Exploration of SAR for novel 2-benzylbenzimidazole analogs as inhibitor of transcription factor NF-κB

Article abstract of DOI:10.1007/s12272-017-0886-1

A novel series of 2-benzylbenzimidazole analogs was designed, synthesized and investigated for their in vitro activities against LPS induced NF-κB inhibition in RAW 264.7 cells using the SEAP assay. Among them, 4-((4-(cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)phenol (6e, >100% inhibition at 30?μM, IC₅₀?=?3.0?μM), 4-((5-(cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)phenol (6j, 96% inhibition at 30?μM, IC₅₀?=?4.0?μM) and 2-((4-(cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)phenol (6k, 95% inhibition at 30?μM, IC₅₀?=?5.0?μM) showed strong inhibitory activity. The structure activity relationship confirmed that the substitution on benzimidazole ring A with hydrophobic cyclohexylmethoxy group at position 4 or 5 markedly enhances the activity. In addition, the hydrophilic hydrogen bonding donor group (OH) at position 2 or 4 on phenyl ring B connected with one methylene spacer to the benzimidazole ring is favorable for the inhibitory activity. However, hydrophobic (–OCH₃ and –Cl) groups on phenyl ring B decrease the activity.

Full text of DOI:10.1007/s12272-017-0886-1

Arch. Pharm. Res.
DOI 10.1007/s12272-017-0886-1
RESEARCH ARTICLE
Exploration of SAR for novel 2-benzylbenzimidazole analogs
as inhibitor of transcription factor NF-jB
PullaReddy Boggu1 Eeda Venkateswararao1 Manoj Manickam1
•
•
•
Youngsoo Kim2 Sang-Hun Jung1
•
Received: 25 October 2016 / Accepted: 4 January 2017
Ó The Pharmaceutical Society of Korea 2017
Abstract A novel series of 2-benzylbenzimidazole ana-
logs was designed, synthesized and investigated for their
in vitro activities against LPS induced NF-jB inhibition
in RAW 264.7 cells using the SEAP assay. Among
them, 4-((4-(cyclohexylmethoxy)-1H-benzo[d]imidazol-2-
Introduction
Nuclear factor kappa B (NF-jB) is a family of transcription
factors and plays a crucial role in inflammation, immune
responses, survival and cell proliferation (Hayden and
Ghosh 2008; Karin 2006; Li and Verma 2002; Hoesel and
Schmid 2013). The NF-jB family is composed of five
members, including RelA, RelB, and c-Rel, the precursor
and processed products of the NFKB1 (p105/p50) and
NFKB2 (p100/p52) genes. Although these proteins form
homodimers or heterodimers in various combinations, the
main complexes that activate transcription are the p50/
RelA and the p52/RelB complexes, which are sequestered
in the cytoplasm by the inhibitor of NF-jB (IjB) family or
a p100 protein, respectively, in unstimulated cells (Oeck-
inghaus et al. 2011). Two distinct pathways have been
proposed for NF-jB activation. The first one is the
canonical pathway that mediates inflammatory responses
and the second one is the non-canonical pathway that is
involved in immune cell differentiation and maturation and
secondary lymphoid organogenesis. The former is depen-
dent on the IjB kinase adaptor molecule NEMO (IKK-c)
and the latter is independent of it (Oeckinghaus et al. 2011;
Razani et al. 2011; Sun 2011).
yl)methyl)phenol (6e,[100% inhibition at 30 lM, IC50
=
3.0 lM), 4-((5-(cyclohexylmethoxy)-1H-benzo[d]imida-
zol-2-yl)methyl)phenol (6j, 96% inhibition at 30 lM,
IC50 = 4.0 lM) and 2-((4-(cyclohexylmethoxy)-1H-benzo
[d]imidazol-2-yl)methyl)phenol (6k, 95% inhibition at
30 lM, IC50 = 5.0 lM) showed strong inhibitory activity.
The structure activity relationship confirmed that the
substitution on benzimidazole ring A with hydrophobic
cyclohexylmethoxy group at position 4 or 5 markedly
enhances the activity. In addition, the hydrophilic hydrogen
bonding donor group (OH) at position 2 or 4 on phenyl ring
B connected with one methylene spacer to the benzimi-
dazole ring is favorable for the inhibitory activity. How-
ever, hydrophobic (–OCH3 and –Cl) groups on phenyl ring
B decrease the activity.
Keywords 2-Benzylbenzimidazole Á LPS Á NF-jB
inhibitor
The expression of multiple genes and effector function
of lymphocytes (Gerondakis and Siebenlist 2010; Sen
2006) have been controlled by the NF-jB. Especially it
modulates the regulation of interleukin-1 (IL-1), inter-
leukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis
factor-a (TNF-a) (Janssens and Burns 2002; Libermann
and Baltimore 1990; Kunsch and Rosen 1993; Schu¨tze
et al. 1995). NF-jB has been acknowledged for its response
to the harmful cellular stimuli, such as physiological stress,
bacterial or viral infection, and other internal proteins, and
it is the first ever among all other transcriptional factors
Electronic supplementary material The online version of this
article (doi:10.1007/s12272-017-0886-1) contains supplementary
material, which is available to authorized users.
& Sang-Hun Jung
jungshh@cnu.ac.kr
1
Present Address: College of Pharmacy and Institute of Drug
Research and Development, Chungnam National University,
Daejeon 34134, South Korea
2
College of Pharmacy, Chungbuk National University,
Cheongju 28644, South Korea
123

P. Boggu et al.
that have recognized to be so. Furthermore, constitutively
active form of NF-jB have been frequently found in can-
cers and in many inflammatory diseases (Hoesel and Sch-
mid 2013; Mantovani et al. 2008) such as inflammatory
bowel disease, arthritis, sepsis, gastritis, asthma and
atherosclerosis. Therefore, the treatment of cancers and
inflammatory diseases, require the regulation or inhibition
of NF-jB and this perspective could be the effective
approach to find the novel immuno-inflammatory agents.
Some of the representative examples for known NF-jB
inhibitors are shown in Fig. 1 (Pan et al. 2000; Kunnu-
makkara et al. 2007; Davis et al. 1999; Li et al. 2005;
Rahman et al. 2007; Ban et al. 2007).
in the JSH compound were considered to be the important
functional groups for the inhibition of LPS induced NF-jB
activation (Roh et al. 2011). Based on these characteristics
of JSH, we designed and synthesized analogs 6a–s (Fig. 2;
Table 1) by introducing the bulky hydrophobic substituents
on the benzimidazole ring A and studied them for their LPS
induced NF-jB inhibitory activity.
Materials and methods
Chemistry
Recently, we discovered a novel small molecule, (E)-1-
(2-hydroxy-6-(isopentyloxy)phenyl)-3-(4-hydroxyphenyl)
prop-2-en-1-one (JSH, Fig. 1) (Roh et al. 2011) and suc-
cessfully proved it as an inhibitor of LPS induced NF-jB
activation in macrophages with an IC50 value of about
10 lM. Later, we explored the structure activity relationship
of JSH analogs related to NF-jB inhibition (Venkateswar-
arao et al. 2012, 2014). Since chalcone JSH and its analogs
are flexible molecules, these analogs have many conforma-
tions and hypothetically isomerize to a rigid flavanone
structure as shown in Fig. 2, which might be an effective
form. These aspects therefore dragged our attention to
investigate the rigid scaffolds like benzimidazole, benzoxa-
zole and benzothiazole retaining HBA and HBD character-
Melting points (mp) were determined on an Electro thermal
1A 9100 MK2 apparatus and are uncorrected. All com-
mercial chemicals were used as obtained and all solvents
were purified by distillation prior to use applying the
standard procedures (Perrin et al. 1982). Thin layer chro-
matography was performed on E Merck silica gel GF-254
pre-coated plates; identification was performed under UV
illumination (k = 254 nm), and colorization with iodine.
Hydrogenation reaction was performed on Parr shaker type
hydrogenator. Flash column chromatography was per-
formed on E Merck silica gel (230–400 mesh). Infrared
1
spectra were recorded on a Nicolet 380 model FTIR. H
NMR and 13C NMR spectra were measured against the
peak of tetramethylsilane using a Bruker Fourier 300 NMR
(300 MHz) and JEOL, JNM-AL400 NMR (400 MHz)
spectrometer. We noted that some quaternary carbon sig-
nals in 13C NMR spectra of benzimidazoles are not
observable possibly due to tautomerism. High resolution
mass spectrum (HRMS) were measured in ESI ionisation
using AB Sciex Triple TOF 5600 LCMS instrument.
istics of chalcone scaffold in JSH. As
a result,
2-benzylbenzimidazoles were identified as novel inhibitors
of NF-jB (Boggu et al. 2016). Thus optimization of this
scaffold is immediately required to obtain more potent
analog for the inhibition of NF-jB. The hydrophobic
isopentyloxy group and 4-hydroxyl group on the phenyl ring
Fig. 1 Representative examples for known NF-jB inhibitors
123

Exploration of SAR for novel 2-benzylbenzimidazole analogs as inhibitor of transcription…
Fig. 2 Structural modification of benzimidazoles as NF-jB inhibitors
Table 1 In vitro NF-jB
inhibitory activity of
Comp. No.
R
R1
n
% of Inhibition
at 30 lM
IC50 valuea
(lM)
benzimidazoles 6a–s
6a
6b
6c
6d
6e
6f
4-(CH2)2CH(CH3)2
4-CH2CH(CH3)2
4-(CH2)2CH3
4-CH3
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
2-OH
4-
1
1
1
1
1
1
1
1
1
1
1
1
77
46
12.0
[30.0
[30.0
[30.0
3.0
30
38
4-CH2Cy
[100
88
4-(CH2)2Cy
4-CH2Ph
17.0
12.0
18.0
7.0
6g
6h
6i
85
4-(CH2)2Ph
5-(CH2)2CH(CH3)2
5-CH2Cy
87
84
6j
96
4.0
6k
6l
4-CH2Cy
95
5.0
4-CH2Cy
71
17.0
OCH3
4-
OCH3
2-
OCH3
6m
6n
5-CH2Cy
4-CH2Cy
1
1
48
90
[30.0
8.0
6o
6p
6q
6r
4-CH2Cy
4-Cl
4-OH
2-OH
4-
1
1
1
1
46
12
19
22
[30.0
[30.0
[30.0
[30.0
–
–
–
OCH3
6s
4-CH2Cy
–
4-OH
–
2
–
90
75
7.0
6.0
1
JSH
10.0
a
IC50 values are taken as a mean from three experiments
Cy cyclohexyl, Ph phenyl
General procedure for the synthesis of compounds 3a–j
cooled, diluted with water, and then extracted with
EtOAc. The combined organic extracts were washed
with water, brine solution, dried over anhydrous Na2SO4
and then concentrated under reduced pressure. The crude
mixture was subjected to flash silica gel (230–400 mesh)
column chromatography to afford the title compounds
3a–j.
To a solution of the corresponding nitro aminophenol
2 (6.49 mmol) in DMF (10 mL), K2CO3 (7.79 mmol)
and the corresponding alkyl (or benzyl) halide
(7.14 mmol) were added. The resulting solution was
stirred at 60 °C for 8–12 h. The reaction mixture was
123

P. Boggu et al.
2-(Isopentyloxy)-6-nitroaniline (3a) Yield 78%; Yellow
1
4-(Cyclohexylmethoxy)-2-nitroaniline (3j) Yield 71%;
Yellow solid;1H NMR (300 MHz, CDCl3) d 7.53 (d,
J = 2.89 Hz, 1H), 7.07 (dd, J = 2.93, 9.08 Hz, 1H), 6.76
(d, J = 9.03 Hz, 1H), 5.88 (br. s., 2H), 3.72 (d,
J = 6.33 Hz, 2H), 1.68–1.90 (m, 6H), 1.14–1.37 (m, 3H),
1.00–1.14 (m, 2H).
solid; H NMR (400 MHz, CDCl3) d 7.72 (dd, J = 1.34,
8.90 Hz, 1H), 6.89 (d, J = 7.56 Hz, 1H), 6.60 (dd,
J = 7.68, 8.90 Hz, 1H), 6.44 (br. s., 2H), 4.07 (t,
J = 6.59 Hz, 2H), 1.81–1.91 (m, 1H), 1.76 (q,
J = 6.75 Hz, 2H), 0.99 (d, J = 6.34 Hz, 6H).
2-Isobutoxy-6-nitroaniline (3b) Yield 87%; Yellow solid;
1H NMR (400 MHz, CDCl3) d 7.73 (dd, J = 0.98,
9.02 Hz, 1H), 6.87 (d, J = 7.81 Hz, 1H), 6.60 (dd,
J = 7.81, 9.02 Hz, 1H), 6.45 (br. s., 2H), 3.81 (d,
J = 6.69 Hz, 2H), 2.18 (quind, J = 6.67, 13.34 Hz, 1H),
1.07 (d, J = 6.67 Hz, 6H).
General procedure for the synthesis of compounds 4a–f,
4h–j and 7
In a 250 mL parr shaker vessel, a mixture of the corre-
sponding alkyl substituted nitro aminophenol
3
(3.99 mmol) in methanol (30 mL) was placed and then
added 10% Pd–C (20% w/w). The resulting solution was
stirred at ambient temperature for 2 h in the presence of
hydrogen gas. After completion of the reaction, the mixture
was filtered through celite bed and washed with methanol,
and then the filtrate was evaporated under vacuum to afford
the title compounds. These compounds were used for the
next step without further purification.
2-Nitro-6-propoxyaniline (3c) Yield 85%; Yellow solid; 1H
NMR (400 MHz, CDCl3) d 7.73 (dd, J = 1.46, 8.78 Hz,
1H), 6.85–6.92 (dd, J = 1.24, 8.78 Hz, 1H), 6.60 (dd,
J = 7.80, 8.78 Hz, 1H), 6.46 (br. s., 2H), 4.01 (t,
J = 6.46 Hz, 2H), 1.82–1.96 (m, 2H), 1.08 (t,
J = 7.44 Hz, 3H).
2-Methoxy-6-nitroaniline (3d) Yield 75%; Light pink solid;
1H NMR (300 MHz, CDCl3) d 7.69–7.78 (dd, J = 0.93,
8.85 Hz, 1H), 6.89 (d, J = 7.71 Hz, 1H), 6.62 (dd,
J = 7.71, 8.85 Hz, 1H), 6.44 (br. s., 2H), 3.93 (s, 3H).
3-(Isopentyloxy)benzene-1,2-diamine (4a) Yield 85%;
Dark brown oil; 1H NMR (400 MHz, DMSO-d6) d 6.36 (t,
J = 7.93 Hz, 1H), 6.17–6.25 (m, 2H), 4.44 (s, 2H), 3.92
(br. s., 2H), 3.90 (t, J = 6.56, 2H), 1.81 (quind, J = 6.69,
13.49 Hz, 1H), 1.61 (q, J = 6.83 Hz, 2H), 0.93 (d,
J = 6.59 Hz, 6H).
2-(Cyclohexylmethoxy)-6-nitroaniline (3e) Yield 67%;
1
Yellow solid; H NMR (300 MHz, CDCl3) d 7.69–7.75
(dd, J = 0.93, 8.80 Hz, 1H), 6.87 (d, J = 7.64 Hz, 1H),
6.59 (dd, J = 7.78, 8.80 Hz, 1H), 6.44 (br. s., 2H), 3.84 (d,
J = 5.96 Hz, 2H), 1.71–1.93 (m, 6H), 1.04–1.39 (m, 5H).
3-Isobutoxybenzene-1,2-diamine (4b) Yield 79%; Black
solid; 1H NMR (400 MHz, CDCl3) d 6.65 (t, J = 8.05 Hz,
1H), 6.38–6.40 (m, 2H), 3.76 (d, J = 6.59 Hz, 2H), 3.45
(br. s., 4H), 2.12 (quind, J = 6.68, 13.26 Hz, 1H), 1.05 (d,
J = 6.83 Hz, 6H).
2-(2-Cyclohexylethoxy)-6-nitroaniline (3f) Yield 82%;
Yellow solid; 1H NMR (400 MHz, CDCl3) d 7.72 (d,
J = 8.78 Hz, 1H), 6.88 (d, J = 7.56 Hz, 1H), 6.54–6.63 (t,
J = 7.80, 8.80 Hz 1H), 6.44 (br. s., 2H), 4.08 (t,
J = 6.59 Hz, 2H), 1.66–1.81 (m, 7H), 1.45–1.55 (m, 1H),
1.15–1.33 (m, 3H), 0.93–1.08 (m, 2H).
3-Propoxybenzene-1,2-diamine (4c) Yield 83%; Black
solid; 1H NMR (400 MHz, CDCl3) d 6.65 (t, J = 8.06 Hz,
1H), 6.36–6.44 (m, 2H), 3.96 (t, J = 6.59 Hz, 2H), 3.45
(br. s., 4H), 1.84 (sxt, J = 7.07 Hz, 2H), 1.06 (t,
J = 7.44 Hz, 3H).
2-(Benzyloxy)-6-nitroaniline (3g) Yield 78%; Yellow solid;
1H NMR (400 MHz, CDCl3) d 7.76 (dd, J = 1.34,
8.90 Hz, 1H), 7.39–7.48 (m, 5H), 6.97 (dd, J = 1.22,
7.80 Hz, 1H), 6.60 (dd, J = 7.80, 8.78 Hz, 1H), 6.45 (br.
s., 1H), 5.14 (s, 2H).
3-Methoxybenzene-1,2-diamine (4d) Yield 87%; Light
brown solid; 1H NMR (300 MHz, CDCl3) d 6.68 (t,
J = 7.8 Hz 1H), 6.41 (td, J = 1.2, 7.5 Hz, 2H), 3.84 (s,
3H), 3.31 (br. s., 4H).
2-Nitro-6-phenethoxyaniline (3h) Yield 75%; Yellow
solid; 1H NMR (300 MHz, CDCl3) d 7.73 (d, J = 8.85 Hz,
1H), 7.28–7.42 (m, 5H), 6.89 (d, J = 7.73 Hz, 1H),
6.54–6.63 (t, J = 8.58 Hz, 1H), 6.33 (br. s., 2H), 4.27 (t,
J = 6.80 Hz, 2H), 3.18 (t, J = 6.80 Hz, 2H).
3-(Cyclohexylmethoxy)benzene-1,2-diamine (4e) Yield
1
92%; Dark brown solid; H NMR (300 MHz, CDCl3) d
6.65 (t, J = 7.83 Hz 1H), 6.40–6.37 (m, 2H), 3.79 (d,
J = 6.05 Hz, 2H), 3.44 (br. s., 4H), 1.66–1.93 (m, 6H),
1.02–1.39 (m, 5H).
4-(Isopentyloxy)-2-nitroaniline (3i) Yield 83%; Yellow
solid; 1H NMR (300 MHz, CDCl3) d 7.55 (d, J = 2.89 Hz,
1H), 7.07 (dd, J = 2.89, 9.03 Hz, 1H), 6.76 (d,
J = 9.03 Hz, 1H), 5.89 (br. s., 2H), 3.96 (t, J = 6.66 Hz,
2H), 1.83 (quind, J = 6.61, 13.42 Hz, 1H), 1.67 (q,
J = 6.74 Hz, 2H), 0.97 (d, J = 6.52 Hz, 6H).
3-(2-Cyclohexylethoxy)benzene-1,2-diamine (4f) Yield
1
82%; Dark brown solid; H NMR (400 MHz, CDCl3) d
6.66 (t, J = 8.05 Hz, 1H), 6.40 (dt, J = 1.10, 7.62 Hz,
2H), 4.03 (t, J = 6.59 Hz, 2H), 3.44 (br. s., 4H), 1.64–1.81
(m, 7H), 1.51 (m, 1H), 1.13–1.31 (m, 3H), 0.99 (m, 2H).
123

Exploration of SAR for novel 2-benzylbenzimidazole analogs as inhibitor of transcription…
3-Phenethoxybenzene-1,2-diamine (4h) Yield 79%; Dark
brown solid; 1H NMR (300 MHz, CDCl3) d 7.22–7.38 (m,
5H), 6.65 (t, J = 8.01 Hz, 1H), 6.38–6.42 (m, 2H), 4.22 (t,
J = 6.89 Hz, 2H), 3.40 (br. s., 4H), 3.13 (t, J = 6.85 Hz,
2H).
4-((4-(Isopentyloxy)-1H-benzo[d]imidazol-2-yl)methyl)
phenol (6a) Yield 71%; Pale yellow solid; mp 195–197 °C;
IR (neat) 3250–2770 (br., peak), 2915, 2843, 1595, 1516,
1441, 1245, 1168, 1098, 1022, 783, 726; 1H NMR
(300 MHz, Methanol-d4) d 7.09–7.15 (m, 2H), 7.03–7.09
(m, 2H), 6.67–6.75 (m, 3H), 4.16 (t, J = 6.66 Hz, 2H), 4.09
(s, 2H), 1.87–2.00 (m, 1H), 1.76 (q, J = 6.80 Hz, 2H), 0.99
(d, J = 6.61 Hz, 6H); 13C NMR (100 MHz, Methanol-d4) d
157.7, 155.1, 149.5, 141.7, 131.0, 129.5, 124.1, 116.6, 108.5,
104.9, 67.9, 39.4, 35.2, 26.1, 23.1; HRMS (ESI) Calcd for
C19H22N2O2 [M?H]? 311.1759, found 311.1784.
4-(Isopentyloxy)benzene-1,2-diamine (4i) Yield 81%; Dark
brown solid; 1H NMR (300 MHz, CDCl3) d 6.63 (d,
J = 8.38 Hz, 1H), 6.33 (d, J = 2.70 Hz, 1H), 6.27 (dd,
J = 2.70, 8.38 Hz, 1H), 3.90 (t, J = 6.71 Hz, 2H), 3.53
(br. s., 2H), 3.06 (br. s., 2H), 1.82 (m, 1H), 1.64 (q,
J = 6.77 Hz, 2H), 0.95 (d, J = 6.61 Hz, 6H).
4-((4-Isobutoxy-1H-benzo[d]imidazol-2-yl)methyl)phenol
(6b) Yield 64%; Off white solid; mp 238–241.5 °C; IR
(neat) 3255–2780 (br., peak), 2917, 2845, 1595, 1518, 1442,
4-(Cyclohexylmethoxy)benzene-1,2-diamine (4j) Yield
1
72%; Dark brown solid; H NMR (300 MHz, CDCl3) d
6.63 (d, J = 8.29 Hz, 1H), 6.33 (d, J = 2.61 Hz, 1H), 6.26
(dd, J = 2.70, 8.38 Hz, 1H), 3.67 (d, J = 6.33 Hz, 2H),
3.51 (br. s., 2H), 3.10 (br. s., 2H), 1.69–1.88 (m, 6H),
1.18–1.34 (m, 3H), 0.95–1.08 (m, 2H).
1
1245, 1165, 1096, 1023, 783, 724; H NMR (300 MHz,
Methanol-d4) d 7.09–7.15 (m, 2H), 7.02–7.09 (m, 2H),
6.64–6.75 (m, 3H), 4.10 (s, 2H), 3.89 (d, J = 6.61 Hz, 2H),
2.17 (quind, J = 6.67, 13.35 Hz, 1H), 1.09 (d, J = 6.80 Hz,
6H); 13C NMR (100 MHz, Methanol-d4) d 157.7, 155.1,
149.5, 131.0, 129.5, 124.1, 116.6, 108.6, 105.0, 76.0, 35.2,
29.7, 19.8; HRMS (ESI) Calcd for C18H20N2O2 [M?H]?
297.1603, found 297.1627.
1
2,3-Diaminophenol (7) Yield 95%; Light brown solid; H
NMR (400 MHz, DMSO-d6) d 8.71 (br. s., 1H), 6.23 (t,
J = 7.80 Hz, 1H), 6.07 (dd, J = 1.59, 7.93 Hz, 2H), 4.36
(br. s., 2H), 3.81 (br. s., 2H).
3-(Benzyloxy)benzene-1,2-diamine (4g) To a solution of the
2-(benzyloxy)-6-nitroaniline 3g (4.09 mmol) in EtOH
(15 mL), SnCl2Á2H2O (20.47 mmol) was added, and stirred
at reflux temperature for 5 h. The reaction mixture was
cooled, quenched with 1N NaOH solution (pH * 9), and
then extracted with EtOAc. The combined organic extracts
were washed with water, brine solution, dried over anhy-
drous Na2SO4 and then concentrated under reduced pressure.
The crude mixture was subjected to flash silica gel (230–400
mesh) column chromatography to afford the title compound
4g. Yield 65%; Brown solid; 1H NMR (400 MHz, DMSO-
d6) d 7.47 (d, J = 7.07 Hz, 2H), 7.36–7.41 (m, 2H),
7.28–7.34 (m, 1H), 6.35 (t, J = 7.80 Hz, 1H), 6.29 (dd,
J = 1.46, 8.04 Hz, 1H), 6.23 (dd, J = 1.46, 7.80 Hz, 1H),
5.03 (s, 2H), 4.49 (br. s., 2H), 4.03 (br. s., 2H).
4-((4-Propoxy-1H-benzo[d]imidazol-2-yl)methyl)phenol (6c)
Yield 69%; White solid; mp 228–230 °C; IR (neat)
3250–2780 (br., peak), 2918, 2846, 1596, 1517, 1445, 1244,
1167, 1097, 1021, 782, 725; 1H NMR (300 MHz, Methanol-
d4) d 7.03–7.15 (m, 4H), 6.66–6.75 (m, 3H), 4.06–4.12 (t,
J = 6.42 Hz, 4H), 1.89 (sxt, J = 7.08 Hz, 2H), 1.10 (t,
J = 7.45 Hz, 3H); 13C NMR (100 MHz, Methanol-d4) d
157.7, 155.1, 131.0, 129.5, 124.1, 116.6, 105.0, 71.2, 35.2,
23.8, 11.0; HRMS (ESI) Calcd for C17H18N2O2 [M?H]?
283.1446, found 283.1466.
4-((4-Methoxy-1H-benzo[d]imidazol-2-yl)methyl)phenol (6d)
Yield 61%; Off white solid; mp 226.5–228 °C; IR (neat)
3250–2780 (br., peak), 2918, 2845, 1595, 1515, 1444,
1245, 1168, 1095, 1023, 835, 782, 725; 1H NMR
(300 MHz, Methanol-d4) d 7.06–7.15 (m, 4H), 6.68–6.75
(m, 3H), 4.09 (s, 2H), 3.95 (s, 3H); 13C NMR (100 MHz,
Methanol-d4) d 157.7, 155.0, 150.2, 141.1, 131.0, 129.3,
124.2, 116.7, 108.3, 104.1, 56.1, 35.2; HRMS (ESI)
Calcd for C15H14N2O2 [M ? H]? 255.1133, found
255.1161.
General procedure for the synthesis of compounds
6a–f and 6h–s
To a solution of the corresponding substituted alkyloxy-
benzene-1,2-diamine 4 (2.26 mmol) in 6N HCl (10 mL),
corresponding acid 5 (6.80 mmol) was added. The resulting
solution was refluxed for 16 h. The reaction mixture was
cooled to 0 °C, diluted with EtOAc, and then pH was
adjusted to *7 by using solid NaHCO3, extracted with
EtOAc. The combined organic extracts were washed with
brine solution, dried over anhydrous Na2SO4 and then con-
centrated under reduced pressure. The crude mixture was
subjected to flash silica gel (230–400 mesh) column chro-
matography to afford the title compounds 6a–f and 6h–s.
4-((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)
phenol (6e) Yield 68%; White solid; mp 118–121 °C; IR
(neat) 3300–2750 (br., peak), 2917, 2848, 1596, 1511, 1441,
1243, 1169, 1097, 1025, 833, 780, 729; 1H NMR (300 MHz,
DMSO-d6) d 12.31 (br. s., 0.5H), 12.11 (br. s., 0.5H), 9.23 (d,
1H), 7.10 (d, J = 7.82 Hz, 2.5H), 6.92–7.03 (m, 1.5H), 6.69
(d, J = 6.15 Hz, 2.5H), 6.59 (d, J = 6.89 Hz, 0.5H), 4.01 (s,
2H), 3.96 (d, J = 5.68 Hz, 1H), 3.90 (d, J = 6.15 Hz, 1H),
1.64–1.94 (m, 6H), 1.18–1.33 (m, 3H), 1.02–1.18 (m, 2H); 1H
123

P. Boggu et al.
NMR (300 MHz, DMSO-d6-D2O-exchange) d 7.12 (d,
J = 8.29 Hz, 2H), 6.90–7.09 (m, 2H), 6.57–6.70 (m, 3H),
3.99 (s, 2H), 3.86–3.92 (m, 2H), 1.60–1.87 (m, 6H),
1.10–1.25 (m, 5H); 1H NMR (300 MHz, Methanol-d4) d 7.12
(d, J = 8.38 Hz, 2H), 7.00–7.09 (m, 2H), 6.72 (d,
J = 8.38 Hz, 2H), 6.62–6.69 (m, 1H), 4.09 (s, 2H), 3.92 (d,
J = 6.24 Hz, 2H), 1.69–2.01 (m, 6H), 1.13–1.40 (m, 5H);
13C NMR (100 MHz, Methanol-d4) d 157.6, 155.1, 149.7,
131.0, 129.5, 124.0, 116.6, 108.5, 104.8, 75.0, 39.2, 35.2,
31.0, 27.7, 27.1; HRMS (ESI) Calcd for C21H24N2O2
[M?H]? 337.1916, found 337.1942.
6.92–6.98 (d, J = 1.82 Hz, 1H), 6.80 (dd, J = 1.82, 8.71 Hz,
1H), 6.69–6.77 (d, J = 8.29 Hz, 2H), 4.07 (s, 2H), 3.76 (d,
J = 6.15 Hz, 2H), 1.68–1.93 (m, 6H), 1.03–1.39 (m, 5H);
13C NMR (100 MHz, Methanol-d4) d 157.8, 157.6, 155.6,
131.7, 131.0, 129.3, 123.2, 116.7, 116.5, 113.4, 99.1, 75.4,
39.3, 35.4, 31.1, 27.8, 27.1; HRMS (ESI) Calcd for
C21H24N2O2 [M?H]? 337.1916, found 337.1944.
2-((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)
phenol (6k) Yield 61%; Off white solid; mp 201–203 °C; IR
(neat) 3300–2700 (br., peak), 2918, 2849, 1599, 1512, 1442,
1243, 1169, 1099, 1025, 833, 780, 732; 1H NMR (300 MHz,
Methanol-d4) d 6.99–7.12 (m, 4H), 6.72–6.84 (m, 2H),
6.63–6.69 (m, 1H), 4.18 (s, 2H), 3.91 (d, J = 6.24 Hz, 2H),
1.66–2.02 (m, 6H), 1.07–1.40 (m, 5H); 13C NMR (100 MHz,
Methanol-d4) d 156.7, 154.8, 131.4, 129.5, 125.2, 124.0,
121.0, 116.4, 105.0, 75.1, 39.3, 31.1, 30.7, 27.8, 27.1; HRMS
(ESI) Calcd for C21H24N2O2 [M?H]? 337.1916, found
337.1937.
4-((4-(2-Cyclohexylethoxy)-1H-benzo[d]imidazol-2-yl)methyl)
phenol (6f) Yield 62%; Purple solid; mp 89–93 °C; IR (neat)
3300–2750 (br., peak), 2918, 2847, 1595, 1512, 1441, 1243,
1169, 1097, 1025, 833, 781, 728; 1H NMR (300 MHz,
Methanol-d4) d 7.05–7.14 (m, 4H), 6.71 (d, J = 8.29 Hz,
3H), 4.17 (t, J = 6.57 Hz, 2H), 4.11 (s, 2H), 1.55–1.82 (m,
8H), 1.17–1.32 (m, 3H), 0.87–1.04 (m, 2H); 13C NMR
(100 MHz, Methanol-d4) d 157.8, 154.9, 149.6, 140.6, 131.5,
131.1, 128.9, 124.6, 116.7, 108.0, 105.3, 67.4, 37.9, 35.5,
34.9, 34.5, 27.8, 27.4; HRMS (ESI) Calcd for C22H26N2O2
[M?H]? 351.2072, found 351.2099.
4-(Cyclohexylmethoxy)-2-(4-methoxybenzyl)-1H-benzo[d]
imidazole (6l) Yield 71%; Light brown solid; 171–173 °C;
IR (neat) 2922, 2845, 1599, 1509, 1448, 1242, 1095, 1025,
1
985, 733; H NMR (300 MHz, CDCl3) d 9.25 (br. s., 1H),
4-((4-Phenethoxy-1H-benzo[d]imidazol-2-yl)methyl)phenol
(6h) Yield 58%; Off white solid; mp 220–222 °C; IR (neat)
3500–2600 (br., peak), 2922, 2851, 1598, 1514, 1445,
1247, 1170, 1099, 1021, 838, 782, 731; 1H NMR
(300 MHz, Methanol-d4) d 7.24–7.35 (m, 4H), 7.06–7.22
(m, 5H), 6.72 (d, J = 7.92 Hz, 3H), 4.35 (t, J = 7.03 Hz,
2H), 4.10 (s, 2H), 3.17 (t, J = 7.08 Hz, 2H); 13C NMR
(100 MHz, Methanol-d4) d 131.6, 131.0, 130.4, 129.7,
129.3, 127.7, 124.3, 116.7, 116.4, 105.5, 70.6, 36.8, 35.1;
HRMS (ESI) Calcd for C22H20N2O2 [M?H]? 345.1603,
found 345.1629.
7.05–7.27 (m, 4H), 6.86 (d, J = 8.66 Hz, 2H), 6.65 (d,
J = 8.57 Hz, 1H), 4.23 (s, 2H), 3.91 (d, J = 5.68 Hz, 2H),
3.80 (s, 3H), 1.65–1.92 (m, 6H), 0.98–1.30 (m, 5H); 13C
NMR (100 MHz, CDCl3) d 158.7, 152.7, 130.1, 128.6,
122.6, 114.2, 103.6, 73.8, 55.2, 37.4, 34.8, 29.8, 26.3, 25.6;
HRMS (ESI) Calcd for C22H26N2O2 [M?H]? 351.2072,
found 351.2093.
5-(Cyclohexylmethoxy)-2-(4-methoxybenzyl)-1H-benzo[d]
imidazole (6m) Yield 75%; Pale yellow solid; mp
140–143 °C; IR (neat) 2921, 2843, 1598, 1508, 1448,
1242, 1095, 1025, 985, 733; 1H NMR (400 MHz,
Methanol-d4) d 7.35 (d, J = 8.54 Hz, 1H), 7.18–7.24 (m,
2H), 6.96 (br. s., 1H), 6.84–6.89 (m, 2H), 6.81 (dd,
J = 2.20, 8.78 Hz, 1H), 4.11 (s, 2H), 3.74–3.78 (m, 5H),
1.83–1.91 (m, 2H), 1.69–1.83 (m, 4H), 1.23–1.36 (m, 3H),
1.05–1.14 (m, 2H); 13C NMR (100 MHz, Methanol-d4) d
160.4, 157.6, 155.4, 131.0, 130.6, 115.4, 113.5, 75.4,
55.8, 39.3, 35.4, 31.1, 27.8, 27.1; HRMS (ESI) Calcd for
C22H26N2O2 [M ? H]? 351.2072, found 351.2098.
4-((5-(Isopentyloxy)-1H-benzo[d]imidazol-2-yl)methyl)phenol
(6i) Yield 74%; Off white solid; mp 237–239 °C; IR (neat)
3350–2770 (br., peak), 2914, 2842, 1594, 1515, 1442, 1243,
1168, 1098, 1022, 783, 726; 1H NMR (300 MHz, Methanol-
d4) d 7.57 (d, J = 9.69 Hz, 1H), 7.18–7.25 (d, J = 8.47 Hz,
2H), 7.11–7.17 (m, 2H), 6.78–6.87 (d, J = 8.47 Hz, 2H),
4.38 (s, 2H), 4.08 (t, J = 6.47 Hz, 2H), 1.79–1.94 (m, 1H),
1.71 (q, J = 6.55 Hz, 2H), 0.98 (d, J = 6.61 Hz, 6H); 13C
NMR (100 MHz, Methanol-d4) d 159.9, 158.9, 154.2, 133.8,
131.6, 126.8, 125.0, 117.7, 117.4, 115.7, 97.9, 68.5, 39.1,
32.9, 26.3, 23.0; HRMS (ESI) Calcd for C19H22N2O2
[M?H]? 311.1759, found 311.1781.
4-(Cyclohexylmethoxy)-2-(2-methoxybenzyl)-1H-benzo[d]
imidazole (6n) Yield 63%; Light brown solid; mp
70–74 °C; IR (neat) 2920, 2842, 1597, 1507, 1447, 1241,
1
1095, 1025, 984, 735; H NMR (300 MHz, Methanol-d4)
4-((5-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)
phenol (6j) Yield 72%; Yellow solid; mp 108–112 °C
(shrinking) and 195–198 °C (liquid); IR (neat) 3300–2700
(br., peak), 2918, 2847, 1597, 1512, 1441, 1243, 1169, 1096,
d 7.19–7.27 (m, 1H), 7.08–7.14 (m, 1H), 7.01–7.08 (m,
2H), 6.95 (d, J = 8.10 Hz, 1H), 6.87 (dt, J = 0.88,
7.43 Hz, 1H), 6.62–6.70 (m, 1H), 4.18 (s, 2H), 3.92 (d,
J = 6.33 Hz, 2H), 3.82 (s, 3H), 1.68–2.01 (m, 6H),
1.09–1.39 (m, 5H); 13C NMR (100 MHz, Methanol-d4) d
159.0, 154.5, 149.7, 131.4, 129.7, 126.8, 123.9, 121.9,
1
1025, 833, 780, 729; H NMR (300 MHz, Methanol-d4) d
7.34 (d, J = 8.85 Hz, 1H), 7.06–7.16 (d, J = 8.29 Hz, 2H),
123

Exploration of SAR for novel 2-benzylbenzimidazole analogs as inhibitor of transcription…
111.8, 108.3, 104.9, 75.1, 56.0, 39.3, 31.1, 30.5, 27.8,
27.1; HRMS (ESI) Calcd for C22H26N2O2 [M?H]?
351.2072, found 351.2095.
(d, J = 6.15 Hz, 2H), 3.04 (m, 4H), 1.69–2.02 (m, 6H),
1.06–1.43 (m, 5H); 13C NMR (100 MHz, Methanol-d4) d
157.1, 155.5, 149.3, 133.1, 130.5, 123.9, 116.4, 108.7, 104.9,
75.0, 39.3, 35.1, 32.3, 31.1, 27.8, 27.1; HRMS (ESI) Calcd
for C22H26N2O2 [M?H]? 351.2072, found 351.2096.
2-(4-Chlorobenzyl)-4-(cyclohexylmethoxy)-1H-benzo[d]
imidazole (6o) Yield 77%; Light yellow solid; mp
82–85 °C; IR (neat) 2920, 2849, 1505, 1490, 1444, 1421,
1241, 1092, 1015, 991, 782, 729; 1H NMR (300 MHz,
Methanol-d4) d 7.28 (s, 4H), 6.99–7.13 (m, 2H), 6.63–6.73
(m, 1H), 4.18 (s, 2H), 3.92 (d, J = 6.24 Hz, 2H), 1.68–2.00
(m, 6H), 1.07–1.39 (m, 5H); 13C NMR (100 MHz, Metha-
nol-d4) d 153.8, 149.6, 137.6, 133.9, 131.5, 129.9, 124.2,
108.6, 104.9, 75.0, 39.2, 35.3, 31.0, 27.7, 27.1; HRMS (ESI)
Calcd for C21H23ClN2O [M ? H]? 355.1577, found
355.1598.
4-((4-(Benzyloxy)-1H-benzo[d]imidazol-2-yl)methyl)phenol
(6g) (Boggu et al. 2016). To a stirred solution of 3-(ben-
zyloxy)benzene-1,2-diamine 4g (4.67 mmol) in xylenes
(10 mL), 2-(4-hydroxyphenyl) acetic acid (9.34 mmol) and
boric acid (0.46 mmol) were added. The resulting solution
was refluxed for 48 h. After cooling to room temperature,
the reaction was concentrated under reduced pressure and
diluted with EtOAc. The organic phase was washed with
saturated NaHCO3 solution, dried over anhydrous Na2SO4
and then concentrated under reduced pressure. The residue
was purified by flash silica gel (230–400 mesh) column
chromatography to afford the title compounds 6g. Yield
22%; White solid; mp 105–109 °C; IR (neat) 3400–2600
(br., peak), 2921, 2850, 1594, 1515, 1442, 1245, 1172,
1098, 1022, 837, 784, 732; 1H NMR (300 MHz, Methanol-
d4) d 7.46–7.57 (m, 2H), 7.26–7.40 (m, 3H), 7.06–7.14 (m,
4H), 6.75–6.80 (m, 1H), 6.68–6.74 (m, 2H), 5.23 (s, 2H),
4.08 (s, 2H); 13C NMR (100 MHz, Methanol-d4) d 157.7,
155.3, 149.1, 138.9, 131.0, 129.7, 129.4, 129.2, 129.1,
124.1, 116.8, 116.7, 108.8, 105.7, 71.6, 35.2; HRMS (ESI)
Calcd for C21H18N2O2 [M ? H]? 331.1446, found
331.1471.
2-(4-Hydroxybenzyl)-1H-benzo[d]imidazol-4-ol (6p) Yield
75%; Dark green solid; mp 285–287 °C; IR (neat)
3450–2750 (br. peak), 2933, 2851, 1499, 1443, 1421, 1248,
1094, 1014, 783, 732; 1H NMR (300 MHz, Methanol-d4) d
7.29–7.37 (t, J = 8.15 Hz, 1H), 7.18–7.23 (m, 2H), 7.12
(d, J = 8.20 Hz, 1H), 6.90 (d, J = 7.92 Hz, 1H),
6.80–6.85 (m, 2H), 4.38 (s, 2H); 13C NMR (75 MHz,
Methanol-d4) d 158.8, 153.7, 146.8, 134.3, 131.3, 128.5,
125.0, 122.6, 117.3, 111.4, 105.1, 32.8; HRMS (ESI) Calcd
for C14H12N2O2 [M?H]? 241.0977, found 241.0997.
2-(2-Hydroxybenzyl)-1H-benzo[d]imidazol-4-ol (6q) Yield
68%; Light brown solid; mp 127–130 °C; IR (neat)
3500–2700 (br. peak), 2923, 2852, 1498, 1442, 1422, 1244,
1093, 1016, 783, 732; 1H NMR (300 MHz, Methanol-d4) d
7.04–7.14 (m, 2H), 6.93–7.00 (m, 2H), 6.74–6.86 (m, 2H),
6.53–6.60 (m, 1H), 4.17 (s, 2H); 13C NMR (100 MHz,
Methanol-d4) d 156.8, 154.6, 147.4, 131.6, 129.5, 125.1,
124.1, 121.0, 116.5, 108.2, 106.7, 30.8; HRMS (ESI) Calcd
for C14H12N2O2 [M?H]? 241.0977, found 241.0999.
Biological study
According to our previous reports, the inhibitory effect
of benzimidazole analogs 6a–s against NF-jB tran-
scriptional activity was evaluated using RAW 264.7 cells
containing pNF-jB-SEAP-neomycin phosphotransferase
(NTP) (Roh et al. 2011). In brief, RAW 264.7 monocytic
cell line stably transfected with NF-jB-dependent
secretory alkaline phosphatase (SEAP) construct (Pas-
tukhov and Ropson 2003; Mancek-Keber and Jerala
2006; Doyle and O’Neill 2006), a reporter gene of NF-
jB transcriptional activity was stimulated with LPS
(30 ng/mL) for 20 h in the presence of sample. Aliquots
of the culture 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 summarized in Table 1, representing the IC50
value and inhibition % at the concentration 30 lM from
three independent experiments using the average values
of triplicate in each experiment.
2-(4-Methoxybenzyl)-1H-benzo[d]imidazol-4-ol (6r) Yield
65%; Light brown solid; mp 58–62 °C; IR (neat)
3200–2700 (br. peak), 2922, 2851, 1491, 1445, 1422,
1
1244, 1093, 1016, 783, 731; H NMR (300 MHz, CDCl3)
d
7.22 (d, J = 8.66 Hz, 2H), 7.07–7.14 (m, 1H),
6.79–6.88 (m, 4H), 4.31 (s, 2H), 3.80 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 158.8, 152.9, 147.6, 136.3, 130.8,
130.2, 127.9, 124.1, 114.4, 108.8, 103.2, 55.2, 34.2;
HRMS (ESI) Calcd for C15H14N2O2 [M ? H]? 255.1133,
found 255.1158.
4-(2-(4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)
ethyl)phenol (6s) Yield 76%; Light yellow solid; mp
118–120 °C; IR (neat) 3350–2700 (br., peak), 2919, 2848,
1598, 1513, 1441, 1242, 1168, 1098, 1024, 833, 780, 731;
1H NMR (300 MHz, Methanol-d4) d 7.04–7.12 (m, 2H),
7.00 (d, J = 8.38 Hz, 2H), 6.67 (d, J = 8.29 Hz, 3H), 3.92
123

P. Boggu et al.
Biological results and SAR
Results and discussion
Chemistry
In the first set of experiments, isopentyloxy analog 6a
(77.0% inhibition at 30 lM, IC50 = 12.0 lM) was pre-
pared and studied for their LPS induced NF-jB inhibition,
which showed almost similar activity as compared to JSH
and less activity than the benzimidazole compound 1
(75.0% inhibition at 30 lM, IC50 = 6.0 lM). For the
confirmation of optimum size of the hydrophobic sub-
stituent at position 4 on benzimidazole ring A of 1, alkoxy
groups were variated as shown in isobutoxy 6b (46.0%
inhibition at 30 lM, IC50 = [ 30.0 lM), propoxy 6c
(30.0% inhibition at 30 lM, IC50 = [ 30.0 lM) and
methoxy 6d (38.0% inhibition at 30 lM, IC50 = [30.0 -
lM) analogs. The decrement in the chain length consid-
erably reduced the activity, which is indicating that bulky
hydrophobic group appears as an important factor for the
inhibition of NF-jB.
Thereafter, to confirm that the optimum size of the
hydrophobic group on benzimidazole ring A of 1, the
bulkier alkoxy groups such as cyclohexylmethoxy 6e
([100.0% inhibition at 30 lM, IC50 = 3.0 lM) and
cyclohexylethoxy 6f (88.0% inhibition at 30 lM,
IC50 = 17.0 lM) were introduced. Surprisingly, analog 6e
showed more potent inhibition than 1, JSH or 6a. How-
ever, increasing the chain length as shown in 6f decreased
the activity. These results confirmed our view point that
bulky hydrophobic group on benzimidazole ring A of 1
should be important for the activity.
The synthetic routes to obtain the target benzimidazole
derivatives (6a-s, Table 1) by cyclisation reactions of
benzene-1,2-diamine 4 (or 7) and substituted phenyl acetic
acid (5) were outlined in Schemes 1 and 2. Commercially
available 2-amino-3-nitrophenol (2a) or 4-amino-3-nitro-
phenol (2b) was transformed into the corresponding
alkoxynitroanilines 3 using alkyl halides with K2CO3 in
N,N-dimethylformamide solvent at 60 °C for 8–16 h.
Compounds 3 was then converted into the alkoxybenzene-
1,2-diamines 4 using 10% Pd–C in the presence of
hydrogen gas in methanol at room temperature for 2 h. The
corresponding benzene-1,2-diamine 4 was reacted with
appropriate phenyl acetic acid 5 in 6N HCl solution at
reflux temperature for 16 h to obtain desired benzimidazole
derivatives (6a–f, 6h–o and 6s). In order to obtain 6g,
compound 2a was reacted with benzyl bromide using
potassium carbonate in N,N-dimethylformamide to give
2-(benzyloxy)-6-nitroaniline 3g, which was reduced by the
treatment with SnCl2ÁH2O in ethanol at reflux temperature
for 5 h to obtain 3-(benzyloxy)benzene-1,2-diamine 4g.
The reaction of 4g with 2-(4-hydroxyphenyl) acetic acid
under the above same condition (6N HCl, reflux) yielded
the debenzylated product 6p instead of 6g. To overcome
this problem, anhydrous condition using boric acid in
refluxing xylene was adopted (Boggu et al. 2016) to get the
desired product 6g. The compounds 6p–r were obtained by
reduction of nitro compound 2a followed by cyclization
with appropriate substituted phenyl acetic acid in 6N HCl.
All these synthesized compounds were characterized by
physical and spectral analysis data that confirmed their
assigned structures.
In the next set of experiments, replacement of cyclo-
hexyl group in 6e and 6f as shown in benzyl analog 6g
(85.0% inhibition at 30 lM, IC50 = 12.0 lM) and phe-
nethyl analog 6h (87.0% inhibition at 30 lM,
IC50 = 18.0 lM) decreased the activity as compared to 6e.
These results indicated that the bulkier cyclohexyl group
have better activity than the phenyl one.
1
Initially the H NMR spectrum of compound 6e was
Next objective of our work was to find the suitable po-
sition of bulky hydrophobic substituent on benzimidazole
ring A of 1. Thus, location of hydrophobic alkoxy groups
was moved to position 5 of benzimidazole as shown in
5-isopentyloxy analog 6i and 5-cyclohexylmethoxy analog
recorded in DMSO-d6 solvent, which showed approxi-
mately 1:1 ratio mixture of two tautomers of benzimidazole
motif (Fig. 3, see supplementary material of NMR data of
1
6e). In order to avoid these kinds of complications in H
NMR, we recorded spectra in methanol-d4 or CDCl3.
Scheme 1 Synthesis of benzimidazole derivatives 6a–o and 6s (substituents are listed in Table 1). Reagents and Conditions: (a) R-X, K2CO3,
DMF, 60 °C, 8–16 h, 67–87%; (b) 10% Pd–C, H2, methanol, rt, 2 h (by using parr shaker), 72–92%; or (c) SnCl2, ethanol, reflux, 5 h (for
R = benzyl), 65%; (d) 6N HCl, reflux, 16 h, 58–77%; or (e) boric acid, xylene, reflux, 48 h, (for R = benzyl), 22%
123

Exploration of SAR for novel 2-benzylbenzimidazole analogs as inhibitor of transcription…
Scheme 2 Synthesis of benzimidazole derivatives 6p–r (substituents are listed in Table 1). Reagents and conditions: (a) 10% Pd–C, H2,
methanol, rt, 2 h (by using parr shaker), 95%; (b) 6N HCl, reflux, 16 h, 65–75%
Fig. 3 Tautomerism of
benzimidazole analog 6e
Fig. 4 SAR analysis of the novel benzimidazole 6 analogs
6j. Analog 6i (84.0% inhibition at 30 lM, IC50 = 7.0 lM)
showed more potent activity compared to 6a, whereas less
potent activity compared to 6e. However, compound 6j
(96.0% inhibition at 30 lM, IC50 = 4.0 lM) showed
almost similar activity as compared to 6e. These results
benzimidazole 6e was shifted to position 2 on phenyl ring
B as shown in analog 6k (95.0% inhibition at 30 lM,
IC50 = 5.0 lM), which resulted in the comparable activity.
Thus, this suggests that the position 2 and 4 of phenyl ring
B for hydroxyl group should be equally suitable for these
benzimidazole analogs in the inhibition of NF-jB.
confirmed that both positions 4 and
5 for bulky
hydrophobic cyclohexylmethoxy group on the benzimida-
zole ring A of 1 are acceptable for the inhibition of LPS
induced NF-jB activity.
The promising activity of 6e led us to shift our focus
toward finding the suitable position for the hydroxyl group
on phenyl ring B, since the hydroxyl group on phenyl ring
in the JSH compound plays an important role for the
inhibition of NF-jB activation as we discussed earlier (Roh
et al. 2011). Therefore, position of hydroxyl group on
To prove the importance of hydroxyl functional group
on phenyl ring B, the hydrogen bonding ability of hydroxyl
function in the highly active compounds 6e, 6j and 6k were
masked by the replacement with methoxy group as shown
in 6l (71.0% inhibition at 30 lM, IC50 = 17.0 lM), 6m
(48.0% inhibition at 30 lM, IC50 = [30.0 lM) and 6n
(90.0% inhibition at 30 lM, IC50 = 8.0 lM) or chloro
group in 6o (46.0% inhibition at 30 lM, IC50 = [30 lM).
Analogs 6l and 6n showed moderate activity while the
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P. Boggu et al.
activity of 6m and 6o were nearly abolished. These results
indicated that the hydrogen bonding ability of hydroxyl
function in ring B should be essential for the NF-jB
inhibitory activity of these benzimidazoles.
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In order to discover the optimum functional groups of
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Acknowledgement This work was supported by Priority Research
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