Indian J Microbiol (Oct–Dec 2017) 57(4):499–502
DOI 10.1007/s12088-017-0675-z
SHORT COMMUNICATION
Antimicrobial Efficacy of Synthetic Pyranochromenones
and (Coumarinyloxy)acetamides
Abhishek K. Singh1 • Suchita Prasad1 • Bipul Kumar2 • Shiv Kumar1 •
Amitesh Anand2 • Shashank S. Kamble2 • Sunil K. Sharma1 • Hemant K. Gautam2
Received: 4 May 2017 / Accepted: 19 September 2017 / Published online: 9 October 2017
Ó Association of Microbiologists of India 2017
Abstract Four (1, 2, 4 and 6) synthetic quaternary
ammonium derivatives of pyranochromenones and
(coumarinyloxy)acetamides were synthesized and investigated for their antimicrobial efficacy on MRSA (Methicillin-resistant Staphylococcus aureus), and multi-drug
resistant Pseudomonas aeruginosa, Salmonella enteritidis
and Mycobacterium tuberculosis H37Rv strain. One of the
four compounds screened i.e. N,N,N-triethyl-10-((4,8,8trimethyl-2-oxo-2,6,7,8-tetrahydropyrano[3,2-g]chromen10-yl)oxy)decan-1-aminium bromide (1), demonstrated
significant activity against S. aureus, P. aeruginosa and M.
tuberculosis with MIC value of 16, 35, and 15.62 lg/ml
respectively. The cytotoxicity evaluation of compound 1 on
A549 cell lines showed it to be a safe antimicrobial
molecule, TEM study suggested that the compound led to
the rupture of the bacterial cell walls.
Keywords Antibacterial Antimycobacterial
Chromenones Cytotoxicity Pyranochromenones
Transmission electron microscopy
Electronic supplementary material The online version of this
article (doi:10.1007/s12088-017-0675-z) contains supplementary
material, which is available to authorized users.
& Sunil K. Sharma
sksharma@chemistry.du.ac.in
& Hemant K. Gautam
hemant@igib.res.in
1
Department of Chemistry, University of Delhi, Delhi 110007,
India
2
CSIR-Institute of Genomics and Integrative Biology,
Sukhdev Vihar, Mathura Road, Delhi 110025, India
The emergence of bacterial strains characterized by multiple antibiotic resistance outlines the legacy of past decades of antimicrobial use and misuse that led to thousands
of deaths every year [1, 2]. The remarkable antibacterial
activity offered by quaternary ammonium derivatives has
been well documented in the literature [3, 4]. In pursuit of
new antimicrobials with improved efficacy, our research
group has been actively involved in the synthesis of
chromenones and quinolinones with the successful incorporation of a quaternary ammonium group which have
been identified as promising antimicrobial agents [5–7].
Furthermore, investigation of pyranochromenones and
chromenones functionalized with quaternary ammonium
(1–3) and amido group (4–6) respectively, against various
pathogenic strains of bacteria and fungus, too, have
revealed them as potential antimicrobial ingredients
(Fig. 1) [8].
The present study deals with the synthesis of the four
quaternary ammonium derivatives of pyranochromenones
and (coumarinyloxy)acetamides (1, 2, 4 and 6) that were
studied for their antimicrobial efficacy against resistant
strains of Staphylococcus aureus (ATCC 43300), Pseudomonas aeruginosa (ATCC 27853), Salmonella enteritidis (ATCC 13076) and Mycobacteria (H37Rv). Also,
these compounds were subjected to the assessment of their
cytotoxicity profile, and transmission electron microscopic
details were used to explore the mechanism of action of the
most potent compound.
The compounds used in the present study were synthesized by following a literature procedure [8] and well
characterized from their physical and spectral data. pIodonitrotetrazolium chloride (INT) used in the determination of MIC was obtained from Sigma-Aldrich Chemicals, USA. TEM grid (3.05 mm (d) 200-mesh copper grid)
was procured from Icon Analytical Equipment Pvt. Ltd.,
123
500
Indian J Microbiol (Oct–Dec 2017) 57(4):499–502
Fig. 1 Active antimicrobial
compounds [8]
Table 1 MIC (lg/ml) of
compounds 1, 2, 4 and 6 against
resistant pathogenic bacterial
strains
Compound
Staphylococcus aureus
Pseudomonas aeruginosa
Salmonella enteritidis
H37Rv
1
16
35
100
15.62
2
50
80
250
62.50
4
200
110
90
[ 250
6
180
90
95
62.50
Gentamicin
100
20
20
–
Neomycin
250
35
35
–
Vancomycin
1.5
250
250
–
Isoniazid
–
–
–
0.30
Mumbai, India. Alamar blue used in anti-mycobacterial
activity was obtained from AbD Serotec, Oxford, UK.
A549 cell lines used to determine the cytotoxicity of
compound 1 were procured from ATCC. Kirby-Bauer disc
susceptibility test was used to determine the bacterial
growth inhibitory potency of the synthesized compounds
[9, 10]. The growth inhibitory potency of compounds 1, 2,
4 and 6 against M. tuberculosis strain H37Rv was assessed
by MDA as described by Swaroop et al. [11]. TEM study
was carried out in order to ascertain the morphological
changes in bacteria, following the protocol of Yadav et al.
[12] and Yuanqing et al. [13]. MTT colorimetric assay as
described by Riss et al. [14] was utilized to evaluate the
in vitro cell cytotoxicity of compound 1 using lung
epithelial cell line (A549).
The initial screening results revealed that compound 1
developed the highest zone of inhibition against resistant S.
aureus and P. aeruginosa in comparison to S. enteritidis
(Table S1, SI). To get a vivid picture about the antibacterial
potential of these compounds, minimum inhibitory concentration (MIC) was evaluated using broth dilution assay.
Compound 1 was observed to inhibit the growth of
pathogenic bacterial strains S. aureus (MRSA strain) and P.
aeruginosa at a concentration of 16 and 35 lg/ml,
respectively. However, on going to a shorter alkyl chain,
from ten carbons to six carbons (in compound 2), MIC
value increased and a concentration of 50 and 80 lg/ml
was required to inhibit the growth of resistant S. aureus and
P. aeruginosa, respectively. On the other hand, moderate
123
activity could be observed for (coumarinyloxy)acetamides
(4/6) against the above mentioned microbes (Table 1).
Encouraged by the antibacterial results of 1, 2, 4 and 6,
the inhibitory potency of these compounds against Mycobacterium tuberculosis strains H37Rv was also examined using the protocol as described by Swaroop et al. [11].
The results revealed that the compound 1 too, displayed
highest antimycobacterial activity (MIC: 15.62 lg/ml) in
comparison to compounds 2, 4 and 6 (Table 1).
To have insight into the mechanism of antimicrobial
action of the most potent compound 1 from all the assays,
morphological changes of untreated and treated resistant S.
aureus and P. aeruginosa with compound 1 at 5X MIC for
15 and 30 min was investigated using transmission electron
microscopy (TEM) (Fig. 2).
The results unraveled that the untreated S. aureus and P.
aeruginosa displayed an even and smooth surface, however
incubation of the bacterial strains with compound 1 led to
the rupture of cell walls and ultimately to cell death. This
substantiated that there was an efficient interaction of
compound 1 with the bacterial cell wall (Fig. 2). These
evidences suggest that quaternary ammonium compounds
(QACs) instigate the disruption of cell membrane with
subsequent leakage of intracellular components and cell
death due to electrostatic and hydrophobic interactions
with phospholipids and hydrophobic membrane core,
respectively.
In vitro cytotoxicity of the most active compound 1 was
examined using lung epithelial cell line (A549). The cell
Indian J Microbiol (Oct–Dec 2017) 57(4):499–502
Fig. 2 TEM images of
untreated and treated S. aureus
and P. aeruginosa with
compound 1 after 15 min and
30 min
501
Untreated
S. aureus
15 min treatment
30 min treatment
Untreated
P. aeruginosa
15 min treatment
30 min treatment
120.865
lines were treated with compound 1 at different multiple
concentrations of its MIC: 1X (16 lg/ml), 2X (32 lg/ml),
4X (64 lg/ml), 6X (96 lg/ml), 8X (128 lg/ml) and 10X
(160 lg/ml) and the viability was evaluated using MTT
assay. The cells were found to be *95–97% viable after
24 h of treatment at 1X, 2X and 4X concentration of its
MIC. However, on further increasing the concentration of
compound 1 (6X), the cell viability reduced to *60–80%,
and even higher reduction observed at 8X. At 10X concentration, the cell viability was reduced by a greater fold
(Figure S1, SI).
Thus, the compound 1 not only exhibit highest
antibacterial activity against resistant strains of S. aureus
and P. aeruginosa with MIC value of 16 and 35 lg/ml,
respectively, but also it was observed to be a safe antimicrobial candidate at its MIC value. Furthermore, the compound 1 was observed to be a good candidate for
antimycobacterial activity due to its capacity to inhibit
pathogenic M. tuberculosis H37Rv strain with MIC value
of 15.62 lg/ml. The inhibitory effect of compound 1 on S.
aureus and P. aeruginosa could be seen through TEM
studies, wherein the morphological changes in bacterial
cells were evident after 15 and 30 min of incubation at 5X
MIC.
Acknowledgements We gratefully acknowledge the financial assistance from the University of Delhi, DU-DST PURSE Grant Phase II
and CSIR-TOUCH (BSC0302) project. The authors are also thankful
to CSIR/UGC, New Delhi for the financial support (JRF/SRF).
Compliance with Ethical standards
Conflict of interest The authors declare that they have no conflict of
interests.
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