Promising bactericidal approach of dihydrazone analogues against bio-film forming Gram-negative bacteria and molecular mechanistic studies

Article abstract of DOI:10.1039/c7ra13661g

Gram-negative members of the ESCAPE family are more difficult to treat, due to the presence of an additional barrier in the form of a lipopolysaccharide layer and the efficiency of efflux pumps to pump out the drugs from the cytoplasm. The development of alternative therapeutic strategies to tackle ESCAPE Gram-negative members is of extreme necessity to provide a solution to the cause of life-threatening infections. The present investigations demonstrated that compounds 17, 20, 25 and 26 possessing the presence of electron donating (OH and OCH₃) groups on the phenyl rings are highly potent; whereas compounds 9, 10, 15, 16, 18, 33 and 36 showed moderate activity against Gram-negative bacteria. An excellent dose-dependent antibacterial activity was established compared to that of the standard antibiotic ampicillin. Significant anti-biofilm properties were measured quantitatively, showing optical density (O.D) values of 0.51 ± 015, 0.63 ± 0.20, 0.38 ± 0.07 and 0.62 ± 0.11 at 492 nm and the leakage of cellular components by the compounds, such as 17, 20, 25 and 26, increased the O.D. of respective treated samples compared to the control. In addition, the implication of experimental results is discussed in the light of the lack of survivability of planktonic bacteria and biofilm destruction in vitro. These results revealed the great significance of the development of a new generation of synthetic materials with greater efficacy in anti-biofilm properties by targeting to lock the bio-film associated protein Bap in Gram-negative bacteria.

Full text of DOI:10.1039/c7ra13661g

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Promising bactericidal approach of dihydrazone
analogues against bio-film forming Gram-negative
bacteria and molecular mechanistic studies†
Cite this: RSC Adv., 2018, 8, 5473
a
b
c
d
K. P. Rakesh,
S. N. A. Bukhari,
H. K. Vivek,
H. M. Manukumar,
C. S. Shantharam,
a
a
e
*
*
Hua-Li Qin and M. B. Sridhara
Gram-negative members of the ESCAPE family are more difficult to treat, due to the presence of an
additional barrier in the form of a lipopolysaccharide layer and the efficiency of efflux pumps to pump
out the drugs from the cytoplasm. The development of alternative therapeutic strategies to tackle
ESCAPE Gram-negative members is of extreme necessity to provide a solution to the cause of life-
threatening infections. The present investigations demonstrated that compounds 17, 20, 25 and 26
possessing the presence of electron donating (OH and OCH3) groups on the phenyl rings are highly
potent; whereas compounds 9, 10, 15, 16, 18, 33 and 36 showed moderate activity against Gram-
negative bacteria. An excellent dose-dependent antibacterial activity was established compared to that
of the standard antibiotic ampicillin. Significant anti-biofilm properties were measured quantitatively,
showing optical density (O.D) values of 0.51 Æ 015, 0.63 Æ 0.20, 0.38 Æ 0.07 and 0.62 Æ 0.11 at 492 nm
and the leakage of cellular components by the compounds, such as 17, 20, 25 and 26, increased the
O.D. of respective treated samples compared to the control. In addition, the implication of experimental
results is discussed in the light of the lack of survivability of planktonic bacteria and biofilm destruction in
vitro. These results revealed the great significance of the development of a new generation of synthetic
materials with greater efficacy in anti-biofilm properties by targeting to lock the bio-film associated
protein Bap in Gram-negative bacteria.
Received 26th December 2017
Accepted 22nd January 2018
DOI: 10.1039/c7ra13661g
rsc.li/rsc-advances
growth of microbial targets while causing minimal or no harm
to the host. Most antimicrobial drugs currently in clinical use
Introduction
because the prokaryotic cell provides a greater variety of unique
targets for selective toxicity, in comparison to fungi, parasites,
and viruses. Each class of antibacterial drugs has a unique
mode of action (the way in which a drug affects microbes at the
cellular level). Based on these facts and the high degree of
resistance of Gram-negative bacteria with the development of
multidrug-resistant (MDR-resistant to three or more classes of
antimicrobials) and extensively drug-resistant (XDR-resistant to
one or two classes of antimicrobials) strains, as well as the
threatening risk of the development of pan-drug resistant (PDR-
resistant to all classes of antimicrobials) strains, researchers
have paid particular attention to the infection rate caused by
this group.2,3 Usually, Gram-negative bacteria are commonly
associated with intra-abdominal infections (IAIs), bacteremia,
ventilator-associated pneumonia (VAP) and urinary tract infec-
tions (UTIs). The main pathogens involved in these infections
are Escherichia coli, Klebsiella pneumoniae and Pseudomonas
aeruginosa. Together, these species account for 70% of all Gram-
negative bacteria causing health care associated infections in
the US.4
Traditional methods employed to discover new antibiotics are
time-consuming and nancially-taxing. This has led researchers
to mine existing libraries of clinical molecules in order to
repurpose old drugs for new applications (as antimicrobials).
Bacterial resistance to antibiotics is a signicant public health
challenge, as infections caused by antibiotic-resistant bacteria
claim the lives of nearly 23 000 people each year in the United
States alone.1 An important quality for an antimicrobial drug is
selective toxicity, meaning that it selectively kills or inhibits the
aDepartment of Pharmaceutical Engineering, School of Chemistry, Chemical
Engineering and Life Science, Wuhan University of Technology, 205 Luoshi Road,
Wuhan, 430073, PR China. E-mail: qinhuali@whut.edu.cn; Fax: +86 27 87749300
bAnalytical Research and Development, Syngene International Ltd, Biocon Park,
Bommasandra Industrial Estate, Bangaluru-560099, Karnataka, India
cDepartment of Studies in Biotechnology, University of Mysore, Manasagangotri,
Mysuru-570006, Karnataka, India
dDepartment of Chemistry, Pooja Bhagavath Memorial Mahajana Education Centre,
Mysuru-570016, Karnataka, India
eDepartment of Chemistry, Rani Channamma University, Vidyasangama, Belagavi-
591156, Karnataka, India. E-mail: sridhara.mb@gmail.com; Tel: +91 9663983459
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra13661g
The nosocomial pathogens includes Acinetobacter bau-
mannii, Enterococcus faecium, Enterobacter species, Klebsiella
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pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus spectral analysis (ESI†). The formation of methyl esters (2) was
are grouped into ESCAPE pathogens due to having overcoming conrmed by the appearance of a singlet at 3.83d for –OCH3 and
1
ability to current antimicrobial agents.5,6 This shows the absence of COOH proton peak at 12.10d in the HNMR spec-
behaviour of these pathogens due to occurrence of antimicro- trum. In the 1H NMR spectrum of 3, two singlet signals dis-
bial resistance genes in their genome carried on its chromo- played at 9.79 ppm and 4.50 ppm corresponding to the NH and
some, plasmid or transposons.7 These drug resistant NH2 protons, respectively. Moreover, the absence of one signal
determinants fall into broad categories by altering the cell at 52.3 ppm of –OCH3 in 13C NMR spectrum conrms the
permeability of the drug lead to decrease in the intracellular formation of isophthalic hydrazide 3 from methyl ester 2. The
accumulation, inactivating/altering the drug, altering the target nal compounds 4–36 were conrmed by their 1H NMR, 13C
binding site, and forming drug resisting protection layer called NMR, and mass spectrum analysis. 1H NMR spectrum of 4
biolm.8,9
showed a singlet for –NH proton at 12.03 ppm and disappear-
The prevalence of biolm formation in infections is esti- ance of the peak for NH2 proton conrms the formation of
mated to be around 65%, with varying degrees of inuence on hydrazones. Further, it showed a singlet at 8.48 ppm for the
the course of the disease. Eliminating biolm-associated newly formed azomethine proton (–N]CH–) conrms the
bacteria, persisting on catheters, a cardiovascular and ortho- formation of the hydrazones. All nal compounds 4–36 showed
pedic implant presents a major problem for clinicians. Biolm carbonyl (C]O) signal in the region of 162–164 ppm and azo-
formation, which can also be considered as a passive resistance methine carbon (–N]CH–) signal in the region of 146–148 ppm
mechanism, is characterized by modied physicochemical in their 13C NMR spectra clearly conrms their formation
microenvironment through the formation of a polysaccharide hydrazones. Further, compound 4 showed a M + 1 peak at
matrix around bacteria, to inhibit the diffusion of antibiotics 371.2414 in their HRMS conrms the formation of nal
(making bacteria incomparably more resistant to them) leading products.
to stationary growth and dormancy (attributed to lower oxygen
and nutrient levels), and to make the bacteriostatic agents quasi
ineffective.10–13
Biology
The motivated by special features of dihydrazones analogs
and our ongoing research program.14–20 With the above back-
ground, we aimed towards the development of new therapeutic
bacteriostatic agents. Herein the synthesis of dihydrazones and
the studies of their biocidal activities were reported and
molecular docking study was also conducted for the under-
standing of the mechanism of the synthetic compounds to their
biocidal activities.
Biocidal activity. The antibacterial activities of synthetic
compounds were evaluated by disc diffusion method22,23 and
minimum inhibitory concentration (MIC) was identied by
studying different concentrations (1–50 mg mLÀ1) against both
Gram-positive (S. aureus and Bacillus cereus) and Gram-negative
(Escherichia coli, Enterobacter aerogenes, Salmonella typhimurium
and Shigella exneri) bacteria in the present study (Tables 1–3).
The compounds 17, 18, 20, 25 and 26 displayed potent activity
against both Gram-positive and Gram-negative bacteria. Among
them compound 25 and 26 are highly potent and exhibited
strong MIC in mg mLÀ1 against S. aureus (22 Æ 0.41 and 20 Æ
0.62), B. cereus (24 Æ 0.52 and 22 Æ 0.42), E. coli (15 Æ 0.71 and
Results and discussion
Chemistry
Syntheses of the dihydrazones were achieved according to the 16 Æ 0.61), E. aerogenes (16 Æ 0.42 and 17 Æ 0.15), S. typhimu-
procedures illustrated in Scheme 1. Isophthalic acid (1) was rium (18 Æ 0.66 and 16 Æ 0.16) and S. exneri (16 Æ 0.82 and 17
methylated using trimethylsilylchloride (TMS-Cl) and methanol Æ 0.18) compared to standard antibiotic ampicillin (19 Æ 0.24,
at room temperature to obtain dimethyl isophthalic (2) which 18 Æ 0.72, 16 Æ 0.14, 16 Æ 0.31, 16 Æ 0.42 and 19 Æ 0.81
upon reaction with an excess of hydrazine hydrate afforded the respectively) (Table 4).
corresponding isophthalic hydrazide 3.21 The obtained hydra-
Anti-biolm activity. In the present study, S. exneri was
zones (4–36) were reacting 3 with different aromatic or aliphatic selected as a model organism to study the anti-biolm and
aldehydes in the presence of catalytic amount of glacial acetic cellular content release experiments for synthesized
acid. All the derivatives were obtained in excellent yields. The compounds. All the 36 compounds were screened for the inhi-
structures of newly synthesized compounds including inter- bition of biolm formation by S. exneri using 96 well plate
mediates were conrmed by 1HNMR, 13CNMR and mass methods.24 Among them, 17, 20, 25 and 26 displayed potent
Scheme 1 Synthesis of target compounds 4–36.
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Table 4 Minimum inhibitory concentration (MIC) of the synthesized compounds against Gram positive and Gram negative bacteria
Paper
Gram positivea
Gram negativea
Compounds
S. aureus
B. cereus
E. coli
E. aerogenes
S. typhimurium
S. exneri
4
5
6
7
8
9
33 Æ 0.02
42 Æ 0.25
32 Æ 0.21
34 Æ 0.04
31 Æ 0.30
30 Æ 0.12
32 Æ 0.31
32 Æ 0.12
40 Æ 0.33
35 Æ 0.14
30 Æ 0.22
31 Æ 0.21
36 Æ 0.41
22 Æ 0.12
21 Æ 0.65
37 Æ 0.31
24 Æ 0.61
28 Æ 0.35
34 Æ 0.24
33 Æ 0.52
35 Æ 0.52
22 Æ 0.41
20 Æ 0.62
—
31 Æ 0.25
39 Æ 0.21
36 Æ 0.37
37 Æ 0.12
30 Æ 0.56
34 Æ 0.18
32 Æ 0.49
33 Æ 0.14
34 Æ 0.85
31 Æ 0.56
29 Æ 0.43
30 Æ 0.72
41 Æ 0.11
20 Æ 0.45
24 Æ 0.31
32 Æ 0.51
26 Æ 0.52
27 Æ 0.63
31 Æ 0.11
33 Æ 0.41
36 Æ 0.16
24 Æ 0.52
22 Æ 0.42
—
28 Æ 0.53
26 Æ 0.52
24 Æ 0.52
23 Æ 0.18
25 Æ 0.19
18 Æ 0.16
17 Æ 0.55
23 Æ 0.24
19 Æ 0.68
26 Æ 0.22
24 Æ 0.23
16 Æ 0.65
17 Æ 0.47
16 Æ 0.51
18 Æ 0.24
25 Æ 0.64
18 Æ 0.71
25 Æ 0.61
20 Æ 0.82
21 Æ 0.24
29 Æ 0.42
15 Æ 0.71
16 Æ 0.61
—
28 Æ 0.29
25 Æ 0.51
26 Æ 0.44
25 Æ 0.56
27 Æ 0.65
16 Æ 0.54
19 Æ 0.65
22 Æ 0.13
18 Æ 0.61
27 Æ 0.24
25 Æ 0.32
18 Æ 0.43
19 Æ 0.52
18 Æ 0.42
17 Æ 0.66
27 Æ 0.44
19 Æ 0.52
23 Æ 0.51
23 Æ 0.15
22 Æ 0.61
28 Æ 0.52
16 Æ 0.42
17 Æ 0.15
—
32 Æ 0.24
26 Æ 0.31
25 Æ 0.32
23 Æ 0.51
25 Æ 0.51
18 Æ 0.52
19 Æ 0.33
22 Æ 0.14
20 Æ 0.63
26 Æ 0.42
27 Æ 0.85
18 Æ 0.96
17 Æ 0.14
19 Æ 0.71
19 Æ 0.27
28 Æ 0.82
16 Æ 0.82
27 Æ 0.34
20 Æ 0.82
23 Æ 0.18
27 Æ 0.61
18 Æ 0.66
16 Æ 0.16
—
31 Æ 0.27
28 Æ 0.85
21 Æ 0.54
24 Æ 0.15
26 Æ 0.61
19 Æ 0.32
20 Æ 0.52
23 Æ 0.24
21 Æ 0.46
24 Æ 0.64
26 Æ 0.73
19 Æ 0.35
18 Æ 0.82
18 Æ 0.18
17 Æ 0.52
26 Æ 0.61
17 Æ 0.42
25 Æ 0.34
21 Æ 0.58
22 Æ 0.31
29 Æ 0.51
16 Æ 0.82
17 Æ 0.18
—
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
A*
—
—
—
—
—
—
—
—
—
—
—
—
35 Æ 0.62
36 Æ 0.64
30 Æ 0.61
28 Æ 0.71
38 Æ 0.71
30 Æ 0.52
26 Æ 0.75
19 Æ 0.24
34 Æ 0.65
38 Æ 0.62
31 Æ 0.32
26 Æ 0.55
37 Æ 0.53
31 Æ 0.35
24 Æ 0.43
18 Æ 0.72
27 Æ 0.61
26 Æ 0.44
25 Æ 0.61
19 Æ 0.81
30 Æ 0.42
28 Æ 0.52
18 Æ 0.72
16 Æ 0.14
25 Æ 0.81
24 Æ 0.19
26 Æ 0.61
18 Æ 0.34
32 Æ 0.13
26 Æ 0.77
18 Æ 0.55
16 Æ 0.31
27 Æ 0.17
24 Æ 0.34
29 Æ 0.17
19 Æ 0.04
28 Æ 0.30
29 Æ 0.52
17 Æ 0.13
16 Æ 0.42
28 Æ 0.61
28 Æ 0.11
30 Æ 0.15
22 Æ 0.07
32 Æ 0.17
30 Æ 0.10
21 Æ 0.53
19 Æ 0.81
a
Values are mean of three determinations, the ranges of which are <5% of the mean in all cases. A*-standard ampicillin (À): not tested, (Æ)
standard deviation.
activity, while 9, 10, 13, 33 and 36 showed moderate activity cytoplasmic membrane leads to damage of membrane
whereas 12, 16, 18, 19, 21, 31 and 32 are less signicant anatomical structure leads to release of potassium ions, DNA,
activity against biolm formation compared to negative and other cellular materials were correlated to the highly potent
control (Fig. 1 and 2). The compounds 17 and 25 exhibited nature of compounds 17, 20, 25 and 26 in time of course was
highly potent anti-biolm nature in different concentrations investigated.23
when assessed by crystal violet method of both
Scanning electron microscopy (SEM). The potent compound
qualitative and quantitative methods. It is clearly deduced 25 was used to study the action on the cell membrane and
that the MIC action of compounds 17 and 25 played an biolm formation of S. exneri. The Fig. 4c showed the MIC
important role in the active destabilization of membrane concentration of compound 25 involved in cell membrane
destruction of the pathogen which leads to failure of damage exerted by the compound on cell and decrease in the
attachment.
biolm formation of S. exneri (Fig. 4b) aer the treatment in
Cellular content release (CCR). As shown in Fig. 3, the both time of course compared to control.22 This evidence clearly
Gram-positive and Gram-negative bacteria subjected to evaluate indicates that the compounds 25 interacted with the cell
the cell damage caused by compounds in a time-dependent membrane and destabilize the membrane integrity to stop the
manner at MIC of compounds and demonstrated the signi- growth of the bacteria.
cant activity with respect to control. Among all the compounds
Discussion. Recently there is an emergence of multi-drug
screened for CCR, compounds 17, 20, 25 and 26 had higher resistant pathogenic bacterial strains and most of the avail-
cellular damage compared to others as represented in Fig. 3.22,23 able antibiotics are not active against these pathogens.25,26
Thus, active molecules having potential interaction with the These drug-resistant pathogens are more pathogenic with high
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Fig. 1 Quantitative determination of anti-biofilm action of synthetic compounds. The inhibitions of biofilm formation by 36 compounds were
screened and showed compounds 17, 20, 25, and 26 as potent anti-biofilm activity against S. flexneri after 24 h of incubation.
mortality rate than that of wild strain. The scientic community antibacterial properties against Gram-positive and Gram-
is continuously searching for the new classes of disinfection negative bacteria. The four compounds 17, 20, 25 and 26 are
systems that could act efficiently against these pathogens. This leading compounds against Gram-negative bacteria compared
study revealed that compounds with electron donating (OH and to Gram-positive bacteria regardless of other compounds.
OCH3) groups on the aromatic rings displayed well to excellent Clinically, most antibacterials are described as potentially being
Fig. 2 Representation of qualitative determination of anti-biofilm activity. The compound 17 and 25 showed dose dependent inhibition of
biofilm formation in 24 h of incubation by S. flexneri. The antimicrobial MIC concentration showed clear indication for anti-biofilm formation
activity.
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Fig. 3 Effect of MIC of compounds on cellular content release from S. flexneri. The MIC of compounds treated to bacteria and results obtained in
time dependent manner exhibiting release of cell contents when cell membrane was ruptured compared to control.
used as both bactericidal and bacteriostatic. This lead was also cell membrane damage, integrity and cell architectural vari-
observed in the present study by evaluating antibacterial and ation in Fig. 4. The inhibition of bacteria and biolm
anti-biolm properties of tested compounds. Even if a bacte- formation is an interesting way to prevent the formation of
ricidal action is preferred in the context of treatment, well-organized attached bacterial biolms and pathogen-
achieving a bacteriostatic effect may advantageously inhibit esis.27 Considering the potential clinical application of our
the pathogen infection.27 The MIC of compound 25 treated to study, additional experiments could be conducted on the
S. xneri and observed for cell membrane integrity, which was combination of natural antibacterial agents and currently
altered to the damage of cell membrane in the Fig. 4a. This used antibiotics to enhance the present management prac-
factor leads to great loss of cell architectural integrity and tices against Gram-negative bacteria.
leakage of critical metabolic molecules, vital ions which
To study the structure activity relationship, the substitu-
regulate the cell division rates ultimately leading to bacterial ents on the phenyl rings play a major role in the antibacterial
cell death.28,29 The microorganisms growing on inert or living activity. Compounds 17, 18, 20, 25 and 26 were found to have
surfaces usually form microbial biolms, which were formed excellent antibacterial activity. The presence of electron
by dense communities of microbial cells surrounded with the donating (OH and OCH3) groups present on the phenyl ring,
self-secreted matrix. Biolm development is one of the increases the antibacterial activity. Compounds 5–8, 11–14
bacterial defense strategies to survive at different conditions. and 21–24 were found to possess moderate antibacterial
The present study revealed that the MIC of 17, 25 and 26 activity against the all tested Gram positive and Gram nega-
exhibited anti-biolm property against S. xneri as a model tive bacterial strains. The presences of electron withdrawing
organism. The failure in the prevention and eradication of (Cl, NO2, Br, and F) groups on the phenyl rings reduce the
microbial biolms might create a number of serious prob- antibacterial activity. Whereas, compounds containing
lems such as bio-deterioration, food contamination and aliphatic (27–29) analogs were displayed nil activities. It is
infectious diseases such as endocarditis, periodontitis and interesting to nd that the more electron donating groups
chronic lung infections in cystic brosis patients being the present on the phenyl ring the better antibacterial activity was
prominent ailments.30 The signicant results of potent observed.
compounds in the present study can be observed through the
Fig. 4 The SEM images of anti-biofilm and cell membrane damage effect of compound 25. (A) The S. flexneri control. (B) The anti-biofilm action
of compound 25 after 12 h of treatment and arrow indicates the loss of bacterial growth after treatment of compound 25, and (C) indicates the
cell membrane damage of bacteria after 24 h of incubation and arrow indicates the cell membrane integrity alterations and anti-bacterial effect
of compound 25 compared to control (A).
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Fig. 5 Molecular docking interactive map of compound 17 (A) and 25 (B) into the DNA Gyrase, binding deep inside the active site, depicting the
best docking pose.
Hence inactivation of MurB causes inhibition of bacterial cell
wall synthesis. In addition to the interaction of compound 15
Glu120, Asp169, and Glu325 via hydrogen bond (Fig. 6A)
whereas compound 26 forms a hydrogen bond with Arg 214,
Ile173 and Ser50 (Fig. 6B). These amino acids are crucial and
reside at the active site of the enzyme, which help in biosyn-
thesis of peptidoglycan, any perturbation in these amino acids
could down-regulate the enzyme activity. Overall docking
result suggest that hydroxyl group present on ortho (15), ortho–
para (17) and ortho–para–meta (25) position is more favorable.
Whereas with methoxy group, present at ortho–para–meta t
into active site of enzyme. Based on XP glide score,
compounds 15, 17, 25 and 26 displayed promising G-scoring
functions, when compared to other structurally related
compounds as tabulated in Table 5. Noteworthy, these data
validate that compounds 15, 17, 25 and 26 is comparatively
potent against standard antibiotic.
Molecular docking
In order to understand possible mechanisms by which the
synthesized compounds exerted their antibacterial activity,
the molecular docking study was conducted.31 The docking
studies demonstrated an interacting map of DNA Gyrase from
Staphylococcus aureus complex, then compound 17 interacting
with Asp437 via a hydrogen bond, metal coordinate with
manganese and with Arg458 it forms a salt bridge, these
interactions are tightly bound to DNA (Fig. 5A). Whereas the
compound 25, also forms a tight interaction with Asn476 via
a hydrogen bond and metal coordinate with manganese
(Fig. 5B) these interactions suggest that the cofactor Manga-
nese, the is chelated, which inhibit the accessibility for DNA
polymerase, thereby inhibiting the further growth of Staphy-
lococcus aureus. The peptidoglycan biosynthesis begins with
the action of two enzymes viz., MurA and MurB, with MurB
catalyzing the second step in the formation of muramyl sugar.
Fig. 6 Molecular docking interactive map of compound 15 (A), and 26 (B) into the MurB, binding deep inside the active site, depicting the best
docking pose.
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Table 5 Molecular docking scores of all the synthesized compounds against DNA Gyrase and MurB as obtained through Glide docking
Paper
2XCT
1MBT
RMSD-
OPLS-2005
Docking
score
Glide
G-score
Glide
E-model
Glide
energy
RMSD-
OPLS-2005
Docking
score
Glide
G-score
Glide
E-model
Glide
energy
Entry
R1
R2
R3
R4
R5
R6
R7
R8
0.006
0.001
0.03
À7.451
À5.97
À5.972
—
À4.434
À6.332
À4.674
À4.232
À4.414
—
À4.69
À4.467
À7.551
À4.396
À6.85
—
À5.035
À6.979
À5.950
À4.955
À4.044
À5.352
À6.697
À6.690
À3.434
À4.809
À5.24
À5.325
À5.808
À5.686
À4.443
À3.984
—
À7.452
À5.97
À5.972
—
À4.435
À6.333
À4.675
À4.233
À4.426
—
À4.691
À4.468
À7.611
À4.397
À6.92
—
À5.035
À6.991
À5.920
À4.956
À4.045
À5.354
À6.751
À6.697
À3.488
À4.86
À5.294
À5.435
À5.88
À5.836
À4.553
À5.633
—
À49.96
À53.784
À59.317
—
À51.844
À34.817
À51.65
À51.818
À44.637
—
À56.647
À55.309
À59.018
À34.725
À62.054
—
À66.84
À60.042
À58.022
À77.14
À56.613
À38.782
À73.632
À45.705
À62.445
À51.594
À79.967
À40.033
À57.928
À64.46
À28.984
À33.271
—
À20.959
À37.654
À41.435
—
À45.592
À41.121
À44.773
À46.244
À40.984
—
À48.568
À50.12
À41.914
À34.372
À51.797
—
0.006
0.001
0.03
0.006
0.014
0.011
0.034
—
0.026
0.014
0.005
0.031
0.044
0.004
0.01
0.021
0.042
0.005
0.002
0.01
0.021
0.005
0.013
0.028
0.006
0.043
0.05
0.007
0.004
0.02
0.002
0.026
0.007
0.042
0.006
À7.105
À5.356
À4.994
À5.12
À7.106
À5.356
À4.994
À5.121
À1.498
À1.094
À3.191
—
À4.886
À6.031
À3.526
À7.372
À3.208
À4.824
À7.911
À3.636
À3.24
À48.427
À43.412
À44.553
À30.307
À7.811
À27.663
À33.114
À33.901
À33.587
À13.048
À6.37
—
0.043
0.014
0.011
0.011
0.034
—
0.026
0.005
0.031
0.044
0.004
—
0.021
0.042
0.040
0.01
0.021
0.005
0.018
0.020
0.006
0.01
À1.496
À1.093
À3.179
—
6.57
À48.633
À40.844
—
—
R9
À4.885
À50.456
À27.401
À36.542
À46.61
À46.619
À36.417
À39.4
R10
R11
R14
R15
R16
R17
R18
R19
R20
R21
R22
R23
R24
R25
R26
R27
R28
R29
R30
R31
R32
R33
R34
R35
R36
Ampicillin
À6.03
À3.525
À7.313
À3.207
À4.753
À7.91
À59.776
À39.168
À43.599
À44.213
À48.255
À44.204
À50.495
À52.475
À44.547
À44.46
À29.191
À65.845
À44.692
À58.385
À52.546
À48.171
À47.151
À35.217
À48.827
À84.32
À3.636
À3.228
À7.396
À5.398
À4.244
À5.06
À56.656
À52.091
À58.291
À60.494
À49.522
À42.542
À42.803
À45.302
À51.999
À41.07
À62.3
À7.391
À5.641
À4.246
À5.061
À6.594
À7.293
À7.512
À3.984
À6.218
À5.552
À5.376
À4.681
À5.028
À5.463
À5.746
À4.232
À3.418
À6.388
À6.593
À7.237
À7.511
À3.929
À4.331
À5.499
À5.266
À4.609
À4.878
À5.353
À4.097
À4.23
À63.562
À44.765
À63.549
À45.311
À48.254
À53.394
À36.778
À49.4
À53.454
À50.895
À55.161
À65.353
À67.599
À38.255
À56.333
À52.878
À60.691
À63.372
À58.572
À56.533
À53.944
0.05
0.007
0.004
0.02
0.002
0.013
—
À35.282
À49.706
À52.57
À31.941
À34.307
—
À43.787
À50.675
À51.775
À50.928
À48.925
À44.146
0.012
0.006
À4.134
À5.107
À63.6
À54.392
À3.114
À6.331
À5.611
À5.668
À61.036
À44.776
antibiotics against ESCAPE pathogens was warranted for the
future research. Molecular docking studies were performed for
all the synthesized compounds among which compounds 15,
17, 25 and 26 showed the highest docking G-scores for anti-
bacterial activity.
Conclusion
The Infectious Diseases Society of America (IDSA) has identied
a few bacterial species as the most threatening pathogens due to
the rapid development of antibiotic resistance in those species.
The ESCAPE, as the name suggests, this class of pathogens can
effectively escape the bactericidal effect of most of the conven-
tional antibiotics especially due to the presence of exclusive
permeability barriers and efflux pumps in Gram-negative
pathogens. To address this, we designed synthetic analogs to
treat Gram-negative bacteria in the present investigation. The
analogs 17, 20, 25 and 26 are potent in nature for Gram-negative
compared to Gram-positive bacteria. The excellent antibacte-
rial, anti-biolm and cell membrane damaging property of
potent molecules, penetrate in to the microbial surface to kill
the life-threatening agents in the study. We reported special
highly potent molecules 17, 25 and 26 as broad-spectrum anti-
biotic agents against resistant ESCAPE pathogens. Further
structural modication and alteration increase antibacterial
resistance towards the development of a new generation of
Conflicts of interest
There are no conicts to declare.
Acknowledgements
We are grateful to the Wuhan applied fundamental research of
Wuhan Science and Technology Bureau (Grant NO.
2017060201010216) and Wuhan University of Technology
providing nancial assist. Rani Channamma University, Bela-
gavi, India for nancial support and continuous encourage-
ment towards the research.
5482 | RSC Adv., 2018, 8, 5473–5483
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RSC Advances
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