Exploration of 2-benzylbenzimidazole scaffold as novel inhibitor of NF-κB

Article abstract of DOI:10.1016/j.bmc.2016.03.012

For finding the novel inhibitor of nuclear factor κB activity, a series of benzimidazole derivatives were rationally designed, synthesized and systematically studied for their in vitro activities against LPS induced NF-κB inhibition in RAW 264.7 cells using the SEAP assay based on the flexible chalcone JSH ((E)-1-(2-hydroxy-6-(isopentyloxy)phenyl)-3-(4-hydroxy phenyl)prop-2-en-1-one) which was previously reported. Although most of the benzimidazole derivatives showed strong inhibitory activity in low micromolar potency, 2-(4-methoxybenzyl)-1H-benzo[d]imidazole (3m; IC₅₀ = 1.7 μM) and 2-(2-methoxybenzyl)-1H-benzo[d]imidazole (3n; IC₅₀ = 2.4 μM) showed the best inhibition. The structure activity relationship revealed that 2-benzylbenzimidazole scaffold with hydrogen bonding acceptor on phenyl ring appears as a pharmacophore.

Full text of DOI:10.1016/j.bmc.2016.03.012

Bioorganic & Medicinal Chemistry 24 (2016) 1872–1878
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Exploration of 2-benzylbenzimidazole scaffold as novel inhibitor
of NF-jB
PullaReddy Boggu a, Eeda Venkateswararao a, Manoj Manickam a, Dajin Kwak b, Youngsoo Kim b,
Sang-Hun Jung a,
⇑
a College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon 305-764, Republic of Korea
b College of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
For finding the novel inhibitor of nuclear factor
jB activity, a series of benzimidazole derivatives were
Received 29 January 2016
Revised 3 March 2016
Accepted 4 March 2016
Available online 4 March 2016
rationally designed, synthesized and systematically studied for their in vitro activities against LPS
induced NF- B inhibition in RAW 264.7 cells using the SEAP assay based on the flexible chalcone JSH
j
((E)-1-(2-hydroxy-6-(isopentyloxy)phenyl)-3-(4-hydroxy phenyl)prop-2-en-1-one) which was previ-
ously reported. Although most of the benzimidazole derivatives showed strong inhibitory activity in
low micromolar potency, 2-(4-methoxybenzyl)-1H-benzo[d]imidazole (3m; IC50 = 1.7
lM) and 2-(2-
Keywords:
2-Benzylbenzimidazole
NF-jB inhibition
Inflammation
methoxybenzyl)-1H-benzo[d]imidazole (3n; IC50 = 2.4 M) showed the best inhibition. The structure
l
activity relationship revealed that 2-benzylbenzimidazole scaffold with hydrogen bonding acceptor on
phenyl ring appears as a pharmacophore.
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
1-(2-hydroxy-6-(isopentyloxy)phenyl)-3-(4-hydroxyphenyl)prop-2-
en-1-one (JSH, Fig. 1)16 and successfully demonstrated it as an
Nuclear factor
j
B (NF-
j
B) transcription factor plays a key role
inhibitor of LPS induced NF-
an IC50 value of about 10 M. It is a non-lipid chalcone which
not only inhibited the TLR4-mediated NF- activating
pathway but also suppresses the NF- B regulated expression of
iNOS, COX-2 or pro-inflammatory cytokine genes. Subsequently,
we explored structure activity relationship of JSH analogs related
jB activation in macrophages with
in immune process and cell proliferation and survival.1,2 NF-
jB
l
controls the expression of multiple genes and effector function of
lymphocytes.3–5 More specifically NF-
B regulates interleukin-1
(IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis
factor- (TNF- B has been
).6 Among transcriptional factors, NF-
jB
j
j
a
a
j
known as the first responder to harmful cellular stimuli, such as
physiological stress, bacterial or viral infection, and other internal
to NF-
jB inhibition and cytotoxicity against human cancer cell
lines.17
proteins. Furthermore, constitutively active form of NF-
jB have
Since JSH is flexible and eventually has many conformations
and hypothetically isomerize to rigid flavanone structure, we
assumed a rigid structure with effective conformation might exert
been frequently found in cancers7 and in many inflammatory dis-
eases,8 such as inflammatory bowel disease, arthritis, sepsis, gastri-
tis, asthma and atherosclerosis. Therefore, the regulation or
better inhibition of NF-jB. Thus, rigid scaffolds like benzimidazole,
inhibition of NF-
j
B transcription should be effective in the treat-
benzoxazole and benzothiazole were designed retaining HBA and
HBD characteristics of chalcone scaffold in JSH (Fig. 2).
ment of cancers7 and inflammatory diseases9 and this approach
could be a highly effective way to find a novel immuno-inflamma-
tory agents.10
2. Chemistry
Some of the representative examples for known chemothera-
peutic agents acting through the inhibition of NF-
jB transcription
Preparation of benzimidazole 3a–y, benzoxazole 4a,b and ben-
zothiazole 5 analogs were outlined in Schemes 1–3 and sub-
stituents are listed in Table 1. Commercially available substituted
benzene-1,2-diamine 1 was reacted with appropriate various sub-
stituted 2-phenylacetic acids 2 in xylene solvent in the presence of
catalytic amount of boric acid18 at reflux temperature for 12–16 h
are shown in Figure 1.11–15 For a decade, our group have explored
the NF-jB inhibitors and discovered a novel small molecule, (E)-
⇑
Corresponding author. Tel.: +82 42 821 5939; fax: +82 42 823 6566.
E-mail address: jungshh@cnu.ac.kr (S.-H. Jung).
http://dx.doi.org/10.1016/j.bmc.2016.03.012
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

P. Boggu et al. / Bioorg. Med. Chem. 24 (2016) 1872–1878
1873
O
O
O
OH
O
O
OH
HO
HO
OH
OCH3
S
O
OCH3
Thiacremonone
Curcumin
DDMP
OH
O
O
OH
O
HO
O
O
OH
O
OH
Simplactone
JSH
OH
Apigenin
Figure 1. Known chemotherapeutic agents through inactivation of NF-
jB.
R4
R3
HBA
HBA
HBA
R2
O
O
O
C
O
O
R
N
B
X
R1
A
n
OH
OH
X = O, NH, S
n = 0,1,or 2
HBA/HBD
OH
HBA/HBD
Flavanone form
JSH (Chalcone form)
Current Design
Figure 2. Design of benzimidazoles, benzoxazoles and benzothiazole as NF-jB inhibitor.
R4
R3
R1
R2
R2
R3
R4
R1
R
NH2
NH2
a
R
O
n
N
n
OH
N
H
1
3a-y
2
Scheme 1. Synthesis of benzimidazole derivatives 3a–y (substituents are listed in Table 1). Reagents and conditions: (a) boric acid, xylene, reflux, 12–16 h, 45–90%.
R2
R3
R2
R1
R4
R1
R4
H
N
R3
R4
R1
R2
R3
NH2
Br
a
b
N
O
O
n
n
n
O
OH
Br
6
2
7
4a, b
Scheme 2. Synthesis of benzoxazoles derivatives 4a,b (substituents are listed in Table 1). Reagents and conditions: (a) EDCÁHCl, DIPEA, DCM, rt, 16 h, 85%; (b) CuI (5 mol %),
1,10-phenanthroline (10 mol %), Cs2CO3, toluene, reflux, 16 h, 75–80%.
to produce desired benzimidazole compounds 3a–y. In order to
obtain benzoxazole 4a and 4b compounds, 2-bromoaniline (6)
was coupled with appropriate substituted phenylacetic acids 2
using EDCÁHCl, DIPEA, DCM, condition at room temperature to give
key intermediate 7. This key intermediate was cyclized with CuI,
1,10-phenanthroline, Cs2CO3, toluene, at reflux temperature for
16 h to afford benzoxazoles 4a,b.19 Benzothiazole 5 was obtained
by refluxing 2-aminobenzenethiol (8) with 4-methoxyphenylacetic
acid in xylene.
O
NH2
SH
O
a
N
S
O
OH
8
2
5
Scheme 3. Synthesis of benzothiazole 5. Reagents and conditions: (a) boric acid,
xylene, reflux, 16 h, 48%.

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P. Boggu et al. / Bioorg. Med. Chem. 24 (2016) 1872–1878
Table 1
In vitro NF-jB inhibitory activity of benzimidazoles 3a–y, benzoxazoles 4a,b and benzothiazole 5
R4
R3
R4
R3
O
R2
C
R2
R
N
N
O
N
S
R1
R1
A
B
n
n
N
H
3a-y
4a-b
R4
5
Compd No.
R
R1
R2
R3
n
% of inhibition at 30
lM
aIC50 value
lM
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
Cl
H
H
H
H
H
H
OCH3
OCH3
H
H
OH
H
H
Cl
OCH3
H
Cl
H
H
Cl
CF3
NO2
SO2CH3
CH3
OCH3
H
OCH3
OCH3
OH
NH2
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
H
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
—
22
12
22
43
85
66
33
63
55
64
43
60
90
85
32
32
75
40
75
31
45
76
61
66
50
33
42
67
>30.0
>30.0
>30.0
18.4
2.4
10.0
>30
15.0
16.4
12.0
28.0
19.0
1.7
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
3y
4a
4b
5
2.4
>30.0
>30.0
6.0
>30.0
6.0
>30
18.0
5.0
>30.0
10.0
>30.0
>30.0
>30.0
9
H
H
H
Cl
OCH3
Cl
OCH3
Cl
H
—
—
—
H
H
H
H
H
H
H
—
H
Cl
H
H
H
H
H
—
OCH3
OCH3
OCH3
H
OCH3
—
H
H
H
—
a
IC50 values are taken as a mean from three experiments.
indicating that mild electron withdrawing or hydrophobic group
should be beneficial for the activity.
3. Bioassay
In order to establish the suitable position of chloro group in the
phenyl ring of 3d, position of chloro group was variated as shown
Inhibitory effect of compounds 3a–y, 4a,b and 5 on NF-jB tran-
scriptional activity was determined as described previously.20 In
brief, RAW 264.7 monocytic cell line stably transfected with NF-
j
in compounds 3f and 3g. Compound 3f (R2 = Cl; IC50 = 10.0
showed reduced activity compared to 3e and increased activity
compared to 3d while compound 3g (R1 = Cl; IC50 = >30.0 M)
lM)
B-secretory alkaline phosphatase (SEAP) construct, a reporter
l
gene of NF- B transcriptional activity, was stimulated with LPS
j
did not show activity. These results indicated that chloro substitu-
(30 ng/mL) for 20 h in the presence of sample. Aliquots of the cul-
tion at position 4 on phenyl ring of 3d as shown in 3e should be
optimum for the inhibition of NF-jB and there is a possibility to
enhance the activity with dichloro substitution on phenyl ring of
3d. Thus, additional chloro group was introduced as shown in 3h
ture supernatants were heated to 65 °C for 5 min and then reacted
with 4-methylumbelliferyl phosphate (500 lM) in the dark. SEAP
activity was measured as relative fluorescence units (RFUs) under
excitation at 360 nm and emission at 450 nm. The results are sum-
marized in Table 1, representing as the IC50 value or inhibition% at
(R2, R3 = Cl, IC50 = 15.0 lM). However, the activity was decreased
compared to mono chloro analogs 3e and 3f.
the concentration 30 lM from three independent experiments
Further, to explore the effect of more electron withdrawing
groups on the phenyl ring we prepared compounds 3i (R3 = CF3,
using the average values of triplicate in each experiment.
IC50 = 16.4
IC50 = 28.0
l
M), 3j (R3 = NO2, IC50 = 12.0
lM) and 3k (R3 = SO2CH3,
4. Results and discussion
l
M). However, all of them showed decrement in activ-
ity. These results suggested that hydrophobic character of 4-chloro
substituent of benzimidazole 3e might more contribute to the inhi-
bition of NF-jB rather than electron withdrawing property.
Initial investigation of 2-phenylbenzimidazole analogs 3a–c
without any spacer between the phenyl and benzimidazole rings
showed very poor activity (Table 1). Thus, methylene groups were
inserted between the benzimidazole and phenyl rings. Surpris-
Thus, to increase the hydrophobic character of benzimidazole
3d, replacement of chloro group with methyl group at R3 position
as shown in 3l (IC50 = 19.0 lM) was performed. However, this ana-
log showed weak inhibitory activity.
ingly, 2-benzylbenzimidazole (3d, inhibition at 30
IC50 = 18.4 M) showed considerably increased activity.
lM = 43%,
l
With these encouraging results, exploration of the effect of sub-
Therefore, we examined the role of electron donating group
stituents on the C ring was attempted. Accordingly, we prepared 4-
with increasing
p constant at R3 position. Replacement of
chloro substituted compound 3e (inhibition at 30
lM = 85%,
methyl with methoxy group as shown in 3m (90% inhibition,
IC50 = 2.4 M) which showed good NF- inhibitory activity
l
jB

P. Boggu et al. / Bioorg. Med. Chem. 24 (2016) 1872–1878
1875
IC50 = 1.7
the methoxy group from para to ortho position as shown in 3n
(R1 = OCH3; IC50 = 2.4 M) retained the activity. These results
might indicate that the electron donating group with hydrogen
bonding acceptor in the phenyl ring of 3d is important. Since ortho-
or para-substituted methoxy analogs showed good activity, addi-
tional methoxy groups on phenyl ring as shown in 3,4-dimethoxy
l
M) remarkably enhanced the inhibitory activity. Shift of
chemicals were used as obtained and all solvents were purified
prior to use applying standard procedures. Thin layer chromatogra-
phy was performed on E Merck silica gel GF-254 pre-coated plates;
identification was performed under UV illumination and coloriza-
tion with 10% phosphomolybdic acid spray followed by heating.
Flash column chromatography was performed on E Merck silica
gel (230–400 mesh). Infrared spectra were recorded on a Nicolet
380 model FTIR. NMR spectra were measured against the peak of
l
3o (IC50 = >30 lM) and 3,4,5-trimethoxy analog 3p (IC50 = >30 lM)
were introduced. However, these analogs abolished the activity.
These results indicated that relatively small binding pocket of the
receptor fits well for a mono methoxy group around the benzyl
ring of benzimidazole 3d scaffold.
Considering hydrogen bonding, we further investigated the
effect of hydrogen bonding donors and therefore, prepared com-
tetramethylsilane using
a
Varian Unity Inova 400 NMR
(400 MHz) spectrometer. We noted that some quaternary carbon
signals in 13C NMR spectra of benzimidazoles 3 are not observable
possibly due to tautomerism.21 High resolution mass spectrum
(HRMS) was recorded on PE SCIEX API 2000 (triple quadrupole)
mass spectrophotometer (Applied Biosystems, Foster City, CA,
USA).
pound 3q (R3 = OH; IC50 = 6.0
activity whereas the amino counterpart 3r (R3 = NH2; IC50 = >30 -
M) did not show any activity. In continuation dihydroxy analog
3s (IC50 = 6.0 M) was prepared and showed the same level of
lM). This analog showed significant
l
6.2. General procedure to synthesis of compounds 3a–y and 5
l
activity as mono hydroxy analog 3q. Addition of hydrogen bonding
donor property did not improve the activity. Therefore, hydrogen
bonding acceptor at R3 position on the phenyl ring of 3d should
be suitable for the activity.
To a stirred solution of benzene-1,2-diamine 1 (1.85 mmol)
in xylenes (10 mL) were added carboxylic acid 2 (2.77 mmol)
and boric acid (0.185 mmol). The resulting solution was
refluxed for 16 h. After cooling to room temperature, the reac-
tion was concentrated under reduced pressure and diluted with
EtOAc (50 mL). The organic phase was washed with saturated
NaHCO3 solution (2 Â 50 mL), dried over anhydrous Na2SO4
and then concentrated under reduced pressure. The residue
was purified by silica gel flash column chromatography (eluting
with 10–15% Ethyl acetate in hexanes) to afford the title com-
pounds 3a–y and 5.
To study the importance of substituents on the A ring of the
benzimidazole, compounds 3t (R = Cl; IC50 >30.0
lM) and 3u
(R = OCH3; IC50 = 18.0 M) were prepared which did not improve
l
the inhibitory activity. Keeping in mind the increment of the activ-
ity by substitution with chloro or methoxy group at R3 position on
phenyl ring of 3d as shown in 3e and 3m, benzimidazoles with
substitution at 5-position and R3 position of phenyl ring as shown
in 3v (R, R3 = Cl, IC50 = 5.0
lM), 3w (R, R3 = OCH3, IC50 >30.0
lM)
and 3x (R = Cl, R3 = OCH3, IC50 = 10.0
l
M) were prepared. However,
6.2.1. 2-Phenyl-1H-benzo[d]imidazole (3a)
none of them improved the activity. These findings strongly
emphasized that the substitution on the benzimidazole ring has
adverse effect on the inhibitory activity.
To further explore the structure activity relationship, we
increased the carbon chain between the two rings as shown in
Yield 60%; Light yellow solid; mp 286–287 °C; IR (KBr) 2950,
2747, 1445, 1421, 1275, 1085, 748 cmÀ1 1H NMR (400 MHz,
;
DMSO-d6) d 12.92 (br s, 1H), 8.15–8.22 (m, 2H), 7.64–7.71 (m,
1H), 7.46–7.59 (m, 4H), 7.16–7.27 (m, 2H); 13C NMR (100 MHz,
DMSO-d6) d 151.3, 143.9, 135.1, 130.3, 129.9, 129.0, 126.6, 126.5,
126.5, 122.6, 121.8, 119.0, 111.4; HRMS calcd for C13H10N2 m/z
194.0825, found 194.0823.
compound 3y (n = 2, R3 = OCH3, IC50 >30.0 lM), which lost the
activity. This result implied that only one methylene group
between the two rings is optimum for the inhibitory activity.
All the above results indicate that the unsubstituted benzimida-
zole with one carbon spacer to the phenyl ring with methoxy
group at R3 position as shown in compound 3m demonstrated as
a pharmacophore for the potent inhibition of NF-
To define the SAR on the B ring of benzimidazole, its isosteres,
benzoxazole derivatives (4a; IC50 >30.0 M and 4b; IC50 >30.0 M)
6.2.2. 2-(4-Chlorophenyl)-1H-benzo[d]imidazole (3b)
Yield 65%; Off white solid; mp 289–291 °C; IR (KBr) 2747, 1448,
1428, 1278, 1089, 830, 742 cmÀ1 1H NMR (400 MHz, DMSO-d6) d
;
jB activity.
13.00 (br s, 1H), 8.16–8.22 (m, 2H), 7.60–7.71 (m, 3H), 7.50–7.58
(m, 1H), 7.17–7.28 (m, 2H); 13C NMR (100 MHz, DMSO-d6) d
150.3, 143.9, 135.1, 134.6, 129.2, 129.2, 128.3, 128.2, 128.2,
122.9, 122.0, 119.1, 111.5; HRMS calcd for
228.0534, found 228.05131.
l
l
and benzothiazole derivative 5 were prepared and these were
found to be inactive. These results are attesting that imidazole
moiety of benzimidazole is critical for NF-kB inhibitory activity.
C13H9ClN2 m/z
6.2.3. 2-(4-Methoxyphenyl)-1H-benzo[d]imidazole (3c)
5. Conclusion
Yield 45%; White solid; mp 224–226 °C; IR (KBr) 2940, 2757,
;
1441, 1426, 1278, 1089, 830, 739 cmÀ1 1H NMR (400 MHz,
On the basis of SAR, the 2-benzylbenzimidazole analogs showed
moderate to good activity, especially hydrophobic hydrogen bond-
ing acceptor (OCH3) substituent on C ring showed potent NF-jB
inhibition. Substitution on the A ring is not favorable for the inhi-
bitory activity. Altogether, we defined the new rigid 2-benzylben-
zimidazole scaffold as a lead from flexible chalcone JSH compound
DMSO-d6) d 12.74 (s, 1H), 8.08–8.16 (m, 2H), 7.58–7.66 (m, 1H),
7.46–7.54 (m, 1H), 7.17 (m, 2H), 7.09–7.13 (m, 2H), 3.34 (s, 3H);
13C NMR (100 MHz, DMSO-d6) d 160.7, 151.5, 144.0, 135.1, 128.1,
128.1, 128.1, 122.8, 122.2, 121.5, 118.6, 114.4, 111.1, 55.3; HRMS
calcd for C14H12N2O m/z 224.0932, found 224.0931.
for finding a novel and potent inhibitor of NF-jB.
6.2.4. 2-Benzyl-1H-benzo[d]imidazole (3d)
Yield 85%; White solid; mp 189–190 °C; IR (KBr) 3001, 2857,
1512, 1456, 1415, 1248, 1175, 1029, 738 cmÀ1
(400 MHz, CHLOROFORM-d) d 9.30–9.49 (m, 1H), 7.64–7.80 (m,
;
1H NMR
6. Materials and methods
6.1. General information
1H), 7.26–7.38 (m, 6H), 7.16–7.25 (m, 2H), 4.25–4.29 (m, 2H);
13C NMR (100 MHz, CHLOROFORM-d)
d 153.4, 136.3, 129.1,
127.3, 122.4, 35.8; HRMS calcd for C14H12N2 m/z 208.1025, found
208.1021.
Melting points (mp) were determined on an Electro thermal 1A
9100 MK2 apparatus and are uncorrected. All commercial

1876
P. Boggu et al. / Bioorg. Med. Chem. 24 (2016) 1872–1878
6.2.5. 2-(4-Chlorobenzyl)-1H-benzo[d]imidazole (3e)
6.2.12. 2-(4-Methylbenzyl)-1H-benzo[d]imidazole (3l)
Yield 85%; Off white solid; mp 191–194 °C; IR (KBr) 2850, 2751,
Yield 78%; Off white solid; mp 196–199 °C; IR (KBr) 2854,
1512, 1451, 1410, 1241, 1181, 1025, 812, 749 cmÀ1
;
1H NMR
2756, 1507, 1451, 1412, 1241, 1183, 1027, 812, 742 cmÀ1 1H
;
(400 MHz, DMSO-d6) d 12.26 (br s, 1H), 7.52 (br s, 1H), 7.31–7.45
(m, 5H), 7.07–7.16 (m, 2H), 4.17 (s, 2H); 13C NMR (100 MHz,
DMSO-d6) d 153.2, 136.7, 134.5, 131.3, 130.8, 128.5, 121.8, 121.1,
118.3, 111.0, 34.1; HRMS calcd for C14H11ClN2 m/z 242.0625, found
242.0621.
NMR (400 MHz, DMSO-d6) d 12.23 (br s, 1H), 7.49–7.55 (m, 1H),
7.37–7.43 (m, 1H), 7.18–7.23 (m, 2H), 7.07–7.15 (m, 4H), 4.11
(s, 2H), 2.26 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 153.9,
143.5, 135.7, 134.7, 134.6, 129.1, 128.8, 121.7, 121.0, 118.3,
111.0, 34.6, 20.6; HRMS calcd for C15H14N2 m/z 222.1225, found
222.1221.
6.2.6. 2-(3-Chlorobenzyl)-1H-benzo[d]imidazole (3f)
Yield 65%; White solid; mp 158–160 °C; IR (KBr) 3010, 2845,
6.2.13. 2-(4-Methoxybenzyl)-1H-benzo[d]imidazole (3m)
Yield 80%; White solid; mp 163–164 °C; IR (KBr) 2840, 2757,
2751, 1509, 1454, 1410, 1241, 1027, 775 cmÀ1
;
1H NMR
(400 MHz, DMSO-d6) d 12.32 (br s, 1H), 7.39–7.58 (m, 3H), 7.26–
7.39 (m, 3H), 7.09–7.16 (m, 2H), 4.19 (s, 2H); 13C NMR (100 MHz,
DMSO-d6) d 153.0, 140.2, 133.1, 130.4, 128.8, 127.7, 126.7, 121.5,
34.4; HRMS calcd for C14H11ClN2 m/z 242.0637, found 242.0631.
1509, 1454, 1410, 1241, 1183, 1027, 813, 749 cmÀ1 1H NMR
;
(400 MHz, CHLOROFORM-d) d 9.38 (br s, 1H), 7.52 (br s, 2H),
7.17–7.24 (m, 4H), 6.87 (d, J = 8.54 Hz, 2H), 4.22 (s, 2H), 3.79 (s,
3H); 13C NMR (100 MHz, CHLOROFORM-d) d 158.9, 153.8, 130.2,
128.2, 122.4, 114.5, 55.3, 35.0; HRMS calcd for C15H14N2O m/z
238.1125, found 238.1123.
6.2.7. 2-(2-Chlorobenzyl)-1H-benzo[d]imidazole (3g)
Yield 69%; Off white solid; mp 220–222 °C; IR (KBr) 3009, 2841,
2758, 1511, 1458, 1410, 1241, 1027, 750 cmÀ1; 1H NMR (400 MHz,
DMSO-d6) d 12.29 (br s, 1H), 7.36–7.55 (m, 4H), 7.28–7.35 (m, 2H),
7.07–7.17 (m, 2H), 4.30 (s, 2H); 13C NMR (100 MHz, DMSO-d6) d
152.3, 143.5, 135.3, 134.5, 133.4, 131.6, 129.4, 128.8, 127.5,
121.8, 121.1, 118.4, 111.1, 32.7; HRMS calcd for C14H11ClN2 m/z
242.0627, found 242.0621.
6.2.14. 2-(2-Methoxybenzyl)-1H-benzo[d]imidazole (3n)
Yield 90%; White solid; mp 197–199 °C; IR (KBr) 3010, 2845,
2751, 1509, 1454, 1410, 1241, 1027, 745 cmÀ1
;
1H NMR
(400 MHz, CHLOROFORM-d) d 9.41 (br s, 1H), 7.61 (br s, 1H),
7.23–7.35 (m, 3H), 7.19 (m, 2H), 6.89–6.99 (m, 2H), 4.27 (s, 2H),
3.95 (s, 3H); 13C NMR (100 MHz, CHLOROFORM-d) d 157.0, 153.9,
131.2, 131.2, 131.1, 128.9, 128.8, 128.8, 125.2, 122.1, 121.5,
6.2.8. 2-(3,4-Dichlorobenzyl)-1H-benzo[d]imidazole (3h)
Yield 65%; Off white solid; mp 189–191 °C; IR (KBr) 2851, 2758,
1508, 1451, 1410, 1241, 1183, 1027, 754 cmÀ1; 1H NMR (400 MHz,
DMSO-d6) d 12.30 (br s, 1H), 7.64 (d, J = 2.20 Hz, 1H), 7.59 (d,
J = 8.05 Hz, 1H), 7.48–7.56 (m, 1H), 7.39–7.48 (m, 1H), 7.33 (dd,
J = 2.19, 8.29 Hz, 1H), 7.08–7.18 (m, 2H), 4.21 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) d 152.7, 138.8, 131.0, 130.7, 129.5, 129.4,
121.5, 33.7; HRMS calcd for C14H10Cl2N2 m/z 276.0225, found
276.0221.
121.5, 111.1, 55.8, 30.9; HRMS calcd for
238.1145, found 238.1141.
C15H14N2O m/z
6.2.15. 2-(3,4-Dimethoxybenzyl)-1H-benzo[d]imidazole (3o)
Yield 75%; Pale yellow solid; mp 160–162 °C; IR (KBr) 3011,
2842, 2751, 1512, 1410, 1241, 1025, 813, 738 cmÀ1 1H NMR
;
(400 MHz, CHLOROFORM-d) d 9.28 (br s, 1H), 7.73 (d, J = 8.54 Hz,
1H), 7.29–7.37 (m, 1H), 7.18–7.26 (m, 2H), 6.78–6.88 (m, 3H),
4.23 (s, 2H), 3.86 (s, 3H), 3.79 (s, 3H); 13C NMR (100 MHz, CHLORO-
FORM-d) d 153.8, 149.3, 148.3, 128.7, 122.4, 121.2, 112.1, 111.4,
55.8, 55.8, 35.5; HRMS calcd for C16H16N2O2 m/z 268.1245, found
268.1241.
6.2.9. 2-(4-(Trifluoromethyl)benzyl)-1H-benzo[d]imidazole (3i)
Yield 59%; Pale yellow solid; mp 213–215 °C; IR (KBr) 2858,
2737, 1514, 1448, 1410, 1241, 1183, 1027, 813, 747 cmÀ1 1H
;
NMR (400 MHz, DMSO-d6) d 12.20–12.50 (br s, 1H), 7.66–7.73
(m, J = 8.54 Hz, 2H), 7.54–7.59 (m, J = 8.29 Hz, 2H), 7.43–7.52 (m,
2H), 7.06–7.20 (m, 2H), 4.29 (s, 2H); 13C NMR (100 MHz, DMSO-
d6) d 152.9, 142.6, 129.8, 127.6, 127.3, 125.8, 125.5, 125.5, 125.5,
6.2.16. 2-(3,4,5-Trimethoxybenzyl)-1H-benzo[d]imidazole (3p)
Yield 76%; Pale yellow solid; mp 151–153 °C; IR (KBr) 3011,
2842, 2758, 1513, 1411, 1241, 1025, 813, 774 cmÀ1 1H NMR
;
(400 MHz, CHLOROFORM-d) d 7.55 (br s, 2H), 7.21–7.27 (m, 2H),
6.49 (s, 2H), 4.21 (s, 2H), 3.81–3.84 (m, 3H), 3.76 (s, 6H); 13C
NMR (100 MHz, DMSO-d6) d 153.7, 153.0, 136.3, 133.2, 121.4,
106.3, 60.0, 55.9, 35.4; HRMS calcd for C17H18N2O3 m/z 298.1347,
found 298.1347.
125.4, 123.1, 121.5, 34.6; HRMS calcd for
276.0925, found 276.0921.
C15H11F3N2 m/z
6.2.10. 2-(4-Nitrobenzyl)-1H-benzo[d]imidazole (3j)
Yield 73%; Brick red solid; mp 217–219 °C; IR (KBr) 2850, 2747,
1505, 1456, 1410, 1241, 1183, 1027, 813, 741 cmÀ1
;
1H NMR
6.2.17. 4-((1H-Benzo[d]imidazol-2-yl)methyl)phenol (3q)
Yield 70%; Off white solid; mp 264–266 °C; IR (KBr) 3258, 1511,
(400 MHz, DMSO-d6) d 12.37 (br s, 1H), 8.18–8.23 (m, 2H), 7.58–
7.65 (m, 2H), 7.51–7.58 (m, 1H), 7.41–7.47 (m, 1H), 7.08–7.19
(m, 2H), 4.35 (s, 2H); 13C NMR (100 MHz, DMSO-d6) d 152.5,
146.5, 145.8, 130.3, 123.8, 121.6, 34.6; HRMS calcd for
1440, 1250, 1027, 786, 736 cmÀ1 1H NMR (400 MHz, DMSO-d6) d
;
12.17 (br s, 1H), 9.26 (s, 1H), 7.41–7.49 (m, 2H), 7.08–7.15 (m,
4H), 6.66–6.73 (m, 2H), 4.03 (s, 2H); 13C NMR (100 MHz, DMSO-
d6) d 156.1, 154.3, 129.8, 127.8, 121.3, 115.3, 34.1; HRMS calcd
for C14H12N2O m/z 224.0935, found 224.0931.
C14H11N3O2 m/z 253.0935, found 253.0931.
6.2.11. 2-(4-(Methylsulfonyl)benzyl)-1H-benzo[d]imidazole (3k)
Yield 55%; White solid; mp 188–190 °C; IR (KBr) 3010, 2845,
2751, 1509, 1454, 1410, 1241, 1027, 813, 748 cmÀ1;1H NMR
(400 MHz, DMSO-d6) d 12.37 (br s, 1H), 7.85–7.92 (m, 2H), 7.57–
7.64 (m, J = 8.54 Hz, 2H), 7.50–7.57 (m, 1H), 7.39–7.46 (m, 1H),
7.07–7.18 (m, 2H), 4.31 (s, 2H), 3.18 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) d 152.7, 143.8, 139.3, 129.9, 127.3, 121.9, 121.2, 118.5,
111.1, 43.6, 34.6; HRMS calcd for C15H14N2O2S m/z 286.0847, found
286.0841.
6.2.18. 4-((1H-Benzo[d]imidazol-2-yl)methyl)aniline (3r)
Yield 75%; Off white solid; mp 211–214 °C; IR (KBr) 3446, 3363,
2889, 1621, 1519, 1409, 1271, 1027, 809, 739 cmÀ1 1H NMR
;
(400 MHz, DMSO-d6) d 12.14 (br s, 1H), 7.37–7.52 (m, 2H), 7.07–
7.13 (m, 2H), 6.93–7.00 (m, J = 8.29 Hz, 2H), 6.44–6.57 (m,
J = 8.29 Hz, 2H), 4.93 (br s, 2H), 3.95 (s, 2H); 13C NMR (100 MHz,
DMSO-d6) d 154.8, 147.4, 129.4, 124.5, 121.3, 114.1, 34.3 HRMS
calcd for C14H13N3 m/z 223.1135, found 223.1131.

P. Boggu et al. / Bioorg. Med. Chem. 24 (2016) 1872–1878
1877
6.2.19. 4-((1H-Benzo[d]imidazol-2-yl)methyl)benzene-1,2-diol
(3s)
122.3, 114.1, 55.2, 33.3, 31.4; HRMS calcd for C16H16N2O m/z
252.1232, found 252.1231.
Yield 60%; Off white solid; mp 258–260 °C; IR (KBr) 3258, 3248,
1512, 1439, 1248, 1026, 812, 738 cmÀ1; 1H NMR (400 MHz, DMSO-
d6) d 12.17 (br s, 1H), 8.87 (s, 1H), 8.75 (s, 1H), 7.45 (br s, 2H), 7.06–
7.14 (m, 2H), 6.62–6.71 (m, 2H), 6.52–6.60 (m, 1H), 3.96 (s, 2H);
13C NMR (100 MHz, DMSO-d6) d 154.4, 145.4, 144.1, 128.5, 121.3,
6.2.26. 2-(4-Methoxybenzyl)benzo[d]thiazole (5)
Yield 48%; Off white solid; mp 60–62 °C; IR (KBr) 3022, 2961,
2929, 2836, 1509, 1450, 1248, 1175, 1126, 1029, 810, 760 cmÀ1
;
1H NMR (400 MHz, CHLOROFORM-d) d 7.97–8.03 (m, 1H), 7.79
(td, J = 0.67, 7.93 Hz, 1H), 7.43–7.49 (m, 1H), 7.28–7.37 (m, 3H),
6.87–6.93 (m, 2H), 4.39 (s, 2H), 3.81 (s, 3H); 13C NMR (100 MHz,
CHLOROFORM-d) d 172.1, 159.0, 153.4, 135.7, 130.3, 129.3, 126.0,
124.8, 122.8, 121.6, 114.3, 55.2, 39.7; HRMS calcd for C15H13NOS
m/z 255.0744, found 255.0741.
119.6, 116.2, 115.6, 34.4; HRMS calcd for
240.0935, found 240.0931.
C14H12N2O2 m/z
6.2.20. 2-Benzyl-5-chloro-1H-benzo[d]imidazole (3t)
Yield 65%; Light orange solid; mp 174–176 °C; IR (KBr) 3004,
2837, 1512, 1410, 1241, 1029, 775, 750 cmÀ1 1H NMR (400 MHz,
;
DMSO-d6) d 12.41–12.54 (m, 1H), 7.35–7.62 (m, 2H), 7.29–7.34
(m, 4H), 7.20–7.29 (m, 1H), 7.10–7.18 (m, 1H), 4.17 (s, 2H); 13C
NMR (100 MHz, DMSO-d6) d 155.3, 137.5, 128.9, 128.7, 126.7,
121.7, 121.6, 34.9; HRMS calcd for C14H11ClN2 m/z 242.0625, found
242.0622.
6.3. General procedure to synthesis of compounds 7
To a stirred solution of 2-bromoaniline 6 (5.81 mmol) in DCM
(10 mL) were added DIPEA (8.72 mmol), EDCÁHCl (6.976 mmol)
and 2-(4-methoxyphenyl)acetic acid 2 (6.39 mmol) at room tem-
perature, and stirred at rt for 16 h. Then the reaction mixture
was diluted with DCM (100 mL), washed with 1 N HCl solution
(2 Â 50 mL), dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was purified by silica gel flash col-
umn chromatography (eluting with 10–15% Ethyl acetate in hex-
anes) to afford the title compounds 7.
6.2.21. 2-Benzyl-5-methoxy-1H-benzo[d]imidazole (3u)
Yield 67%; Off white solid; mp 109–111 °C; IR (KBr) 3009, 2847,
1510, 1466, 1410, 1248, 1175, 1029, 775 cmÀ1; 1H NMR (400 MHz,
DMSO-d6) d 12.04–12.14 (m, 1H), 7.18–7.44 (m, 6H), 6.91 – 7.07
(m, 1H), 6.70–6.79 (m, 1H), 4.12 (s, 2H), 3.75 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) d 155.5, 137.9, 128.8, 128.5, 126.6, 118.7,
110.1, 101.3, 94.5, 55.4, 34.9; HRMS calcd for C15H14N2O m/z
238.1182, found 238.1180.
Yield 85%; off white solid; 1H NMR (400 MHz, CHLOROFORM-d)
d 8.37 (dd, J = 1.46, 8.29 Hz, 1H), 7.59–7.71 (m, 1H), 7.27–7.49 (m,
7H), 6.94 (tt, J = 1.46, 7.68 Hz, 1H), 3.81 (s, 2H).
6.2.22. 5-Chloro-2-(4-chlorobenzyl)-1H-benzo[d]imidazole (3v)
Yield 59%; Light grey solid; mp 155–158 °C; IR (KBr) 3004, 2832,
6.4. General procedure to synthesis of compounds 4a–b
1510, 1466, 1410, 1242, 1029, 813, 769 cmÀ1
;
1H NMR (400 MHz,
To a stirred solution of N-(2-bromophenyl)-2-(4-methoxyphe-
nyl)acetamide 7 (1.56 mmol) in toluene (10 mL) were added Cs2-
CO3 (3.12 mmol), 1,10-phenanthroline (0.156 mmol) and CuI
(0.078 mmol) at room temperature, and refluxed for 16 h. After
cooling to rt, the reaction was concentrated under reduced pres-
sure and diluted with EtOAc (50 mL). The organic phase was
washed with water (2 Â 30 mL), dried over anhydrous Na2SO4
and then concentrated under reduced pressure. The residue was
purified by silica gel flash column chromatography (eluting with
10–15% Ethyl acetate in hexanes) to afford the title compounds
4a–b.
DMSO-d6) d 12.52 (br s, 1H), 7.46–7.58 (m, 2H), 7.33–7.45 (m,
4H), 7.16 (td, J = 1.71, 8.54 Hz, 1H), 4.19 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) d 154.9, 136.4, 131.5, 130.8, 128.6, 125.9,
121.7, 34.1; HRMS calcd for C14H10Cl2N2 m/z 276.0242, found
276.0240.
6.2.23. 5-Methoxy-2-(4-methoxybenzyl)-1H-benzo[d]imidazole
(3w)
Yield 61%; Off white solid; mp 160–161 °C; IR (KBr) 3019, 2857,
1512, 1461, 1410, 1247, 1165, 1028, 813, 776 cmÀ1 1H NMR
;
(400 MHz, METHANOL-d4) d 7.36 (d, J = 8.78 Hz, 1H), 7.18–7.24
(m, 2H), 6.96–7.01 (m, 1H), 6.84–6.90 (m, 2H), 6.82 (dd, J = 2.44,
8.78 Hz, 1H), 4.11 (s, 2H), 3.80 (s, 3H), 3.75 (s, 3H); 13C NMR
(100 MHz, METHANOL-d4) d 160.3, 157.9, 155.6, 131.0, 130.6,
115.4, 112.9, 56.4, 55.9, 35.4; HRMS calcd for C16H16N2O2 m/z
268.1256, found 268.1251.
6.4.1. 2-Benzylbenzo[d]oxazole (4a)
Yield 80%; Pale yellow oil; IR (KBr) 2923, 2831, 1512, 1452,
1242, 1176, 1032, 815, 738 cmÀ1 1H NMR (400 MHz, CHLORO-
;
FORM-d) d 7.67–7.73 (m, 1H), 7.45–7.50 (m, 1H), 7.27–7.42 (m,
7H), 4.29 (s, 2H); 13C NMR (100 MHz, CHLOROFORM-d) d 165.3,
151.1, 141.4, 134.8, 129.0, 128.9, 127.4, 124.7, 124.2, 119.9,
110.5, 35.2; HRMS calcd for C14H11NO m/z 209.0841, found
209.0840.
6.2.24. 5-Chloro-2-(4-methoxybenzyl)-1H-benzo[d]imidazole
(3x)
Yield 68%; Off white solid; mp 166–168 °C; IR (KBr) 3005, 2837,
1512, 1468, 1410, 1242, 1177, 1029, 813 771 cmÀ1
;
1H NMR
6.4.2. 2-(4-Methoxybenzyl)benzo[d]oxazole (4b)
(400 MHz, DMSO-d6) d 12.33–12.47 (m, 1H), 7.35–7.61 (m, 2H),
7.21–7.27 (m, 2H), 7.14 (td, J = 1.25, 8.48 Hz, 1H), 6.86–6.91 (m,
2H), 4.10 (s, 2H), 3.70–3.73 (m, 3H); 13C NMR (100 MHz, DMSO-
d6) d 158.2, 155.8, 155.8, 130.0, 129.3, 125.8, 125.7, 125.7, 121.6,
Yield 75%; Light brown oil; IR (KBr) 2933, 2835, 1510, 1454,
1242, 1176, 1032, 813, 743 cmÀ1 1H NMR (400 MHz, CHLORO-
;
FORM-d) d 7.66–7.72 (m, 1H), 7.44–7.50 (m, 1H), 7.27–7.34 (m,
4H), 6.86–6.92 (m, 2H), 4.22 (s, 2H), 3.80 (s, 3H); 13C NMR
(100 MHz, CHLOROFORM-d) d 165.7, 158.9, 151.1, 141.4, 130.1,
126.8, 124.7, 124.2, 119.8, 114.3, 110.5, 55.2, 34.4; HRMS calcd
for C15H13NO2 m/z 239.0942, found 224.0941.
121.6, 114.0, 55.1, 34.0; HRMS calcd for
272.0728, found 272.0722.
C15H13ClN2O m/z
6.2.25. 2-(4-Methoxyphenethyl)-1H-benzo[d]imidazole (3y)
Yield 55%; White solid; mp 195–196 °C; IR (KBr) 2991, 2838,
Acknowledgment
2746, 1510, 1452, 1236, 1178, 1035, 818, 748, 738 cmÀ1 1H
;
NMR (400 MHz, CHLOROFORM-d) d 7.53 (br s, 2H), 7.23 (dd,
J = 3.17, 6.10 Hz, 2H), 7.07–7.12 (m, J = 8.78 Hz, 2H), 6.80–6.85
(m, 2H), 3.79 (s, 3H), 3.17–3.23 (m, 2H), 3.09–3.15 (m, 2H); 13C
NMR (100 MHz, CHLOROFORM-d) d 158.3, 154.1, 132.6, 129.4,
This work was supported by Priority Research Center Program
through the National Research Foundation (NRF) funded by the
Ministry of Education, Science and Technology (2009-0093815),
South Korea.

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13. Ban, J. O.; Lee, H. S.; Jeong, H.-S.; Song, S.; Hwang, B. Y.; Moon, D. C.; Han, S. B.;
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