Novel trisubstituted benzimidazoles, targeting Mtb FtsZ, as a new class of antitubercular agents

Article abstract of DOI:10.1021/jm1012006

Libraries of novel trisubstituted benzimidazoles were created through rational drug design. A good number of these benzimidazoles exhibited promising MIC values in the range of 0.5-6 μg/mL (2-15 μM) for their antibacterial activity against Mtb H37Rv strai

Full text of DOI:10.1021/jm1012006

374 J. Med. Chem. 2011, 54, 374–381
DOI: 10.1021/jm1012006
Novel Trisubstituted Benzimidazoles, Targeting Mtb FtsZ, as a New Class of
Antitubercular Agents
Kunal Kumar,† Divya Awasthi,† Seung-Yub Lee,‡ Ilaria Zanardi,‡ Bela Ruzsicska,‡ Susan Knudson,§ Peter J. Tonge,†,‡
Richard A. Slayden,§ and Iwao Ojima*,†,‡
†Department of Chemistry, and ‡Institute of Chemical Biology and Drug Discovery, State University of New York at Stony Brook, Stony Brook,
New York 11794-3400, United States, and §Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins,
Colorado 80523-1682, United States
Received September 14, 2010
Libraries of novel trisubstituted benzimidazoles were created through rational drug design. A good
number of these benzimidazoles exhibited promising MIC values in the range of 0.5-6 μg/mL (2-15 μM)
for their antibacterial activity against Mtb H37Rv strain. Moreover, five of the lead compounds also
exhibited excellent activity against clinical Mtb strains with different drug-resistance profiles. All lead
compounds did not show appreciable cytotoxicity (IC50>200 μM) against Vero cells, which inhibited Mtb
FtsZ assembly in a dose dependent manner. The two lead compounds unexpectedly showed enhance-
ment of the GTPase activity of Mtb FtsZ. The result strongly suggests that the increased GTPase activity
destabilizes FtsZ assembly, leading to efficient inhibition of FtsZ polymerization and filament forma-
tion. The TEM and SEM analyses of Mtb FtsZ and Mtb cells, respectively, treated with a lead compound
strongly suggest that lead benzimidazoles have a novel mechanism of action on the inhibition of Mtb
FtsZ assembly and Z-ring formation.
Introduction
pressing need for the development of novel TB drugs that are
effective against both drug sensitive and resistant Mtb strains.
FtsZ, a tubulin homologue, is a highly conserved and ubiq-
uitous bacterial cell division protein. Similar to the process of
microtubule formation by tubulin, FtsZ polymerizes in a GTP-
dependent manner, forming a highly dynamic cytokinetic struc-
ture, designated as the Z-ring, at the midpoint of the cell.4,5
The recruitment of the other cell division proteins leads to
Z-ring contraction, resulting in septum formation. Because of
the requirement of FtsZ in mycobacterial cytokinesis, inhibi-
tion of FtsZ is a promising target for antituberculosis drug
discovery.6-9 While tubulin and FtsZ share structural and
functional homology and tubulin inhibitors are known to
affect FtsZ assembly,6-8 the limited sequence homology at the
protein level affords an opportunity to discover FtsZ-specific
compounds with limited cytotoxicity to eukaryotic cells.10 Since
FtsZ is a novel drug target, compounds targeting FtsZ are
expected to be active against drug resistant Mtb strains.11
Furthermore, the validation of FtsZ as a novel antibacterial
drug target has been confirmed by the work of various groups
including ours.12-24
Tuberculosis (TBa) is one of the leading infectious diseases
and remains a major global health problem. According to
WHO, 9.2 million new cases and 1.7 million deaths from TB
have been reported.1 With the emergence of HIV, TB has
become the most common opportunistic infection afflicting
patients living with AIDS. There are 0.7 million HIV-positive
people infected with TB, contributing to 0.2 million deaths
worldwide.1 The lethal combination of TB and HIV is fueling
the TB epidemic in many parts of the world, especially Africa.1
Poor chemotherapeutics and the inadequate administration of
drugs have led to the development of multidrug resistant TB
(MDR-TB),2 the treatment of which requires administration
of more expensive, second line antibiotics for up to 2 years. In
addition, even more alarming cases of extensively drug resis-
tant strains of TB (XDR-TB) that are resistant to both first and
second line drugs have been reported.3 Recent findings
by WHO from 2000 to 2004 suggested that 4% of MDR-TB
cases meet the criteria for XDR-TB. Consequently, there is a
Researchers at the Southern Research Institute screened
known tubulin inhibitors against Mtb and identified several
pyridopyrazine and pteridine based FtsZ inhibitors with anti-
TB activity.12-14 Later, Slayden et al. found that thiabenda-
zole and albendazole, known tubulin inhibitors, interfered
and delayed the Mtb cell division processes.8 Taking into
account the structuralsimilarityofthe pyridopyrazine moiety,
pteridine moiety, albendazole, and thiabendazole,12-14 we
envisioned that the benzimidazole scaffold would be a good
starting point for the development of novel FtsZ inhibitors,
which will have activity against both drug sensitive and drug
resistant Mtb. Therefore, trisubstituted benzimidazoles were
investigated for antibacterial activity. Specifically we report
*To whom correspondences should be addressed. Phone: 631-632-
1339. Fax: 631-632-7942. E-mail: iojima@notes.cc.sunysb.edu.
a Abbreviations: ACN, acetonitrile; AIDS, acquired immuno deficiency
syndrome; BCA, bicinchoninic acid; DIPEA, diisopropyl ethylamine;
DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; E. coli,
Escherichia coli; FtsZ, filamenting temperature sensitive mutant Z; GTP,
guanosine triphosphate; GTPase, guanosine triphosphatase; HIV, human
immunodeficiency virus; MABA, microplate Alamar Blue assay; MDR-
TB, multidrug resistant tuberculosis; MIC, minimum inhibitory concen-
tration; Mtb, Mycobacterium tuberculosis; OADC, oleic acid/albumin/
catalase ; /catalase ; PBS, phosphate buffered saline; Pi, inorganic phos-
phate; RT, room temperature, SAR, structure-activity relationship;
SEM, scanning electron microscopy; TB, tuberculosis; TEM, transmission
electron microscopy; THF, tetrahydrofuran; TLC, thin layer chromatog-
raphy; WHO, World Health Organization; XDR-TB, extremely drug
resistant tuberculosis.
r
pubs.acs.org/jmc
Published on Web 12/02/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 375
Scheme 2. Synthesis of 2,5,7-Trisubstituted Benzimidazolesa
Figure 1. Proposed 2,5,6- and 2,5,7-trisubstituted benzimidazoles.
Scheme 1. Synthesis of 2,5,6-Trisubstituted Benzimidazolesa
a Reagents and conditions: (i) 6 N HCl,reflux, 1.5 days; (ii) SOCl2,
reflux, overnight; (iii) NaN3, acetone, 0 °C, 30 min; (iv) toluene, reflux, 4 h;
1
(v) R0 OH, RT, 1 day; (vi) 10% Pd/C-HCO2NH4, EtOH, 1 h, RT;
2
3
(vii) R0 CH(OH)SO3Na, EtOH/H2O (1:1); (viii) (a) R0 COCl or
3
R0 OCOCl, DMF, overnight, RT; (b) aminomethylated polystyrene
resin, DMF, overnight, RT.
1
1
corresponding carbamates 9a (R0 =Et) or 9b (R0 =Me) in
78-85% yield (for five steps) as a bright red solid. The
reduction of 9a,b followed by cyclocondensation with the
bisulfite salts of different aldehydes afforded 7-aminobenz-
imidazoles 10a-A-F and 10b. The final acylation of 10a,b in
the same manner as that for the library of 1a gave the
77-compound library of 2,5,7-trisubstituted benzimidazoles 2
with good to high purity (see the Supporting Information for
the detailed experimental procedures, structures of intermedi-
ates, and library).
a Reagents and conditions: (i) Et2NH, DIPEA, THF, 2 h, room tem-
perature (RT); (ii) R3COCl, pyridine, reflux, overnight; (iii) 10%
Pd/C-HCO2NH4, dioxane, EtOH, 1 h, RT; (iv) 4 N HCl, reflux,4 h;
(v) (a) R4COCl or R4COOCl, CH2Cl2, overnight, RT; (b) aminomethy-
lated polystyrene resin, CH2Cl2, overnight, RT.
the activity of 2,5,6- and 2,5,7-trisubstituted benzimidazoles
against Mtb (Figure 1). Importantly, this work substantiates
trisubstituted benzimidazoles for the development of next gener-
ation antimycobacterial inhibitors with activity against diffi-
cult to treat clinical strains.
Results and Discussion
The libraries of 2,5,6- and 2,5,7-trisubstituted benzimida-
zoles 1a and 2 (349 compounds) were screened for their activity
against Mtb H37Rv using the “microplate Alamar Blue assay
(MABA)”25 in a 96-well format (single point assay in trip-
licates). Of these, 26 compounds were identified to inhibit the
growth of Mtb H37Rv with MIC values of e5 μg/mL.
Furthermore, among the 26 hits, the resynthesized pure 9 com-
pounds showed MIC values of 0.50-6.1 μg/mL (1.5-15 μM)
(Table 1).
Preliminary structure-activity relationship studies of hits
from the library of 1a indicated that cyclohexyl and diethyl-
amino groups at the 2 and 6 positions, respectively, were criti-
cally important for antibacterial activity (Figure 2). Accord-
ingly, another library of 1 (1-G: 238 compounds) was synthe-
sized with different dialkylamino substituents at the 6-position
but retaining the cyclohexyl group at the 2-position using the
procedures similar to those for the synthesis of the libraries of
1a (Scheme 1). (See Supporting Information for the detailed
experimental procedures, structures of the intermediates, and
library.)
Chemistry
The 2,5,6-trisubstituted benzimidazoles were synthesized
as outlined in Scheme 1. The aromatic nucleophilic substitu-
tion of commercially available 2,4-dinitro-5-fluoroaniline (3)
with diethylamine afforded 5-diethylaminodinitroaniline 4a
(R1=R2=Et) in quantitative yield. The acylation of 4a with
different acyl chlorides afforded the corresponding N-acyl-
anilines 5a-1-8 in 75-90% yields. The reduction and subse-
quent acid-catalyzed cyclization gave 5-aminobenzimidazoles
6a-1-8 in 40-60% yield. The advanced intermediates 6a-1-8
were dissolved in dichloromethane and transferred to a
96-well plate. Subsequently, 33 different acyl chlorides and/or
alkyl chloroformates were added to 6a-1-8 and reacted at
room temperature overnight using a shaker. To the reaction
mixture was added an aminomethylated polystyrene resin to
scavenge excess or unreacted acyl chlorides and/or alkyl chlo-
roformates. After the mixture was reacted overnight with gentle
shaking, the resin was filtered to afford a library of 272 2,5,6-
trisubstituted benzimidazoles 1a (R1 = R2 = Et) with good to
high purity based on LC-MS/UV analysis (see the Support-
ing Information for the detailed experimental procedures,
structures of intermediates, and library).
On the basis of the MABA screening (single point assay in
triplicates), 54 compounds were identified as hits, which
inhibited the cell growth with MIC values of 5 μg/mL or less.
Among the 54 hits, resynthesized pure 1b-G1 and 1b-G2
showed MIC values of 0.39 μg/mL (1.0 μM) and 1.56 μg/mL
(3.7 μM), respectively(Table1). Itisnoted thatall 11purelead
The 2,5,7-trisubstituted benzimidazoles were synthesized as
outlined in Scheme 2. Commercially available 5-amino-2,4-
dinitrobenzamide (7) was hydrolyzed to give 4-amino-3,5-
dinitrobenzoic acid, which was converted to acyl chloride 8.
The reaction of 8 with sodium azide afforded the correspond-
ing acyl azide, which was subjected to the Curtius rearrange-
ment to give the corresponding isocyanate. The isocyanate
was subsequently reacted with methanol or ethanol to give the
compounds did not show appreciable cytotoxicity (IC50
200 μM) against Vero cells (Table 1).
>
Five lead compounds, 1a-G4, 1a-G7, 1b-G1, 1b-G2, and
2b-1, were also assayed for their activities against clinical
isolates of Mtb strains W210,26 NHN20,26 NHN335-2
(isoniazide-resistant, KasA G269S mutation),27 NHN382

376 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Kumar et al.
Table 1. Antimicrobial Activities of Selected Trisubstituted Benzimidazoles against Drug-Sensitive and Drug-Resistant Strains of Mtba
a Mtb H37Rv is a standard drug sensitive strain. Mtb W210, NHN 20, NHN335, NHN382, and TN587 are clinical strains with differing drug resistant
profiles.
(isoniazide-resistant, Kat G 315T mutation),26 and TN587
amount of inorganic phosphate (Pi) released upon treatment
of Mtb FtsZ with two lead compounds, 1a-G4 and 1a-G7. On
treatment of Mtb FtsZ with these two compounds, an en-
hancement in GTPase activity was observed (Figure 4). This
unique behavior is analogous to the effect that curcumin
exhibited on recombinant E.coli FtsZ.28 Enhancement in the
GTPase activity together with the fact that these compounds
inhibit polymerization of Mtb FtsZ (Figure 3) leads us to con-
clude that the increased GTPase activity causes the instability
of the Mtb FtsZ polymer. As a result of the instability, Mtb
FtsZ is unable to polymerize normally, leading to the efficient
inhibition of the formation of sizable Mtb FtsZ polymers and
filaments.
To further investigate the effect of the hit compounds on
polymerization of the FtsZ protein, transmission electron micros-
copy (TEM) imaging of the treated and untreated Mtb FtsZ
protein was carried out. Mtb FtsZ (5 μM) polymerized in the
presence of GTP (25 μM) was diluted 5 times with polymer-
ization buffer and immediately transferred onto a carbon
coated copper-mesh grid for TEM imaging. Mtb FtsZ protein
treated with GTP and 40 and 80 μM 1a-G7 were visualized
(Figure 5). The protein treated with colchicine, which is
(isoniazide-resistant, KatGS315T mutation),26 which showed
no difference between the drug-sensitive and drug-resistant
strains, as anticipated, exhibiting excellent MIC values
in the range of 0.39-1.56 μg/mL (1-4.6 μM). These com-
pounds have been advanced to the in vivo rapid model assay
in mice.
FtsZ polymerization assay28 was carried out to validate the
hypothesis that these lead compounds exhibit antibacterial
activity by interacting with FtsZ. Three benzimidazoles, selected
from the 11 lead compounds shown in Table 1, have been
evaluated for their ability to inhibit the polymerization of the
wild-type FtsZ. 1a-G1 (MIC=7.9 μM) inhibited FtsZ poly-
merization in a dose-dependent manner, and ∼80% inhibi-
tion was achieved at 10 μM (FtsZ concentration of 15 μM)
(Figure 3b). A similar result was obtained for 1a-G7 (MIC=
2 μM) (Figure 3a). 1a-G1 and 1a-G7 inhibited FtsZ polymeri-
zation with IC50 values of 6.21 and 7.69 μM, respectively. 1a-G4
(MIC 4.2 μM), showed ∼45% inhibition at 10 μM (Figure 3c).
The assembly and disassembly of FtsZ protein have been
shown to be GTP dependent. Since FtsZ has GTPase activity,
the Malachite Green assay29 was performed to monitor the

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 377
Figure 4. Enhancement of GTPase activity of FtsZ by lead benz-
imidazoles 1a-G4 (red) and 1a-G7 (blue).
there was a considerable reduction in the extent of FtsZ proto-
filament formation upon treatment with 1a-G7 compared to
the control experiment. At 40 μM 1a-G7, the density of Mtb
FtsZ protofilaments was substantially reduced (Figure 5B,C).
Furthermore, the protofilaments were short and very thin. At
80 μM, the formation of protofilaments was drastically reduced
and only numerous tiny aggregates were formed (Figure 5D,E).
In sharp contrast, the Mtb FtsZ protein treated with a high
concentration (200 μM) of colchicine showed decreased and
thinner protofilaments (Figure 5G,H) but with more or less
similar morphology compared with that of GTP-initiated poly-
merization and protofilaments (Figure 5A,B). These results
clearly indicate the highly efficient inhibition of Mtb FtsZ
assembly by 1a-G7 in a dose-dependent manner with a novel
mechanism of action.
Figure 2. Preliminary SAR of 2,5,6-trisubstituted benzimidazole
hit compounds 1a.
Visualization of bacteria treated with 2 ꢀ MIC and 4 ꢀ MIC
1a-G7 for 2 days using scanning electron microscopy (SEM)
revealed altered cell division lengths and aberrant division
indicative of the inhibition of FtsZ assembly and cell division
(Figure 6). Specifically, cell division and elongation were inhib-
ited in a dose dependent manner resulting in shorter bacterial
cells withincreasedcircumferences andalteredpolarcaps. The
observed altered bacterial morphology upon exposure to
1a-G7 is thought to result from inhibition of FtsZ leading to
disruption in septum formation and recruitment of septum
associated proteins involved in later steps of division and
septum resolution.
Conclusions
We have created a library of novel trisubstituted benzimid-
azoles through rational drug design. As Table 1 shows, a
good number of these compounds exhibited promising MIC
values in the range of 0.5-6.1 μg/mL (1.0-15 μM) for their
antibacterial activity against Mtb H37Rv strain. Moreover,
1b-G4, 1b-G7, 1b-G1, 1b-G2, and 2a-1 were also found to
exhibit excellent activity against clinical Mtb strains W210,
NHN20, NHN335, NHN382, and TN587, with different drug-
resistance profiles. All lead compounds do not show appre-
ciable cytotoxicity (IC50 >200μM) against Vero cells. The stan-
dard light scattering assay to assess the effect of these compounds
on the polymerization of FtsZ clearly showed the inhibition of
FtsZassembly ina dose dependent manner. Asthe duration of
steady state of FtsZ polymer is dependent on the rate of GTP
hydrolysis, the effect of lead compounds 1a-G4 and 1a-G7
on GTPase activity was examined. Unexpectedly, instead of
Figure 3. Inhibition of FtsZ polymerization by (a) 1a-G7, (b) 1a-G1,
and (c) 1a-G4.
known to inhibit FtsZ polymerization,13 was used as a refer-
ence in this set of experiments (Figure 5F,G). As anticipated,

378 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Kumar et al.
Figure 5. Transmission electron microscope (TEM) images of FtsZ. FtsZ (5 μM) was polymerized by GTP in the absence (A, B) and presence
of 1a-G7 at 40 μM (C, D) and 80 μM (E, F). TEM images of the inhibition of FtsZ polymerization in the presence of colchicine at 200 μM (G, H)
are also shown for comparison. Images A, C, E, G are at 30000ꢀ magnification (scale bar 500 nm), and images B, D, F, H are at 68000ꢀ
magnification (scale bar 200 nm).
evaluation of these highly promising lead compounds are
actively underway in these laboratories.
Experimental Section
Methods. 1H and 13C NMR spectra were measured on a
Varian 300, 400, or 500 MHz NMR spectrometer. Melting
points were measured on a Thomas-Hoover capillary melting
point apparatus and are uncorrected. TLC was performed on
Merck DC Alufolien with Kieselgel 60F-254, and column
chromatography was carried out on silica gel 60 (Merck, 230-
400 mesh ASTM). High-resolution mass spectra were obtained
from Mass Spectrometry Laboratory, University of Illinois at
Urbana Champaign, Urbana, IL. Purity was determined by
;
an Agilent 1100 series HPLC assembly. Two analytical condi-
tions were used and noted as a part of the characterization data
˚
for resynthesized compounds. HPLC1: Luna PFP, 3 μm, 100 A
(pore size), 3.0 mm ꢀ 150 mm column, solvent A of H2O/ACN,
95:5 (20 mM ammonium acetate, pH 6.5), solvent B of H2O/
ACN, 5:95 (20 mM ammonium acetate, pH 6.5), temperature of
30 °C, flow rate of 0.6 mL/min, t = 0-13.5 min, gradient of
˚
15-90% solvent B. HPLC2: Kinetex PFP, 2.6 μm, 100 A (pore
size), 4.6 mm ꢀ 100 mm column, solvent A of H2O/ACN, 95:5
(25 mM ammonium acetate, pH 6.5), solvent B of H2O/ACN,
5:95 (25 mM ammonium acetate, pH 6.5), temperature of 25 °C,
flow rate of 1.0 mL/min, t = 0-15 min, gradient of 20-95%
solvent B. SensoLyte MG phosphate assay kit from Anaspec
was used for Malachite Green assay. Light scattering assays
were performed using a Fluorolog fluorimeter from ISA. Scan-
ning electron micrographs were obtained with a JEOL JSM-
6500F scanning electron microscope. FEI Tecnai12 BioTwinG
transmission electron microscope with an AMT XR-60 CCD
digital camera system was used to acquire transmission electron
microscopy images.
Materials. BL21(DE3)pLysS cells, His-Bind protein purifica-
tion resin, and the buffers were purchased from Novagen. BCA
kit for protein concentration determination was purchased from
Sigma. Buffer salts (reagent grade or better), solvents (HPLC
grade or better), and all the other chemicals were purchased
from Fisher Scientific Co. (Pittsburgh, PA). The chemicals were
purchased from Aldrich Co., Synquest Inc., and Sigma and
purified before use by standard methods. Tetrahydrofuran was
freshly distilled from sodium metal and benzophenone. Dichloro-
methane was also distilled immediately prior to use under nitro-
gen from calcium hydride. Aminomethylated polystyrene resin
EHL (200-400 mesh) 2% DVB was purchased from Novagen-
biochem.
Figure 6. SEM of Mtb cells treated with 1a-7 (20000ꢀ): (A) con-
trol; (B) 2 ꢀ MIC; (C) 4 ꢀ MIC; (D) distribution of bacterial cell
length, showing control (black), 2 ꢀ MIC (gray), 4 ꢀ MIC (light
gray).
inhibiting, these compounds enhanced the GTPase activity of
Mtb FtsZ by 3- to 4-fold. The result strongly suggests that the
increased GTPase activity destabilizes FtsZ polymer, leading
to efficient inhibition of FtsZ polymerization and filament
formation. The effect of 1a-G7 on FtsZ polymerization was
examined by TEM, which revealed an impressive dose-depen-
dent inhibition of Mtb FtsZ assembly and a dramatic suppres-
sion of FtsZ protofilament formation at higher concentration
of 1a-G7. The SEM images of Mtb cells treated with 1a-G7
showed the absence of septum formation and shorter bacterial
cellswithincreased circumferencesandaltered polarcaps. The
unique bacterial morphology indicates that the disruption of
septum formation by 1a-G7 may recruit other septum asso-
ciated proteins involved in later steps of division and septum
resolution. The TEM and SEM analyses strongly suggest that
1a-G7 and very likely other lead benzimidazoles have a novel
mechanism of action on the inhibition of Mtb FtsZ assembly
and Z-ring formation. Further optimization and in vivo
Chemical Synthesis. Novel 2,5,6-trisubstituted benzimida-
zoles 1 and 2,5,7-trisubstituted benzimidazoles were synthe-
sized in accordance with the general procedures illustrated in

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 379
Schemes 1 and 2. The detailed procedures for the syntheses of
intermediates 4, 5, 6, 8, 9, and 10 as well as their characterization
data are described in the Supporting Information.
162.6, 164.9; HRMS (FAB) m/z calcd for C25H32N4O2Hþ:
421.2601. Found: 421.2604 (Δ = -0.7 ppm). HPLC(2): 9.5 min,
purity >99%.
General Procedure for the Synthesis of the Libraries of Trisub-
stituted Benzimidazoles 1 and 2. 5-Aminobenzimidazole 6 or
7-aminobenzimidazole 10 (0.005 mmol) was dissolved in di-
chloromethane and transferred into a 96-well plate. To these wells
were added 33 different acyl chlorides (1.1 equiv) or alkyl chloro-
formates (1 equiv), and they were reacted overnight on a shaker.
Aminomethylated polystyrene resin EHL (200-400 mesh) 2%
DVB (10 equiv) was added to scavenge excess or unreacted acyl
chlorides or alkyl chloroformates. After reacting overnight
using the shaker, the resin was filtered to provide a library of
trisubstituted benzimidazoles 1 or 2. The product in each well
was analyzed by LC-MS/UV for purity and confirmation of the
structure. The purity was in the range of 80-90%.
General Procedure for the Synthesis of Analytically Pure
Trisubstituted Benzimidazoles 1 and 2. A typical procedure is
described for the synthesis of 5-butoxycarbonylamino-2-cyclo-
hexyl-6-N,N-diethylamino-1H-benzo[d ]imidazole (1a-G7).
To a solution of 2-cyclohexyl-5-N,N-diethylamino-6-amino-
benzimidazole (6a-G) (486 mg, 1.71 mmol) in dichloromethane
(20 mL) was added a solution of N-butoxycarbonyloxysuccini-
mide (340 mg, 1.71 mmol) in dichloromethane (20 mL) dropwise
at room temperature. After the addition, the reaction mixture
was stirred at room temperature overnight. After the comple-
tion of the reaction, the reaction mixture was concentrated. The
crude was purified via flash chromatography on silica gel
(gradient 20-40% EtOAc/hexanes) to give 1a-G7 as a white
solid (336 mg, 51% yield): mp 171-172 °C; 1H NMR (500 MHz,
CDCl3) δ 0.91 (t, 6 H, J=7.2 Hz), 0.96 (t, 3 H, J = 7.5 Hz),
1.23-1.46 (m, 5 H), 1.57-1.72 (m, 5 H), 1.81 (m, 2 H), 2.07
(m, 2 H), 2.85 (m, 1 H), 2.92 (q, 4 H, J = 7.2 Hz), 4.19 (t, 2 H,
J = 6.5 Hz), 7.46 (s, 1 H), 8.24 (s, 1 H), 8.51 (s, 1 H); 13C NMR
(125 MHz, CDCl3) δ 12.72, 13.75, 19.11, 25.81, 26.02, 31.07,
31.78, 38.46, 50.43, 64.87, 98.88, 107.3, 113.3, 131.2, 132.2,
134.3, 138.5, 154.1, 158.8; HRMS (FAB) m/z calcd for
C22H34N4O2Hþ: 387.2763. Found: 387.2760 (Δ = 0.8 ppm).
HPLC(2): 10.6 min, purity >99%.
5-But-3-enoxycarbonylamino-2-cyclohexyl-6-N,N-diethylamino-
1H-benzo[d]imidazole (1a-G4). White solid; 51% yield; mp
143-145 °C; 1H NMR (500 MHz, CDCl3) δ 0.91 (t, J =
7 Hz, 6 H), 1.16-1.31 (m, 3 H), 1.56-1.66 (m, 3 H), 1.77 (d, 2 H,
J = 13.5 Hz), 2.04 (d, 2 H, J = 12.5 Hz), 2.47 (t, 2 H, J =
6.5 Hz), 2.83 (m, 1 H), 2.88 (q, 4 H, J = 7.5 Hz), 4.23 (t, 2 H, J =
7 Hz), 5.11 (m, 2 H), 5.84 (m, 1 H), 7.41 (s, 1 H), 8.24 (s, 1 H),
8.52 (s, 1 H); 13C NMR (125 MHz, CDCl3) δ 12.66, 25.72, 25.96,
31.76, 33.47, 50.39, 63.98, 99.97, 112.1, 117.1, 131.9, 132.0,
131.9, 133.9, 134.3, 137.3, 153.9, 159.2; HRMS (FAB) m/z calcd
for C22H32N4O2Hþ: 385.2604. Found: 385.2604 (Δ = 0.0 ppm).
HPLC(2): 10.1 min, purity >99%.
5-(4-Chlorobenzamido)-2-cyclohexyl-6-N,N-diethylamino-1H-
benzo[d]imidazole (1a-G5). White solid; 64% yield; mp 216-
1
217 °C (turned brown); H NMR (400 MHz, CDCl3) δ 0.96
(t, 6 H, J = 7.2 Hz), 1.21-1.32 (m, 2 H), 1.59-1.81 (m, 5 H), 2.08
(m, 2 H), 2.79 (m, 1 H), 3.02 (q, 4 H, J = 7.2 Hz), 7.50 (d, 2 H,
J = 6.8 Hz), 7.58 (s, 1 H), 7.88 (d, 2 H, J = 8.8 Hz), 8.82
(s, 1 H),10.31 (s, 1 H); 13C NMR (125 MHz, CDCl3) δ 13.12,
25.75, 25.97, 29.69, 31.76, 38.47, 50.78, 100.5, 113.6, 128.3,
129.1, 131.3, 131.8, 133.9, 135.2, 137.9, 139.6, 159.5, 163.7;
HRMS (FAB) m/z calcd for C24H29N4OClHþ: 425.2108.
Found: 425.2108 (Δ = -0.0 ppm). HPLC(2): 10.5 min, purity
>99%.
2-Cyclohexyl-6-N,N-diethylamino-5-(4-methylbenzamido)-
1H-benzo[d]imidazole (1a-G6). White solid; 65% yield; mp
183-184 °C; 1H NMR (300 MHz, CDCl3) δ 0.97 (t, 6 H, J =
6 Hz), 1.2 (m, 3 H), 1.58-1.78 (m, 5 H), 2.06 (m, 2 H), 2.45
(s, 3 H), 2.76 (m, 1 H), 3.03 (q, 4 H, J = 7.2 Hz), 7.34 (d, 2 H, J =
8.4 Hz), 7.59 (s, 1 H), 7.87 (d, 2 H, J=8.1 Hz), 8.91 (s, 1 H), 10.31
(s, 1 H); 13C NMR (100 MHz, CDCl3) δ 13.09, 21.48, 25.63,
25.92, 31.72, 38.40, 50.75, 100.9, 113.1, 126.8, 129.6, 131.8,
132.7, 135.0, 139.4, 142.1, 159.8, 165.1; HRMS (FAB) m/z calcd
for C25H32N4OHþ: 405.2654. Found: 405.2654 (Δ = -0.0 ppm).
HPLC(2): 10.1 min, purity >99%.
Benzyloxycarbonylamino-2-cyclohexyl-6-N,N-diethylamino-
1H-benzo[d]imidazole (1a-G8). White solid; 61% yield; mp
208-209 °C; 1H NMR (300 MHz, CDCl3) δ 0.90 (t, 6 H, J =
7.2 Hz), 1.38 (m, 4 H), 1.84-1.62 (m, 5 H), 2.11 (m, 2 H), 2.84 (m,
1 H), 2.91 (q, 4 H, J = 7.2 Hz), 5.22 (s, 2 H), 7.45-7.35 (m, 5 H),
8.25 (s, 1 H), 8.61 (s, 1 H); 13C NMR (125 MHz, CDCl3) δ 12.62,
25.77, 25.97, 31.77, 38.46, 50.41, 66.68, 99.02, 113.2, 128.1,
128.5, 131.3, 132.0, 134.2, 136.3, 138.2, 153.7, 159.0; HRMS
(FAB) m/z calcd for C25H32N4O2Hþ: 421.2601. Found:
421.2604 (Δ = -0.5 ppm). HPLC(2): 10.7 min, purity >99%.
5-Butoxycarbonylamino-2-cyclohexyl-6-(pyrrolidin-1-yl)-1H-
benzo[d]imidazole (1b-G1). White solid; 80% yield; mp 128-129 °C;
1H NMR (500 MHz, CDCl3) δ 0.94 (t, 3 H, J = 6.5 Hz), 1.26-1.19
(m, 5 H), 1.73-1.55 (m, 7 H), 1.92 (bs, 4 H), 2.00 (m, 2 H), 2.76 (m,
1 H), 2.93 (bs, 4 H), 4.18 (t, J = 6.5 Hz, 2 H), 7.40 (s, 1 H), 7.95 (s,
1 H), 8.17 (s,1 H); 13C NMR (125 MHz, CDCl3) δ 13.68, 19.03,
24.24, 25.71, 25.94,30.99, 31.75, 38.45, 53.41, 64.90, 101.3, 109.80,
129.3, 131.8, 135.6, 137.5, 154.3, 159.1. HRMS (FAB) m/z calcd for
C22H32N4O2Hþ: 385.2594. Found: 385.2604 (Δ = -1.1 ppm).
HPLC(2): 10 min, purity >99%.
5-Benzyloxycarbonylamino-2-cyclohexyl-6-(pyrrolidin-1-yl)-
1H-benzo[d]imidazole (1b-G2). White solid; 68% yield; mp
86-87 °C; 1H NMR (500 MHz, CDCl3) δ 1.21-1.28 (m, 4 H),
1.59-1.65 (m, 4 H), 1.76 (m, 2 H), 1.91 (s, 4 H), 2.04 (d, 2H, J =
12.5 Hz), 2.82 (m, 1 H), 2.93 (s, 4 H), 5.22 (s, 2 H), 7.34-7.48 (m,
5 H aromatic), 8.08 (s, 1 H), 8.22 (s, 1 H); 13C NMR (125 MHz,
CDCl3) δ 24.29, 25.69, 25.91, 31.72, 38.31, 53.54, 66.75, 101.5,
108.7, 128.1, 128.5, 129.6, 131.9, 136.0, 136.2, 153.8, 158.9.
HRMS (FAB) m/z calcd for C25H30N4O2Hþ: 419.2448. Found:
419.2447 (Δ = 0.2 ppm). HPLC(2): 10.1 min, purity >99%.
7-Acetylamino-5-ethoxycarbonylamino-2-phenyl-1H-benzo-
[d]imidazole (2a-1). White solid; 84% yield: mp 158-160 °C;
In a similar manner, other lead benzimidazoles were synthe-
sized and characterized.
5-Benzamido-2-cyclohexyl-6-N,N-diethylamino-1H-benzo-
[d]imidazole (1a-G1). White solid; 74% yield; mp 180-181 °C;
1H NMR (400 MHz, CDCl3) δ 0.97 (t, 6 H, J = 7.2 Hz), 1.16
(m, 3 H), 1.65 (m, 5 H), 1.98 (m, 2 H), 2.71 (m, 1 H), 3.03 (m, 4 H,
J = 6.8 Hz), 7.57 (m, 4 H), 7.98 (d, 2 H, J = 6.2 Hz), 8.97
(s, 1 H),10.4 (s, 1 H); 13C NMR (100 MHz, CDCl3) δ 13.09,
21.48, 25.64, 25.91, 31.73, 38.40, 50.77, 100.9, 113.1, 126.9,
129.5, 131.6, 132.7, 135.15, 139.5, 159.8, 165.1; HRMS (FAB)
m/z calcd for C24H30N4OHþ: 391.2486. Found: 391.2498
(Δ = -3.1 ppm). HPLC(2): 9.50 min, purity >99%.
2-Cyclohexyl-6-N,N-diethylamino-5-propoxycarbonylamino-
1H-benzo[d]imidazole (1a-G2). White solid; 63% yield; mp 80-
181 °C; 1H NMR (300 MHz, CDCl3) δ 0.92 (t, 6 H, J = 7.2 Hz),
0.99 (t, 3 H, J=7.5 Hz), 1.25-1.41 (m, 4 H), 1.60-1.76 (m,
5 H), 1.86 (m, 2 H), 2.10 (m, 2 H), 2.87 (m, 1 H), 2.92 (q, 4 H, J =
7.2 Hz), 4.14 (t, 2 H, J = 6.6 Hz), 7.47 (s, 1 H), 8.23 (s, 1 H), 8.51
(s, 1 H); 13C NMR (125 MHz, CDCl3) δ 10.28, 12.67, 22.28,
25.71, 25.96, 31.75, 38.44, 50.38, 66.56, 100.0, 112.0, 132.1,
132.4, 134.3, 136.92, 154.1, 159.0; HRMS (FAB) m/z calcd for
C21H32N4O2Hþ: 373.2601. Found: 373.2604 (Δ=-0.8 ppm).
HPLC(2): 9.98 min, purity >99%.
2-Cyclohexyl-6-N,N-diethylamino-5-(4-methoxybenzamido)-
1H-benzo[d]imidazole (1a-G3). White solid; 79%; mp 198-199 °C;
1H NMR (400 MHz, CDCl3) δ 0.97 (t, 6 H, J = 7.2 Hz), 1.16 (m, 3
H), 1.53-1.68 (m, 5 H), 1.98 (m, 2 H), 2.71 (m, 1 H), 3.02 (q, 4 H,
J = 7.2 Hz), 3.9 (s, 3 H), 7.05 (d, 2 H, J = 8.8 Hz), 7.55 (s, 1 H),
7.95 (d, 2 H, J = 8.8 Hz), 8.95 (s, 1 H), 10.32 (s, 1 H); 13C NMR
(100 MHz, CDCl3) δ 13.36, 25.85, 26.18, 31.96, 38.67, 51.02, 55.75,
101.2, 113.2, 114.2, 127.9, 128.8, 129.0, 132.1, 139.3, 135.2, 160.0,

380 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Kumar et al.
1H NMR (400 MHz, CD3OD) δ 1.32 (t, J = 7.2 Hz, 3H), 2.26
(s, 3H), 4.20 (q, J=7.2 Hz, 2H), 7.54 (m, 3H), 7.69 (bs, 1H), 7.79
(bs, 1H), 8.05 (m, 2H); 13C NMR (100 MHz, DMSO) δ 14.59,
23.99, 59.92, 96.14, 105.46, 126.18, 128.87, 129.16, 129.48,
130.08, 131.28, 131.82, 135.21, 149.61, 153.74, 168.61. HRMS
(FAB) m/z calcd for C18H18N4O3Hþ: 339.1450. Found: 339.1457
(Δ=-2.1 ppm). HPLC(2): 4.95 min, purity >99%.
Bacterial Strains and Growth. H37Rv, the drug sensitive labo-
ratory strain of Mtb, and clinical Mtb strains W210, NHN20,
HN355, NHN382, and TN587 exhibit different resistant profiles
to isoniazid and rifampicin.8,27 For evaluation of drug sensi-
tivity all strains were grown in 7H9 media containing 10% oleic
acid/albumin/catalase ; /catalase (OADC) enrichment and 0.05%
Tween-80 and assessed at mid log phase growth.
Antibacterial Activity. The minimum inhibitory concentra-
tion (MIC) was determined by the microplate Alamar Blue assay
(MABA) as described previously.25 Briefly, stock solutions of
the compounds were prepared in DMSO and were serially diluted
2-fold in 96-well microtiter plates, and each Mtb strain was
added to each well to an OD600 of 0.005. Plates were incubated
for 6 days at 37 °C. Alamar Blue (Invitrogen) was added to the
plates, and the plates were incubated for an additional 24 h at
37 °C. Plates were monitored for color change, and MIC99 was
determined in triplicate.
Cytotoxicity Assay. The cytotoxicity of the compounds was
tested against VERO cells. Epithelial cells from the kidneys of
the African Green Monkey were used to start the VERO cell
line. Vero cells were grown in L15 media without CO2. Serial
2-fold dilutions of the drugs were prepared in the 96-well micro-
titer plates. The cells were added to the plates in media contain-
ing the appropriate amount of Alamar Blue. The cells were
added to a final concentration of 2.5 ꢀ 104/well. The plates were
incubated for 3 days at 37 °C. The IC50 was calculated accord-
ing to manufacturer directions.
Transmission Electron Microscopy (TEM) Analysis. Mtb FtsZ
(5 μM) was incubated with 40 or 80 μM 1a-G7 in the polymer-
ization buffer (50 mM MES, 5 mM MgCl2, 50 mM KCl, pH 6.5)
for 15-20 min. To each solution was added GTP to the final
concentration of 25 μM. The resulting solution was incubated at
37 °C for 30 min. The incubated solution was diluted 5 times
with the polymerization buffer and immediately transferred
to carbon coated 300 mesh formvar copper grid and negatively
stained with 1% uranyl acetate. The samples were viewed with a
FEI Tecnai12 BioTwinG transmission electron microscope at
80 kV.19 Digital images were acquired with an AMT XR-60
CCD digital camera system.
Ultrastructural Analysis via Scanning Electron Micrscopy
(SEM). Mtb H37Rv bacteria were exposed to 2 ꢀ MIC and
4 ꢀ MIC 1a-G7 for 2 days. The bacteria were prepared for
scanning electron microscopy as described by Slayden et al.8
Briefly, bacteria were prepared for SEM treatment with 2.5%
glutaraldehyde in 0.1 M sodium cacodyte (pH 7.2), 5 mM CaCl2,
and 5 mM MgCl2 and incubated at room temperature for 1-2 h.
The fixed bacteria were harvested by centrifugation and washed
in PBS and subjected to 2.5% glutaraldehyde overnight at 4 °C.
Final preparation for SEM was achieved by treatment with 1%
OsO4 in sodium cacodylate buffer and dehydration in a graded
alcohol series. The bacteria were then examined using a JEOL
JSM-6500F scanning electron microscope. Bacteria from all
treatment groups were measured for their lengths. Size fre-
quency graphs were prepared for each treatment group.
Acknowledgment. This research is supported by grants
from NYSTAR (to I.O.) and the National Institutes of Health
(Grant AI078251 to I.O.). The authors acknowledge
Dr. Edward Melief for the helpful discussions. The authors
gratefully acknowledge the technical support of Susan Van
Horn for TEM operation at the Microscopy Imaging Center at
Stony Brook University, NY.
FtsZ Polymerization Inhibitory Assay.12 The inhibitory activ-
ity of lead benzamidazoles for Mtb FtsZ polymerization was
determined by means of light scattering on a PTI Fluorescence
Master system. The 90° light scattering was measured at 30 °C,
using excitation and emission wavelength of 400 nm with slit
width of 2 nm. The gain was set at 950 V. Mtb FtsZ (15 μM) was
incubated in the polymerization buffer (50 mM MES, 5 mM
MgCl2, 50 mM KCl, pH 6.5) to establish a baseline. Then 5 mM
GTP (20 μL) was added to make the final volume 1000 μL in a
1.5 mL cuvette. The light scattering was measured for a period of
30-60 min. When DMSO was used as a solvent for the inhibitor,
the control also contained the same amount of DMSO. For
inhibition studies, a lead benzimidazole at various concentra-
tions was incubated with the polymerization buffer to establish a
baseline. This was followed by addition of FtsZ (15 μM). Once
the baseline was stabilized, 5 mM GTP (20 μL) was added to
make the final volume 1000 μL in a 1.5 mL cuvette and the light
scattering was measured.
Supporting Information Available: Synthetic procedures and
characterization data for new benzimidazole intermediates as
well as Mtb FtsZ protein preparation. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) World Health Organization. Tuberculosis: Data and Country
Profiles. http://www.who.int/tb/country/en/.
(2) Raviglione, M. C. Issues facing TB control (7). Multiple drug-
resistant tuberculosis. Scott. Med. J. 2000, 45 (5, Suppl.), 52-55,
discussion 56.
(3) Gandhi, N. R.; Moll, A.; Sturm, A. W.; Pawinski, R.; Govender,
T.; Lalloo, U.; Zeller, K.; Andrews, J.; Friedland, G. Extensively
drug-resistant tuberculosis as a cause of death in patients co-
infected with tuberculosis and HIV in a rural area of South Africa.
Lancet 2006, 368 (9547), 1575–1580.
(4) Errington, J.; Daniel, R. A.; Scheffers, D.-J. Cytokinesis in bacter-
GTPase Assay. The amount of inorganic phosphate (Pi)
released during the assembly of FtsZ was measured using a stan-
dard Malachite Green/ammonium molybdate assay.29 Briefly,
FtsZ protein (10 μM) was incubated without or with a benz-
imidazole at different concentrations (0, 20, 40, and 80 μM) in
polymerization buffer (50 mM MES, 5 mM MgCl2, 50 mM KCl,
pH 6.5) at room temperature for 15 min. Then 50 μM GTP was
added to the reaction mixture and incubated at 37 °C to start the
hydrolysis reaction. After 30 min of incubation, Malachite Green
reagent (20% v/v) was added to the reaction mixtures to quench
the reaction. The reaction mixtures were centrifuged at 13 000
rpm for 90 s to remove the protein debris. The samples (100 μL)
were transferred to a 96-well plate, and the absorbance of each
well was measured at 620 nm. The background absorbance was
subtracted from all the readings. A phosphate standard curve
was prepared using phosphate standard provided with a Mala-
chite Green assay kit (SensoLyte). All the solutions were pre-
pared in polymerization buffer.
ia. Microbiol. Mol. Biol. Rev. 2003, 67 (1), 52–65.
(5) Bi, E.; Lutkenhaus, J. FtsZ Ring structure associated with division
in Escherichia coli. Nature 1991, 354, 161–164.
(6) Vollmer, W. The prokaryotic cytoskeleton: a putative target for
inhibitors and antibiotics? Appl. Microbiol. Biotechnol. 2006, 73 (1),
37–47.
(7) Huang, Q.; Tonge, P. J.; Slayden, R. A.; Kirikae, T.; Ojima, I. FtsZ:
a novel target for tuberculosis drug discovery. Curr. Top. Med.
Chem. (Sharjah, United Arab Emirates) 2007, 7 (5), 527–543.
(8) Slayden, R. A.; Knudson, D. L.; Belisle, J. T. Identification of cell
cycle regulators in Mycobacterium tuberculosis by inhibition of
septum formation and global transcriptional analysis. Microbiol-
ogy (Reading, U. K.) 2006, 152 (6), 1789–1797.
(9) Respicio, L.; Nair, P. A.; Huang, Q.; Burcu, A. B.; Tracz, S.; Truglio,
J. J.; Kisker, C.; Raleigh, D. P.; Ojima, I.; Knudson, D. L.; Tonge,
P. J.; Slayden, R. A. Characterizing septum inhibition in Mycobac-
terium tuberculosis for novel drug discovery. Tuberculosis 2008, 88,
420–429.
(10) Nogales, E.; Wang, H. W. Structural mechanisms underlying
nucleotide-dependent self-assembly of tubulin and its relatives.
Curr. Opin. Struct. Biol. 2006, 16, 221–229.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 381
(11) Farmer, P.; Bayona, J.; Becerra, M.; Furin, J.; Henry, C.; Hiatt, H.;
Kim, J. Y.; Mitnick, C.; Nardell, E.; Shin, S. The dilemma of
MDR-TB in the global era. Int. J. Tuberc. Lung Dis. 1998, 2 (11),
869–876.
(12) White, E. L.; Ross, L. J.; Reynolds, R. C.; Seitz, L. E.; Moore,
G. D.; Borhani, D. W. Slow polymerization of Mycobacterium
tuberculosis FtsZ. J. Bacteriol. 2000, 182, 4028–4034.
(21) Ito, H.; Ura, A.; Oyamada, Y.; Tanitame, A.; Yoshida, H.;
Yamada, S.; Wachi, M.; Yamagishi, J.-i. A 4-aminofurazan deriv-
ative-A189-inhibits assembly of bacterial cell division protein
FtsZ in vitro and in vivo. Microbiol. Immunol. 2006, 50 (10), 759–
764.
(22) Paradis-Bleau, C.; Beaumont, M.; Sanschagrin, F.; Voyer, N.;
Levesque, R. C. Parallel solid synthesis of inhibitors of the essential
cell division FtsZ enzyme as a new potential class of antibacterials.
Bioorg. Med. Chem. 2007, 15 (3), 1330–1340.
(23) Huang, Q.; Kirikae, F.; Kirikae, T.; Pepe, A.; Amin, A.; Respicio,
L.; Slayden, R. A.; Tonge, P. J.; Ojima, I. Targeting FtsZ for
antituberculosis drug discovery: noncytotoxic taxanes as novel
antituberculosis agents. J. Med. Chem. 2006, 49 (2), 463–466.
(24) Kumar, K.; Lee, S.-Y.; Zanardi, I.; Slayden, R. A.; Ojima, I.,
Synthesis and Optimization of a Library of Novel Benzimidazole
Leads for Antituberculosis Drug Discovery. Presented at the 234th
National Meeting of the American Chemical Society, Boston, MA,
United States, August 19-23, 2007; MEDI-205.
(25) Collins, L.; Franzblau, S. G. Microplate Alamar blue assay versus
BACTEC 460 system for high-throughput screening of compounds
against Mycobacterium tuberculosis and Mycobacterium avium.
Antimicrob. Agents Chemother. 1997, 41 (5), 1004–1009.
(26) Boyne, M. E.; Sullivan, T. J.; amEnde, C. W.; Lu, H.; Gruppo, V.;
Heaslip, D.; Amin, A. G.; Chatterjee, D.; Lenaerts, A.; Tonge, P. J.;
Slayden, R. A. Targeting fatty acid biosynthesis for the develop-
ment of novel chemotherapeutics against Mycobacterium tubercu-
losis: evaluationof A-ring-modified diphenyl ethers as high-affinity
InhA inhibitors. Antimicrob. Agents Chemother. 2007, 51, 3562–
3567.
(13) White, E. L.; Suling, W. J.; Ross, L. J.; Seitz, L. E.; Reynolds, R. C.
2-Alkoxycarbonylaminopyridines: inhibitors of Mycobacterium
tuberculosis FtsZ. J. Antimicrob. Chemother. 2002, 50 (1), 111–114.
(14) Reynolds, R. C.; Srivastava, S.; Ross, L. J.; Suling, W. J.; White,
E. L. A new 2-carbamoyl pteridine that inhibits mycobacterial
FtsZ. Bioorg. Med. Chem. Lett. 2004, 14 (12), 3161–3164.
(15) Margalit, D. N.; Romberg, L.; Mets, R. B.; Hebert, A. M.;
Mitchison, T. J.; Kirschner, M. W.; RayChaudhuri, D. Targeting
cell division: small-molecule inhibitors of FtsZ GTPase perturb
cytokinetic ring assembly and induce bacterial lethality. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101 (38), 13969; erratum to document cited in
CA141:271048 .
(16) Urgaonkar, S.; La Pierre, H. S.; Meir, I.; Lund, H.; Ray Chaud-
huri, D.; Shaw, J. T. Synthesis of antimicrobial natural products
targeting FtsZ: (þ-)-dichamanetin and (þ-)-2000-hydroxy-500-
benzylisouvarinol-B. Org. Lett. 2005, 7 (25), 5609–5612.
(17) Rai, D.; Singh, J. K.; Roy, N.; Panda, D. Curcumin inhibits FtsZ
assembly: an attractive mechanism for its antibacterial activity.
Biochem. J. 2008, 410 (1), 147–155.
(18) Domadia, P.; Swarup, S.; Bhunia, A.; Sivaraman, J.; Dasgupta, D.
Inhibition of bacterial cell division protein FtsZ by cinnamalde-
hyde. Biochem. Pharmacol. 2007, 74 (6), 831–840.
(19) Jaiswal, R.; Beuria, T. K.; Mohan, R.; Mahajan, S. K.; Panda, D.
Totarol inhibits bacterial cytokinesis by perturbing the assembly
dynamics of FtsZ. Biochemistry 2007, 46 (14), 4211–4220.
(20) Stokes, N. R.; Sievers, J.; Barker, S.; Bennett, J. M.; Brown, D. R.;
Collins, I.; Errington, V. M.; Foulger, D.; Hall, M.; Halsey, R.;
Johnson, H.; Rose, V.; Thomaides, H. B.; Haydon, D. J.; Czaplewski,
L. G.; Errington, J. Novel inhibitors of bacterial cytokinesis identified
by a cell-based antibiotic screening assay. J. Biol. Chem. 2005, 280 (48),
39709–39715.
(27) Slayden, R. A.; Lee, R. E.; Barry, C. E. Isoniazid effects multiple
components of the type II fatty acid synthase system of Mycobac-
terium tuberculosis. Mol. Microbiol. 2000, 38 (3), 514–525.
(28) Mukherjee, A.; Lutkenhaus, J. Analysis of FtsZ assembly by light
scattering and determination of the role of divalent metal cations.
J. Bacteriol. 1999, 181 (3), 823–832.
(29) Geladopoulos, T. P.; Sotiroudis, T. G.; Evangelopoulos, A. E. A
Malachite Green colorimetric assay for protein phosphatase activ-
ity. Anal. Biochem. 1991, 192, 112–116.