Synthesis and evaluation of thiophene based small molecules as potent inhibitors of Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2020.112772

Herein, we report the synthesis and anti-tubercular studies of novel molecules based on thiophene scaffold. We identified two novel small molecules 4a and 4b, which demonstrated 2-fold higher in vitro activity (MIC₉₉: 0.195 μM) compared to firs

Full text of DOI:10.1016/j.ejmech.2020.112772

European Journal of Medicinal Chemistry 208 (2020) 112772
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis and evaluation of thiophene based small molecules as
potent inhibitors of Mycobacterium tuberculosis
Chhuttan L. Meena 1, Padam Singh 1, Ravi P. Shaliwal, Varun Kumar, Arun Kumar,
Anoop Kumar Tiwari, Shailendra Asthana, Ramandeep Singh**, Dinesh Mahajan*
Translational Health Science and Technology Institute (THSTI), NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurugram Expressway, Faridabad,
121001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we report the synthesis and anti-tubercular studies of novel molecules based on thiophene
Received 27 March 2020
Received in revised form
18 June 2020
Accepted 17 August 2020
Available online 23 August 2020
scaffold. We identified two novel small molecules 4a and 4b, which demonstrated 2-fold higher in vitro
activity (MIC99: 0.195
mM) compared to first line TB drug, isoniazid (0.39 mM). The identified leads
demonstrated additive effect with front line TB drugs (isoniazid, rifampicin and levofloxacin) and syn-
ergistic effect with a recently FDA-approved drug, bedaquiline. Mechanistic studies (i) negated the role of
Pks13 and (ii) suggested the involvement of KatG in the anti-tubercular activity of these identified leads.
© 2020 Elsevier Masson SAS. All rights reserved.
Keywords:
Tuberculosis
Thiophene
Pks13
KatG
Synergy
1. Introduction
drug-resistant strains and failure of BCG vaccine to impart protec-
tion against adulthood TB [1]. However, the combination of
Tuberculosis (TB) has decimated and impacted countless human
lives for tens of thousands of years and is ranked among the top 10
causes of the deaths worldwide in modern era [1]. According to
World Health Organization (WHO) reports, nearly 1.2 million and
0.25 million deaths occurred due to TB infection and HIV-TB co-
infection, respectively in 2018 [1]. The current efforts to control TB
are focused on the development of potent chemotherapeutics,
sensitive diagnostics and efficacious vaccines. There is an urgent
need to develop next-generation chemotherapeutics that possess a
novel mechanism of action and are compatible with the current
regimens. The new chemical entities should have low toxicological
profile and also be effective against drug-resistant M. tuberculosis.
Isoniazid (INH), pyrazinamide (PZA), rifampicin (RIF) and etham-
butol (EMB) are the four first line TB drugs which were identified in
1951, 1952, 1957 and 1962, respectively [2]. The current situation is
further aggravated due to the emergence of multi- and extensively
phenotypic screening and next generation sequencing has resulted
in identification of bedaquiline (BDQ), delamanid and pretomanid
(PA-824) that have been approved by FDA for use to treat in-
dividuals with MDR-TB [3e8]. Further, resurgence in TB drug dis-
covery have also led to identification of various small molecules
that possess a novel mechanism of action and are being evaluated
for safety in different stages of clinical trials [9e13].
Thiophene based compounds have been reported to possess
antimicrobial, analgesic, anti-inflammatory, antihypertensive and
antitumor activity [14e17]. Thiophene based compounds (Fig. 1)
have also been reported for their anti-tubercular activity [17e24].
Wilson et al. screened a NIH library of 1113 compounds in iniBAC
reporter assay and identified two lead compounds A and B (Fig. 1)
having capability to inhibit mycobacterial growth by targeting
Pks13 enzyme [18]. In another study, Aggarwal et al. showed that
TAM16, a benzofuran scaffold inhibits thioesterase activity associ-
ated with Pks13 [23]. A small molecule C was identified using NMR
based fragment screening of antigen 85C [20]. However the iden-
tified molecule possesses weak activity against M. tuberculosis.
Ibrahim et al. reported that ethyl 2-amino-4,5,6,7-tetrahydrobenzo
[b]thiophene-3-carboxylate (compound D) inhibits mycolyl trans-
ferase activity of Ag85C, however, the lead compound displayed
* Corresponding author.
** Corresponding author.
E-mail addresses: ramandeep@thsti.res.in (R. Singh), dinesh.mahajan@thsti.res.
in (D. Mahajan).
1
Authors contributed equal to the study.
https://doi.org/10.1016/j.ejmech.2020.112772
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
C.L. Meena et al. / European Journal of Medicinal Chemistry 208 (2020) 112772
MIC99 values of 1.8
m
M against M. tuberculosis in whole cell based
analogues 2c (dimethylamine) and 2e (pyrrolidine) were inactive
with MIC99: >10 M. In concordance, compounds substituted with
higher structural homologs of pyrrolidine i.e. piperidine (2f) and
morpholine (2j) were also found to be inactive (MIC99: >10 M). In
continuation, loss of anti-mycobacterial activity was also observed
for analogues synthesized from more bulky amines such as aniline
and benzylamine i.e. 2k and 2l respectively. This data indicates the
possibility of steric clashes between target structure and ligands
possessing bulky substituents. In contrast, we noticed that the
assays [21]. In a target-based screening study against tyrosine
phosphatase activity of PtpB, thiophenesulfonamide (compound E)
was identified [22]. In a whole cell based screening against
M. tuberculosis, another thiophene based compound F was identi-
fied [19].
In the present study, we are reporting the synthesis and
screening of a novel library of thiophene based compounds for
activity against M. tuberculosis. We have identified few novel
compounds that are more potent than INH and also exhibit synergy
with BDQ, a recently FDA-approved drug for individuals suffering
with MDR-TB. The identified compounds also possess activity
against intracellular bacteria in THP-1 macrophages.
m
m
replacement of diethylamine (2d; 3.12
mM) with isopropylamine
(2g; 1.56 M) at R2 position resulted in 2.0-fold increase of anti-
m
mycobacterial activity (Table 1). Also, anti-mycobacterial activity
of the compounds was retained when isopropylamine (2g;
1.56
formationally restricted cyclopropylamine (2h). The synthesized
compounds were non-cytotoxic even at 50 M in our cell viability
mM) or diethylamine (2d; 3.12 mM) was replaced with con-
2. Results and discussion
m
assays (Table 1). Taken together, SAR understanding from series 1
and 2 suggested that these compounds inhibit bacterial growth by
interacting with a specific target. Despite our lack of understanding
for the molecular target of this series of compounds, the SAR
studies suggested that the anti-tubercular activity is sensitive to the
right-hand side derivatization on amine. The in vitro anti-
tubercular activity was observed to be sensitive for size/steric
bulk as well as conformation of the substituents at N-atom. This
advocates the presence of a well-defined three-dimensional pocket
in the molecular target. Based on these results, we further designed
new analogues of the two most active molecules 1h and 2h as
described in Schemes 3 and 4 (vide infra).
The synthesis of second generation analogues was initiated as
mentioned in Scheme 3. A suitable protection of methyl 2-
aminothiophene-3-carboxylate (8) was achieved by utilizing
(Boc)2O. Further, the protected intermediate was reacted with N-
bromosuccinimide to form compound 9 in good yield and purity.
The compound 9 was used for the synthesis of two new key in-
termediates 10 (Scheme 3) and 11 (Scheme 4) which served as
precursors for synthesis of 3a-e and 4a-h, respectively. The inter-
mediate 10 was reacted with triphosgene and desired amine to get
different urea derivatives i.e. 3a-e. The intermediate 11 was reacted
with different acid chlorides to prepare corresponding amides 4a-
h. The compounds 3a-e and 4a-h were evaluated for their anti-
tubercular activity (Table 2). All newly synthesized urea ana-
logues (3a-e) of compound 2h were found to be inactive. The lack of
activity of compounds 3b, 3c was explainable based on the struc-
tural similarity of these molecules to compounds 2e and 2f as
mentioned in Table 1. However, the complete loss of activity for
compounds 3a, 3d and 3e which are fluorine-substituted de-
rivatives of active compounds 2d, 2g and 2h, respectively was
The chemical synthesis of various thiophene analogues was
performed by diversification of a key intermediate 7. The synthesis
of intermediate 7 was achieved by reaction of ethyl cyanoacetate 6,
with elemental sulfur [S8] in the presence of 2-phenylacetaldehyde
(5) under mild condition as mentioned in the Scheme 1 [25]. The
intermediate 7 was subsequently derivatized by coupling with
different carboxylic acids leading to the synthesis of corresponding
amides 1a-h (Scheme 1). In a typical procedure, the carboxylic acid
was activated by methanesulfonyl chloride (MsCl), followed by the
addition of intermediate 7 as a solution in dichloromethane (DCM),
as mentioned in Scheme 1 (vide infra). Additionally, the key inter-
mediate 7 was used for the synthesis of urea derivatives 2a-k. To
achieve this synthesis, 7 was reacted with triphosgene, followed by
addition of corresponding amine to form 2a-k as mentioned in
Scheme 2 (vide infra).
All newly synthesized molecules of both the series i.e. 1a-h and
2a-k were evaluated for whole-cell anti-tubercular activity in
triplicates using INH as a positive control. MIC99 values were
determined against M. tuberculosis H37Rv and shown in Table 1
(vide infra). We observed that most of the amide analogues in se-
ries 1 (except 1h; MIC99: 0.78
M. tuberculosis growth even at 10
m
M) were unable to inhibit
m
M concentration. However, the
screening data of urea analogues of series 2 was encouraging. Three
new molecules i.e. 2d, 2g and 2h showed anti-mycobacterial ac-
tivity in the range of 1.56e3.12 mM. The analysis of activity data of
series 1 and 2 provided some intriguing insights on structure ac-
tivity relationship (SAR). The intermediate 7 itself was observed to
be inactive. It was observed that minor modifications in the
structures resulted in considerable alteration in anti-mycobacterial
activity. For example, compound 2d, a urea analogue with dieth-
ylamine is active (MIC99
: 3.12 mM), whereas the associated
Fig. 1. Reported small molecules based on thiophene scaffold with their anti-tubercular activity.

C.L. Meena et al. / European Journal of Medicinal Chemistry 208 (2020) 112772
3
Scheme 1. Reagents and conditions: (i) Et3N, S8, EtOH, rt, 24 h (ii) DCM, MsCl, DIPEA, 0 ꢀC to rt, 2 h.
Scheme 2. Reagents and conditions: (i) Triphosgene, Toluene, reflux, 12 h (ii) Desired amine, DIPEA, rt, 2e6 h.
surprising. The fluorine atom has a bigger size (van der Waals
radius of 1.47 Å), comparable to hydrogen (van der Waals radius of
1.20 Å), but the associated complete loss of activity was not antic-
ipated owing to such a minor steric perturbation. However, it may
not be wise to disregard the contribution of significant change in
electronic nature of the molecule due to substitution of fluorine as
previously reported [26]. This certainly needs further study for a
better understanding. However, we also observed a 4.0-fold in-
crease in potency with the chloro analogue 4a in comparison to the
analogue with bromine substitution, 1h. This increase in potency
instigated to the synthesis of new set of molecules (4a and 4c-g)
around 4b with structural changes on R1 (Table 2). Unlike urea
analogues, the changes on the R1 position demonstrated very
minimal effect on the activity of 4b. Consistent to earlier findings
from data of series 3, flouro substitution demonstrated a detri-
mental effect on activity (4b vs 4c) in series 4 also. However, this
loss was recovered by altering the position of flouro substitution
from -para to -meta (4c vs 4d). In our cell viability experiments, the
analogues synthesized in Scheme 3 and Scheme 4 were inactive
identification of four leads 4a, 4b, 4d and 4f which exhibited better
or comparable activity to INH, a first-line TB drug. Since the active
compounds 4a-g, possess electrophilic a-chloroacetamide moiety,
so there is a possibility that these molecules inhibit mycobacterial
growth by covalently binding to target protein.
Subsequent experiments were performed using 4a and 4b, the
two most active compounds identified in our screen. We next
determined the mode of M. tuberculosis killing by selected com-
pounds 4a and 4b in vitro and in macrophages. As shown in Fig. 2A,
we observed that exposure of M. tuberculosis early-logarithmic
cultures to these compounds at 10x MIC99 resulted in growth in-
hibition. We noticed that in comparison to untreated samples, 10.0-
fold less bacterial numbers were observed upon exposure of
M. tuberculosis to 10x MIC99 concentration of both 4a and 4b for 7
days (Fig. 2A). As expected, >4 log10 killing was observed upon
exposure of early-log phase culture to INH (Fig. 2A). We also
determined the ability of both 4a and 4b to inhibit growth of
intracellular bacteria in THP-1 macrophages. We observed that
exposure to 4a and 4b for 4 days inhibited the growth of intracel-
lular bacteria by 2.5- and 5-fold, respectively, in THP-1 macro-
phages (Fig. 2B). As expected, exposure to INH for 4 days inhibited
even at 50
mM concentration (Table 2). In conclusion, the screening
of different analogues for anti-mycobacterial activity resulted in

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C.L. Meena et al. / European Journal of Medicinal Chemistry 208 (2020) 112772
Table 1
MIC99values of various amides/ureas against M. tuberculosis H37Rv.
Sr. No
Compound No
R2
MIC99
[mM]
Cytotoxicity [mM]
1
2
7
1a
NA
>10
>10
>50
>50
3
4
1b
1c
>10
>10
>50
>50
5
6
1d
1e
>10
>10
>50
>50
7
1f
>10
>50
8
9
1g
1h
>10
>50
>50
0.78
10
11
2a
2b
>10
>10
>50
>50
12
13
2c
>10
>50
>50
2d
3.12
14
15
16
2e
2f
>10
>10
1.56
>50
>50
>50
2g
17
2h
1.56
>50
18
19
2i
2j
>10
>10
>50
>50
20
2k
>10
>50
21
22
2l
>10
>50
>50
INH
e
0.39
the growth of intracellular bacteria by ~200 fold (Fig. 2B).
improved their individual MIC99 by 4.0-fold and 8.0 fold, respec-
tively (Fig. 2C). We also observed the additive effect of 4a and 4b
when used in combination with other TB drugs. The FICI values of
4a and 4b with RIF were 0.75 and 0.625 with INH were 0.75 and
0.562 and with LEVO were 0.562 and 0.562 and with PA-824 were
1.0 and 0.625, respectively (Fig. 2C). Previously, it has been shown
that BDQ shows synergy with drugs involved in lipid transport and
arabinan biosynthesis such as BTZ043 and SQ-109 [29,30]. The
observed synergy between 4a, 4b and BDQ could be attributed to
A new drug lead possessing capability to decrease dosing con-
centration of existing drugs, when used in combination, can
potentially reduce dose associated side effects. Therefore, we
determined the activity of 4a, 4b alone or in combination with RIF,
INH, LEVO, BDQ or PA-824 using two-drug checkerboard assay as
previously described [27,28]. As shown in Fig. 2C, 4a and 4b syn-
ergised with BDQ against M. tuberculosis with the FICI value of 0.5
and 0.25 respectively. The combination of 4a and 4b with BDQ

C.L. Meena et al. / European Journal of Medicinal Chemistry 208 (2020) 112772
5
Scheme 3. Reagents and conditions: (i) DCM, (BOC)2O, 4-(Dimethylamino)pyridine, rt, 24h (ii) NBS, AcOH:DCM, (1:1) rt, 3h (iii) 4-Flurophenyl boronic acid, aq.K2CO3, Pd(PPh3)4, 1,4-
dioxane, N2(g), 80 ꢀC, 3e4h (iv) 4M HCl in 1,4-Dioxane, rt (v) Triphosgene, Toluene, reflux, 12 h, (vi) Desired amine, DIPEA, Dry DCM, rt. 2h
Scheme 4. Reagents and conditions: (i) AreB(OH)2, Pd(PPh3)4, 80 ꢀC, 3e4h (ii). 4M HCl in 1,4-Dioxane (iii) ClCH2COOH., Dry DCM, MsCl, DIPEA, rt, 2h
the fact that these molecules either interact with a complementary
off target or facilitate disruption of the cell wall architecture leading
to increased BDQ intracellular levels. Taken together, the data
presented suggests that the lead molecules have the potential to
reduce the dosing concentration of BDQ in clinical settings. The use
of combination therapy might result in (i) faster clearance of bac-
teria (ii) better patient compliance and (iii) minimize the emer-
gence of drug-resistant strains.
It has been shown that the over expression of the drug target
involved in mechanism of action may result in resistance to the
corresponding drug. An increase in anti-tubercular activity for TB
drugs i.e. INH, PZA and cycloserine (CS) have been reported when
their corresponding targets inhA, rpsA and alrA have been overex-
pressed [31e33]. Previous studies have shown that thiophene
based scaffolds inhibit mycobacterial Pks13 and an increase in MICs
was observed upon overexpression of Pks13 in M. tuberculosis [18].
Therefore, the active compounds identified from the present study
were further evaluated for their inhibitory activity in Pks13 over-
expressing strain of M. tuberculosis. We observed that over-
expressing of Pks13 did not alter the activity of these analogues;
thereby suggesting that Pks13 is not the cellular target for these
identified leads (Table 3).
M. tuberculosis KatG activates the INH by facilitating its hydrolysis
[34]. Surprisingly, all active compounds were unable to inhibit the
growth of INH resistant strain (Table 3). These observations suggest
that KatG is involved in the mechanism of action of these identified
drug leads. Since the lead compounds 4a and 4b were inactive in
resistant strains harbouring mutations in KatG, we performed
molecular docking studies to study the interaction of these thio-
phene analogues with KatG. The interaction maps of compounds 4a
and 4b with KatG was also compared with the reported interaction
map of KatG and INH (Fig. 3). The molecular docking of molecules
was performed on the reported crystal structure of KatG (PDB-ID:
1SJ2) [35]. For optimum understanding of the INH binding site as
well as key interacting residues, another co-crystal structure i.e.
3WXO was also referred in the present study (Fig. 3A and B). Three
binding sites have been reported by Kamachi et al. as potential sites
of interactions with INH [36]. We also performed docking of INH in
the crystal structure of KatG and observed an overlay between the
docked pose with reported co-crystal pose of INH (Fig. 3B). In
concordance with previous reports, we found residues W77, W78,
K130, L291, W293, I294, N295, G300 and I301 at binding site 2 are
the main INH interacting residues (Fig. 3C). Therefore, the docked
pose of INH was considered as grid centre to dock compounds 4a
and 4b at binding site 2. The molecular docking was conducted for
all the three reported INH potential interaction sites; however we
observed that both the compounds 4a and 4b have shown
considerably good docking scores at site 2 in comparison to site 1
and site 3. Therefore, only site 2 was considered for further
extensive focused docking studies to obtain the most likely
Due to the emergence of drug-resistant strains, there is an ur-
gent need to identify small molecules that possess inhibitory ac-
tivity against drug-resistant strains. Therefore, we next evaluated
the activity of the active compounds against INH resistant
M. tuberculosis strain, which harbours a mutation in activating
enzyme, KatG gene. Previously, it has been reported that

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C.L. Meena et al. / European Journal of Medicinal Chemistry 208 (2020) 112772
Table 2
MIC99values of compounds against M. tuberculosis H37Rv.
Sr. No
23
Compound No
3a
R1
R2
MIC99
[m
M]
Cytotoxicity [mM]
>10
>50
24
25
3b
3c
>10
>10
>50
>50
26
27
3d
3e
>10
>10
>50
>50
28
29
4a
4b
H
0.195
0.195
>50
>50
30
4c
0.78
>50
31
32
4d
4e
0.195
0.78
>50
>50
33
4f
0.39
>50
34
35
4g
0.78
0.39
>50
>50
INH
e
e
orientation of compounds. The docking energy of compound 4b
was ꢁ9.3 kcal/mol with 113 conformations and binding free energy
3. Conclusions
(DG) of - 54.2 kcal/mol, while the docking energy of compound 4a
In summary, this work led to the identification of a new class of
inhibitors that possess potent in vitro anti-tubercular activity.
Compound 4a and 4b were found to be more potent than INH, a
first line drug in our MIC99 determination assays. The two leads
demonstrated additive effect with front line TB drugs (INH, RIF and
LEVO) and synergistic effect with a recently approved drug BDQ,
when used in combination. The identified compounds were also
able to clear the growth of intracellular M. tuberculosis in THP-1
macrophages. In vitro studies suggest the involvement of KatG in
the mechanism of action of 4a and 4b.
was ꢁ8.4 kcal/mol with 89 conformations and
mol. However, the docking energy of INH was found to be ꢁ7.2 kcal/
mol with 91 conformations along with
G ¼ ꢁ41.3 kcal/mol. Apart
DG ¼ ꢁ43.8 kcal/
D
from sharing common INH interacting residues, the compound 4b
was also found to interact with residues N127, L277 G292. Unlike
compound 4b, compound 4a interacted with residue Q298 only and
did not show any interaction with residues K130 and L291 (Fig. 3D
and E). This study suggested that the binding site of 4a and 4b
overlapped with that of INH and these three compounds potentially
bind in same orientation. Additionally, for both 4a and 4b, the ar-
omatic residues of the binding site appear as the major contributors
for net binding affinity followed by hydrophobic contacts. We
observed that both the compounds 4a and 4b also established the
hydrogen bonds (HBs) with key residues. The compound 4a
showed HBs with residues N295 and G300, while compound 4b
formed three HBs with residues N127, K130 and N295. However, no
HBs were found in the case of INH. Overall, from this study, it may
be concluded that compounds 4a and 4b possibly mimic the
binding pattern and orientation of INH in KatG for their
interactions.
4. Experimental section
4.1. General methods for the synthesis
Unless otherwise stated, all the chemicals, silica and analytical
TLC were purchased from Sigma-Aldrich, Avra Pvt Ltd., Spec-
trochem and Merck. All intermediates and final compounds were
routinely checked for their purity on pre-coated silica gel G254 TLC
plates (Merck), Visualization of spot on TLC was achieved by the use
of UV light (254 nm) or by iodine vapours. All the synthesized

C.L. Meena et al. / European Journal of Medicinal Chemistry 208 (2020) 112772
7
Fig. 2. (A) In vitro killing of selected hits in liquid cultures. Early-logarithmic phase (OD600nm ~ 0.2) culture of M. tuberculosis was exposed to 10x MIC99 of various compounds for
7 days followed by CFU enumeration. (B) Intracellular activity of the selected hits; THP-1 cells were infected with M. tuberculosis followed by treatment with 4a, 4b or INH for 4
days. At designated time points, the infected macrophages were lysed with 0.1% PBST. For CFU enumeration, tenfold serial dilutions of the samples were plated on MB 7H11 agar and
incubated at 37 ꢀC for 3e4 weeks. (C) In vitro drug combination of selected hits with TB drugs against M. tuberculosis. In order to determine drug-drug interactions, check-
erboard assays were performed. Two fold serial dilutions of the lead compounds 4a and4b were prepared horizontally. This was followed by 2.0 fold cross-dilution with TB drugs to
make various combinations. FICI values for each combination were calculated as described in Materials and Methods.
Table 3
MIC99 of thiophene analogues against parental, Pks13 overexpressing and INH resistant M. tuberculosis strains.
Treatment
M. tuberculosis wild type
Sensitivity Profilea
M. tuberculosis-pMV306
M. tuberculosis-pMV306-pks13
INHR-mc2 497
4f
0.39
0.19
0.78
0.78
0.78
0.19
0.19
0.78
0.19
0.007
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
R
R
R
R
R
R
R
R
S
4d
4g
4c
4e
4b
4a
1h
INH
RIF
S
S
ND
NDb
a
In comparison to parental strain, MIC99 values of equal to 2x MIC99 or less are denoted by S; Sensitive and MIC99 values of equals to 4x MIC99 or more denoted as R;
Resistant.
b
ND; not done.
compounds were purified on column chromatographic by using of
100e200, 230e400 Mesh Silica gel. Automated flash column
chromatography was undertaken on SiO2 (230e400 mesh). The 1H
and 13C NMR spectra were recorded either using a Bruker Avance
DPX 400 (400 MHz for 1H and 100 MHz for 13C) or Bruker Avance
300 (300 MHz for 1H and 75 MHz for 13C) spectrometer in CDCl3 or
DMSO‑d6 using TMS as an internal standard. Proton and carbon
Exactive, Accela 1250 pump.
4.1.1. Ethyl 2-amino-5-phenylthiophene-3-carboxylate (7)
The mixture of ethyl cyanoacetate (107 mL, 1.0 mol), elemental
sulfur (S8) (32 gm, 1.0 mol) and 2-phenylacetaldehyde (116 mL,
1.0 mol) in ethanol (200 mL) was stirred at room temperature. This
was followed by dropwise addition of diethylamine (80 mL) with
stirring. The reaction mixture was left at room temperature for 14h;
the precipitate was filtered under vacuum, washed by ethanol and
chemical shifts are expressed in parts per million (ppm,
were reference to NMR solvent CDCl3 d 7.26, 77 ppm, and DMSO‑d6
2.5, 39.8 ppm respectively. The following abbreviations were used
d scale) and
d
dried in air. Yield: 89%; 1H NMR (300 MHz, DMSO‑d6)
d 7.48e7.44
to describe peak patterns when appropriate: bs ¼ broad singlet,
s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet and m ¼ multiplet.
HRMS mass spectra were recorded on a Thermo Scientific Q-
(m, 4H, AreH þ NH2), 7.33 (t, J ¼ 7.8 Hz, 2H, AreH), 7.24 (s, 1H,
AreH), 7.21e7.17 (m, 1H, AreH), 4.24e4.19 (m, 2H, CH2), 1.25e1.30,
(m, 3H, CH3); 13C NMR (75 MHz, CDCl3)
d 165.44, 162.07, 134.00,

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C.L. Meena et al. / European Journal of Medicinal Chemistry 208 (2020) 112772
C
20H17NO3S, 351.0929, found, 352.1002 [MþH]þ.
4.1.3. Ethyl 2-(4-methylbenzamido)-5-phenylthiophene-3-
carboxylate (1b)
The synthetic method of 1a was adopted to synthesize 1b. Yield:
53%; 1H NMR (400 MHz, DMSO‑d6)
d 11.80 (s, 1H, NH), 7.90 (m, 2H,
AreH), 7.70 (m, 2H, AreH), 7.60 (s, 1H, AreH), 7.50e7.40 (m, 5H,
AreH), 4.38e4.36 (m, 2H, CH2) 2.43 (s, 3H, CH3), 1.39 (m, 3H, CH3),
13C NMR (75 MHz, CDCl3)
d 166.01, 163.58, 148.73, 143.55, 133.83,
129.72, 129.20, 128.98, 127.57, 127.44, 125.49, 119.29, 113.89, 60.97,
21.67, 14.45; HRMS (ESI-TOF): Calculated m/z for C21H20NO3S,
366.1164, found 366.1158 [MþH]þ.
4.1.4. Ethyl 2-(nicotinamide)-5-phenylthiophene-3-carboxylate
(1c)
The synthetic method of 1a was adopted to synthesize 1c. Yield:
56%; 1H NMR (300 MHz, DMSO‑d6)
d 11.80 (s, 1H, NH), 8.90e9.10
(m, 2H, AreH), 8.30 (m, 1H), 7.69 (d, J ¼ 7.2 Hz, 3H, AreH), 7.62 (s,
1H, AreH), 7.45e7.42 (m, 2H, AreH), 7.33 (m, 1H, AreH), 4.40e4.38
(m, 2H, CH2), 1.40e1.36 (m, 3H, CH3), 13C NMR (75 MHz, CDCl3);
166.02, 161.84, 153.34, 148.93, 147.75, 135.10, 133.56, 129.04, 127.89,
127.60, 125.55, 123.72, 119.31, 114.68, 61.20, 14.41; HRMS (ESI-TOF):
Calculated m/z for C19H16N2O3S, 352.0882 found 353.0954 [MþH]þ.
4.1.5. Ethyl 2-acetamido-5-phenylthiophene-3-carboxylate (1d)
The synthetic method of 1a was adopted to synthesize 1d. Yield:
59%; 1H NMR (300 MHz, DMSO‑d6)
d 10.87 (s, 1H, NH), 7.64 (t,
J ¼ 4.2 Hz, 2H, AreH), 7.52 (s, 1H, CH), 7.41 (t, J ¼ 7.8 Hz, 2H, AreH),
7.30 (t, J ¼ 3.4 Hz, 2H, AreH), 4.37e4.31 (m, 2H, CH2), 2.29 (s, 3H,
CH3), 1.37e1.34 (m, 3H, CH3); 13C NMR (75 MHz, CDCl3);
d 167.07,
Fig. 3. Interaction map of compounds with M. tuberculosis KatG;(A) INH (cen-
tred:thick:darkgreen), 4a (light green) and 4b (opaque green) are shown in the binding
site of M. tuberculosis KatG. The binding site is shown in surface view and the mole-
cules are rendered in licorice. (B) The zoom-in inset view is highlighting the overlay
between INH and compounds 4a and 4b. The orange dotted line highlights the
alignment of core chemical moiety. (C) The interacting residues within 3.5 Å from the
centre of the INH (co-crystal pose from PDB ID: 3WXO). The residues of INH binding
sites which were reported earlier are highlighted in green circles. (D and E) The
interacting residues of compound 4a (D) and 4b (E) with KatG of M. tuberculosis are
shown. For the entire image panel the rendering was done as per atom wise like O: red,
N: blue, C: green (different shades) and S:yellow. The KatG protein is shown in cartoon
and in ghost view. (For interpretation of the references to colour in this figure legend,
the reader is referred to the Web version of this article.)
165.64, 148.09, 133.75, 133.65, 128.95, 127.43, 125.48, 119.03, 113.37,
60.96, 31.95, 31. 64, 29.72, 23.58, 22.72; HRMS (ESI-TOF): Calculated
m/z for C15H15NO3S, 289.0773, found 290.0845 [MþH]þ 312.0845
[MþNa]þ.
4.1.6. Ethyl 2-(4-fluorobenzamido)-5-phenylthiophene-3-
carboxylate (1e)
The synthetic method of 1a was adopted to synthesize 1e. Yield:
49%; 1H NMR (400 MHz, DMSO‑d6)
d 11.77 (s, 1H, NH), 8.03e8.00
(m, 2H, AreH), 7.67 (t, J ¼ 4.2 Hz, 2H, AreH), 7.59 (s, 1H, AreH),
7.50e7.43 (m, 2H, AreH), 7.41e7.40 (m, 2H, CH2), 7.31 (m, 1H,
AreH), 4.38e4.36 (m, 2H, CH2), 1.38e1.34 (m, 3H, CH3); 13C NMR
128.92, 126.69, 124.88, 124.71, 121.23, 107.97, 59.90, 14.57; HRMS
(ESI-TOF): Calculated m/z for C13H13NO2S 247.0667, found 247.0659.
1
(75 MHz, CDCl3);
d
165.52 (d, JCF ¼ 252.75 Hz), 166.10, 162.47,
3
148.44, 134.11, 133.70, 130.04 (d, JCF ¼ 9.0 Hz), 129.00, 128.28 (d,
4JCF ¼ 3.0 Hz), 127.55, 125.50, 119.27, 116.23 (d, JCF ¼ 22.5 Hz),
2
4.1.2. Ethyl 2-benzamido-5-phenylthiophene-3-carboxylate (1a)
To a stirred solution of benzoic acid (244 mg, 1 equiv, 2 mmol) in
dry DCM at 0 ꢀC, methanesulfonyl chloride (0.184 mL, 1.2 equiv,
2.4 mmol) and triethylamine (0.612 mL, 2.2 equiv, 4.4 mmol) were
added, and stirred for 30 min and followed by addition of inter-
mediate 7 (543 mg, 1.1 equiv, 2.2 mmol) and stirred for 2h at room
temperature. The resulting solution was diluted with DCM (20 mL),
subsequently washed by 1N HCl (20 mL), saturated NaHCO3 solu-
tion (10 mL) and brine solution (10 mL). Organic layer was sepa-
rated and dried using anhydrous Na2SO4 and concentrated to dry
under vacuum. The crude product thus obtained was purified using
silica gel column chromatography employing DCM: Hexane (80:20)
as eluent. The purified fraction was concentrated under reduced
pressure to afford 1a as a white solid. Yield: 60%; 1H NMR
114.15, 61.08, 14.43; HRMS (ESI-TOF): Calculated m/z for
C
20H16FNO3S, 369.0853, found 370.0909 [MþH]þ.
4.1.7. Ethyl 2-(4-cyanobenzamido)-5-phenylthiophene-3-
carboxylate (1f)
The synthetic method of 1a was adopted to synthesize 1f. Yield:
69%; 1H NMR (300 MHz, DMSO‑d6)
d 11.90 (s, 1H, NH), 8.15 (m, 4H,
AreH), 7.70 (m, 2H, AreH), 7.65 (s, 1H, AreH), 7.40 (m, 2H, AreH),
7.32 (m, 1H, AreH), 4.41e4.39 (m, 2H, CH2), 1.40e1.37 (m, 3H, CH3);
13C NMR (75 MHz, CDCl3)
d 166.13, 161.59, 147.66, 135.86, 134.78,
133.47, 132.84, 129.06, 128.14, 127.77, 125.56, 119.35, 117.84, 116.19,
114.85, 61.29, 14.41; MS (APCIþ): Calculated m/z for C21H16NO3S,
377.8, found, 377.9.
(300 MHz, DMSO‑d6):
d
11.82 (s, 1H, NH), 7.96 (t, J ¼ 2.2 Hz, 2H,
4.1.8. Ethyl-5-phenyl-2-(2-phenylacetamido) thiophene-3-
carboxylate (1g)
AreH), 7.69e7.64 (m, 5H, AreH), 7.60 (s, 1H, AreH), 7.42 (m, 2H,
AreH), 7.30 (m, 1H, AreH), 4.39e4.37 (m, 2H, CH2), 1.38e1.35 (m,
The synthetic method of 1a was adopted to synthesize 1g. Yield:
3H, CH3), 13C NMR (75 MHz, CDCl3)
d
166.01, 163.58, 148.54, 134.00,
62%; H NMR (300 MHz, DMSO‑d6)
d
10.87 (s, 1H, NH), 7.64e7.62
1
133.78, 132.78, 132.03, 129.05, 128.99, 127.54, 127.49, 125.51, 119.31,
114.10, 61.01, 14.44; HRMS (ESI-TOF): Calculated m/z for
(m, 2H, AreH), 7.52 (s, 1H, AreH), 7.41e7.39 (m, 6H, AreH), 7.32 (m,
2H, AreH), 4.29e4.27 (m, 2H, CH2), 3.98 (s, 2H, CH2), 1.32e1.29,(m,

C.L. Meena et al. / European Journal of Medicinal Chemistry 208 (2020) 112772
9
3H, CH3), 13C NMR (75 MHz, DMSO‑d6)
d
)d
164.67, 154.02, 151.35,
AreH), 4.41e4.34 (m, 2H, CH2), 3.51e3.44 (m, 4H, 2CH2), 1.45e1.40
(m, 3H, CH3), 1.33e1.28 (m, 6H, 2CH3), 13C NMR (75 MHz, CDCl3)
139.73, 133.93, 130.81, 129.58, 128.90, 127.79, 127.55, 127.48, 125.11,
119.64, 110.64, 60.62, 43.64, 14.78; HRMS (ESI-TOF): Calculated m/z
for: C21H19NO3S, 366.1158, found 367.1158 [MþH]þ.
d
166.27,152.94,152.31,134.19,131.87,131.87,128.86,126.95,126.95,
125.15, 118.97, 111.04, 60.55, 41.88, 14.45, 13.70; HRMS (ESI-TOF):
Calculated m/z for: C18H22N2O3S, 346.1351 found, 347.1424 [MþH]þ.
4.1.9. Ethyl 2-(2-bromoacetamido)-5-phenylthiophene-3-
carboxylate (1h)
4.1.14. Ethyl 5-phenyl-2-(pyrrolidine-1-carboxamido) thiophene-3-
carboxylate (2e)
The synthetic method of 1a was adopted to synthesize 1h. Yield:
53%; 1H NMR (300 MHz, DMSO‑d6):
d
11.50 (s, 1H, NH), 7.67 (d, J ¼
The synthetic method of 2a was adopted to synthesize 2e. Yield:
92%; 1H NMR (300 MHz, DMSO‑d6)
4.2 Hz, 2H, AreH), 7.46 (s, 1H, AreH), 7.39 (t, J ¼ 7.5 Hz, 2H, AreH),
7.30e7.27 (m,1H, AreH), 4.33e4.31 (m, 2H, CH2), 3.40 (s, 4H, 2CH2),
2.00 (s, 4H, 2CH2), 1.36e1.31 (m, 3H, CH3), 13C NMR (75 MHz, CDCl3)
d
10.31 (s, 1H, NH), 7.60 (t, J ¼
7.2 Hz, 2H, AreH), 7.59 (s, 1H, AreH), 7.43e7.41 (m, 2H, AreH), 7.30
(m, 1H, AreH), 4.67 (s, 2H, CH2), 4.37e4.36 (m, 2H, CH2), 1.37e1.34,
(m, 3H, CH3); 13C NMR (75 MHz, DMSO‑d6); 164.84, 164.53, 164.36,
146.42, 146.26, 133.71, 133.23, 129.69, 128.26, 125.61, 120.03, 115.04,
61.41, 43.00, 29.49, 14.67; HRMS (ESI-TOF): Calculated m/z for
d
166.24, 152.07, 134.16, 131.88, 128.87, 126.98, 125.17, 118.92, 111.01,
C
15H15BrNO3S, 366.9878, found 367.994 [MþH]þ.
60.55, 45.78, 29.71, 14.46; HRMS (ESI-TOF): Calculated m/z for
C
18H20N2O3S, 344.1195, found, 345.1267 [MþH]þ.
4.1.10. Ethyl 5-phenyl-2-ureidothiophene-3-carboxylate (2a)
To a solution of intermediate 7 (1 gm, 4 mmol, 1.0 equiv.) in
toluene, triphosgene (1.26 gm, 4 mmol, 1.05 equiv.) was added and
refluxed for 12 h. The mixture was concentrated under vacuum and
resulting semisolid residue was used for the next step without
purification. The residue (0.2 gm, 0.490 mmol, 1equiv.) was dis-
solved in dry DCM followed by addition of DIPEA (0.255 mL,
1.47 mmol, 3.0 equiv.) and ammonium hydroxide solution (1.5 mL)
and stirred for 2e6 h at room temperature. The reaction mixture
was diluted with DCM (20 mL) and subsequently washed by diluted
HCl, followed by saturated NaHCO3 solution. The organic layer was
dried over anhydrous Na2SO4 and concentrated under vacuum. The
residue was purified by column chromatography using DCM: hex-
ane (90:20) as eluent, resulting in 2a as a white solid. Yield: 91%; 1H
4.1.15. Ethyl 5-phenyl-2-(piperidine-1-carboxamido) thiophene-3-
carboxylate (2f)
The synthetic method of 2a was adopted to synthesize 2f. Yield:
83%; 1H NMR (300 MHz, DMSO‑d6)
d 10.65 (s, 1H, NH),7.62e7.59 (m,
2H, AreH), 7.47 (s, 1H, AreH), 7.39 (t, J ¼ 7.5 Hz, 2H, AreH),
7.30e7.28 (m, 1H, AreH), 4.34e4.32 (m, 2H, CH2), 3.46e3.42 (bs,
4H, 2CH2), 1.59 (bs, 6H, 3CH2), 1.36e1.31 (m, 3H, CH3), 13C NMR
(75 MHz, CDCl3)
d 166.38, 152.79, 152.46, 134.16, 132.03, 128.96,
128.87, 126.98, 125.33, 118.99, 111.04, 60.59, 45.08, 25.68, 24.25,
14.45; HRMS (ESI-TOF) Calculated m/z for C19H22N2O3S, 358.1351
found, 359.1424 [MþH]þ.
4.1.16. Ethyl 2-(3-isopropylureido)-5-phenylthiophene-3-
carboxylate (2g)
NMR (300 MHz, DMSO‑d6)
d
10.18 (s, 1H, NH), 7.59 (d, J ¼ 7.2 Hz, 2H,
AreH), 7.42 (s, 1H, AreH), 7.40e7.36 (m, 2H, AreH), 7.28e7.24, (m,
1H, AreH), 7.20 (bs, 1H, NH), 4.34e4.29 (m, 2H, CH2), 1.35e1.32 (m,
The synthetic method of 2a was adopted to synthesize 2g. Yield:
51%; 1H NMR (300 MHz, DMSO‑d6)
d 10.14 (s,1H, NH), 7.95e7.92 (bs,
2H, CH2); 13C NMR (75 MHz, DMSO‑d6)
d 164.69, 164.65, 154.76,
1H, NH), 7.59e7.57 (m, 2H, AreH), 7.41e7.36 (m, 3H, AreH),
7.38e7.36 (m, 1H, AreH), 7.28e7.23 (t, 2H, AreH), 4.34e4.27 (m,
2H, CH2), 3.76 (m, 1H, CH), 1.33 (m, 3H, CH3), 1.12 (d, J ¼ 6.6 Hz, 6H,
154.72, 154.68, 151.52, 151.36, 133.95, 130.92, 129.57, 127.52, 125.13,
119.55,110.55,100.00, 60.60,14.77; HRMS (ESI-TOF): Calculated m/z
for C14H14N2O3S 291.0725, found 291.0843[MþH]þ.
3CH3); 13C NMR (75 MHz, CDCl3)
d 166.09, 152.64, 151.69, 134.06,
131.93, 128.87, 127.03, 125.18, 118.95, 110.91, 60.57, 43.04, 23.16,
14.42; HRMS (ESI-TOF); Calculated m/z for C17H20N2O3S, 332.1195,
found 333.1262 [MþH]þ 355.1083 (M þ Na)þ.
4.1.11. Ethyl 2-(3-methylureido)-5-phenylthiophene-3-carboxylate
(2b)
The synthetic method of 2a was adopted to synthesize 2b. Yield:
73%; 1H NMR (400 MHz, DMSO‑d6)
d 10.20 (s, 1H, NH), 7.80 (bs, 1H,
4.1.17. Ethyl 2-(3-cyclopropylureido)-5-phenylthiophene-3-
carboxylate (2h)
NH), 7.65e0.760 (m, 2H, AreH), 7.41 (m, 3H, AreH), 7.24e7.20 (m,
1H, AreH), 4.32e4.30 (m, 2H, CH2), 2.71 (d, J ¼ 4.8 Hz, 3H, CH3),
1.35e1.33, (m, 3H, CH3); 13C NMR (75 MHz, CDCl3)
d 166.04, 154.26,
The synthetic method of 2a was adopted to synthesize 2h. Yield:
81%; 1H NMR (300 MHz, DMSO‑d6)
d 10.18 (s, 1H, NH), 8.09 (s, 1H,
151.67, 134.02, 131.96, 128.90, 127.08, 125.19, 119.00, 111.03, 60.59,
27.37, 14.42; HRMS (ESI-TOF): Calculated m/z for C15H17N2O3S
304.0882, found 305.0950 [MþH]þ, 327.0769 [MþNa]þ.
NH), 7.60e7.58 (m, 2H, AreH), 7.43 (m, 3H, AreH), 7.29e7.27 (m,
1H, AreH), 4.35e4.28 (m, 2H, CH2), 2.62 (m, 1H, CH), 1.35e1.30 (m,
3H, CH3), 0.40e0.80 (bs, 4H, 2CH2); 13C NMR (75 MHz, CDCl3)
d
165.83, 155.00, 150.93, 134.05, 132.35, 128.88, 127.10, 125.26,
4.1.12. Ethyl 2-(3, 3-dimethylureido)-5-phenylthiophene-3-
carboxylate (2c)
119.14, 111.90, 60.54, 22.56, 14.46, 7.71; HRMS (ESI-TOF): Calculated
m/z for C17H18N2O3S, 330.1038 found, 331.1111[MþH]þ 353.0922
[MþNa]þ.
The synthetic method of 2a was adopted to synthesize 2c. Yield:
45%; 1H NMR (300 MHz, DMSO‑d6)
d
10.50 (s, 1H, NH), 7.58 (d, J ¼
7.6 Hz, 2H, AreH), 7.44 (s, 1H, AreH), 7.38e7.36 (m, 2H, AreH),
7.23e7.20 (m,1H, AreH), 4.32e4.30 (m, 2H, CH2), 2.99 (s, 6H, 2CH3),
4.1.18. Ethyl 2-(4-methylpiperazine-1-carboxamido)-5-
phenylthiophene-3-carboxylate (2i)
1.34e1.30, (m, 3H, CH3); 13C NMR (75 MHz, DMSO‑d6)
d 165.55,
153.42, 151.76, 133.82, 131.28, 131.28, 129.59, 127.65, 125.16, 119.46,
The synthetic method of 2a was adopted to synthesize 2i. Yield:
111.12, 61.00, 36.19, 14,70; HRMS (ESI-TOF): Calculated m/z for
70%; 1H NMR (400 MHz, DMSO‑d6)
d
10.63 (s, 1H, NH), 7.61 (t, J ¼
C
16H18N2O3S, 318.1038, found, 319.1111 [MþH]þ.
4.2 Hz, 2H, AreH), 7.47 (s, 1H, AreH), 7.42e7.38 (m, 2H, AreH),
7.30e7.26 (m,1H, AreH), 4.36e4.30 (m, 2H, CH2), 3.47e3.44 (m, 4H,
2CH2), 2.40, (t, 4H, 2CH2), 2.09 (s, 3H, CH3), 1.36e1.32, (m, 3H, CH3);
4.1.13. Ethyl 2-(3, 3-diethylureido)-5-phenylthiophene-3-
carboxylate (2d)
13C NMR (75 MHz, DMSO‑d6)
d 165.60,152.52,151.50,133.76,131.60,
The synthetic method of 2a was adopted to synthesize 2d. Yield:
131,60, 129.61, 127.73, 125.21, 119.56, 111.43, 61.09, 61.09, 54.40,
71%; 1H NMR (300 MHz, CDCl3)
d
10.77 (s, 1H, NH), 7.60 (t, J ¼
46.07, 43.80, 14.70; HRMS (ESI-TOF): Calculated m/z for
4.5 Hz, 2H, AreH), 7.40e7.35 (m, 3H, AreH), 7.28e7.23 (m, 1H,
C
19H23N3O3S, 373.1460 found, 374.1533 [MþH]þ.

10
C.L. Meena et al. / European Journal of Medicinal Chemistry 208 (2020) 112772
4.1.19. Ethyl 2-(morpholine-4-carboxamido)-5-phenylthiophene-3-
carboxylate (2j)
(m, 2H, AreH), 7.43 (s, 1H, AreH), 7.24e7.18 (m, 2H, AreH), 3.83 (s,
3H, CH3), 3.25e3.20 (m, 4H, 2CH2), 1.90 (m, 4H, 2CH2); 13C NMR
1
The synthetic method of 2a was adopted to synthesize 2j. Yield:
(75 MHz, CDCl3)
d
166.56, 162.03 (d, JCF ¼ 245.25 Hz), 152.15,
83%; 1H NMR (400 MHz, DMSO‑d6)
7.6 Hz, 2H, AreH), 7.44 (s, 1H, AreH), 7.38e7.34 (m, 2H, AreH),
7.26e7.24 (m, 1H, AreH), 4.32e4.26 (m, 2H, CH2), 3.65e3.63, (m,
4H, 2CH2), 3.42e3.40, (m, 4H, 2CH2), 1.32e1.28 (m, 3H, CH3), 13C
d
10.57 (s, 1H, NH), 7.57 (d, J ¼
152.01, 130.97, 130.37 (d, JCF ¼ 3.0 Hz), 126.81 (d, JCF ¼ 8.25 Hz),
4
3
2
118.72, 115.83 (d, JCF ¼ 21.0 Hz), 110.62, 51.62, 50.81, 45.82, 25.34,
23.74; HRMS (ESI-TOF): Calculated m/z for C17H17FN2O3S 348.0944,
found, 349.1017 [MþH]þ.
NMR (75 MHz, CDCl3)
d 166.47, 152.99, 151.75, 133.96, 132.44,
128.92, 127.17, 125.23, 118.99, 111.54, 66.41, 60.77, 43.98, 14.44;
HRMS (ESI-TOF): Calculated m/z for C18H20N2O4S, 360.1144 found,
361.1217 [MþH]þ.
4.1.24. Methyl 5-(4-fluorophenyl)-2-(piperidine-1-carboxamido)
thiophene-3-carboxylate (3c)
The synthetic method of 3a was adopted to synthesize 3c. Yield:
72%; 1H NMR (300 MHz, DMSO‑d6)
d 10.63 (s,1H, NH), 7.64e759 (m,
4.1.20. Ethyl 5-phenyl-2-(3-phenylureido) thiophene-3-carboxylate
(2k)
2H, AreH), 7.41 (s, 1H, AreH), 7.23e7.17 (m, 2H, AreH), 3.83 (s, 3H,
CH3), 3.43e3.42, (m, 4H, 2CH2), 1.56 (s, 6H, 3CH2); 13C NMR
The synthetic method of 2a was adopted to synthesize 2k.
(75 MHz, DMSO‑d6); d166.12, 160.00, 152.23, 151.98, 130.46, 127.23
Yield:85%; 1H NMR (400 MHz, DMSO‑d6)
d 10.53 (s, 1H, NH), 10.32
3
2
(d, JCF ¼ 8.25 Hz), 119.53, 116.46 (d, JCF ¼ 21.0 Hz), 110.84, 52.35,
(s, 1H, NH), 7.60 (d, J ¼ 7.2 Hz, 2H, AreH), 7.51e7.46 (m, 2H, AreH),
7.46e7.40 (m, 2H, AreH), 7.33e7.26 (m, 4H, AreH), 7.03 (m, 1H,
AreH), 4.34e4.32 (m, 2H, CH2), 1.35e1.32 (m, 3H, CH3); 13C NMR
44.95, 25.59, 24.06; HRMS (ESI-TOF): Calculated m/z for:
C
18H19FN2O3S, 362.1100, found, 363.1173 [MþH]þ.
(75 MHz, DMSO‑d6)
d 164.79, 151.45, 150.38, 139.33, 133.79, 131.50,
4.1.25. Methyl 2-(3-cyclopropylureido)-5-(4-fluorophenyl)
thiophene-3-carboxylate (3d)
129.62, 129.45, 127.70, 125.21, 123.25, 119.69, 118.90, 111.48, 60.90,
14.77; HRMS (ESI-TOF): Calculated m/z for C20H18N2O3S, 366.1038,
found, 367.1111 [MþH]þ 389.0930 [MþNa]þ.
The synthetic method of 3a was adopted to synthesize 3d. Yield:
82%; (1H NMR (300 MHz, DMSO‑d6)
d 10.07 (bs, 1H, NH), 8.10 (bs,
1H, NH), 7.65e7.60 (m, 2H, AreH), 7.41 (s, 1H, AreH), 7.24e7.18 (m,
2H, AreH), 3.82 (s, 3H, CH3), 2.60 (m, 1H, CH), 0.69 (m, 2H, CH2),
4.1.21. Ethyl 2-(3-benzylureido)-5-phenylthiophene-3-carboxylate
(2l)
0.45 (m, 2H, CH2); 13C NMR (75 MHz, DMSO‑d6);
d 161.78
The synthetic method of 2a was adopted to synthesize 2l. Yield:
(1JCF ¼ 242.25 Hz), 154.70, 151.13, 130.48, 127.18 (d, 3JCF ¼ 8.25 Hz),
62%; 1H NMR (400 MHz, DMSO‑d6)
d 10.30 (bs,1H, NH), 8.50 (bs,1H,
2
119.69, 116.45 (d, JCF ¼ 21.75 Hz), 52.03, 29.42, 6.63; HRMS (ESI-
NH), 7.59 (t, J ¼ 4.2 Hz, 2H, AreH), 7.43 (s, 1H, AreH), 7.41e7.32 (m,
TOF): Calculated m/z for C16H15FN2O3S, 334.0787, found 335.0.860
[MþH]þ.
6H, AreH), 7.28e7.26 (m, 2H, AreH), 4.37e4.30 (m, 4H, 2CH2),
1.35e1.31, (m, 3H, CH3); 13C NMR (75 MHz, DMSO‑d6)
d 164.67,
154.02, 151.35, 139.73, 133.93, 130.81, 129.58, 128.90, 127.79, 127.55,
127.48, 125.11, 119.64, 110.64, 60.62, 43.64, 14.70. HRMS (ESI-TOF):
Calculated for C21H20N2O3S, 380.1195, found, 381.1267 [MþH]þ
391.1087[MþNa]þ.
4.1.26. Methyl 5-(4-fluorophenyl)-2-(3-isopropylureido)thiophene-
3-carboxylate (3e)
The synthetic method of 3a was adopted to synthesize 3e. Yield:
91%; 1H NMR (300 MHz, DMSO‑d6)
d 10.07 (s, 1H, NH), 7.94 (m, 1H,
NH), 7.91e7.59 (m, 2H, AreH), 7.38 (s, 1H, AreH), 7.23e7.18 (m, 2H,
AreH), 3.89 (s, 3H, CH3), 3.79e3.73 (bs, 1H, CH), 1.11 (d, J ¼ 6.6 Hz,
4.1.22. Methyl 2-(3, 3-diethylureido)-5-(4-fluorophenyl)
thiophene-3-carboxylate (3a)
6H, 2CH3); 13C NMR (75 MHz, CDCl3);
d 166.35, 162.06 (d,
To a stirred solution of intermediate 10 (1 gm, 3.98 mmol,
1equiv.) in toluene, triphosgene (1.23 gm, 4.18 mmol, 1.05 equiv.)
was added and refluxed for 12 h. The solvent was concentrated
under vacuum and resulting residue was used for the next syn-
thetic step without any purification. This residue (0.2 gm,
0.487 mmol, 1 equiv.) was dissolved in dry DCM and DIPEA
(0.254 mL, 1.46 mmol, 3equiv.) was added followed by diethyl-
amine (0.071 mL, 0.975 mmol, 2equiv.) and stirred for 2e6 h at
room temperature. The reaction mixture was diluted with DCM
(20 mL) and subsequently washed with diluted HCl and saturated,
NaHCO3 solution. Organic layer was separated and dried using
anhydrous Na2SO4 and concentrated under vacuum. The resulting
residue was purified by column chromatography using DCM: hex-
ane (90:20) as eluent resulting in 3a as a white solid. Yield: 70%; 1H
4
1JCF ¼ 244.5 Hz), 152.61, 151.71, 131.02, 130.26 (d, JCF ¼ 3.75 Hz),
126.82 (d, 3JCF ¼ 8.25 Hz), 118.80, 115.84 (d, 2JCF ¼ 21.75 Hz), 110.55,
51.63, 43.08, 23.14; HRMS (ESI-TOF):Calculated m/z for
C
16H17FN2O3S, 336.0944, found, 337.1017[MþH]þ 359.0836
[MþNa]þ.
4.1.27. Methyl 2-(2-chloroacetamido) thiophene-3-carboxylate
(4a)
To a solution of 2-chloroacetic acid (0.1 g, 1.07 mmol, 1equiv.) in
dry DCM (10 mL), a solution of methanesulfonyl chloride (0.099 mL,
1.29 mmol, 1.2 equiv. in 2 mL dry DCM) and triethylamine
(0.329 mL, 2.36 mmol, 2.2 equiv) was added at 0 ꢀC. The reaction
temperature was maintained and stirred for 30 min. This was fol-
lowed by an addition of methyl 2-aminothiophene-3-carboxylate 8
(184 mg, 1.18 mmol, 1.2 equiv.). The reaction mixture was stirred
further at room temperature for additional 2h and progress of the
reaction was monitored by TLC. The reaction was diluted with DCM
and subsequently washed with brine solution, dried over anhy-
drous Na2SO4 and concentrated under vacuum to obtain crude
product. The crude product was purified by column chromatog-
raphy using DCM: Hexane (80:20) as eluent to afford 4a as a white
NMR (300 MHz, DMSO‑d6)
d 10.55 (bs, 1H, NH), 7.66e7.61 (m, 2H,
AreH), 7.44 (s, 1H, AreH), 7.25e7.19 (m, 2H, AreH), 3.85 (s, 3H,
CH3), 3.41e3.36 (m, 4H, 2CH2), 1.23e1.15 (s, 6H, 2CH3); 13C NMR
1
(75 MHz, CDCl3)
d
166.58, 162.02 (d, JCF ¼ 245.25 Hz), 152.89,
4
3
152.40, 130.97, 130.4 (d, JCF ¼ 3.0 Hz), 126.78 (d, JCF ¼ 8.25 Hz),
2
118.76, 115.83 (d, JCF ¼ 21.0 Hz), 110.65, 51.66, 48.38, 41.91, 29.70,
14.99, 13.68; HRMS (ESI-TOF): Calculated m/z for: C17H19FN2O3S,
350.1100 found, 351.1173 [MþH]þ.
solid. Yield: 54%;1H NMR (400 MHz, DMSO‑d6)
7.23 (d, J ¼ 5.6 Hz, 1H, CH), 7.12 (d, J ¼ 5.6 Hz, 1H, CH), 4.64 (s, 2H,
d 11.50 (s, 1H, NH),
4.1.23. Methyl 5-(4-fluorophenyl)-2-(pyrrolidine-1-carboxamido)
thiophene-3-carboxylate (3b)
CH2), 3.86 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3)
d 165.70, 163.69,
The synthetic method of 3a was adopted to synthesize 3b. Yield:
147.42, 124.06, 116.92, 114.09, 52.00, 42.16, 29.70; MS (APCIþ):
86%; 1H NMR (300 MHz, DMSO‑d6)
d
10.30 (s, 1H, NH), 7.65e7.60
Calculated m/z for C8H9ClNO3S 233.999, found 233.9.

C.L. Meena et al. / European Journal of Medicinal Chemistry 208 (2020) 112772
11
4.1.28. Methyl 2-(2-chloroacetamido)-5-phenylthiophene-3-
364.99 found 365.85.
carboxylate (4b)
Yield: 67%; The synthetic method of 4a was adopted to syn-
4.2. MIC99 determination assays
thesize 4b:1H NMR (300 MHz, DMSO‑d6)
d 11.50 (bs, 1H, NH), 7.70
(m, 2H, AreH), 7.60 (s, 1H, AreH), 7.40 (m, 2H, AreH), 7.30 (m, 1H,
Mycobacterium tuberculosis H37Rv (M. tuberculosis) was cultured
in Middlebrook 7H9 broth (MB broth) supplemented with 0.05%
Tween-80, 0.2% glycerol and 1x albumin-dextrose-saline (ADS) at
37 ꢀC with shaking (200 rpm). MIC99 determination assays, two-
drug checkerboard assays and in vitro killing experiments were
performed as previously described [28,37].
AreH), 4.64 (s, 2H, CH2), 3.86 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3)
d
165.62, 163.68, 146.54, 134.79, 133.41, 129.02, 127.74, 125.56,
119.31, 114.85, 52.08, 42.16, 40.49, 29.71. HRMS (ESI-TOF): Calcu-
lated m/z for C14H12ClNO3S, 309.0226 found, 310.0295 [MþH]þ
332.0119 [MþNa]þ.
4.1.29. Methyl 2-(2-chloroacetamido)-5-(4-fluorophenyl)
thiophene-3-carboxylate (4c)
4.3. Cell viability experiments
The synthetic method of 4a was adopted to synthesize 4c. Yield:
Briefly, THP-1 cells were seeded in 96 well cell culture plates at
the density of 20,000/well in the presence of 20 ng/ml of phorbol
12-myristate-13-acetate (PMA). Following 48 h incubation, cells
were washed with 1x PBS and overlaid for 24 h in RPMI complete
medium (with 10% FBS). The drug dilutions were prepared in RPMI
complete medium and added to the designated wells in triplicates.
After 96 h of incubation, water soluble tetrazolium salt (WST)-1 cell
proliferation reagent was added to each well following incubation
for 30 min. Absorbance were taken at 450 nm and 630 nm and %
cell viability following drug treatment was calculated as per man-
ufacturer’s recommendations.
63% 1H NMR (400 MHz, DMSO‑d6)
d
11.52 (s, 1H, NH), 7.74e7.70 (m,
2H, AreH), 7.58 (s, 1H, AreH), 7.27e7.23 (m, 2H, AreH), 4.67 (s, 2H,
CH2), 3.89 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3)
d 165.55, 163.69,
160.80, 146.49, 139.30, 135.06, 133.71, 129.69 (d, 4JCF ¼ 3.0 Hz), 127.3
3
2
(d, JCF ¼ 7.5 Hz), 119.29, 116.03 (d, JCF ¼ 21.75 Hz), 114.84, 52.10,
42.14, 33.41, 31.94; HRMS (ESI-TOF): Calculated m/z for
C
14H11ClNO3S, 328.0132 found 327.0205. [MþH]þ 350.0024
[MþNa]þ.
4.1.30. Methyl 2-(2-chloroacetamido)-5-(3-fluorophenyl)
thiophene-3-carboxylate (4d)
The synthetic method of 4a was adopted to synthesize 4d. Yield:
4.4. Macrophage CFU determination experiments
56% 1H NMR (300 MHz, DMSO‑d6)
d 11.50 (s, 1H, NH), 7.72 (s, 1H,
AreH), 7.60e7.58 (m, 1H, AreH), 7.50e7.43 (m, 2H, AreH),
THP-1 cells (human monocytic cell line) were cultured in RPMI
medium supplemented with 10% heat inactivated-foetal bovine
serum (HI-FBS) and differentiated into macrophages with the
addition of 20 ng/ml phorbol myristate acetate (PMA). For the
intracellular killing experiment, macrophages (0.2 million/well/ml;
24 well plates) were infected with single-cell M. tuberculosis sus-
pension at an MOI of 1:10. Following 4 h infection extracellular
bacteria were removed by overlaying macrophages with RPMI
7.17e7.13 (m, 1H, AreH), 4.68 (s, 2H, CH2), 3.90 (s, 3H, CH3); 13C
NMR (75 MHz, CDCl3)
d
165.49, 163.76, 163.16 (d, 1JCF ¼ 245.25 Hz),
146.91, 135.61, 133.27, 130.64, 121.23, 114.85, 112.52, 52.14, 42.14,
29.53, 22.71; HRMS (ESI-TOF): Calculated m/z for C14H11ClNO3S,
328.0132 found 327.0202. [MþH]þ 350.0022 [MþNa]þ.
4.1.31. Methyl 2-(2-chloroacetamido)-5-(3, 5-difluorophenyl)
thiophene-3-carboxylate (4e)
containing 200 mg/ml of amikacin. Further, cells were washed twice
The synthetic method of 4a was adopted to synthesize 4e. Yield:
with 1xPBS and infected macrophages were overlaid with RPMI
containing drugs for 4 days. The infected macrophages were lysed
on day 0 and day 4 post-exposure using 1x PBST (0.1% Triton X-
100). For CFU enumeration 10.0 fold serial dilutions was prepared
and 100
weeks.
77%; 1H NMR (400 MHz, DMSO‑d6)
d
11.53 (s, 1H, NH), 7.79 (s, 1H,
AreH), 7.46e7.40 (m, 2H, AreH), 7.17e7.11 (m, 1H, AreH), 4.67 (s,
2H, CH2) 3.86 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3)
165.14,
d
163.86, 147.26, 136.54, 121.02, 114.89, 108.53, 103.21, 102.87, 102.53,
m
l was plated on MB7H11 agar plates at 37 ꢀC for 3e4
52.21, 42.10, 31.94, 29.71. HRMS (ESI-TOF): Calculated m/z for
C
14H10ClF2NO3S, 345.0038 found 345.0012 [MþH]þ, 367.9932
[MþNa]þ.
4.5. Computational molecular docking studies
4.1.32. Methyl 2-(2-chloroacetamido)-5-(4-(trifluoromethyl)
phenyl) thiophene-3-carboxylate (4f)
4.5.1. Protein structure preparations
The KatG crystal 1SJ2and 3WXO was obtained from Protein Data
Bank (www.pdb.org) [35,36]. The crystal is found to be bounded
with heme and glucose. The protein structure was optimized and
subsequently minimized using Protein Preparation Wizard module
of Maestro in which OPLS3 force field was used [38]. An additional
crystal PDB ID: 3WXO, which is heme and INH bound was taken to
observe the structural changes with different substrates.
The synthetic method of 4a was adopted to synthesize 4f. Yield:
63%; 1H NMR (300 MHz, DMSO‑d6)
d
11.57 (s, 1H, NH), 7.91 (d, J ¼
8.0 Hz, 2H, AreH), 7.80 (s, 1H, AreH), 7.75 (d, J ¼ 8.4 Hz, 2H, AreH),
4.69 (s, 2H, CH2), 3.91 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3)
d
165.41, 163.87, 147.37, 136.87, 132.81, 129.64, 126.04, 125.99,
122.24, 120.93, 115.03, 52.20, 42.12, 29.71; HRMS (ESI-TOF):
Calculated m/z for C15H11ClF3NO3S, 377.0100 found, 378.0172
[MþH]þ 399.9992 [MþNa]þ
4.5.2. Compounds preparation
€
The molecules 4a, and 4b were prepared using Schrodinger’s
4.1.33. Methyl 5-(benzo[b]thiophen-2-yl)-2-(2-chloroacetamido)
thiophene-3-carboxylate (4g)
module LIGPREP (version 2017e2), which generates tautomer’s,
and possible ionization states at the pH range 7 2 using Epikand,
also generates all the stereoisomers of the compound if necessary.
The optimization was done using the OPLS3 force field [38].
The synthetic method of 4a was adopted to synthesize 4g. Yield:
96%; 1H NMR (300 MHz, DMSO‑d6)
d 11.50 (s, 1H, NH), 7.90 (m, 1H,
AreH), 7.80 (m, 1H, AreH),7.70 (s, 1H, AreH), 7.40 (s, 1H, AreH),
7.30 (m, 2H, AreH), 4.71e4.69 (s, 2H, CH2), 3.89 (s, 3H, CH3); 13C
4.5.3. Binding site identification
NMR (75 MHz, CDCl3)
d
165.37, 163.80, 146.80, 140.26, 138.99,
The most likely binding site of the compounds 4a, and 4b was
identified using ligand independent (sitemap) and ligand depen-
dent (molecular docking) methods.
136.23, 128.31, 124.82, 124.69, 123.53, 122.11, 121.19, 114.5, 52.19,
42.11, 29.72, 22.72; MS (APCIþ): Calculated m/z for C16H12ClNO3S2,

12
C.L. Meena et al. / European Journal of Medicinal Chemistry 208 (2020) 112772
4.5.4. SitMap analysis
Author’s contribution
Site-Map program of Schrodinger Suite was used for calculating
binding site of 4a, and 4b [38,39]. The different parameters such as:
site score, size, exposure score, enclosure, hydrophobic/hydrophilic
character, contact, and donor/acceptor character were used for
calculation of potential binding site [39,40]. Drugability of the site is
denoted by D-score. The OPLS-2003 force field was employed, and a
standard grid was used with 15 site points per reported site and
cropped at 4.0 Å from the nearest site point.
DM and RS conceived the idea, supervised the project and
analysed the results. PS and RPS performed microbiology experi-
ments. CLM, VK and AK contributed towards the synthesis of
molecules. SA and AKT performed the computational studies.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4.5.5. Molecular docking
Molecular docking was used to assess the robustness of results
provided by Sitemap. The docking studies were performed using
AUTODOCK 4.2 [40]. The tools was required to identify the putative
binding site as no co-crystal (with inhibitor) is reported for
M. tuberculosis-katG, indeed, the INH bounded crystal is reported
recently, which have shown more than one binding site [36]. The
docking with AUTODOCK was performed using two different pro-
tocols: 1) Blind docking to explore the possible binding sites at the
surface of KatG to rule out any biasedness, 2) Focused docking was
performed on the most populated cluster obtained from blind
docking by entering the docking grid on the center of mass of
cluster representative conformation of INH. Two different ap-
proaches were performed as the most likely site identified by
sitemap followed by the most populated cluster obtained through
blind docking [40,41]. In all docking protocols, 200 conformers
were generated separately to observe their convergence at the
catalytic site. The grid coordinates, energy evaluations and gener-
ations of all three protocols were performed as previously
described [40,41].
Acknowledgements
We would like to thank the Translational Health Science and
Technology Institute (THSTI) for intramural research funding. PS
acknowledges Department of Science and Technology for his
fellowship (PDF/2018/002454). RS acknowledges the funding
received from Department of Biotechnology (BT/PR29075/BRB/10/
1699/2018).The authors are also thankful to Dr. David Alland and
Dr. Pradeep Kumar for sharing Pks13 overexpression constructs. Dr.
Bill Jacobs is also acknowledged for sharing INH resistant
M. tuberculosis strain.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112772.
References
4.5.6. Energetic analysis
[1] P. Glaziou, K. Floyd, M.C. Raviglione, Global epidemiology of tuberculosis,
Epub 2018/08/03, Semin. Respir. Crit. Care Med. 39 (3) (2018) 271e285,
https://doi.org/10.1055/s-0038-1651492. PubMed PMID: 30071543.
[2] S. Chakraborty, K.Y. Rhee, Tuberculosis drug development: history and evo-
lution of the mechanism-based paradigm, Epub 2015/04/17, Cold Spring Harb
Free energy was carried out using the MMGBSA python script of
AMBER tools and AMBER16 [41]. For this, the frames were extracted
from the most stable state from the 300ns trajectory which was
performed using the visual molecular dynamics tool (VMD). So,
overall 1000 frames from all the complexes (COM1 and COM2)
were subjected to energy calculations. The binding free energy
Perspect Med
5
(8) (2015) a021147, https://doi.org/10.1101/cshper-
spect.a021147. PubMed PMID: 25877396; PubMed Central PMCID:
PMCPMC4526730.
[3] C.K. Stover, P. Warrener, D.R. VanDevanter, D.R. Sherman, T.M. Arain,
M.H. Langhorne, et al., A small-molecule nitroimidazopyran drug candidate
for the treatment of tuberculosis, Epub 2000/07/06, Nature 405 (6789) (2000)
962e966, https://doi.org/10.1038/35016103. PubMed PMID: 10879539.
[4] R. Singh, U. Manjunatha, H.I. Boshoff, Y.H. Ha, P. Niyomrattanakit, R. Ledwidge,
et al., PA-824 kills nonreplicating Mycobacterium tuberculosis by intracellular
NO release, Epub 2008/11/29, Science 322 (5906) (2008) 1392e1395, https://
doi.org/10.1126/science.1164571. PubMed PMID: 19039139; PubMed Central
PMCID: PMCPMC2723733.
[5] Y. Li, F. Sun, W. Zhang, Bedaquiline and delamanid in the treatment of
multidrug-resistant tuberculosis: promising but challenging, Epub 2018/12/
15, Drug Dev. Res. 80 (1) (2019) 98e105, https://doi.org/10.1002/ddr.21498.
PubMed PMID: 30548290; PubMed Central PMCID: PMCPMC6590425.
[6] A.H. Diacon, A. Pym, M.P. Grobusch, J.M. de los Rios, E. Gotuzzo, I. Vasilyeva, et
al., Multidrug-resistant tuberculosis and culture conversion with bedaquiline,
Epub 2014/08/21, N. Engl. J. Med. 371 (8) (2014) 723e732, https://doi.org/
10.1056/NEJMoa1313865. PubMed PMID: 25140958.
[7] H. Li, D.H. Salinger, D. Everitt, M. Li, A. Del Parigi, C. Mendel, et al., Long-term
effects on QT prolongation of pretomanid, alone and in combinations, in pa-
tients with tuberculosis, Epub 2019/07/31, Antimicrob. Agents Chemother.
(2019), https://doi.org/10.1128/AAC.00445-19. PubMed PMID: 31358590.
[8] A. Bahuguna, D.S. Rawat, An overview of new antitubercular drugs, drug
candidates, and their targets, Epub 2019/06/30, Med. Res. Rev. 40 (1) (2020)
263e292, https://doi.org/10.1002/med.21602. PubMed PMID: 31254295.
[9] I.D. Olaru, F. von Groote-Bidlingmaier, J. Heyckendorf, W.W. Yew, C. Lange,
K.C. Chang, Novel drugs against tuberculosis: a clinician’s perspective, Epub
2014/11/29, Eur. Respir. J. 45 (4) (2015) 1119e1131, https://doi.org/10.1183/
09031936.00162314. PubMed PMID: 25431273.
(D
Gbind) on each system was evaluated as follows:
DGbind ¼ Gcom e (Grec þ Glig
)
(1)
where Gcom, Grec and Glig are the absolute free energies of complex,
receptor and compounds, respectively, arranged over the equilib-
rium trajectory. According to free energy calculating method (MM/
PBSA), the free energy difference can be decomposed as
D
G ¼
molecular mechanics energy,
(including an entropic contribution), and
D
EMM
þ
D
Gsolv e T
D
Sconf, where
Gsolv the solvation energy
Sconf the solute
DEMM is the difference in
D
T
D
configurational entropy (including the loss of translational and
rotational entropy due to binding, as well as change in the vibra-
tional entropy). The first two terms are calculated with the
following equations:
D
EMM
Gsolv
¼
¼
D
Ebond
GPB
þ
D
Eangle
þ
D
Etorsion
þ
D
Evdw
þ
D
Eelect
(2)
(3)
D
D
þ
D
GSA
where EMM includes the molecular mechanics energy contributed
by bonded (Ebond, Eangle, and Etorsion) and nonbonded (Evdw and
[10] R. Dhiman, R. Singh, Recent advances for identification of new scaffolds and
drug targets for Mycobacterium tuberculosis, Epub 2018/05/16, IUBMB Life 70
(9) (2018) 905e916, https://doi.org/10.1002/iub.1863. PubMed PMID:
29761628.
[11] R.S. Wallis, M. Maeurer, P. Mwaba, J. Chakaya, R. Rustomjee, G.B. Migliori, et
al., Tuberculosis–advances in development of new drugs, treatment regimens,
host-directed therapies, and biomarkers, Epub 2016/04/03, Lancet Infect. Dis.
E
elect) terms of the force field and
ergy, which has an electrostatic
PoissonꢁBoltzmann equation) and
GSA
gDSA þ
DGsolv is the solvation free en-
(
D
GPB
a
,
evaluated using the
nonpolar contribution
SA).
(D
¼
b
) proportional to the surface area (
D

C.L. Meena et al. / European Journal of Medicinal Chemistry 208 (2020) 112772
13
16 (4) (2016) e34ee46, https://doi.org/10.1016/S1473-3099(16)00070-0.
PubMed PMID: 27036358.
[12] D.T. Hoagland, J. Liu, R.B. Lee, R.E. Lee, New agents for the treatment of drug-
resistant Mycobacterium tuberculosis, Epub 2016/05/08, Adv. Drug Deliv. Rev.
102 (2016) 55e72, https://doi.org/10.1016/j.addr.2016.04.026. PubMed
PMID: 27151308; PubMed Central PMCID: PMCPMC4903924.
[13] T. Yuan, N.S. Sampson, Hit generation in TB drug discovery: from genome to
granuloma, Epub 2018/02/01, Chem. Rev. 118 (4) (2018) 1887e1916, https://
doi.org/10.1021/acs.chemrev.7b00602. PubMed PMID: 29384369; PubMed
Central PMCID: PMCPMC5832989.
[14] F. Zani, P. Vicini, M. Incerti, Synthesis and antimicrobial properties of 2-
(benzylidene-amino)-benzo[d]isothiazol-3-ones, Epub 2004/02/28, Eur. J.
[27] F.C. Odds, Synergy, antagonism, and what the chequerboard puts between
them, Epub 2003/06/14, J. Antimicrob. Chemother. 52 (1) (2003) 1, https://
doi.org/10.1093/jac/dkg301. PubMed PMID: 12805255.
[28] G. Arora, Behura A. Gagandeep, T.P. Gosain, R.P. Shaliwal, S. Kidwai, et al., NSC
18725, a pyrazole derivative inhibits growth of intracellular Mycobacterium
tuberculosis by induction of autophagy, Epub 2020/02/18, Front. Microbiol. 10
(2019) 3051, https://doi.org/10.3389/fmicb.2019.03051. PubMed PMID:
32063889; PubMed Central PMCID: PMCPMC6999026.
[29] B. Lechartier, R.C. Hartkoorn, S.T. Cole, In vitro combination studies of ben-
zothiazinone lead compound BTZ043 against Mycobacterium tuberculosis,
Epub 2012/08/29, Antimicrob. Agents Chemother. 56 (11) (2012) 5790e5793,
https://doi.org/10.1128/AAC.01476-12. PubMed PMID: 22926573; PubMed
Central PMCID: PMCPMC3486603.
Med.
Chem.
39
(2)
(2004)
135e140,
https://doi.org/10.1016/
j.ejmech.2003.11.004. PubMed PMID: 14987822.
[30] V.M. Reddy, L. Einck, K. Andries, C.A. Nacy, In vitro interactions between new
antitubercular drug candidates SQ109 and TMC207, Epub 2010/04/14, Anti-
microb. Agents Chemother. 54 (7) (2010) 2840e2846, https://doi.org/
10.1128/AAC.01601-09. PubMed PMID: 20385864; PubMed Central PMCID:
PMCPMC2897281.
[15] I.M. Fakhr, M.A. Radwan, S. el-Batran, O.M. Abd el-Salam, S.M. el-Shenawy,
Synthesis and pharmacological evaluation of 2-substituted benzo[b]thio-
phenes as anti-inflammatory and analgesic agents, Epub 2008/04/25, Eur. J.
Med. Chem. 44 (4) (2009) 1718e1725, https://doi.org/10.1016/
j.ejmech.2008.02.034. PubMed PMID: 18433939.
[16] R.J. Mourey, B.L. Burnette, S.J. Brustkern, J.S. Daniels, J.L. Hirsch, W.F. Hood, et
al., A benzothiophene inhibitor of mitogen-activated protein kinase-activated
protein kinase 2 inhibits tumor necrosis factor alpha production and has oral
anti-inflammatory efficacy in acute and chronic models of inflammation, Epub
2010/03/20, J. Pharmacol. Exp. Therapeut. 333 (3) (2010) 797e807, https://
doi.org/10.1124/jpet.110.166173. PubMed PMID: 20237073.
[17] W. Shi, T.L. Lowary, Structure-activity relationships in glycosylated 2-phenyl-
indoles, 2-phenyl-benzo[b]thiophenes and 2-phenyl-benzo[b]furans as DNA
binding and potential antitumor agents, Epub 2011/02/08, Bioorg. Med. Chem.
19 (5) (2011) 1779e1789, https://doi.org/10.1016/j.bmc.2011.01.014.
PubMed PMID: 21296580.
[31] C. Vilcheze, W.R. Jacobs Jr., Resistance to isoniazid and ethionamide in
Mycobacterium tuberculosis: genes, mutations, and causalities, MGM2-0014-
2013. Epub 2015/06/25, Microbiol. Spectr.
10.1128/microbiolspec.MGM2-0014-2013. PubMed
PubMed Central PMCID: PMCPMC6636829.
2
(4) (2014), https://doi.org/
PMID: 26104204;
[32] W. Shi, X. Zhang, X. Jiang, H. Yuan, J.S. Lee, C.E. Barry 3rd, et al., Pyrazinamide
inhibits trans-translation in, Epub 2011/08/13, Mycobacterium Tuberculosis.
Sci. 333 (6049) (2011) 1630e1632, https://doi.org/10.1126/science.1208813.
PubMed PMID: 21835980; PubMed Central PMCID: PMCPMC3502614.
[33] N.E. Caceres, N.B. Harris, J.F. Wellehan, Z. Feng, V. Kapur, R.G. Barletta, Over-
expression of the D-alanine racemase gene confers resistance to D-cycloserine
in Mycobacterium smegmatis, Epub 1997/08/01, J. Bacteriol. 179 (16) (1997)
[18] R. Wilson, P. Kumar, V. Parashar, C. Vilcheze, R. Veyron-Churlet,
J.S. Freundlich, et al., Antituberculosis thiophenes define a requirement for
Pks13 in mycolic acid biosynthesis, Epub 2013/06/19, Nat. Chem. Biol. 9 (8)
(2013) 499e506, https://doi.org/10.1038/nchembio.1277. PubMed PMID:
23770708; PubMed Central PMCID: PMCPMC3720791.
[19] P. Singh, S.K. Kumar, V.K. Maurya, B.K. Mehta, H. Ahmad, A.K. Dwivedi, et al.,
S-enantiomer of the antitubercular compound S006-830 complements ac-
tivity of frontline TB drugs and targets biogenesis of Mycobacterium tuber-
culosis cell envelope, Epub 2018/07/20, ACS Omega 2 (11) (2017) 8453e8465,
https://doi.org/10.1021/acsomega.7b01281. PubMed PMID: 30023583;
PubMed Central PMCID: PMCPMC6045410.
[20] C. Scheich, V. Puetter, M. Schade, Novel small molecule inhibitors of MDR
Mycobacterium tuberculosis by NMR fragment screening of antigen 85C, Epub
2010/11/16, J. Med. Chem. 53 (23) (2010) 8362e8367, https://doi.org/
10.1021/jm100993z. PubMed PMID: 21073150.
[21] D.A. Ibrahim, J. Boucau, D.H. Lajiness, S.K. Veleti, K.R. Trabbic, S.S. Adams, et al.,
Design, synthesis, and X-ray analysis of a glycoconjugate bound to Myco-
bacterium tuberculosis antigen 85C, Epub 2012/11/30, Bioconjugate Chem. 23
(12) (2012) 2403e2416, https://doi.org/10.1021/bc3004342. PubMed PMID:
23190459; PubMed Central PMCID: PMCPMC3548330.
[22] C. Grundner, D. Perrin, R. Hooft van Huijsduijnen, D. Swinnen, J. Gonzalez,
C.L. Gee, et al., Structural basis for selective inhibition of Mycobacterium
tuberculosis protein tyrosine phosphatase PtpB, Epub 2007/04/18, Structure
15 (4) (2007) 499e509, https://doi.org/10.1016/j.str.2007.03.003. PubMed
PMID: 17437721; PubMed Central PMCID: PMCPMC2775457.
[23] A. Aggarwal, M.K. Parai, N. Shetty, D. Wallis, L. Woolhiser, C. Hastings, et al.,
Development of a novel lead that targets M. tuberculosis polyketide synthase
13, e25. Epub 2017/07/04, Cell 170 (2) (2017) 249e259, https://doi.org/
10.1016/j.cell.2017.06.025. PubMed PMID: 28669536; PubMed Central
PMCID: PMCPMC5509550.
[24] V.N. Khachatryan Dso, R.A. Sandu, V.N. Danilenko, A.V. Kolotaev,
O.A. Zhdanovich, O.B. Bekker, K.R. Matevosyan, Quaternary Ammonium De-
rivatives of 2-Aminothiophen-3-Carboxylates Having Anti-tuberculosis Ac-
tivity, 2017. RU 2629369 C1 20170829.
[25] G. Nikolakopoulos, H. Figler, J. Linden, P.J. Scammells, 2-Aminothiophene-3-
carboxylates and carboxamides as adenosine A1 receptor allosteric en-
hancers, Epub 2005/11/30, Bioorg. Med. Chem. 14 (7) (2006) 2358e2365,
https://doi.org/10.1016/j.bmc.2005.11.018. PubMed PMID: 16314104.
[26] S. Purser, P.R. Moore, S. Swallow, V. Gouverneur, Fluorine in medicinal
chemistry, Epub 2008/01/17, Chem. Soc. Rev. 37 (2) (2008) 320e330, https://
doi.org/10.1039/b610213c. PubMed PMID: 18197348.
5046e5055,
https://doi.org/10.1128/jb.179.16.5046-5055.1997. PubMed
PMID: 9260945; PubMed Central PMCID: PMCPMC179361.
[34] J. Suarez, K. Ranguelova, A.A. Jarzecki, J. Manzerova, V. Krymov, X. Zhao, et al.,
An oxyferrous heme/protein-based radical intermediate is catalytically
competent in the catalase reaction of Mycobacterium tuberculosis catalase-
peroxidase (KatG), Epub 2009/01/14, J. Biol. Chem. 284 (11) (2009)
7017e7029, https://doi.org/10.1074/jbc.M808106200. PubMed PMID:
19139099; PubMed Central PMCID: PMCPMC2652337.
[35] T. Bertrand, N.A. Eady, J.N. Jones, Jesmin, J.M. Nagy, B. Jamart-Gregoire, et al.,
Crystal structure of Mycobacterium tuberculosis catalase-peroxidase, Epub
2004/07/03, J. Biol. Chem. 279 (37) (2004) 38991e38999, https://doi.org/
10.1074/jbc.M402382200. PubMed PMID: 15231843.
[36] S. Kamachi, K. Hirabayashi, M. Tamoi, S. Shigeoka, T. Tada, K. Wada, The crystal
structure of isoniazid-bound KatG catalase-peroxidase from Synechococcus
elongatus PCC7942, Epub 2014/10/11, FEBS J. 282 (1) (2015) 54e64, https://
doi.org/10.1111/febs.13102. PubMed PMID: 25303560.
[37] S. Kidwai, R. Bouzeyen, S. Chakraborti, N. Khare, S. Das, T. Priya Gosain, et al.,
NU-6027 inhibits growth of Mycobacterium tuberculosis by targeting protein
kinase D and protein kinase G, Epub 2019/07/10, Antimicrob. Agents Che-
mother. 63 (9) (2019), https://doi.org/10.1128/AAC.00996-19. PubMed PMID:
31285226; PubMed Central PMCID: PMCPMC6709498.
[38] S. Asthana, S. Shukla, A.V. Vargiu, M. Ceccarelli, P. Ruggerone, G. Paglietti, et
al., Different molecular mechanisms of inhibition of bovine viral diarrhea vi-
rus and hepatitis
C virus RNA-dependent RNA polymerases by a novel
benzimidazole, Epub 2013/05/01, Biochemistry 52 (21) (2013) 3752e3764,
https://doi.org/10.1021/bi400107h. PubMed PMID: 23627712.
[39] S. Kant, S. Asthana, D. Missiakas, V. Pancholi, A novel STK1-targeted small-
molecule as an "antibiotic resistance breaker" against multidrug-resistant
Staphylococcus aureus, Epub 2017/07/13, Sci. Rep.
https://doi.org/10.1038/s41598-017-05314-z. PubMed PMID: 28698584;
PubMed Central PMCID: PMCPMC5505960.
7 (1) (2017) 5067,
[40] S. Asthana, P. Zucca, A.V. Vargiu, E. Sanjust, P. Ruggerone, A. Rescigno,
Structure-activity relationship study of hydroxycoumarins and mushroom
tyrosinase, Epub 2015/08/12, J. Agric. Food Chem. 63 (32) (2015) 7236e7244,
https://doi.org/10.1021/acs.jafc.5b02636. PubMed PMID: 26263396.
[41] M. Srivastava, C. Suri, M. Singh, R. Mathur, S. Asthana, Molecular dynamics
simulation reveals the possible druggable hot-spots of USP7, Epub 2018/10/
23, Oncotarget 9 (76) (2018) 34289e34305, https://doi.org/10.18632/onco-
target.26136. PubMed PMID: 30344943; PubMed Central PMCID:
PMCPMC6188144.