Gyrase ATPase domain as an antitubercular drug discovery platform: Structure-based design and lead optimization of nitrothiazolyl carboxamide analogues

Article abstract of DOI:10.1002/cmdc.201402035

In this study, we explored the pharmaceutically underexploited mycobacterial gyrase ATPase (GyrB) domain as a template for a structure-based virtual screening of our in-house (BITS Pilani) compound collection to discover new inhibitors targeting Mycobacterium tuberculosis (M.tb.) The hit identified was further customized by using a combination of molecular docking and medicinal chemistry strategies to obtain an optimized analogue displaying considerable in vitro enzyme efficacy and bactericidal properties against the M.tb. H ₃₇Rv strain. The binding affinity of the ligand toward the GyrB domain was reascertained by differential scanning fluorimetry experiments. Further evaluation of the hERG toxicity (a major limitation among the previously reported N-linked aminopiperidine analogues) indicated these molecules to be completely devoid of cardiotoxicity, a significant achievement within this class.

Full text of DOI:10.1002/cmdc.201402035

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DOI: 10.1002/cmdc.201402035
Gyrase ATPase Domain as an Antitubercular Drug
Discovery Platform: Structure-Based Design and Lead
Optimization of Nitrothiazolyl Carboxamide Analogues
Variam Ullas Jeankumar,[a] Janupally Renuka,[a] Sonali Kotagiri,[a] Shalini Saxena,[a]
Shruti Singh Kakan,[a] Jonnalagadda Padma Sridevi,[a] Swapna Yellanki,[b, c]
Pushkar Kulkarni,[b, c] Perumal Yogeeswari,[a] and Dharmarajan Sriram*[a]
In this study, we explored the pharmaceutically underexploited
mycobacterial gyrase ATPase (GyrB) domain as a template for
a structure-based virtual screening of our in-house (BITS Pilani)
compound collection to discover new inhibitors targeting My-
cobacterium tuberculosis (M.tb.) The hit identified was further
customized by using a combination of molecular docking and
medicinal chemistry strategies to obtain an optimized ana-
logue displaying considerable in vitro enzyme efficacy and bac-
tericidal properties against the M.tb. H37Rv strain. The binding
affinity of the ligand toward the GyrB domain was reascer-
tained by differential scanning fluorimetry experiments. Further
evaluation of the hERG toxicity (a major limitation among the
previously reported N-linked aminopiperidine analogues) indi-
cated these molecules to be completely devoid of cardiotoxici-
ty, a significant achievement within this class.
Introduction
Resistance, the man-made amplification of a natural phenom-
enon, has allowed the resurgence of Mycobacterium tuberculo-
sis (M.tb.) in the new virulent forms of multidrug-resistant and
extremely drug-resistant strains, which render the presently
available antitubercular drug regime inadequate to address
the many inherent and emerging challenges of treatment. Sub-
stantial progress has been made in the research and develop-
ment of new antitubercular drugs during the past decade and
a portfolio of promising new leads are on the horizon, includ-
ing six new compounds specifically developed for tuberculosis
(TB).[1] Despite this progress, the global drug pipeline for tuber-
culosis is still insufficient to address the unmet needs of treat-
ment.[2]
agents.[3] DNA gyrase is an essential enzyme that introduces
negative supercoils into DNA and regulates the superhelical
state of the bacterial chromosomes. Quinolones preferably
target the GyrA subunit and have made quite successful prog-
ress in the TB drug discovery pipeline, with gatifloxacin and
moxifloxacin currently undergoing phase III clinical trials for
the treatment of TB. However, the increasing resistance to the
quinolone class of drugs[3] calls for novel research in this field.
Novel (non-fluoroquinolone) bacterial type II topoisomerase
inhibitors (NBTIs), a recent class of DNA gyrase inhibitors with
promising antibacterial potency, were highly regarded as
a probable solution to the fluoroquinolone-resistant strains
due to their distinct structure and mechanism.[4] However, their
encouraging development has been significantly hampered by
considerable cardiovascular safety risks arising from their
potent hERG inhibition and QTc prolongation in vivo, which
limits their translation into future clinical candidates.[4]
The recent success of moxifloxacin, a fluoroquinolone drug
that demonstrated promising activity against both drug-sensi-
tive and drug-resistant strains of M.tb., has validated DNA
gyrase as a potential target for developing novel antitubercular
Researchers are now focusing their attention on the ATPase
activity of bacterial DNA gyrase that resides in the GyrB sub-
unit.[3,5,6] Research efforts from both industry and academia fo-
cused on the development of antibacterial DNA GyrB inhibitors
have identified many potent inhibitors belonging to the chem-
ical classes of pyrrolamides,[7] pyrazolthiazoles,[8] indolinones,[9]
indazoles,[10] benzimidazoleureas,[11] azaindoles,[12] bithiazoles,[13]
rhodanines,[14] and pyrimidines[15] as potential hits. GyrB has
been genetically demonstrated to be a bactericidal drug target
in M.tb. but there have not been any effective therapeutics de-
veloped against this target for TB.[3]
[a] V. U. Jeankumar, J. Renuka, S. Kotagiri, S. Saxena, S. S. Kakan, J. P. Sridevi,
Prof. P. Yogeeswari, Prof. D. Sriram
Department of Pharmacy, Birla Institute of Technology & Science Pilani
Hyderabad Campus, Shameerpet, R.R. District
Hyderabad 500078, Andhra Pradesh (India)
E-mail: dsriram@hyderabad.bits-pilani.ac.in
[b] S. Yellanki, P. Kulkarni
Dr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus
Gachibowli, Hyderabad 500046 (India)
[c] S. Yellanki, P. Kulkarni
Zephase Therapeutics (an incubated company at Dr. Reddy’s Institute of
Life Sciences), University of Hyderabad Campus
Gachibowli, Hyderabad 500046 (India)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402035.
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Results and Discussion
Information on the common properties of the binding groups
is essential for resolving the type of inhibitor binding to the
target protein. The major aim of drug design is the identifica-
tion and development of new ligands with high affinities of
binding toward the protein receptor. A very useful model for
achieving this goal is structure-based drug design. In the pres-
ent study, the crystal structure of the Mycobacterium smegma-
tis gyrase B protein co-crystallized with a ligand (Protein Data
Bank (PDB) file 4B6C; http://www.rcsb.org)[6] was used as
a structural framework for virtual screening of the BITS Pilani
in-house database to explore new classes of GyrB inhibitor.
The basic goal of the virtual screening was to decrease the
enormous virtual chemical space of small organic molecules to
a manageable number of compounds that could inhibit the
protein with the highest chance to lead to a drug candidate.
The virtual screening options for high-throughput virtual
screening (HTVS), standard precision (SP), and extra precision
(XP) docking were executed. The Glide XP module of Schrç-
dinger 9.2[16] was used finally for docking because it combined
accurate, well-defined physics-based scoring terms and thor-
ough sampling of the terms.
An initial validation of the active site pocket was performed
by re-docking the crystal ligand (6-(3,4-dimethylphenyl)-
3-[[4-[3-(4-methylpiperazin-1-yl)propoxy]phenyl]amino]pyrazine-
2-carboxamide) with the active site residues of the M. smegma-
tis GyrB protein. Re-docking results showed that the com-
pound exhibited similar interactions to those in the original
crystal structure, which was further confirmed with a root
mean square deviation (RMSD) of 0.86 ꢁ. Later, the BITS Pilani
in-house database collection, which consists of a little more
than 3000 compounds, was subjected to a high-throughput
virtual screening. The ligands that displayed a docking score of
ꢀꢁ3.0 kcalmolꢁ1 were regarded as potential hits and were fur-
ther flexibly docked together with the crystal ligand to evalu-
ate precisely the hydrogen-bond interactions, electrostatic in-
teractions, hydrophobic enclosure, and p–p stacking interac-
tions. A final short list of possible lead compounds was based
on visual inspection of the important amino acid residues in-
volved in binding that included hydrogen bonding with Asp79
and Arg 141 and p stacking with Arg82, analogous to that ob-
served with the crystal ligand. Finally, a small subset of 40
compounds was shortlisted from the Glide XP docking study.
The working outline used for identifying the inhibitors has
been depicted in Figure 1 and is described in more detail in
the Supporting Information.
Figure 1. Protocol used for high-throughput screening of the BITS com-
pound collection.
tis. Novobiocin, which has been previously demonstrated to be
a potent inhibitor of DNA GyrB, was used as a positive control
in this study. Negative controls (0% inhibition) did not contain
any inhibitory compounds. The fluoroquinolone drug moxiflox-
acin was also tested; however, as expected, it did not have any
significant inhibitory effect in the GyrB assay. Compounds were
also tested in the presence of Brij-35, a nonanionic detergent,
to ascertain whether these inhibitions were an artifact due to
sequestration of the enzyme by drug aggregates. Other arti-
facts, like autoabsorbance of the drug, were also ruled out by
nullifying its absorbance during the reaction. Compounds in-
hibiting GyrB activity by ꢂ50% at this stage were rescreened
subsequently at 75 mm and 50 mm concentrations and finally
for a dose–response estimation in more detail.
The selected hits retrieved from the BITS Pilani database
were experimentally evaluated for their in vitro M. smegmatis
GyrB ATPase inhibitory potency at a single concentration of
100 mm in triplicate by using a malachite green based assay
adapted to a 96-well plate format as described previously[6]
and detailed extensively in the Supporting Information. The
use of the M. smegmatis GyrB protein as a surrogate for the
GyrB protein from M.tb. has been well demonstrated in the lit-
erature[5,6] because the activity of GyrB protein from M.tb. was
found to be very low when compared with that of M. smegma-
The active hits were subjected to a number of secondary
screenings (described in more detail in the Supporting Infor-
mation) that included the DNA supercoiling assay[17] with DNA
gyrase from M.tb. (Inspiralis, Norwich) to reconfirm their activi-
ty, because any inhibition of ATPase activity conferred by the
DNA GyrB subunit should also inhibit the supercoiling activity
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performed by the GyrA domain.
Moxifloxacin and novobiocin
were used as positive controls
in the study. This was followed
by whole cell screening against
the M.tb. H37Rv strain to under-
stand the bactericidal potency
of the hits by using the alamar
blue assay.[18] Subsequently, the
safety profiles of these mole-
cules were evaluated by check-
ing the in vitro cytotoxicity
against RAW 264.7 cell lines
(mouse macrophage) at the
100 mm concentration by the 3-
(4,5-dimethylthiazol-2-yl)-2,5-di-
Table 1. Biological evaluation of the designed analogues.
Compd
R
IC50 [mm][a]
MIC [mm][c]
Inhib. [%][d]
GyrB
Supercoil
5
6
11.56ꢃ0.21
5.56ꢃ0.29
38.7
36.9
21
31.88ꢃ1.52
1.72ꢃ0.14
14.16ꢃ0.28
3.32ꢃ0.31
35.2
7
19.06
30.8
subset 1
8
9
7.2ꢃ0.31
5.9ꢃ0.22
4.9ꢃ0.44
5.1ꢃ0.23
37.16
35.5
35.6
25.2
phenyltetrazolium
bromide
(MTT) assay.[19] The leads ob-
tained were then clustered
based on scaffold similarity and
segregated based on novelty
search. Finally, the ease/feasibili-
ty of synthesis was also taken
into consideration to arrive at
the lead modification step.
10
11
3.12ꢃ0.17
4.9ꢃ0.18
18.6
39.3
4.8ꢃ0.82
2.3ꢃ0.61
6.2ꢃ0.23
3.6ꢃ0.19
34.1
32.6
58.5
12
45.89
Among the small number of
leads identified, the nitrothiazol-
yl carboxamides 5, 6, and 7
(Table 1) emerged as the most
promising series. The most
potent compound from the vir-
tual high-throughput screening,
subset 2
13
14
21.66ꢃ0.92
0.49ꢃ0.17
19.23ꢃ0.22
1.56ꢃ0.2
38.41
7.51
39.6
35
compound
1.72 mm), displayed
docking score of ꢁ4.63 kcal
molꢁ1
Compound (N-[1-(5-
7
(GyrB IC50 of
a
good
15
39.04ꢃ1.4
>25
50.7
37.72
.
7
nitro-1,3-thiazol-2-yl)piperidin-4-
yl]-1H-pyrrole-2-carboxamide)
16
17
11.58ꢃ0.72
0.81ꢃ0.32
9.32ꢃ0.21
2.16ꢃ029
18.7
33.26
37
was analyzed in more detail to
understand its binding pattern.
As shown in Figure S2 of the
Supporting Information, com-
pound 7 showed two hydrogen-
bonding interactions and one
cation–p interaction with the
relevant amino acid residues.
The NH group on the pyrrole
ring of the right-hand core was
involved in a hydrogen-bonding
interaction with Asp79 analo-
gous to the one observed in the
crystal ligand and considered as
a crucial amino acid interaction
for enzyme activity. In addition,
the thiazole ring on the left-
hand side formed a p-stacking
interaction with Arg82 similar to
16.82
18
19
20
21
22
28.13ꢃ1.2
25.5
32.1
7.7
36
32
36
33
28
4.628ꢃ0.82
32.1ꢃ1.55
7.85ꢃ0.31
0.86ꢃ0.33
1.56ꢃ0.17
>25
62.3
16.2
30.9
7.1ꢃ0.41
8.2ꢃ0.33
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The in vitro GyrB and super-
coiling results of the small
subset synthesized along with
the virtual screening hits again
validated the earlier findings.
One of the most potent ana-
logues in this set also belonged
to the pyrrolamide class (10).
The importance of the hydro-
gen-bonding interaction be-
tween the NH group on the pyr-
role ring with Asp79 for activity
determination was again validat-
ed by compound 10 in subset 1
(Figure S5 in the Supporting In-
formation), which was analogous
to the most potent HTVS lead
compound 7. The docking pat-
tern was similar to that of com-
pound 7 and retained the two
Table 1. (Continued)
Compd
R
IC50 [mm][a]
MIC [mm][c]
Inhib. [%][d]
GyrB
Supercoil
23
24
8.31ꢃ0.21
9.31ꢃ0.6
14.9
37
29
4.3ꢃ0.35
7.32ꢃ0.87
30.05
novobiocin
isoniazid
rifampicin
moxifloxacin
0.18ꢃ0.003.9
0.046ꢃ0.01
ND
ND
11.2ꢃ0.36
ND
ND
ND
ND
ND
ND
ND
0.66
0.23
1.2
>50
[a] Half-maximal inhibitory concentration for M. smegmatis GyrB ATPase activity. [b] Half-maximal inhibitory con-
centration for M.tb. DNA gyrase supercoiling activity. [c] Minimum inhibitory concentration for in vitro M.tb. ac-
tivity. [d] Cytotoxicity measured at 100 mm against RAW 264.7 cells. ND: not determined.
that of the crystal ligand. Also, the NO2 group on the thiazole
ring was found to be involved in an additional hydrogen-
bonding interaction with Arg141 relative to the reference
ligand. The orientation of compound 7 was found to be similar
to that of the crystal ligand and exhibited a very good fit in
the active site pocket, which made this compound more
potent (IC50 =1.72 mm; Table 1).
The other two HTVS hit compounds, 5 and 6 (IC50 values of
11.56 and 31.88 mm, respectively), oriented nicely in the active
site pocket but failed to retain the crucial hydrogen-bonding
interaction with Asp79, which thus explains their reduced ac-
tivity. Compound 5, however, retained a hydrogen-bonding in-
teraction with Arg 141, in addition to the p-stacking interac-
tion with Arg82 (Figure S3 in the Supporting Information), but
compound 6 could only maintain the p-stacking interaction
with Arg 82 (Figure S4 in the Supporting Information), which
again explains the difference in their activity profiles.
Scheme 1. Synthetic pathway adopted for lead optimization. Reagents and
conditions: a) NaNO2/HBr, 08C!RT, 4 h, 65%; b) N-Boc-aminopyridine, K2CO3,
CH3CN, RT, 3 h, 84%; c) HCl in dioxane, 08C!RT, 6 h, 83.4%; d) RCOOH, pro-
pylphosphonic anhydride, (C2H5)3N, CH2Cl2, 08C!RT, 6 h.
A final check was then performed by resynthesizing the
HTVS leads and reinvestigating their exhibited biological po-
tency as a measure of the reproducibility of the results ob-
tained. A quick synthesis of a small subset (subset 1) compris-
ing a modified right-hand portion (8–12) with the introduction
of electron-withdrawing and -donating groups on the hetero-
cyclic moiety was also attempted along with the synthesis of
the HTVS leads to identify the ideal sites for introducing chem-
ical diversity and to have a clear understanding of the role of
these substituents toward bioactivity. The results are tabulated
in Table 1.
important hydrogen-bonding interactions observed with com-
pound 7.
With these interactions and considerations in mind,
a second library (subset 2 in Table 1) retaining the NH group
on the right-hand side was designed and synthesized by using
the same synthetic protocol as above (Scheme 1) as a step
toward lead optimization and the derivation of structure–activ-
ity relationships. The results are summarized in Table 1.
Among the 12 synthesized derivatives, seven compounds in-
hibited GyrB enzyme activity with an IC50 value of less than
10 mm, with compound 14 (3,4-dichloro-5-methyl-N-[1-(5-nitro-
1,3-thiazol-2-yl)piperidin-4-yl]-1H-pyrrole-2-carboxamide) exhib-
iting the most promising potency with an GyrB IC50 value of
0.49 mm, which correlates equally well with a DNA supercoiling
IC50 value of 1.56 mm, a mycobacterial MIC of 7.51 mm, and a rel-
atively good selectivity index. This was well supported by
a very good interaction profile in the docking studies as well,
with a Glide docking score of ꢁ5.88 and a similar orientation
The construction of the target molecules began by condens-
ing 2-bromo-5-nitrothiazole (2) with commercially available 4-
N-tert-butoxycarbonyl-aminopiperidine through a SnAr dis-
placement to give the nitrogen-linked analogue 3 in good
yield. Subsequent tert-butoxycarbonyl (Boc) deprotection fol-
lowed by coupling of the so-obtained primary amine with vari-
ous heterocyclic acids afforded the target compounds in good
yields (Scheme 1).
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observed at the enzyme level.[6] This probably would also ex-
plain why compounds 21–24 displayed excellent to good in-
hibitory potencies in spite of the NH group being masked by
a methyl tail (Figure S9 in the Supporting Information). Two-di-
mensional representations of the interaction profiles of a few
representative analogues, along with their surface pictures, are
given in the Supporting Information (Figures S2–S9) for
a better understanding.
Furthermore, all of the active GyrB leads (subsets 1 and 2)
were found to show equally good activity profiles in the subse-
quent secondary assays as well and exhibited well-correlated
in vitro gyrase supercoiling activity, in vitro bactericidal proper-
ties, and good safety profiles (Table 1). However, a lack of
translation of the promising in vitro gyrase activity with the
bactericidal properties (MIC values), relative to that of the
standards tested (Table 1), could probably be due to cell wall
permeability issues or due to efflux pump transportation.
The binding affinities of the most potent ligands (com-
pounds 7 and 14) were evaluated by measuring the fluores-
cence of the native protein and the protein–ligand complexes
in the presence of a fluorescent dye, the fluorescence of which
increases upon exposure to the nonpolar residues of the pro-
tein and reaches a maximum when the protein denatures.[20,21]
A higher or positive shift of the melting temperature (Tm) of
the protein–ligand complex relative to that of the native pro-
tein signifies a better stabilization of the protein–ligand com-
plex, which in turn would reflect on the inhibitor binding. The
most potent analogue from the HTVS, compound 7, displayed
a Tm shift of 2.28C (Figure S12 in the Supporting Information),
whereas the optimized analogue compound 14 displayed a Tm
shift of 2.98C (Figure 3), which reascertained their affinity
toward the GyrB protein.
Figure 2. a) The binding mode of compound 14 and b) its interaction pat-
tern in the active site of the M. smegmatis GyrB protein.
for the crystal ligand and the lead molecule (compound 7).
Compound 14 retained the two hydrogen-bonding interac-
tions analogous to those observed with the lead molecule 7,
for which interactions with Arg141 and Asp79 were considered
to be crucial for bioactivity (Figure 2). Furthermore, the di-
chloro-substituted pyrrole ring oriented deeply into the hydro-
phobic pocket and was found to be stabilized by hydrophobic
interactions with the Val49, Met100, Ile171, Val77, Ile84 and
Pro38 residues.
However, it was surprising to note that replacement of the
two chloro substituents (compound 14) with two bromo sub-
stituents (compound 15) at the 3 and 4 positions of the pyr-
role core led to a drastic reduction in the activity, as indicated
in interaction profiles, in which compound 15 was found to be
in a slightly different orientation/pose (probably be due to
bulkiness of the bromo group) relative to that of compound
14; it thereby lost the hydrogen-bonding interaction of the
nitro group on the left-hand thiazole core with Arg141 (Fig-
ure S6 in the Supporting Information). A similar profile was
also observed with the saturated analogue, compound 13.
Further replacement of pyrrole core on the right-hand side
with an indole nucleus also resulted in compounds with good
GyrB inhibitory potency (IC50 =
As some of the previously reported DNA gyrase inhibitors
exhibited serious hERG toxicity, which limits their development
as prospective clinical antibacterial candidates,[4,21] the potent
compounds from our study were also evaluated for their cardi-
otoxicity profile in a zebrafish model by using a previously re-
ported protocol.[21,22] All of the tested leads (compounds 7, 14,
and 17) were found to be completely devoid of cardiotoxicity
0.81–28.13 mm). In silico analysis
of these compounds indicated
that the molecules were orient-
ed in a similar manner to that of
the pyrrolamide analogues, with
the hydrogen bonding with
Arg141 retained but without any
interaction with Asp79. However,
their good GyrB potency could
be explained by the hydropho-
bic interaction that the indole
core maintained with the amino
acid residues in the hydrophobic
pocket (Figure S7 in the Sup-
porting Information), which was
earlier demonstrated to be cru-
Figure 3. Differential scanning fluorimetry curve for compound 14 (protein–ligand complex, inner curve) showing
cial for activity and specificity an increase of 2.98C in the Tm value relative to that of the native GyrB protein (outer curve).
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novel DNA GyrB inhibitor class reported in this study
would provide interesting potential for further opti-
mization.
Experimental Section
General: All commercially available chemicals and sol-
vents were used without further purification. TLC experi-
ments were performed on alumina-backed silica gel 40
F254 plates (Merck, Darmstadt, Germany). The homoge-
neity of the compounds was monitored by thin layer
chromatography (TLC) on silica gel 40 F254 coated onto
aluminum plates, visualized by UV light and KMnO4
treatment. Flash chromatography was performed on a Bi-
otage Isolera apparatus with prepackaged disposable
1
normal-phase silica columns. All H and 13C NMR spectra
were recorded on
a
Bruker AM-300 (1H NMR:
300.12 MHz; 13C NMR: 75.12 MHz) NMR spectrometer
(Bruker BioSpin Corp, Germany). Chemical shifts (d) are
reported in ppm with reference to the internal standard
tetramethylsilane. The signals were designated as fol-
lows: br: broad; s: singlet; d: doublet; dd: doublet of
doublets; t: triplet; m: multiplet. The molecular weights
of the synthesized compounds were checked with an
LCMS 6100B series instrument from Agilent Technology.
Elemental analyses were carried out on an automatic
Flash EA 1112 series CHN Analyzer (Thermo). The purity
of the final compounds was examined by HPLC (Shimad-
zu, Japan; with a Phenomenex C8 (150ꢂ4.6 mm, 5 mm,
100 ꢁ) double-end-capped reversed-phase (RP) HPLC
column) and was greater than 95%.
Figure 4. Evaluation of cardiotoxicity of the potent analogues in a zebrafish model.
a) Heart rates in the atria and ventricles: values represent the mean ꢃSEM (n=5 per
group) for treatment groups of compounds 7, 14, and 17 (*p<0.05, **p<0.01, and
***p<0.001); statistical significance was analyzed with respect to the control group.
b) Atrio-venticular beat ratio: values represent the mean ꢃSEM (n=5 per group) for
treatment groups of compounds 7, 14, and 17 (***p<0.001); statistical significance was
analyzed with respect to the control group.
tert-Butyl
(1-(5-nitrothiazol-2-yl)piperidin-4-yl)carba-
mate (3): 4-N-Boc-aminopiperidine (0.96 g, 4.78 mmol)
was added portionwise to a well-stirred suspension of 2-
bromo-5-nitrothiazole (1 g, 4.78 mmol) and potassium
carbonate (0.79 g, 5.74 mmol) in acetonitrile (15 mL). The
reaction mixture was then stirred at room temperature
for 3 h (monitored by TLC and LCMS for completion),
(Figure 4) up to 30 mm concentrations, which makes them rela-
tively safe among the previously reported cardiotoxic N-linked
aminopiperidine analogues.
and the solvent was evaporated under reduced pressure. The resi-
due was further diluted with water (10 mL) and ethyl acetate
(20 mL), and the layers were separated. The aqueous layer was re-
extracted with ethyl acetate (2ꢂ15 mL), and the combined organic
layers were washed with brine (15 mL), dried over anhydrous
sodium sulfate, and evaporated under reduced pressure. The resi-
due was purified by column chromatography with hexane/EtOAc
as the eluent to give the desired product (1.32 g, 84%) as a yellow
Furthermore, the most potent analogues (compounds 5, 7,
10, 14, 17, and 22) were also evaluated for their absorption,
distribution, metabolism, excretion, and toxicity (ADMET) prop-
erties, which were predicted by using the QikProp 3.5 module
of Schrçdinger software.[16] A preliminary view of pharmacoki-
netic parameters indicated that all of the hits preserved the
basic criterion of drug likeness, with characteristics (Table S1 in
the Supporting Information) of promising drug candidates to
be worked for rational drug design against M.tb. GyrB from
a pharmaceutical point of view.
1
solid. H NMR ([D6]DMSO): d=1.39 (s, 9H, Boc H), 1.44–1.49 (m, 2H,
linker H), 1.84–1.89 (m, 2H, linker H), 3.39–4.03 (m, 5H, linker H),
6.89 (br, 1H, NH), 8.38 ppm (s, 1H, thiazole H); 13C NMR
([D6]DMSO): d=171.9, 154.8, 147.6, 135.7, 77.8, 47.1 (2C), 46.1, 30.7
(2C), 28.2 (3C); ESI MS: m/z 329.1 [M+H]+; elemental analysis:
calcd for C13H20N4O4S: C 47.55, H 6.14, N 17.06, found: C 47.49, H
6.11, N 17.09.
1-(5-Nitrothiazol-2-yl)piperidin-4-amine (4): HCl in dioxane (2m,
15 mL) was added dropwise to a solution of tert-butyl (1-(5-nitro-
thiazol-2-yl)piperidin-4-yl)carbamate (1 g, 3.05 mmol) in dry diox-
ane (20 mL) at 08C over a period of 10 min. The reaction was
slowly warmed to room temperature and stirred at that tempera-
ture for 6 h (monitored by TLC and LCMS for completion). The sol-
vent was then removed under reduced pressure, and the residue
was diluted with water (5 mL). The solution was adjusted to pH 8
with solid sodium bicarbonate. The aqueous phase was back ex-
Conclusions
In conclusion, we have disclosed a new class of compounds
that target selectively the mycobacterial DNA gyrase enzyme
with promising attributes of synthetic accessibility, good struc-
ture–activity relationships, and antitubercular activity. With
new anti-TB agents desperately needed, we believe that the
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tracted with dichloromethane (3ꢂ25 mL) and dried over sodium
sulfate. The combined organic phases were concentrated under re-
duced pressure to give the deprotected product (0.58 g, 83.4%) as
yellow solid, which was immediately taken to the next step.
0.438 mmol), triethylamine (0.089 g, 0.88 mmol), propylphosphonic
anhydride solution (0.35 g, 1.1 mmol), and 5-methylfuran-2-carbox-
ylic acid (0.055 g, 0.438 mmol) to afford 8 (0.12 g, 81.6%) as a pale-
yellow solid; mp: 194–1968C; 1H NMR ([D6]DMSO): d=1.51–1.56
(m, 2H, linker H), 1.88–1.93 (m, 2H, linker H), 2.31 (s, 3H, CH3),
3.35–3.43 (m, 2H, linker H), 4.03–4.14 (m, 3H, linker H), 6.47 (d, 1H,
furan H), 7.29 (d, 1H, furan H), 7.63 (br, 1H, NH), 8.42 ppm (s, 1H,
thiazole H); 13C NMR ([D6]DMSO): d=169.6, 163.5, 159.9, 147.6,
145.3, 135.6, 113.9, 108.2, 47.8 (2C), 45.2, 30.7 (2C), 13.2 ppm; ESI
MS: m/z 337.2 [M+H]+; elemental analysis: calcd for C14H16N4O4S:
C 49.99, H 4.79, N 16.66, found: C 50.03, H 4.82, N 16.62.
General procedure for the synthesis of amide derivatives 5–24:
Triethylamine (2 mmol) and the appropriate acid (1 equiv) were
added to
a solution of 1-(5-nitrothiazol-2-yl)piperidin-4-amine
(1 mmol) in dry dichloromethane (3 mL) at 08C. Propylphosphonic
anhydride solution (ꢂ50 wt% in ethyl acetate; 2.5 mmol) was then
added dropwise to the reaction mixture, which was stirred at room
temperature for 6 h (monitored by TLC and LCMS for completion).
The reaction mixture was then washed with water (2 mL) and
brine (2 mL), dried over anhydrous sodium sulfate, and evaporated
in vacuo. The residue obtained was then recrystallized from diethyl
ether.
5-Methyl-N-(1-(5-nitrothiazol-2-yl)piperidin-4-yl)thiophene-2-car-
boxamide (9): The compound was synthesized according to the
general procedure by using 1-(5-nitrothiazol-2-yl)piperidin-4-amine
(0.1 g, 0.438 mmol), triethylamine (0.089 g, 0.88 mmol), propylphos-
phonic anhydride solution (0.35 g, 1.1 mmol), and 5-methylthio-
phene-2-carboxylic acid (0.062 g, 0.438 mmol) to afford 9 (0.1 g,
N-(1-(5-Nitrothiazol-2-yl)piperidin-4-yl)furan-2-carboxamide (5):
The compound was synthesized according to the general proce-
dure by using 1-(5-nitrothiazol-2-yl)piperidin-4-amine (0.1 g,
0.438 mmol), triethylamine (0.089 g, 0.88 mmol), propylphosphonic
anhydride solution (0.35 g, 1.1 mmol), and 2-furoic acid (0.049 g,
0.438 mmol) to afford 5 (0.1 g, 71.4%) as a pale-yellow solid; mp:
208–2108C; 1H NMR ([D6]DMSO): d=1.52–1.59 (m, 2H, linker H),
1.87–1.93 (m, 2H, linker H), 3.37–3.45 (m, 2H, linker H), 4.03–4.14
(m, 3H, linker H), 6.99 (t, 1H, furan H), 7.29 (d, 1H, furan H), 7.68
(br, 1H, NH), 8.13–8.42 ppm (s, 2H, 1 furan and 1thiazole H);
13C NMR ([D6]DMSO): d=169.8, 160.0, 147.6, 146.9, 143.5, 135.8,
111.9, 111.5, 47.7 (2C), 45.2, 30.8 (2C) ppm; ESI MS: m/z 323.3 [M+
H]+; elemental analysis: calcd for C13H14N4O4S: C 48.44, H 4.38, N
17.38, found: C 48.39, H 4.35, N 17.39.
1
64.9%) as a yellow solid; mp: 187–1898C; H NMR ([D6]DMSO): d=
1.56–1.63 (m, 2H, linker H), 1.94–2.03 (m, 2H, linker H), 2.33 (s, 3H,
CH3), 3.34–3.45 (m, 2H, linker H), 4.05–4.19 (m, 3H, linker H), 7.03
(d, 1H, thiophene H), 7.62 (d, 1H, thiophene H), 7.83 (br, 1H, NH),
8.37 ppm (s, 1H, thiazole H); 13C NMR ([D6]DMSO): d=173.1, 160.0,
153.3, 147.4, 136.7, 135.6, 129.7, 128.3, 47.6 (2C), 44.2, 30.7 (2C),
14.2. ppm; ESI MS: m/z 353.5 [M+H]+; elemental analysis: calcd
for C14H16N4O3S2: C 47.71, H 4.58, N 15.90, found: C 47.68, H 4.56; N
15.87.
5-Methyl-N-(1-(5-nitrothiazol-2-yl)piperidin-4-yl)-1H-pyrrole-2-
carboxamide (10): The compound was synthesized according to
the general procedure by using 1-(5-nitrothiazol-2-yl)piperidin-4-
amine (0.1 g, 0.438 mmol), triethylamine (0.089 g, 0.88 mmol), pro-
pylphosphonic anhydride solution (0.35 g, 1.1 mmol), and 5-meth-
ylpyrrole-2-carboxylic acid (0.055 g, 0.438 mmol) to afford 10
N-(1-(5-Nitrothiazol-2-yl)piperidin-4-yl)thiophene-2-carboxamide
(6): The compound was synthesized according to the general pro-
cedure by using 1-(5-nitrothiazol-2-yl)piperidin-4-amine (0.1 g,
0.438 mmol), triethylamine (0.089 g, 0.88 mmol), propylphosphonic
anhydride solution (0.35 g, 1.1 mmol), and thiophene-2-carboxylic
acid (0.056 g, 0.438 mmol) to afford 6 (0.11 g, 74.3%) as a pale-
yellow solid; mp: 201–2038C; 1H NMR ([D6]DMSO): d=1.54–1.61
(m, 2H, linker H), 1.89–1.97 (m, 2H, linker H), 3.35–3.45 (m, 2H,
linker H), 4.04–4.18 (m, 3H, linker H), 7.24 (t, 1H, thiophene H), 7.83
(br, 1H, NH), 8.13–8.47 ppm (m, 3H, 2thiophene and 1thiazole H);
13C NMR ([D6]DMSO): d=173.3, 160.0, 147.4, 137.6, 135.3, 131.6,
130.1, 129.2, 47.4 (2C), 44.1, 30.6 (2C) ppm; ESI MS: m/z 339.4 [M+
H]+; elemental analysis: calcd for C13H14N4O3S2: C 46.14, H 4.17, N
16.56, found: C 46.16, H 4.13, N 16.62.
(0.09 g, 61.2%) as
a
brown solid; mp: 228–2308C; 1H NMR
([D6]DMSO): d=1.53–1.59 (m, 2H, linker H), 1.92–1.99 (m, 2H, linker
H), 2.11 (s, 3H, CH3), 3.36–3.46 (m, 2H, linker H), 4.06–4.2 (m, 3H,
linker H), 5.82 (br, 1H, NH), 6.03 (t, 1H, pyrrole H), 7.12 (d, 1H, pyr-
role H), 7.79 (br, 1H, NH), 8.43 ppm (s, 1H, thiazole H); 13C NMR
([D6]DMSO): d=171.9, 160.1, 147.7, 138.5, 135.8, 126.3, 125.5,
108.6, 47.6 (2C), 44.8, 30.8 (2C), 16.3 ppm; ESI MS: m/z 336.5 [M+
H]+; elemental analysis: calcd for C14H17N5O3S: C 50.14, H 5.11, N
20.88, found: C 50.16, H 5.09, N 20.85.
5-Nitro-N-(1-(5-nitrothiazol-2-yl)piperidin-4-yl)furan-2-carboxa-
mide (11): The compound was synthesized according to the gener-
al procedure by using 1-(5-nitrothiazol-2-yl)piperidin-4-amine
(0.1 g, 0.438 mmol), triethylamine (0.089 g, 0.88 mmol), propylphos-
phonic anhydride solution (0.35 g, 1.1 mmol), and 5-nitro-2-furoic
acid (0.069 g, 0.438 mmol) to afford 11 (0.11 g, 68.7%) as an
orange solid; mp: 227–2298C; 1H NMR ([D6]DMSO): d=1.54–1.63
(m, 2H, linker H), 1.91–1.98 (m, 2H, linker H), 3.35–3.46 (m, 2H,
linker H), 4.09–4.18 (m, 3H, linker H), 7.83–8.47 ppm (m, 4H, 1NH,
2 furan, and 1thiazole H); 13C NMR ([D6]DMSO): d=169.7, 160.0,
154.5, 151.2, 147.6, 135.8, 116.3, 115.9, 47.7 (2C), 45.1, 30.7
(2C) ppm; ESI MS: m/z 368.1 [M+H]+; elemental analysis: calcd for
C13H13N5O6S: C 42.51, H 3.57, N 19.07, found: C 42.49, H 3.58, N
19.04.
N-(1-(5-Nitrothiazol-2-yl)piperidin-4-yl)-1H-pyrrole-2-carboxa-
mide (7): The compound was synthesized according to the general
procedure by using 1-(5-nitrothiazol-2-yl)piperidin-4-amine (0.1 g,
0.438 mmol), triethylamine (0.089 g, 0.88 mmol), propylphosphonic
anhydride solution (0.35 g, 1.1 mmol), and pyrrole 2 carboxylic acid
(0.048 g, 0.438 mmol) to afford 7 (0.09 g, 63.8%) as a buff-colored
solid; mp: 211–2138C; 1H NMR ([D6]DMSO): d=1.54–1.61 (m, 2H,
linker H), 1.91–1.97 (m, 2H, linker H), 3.35–3.44 (m, 2H, linker H),
4.05–4.19 (m, 3H, linker H), 5.32 (br, 1H, NH), 6.09 (t, 1H, pyrrole
H), 6.92 (d, 1H, pyrrole H), 7.84 (d, 1H, pyrrole H), 7.91 (br, 1H, NH),
8.43 ppm (s, 1H, thiazole H); 13C NMR ([D6]DMSO): d=172.0, 160.0,
147.6, 135.8, 126.1, 121.3, 110.0, 108.4, 47.5 (2C), 44.8, 30.7
(2C) ppm; ESI MS: m/z 322.3 [M+H]+; elemental analysis: calcd for
C13H15N5O3S: C 48.59, H 4.70, N 21.79, found: C 48.62, H 4.66, N
21.75.
5-Nitro-N-(1-(5-nitrothiazol-2-yl)piperidin-4-yl)thiophene-2-car-
boxamide (12): The compound was synthesized according to the
general procedure by using 1-(5-nitrothiazol-2-yl)piperidin-4-amine
(0.1 g, 0.438 mmol), triethylamine (0.089 g, 0.88 mmol), propylphos-
phonic anhydride solution (0.35 g, 1.1 mmol), and 5-nitrothio-
phene-2-carboxylic acid (0.075 g, 0.438 mmol) to afford 12 (0.11 g,
5-Methyl-N-(1-(5-nitrothiazol-2-yl)piperidin-4-yl)furan-2-carboxa-
mide (8): The compound was synthesized according to the general
procedure by using 1-(5-nitrothiazol-2-yl)piperidin-4-amine (0.1 g,
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65.5%) as
a
reddish orange solid; mp: 224–2268C; 1H NMR
m/z 334.2 [M+H]+; elemental analysis: calcd for C14H15N5O3S: C
([D6]DMSO): d=1.58–1.69 (m, 2H, linker H), 1.954–2.031 (m, 2H,
linker H), 3.38–3.46 (m, 2H, linker H), 4.11–4.27 (m, 3H, linker H),
8.02–8.45 ppm (m, 4H, 1NH, 2thiophene, and 1thiazole H);
13C NMR ([D6]DMSO): d=173.4, 164.8, 160.2, 147.6, 146.6, 138.8,
135.8, 130.6, 47.6 (2C), 44.2, 30.6 (2C) ppm; ESI MS: m/z 368.1 [M+
H]+; elemental analysis: calcd for C13H13N5O5S2: C 40.72, H 3.42, N
18.27, found: C 40.75, H 3.40, N 18.25.
50.44, H 4.54, N 21.01, found: C 50.42, H 4.51, N 21.03.
N-(1-(5-Nitrothiazol-2-yl)piperidin-4-yl)-1H-indole-2-carboxamide
(17): The compound was synthesized according to the general pro-
cedure by using 1-(5-nitrothiazol-2-yl)piperidin-4-amine (0.1 g,
0.438 mmol), triethylamine (0.089 g, 0.88 mmol), propylphosphonic
anhydride solution (0.35 g, 1.1 mmol), and indole-2-carboxylic acid
(0.071 g, 0.438 mmol) to afford 17 (0.13 g, 81.3%) as a yellow solid;
mp: 178–1808C; 1H NMR ([D6]DMSO): d=1.63–1.71 (m, 2H, linker
H), 1.93–2.06 (m, 2H, linker H), 3.43–3.54 (m, 2H, linker H), 4.06–
4.23 (m, 3H, linker H), 7.04–7.64 (m, 5H, Ar H), 8.01 (br, 1H, NH),
8.41 (s, 1H, thiazole H), 11.31 ppm (br, 1H, NH); 13C NMR
([D6]DMSO): d=172.1, 160.5, 147.6, 136.4, 135.9, 131.5, 127, 123.3,
121.4, 119.7, 112.3, 102.7, 47.5 (2C), 45.3, 30.6 (2C) ppm; ESI MS:
m/z 372.2 [M+H]+; elemental analysis: calcd for C17H17N5O3S: C
54.97, H 4.61, N 18.86, found: C 54.95, H 4.58, N 18.83.
N-(1-(5-Nitrothiazol-2-yl)piperidin-4-yl)pyrrolidine-2-carboxa-
mide (13): The compound was synthesized according to the gener-
al procedure by using 1-(5-nitrothiazol-2-yl)piperidin-4-amine
(0.1 g, 0.438 mmol), triethylamine (0.089 g, 0.88 mmol), propylphos-
phonic anhydride solution (0.35 g, 1.1 mmol), and proline (0.05 g,
0.438 mmol) to afford 13 (0.08 g, 56.3%) as a yellow solid; mp:
172–1748C; 1H NMR ([D6]DMSO): d=1.59–4.24 (m, 17H, 8pyrroli-
dine and 9linker H), 7.94 (br, 1H, NH), 8.35 ppm (s, 1H, thiazole H);
13C NMR ([D6]DMSO): d=178.6, 160.0, 147.6, 135.8, 62.9, 47.4 (2C),
44.3, 42.1, 31.4, 30.6 (2C), 24.8 ppm; ESI MS: m/z 326.3 [M+H]+; el-
emental analysis: calcd for C13H19N5O3S: C 47.99, H 5.89, N 21.52,
found: C 48.02, H 5.86, N 21.49.
5-Fluoro-N-(1-(5-nitrothiazol-2-yl)piperidin-4-yl)-1H-indole-2-car-
boxamide (18): The compound was synthesized according to the
general procedure by using 1-(5-nitrothiazol-2-yl)piperidin-4-amine
(0.1 g, 0.438 mmol), triethylamine (0.089 g, 0.88 mmol), propylphos-
phonic anhydride solution (0.35 g, 1.1 mmol), and 5-fluoroindole-2-
carboxylic acid (0.078 g, 0.438 mmol) to afford 18 (0.12 g, 72.9%)
as a pale-yellow solid; mp: 186–1888C; 1H NMR ([D6]DMSO): d=
1.64–1.73 (m, 2H, linker H), 1.91–2.03 (m, 2H, linker H), 3.43–3.54
(m, 2H, linker H), 4.05–4.23 (m, 3H, linker H), 7.18–7.54 (m, 4H, Ar
H), 8.03 (br, 1H, NH), 8.39 (s, 1H, thiazole H), 11.38 ppm (br, 1H,
NH); 13C NMR ([D6]DMSO): d=172.0, 160.5, 151.6, 147.5, 136.4,
135.8, 135.6, 132.6, 115, 114.6, 108.6, 108.4, 47.5 (2C), 45.3, 30.5
(2C) ppm; ESI MS: m/z 390.1 [M+H]+; elemental analysis: calcd for
C17H16FN5O3S: C 52.43, H 4.14, N 17.98, found: C 52.40, H 4.16, N
17.94.
3,4-Dichloro-5-methyl-N-(1-(5-nitrothiazol-2-yl)piperidin-4-yl)-
1H-pyrrole-2-carboxamide (14): The compound was synthesized
according to the general procedure by using 1-(5-nitrothiazol-2-yl)-
piperidin-4-amine (0.1 g, 0.438 mmol), triethylamine (0.089 g,
0.88 mmol), propylphosphonic anhydride solution (0.35 g,
1.1 mmol), and 3,4-dichloro-5-methyl-1H-pyrrole-2-carboxylic acid
(0.085 g, 0.438 mmol) to afford 14 (0.12 g, 66.6%) as a pale-yellow
solid; mp: 214–2168C; 1H NMR ([D6]DMSO): d=1.53–1.62 (m, 2H,
linker H), 1.87–1.95 (m, 2H, linker H), 2.13 (s, 3H, CH3), 3.35–3.43
(m, 2H, linker H), 4.08–4.25 (m, 3H, linker H), 5.96 (br, 1H, NH), 7.97
(br, 1H, NH), 8.45 ppm (s, 1H, thiazole H); 13C NMR ([D6]DMSO): d=
172.0, 160.0, 147.6, 138.9, 135.8, 133.0, 117.8, 112.3, 47.4 (2C), 44.6,
30.7 (2C), 12.9 ppm; ESI MS: m/z 403 [M]+; elemental analysis:
calcd for C14H15Cl2N5O3S: C 41.59, H 3.74, N 17.32, found: C 41.61, H
3.75, N 17.29.
5-Chloro-N-(1-(5-nitrothiazol-2-yl)piperidin-4-yl)-1H-indole-2-car-
boxamide (19): The compound was synthesized according to the
general procedure by using 1-(5-nitrothiazol-2-yl)piperidin-4-amine
(0.1 g, 0.438 mmol), triethylamine (0.089 g, 0.88 mmol), propylphos-
phonic anhydride solution (0.35 g, 1.1 mmol), and 5-chloroindole-2-
carboxylic acid (0.085 g, 0.438 mmol) to afford 19 (0.11 g, 61.8%)
as a pale-yellow solid; mp: 212–2148C; 1H NMR ([D6]DMSO): d=
1.63–1.71 (m, 2H, linker H), 1.92–1.98 (m, 2H, linker H), 3.39–3.47
(m, 2H, linker H), 4.08–4.19 (m, 3H, linker H), 6.94–8.03 (m, 5H,
1NH and 4Ar H), 8.44 (s, 1H, thiazole H), 11.47 ppm (br, 1H, NH);
13C NMR ([D6]DMSO): d=172.1, 160.0, 147.6, 136.8, 136.4, 135.5,
132.6, 125.2, 122.1, 121.5, 114.3, 113.5, 47.5 (2C), 44.7, 30.6
(2C) ppm; ESI MS: m/z 405.4 [M]+; elemental analysis: calcd for
C17H16ClN5O3S: C 50.31, H 3.97, N 17.26, found: C 50.27, H 3.94, N
17.24.
3,4-Dibromo-5-methyl-N-(1-(5-nitrothiazol-2-yl)piperidin-4-yl)-
1H-pyrrole-2-carboxamide (15): The compound was synthesized
according to the general procedure by using 1-(5-nitrothiazol-2-yl)-
piperidin-4-amine (0.1 g, 0.438 mmol), triethylamine (0.089 g,
0.88 mmol), propylphosphonic anhydride solution (0.35 g,
1.1 mmol), and 3,4-dibromo-5-methyl-1H-pyrrole-2-carboxylic acid
(0.12 g, 0.438 mmol) to afford 15 (0.11 g, 50.9%) as a pale-brown
solid; mp: 188–1908C; 1H NMR ([D6]DMSO): d=1.56–1.69 (m, 2H,
linker H), 1.93–1.99 (m, 2H, linker H), 2.16 (s, 3H, CH3), 3.37–3.46
(m, 2H, linker H), 4.08–4.24 (m, 3H, linker H), 6.13 (br, 1H, NH), 7.89
(br, 1H, NH), 8.42 ppm (s, 1H, thiazole H); 13C NMR ([D6]DMSO): d=
172.2, 159.9, 147.6, 138.6, 135.7, 132.9, 108.6, 98.7, 47.7 (2C), 44.8,
30.8 (2C), 15.3 ppm; ESI MS: m/z 495.1 [M]+; elemental analysis:
calcd for C14H15Br2N5O3S: C 34.10, H 3.07, N 14.20, found: C 34.08,
H 3.09, N 14.16.
5-Methoxy-N-(1-(5-nitrothiazol-2-yl)piperidin-4-yl)-1H-indole-2-
carboxamide (20): The compound was synthesized according to
the general procedure by using 1-(5-nitrothiazol-2-yl)piperidin-4-
amine (0.1 g, 0.438 mmol), triethylamine (0.089 g, 0.88 mmol), pro-
pyphosphonic anhydride solution (0.35 g, 1.1 mmol), and 5-me-
thoxyindole-2-carboxylic acid (0.085 g, 0.438 mmol) to afford 20
N-(1-(5-Nitrothiazol-2-yl)piperidin-4-yl)picolinamide (16): The
compound was synthesized according to the general procedure by
using 1-(5-nitrothiazol-2-yl)piperidin-4-amine (0.1 g, 0.438 mmol),
triethylamine (0.089 g, 0.88 mmol), propylphosphonic anhydride
solution (0.35 g, 1.1 mmol), and pyridine-2-carboxylic acid (0.054 g,
0.438 mmol) to afford 16 (0.1 g, 68.5%) as a pale-yellow solid; mp:
192–1948C; 1H NMR ([D6]DMSO): d=1.59–1.65 (m, 2H, linker H),
1.91–1.98 (m, 2H, linker H), 3.36–3.45 (m, 2H, linker H), 4.07–4.19
(m, 3H, linker H), 7.81–8.65 ppm (m, 6H, 1NH, 4Ar H, and 1thia-
zole H); 13C NMR ([D6]DMSO): d=172.1, 160.2, 152.1, 147.9, 147.5,
137.2, 135.6, 126.7, 121.8, 47.6 (2C), 44.7, 30.5 (2C) ppm; ESI MS:
(0.12 g, 67.4%) as
a
yellow solid; mp: 260–2628C; 1H NMR
([D6]DMSO): d=1.62–1.68 (m, 2H, linker H), 1.89–1.98 (m, 2H, linker
H), 3.35–3.46 (m, 2H, linker H), 3.99 (s, 3H, OCH3), 4.01–4.17 (m,
3H, linker H), 6.89–7.51 (m, 4H, Ar H), 7.93 (br, 1H, NH), 8.39 (s, 1H,
thiazole H), 11.29 ppm (br, 1H, NH); 13C NMR ([D6]DMSO): d=172.0,
160.0, 157.9, 147.6, 136.5, 135.6, 132.2, 131.9, 114.6, 112.1, 111.9,
111.2, 53.6, 47.6 (2C), 44.8, 30.7 (2C) ppm; ESI MS: m/z 402.1 [M+
H]+; elemental analysis: calcd for C18H19N5O4S: C 53.85, H 4.77, N
17.45, found: C 53.82, H 4.74, N 17.48.
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1-Methyl-N-(1-(5-nitrothiazol-2-yl)piperidin-4-yl)-1H-indole-2-car-
boxamide (21): The compound was synthesized according to the
general procedure by using 1-(5-nitrothiazol-2-yl)piperidin-4-amine
(0.1 g, 0.438 mmol), triethylamine (0.089 g, 0.88 mmol), propyphos-
phonic anhydride solution (0.35 g, 1.1 mmol), and 1-methyl-1H-
indole-2-carboxylic acid (0.076 g, 0.438 mmol) to afford 21 (0.1 g,
calcd for C19H21N5O4S: C 54.93, H 5.09, N 16.86, found: C 54.91, H
5.06, N 16.82.
Supporting Information: The Supporting Information contains in-
formation about the computational protocols used, figures of the
binding modes and interactions patterns of representative com-
pounds, and full details of the biological assays.
1
59.2%) as a yellow solid; mp: 219–2218C; H NMR ([D6]DMSO): d=
1.66–1.72 (m, 2H, linker H), 1.94–2.06 (m, 2H, linker H), 3.44–3.54
(m, 2H, linker H), 4.03 (s, 3H, N-CH3), 4.06–4.25 (m, 3H, linker H),
7.12–7.54 (m, 5H, Ar H), 7.88 (br, 1H, NH), 8.41 ppm (s, 1H, thiazole
H); 13C NMR ([D6]DMSO): d=172.0, 160.5, 147.6, 141.6, 136.3, 135.6,
126.3, 121.4, 120.5, 119.6, 114.9, 109.3, 47.6 (2C), 44.9, 32.8, 30.7
(2C) ppm; ESI MS: m/z 386.5 [M+H]+; elemental analysis: calcd for
C18H19N5O3S: C 56.09, H 4.97, N 18.17, found: C 56.11, H 4.95, N
18.19.
Acknowledgements
D.S. acknowledges the University Grant Commission, India for
a UGC research award.
Keywords: DNA gyrase · enzymes · inhibitors · tuberculosis ·
5-Fluoro-1-methyl-N-(1-(5-nitrothiazol-2-yl)piperidin-4-yl)-1H-
indole-2-carboxamide (22): The compound was synthesized ac-
cording to the general procedure by using 1-(5-nitrothiazol-2-yl)pi-
peridin-4-amine (0.1 g, 0.438 mmol), triethylamine (0.089 g,
0.88 mmol), propyphosphonic anhydride solution (0.35 g,
1.1 mmol), and 5-fluoro-1-methyl-1H-indole-2-carboxylic acid
(0.085 g, 0.438 mmol) to afford 22 (0.11 g, 62.2%) as a pale-yellow
solid; mp: 198–2008C; 1H NMR ([D6]DMSO): d=1.68–1.75 (m, 2H,
linker H), 1.92–2.01 (m, 2H, linker H), 3.39–3.50 (m, 2H, linker H),
3.97 (s, 3H, N-CH3), 4.04–4.19 (m, 3H, linker H), 7.19–7.73 (m, 4H,
Ar H), 7.92 (br, 1H, NH), 8.42 ppm (s, 1H, thiazole H); 13C NMR
([D6]DMSO): d=172.1, 160.4, 151.5, 147.6, 141.8, 135.6, 133.1,
131.3, 115.0, 114.8, 112.3, 109.7, 47.6 (2C), 44.8, 32.7, 30.6
(2C) ppm; ESI MS: m/z 404.2 [M+H]+; elemental analysis: calcd for
C18H18FN5O3S: C 53.59, H 4.50, N 17.36, found: C 53.62, H 4.49, N
17.35.
virtual screening
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5-Chloro-1-methyl-N-(1-(5-nitrothiazol-2-yl)piperidin-4-yl)-1H-
indole-2-carboxamide (23): The compound was synthesized ac-
cording to the general procedure by using 1-(5-nitrothiazol-2-yl)pi-
peridin-4-amine (0.1 g, 0.438 mmol), triethylamine (0.089 g,
0.88 mmol), propyphosphonic anhydride solution (0.35 g,
1.1 mmol), and 5-chloro-1-methyl-1H-indole-2-carboxylic acid
(0.092 g, 0.438 mmol) to afford 23 (0.13 g, 70.6%) as a yellow solid;
mp: 208–2108C; 1H NMR ([D6]DMSO): d=1.68–1.75 (m, 2H, linker
H), 1.89–2.03 (m, 2H, linker H), 3.38–3.47 (m, 2H, linker H), 4.02 (s,
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8.11 (br, 1H, NH), 8.39 ppm (s, 1H, thiazole H); 13C NMR
([D6]DMSO): d=171.9, 160.4, 147.6, 141.9, 135.6, 133.9, 132.9,
125.1, 122.2, 121.3, 115.2, 112.1, 47.6 (2C), 44.9, 32.8, 30.7
(2C) ppm; ESI MS: m/z 420 [M]+; elemental analysis: calcd for
C18H18ClN5O3S: C 51.49, H 4.32, N 16.68, found: C 51.51, H 4.30, N
16.63.
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5-Methoxy-1-methyl-N-(1-(5-nitrothiazol-2-yl)piperidin-4-yl)-1H-
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GyrB for TB: By using the pharmaceuti-
cally underexploited mycobacterial DNA
gyrase GyrB domain as a template,
a high-throughput virtual screening
(HTVS) exploration of the BITS Pilani in-
house chemical library led to the iden-
tification of a novel series of Mycobacte-
rium tuberculosis (M.tb.) GyrB inhibitors
displaying considerable in vitro enzyme
efficacy and bactericidal properties
against the M.tb. H37Rv strain.
V. U. Jeankumar, J. Renuka, S. Kotagiri,
S. Saxena, S. S. Kakan, J. P. Sridevi,
S. Yellanki, P. Kulkarni, P. Yogeeswari,
D. Sriram*
&& – &&
Gyrase ATPase Domain as an
Antitubercular Drug Discovery
Platform: Structure-Based Design and
Lead Optimization of Nitrothiazolyl
Carboxamide Analogues
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