Thiazole-aminopiperidine hybrid analogues: Design and synthesis of novel Mycobacterium tuberculosis GyrB inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.09.025

A series of ethyl-4-(4-((substituted benzyl)amino)piperidin-1-yl)-2- (phenyl/pyridyl)thiazole-5-carboxylates was designed by molecular hybridization and synthesized from aryl thioamides in five steps. The compounds were evaluated for their in vitro Mycoba

Full text of DOI:10.1016/j.ejmech.2013.09.025

European Journal of Medicinal Chemistry 70 (2013) 143e153
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Thiazoleeaminopiperidine hybrid analogues: Design and synthesis of
novel Mycobacterium tuberculosis GyrB inhibitors
Variam Ullas Jeankumar, Janupally Renuka, Peddi Santosh, Vijay Soni,
Jonnalagadda Padma Sridevi, Priyanka Suryadevara, 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:
Received 12 June 2013
Received in revised form
A series of ethyl-4-(4-((substituted benzyl)amino)piperidin-1-yl)-2-(phenyl/pyridyl)thiazole-5-carboxy
lates was designed by molecular hybridization and synthesized from aryl thioamides in five steps. The
compounds were evaluated for their in vitro Mycobacterium smegmatis (MS) GyrB ATPase assay, Myco-
bacterium tuberculosis (MTB) DNA gyrase super coiling assay, antituberculosis activity and cytotoxicity.
Among the twenty four compounds studied, ethyl-4-(4-((4-fluorobenzyl)amino)piperidin-1-yl)-2-
phenylthiazole-5-carboxylate (14) was found to be the promising compound which showed activity
6
September 2013
Accepted 7 September 2013
Available online 3 October 2013
against all test with MS GyrB IC50 of 24.0 ꢀ 2.1
m
M, 79% inhibition of MTB DNA gyrase at 50
mM, MTB MIC
Keywords:
of 28.44 M, and not cytotoxic at 50 M.
m
m
Tuberculosis
DNA gyrase
GyrB subunit
Thiazole
Ó 2013 Elsevier Masson SAS. All rights reserved.
Amino piperidine
1
. Introduction
(bedaquiline) has recently been granted licensure by the U.S. Food
and Drug Administration under its accelerated approval procedure.
The results of the studies with moxifloxacin, a potent inhibitor of
DNA gyrase subunit A (GyrA), suggest that DNA gyrase may be a
good target for reducing the length of TB treatment regimens. DNA
gyrase is an essential enzyme that introduces negative supercoils
into DNA and regulates the super helical state of the bacterial
chromosomes. Gyrase consists of two subunits, GyrA and GyrB,
which form an A2B2 complex in the active enzyme in which a
segment of DNA is wrapped around the protein. The GyrA subunit
interacts with DNA and contains the active-site tyrosine respon-
sible for DNA cleavage and the formation of a protein-DNA covalent
bond during the reaction cycle. GyrB also interacts with DNA and
contains the ATPase active site [2]. GyrA has been well understood
and all fluoroquinolones target this subunit and not much has been
explored on the GyrB subunit except one approved antibiotic,
novobiocin being withdrawn from the market due to safety con-
cerns and poor pharmacological properties. GyrB has been genet-
ically demonstrated to be a bactericidal drug target in MTB, but
there have not been any effective therapeutics developed against
this target for TB [3]. This enzyme is present in all prokaryotes and
is the only type II topoisomerase enzyme in MTB. Thus it seems to
be an attractive target for developing novel drugs against TB; as the
Tuberculosis (TB) is contagious and airborne infection caused by
Mycobacterium tuberculosis (MTB). In 2011, there were an estimated
.7 million new cases of TB (13% co-infected with HIV) and 1.4
8
million people died from TB, including almost one million deaths
among HIV-negative individuals and 430,000 among people who
were HIV-positive. Worldwide, 3.7% of new cases and 20% of pre-
viously treated cases were estimated to have multi drug-resistant
TB (MDR-TB). Extensively drug-resistant TB, or XDR-TB, has been
reported by 84 countries; the average proportion of MDR-TB cases
with XDR-TB is 9.0% [1]. There are reports on Phase II and III trials
on fluoroquinolones and some new dosages of known drugs
(
64 4
e.g. rifamycin (C37H46NO12), rifapentine (C47H N O12)). Novel
drugs are being evaluated in Phase IIb and III trials, including two
drugs that are being tested for the treatment of MDR-TB (beda-
quiline (C32
2 2 25 3 4 6
H31BrN O ) and delamanid (C25H F N O )), with dos-
siers submitted to drug regulatory authorities. One of these
*
Corresponding author.
E-mail addresses: drdsriram@yahoo.com, dsriram@hyderabad.bits-pilani.ac.in
(
D. Sriram).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.025

144
V.U. Jeankumar et al. / European Journal of Medicinal Chemistry 70 (2013) 143e153
GyrB ATPase domain has not been explored extensively. Hence in
the present work we initiated drug discovery program targeting
GyrB of MTB and here we describe the design and development of
thiazoleepiperidine hybrids as novel compounds displaying
unique activity against MTB GyrB ATPase and promising antitu-
bercular activity in vitro.
hand substituent. Dichloro substitution and an heteroaryl, nitro
furanyl substituent were also attempted in synthesis as steps to-
wards the derivation of structureeactivity relationships (SAR) and
lead optimization.
The synthetic pathway used to achieve the target compounds
has been delineated in Scheme 1. The construction of target
molecule began by condensing commercially available aryl (phenyl,
2-pyrdiyl and 4-pyridyl) thioamides (8aec) with diethyl bromo-
malonate in toluene under refluxing conditions as previously re-
ported by Kerdesky et al. [21], to form the corresponding 4-hydroxy
thiazole derivatives (9aec). Compounds 9aec on further treatment
with trifluoromethanesulfonic anhydride and pyridine afforded the
corresponding triflate (10aec) in good yield. This was further
condensed with 4-N-Boc-aminopiperidine via a SnAr displacement
to give nitrogen-linked C-4 thiazole analogues (11aec) [10]. Sub-
sequent Boc deprotection of the primary amine followed by
reductive amination of the so obtained scaffold (12aec) with
various substituted benzaldehydes (RHS: as shown in Table 1)
afforded the titled compound (13e36) in good yields [18]. It was
observed that reductive amination gave the best results when
performed with the free base of amine (12aec) in presence of
freshly activated molecular sieves. A series of 24 derivatives were
prepared using the above method for the present study; both
2
. Results and discussion
2.1. Design and synthesis
Molecular hybridization is a relatively new terminology in the
field of drug design and development involving the fusion/hy-
bridization of two or more pharmacophoric subunits from the
molecular structure of ligands/prototypes previously reported to
have an inhibitory effect/potency against the targeted protein/
disease [4]. The newly designed architecture can lead to com-
pounds having improved affinity and efficacy than the parent
compounds with reduced side effects, while retaining the desired
characteristics of the original template. Various literature have
explored this methodology in designing newer analogues as po-
tential candidates for biological evaluation [5]. Encouraged by our
previous successful research efforts in this regard [6,7], it was
decided to further extend the above methodology to identify novel
starting points to design inhibitors for ATPase domain of myco-
bacterial gyrase. Research efforts from both industry and academia
focussed on the development of antibacterial DNA gyraseB in-
hibitors have identified many potent inhibitors belonging to the
chemical class of pyrrolamides [8,9], pyrazolthiazoles [10] indoli-
nones [11] indazoles [12], benzimidazole ureas [13] azaindole [14]
and bithiazoles [15] as potential hits. In spite of a quite successful
progress with respect to the bacterial target, very little has been
achieved when it comes to mycobacterial gyrase and scarcely
anything with regard to mycobacterial GyrB as a target. One
important class among the previously identified antibacterial hits
are the Novel (non-fluoroquinolone) bacterial type II topoisomer-
ase inhibitors (NBTIs) as they act through a novel mechanism of
action and hence are not impacted by target mutation that cause
resistance to fluoroquinolones. Their overall chemical structure
topology as described by GSK researchers incorporates an N-linked
1
13
analytical and spectral data ( H NMR, C NMR, and mass spectra) of
all the synthesized compounds were in full agreement with the
proposed structures.
2.2. Pharmacological evaluation
In the preliminary screening, synthesized compounds were
evaluated for their in vitro Mycobacterium smegmatis (MS) GyrB
ATPase assay. ATPase activity of GyrB protein from MTB was found
to be very low when compared to MS [20]. The reason for less ac-
tivity of GyrB MTB was known because of slow growing mechanism
of MTB when compared to MS. While performing the assay we
considered novobiocin as a standard inhibitor, further it gave a
correlation of approximately 3e5 fold variation in IC50 between the
ATPase activity of MS and supercoiling activity of MTB, this has
given us enough confidence to use MS as a surrogate enzyme for
the DNA GyrB ATPase activity, the other parameters which we
considered were the sequence alignment between MS and MTB
gyrase proteins; which showed almost 87% identity, that proved a
high degree of conservation in the ATP binding pocket of the two
organisms [22].
‘
4-aminopiperidine’ linker fused between a bicyclic left hand nu-
cleus (LHS) and an aryl/heteroaryl right-hand-core (RHS) [16e18].
Research group from GSK have also found success by extending the
above antibacterial aminopiperidine class to the antimicrobial
target as well, with many compounds exhibiting promising inhi-
bition of MTB gyrase [19].
The MS GyrB was cloned into pQE2 vector and was expressed in
BL21 (DE3) pLysS cells, the expressed protein was induced with a
In the present methodology we hybridized in one molecular
platform the aminopiperidine linker (4e6) to thiazole (1e3) core to
give the designed compound (7). The designed ligand thus has
three parts: an N-linked aminopiperidine sandwiched between a
left hand thiazole core and a right hand substituted benzyl moiety
as show in Fig. 1. The presence of a thiazole nucleus in many of the
previously reported GyrB inhibitors (1e3 in Fig. 1) made it an
automatic choice as the left hand core. The selection of the right
hand substituent was purely based on the SAR inputs from the
previous GSK efforts [19], wherein it was found that the TB activity
which did not correlate with broad antibacterial activity could be
optimized at the right hand side. As the most potent analogues in
the study had fluoro, trifluoromethyl, methoxy and nitro
substituted aryl rings on their right hand side, it was decided to
retain fluoro, trifluoromethyl, and methoxy substituted benzyl core
as the right hand analogues in the initial stage of our study as well.
The presence of the hydrophobic pocket in the GyrB domain as
evidenced by the recent crystal structure [20] encouraged us to
attempt a more hydrophobic benzyloxy group as one of the right
final concentration of 0.2 mM IPTG (Isopropyl-
anoside) and was purified using Ni-NTA column and identified by
SDS-PAGE. At 50 M concentration, out of twenty four compounds;
fifteen compounds showed more than 50% inhibition (Table 1) and
at 25 M concentration, only compound 14 showed more than 50%
inhibition. Compounds which showed more than 80% inhibition at
50 M, were regarded as potential hits and were carried forward for
the dose response study. Compound 14 was found to be most active
one with an IC50 of 24.00 ꢀ 1.2 M. Other compounds like 18, 26,
and 36 showed MS GyrB ATPase IC50 of 26.9 ꢀ 2.1 M, 29.1 ꢀ1.7 M,
and 27.12 ꢀ 2.6 M respectively. The standard compound novobi-
ocin showed 96% inhibition at 25
b-D thiogalactopyr-
m
m
m
m
m
m
m
m
M and its IC50 is 200 ꢀ 46 nM.
These molecules selectively inhibited mycobacterial GyrB and were
not active against Staphylococcus aureus GyrB.
In order to analyse the interaction profile of the molecules re-
ported from the assay, compounds were docked to the GyrB ATPase
domain of MS retrieved from protein data bank (PDB IDe4B6C) [20]
using extra precision mode (XP) of Glide module [23]. The inter-
action pattern of the compounds was studied by starting with the

V.U. Jeankumar et al. / European Journal of Medicinal Chemistry 70 (2013) 143e153
145
Fig. 1. Chemical structure of previously reported synthetic inhibitors of DNA Gyrase bearing thiazole nucleus (1e3), aminopiperidine nucleus (4e6) and the inhibitor designed
through molecular hybridization (7).
Scheme 1. .

146
V.U. Jeankumar et al. / European Journal of Medicinal Chemistry 70 (2013) 143e153
Table 1
In vitro evaluation of the synthesized derivatives.
Compd.
Ar
R
1
R
2
R
3
MS GyrB assay (% inhib.)
MTB supercoiling
assay (% inhib.)
MTB MIC
m
M
RAW264.7 Cytotoxicity
at 50 mM (% inhib.)
50
m
M
25
m
M
50
m
M
25 mM
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
2-Pyridyl
2-Pyridyl
2-Pyridyl
2-Pyridyl
2-Pyridyl
2-Pyridyl
2-Pyridyl
2-Pyridyl
4-Pyridyl
4-Pyridyl
4-Pyridyl
4-Pyridyl
4-Pyridyl
4-Pyridyl
4-Pyridyl
4-Pyridyl
Novobiocin
H
H
H
H
H
H
H
H
H
H
H
H
Fluoro
62.32 ꢀ 0.53
96.73 ꢀ 0.74
51.78 ꢀ 0.53
64.57 ꢀ 0.8
31.35 ꢀ 0.85
95.90 ꢀ 0.97
58.3 ꢀ 0.7
28.57 ꢀ 0.5
27.98
79
18.10
24
19.60
34.50
22
38.70
29.99
51.79
31.10
22.37
27.58
66.99
53
26.57
56.90
29.57
19.79
31.78
16.99
39.66
39.77
45.79
11.68
43.89
7.78
9.68
7.97
118.61
28.44
>110.73
47.38
102.13
102.13
>101.95
50.97
59.17
28.37
55.24
47.28
50.96
25.48
>101.74
50.88
59.17
56.75
110.48
47.28
101.93
50.96
101.74
25.44
11.66
6.23
8.40
18.42
22.72
28.12
18.10
9.43
51.22 ꢀ 1.08
28.57 ꢀ 2.63
29.33 ꢀ 0.34
19.17 ꢀ 1.99
48.21 ꢀ 0.86
16.12 ꢀ 0.97
24.13 ꢀ 0.96
15.64 ꢀ 0.49
32.15 ꢀ 0.14
16.99 ꢀ 1.07
27.93 ꢀ 0.77
10.65 ꢀ 0.90
45.15 ꢀ 1.63
29.45 ꢀ 0.49
31.15 ꢀ 0.53
11.66 ꢀ 1.28
26.24 ꢀ 0.53
35.12 ꢀ 0.26
35.15 ꢀ 2.37
15.90 ꢀ 0.46
39.45 ꢀ 1.84
36.16 ꢀ 2.20
45.13 ꢀ 0.91
96.00 ꢀ 0.94
Methoxy
Benzyloxy
Trifluoromethyl
H
Chloro
Chloro
H
H
Trifluoromethyl
Chloro
H
H
H
H
H
H
Trifluoromethyl
Chloro
H
H
H
H
H
H
20.90
6.48
Chloro
H
H
H
H
H
H
H
Chloro
H
H
H
H
H
H
H
55.61 ꢀ 0.97
27.98 ꢀ 0.74
51.53 ꢀ 0.57
38.73 ꢀ 0.64
42.43 ꢀ 0.38
22.28 ꢀ 1.79
84.88 ꢀ 0.56
46.37 ꢀ 0.96
53.30 ꢀ 0.64
22.29 ꢀ 0.37
48.72 ꢀ 0.78
52.72 ꢀ 0.47
73.29 ꢀ 0.76
18.68 ꢀ 0.69
76.38 ꢀ 1.63
52.28 ꢀ 0.65
92.73 ꢀ 0.64
98.2 ꢀ 1.04
28.12
13.88
23.74
13.75
13.88
10.54
34.87
30.68
10.88
30.81
14.34
7.92
18.39
7.74
22.84
15.98
30.69
14.64
12.6
4.62
Fluoro
Methoxy
Benzyloxy
Trifluoromethyl
H
Chloro
Chloro
H
12.84
14.60
19.16
30.12
18.63
10.8
17.45
16.12
8.04
18.12
14.32
32.16
18.42
e
Fluoro
Methoxy
Benzyloxy
Trifluoromethyl
H
Chloro
Chloro
Trifluoromethyl
Chloro
H
Chloro
100
100
>200
analysis of co-crystallized ligand docking pattern. The crystal ligand
6-(3,4-dimethylphenyl)-3-[[4-[3-(4-methylpiperazin-1-yl)pro-
Arg82. The hydrophobic pocket was occupied by the highly lipo-
philic 4-fluorobenzyl core where it was found to be stabilized by
non-polar interactions with Val99 and Met100. The presence of the
fluoro group in the benzyl ring resulted in inductive effect which
(
poxy]phenyl]amino]pyrazine-2-carboxamide) was involved in two
prominent H-bonding interactions, the first one between the amino
group of the carboxamide moiety and the oxygen atom of Asp79 as
shown in Fig. 2. The second hydrogen bond was observed between
the nitrogen atom of piperazine ring and hydrogen atom present on
the guanidine moiety of Arg82. A close analysis of the orientation of
this ligand in the protein active site revealed the presence of a
hydrophobic pocket where the 3,4-dimethyl phenyl moiety was
stabilized by the non-polar interactions with Val99, Met100 and
Val128. Earlier studies have revealed that it’s these hydrophobic
interactions which were detrimental in bringing the selectivity
observed in the enzyme level [20]. In the docking studies too,
compound 14, the most potent analogue from the in vitro GyrB
assay, exhibited good docking score with significant polar and non-
polar contacts. The interaction profile of compound 14 in the pro-
tein active site revealed the presence of a polar contact with Asn52
as shown in Fig. 3. The hydrogen atom present on the secondary
amine which connects the aminopiperidine linker to the 4-flouro
benzyl moiety interacts with the carbamoyl oxygen present on
deactivated the
p cloud and hence deactivated the ring. The ring
thus acts as electron acceptor at the active site thereby increasing
the affinity towards the active site residues. Such hydrophobic in-
teractions contribute towards the specificity of molecule with this
protein, attributing to its activity. All these above illustrations
support the excellent activity profile of compound 14.
The docking studies also gave us an insight into the differences
in the binding pattern of the molecules used in the present study
at the active site of the protein which probably explains the
diverse activity profile displayed by these molecules. With
respect to structureeactivity relationship study, on the left hand
core we have attempted phenyl (13e20), 2-pyridyl (21e28) and
4-pyridyl (29e36) ring system in the second position of thiazole
nucleus. Among these, phenyl derivatives displayed better ac-
tivity profile than the corresponding pyridyl substituted ana-
logues. With the phenyl substitution, the molecules were
observed to have a consistent activity range as evidenced from
GyrB assay, with seven out of the eight derivatives attempted
the side chain of Asn52. A cation-
p interaction was also observed
between the
p
-electron environment of thiazole ring of compound
showing more than 50% inhibition at 50
profile displayed by the phenyl analogues (13e20) compared to
mM. The high activity
þ
14 and the ammonium ion (NH
3
) present in the guanidine group of

V.U. Jeankumar et al. / European Journal of Medicinal Chemistry 70 (2013) 143e153
147
Fig. 2. Aminopyrazinamide derivative interaction profile with GyrB ATPase domain of M smegmatis. The polar contacts are represented with red dots. The hydrophobic pocked is
highlighted with the residues interacting with the crystal ligand.
the pyridyl derivatives (21e36) can perhaps be due to more
lipophilic nature of the phenyl ring system compared to the
pyridyl ring, which as evident from their interaction profile
substitution attempted on the right handed benzyl core, the 4-
benzyloxy 3-trifluoromethyl, and 4-fluoro analogues were the
most promising leads. Their interaction map of the 4-benzyloxy
analogues revealed these compounds to be involved in significant
polar interactions with Glu48 and Asn52. These interactions orients
the right hand benzyl oxy core deep into the hydrophobic cleft
inside the adenine binding pocket where it is found to stabilized by
the hydrophobic interactions with the active site residues. A similar
trend was also observed in the case of 3-trifluoromethyl and 4-
fluoro substituted analogues as well attributing to their high ac-
tivity profile. On the contrary, the introduction of groups like tri-
fluoromethyl, methoxy etc on the para position of the right hand
benzyl core, oriented the molecules towards the solvent taking it
away from the active site thereby losing key hydrophobic in-
teractions which probably results in their diminished activity.
(
Fig. S1 in Supplementary information) establishes a much better
non-polar interactions in hydrophobic pocket of the active site
than the less hydrophobic pyridyl system. A closer look on the
interaction diagram of a representative example from each series
(
compound 13, 21, and 29, Fig. S1 in Supplementary information)
shows the terminal nitrogen atom of the aminopiperdine linker
of the phenyl analogue to be involved in two hydrogen bonds
with Glu56 and Arg82 whereas the pyridyl derivatives could
establish only one prominent hydrogen bond which further
supports their activity.
Effect of hydrophobicity on activity determination was also
evident on the right hand substitutions as well. Among the various
Fig. 3. Compound 14 interaction profile with GyrB ATPase domain of M smegmatis. The various polar contacts, cation-
4 are marked.
p interaction and the hydrophobic interaction with compound
1

148
V.U. Jeankumar et al. / European Journal of Medicinal Chemistry 70 (2013) 143e153
These differences were also clearly evident in their docking scores
as well, with compound 18, a 3-trifluormethyl substituted deriva-
tive displaying a good docking score of ꢁ5.838 compared to a
docking score of ꢁ2.982 for the 4-trifluoromethyl analogue (17).
Among the dichloro substituted analogues compound 36 demon-
strated the best activity profile. The fingerprint profile of this
molecule was similar to that of earlier described active analogues
exhibiting a docking score of ꢁ5.029. The 2D representation of the
interaction profile of few active and inactive analogues has been
provided in the supplementary information (Fig. S1 & S2) for a
better understanding. In general it can be concluded that, it’s the
overall orientation along with the non polar interactions that these
molecule maintained in the hydrophobic pocket of the active site
that was detrimental in bioactivity.
3. Conclusion
In conclusion, we disclose a new class of compounds that target
selectively the mycobacterial DNA gyrase enzyme with promising
attributes of synthetic accessibility, good SAR, and antitubercular
activity. With new antiTB agents desperately needed, we believe
that the present class of DNA GyrB inhibitors reported in this work,
provide an interesting potential for further optimization. Further
improvements to this series will be communicated in due course.
4. Experimental section
4.1. Chemistry
As many of the synthesized compounds showed good inhibition
in MS GyrB ATPase activity, we also tested the ability of these
compounds to inhibit DNA supercoiling using DNA gyrase from
MTB (Inspiralis, Norwich). Novobiocin was used as positive control
in these assays, as it has been shown to be a potent inhibitor of DNA
supercoiling of mycobacterial DNA gyrase. Each of the new com-
4.1.1. General
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
pounds tested showed dose-dependent inhibition. At 50
centration, four compounds showed more than 50% inhibition
Table 1) and at 25 M concentration, only compound 14 showed
4% inhibition with an IC50 of 27.10 ꢀ 2.1 M (Fig. 4). The standard
compound novobiocin showed 100% inhibition at 50 and 25
m
M con-
aluminium plates, visualized by UV light and KMnO
H and C NMR spectra were recorded on a Bruker AM-300
(300.12 MHz, 75.12 MHz) NMR spectrometer, Bruker BioSpin
Corp, Germany. Molecular weights of the synthesized compounds
were checked by LCMS 6100B series Agilent Technology. Chemical
4
treatment. All
1
13
(
4
m
m
mM
and its IC50 was 50 ꢀ 10 nM. These molecules did not inhibit DNA
supercoiling activity of S. aureus, Escherichia coli and Pseudomonas
aeruginosa DNA gyrases.
The compounds were further screened for their in vitro anti-
mycobacterial activity against M. tuberculosis H37Rv by microplate
Alamar blue assay method [24]. Six compounds inhibited MTB with
shifts are reported in ppm (d) with reference to the internal stan-
dard TMS. The signals are designated as follows: s, singlet; d,
doublet; dd, doublet of doublets; t, triplet; m, multiplet. Elemental
analyses were carried out on an automatic Flash EA 1112 Series,
CHN Analyzer (Thermo).
MIC less than 50
inactive at 200 M. In general, compounds with 4-fluorophenyl (14,
2, and 30), 4-benzyloxyphenyl (16, 24, and 32) and 2,4-
dichlorophenyl (20, 28 and 36) as their right hand substituent
showed good antitubercular activity. Compound 14 was found to be
most promising and showed good correlation in GyrB ATPase assay,
DNA gyrase super coiling assay and in vitro MIC. When compared to
m
M. The standard compound novobiocin was
4.1.2. General procedure for synthesis of ethyl 4-hydroxy-2-(phenyl/
pyridyl)-thiazole-5-carboxylate (9aec)
m
2
The synthesis followed the literature procedure [21]. Diethyl-2-
bromomalonate (7.3 mmol) was added drop wise to a well stirred
solution of the corresponding phenyl/pyridyl thioamide (7.3 mmol)
and pyridine (29.2 mmol) in toluene (60 mL) at room temperature
(rt). The reaction mixture was then heated to reflux for
2 h (monitored by TLC & LCMS for completion) and allowed to cool.
The precipitate formed was filtered and re-crystallized form
ethanol to afford the desired product in good yield.
first line antitubercular drugs like isoniazid (MIC: 0.66
rifampicin (MIC: 0.23 M) and ethambutol (MIC: 15.31 M), com-
pound 14 was less active against MTB in vitro.
The safety profile of all the compounds were accessed by testing
their in vitro cytotoxicity against RAW 264.7 cells at 50 M con-
mM),
m
m
m
4.1.2.1. Ethyl 4-hydroxy-2-(pyridin-2-yl)thiazole-5-carboxylate (9b).
ꢂ
1
centration by (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay [25]. Percentage inhibitions of cells are re-
ported in Table 1. The most promising antiTB compound 14 dis-
Pale green solid; Yield (71%); M.P: 140e142 C. H NMR (CDCl ):
3
d
H.1.33 (t, 3H, J ¼ 6.97 Hz), 4.24 (q, 2H, J ¼ 6.97 Hz), 7.37e8.43 (m,
13
4H). C NMR (CDCl
3
):
d
C. 166.9, 159.9, 157.1, 149.3, 148, 143, 136.9,
þ
played a good safety profile showing only 6.2% inhibition at 50
mM.
124.1, 123.3, 61.1, 14.3. ESIeMS m/z 251.05 (M þ H) . Anal Calcd for
11 10 2 3
C H N O S C, 52.79; H, 4.03; N, 11.19; Found: C, 52.82; H, 4.03; N,
11.21.
4
.1.2.2. Ethyl 4-hydroxy-2-(pyridin-4-yl)thiazole-5-carboxylate (9c).
ꢂ
1
3
Pale yellow solid; Yield (71%); M.P: 157e159 C. H NMR (CDCl ):
d
H.1.31 (t, 3H, J ¼ 7.01 Hz), 4.30 (q, 2H, J ¼ 7.01 Hz), 7.96e7.98 (m,
13
2
1
H), 8.81e8.83 (m, 2H). C NMR (CDCl
3
):
d
C. 169.3, 160.3, 149.6,
þ
47.5, 144, 143.2, 121, 61.3, 14.2. ESIeMS m/z 251.07 (M þ H) . Anal
10 2 3
Calcd for C11H N O S C, 52.79; H, 4.03; N, 11.19; Found: C, 52.80; H,
4.01; N, 11.17.
4
4
.1.3. General procedure for synthesis of ethyl-2-(phenyl/pyridyl)-
-(((trifluoromethyl)sulfonyl)oxy)thiazole-5-carboxylate (10aec)
Trifluoromethanesulfonic anhydride (4.8 mmol) was added
drop wise to a well stirred solution of Step-1 product (4 mmol) and
ꢂ
pyridine (6 mmol) in dichloromethane (20 mL) at 0 C. The reaction
Fig. 4. Depicting the supercoiling assay picture of compound 14 at three different
concentrations of 50 M, 25 M, 10
M, where R-Relaxed DNA substrate þ DMSO, C-
Relaxed DNA substrate with DNA Gyrase enzyme þ DMSO respectively.
mixture was slowly warmed to room temperature (rt) and stirred at
rt for about 6 h (monitored by TLC & LCMS for completion). The
m
m
m

V.U. Jeankumar et al. / European Journal of Medicinal Chemistry 70 (2013) 143e153
149
reaction mixture was diluted with water (10 mL) and the layers
separated. The aqueous layer was re-extracted with dichloro-
methane (2 ꢃ 15 mL). The combined organic layer was washed with
brine (10 mL), dried over anhydrous sodium sulphate and evapo-
rated under reduced pressure and the residue. The residue was
further re-crystallized from ethanol to afford the desired product in
good yield.
Anal Calcd for C21
58.29; H, 6.51; N, 12.96.
28 4 4
H N O S: C, 58.31; H, 6.52; N, 12.95; Found: C,
4.1.5. General procedure for ethyl 4-(4-aminopiperidin-1-yl)-2-
(phenyl/pyridyl)-thiazole-5-carboxylate (12aec)
To a solution of corresponding Step-3 product (2.3 mmol) in dry
dichloromethane (20 mL) at 0 C was added trifluoroacetic acid
ꢂ
(5 mL) drop wise over a period of 10 min. The reaction was slowly
4
.1.3.1. Ethyl-2-phenyl-4-(((trifluoromethyl)sulfonyl)oxy)thiazole-5-
warmed to room temperature (rt) and stirred at rt for
4 h (monitored by TLC & LCMS for completion). The solvent was
then removed under reduced pressure and the residue co distilled
once with dichloromethane (15 mL), the residue was diluted with
water (5 mL) and pH adjusted to 8 with solid sodium bicarbonate.
The aqueous phase was back-extracted with dichloromethane
(3 ꢃ 25 mL) and the combined organic layer was washed with brine
ꢂ
1
carboxylate (10a). Yellow solid; Yield (88%); M.P: 88e90 C.
NMR (CDCl ):
.37e8.01 (m, 5H). C NMR (CDCl
H
3
d
H.1.31 (t, 3H, J ¼ 7.01 Hz), 4.29 (q, 2H, J ¼ 7.01 Hz),
13
7
3
):
d
C. 169.4, 160.2, 148.3, 144.3,
þ
143,130.6,129.3,128.7,117.5, 61,14.3. ESIeMS m/z 381.01 (M) . Anal
Calcd for C13
H, 2.62; N, 3.65.
10 3 5 2
H F NO S : C, 40.94; H, 2.64; N, 3.67; Found: C, 40.95;
(15 mL), dried over anhydrous sodium sulphate and evaporated
4
.1.3.2. Ethyl-2-(pyridin-2-yl)-4-(((trifluoromethyl)sulfonyl)oxy)
under reduced pressure give the de-protected product which was
immediately taken to next step.
thiazole-5-carboxylate (10b). Pale green solid; Yield (79%); M.P:
ꢂ
1
1
10e112 C. H NMR (CDCl
J ¼ 7.0 Hz), 7.36e8.41 (m, 4H). C NMR (CDCl
57.3, 148.9, 148.1, 143.1, 137.1, 124.2, 123.4, 117.6, 61.1, 14.2. ESIeMS
3
):
d
H.1.32 (t, 3H, J ¼ 7.0 Hz), 4.29 (q, 2H,
13
3
):
d
C. 167.2, 159.6,
4.1.6. General procedure for the synthesis of ethyl 4-(4-(sub: benzyl)
piperidin-1-yl)-2-(phenyl/pyridyl)thiazole-5-carboxylate (13e36)
To a solution of Step-4 product (2.3 mmol) in dry dichloroethane
(10 mL) was added the corresponding aldehyde (2.3 mmol), freshly
1
þ
9 3 2 5 2
m/z 381.99 (M) . Anal Calcd for C12H F N O S C, 37.70; H, 2.37; N,
7.33; Found: C, 37.68; H, 2.38; N, 7.31.
ꢀ
activated 3 A molecular sieves (0.75 g) and catalytic amount of
4
.1.3.3. Ethyl-2-(pyridin-4-yl)-4-(((trifluoromethyl)sulfonyl)oxy)
acetic acid. The reaction mixture was stirred at room temperature
thiazole-5-carboxylate (10c). Pale yellow solid; Yield (84%); M.P:
2
for 6 h and filtered through celite bed (under N atmosphere). The
ꢂ
1
1
17e119 C. H NMR (CDCl
3
):
d
H.1.35 (t, 3H, J ¼ 7.01 Hz), 4.32 (q, 2H,
solvent was removed under reduced pressure and residue further
13
ꢂ
J ¼ 7.01 Hz), 7.94e7.96 (m, 2H), 8.85e8.87 (m, 2H). C NMR
diluted with dry methanol (10 mL) and cooled to 0 C. Sodium
(
CDCl
3
):
dC. 169.1, 160.1, 150, 147.8, 144.3, 143.5, 121.2, 117.9, 61.4,
triacetoxy borohydride (7.5 mmol) was added (portion-wise) and
the reaction was stirred at room temperature for 6 h (monitored by
TLC & LCMS for completion for completion). The solvent was then
removed under reduced pressure and the residue diluted with
water (10 mL) and extracted with dichloromethane (1 ꢃ 30 mL).
The aqueous phase was back-extracted with dichloromethane
(3 ꢃ 25 mL) and the combined organic layer was washed with brine
(15 mL), dried over anhydrous sodium sulphate and evaporated
under reduced pressure. The residue obtained was purified by
column chromatography using hexane: ethylacetate as eluent to
give the desired product in good yield.
þ
14.3. ESIeMS m/z 382.01 (M) . Anal Calcd for C12
H F
9 3
N
2
O
5
S
2
C,
37.70; H, 2.37; N, 7.33; Found: C, 37.72; H, 2.38; N, 7.34.
4
.1.4. General procedure for synthesis of ethyl 4-(4-(tert-
butoxycarbonyl)amino)piperidin-1-yl-2-(phenyl/pyridyl)-thiazole-
-carboxylate (11aec)
-N-Boc-aminopiperidine (2.6 mmol) was added to a well stir-
5
4
red solution of Step 3 product (2.6 mmol) in toluene (25 mL). The
ꢂ
reaction mixture was then heated to 90 C for about 4 h (monitored
by TLC & LCMS for completion), allowed to cool and solvent evap-
orated under reduced pressure. The residue was further diluted
with water (10 mL) and dichloromethane (20 mL) and the layers
separated. The aqueous layer was re-extracted with dichloro-
methane (2 ꢃ 15 mL) and the combined organic layer was washed
with brine (15 mL), dried over anhydrous sodium sulphate and
evaporated under reduced pressure and the residue purified by
column chromatography using hexane: ethylacetate as eluent to
give the desired product.
4.1.6.1. Ethyl-4-(4-(benzylamino)piperidin-1-yl)-2-phenylthiazole-5-
ꢂ
1
carboxylate (13). Yellow solid; Yield (81%); M.P: 89e91 C. H NMR
(CDCl ):
H.1.33 (t, 3H, J ¼ 6.95 Hz), 1.79e1.83 (m, 2H), 2.14e2.18 (m,
2H), 2.64e2.67 (m, 1H), 3.52e3.64 (m, 4H), 3.86 (s, 2H), 4.29 (q, 2H,
3
d
13
J ¼ 6.96 Hz), 7.28e7.34 (m, 5H), 7.78e7.93 (m, 5H). C NMR
(CDCl ): . 169.7, 161.8, 160.4, 140.8, 132, 130.8, 129.3, 128.2, 128,
3
d
C
126.9, 126.5, 100.6, 61.5, 54.6, 52.2, 48.2, 32.6, 14.2. ESIeMS m/z
þ
4
22.19 (M þ H) . Anal Calcd for C24
27 3 2
H N O S: C, 68.38; H, 6.46; N,
4
.1.4.1. Ethyl-4-(4-((tert-butoxycarbonyl)amino)piperidin-1-yl)-2-
9.97; Found: C, 68.41; H, 6.45; N, 9.96.
(
(
1
5
pyridin-2-yl)thiazole-5-carboxylate (11b). Pale green solid; Yield
ꢂ
1
71%); M.P: 139e141 C. H NMR (CDCl
.41 (s, 9H), 1.96e1.98 (m, 2H), 2.29e2.32 (m, 2H), 3.61e3.79 (m,
3
):
d
H. 1.29 (t, 3H, J ¼ 7.02 Hz),
4.1.6.2. Ethyl-4-(4-((4-methoxybenzyl)amino)piperidin-1-yl)-2-
phenylthiazole-5-carboxylate (15). Off white solid; Yield (71%); M.P:
13
ꢂ
1
H), 4.30 (q, 2H, J ¼ 7.02 Hz), 7.38e8.46 (m, 4H). C NMR (CDCl
3
):
96e98 C. H NMR (CDCl
3
):
d
H.1.31 (t, 3H, J ¼ 6.97 Hz), 1.74e1.79 (m,
d
C. 167.6, 163.5, 19.5, 154.5, 148.7, 148.3, 138.3, 123.7, 122.3, 101.7,
2H), 2.11e2.17 (m, 2H), 2.51e2.54 (m, 1H), 3.54e3.63 (m, 4H), 3.82
(s, 2H), 3.86 (s, 3H), 4.28 (q, 2H, J ¼ 6.95 Hz), 6.84e6.86 (m, 2H),
þ
7
8.6, 61.6, 47.8, 47.2, 32, 28.5, 14.1. ESIeMS m/z 433.19 (M þ H) .
13
Anal Calcd for C21
H
28
N
4
O
4
S: C, 58.31; H, 6.52; N, 12.95; Found: C,
3
7.24e7.27 (m, 2H). 7.76e7.95 (m, 5H). C NMR (CDCl ): dC. 169.8,
5
8.33; H, 6.51; N, 12.94.
161.7, 160.3, 158.8, 132.4, 132, 130.8, 128.9, 128.7, 126.3, 113.4, 100.7,
þ
61.4, 55.3, 54.4, 52.4, 48.3, 32.5, 14.2. ESIeMS m/z 452.21 (M þ H) .
4
.1.4.2. Ethyl-4-(4-((tert-butoxycarbonyl)amino)piperidin-1-yl)-2-
29 3 3
Anal Calcd for C25H N O S: C, 66.49; H, 6.47; N, 9.31; Found: C,
(
(
1
pyridin-4-yl)thiazole-5-carboxylate (11c). Yellow solid; Yield
66.47; H, 6.45; N, 9.29.
ꢂ
1
69%); M.P: 147e149 C. H NMR (CDCl
3
):
d
H.1.34 (t, 3H, J ¼ 6.98 Hz),
.39 (s, 9H), 1.94e1.96 (m, 2H), 2.30e2.33 (m, 2H), 3.49e3.81 (m,
4.1.6.3. Ethyl-4-(4-((4-(benzyloxy)benzyl)amino)piperidin-1-yl)-2-
5
H), 4.32 (q, 2H, J ¼ 6.98 Hz), 7.94e7.96 (m, 2H), 8.85e8.87 (m, 2H).
phenylthiazole-5-carboxylate (16). Buff coloured solid; Yield (77%);
13
ꢂ
1
C NMR (CDCl
3
):
d
C..169.6, 161.3, 160.4, 154.5, 151.3, 137.7, 120.4,
M.P: 87e89 C. H NMR (CDCl
3
):
dH.1.32 (t, 3H, J ¼ 6.96 Hz), 1.77e
þ
102, 79, 61.6, 47.4, 47, 31.8, 28.3, 13.9. ESIeMS m/z 433.21 (M þ H) .
1.82 (m, 2H), 2.13e2.16 (m, 2H), 2.53e2.57 (m, 1H), 3.54e3.65 (m,

150
V.U. Jeankumar et al. / European Journal of Medicinal Chemistry 70 (2013) 143e153
4
H), 3.81 (s, 2H), 4.30 (q, 2H, J ¼ 6.95 Hz), 5.04 (s, 2H), 6.89e6.92
4H), 3.81 (s, 2H), 4.32 (q, 2H, J ¼ 6.93 Hz), 7.01e7.03 (m, 2H), 7.28e
7.30 (m, 2H), 7.38 (dd, 1H, J ¼ 7.6 Hz, J ¼ 4.8 Hz) 7.88 (t, 1H,
13
(
1
1
m, 2H), 7.26e7.37 (m, 7H). 7.74e7.93 (m, 5H). C NMR (CDCl
69.6, 161.8, 160.4, 157.4, 136.7, 132.6, 132.1, 130.8, 129.1, 128.9, 128,
27.7, 127.3, 126.4, 114.7, 100.7, 69.7, 61.3, 54.4, 52.3, 48.2, 32.5, 14.1.
3 C.
): d
13
J ¼ 8.0 Hz), 8.16 (t, 1H, J ¼ 8.0 Hz), 8.44 (t, 1H, J ¼ 4.81 Hz). C NMR
3
(CDCl ): dC. 168, 163.6, 161.8, 159.5, 148.6, 148.3, 138.5, 136.6, 129.7,
þ
ESIeMS m/z 528.22 (M þ H) . Anal Calcd for C31
H N
33 3
O
3
S: C, 70.56;
123.7, 122.4, 114.9, 101.5, 61.5, 54.5, 52.2, 48.6, 32.5, 14.2. ESIeMS m/
þ
H, 6.30; N, 7.96; Found: C, 70.57; H, 6.28; N, 7.94.
z 441.19 (M þ H) . Anal Calcd for C23
4 2
H25FN O S: C, 62.71; H, 5.72; N,
1
2.72; Found: C, 62.73; H, 5.70; N, 12.70.
4
.1.6.4. Ethyl-4-(4-((4-(trifluoromethyl)benzyl)amino)piperidin-1-
yl)-2-phenylthiazole-5-carboxylate (17). Pale yellow solid; M.P: 93e
4.1.6.10. Ethyl-4-(4-((4-methoxybenzyl)amino)piperidin-1-yl)-2-
(pyridin-2-yl)thiazole-5-carboxylate (23). Pale yellow solid; Yield
ꢂ
1
9
1
4
7
5 C Yield (79%). H NMR (CDCl
.83 (m, 2H), 2.14e2.20 (m, 2H), 2.57e2.60 (m, 1H), 3.56e3.68 (m,
H), 3.92 (s, 2H), 4.34 (q, 2H, J ¼ 6.99 Hz), 7.34e7.56 (m, 4H), 7.76e
.92 (m, 5H).13C NMR (CDCl
): C. 170,161.9,160.4,141.5,132.1,130.8,
29.1, 128, 127.2, 126.1, 125, 124.4, 100.8, 61.3, 54.3, 52.8, 48.3, 32.4,
3
):
d
H.1.37 (t, 3H, J ¼ 6.98 Hz), 1.78e
ꢂ
1
(64%); M.P: 85e88 C. H NMR (CDCl
3
):
d
H.1.33 (t, 3H, J ¼ 6.98 Hz),
1.73e1.79 (m, 2H), 2.10e2.16 (m, 2H), 2.53e2.56 (m, 1H), 3.65e3.74
(m, 4H), 3.82 (s, 2H), 3.84 (s, 3H), 4.31 (q, 2H, J ¼ 6.99 Hz), 6.86e6.88
(m, 2H), 7.27e7.29 (m, 2H), 7.36 (dd, 1H, J ¼ 7.56 Hz, J ¼ 4.79 Hz)
7.89 (t,1H, J ¼ 8.0 Hz), 8.15 (t,1H, J ¼ 8.0 Hz), 8.47 (t,1H, J ¼ 4.79 Hz).
3
d
1
þ
14.2. ESIeMS m/z 490.19 (M þ H) . Anal Calcd for C25
26 3 3 2
H F N O S: C,
13
61.34; H, 5.35; N, 8.58; Found: C, 61.33; H, 5.32; N, 8.59.
3
C NMR (CDCl ): dC. 167.9, 163.5, 159.5, 158.9, 148.6, 148.3, 138.5,
1
32.4, 129, 123.5, 122.5, 113.4, 101.4, 61.5, 55.3, 54.5, 52.4, 48.7, 32.5,
þ
4
.1.6.5. Ethyl-4-(4-((3-(trifluoromethyl)benzyl)amino)piperidin-1-
14.2. ESIeMS m/z 453.21 (M þ H) . Anal Calcd for C24
28 4 3
H N O S: C,
yl)-2-phenylthiazole-5-carboxylate (18). Yellow solid; Yield (70%);
63.69; H, 6.24; N, 12.38; Found: C, 63.71; H, 6.25; N, 12.35.
ꢂ
1
M.P: 96e98 C. H NMR (CDCl
(
3
):
d
H.1.36 (t, 3H, J ¼ 7.0 Hz), 1.82e1.87
m, 2H), 2.15e2.21 (m, 2H), 2.59e2.63 (m, 1H), 3.57e3.67 (m, 4H),
4.1.6.11. Ethyl-4-(4-((4-(benzyloxy)benzyl)amino)piperidin-1-yl)-2-
3
7
.83e3.85 (m, 2H), 4.30 (q, 2H, J ¼ 7.01 Hz), 7.36 (d, 1H, J ¼ 7.56),
(pyridin-2-yl)thiazole-5-carboxylate (24). Buff coloured solid; Yield
.43e7.48 (m, 2H), 7.70e7.93 (m, 6H). 13C NMR (CDCl
): C. 169.9,
d
(74%); M.P: 92e94 C. H NMR (CDCl
ꢂ
1
):
d
H. 1.35 (t, 3H, J ¼ 7.01 Hz),
3
3
161.8, 160.3, 140, 132.3, 131, 129, 128.8, 128.6, 128.5, 126.3, 125, 124,
1.78e1.84 (m, 2H), 2.13e2.17 (m, 2H), 2.51e2.55 (m, 1H), 3.67e3.78
(m, 4H), 3.83 (s, 2H), 4.30 (q, 2H, J ¼ 7.03 Hz), 5.06 (s, 2H), 6.89e6.91
(m, 2H), 7.29e7.40 (m, 8H), 7.91 (t, 1H, J ¼ 8.0 Hz), 8.17 (t, 1H,
123.9, 100.6, 61.4, 54.6, 52.5, 48.3, 32.6, 14.3. ESIeMS m/z 490.24
þ
(
M þ H) . Anal Calcd for C25
26 3 3 2
H F N O S: C, 61.34; H, 5.35; N, 8.58;
13
Found: C, 61.36; H, 5.36; N, 8.59.
J ¼ 8.0 Hz), 8.46 (t, 1H, J ¼ 4.8 Hz). C NMR (CDCl
3
): dC. 167.7, 163.4,
159.5, 157.4, 148.6,148.3, 138.5,136.8,132.7, 129.1, 127.9, 127.7, 127.3,
4
.1.6.6. Ethyl-4-(4-((3,4-dichlorobenzyl)amino)piperidin-1-yl)-2-
123.5, 122.4, 114.7, 101.4, 69.70, 61.4, 54.5, 52.4, 48.5, 32.5, 14.1. ESIe
þ
phenylthiazole-5-carboxylate (19). Off white solid; Yield (67%); M.P:
MS m/z 529.23 (M þ H) . Anal Calcd for C30
32 4 3
H N O S: C, 68.16; H,
6.10; N, 10.60; Found: C, 68.15; H, 6.09; N, 10.64.
ꢂ
1
8
2
3
6e88 C. H NMR (CDCl
3
):
d
H.1.37 (t, 3H, J ¼ 6.98 Hz), 1.83e1.88 (m,
H), 2.17e2.23 (m, 2H), 2.71e2.75 (m, 1H), 3.58e3.66 (m, 4H),
.90e3.92 (m, 2H), 4.28 (q, 2H, J ¼ 6.95 Hz), 7.15 (d, 1H, J ¼ 8.1 Hz),
4.1.6.12. Ethyl-4-(4-((4-(trifluoromethyl)benzyl)amino)piperidin-1-
yl)-2-(pyridin-2-yl)thiazole-5-carboxylate (25). Pale yellow solid;
13
7
.31 (d, 1H, J ¼ 8.1 Hz), 7.49 (s, 1H), 7.76e7.95 (m, 5H). C NMR
ꢂ
1
(
CDCl
3
):
dC. 169.7, 161.7, 160.3, 141.4, 132.4, 132, 131.4, 130.8, 129.7,
Yield (69%); M.P: 104e106 C. H NMR (CDCl ): dH. 1.37 (t, 3H,
3
1
29.6, 128.9, 127.7, 126.5, 100.8, 61.5, 54.2, 52.9, 48.2, 32.4, 14. ESIe
J ¼ 6.96 Hz), 1.76e1.83 (m, 2H), 2.12e2.17 (m, 2H), 2.53e2.57 (m,
1H), 3.64e3.76 (m, 4H), 3.95 (s, 2H), 4.32 (q, 2H, J ¼ 6.97 Hz), 7.35
(dd, 1H, J ¼ 7.53 Hz, J ¼ 4.82 Hz) 7.39e7.41 (m, 2H), 7.60e7.62 (m,
þ
2 3 2
MS m/z 489.10 (M) . Anal Calcd for C24H25Cl N O S: C, 58.77; H,
5
.14; N, 8.57; Found: C, 58.75; H, 5.13; N, 8.55.
2
H), 7.85 (t, 1H, J ¼ 8.0 Hz), 8.17 (t, 1H, J ¼ 8.0 Hz), 8.44 (t, 1H,
13
4
.1.6.7. Ethyl-4-(4-((2,4-dichlorobenzyl)amino)piperidin-1-yl)-2-
J ¼ 4.81 Hz). C NMR (CDCl
3
): dC. 168.1, 163.6, 159.6, 148.6, 148.3,
phenylthiazole-5-carboxylate (20). Off white solid; Yield (75%);
M.P: 82e84 C. H NMR (CDCl
141.4, 138.5, 127.9, 127.3, 125.1, 124.4, 123.5, 122.5, 101.5, 61.5, 54.4,
ꢂ
1
þ
3
):
d
H.1.33 (t, 3H, J ¼ 6.92 Hz), 1.69e
52.8, 48.6, 32.6, 14.3. ESIeMS m/z 491.17 (M þ H) . Anal Calcd for
1
4
1
.74 (m, 2H), 2.19e2.24 (m, 2H), 2.57e2.60 (m, 1H), 3.56e3.65 (m,
24 25 3 4 2
C H F N O S: C, 58.76; H, 5.14; N, 11.42; Found: C, 58.74; H, 5.13;
H), 4.08 (s, 2H), 4.32 (q, 2H, J ¼ 6.93 Hz), 7.17e7.21 (m, 2H), 7.54 (s,
N, 11.39.
H), 7.78e7.96 (m, 5H). 1 C NMR (CDCl
3
3
): dC. 169.6, 161.7, 160.1, 135,
1
5
C
34.2, 132.1, 131.8, 130.7, 130.1, 128.9, 127.9, 126.5, 126.1, 100.6, 61.4,
4.1.6.13. Ethyl-4-(4-((3-(trifluoromethyl)benzyl)amino)piperidin-1-
yl)-2-(pyridin-2-yl)thiazole-5-carboxylate (26). Pale yellow solid;
þ
3.9, 48.3, 48.1, 32.2, 14. ESIeMS m/z 489.14 (M) . Anal Calcd for
S: C, 58.77; H, 5.14; N, 8.57; Found: C, 58.78; H, 5.17;
ꢂ
1
24
H
25Cl
2
N
3
O
2
Yield (65%); M.P: 86e88 C. H NMR (CDCl
J ¼ 7.01 Hz), 1.79e1.87 (m, 2H), 2.10e2.16 (m, 2H), 2.57e2.61 (m,
H), 3.63e3.72 (m, 4H), 3.83e3.84 (m, 2H), 4.29 (q, 2H, J ¼ 7.04 Hz),
3
): dH.1.33 (t, 3H,
N, 8.56.
1
13
4
.1.6.8. Ethyl-4-(4-(benzylamino)piperidin-1-yl)-2-(pyridin-2-yl)
7.32e7.36 (m, 2H), 7.43e7.47 (m, 2H), 7.71e8.43 (m, 4H). C NMR
(CDCl ): C. 168, 163.5, 159.5, 148.6, 148.3, 139.9, 138.6, 128.7, 128.6,
thiazole-5-carboxylate (21). Yellow solid; Yield (76%); M.P: 77e
7
2
3
d
ꢂ
1
9
C. H NMR (CDCl
3
):
d
H.1.36 (t, 3H, J ¼ 6.97 Hz), 1.78e1.85 (m,
128.4, 125.1, 124, 123.9, 123.4, 122.5, 100.6, 61.4, 54.7, 52.5, 48.6,
þ
H), 2.12e2.18 (m, 2H), 2.62e2.66 (m, 1H), 3.61e3.72 (m, 4H), 3.87
32.5, 14.2. ESIeMS m/z 491.19 (M
þ
H) . Anal Calcd for
(
s, 2H), 4.30 (q, 2H, J ¼ 6.97 Hz), 7.26e7.38 (m, 6H), 7.85 (t, 1H,
24 25 3 4 2
C H F N O S: C, 58.76; H, 5.14; N, 11.42; Found: C, 58.75; H, 5.12;
13
J ¼ 7.96 Hz), 8.13 (t, 1H, J ¼ 8.0 Hz), 8.46 (t, 1H, J ¼ 4.83 Hz). C NMR
CDCl ): C. 167.8, 163.6, 159.7, 148.5, 148.2, 140.8, 138.5, 128.4, 128,
26.9, 123.6,122.5,101.5, 61.5, 54.7, 52.3, 48.6, 32.6, 14.3. ESIeMS m/
N, 11.45.
(
1
3
d
4.1.6.14. Ethyl-4-(4-((3,4-dichlorobenzyl)amino)piperidin-1-yl)-2-
þ
z 423.18 (M þ H) . Anal Calcd for C23
26
H N
4
O
2
S: C, 65.38; H, 6.20; N,
(pyridin-2-yl)thiazole-5-carboxylate (27). Pale yellow solid; Yield
ꢂ
1
13.26; Found: C, 65.36; H, 6.19; N, 13.24.
(78%); M.P: 99e101 C. H NMR (CDCl
1
3
):
d
H.1.39 (t, 3H, J ¼ 7.01 Hz),
.81e1.89 (m, 2H), 2.13e2.19 (m, 2H), 2.70e2.74 (m, 1H), 3.68e3.76
4
.1.6.9. Ethyl-4-(4-((4-fluorobenzyl)amino)piperidin-1-yl)-2-(pyr-
(m, 4H), 3.89e3.91 (m, 2H), 4.33 (q, 2H, J ¼ 7.03 Hz), 7.16 (d, 1H,
idin-2-yl)thiazole-5-carboxylate (22). Pale green solid; Yield (71%);
M.P: 80e82 C. H NMR (CDCl
J ¼ 8.1 Hz), 7.34 (d, 1H, J ¼ 8.1 Hz), 7.39 (dd, 1H, J ¼ 7.55 Hz,
ꢂ
1
13
3
):
d
H.1.38 (t, 3H, J ¼ 6.93 Hz), 1.76e
J ¼ 4.8 Hz), 7.50 (s, 1H), 7.84e8.44 (m, 3H). C NMR (CDCl
3 C.
): d
1.83 (m, 2H), 2.09e2.16 (m, 2H), 2.52e2.55 (m, 1H), 3.63e3.74 (m,
167.9, 163.6, 159.5, 148.5, 148.2, 141.6, 138.5, 132.5, 131.7, 129.6,

V.U. Jeankumar et al. / European Journal of Medicinal Chemistry 70 (2013) 143e153
151
1
29.4, 127.5, 123.6, 122.6, 101.6, 61.5, 54.3, 53, 48.7, 32.4, 14.2. ESIe
3
(CDCl ): dC. 169.6, 161.4, 160.4, 151.3, 141.5, 137.7, 127.9, 127.1, 125,
þ
MS m/z 490.13 (M) . Anal Calcd for C23
H24Cl
2
N
4
O
2
S: C, 56.21; H,
124.4, 120.1, 101.9, 61.6, 54.6, 53, 48.3, 32.4, 13.9. ESIeMS m/z 491.20
þ
4
.92; N, 11.40; Found: C, 56.23; H, 4.93; N, 11.37.
(M þ H) . Anal Calcd for C24
25 3 4 2
H F N O S: C, 58.76; H, 5.14; N, 11.42;
Found: C, 58.78; H, 5.14; N, 11.43.
4
.1.6.15. Ethyl-4-(4-((2,4-dichlorobenzyl)amino)piperidin-1-yl)-2-
(
pyridin-2-yl)thiazole-5-carboxylate (28). Pale yellow solid; Yield
4.1.6.21. Ethyl-4-(4-((3-(trifluoromethyl)benzyl)amino)piperidin-1-
yl)-2-(pyridin-4-yl)thiazole-5-carboxylate (34). Pale yellow solid;
ꢂ
1
(
1
(
7
67%); M.P:109e111 C. H NMR (CDCl
.65e1.71 (m, 2H), 2.16e2.23 (m, 2H), 2.54e2.57 (m, 1H), 3.67e3.78
m, 4H), 4.06 (s, 2H), 4.34 (q, 2H, J ¼ 6.97 Hz), 7.18e7.36 (m, 3H),
3
):
d
H.1.37(t, 3H, J ¼ 6.98 Hz),
ꢂ
1
Yield (70%); M.P: 126e128 C. H NMR (CDCl ): dH. 1.36 (t, 3H,
3
J ¼ 7.06 Hz), 1.86e1.94 (m, 2H), 2.18e2.27 (m, 2H), 2.60e2.63 (m,
1H), 3.53e3.69 (m, 4H), 3.81e3.83 (m, 2H), 4.35 (q, 2H, J ¼ 7.04 Hz),
1
3
.58 (s, 1H), 7.86e8.46 (m, 3H). C NMR (CDCl
59.4, 148.4, 148.2, 138.5, 134.9, 134.3, 132.2, 130.1, 128, 126.5, 123.4,
22.5, 101.6, 61.5, 53.8, 48.7, 48.3, 32.2, 14.1. ESIeMS m/z 490.09
3
): dC. 167.6, 163.4,
13
1
1
7.36e7.51 (m, 3H), 7.72e7.99 (m, 3H), 8.83e8.86 (m, 2H). C NMR
3
(CDCl ): dC. 169.7, 161.3, 160.4, 151.2, 140, 137.6, 128.8, 128.7, 128.6,
þ
(
M) . Anal Calcd for C23
H24Cl
2
N
4
O
2
S: C, 56.21; H, 4.92; N, 11.40;
125, 123.9, 123.7, 120.1, 101.9, 61.6, 54.9, 52.8, 48.3, 32.3, 14.2. ESIe
þ
Found: C, 56.21; H, 4.91; N, 11.39.
MS m/z 491.23 (M þ H) . Anal Calcd for C24
25 3 4 2
H F N O S: C, 58.76; H,
5
.14; N, 11.42; Found: C, 58.77; H, 5.13; N, 11.44.
4
.1.6.16. Ethyl-4-(4-(benzylamino)piperidin-1-yl)-2-(pyridin-4-yl)
thiazole-5-carboxylate (29). Yellow solid; Yield (82%); M.P: 95e
4.1.6.22. Ethyl-4-(4-((3,4-dichlorobenzyl)amino)piperidin-1-yl)-2-
ꢂ
1
9
2
2
8
7 C. H NMR (CDCl
3
):
d
H.1.38 (t, 3H, J ¼ 7.05 Hz),1.83e1.89 (m, 2H),
(pyridin-4-yl)thiazole-5-carboxylate (35). Pale green solid; Yield
ꢂ
1
.16e2.21 (m, 2H), 2.67e2.71 (m, 1H), 3.49e3.60 (m, 4H), 3.84 (s,
H), 4.32 (q, 2H, J ¼ 7.06 Hz), 7.28e7.35 (m, 5H), 7.94e7.96 (m, 2H),
(81%); M.P: 131e133 C. H NMR (CDCl
3
):
d
H.1.33 (t, 3H, J ¼ 6.92 Hz),
1.84e1.92 (m, 2H), 2.14e2.23 (m, 2H), 2.70e2.74 (m, 1H), 3.56e3.67
(m, 4H), 3.93e3.95 (m, 2H), 4.31 (q, 2H, J ¼ 6.93 Hz), 7.13 (d,
.79e8.81 (m, 2H). 13C NMR (CDCl
): dC. 169.5, 161.2, 160.1, 151.1,
3
1
3
C
40.3, 137.6, 128.2, 127.7, 126.6, 120.2, 101.9, 61.6, 54.8, 52.4, 48.3,
J ¼ 8.1 Hz, 1H), 7.30 (d, J ¼ 8.1 Hz, 1H), 7.51 (s, 1H), 7.96e8.83 (m,
þ
13
2.4, 13.8. ESIeMS m/z 423.23 (M
þ
H) . Anal Calcd for
4H). C NMR (CDCl
3
):
d
C. 169.6, 161.2, 160.1, 151.2, 141.4, 137.5, 132.4,
H
23 26
N
4
O
2
S: C, 65.38; H, 6.20; N,13.26; Found: C, 65.39; H, 6.18; N,
131.5, 129.5, 129.3, 127.4, 120.1, 102, 61.6, 54.3, 53.1, 48.3, 32.3, 14.
þ
13.29.
ESIeMS m/z 490.13 (M) . Anal Calcd for C23
H
24Cl
2
N
4
O
2
S: C, 56.21;
H, 4.92; N, 11.40; Found: C, 56.19; H, 4.90; N, 11.42.
4
.1.6.17. Ethyl-4-(4-((4-fluorobenzyl)amino)piperidin-1-yl)-2-(pyr-
idin-4-yl)thiazole-5-carboxylate (30). Green solid; Yield (74%); M.P:
4.1.6.23. Ethyl-4-(4-((2,4-dichlorobenzyl)amino)piperidin-1-yl)-2-
ꢂ
1
1
13e115 C. H NMR (CDCl
3
):
d
H.1.36 (t, 3H, J ¼ 6.99 Hz), 1.79e1.86
(pyridin-4-yl)thiazole-5-carboxylate (36). Pale green solid; Yield
ꢂ
1
(
m, 2H), 2.13e2.19 (m, 2H), 2.55e2.59 (m, 1H), 3.52e3.63 (m, 4H),
(79%); M.P: 122e124 C. H NMR (CDCl
3
):
d
H.1.29 (t, 3H, J ¼ 6.95 Hz),
3
.79 (s, 2H), 4.29 (q, 2H, J ¼ 7.01 Hz), 7.04e7.07 (m, 2H), 7.32e7.34
1.67e1.73 (m, 2H), 2.21e2.27 (m, 2H), 2.58e2.61 (m, 1H), 3.54e3.66
(m, 4H), 4.10 (s, 2H), 4.32 (q, 2H, J ¼ 6.97 Hz), 7.18e7.21 (m, 2H), 7.56
13
(
m, 2H), 7.96e7.98 (m, 2H), 8.81e8.83 (m, 2H). C NMR (CDCl
3 C.
): d
13
1
69.6, 161.8, 161.3, 160.2, 151.2, 137.6, 136.5, 129.6, 120, 114.9, 101.8,
3 C.
(s, 1H), 7.98e8.03 (m, 2H), 8.81e8.85 (m, 2H). C NMR (CDCl ): d
þ
6
1.5, 54.6, 52.3, 48.4, 32.4, 14. ESIeMS m/z 441.17 (M þ H) . Anal
Calcd for C23 S: C, 62.71; H, 5.72; N, 12.72; Found: C, 62.70;
H, 5.71; N, 12.75.
169.5, 161.2, 160.3, 151.1, 137.6, 134.9, 134.3, 132.1, 129.9, 127.8, 126.5,
H25FN
4
O
2
120.1, 101.8, 61.6, 53.8, 48.5, 48.3, 32.4, 13.9. ESIeMS m/z 490.11
þ
(M) . Anal Calcd for C23
H24Cl
2
N
4
O
2
S: C, 56.21; H, 4.92; N, 11.40;
Found: C, 56.18; H, 4.91; N, 11.36.
4
.1.6.18. Ethyl-4-(4-((4-methoxybenzyl)amino)piperidin-1-yl)-2-
(
(
1
(
pyridin-4-yl)thiazole-5-carboxylate (31). Pale green solid; Yield
4.2. Cloning and purification
ꢂ
1
68%); M.P: 97e99 C. H NMR (CDCl
.81e1.87 (m, 2H), 2.11e2.18 (m, 2H), 2.58e2.61 (m, 1H), 3.54e3.66
3
):
d
H.1.39 (t, 3H, J ¼ 7.02 Hz),
Cloning of M. smegmatis gyrB was done by amplifying the
gene from the genomic DNA of mc 155 using specific forward
and reverse primer with the sequence CACCCATATGGTGGCTGCC
CAGAAGAACAA (NdeI), AGCTAAGCTTTTAAACATCCAGGAAGCGAA
(Hind III) respectively. The final PCR amplicons were cloned in
E. coli expression vector pQE2 (Qiagen). His-tagged cloned vector
2
m, 4H), 3.81 (s, 2H), 3.87 (s, 3H), 4.34 (q, 2H, J ¼ 7.01 Hz), 6.87e6.89
(
m, 2H), 7.29e7.32 (m, 2H), 7.98e8.01 (m, 2H), 8.84e8.86 (m, 2H).
1
3
3
C NMR (CDCl ): dC. 169.4, 161.3, 160.2, 159, 150.9, 137.5, 132.4, 129,
1
4
1
20.2,113.6, 101.9, 61.6, 55.4, 54.5, 52.6, 48.4, 32.3, 13.9. ESIeMS m/z
þ
53.19 (M þ H) . Anal Calcd for C24
28 4 3
H N O S: C, 63.69; H, 6.24; N,
2.38; Found: C, 63.69; H, 6.23; N, 12.39.
was then transformed into BL21 (DE3) pLysS cells. The transformed
cells were later grown at 37 C in LB broth containing 50
ꢂ
mg/mL
4
.1.6.19. Ethyl-4-(4-((4-(benzyloxy)benzyl)amino)piperidin-1-yl)-2-
ampicillin to an optical density (OD) of 0.6 (A595). Once bacterial
cells reached exponential growth phase they were induced with
Isopropyl-b-D-thiogalactopyranoside at a final concentration of
(
pyridin-4-yl)thiazole-5-carboxylate (32). Yellow solid; Yield (71%);
ꢂ
1
M.P: 120e122 C. H NMR (CDCl
3
):
d
H.1.36 (t, 3H, J ¼ 7.04 Hz), 1.76e
1
4
2
.84 (m, 2H), 2.09e2.16 (m, 2H), 2.52e2.55 (m, 1H), 3.49e3.58 (m,
0.2 mM and the cell growth was further continued for another 12 h
ꢂ
H), 3.86 (s, 2H), 4.34 (q, 2H, J ¼ 7.03 Hz), 5.08 (s, 3H), 6.91e6.93 (m,
at 18 C. The bacterial cells were then centrifuged at 10,000ꢃ g and
13
ꢂ
H), 7.29e7.38 (m, 7H), 8.03e8.82 (m, 4H), C NMR (CDCl
3
):
d
C.
4 C for 15 min. The cell pellet was resuspended in PBSG buffer (PBS
1
69.5, 161.2, 160.3, 157.4, 151, 137.6, 136.7, 132.6, 129.1, 127.9, 127.5,
containing 5% glycerol). Cells were then lysed using sonicator
(20 s pulse and 45 s halt) and centrifuged the crude lysate at
127.1, 120, 114.5, 101.8, 69.8, 61.6, 54.5, 52.6, 48.3, 32.3, 14.1. ESIeMS
þ
ꢂ
m/z 529.24 (M þ H) . Anal Calcd for C30
H
32
N
4
O
3
S: C, 68.16; H, 6.10;
8000 rpm at 4 C for 10 min, further centrifugation was repeated for
ꢂ
N, 10.60; Found: C, 68.15; H, 6.09; N, 10.64.
the supernatant of previous step at 10,000 rpm at 4 C for 35 min.
The cell extract was later applied to Ni-NTA column (Biorad). After
washing the column with wash buffer (5% glycerol in PBS and
500 mM NaCl) desired protein was eluted using different concen-
tration of imidazole ranging from 10 mM to 200 mM in the elution
buffer (5% Glycerol, 140 mM NaCl in 25 mM Tris-Cl (pH 8.0)).
Fractions containing the GyrB subunit were identified by sodium
dodecyl sulphate-polyacrylamide gel electrophoresis; pooled;
4
.1.6.20. Ethyl-4-(4-((4-(trifluoromethyl)benzyl)amino)piperidin-1-
yl)-2-(pyridin-4-yl)thiazole-5-carboxylate (33). Pale green solid;
Yield (73%); M.P: 115e117 C. 1H NMR (CDCl
J ¼ 6.96 Hz), 1.78e1.86 (m, 2H), 2.12e2.19 (m, 2H), 2.53e2.57 (m,
H), 3.52e3.64 (m, 4H), 3.94 (s, 2H), 4.36 (q, 2H, J ¼ 6.98 Hz), 7.39e
ꢂ
): dH.1.34 (t, 3H,
3
1
13
7.58 (m, 4H), 7.98e8.02 (m, 2H), 8.79e8.82 (m, 2H). C NMR

152
V.U. Jeankumar et al. / European Journal of Medicinal Chemistry 70 (2013) 143e153
dialysed against 15% Glycerol, 140 mM NaCl in 25 mM Tris-Cl (pH
the concentration of 100 mM. After 72 h of exposure, viability was
ꢂ
7.4); frozen in liquid nitrogen; and stored at ꢁ80 C.
assessed on the basis of cellular conversion of MTT into a formazan
product using the Promega Cell Titer 96 non-radioactive cell pro-
liferation assay [25].
4.3. MS GyrB ATPase assay
The ATPase assay was performed similar to the previously re-
4
.7. Molecular modelling studies
ported protocol [20]. The assay was carried out in 30
h at 25 C in reaction buffer containing 60 mM HEPES-KOH pH
m
L volume for
ꢂ
2
7
The molecules which were proved to be active in gyrB and
.7, 250 mM potassium glutamate, 200 mM KCl, 2 mM magnesium
chloride, 1 mM DTT, 2% Glycerol, 4% DMSO, 0.001% BriJ, 0.65 mM
ATP, 40 nM GyrB. All the test compounds were diluted in DMSO at
supercoiling assay were docked onto the active pocket of the GyrB
ATPase domain of M. smegmatis retrieved from Protein Data Bank
(PDB ID e 4B6C). The protein was initially processed using the
3
0 times the final assay concentrations. Assay was performed in 96
well (Polystyrene untreated) flat bottomed plates. Initially 1 L of
test compound were placed in the assay well, which was followed
by 15
L of 2ꢃ assay buffer containing purified GyrB enzyme and
substrate mix, later the enzyme reaction was initiated by adding
L of MgCl solution. Then the reaction was allowed to proceed
Protein Preparation Wizard of Schrödinger Suite 2012 [26]. The
optimization of the hydrogen-bonding network and the ligands to
be docked were sketched in Maestro panel of Schrödinger and
optimized with OPLS force field [27]. The docking of these mole-
cules with the protein was done using Glide v5.8 [28].
m
m
14
m
2
for 2 h at room temperature. Later the reaction was quenched by
adding malachite green reagent (Bioassay systems). Inorganic
phosphates released (Pi) after the reaction was measured at
Acknowledgements
This work was supported by the Council of Scientific and In-
dustrial Research, [01 (2394)/10/EMR-II] India.
6
35 nm after incubating with malachite reagent for 20 min.
4.4. MTB DNA super coiling assay
Appendix A. Supplementary data
Supercoiling assay was performed using MTB DNA supercoiling
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.09.025.
assay kit (Inspiralis Limited, Norwich), the assay was done in 30
mL
ꢂ
reaction volume for 30 min at 37 C in assay buffer containing
5
1
0
0 mM HEPES. KOH (pH 7.9), 6 mM magnesium acetate, 4 mM DTT,
mM ATP, 100 mM potassium glutamate, 2 mM spermidine and
.05 mg/mL of albumin. During the assay 1 unit of DNA gyrase was
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chloroform:isoamylalcohol (24:1) and STEB (40% sucrose, 100 mM
TriseHCl (pH 8.0), 100 mM EDTA and 0.5 mg/mL bromophenol
blue) brief vortexing was done followed by centrifugation. The
products were analysed by electrophoresis on 1% agarose gels and
stained with ethidium bromide. Using Image lab software (Biorad)
intensity of bands were measured and analysed to know the
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.5. In vitro MTB MABA assay
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7] T. Yempala, D. Sriram, P. Yogeeswari, S. Kantevari, Bioorg. Med. Chem. Lett. 22
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[OADC]), adjusted to a McFarland tube No. 1, and diluted 1:20;
100 L was used as inoculum. Each drug stock solution was thawed
m
[
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