Full text of DOI:10.1039/d0md00256a

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RESEARCH ARTICLE
Structure–activity relationship studies on 2,5,6-
trisubstituted benzimidazoles targeting Mtb-FtsZ
as antitubercular agents†
Cite this: DOI: 10.1039/
d0md00256a
ab
b
b
b
b
Krupanandan Haranahalli, Simon Tong, Saerom Kim, Monaf Awwa, Lei Chen,
c
c
d
d
Susan E. Knudson, Richard A. Slayden, Eric Singleton, Riccardo Russo,
Nancy Connell and Iwao Ojima
de
ab
*
Filamenting temperature sensitive protein Z (FtsZ) is an essential bacterial cell division protein and a
promising target for the development of new antibacterial therapeutics. As a part of our ongoing SAR
studies on 2,5,6-trisubstituted benzimidazoles as antitubercular agents targeting Mtb-FtsZ, a new library of
compounds with modifications at the 2 position was designed, synthesized and evaluated for their activity
Received 22nd July 2020,
Accepted 14th September 2020
against Mtb-H37Rv. This new library of trisubstituted benzimidazoles exhibited MIC values in the range of
−1
0
.004–50 μg mL . Compounds 6b, 6c, 20f and 20g showed excellent growth inhibitory activities ranging
−
1
from 0.004–0.08 μg mL . This SAR study has led to the discovery of a remarkably potent compound 20g
DOI: 10.1039/d0md00256a
−
1
−1
(
MIC 0.0039 μg mL ; normalized MIC 0.015 μg mL ). Our 3DQSAR model predicted 20g as the most
rsc.li/medchem
potent compound in the library.
drugs are associated with safety concerns. Bedaquiline and
delamanid are known to cause QT prolongation, which if left
Introduction
9
,10
Tuberculosis (TB) an infectious disease caused by
unmonitored can lead to cardiac arrest.
It is imperative that
Mycobacterium tuberculosis (Mtb), was one of the top 10 killers
patients receiving either of these two medications need to
monitored very closely and for the same reason, their
combined use has not yet been recommended by WHO.
1
,2
claiming 1.6 million lives in 2017 alone.
estimated 10 million new TB cases were reported in 2017. TB
being the most common opportunistic infection in HIV-
Globally, an
1
10
Pretomanid is associated with side effects such as nerve
1
,3
11
positive patients, resulted in 0.3 million deaths in 2017.
damage, hypoglycemia and lower respiratory tract infections.
Nearly one fourth of the world's population is afflicted with
Pretomanid has been approved by FDA only in cases of
pulmonary XDR-TB and treatment intolerant or non-responsive
MDR-TB in combination with other anti-TB drugs.
4
latent TB. Once an individual is infected with TB, there is a
1
12
1
0% probability of developing an active infection.
With the increase in emergence of drug resistant strains of
With the increase in the number of cases of multidrug-
resistant TB (MDR-TB) and extensively drug-resistant TB
(XDR-TB) and new drugs displaying life threatening side
effects, there is a dire need for the development of new anti-
tubercular agents with novel targets, that would not only
make it difficult for the bacteria to develop resistance, but are
also safe. In this context, filamentous temperature sensitive
TB, there is an urgent need for newer drugs with novel
5
mechanisms of action. In the past decade, 3 new drugs
bedaquiline, delamanid and pretomanid have been approved
6–8
to treat resistant forms of TB.
Although recently there has
been progress in antitubercular drug discovery, the newer
protein Z (FtsZ), a bacterial cell division protein seems to be a
a
13,14
Institute of Chemical Biology and Drug Discovery, Stony Brook University, Stony
promising target.
FtsZ, a guanosine triphosphate (GTP)
Brook, NY, USA. E-mail: iwao.ojima@stonybrook.edu
b
dependent prokaryotic cell division protein, is a homolog of
Department of Chemistry, Stony Brook University, Stony Brook, NY, USA
1
5
c
eukaryotic tubulin. In the presence of GTP, FtsZ first
polymerizes into protofilaments which then assemble to form
Department of Microbiology, Immunology and Pathology, Colorado State
University, Fort Collins, Colorado 80523-1682, USA
d
16,17
Department of Medicine, Center for Emerging and Re-emerging Pathogens, Rutgers
a highly dynamic Z-ring at the center of the bacterial cell.
University, Newark, New Jersey, 07103, USA
e
With the aid of other cell division proteins the Z-ring then
Department of Physiology, Rutgers University, Newark, New Jersey, 07103, USA
18
forms a septum, which eventually results in cytokinesis.
†
Electronic supplementary information (ESI) available: Synthetic procedures
1
13
From a drug discovery perspective, targeting FtsZ offers
several advantages. First, FtsZ is highly conserved among
and characterization data for all key synthetic intermediates; H and C NMR
spectra of all new 2,5,6-trisubstituted benzimidazoles. See DOI: 10.1039/
d0md00256a
1
9
prokaryotes. Thus, developing agents that target FtsZ could
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potentially lead to broad spectrum antibiotics. Second, FtsZ
and tubulin share only ∼7% sequence identity but high
20,21
structural similarity.
This would help design inhibitors
that specifically target FtsZ and reduce the chances of toxicity
to mammalian cells. Lastly, since there are no FtsZ inhibitors
as drugs in the market, there are less chances of cross
resistance within the drug resistant bacterial species that
2
2
already exist.
Libraries of 2,5,6- and 2,5,7-trisubstituted benzimidazoles
have been designed and synthesized as Mtb-FtsZ inhibitors in
our laboratories, and evaluated for their antitubercular (anti-
1
3,14,23
TB) activities against Mtb-H37Rv strain.
In general,
2
,5,6-trisubstituted benzimidazoles exhibited better anti-TB
1
4
activities than the 2,5,7-trisubstituted counterparts. Some
of the lead 2,5,6-trisubstituted benzimidazoles, including SB-
P17G-C2, SB-P17G-A20, SB-P17G-A38 and SB-P17G-A42
−
1
(Fig. 1), exhibited excellent MIC values (0.06–0.31 μg mL )
Fig. 2 New 2,5,6-trisusbstituted benzimidazoles in the present SAR study.
1
3,24
against Mtb H37Rv.
Selected lead compounds were found
to inhibit the assembly of Mtb FtsZ in a dose dependent
1
3,14
manner by increasing the GTPase activity.
When
different acyl chlorides, hydroxysuccinimide esters and
chloroformates (1.1 eq.) in dichloromethane in 96 well plates.
The plates were gently shaken at room temperature for 24 h,
followed by treatment with aminomethylated polystyrene
resin EHL/2% DVB (200–400 mesh) (10 equiv.) in order to
scavenge any unreacted acyl chlorides, succinate esters or
chloroformates. Then, the reaction mixture was filtered to give
245 novel 2,5,6-trisubstituted benzimidazoles, 5 and 6. In
the case of trisubstituted benzimidazoles with N,N-
dialkylaminomethyl substitution at the 2-position (Scheme 2),
commercially available 2,4-dinitro-5-fluoroaniline (1) was
subjected to nucleophilic aromatic substitution with
dimethylamine to give 5-dimethylaminodinitroaniline (2) in
evaluated their efficacies in vivo against immune
compromised GKO mice, SB-P17G-A38 and SB-P17G-A42
reduced the bacterial load in the lung by 5.7–6.3 log10 CFU
2
4
and in the spleen by 3.9–5.0 log10 CFU, respectively.
Our previous structure activity relationship (SAR) studies
on these 2,5,6-trisubstituted benzimidazoles for optimization
of potency and metabolic properties were conducted on the 5
and 6 positions, wherein a cyclohexyl group was kept at the 2
position intact. In our present SAR study, we fixed the 6
position as a dimethylamino group and investigated the
effects of various substituents at the 2 and 5 positions on the
potency and pharmacological properties (Fig. 2).
9
1
7% yield. Acylation of 2 with chloroacetyl chloride afforded
-chloromethylcarboxamido-5-N,N-dimethylamino-2,4-
Results and discussion
Chemical synthesis
dinitrobenzene (7) in 91% yield. Nucleophilic substitution of 7
with a variety of dialkylamines afforded compounds 8a–e in
71–93% yields. This was followed by one-pot reduction and
cyclization using stannous chloride dihydrate and 4 M
hydrochloric acid to give the key intermediates 9a–e. Then,
5-aminobenzimidazoles 9a–e were converted to either
benzamides 10 or carbamates 11. For the synthesis of
trisubstituted benzimidazoles with 2-sulfanyl substitution
Synthesis of 2,5,6-trisubstituted benzimidazoles with
modifications at the 2 position are outlined in Schemes 1–3.
Compounds shown in Scheme
the method previously
-aminobenzimidazoles 4 were further derivatized with 35
1 were synthesized by
1
3,14
described.
The
5
(Scheme
3),
commercially
available
1,2-diamino-4-
fluorobenzene 12 was protected, using p-toluenesulfonyl
chloride to yield 13 in 77% yield, and the subsequent nitration
of 13 afforded 14 in 52% yield. Aromatic nucleophilic
substitution of 14 with dimethylamine gave 15. Deprotection of
the tosyl groups in 15 afforded 1,2-diamino-4-dimethylamino-5-
nitrobenzene (16) in 69% yield for two steps. The reaction of 16
with carbon disulfide to give 6-dimethylamino-5-nitro-2-thio-
1
H-benzo[d]imidazole (17) in 76% yield. Compound 17 was
alkylated, using a variety of alkyl or aromatic halides, to afford
8a–g in 31–84% yields. The reduction of 18a–g with stannous
1
chloride dihydrate gave 19a–g in 44–89% yields. Finally, the
amine moiety of 19a–g was converted to the corresponding
n-butyl carbamates 20a–g in 61–77% yields.
Fig.
1
Lead
compounds
arising
from
3,24
2,5,6-trisubstituted
1
benzimidazole libraries previously studied.
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Scheme 1 Synthesis of 2,5,6-trisusbstituted benzimidazoles 5 and 6. Reagents and conditions: a) dimethylamine, DIPEA, THF, 2 h, room
1
1
temperature (rt); b) R COCl, pyridine, reflux, overnight; c) R COOH, PCl
3
, acetonitrile, reflux, overnight; d) SnCl
2
·2H
2
O, 4 M HCl, EtOH, reflux, 4 h;
2
2
3 2 2
2
2
e) R COCl, Et
aminomethylated polystyrene resin, DCM, RT, 6 h; h) (i) 1,1′-carbonyldiimidazole, CH
N, 0 °C to rt, 4 h; f) R COOH, EDC.HCl, DMAP, CH Cl , reflux, overnight; g) (i) R C(O)Cl or R (O)Su, DCM, RT, 24 h; (ii)
2
Cl
2
, reflux, 4 h; (ii) n-butanol, CH
2
Cl
2
, reflux, overnight.
−
1
25,26
Evaluation of antibacterial activities against Mtb H37Rv and
SAR analysis
mL using microplate alamar blue assay (MABA).
Out of
−
1
245 compounds, 53 of them showed MIC ≤ 5 μg mL .
Furthermore, when 37 out of the 53 hits were resynthesized
in analytically pure form and examined for their accurate
The library of 2,5,6-trisubstiutted benzimidazoles 5 and 6
(245 compounds) were screened against Mtb H37Rv at 5 μg
Scheme 2 Synthesis of 2,5,6-trisusbstituted benzimidazoles 10 and 11. Reagents and conditions: a) dimethylamine, DIPEA, THF, 2 h, rt; b)
chloroacetyl chloride, TEA, CH Cl , 0 °C to rt, overnight; c) dialkylamine, DIPEA, THF, reflux, overnight; d) SnCl ·2H O, 4 M HCl, EtOH, reflux, 4 h;
N, 0 °C to rt, 4 h; f) R COOH, EDC.HCl, DMAP, CH , reflux, overnight; g) (i) 1,1′-carbonyldiimidazole, CH Cl , reflux, 4 h; (ii)
Cl , reflux, overnight.
2
2
2
2
2
2
e) R COCl, Et
n-Butanol, CH
3
2
Cl
2
2
2
2
2
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Scheme 3 Synthesis of 2,5,6-trisusbstituted benzimidazoles 20. Reagents and conditions: a) p-toluenesulfonyl chloride, pyridine, 85 °C, overnight;
b) fuming nitric acid, acetic acid, 65 °C, 40 minutes; c) dimethylamine, TEA, THF, 50 °C, 48 h; d) H SO , water, 85 °C, 1 h; e) carbon disulfide, TEA,
MeOH, THF, 50 °C, 4 h; f) R–X, KOH, water, EtOH, rt, overnight; g) Ar–I, K CO , CuI, 1,10-phenanthroline, DMF, 140 °C, 22 h, pressure vessel; h)
SnCl ·2H O, 4 M HCl, EtOH, reflux, 3 h; i) (i) 1,1′-carbonyldiimidazole, CH Cl , reflux, 4 h; (ii) n-butanol, CH Cl , reflux, overnight.
2
4
2
3
2
2
2
2
2
2
MIC values, there were a few compounds that exhibited
Next, we examined the effect of N,N-dialkylaminomethyl
substitution at the 2-position. All the compounds were
−
1
higher MIC values than the initial cut off of 5 μg mL . This
could be attributed to the fact that the actual weight and
purity of test compounds are not very accurate in a 96-well
plate and could also be due to false positives in high
evaluated for their activity against Mtb H37Rv using
2
6,27
MABA.
Compounds with an N,N-methyl(phenyl)
aminomethyl group at the 2 position (10e-1–10e-5, 11e) were
far less active compared to ones with a pent-3-yl group
with an exception of 10e-6. Compound with N,N-
dipropylaminomethyl (11a) showed a high activity (MIC 0.16
throughput (HTP) screening. As Table
1 shows, the
resynthesized compounds showed MIC values ranging from
−
1
0
.078 to 50 μg mL [note: MIC values were determined by
−
1
two different laboratories using MABA with slightly different
protocols, “A” and “B”. Thus, Table 1 shows normalized MIC
values with asterisks and also lists the original values in
parentheses with superscript “b” marks].
μg mL ). Morpholin-4-ylmethyl (11b) and pyrrolidin-1-
ylmethyl (11d) groups at the position resulted in
2
diminished activities. 2,5,6-Trisubstituted benzimidazole with
a n-butoxycarbonylamino group at the 5 position and a
piperidin-1-ylmethyl group at the 2 position (11c) showed a
Preliminary SAR of the hits from the library indicated that
cyclopentyl, pent-3-yl, isopropyl and benzyl substituents at
the 2 position were preferred. None of the compounds
bearing a phenyl or 2-furyl substituents at the 2 position
made the cut-off line in the initial screening. Although there
was one hit with thien-2-yl substituent at the 2 position, this
compound 6f showed poor cell growth inhibitory activity
−
1
high activity (MIC 0.09 μg mL ; normalized MIC 0.14 μg
−
1
mL ).
In order to further diversify our library, a small series of
2,5,6-trisusbstituted benzimidazoles with alkyl/arylsulfanyl
groups at the 2 position and n-butyl carbamate at the 5
position were designed and synthesized. All these sulfide-
−
1
(
MIC >10 μg mL ) when assayed with the resynthesized
containing compounds were evaluated for their activities
3
26,27
compound. These results indicate that an sp hybridized
carbon is preferred over an sp carbon at the 2 position.
against Mtb H37Rv using MABA.
Results are summarized
2
in Table 2.
Also, compounds bearing substituted benzamides at the 5
position fared better than their aliphatic amide counter
parts.
Compounds 20b and 20e, bearing a benzylsulfanyl and a
phenylsulfanyl groups, respectively, exhibited good activity
−
1
(MIC 0.31 μg mL ). In the 2-alkylsulfanyl series of analogs,
the size of alkyl groups is critical to their activities. Thus,
cyclohexyl analog 20f exhibited high activity (MIC <0.0081 μg
Compounds bearing a cyclopentyl substituent at the 2
position (5a-1–5a-8, 6a exhibited good inhibitory activity. The
MIC values of trisubstituted benzimidazoles with pent-3-yl
substituent at the 2 position (5c-1–5c-7, c6b) were comparable
to the previously reported compounds bearing a cyclohexyl
−
1
−1
mL ; normalized MIC <0.031 μg mL ) and isopropyl analog
−
1
20c retained good activity (MIC 0.31 μg mL ), while ethyl
analog 20d displayed moderate activity (normalized MIC 1.2
μg mL ), a significant decrease in activity was observed for
the methyl analog 20a (normalized MIC 9.6 μg mL ). The
introduction of a methylene group between the sulfur and
cyclohexyl unexpectedly increased the activity. Thus,
cyclohexylmethylsulfanyl analog 20g exhibited remarkably
enhanced activity (MIC 0.0039 μg mL ; normalized MIC
0.015 μg mL ), which marks the highest inhibitory activity
so far to date.
1
3
−1
substituent at the 2 position. These results suggest that
pent-3-yl group is a close mimic of the cyclohexyl group at
the 2 position. Compounds with isopropyl substitution at the
−
1
2
position (-15d, 6d) were found to be less active than the
ones with pent-3-yl substituent, suggesting the importance of
the carbon chain length. In the case of 4-tetrahydropyranyl
substituent at the 2 position (5h-1, 5h-2 and 6h), there was a
significant loss of inhibitory activity when compared to the
cyclohexyl counterparts, indicating that the presence of an
oxygen atom within the cyclohexyl moiety is unfavorable.
−
1
−
1
Cytotoxicity in human cells (MTT assay). The cytotoxicity
of top 10 compounds was evaluated against two human cell
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−1
Table 1 Anti-TB activity of 2,5,6-trisusbstituteid benzimidazoles 5, 6, 10 and 11 against Mtb H37Rv (MIC μg mL )
1
2
a
1
2
a
Compound
SB-P17G-C2
R
R
MIC
Compound
R
R
MIC
0.06
(
0
5c-3
1.56
b
0.008 )
c
.064
SB-P17G-A20
SB-P17G-A38
SB-P17G-A42
0.16
5c-4
5c-5
5c-6
0.50
0.71
0.78
0.16
b
(
0
0.019 )
.15
c
0.16
1.56
0.78
5
5
a-1
a-2
5c-7
5d-1
0.31
0.78
5
a-3
a-4
1.25
12.5
5h-1
5h-2
>10
5
10
5a-5
5a-6
5a-7
5a-8
0.625
0.625
0.78
6a
6b
6c
6d
0.31
<0.078
0.078
1.25
1.56
5
5
5
a-9
50
6f
>10
>2.5
2.5
a-10
a-11
>10
>50
6h
10e-1
5
5
b-1
b-2
1.25
0.33
10e-2
10e-3
1.25
1.25
5b-3
1.25
10e-4
>10
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Table 1 (continued)
1
2
a
1
2
a
Compound
R
R
MIC
0.63
Compound
R
R
MIC
5
5b-4
5b-5
5b-6
10e-5
c
1.56
0.63
10e-6
11a
0.29
b
(
0.19 )
0.16
c
5b-7
1.25
11b
2.4
b
(
1.6 )
c
5b-8
5b-9
5c-1
>10
>10
1.56
11c
11d
11e
0.14
(
b
0.09 )
c
2.4
b
(
1.6 )
0.16
5c-2
12.5
a
MIC: minimum concentration of the compound required to inhibit growth of 99% of Mtb H37Rv cells. MIC determined by protocol A. See
b
c
Experimental section. MIC determined by protocol B. See Experimental section. Normalized MIC from the MIC determined by protocol B to
the MIC by protocol A.
2
9
lines, WI38 (normal lung fibroblast) and HepG2 (liver
cancer). The level of cytotoxicity was determined as%
inhibition of cell growth, which gave sufficient information
about potential systemic toxicity. Results are summarized in
Table 3. The IC50 of 20g appears to be very close to 20 μM
since it shows 52% of cell growth inhibition against WI38
reported by Li et al. in 2016, which used 67 compounds in
1
3
our publication. Since we have investigated a more diverse
library of 2,5,6-trisubstituted benzimidazoles and obtained
relevant data, we expanded the database to 148 compounds
for building to a more robust 3D-QSAR model by building
upon Li′s model. First, Li′s model was reproduced and
2
2
human normal cell line. Thus, the therapeutic window (IC50
/
confirmed with the 67 compounds he used (R = 0.90; Q =
MIC) of 20g is >100. In comparison, 20f is considerably more
cytotoxic (87% inhibition at 20 μM) than 20g. Also, it is
interesting that 20g did not show any cytotoxicity against
HepG2 human liver cancer cell line at 20 μM concentration.
The least cytotoxic compound is 11a (only 4% inhibition at
0.97; RMSE = 0.19) using Schrödinger's AutoQSAR program.
Then, we expanded it to 148 compounds, which also gave
excellent results (R = 0.90; Q = 0.90; RMSE = 0.21) as
illustrated in Fig. 3.
2
2
Next, we examined the validity of this 3D-QSAR model by
applying it for the prediction of the MIC values of the top 12
and other two new benzimidazoles in the present work, as
well as four of the lead compounds from our previously
published work. The results are summarized in Table 4. As
Table 4 shows, the predicted MIC values are all very close to
those determined experimentally, except for 20f (entry 6).
It should be noted that the most potent compound, 20g,
in this series, is actually predicted as the best compound
(entry 5). The results clearly indicate that the prediction
2
0 μM), while 11e is most cytotoxic (96% inhibition) followed
by 6b (93% inhibition). It is interesting to note that 11a and
1c did not inhibit the cell growth of HepG2 cells, but even
1
enhanced the cell growth at 20 μM.
Computational analysis of SAR and toxicity prediction
3
D-QSAR analysis. The first 3D-QSAR study on 2,5,6-
trisubstituted benzimidazole series was conducted and
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Table 2 Anti-TB activity of 2-alkyl/arylsulfanylbenzimidazoles 20 against
Mtb H37Rv (MIC μg mL )
2
2
0e, 20f and 20g) that were highly potent. Overall SAR of
,5,6-trisusbstituted benzimidazoles is summarized in Fig. 4.
−1
We built a robust 3DQSAR model by following up Li's
2
9
model, and used this updated model to predict the MIC of
the top 13 compounds. Since almost all of the predicted MIC
values are very close to those determined experimentally, this
model can be used for the selection of promising compounds
from the designed compounds in silico for chemical synthesis
and biological evaluations in the future. Compound 20g was
found to be the most potent compound (MIC 0.0039 μg
1
a
Compound
SB-P17G-C2
R
MIC
0.06
0.008 )
b
(
(
c
2
0a
0b
9.6
1.28 )
0.31
−
1
−1
b
mL ; normalized MIC 0.015 μg mL ) from the current
series, both experimentally and computationally from the
2
3DQSAR model. Not only was 20g the most potent compound
2
0c
0.31
of the series, but also it was more potent than the previous
−
1
lead SB-P17G-C2 (MIC 0.06 μg mL ). Also, the cytotoxicity
IC ) of 20g is found to be ca. 20 μM, which indicates the
c
2
0d
0e
1.2
(
5
0
b
(
0.16 )
therapeutic index (IC /MIC) of 20g is >100. Based on our
5
0
2
0.31
current
results,
systematic
SAR
study
on
2
-alkylsulfanylbenzimidazoles with varying substituents at
c
2
2
0f
<0.031
(
b
the 5 and 6 positions is clearly warranted.
<0.0081 )
c
0g
0.015
b
Experimental section
(
0.0039 )
General methods
a
b
MIC determined by protocol A. See Experimental section. MIC
c
Melting points were measured on a Thomas Hoover Capillary
melting point apparatus and are uncorrected. NMR spectra
determined by protocol B. See Experimental section. Normalized
MIC from the MIC determined by protocol B to the MIC by protocol
A.
were recorded on
operating at 700 MHz for H and 175 MHz for C, a Bruker
00 Advance spectrometer operating at 500 MHz and 125
a Bruker Ascend 700 spectrometer
1
13
5
1
13
based on the AutoQSAR model developed in the present work
can be used for the selection of promising compounds from
the designed compounds in silico for chemical synthesis and
biological evaluations. Table 4 also lists calculated log P
values using the ChemDraw Pro program. All compounds
show the Clog P values of 3–5, which are in a good range in
MHz for H and C, respectively, or a Bruker 400 Nanobay
spectrometer operating at 400 MHz, 100 MHz, and 376 MHz
1
13
19
for H, C, and F, respectively or a Bruker 300 Nanobay
1
spectrometer operating at 300 MHz of H. Chemical shifts
were referenced to the residual proton and carbon-13 peaks
1
13
1
of solvents used for H and C NMRs, respectively ( H:
3
0
13
1
13
the Lipinski's rule of 5.
3 3 6
CDCl , δ 7.26; C: CDCl , δ 77.23; H: DMSO-d , δ 2.50; C:
We also carried out computational analysis of toxicity
prediction using pKCSM program (Table S1†), as well as
docking analysis of compound 20g in the putative binding
site of Mtb FtsZ (Fig. S1†). This information is available in
the ESI.†
DMSO-d , δ 39.51). Signals are listed in ppm, and multiplicity
6
identified as s (singlet), bs (broad singlet), d (doublet), dd
(doublet of doublets), t (triplet), q (quartet), m (multiplet); J -
coupling constants in Hz, and integration. Low resolution
mass spectrometry (LRMS) analysis was carried out on an
Agilent LC-MSD mass spectrometer and high resolution mass
spectrometry (HRMS) analysis was carried out on an Agilent
LC-UV-TOF mass spectrometer at the Institute of Chemical
Biology and Drug Discovery, Stony Brook. Purity of the
synthesized compounds was determined by a Shimadzu LC-
2010A HT series HPLC assembly or Agilent 1100 series HPLC
assembly. Four analytical conditions were used and noted as
Conclusions
A library of 2,5,6-trisubstituted benzimidazoles with a variety
of modifications at the 2 position were synthesized and
tested for their antitubercular activities against Mtb H37Rv.
Among the compounds with different functional groups at
the 2 position, those having a butyl carbamate at the 5
position resulted in the most active compound within each
series (6a, 6b, 6c, 6d, 11e), following a trend similar to our
previous series of 2,5,6-trisubstituted benzimidazoles with a
cyclohexyl substitution at the 2 position. Functional groups
a
part of the characterization data for synthesized
compounds. HPLC (1): adsorbosphere silica 5 μm, 250 mm Å
∼ 4.6 mm column, isopropanol and hexanes, flow rate of 1
−
1
mL min , t = 0–40 min, gradient of 5–50% isopropanol. (2):
Kinetex PFP, 2.6 μm, 4.6 mm Å ∼ 100 mm column,
3
−1
with an sp carbon at the 2 position were preferred over ones
isopropanol and hexanes, flow rate of 0.3 mL min ,, t = 0–25
2
with an sp carbon. From this series, we identified 13
min, gradient of 5–95% isopropanol. (3): Kinetex PFP, 2.6
μm, 4.6 mm Å ∼ 100 mm column, isopropanol and hexanes,
compounds (5c-7, 6a, 6b, 6c, 10e-6, 11a, 11c, 11e, 20b, 20c,
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Table 3 Cytotoxicity (% inhibition at 20 μM) of select 10 compounds with high potency
1
2
a
−1
d
d
Entry
1
Compound
R
R
MIC (μg mL
)
WI38 % inhibition at 20 μM
HepG2 % inhibition at 20 μM
SB-P17G-A38
0.16
78
57
c
2
3
4
5
20g
20f
6b
0.015
(
52
87
93
77
0
b
0.0039 )
a,c
<0.031
50
80
32
b
(
<0.0081 )
<0.078
6c
0.078
c
e
6
7
11c
11a
0.14
(
60
0
b
0.09 )
e
0.16
4.0
0
8
9
11e
0.16
0.29
96
59
54
30
10e-6
10
6a
0.31
86
42
a
b
c
MIC determined by protocol A. See Experimental section. MIC determined by protocol B. See Experimental section. Normalized MIC from
d
e
the MIC determined by protocol B to the MIC by protocol A. Average value of MTT assay in triplicate. Enhancement of cell growth was
observed.
−
1
flow rate of 0.3 mL min , t = 0–20 min, gradient of 5–80%
isopropanol. (4): Kinetex PFP, 2.6 μm, 4.6 mm Å ∼ 100 mm
Materials
−
1
column, isopropanol and hexanes, flow rate of 0.3 mL min ,
t = 0–30 min, gradient of 5–80% isopropanol. Measurements
were made at 254 and 303 nm.
All air- and moisture-insensitive reactions were carried out
under an ambient atmosphere, magnetically stirred, and
monitored by thin layer chromatography (TLC) using Agilent
Technologies TLC plates pre-coated with 250 μm thickness
silica gel 60 F254 plates and visualized by fluorescence
quenching under UV light. Flash chromatography was
performed on SiliaFlash® Silica Gel 40–63 μm 60 Å particle
size using a forced flow of eluent at 0.3–0.5 bar pressure. All
air- and moisture-sensitive manipulations were performed
using oven-dried glassware using the standard Schlenk
techniques under nitrogen. Diethyl ether and tetrahydrofuran
(
THF) were distilled from deep purple sodium benzopheone
ketyl. Dichloromethane (DCM), chloroform and acetonitrile
were dried over calcium hydride and distilled. DCM was
degassed via three freeze-pump-thaw cycles. All other
chemicals were used as received. All deuterated solvents were
purchased from Cambridge Isotope Laboratories. 5-N,N-
Dimethylamino-2,4-dinitroaniline (2) was synthesized using
Fig. 3 Plot of experimental vs. predicted activities for the training set
and test set compounds (AutoQSAR).
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Table 4 Prediction of MIC values of top 13 compounds and other selected compounds using AutoQSAR
1
2
a
Entry
1
Compound
SB-P17G-C2
R
R
MIC
Predicted MIC
Clog P
0.06
0.16
4.456
2
3
4
SB-P17G-A20
SB-P17G-A38
SB-P17G-A42
0.16
0.16
0.16
0.24
4.936
4.669
4.524
0.28
0.21
c
5
6
20g
20f
0.015
(
0.03
0.62
5.675
5.056
b
0.0039 )
0.031
(
b
<0.0081 )
7
8
9
6b
6c
<0.078
0.17
0.08
0.22
3.903
4.321
3.093
0.078
c
11c
0.14
b
(
0.09 )
10
11a
0.16
0.26
4.016
11
12
13
11e
0.16
0.29
0.13
0.10
0.43
3.999
4.212
4.801
c
10e-6
5c-7
b
(
0.19 )
0.31
1
1
4
5
6a
0.31
0.31
0.60
0.31
3.897
4.593
20b
1
1
6
7
20c
20e
0.31
0.31
0.68
0.67
3.863
4.804
18
5b-2
0.33
0.33
5.181
c
1
9
0
20d
20a
1.2
(
9.6
(
0.5
3.554
3.025
b
0.16 )
c
2
3.96
b
1.28 )
a
b
c
MIC determined by Protocol A. See Experimental Section. MIC determined by Protocol B. See Experimental Section. Normalized MIC from
the MIC determined by Protocol B to the MIC by Protocol A.
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give a crude product. The crude product was purified by
column chromatography on alumina, using 1 : 1 AcOEt :
hexanes as eluent to afford 5a-2 as beige solid (132 mg, 80%
1
yield); mp 207–210 °C; H NMR (400 MHz, CDCl ) δ 1.38 (s, 9
3
H), 1.65–1.70 (m, 2 H), 1.82–1.85 (m, 2 H), 1.87–1.93 (m, 4 H),
2
8
.73 (s, 6 H), 3.03–3.12 (quintet, J = 8.4 Hz, 1 H), 7.56 (d, J =
.4 Hz, 2 H), 7.61 (s, 1 H), 7.91 (d, J = 8.4 Hz, 2 H), 8.96 (s, 1
1
3
H), 9.96 (s, 1 H), 11.39 (s, 1H); C NMR (100 MHz, CDCl ) δ
3
2
1
5.6, 31.4, 32.5, 35.2, 39.9, 46.2, 101.7, 110.4, 126.0, 127.0,
29.1, 131.9, 133.1, 139.2, 139.9, 155.5, 159.6, 165.9; HRMS
+
(
ESI-TOF) m/z calcd for C H N OH : 405.2649, found:
2
5
32 4
405.2641 (Δ = 1.87 ppm). HPLC (1): t = 9.98 min, purity >99%.
The same procedure was used for the synthesis and
characterization of compounds 5a-4, 5a-5, 5a-6, 5a-9, 5a-10,
5
5
a-11, 5b-2, 5b-4, 5b-5, 5b-8, 5b-9, 5c-3, 5c-4, 5c-5, 5c-6, 5c-7,
d-1, 10e-1, 10e-2, 10e-3, 10e-4 and 10e-5.
Fig. 4 SAR of 2,5,6-trisubstituted benzimidazoles.
2-Cyclopentyl-6-dimethylamino-5-(4-methoxy-benzamido)-
1
H-benzo[d]-imidazole (5a-3). White solid (53% yield); mp
1
3
1
the procedure previously published by us. 4-Fluoro-1,2-di(4-
205–208 °C; H NMR (400 MHz, CDCl ) δ 1.60–1.65 (m, 2 H),
3
methylbenzenesulfonamido)benzene (13) and 4-fluoro-5-
nitro-1,2-di(4-methylbenzene-sulfonamido)benzene (14) were
1.71–1.81 (m, 2 H), 1.87–1.95 (m, 2 H), 2.04 2.06 (m, 2 H),
2.73 (s, 6 H), 3.18 (quintet, J = 6.7 Hz, 1 H), 3.89 (s, 3 H), 7.03
(d, J = 7.0 Hz, 2 H), 7.59 (s, 1 H), 7.93 (d, J = 7 Hz, 2 H), 8.81
3
1
synthesized by literature methods.
-Benzamido-2-cyclopentyl-6-dimethylamino-1H-benzo[d]
1
3
5
(s, 1 H), 9.80 (s, 1 H), 10.07 (bs, 1 H); C NMR (100 MHz,
CDCl ) δ 25.7, 32.5, 39.9, 46.2, 55.7, 101.4, 110.9, 114.3,
128.0, 129.0, 129.5, 139.5, 159.1, 162.6, 165.1; HRMS (ESI-
imidazole (5a-1). Compound 4a (0.100 g, 0.4 mmol), benzoic
acid (0.060 g, 1.2 mmol), 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC·HCl) (0.100 g, 0.53 mmol)
and N,N-4-dimethylaminopyridine (DMAP) (0.065 g, 1.3
mmol) were dissolved in DCM (2 mL) and the reaction
mixture was refluxed overnight. Then, the reaction mixture
was cooled to room temperature and washed with aqueous
3
+
26 4 2
TOF) m/z calcd for C22H N O H : 379.2129, found: 379.2129
(Δ = −0.19 ppm). HPLC (1): t = 15.1 min, purity >99%.
2-Cyclopentyl-6-dimethylamino-5-(2-methoxybenzamido)-
1H-benzo[d]imidazole (5a-4). White solid (76% yield); mp
1
214–217 °C; H NMR (400 MHz, CDCl
3
) δ 1.39–1.48 (m, 2 H),
saturated NaHCO (20 mL × 2) followed by water (20 mL × 2).
The organic layer was separated, dried with MgSO , filtered
4
and concentrated on a rotary evaporator to give a crude
product. The crude product was purified by column
chromatography on silica get, using 1 : 1 AcOEt : hexanes as
1.57–1.65 (m, 2 H), 1.80–1.89 (m, 2 H), 1.91 1.99 (m, 2 H),
2.73 (s, 6 H), 3.09 (quintet, J = 8.5 Hz 1 H), 4.07 (s, 3 H), 7.06
(d, J = 7.5 Hz, 1 H), 7.14 (t, J = 7.5 Hz, 1 H), 7.51 (t, J = 7.8 Hz,
1 H), 7.57 (s, 1 H), 8.37 (dd, J = 7.8, 1.8 Hz, 1 H), 9.04 (s, 1 H),
3
1
3
3
11.19 (s, 1 H), 11.39 (s, 1 H); C NMR (100 MHz, CDCl ) δ
eluent to afford 5a-1 as an off-white solid (134 mg, 94%
25.5, 32.5, 39.9, 46.1, 55.9, 102.6, 110.4, 111.6, 121.4, 122.6,
1
yield); mp 176–180 °C; H NMR (500 MHz, CDCl ) δ 1.46–1.48
129.9, 131.7, 132.4, 133.3, 139.7, 139.9, 157.6, 159.5, 163.6;
3
+
(
m, 2 H), 1.62–1.68 (m, 2 H), 1.83–1.96 (m, 4 H), 2.72 (s, 6 H),
.09 (quintet, J = 8.5 Hz, 1 H), 7.52–7.58 (m, 3 H), 7.61 (s, 1
H), 7.97 (d, J = 7.1 Hz, 2 H), 8.94 (s, 1 H), 9.98 (s, 1 H), 11.66
HRMS (ESI-TOF) m/z calcd for C22
H
26
N
4
O
2
H : 379.2129,
3
found: 379.2119 (Δ = 2.62 ppm). HPLC (1): t = 12.7 min,
purity >99%.
1
3
(
1
1
s, 1 H); C NMR (125 MHz, CDCl
3
) δ 25.5, 32.5, 39.9, 46.1,
01.8, 110.6, 127.1, 128.9, 129.0, 131.8, 131.9, 135.8, 139.3,
39.9, 159.74, 165.7; HRMS (ESI-TOF) m/z calcd for C H N -
2-Cyclopentyl-5-(2,4-difluorobenzamido)-6-dimethylamino-
1H-benzo[d]imidazole (5a-5). White solid (92% yield); mp
1
213–214 °C H NMR (500 MHz, CDCl ) δ 1.58–1.61 (m, 2 H),
2
1
24
4
3
+
OH : 349.2023, found: 349.2013 (Δ = 2.78 ppm); HPLC (1): t =
1.73–1.77 (m, 2 H), 1.87–1.94 (m, 2 H), 2.03–2.07 (m, 2 H),
2.71 (s, 6 H), 3.19 (quintet, J = 8.0 Hz, 1 H), 6.94–6.98 (m, 2
H), 7.03–7.07 (m, 1 H), 7.60 (s, 1 H), 8.21–8.26 (m, 1 H), 8.82
1
0.0 min, purity >96%.
The same procedure was used for the synthesis and
characterization of 5a-3, 5a-7, 5a-8, 5b-1, 5b-3, 5b-6, 5b-7, 5c-
1
3
(s, 1 H), 10.29 (s, 1 H), 10.33 (s, 1H); C NMR (125 MHz,
CDCl ) δ 25.6, 32.5, 39.9, 46.0, 102.1, 104.5, 104.8, 105.0,
1
, 5c-2, 5h-1, 5h-2 and 10e-6.
-(4-tert-Butylbenzamido)-2-cyclopentyl-6-dimethyl-amino-
H-benzo[d]imidazole (5a-2). A solution of 4a (0.100 g, 0.4
3
5
110.9, 112.5, 112.5, 112.69, 112.71, 118.91, 118.94, 119.01,
119.03, 129.3, 131.5, 133.7, 133.8, 139.7, 140.0, 159.6, 159.9,
1
1
9
mmol) and 4-tert-butylbenzoyl chloride (0.072 g, 0.36 mmol)
in DCM (4 mL) was stirred at 0 °C for 2 h. After the
completion of the reaction, the reaction mixture was washed
160.0, 160.5, 161.9, 162.0, 164.0, 164.1, 166.0, 166.1; F NMR
(376 MHz, CDCl ) δ −103.58 (d, 1 F), −108.92 (d, 1 F); HRMS
3
+
(ESI-TOF) m/z calcd for C21
H
22
F
2
N
4
OH : 385.1834, found:
with saturated aqueous NaHCO
water (20 mL × 2). The organic layer was separated, dried over
MgSO , filtered and concentrated on a rotary evaporator to
3
(20 mL × 2) followed by
385.1829 (Δ = 1.46 ppm). HPLC (1): t = 8.5 min, purity >99%.
2-Cyclopentyl-6-dimethylamino-5-(4-methylbenzamido)-1H-
benzo[d]imidazole (5a-6). White solid (76% yield); mp 187–
4
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1
1
1
90 °C; H NMR (500 MHz, CDCl ) δ 1.47–1.48 (m, 2 H),
212 °C; H NMR (400 MHz, CDCl ) δ 0.99 (t, J = 7.4 Hz, 6 H),
3
3
1
.66–1.76 (m, 2 H), 1.87–1.93 (m, 4 H), 2.44 (s, 3 H), 2.72 (s, 6
1.59–1.79 (m, 6 H), 1.81–1.86 (m, 2 H), 1.92–2.01 (m, 2 H),
2.09–2.21 (m, 3 H), 2.67 (s, 6 H), 3.22–3.30 (m, 1 H), 7.57 (s, 1
H), 8.59 (s, 1 H), 8.91 (s, 1 H), 9.84 (s, 1 H); C NMR (100
H), 3.04–3.11 (quintet, J = 8.3 Hz, 1 H), 7.34 (d, J = 7.7 Hz, 2
H), 7.60 (s, 1 H), 7.87 (d, J = 7.7 Hz, 2 H), 8.92 (s, 1 H), 9.92
1
3
1
3
(s, 1 H), 11.52 (s, 1 H); C NMR (125 MHz, CDCl ) δ 21.7,
MHz, CDCl ) δ 12.4, 25.8, 26.2, 32.5, 39.8, 46.0, 53.1, 101.5,
3
3
2
1
5.5, 32.5, 39.9, 46.1, 101.7, 110.6, 127.1, 128.9, 129.7, 131.8,
110.7, 129.2, 131.2, 139.2, 139.5, 159.1, 174.3; HRMS (ESI-
+
33.0, 139.2, 139.8, 142.4, 159.6, 165.8; HRMS (ESI-TOF) m/z
TOF) m/z calcd for C H N OH : 343.2411, found: 343.2483
2
0
30 4
+
calcd for C H N OH : 363.2179, found: 363.2171 (Δ = 2.3
(Δ = 2.44 ppm). HPLC (1): t = 10.3 min, purity >99%.
5-Benzamido-2-benzyl-6-dimethylamino-1H-benzo[d]
2
2
26 4
ppm). HPLC (1): t = 12.98 min, purity >99%.
-Cyclopentyl-6-dimethylamino-5-(4-fluoro-benzamido)-1H-
benzo[d]imidazole (5a-7). White solid (75% yield); mp 198–
2
imidazole (5b-1). Light yellow solid (57% yield); mp 212–213
1
°C; H NMR (500 MHz, CDCl ) δ 2.72 (s, 6 H), 4.12 (s, 2 H),
3
1
1
99 °C; H NMR (500 MHz, CDCl
3
) δ 1.47–1.54 (m, 2 H), 1.68–
7.13–7.23 (m, 5 H,), 7.48–7.51 (m, 2 H), 7.54–7.57 (m, 2 H),
7.92–7.93 (m, 2 H), 8.84 (s, 1 H), 9.89 (s, 1 H), 10.99 (bs, 1 H);
1
.70 (m, 2 H), 1.84–1.88 (m, 2 H), 1.90–1.96 (m, 2 H), 2.71 (s, 6
1
3
H), 3.09–3.15 (quintet, J = 8.6 Hz, 1 H), 7.21 (t, J = 8.3 Hz, 2 H),
C NMR (125 MHz, CDCl ) δ 35.9, 46.1, 101.8, 111.0, 127.0,
3
7
1
4
1
.59 (s, 1 H), 7.95–7.98 (m, 2 H), 8.82 (s, 1 H), 9.90 (s, 1 H),
127.1, 128.88, 128.90, 129.1, 129.4, 131.9, 135.8, 137.0, 139.7,
1
3
1.36 (bs, 1H); C NMR (125 MHz, CDCl ) δ 25.6, 32.5, 39.9,
140.1, 154.0, 165.5; HRMS (ESI-TOF) m/z calcd for C H N -
3
23 22 4
+
6.1, 101.7, 110.7, 116.0, 116.2, 128.8, 129.4, 129.5, 131.7,
OH : 371.1866, found: 371.1864 (Δ = 0.7 ppm). HPLC (1): t =
12.67 min, purity >98%.
1
9
31.88, 131.90, 139.3, 139.9, 159.7, 164.0, 164.5, 166.0;
F
NMR (376 MHz, CDCl ) δ −107.42 (s, 1 F); HRMS (ESI-TOF) m/
2-Benzyl-5-(4-tert-butylbenzamido)-6-dimethylamino-1H-
3
+
z calcd for C21
H
23FN
4
OH : 367.1929, found: 367.1919 (Δ =
benzo[d]imidazole (5b-2). Beige solid (79% yield); mp 164–
1
2
.51 ppm). HPLC (1): t = 12.0 min, purity >99%.
-Cyclopentyl-6-dimethylamino-5-(4-trifluoromethoxy-
benzamido)-1H-benzo[d] imidazole (5a-8). White solid (50%
167 °C; H NMR (400 MHz, CDCl
3
) δ 1.35 (s, 9 H), 2.73 (s, 6
2
H), 4.16 (s, 2 H), 7.15–7.25 (m, 5 H,), 7.51 (d, J = 8.5 Hz, 2 H),
7.59 (s, 1 H), 7.86 (d, J = 8.5 Hz, 2 H), 8.82 (s, 1 H), 9.85 (s, 1
1
13
yield); mp 182–186 °C; H NMR (500 MHz, CDCl
3
) δ 1.53–1.57
m, 2 H), 1.68–1.75 (m, 2 H), 1.86–1.93 (m, 2 H), 1.97–2.03
m, 2 H), 2.73 (s, 6 H), 3.11–3.18 (m, 1 H), 7.38 (d, J = 8.3 Hz,
H), 7.62 (s, 1 H), 8.0 (d, J = 8.3 Hz, 2 H), 8.81 (s, 1 H), 9.94
3
H), 10.45 (s, 1 H); C NMR (100 MHz, CDCl ) δ 31.4, 35.2,
(
(
2
36.0, 46.1, 101.6, 111.0, 126.1, 127.0, 127.1, 128.9, 129.0,
129.7, 131.8, 132.9, 136.9, 139.7, 139.9, 153.7, 155.5, 165.5;
+
HRMS (ESI-TOF) m/z calcd for C27
H
30
N
4
OH : 427.2492,
1
3
(s, 1 H), 10.77 (s, 1 H); C NMR (125 MHz, CDCl ) δ 25.6,
found: 427.2496 (Δ = 1.51 ppm). HPLC (1): t = 10.5 min,
purity >98%.
3
3
1
2.5, 39.9, 46.2, 101.6, 111.0, 117.5, 119.5, 121.1, 121.6, 123.6,
31.6, 134.2, 139.4, 140.0, 151.8, 159.6, 164.2; F NMR (376
1
9
2-Benzyl-6-dimethylamino-5-(4-methoxybenzamido)-1H-
MHz, CDCl ) δ −57.69 (s, 3 F). HRMS (ESI-TOF) m/z calcd for
benzo[d]imidazole (5b-3). Light yellow solid (55% yield); mp
3
+
1
C
22
H
23
F
3
N
4
O
2
H : 433.1846, found: 433.1843 (Δ = 0.77 ppm).
HPLC (1): t = 11.1 min, purity >98%.
-Cyclopentyl-6-dimethylamino-5-(pivalamido)-1H-benzo[d]
204–206 °C; H NMR (400 MHz, CDCl
3
) δ 2.71 (s, 6 H), 3.85
(s, 3 H), 4.11 (s, 2 H), 6.97 (d, J = 8.8 Hz, 2 H), 7.12–7.20 (m, 5
2
H,), 7.55 (s, 1 H), 7.88 (d, J = 8.8 Hz, 2 H), 8.82 (s, 1 H), 9.80
1
13
imidazole (5a-9). White solid (94% yield); mp >220 °C;
H
3
(s, 1 H), 11.26 (bs, 1 H); C NMR (100 MHz, CDCl ) δ 35.8,
NMR (500 MHz, CDCl ) δ 1.34 (s, 9H), 1.55–1.57 (m, 2 H),
3
46.0, 55.6, 101.7, 110.8, 114.3, 126.9, 127.9, 128.8, 129.0,
1
2
.68–1.76 (m, 2 H), 1.85–1.90 (m, 2 H), 1.99–2.05 (m, 2 H),
.64 (s, 6 H), 3.19 (quintet, J = 8.4 Hz, 1 H), 7.46 (s, 1 H), 8.53
129.5, 131.8, 137.1, 139.6, 139.9, 153.9, 162.6, 165.1; HRMS
+
(ESI-TOF) m/z calcd for C24
H
24
N
4
O
2
H : 401.1972, found:
1
3
(s, 1 H), 9.25 (s, 1 H); C NMR (125 MHz, CDCl
3
) δ 25.6,
401.1967 (Δ = 1.26 ppm). HPLC (1): t = 14.6 min, purity
>98%.
2
1
3
7.8, 32.4, 39.7, 40.1, 45.7, 102.0, 110.0, 128.8, 131.7, 139.3,
+
59.4, 176.7; HRMS (ESI-TOF) m/z calcd for C19
H
28
N
4
OH :
2-Benzyl-6-dimethylamino-5-(2,4-difluorobenzamido)-1H-
29.2336, found: 329.2327 (Δ = 2.83 ppm). HPLC (1): t = 10.6
benzo[d]imidazole (5b-4). Light yellow solid (92% yield); mp
1
min, purity >99%.
-Cyclobutanecarboxamido-2-cyclopentyl-6-dimethylamino-
180–182 °C; H NMR (500 MHz, CDCl ) δ 2.68 (s, 6 H), 4.11
3
5
(s, 2 H), 6.90–6.94 (m, 1 H), 6.95–6.99 (m, 1 H), 7.11–7.16 (m,
1
2
1
2
3
H-benzo[d]imidazole (5a-10). Brown solid (75% yield); mp
5 H), 7.49 (s, 1 H), 8.13–8.18 (m, 1 H), 8.80 (s, 1 H), 10.27 (s,
1
13
06–209 °C; H NMR (400 MHz, CDCl ) δ 1.64–1.69 (m, 2 H),
1 H); C NMR (125 MHz, CDCl ) δ 36.0, 45.9, 102.1, 104.4,
3
3
.76–1.84 (m, 2 H), 1.88–1.99 (m, 3 H), 2.01–2.13 (m, 3 H),
.26–2.34 (m, 2 H), 2.37–2.47 (m, 2 H), 2.64 (s, 6 H), 3.21–
.34 (m, 2 H), 7.51 (s, 1 H), 8.59 (s, 1 H), 8.87 (s, 1 H), 10.63
104.7, 104.9, 111.2, 112.6, 112.6, 112.72, 112.74, 118.8, 118.9,
127.3, 129.0, 129.11, 130.0, 134.0, 136.7, 140.0, 153.8, 159.90,
1
9
159.97, 160.3, 161.9, 162.0, 164.0, 164.1, 166.0, 166.1;
NMR (376 MHz, CDCl ) δ −103.53 (d, 1 F), −108.97 (d, 1 F);
HRMS (ESI-TOF) m/z calcd for C23
F
1
3
(s, 1 H); C NMR (100 MHz, CDCl
3
) δ 18.3, 25.76, 25.80, 32.5,
3
+
3
9.8, 41.6, 45.8, 101.7, 110.5, 128.9, 131.4, 139.1, 159.3, 176.3;
20 2 4
H F N OH : 407.1678,
+
HRMS (ESI-TOF) m/z calcd for C H N OH : 327.2179,
found: 327.2171 (Δ = 2.65 ppm). HPLC (3): t = 10.3 min,
purity >98%.
found: 407.1672 (Δ = 1.49 ppm). HPLC (1): t = 10.9 min,
purity >99%.
1
9
26 4
2-Benzyl-6-dimethylamino-5-(4-methylbenzamido)-1H-
2
-Cyclopentyl-6-dimethylamino-5-(2-ethylbutamido)-1H-
benzo[d]imidazole (5b-5). Off white solid (68% yield); mp
1
benzo[d]imidazole (5a-11). Beige solid (66% yield); mp 210–
218–220 °C; H NMR (400 MHz, CDCl
3
) δ 2.43 (s, 3 H), 2.73
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1
3
(s, 6 H), 4.12 (s, 2 H), 7.12–7.22 (m, 5 H,), 7.29 (d, J = 8.1 Hz,
(s, 1 H), 10.99 (s, 1 H); C NMR (100 MHz, CDCl ) δ 12.2,
3
2
H), 7.58 (s, 1 H), 7.82 (d, J = 8.1 Hz, 2 H), 8.82 (s, 1 H), 9.85
27.5, 44.1, 46.1, 101.8, 110.9, 127.2, 129.0, 129.1, 131.3, 131.9,
1
3
(s, 1 H), 11.02 (s, 1 H); C NMR (100 MHz, CDCl
3
) δ 21.7,
5.9, 46.1, 101.8, 110.9, 127.0, 127.1, 128.9, 129.5, 129.8,
31.8, 132.9, 137.06, 139.6, 140.0, 142.5, 153.9, 165.6; HRMS
135.9, 139.3, 140.1, 159.3, 165.8; HRMS (ESI-TOF) m/z calcd
+
3
1
for C H N OH : 351.2179, found: 351.2173 (Δ = 1.94 ppm).
2
1
26 4
HPLC (1): t = 8.8 min, purity >99%.
+
(
3
ESI-TOF) m/z calcd for C24
H
24
N
4
OH : 385.2023, found:
85.2023 (Δ = 0.08 ppm). HPLC (1): t = 11.4 min, purity
96%.
-Benzyl-6-dimethylamino-5-(4-fluorobenzamido)-1H-
benzo[d]imidazole (5b-6). Yellow solid (89% yield); mp 169–
5-(3,4-Dimethoxybenzamido)-2-(pent-3-yl)-6-
dimethylamino-1H-benzo[d]imidazole (5c-2). White solid
1
>
(58% yield); mp 184–186 °C; H NMR (500 MHz, CDCl ) δ
3
2
0.77 (t, J = 7.4 Hz, 6 H,), 1.64–1.79 (m, 4 H), 2.56–2.62 (m, 1
H), 2.73 (s, 6 H), 3.97 (s, 6 H), 6.99 (d, J = 8.4 Hz, 1 H), 7.49
(d, J = 8.4 Hz, 1 H,), 7.60 (s, 2 H), 8.80 (s, 1 H), 9.85 (s, 1 H),
1
1
71 °C; H NMR (400 MHz, CDCl ) δ 2.71 (s, 6 H), 4.16 (s, 2
3
1
3
H), 7.14–7.25 (m, 7 H), 7.56 (s, 1 H), 7.89–7.93 (m, 2 H), 8.73
3
10.40 (s, 1 H); C NMR (125 MHz, CDCl ) δ 12.3, 27.6, 44.2,
1
3
(s, 1 H), 9.79 (s, 1 H), 10.62 (bs, 1 H); C NMR (100 MHz,
46.1, 56.2, 56.3, 101.5, 110.80, 110.84, 110.9, 119.7, 128.3,
CDCl ) δ 35.9, 46.1, 101.8, 111.1, 116.0, 116.2, 127.2, 128.9,
129.2, 131.1, 139.3, 139.8, 149.4, 152.2, 158.9, 165.2; HRMS
3
+
1
1
29.0, 129.4, 129.5, 131.79, 131.81, 136.9, 139.7, 153.9, 163.8,
(ESI-TOF) m/z calcd for C23
H
30
N
4
O
3
H : 411.2391, found:
1
9
64.3, 166.3; F NMR (376 MHz, CDCl ) δ −107.42 (s, 1 F);
411.2387 (Δ = 0.79 ppm). HPLC (1): t = 14.1 min, purity
>79%.
3
+
HRMS (ESI-TOF) m/z calcd for C H FN OH : 389.1772,
2
3
21
4
found: 389.1764 (Δ = 2.18 ppm). HPLC (1): t = 13.2 min,
purity >99%.
2-(Pent-3-yl)-6-dimethylamino-5-(2-methoxybenzamido)-
1H-benzo[d]imidazole (5c-3). Light brown solid (60% yield);
1
2
-Benzyl-6-dimethylamino-5-(4-trifluoromethoxy-
mp 214–215 °C; H NMR (500 MHz, CDCl
3
) δ 0.75 (t, J = 7.3
benzamido)-1H-benzo[d]imidazole (5b-7). Light yellow solid
Hz, 6 H), 1.59–1.77 (m, 4 H), 2.53–2.59 (m, 1 H), 2.74 (s, 6 H),
4.08 (s, 3 H), 7.07 (d, J = 7.9 Hz, 1 H), 7.15 (t, J = 7.9 Hz, 1 H),
7.52 (t, J = 7.8 Hz, 1 H), 7.58 (s, 1 H), 8.37 (dd, J = 7.8, 1.8 Hz,
1
(
66% yield); mp 196–197 °C; H NMR (500 MHz, CDCl ) δ
3
2
.71 (s, 6 H), 4.12 (s, 2 H), 7.11–7.19 (m, 5 H,), 7.32 (d, J = 8.5
13
Hz, 2 H), 7.53 (s, 1 H), 7.94 (d, J = 8.5 Hz, 2 H), 8.76 (s, 1 H),
1 H), 9.01 (s, 1 H), 10.60 (s, 1 H), 11.36 (s, 1 H); C NMR
1
3
9
3
1
1
.85 (s, 1 H), 11.39 (bs, 1 H); C NMR (125 MHz, CDCl ) δ
(125 MHz, CDCl ): δ 12.2, 27.5, 44.1, 46.0, 56.0, 102.6, 110.5,
3
3
5.6, 45.9, 101.7, 110.8, 117.3, 119.3, 120.9, 121.4, 123.5,
26.9, 128.7, 129.0, 131.7, 133.8, 136.8, 139.6, 139.8, 151.6,
54.0, 163.8; F NMR (376 MHz, CDCl ) δ −57.68 (s, 3 F);
HRMS (ESI-TOF) m/z calcd for C24
111.7, 121.5, 122.7, 130.0, 131.1, 132.6, 133.2, 139.7, 140.0,
157.7, 158.95, 163.6; HRMS (ESI-TOF) m/z calcd for C22
O H : 381.2285, found: 381.228 (Δ = 1.24 ppm).
2
H
28
N
4
-
1
9
+
3
+
H
21
F
3
N
4
O
2
H : 455.1619,
2-(Pent-3-yl)-6-dimethylamino-5-(4-methylbenzamido)-1H-
found: 455.1690 (Δ = −0.42 ppm). HPLC (1): t = 9.7 min,
purity >98%.
benzo[d]imidazole (5c-4). White solid (61% yield); mp >230
1
°C; H NMR (500 MHz, CDCl ) δ 0.76 (t, J = 7 Hz, 6 H), 1.61–
3
2
-Benzyl-6-dimethylamino-5-(pivalamido)-1H-benzo[d]
1.75 (m, 4 H), 2.45 (s, 3 H), 2.54–2.60 (m, 1 H), 2.73 (s, 6 H),
7.36 (d, J = 7.8 Hz, 2 H), 7.61 (s, 1 H), 7.86 (d, J = 7.8 Hz, 2
1
imidazole (5b-8). White solid (92% yield); mp >220 °C;
NMR (400 MHz, CDCl ) δ 1.33 (s, 9 H), 2.65 (s, 6 H), 4.12 (s, 2
H), 7.16–7.18 (m, 5 H), 7.47 (s, 1 H), 8.54 (s, 1 H), 9.29 (s, 1
H
1
3
H), 8.89 (s, 1 H), 9.89 (s, 1 H), 10.91 (s, 1 H); C NMR (125
MHz, CDCl ) δ 12.2, 21.7, 27.5, 44.0, 46.1, 101.7, 110.8, 127.2,
3
3
1
3
H), 11.24 (s, 1 H); C NMR (100 MHz, CDCl
3
) δ 27.9, 35.8,
0.1, 45.7, 101.7, 110.5, 126.9, 128.8, 128.9, 129.4, 131.6,
37.1, 139.5, 153.8, 176.7; HRMS (ESI-TOF) m/z calcd for C21
129.1, 129.7, 131.3, 133.0, 139.3, 140.0, 142.5, 159.2, 165.8;
+
4
1
H
HRMS (ESI-TOF) m/z calcd for C H N OH : 365.2336,
2
2
28 4
-
found: 365.2332 (Δ = 1.02 ppm). HPLC (1): t = 10.2 min,
purity >97%.
+
26
N
4
OH : 351.2179, found: 351.2172 (Δ = 2.18 ppm). HPLC
1): t = 11.1 min, purity >98%.
-Benzyl-6-dimethylamino-5-(2-ethylbutanamido)-1H-
benzo[d]imidazole (5b-9). White solid (67% yield); mp 175–
(
5-(2,4-Difluorobenzamido)-6-dimethylamino-2-(pent-3-yl)-
2
1H-benzo[d]imidazole (5c-5). Beige solid (62% yield); mp
1
207–210 °C; H NMR (500 MHz, CDCl
3
) δ 0.81 (t, J = 7.2 Hz, 6
1
1
1
2
8
77 °C; H NMR (500 MHz, CDCl ) δ 0.95 (t, J = 7.4 Hz, 6 H),
.53–1.62 (m, 2 H), 1.66–1.75 (m, 2 H), 2.09–2.16 (m, 1 H),
.67 (s, 6 H), 4.24 (s, 2 H), 7.21–7.30 (m, 5 H), 7.54 (s, 1 H),
H), 1.69–1.78 (m, 4 H), 2.59–2.66 (m, 1 H), 2.70 (s, 6 H), 6.96
(t, J = 9.5 Hz, 1 H), 7.05 (t, J = 9.5 Hz, 1 H), 7.60 (s, 1 H),
3
1
3
8.21–8.26 (m, 1 H), 8.83 (s, 1 H), 10.32–10.37 (m, 2 H);
C
1
3
.58 (s, 1 H), 8.91 (s, 1 H), 10.09 (s, 1 H); C NMR (125 MHz,
) δ 12.4, 26.3, 36.1, 45.9, 53.1, 101.7, 110.8, 127.2,
29.0, 129.1, 129.5, 131.5, 136.9, 139.4, 139.7, 153.5, 174.4;
NMR (125 MHz, CDCl ) δ 12.3, 27.6, 44.2, 45.9, 102.1, 104.5,
3
CDCl
3
104.7, 105.00, 111.0, 112.5, 112.6, 112.7, 118.97, 119.03,
129.4, 131.0, 133.9, 139.7, 140.1, 159.0, 159.88, 159.98, 160.5,
1
+
19
HRMS (ESI-TOF) m/z calcd for C H N OH : 365.2336,
161.9, 162.0, 164.0, 164.1, 166.05, 166.14; F NMR (376 MHz,
2
2
28 4
found: 365.2331 (Δ = 1.42 ppm). HPLC (1): t = 11.3 min,
purity >97%.
CDCl
m/z calcd for C21
3
) δ −103.63 (d, 1 F), −108.95 (d, 1 F); HRMS (ESI-TOF)
+
24 2 4
H F N OH : 387.1991, found: 387.1988 (Δ =
5
-Benzamido-6-dimethylamino-2-(pent-3-yl)-1H-benzo[d]
0.76 ppm). HPLC (1): t = 8.98 min, purity >98%.
1
imidazole (5c-1). White solid (85% yield); mp 208–210 °C; H
NMR (400 MHz, CDCl ) δ 0.76 (t, J = 7.40 Hz, 6 H), 1.58–1.78
m, 4 H), 2.54–2.59 (m, 1 H), 2.74 (s, 6 H), 7.52–7.60 (m, 3 H),
6-Dimethylamino-5-(4-fluorobenzamido)-2-(pent-3-yl)-1H-
3
benzo[d]imidazole (5c-6). White solid (44% yield); mp 192–
1
(
195 °C; H NMR (500 MHz, CDCl ) δ 0.77 (t, J = 7.4 Hz, 6 H),
3
7
.63 (s, 1 H), 7.97 (dd, J = 7.8, 1.4 Hz, 2 H), 8.91 (s, 1 H), 9.94
1.63–1.78 (m, 4 H), 2.58–2.62 (m, 1 H), 2.71 (s, 6 H), 7.19–
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7
9
1
1
1
.25 (m, 2 H), 7.59 (s, 1 H), 7.95–7.98 (m, 2 H), 8.83 (s, 1 H),
in DCM (2 mL), carbonyldiimidazole (73.2 mg, 0.45 mmol)
was added. The reaction mixture was refluxed for 4 h,
followed by the addition of n-BuOH (66 μL), and the reaction
mixture was refluxed overnight. After the completion of the
1
3
.86 (s, 1 H), 11.10 (bs, 1 H); C NMR (125 MHz, CDCl ) δ
3
2.2, 27.5, 44.1, 46.1, 101.8, 110.7, 116.0, 116.2, 128.8, 129.4,
29.5, 131.3, 131.89, 131.91, 139.3, 140.0, 159.3, 164.0, 164.5,
1
9
66.0; F NMR (376 MHz, CDCl ) δ −107.54 (s, 1 F); HRMS
reaction, the reaction mixture was washed with NaHCO (50
3
3
+
(
ESI-TOF) m/z calcd for C21
H
25FN
4
OH : 369.2085, found:
69.2082 (Δ = 0.84 ppm). HPLC (1): t = 10.7 min, purity
99%.
-Dimethylamino-2-(pent-3-yl)-5-(4-trifluoromethoxy-
mL × 2) and water (50 mL × 2). The organic layer was
3
>
separated, dried over MgSO , filtered and concentrated on a
4
rotary evaporator to give a crude product. The crude product
was purified by column chromatography on alumina, using
6
benzamido)-1H-benzo[d]imidazole (5c-7). White solid (76%
1 : 1 AcOEt : hexanes as eluent to afford 6a as a white solid (98
1
1
yield); mp 194–198 °C; H NMR (500 MHz, CDCl ) δ 0.80 (t, J
mg, 70% yield); mp 174–175 °C; H NMR (500 MHz, CDCl ) δ
3
3
=
7.4 Hz, 6 H,), 1.67–1.80 (m, 4 H), 2.58–2.64 (m, 1 H), 2.74
0.95 (t, J = 7.4 Hz,3 H), 1.39–1.47 (m, 2 H), 1.63–1.71 (m, 4
H), 1.75–1.82 (m, 2 H), 1.87–1.95 (m, 2 H), 2.07–2.13 (m, 2
H), 2.62 (s, 6 H), 3.23 (quintet, J = 8.4 Hz,1 H,), 4.18 (t, J = 6.7
(s, 6 H), 7.38 (d, J = 8.5 Hz, 2 H), 7.64 (s, 1 H), 8.00 (d, J = 8.5
13
Hz, 2 H), 8.79 (s, 1 H), 9.9 (s, 1 H), 10.26 (bs, 1 H); C NMR
125 MHz, CDCl ) δ 12.3, 27.6, 44.2, 46.2, 101.6, 111.1, 119.5,
21.2, 121.6, 129.1, 131.1, 134.2, 139.4, 140.1, 151.8159.1,
13
(
3
Hz, 2 H), 7.5 (s, 1 H), 8.19 (s, 2 H), 9.99 (s, 1 H); C NMR
1
1
(125 MHz, CDCl ) δ 14.0, 19.3, 25.7, 31.3, 32.5, 39.8, 45.8,
3
1
9
64.2; F NMR (376 MHz, CDCl ) δ −57.70 (s, 3 F); HRMS
65.1, 99.7, 110.7, 129.5, 131.3, 138.8, 154.4, 158.7; HRMS
3
+
+
(ESI-TOF) m/z calcd for C22
H
25
F
3
N
4
O
2
H : 435.2002, found:
35.2 (Δ = 0.62 ppm). HPLC (1): t = 10.8 min, purity >97%.
-(4-tert-Butylbenzamido)-2-isopropyl-6-dimethylamino-
(ESI-TOF) m/z calcd for C19
H
28
N
4
O
2
H : 345.2285, found:
4
345.2279 (Δ = 1.87 ppm). HPLC (2): t = 22.1 min, purity
>96%.
5
1
1
6
H-benzo[d]imidazole (5d-1). White solid (87% yield); mp
Compounds 6b–6d, 6f, 6h, 11a–11e and 20a–20h were
synthesized in a similar manner and characterized.
2-Benzyl-5-butoxycarbonylamino-6-dimethylamino-1H-
1
98–200 °C; H NMR (500 MHz, CDCl ) δ 1.30 (d, J = 6.1 Hz,
3
H), 1.38 (s, 9 H), 2.72 (s, 6 H), 3.01–3.03 (m, 1 H), 7.55–7.62
(
m, 3 H), 7.90 (d, J = 8.1 Hz, 2 H), 8.90 (s, 1 H), 9.96 (s, 1 H),
benzo[d]imidazole (6b). Light brown solid (44% yield); mp
1
3
1
1
3
1
0.87 (s, 1H); C NMR (125 MHz, CDCl ) δ 21.7, 29.1, 31.4,
193–196 °C; H NMR (400 MHz, CDCl ) δ 0.95 (t, J = 9.2 Hz, 3
3
3
5.2, 46.2, 101.8, 110.9, 126.0, 127.1, 129.3, 131.6, 133.0,
H), 1.37–1.51 (m, 2 H), 1.63–1.70 (m, 2 H), 2.63 (s, 6 H), 4.14–
39.4, 139.7, 155.5, 160.7, 165.9; HRMS (ESI-TOF) m/z calcd
4.19 (m, 4 H), 7.21–7.30 (m, 5 H,), 7.44 (s, 1 H), 8.15 (s, 1 H),
+
13
for C H N OH : 379.2492, found: 379.2495 (Δ = 1.89 ppm).
9.71 (s, 1 H); C NMR (125 MHz, CDCl ) δ 14.0, 19.3, 31.3,
2
3
30
4
3
6
-Dimethylamino-2-tetrahydropyranyl-5-(4-trifluoro-
36.1, 45.8, 65.1, 99.6, 111.1, 127.4, 129.1, 129.2, 130.1, 136.7,
methoxybenzamido)-1H-benzo[d]imidazole (5h-1). White
26 4
139.1, 153.1, 154.3; HRMS (ESI-TOF) m/z calcd for C21H N -
O H : 367.2129, found: 367.2122 (Δ = 1.82 ppm). HPLC (1): t
2
1
+
solid (75% yield); mp 210–213 °C; H NMR (500 MHz, CDCl )
3
δ 1.96–2.07 (m, 4 H), 2.73 (s, 6 H), 3.09–3.14 (m, 1 H), 3.42–
= 10.3 min, purity >98%.
3
7
1
1
1
.47 (m, 2 H), 4.00–4.03 (m, 2 H), 7.40 (d, J = 8.1 Hz, 2 H),
5-Butoxycarbonylamino-6-dimethylamino-2-(3-pentyl)-1H-
.59 (bs, 1 H), 8.00 (d, J = 8.1 Hz, 2 H), 8.80 (s, 1 H), 9.88 (bs,
benzo[d]imidazole (6c). Light brown solid (57% yield); mp
1
3
1
H); C NMR (100 MHz, CDCl
3
) δ 31.5, 36.1, 46.1, 67.7,
208–210 °C; H NMR (500 MHz, CDCl
3
) δ 0.83 (t, J = 7.4 Hz, 6
02.5, 110.5, 119.5, 121.2, 121.6, 123.6, 129.0, 129.3, 134.1,
H,), 0.95 (t, J = 7.3 Hz, 3 H), 1.40–1.46 (m, 2 H), 1.65–1.70 (m,
2 H), 1.72–1.81 (m, 4 H), 2.62 (s, 6 H), 2.67–2.69 (m, 1 H),
1
9
39.8, 151.90, 151.92, 158.0, 164.3; F NMR (376 MHz,
) δ −57.66 (s, 3 F); HRMS (ESI-TOF) m/z calcd for C22
CDCl
3
-
4.19 (t, J = 6.7 Hz, 2 H), 7.25 (s, 1 H), 7.51 (s, 1 H), 8.19 (s, 1
+
13
H
23
F
3
N
4
O
3
H : 449.1795, found: 449.1792 (Δ = 0.57 ppm).
HPLC (3): t = 12.3 min, purity >98%.
-Dimethylamino-5-(2-fluoro-4-trifluoromethoxy-
3
H), 10.18 (s, 1 H); C NMR (125 MHz, CDCl ) δ 12.3, 14.0,
19.3, 27.7, 31.2, 44.2, 45.8, 65.2, 99.8, 110.8, 129.4, 131.0,
138.7, 138.8, 154.5, 158.3; HRMS (ESI-TOF) m/z calcd for C19
6
-
+
benzamido)-2-tetrahydropyranyl-1H-benzo[d]imidazole (5h-2).
White solid (73% yield); mp 213–214 °C; H NMR (500 MHz,
H
30
N
4
O
2
H : 347.2442, found: 347.2434 (Δ = 2.12 ppm).
5-Butoxycarbonylamino-6-dimethylamino-2-isopropyl-1H-
benzo[d]imidazole (6d). Brown solid (79% yield); mp 112–115
1
CDCl
.47–3.54 (m, 2 H), 4.03–4.07 (m, 2 H), 7.12 (dd, J = 14.5, 1.4
Hz, 1 H), 7.22 (d, J = 11.1 Hz, 1 H), 7.58 (s, 1 H), 8.26 (t, J =
3
) δ 1.59–2.15 (m, 4 H), 2.71 (s, 6 H), 3.12–3.20 (m, 1 H),
1
3
3
°C; H NMR (500 MHz, CDCl ) δ 0.90 (t, J = 7.4 Hz, 3 H),
1.34–1.41 (m, 2 H), 1.58–1.64 (m, 8 H), 2.61 (s, 6 H), 3.76
(quintet, J = 7.0 Hz, 1 H), 4.09 (t, J = 6.7 Hz, 2 H), 7.64 (s, 1
1
3
1
1.1 Hz, 1 H), 8.82 (s, 1 H), 10.33 (d, J = 14.5 Hz, 1 H);
C
1
3
NMR (100 MHz, CDCl
3
) δ 31.5, 36.1, 46.0, 67.7, 109.1, 109.4,
H), 8.01 (s, 1 H), 8.48 (s, 1 H), 14.7 (s, 1 H); C NMR (125
1
1
17.1, 119.1, 120.9, 121.0, 121.7, 129.8, 133.6, 134.0, 140.2,
52.3, 152.4, 157.8, 159.3, 160.20, 160.24, 161.8; F NMR
MHz, CDCl ) δ 13.9, 19.3, 21.2, 27.8, 31.1, 45.3, 65.5, 102.6,
3
1
9
106.3, 126.4, 128.4, 132.8, 142.0, 153.7, 157.8; HRMS (ESI-
+
(376 MHz, CDCl
3
) δ −57.66 (s, 3 F), −109.38 (s, 1 F); HRMS
TOF) m/z calcd for C17
H
26
N
4
O
2
H : 319.2129, found: 319.2121
+
(TOF) m/z calcd for C H F N O H : 467.1701, found:
(Δ = 2.27 ppm). HPLC (3): t = 7.3 min, purity >99%.
5-Butoxycarbonylamino-6-dimethylamino-2-thienyl-1H-
2
2
22 4 4 3
4
>
67.1699 (Δ = 0.47 ppm). HPLC (4): t = 14.7 min, purity
97%.
-Butoxycarbonylamino-2-cyclopentyl-6-dimethylamino-1H-
benzo[d]imidazole (6f). White solid (57% yield); mp 175–177
1
5
°C H NMR (500 MHz, CDCl ) δ 0.94 (t, J = 7.4 Hz, 3 H), 1.38–
3
benzo[d]imidazole (6a. To a solution of 4 (100 mg, 0.4 mmol)
1.45 (m, 2 H,), 1.64–1.70 (m, 2 H), 2.64 (s, 6 H), 4.18 (t, J = 6.7
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Hz, 2 H), 7.03–7.05 (m, 1 H), 7.37 (dd, J = 5.0, 1.1 Hz, 1 H),
Yellow solid (29% yield); H NMR (500 MHz, CDCl ) δ ppm
3
7
.53–7.56 (m, 2 H), 8.23 (s, 1 H), 8.26 (s, 1 H), 10.32 (s, 1 H);
2.36 (s, 6 H), 3.00 (s, 3 H), 3.66 (s, 2 H), 3.83 (s, 3 H), 4.65 (s,
1
3
C NMR (125 MHz, CDCl
3
) δ 14.1, 19.3, 31.2, 45.8, 65.3, 99.7,
2 H), 6.73–6.84 (m, 3 H), 6.93 (d, J = 8.5 Hz, 2 H), 7.14–7.26
1
3
1
1
3
11.2, 125.9, 127.8, 128.1, 130.6, 131.7, 133.7, 139.5, 139.7,
(m, 4 H), 7.38 (bs, 1 H), 8.58 (bs, 1 H), 8.85 (bs, 1 H);
C
+
47.2, 154.5. HRMS (ESI-TOF) m/z calcd for C H N O SH :
NMR (500 MHz, CDCl ) δ 39.4, 44.4, 45.2, 52.5, 55.3, 113.4,
1
8
22
4
2
3
59.1536, found: 359.1537 (Δ = −0.13 ppm). HPLC (1): t = 8.8
114.3, 118.3, 126.7, 129.4, 129.5, 130.8, 139.4, 149.5, 152.7,
min, purity >98%.
-Butoxycarbonylamino-6-dimethylamino-2-
tetrahydropyranyl-1H-benzo[d] imidazole (6h). White solid
159.0, 169.6; HRMS (ESI-TOF) m/z calculated for C H N O -
2
6
29 5 2
+
5
H : 444.2394, found 444.2374 (Δ 4.6 ppm). HPLC (3): t = 8.9
min, purity >99%.
1
(59% yield); mp 176–178 °C; H NMR (500 MHz, CDCl ) δ
6-Dimethylamino-2-(N-methyl-N-phenylamino)methyl-5-(4-
3
0
.96 (t, J = 7.4 Hz, 3 H,), 1.41–1.48 (m, 2 H), 1.67–1.72 (m, 2
propylbenzamido)-1H-benzo[d]imidazole (10e-5). Yellow solid
1
H), 2.00–2.03 (m, 4 H), 2.64 (s, 6 H), 3.14–3.20 (m, 1 H), 3.52–
(66% yield); mp 164–166 °C; H NMR (500 MHz, CDCl
3
) δ
3
7
.57 (m, 2 H), 4.06–4.09 (m, 2 H), 4.20 (t, J = 6.7 Hz, 2 H,),
0.95 (t, J = 7.5 Hz, 3 H), 1.67 (sextet, J = 7.5 Hz, 2 H), 2.64 (t, J
= 7.5 Hz, 2 H), 2.71 (s, 6 H), 2.94 (s, 3 H), 4.59 (s, 2 H), 6.65–
6.78 (m, 3 H), 7.13–7.16 (m, 2 H), 7.24–7.31 (m, 2 H), 7.55
1
3
.50 (s, 1 H), 8.17 (s, 1 H), 8.25 (s, 1H); C NMR (100 MHz,
) δ 14.0, 19.4, 31.3, 31.5, 35.9, 45.8, 65.25, 67.7, 130.2,
39.4, 154.4, 156.8; HRMS (ESI-TOF) m/z calcd for C H N -
CDCl
1
3
(bs, 1 H), 7.82 (d, J = 8.2 Hz, 2 H), 8.72 (bs, 1 H), 9.72 (bs, 1
1
9
28 4
+
13
O H : 361.2234, found: 361.2229 (Δ = 1.56 ppm). HPLC (4): t
H), 10.80 (bs, 1 H); C NMR (126 MHz, CDCl ) δ 13.7, 24.2,
3
3
=
14.8 min, purity > 98%.
-(4-tert-Butylbenzamido)-6-dimethylamino-2-(N-methyl-N-
phenylamino)-methyl-1H-benzo[d]imidazole (10e-1). Yellow
37.8, 39.3, 45.7, 52.2, 101.8, 110.4, 113.3, 117.9, 126.9, 129.2,
5
129.5, 132.7, 139.6, 146.7, 149.4, 153.0, 165.0; HRMS (ESI-
+
TOF) m/z calculated for C27
H
31
N
5
OH : 442.2601, found
1
solid (75% yield); mp 111–112 °C H NMR (500 MHz, CDCl
ppm 1.37 (s, 9 H), 2.75 (s, 6 H), 3.02 (s, 3 H), 4.68 (s, 2 H), 6.76–
3
) δ
442.2602 (Δ −0.12 ppm). HPLC (3): t = 67.0 min, purity >99%.
6-Dimethylamino-5-(2-fluoro-4-trifluoro-
6
2
.84 (m, 3 H), 7.23 (dd, J = 8.9, 7.3 Hz, 2 H), 7.52 (d, J = 8.5 Hz,
H), 7.60 (bs, 1 H), 7.87 (d, J = 8.5 Hz, 2 H), 8.70 (bs, 1 H), 9.76
methoxybenzamido)-2-(N-methyl-N-phenylamino)methyl-1H-
benzo[d]imidazole (10e-6). Beige solid (73% yield); mp 73–75
13
1
(bs, 1 H), 9.89 (bs, 1 H); C NMR (500 MHz, CDCl ) δ 31.2,
°C; H NMR (500 MHz, CDCl ) δ 2.72 (s, 6 H), 2.99 (s, 3 H),
3
3
3
5.0, 39.6, 45.8, 52.6, 101.5, 110.8, 113.5, 118.4, 125.8, 126.9,
4.66 (s, 2 H), 6.73–6.80 (m, 3 H), 7.07 (d, J = 12.2 Hz, 1 H),
7.14 (d, J = 8.9 Hz, 1 H), 7.17–7.23 (m, 2 H), 7.52 (s, 1 H), 8.20
(t, J = 8.9 Hz, 1 H), 8.75 (s, 1 H), 10.23 (d, J = 12.2 Hz, 1 H);
129.5, 130.0, 132.6, 139.8, 149.5, 152.9, 155.1, 165.0; HRMS
+
(ESI-TOF) m/z calculated for C H N OH : 456.2758, found
2
8
33 5
1
3
456.2757 (Δ 0.13 ppm). HPLC (3): t = 6.5 min, purity >99%.
3
C NMR (126 MHz, CDCl ) δ 39.4, 45.6, 52.3, 103.7, 108.7,
6
-Dimethylamino-5-(4-methylbenzamido)-2-(N-methyl-N-
108.9, 109.2, 113.3, 116.7, 117.1, 118.3, 119.1, 120.5, 120.6,
121.2, 123.2, 129.4, 129.8, 132.5, 133.5, 133.5, 137.7, 140.1,
149.3, 151.8, 151.9, 153.2, 159.2, 159.6, 159.7, 161.2; F NMR
phenylamino)methyl-1H-benzo[d]imidazole (10e-2). Yellowish
brown solid (50% yield); mp 178–181 °C; H NMR (500 MHz,
1
19
CDCl
H), 6.66–6.86 (m, 3 H), 7.12–7.24 (m, 2 H), 7.29 (d, J = 8.1
Hz, 2 H), 7.51 (bs, 1 H) 7.82 (d, J = 8.1 Hz, 2 H), 8.72 (s, 1 H),
3
) δ ppm 2.43 (s, 3 H), 2.72 (s, 6 H), 2.99 (s, 3 H), 4.64 (s,
(376 MHz, CDCl
(ESI-TOF) m/z calculated for C H F N O H : 502.1861,
found 502.1859 (Δ 0.23 ppm). HPLC (3): t = 6.4 min, purity
>95%.
5-Butoxycarbonylamino-6-dimethylamino-2-N,N-
dipropylaminomethyl-1H-benzo[d]imidazole
brown solid (54% yield); mp 176–178 °C; H NMR (500 MHz,
CDCl3) δ 0.80 (t, J = 7.3 Hz, 6 H), 0.89 (t, J = 7.5 Hz, 3 H),
1.31–1.48 (m, 6 H), 1.59–1.65 (m, 2 H), 2.30–2.46 (m, 4 H),
2.58 (s, 6 H), 3.75 (s, 2 H), 4.12 (t, J = 6.7 Hz, 2 H), 7.43 (bs, 1
3
) δ −57.85 (s, 3 F), −109.42 (s, 1 F); HRMS
+
2
2
5
23 4 5 2
1
3
9
5
1
3
.71 (bs, 1 H); C NMR (500 MHz, CDCl ) δ 21.5, 39.5, 45.8,
2.4, 102.6, 109.9, 113.4, 118.2, 127.0, 129.4, 129.5, 129.7,
32.6, 139.8, 142.1, 149.5, 153.0, 165.1; HRMS (ESI-TOF) m/z
(11a).
Light
+
1
calculated for C25
H
27
N
5
OH : 414.2288, found 414.229 (Δ
−
0.29 ppm). HPLC (3): t = 7.7 min, purity >99%.
6
-Dimethylamino-5-(2,4-difluorobenzamido)-2-(N-methyl-
N-phenylamino)methyl-1H-benzo[d]imidazole (10e-3). Yellow
1
13
solid (49% yield); mp 130–134 °C; H NMR (500 MHz, CDCl )
H), 8.12 (s, 1 H), 8.15 (s, 1 H), 9.52 (bs, 1 H); C NMR (126
3
δ ppm 2.71 (s, 6 H), 2.98 (s, 3 H), 4.63 (s, 2 H), 6.63–6.82 (m,
3
MHz, CDCl ) δ 11.8, 13.8, 19.1, 20.3, 31.1, 45.6, 53.1, 56.8,
3
8
H), 6.85–7.05 (m, 2 H), 7.18–7.21 (m, 2 H), 7.51 (bs, 1 H),
64.9, 99.4, 110.8, 129.9, 130.5, 138.6, 138.9, 154.0, 154.2;
1
3
+
.07–8.26 (m, 1 H), 8.76 (bs, 1 H) 10.18–10.22 (m, 1 H);
) δ 39.3, 45.5, 52.2, 104.0, 104.2, 104.3,
04.5, 112.2, 112.3, 112.4, 113.1, 117.9, 118.4, 118.5, 129.1,
29.6, 133.5, 133.6, 133.7, 139.9, 149.3, 153.3, 159.5, 159.6,
C
HRMS (ESI-TOF) m/z calculated for C H N O H : 390.2864,
2
1
35 5 2
NMR (126 MHz, CDCl
3
found 390.2864 (Δ −0.09 ppm). HPLC (3): t = 6.0 min, purity
>95%.
1
1
1
5-Butoxycarbonylamino-6-dimethylamino-2-morpholin-1-
1
9
59.90, 159.93, 161.5, 161.6, 163.6, 163.7, 165.6, 165.7;
F
ylmethyl-1H-benzo[d]imidazole (11b). Light brown solid (54%
1
NMR (376 MHz, CDCl
HRMS (ESI-TOF) m/z calculated for C H F N OH :
3
) δ −103.61 (d, 1 F), −109.01 (d, 1 F);
yield); mp 146–147 °C; H NMR (500 MHz, CDCl
3
) δ 0.97 (t, J
+
= 7.3 Hz, 3 H), 1.41–1.46 (m, 2 H), 1.64–1.76 (m, 2 H), 2.45–
2.60 (m, 4 H), 2.66 (s, 6 H), 3.54–3.76 (m, 4 H), 3.78 (s, 2 H),
4.20 (t, J = 6.7 Hz, 2 H), 7.52 (bs, 1 H), 8.21 (bs, 2 H), 9.72 (bs,
2
4
23 2 5
4
36.1943, found 436.1946 (Δ −0.55 ppm). HPLC (3): t = 6.8
min, purity >99%.
-Dimethylamino-2-(N-methyl-N-phenylamino)methyl-5-(4-
1
3
6
1 H); C NMR (126 MHz, CDCl ) δ 14.0, 19.3, 31.3, 45.8, 54.0,
3
methoxyphenyl)-ethanamido-1H-benzo[d]imidazole
(10e-4).
57.1, 65.1, 67.1, 99.6, 111.2, 130.4, 131.1, 138.7, 139.1, 151.5,
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+
1
3
54.3; HRMS (ESI-TOF) m/z calculated for C H N O H :
5-Butoxycarbonylamino-6-dimethylamino-2-
(isopropylsulfanyl)-1H-benzo[d]-imidazole (20c). White solid
(62% yield); mp 165–166 °C; H NMR (500 MHz, CDCl ) δ
3
1
9
29 5 3
76.2343, found 376.2347 (Δ −0.89 ppm). HPLC (3): t = 8.1
min, purity >99%.
-Butoxycarbonylamino-6-dimethylamino-2-piperidin-1-
ylmethyl-1H-benzo[d]imidazole (11c). Beige solid (63% yield);
) δ 0.97 (t, J = 7.3 Hz,
H), 1.39–1.52 (m, 4 H), 1.58–1.60 (m, 4 H), 1.66–1.74 (m, 2 H),
.47 (bs., 4 H), 2.66 (s, 6 H), 3.73 (s, 2 H), 4.20 (t, J = 6.7 Hz, 2
1
5
0.98 (t, J = 7.3 Hz, 3 H), 1.39 (d, J = 7.02 Hz, 6 H), 1.43–1.54
(m, 2 H), 1.71–1.75 (m, 2 H), 2.65 (s, 6 H), 3.91–3.97 (m, 1 H),
4.30 (t, J = 6.4 Hz, 2 H), 7.53 (bs, 1 H), 8.32 (bs, 2 H), 11.05
1
mp 160–161 °C; H NMR (500 MHz, CDCl
3
2
3
1
3
(bs, 1 H); C NMR (126 MHz, CDCl ) δ 13.8, 19.2, 23.6, 31.0,
3
38.9, 45.7, 65.4, 99.3, 110.1, 129.5, 132.2, 138.8, 139.9, 148.7,
13
+
H), 7.51 (bs, 1 H), 8.20 (bs, 1 H), 9.73 (bs, 1 H); C NMR (126
154.4; HRMS (ESI-TOF) m/z calculated for C17
H
26
N
4
O
2
SH :
MHz, CDCl ) δ 13.8, 19.1, 23.9, 26.0, 31.1, 45.6, 54.9, 57.2, 64.9,
351.1849, found 351.1846 (Δ 0.79 ppm). HPLC (3): t = 3.9
min, purity >99%.
3
9
9.4, 110.9, 129.0, 130.0, 130.8, 138.7, 152.5, 154.0; HRMS (ESI-
+
TOF) m/z calculated for
74.2551 (Δ −0.23 ppm). HPLC (3): t = 6.1 min, purity >99%.
-Butoxycarbonylamino-6-dimethylamino-2-pyrrolidin-1-
20 31 5 2
C H N O H : 374.2551, found
5-Butoxycarbonylamino-6-dimethylamino-2-(ethylsulfanyl)-
3
1H-benzo[d]imidazole (20d). Beige solid (76% yield); mp 136–
1
5
138 °C; H NMR (500 MHz, CDCl ) δ 0.98 (t, J = 7.2 Hz, 3 H),
3
ylmethyl-1H-benzo[d]imidazole (11d). Light brown solid (41%
1.40 (t, J = 7.5 Hz, 3 H), 1.47–1.51 (m, 2 H), 1.62–1.81 (m, 2
H), 2.65 (s, 6 H), 3.28 (q, J = 7.5 Hz, 2 H), 4.29 (bs, 2 H), 7.53
1
yield); mp 135–137 °C; H NMR (500 MHz, CDCl ) δ 0.96 (t, J
3
1
3
=
1
6
7.5 Hz, 3 H), 1.38–1.50 (m, 2 H), 1.63–1.73 (m, 2 H), 1.81–
(bs, 1 H), 8.30 (s, 2 H), 10.96 (bs, 1 H); C NMR (126 MHz,
CDCl ) δ 13.8, 15.0, 19.1, 27.1, 31.0, 45.6, 65.4, 99.2, 109.9,
129.2, 132.4, 138.5, 139.5, 149.5, 154.4; HRMS (ESI-TOF) m/z
.83 (m, 4 H), 2.55–2.72 (m, 10 H), 3.91 (s, 2 H), 4.19 (t, J =
3
1
3
.7 Hz, 2 H), 7.50 (bs, 1 H), 8.18 (bs, 2 H), 10.19 (bs, 1 H);
) δ 14.0, 19.3, 23.9, 31.3, 45.8, 54.1,
4.6, 65.1, 99.7, 111.1, 130.1, 131.2, 139.0, 152.8, 154.2;
C
+
NMR (126 MHz, CDCl
5
HRMS (ESI-TOF) m/z calculated for C H N O H : 360.2394,
3
24 4 2
calculated for C16H N O SH : 337.1693, found 337.1692 (Δ
0.35 ppm). HPLC (3): t = 6.5 min, purity >95%.
+
5-Butoxycarbonylamino-6-dimethylamino-2-
1
9
29 5 2
found 360.2396 (Δ −0.58 ppm). HPLC (3): t = 6.2 min, purity
95%.
-Butoxycarbonylamino-6-dimethylamino-2-(N-methyl-N-
(phenylsulfanyl)-1H-benzo[d]imidazole (20e). White solid
1
>
(77% yield); mp 172–173 °C; H NMR (500 MHz, CDCl
3
) δ
5
0.95 (t, J = 7.3 Hz, 3 H), 1.33–1.51 (m, 2 H), 1.57–1.77 (m, 2
H), 2.63 (s, 6 H), 4.21 (t, J = 6.7 Hz, 2 H), 7.14–7.25 (m, 3 H),
phenylamino)methyl-1H-benzo[d]imidazole
(11e).
Light
1
13
brown solid (59% yield); H NMR (500 MHz, CDCl
3
) δ 0.96 (t,
7.39–7.45 (m, 3 H), 8.20 (bs, 1 H), 8.28 (bs, 1 H); C NMR
J = 7.5 Hz, 3 H), 1.36–1.54 (m, 2 H), 1.57–1.77 (m, 2 H), 2.65
s, 6 H), 3.00 (s, 3 H), 4.16 (t, J = 6.7 Hz, 2 H), 4.63 (s, 2 H),
.70–6.87 (m, 3 H), 7.18–7.26 (m, 2 H), 7.45 (bs, 1 H), 8.13
(126 MHz, CDCl ) δ 13.7, 19.1, 30.9, 45.4, 65.1, 77.2, 127.9,
3
(
6
129.3, 130.0, 131.2, 131.8, 139.2, 146.4, 154.2; HRMS (ESI-
+
TOF) m/z calculated for C20
H
24
N
4
O
2
SH : 385.1693, found
1
3
(
bs, 1 H), 8.19 (bs, 1 H); C NMR (125 MHz, CDCl ) δ 13.9,
385.1691 (Δ 0.52 ppm). HPLC (3): t = 4.5 min, purity >95%.
3
1
1
9.3, 31.2, 39.7, 45.7, 52.6, 65.1, 99.9, 110.7, 113.4, 118.4,
5-Butoxycarbonylamino-2-(cyclohexylsulfanyl)-6-
29.6, 130.1, 131.1, 139.1, 139.3, 149.6, 152.7, 154.2; HRMS
dimethylamino-1H-benzo[d]imidazole (20f). White solid (73%
+
1
(ESI-TOF) m/z calculated for C H N O H : 396.2394, found
yield); mp 129–132 °C; H NMR (500 MHz, CDCl ) δ 0.98 (t, J =
2
2
29
5
2
3
3
96.2395 (Δ −0.16 ppm). HPLC (3): t = 6.6 min, purity >99%.
-Butoxycarbonylamino-6-dimethylamino-2-(methylthio)-
H-benzo[d]imidazole (20a). White solid (66% yield); mp
7.3 Hz, 3 H), 1.19–1.31 (m, 1 H), 1.31–1.42 (m, 2 H), 1.42–1.53
(m, 4 H), 1.56–1.59 (m, 1 H), 1.66–1.80 (m, 4 H), 2.01–2.15 (m,
5
1
2 H), 2.66 (s, 6 H), 3.65–3.81 (m, 1 H), 4.30 (s, 2 H), 7.54 (bs, 1
1
13
1
59–160 °C; H NMR (500 MHz, CDCl
3
) δ 0.97 (t, J = 7.3 Hz, 3
3
H), 8.29 (bs, 2 H), 10.55 (bs, 1 H); C NMR (126 MHz, CDCl )
H), 1.34–1.55 (m, 2 H), 1.58–1.82 (m, 2 H), 2.64 (bs, 6 H), 2.72
δ 13.8, 19.1, 25.5, 25.8, 31.0, 33.6, 45.6, 46.7, 65.3, 99.1, 110.2,
(
(
1
1
H
s, 3 H), 4.28–4.30 (m, 2 H), 7.52 (bs, 1 H), 8.30 (s, 1 H), 8.28
129.5, 132.1, 138.6, 139.6, 148.3, 154.4; HRMS (TOF) m/z
1
3
+
s, 1 H), 11.30 (bs, 1 H); C NMR (126 MHz, CDCl
3
) δ 13.7,
4.8, 19.1, 30.9, 45.6, 65.3, 99.2, 109.7, 129.1, 132.7, 138.4,
39.3, 150.8, 154.4; HRMS (ESI-TOF) m/z calculated for C -
calculated for C20
H
30
N
4
O
2
SH : 391.2162, found 391.2161 (Δ
0.23 ppm). HPLC (3): t = 12.6 min, purity >99%.
5-Butoxycarbonylamino-2-(cyclohexylmethylsulfanyl)-6-
1
5
+
22
N
4
O
2
SH : 323.1536, found 323.1533 (Δ 0.86 ppm). HPLC
dimethylamino-1H-benzo[d]imidazole (20g). White solid
1
(
2): t = 4.4 min, purity >95%.
-Benzylsulfanyl-5-butyloxycarbonylamino-6-
dimethylamino-1H-benzo[d]imidazole (20b). White solid
(61% yield); mp 109–110 °C; H NMR (500 MHz, CDCl
3
) δ
2
0.91–1.06 (m, 5 H), 1.07–1.29 (m, 3 H),1.37–1.55 (m, 2 H),
1.56–1.79 (m, 6 H), 1.84–1.86 (m, 2 H), 2.59 (bs, 1 H), 2.65 (s,
5H), 3.22 (d, J = 7.0 Hz, 2 H), 4.31 (t, J = 6.6 Hz, 2 H), 7.52 (s,
1
(
77% yield); mp 126–127 °C; H NMR (500 MHz, CDCl
.95 (t, J = 7.3 Hz, 3 H), 1.41–1.44 (m, 2 H), 1.57–1.78 (m, 2
H), 2.66 (s, 6 H), 4.26 (t, J = 6.7 Hz, 2 H), 4.52 (s, 2 H), 7.20–
.31 (m, 3 H), 7.32–7.40 (m, 2 H), 7.56 (bs, 1 H), 8.30 (bs, 2
3
) δ
13
0
1 H), 8.27 (bs, 1 H), 8.31 (s, 1 H), 10.81 (s, 1 H); C NMR
(126 MHz, CDCl ) δ 13.8, 19.1, 25.9, 26.2, 31.0, 32.5,
3
7
37.7,39.7, 45.6, 65.4, 99.1, 109.8, 129.1, 132.4, 138.4, 139.4,
13
H), 10.84 (bs, 1 H); C NMR (126 MHz, CDCl ) δ 13.7, 19.1,
3
1
calculated for C H N O SH : 399.1849, found 399.1848 (Δ
0
150.3, 154.4; HRMS (ESI-TOF) m/z calculated for C H N O -
3
21 32 4 2
+
0.9, 37.4, 45.6, 65.3, 99.2, 110.0, 127.5, 128.6, 128.9, 129.4,
SH : 405.2319, found 405.2317 (Δ 0.43 ppm). HPLC (3): t =
18.6 min, purity >96%.
32.4, 136.8, 138.7, 139.4, 149.0, 154.3; HRMS (ESI-TOF) m/z
+
In silico predictions of pharmacological properties with
pkCSM. All of the tested compounds were drawn and
2
1
26 4 2
.25 ppm). HPLC (3): t = 3.97 min, purity >95%.
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−
1
−1
translated into the SMILES format using the ChemDraw
Prime 16.0 program. Then, the compounds in the SMILES
format were processed and analyzed by the pkCSM online
mL to 0.488 ng mL . M. tuberculosis strain H37Rv at the
midlogarithmic stage of growth (OD580 = 0.4) was diluted 1 : 100,
and 0.1 mL was added to each well of a 96-well plate along with
0.1 mL of test compound solution. After 6 days of incubation at
37 °C, Alamar blue (Invitrogen) reagent was added along with
12.5 mL of 20% Tween 80 (Sigma) to evaluate bacterial cell
viability. Plates were scanned 24 h later at 570 nm with a
reference wavelength of 600 nm utilizing a Biotek Instruments
ELX 808. Inoculum control wells of untreated H37Rv were used
to create a survival-inhibition curve with each assay. Rifampicin
2
8
tool. Results are summarized in Table S1 in the ESI.†
D-QSAR. All 2,4,6-trisubstituted benzimidazoles we have
3
studied are fully characterized and MIC values were determined
by the standard assay method. All these benzimidazoles are
assumed to bind the same putative allosteric site in a similar
manner. For the 3D-QSAR analysis, the MIC value of each
compound was converted to the negative logarithmic scale (−log
pM). Then, all benzimidazole molecules were drawn using the
ChemDraw Prime 16.0 program and saved individually as MOL
files. Each compound's structural and biological information
−1
was used as a positive control (MIC = 0.0125–0.05 μg mL ).
Cell growth inhibition assay. Human liver cancer cell line
HepG2 was cultured and maintained in Roswell Park
Memorial Institute growth medium RPMI1640 (with
L-glutamine) supplemented by 10% fetal bovine serum (FBS)
and 1% penicillin (PenStrip). Normal human lung fibroblast
cell line WI-38 was cultured and maintained in Dulbecco's
modified Eagle medium (DMEM) supplemented by 10% fetal
bovine serum (FBS) and 1% penicillin (PenStrip). Cells were
plated at a density of 2500 cells per well in 96-well plates and
allowed to attach overnight and reach 70–90% confluency.
Benzimidazoles were dissolved in sterilized DMSO and
further diluted with either RPMI-1640 medium or DMEM
medium to reach the concentration of 20 uM. The
concentration of DMSO was kept at 0.5% (i.e., 99.5% culture
medium and 0.5% DMSO). After 72 h of incubation with test
compounds, supernatant was aspirated to remove the
medium. Then MTT solution was added into each well.
Following 4 h incubation avoiding light, the remaining MTT
solution was aspirated. The dye was dissolved in DMSO with
100 μL per well. Then, the 96-well plate was shaken for 10
min. Optical density (OD) was measured at 570 nm. The
percentage inhibition was calculated as follows: % inhibition
= 1 − (OD of compound/OD of DMSO control) × 100.
32
was transferred to the Schödinger's AutoQSAR program. The
original dataset was divided into training and test sets by given
ratio. The sampling process of these training and test sets
occurs randomly selected, while the ratio between training and
test sets remains the same. With given information along with a
default set of descriptors, a large number of models were built
using the AutoQSAR. Then, their predictive powers were
estimated using test set to choose 10 best models. All the
predictions were proceeded using all top ten models and the
predicted values were simply arithmetically averaged from these
10 different models has been used.
Molecular docking. The 2,4,6-trisubstituted benzimidazole
molecules were drawn as 2D structures using ChemDraw
Prime 16.0. Then, the Avogadro program was used to build
3
D structures and to optimize the internal energy of each
compounds. The force field chosen to perform during the
energy optimization was Merck Molecule Force Field
(MMFF94). Once this was completed, individual compounds
were saved as MOL2 files. Molecular docking was performed
with the AutoDock 4.2 program (with default settings) and
each compound was docked on the Mtb-FtsZ homology
2
9
model, reproduced from the published Li′s model. Analysis
of predicted binding poses and their associated scores was Author contributions
performed with the UCSF Chimera program. The results on
K. H.: experimental design and synthesis, data collections, data
analysis, manuscript preparation; S. T. design, synthesis, data
collections, partial manuscript preparation; S. K.: 3D-QSAR
model building and analysis, pkCSM analysis, data collections,
partial manuscript preparation; M. A.: molecular docking
analysis, partial 3D-QSAR model building; L. C.: cytotoxicity
assay; S. E. K., E. S. and R. R. MABA assay, MIC determination;
R. A. S. and N. C. supervision of MABA assays. I. O.: research
design, supervision of whole project, manuscript preparation.
2
0g are shown in Fig. S1 in the ESI.†
Bacterial strains and growth.
1
4,33
For evaluation of drug
sensitivity, Mtb H37Rv was grown in 7H9 media containing
1
0
0% oleic acid/albumin/catalase (OADC) enrichment and
.05% Tween-80 and assessed at mid log phase growth.
2
5
Antibacterial activity. Protocol A:
The minimum
inhibitory concentration (MIC) was determined by the
microplate Alamar Blue assay (MABA) as described
2
5
previously.
Stock solutions of the compounds were
prepared in DMSO and were serially diluted 2-fold in 96-well
microtiter plates. Mtb H37Rv was added to each well to an
OD600 of 0.005. The 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. The
Conflicts of interest
The authors declare no competing financial interest.
Acknowledgements
−
1
MIC was the lowest concentration (μg mL ) of compound
that inhibited bacterial growth and prevented a color change.
This research is supported by grants from the National
Institutes of Health (Grant AI078251 to I. O. and Grant
AI082164 to R. A. S.). The authors thank Adam Taouil for his
technical assistance on computational analyses and Dr. Bela
27
Protocol B: Each compound was dissolved in DMSO at a
−1
final concentration of 12 mg mL and serial dilutions were
performed to generate test concentrations ranging from 32 mg
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