Synthesis and biological evaluation of aminobenzimidazole derivatives with a phenylcyclohexyl acetic acid group as anti-obesity and anti-diabetic agents

Article abstract of DOI:10.1016/j.bmcl.2013.05.081

A series of benzimidazole derivatives with a phenylcyclohexyl acetic acid group as DGAT-1 inhibitors was developed. Among the benzimidazole series, compound 5k showed submicromolar in vitro activity toward human and mouse DGAT-1, good selectivity toward D

Full text of DOI:10.1016/j.bmcl.2013.05.081

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4713–4718
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and biological evaluation of aminobenzimidazole
derivatives with a phenylcyclohexyl acetic acid group as anti-obesity
and anti-diabetic agents
Hyun Jung Kwak a, Yu Mi Pyun b, Ji Young Kim a,c, Haushabhau S. Pagire a,c, Ki Young Kim a, Kwang Rok Kim a,
Sang Dal Rhee a, Won Hoon Jung a, Jin Sook Song a, Myung Ae Bae a, Duck Hyung Lee b, Jin Hee Ahn a,c,
⇑
a Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea
b Department of Chemistry, Sogang University, Seoul 121-742, Republic of Korea
c Department of Medicinal and Pharmaceutical Chemistry, University of Science and Technology, Daejeon 305-333, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzimidazole derivatives with a phenylcyclohexyl acetic acid group as DGAT-1 inhibitors was
developed. Among the benzimidazole series, compound 5k showed submicromolar in vitro activity
toward human and mouse DGAT-1, good selectivity toward DGAT-2, human liver metabolic stability,
and pharmacokinetic (PK) and safety profiles such as hERG, CYP and acute toxicity. Additionally, 5k
showed good in vivo efficacy in 4 weeks study with DIO mouse model.
Received 7 February 2013
Revised 21 May 2013
Accepted 24 May 2013
Available online 4 June 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Diacylglycerol acyltransferase-1
DGAT-1
Triglycerides
TG
Obesity
Diabetes
Aminobenzimidazole
Triacylglycerols (triglycerides, TG), a major type of neutral lipid,
are a heterogeneous group of molecules with a glycerol backbone
and three fatty acids (FAs) attached by ester bonds. Excess accumu-
lation of triglycerides can participate in the pathogenesis of several
metabolic disorders such as obesity, insulin resistance and type II
diabetes and cardiovascular disease.1,2
Since the initial disclosure of DGAT-1 inhibitors by Bayer9 and
the Tularik/Japan Tobacco group,10 several companies have re-
ported orally active inhibitors of this enzyme that recapitulate sev-
eral metabolic aspects of the DGAT-1 knockout mice.11 In addition,
Novartis12 and Sankyo have advanced DGAT-1 inhibitors into the
clinic for dyslipidemia, diabetes and obesity.
Dietary TG is cleaved to monoacylglycerol and free fatty acids
(FFAs) by lipases in the gut lumen, which is moved to the entero-
cytes and reorganized by diverse enzymes.3 DGAT (acyl-CoA: diac-
ylglycerol acyltransferase), which has two isoforms (DGAT-1 and
DGAT-2), is a transmembrane enzyme that functions in the final
step of TG biosynthesis. DGAT-1 inhibition is expected to impair
the TG and chylomicrons formation in the enterocytes to reduce
postprandial TG levels in the blood.4
During the last few years, several classes of DGAT-1 inhibitors
have been reported.13 Among the classes of DGAT-1 inhibitors,
the phenylcyclohexyl acetic acid group is one of the most popular
and promising skeletons.14 Therefore, we looked for a new DGAT-1
inhibitor with a phenylcyclohexyl acetic acid group and identified
an aminobenzimidazole compound with an IC50 value of 1.06 lM
as a hit scaffold (Fig. 1). Herein, we report the synthesis of amino-
benzimidazole derivatives with a phenylcyclohexyl acetic acid
group and their biological evaluation as DGAT-1 inhibitors.
DGAT-1 deficient (Dgat-1-) mice are viable and resistant to
weight gain when fed a high-fat diet and show increased insulin
and leptin sensitivity.5–7 These findings have lead a number of
companies to pursue selective DGAT-1 inhibitors for the treatment
of obesity and related metabolic disorders.8
O
O2N
OEt
N
N
H
⇑
Corresponding author. Tel.: +82 42 860 7076.
E-mail address: jhahn@krict.re.kr (J.H. Ahn).
Figure 1. Hit compound (IC50 = 1.06
lM against human DGAT-1).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.081

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H. J. Kwak et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4713–4718
a, b
O
O
EtO
2
1
c
O
H
H2N
N
d, e
O
N
H
O
EtO
EtO
3
4
f, g
H
N
N
R1
N
H
O
HO
5a - p
Scheme 1. General synthetic procedure for compounds 5a–p; Reagent and condition: (a) triethylphosphonoacetate, NaH, THF, 0 °C, 4 h, 96%; (b) 10% Pd/C, H2, EtOH, room
temperature, 18 h, 99%; (c) Cl2CHOCH3, 1 M-TiCl4, CH2Cl2, from 0 °C to room temperature, 4 h, 80%; (d) 5-nitrophenylenediamine, oxone, DMF/H2O(10/1), 25 °C, 24 h, 69%; (e)
10% Pd/C, H2, EtOH, room temperature, 18 h, 79%; (f) electrophiles, EDCI, HOBT, TEA or DIPEA, CH2Cl2, room temperature; (g) LiOHÁH2O, THF/H2O/EtOH, 25 °C.
Table 1
Activity of aminobenzimdazole derivatives on human DGAT-1
b
Compound
Structurea
hDGAT-1 inhibition IC50
(lM)
O
H
N
H
N
OH
N
5a
>10
O
N
H
O
H
N
H2N
OH
N
5b
5c
>10
>10
S
N
H
O
H
N
H
N
OH
N
S
N
H
O
H
N
OH
N
5d
>10
O
N
H
S
O
O
H
N
OH
OH
N
5e
5f
>10
>10
O
O
N
H
O
H
N
N
N
H
O
H
5g
0.33
N
OH
N
O
N
H
PF4620110
0.090
a
A mixture of cis/trans (cyclohexane ring).
IC50 values were determined from concentration-dependent inhibition curves of triplicate experiments by GraphPad Prism software.
b

H. J. Kwak et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4713–4718
4715
A series of aminobenzimidazole derivatives was prepared as
depicted in Scheme 1. Commercially available 4-phenylcyclo-
compound (5g) showed moderate inhibitory activity with an IC50
value of 330 nM.
hexanone
1
reacted with triethylphosphonoacetate in the
,b-
We focused our attention on biaryl amide derivatives and deriv-
atized them with mainly biaryl acids. As shown in Table 2, fused
bicyclic amide compounds (5h and 5i) showed no inhibitory activ-
presence of NaH to afford the Horner–Emmons product,
a
unsaturated ester followed by hydrogenation led to intermediate
2. The ester compound 2 was formylated using a TiCl4 and
dichloromethyl methyl ether to produce compound 3. Compound
3 was cyclized with nitrophenylenediamine in the presence of
oxidizing reagent oxone at room temperature to afford the
nitrobenzimidazole, which was hydrogenated to produce
ities (IC50 value >10 lM). However, the 5j–5l, phenyl-substituted
5-membered heteroaryl amide derivatives, demonstrated good
inhibitory activity comparable to that of the reference in the hu-
man DGAT-1 enzyme assay. To assess their in vivo efficacy, plasma
TG level was measured in mouse oral lipid tolerance test (OLTT).
We found that 5k showed favorable in vivo efficacy in mouse OLTT
compared with others (5j and 5l) (data not shown).
On the basis of these results, we further evaluated substituted
5-membered oxazole amide derivatives and the results are sum-
marized in Table 3. Compound 5k showed submicromolar inhibi-
tory activity on both human and mouse DGAT-1 with IC50 values
of 112 and 568 nM, respectively. Oxazole compound 5m, the
5-methyl instead of CF3 group, and 5n, the methyl substituted
oxazole, showed no inhibitory activities. Although 5o and 5p have
better mouse DGAT-1 inhibitory activities than 5k, compound 5k
showed at least 10-fold more human DGAT-1 inhibition than those
of 5o and 5p. Thus 5k was further assessed for its long term effi-
cacy, safety and PK profiles.
amino-benzimidazole 4. Compound
4 was derivatized with
diverse electrophiles such as isocyanate, isothiocyanate, sulfonyl
chloride, or an acid equivalent to give coupled products, which
were hydrolyzed by lithium hydroxide (LiOHÁH2O) to the final
acid compounds 5a–p.
The newly synthesized compounds in this study were evaluated
for inhibitory activity against DGAT-1. The Pfizer compound was
used as a standard DGAT-1 inhibitor (PF4620110).15
The hit compound in Figure 1 showed micromolar inhibitory
activity with an IC50 value of 1.06 lM toward human DGAT-1. It
had a metabolically unstable nitro and ester group. Therefore, we
modified the hit scaffold by changing the nitro and ester group.
The nitro group was reduced to amine and derivatized with various
electrophiles, and the ester group was hydrolyzed to the metabol-
ically stable corresponding acid. As shown in Table 1, urea, thio-
urea, sulfonamide and most of the amide derivatives showed
weak or no inhibitory activity. Fortunately, the biphenyl amide
As shown in Table 4, 5k exhibited good selectivity against
DGAT-2 and stability in human and mouse liver microsomes. Com-
pound 5k is non cytotoxic and does not interact with the human
ether-a-go-go-related gene (hERG) channel (IC50 >100 lM).
Table 2
Activity of aminobenzimdazole derivatives on human DGAT-1
a
Compound
Structure
hDGAT-1 inhibition IC50
(l
M)
O
H
N
5g
0.33
OH
N
O
N
H
S
O
O
H
N
OH
N
5h
>10
O
O
N
H
O
H
N
OH
O
N
5i
>10
N
H
O
N
H
N
OH
N
N
5j
0.090
0.112
O
N
H
CF3
O
O
H
N
OH
N
N
5k
O
N
H
CF3
N
O
H
N
N
OH
N
5l
0.049
0.090
O
N
H
PF4620110
a
IC50 values were determined from concentration-dependent inhibition curves of triplicate experiments by GraphPad Prism software.

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H. J. Kwak et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4713–4718
Table 3
Activity of aminobenzimdazole derivatives on human and mouse DGAT-1
a
Compound
Structure
hDGAT-1 inhibition IC50
(l
M)
mDGAT-1 inhibition IC50 (lM)
CF3
O
O
O
H
N
OH
OH
N
N
5k
0.112
0.568
O
N
H
CH3
O
H
N
N
N
5m
5n
5o
>10
>10
1.07
—
O
N
H
CF3
O
N
O
H
N
OH
O
N
—
O
O
N
H
CF3
H
N
OH
N
N
0.277
Cl
O
N
H
CF3
O
O
H
N
OH
N
N
5p
2.01
0.201
0.088
CF3
O
N
H
PF4620110
0.090
a
IC50 values were determined from concentration-dependent inhibition curves of triplicate experiments by GraphPad Prism software.
Table 4
Table 5
In vivo PK and OLTT with 5k
Selectivity, microsomal stability, cell cytotoxicity, CYP inhibition and induction, hERG
and acute toxicity
Assay
Results
Assay
Results
>20 lM
Human >99%
Mouse >99%
Hepatocyte >57.6 lM
NIH 3T3 >100
L929 >69.3
VERO >100
HFL-1 >100
Mouse PK (iv: 1 mg/kg po: 5 mg/kg)
t1/2 a= 5.3 h
hDGAT-2 (IC50
Microsomal stability (after 30 min incubation)
)
AUCb
(
l
g h/mL) 8.04 (oral)
CLc = 0.506 L/kg/h
d = 61.9%
>90% Inhibition (10 mg/kg)
F
Cell cytotoxicitya (IC50
)
Oral lipid tolerance test (OLTT)
l
lM
M
M
M
a
t1/2: Half life.
AUC: Area under curve.
CL: Clearance.
F: Bioavailability.
b
l
l
c
d
CHO-K1 >100
>100
lM
hERG (binding, IC50
)
lM
CYP inhibition (IC50
)
1A2 >100
lM
2C9 >100
lM
2C19 >69.3 lM
gle intravenous 1 mg/kg and oral 5 mg/kg doses. 5k showed t1/2
:
2D6 >100
3A4 >100
1A2 >100
2B6 >100
2C9 >100
2E1 >100
3A4 >100
l
l
l
M
M
M
5.3 h, AUC: 8.04 g h/mL, CL: 0.51 L/kg/h, and the bioavailability
l
of 62%. To evaluate 5k in vivo efficacy, the oral lipid tolerance test
(OLTT), an acute lipid challenge model measuring TG change after a
corn oil bolus dosing, was performed. Mice were treated with vehi-
cle (0.5% carboxymethyl cellulose, CMC) or 10 mg/kg of 5k 30 min
prior to corn oil dosing, blood samples for TG measurement were
taken at 2 h post-lipid challenge. Compound 5k reduced plasma
TG levels in the blood to over 90% at an oral dose of 10 mg/kg.
Compound 5k was tested for in vivo efficacy on body weight
gain reduction and glucose lowering effect in a diet-induced obes-
ity (DIO) mouse model (Fig. 2). Treatment for 4 weeks resulted in a
statistically significant reduction in body weight gain compared to
the control mice on the high-fat diet (HFD). On the basis of the con-
trol group, compound 5k administered at a dose of 30 mg/kg to the
HFD group was observed to reduce body weight gain by 8% without
affecting food intake (A). After 4 weeks of treatment with 5k, an
oral glucose tolerance test (OGTT) was performed, and 5k reduced
the glucose AUC by 54% (B). Also, 5k showed improved insulin sen-
CYP induction (EC50
)
l
l
M
M
l
M
lM
Acute toxicity (LD50
)
>500 mg/kg
a
NIH 3T3 mouse embryonic fibroblast cell line, L929: NCTC clone 929, mouse
fibroblast cell line, VERO: African green monkey kidney cell line, HFL-1: human
embryonic lung cell line, CHO-K1: Chinese hamster ovary cell line.
Compound 5k showed no significant inhibition in any of the major
CYP isoforms (CYP450 enzymes, 1A2, 2C9, 2C19, 2D6, and 3A4) and
similar results in the CYP induction assays (CYP450 enzymes, 1A2,
2B6, 2C9, 2E1, and 3A4). Moreover, it showed LD50 value over
500 mg/kg.
The pharmacokinetic (PK) profiling was performed in the
C57BL/6 J mice (Table 5). The PK profiles were investigated in a sin-

H. J. Kwak et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4713–4718
4717
(A) The body weight change analysis
(B) OGTT
(C) ITT
3000
14
12
10
8
51.3%**
54.0%**
2000
1000
0
6
4
2
0
Vehicle
Lean
5k
Vehicle
Lean
Treatment
5k
Treatment
Figure 2. The effect of repeated oral administration of compound 5k in diet-induced obese (DIO) mouse model. Compound 5k was administered orally QD to DIO C57BL/6 J
mice for 4 weeks [initial body weights in the groups of vehicle (HFD), 5k (30 mg/kg, HFD) and Lean (normal diet) were 42.16, 42.75, 30.21 g, respectively]. (A) The body
weight change analysis (B) oral glucose tolerance test (OGTT) (C) insulin tolerance test (ITT). Data are expressed as means SEM (n = 10 mice). ⁄P <0.05, and ⁄⁄P <0.01 versus
vehicle by Student’s t-test.
sitivity in an insulin tolerance test (ITT), carried out after 4 weeks
treatment, by reducing the glucose AUC by 51%.
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