Identification of 2-[2-(4-tert-butylphenyl)ethyl]-N-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide (29) as an orally available MGAT2 inhibitor

Article abstract of DOI:10.1016/j.bmc.2015.06.065

MGAT2 (monoacylglycerol acyltransferase 2) is expected to be an attractive target for the drug treatment of obesity, diabetes, and other disease. We describe our exploration and structure-activity relationship (SAR) study of 2,3-dihydro-1H-isoindole-5-sul

Full text of DOI:10.1016/j.bmc.2015.06.065

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of 2-[2-(4-tert-butylphenyl)ethyl]-N-(4-fluorophenyl)-
1,2,3,4-tetrahydroisoquinoline-6-sulfonamide (29) as an orally
available MGAT2 inhibitor
a
a
b
b
Tsuyoshi Busujima a, , Hiroaki Tanaka , Yoshihisa Shirasaki , Eiji Munetomo , Masako Saito ,
⇑
Kiyokazu Kitano b, Toshiya Minagawa c, Koji Yoshida c, Naoto Osaki d, Nagaaki Sato a
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, Japan
c Drug Safety and Pharmacokinetics Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku, Saitama 331-9530, Japan
d Pharmaceutical Science Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku, Saitama 331-9530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
MGAT2 (monoacylglycerol acyltransferase 2) is expected to be an attractive target for the drug treatment
of obesity, diabetes, and other disease. We describe our exploration and structure–activity relationship
(SAR) study of 2,3-dihydro-1H-isoindole-5-sulfonamide derivatives. In this study, we identified 29 as
an orally available inhibitor of MGAT2 through optimization especially in terms of solubility. This
compound exhibited moderate potency in the enzyme inhibitory assay (IC50 = 1522 nM) and significant
suppression of fat absorption (57% inhibition) in mice oral lipid tolerance test.
Received 2 May 2015
Revised 23 June 2015
Accepted 24 June 2015
Available online xxxx
Keywords:
Monoacylglycerol acyltransferase
MGAT2
Ó 2015 Elsevier Ltd. All rights reserved.
Fat absorption
Oral lipid tolerance test
Tetrahydroisoquinoline
1. Introduction
abnormalities. After these mice were orally given dietary oil (oral
lipid tolerance test), plasma TG levels of these mice were signifi-
Dietary-derived fats are degraded in the gastrointestinal tract
and then taken up by small-intestinal epithelial cells. After re-syn-
thesis into neutral fats in the cells,1 the neutral fats are packaged
into chylomicrons, secreted into the lymphatics, and taken up into
body. Acyl CoA:monoacylglycerol acyltransferase (MGAT), which
catalyzes the synthesis of diacylglycerol from monoacylglycerol
and acyl-CoA, plays a crucial role in triglyceride (TG) re-synthesis
in the small intestine.2 In 2009, MGAT2 deficient mice were
reported and showed resistance to obesity, glucose intolerance,
cantly lower than that of the wild type, suggesting a reduced rate
of fat absorption. In addition, these mice demonstrated increased
oxygen consumption without high-fat feeding, and MGAT2 was
expected to modulate energy expenditure.5 These phenotyping
studies have shown that inhibition of MGAT2 could be an
attractive mechanism for the reduction of fat absorption and the
treatment of obesity and associated metabolic disorders.
Structurally distinct MGAT inhibitors6–9 disclosed previously
are depicted in Figure 1. These compounds (1–4), which have been
disclosed in patents, were reported to show good potency with IC50
values of nM order for MGAT inhibition in an in vitro enzymatic
assay. However, there was no in vivo data as a MGAT2 inhibitor.
On the other hand, AstraZeneca’s compound 5 reported in the lit-
erature demonstrated potent MGAT2 inhibitory activity with a
pIC50 value of 8.2 and a significant reduction of plasma TG concen-
tration after an oral administration at a dose of 150 mg/kg in mice
lipid tolerance test.10
and hypercholesterolemia induced by
a
high fat diet.3
Furthermore, it was reported that high-fat feeding increased
MGAT2 expression and its activity in the small intestine,4 and
MGAT2 knock-out mice were viable and fertile without apparent
Abbreviations: MGAT2, monoacylglycerol acyltransferase 2; TG, triglyceride;
FeSSIF, Fed State Simulated intestinal Fluid; MS, metabolic stability; TFAA,
trifluoroacetic anhydride; EDCÁHCl, 1-(3-dimethylaminopropyl)-3-ethylcarbodi-
imide hydrochloride; HOBtÁH2O, 1-hydroxy-1H-benzotriazole hydrate; TFA, triflu-
oroacetic acid.
Our hit compound 6 identified by our high throughput screen-
ing campaign exhibited moderate potency against both human
and mice MGAT2 enzymes, while its aqueous solubility was very
⇑
Corresponding author. Tel.: +81 48 669 3107; fax: +81 48 652 7254.
E-mail address: t-busujima@so.taisho.co.jp (T. Busujima).
http://dx.doi.org/10.1016/j.bmc.2015.06.065
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Busujima, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.06.065

2
T. Busujima et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
N
S
N
O
O
O
O
COOEt
H
N
S
N
O
N
N
S
N
O
N
H
N
N
N
N
H
F
F
O
O
CF3
F
1 (Dainippon-sumitomo)
2 (Merck-banyu)
3 (BMS)
F
F
N
O
O
O
H
O O
N
S
NH
S
HN
O
S
N
N
H
H
N
O
O
F
F
F
N
F
H
O
4 (Eli Lilly)
5 (AstraZeneca)
6
hMGAT2 : 594 nM
mMGAT2 : 1310 nM
Figure 1. Chemical structures of reported MGAT2 inhibitors and our HTS hit compound 6.
poor (vide infra). The identification of hit compound 6 promoted us
to explore its structure-activity relationship and address the poor
solubility issue, because it can be said to be a novel chemical class
compared to other reported MGAT2 inhibitors except for
compound 5.
Herein, we describe the synthesis, structure–activity relation-
ship (SAR) study, and improvement of the aqueous solubility of
2,3-dihydro-1H-isoindole-5-sulfonamide derivatives, leading to
the identification of an orally available inhibitor of MGAT2.
chlorosulfonylation to afford 9. The sulfonyl chloride 9 was
coupled with the desired anilines to give 10a–10j. Sulfonamide
intermediates 10a–10j were exposed to a basic condition to
remove trifluoroacetyl group, followed by coupling with 4-tert-
butylphenyl isocyanate to give isoindoline urea derivatives 6 and
12b–12j.
The secondary amine 11a was condensed with (4-tert-butyl-
phenyl)acetic acid to yield the amide compound 13. N-Alkyl
derivative 14 was prepared by reduction of 13 using borane-THF
complex as a reducing reagent.
The preparation of compounds 19–21 were shown in Scheme 2.
The N-protected sulfonamide derivative 17 was obtained by con-
densation of the sulfonyl chloride 9 and the corresponding aniline
16 protected by 2,4-dimethoxybenzyl group. The trifluoroacetyl
group of 17 was removed by hydrolysis under a basic condition,
2. Chemistry
Syntheses of 2,3-dihydro-1H-isoindole-5-sulfonamide deriva-
tives were shown in Scheme 1. The secondary amine of 7 was
protected
with
trifluoroacetic
anhydride,
followed
by
O
S
O
O O
R1
S
CF3
O
CF3
O
CF3
O
a
b
c
N
Cl
NH
N
N
N
H
7
10a - 10j
8
9
O
O
O
O
S
d
R1
S
R1
e
HN
N
N
N
NH
H
H
O
11a - 11j
6, 12b-12j
F
F
F
O
O
g
O
O
S
O O
f
HCl
N
S
S
N
H
N
N
N
O
NH
H
H
11a
13
14
Scheme 1. Preparation of isoindoline derivatives. Reagents and conditions: (a) TFAA, pyridine, CHCl3, rt, 99%; (b) ClSO3H, CHCl3, rt, 71%; (c) R1NH2, pyridine, CHCl3, rt, 34–
95%; (d) KOH aq., EtOH, rt; (e) 1-(tert-butyl)-4-isocyanatobenzene, Et3N, CHCl3-DMSO, rt, 32–84% in two steps; (f) 2-(4-(tert-butyl)phenyl)acetic acid, EDCÁHCl, HOBtÁH2O,
CHCl3, rt, 29%; (g) BH3ÁTHF, THF, 90 °C, then 4N HCl/EtOAc, EtOAc, rt, 30%.
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T. Busujima et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
HO
F
HO
F
a
NH
NH2
MeO
OMe
15
16
HO
F
O
S
O
O
O
S
CF3
O
b
CF3
O
c
Cl
N
N
N
MeO
OMe
9
17
HO
F
O
O
O
F
S
R1
d
HN
O
O
N
N
S
HN
O
N
H
O
N
MeO
OMe
18
19-21
Scheme 2. Preparation of aniline and sulfonamide derivatives. Reagents and conditions; (a) 2, 4-dimethoxybenzaldehyde, NaBH(OAc)3, AcOH, THF, rt, 92%; (b) 16, pyridine,
CHCl3, rt, 52%; (c) KOH aq, EtOH, rt, then 1-(tert-butyl)-4-isocyanatobenzene, Et3N, CHCl3-DMSO, rt, 49%; (d) R1X, K2CO3, DMF, rt, then 10% TFA, CHCl3, rt, 46–68%.
followed by treatment with 4-tert-butylphenyl isocyanate to afford
the intermediate 18. Alkylation of 18 with the corresponding alkyl
halides and subsequent deprotection of the 2,4-dimethoxybenzyl
group by treating with TFA produced O-alkyl derivatives 19–21.
As shown in Scheme 3, tetrahydroisoquinoline derivatives were
synthesized by using tetrahydroisoquinoline 22 as a starting mate-
rial in a similar manner. Protection of the secondary amine and
subsequent chlorosulfonylation reaction gave 24 and 25 as a mix-
ture of regioisomer,11 which were isolated by silica gel
chromatography. Further modification of these intermediates (24
and 25) was carried out as described for the preparation of 14 to
afford 27 and 29, respectively.
3. Result and discussion
All compounds were evaluated for their MGAT2 inhibitory
activity using a human recombinant enzyme assay (hMGAT2). As
O
O
S
O
O
O
S
a
b
Cl
N
CF3
+
Cl
N
CF3
N
NH
CF3
O
F
O
22
23
24
25
O
F
O
S
O
O
O
O
d, e, f
d, e, f
O
O
S
c
S
Cl
N
CF3
N
N
CF3
N
N
H
H
HCl
24
25
26
27
29
F
F
O
O
O
S
O
O
O
S
HCl
N
H
S
c
Cl
N
H
N
N
CF3
N
CF3
O
O
28
Scheme 3. Preparation of tetrahydroisoquinoline derivatives. Reagents and conditions; (a) TFAA, pyridine, CHCl3, rt, 99%; (b) ClSO3H, CHCl3, rt; (c) 4-fluoroaniline, pyridine,
CHCl3, rt, 92%; (d) KOH aq., EtOH, rt; (e) 2-(4-(tert-butyl)phenyl)acetic acid, EDCÁHCl, HOBtÁH2O, CHCl3, rt; (f) BH3ÁTHF, THF, 90 °C, then 4N HCl/EtOAc, EtOAc, rt, 36–50% in
three steps.
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T. Busujima et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
sulf onamide phenyl ring moiety
F
O
O
S
HN
O
N
N
H
isoindoline scaf f old and urea moiety
6
hMGAT2 : 594 nM
mMGAT2 : 1310 nM
µ
Solubility (water) : < 0.07 g/mL
Figure 2. MGAT2 inhibitory activity and solubility of HTS hit compound 6.
Table 1
Structure–activity
derivatives
shown in Figure 2, HTS hit compound 6 showed a moderate
MGAT2 inhibition, but it was found that its aqueous solubility
was extremely low. Hence, our strategy for structural modification
was focused on improving its aqueous solubility in parallel with
enhancing inhibitory activity against MGAT2 enzyme. Therefore,
firstly, we examined the effect of substituents at the ortho position
of the sulfonamide phenyl ring moiety on the inhibition activity
and aqueous solubility, and secondly modified the isoindoline
scaffold and the urea moiety.
relationships
of
2,3-dihydro-1H-isoindole-5-sulfonamide
R1
R2
O
O
S
HN
N
N
H
O
R1
R2
hMGAT2 IC50 (nM)
a
Compound
Our preliminary SAR studies suggested that the substituents at
the ortho and/or para positions (R1 and R2) of the aryl ring were
tolerable in terms of the enzyme inhibition. This result was closely
similar to those of sulfonamide compounds reported by
AstraZeneca,10 suggesting that both inhibitors would occupy the
same binding pockets in MGAT2. At first, we introduced several
kinds of substituents at the ortho and para positions of the phenyl
ring. The results on the enzyme inhibition activity were shown in
Table 1.
Introduction of fluorine atom at the ortho position, 2,4-difluoro
phenyl derivative (12b), was tolerable, but 2-chloro-4-fluoro
phenyl and 2-ethyl-4-fluoro phenyl derivatives (12c and 12d,
respectively) resulted in 2 to 5 fold decrease of the potency.
However, it was found that 4-fluoro-2-methoxy phenyl derivative
(12e) was 3-fold more potent than compound 6. Extension of the
length of the alkoxy group such as ethoxy and n-propoxy resulted
in decrease of the potency (12f and 12g). 2,4-Dimethoxy phenyl
derivative (12h) showed a significant decrease compared to 12e,
but interestingly 2-fluoro-4-methoxy phenyl analog (12i) was as
potent as 12e. Replacement of the methoxy group of 12i with the
ethoxy group (19) led to a significant reduction in the enzyme inhi-
bitory activity. This result was the same as the extension of alkoxy
group at the ortho position. While introduction of n-propoxy group
at the ortho position resulted in more decrease of the potency than
the corresponding ethoxy analog (12f vs 12g), the inhibitory
activity of n-propoxy analog at the para position was better than
that of ethoxy analog (19 vs 20). Moreover, the longer the length
of the alkoxy chain was extended, the more it improved the
potency (21 and 12j).
Selected isoindoline derivatives (6, 12e, 12i, and 12j) were
evaluated for their solubility in water and FeSSIF12 (Table 2). As a
result, the solubility of compound 12e was improved in compar-
ison with compound 6, while 12i and 12j showed no significant
improvement in terms of the aqueous solubility. These results
suggested that introduction of a small and hydrophilic group at
the ortho position might have a positive effect on the improvement
of aqueous solubility.
The exposure level in plasma of 12e after oral administration in
mice at a dose of 100 mg/kg was measured. As shown in Figure 3,
the plasma exposure of 12e was still low (Cmax: 94.3 ng/mL,
AUC0–4 h: 134 ng h/mL) partly because of its insufficient solubility
6
F
F
F
F
F
F
F
H
F
Cl
Et
MeO
EtO
n-PrO
MeO
F
F
F
F
F
594
685
1110
2490
168
12b
12c
12d
12e
12f
12g
12h
12i
19
733
2300
3160
164
5200
1980
547
MeO
MeO
EtO
n-PrO
n-BuO
20
21
12j
n-PenO
147
a
Values are the means of two or more separate experiments.
Table 2
Inhibitory activity for MGAT2 enzyme, solubility, and metabolic stability in micro-
somes of selected compounds
MSc (h/m) (%)
a
Compound
hMGAT2 IC50 (nM)
Solubility (
lg/mL)
[water/FeSSIF]b
6
594
168
164
147
<0.07/NTd
0.607/28.3
<0.06/0.783
<0.06/10.2
12.3/70.9
43.1/60.6
20.6/51.1
0.1/12.6
12e
12i
12j
a
b
c
Values are the means of two or more separate experiments.
Fed State Simulated intestinal Fluid, pH 5.0.
Metabolized percentage after 15 min of incubation with human/mouse liver
microsomes.
d
NT: not tested.
(low absorption) and/or its susceptibility to metabolism by mice
microsomes.
Based on this result, our chemical efforts were focused on
modifying the isoindoline scaffold and the urea linker moiety of
compound 6. Table 3 showed the effect of the modifications on
the inhibition activity and the solubility.
Although replacement of the urea linker with the amide linker
did not affect the solubility in water (6 vs 13), ethylene linker
derivative 14 showed dramatic increase of the solubility with
4-fold decrease of the potency. Keeping the ethylene as a linker,
replacement of the isoindoline scaffold with tetrahydroisoquino-
line scaffold led to identification of more water-soluble analog,
6-tetrahydoroisoquinoline compound 29 with as potent as 14.
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T. Busujima et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Table 4
Inhibitory activity for MGAT2 enzyme, metabolic stability in microsomes, and
solubility of compound 29
Compound
hMGAT2
IC50 (nM)
mMGAT2
IC50 (nM)
MSb (h/m)
(%)
Solubility (lg/mL)
water/FeSSIFc
641/385
a
a
29
1522
1170
14.6/47.8
a
Values are the means of two or more separate experiments.
Metabolized percentage after 15 min of incubation with human/mouse liver
b
microsomes.
c
Fed State Simulated intestinal Fluid, pH 5.0.
Figure 3. Plasma exposure for 12e after orally dosing at 100 mg/kg as a 0.5% methyl
cellulose (MC) solution to mice; n = 3 per group. Values are means SEM.
Table 3
Inhibitory activity for MGAT2 and solubility of 2,3-dihydro-1H-isoindole-5-sulfon-
amide analogs with the linker and scaffold modification of compound 6
F
O
O
S
m
n
N
N
linker
H
Compound
m
n
Linker
hMGAT2
IC50 (nM)
Solubility (lg/mL)
water/FeSSIFb
a
6
13
14
27
29
0
0
0
0
1
0
0
0
1
0
–CONH–
594
8070
2120
3060
1522
<0.07/NTc
<0.11/NTc
270/NTc
NTc/NTc
Figure 5. Fat absorption inhibition of compound 29; n = 7 per group. Values are
means SEM (##, P <0.01 versus vehicle group in the Steel’s test).
–COCH2–
–CH2CH2–
–CH2CH2–
–CH2CH2–
641/385
a
b
c
Values are the means of two or more separate experiments.
Fed State Simulated intestinal Fluid, pH 5.0.
NT: not tested.
much higher than that of 12e (Cmax of 29: 1200 ng/mL, AUC0–4
of 29: 1808 ng h/mL).
h
Compound 29 was evaluated for its inhibition activity for
mouse MGAT2 enzyme (mMGAT2) and metabolic stability in
human and mouse microsomes (Table 4). The inhibition activity
in the mouse recombinant enzyme assay was as potent as in
human. In addition, compound 29 was metabolically more stable
in human microsomes than 12e, while its stability in mouse
microsomes was not significantly improved in comparison with
that of 12e. This result suggested that the solubility was one of
the main factors for improvement of plasma exposure in mice.
Compound 29 was evaluated in mice oral lipid tolerance test. As
shown in Figure 5, the AUC0–4 h level of TG was significantly lower
than that of the vehicle group after an oral administration of 29 at a
dose of 100 mg/kg, indicating that 29 significantly suppressed fat
absorption.
4. Conclusion
In summary, HTS hit compound 6 was modified to improve its
aqueous solubility with enhancing the potency against MGAT2
enzyme. The introduction of substituents at the ortho position of
sulfonamide phenyl ring moiety resulted in increase of the
potency, but having a low impact on improvement of its solubility.
However, modification of the isoindoline scaffold and the urea
moiety led to the identification of 6-tetrahydroisoquinoline com-
pound 29, of which the solubility and plasma exposure levels were
improved drastically. Compound 29 demonstrated the significant
reduction of plasma TG levels in mice oral lipid tolerance test after
Figure 4. Plasma exposure for 29 after orally dosing at 100 mg/kg as a 0.5% methyl
cellulose (MC) solution to mice; n = 3 per group. Values are means SEM.
Having obtained more soluble MGAT2 inhibitor 29, we tested
its plasma exposure level after oral administration in mice at a
dose of 100 mg/kg (Fig. 4). As expected, its exposure level was
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T. Busujima et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
an oral administration. Optimization of 29 to discover more potent
MGAT2 inhibitors will be reported in due course.
concentrated under reduced pressure to afford crude 9 (5.15 g,
71%) as a brown oil: 1H NMR (300 MHz, CDCl3) d 5.04 (s, 2H),
5.16 (s, 2H), 7.53–7.65 (m, 1H), 7.93–8.09 (m, 2H).
5. Experimental
5.1. Chemistry
5.1.3. N-(4-Fluorophenyl)-2-(trifluoroacetyl)-2,3-dihydro-1H-
isoindole-5-sulfonamide (10a)
Pyridine (2.57 mL, 31.9 mmol) and N,N-dimethyl-4-aminopy-
ridine (195 mg, 1.59 mmol) were added to a solution of 4-fluo-
roaniline (1.86 g, 16.7 mmol) in chloroform (46 mL) at room
temperature. The mixture was cooled to 0 °C, and 9 (5.00 g,
15.9 mmol) was added to the mixture. The reaction mixture was
warmed to room temperature and stirred for 15 h. The reaction
mixture was concentrated under reduced pressure, and the residue
was diluted with ethyl acetate. The mixture was washed with
1 mol/L hydrochloric acid, and then brine. The organic layer was
dried over anhydrous MgSO4, filtered, and concentrated under
reduced pressure. To the resulting residue was added diethyl ether,
and the resulting powder was collected by filtration to afford 10a
All commercially available starting materials and reagents were
used without further purification unless otherwise noted. Thin
layer chromatography was performed to monitor reactions using
Merck silica gel 60 F254 plates or Fuji Silysia chromatorex NH
plates. Silica gel column chromatography was performed using
WakogelÒ C–200, or NH–silica gel Fuji Silysia chromatorexÒ
DM1020, or an appropriately sized pre-packed silica cartridge on
a Biotage system. 1H NMR spectra were recorded on a Varian
Instruments INOVA–300 spectrometer at 300 MHz or a JEOL
ECA–600 spectrometer at 600 MHz, and are referenced to an inter-
nal standard of trimethylsilane (TMS, d 0.00 ppm). Chemical shifts
are reported in parts per million (ppm) in the indicated solvent.
Multiplicity was defined as s (singlet), d (doublet), t (triplet), q
(quartet), dd (double doublet), m (multiplet), br s (broad singlet),
br d (broad doublet), or br t (broad triplet). Mass spectra (MS) were
recorded on a Shimadzu LCMS-IT-TOF mass spectrometer or a
Shimadzu LCMS-2010EV mass spectrometer with an electrospray
ionization (ESI)/atmospheric pressure chemical ionization (APCI)
Dual source. High resolution mass spectra (HRMS) were recorded
on a Shimadzu LCMS-IT-TOF mass spectrometer with an electro-
spray ionization (ESI)/atmospheric pressure chemical ionization
(APCI) Dual source. Elemental analyses were performed using a
Perkin–Elmer 2400II or a Yanaco MT–6, and the results were
within 0.4% of the calculated values. The preparative HPLC purifi-
cation condition was as follows: Gilson preparative HPLC system;
column waters ODS sunfire, 50 mm  30 mm; eluent A,
water + 0.1% CF3CO2H; eluent B, acetonitrile + 0.1% CF3CO2H; 10%
B up to 95% B in 12 min; Flow rate 40 mL/min.
(5.85 g, 95%) as
DMSO-d6) 4.86 (s, 2H), 5.06 (s, 2H), 7.04–7.14 (m, 4H),
a
pale pink powder: 1H NMR (300 MHz,
d
7.47–7.59 (m, 1H), 7.64–7.72 (m, 1H), 7.75–7.82 (m, 1H), 10.31
(br s, 1H); MS (ESI/APCI Dual): m/z 387 [MÀH]À.
Compound 10b to 10j were prepared from the corresponding
anilines in the same procedure described for 10a.
5.1.4. N-(2,4-Difluorophenyl)-2-(trifluoroacetyl)-2,3-dihydro-
1H-isoindole-5-sulfonamide (10b)
Colorless powder (yield 34%): 1H NMR (300 MHz, DMSO-d6) d
4.81–4.93 (m, 2H), 5.03–5.13 (m, 2H), 6.98–7.07 (m, 1H), 7.18–
7.29 (m, 2H), 7.52–7.61 (m, 1H), 7.63–7.71 (m, 1H), 7.73–7.78
(m, 1H), 10.20 (s, 1H); MS (ESI/APCI Dual): m/z 405 [MÀH]À.
5.1.5. N-(2-Chloro-4-fluorophenyl)-2-(trifluoroacetyl)-2,3-
dihydro-1H-isoindole-5-sulfonamide (10c)
Colorless amorphous (yield 87%): 1H NMR (300 MHz, CDCl3) d
4.88–5.09 (m, 4H), 6.79 (s, 1H), 6.97–7.07 (m, 2H), 7.31–7.44 (m,
1H), 7.63–7.74 (m, 3 H); MS (ESI/APCI Dual): m/z 421 [MÀH]À.
5.1.1. 1-(1,3-Dihydro-2H-isoindol-2-yl)-2,2,2-trifluoroethanone
(8)
5.1.6. N-(2-Ethyl-4-fluorophenyl)-2-(trifluoroacetyl)-2,3-
dihydro-1H-isoindole-5-sulfonamide (10d)
Pyridine (6.56 mL, 81.1 mmol) and N,N-dimethyl-4-aminopy-
ridine (248 mg, 2.03 mmol) were added to a solution of 2,3-dihy-
dro-1H-isoindole (8.08 g, 40.6 mmol) in chloroform (120 mL) at
room temperature. The mixture was cooled to 0 °C, and trifluo-
roacetic anhydride (6.77 mL, 48.7 mmol) was added dropwise to
the mixture. After the addition, the reaction mixture was warmed
to room temperature and stirred for 4 h. The reaction mixture was
concentrated under reduced pressure, and the residue was diluted
with ethyl acetate. The mixture was then washed with 1 mol/L
hydrochloric acid, saturated aqueous NaHCO3, and brine. The
organic layer was dried over anhydrous MgSO4 and then concen-
trated under reduced pressure. The resulting residue was purified
using a silica gel column eluted with 10% ethyl acetate/n-hexane
to afford 8 (8.66 g, 99%) as a light brown powder: 1H NMR
(300 MHz, CDCl3) d 4.93 (s, 2H), 5.04 (s, 2H), 7.22–7.43 (m, 4H);
MS (ESI/APCI Dual): m/z 216 [M+H]+.
Pale yellow amorphous (yield 65%): 1H NMR (300 MHz, CDCl3) d
1.07 (t, J = 7.6 Hz, 3H), 2.40 (q, J = 7.6 Hz, 2H), 4.90–5.14 (m, 4H),
6.18 (s, 1H), 6.79–6.92 (m, 2H), 7.12–7.18 (m, 1H), 7.35–7.46 (m,
1H), 7.63–7.74 (m, 2H); MS (ESI/APCI Dual): m/z 415 [MÀH]À.
5.1.7. N-(4-Fluoro-2-methoxyphenyl)-2-(trifluoroacetyl)-2,3-
dihydro-1H-isoindole-5-sulfonamide (10e)
Colorless amorphous (yield 85%): 1H NMR (300 MHz, CDCl3) d
3.63 (s, 3H), 4.87–5.08 (m, 4H), 6.46–6.53 (m, 1H), 6.59–6.69 (m,
1H), 6.80 (s, 1H), 7.29–7.42 (m, 1H), 7.45–7.53 (m, 1H), 7.65–
7.76 (m, 2H); MS (ESI/APCI Dual): m/z 417 [MÀH]À.
5.1.8. N-(2-Ethoxy-4-fluorophenyl)-2-(trifluoroacetyl)-2,3-
dihydro-1H-isoindole-5-sulfonamide (10f)
Colorless amorphous (yield 91%): 1H NMR (300 MHz, CDCl3) d
1.23–1.30 (m, 3H), 3.82 (q, J = 6.9 Hz, 2H), 4.86–5.07 (m, 4H),
6.42–6.49 (m, 1H), 6.59–6.67 (m, 1H), 6.78–6.82 (m, 1H), 7.29–
7.41 (m, 1H), 7.46–7.54 (m, 1H), 7.62–7.74 (m, 2H); MS (ESI/APCI
Dual): m/z 431 [MÀH]À.
5.1.2. 2-(Trifluoroacetyl)-2,3-dihydro-1H-isoindole-5-sulfonyl
chloride (9)
A solution of 8 (5.00 g, 23.2 mmol) in chloroform (80 mL) was
cooled at –78 °C, and chlorosulfonic acid (10.1 mL, 152 mmol)
was added dropwise. The reaction mixture was warmed to room
temperature and stirred overnight. The reaction mixture was
poured into an ice water. The aqueous layer was extracted twice
with chloroform, and the combined organic layers were washed
with water, dried over anhydrous MgSO4, filtered, and
5.1.9. N-(4-Fluoro-2-propoxyphenyl)-2-(trifluoroacetyl)-2,3-
dihydro-1H-isoindole-5-sulfonamide (10g)
Colorless amorphous (yield 93%): 1H NMR (300 MHz, CDCl3) d
0.92 (t, J = 7.4 Hz, 3H), 1.60–1.73 (m, 2H), 3.71 (t, J = 6.6 Hz, 2H),
4.86–5.06 (m, 4H), 6.44–6.50 (m, 1H), 6.59–6.67 (m, 1H),
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T. Busujima et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
6.76–6.80 (m, 1H), 7.29–7.39 (m, 1H), 7.47–7.55 (m, 1H), 7.63–7.73
methanol/chloroform. The fractions including compound 6 were
collected and evaporated by rotary evaporation. To the residue
was added ethyl acetate, and the resulting precipitates were col-
lected by filtration to afford 6 (124 mg, 32%) as a colorless powder:
mp 238–240 °C; 1H NMR (300 MHz, DMSO-d6) d 1.26 (s, 9H), 4.77
(s, 4H), 7.07–7.12 (m, 4H), 7.23–7.30 (m, 2H), 7.41–7.47 (m, 2H),
7.49–7.55 (m, 1H), 7.61–7.67 (m, 1H), 7.70 (s, 1H), 8.30 (s, 1H);
HRMS (ESI/APCI Dual): m/z Calcd for
468.1752; Found 468.1757.
Compounds 12b to 12j were prepared from the corresponding
starting materials in the same procedure described for 6.
(m, 2H); MS (ESI/APCI Dual): m/z 445 [MÀH]À.
5.1.10. N-(2,4-Dimethoxyphenyl)-2-(trifluoroacetyl)-2,3-
dihydro-1H-isoindole-5-sulfonamide (10h)
Colorless amorphous (yield 95%): 1H NMR (300 MHz, CDCl3) d
3.56 (s, 3H), 3.76 (s, 3H), 4.85–5.06 (m, 4H), 6.27–6.32 (m, 1H),
6.41–6.48 (m, 1H), 6.67 (s, 1H), 7.28–7.39 (m, 1H), 7.41–7.48 (m,
1H), 7.62–7.74 (m, 2H); MS (ESI/APCI Dual): m/z 429 [MÀH]À.
C
25H26FN3O3S [M+H]+,
5.1.11. N-(2-Fluoro-4-methoxyphenyl)-2-(trifluoroacetyl)-2,3-
dihydro-1H-isoindole-5-sulfonamide (10i)
Pale pink powder (yield 87%): 1H NMR (300 MHz, CDCl3) d 3.76
(s, 3H), 4.84–5.14 (m, 4H), 6.37 (s, 1H), 6.46–6.56 (m, 1H), 6.65–
6.76 (m, 1H), 7.30–7.55 (m, 2H), 7.63–7.74 (m, 2H); MS (ESI/APCI
Dual): m/z 417 [MÀH]À.
5.1.15. N-(4-tert-Butylphenyl)-5-[(2,4-difluorophenyl)-
sulfamoyl]-1,3-dihydro-2H-isoindole-2-carboxamide (12b)
Colorless powder (yield 45%): mp 232–234 °C; 1H NMR
(300 MHz, DMSO-d6) d 1.26 (s, 9H), 4.73–4.85 (m, 4H), 6.98–7.09
(m, 1H), 7.18–7.31 (m, 4H), 7.41–7.48 (m, 2H), 7.50–7.57 (m,
1H), 7.60–7.70 (m, 2H), 8.31 (s, 1H), 10.14 (s, 1H); HRMS
(ESI/APCI Dual): m/z Calcd for C25H25F2N3O3S [M+H]+, 486.1657.
Found 486.1657.
5.1.12. N-[2-Fluoro-4-(pentyloxy)phenyl]-2-(trifluoroacetyl)-2,
3-dihydro-1H-isoindole-5-sulfonamide (10j)
Colorless amorphous (yield 84%): 1H NMR (300 MHz, CDCl3) d
0.88–0.95 (m, 3H), 1.33–1.44 (m, 4H), 1.69–1.82 (m, 2H), 3.88 (t,
J = 6.5 Hz, 2H), 4.87–5.09 (m, 4H), 6.34 (s, 1H), 6.44–6.53 (m, 1H),
6.63–6.70 (m, 1H), 7.30–7.51 (m, 2H), 7.62–7.72 (m, 2H); MS
(ESI/APCI Dual): m/z 473 [MÀH]À.
5.1.16. N-(4-tert-Butylphenyl)-5-[(2-chloro-4-fluorophenyl)-
sulfamoyl]-1,3-dihydro-2H-isoindole-2-carboxamide (12c)
Colorless powder (yield 64%): mp 207–209 °C; 1H NMR
(300 MHz, DMSO-d6) d 1.26 (s, 9H), 4.75–4.84 (m, 4H), 7.14–7.31
(m, 4H), 7.40–7.48 (m, 3H), 7.51–7.57 (m, 1H), 7.60–7.69 (m,
2H), 8.32 (s, 1H), 10.1 (s, 1H); MS (ESI/APCI Dual): m/z 502
[M+H]+, 500 [MÀH]À; Anal. Calcd for C25H25ClFN3O3S: C, 59.81;
H, 5.02; N, 8.37. Found: C, 59.79; H, 5.01; N, 8.32.
5.1.13. N-(4-Fluorophenyl)-2,3-dihydro-1H-isoindole-5-
sulfonamide (11a)
A solution of potassium hydroxide (1.69 g, 30.1 mmol) in H2O
(30 mL) was added to a solution of 10a (5.85 g, 15.1 mmol) in etha-
nol (120 mL) at room temperature, and the reaction mixture was
stirred for 3 h. After the reaction mixture was concentrated by dis-
tillation under reduced pressure, and the residue was diluted with
water. The resulting mixture was acidified by addition of 3 mol/L
hydrochloric acid, and then neutralized by addition of saturated
aqueous NaHCO3. The mixture was extracted three times with
10% methanol/chloroform. The combined organic layers were dried
over anhydrous MgSO4, filtered, and concentrated under reduced
pressure. To the residue was added 50% ethyl acetate/n-hexane,
and the resulting precipitates were collected by filtration to afford
crude 11a (4.09 g, 93%) as a pale yellow powder: 1H NMR
(300 MHz, DMSO-d6) d 4.18 (s, 3H), 7.05–7.11 (m, 4H), 7.41–7.47
(m, 1H), 7.55–7.61 (m, 1H), 7.64–7.67 (m, 1H); MS (ESI/APCI
Dual): m/z 293 [M+H]+, 291 [MÀH]À.
5.1.17. N-(4-tert-Butylphenyl)-5-[(2-ethyl-4-fluorophenyl)-
sulfamoyl]-1,3-dihydro-2H-isoindole-2-carboxamide (12d)
Colorless powder (yield 61%): mp 229–231 °C; 1H NMR
(300 MHz, DMSO-d6) d 0.98 (t, J = 7.5 Hz, 3H), 1.26 (s, 9H), 3.26–
3.39 (m, 2H), 4.75–4.84 (m, 4H), 6.80–6.93 (m, 2H), 7.02–7.08
(m, 1H), 7.24–7.30 (m, 2H), 7.42–7.47 (m, 2H), 7.52–7.56 (m,
1H), 7.59–7.63 (m, 2H), 8.32 (s, 1H), 9.60 (s, 1H); MS (ESI/APCI
Dual): m/z 496 [M+H]+, 494 [MÀH]À; Anal. Calcd for
C27H30FN3O3S: C, 65.43; H, 6.10; N, 8.48. Found: C, 65.23; H,
6.08; N, 8.38.
5.1.18. N-(4-tert-Butylphenyl)-5-[(4-fluoro-2-methoxyphenyl)-
sulfamoyl]-1,3-dihydro-2H-isoindole-2-carboxamide (12e)
Colorless powder (yield 60%): mp 175–177 °C; 1H NMR
(300 MHz, DMSO-d6) d 1.26 (s, 9H), 3.46 (s, 3H), 4.74–4.82 (m,
4H), 6.64–6.75 (m, 1H), 6.78–6.88 (m, 1H), 7.14–7.22 (m, 1H),
7.23–7.30 (m, 2H), 7.41–7.51 (m, 3H), 7.56–7.66 (m, 2H), 8.31 (s,
1H); MS (ESI/APCI Dual): m/z 498 [M+H]+, 496 [MÀH]À; Anal.
Calcd for C26H28FN3O4S: C, 62.76; H, 5.67; N, 8.44. Found: C,
62.56; H, 5.70; N, 8.37.
5.1.14. N-(4-tert-Butylphenyl)-5-[(4-fluorophenyl)sulfamoyl]-1,
3-dihydro-2H-isoindole-2-carboxamide (6)
Potassium hydroxide (286 mg, 5.10 mmol) was added to a solu-
tion of 10a (990 mg, 2.55 mmol) in 20% water/ethanol (13 mL) at
room temperature and the mixture was stirred overnight. The
reaction mixture was distilled off under reduced pressure, and
water was added to the residue. The mixture was neutralized by
addition of 1 mol/L hydrochloric acid, and the mixture was
extracted three times with ethyl acetate. The combined organic
layers were dried over anhydrous MgSO4, filtered, and concen-
trated under reduced pressure to afford crude 11a (615 mg) as a
purple amorphous.
5.1.19. N-(4-tert-Butylphenyl)-5-[(2-ethoxy-4-fluorophenyl)-
sulfamoyl]-1,3-dihydro-2H-isoindole-2-carboxamide (12f)
Colorless powder (yield 36%): mp 196–198 °C; 1H NMR
(300 MHz, DMSO-d6) d 1.07 (t, J = 6.9 Hz, 3H), 1.26 (s, 9H), 3.73
(q, J = 6.9 Hz, 2H), 4.67–4.84 (m, 4H), 6.65–6.84 (m, 2H), 7.18–
7.31 (m, 3H), 7.40–7.51 (m, 3H), 7.55–7.64 (m, 2H), 8.31 (s, 1H);
MS (ESI/APCI Dual): m/z 512 [M+H]+, 510 [MÀH]À; Anal. Calcd
for C27H30FN3O4S: C, 63.39; H, 5.91; N, 8.21. Found: C, 63.32; H,
5.93; N, 8.19.
To a solution of crude 11a (200 mg) in 10% chloroform/DMSO
(3.5 mL) were added triethylamine (95.4
lL, 0.684 mmol) and 4-
tert-butylphenyl isocyanate (122 L, 0.684 mmol) at room temper-
l
ature, and the reaction mixture was stirred for 2 h. The reaction
was quenched by addition of 1 mol/L hydrochloric acid, and the
mixture was extracted three times with ethyl acetate. The com-
bined organic layers were washed with brine, dried over anhy-
drous MgSO4, filtered, and concentrated under reduced pressure.
The resulting residue was purified using a silica gel column eluted
5.1.20. N-(4-tert-Butylphenyl)-5-[(4-fluoro-2-propoxyphenyl)-
sulfamoyl]-1,3-dihydro-2H-isoindole-2-carboxamide (12g)
Colorless powder (yield 37%): mp 203–205 °C; 1H NMR
(300 MHz, DMSO-d6) d 0.82 (t, J = 7.5 Hz, 3H), 1.26 (s, 9H), 1.37–
1.53 (m, 2H), 3.62 (t, J = 6.6 Hz, 2H), 4.71–4.82 (m, 4H), 6.64–6.75
with
33%
ethyl
acetate/n-hexane
and
then
10%
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8
T. Busujima et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
(m, 1H), 6.77–6.85 (m, 1H), 7.18–7.30 (m, 3 H), 7.40–7.51 (m, 3H),
7.53–7.63 (m, 2H), 8.31 (s, 1H); MS (ESI/APCI Dual): m/z 526
[M+H]+, 524 [MÀH]À; Anal. Calcd for C28H32FN3O4S: C, 63.98; H,
6.14; N, 7.99. Found: C, 63.91; H, 6.11; N, 7.97.
temperature, and the mixture was heated at reflux temperature
for 2 h. After cooling to room temperature, 6 mol/L hydrochloric
acid (5 mL) was added, and the mixture was stirred at 90 °C for
1 h. The mixture was cooled to 0 °C, 6 mol/L aqueous sodium
hydroxide was added to adjust the pH to 8 to 9, and the mixture
was extracted with ethyl acetate. The organic layer was washed
with brine, dried over anhydrous MgSO4, filtered, and concentrated
under reduced pressure. The resulting residue was purified using a
silica gel column eluted with 33% ethyl acetate/n-hexane to afford
14 (53.9 mg) as a colorless amorphous. To a solution of 14 in ethyl
acetate (3 mL) was added 4 mol/L hydrogen chloride–ethyl acetate
(0.5 mL), and the mixture was stirred at room temperature for
0.5 h. After the volatiles were removed by rotary evaporation, ethyl
acetate/diethyl ether was added to the residue, and the resulting
precipitates were collected by filtration to afford the monohy-
drochloride salt of 14 (38.2 mg, 30%) as a gray powder: mp 142–
148 °C; 1H NMR (600 MHz, DMSO-d6) d 1.27 (s, 9H), 2.95–3.02
(m, 2H), 3.49–3.65 (m,2H), 4.51–4.70 (m, 2H), 4.78–4.98 (m, 2H),
7.04–7.15 (m, 4H), 7.22 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.3 Hz, 2H),
7.53–7.62 (m, 1H), 7.70–7.76 (m, 1H), 7.79 (s, 1H), 10.38 (s, 1H),
11.32 (br s, 1H); HRMS (ESI/APCI Dual): m/z Calcd for
5.1.21. N-(4-tert-Butylphenyl)-5-[(2,4-dimethoxyphenyl)-
sulfamoyl]-1,3-dihydro-2H-isoindole-2-carboxamide (12h)
Colorless powder (yield 69%): mp 176–178 °C; 1H NMR
(300 MHz, DMSO-d6) d 1.26 (s, 9H), 3.40 (s, 3H), 3.71 (s, 3H),
4.74–4.82 (m, 4H), 6.42–6.49 (m, 2H), 7.04–7.11 (m, 1H), 7.23–
7.31 (m, 2H), 7.41–7.51 (m, 3H), 7.53–7.63 (m, 2H), 8.31 (s, 1H),
9.28 (br s, 1H); MS (ESI/APCI Dual): m/z 510 [M+H]+, 508
[MÀH]À; Anal. Calcd for C27H31N3O5S: C, 63.63; H, 6.13; N, 8.25.
Found: C, 63.55; H, 6.10; N, 8.11.
5.1.22. N-(4-tert-Butylphenyl)-5-[(2-fluoro-4-methoxyphenyl)-
sulfamoyl]-1,3-dihydro-2H-isoindole-2-carboxamide (12i)
Colorless powder (yield 84%): mp 200–202 °C; 1H NMR
(300 MHz, DMSO-d6) d 1.26 (s, 9H), 3.71 (s, 3H), 4.75–4.83 (m,
4H), 6.66–6.72 (m, 1H), 6.76–6.83 (m, 1H), 7.03–7.09 (m, 1H),
7.27 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 8.6 Hz, 2H), 7.49–7.55 (m, 1H),
7.59–7.66 (m, 2H), 8.32 (s, 1H), 9.85 (br s, 1H); MS (ESI/APCI
Dual): m/z 498 [M+H]+, 496 [MÀH]À; Anal. Calcd for
C
26H29FN2O2S [M+H]+, 453.2007; Found 453.2001.
C26H28FN3O4S: C, 62.76; H, 5.67; N, 8.44. Found: C, 62.55; H,
5.1.26. 4-[(2,4-Dimethoxybenzyl)amino]-3-fluorophenol (16)
To a solution of 4-amino-3-fluorophenol (5.00 g, 39.3 mmol) in
tetrahydrofuran (120 mL) were added acetic acid (1.62 mL,
283 mmol), 2,4-dimethoxybenzaldehyde (7.84 g, 47.2 mmol) and
sodium triacetoxyborohydride (25.0 g, 118 mmol). The reaction
mixture was stirred at room temperature for 5 h. After the volatiles
were removed by rotary evaporation, saturated aqueous NaHCO3
(500 mL) was added to the residue, and the mixture was extracted
three times with ethyl acetate. The organic layer was dried over
anhydrous MgSO4, filtered, and concentrated under reduced pres-
sure. The resulting residue was purified using a silica gel column
eluted with 30–45% ethyl acetate/n-hexane to afford 16 (12.0 g,
92%) as an orange oil: 1H NMR (300 MHz, CDCl3) d 3.79 (s, 3H),
3.83 (s, 3H), 4.22 (s, 2H), 6.33–6.73 (m, 5H), 7.16 (d, J = 8.1 Hz,
1H); MS (ESI/APCI Dual): m/z 276 [MÀH]À.
5.72; N, 8.32.
5.1.23. N-(4-tert-Butylphenyl)-5-{[2-fluoro-4-(pentyloxy)phenyl]-
sulfamoyl}-1,3-dihydro-2H-isoindole-2-carboxamide (12j)
Colorless powder (yield 65%): mp 145–146 °C; 1H NMR
(300 MHz, DMSO-d6) d 0.82–0.92 (m, 3H), 1.26 (s, 9H), 1.28–1.39
(m, 4H), 1.60–1.73 (m, 2H), 3.90 (t, J = 6.5 Hz, 2H), 4.74–4.85 (m,
4H), 6.65–6.72 (m, 1H), 6.74–6.82 (m, 1H), 7.00–7.10 (m, 1H),
7.24–7.32 (m, 2H), 7.42–7.48 (m, 2H), 7.50–7.55 (m, 1H), 7.58–
7.67 (m, 2H), 8.31 (s, 1H), 9.84 (br s, 1H); MS (ESI/APCI Dual):
m/z 554 [M+H]+, 552 [MÀH]À; Anal. Calcd for C30H36FN3O4S: C,
65.08; H, 6.55; N, 7.59. Found: C, 65.04; H, 6.52; N, 7.47.
5.1.24. 2-[(4-tert-Butylphenyl)acetyl]-N-(4-fluorophenyl)-2,3-
dihydro-1H-isoindole-5-sulfonamide (13)
To a suspension of 11a (190 mg, 0.650 mmol) in chloroform
(3 mL) were added (4-tert-butylphenyl)acetic acid (150 mg,
0.780 mmol), 1-hydroxybenzotriazole monohydrate (149 mg,
0.975 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide hydrochloride (187 mg, 0.975 mmol). The reaction mixture
was stirred at room temperature overnight. Water was added,
and the mixture was extracted with ethyl acetate. The organic
layer was washed with brine and then saturated aqueous NH4Cl,
dried over anhydrous MgSO4, filtered, and concentrated under
reduced pressure. The resulting residue was purified using a silica
gel column eluted with 33% ethyl acetate/n-hexane and then 10%
methanol/chloroform. The fractions including compound 13 were
collected and evaporated by rotary evaporation. To the residue
was added ethyl acetate, and the resulting precipitates were col-
lected by filtration to afford 13 (88.2 mg, 29%) as a colorless pow-
der: mp 249–251 °C; 1H NMR (300 MHz, DMSO-d6) d 1.23–1.29 (m,
9H), 3.67 (s, 2H), 4.67 (s, 2H), 4.92 (s, 2H), 7.04–7.11 (m, 4H), 7.16–
7.22 (m, 2H), 7.29–7.36 (m, 2H), 7.45–7.55 (m, 1H), 7.58–7.67 (m,
1H), 7.68–7.77 (m, 1H); MS (ESI/APCI Dual): m/z 467 [M+H]+, 465
[MÀH]À; Anal. Calcd for C26H27FN2O3S: C, 66.93; H, 5.83; N, 6.00.
Found: C, 66.96; H, 5.80; N, 6.04.
5.1.27. N-(2,4-Dimethoxybenzyl)-N-(2-fluoro-4-hydroxy-
phenyl)-2-(trifluoroacetyl)-2,3-dihydro-1H-isoindole-5-
sulfonamide (17)
Compound 17 was prepared by the same procedure described
for 10a using compound 16 instead of 4-fluoroaniline.
Colorless powder (yield 52%): 1H NMR (300 MHz, DMSO-d6) d
3.56 (s, 3H), 3.70 (s, 3H), 4.56 (s, 2H), 4.87–5.19 (m, 4H), 6.37–
6.53 (m, 4H), 6.70–6.81 (m, 1H), 7.04–7.12 (m, 1H), 7.56–7.71
(m, 2H), 7.74–7.83 (m, 1H), 10.05 (s, 1H).
5.1.28. N-(4-tert-Butylphenyl)-5-[(2,4-dimethoxybenzyl)(2-
fluoro-4-hydroxyphenyl)sulfamoyl]-1,3-dihydro-2H-isoindole-
2-carboxamide (18)
Compound 18 was prepared by the same procedure described
for 6 using compound 17 instead of 10a.
Colorless powder (yield 49%): 1H NMR (300 MHz, DMSO-d6) d
1.23 (s, 9H), 3.56 (s, 3H), 3.70 (s, 3H), 4.56 (s, 2H), 4.84 (d,
J = 12 Hz, 4H), 6.32–6.56 (m, 4H), 6.69–6.80 (m, 1H), 7.04–7.11
(m, 1H), 7.28 (d, J = 8.6 Hz, 2H), 7.46 (d, J = 8.6 Hz, 2H), 7.51–7.78
(m, 3H), 8.35 (s, 1H), 10.04 (br s, 1H).
5.1.29. N-(4-tert-Butylphenyl)-5-[(4-ethoxy-2-fluorophenyl)-
sulfamoyl]-1,3-dihydro-2H-isoindole-2-carboxamide (19)
To a solution of 18 (40.0 mg, 0.0631 mmol) in N,N-dimethylfor-
5.1.25. 2-[2-(4-tert-Butylphenyl)ethyl]-N-(4-fluorophenyl)-2,3-
dihydro-1H-isoindole-5-sulfonamide (14)
A borane-tetrahydrofuran complex (0.9 mol/L tetrahydrofuran
solution, 0.538 mL, 0.484 mmol) was added to a solution of 13
(113 mg, 0.242 mmol) in tetrahydrofuran (3 mL) at room
mamide (400 lL) were added iodoethane (7.69 lL, 0.0946 mmol)
and potassium carbonate (17.0 mg, 0.126 mmol), and the mixture
was stirred at room temperature for 3 days. To the reaction
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T. Busujima et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
mixture was added water, and the mixture was extracted three
times with ethyl acetate. The combined organic layers were con-
centrated under reduced pressure. After the resulting residue
was dissolved in chloroform (1.8 mL), trifluoroacetic acid (0.2 mL)
was added, and the mixture was stirred at room temperature for
1 h. The reaction mixture was concentrated under reduced pres-
sure, and the resulting residue was purified by preparative HPLC
to afford 19 (14.6 mg, 46%) as a colorless powder: 1H NMR
(600 MHz, DMSO-d6) d 1.26 (s, 9H), 1.28 (t, J = 7.0 Hz, 2H), 3.97
(d, J = 7.0 Hz, 3H), 4.75–4.83 (m, 4H), 6.66–6.71 (m, 1H),
6.75–6.80 (m, 1H), 7.01–7.08 (m, 1H), 7.27 (d, J = 8.7 Hz, 2H),
7.44 (d, J = 8.7 Hz, 2H), 7.50–7.54 (m, 1H), 7.59–7.63 (m, 1H),
7.64 (s, 1H), 8.32 (s, 1H), 9.83 (br s, 1H); HRMS (ESI/APCI Dual):
m/z Calcd for C27H30FN3O4S [M+H]+, 512.2014; Found 512.2005.
Compound 20 and 21 were prepared by the same procedure
described for 19 using 1-iodopropane and 1-iodobutane respec-
tively instead of iodoethane.
Colorless amorphous (yield 92%): 1H NMR (300 MHz, CDCl3) d
2.93–3.08 (m, 2H), 3.80–3.96 (m, 2 H), 4.67–4.85 (m, 2H), 6.52–
6.71 (m, 1H), 6.90–7.12 (m, 4H), 7.20–7.33 (m, 1H), 7.45–7.62
(m, 2H); MS (ESI/APCI Dual): m/z 401 [MÀH]À.
5.1.35. N-(4-Fluorophenyl)-2-(trifluoroacetyl)-1,2,3,4-tetra-
hydroisoquinoline-6-sulfonamide (28)
Compound 28 was prepared by the same procedure described
for 10a using compound 25 instead of 9.
Colorless amorphous (yield 92%): 1H NMR (300 MHz, CDCl3) d
2.90–3.02 (m, 2H), 3.80–3.95 (m, 2H), 4.75–4.87 (m, 2H), 6.63–
6.74 (m, 1H), 6.90–7.10 (m, 4H), 7.18–7.29 (m, 1H), 7.51–7.61
(m, 2H); MS (ESI/APCI Dual): m/z 401 [MÀH]À.
5.1.36. 2-[2-(4-tert-Butylphenyl)ethyl]-N-(4-fluorophenyl)-1,2,
3,4-tetrahydroisoquinoline-7-sulfonamide (27)
A solution of potassium hydroxide (254 mg, 4.53 mmol) in H2O
(2 mL) was added to a solution of 26 (1.22 g, 3.02 mmol) in ethanol
(18 mL) at room temperature and the reaction mixture was stirred
for 14 h. A solution of potassium hydroxide (254 mg, 4.53 mmol) in
H2O (2 mL) was added to the mixture, and the reaction mixture
was stirred for 3 h. After the reaction mixture was concentrated
by distillation under reduced pressure, and the residue was diluted
with water. The resulting mixture was acidified by addition of
1 mol/L hydrochloric acid and then neutralized by addition of sat-
urated aqueous NaHCO3. The resulting precipitates were collected
by filtration to afford crude product (849 mg, 92%) as a colorless
powder.
5.1.30. N-(4-tert-Butylphenyl)-5-[(2-fluoro-4-propoxyphenyl)-
sulfamoyl]-1,3-dihydro-2H-isoindole-2-carboxamide (20)
Colorless powder (yield 55%): 1H NMR (300 MHz, DMSO-d6) d
0.94 (t, J = 7.4 Hz, 3H), 1.26 (s, 9 H), 1.62–1.75 (m, 2H), 3.87 (t,
J = 6.5 Hz, 2H), 4.71–4.86 (m, 4 H), 6.65–6.83 (m, 2H), 6.99–7.10
(m, 1H), 7.27 (d, J = 8.7 Hz, 2H), 7.44 (d, J = 8.7 Hz, 2H), 7.50–7.56
(m, 1H), 7.58–7.69 (m, 2H), 8.31 (s, 1H), 9.83 (br s, 1H); HRMS
(ESI/APCI Dual): m/z Calcd for C28H32FN3O4S [M+H]+, 526.2170;
Found 526.2166.
5.1.31. 5-[(4-Butoxy-2-fluorophenyl)sulfamoyl]-N-(4-tert-
butylphenyl)-1,3-dihydro-2H-isoindole-2-carboxamide (21)
Colorless powder (yield 68%): 1H NMR (600 MHz, DMSO-d6) d
0.90 (t, J = 7.4 Hz, 3H), 1.26 (s, 9H), 1.35–1.43 (m, 2H), 1.61–1.68
(m, 2H), 3.91 (t, J = 6.6 Hz, 2H), 4.75–4.83 (m, 4H), 6.65–6.72 (m,
1H), 6.75–6.81 (m, 1H), 7.00–7.08 (m, 1H), 7.24–7.30 (m, 2H),
7.42–7.47 (m, 2H), 7.50–7.54 (m, 1H), 7.60–7.63 (m, 1H), 7.65 (s,
1H), 8.32 (s, 1H), 9.83 (br s, 1H); HRMS (ESI/APCI Dual): m/z
Calcd for C29H34FN3O4S [M+H]+, 540.2327; Found 540.2331.
To a suspension of the crude product (153 mg, 0.500 mmol) in
chloroform (10 mL) were added (4-tert-butylphenyl)acetic acid
(106 mg, 0.650 mmol), 1-hydroxybenzotriazole monohydrate
(100 mg, 0.650 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)car-
bodiimide hydrochloride (125 mg, 0.650 mmol). The reaction mix-
ture was stirred at room temperature for 15 h. Water was added,
and the mixture was extracted with ethyl acetate. The organic
layer was washed with brine, dried over anhydrous MgSO4, fil-
tered, and concentrated under reduced pressure. The resulting resi-
due was purified using a silica gel column eluted with 25% and
then 60% ethyl acetate/n-hexane. The fractions including product
were collected and evaporated by rotary evaporation. To the
residue was added ethyl acetate/IPE/n-hexane, and the resulting
precipitates were collected by filtration to afford the target inter-
mediate (168 mg, 70%) as a colorless powder.
A borane-tetrahydrofuran complex (0.9 mol/L tetrahydrofuran
solution, 0.770 mL, 0.696 mmol) was added to a solution of the
above intermediate (167 mg, 0.348 mmol) in tetrahydrofuran
(8 mL) at 0 °C, and the mixture was heated at reflux temperature
for 6 h. After cooling to room temperature, 6 mol/L hydrochloric
acid (6 mL) was added, and the mixture was stirred at reflux tem-
perature for 3 h. The mixture was cooled to 0 °C, 6 mol/L aqueous
sodium hydroxide was added to adjust the pH to 8 to 9, and the
mixture was extracted with ethyl acetate. The organic layer was
washed with brine, dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The resulting residue was
purified using a silica gel column eluted with 0–5% methanol/chlo-
roform to afford 27 (109 mg, 67%) as a colorless amorphous. To a
solution of 27 in ethyl acetate (4 mL) was added 4 mol/L hydrogen
chloride-ethyl acetate (1 mL), and the mixture was stirred at room
temperature for 15 h. After the volatiles were removed by rotary
evaporation, diethyl ether was added to the residue, and the result-
ing precipitates were collected by filtration to afford the monohy-
drochloride salt of 27 (97.8 mg, 83%) as a Colorless powder: 1H
NMR (300 MHz, DMSO-d6) d 1.27 (s, 9H), 2.94–3.24 (m, 5H),
3.34–3.50 (s, 2H), 3.72–3.84 (m, 1H), 4.33–4.48 (m, 1H), 4.63–
4.80 (m, 1H), 7.07–7.15 (m, 4H), 7.18–7.25 (m, 2H), 7.34–7.40
(m, 2H), 7.42–7.48 (m, 1H), 7.59–7.68 (m, 2H), 10.36 (s, 1 H),
5.1.32. 1-(3,4-Dihydroisoquinolin-2(1H)-yl)-2,2,2-
trifluoroethanone (23)
Compound 23 was prepared by the same procedure described
for 8 using 1,2,3,4-tetrahydroisoquinoline instead of 2,3-dihydro-
1H-isoindole.
Colorless oil (yield 96%): 1H NMR (300 MHz, CDCl3) d 2.90–3.01
(m, 2H), 3.82–3.94 (m, 2H), 4.70–4.85 (m, 2H), 7.09–7.29 (m, 4H);
MS (ESI/APCI Dual): m/z 230 [M+H]+.
5.1.33. 2-(Trifluoroacetyl)-1,2,3,4-tetrahydroisoquinoline-7-
sulfonyl chloride (24) and 2-(trifluoroacetyl)-1,2,3,4-
tetrahydroisoquinoline-6-sulfonyl chloride (25)
Compounds 24 and 25 were prepared from compound 23 in a
similar manner described for 9. The crude mixture was purified
using a silica gel column eluted with 20% ethyl acetate/n-hexane
to afford 24 (3.51 g, 20%) as a colorless powder and 25 (31.5 mg,
0.18%) as a colorless amorphous.
Compound 24: 1H NMR (300 MHz, CDCl3) d 3.02–3.17 (m, 2H),
3.86–4.02 (m, 2H), 4.80–4.97 (m, 2H), 7.37–7.52 (m, 1H), 7.79–
7.99 (m, 2H).
Compound 25: 1H NMR (300 MHz, CDCl3) d 3.05–3.14 (m, 2H),
3.87–4.00 (m, 2H), 4.81–4.99 (m, 2H), 7.36–7.48 (m, 1H), 7.85–
7.96 (m, 2H).
5.1.34. N-(4-Fluorophenyl)-2-(trifluoroacetyl)-1,2,3,4-
tetrahydroisoquinoline-7-sulfonamide (26)
Compound 26 was prepared by the same procedure described
for 10a using compound 24 instead of 9.
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10
T. Busujima et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
10.65 (br s, 1H); MS (ESI/APCI Dual): m/z 467 [M+H]+, 465 [MÀH]À;
Anal. Calcd for C27H31FN2O2S HCl: C, 64.46; H, 6.41; N, 5.57. Found:
C, 64.33; H, 6.37; N, 5.51.
100 mM Tris–HCl (pH 7.5), 100 mM sucrose, 5 mM MgCl2] and
recombinant MGAT2 and test compound were added to each well
of a 96-well black plate(Corning). Substrate solution [final concen-
tration: 5.3 lM oleoyl-CoA, 0.78 lM 2-oleoylglycerol, 7.5 lM
5.1.37. 2-[2-(4-tert-Butylphenyl)ethyl]-N-(4-fluorophenyl)-
1,2,3,4-tetrahydroisoquinoline-6-sulfonamide (29)
phosphatidylcholine] were added to start the reaction, which
was allowed to proceed for 20 min. The reaction was terminated
Compound 29 was prepared by the same procedure described
for 27 using compound 28 instead of compound 26.
upon the addition of CPM with final concentration 5 lM. The plates
were sealed, incubated for 20 min. Fluorescence that emits at
460 nm when excited at 380 nm was counted on a SpectraMax
Plate Reader (Molecular Devices, LLC). Oleoyl-CoA, 2-oleoylglycerol
and phosphatidylcholine were obtained from Sigma-Aldrich and
CPM from Life Technologies.
Colorless powder (total yield 50%): 1H NMR (300 MHz, DMSO-
d6) d 1.29 (s, 9H), 2.91–3.27 (m, 5H), 3.34–3.48 (m, 2H), 3.73–
3.87 (m, 1H), 4.33–4.50 (m, 1H), 4.60–4.76 (m, 1H), 7.08–7.14
(m, 4H), 7.19–7.25 (m, 2H), 7.34–7.43 (m, 3H), 7.57–7.69 (m,
2H), 10.35 (s, 1H), 10.82 (br s, 1H); MS (ESI/APCI Dual): m/z 467
[M+H]+, 465 [MÀH]À; Anal. Calcd for C27H31FN2O2S HCl: C, 64.46;
H, 6.41; N, 5.57. Found: C, 64.31; H, 6.41; N, 5.49.
5.4.2. Oral lipid tolerance test in mice
C57BL/6 J male mice (10 weeks old, n = 7 to 8) were used. To
inhibit the clearance of plasma triacylglycerol, we administered
5.2. Solubility
100 lL of the surfactant tyloxapol (10% in PBS) through a tail vein.
Immediately, we administered orally 4 mL/kg of test compound or
0.5% methylcellulose solution as vehicle. After 30 min, we had
challenged orally with 4.4 mL/kg of triolein containing
0.1 mCi/mL 3H triolein. We collected blood from tail vein at 2 h,
3 h and 4 h after lipid challenge and counted scintillation.
Efficacy was calculated as the percentage reduction in area under
the curve of plasma scintillation for 4 h (AUC0–4 h) compared to
vehicle-treated control animals. Steel’s test was used to establish
statistical significance of AUC0–4h between vehicle and treated
mice.
An excess amount of each compound was added to Water and
shaken on a shaker (model SR-2s; Yamato Kagaku) at 25 °C for
24 h.
An excess amount of each compound was added to FeSSIF (Fed
State Simulated intestinal Fluid,12 pH 5.0) and shaken on a shaker
(model SR-2s; Yamato Kagaku) at 25 °C for 2 h and keep on 37 °C
for 22 h on a water bath (model LT-10s; Yamato Kagaku).
The suspensions were centrifuged at 3000 and 11,000 rpm for
10 min. and supernatant was diluted with 50% aqueous acetonitrile
or 50% aqueous methanol solution. The concentrations were mea-
sured by HPLC. The HPLC analysis was performed using a Shimadzu
HPLC system composed of a LC-20AD, SPD-20A and SIL-20AC. The
conditions for HPLC were as follows: mobile phase, 0.1%
Phosphoric acid aqueous solution/acetonitrile; flow rate,
Acknowledgments
We are grateful to Dr. Yoshizumi for encouragement and edito-
rial comments on this manuscript. We are grateful to Dr. Ohtake
for encouragement and helpful advice. We are grateful to Mr.
Higuchi for analytical support (the high-resolution mass spectra).
0.8 mL/min.; column, reversed-phase (Shimpack XR-ODS, 2.2 lm,
2.0 Â 75 mm; Shimadzu) at 40 °C; and detection wavelength,
210 nm.
References and notes
5.3. Metabolic stability in liver microsomes
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489.
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Test substances (5 lM) were incubated at 37 °C in 1 mg/mL
human or mouse microsomes supplemented with 1.5 mM glu-
cose-6-phosphate, 0.16 mM ß-nicotinamide-adenine dinucleotide
phosphate, 0.18 units/mL glucose-6-phosphate dehydrogenase,
2.4 mM magnesium chloride and 69 mM potassium chloride in
250 mM phosphate buffer (pH 7.4). Concentrations of the test com-
pounds were determined by LC–MS/MS. The metabolized % was
determined by comparing a peak area of the test substance at
15 min incubation with the peak area at 0 min.
8. (a) Turdi, H.; Hangeland, J. J.; Lawrence, R. M.; Cheng, D.; Ahmad, S.; Meng, W.;
Brigance, R. P.; Devasthale, P. WO 2013/082345; Chem. Abstr. 2013, 159,
75803; (b) Ahmad, S.; Megash, L. A. WO 2014/193884; Chem. Abstr. 2014, 162,
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112323; Chem. Abstr. 2013, 159, 305466.
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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. 2013, 23, 2721.
11. Danilewicz, J.; Ellis, D.; Kobylecki, R. J., WO 95/13274; Chem. Abstr. 1995, 123,
314532.
5.4. Biology
5.4.1. MGAT2 assay
This assay detects CoA, a product of the MGAT2-catalyzed dea-
cylation of oleoyl-CoA. The free thiol of CoA can react with 7-di-
ethylamino-3-(4’-maleimidylphenyl)-4-methylcoumarin (CPM), a
pro-fluorescent coumarin maleimide derivative that becomes fluo-
rescent upon reaction with thiols.
In this assay, recombinant human and mouse MGAT2 were pro-
duced in the baculovirus expression system. The MGAT2 activity
was determined as follows: Assay buffer [final concentration:
12. Galia, E.; Nicolaides, E.; Horter, D.; Lobenberg, R.; Reppas, C.; Dressman, J. B.
Pharma. Res. 1998, 15, 698.
Please cite this article in press as: Busujima, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.06.065