Arylsulfonylamino-benzanilides as inhibitors of the apical sodium-dependent bile salt transporter (SLC10A2)

Article abstract of DOI:10.3390/molecules18066883

The apical sodium-dependent bile salt transporter (ASBT) plays a pivotal role in maintaining bile acid homeostasis. Inhibition of ASBT would reduce bile acid pool size and lower cholesterol levels. In this report, a series of novel arylsulfonylaminobenzan

Full text of DOI:10.3390/molecules18066883

Molecules 2013, 18, 6883-6897; doi:10.3390/molecules18066883
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Arylsulfonylamino-Benzanilides as Inhibitors of the Apical
Sodium-Dependent Bile Salt Transporter (SLC10A2)
Hong-Tao Liu 1, Hong-Wei He 1, Xiao-Guang Bai 1, Ju-Xian Wang 1, Chang-Liang Xu 1,
Shi-Ying Cai 2, Rong-Guang Shao 1,* and Yu-Cheng Wang 1,*
1
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, China
2
Department of Internal Medicine and Liver Center, School of Medicine, Yale University,
New Haven, CT 06520, USA
* Authors to whom correspondence should be addressed; E-Mails: shaor@bbn.cn (R.G.S.);
wyc9999@gmail.com (Y.C.W.); Tel./Fax: +86-10-6302-6956 (R.G.S.);
Tel./Fax: +86-10-6316-5263 (Y.C.W.).
Received: 2 May 2013; in revised form: 4 June 2013 / Accepted: 5 June 2013 /
Published: 10 June 2013
Abstract: The apical sodium-dependent bile salt transporter (ASBT) plays a pivotal role in
maintaining bile acid homeostasis. Inhibition of ASBT would reduce bile acid pool size and
lower cholesterol levels. In this report, a series of novel arylsulfonylaminobenzanilides were
designed and synthesized as potential inhibitors of ASBT. Most of them demonstrated great
potency against ASBT’s bile acid transport activity. In particular, compound 5g2 inhibited
ASBT activity with an IC50 value of 0.11 μM. These compounds represent potential
cholesterol-lowering drugs.
Keywords: ASBT inhibitors; bile acids; arylsulfonylaminobenzanilides; cholesterol
lowering drug
1. Introduction
Coronary artery disease (CAD) is a leading cause of death around the World [1]. High levels of
cholesterol are one of the main causes of CAD, which greatly increases the risk of formation of plaques
and atherosclerosis [2]. As a result, lowering cholesterol is beneficial to the prevention of CAD. Bile
acids are metabolites of cholesterol. Bile acids are synthesized in the liver and released into the

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duodenum after a meal to facilitate lipid absorption. In the ileum, most bile acids are reabsorbed by the
apical sodium-dependent bile salt transporter (ASBT, SLC10A2) and transported back to the liver
through enterohepatic circulation. However, there is a fraction of bile acids that escape intestinal
reabsorption, and are excreted with feces. The loss of bile acid in feces triggers de novo bile acids
synthesis from cholesterol in order to maintain the bile acid pool size. This process represents the major
route for the elimination of cholesterol from the body [3].
The argument that the increase of bile acids excretion can reduce hepatic and serum cholesterol was
proven by the usage of bile acid sequestrants (BASs) [4]. As one of the most commonly used drugs for
treating hypercholesterolemia and hyperlipidemia, BASs bind to bile acids and prevent their
re-absorption in the intestine. Although BASs have a good safety record and synergistic effects when
combined with statins, they still suffer from poor patient compliance due to their high dosages and bad
palatability [5]. Therefore, the development of new drugs with similar physiological response to BASs,
but with improved palatability, is in demand for lowering cholesterol.
ASBT plays a critical role in maintaining the bile acids pool size by reabsorbing bile acids in the
ileum [6–8]. Ablation of ASBT function reduces bile acid pool size in mouse. Lower serum cholesterol
levels were also observed in humans with ASBT mutations [9]. Therefore, ASBT is an attractive target
for developing new cholesterol-lowering drugs [10]. Inhibition of ASBT function can increase bile acid
fecal loss, which in turn stimulates hepatic conversion of cholesterol into bile acids [11]. Because ASBT
is localized on the apical membrane of the lumen in the ileum, its inhibitors can block ASBT activity
without entering the circulation system. This non-systemic character of ASBT inhibitors implies a low
risk of potential systemic toxicity and drug–drug interactions [12,13]. So far, a number of ASBT
inhibitors having various structural characteristics have been synthesized. Among of them, three
candidates—264W94, SC-435 and R-146224 (Figure 1) were reported to block bile acid re-absorption
and reduce cholesterol levels significantly in animal models [14–16]. In addition, it has recently been
demonstrated in a Phase Ⅲ trial that A3309 (Figure 1), another ASBT inhibitor, can be used to treat
patients with chronic idiopathic constipation (CIC).
Figure 1. Structures of ASBT inhibitors.
O
O
S
O
O
S
O
O
N
OH
NH
Cl-
N+
O
N
264W94
SC-435
HOOC
HN
HN
O
F
O
S
O
O
N
O
N
O
O
O
Br
N
F
S
N
O
O
A3309
R-146224

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Baringhaus et al. developed a reliable 3D QSAR pharmacophore model for ASBT and screened a
novel compound S-1647 (Figure 2) with considerable inhibition against ASBT (IC50: 4 μM) [17]. The
simpler structure of S-1647 containing the three benzene rings A, B and C, compared with 264W94,
SC-435 and R-146224, attracted our attention. We decided to make structural modifications on S-1647.
In this study structure–activity relationships (SAR) of the relative positions of the ring C carbamyl
group to ring B were investigated first, leading to three classes of compounds, and then various
substitutions of rings A and C were added (Figure 2). Our primary objective was to optimize the
potency of S-1647 against ASBT and a preliminary SAR was also explored to facilitate the further study
of this class of compounds.
Figure 2. Design of arylsulfonylaminobenzanilides.
2. Result and Discussion
2.1. Chemistry
The synthetic pathways to this series of target compounds were shown in Scheme 1. Nucleophilic
substitution of substituted sulfonyl chlorides 1a–e with various aminobenzoates 2a–c in the presence
of pyridine in tetrahydrofuran (THF) gave arylsulfonylaminobenzoates 3a–g. Hydrolysis of the
benzoates 3a–g in a NaOH-H2O-EtOH system yielded the corresponding arylsulfonylaminobenzoic acids
4a–g. Coupling of the benzoic acids 4a–g with commercially available substituted anilines in the
presence of 1-hydroxybenzotrizole (HOBt), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
chloride (EDC.HCl) and ethyldiisopropylamine (DIEA) in dimethylformamide (DMF) afforded the
target compounds 5a–g.
Scheme 1. The synthesis of arylsulfonylamino-benzanilides 5a–g.
Reagents and conditions: (a) Pyridine, THF, r. t. (b) NaOH, EtOH, reflux; (c) selected amine, HOBt, EDC HCl,
DIEA, DMF, r.t.

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2.2. Biological Activity
In vitro inhibitory activity of all target compounds against ASBT was evaluated using a
radioisotope-based assay. All the newly synthesized derivatives were initially tested at 10 μM
concentration (Table 1).
Table 1. The structures and ASBT inhibitory rate of 5a1–a4, 5b1–b3 and 5c1–c2.
Inhibition
(%) a
Inhibition
(%) a
Compd.
R1
Compd.
R1
5a1 (S-1647)
3,4-dichloro
79.3 ± 5.2
95.4 ± 3.3
89.0 ± 2.6
83.8 ± 3.4
66.3 ± 5.0
5b2
5b3
5c1
5c2
3-difluoromethoxy
3,4-dichloro
30.6 ± 4.3
62.4 ± 2.9
29.6 ± 3.5
34.8 ± 3.7
5a2
5a3
5a4
3-chloro-4-fluoro
3-difluoromethoxy
3-trifluoromethoxy
3-chloro-4-fluoro
3- trifluoromethoxy
3,4-dichloro
5b1
a
Values represent the percent inhibition of ASBT at 10 µM of the test compounds and are the average of
three independent experiments.
The results suggest that the activity against ASBT decreased while the relative distance of the ring C
carbamyl group to ring B increased. For example, ortho position compounds 5a1–a4 exhibited better
activity than the corresponding meta position compounds 5b1–b3 and para position compounds 5c1–c2,
so the carbamyl group in the ortho position with respect to the ring B is preferably for activity. Then, we
explored the nitro group position in the ring A, and prepared two types of compounds (Table 2).
Table 2. The structures and ASBT inhibitory rate of 5a5–a10 and 5d1–d6.
Inhibition
(%) a
Inhibition
(%) a
Compd.
R1
Compd.
R1
5a5
5a6
5a7
5a8
5a9
3,5-difluoro
3-hydroxy-4-methoxy
3,5-dichloro
21.6 ± 4.1
32.1 ± 3.3
56.3 ± 2.8
99.1 ± 2.3
46.9 ± 3.6
5d1
5d2
5d3
5d4
5d5
5d6
2,4- dichloro
3-chloro-4-fluoro
66.9 ± 3.5
75.5 ± 4.4
73.7 ± 3.8
58.8 ± 4.3
66.7 ± 3.9
73.9 ± 2.2
3-trifluoromethyl-4-methyl
3-difluoromethoxy
3- trifluoromethoxy
3,4- dichloro
2,4-dichloro
3- chloro
5a10
3-trifluoromethyl-4-methyl 90.7 ± 4.2
a
Values represent the percent inhibition of ASBT at 10 µM of the test compounds and are the average of
three independent experiments.

Molecules 2013, 18
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The position of the nitro group was considered an important factor in the activity. Apparently,
compounds 5a1–a4, 5a8, and 5a10 with 3-nitro groups showed better activity than the corresponding
4-nitro analogues 5d1–d6, respectively. Finally, we investigated the R2 group in ring A, and designed
three types of compounds (Table 3).
Table 3. The structures and ASBT inhibitory rate of 5e1–e5, 5f1–f5 and 5g1–g5.
Inhibition
(%) a
Inhibition
(%) a
Compd.
R1
Compd.
R1
5e1
5e2
5e3
5e4
5e5
5f1
5f2
2,4-dichloro
3-chloro-4-fluoro
3-trifluoromethyl-4-methyl
3-trifluoromethoxy
3,4-dichloro
42.6 ± 3.4
63.2 ± 3.8
57.6 ± 2.9
45.7 ± 3.6
40.6 ± 2.8
21.2 ± 3.0
59.5 ± 3.6
43.0 ± 3.2
5f4
5f5
3-trifluoromethoxy
3,4-dichloro
25.8 ± 2.1
20.1 ± 1.9
89.3 ± 3.3
98.6 ± 2.6
91.8 ± 3.5
88.1 ± 2.7
80.8 ± 4.3
5g1
5g2
5g3
5g4
5g5
2,4-dichloro
3-chloro-4-fluoro
3-trifluoromethyl-4-methyl
3-trifluoromethoxy
3,4-dichloro
2,4-dichloro
3-chloro-4-fluoro
3-trifluoromethyl-4-methyl
5f3
a
Values represent the percent inhibition of ASBT at 10 µM of the test compounds and are the average of
three independent experiments.
The electronic properties of the R2 group were also an important factor in the activity.
Electron- withdrawing groups exhibited better activity than electron-donating groups. Compounds 5a,
5e and 5g showed better activity than 5f. Compounds where the R2 group was 3-trifluoromethoxy
showed the best inhibitory effect.
Table 4. IC50 values of target compounds.
Compd.
5a2
R1
R2
3-nitro
3-nitro
3-nitro
3-nitro
3-nitro
Inhibition (%) a
95.4 ± 3.3
89.0 ± 2.6
83.8 ± 3.4
99.1 ± 2.3
90.7 ± 4.2
89.3 ± 3.3
98.6 ± 2.6
91.8 ± 3.5
IC50 (µM)
1.32 ± 0.28
2.84 ± 0.52
1.23 ± 0.12
0.37 ± 0.08
1.49 ± 0.09
0.91 ± 0.18
0.11 ± 0.05
0.83 ± 0.11
3-chloro-4-fluoro
3-difluoromethoxy
3-trifluoromethoxy
2,4-dichloro
5a3
5a4
5a8
5a10
5g1
3-trifluoromethyl-4-methyl
2,4-dichloro
3-chloro-4-fluoro
3-trifluoromethyl-4-methyl
3-trifluoromethoxy
3-trifluoromethoxy
3-trifluoromethoxy
5g2
5g3

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Table 4. Cont.
Compd.
R1
R2
Inhibition (%) a
88.1 ± 2.7
IC50 (µM)
5g4
5g5
3-trifluoromethoxy
3,4-dichloro
3-trifluoromethoxy
3-trifluoromethoxy
3-nitro
1.03 ± 0.16
1.22 ± 0.20
1.52 ± 0.12
80.8 ± 4.3
S-1647
3,4-dichloro
79.3 ± 5.2
a
Values represent the percent inhibition of ASBT at 10 µM of the test compounds and are the average of
three independent experiments.
Finally, the compounds with inhibition rate above 80% were further assessed based on IC50 values
and the results are listed in Table 4. All of the tested compounds showed considerable activity
(IC50: 0.11–2.84 μM). Among of them, 5a8, 5g1, 5g2 and 5g3 were more active than the lead compound
S-1647. Particularly, compound 5g2 which had the best inhibitory activity (IC50: 0.11 μM) was found to
be 14- fold more active than S-1647 (IC50: 1.52 μM).
3. Experimental
3.1. General
All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik,
Flawil, Switzerland) and are uncorrected. Mass spectra (MS) were taken in ESI mode on an Agilent
1100 LC-MS system (Agilent, Palo Alto, CA, USA). Nuclear magnetic resonance spectroscopy was
performed using a 400 MHz Bruker ARX-400 spectrometers (Bruker Bioscience, Billerica, MA, USA)
with DMSO-d6 as solvent and TMS as an internal standard. All the starting materials were obtained from
commercially available sources and used without further purification, unless otherwise specified. Yields
were not optimized.
Methyl 2-(3-Nitrophenylsulfonamido)benzoate (3a). To a solution of 1a (5.0 g, 21.4 mmol) in THF
(60 mL) was added methyl 2-aminobenzoate (2a, 2.7 mL, 21.4 mmol) and then pyridine (1.7 mL,
21.4 mmol). The reaction mixture was stirred for 9 h at room temperature and then concentrated. To
the residue was added water and then 5% HCl. The mixture was stirred for 0.5h and filtered. The filter
cake was washed with water, dried and gave 3a as a red solid (74.0% yield); m.p.: 127.1–128.0 °C.
1H-NMR δ: 3.75 (3H, s), 7.25 (1H, t, J = 6.0 Hz), 7.39 (1H, d, J = 6.0 Hz), 7.57 (1H, t, J = 6.0 Hz),
7.79 (1H, dd, J1 = 6.0 Hz, J2 = 1.2 Hz), 7.85 (1H, t, J = 6.4 Hz), 8.15 (1H, d, J = 6.4 Hz), 8.44–8.47
(2H, m), 10.49 (1H, s). MS m/z: 359.07 [M+Na]+.
Methyl 3-(3-Nitrophenylsulfonamido)benzoate (3b). Compound 3b was obtained as a white solid
(78.7% yield) from compounds 1a and 2b as described for 3a; m.p.: 153.5–154.4 °C. 1H-NMR δ: 3.82
(3H, s) , 7.37–7.45 (2H, m), 7.66 (1H, d, J = 7.6 Hz), 7.72 (1H, s), 7.86 (1H, t, J = 8.0 Hz), 8.13 (1H, d,
J = 7.6 Hz), 8.45 (1H, dd, J1 = 8.0 Hz, J2= 2.0 Hz), 8.49 (1H, t, J = 2.0 Hz), 10.80 (1H, s). MS m/z:
335.16 [M-H]−.
Methyl 4-(3-Nitrophenylsulfonamido)benzoate (3c). Compound 3c was obtained as a white solid
(76.3% yield) from compounds 1a and 2c as described for 3a; m.p.: 209.9–211.0 °C. 1H-NMR δ: 3.78

Molecules 2013, 18
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(3H, s), 7.25 (2H, d, J = 8.8 Hz), 7.84–7.89 (3H, m), 8.20 (1H, d, J = 8.0 Hz), 8.45 (1H, dd, J1 = 8.0
Hz, J2= 2.0 Hz), 8.54 (1H, t, J = 2.0 Hz), 11.10 (1H, s). MS m/z: 335.16 [M−H]−.
Methyl 2-(4-Nitrophenylsulfonamido)benzoate (3d). Compound 3d was obtained as a yellow solid
(67.7% yield) from compounds 1b and 2a as described for 3a; m.p.: 154.9–155.7 °C. 1H-NMR δ: 3.76
(3H, s), 7.25 (1H, t, J = 6.0 Hz), 7.40 (1H, d, J = 6.8 Hz), 7.58 (1H, t, J = 6.0 Hz), 7.80 (1H, dd,
J1 = 6.4 Hz, J2 = 0.8 Hz), 8.01 (2H, d, J = 7.2 Hz), 8.35 (2H, d, J = 7.2 Hz), 10.55 (1H, s). MS m/z:
335.08 [M−H]−.
Methyl 2-(3-Fluorophenylsulfonamido)benzoate (3e). Compound 3e was obtained as a white solid
(72.6% yield) from compounds 1c and 2a as described for 3a; m.p.: 122.3–123.5 °C. 1H-NMR δ: 3.80
(3H, s), 7.22 (1H, t, J = 8.0 Hz), 7.43 (1H, d, J = 8.0 Hz), 7.51-7.63 (5H, m), 7.83 (1H, dd, J1 = 8.0 Hz,
J2 = 1.6 Hz), 10.42 (1H, s). MS m/z: 310.95 [M+H]+.
Methyl 2-(3-Methoxyphenylsulfonamido)benzoate (3f). Compound 3f was obtained as a white solid
(73.3% yield) from compounds 1d and 2a as described for 3a; m.p.: 123.7–124.9 °C. 1H-NMR δ: 3.76
(3H, s), 3.81 (3H, s), 7.17–7.24 (3H, m), 7.34 (1H, d, J = 7.6 Hz), 7.47 (2H, t, J = 8.0 Hz), 7.58 (1H, t,
J = 8.0 Hz), 7.84 (1H, dd, J1 = 8.0 Hz, J2 = 1.6 Hz), 10.37 (1H, s). MS m/z: 320.05 [M−H]−.
Methyl 2-(3-(Trifluoromethoxy)phenylsulfonamido)benzoate (3g). To a solution of 1e (8.6 g,
33.1 mmol) in THF (60 mL) was added methyl 2-aminobenzoate (2a, 5.0 g, 33.1 mmol) and then
pyridine (3.2 g, 39.7 mmol). The reaction mixture was stirred for 9 h at room temperature. To the
residue was added water and then 5% HCl. The mixture was stirred for 0.5 h and extracted with
dichloromethane. The dichloromethane layer was dried over MgSO4, concentrated in vacuo to afford
3g as a red liquid (75.2% yield) that was used directly for the next reaction without further purification.
2-(3-Nitrophenylsulfonamido)benzoic acid (4a). To a solution of 3a (5.7 g, 17.0 mmol) in the ethanol
(20 mL) was added 10% aqueous sodium hydroxide (12 mL). The mixture was heated to 80 °C for 8 h
and cooled to room temperature. The solution was concentrated and dissolved in water (50 mL). The
mixture was adjusted to pH 2 with 6 N hydrochloric acid to give a white precipitate. The precipitate
was filtered and washed with water to pH 7. The filter cake was dried to give 4a (86.2% yield) as a
white solid; m.p.: 219.8–220.5 °C. 1H-NMR δ: 7.17 (1H, t, J = 7.6 Hz), 7.47 (1H, d, J = 8.0 Hz), 7.56
(1H, t, J = 8.4 Hz), 7.82–7.88 (2H, m), 8.19 (1H, d, J = 7.6 Hz), 8.45–8.47 (2H, m), 11.28 (1H, s). MS
m/z: 321.08 [M−H]−.
3-(3-Nitrophenylsulfonamido)benzoic acid (4b). Obtained as a white solid (86.1% yield) from
compound 3b as described for 4a; m.p.: 249.0–250.2 °C. 1H-NMR δ: 7.34–7.41 (2H, m), 7.63 (1H, d,
J = 7.2 Hz), 7.69 (1H, s), 7.86 (1H, t, J = 8.0 Hz), 8.12 (1H, d, J = 8.0 Hz), 8.45 (1H, dd, J1 = 8.0 Hz,
J2 = 2.0 Hz,), 8.49 (1H, t, J = 2.0 Hz), 10.80 (1H, s), 13.10 (1H, s). MS m/z: 321.07 [M−H]−.
4-(3-Nitrophenylsulfonamido)benzoic acid (4c). Obtained as a white solid (83.9% yield) from
1
compound 3c as described for 4a; m.p.: 269.4–270.6 °C. H-NMR δ: 7.22 (2H, d, J = 8.8 Hz),
7.81–7.89 (3H, m), 8.20 (1H, d, J = 8.0 Hz), 8.45 (1H, dd, J1 = 8.0 Hz, J2= 2.0 Hz), 8.54 (1H, t,
J = 2.0 Hz), 11.06 (1H, s), 12.80 (1H, s). MS m/z: 321.08 [M−H]−.

Molecules 2013, 18
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2-(4-Nitrophenylsulfonamido)benzoic acid (4d). Obtained as a yellow solid (82.5% yield) from
compound 3d as described for 4a; m.p.: 238.1–239.2 °C. 1H-NMR δ: 7.16 (1H, t, J = 6.0 Hz), 7.47 (1H,
d, J = 6.4 Hz), 7.55 (1H, t, J = 6.8 Hz), 7.89 (1H, dd, J1 = 6.0 Hz, J2 = 1.2 Hz), 8.05 (2H, d, J = 7.2 Hz),
8.34 (2H, d, J = 7.2 Hz), 10.39 (1H, s). MS m/z: 321.00 [M−H]−.
2-(3-Fluorophenylsulfonamido)benzoic acid (4e). Obtained as a white solid (88.3% yield) from
compound 3e as described for 4a; m.p.: 187.1–188.2 °C. 1H-NMR δ: 7.16 (1H, t, J = 8.0 Hz), 7.48 (1H,
d, J = 8.0 Hz), 7.51–7.66 (5H, m), 7.90 (1H, dd, J1 = 8.0 Hz, J2 = 1.6 Hz), 11.22 (1H, s). MS m/z:
294.09 [M−H]−.
2-(3-Methoxyphenylsulfonamido)benzoic acid (4f). Obtained as a white solid (82.7% yield) from
1
compound 3f as described for 4a; m.p.: 157.1–158.3 °C. H-NMR δ: 3.76 (3H, s), 7.12 (1H, t,
J = 8.0 Hz), 7.20 (1H, dd, J1 = 8.0 Hz, J2 = 2.4 Hz), 7.26 (1H, t, J = 2.0 Hz), 7.35 (1H, d, J = 8.0 Hz),
7.47 (1H, t, J = 8.0 Hz), 7.52-7.58 (2H, m), 7.89 (1H, dd, J1 = 8.0 Hz, J2 = 1.6 Hz), 11.08 (1H, s),
13.97 (1H, s). MS m/z: 306.12 [M−H]−.
2-(3-(Trifluoromethoxy)phenylsulfonamido)benzoic acid (4g). Obtained as a white solid (85.1% yield)
1
from compound 3g as described for 4a; m.p.: 135.0–136.2 °C. H-NMR δ: 7.17 (1H, t, J = 8.0 Hz),
7.50 (1H, d, J = 8.0 Hz), 7.57 (1H, t, J = 8.0 Hz), 7.67-7.74 (3H, m), 7.84 (1H, d, J = 7.6 Hz), 7.89 (1H,
dd, J1 = 7.6 Hz, J2 = 1.2 Hz), 11.17 (1H, s). MS m/z: 360.11 [M−H]−.
3.2. General Procedure for the Synthesis of Arylsulfonylaminobenzanilides
To a solution of 4a–g (1 mmol) in dry DMF (10 mL) was added HOBt (1.5 mmol) and EDC.HCl
(1.5 mmol). The reaction mixture was stirred at room temperature for 2 h, and then the substituted
arylamines (2.0 mmol) and DIEA (2.0 mmol) were added, and then stirred at room temperature for 12 h,
poured into ice-cold water. The precipitate was filtered, washed with water, and then recrystallized with
ethyl acetate or purified by column chromatography (silica gel) to give the title compounds.
N-(3,4-Dichlorophenyl)-2-(3-nitrophenylsulfonamido)benzamide (5a1). White solid, 76.7% yield, m.p.:
1
185.0–186.3 °C. H-NMR δ: 7.30–7.34 (2H, m), 7.48–7.51 (2H, m), 7.57–7.62 (2H, m), 7.73 (1H, t,
J = 8.0 Hz), 7.95 (1H, s), 8.07 (1H, d, J = 7.6 Hz), 8.32 (1H, d, J = 8.0 Hz), 8.39 (1H, s), 10.34 (1H, s),
10.41 (1H, s). MS m/z: 464.18 [M−H]−.
N-(3-Chloro-4-fluorophenyl)-2-(3-nitrophenylsulfonamido)benzamide (5a2). White solid, 80.6% yield,
1
m.p.: 203.4–204.1 °C. H-NMR δ: 7.33–7.40 (3H, m), 7.45–7.53 (2H, m), 7.61 (1H, d, J = 7.2 Hz),
7.72 (1H, t, J =8.0 Hz), 7.87 (1H, dd, J1 = 6.8 Hz, J2 = 2.4 Hz), 8.06 (1H, d, J = 8.4 Hz), 8.32 (1H, dd,
J1 = 8.4 Hz, J2 = 1.6 Hz), 8.38 (1H, t, J =4.0 Hz), 10.35 (1H, s), 10.38 (1H, s). MS m/z: 448.07 [M−H]−.
N-(3-(Difluoromethoxy)phenyl)-2-(3-nitrophenylsulfonamido)benzamide (5a3). Yellow solid, 43.3%
1
yield, m.p.: 127.4–128.2 °C. H-NMR δ: 6.91 (1H, d, J = 8.0 Hz), 7.19 (1H, s), 7.31–7.42 (4H, m),
7.49–7.53 (2H, m), 7.63 (1H, d, J = 7.6 Hz), 7.71 (1H, t, J = 8.0 Hz), 8.08 (1H, d, J = 8.0 Hz), 8.31
(1H, dd, J1 = 8.0 Hz, J2 = 1.2 Hz), 8.38 (1H, t, J = 2.0 Hz), 10.33 (1H, s), 10.41 (1H, s). MS m/z:
462.15 [M−H]−.

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2-(3-Nitrophenylsulfonamido)-N-(3-(trifluoromethoxy)phenyl)benzamide (5a4). White solid, 47.6%
yield, m.p.: 141.4–142.0 °C. 1H-NMR δ: 7.08 (1H, d, J = 8.0 Hz), 7.33 (2H, t, J = 8.0 Hz), 7.44 (1H, t,
J = 8.0 Hz), 7.49–7.54 (2H, m), 7.63 (1H, d, J = 7.6 Hz), 7.70 (2H, t, J = 8.0 Hz), 8.07 (1H, d, J = 7.6 Hz),
8.31 (1H, dd, J1 = 8.4 Hz, J2 = 1.6 Hz), 8.39 (1H, t, J = 2.0 Hz), 10.37 (1H, s), 10.42 (1H, s). MS m/z:
504.12 [M+Na]+.
N-(3,5-Difluorophenyl)-2-(3-nitrophenylsulfonamido)benzamide (5a5). White solid, 78.0% yield, m.p.:
1
181.1–181.9 °C. H-NMR δ: 7.01 (1H, d, J = 6.0 Hz), 7.23 (1H, t, J = 6.0 Hz), 7.35–7.41 (3H, m),
7.44–7.46 (1H, m), 7.48–7.52 (2H, m), 8.03 (1H, t, J = 6.4 Hz), 8.45 (1H, d, J = 6.4 Hz), 8.67 (1H, dd,
J1 = 6.8 Hz, J2 = 1.2 Hz), 8.73 (1H, s), 10.24 (1H, s). MS m/z: 432.12 [M−H]−.
N-(3-Hydroxy-4-methoxyphenyl)-2-(3-nitrophenylsulfonamido)benzamide (5a6). Yellow solid, 65.1%
yield, m.p.: 168.9–170.1 °C. 1H-NMR δ: 3.30 (3H, s), 7.01 (1H, d, J = 6.0 Hz), 7.23 (1H, t, J = 6.0 Hz),
7.35–7.40 (3H, m), 7.44–7.47 (1H, m), 7.49–7.52 (2H, m), 8.03 (1H, t, J = 6.4 Hz), 8.45 (1H, d,
J = 6.4 Hz), 8.67 (1H, d J = 6.4 Hz), 8.73 (1H, s), 10.24 (1H, s). MS m/z: 442.08 [M−H]−.
N-(3,5-Dichlorophenyl)-2-(3-nitrophenylsulfonamido)benzamide (5a7). White solid, 72.3% yield, m.p.:
209.9–210.4 °C. 1H-NMR δ: 7.30-7.36 (3H, m), 7.51 (1H, t, J = 8.0 Hz), 7.60 (1H, d, J = 7.2 Hz), 7.64
(2H, d, J = 2.0 Hz), 7.73 (1H, t, J = 8.0 Hz), 8.06 (1H, d, J = 8.4 Hz), 8.31-8.33 (1H, m), 8.40–8.41
(1H, m), 10.31 (1H, s), 10.45 (1H, s). MS m/z: 464.07 [M−H]−.
N-(2,4-Dichlorophenyl)-2-(3-nitrophenylsulfonamido)benzamide (5a8). White solid, 49.6% yield, m.p.:
203.6–204.1 °C. 1H-NMR δ: 7.30–7.35 (2H, m), 7.49–7.59 (3H,m), 7.72 (1H, d, J = 2.0 Hz),7.83 (1H,
t, J = 8.0 Hz), 7.93 (1H, d, J = 8.0 Hz), 8.14 (1H, dd, J1 = 8.0 Hz, J2 = 2.0 Hz), 8.38 (1H, t, J = 2.0 Hz),
8.45 (1H, d, J = 8.0 Hz), 10.24 (1H, s), 10.88 (1H, s). MS m/z: 464.10 [M−H]−.
N-(3-Chlorophenyl)-2-(3-nitrophenylsulfonamido)benzamide (5a9). White solid, 70.2% yield, m.p.:
1
167.3–168.1 °C. H-NMR δ: 7.15 (1H, d, J = 8.0 Hz), 7.31–7.35 (3H, m), 7.46 (1H, d, J = 8.4 Hz),
7.50 (1H, t, J = 8.0 Hz), 7.63 (1H, d, J = 7.6 Hz), 7.72 (1H, t, J = 8.0 Hz), 7.77 (1H, s), 8.07 (1H, d,
J = 8.0 Hz), 8.32 (1H, dd, J1 = 8.0 Hz, J2 = 1.6 Hz), 8.39 (1H, t, J = 2.4 Hz), 10.33 (1H, s), 10.40 (1H,
s). MS m/z: 430.09 [M−H]−.
N-(4-Methyl-3-(trifluoromethyl)phenyl)-2-(3-nitrophenylsulfonamido)benzamide (5a10). White solid,
40.3% yield, m.p.: 166.4–167.4 °C 1H-NMR δ: 2.40 (3H, s), 7.31–7.38 (3H, m), 7.51 (1H, t, J = 7.6 Hz),
7.64 (1H, d, J = 7.6 Hz), 7.70 (2H, t, J = 8.0 Hz), 7.97 (1H, d, J = 1.2 Hz), 8.06 (1H, d, J = 8.0 Hz),
8.27–8.30 (1H, m), 8.37 (1H, t, J = 2.0 Hz), 10.36 (1H, s), 10.42 (1H, s). MS m/z: 478.10 [M−H]−.
N-(3-Chloro-4-fluorophenyl)-3-(3-nitrophenylsulfonamido)benzamide (5b1). White solid, 55.3% yield,
1
m.p.: 161.3–162.6 °C. H-NMR δ: 7.31–7.33 (1H, m), 7.37–7.45 (2H, m), 7.63–7.67 (3H, m), 7.86
(1H, t, J = 8.0 Hz), 8.01 (1H, dd, J1 = 7.2 Hz, J2= 2.8 Hz), 8.13–8.15 (1H, m), 8.43–8.46 (1H, m), 8.51
(1H, t, J = 2.0 Hz), 10.41 (1H, s), 10.80 (1H, s). MS m/z: 448.75 [M−H]−.
N-(3-(Difluoromethoxy)phenyl)-3-(3-nitrophenylsulfonamido)benzamide (5b2). White solid, 57.7%
1
yield, m.p.: 159.8–160.6 °C. H-NMR δ: 6.90 (1H, d, J = 8.0 Hz), 7.19 (1H, s), 7.31–7.44 (3H, m),

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7.59 (1H, d, J = 8.0 Hz), 7.66 (3H, d, J = 8.0 Hz), 7.85 (1H, t, J = 8.0 Hz), 8.15 (1H, d, J = 8.0 Hz),
8.45 (1H, d, J = 8.0 Hz), 8.51 (1H, s), 10.39 (1H, s), 10.80 (1H, s). MS m/z: 462.49 [M−H]−.
N-(3,4-dichlorophenyl)-3-(3-nitrophenylsulfonamido)benzamide (5b3). Yellow solid, 60.8% yield, m.p.:
192.5–193.7 °C. 1H-NMR δ: 7.32 (1H, d, J = 8.0 Hz), 7.43 (1H, t, J = 8.0 Hz), 7.59–7.70 (4H, m), 7.85
(1H, t, J = 8.0 Hz), 8.09 (1H, d, J = 2.4 Hz), 8.15 (1H, d, J = 8.0 Hz), 8.45 (1H, d, J = 8.0 Hz), 8.51
(1H, s), 10.48 (1H, s), 10.80 (1H, s). MS m/z: 464.91 [M−H]−.
4-(3-Nitrophenylsulfonamido)-N-(3-(trifluoromethoxy)phenyl)benzamide (5c1). White solid, 51.3%
yield, m.p.: 177.3–178.2 °C. 1H-NMR δ: 7.09 (1H, d, J = 8.8 Hz), 7.26 (2H, d, J = 8.0 Hz), 7.46 (1H, t,
J = 8.0 Hz), 7,71 (1H, d, J = 8.0 Hz), 7.85–7.91 (4H, m), 8.23 (1H, d, J = 8.0 Hz), 8.47 (1H, dd,
J1 = 8.0 Hz, J2= 2.0 Hz), 8.57 (1H, s), 10.37 (1H, s), 11.04 (1H, s). MS m/z: 480.57 [M−H]−.
N-(3,4-Dichlorophenyl)-4-(3-nitrophenylsulfonamido)benzamide (5c2). White solid, 57.8% yield, m.p.:
189.2–190.5 °C. 1H-NMR δ: 7.25 (2H, d, J = 8.0 Hz), 7.59 (1H, d, J = 8.8 Hz), 7.69 (1H, dd, J1 = 8.8
Hz, J2= 2.4 Hz), 7.83–7.90 (3H, m), 8.09 (1H, d, J = 2.0 Hz), 8.22 (1H, d, J = 8.0 Hz), 8.47 (1H, dd,
J1 = 8.0 Hz, J2= 2.0 Hz), 8.56 (1H, s), 10.34 (1H, s), 11.04 (1H, s). MS m/z: 464.65 [M−H]−.
N-(2,4-Dichlorophenyl)-2-(4-nitrophenylsulfonamido)benzamide (5d1). White solid, 76.1% yield, m.p.:
220.6–221.3 °C. 1H-NMR δ: 7.31–7.37 (2H, m), 7.46–7.57 (3H, m), 7.72 (1H, d, J = 2.0 Hz), 7.97 (2H,
d, J = 8.8 Hz), 8.33 (3H, m), 10.25 (1H, s), 10.96 (1H, s). MS m/z: 464.08 [M−H]−.
N-(3-Chloro-4-fluorophenyl)-2-(4-nitrophenylsulfonamido)benzamide (5d2). White solid, 77.5% yield,
1
m.p.: 226.4–227.1 °C. H-NMR δ: 7.35–7.40 (3H, m), 7.48–7.54 (2H, m), 7.64 (1H, d, J = 7.2 Hz),
7.85 (1H, dd, J1 = 7.2 Hz, J2 = 2.4 Hz), 7.91 (2H, d, J = 8.8 Hz), 8.21 (2H, d, J = 8.8 Hz), 10.33 (1H, s),
10.39 (1H, s). MS m/z: 448.07 [M−H]−.
N-(4-Methyl-3-(trifluoromethyl)phenyl)-2-(4-nitrophenylsulfonamido)benzamide (5d3). White solid,
1
53.7% yield, m.p.: 198.8–199.6 °C. H-NMR δ: 2.41 (3H, s), 7.33–7.40 (3H, m), 7.52–7.56 (1H, m),
7.67 (1H, d, J = 7.6 Hz), 7.74 (1H, d, J = 7.6 Hz),7.90 (2H, d, J = 8.8 Hz), 7.95 (1H, s), 8.18 (2H, d,
J = 8.8 Hz), 10.36 (1H, s), 10.43 (1H, s). MS m/z: 478.21 [M−H]−.
N-(3-(Difluoromethoxy)phenyl)-2-(4-nitrophenylsulfonamido)benzamide (5d4). White solid, 69.1%
1
yield, m.p.: 203.9–205 °C. H-NMR δ: 6.91 (1H, d, J = 8.8 Hz), 7.19 (1H, s), 7.33–7.41 (3H, m),
7.49–7.54 (2H, m), 7.65 (1H, d, J = 7.2 Hz), 7.92 (2H, d, J = 8.8 Hz), 8.21 (2H, d, J = 8.8 Hz), 10.33
(1H, s), 10.44 (1H, s). MS m/z: 462.15 [M−H]−.
2-(4-Nitrophenylsulfonamido)-N-(3-(trifluoromethoxy)phenyl)benzamide (5d5). White solid, 70.3%
yield, m.p.: 198.8–199.4 °C. 1H-NMR δ: 7.08 (1H, d, J = 7.6 Hz), 7.33 (2H, d, J = 8.0 Hz), 7.44 (1H, t,
J = 8.0 Hz), 7.53 (2H, d, J = 8.0 Hz), 7.65 (1H, d, J = 7.2 Hz), 7.73 (1H,s), 7.91 (2H, d, J = 8.8 Hz),
8.20 (2H, d, J = 8.8 Hz), 10.38 (1H, s), 10.40 (1H, s). MS m/z: 480.21 [M−H]−.
N-(3,4-Dichlorophenyl)-2-(4-nitrophenylsulfonamido)benzamide (5d6). White solid, 65.2% yield, m.p.:
1
243.4–243.6 °C. H-NMR δ: 7.32–7.34 (2H, m), 7.52 (1H, dd, J1 = 8.4 Hz, J2 = 2.4 Hz), 7.56 (2H, t,

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J = 8.8 Hz), 7.62 (1H, d, J = 7.2 Hz), 7.90 (2H, d, J = 8.8 Hz),7.92 (1H,s), 8.21 (2H, d, J = 8.8 Hz),
10.35 (1H, s), 10.39 (1H, s). MS m/z: 464.08 [M−H]−.
N-(2,4-Dichlorophenyl)-2-(3-fluorophenylsulfonamido)benzamide (5e1). White solid, 67.6% yield,
1
m.p.: 146.3–147.6 °C. H-NMR δ: 7.29 (1H, t, J = 7.2 Hz), 7.40 (1H, d, J = 8.0 Hz), 7.50–7.64
(7H, m), 7.75 (1H, d, J = 2.0 Hz), 7.88 (1H, d, J = 8.0 Hz), 10.34 (1H, s), 10.90 (1H, s). MS m/z:
438.05 [M−H]−.
N-(3-Chloro-4-fluorophenyl)-2-(3-fluorophenylsulfonamido)benzamide (5e2). White solid, 52.1% yield,
1
m.p.: 178.6–179.9 °C. H-NMR δ: 7.29 (1H, t, J = 7.6 Hz), 7.35 (1H, d, J = 8.0 Hz), 7.41–7.59
(7H, m), 7.70 (1H, d, J = 7.6 Hz), 7.95 (1H, dd, J1 = 6.8 Hz, J2= 2.4 Hz), 10.41 (1H, s), 10.47 (1H, s).
MS m/z: 421.28 [M−H]−.
2-(3-Fluorophenylsulfonamido)-N-(4-methyl-3-(trifluoromethyl)phenyl)benzamide (5e3). Yellow solid,
1
55.8% yield, m.p.: 168.8–169.6 °C. H-NMR δ: 2.41 (3H, s), 7.27 (1H, t, J = 7.3 Hz), 7.35 (1H, d,
J = 8.4 Hz), 7.42 (2H, d, J = 8.1 Hz), 7.48–7.56 (3H, m), 7.72 (1H, d, J = 6.8 Hz), 7.78 (1H, d,
J = 8.0 Hz), 8.03 (1H, s), 10.46 (1H, s), 10.48 (1H, s). MS m/z: 451.30 [M−H]−.
2-(3-Fluorophenylsulfonamido)-N-(3-(trifluoromethoxy)phenyl)benzamide (5e4). White solid, 61.7%
1
yield, m.p.: 166.8–168.7 °C. H-NMR δ: 7.11 (1H, d, J = 8.4 Hz), 7.26–7.34 (2H, m), 7.39–7.57
(6H, m), 7.62 (1H, d, J = 8.4 Hz), 7.70 (1H, d, J = 7.2 Hz), 7.79 (1H, s), 10.37 (1H, s), 10.52 (1H, s).
MS m/z: 453.68 [M−H]−.
N-(3,4-Dichlorophenyl)-2-(3-fluorophenylsulfonamido)benzamide (5e5). White solid, 58.5% yield,
1
m.p.: 179.1–180.6 °C. H-NMR δ: 7.27–7.33 (2H, m), 7.42–7.58 (5H, m), 7.60–7.64 (2H, m), 7.69
(1H, d, J = 8.0 Hz), 8.03 (1H, d, J = 1.6 Hz), 10.34 (1H, s), 10.53 (1H, s). MS-ESI m/z: 437.34
[M−H]−.
N-(2,4-Dichlorophenyl)-2-(3-methoxyphenylsulfonamido)benzamide (5f1). White solid, 51.5% yield,
1
m.p.: 138.8–139.7 °C. H-NMR δ: 3.73 (3H, s), 7.19–7.30 (4H, m), 7.43–7.54 (4H, m), 7.61 (1H, d,
J = 8.4 Hz), 7.75 (1H, d, J = 1.6 Hz), 7.88 (1H, d, J = 8.0 Hz), 10.35 (1H, s), 10.85 (1H, s). MS m/z:
449.44 [M−H]−.
N-(3-Chloro-4-fluorophenyl)-2-(3-methoxyphenylsulfonamido)benzamide (5f2). White solid, 57.8%
1
yield, m.p.: 181.9–183.2 °C. H-NMR δ: 3.67 (3H, s), 7.12 (1H, dd, J1 = 8.0 Hz, J2= 2.4 Hz),
7.25–7.29 (2H, m), 7.36–7.45 (3H, m), 7.51 (1H, t, J = 7.6 Hz), 7.56–7.60 (1H, m), 7.71 (1H, d,
J = 7.6 Hz), 7.94 (1H, dd, J1 = 6.8 Hz, J2= 2.4 Hz), 10.36 (1H, s), 10.46 (1H, s). MS m/z: 433.65
[M−H]−.
2-(3-Methoxyphenylsulfonamido)-N-(4-methyl-3-(trifluoromethyl)phenyl)benzamide(5f3).White solid,
1
56.2% yield, m.p.: 165.7–166.6 °C. H-NMR δ: 2.42 (3H, s), 3.63 (3H, s), 7.11 (1H, d, J = 8.0 Hz),
7.21 (1H, s), 7.26 (2H, t, J = 8.7 Hz), 7.35–7.44 (3H, m), 7.51 (1H, t, J = 7.7 Hz), 7.74 (1H, d, J = 7.7 Hz),
7.80 (1H, d, J = 8.0 Hz), 8.04 (1H, s), 10.42 (1H, s), 10.49 (1H, s). MS m/z: 463.58 [M−H]−.

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2-(3-Methoxyphenylsulfonamido)-N-(3-(trifluoromethoxy)phenyl)benzamide (5f4). White solid, 63.5%
1
yield, m.p.: 149.3–150.8 °C. H-NMR δ: 3.64 (3H, s), 7.09–7.27 (5H, m), 7.34–7.41 (2H, m),
7.46–7.53 (2H, m), 7.63 (1H, d, J = 7.6 Hz), 7.71 (1H, d, J = 7.6 Hz), 7.78 (1H, s), 10.33 (1H, s),
10.52 (1H, s). MS m/z: 465.22 [M−H]−.
N-(3,4-Dichlorophenyl)-2-(3-methoxyphenylsulfonamido)benzamide (5f5). White solid, 67.9% yield,
1
m.p.: 160.3–161.4 °C. H-NMR δ: 3.66 (3H, s), 7.11 (1H, dd, J1 = 8.4 Hz, J2= 2.4 Hz), 7.28–7.30
(3H, m), 7.35–7.39 (2H, m), 7.50 (1H, d, J = 8.0 Hz), 7.57–7.63 (2H, m), 7.69 (1H, d, J = 8.0 Hz),
8.01 (1H, d, J = 2.0 Hz), 10.27 (1H, s), 10.51 (1H, s). MS m/z: 449.57 [M−H]−.
N-(2,4-Dichlorophenyl)-2-(3-(trifluoromethoxy)phenylsulfonamido)benzamide (5g1). White solid,
1
69.6% yield, m.p.: 151.6–152.8 °C. H-NMR δ: 7.30 (1H, t, J = 7.6 Hz), 7.35 (1H, d, J = 8.0 Hz),
7.49–7.51 (2H, m), 7.62–7.78 (6H, m), 7.88 (1H, d, J = 7.6 Hz), 10.31 (1H, s),10.94 (1H, s). MS m/z:
503.12 [M−H]−.
N-(3-Chloro-4-fluorophenyl)-2-(3-(trifluoromethoxy)phenylsulfonamido)benzamide (5g2). White solid,
68.1% yield, m.p.: 157.3–158.6 °C. 1H-NMR δ: 7.28–7.34 (2H, m), 7.42 (1H, t, J = 8.8 Hz), 7.50 (1H,
t, J = 8.0 Hz), 7.54–7.74 (6H, m), 7.96 (1H, dd, J1 = 6.8 Hz, J2= 2.4 Hz), 10.46 (2H, s). MS m/z:
487.85 [M−H]−.
N-(4-Methyl-3-(trifluoromethyl)phenyl)-2-(3-(trifluoromethoxy)phenylsulfonamido)benzamide (5g3).
1
White solid, 55.7% yield, m.p.: 179.4–180.5 °C. H-NMR δ: 2.42 (3H, s), 7.31 (1H, d, J = 7.6 Hz),
7.35 (1H, d, J = 8.4 Hz), 7.42 (1H, d, J = 8.4 Hz), 7.51 (1H, t, J = 8.0 Hz), 7.56–7.62 (2H, m), 7.64
(1H, s), 7.70–7.73 (2H, m), 7.77 (1H, d, J = 8.4 Hz), 8.06 (1H, s), 10.47 (1H, s), 10.51 (1H, s). MS m/z:
517.52 [M−H]−.
N-(3-(Trifluoromethoxy)phenyl)-2-(3-(trifluoromethoxy)phenylsulfonamido)benzamide (5g4). White
1
solid, 62.3% yield, m.p.: 169.1–170.4 °C. H-NMR δ: 7.11 (1H, d, J = 8.4 Hz), 7.29–7.34 (2H, m),
7.46–7.51 (2H, m), 7.57–7.62 (3H, m), 7.66 (1H, s), 7.71 (2H, t, J = 7.2 Hz), 7.81 (1H, s), 10.43
(1H, s), 10.52 (1H, s). MS m/z: 519.12 [M−H]−.
N-(3,4-Dichlorophenyl)-2-(3-(trifluoromethoxy)phenylsulfonamido)benzamide (5g5). White solid,
1
66.7% yield, m.p.: 160.5–161.6 °C. H-NMR δ: 7.32 (2H, t, J = 8.0 Hz), 7.51 (1H, t, J = 8.0 Hz),
7.58–7.74 (7H, m), 8.05 (1H, d, J = 2.0 Hz), 10.40 (1H, s), 10.53 (1H, s). MS m/z: 503.75 [M−H]−.
3.3. ASBT Inhibition Assay
HEK293T was obtained from the American Type Culture Collection (Manassas, VA, USA), and
grown in MEM supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin and 10%
heat-inactivated fetal bovine serum. Human ASBT expression construct was prepared as previously
described [18]. HEK293T cells were seeded in 12 well plates and transiently transfected
with 0.5 μg/well pcDNA3.1/ASBT or negative control plasmid pcDNA3.1 using lipofactamine 2000
(Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Twenty-four hours after
transfection, HEK293T cells were assayed for taurocholic acid uptake as previously described with

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minor modifications [19]. Briefly, cells were washed twice with warm wash & uptake buffer (116 mM
NaCl, 5.3 mM KCl, 1.1 mM KH2PO4, 0.8 mM MgSO4, 1.8 mM CaCl2, 11 mM D-dextrose/D-glucose,
and 10 mM HEPES, pH 7.4), then cells were incubated with the same buffer containing the indicated
concentrations of test compounds (dissolved in dimethyl sulfoxide) and 1 μCi/mL of [3H]-taurocholic
acid(TCA, 0.2 μM) (PerkinElmer Life Sciences) for 10 min. To terminate the transport process, the
plates were chilled on ice and the cells were immediately washed with ice-cold buffer three times. Cells
were lysed with 0.3 mL lysis buffer (0.5% triton x-100) and shaken vigorously for 20 min. The
radioactivity of the cell lysate was counted using a MicroBeta2 Liquid Scintillation and Luminescence
Counter (PerkinElmer Life Sciences). Protein concentration of the lysate was used to normalize
uptake activity.
The inhibition rate was calculated using the following formula:
Inhibition Rate (%) = [(1 − (A − C)/(B − C)] × 100
A, [3H] uptake value of test compound in [3H]-TCA buffer added to pcDNA3.1/ASBT transfected
cells; B, [3H] uptake value of DMSO (without inhibitor) in [3H]-TCA buffer added to pcDNA3.1/ASBT
transfected cells; C, [3H] uptake value of Blank sample (DMSO) in [3H]-TCA buffer added to
empty vector (pcDNA3.1) transfected cells. The IC50 value was defined as the inhibitor concentration
that gives 50% taurocholate uptake compared to the no inhibitor control and calculated using
SigmaPlot 10.0 software.
4. Conclusions
A series of novel arylsulfonylaminobenzanilide derivatives were designed and synthesized. Their
inhibitory activities against ASBT were assessed. In general, most of them had considerable ASBT
inhibitory activity. In particular, four compounds (5a8, 5g1, 5g2 and 5g3) were superior to the lead
compound S-1647, especially compound 5g2 which exhibited the most inhibitory effect on ASBT
transport activity with an IC50 value of 0.11 μM, or 14-fold more potent than S-1647. It is not known if
these compounds can pass the cell membrane using a transporter or permissively, so future studies will
address this issue in vitro in cells and in vivo in animal models. It is unlikely that these compounds will have
much cytotoxicity as both HEK293T and CACO2 cells showed no morphological differences when treated
with 20 μM of the compounds for 24 h.
Acknowledgments
We are grateful to the financial support from the National S&T Major Special Project on Major New
Drug Innovation (2012ZX093101002-001-017) and the National Natural Science Foundation of China
(No. 81072577 and 81102376).
Conflict of Interest
The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 5a9 and 5a10 are available from the authors.
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