Discovery of benzimidazole analogs as a novel interleukin-5 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.111574

A series of novel hydroxyethylaminomethylbenzimidazole analogs 5a-y were synthesized and evaluated for their IL-5 inhibitory activity using pro-B Y16 cell line. Among them, 2-(((4-(cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)amino)butan-1-ol (5e, 94.3% inhibition at 30 μM, IC₅₀ = 3.5 μM, cLogP = 4.132) and 3-cyclohexyl-2-(((4-(cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)amino) propan-1-ol (5k, 94.7% inhibition at 30 μM, IC₅₀ = 5.0 μM, cLogP = 6.253) showed the most potent inhibitory activity. The essential feature of SAR (Fig. 5) indicated that the chromenone ring can be replaced by a benzimidazole ring to maintain the inhibitory activity. In addition, the hydroxyethylaminomethyl group was suitable for the IL-5 inhibitory activity. Moreover, the hydrophobic substituents on carbon play an important role in the IL-5 inhibitory activity of these analogs. However, N-substituted analogs did not improve inhibitory activity. In addition, MTT assay of 5e and 5k with normal B lymphoblasts revealed that they had no significant effects on cell viability.

Full text of DOI:10.1016/j.ejmech.2019.111574

Accepted Manuscript
Discovery of benzimidazole analogs as a novel interleukin-5 inhibitors
Pulla Reddy Boggu, Youngsoo Kim, Sang-Hun Jung
PII:
S0223-5234(19)30704-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.111574
Article Number: 111574
Reference:
EJMECH 111574
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 May 2019
Revised Date: 19 July 2019
Accepted Date: 29 July 2019
Please cite this article as: P.R. Boggu, Y. Kim, S.-H. Jung, Discovery of benzimidazole analogs
as a novel interleukin-5 inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111574.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of benzimidazole analogs as a novel interleukin-5 inhibitors
a,b
c
a
Pulla Reddy Boggu , Youngsoo Kim , Sang-Hun Jung *
a
College of Pharmacy and Institute of Drug Research and Development, Chungnam
National University, Daejeon 34134, Republic of Korea
b
School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do 16419, Republic of
Korea
c
College of Pharmacy, Chungbuk National University, Chinju 19421, Republic of Korea
Abstract
A series of novel hydroxyethylaminomethylbenzimidazole analogs 5a-y were synthesized
and evaluated for their IL-5 inhibitory activity using pro-B Y16 cell line. Among them, 2-
(
((4-(cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)amino)butan-1-ol (5e, 94.3%
inhibition at 30 µM, IC50 = 3.5 µM, cLogP = 4.132) and 3-cyclohexyl-2-(((4-
cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)amino) propan-1-ol (5k, 94.7%
(
inhibition at 30 µM, IC50 = 5.0 µM, cLogP = 6.253) showed the most potent inhibitory
activity. The essential feature of SAR (Fig. 5) indicated that the chromenone ring can be
replaced by benzimidazole ring to maintain the inhibitory activity. In addition, the
hydroxyethylaminomethyl group was suitable for the IL-5 inhibitory activity. Moreover, the
hydrophobic substituents on carbon play an important role in the IL-5 inhibitory activity of
these analogs. However, N-substituted analogs did not improve the inhibitory activity. In
addition, MTT assay of 5e and 5k with normal B lymphoblasts revealed that they had no
significant effects on cell viability.

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Key words: Benzimidazole, Interleukin-5, Inhibitor, SAR
*
Corresponding author: E-mail: jungshh@cnu.ac.kr
1
. Introduction
In the past decades have noticed a worldwide increase in allergic diseases, including asthma,
atopic dermatitis, allergic rhinitis, and food allergy [1,2]. It is believed that several
environmental factors interacting with genetic factors contribute to sensitization to
environmental allergens and to suffering from allergic diseases [3,4]. The pivotal roles of T
helper type 2 (Th2) cytokines have been well defined in these diseases and therefore
therapeutic strategies that target the Th2 cytokines are of potential benefit in allergic disease
[
5–7]. In these regards, glucocorticoids with immunosuppressive activities such as
dexamethasone have been used as the most fruitful treatment accessible for hypersensitivity
diseases including asthma, atopic dermatitis and food allergy [8]. Nevertheless, during long
term treatment, systemic side effects are identified [9]. Consequently, this issue resulted in
many efforts to discover novel potential therapies, which is safer than corticoids.
In the 1990s, eosinophilic inflammation is well established as fundamental causes of allergic
diseases. Excessive release of eosinophils from the bone marrow into circulation by the
stimulation of allergens results in rapid accumulation in the tissue, where they produce lipid
mediators, enzymes and proteins to cause tissue damage and eventually adverse allergic
reactions such as nasal rhinitis, asthma, and atopic dermatitis. Thus selective inhibition of
eosinophil is an ideal target for reducing tissue damage as well as allergic diseases without
inducing entire immunosuppressive consequences. [10–14].

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In these considerations, the IL-5 antagonist with small molecular weight has been
investigated. The small isothiazolone molecules by the modification of Cys 66 residue in IL-5
was originally tested as its antagonists [15]. In an effort to screening the natural compounds
had resulted in the discovery of sophoricoside (SOP, 1) [16] isolated from Sophora japonica,
which showed specific inhibition in the mIL-5-dependent Y16 proliferation over IL-3 and
GM-CSF stimulation [17]. However, SOP (1) is chemically and metabolically unstable
glycoside and therefore the formation of stable analogs as a candidate was inevitable. Thus,
the structural necessities of this isoflavone for its inhibitory effect against IL-5 were
investigated [18,19]. The structural requirements of these isoflavone analogs comprise a
th
planar chromen-4-one ring, the existence of hydroxyl group at the 4 position of B ring, and
th
introduction of hydrophobic units at 5 position, which might regulate the permeability of
these isoflavones. Fortunately, the metabolically unstable glycopyranosyl group of SOP is not
needed for the inhibitory activity [18,19]. Our further studies explored the exact role of B
ring and the optimum connection between the chromenone core and phenyl ring B. Thus, a
series of hydroxyethylaminomethylchromen-4-one (2 and 3, Fig. 1) [20–22] were discovered
as IL-5 inhibitor.
Recently, our efforts focused to find a novel bioisostere of the chromen-4-one ring of SOP.
Thus, a novel 2-benzyl-1-indanone scaffold (4, Fig. 1) was tested as IL-5 inhibitor [23].
However, the 1-indanone compounds showed similar activity as compared to chromen-4-one
compounds. Therefore, to get more potency than chromenone and 1-indanone analogs we
synthesized a novel hybrid scaffold 5 (Fig. 2), which is designed from the chromen-4-one (2
&
3) and 1-indanone (4) scaffolds, and evaluated the IL-5 inhibitory effect on IL-5 bioassay.

ACCEPTED MANUSCRIPT
Figure 1. Representative IL-5 inhibitors.
Figure 2. Design of novel benzimidazole analogs as IL-5 inhibitor.
2
. Chemistry

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The synthetic routes to obtain the target benzimidazole derivatives 5a-y by cyclization
reactions of 1,2-diamines and 2-chloroacetic acid followed by nucleophilic substitution with
various substituted amino alcohols are outlined in Scheme 1, and substituents are denoted in
Table 1. The synthesis of 1,2-diamine 6 was adopted by previously reported literature [24].
The intermediate 6 was reacted with 2-chloroacetic acid in the presence of aq. 6N HCl to
afford the corresponding cyclized chloro intermediate 7. The final compounds 5a-y were
obtained from the nucleophilic substitution reaction of chloro intermediate 7 with the
appropriate amino alcohol in the presence of potassium carbonate (K CO ) in N,N-
2
3
dimethylformamide (DMF). The intermediate 9 was synthesized from the ethanolamine by
reaction with alkyl bromide in the presence of K CO in DMF in excellent yields. All the
2
3
1
13
synthesized compounds were mainly characterized by H and C NMR spectroscopy
techniques.
1
Interestingly, on comparing the H NMR spectra of linear chain benzimidazoles 5a-d, C-
substituted benzimidazoles 5e, 5f, and 5h-k and N-substituted benzimidazoles 5l-u, we
observed doublet of doublet (dd) signals of benzylic methylene (–CH -NH) protons and dd or
2
multiplet (m) signals of alcoholic methylene (–CH -OH) protons for the C-substituted
2
benzimidazoles, however, the linear chain and N-substituted benzimidazoles showed singlet
(s) of benzylic methylene and triplet (t) of alcoholic methylene protons at room temperature
1
(
see the supplementary material). Distinct features of H NMR spectra for the benzylic
methylene protons and alcoholic methylene protons of the benzimidazoles of 5a, 5e, 5g and
m are illustrated in Fig. 3 and their chemical shift values are listed in Table 2. These data
clearly indicate that the chiral center as well as tautomerism of C-substituted benzimidazole
e is effecting the splitting pattern (geminal and vicinal coupling) of the benzylic methylene
5
5
and alcoholic methylene protons as shown in Fig. 3a & 3b, which can be detected as separate
1
sets of signals in H NMR. However, the linear chain benzimidazole 5a, C-disubstituted

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benzimidazole 5g, and N-substituted benzimidazole 5m did not detect separate sets of signals.
1
Moreover, on comparing the H NMR spectra of linear chain benzimidazoles 5a-d, C-
substituted benzimidazoles 5e-k and N-substituted benzimidazoles 5l-u, we observed doublet
(d) of o- proton and triplet (t) of m- proton and broad singlet (br. s.) of p- proton of the N-
substituted benzimidazoles, however, the linear chain and C-substituted benzimidazoles
showed doublet (d) of o- proton and multiplet (m) of m- and p- protons at room temperature
(see the supplementary material). As shown in Fig. 3c, the aromatic p- proton of the
compound 5m showed broad singlet pattern. This outcome clearly indicates that the
tautomerism of N-substituted benzimidazole 5m is causing the splitting pattern of the
aromatic p- proton. Additionally, we also observed that some of the quaternary carbon signals
13
in C NMR spectra of benzimidazoles are not observable possibly due to tautomerism [24]
(see the supplementary material).

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Scheme 1. Synthesis of 5a-y (substituents are denoted in Table 1). Reagents and Conditions:
(
a) Chloroacetic acid, 6N HCl, reflux, 16 h, 72%; (b) Amine 8 (or) 9, K CO , DMF, 60 °C, 3
2 3
3
h, 52 - 76%; (c) Amine 10, K CO , DMF, 60 °C, 3 h, 71 - 78%; (d) R -Br, K CO , DMF, 60
2
3
2
3
°
C, 3 h, 81 - 89%.
Table 1. Substituents, cLog P values and IL-5 activity results of synthesized
hydroxyethylaminomethylbenzimidazoles 5a-y
Substituents
IL-5
Comp.
No.
a
b
CLogP %Inhibition
IC50
1
2
3
R
R
R
n
b
at 30 µM
(µM)
20.0
28.5
30.0
>30.0
3.5
c
5
a
CH
2
Cy
H
H
H
H
H
H
H
H
H
H
H
1
2
3
4
1
1
1
3.294
3.624
3.487
4.016
4.133
4.662
4.003
63.3
52.7
44.3
23.7
94.3
73.3
54.7
5
b
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
Cy
5
c
Cy
Cy
Cy
Cy
Cy
5
d
5
e
5
f
7.5
5
g
(CH
3
)
2
27.5
5
h
CH
CH
CH
2
2
2
Cy
Cy
Cy
H
H
H
1
2
1
4.339
4.644
5.172
77.7
64.7
81.0
20.0
22.0
12.0
5
i
5
j

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5
k
CH
2
Cy
H
1
6.254
94.7
5.0
5
l
CH
CH
CH
2
2
2
Cy
Cy
Cy
H
H
H
1
1
1
4.894
4.365
3.836
63.0
34.7
42.0
20.0
>30.0
>30.0
5
m
5
n
CH
3
5
o
CH
2
2
Cy
Cy
H
H
1
1
4.674
5.867
10.0
24.0
>30.0
>30.0
5
p
CH
5
q
CH
2
Cy
H
1
6.486
76.7
21.0
5
r
s
CH
2
2
Cy
Cy
H
H
1
1
5.552
4.055
62.3
43.3
25.0
5
CH
>30.0
5
t
CH
2
Cy
H
1
6.265
96.0
13.0
5
u
CH
2
2
Cy
Cy
H
-
1
-
5.471
5.045
73.3
49.3
19.0
5
v
CH
>30.0
5
w
CH
CH
CH
2
2
2
Cy
Cy
Cy
-
-
-
-
-
-
4.965
5.466
5.568
43.3
25.0
20.3
>30.0
>30.0
>30.0
5
5
x
y
2a[20]
2b[20]
3a[21]
4.791
5.873
5.172
72.0
93.0
94.8
25.7
17.3
16.1

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3
b[21]
6.106
5.856
94.7
12.4
4.0
4a [23]
100.0
e
Sophoricoside (1)
79.1
10.6
26.2
e
Budesonide
70.2
a
®
b
ClogP calculated by Chemdraw (ver. 15.1); % of inhibition and IC values are taken as a
5
0
c
mean from three independent experiments; Cy = Cyclohexyl.
1
Figure 3. Comparison among H NMR data of compound 5a (linear chain benzimidazole),
5
e (C-substituted benzimidazole), and 5g (C-disubstituted benzimidazole), 5m (N-substituted

ACCEPTED MANUSCRIPT
benzimidazole). (a) Proton peaks of benzylic methylene group, (b) Proton peaks of alcoholic
methylene group, (c) Proton peaks of aromatic ring (o-, m-, and p-).
1
Table 2. H NMR chemical shifts (δ) of the benzylic methylene, alcoholic methylene, and
aromatic ring protons of 5a, 5e, 5g, and 5m
Chemical shift
Benzylic –CH2
5a
5e
5g
5m
4.18 (s, 2H)
4.21 (d, J = 15.65 Hz, 1H)
4.05 (s, 2H)
3.98 (s)
4
.08 (d, J = 15.55 Hz, 1H)
3.72 (dd, J = 3.59, 11.13 Hz, 1H)
.45 (dd, J = 6.80, 11.18 Hz, 1H)
Alcoholic –CH2
Aromatic ring
3.70 (t, J = 4.0 Hz, 2H)
7.07 - 7.17 (m, 2H),
3.39 (s, 2H)
3.73 (t, J = 5.12 Hz, 2H)
3
m- & p-
o-
7.07 - 7.19 (m, 2H),
7.07 - 7.20 (m, 2H),
6.66 (d, J = 8.38 Hz, 1H),
7.11 (t, J = 8.0 Hz, 1H, m-),
6
.87-7.26 (br.s., 1H, p-)
6.64 (d, J = 7.56 Hz, 1H)
6.66 (d, J = 7.54 Hz, 1H)
6.66 (d, J = 7.80 Hz, 1H)
3
. Pharmacology
Inhibitory activity of the benzimidazole derivatives 5 against IL-5 was evaluated using the
IL-5 dependent pro-B Y16 cell line according to the previously described procedure [16]. The
Y16 cells were incubated with 3 units/mL mIL-5 for 48 h in the presence or absence of the
compound, and then cell metabolism was calculated as an index of proliferation, using WST-
1
. The outcomes of biological screening of benzimidazoles against IL-5 are noted in Table 1
as % inhibition at 30 µM and also IC values. To exclude any cytotoxic effects, the
5
0
most potent compounds 5e and 5k were subjected to MTT assay with normal B lymphoblasts
such as HCC1395 BL and NCI-BL 1184 that were incubated in the presence of serum but not
IL-5 (Table 3). The detailed assay protocols are designated in the experimental section.

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Table 3. MTT assay in normal B lymphoblasts
%
Inhibition at 30 µM
Comp. No.
HCC1395 BL
7.5 ± 4.6
NCI-BL1184
10.2 ± 8.8
9.8 ± 5.1
5
5
e
k
12.5 ± 9.3
Each value represents the mean ± SD of the three independent measurements.
4
. Conformational analysis and alignment studies of 5k and 5q
®
Molecular models of the benzimidazole analogs 5k and 5q were constructed using SYBYL -
x2.0 program package (Tripos Associates Inc.)[25] and their geometry were optimized
(Powell conjugate gradient minimization, termination at a gradient of 0.0005 kcal/mol)[26]
using the Tripos standard force field and Gasteiger-Huckel atomic partial charges. All the
molecules were aligned by an atom-by-atom least-square fit and used the 4-
cyclohexymethoxybenzimidazole structure as a template as represented. The 3D structures of
the analyzed compounds were assumed to be a bioactive conformation and were aligned
according to a 4-cyclohexylmethoxybenzimidazole template as shown in Fig. 4. The selected
dihedral angles and atomic distances are listed in Table 4 and 5 respectively.

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Figure 4. Alignment of 5k (Salmon color) and 5q (Light pink color)
Table 4. Torsion angle (°) and total energy (kcal/mol) of 5k and 5q
a
a
Compound 5k (27.138 kcal/mol)
Compound 5q (25.967 kcal/mol)
∠
∠
∠
N -C -C -N
252.9
199.5
295.3
66.2
∠N -C -C -N
179.6
359.8
66.3
3
2
6
7
3
2
6
7
C -C -N -C
∠C -C -N -C
2
6
7
8
2
6
7
8
C -N -C -C
∠C -N -C -C
6
7
8
9
6
7
8
9
∠
N -C -C -O
∠N -C -C -O
173.9
7
8
9
10
7
8
9
10
a
Numbers on the atoms of 5k and 5q are presented in Fig. 4.
Table 5. Distance (Å) in 5k and 5q
Compound 5k
a
a
Compound 5q
O -O
7.976
5.133
O -O
5.362
4.225
5
10
5
10
N -O
N -O
3
10
3
10
a
Numbers on the atoms of 5k and 5q are presented in Fig. 4.
4
. Results and discussion

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We designed and synthesized a novel series of benzimidazole derivatives 5a-y (Table 1) with
aminoalcohol group to evaluate which moiety plays an important role in the IL-5 inhibitory
activity. Initially, analog 5a (63.3% inhibition at 30 µM, IC = 20.0 µM, cLogP = 3.294)
5
0
showed similar inhibitory activity as compared to budesonide (IC50 = 26.2 µM) and
chromenone 2a (IC = 25.7 µM). This result suggested that the chromenone ring can be
5
0
replaced by benzimidazole ring.
The above result encouraged us to find a potent bezimidazole analog as IL-5 inhibitor. Thus
to find out the optimum length of aminoalcohol moiety between amino (NH) and hydroxyl
(
OH) function in 5a, methylene linkage was increased. As a result, decrement in the
inhibitory activity was observed in the case of three and four methylene unit analogs 5b (n =
, 52.7% inhibition at 30 µM, IC50 = 28.5 µM, cLogP = 3.624) and 5c (n = 3, 44.3%
inhibition at 30 µM, IC = 30.0 µM, cLogP = 3.487), and complete loss of activity was
2
5
0
observed in five methylene unit analog 5d (n =4, 23.7% inhibition at 30 µM, IC = >30.0
5
0
µM, cLogP = 4.016). This result supports that chain length of amino alcohol plays an
important role in the inhibitory activity of benzimidazole analogs and therefore
hydroxyehylamino group is optimum.
1
2
Interestingly, branched aminoethanol analogs as shown in 5e (n = 1, R = ethyl, R = H,
1
2
9
4.3% inhibition at 30 µM, IC = 3.5 µM, cLogP = 4.132) and 5f (n = 1, R = propyl, R =
50
H, 73.3% inhibition at 30 µM, IC = 7.5 µM, cLogP = 4.661) showed 5.7-fold and 2.7-fold
5
0
more active than unbranched analog 5a, respectively. However, dimethyl branched analog 5g
54.7% inhibition at 30 µM, IC = 27.5 µM, cLogP = 4.002) did not improve the inhibitory
(
5
0
activity. Thus, only straight chain hydrophobic group as a branch in amino ethanol should be
more favourable for the inhibitory activity.
In the next set of experiments, phenyl substituted aminoethanol analog 5h (77.7% inhibition
at 30 µM, IC = 20.0 µM, cLogP = 4.339) was prepared and exhibited moderate inhibitory
5
0

ACCEPTED MANUSCRIPT
activity as compared to 5a (IC = 27.0 µM). This result suggested that the planar group as a
5
0
branch is still applicable to maintain the IL-5 inhibitory activity. In addition, we increased
methylene unit between amino and hydroxyl function of 5h. As a result, decrement in the
activity was observed in 5i (64.7% inhibition at 30 µM, IC = 22.0 µM, cLogP = 4.64367).
5
0
This result again confirmed that aminoethanol group is optimum for the activity. However,
insertion of methylene unit (one methylene unit) between amino alcohol chain and the phenyl
group in 5h as shown in 5j (81.0% inhibition at 30 µM, IC = 12.0 µM, cLogP = 5.171)
5
0
enhanced the inhibitory activity. Interestingly, replacement of phenyl group of 5j with
cyclohexyl moiety as shown in 5k (94.7% inhibition at 30 µM, IC = 5.0 µM, cLogP =
5
0
6
.253) led to slight enhancement of inhibitory activity. This implied that the influence of
bulkiness of the hydrophobic group in this region should be marginal.
For determining more detail SAR of 5a, hydrophobic substituents at the amino group were
introduced. Accordingly, we prepared n-propyl analog 5l (63.0% inhibition at 30 µM, IC =
5
0
2
0.0 µM, cLogP = 4.894), ethyl analog 5m (34.7% inhibition at 30 µM, IC = >30.0 µM,
50
cLogP = 4.365), and methyl analog 5n (42.0% inhibition at 30 µM, IC = >30 µM, cLogP =
5
0
3
.836), which did not turn up the IL-5 inhibitory activity. In another set of experiments, the
bulky hydrophobic groups on the nitrogen of 5a were introduced. The resulted isopropyl 5o
10.0% inhibition at 30 µM, IC = >30.0 µM, cLogP = 4.674) and cyclohexyl 5p (24.0%
(
5
0
inhibition at 30 µM, IC = >30.0 µM, cLogP = 5.867) analogs showed almost diminished the
5
0
inhibitory activity. Insertion of a methylene unit between amino nitrogen and bulky
cyclohexyl group as in analog 5q (76.7% inhibition at 30 µM, IC = 21.0 µM, cLogP =
5
0
6
.486) showed better inhibitory activity than 5p and 5a. Therefore, directly attached bulky
hydrophobic groups on amino nitrogen are not suitable for the IL-5 inhibitory activity.
Replacement of cyclohexyl group in 5q with planar phenyl ring as shown in benzyl analog 5r
(
62.3% inhibition at 30 µM, IC = 25.0 µM, cLogP = 5.552) showed similar activity as
50

ACCEPTED MANUSCRIPT
compared to 5q. Installing basic function with pyridine-4-ylmethyl as shown in 5s (43.3%
inhibition at 30 µM, IC = >30.0 µM, cLogP = 4.055) gave the adverse effect on the activity.
5
0
To further investigate the effect of hydrophobic substituents at position 4 of the phenyl ring
of 5r, analogs 5t and 5u were prepared. Slight increment in the inhibitory activity was
observed in 4-chlorobenzyl 5t (96.0% inhibition at 30 µM, IC = 13.0 µM, cLogP = 6.265)
5
0
and 4-methoxybenzyl 5u (73.3% inhibition at 30 µM, IC = 19.0 µM, cLogP = 5.471). These
5
0
efforts marginally variated the activity.
In another set of experiment, we removed hydroxyethyl group of 5r and 5u. The resulted
compounds 5v (49.3% inhibition at 30 µM, IC = >30.0 µM, cLogP = 5.045) and 5w (43.3%
5
0
inhibition at 30 µM, IC = >30.0 µM, cLogP = 4.964) much decreased the activity. Further,
5
0
we reduced the methylene unit between amino and phenyl ring of 5v and 5w as shown in
analog 5x (25.0% inhibition at 30 µM, IC50 = >30 µM, cLogP = 5.467) and 5y (20.3%
inhibition at 30 µM, IC50 = >30.0 µM, cLogP = 5.568) which did not improve the IL-5
inhibitory activity. These results imply that the hydroxyethyl group is essential for the
activity.
Lipophilicity of analogs 5a-y as indicated with cLogP does not correlate with the activity.
However, increasing the hydrophobic binding ability of substituent on terminal carbon is
important for the enhancement of the activity.
To investigate the effective conformations for the potent IL-5 inhibitor, we compared the 3D
structural sketches of active C-substituted analog 5k and less active N-substituted analog 5q
(
Fig. 4 and Table 4, 5) and observed a dramatic difference in the region of the side chain at
position 2 of benzimidazoles. As shown in Table 4, the dihedral angle ( C -C -N -C ) of
analog 5k is 199.5°, which indicates that the hydroxyethylaminomethyl group at position 2 of
k is located out of the plane of the benzimidazole ring. In addition, the longer distance (O5-
O = 7.976 Å, Table 5) from the oxygen (O ) of the benzimidazole ring to the oxygen (O )
∠
2
6
7
8
5
1
0
5
10

ACCEPTED MANUSCRIPT
of hydroxyethylaminomethyl group of 5k also supports this stretched conformation. The
∠
corresponding dihedral angle ( C -C -N -C , Table 4) of 5q is 359.8°, which depicts that the
2 6 7 8
hydroxyethanolaminemethyl chain oxygen (O ) is close to cyclohexylmethoxy oxygen (O )
1
0
5
of benzimidazole ring. Therefore, the shorter distance (O -O = 5.362 Å, Table 5) from the
5
10
oxygen (O ) of benzimidazole ring to the oxygen (O ) of hydroxyethylaminomethyl group of
5
10
5
q compared to that of 5k clearly indicates the folded conformation. Therefore, these studies
indicate that the C-substituted analog 5k could be much closer to the effective conformation
for binding to the putative receptor.
For confirmation of IL-5 activity related to the cytotoxicity, an MTT assay of the active
compounds was carried out with normal B lymphoblasts. As shown in Table 3, none of the
compounds 5e and 5k showed significant effects on cell viability. These results implied
that the IL-5 inhibitory activity of 5e and 5k could be originated from the inhibition of
proliferation of Y-16 cells by blocking the stimulatory activity of IL-5 rather than cytotoxic
effect. Thus this could be effective and safe as drug candidate for the treatment of
eosinophilia and other allergic disorders.
5
. Conclusion
A series of novel hydroxyethylaminomethylbenzimidazole analogs 5a-y were synthesized
and evaluated for their IL-5 inhibitory activity. Among them, C-substituted benzimidazole
analogs 5e (IC50 = 3.5 µM) and 5k (IC50 = 5.0 µM) showed the most potent inhibitory
activity than the previously reported chromenone analog 2b (IC50 = 17.3 µM) and
Budesonide (IC50 = 26.2 µM). The essential feature of SAR (Fig. 5) indicated that the
chromenone ring can be replaced by benzimidazole ring to maintain the inhibitory activity. In
addition, the hydroxyethylaminomethyl group was suitable for the IL-5 inhibitory activity.
Moreover, the hydrophobic substituents on carbon play an important role in the IL-5

ACCEPTED MANUSCRIPT
inhibitory activity of these analogs. However, N-substituted analogs did not improve the
inhibitory activity. In addition, MTT assay of 5e and 5k with normal B lymphoblasts revealed
that they had no significant effects on cell viability. Taken together, current study identifies
the benzimidazole molecules as a novel scaffold for inhibiting the IL-5 receptor and makes
them potential therapeutic agents against eosinophilia and other allergic disorders.
Figure 5. SAR analysis of the novel benzimidazole analogs 5
6
. Materials and methods
6
.1. Chemistry
All commercial chemicals were used as obtained and all solvents were purified by distillation
prior to use applying the standard procedures [27]. Thin layer chromatography (TLC) was
performed on E Merck silica gel GF-254 precoated plates, visualization was performed under
UV illumination (λ = 254 nm), and colorization with KMnO and I . All derivatives were
4
2
purified by flash column chromatography which was done on E Merck silica gel (230–400
1
13
mesh). H and C NMR spectra was measured against the peak of tetramethylsilane (TMS)
using a JEOL, JNM-AL400 NMR (400 MHz) and Bruker Fourier 300 NMR (300 MHz)

ACCEPTED MANUSCRIPT
spectrometer. Melting points (mp) of the synthesized compounds were determined on an
Electro thermal 1A 9100 MK2 apparatus and are uncorrected. Infrared (IR) spectra was noted
on a Nicolet 380 model FTIR. HRMS was measured in ESI ionization by using AB Sciex
Triple TOF 5600 LCMS instrument.
6
.1.1. General procedure for the synthesis of compounds 5a-y
To a solution of 2-(chloromethyl)-4-(cyclohexylmethoxy)-1H-benzo[d]imidazole 7 (0.72
mmol) in DMF (5 mL), K CO (0.79 mmol) and appropriate amine 8 or 9 or 10 (0.79 mmol)
2
3
were added. The resulting solution was stirred at 60 °C for 3 h. The mixture was cooled,
diluted with water, and then extracted with EtOAc. The combined organic extracts were
washed with water, brine solution, dried over anhydrous Na SO and then concentrated under
2
4
reduced pressure. The crude mixture was subjected to flash silica gel (230-400 mesh) column
chromatography (eluting with 0 - 2% MeOH in dichloromethane) to afford the title
compounds 5.
6
.1.1.1.
2-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)amino)ethan-1-ol
(5a). Yield 52%; Off white solid; mp 116 – 118 °C; IR (neat) 3400 - 2600 (br., peak), 2920,
-
1 1
2
850, 1597, 1537, 1445, 1425, 1244, 1189, 1094, 1079, 984, 881, 781, 728 cm ; H NMR
7.07 - 7.17 (m, 2H), 6.64 (d, J = 7.56 Hz, 1H), 4.18 (s, 2H), 3.89 (d, J =
.34 Hz, 2H), 3.70 (t, J = 4.0 Hz, 2H), 2.84 - 2.91 (t, J = 4.0 Hz, 2H), 1.62 - 1.88 (m, 6H),
(400 MHz, CDCl3) δ
6
0
1
1
3
.96 - 1.28 (m, 5H); C NMR (100 MHz, CDCl )
δ
152.6, 148.2, 140.2, 128.6, 122.8, 107.7,
03.7, 73.8, 60.6, 51.2, 46.9, 37.5, 29.8, 26.3, 25.6; HRMS (ESI) calculated for C H N O
2
3
1
7
25
3
+
[
M+H] 304.2025, found 304.2046.
6
.1.1.2.
3-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)amino)propan-1-ol
(5b). Yield 56%; Colorless sticky oil; IR (neat) 3500 - 2500 (br., peak), 2921, 2849, 1595,
-1 1
1
541, 1443, 1325, 1255, 1240, 1102, 1065, 995, 781, 725 cm ; H NMR (300 MHz, CDCl )
3

ACCEPTED MANUSCRIPT
δ
7.08 - 7.20 (m, 2H), 6.62 - 6.70 (m, 1H), 4.12 (s, 2H), 3.92 (d, J = 6.15 Hz, 2H), 3.82 (t, J =
1
3
5
.54 Hz, 2H), 2.92 (t, J = 6.01 Hz, 2H), 1.61 - 1.92 (m, 8H), 1.00 - 1.30 (m, 5H); C NMR
(100 MHz, CDCl3)
δ
151.9, 148.3, 139.9, 128.8, 122.8, 107.5, 103.6, 73.7, 61.6, 47.7, 46.9,
+
3
3
7.4, 31.0, 29.7, 26.3, 25.5; HRMS (ESI) calculated for C H N O [M+H] 318.2181, found
1
8
27
3
2
18.2202.
6
.1.1.3.
4-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)amino)butan-1-ol
(5c). Yield 63%; Light yellow sticky oil to solid; mp 55 – 57 °C; IR (neat) 3500 - 2500 (br.,
-1 1
peak), 2922, 2849, 1623, 1594, 1541, 1443, 1325, 1256, 1241, 1102, 1065, 781, 725 cm ; H
NMR (300 MHz, CDCl3) 7.09 - 7.20 (m, 2H), 6.66 (dd, J = 1.12, 7.45 Hz, 1H), 4.15 (s,
H), 3.92 (d, J = 6.15 Hz, 2H), 3.63 (t, J = 4.84 Hz, 2H), 2.74 (t, J = 5.45 Hz, 2H), 1.63 -
δ
2
1
1
1
3
.95 (m, 10H), 1.02 - 1.30 (m, 5H); C NMR (100 MHz, CDCl )
δ 151.2, 148.3, 140.0,
3
28.8, 122.9, 107.6, 103.6, 73.7, 62.0, 48.9, 46.7, 37.4, 31.1, 29.7, 27.0, 26.3, 25.6; HRMS
+
(
ESI) calculated for C H N O [M+H] 332.2338, found 332.2361.
1
9
29
3
2
6
.1.1.4.
5-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)amino)pentan-1-ol
(5d). Yield 63%; Colorless sticky oil; IR (neat) 3500 - 2500 (br., peak), 2920, 2851, 1625,
-1 1
1
593, 1446, 1330, 1243, 1102, 1045, 1006, 780, 721 cm ; H NMR (300 MHz, CDCl ) δ
3
7
.08 - 7.22 (m, 2H), 6.67 (d, J = 7.64 Hz, 1H), 4.11 (s, 2H), 3.93 (d, J = 6.15 Hz, 2H), 3.65 (t,
J = 6.19 Hz, 2H), 2.69 (t, J = 6.85 Hz, 2H), 1.82 - 1.98 (m, 3H), 1.64 - 1.80 (m, 3H), 1.36 -
1
3
1
.62 (m, 6H), 0.97 - 1.35 (m, 5H); C NMR (100 MHz, CDCl ) δ 152.1, 148.1, 140.3, 128.5,
3
1
22.8, 107.9, 103.7, 73.8, 61.9, 49.1, 47.2, 37.5, 32.0, 29.8, 28.9, 26.3, 25.6, 23.1; HRMS
+
(
ESI) calculated for C H N O [M+H] 346.2494, found 346.2518.
2
0
31
3
2
6
.1.1.5.
2-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)amino)butan-1-ol
(5e). Yield 61%; White solid; mp 84 – 86 °C; IR (neat) 3500 - 2500 (br., peak), 2920, 2851,
-1 1
1
626, 1594, 1531, 1447, 1331, 1246, 1102, 1046, 1007, 781, 727 cm ; H NMR (300 MHz,

ACCEPTED MANUSCRIPT
CDCl3)
δ
7.07 - 7.19 (m, 2H), 6.66 (d, J = 7.54 Hz, 1H), 4.21 (d, J = 15.65 Hz, 1H), 4.08 (d,
J = 15.55 Hz, 1H), 3.91 (d, J = 6.15 Hz, 2H), 3.72 (dd, J = 3.59, 11.13 Hz, 1H), 3.45 (dd, J =
.80, 11.18 Hz, 1H), 2.60 - 2.70 (m, 1H), 1.62 - 1.94 (m, 6H), 1.38 - 1.61 (m, 2H), 1.00 - 1.36
6
1
3
(
m, 5H), 0.93 (t, J = 7.54 Hz, 3H); C NMR (100 MHz, CDCl )
δ
153.2, 148.2, 140.0, 128.6,
3
1
22.7, 107.5, 103.6, 73.8, 62.8, 60.6, 44.7, 37.4, 29.8, 26.3, 25.6, 24.1, 10.3; HRMS (ESI)
+
calculated for C H N O [M+H] 332.2338, found 332.2359.
1
9
29
3
2
6
.1.1.6.
2-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)amino)pentan-1-ol
(5f). Yield 58%; Colorless oil; IR (neat) 3500 - 2500 (br., peak), 2921, 2850, 1625, 1593,
-
1 1
1
7
1
530, 1446, 1245, 1102, 1046, 1007, 781, 727 cm ; H NMR (300 MHz, CDCl ) δ 7.08 -
3
.20 (m, 2H), 6.66 (d, J = 7.92 Hz, 1H), 4.21 (d, J = 15.65 Hz, 1H), 4.08 (d, J = 15.55 Hz,
H), 3.92 (d, J = 6.05 Hz, 2H), 3.72 (dd, J = 3.40, 11.13 Hz, 1H), 3.43 (dd, J = 6.61, 11.18
Hz, 1H), 2.67 - 2.78 (m, 1H), 1.81 - 1.96 (m, 3H), 1.63 - 1.80 (m, 3H), 1.02 - 1.50 (m, 9H),
1
3
0
.86 - 0.97 (m, 3H); C NMR (100 MHz, CDCl ) δ 153.2, 148.2, 140.2, 128.4, 122.7, 107.7,
3
1
03.7, 73.8, 63.2, 59.0, 44.7, 37.5, 33.7, 29.8, 26.3, 25.6, 19.2, 14.1; HRMS (ESI) calculated
+
for C H N O [M+H] 346.2494, found 346.2515.
2
0
31
3
2
6
.1.1.7.
2-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)amino)-2-
methylpropan-1-ol (5g). Yield 65%; White solid; mp 121 – 123 °C; IR (neat) 3500 - 2500
(br., peak), 2915, 2848, 1600, 1548, 1450, 1425, 1330, 1276, 1248, 1102, 1055, 995, 852,
-
1 1
7
4
1
3
81, 727 cm ; H NMR (300 MHz, CDCl ) δ 7.07 - 7.20 (m, 2H), 6.66 (d, J = 8.38 Hz, 1H),
3
.05 (s, 2H), 3.91 (d, J = 6.24 Hz, 2H), 3.39 (s, 2H), 1.62 - 1.93 (m, 6H), 0.95 - 1.34 (m,
1
1H); H NMR (400 MHz, CD OD)
δ 7.07 - 7.12 (m, 2H), 6.66 - 6.72 (m, 1H), 3.97 (s, 2H),
3
.92 (d, J = 6.34 Hz, 2H), 3.41 (s, 2H), 1.84 - 2.00 (m, 3H), 1.69 - 1.82 (m, 3H), 1.24 - 1.37
1
3
(
m, 3H), 1.09 - 1.19 (m, 9H); C NMR (100 MHz, CD OD) δ 154.9, 149.7, 124.3, 108.6,
3

ACCEPTED MANUSCRIPT
1
05.1, 75.1, 69.4, 55.6, 41.0, 39.3, 31.1, 27.8, 27.1, 23.7; HRMS (ESI) calculated for
+
C H N O [M+H] 332.2338, found 332.2362.
19
29
3
2
6
.1.1.8.
2-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)amino)-2-
phenylethan-1-ol (5h). Yield 75%; White solid; mp 85 – 87 °C; IR (neat) 3500 - 2500 (br.,
-1 1
peak), 2919, 2850, 1596, 1538, 1444, 1246, 1097, 1061, 1021, 729, 699 cm ; H NMR (300
MHz, CDCl3) 7.28 - 7.37 (m, 5H), 7.08 - 7.23 (m, 2H), 6.67 (d, J = 8.38 Hz, 1H), 3.97 -
.09 (m, 2H), 3.92 (d, J = 5.96 Hz, 2H), 3.78 - 3.90 (m, 2H), 3.64 - 3.73 (m, 1H), 1.63 - 1.96
δ
4
13
(
m, 6H), 1.00 - 1.31 (m, 5H); C NMR (100 MHz, CDCl )
δ
152.7, 139.9, 128.7, 127.8,
3
1
27.3, 122.8, 103.8, 73.8, 66.7, 64.5, 45.1, 37.5, 29.8, 26.4, 25.6; HRMS (ESI) calculated for
+
C H N O [M+H] 380.2338, found 380.2359.
23
29
3
2
6
.1.1.9.
phenylpropan-1-ol (5i). Yield 72%; Off white solid; mp 77 – 79 °C; IR (neat) 3500 - 2500
br., peak), 2920, 2850, 1596, 1538, 1504, 1445, 1247, 1097, 1061, 1020, 992, 781, 729, 699
3-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-
(
-1 1
cm ; H NMR (300 MHz, CDCl )
δ
7.27 - 7.36 (m, 4H), 7.22 - 7.26 (m, 1H), 7.09 - 7.21 (m,
3
2
H), 6.67 (d, J = 8.38 Hz, 1H), 3.77 - 4.05 (m, 7H), 1.67 - 2.06 (m, 8H), 0.96 - 1.35 (m, 5H);
1
3
C NMR (100 MHz, CDCl3)
δ
152.7, 142.9, 128.6, 127.3, 126.8, 122.7, 103.7, 73.7, 61.5,
+
6
0.9, 44.7, 39.2, 37.4, 29.7, 26.3, 25.6; HRMS (ESI) calculated for C H N O [M+H]
24 31 3 2
3
94.2494, found 394.2515.
.1.1.10. 2-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)amino)-3-
6
phenylpropan-1-ol (5j). Yield 71%; Colorless sticky oil; IR (neat) 3500 - 2500 (br., peak),
-
1 1
2
920, 2850, 1594, 1536, 1503, 1446, 1246, 1096, 1062, 1021, 781, 729, 698 cm ; H NMR
300 MHz, CDCl3) 7.27 - 7.35 (m, 2H), 7.01 - 7.25 (m, 5H), 6.65 (d, J = 8.75 Hz, 1H), 4.16
d, J = 16.11 Hz, 1H), 4.02 (d, J = 16.11 Hz, 1H), 3.91 (d, J = 6.33 Hz, 2H), 3.72 (dd, J =
.54, 11.08 Hz, 1H), 3.49 (dd, J = 5.59, 10.99 Hz, 1H), 2.94 (td, J = 5.62, 10.83 Hz, 1H),
(
(
δ
3

ACCEPTED MANUSCRIPT
1
3
2
.78 (d, J = 6.98 Hz, 2H), 1.69 - 1.94 (m, 6H), 0.90 - 1.30 (m, 5H); C NMR (100 MHz,
CDCl3)
δ
153.2, 138.8, 129.4, 128.6, 126.5, 122.8, 103.7, 73.8, 62.9, 60.7, 44.9, 38.3, 37.5,
+
2
9.9, 26.4, 25.6; HRMS (ESI) calculated for C H N O [M+H] 394.2494, found 394.2515.
2
4
31
3
2
6
.1.1.11.
3-Cyclohexyl-2-(((4-(cyclohexylmethoxy)-1H-benzo[d]imidazol-2-
yl)methyl)amino)propan-1-ol (5k). Yield 63%; Colorless sticky oil; IR (neat) 3500 - 2500
-
1 1
(
br., peak), 2920, 2848, 1623, 1590, 1535, 1444, 1243, 1101, 1046, 1007, 781, 727 cm ; H
NMR (300 MHz, CDCl3) 7.07 - 7.21 (m, 2H), 6.66 (d, J = 8.48 Hz, 1H), 4.20 (d, J = 15.65
Hz, 1H), 4.07 (d, J = 15.65 Hz, 1H), 3.93 (d, J = 6.15 Hz, 2H), 3.71 (dd, J = 3.49, 11.04 Hz,
δ
1
1
5
H), 3.40 (dd, J = 6.47, 11.13 Hz, 1H), 2.82 (dq, J = 3.73, 6.40 Hz, 1H), 1.59 - 1.95 (m,
1
3
1H), 0.86 - 1.40 (m, 13H); C NMR (100 MHz, CDCl )
δ 153.2, 122.7, 103.7, 73.8, 63.6,
3
6.5, 44.7, 39.6, 37.5, 34.3, 33.5, 33.5, 29.8, 26.4, 26.1, 26.1, 25.6; HRMS (ESI) calculated
+
for C H N O [M+H] 400.2964, found 400.2987.
2
4
37
3
2
6
.1.1.12.
2-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-
yl)methyl)(propyl)amino)ethan-1-ol (5l). Yield 61%; Light brown solid; mp 107 – 109 °C; IR
(neat) 3500 - 2500 (br., peak), 2921, 2849, 1621, 1597, 1537, 1446, 1423, 1326, 1248, 1099,
-
1 1
1
7
3
-
058, 892, 782, 729 cm ; H NMR (400 MHz, CDCl ) δ 10.35 (br. s., 1H), 7.22 (br. s., 1H),
3
.12 (t, J = 7.93 Hz, 1H), 6.66 (d, J = 8.29 Hz, 1H), 3.98 (s, 2H), 3.93 (d, J = 6.34 Hz, 2H),
.74 (t, J = 5.12 Hz, 2H), 2.76 (t, J = 5.12 Hz, 2H), 2.53 - 2.60 (m, 2H), 1.92 (br. s., 3H), 1.66
1.80 (m, 3H), 1.52 (sxt, J = 7.41 Hz, 2H), 1.17 - 1.36 (m, 3H), 0.99 - 1.13 (m, 2H), 0.88 (t, J
1
3
=
7.32 Hz, 3H); C NMR (100 MHz, CDCl )
δ
152.6, 122.5, 103.6, 73.8, 59.5, 56.9, 56.5,
3
+
5
2.6, 37.4, 29.9, 26.4, 25.6, 19.9, 11.5; HRMS (ESI) calculated for C H N O [M+H]
20 31 3 2
3
46.2494 found 346.2517.
6
1
.1.1.13. 2-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)(ethyl)amino)ethan-
-ol (5m). Yield 68%; Light yellow solid; mp 99-102 °C; IR (neat) 3500 - 2500 (br., peak),

ACCEPTED MANUSCRIPT
2
919, 2847, 1596, 1538, 1446, 1426, 1324, 1259, 1245, 1099, 1056, 994, 891, 867, 781, 728
-1 1
cm ; H NMR (400 MHz, CDCl )
δ 10.36 (br. s., 1H), 6.87 - 7.26 (m, 2H), 6.66 (d, J = 7.80
3
Hz, 1H), 3.98 (s, 2H), 3.92 (d, J = 6.34 Hz, 2H), 3.73 (t, J = 5.12 Hz, 2H), 2.77 (t, J = 5.12
Hz, 2H), 2.71 (q, J = 7.07 Hz, 2H), 1.81 - 2.02 (m, 3H), 1.64 - 1.80 (m, 3H), 1.17 - 1.35 (m,
1
3
3
5
3
H), 0.99 - 1.13 (m, 5H); C NMR (100 MHz, CDCl )
δ
152.5, 122.6, 103.6, 73.8, 59.4,
3
+
5.8, 52.0, 48.4, 37.4, 29.9, 26.4, 25.6, 11.3; HRMS (ESI) calculated for C H N O [M+H]
19
29
3
2
32.2338 found 332.2359.
6
.1.1.14.
2-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-
yl)methyl)(methyl)amino)ethan-1-ol (5n). Yield 76%; Colorless sticky oil; IR (neat) 3500 -
500 (br., peak), 2920, 2846, 1595, 1538, 1445, 1426, 1258, 1245, 1099, 1055, 867, 781, 728
2
-1 1
cm ; H NMR (400 MHz, CDCl )
δ
7.24 (br. s., 1H), 7.12 (t, J = 7.93 Hz, 1H), 6.67 (d, J =
3
8
.78 Hz, 1H), 3.89 - 3.95 (m, 4H), 3.75 - 3.80 (m, 2H), 2.67 - 2.73 (m, 2H), 2.38 (s, 3H), 1.92
1
3
(
d, J = 12.44 Hz, 3H), 1.65 - 1.79 (m, 3H), 1.16 - 1.35 (m, 3H), 0.98 - 1.12 (m, 2H); C
NMR (100 MHz, CDCl3)
δ
151.8, 148.2, 122.6, 108.1, 103.6, 73.7, 59.4, 58.9, 55.8, 42.5,
+
3
3
7.4, 29.8, 26.3, 25.6; HRMS (ESI) calculated for C H N O [M+H] 318.2181, found
1
8
27
3
2
18.2205.
6
.1.1.15.
2-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-
yl)methyl)(isopropyl)amino)ethan-1-ol (5o). Yield 61%; Light yellow solid; mp 75 – 77 °C;
IR (neat) 3500 - 2500 (br., peak), 2919, 2848, 1622, 1598, 1538, 1445, 1424, 1326, 1248,
-
1 1
1
8
5
3
099, 1057, 892, 782, 729 cm ; H NMR (400 MHz, CDCl ) δ 10.48 (br. s., 1H), 7.11 (t, J =
3
.05 Hz, 1H), 6.99 (br. s., 1H), 6.65 (d, J = 8.05 Hz, 1H), 3.88 - 3.98 (m, 4H), 3.68 (t, J =
.24 Hz, 2H), 2.98 - 3.09 (m, 1H), 2.76 (t, J = 5.00 Hz, 2H), 1.92 (br. s., 3H), 1.66 - 1.80 (m,
13
H), 1.19 - 1.34 (m, 3H), 1.01 - 1.11 (m, 8H); C NMR (100 MHz, CDCl )
δ 153.7, 122.5,
3

ACCEPTED MANUSCRIPT
1
03.6, 73.8, 59.7, 52.6, 51.4, 48.1, 37.4, 29.9, 26.4, 25.6, 17.9; HRMS (ESI) calculated for
+
C H N O [M+H] 346.2494 found 346.2517.
20
31
3
2
6
.1.1.16.
2-(Cyclohexyl((4-(cyclohexylmethoxy)-1H-benzo[d]imidazol-2-
yl)methyl)amino)ethan-1-ol (5p). Yield 61%; White solid; mp 159 – 161 °C; IR (neat) 3500 -
2
1
7
500 (br., peak), 2921, 2849, 1619, 1598, 1541, 1500, 1449, 1426, 1356, 1324, 1241, 1101,
-1 1
070, 1015, 994, 890, 870, 781, 730 cm ; H NMR (400 MHz, CDCl )
δ
10.47 (br. s., 1H),
3
.10 (t, J = 8.05 Hz, 1H), 6.98 (br. s., 1H), 6.65 (d, J = 8.29 Hz, 1H), 4.00 (br. s., 2H), 3.93
(br. s., 2H), 3.67 (t, J = 5.12 Hz, 2H), 2.82 (t, J = 5.24 Hz, 2H), 2.47 - 2.62 (m, 1H), 1.59 -
1
3
2
.00 (m, 11H), 1.04 - 1.32 (m, 10H); C NMR (100 MHz, CDCl ) δ 154.1, 122.4, 103.6,
3
7
3.9, 60.9, 60.1, 53.4, 48.7, 37.4, 29.9, 28.8, 26.4, 26.0, 25.9, 25.7; HRMS (ESI) calculated
+
for C H N O [M+H] 386.2807 found 386.2828.
2
3
35
3
2
6
.1.1.17.
2-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-
yl)methyl)(cyclohexylmethyl)amino)ethan-1-ol (5q). Yield 66%; Off white solid; mp 83 – 85
°
C; IR (neat) 3500 - 2500 (br., peak), 2920, 2848, 1619, 1599, 1543, 1501, 1448, 1426, 1356,
-
1 1
1
1
2
324, 1241, 1102, 1069, 1015, 782, 730 cm ; H NMR (400 MHz, CDCl ) δ 7.23 (br. s.,
3
H), 7.12 (t, J = 7.93 Hz, 1H), 6.67 (d, J = 8.05 Hz, 1H), 3.97 (s, 4H), 3.73 (t, J = 5.00 Hz,
H), 2.73 (br. s., 2H), 2.39 (d, J = 7.32 Hz, 2H), 1.56 - 2.03 (m, 12H), 0.90 - 1.32 (m, 10H);
1
3
C NMR (100 MHz, CDCl3)
δ
152.6, 122.6, 103.7, 73.9, 62.2, 59.8, 57.1, 53.3, 37.5, 35.9,
+
3
1.6, 29.9, 26.6, 26.5, 25.9, 25.7; HRMS (ESI) calculated for C H N O [M+H] 400.2964
2
4
37
3
2
found 400.2987.
6
1
1
.1.1.18. 2-(Benzyl((4-(cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)amino)ethan-
-ol (5r). Yield 68%; Colorless sticky oil; IR (neat) 3500 - 2500 (br., peak), 2920, 2850,
-1 1
619, 1595, 1538, 1508, 1448, 1355, 1324, 1242, 1100, 1065, 1030, 783, 730 cm ; H NMR
7.28 - 7.38 (m, 5H), 7.22 - 7.27 (m, 1H), 7.11 (t, J = 8.01 Hz, 1H), 6.66
(300 MHz, CDCl3)
δ

ACCEPTED MANUSCRIPT
(d, J = 8.57 Hz, 1H), 3.99 (s, 2H), 3.93 (d, J = 5.96 Hz, 2H), 3.73 - 3.80 (m, 4H), 2.82 (t, J =
1
3
5
.03 Hz, 2H), 1.85 - 1.99 (m, 3H), 1.67 - 1.81 (m, 3H), 1.03 - 1.34 (m, 5H); C NMR (100
MHz, CDCl3)
δ
152.3, 138.1, 128.9, 128.4, 127.3, 122.6, 103.7, 73.8, 59.4, 58.9, 56.1, 52.0,
+
3
3
7.4, 29.8, 26.3, 25.6; HRMS (ESI) calculated for C H N O [M+H] 394.2494, found
2
4
31
3
2
94.2515.
6
.1.1.19.
2-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)(pyridin-4-
ylmethyl)amino)ethan-1-ol (5s). Yield 56%; Light brown sticky oil; IR (neat) 3500 - 2500
(br., peak), 3079, 2920, 2855, 1633, 1594, 1535, 1509, 1448, 1375, 1354, 1235, 1105, 1069,
-
1 1
1
031, 981, 782, 729 cm ; H NMR (400 MHz, CDCl )
δ
8.44 (d, J = 6.10 Hz, 2H), 7.29 (d, J
6.20 Hz, 2H), 7.19 (br. s., 1H), 7.09 - 7.14 (m, 1H), 6.66 (d, J = 8.54 Hz, 1H), 4.01 (s, 2H),
.91 (d, J = 6.10 Hz, 2H), 3.81 (t, J = 5.00 Hz, 2H), 3.77 (s, 2H), 2.85 (t, J = 5.00 Hz, 2H),
3
=
3
1
1
3
.87 (d, J = 11.22 Hz, 3H), 1.65 - 1.74 (m, 3H), 1.13 - 1.29 (m, 4H), 0.97 - 1.08 (m, 2H); C
NMR (100 MHz, CDCl3)
δ
151.7, 149.4, 148.4, 123.7, 122.8, 103.8, 73.8, 59.5, 57.9, 56.7,
+
5
3
2.0, 37.5, 29.8, 26.3, 25.6; HRMS (ESI) calculated for C H N O [M+H] 395.2447, found
2
4
31
3
2
95.2468.
6
.1.1.20.
2-((4-Chlorobenzyl)((4-(cyclohexylmethoxy)-1H-benzo[d]imidazol-2-
yl)methyl)amino)ethan-1-ol (5t). Yield 62%; Yellow solid; mp 78 – 81 °C; IR (neat) 3500 -
-
1 1
2
500 (br., peak), 2921, 2851, 1594, 1538, 1446, 1252, 1090, 1014, 986, 783, 729 cm ; H
NMR (400 MHz, CDCl3) 7.27 - 7.29 (m, 1H), 7.23 - 7.27 (m, 4H), 7.17 (d, J = 6.83 Hz,
H), 7.09 - 7.14 (m, 1H), 6.66 (d, J = 8.78 Hz, 1H), 3.97 (s, 2H), 3.92 (d, J = 6.10 Hz, 2H),
.75 - 3.80 (m, 2H), 3.72 (s, 2H), 2.77 - 2.82 (m, 2H), 1.83 - 1.96 (m, 3H), 1.66 - 1.79 (m,
δ
1
3
3
1
13
H), 1.12 - 1.34 (m, 3H), 0.98 - 1.12 (m, 2H); C NMR (100 MHz, CDCl )
δ 152.1, 136.7,
3
32.9, 130.2, 128.4, 122.7, 103.7, 73.8, 59.3, 58.2, 56.1, 51.8, 37.4, 29.7, 26.3, 25.6; HRMS
+
(
ESI) calculated for C H ClN O [M+H] 428.2105, found 428.2126.
2
4
30
3
2

ACCEPTED MANUSCRIPT
6
.1.1.21.
2-(((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)(4-
methoxybenzyl)amino)ethan-1-ol (5u). Yield 67%; Light yellow crystals; mp 68 – 70 °C; IR
(neat) 3500 - 2500 (br., peak), 2920, 2850, 1595, 1538, 1509, 1446, 1243, 1099, 1033, 988,
-1 1
7
83, 729 cm ; H NMR (300 MHz, CDCl ) δ 10.14 (br. s., 1H), 7.18 - 7.27 (m, 3H), 7.11 (t,
3
J = 8.01 Hz, 1H), 6.82 - 6.86 (m, 2H), 6.66 (d, J = 8.66 Hz, 1H), 3.98 (s, 2H), 3.94 (d, J =
.59 Hz, 2H), 3.78 (s, 3H), 3.73 - 3.77 (m, 2H), 3.70 (s, 2H), 2.80 (t, J = 4.89 Hz, 2H), 1.93
5
1
3
(
d, J = 11.18 Hz, 3H), 1.70 - 1.85 (m, 3H), 1.05 - 1.33 (m, 5H); C NMR (100 MHz, CDCl )
3
δ
5
4
158.9, 158.9, 152.3, 130.2, 130.0, 129.9, 122.6, 113.8, 113.8, 103.7, 73.8, 59.5, 58.3, 56.0,
+
5.1, 55.1, 51.9, 37.4, 29.8, 26.4, 25.6; HRMS (ESI) calculated for C H N O [M+H]
2
5
33
3
3
24.2600, found 424.2621.
.1.1.22. N-benzyl-1-(4-(cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methanamine (5v).
6
Yield 76%; Light brown sticky oil; IR (neat) 3500 - 2500 (br., peak), 2918, 2850, 1603, 1506,
-1 1
1
445, 1244, 1097, 1016, 993, 782, 729 cm ; H NMR (300 MHz, CDCl ) δ 7.28 (m, 2H),
3
7
.18 - 7.26 (m, 3H), 7.02 - 7.18 (m, 2H), 6.61 (d, J = 8.20 Hz, 1H), 4.07 (s, 2H), 3.88 (d, J =
.96 Hz, 2H), 3.80 (s, 2H), 1.87 (d, J = 10.43 Hz, 3H), 1.62 - 1.79 (m, 3H), 0.97 - 1.29 (m,
5
1
3
5
H); C NMR (100 MHz, CDCl )
δ
152.5, 139.3, 128.6, 128.2, 127.3, 122.7, 103.7, 73.8,
3
+
5
3.5, 46.8, 37.5, 29.9, 26.4, 25.6; HRMS (ESI) calculated for C H N O [M+H] 350.2232,
2
2
27
3
found 350.2255.
6
.1.1.23.
1-(4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)-N-(4-
methoxybenzyl)methanamine (5w). Yield 78%; Colorless oil; IR (neat) 3500 - 2500 (br.,
-1 1
peak), 2921, 2849, 1616, 1595, 1510, 1446, 1234, 1096, 1033, 987, 817, 782, 729 cm ; H
NMR (300 MHz, CDCl3) 7.25 (d, J = 8.47 Hz, 2H), 7.19 (br. s., 1H), 7.10 - 7.16 (m, 1H),
.87 (d, J = 8.57 Hz, 2H), 6.67 (d, J = 8.01 Hz, 1H), 4.11 (s, 2H), 3.94 (d, J = 6.15 Hz, 2H),
δ
6
3
1
3
.80 (s, 5H), 1.70 - 1.96 (m, 6H), 1.07 - 1.34 (m, 5H); C NMR (75 MHz, CDCl )
δ 158.9,
3

ACCEPTED MANUSCRIPT
1
31.5, 129.4, 113.9, 103.7, 73.8, 55.3, 53.1, 46.9, 37.5, 30.0, 26.5, 25.8; HRMS (ESI)
+
calculated for C H N O [M+H] 380.2338, found 380.2362.
2
3
29
3
2
6
7
1
.1.1.24. N-((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)aniline (5x). Yield
1%; Off white solid; mp 78 – 80 °C; IR (neat) 3500 - 2500 (br., peak), 3396, 2918, 2850,
-1 1
602, 1505, 1445, 1422, 1313, 1245, 1097, 1016, 992, 891, 870, 782, 732 cm ; H NMR
7.11 - 7.22 (m, 4H), 6.79 (t, J = 7.36 Hz, 1H), 6.62 - 6.72 (m, 3H), 4.64
s, 2H), 4.44 (br. s., 1H), 3.93 (d, J = 6.33 Hz, 2H), 1.91 (d, J = 9.97 Hz, 3H), 1.65 - 1.78 (m,
(300 MHz, CDCl3) δ
(
1
3
3
H), 0.99 - 1.33 (m, 6H); C NMR (100 MHz, CDCl )
δ
152.2, 147.5, 129.4, 122.9, 118.5,
3
1
13.2, 103.8, 73.9, 42.9, 37.4, 29.8, 26.3, 25.6; HRMS (ESI) calculated for C H N O
2
1
25
3
+
[
M+H] 336.2076, found 336.2099.
6
.1.1.25.
N-((4-(Cyclohexylmethoxy)-1H-benzo[d]imidazol-2-yl)methyl)-4-methoxyaniline
(5y). Yield 74%; Yellow solid; mp 70 – 72 °C (shrinking), 147 – 150 °C; IR (neat) 3500 -
2
7
500 (br., peak), 2920, 2849, 1618, 1590, 1510, 1444, 1312, 1234, 1097, 1033, 987, 816,
-1 1
82, 732 cm ; H NMR (300 MHz, CDCl ) δ 9.65 (br.s., 1H), 7.21 (br. s., 1H), 7.11 - 7.17
3
(m, 1H), 6.73 - 6.80 (m, 2H), 6.59 - 6.72 (m, 3H), 4.60 (s, 2H), 4.18 (br.s., 1H), 3.89 - 3.97
1
3
(
m, 2H), 3.73 (s, 3H), 1.92 (d, J = 9.59 Hz, 3H), 1.67 - 1.84 (m, 3H), 1.02 - 1.33 (m, 5H); C
NMR (100 MHz, CDCl ) δ 152.9, 152.5, 141.5, 122.9, 114.9, 114.6, 114.5, 103.8, 73.9, 55.6,
3
+
4
3.9, 37.4, 29.9, 29.8, 26.3, 25.6; HRMS (ESI) calculated for C H N O [M+H] 366.2181,
2
2
27
3
2
found 366.2202.
6
.1.2. Synthesis of 2-(chloromethyl)-4-(cyclohexylmethoxy)-1H-benzo[d]imidazole (7). This
compound was prepared from 2-chloroacetic acid and 3-(cyclohexylmethoxy)benzene-1,2-
diamine (6) in a manner similar to the preparation of 5. Yield 72%; Light yellow solid; mp
1
25 – 127 °C; 1H NMR (300 MHz, CDCl3) δ 7.14 - 7.25 (m, 2H), 6.68 - 6.74 (m, 1H), 4.85 -

ACCEPTED MANUSCRIPT
4
.89 (m, 2H), 3.94 (d, J = 6.15 Hz, 2H), 1.64 - 1.96 (m, 6H), 0.96 - 1.36 (m, 5H); HRMS
+
(
ESI) calculated for C H ClN O [M+H] 279.1264, found 279.1285.
1
5
19
2
6
6
.2. Biology
.2.1. In vitro study: IL-5 bioassay, mIL-5-dependent Y16 proliferation [28].
Y16 cell line was originated from a murine early B cell. The cell line was grown in RPMI-
640 media containing 8% FBS and 3 units/mL mIL-5 at 37 °C with 5% CO . The Y16 cells
1
2
grown were harvested by centrifugation at 250 x g for 10 min at 4 °C, washed 2-times with
Hanks’ solution, and resuspended in a lesser volume of RPMI-1640 media containing 8%
4
FBS. Numbers of the cells were counted after trypan blue exclusion and the diluted to 1 x 10
cells/mL with RPMI-1640 media containing 8% FBS. Viability of the cells were more than
4
9
5% in all preparations. One hundred µL of (1 x 10 numbers) Y16 cells were allotted to each
well of a 96-well microplate, and 50 µL of 3 units/mL mIL-5 and 50 µL of sample was
added. Control group was treated with RPMI-1640 media containing 8% FBS instead of
sample, and blank group with RPMI-1640 media containing 8% FBS instead of mIL-5. After
incubation at 37 °C with 5% CO for 48 h, Y16 cells in each well were treated with 20 µL of
2
WST-1 solution. Absorbance at wavelength 450 nm (A ) was measured by using a
450
microplate reader after incubation at 37 °C with 5% CO for 2 - 4 h.
2
6.2.2. MTT assay
Normal B lymphocytes such as HCC1395 BL and NCI-BL1184 were cultured in RPMI-
1640 medium or IMDM (Iscove’s modified Dulbecco’s medium) containing 10% FBS at 37
°
C with 5% CO2. The cells were incubated with the compound 5e or 5k for 48 h and reacted
diphenyltetrazolium bromide (MTT) for
with 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-
another 3 h. Formazan crystals were dissolved in 99% DMSO, and absorbance values were
measured at 590 nm.