A novel class of apical sodium-dependent bile acid transporter inhibitors: The amphiphilic 4-oxo-1-phenyl-1,4-dihydroquinoline derivatives

Article abstract of DOI:10.1016/j.bmcl.2003.12.063

A series of 4-oxo-1-phenyl-1,4-dihydroquinolines possessing a linker and an ammonio moiety were synthesized and found to inhibit the apical sodium-dependent bile acid transporter (ASBT). The potency of ASBT inhibition varied with the position and length o

Full text of DOI:10.1016/j.bmcl.2003.12.063

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1183–1186
A novel class of apical sodium-dependent bile acid transporter
inhibitors: the amphiphilic 4-oxo-1-phenyl-1,4-dihydroquinoline
derivatives
Hitoshi Kurata,* Sayaka Suzuki, Yasuo Ohhata, Takuya Ikeda, Toru Hasegawa,
Ken Kitayama, Toshimori Inaba, Keita Kono and Takafumi Kohama
Research Laboratories, Sankyo Co., Ltd, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
Received 7October 2003; accepted 15 December 2003
Abstract—A series of 4-oxo-1-phenyl-1,4-dihydroquinolines possessing a linker and an ammonio moiety were synthesized and
found to inhibit the apical sodium-dependent bile acid transporter (ASBT). The potency of ASBT inhibition varied with the posi-
tion and length of the linking tether. Compound 21e effectively lowered the total serum cholesterol levels in hamsters.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
compounds have been identified as candidates for
ASBT inhibitors.4 Researchers at Shionogi reported the
substituted naphthol 1(S-8921)5 as an ASBT inhibitor
and showed that 1 was able to lower the total serum
cholesterol levels in animals.6 Evaluation of S-8921 and
S-8921 glucuronide 2 (one of the metabolites of 17) for
ASBT inhibitory activity by the hamster ileal ring
method revealed that S-8921 glucuronide (IC50=2.8 mM)
has higher potency than S-8921 in inhibiting ASBT
(IC50>185 mM). Similarly, Searle’s ASBT inhibitor 38
(IC50=0.89 mM) was found to be more efficacious than
4 (IC50>63 mM). These findings suggest that the
amphiphilic property of these compounds (2 and 3)
plays an important role in effective ASBT inhibition. As
we have been working on the synthesis of 4-oxo-1-
phenyl-1,4-dihydroquinolines (e.g., 5, 6) as ASBT inhi-
bitors, we decided to prepare 4-oxo-1-phenyl-1,4-dihy-
droquinolines possessing a linker and an ammonio
moiety in order to enhance the ASBT inhibitory activ-
ity. We report here the synthesis of a series of novel
amphiphilic 4-oxo-1-phenyl-1,4-dihydroquinolines and
their biological activities.
Bile acid sequestrants (BAS) such as anion exchanging
resins have been used to treat hypercholesterolemia and
hyperlipidemia with a good record of safety for over 30
years.1 Even since the introduction of the prevalent
therapy with HMG-CoA reductase inhibitors (statins),
BASs have remained useful in some patients such as
young children and fertile women due to the potential
risks posed by systemic exposure to the alternative lipid-
altering drugs. Furthermore, the combination therapy
with statin and BAS is often required for patients with
severe dyslipidemia. In spite of these merits of BAS,
several demerits can lead to its therapeutic failure,
including poor patient compliance due to high dosage,
unpalatability, and gastrointestinal symptoms, and
altered absorption of vitamins, minerals, and co-admin-
istered drugs.2 For this reason, physicians have hoped
for an alternative method for bile acid sequestration.
The cloning and characterization of apical sodium-
dependent bile salt transporter (ASBT/SLC10A2),3
otherwise known as ileal bile acid transporter (IBAT),
greatly accelerated the development of specific inhibi-
tors of bile acid reabsorption, potentially a new class
of plasma cholesterol lowering drugs. Several types of
2. Chemistry
In the course of our ASBT inhibitor research program
at Sankyo, we discovered that a series of 4-oxo-1-
phenyl-1,4-dihydroquinolines inhibited ASBT, and fur-
ther, that the potency of these dihydroquinolines was
very sensitive to both the position and nature of the
Keywords: Apical sodium-dependent bile acid transporter (ASBT);
Ileal bile acid transporter (IBAT).
* Corresponding author. Tel.: +81-3-3492-3131; fax: +81-3-5436-
8563; e-mail: hitosi@shina.sankyo.co.jp
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.063

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H. Kurata et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1183–1186
Scheme 1. (a) NaH, CO(OMe)2 (79%); (b) Me2NCH(OMe)2 (68%);
(c) 4-benzyloxyaniline, K2CO3, DMF (27%); (d) aq NaOH, EtOH
(94%); (e) ClCOOi-Bu, Et3N, 3,5-difluoroaniline (quant); (f) NaH,
EtI, DMF (83%); (g) H2/Pd–C, EtOH (95%); (h) a,a0-dichloro-p-
xylene, K2CO3, DMF (54%); (i) DABCO, MeCN (75%).
substituent (data not shown). As a result of optimiza-
tion of the substituents, compounds such as 5 and 6
were found to exhibit potent ASBT inhibitory activity in
vitro. Consequently, we first sought to replace the
methyl of the methoxy group in 5 and 6 with a linker
and an ammonio moiety in order to preserve the opti-
mized substituents.
Based on the structures of 3 and 5, we designed 11 and
synthesized it by the process shown in Scheme 1.9
Methoxycarbonylation of 7 and subsequent condensa-
tion with N,N-dimethylformamide dimethyl acetal were
carried out to give 8, which was treated with 4-benzyl-
oxyaniline and cyclized to furnish 9. In this cyclization
reaction, the methoxy group was found to work as a
good leaving group, and this methodology greatly facili-
tated preparation of 4-oxo-1-phenyl-1,4-dihydroquino-
line derivatives. N-Ethyl N-(3,5-difluorophenyl) amide
was then installed through a conventional saponifica-
tion/amidation/ethylation sequence, followed by depro-
tection of benzyl ether under a hydrogenation condition
to give 10. Etherification of 10 with a,a0-dichloro-p-
xylene in the presence of K2CO3 gave the corresponding
ether, which was treated with DABCO in acetonitrile to
afford the desired ammonium compound 11.
Scheme 2. (a) NaH, BnBr, DMF (39%); (b) K2CO3, MeI, DMF
(96%); (c) aq NaOH, EtOH (95%); (d) DPPA, Et3N, toluene, t-BuOH
(90%); (e) HCl–AcOEt (78%); (f) 8, K2CO3, DMF (64%); (g) aq
NaOH, MeOH (97%); (h) (COCl)2, N-ethyl 3,5-difluoroaniline (62%);
(i) H2/Pd–C, EtOH (92%); (j) alkylene dihalide, NaH, DMF (31–
82%); (k) DABCO, MeCN (77–85%).
Monobenzylation and subsequent methylation of 12
provided the resulting ether, which was subjected to
saponification and Curtius rearrangement using DPPA
to give 13. Treatment of 13 with 8 in DMF followed by
cyclization led to 14 in moderate yield. Saponification of
14 followed by amidation with N-ethyl 3,5-difluoroani-
line via acid chloride gave an amide, and removal of the
Next, based on the structures of 3 and 6, we designed
16a, and started the synthesis from methyl 3,5-dihy-
droxybenzoate 12 (Scheme 2).

H. Kurata et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1183–1186
1185
benzyl protecting group provided 15. Etherification of
15 with a,a0-dichloro-p-xylene and subsequent treat-
ment with DABCO afforded the desired 16a. In order to
investigate SAR, carbon chain analogues 16b–d were also
prepared by employing 1,6-diiodohexane, 1,7-dibromo-
heptane and 1,8-dibromooctane as linkers.
Finally, compounds 21a–f were designed and synthe-
sized as outlined in Scheme 3. Selective monobenzyla-
tion successfully proceeded to give a monobenzyl ether,
and subsequent methylation provided 18 in good yield.
Compound 18 was converted to 21a–f under the same
conditions as those described above.
All the compounds synthesized here have no chiral cen-
ters, and their structures are so simple that they can be
easily prepared even on a large scale.
3. Results and discussion
ASBT inhibitory activities of the synthesized com-
pounds are summarized in Table 1. The in vitro activity
was determined by measuring the uptake of [3H]-tauro-
cholate in hamster ileal rings.10
Scheme 3. (a) K2CO3, BnBr, acetone (90%); (b) K2CO3, MeI, DMF
(98%); (c) NaH, CO(OMe)2 (86%); (d) Me2NCH(OMe)2; (e) 3,5-
dimethoxyaniline, K2CO3, DMF (73% for two steps); (f) aq NaOH,
EtOH (quant); (g) (COCl)2, N-ethyl 3,5-difluoroaniline (97%); (h) H2/
Pd–C, EtOH (quant); (i) alkylene dihalide, K2CO3, DMF (67–97%);
(j) DABCO, MeCN (53–81%).
Although compound 11 was initially thought to be
structurally close to 3 and expected to inhibit ASBT
potently, it turned out to be a relatively weak inhibitor
of ASBT. On the other hand, 16a was actually more
potent than the non-amphiphilic 6. Substitution of the
linker of 16a with C6–C8 chain linkers led to an addi-
tional improvement in ASBT inhibition, although the
results did not suggest clear relationships between
ASBT inhibition and the length of the tether. Com-
pound 21a prepared by modification at the 7position of
the quinoline ring showed slightly improved potency
compared to 6. In the carbon chain linker analogues
(21b–f), the potency was apparently dependent on the
length of the linker. The heptamethylene derivative 21e
exhibited an optimal improvement and was found to
have IC50=0.77 mM.
Table 1. In vitro assay of compounds that inhibit IBAT-mediated
uptake of [3H]-taurocholate
Compd
Xꢀ
IC50 (mM)
% inhibition
@ 30 mg/mL
mp
3
0.89
5
6
11
64
64
41
71
83
86
83
71
194
193
182
167
Clꢀ
Clꢀ
Iꢀ
16a
16b
16c
16d
21a
21b
21c
21d
21e
21f
147
Brꢀ
Brꢀ
Clꢀ
Iꢀ
Foam
Foam
180–182
146–149
We next assessed the cholesterol-lowering effect of 21e
in hamsters.
24
Iꢀ
–140 67137
88
100
Iꢀ
130–133
130–132
Brꢀ
Brꢀ
0.77
1.0794
Table 2 shows the effects of Compound 21e on serum
total cholesterol, non HDL-cholesterol, HDL-cholesterol,
128–130
Table 2. Effect of compound 21e on serum lipids in hamsters
Group
Dose
(mg/kg)
TC
(mg/dL)
Non HDL-C
(mg/dL)
HDL-C
(mg/dL)
Ratio:
non HDL-C/HDL-C
TG
(mg/dL)
Vehicle
Compd 21e
0
0.3
1.0
3.0
10.0
30.0
100.0
183.4ꢁ1.0
103.5ꢁ3.4
75.7ꢁ6.5**
85.2ꢁ1.5
ꢁ4.4
79.9ꢁ3.6
81.8ꢁ4.2
81.7ꢁ3.9
80.2ꢁ3.2
69.8ꢁ3.2
63.9ꢁ4.6*
61.9ꢁ2.0**
1.3ꢁ0.1
0.9ꢁ0.1
1.1ꢁ0.1
1.2ꢁ0.1
1.1ꢁ0.1
1.3ꢁ0.1
0.9ꢁ0.1*
301.7ꢁ28.7
142.7ꢁ37.0**
249.3ꢁ26.4
270.8ꢁ48.4
217.6ꢁ30.6
235.0ꢁ24.4
217.7ꢁ32.2
157.4ꢁ7.5*
166.9ꢁ3.8
174.3ꢁ4.794.1
144.1ꢁ6.6***
142.8ꢁ7.1***
118.9ꢁ7.8***
74.3ꢁ6.1**
78.9ꢁ5.4*
57.0ꢁ7.1***
Each value represents the meanꢁSEM (n=3–5/group). *p<0.05, **p<0.01 and ***p<0.001, as compared with the vehicle-treated group using
Dunnett’s Test.

1186
H. Kurata et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1183–1186
the cholesterol ratio, and triglycerides in chow-fed male
Syrian golden hamsters following 14 days of treat-
ment.11 Compound 21e treatment reduced serum total
cholesterol by 35%, non HDL-cholesterol by 45%,
HDL-cholesterol by 23%, the cholesterol ratio by 30%
maximum. Compound 21e significantly reduced trigly-
cerides without apparent dose-dependency. Compound
21e did not affect the body weights of any of the groups
throughout the treatment (data not shown). Accord-
ingly, compound 21e was concluded to be useful for
lipid lowering and improving the cholesterol ratio in the
blood. The biological activities of the other derivatives
will be reported in a full account in due course.
Zhi, B. PCT Int. Appl. WO01/68637. (b) Searle has also
disclosed 1-[4-[4-[(4R,5R)-3,3-dibutyl-7-(dimethylamino)-
2,3,4,5-tetrahydro-4-hydroxy-1,1-dioxido-1-benzothiepin-
5-yl]phenoxy]butyl] - 4 - aza - 1 - azoniabicyclo[2.2.2]octane
methanesulfonate (SC-435) and its biological activities:
West, K. L.; Ramjiganesh, T.; Roy, S.; Keller, B. T.;
Fernandez, M. L. J. Pharmacol. Exp. Ther. 2002, 303,
293.
9. All new compounds are fully characterized by their spec-
troscopic and analytical data.
10. The freshly obtained ileum segment from a hamster (Syr-
ian, male, 5–7weeks old; Japan SLC, Inc.) was everted by
a stainless steel rod and washed in ice-cold oxygenated
Krebs Ringer solution (115 mM NaCl, 5.9 mM KCl, 1.2
mM MgCl2, 1.2 mM NaH2PO4, 2.5 mM CaCl2, 1.2 mM
Na2SO4 and 25 mM NaHCO3, pH=7.4). The everted
ileum was divided into about 5-mm pieces, which were
randomized into several groups based on the gradient of
ASBT activities. Next, tissue pieces were incubated for 5
min at 37 ꢂC in oxygenated Krebs Ringer solution sup-
plemented with 37 mM [3H]-taurocholate (TCA, 74 GBq/
mmol; NEN/Perkin–Elmer), 2 mg/mL bovine serum albu-
min (BSA, essential fatty acid free; Sigma) and 30 mg/mL
of each test compound. Nonspecific uptake was measured
by the addition of 37mM of non-radiolabeled TCA. After
incubation, the pieces were washed 3 times in ice-cold
Krebs Ringer solution containing 2 mg/mL BSA, weigh-
ted, and solubilized in NCS-II tissue solubilizer (Amer-
sham Pharmacia). Thereafter, each radioactivity (dpm)
was determined in a liquid scintillation counter. TCA-
uptake was corrected for nonspecific counts and expressed
as dpm/mg of wet weight. Values obtained for the test
compounds were the mean of triplicate incubations.
11. Compound 21e was administered daily at doses of 0.3, 1,
3, 10, 30, and 100 mg/kg/day to chow-fed male Syrian
golden hamsters on 14 consecutive evenings by oral
gavage in 0.5% carboxymethylcellulose (dosing vehicle).
The animals were allowed free access to rodent chow and
water throughout the study. The animals were weighed
daily and sacrificed on the morning after the 14th dose.
Blood serum was assayed for serum triglycerides (TG),
total cholesterol (TC), and lipoprotein cholesterol pro-
files; very low density lipoprotein (VLDL) plus low den-
sity lipoprotein (LDL), high density lipoprotein (HDL)
cholesterol, and the ratio of non HDL-cholesterol (VLDL
plus LDL cholesterol) to HDL-cholesterol.
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