.56
–
20.8 0.62
21.6 0.70
22.4 0.47
20.3 0.38
21.8 0.66
20.9 0.81
22.2 0.44
20.9 0.36
20.6 0.42
24.8 0.34
21.4 0.72
21.2 0.56
–
*aMP
*Flc
19.6 0.34
20.2 0.45
Notes:
*reference antibiotic; aMP, ampicillin; Flc, fluconazole; –, not done. data with mean standard deviation from 6 replica-
tions.
2.2. In vitro antibacterial and antifungal study
In vitro antibacterial study against 4 isolated MDR bacterial strains, diameter values of inhi-
bition zones of thymol derivatives ranged from 13 to 23 mm, while the standard antibiotic,
amikacin had inhibition zones 16–18 mm (Table 1). Derivatives, 4a, 4b, 4c, 4e, 4i and 4j
caused inhibition zone sizes as 18–22 mm, and 4j had the highest inhibition zone as 22.9 mm
against MRSA. Similarly, inhibition zone sizes of 12 derivatives were > 20 mm against E. coli,
and inhibition zones against P. aeruginosa were > 22 mm (Table 1). It was clear that 4j was
the most effective derivative against clinically isolated MDR bacterial strains. Furthermore,
the recorded antifungal activity of derivatives as diameter values of inhibition zones ranged
from 19 to 25 mm, and 4j had the highest inhibition zones, 25 and 24 mm against A. fumig-
atus and C. albicans, respectively (Table 1). Minimum inhibitory concentration (MIC) values
of 12 synthesized conjugates were within the range, 40–80 μg/ml; and particularly conjugate,
4a, 4b, 4c, 4e, 4 g and 4j had 40–80 μg/ml as the lowest minimum bactericidal concentration
(MBC) value against S. aureus (Table S1). The recoded MIC and MBC values of the reference
antibiotic, gentamicin were 80 and > 100 μg/ml, respectively. Furthermore, 4a, 4d, 4f, 4i,
4j, 4 k and gentamicin had MIC values 40 μg/ml, and the rest conjugates had MIC val-
ues > 80 μg/ml, against E. coli; and MIC values of 4b, 4c, 4d, 4i, 4j and 4 l were 40 μg/ml and
corresponding MBC values were 160 μg/ml against K. pneumoniae. Similarly against P. aerug-
inosa, MIC values of thymol derivatives, 4b, 4 g, 4i, 4j and 4 k were 40 μg/ml and the corre-
sponding MBC values were within the range, 80 to > 100 μg/ml. (Table S1). Thus, these thymol
derivatives could be used as alternatively/ synergistically along with a class of antibiotics for
the control MDR bacterial strains. Moreover, the emergence of resistance of pathogenic
bacteria to drugs/ antibiotics has now become increasingly commonplace, due to two obvi-
ous reasons, the versatility of the microbial genetic system and the lack of stringent antibiotic
policy worldwide (Davies and Davies 2010; Canton and Morosini 2011). In this perspective,
chemical manipulation of phytochemicals were considered suitable for the use as derivative/

4
S. S. SWAIN ET AL.
Figure 1. target-ligand interactions visualized through PyMol software. A and B represent the interaction
of derivative 4b with β-lactamase of K. pneumoniae (PdB Id: 1N9B); C and D represent the interaction of
derivative 4j with sterol α-demethylase of A. fumigatus (PdB Id: 4uYl) during docking.
semi-synthetic drugs, like attempted examples for malaria and pathogenic bacteria (Brown
and Dawson 2015; Cochrane et al. 2009; Machado et al. 2018; Swain et al. 2017).
Moreover, the advance bioinformatics tools are not used only for optimization and selection
of effective agents through binding affinity through molecular docking (Table S2; Figure 1),
but it could predict biological characteristics, absorption and permeability properties of syn-
thesized thymol derivatives. Herein, the calculated estimates of oral absorption of derivatives
are > 80 %, which means chemicals are well soluble and have permeableproperties(Table2).
Moreover, it was too clear that except 4b, computational prediction for LD50 values of all deriva-
tives were > 2000 mg/kg body weight, confirming by PASS prediction that the synthesized
derivatives are safe as possible antimicrobial agents (Table S3). Thus, the computational
study too supports to in vitro study and helped to conclude that, thymol derivative have
comparatively more effective drug likeness properties to control MDR pathogens. In PASS
prediction, a chemical with‘a higher possible active (Pa) value than the corresponding pos-
sible inactive (Pi) value’was considered as the possible drug molecule for a specific biological
activity (Khurana et al. 2011). Moreover, Lipinski rule of five (RO5), based on molecular prop-
erties, molecular weight, numbers of hydrogen acceptors (H-ba), numbers of hydrogen
donors (H-bd) and cLogP (partition coefficient between n-octanol and water) values were
widely used in the selection-criterion for an active drug molecule (Lipinski et al. 2001); and
all derivatives are effective under RO5. Indeed, the ‘highest occupied molecular orbitals’
(HOMO) energy and the ‘lowest unoccupied molecular orbital’ (LUMO) energy describe

NATURAL PRODUCT RESEARCH
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6
S. S. SWAIN ET AL.
electron donor and accepter regions, respectively of an actively participating ligand during
the formation of a protein-ligand complex (Figures S4 and S5). Today, computational tools
are used to predict possible effective drug likeness molecule in current drug development
method to guide or avoid of synthesis unwanted chemical and save the time and resources
in early drug discovery (Cochrane et al. 2009; Hughes et al. 2011; Sliwoski et al. 2013; Swain
et al. 2015, 2017).
The molecular docking is used for deciphering the interaction between a small molecule
as the ligand with a target enzyme related to a particular disease (Leelananda and Lindert
2016). Advances computational strategies are more beneficial for locating effective drugable
candidate or ‘lead optimization’ thought virtual screening and dynamic techniques in the
current drug discovery process in comparison with traditional experimental rather, traditional
screening. Particularly, low cost and effective screening is the most advantage of a docking
process and it serves as an important tool in pharmaceutical research (Leelananda and
Lindert 2016; Swain et al. 2017).
3. Experimental
3.1. Syntheses of thymol derivatives
A series of azoaryl substituted derivatives (4a-4 l) were synthesized by coupling of diazonium
salt of aniline derivatives 2 (a to l) with thymol in the presence of 10 % sodium hydroxide
(see, Supplementary files for characterization reports of 12 thymol derivatives in Scheme-1).
+
The coupling component, thymol was attached by strong diazonium electrophilic N2 to
produce azo- substituted thymol, as thymol was the neutral nucleophile agent in the reac-
tion, as described (Sahoo and Paidesetty 2016; Swain et al. 2017). The obtained products
were filtered and washed with pre-cooled distilled water, and the final products were dried
at 30 °C and were re-crystallized by ethanol. High-performance liquid chromatography
(HPLC) study was carried out for checking purity of derivatives, using Shimadzu (Scientific
Instruments, Kyoto, Japan), by a reverse-phase Grace Alltima HP Amide analytical column
(150 × 4.6 mm; 3 μm) with the injection of an aliquot of 20 μL of each sample. Spectral char-
acterizations of derivatives were interpreted by Fourier transform infrared spectroscopy (FTIR)
using KBr discs with JASCO FTIR 4100 Spectrophotometer. Proton nuclear magnetic reso-
nance (1H NMR) spectra of each derivative was studied using tetramethylsilane as the internal
standard with Bruker 1H NMR 400 MHZ using deuterated chloroform (CDCl3) as solvent. Mass
spectrometry (MS) using Shimadzu-Mass Spectrometer and differential scanning calorimetry
with Mettler Toledo Stare system were carried out. Furthermore, elemental analyses of deriv-
atives were carried out by Perkin Elmer-2400 CHNO/S analyzer system.
3.2. In vitro antibacterial and antifungal study
Antibacterial activities of thymol derivatives were evaluated by the agar-well diffusion method
against 4 pathogenic bacterial (Dubey and Padhy 2013; Swain et al. 2017) and 2 antifungal
resistant pathogenic fungal strains isolated from clinical samples using Muller-Hinton agar
(MHA) and potato dextrose agar (PDA) media, respectively (Fan et al. 2011; [CLSI] Clinical and
Laboratory Standards Institute 2013; Swain and Padhy 2015). The MIC and MBC values of
individual derivatives were determined. The original stock solution (mg/ml) of individual

NATURAL PRODUCT RESEARCH
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derivatives along with the reference antibiotic was prepared in 10% DMSO, thereafter were
converted to different concentrations (μg/ml) of each tested sample by serial dilution method.
Using 96-well (12 × 6) micro titer plate MIC values as the lowest concentration that completely
inhibited bacterial growth by each sample was calculated (Dubey and Padhy 2013; Swain
et al. 2017). Bacteria from each well of the microtiter plate were sub-cultured on a nutrient
agar plates for the determination of the MBC value of each chemical.
3.3. Computational analysis
In present docking attempts, the bacterial β-lactamase species of S. aureus (Protein Data
Bank or PDB ID: 1MWS), Escherichia coli (PDB ID: 1XPB), Klebsiella pneumoniae (PDB ID: 1N9B)
and P. aeruginosa (PDB ID: 4HEF), while fungal targets sterol α-demethylase of Aspergillus
fumigatus (PDB ID: 4UYL) and glucosamine-6-phosphate synthase of Candida albicans (PDB
ID: 2POC) were used as putative drug targets for evaluating docking scores of each thymol
derivative (Nichols et al. 2014; Hargrove et al. 2015). Derivatives were designed by the soft-
ware ChemDraw Ultra for the intended docking study by software AutoDock Vina (Swain
et al. 2015, 2017). Moreover, interactions of protein-ligand complexes were visualized by
PyMOL software. Possible antiseptic, antiinfective, antiparasitic, antimycobacterial and anti-
protozoal activities of thymol derivatives were evaluated using PASS tool (http://www.phar-
maexpert.ru/passonline/). Molecular properties of derivatives were standardized in RO5,
using the software, ChemDraw Ultra along with Molsoft (http://molsoft.com/mprop/) and
Molinspiration (http://www.molinspiration.com/) tools. Additionally, molecular orbital sur-
faces, HOMO, LUMO and‘energy electrostatic potential charges’plots of 12 derivatives were
visualized using the software, Argus Lab.
4. Conclusion
From inhibition zone sizes, MIC and MBC values of all derivatives, 4a, 4b, 4c, 4 g, 4i, 4j and
4k were selected as the effective-most antimicrobial agents; nevertheless, all derivatives
had controlling capacity on MDR strains of GN bacteria along with fungal strains, in vitro and
could be used as newer anti-bacterial and antifungal agents after due pharmacologic vali-
dation. Furthermore, advanced computational tools, PASS, molecular docking, RO5 sup-
ported the in vitro work and could help assess several possible drugable information for
these synthesized aryl-azo-thymol derivatives. Thus, the computational tools could be con-
sidered as effective system in saving time and resources, in modern drug development era
that encourages for the synthesis of comparatively more effective antibacterial drugs.
Supplementary material
Synthesis procedure, antibacterial activity procedure with NMR data of thymol derivatives (4a to 4 l)
with more figures and tables are available in the supplementary material which are cited in appropriate
place in text. https://doi.org/10.1080/14786419.2018.1474465.

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S. S. SWAIN ET AL.
Acknowledgments
We are grateful to Deans, SPS and IMS and Sum Hospital, Siksha ‘O’ Anusandhan (Deemed to be
University), for facilities and encouragements. NMR Spectroscopy was done at NISER, Bhubaneswar,
Odisha and the HPLC study at Raghu College of Pharmacy, Visakhapatnam, India.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by Siksha ‘O’ Anusandhan (Deemed to be University) to SS Swain, for carry
out his PhD work.
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