Synthesis and mechanistic studies of diketo acids and their bioisosteres as potential antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2018.11.053

A series of diketo esters and their pertinent bioisosteres were designed and synthesized as potent antibacterial agents by targeting methionine amino peptidases (MetAPs). In the biochemical assay against purified MetAPs from Streptococcus pneumoniae (SpMe

Full text of DOI:10.1016/j.ejmech.2018.11.053

Accepted Manuscript
Synthesis and mechanistic studies of diketo acids and their bioisosteres as potential
antibacterial agents
Phool Hasan, Vijay K. Pillalamarri, Babita Aneja, Mohammad Irfan, Mudsser Azam,
Ahmad Perwez, Ronan Maguire, Umesh Yadava, Kevin Kavanagh, Constantin G.
Daniliuc, Md Belal Ahmad, M. Moshahid A. Rizvi, Qazi Mohd Rizwanul Haq, Anthony
Addlagatta, Mohammad Abid
PII:
S0223-5234(18)31012-2
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.053
EJMECH 10911
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 July 2018
Revised Date: 13 October 2018
Accepted Date: 21 November 2018
Please cite this article as: P. Hasan, V.K. Pillalamarri, B. Aneja, M. Irfan, M. Azam, A. Perwez, R.
Maguire, U. Yadava, K. Kavanagh, C.G. Daniliuc, M.B. Ahmad, M.M.A. Rizvi, Q.M. Rizwanul Haq,
A. Addlagatta, M. Abid, Synthesis and mechanistic studies of diketo acids and their bioisosteres
as potential antibacterial agents, European Journal of Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.ejmech.2018.11.053.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and mechanistic studies of diketo acids and their
bioisosteres as potential antibacterial agents
Phool Hasana,b, Vijay K. Pillalamarric, Babita Anejaa, Mohammad Irfana, Mudsser
Azamd, Ahmad Perweze, Ronan Maguiref, Umesh Yadavag, Kevin Kavanaghf,
Constantin G. Daniliuch, Md. Belal Ahmadb, M. Moshahid A. Rizvie, Qazi Mohd.
Rizwanul Haqd, Anthony Addlagattac, Mohammad Abid*a
aMedicinal Chemistry Laboratory, Department of Biosciences, Jamia Millia Islamia, Jamia
Nagar, New Delhi 110025, India
bDepartment of Chemistry, TNB College, TM Bhagalpur University, Bhagalpur 812007,
India
cCentre for Chemical Biology, Indian Institute of Chemical Technology, Tarnaka, Hyderabad
500607, India
dMicrobiology Research Laboratory, Department of Biosciences, Jamia Millia Islamia, Jamia
Nagar, New Delhi 110025, India,
eGenome Biology Laboratory, Department of Biosciences, Jamia Millia Islamia, Jamia
Nagar, New Delhi 110025, India
fDepartment of Biology, Maynooth University, Co. Kildare W23 F2H6 , Ireland.
gDepartment of Physics, Deen Dayal Upadhyay Gorakhpur University, Gorakhpur, Uttar
Pradesh 273009, India
hOrganisch-Chemisches Institut, Westfälische Wilhelm-Universität, Münster, Germany
*Corresponding author:
E-mail address: mabid@jmi.ac.in (Mohammad Abid)
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Highlights
‹ Various diketoesters and their bioisosteres were designed as inhibitors of bacterial
methionine aminopeptidases (MetAPs).
‹ Compounds 4a and 5a showed more than 85% inhibition of all the MetAPs as well as
multidrug resistant Escherichia coli strains.
‹ In vitro and in vivo cytotoxicity studies confirmed non-toxic nature of 5a at its
bacterial minimum inhibitory concentration (MIC).
‹ 5a also inhibited biofilm formation as visualized by SEM micrographs.
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Abstract
A series of diketo esters and their pertinent bioisosteres were designed and
synthesized as potent antibacterial agents by targeting methionine amino peptidases
(MetAPs). In the biochemical assay against purified MetAPs from Streptococcus pneumoniae
(SpMetAP1a), Mycobacterium tuberculosis (MtMetAP1c), Enterococcus faecalis
(EfMetAP1a) and human (HsMetAP1b), compounds 3a, 4a and 5a showed more than 85%
inhibition of all the tested MetAPs at 100 µM concentration. Compounds 4a and 5a also
exhibited antibacterial potential with MIC values 62.5 µg/mL (S. pneumoniae), 31.25 µg/mL
(E. faecalis), 62.5 µg/mL (Escherichia coli) and 62.5 µg/mL (S. pneumoniae), 62.5 µg/mL
(E. coli), respectively. Moreover, 5a also significantly inhibited the growth of multidrug
resistant E. coli strains at 512 µg/mL conc., while showing no cytotoxic effect towards
healthy CHO cells and thus being selected. Growth kinetics study showed significant
inhibition of bacterial growth when treated with different conc. of 5a. TEM analysis also
displayed vital damage to bacterial cells by 5a at MIC conc. Moreover, significant inhibition
of biofilm formation was observed in bacterial cells treated with MIC conc. of 5a as
visualized by SEM micrographs. Interestingly, 5a did not cause an alteration in the hemocyte
density in Galleria mellonella larvae which is considered in vivo model for antimicrobial
studies and was non-toxic up to a conc. of 2.5 mg/mL.
Keywords: diketo acids, bioisosteres, cytotoxicity, methionine aminopeptidase, antibacterial,
Galleria mellonella.
1. Introduction
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Increase in consumption of antibiotics due to the emergence of dangerous and
multidrug resistant strains has become a serious worldwide medical crisis in recent decades
[1,2]. India leads in consumption of antibiotics, expressed in defined daily doses (DDDs),
among low middle income countries (LMICs) with 103% rise from 2000 to 2015 [2]. Recent
reports from the Center for Disease Control and Prevention (CDC) suggested that multi-drug
resistant bacterial strains are affecting at least 2 million people and are responsible for
approximately 23,000 deaths each year in the United States alone [3]. This escalating
evolution of resistance coupled with a diminished antibiotic pipeline has increased the
urgency of the search for new antibiotics and enzyme targets [4]. In an attempt to identify
potent inhibitors against bacterial infections, we have directed our drug-discovery approach
towards methionine aminopeptidase (MetAP) which is crucial for the viability of bacterial
cells [5].
MetAPs are ubiquitous metalloproteases present in prokaryotic and eukaryotic cells [6].
This enzyme cleaves the initiated methionine from the newly synthesized proteins, co-
translationally [7,8]. MetAPs are typically classified into two types, type I (MetAP1) and type
II (MetAP2), differentiated by the presence of a 60 amino acid insert in the latter. MetAP1
enzymes are further classified into types Ia, Ib, Ic, and Id. Types Ib, Id, and II MetAPs are
expressed in eukaryotes, while types Ia and Ic are present in prokaryotes [9]. Deletion of the
MetAP gene is lethal in E. coli and Salmonella typhimurium demonstrating its crucial role in
bacterial survival, and highlighting its potential as a target for the development of novel
antibiotics [10,11]. Additionally, inhibition of various MetAPs may have great potential in
the treatment of such pathologies as cancer, rheumatoid arthritis, malaria, and atherosclerosis
in humans [12].
It has been reported that diketo acid (DKA) derivatives interact with numerous viral
enzymes, i.e., HIV-1 integrase [13], HIV-1 RNase [14], influenza endonuclease [15] and
hepatitis C RNA polymerase [16]. It might be due to the coordination of diketo functionality
with dinuclear Mn(II) or Mg(II) metal ions present in the active site of these enzymes [17].
Since MetAPs also require divalent metal ions for catalysis, it is believed that diketo acid
might play a promising role in the inhibition of MetAPs [18]. Based on this hypothesis, we
previously reported amino acid/dipeptidic analogues of β-diketo acids and their activity
against bacterial MetAPs. The diketo acid derivatives emerged as potent antibacterial agents
acting via inhibition of MetAPs [19]. Recently, we also developed LiHMDS catalyzed
protocol for C-N bond formation in one step via in situ cyclo-condensation of diketo ester to
give novel N-substituted 2-carboxy-4-quinolones (Fig. 1) [20]. In this study, we report the
further optimization of β-diketo acids as potent MetAP inhibitor as well as antibacterial
agents. We also synthesized constrained isoxazole ring as biological isosteric analogue of β-
diketo acid to investigate rational structure–activity relationship among these compounds. All
the synthesized β-diketo acids and their bioisosteres were evaluated against purified MetAPs
from S. pneumoniae (SpMetAP1a), M. tuberculosis (MtMetAP1c), E. faecalis (EfMetAP1a)
and human (HsMetAP1b). We observed that some of these derivatives showed improved
potency against bacterial MetAPs and displayed considerable antibacterial activity.
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Fig. 1. β-diketo pharmacophore and its bioisosteres.
2. Results and discussion
2.1. Chemistry
The synthesis of diketo acids and their bioisosteres was accomplished as illustrated in
Schemes 1-3. Initially, 1-(5-bromo-2-hydroxyphenyl)ethanone on etherification with
chloroacetone in the presence of potassium carbonate followed by cyclisation under thermal
condition gave benzofuran ketone (1a) in good yield (Scheme 1).
OH
O
O
a
O
Br
Br
1a
Scheme 1. Synthesis of benzofuran ketone. Reagents and conditions (a) K2CO3, DMF, chloroacetone,
80 °C.
The benzofuran ketone (1a) and other commercially available aryl methyl ketones (1b
and 1c) were subjected to oxalylation by diethyl oxalate in the presence of freshly prepared
sodium ethoxide to yield β-diketoesters (2a-c) in good yield except diketoester 2a. It might
be due to the inability of sodium ethoxide to abstract α-hydrogen atom of benzofuran ketone
(1a) with ease due to steric hindrance. Therefore, a much stronger base lithium
hexamethyldisilamide (LiHMDS) was employed to effectively execute a sterically hindered
Claisen condensation to get the diketoester (2a) in excellent yield. Selected β-diketoesters
(2a-b) were then treated with lithium hydroxide in THF/H2O mixture (3:1) for 2 h at room
temperature to yield corresponding β-diketo acids (3a-b) in quantitative yields. Coupling of
the β-diketo acids (3a-b) with methyl ester of L-alanine using hexafluorophosphate
azabenzotriazole tetramethyl uronium (HATU) as coupling reagent and N,N
diisopropylethylamine (DIPEA) as a base at room temperature gave L-alanine-methyl ester
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conjugate of diketo acid (4a-b) which was further hydrolyzed by lithium hydroxide to yield
corresponding acids (5a-b) in quantitative yields (Scheme 2).
O
OH
O
OH
O
O
OH
b
a
O
O
Ar
Ar
O
Ar
CH3
1a-c
O
O
O
3a-b
2a-c
c
Ar
O
1a
1b
1c
O
OH
O
O
OH
O
Br
H
N
H
d
N
Ar
O
Ar
OH
O
O
4a-b
5a-b
Cl
Scheme 2. Synthesis of L-alanine conjugate of diketo acid (5a-b). Reagents and conditions (a)
LiHMDS (1 M in THF), diethyl oxalate, THF, -50 °C to 0 °C, 1 h (for 2a); Na metal, C2H5OH, 0 °C-
r.t., 3-4 h (2b and 2c); (b) LiOH.H2O, THF:H2O (3:1), r.t., 4 h; (c) HATU/DIPEA, THF, L-alanine
methyl ester, r.t., 16 h; (d) LiOH.H2O, THF:H2O (3:1), r.t., 4 h.
In another set of reactions to achieve bioisosteres of β-diketo functionality, the diketo
esters (2a-c) on reaction with hydroxylamine hydrochloride in absolute ethanol at reflux
condition underwent [3+2] cyclocondensation reaction to give isoxazole methyl esters (6a-c).
Basic hydrolysis of isoxazole methyl esters (6a-c) using freshly prepared LiOH solution (2M)
in THF/H2O mixture (3:1) for 4 h gave corresponding acids (7a-c) in quantitative yield. L-
alanine methyl ester on coupling with isoxazole acids (7a-c) in the presence of HATU and
DIPEA gave the corresponding isoxazole-alanine methyl ester conjugates (8a-c). Alkaline
hydrolysis of these isoxazole-alanine methyl ester derivatives (8a-c) by lithium hydroxide
afforded corresponding isoxazole-alanine acids (9a-c) in quantitative yields (Scheme 3).
O
O
O
OH
b
a
Ar
Ar
O
O
OH
Ar
O
N
O
N
O
6a-c
7a-c
2a-c
Ar
c
O
2a
2b
2c
Br
O
O
d
Ar
Ar
OH
O
N
N
H
H
O
O
N
N
O
O
9a-c
8a-c
Cl
Scheme 3. Synthesis of isoxazole-L-alanine acid conjugates. Reagents and conditions (a)
NH2OH.HCl, EtOH, reflux, 4 h; (b) LiOH.H2O, THF:H2O (3:1), r.t., 4 h; (c) HATU/DIPEA, THF, L-
alanine methyl ester, r.t., 16 h; (d) LiOH.H2O, THF:H2O (3:1), r.t., 4 h.
The physico-chemical parameters for all the synthesized compounds were evaluated
using QikProp version 3.2 from Schrödinger software. All the synthesized compounds were
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found within the recommended range of important physico-chemical parameters. None of
them was found violating the Lipinski’s rule of five (Supplementary File).
2.2. X-ray crystallographic analysis
Single X-ray crystal structure analysis of isoxazole acid (7b) was carried out to
establish the authenticity of the basic structure. X-ray and structure refinement statistics are
presented in Table 1. A colorless plate-like specimen of compound 7b (C11H9NO3) with
approximate dimensions 0.050 × 0.140 × 0.160 mm was used for X-ray diffraction. The
compound crystallized in the monoclinic crystal system, using the space group P21/c, with
four molecules in the unit cell. The structure was determined in the monoclinic space group,
P21/c with four molecules in the asymmetric unit. The XP diagram of compound 7b with
thermal ellipsoids drawn at 30% probability level and with atomic numbering scheme is
shown in Fig. 2A. The o-tolyl substituent is orientated almost planar compared with the five
membered ring (C3-N1-O1-C4-C2), with a deviation angle of 9°. Strong hydrogen bonding
interactions involving two adjacent carboxyl groups [O3-H1...O2 2.639(2) Å and 177(3) °]
leads to the formation of dimeric units in the solid state of 7b (Fig. 2B).
Table 1
Crystal data and structure refinement details for 7b.
Identification code
Formula
MW (g/mol)
dcalcd. (g cm–3)
Crystal size (mm)
T/K
7b
C11H9NO3
203.19
1.446
0.050 × 0.140 × 0.160
100(2)
λ (Å)
F(000)
1.54178
424
Crystal System
Space Group
a(Å)
b(Å)
c(Å)
Monoclinic
P21/c
5.5856(12)
10.373(2)
16.309(3)
90
α (°)
β (°)
98.969(12)
90
933.4(3)
4
γ (°)
V (Å3)
Z
θ range (°)
Collected reflections
Unique reflections; Rint
µ/mm–1
5.07 –66.68
12118
1638 [Rint = 0.0767]
0.891
R(Fo), [Iꢀ>ꢀ2σ(I)]
0.0429
0.0595
1.044
0.203/–0.283
1415262
2
Rw (Fo ) (all data)
GoF (F2)
∆ρ [e Å–3]
CCDC number
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Notable is the almost planar structure of this dimeric unit. In the packing diagram, the
formation of a linear chain of dimeric units along a-axis involving π...π interactions was
observed. Shortest distances between these dimeric units are C2...O2 3.226 Å and C2...C10
3.294 Å (Fig. 2C and 2D). An overlapping mode of these dimeric units is shown in Fig. 2E.
Additionally, non-covalent C-H...O and C-H...N interactions between the o-tolyl group and the
five membered ring stabilized the crystal structure of 7b [C9-H9...N1 2.699 Å and 146.5 °;
C10-H10...O1 2.771 Å and 131.5 °; C7-H7...N1 2.834 Å and 171.2 °; C11-H11A...O3 2.701 Å
and 126.1 °] (Fig. 2F).
Fig. 2. (A) XP diagram of compound 7b with atomic numbering scheme (30% probability level of
thermal ellipsoids); (B) Dimeric unit formation trough O-H...O hydrogen bonds; (C) Dimer formation
trough π...π interactions in the solid state of compound 7b. (D) Excerpt of the packing diagram of
compound 7b presenting the linear chain formation along a-axis; (E) Overlapping mode between two
dimeric units in 7b; (F) Excerpt of the packing diagram of 7b presenting the C-H...O and C-H...N
interactions between the dimeric units.
2.3. Biochemical assays: In vitro methionine aminopeptidases (MetAPs) inhibition studies
Mounting evidence suggested that MetAP plays an important role in co- or post-
translational modification of proteins thus is crucial for the metabolism and survival of
bacterial cells [21]. As we reported earlier, β-diketo esters and their amino acids/dipeptidic
analogues emerged as potent antibacterial agents acting via inhibition of bacterial MetAPs. In
this work, we designed some diketo esters and their bioisosters and evaluated their effect on
inhibition of MetAPs from different origins viz. H. sapiens, E. feacalis, M. tuberculosis and
S. pneumoniae. The inhibitory results (in percentage) and Ki values of selected compounds
are summarized in Table 2 and Table 3, respectively. Results presented here clearly
demonstrated that the transformation of diketoesters into isoxazole resulted in the loss of
inhibitory potential against bacterial MetAPs. However, compound 9a emerged as potent
inhibitor of HsMetAP1b among all isoxazole derivatives in the series with 93.39% inhibition
(Ki = 3.11 µM) indicating selectivity over bacterial enzymes. Among all compounds,
benzofuran-containing compounds (2a, 3a, 4a, and 5a) displayed the highest inhibition
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against both human and bacterial MetAPs with better preference to E. faecalis. Compound 4a
displayed better selectivity towards S. pneumonia MetAP1a. Surprisingly compound 9a
showed inhibition only against human MetAP1b. Repeated attempts to determine the enzyme
inhibitor complexes with all these lead compounds and all enzymes in this study did not
succeed which limited our ability to pinpoint the exact molecular basis for this observed
selectivity. Based on enzyme inhibition studies, compounds 4a and 5a could be considered as
potent antibacterial though having some selectivity towards bacterial MetAPs over human
enzyme. To better understand the molecular basis, we carried out molecular docking studies.
Table 2
In vitro inhibition study of title compounds against different MetAPs.
Compound % Inhibition at 100 µM inhibitor concentration
HsMetAP1b EfMetAP1a
MtMetAP1c SpMetAP1a
85.96±0.85
61.36±2.88
61.01±2.58
88.15±0.19
52.27±3.44
89.09±0.76
51.88±3.73
87.45±0.66
59.24±2.68
43.57±1.64
48.85±3.78
66.29±3.06
47.26±3.16
48.97±2.96
43.87±5.72
45.08±6.55
51.09±6.02
43.13±3.91
93.39±0.31
50.22±2.34
47.25±1.58
93.31±0.35
43.5±2.45
91.63±041
55.83±1.24
70.51±0.54
93.47±0.34
65.69±0.34
99.59±0.02
67.07±0.3
87.65±0.23
47.7±2.62
41.32±0.68
47.09±6.95
47.43±0.5
39.12±2.8
43.72±1.21
58.21±0.98
36.2±1.12
73.05±0.39
50.29±3.02
55.32±1.5
93.4±0.04
62.84±4.6
97.44±0.08
56.96±2.49
98.16±0.07
58.05±1.38
40.59±1.54
41.39±2.35
40.3±0.24
43.24±1.48
42.41±1.47
39.66±2.06
36.47±1.21
36.95±1.01
43.76±2.62
42.8±2.24
44.65±1.22
43.69±2.05
2a
2b
2c
3a
3b
4a
4b
5a
5b
6a
6b
6c
7a
7b
7c
8a
8b
8c
9a
9b
9c
27.34±2.39
91.90±1.04
54.7±3.68
93.86±0.23
59.61±1.37
92.68±0.08
63.23±0.19
21.08±0.27
27.65±1.21
19.43±5.86
40.28±0.43
32.01±1.23
20.01±2.02
18.82±065
24.926±1.16 45.91±0.54
15.37±3.96
34.62±0.21
43.42±0.67
23.65±2.96
53.16±0.46
33.2±0.62
40.91±1.71
51.66±0.54
Table 3
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Ki values of potent MetAP inhibitors
Compound
Ki value (in µM)
EfMetAP1a MtMetAP1c
10.79±1.42 18.16±2.17
HsMetAP1b
19.75±1.75
74.31±4.9
56.1±2.8
SpMetAP1a
68.24±4.62
98.49±8.94
62.61±3.1
27.47±1.24
87.5±7.77
8.78±0.89
51.91±3.17
28.32±1.8
59.87±7.55
ND
2a
2b
2c
3a
3b
4a
4b
5a
5b
6b
6c
7b
7c
8b
8c
9a
9b
9c
ND
36.74±109
39.5±3.23
26.28±2.3
ND
32.72±2.53
73.8±5.44
22.14±2.2
112.82±6.86
22.1±1.06
54±2.72
17.67±1.9
62.56±3.96 37.54±1.67
10.42±1.09 17.59±1.45
60.35±7.8
32.62±1
11.89±1.34 9.89±0.66
63.96±4.89 ND
122.58±7.98
ND
ND
160.43±10.16 ND
152.15±4.9
ND
ND
103.92±6.62
ND
ND
ND
ND
ND
ND
ND
ND
ND
71.81±8.31
ND
ND
136.7±10.35
ND
ND
134.31±9.83 ND
3.11±0.4
86.85±4.7
ND
ND
ND
ND
ND
ND
94.62±6.02
2.4. Docking Studies
Glide XP docking of the compounds (4a and 5a) within the active site of different
targets show variations in the glide score, model energies and glide energies. The penalty of
buried ligands in the cavity makes a very small contribution in all the docking conditions.
Among all the energy parameters the largest contribution of the energy comes from
electrostatic and van der Waals interactions.
XP docking within the binding site of methionine aminopeptidase Type 1c from M.
tuberculosis (PDB ID: 1YJ3) indicates the better binding capability of compound 4a within
the binding site compared to 5a. The model energies of the binding of ligands have been
found to be -47.513 and -41.015 kcal/mol respectively. The various non-covalent interactions
with the ligands in the binding site of 1YJ3 have been shown in Fig. 3. It has been observed
that the residues LYS98, HIS212 and THR203 form N-H…O type hydrogen bonding
interactions with both ligands 4a and 5a. Hydrophobic and other polar interactions are also
similar in both types of ligands. Residues TYR97, TRP255, ILE250, PHE202, MET240 form
a hydrophobic enclosure to the ligands. Polar interactions are exhibited by HIS114, HIS212,
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HIS205 and THR203 residues while ASP201 and GLU238 residues show charge negative
interactions.
Docking of ligands within the binding site of E. faecalis Methionine aminopeptidase
(PDB ID:3TB5) also exhibit the better binding capability of 4a with Glide score and model
energy -5.391 and -58.622 kcal/mol compared to 5a with Glide score and Glide energy of -
5.056 and -46.471 kcal/mol respectively. The contribution of van der Waal and Coulomb
energies play significant roles in the binding of ligands. Fig. 3 depicts the interactions of the
ligands within the binding site. It can be deduced that several hydrogen bonding and other
noncovalent interactions stabilize the compounds within the binding site of 3TB5. N-H…O
type hydrogen bonding interactions involves residues GLU201, GLN57, and VAL166.
GLU201 also exhibits classical strong O-H.O hydrogen bond formation in the case of binding
with 4a. π-π stacking interactions are displayed by a PHE165 residue with the aromatic
moiety of the ligands. Hydrophobic interactions are shown within the binding site by residues
TRP218, PHE165, VAL166, CYS96, TYR60, ILE174, and MET178. Residues HIS168,
HIS77, GLN57, HIS175, THR199, and GLN230 display polar interactions whereas residues
ASP164, ASP94, GLU232, ASP105 and GLU201 exhibit electrostatic interactions.
The Glide score and model energies of the 4a and 5a as obtained through extra
precision docking within binding site of methionine aminopeptidase from S. pneumoniae
(PDB ID: 4KM3) were found to be -4.620 and -51.377 kcal/mol; -4.579 and -41.922 kcal/mol
respectively. The non-covalent interactions are exhibited in Fig. 3. Residues HIS199,
VAL197 and ASP195 form N-H…O hydrogen bonding interactions with both the ligands.
Interestingly, His252 residue exhibit C-H…π weak hydrogen bonding interaction in the case
of both ligands. Additionally, aromatic π-π interactions are also shown in the best docking
pose of 5a involving TRP250 and an aromatic moiety of the compound. The hydrophobic
enclosure is formed by the residues PRO203, ALA63, TRP250, LEU196, VAL197, and
TYR214. HIS252, THR244, and HIS199 are involved in the polar interactions. ASP195 also
exhibit electrostatic interactions within the binding site.
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Fig. 3. Docking studies of compounds 4a and 5a with MetAPs from M. tuberculosis (PDB ID: IYJ3),
E. faecalis (PDB ID: 3TB5) and S. pneumoniae (PDB ID:4KM3) showing their interaction with key
residues.
2.5. In vitro antibacterial activity
Eleven compounds were selected on the basis of MetAP inhibition studies for further
evaluation of their antibacterial potential against P. aeruginosa, S. typhimurium, S.
pneumoniae, E. faecalis, K. pneumoniae and E. coli. The results in terms of minimum
inhibitory concentration (MIC) are presented in Table 4. The results showed an irregular
pattern in the antibacterial activity of the compounds. The benzofuran based diketo ester (2a)
showed a moderate inhibitory effect against Gram-positive S. pneumoniae and E. faecalis and
Gram-negative E. coli with MIC value of 250 µg/mL, although its activity against P.
aeruginosa and S. typhimurium was very poor. The corresponding acid, 3a showed better
activity as it was found active against four bacterial strains viz. P. aeruginosa, S.
typhimurium, S. pneumoniae and E. faecalis with MIC values of 62.5-500 µg/mL. The 2-
methylphenyl diketo ester (2b) and its acid 3b exhibited moderate effect on S. pneumoniae,
E. faecalis and E. coli with MIC ranging from 250-500 µg/mL. Similarly, L-alanine methyl
ester conjugate of 2-methylphenyl based diketo ester 4b and its corresponding acid, 5b were
found more active against Gram-positive bacteria S. pneumoniae and E. faecalis with good to
moderate antibacterial activity (MIC ranges between 125-500 µg/mL) while no significant
inhibition was observed in the case of Gram-negative bacterial strains. p-Chlorophenyl diketo
acid, 2c and benzofuran isoxazole based alanine derivative, 9a exhibited good to moderate
inhibition ranging from 125-500 µg/mL against all the tested strains except K. pneumoniae.
Benzofuran-L-alanine methyl ester conjugate (4a) and its corresponding acid, (5a) emerged
as potent inhibitors among these selected synthesized derivatives as they showed better
inhibitory activity against all the bacterial strains used in the study. Both these compounds
exhibited equal potency with MIC values of 500, 500, 62.5 and 62.5 µg/mL against S.
typhimurium, K. pneumoniae, S. pneumoniae and E. coli, respectively. However, compound
4a was found to be a better inhibitor than compound 5a against P. aeruginosa and E. faecalis
in terms of MIC values.
Table 4
In vitro antibacterial activity of diketo acids and their bioisosteres
Compounds
Minimum Inhibitory Concentration (µg/mL)
S. S. E. K.
aeruginosa typhimurium pneumoniae faecalis pneumoniae
P.
E.
coli
1000
1000
500
125
1000
125
1000
1000
500
500
1000
500
250
250
500
62.5
500
62.5
125
62.5
250
250
250
62.5
500
31.25
500
500
1000
>1000
1000
1000
500
250
250
500
1000
250
62.5
1000
62.5
2a
2b
2c
3a
3b
4a
4b
5a
1000
250
1000
500
1000
500
125
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1000
500
1000
0.25
1000
>1000
500
500
1000
250
500
500
125
1000
1000
>1000
0.25
1000
500
500
5b
6c
9a
0.0156
0.312
0.0625
0.125
CIP
Based on the results obtained on sensitive bacterial strains, compounds 4a and 5a were
selected for further assessment of their antibacterial activity on eight multidrug resistant E.
coli strains. It was observed that compound 5a at a concentration of 512 µg/mL exhibited
almost 100% inhibition of E. coli MRA11, MRC17, MRC24, MRAE32, and MROB11
strains while at the same concentration compound 4a caused only 65%, 60%, 76%, 88% and
87% inhibition of the respective strains. Therefore, compound 5a was found to be more
effective than compound 4a against MDR E. coli strains. Even at a concentration of 1024
µg/mL, compound 5a exhibited more efficacy than compound 4a and showed more than 95%
inhibition of E. coli MRAE26 and MRAE33 strains while compound 4a was able to inhibit
these strains only by 54% and 50%, respectively. However, at a concentration of 1024 µg/mL
both these compounds were found to be almost equally potent against E. coli MRAE44 strain
(Fig. 4).
Fig. 4. In vitro antibacterial activity of 4a, 5a and ciprofloxacin (µg/mL) against multidrug-resistant
E. coli strains: MRA11, MRC17, MRC24, MRAE26, MRAE32, MRAE33, MRAE44 and MROB11.
The numbers written above the respective columns corresponds to the concentrations of 4a, 5a and
CIP at which maximum inhibition was observed, respectively.
These compounds were further evaluated for their in vitro synergistic effect in
combination with ciprofloxacin against E. coli MRA11, MRC17 and MRAE33 strains. The
results showed that the antibacterial efficacy of these compounds against the selected strains
MRA11 and MRC17 was not affected in combination with ciprofloxacin while a minor
increase in activity was observed against MRAE33. The fractional inhibitory concentration
index (FICI) values of compounds 4a and 5a suggested that these compounds showed no
significant synergy in combination with ciprofloxacin against any of the tested strains (Table
5 and 6).
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Table 5
In vitro synergistic antibacterial activity of 4a.
MDR E. coli MIC alone (µg/mL) MIC
in FICI*
Mode
of
strains
combination
interaction
(µg/mL)
4a
1024
1024
16
4a
CIP
128
128
2
CIP
128
64
1024
1024
1024
2.0
1.5
1.0156
Indifferent
Indifferent
Indifferent
MRA11
MRC17
MRAE33
2
Table 6
In vitro synergistic antibacterial activity of 5a
Multidrug
resistant E.
coli strains
MIC alone (µg/mL) MIC
combination
in FICI*
Mode
interaction
of
(µg/mL)
5a
5a
CIP
128
128
2
CIP
16
64
2
512
512
1024
512
512
16
1.125
1.5
1.0156
Indifferent
Indifferent
Indifferent
MRA11
MRC17
MRAE33
2.6. Cytotoxicity assessment of lead inhibitors by MTT assay
Cytotoxicity of selected compounds 4a and 5a was determined by cell proliferation assay
(MTT) on Chinese hamster ovary (CHO) cell line. The study revealed that compounds 4a and
5a were found to be non-toxic at a concentration of 50 µg/mL to CHO cells. However, on
increasing the concentration of compounds to 100 µg/mL, compound 4a exhibited more
toxicity compared to compound 5a on CHO cells with only 50% cell viability while later
displayed more than 70% cell viability. The cell viability was further decreased on increasing
the concentration of these compounds to 300 µg/mL indicated toxic nature of these
compounds at high concentration. Compound 5a can be considered non-toxic at its MIC
values thus selected for further biological evaluation (Fig. 5).
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Fig. 5. Cytotoxicity of compounds determined by MTT assay on CHO cell line.
2.7. Growth kinetic studies
Based on the MetAP inhibition as well as the antibacterial effect on sensitive/resistant
bacterial strains, compound 5a was evaluated for its effect on the growth of P. aeruginosa, E.
coli, S. pneumoniae and E. faecalis bacterial strains. The results showed that 5a exerted
significant effect on the growth pattern of these strains and completely inhibited the growth
of S. pneumoniae and E. faecalis even at sub-MIC values with a continuous lag-phase of 24
h. Furthermore, at MIC and 2MIC values no growth was observed in case of P. aeruginosa
and E. coli even after 24 h of incubation. However, at sub-MIC concentration very negligible
growth of E. coli was observed after 22 h while significant growth of P.aeruginosa was
observed after 10 h. Therefore, it can be concluded that 5a is bacteriostatic in nature and
effectively arrests the bacterial growth (Fig. 6).
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Fig. 6. The effect of different concentrations of compound 5a on the growth kinetic studies.
2.8. Assessment of biofilm formation
To see the effect of lead inhibitor (5a) on biofilm formation, XTT assay was also performed
on P. aeruginosa, E. coli, K. pneumoniae and E. faecalis strains. Compound 5a exhibited
significant anti-biofilm activity on E. coli and P. aeruginosa with IC50 values of 0.404 and
1.69 µg/mL, respectively. However, this compound showed moderate efficacy on K.
pneumoniae and E. faecalis with IC50 values of 113.21and 1243.08 µg/mL, respectively
(Table 7).
Table7
Anti-biofilm activity of compound 5a; IC50 values (µg/mL).
Strain
IC50 (µg/mL)
1.69±0.084
0.404±0.02
P. aeruginosa
E. coli
113.21±5.66
1243.08±62.15
K. pneumoniae
E. faecalis
SEM analysis was also performed to visualize the effect of 5a on biofilm formation
using E. coli strain. Significant inhibition of biofilm development was observed in E. coli on
treatment with 2MIC concentration of compound 5a. Clustering of cells which signifies the
formation of biofilm was observed in untreated samples while scattered cells with poor cell
density were observed in treated cells. So, it can be concluded that the 5a may have the
potential to be an anti-biofilm agent as well as an antibacterial agent (Fig. 7).
Fig. 7. Scanning electron microscopy of biofilm: (a) Untreated, (b) treated with 2MIC concentration
of compound 5a.
2.9. Transmission electron microscopic (TEM) analysis
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Structural changes induced by 5a on E. coli and S. pneumoniae were characterized using
TEM micrographs. The control cells and cells treated with MIC value of 5a were used for
TEM analysis. The untreated cells were observed with normal cell morphology with intact
cell membrane and uniform cell density while treated cells showed significant cell damage
with several deformations. Distorted cell membrane with leakage of cell cytoplasm was
observed in 5a treated cell micrographs. These results confirmed the cell damaging effect of
5a on both bacterial strains. (Fig. 8).
Fig. 8. TEM images showing cellular alteration in E. coli and S. pneumoniae treated with MIC
concentration of 5a.
2.10. In vivo toxicity evaluation of 5a on Galleria mellonella larvae
It has been reported previously that G. mellonella larvae may be utilized as an in vivo model
for the assessment of toxicity of novel antimicrobial agents due to remarkable similarities
between the innate immune responses of insects and mammals. Therefore, G. mellonella
larvae were chosen to determine the in vivo toxicity of the lead compound. The results clearly
indicated the non-toxic behavior of the compound 5a up to the concentration of 2.5 mg/mL.
Moreover, the hemocyte (immune cell) density of the larvae was not affected at the same
concentration indicating the lack of an immune response (Fig. 9).
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Fig. 9. Effect of (A) DMSO and (B) 5a on the percentage viability of G. mellonella; (D) Haemocyte
densities of larvae.
Overall, the study reveals that compound 5a which is β-diketo acid-alanine conjugate
derived from benzofuran ketone conjugated with L-alanine methyl acid was evolved as potent
antibacterial agent validated by biochemical and other pharmacological assays. As the metal
chelating property of β-diketo functionality disrupt the biological function of MetAPs,
compound 5a significantly inhibited the activity of bacterial MetAPs. Conversion of diketo
moiety into a constrained isoxazole ring led to decrease in MetAP inhibition potential, which
further confirms diketo moiety is endowed with metal chelating property and essential for
MetAP inhibition. Moreover, the selectivity of compounds towards various MetAPs was also
determined. Repeated attempts to determine the enzyme-inhibitor complexes with the lead
compound and all enzymes in this study did not succeed which limited our ability to pinpoint
the exact molecular basis for this observed selectivity. The study on multidrug resistance
(MDR) strains of E. coli as well as cytotoxicity data support the candidature of compound 5a
as the lead inhibitor. Further pharmacological experiments were in favour of preliminary
biological results which indicate 5a as potent antibacterial agent.
3. Conclusion
To summarize, a series of diketo esters and their bioisosteres were designed as
methionine aminopeptidase (MetAP) inhibitors and evaluated for their antibacterial potential
against a panel of Gram-negative and Gram-positive bacteria. Among all, compounds 4a and
5a exhibited more than 85% inhibition against SpMetAP, MtMetAP, EfMetAP and
HsMetAP, at 100 µM. Moreover, these compounds displayed considerable potency against
several Gram-positive as well as Gram-negative bacterial strains. The considerable efficacy
and non-toxic nature of compound 5a encouraged us to investigate its pharmacological
potential. Compound 5a also inhibited the growth of various bacterial strains at different
concentrations as signified by growth kinetics study. Compound 5a also inhibited the
formation of biofilm in E. coli as seen in SEM micrographs. TEM micrographs showed that
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compound 5a exhibited significant membrane disruption and cell-wall damage in bacterial
cells (S. pneumoniae and E. coli). Besides, this compound did not cause alteration in
hemocyte density in G. mellonella larvae and was non-toxic up to a concentration of 2.5
mg/mL. Furthermore, efforts in modifying these and other lead structures with the aim of
improving potency as well as specificity are in progress.
4. Experimental Section
4.1. Chemical synthesis
All the reagents and solvents (analytical grade) were procured from Sigma-Aldrich,
USA. Reactions were monitored by thin-layer chromatographic analysis on pre-coated Merck
silica gel 60 F254 TLC aluminum sheets. Infrared spectra were recorded on Agilent Cary 630
FT-IR spectrometer; wave numbers are given in cm-1. 1H and 13C NMR spectra were obtained
on BrukerSpectrospin DPX-300 spectrometer at 300 MHz and 75 MHz, respectively or a
1
13
BrukerAvance II 400 spectrometer at 400 MHz. H and C NMR chemical shifts (δ) values
are reported in parts per million (ppm) relative to residual solvent CDCl3, δ 7.26 and 77.16;
DMSO-d6, δ 2.54 and 39.5, respectively with tetramethylsilane (TMS) as an internal
standard. Splitting patterns are designated as follows: s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet) or brs (broad) and coupling constants (J) are expressed in Hertz (Hz).
Mass spectra were recorded on Agilent RRLC MS 6320 ion trap spectrometer fitted with
electrospray ionization (ESI) interface or Waters ZQ Single Quadrupole Detector (SQD)
electron spray ionization mass spectrometer. Ultra performance liquid chromatography was
performed on API 2000 triple quadrupole detector (TQD) mass spectrometer (UPLC-
MS/MS). Melting points were recorded on a digital Buchi melting point apparatus (M-560)
and are uncorrected. Purification of the compounds was carried out by flash column
chromatography (silica gel, 230-400 mesh size, Merck, Germany) with the indicated solvent.
4.1.1. Synthesis of benzofuran ketone (1a)
To the stirred solution of 1-(5-bromo-2-hydroxyphenyl)ethanone (5.0 g, 23.25 mmol,
1.0 eq) in DMF (2mL/mmol) was added K2CO3 (9.62 g, 69.76 mmol, 3.0 eq) and the mixture
was stirred at room temperature for 30 min. Chloroacetone (2.0 eq) was added and the
reaction mixture was refluxed at 80 °C for 4 h. After completion of reaction (monitored by
TLC), the reaction mixture was cooled, quenched with water and extracted with diethyl ether.
The combined organic layers were washed with brine solution, dried over anhydrous Na2SO4,
filtered and concentrated under vacuo. The crude was purified by column chromatography
over 230-400 silica gel eluting with 10% ethyl acetate in hexane to afford off white
benzofuran ketone [22].
4.1.1.1. 1-(5-bromo-3-methylbenzofuran-2-yl) ethanone (1a). Off white solid, yield: 66%, mp
115-117 °C, Rf (hexane: ethyl acetate = 7:3): 0.83, 1H NMR (400 MHz, DMSO-d6) (δ, ppm):
13
8.09 (s, 1H, Ar-H), 7.68 (s, 2H, Ar-H), 2.56 (s, 3H, CH3), 2.53 (s, 3H, CH3). C NMR (75
MHz, CDCl3) (δ, ppm): 191.16, 152.57, 148.96, 131.29, 131.06, 124.20, 123.23, 116.32,
113.68, 27.83, 9.37. LC-MS (m/z): 253.08 [M + H]+. IR (neat): 1687, 1577, 1361, 1272 cm-1.
4.1.2. General synthesis of diketo esters (2a-c)
A solution of ketone (1a-c) (1.0 eq) in anhydrous THF (5 mL/ mmol) was added
dropwise to a solution of LiHMDS (1 M solution in THF) (2.5 eq) at -50 °C and the reaction
19

ACCEPTED MANUSCRIPT
mixture was stirred at the same temperature for 30 min. A solution of diethyl oxalate (1.1 eq)
in THF (1 mL/ mmol) was added and the reaction mixture was stirred at 0 °C for 1 h. After
completion of reaction, the reaction mixture was quenched by adding water, acidified with 1
N HCl up to pH 2~3 and extracted with ethyl acetate (3×100 mL). The combined organic
layers were washed with brine, dried over anhyd. Na2SO4, filtered and concentrated under
vacuo. The crude was purified by column chromatography over 230-400 silica gel eluted with
10% ethyl acetate in hexane to afford diketo esters [23].
4.1.2.1. (Z)-ethyl 4-(5-bromo-3-methylbenzofuran-2-yl)-2-hydroxy-4-oxobut-2-enoate (2a).
1
Light yellow solid, mp 169-171 °C, yield: 59%, Rf (hexane: ethyl acetate = 7:3): 0.17, H
NMR (400 MHz, DMSO-d6) (δ, ppm): 8.14 (s, 1H, Ar-H), 7.73 (s, 2H, Ar-H, =CH), 6.98 (s,
13
1H, Ar-H), 4.33 (q, 2H, J = 7.05, CH2), 2.62 (s, 3H, CH3), 1.31 (t, 3H, J = 7.1, CH2). C
NMR (75 MHz, CDCl3) (δ, ppm): 183.55, 167.74, 161.99, 153.35, 146.76, 131.68, 131.41,
126.07, 124.11, 116.69, 113.70, 99.85, 62.68, 14.12, 9.61. x LC-MS (m/z): 352.9 [M+H]+. IR
(neat): 1726, 1570, 1145 cm-1.
4.1.2.2. (Z)-ethyl 2-hydroxy-4-oxo-4-(2-methylphenyl)-but-2-enoate (2b). Yellow oil, yield:
65%, Rf (hexane: ethyl acetate = 7:3): 0.66, 1H NMR (300 MHz, DMSO-d6) (δ, ppm): 12.79
(brs, 1H, OH), 7.62 (d, 1H, J = 7.6 Hz, Ar-H), 7.41 (t, 1H, J = 7.0 Hz, Ar-H), 7.30-7.25 (m,
2H, Ar-H), 6.82 (s, 1H, Ar-H), 4.38 (q, 2H, J = 7.2, CH2), 2.54 (s, 3H, CH3), 1.39 (t, 3H, J =
7.2 Hz, CH3). LC-MS: m/z [M +H]+ 235.5. IR (neat): 2985, 1734, 1611, 1261, 1019 cm-1.
4.1.2.3. (Z)-ethyl 4-(4-chlorophenyl)-2-hydroxy-4-oxobut-2-enoate (2c). Yellow solid, mp
189-191 °C, yield: 63%, Rf (hexane: ethyl acetate = 7:3): 0.66, 1H NMR (300 MHz, DMSO-
d6) (δ, ppm): 14.79 (brs, 1H, OH), 8.09 (d, 2H, J = 7.4 Hz, Ar-H), 7.64 (d, 2H, J = 7.8 Hz,
Ar-H), 7.12 (s, 1H, Ar-H), 4.32 (q, 2H, J = 6.9, CH2), 1.31 (t, 3H, J = 7.1 Hz, CH3). LC-MS
(m/z): 255.3 [M+H]+. IR (neat): 1734, 1592, 1261 cm-1.
4.1.3. General synthesis of diketo acids (DKA) (3a-b)
To the stirred solution of diketoester (1.0 eq) in the mixture of THF and water (3:1,
5mL/mmol) was added lithium hydroxide (2.2 eq) and the reaction mixture was stirred at
room temperature for 4 h. The reaction mixture was concentrated under vacuo, water was
added, acidified with 1 N HCl up to pH-2~3 and extracted with ethyl acetate. The combined
organic layers were washed with brine solution, dried over anhyd. Na2SO4, filtered and
concentrated under vacuo to afford pure diketo acid [24].
4.1.3.1. (Z)-4-(5-bromo-3-methylbenzofuran-2-yl)-2-hydroxy-4-oxobut-2-enoic acid (3a).
Yellow solid, mp 189-191 °C (decomposed), yield: 86%, Rf (hexane: ethyl acetate = 7:3):
1
0.0, H NMR (400 MHz, DMSO-d6) (δ, ppm): 8.11 (s, 1H, Ar-H), 7.70 (s, 2H, Ar-H, =CH),
6.87 (s, 1H, Ar-H), 2.60 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3) (δ, ppm): 183.26, 167.94,
163.18, 152.79, 146.41, 131.07, 130.93, 125.25, 123.57, 116.09, 113.24, 99.41, 9.10. LC-MS
(m/z): 323.2 [M-2H]-. IR (neat): 1741, 1719, 1577, 1249, 1089 cm-1.
4.1.3.2. (Z)-2-hydroxy-4-oxo-4-(2-methylphenyl)-but-2-enoic acid (3b). Yellow solid, mp
108-110°C, yield: 84%, Rf (hexane: ethyl acetate = 7:3): 0.0, 1H NMR (300 MHz, CDCl3) (δ,
ppm): 7.66 (d, 1H, J = 7.5 Hz, Ar-H), 7.44 (d, 1H, J = 6.9 Hz, Ar-H), 7.31-7.26 (m, 1H, Ar-
H), 6.93 (s, 1H, =CH), 6.09 (s, 1H, OH), 2.57 (s, 3H, CH3). LC-MS (m/z): 206.9 [M+H]+. IR
(neat): 1711, 1633, 1275, 1037 cm-1.
20

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4.1.4. General synthesis of coupling of diketo acid with L-alanine methyl ester (L-Ala-OCH3)
(4a-b)
To the stirred solution of diketo acid (1.0 eq) in dry THF (5 mL/mmol) was added N,N-
diisopropylethylamine (DIPEA) (2 eq) and HATU (1.5 eq) and the reaction mixture was
stirred at room temperature for 20 min. L-alanine methyl ester (1.1 eq) was added and the
reaction mixture was stirred at room temperature for 16 h. After completion, the reaction
mixture was quenched with water and extracted with ethyl acetate. The combined organic
layers were washed with brine solution, dried over anhyd. Na2SO4, filtered and concentrated
under vacuo. The crude obtained was purified with column chromatography eluted with 20%
ethyl acetate in hexane to afford the title compounds [25].
4.1.4.1.
(R,Z)-methyl
2-(4-(5-bromo-3-methylbenzofuran-2-yl)-2-hydroxy-4-oxobut-2-
enamido)propanoate (4a). Off white solid, mp 163-165 °C, yield: 67%, Rf (hexane: ethyl
acetate = 7:3): 0.17, 1H NMR (400 MHz, DMSO-d6) (δ, ppm): 9.21 (d, 1H, J = 7.2 Hz, NH),
8.13 (s, 1H, Ar-H), 7.74 (s, 2H, Ar-H, =CH), 6.98 (s, 1H, Ar-H), 4.46 (q, 1H, J = 6.76 Hz,
CH), 3.65 (s, 3H, OCH3), 2.62 (s, 3H, Ar-CH3), 1.39 (d, 3H, J = 7.2 Hz, CH3). 13C-NMR (75
MHz, CDCl3) (δ, ppm): 179.63, 175.49, 172.53, 160.62, 153.51, 146.11, 131.43, 125.46,
124.37, 123.87, 116.58, 113.62, 96.26, 52.67, 48.22, 18.22, 9.58. LC-MS (m/z): 411.9 [M +
H]+. IR (neat): 3432,1756, 1633, 1208, 1138, 1086 cm-1.
4.1.4.2 (Z)-methyl 2-(2-hydroxy-4-oxo-4-(2-methylphenyl)-but-2-enamido) propanoate (4b).
1
Yellow oil, yield: 59%, Rf (hexane: ethyl acetate = 7:3): 0.56, H-NMR (400 MHz, DMSO-
d6) (δ, ppm): 9.19 (d, 1H, J = 6.6 Hz, NH), 7.68 (d, 1H, J = 7.4 Hz, Ar-H), 7.49-7.48 (m, 1H,
Ar-H), 7.37-7.35 (m, 2H, Ar-H), 6.72 (s, 1H, =CH), 4.47-4.41 (m, 1H, CH), 3.64 (s, 3H,
13
OCH3), 2.43 (s, 3H, CH3), 1.38 (d, 3H, J = 7.2 Hz, CH3). C-NMR (75 MHz, CDCl3) (δ,
ppm): 162.51, 143.24, 136.12, 134.88, 129.73, 129.29, 128.71, 127.99, 125.98, 121.44,
117.76, 57.61, 31.69, 28.75, 21.42. LC-MS (m/z): 292.4 [M + H]+. IR (neat): 3346, 2959,
1745, 1685, 1514, 1454 cm-1.
4.1.5. General hydrolysis of diketo acid-L-alanine methyl ester (DKA-Ala-OCH3) conjugates
(5a-b)
To the stirred solution of DKA-Ala-OCH3 (1.0 eq) in the mixture of THF and water
(3:1, 5mL/mmol) was added lithium hydroxide (2.2 eq) and the reaction mixture was stirred
at room temperature for 4 h. The reaction mixture was concentrated under vacuo, water was
added, acidified with 1 N HCl up to pH-2~3 and extracted with ethyl acetate (3 × 100 mL).
The combined organic layers were washed with brine solution, dried over anhyd. Na2SO4,
filtered and concentrated under vacuo to afford desired compounds [24].
4.1.5.1.
(R,Z)-2-(4-(5-bromo-3-methylbenzofuran-2-yl)-2-hydroxy-4-oxobut-2-enamido)
propanoic acid (5a). Off-white solid, mp 196-198 °C (decomposed), yield: 78%, Rf (hexane:
1
ethyl acetate = 7:3): 0.0, H NMR (400 MHz, DMSO-d6) (δ, ppm): 12.05 (s, 1H, OH), 9.01
(d, 1H, J = 7.5 Hz, NH), 8.12 (s, 1H, Ar-H), 7.72 (s, 2H, Ar-H, =CH), 6.98 (s, 1H, Ar-H),
13
4.39-4.36 (m, 1H, CH), 2.62 (s, 3H, Ar-CH3), 1.39 (d, 3H, J = 7.2 Hz, CH3). C NMR (75
MHz, CDCl3) (δ, ppm): 175.32, 173.22, 159.95, 152.47, 145.45, 130.74, 124.84, 123.25,
115.86, 113.02, 95.59, 47.62, 17.38, 8.95. LC-MS (m/z): 397.7 [M + H]+. IR (neat): 3335,
1722, 1659, 1536, 1086 cm-1.
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4.1.5.2. (R,Z)-2-(2-hydroxy-4-oxo-4-(2-methylphenyl)-but-2-enamido) propanoic acid (5b).
1
Yellow solid, mp 101-103 °C, yield: 68%, Rf (hexane: ethyl acetate = 7:3): 0.0, H NMR
(400 MHz, DMSO-d6) (δ, ppm): 12.76 (brs, 1H, OH), 8.98 (d, 1H, J = 7.2 Hz, NH), 7.68 (d,
1H, J = 7.2 Hz, Ar-H), 7.49-7.47 (m, 1H, Ar-H), 7.37-7.36 (m, 2H, Ar-H), 6.72 (s, 1H, =CH),
4.40-4.36 (m, 1H, CH), 2.41 (s, 3H, CH3), 1.38 (d, 3H, J = 7.2 Hz, CH3). 13C NMR (75 MHz,
CDCl3) (δ, ppm): 192.07, 176.64, 175.37, 161.29, 138.20, 134.73, 132.01, 131.87, 129.19,
126.04, 98.42, 48.15, 21.14, 17.88. LC-MS (m/z): 278.3 [M + H]+. IR (neat): 3335, 1741,
1659, 1532, 1197 cm-1.
4.1.6. General method for synthesis of isoxazole methyl ester (6a-c)
To the stirred solution of diketo ester (1.0 eq) in ethanol (5 mL/mmol) was added
hydroxylamine hydrochloride (3 eq) and the reaction mixture was refluxed for 4 h. After
completion of the reaction, the reaction mixture was cooled to room temperature. The solid
precipitate obtained was filtered over sintered funnel and washed with ethanol. The solid
product was dried under vacuo to afford isoxazole methyl ester [26].
4.1.6.1. Ethyl 3-(5-bromo-3-methylbenzofuran-2-yl) isoxazole-5-carboxylate (6a). Creamish
solid, mp 170-172 °C, yield: 74%, Rf (hexane: ethyl acetate = 7:3): 0.86, 1H NMR (400 MHz,
DMSO-d6) (δ, ppm): 8.07 (s, 1H, Ar-H), 7.67 (d, 1H, J = 8.4 Hz, Ar-H), 7.62 (d, 1H, J = 8.8
Hz, Ar-H), 7.34 (s, 1H, Ar-H), 4.42 (q, 2H, J = 6.8 Hz, CH2), 2.53 (s, 3H, CH3), 1.35 (t, 3H, J
= 7.0 Hz, CH3).13C NMR (75 MHz, CDCl3) (δ, ppm): 163.69, 159.60, 156.55, 153.39,
140.58, 131.46, 129.56, 123.16, 117.73, 116.45, 113.06, 101.93, 62.42, 14.15, 8.62. LC-MS
(m/z): 352.2 [M + 2H]+. IR (neat): 3093, 2985, 1726, 1618, 1443, 1261 cm-1.
4.1.6.2. Ethyl 3-(2-methylphenyl)-isoxazole-5-carboxylate (6b). Light yellow solid, mp 60-62
°C, yield: 70%, Rf (hexane: ethyl acetate = 7:3): 0.86, 1H NMR (300 MHz, CDCl3) (δ, ppm):
7.74 (d, 1H, J = 8.4 Hz, Ar-H), 7.41-7.27 (m, 3H, Ar-H), 6.84 (s, 1H, Ar-H), 4.48 (q, 2H, J =
10.6 Hz, CH2), 2.52 (s, 3H, CH3), 1.45 (t, 3H, J = 7.0 Hz, CH3). 13C-NMR (75 MHz, CDCl3)
(d, ppm): 168.54, 162.43, 157.87, 136.4, 130.32, 129.56, 128.65, 127.61, 125.92, 101.23,
60.41, 17.72, 14.59. LC-MS: m/z [M + H]+232.4. IR (neat): 2988, 1741, 1432, 1227 cm-1.
4.1.6.3. Ethyl 3-(4-chlorophenyl) isoxazole-5-carboxylate (6c). White solid, mp 62-64 °C,
yield: 74%, Rf (hexane: ethyl acetate = 7:3): 0.83, 1H NMR (400 MHz, DMSO-d6) (δ, ppm):
8.00 (d, 2H, J = 8.4 Hz, Ar-H), 7.64 (d, 2H, J = 8.4 Hz, Ar-H), 7.57 (s, 1H, Ar-H), 4.40 (q,
13
2H, J = 7.1 Hz, CH2), 1.34 (t, 3H, J = 7.1 Hz, CH3). C NMR (75 MHz, CDCl3) (δ, ppm):
170.56, 159.85, 157.06, 136.97, 129.50, 127.19, 125.09, 100.24, 62.31, 14.15. LC-MS (m/z):
252.23 [M + H]+. IR (neat): 3134, 2988, 1726, 1451 cm-1.
4.1.7. General method for the hydrolysis of isoxazole methyl ester (7a-c)
To the stirred solution of isoxazole methyl ester (1.0 eq) in the mixture of THF and
water (3:1, 5mL/ mmol) was added lithium hydroxide (2.2 eq) and the reaction mixture was
allowed to stir at room temperature for 4 h. After completion of the reaction, the mixture was
concentrated under vacuo, added water, acidified with 1 N HCl upto pH-2~3 and extracted
with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine
solution, dried over anhydrous Na2SO4, filtered and concentrated under vacuo to afford
isoxazole acid [24].
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4.1.7.1. 3-(5-bromo-3-methylbenzofuran-2-yl) isoxazole-5-carboxylic acid (7a). Creamish
solid, mp 185-187 °C, yield: 82%, Rf (hexane: ethyl acetate = 7:3): 0.0, 1H NMR (300 MHz,
DMSO-d6) (δ, ppm): 8.00 (s, 1H, Ar-H), 7.59 (q, 2H, J = 8.9 Hz, Ar-H), 7.19 (s, 1H, Ar-H),
2.51 (s, 3H, CH3).13C-NMR (75 MHz, CDCl3) (δ, ppm): 164.29, 159.71, 155.85, 153.87,
141.08, 131.73, 130.86, 123.56, 117.45, 115.95, 114.86, 101.47, 8.72. LC-MS (m/z): 276.0
[M -COOH]-. IR (neat): 2880,1715, 1644, 1447 cm-1.
4.1.7.2. 3-(2-methylphenyl)-isoxazole-5-carboxylic acid (7b). White solid, mp 148-150 °C,
yield: 86%, Rf (hexane: ethyl acetate = 7:3): 0.0, 1H NMR (300 MHz, CDCl3) (δ, ppm): 7.76
(d, 1H, J = 8.7 Hz, Ar-H), 7.43-7.33 (m, 3H, Ar-H), 6.91 (s, 1H, Ar-H), 5.68 (brs, 1H, OH),
13
2.54 (s, 3H, CH3). C-NMR (75 MHz, CDCl3) (d, ppm): 167.84, 161.83, 157.37, 135.76,
131.02, 129.66, 127.95, 127.31, 125.82, 101.54, 17.52. LC-MS (m/z): 204.2 [M + H]+. IR
(neat): 2955, 2869, 1704, 1264 cm-1.
4.1.7.3. 3-(4-chlorophenyl) isoxazole-5-carboxylic acid (7c). White solid, mp 201-203 °C,
1
yield: 75%, Rf (hexane: ethyl acetate = 7:3): 0.0, H NMR (400 MHz, DMSO-d6) (δ, ppm):
14.11 (brs, 1H, -COOH), 7.98 (d, 2H, J = 8.4 Hz, Ar-H), 7.64 (d, 2H, J = 8.5 Hz, Ar-H), 7.48
(s, 1H, Ar-H). 13C-NMR (75 MHz, CDCl3) (δ, ppm): 170.15, 161.17, 158.37, 136.04, 129.88,
128.05, 125.55, 101.88. LC-MS (m/z): 178.1 [M - COOH]-, 222.1 [M - H]-. IR (neat): 2858
1707, 1443, 1264 cm-1.
4.1.8. General synthesis of coupling of isoxazole acid with L-Ala-OCH3 (8a-c)
To the stirred solution of isoxazole acid (1.0 eq) in dry THF (5 mL/ mmol) was added
N,N-DIPEA (2 eq) and HATU (1.5 eq) and the reaction mixture was stirred at room
temperature for 20 min. L-alanine methyl ester (1.1 eq) was added to the reaction mixture and
was stirred at room temperature for 16 h. After completion of reaction, the reaction mixture
was quenched with water and extracted with ethyl acetate. The combined organic layers were
washed with brine solution, dried over anhyd. Na2SO4, filtered and concentrated under vacuo.
The crude obtained was purified with column chromatography with 20% ethyl acetate in
hexane to afford the title compound.
4.1.8.1.
(2R)-Methyl
2-(3-(5-bromo-3-methylbenzofuran-2-yl)isoxazole-5-
carboxamido)propanoate (8a). Brown solid, mp 160-162 °C, yield: 67%, Rf (hexane: ethyl
1
acetate = 7:3): 0.63, H-NMR (400 MHz, CDCl3) (δ, ppm): 7.73 (s, 1H, NH), 7.49-7.47 (m,
1H, Ar-H), 7.39-7.34 (m, 2H, Ar-H), 7.03 (s, 1H, Ar-H), 4.83-4.76 (m, 1H, CH), 3.79 (s, 3H,
OCH3), 2.53 (s, 3H, CH3), 1.55 (s, 3H, CH3).13C-NMR (75 MHz, CDCl3) (δ, ppm): 172.55,
163.49, 158.25, 157.94, 153.38, 140.66, 131.41, 129.51, 123.08, 117.54, 116.42, 113.10,
101.20, 52.67, 48.25, 18.29, 8.62. LC-MS (m/z): [M + 2H]+409.3. IR (neat): 3409, 2959,
1737, 1685, 1529, 1208 cm-1.
4.1.8.2. (2R)-methyl 2-(3-o-tolylisoxazole-5-carboxamido) propanoate (8b). Creamish yellow
1
solid, mp 68-70 °C, yield: 59%, Rf (hexane: ethyl acetate = 7:3): 0.64, H NMR (400 MHz,
DMSO-d6) (δ, ppm): 9.22 (d, 1H, J = 6.84 Hz, NH), 7.76 (d, 1H, J = 7.36 Hz, Ar-H), 7.47-
7.37 (m, 3H, Ar-H), 7.14 (s, 1H, Ar-H), 4.52 (q, 1H, J = 7.2 Hz, CH), 3.66 (s, 3H, OCH3),
2.49 (s, 3H, CH3), 1.42 (d, 3H, J = 7.2 Hz, CH3). 13C NMR (75 MHz, CDCl3) (δ, ppm):
172.63, 171.66, 158.59, 136.40, 131.48, 130.53, 128.48, 126.37, 102.19, 52.66, 48.17, 21.43,
18.31. LC-MS (m/z): 289.3 [M + H]+. IR (neat): 3286, 3339, 1748, 1663 cm-1.
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4.1.8.3. (R)-methyl 2-(3-(4-chlorophenyl) isoxazole-5-carboxamido) propanoate (8c). Off-
white solid, mp 144-146 °C, yield: 64%, Rf (hexane: ethyl acetate = 7:3): 0.55, 1H NMR (300
MHz, CDCl3) (δ, ppm): 7.73 (d, 2H, J = 8.4 Hz, Ar-H), 7.47 (d, 2H, J = 8.4 Hz, Ar-H), 7.34
(s, 1H, NH), 6.94 (s, 1H, Ar-H), 4.84-4.74 (m, 1H, CH), 3.80 (s, 3H, CH3), 1.55 (t, 3H, J =
7.2 Hz, CH3). 13C NMR (75 MHz, CDCl3) (δ, ppm): 171.60, 163.38, 161.54, 158.67, 135.24,
130.65, 129.65, 128.76, 101.88, 52.10, 49.5, 17.2. LC-MS: m/z [M + H]+309.2. IR (neat):
3312, 1745, 1447 cm-1.
4.1.9. General synthesis of hydrolysis of isoxazole-Ala-OCH3 (9a-c)
To the stirred solution of isoxazole-Ala-OCH3 (0.48 mmol) in the mixture of THF (6
mL) and water (2mL) was added lithium hydroxide (2.2 eq) and the reaction mixture was
stirred at room temperature for 4h. After completion of reaction (monitored by TLC) the
reaction mixture was concentrated under reduced pressure, added water, acidified with
1NHCl up to pH-2~3 and extracted with ethyl acetate (3x100 mL). The combined organic
layer was washed with brine solution, dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure to afford white acid.
4.1.9.1. (2R)-2-(3-(5-bromo-3-methylbenzofuran-2-yl) isoxazole-5-carboxamido) propanoic
acid (9a). White soild, mp 201-203 °C (decomposed), yield: 74%, Rf(hexane: ethylacetate =
7: 3): 0.0, 1H-NMR (300 MHz, DMSO-d6) (δ, ppm): 12.74 (brs, 1H, -COOH), 9.12 (d, 2H, J
= 6.9 Hz, NH), 8.01 (d, 2H, J = 7.8 Hz, Ar-H), 7.65-7.56 (m, 2H, Ar-H), 7.32-7.27 (m, 2H,
Ar-H), 4.45 (q, 1H, J = 6.5 Hz, CH), 2.49 (s, 3H, CH3),1.31 (s, 3H, CH3). 13C-NMR (75
MHz, CDCl3) (δ, ppm): 173.81, 162.07, 159.26, 158.21, 153.21, 140.60, 131.55, 129.93,
123.92, 118.19, 116.40, 114.05, 102.05, 48.46, 17.15, 8.82. LC-MS: m/z [M + H]+393.4.IR
(neat): 3432, 1722, 1689, 1532, 1451 cm-1.
4.1.9.2. (R)-2-(3-(4-chlorophenyl) isoxazole-5-carboxamido) propanoic acid (9b). White
1
solid, mp177-179 °C (decomposed), yield: 70%, Rf (hexane: ethylacetate = 7: 3): 0.0, H-
NMR (300 MHz, DMSO-d6) (δ, ppm): 12.79 (brs, 1H, -COOH), 9.02 (d, 2H, J = 7.5 Hz,
NH), 7.98 (d, 2H, J = 8.4 Hz, Ar-H), 7.64 (d, 2H, J = 8.4 Hz, Ar-H), 7.45 (s, 1H, Ar-H), 4.48
13
(q, 1H, J = 7.2 Hz, CH), 1.35 (s, 3H, CH3). C-NMR (75 MHz, CDCl3) (δ, ppm): 173.81,
169.78, 159.84, 158.69, 136.01, 129.90, 128.07, 125.60, 100.93, 48.37, 17.16.LC-MS: m/z
[M + H]+295.4.IR (neat): 3417, 3398, 3123, 1715, 1678, 1540, 1451, 1097 cm-1
4.1.9.3. 2-(3-(2-methylphenyl)isoxazole-5-carboxamido) propanoic acid (9c). White solid,
M.pt.: 146 °C, yield: 74%, Rf (hexane: ethylacetate = 7: 3): 0.0, 1H-NMR (400 MHz, DMSO-
d6) (δ, ppm): 7.72 (d, 1H, J = 5.7 Hz, NH), 7.39-7.38 (m, 2H, Ar-H), 7.33-7.30 (m, 2H, Ar-
H), 6.88 (s, 1H, Ar-H), 4.38 (q, 1H, J = 8.1 Hz, CH), 2.51 (s, 3H, CH3), 1.61 (d, 3H, J = 5.4
Hz, CH3). 13C-NMR (75 MHz, CDCl3) (δ, ppm): 176.63, 171.85, 158.95, 158.22, 136.46,
131.49, 130.59, 128.51, 126.37, 102.29, 48.16, 21.39, 17.94. LC-MS: m/z [M + H]+275.5. IR
(neat): 3361,3324, 1734, 1644, 1551, 1443, 1302cm-1.
4.2. X-ray crystallographic analysis
The structure of isoxazole acid (7b) was established by X-ray crystallographic analysis.
Single crystal of the compound was obtained through the slow evaporation of its
tetrahydrofuran solution. Suitable crystal was selected and analyzed on an APEX II CCD
Bruker
diffractometer equipped with rotating anode generator, the radiation was
monochromated with a Montel mirror (Cu Kα = 1.54178 Å). Intensity data were collected at
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100 K using θ-scan method. No significant loss in intensities was observed during data
collection. Multi-scan absorption corrections were applied to the intensity data empirically
using SADABS [27]. Data collection, reduction and refinement were performed using APEX
II [27] and SHELX [28] software. Crystal structures were solved by direct methods using
SHELXS and refined with full-matrix least-squares based on F2 using SHELXL [28]. All
non-hydrogen atoms were refined anisotropically. Hydrogens were first located in the Fourier
difference map, then positioned geometrically and allowed to ride on their respective parent
atoms. The molecular graphics and crystallographic illustrations were prepared using XP
[29].
4.3. Enzymatic inhibition assay
All compounds were dissolved in DMSO to a stock concentration of 10 mM. Initially
the compounds were screened for inhibition with each of the four MetAP's (HsMetAP1b,
MtMetAP1c, SpMetAP1a, and EfMetAP1a). Expression, purification and biochemical assays
are performed as reported previously. The enzyme assays were performed using Met-AMC as
substrate. % Inhibition were determined by using compound concentration range of 1 µM to
120 µM. The Ki values were determined by Dixon method. The reaction mixture contained
50 mM Hepes (pH-7.5 for SpMetAP1a, EfMetAP1a, MtMetAP1c and pH-8.0 for
HsMetAP1b), 150 mM KCl, CoCl2 (three molar equivalents of the enzyme concentration), 4
µM enzymes (HsMetAP1b, MtMetAP1c, SpMetAP1a or EfMetAP1a) and 50, 100, 150 µM
concentrations of L-Met-AMC. All reactions were performed in triplicate. 2,2’-Bipyridine
was used as positive control [9,19].
4.4. Glide Docking
In order to perform structure-function studies, docking of compounds 4a and 5a was
carried out within the active sites of the methionine aminopeptidase from M. tuberculosis
(PDB ID: 1YJ3), E. faecalis (PDB ID:3TB5) and S. pneumoniae (PDB ID: 4KM3) utilizing
GLIDE5.8 [30] in extra precision (XP) mode as implemented in the Schrödinger Suite.
During dockings, the receptors were kept rigid while ligands were treated as flexible, which
enables to dock the ligand at the receptor’s binding site.
The protein complexes were prepared using the protein-preparation wizard. Water
molecules were eliminated as no water bridges observed in the complexes. After assignment
of bond orders, polar hydrogens and adding missing residues, the whole systems were
optimized to an RMSD of 0.30 Å. Subsequently, electron affinity grids were generated for
each protein. Both the legands (4a and 5a) were optimized and prepared using methods as
mentioned in our earlier reports [31]. After ligand preparation, docking was performed using
extra precision mode of GLIDE. To improve the geometry, post-docking minimization of the
poses were accomplished.
4.5. In vitro antibacterial activity
The compounds which showed more than 60% enzyme inhibition (2a-c, 3a-b, 4a-b, 5a-
b, 6c and 9a) were screened for antibacterial activity against Gram-positive bacteria
including S. pneumoniae (MTCC 655), E. faecalis (MTCC 439) and Gram-negative bacteria
including P. aurogenosa (MTCC 2453), S. typhimurium (MTCC 3224), K. pneumoniae
(ATCC 700603), E. coli (ATCC 25922) and multidrug resistant strains viz. E. coli MRA11
(GenBank Accession no. KJ957160), MRC17 (GenBank Accession no. KJ906623), MRC24
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(GenBank Accession no. KM822765), MRAE26 (GenBank Accession no. KJ923014),
MRAE32 (GenBank Accession no. KJ923017), MRAE33 (GenBank Accession no.
KM822768), MRAE44 (GenBank Accession no. KJ923018) and MROB11 (GenBank
Accession no. KC963018) using broth dilution technique according to standard protocol for
antibacterial assessment by NCCLS [32]. Ciprofloxacin (CIP) was used as positive control.
DMSO was used as solvent with less than 4% concentration in final volume used for the
assay. The test compounds were serially diluted (1000 to 7.8 µg/mL) in 96 well plate
containing (Tarson) 100 µL nutrient broth medium. Each well of plates was inoculated with
100 µL of fresh inoculums containing approximately 2.5 × 106 cells/mL and incubated at 37
°C with constant stirring of 150 rpm for 24 h. After incubation period, minimum inhibitory
concentration (MIC) was determined as the lowest concentration of compound/drug at which
no visible growth appeared.
However, in case of multi-drug resistant isolates, varying concentrations (8 to 1024
µg/mL for 4a and 5a) and (0.49 to 63.3 µg/mL for ciprofloxacin) were dispensed into a 96-
well plate (Tarson) in luria broth medium. Then, the medium was inoculated with 10 µl of
1000 times diluted 0.1 OD600 culture and incubated overnight at 37 °C. The OD was
measured at 600 nm using a microplate reader (Thermo Scientific MultiskanGo). Percentage
inhibition of these resistant isolates was calculated using the formula:
Percentage inhibition = a−b ̸ a×100
Where a = pure culture reading – media reading
b = test reading – plain compound reading (if the compound is colored solution)
4.6. Synergistic study
Synergistic or antagonistic property of lead inhibitors 4a and 5a with standard drug
ciprofloxacin was determined as fractional inhibitory concentration index (FICI) according to
the checkerboard method [33]. A concentration gradient ranging from 1024-16 µg/mL of
compounds 4a and 5a were maintained in 96 well plate followed by the addition of the
ciprofloxacin with concentration ranging from 128-2 µg/mL in 100 µL of Mueller-Hinton
broth. Then, 100 µL of a suspension of 5 × 105 CFU/mL was added in each well as inoculums
incubated for 18 h at 37 °C. The fractional inhibitory concentration (FIC) index is defined as
the sum of the MIC of each drug when used in combination divided by the MIC of the drug
used alone. Synergy and antagonism were defined by FICI ≤ 0.5 and > 4, respectively.
Partially synergistic was denoted by 0.5> FICI <1 while Indifferent was defined by 1 < FICI
≤ 4 [34].
4.7. Cytotoxicity by MTT assay
MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyl tetrazolium bromide), Dulbecco’s modified
Eagle’s medium (DMEM), 0.25% trypsin and a 0.02% EDTA mixture were purchased from
Hi Media (Mumbai, India). Fetal bovine serum (FBS) was obtained from Gibco (Grand
Island, NY). The human embryonic kidney (HEK293) cell line was procured from National
Centre for Cell Sciences (NCCS), Pune, India. The cells were cultured and maintained as a
monolayer in DMEM supplemented with 10% FBS and antibiotics (100 units/mL penicillin
and 100 µg/mL streptomycin) at 37 °C in humidified atmosphere of 5% CO2 in T-25 flasks.
The cells were subcultured twice in a week. A cell count of approximately 2 × 104 cells per
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well were seeded in 96-well plate (150 µL per well) and incubated for 24 h before treatment.
The cells were then treated with varying concentrations (10-400 µg/mL) of the test
compounds. After 48 h of incubation at 37 °C, the exhausted serum supplemented medium
was removed and serum free media (50 µL) was added into each well. After that, 20 µL per
well of MTT at a concentration of 5 mg/mL in PBS was added to each well and the plates
were incubated for 4 h at 37 °C. Formazan crystals, the metabolized MTT product, were
solubilized in DMSO (150 µL per well) and were quantified by reading the absorbance at 570
nm after incubation of 10 min on iMark Microplate Reader (Bio-Rad, Hercules, CA). All
assays were performed in triplicate. Percent viability was taken as the relative absorbance of
treated versus untreated control cells [35].
4.8. Growth kinetics studies
On the basis of preliminary studies and cytotoxicity, compound 5a was selected as lead
inhibitor and explored for further biological activities. Growth kinetics study of S.
pneumoniae and E. coli was determined in the presence of lead inhibitors 5a along with
standard drug ciprofloxacin. 50 mL of sterile nutrient broth medium was inoculated with
freshly prepared bacterial culture containing approx. 2×106 cells/mL. After addition of
different concentrations (2MIC, MIC and ½ MIC) of compound 5a, flasks were incubated at
37 °C and 160 rpm. Ciprofloxacin at a concentration of 10 µg/mL was used as positive
control. Aliquots (1 mL) of culture were removed from each test sample at time interval of
two hours (i.e., time point of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 h) and growth was
measured turbidometrically at 600 nm using Thermo MultiskanGo Spectrophotometer.
Optical density was recorded for each concentration against time (h) [36].
4.9. Transmission electron microscopic (TEM) analysis
The morphology of S. pneumoniae and E. coli cells were analysed using TEM
following the standard protocol [37]. Briefly, the cells were harvested, standardized (A600
≈
0.1) and exposed to MIC conc. of test inhibitor 5a for 1 h. Then, the cells were washed thrice
with PBS to remove the residual medium and fixed overnight in 2.5% glutaraldehyde in
phosphate/magnesium buffer (40 mM K2HPO4/KH2PO4, pH 6.5, 0.5 mM MgCl2). The cells
were washed twice for 15 min in 0.1 M sodium phosphate buffer (pH 6.0) and post-fixed for
2 h in 2% osmium tetroxide. Again, the cells were washed twice for 15 min in distilled water
and then en bloc stained with 1% uranyl acetate (aqueous) for 30 min. After two further
washes, cells were dehydrated in 95% and 100% ethanol. The cells were exposed to
propylene oxide for 2×10 min and infiltrated for 1 h in 1:1 propylene/epoxy embedding
material (Epon) mixture and then overnight in fresh Epon. After polymerization for 48 h at 60
°C, ultrathin sections were cut using a microtome (LeicaEM UC6) and transferred onto a
copper grid. The samples were stained with uranyl acetate (saturated solution of uranyl
acetate in 50% alcohol) followed by lead citrate. The samples were washed three times in
Milli-Q (MQ) water and dried by touching with Whatman filter paper. The sections were
examined with a Tecnai G2 20 high resolution transmission electron microscope (Fei
Company, The Netherlands) at 200 kV.
4.10. Anti-biofilm assessment
Biofilm formation is an important virulence factor of most of the pathogenic micro-
organism which is an initial step to establish an infection. Anti-biofilm activity of compound
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5a was determined by biochemical assay using XTT as well as by scanning electron
microscopy (SEM).
4.10.1. Anti-biofilm activity by XTT assay
XTT (2, 3-bis (2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-
tetrazolium-hydroxide) reduction assay was used to determine the metabolic activity of
biofilm formation in bacteria [38]. Compound 5a was serially diluted with concentration
range from 1000 µg/mL to 7.8 µg/mL in microtitre plate having tryptic soy broth. Aliquots of
100 µL of tryptic soy broth containing bacterial inoculums of approximately 5×105 CFU/mL
were added to each well and incubated at 37 °C for 24 h under static conditions. The medium
was discarded and washed with phosphate buffer saline (PBS) to remove the non-adherent
bacteria. To each well 50 µL of prepared XTT salt solution (HiMedia, INDIA) was added and
plates were incubated at 37 °C in dark for 90 min. Bacterial dehydrogenase activity reduces
XTT tetrazolium salt to XTT formazan, resulting in colorimetric change (turns to orange) that
was correlated with cell viability. The colorimetric changes were measured
spectrophotometrically at 490 nm [39]. The inhibition data were interpreted from dose–
response curves, where an IC50 value is defined as the concentration of the inhibitor required
to inhibit 50% of biofilm formation under the above assay conditions.
4.10.2. Scanning electron microscopic (SEM) analysis for biofilm formation
Scanning electron microscopic (SEM) analysis was performed to determine the biofilm
formation by E. coli. The fresh bacterial cultures were prepared and inoculated into six-well
cell culture plates containing 3 mL of tryptic soy broth (TSB) (containing 2.5 g/L of glucose).
Glass coverslips (8 mm dia.) were dispensed into each well for biofilm formation on the
surface and incubated at 37 °C for 24 h. Post incubation, plates were removed and the test
compound was added to determine the anti-biofilm effect. Parafilm sealed plates were further
incubated for the next 24 h. Coverslips were removed after total incubation of 24 h and
washed with 0.1 M PBS. The biofilm that formed on the coverslips were placed in fixative
(4%, v/v, formaldehyde in PBS) overnight. Samples were again washed with PBS and then
coverslips were left for drying and later examined under scanning electron microscope [40].
4.11. In vivo toxicity evaluation of 5a in G. mellonella larvae
The larvae of the sixth developmental stage of G. mellonella were obtained from the
Meal Worm Company (Sheffield, England). The larvae of G. mellonella were stored in wood
shavings in the dark at 15 °C prior to use. The larvae that were chosen for experiments
weighed on 0.21 g and were used within 3–4 weeks of receipt. Ten healthy larvae were
placed in sterile 9 cm Petri dishes with Whatman filter paper inserted inside. A culture of E.
coli was grown to the stationary phase (1–2×108/mL) in nutrient broth at 30 °C and 200 rpm.
The cells were harvested by centrifugation (2,056 g for 5 minutes on a Beckmann GS-6
bench centrifuge) washed in PBS and resuspended in PBS at a cell density 5 × 105 per 20 µL.
The larvae were inoculated by injecting 20 µL through the last left pro-leg into the haemocoel
using a Myjector syringe (Terumo Europe) and incubated for 1 h placed at 30 °C in the dark.
After the incubation period, the larvae were inoculated with test compound 5a at a
concentrations of 2.5 mg suspended in PBS, supplemented with 12.5% DMSO (v/v), through
the last right pro-leg. The larvae injected with 20 µL of PBS supplemented with 12.5%
DMSO (v/v) were used as controls. For assessment of larval viability, the larvae were gently
probed with a needle and if no response was observed, the larvae were considered to be dead.
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