Synthesis and diacylglycerol acyltransferase-1 inhibition of azabicyclo[3.1.0]hexane derivatives

Article abstract of DOI:10.1002/bkcs.10302

We identified azabicyclo[3.1.0]hexane derivatives that are active diacylglycerol acyltransferase-1 (DGAT)-1 inhibitor. Among the azabicyclo[3.1.0]hexane series, compound 6b showed good in vitro activity toward human DGAT-1, selectivity toward DGAT-2, and

Full text of DOI:10.1002/bkcs.10302

Article
DOI: 10.1002/bkcs.10302
BULLETIN OF THE
S.-J. Han et al.
KOREAN CHEMICAL SOCIETY
Synthesis and Diacylglycerol Acyltransferase-1 Inhibition of
Azabicyclo[3.1.0]hexane Derivatives
†
,††
†,††
†
†,‡
†
Seo-Jung Han,
Gwi Bin Lee,
Hyun Jung Kwak, Suvarna H. Pagire, Ji Young Kim, †
†
†,‡ † †
Haushabhau S. Pagire, Sung Bum Park, Chong Hak Chae, Joo Yun Lee, Ki Young Kim,
†
†
†
†
§
§
Sang Dal Rhee, Hee Youn Kim, Sun Hye Shin, Myung Ae Bae, Mi-jin Park, Dooseop Kim,
¶
†,‡,
*
Duck Hyung Lee, and Jin Hee Ahn
†
Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of
Korea. *E-mail: jhahn@krict.re.kr
‡
Department of Medicinal and Pharmaceutical Chemistry, University of Science and Technology, Daejeon
05-333, Republic of Korea
Handok Pharmaceuticals Co., Ltd., Seoul 135-755, Republic of Korea
Department of Chemistry, Sogang University, Seoul 121-742, Republic of Korea
Received January 6, 2015, Accepted February 12, 2015, Published online June 5, 2015
3
§
¶
We identified azabicyclo[3.1.0]hexane derivatives that are active diacylglycerol acyltransferase-1 (DGAT)-1
inhibitor. Among the azabicyclo[3.1.0]hexane series, compound 6b showed good in vitro activity toward
human DGAT-1, selectivity toward DGAT-2, and liver microsomal stability. Compound 6b exhibited no
CYP inhibition, hERG binding, or cell cytotoxicity. Additionally, compound 6b reduced the level of plasma
triglyceride after oral administration in mice.
Keywords: Diacylglycerol acyltransferase-1, Triglycerides, 3-Azabicyclo[3.1.0]hexane,
Oral lipid tolerance test
5
Introduction
efficacy. Other DGAT-1 inhibitors developed by Pfizer (PF-
0
6
7
4620110) and AstraZeneca (AZD-7687) reduced post-
Triacylglycerols (triglycerides, TG), a major type of neutral
lipids, are a heterogeneous group of molecules with a glycerol
backbone and three fatty acids (FAs). Excess accumulation of
TG can participate in the pathogenesis of several metabolic
diseases such as obesity, insulin resistance, type II diabetes,
prandial TG excursion in human trials, but their development
as drug candidates was discontinued due to side effects
(Figure 1).
During the last few years, small molecule inhibitors for
8
DGAT-1 have been reported. Based on structures reported
1
and cardiovascular disease.
in literatures and patents, we envisioned a bicyclic acid (aza-
bicyclo[3.1.0]hexane carboxylic acid) as a potential alterna-
tive skeleton (Figure 2).
Herein, we report the synthesis of 3-azabicyclo[3.1.0]hex-
ane derivatives and their biological evaluation as DGAT-1
inhibitors.
DGAT (acyl-CoA: diacylglycerol acyltransferase) has two
isoforms, DGAT-1 and DGAT-2. It is a transmembrane
enzyme that functions in the final step of TG biosynthesis.
DGAT-1 inhibition is expected to impair TG and chylomi-
crons formation in enterocytes to reduce postprandial TG
2
levels in the blood.
−/−
The DGAT-1 knockout (Dgat ) mice are resistant to diet-
induced obesity, exhibit increased insulin sensitivity relative
to wild-type littermates, and have decreased adiposity. These
findings have led a number of companies to pursue potent
DGAT-1 inhibitors for the treatment of obesity and related
Results and Discussion
3-Azabicyclo[3.1.0]hexane derivatives were prepared as
depicted in Scheme 1. Commercially available 3-pyrroline 1
was protected with benzyl chloroformate (CbzCl) in the pres-
ence of triethylamine (TEA) to provide benzyl 2,5-dihydro-
1H-pyrrole-1-carboxylate, which was cyclized via
rhodium(II) acetate-mediated cyclopropanation in the pres-
ence of ethyl diazoacetate. This reaction produced a mixture
ofexo-andendo-3-azabicyclo[3.1.0]hexanes2aand2b(ratio
= 2.3:1), respectively, which were separated by silica gel chro-
3
metabolic disorders.
Recently, Novartis reported findings from their clinical
studies. Pradigastat (LCQ-908) is the most advanced clinical
candidate and is being evaluated in Phase II and Phase III
stages in patients with dyslipidemia, diabetes, and familial
4
chylomicronemia syndrome (FCS). The candidate P-7435
9
developed by Piramal was evaluated in a Phase I study in India
as well as in the United States to determine its safety and
matography. We selected the isomer 2a, which showed better
DGAT-1 inhibitory activity than derivatives from 2b moiety
(data not shown), for further derivatization. Compound 2a
†
† These authors contributed equally to this work.
was deprotected under H and 10% Pd/C in MeOH to give
2
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Azabicyclo-[3.1.0]hexane Derivatives
KOREAN CHEMICAL SOCIETY
OH
O
OH
O
OH
O
O
NH O
F3C
N
N
2
N
N
H N
N
2
N
N
H
N
O
Pradigastat (LCQ-908)
PF-04620110
AZD-7687
Figure 1. DGAT-1 inhibitors in clinical trials.
O
derivative (6b) showed good inhibitory activity with an
IC50 value of 29 nM. Furthermore, the (D)-valine-
azabicyclohexyl derivative (6c) exhibited weaker inhibition
activity than 6b. The (L)-leucine (6e) and cyclopropylgly-
cine-azabicyclohexyl (6f)derivatives alsoshowedgoodinhib-
itory activities with IC values of 32 and 31 nM, respectively.
H
R2
OH
R1
N
H
R5
N
H
R4
50
R3
However, (L)-isoleucine-azabicyclohexylcompound(6d)was
not active in DGAT-1 in vitro. The oral lipid tolerance test
Figure 2. 3-Azabicyclo[3.1.0]hexane skeleton.
(
(
OLTT) was performed with the three active compounds
6b, 6e, and 6f). Based on the TG reduction efficacy in vivo
compound 3. It was coupled with 1-fluoro-4-nitrobenzene (or
(data not shown), compound 6b was determined as the most
active derivative, and was thus selected for further
modification.
substituted 1-fluoro-4-nitrobenzene)inthepresenceofK CO3
2
to obtain phenyl azabicyclohexane, followed by hydrogena-
tion using 10% Pd/C under H to produce amino ester 4. Com-
pound 4 was derivatized with diverse acids to give coupled
We further evaluated the substituted five-membered oxa-
zole amide (L)-valine-azabicyclohexyl derivatives and the
results are summarized in Table 3. Compound 6b showed
good human and moderate mouse DGAT-1 inhibitory activ-
ities with IC50 values of 29 and 338 nM, respectively. In con-
trast, methyl substitution decreased the in vitro activity (6g).
The 2-trifluoromethyl (6h) and 2,6-difluoro (6i) (L)-valine-
azabicyclohexyl derivatives showed good inhibitory activities
against human DGAT-1; however, 6b was more promising
mouse DGAT-1 inhibitor. The 3,5-dimethyl (6j) and pyrimi-
dine (6k) derivatives were not active. Compound 6b was fur-
ther tested for its pharmacokinetics properties, druggability,
and in vivo efficacy.
2
products, which were hydrolyzed by LiOHÁH O to produce
2
acids 5a–e.
The obtained 3-azabicyclo[3.1.0]hexane derivatives
were evaluated in vitro for DGAT-1 inhibitory activity, and
the results are summarized in Table 1. And PF-04620110
6
(Pfizer) was used as the standard DGAT-1 inhibitor.
We introduced diverse acids and acid equivalents such
as urea, thiourea, and sulfonamide group (data not shown)
5
at R position, and identified the aryl substituted oxazole
amide 5a as a potential hit with an IC50 value of 1.71 μM.
Therefore, we focused on aryl-substituted oxazole amide deri-
vatives and the results are summarized in Table 1. 2-Phenyl-5-
As shown in Table 4, 6b exhibited good selectivity against
DGAT-2 and it was stable in human and mouse liver micro-
somes. Furthermore, it showed no cytotoxicity in the five cell
lines tested. It did not interact with the human ether-a-go-go-
(
trifluoromethyl)oxazole amide (5a) exhibited the micromolar
inhibition with an IC50 value of 1.71 μM. However, 2-phenyl-
-(trifluoromethyl)oxazole amide (5b) did not show any
4
inhibitory activity. Therefore, we further derivatized 2-
phenyl-5-(trifluoromethyl)oxazole group and indentified potent
compounds that showed good in vitro activities with IC values
related gene (hERG) channel in ligand binding assay (IC50 >
100 μM). And then it did not significantly inhibit any of the
major CYP isoforms assays (CYP450 enzymes, 1A2, 2C9,
2D6, and 3A4 at 10 μM).
To evaluate the in vivo efficacy of compound 6b, we per-
formed OLTT in male C57BL/6 mice. Compound 6b was
orally treated and exhibited 82 and 99% TG reduction at 3
and 10 mg/kg doses, respectively (Figure 3).
The crystal structure of DGAT-1 has not been reported till
date. Hence, we superimposed the 3D structure of the active
compound 6b with known DGAT-1 inhibitors such as PF-
04620110 and Pradigastat (LCQ-908), to validate its 3D-
pharmacophore alignment. Key elements of the plausible
50
of 104 (5c) and 62 nM (5d) having 4-substituent. However,
the biphenyl derivative (5e) was not an effective inhibitor.
Amino acid extension of the acid part for effective DGAT-1
1
0
inhibition has been reported previously. And we introduced
amino acid groups, and analogs were synthesized by the syn-
thetic route outlined in Scheme 2. Compound 5 was derivatized
with diverse amino acid esters to give coupled products, which
were hydrolyzed by LiOHÁH O to give final products 6a–j.
2
As shown in Table 2, the glycine-azabicyclohexyl deriva-
tive (6a) was not active, but the (L)-valine-azabicyclohexyl
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Azabicyclo-[3.1.0]hexane Derivatives
KOREAN CHEMICAL SOCIETY
CO Et
CO Et
CO
2
Et
2
2
a, b
c
H
H
H
H
H
H
+
N
H
N
N
N
H
Cbz
Cbz
1
2a
2b
3
d, e
O
O
H
H
R2
OEt
R2
OH
f, g
R1
R1
N
N
H
H
O
R4
R5
R4
H N
N
H
2
R3
R3
5a-e
4
Scheme 1. Reagents and conditions: (a) benzyl chloroformate, triethylamine, CH Cl , room temperature (rt), 3 h; (b) Rh(OAc) , ethyl diazoa-
2
2
2
ꢀ
cetate, CH Cl , rt, 72 h; (c) 10% Pd/C, H , MeOH, rt, 20 h; (d) 1-fluoro-4-nitrobenzene, K CO , DMF, 90 C, 13 h; (e) 10% Pd/C, H , EtOH, rt,
2
2
2
2
3
2
1
6 h; (f ) carboxylic acid, EDCI, HOBT, TEA, CH Cl , rt, 12 h; (g) LiOHÁH O, THF/MeOH/H O, rt, 12 h.
2
2
2
2
pharmacophore-based binding areas are illustrated in
obtained on the Autospec magnetic sector mass spectrometer
(Micromass, Manchester, UK).
Chemistry. 3-Benzyl 6-ethyl (1α,5α,6α)-3-azabicyclo
[3.1.0]hexane-3,6-dicarboxylate (exo-2a) and benzyl 2,5-
dihydro-1H-pyrrole-1-carboxylate. To a solution of 3-
pyrroline 1 (7.7 g, 72.3 mmol) and TEA (15 mL, 144.6 mmol)
11
Figure 4. Pharmacophore structure of 6b consists of two
hydrophobic-aromatics, one hydrophobic, two hydrogen-
acceptors, and one hydrogen-donor. The pharmacophore fea-
tures of 6b and the models showed good alignment except for
the hydrogen-acceptor feature on the oxazepan-5-one and pyr-
idine rings of reference compounds. The hydrophobic-
aromatic and hydrogen-acceptor/donor features mapped to
the benzene rings and amide group of 6b and the hydrophobic
features originated from thecyclohexanerings of thereference
compounds aligned with the azabicyclo[3.1.0]hexane ring of
in CH Cl2 was added benzyl chloroformate (16 mL,
2
ꢀ
108.5 mmol)at0 Candthemixturewaswarmedtoroomtem-
perature. After 3 h, the reaction mixture was quenched
with water, washed with brine, and dried over magnesium
sulfate. The solvent was filtered, and concentrated in vacuo
to afford an oily residue. The residue was purified by
silica gel column chromatography to afford benzyl 2,5-dihy-
6b (Figure 4).
1
Conclusions
dro-1H-pyrrole-1-carboxylate (9.4 g, 98%). H NMR (300
MHz, CDCl ) δ 7.37–7.30 (m, 5H), 5.79–5.77 (m, 2H),
3
In conclusion, we developed a series of 3-azabicyclo[3.1.0]
hexane derivatives as DGAT-1 inhibitors. Compound 6b
showed nanomolar in vitro activity toward human DGAT-1,
good selectivity toward DGAT-2, liver microsomal stability,
and acceptable safety profiles with respect to hERG and CYP
inhibition. Inaddition, 6balsoshowedgoodinvivoplasmaTG
reduction after oral administration in mice.
5.16 (s, 2H), 4.20–4.08 (m, 4H); LCMS (ESI) m/z 204.05
+
(M + H) .
To
a solution of benzyl 2,5-dihydro-1H-pyrrole-1-
carboxylate (7.6 g, 37.5 mmol) and rhodium acetate (2.5 g,
5.6 mmol) in CH Cl was added slowly a solution of ethyl dia-
2
2
zoacetate(9.7 mL, 93.7 mmol) inCH Cl (75 mL)usingsyringe
2
2
pump for 72 h. At the end of the addition, the reaction mixture
was filtered through celite and the filtrate was concentrated to
give yellow residue. The residue was purified by silica gel col-
umn chromatography to afford exo-2a, 3-benzyl 6-ethyl
(1α,5α,6α)-3-azabicyclo[3.1.0]hexane-3,6-dicarboxylate (2.1
Experiments
Synthesis in General. All the commercial chemicals and sol-
ventswerereagentgradeandusedwithoutfurtherpurification.
Products werepurifiedbyflashcolumnchromatographyusing
the silica gel 60 (230–400 mesh Kieselgel 60) or crystalliza-
g, 19%) and endo-2b, 3-benzyl 6-ethyl (1α,5α,6β)-3-azabicy-
1
clo[3.1.0]hexane-3,6-dicarboxylate (0.9 g, 8%). exo-2a:
H
NMR (300 MHz, CDCl ) δ 7.35–7.34 (m, 5H), 5.09 (s, 2H),
3
1
tion. H NMR spectra were obtained on FT-NMR Varian
4.12 (q, J = 7.1 Hz, 2H), 3.74 (d, J = 11.3 Hz, 1H), 3.70 (d,
J = 11.2 Hz, 1H), 3.51–3.46 (m, 2H), 2.10–2.09 (m, 2H), 1.48
(t, J = 3.1 Hz, 1H), 1.29 (t, J = 7.1 Hz, 3H); LCMS (ESI) m/z
Gemini-300 FT (Palo Alto, CA, USA) or Bruker
AVANCE-300 (Billerica, MA, USA) with trimethylsilane
as internal reference. LC/MS spectra were obtained on the
Agilent and high-resolution mass spectrum (HRMS) was
+
1
290.03 (M + H) ; endo-2b: H NMR (300 MHz, CDCl ) δ
3
7.35–7.28 (m, 5H), 5.10 (s, 2H), 4.10 (q, J = 7.1 Hz, 2H),
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Azabicyclo-[3.1.0]hexane Derivatives
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3
2
.84 (d, J = 11.3 Hz, 2H), 3.56–3.54 (m, 2H), 1.96–1.94 (m,
H), 1.79 (t, J = 3.1 Hz, 1H), 1.18 (t, J = 7.1 Hz, 3H).
Ethyl (1α,5α,6α)-3-azabicyclo[3.1.0]hexane-6-carboxylate
Table2. ActivityofazabicyclohexanederivativesonhumanDGAT-1.
hDGAT-1
a
Compound
Structure
IC50 (nM)
(
3). To a solution of 3-benzyl 6-ethyl (1α,5α,6α)-3-azabicy-
O
O
O
clo[3.1.0]hexane-3,6-dicarboxylate (2.1 g, 7.3 mmol) in
H
H
H
OH
OH
OH
N
H
O
O
O
N
H
H
H
6a
6b
6c
O
NA
Table 1. Activity of azabicyclohexane derivatives on human
DGAT-1.
N
N
H
O
CF3
hDGAT-1
a
Compound
Structure
IC50 (nM)
N
H
O
H
N
O
29.1
438
OH
N
O
N
H
N
H
5
5
5
5
5
a
b
c
O
1710
CF3
N
N
H
CF3
O
N
H
O
H
N
O
OH
N
O
N
N
H
CF3
H
O
NA
104
62
O
N
H
N
CF3
O
H
O
OH
H
N
H
OH
6d
6e
N
O
NA
H
O
N
H
O
N
O
N
N
H
MeO
N
H
CF3
O
CF3
O
O
H
H
OH
OH
N
H
N
N
O
32.0
H
H
d
e
O
O
N
F
N
H
N
O
N
H
O
CF3
CF3
O
O
H
H
OH
OH
N
H
N
N
O
H
H
O
NA
90
6f
O
30.8
90
N
N
O
N
N
H
H
O
CF3
CF3
PF-04620110
PF-04620110
NA, not active.
NA, not active.
a
a
IC50 values were determined from concentration-dependent inhibition
curves of triplicate experiments by the GraphPad Prism software.
IC50 values were determined from concentration-dependent inhibition
curves of triplicate experiments by the GraphPad Prism software.
O
H
O
H
X
OH
N
R2
R2
OH
H
O
R1
N
H
R1
a, b
O
N
H
O
R5
N
H
R4
R5
R4
N
H
R3
R3
6
a-j
5
Scheme2. Reagentsandconditions:(a)aminoacidester, EDCI, HOBT, TEA, CH
O, rt, 24 h.
2
Cl
2
, roomtemperature(rt), 12 h;(b)LiOHÁH
2
O, THF/MeOH/
H
2
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Azabicyclo-[3.1.0]hexane Derivatives
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Table 3. Activity of azabicyclohexane derivatives on human and mouse DGAT-1.
a
a
Compound
Structure
hDGAT-1 IC50 (nM)
mDGAT-1 IC50 (nM)
O
O
O
O
O
H
H
H
OH
OH
OH
OH
OH
N
H
O
O
O
O
O
N
H
H
H
H
H
6
6
6
6
6
b
g
h
i
O
29.1
338
N
O
N
H
CF3
N
H
N
O
109.5
1781
488
O
N
N
H
CF3
N
H
N
CF3
O
32
N
O
N
H
CF3
H
N
H
F
N
O
40
1310
N
O
N
F
H
CF3
H
N
H
N
j
O
NA
90
NA
88
N
O
N
H
CF3
PF-04620110
NA, not active.
a
IC50 values were determined from concentration-dependent inhibition curves of triplicate experiments by the GraphPad Prism software.
Table 4. Selectivity, microsomal stability, cell cytotoxicity, hERG, and CYP inhibition properties of compound 6b.
Assay
Results
Selectivity (hDGAT-2, IC50
)
>20 μM
Microsomal stability (after 30 min incubation)
>99% (human)>99% (mouse)
NIH3T3: >100 μML929: >100 μMVERO: >100 μMHFL-1: >100 μMCHO-K1: >100 μM
26.4%
a
Cell cytotoxicity (IC )
5
0
b
hERG (% inhibition at 10 μM)
CYP inhibition (% inhibition at 10 μM)
1A2: 42%2C9: 1%2D6: 0%3A4: 0%
a
NIH3T3:mouseembryonicfibroblastcellline;L929:NCTCclone929,mousefibroblastcellline;VERO:Africangreenmonkeykidneycellline;HFL-
: human embryonic lung cell line; CHO-K1: Chinese hamster ovary cell line.
hERG: ligand binding assay.
1
b
1
MeOH (72 mL) was added 10% Pd/C (77 mg, 0.7 mmol). The
mixture wasstirredatroomtemperature 20 hunderahydrogen
atmosphere. The catalyst was removed by filtration through
celite with MeOH, and the filtrate was concentrated in vacuo
to afford ethyl (1α,5α,6α)-3-azabicyclo[3.1.0]hexane-6-car-
boxylate, which was used for the next reaction without further
purification. H NMR (300 MHz, CDCl ) δ 4.04 (q, J = 7.1
3
Hz, 2H), 3.07 (d, J = 11.7 Hz, 2H), 2.96 (d, J = 11.7 Hz,
2H), 2.01–2.00 (m, 2H), 1.44–1.42 (m, 1H), 1.26 (t, J = 6.9
Hz, 3H); LCMS (ESI) m/z 156.08 (M + H) .
+
Ethyl
(1α,5α,6α)-3-(4-aminophenyl)-3-azabicyclo
[3.1.0] hexane-6-carboxylate (4) and ethyl (1α,5α,6α)-3-
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Azabicyclo-[3.1.0]hexane Derivatives
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3
3
2
2
1
1
50
00
50
00
50
00
through celite, and the filtrate was concentrated in vacuo to
afford ethyl (1α,5α,6α)-3-(4-aminophenyl)-3-azabicyclo
1
[
3.1.0]hexane-6-carboxylate as a product. H NMR (300
MHz, CDCl ) δ 6.65 (d, J = 8.7 Hz, 2H), 6.43 (d, J = 8.4
3
Hz, 2H), 4.14 (q, J = 7.2 Hz, 2H), 3.59 (d, J = 9.0 Hz, 2H),
3
.18 (d, J = 9.0 Hz, 2H), 2.21–2.19 (m, 2H), 1.76 (t, J = 3.0
Hz, 1H), 1.27 (t, J = 7.2 Hz, 3H); LCMS (ESI) m/z 247.07
*
+
(
M + H) .
**
(
1α,5α,6α)-3-(4-(2-Phenyl-4-(trifluoromethyl)oxazole-5-
50
carboxamido)phenyl)-3-azabicyclo[3.1.0]hexane-6-
0
carboxylic acid (5b). To a solution of 2-phenyl-5-(trifluoro-
methyl)oxazole-4-carboxylic acid (100 mg, 0.39 mmol) in
CH Cl (2 mL) were sequentially added 1-ethyl-3-(3-dimethy-
laminopropyl)carbodiimide (EDCI, 111 mg, 0.58 mmol),
hydroxylbenzotriazole (HOBT, 78 mg, 0.58 mmol), ethyl
V
V-C
6b 3mpk
6b 10mpk
Figure 3. Effect of compound 6b on plasma TG in an acute OLTT in
C57BL/6 mice. Data are expressed as means ± S.E.M. (n = 10 mice).
p < 0.05 and p < 0.01 vs. vehicle by Student's t-test.
2
2
∗
∗∗
(1α,5α,6α)-3-(4-aminophenyl)-3-azabicyclo[3.1.0]hexane-6-
carboxylate 4 (123 mg, 0.47 mmol) and TEA (118 mg, 1.17
mmol). The mixture was stirred for 12 h, and then the reaction
mixture was diluted with water, extracted with EtOAc. The
organic layer was washed with brine and dried over magnesium
sulfate, filtered and concentrated in vacuo to obtain an oily res-
idue.Theresiduewaspurifiedbyflashcolumnchromatography
to afford ethyl (1α,5α,6α)-3-(4-(2-phenyl-5-(trifluoromethyl)
oxazole-4-carboxamido)phenyl)-3-azabicyclo[3.1.0]hexane-
1
6
8
6
-carboxylate (148 mg, 79%). H NMR (300 MHz, CDCl ) δ
3
.78 (s, 1H), 8.13 (d, J = 9.0 Hz, 2H), 7.60–7.55 (m, 5H),
.54 (d, J = 9.0 Hz, 2H), 4.15 (q, J = 7.2 Hz, 2H), 3.65 (d, J
Figure 4. The 3D-pharmacophore alignment of 6b (green) with
known compounds (PF-4620110: cyan, Pradigastat, LCQ-908:
magenta); hydrophobic-aromatic and hydrophobic (orange sphere),
hydrogen acceptor (cyan sphere), and hydrogen-donor (red sphere)
features.
=
9.3 Hz, 2H), 3.33 (d, J = 9.0 Hz, 2H), 2.26 (m, 2H), 1.71 (t,
J = 3.0 Hz, 1H), 1.28 (t, J = 7.2 Hz, 3H).
To a solution of ethyl (1α,5α,6α)-3-(4-(2-phenyl-5-(tri-
fluoromethyl)oxazole-4-carboxamido)phenyl)-3-azabicyclo
[
(
3.1.0]hexane-6-carboxylate (146 mg, 0.30 mmol) in THF
1.5 mL), MeOH (1.5 mL), and H O (0.5 mL) was added
2
(
4-nitrophenyl)-3-azabicyclo[3.1.0]hexane-6-carboxylate.
LiOHÁH O (51 mg, 1.2 mmol). The reaction mixture was stir-
2
To a stirred solution of ethyl (1α,5α,6α)-3-azabicyclo[3.1.0]
hexane-6-carboxylate 3 (195 mg, 1.3 mmol) in DMF (6 mL)
were added 1-fluoro-4-nitrobenzene (231 mg, 1.6 mmol)
and K CO (261 mg, 1.9 mmol) and the resulting mass was
stirredat90 Cfor13 h.Followingcompletion,thereactionmix-
ture was quenched by the addition of water and extracted
with EtOAc. The combined organic layers were washed
with brine, and dried over magnesium sulfate. The mixture
was filtered, and concentrated in vacuo to give an oil residue.
The residue was purified by silica gel column chromatography
red for 12 h, and then concentrated under reduced pressure.
More water was added and the aqueous phase was acidified
to pH 2–3, then the resulting mixture was extracted with
EtOAc. The organic layer was washed with brine, dried over
magnesium, filtered, and concentrated under reduced pres-
sure. The residue was crystallized with ether to afford
(1α,5α,6α)-3-(4-(2-phenyl-5-(trifluoromethyl)oxazole-4-car-
boxamido)phenyl)-3-azabicyclo[3.1.0]hexane-6-carboxylic
2
3
ꢀ
1
acid as a brown solid (104 mg, 76%). H NMR (300 MHz,
DMSO-d ) δ 10.3 (s, 1H), 8.14 (d, J = 8.1 Hz, 2H),
6
to afford ethyl (1α,5α,6α)-3-(4-nitrophenyl)-3-azabicyclo
7.67–7.58 (m, 5H), 6.56 (d, J = 8.7 Hz, 2H), 3.61 (d, J = 9.9
Hz, 2H), 3.22 (d, J = 9.3 Hz, 2H), 2.15 (m, 2H), 1.46 (br,
1
[3.1.0]hexane-6-carboxylate as a product (160 mg, 46%). H
+
NMR (300 MHz, CDCl ) δ 8.11 (d, J = 9.3 Hz, 2H), 6.47 (d,
J = 9.3 Hz, 2H), 4.12 (q, J = 7.1 Hz, 2H), 3.70 (d, J = 10.2 Hz,
2
=
1H); LCMS (ESI) m/z 457.89 (M + H) .
3
((1α,5α,6α)-3-(4-(2-Phenyl-5-(trifluoromethyl)oxazole-
4-carboxamido)phenyl)-3-azabicyclo[3.1.0]hexane-6-car-
bonyl)-L-valine (6b) and ethyl ((1α,5α,6α)-3-(4-(2-phenyl-
H), 3.55 (d, J = 10.2 Hz, 2H), 2.35–2.33 (m, 2H), 1.59 (t, J
3.1 Hz, 1H), 1.27 (t, J = 7.1 Hz, 3H); LCMS (ESI) m/z
+
2
77.04 (M + H) .
5-(trifluoromethyl)oxazole-4-carboxamido)
phenyl)-3-
To a solution of ethyl (1α,5α,6α)-3-(4-nitrophenyl)-3-aza-
azabicyclo[3.1.0]hexane-6-carbonyl)-L-valinate. To
a
bicyclo[3.1.0]hexane-6-carboxylate (160 mg, 0.6 mmol) in
EtOH (1 mL) was added 10% Pd/C (6 mg, 0.1 mmol). The
mixture was stirred at room temperature for 16 h under a
hydrogen atmosphere. The reaction mixture was filtered
stirred solution of ((1α,5α,6α)-3-(4-(2-phenyl-5-(trifluoro-
methyl)oxazole-4-carboxamido)phenyl)-3-azabicyclo[3.1.0]
hexane-6-carboxylic acid 5b (60 mg, 0.13 mmol) in
CH Cl2 (1 mL) were sequentially added 1-ethyl-3-(3-
2
Bull. Korean Chem. Soc. 2015, Vol. 36, 1586–1593
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BULLETIN OF THE
Article
Azabicyclo-[3.1.0]hexane Derivatives
KOREAN CHEMICAL SOCIETY
dimethylaminopropyl)carbodiimide (30 mg, 0.16 mmol) and
hydroxybenzotriazole (21 mg, 0.16 mmol), (L)-Valine methyl
ester hydrochloride (26 mg, 0.16 mmol) and TEA (16 mg,
(didecanoyl glycerol) and 7.5 μM radiolabeled acyl-CoA sub-
14
strate ([ C]decanoyl-CoA) was added to each well of a phos-
pholipid FlashPlate (PerkinElmer Life Sciences, Waltham,
MA, USA). A small aliquot of lysate (5 μg/well) or mouse
liver microsome (0.1 mg/well) was added to start the reaction,
which was allowed to proceed for 60 min. The reaction was
terminated upon the addition of an equal volume (100 μL)
of isopropanol. The plates were sealed, incubated overnight,
and counted the next morning on a Wallac 1450 Microbeta
Trilux Liquid Scintillation Counter and Luminometer
(PerkinElmer Life Science). DGAT-1 catalyzes the transfer
of the radiolabeled decanoyl group onto the sn-3 position of
didecanoyl glycerol. The resultant-radiolabeled tridecanoyl
glycerol (tricaprin) preferentially binds to the hydrophobic
coating on the phospholipid FlashPlate. The proximity of
the S15-radiolabeled product to the solid scintillant incorpo-
rated into the bottom of the FlashPlate induced fluorescence
release from the scintillant, which was measured in the Wallac
1450 Microbeta Trilux Liquid Scintillation Counter and
Luminometer. Various concentrations (e.g., 0.0008, 0.004,
0.02, 0.1, 0.5, 2.5, and 10.0 μM) of the representative com-
pounds were added to individual wells prior to the addition
of lysates. IC50 values of compounds were determined from
concentration-dependent inhibition curves oftriplicate experi-
ments by GraphPad Prism software (GraphPad Software Inc.,
La Jolla, CA, USA).
0.16 mmol). The reaction mixture was stirred for 12 h, and
then diluted with water and extracted with EtOAc. The organic
layer was washed with brine, dried over MgSO , filtered and
4
concentrated in vacuo. The residue was purified by silica gel
column chromatography to produce methyl ((1α,5α,6α)-3-(4-
(2-phenyl-5-(trifluoromethyl)oxazole-4-carboxamido)
phenyl)-3-aza-bicyclo[3.1.0]hexane-6-carbonyl)-L-valinate
1
(
8
=
(
60 mg, 81%). H NMR (300 MHz, CDCl ) δ 8.77 (s, 1H),
3
.13 (dd, J = 7.7, 1.4 Hz, 2H), 7.61–7.51 (m, 5H), 6.54 (d, J
9.0 Hz, 2H), 6.11 (d, J = 8.7 Hz, 1H), 4.58 (m, 1H), 3.75
s, 3H), 3.64 (m, 2H), 3.32 (m, 2H), 2.22 (m, 2H), 2.15 (m,
H), 1.54 (t, J = 2.9 Hz, 1H), 0.94 (dd, J = 9.2, 6.9 Hz, 6H);
1
+
LCMS (ESI) m/z 570.96 (M + H) ; HRMS (C H F N O )
2
9 29 3 4 5
m/z calcd for 570.2090, found 570.2085.
To a solution of methyl ((1α,5α,6α)-3-(4-(2-phenyl-5-(tri-
fluoromethyl)oxazole-4-carboxamido)phenyl)-3-azabicyclo
[3.1.0]hexane-6-carbonyl)-L-valinate (60 mg, 0.11 mmol) in
THF (0.5 mL), MeOH (0.5 mL), and H O (0.2 mL) was added
2
LiOHÁH O(23 mg, 0.55 mmol) inH O(0.2 mL). Thereaction
2
2
mixture was stirred for 24 h, and then concentrated under
reduced pressure. More water was added and the aqueous
phase was acidified to pH 2–3, the resulting mixture was
extracted with EtOAc. The organic layer was washed with
brine, dried over magnesium sulfate, filtered, and concen-
trated in vacuo. The residue was crystallized with ether to
afford ((1α,5α,6α)-3-(4-(2-phenyl-5-(trifluoromethyl)oxa-
zole-4-carboxamido)phenyl)-3-azabicyclo[3.1.0]hexane-6-
In vivo OLTT Assay. Animalswereweighedregularlytoallow
accurate dosing withdrugs. Allmice (8-week-old maleC57BL/
6) were fasted for 16 h and basal blood samples were collected
from ophthalmic venous plexus using heparin-coated capillary
tubes. The test compound 6b was formulated as a suspension in
0.5% carboxymethyl cellulose (CMC, vehicle) and orally trea-
ted with chemical dosing (10 mL/kg). A 30-min later, a bolus
dose of oil (5 mL/kg, 0.5% CMC) was given to the mice. After
oil treatment, blood samples were collected at 2 h. The blood
samples were immediately centrifuged for 10 min at 1000 × g
1
carbonyl)-L-valine as a brown solid (38 mg, 63%). H NMR
(
2
2
300 MHz, DMSO-d ) δ 10.3 (s, 1H), 8.15 (d, J = 8.2 Hz,
6
H), 7.68–7.59 (m, 5H), 6.59 (d, J = 9.0 Hz, 2H), 4.15 (m,
H), 3.61(d, J = 9.9 Hz, 2H), 3.22 (d, J = 9.2 Hz, 2H), 2.02
(
5
5
m, 2H), 1.85 (m, 1H), 0.91–0.86 (m, 6H); LCMS (ESI) m/z
56.86 (M + H) ; HRMS (C H F N O ) m/z calcd for
29 27 3 4 5
56.1934, found 556.1926.
+
ꢀ
and resulting plasma samples were stored at −20 C until
Biological Assay
assayed. Plasma concentration of TG was measured by a color-
imetric assay using an automatic biochemical analyzer, the
Selectra 2 (Vital Scientific N. V., Dieren, The Netherlands).
InvitroDGAT-1 Enzyme Activity Assay. Theidentificationof
compounds as DGAT-1 inhibitors was readily achieved using
a FlashPlate assay. In this assay, pCMV6-recombinant human
DGAT-1 plasmid (Origene No. RC220595)transfected for 24
h in Hep3B cells. After transfection of the plasmid, human
DGAT-1 expression was analyzed by Western blot or RT-
PCR using whole cell extracts. For establishment of a human
DGAT-1 overexpressed stable cell line, cells were grown and
maintained in Dulbecco's modified Eagle's medium high glu-
cose containing 10% fetal bovine serum and 200 μg/mL G-
Acknowledgments. This research was supported by the
National Research Foundation of Korea (NRF) funded by
the Ministry of Science, ICT, & Future Planning (2012-
0
019773), and the Ministry of Health & Welfare (A121102).
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Bull. Korean Chem. Soc. 2015, Vol. 36, 1586–1593
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de 1592

BULLETIN OF THE
Article
Azabicyclo-[3.1.0]hexane Derivatives
KOREAN CHEMICAL SOCIETY
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Bull. Korean Chem. Soc. 2015, Vol. 36, 1586–1593
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de 1593