Benzimidazole-based compounds kill Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2014.01.039

Tuberculosis remains one of the deadliest infectious diseases, killing 1.4 million people annually and showing a rapid increase in cases resistant to multiple drugs. New antibiotics against tuberculosis are urgently needed. Here we describe the design, sy

Full text of DOI:10.1016/j.ejmech.2014.01.039

European Journal of Medicinal Chemistry 75 (2014) 336e353
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Benzimidazole-based compounds kill Mycobacterium tuberculosis
,
,e,
Yaling Gong a 1, Selin Somersan Karakaya b 1, Xiaoyong Guo a, Purong Zheng a, Ben Gold b,
Yao Ma a, David Little b, Julia Roberts b, Thulasi Warrier b, Xiuju Jiang b, Maneesh Pingle b,
**
Carl F. Nathan b , Gang Liu
,
a,c,d,
*
a Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, 2A Nanwei Road, Xuanwu District, Beijing 100050, PR
China
b Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065, USA
c Tsinghua-Peking Center for Life Sciences and School of Medicine, Tsinghua University, Haidian Dist., Beijing 100084, PR China
d Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Haidian Dist., Beijing 100084, PR China
e Division of Infectious Diseases, Department of Medicine, Weill Cornell Medical College, New York, NY 10065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Tuberculosis remains one of the deadliest infectious diseases, killing 1.4 million people annually and
showing a rapid increase in cases resistant to multiple drugs. New antibiotics against tuberculosis are
urgently needed. Here we describe the design, synthesis and structureeactivity relationships of a series
of benzimidazole-based compounds with activity against Mycobacterium tuberculosis (Mtb) in a repli-
cating state, a physiologically-induced non-replicating state, or both. Compounds 49, 67, 68, 69, 70, and
72, which shared a 5-nitrofuranyl moiety, exhibited high potency and acceptable selectivity indices (SI).
Received 30 October 2013
Received in revised form
16 January 2014
Accepted 17 January 2014
Available online 31 January 2014
As illustrated by compound 70 (MIC90 < 0.049 mg/mL, SI > 512), the 5-nitrofuranyl group was compatible
Keywords:
Benzimidazole
Drug
Mycobacterium tuberculosis
Nitrofuran
Tuberculosis
with minimal cytotoxicity and good intra-macrophage killing, although it lacked non-replicating activity
when assessed by CFU assays. Compound 70 had low mutagenic potential by SOS Chromotest assay,
making this class of compounds good candidates for further evaluation and target identification.
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
and two of them (INH and RIF) for an additional 4 months. When
Mtb is resistant to INH and RIF, optimal treatment with existing
Tuberculosis (TB), which is caused predominantly by Mycobac-
terium tuberculosis (Mtb), is the leading cause of death from a
curable infectious disease, and has been identified by the World
Health Organization (WHO) as one of the three priority diseases for
drug research and development [1]. One-third of the world’s pop-
ulation is estimated to be infected with Mtb, and in 2010 there were
an estimated 8.8 million incident cases of TB globally, equivalent to
128 cases per 100,000 people.
drugs averages 25e27 months with a total of 7 other drugs, a 21%
rate of adverse effects serious enough to interrupt therapy, and an
apparent short-term cure rate of only 54% [2]. The requirement for
such prolonged treatment may be due in part to the presence of
subpopulations of bacteria that are non-replicating (NR-Mtb) [3,4].
The lengthy course of treatment contributes to poor patient
compliance, a high incidence of side effects, and the emergence of
drug-resistant Mtb strains [5]. Most current drugs target replicating
Mtb (R-Mtb), with only RIF, PZA and fluoroquinolones showing
activity against NR-Mtb [6e8].
The current TB treatment regimen requires patients to take a
combination of four drugs, namely, isoniazid (INH), rifampicin
(RIF), pyrazinamide (PZA), and ethambuthol (EMB), for 2 months
For these reasons, we have conducted an anti-Mtb whole-cell
screening campaign using both R-Mtb and NR-Mtb, as recently
described for a compound collection of known drugs [9]. Mtb was
rendered non-replicating by
a combination of four stresses
* Corresponding author. Department of Pharmacology and Pharmaceutical Sci-
ences, School of Medicine, Tsinghua University, Haidian Dist., Beijing 100084, PR
China. Tel./fax: þ86 10 62797740.
designed to mimic an activated macrophage or a granuloma: mild
acidity (pH 5.0), hypoxia (1% O2), a fatty acid as the carbon source
(butyrate), and mild nitrosative stress (a flux of nitric oxide). Here,
we applied these same screening methods to a privileged-structure
library centered on novel benzimidazoles. Compounds 1, 2, and 3
** Corresponding author. Tel.: þ1 212 746 6505; fax: þ1 212 746 8587.
E-mail addresses: cnathan@med.cornell.edu (C.F. Nathan), gliu@imm.ac.cn,
gangliu27@biomed.tsinghua.edu.cn, gangliu27@tsinghua.edu.cn (G. Liu).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.039

Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
337
Fig. 1. Structures of active compounds in our benzimidazole library.
(Fig. 1) were identified to be potent actives against NR-Mtb, R-Mtb,
or both. The common structural features of these active compounds
were the benzimidazole core and a 5-nitrofuranyl substituent.
The benzimidazole moiety has long been recognized as a
“privileged substructure” for drug design, and has been incorpo-
rated into many drugs with various therapeutic applications,
including antiviral [10], antifungal [11], antitumor [12], and anti-
histaminic [13] agents. The benzimidazole scaffold has also shown
antimycobacterial activity in vitro [14,15], making it a promising
starting point for the discovery of new anti-TB drugs. Below we
report the design, synthesis and biological actions of derivatives of
three benzimidazole-based scaffolds (I, II, III, Fig. 2).
oxidized. We herein adopted a one-pot strategy without purifying
compound 30 [16], in which the reaction solution was directly
filtered into another reaction vessel containing the aldehyde
(R3CHO) to yield compound 31. The free aromatic amino group of
compound 31 was modified via acylation with different acyl chlo-
rides, isocyanates, or isothiocyanates to produce the expected
1,2,5,6-tetrasubstituted benzimidazoles II (3, 32e74).
2.3. The synthesis of 6H-imidazo[40,50:3,4]benzo[1,2-d]thiazole
(Scaffold III)
6H-imidazo[40,50:3,4]benzo[1,2-d]thiazole (Scaffold III) was
synthesized as outlined in Scheme 3. The intermediate 75 was
obtained by reaction of compound 31 with potassium thiocyanate
(KSCN) and bromine (Br2) in acetic acid. Oxidation of the free ar-
omatic amino group on compound 75 with sodium nitrite (NaNO2)
in acetic acid and water resulted in the desired compound 76.
Acylation of 75 with acyl chloride produced the expected com-
pounds 77 and 78.
2. Chemistry
2.1. The synthesis of 1H-imidazo[4,5-g]quinoxalin-6(5H)-ones
(Scaffold I)
1H-Imidazo[4,5-g]quinoxalin-6(5H)-ones (Scaffold I) were
synthesized as outlined in Scheme 1 [16]. In the presence of dii-
sopropylethylamine (DIPEA), one of the fluorine atoms of 1,5-
difluoro-2,4-dinitrobenzene (DFDNB) was substituted by an
3. Results and discussion
equivalent
a-amino acid methyl or ethyl ester hydrochlorate to
afford compound 4, and then the other fluorine atom of DFDNB was
substituted by a series of primary amines to give compound 5 in
excellent yields. After reduction of the two nitro-groups using 10%
palladium on activated carbon and subsequent acid-catalyzed
cyclization, the expected 1H-imidazo[4,5-g]quinoxalin-6(5H)-ones
I (1, 2, 6e27) were obtained with total yields of 15e35%.
3.1. Chemistry
Twenty-four compounds of scaffold
I were synthesized
(Table 1). These compounds contained diversified substituent
groups in which R1 was derived from various methyl esters of
natural amino acids, including alanine, glycine, leucine, isoleucine,
phenylalanine, and valine. R2 was introduced by various primary
amines bearing alkyl, aryl, arylalkyl, and cycloalkyl groups. Four
compounds (1, 6, 7 and 9) showed antibacterial activity, with MIC90
2.2. The synthesis of 1,2,5,6-tetrasubstituted benzimidazoles
(Scaffold II)
values ranging from 0.78 to 6.3 mg/mL (Table 1). However, these
1,2,5,6-Tetrasubstituted benzimidazoles (Scaffold II) were ob-
tained according to the synthetic route shown in Scheme 2. In the
first step, secondary amines or alcohols were used as nucleophiles
to replace quantitatively one fluorine atom of DFDNB in the pres-
ence of organic base. Then the remaining fluorine was substituted
by primary amines to obtain compound 29. Simultaneous reduc-
tion of the aromatic m-dinitro group to generate 30 was achieved
by use of HCOONH4 and Pd/C or sodium hydrosulfite in a mixture of
THF and EtOH. 2,4,5-Benzenetriamine 30 was found to be readily
active compounds were also toxic to human hepatoma HepG2 cells.
Eighteen compounds of scaffold II were then synthesized
(Table 2). The diversity of R1 was introduced by using primary
amines; R2 was from secondary amines or alcohols; R3 was kept
constant as a 5-nitrofuran pharmacophore that appears in some
antibiotics [17e19] although the 5-nitrofuran moiety may have off-
target toxicity mediated by human aldehyde dehydrogenase
(ALDH) 2 [20]; and R4 was derived from acyl chlorides, isocyanates,
or isothiocyanates (Table 2).
Fig. 2. Benzimidazole-based scaffolds.

338
Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
MIC90 ¼ 3.1
(NR-Mtb: MIC90 ¼ 0.78
SI(R) > 16) and 59 (NR-Mtb: MIC90 ¼ 0.39
MIC90 < 0.098 g/mL; SI(R) > 32) with significantly improved
m
g/mL; R-Mtb: MIC90 ¼ 0.2
g/mL; R-Mtb: MIC90 < 0.098
g/mL; R-Mtb:
m
g/mL; SI(R) ¼ 64), 50
m
m
g/mL;
m
m
MIC and SI values from scaffold II (Table 3). Similar compounds
with a thiazolobenzimidazole core were inactive and toxic to
HepG2 cells (Table 4).
Subsequently, eight modified analogs of 49, 50, and 59 were
designed and synthesized (Table 5). The introduced substituents
included an aliphatic chain (R1), such as tert-butyl, isopropyl,
methyl, 1-ethoxybutanyl and 1,3-dimethylbutanyl, morpholine
or morpholine mimics such as thiomorpholine for R2, and
methyl for R3. All of the new compounds were highly active
against Mtb with significantly improved SI values, especially
Scheme 1. Synthesis of 1H-imidazo[4,5-g]quinoxalin-6(5H)-onesa. aReagents and
conditions: (i) DIPEA, THF, rt; (ii) R2NH2, DIPEA, THF, rt; (iii) Pd/CeHCOONH4, THFe
EtOH, rt; (iv) 5-nitrofuraldehyde or 5-nitrothiophenolaldehyde, dioxane, THFeEtOH,
5% AcOH, rt; (v) 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), reflux.
compound 70 (NR-Mtb: MIC90
¼
0.20
mg/mL; R-Mtb:
MIC90 < 0.049 g/mL; SI(R) > 512) (Table 5). For these com-
m
pounds, the 5-nitrofuran pharmacophore introduced at the 6-
position of benzimidazole was particularly useful when R1 was
an aliphatic chain, R2 was a morpholinyl or thiomorpholinyl
substitution, and R3 was a methyl group. The activity of selected
compounds (49, 70) was confirmed with wild-type (wt) Mtb,
and minimal inhibitory concentrations (MICs) were within 2e4
fold of those seen with the
strain as described [9].
DpanCDDlysA double auxotroph
3.2. Biology
3.2.1. Cidality of benzimidazoles against wild-type M. tuberculosis
(Mtb)
Our screening assay has the potential to assign provisional NR
activity to a compound whose R activity is potent enough to be
manifest after dilution from the NR screening phase into the R
outgrowth phase. To definitively characterize activity as being
manifest against NR-Mtb, R-Mtb or both, we tested selected benz-
imidazoles’ (49 and 70) cidal activity in both conditions by a CFU-
based assay (Fig. 3). 49 was cidal against replicating wt Mtb,
Scheme 2. Synthesis of 1,2,5,6-tetrasubstituted benzimidazolesa. aReagents and con-
ditions: (i) secondary amine or hydroxyalkyl, base, THF, rt; (ii) R1NH2, DIPEA, THF, rt;
(iii) reducing agent, THFeEtOH; (iv) R3CHO, dioxane, THFeEtOH, 5% AcOH, rt; (v)
RCOCl or RNCO, or RNCS, Et3N, CH2Cl2, rt.
reducing viable bacilli 1.4 log10 at 0.74
mg/mL (Fig. 3B) but was
inactive against NR-Mtb by a CFU assay (Fig. 3C). In one experiment,
compound 70 likewise killed R-Mtb but was inactive against NR-
Mtb.
Table 2 indicates that compounds 33 (NR-Mtb: MIC90 ¼ 6.3
mL; R-Mtb: MIC90 ¼ 1.6 g/mL; SI(R) ¼ 8.0) and 34 (NR-Mtb:
MIC90 ¼ 13 g/mL; R-Mtb: MIC90 ¼ 6.3 g/mL; SI(R) > 8.0)
mg/
m
m
m
showed activity against both R-Mtb and NR-Mtb with improved SI
values. Compound 35 was only active against R-Mtb. While these
data suggested that presence of the 5-nitrofuran pharmacophore
at the 2-position of the benzimidazole core was effective, we also
tested placement at the 6-position of the benzimidazole core
(Table 3). These efforts resulted in compounds 49 (NR-Mtb:
3.2.2. Benzimidazoles kill wild-type Mtb infecting human
macrophages
Given their cidality in vitro against wild-type Mtb, we next
explored the activity of these compounds against Mtb in primary
human macrophages, cells that provide an important niche for Mtb
in vivo. Macrophages were differentiated in vitro from normal do-
nors’ blood monocytes and immunologically activated as reported
[21]. At 2.5
in macrophages when compared to the DMSO control (Fig. 4A). This
killing activity was similar to that of rifampicin at 2.5
m
g/mL (8ꢀ MIC), compound 49 killed 1.9 log10 of wt Mtb
mg/mL (30ꢀ
MIC), the most potent of the front-line drugs used to treat TB. Based
on these results for compound 49, we synthesized the compounds
in Table 5. Given its improved potency and SI, compound 70 was
also tested in human macrophages, and concentration-dependent
bacterial killing was observed (Fig. 4B).
3.2.3. Selectivity of benzimidazoles
Compound 70 was tested against several different species of
Gram-positive and Gram-negative bacteria to evaluate its selec-
tivity. No activity was observed against Escherichia coli (Fig. 5A),
Salmonella spp (Fig. 5B), Staphylococcus aureus (Fig. 5C) or Pseudo-
Scheme 3. Synthesis of 4,6,7-trisubstituted-2-nitro-6H-imidazo[40,50:3,4]benzo[1,2-d]
thiazole or N-(4,6,7-trisubstituted-6H-imidazo[40,50:3,4]benzo[1,2-d]thiazol-2-yl)acet-
amidea. aReagents and conditions: (i) KSCN, Br2, HOAc, rt; (ii) NaNO2, HOAc:H2O ¼ 1:1;
(iii) RCOCl, Et3N, CH2Cl2, rt.
monas aeruginosa (Fig. 5D) at concentrations up to 10
mg/mL
(ꢁ250ꢀ MIC for Mtb). In contrast, both 49 and 70 were active
against Mycobacterium smegmatis with MICs comparable to those

Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
339
Table 1
Scaffold I compounds and their antibacterial activity.
Compound
R1
R2
X
MIC90
(
mg/mL)
MIC90
(
mg/mL)
Toxicitya
SI(R)b
NR-Mtb
0.78
R-Mtb
1
H
H
H
O
O
50
1.6
0.031
NDd
2c
5.0
2.5
NDd
6
7
O
O
1.6
6.3
1.6
1.6
0.26
0.50
0.78
0.78
8
9
S
>50
>50
50
<1.0
H
H
O
6.3
6.3
6.3
1.0
10
11
O
O
>50
>10
>50
>10
1.6
1.3
<0.031
<0.13
H
12
13
14
H
H
O
O
O
>50
>50
>10
>50
>50
>10
>50
>50
10
NCc
NCe
<1.0
15
O
>50
>50
>50
NCe
16
17
18
19
O
O
O
O
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.78
3.1
NCe
NCe
<0.016
<0.062
20
21
O
O
>25
>50
>25
>50
>25
NCe
25
<0.50
22
23
O
O
>50
>50
>50
>50
1.6
25
<0.031
<0.50
24
O
>25
>25
25
<1.0
25
26
O
O
O
>10
>50
>25
>10
>50
>25
>10
25
NCe
<0.50
<0.12
27
3.1
a
Toxicity ¼ lethal dose (
mg/mL) for 50% of HepG2 cells (LD50).
SI ¼ selectivity index, lethal dose for 50% of HepG2 cells (LD50)/MIC for R-Mtb. All values are rounded to two significant digits.
b
c
This compound was tested in 96 well format vs. 384 well format for all the other compounds.
ND: not determined.
NC ¼ cannot be calculated.
d
e

340
Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
Table 2
Scaffold II compounds and their antibacterial activity.
Compound
R1
R2
R4
MIC90
(m
g/mL)
MIC90
(m
g/mL)
Toxicitya
SI(R)b
NR-Mtb
R-Mtb
3
50
13
6.3
25
0.50
32
>50
>50
<0.50
33
6.3
1.6
13
8.0
34
35
13
6.3
6.3
>50
>50
>8.0
>8.0
>50
36
>50
>10
>25
>50
>50
>10
>25
>50
>50
>10
25
NCc
NCc
<1.0
NCc
37d
38d
39
>50
40
13
13
13
1.0
41
42
>50
>50
>50
>50
50
<1.0
1.6
<0.031
43
>50
6.3
1.6
0.25

Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
341
Table 2 (continued )
Compound
R1
R2
R4
MIC90
(
m
g/mL)
MIC90
(
mg/mL)
Toxicitya
SI(R)b
0.25
NR-Mtb
R-Mtb
44
50
50
13
45
46
>50
>50
>50
>50
25
<0.50
<0.13
6.3
47
48
>50
25
6.3
3.1
0.25
0.50
50
6.3
a
Toxicity ¼ lethal dose (
mg/mL) for 50% of HepG2 cells (LD50).
SI ¼ selectivity index, lethal dose for 50% of HepG2 cells (LD50)/MIC for R-Mtb. All values are rounded to two significant digits.
b
c
NC ¼ cannot be calculated.
d
5-nitrothiophenolaldehyde was used instead of 5-nitrofuraldehyde.
against Mtb: <0.039
(Fig. 5E), respectively.
mg/mL and 0.31e0.63
mg/mL, for 70 and 49
compounds good candidates for further evaluation and target
identification.
3.2.4. Evaluation of mutagenic potential of benzimidazoles
Mutagenicity is concern in drug development efforts
5. Experimental protocols
a
involving a nitrofuran moiety [22,23]. A convenient method for
assessing the potential of a chemical compound to induce ge-
netic damage is the SOS Chromotest [24e26]. This test was
developed as a complement or alternative to the traditional
Ames test for genotoxicity, and is a good screening tool given its
quick turnaround time. We selected potent study compounds 67,
70 for SOS Chromotest and clinical drugs metronidazole and
isoniazid were evaluated as controls in the absence and presence
of the metabolic activator (S9). The induction factors are shown
in Fig. 6A (-S9) and Fig. 6B (þS9). Metronidazole and 67 (in-
duction factor, IF > 2) were genotoxic either in the presence or in
the absence of an exogenous metabolizing system from rat liver
S9-mix. Isoniazid and 70 were classified as marginally genotoxic
(IF ¼ 1.5e2) in the absence of S9-mix [27]. The LOEC (lowest
5.1. Biology
5.1.1. Bacterial strains and growth conditions
Mycobacterial strains used were M. tuberculosis H37Rv, which
was grown in Middlebrook 7H9 supplemented with 0.2% glycerol,
0.02% tyloxapol, and 10% ADN (albumin, dextrose, NaCl) supple-
ment. M. tuberculosis strain mc26220
DpanCDDlysA was grown in
Middlebrook 7H9 supplemented with 0.5% glycerol, 0.02% tylox-
apol, 10% OADC (oleic acid, albumin, dextrose, catalase), 0.05%
casein hydrolyzate(CAS) amino acids, 240 mg/mL L-lysine and 24 mg/
mL pantothenate. For R conditions, bacteria were plated in their
respective growth media at 37 ꢂC with 20% O2 and 5% CO2. NR
media included 0.5 g of MgSO4, 0.05 g of ferric ammonium citrate,
0.5 g of KH2PO4, 0.5% BSA, 0.085% NaCl, 0.02% tyloxapol, 50
butyrate and 0.5 mM NaNO2 at ph5.0. 240 g/mL -lysine and 24
mL pantothenate was added for mc26220
panCD lysA. For NR
mM
observed effect concentration) value of 70 was 333.3
m
g/mL,
m
L
mg/
which far exceeded its MIC value and was comparable to the
LOEC value of isoniazid [28]. In the presence of S9-mix, 70
revealed no genotoxicity (IF < 1.5) while isoniazid was margin-
ally genotoxic [27]. These results demonstrated a very low
mutagenic potential of 70.
D
D
conditions, bacteria were incubated at 37 ꢂC with 1% O2 and 5% CO2.
M. smegmatis was grown in Middlebrook 7H9 supplemented with
0.2% glycerol, 0.02% tyloxapol at 37 ꢂC in a shaking incubator. Other
bacterial strains used included Salmonella spp (Luria Broth), uro-
pathogenic E. coli (Luria Broth), P. aeruginosa PAO1 (Luria Broth) and
Staphyloccus aureus ATCC 29213 (MuellereHinton broth).
4. Conclusions
In summary, a series of novel benzimidazole-based com-
pounds were designed and synthesized in this study. A struc-
tureeactivity relationship study evaluating activity against
replicating and non-replicating M. tuberculosis (Mtb) was carried
out and potent compounds with acceptable selectivity indices
(SI) were discovered. Several nitrofuranyl benzimidazoles were
selectively bactericidal for mycobacteria and killed Mtb in pri-
mary human macrophages, making them good leads for further
study. Compound 70 potently killed replicating Mtb, although it
lacked non-replicating activity when assessed by a CFU assay.
Data further indicated that 70 had a very low mutagenic po-
tential by SOS Chromotest assay, making this class of
5.1.2. Testing activity of compounds
High throughput screening (HTS) was performed as described
before [9]. Briefly, for R activity, log phase bacteria were diluted to
an OD 0.01 and added (50 mL/well) to 384 well plates. Compound
(500 nL in DMSO) was added (ꢃ1% final DMSO concentration).
Plates were incubated for 7 days, after which the OD580 was read. To
test NR activity, log phase bacteria were washed 2ꢀ with PBS/0.02%
tylaxopol, resuspended in NR medium (as described above) and
cells at a 0.1 OD580 were dispensed into 384 well plates at 15 mL/
well. Compound (150 nl) was added. Plates were incubated for 3
days at 37 ꢂC with 1% O2 and 5% CO2. An outgrowth stage was
performed by addition of 60 mL of R medium and incubation at

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Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
Table 3
Scaffold II compounds and their antibacterial activity.
Compound
R1
R2
R3
MIC90
(
m
g/mL)
MIC90
(
m
g/mL)
Toxicitya
SI(R)b
NR-Mtb
R-Mtb
49
50
51
52
53
54c
3.1
0.20
<0.098
>50
6.3
25
13
64
H
0.78
>50
25
1.6
25
>16
<0.50
0.12
0.12
<1.0
0.78
3.1
50
50
H
>50
>50
>50
55c
56
57
58
59
60
61
62
63c
64
65
H
H
>50
13
>50
3.1
NCd
1.6
2.0
25
13
3.1
0.25
0.12
>32
NCd
>50
0.39
>50
>50
>10
>50
>50
>25
25
3.1
<0.098
3.1
>50
>50
6.3
>50
>10
>50
>50
>25
<0.12
NCd
>10
>50
3.1
NCd
<0.062
<0.50
13
66
25
13
1.6
0.12
a
Toxicity ¼ lethal dose (
mg/mL) for 50% of HepG2 cells (LD50).
SI ¼ selectivity index, lethal dose for 50% of HepG2 cells (LD50)/MIC for R-Mtb. All values are rounded to two significant digits.
b
c
5-nitrofuran was replaced by furan.
NC ¼ cannot be calculated.
d
37 ꢂC with 20% O2 and 5% CO2. OD580 was read after 7 days. Initial
screening was done using the mc26220
panCD lysA strain. For
select compounds, activity was confirmed against wt Mtb. MIC90
(minimal inhibitory concentration) is the measure of activity of
compound against Mtb, defined as 90% inhibition of growth when
compared to vehicle control (DMSO).
To assay colony-forming units (CFU), wt Mtb (200
m
L at OD
D
D
0.01 or 0.1 for R and NR conditions, respectively) was plated in 96
well plates and incubated with compound. After 7 days, bacteria
were diluted and plated onto Middlebrook 7H11 agar plates
containing 0.5% glycerol and 10% OADC. Colonies were counted
after 3 weeks.

Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
343
Table 4
Scaffold III-a and III-b compounds and their antibacterial activity.
Compound
R1
R2
R3
MIC90
(
mg/mL)
MIC90
(
mg/mL)
Toxicitya
SI(R)b
NR-Mtb
>50
R-Mtb
76
77
50
3.1
1.6
0.062
>50
>50
>50
<0.031
<0.016
78
>50
0.78
a
Toxicity ¼ lethal dose (
mg/mL) for 50% of HepG2 cells (LD50).
SI ¼ selectivity index, lethal dose for 50% of HepG2 cells (LD50)/MIC for R-Mtb. All values are rounded to two significant digits.
b
Table 5
Compounds 49, 50, and 59 analogs.
Compound
R1
R2
R3
MIC90
(m
g/mL)
MIC90
(m
g/mL)
Toxicitya
SI(R)b
NR-Mtb
R-Mtb
67
68
69
70
71
72
73
0.78
<0.049
<0.098
0.098
>25
6.3
>512
>64
0.39
0.78
0.20
0.20
0.63
0.20
>25
>25
1.6
>256
>512
>16
<0.049
<0.10
0.039
H
H
>10
3.1
>256
>32
<0.10
74
6.3
0.78
25
32
a
Toxicity ¼ ethal dose (
mg/mL) for 50% of HepG2 cells (LD50).
SI ¼ selectivity index, lethal dose for 50% of HepG2 cells (LD50)/MIC for R-Mtb. All values are rounded to two significant digits.
b

344
Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
Fig. 3. Cidality of 49 against wt Mtb. Compound 49 (A) or vehicle (DMSO) was incubated for 7 days with wt Mtb was plated in R conditions (200
m
L of 5 ꢀ 106/mL) (B) or NR
conditions (200
m
L of 5 ꢀ 107/mL) (C). After 7 days bacteria were resuspended and enumerated on 7H11 agar plates containing OADC and glycerol. Colonies were counted 2.5e3
weeks later. Results are means ꢅ SD of triplicates in one experiment representative of 2 (B) or 3 (C) independent experiments.
5.1.3. Toxicity assays
Aminoanthracene) were included as direct acting and metabolism
dependent positive controls. All the test agents were dissolved in
DMSO at 5 mg/mL and from this eight serial 1:3 dilutions were
prepared.
HepG2 cells, a human hepatocellular line, were grown in Dul-
becco’s Modified Eagle Medium (DMEM) containing 10% fetal
bovine serum (FBS) supplemented with pyruvate, glutamine and
non-essential amino acids. Three thousand cells per well in 384-
well plates were incubated with compound (ꢃ1% final DMSO
concentration) for 2 days. Viability of HepG2 cells was measured by
quantification of ATP content with the CellTiter-Glo kit from
Promega. LD50 was defined as the concentration of compound that
caused a 50% decrease in the ATP signal when compared to the
DMSO control. Selectivity index (SI) was calculated as LD50 divided
In a 96-well plate 50 mL of LB dosing stock was added to 50 mL of
bacteria with or without S9 (þ/ꢄS9). For each dose concentration
duplicate cultures þ/ꢄS9 were tested for mutagenicity as well as a
toxicity control. The plates were incubated at 37 ꢂC with 100 rpm
shaking for 2 h, protected from light.
Test agent mutagenicity and toxicity were determined by
measuring reporter gene activity. Bacteria were lysed and enzyme
activity determined after reaction with a colorimetric substrate.
by MIC90
.
100
mL/well of ONPG Chromobuffer was added to all mutagenicity
plates and 100
mL/well of PNPP Chromobuffer was added to
5.1.4. Macrophage assays
cytotoxicity plates. The plates were returned to the orbital shaker
for 60 min (mutagenicity) or 30 min (toxicity) then absorbance
was determined at 450 nm.
Human macrophages were generated as described [21]. Briefly,
monocytes were isolated from healthy donors under an institu-
tional review board-approved protocol and differentiated in 40%
human plasma with 0.5 ng/mL GM-CSF and 0.5 ng/mL TNF-alpha
under 10% O2, 5% CO2. After 2 weeks, cells were stimulated with
5 ng/mL IFN-gamma and infected with wt Mtb at multiplicity of
infection (MOI) of 0.1. 24 h later, compound was added. After 7 days
of co-incubation, macrophages were visually inspected for viability
and lysed with 0.55% triton-X. Lysates were plated for CFU.
For each dose concentration the ratio of
to alkaline phosphatase units was calculated. This ratio reflects the
induction of the SOS repair gene. -galactosidase activity is
b-galactosidase units
b
directly proportional to the extent of SOS gene induction and this
value is divided by the constitutively expressed alkaline phos-
phatase activity to correct for inhibition of protein synthesis
resulting from cytotoxicity.
5.1.5. Evaluation of mutagenic potential of benzimidazoles
Experimental compounds were screened for microbial muta-
genicity activity using the SOS Chromotest in the presence and
absence of an Aroclor 1254-induced rat-liver metabolic (S9) acti-
vation system. Dilutions of all test agents were prepared in DMSO.
Reference compounds 4NQO (4-nitroquinoline oxide) and 2AA (2-
OD450b ꢀ TP
R ¼
OD450P ꢀ Tb
where OD450b and Tb ¼ absorbance and reaction times for the
b-
galactosidase assay and OD450 P and TP ¼ absorbance and reaction
Fig. 4. Activity of 49 and 70 against wt Mtb infecting human macrophages. Differentiated and activated primary human macrophages were infected with Mtb at a multiplicity of
infection (MOI) of 0.1 and treated with or without compounds for 7 days. Concentrations indicated in parenthesis are in g/mL. At the end of 7 days, macrophages showed no
m
morphologic evidence of toxicity. Excess compound was removed by washing 2ꢀ with PBS, and macrophages were lysed to release intracellular bacteria. Mtb was enumerated by
plating serial dilutions on 7H11 agar plates containing OADC and glycerol. Colonies were counted after 2.5e3 weeks incubation. Input was calculated by plating bacterial content of
macrophages lysed at 24 h post infection, prior to adding any compound. Results are means ꢅ SD of triplicates of one of 3 similar experiments.

Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
345
Fig. 5. Selectivity of benzimidazoles 49 and 70. Log phase Gram-positive (S. aureus) and Gram-negative (Salmonella spp, E. coli, P. aeruginosa) bacteria were diluted to an OD 0.01 and
co-incubated with compounds for 6e10 h at doses up to 10
m
g/mL. Compound 70 was inactive against these bacteria (AeD) when tested at >250ꢀ MIC for Mtb. In contrast,
compounds 49 and 70 were potently active against Mycobacterium smegmatis after an overnight incubation, with an MIC <0.04
Results are means ꢅ SD of triplicates and are representative of two similar experiments.
mg/mL and 0.31e0.625 mg/mL respectively (E).
times for the alkaline phosphatase assay. To compare results be-
RðdÞ
I ¼
tween experiments or between þS9 and ꢄS9 metabolic activation,
R is normalized by dividing R at particular concentration by R(0), R
at zero concentration. This is called the induction factor and is
calculated for each dose concentration R(d).
Rð0Þ
A compound is classified as “not genotoxic” if the induction
factor remains <1.5, as “marginal” if the induction factor is between
Fig. 6. Evaluation of the mutagenic potential of benzimidazoles 67 and 70. The compounds 67 and 70 were evaluation in the absence or presence of metabolic activation (S9). In the
absence of S9 (Fig. 6A), 4NQO was used as positive control. In the presence of S9 (Fig. 6B), 2AA was used as positive control.

346
Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
1.5 and 2.0, and as “genotoxic” if the induction factor exceeds 2.0
and there’s a dose-dependent increase in -galactosidase activity is
found [27]. The LOEC (lowest observed effect concentration) values
and NOEC (no observed effect concentration) values were calcu-
lated as described [28].
J ¼ 3.9 Hz, 1H), 7.50 (s, 1H), 4.53e4.46 (m, 2H), 2.09e1.98 (m, 1H),
1.45e1.20 (m, 2H), 0.89e0.83 (m, 6H). HRMS (ESI): m/z (M þ Hþ)
calcd for C18H18N5O4, 368.1353; found 368.1350.
b
5.2.1.2. 1-Cyclopentyl-2-(5-nitrofuran-2-yl)-1H-imidazo[4,5-g]qui-
noxalin-6(5H)-one (2). Yellow powder, total yield 21%, m.p. 241e
5.2. Chemistry
243 ꢂC 1H NMR (300 MHz, DMSO-d6)
d 12.33 (s, 1H), 8.15 (d,
J ¼ 1.8 Hz,1H), 8.04 (s,1H), 7.93 (d, J ¼ 3.9 Hz,1H), 7.60 (d, J ¼ 3.9 Hz,
Unless otherwise noted, all reagents and starting materials were
purchased from commercial suppliers and used without further
purification. NMR experiments were carried out on a Varian Mer-
cury 300 or 400 or 600 MHz NMR spectrometer using DMSO-d6 as
1H), 7.57 (s, 1H), 5.47e5.36 (m, 1H), 2.25e2.20 (m, 4H), 2.05e2.03
(m, 2H), 1.79e1.76 (m, 2H). 13C NMR (150 MHz, DMSO-d6)
d 154.5,
152.2, 150.7, 145.7, 145.0, 144.7, 130.5, 129.9, 128.8, 116.8, 114.1, 111.9,
104.1, 57.7, 30.0, 24.7. HRMS (ESI): m/z (M þ Hþ) calcd for
the solvent. Chemical shifts were reported in ppm (
d
) relative to the
C18H16N5O4, 366.1202; found 366.1194.
solvent and coupling constants (J) were reported in Hz. Melting
points were determined without correction with a Yanaco micro-
melting point apparatus. Automatic HPLC-MS analysis was per-
formed on a Thermo Finnigan LCQ-Advantage mass spectrometer
equipped with an Agilent pump, an Agilent detector, an Agilent
liquid handler, and a fluent splitter. The column was a Kromasil C18
5.2.1.3. 1-(3-Methoxypropyl)-2-(5-nitrofuran-2-yl)-1H-imidazo[4,5-
g]quinoxalin-6(5H)-one (6). Brown powder, total yield: 23%, m.p.
268e270 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 12.33 (s, 1H), 8.16 (d,
J ¼ 2.4 Hz,1H), 8.14 (s, 1H), 7.95 (d, J ¼ 3.9 Hz,1H), 7.63 (d, J ¼ 3.9 Hz,
1H), 7.55 (s, 1H), 4.70 (t, J ¼ 6.6 Hz, 2 H), 3.34e3.33 (m, 2 H), 3.14 (s,
column (4.6
m
m, 4.6 mm ꢀ 50 mm) from DIKMA for analysis. The
3 H), 2.11 (t, J ¼ 6.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6)
d 154.5,
eluent was a mixture of acetonitrile and water containing 0.05%
HCOOH with a linear gradient from 5:95 (v/v) to 95:5 (v/v) of
acetonitrileewater within 5 min at a 1.0 mL/min flow rate for
analysis. The UV detection was carried out at a wavelength of
254 nm. The 5% of the eluent was split into the MS system. Mass
spectra were recorded in positive ion mode using electrospray
ionization (ESI). High resolution LC-MS was carried out by Agilent
LC/MSD TOF using a column of Agilent ZORBAX SB-C18 (rapid
152.0,150.4,146.1, 144.3,143.9,133.1,130.4,129.0,116.0,114.3,110.3,
103.4, 68.6, 57.8, 42.3, 29.5. HRMS (ESI): m/z (M þ Hþ) calcd for
C17H16N5O5, 370.1146; found 370.1148.
5.2.1.4. 1-(3-Methoxypropyl)-7-methyl-2-(5-nitrofuran-2-yl)-1H-
imidazo[4,5-g]quinoxalin-6(5H)-one (7). Brown powder, total yield:
27%, m.p. >280 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 12.20 (s, 1H),
8.00 (s, 1H), 7.92 (d, J ¼ 3.6 Hz, 1H), 7.58 (d, J ¼ 3.6 Hz, 1H), 7.48 (s,
resolution, 3.5
m
m, 2.1 mm ꢀ 30 mm) at a flow of 0.40 mL/min. The
1H), 4.65e4.62 (m, 2H), 3.34e3.31 (m, 2H), 3.15 (s, 3H), 2.41 (s, 3H),
solvent was methanol/water ¼ 75:25 (v/v) containing 5 mmol/L
ammonium formate. The ion source was electrospray ionization
(ESI). Flash column chromatography was performed with silica gel
60 (200e300 mesh) from Qingdao Haiyang Chemical Factory. The
purities of all tested compounds were ꢁ95%, detected by HPLC
under UV 254 nm wavelength, NMR, melting point, and HPLC-MS.
2.09e2.07 (m, 2H). 13C NMR (150 MHz, DMSO-d6)
d 157.9, 154.5,
151.9, 146.2, 143.7, 142.9, 132.9, 130.1, 129.3, 115.6, 114.4, 109.1, 103.0,
68.7, 57.8, 42.2, 29.4, 20.6. HRMS (ESI): m/z (M þ Hþ) calcd for
C18H18N5O5, 384.1302; found 384.1304.
5.2.1.5. 7-Isobutyl-1-(3-methoxypropyl)-2-(5-nitrothiophen-2-yl)-
1H-imidazo[4,5-g]quinoxalin-6(5H)-one (8). Yellow powder, total
5.2.1. General procedure for the synthesis of compounds 1, 6e27
To a stirred solution of 1,5-difluoro-2,4-dinitrobenzene (DFDNB,
2.04 g, 10 mmol) in THF (50 mL), DIPEA (20 mmol) and NH2(R1)
CH2COOCH3$HCl (10 mmol) was added. After vigorous stirring at
room temperature until the total disappearance of DFDNB, com-
pound 4 was obtained without purification. Then primary amine
(R2NH2) (10 mmol) and DIPEA (10 mmol) were added and stirred
under room temperature or refluxed conditions. The solvent was
removed under reduced pressure to give crude compound 5
without purification. The two reactions above were traced by a fast
LC-MS system until the all of the starting material disappeared.
Compound 5 (1 g) was dissolved in a mixed solvent of THF (30 mL)
and EtOH (30 mL), followed by addition of 10% Pd/C (1 g) and
HCOONH4 (2 g). The reaction mixture stirred at room temperature
for 2 h. Then the reaction solution was directly filtered into another
reaction vessel containing 5-nitro-furaldehyde (1.2 equiv) or 5-
nitro-thiophenolaldehyde (1.2 equiv) in dioxane with 5% AcOH.
The reaction mixture stirred at room temperature or refluxed
conditions for 3e5 h. Then 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ) was added to the reaction mixture under
reflux for 3e5 h and the expected compounds 1, 6e27 were ob-
tained. Following evaporation of the solvent in vacuo, the final
products were characterized after purification by silica gel column
chromatography.
yield: 32%, m.p. >280 ꢂC. 1H NMR (400 MHz, DMSO-d6)
d 12.24 (s,
1H), 8.26 (d, J ¼ 4.4 Hz, 1H), 8.11 (s, 1H), 7.88 (d, J ¼ 4.4 Hz, 1H), 7.49
(s, 1H), 4.66 (t, J ¼ 6.7 Hz, 2H), 3.36e3.33 (m, 2H), 3.19 (s, 3H), 2.70
(d, J ¼ 7.0 Hz, 2H), 2.35e2.22 (m, 1H), 2.07 (t, J ¼ 6.1 Hz, 2H), 0.97 (d,
J ¼ 6.6 Hz, 6H). HRMS (ESI): m/z (M þ Hþ) calcd for C21H24N5O4S,
442.1544; found 442.1543.
5.2.1.6. 1-(tert-Butyl)-2-(5-nitrofuran-2-yl)-1H-imidazo[4,5-g]qui-
noxalin-6(5H)-one (9). Brown powder, total yield: 27%, m.p.
>270 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 12.32 (s, 1H), 8.37 (s, 1H),
8.18 (s, 1H), 7.91 (d, J ¼ 3.6 Hz, 1H), 7.55 (s, 1H), 7.39 (d, J ¼ 3.6 Hz,
1H), 1.71 (s, 9H). HRMS (ESI): m/z (M þ Hþ) calcd for C17H16N5O4,
354.1197; found 354.1203.
5.2.1.7. 2-(5-Nitrofuran-2-yl)-1-(2-phenylpropyl)-1H-imidazo[4,5-g]
quinoxalin-6(5H)-one (10). Yellow powder, total yield: 31%,
m.p.277e279 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 12.28 (s, 1H), 8.11
(d, J ¼ 1.5 Hz, 1H), 8.04 (s, 1H), 7.86 (d, J ¼ 3.6 Hz, 1H), 7.46e7.45 (m,
2H), 7.08e7.04 (m, 5H), 4.88e4.69 (m, 2H), 3.39e3.35 (m, 1H), 1.31
(d, J ¼ 7.2 Hz, 3H). 13C NMR (150 MHz, DMSO-d6)
d 154.4, 151.7,
150.3, 146.1, 144.2, 143.7, 142.5, 133.0, 130.2, 128.8, 128.0, 127.2,
126.6, 116.0, 114.2, 111.0, 103.1, 51.5, 18.0. HRMS (ESI): m/z (M þ Hþ)
calcd for C22H18N5O4, 416.1353; found 416.1354.
5.2.1.8. 1-(3,4-Dimethoxyphenethyl)-2-(5-nitrofuran-2-yl)-1H-imi-
5.2.1.1. 1-(2-Methylbutyl)-2-(5-nitrofuran-2-yl)-1H-imidazo[4,5-g]
dazo[4,5-g]quinoxalin-6(5H)-one (11). Yellow powder, total yield:
quinoxalin-6(5H)-one (1). Yellow powder, total yield: 23%,
34%, m.p. 279e281 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 12.33 (s, 1H),
m.p.282e283 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d
12.30 (s, 1H), 8.17
8.21 (s, 1H), 8.16 (s, 1H), 7.86 (d, J ¼ 3.9 Hz, 1H), 7.53 (s, 1H), 7.45 (d,
(s, 1H), 8.13 (d, J ¼ 1.5 Hz, 1H), 7.91 (d, J ¼ 3.6 Hz, 1H), 7.61 (d,
J ¼ 3.9 Hz,1H), 6.70e6.67 (m, 2H), 6.56 (d, J ¼ 7.8 Hz,1H), 4.87e4.85

Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
347
(m, 2H), 3.62 (s, 3H), 3.59 (s, 3H), 3.04e3.02 (m, 2H). 13C NMR
(150 MHz, DMSO-d6) 154.5, 151.9, 150.4, 148.5, 147.6, 146.3, 144.4,
114.3, 109.6, 102.8, 44.9, 31.1, 29.8, 29.7, 28.1, 25.9, 21.9, 20.1, 13.8.
HRMS (ESI): m/z (M þ Hþ) calcd for C23H28N5O4, 438.2136; found
438.2144.
d
144.0, 132.7, 130.3, 129.8, 129.1, 121.0, 115.9, 114.2, 112.7, 111.7, 110.8,
103.3, 55.5, 55.2, 46.6, 35.1. HRMS (ESI): m/z (M þ Hþ) calcd for
C
23H20N5O6, 462.1408; found 462.1406.
5.2.1.15. 1-Cyclohexyl-7-isopropyl-2-(5-nitrofuran-2-yl)-1H-imidazo
[4,5-g]quinoxalin-6(5H)-one (18). Yellow powder, total yield: 12%,
5.2.1.9. 2-(5-Nitrofuran-2-yl)-1-nonyl-1H-imidazo[4,5-g]quinoxalin-
m.p. 251e253 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 12.22 (s, 1H), 8.18
6(5H)-one (12). Yellow powder, total yield: 25%, m.p.233e235 ꢂC.
(s, 1H), 7.91 (d, J ¼ 3.9 Hz, 1H), 7.53 (d, J ¼ 3.9 Hz, 1H), 7.51 (s, 1H),
5.00e4.87 (m, 1H), 3.48e3.45 (m, 1H), 2.49e2.48 (m, 2H), 2.07e
1.90 (m, 4H), 1.78e1.75 (m, 1H), 1.53e1.51 (m, 3H), 1.24 (d,
1H NMR (300 MHz, DMSO-d6)
d 12.32 (s, 1H), 8.19 (s, 1H), 8.14 (d,
J ¼ 2.1 Hz, 1H), 7.93 (d, J ¼ 3.9 Hz, 1H), 7.61 (d, J ¼ 3.9 Hz,1H), 7.52 (s,
1H), 4.62 (t, J ¼ 7.2 Hz, 2H), 1.83e1.81 (m, 2H), 1.33e1.18 (m, 12H),
J ¼ 6.6 Hz, 6H). 13C NMR (150 MHz, DMSO-d6)
d 164.5, 153.7, 151.9,
146.0, 143.8, 143.7, 131.1, 129.5, 128.6, 116.4, 114.1, 111.7, 103.2, 57.3,
0.81 (d, J ¼ 6.5 Hz, 3H). 13C NMR (150 MHz, DMSO-d6)
d
154.5,152.0,
150.3,146.1, 144.1,143.9,133.0,130.3,129.0,116.0,114.3,110.4,103.3,
44.9, 31.1, 29.6, 28.8, 28.5, 28.4, 25.9, 21.9, 13.8. HRMS (ESI): m/z
(M þ Hþ) calcd for C22H26N5O4, 424.1979; found 424.1985.
30.7, 29.8, 25.4, 24.2, 20.1. HRMS (ESI): m/z (M þ Hþ) calcd for
C22H24N5O4, 422.1823; found 422.1825.
5.2.1.16. 7-Isobutyl-1-(3-methoxypropyl)-2-(5-nitrofuran-2-yl)-1H-
5.2.1.10. 1-(3,3-Diphenylpropyl)-2-(5-nitrofuran-2-yl)-1H-imidazo
imidazo[4,5-g]quinoxalin-6(5H)-one (19). Yellow powder, total
[4,5-g]quinoxalin-6(5H)-one (13). Yellow powder, total yield: 26%,
yield: 23%, m.p. 249e250 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 12.23
m.p.>260 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d
12.31 (s, 1H), 8.14 (d,
(s, 1H), 8.08 (s, 1H), 7.95 (d, J ¼ 3.9 Hz, 1H), 7.61 (d, J ¼ 3.9 Hz, 1H),
7.52 (s, 1H), 4.68 (t, J ¼ 6.9 Hz, 2H), 3.38e3.32 (m, 2H), 3.16 (s, 3H),
2.70 (d, J ¼ 6.6 Hz, 2H), 2.31e2.23 (m, 1H), 2.13e2.09 (m, 2H), 0.97
(d, J ¼ 6.9 Hz, 6H). HRMS (ESI): m/z (M þ Hþ) calcd for C21H24N5O5,
426.1772; found 426.1776.
J ¼ 2.1 Hz, 1H), 7.90 (s, 1H), 7.84 (d, J ¼ 4.2 Hz, 1H), 7.50 (s, 1H), 7.42
(d, J ¼ 4.2 Hz, 1H), 7.30e7.10 (m, 10H), 4.54 (t, J ¼ 7.0 Hz, 2H), 4.06 (t,
J ¼ 7.8 Hz, 1H), 2.66 (q, J ¼ 7.5 Hz, 2H). 13C NMR (150 MHz MHz,
DMSO-d6)
d 154.5, 151.9, 150.4, 145.9, 144.2, 144.0, 143.9, 132.6,
130.2, 129.0, 128.5, 127.4, 126.2, 116.1, 114.2, 110.4, 103.5, 48.1, 43.9,
34.3. HRMS (ESI): m/z (M þ Hþ) calcd for C28H22N5O4, 492.1666;
found 492.1668.
5.2.1.17. 1-(2-(Cyclohex-1-en-1-yl)ethyl)-7-isobutyl-2-(5-nitrofuran-
2-yl)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one (20). Yellow powder,
total yield: 13%, m.p. 255e256 ꢂC. 1H NMR (300 MHz, DMSO-d6)
5.2.1.11. 7-Methyl-2-(5-nitrofuran-2-yl)-1-phenethyl-1H-imidazo
d
12.22 (s,1H), 8.06 (s,1H), 7.95 (d, J ¼ 4.2 Hz,1H), 7.60 (d, J ¼ 4.2 Hz,
[4,5-g]quinoxalin-6(5H)-one (14). Yellow powder, total yield: 18%,
1H), 7.49 (s, 1H), 5.04 (s, 1H), 4.71 (t, J ¼ 6.0 Hz, 2H), 2.68 (d,
J ¼ 6.9 Hz, 2H), 2.38e2.36 (m, 2H), 2.30e2.23 (m, 1H), 1.95e1.92 (m,
2H), 1.68e1.65 (m, 2H), 1.33e1.31 (m, 4H), 0.95 (d, J ¼ 6.3 Hz, 6H).
m.p. 255e256 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 12.23 (s, 1H), 8.11
(s, 1H), 7.86 (d, J ¼ 4.2 Hz, 1H), 7.49 (s, 1H), 7.44 (d, J ¼ 4.2 Hz, 1H),
7.17e7.14 (m, 5H), 4.85 (t, J ¼ 6.6 Hz, 2H), 3.13e3.11 (m, 2H), 2.40 (s,
3H). HRMS (ESI): m/z (M þ Hþ) calcd for C22H18N5O4, 416.1353;
found 416.1359.
13C NMR (150 MHz, DMSO-d6)
d 159.8, 154.2, 151.6, 146.4, 143.6,
142.8, 133.3, 132.5, 129.8, 128.9, 123.8, 115.5, 114.3, 109.6, 102.7, 43.7,
41.3, 37.2, 27.4, 26.0, 24.4, 22.3, 21.9, 21.2. HRMS (ESI): m/z (M þ Hþ)
calcd for C25H28N5O4, 462.2136; found 462.2139.
5.2.1.12. 1-(3,4-Dimethoxyphenethyl)-7-isopropyl-2-(5-nitrofuran-2-
yl)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one (15). Yellow powder,
total yield: 23%, m.p. 265e267 ꢂC. 1H NMR (300 MHz, DMSO-d6)
5.2.1.18. 1-(1-Benzylpiperidin-4-yl)-7-isobutyl-2-(5-nitrofuran-2-
yl)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one (21). Yellow powder,
d
12.23 (s, 1H), 8.16 (s, 1H), 7.84 (d, J ¼ 4.2 Hz, 1H), 7.50 (s, 1H), 7.40
total yield: 21%, m.p. >260 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 12.24
(d, J ¼ 4.2 Hz, 1H), 6.69e6.53 (m, 3H), 4.86 (t, J ¼ 6.9 Hz, 2H), 3.62 (s,
3H), 3.59 (s, 3H), 3.52e3.45 (m, 1H), 3.03 (t, J ¼ 6.9 Hz, 2H), 1.05 (d,
(s, 1H), 8.07 (s, 1H), 7.93 (d, J ¼ 3.9 Hz, 1H), 7.55 (d, J ¼ 3.9 Hz, 1H),
7.53 (s, 1H), 7.41e7.26 (m, 5H), 5.03e5.01 (m, 1H), 3.59 (s, 2H),
3.05e3.04 (m, 2H), 2.72 (d, J ¼ 6.9 Hz, 2H), 2.58e2.56 (m, 2H), 2.31e
2.22 (m, 3H), 2.02e2.00 (m, 2H), 0.97 (d, J ¼ 6.6 Hz, 6H). HRMS
(ESI): m/z (M þ Hþ) calcd for C29H31N6O4, 527.2401; found
527.2390.
J ¼ 6.9 Hz, 6H). 13C NMR (150 MHz, DMSO-d6)
d 164.8, 154.3, 152.3,
149.0, 148.1, 147.0, 144.4, 143.6, 133.1, 130.4, 130.3, 129.5, 121.5, 116.1,
114.8, 113.3, 112.2, 110.5, 103.4, 56.0, 55.7, 46.9, 35.6, 30.4, 20.7.
HRMS (ESI): m/z (M þ Hþ) calcd for C26H26N5O6, 504.1878; found
504.1882.
5.2.1.19. 1-(Heptan-2-yl)-7-isobutyl-2-(5-nitrofuran-2-yl)-1H-imi-
5.2.1.13. 7-Isopropyl-2-(5-nitrofuran-2-yl)-1-(3-phenylpropyl)-1H-
dazo[4,5-g]quinoxalin-6(5H)-one (22). Yellow powder, total yield:
imidazo[4,5-g]quinoxalin-6(5H)-one (16). Yellow powder, total
14%, m.p. 219e220 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 12.23 (s, 1H),
yield: 22%, m.p. 269e270 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d
12.22
8.17 (s, 1H), 7.93 (d, J ¼ 4.2 Hz, 1H), 7.57 (d, J ¼ 4.2 Hz, 1H), 7.53 (s,
1H), 5.15e5.10 (m, 1H), 2.68 (d, J ¼ 6.9 Hz, 2H), 2.28e2.18 (m, 2H),
1.99e1.95 (m, 1H), 1.70 (d, J ¼ 6.9 Hz, 3H), 1.15e1.14 (m, 4H), 0.96e
0.94 (m, 8H), 0.71 (t, J ¼ 6.6 Hz, 3H). 13C NMR (150 MHz, DMSO-d6)
(s, 1H), 8.13 (s, 1H), 7.89 (d, J ¼ 4.2 Hz, 1H), 7.52 (d, J ¼ 4.2 Hz, 1H),
7.50 (s, 1H), 7.23e7.19 (m, 5H), 4.66 (t, J ¼ 6.0 Hz, 2H), 3.51e3.47 (m,
1H), 2.74e2.71 (m, 2H), 2.17e2.15 (m, 2H), 1.24 (d, J ¼ 6.6 Hz, 6H).
13C NMR (150 MHz, DMSO-d6)
d
164.3, 153.7, 152.0, 146.1, 143.6,
d 160.3, 154.4, 152.0, 145.9, 144.1, 143.8, 130.7, 129.7, 128.8, 116.6,
143.0, 141.0, 132.8, 129.9, 129.0, 128.2, 128.1, 125.8, 115.6, 114.3,
109.6, 102.9, 44.7, 32.1, 31.4, 29.8, 20.1. HRMS (ESI): m/z (M þ Hþ)
calcd for C25H24N5O4, 458.1823; found 458.1831.
114.1, 111.4, 103.4, 53.7, 41.5, 34.0, 30.5, 26.4, 25.4, 22.6, 21.7, 19.5,
13.6. HRMS (ESI): m/z (M þ Hþ) calcd for C24H30N5O4, 452.2292;
found 452.2300.
5.2.1.14. 1-Heptyl-7-isopropyl-2-(5-nitrofuran-2-yl)-1H-imidazo
5.2.1.20. 1-(Benzo[d][1,3]dioxol-5-ylmethyl)-7-isobutyl-2-(5-
[4,5-g]quinoxalin-6(5H)-one (17). Yellow powder, total yield: 16%,
nitrofuran-2-yl)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one
Yellow powder, total yield: 27%，m.p. >260 ꢂC. 1H NMR (300 MHz,
DMSO-d6)
(23).
m.p. 210e211 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 12.23 (s, 1H), 8.14
(s, 1H), 7.93 (d, J ¼ 4.2 Hz, 1H), 7.59 (d, J ¼ 4.2 Hz, 1H), 7.50 (s, 1H),
4.62 (t, J ¼ 7.2 Hz, 2 H), 3.51e3.45 (m, 1 H), 1.83e1.80 (m, 2 H), 1.36e
1.01 (m, 11 H), 0.83 (d, J ¼ 7.2 Hz, 6H). 13C NMR (150 MHz, DMSO-d6)
d
12.21 (s,1H), 8.08 (s,1H), 7.85 (d, J ¼ 4.2 Hz,1H), 7.52 (d,
J ¼ 4.2 Hz, 1H), 7.50 (s, 1H), 6.80e6.60 (m, 3H), 5.89 (s, 2H), 5.78 (s,
2H), 2.62 (d, J ¼ 6.6 Hz, 2H), 2.27e2.18 (m, 1H), 0.90 (d, J ¼ 6.6 Hz,
d
164.3, 153.7, 151.9, 146.2, 143.5, 142.9, 132.9, 129.9, 129.0, 115.7,
6H). 13C NMR (150 MHz, DMSO-d6)
d 160.2, 154.3, 151.9, 147.5, 146.7,

348
Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
145.8,143.7,143.0,132.9,130.1,130.0,129.3,120.1,116.0,114.3,109.8,
108.4, 107.3, 103.1, 101.1, 47.6, 41.5, 26.2, 22.5. HRMS (ESI): m/z
(M þ Hþ) calcd for C25H22N5O6, 488.1565; found 488.1566.
expected product 3, 32e38, 41, 45, 46, 49e51, 59e64, 67, 70e74
were obtained. After the reaction was completed, the solvent was
evaporated in vacuo. The final products were characterized after
purification by silica gel column chromatography.
5.2.1.21. 1-Benzyl-7-isobutyl-2-(5-nitrofuran-2-yl)-1H-imidazo[4,5-
g]quinoxalin-6(5H)-one (24). Yellow powder, total yield: 30%, m.p.
5.2.2.2. General procedure B for the synthesis of compounds 42e44.
According to the general procedure A, the intermediate 31 was
obtained. To a stirred solution of compound 31 (1 mmol) (without
purification) in dry CH2Cl2, various isocyanates (2 mmol) or iso-
thiocyanates (2 mmol) were added. The reaction mixture was
refluxed for 3e5 h, the expected product 42e44 was obtained. After
the reaction was completed, the solvent was evaporated in vacuo.
The final products were characterized after purification by silica gel
column chromatography.
>260 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 12.25 (s, 1H), 8.11 (s, 1H),
7.88 (d, J ¼ 3.9 Hz, 1H), 7.54 (d, J ¼ 3.9 Hz, 1H), 7.53 (s, 1H), 7.34e7.18
(m, 5H), 5.97 (s, 2H), 2.66 (d, J ¼ 6.9 Hz, 2H), 2.28e2.30 (m, 1H), 0.90
(d, J ¼ 6.6 Hz, 6H). HRMS (ESI): m/z (M þ Hþ) calcd for C24H22N5O4,
444.1666; found 444.1668.
5.2.1.22. 7-Benzyl-1-cyclohexyl-2-(5-nitrofuran-2-yl)-1H-imidazo
[4,5-g]quinoxalin-6(5H)-one (25). Yellow powder, total yield: 24%,
m.p. 237e239 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 12.32 (s, 1H), 8.25
(s, 1H), 7.92 (d, J ¼ 3.9 Hz, 1H), 7.54 (d, J ¼ 3.9 Hz, 1H), 7.53 (s, 1H),
7.38e7.13 (m, 5H), 4.83e4.88 (m, 1H), 4.15 (s, 2H), 2.38e2.34 (m,
2H), 1.99e1.90 (m, 4H), 1.69e1.67 (m, 1H), 1.51e1.48 (m, 3H). 13C
5.2.2.3. General procedure C for the synthesis of compounds 65, 68
and 69. To a stirred solution of DFDNB (2.04 g, 10 mmol) in THF
(50 mL), DIPEA (10 mmol) and thiomorpholine (10 mmol) were
added. After vigorous stirring at room temperature until DFDNB
completely disappeared, compound 28 (R2 ¼ thiomorpholine) was
obtained without purification. Then primary amine R1NH2
(10 mmol) and DIPEA (10 mmol) were added and stirred at room
temperature overnight. The solvent was removed under reduced
pressure to give compound 29. Compound 29 (1 mmol) was dis-
solved in a mixed solvent of THF (30 mL) and EtOH (30 mL), fol-
lowed by the addition of sodium hydrosulfite (Na2S2O4, 20 mmol),
K2CO3 (20 mmol) and H2O (30 mL) at 50 ꢂC, the reaction mixture
was stirred at 50 ꢂC for 2 h. The formation of compound 30 was
traced by a fast LC-MS system. The solvent was evaporated in vacuo
and extracted with EtOAc (3 ꢀ 30 mL). The organic layer was
washed with saturated NaCl, dried with anhydrous Na2SO4 and
evaporated. The residual solid 30 (1 mmol) was dissolved in a
mixed solvent of THF (25 mL), EtOH (25 mL) after which dioxane
(10 mL), aldehyde (R3CHO, 1.2 mmol) and 5% HOAc were added. The
mixture was neutralized with NaHCO3 and extracted with CH2Cl2.
The organic layer was washed with H2O, dried with anhydrous
Na2SO4 and evaporated, resulting in 31. To a stirred solution of
compound 31 (1 mmol) (without purification) in dry CH2Cl2,
various acyl chlorides (1.2 mmol) and Et3N (1.2 mmol) were added.
The reaction mixture was stirred at room temperature for 3e5 h
and the expected product 65, 68 and 69 was obtained. After the
reaction was completed, the solvent was evaporated in vacuo. The
final products were characterized after purification by silica gel
column chromatography.
NMR (150 MHz, DMSO-d6)
d 159.0, 154.0, 151.8, 145.8, 143.8, 143.7,
137.3, 131.0, 129.4, 128.8, 128.7, 128.1, 126.1, 116.4, 113.9, 111.7, 103.1,
57.1, 30.5, 25.3, 24.0. HRMS (ESI): m/z (M þ Hþ) calcd for
C
26H24N5O4, 470.1823; found 470.1825.
5.2.1.23. 7-Benzyl-2-(5-nitrofuran-2-yl)-1-nonyl-1H-imidazo[4,5-g]
quinoxalin-6(5H)-one (26). Yellow powder, total yield: 26%, m.p.
230e231 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 12.31 (s, 1H), 8.14 (s,
1H), 7.93 (d, J ¼ 4.2 Hz, 1H), 7.61 (d, J ¼ 4.2 Hz, 1H), 7.50 (s, 1H),
7.36e7.18 (m, 5H), 4.60 (t, J ¼ 6.9 Hz, 2H), 4.14 (s, 2H), 1.81e1.79 (m,
2H), 1.36e1.18 (m, 12H), 0.80 (d, J ¼ 6.3 Hz, 3H). HRMS (ESI): m/z
(M þ Hþ) calcd for C29H32N5O4, 514.2449; found 514.2445.
5.2.1.24. 7-Benzyl-1-(2-(cyclohex-1-en-1-yl)ethyl)-2-(5-nitrofuran-
2-yl)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one (27). Yellow powder,
total yield: 18%, m.p. 135e137 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d
12.30 (s, 1H), 8.07 (s, 1H), 7.94 (d, J ¼ 4.2 Hz,1H), 7.61 (d, J ¼ 4.2 Hz,
1H), 7.50 (s, 1H), 7.35e7.17 (m, 5H), 5.02 (s, 1H), 4.70 (t, J ¼ 6.6 Hz,
2H), 4.14 (s, 2H), 2.36e2.35 (m, 2H), 1.91e1.90 (m, 2H), 1.65e1.63
(m, 2H), 1.31e1.27 (m, 4H). 13C NMR (150 MHz, DMSO-d6)
d 159.1,
154.2, 151.9, 146.6, 144.0, 143.3, 137.5, 133.5, 132.8, 130.0, 129.3,
129.2, 128.3, 126.3, 124.1, 115.8, 114.5, 110.0, 103.0, 44.0, 37.4, 32.4,
27.7, 24.6, 22.2, 21.4. HRMS (ESI): m/z (M þ Hþ) calcd for
C
28H26N5O4, 496.1979; found 496.1971.
5.2.2. General procedure for 1H-benzo[d]imidazole compounds
5.2.2.1. General procedure A for the synthesis of compounds 3, 32e
38, 41, 45, 46, 49e51, 59e64, 67, 70e74. To a stirred solution of
DFDNB (2.04 g, 10 mmol) in THF (50 mL), DIPEA (10 mmol) and
secondary amine (10 mmol) were added. After vigorous stirring at
room temperature until the total disappearance of DFDNB, com-
pound 28 was obtained without purification. Then primary amine
R1NH2 (10 mmol) and DIPEA (10 mmol) were added and stirred at
room temperature overnight. The solvent was removed under
reduced pressure to yield compound 29. The above two reactions
were traced by a fast LC-MS system until all of the starting material
was changed to the anticipated compound. Compound 29 (1 g) was
dissolved in a mixed solvent of THF (30 mL) and EtOH (30 mL),
followed by the addition of 10% Pd/C (1 g) and HCOONH4 (2 g). The
formation of compound 30 was traced by a fast LC-MS system. Then
the reaction solution was directly filtered into another reaction
vessel containing the aldehyde (R3CHO, 1.2 equiv) in dioxane with
5% HOAc to yield compound 31. After the reaction was completed,
the solvent was removed in vacuo. To a stirred solution of com-
pound 31 (1 mmol) (without purification) in dry CH2Cl2, various
acyl chlorides (1.2 mmol) and Et3N (1.2 mmol) were added. The
reaction mixture was stirred at room temperature for 3e5 h and the
5.2.2.4. General procedure D for the synthesis of compounds 47, 48,
52e58 and 66. To a stirred solution of alcohols (1 mmol) in THF
NaH (1 mmol) was added at 0 ꢂC, then the reaction mixture was
refluxed for 2 h to give the sodium alcoholate. To a solution of
DFDNB (1 mmol) in THF (20 mL), the above sodium alcoholate
solvent was added dropwise. The reaction mixture was refluxed for
3e5 h with stirring and then washed with saturated NH4Cl
aqueous, followed by extraction with DCM. The organic layer was
condensed in vacuo and dried with anhydrous Na2SO4. Compound
28 (R2 ¼ alcohol) was obtained after purification. To a solution of 28
(1 mmol) in THF (20 mL), various primary amines (10 mmol) and
DIPEA (10 mmol) were added. The mixture was stirred at room
temperature overnight and compound 29 was obtained after
evaporating the solvent in vacuo. Compound 29 (1 g) was dissolved
in a mixed solvent of THF (30 mL) and EtOH (30 mL), followed by
the addition of 10% Pd/C (1 g) and HCOONH4 (2 g). The formation of
compound 30 was monitored by a fast LC-MS system. Then the
reaction solution was directly filtered into another reaction vessel
containing the aldehydes (R3CHO, 1.2 equiv) in dioxane with 5%
HOAc to yield intermediate 31. After the reaction was completed,

Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
349
the solvent was condensed in vacuo. To a stirred solution of inter-
mediate 31 (1 mmol) (without purification) in dry CH2Cl2, various
acyl chlorides (1.2 mmol) and Et3N (1.2 mmol) were added. The
reaction mixture was stirred at room temperature for 3e5 h, and
the expected products 47, 48, 52e58 and 66 were obtained. After
the reaction was completed, the solvent was evaporated in vacuo.
The final products were characterized after purification by silica gel
column chromatography.
J ¼ 4.2 Hz, 1H), 4.63 (t, J ¼ 6.3 Hz, 2H), 3.79e3.78 (m, 4H), 3.34e
3.31 (m, 2H), 3.17 (s, 3H), 2.96-2.95 (m, 4H), 2.12-2.07 (m, 2H). 13
NMR (150 MHz, DMSO-d6) 162.8, 151.7, 146.9, 142.8, 141.2, 139.0,
C
d
135.7, 135.1, 133.5, 131.8, 131.0, 130.2, 129.2, 118.2, 114.7, 114.4,
112.8, 111.9, 103.2, 68.6, 66.7, 58.0, 52.6, 41.9, 29.6. HRMS (ESI): m/
z (M þ Hþ) calcd for C27H27N6O6, 531.1987; found 531.1997.
5.2.2.5.6. 3-(2-Chlorophenyl)-N-(1-(3-methoxypropyl)-6-
morpholino-2-(5-nitrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)prop-
anamide (36). Procedure A was used. Yellow powder, total yield:
5.2.2.5. General procedure E for the synthesis of compounds 39 and
40. According to the general procedure D, intermediate 31
(R2 ¼ alcohol) was obtained. To a stirred solution of compound 31
(1 mmol) (without purification) in dry CH2Cl2, various isocyanates
(2 mmol) or isothicyanates (2 mmol) were added. The reaction
mixture was refluxed for 3e5 h, and the expected product 39 and
40 were obtained. After the reaction was completed, the solvent
was condensed in vacuo. The final products were characterized
after purification by silica gel column chromatography.
21%, m.p. 253e254 ꢂC. 1H NMR (400 MHz, DMSO-d6)
d 9.33 (s, 1H),
8.53 (s, 1H), 7.95e7.91 (m, 3H), 7.58e7.52 (m, 3H), 7.47e7.45 (m,
2H), 7.26 (d, J ¼ 15.6, 1H), 4.62 (t, J ¼ 6.7 Hz, 2H), 3.91e3.89 (m, 4H),
3.35e3.33 (m, 2H), 3.19 (s, 3H), 2.95e2.93 (m, 4H), 2.13-2.07 (m,
2H). 13C NMR (150 MHz, DMSO-d6)
d 162.9, 151.6, 147.0, 141.7, 140.9,
138.9, 135.2, 133.4, 132.8, 132.5, 131.1, 129.9, 129.3, 127.8, 127.6,
125.6,114.6,114.2,111.5,102.5, 68.5, 66.1, 57.9, 52.6, 41.7, 29.6. HRMS
(ESI): m/z (M þ Hþ) calcd for C28H29ClN5O6, 566.1801; found
566.1807.
5.2.2.5.1. N-(6-(4-Methylpiperazin-1-yl)-2-(5-nitrofuran-2-yl)-1-
pentyl-1H-benzo[d]imidazol-5-yl)acetamide (3). Procedure A was
used. Yellow powder, total yield: 33%, m.p. 186e187 ꢂC. 1H NMR
5.2.2.5.7. 3-(2-Chlorophenyl)-N-(1-(3-methoxypropyl)-6-
morpholino-2-(5-nitrothiophen-2-yl)-1H-benzo[d]imidazol-5-yl)
propanamide (37). Procedure A was used. Brown powder, total
(300 MHz, DMSO-d6)
d
8.87 (s, 1H), 8.23 (s, 1H), 7.91 (d, J ¼ 3.9 Hz,
yield: 26%, m.p. 280 ꢂC decom. 1H NMR (300 MHz, DMSO-d6)
d 9.32
1H), 7.47 (d, J ¼ 3.9 Hz, 2H), 4.54 (t, J ¼ 7.4 Hz, 2H), 2.91 (br, 4H),
2.58e2.56 (m, 4H), 2.28 (s, 3H), 2.14 (s, 3H), 1.81e1.80 (m, 2H),
1.34e1.32 (m, 4H), 0.85 (t, J ¼ 6.9 Hz, 3H). HRMS (ESI): m/z (M þ Hþ)
calcd for C23H31N5O4, 454.2329; found 454.2335.
(s, 1H), 8.48 (s, 1H), 8.22e8.21 (m, 1H), 7.94e7.89 (m, 2H), 7.76e7.75
(m, 1H), 7.54e7.52 (m, 2H), 7.45e7.43 (m, 2H), 7.27e7.22 (m, 1H),
4.58e4.57 (m, 2H), 3.89e3.88 (m, 4H), 3.32e3.31 (m, 2H), 3.21 (s,
3H), 2.92e2.91 (m, 4H), 2.04e2.02 (m, 2H). HRMS (ESI): m/z
(M þ Hþ) calcd for C28H29ClN5O5S, 582.1572; found 582.1574.
5.2.2.5.8. 3-Cyano-N-(1-(3-methoxypropyl)-6-morpholino-2-
(5-nitrothiophen-2-yl)-1H-benzo[d]imidazol-5-yl)benzamide (38).
Procedure A was used. Brown powder, total yield: 23%, m.p. 255e
5.2.2.5.2. N-(1-(3-Methoxypropyl)-6-morpholino-2-(5-
nitrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)butyramide
Procedure A was used. Yellow powder, total yield: 23%, m.p. 182e
183 ꢂC. 1H NMR (300 MHz, DMSO-d6)
8.95 (s, 1H), 8.27 (s, 1H), 7.91
(32).
d
(d, J ¼ 3.9 Hz, 1H), 7.49 (d, J ¼ 3.9 Hz, 1H) 7.47 (s, 1H), 4.59 (t,
J ¼ 6.8 Hz, 2H), 3.84e3.82 (m, 4H), 3.31e3.28 (m, 2H), 3.16 (s, 3H),
2.90e2.80 (m, 4H), 2.41 (t, J ¼ 7.4 Hz, 2H), 2.09e2.05 (m, 2H), 1.68e
1.61 (m, 2H), 0.93 (t, J ¼ 7.4 Hz, 3H). HRMS (ESI): m/z (M þ Hþ) calcd
for C23H30N5O6, 472.2191; found 472.2195.
256 ꢂC decom. 1H NMR (400 MHz, DMSO-d6)
d 9.96 (s, 1H), 8.40 (s,
1H), 8.34 (s, 1H), 8.29 (d, J ¼ 7.8 Hz, 1H), 8.24 (d, J ¼ 4.5 Hz, 1H),
8.10 (d, J ¼ 7.8 Hz, 1H), 7.84e7.79 (m, 2H), 7.62 (s, 1H), 4.61 (t,
J ¼ 6.9 Hz, 2H), 3.80 (t, J ¼ 4.2 Hz, 4H), 3.34e3.32 (m, 2H), 3.21 (s,
3H), 2.96 (t, J ¼ 4.2 Hz, 4H), 2.05 (t, J ¼ 6.3 Hz, 2H). HRMS (ESI): m/z
(M þ Hþ) calcd for C27H27N6O5S, 547.1758; found 547.1759.
5.2.2.5.9. 1-(6-Methoxy-1-(3-methoxypropyl)-2-(5-nitrofuran-
2-yl)-1H-benzo[d]imidazol-5-yl)-3-(4-methoxyphenyl)urea (39).
Procedure E was used. Brown powder, total yield: 17%, m.p. 255e
5.2.2.5.3. 4-Fluoro-N-(1-(3-methoxypropyl)-6-morpholino-2-
(5-nitrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)benzamide
Procedure A was used. Yellow powder, total yield: 18%, m.p. 221e
222 ꢂC. 1H NMR (300 MHz, DMSO-d6)
9.79 (s, 1H), 8.44 (s, 1H),
(33).
d
8.06 (dd, J ¼ 8.7 Hz, 5.4 Hz, 2H), 7.92 (d, J ¼ 4.2 Hz, 1H), 7.63 (s, 1H),
7.52 (d, J ¼ 4.2 Hz, 1H), 7.43 (t, J ¼ 8.9 Hz, 2H), 4.62 (t, J ¼ 6.8 Hz,
2H), 3.81e3.80 (m, 4H), 3.34e3.31 (m, 2H), 3.17 (s, 3H), 2.96e2.94
256 ꢂC. 1H NMR (400 MHz, DMSO-d6)
d 9.15 (s, 1H), 8.42 (s, 1H),
8.20 (s, 1H), 7.90 (d, J ¼ 4.0 Hz, 1H), 7.44 (d, J ¼ 4.0, 1H), 7.37 (d,
J ¼ 9.0 Hz, 2H), 7.28 (s, 1H), 6.87 (d, J ¼ 9.0 Hz, 2H), 4.59 (t,
J ¼ 6.8 Hz, 2H), 4.01 (s, 3H), 3.71 (s, 3H), 3.34e3.31 (m, 2H), 3.17 (s,
(m, 4H), 2.11e2.07 (m, 2H). 13C NMR (150 MHz, DMSO-d6)
d 163.3,
151.7, 146.9, 142.2, 141.1, 139.2, 133.1, 131.1, 129.8 (d, 3JC-F ¼ 9.0 Hz)
129.6, 115.9, 115.8, 114.7, 114.4, 111.7, 103.4, 68.6, 66.8, 58.0, 52.7,
41.9, 29.7. HRMS (ESI): m/z (M þ Hþ) calcd for C26H27FN5O6,
524.1940; found 524.1944.
3H), 2.11e2.04 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
d 154.4,
152.7, 151.4, 147.5, 147.4, 139.3, 136.5, 132.9, 130.9, 126.6, 119.7,
114.8, 114.0, 113.4, 108.0, 92.3, 68.6, 57.9, 56.4, 55.2, 41.7, 29.7.
HRMS (ESI): m/z (M þ Hþ) calcd for C24H26N5O7, 496.1827; found
496.1821.
5.2.2.5.4. N-(1-(3-Methoxypropyl)-6-morpholino-2-(5-
nitrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)furan-2-carboxamide
(34). Procedure A was used. Yellow powder, total yield: 33%, m.p.
5. 2. 2. 5.10. 1-(4-Fluorophenyl)-3-(6-methoxy-1-(3-
methoxypropyl)-2-(5-nitrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)
urea (40). Procedure E was used. Brown powder, total yield: 25%,
226e227 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 9.73 (s, 1H), 8.58 (s,
1H), 8.02 (s, 1H), 7.92 (d, J ¼ 3.9 Hz, 1H), 7.65 (s, 1H), 7.51 (d,
J ¼ 3.9 Hz, 1H), 7.29 (d, J ¼ 3.0 Hz, 1H), 6.74e6.73 (m, 1H), 4.61e4.60
(m, 2H), 3.87e3.86 (m, 4H), 3.32e3.31 (m, 2H), 3.17 (s, 3H), 2.96e
2.95 (m, 4H), 2.09e2.07 (m, 2H). 13C NMR (150 MHz, DMSO-d6)
m.p. 204 ꢂC decom. 1H NMR (300 MHz, DMSO-d6)
d 9.39 (s, 1H),
8.43 (s, 1H), 8.28 (s, 1H), 7.92 (d, J ¼ 3.0 Hz, 1H), 7.48e7.46 (m, 3H),
7.30 (s, 1H), 7.14 (t, J ¼ 8.6 Hz, 2H), 4.60 (t, J ¼ 6.0 Hz, 2H), 4.02 (s,
3H), 3.32e3.31 (m, 2H), 3.18 (s, 3H), 2.09 (t, J ¼ 6.0 Hz, 2H). 13C NMR
1
d
155.2, 151.7, 147.6, 146.9, 145.9, 141.2, 140.6, 139.3, 132.6, 129.3,
(150 MHz, DMSO-d6)
d
157.2 (d, JCeF ¼ 236.4 Hz), 152.5, 151.3,
114.8, 114.7, 114.4, 112.7, 109.3, 103.7, 68.6, 66.8, 58.0, 52.7, 41.9, 29.7.
HRMS (ESI): m/z (M þ Hþ) calcd for C24H26N5O7, 496.1827; found
496.1830.
147.42, 147.4, 139.4, 136.4, 136.1 (d, 4JC-F ¼ 1.7 Hz), 131.0, 126.3, 119.5
(d, 3JCeF ¼ 7.5 Hz), 115.2 (d, 2JCeF ¼ 21.9 Hz), 114.8, 113.4, 108.1, 92.4,
68.5, 57.9, 56.4, 41.7, 29.6. HRMS (ESI): m/z (M þ Hþ) calcd for
5.2.2.5.5. 3-Cyano-N-(1-(3-methoxypropyl)-6-morpholino-2-
C
23H23FN5O6, 484.1627; found 484.1629.
(5-nitrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)benzamide
Procedure A was used. Yellow powder, total yield: 19%, m.p. 226e
227 ꢂC. 1H NMR (300 MHz, DMSO-d6)
9.95 (s, 1H), 8.38 (s, 1H),
(35).
5 . 2 . 2 . 5 .11. N - ( 6 - ( C yc l o h e x yl ( m e t hyl ) a m i n o ) - 1 - ( 3 -
methoxypropyl)-2-(5-nitrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)-4-
d
fluorobenzamide (41). Procedure A was used. Yellow powder, total
8.35 (s, 1H), 8.28 (d, J ¼ 8.1 Hz, 1H), 8.09 (d, J ¼ 8.1 Hz, 1H), 7.92 (d,
yield: 21%, m.p. 183e184 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d
9.83 (s,
J ¼ 4.2 Hz, 1H), 7.81 (t, J ¼ 8.1 Hz, 1H), 7.61 (s, 1H), 7.52 (d,
1H), 8.43 (s, 1H), 8.02 (dd, J ¼ 8.7 Hz, 5.4 Hz, 2H), 7.92 (d, J ¼ 3.9 Hz,

350
Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
1H), 7.59 (s, 1H), 7.52 (d, J ¼ 3.9 Hz, 1H), 7.41 (t, J ¼ 8.9 Hz, 2H), 4.61
(t, J ¼ 6.6 Hz, 2H), 3.34e3.31 (m, 2H), 3.17 (s, 3H), 2.81e2.77 (m,1H),
2.73 (s, 3H), 2.10e2.06 (m, 2H), 1.76e1.72 (m, 2H), 1.64e1.62 (m,
2H), 1.50e1.49 (m, 2H), 1.33e1.30 (m, 2H), 1.07e1.04 (m, 2H). 13C
2H), 4.25e4.23 (m, 2H), 3.33e3.31 (m, 4H), 2.79e2.76 (m, 2H), 2.37
(t, J ¼ 7.2 Hz, 2H), 2.08e1.98 (m, 4H), 1.66e1.41 (m, 8H), 1.03 (t,
J ¼ 6.9 Hz, 3H), 0.93 (t, J ¼ 7.5 Hz, 3H), 0.84e0.82 (m, 2H). HRMS
(ESI): m/z (M þ Hþ) calcd for C27H38N5O6, 528.2817; found
528.2816.
5.2.2.5.18. N-(1-(4-Methylpentan-2-yl)-2-(5-nitrofuran-2-yl)-6-
(2-(piperidin-1-yl)ethoxy)-1H-benzo[d]imidazol-5-yl)cyclo-
propanecarboxamide (48). Procedure D was used. Brown powder,
total yield: 23%, m.p. 109e111 ꢂC. 1H NMR (400 MHz, DMSO-d6)
NMR (150 MHz, DMSO-d6)
d
164.0 (d, 1JCeF ¼ 247.9 Hz), 163.1, 151.6,
147.0, 142.0, 140.9, 138.9, 132.9, 131.1 (d, 4JCeF ¼ 2.4 Hz), 130.6, 129.5
(d, 3JCeF ¼ 9.3 Hz), 115.7 (d, 2JCeF ¼ 21.9 Hz), 114.6, 114.2, 111.1, 105.0,
68.6, 62.0, 57.9, 41.7, 37.1, 29.7, 29.5, 25.3, 24.8. HRMS (ESI): m/z
(M þ Hþ) calcd for C29H33FN5O5, 550.2460; found 550.2463.
5.2.2.5.12. 1-(4-Methoxyphenyl)-3-(1-(3-methoxypropyl)-6-(4-
methylpiperazin-1-yl)-2-(5-nitrofuran-2-yl)-1H-benzo[d]imidazol-5-
yl)urea (42). Procedure B was used. Red powder, total yield: 24%,
d
9.31 (s, 1H), 8.25 (s, 1H), 7.91 (d, J ¼ 3.9 Hz, 1H), 7.44 (d, J ¼ 3.9 Hz,
1H), 7.41 (s,1H), 5.15e5.09 (m,1H), 4.31 (t, J ¼ 5.8 Hz, 2H), 2.79 (br.s,
2H), 2.51e2.50 (m, 2H), 2.09e1.99 (m, 1H), 1.83e1.76 (m, 1H), 1.67
(d, J ¼ 6.8 Hz, 3H), 1.55e1.52 (m, 4H), 1.41e1.40 (m, 2H), 1.23e1.21
(m, 2H), 0.84e0.75 (m, 12H). HRMS (ESI): m/z (M þ Hþ) calcd for
m.p. 221e222 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 9.27 (s, 1H), 8.32
(s, 1H), 8.00 (s, 1H), 7.90 (d, J ¼ 3.6 Hz, 1H), 7.47 (d, J ¼ 3.6 Hz, 1H),
7.44 (s, 1H), 7.39 (d, J ¼ 8.4 Hz, 2H), 6.90 (d, J ¼ 8.4 Hz, 2H), 4.57 (m,
2H), 3.72 (s, 3H), 3.33e3.31 (m, 2H), 3.17 (s, 3H), 2.88 (s, 4H), 2.58 (s,
4H), 2.28 (s, 3H), 2.07e2.05 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
C
28H38N5O5, 524.2867; found 524.2859.
5.2.2.5.19. N-(1-(tert-Butyl)-2-methyl-6-morpholino-1H-benzo
[d]imidazol-5-yl)-5-nitrofuran-2-carboxamide (49). Procedure
was used. Yellow powder, total yield: 21%, m.p. 240 ꢂC decom. 1H
NMR (300 MHz, DMSO-d6) 10.15 (s, 1H), 8.36 (s, 1H), 7.85 (d,
A
d
154.7, 152.9, 151.5, 147.3, 140.9, 140.3, 139.3, 132.7, 131.4, 130.9,
120.9, 114.7, 114.1, 113.9, 108.9, 102.1, 68.6, 58.0, 55.1, 54.6, 52.1, 45.7,
41.6, 29.7. HRMS (ESI): m/z (M þ Hþ) calcd for C28H34N7O6,
564.2565; found 564.2566.
d
J ¼ 3.9 Hz, 1H), 7.62 (s, 1H), 7.55 (d, J ¼ 3.9 Hz, 1H), 3.89e3.87 (m,
4H), 2.95e2.93 (m, 4H), 2.70 (s, 3H), 1.78 (s, 9H). 13C NMR (150 MHz,
5.2.2.5.13. 1-(4-Fluorophenyl)-3-(1-(3-methoxypropyl)-6-(4-
methylpiperazin-1-yl)-2-(5-nitrofuran-2-yl)-1H-benzo[d]imidazol-5-
yl)urea (43). Procedure B was used. Red powder, total yield: 27%,
DMSO-d6) d 153.3, 152.4, 151.1, 147.9, 139.8, 136.9, 131.9, 126.8, 116.4,
113.9, 108.6, 107.7, 66.9, 58.4, 52.8, 30.1, 19.7. HRMS (ESI): m/z
(M þ Hþ) calcd for C21H26N5O5, 428.1928; found 428.1931. Anal.
Calcd for C21H25N5O5: C, 59.01; H, 5.90; N, 16.38. Found: 58.98; H,
6.19; N, 15.80.
5.2.2.5.20. N-(1-(tert-Butyl)-6-morpholino-1H-benzo[d]imidazol-
5-yl)-5-nitrofuran-2-carboxamide (50). Procedure A was used. Yel-
low powder, total yield: 27%, m.p. 225 ꢂC decom. 1H NMR (300 MHz,
m.p. 207e208 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 9.52 (s, 1H), 8.29
(s,1H), 8.04 (s,1H), 7.89 (d, J ¼ 3.3 Hz,1H), 7.50e7.46 (m, 4H), 7.13 (t,
J ¼ 8.7 Hz, 2H), 4.58e4.57 (m, 2H), 3.30e3.28 (m, 2H), 3.18 (s, 3H),
2.90 (br.s, 4H), 2.63 (br.s, 4H), 2.30 (s, 3H), 2.07e2.05 (m, 2H). 13
C
NMR (150 MHz, DMSO-d6)
d
157.3 (d, 1JCeF ¼ 236.6 Hz), 152.6, 151.5,
147.2, 141.0, 140.4, 139.2, 136.1, 131.5, 130.5, 120.1(d, 3JCeF ¼ 7.4 Hz),
115.2 (d, 2JCeF ¼ 22.1 Hz), 114.7, 114.0, 109.2, 102.0, 68.5, 57.9, 54.5,
52.0, 45.7, 41.6, 29.6. HRMS (ESI): m/z (M þ Hþ) calcd for
DMSO-d6)
1H), 7.72 (s, 1H), 7.57 (d, J ¼ 3.9 Hz, 1H), 3.91e3.88 (m, 4H), 2.99e
2.96 (m, 4H), 1.70 (s, 9H). 13C NMR (100 MHz, DMSO)
153.4, 151.1,
d
10.16 (s, 1H), 8.52 (s, 1H), 8.25 (s, 1H), 7.85 (d, J ¼ 3.9 Hz,
d
C
27H31FN7O5, 552.2365; found 552.2361.
148.0, 142.4, 141.8, 138.1, 129.5, 127.3, 116.4, 113.8, 109.8, 106.8, 66.9,
56.1, 52.8, 28.9. HRMS (ESI): m/z (M þ Hþ) calcd for C20H24N5O5,
414.1772; found 414.1771.
5.2.2.5.14. 1-(4-Methoxyphenyl)-3-(1-(3-methoxypropyl)-6-(4-
methylpiperazin-1-yl)-2-(5-nitrofuran-2-yl)-1H-benzo[d]imidazol-5-
yl)thiourea (44). Procedure B was used. Yellow powder, total yield:
5.2.2.5.21. N-(1-(tert-Butyl)-6-morpholino-2-propyl-1H-benzo[d]
imidazol-5-yl)-5-nitrofuran-2-carboxamide (51). Procedure A was
used. Yellow powder, total yield: 32%, m.p. 193e194 ꢂC. 1H NMR
21%, m.p. 186e187 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 9.94 (s, 1H),
9.19 (s, 1H), 8.82 (s, 1H), 7.92 (s, 1H), 7.51e7.32 (m, 4H), 7.00e6.98
(m, 2H), 4.58 (br.s, 2H), 3.77 (s, 3H), 3.30 (s, 2H), 3.16 (s, 3H), 2.79 (s,
4H), 2.49 (s, 4H), 2.18e2.07 (m, 5H). HRMS (ESI): m/z (M þ Hþ) calcd
for C28H34N7O5S, 580.2337; found 580.2329.
(400 MHz, DMSO-d6)
d
10.12 (s, 1H), 8.40 (s, 1H), 7.86 (d, J ¼ 3.9 Hz,
1H), 7.67 (s, 1H), 7.57 (d, J ¼ 3.9 Hz, 1H), 3.89 (t, J ¼ 4.2 Hz, 4H), 3.04
(t, J ¼ 7.5 Hz, 2H), 2.96e2.94 (m, 4H),1.88e1.83 (m, 2H),1.81 (s, 9 H),
1.01 (t, J ¼ 7.4 Hz, 3H). HRMS (ESI): m/z (M þ Hþ) calcd for
5.2.2.5.15. N-(1-(3-Methoxypropyl)-6-(4-methylpiperazin-1-
yl)-2-(5-nitrofuran-2-yl)-1H-benzo[d]imidazol-5-yl)acetamide
(45). Procedure A was used. Yellow powder, total yield: 19%, m.p.
C23H30N5O5, 456.2241; found 456.2243.
5.2.2.5.22. N-(1-(tert-Butyl)-6-methoxy-2-methyl-1H-benzo[d]
186e187 ꢂC. 1H NMR (400 MHz, DMSO-d6)
d
8.86 (s, 1H), 8.21 (s,
imidazol-5-yl)-5-nitrofuran-2-carboxamide (52). Procedure D was
1H), 7.91 (d, J ¼ 4.0 Hz, 1H), 7.48 (d, J ¼ 4.0 Hz, 1H), 7.42 (s, 1H),
4.58 (t, J ¼ 6.7 Hz, 2H), 3.33e3.31 (m, 2H), 3.16 (s, 3H), 2.91e2.89
(m, 4H), 2.60e2.58 (m, 4H), 2.28 (s, 3H), 2.13 (s, 3H), 2.08e2.05
(m, 2H). HRMS (ESI): m/z (M þ Hþ) calcd for C22H29N6O5,
457.2194; found 457.2198.
used. Brown powder, total yield: 24%, m.p. 192e193 ꢂC. 1H NMR
(400 MHz, DMSO-d6)
d
10.00 (s, 1H), 8.11 (s, 1H), 7.84 (d, J ¼ 3.9 Hz,
1H), 7.71 (d, J ¼ 3.9 Hz, 1H), 7.49 (s, 1H), 4.02 (s, 3H), 2.94 (s, 3H),
1.88 (s, 9H). HRMS (ESI): m/z (M þ Hþ) calcd for C18H21N4O5,
373.1506; found 373.1503.
5.2.2.5.16. N-(2-(5-Nitrofuran-2-yl)-1-pentyl-6-(piperidin-1-yl)-
1H-benzo[d]imidazol-5-yl)acetamide (46). Procedure A was used.
Yellow powder, total yield: 6%, m.p. 149e150 ꢂC. 1H NMR (300 MHz,
5.2.2.5.23. N-(1-(3-Ethoxypropyl)-6-(2-morpholinoethoxy)-2-
propyl-1H-benzo[d]imidazol-5-yl)-5-nitrofuran-2-carboxamide (53).
Procedure D was used. Brown oil, total yield: 28%. 1H NMR
DMSO-d6)
d
8.88 (s, 1H), 8.25 (s, 1H), 7.90 (d, J ¼ 3.9 Hz, 1H), 7.48 (s,
(400 MHz, DMSO-d6)
d
9.72 (s, 1H), 7.96 (s, 1H), 7.84 (d, J ¼ 3.9 Hz,
1H), 7.45 (d, J ¼ 3.9 Hz, 1H), 4.53 (t, J ¼ 6.9 Hz, 2H), 2.84 (s, 4H), 2.15
1H), 7.58 (d, J ¼ 3.9 Hz, 1H), 7.27 (s, 1H), 4.26e4.19 (m, 4H), 3.51 (t,
J ¼ 4.4 Hz, 4H), 3.41e3.29 (m, 4H), 2.82e2.77 (m, 4H), 2.54 (s, 4H),
1.96e1.93 (m, 2H), 1.85e1.75 (m, 2H), 1.14 (t, J ¼ 7.0 Hz, 3H), 1.00 (t,
J ¼ 7.6 Hz, 3H). HRMS (ESI): m/z (M þ Hþ) calcd for C26H36N5O7,
530.2609; found 530.2603.
5.2.2.5.24. N-(6-(2-Methoxyethoxy)-1-(4-methylpentan-2-yl)-
1H-benzo[d]imidazol-5-yl)furan-2-carboxamide (54). Procedure D
was used. Brown oil, total yield: 18%. 1H NMR (300 MHz, DMSO-d6)
(s, 3H), 1.76e1.74 (m, 6H), 1.58e1.56 (m, 2H), 1.32e1.29 (m, 4H),
0.84 (t, J ¼ 6.9 Hz, 3H). 13C NMR (150 MHz, DMSO-d6)
d 168.0, 151.6,
147.3, 143.2, 140.3, 138.7, 132.5, 129.6, 114.8, 114.1, 111.3, 102.1, 53.5,
44.5, 29.6, 28.3, 25.9, 24.2, 23.8, 21.7, 13.9. HRMS (ESI): m/z
(M þ Hþ) calcd for C23H30N5O4, 440.2292; found 440.2291.
5.2.2.5.17. N-(1-(3-Ethoxypropyl)-2-(5-nitrofuran-2-yl)-6-(2-
(piperidin-1-yl)ethoxy)-1H-benzo[d]imidazol-5-yl)butyramide (47).
Procedure D was used. Yellow powder, total yield: 18%, m.p. 123e
d
9.18 (s, 1H), 8.32 (s, 1H), 8.25 (s, 1H), 7.96 (m, 1H), 7.44 (s, 1H), 7.27
125 ꢂC. 1H NMR (300 MHz, DMSO-d6)
(d, J ¼ 3.9 Hz, 1H), 7.47 (d, J ¼ 3.9 Hz, 1H), 7.36 (s, 1H), 4.61e4.57 (m,
d
8.94 (s, 1H), 8.22 (s, 1H), 7.91
(d, J ¼ 3.3 Hz,1H), 6.73 (m,1H), 4.67e4.64 (m,1H), 4.30 (t, J ¼ 4.5 Hz,
2H), 3.72 (t, J ¼ 4.5 Hz, 2H), 3.33 (s, 3H), 2.01e1.93 (m, 2H), 1.70e

Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
351
1.62 (m, 1H), 1.49 (d, J ¼ 6.9 Hz, 3H), 0.89 (d, J ¼ 6.9 Hz, 3H), 0.83 (d,
J ¼ 6.3 Hz, 3H). HRMS (ESI): m/z (M þ Hþ) calcd for C21H27N3O4,
386.2074; found 386.2077.
5.2.2.5.25. N-(1-(4-Methylpentan-2-yl)-6-(2-morpholinoethoxy)-
1H-benzo[d]imidazol-5-yl)furan-2-carboxamide (55). Procedure D
was used. Brown powder, total yield: 32%, m.p. 60e62 ꢂC. 1H NMR
d 158.9, 153.2, 151.0, 148.0, 139.8, 137.4, 129.8, 126.8, 116.3, 113.8,
109.1, 105.3, 66.8, 52.7, 46.5, 35.5, 31.6, 25.5, 25.4, 21.0. HRMS (ESI):
m/z (M þ Hþ) calcd for C25H32N5O5, 482.2398; found 482.2394.
5.2.2.5.31. N-(2-Cyclohexyl-1-methyl-6-morpholino-1H-benzo[d]
imidazol-5-yl)-5-nitrofuran-2-carboxamide (61). Procedure A was
used. Pale yellow powder, total yield: 18%, m.p. 260 ꢂC decom. 1H
(300 MHz, DMSO-d6)
d
9.33 (s, 1H), 8.27 (s, 1H), 8.18 (s, 1H), 7.95 (m,
NMR (300 MHz, DMSO-d6) d 10.13 (s, 1H), 8.43 (s, 1H), 7.86 (d,
1H), 7.42 (s, 1H), 7.30 (d, J ¼ 3.6 Hz, 1H), 6.73 (dd, J1 ¼ 3 Hz,
J2 ¼ 1.8 Hz, 1H), 4.69e4.66 (m, 1H), 4.36e4.33 (m, 2H), 3.59e3.56
(m, 4H), 2.92e2.66 (m, 6H), 2.02e1.94 (m, 2H), 1.70e1.61 (m, 1H),
1.50 (d, J ¼ 6.6 Hz, 3H), 0.89 (d, J ¼ 6.6 Hz, 3H), 0.83 (d, J ¼ 6.3 Hz,
3H). HRMS (ESI): m/z (M þ Hþ) calcd for C24H33N4O4, 441.2496;
found 441.2495.
J ¼ 3.6 Hz,1H), 7.58 (s, 1H), 7.56 (d, J ¼ 3.6 Hz,1H), 3.90 (m, 4H), 3.77
(s, 3H), 2.99e2.93 (m, 5H), 1.94e1.26 (m, 10H). 13C NMR (150 MHz,
DMSO-d6)
d 159.6, 153.2, 151.0, 148.0, 138.9, 137.9, 132.5, 126.8,
116.2, 113.8, 108.8, 103.4, 66.8, 52.9, 35.0, 31.0, 29.4, 25.5, 25.4.
HRMS (ESI): m/z (M þ Hþ) calcd for C23H28N5O5, 454.2085; found
454.2088.
5 . 2 . 2 . 5 . 2 6 . N - ( 1 - ( 4 - M e t h y l p e n t a n - 2 - y l ) - 6 - ( 2 -
morpholinoethoxy)-1H-benzo[d]imidazol-5-yl)-5-nitrofuran-2-
carboxamide (56). Procedure D was used. Yellow powder, total
5.2.2.5.32. N-(1-(tert-Butyl)-2-cyclohexyl-6-morpholino-
1H-benzo[d]imidazol-5-yl)-5-nitrofuran-2-carboxamide
Procedure A was used. Pale yellow powder, total yield: 22%, m.p.
270 ꢂC decom. 1H NMR (300 MHz, DMSO-d6)
10.09 (s, 1H), 8.36
(62).
yield: 26%, m.p. 88e90 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d
9.70 (s,
d
1H), 8.28 (s, 1H), 8.08 (s, 1H), 7.85 (d, J ¼ 3.6 Hz, 1H), 7.60 (d,
J ¼ 3.6 Hz, 1H), 7.43 (s, 1H), 4.69e4.65 (m, 1H), 4.29e4.25 (m, 2H),
3.52e3.48 (m, 4H), 2.79e2.76 (m, 2H), 2.48e2.46 (m, 4H), 1.99e
1.94 (m, 2H), 1.69e1.64 (m, 1H), 1.50 (d, J ¼ 6.6 Hz, 3H), 0.89 (d,
J ¼ 6.6 Hz, 3H), 0.83 (d, J ¼ 6.3 Hz, 3H). 13C NMR (150 MHz, DMSO-
(s, 1H), 7.86 (d, J ¼ 3.9 Hz, 1H), 7.65 (s, 1H), 7.57 (d, J ¼ 3.9 Hz, 1H),
3.89e3.87 (m, 4H), 3.18e3.15 (m, 1H), 2.93e2.91 (m, 4H), 1.91e
1.70 (m, 15H), 1.41e1.34 (m, 4H). 13C NMR (150 MHz, DMSO-d6)
d
161.0, 153.2, 151.0, 147.9, 139.8, 136.9, 131.1, 126.5, 116.3, 113.8,
108.7, 108.0, 66.8, 58.6, 52.8, 40.0, 33.0, 30.9, 25.8, 25.5. HRMS
(ESI): m/z (M þ Hþ) calcd for C26H34N5O5, 496.2554; found
496.2552.
5.2.2.5.33. N-(2-Cyclohexyl-1-isopropyl-6-morpholino-1H-1H-
benzo[d]imidazol-5-yl)furan-2-carboxamide (63). Procedure A was
used. White powder, total yield: 26%, m.p. 255e256 ꢂC. 1H NMR
d6) d 154.3, 151.4, 148.0, 147.5, 141.8, 136.9, 131.2, 121.6, 116.4, 114.0,
113.7, 95.2, 67.5, 66.0, 57.0, 53.5, 49.4, 44.5, 24.6, 22.6, 22.0, 21.5.
HRMS (ESI): m/z (M þ Hþ) calcd for C24H32N5O6, 486.2347; found
486.2349.
5.2.2.5.27. N-(1-(4-Methylpentan-2-yl)-6-(2-morpholinoethoxy)-
2-propyl-1H-benzo[d]imidazol-5-yl)-5-nitrofuran-2-carboxamide
(57). Procedure D was used. Brown powder, total yield: 23%, m.p.
(300 MHz, DMSO-d6) d 9.77 (s, 1H), 8.44 (s, 1H), 8.01 (s, 1H), 7.54 (s,
1H), 7.26 (d, J ¼ 3 Hz, 1H), 6.74e6.73 (m, 1H), 4.82e4.75 (m, 1H),
3.86e3.84 (m, 4H), 2.99e2.93 (m, 5H), 1.90e1.65 (m, 6H), 1.58 (d,
J ¼ 7.2 Hz, 6H), 1.50e1.25 (m, 4H). 13C NMR (150 MHz, DMSO-d6)
40e42 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 9.68 (s, 1H), 7.99 (s, 1H),
7.85 (d, J ¼ 3.9 Hz, 1H), 7.59 (d, J ¼ 3.9 Hz, 1H), 7.33 (s, 1H), 4.63e
4.59 (m, 1H), 4.27 (t, J ¼ 5.3 Hz, 2H), 3.49 (t, J ¼ 4.7 Hz, 4H), 2.85e
2.74 (m, 4H), 2.48e2.46 (m, 4H), 2.17e1.97 (m, 2H), 1.84e1.74 (m,
3H),1.54 (d, J ¼ 6.6 Hz, 3H),1.00 (t, J ¼ 7.4 Hz, 3H), 0.91 (d, J ¼ 6.3 Hz,
3H), 0.79 (d, J ¼ 6.6 Hz, 3H). HRMS (ESI): m/z (M þ Hþ) calcd for
d
158.5, 154.9, 147.8, 145.6, 139.8, 137.1, 129.3, 127.3, 114.4, 112.5,
108.9, 104.8, 66.9, 52.7, 46.4, 35.5, 31.6, 25.5, 25.4, 21.0. HRMS (ESI):
m/z (M þ Hþ) calcd for C25H33N4O3, 437.2547; found 437.2545.
5.2.2.5.34. N-(1-(tert-Butyl)-6-morpholino-2-(pyridin-4-
yl)-1H-benzo[d]imidazol-5-yl)-5-nitrofuran-2-carboxamide (64).
Procedure A was used. Yellow powder, total yield: 19%, m.p.
C
27H38N5O6, 528.2817; found 528.2808.
5.2.2.5.28. N-(1-(4-Methylpentan-2-yl)-6-(2-(piperidin-1-yl)
ethoxy)-2-propyl-1H-benzo[d]imidazol-5-yl)-5-nitrofuran-2-
260 ꢂC decom. 1H NMR (300 MHz, DMSO-d6)
d 10.19 (s, 1H), 8.70
carboxamide (58). Procedure D was used. Brown oil, total yield:
(d, J ¼ 6.0 Hz, 2H), 8.51 (s, 1H), 7.87 (d, J ¼ 3.9 Hz, 1H), 7.80 (s, 1H),
7.60e7.58 (m, 3H), 3.94e3.92 (m, 4H), 3.02e3.00 (m, 4H), 1.59 (s,
9H). HRMS (ESI): m/z (M þ Hþ) calcd for C25H27N6O5, 491.2037;
found 491.2034.
18%. 1H NMR (400 MHz, DMSO-d6)
d 9.73 (s,1H), 8.01 (s,1H), 7.85 (d,
J ¼ 3.9 Hz, 1H), 7.60 (d, J ¼ 3.9 Hz, 1H), 7.34 (s, 1H), 4.61 (br.s, 1H),
4.27 (t, J ¼ 2.7 Hz, 2H), 2.88e2.80 (m, 2H), 2.79e2.75 (m, 2H), 2.51e
2.50 (m, 2H), 2.06e1.99 (m, 1H), 1.85e1.73 (m, 4H), 1.54 (d,
J ¼ 6.8 Hz, 3H), 1.41e1.31 (m, 4H), 1.29e1.23 (m, 4H), 1.00 (t,
J ¼ 7.3 Hz, 3H), 0.91 (d, J ¼ 6.6 Hz, 3H), 0.79 (d, J ¼ 6.6 Hz, 3H). HRMS
(ESI): m/z (M þ Hþ) calcd for C28H40N5O5, 526.3024; found
526.3019.
5.2.2.5.35. N-(1-(tert-Butyl)-2-cyclohexyl-6-thiomorpholino-
1H-benzo[d]imidazol-5-yl)-5-nitrofuran-2-carboxamide
Procedure C was used. Yellow powder, total yield: 21%, m.p. 257e
259 ꢂC. 1H NMR (300 MHz, DMSO-d6)
10.03 (s, 1H), 8.36 (s, 1H),
(65).
d
7.86 (d, J ¼ 3.6 Hz, 1H), 7.64 (s, 1H), 7.58 (d, J ¼ 3.6 Hz, 1H), 3.21e
5.2.2.5.29. N-(1-(3-Ethoxypropyl)-2-methyl-6-morpholino-1H-
3.18 (m, 1H), 3.14e3.12 (m, 4H), 2.93e2.91 (m, 4H), 1.92e1.67 (m,
benzo[d]imidazol-5-yl)-5-nitrofuran-2-carboxamide
Procedure A was used. Yellow powder, total yield: 21%, m.p. 235e
237 ꢂC. 1H NMR (400 MHz, DMSO-d6)
10.11 (s, 1H), 8.41 (s, 1H),
(59).
15H), 1.46e1.27 (m, 4H). 13C NMR (150 MHz, DMSO-d6)
d 161.1,
153.3, 151.1, 147.9, 139.8, 138.2, 131.2, 126.5, 116.4, 113.8, 108.7,
108.6, 58.7, 54.7, 40.1 33.1, 30.1, 28.2, 25.9, 25.6. HRMS (ESI): m/z
(M þ Hþ) calcd for C26H34N5O4S, 512.2326; found 512.2332.
5.2.2.5.36. N-(6-(2-Methoxyethoxy)-1-(4-methylpentan-2-yl)-2-
propyl-1H-benzo[d]imidazol-5-yl)-5-nitrofuran-2-carboxamide (66).
Procedure D was used. Yellow powder, total yield: 25%, m.p. 78e
d
7.85 (d, J ¼ 3.9 Hz, 1H), 7.54 (d, J ¼ 3.9 Hz, 1H), 7.53 (s, 1H), 4.22 (t,
J ¼ 6.8 Hz, 2H), 3.89 (t, J ¼ 4.2 Hz, 4H), 3.28e3.26 (m, 2H), 3.25 (s,
3H), 2.93 (t, J ¼ 4.2 Hz, 4H), 2.51 (s, 3H), 1.97e1.94 (m, 2H). 13C
NMR (100 MHz, DMSO-d6)
d 153.2, 152.5, 151.1, 148.1, 139.1, 137.9,
132.1, 127.0, 116.3, 113.8, 108.7, 103.2, 68.4, 66.8, 58.0, 53.0, 29.1,
21.0, 13.3. HRMS (ESI): m/z (M þ Hþ) calcd for C21H26N5O6,
444.1878; found 444.1881.
80 ꢂC. 1H NMR (400 MHz, DMSO-d6)
d 9.62 (s, 1H), 8.08 (s, 1H), 7.84
(d, J ¼ 3.9 Hz, 1H), 7.59 (d, J ¼ 3.9 Hz, 1H), 7.40 (s, 1H), 4.62e4.61 (m,
1H), 4.29 (t, J ¼ 4.4 Hz, 2H), 3.71 (t, J ¼ 4.4 Hz, 2H), 3.30 (s, 3H),
2.91e2.78 (m, 2H), 2.04e1.97 (m, 1H), 1.85e1.77 (m, 3H), 1.54 (d,
J ¼ 6.8 Hz, 3H), 1.34e1.24 (m, 1H), 1.00 (t, J ¼ 7.3 Hz, 3H), 0.91 (d,
J ¼ 6.6 Hz, 3H), 0.79 (d, J ¼ 6.6 Hz, 3H). 13C NMR (100 MHz, DMSO-
5.2.2.5.30. N-(2-Cyclohexyl-1-isopropyl-6-morpholino-1H-benzo
[d]imidazol-5-yl)-5-nitrofuran-2-carboxamide (60). Procedure
was used. Yellow powder, total yield: 14%, m.p. 280 ꢂC decom. 1H
NMR (300 MHz, DMSO-d6) 10.16 (s, 1H), 8.43 (s, 1H), 7.86 (d,
A
d
d6) d 158.4, 154.2, 151.4, 147.9, 146.8, 146.4, 122.3, 118.4, 116.4, 113.6,
J ¼ 3.6 Hz, 1H), 7.61 (s, 1H), 7.57 (d, J ¼ 3.6 Hz, 1H), 4.83e4.79 (m,
1H), 3.89e3.87 (m, 4H), 2.96e2.94 (m, 5H), 1.90e1.65 (m, 6H), 1.58
(d, J ¼ 6.6 Hz, 6H), 1.50e1.25 (m, 4H). 13C NMR (150 MHz, DMSO-d6)
113.1, 111.9, 70.4, 70.1, 58.4, 50.0, 42.9, 29.0, 24.9, 22.4, 20.6, 19.6,
13.7. HRMS (ESI): m/z (M þ Hþ) calcd for C24H33N4O6, 473.2395;
found 473.2398.

352
Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
5.2.2.5.37. N-(1-Cyclobutyl-2-methyl-6-morpholino-1H-benzo[d]
108.6, 103.3, 66.8, 52.9, 29.7, 13.4. HRMS (ESI): m/z (M þ Hþ) calcd
for C18H20N5O5, 385.1459; found 386.1458.
5.2.2.5.44. N-(1-Isopropyl-2-methyl-6-morpholino-1H-benzo[d]
imidazol-5-yl)-5-nitrofuran-2-carboxamide (74). Procedure A was
used. Yellow powder, total yield: 25%, m.p. 271e272 ꢂC. 1H NMR
imidazol-5-yl)-5-nitrofuran-2-carboxamide (67). Procedure A was
used. Yellow powder, total yield: 28%, m.p. 280 ꢂC decom. 1H NMR
(400 MHz, DMSO-d6)
d
10.12 (s, 1H), 8.40 (s, 1H), 7.84 (d, J ¼ 3.9 Hz,
1H), 7.61 (s, 1H), 7.54 (d, J ¼ 3.9 Hz, 1H), 5.02e4.93 (m, 1H), 3.89 (t,
J ¼ 4.3 Hz, 4H), 2.95 (t, J ¼ 4.3 Hz, 4H), 2.81e2.71 (m, 2H), 2.52 (s,
(300 MHz, DMSO-d6)
d
10.14 (s, 1H), 8.40 (s, 1H), 7.84 (d, J ¼ 3.9 Hz,
3H), 2.51e2.49 (m, 2H), 2.02e1.90 (m, 1H), 1.89e1.84 (m, 1H). 13
C
1H), 7.60 (s, 1H), 7.54 (d, J ¼ 3.9 Hz, 1H), 4.78e4.69 (m, 1H), 3.89e
3.87 (m, 4H), 2.94e2.92 (m, 4H), 2.53 (s, 3H), 1.55 (d, J ¼ 7.2 Hz, 6H).
NMR (150 MHz, DMSO-d6)
d 153.1, 152.2, 150.9, 147.8, 139.2, 137.4,
131.1, 126.8, 116.1, 113.6, 108.6, 104.3, 66.6, 52.5, 49.0, 28.7, 14.6, 14.4.
HRMS (ESI): m/z (M þ Hþ) calcd for C21H24N5O5, 426.1772; found
426.1769.
13C NMR (150 MHz, DMSO-d6)
d 153.2, 151.8, 151.0, 148.0, 139.7,
137.4, 130.3, 126.8, 116.2, 113.8, 108.7, 104.8, 66.8, 52.7, 47.2, 20.8,
14.5. HRMS (ESI): m/z (M þ Hþ) calcd for C20H24N5O5, 414.1772;
found 414.1773.
5.2.2.5.38. N-(1-(tert-Butyl)-2-methyl-6-thiomorpholino-
1H-benzo[d]imidazol-5-yl)-5-nitrofuran-2-carboxamide
Procedure C was used. Pale yellow powder, total yield: 23%, m.p.
259e260 ꢂC. 1H NMR (300 MHz, DMSO-d6)
10.04 (s, 1H), 8.34 (s,
1H), 7.84 (d, J ¼ 3.9 Hz, 1H), 7.61 (s, 1H), 7.54 (d, J ¼ 3.9 Hz, 1H),
3.16e3.14 (m, 4H), 2.91e2.89 (m, 4H), 2.70 (s, 3H), 1.77 (s, 9H). 13
NMR (150 MHz, DMSO-d6) 153.2, 152.3, 151.0, 147.9, 139.7, 138.1,
131.8, 126.5, 116.3, 113.7, 108.4, 108.2, 58.3, 54.6, 30.1, 28.1, 19.6.
HRMS (ESI): m/z (M þ Hþ) calcd for C21H26N5O4S, 444.1700; found
444.1702.
(68).
5.2.3. General procedure for the synthesis of compounds 76e78
Reaction of compound 31 (1 mmol) with KSCN (4 mmol) and Br2
(1 mmol) was carried out in AcOH (20 mL) at room temperature
overnight. The mixture was evaporated in vacuo, and the inter-
mediate 75 was obtained. Compound 75 (1 mmol) in AcOH and H2O
(1:1) (20 mL) was added dropwise into the solution of NaNO2
(45 equiv) in H2O (20 mL). The reaction mixture was stirred at room
temperature for 5e8 h, neutralized with 1 M NaOH, extracted with
CH2Cl2, washed with H2O, evaporated in vacuo, and purified with
column chromatography to give the expected compound 76.
Reaction of intermediate 75 (1 mmol) with acyl chloride
(1.2 mmol) and Et3N (1.2 mmol) was carried out in CH2Cl2 at room
temperature for 3e5 h. After evaporation of the solvent, the residue
waspurified by column chromatography to give compound 77 and 78.
d
C
d
5.2.2.5.39. N-(1-Isopropyl-2-methyl-6-thiomorpholino-1H-benzo
[d]imidazol-5-yl)-5-nitrofuran-2-carboxamide (69). Procedure
was used. Yellow powder, total yield: 31%, m.p. 229e231 ꢂC. 1H
NMR (300 MHz, DMSO-d6) 10.08 (s, 1H), 8.38 (s, 1H), 7.84 (d,
C
d
J ¼ 3.9 Hz, 1H), 7.61 (s, 1H), 7.55 (d, J ¼ 3.9 Hz, 1H), 4.76e4.71 (m,
1H), 3.17e3.15 (m, 4H), 2.92e2.90 (m, 4H), 2.53 (s, 3H), 1.55 (d,
J ¼ 6.9 Hz, 6H). 13C NMR (150 MHz, DMSO-d6)
d 153.2, 151.9, 151.1,
147.9, 139.7, 138.6, 130.3, 126.7, 116.2, 113.7, 108.7, 105.3, 54.6, 47.2,
28.1, 20.8, 14.5. HRMS (ESI): m/z (M þ Hþ) calcd for C20H24N5O4S,
430.1544; found 430.1541.
5.2.3.1. 4-(2-((7-Methyl-6-(4-methylpentan-2-yl)-2-nitro-6H-imi-
dazo[40,50:3,4]benzo[1,2-d]thiazol-4-yl)oxy)ethyl)morpholine
(76).
Brown powder, total yield: 8%, m.p. 205e207 ꢂC. HRMS (ESI): m/z
(M þ Hþ) calcd for C22H30N5O3S, 446.2220; found 446.2211.
5.2.2.5.40. N-(1-Isopentyl-2-methyl-6-morpholino-1H-benzo[d]
imidazol-5-yl)-5-nitrofuran-2-carboxamide (70). Procedure A was
used. Yellow powder, total yield: 24%, m.p. 271e272 ꢂC. 1H NMR
5.2.3.2. N-(4-Morpholino-7-propyl-6-(2,2,2-trifluoroethyl)-6H-imi-
dazo[40,50:3,4]benzo[1,2-d]thiazol-2-yl)-5-nitrofuran-2-carboxamide
(77). Orange powder, total yield: 18%, m.p. >270 ꢂC. 1H NMR
(400 MHz, DMSO-d6)
d
10.13 (s, 1H), 8.41 (s, 1H), 7.85 (d, J ¼ 3.9 Hz,
1H), 7.56 (d, J ¼ 3.9 Hz, 1H), 7.55 (s, 1H), 4.17 (t, J ¼ 7.5 Hz, 2H), 3.89
(t, J ¼ 4.1 Hz, 4H), 2.94 (t, J ¼ 4.1 Hz, 4H), 2.52 (s, 3H), 1.68e1.55 (m,
(400 MHz, DMSO-d6)
d
13.26 (s, 1H), 7.93 (d, J ¼ 4.0 Hz, 1H), 7.84 (d,
3H), 0.97 (d, J ¼ 6.3 Hz, 6H). 13C NMR (150 MHz, DMSO-d6)
d
153.3,
J ¼ 4.0 Hz,1H), 7.27 (s,1H), 5.36 (q, J ¼ 8.9 Hz, 2H), 3.86 (t, J ¼ 4.3 Hz,
4H), 3.32e3.31 (m, 4H), 2.86 (t, J ¼ 7.5, 2H), 1.91e1.82 (m, 2H), 1.02
(t, J ¼ 7.4 Hz, 3H). HRMS (ESI): m/z (M þ Hþ) calcd for
152.3, 151.1, 148.1, 139.1, 137.9, 131.9, 126.9, 116.3, 113.9, 108.7, 103.3,
66.9, 52.9, 41.4, 38.1, 25.4, 22.3, 13.5. HRMS (ESI): m/z (M þ Hþ)
calcd for C22H28N5O5, 442.2085; found 442.2087.
C22H22F3N6O5S, 539.1319; found 539.1316.
5.2.2.5.41. N-(1-Isopropyl-6-morpholino-1H-benzo[d]imidazol-5-
yl)-5-nitrofuran-2-carboxamide (71). Procedure A was used. Yellow
powder, total yield: 27%, m.p. 285e286 ꢂC. 1H NMR (400 MHz,
5.2.3.3. N-(6-(tert-Butyl)-4-(2-methoxyethoxy)-7-methyl-6H-imi-
dazo[40,50:3,4]benzo[1,2-d]thiazol-2-yl)-5-nitrofuran-2-carboxamide
(78). Yellow powder, total yield: 15%, m.p. 237e239 ꢂC. 1H NMR
DMSO-d6)
d
10.13 (s, 1H), 8.51 (s, 1H), 8.33 (s, 1H), 7.85 (d, J ¼ 3.9 Hz,
1H), 7.68 (s, 1H), 7.56 (d, J ¼ 3.9 Hz, 1H), 4.80e4.73 (m, 1H), 3.89 (t,
(400 MHz, DMSO-d6)
d
13.59 (s, 1H), 7.92 (d, J ¼ 4.2 Hz, 1H), 7.83 (d,
J ¼ 4.2 Hz, 4H), 2.94 (t, J ¼ 4.2 Hz, 4H), 1.53 (d, J ¼ 6.7 Hz, 6H). 13
C
J ¼ 4.2 Hz, 1H), 7.30 (s, 1H), 4.34 (t, J ¼ 4.5 Hz, 2H), 3.76 (t, J ¼ 4.5 Hz,
2H), 3.34 (s, 3H), 2.74 (s, 3H), 1.82 (s, 9H). HRMS (ESI): m/z (M þ Hþ)
calcd for C21H24N5O6S, 474.1442; found 474.1445.
NMR (150 MHz, DMSO-d6)
d 153.3, 151.1, 147.9, 142.1, 140.2, 138.6,
130.1, 127.3, 116.3, 113.8, 109.7, 104.1, 66.8, 52.8, 46.8, 22.2. HRMS
(ESI): m/z (M þ Hþ) calcd for C19H22N5O5, 400.1615; found
400.1614.
Acknowledgment
5.2.2.5.42. N-(1-Methyl-6-morpholino-1H-benzo[d]imidazol-5-
yl)-5-nitrofuran-2-carboxamide (72). Procedure A was used. Yellow
powder, total yield: 23%, m.p. 285 ꢂC decom. 1H NMR (400 MHz,
The research was supported by the Bill & Melinda Gates Foun-
dation TB Drug Accelerator grant OPP1024029 and by the Milstein
Program in Chemical Biology and Translational Medicine. We also
thank the National Natural Science Foundation of China (No.
81001364) for support and W.R. Jacobs, Jr., for the gift of Mtb
mc26220. The Department of Microbiology and Immunology is
supported by the William Randolph Hearst Foundation. SSK was
partially supported by the training grant T32 AI007613. We thank
Amy Cunningham-Bussel for sharing her expertise in preparation
of human macrophages. We are also very grateful to Dr. Jennifer
Price and Dr. Bruce Car at Bristol-Myers Squibb for their help in
mutagenic potential evaluation of benzimidazoles by using the SOS
Chromotest.
DMSO-d6)
d
10.12 (s, 1H), 8.52 (s, 1H), 8.16 (s, 1H), 7.85 (d, J ¼ 3.8 Hz,
1H), 7.64 (s, 1H), 7.57 (d, J ¼ 3.8 Hz, 1H), 3.89e3.87 (m, 4H), 3.83 (s,
3H), 2.94e2.92 (m, 4H). HRMS (ESI): m/z (M þ Hþ) calcd for
C
17H18N5O5, 372.1302; found 372.1304.
5.2.2.5.43. N-(1,2-Dimethyl-6-morpholino-1H-benzo[d]imidazol-
5-yl)-5-nitrofuran-2-carboxamide (73). Procedure A was used. Yel-
low powder, total yield: 28%, m.p. >280 ꢂC. 1H NMR (400 MHz,
DMSO-d6)
d
10.11 (s, 1H), 8.40 (s, 1H), 7.83 (d, J ¼ 3.8 Hz, 1H), 7.55 (s,
1H), 7.53 (d, J ¼ 3.8 Hz, 1H), 3.88 (t, J ¼ 4.3 Hz, 4H), 3.72 (s, 3H), 2.92
(t, J ¼ 4.3 Hz, 4H), 2.50 (s, 3H). 13C NMR (150 MHz, DMSO-d6)
d
153.2, 152.9, 151.1, 148.1, 138.9, 137.8, 132.7, 126.9, 116.3, 1113.9,

Y. Gong et al. / European Journal of Medicinal Chemistry 75 (2014) 336e353
353
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Abbreviations
TB
Mtb
tuberculosis
M. tuberculosis
NR-Mtb non-replicating Mtb
R-Mtb
DOS
DIPEA
replicating Mtb
directly observed therapy short-course
diisopropylethylamine
DFDNB 1,5-difluoro-2,4-dinitrobenzene
RIF
rifampicin
PZA
INH
EMB
LD50
MIC
ALDH
SI
pyrazinamide
isoniazid
ethambuthol
concentration lethal to 50% of mammalian cells
minimal inhibitory concentration
human aldehyde dehydrogenase
selectivity index.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.01.039.
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