Development of novel tetrahydrothieno[2,3-c]pyridine-3-carboxamide based Mycobacterium tuberculosis pantothenate synthetase inhibitors: Molecular hybridization from known antimycobacterial leads

Article abstract of DOI:10.1016/j.bmc.2014.01.030

Twenty six 2,6-disubstituted 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3- carboxamide derivatives were designed by molecular hybridization approach using and synthesized from piperidin-4-one by five step synthesis. Compounds were evaluated for Mycobacterium tuberculosis (MTB) pantothenate synthetase (PS) inhibition study, in vitro activities against MTB, cytotoxicity against RAW 264.7 cell line. Among the compounds, 6-(4-nitrophenylsulfonyl)-2-(5- nitrothiophene-2-carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c] pyridine-3-carboxamide (11) was found to be the most active compound with IC₅₀ of 5.87 ± 0.12 μM against MTB PS, inhibited MTB with MIC of 9.28 μM and it was non-cytotoxic at 50 μM. The binding affinity of the most potent inhibitor 11 was further confirmed biophysically through differential scanning fluorimetry.

Full text of DOI:10.1016/j.bmc.2014.01.030

Bioorganic & Medicinal Chemistry 22 (2014) 1938–1947
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of novel tetrahydrothieno[2,3-c]pyridine-3-carboxamide
based Mycobacterium tuberculosis pantothenate synthetase
inhibitors: Molecular hybridization from known antimycobacterial
leads
Ganesh Samala, Parthiban Brindha Devi, Radhika Nallangi, Jonnalagadda Padma Sridevi, Shalini Saxena,
⇑
Perumal Yogeeswari, Dharmarajan Sriram
Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, Shameerpet, R.R. District, Hyderabad 500078, Andhra Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty six 2,6-disubstituted 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide derivatives were
designed by molecular hybridization approach using and synthesized from piperidin-4-one by five step
synthesis. Compounds were evaluated for Mycobacterium tuberculosis (MTB) pantothenate synthetase
(PS) inhibition study, in vitro activities against MTB, cytotoxicity against RAW 264.7 cell line. Among
the compounds, 6-(4-nitrophenylsulfonyl)-2-(5-nitrothiophene-2-carboxamido)-4,5,6,7-tetrahydrothie-
no[2,3-c]pyridine-3-carboxamide (11) was found to be the most active compound with IC50 of
5.87 0.12 lM against MTB PS, inhibited MTB with MIC of 9.28 lM and it was non-cytotoxic at 50 lM.
The binding affinity of the most potent inhibitor 11 was further confirmed biophysically through differ-
ential scanning fluorimetry.
Received 19 November 2013
Revised 18 January 2014
Accepted 20 January 2014
Available online 6 February 2014
Keywords:
Tuberculosis
Pantothenate synthetase
Cytotoxicity
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
to form pantothenate.7 Pantothenate is a major precursor of coen-
zyme A and acyl carrier protein, essential for many intracellular
Mycobacterium tuberculosis (MTB) is the causative agent of
tuberculosis (TB), a disease which latently affects over 1.7 billion
people worldwide.1 The incidence of TB has been increasing during
the last 20 years; it is now the first cause of mortality among infec-
tious diseases in the world.2 The combination of four active drugs
(rifampicin, isoniazid, pyrazinamide, and ethambutol or strepto-
mycin) is currently used in MTB treatment, but this has led to
the appearance of resistant bacterial strains.3 These resistant
strains are alarming for two reasons. First, as there are only a
few effective drugs available, infection with drug-resistant strains
could give rise to a potentially untreatable form of the disease.4
Second, although only 5% of immune competent people infected
with MTB succumb to the disease, it is nevertheless highly conta-
gious.5 Therefore, a large effort is necessary to identify potential
new targets and inhibitors. The pantothenate biosynthetic path-
way involves four steps catalyzed by enzymes encoded by panB,
panC, panD, and panE genes.6 PanC encodes a pantothenate synthe-
tase (PS), which catalyzes the last step of pantothenate biosynthe-
processes including fatty acid metabolism, cell signaling, and syn-
thesis of polyketides and non-ribosomal peptides. Microorganisms
must synthesize pantothenate, while mammals must obtain it
from their diet, and its biosynthetic pathway is not present. There
is evidence that the pantothenate biosynthesis pathway is a
potential drug target and is essential for persistent growth and vir-
ulence of MTB and PS is an appropriate target for developing new
therapeutics to treat TB.8 Till date few of the MTB PS inhibitors
were reported (Fig. 1) 3-phenyl-4,5,6,7-tetrahydro-1H-pyrazol-
o[4,3-c]pyridine derivatives (A),9 5-tert-butyl-N-pyrazol-4-yl-
4,5,6,7-tetrahydrobenzo[d]isoxazole-3-carboxamide
derivatives
(B),10 actinomycin D (IC50 of 250.72
l
M),11 nafronyl oxalate (D),12
benzofuran derivatives (C).13 In this work, we designed and devel-
oped novel MTB PS inhibitors by molecular hybridization approach
using two known antimycobacterial agents.
2. Results and discussion
2.1. Design and chemistry
sis, the ATP-dependent condensation of D-pantoate, and b-alanine
The molecular hybridization is a strategy of rational design of new
ligands or prototypes based on the recognition of pharmacophoric
⇑
Corresponding author. Tel.: +91 40 66303506.
E-mail address: dsriram@hyderabad.bits-pilani.ac.in (D. Sriram).
http://dx.doi.org/10.1016/j.bmc.2014.01.030
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

G. Samala et al. / Bioorg. Med. Chem. 22 (2014) 1938–1947
1939
O
O
CH3
CH3
O
S
O
NH
CH3
NH
O
N
N
O
H3C
N
N
N
N
O
O
O
NH
OH
N
O
O
D
O
B
IC50: 90 nM
Nafronyl oxalate
A
C
MTB PS active at 10 ug/mL
MTB: 43%inhibition
at 128 uM
IC50: 21.8 uM
MTB MIC 26.7 uM
KD 1.8 uM
NO2
MTB MIC not active at 6.25 ug/mL
Figure 1. MTB PS inhibitors.
sub-units in the molecular structure of two or more known
bioactive derivatives which, through the adequate fusion of these
sub-units, lead to the design of new hybrid architectures that
maintain pre-selected characteristics of the original templates. In
this work we designed novel MTB inhibitors by hybridizing our
reported MTB PS inhibitor compound A (1-benzoyl-N-(4-nitro-
phenyl)-3-phenyl-6,7-dihydro-1H-pyrazolo[4,3-c]pyridine-5(4H)-
carboxamide)9 and TAACF inhibitor SID 9209788014 (Fig. 2). This
approach may lead to development of novel lead compound for
MTB PS inhibition with potential MTB MIC. The designed target
molecules were synthesised by following six step synthetic
schemes starting with commercially available, less expensive
4-piperidone hydrochloride monohydrate (I) (Scheme 1). In the
first step we protected the amine group of starting material with
Boc-protection. Here we used the combination of methanol/dichlo-
romethane as reaction solvents in order to achieve greater yields.
Then the obtained N-Boc-piperidone (II) was treated with cyano-
acetamide (III), sulfur powder and morpholine in ethanol (Gewald
reaction)15 yielded the 2-aminothiophene derivative (IV). We ini-
tially plan to synthesise compound IV, in linear synthesis fashion
using ethylcyanoacetate. That is, reaction of compound II with eth-
ylcyanoacetate, sulfur powder, morpholine under Gewald reaction
conditions to produce the intermediate ‘6-tert-butyl 3-ethyl 2-ami-
no-4,5-dihydrothieno[2,3-c]pyridine-3,6(7H)-dicarboxylate’; later
the conversion of ester to amide to achieve compound IV. Here
we succeeded in synthesising ‘6-tert-butyl 3-ethyl 2-amino-4,5-
dihydrothieno[2,3-c]pyridine-3,6(7H)-dicarboxylate’, but conver-
sion of ester group to amide was not successful. This might be
due to prevention of attacking nucleophile (NH3) on ester by the
steric bulkiness of Boc-protected six-membered ring. Hence we
followed the convergent synthesis; we made reacted the ethyl-
cyanoacetate with ammonia to produce cyanoacetamide (III). Then
treating cyanoacetamide with compound II under Gewald reac-
tions conditions produced compound IV. The free amino group of
compound IV reacts with 5-nitrofuran-2-carbonyl chloride &
5-nitrothiophene-2-carbonyl chloride to yield compound VIa & VIb.
Here we converted the carboxylic group into acid chloride using
oxalyl chloride, since the attempts to couple the carboxylic acid
group with the amine using coupling agents (EDCI/HOBt and
HATU) failed to obtain desired product. Then the deprotection of
Boc using trifluoroacetic acid yielded free secondary amino func-
tion VIIa–b; followed by reaction with various substituted aryli-
socyantes, arylisothiocyanates and arylsulfonyl chloride yielded
corresponding urea, thiourea and sulfonamide derivatives (1–26).
The purity of the synthesized compounds was checked by
LC–MS, elemental analyses and the structures were confirmed by
spectral data. In the nuclear magnetic resonance spectra (1H
NMR and 13C NMR), the signals of the respective protons of the
prepared derivatives were verified on the basis of their chemical
shifts, multiplicities, and coupling constants. The elemental
analysis results were within 0.4% of the theoretical values.
2.2. In vitro MTB screening
All the synthesized compounds were screened for their in vitro
anti-tubercular activity against Mycobacterium tuberculosis H37Rv
(ATCC27294) using an agar dilution method using drugconcentration
from 50 lg/mL to 0.78 lg/mL in duplicates. The minimum inhibitory
concentration (MIC) was determined for each compound. The MIC is
defined as the minimum concentration of compound required to
completely inhibit the bacterial growth. Isoniazid, ethambutol, com-
pound A and SID 92097880 were used as a reference compound for
comparison. The MIC values of the synthesized compounds along
with the standard drug for comparison were reported in Table 1. All
the synthesized compounds showed activity against MTB with MIC
ranging from 9.28 to 51
lM. Eight compounds (1–2, 5–9 and 11)
inhibited MTB with MIC of >20
lM. Compound 11 (6-(4-nitrophenyl-
sulfonyl)-2-(5-nitrothiophene-2-carboxamido)-4,5,6,7-tetrahydro
thieno[2,3-c]pyridine-3-carboxamide) was found to be the most
active compound in vitro with MICs of 9.28 lM against log-phase
culture of MTB. All the synthesized compounds were less potent
than standard antitubercular compounds like isoniazid and etham-
butol. Only compound 11 showed equipotent as one of the lead com-
pound SID 92097880 (MIC 9.15
lead compound A (MIC 21.8 M), eight compounds were found to
be more active (up to 2.3 times). Structural changes at 2nd position
lM). When compared to another
l
Figure 2. Molecular hybridization strategy.

1940
G. Samala et al. / Bioorg. Med. Chem. 22 (2014) 1938–1947
R1
O
O
HN
S
H2N
N
R2
O
N
H
O
R1-COOH
f
1 - 4
NC
NC
14 - 17
OEt
(COCl)2, DMF (Cat)
CH2Cl2
b
R1
O
O
R1
O
R1
O
O
O
HN
O
O
H2N
NH2
S
HN
S
O
HN
S
O
R1
Cl H2N
H2N
H2N
NH2
O
S
III
g
e
V
a
c
d
N
N
N
H.HCl
Boc
N
N
R2
N
H2O
Boc
Boc
H
S
N
H
VIa: R1 = 5-nitrothiophen-2-yl;
VIb: R1 = 5-nitrofuran-2-yl;
VIIa
VIIb
I
II
IV
5 - 8
18 - 21
h
R1
O
O
HN
H2N
S
N
O
S
O
R2
9 - 13
22 - 26
Scheme 1. Synthetic protocol of the compounds. Reagents and conditions: (a) (Boc)2O, Et3N, CH2Cl2/MeOH, rt, 16 h; (b) aq NH3, THF; (c) Morpholine, Sulphur powder, EtOH,
rt, 7 h; (d) DIPEA, DCM, rt, 6 h; (e) TFA, DCM, rt, 2 h; (f) R2NCO, Et3N, DMF, rt, 3 h; (g) R2NCS, Et3N, DMF, rt, 3 h; (h) R2SO2Cl, Et3N, rt, 4 h.
alter the activity appreciably, 5-nitrothiophene-2-carboxamido side
chain containing compounds (1–13) were found to be more potent
than 5-nitrofuran-2-carboxamido counterpart (14–26). Among
5-nitrothiophene-2-carboxamido side chain containing compounds
(1–13); we had ureas (1–4), thioureas (5–8) and sulfonamides
(9–13) and we have found not much difference in activity. But
substituent with electro withdrawing groups like halogen and nitro
group showed better activity than electron donating groups.
hydrogen-bonding interactions between the sulfone oxygen atom
and both the backbone amide group of Met40 and the side-chain
nitrogen atom of His47. It was also found to have hydrogen bonding
interactions with Val187, Ser196, Ser197 and His44. The protein crys-
tal structure has two hydrophobic pockets-one consists of Met40,
Leu50, Phe157, Thr39 and Pro38 whereas the other consists of
Lys160, His44 and Gly46. The reference ligand 2-(2-(benzofuran-2-
ylsulfonylcarbamoyl)-5-methoxy-1H-indol-1-yl)acetic acid was re-
docked with the active site residues of the MTB PS to validate the
active site cavity. The ligand exhibited a highest Glide score of
À9.03 kcal/mol and was found in the vicinity of amino acids HIE47,
Met40, Val143, Val139, Pro38, Val142, Leu146, Asp161, Tyr82,
Lys160,Ser196, Ser197, HIE44, Thr186,Val187, Leu50, Ala49, Gly46,
and Gly158 amino acid residues. Re-docking results showed that
the compound exhibited similar interactions as that of the original
crystal structure and showed a RMSD of 1.18 A°.
Our docking studies confirmed that the structural changes at 2nd
position with 5-nitrothiophene-2-carboxamido side chain (Com-
pounds 1–13) were found to be more potent than 5-nitrofuran-2-
carboxamido counterpart (compounds 14–26). In the first series
the compounds is substituted with ureas (1–4), thioureas (5–8)
and sulfonamides (9–13); in case of substituted urea the compound
2 [6-N-(4-nitrophenyl)-2-C-5-nitrothiophene-2-4H,5H,6H,7H-thie-
no[2,3-c]pyridine-2,3,6-tricarboxamide] where R2 is substituted
with 4-nitrophenyl is showing good activity IC50 9.68 0.28 as com-
pare to other substituted urea (1, 3 and 4) where the R2 is substituted
with 4-chlorophenyl, benzyl, and 4-methoxyphenyl. The activity
profile of all the four compounds from this series is well correlated
with the binding energy of the molecules. The compound 2 is show-
ing high docking score (À7.82 kcal/mol) while the other compounds
(1, 3 and 4) are showing comparatively low docking score (À7.69,
À7.29 and À7.50 kcal/mol). A close analysis of the orientation of
compound 2 in the protein active site revealed that it makes four
2.3. Pantothenate synthetase enzyme inhibition and docking
studies
Synthesized compounds were assayed for MTB PS inhibition
study, that couples the AMP produced in the condensation of
b-alanine and pantoate with the reduction of NADH to NAD+
through myokinase, pyruvate kinase and lactate dehydrogenase.7
The decrease in NADH can be monitored spectrophotometrically at
340 nm. Initial screening at 25 lM, compounds showed inhibition
ranged from 18.0% to 82.6%. Twenty one compounds showed >50%
inhibition against MTB PS were further selected for IC50 calculation
(the graph was plotted against concentration (dose) of the com-
pound and % inhibition was plotted using Graphpad prism software
and thereby IC50 value was calculated) and compounds showed IC50
ranged from 5.77 0.12
All the twenty one compounds were found to be more potent than
our earlier reported lead compound A (IC50 of 21.8 M). Compound
11 which showed good MIC in the in vitro MTB screening, was found
to be most potent MTB PS inhibitor with IC50 of 5.77 0.12 M.
lM to 20.86 1.25 lM (Table 1 and Fig. 3).
l
l
Further we have taken all the twenty six compounds for docking
studies for establishing the structure–activity relationship using
crystal structure of MTB PS co-crystallized with 2-(2-(benzofuran-
2-ylsulfonylcarbamoyl)-5-methoxy-1H-indol-1-yl) acetic acid [PDB:
3IVX]. Analysis of the crystal structure of 3IVX revealed two key

G. Samala et al. / Bioorg. Med. Chem. 22 (2014) 1938–1947
1941
Table 1
Biological activities of synthesized compounds
R1
O
R1
O
R1
O
O
O
O
HN
S
HN
S
HN
S
H2N
H2N
H2N
N
N
N
O
S
R2
O
O
NH
R2
S
NH
R2
1 - 4
14 - 17
5 - 8
18 - 21
9 - 13
22 - 26
Compound
R1
R2
MTB MIC in
l
M
PanC IC50
Cytotoxicity at 50 lM (RAW 264.7 cells)% inhibition
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
5-Nitrothiophen-2-yl
5-Nitrothiophen-2-yl
5-Nitrothiophen-2-yl
5-Nitrothiophen-2-yl
5-Nitrothiophen-2-yl
5-Nitrothiophen-2-yl
5-Nitrothiophen-2-yl
5-Nitrothiophen-2-yl
5-Nitrothiophen-2-yl
5-Nitrothiophen-2-yl
5-Nitrothiophen-2-yl
5-Nitrothiophen-2-yl
5-Nitrothiophen-2-yl
5-Nitrofuran-2-yl
5-Nitrofuran-2-yl
5-Nitrofuran-2-yl
5-Nitrofuran-2-yl
5-Nitrofuran-2-yl
5-Nitrofuran-2-yl
5-Nitrofuran-2-yl
5-Nitrofuran-2-yl
5-Nitrofuran-2-yl
4-Chlorophenyl
4-Nitrophenyl
Benzyl
4-Methoxyphenyl
4-Fluorophenyl
4-Chlorophenyl
4-Nitrophenyl
Benzyl
4-Fluorophenyl
4-Bromophenyl
4-Nitrophenyl
4-Acetylphenyl
4-Methoxyphenyl
4-Chlorophenyl
4-Nitrophenyl
Benzyl
4-Methoxyphenyl
4-Fluorophenyl
4-Chlorophenyl
4-Nitrophenyl
Benzyl
12.32
12.09
25.72
24.90
12.34
11.95
11.72
19.92
12.23
21.89
9.28
10.22 0.8
9.68 0.28
18.16 0.8
14.5 0.66
10.16 0.56
6.28 0.32
9.11 0.43
13.24 1.2
11.72 0.64
14.22 0.92
5.87 0.12
>25
24.42
33.63
26.12
20.86
26.76
21.42
28.62
22.16
30.16
26.82
29.16
ND
24.50
ND
36.12
24.31
ND
28.12
24.69
28.21
18.12
22.06
ND
ND
ND
19.43
ND
ND
46.73
23.90
51.0
19.05 0.86
>25
24.95
26.60
53.19
25.51
24.70
24.18
26.48
25.25
45.04
47.89
48.16
24.65
0.72
19.79 1.1
14.08 0.67
15.06 0.53
20.86 1.25
19.16 0.96
18.72 0.64
17.02 0.73
17.56 0.94
>25
>25
>25
20.33 1.4
>25
>25
4-Fluorophenyl
4-Bromophenyl
4-Nitrophenyl
4-Acetylphenyl
4-Methoxyphenyl
5-Nitrofuran-2-yl
5-Nitrofuran-2-yl
5-Nitrofuran-2-yl
5-Nitrofuran-2-yl
25
26
Isoniazid
Ethambutol
SID 92097880
Compound A
7.64
9.15
26.7
>25
21.8 0.8
ND
0
ND indicates that not determined.
Figure 3. Dose response curves for selected MTB PS inhibitors.
hydrogen bonding interactions with the relevant amino acid resi-
dues such as Ser196, Ser197 Asp161, and Gly158. In addition to
hydrogen bonding interaction, the compound is further stabilized
here also 4-chlorophenyl is inserted into the hydrophobic pocket
and making contacts with Leu50, Ala49, Val187 and Val184 amino
acid residues (Fig. in Supplementary information). In case of
compound 3 and 4 where R2 is substituted with benzyl, and
by
p–p stacking interaction with Hie44 amino acid residue. The
NO2 group on phenyl ring is stabilized by making contacts with pos-
itively charged amino acid such as Arg198, Arg278 and also it has
some hydrophobic interaction with Leu280 and Leu279 amino acids,
while the compound 1 where R2 is substituted with 4-chlorophenyl
is also showing good activity in PS enzyme activity (IC50 10.22 0.8),
4-methoxyphenyl showing moderately activity IC50 = 18.16 0.8 lM
and 14.5 0.66 as compare to compound 2 and 1. Both the
compounds having comparatively low binging energy (7.29 and
À7.40 kcal/mol), the binding orientation is also changed in the ac-
tive site of the protein this is may be because of the bulky substitu-

1942
G. Samala et al. / Bioorg. Med. Chem. 22 (2014) 1938–1947
Figure 4. Binding pattern of most active compound 11 and the ligand interaction with the protein.
tions; thus it was not fully occupy the hydrophobic cavity (Fig. in
Supplementary information).
Similarly the next series is with substituted urea (5–8) in that
substitution at R2 position with 4-fluorophenyl, chlorophenyl,
nitrophenyl and benzyl is also showing good activity IC50 ranging
ing contours, (where three correspond to the reference inhibitor 2-
(2-(benzofuran-2-ylsulfonylcarbamoyl)-5-methoxy-1H-indol-1-yl)
acetic acid); one with Val187, one with HIE44 and one with Ser196
with a docking score of À9.22, suggests that this compound has
been well-docked in the active site and it shows a good MTB PS
inhibitory activity. The binding pattern of the most active com-
pound 11 is represented in Figure 4. In this series the substitution
with 4-acetylphenyl at R2 position (compound 12) has showing
from 6.01 0.32 to 13.24 1.2 lM. Here; the most active compound
6 is the substitution with 4-chlorophenyl at R2 position (IC50
6.01 0.32) showing the good docking score À7.53 kcal/mol sug-
gests that this compound has been well-docked in the active site
and also involved in two hydrogen bonding interaction with
Gln72, and Ser197. The presence of chloro group on the phenyl ring
increase its hydrophobicity towards the protein and it was buried in
the hydrophobic cavity surrounded by Val142, Val143, Val139,
Met40 and Pro38 amino acid residues (Fig. in Supplementary infor-
mation). While the substitution with benzyl group (compound 8)
again showing moderately activity in the enzyme IC50 13.24 1.2,
it was also showing low docking score of À6.66 kcal/mol. The close
analysis revealed that the compound 8 orientation is change in the
protein and the benzyl group is away from the active site (Fig. in
Supplementary information).
After the next series is with substituted sulfonamides (9–13)
here 4-nitrophenyl substitution (compound 11) is showing best
activity in the PS enzyme. With the aim of getting insights into
the structural basis for its activity, most active compound 11 was
analysed in more detail. The compound showed good docking
score of À9.22 kcal/mol, and it makes six hydrogen bonding inter-
actions with the relevant amino acid residues such as Val187,
Gln72, HIE44, Ser196, Ser197 and Lys160. In addition to hydrogen
inactivity in the enzyme having IC50 > 25 lM; here also because
of the bulky substitution is slightly away from the active site.
The activity of compound is correlated with the low binding energy
À6.22 kcal/mol as compare to compound 11 (Fig. in Supplemen-
tary information). From this analysis we observed that the hydro-
phobicity is important for retaining the activity if the substituted
group is occupies the hydrophobic cavity it was showing activity
and if the substituted group is away from the cavity the activity
of the compound is decreased.
The structural changes at 2nd position with 5-nitrofuran-2-car-
boxamido (compounds 14–26) are not showing activity in the PS
enzyme. To know the more about the binding details of the less ac-
tive compounds 23, 24, 25 in this series we analyse it in more de-
tail Figure 5. Binding analysis of all the three compounds in the
active site revealed that the compounds are not occupied in the
hydrophobic cavity of the protein. As shown in the Figure 5 all
the substitution at R2 position with bromo, nitro and acetylphenyl
are slightly away from the active site. Apart from that all the com-
pounds having low docking score in the range of À5.16 to À6.24 as
camper to other active compounds.
bonding interaction, the compound is further stabilized by
interaction with Hie44 and His135 amino acid residues. The NO2
group on thiophene ring interacts with the side chain of Val187.
His44 interacts with carbonyl oxygen of the amide and has a p–p
interaction with the thiophene ring, as well. The carbonyl oxygen
of the amide group on 4,5,6,7-tetrahydrothieno[2,3-c]pyridine ring
interacts with both Ser196 and Ser197 amino acid residues. One of
the O-atom of sulfonyl group interacts with Gln72 amino acid. The
p
–
p
Analysis of all the compounds in the active site of panC protein
revealed that the hydrophobic interaction is important for retain-
ing the activity of compounds and we have found not much differ-
ence in activity. But substituent with electro withdrawing groups
like halogen and nitro group showed better activity than electron
donating groups.
2.4. Biophysical characterization
Lys160 and His135 both have cation–p and p–p interaction with
nitro phenyl group but Lys160 also forms hydrogen bond with
the NH2 of the amide group on the 4,5,6,7-tetrahydrothieno[2,3-
c]pyridine. Apart from hydrogen bonding interaction, the com-
pound also makes some hydrophobic interaction with Met40,
Thr82, Met195, Ph3136, Val139 and Ala49 amino acid residues.
The fact that the most active compound 11 has six hydrogen bond-
Differential scanning flourimetry (DSF) measures the thermal
stability of a target protein and a subsequent increase in protein
melting temperature due to the binding of a ligand to the protein.
Protein complexes with ligand where heated from 25 °C to 95 °C in
steps of 0.1 °C in the presence of dye called sypro orange. The fluo-
rescence will increases when the protein get interacts with hydro-

G. Samala et al. / Bioorg. Med. Chem. 22 (2014) 1938–1947
1943
Figure 5. Binding mode of the compound 23, 24, 25 and the interacting pattern in the active site of the panC protein.
Figure 6. DSF experiment for compound 11 showing an increase in thermal stability between the native MTB PS protein (pink) and MTB PS proteinÀcompound 11 complex
(blue).
phobic residues of the protein. Positive shift of Tm corresponding to
native protein says that stability is increased due to inhibitor bind-
ing. The Figure 6 shows the melting curve of compound 11. The
protein MTB PS has the melting temperature of 46.70 °C, whereas
with ligand the corresponding Tm is 50.40 °C. The difference in
the Tm shows the stability of the native protein when it binds with
inhibitor.
synthesizing new compounds that possess better activity. Further
structure–activity, biophysical, pharmacokinetic and metabolism
studies should prove fruitful.
4. Experiments
4.1. Chemistry
2.5. In vitro cytotoxicity study
4.1.1. Preparation of tert-butyl 4-oxopiperidine-1-carboxylate
(II)
Some of the selected compounds were also tested for in vitro
cytotoxicity against RAW 264.7 cells at 50 lM concentration by
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. Percentage inhibition of cells was reported in Table 1.
Compounds inhibited with 18.12–33.63%. The most promising
Et3N (26.45 mL, 185.17 mmol) was added drop wise to a stirred
solution of compound I (10.0 g, 74.07 mmol) in CH2Cl2 (100 mL)
and MeOH (10 mL) at 0 °C, then (Boc)2O (19.37 mL, 88.88 mmol)
was added drop wise to the reaction mixture at same temperature
and allowed to stir at room temperature for 16 h. The reaction mix-
ture was diluted with H2O (100 mL) and extracted with CH2Cl2
(3 Â 200 mL). The separated organic layer was concentrated under
reduced pressure and the crude residue was washed with hexanes
to get compound II (11.5 g, 78%) as an off-white solid. ESI-MS
found 200 [M+H]+ and carried to next step.
anti-TB compound 11 showed 29.16% cytotoxicity at 50 lM.
3. Conclusions
In this work we designed novel 2,6-disubstituted 4,5,6,7-tetrahy-
drothieno[2,3-c]pyridine-3-carboxamide derivatives by molecular
hybridization approach using reported antitubercular compounds
and synthesized twenty six analogues. Many of the compounds
showed better MTB PS inhibition and MTB MIC than the one of the
lead compound A. In conclusion, it has been shown that the potency
and low cytotoxicity of these compounds make them valid leads for
4.1.2. Preparation of 2-cyanoacetamide (III)
To the stirred solution of ethylcyanoacetate (3.0 g) in THF
(10 mL) was added aqueous ammonia (15.0 mL), and allowed to
stir at room temperature in a closed vessel for 2 h. The reaction
mixture was concentrated under reduced pressure and obtained

1944
G. Samala et al. / Bioorg. Med. Chem. 22 (2014) 1938–1947
solids were filtered, dried in vacuum oven to get 2-cyanoacetamide
(2.1 g, 93%) as white solid. ESI-MS found 85 [M+H]+.
139.6, 136.9, 135.6, 134.4, 132.6(2C), 130.4, 129.5(2C), 124.5,
116.6, 49.9, 44.6, 21.9. Anal. Calcd for C20H16ClN5O5S2: C, 47.48;
H, 3.19; N, 13.84. Found: C, 47.66; H, 3.24; N, 13.92.
4.1.3. Preparation of tert-butyl 2-amino-3-carbamoyl-4,5-
dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate (IV)
4.1.9. 6-(4-Nitrophenylcarbamoyl)-2-(5-nitrothiophene-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (2)
To the stirred solution of compound II (3.0 g, 15.07 mmol), 2-
cyanoacetamide (1.51 g, 18.09 mmol), sulfur powder (1.06 g,
15.07 mmol) in ethanol (40 mL) was added morpholine
(3.21 mL, 33.15 mmol) and stirred the reaction mixture at room
temperature for 9 h. the reaction mixture was concentrated, di-
luted with EtOAc and washed the organic layer with H2O
(2 Â 30 mL). The separated organic layer was dried over anhy-
drous Na2SO4, evaporated and purified by column chromatogra-
phy to get compound 4 (3.67 g, 82%) as an light yellow solid.
ESI-MS found 298 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d
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).
Yield: 79%; MS(ESI) m/z 517 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.42 (s, 1H), 11.26 (s, 1H), 8.41 (d, J = 7.2 Hz, 1H), 8.22 (d,
J = 7.2 Hz, 1H), 7.87–7.69 (m, 6H), 4.72 (s, 2H), 4.03 (t, J = 6.8 Hz,
2H), 3.31 (t, J = 6.8 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) d
180.1, 171.4, 165.2, 164.9, 156.3, 146.4, 142.6, 139.9, 137.6,
133.4, 132.6(2C), 129.9, 129.1(2C), 127.4, 109.6, 47.9, 42.3, 20.1.
Anal. Calcd for C20H16N6O7S2: C, 46.51; H, 3.12; N, 16.27. Found:
C, 46.66; H, 3.24; N, 16.33.
4.1.10. 6-(Benzylcarbamoyl)-2-(5-nitrothiophene-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (3)
4.1.4. General procedure for the preparation of compound V
5-Nitrothiophene-2-carboxylic acid (2.0 g) was taken in
a
Yield: 81%; MS(ESI) m/z 486 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.42 (s, 1H), 11.62 (s, 1H), 8.31 (d, J = 7.2 Hz, 1H), 8.13 (d,
J = 7.2 Hz, 1H), 7.81–7.63 (m, 7H), 5.11 (s, 2H), 4.63 (s, 2H), 4.03
(t, J = 6.8 Hz, 2H), 3.01 (t, J = 6.8 Hz, 2H); 13C NMR (100 MHz, CDCl3)
d 178.3, 172.8, 166.2, 163.9, 158.3, 152.5, 147.2, 142.6, 133.9,
135.5(2C), 133.4, 130.3(2C), 128.4, 126.9, 116.7, 52.4, 46.9, 42.6,
19.2. Anal. Calcd for C21H19N5O5S2: C, 51.95; H, 3.94; N, 14.42.
Found: C, 52.00; H, 3.97; N, 14.54.
100 mL single neck RB flask equipped with N2-inlet, to this was
added CH2Cl2 (30 mL), OxallylChloride (6.0 mL, 3 V) at 0 °C and
stirred for few minutes. Then DMF (few drops) was added slowly
(drop wise) at same temperature until evolution of bubbles in
the reaction mixture was stopped, and allowed to stir at room tem-
perature for 3 h. The reaction mixture was concentrated re-dis-
solved in dry CH2Cl2 and used in next step.
4.1.5. General procedure for the preparation of compound VI
To the stirred solution of compound IV (1.00 equiv), in dichloro-
methane at 0 °C, was added DIPEA (2.50 equiv) stirred for few min-
utes then added compound V (1.20 equiv) drop wise under N2 atm
at same temperature, and allowed to stir the reaction mixture at rt
for 6 h. the reaction mixture was diluted with DCM and washed
with H2O, the separated organic layer was dried over anhy Na2SO4
and evaporated under vacuo, the obtained product was purified by
column chromatography using EtOAc/Hexanes as eluent.
4.1.11. 6-(4-Methoxyphenylcarbamoyl)-2-(5-nitrothiophene-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (4)
Yield: 69%; MS(ESI) m/z 502 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.32 (s, 1H), 11.54 (s, 1H), 8.22 (d, J = 7.6 Hz, 1H), 8.08 (d,
J = 7.2 Hz, 1H), 7.83 (d, J = 7.6 Hz, 2H), 7.77 (s, 2H), 7.72 (d,
J = 8.0 Hz, 2H), 4.58 (s, 2H), 4.03 (t, J = 6.8 Hz, 2H), 3.94 (s, 3H),
3.21 (t, J = 6.8 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) d 181.1,
177.8, 164.2, 161.9, 158.3, 154.5, 148.2, 141.6, 136.9, 136.0,
133.4, 132.3(2C), 132.4 (2C), 128.5, 118.5, 56.4, 47.9, 43.6, 21.0.
Anal. Calcd for C21H19N5O6S2: C, 50.29; H, 3.82; N, 13.96. Found:
C, 50.33; H, 3.91; N, 13.99.
4.1.6. General procedure for the preparation of compounds VIIa/
VIIb
To the stirred solution of compound VIa/VIb in DCM at 0 °C un-
der N2 atm, was added TFA (2 volumes) and allowed to stir at room
temperature for 2 h. The reaction mixture was concentrated to dry-
ness and the obtained solids were washed with hexanes to afford
compound VIIa/VIIb respectively.
4.1.12. General procedure for the synthesis of final compounds
5–8, 18–21
To the stirred solution of compound VIIa (for 5–8)/VIIb (for
18–21) in DMF at 0 °C under N2 atm, was added Et3N
(2.0 equiv) followed by addition of arylisothiocyanate (1.2 equiv)
and allowed to stir at room temperature for 3 h. The reaction
mixture was diluted with EtOAc and washed with Brine solu-
tion and H2O. The separated organic layer was dried over anhy
Na2SO4 and evaporated under vacuo; the obtained product was
purified by column chromatography using EtOAc/Hexanes as
eluent.
4.1.7. General procedure for the synthesis of final compounds
1–4, 14–17
To the stirred solution of compound VIIa (for 1–4)/VIIb (for
14–17) in DMF at 0 °C under N2 atm, was added Et3N
(2.0 equiv) followed by addition of Arylisocyanate (1.2 equiv)
and allowed to stir at room temperature for 3 h. The reaction
mixture was diluted with EtOAc and washed with Brine solu-
tion and H2O. The separated organic layer was dried over anhy
Na2SO4 and evaporated under vacuo; the obtained product was
purified by column chromatography using EtOAc/Hexanes as
eluent.
4.1.13. 6-(4-Fluorophenylcarbamothioyl)-2-(5-nitrothiophene-
2-carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (5)
Yield: 76%; MS(ESI) m/z 506 [M+H]+. 1H NMR (400 MHz,
DMSO-d6): d 12.43 (s, 1H), 8.31 (d, J = 7.2 Hz, 1H), 8.03 (d,
J = 7.2 Hz, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.87 (d, J = 8.0 Hz, 2H),
7.81–7.74 (m, 3H), 4.82 (s, 2H), 3.89 (t, J = 7.2 Hz, 2H), 3.04 (t,
J = 7.2 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) d 180.8, 180.0,
171.9, 164.1, 160.9, 158.2, 146.2, 142.4, 141.5, 139.4, 134.3(2C),
133.8, 130.0(2C), 129.0, 116.1, 47.3, 43.2, 22.3. Anal. Calcd for
4.1.8. 6-(4-Chlorophenylcarbamoyl)-2-(5-nitrothiophene-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (1)
Yield: 82%; MS(ESI) m/z 506 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.21 (s, 1H), 11.23 (s, 1H), 8.31 (d, J = 7.6 Hz, 1H), 8.10 (d,
J = 7.2 Hz, 1H), 7.90 (d, J = 8.0 Hz, 2H), 7.81–7.72 (m, 4H), 4.66 (s,
2H), 3.91 (t, J = 6.8 Hz, 2H), 3.13 (t, J = 6.8 Hz, 2H); 13C NMR
(100 MHz, DMSO-d6) d 179.1, 172.8, 165.2, 162.9, 150.3, 148.4,
C20H16FN5O4S3: C, 47.51; H, 3.19; N, 13.85. Found: C, 47.61; H,
3.23; N, 13.88.

G. Samala et al. / Bioorg. Med. Chem. 22 (2014) 1938–1947
1945
4.1.14. 6-(4-Chlorophenylcarbamothioyl)-2-(5-nitrothiophene-
2-carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (6)
J = 6.8 Hz, 2H), 3.04 (t, J = 6.8 Hz, 2H); 13C NMR (100 MHz, DMSO-
d6) 171.1, 166.7, 166.3, 163.4, 157.6, 146.5, 142.3, 138.6,
137.2(2C), 135.1(2C), 132.3, 128.5, 124.4, 120.4, 49.3, 46.2, 21.6.
Anal. Calcd for C19H15BrN4O6S3: C, 39.93; H, 2.65; N, 9.80. Found:
C, 39.99; H, 2.71; N, 9.88.
d
Yield: 64%; MS(ESI) m/z 523 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.54 (s, 1H), 8.29 (d, J = 7.2 Hz, 1H), 8.16 (d, J = 7.2 Hz, 1H),
7.86 (d, J = 8.0 Hz, 2H), 7.83–7.74 (m, 5H), 4.72 (s, 2H), 3.96 (t,
J = 7.2 Hz, 2H), 3.11 (t, J = 7.2 Hz, 2H); 13C NMR (100 MHz, DMSO-
d6) d 178.8, 177.1, 168.8, 161.1, 159.3, 148.2, 144.2, 143.4, 141.9,
138.4, 136.3(2C), 133.2, 132.7(2C), 126.0, 116.5, 49.3, 42.3, 21.0.
Anal. Calcd for C20H16ClN5O4S3: C, 46.02; H, 3.09; N, 13.42. Found:
C, 46.09; H, 3.11; N, 13.49.
4.1.20. 6-(4-Nitrophenylsulfonyl)-2-(5-nitrothiophene-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (11)
Yield: 78%; MS(ESI) m/z 538 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.99 (s, 1H), 8.41 (d, J = 8.4 Hz, 2H), 8.10 (d, J = 8.4 Hz, 2H),
7.94 (s, 1H), 7.82 (d, J = 7.2 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H), 4.41 (s,
4.1.15. 6-(4-Nitrophenylcarbamothioyl)-2-(5-nitrothiophene-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (7)
2H), 3.46 (t, J = 6.4 Hz, 2H), 2.92 (t, J = 6.4 Hz, 2H), 2.51 (s, 1H); 13
C
NMR (100 MHz, CDCl3) d 166.9, 152.5, 151.2, 150.0, 146.2, 142.6,
142.0, 128.9(2C), 127.9, 124.7(2C), 122.3, 117.8, 116.8, 113.7,
44.3, 43.1, 25.2. Anal. Calcd for C19H15N5O8S3: C, 42.45; H, 2.81;
N, 13.03. Found: C, 42.51; H, 2.88; N, 13.06.
Yield: 79%; MS(ESI) m/z 533 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.38 (s, 1H), 11.22 (s, 1H), 8.37 (d, J = 7.2 Hz, 1H), 8.18 (d,
J = 7.2 Hz, 1H), 8.01 (d, J = 7.6 Hz, 2H), 7.83–7.72 (m, 4H), 4.69 (s,
2H), 3.94 (t, J = 6.8 Hz, 2H), 3.23 (t, J = 6.8 Hz, 2H); 13C NMR
(100 MHz, DMSO-d6) d 179.8, 178.9, 168.2, 161.9, 158.3, 148.2,
143.6, 141.9, 138.9, 136.4, 135.9(2C), 134.1, 133.9(2C), 127.0,
120.6, 50.2, 45.0, 22.5. Anal. Calcd for C20H16N6O6S3: C, 45.10; H,
3.03; N, 15.78. Found: C, 45.21; H, 3.09; N, 15.81.
4.1.21. 6-(4-Acetylphenylsulfonyl)-2-(5-nitrothiophene-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (12)
Yield: 81%; MS(ESI) m/z [M+H]+. 1H NMR (400 MHz, DMSO-d6):
d 11.91 (s, 1H), 8.01 (d, J = 7.2 Hz, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.81–
7.72 (m, 6H), 4.32 (s, 2H), 4.03 (t, J = 6.8 Hz, 2H), 2.99 (t, J = 6.8 Hz,
2H), 2.23 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 172.9, 169.9,
167.4, 162.9, 160.3, 144.4, 144.0, 142.9, 141.6, 138.9 (2C), 136.2,
131.4(2C), 125.2, 121.2, 114.6, 48.2, 44.4, 24.2, 21.4. Anal. Calcd
for C21H18N4O7S3: C, 47.18; H, 3.39; N, 10.48. Found: C, 47.21; H,
3.44; N, 10.53.
4.1.16. 6-(Benzylcarbamothioyl)-2-(5-nitrothiophene-2-carbo
xamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carbo
xamide (8)
Yield: 67%; MS(ESI) m/z 502 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.24 (s, 1H), 10.06 (s, 1H), 8.52 (t, J = 6.4 Hz, 1H), 8.01 (d,
J = 7.2 Hz, 1H), 7.62–7.45 (m, 7H), 5.19 (s, 2H), 4.81 (s, 2H), 4.12
(t, J = 7.2 Hz, 2H), 3.01 (t, J = 7.2 Hz, 2H); 13C NMR (100 MHz,
DMSO-d6) d 180.7, 171.9, 161.1, 160.9, 149.3, 139.4, 138.6, 136.9,
134.6, 133.9 (2C), 130.7 (2C), 127.4, 126.4, 116.6, 112.6, 49.2,
43.8, 22.5, 20.5. Anal. Calcd for C21H19N5O4S3: C, 50.28; H, 3.82;
N, 13.96. Found: C, 50.31; H, 3.87; N, 14.04.
4.1.22. 6-(4-Methoxyphenylsulfonyl)-2-(5-nitrothiophene-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (13)
Yield: 63%; MS(ESI) m/z 523 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 11.56 (s, 1H), 8.31 (d, J = 7.2 Hz, 1H), 7.92 (d, J = 7.2 Hz, 1H),
7.81 (d, J = 7.6 Hz, 2H), 7.74 (s, 2H), 7.69 (d, J = 8.0 Hz, 2H), 4.63 (s,
4.1.17. General procedure for the synthesis of final compounds
9–13, 22–26
2H), 4.12 (t, J = 6.8 Hz, 2H), 3.89 (s, 3H), 3.12 (t, J = 6.8 Hz, 2H); 13
C
NMR (100 MHz, DMSO-d6) d 176.1, 172.0, 169.2, 164.2, 161.9,
152.2, 148.2, 141.4, 134.4, 133.2(2C), 132.1, 129.9(2C), 124.4,
119.2, 62.1, 49.3, 45.6, 24.0. Anal. Calcd for C20H18N4O7S3: C,
45.97; H, 3.47; N, 10.72. Found: C, 45.99; H, 3.51; N, 10.79.
To the stirred solution of compound VIIa (for 9–13)/VIIb (for
22–26) in DMF at 0 °C under N2 atm, was added Et3N (2.0 equiv)
followed by addition of ArylsulfonylChloride (1.2 equiv) and al-
lowed to stir at room temperature for 4 h. The reaction mixture
was diluted with EtOAc and washed with Brine solution and
H2O. The separated organic layer was dried over anhy Na2SO4
and evaporated under vacuo; the obtained product was purified
by column chromatography using EtOAc/Hexanes as eluent.
4.1.23. 6-(4-Chlorophenylcarbamoyl)-2-(5-nitrofuran-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (14)
Yield: 71%; MS(ESI) m/z 490 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.32 (s, 1H), 10.44 (s, 1H), 8.19 (d, J = 7.6 Hz, 1H), 8.01 (d,
J = 7.2 Hz, 1H), 7.81 (d, J = 8.0 Hz, 2H), 7.72 (d, J = 8.0 Hz, 2H),
7.63 (s, 2H), 4.45 (s, 2H), 3.94 (t, J = 7.2 Hz, 2H), 3.04 (t, J = 6.8 Hz,
2H); 13C NMR (100 MHz, DMSO-d6) d 183.2, 172.5, 166.4, 154.2,
151.2, 149.5, 142.5, 139.5, 136.8, 132.6(2C), 130.7, 128.5(2C),
126.4, 123.3, 114.4, 46.7, 45.9, 21.4. Anal. Calcd for C20H16ClN5O6S:
C, 49.03; H, 3.29; N, 14.30. Found: C, 49.16; H, 3.34; N, 14.41.
4.1.18. 6-(4-Fluorophenylsulfonyl)-2-(5-nitrothiophene-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (9)
Yield: 67%; MS(ESI) m/z 511 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.03 (s, 1H), 8.22 (d, J = 7.6 Hz, 1H), 7.99 (d, J = 7.2 Hz, 1H),
7.92 (d, J = 8.0 Hz, 2H), 7.81–7.69 (m, 4H), 4.61 (s, 2H), 3.99 (t,
J = 6.8 Hz, 2H), 3.01 (t, J = 6.8 Hz, 2H); 13C NMR (100 MHz, CDCl3)
d 172.1, 169.7, 166.3, 164.1, 162.6, 146.2, 145.3, 142.5, 139.4(2C),
136.3, 133.2(2C), 130.5, 126.0, 119.4, 48.4, 44.3, 22.2. Anal. Calcd
for C19H15FN4O6S3: C, 44.70; H, 2.96; N, 10.97. Found: C, 44.73;
H, 3.03; N, 10.98.
4.1.24. 6-(4-Nitrophenylcarbamoyl)-2-(5-nitrofuran-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (15)
Yield: 66%; MS(ESI) m/z 501 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.23 (s, 1H), 11.11 (s, 1H), 8.31 (d, J = 7.2 Hz, 1H), 8.19 (d,
J = 7.2 Hz, 1H), 7.92 (d, J = 7.6 Hz, 2H) 7.83–7.67 (m, 4H), 4.74 (s,
2H), 4.12 (t, J = 7.2 Hz, 2H), 3.21 (t, J = 6.8 Hz, 2H); 13C NMR
(100 MHz, DMSO-d6) d 180.7, 166.5, 162.3, 155.4, 152.1, 148.2,
145.4, 142.1, 136.2, 133.2, 130.4(2C), 127.8, 126.5(2C), 124.5,
115.4, 47.4, 45.4, 21.4. Anal. Calcd for C20H16N6O8S: C, 48.00; H,
3.22; N, 16.79. Found: C, 48.03; H, 3.24; N, 16.82.
4.1.19. 6-(4-Bromophenylsulfonyl)-2-(5-nitrothiophene-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (10)
Yield: 74%; MS(ESI) m/z 571 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 11.82 (s, 1H), 8.19 (d, J = 7.2 Hz, 1H), 7.92 (d, J = 7.2 Hz, 1H),
7.87 (d, J = 8.0 Hz, 2H), 7.83–7.72 (m, 4H), 4.59 (s, 2H), 4.12 (t,

1946
G. Samala et al. / Bioorg. Med. Chem. 22 (2014) 1938–1947
4.1.25. 6-(Benzylcarbamoyl)-2-(5-nitrofuran-2-carboxamido)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide (16)
Yield: 67%; MS(ESI) m/z 470 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.25 (s, 1H), 11.31 (s, 1H), 8.41 (d, J = 7.2 Hz, 1H), 8.08 (d,
J = 7.2 Hz, 1H), 7.87–7.72 (m, 7H), 5.22 (s, 2H), 4.52 (s, 2H), 4.11
(t, J = 6.8 Hz, 2H), 3.04 (t, J = 6.8 Hz, 2H); 13C NMR (100 MHz,
DMSO-d6) d 182.0, 169.2, 162.3, 157.5, 150.4, 148.3, 143.4, 137.2,
133.4, 133.0(2C), 132.6, 130.3(2C), 127.8, 126.9, 112.7, 48.1, 46.4,
43.6, 19.9. Anal. Calcd for C21H19N5O6S: C, 53.73; H, 4.08; N,
14.92. Found: C, 53.81; H, 4.16; N, 14.96.
J = 7.2 Hz, 1H), 7.81 (d, J = 7.6 Hz, 2H), 7.74–7.63 (m, 5H), 5.11 (s,
2H), 4.39 (s, 2H), 4.13 (t, J = 6.8 Hz, 2H), 2.9 (t, J = 6.8 Hz, 2H); 13C
NMR (100 MHz, DMSO-d6) d 178.3, 172.6, 166.2, 160.9, 155.4,
154.1, 145.7, 144.9, 143.4, 137.4, 135.5, 132.6(2C), 129.4,
127.4(2C), 117.7, 63.0, 49.2, 46.7, 20.6. Anal. Calcd for C21H19N5O5-
S2: C, 51.95; H, 3.94; N, 14.42. Found: C, 52.06; H, 3.91; N, 14.56.
4.1.31. 6-(4-Fluorophenylsulfonyl)-2-(5-nitrofuran-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (22)
Yield: 74%; MS(ESI) m/z 495 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.13 (s, 1H), 8.31 (d, J = 7.6 Hz, 1H), 8.09 (d, J = 7.2 Hz, 1H),
7.89 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 7.6 Hz, 2H), 7.69 (s, 2H), 4.51 (s,
2H), 4.09 (t, J = 7.2 Hz, 2H), 3.04 (t, J = 6.8 Hz, 2H); 13C NMR
(100 MHz, DMSO-d6) d 183.6, 169.9, 168.3, 163.6, 149.9, 149.2,
141.4, 138.6, 136.5, 132.0(2C), 129.4, 127.6(2C), 123.2, 111.9,
48.3, 47.3, 21.4. Anal. Calcd for C19H15FN4O7S2: C, 46.15; H, 3.06;
N, 11.33. Found: C, 46.23; H, 3.13; N, 11.38.
4.1.26. 6-(4-Methoxyphenylcarbamoyl)-2-(5-nitrofuran-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (17)
Yield: 74%; MS(ESI) m/z 486 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.34 (s, 1H), 11.76 (s, 1H), 8.32 (d, J = 7.2 Hz, 1H), 8.21 (d,
J = 7.2 Hz, 1H), 7.92 (d, J = 7.6 Hz, 2H), 7.81 (s, 2H), 7.72 (d,
J = 7.6 Hz, 2H), 4.48 (s, 2H), 4.08 (t, J = 6.8 Hz, 2H), 3.94 (s, 3H),
3.13 (t, J = 6.8 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) d 181.7,
168.9, 164.3, 153.9, 151.0, 148.6, 144.8, 141.6, 137.6, 136.4,
133.6(2C), 130.5(2C), 126.9, 124.6, 114.4, 61.2, 49.0, 47.6, 20.7.
Anal. Calcd for C21H19N5O7S: C, 51.95; H, 3.94; N, 14.43. Found:
C, 52.03; H, 3.92; N, 14.58.
4.1.32. 6-(4-Bromophenylsulfonyl)-2-(5-nitrofuran-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (23)
Yield: 76%; MS(ESI) m/z 555 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.22 (s, 1H), 8.21 (d, J = 7.2 Hz, 1H), 8.02 (d, J = 7.2 Hz, 1H),
7.92 (d, J = 8.0 Hz, 2H), 7.81–7.69 (m, 4H), 4.47 (s, 2H), 4.04 (t,
J = 6.8 Hz, 2H), 2.89 (t, J = 6.8 Hz, 2H); 13C NMR (100 MHz, DMSO-
d6) d 180.7, 176.6, 172.7, 162.6, 160.2, 147.2, 144.6, 133.2(2C),
132.6, 132.0(2C), 127.3, 126.9, 124.2, 117.2, 48.4, 47.3, 21.6. Anal.
Calcd for C19H15BrN4O7S2: C, 41.09; H, 2.72; N, 10.09. Found: C,
41.17; H, 2.81; N, 10.17.
4.1.27. 6-(4-Fluorophenylcarbamothioyl)-2-(5-nitrofuran-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (18)
Yield: 70%; MS(ESI) m/z 490 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.23 (s, 1H), 10.24 (s, 1H), 8.26 (d, J = 7.2 Hz, 1H), 7.98 (d,
J = 7.2 Hz, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.84 (d, J = 8.0 Hz, 2H),
7.78 (s, 2H), 4.69 (s, 2H), 3.93 (t, J = 7.2 Hz, 2H), 3.00 (t, J = 7.2 Hz,
2H); 13C NMR (100 MHz, DMSO-d6) d 179.1, 176.4, 164.2, 158.4,
152.2, 150.6, 147.4, 145.3, 141.4, 139.4, 134.4, 133.2(2C), 130.4,
126.6(2C), 114.3, 47.3, 46.1, 19.9. Anal. Calcd for C20H16FN5O5S2:
C, 49.07; H, 3.29; N, 14.31. Found: C, 49.11; H, 3.31; N, 14.40.
4.1.33. 2-(5-Nitrofuran-2-carboxamido)-6-(4-
nitrophenylsulfonyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-
3-carboxamide (24)
Yield: 79%; MS(ESI) m/z 522 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.97 (s, 1H), 8.41 (d, J = 7.5 Hz, 2H), 8.09 (d, J = 7.2 Hz, 2H),
7.94–7.56 (m, 4H), 4.35 (s, 2H), 3.03 (t, J = 7.2 Hz, 2H), 2.72 (t,
J = 6.9 Hz, 2H); 13C NMR (75 MHz, CDCl3) d 166.9, 162.3, 152.5,
149.3, 146.8, 143.2, 142.8, 128.2(2C), 127.2, 123.8(2C), 121.9,
117.2, 116.4, 114.2, 45.0, 44.2, 24.9. Anal. Calcd for C19H15N5O9S2:
C, 43.76; H, 2.90; N, 13.43. Found: C, 43.81; H, 2.93; N, 13.50.
4.1.28. 6-(4-Chlorophenylcarbamothioyl)-2-(5-nitrofuran-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (19)
Yield: 62%; MS(ESI) m/z 506 [M+H]+. 1H NMR (300 MHz, DMSO-
d6): d 13.01 (s, 1H), 9.55 (s, 1H), 7.87 (d, J = 3.6 Hz, 1H), 7.58 (d,
J = 3.9 Hz, 1H), 7.45–7.27 (m, 6H), 5.10 (s, 2H), 4.11 (t, J = 6.9 Hz,
2H), 3.03 (t, J = 6.9 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) d 181.1,
167.1, 152.5, 151.6, 146.3, 142.4, 139.8, 129.0, 128.6, 127.9(2C),
127.2(2C), 124.2, 117.8, 117.0, 113.8, 47.2, 45.4, 25.3. Anal. Calcd
for C20H16ClN5O5S2: C, 47.48; H, 3.19; N, 13.84. Found: C, 47.52;
H, 3.24; N, 13.89.
4.1.34. 6-(4-Acetylphenylsulfonyl)-2-(5-nitrofuran-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (25)
Yield: 72%; MS(ESI) m/z 519 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.21 (s, 1H), 8.11 (d, J = 7.2 Hz, 1H), 7.99 (d, J = 7.2 Hz, 1H),
7.92 (d, J = 7.6 Hz, 2H), 7.81–7.72 (m, 4H), 4.26 (s, 2H), 4.07 (t,
J = 6.8 Hz, 2H), 3.09 (t, J = 6.8 Hz, 2H), 2.20 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) d 184.1, 172.6, 169.8, 160.4, 146.7, 144.2,
142.4, 139.5, 136.8, 134.2(2C), 132.4(2C), 128.4, 125.3, 123.6,
114.3, 47.5, 46.9, 22.6, 20.7. Anal. Calcd for C21H18N4O8S2: C,
48.64; H, 3.50; N, 10.81. Found: C, 48.71; H, 3.57; N, 10.89.
4.1.29. 2-(5-Nitrofuran-2-carboxamido)-6-(4-nitrophenyl
carbamothioyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (20)
Yield: 69%; MS(ESI) m/z 517 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.37 (s, 1H), 11.18 (s, 1H), 8.34 (d, J = 7.2 Hz, 1H), 8.10 (d,
J = 7.2 Hz, 1H), 7.81–7.72 (m, 6H), 4.66 (s, 2H), 4.12 (t, J = 6.8 Hz,
2H), 3.22 (t, J = 6.8 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) d
180.2, 171.9, 167.8, 158.4, 156.7, 144.8, 141.6, 135.7, 134.2,
133.3(2C), 132.7(2C), 130.5, 126.3, 124.2, 112.4, 48.1, 46.6, 19.8.
Anal. Calcd for C20H16N6O7S2: C, 46.51; H, 3.12; N, 16.27. Found:
C, 46.60; H, 3.18; N, 16.31.
4.1.35. 6-(4-Methoxyphenylsulfonyl)-2-(5-nitrofuran-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (26)
Yield: 74%; MS(ESI) m/z 507 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.06 (s, 1H), 8.21 (d, J = 7.2 Hz, 1H), 7.99 (d, J = 7.2 Hz, 1H),
7.80 (d, J = 7.6 Hz, 2H), 7.72–7.58 (m, 4H), 4.53 (s, 2H), 4.04 (t,
J = 6.8 Hz, 2H), 3.90 (s, 3H), 3.04 (t, J = 6.8 Hz, 2H); 13C NMR
(100 MHz, DMSO-d6) d 177.1, 172.6, 169.9, 164.3, 163.8, 144.9,
138.3, 135.4, 132.4, 129.4, 125.1(2C), 124.4(2C), 123.5, 116.3,
60.5, 47.4, 45.9, 22.0. Anal. Calcd for C20H18N4O8S2: C, 47.43; H,
3.58; N, 11.06. Found: C, 47.49; H, 3.60; N, 11.09.
4.1.30. 6-(Benzylcarbamothioyl)-2-(5-nitrofuran-2-
carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (21)
Yield: 66%; MS(ESI) m/z 486 [M+H]+. 1H NMR (400 MHz, DMSO-
d6): d 12.19 (s, 1H), 10.74 (s, 1H), 8.29 (d, J = 7.6 Hz, 1H), 8.13 (d,

G. Samala et al. / Bioorg. Med. Chem. 22 (2014) 1938–1947
1947
4.2. Biological activity
concentration of compound required to give complete inhibition
ofbacterial growth. This method is similar to that recommended
by the National Committee for Clinical Laboratory Standards for
the determination of MIC in triplicate.
4.2.1. Protein expression and MTB PS screening
The MTB panC gene (Rv3602c) encoding the pantothenate syn-
thetase was cloned and transformed into BL21 (DE3) cells and the
expression of the protein was performed as reported in literature.7
4.2.4. In vitro cytotoxicity screening
For the assay, in a 96-well plate, 60
l
L of PS reagent mix containing
Some compounds were further examined for toxicity in a RAW
NADH, pantoic acid, b-alanine, ATP, phosphoenol pyruvate, MgCl2,
264.7 cell line at the concentration of 50 lM. After 72 h of expo-
myokinase, pyruvate kinase, and lactate dehydrogenase in buffer
sure, 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.
was added. compounds were then added to plates in 1-
lL volumes.
The reaction was initiated with the addition of 39 L of PS, diluted
l
in buffer. The test plate was immediately transferred to a micro
plate reader, and absorbance was measured at 340 nm every 12 s
for 120 s.7 Percentage inhibition was calculated using following
formula, 100 ⁄1 À compoundrate – background rate/full reaction
rate À background rate.
Acknowledgments
The authors are thankful to Department of Science & Technol-
ogy (SR/S1/OC-70/2010), New Delhi, India for their financial assis-
tances. G.S. thankful to CSIR-India. We also acknowledge Dr David
Eisenberg and Dr Shuishu Wang, Department of Biochemistry &
Molecular biology, USUHS, Maryland for providing MTB PS clone.
DS acknowledges University Grants Commission, New Delhi, India
for UGC Research Award.
4.2.2. Molecular docking
The computation was carried out using Schrodinger 2011
molecular modeling software package. Docking was performed
by using the Glide v5.7, integrated with Maestro 9.2 (Schrödinger,
LLC, 2011) interface on the linux operating system. The crystal
structure of pantothenate synthetase co-crystallized with 2-(2-
(benzofuran-2-ylsulfonylcarbamoyl)-5-methoxy-1H-indol-1-yl) acetic
acid [PDB: 3IVX] was retrieved from the Protein Data Bank
(www.rcsb.org). The reported crystal structure is a dimer, having
two asymmetric units (A and B) with the inhibitor bound to both
of them. In this study, the asymmetric unit A was isolated and used
further for docking. The protein was prepared using protein prep-
aration wizards and the binding site was defined by a rectangular
box surrounding the ligand in the X-ray structure. Ligands were
refined using the ‘Refine’ option in Glide, and the Glide XP (extra
precision) descriptor was chosen. Default settings were used for
refinement and scoring.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.01.030.
References and notes
1. Dye, C.; Scheele, S.; Dolin, P.; Pathania, V.; Raviglione, M. C. J. Am. Med. Assoc.
1999, 282, 677.
2. Stokstad, E. Science 2000, 287, 239.
3. Lynch, J. B. Med. Clin. North Am. 2013, 97, 553.
4. Palomino, J. C.; Martin, A. Curr. Med. Chem. 2013, 20, 3785.
5. Tsuyuguchi, I. Tuberculosis 2001, 81, 221.
6. Merkel, W. K.; Nichols, B. P. FEMS Microbiol. Lett. 1996, 143, 247.
7. Zheng, R.; Blanchard, J. S. Biochemistry 2001, 40, 12904.
8. Wang, S.; Eisenberg, D. Protein Sci. 2003, 12, 1097.
9. Samala, G.; Devi, P. B.; Nallangi, R.; Yogeeswari, P.; Sriram, D. Eur. J. Med. Chem.
2013, 69, 356.
4.2.3. In vitro MTB screening
Two-fold serial dilutions of each test compound/drug were pre-
pared and incorporated into Middle-brook 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,
10. Velaparthi, S.; Brunsteiner, M.; Uddin, R.; Wan, B.; Franzblau, S. G.; Petukhov, P.
A. J. Med. Chem. 2008, 51, 1999.
11. Yang, Y.; Gao, P.; Liu, Y.; Ji, X.; Gan, M.; Guan, Y.; Hao, X.; Li, Z.; Xiao, C. Bioorg.
Med. Chem. Lett. 2011, 21, 3943.
12. White, E. L.; Southworth, K.; Ross, L.; Cooley, S.; Gill, R. B.; Sosa, M. I.;
Manouvakhova, A.; Rasmussen, L.; Goulding, C.; Eisenberg, D.; Fletcher, T. M. J.
Biomol. Screen. 2007, 12, 100.
13. Hung, A. W.; Silvestre, H. L.; Wen, S.; Ciulli, A.; Blundell, T. L.; Abell, C. Angew.
Chem. 2009, 48, 8452.
14. Reynolds, R. C.; Ananthan, S.; Faaleolea, E.; Hobrath, J. V.; Kwong, C. D.;
Maddox, C.; Rasmussen, L.; Sosa, M. I.; Thammasuvimol, E.; White, E. L.; Zhang,
W., ; Secrist, J. A., III Tuberculosis 2012, 92, 72.
1.56, and 0.78 lg/mL. Inoculum of M. tuberculosis H37Rv ATCC
27294 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 10À2 to give a con-
centration of ꢀ107 cfu/mL. Five microliters of this bacterial suspen-
sion 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
lg/mL) were
15. Huang, Y.; Dömling, A. Mol. Divers. 2011, 15, 3.
determined after 28 days. The MIC is defined as the minimum