Design and development of new class of Mycobacterium tuberculosis L-alanine dehydrogenase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.07.051

Mycobacterium tuberculosis L-alanine dehydrogenase (MTB L-AlaDH) is one of the important drug targets for treating latent/persistent tuberculosis. In this study we used crystal structure of the MTB L-AlaDH bound with cofactor NAD⁺as a structura

Full text of DOI:10.1016/j.bmc.2016.07.051

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and development of new class of Mycobacterium tuberculosis
L-alanine dehydrogenase inhibitors
Rudraraju Srilakshmi Reshma, Shalini Saxena, Karyakulam Andrews Bobesh, Variam Ullas Jeankumar,
⇑
Saritha Gunda, Perumal Yogeeswari, Dharmarajan Sriram
Drug Discovery Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, Hyderabad 500078, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Mycobacterium tuberculosis
for treating latent/persistent tuberculosis. In this study we used crystal structure of the MTB
L
-alanine dehydrogenase (MTB
L
-AlaDH) is one of the important drug targets
-AlaDH
bound with cofactor NAD+ as a structural framework for virtual screening of our in-house database to
identified new classes of -AlaDH inhibitor. We identified azetidine-2,4-dicarboxamide derivative as
one of the potent inhibitor with IC50 of 9.22 0.72 M. Further lead optimization by synthesis leads to
compound 1-(isonicotinamido)-N2,N4-bis(benzo[d]thiazol-2-yl)azetidine-2,4-dicarboxamide (18) with
-AlaDH IC50 of 3.83 0.12 M, 2.0log reduction in nutrient starved dormant MTB model and MIC of
11.81 M in actively replicative MTB.
Received 25 June 2016
Revised 22 July 2016
Accepted 22 July 2016
Available online xxxx
L
L
l
Keywords:
Tuberculosis
Mycobacterium tuberculosis
Alanine dehydrogenase
Azetidine-2,4-dicarboxamide
L
l
l
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
It is generally assumed that the microenvironment inside the
granulomas is characterized by hypoxia, nutrient starvation, and
reactive oxygen and nitrogen species.5 Comparison of gene expres-
sion profiles and proteome analyses of active versus non-replicat-
ing bacteria have identified a number of up-regulated genes in
persistent MTB.6 One of these genes, Rv2780 was found to be over-
Tuberculosis (TB) is a major cause of death around the world,
and is caused by Mycobacterium tuberculosis (MTB), an acid-fast
bacillus that is transmitted primarily via the respiratory route.
Infection with MTB is believed to occur in an alveolar macrophage
initially. MTB replicate within the macrophage and induces cytoki-
nes that initiate the inflammatory response in the lungs. Macro-
phages and lymphocytes migrate to the site of infection and form
a granuloma.1 The function of the granuloma is to segregate the
infection to prevent spread to the remainder of the lung and to
other organs, as well as to concentrate the immune response
directly at the site of infection. The granuloma is maintained in a
persistently infected host, probably due to chronic stimulation of
the immune cells, and forms the basis for a TB lesion. Live bacilli
have been isolated from granulomas or tubercles in the lungs of
persons with clinically inactive TB, indicating that the MTB can
persist in a granulomatous lesion for many years.2 About one-third
of the world’s population has dormant TB, which means people
have been infected by MTB but are not (yet) ill with the disease
and cannot transmit the disease.3 Dormant MTB is insensitive to
isoniazid, but some inhibition is induced by rifampin, moxi-
floxacin, amikacin, capreomycin, and metronidazole;4 thus there
is an urgent need for a new anti-TB drug to cure dormant TB.
expressed under nutrient starvation7 regimes that encode
nine dehydrogenase ( -AlaDH). Increased levels of this enzyme
L-ala-
L
has been linked to the generation of alanine for peptidoglycan
biosynthesis6 and the maintenance of the NAD+ pool under condi-
tions when the terminal electron acceptor oxygen become limit-
ing.8 NAD(H)-dependent
deamination of
tion) or, in the reverse direction, the reductive amination of pyru-
vate to -alanine (biosynthetic reaction). recent analysis
L-AlaDH catalyze the oxidative
L-alanine to pyruvate and ammonia (catabolic reac-
L
A
involving microarray and other data had identified this enzyme
among the top three drug targets, especially against persistence.9
Earlier we have reported structure based design of inhibitors
(Fig. 1) using crystal structure of MTB L-AlaDH N6-methyl adeno-
sine (PDB: 4 LMP)10 and with NADH (PDB: 2VHW).11 To further
work identifying new class of inhibitors we used enzyme bound
NAD+ (Protein Data Bank (PDB: 2VOJ); http://www.rcsb.org) struc-
ture for identification and development of new ligands with high
affinities of binding toward the protein. One of the most potent
lead compound was derivatized by synthesis and carried out
biological evaluation.
⇑
Corresponding author.
E-mail address: dsriram@hyderabad.bits-pilani.ac.in (D. Sriram).
http://dx.doi.org/10.1016/j.bmc.2016.07.051
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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2
R. S. Reshma et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
ON
based on the spectrophotometric determination in a 96-well plate
using Perkin Elmer Victor V3 spectrophotometer.
N
O
S
O2N
Photometric determination of L-AlaDH activity was accom-
S
O
N
plished by measuring the rate of production of NADH that accom-
panied the conversion of alanine into pyruvate in the oxidative
deamination. The five hit candidates exhibited IC50 values in the
HN
S
H2N
S
O
HN
range of 9.21–23.26
lM and the IC50 of most active compound
O
(Lead 1) was found to be 9.21
l
M (Fig. 3). This compound was
N
the ones that showed high docking score and number of H-bond
interaction (Table 1) which thus correlated with our in vitro enzy-
matic inhibition studies.
O
NH
OCH3
IC50: 6.77 µM
IC50: 8.17 µM
To understand its binding pattern Lead 1 was analyzed in more
detail (Fig. 4). In Lead 1 phenyl rings is attached via amide bond to
azetidine ring, where it showed five hydrogen bonding interactions
with the relevant amino acid residues. The NH group on the phenyl
ring of the right-hand core was involved in a hydrogen-bonding
interaction with Asp198 similar to that of the crystal ligand, while
the another NH group on the phenyl ring of the left hand side inter-
act with Ala238 amino acid residue. In addition, the NH group on
azetidine ring and O atom formed another hydrogen bonding inter-
action with Leu240. Also, the O atom in the carbonyl group
attached to pyridyl ring was found to be involved in hydrogen
bonding interaction with Lys203 analogous to one observed in ref-
erence molecule also. Apart from hydrogen bonding interactions
Lead 1 was also involved in various hydrophobic interactions with
Pro242, Val241, Ala176, Leu130, Ile267, Ala179, Ile199 and Ile174
amino acid residues which resulted in one of the highest affinities
for the enzyme (docking score À7.80 kcal/mol), as a result this
compound is showing more potency as compare to other lead
OH
OH
O
NH
OH
HO
HO
OH
OH
HN
O
HO
IC50: 35.54 µM
Figure 1. Earlier reported MTB
L
AlaDH inhibitors.
2. Results and discussion
2.1. Designing of the molecules
In the present study, the crystal structure of the MTB L-AlaDH
molecules (IC50 = 9.21 lM; Fig. 3).
bound with cofactor NAD+ was used as a structural framework
for virtual screening of our (BITS Pilani) in-house database to
2.2. Chemistry
explore new classes of L-AlaDH inhibitor. The basic goal of the vir-
tual screening is to decrease the enormous virtual chemical space
of small organic molecules to a manageable number of compounds
that could inhibit the protein with the highest chance to lead to a
drug candidate. The virtual screening options for high-throughput
virtual screening (HTVS), standard precision (SP), and extra preci-
sion (XP) docking were executed. The Glide XP module of Schrodin-
ger 9.2 was used finally for docking because it combined accurate,
well-defined physics-based scoring terms and thorough sampling
of the terms.
The identified Lead 1was taken as a starting point and subse-
quently a library of 12 analogues (Table 2) were designed and
taken for synthesis as the steps towards the derivation of struc-
ture–activity relationship (SAR) and lead optimization. A series of
12 molecules were synthesized using the protocol described in
Scheme 1.
Glutaric anhydride (1) on treatment with bromine followed by
acidic workup gave 2,4-dibromopentanedioic acid (2). The di acid
(2) was then converted to their corresponding tert-butyl ester (3)
by utilizing isobutylene gas in presence of catalytic amount of sul-
furic acid. The acid labile tert-butyl ester was introduced to avoid
the possible racemisation which may occur if a base a catalyzed
hydrolysis was employed in the later stage of the synthesis. The
obtained di-tert-butyl 2,4-dibromopentanedioate (3) was then
converted into the respective azetidine analogues (4 and 5) by
heating with their respective acid hydrazide in DMF, which on acid
hydrolysis followed by coupling of the so obtained di-carboxylic
acid (6 and 7)with desired amines gave the titled compounds 8–
19 in very good yield and purity. An overview of the various mod-
ification/substituent attempted in synthesis has been outlined in
Table 2. The analytical and spectral data (1H NMR, 13C NMR, and
mass spectra) of all the synthesized compounds were in full agree-
ment with the proposed structures.
An initial validation of the active site pocket was performed by
re-docking the crystal ligand NAD+ with the active site residues of
the L-AlaDH protein. The ligand exhibited a highest Glide score of
À11.42 kcal molÀ1 and was found in the vicinity of important
amino acids like Asp270, Val298, Thr178, Ser220, Lys203,
Asp198, Met301, Ile267, Ser134, Leu240 and Ala179. Re-docking
results showed that the compound exhibited similar interactions
to those in the original crystal structure, which was further con-
firmed with a root mean square deviation (RMSD) of 0.897 Å
(Fig. 2).
Later, our in-house database collection, which consists of a little
more than 3000 compounds, was subjected to a high-throughput
virtual screening. The ligands that displayed a docking score of
P7.00 kcal/mol were regarded as potential hits and were further
flexibly docked together with the crystal ligand to evaluate pre-
cisely the hydrogen-bond interactions, electrostatic interactions,
hydrophobic enclosure, and cation–pi stacking interactions. A final
short list of possible lead compounds was based on visual inspec-
tion of the important amino acid residues involved in binding.
Finally, a small subset of 5 compounds was shortlisted from the
Glide XP docking study (Fig. 3).
2.3. MTB
analysis
L-AlaDH enzyme inhibition studies and docking
The synthesized compounds were assayed for the inhibition of
MTB -AlaDH at concentrations ranging from 25 M to 0.5 M.
All the synthesized compounds (except 14 and 19) showed activity
with IC50 ranging from 3.83 0.12 M to 14.63 0.32 M (Table 2).
L
l
l
The top hits were screened initially at 50
lM concentration and
l
l
later at 25, 10, 5 and 1 M against MTB- -AlaDH using an assay
l
L
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R. S. Reshma et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Figure 2. (a) XP docking showing interactions of NAD+ with the active site residues of MTB -AlaDH protein. (b) Superimposition of docked pose of the NAD+ to the original
L
pose of the NAD+.
Figure 3. 2D representation of top hits obtained from virtual screening.
With respect to the structure–activity relationship study,
modifications are done are as follows:
Table 1
The docking score, no. of hydrogen bond and important interactions of best fit ligands
Compound
name
Docking
score
No. of
H-bond
Important interactions
(1) Increasing chain length between amide group and phenyl
ring (Lead 1, 8 and 9).
Lead 1
Lead 2
Lead 3
Lead 4
Lead 5
À7.80
À7.15
À7.00
À7.34
À7.56
5
4
4
4
4
Asp198, Lys203, 2Leu240, Ala238
Asp198, Ser220, Leu240, Ser134
Asp198, Thr178, Ser220, Leu240
Asp198, Ala238, Ser220, Leu240
Asp198, Ala238, Ser220, Leu240
(2) Introducing electron withdrawing and donating groups on
phenyl ring (10–15).
(3) Replacement of phenyl ring with hetero-aromatic groups
(16, 17 and 18).
Our SAR study started with exploring the various chain lengths
between amide group and phenyl ring by substituting with ethyl
and methyl (8 and 9) linker. Here by increasing chain length by
Four compounds were found to be more potent than the initially
identified Lead 1 (IC50 of 9.22 0.72 M). Further to support the
l
linking with ethyl group (IC50 = 4.75
compare to methyl linker (IC50 = 10.24
l
M) showed good activity as
M). Binding analysis of
activity, we performed docking analyses of all the synthesized
compounds together with lead compound using extra precision
mode (XP) of Glide module; as molecular docking study is a
well-established technique to determine the interaction of two
molecules and find the best orientation of ligand would form a
complex with overall minimum energy.
l
both the compounds revealed that both the compounds were
bounded well with the proteins and showed good binding energy
toward the target protein ranging from À8.82 to À8.15 kcal/mol.
It was observed that the most active compound 9 was predicted
to be most active as compare to compound 8 in silico too due to
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4
R. S. Reshma et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Ala238, Leu240 and Thr178 amino acid residues along with various
hydrophobic interactions with the protein. The binding energy of
these set of compounds were found in the range of À8.90 to
À7.71 kcal/mol (Fig. 6).
Further the effect of various electron withdrawing and donating
groups on phenyl ring was investigated (10–15). Here compound
substituted with 2,4 dimethyl and 4-chloro derivatives (10 and
14) showed good activity with IC50 of 4.38 and 5.56
tively, while substitution with 4-bromo derivative (13) led to dras-
tic reduction in the activity (IC50 = >25 M). The other substitution
lM respec-
l
with 2,6-dimethyl (11), 2,4-dimethoxy (12) and 3,4-dichloro (16)
derivatives showed moderate activity. Both the active compounds
(10 and14) from this set exhibited good docking score (À8.83 and
À8.50 kcal/mol) with significant polar and non-polar contacts,
hydrogen bond interactions with the relevant amino acids and var-
ious hydrophobic interactions (Fig. 7).
However it was surprising to note that replacement of 4-bromo
derivative (13) led to a drastic reduction in the activity as indicated
in their interaction profile where the molecule was found to be in a
slightly different orientation/pose thereby losing the hydrogen
bonding interaction with as presented in Figure 8.
Another modification was attempted by replacing pyridyl ring
with phenyl ring (19), the binding analysis of compound 19
demonstrated that it was involved in two hydrogen bonding inter-
action with Ser134; while the binding analysis of compound 18
showed that it was involved in five hydrogen bonding interaction
with three with Ser134, another one is with Thr178 and Ala238
amino acid residues. Compound 19 failed to show hydrogen bond-
ing interactions with Thr178 and Ala238 amino acids, which
explained its in-activity and also the orientation of the molecule
was different. Ligand binding analysis also showed hydrophobic
interactions between the phenyl ring and some hydrophobic
amino acid residues (Fig. 9). This difference in the activity profile
was also supported by docking score (À4.15 kcal/mol).
Figure 4. The interaction profile picture of Lead 1 at the active pocket of MTB
L-AlaDH.
little difference in the binding energy (Fig. 5) which is due to
hydrophobic and hydrogen bond interactions with amino acids of
targeted protein.
Next substitution was made by replacing the phenyl ring with
hetero aromatic groups such as 2-pyridyl, 3-methyl-2-pyridyl
and benzothiazole groups (16, 17and 18). In this set of compounds
benzothiazole substituted group (18) showed best activity with
IC50 of 3.83
IC50 = 9.47 and 14.63
the active site of MTB
tional cation–pi interactions with Lys203 of compound 18 showed
good activity as compare to 16 and 17. Though; compounds 16 and
17 showed four hydrogen bonding interactions with the Lys203,
l
M among the other two compounds (16 and 17;
M). In silico studies of these compounds in
-AlaDH protein revealed that due to addi-
2.4. In vitro MTB screening
l
Betts et al. earlier established an in vitro MTB dormant model,7
in which nutrient starvation caused MTB to arrest growth,
minimized aerobic metabolism and became resistant to existing
anti-TB drugs while maintaining viability. Nutrient starvation
L
Table 2
Structures of the synthesized compounds and its biological activities
HN
O
R
O
N
NH
R
NH
O
8-19
X
Compound
X
R
MTB MIC (
l
M)
Cytotoxicity at 25 lM (% Inhib.)
L
-AlaDH IC50 (lM)
Lead 1
8
9
N
N
N
N
N
N
N
N
N
N
N
N
CH
Phenyl
Benzyl
Phenethyl
9.22 0.72
10.24 0.63
4.75 0.31
4.38 0.28
9.69 0.54
11.65 0.86
>25
5.56 0.25
13.65 0.78
9.47 0.91
14.63 0.32
3.83 0.12
>25
15.06
28.22
26.54
13.27
3.31
46.73
2.72
51.65
1.41
3.74
3.51
11.81
5.93
19.81
49.34
42.26
19.29
26.61
48.87
19.69
32.92
49.45
7.68
10
11
12
13
14
15
16
17
18
19
2,4-Dimethyl phenyl
2,6-Dimethyl phenyl
2,4-Dimethoxy phenyl
4-Bromo phenyl
4-Chloro phenyl
3,4-Dichloro phenyl
2-Pyridyl
3-Methyl-2-pyridyl
2-Benzothiazolyl
2-Benzothiazolyl
3.89
34.73
12.33
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R. S. Reshma et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
CH3
CH3
CH3
O
O
CH3
CH3
O
O
O
O
O
a
b
c
CH3
O
O
OH
OH
Br
Br
Br
Br
3
1
2
CH3
CH3
CH3
O
O
O
O
OH
O
NH R
O
O
O
CH3
CH3
CH3
N
NH
N
NH
N
NH
OH
O
NH
O
R
d
O
e
4-5
X
6-7
8-19
X
X
Scheme 1. Synthetic protocol of target compounds.12 Reagents and conditions: (a) (i) bromine, 100 °C; (ii) formic acid, 100 °; (b) isobutylene (g), H2SO4, DCM, 30 °C; (c)
benzohydrazide or isonicotinic hydrazide, DMF, 80 °C; (d) DCM, TFA, 30 °C; (e) R-NH2, EDCÁHCl, HOBT, TEA, DCM, 30 °C.
Figure 5. The interaction profile pictures of compound 8 and 9 at the active pocket of MTB L-AlaDH.
Figure 6. The interaction profile pictures of compounds 16, 17 and 18 at the active pocket of MTB L-AlaDH.
may therefore mimic some of the features of MTB during the per-
sistent 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 synthesized AlaDH inhibitors. In this
model, cultures of in vitro grown MTB bacteria were subjected to
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6
R. S. Reshma et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 7. The interaction profile pictures of compounds 10 and 14 at the active pocket of MTB L-AlaDH.
Figure 9. The interaction profile picture of compound 19 at the active pocket of
MTB -AlaDH.
L
Figure 8. The interaction profile picture of compound 13 at the active pocket of
MTB L-AlaDH.
activity with ꢀ2.8log bacterial reduction. When compared to iso-
niazid, ten synthesized compounds and Lead 1 showed better
activity with ꢀ1.6 to 2.0log bacterial reduction. Two compounds
(13 and 19) does not showed any significant activity which were
also inactive in MTB AlaDH enzyme screening and these results
supports that these persistent bacteria were killed through AlaDH
enzyme. The compound which showed good MTB AlaDH activity
showed maximum activity with ꢀ2.0log bacterial reduction.
As most of these molecules (Lead 1, 8–18)) contains isoniazid
fragment, we also tested the synthesised compounds against
in vitro anti-TB activity against replicative MTBH37Rv
(ATCC27294) using MABA assay method14 with drug concentra-
nutrient depletion by growing the culture in phosphate buffer
saline (PBS) for 6 weeks. Nutrient starvation using this method
triggered a dormancy response in the bacilli that was termed
non-replicating persistence (NRP), a physiological state thought
to mimic the one exhibited by MTB during various stages of
persistent infection. After 6 weeks, the culture was treated with
the synthesized compounds and standard drugs at a concentration
of 10 lg/ml in a tube and incubated at 37 °C for 7 days. The treated
cell suspensions were diluted 10-fold up to 10À6 using Middle-
brook 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 stan-
dard statistical methods.13
tions from 25 lg/mL to 0.2 lg/mL. The minimum inhibitory con-
centration (MIC) was determined for each compound which was
measured as the minimum concentration of compound required
to completely inhibit the bacterial growth (Table 2). Isoniazid
At 10 lg/ml concentration (Fig. 10), standard first line anti-TB
drugs isoniazid and rifampicin reduced ꢀ1.5 and 2.2log bacterial
(MIC of 0.72
and moxifloxacin (1.24
l
M), ethambutol (7.64
lM), rifampicin (0.15 lM)
reduction respectively whereas moxifloxacin showed highest
lM) were used as reference compounds
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R. S. Reshma et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4. Experimental section
7
7
6
5
4
3
2
1
0
4.1. Chemistry
All commercially available chemicals and solvents were used
without further purification. TLC experiments were performed on
alumina-backed silica gel 40 F254 plates (Merck, Darmstadt, Ger-
many). The homogeneity of the compounds was monitored by thin
layer chromatography (TLC) on silica gel 40 F254 coated on alu-
minum plates, visualized by UV light and KMnO4 treatment. All
1H and 13C NMR spectra were recorded on
a Bruker AM
(400 MHz, 100 MHz) NMR spectrometer, Bruker BioSpin Corp, Ger-
many. Molecular weights of the synthesized compounds were
checked by LCMS 6100B series Agilent Technology with electro-
spray ionization (ESI+). Chemical shifts are reported in ppm (d)
with reference to the internal standard TMS. The signals are desig-
nated as follows: s, singlet; d, doublet; dd, doublet of doublets; t,
triplet; m, multiplet. Elemental analyses were carried out using
CHN Analyzer (Vario MICRO cube in CHN mode).
l
I
8
9
5
8
1
F
r
H
1
10 11 12 13 14
1
16 17
19
RI
Ct IN
ad
MOX
Le
compounds
Figure 10. Biological activities of the synthesised compounds against nutrient
starved MTB model. Bacterial count estimation (Mean SD, n = 3) for control and
treated groups conducted by using MPN (most probable number) assay. The
statistical significance (p <0.0001) with respect to control has been analyzed by
Two-way ANOVA using GraphPad Prism Software.
4.1.1. Synthesis of 2,4-dibromopentanedioic acid (2)
Bromine (4.49 ml, 87.6 mmol) was added drop wise to glutaric
anhydride (5.0 g, 43.8 mmol) in a three-necked flask fitted with
reflux condenser and CaCl2 guard tube at 30 °C. After the addition
of bromine, the reaction mixture was heated to 90 °C for 1 h and
then at 100 °C for another 1 h. The reaction mixture was cooled
to 30 °C and formic acid (4 ml) was added slowly. The content of
the flask was heated to 100 °C for 1 h. The reaction mixture was
quenched with water and extracted with ethyl acetate
(4 Â 100 ml). The combined organic layer was dried over sodium
sulfate and evaporated to give white gum. The trituration of the
white gum with diethyl ether give 2,4-dibromopentanedioic acid
as white solid (2.7 g, 20.5%). 1H NMR (DMSO-d6): dH 11.89 (s,
2H), 4.36 (t, J = 7.2 Hz,2H), 2.54 (t, J = 7 Hz, 2H). 13C NMR (DMSO-
d6): dC 174.6 (2C), 40.4 (2C), 39.3. ESI-MS m/z 288.99 (MÀH)+. Anal
Calcd for C5H6Br2O4; C, 20.71; H, 2.09; O, 22.08; Found: C, 20.77; H,
2.08; O, 22.03.
for comparison. The MIC values of the synthesized compounds
were presented in Table 2. All the synthesized compounds (Lead
1 and 8–19) showed activity against replicative MTB with MIC
ranging from 1.41 to 51.65
and 17) inhibited MTB with MIC of <5
ethambutol (MIC of 7.64 M). All the compounds were less potent
lM. Five compounds (11, 13, 15, 16
lM and more potent than
l
than isoniazid, rifampicin and moxifloxacin. Surprisingly the com-
pound 19 which does not contain isoniazid fragment also showed
appreciable activity with MIC of 5.93 lM indicates that these
molecules may act through different target/mechanism than isoni-
azid in replicative MTB.
2.5. In vitro cytotoxicity studies
The safety profile of all the synthesized compounds was evalu-
ated by testing there in vitro cytotoxicity in mouse macrophage
cell line (RAW 264.7) cells at 25 lM concentration using (4,5-
4.1.2. Synthesis of di-tert-butyl 2,4-dibromopentanedioate (3)
To
a
solution of 2,4-dibromopentanedioic acid (1.0 g,
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay.15 Since MTB reside inside the macrophage, the monocyte
macrophage cell lines (RAW 264.7) were mostly used for tubercu-
losis research; in order to check whether the screened compounds
were not toxic towards macrophages but toxic to the bacteria.
Compounds inhibited within a range of 3.89–49.45%. Furthermore,
the most active compound 18 showed 34.73% inhibitions at 25 lM
concentration, reflecting its safety profile in the eukaryotes, cyto-
toxic results of all the compounds are illustrated in the Table 2.
3.33 mmol) in dry dichloromethane (10 ml), isobutylene gas and
2–3 drops of concentrated sulfuric acid was added. The reaction
mixture was stirred in an autoclave at 30 °C for 12 h. The reaction
mixture was washed with water, saturated aq NaHCO3 solution,
brine and dried over Na2SO4. After evaporation of the solvent, the
crude product was purified by column chromatography to give
di-tert-butyl 2,4-dibromopentanedioate as colorless oil (0.069 g,
50%).1H NMR (DMSO-d6): dH 4.38 (t, J = 7.2 Hz, 2H), 2.56 (t,
J = 7 Hz, 2H), 1.39 (s, 18H). 13C NMR (DMSO-d6): dC 166.4 (2C),
78.1 (2C), 40.7, 37.9 (2C), 27.5 (6C). ESI-MS m/z 402.19 (M). Anal
Calcd for C13H22Br2O4; C, 38.83; H, 5.51; O, 15.92; Found: C,
38.90; H, 5.52; N, 15.90.
3. Conclusion
The main objective of the present study was to identify the new
class of inhibitors for the MTB-
crystal structure. We identified novel azetidine-2,4-dicarboxamide
derivative as hit with IC50 of 9.22 M. Hit expansion by five step
L-AlaDH enzyme by using enzyme
4.1.3. General procedure for synthesis of azetidine-2,4-dicarbo
xylate derivatives
l
To
a solution of di-tert-butyl 2,4-dibromopentanedioate
synthetic protocol provided twelve analogues and we found that
compound 18 (1-(isonicotinamido)-N2,N4-bis(benzo[d]thiazol-2-
yl)azetidine-2,4-dicarboxamide) as the most potent compound
(1 equiv) in dry DMF (10 ml), hydrazide derivative (3 equiv) was
added and heated to 80 °C for 2 h. The reaction mixture was con-
centrated to remove DMF and the residue was dissolved in dichlor-
omethane. The organic layer was washed with saturated aq
NaHCO3 solution, water, brine, dried over Na2SO4 and concen-
trated. The crude product was purified by column chromatography
to afford respective azetidine-2,4-dicarboxylate as mentioned
below.
against MTB
L
-AlaDH IC50 of 3.83 0.12
lM, 2.0log reduction in
nutrient starved dormant MTB model and MIC of 11.81
l
M in
actively replicative MTB. Owing to the activity against active and
persistent phases of mycobacterium compound can be further
developed as newer antibacterial agent.
Please cite this article in press as: Reshma, R. S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.051

8
R. S. Reshma et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4.1.4. Synthesis of di-tert-butyl 1-(isonicotinamido)azetidine-2,
4-dicarboxylate (4)
(6 equiv) were added at 0 °C and stirred at rt for 16 h. The reaction
mixture was quenched with water and extracted with dichloro-
methane. The combined organic layers were washed with brine,
dried over sodium sulfate and concentrated. The crude products
were purified by column chromatography to afford respective
products.
The compound was synthesized according to above general pro-
cedure using di-tert-butyl 2,4-dibromopentanedioate (0.50 g,
1.24 mmol) and isoniazid (0.51 g, 3.73 mmol) to afford di-tert-
butyl 1-(isonicotinamido)azetidine-2,4-dicarboxylate (0.2 g, 47%)
as pale yellow thick oil. 1H NMR (DMSO-d6): dH 9.01 (s, 1H),
8.86–7.84 (m, 4H), 4.08 (t, J = 7.2 Hz, 2H), 3.10–2.93 (m, 2H), 1.38
(s, 18H). 13C NMR (DMSO-d6): dC 169.4 (2C), 164.2, 147.8 (2C),
139.8, 118.5 (2C), 80.8 (2C), 70.4 (2C), 27.4 (6C), 17.2. ESI-MS m/z
378.39 (M+H)+. Anal Calcd for C19H27N3O5; C, 60.46; H, 7.21; N,
11.13; Found: C, 60.31; H, 7.22; N, 11.11.
4.1.10. 1-(Isonicotinamido)-N2,N4-diphenylazetidine-2,4-dicarb
oxamide (Lead 1)
The compound was synthesized according to above general pro-
cedure using 1-(isonicotinamido)azetidine-2,4-dicarboxylic acid
(0.1 g, 0.38 mmol), aniline (0.08 g, 0.84 mmol), EDCÁHCl (0.22 g,
1.14 mmol), HOBt (0.18 g, 1.14 mmol) and triethylamine (0.23 g,
2.28 mmol) to afford 1-(isonicotinamido)-N2,N4-diphenylaze-
tidine-2,4-dicarboxamide (0.064 g, 40.89%). 1H NMR (DMSO-d6):
dH, 9.12 (s, 1H), 8.94 (d, J = 8.0 Hz, 2H), 8.56 (s, 2H), 7.84 (d,
J = 7.8 Hz, 2H), 7.53–7.15 (m, 10H), 4.04 (t, J = 7.0 Hz, 2H), 3.05–
2.89 (m, 2H).13C NMR (DMSO-d6): dC 170.2 (2C), 165.4, 148.3
(2C), 139.7, 137.6 (2C), 129.1 (4C), 128.3 (2C), 122.2 (2C), 122.0
(4C), 68.1 (2C), 17.5. ESI-MS m/z 416.17 (M+H)+. Anal Calcd for
4.1.5. Synthesis of di-tert-butyl 1-benzamidoazetidine-2,4-dicar
boxylate (5)
The compound was synthesized according to above general
procedure
(0.5 g, 1.24 mmol) and benzohydrazide (0.51 g, 3.73 mmol) to
afford di-tert-butyl 1-benzamidoazetidine-2,4-dicarboxylate
using
di-tert-butyl
2,4-dibromopentanedioate
(0.34 g, 72.34%) as pale yellow thick oil. 1H NMR (DMSO-d6): dH
8.97 (s, 1H), 7.98–7.56 (m, 5H), 4.09 (t, J = 7.5 Hz, 2H), 3.12–2.95
(m, 2H), 1.39 (s, 18H). 13C NMR (DMSO-d6): dC 173.5 (2C), 167.3,
132.7, 132.5, 130.2 (2C), 128.4 (2C), 80.8 (2C), 70.5 (2C), 27.3
(6C), 17.2. ESI-MS m/z 377.29 (M+H)+. Anal Calcd for C20H28N2O5;
C, 63.81; H, 7.50; N, 7.44; Found: C, 63.92; H, 7.49; N, 7.45.
C23H21N5O3; C, 66.49; H, 5.09; N, 16.86; Found: C, 66.41; H, 5.10;
N, 16.88.
4.1.11. 1-(Isonicotinamido)-N2,N4-dibenzylazetidine-2,4-dicarb
oxamide (8)
The compound was synthesized according to above general pro-
cedure using 1-(isonicotinamido)azetidine-2,4-dicarboxylic acid
(0.10 g, 0.38 mmol), benzylamine (0.09 g, 0.84 mmol), EDCÁHCl
(0.22 g, 1.14 mmol), HOBt (0.18 g, 1.14 mmol) and triethylamine
(0.23 g, 2.28 mmol) to afford 1-(isonicotinamido)-N2,N4-dibenzy-
lazetidine-2,4-dicarboxamide (0.059 g, 35.28%). 1H NMR (DMSO-
d6): dH 9.09 (s, 1H), 8.91 (d, J = 7.8 Hz, 2H), 8.73 (s, 2H), 7.86 (d,
J = 7.6 Hz, 2H), 7.32–7.18 (m, 10H), 5.12 (s, 4H), 4.15 (t, J = 7.5 Hz,
2H), 3.14–2.92 (m, 2H). 13C NMR (DMSO-d6): dC 169.4 (2C),
167.2, 148.7 (2C), 139.2, 136.4 (2C), 126.3 (4C), 124.3 (4C),123.9
(2C), 120.3 (2C), 70.4 (2C), 43.6 (2C), 17.4. ESI-MS m/z 444.20 (M
+H)+. Anal Calcd for C25H25N5O3; C, 67.70; H, 5.68; N, 15.79; Found:
C, 67.64; H, 5.67; N, 15.77.
4.1.6. General procedure for synthesis of azetidine-2,4-dicarbo
xylic acid derivatives
The corresponding ester (1 equiv) was dissolved in dry dichlor-
omethane (5 ml) and trifluoro acetic acid (3 equiv) was added. The
reaction mixture was stirred at 30 °C for 48 h and concentrated
under high vacuum. The crude product was purified by column
chromatography to afford the respective azetidine-2,4-dicar-
boxylic acid as mentioned below.
4.1.7. Synthesisof1-(isonicotinamido)azetidine-2,4-dicarboxylic
acid(6)
The compound was synthesized according to above general pro-
cedure using di-tert-butyl 1-(isonicotinamido)azetidine-2,4-dicar-
boxylate (0.25 g, 0.53 mmol) and trifluoro acetic acid (0.12 ml,
1.59 mmol) to afford 1-(isonicotinamido)azetidine-2,4-dicar-
boxylic acid (0.08 g, 57.14%). 1H NMR (DMSO-d6): dH 12.32 (s,
2H), 9.05 (s, 1H), 8.87–7.84 (m, 4H), 4.07 (t, J = 7.2 Hz, 2H),
3.08–2.92 (m, 2H). 13C NMR (DMSO-d6): dC 174.9 (2C), 165.2,
150.8 (2C), 141.2, 119.0 (2C), 71.3 (2C), 17.1. ESI-MS m/z 264.12
(MÀH)+. Anal Calcd for C11H11N3O5; C, 49.81; H, 4.18; N, 15.84;
Found: C, 49.72; H, 4.19; N, 15.86.
4.1.12. 1-(Isonicotinamido)-N2,N4-diphenethylazetidine-2,4-
dicarboxamide (9)
The compound was synthesized according to above general pro-
cedure using 1-(isonicotinamido)azetidine-2,4-dicarboxylic acid
(0.10 g, 0.38 mmol), phenethylamine (0.10 g, 0.84 mmol), EDCÁHCl
(0.22 g, 1.14 mmol), HOBt (0.18 g, 1.14 mmol) and triethylamine
(0.23 g, 2.28 mmol) to afford 1-(isonicotinamido)-N2,N4-
diphenethylazetidine-2,4-dicarboxamide (0.044 g, 24.75%). 1H
NMR (DMSO-d6): dH 9.13 (s, 1H), 8.94 (d, J = 8.0 Hz, 2H), 8.75 (s,
2H), 7.83 (d, J = 7.8 Hz, 2H), 7.36–7.22 (m, 10H), 4.18 (t, J = 7.4 Hz,
2H), 3.16 (t, J = 8.0 Hz, 4H), 3.07–2.90 (m, 2H), 2.78 (t, J = 8.2 Hz,
4H). 13C NMR (DMSO-d6): dC 170.6 (2C), 165.8, 147.5 (2C), 140.6,
138.2 (2C), 127.1 (4C), 125.7 (4C), 123.3 (2C), 121.3 (2C), 70.1 (2C),
39.8 (2C), 34.2 (2C), 17.6. ESI-MS m/z 472.23 (M+H)+. Anal Calcd
for C27H29N5O3; C, 68.77; H, 6.20; N, 14.85; Found: C, 68.69; H,
6.21; N, 14.87.
4.1.8. Synthesis of 1-benzamidoazetidine-2,4-dicarboxylic acid
(7)
The compound was synthesized according to above general pro-
cedure using di-tert-butyl 1-benzamidoazetidine-2,4-dicarboxy-
late (0.2 g, 0.53 mmol) and trifluoro acetic acid (0.12 ml,
1.59 mmol) to afford 1-benzamido azetidine-2,4-dicarboxylic acid
(0.07 g, 50%). 1H NMR (DMSO-d6): dH 2.93–3.10 (m, 2H), 4.08
(t, J = 7.4 Hz, 2H), 7.57–8.00 (m, 5H), 9.01 (s, 1H), 11.93 (s, 2H).
13C NMR (DMSO-d6): dC 175.5 (2C), 167.3, 130.8, 130.5, 127.6
(2C), 126.8 (2C), 71.2 (2C), 17.2. ESI-MS m/z 263.09 (MÀH)+. Anal
Calcd for C12H12N2O5; C, 54.55; H, 4.58; N, 10.60; Found: C,
54.48; H, 4.57; N, 10.58.
4.1.13. 1-(Isonicotinamido)-N2,N4-bis(2,4-dimethylphenyl)
azetidine-2,4-dicarboxamide (10)
The compound was synthesized according to above general
procedure using 1-(isonicotinamido)azetidine-2,4-dicarboxylic acid
(0.10 g, 0.38 mmol), 2,4-dimethylphenylamine (0.10 g, 0.84 mmol),
EDCÁHCl (0.22 g, 1.14 mmol), HOBt (0.18 g, 1.14 mmol) and triethy-
lamine (0.23 g, 2.28 mmol) to afford 1-(isonicotinamido)-N2,N4-bis
(2,4-dimethylphenyl)azetidine-2,4-dicarboxamide (0.052 g, 29.25%).
1H NMR (DMSO-d6): dH 9.02 (s, 1H), 8.92 (d, J = 7.6 Hz, 2H),
8.62 (s, 2H), 7.87 (d, J = 8.0 Hz, 2H), 7.18–6.97 (m, 6H), 4.21
4.1.9. General procedure for synthesis of amide derivatives
To a solution of 1-isonicotinamidoazetidine-2,4-dicarboxylic
acid (1 equiv) in dry dichloromethane (10 ml), various amines
(2.2 equiv) was added at 0 °C and stirred under nitrogen atmo-
sphere. EDCÁHCl (3 equiv), HOBt (3 equiv) and triethylamine
Please cite this article in press as: Reshma, R. S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.051

R. S. Reshma et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
(t, J = 7.8 Hz, 2H), 3.04–2.89 (m, 2H), 2.29 (s, 6H), 2.07 (s, 6H). 13C
NMR (DMSO-d6): dC 169.3 (2C), 164.3, 148.6 (2C), 142.6 (2C),
139.8, 136.4 (2C), 131.0 (2C), 129.3 (2C), 124.7 (2C), 119.6 (2C),
115.4 (2C), 69.7 (2C), 20.9 (2C), 17.1, 15.1 (2C). ESI-MS m/z 472.23
(M+H)+. Anal Calcd for C27H29N5O3; C, 68.77; H, 6.20; N, 14.85;
Found: C, 68.86; H, 6.21; N, 14.83.
2H), 3.02–2.87 (m, 2H).13C NMR (DMSO-d6): dC 170.6 (2C), 165.3,
148.4 (2C), 142.1, 138.7 (2C), 132.1 (2C), 130.5 (4C), 119.8 (2C),
118.4 (4C), 69.5 (2C), 17.2. ESI-MS m/z 485.08 (M+H)+. Anal Calcd
for C23H19Cl2N5O3; C, 57.04; H, 3.95; N, 14.46; Found: C, 56.93;
H, 3.96; N, 14.44.
4.1.18. 1-(Isonicotinamido)-N2,N4-bis(3,4-dichlorophenyl)azetid
ine-2,4-dicarboxamide (15)
4.1.14. 1-(Isonicotinamido)-N2,N4-bis(2,6-dimethylphenyl)azeti
dine-2,4-dicarboxamide (11)
The compound was synthesized according to above general pro-
cedure using 1-(isonicotinamido)azetidine-2,4-dicarboxylic acid
(0.10 g, 0.38 mmol), 3,4-dichloro aniline (0.14 g, 0.84 mmol),
EDCÁHCl (0.22 g, 1.14 mmol), HOBt (0.18 g, 1.14 mmol) and triethy-
lamine (0.23 g, 2.28 mmol) to afford 1-(isonicotinamido)-N2,N4-bis
(3,4-dichlorophenyl)azetidine-2,4-dicarboxamide (0.092 g, 44.10%).
1H NMR (DMSO-d6): dH 9.11 (s, 1H), 8.85 (d, J = 6.8 Hz, 2H), 8.56 (s,
2H), 8.01–7.48 (m, 8H), 4.18 (t, J = 7.2 Hz, 2H), 3.04–2.88 (m, 2H).
13C NMR (DMSO-d6): dC 172.2 (2C), 164.3, 149.2 (2C), 138.9,
137.2 (2C), 129.4 (2C), 128.7 (2C), 123.7 (2C), 120.3 (2C), 119.7
(2C), 118.2 (2C), 69.3 (2C), 17.3. ESI-MS m/z 554.82 (M+H)+. Anal
Calcd for C23H17Cl4N5O3; C, 49.93; H, 3.10; N, 12.66; Found: C,
50.01; H, 3.09; N, 12.64.
The compound was synthesized according to above general pro-
cedure using 1-(isonicotinamido)azetidine-2,4-dicarboxylic acid
(0.10 g, 0.38 mmol), 2,6-dimethylphenylamine (0.10 g, 0.84 mmol),
EDCÁHCl (0.22 g, 1.14 mmol), HOBt (0.18 g, 1.14 mmol) and triethy-
lamine (0.23 g, 2.28 mmol) to afford 1-(isonicotinamido)-N2,N4-bis
(2,6-dimethylphenyl)azetidine-2,4-dicarboxamide (0.71 g, 39.94%).
1H NMR (DMSO-d6): dH 8.98 (s, 1H), 8.82 (d, J = 7.8 Hz, 2H), 8.58
(s, 2H), 7.76 (d, J = 7.6 Hz, 2H), 7.15–7.12 (m, 6H), 4.13 (t,
J = 7.2 Hz, 2H), 3.01–2.86 (m, 2H), 2.08 (s, 12H). 13C NMR
(DMSO-d6): dC 169.5 (2C), 165.3, 147.5 (2C), 139.2, 135.3 (2C),
129.2 (4C), 125.7 (4C), 125.4 (2C), 119.0 (2C), 69.5 (2C), 17.7,
17.0 (4C). ESI-MS m/z 472.23 (M+H)+. Anal Calcd for C27H29N5O3;
C, 68.77; H, 6.20; N, 14.85; Found: C, 68.69; H, 6.21; N, 14.86.
4.1.19. 1-(Isonicotinamido)-N2,N4-di(pyridin-2-yl)azetidine-2,4-
dicarboxamide (16)
4.1.15. 1-(Isonicotinamido)-N2,N4-bis(2,4-dimethoxyphenyl)
azetidine-2,4-dicarboxamide (12)
The compound was synthesized according to above general pro-
cedure using 1-(isonicotinamido)azetidine-2,4-dicarboxylic acid
(0.10 g, 0.38 mmol), 2-pyridylamine (0.08 g, 0.84 mmol), EDCÁHCl
(0.22 g, 1.14 mmol), HOBt (0.18 g, 1.14 mmol) and triethylamine
(0.23 g, 2.28 mmol) to afford 1-(isonicotinamido)-N2,N4-di(pyri-
din-2-yl)azetidine-2,4-dicarboxamide (0.064 g, 40.66%). 1H NMR
(DMSO-d6): dH 9.27 (s, 2H), 9.10 (s, 1H), 8.93 (d, J = 7.0 Hz, 2H),
8.51–7.32 (m, 10H), 4.24 (t, J = 7.6 Hz, 2H), 3.05–2.90 (m, 2H). 13C
NMR (DMSO-d6): dC 170.2 (2C), 164.8, 148.1 (2C), 146.2 (2C),
143.1 (2C), 138.3, 137.7 (2C), 123.5 (2C), 120.6 (2C), 116.3 (2C),
66.3 (2C), 17.2. ESI-MS m/z 418.16 (M+H)+. Anal Calcd for
The compound was synthesized according to above general
procedure using 1-(isonicotinamido)azetidine-2,4-dicarboxylic
acid (0.10 g, 0.38 mmol), 2,4-dimethoxyphenylamine (0.13 g,
0.84 mmol), EDCÁHCl (0.22 g, 1.14 mmol), HOBt (0.18 g,
1.14 mmol) and triethylamine (0.23 g, 2.28 mmol) to afford
1-(isonicotinamido)-N2,N4-bis(2,4-dimethoxyphenyl)azetidine-2,4-
dicarboxamide (0.082 g, 40.61%). 1H NMR (DMSO-d6): dH 9.05
(s, 1H), 8.80 (d, J = 7.4 Hz, 2H), 8.54 (s, 2H), 8.03–6.62 (m, 8H),
4.08 (t, J = 7.2 Hz, 2H), 3.86 (s, 12H), 3.00–2.83 (m, 2H). 13C NMR
(DMSO-d6): dC 170.2 (2C), 167.3 (2C), 164.2, 150.1 (2C), 148.6
(2C), 139.3, 120.3 (2C), 118.4 (2C), 116.5 (2C), 104.7 (2C), 98.9
(2C), 69.5 (2C), 53.4 (4C), 17.6. ESI-MS m/z 536.21 (M+H)+. Anal
Calcd for C27H29N5O7; C, 60.55; H, 5.46; N, 13.08; Found: C,
60.67; H, 5.45; N, 13.06.
C21H19N7O3; C, 60.42; H, 4.59; N, 23.49; Found: C, 60.50; H, 4.58;
N, 23.51.
4.1.20. 1-(Isonicotinamido)-N2,N4-bis(6-methylpyridin-2-yl)azet
idine-2,4-dicarboxamide (17)
The compound was synthesized according to above general pro-
cedure using 1-(isonicotinamido)azetidine-2,4-dicarboxylic acid
(0.10 g, 0.38 mmol), 6-methyl-2-pyridylamine (0.09 g, mmol),
EDCÁHCl (0.22 g, 1.14 mmol), HOBt (0.18 g, 1.14 mmol) and triethy-
lamine (0.23 g, 2.28 mmol) to afford 1-(isonicotinamido)-N2,N4-
4.1.16. 1-(Isonicotinamido)-N2,N4-bis(4-bromophenyl)azetid
ine-2,4-dicarboxamide (13)
The compound was synthesized according to above general pro-
cedure using 1-(isonicotinamido)azetidine-2,4-dicarboxylic acid
(0.10 g, 0.38 mmol), 4-bromo aniline (0.14 g, 0.84 mmol), EDCÁHCl
(0.22 g, 1.14 mmol), HOBt (0.18 g, 1.14 mmol) and triethylamine
(0.23 g, 2.28 mmol) to afford 1-(isonicotinamido)-N2,N4-bis(4-bro-
mophenyl)azetidine-2,4-dicarboxamide (0.076 g, 35.16%). 1H NMR
(DMSO-d6): dH 9.11 (s, 1H), 8.91 (d, J = 7.4 Hz, 2H), 8.55 (s, 2H), 7.85
(d, J = 7.2 Hz, 2H), 7.65–7.52 (m, 8H), 4.20 (t, J = 7.4 Hz, 2H),
3.03–2.89 (m, 2H). 13C NMR (DMSO-d6): dC 170.5 (2C), 165.2,
148.8 (2C), 139.2, 136.5 (2C), 128.7 (4C), 120.1 (2C), 119.3 (4C),
118.9 (2C), 69.6 (2C), 17.4. ESI-MS m/z 574.21 (M+H)+. Anal Calcd
for C23H19Br2N5O3; C, 48.19; H, 3.34; N, 12.22; Found: C, 48.10;
H, 3.35; N, 12.24.
bis(6-methylpyridin-2-yl)azetidine-2,4-dicarboxamide
(0.082 g,
48.82%). 1H NMR (DMSO-d6): dH 9.26 (s, 2H), 9.07 (s, 1H), 8.89 (d,
J = 7.2 Hz, 2H), 8.06–7.42 (m, 8H), 4.20 (t, J = 7.4 Hz, 2H),
3.01–2.88 (m, 2H), 2.46 (s, 6H). 13C NMR (DMSO-d6): dC 170.6
(2C), 165.1, 149.9 (2C), 147.5 (4C), 147.0 (2C), 138.2, 125.6 (2C),
119.6 (2C), 111.7 (2C), 69.3 (2C), 25.2 (2C), 17.4. ESI-MS m/z
446.19 (M+H)+. Anal Calcd for C23H23N7O3; C, 62.01; H, 5.20; N,
22.01; Found: C, 61.95; H, 5.21; N, 22.02.
4.1.21. 1-(Isonicotinamido)-N2,N4-bis(benzo[d]thiazol-2-yl)azeti
dine-2,4-dicarboxamide (18)
4.1.17. 1-(Isonicotinamido)-N2,N4-bis(4-chlorophenyl)azetidi
ne-2,4-dicarboxamide (14)
The compound was synthesized according to above general pro-
cedure using 1-(isonicotinamido)azetidine-2,4-dicarboxylic acid
(0.10 g, 0.38 mmol), benzo[d]thiazole-2-amine (0.13 g, 0.84 mmol),
EDCÁHCl (0.22 g, 1.14 mmol), HOBt (0.18 g, 1.14 mmol) and triethy-
lamine (0.23 g, 2.28 mmol) to afford 1-(isonicotinamido)-N2,N4-bis
(benzo[d]thiazol-2-yl)azetidine-2,4-dicarboxamide (0.093 g, 46.58%).
1H NMR (CDCl3): dH 9.28 (s, 2H), 9.08 (s, 1H), 8.92 (d, J = 7.4 Hz, 2H),
8.05–7.57 (m, 10H), 4.23 (t, J = 7.4 Hz, 2H), 3.03–2.84 (m, 2H). 13C
NMR (DMSO-d6): dC 175.6 (2C), 170.5 (2C), 164.3, 155.2 (2C),
148.6 (2C), 140.3, 130.2 (2C), 124.5 (2C), 123.1 (2C), 119.8 (4C),
The compound was synthesized according to above general pro-
cedure using 1-(isonicotinamido)azetidine-2,4-dicarboxylic acid
(0.10 g, 0.38 mmol), 4-chloro aniline (0.11 g, 0.84 mmol), EDCÁHCl
(0.22 g, 1.14 mmol), HOBt (0.18 g, 1.14 mmol) and triethylamine
(0.23 g, 2.28 mmol) to afford 1-(isonicotinamido)-N2,N4-bis(4-
chlorophenyl)azetidine-2,4-dicarboxamide (0.051 g, 27.93%). 1H
NMR (DMSO-d6): dH 9.09 (s, 1H), 8.88 (d, J = 7.0 Hz, 2H), 8.53 (s,
2H), 7.82 (d, J = 7.0 Hz, 2H), 7.70–7.43 (m, 8H), 4.21 (t, J = 7.4 Hz,
Please cite this article in press as: Reshma, R. S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.051

10
R. S. Reshma et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
117.3 (2C), 69.4 (2C), 17.3. ESI-MS m/z 530.10 (M+H)+. Anal Calcd
for C25H19N7O3S2; C, 56.70; H, 3.62; N, 18.51; Found: C, 56.79; H,
3.61; N, 18.49.
tested. Serial two-fold dilutions of each drug were prepared
directly in a sterile 96-well microtiter plate using 100 l 7H9-S.
A growth control containing no antibiotic and a sterile control
were also prepared on each plate. Sterile water was added to all
perimeter wells to avoid evaporation during the incubation. The
plate was covered, sealed in plastic bags and incubated at 37 °C
l
4.1.22. 1-(Benzamido)-N2,N4-bis(benzo[d]thiazol-2-yl)azetidine-
2,4-dicarboxamide (19)
The compound was synthesized according to above general pro-
cedure using (2R,4R)-1-benzamido azetidine-2,4-dicarboxylic acid
(0.10 g, 0.38 mmol), benzo[d]thiazole-2-amine (0.13 g, 0.84 mmol),
EDCÁHCl (0.22 g, 1.14 mmol), HOBt (0.18 g, 1.14 mmol) and triethy-
lamine (0.23 g, 2.28 mmol) to afford 1-(benzamido)-N2,
N4-bis(benzo[d]thiazol-2-yl)azetidine-2,4-dicarboxamide (0.087 g,
43.57%). 1H NMR (DMSO-d6): dH 9.31 (s, 2H), 9.10 (s, 1H),
8.15–7.50 (m, 13H), 4.25 (t, J = 7.2 Hz, 2H), 2.99–2.81 (m, 2H). 13C
NMR (DMSO-d6): dC 175.8 (2C), 170.2 (2C), 164.1, 150.7 (2C),
131.4 (2C), 129.7 (2C), 128.3 (2C), 126.3 (2C), 124.7 (2C), 123.2
(2C), 119.2 (2C), 116.8 (2C), 69.6 (2C), 17.5. ESI-MS m/z 529.11
(M+H)+. Anal Calcd for C26H20N6O3S2; C, 59.08; H, 3.81; N, 15.90;
Found: C, 59.15; H, 3.82; N, 15.92.
in normal atmosphere. After 7 days incubation, 50 ll of alamar
blue solution was added to each well, and the plate was re-incu-
bated overnight. A change in color from blue (oxidized state) to
pink (reduced) indicated the growth of bacteria, and the MIC was
defined as the lowest concentration of drug that prevented this
change in color.
4.3. In vitro cytotoxicity screening
The synthesized compounds were further examined for its cyto-
toxicity in mouse macrophage cell line (RAW 264.7) at 50 lM con-
centration. 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.
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% conflu-
ency. Cells were scraped and seeded into wells approx 5000 cells
4.2. 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
per well in poly-L-lysine coated plates. The microtiter plates were
reaction product NADH that accompanies the conversion of
L
-ala-
incubated at 37 °C, 5% CO2, 95% air and 100% relative humidity
nine into pyruvate in the oxidative deamination was measured at
340 nm in heat controlled microplate reader PerkinElmer Victor
X3 instrument. The synthesized compounds were added to the
for 24 h prior to addition of experimental drugs. The test com-
pounds at 50
lM concentration were then added to cells and incu-
bated at 37 °C for 48 h; later 10
lL of 0.5 mg/ml concentration of
plate with different concentrations from 25
to determine IC50 values for the all the synthesized compounds.
The reaction mixture except MTB -AlaDH was added and back-
l
M to 0.5
l
M in order
MTT was added and incubated for 3 h at 37 °C and the final product
formazan crystals were measured at 595 nm.
L
ground reactions were measured. Reactions were carried out at
37 °C in a heat-controlled Perkin elmer Victor X3 Spectrophotome-
ter. Further the IC50 values were calculated using Graph Pad Prism
analysis software.
Acknowledgements
D.S. is thankful to Department of Biotechnology, Government of
India for the Tata innovation fellowship. R.S.R. is thankful to
Department of Science and Technology, Government of India for
the Inspire fellowship.
4.2.1. 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 Buf-
fer Saline, HiMedia Laboratories). The pellet was re suspended in
PBS in sealed bottles and is incubated at 37 °C for 6 weeks. Six
week starved cultures were treated with standard drugs like Isoni-
azid, Rifampicin and Moxifloxacin along with synthesized drugs for
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7 days at a concentration of 10
were diluted 10-fold up to 10À6 using Middlebrook 7H9 medium
supplemented with OADC and 100 l of each dilutions were plated
in 48 well plates in triplicates along with 450 l of Middlebrook
lg/ml. The treated cell suspensions
l
l
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100
ll was used as inoculum. Each drug stock solution was thawed
and diluted in 7H9-S at four-fold the final highest concentration
Please cite this article in press as: Reshma, R. S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.051