Design, synthesis and evaluation of novel 2,5,6-trisubstituted benzimidazoles targeting FtsZ as antitubercular agents

Article abstract of DOI:10.1016/j.bmc.2014.03.035

Filamenting temperature-sensitive protein Z (FtsZ), an essential cell division protein, is a promising target for the drug discovery of new-generation antibacterial agents against various bacterial pathogens. As a part of SAR studies on benzimidazoles, we

Full text of DOI:10.1016/j.bmc.2014.03.035

Bioorganic & Medicinal Chemistry 22 (2014) 2602–2612
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of novel 2,5,6-trisubstituted
benzimidazoles targeting FtsZ as antitubercular agents
Bora Park a, Divya Awasthi a, Soumya R. Chowdhury b, Eduard H. Melief b, Kunal Kumar b
Susan E. Knudson c, Richard A. Slayden c, Iwao Ojima b,
⇑
a Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400, United States
b Institute of Chemical Biology and Drug Discovery, Stony Brook University, Stony Brook, NY 11794-3400, United States
c Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-0922, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Filamenting temperature-sensitive protein Z (FtsZ), an essential cell division protein, is a promising target
for the drug discovery of new-generation antibacterial agents against various bacterial pathogens. As a
part of SAR studies on benzimidazoles, we have synthesized a library of 376 novel 2,5,6-trisubstituted
benzimidazoles, bearing ether or thioether linkage at the 6-position. In a preliminary HTP screening
Received 16 January 2014
Revised 12 March 2014
Accepted 21 March 2014
Available online 1 April 2014
against Mtb H37Rv, 108 compounds were identified as hits at a cut off concentration of 5
those hits, 10 compounds exhibited MIC values in the range of 0.63–12.5 g/mL. Light scattering assay
and TEM analysis with the most potent compound 5a clearly indicate that its molecular target is
Mtb-FtsZ. Also, the Kd of 5a with Mtb-FtsZ was determined to be 1.32 M.
lg/mL. Among
l
Keywords:
Tuberculosis
Antibacterial
FtsZ
l
Ó 2014 Elsevier Ltd. All rights reserved.
Benzimidazole
1. Introduction
inactivation of FtsZ is an attractive target for novel drug
discovery.10,11
Tuberculosis (TB), which is caused by Mycobacterium tuberculo-
sis (Mtb), remains a leading single infectious disease killer. In 2012,
the World Health Organization (WHO) estimated that there were
8.6 million new cases of TB globally (13% co-infected with HIV)
resulting in 1.3 million deaths.1 In addition, multidrug-resistant
(MDR-TB) and extensively drug resistant TB (XDR-TB) are a signif-
icant public health threat for TB control efforts.2,3 Emergence of
drug resistant strains of Mtb makes many of the currently available
anti-TB drugs much less effective.3 Despite efforts in last 50 years,
development of new TB treatments have been limited to drug tar-
gets like cell wall biosynthesis, ATP synthesis, RNA synthesis etc.,
leading to resistance in these areas.4,5 Hence, we need to discover
novel drugs that target other bacterial processes in order to coun-
ter the developed bacterial resistance.
Filamenting temperature-sensitive protein Z (FtsZ), a tubulin
homologue,6 is an essential and the most abundant bacterial cell
division protein. FtsZ polymerizes in the presence of GTP to form
a highly dynamic structure, the Z-ring at the division site.7,8 With
the recruitment of several other cell division proteins, Z-ring con-
striction proceeds resulting in septum formation and subsequent
cell division.9 Due to the crucial role of FtsZ in bacterial cytokinesis,
Since FtsZ is a homologue of tubulin with less than 10% se-
quence identity12, known tubulin inhibitors could be a good start-
ing point for developing FtsZ specific inhibitor. Previously, various
groups have explored known tubulin inhibitors based on the
importance of FtsZ assembly in cell division to identify their ability
to inhibit FtsZ polymerization or depolymerization.10,13,14
Following on this principle, albendazole and thiabendazole,
known tubulin inhibitors, were tested for their anti-TB activities.15
Slayden and co-workers found that these two compounds inhibit
FtsZ polymerization that led to the absence of septum formation
based on ultra structural analysis and gene expression profiling.
As both of these compounds share a common benzimidazole moi-
ety, we chose benzimidazole as the scaffold for development of no-
vel anti-TB agents. In our previous work,16,17 based on rational
drug design, libraries of 2,5,6- and 2,5,7-trisubstituted benzimidaz-
oles were synthesized and evaluated for anti-TB activities. A large
number of compounds were identified with MICs in the range of
0.38–6.2 lg/mL against drug sensitive as well as drug resistant
Mtb strains (Fig. 1). In the light scattering experiment, some of
these novel lead compounds exhibited inhibition of FtsZ assembly
in a dose dependent manner while enhancing the GTPase activity18
of Mtb-FtsZ. These results confirmed the hypothesis that the lead
benzimidazoles target FtsZ.
⇑
The preliminary SAR studies of lead compounds indicate that
cyclohexyl group at the 2-position and diethyl amino/dimethyl
Corresponding author. Tel.: +1 631 632 1339; fax: +1 631 632 7942.
E-mail address: iwao.ojima@stonybrook.edu (I. Ojima).
http://dx.doi.org/10.1016/j.bmc.2014.03.035
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

B. Park et al. / Bioorg. Med. Chem. 22 (2014) 2602–2612
2603
O
N
N
N
N
∗
∗
∗
∗
∗
R'
N
N
H
N
O
N
N
O
N
6
∗
∗
∗
R''
R2
*
∗
∗
O
2
S
∗
N
N
N
N
N
HN
5
∗
∗
*
R3
O
∗
N
∗
S
R
O
R
R
∗
H
N
H
H
N
N
N
N
N
N
N
N
HN
HN
HN
O
O
O
O
O
O
SB-P3G2
SB-P1G10
SB-P8B2
MIC = 0.39 μg/mL
μ
MIC = 0.78 g/mL
μ
MIC = 3.1 g/mL
Figure 1. Previously reported anti-TB 2,5,6-trisubstituted benzimidazoles.
amino group at the 6-position play important role for antibacterial
activity.17,18 Building upon three representative compounds bear-
ing alkyl carbamate or benzamide at the 5-position, we planned
to expand our novel trisubstituted benzimidazole libraries with a
substitution pattern different from the previous series for high
throughput (HTP) screening.19 [Note: In the 6-amino series, we
have very recently found that the 6-dimethylamino series exhibit
Compounds 3a–3d were treated with tin(II) chloride dihydrate
while 3e–3h were reacted with tin(II) chloride dihydrate and 4 M
HCl to afford benzimidazoles 4a–h in 56–69% yields. 5-Amino-
benzimidazoles 4a–h (0.01 mM) were dissolved in dichlorometh-
ane and transferred into 96 well plates. Then, 47 different acyl
chlorides, hydroxysuccinimide esters of chloroformates, isocya-
nates, isothiocyanates and sulfonyl chlorides (1.1 equiv) in dichlo-
romethane were added to the individual wells. These 47 different
reagents are shown in the Supporting information. The plates were
gently shaken for a day. Then, aminomethylated polystyrene resin
EHL/2% DVB (200–400 mesh) (10 equiv) was added to scavenge
excess or unreacted acyl chloride, isocyanates, isothiocyanate
and sulfonyl chlorides. After reacting for 24 h, the resin was fil-
excellent activities up to the MIC value of 0.06 l ]
g/mL.17
In order to investigate the effect of substituents other than
amines at the 6-postion on antibacterial activity, a new series of
2,5,6-trisubstituted benzimidazole library was designed and syn-
thesized with ether/thioether groups at the 6-position (Fig. 2).
Based on previous SAR studies, the cyclohexyl group at the 2-pos-
iton was fixed and various substituents at the 5-position were
examined.
tered to afford
a library of 376 novel 2,5,6-tribsustituted
benzimidazoles 5.
2. Chemical synthesis
3. Results and discussion
General procedure for the synthesis of 2,5,6-trisubstituted
benzimidazoles bearing an ether/thioether moiety at the 6-posi-
tion is illustrated in Scheme 1.20 The first step was the nucleophilic
aromatic substitution of commercially available 2,4-dinitro-5-fluo-
roaniline (1) with various alkyl or aryl alcohol/thiols. Compounds
2a, 2b and 2e were prepared by using 1 M KOH while 2c, 2d and
2f–2h were obtained by using 1 M K2CO3 to afford compounds
2a–2h in 91–100% yields. The acylation of compounds 2a–h with
the cyclohexanecarbonyl chloride gave 3a–h in 82–89% yields.
3.1. In vitro preliminary screening of the library of 2,5,6-
trisubstituted benzimidazoles 5 against Mtb-H37Rv
The library of 2,5,6-trisubstituted benzimidazoles 5 (376 com-
pounds) was screened against drug sensitive Mtb H37Rv strain
using ‘Microplate Alamar Blue Assay (MABA)’15 and then, growth
inhibition was measured in percentage. Among these compounds,
108 compounds were identified to inhibit the growth of Mtb H37Rv
by 22–79% at 5
lg/mL concentration and 22 compounds (see
Table 1) exhibited 28–65% growth inhibition at 1.0
lg/mL concen-
tration. From the preliminary screening, the butylthio group, fol-
lowed by the benzylthio group at the 6 position appeared to be
rather preferred, but 4-fluorophenoxy, 4-fluorophenylthio, and
phenylthio groups did not seem to be much different. However, no
compounds with a phenoxy group at the 6 position were included
inthehitlist. Also, nobenzimidazolesbearingsulfoxide, ureaorthio-
urea groups at the 5 position were found in the hit list. Thus, only
amide or carbamate groups appear to be preferred at this position.
These hit compounds were resynthesized in analytically pure
form and examined for their accurate MIC values. Then, it turned
out that the MIC values did not necessarily correlate with the per-
cent inhibition at the fixed concentration of the test compounds, as
O
O
∗
∗
F
O
O
∗
∗
H
N
X
S
R1
S
S
∗
F
∗
∗
S
N
∗
HN
R2
∗
R
R
∗
O
Figure 2. Novel 2,5,6-trisubstituted benzimidazoles bearing an ether or thioether
substituent at the 6 position.

2604
B. Park et al. / Bioorg. Med. Chem. 22 (2014) 2602–2612
O
a
b
F
NH2
NO2
X
NH2
NO2
X
NH
R1
R1
91-100%
82-89%
O2N
O2N
O2N
NO2
1
2
3
2a: R1X = ethoxy
3a: R1X = ethoxy
1
1
2b
2c
: R X = butoxy
3b
3c
: R X = butoxy
1
1
: R X = phenoxy
: R X = phenoxy
2d: R1X = 4-flurophenoxy
3d: R1X = 4-flurophenoxy
1
1
2e
2f
2g
: R X = butylthio
: R X = phenlythio
3e
3f
3g
: R X = butylthio
: R X = phenlythio
1
1
1
1
: R X = 4-fluorophenylthio
: R X = 4-fluorophenylthio
2h: R1X = benzylthio
3h: R1X = benzylthio
H
N
X
H
N
c
d
X
R1
R1
N
HN
56-69%
N
RC(O)Cl
H2N
R2
5
RC(O)OSu
R2NC(O)Cl
R2NC(S)Cl
RSO2Cl
4
1
4a
: R X = ethoxy
4b: R1X = butoxy
RN=C=O
RN=C=S
1
4c
4d
4e
: R X = phenoxy
: R X = 4-flurophenoxy
1
1
: R X = butylthio
4f : R1X = phenlythio
1
4g
4h
: R X = 4-fluorophenylthio
: R X = benzylthio
1
Scheme 1. Library synthesis of 2,5,6-trisubstituted benzimidazoles. Reagents and conditions: (a) R1OH/ R1SH, 1 M KOH, THF, or K2CO3, Acetone, room temperature (rt), 1 h;
(b) cyclohexanecarbonyl chloride, pyridine, reflux, overnight; (c) (i) SnCl2Á2H2O, EtOH, reflux, 1–6 h or (ii) SnCl2Á2H2O, 4 M HCl, EtOH, reflux, 1–6 h; (d) (i) RC(O)Cl, RC(O)OSu,
R2NC(O)Cl, R2NC(S)Cl, RSO2Cl, RN@C@O or RN@C@S (1.0 equiv), CH2Cl2, overnight, rt, aminomethylated polystyrene resin, filtration and concentration in vacuo.
Table 1
Hit compounds 5 from the preliminary screening against Mtb H37Rv strain at 1.0
l
g/mL concentration
H
N
X
R1
N
HN
R2
5
Compound
R1X
R2
% Growth inhibition
Compound
R1X
R2
% Growth inhibition
1
2
3
4
5
6
7
8
EtO
BuO
4-FC6H4O
4-FC6H4O
4-FC6H4O
BuS
BuS
BuS
BuS
BuS
4-MeC6H4CO
CH2@CH(CH2)2CO
2,4-F2C6H3CO
4-MeC6H4CO
CH2@CH(CH2)2CO
2,4-F2C6H3CO
CH3(CH2)2OCO
PhSO2
65
28
44
36
54
51
38
42
45
53
52
12
13
14
15
16
17
18
19
20
21
22
BuS
PhS
PhS
PhS
4-FC6H4S
4-FC6H4S
4-FC6H4S
PhCH2S
PhCH2S
PhCH2S
PhCH2S
PH(CH2)2CO
33
31
42
56
46
30
41
28
30
52
64
4-MeC6H4CO
4-t-BuC6H4CO
CH2@CH(CH2)2CO
CH3(CH2)2OCO
PhSO2
CH2@CH(CH2)2CO
CH3(CH2)2OCO
PhSO2
9
10
11
4-t-BuC6H4CO
4-MeC6H4CO
CH2@CH(CH2)2CO
4-t-BuC6H4CO
CH2@CH(CH2)2CO
BuS
anticipated. This would be due to, for example, inaccuracy in the
actual weight and purity of a test compound in a 96-well plate,
as well as false positives in the HTP screening. As Table 2 shows,
some of the hit compounds with a 4-fluorophenoxy or buthylthio
group exhibit promising activities, but those with a 6-phenylthio
or 6-benzylthio group appear to be less potent among the com-
pounds examined so far.
exhibited the best potency (MIC 0.63
in this series (Table 2).
l
g mL) against Mtb H37Rv
The cytotoxicity of 5a–5j was evaluated in vitro against Vero
cells using the MTT assay.21 Compounds 5a, 5b, 5c and 5j showed
cytotoxicity with IC50 values in the range of 26–75 lM. However,
most of the analytically pure compounds did not show appreciable
cytotoxicity against Vero cells.
Although 5a, bearing a n-butoxycarbonylamino group at the 5
position, was not among the 22 hit compounds, we added this
compound for the MIC determination, since this carbamate group
gave the best potency in the 6-dialkyamino series of 2,5,6-trisub-
stituted benzimidazoles in our another study,17,18 Indeed, 5a
3.2. FtsZ polymerization assay
Two benzimidazoles 5a and 5d were evaluated for their ability
to inhibit the Mtb-FtsZ polymerization.22 A light scattering assay

B. Park et al. / Bioorg. Med. Chem. 22 (2014) 2602–2612
2605
Table 2
MIC of selected hit compounds against Mtb H37Rv strain
H
X
N
N
R1
HN
R2
5
Compound
R1X
R2
MIC (l
g/mL) Mtb H37Rv
Cytotoxicity (lM) vero cells
O
O
O
O
∗
∗
5a
0.63
60
∗
∗
O
F
F
5b
5c
5d
3.13
1.56
12.5
40
O
O
O
O
∗
∗
26
∗
F
F
O
∗
>200
F
F
O
O
∗
5e
5f
12.5
6.25
>200
>200
∗
F
S
O
∗
∗
O
S
S
∗
∗
5g
5h
12.5
1.25
>200
>200
O
∗
∗
F
F
S
S
O
∗
∗
5i
5j
1.25
1.25
>200
75
∗
O
∗
O
was carried out to examine the effect of these compounds on inhi-
bition of the FtsZ polymerization.17,18 The amount of the FtsZ poly-
mer formed after addition of GTP was monitored by the intensity of
light scattered by the sample. As Figure 3 shows, 5a and 5d inhib-
ited FtsZ polymerization in a dose-dependent manner.
3.4. Dissociation constant of compound 5a with FtsZ
The fluorescence anisotropy of 5a, the most active compound,
was used to determine its dissociation constant (Kd) with Mtb-
FtsZ.23 Compound 5a has an emission maximum at 427.9 nm with
the excitation maximum at 316 nm. The fluorescence anisotropy of
5a was measured in the presence of increasing FtsZ concentrations
to plot the resulting anisotropy profile (Fig. 5), which showed a
concentration-dependent change. The change in fluorescence
3.3. Transmission electron microscopy (TEM) imaging of FtsZ
with compound 5a
Transmission electron microscopy (TEM) imaging of Mtb-FtsZ
treated with 5a exhibited the ability of the compound to inhibit
(
D
D
F) at 427.9 nm was applied to the standard equation
F = (
Fmax  L)/(Kd + L) and the Kd of 5a was determined to be
1.32 0.5 M.
D
FtsZ polymerization and aggregation.17,18 Mtb-FtsZ (5
l
M) incu-
M concentrations in the presence
M) showed shorter and thinner FtsZ polymers as com-
l
bated with 5a at 40
of GTP (25
lM and 80 l
l
4. Conclusion
pared to Mtb-FtsZ in the absence of compound 5a. In the absence of
inhibitor, Mtb-FtsZ formed a dense network of long polymers
which tend to aggregate (Fig. 4A and B) while in the presence of
New library of 2,5,6-trisubstituted benzimidazoles bearing sul-
fide and ether linkage at the 6 position have been synthesized. A
number of hit compounds have been identified against Mtb H37Rv
5a (40
lM), the length, density and aggregation was visibly re-
duced (Fig. 4C and D). The effect was more apparent at 80
lM
strain with good MIC values in the range of 0.63–12.5 lg/mL. Com-
treatment where very short and dispersed FtsZ polymers were ob-
served (Fig. 4E and F). Together with the light scattering assay, TEM
images confirm the target of 5a as Mtb-FtsZ and gives insight into
the mode of action of the new series of trisubstituted benzimidaz-
oles bearing an ether or thioether linkage at 6-postion.
pound 5a and 5d were chosen for FtsZ polymerization assays and
compound 5a was used for TEM images for target validation. These
results showed that the two selected compounds inhibit FtsZ
assembly in a dose dependent manner. The dissociation constant
(Kd) of 5a was determined to be 1.32 lM based on its fluorescent

2606
B. Park et al. / Bioorg. Med. Chem. 22 (2014) 2602–2612
Tetrahydrofuran was freshly distilled from sodium metal and ben-
A
zophenone. Dichloromethane was also distilled immediately prior
to use under nitrogen from calcium hydride. 1H and 13C NMR spec-
tra were measured on a Bruker 400 or 500 MHz NMR spectrometer.
Melting points were measured on a Thomas Hoover Capillary melt-
ing point apparatus and are uncorrected. TLC was performed on
Sorbtech with UV254 and column chromatography was carried
out on silica gel 60 (Merck; 230–400 mesh ASTM). High-resolution
mass spectra were obtained on Agilent-TOF instrument. The opti-
cal density was determined from the resulting solutions using
the Acsent Multiskan optical density reader. Purity of synthesized
compounds was determined by HPLC analysis at 244 and 303 nm
wavelengths with a Shimadzu LC-2010A HT series HPLC assembly
or Agilent 1100 series HPLC assembly. For the HPLC analysis, an
Adsorbosphere silica 5
l
m, 250 mm  4.6 mm column was used
with isopropanol–hexanes (5–50% isopropanol in gradient) as elu-
ent at the flow rate of 1 mL/min, t = 0–40 min. Light scattering as-
says were performed using a PTI-QM4 spectrofluorimeter. FEI
Tecnai12 BioTwinG transmission electron microscope with an
AMT XR-60 CCD digital camera system was used to acquire trans-
mission electron microscopy images.
B
5.1. 2,4-Dinitro-5-ethoxyaniline (2a)
To a magnetically stirred solution of 2,4-dinitro-5-fluoroaniline
(1) (4.0 g, 19.9 mmol) in 50 mL of THF and excess ethanol was
added 1 M KOH aqueous solution dropwise until a yellow precipi-
tate appeared. The reaction mixture was stirred for additional 1 h.
The solution was concentrated to dryness and then extracted with
ethyl acetate. The organic layer was collected, dried over anhy-
drous magnesium sulfate and concentrated in vacuo to give 2a as
a yellow solid (4.8 g, 100% yield): mp 162–165 °C; 1H NMR
(500 MHz, CDCl3) d 1.53 (t, 3H, J = 7.0 Hz), 4.18 (q, 2H, J = 7.0 Hz),
6.23 (s, 1H), 8.95 (s, 1H); 13C NMR (125 MHz, CDCl3) d 14.3, 66.1,
99.7, 127.2, 148.8, 158.1; HRMS (ESI) m/z calcd for C8H10N3O5+
228.0615 found: 228.0615 (
D = 0.0 ppm).
Figure 3. Inhibition of FtsZ polymerization by (A) 5a, (B) 5d.
In a similar manner, intermediate 2b, 2e were synthesized and
characterized.
anisotropy. This result provides direct evidence for the binding
interaction between this benzimidazole and Mtb-FtsZ protein,
which could be applicable to all potent benzimidazoles in this ser-
ies. Further SAR study is necessary to obtain more detailed infor-
mation for the substituent effects at 5 and 6 positions of the
2,5,6-trisubstituted benzimidazoles in this series. Nevertheless,
4-fluorophenoxy and butylthio groups were found to be preferred
substituents at the 6 position. For the compounds, bearing a 4-flu-
orophenoxy group at the 6 position, a carbamate group at the 5 po-
sition gave the most potent compound (5a), but there is no
difference between a carbamate group and benzamide groups for
their potency (5j vs 5h and 5i) for the compounds, bearing a butyl-
thio group at the 6 position. This is a unique feature in this series of
benzimidazoles since a carbamate group at the 5 position provides,
in general, more potent compounds than the corresponding 5-
amidobenzimidazoles in the 5-dialkylamino-benzimidazole
series.17,18 Further optimization of the lead compounds for their
anti-TB activities is actively underway in our laboratory. Also bio-
logical evaluations of the hit/lead compounds of this series against
various other pathogens will be carried out to investigate their
pathogen specific as well as broad spectrum antibacterial
activities.
5.2. 5-Butoxy-2,4-dinitroaniline (2b)
Yellow solid; 76% yield; mp 176–178 °C; 1H NMR (500 MHz,
CDCl3) d 0.99 (t, 3H, J = 7.4 Hz), 1.21–1.33 (m, 3H), 1.41–1.46 (m,
2H), 1.53 (dd, 2H, J = 15.1, 7.5 Hz), 1.63–1.66 (m, 2H), 1.75–1.78
(m, 2H), 1.86 (t, 2H, J = 7.6 Hz), 1.92–1.95 (m, 2H), 2.32–2.36 (m,
1H), 4.10 (t, 2H, J = 6.4 Hz), 6.24 (s, 1H), 8.95 (s, 1H); 13C NMR
(125 MHz, CDCl3) d 13.7, 19.0, 25.3, 25.7, 28.8, 30.6, 42.8, 70.0,
99.7, 124.5, 127.2, 130.2, 148.8, 158.2; HRMS (ESI) m/z calcd for
C
10H14N3O+5 256.0928 found: 256.0931 (
D = 1.17 ppm).
5.3. 2,4-Dinitro-5-phenoxyaniline (2c)
To a magnetically stirred solution of 2,4-dinitro-5-fluoroaniline
1 (3.0 g, 14.9 mmol) in 45 mL of acetone were added phenol
(1.68 g, 17.9 mmol) and anhydrous K2CO3 (4.12 g, 29.8 mmol).
The reaction mixture was stirred mechanically at room tempera-
ture for at least 16 h until the total disappearance of 1 was con-
firmed by MS (FIA) analysis. The solution was concentrated to
dryness and then extracted with ethyl acetate. The organic layer
was collected, dried over anhydrous magnesium sulfate, and con-
centrated in vacuo to give 2c as a yellow solid (3.7 g, 91% yield):
mp 148–150 °C; 1H NMR (500 MHz, CDCl3) d 5.97 (s, 1H), 7.56
(m, 3H), 7.60 (dd, 2H, J = 7.57, 1.83 Hz), 9.05 (s, 1H); 13C NMR
(125 MHz, CDCl3) d 114.6, 126.4, 128.4, 129.7, 130.5, 130.9,
134.7, 136.2, 146.3, 148.7; HRMS (ESI) m/z calcd for C12H10N3O5+
5. Experimental
The chemicals were purchased from Sigma Aldrich Co., Syn-
quest Inc., Alfa Aesar and purified before use by standard methods.
276.0615 found: 275.0358 (
D
= À29.0 ppm).

B. Park et al. / Bioorg. Med. Chem. 22 (2014) 2602–2612
2607
Figure 4. Transmission electron microscopy (TEM) Images of FtsZ. FtsZ (5
lM) was polymerized by GTP (25
lM) in the absence (A and B) and presence of 5a at 40
lM (C and
D) and 80
lM (E and F). Images (A, C, E) are at 23,000Â magnification (scale bar 500 nm) and (B, D, F) are at 49,000Â magnification (scale bar 500 nm).
In a similar manner, intermediate 2d, 2f, 2g, and 2h were syn-
thesized and characterized.
(100 MHz, CDCl3) d 13.7, 22.3, 29.2, 32.4, 112.6, 126.6, 127.8,
135.4, 146.3, 147.8; HRMS (ESI) m/z calcd for C10H14N3O4S+
272.0700 found: 272.0701 (
D = 0.35 ppm).
5.4. 2,4-Dinitro-5-(4-fluorophenoxy)aniline (2d)
5.6. 2,4-Dinitro-5-(phenylthio)aniline (2f)
Yellow solid; 93% yield; mp 164–165.5 °C; 1H NMR (500 MHz,
CDCl3) d 6.01 (s, 1H), d 7.11–7.19 (m, 4H), d 9.05 (s, 1H); 13C
NMR (125 MHz, CDCl3) d 103.7, 117.2, 117.4, 122.5, 122.6, 127.6,
148.5, 149.2, 157.8, 159.5, 161.5; HRMS (ESI) m/z calcd for C12H9-
Yellow solid; 100% yield; mp 214–217 °C; 1H NMR (400 MHz,
CDCl3) d 5.97 (s, 1H), 7.21–7.24 (m, 3H), 7.28–7.32 (m, 2H), 9.02
(s, 1H); 13C NMR (100 MHz, CDCl3) d 126.4, 127.2, 127.5, 129.1,
130.5, 130.8, 136.2, 137.0; HRMS (ESI) m/z calcd for C12H10N3O4S+
FN3O+5 294.0521 found: 294.0521 (
D = 0.0 ppm).
292.0387 found: 292.0387 (
D = 0.0 ppm).
5.5. 5-(Butylthio)-2,4-dinitroaniline (2e)
5.7. 2,4-Dinitro-5-(4-fluorophenylthio)aniline (2g)
Yellow solid; 99% yield; mp 148–149 °C; 1H NMR (400 MHz,
CDCl3) d 0.99 (t, 3H, J = 7.5 Hz), 1.53–1.57 (m, 2H), 1.74–2.05 (m,
2H), 2.01 (t, 2H, J = 7.5 Hz), 6.55 (s, 1H), 9.18 (s, 1H); 13C NMR
Yellow solid; 100% yield; mp 217–219 °C; 1H NMR (500 MHz,
CDCl3) d 5.96 (s, 1H), 7.04 (t, 1H, J = 8.6 Hz), 7.27 (d, 1H, J = 8.53),

2608
B. Park et al. / Bioorg. Med. Chem. 22 (2014) 2602–2612
diluted with ethyl acetate, and washed with water three times.
The organic layers were dried over sodium sulfate, filtered, and
concentrated to afford 3a as a yellow solid (0.9 g, 89% yield): mp
109–109.5 °C; 1H NMR (400 MHz, CDCl3) d 1.28–1.31 (m, 1H),
1.38–1.42 (m, 2H), 1.51 (t, 3H, J = 7.41 Hz), 1.59–1.60 (m, 2H),
1.76–1.79 (m, 1H), 1.88–1.93 (m, 2H), 2.05–2.09 (m, 2H), 2.44 (tt,
1H, J = 11.6, 3.5 Hz), 3.12 (q, 2H, J = 7.44 Hz), 9.19 (s, 1H), 9.24 (s,
1H), 10.9 (s, 1H); 13C NMR (100 MHz, CDCl3) d 12.2, 25.4, 25.5,
27.1, 29.4, 47.4, 116.9, 124.9, 130.7, 138.1, 149.7, 176.0; HRMS
(ESI) m/z calcd for
= 1.18 ppm).
In a similar manner, compounds 3b–3h were synthesized and
C
15H20N3O+6 338.1347 found: 338.1351
(D
characterized.
5.10. 1-(Cyclohexanecarboxamido)-5-butoxy-2,4-dinitro-
benzene (3b)
Yellow solid; 100% yield; mp 187.5–189 °C; 1H NMR (500 MHz,
CDCl3) d 0.99 (t, 3H, J = 7.4 Hz), 1.21–1.33 (m, 3H), 1.45 (d, 2H,
J = 11.5 Hz), 1.50–1.56 (m, 2H), 1.6401.66 (m, 1H), 1.75–1.78 (m,
2H), 1.86 (dd, 2H, J = 8.4, 6.8 Hz), 1.93 (d, 2H, J = 12.8 Hz), 2.32–
2.36 (m, 1H), 4.09 (t, 2H, J = 6.4 Hz), 6.24 (s, 1H), 8.95 (s, 1H);13
C
NMR (125 MHz, CDCl3) d 13.7, 19.0, 25.3, 25.3, 28.8, 30.6, 42.8,
70.0, 99.7, 124.5, 127.2, 130.2, 148.8, 158.2; MS (ESI) m/z 366.1
(M+1).
5.11. 1-(Cyclohexanecarboxamido)-5-phenoxyphenyl-2,4-
dinitro-benzene (3c)
Yellow solid; 97% yield; mp 150.5–152 °C; 1H NMR (400 MHz,
CDCl3) d 1.21 (tt, 1H, J = 12.3, 3.2 Hz), 1.26–1.35 (m, 2H), 1.43
(qd, 2H, J = 12.3, 3.1), 1.68–1.72 (m, 1H), 1.81 (dt, 2H, J = 13.1,
3.3 Hz), 1.95 (dd, 2H, J = 13.5, 1.9 Hz), 2.23–2.33 (m, 1H), 7.15–
7.17 (m, 2H), 7.35 (t, 1H, J = 7.5 Hz), 7.49–7.52 (m, 2H), 8.53 (s,
1H), 9.06 (s, 1H), 10.8 (s, 1H); 13C NMR (125 MHz, CDCl3) d 25.5,
29.3, 47.3, 108.8, 120.6, 121.6, 125.6, 126.7, 128.8, 129.4, 130.6,
133.2, 140.4, 153.2, 157.7, 175.4; HRMS (ESI) m/z calcd for
Figure 5. Determination of binding parameter of 5a with Mtb-FtsZ. (A) Fixed
concentration of 5a (100 lM) was excited at 316 nm with varying concentration of
Mtb-FtsZ (as shown in the graph) and fluorescence monitored at 427.9 nm and
plotted against wavelength. Increase in fluorescence intensity observed on addition
of increasing concentration of protein. (B) Fluorescence profiles of emission
intensity at 427 nm for the titration of Mtb-FtsZ. The peak saturation was observed
C
19H20N3O+6 386.1347 found: 386.1345 (
D
= À0.52 ppm).
5.12. 1-(Cyclohexanecarboxamido)-5-(4-fluorophenoxy)-2,4-
dinitrobenzene (3d)
at 2.5
lM and then there is a progressive decrease in fluorescence emission. The Kd
of 5a was calculated to be 1.32 0.5
lM/L.
Yellow solid; mp 147–149 °C; 1H NMR (500 MHz, CDCl3) d 1.18–
1.20 (m, 1H), 1.27–1.32 (m, 2H), 1.40 (d, 2H, J = 11.6 Hz), 1.71 (dd,
1H, J = 1.69, 1.42 Hz), 1.78–1.83 (m, 2H), 1.90–1.94 (m, 2H), 2.22–
2.30 (m, 1H), 7.23–2.30 (m, 2H), 7.58–7.62 (m, 2H), 8.47 (s, 1H),
d 9.21 (s, 1H), d 10.6 (s, 1H); 13C NMR (125 MHz, CDCl3) d 25.2,
25.4, 29.1, 29.3, 47.2, 117.9, 118.1, 119.0, 123.9, 124.1, 124.7,
138.1, 138.2,150.4, 165.8, 174.8; MS (ESI) m/z 404.0 (M+1)+.
7.49–7.46 (m, 1H), 7.62 (dd, 1H, J = 8.5, 5.3 Hz), 9.23 (s, 1H); 13C
NMR (125 MHz, CDCl3) d 114.4, 116.2, 116.4, 117.8, 118.0, 126.5,
131.2, 131.3, 138.3, 138.4, 146.3; HRMS (ESI) m/z calcd for
C
12H9FN3O4S+ 310.0292 found: 310.0292 (
D = 0.0 ppm).
5.8. 5-(Benzylthio)-2,4-dinitroaniline (2h)
Yellow solid; 100% yield; mp 188.5–190 °C; 1H NMR (400 MHz,
CDCl3) d 4.17 (s, 2H), d 6.62 (s, 1H), d 7.36–7.44 (m, 5H), 9.19 (s,
1H); 13C NMR (100 MHz, CDCl3) d 37.8, 113.0, 126.5, 128.2,
129.0, 129.1, 133.7; HRMS (ESI) m/z calcd for C12H12N3O4S+
5.13. 1-(Cyclohexanecarboxamido)-5-(butylthio)-2,4-dinitro-
benzene (3e)
Yellow solid; 100% yield; mp 118–119 °C; 1H NMR (500 MHz,
CDCl3) d 0.99 (t, 3H, J = 7.3 Hz), 1.25–1.32 (m, 1H), 1.33–1.42 (m,
2H), 1.55–1.59 (m, 4H), 1.74–1.78 (m, 1H), 1.80–1.83 (m, 2H), 1.88
(dt, 2H, J = 13.3, 3.4 Hz), 2.05 (dd, 2H, J = 13.4, 2.2 Hz), 2.42 (tt, 1H,
J = 11.7, 3.5 Hz), 3.07 (t, 2H, J = 7.3 Hz), 9.16 (s, 1H), 9.21 (s, 1H), 10.9
(s, 1H); 13C NMR (125 MHz, CDCl3) d 13.7, 22.1, 25.5, 29.5, 32.7,
47.5, 117.0, 124.9, 138.1, 150.0, 175.9; MS (ESI) m/z 382.1 (M+1)+.
306.0543 found: 306.0543 (
D = 0.0 ppm).
5.9. 1-(Cyclohexanecarboxamido)-5-ethoxy-2,4-dinitro-
benzene (3a)
To a solution of 2a (0.71 g, 3.13 mmol) in 12 mL of pyridine was
added cyclohexanecarbonyl chloride (0.54 mL, 1.3 equiv), and the
mixture was magnetically stirred and refluxed overnight. After
completion of the reaction by TLC analysis, the reaction mixture
was concentrated under reduced pressure, diluted with ethyl ace-
tate, and then washed with CuSO4 solution twice to get rid of the
leftover pyridine. The reaction mixture was washed with brine,
5.14. 1-(Cyclohexanecarboxamido)-5-(phenylthio)-2,4-
dinitrobenzene (3f)
Yellow solid; 100% yield; mp 217–218 °C; 1H NMR (400 MHz,
CDCl3) d 1.18–1.22 (m, 2H), 1.24–1.32 (m, 2H), 1.37(qd, 2H,

B. Park et al. / Bioorg. Med. Chem. 22 (2014) 2602–2612
2609
J = 12.2, 2.8 Hz), 1.67 (dd, 1H, J = 12.8, 0.8 Hz), 1.78–1.82 (m, 2H),
5.19. 5-Amino-6-phenoxy-2-cyclohexyl-1H-benzo[d]imidazole
(4c)
1.89–1.92 (m, 2H), 2.24 (tt, 1H, J = 11.7, 3.5 Hz), 7.60 (q, 5H,
J = 6.1 Hz), 8.46 (s, 1H), 9.22 (s, 1H), 10.6 (s, 1H); 13C NMR
(100 MHz, CDCl3) d 25.4, 29.3, 30.9, 47.3, 119.1, 124.7, 128.6,
130.6, 131.1, 135.9, 137.5, 138.0, 150.7, 174.8; MS (ESI) m/z
402.1 (M+1)+.
Pale yellow solid; 46% yield; mp 136–138 °C; 1H NMR
(400 MHz, CDCl3) d 1.25–1.32 (m, 1H), 1.35–1.44 (m, 2H), 1.56–
1.66 (m, 2H), 1.72–1.77 (m, 1H), 1.83–1.88 (m, 2H), 2.09–2.14
(m, 2H), 2.80–2.86 (m, 1H), 3.73 (br, 2H), 6.93–6.97 (m, 2H),
7.01–7.05 (m, 1H), 7.12 (s, 1H), 7.26–7.29 (m, 2H); 13C NMR
(100 MHz, CDCl3) d 25.9, 26.0, 31.9, 38.5, 116.7, 122.4, 129.7,
5.15. 1-(Cyclohexanecarboxamido)-5-(4-fluorophenylthio)-2,4-
dinitrobenzene (3g)
135.3, 140.3, 158.1; HRMS (ESI) m/z calcd for
308.1757 found: 308.1758 ( = 0.32 ppm).
C
19H22N3O+
Yellow solid; 96% yield; mp 186–189 °C; 1H NMR (400 MHz,
CDCl3) d 1.16–1.23 (m, 1H), 1.25–1.31 (m, 2H), 1.34–1.42 (m,
2H), 1.68–1.74 (m, 1H), 1.79–1.83 (m, 2H), 1.94 (dd, 2H, J = 12.9,
2.1 Hz), 2.25–2.31 (m, 1H), 7.25–7.29 (m, 2H), 7.62 (dd, 2H,
J = 8.8, 5.2 Hz), 8.49 (s, 1H), 9.24 (s, 1H), 10.6 (s, 1H); 13C NMR
(100 MHz, CDCl3) d 25.5, 29.3, 31.0, 47.3, 119.1, 124.7, 128.7,
130.6, 131.1, 131.5, 135.9, 137.6, 138.1, 150.8, 174.8; MS (ESI) m/z
420.1 (M+1)+.
D
5.20. 5-Amino-6-(4-fluorophenoxy)-2-cyclohexyl-1H-
benzo[d]imidazole (4d)
Beige solid; 70% yield; mp 195–196 °C; 1H NMR (400 MHz,
CDCl3) d 1.26–2.30 (m, 1H), 1.38–1.43 (m, 2H), 1.61 (dd, 2H,
J = 12.4, 3.1 Hz), 1.72–1.77(m, 1H), 1.86 (dt, 2H, J = 13.1, 3.3 Hz),
2.12 (dd, 2H, J = 13.6, 2.0 Hz), 2.83 (tt, 1H, J = 11.8, 3.6 Hz), 3.74
(br, 2H), 6.95 (m, 5H), 8.89 (s, 1H); 13C NMR (100 MHz, CDCl3) d
25.8, 26.0, 31.8, 38.5, 116.0, 116.2, 118.1, 118.2, 154.0, 157.1,
159.5; HRMS (ESI) m/z calcd for C19H23FN3O+ 326.1663 found:
5.16. 1-(Cyclohexanecarboxamido)-5-(benzylthio)-2,4-
dinitrobenzene (3h)
326.1667 (
D = 1.2 ppm).
Yellow solid; 96% yield: mp 150–153 °C; 1H NMR (500 MHz,
CDCl3) d 1.27–1.30 (m, 1H), 1.37–1.44 (m, 2H), 1.52–1.62 (m,
2H), 1.74–1.77 (m, 1H), 1.87–1.90 (m, 2H), 2.04–2.07 (m, 2H),
2.39–2.47 (m, 1H), 4.30 (s, 2H), 7.26–7.37 (m, 3H), 7.45 (d, 2H,
J = 6.8 Hz), 9.21 (s, 1H), 9.29 (s, 1H), 10.9 (s, 1H); 13C NMR
(125 MHz, CDCl3) d 25.4, 25.5, 29.4, 38.1, 43.3, 47.4, 117.1, 124.8,
127.4, 128.2, 128.5, 128.8, 129.4, 129.6, 130.9, 133.5, 137.4,
138.1, 138.2, 149.1, 175.9; MS (ESI) m/z 416.0 (M+1)+.
5.21. 5-Amino-6-(butylthio)-2-cyclohexyl-1H-
benzo[d]imidazole (4e)
A solution of 3e (1.97 g, 5.16 mmol), tin(II) chloride dihydrate
(15.5 g, 36.1 mmol), and conc. HCl (80 mL) in 200 mL of ethanol
was magnetically stirred and refluxed for 4 h. The reaction mixture
was cooled, quenched with 1 M NaOH, and pH was adjusted to
ꢀ10. Tin salts precipitated in solution upon addition of 1 M NaOH.
The reaction mixture was filtered to remove the tin salts. The solu-
tion was diluted with ethyl acetate and washed with water three
times. The organic layers were dried over magnesium sulfate, fil-
tered, and concentrated in vacuo. The residue was purified by flash
chromatography on silica gel (gradient 20–40% ethyl acetate/hex-
5.17. 5-Amino-6-ethoxy-2-cyclohexyl-1H-benzo[d]imidazole
(4a)
A solution of 3a (100 mg, 0.30 mmol), tin(II) chloride dihydrate
(0.47 g, 2.1 mmol) in 10 mL of EtOH was magnetically stirred and
refluxed at 90 °C under nitrogen for 1 h. The reaction mixture
was cooled, quenched with 30% KOH, and pH adjusted to ꢀ13.
The solution was diluted with dichloromethane and washed with
water three times. The organic layers were dried over sodium sul-
fate, filtered, and concentrated. The residue was purified by flash
chromatography on silica gel (gradient 20–40% ethyl acetate/hex-
anes) to afford compound 4a as a pale red color solid (69 g, 89%
yield): mp 89–90 °C; 1H NMR (500 MHz, CDCl3) d 1.23 (ddd, 1H,
J = 14.2, 10.8, 3.3 Hz), 1.32 (ddd, 2H, J = 14.2, 11.1, 3.1 Hz),
1.37–1.42 (m, 3H), 1.60 (qd, 2H, J = 12.4, 3.1 Hz), 1.69–1.71
(m, 1H), 1.79–1.81 (m, 2H), 2.08 (d, 2H, J = 12.4 Hz), 2.80 (tt, 1H,
J = 11.8, 3.51 Hz), 3.79 (br, 2H), 3.99 (dd, 2H, J = 8.3, 3.3 Hz), 6.78
(s, 1H), 6.96 (s, 1H); 13C NMR (125 MHz, CDCl3) d 15.0, 25.9, 26.1,
32.0, 38.5, 64.4, 132.9, 133.0, 144.5, 157.0; HRMS (ESI) m/z calcd
anes) to afford compound 4e as
a pale greenish gray solid
(0.413 g): 71% yield; mp 110–112 °C; 1H NMR 500 MHz, CDCl3) d
0.88 (t, 3H, J = 7.3 Hz), 1.25–1.30 (m, 1H), 1.36–1.42 (m, 4H), 1.55
(dt, 2H, J = 14.0, 7.4 Hz), 1.64 (qd, 2H, J = 12.4, 3.2 Hz), 1.72–1.76
(m, 1H), 1.85 (dt, 2H, J = 13.2, 3.3 Hz), 2.13 (dd, 2H, J = 13.7, 1.9 Hz),
2.72 (t, J = 7.4 Hz), 2.86 (tt, 1H, J = 11.8, 3.5 Hz), 4.35 (br, 2H), 6.83
(s, 1H), 7.66 (s, 1H); 13C NMR (100 MHz, CDCl3) d 13.7, 21.9, 25.8,
26.0, 31.6, 31.9, 35.5, 38.5, 114.5, 144.0, 158.8; HRMS (ESI) m/z calcd
for C17H26N3S+ 304.1842 found: 304.1842 (
In a similar manner, compounds 4f–4h were synthesized and
characterized.
D = 0.0 ppm).
5.22. 5-Amino-6-(phenylthio)-2-cyclohexyl-1H-
benzo[d]imidazole (4f)
for C15H22N3O+ 260.1757 found: 260.1758 (
D = 0.38 ppm).
Pale brown solid; 57% yield; mp 116–118 °C; 1H NMR (500 MHz,
CDCl3) d 1.27–1.35 (m, 1H), 1.42 (qt, 2H, J = 12.79, 3.29 Hz), 1.66 (qd,
2H, J = 12.4, 3.29 Hz), 1.76–1.80 (m, 1H), 1.89 (dt, 2H, J = 13.3,
3.38 Hz), 2.16 (dd, 2H, J = 13.7, 2.01 Hz), 2.88 (tt, 1H, J = 11.8,
3.55 Hz), 4.22 (br, 2H), 6.92 (s, 1H), d 7.08–7.13 (m, 3H), d 7.20–
7.23 (m, 2H), d 7.67 (s, 1H); 13C NMR (125 MHz, CDCl3) d 25.8,
26.0, 31.8, 38.5, 110.6, 125.3, 126.1, 128.9, 137.6, 144.6; HRMS (ESI)
In a similar manner, compounds 4b–4d were synthesized and
characterized.
5.18. 5-Amino-6-butoxy-2-cyclohexyl-1H-benzo[d]imidazole
(4b)
Pale red solid; 69% yield; mp 113–115 °C; 1H NMR (400 MHz,
CDCl3) d 0.97 (t, 3H, J = 7.4 Hz), 1.23–1.28 (m, 1H), 1.35–1.40 (m,
2H), 1.50 (dd, 2H, J = 15.0, 7.5 Hz), 1.60 (qd, 2H, J = 12.4, 3.2 Hz),
1.70–1.75 (m, 1H), 1.76–1.86 (m, 4H), 2.08–2.12 (m, 2H),
2.78–2.84 (m, 1H), 3.76 (br, 2H), 3.98 (t, 2H, J = 6.5 Hz), 6.80 (s,
1H), 7.00 (s, 1H); 13C NMR (100 MHz, CDCl3) d 13.9, 19.4, 25.9,
26.1, 31.4, 32.0, 38.4, 68.6, 133.1, 144.6, 156.7; HRMS (ESI) m/z
m/z calcd for C19H22N3S+ 324.1529 found: 324.1529 (
D = 0.0 ppm).
5.23. 5-Amino-6-(4-fluorophenylthio)-2-cyclohexyl-1H-
benzo[d]imidazole (4g)
Pale green solid; 61% yield; mp 107–108 °C; 1H NMR (500 MHz,
CDCl3) d 1.25 (ddd, 1H, J = 13.8, 7.1, 3.6 Hz), 1.33–1.40 (m, 2H), 1.62
calcd for C17H26N3O+ 288.2070 found: 288.2071 (
D = 0.35 ppm).

2610
B. Park et al. / Bioorg. Med. Chem. 22 (2014) 2602–2612
(qd, 2H, J = 12.4, 3.1 Hz), 1.71–1.74 (m, 1H), 1.82–1.84 (m, 2H), 2.85
(tt, 1H, J = 11.8, 3.5 Hz), 6.86–6.89 (m, 3H), 7.02–7.05 (m, 2H), 7.70
(s, 1H); 13C NMR (125 MHz, CDCl3) d 25.8, 26.0, 31.8, 38.5, 98.8,
111.2, 115.9, 116.1, 124.2, 128.2, 132.4, 144.4, 159.3, 160.2,
162.1; HRMS (ESI) m/z calcd for C19H22N3S+ 342.1435 found:
0.31 mmol) in 6 mL of dichloromethane, and magnetically stirred
in the ice bath. The reaction mixture was slowly warmed up to
room temperature and stirred for 16 h. The solution was diluted
with dichloromethane, basified with NaHCO3 and then washed
with water three times. The organic layers were dried over sodium
sulfate, filtered, and concentrated. The residue was purified by
flash chromatography on silica gel (gradient 20–40% ethyl ace-
tate/hexanes) to afford compound 5b as an off-white solid
(152 mg, 92% yield): mp >230 °C; 1H NMR (400 MHz, CDCl3) d
1.22–1.33 (m, 3H), 1.59 (dd, 2H, J = 12.1, 2.9 Hz), 1.68–1.71 (m,
1H), 1.78–1.81 (m, 2H), 2.01–2.08 (m, 2H), 2.76–2.82 (m, 1H),
3.87 (s, 3H), 6.96 (d, 2H, J = 8.8 Hz), 7.03 (d, 3H, J = 6.3 Hz),
7.78 (d, 2H, J = 8.8 Hz), 8.55 (s, 1H), 8.83 (s, 1H), 9.81 (s, 1H); 13C
342.1435 (D = 0.0 ppm).
5.24. 5-Amino-6-(benzylthio)-2-cyclohexyl-1H-
benzo[d]imidazole (4h)
Pale beige solid; 56% yield; mp 129.5–131 °C; 1H NMR
(500 MHz, CDCl3) d 1.18–1.23 (m, 1H), 1.27–1.35 (m, 2H), 1.60
(qd, 2H, J = 12.4, 3.2 Hz), 1.69 (d, 1H, J = 12.7 Hz), 1.78 (dt, 2H,
J = 13.1, 3.0 Hz), 2.07 (dd, 2H, J = 14.2, 2.5 Hz), 2.80–1.85 (m, 1H),
3.86 (s, 2H), 6.77 (s, 1H), 7.10–7.12 (m, 2H), 7.17 (td, 3H, J = 6.5,
2.8 Hz), 7.49 (s, 1H); 13C NMR (125 MHz, CDCl3) d 25.7, 26.0,
31.7, 38.4, 40.7, 64.4, 98.4, 114.0, 123.1, 126.9, 128.3, 128.7,
132.8, 138.2, 139.1, 144.2, 159.1, 176.3; HRMS (ESI) m/z calcd for
NMR (100 MHz, CDCl3)
38.5, 65.3, 116.5, 119.5, 125.6, 142.3, 153.1, 154.0, 157.8,
159.5, 159.6, 159.8; HRMS (ESI) m/z calcd for
27H27FN3O+3
= À0.7 ppm). HPLC: t = 9.4 min, pur-
d 13.8, 19.1, 25.8, 26.0, 31.0, 31.8,
C
460.2031 found: 460.2028 (
ity >99%.
D
C
20H24N3S+ 338.1685 found: 338.1686 (
D
= 0.3 ppm).
In a similar manner, compounds 5c–5g were synthesized and
characterized.
5.25. 5-Butoxycarbonylamino-2-cyclohexyl-6-(4-
fluorophenoxy)-1H-benzo[d]imidazole (5a)
5.28. 2-Cyclohexyl-6-(4-fluorophenoxy)-5-(4-methxyl-
benzamido)-1H-benzo[d]imidazole (5c)
To a solution of 4a (100 mg, 0.31 mmol) in 6 mL of dichloro-
methane was added N-butoxycarbonyloxysuccinimide (68 mg,
0.31 mmol) in 6 mL of dichloromethane and the mixture was mag-
netically stirred under nitrogen atmosphere in an ice bath. The
reaction mixture was slowly warmed up to room temperature
and stirred for 16 h. The solution was diluted with dichlorometh-
ane and basified with NaHCO3 and then washed with water three
times. The organic layers were dried over sodium sulfate, filtered,
and concentrated. The residue was purified by flash chromatogra-
phy on silica gel (gradient 20–40% ethyl acetate/hexanes) to afford
compound 5a as an off-white solid (54 mg, 47% yield): mp 91–
92 °C; 1H NMR (400 MHz, CDCl3) 0.97 (t, 3H, J = 7.4 Hz), 1.28–
1.30 (m, 1H), 1.39–1.45 (m, 4H), 1.61–1.65 (m, 2H), 1.67 (t, 2H,
J = 7.5 Hz), 1.76 (d, 1H, J = 12.5 Hz), 1.85–1.88 (m, 2H), 2.12 (d,
2H, J = 12.5 Hz), 2.83–2.88 (m, 1H), 4.19 (t, 2H, J = 6.7 Hz), 6.96–
7.05 (m, 4H), 7.13 (s, 1H), 8.23 (s, 1H); 13C NMR (100 MHz, CDCl3)
d 13.8, 19.1, 25.8, 26.0, 31.0, 31.8, 38.5, 65.3, 116.5, 119.5, 125.6,
142.3, 153.1, 154.0, 157.8, 159.5, 159.6, 159.7; HRMS (ESI) m/z
Off-white solid; 47% yield; mp 166–168 °C; 1H NMR (500 MHz,
CDCl3) d 1.81–1.23 (m, 1H), 1.26–1.34 (m, 2H), 1.56–1.64 (qd, 2H,
J = 12.4, 2.6 Hz), 1.71 (m, 1H), 1.78–1.81 (d, 2H, J = 12.8 Hz), 2.01
(dd, 2H, J = 12.5, 0.6 Hz), 2.45 (t, 3H), 2.79 (t, 1H, J = 11.5 Hz),
7.05 (d, 3H, J = 6.4 Hz), 7.24 (s, 1H), 7.31 (d, 2H, J = 7.8 Hz), 7.74
(d, 2H, J = 8.1 Hz), 8.63 (s, 1H), 8.90 (s, 1H), 10.2 (s, 1H); 13C NMR
(100 MHz, CDCl3) d 21.5, 25.7, 25.9, 29.7, 31.7, 38.5, 116.4, 116.6,
119.4, 119.5, 125.3, 126.9, 132.2, 142.6, 153.0, 153.1, 137.9,
159.9, 160.2, 165.8; HRMS (ESI) m/z calcd for
C
27H27FN3O+2
D = 0.0 ppm). HPLC: t = 7.7 min, purity
442.2082 found: 442.2082 (
>99%.
5.29. 2-Cyclohexyl-5-(2,4-difluorobenzamido)-6-(4-
fluorophenoxy)-1H-benzo[d]imidazole (5d)
Off-white solid; 92% yield; mp 184–185 °C; 1H NMR (500 MHz,
CDCl3) 0.83–0.88 (m, 1H), 1.25–1.32 (m, 2H), 1.59 (dd, 2H, J = 12.3,
3.0 Hz), 1.68–1.70 (m, 1H), 1.78–1.80 (m, 2H), 2.07 (d, 2H,
J = 12.8 Hz), 2.80–2.84 (m, 1H), 6.90 (ddd, 1H, J = 11.7, 8.8,
2.6 Hz), 6.99 (d, 3H, J = 6.3 Hz), 7.02–7.06 (m, 1H), 7.21 (s, 1H),
8.21 (td, 1H, J = 8.9, 6.6 Hz), 8.81 (s, 1H), 9.26 (d, 1H, J = 15.7 Hz);
13C NMR (100 MHz, CDCl3) d 25.9, 26.2, 31.9, 38.2, 101.1, 105.2,
112.5, 112.6, 112.7, 116.5, 116.7, 120.8 120.9, 126.4, 128.8, 138.4,
144.8, 151.7, 158.4, 161.0, 163.1; HRMS (ESI) m/z calcd for
calcd for C24H29FN3O+3 426.2187 found: 426.2187 (
D = 0.0 ppm).
HPLC: t = 7.2 min, purity >98%.
In
a similar manner, compound 5j was synthesized and
characterized.
5.26. 6-(Butylthio)-2-cyclohexyl-5-propoxycarbonylamino-1H-
benzo[d]imidazole (5j)
C
26H23F3N3O+2 466.1737 found: 466.1743 (
t = 4.6 min, purity >96%.
D = 1.29 ppm). HPLC:
Off-white solid; 62% yield; mp 133–134.5 °C; 1H NMR
(500 MHz, CDCl3)
d 0.87 (t, 3H, J = 7.32 Hz), 1.00 (t, 3H,
J = 7.43 Hz), 1.30 (dt, 1H, J = 3.51, 12.5 Hz), 1.38–1.43 (m, 3H),
1.50–1.53 (m, 2H), 1.59–1.67 (m, 4H), 1.74 (q, 2H, J = 7.11 Hz),
1.88 (dt, 2H, J = 3.41, 13.3 Hz), 2.13 (dd, 2H, J = 2.29, 13.4 Hz),
2.69 (t, 2H, J = 7.35 Hz), 2.84–2.89 (m, 1H), 4.26 (t, 2H,
J = 6.74 Hz), 7.88 (s, 1H), 8.20 (s, 1H), 8.27 (s, 1H), 8.95 (s, 1H);
13C NMR (125 MHz, CDCl3) d 10.4, 13.6, 21.8, 22.3, 25.8, 26.0,
29.7, 31.4, 31.7, 36.9, 38.4, 66.8, 98.5, 100.2, 126.5, 126.6, 134.9,
153.9; HRMS (ESI) m/z calcd for C21H32N3O2S+ 390.2210 found:
5.30. 2-Cyclohexyl-6-(4-fluorophenoxy)-5-(pent-4-enimido)-
1H-benzo[d]imidazole (5e)
White solid; 63% yield; mp 180.5–181.5 °C; 1H NMR (500 MHz,
CDCl3) d 1.24–1.30 (m, 2H), 1.36–1.44 (m, 2H), 1.59–1.64 (m, 2H),
1.73–1.76 (m, 1H), 1.86 (dq, 2H, J = 3.34, 9.98 Hz), 2.11 (dd, 2H,
J = 2.28, 13.3 Hz), 2.48 (dt, 3H, J = 5.88, 11.7 Hz), 2.82–2.87 (m,
1H), 4.99 (d, 1H, J = 10.2 Hz), 5.07 (d, 1H, J = 16.1 Hz), 5.82 (ddt,
1H, J = 6.27, 10.5, 16.9 Hz), 6.95–7.04 (m, 3H), 7.17 (s, 1H), 7.87
(s, 1H), 8.58 (s, 1H), 9.58 (s, 1H); 13C NMR (125 MHz, CDCl3) d
14.2, 25.8, 26.0, 29.5, 31.7, 37.3, 38.5, 60.4, 102.7, 108.0, 116.1,
116.3, 116.5, 119.5, 125.2, 136.4, 142.4,157.9, 159.8, 170.7; HRMS
390.2214 (D = 1.0 ppm). HPLC: t = 11.3 min, purity >94%.
5.27. 2-Cyclohexyl-6-(4-fluorophenoxy)-5-(4-methoxy-
benzamido)-1H-benzo[d]imidazole (5b)
(ESI) m/z calcd for
= 1.96 ppm). HPLC: t = 10.3 min, purity >97%.
C
24H26FN3O+2 408.2082 found: 408.2090
To a solution of 4d (100 mg, 0.31 mmol) in 6 mL of dichloro-
methane was added 4-methoxybenzoyl chloride (42 lL,
(D

B. Park et al. / Bioorg. Med. Chem. 22 (2014) 2602–2612
2611
5.31. 2-Cyclohexyl-5-(4-methxylbenzamido)-6-(phenylthio)-
1H-benzo[d]imidazole (5f)
previously. Briefly, stock solutions of the compounds were pre-
pared in DMSO and were serially diluted 2-fold in 96-well micro-
titer plates, and Mtb H37Rv 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.
Off-white solid; 79% yield; mp 184.5–186 °C; 1H NMR
(500 MHz, CDCl3) d 1.06–1.20 (m, 3H), 1.53–1.63 (m, 3H), 1.70 (d,
2H, J = 9.5 Hz), 2.00 (d, 2H, J = 11.3 Hz), 2.44 (s, 3H), 2.73 (t, 1H,
J = 9.6 Hz), 7.14 (t, 2H, J = 7.62 Hz), 7.22–7.27 (m, 4H), 7.59 (d,
2H, J = 8.0 Hz), 8.06 (s, 1H), 9.01 (s, 1H), 9.39 (s, 1H), 11.0 (s,
1H);13C NMR (125 MHz, CDCl3) d 21.5, 25.6, 25.9, 29.7, 31.6, 38.5,
103.3, 113.6, 126.0, 126.3, 126.4, 126.9, 129.0, 129.3, 129.6,
132.2, 134.2, 136.6, 142.6, 160.9, 166.1; HRMS (ESI) m/z calcd for
5.37. Cytotoxicity assay
The cytotoxicity of the compounds was tested against Vero cells
using MTT assay.21,24 The cells were grown in DMEM media sup-
plemented with 5% Bovine Serum and 1% Penn Strip and incubated
at 37 °C with 5% CO2. Then, 5000 cells were added to each well of a
C
26H34N3OS+ 436.2417 found: 436.2417
t = 4.8 min, purity >99%.
(D = 0.0 ppm). HPLC:
5.32. 6-(Benzylthio)-2-cyclohexyl-5-(4-tert-butylbenzamido)-
1H-benzo[d]imidazole (5g)
96-well plate in 200
for 1–2 days. A serial dilution of benzimidazoles 5 dissolved in
sterile DMSO was added to the 96-well plates in 200 L aliquots.
The plates were incubated at 37 °C for 3 days. The medium was
aspirated and then 40 L of 0.5 mg/mL MTT in DPBS was added
to each well. The plates were then incubated at 37 °C for 3 h. Then,
40 L of 0.8 M HCl solution were added to dissolve the remaining
crystals. Each experiment was run in triplicate. The optical density
data was used to calculate IC50 values using the Hill slope equation.
The IC50 values and their standard errors were calculated from the
viability-concentration curve using the Four Parameter Logistic
Model of Sigmaplot.
lL aliquots. The cells were incubated at 37 °C
l
Off-white solid; 92% yield; mp 173–173.5 °C; 1H NMR (500 MHz,
CDCl3) d 1.06–1.23 (m, 3H), 1.40 (s, 9 H), 1.51–1.59 (m, 3H), 1.68 (d,
2H, J = 11.3 Hz), 1.97 (d, 2H, J = 11.8 Hz), 2.67–2.72 (m, 1H), 3.90 (s,
2H), 6.98 (dd, 2H, J = 7.1, 2.3 Hz), 7.04 (dd, 3H, J = 5.0, 1.9 Hz), 7.52
(d, 2H, J = 8.4 Hz), 7.70 (d, 2H, J = 8.4 Hz), 7.96 (s, 1H), 8.93 (s, 1H),
9.38 (s, 1H), 11.1 (s, 1H); 13C NMR (125 MHz, CDCl3) d 25.6, 25.9,
31.2, 31.7, 35.1, 38.5, 43.4, 102.2, 116.4, 125.8, 126.9, 127.1, 127.3,
128.5, 132.3, 135.3, 138.0, 140.2, 155.5, 160.8, 165.8; HRMS (ESI)
l
l
m/z calcd for
= 0.0 ppm). HPLC: t = 13.8 min, purity >95%.
C
31H36N3OS+ 498.2574 found: 498.2574
(D
5.38. Mtb-FtsZ protein expression and preparation18,25
5.33. 6-(Butylthio)-5-(2,4-difluorobenzamido)-2-cyclohexyl-1H-
benzo[d]imidazole (5h)
Escherichia coli expression plasmid encoding the ftsz gene (pET
15b vector) was transformed into 100
transformed cells were plated onto LB plates, containing 100
l
L of BL21 (DE3) cells. The
Off-white solid; 93% yield; mp 170–170.5 °C; 1H NMR
(400 MHz, CDCl3) d 0.83 (t, 3H, J = 7.33 Hz), 1.17–1.29 (m, 3H),
1.36 (dd, 2H, J = 15.0, 7.37 Hz), 1.49–1.60 (m, 4H), 1.67 (d, 1H,
J = 12.1 Hz), 1.77 (d, 2H, J = 12.3 Hz), 2.06 (d, 2H, J = 11.9 Hz), 2.73
(t, 2H, J = 7.42 Hz), 2.79 (m, 1H), 6.96–7.09 (m, 2H), 7.93 (s, 1H),
8.23 (td, 1H, J = 8.85, 6.54 Hz), 8.87 (s, 1H), 10.1 (s, 1H), 10.3 (s,
1H); 13C NMR (100 MHz, CDCl3) d 13.6, 21.7, 25.7, 25.9, 31.3,
31.7, 37.0, 38.5, 104.7, 112.5, 112.7, 117.8, 118.3, 118.4, 133.8,
134.3, 160.5; HRMS (ESI) m/z calcd for C24H28F2N3OS+ 444.1916
lg/mL
ampicilin. The antibiotic concentration was kept the same for the
following steps. The plates were incubated overnight at 37 °C.
The colonies were picked and grown in 10 mL of LB media at
37 °C at 250 rpm. The inoculum was transferred to 1 L of LB media
in a 4 L flask and grown to an OD of 0.6 at A600. Then, 1 mM IPTG
was added to induce protein expression overnight at 20 °C at
250 rpm. Cells were then pelleted by centrifugation at 3000g, flash
frozen in liquid nitrogen, and stored at À80 °C until further purifi-
cation steps. Thawed cells were suspended in 40 mL 50 mM Tris
pH 7.5, 500 mM NaCl, 100 mM KCl, 0.1% NP-40 per liter cell culture
growth and passed through 3 rounds of cell disrupter (French
press) at 27 psi to disrupt cells. Lysed cells were centrifuged at
27,000g for 20 min to clear insoluble cellular components and cell
wall fractions.
For polymerization assay, protein was purified as follows.
Thawed cells were re-suspended in 40 mL of buffer containing
50 mM sodium phosphate pH 7.5, 300 mM sodium chloride,
10 mM imidazole, per liter cell culture growth and sonicated at
15 W 6 times for 30 s each with 1 min pauses in between to disrupt
cells. Lysed cells were centrifuged at 44,000g for one hour to clear
insoluble cellular components. Cleared lysate was applied to Ni+2
charged His-bind resin and washed with double the volume of
resuspension buffer of 50 mM sodium phosphate pH 7.5, 300 mM
sodium chloride, then double the volume of re-suspension buffer
of 50 mM sodium phosphate pH 7.5, 300 mM sodium chloride,
60 mM imidazole. FtsZ protein was eluted with 4 Â 5 mL portions
of 50 mM sodium phosphate pH 7.5, 300 mM NaCl, 500 mM imid-
azole. Protein was loaded onto a Sephadex G25 size exclusion col-
umn to remove excess imidazole and to exchange protein into
25 mM HEPES pH 7.2, 1 mM DTT, 0.1 mM EDTA, 10% glycerol (or
other buffer as indicated). Resulting protein fractions were pooled
and the N-terminal 6ÂHis affinity tag was removed by biotin
tagged thrombin treatment overnight at 4 °C (0.25 Units biotinyla-
ted thrombin per mg tagged FtsZ protein). Successive passes
found: 444.1922 (
D = 1.35 ppm). HPLC: t = 7.0 min, purity >97%.
5.34. 6-(Butylthio)-5-(4-metylbenzamido)-2-cyclohexyl-1H-
benzo[d]imidazole (5i)
Off-white solid; 34% yield; mp 155–156 °C; 1H NMR (400 MHz,
CDCl3) d 0.81 (t, 3H, J = 7.38 Hz), 1.05–1.15 (m, 3H), 1.24 (s, 1H),
1.34 (q, 2H, J = 7.34 Hz), 1.49–1.60 (m, 4H), 1.66 (d, 2H,
J = 12.1 Hz), 1.97 (d, 2H, 11.9 Hz), 2.46 (s, 3H), 2.71–2.75 (m, 3H),
7.36 (d, 2H, J = 7.79 Hz), 7.92 (d, 3H, J = 7.73 Hz), 8.99 (s, 1H),
9.84 (s, 1H), 11.4 (s, 1H); 13C NMR (100 MHz, CDCl3) d; 13.5,
21.5, 21.8, 25.5, 25.8, 29.6, 31.4, 31.6, 37.2, 38.4, 102.7, 117.1,
126.2, 127.0, 129.7, 132.3, 133.8, 135.2, 140.1, 142.6, 160.7,
165.7; HRMS (ESI) m/z calcd for C25H32N3OS+ 422.2261 found:
422.2265 (D = 0.9 ppm). HPLC: t = 5.2 min, purity >97%.
5.35. Bacterial strains and growth
For evaluation of drug sensitivity, Mtb H37Rv was grown in 7H9
media containing 10% oleic acid/albumin/catalase (OADC) enrich-
ment and 0.05% Tween-80 and assessed at mid log phase growth.
5.36. Antibacterial activity
The minimum inhibitory concentration (MIC) was determined
by the microplate Alamar Blue assay (MABA)19 as described

2612
B. Park et al. / Bioorg. Med. Chem. 22 (2014) 2602–2612
through streptavidin agarose and fresh Ni+2 charged His-bind resin
removed biotinylated thrombin, uncut FtsZ protein, and free cut off
affinity tag. A final cleanup was performed through an Akta driven
Sephadex 200 60/16 size exclusion column in 25 mM HEPES pH
7.2, 1 mM DTT, 0.1 mM EDTA, 10% glycerol or other buffer as indi-
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. Digital
images were acquired with an AMT XR-60 CCD digital camera
system.18
cated. Protein was then concentrated to 10 mg/mL (ꢀ250
centrifugal 30 kDa molecular weight cutoff filters, aliquoted
150
L, flash frozen in liquid nitrogen, and stored at À80 °C.
lM) with
5.41. Binding studies of 5a with Mtb-FtsZ
l
For Kd studies the protein was purified as follows. Cleared lysate
was applied to Ni+2 charged His-bind resin and made to equilibrate
for one hour followed by washing in two volumes of wash buffer
(50 mM Tris pH 7.5, 300 mM NaCl, 100 mM KCl, 0.1% NP-40, and
10 mM Imidazole). Bound protein was eluted with 10 mL of Elution
buffer (50 mM Tris pH 7.5, 500 mM NaCl, 100 mM KCl, and
500 mM imidazole). The eluted protein was first dialyased against
2 L Storage buffer (50 mM Tris pH 7.8, 200 mM NaCl, 100 mM KCl),
overnight followed by 3 h dialysis against Storage buffer with 10%
glycerol. Post dialysis, the concentration of the protein was
checked by Bradford assay and purity of the purified protein deter-
mined by SDS page. If necessary, protein was further concentrated
The fluorescence anisotropy of the compound was measured in
the presence of increasing FtsZ concentrations, in 50 mM MES,
100 mM KCl, 5 mM MgCl2 in absence of GTP by exciting the com-
pound at 316 nm and monitoring the change of fluorescence at
427.9 nm using a PTI-QM4 spectrofluorometer. The change in fluo-
rescence
F = (
(DF) at 427.9 nm was fitted into the equation
D
D
Fmax  L)/(Kd + L).23
Acknowledgments
This research was supported by Grants from the National Insti-
tutes of Health (Grant AI078251 to I.O.) and (U01082164 to R.A. S.).
The authors gratefully acknowledge the technical support of Dr.
Susan Van Horn for TEM operation at the Microscopy Imaging Cen-
ter at Stony Brook University, NY
to 10 mg/mL (ꢀ250
l
M) with centrifugal 10 kDa molecular weight
cutoff filters, aliquoted 500
l
L, and flash frozen in liquid nitrogen,
and stored at À80 °C till further use.
For TEM analysis the following procedure was followed for pro-
tein purification. The cells were suspended in approximately
20–30 mL binding buffer (500 mM NaCl, 20 mM sodium phos-
phate, pH 7.8) and lysed using cell disruptor. The lysate was centri-
fuged in an ultracentrifuge at 126,603g, 4 °C for 90 min. The
supernatant was filtered and loaded onto Ni2+-NTA column,
washed with 50 mL of binding buffer and eluted using a gradient
of binding buffer with 30–500 mM imidazole. The eluted protein
was dialyzed against buffer containing 50 mM Tris, 5 mM MgCl2,
50 mM KCl, pH 7.2 followed by buffer containing 10% v/v glycerol.
The protein after dialysis was concentrated and stored at À80 °C
for further use. Since the number of aromatic residues in Mtb-FtsZ
protein are low (Tyr: 1, Trp: 0), it is not reliable to follow concen-
tration of protein by scanning at A280. The concentration of pro-
tein was therefore ascertained using the Bradford kit from Sigma.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.03.035.
References and notes
1. World Health Organization Global Tuberculosis Report 2013, http://apps.who.int/
iris/bitstream/10665/75938/10661/9789241564502_eng.pdf.
2. Raviglione, M. C. Scott. Med. J. 2000, 45, 52.
3. Gandhi, N. R.; Moll, A.; Sturm, A. W.; Pawinski, R.; Govender, T.; Lalloo, U.;
Zeller, K.; Andrews, J.; Friedland, G. Lancet 2006, 368, 1575.
4. Huang, Q.; Tonge, P. J.; Slayden, R. A.; Kirikae, T.; Ojima, I. Curr. Top. Med. Chem.
2007, 7, 527.
5. Kumar, K.; Awasthi, D.; Berger, W. T.; Tonge, P. J.; Slayden, R. A.; Ojima, I. Future
Med. Chem. 2010, 2, 1305.
6. van den Ent, F.; Amos, L.; Lowe, J. Curr. Opin. Microbiol. 2001, 4, 634.
7. Ben-Yehuda, S.; Losick, R. Cell 2002, 109, 257.
5.39. Mtb-FtsZ polymerization inhibitory assay
8. Bi, E. F.; Lutkenhaus, J. Nature 1991, 354, 161.
The inhibitory activity of lead benzamidazoles for Mtb-FtsZ
polymerization was determined by means of light scattering on a
PTI-QM4 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 875 V. Mtb-
9. Errington, J.; Daniel, R. A.; Scheffers, D. J. Microbiol. Mol. Biol. Rev. 2003, 67, 52.
10. Slayden, R. A.; Knudson, D. L.; Belisle, J. T. Microbiology 2006, 152, 1789.
11. Respicio, L.; Nair, P. A.; Huang, Q.; Anil, B.; Tracz, S.; Truglio, J. J.; Kisker, C.;
Raleigh, D. P.; Ojima, I.; Knudson, D. L.; Tonge, P. J.; Slayden, R. A. Tuberculosis
(Edinb) 2008, 88, 420.
12. Romberg, L.; Levin, P. A. Annu. Rev. Microbiol. 2003, 57, 125.
13. White, E. L.; Suling, W. J.; Ross, L. J.; Seitz, L. E.; Reynolds, R. C. J. Antimicrob.
Chemother. 2002, 50, 111.
14. Reynolds, R. C.; Srivastava, S.; Ross, L. J.; Suling, W. J.; White, E. L. Bioorg. Med.
Chem. Lett. 2004, 14, 3161.
15. Sarcina, M.; Mullineaux, C. W. FEMS Microbiol. Lett. 2000, 191, 25.
16. Awasthi, D.; Kumar, K.; Ojima, I. Expert. Opin. Ther. Pat. 2011, 21, 657.
17. Awasthi, D.; Kumar, K.; Knudson, S. E.; Slayden, R. A.; Ojima, I. J. Med. Chem.
2013, 56, 9756.
FtsZ (15
MES pH 6.5, 100 mM KCl, 5 m M MgCl2) for up to 300 s. Polymeri-
zation was initiated with 100 M GTP and monitored for up to
lM) was incubated in the polymerization buffer (50 mM
l
30 min. Benzimidazole stocks were prepared in DMSO and incu-
bated with FtsZ enzyme prior to initiation of polymerization with
GTP.
18. Kumar, K.; Awasthi, D.; Lee, S. Y.; Zanardi, I.; Ruzsicska, B.; Knudson, S.; Tonge,
P. J.; Slayden, R. A.; Ojima, I. J. Med. Chem. 2011, 54, 374.
19. Collins, L.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997, 41, 1004.
20. Li, L.; Liu, G.; Wang, Z.; Yuan, Y.; Zhang, C.; Tian, H.; Wu, X.; Zhang, J. J. Comb.
Chem. 2004, 6, 811.
5.40. Transmission electron microscopy (TEM) analysis
Stock solution of compound 5a was prepared in ethanol. Mtb-
FtsZ (5 lM) was incubated with 40 or 80 lM of 5a in the polymer-
ization buffer (50 mM MES, 5 mM MgCl2, 100 mM KCl, pH 6.5) for
15 min on ice. To each solution was added GTP to the final concen-
tration of 25 lM. The resulting solution was incubated at 37 °C for
30 min. The incubated solution was diluted 2 times with the
polymerization buffer and immediately transferred to carbon
21. Mosmann, T. J. Immunol. Methods. 1983, 65, 55.
22. Mukherjee, A.; Lutkenhaus, J. J. Bacteriol. 1999, 181, 823.
23. Kelley, C.; Zhang, Y.; Parhi, A.; Kaul, M.; Pilch, D. S.; LaVoie, E. J. Bioorg. Med.
Chem. 2012, 20, 7012.
24. Chen, S.; Zhao, X.; Chen, J.; Chen, J.; Kuznetsova, L.; Wong, S. S.; Ojima, I.
Bioconjug. Chem. 2010, 21, 979.
25. White, E. L.; Ross, L. J.; Reynolds, R. C.; Seitz, L. E.; Moore, G. D.; Borhani, D. W. J.
Bacteriol. 2000, 182, 4028.