Discovery of potent anti-tuberculosis agents targeting leucyl-tRNA synthetase

Article abstract of DOI:10.1016/j.bmc.2016.01.028

Tuberculosis is a serious infectious disease caused by human pathogen bacteria Mycobacterium tuberculosis. Bacterial drug resistance is a very significant medical problem nowadays and development of novel antibiotics with different mechanisms of action is an important goal of modern medical science. Leucyl-tRNA synthetase (LeuRS) has been recently clinically validated as antimicrobial target. Here we report the discovery of small-molecule inhibitors of M. tuberculosis LeuRS. Using receptor-based virtual screening we have identified six inhibitors of M. tuberculosis LeuRS from two different chemical classes. The most active compound 4-{[4-(4-Bromo-phenyl)-thiazol-2-yl]hydrazonomethyl}-2-methoxy-6-nitro-phenol (1) inhibits LeuRS with IC₅₀ of 6 μM. A series of derivatives has been synthesized and evaluated in vitro toward M. tuberculosis LeuRS. It was revealed that the most active compound 2,6-Dibromo-4-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol inhibits LeuRS with IC₅₀ of 2.27 μM. All active compounds were tested for antimicrobial effect against M. tuberculosis H37Rv. The compound 1 seems to have the best cell permeability and inhibits growth of pathogenic bacteria with IC₅₀ = 10.01 μM and IC₉₀ = 13.53 μM.

Full text of DOI:10.1016/j.bmc.2016.01.028

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of potent anti-tuberculosis agents targeting leucyl-tRNA
synthetase
Olga I. Gudzera a, Andriy G. Golub b, Volodymyr G. Bdzhola a, Galyna P. Volynets a, Sergiy S. Lukashov a,
Oksana P. Kovalenko a, Ivan A. Kriklivyi a, Anna D. Yaremchuk a, Sergiy A. Starosyla a,
a
Sergiy M. Yarmoluk a, , Michail A. Tukalo
⇑
a Institute of Molecular Biology and Genetics, NAS of Ukraine, 150 Zabolotnogo St., 03680 Kyiv, Ukraine
b OTAVA Ltd, 400 Applewood Crescent, Unit 100, Vaughan, Ontario L4K0C3, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Tuberculosis is a serious infectious disease caused by human pathogen bacteria Mycobacterium tubercu-
losis. Bacterial drug resistance is a very significant medical problem nowadays and development of novel
antibiotics with different mechanisms of action is an important goal of modern medical science. Leucyl-
tRNA synthetase (LeuRS) has been recently clinically validated as antimicrobial target. Here we report the
discovery of small-molecule inhibitors of M. tuberculosis LeuRS. Using receptor-based virtual screening
we have identified six inhibitors of M. tuberculosis LeuRS from two different chemical classes. The most
active compound 4-{[4-(4-Bromo-phenyl)-thiazol-2-yl]hydrazonomethyl}-2-methoxy-6-nitro-phenol
Received 5 October 2015
Revised 23 December 2015
Accepted 15 January 2016
Available online xxxx
Keywords:
Mycobacterium tuberculosis
Leucyl-tRNA synthetase
Inhibitor
Virtual screening
Aminoacylation assay
(1) inhibits LeuRS with IC50 of 6
in vitro toward M. tuberculosis LeuRS. It was revealed that the most active compound 2,6-Dibromo-4-
{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol inhibits LeuRS with IC50 of 2.27 M. All
lM. A series of derivatives has been synthesized and evaluated
l
active compounds were tested for antimicrobial effect against M. tuberculosis H37Rv. The compound 1
seems to have the best cell permeability and inhibits growth of pathogenic bacteria with
IC50 = 10.01 lM and IC90 = 13.53 lM.
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
At present time it is known several mechanisms of aaRSs inhi-
bition. Most inhibitors interact with the highly conservative syn-
thetic active site and act as amino acid competitive inhibitors or
mimic aminoacyl-adenylate. Among the inhibitors which compete
with amino acids, there were reported analogs of isoleucine such
as cispentacin7 and icofungipen,8 tryptophan analog—indolmycin,9
proline analog—halofuginone,10 phenylalanine analog—ochratoxin
A.11 A substantial number of inhibitors mimic aminoacyl-adeny-
late. For example, mupirocin (pseudomonic acid) is a natural
inhibitor of isoleucyl-tRNA synthetase (IleRS) from gram-positive
bacteria.12 There were designed a number of synthetic inhibitors
which compete with aminoacyl-adenylate with nanomolar and
picomolar affinities toward aaRSs.13–17 Also, a number of inhibitors
bind to other regions of the aaRSs outside the active site, either
allosterically affecting the synthetic active site or binding to
alternative active sites, such as the editing domain.18,19
Tuberculosis is one of the world’s deadliest diseases and a
serious public health problem. A major problem of tuberculosis
treatment is increasing resistance to antibiotics. This provides
the stimulus for search of novel antibiotics with different mecha-
nisms of action. The inhibition of bacterial protein synthesis repre-
sents ideal target for drug discovery. Aminoacyl-tRNA synthetases
(aaRSs) play an indispensable role in protein synthesis and their
structures have some differences in prokaryotes and eukaryotes.1,2
This structural divergence can be exploited for the development of
drugs that inhibit a pathogen synthetase but not its human cell
counterpart. It is known that substitution of key amino acid
residues in the enzyme active site leads to the drug resistance.
Since aaRSs don’t mutate very easily these enzymes are less likely
to become drug-resistant. Therefore, aaRSs are reported to be
promising drug targets.3–6
LeuRS plays an essential role in cellular translation and recently
has been clinically validated as antimicrobial target. Earlier, the
inhibitor of LeuRS—AN2690, was proposed as a new class of
antifungal agent for the topical treatment of onychomycosis.18
Recently, there were published inhibitors of Streptococcus pneumo-
nia20 and Trypanosoma brucei LeuRS.14,15,21 However, to the best
Abbreviations: LeuRS, leucyl-tRNA synthetase; aaRSs, aminoacyl-tRNA
synthetases; IleRS, isoleucyl-tRNA synthetase.
⇑
Corresponding author. Tel.: +38 044 522 2458; fax: +38 044 200 0335.
E-mail address: sergiy@yarmoluk.org.ua (S.M. Yarmoluk).
http://dx.doi.org/10.1016/j.bmc.2016.01.028
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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2
O. I. Gudzera et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
of our knowledge none of the small molecule inhibitors of
Mycobacterium tuberculosis LeuRS have been reported in scientific
literature so far. Therefore, the main aim of this research is to iden-
tify the small molecule inhibitors of M. tuberculosis LeuRS.
In this study the virtual screening experiments were carried out
targeting the active site of M. tuberculosis LeuRS by screening the
compound library of 100,000 organic compounds. After the dock-
ing followed by visual inspection of the best-scored complexes
having score less than ꢀ40 kcal/mol, 270 compounds were
selected for biochemical testing of their inhibitory activity toward
M. tuberculosis LeuRS. In vitro experiments revealed that six com-
pounds from two different chemical classes demonstrated inhibi-
tory activity toward M. tuberculosis LeuRS (Table 1).
Several important structural features of these compounds
can be identified from analysis of their activity on LeuRS
M. tuberculosis.
Compounds 1, 2, 3 and 4 belong to the one chemical class—
derivatives of N-Benzylidene-N0-thiazol-2-yl-hydrazine. Interest-
ingly, other compounds with 4-thiazolidinon core have been found
to have anti-tubercular activity.27,28
2. Results and discussion
In order to discover inhibitors of M. tuberculosis LeuRS we have
performed screening program, using computer modeling methods
and in vitro approaches. The system based on DOCK package was
applied to perform receptor–ligand flexible docking.22–25 The three
dimensional structure of M. tuberculosis LeuRS was modeled using
the known Thermus thermophilus LeuRS structure as a template.18,26
Amino acid sequence identity of the full length M. tuberculosis LeuRS
and T. thermophilus LeuRS is 37%. However, the synthetic active sites
of two enzymes share about 95% sequence homology. Therefore, the
M. tuberculosis LeuRS synthetic active site should serve as a quite
acceptable model for the following virtual screening.
We have performed pairwise alignment of amino acid
sequences for M. tuberculosis and human LeuRS using ClustalW
program. It was revealed that these two enzymes have significant
difference in the amino acid sequences of their active sites (Fig. 1).
This structural divergence can be basis for development of inhibi-
tors with selective action toward M. tuberculosis LeuRS.
Compounds 1 and 2 differ from each other by the nature of sub-
stituent at the fourth position in the benzene ring which accord-
ingly to binding mode obtained with molecular docking interacts
with amino acid residues Tyr99, Trp628, Ser631, His681 and
His685 in the adenine-binding region (Fig. 2). A substitution of bro-
mine atom (compound 1) with a nitro group (compound 2) results
in slightly decreasing of inhibitory activity (IC50 = 6 and 8
lM,
respectively). Apparently, it can be caused by the ability of bromine
Figure 1. Alignment of amino acid sequences for M. tuberculosis and human LeuRS.
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O. I. Gudzera et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Table 1
Compounds chemical structure and in vitro inhibitory activity toward M. tuberculosis leucyl-tRNA
synthetase
S
S
O
O
O
O
N+
Br
OH
OH
N
N
NH
N
NH
N
O
O
N+
O
N+
O
Compound 1 (IC50 = 6 μM)
Compound 2 (IC50 = 8 μM)
S
S
O
O
N+
OH
OH
N
N
NH
N
NH
N
O
N+
O
O
N+
O
Compound 3 (IC50 = 20 μM)
Compound 4 (IC50 = 20 μM)
O
O
O
OH
O
N
N
OH
S
S
O
N
N
H
S
H
S
OH
Compound 5 (IC50 = 22.2 μM)
Compound 6 (IC50 > 50 μM)
atom to form hydrophobic interactions with the amino acid resi-
dues in adenine-binding region.
hydrophobic interactions of methoxy group with active site of
enzyme.
The LeuRS inhibitory activity of compounds in this class
depends on the chemical structure of substituents on another ben-
zene ring which accordingly to computer simulation, interacts
with amino acid residues Gly108, His109, Leu111, Gly112,
Gln714, Gly715, Tyr716 and Ile717 in leucyl-binding region of
LeuRS active site (Fig. 2). As shown in Table 1, the substitution of
methoxy group at the third position (compound 2) with hydrogen
(compound 3) results in over 3-fold decrease in activity (IC50 equal
The compounds 5 and 6 belong to other chemical class—
derivatives of 3-[5-(2-methyl-3-phenyl-allidene)-4-oxo-2-thioxo-
thizolidin-3-yl]-N-phenyl-propionamide. These inhibitors have
different substituents on the phenyl ring: compound 5 has hydroxyl
group at the second position, while compound 6 has carboxyl group
at this place and hydroxyl group at the third position. Such structure
change results in more than 2-fold decreasing of inhibitory activity
(IC50 = 50
lM and 22.2 lM, respectively). Since compounds 5 and 6
to 6 and 20 lM, correspondingly). It can be explained by additional
possess low inhibitory activity toward M. tuberculosis LeuRS and
Figure 2. The binding mode of compound 1 in the active site of LeuRS M. tuberculosis. Hydrogen bonds are shown by the dotted lines.
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4
O. I. Gudzera et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
belong to pan-assay interference (PAINS) compounds we did not
consider them further.
The antimicrobial effect of compounds 1, 2, 3, 4, 7, 8, 9, 11 and
24 against Mycobacterium tuberculosis H37Rv grown under aerobic
conditions was assessed by determining the minimum inhibitory
concentration (MIC) and the concentrations which resulted in
50% and 90% inhibition of growth (IC50 and IC90, respectively).
The assay is based on measurement of growth in liquid medium
of a fluorescent reporter strain of H37Rv where the readout is
either optical density (OD) or fluorescence.29–31 The use of two
readouts minimizes problems caused by compound precipitation
or autofluoresence. A linear relationship between OD and fluores-
cence readout has been established justifying the use of fluores-
cence as a measure of bacterial growth. MIC, IC50 and IC90 values
generated from the OD are presented in Table 4.
In order to find more active inhibitors of M. tuberculosis LeuRS
we have selected 26 derivatives of N-Benzylidene-N0-thiazol-2-yl-
hydrazine from compound library containing about 100,000 com-
pounds and tested in vitro. We have found five molecules able to
inhibit M. tuberculosis LeuRS including compounds 7 (IC50 = 2.27
lM), 8 (IC50 = 19.3
lM), 9 (IC50 = 15.9
lM), 11 (IC50 = 15.9 lM)
and 24 (IC50 = 11.4
lM) (Table 2).
The substitution of methoxy group and nitro group (compound
2) with bromine atoms in R3 phenyl ring (compound 7) results in
significant increasing of inhibitory activity (corresponding IC50 val-
ues are 6 lM and 2.27 lM). The substitution of nitro group in the
structure of R1 substituent and bromine atom in R3 phenyl ring
(compound 7) accordingly with chlorine atom and methoxy group
(compound 9) results in two-fold decreasing of inhibitory activity
The most active compound 1, which seems to have the best cell
permeability, was selected for intracellular (macrophage) drug
screening assay, which evaluates intracellular drug effectiveness.
This is important because M. tuberculosis can survive inside macro-
phages which contributes to treatment failure and disease relapse.
Intracellular drug activity for compound 1 is reported as log
reduction values calculated as reduction in M. tuberculosis concen-
tration from zero hour to 7 days post-infection. The three concen-
(IC50 = 2.27 lM and 4.54 lM, respectively). The presence of nitro
group in meta-position of R1 substituent (compound 11) instead
of chlorine atom in para-position (compound 9) and replacement
of bromine atom to hydrogen atom in R3 phenyl ring causes
decreasing of inhibitory activity toward M. tuberculosis LeuRS in
more than three times. It can be explained by the fact that bromine
atoms take part in strong hydrophobic interactions with adenine-
binding region of LeuRS active site (Fig. 3). Also, it should be noted
that the presence of hydroxy group in para-position of R3 ring com-
pound 7 is very important for inhibitory activity, because the com-
pound 8 with hydroxy group in ortho-position of R3 substituent has
significantly less inhibitory efficiency toward M. tuberculosis LeuRS.
The most active compounds 1, 2, 7, 9, and 24 were tested
against human LeuRS (Table 3). The inhibitory activity of com-
pounds toward pathogenic LeuRS is significantly better, than for
eukaryotic enzyme. Therefore, the inhibitors from this chemical
class seem to be selective toward M. tuberculosis LeuRS.
trations chosen (80 lg/mL, 8.0 lg/mL and 0.8 lg/mL) were based
on the MIC data generated in the HTS primary screen. The mid con-
centration bracketed the reported MIC with the lower concentra-
tion 10-fold below the mid and the higher concentration 10-fold
above the mid. Drug cytotoxicity is reported as cell proliferation,
or percentage of viability. These data are presented in Table 5.
3. Conclusions
Using rational design approach of enzyme inhibitors we have found
small-molecule compounds able to inhibit M. tuberculosis LeuRS
among the derivatives of N-Benzylidene-N0-thiazol-2-yl-hydrazine.
All active compounds were tested for antimicrobial effect against M.
tuberculosis H37Rv. The compound 4-{[4-(4-Bromo-phenyl)-thiazol-
2-yl]hydrazonomethyl}-2-methoxy-6-nitro-phenol seems to have the
best cell permeability and inhibits growth of pathogenic bacteria with
Table 2
Structures and in vitro inhibitory activity for N-Benzylidene-N0-thiazol-2-yl-hydra-
zine derivatives toward M. tuberculosis LeuRS
IC50 = 10.01 lM and IC90 = 13.53 lM.
H
N
N
R3
N
R1
S
4. Experimental
R2
4.1. Synthetic chemistry
R1
R2
R3
IC50 (lM)
Starting materials and solvents were purchased from commer-
cial suppliers and used without further purification. 1H NMR spec-
tra were recorded on a Varian VXR 400 instrument at 400 MHz.
Chemical shifts are described as parts per million (d) downfield
from an internal standard of tetramethylsilane, and spin multiplic-
ities are given as s (singlet), d (doublet), dd (double doublet), t (tri-
plet), q (quartet), quintet (quintet), sext (sextet), sep (septet) or m
(multiplet). HPLC–MS analysis was performed using the Agilent
1100 LC/MSD SL separations module and Mass Quad G1956B mass
detector with electrospray ionization (+ve or ꢀve ion mode as indi-
cated), and HPLC was performed using a Zorbax SB-C18, Rapid Res-
7
8
9
4NO2Ph
4NO2Ph
4ClPh
4BrPh
3NO2Ph
3NO2Ph
3NO2Ph
3NO2Ph
3NO2Ph
2,4dClPh
Ph
Ph
Ph
Ph
Ph
CH2CO2Et
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
3,5dBr-4OH-Ph
3,5dBr-2OH-Ph
3Br-4OH-5MeO-Ph
3,4,5-tMeO-Ph
3MeO-4OH-Ph
4OH-3,5dMeO-Ph
3OH-Ph
2.27
19.3
4.54
>20
15.9
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
11.4
>20
>20
>20
>20
>20
>20
>20
>20
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
2OH-Ph
H
H
3Br-4OH-5MeO-Ph
3MeO-4OH-Ph
4MeO-3OH-Ph
4OH-3,5dMeO-Ph
4OH-3,5dMeO-Ph
4OH-Ph
3Br-4OH-5MeO-Ph
3,5dBr-4OH-Ph
2OH-5Br-Ph
3MeO-4OH-Ph
3Br-4OH-5MeO-Ph
3Br-4OH-5MeO-Ph
4OH-Ph
4OH-3,5dMeO-Ph
3Br-4OH-5MeO-Ph
3MeO-4OH-Ph
2OH-5Br-Ph
Ph
Ph
H
Ph
H
olution HT cartridge, 4.6 mm ꢁ 30 mm, 1.8
lm i.d. column (Agilent
P/N 823975-902) at a temperature of 40 °C with gradient elution of
0–100% CH3CN (with 1 mL/L HCOOH)/H2O (with 1 mL/L HCOOH) at
a flow rate of 3 mL/min and a run time of 2.8 min. Compounds
were detected at 215 nm using a diode array G1315B detector.
All tested compounds gave P95% purity as determined by these
methods.
H
COCH3
CO2Et
H
COCH3
CH3
H
H
H
CH3
H
H
H
H
H
N-Benzylidene-N0-thiazol-2-yl-hydrazine derivatives were syn-
thesized in one pot manner (Scheme 1). Primarily 1,3-thiazole-2-
H
H
hydrazine salt formed in reaction of correspondent
a-chloro- or
4CO2H-Ph
bromoketone with thiosemicarbazide in iPrOH solution.32,33
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O. I. Gudzera et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Figure 3. The binding mode of compound 7 in the active site of LeuRS M. tuberculosis. Hydrogen bonds are shown by the dotted lines.
Table 3
ganic solid material. Mixture was allowed to cool to room temper-
ature, and then precipitate of product was filtered and dried on the
air. Following substances were obtained:
Inhibitory activities for the five selected compounds with human cytoplasmic LeuRS
(HsLeuRS) and M. tuberculosis LeuRS (MtLeuRS)
Compound
IC50 (lM)
4.1.1.1. 4-{[4-(4-Bromo-phenyl)-thiazol-2-yl]-hydrazonomethyl}-
HsLeuRS
MtLeuRS
2-methoxy-6-nitro-phenol (1).
Yield 3.45 g (77%) as wine
1
2
7
9
115.7
45.8
18.7
27.6
116.3
6.00
8.20
2.27
4.54
11.50
powder mp 217–218 °C. LC–MS m/z 449 [M+H+], tR = 1.128 min. 1H
NMR (DMSO-d6) d: 12.00 (br s, 1H), 10.00 (br s, 1H), 8.01 (s, 1H),
7.80 (d, J = 8.4 Hz, 2H), 7.71 (s, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.51 (d,
J = 1.8 Hz, 1H), 7.39 (s, 1H), 3.94 (s, 3H).
24
4.1.1.2. 2-Methoxy-6-nitro-4-{[4-(4-nitro-phenyl)-thiazol-2-yl]-
Hydrazine salt without isolation was basified with sodium carbon-
ate, and then correspondent aldehyde was added. Unsubstituted 2-
hydrazinothiazole hydrochloride was prepared separately starting
from 2-aminothiazole.34
hydrazonomethyl}-phenol (2).
Yield 3.35 g (81%) as wine
powder mp 231–232 °C. LC–MS m/z 416 [M+H+], tR = 1.055 min.
1H NMR (DMSO-d6) d: 12.00 (br s, 1H), 10.00 (br s, 1H), 8.26 (d,
J = 9.0 Hz, 2H), 8.13 (d, J = 9.0 Hz, 2H), 7.93 (s, 1H), 7.70 (br s,
1H), 7.63 (br s, 1H), 7.16 (s, 1H), 3.82 (s, 3H).
4.1.1. General procedure of N-Benzylidene-N0-thiazol-2-yl-
hydrazine derivatives synthesis
4.1.1.3.
2-Nitro-4-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydra-
12 mmol of correspondent halogenoketone and 0.91 g
(10 mmol) of thiosemicarbazide were mixed in 30 ml of iPrOH
and refluxed for 20 min. until bulky precipitate of 2-hydrazinothia-
zole salt formed. Then 2.12 g (20 mmol) of sodium carbonate were
added. Reaction mixture was refluxed for additional 1–2 min,
10 mmol of correspondent aldehyde were added and refluxing
was continued for 1–2 h. During that time heavy bulky precipitate
substantially decreased in volume. After evaporation of solvent
20 ml of acetonitrile were added, mixture was heated to reflux
and resulted solution or suspension was decanted from heavy inor-
zonomethyl}-phenol (3).
Yield 1.79 g (46%) as orange brown
powder mp 277–278 °C. LC–MS m/z 386 [M+H+], tR = 1.077 min. 1H
NMR (DMSO-d6) d: 12.25 (br s, 1H), 10.00 (br s, 1H), 8.26 (d,
J = 8.6 Hz, 2H), 8.12 (d, J = 8.6 Hz, 2H), 8.02 (d, J = 1.2 Hz, 1H),
7.80 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 7.70 (s, 1H), 7.11 (d, J = 8.2 Hz, 1H).
4.1.1.4.
(4).
2-Nitro-4-(thiazol-2-yl-hydrazonomethyl)-phenol
Compound (4) was synthesized starting from 2-
hydrazinothiazole hydrochloride that was prepared separately
from 2-aminothiazole.31 Yield 0.79 g (30%) as brown powder mp
246–247 °C. LC–MS m/z 265 [M+H+], tR = 0.811 min. 1H NMR
(DMSO-d6) d: 11.00 (br s, 2H), 7.99 (d, J = 1.4 Hz, 1H), 7.92 (s,
1H), 7.68 (dd, J = 7.8 Hz, 1.4 Hz, 1H), 7.19 (d, J = 6.5 Hz, 1H), 6.96
(d, J = 8.0 Hz, 1H), 6.81 (d, J = 6.5 Hz, 1H).
Table 4
Compounds antimicrobial activity against Mycobacterium tuberculosis H37Rv (lM)
Compound
MIC
IC50
IC90
1
2
3
4
7
8
9
11
24
25
50
NA
NA
>200
>200
>200
>200
81
10.01
20.594
>100
59.36
130
86
200
>200
59
13.53
28.847
>100
>100
>200
>200
>200
>200
>200
4.1.1.5. N-(4-Hydroxy-phenyl)-3-[5-(2-methyl-3-phenyl-allyli-
dene)-4-oxo-2-thioxo-thiazolidin-3-yl]-propionamide
(5).
Compound (5) was synthesized in a similar manner with
Maga et al.35 as orange powder mp 216–217 °C. LC–MS m/z 425 [M
+H+], tR = 1.061 min. 1H NMR (DMSO-d6) d: 9.80 (br s, 1H), 9.20 (s,
1H), 7.56 (d, J = 1.6 Hz, 1H), 7.47 (m, 6H), 7.30 (d, J = 8.8 Hz, 2H),
6.70 (d, J = 9.0 Hz, 2H), 4.30 (t, J = 6.7 Hz, 2H), 2.66 (t, J = 6.5 Hz,
2H), 2.21 (br. s., 3H).
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O. I. Gudzera et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 5
Macrophage and MTT results for compound 1
Compd
Macrophage log
reduction (low concn)
Macrophage log reduction Macrophage log reduction MTT % viability
MTT % viability
(mid concn)
MTT % viability
(high concn)
(mid concn)
(high concn)
(low concn)
1
1.96
1.06
1.42
2.39
1.20
2.86
100
95
<10
85
10
73
Rifampin (pos
control)
H
H
N
N
N
NH2
+
R1
R2
O
N
NH2
R3
N
R1
R1
+
NH2
O
R3
S
N
H
S
S
Cl or Br
R2
R2
Scheme 1. Synthesis of N-Benzylidene-N0-thiazol-2-yl-hydrazine derivatives.
4.1.1.6.
2-Hydroxy-4-{3-[5-(2-methyl-3-phenyl-allylidene)-4-
4.1.1.13. 3-{[4-(3-Nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-
oxo-2-thioxo-thiazolidin-3-yl]-propionylamino}-benzoic acid
phenol (13).
Yield 1.67 g (49%) as orange powder, mp 238–
(6).
Compound (6) was synthesized analogously to (5) orange
240 °C. LC–MS m/z 341 [M+H+], tR = 0.91 min. 1H NMR (DMSO-d6)
d: 10.50 (br s, 2H), 8.69 (d, J = 2.2 Hz, 1H), 8.31 (d, J = 8.8 Hz, 1H),
8.15 (dd, J = 8.6 Hz, 1.6 Hz, 1H), 7.97 (s, 1H), 7.69 (t, J = 8.8 Hz, 1H),
7.65 (s, 1H), 7.24 (t, J = 8.4 Hz, 1H), 7.14 (s, 1H), 7.09 (d, J = 7.8 Hz,
1H), 6.82 (d, J = 8.6 Hz, 1H).
powder mp 257–258 °C. LC–MS m/z 469 [M+H+], tR = 0.942 min. 1H
NMR (DMSO-d6) d: 13.50 (br. s., 1H), 11.38 (br s, 1H), 10.32 (s, 1H),
7.70 (d, J = 7.8 Hz, 1H), 7.56 (s, 1H), 7.47 (m, 7H), 7.03 (d, J = 8.4 Hz,
1H), 4.33 (t, J = 7.0 Hz, 2H), 2.76 (t, J = 6.8 Hz, 2H), 2.21 (s, 3H).
4.1.1.7. 2,6-Dibromo-4-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydra-
4.1.1.14. 2-{[4-(3-Nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-
zonomethyl}-phenol (7).
Yield 1.59 g (32%) as light brown
phenol (14).
Yield 1.79 g (53%) as yellow powder, mp 231–
powder, mp 230–232 °C. LC–MS m/z 497 [M+H+], tR = 1.107 min.
1H NMR (DMSO-d6) d: 12.65 (br s, 1H), 10.41 (br s, 1H), 8.26 (d,
J = 6.5 Hz, 1H), 8.14 (d, J = 7.4 Hz, 2H), 7.89 (d, J = 7.4 Hz, 2H), 7.78
(m, 3H).
233 °C. LC–MS m/z 341 [M+H+], tR = 1.089 min. 1H NMR (DMSO-d6)
d: 12.15 (br s, 1H), d 10.0 (br s, 1H), 8.66 (d, J = 2.4 Hz, 1H), 8.34 (s,
1H), 8.29 (d, J = 9.0 Hz, 1H), 8.12 (dd, J = 8.8 Hz, 1.5 Hz, 1H), 7.70 (t,
J = 9.0 Hz, 1H), 7.62 (t, J = 8.6 Hz, 1H), 7.22 (t, J = 8.4 Hz, 1H), 6.90
(m, 2H).
4.1.1.8. 2,4-Dibromo-6-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydra-
zonomethyl}-phenol (8).
Yield 0.032 g (7%) as mustard pow-
4.1.1.15. 2-Bromo-6-methoxy-4-{[4-(3-nitro-phenyl)-thiazol-2-
der, mp 247–249 °C. LC–MS m/z 497 [M+H+], tR = 1.112 min. 1H
NMR (DMSO-d6) d: 12.63 (br s, 1H), 10.75 (br s, 1H), 8.27 (m, 3H),
8.13 (d, J = 7.6 Hz, 2H), 7.77 (m, 3H).
yl]-hydrazonomethyl}-phenol (15).
Yield 2.37 g (53%) as light
brown powder, mp 121–122 °C. LC–MS m/z 449 [M+H+], tR =
1.114 min. 1H NMR (DMSO-d6) d: 12.24 (br s, 1H), 9.90 (br s, 1H),
8.68 (d, J = 2.0 Hz, 1H), 8.33 (d, J = 8.5 Hz, 1H), 8.13 (dd, J = 8.4 Hz,
1.8 Hz, 1H), 7.94 (s, 1H), 7.73 (d, J = 9.2 Hz, 1H), 7.64 (t, J = 8.4 Hz,
1H), 7.41 (d, J = 2.6 Hz, 1H), 7.27 (d, J = 2.8 Hz, 1H), 3.90 (s, 3H).
4.1.1.9. 2-Bromo-4-{[4-(4-chloro-phenyl)-thiazol-2-yl]-hydra-
zonomethyl}-6-methoxy-phenol (9).
Yield 0.70 g (17%) as
cocoa powder, mp 253–255 °C. LC–MS m/z 438 [M+H+], tR =
0.994 min. 1H NMR (DMSO-d6) d: 12.37 (br s, 1H), 9.93 (br s, 1H),
7.90 (s, 1H), 7.76 (d, J = 7.6 Hz, 2H), 7.40 (d, J = 7.6 Hz, 2H), 7.36
(s, 2H), 7.21 (s, 1H), 3.86 (s, 3H).
4.1.1.16.
4-{[4-(2,4-Dichloro-phenyl)-thiazol-2-yl]-hydra-
Yield 0.6 g (15%) as
zonomethyl}-2-methoxy-phenol (16).
light brown powder, mp 127–128 °C. LC–MS m/z 394 [M+H+],
tR = 1.054 min. 1H NMR (DMSO-d6) d: 11.80 (br s, 1H), 9.90 (br s,
1H), 7.92 (d, J = 8.7 Hz, 1H), 7.89 (s, 1H), 7.45 (d, J = 2.0 Hz, 1H),
7.36 (dd, J = 8.5 Hz, 1.8 Hz, 1H), 7.20 (m, 2H), 6.99 (d, J = 9.2 Hz,
1H), 6.78 (d, J = 9.0 Hz, 1H), 3.88 (s, 3H).
4.1.1.10. N-[4-(4-Bromo-phenyl)-thiazol-2-yl]-N0-(3,4,5-trimethoxy-
benzylidene)-hydrazine (10).
Yield 3.14 g (70%) as creamy pow-
der, mp 170–171 °C. LC–MS m/z 448 [M+H+], tR = 1.104 min. H NMR
(DMSO-d6) d: 12.15 (br s, 1H), 7.97 (s, 1H), 7.78 (d, J = 7.8 Hz, 2H),
7.62 (d, J = 7.8 Hz, 2H), 7.38 (s, 1H), 6.96 (s, 2H), 3.84 (s, 6H), 3.70 (s,
3H).
1
4.1.1.17.
5-[(4,5-Diphenyl-thiazol-2-yl)-hydrazonomethyl]-2-
methoxy-phenol (17).
Yield 1.83 g (46%) as sandy powder,
mp 165–167 °C. LC–MS m/z 402 [M+H+], tR = 1.011 min. 1H NMR
(DMSO-d6) d: 11.98 (br s, 1H), 9.21 (br s, 1H), 7.91 (s, 1H), 7.31
(m, 10H), 7.21 (s, 1H), 6.96 (q, J = 7.6 Hz, 2H), 3.80 (s, 3H).
4.1.1.11. 2-Methoxy-4-{[4-(3-nitro-phenyl)-thiazol-2-yl]-hydra-
zonomethyl}-phenol (11).
Yield 2.09 g (57%) as brown pow-
der, mp 200–202 °C. LC–MS m/z 371 [M+H+], tR = 0.99 min. 1H NMR
(DMSO-d6) d: 12.10 (br s, 1H), 9.65 (br s, 1H), 8.68 (s, 1H), 8.29 (d,
J = 10.0 Hz, 1H), 8.14 (dd, J = 10.4 Hz, 1.8 Hz, 1H), 7.95 (s, 1H), 7.71
(t, J = 10.0 Hz, 1H), 7.63 (s, 1H), 7.25 (d, J = 1.5 Hz, 1H), 7.08 (dd,
J = 10.0 Hz, 1.5 Hz, 1H), 6.84 (d, J = 10.0 Hz, 1H), 3.84 (s, 3H).
4.1.1.18. 4-[(4,5-Diphenyl-thiazol-2-yl)-hydrazonomethyl]-2,6-
dimethoxy-phenol (18).
Yield 2.63 g (61%) as beige powder,
mp 222–223 °C. LC–MS m/z 432 [M+H+], tR = 1.074 min. 1H NMR
(DMSO-d6) d: 10.0 (br s, 2H), 7.93 (s, 1H), 7.43 (m, 2H), 7.32 (m,
8H), 6.94 (s, 2H), 3.81 (s. 6H).
4.1.1.12.
hydrazonomethyl}-phenol (12).
2,6-Dimethoxy-4-{[4-(3-nitro-phenyl)-thiazol-2-yl]-
Yield 1.54 g (39%) as brown
4.1.1.19.
zonomethyl]-phenol (19).
2,6-Dimethoxy-4-[(4-phenyl-thiazol-2-yl)-hydra-
Yield 0.87 g (25%) as sandy pow-
powder, mp 155–157 °C. LC–MS m/z 401 [M+H+], tR = 1.079 min.
1H NMR (DMSO-d6) d: 12.15 (br s, 1H), 8.81 (br s, 1H), 8.68 (d,
J = 2.4 Hz, 1H), 8.29 (dd, J = 8.6 Hz, 1.8 Hz, 1H), 8.16 (dd, J = 8.2 Hz,
1.7 Hz, 1H), 7.95 (d, J = 1.4 Hz, 1H), 7.71 (t, J = 8.7 Hz, 1H), 7.63 (s,
1H), 6.96 (s, 2H), 3.83 (s, 6H).
der, mp 212–213 °C. LC–MS m/z 356 [M+H+], tR = 0.954 min. 1H
NMR (DMSO-d6) d: 10.00 (br s, 2H), 7.94 (s, 1H), 7.85 (d,
J = 7.8 Hz, 2H), 7.41 (t, J = 7.7 Hz, 2H), 7.29 (m, 2H), 6.95 (s, 2H),
3.82 (s. 6H).
Please cite this article in press as: Gudzera, O. I.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.028

O. I. Gudzera et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
4.1.1.20. 4-[(4,5-Diphenyl-thiazol-2-yl)-hydrazonomethyl]-phe-
LC–MS m/z 250 [M+H+], tR = 0.644 min. 1H NMR (DMSO-d6) d:
10.00 (br s, 2H), 7.92 (s, 1H), 7.22 (s, 2H), 7.03 (d, J = 6.8 Hz, 1H),
6.81 (m, 2H), 3.82 (s, 3H).
nol (20).
Yield 2.71 g (73%) as beige powder, mp 126–127 °C.
LC–MS m/z 372 [M+H+], tR = 1.064 min. 1H NMR (DMSO-d6) d:
11.96 (br s, 1H), 9.86 (br s, 1H), 7.96 (s, 1H), 7.49 (m, 4H), 7.31
(m, 8H), 6.85 (d, J = 8.4 Hz, 2H).
4.1.1.31.
(31).
4-Bromo-2-(thiazol-2-yl-hydrazonomethyl)-phenol
Yield 0.65 g (22%) as dark gray powder, mp 236–
4.1.1.21. 2-Bromo-6-methoxy-4-[(4-phenyl-thiazol-2-yl)-hydra-
237 °C. LC–MS m/z 298 [M+H+], tR = 0.683 min. 1H NMR (DMSO-
d6) d: 12.00 (br s, 1H), 10.50 (s, 1H), 8.25 (s, 1H), 7.72 (d,
J = 1.2 Hz, 1H), 7.33 (d, J = 7.0 Hz, 1H), 7.21 (s, 1H), 6.88 (m, 2H).
zonomethyl]-phenol (21).
Yield 0.49 g (12%) as brown
prisms, mp 196–197 °C. LC–MS m/z 404 [M+H+], tR = 0.973 min.
1H NMR (DMSO-d6) d: 12.05 (br s, 1H), 9.99 (br s, 1H), 7.94 (s,
1H), 7.85 (d, J = 7.6 Hz, 2H), 7.41 (m, 3H), 7.30 (m, 3H), 3.89 (s, 3H).
4.1.1.32.
(32).
4-(Thiazol-2-yl-hydrazonomethyl)-benzoic
acid
Yield 1.09 g (43%) as mustard powder, mp 277–278 °C
4.1.1.22. {2-[N0-(3,5-Dibromo-4-hydroxy-benzylidene)-hydra-
(subl.). LC–MS m/z 248 [M+H+], tR = 0.706 min. 1H NMR (DMSO-
d6) d: 11.30 (br s, 3H), 8.44 (s, 1H), 8.02 (d, J = 8.7 Hz, 2H), 7.92
(d, J = 8.6 Hz, 2H), 7.49 (d, J = 6.0 Hz, 1H), 7.17 (d, J = 6.0 Hz, 1H).
zino]-thiazol-4-yl}-acetic acid ethyl ester (22).
Yield 4.10 g
(89%) as ivory powder, mp 187–188 °C. LC–MS m/z 462 [M+H+],
tR = 0.847 min. 1H NMR (DMSO-d6) d: 11.00 (br s, 2H), 7.84 (s,
1H), 7.80 (s, 2H), 6.64 (s, 1H), 4.07 (q, J = 7.7 Hz, 2H), 3.58 (s, 2H),
1.19 (t, J = 7.5 Hz, 3H).
4.2. Receptor preparation for flexible docking
Homology model of Mycobacterium tuberculosis LeuRS has been
built using SWISS-MODEL workspace.36 As a template for homol-
ogy modeling was used crystal structure of Thermus thermophilus
LeuRS (PDB ID: 2V0C).18
4.1.1.23. 1-{2-[N0-(5-Bromo-2-hydroxy-benzylidene)-hydrazino]-
4-methyl-thiazol-5-yl}-ethanone (23).
Yield 2.89 g (82%) as
golden prisms, mp 262–263 °C. LC–MS m/z 354 [M+H+], tR =
0.779 min. 1H NMR (DMSO-d6) d: 12.53 (br s, 1H), 10.59 (br s, 1H),
8.36 (s, 1H), 7.76 (d, J = 1.4 Hz, 1H), 7.38 (dd, J = 8.2 Hz, 1.4 Hz, 1H),
6.91 (d, J = 8.4 Hz, 1H), 2.50 (s, 3H), 2.42 (s, 3H).
A receptor molecule has been minimized in water with GRO-
MACS molecular dynamics simulation package (GROMACS force
field, steepest descent algorithm, 1000 steps, em_tolerance = 100,
em_step = 0,001). The partial atom charges of the receptor mole-
cule were taken from the Amber force field. Active site spheres
were calculated with DOCK sphgen software. The spheres with
the positions outside of the active site of LeuRS were deleted man-
ually. Connolly MS and Grid programs from DOCK package were
used to generate receptor Connolly surface and energy grids. Sur-
face and grid calculations were performed with parameter settings
detailed in the reference.37 The grid spacing was set to 0.3.
4.1.1.24. 2-[N0-(4-Hydroxy-3-methoxy-benzylidene)-hydrazino]-
4-methyl-thiazole-5-carboxylic acid ethyl ester (24).
Yield
1.05 g (32%) as ivory powder, mp 178–179 °C. LC–MS m/z 336 [M
+H+], tR = 0.727 min. 1H NMR (DMSO-d6) d: 12.30 (br s, 1H), 9.52
(br s, 1H), 7.98 (s, 1H), 7.25 (d, J = 1.2 Hz, 1H), 7.10 (d, J = 9.3 Hz,
1H), 6.84 (dd, J = 9.4 Hz, 1.0 Hz, 1H), 4.21 (q, J = 6.8 Hz, 2H), 3.84
(s, 3H), 2.46 (s, 3H), 1.27 (t, J = 7.0 Hz, 3H).
4.1.1.25. 2-Bromo-6-methoxy-4-[(4-methyl-thiazol-2-yl)-hydra-
4.3. Ligand database processing
zonomethyl]-phenol (25).
Yield 0.37 g (11%) as gray powder,
mp 176–178 °C. LC–MS m/z 342 [M+H+], tR = 0.672 min. 1H NMR
(DMSO-d6) d: 10.00 (br s, 2H), 7.99 (s, 1H), 7.44 (s, 1H), 7.29 (s,
1H), 6.46 (s, 1H), 3.90 (s, 3H), 2.21 (s, 1H).
Calculations of ligand geometry were performed using YFF force
field described in the reference.38 Partial atomic charges of the
ligands were calculated with Kirchhoff method.39
4.1.1.26. 1-{2-[N0-(3-Bromo-4-hydroxy-5-methoxy-benzylidene)-
4.4. Flexible docking
hydrazino]-4-methyl-thiazol-5-yl}-ethanone
(26).
Yield
2.34 g (61%) as mustard powder, mp 215–216 °C. LC–MS m/z 384
[M+H+], tR = 0.731 min. 1H NMR (DMSO-d6) d: 7.97 (s, 1H), 7.38 (s,
1H), 7.23 (s, 1H), 5.00 (br s, 2H), 3.86 (s, 3H), 2.48 (s, 3H), 2.39 (s, 3H).
DOCK program has been used for receptor-ligand flexible dock-
ing. DOCK input parameters have been set as described previ-
ously.37 In our virtual screening experiments ‘multiple anchors’
parameter was set as following: the minimum of heavy atoms in
the anchor was set to 6, the maximum number of orientations
was set to 1000, and the ꢂall atomsꢃ model has been chosen.
270 compounds with scores less than ꢀ40 kcal/mol have been
taken for the in vitro analysis of their inhibitory activity.
The structures were visualized using the Discovery Studio
Visualizer 4.0.40
4.1.1.27. 4-[(4,5-Dimethyl-thiazol-2-yl)-hydrazonomethyl]-phe-
nol (27).
Yield 1.45 g (59%) as beige powder, mp 145–147 °C.
LC–MS m/z 248 [M+H+], tR = 0.608 min. 1H NMR (DMSO-d6) d:
11.00 (br s, 1H), 9.77 (s, 1H), 7.88 (s, 1H), 7.43 (d, J = 9.0 Hz, 2H),
6.81 (d, J = 9.0 Hz, 2H), 2.15 (s, 3H), 2.05 (s, 3H).
4.1.1.28. 2,6-Dimethoxy-4-(thiazol-2-yl-hydrazonomethyl)-phe-
nol (28).
Yield 2.17 g (78%) as cocoa powder, mp 207–208 °C.
4.5. Purification of Mycobacterium tuberculosis LeuRS
LC–MS m/z 280 [M+H+], tR = 0.763 min. 1H NMR (DMSO-d6) d:
10.00 (br s, 2H), 7.91 (s, 1H), 7.20 (d, J = 6.5 Hz, 1H), 6.92 (s, 2H),
6.80 (d, J = 6.3 Hz, 1H), 3.81 (s, 6H).
The plasmid pET28a encoding the gene for M. tuberculosis LeuRS
(the plasmid was the kind gift of Stephen Cusack and Andres Palen-
cia) was used to express the protein in Escherichia coli strain
Rosetta (DE3). A single colony was used to inoculate LB medium
containing 50 mg/L kanamycin and 36 mg/L chloramphenicol,
which was then incubated overnight at 37 °C. A culture was
induced to a final concentration of 1 mM IPTG and was incubated
for 7 h at 23 °C. The cells were harvested by centrifugation.
The cells pellet was resuspended in 20–25 mL of buffer contain-
ing 20 mM Tris–HCl (pH 8.0), 100 mM NaCl, 5 mM MgCl2, 1 mM b-
mercaptoethanol and 1 tablet of Complete EDTA-free protease
4.1.1.29. 2-Bromo-6-methoxy-4-(thiazol-2-yl-hydrazonomethyl)-
phenol (29).
Yield 1.40 g (43%) as sandy powder, mp 202–
203 °C. LC–MS m/z 328 [M+H+], tR = 0.796 min. 1H NMR (DMSO-d6)
d: 10.00 (br s, 2H), 7.89 (s, 1H), 7.36 (s, 1H), 7.22 (m, 2H), 6.83 (d,
J = 6.0 Hz, 1H), 3.87 (s, 6H).
4.1.1.30. 2-Methoxy-4-(thiazol-2-yl-hydrazonomethyl)-phenol
(30).
Yield 0.75 g (30%) as black prisms, mp 158–160 °C.
Please cite this article in press as: Gudzera, O. I.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.028

8
O. I. Gudzera et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
cocktail inhibitors and was sonicated for 10 min (in total) with 30 s
of active cycle and 60 s of cool down. The lysate was cleared by cen-
trifugation for 30 min at 16,000 rpm. The imidazole was added to
supernatant to reach 15 mM in final and was mixed with 5 mL of
Ni-NTA in binding buffer A (20 mM Tris–HCl (pH 8.0), 300 mM
NaCl, 5 mM MgCl2, 15 mM imidazole, 1 mM b-mercaptoethanol,
0.1 mM PMSF). The mixture was stirred for 1 h at 4 °C and was
applied into the empty column. The column was washed by
15 mL of buffer B (20 mM Tris–HCl (pH 8.0), 0.3 M NaCl, 5 mM
MgCl2, 15 mM imidazole, 1 mM b-mercaptoethanol, 0.1 mM PMSF).
Elution of protein was carried by buffer E (20 mM Tris–HCl (pH 8.0),
0.3 M NaCl, 5 mM MgCl2, 0.2 M imidazole, 5 mM b-mercap-
toethanol, 0.1 mM PMSF). The homogeneity of the protein was
tested by 12.5% SDS–PAGE. The fractions were pooled and dialyzed
for 2 h with 20 mM Tris–HCl (pH 8.0), 100 mM NaCl, 5 mM MgCl2,
5 mM mercaptoethanol and overnight with 20 mM Tris–HCl (pH
7.4), 100 mM NaCl, 5 mM MgCl2, 5 mM mercaptoethanol. The puri-
fied protein was concentrated using a Centricon-30 (Amicon).
were prepared as 10-point two-fold serial dilutions in DMSO and
diluted into 7H9-Tw-OADC medium in 96-well plates with a final
DMSO concentration of 2%. The highest concentration of com-
pound was 200 lM where compounds were soluble in DMSO at
10 mM. For compounds with limited solubility, the highest con-
centration was 50ꢁ less than the stock concentration, for example,
100 lM for 5 mM DMSO stock, 20 lM for 1 mM DMSO stock. For
potent compounds, assays were repeated at lower starting concen-
trations. Each plate included assay controls for background (med-
ium/DMSO only, no bacterial cells), zero growth (100 lM
rifampicin) and maximum growth (DMSO only), as well as a rifam-
picin dose response curve. Plates were inoculated with M. tubercu-
losis and incubated for 5 days: growth was measured by OD590 and
fluorescence (Ex 560/Em 590) using a BioTekTM Synergy 4 plate
reader. Growth was calculated separately for OD590 and RFU. To
calculate the MIC, the 10-point dose response curve was plotted
as % growth and fitted to the Gompertz model using GraphPad
Prism 5. The MIC was defined as the minimum concentration at
which growth was completely inhibited and was calculated from
the inflection point of the fitted curve to the lower asymptote (zero
growth). In addition dose response curves were generated using
the Levenberg–Marquardt algorithm and the concentrations that
resulted in 50% and 90% inhibition of growth were determined
(IC50 and IC90 respectively).
4.6. Purification of human cytoplasmic LeuRS
The plasmid pET16 encoding humancytoplasmicLeuRS, C-termi-
nal histidine tag (the plasmid was the kind gift of Dr. Michael Alley),
was transformed in E. coli strain Rosetta DE3. A single colony was
inoculated into 10 ml of 2xYT medium contained 69 mg/L chloram-
phenicol, 100 mg/L ampicillin and incubated at 37 °C overnight.
10 ml overnight culture was transferred to 1 l of 2xYT medium
and grown at 37 °C until OD600 = 0.6. A culture was then induced
with 0.4 mM IPTG and further grown at 20 °C for 5 h. The cells were
pelleted by centrifugation at 5000 rpm for 15 min at 4 °C. The cells
pellet was lysed with 20 ml buffer A (20 mM Na-p (pH 7.9), contain-
ing 300 mM NaCl, 5 mM imidazole, 5% glycerol, 1 mM b-mercap-
toethanol and 1 tablet of Complete EDTA-free protease cocktail
inhibitors). The suspended cells were sonicated at 40% power
3 min using an ultrasonic processor XL-Sonicator and centrifuged
at 14,000 rpm for 30 min at 4 °C. The supernatant was combined
with 5 ml Ni-NTA agarose (pre-equilibrated with buffer A). The gel
and lysate were mixed 1 h at 4 °C, and loaded on column. The col-
umn was then washed with 5 column volumes of buffer A and eluted
with 250 mM imidazole in buffer A. The eluted fractions containing
4.9. Intracellular drug activity
The murine J774 cell line was propagated in RPMI 1640 supple-
mented with L-glutamine and fetal bovine serum (FBS). Cells were
maintained in tissue culture flasks at 37 °C in the presence of 5%
CO2. For infection studies, J774 cells were transferred to 12-well tis-
sue culture chambers in 1 mL volumes at a density of 2.0 * 105 in
the presence of 10% FBS. After overnight incubation, the medium
was replaced with fresh medium containing 1% FBS to stop macro-
phage division while maintaining cell viability. Twenty-four hours
later, the macrophage monolayer was enumerated with an ocular
micrometer for total number of cells per well to determine the
infection ratio. The medium was removed and replaced with 1 mL
of fresh medium with 1% FBS containing M. tuberculosis at a multi-
plicity of infection (MOI) of 5 Mycobacteria/macrophage. The cells
are infected for 4 h after which time nonphagocytosed Mycobacteria
were washed from the monolayers and fresh medium added. Drugs
were then added, using 3 concentrations, and infection allowed to
proceed for 7 days. At 0 and 7 days, the macrophages were lysed
with sodium dodecyl sulfate, treated with DNAase, diluted and pla-
ted onto 7H10 agar to determine the cell number or colony forming
units (CFU). Each drug concentration was tested in duplicate and
rifampin was used as the positive control drug. A drug cytotoxicity
control plate assay (MTT proliferation) was also conducted in paral-
lel using uninfected macrophages to confirm that concentrations
utilized for testing were not toxic to the macrophages.
desired protein were pooled and dialyzed against buffer
B
(100 mM K-p (pH 6.8), 10 mM mercaptoethanol and 20% glycerol).
Protein was concentrated using a Centricone-30 (Amicon).
4.7. Aminoacylation assay
The standard aminoacylation assays were performed in
100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 2 mM DTT, 5 mM ATP,
10 mM KCl, 90 lM [ L-leucine (238 mCi/mmol), 4 mg/ml
14C]-
E. coli tRNA and 25 nM M. tuberculosis LeuRS. The reaction was
incubated at 37 °C and aliquots were quenched by 10% trichlo-
racetic acid, and the level of aminoacylation of tRNA was deter-
mined by scintillation counting.
Acknowledgements
For the inhibitory studies in aminoacylation reaction 20
containing 25 nM M. tuberculosis LeuRS, 100 mM Tris–HCl (pH 8.0),
10 mM MgCl2, 90 -leucine (238 mCi/mmol), 2 mM DTT,
M [14C]-
4 mg/ml E. coli tRNA and appropriate concentrations of inhibitor (dis-
solved in DMSO) were incubated for 5 min at 37 °C. Reactions were ini-
tiated by addition of ATP to final concentration of 2 mM. At least
triplicates were averaged to generate an IC50 value using Origin 7.0.
ll solution
We thank Dr. Stephen Cusack and Dr. Andres Palencia (EMBL
Grenoble Outstation, France) for the gift of clone for expression
and purification of M. tuberculosis LeuRS.
We thank Dr. Michael Alley (Anacor Pharmaceuticals, USA) for
the gift of plasmid for expression and purification of human cyto-
plasmic LeuRS.
This work was supported by the Science and Technology Center
in Ukraine (contract No. 5218), the National Agency of Science,
Innovation and Informatization of Ministry of Education and
Science of Ukraine (contract No. DP/337-2013) and by National
Institutes of Health and the National Institute of Allergy and Infec-
tious Diseases (contract No. HHSN272201100009I).
l
L
4.8. Minimal inhibitory concentration (MIC)
The MIC of compound was determined by measuring bacterial
growth after 5 d in the presence of test compounds. Compounds
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Please cite this article in press as: Gudzera, O. I.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.028