Design and development of novel Mycobacterium tuberculosis l-alanine dehydrogenase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.12.046

In the present study, we used crystal structure of MTB L-AlaDH protein complex with N₆-methyl adenosine for structure based virtual screening of in house database to identify new small molecule inhibitors for MTB-L-AlaDH. Two molecules identified as better leads and were modified synthetically to obtain thirty novel analogues belonging to 2-iminothiazolidine-4-ones and 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamides. Among the screened compounds four (4n, 4o, 12 and 14) emerged as potent inhibitors displaying IC₅₀ values ranging from 0.58 ± 0.02 to 1.74 ± 0.03 μM against MTB-L-AlaDH and were non-cytotoxic at 50 μM. Some of these synthesized compounds also exhibited good activity against nutrient starved dormant MTB cells. The most potent inhibitors were found to stabilize the protein which was confirmed biophysically through differential scanning fluorimetry.

Full text of DOI:10.1016/j.ejmech.2014.12.046

Accepted Manuscript
Design and development of novel Mycobacterium tuberculosis L-alanine
dehydrogenase inhibitors
Shalini Saxena, Ganesh Samala, Jonnalagadda Padma Sridevi, Parthiban Brindha
Devi, Perumal Yogeeswari, Dharmarajan Sriram, Chair Professor
PII:
S0223-5234(14)01155-6
10.1016/j.ejmech.2014.12.046
EJMECH 7606
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 September 2014
Revised Date: 13 November 2014
Accepted Date: 26 December 2014
Please cite this article as: S. Saxena, G. Samala, J.P. Sridevi, P.B. Devi, P. Yogeeswari, D. Sriram,
Design and development of novel Mycobacterium tuberculosis L-alanine dehydrogenase inhibitors,
European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2014.12.046.
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Design and development of novel Mycobacterium tuberculosis
L-alanine dehydrogenase inhibitors
Shalini Saxena, Ganesh Samala, Jonnalagadda Padma Sridevi, Parthiban Brindha Devi, Perumal
Yogeeswari, Dharmarajan Sriram*
Centre for Infectious Disease Research, Department of Pharmacy, Birla Institute of Technology &
Science-Pilani, Hyderabad campus, Jawaharnagar, Shameerpet, Hyderabad-500078, India
Compound tert-butyl 3-carbamoyl-2-(4-chlorobenzamido)-4,5-dihydrothieno[2,3-c]pyridine-6(7H)-
carboxylate (14) was the most promising potent inhibitor with an IC50 of 0.58±0.02 µM against
Mycobacterium tuberculosis L-AlaDH enzyme, with a good MTB MIC of 1.79 µM, and was not
cytotoxic at 50 µM in eukaryotic cell lines.

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Design and development of novel Mycobacterium tuberculosis L-alanine
dehydrogenase inhibitors
Shalini Saxena, Ganesh Samala, Jonnalagadda Padma Sridevi, Parthiban Brindha Devi,
Perumal Yogeeswari, Dharmarajan Sriram*
Centre for Infectious Disease Research, Department of Pharmacy, Birla Institute of
Technology & Science-Pilani, Hyderabad campus, Jawaharnagar, Shameerpet, Hyderabad-
500078, India
ABSTRACT
In the present study, we used crystal structure of MTB L-AlaDH protein complex with N6-
methyl adenosine for structure based virtual screening of in house database to identify new
small molecule inhibitors for MTB-L-AlaDH. Two molecules identified as better leads and
were modified synthetically to obtain thirty novel analogues belonging to 2-
iminothiazolidine-4-ones
and
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamides.
Among the screened compounds four (4n, 4o, 12 and 14) emerged as potent inhibitors
displaying IC50 values ranging from 0.58±0.02 to 1.74±0.03 µM against MTB-L-AlaDH and
were non-cytotoxic at 50 µM. Some of these synthesized compounds also exhibited good
activity against nutrient starved dormant MTB cells. The most potent inhibitors were found to
stabilize the protein which was confirmed biophysically through differential scanning
fluorimetry.
Keywords: Tuberculosis, Dormant tuberculosis, L-Alanine dehydrogenase, 2-
iminothiazolidine-4-ones, 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamides.
*For correspondence: D. Sriram, Chair Professor, Department of Pharmacy,
Birla Institute of Technology & Science-Pilani, Hyderabad Campus,
Jawahar Nagar, Hyderabad- 500 078. INDIA
Telephone: +91-40663030506; Fax: +91-4066303998
Email: dsriram@hyderabad.bits-pilani.ac.in

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1. Introduction
Tuberculosis (TB) infected by Mycobacterium tuberculosis (MTB), is an infectious
disease that usually affects the lungs of infected individuals for decades by switching to a
dormant or latent phases. It is the second greatest killer due to a single infectious agent
worldwide, and in 2012 around 1.3 million people died from this disease, with 8.6 million
falling ill [1]. Among the infected individuals, approximately eight million develop active
TB, and almost two million of these die from this disease. Of new TB cases 95% occur in
developing countries every year [2]. Due to the emergence of multidrug-resistant TB
(MDR-TB), high incidence of HIV/TB co-infection and lack of new anti-tubercular drugs,
TB is declared a global health emergency by WHO (World Health Organization).
Currently available antitubercular drugs were found to be ineffective to treat dormant
MTB; therefore, the development of new antimicrobial drugs for treating this disease is
urgently desired [3] Current chemotherapeutic treatments are based on the use of
antibiotics such as isoniazid (INH), rifampicin, pyrazinamide, ethambutol and
streptomycin. The effectiveness of current anti-tuberculosis drugs to combat this infection
is severely compromised by the emergence of multi and extensively drug resistant TB [4-
5]. To treat MDR-TB, the WHO recommended using second-line drugs such as
fluoroquinolones, ethionamide and cycloserin. These agents besides being unsuitable for
short-course treatment could also be less effective and more toxic [6]. Dormancy has been
associated with non-replicating or very slow growth of MTB that resides in granulomas, a
heterogeneous assembly of macrophages in the lungs of infected individuals [7] Although
MTB is able to evade in immune system and can survive in human host for decades in a
persistent state, when the pathogens are under anaerobic growth conditions or nutrient
starvation regimes, one of gene, Rv2780 encoding L-alanine dehydrogenase (L-AlaDH)
was found to be up-regulated. When the terminal electron acceptor oxygen becomes

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limiting, the production and activity of this enzyme were observed to increase in MTB [8-
9]. NAD(H)-dependent L-AlaDH catalyze the oxidative deamination of L-alanine to
pyruvate and ammonia (catabolic reaction) or, in the reverse direction, the reductive
amination of pyruvate to L-alanine (biosynthetic reaction) [10] AlaDH are well-studied
enzymes found in a wide range of bacterial species. In mycobacteria, it was first identified
as an enzyme absent from the vaccine strains of M. bovis BCG but present in
virulent MTB [11] Recent analysis involving microarray and other data has identified this
enzyme to be among the top three drug targets, especially against persistence [12]. In our
previous study, we have reported the first set of novel diverse inhibitors for MTB L-
AlaDH through structure based drug design [3], but those compounds were not
synthetically feasible to develop further analogues and also they were not drug like
compounds; hence in the current study we took another MTB L-AlaDH protein complex
with N6-methyl adenosine to design some small molecules for MTB L-AlaDH inhibition.
The aim was to identify small novel inhibitors which could inhibit the MTB L-AlaDH
enzymatic activity through structure based virtual screening.
2. Results and Discussion
2.1. Designing of the molecules
In the present study, crystal structure of the mycobacterial MTB L-AlaDH in complex with
N6-methyl adenosine (PDB: 4LMP) [13] having resolution of 1.95A0 was used as a
framework for structure based virtual screening of in house database consisting of 3000
diverse structured compounds to identify lead compounds against this enzyme. E-
pharmacophore hypotheses were developed by mapping Glide XP energetic terms onto
pharmacophore sites, and these energies were calculated based on the structural and energetic
information between the protein and the ligand using Phase module having the default set of
chemical features [14] The number of pharmacophore sites was set to ten and the total

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number of pharmacophore sites derived was six with three donors (D), two ring aromatic (R)
and one acceptor (A). The six point e-pharmacophore shown in Fig. 1 was then utilized for
the virtual screening of in house database.
Fig. 1
Compounds retrieved by e-pharmacophore filter using Phase with a fit value above 1.0 were
considered as potential hits and were carried forward for high-throughput virtual screening.
Compounds resulting with a score of ≤ -4.0 kcal mol-1 were subjected to another round of
docking by Glide XP. Glide XP combine accurately, physics-based scoring terms and
thorough sampling and resulted in compounds with docking scores between -6.649 to -
5.45kcal mol-1. Final shortlisting of hit compounds was based on visual inspection of the
important amino acid residues involved in binding that included hydrogen bonding with
Ser220, Leu240, and Asp198. Thus we selected top five compounds from the Glide XP
docking study with the best Glide scores (-6.50 to -7.50kcal/mol) suggesting strong protein-
ligand interactions. The chemical structures of these lead compounds (Lead 1 to Lead 5) are
illustrated in Fig. 2. Further to prove the design concept, these hits were subjected to
biological assay.
Fig. 2
We identified (Z)-5-(4-benzyloxy)benzylidene-2-imino-3-(5-nitrothiazol-2-yl)thiazolidin-4-
one
(Lead
1)
and
2-(5-nitrothiophene-2-carboxamido)-4,5-dihydro-N6-(4-
(Lead 2) as potential
methoxyphenyl)thieno[2,3-c]pyridine-3,6(7H) dicarboxamide
inhibitors in the MTB L-AlaDH site interacting with amino acid residues Ser220, Ser134, and
Lys203 with a docking score of -6.92 and -7.21kcal/mol. Lead 1 and 2 showed an IC50 of
6.77ꢀM and 8.17ꢀM when tested for MTB L-AlaDH activity. Based on the two identified
hits, we undertook synthesis of various analogues and evaluated for its biological activity
against MTB L-AlaDH for establishing structure-activity relationship (SAR), against active

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and dormant MTB and cytotoxicity. Docking of the Lead 1 within the active site of the MTB
L-AlaDH protein is illustrated in Fig. 3. Lead 1 showed one hydrogen bonding with Ser134
with a well fitted pose in the active site in the hydrophobic pocket within the vicinity of
Ile199, Pro247, Leu255, Leu249, Leu197, Ile174, Ala126, Leu127, Ala179, Leu130, Ala176
and few polar amino acid residues Gln121, Asp198, Thr178, Ser220 respectively. The
binding pattern within the active site pocket of the crystal ligand and reference ligand was
quite similar and additionally the cation-pi interaction between Lys203 and the ligand
constituted for a stable binding profile of the molecule.
Fig. 3
The docking orientation of Lead 2 is representation in Fig. 4, where the following
interactions were observed at the binding site. The NO2 group on thiophene ring was
involved in hydrogen bonding interaction with Ser220, which was also observed with
reference ligand. Further the NH2 group on pyridine was stabilized by two hydrogen bonding
interaction with Leu240 and Ala238 amino acid residues. The compound was found placed
proximal to various hydrophobic amino acids such as Ile199, Pro247, Leu255, Leu249, Leu197,
Ile174, Ala126, Leu127, Ala179, Leu130, and Ala176 and hence exhibited hydrophobic
interactions. Apart from these interactions the compound was further stabilized by cation-pi
interaction with Lys203.
Fig. 4
2.2. Chemistry
A library of 30 molecules, comprising of fifteen iminothiazolidine-4-one derivatives
(compound 4a-o) based on Lead 1 and another fifteen 4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide derivatives (compound 11-25) based on Lead 2 were synthesised
as shown in Schemes 1 and 2 respectively. Synthesis of chloroacetamides 2a-o was carried
out by treating different substituted aromatic amines with chloroacetyl chloride under reflux

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conditions in benzene. Further cyclisation of substituted chloroacetamide was accomplished
with KSCN in acetone under reflux conditions to yield iminothiazolidine-4-one (3 a-o).
Finally, the target molecules 4 (a-o) were obtained by reaction of compounds 3 a-o with 4-
benzyloxybenzaldehyde using sodium acetate in acetic acid under thermal conditions.
The conversion of N-Boc protected 4-piperdone (5) to tert-butyl 2-amino-3-carbamoyl-4,5-
dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate (6) was based on Gewald reaction, which
was performed by treating compound 5 with cyanoacetamide, sulphur powder and
morpholine using EtOH as solvent at room temperature. Compound 6 on direct reaction with
substituted benzoyl chloride did not yield the output; hence we used mixed anhydride instead
of benzoyl chloride derivatives. The mixed anhydride (8 a-c) was synthesised by treating
compound corresponding benzoic acids (7 a-c) with ethyl chloroformate, and in next step the
synthesised mixed anhydrides were reacted with compound 6 under microwave reaction
conditions using NaH as base and dry THF as solvent to yield corresponding 2-carboxamides
(9 a-c). Further deprotection of Boc was carried out by treating with TFA in dichloromethane
at room temperature. Finally, the secondary free amines were treated with acetyl
chloride/benzoyl chloride using Et3N in dichloromethane and with CH3I/PhCH2Br using NaH
as base under microwave irradiation to get titled compounds.
Schemes 1 and 2
2.3. Enzyme kinetics of MTB L-AlaDH
Kinetic and mechanistic studies of L-AlaDH from several species showed that the enzyme
followed a predominantly ordered mechanism, with NAD+ and NADH binding occurring first
in the oxidative deamination and reductive amination direction, respectively [15]. Initially we
measured the concentration by range finding experiment at which the MTB L-AlaDH enzyme
works, later substrate alanine and NAD+ concentration were also measured. Then, we
performed Michaelis–Menten kinetics to determine Km and Vmax for alanine and NAD+. The

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Km and Vmax were found to be 0.049 mM and 1.071 absorbance units (AU); similarly for
NAD+ the Km and Vmax were found to be 0.53 mM and 0.37 AU respectively (Fig. 5).
Fig. 5
2.4. MTB L-AlaDH enzyme inhibition studies
The synthesized compounds were assayed for the inhibition of MTB L-AlaDH at
concentrations ranging from 25µM to 0.5µM. In the initial screening all the synthesized
compounds showed good percentage inhibition in the range of 40.0 to 86.6%. Thus IC50
values were calculated using non-linear regression analysis using GraphPad Prism software
for all the compounds from both series. All the synthesized compounds showed activity with
IC50 ranging from 0.58±0.02 ꢀM to 22.31±0.06 ꢀM (Table 1 and 2). Twenty three
compounds out of thirty were found to be more potent than the initially identified Lead 1 and
2 (IC50 of 6.77± 0.12 ꢀM and 8.17± 0.18 ꢀM respectively). From the first series of
iminothiazolidine-4-one derivatives, compounds 4n and 4o were found to be most potent
MTB L-AlaDH inhibitor with IC50 1.34±0.01 and 1.74±0.03 ꢀM respectively, while the
second series 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide derivatives compounds
12 and 14 were found to be most active with IC50 of 0.58±0.02 and 0.64±0.06 ꢀM
respectively. Dose-response curves were plotted for the two most potent compounds (4n and
4o) from series 1 and another two most active compound (12 and 14) from series 2 using
GraphPad Prism software (GraphPad Software Inc., La Jolla, CA) by taking log (inhibitor
concentration) on the X-axis and response (% inhibition) on the Y-axis as shown in Fig. 6A
and 6B.
Fig. 6
Further to support the activity, we performed docking analyses. As shown in activity Table 1,
most of the compounds exhibited good IC50 in the range of 1.34 to 22.31ꢀM against AlaDH,
six compounds showed better activity than the lead compound 1 (IC50= 6.77ꢀM).

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Additionally, all of these inhibitors occupied a narrow potency range, indicating tolerability
for a wide variety of substituent’s on the R1 position. The 4-chlorophenyl, 4-flourophenyl
substituents (4b and 4c) at the R1 position displayed good MTB L-AlaDH inhibitory activity,
while 4-bromophenyl substituted group (4a) showed moderate activity. Compounds
containing the combination of the nitrophenyl group at meta and para position at R1 (4e and
4f) provided good MTB L-AlaDH inhibitory activity, while introduction of 2-nitrophenyl
group at R1 position (4d) provided moderately inhibitory activity. The compounds substituted
with 2,4-dichlorophenyl, 3-chloro-2-methylphenyl, pyrimidin-2-yl, pyridin-2-yl, 6-
methylpyridin-2-yl and simple phenyl group at R1 (4g-4l) resulted in good in-vitro MTB L-
AlaDH inhibitory activity in the range of 3.49±0.06 to 2.27±0.04 ꢀM, while substitutions
with 2,5-dimethylphenyl and 2,6-dimethylphenyl (4n-4o) led to most potent compounds
compared to other compounds from this series. This was well supported by the interaction
profile of the molecules in the docking studies. Compound 4n (Z)-5-(4-
(benzyloxy)benzylidene)-2-imino-3-(2,5-dimethylphenyl)thiazolidin-4-one from the series 1
showed highest docking score of -7.50 kcal/mol which correlated well with its potency in the
enzyme assay (IC50=1.34±0.01ꢀM). Closer analysis of this compound in the protein active
site revealed hydrogen bonding interactions with the Thr178. In addition to hydrogen
bonding interactions, the compound was further stabilized by cation-pi stacking interaction
with the phenyl ring of Lys203. The compound also showed hydrophobic interaction with
Leu130, Ala131, Leu127, Pro128, Ala126, Pro242, Ala238, Val239, Leu197, Leu249, Ile174,
Ile199, Leu225, Ala176 and Ala179 amino acid residues. Some polar contacts were also
observed such as Asn200, Ser220, Ser134 and Gln271 (Fig. 7). Further in silico investigation
into binding profile of the molecules in the MTB L-AlaDH domain revealed the importance
of the hydrophobic interactions in increasing the specificity of the molecule towards the
protein.

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Fig. 7
The other active compound 4o (Z)-5-(4-(benzyloxy)benzylidene)-2-imino-3-(2,6-
dimethylphenyl)thiazolidin-4-one from this series also showed good binding energy of -6.90
kcal/mol which was further analyzed in more detail to understand its binding pattern. The
oxygen atom of benzyloxy group showed interaction with Ser220 amino acid, analogous to
the one observed in the reference ligand. Apart from that, 2,6-dimethylphenyl ring was found
to interact with Lys203 via cation-pi interaction. In the active site cavity the compound also
made some polar contacts with Thr178, Asn200, Ser221 and Asp198 amino acid residues as
well as hydrophobic interactions with Leu130, Ala131, Leu127, Pro128, Ala126, Pro242,
Ala238, Val239, Leu197, Leu249, Ile174, Ile199, Leu225, Ala176 and Ala179 amino acid
residues (Fig. 8).
Fig. 8
The second Lead 2 was also selected for further optimization, on which the structural
modification was carried out in order to increase the binding ability. Thus, we synthesized a
series of 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide derivatives. Among the
synthesised derivatives twelve compounds inhibited MTB L-AlaDH with an IC50 of less than
1µM (Table 2). Binding analysis of two most active compounds from this series (12 and 14)
showed that both the compounds were well docked in the active site cavity of AlaDH protein
with the docking score of -7.50 and -7.89 kcal/mol. From the docking results, the formation
of hydrogen bonds and hydrophobic interactions with the active site were predicted to be the
most important factors affecting the inhibitory potency of these compounds. Compound 12
was found to be involved in two hydrogen bonding interactions with Ser220, and Leu197
amino acid residues. Apart from that the compound was further stabilized by cation-pi
interaction with Lys203 amino acid residue. The acetyl and 4-chloro group is making polar
contact with Ser221 and Thr178 amino acid residue. The compound was well placed in to the

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hydrophobic cavity where it was stabilized by various hydrophobic amino acid such as
Ile174, Ile199, Leu225, Leu249, Ala22, Pro247, Val239, Pro242, Val241, Leu241, Leu240
Ala179, Ala238 and Ala176 (Fig. 9).
Fig. 9
The predicted binding pose of the second most active compound (14) suggested that the
observed improved potency arised due to the extensive hydrophobic interactions predicted to
be formed with the side chains of Ile174, Ile199, Leu225, Leu249, Ala22, Pro247, Val239,
Pro242, Val241, Leu241, Leu240 Ala179, Ala238 and Ala176 amino acid residues (Fig. 10).
The binding energy of the compound 14 was found to be of -7.89 kcal/mol. The cation-pi
interaction between the pyridine ring of compound 14 and Lys203 resulted to its good
activity, and the additional hydrogen-bond interactions was observed with amino acid
residues Ser220 which made the compound more fit in to the active site of protein.
Fig. 10
2.5. In vitro dormant MTB model
J.C. Betts et al. earlier established an in vitro model [16], in which nutrient starvation caused
M. tuberculosis to arrest growth, minimized aerobic metabolism and became resistant to
existing antitubercular drugs while maintaining viability. Nutrient starvation may therefore
mimic some of the features of M. tuberculosis during the persistent state. Moreover their
protein expression profile of their model indicated that AlaDH was over expressed 6.04
times. Owing to its simplicity, reproducibility and ease of handling, we utilized this model for
testing our novel potent AlaDH inhibitors aimed at persistent bacteria. In this model, cultures
of in vitro grown MTB bacteria were subjected to nutrient depletion by growing the culture in
PBS (Phosphate buffer saline) for 6 weeks. Nutrient starvation using this method triggered a
dormancy response in the bacilli that wastermed non-replicating persistence (NRP), a
physiological state thought to mimic the one exhibited by MTB during various stages of

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persistent infection. After 6 weeks, the culture was treated with the synthesized and standard
drugs at a concentration of 10 µg/ml in a tube and incubated at 37°C for 7 days. The treated
cell suspensions were diluted 10-fold up to 10-6 using Middlebrook 7H9 medium
supplemented with OADC and were plated in 48 well plates in triplicates. The plates were
incubated at 37°C for 4 weeks and the wells with visible bacterial growth were counted as
positive and MPN values were calculated using standard statistical methods [17] At 10 µg/ml
concentration, standard first line anti-TB drugs isoniazid and rifampicin reduced ~1.2 and 2.0
log bacterial reduction respectively whereas and DNA gyrase inhibitor moxifloxacin showed
high activity with ~2.5 log bacterial reduction. When compared to isoniazid, all the eighteen
screened compounds showed better activity with ~2.0 to 3.2 log bacterial reduction.
Seventeen compounds were found to be more potent than rifampicin, and nine compounds
(4c, 4g, 4o, 11, 16, 19, 23-25) were found to be more potent than moxifloxacin. Compound
24 (tert-butyl 2-(4-(benzyloxy)benzamido)-3-carbamoyl-4,5-dihydrothieno[2,3-c]pyridine-
6(7H)-carboxylate)
and
25
(6-benzyl-2-(4-(benzyloxy)benzamido)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide) were found to be more promising with ~3.2
log bacterial reduction (Fig. 11 and 12). These compounds also inhibited MTB AlaDH with
IC50 of < 1 µM.
Fig. 11
Fig. 12
2.6. In vitro active MTB model
All the synthesized compounds were also screened for their in vitro anti-tubercular activity
against MTB H37Rv (ATCC27294) using agar dilution method [18] with drug concentrations
from 50 µg/mL to 0.2 µg/mL in duplicates. The minimum inhibitory concentration (MIC)
was determined for each compound which was measured as the minimum concentration of
compound required to completely inhibit the bacterial growth. Isoniazid, ethambutol,

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rifampicin and ofloxacin were used as reference compounds for comparison. The MIC values
of the synthesized compounds along with the standard drug for comparison are presented in
Table 1 and 2. All the synthesized compounds showed activity against MTB with MIC
ranging from 1.53 to 60.38 µM. Six compounds (4k, 4m, 14, 18, 22 and 24) inhibited MTB
with MIC of < 2 µM. All the compounds were less potent than isoniazid, rifampicin and
moxifloxacin. Sixteen compounds were found to be more potent than ethambutol (MIC of
7.64 µM).
2.7. In vitro cytotoxicity studies
The safety profile of all the synthesized compounds was evaluated by testing their in vitro
cytotoxicity in mouse macrophage cell line (RAW 264.7) cells at 50µM concentration
using (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [19].
Since MTB reside inside the macrophage, the monocyte macrophage cell lines (RAW
264.7) were mostly used for tuberculosis research; in order to check whether the screened
compounds were not toxic towards macrophages but toxic to the bacteria. Almost all the
tested compounds demonstrated a good safety profile with very low inhibitory potential.
Compounds inhibited within a range of 4.71-59.67%. Furthermore, the most active
compound 4n and 4o from first series and compounds 14 and 12 from second series had
38.67 and 45.81 and 37.32 and 38.43% inhibitions respectively at 50 µM concentration of
the drug, reflecting its safety profile in the eukaryotes, cytotoxic results of all the
compounds are illustrated in the Table 1 and 2.
2.8. Differential scanning fluorimetry studies
The stabilization of two active compounds from both series were investigated using
biophysical technique called DSF (Differential Scanning Fluorimetry) by measuring the
fluorescence of the native protein and protein-ligand complexes in presence of a fluorescent
dye whose fluorescence increase when exposed to non polar residues of the protein and reach

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the maximum when the protein denature [20]. A higher or positive shift of melting
temperature (Tm) of protein-ligand complex compared to the native protein Tm signify a better
stabilization of the protein-ligand complex, which in turn would reflect on the inhibitor
binding. The most potent compound 4n and 4o from first series display good positive shifts
towards the MTB L-AlaDH protein (4.6°C and 1.4°C), whereas the second series compound
12 and 14 shows 1.7°C and 1.8°C positive shifts towards the MTB L-AlaDH protein which
further was re-ascertained for their stabilization towards the protein (Fig. 13). The native
protein melting temperature is 46.20°C; whereas the protein complexed with ligand showed
melting temperature of 49.90°C to 51.80°C proves the stabilization of the ligand towards the
desired protein MTB L-AlaDH.
Fig. 13
3. Conclusion
In summary, we identified and synthesized a novel thiazolidine and ‘2-aminothiophene’
derivatives from a structure based virtual screening of in house database consist of 2500
compounds. Most of the compounds showed potent MTB L-AlaDH inhibition and MTB MIC
in active as well as dormant model. Compound 4n and 4o from first series and compound 12
and 14 from second series showed potency, selectivity, and no cytotoxicity up to 50 ꢀM and
emerged as valid lead for further development. Furthermore, exploring the binding affinity
and thermal stability of the most active compounds from both series to the MTB L-AlaDH
protein is done by DSF experiments.
4. Experimental Section
4.1. Computational details
All computations works were carried out on an Intel Core 2 Duo 63 E7400 2.80 GHz
capacity processor with memory of 2 GB RAM 64 running with the RHEL 5.2 operating

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system. In the Maestro 9.3 software package (Schro dinger, LLC) [21]; PHASE 3.3 was
used to derive the e-pharmacophore. For each prepared complexes, glide energy were
generated. The binding site was defined by a rectangular box surrounding the reference
ligand in the X-ray structure. Ligands were refined using the “Refine” option in Glide, and
the Glide XP (extra precision) descriptor was chosen (Glide v5.7, Schrodinger, LLC, New
York, NY).
4.2. Chemistry
4.2.1. General procedure for synthesis of N-substituted chloroacetamides (2a-o)
Compound 1 (1.0 equiv.) and chloroacetyl chloride (1.0 equiv.) were taken in benzene and
the reaction mixture was refluxed for 6 h. after completion of the reaction monitored by TLC,
the reaction mixture was diluted with EtOAc and washed with sat NaHCO3, H2O and brine.
The combined organic layer was dried over anhyd. Na2SO4, evaporated to get compound
corresponding 2a-o.
4.2.2. General procedure for synthesis of 3-substituted-2-iminothiazolidine-4-one (3a-o)
Compound 2a-o (1.0 equiv) and KSCN (1.6 equiv) were taken in dry acetone and refluxed
for 2 h. The reaction mixture was concentrated and obtained solid was washed with H2O and
dried in vacuum oven to get corresponding 3-substituted-2-iminothiazolidine-4-one 3a-o.
4.2.3. General procedure for synthesis of compounds 4 (a-o)
Compound 3a-o (1.0 equiv), NaOAc (2.0 equiv) and 4-benzyloxybenzaldehyde (1.1 equiv)
were taken in acetic acid and heated at 100 °C for 3 hours, the solids formed in the reaction
mixture were filtered and washed with water, little amount of ethanol and hexanes to afford
title compounds 4 (a-o) as an off-white solid.
4.2.4. 5-(4-(Benzyloxy)benzylidene)-3-(4-bromophenyl)-2-iminothiazolidin-4-one (4a)
ESI-MS 465 [M+H]+. 1H NMR (300 MHz, CDCl3): δ 10.44 (s, 1H), 8.35 (d, J = 7.5 Hz, 2H),
7.72–7.29 (m, 5H), 7.11–6.88 (m, 7H), 5.20 (s, 2H); 13C NMR (75 MHz, CDCl3) δ 176.2,

ACCEPTED MANUSCRIPT
163.9, 156.2, 144.3, 137.6, 136.6, 133.9(2C), 131.4, 130.5(2C), 128.4(2C), 126.4(2C), 126.0,
125.8(2C), 123.3(2C), 121.4, 117.7, 72.9. Anal calcd for C23H17BrN2O2S: C, 59.36; H, 3.68;
N, 6.02 % Found C, 59.42; H, 3.77; N, 6.12 %.
4.2.5. 5-(4-(Benzyloxy)benzylidene)-3-(4-chlorophenyl)-2-iminothiazolidin-4-one (4b)
ESI-MS 421 [M+H]+. 1H NMR (300 MHz, CDCl3): δ 10.71 (s, 1H), 8.12 (d, J = 7.8 Hz, 2H),
13
7.90 (s, 1H), 7.63–7.45 (m, 9H), 7.27 (d, J = 7.8 Hz, 2H), 5.22 (s, 2H); C NMR (75 MHz,
CDCl3) δ 177.3, 164.5, 156.6, 145.4, 138.4, 135.9, 134.0(2C), 132.2(2C), 130.8(2C),
129.6(2C), 128.9(2C), 127.2, 126.7(2C), 124.8, 122.4, 118.6, 73.2. Anal calcd for
C23H17ClN2O2S: C, 65.63; H, 4.07; N, 6.66 % Found C, 65.72; H, 3.97; N, 6.79 %.
4.2.6. 5-(4-(Benzyloxy)benzylidene)-3-(4-fluorophenyl)-2-iminothiazolidin-4-one (4c)
1
ESI-MS 405 [M+H]+. H NMR (300 MHz, DMSO-d6): δ 8.44 (d, J = 8.1 Hz, 2H), 7.82 (s,
1H), 7.72 (s, 1H), 7.68–7.49 (m, 4H), 7.38–7.21 (m, 5H), 7.09 (d, J = 7.8 Hz, 2H), 5.24 (s,
2H); 13C NMR (75 MHz, DMSO-d6) δ 179.1, 165.4, 158.3, 146.3, 139.4, 136.8, 135.2(2C),
133.2(2C), 130.3(2C), 130.3(2C), 129.0(2C), 128.2, 127.8(2C), 125.3, 123.6, 119.7, 74.2.
Anal calcd for C23H17FN2O2S: C, 68.30; H, 4.24; N, 6.93% Found C, 68.42; H, 4.37; N, 7.09
%.
4.2.7. 5-(4-(Benzyloxy)benzylidene)-2-imino-3-(2-nitrophenyl)thiazolidin-4-one (4d)
MS(ESI) m/z 432 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 12.63 (s, 1H), 8.04 (d, J = 2.4
Hz, 1H), 7.78–7.65 (m, 2H), 7.51–7.25 (m, 9H), 7.15–7.09 (m, 2H), 5.16 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) δ 167.2, 159.8, 152.8, 142.0, 141.9, 136.5, 134.5, 131.7(2C), 130.6,
128.5(2C), 128.0, 127.8(2C), 125.7, 125.3, 124.9, 123.5, 119.1, 115.6(2C), 69.4. Anal calcd
for C23H17N3O4S: C, 64.03; H, 3.97; N, 9.74 % Found C, 64.12; H, 3.99; N, 9.89 %.
4.2.8. 5-(4-(Benzyloxy)benzylidene)-2-imino-3-(3-nitrophenyl)thiazolidin-4-one (4e)
ESI-MS 432 [M+H]+. 1H NMR (300 MHz, DMSO-d6): δ 9.99 (s, 1H), 8.91 (s, 1H), 8.29 (d, J
= 7.2 Hz, 1H), 8.01–7.89 (m, 3H), 7.81–7.64 (m, 4H), 7.60–7.24 (m, 3H), 7.11 (d, J = 7.8 Hz,

ACCEPTED MANUSCRIPT
2H), 5.24 (s, 2H); 13C NMR (75 MHz, DMSO-d6) δ 176.3, 164.8, 154.6, 147.3, 145.2, 142.8,
138.2, 137.1(2C), 135.6(2C), 133.3(2C), 133.0, 132.1, 129.6, 129.2, 127.2, 125.4, 125.0(2C),
118.8, 73.1. Anal calcd for C23H17N3O4S: C, 64.03; H, 3.97; N, 9.74 % Found C, 64.16; H,
4.05; N, 9.85 %.
4.2.9. 5-(4-(Benzyloxy)benzylidene)-2-imino-3-(4-nitrophenyl)thiazolidin-4-one (4f)
1
ESI-MS 432 [M+H]+. H NMR (300 MHz, DMSO-d6): δ 10.20 (s, 1H), 8.43 (d, J = 7.5 Hz,
2H), 7.88 (d, J = 7.2 Hz, 2H), 7.74–7.54 (m, 6H), 7.51–7.39 (m, 4H), 5.21 (s, 2H); 13C NMR
(75 MHz, DMSO-d6) δ 177.4, 162.9, 156.6, 148.4, 146.2, 141.5, 139.0, 138.2(2C),
134.6(2C), 133.9(2C), 133.3, 132.1(2C), 130.5, 129.7(2C), 127.4, 125.4(2C), 119.1, 73.1.
Anal calcd for C23H17N3O4S: C, 64.03; H, 3.97; N, 9.74 % Found C, 64.16; H, 4.05; N, 9.85
%.
4.2.10. 5-(4-(Benzyloxy)benzylidene)-3-(2,4-dichlorophenyl)-2-iminothiazolidin-4-one (4g)
ESI-MS 455 [M+H]+. 1H NMR (300 MHz, CDCl3): δ 9.49 (s, 1H), 8.13 (s, 1H), 7.92 (d, J =
7.2 Hz, 1H), 7.76 (s, 1H), 7.67–7.56 (m, 4H), 7.49–7.37 (m, 6H), 5.29 (s, 2H); 13C NMR (75
MHz, CDCl3) δ 172.3, 162.3, 155.6, 142.4, 138.4, 137.2, 136.4, 135.2(2C), 133.9(2C), 132.6,
133.3, 132.4(2C), 130.9, 128.4(2C), 126.6, 125.2, 123.4, 118.2, 74.9. Anal calcd for
C23H16Cl2N2O2S: C, 60.67; H, 3.54; N, 6.15 % Found C, 60.76; H, 3.65; N, 6.25 %.
4.2.11.
5-(4-(Benzyloxy)benzylidene)-3-(3-chloro-2-methylphenyl)-2-iminothiazolidin-4-
one (4h)
1
ESI-MS 435 [M+H]+. H NMR (300 MHz, CDCl3): δ 10.45 (s, 1H), 7.92 (s, 1H), 7.80–7.56
(m, 6H), 7.47–7.11 (m, 6H), 5.23 (s, 2H), 2.25 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 173.4,
163.5, 154.7, 141.3, 139.2, 138.1, 137.2, 136.0(2C), 134.2(2C), 133.5, 134.2, 130.2(2C),
129.4, 127.2(2C), 125.3, 124.6, 121.5, 117.9, 74.3, 16.2. Anal calcd for C24H19ClN2O2S: C,
66.28; H, 4.40; N, 6.44 % Found C, 66.36; H, 4.45; N, 6.54 %.
4.2.12. 5-(4-(Benzyloxy)benzylidene)-2-imino-3-(pyrimidin-2-yl)thiazolidin-4-one (4i)

ACCEPTED MANUSCRIPT
1
ESI-MS 389 [M+H]+. H NMR (300 MHz, DMSO-d6): δ 9.45 (s, 1H), 8.41 (d, J = 8.4 Hz,
2H), 7.88 (s, 1H), 7.78–7.54 (m, 4H), 7.39 (d, J = 8.1 Hz, 2H), 7.30–7.09 (m, 4H), 5.19 (s,
2H); 13C NMR (75 MHz, CDCl3) δ 174.6, 166.5, 158.2, 142.2(2C), 136.3, 133.0, 129.9(2C),
128.2(2C), 127.5, 126.2, 124.8(2C), 123.6, 123.0(2C), 121.6, 118.4, 73.6. Anal calcd for
C21H16N4O2S: C, 64.93; H, 4.15; N, 14.42 % Found C, 64.96; H, 4.25; N, 14.51 %.
4.2.13. 5-(4-(Benzyloxy)benzylidene)-2-imino-3-(pyridin-2-yl)thiazolidin-4-one (4j)
1
ESI-MS 388 [M+H]+. H NMR (300 MHz, DMSO-d6): δ 9.22 (s, 1H), 8.02 (d, J = 8.1 Hz,
1H), 7.84 (s, 1H), 7.72–7.63 (m, 4H), 7.54 (d, J = 8.1 Hz, 2H), 7.44–7.34 (m, 3H), 7.18–6.88
(m, 3H), 5.18 (s, 2H); 13C NMR (75 MHz, DMSO-d6) δ 171.6, 164.6, 160.2, 144.9, 137.4,
134.6, 133.9(2C), 133.0, 129.0(2C), 128.3, 127.4, 126.6, 125.4(2C), 124.3, 123.4, 121.0(2C),
117.7, 74.2. Anal calcd for C22H17N3O2S: C, 68.20; H, 4.42; N, 10.85 % Found C, 68.26; H,
4.54; N, 10.91 %.
4.2.14.
(4k)
5-(4-(Benzyloxy)benzylidene)-2-imino-3-(6-methylpyridin-2-yl)thiazolidin-4-one
ESI-MS 402 [M+H]+. 1H NMR (300 MHz, DMSO-d6): δ 8.94 (s, 1H), 7.81 (s, 1H), 7.72 (d, J
= 8.1 Hz, 2H), 7.53–7.39 (m, 6H), 7.24–7.02 (m, 4H), 5.21 (s, 2H), 2.48 (s, 3H); 13C NMR
(75 MHz, DMSO-d6) δ 166.4, 163.6, 157.2, 153.5, 146.4, 139.5, 137.4, 134.4(2C), 133.2,
129.4(2C), 127.4, 126.0, 124.2(2C), 123.2, 121.4, 120.6(2C), 118.8, 73.6, 22.5. Anal calcd
for C23H19N3O2S: C, 68.81; H, 4.77; N, 10.47 % Found C, 68.96; H, 4.94; N, 10.51 %.
4.2.15. 5-(4-(Benzyloxy)benzylidene)-2-imino-3-phenylthiazolidin-4-one (4l)
1
ESI-MS 387 [M+H]+. H NMR (300 MHz, CDCl3): δ 8.88 (s, 1H), 7.78 (s, 1H), 7.63–7.51
(m, 4H), 7.36–7.20 (m, 5H), 7.11–6.93 (s, 5H), 5.18 (s, 2H); 13C NMR (75 MHz, CDCl3) δ
171.3, 162.9, 154.2, 143.3, 136.6, 134.3(2C), 133.6(2C), 132.4, 130.4(2C), 129.1(2C),
127.6(2C), 126.1, 125.3(2C), 124.1, 123.4, 117.9, 71.8. Anal calcd for C23H18N2O2S: C,
71.48; H, 4.69; N, 7.25 % Found C, 71.56; H, 4.74; N, 7.31 %.

ACCEPTED MANUSCRIPT
4.2.16. 5-(4-(Benzyloxy)benzylidene)-3-(2,4-dimethylphenyl)-2-iminothiazolidin-4-one (4m)
1
ESI-MS 415 [M+H]+. H NMR (300 MHz, CDCl3): δ 7.81 (s, 1H), 7.72–7.62 (m, 4H), 7.58
(s, 1H), 7.42–7.18 (m, 5H), 7.02–6.90 (m, 3H), 5.24 (s, 2H), 2.42 (s, 3H), 2.33 (s, 3H); 13C
NMR (75 MHz, CDCl3) δ 169.9, 161.6, 153.6, 144.7, 137.5, 135.1(2C), 134.2(2C), 133.2,
130.9(2C), 129.3, 128.4, 126.4(2C), 126.1, 125.4, 124.7, 124.1, 123.6, 119.7, 73.3, 25.1,
23.4. Anal calcd for C25H22N2O2S: C, 72.44; H, 5.35; N, 6.76 % Found C, 72.56; H, 5.44; N,
6.81 %.
4.2.17. 5-(4-(Benzyloxy)benzylidene)-3-(2,5-dimethylphenyl)-2-iminothiazolidin-4-one (4n)
1
ESI-MS 415 [M+H]+. H NMR (300 MHz, CDCl3): δ 9.45 (s, 1H), 7.78 (s, 1H), 7.73–7.65
(m, 4H), 7.46–7.21 (m, 4H), 7.06–6.93 (m, 4H), 5.22 (s, 2H), 2.40 (s, 3H), 2.29 (s, 3H); 13C
NMR (75 MHz, CDCl3) δ 172.9, 162.5, 154.6, 146.3, 138.6, 136.3(2C), 135.6(2C), 134.4,
132.3(2C), 130.6, 129.6, 127.6(2C), 125.6, 124.6, 124.0, 123.5, 121.4, 120.1, 74.2, 23.2,
18.9. Anal calcd for C25H22N2O2S: C, 72.44; H, 5.35; N, 6.76 % Found C, 72.49; H, 5.40; N,
6.88 %.
4.2.18. 5-(4-(Benzyloxy)benzylidene)-3-(2,6-dimethylphenyl)-2-iminothiazolidin-4-one (4o)
1
ESI-MS 415 [M+H]+. H NMR (300 MHz, CDCl3): δ 8.91 (s, 1H), 7.81 (s, 1H), 7.69–7.54
(m, 5H), 7.38–7.20 (m, 4H), 7.09–6.91 (m, 3H), 5.17 (s, 2H), 2.46 (s, 3H), 2.38 (s, 3H); 13C
NMR (75 MHz, CDCl3) δ 171.7, 163.7, 156.4, 147.6, 139.4, 137.4(2C), 136.3(2C),
134.4(2C), 133.3(2C), 132.4, 130.5(2C), 127.4(2C), 126.3, 125.2, 124.4, 123.4, 118.1, 73.6,
19.8. Anal calcd for C25H22N2O2S: C, 72.44; H, 5.35; N, 6.76 % Found C, 72.60; H, 5.46; N,
6.84 %.
4.2.19. Preparation of tert-butyl 2-amino-3-carbamoyl-4,5-dihydrothieno[2,3-c]pyridine-
6(7H)-carboxylate (6)
To the stirred solution of Compound 5 (3.0 g, 15.07 mmol), 2-cyanoacetamide (1.51 g, 18.09
mmol), sulphur powder (1.06 g, 15.07 mmol) in Ethanol (40 mL) was added morpholine

ACCEPTED MANUSCRIPT
(3.21 mL, 33.15 mmol) and stirred the reaction mixture at room temperature for 9 h. the
reaction mixture was concentrated, diluted with EtOAc and washed the organic layer with
H2O (3 × 40 mL). The separated organic layer was dried over anhydrous Na2SO4, evaporated
and purified by column chromatography to get Compound 6 (3.67 g, 82%) as a light yellow
solid. ESI-MS found 298 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 11.25 (s, 2H), 7.90 (s,
2H), 4.32 (s, 2H), 3.51 (t, J = 6.8 Hz, 2H), 3.04 (t, J = 6.8 Hz, 2H).
4.2.20. General procedure for the preparation of compounds 8 a-c
4-Phenoxybenzoic acid (2.0 g, 9.34 mmol) was taken in a 100 mL single neck RB flask
equipped with N2-inlet, to this was added THF (30 mL), Et3N (2.69 mL, 18.68 mmol) and
Ethylchloroformate (0.93 mL, 9.80 mmol) at 0 °C and reaction mixture was allowed to stir at
room temperature for 4 h. The reaction mixture was concentrated, diluted with ethyl acetate
and washed the organic phase with H2O (3 × 40 mL). The separated organic layer was dried
over anhydrous Na2SO4, evaporated to get solid compound. The solids were washed with
hexanes to get compound 8b (2.60 g, 97%) as an Off-white solid. Similar procedure was
followed for the synthesis of 8a and 8c starting with 4-chloro benzoic acid and 4-benzyloxy
benzoic acid respectively. ESI-MS showed desired mass and carried to next step.
4.2.21. Synthesis of compound 9a
To the stirred solution of compound 6 (3.0 g, 10.10 mmol) in CH2Cl2 at 0 °C under N2 atm,
was added 4-chlorobenzoylchloride (1.42 mL, 11.11 mmol) followed Et3N (2.91 mL, 20.20
mmol) and allowed to stir at room temperature for 7 h. The reaction mixture was
concentrated and dissolved in EtOAc, washed with sat. NaHCO3 (2 × 40 mL) and H2O (2 ×
60 mL). The separated organic layer was concentrated under vaccum and purified by flash
chromatography to get compound 9a (3.6 g, 82%) as light yellow solid.
4.2.22. General procedure for the preparation of compounds 9 a-c

ACCEPTED MANUSCRIPT
Compound 6 (1.00 equiv) in THF was taken in a microwave vial and added NaH (1.20
equiv), respective mixed anhydride (8 a-c) and subjected to microwave irradiation [Biotage
initiator, temperature: 120 °C] for 20 min. The reaction mixture was quenched with sat.
NH4Cl and extracted into EtOAc. The organic layer was evaporated and the obtained solid
product was triturated with CH2Cl2/Hexanes to get pure product.
4.2.23. Synthesis of compounds 10 (a-c)
To the stirred solution of compound 9 (a-c) in CH2Cl2 at 0 °C under N2 atm, was added TFA
(2 volumes) and allowed to stir at room temperature for 2 h. The reaction mixture was
concentrated to dryness and the obtained solids were washed with hexanes to afford
compounds 10 (a-c) respectively.
4.2.24. General procedure for the synthesis of compounds 11-15, 16-20 and 20-25
To the stirred solution of 2-(4-chlorobenzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (10a) in DMF at 0 °C under N2 atm was added Et3N (1.5 equiv) followed by
benzoyl chloride/acetyl chloride (1.2 equiv) and stirred the reaction mixture at room
temperature for 3 h. The reaction mixture was added ice and obtained solids were filtered and
washed with H2O, dried and purified by column chromatography using EtOAc/Hexanes as
eluent to get compounds 11/12 respectively.
To the stirred solution of 2-(4-chlorobenzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (10a) in MeOH at 0 °C under N2 atm was added formaldehyde (for compound
13)/Benzaldehyde (for compound 15) (2.0 equiv) followed by NaCNBH3 (1.2 equiv) and
stirred the reaction mixture at room temperature for 3 h. The reaction mixture was quenched
with ice, extracted with EtOAc. The organic layer was dried over anhy Na2SO4 and
evaporated to get crude compound. Purified by column chromatography using
EtOAc/Hexanes as eluent. Similar procedure was followed with 10b and 10c to get final
molecules.

ACCEPTED MANUSCRIPT
4.2.25.
6-Benzoyl-2-(4-chlorobenzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (11)
1
Yield: 78%; MS(ESI) m/z 440 [M+H]+. H NMR (300 MHz, DMSO-d6): δ 10.42 (s, 1H),
9.63 (s, 2H), 8.01 (d, J = 7.2 Hz, 2H), 7.81 (d, J = 7.5 Hz, 2H), 7.72–7.63 (m, 5H), 4.72 (s,
2H), 3.93 (t, J = 7.8 Hz, 2H), 3.01 (t, J = 7.8 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) δ
178.6, 169.8, 166.2, 161.9, 144.3, 140.5, 138.2, 137.6(2C), 136.9(2C), 135.5(2C), 133.4,
132.8(2C), 126.4, 125.9, 117.7, 47.9, 43.8, 19.6. Anal calcd for: C22H18ClN3O3S: C, 60.07;
H, 4.12; N, 9.55 % Found C, 60.10; H, 4.27; N, 9.64%.
4.2.26.
6-Acetyl-2-(4-chlorobenzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (12)
Yield: 79%; MS(ESI) m/z 378 [M+H]+. 1H NMR (300 MHz, DMSO-d6): δ 9.42 (s, 1H), 8.64
(s, 2H), 7.92 (d, J = 8.1 Hz, 2H), 7.74 (d, J = 7.8 Hz, 2H), 4.84 (s, 2H), 3.90 (t, J = 7.8 Hz,
2H), 3.09 (t, J = 7.8 Hz, 2H), 2.25 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 179.3, 170.1,
166.9, 162.6, 141.3, 137.5, 133.6(2C), 131.4(2C), 130.4, 126.0, 118.1, 46.4, 42.7, 22.5, 20.1.
Anal calcd for: C17H16ClN3O3S: C, 54.04; H, 4.27; N, 11.12 % Found C, 54.10; H, 4.34; N,
11.24%.
4.2.27.
2-(4-Chlorobenzamido)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (13)
1
Yield: 81%; MS(ESI) m/z 350 [M+H]+. H NMR (300 MHz, DMSO-d6): δ 10.12 (s, 1H),
7.89 (d, J = 8.1 Hz, 2H), 7.76 (d, J = 7.8 Hz, 2H), 7.47 (s, 2H), 4.60 (s, 2H), 3.78 (t, J = 7.8
Hz, 2H), 2.97 (t, J = 7.8 Hz, 2H), 2.61 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 176.3,
165.4, 163.7, 140.3, 133.2, 131.9(2C), 129.6(2C), 124.5, 121.5, 117.9, 49.4, 44.8, 42.3, 18.0.
Anal calcd for: C16H16ClN3O2S: C, 54.93; H, 4.61; N, 12.01 % Found C, 55.01; H, 4.74; N,
12.16%.

ACCEPTED MANUSCRIPT
4.2.28. tert-Butyl 3-carbamoyl-2-(4-chlorobenzamido)-4,5-dihydrothieno[2,3-c]pyridine-
6(7H)-carboxylate (14)
1
Yield: 84%; MS(ESI) m/z 436 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 12.95 (s, 1H),
7.89 (d, J = 9.6 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H), 4.50 (s, 2H), 3.58 (t, J = 6.0 Hz, 2H), 2.85
(t, J = 6.4 Hz, 2H), 1.43 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ 167.4, 161.5, 153.8,
143.8, 137.4, 130.9, 129.2(2C), 129.0, 128.9(2C), 128.4, 116.0, 79.3, 40.6, 40.0, 28.1(3C),
25.2. Anal calcd for: C20H22ClN3O4S: C, 55.10; H, 5.09; N, 9.64 % Found C, 55.16; H, 5.13;
N, 9.74%.
4.2.29.
6-Benzyl-2-(4-chlorobenzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (15)
1
Yield: 72%; MS(ESI) m/z 426 [M+H]+. H NMR (300 MHz, DMSO-d6): δ 10.64 (s, 1H),
9.27 (s, 2H), 7.94 (d, J = 8.1 Hz, 2H), 7.72 (d, J = 7.8 Hz, 2H), 7.54–7.46 (m, 5H), 3.72 (s,
2H), 3. 69 (s, 2H), 3.33 (t, J = 7.8 Hz, 2H), 2.91 (t, J = 7.8 Hz, 2H); 13C NMR (75 MHz,
DMSO-d6) δ 180.6, 166.8, 162.2, 142.3, 141.5, 136.4, 133.2(2C), 132.9(2C), 130.5(2C),
130.0, 128.4, 127.8(2C), 125.4, 117.7, 63.9, 49.9, 46.4, 20.7. Anal calcd for: C22H20ClN3O2S:
C, 62.04; H, 4.73; N, 9.87 % Found C, 62.10; H, 4.87; N, 9.94%.
4.2.30.
6-Benzoyl-2-(4-phenoxybenzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (16)
1
Yield: 76%; MS(ESI) m/z 498 [M+H]+. H NMR (300 MHz, DMSO-d6): δ 10.33 (s, 2H),
8.12–7.88 (m, 4H), 7.72–7.36 (m, 6H), 7.31–7.11 (m, 5H), 4.63 (s, 2H), 3.96 (t, J = 7.8 Hz,
2H), 3.12 (t, J = 7.8 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) δ 181.6, 172.8, 169.2, 166.4,
162.5, 153.3, 137.5, 136.2, 135.3(2C), 133.9(2C), 132.4, 132.0, 130.5(2C), 128.6, 127.2(2C),
126.0(2C), 124.2(2C), 121.5, 118.2, 46.9, 43.4, 20.7. Anal calcd for: C28H23N3O4S: C, 67.59;
H, 4.66; N, 8.45 % Found C, 67.70; H, 4.72; N, 8.53%.

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4.2.31.
6-Acetyl-2-(4-phenoxybenzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (17)
Yield: 81%; MS(ESI) m/z 436 [M+H]+. 1H NMR (300 MHz, DMSO-d6): δ 9.66 (s, 1H), 8.08
(d, J = 8.1 Hz, 2H), 7.92–7.45 (m, 6H), 7.39–7.26 (m, 3H), 4.60 (s, 2H), 3.90 (t, J = 7.8 Hz,
2H), 3.15 (t, J = 7.8 Hz, 2H), 2.27 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 180.7, 172.6,
168.3, 166.4, 160.5, 158.4, 136.8(2C), 135.2(2C), 134.8, 130.0(2C), 129.0, 126.9, 124.4(2C),
121.5, 117.0, 47.9, 44.4, 22.5, 20.0. Anal calcd for: C23H21N3O4S: C, 63.43; H, 4.86; N, 9.65
% Found C, 63.49; H, 4.92; N, 9.73%.
4.2.32.
6-Methyl-2-(4-phenoxybenzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (18)
Yield: 84 %; MS(ESI) m/z 408 [M+H]+. 1H NMR (300 MHz, CDCl3): δ 9.43 (s, 1H), 8.12 (d,
J = 8.1 Hz, 2H), 7.72–7.63 (m, 3H), 7.44–7.26 (m, 6H), 4.12 (s, 2H), 3.60 (t, J = 7.8 Hz, 2H),
3.04 (t, J = 7.8 Hz, 2H), 2.32 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 180.9, 174.3, 166.3,
161.4, 159.3, 137.6(2C), 136.4, 135.8(2C), 132.0(2C), 128.6, 127.6, 126.0, 123.3(2C), 116.1,
52.9, 48.4, 42.3, 19.2. Anal calcd for: C22H21N3O3S: C, 64.85; H, 5.19; N, 10.31 % Found C,
64.89; H, 5.22; N, 10.43%.
4.1.33. tert-Butyl 3-carbamoyl-2-(4-phenoxybenzamido)-4,5-dihydrothieno[2,3-c]pyridine-
6(7H)-carboxylate (19)
Yield: 79 %; MS (ESI) m/z 494 [M+H]+. 1H NMR (300 MHz, DMSO-d6): δ 8.01 (d, J = 8.4
Hz, 2H), 7.84 (s, 1H), 7.63–7.44 (m, 6H), 7.39–7.27 (m, 3H), 4.47 (s, 2H), 3.81 (t, J = 8.1
Hz, 2H), 3.21 (t, J = 8.1 Hz, 2H), 1.42 (s, 9H); 13C NMR (75 MHz, DMSO-d6) δ 180.7,
167.4, 164.4, 162.6, 158.7, 156.3, 134.6(2C), 133.3(2C), 130.4, 129.4, 127.2(2C), 126.0,
124.2(2C), 123.4, 117.9, 79.9, 49.3, 46.6, 27.9(3C), 18.7. Anal calcd for: C26H27N3O5S: C,
63.27; H, 5.51; N, 8.51 % Found C, 63.36; H, 5.63; N, 8.74%.

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4.1.34.
6-Benzyl-2-(4-phenoxybenzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (20)
Yield: 79 %; MS(ESI) m/z 484 [M+H]+. 1H NMR (300 MHz, DMSO-d6): δ 9.97 (s, 1H), 8.08
(d, J = 7.8 Hz, 2H), 7.78–7.63 (m, 6H), 7.54–7.39 (m, 4H), 7.33–7.24 (m, 4H), 3.79 (s, 2H),
13
3.66 (s, 2H), 3.19 (t, J = 7.8 Hz, 2H), 3.01 (t, J = 7.8 Hz, 2H); C NMR (75 MHz, DMSO-
d6) δ 179.4, 169.8, 164.6, 162.4, 160.2, 139.3, 138.4(2C), 136.4, 134.4(2C), 133.0,
132.7(2C), 130.3(2C), 129.0(2C), 127.6, 126.4(2C), 123.3, 125.4, 116.9, 64.8, 48.6, 46.6,
18.7. Anal calcd for: C28H25N3O3S: C, 69.54; H, 5.21; N, 8.69 % Found C, 69.60; H, 5.27; N,
8.84%.
4.1.35. 6-Benzoyl-2-(4-(benzyloxy)benzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (21)
Yield: 79%; MS(ESI) m/z 512 [M+H]+. 1H NMR (300 MHz, DMSO-d6): δ 9.34 (s, 1H), 8.01
(d, J = 7.8 Hz, 2H), 7.92–7.84 (m, 6H), 7.77–7.44 (m, 4H), 7.33–7.06 (m, 4H), 5.22 (s, 2H),
13
4.68 (s, 2H), 3.96 (t, J = 8.1 Hz, 2H), 3.13 (t, J = 7.8 Hz, 2H); C NMR (75 MHz, DMSO-
d6) δ 180.7, 171.9, 168.4, 164.7, 160.9, 141.3, 139.6, 137.5(2C), 135.4, 134.5, 133.9(2C),
131.3(2C), 129.9, 128.2, 127.6(2C), 126.0, 125.1(2C), 124.5, 124.0, 117.3, 72.9, 48.4, 44.8,
19.6. Anal calcd for: C29H25N3O4S: C, 68.08; H, 4.93; N, 8.21 % Found C, 68.20; H, 4.98; N,
8.33%.
4.2.36.
6-Acetyl-2-(4-(benzyloxy)benzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (22)
1
Yield: 81%; MS(ESI) m/z 450 [M+H]+. H NMR (300 MHz, DMSO-d6): δ 10.06 (s, 1H),
7.98 (d, J = 8.1 Hz, 2H), 7.81–7.64 (m, 7H), 7.29 (d, J = 8.1 Hz, 2H), 5.24 (s, 2H), 4.63 (s,
2H), 3.91 (t, J = 7.8 Hz, 2H), 3.18 (t, J = 8.1 Hz, 2H), 2.34 (s, 3H); 13C NMR (75 MHz,
DMSO-d6) δ 181.8, 171.6, 169.2, 165.3, 160.3, 138.4, 134.6(2C), 133.6(2C), 132.6,

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130.5(2C), 128.6, 126.7, 123.4(2C), 123.0, 116.9, 71.8, 48.4, 46.6, 21.6, 19.8. Anal calcd for:
C24H23N3O4S: C, 64.13; H, 5.16; N, 9.35 % Found C, 64.22; H, 5.22; N, 9.43%.
4.2.37.
2-(4-(Benzyloxy)benzamido)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (23)
1
Yield: 84 %; MS(ESI) m/z 422 [M+H]+. H NMR (300 MHz, CDCl3): δ 10.42 (s, 1H), 7.99
(d, J = 8.1 Hz, 2H), 7.92 (s, 2H), 7.60–7.42 (m, 5H), 7.27 (d, J = 8.1 Hz, 2H), 5.23 (s, 2H),
4.09 (s, 2H), 3.43 (t, J = 8.1 Hz, 2H), 3.03 (t, J = 8.4 Hz, 2H), 2.36 (s, 3H); 13C NMR (75
MHz, CDCl3) δ 181.4, 172.3, 169.3, 160.4, 142.5, 139.3, 137.6(2C), 136.2(2C), 135.4(2C),
134.0, 130.5(2C), 128.1, 127.3, 118.2, 74.0, 54.9, 49.5, 43.3, 18.0. Anal calcd for:
C23H23N3O3S: C, 65.54; H, 5.50; N, 9.97 % Found C, 65.69; H, 5.62; N, 10.03%.
4.2.38.
tert-Butyl
2-(4-(benzyloxy)benzamido)-3-carbamoyl-4,5-dihydrothieno[2,3-
c]pyridine-6(7H)-carboxylate (24)
1
Yield: 84 %; MS (ESI) m/z 508 [M+H]+. H NMR (300 MHz, DMSO-d6): δ 9.45 (s, 2H),
9.22 (s, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.1 Hz, 2H), 7.58–7.49 (m, 3H), 7.33 (d, J
= 8.7 Hz, 2H), 5.26 (s, 2H), 4.46 (s, 2H), 3.91 (t, J = 8.4 Hz, 2H), 3.18 (t, J = 8.1 Hz, 2H),
1.44 (s, 9H); 13C NMR (75 MHz, DMSO-d6) δ 182.7, 170.1, 166.6, 164.3, 156.6, 141.3,
136.2(2C), 135.6(2C), 133.9, 129.5(2C), 127.0, 126.4, 124.4(2C), 123.4, 118.8, 83.6, 74.0,
48.3, 45.4, 29.1(3C), 19.2. Anal calcd for: C27H29N3O5S: C, 63.89; H, 5.76; N, 8.28 % Found
C, 63.96; H, 5.93; N, 8.44%.
4.2.39.
6-Benzyl-2-(4-(benzyloxy)benzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (25)
Yield: 74 %; MS(ESI) m/z 498 [M+H]+. 1H NMR (300 MHz, DMSO-d6): δ 9.54 (s, 1H), 7.94
(d, J = 7.8 Hz, 2H), 7.81–7.74 (m, 4H), 7.63–7.42 (m, 6H), 7.36–7.22 (m, 4H), 5.21 (s, 2H),
3.76 (s, 2H), 3.42 (s, 2H), 3.06 (t, J = 8.1 Hz, 2H), 2.99 (t, J = 8.1 Hz, 2H); 13C NMR (75
MHz, DMSO-d6) δ 180.6, 168.3, 162.6, 161.1, 138.6, 136.2, 134.7(2C), 132.4(2C), 133.2,

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130.7(2C), 129.2, 128.2, 127.6(2C), 127.0(2C), 126.4(2C), 124.3, 123.4, 117.4, 72.9, 63.9,
51.6, 48.3, 19.2. Anal calcd for: C29H27N3O3S: C, 70.00; H, 5.47; N, 8.44 % Found C, 70.11;
H, 5.52; N, 8.55%.
4.3. Biological activity
4.3.1. Enzyme kinetics of MTB L-AlaDH
MTB L-AlaDH enzyme assay was performed in 100ꢀl reaction in 96 well microtitre plate
containing 125mM Glycine-KOH buffer (pH-10.2), 1mM NAD+, 50mM of L-Alanine as
substrate and various concentrations of MTB L-AlaDH for 20min at 37°C. The MTB L-
AlaDH enzyme activity was continuously monitored and the end product NADH was
observed at 340nm. Concentration of enzyme was later determined based on the range
finding experiments. In order to determine the kinetic parameters Km and Vmax, the substrate
L-Alanine was varied from 1mM to 200mM, similarly NAD+ concentrations were also varied
from 0.1mM to 1mM and Km and Vmax were determined using non-linear regression analysis
from GraphPad prism software.
4.3.2. In vitro MTB l-AlaDH enzyme inhibition assay
The enzyme inhibition studies were performed in microtitre plate containing desired substrate
and enzyme concentration as explained above. The spectrophotometric determination of the
reaction product NADH that accompanies the conversion of L-alanine into pyruvate in the
oxidative deamination was measured at 340nm in heat controlled microplate reader
PerkinElmer Victor X3 instrument. The synthesized compounds were added to the plate with
different concentrations from 25µM to 0.5µM in order to determine IC50 values for the all the
synthesized compounds. The reaction mixture except MTB L-AlaDH was added and
background reactions were measured. Reactions were carried out at 37°C in a heat-controlled
Perkinelmer Victor X3 Spectrophotometer. Further the IC50 values were calculated using
GraphPad Prism analysis software.

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4.3.3. In vitro dormant MTB model
Culture of Mycobacterium tuberculosis H37RV were grown in Middlebrook 7H9 medium
supplemented with OADC (nutrient rich medium) was pelleted and washed twice with PBS
(Phosphate Buffer Saline, HiMedia Laboratories). The pellet was resuspended in PBS in
sealed bottles and is incubated at 37°C for 6 weeks. Six week starved cultures were treated
with standard drugs like Isoniazide, Rifampicin and Moxifloxacin along with synthesized
drugs for 7 days at a concentration of 10ug/ml. The treated cell suspensions were diluted 10-
fold up to 10-6 using Middlebrook 7H9 medium supplemented with OADC and 100 µl of
each dilutions were plated in 48 well plates in triplicates along with 900 µl of Midllebrook
7H9 medium (HiMedia Laboratories) supplemented with OADC (HiMedia Laboratories).
The microplates were incubated at 37°C for 28 days without agitation. Wells with visible
bacterial growth were counted as positive, and MPN values were calculated using standard
statistical methods.
4.3.4. In vitro active MTB model
Two-fold serial dilutions of each test compound/drug were prepared and incorporated into
Middlebrook 7H11 agar medium with oleic acid, albumin, dextrose, and catalase (OADC)
growth supplement to get final concentrations of 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78
µg/mL. Inoculum of MTB H37Rv ATCC 27294/XDR-TB was prepared from fresh
Middlebrook 7H11 agar slants with OADC (Difco) growth supplement adjusted to 1 mg/mL
(wet weight) in Tween 80 (0.05%) saline diluted to 10-2 to give a concentration of ~107
cfu/mL. Five microliters of this bacterial suspension was spotted onto 7H11 agar tubes
containing different concentrations of the drug as discussed above. The tubes were incubated
at 37 °C, and final readings (as MIC in mg/mL) were determined after 28 days. This method

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is similar to that recommended by the National Committee for Clinical Laboratory Standards
for the determination of MIC in duplicate.
4.3.5. In vitro cytotoxicity screening
The synthesized compounds were further examined for its cytotoxicity in mouse macrophage
cell line (RAW 264.7) at 50µM concentration. After 48 h of exposure, viability was assessed
on the basis of cellular conversion of MTT into a formazan product using the Promega Cell
Titer 96 non-radioactive cell proliferation assay [12]. Mouse macrophages were grown in
RPMI medium supplemented with 10% fetal bovine serum (FBS), 10,000 units’ penicillin
and 10 mg streptomycin per ml in T25 flasks to attain 80-90% confluency. Cells were
scraped and seeded into wells approx 5,000 cells per well in poly-L-lysine coated plates. The
microtiter plates were incubated at 37o C, 5% CO2, 95% air and 100% relative humidity for
24h prior to addition of experimental drugs. The test compounds at 50µM concentration were
then added to cells and incubated at 37°C for 48 hrs; later 10 ꢀL of 0.5mg/ml concentration
of MTT was added and incubated for 3 h at 37oC and the final product formazon crystals
were measured at 595nm.
4.4. Differential scanning fluorimetry
A thermal shift assay, also called Differential Scanning Fluorimetry (DSF) is a thermal-
denaturation assay that measures the thermal stability of a target protein and a subsequent
increase in protein melting temperature upon binding of a ligand to the protein [13]. We used
Sypro Orange dye (Sigma-aldrich) in a Real time PCR instrument (Bio-Rad iCycle5), which
binds to exposed core residues of a denatured protein and results in an increased fluorescence
signal. The instrument was programmed to equilibrate the samples at 25°C for 3min and
further rise in temperature till 95°C, with every 0.1°C rice using a LED/Photodiode set
matched with dye excitation and emission wavelengths. Required concentration of MTB L-