Identification of 2-[2-(4-tert-Butylphenyl)ethyl]-N-[4-(3-cyclopentylpropyl)- 2-fluorophenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide as an Orally Active MGAT2 Inhibitor

Article abstract of DOI:10.1248/cpb.c15-00803

We previously reported 2-[2-(4-tert-butylphenyl)ethyl]-N-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinoline- 6-sulfonamide 2 as on orally available monoacylglycerol acyltransferase 2 (MGAT2) inhibitor which exhibited an in vivo efficacy at an oral dose of 100 mg/kg in a mouse oral lipid tolerance test. Further optimization of compound 2 to improve the intrinsic potency culminated in the identification of compound 11. Compound 11 showed a >50-fold lower IC50 against human MGAT2 enzyme than 2. Oral administration of 11 at a dose of 3 mg/kg in the oral lipid tolerance test resulted in significant suppression of triglyceride synthesis.

Full text of DOI:10.1248/cpb.c15-00803

228
Chem. Pharm. Bull. 64, 228–238 (2016)
Vol. 64, No. 3
Regular Article
Identification of 2-[2-(4-tert-Butylphenyl)ethyl]-N-[4-(3-cyclopentylpropyl)-
2-fluorophenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide as an
Orally Active MGAT2 Inhibitor
,a
Tsuyoshi Busujima,* Hiroaki Tanaka,a Kanako Iwakiri,a Yoshihisa Shirasaki,a Eiji Munetomo,b
Masako Saito,b Aiko Masuko,b Kiyokazu Kitano,b Fusayo Io,b Koji Kato,c Shunsuke Kamigaso,c
Akiko Nozoe,d and Nagaaki Satoa
a Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd.; 1–403 Yoshino-cho, Kita-ku, Saitama 331–9530, Japan:
b Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd.; 1–403 Yoshino-cho, Kita-ku, Saitama 331–9530,
c
Japan: Drug Safety and Pharmacokinetics Laboratories, Taisho Pharmaceutical Co., Ltd.; 1–403 Yoshino-cho, Kita-
d
ku, Saitama 331–9530, Japan: and Pharmaceutical Science Laboratories, Taisho Pharmaceutical Co., Ltd.; 1–403
Yoshino-cho, Kita-ku, Saitama 331–9530, Japan.
Received October 20, 2015; accepted December 15, 2015
We previously reported 2-[2-(4-tert-butylphenyl)ethyl]-N-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquino-
line-6-sulfonamide 2 as on orally available monoacylglycerol acyltransferase 2 (MGAT2) inhibitor which
exhibited an in vivo efficacy at an oral dose of 100mg/kg in a mouse oral lipid tolerance test. Further optimi-
zation of compound 2 to improve the intrinsic potency culminated in the identification of compound 11. Com-
pound 11 showed a >50-fold lower IC50 against human MGAT2 enzyme than 2. Oral administration of 11 at
a dose of 3mg/kg in the oral lipid tolerance test resulted in significant suppression of triglyceride synthesis.
Key words monoacylglycerol acyltransferase 2; fat absorption; oral lipid tolerance test; tetrahydroisoquinoline
A rapid increases of obesity, type 2 diabetes and cardiovas- of these mice were significantly lower than those of the wild
cular disease have become a huge issue in the world’s indus- type. In addition, MGAT2 knock-out mice showed resistance
trialized countries. The unmet medical need for the treatment to obesity, glucose intolerance, and hypercholesterolemia in-
of these diseases has promoted the identification of many new duced by a high fat diet. Furthermore, these mice demonstrat-
targets, one of which is the inhibition of triacylglycerol syn- ed increased oxygen consumption without high-fat feeding,
thesis.
and MGAT2 was expected to modulate energy expenditure.7,8)
Dietary-derived fats are degraded in the gastrointestinal These results implied that inhibition of MGAT2 could be an
tract and then taken up by small-intestinal epithelial cells. attractive mechanism for reduction of fat absorption and treat-
Acyl CoA:monoacylglycerol acyltransferase (MGAT), which ment of obesity and associated metabolic disorders.
catalyzes the synthesis of diacylglycerol from monoacylglyc-
Some small molecule MGAT2 inhibitors have been dis-
erol and acyl-CoA, plays a crucial role in triglyceride (TG) closed.9–15) As shown in Fig. 1, AstraZeneca’s compound 1
re-synthesis in the small intestine.1) After re-synthesis into reported in the literature showed potent MGAT2 inhibitory
neutral fats in the cells,2) the neutral fats are packaged into activity and a significant reduction of plasma TG concentra-
chylomicrons, secreted into the lymphatics, and taken up into tion after an oral administration at a dose of 150mg/kg in
body. Therefore, the inhibition of activity of this enzyme is mice oral lipid tolerance test.16–18) We have also reported an
expected to suppress re-synthesis of TG and lead to reduce fat orally available MGAT2 inhibitor,19) 2-[2-(4-tert-butylphenyl)-
absorption.
ethyl]-N-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinoline-6-
Three isoforms of MGAT enzyme have been identified,3–5) sulfonamide 2, which demonstrated the significant suppression
and MGAT2 is highly expressed in small intestine in both hu- of fat absorption in mice oral lipid tolerance test at an oral
mans and mice.1) The phenotype of MGAT2 deficient mice has dose of 100mg/kg.
been already reported.6) These mice were viable and fertile
The identification of compound 2 promoted us to explore
without apparent abnormalities. After these mice were orally a more potent and orally available MGAT2 inhibitor, and our
given dietary oil (oral lipid tolerance test), plasma TG levels efforts have been focused on optimization of compound 2. As
Fig. 1. MGAT2 Inhibitor 1 and Our Novel Class Tetrahydroisoquinoline Compounds
*To whom correspondence should be addressed. e-mail: t-busujima@so.taisho.co.jp
© 2016 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
229
Reagents and conditions: (a) TFAA, pyridine, DMAP, CHCl3, rt; (b) ClSO3H, CHCl3, 60°C, 62% in two steps; (c) R1R2NH, pyridine, CHCl3, rt; (d) H2, 10% Pd–C,
MeOH–EtOAc, rt; (e) KOH aq., EtOH, rt, 61–94% in three steps; (f) 2-(4-(tert-butyl)phenyl)acetic acid, EDC·HCl, HOBt·H2O, CHCl3, rt, 59% for 17a; (g) 10% TFA,
CHCl3, rt, 54% in two steps for 17b; (h) BH3–THF, THF, relux; (i) 4N HCl/EtOAc, EtOAc, rt, 91 and 45% in two steps for 2 and 9, 65 and 63% in three steps for 5 and 8,
56–84% in two steps for 10–13; (j) 2-(4-(tert-butyl)phenyl)acetaldehyde, NaBH(OAc)3, ClCH2CH2Cl, rt.
Chart 1
Reagents and conditions: (a) RX, K2CO3, DMF, rt; (b) H2, 10% Pd–C, EtOH, rt, 55–81% in two steps; (c) 2,4-dimethoxybenzaldehyde, NaBH(OAc)3, AcOH, THF, rt,
34%-quant.; (d) olefin or borane reagent, Pd catalyst, solvent, reflux, 69–92%; (e) PPh3, I2, imidazole, CHCl3, rt; (f) PPh3, CH3CN, reflux, then 3-fluoro-4-nitrobenzalde-
hyde, KHMDS, THF, rt, 37% in two steps; (g) H2,10% Pd–C, EtOH, rt, 57%.
Chart 2
a result, introduction of the substituents at the para position 15. Intermediates 16a–h were synthesized by condensation
of the sulfonamide phenyl ring of compound 2 led to identify with the corresponding anilines, debromination by hydroge-
compound 11 (Fig. 1).
nolysis, and subsequent removal of trifluoroacetyl group. The
Herein, we describe the synthesis and biological properties of secondary amines 16a and b were condensed with (4-tert-
2-[2-(4-tert-butylphenyl)ethyl]-N-[4-(3-cyclopentylpropyl)-2- butylphenyl)acetic acid, followed by deprotection of the
fluorophenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide 2,4-dimethoxybenzyl group by treating with trifluoroacetic
(11) as a more potent MGAT2 inhibitor and its derivatives.
acid (TFA) to afford the amide compounds 17a and b, respec-
tively. N-Alkyl derivatives 2 and 9 were prepared by reduc-
tion of these amide compounds using borane–tetrahydrofuran
Chemistry
Synthesis of 1,2,3,4-tetrahydroisoquinoline-6-sulfonamide (THF) complex as a reducing reagent. Other intermediates
derivatives (compounds 2, 5, 8–13) were shown in Chart 1. 16c–h were converted into the corresponding N-alkylated
The secondary amine of 14 was protected with trifluoroacetic compounds by reductive amination with 2-(4-(tert-butyl)-
anhydride (TFAA), followed by chlorosulfonylation to afford phenyl)acetaldehyde.

230
Chem. Pharm. Bull.
Vol. 64, No. 3 (2016)
Reagents and conditions: (a) 2,4-dimethoxybenzaldehyde, NaBH(OAc)3, AcOH, THF, rt, 92%; (b) 27, pyridine, CHCl3, rt; (C) H2, 10% Pd–C, Et3N, MeOH–EtOAc, rt,
70% in two steps; (d) RX, K2CO3, DMF, rt; (e) KOH aq., EtOH, rt; (f) 2-(4-(tert-butyl)phenyl)acetaldehyde, NaBH(OAc)3, CHCl3, rt, 36–68% in three steps; (g) 20% TFA,
CHCl3, rt; (h) 4N HCl/EtOAc, EtOAc, rt, 2–33% in two steps.
Chart 3
Fig. 2. MGAT2 Inhibitory Activity of Tetrahydroisoquinoline and Dihydro-1H-isoindole Sulfonamide Derivatives
Preparation of the anilines (20a–22c) was accomplished by
the synthetic routes as depicted in Chart 2. Anilines 19a–c
Results and Discussion
As we previously reported,19) the structure–activity relation-
were synthesized by alkylation with the corresponding alkyl ship (SAR) study of 2,3-dihydro-1H-isoindole-5-sulfonamide
halides using 3-fluoro-4-nitrophenol 18 as a starting material derivative 3 indicated that the longer the alkoxy chain at the
and subsequent reduction of the nitro group. These anilines para positions of sulfonamide-substituted phenyl ring was,
were protected with 2,4-dimethoxybenzyl group to give the more it improved the enzyme inhibitory activity (3a vs.
20a–c.
b in Fig. 2). The plasma exposure level of these isoindoline
Aniline 21 was coupled with the corresponding olefins or derivatives after an oral administration to mice, however,
borane reagents using Pd-catalyst to afford 22e–g. Aniline 25 was too low due to its insufficient solubility (low absorption),
was prepared from alcohol 23 by iodination, Wittig reaction while compound 2 was orally available because its solubility
with 3-fluoro-4-nitrobenzaldehyde, and reduction of the nitro was improved by replacing the isoindolyl-urea scaffold with
group.
tetrahydroisoquinoline. Therefore, to identify a more potent
As shown in Chart 3, O-alkyl derivatives 4, 6, and 7 were and orally available MGAT2 inhibitor, we focused on the opti-
synthesized using aniline 27 protected with 2,4-dimethoxy- mization of the substituents at the para position of the phenyl
benzyl group. The N-protected sulfonamide intermediate 28 ring and applied these findings to the tetrahydroisoquinoline
was obtained by condensation of the sulfonyl chloride 15 scaffold (Table 1).
with aniline 27, and subsequent hydrogenolysis to remove
As expected, compound 4 possessing the same substituents
the bromo group. Compound 28 was alkylated with the cor- as compound 3b was three times more potent than 2. It was
responding alkyl halides, followed by the same procedure as found that further extension of the length of alkoxy group (to
described for the preparation of 5 to afford 4, 6, and 7.
n-hexyloxy and n-heptyloxy) resulted in increase of the poten-

Vol. 64, No. 3 (2016)
Chem. Pharm. Bull.
231
cy (5 and 6), while n-octyloxy analog (7) was as potent as 6. and in vivo efficacy in mice oral lipid tolerance test (OLTT)
Incorporation of benzene ring at the terminal position of the (Table 2). These compounds showed relatively higher potency
side chain was tolerable like 8. Replacement of the benzene in mouse recombinant enzyme assay than that in human.
ring with cyclohexyl group enhanced the potency (8 vs. 9). In Their plasma exposure levels at 0.5h were measured after oral
addition, replacement of the ether linker with the methylene administration in mice at a dose of 30mg/kg. The exposure
linker was also found to be tolerable (9 vs. 10). Introduction levels were well correlated with their solubility in FeSSIF.
of the cyclopentyl group at the terminal position (11) led to
Their in vivo efficacy was tested using mice oral lipid toler-
the most potent MGAT2 inhibitor among them with an IC50 ance test. Triglyceride (TG) was administrated at 0.5h after
value of 28nM. Replacement of the cyclopentyl group with oral dosing (3, 10 or 30mg/kg) of the compounds. Then the
cyclopropyl or contraction of the carbon chain to ethylene values of plasma levels of TG were measured at 0.5, 1, 2 and
linker resulted in decrease of the potency drastically (11 vs. 4h post triglyceride dose and calculated its area under the
12 and 13).
curve (AUC)0–4h value. Compound 2 demonstrated the signifi-
Having obtained more potent MGAT2 inhibitors than com- cant suppression of fat absorption in mice oral lipid tolerance
pound 2, selected compounds (2, 7, 8, and 11), which have dis- test at an oral dose of 100mg/kg, while 2 did not show any
tinctive substituents such as n-alkyl group, phenyl group, and effects on plasma AUC0–4h levels of TG at 30mg/kg per os
cycloalkyl group, were evaluated for their inhibition activity (p.o.) dosing despite the highest exposure level among these
for mice MGAT2 enzyme, metabolic stability (MS) in human compounds. This result indicated that its intrinsic potency in
and mice microsomes, and solubility in Fed State Simulated MGAT2 inhibitory activity was insufficient to show in vivo
Intestinal Fluid (FeSSIF),20) plasma exposure level in mice, efficacy at less than 30mg/kg dosing. On the other hand, other
compounds exhibited significant suppression of fat absorption
at an oral dose of 30mg/kg. At lower dose of 30mg/kg, 7 and
Table 1. Structure–Activity Relationships of Tetrahydroisoquinoline De-
8 resulted in decrease of the efficacy. However, 11 demon-
rivatives with a Variety of Substituents at the para Position
strated significant reduction of fat absorption even at a dose
of 3mg/kg.
Having showed a significant suppression in plasma TG lev-
els of 11, we studied its pharmacokinetics (PK) in mice after
intravenous (i.v.) administration at a dose of 1mg/kg and oral
administration at a dose of 3mg/kg (Table 3). The Cmax and
AUC of 11 were 127ng/mL and 402ng·h/mL, respectively,
a)
Compound
R1
R2
hMGAT2 IC50 (nM)
and the bioavailability was 21.4%. In this study, the plasma
concentration of 11 after 4h of the TG dosing was 43.5ng/mL
(75 nM) and 18-fold higher than its IC50 value (4nM).
2
4
F
H
F
F
F
F
F
F
F
F
F
F
1522
454
133
40
n-PenO
5
n-HexO
6
n-HepO
Conclusion
7
n-OctO
45
In summary, through an optimization of substitutions
at the para position of the phenyl ring of compound 2,
2-[2-(4-tert-butylphenyl)ethyl]-N-[4-(3-cyclopentylpropyl)-2-
fluorophenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide
(11) as an orally active MGAT2 inhibitor was identified. Com-
pound 11 exhibited the significant reduction of TG levels in
plasma even at an oral dose of 3mg/kg in mice oral lipid tol-
erance test. Further in vivo evaluation of compound 11, such
8
PhCH2CH2CH2O
c-HexCH2CH2O
c-HexCH2CH2CH2
c-PenCH2CH2CH2
c-PrCH2CH2CH2
c-PenCH2CH2
173
76
9
10
11
12
13
62
28
379
10000
a) Values are the means of two or more separate experiments.
Table 2. Profiles of Representative Compounds
a)
MGAT2 IC50
(nM)
Solubility
Plasma exposure
% Reduction of in OLTTe)
MSb) (%)
(h/m)
Compound
(µg/mL)
at 0.5hd)
FeSSIFc)
(ng/mL)
1 (mg/kg)
3 (mg/kg) 10 (mg/kg) 30 (mg/kg) 100 (mg/kg)
(h/m)
2
7
1522/1170
45/20
14.6/47.8
−5.2/−3.5
23.6/14.3
385
207
68.3
288
135
NTf)
NTf)
1%
NTf)
−10%
5%
NTf)
2%
57%##
NTf)
21.5
13.9
54.3
22%##
28%$$
29%*
38%***
8
173/25
28/4
14%
NTf)
11
−9.6/−4.3
19%
25%**
35%***
41%***
a) Values are the means of two or more separate experiments. b) % metabolized after 15min of incubation with human/mouse liver microsomes. c) Fed State Simulated
Intestinal Fluid, pH 5.0. d) Orally dosed at 30mg/kg as a 0.5% methyl cellulose (MC) solution to mice. e) Data are the mean standard error of the mean averaged from 7–8
studies, expressed as percentage reduction of plasma AUC0–4h levels of TG for 4h after an oral administration in mice fat load test (*p<0.05, **p<0.01, ***p<0.001 vs. ve-
hicle (Dunnett’s test), ## p<0.01 vs. vehicle (Steel’s test), $$ p<0.01 vs. vehicle (Student’s t-test)). f) NT: Not tested.
Table 3. PK Profiles of Compound 11 in Mice
CL (mL/h/kg)
Vdss (mL/kg)
T1/2 (h) AUClast (ng·h/mL) Cmax (ng/mL)
Tmax (h) AUC0–24h (ng·h/mL) BA (%)
i.v.-1mg/kg
1560
—
6930
—
7.1
—
625
—
—
—
—
—
p.o.-3mg/kg
127
1.67
402
21.4

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Chem. Pharm. Bull.
Vol. 64, No. 3 (2016)
as C57BL/6 mice on a high-fat diet and other pharmacological (2H, m), 7.62–7.70 (1H, m), 7.97–8.06 (1H, m).
studies will be reported in due course.
N-(4-Fluorophenyl)-1,2,3,4-tetrahydroisoquinoline-
6-sulfonamide (16a) Pyridine (3.09mL, 38.4mmol) was
added to a solution of 4-fluoroaniline (3.73g, 33.6mmol)
Experimental
Chemistry All commercially available starting materials in chloroform (107mL). After cooling to 0°C, 15 (13.0g,
and reagents were used without further purification unless 32.0mmol) was added to the mixture. The mixture was
otherwise note. Thin layer chromatography was performed to warmed to room temperature and stirred for 13h. To the mix-
monitor reactions using Merck silica gel 60F254 plates or Fuji ture was added 1mol/L hydrochloric acid, and the mixture
Silysia chromatorex NH plates. Silica gel column chromatog- was extracted with chloroform. The organic layer was concen-
raphy was performed using Wakogel® C-200, or NH-silica gel trated under reduced pressure, and to the residue was added
Fuji Silysia chromatorex® DM1020, or an appropriately sized 20% ethyl acetate–n-hexane. The resulting precipitates were
pre-packed silica cartridge on a Biotage system. 1H-NMR collected by filtration to afford crude product (14.5g, 94%) as
spectra were recorded on a Varian Instruments INOVA-300 an orange powder.
spectrometer at 300MHz or a JEOL ECA-600 spectrometer
To a solution of the above product (14.5g, 30.2mmol) in
at 600MHz, and are referenced to an internal standard of tri- methanol (200mL) and ethyl acetate (100mL) was added
methylsilane (TMS, δ 0.00ppm). Chemical shifts are reported 10% palladium activated carbon (4.36g), and the mixture was
in parts per million (ppm) in the indicated solvent. Multiplic- stirred under a hydrogen atmosphere at room temperature for
ity was defined as s (singlet), d (doublet), t (triplet), q (quartet), 15h. Then to the mixture was added 10% palladium activated
dd (double doublet), m (multiplet), brs (broad singlet), brd carbon (2.00g), and the mixture was stirred under a hydrogen
(broad doublet) or brt (broad triplet). Mass spectra (MS) were atmosphere at room temperature for 5h. The mixture was
recorded on a Shimadzu LCMS-2010EV mass spectrometer, filtered through a pad of Celite® and the filtrate was concen-
LCMS-iontrap-time-of-flight (LCMS-IT-TOF) mass spectrom- trated under reduced pressure. Ethanol was added to the resi-
eter, a Waters Micromass Platform LC mass spectrometer, or due, and the resulting precipitates were collected by filtration
a ThermoFisher Scientific LCQ Deca XP with an electrospray to afford the intermediate (10.6g, 88%) as a colorless powder.
ionization (ESI) or an ESI/atmospheric pressure chemical
An aqueous solution (10mL) of potassium hydroxide (2.24g,
ionization (APCI) dual source. High resolution mass spectra 40.0mmol) was added to a suspension of the above intermedi-
(HR-MS) were recorded on a Shimadzu LCMS-IT-TOF mass ate (8.05g, 20.0mmol) in ethanol (40mL), and the mixture
spectrometer with an ESI/APCI dual source. Elemental analy- was stirred at room temperature for 15h. The mixture was
ses were performed using a Perkin-Elmer 2400II or a Yanaco concentrated under reduced pressure, and the resulting residue
MT-6, and the results were within 0.4% of the calculated was diluted with water. After cooling to 0°C, 3mol/L hydro-
values.
chloric acid was added dropwise to adjust the pH to 7 to 8.
7-Bromo-2-(trifluoroacetyl)-1,2,3,4-tetrahydroisoquino- The resulting precipitates were collected by filtration to afford
1
line-6-sulfonyl Chloride (15) Pyridine (18.2mL, 226mmol) 16a (6.04g, 99%) as a colorless powder: H-NMR (300MHz,
and 4-N,N-dimethylaminopyridine (184mg, 1.51mmol) were DMSO-d6) δ: 2.65–2.72 (2H, m), 2.87–2.94 (2H, m), 3.84 (2H,
added to a solution of 7-bromo-1,2,3,4-tetrahydroisoquinoline s), 7.05–7.10 (4H, m), 7.16 (1H, d, J=8.1Hz), 7.38–7.45 (2H,
(32.0g, 151mmol) in chloroform (280mL). After cooling to m); MS (ESI/APCI dual) m/z: 307 [M+H]+.
0°C, trifluoroacetic acid anhydride (21.9mL, 158mmol) was
added dropwise to the mixture. The mixture was warmed to aniline in the same procedure described for 16a.
room temperature and stirred for 20h. The mixture was con-
N-[4-(2-Cyclohexylethoxy)-2-fluorophenyl]-N-(2,4-di-
Compounds 16b to h were prepared from the corresponding
centrated under reduced pressure, and the resulting residue methoxybenzyl)-1,2,3,4-tetrahydroisoquinoline-6-sulfon-
was diluted with ethyl acetate. One mole/liter hydrochloric amide (16b) Colorless amorphous (yield 68%): 1H-NMR
acid was added to the mixture, and the mixture was ex- (600MHz, CDCl3) δ: 0.89–1.00 (2H, m), 1.11–1.30 (4H, m),
tracted with ethyl acetate. The organic layer was washed with 1.40–1.50 (1H, m), 1.54–1.77 (6H, m), 2.79–2.86 (2H, m),
saturated aqueous sodium hydrogen carbonate solution and 3.15–3.17 (2H, m), 3.57 (3H, s), 3.75 (3H, s), 3.85–3.95 (2H,
then brine, dried over anhydrous MgSO4, filtered, and con- m), 4.08 (2H, s), 4.67 (2H, s), 6.25–6.29 (1H, m), 6.35–6.40
centrated under reduced pressure. The resulting residue was (1H, m), 6.45–6.52 (2H, m), 6.87–6.93 (1H, m), 7.07–7.12 (1H,
purified using a silica gel column eluted with 10 to 30% ethyl m), 7.19–7.25 (1H, m), 7.46–7.50 (2H, m); MS (ESI): m/z 583
acetate–n-hexane to afford the intermediate (42.4g, 91%) as a [M+H]+.
pale yellow oil.
N-(2,4-Dimethoxybenzyl)-N-[2-fluoro-4-(hexyloxy)-
Chlorosulfonic acid (35.6mL, 535mmol) was added phenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide (16c)
1
dropwise to a solution of the above intermediate (25.4g, Pale yellow oil (yield 81%): H-NMR (300MHz, CDCl3) δ:
82.3mmol) in chloroform (35mL) at 0°C, and the mixture 0.85–0.94 (3H, m), 1.24–1.50 (4H, m), 1.63–1.81 (4H, m), 2.82
was stirred at room temperature for 1h and at 60°C for 4h. (2H, t, J=5.8Hz), 3.16 (2H, t, J=5.8Hz), 3.57 (3H, s), 3.75 (3H,
After cooling to room temperature, the reaction mixture was s), 3.86 (2H, t, J=6.5Hz), 4.08 (2H, s), 4.67 (2H, s), 6.27 (1H,
added dropwise to ice water, and the mixture was extracted d, J=2.3Hz), 6.37 (1H, dd, J=8.2, 2.3Hz), 6.43–6.52 (2H,
with chloroform. The organic layer was dried over anhydrous m), 6.84–6.94 (1H, m), 7.08–7.13 (1H, m), 7.20–7.27 (1H, m),
MgSO4, filtered, and concentrated under reduced pressure. To 7.44–7.52 (2H, m); MS (ESI/APCI dual) m/z: 557 [M+H]+.
the residue was added 18% ethyl acetate–n-hexane, and the re-
N-(2,4-Dimethoxybenzyl)-N-[2-fluoro-4-(3-phenylpro-
sulting precipitates were collected by filtration to afford crude poxy)phenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide
1
1
15 (22.7g, 68%) as a pale yellow powder: H-NMR (300MHz, (16d) Colorless amorphous (yield 70%): H-NMR (300MHz,
CDCl3) δ: 2.96–3.10 (2H, m), 3.83–4.02 (2H, m), 4.76–4.93 CDCl3) δ: 1.97–2.16 (2H, m), 2.71–2.86 (4H, m), 3.11–3.20 (2H,

Vol. 64, No. 3 (2016)
Chem. Pharm. Bull.
233
m), 3.57 (3H, s), 3.75 (3H, s), 3.83–3.89 (2H, m), 4.08 (2H, s), 1.04mmol). The reaction mixture was stirred at room tem-
4.67 (2H, s), 6.26–6.28 (1H, m), 6.35–6.40 (1H, m), 6.43–6.51 perature for 19h. Water was added, and the mixture was ex-
(2H, m), 6.86–6.94 (1H, m), 7.06–7.34 (7H, m), 7.44–7.52 (2H, tracted with ethyl acetate. The organic layer was washed with
m); MS (ESI/APCI dual) m/z: 591 [M+H]+.
saturated aqueous NaHCO3 and brine, dried over anhydrous
N-[4-(3-Cyclohexylpropyl)-2-fluorophenyl]-1,2,3,4-tetra- MgSO4, filtered, and concentrated under reduced pressure.
hydroisoquinoline-6-sulfonamide (16e) Colorless powder The resulting residue was purified using a silica gel column
1
(yield 66%): H-NMR (300MHz, DMSO-d6) δ: 0.73–0.88 (2H, eluted with 30 to 50% ethyl acetate–n-hexane to afford the
m), 1.01–1.26 (6H, m), 1.42–1.73 (7H, m), 2.42–2.51 (2H, m), intermediate (530mg, 80%) as a colorless powder.
2.65–2.75 (2H, m), 2.90–3.00 (2H, m), 3.89 (2H, s), 6.86–7.02
(2H, m), 7.05–7.20 (2H, m), 7.34–7.46 (2H, m); MS (ESI/APCI 0.661mmol) in chloroform (1.8mL) was added trifluoro-
dual) m/z: 431 [M+H]+.
acetic acid (1.77mL), and the mixture was stirred at room
To
a
solution of the above intermediate (500mg,
N-[4-(3-Cyclopentylpropyl)-2-fluorophenyl]-1,2,3,4-tetra- temperature for 1h. The reaction mixture was concentrated
hydroisoquinoline-6-sulfonamide (16f) Colorless powder under reduced pressure, and ethyl acetate–n-hexane was
1
(yield 61%): H-NMR (300MHz, DMSO-d6) δ: 0.93–1.09 (2H, added to the residue. The resulting precipitate was collected
m), 1.17–1.29 (2H, m), 1.40–1.60 (6H, m), 1.63–1.78 (3H, m), by filtration to afford 17b (270mg, 67%) as a colorless pow-
2.40–2.53 (2H, m), 2.65–2.75 (2H, m), 2.90–2.97 (2H, m), 3.88 der: 1H-NMR (300MHz, DMSO-d6) δ: 0.80–1.02 (2H, m),
(2H, s), 6.87–6.99 (2H, m), 7.05–7.19 (2H, m), 7.37–7.44 (2H, 1.09–1.27 (2H, m), 1.26 (9H, s), 1.34–1.46 (1H, m), 1.49–1.78
m); MS (ESI/APCI dual) m/z: 417 [M+H]+.
(8H, m), 2.74–2.84 (2H, m), 3.64–3.81 (4H, m), 3.88–3.99 (2H,
N-[4-(3-Cyclopropylpropyl)-2-fluorophenyl]-1,2,3,4-tet- m), 4.64–4.81 (2H, m), 6.65–6.81 (2H, m), 6.97–7.08 (1H, m),
rahydroisoquinoline-6-sulfonamide (16g) Colorless pow- 7.11–7.20 (2H, m), 7.24–7.41 (3H, m), 7.43–7.51 (2H, m), 9.77
1
der (yield 91%): H-NMR (300MHz, DMSO-d6) δ: 0.07–0.01 (1H, s); MS (ESI) m/z: 607 [M+H]+.
(2H, m), 0.33–0.40 (2H, m), 0.59–0.72 (1H, m), 1.09–1.19 (2H,
m), 1.54–1.66 (2H, m), 2.48–2.57 (2H, m), 2.81–2.89 (2H, m), 1,2,3,4-tetrahydroisoquinoline-6-sulfonamide (2)
2-[2-(4-tert-Butylphenyl)ethyl]-N-(4-fluorophenyl)-
bo-
A
3.12–3.20 (2H, m), 4.10 (2H, s), 6.91–7.03 (2H, m), 7.08–7.16 rane–THF complex (0.9mol/L THF solution, 0.550mL,
(1H, m), 7.26–7.32 (1H, m), 7.46–7.52 (2H, m); MS (ESI/APCI 0.500mmol) was added to a solution of 17a (120mg,
dual) m/z: 389 [M+H]+.
0.250mmol) in THF (10mL) at 0°C, and the mixture was
N-[4-(2-Cyclopentylethyl)-2-fluorophenyl]-1,2,3,4-tetra- heated at reflux temperature for 5h. After cooling to room
hydroisoquinoline-6-sulfonamide (16h) Colorless powder temperature, 6mol/L hydrochloric acid (6mL) was added,
1
(yield 94%): H-NMR (300MHz, DMSO-d6) δ: 0.99–1.16 (2H, and the mixture was stirred at reflux temperature for 3h.
m), 1.37–1.79 (9H, m), 2.45–2.56 (2H, m), 2.80–2.89 (2H, m), The mixture was cooled to 0°C, 6mol/L aqueous sodium
3.09–3.20 (2H, m), 4.10 (2H, s), 6.90–7.03 (2H, m), 7.06–7.16 hydroxide was added to adjust the pH to 8 to 9, and the mix-
(1H, m), 7.28 (1H, d, J=8.7Hz), 7.45–7.53 (2H, m); MS (ESI/ ture was extracted with ethyl acetate. The organic layer was
APCI dual) m/z: 403 [M+H]+.
washed with brine, dried over anhydrous MgSO4, filtered, and
2-[(4-tert-Butylphenyl)acetyl]-N-(4-fluorophenyl)-1,2,3,4- concentrated under reduced pressure. The resulting residue
tetrahydroisoquinoline-6-sulfonamide (17a) To a suspen- was purified using a silica gel column eluted with chloro-
sion of 16a (153mg, 0.500mmol) in chloroform (10mL) were form to 5% methanol–chloroform to afford 2 (115mg, 99%)
added (4-tert-butylphenyl)acetic acid (106mg, 0.650mmol), as a colorless amorphous. To a solution of 2 in ethyl acetate
1-hydroxybenzotriazole monohydrate (100mg, 0.650mmol) (4mL) was added 4mol/L hydrogen chloride in ethyl acetate
and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro- (1mL), and the mixture was stirred at room temperature for
chloride (125mg, 0.650mmol). The reaction mixture was 15h. After the volatiles were removed by rotary evaporation,
stirred at room temperature for 15h. Water was added, and the diethyl ether–ethyl acetate was added to the residue, and the
mixture was extracted with ethyl acetate. The organic layer resulting precipitates were collected by filtration to afford the
was washed with brine, dried over anhydrous MgSO4, fil- monohydrochloride salt of 2 (115mg, 92%) as a colorless pow-
1
tered, and concentrated under reduced pressure. The resulting der: H-NMR (600MHz, DMSO-d6) δ: 1.27 (9H, s), 3.04–3.16
residue was purified using a silica gel column eluted with 25% (3H, m), 3.20–3.31 (2H, m), 3.34–3.48 (2H, m), 3.72–3.82
and then 60% ethyl acetate–n-hexane. The fractions incruding (1H, m), 4.35–4.45 (1H, m), 4.63–4.73 (1H, m), 7.06–7.15 (4H,
product were collected and evaporated by rotary evaporation. m), 7.18–7.25 (2H, m), 7.33–7.42 (3H, m), 7.57–7.68 (2H, m),
To the residue was added ethyl acetate–diisopropylether–n- 10.35 (1H, s), 11.01 (1H, brs); MS (ESI/APCI dual) m/z: 467
hexane, and the resulting precipitates were collected by [M+H]+, 465 [M−H]−; Anal. Calcd for C27H31FN2O2S HCl: C,
filtration to afford 17a (142mg, 59%) as a colorless powder: 64.46; H, 6.41; N, 5.57. Found: C, 64.31; H, 6.41; N, 5.49.
1H-NMR (300MHz, DMSO-d6) δ: 1.26 (9H, s), 2.73–2.83 (2H,
2-[2-(4-tert-Butylphenyl)ethyl]-N-[4-(2-cyclohexylethoxy)-
m), 3.62–3.76 (4H, m), 4.62–4.78 (2H, m), 7.04–7.19 (6H, m), 2-fluorophenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfon-
7.24–7.39 (3H, m), 7.47–7.56 (2H, m), 10.20 (1H, brs); MS amide (9) Compound 9 was prepared by the same procedure
(ESI/APCI dual) m/z: 481 [M+H]+.
2-[(4-tert-Butylphenyl)acetyl]-N-[4-(2-cyclohexylethoxy)-
described for 2 using 17b instead of 17a.
Colorless powder (yield 45%): 1H-NMR (600MHz,
2-fluorophenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfon- DMSO-d6) δ: 0.88–0.98 (2H, m), 1.08–1.34 (4H, m), 1.27
amide (17b) To a suspension of 16b (468mg, 0.803mmol) (9H, s), 1.36–1.46 (1H, m), 1.52–1.74 (7H, m), 3.04–3.17 (3H,
in N,N-dimethylformamide (10mL) were added (4-tert- m), 3.18–3.50 (3H, m), 3.75–3.84 (1H, m), 3.87–4.01 (2H,
butylphenyl)acetic acid (132mg, 0.879mmol), 1-hydroxyben- m), 4.38–4.49 (1H, m), 4.67–4.78 (1H, m), 6.66–6.74 (1H,
zotriazole monohydrate (207mg, 1.04mmol) and 1-ethyl-3- m), 6.75–6.82 (1H, m), 7.02–7.09 (1H, m), 7.18–7.28 (2H,
(3-dimethylaminopropyl)carbodiimide hydrochloride (199mg, m), 7.32–7.44 (3H, m), 7.52–7.64 (2H, m), 9.90 (1H, brs),

234
Chem. Pharm. Bull.
Vol. 64, No. 3 (2016)
10.79 (1H, brs); HR-MS (ESI/APCI Dual) m/z: Calcd for 1.27 (9H, s), 1.48–1.54 (2H, m), 1.56–1.68 (5H, m), 2.44–2.53
C35H45FN2O3S [M+H]+; 593.3208. Found 593.3208.
(2H, m), 3.04–3.17 (3H, m), 3.19–3.48 (4H, m), 3.75–3.83 (1H,
2-[2-(4-tert-Butylphenyl)ethyl]-N-[2-fluoro-4-(hexyloxy)- m), 4.40–4.48 (1H, m), 4.68–4.75 (1H, m), 6.92–6.96 (1H,
phenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide (5) m), 6.97–7.02 (1H, m), 7.10–7.14 (1H, m), 7.20–7.25 (2H, m),
i) To a solution of 16c (520mg, 0.935mmol) in 1,2-dichlo- 7.34–7.43 (3H, m), 7.57–7.65 (2H, m), 10.10 (1H, s), 10.60 (1H,
roethane (3mL) were added a solution of 2-(4-(tert-butyl)- brs); MS (ESI/APCI dual) m/z: 591 [M+H]+, 589 [M−H]−;
phenyl)acetaldehyde (181mg, 1.03mmol) in 1,2-dichloroethane Anal. Calcd for C36H47FN2O2S HCl: C, 68.93; H, 7.71; N, 4.47.
(2mL) and sodium triacetoxyborohydride (297mg, 1.40mmol), Found: C, 68.83; H, 7.66; N, 4.42.
and the mixture was stirred overnight at room temperature.
2-[2-(4-tert-Butylphenyl)ethyl]-N-[4-(3-cyclopentylpropyl)-
Saturated aqueous NaHCO3 was added to the reaction mix- 2-fluorophenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfon-
ture, and the mixture was extracted with ethyl acetate. The amide (11) Colorless powder (yield 60%): 1H-NMR
organic layer was washed with brine, dried over anhydrous (600MHz, DMSO-d6) δ: 0.94–1.06 (2H, m), 1.20–1.29 (2H, m),
MgSO4, filtered, and concentrated under reduced pressure. 1.27 (9H, s), 1.41–1.59 (6H, m), 1.66–1.76 (3H, m), 2.49–2.54
The resulting residue was purified using a silica gel column (2H, m), 3.06–3.19 (3H, m), 3.28–3.45 (4H, m), 3.73–3.81 (1H,
eluted with 20 to 34% ethyl acetate–n-hexane to afford the m), 4.39–4.48 (1H, m), 4.66–4.75 (1H, m), 6.92–6.97 (1H, m),
intermediate (622mg, 93%) as a pale yellow oil.
6.98–7.03 (1H, m), 7.08–7.13 (1H, m), 7.22 (2H, d, J=8.3Hz),
ii) To a solution of the above intermediate (593mg, 7.34–7.42 (3H, m), 7.57–7.65 (2H, m), 10.12 (1H, s), 11.64 (1H,
0.827mmol) in chloroform (5mL) was added anisole (5mL), brs); MS (ESI/APCI dual) m/z: 577 [M+H]+, 575 [M−H]−;
the mixture was cooled to 0°C. To the mixture was added Anal. Calcd for C35H45FN2O2S HCl: C, 68.55; H, 7.56; N, 4.57.
trifluoroacetic acid (1mL), and the mixture was stirred at Found: C, 68.46; H, 7.57; N, 4.55.
room temperature for 21h. Saturated aqueous NaHCO3 was
2-[2-(4-tert-Butylphenyl)ethyl]-N-[4-(3-cyclopropylpropyl)-
added to the reaction mixture, and the mixture was extracted 2-fluorophenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfon-
with chloroform. The organic layer was washed with brine, amide (12) Colorless powder (yield 84%): 1H-NMR
dried over anhydrous MgSO4, filtered, and concentrated under (600MHz, DMSO-d6) δ: −0.05–0.02 (2H, m), 0.33–0.40
reduced pressure. The resulting residue was purified using a (2H, m), 0.62–0.70 (1H, m), 1.11–1.18 (2H, m), 1.27 (9H, s),
NH silica gel column eluted with 50 to 100% ethyl acetate–n- 1.56–1.64 (2H, m), 2.44–2.57 (2H, m), 3.02–3.49 (7H, m),
hexane to afford 5 (378mg, 76%) as a pale yellow amorphous. 3.75–3.84 (1H, m), 4.40–4.49 (1H, m), 4.68–4.76 (1H, m),
iii) To a solution of 5 in ethyl acetate (5mL) was added 6.92–7.03 (2H, m), 7.10–7.16 (1H, m), 7.19–7.26 (2H, m),
4mol/L hydrogen chloride in ethyl acetate (1mL), and the 7.35–7.43 (3H, m), 7.56–7.66 (2H, m), 10.11 (1H, s), 10.62 (1H,
mixture was stirred overnight at room temperature. To the brs); HR-MS (ESI/APCI dual) m/z: Calcd for C33H41FN2O2S
reaction mixture was added n-hexane, and the mixture was [M+H]+; 549.2946. Found 549.2935.
stirred for 1h. The resulting precipitates were collected by
2-[2-(4-tert-Butylphenyl)ethyl]-N-[4-(2-cyclopentylethyl)-
filtration to afford the monohydrochloride salt of 5 (376mg, 2-fluorophenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfon-
1
92%) as a colorless powder: H-NMR (600MHz, DMSO-d6) δ: amide (13) Colorless powder (yield 72%): 1H-NMR
0.80–0.92 (3H, m), 1.25–1.30 (4H, m), 1.27 (9H, s), 1.33–1.42 (600MHz, DMSO-d6) δ: 1.04–1.12 (2H, m), 1.27 (9H, s),
(2H, m), 1.62–1.70 (2H, m), 3.02–3.28 (4H, m), 3.34–3.50 (3H, 1.41–1.60 (6H, m), 1.63–1.74 (3H, m), 2.45–2.55 (2H, m),
m), 3.76–3.85 (1H, m), 3.91 (2H, t, J=6.4Hz), 4.40–4.52 (1H, 3.05–3.16 (3H, m), 3.20–3.46 (4H, m), 3.74–3.83 (1H, m),
m), 4.68–4.79 (1H, m) 6.67–6.72 (1H, m), 6.75–6.79 (1H, m), 4.40–4.48 (1H, m), 4.66–4.76 (1H, m), 6.93–6.97 (1H, m),
7.03–7.10 (1H, m), 7.23 (2H, d, J=8.3Hz), 7.35–7.43 (3H, m), 6.98–7.03 (1H, m), 7.09–7.15 (1H, m), 7.20–7.24 (2H, m),
7.55–7.62 (2H, m), 9.90 (1H, s), 10.46 (1H, brs); MS (ESI) m/z: 7.35–7.42 (3H, m), 7.57–7.64 (2H, m), 10.10 (1H, s), 10.82 (1H,
607 [M+H]+; Anal. Calcd for C33H43FN2O3S HCl: C, 65.71; H, brs); HR-MS (ESI/APCI dual) m/z: Calcd for C34H43FN2O2S
7.35; N, 4.64. Found: C, 65.53; H, 7.29; N, 4.53.
Compound 8 was prepared from the corresponding 16d in
[M+H]+; 563.3102. Found 563.3089.
4-(2-Cyclohexylethoxy)-2-fluoroaniline (19a) Potassium
the same procedure described for 5. Compounds 10 to 13 were carbonate (4.15g, 30.0mmol) and (2-bromoethyl)cyclohexane
prepared from the corresponding 16e to h in the same proce- (3.76mL, 24.0mmol) were added to a solution of 3-fluoro-
dure described for 5-(i) and -(iii).
4-nitrophenol (3.14g, 20.0mmol) in N,N-dimethylformamide
2-[2-(4-tert-Butylphenyl)ethyl]-N-[2-fluoro-4-(3-phen- (50mL) at room temperature, and the mixture was stirred
ylpropoxy)phenyl]-1,2,3,4-tetrahydroisoquinoline-6-sul- overnight. Water was added to the reaction mixture and the
fonamide (8) Colorless powder (yield 63%): 1H-NMR mixture was extracted three times with ethyl acetate. The
(600MHz, DMSO-d6) δ: 1.27 (9H, s), 1.94–2.02 (2H, m), organic layer was washed with 1mol/L hydrochloric acid and
2.68–2.73 (2H, m), 3.02–3.24 (4H, m), 3.35–3.51 (3H, m), brine, dried over anhydrous MgSO4, filtered, and concentrated
3.76–3.84 (1H, m), 3.89–3.96 (2H, m), 4.41–4.52 (1H, m), under reduced pressure. The resulting residue was purified
4.69–4.79 (1H, m), 6.68–6.82 (2H, m), 7.03–7.12 (1H, m), using a silica gel column eluted with 10 to 20% ethyl ace-
7.15–7.31 (7H, m), 7.35–7.43 (3H, m), 7.55–7.64 (2H, m), 9.91 tate–n-hexane to afford the target intermediate (2.31g, 43%)
(1H, s), 10.28 (1H, brs); MS (ESI) m/z: 601 [M+H]+; Anal. as a pale yellow oil.
Calcd for C36H41FN2O3S HCl: C, 67.85; H, 6.64; N, 4.40.
Found: C, 67.83; H, 6.63; N, 4.32.
To a solution of the above intermediate (2.28g, 8.52mmol)
in ethanol (25mL) was added 10% palladium activated carbon
2-[2-(4-tert-Butylphenyl)ethyl]-N-[4-(3-cyclohexylpropyl)- (2.28g) at room temperature, and the mixture was stirred
2-fluorophenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfon- overnight under a hydrogen atmosphere. The reaction mixture
amide (10) Colorless powder (yield 56%): 1H-NMR was filtered through a pad of Celite®, and the filtrate was
(300MHz, DMSO-d6) δ: 0.77–0.86 (2H, m), 1.05–1.23 (6H, m), concentrated under reduced pressure. The resulting residue

Vol. 64, No. 3 (2016)
Chem. Pharm. Bull.
235
was purified using a silica gel column eluted with 10 to 20% m), 3.67 (2H, brs), 5.96–6.07 (1H, m), 6.16–6.24 (1H, m),
ethyl acetate–n-hexane to afford 19a (1.63g, 81%) as a red oil: 6.65–6.72 (1H, m), 6.87–6.93 (1H, m), 6.96–7.04 (1H, m); MS
1H-NMR (300MHz, CDCl3) δ: 0.84–1.05 (2H, m), 1.09–1.34 (ESI/APCI dual) m/z: 234 [M+H]+.
(3H, m), 1.40–1.53 (1H, m), 1.57–1.81 (7H, m), 3.41 (2H, brs),
3.90 (2H, t, J=6.7Hz), 6.48–6.76 (3H, m); MS (ESI/APCI (22f) 1,4-Dioxane (5mL) and water (1.2mL) were added to
dual) m/z: 238 [M+H]+.
a mixture of 4-bromo-2-fluoroaniline (285mg, 1.50mmol),
Compounds 19b and c were prepared from the correspond- 2-[(1E)-3-cyclopentylprop-1-en-1-yl]-4,4,5,5-tetramethyl-1,3,2-
ing alkyl halide in the same procedure described for 19a. dioxaborolane (425mg, 1.80mmol), tris(dibenzylideneacetone)-
4-[(1E)-3-Cyclopentylprop-1-en-1-yl]-2-fluoroaniline
2-Fluoro-4-(hexyloxy)aniline (19b) Pale yellow oil (yield dipalladium (137mg, 0.15mmol), tri-2-furylphosphine (210mg,
55%): 1H-NMR (300MHz, CDCl3) δ: 0.82–1.01 (3H, m), 0.90mmol) and cesium carbonate (976mg, 3.00mmol). The
1.20–1.52 (6H, m), 1.63–1.83 (2H, m), 3.42 (2H, brs), 3.86 (2H, mixture was stirred at reflux temperature for 6h under an
t, J=6.6Hz), 6.45–6.84 (3H, m); MS (ESI/APCI dual) m/z: 212 argon atmosphere. After cooling to room temperature, the
[M+H]+.
mixture was diluted with ethyl acetate, washed with brine.
2-Fluoro-4-(3-phenylpropoxy)aniline (19c) Brown oil The organic layer was dried over anhydrous MgSO4, filtered,
1
(yield 64%): H-NMR (300MHz, CDCl3) δ: 1.95–2.14 (2H, and concentrated under reduced pressure. The resulting
m), 2.74–2.83 (2H, m), 3.42 (2H, brs), 3.87 (2H, t, J=6.3Hz), residue was purified using a silica gel column eluted with
6.48–6.77 (3H, m), 7.12–7.34 (5H, m); MS (ESI/APCI dual) 10% ethyl acetate–n-hexane to afford 22f (302mg, 92%) as
1
m/z: 246 [M+H]+.
a pale yellow oil: H-NMR (300MHz, CDCl3) δ: 1.10–1.24
4-(2-Cyclohexylethoxy)-N-(2,4-dimethoxybenzyl)-2-fluo- (2H, m), 1.44–1.67 (4H, m), 1.69–1.82 (2H, m), 1.83–2.00
roaniline (20a) To a solution of 19a (1.39g, 5.84mmol) in (1H, m), 2.12–2.22 (2H, m), 3.68 (2H, brs), 5.96–6.09 (1H,
THF (20mL) were added acetic acid (2.01mL, 35.1mmol), m), 6.17–6.28 (1H, m), 6.64–6.74 (1H, m), 6.86–6.94 (1H, m),
2,4-dimethoxybenzaldehyde (1.17g, 7.01mmol) and sodium 6.96–7.05 (1H, m); MS (ESI/APCI dual) m/z: 220 [M+H]+.
triacetoxyborohydride (3.72g, 17.6mmol). The reaction mix-
4-(2-Cyclopentylethyl)-2-fluoroaniline (22g) 9-Borabicyclo-
ture was stirred overnight at room temperature. To the reac- [3,3,1]nonane (9-BBN) (THF solution, 0.5mol/L, 33mL,
tion mixture was added 1mol/L aqueous sodium hydroxide 16.5mmol) was added dropwise to a solution of vinylcyclo-
to adjust the pH to 8 to 9, and the mixture was extracted pentane (1.59g, 16.5mmol) in THF (7.5mL) at 0°C under a
with ethyl acetate. The organic layer was washed with brine, nitrogen atomosphere. The mixture was warmed gradually to
dried over anhydrous MgSO4, filtered, and concentrated under room temperature and stirred for 15h. To the reaction mixture
reduced pressure. The resulting residue was purified using a were added dichloro[1,1′-bis(diphenylphosphino)ferrocene]-
silica gel column eluted with 10% ethyl acetate–n-hexane to palladium(II) (367mg, 0.45mmol), 4-bromo-2-fluoroaniline
1
afford 20a (2.28g, quant.) as a purple oil: H-NMR (300MHz, (2.85g, 15.0mmol) and 3mol/L aqueous sodium hydroxide
CDCl3) δ: 0.84–1.06 (2H, m), 1.07–1.34 (3H, m), 1.36–1.87 (15mL, 45.0mmol), and the mixture was heated at reflux
(8H, m), 3.79 (3H, s), 3.83 (3H, s), 3.88 (2H, t, J=6.7Hz), temperature for 10h. After cooling to room temperature, the
4.01 (1H, brs), 4.23 (2H, s), 6.34–6.76 (5H, m), 7.17 (1H, d, mixture was diluted with ethyl acetate and filtered through a
J=8.2 Hz).
Compounds 20b and c were prepared from the correspond- pressure. Water was added to the residue, and the mixture
ing aniline in the same procedure described for 20a. was extracted with ethyl acetate. The organic layer was
pad of Celite®. The filtrate was concentrated under reduced
N-(2,4-Dimethoxybenzyl)-2-fluoro-4-(hexyloxy)aniline washed with brine, dried over anhydrous MgSO4, filtered, and
(20b) Light brown oil (yield 34%): 1H-NMR (300MHz, concentrated under reduced pressure. The resulting residue
CDCl3) δ: 0.84–0.96 (3H, m), 1.26–1.48 (6H, m), 1.64–1.81 was purified using a silica gel column eluted with 10% ethyl
(2H, m), 3.80 (3H, s), 3.84 (3H, s), 3.82–3.88 (2H, m), 4.01 (1H, acetate–n-hexane to afford 22g (3.01g, 88%) as a pale yellow
brs), 4.23 (2H, s), 6.32–6.78 (5H, m), 7.17 (1H, d, J=8.2 Hz).
oil. To a solution of 22g (3.01g) in ethyl acetate (75mL) was
N-(2,4-Dimethoxybenzyl)-2-fluoro-4-(3-phenylpropoxy)- added 4mol/L hydrogen chloride in ethyl acetate (8mL), and
aniline (20c) Pale yellow oil (yield 90%): 1H-NMR the mixture was stirred at room temperature for 3h. After the
(300MHz, CDCl3) δ: 1.99–2.11 (2H, m), 2.73–2.82 (2H, m), volatiles were removed by rotary evaporation, diethyl ether
3.79 (3H, s), 3.83 (3H, s), 3.82–3.89 (2H, m), 4.03 (1H, brs), was added to the residue, and the resulting precipitates were
4.23 (2H, s), 6.38–6.73 (5H, m), 7.14–7.22 (4H, m), 7.25–7.32 collected by filtration to afford the monohydrochloride salt of
1
(2H, m).
22g (2.34g, 64%) as a colorless powder: H-NMR (300MHz,
4-[(1E)-3-Cyclohexylprop-1-en-1-yl]-2-fluoroaniline (22e) DMSO-d6) δ: 1.02–1.17 (2H, m), 1.40–1.79 (9H, m), 2.53–2.60
Toluene (5mL) was added to 4-bromo-2-fluoroaniline (950mg, (2H, m), 6.99–7.04 (1H, m), 7.11–7.27 (2H, m); MS (ESI/APCI
5.00mmol), allylcyclohexane (1.15mL, 7.50mmol), palladium dual) m/z: 208 [M+H]+.
acetate (112mg, 0.50mmol), tris(2-methylphenyl)phosphine
4-[(1E)-3-Cyclopropylprop-1-en-1-yl]-2-fluoro-1-nitro-
(457mg, 1.50mmol) and triethylamine (2.09mL, 15.0mmol), benzene (24) Iodine (3.05g, 24.0mmol) and imidazole
and the mixture was stirred at reflux temperature for 10h. (1.63g, 24.0mmol) were added to a solution of triphenylphos-
After cooling to room temperature, the mixture was diluted phine (6.29g, 24.0mmol) in chloroform (50mL) at 0°C under a
with ethyl acetate and filtered through a pad of Celite®. The nitrogen atmosphere, and the mixture was stirred at the same
filtrate was concentrated under reduced pressure. The result- temperature for 15min. A solution of 2-cyclopropylethanol
ing residue was purified using a silica gel column eluted with (1.72g, 20.0mmol) in chloroform (50mL) was added dropwise
10% ethyl acetate–n-hexane to afford 22e (805mg, 69%) as to the reaction mixture, and the mixture was stirred at room
1
a pale yellow oil: H-NMR (300MHz, CDCl3) δ: 0.82–1.02 temperature for 3h. To the reaction mixture were added satu-
(2H, m), 1.03–1.44 (5H, m), 1.58–1.80 (4H, m), 2.01–2.10 (2H, rated aqueous sodium thiosulfate solution (60mL) and water

236
Chem. Pharm. Bull.
Vol. 64, No. 3 (2016)
(60mL), and the mixture was extracted with chloroform. The amide (28) Pyridine (2.22mL, 27.4mmol) was added to a
organic layer was concentrated under reduced pressure, and solution of 27 (3.80g, 13.7mmol) in chloroform (50mL), and
the resulting residue was purified using a silica gel column 15 (5.07g, 12.5mmol) was added to the mixture. The mixture
eluted with 100% n-hexane to afford the desired product was stirred at room temperature for 5h. The mixture was
(2.14g, 55%) as a pale yellow oil.
concentrated under reduced pressure, and the resulting resi-
Triphenylphosphine (2.86g, 10.9mmol) was added to a so- due was purified using a silica gel column eluted with 30 to
lution of the above product (2.14g, 10.9mmol) in acetonitrile 50% ethyl acetate–n-hexane to afford the target intermediate
(5mL), and the mixture was heated at reflux temperature for (6.52g, 81%) as a colorless amorphous.
15h. After cooling to room temperature, diethyl ether was
To a solution of the above intermediate (6.50g, 10.0mmol)
added to the mixture, and the resulting precipitates were col- in methanol (50mL) and ethyl acetate (50mL) was added
lected by filtration to afford the target intermediate (3.87g, triethylamine (1.68mL, 12.1mmol) and 10% palladium acti-
77%) as a colorless powder.
To
vated carbon (650mg), and the mixture was stirred under a
a
suspension of the above intermediate (3.83g, hydrogen atmosphere at room temperature for 4h. The mix-
8.36mmol) in THF (60mL) was added dropwise potassium ture was filtered through a pad of Celite®, and the filtrate was
hexamethyldisilazane (toluene solution, 0.5mol/L) (16.7mL, concentrated under reduced pressure. The resulting residue
8.36mmol) at 0°C under a nitrogen atmosphere, and the mix- was purified using a silica gel column eluted with 2 to 50%
ture was stirred at room temperature for 1h. After cooling ethyl acetate–n-hexane to afford 28 (4.94g, 87%) as a color-
1
to 0°C, a solution of 3-fluoro-4-nitrobenzaldehyde (1.23g, less amorphous: H-NMR (300MHz, DMSO-d6) δ: 2.90–3.10
7.27mmol) in THF (10mL) was added dropwise to the reac- (2H, m), 3.54 (3H, s), 3.69 (3H, s), 3.80–3.91 (2H, m), 4.54
tion mixture, and the mixture was stirred at room temperature (2H, s), 4.83–4.93 (2H, m), 6.37–6.53 (4H, m), 6.75–6.80 (1H,
for 1h. Saturated aqueous ammonium chloride solution was m), 7.04–7.13 (1H, m), 7.43–7.63 (3H, m), 10.05 (1H, brs); MS
added to the reaction mixture, and the mixture was extracted (ESI/APCI dual) m/z: 591 [M+Na]+.
twice with ethyl acetate. The organic layer was washed with
2-[2-(4-tert-Butylphenyl)ethyl]-N-(2,4-dimethoxybenzyl)-
brine, dried over anhydrous MgSO4, filtered, and concentrated N-[2-fluoro-4-(pentyloxy)phenyl]-1,2,3,4-tetrahydroiso-
under reduced pressure. The resulting residue was purified quinoline-6-sulfonamide (29a) To a solution of 28 (400mg,
using a silica gel column eluted with 25% ethyl acetate–n- 0.704mmol) in N,N-dimethylformamide (1mL) were added
1
hexane to afford 24 (1.42g, 88%) as a brown oil: H-NMR 1-iodopentane (119µL, 0.915mmol) and potassium carbon-
(300MHz, CDCl3) δ: 0.07–0.21 (2H, m), 0.45–0.60 (2H, m), ate (146mg, 1.06mmol), and the mixture was stirred at room
0.74–0.92 (1H, m), 2.14–2.30 (2H, m), 5.96–6.11 (1H, m), temperature overnight. To the reaction mixture was added
6.36–6.45 (1H, m), 7.12–7.31 (2H, m), 7.98–8.08 (1H, m).
saturated aqueous NaHCO3, and the mixture was extracted
4-(3-Cyclopropylpropyl)-2-fluoroaniline (25) To a solu- with ethyl acetate. The organic layer was filtered through a
tion of 24 (1.42g, 6.42mmol) in ethanol (40mL) was added phase separator and concentrated under reduced pressure. The
10% palladium activated carbon (150mg) at room tempera- resulting residue was purified using a silica gel column eluted
ture, and the mixture was stirred under a hydrogen atmo- with 15 to 30% ethyl acetate–n-hexane to afford the target in-
sphere for 5h. The reaction mixture was filtered through a termediate (307mg, 68%) as a colorless amorphous.
pad of Celite®, and the filtrate was concentrated under reduced
An aqueous solution (0.5mL) of potassium hydroxide
pressure. The resulting residue was purified using a silica (59mg, 1.06mmol) was added to a solution of the above in-
gel column eluted with 25% ethyl acetate–n-hexane to af- termediate (307mg, 0.48mmol) in ethanol (3mL), and the
1
ford 25 (709mg, 57%) as a brown oil: H-NMR (300MHz, mixture was stirred at room temperature for 5h. The reac-
CDCl3) δ: 0.04–0.02 (2H, m), 0.36–0.43 (2H, m), 0.60–0.73 tion mixture was concentrated under reduced pressure, and
(1H, m), 1.16–1.25 (2H, m), 1.60–1.72 (2H, m), 2.48–2.55 (2H, the resulting residue was diluted with water. The mixture
m), 6.65–6.77 (2H, m), 6.78–6.85 (1H, m); MS (ESI) m/z: 194 was extracted with ethyl acetate, and the organic layer was
[M+H]+.
filtered through a phase separator, concentrated under reduced
4-[(2,4-Dimethoxybenzyl)amino]-3-fluorophenol (27) To pressure to afford the crude product (252mg) as a colorless
a solution of 4-amino-3-fluorophenol (5.00g, 39.3mmol) in amorphous.
THF (120mL) were added acetic acid (16.2mL, 283mmol),
To a solution of the above product (125mg, 0.23mmol) in
2,4-dimethoxybenzaldehyde (7.84g, 47.2mmol) and sodium chloroform (1mL) were added a solution of 2-(4-(tert-butyl)-
triacetoxyborohydride (25.0g, 118mmol). The reaction mix- phenyl)acetaldehyde (50.0mg, 0.28mmol) in chloroform
ture was stirred at room temperature for 5h. After the vola- (1mL) and sodium triacetoxyborohydride (146mg, 0.69mmol),
tiles were removed by rotary evaporation, saturated aqueous and the mixture was stirred at room temperature for 19h. Sat-
NaHCO3 (500mL) was added to the residue, and the mixture urated aqueous NaHCO3 was added to the reaction mixture,
was extracted three times with ethyl acetate. The organic and the mixture was extracted with chloroform. The organic
layer was dried over anhydrous MgSO4, filtered, and con- layer was filtered through a phase separator and concentrated
centrated under reduced pressure. The resulting residue was under reduced pressure to afford 29a (162mg, quant.) as a col-
1
purified using a silica gel column eluted with 30 to 45% ethyl orless amorphous: H-NMR (600MHz, CDCl3) δ: 0.88–0.94
acetate–n-hexane to afford 27 (12.0g, 92%) as an orange oil: (3H, m), 1.31 (9H, s), 1.32–1.42 (4H, m), 1.69–1.77 (2H, m),
1H-NMR (300MHz, CDCl3) δ: 3.79 (3H, s), 3.83 (3H, s), 4.22 2.76–2.86 (4H, m), 2.86–2.92 (2H, m), 2.92–2.97 (2H, m), 3.56
(2H, s), 6.33–6.73 (5H, m), 7.16 (1H, d, J=8.1Hz); MS (ESI/ (3H, s), 3.74 (3H, s), 3.76 (2H, s), 3.84 (2H, t, J=6.6Hz), 4.65
APCI dual) m/z: 276 [M−H]−.
(2H, s), 6.24–6.27 (1H, m), 6.34–6.39 (1H, m), 6.42–6.49 (2H,
N-(2,4-Dimethoxybenzyl)-N-(2-fluoro-4-hydroxyphenyl)- m), 6.83–6.88 (1H, m), 7.09–7.14 (1H, m), 7.15–7.23 (3H, m),
2-(trifluoroacetyl)-1,2,3,4-tetrahydroisoquinoline-6-sulfon- 7.29–7.37 (2H, m), 7.43–7.52 (2H, m); MS (ESI/APCI dual)

Vol. 64, No. 3 (2016)
Chem. Pharm. Bull.
237
m/z: 703 [M+H]+.
(1H, s), 10.67 (1H, brs); HR-MS (ESI/APCI dual) m/z: Calcd
Compounds 29b and c were prepared from 28 using 1-iodo- for C34H45FN2O3S [M+H]+, 581.3208; Found 581.3204.
heptane and 1-iodooctane, respectively, in the same procedure
2-[2-(4-tert-Butylphenyl)ethyl]-N-[2-fluoro-4-(octyloxy)-
described for 29a.
phenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide (7)
1
2-[2-(4-tert-Butylphenyl)ethyl]-N-(2,4-dimethoxybenzyl)- Colorless amorphous (total yield 33%): H-NMR (600MHz,
N-[2-fluoro-4-(heptyloxy)phenyl]-1,2,3,4-tetrahydroiso- DMSO-d6) δ: 0.81–0.90 (3H, m), 1.22–1.32 (8H, m), 1.27 (9H,
quinoline-6-sulfonamide (29b) Colorless amorphous (total s), 1.34–1.39 (2H, m), 1.62–1.70 (2H, m), 3.00–3.48 (7H, m),
1
yield 36%): H-NMR (300MHz, CDCl3) δ: 0.83–0.95 (3H, m), 3.76–3.85 (1H, m), 3.88–3.93 (2H, m), 4.40–4.51 (1H, m),
1.18–1.44 (8H, m), 1.32 (9H, s), 1.67–1.80 (2H, m), 2.76–3.02 4.67–4.78 (1H, m), 6.66–6.82 (2H, m), 7.01–7.09 (1H, m),
(8H, m), 3.57 (3H, s), 3.75 (3H, s), 3.78 (2H, s), 3.81–3.89 7.20–7.25 (2H, m), 7.34–7.44 (3H, m), 7.53–7.64 (2H, m), 9.90
(2H, m), 4.67 (2H, s), 6.25–6.29 (1H, m), 6.34–6.41 (1H, m), (1H, s), 10.80 (1H, brs); HR-MS (ESI/APCI dual) m/z: Calcd
6.43–6.53 (2H, m), 6.84–6.93 (1H, m), 7.08–7.25 (4H, m), for C35H47FN2O3S [M+H]+; 595.3364. Found 595.3346.
7.31–7.39 (2H, m), 7.44–7.55 (2H, m); MS (ESI) m/z: 731
Solubility An excess amount of each compound was
[M+H]+.
added to FeSSIF (Fed State Simulated Intestinal Fluid,20) pH
2-[2-(4-tert-Butylphenyl)ethyl]-N-(2,4-dimethoxybenzyl)- 5.0) and shaken on a shaker (model SR-2s; Yamato Kagaku) at
N-[2-fluoro-4-(octyloxy)phenyl]-1,2,3,4-tetrahydroisoquin- 25°C for 2h and keep on 37°C for 22h on a water bath (model
oline-6-sulfonamide (29c) Colorless amorphous (total yield LT-10s; Yamato Kagaku).
42%): 1H-NMR (600MHz, CDCl3) δ: 0.85–0.91 (3H, m),
The suspensions were centrifuged at 3000 and 11000rpm
1.23–1.35 (8H, m), 1.31 (9H, s), 1.36–1.43 (2H, m), 1.69–1.75 for 10min, and the supernatant was diluted with 50% aqueous
(2H, m), 2.76–2.99 (8H, m), 3.56 (3H, s), 3.74 (3H, s), 3.77 acetonitrile or 50% aqueous methanol solution. The concen-
(2H, s), 3.82–3.86 (2H, m), 4.66 (2H, s) 6.24–6.28 (1H, m) trations were measured by HPLC. The HPLC analysis was
6.33–6.39 (1H, m), 6.43–6.51 (2H, m), 6.82–6.91 (1H, m), performed using a Shimadzu HPLC system composed of a
7.10–7.14 (1H, m) 7.16–7.23 (3H, m), 7.31–7.36 (2H, m), LC-20AD, SPD-20A and SIL-20AC. The conditions for HPLC
7.44–7.53 (2H, m); MS (ESI) m/z: 745 [M+H]+.
were as follows: mobile phase, 0.1% phosphoric acid aqueous
2-[2-(4-tert-Butylphenyl)ethyl]-N-[2-fluoro-4-(pentyloxy)- solution–acetonitrile; flow rate, 0.8mL/min; column, reversed-
phenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide (4) phase (Shimpack XR-ODS, 2.2µm, 2.0×75mm; Shimadzu) at
To a solution of 29a (162mg, 0.230mmol) in chloroform 40°C; and detection wavelength, 210nm.
(4mL) was added trifluoroacetic acid (880µL), and the mix-
Metabolic Stability in Liver Microsomes Test sub-
ture was stirred overnight at room temperature. Saturated stances (5µM) were incubated at 37°C in 1mg/mL human
aqueous NaHCO3 was added to the reaction mixture, and the or mouse microsomes supplemented with 1.5mM glucose-
mixture was extracted with ethyl acetate. The organic layer 6-phosphate, 0.16mM β-nicotinamide-adenine dinucleotide
was washed with brine, filtered through a phase separator, and phosphate, 0.18units/mL glucose-6-phosphate dehydrogenase,
concentrated under reduced pressure. The resulting residue 2.4mM magnesium chloride and 69mM potassium chloride in
was purified using a silica gel column eluted with 30 to 70% 250mM phosphate buffer (pH 7.4). Concentrations of the test
ethyl acetate–n-hexane to afford 4 (20mg, 16%) as a colorless compounds were determined by LC-MS/MS. The metabolized
amorphous.
% was determined by comparing a peak area of the test sub-
To a solution of 4 (20mg, 0.036mmol) in ethyl acetate stance at 15min incubation with the peak area at 0min.
(0.5mL) was added 4mol/L hydrogen chloride in ethyl acetate
(90 µL), and the mixture was stirred at room temperature for
5min. After the volatiles were removed by rotary evapora-
Biology
MGAT2 Assay
This assay detects CoA, a product of the MGAT2-cat-
tion, ethyl acetate was added to the residue, and the resulting alyzed deacylation of oleoyl-CoA. The free thiol of CoA
precipitates were collected by filtration to afford the mono- can react with 7-diethylamino-3-(4′-maleimidylphenyl)-4-
hydrochloride salt of 4 (3.0mg, 14%) as a colorless powder: methylcoumarin (CPM),
a pro-fluorescent coumarin ma-
1H-NMR (600MHz, DMSO-d6) δ: 0.84–0.92 (3H, m), 1.27 leimide derivative that becomes fluorescent upon reaction with
(9H, s), 1.29–1.38 (4H, m), 1.64–1.70 (2H, m), 3.02–3.50 (7H, thiols.
m), 3.76–3.84 (1H, m), 3.91 (2H, t, J=6.4Hz), 4.41–4.48 (1H,
In this assay, recombinant human and mouse MGAT2 were
m), 4.68–4.77 (1H, m), 6.67–6.73 (1H, m), 6.75–6.81 (1H, m), produced in the baculovirus expression system. The MGAT2
7.02–7.09 (1H, m), 7.23 (2H, d, J=7.8Hz), 7.35–7.44 (3H, m), activity was determined as follows: Assay buffer [final con-
7.54–7.61 (2H, m), 9.91 (1H, s), 10.79 (1H, brs); HR-MS (ESI/ centration: 100mM Tris–HCl (pH 7.5), 100mM sucrose, 5mM
APCI dual) m/z: Calcd for C32H41FN2O3S [M+H]+, 553.2895; MgCl2] and recombinant MGAT2 and test compound were
Found 553.2883.
Compounds 6 and 7 were prepared from 29b and c, respec- strate solution [final concentration: 5.3µM oleoyl-CoA, 0.78µM
tively, in the same procedure described for 4. 2-oleoylglycerol, 7.5µM phosphatidylcholine] were added to
added to each well of a 96-well black plate (Corning). Sub-
2-[2-(4-tert-Butylphenyl)ethyl]-N-[2-fluoro-4-(heptyloxy)- start the reaction, which was allowed to proceed for 20min.
phenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide (6) The reaction was terminated upon the addition of CPM with
1
Colorless amorphous (total yield 29%): H-NMR (600MHz, final concentration 5µM. The plates were sealed, incubated
DMSO-d6) δ: 0.83–0.89 (3H, m), 1.24–1.32 (6H, m), 1.27 for 20min. Fluorescence that emits at 460nm when excited at
(9H, s), 1.33–1.40 (2H, m), 1.63–1.70 (2H, m), 3.04–3.49 (7H, 380nm was counted on a SpectraMax Plate Reader (Molecu-
m), 3.77–3.85 (1H, m), 3.88–3.94 (2H, m), 4.40–4.51 (1H, m), lar Devices, LLC, Japan). Oleoyl-CoA, 2-oleoylglycerol and
4.66–4.78 (1H, m), 6.66–6.81 (2H, m), 7.02–7.10 (1H, m), phosphatidylcholine were obtained from Sigma-Aldrich and
7.19–7.27 (2H, m), 7.33–7.45 (3H, m), 7.52–7.63 (2H, m), 9.90 CPM from Life Technologies.

238
Chem. Pharm. Bull.
J. Biol. Chem., 278, 13611–13614 (2003).
Vol. 64, No. 3 (2016)
Oral Lipid Tolerance Test in Mice
6) Yen C.-L. E., Cheong M.-L., Grueter C., Zhou P., Moriwaki J.,
Wong J. S., Hubbard B., Marmor S., Farese R. V. Jr., Nat. Med., 15,
442–446 (2009).
7) Nelson D. W., Gao Y., Spencer N. M., Banh T., Yen C.-L. E., J.
Lipid Res., 52, 1723–1732 (2011).
8) Yen C.-L. E., Nelson D. W., Yen M.-I., J. Lipid Res., 56, 489–501
(2015).
9) Nakamura K., Nakagawa H., Kan Y., PCT Int. Appl., WO
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PCT Int. Appl., WO 2013112323 (2013) [Chem. Abstr., 159, 305466
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15) Berger R., Blizzard T. A., Campbell B. T., Chen H. Y., Debenham
J. S., Dewnani S. V., Dubois B., Gude C., Guo Z., Harper B., Hu
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C57BL/6J male mice (10 weeks old, n=7 to 8) were used.
To inhibit the clearance of plasma triacylglycerol, we admin-
istered 100µL of the surfactant tyloxapol (10% in phosphate
buffered saline (PBS)) through a tail vein. Immediately, we
administered orally 4mL/kg of test compound or 0.5% meth-
ylcellulose solution as vehicle. After 30min, we had chal-
lenged orally with 4.4mL/kg of triolein containing 0.1mCi/
mL 3H triolein. We collected blood from tail vein at 2, 3 and
4h after lipid challenge and counted scintillation. Efficacy was
calculated as the percentage reduction in area under the curve
of plasma scintillation for 4h (AUC0–4h) compared to vehicle-
treated control animals. Bartlett’s test was applied to evaluate
the equality of variance for AUC0–4h between vehicle and
treated mice. If the equality of variance isn’t statistically sig-
nificant in Bartlett’s test, Dunnett’s test is applied to establish
statistical significance of AUC0–4h between vehicle and treated
mice. If the equality of variance is statistically significant in
Bartlett’s test, Steel’s test is applied to establish statistical
significance of AUC0–4h between vehicle and treated mice.
Student’s t-test was used to establish statistical significance in
the comparison between 2 groups.
Acknowledgments We are grateful to Dr. N. Ohtake and
Dr. T. Yoshizumi for their helpful and critical reading of the
manuscript. We acknowledge the analytical support (the high-
resolution mass spectra) by Mr. A. Higuchi.
16) Barlind J. G., Buckett L. K., Crosby S. G., Davidsson Ö., Emtenäs
H., Ertan A., Jurva U., Lemurell M., Gutierrez P. M., Nilsson K.,
O’Mahony G., Petersson A. U., Redzic A., Wågberg F., Yuan Z.-Q.,
Bioorg. Med. Chem. Lett., 23, 2721–2726 (2013).
Conflict of Interest The authors declare no conflict of
interest.
17) Recently N-phenylindoline-5-sufonamide derivatives as MGAT2
inhibitor has been reported. Sato K., Takahagi H., Yoshikawa T.,
Morimoto S., Takai T., Hidaka K., Kamaura M., Kubo O., Adachi
R., Ishii T., Maki T., Mochida T., Takekawa S., Nakakariya M.,
Amano N., Kitazaki T., J. Med. Chem., 58, 3892–3909 (2015).
18) Sato K., Takahagi H., Kubo O., Hidaka K., Yoshikawa T., Kam-
aura M., Nakakariya M., Amano N., Adachi R., Maki T., Take K.,
Takekawa S., Kitazaki T., Maekawa T., Bioorg. Med. Chem., 23,
4544–4560 (2015).
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