Identification of: N -benzothiazolyl-2-benzenesulfonamides as novel ABCA1 expression upregulators

Article abstract of DOI:10.1039/c9md00556k

ATP binding cassette transporter A1 (ABCA1) is a critical transporter that mediates cellular cholesterol efflux from macrophages to apolipoprotein A-I (ApoA-I). Therefore, increasing the expression level of ABCA1 is anti-atherogenic and ABCA1 expression upregulators have become novel choices for atherosclerosis treatment. In this study, a series of N-benzothiazolyl-2-benzenesulfonamides, based on the structure of WY06 discovered in our laboratory, were designed and synthesized as novel ABCA1 expression upregulators. Based on an in vitro ABCA1 upregulatory cell model, ABCA1 upregulation of target compounds was evaluated. Compounds 6c, 6d, and 6i have good upregulated ABCA1 expression activities, with EC₅₀ values of 0.97, 0.37, and 0.41 ΜM, respectively. A preliminary structure-activity relationship is summarized. Replacing the methoxy group on the benzothiazole moiety of WY06 with a fluorine or chlorine atom and exchanging the ester group with a cyano group resulted in more potent ABCA1 upregulating activity. Moreover, compound 6i increased ABCA1 mRNA and protein expression and significantly promoted cholesterol efflux in RAW264.7 cells. In conclusion, N-benzothiazolyl-2-benzenesulfonamides were identified as novel ABCA1 expression upregulators.

Full text of DOI:10.1039/c9md00556k

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RESEARCH ARTICLE
Identification of N-benzothiazolyl-2-
benzenesulfonamides as novel ABCA1 expression
Cite this: DOI: 10.1039/c9md00556k
upregulators†
Hongtao Liu,‡ab Xinhai Jiang,‡c Xinfeng Gao,‡b Wenhua Tian,b Chen Xu,b
b
c
b
Ruizhi Wang, Yanni Xu, Liping Wei,d Feng Caoe and Wenyan Li
*
*
ATP binding cassette transporter A1 (ABCA1) is a critical transporter that mediates cellular cholesterol efflux
from macrophages to apolipoprotein A-I (ApoA-I). Therefore, increasing the expression level of ABCA1 is
anti-atherogenic and ABCA1 expression upregulators have become novel choices for atherosclerosis
treatment. In this study, a series of N-benzothiazolyl-2-benzenesulfonamides, based on the structure of
WY06 discovered in our laboratory, were designed and synthesized as novel ABCA1 expression
upregulators. Based on an in vitro ABCA1 upregulatory cell model, ABCA1 upregulation of target
compounds was evaluated. Compounds 6c, 6d, and 6i have good upregulated ABCA1 expression activities,
with EC50 values of 0.97, 0.37, and 0.41 μM, respectively. A preliminary structure–activity relationship is
summarized. Replacing the methoxy group on the benzothiazole moiety of WY06 with a fluorine or
chlorine atom and exchanging the ester group with a cyano group resulted in more potent ABCA1
upregulating activity. Moreover, compound 6i increased ABCA1 mRNA and protein expression and
significantly promoted cholesterol efflux in RAW264.7 cells. In conclusion, N-benzothiazolyl-2-
benzenesulfonamides were identified as novel ABCA1 expression upregulators.
Received 26th November 2019,
Accepted 11th February 2020
DOI: 10.1039/c9md00556k
rsc.li/medchem
macrophages in the arterial wall and transports it back to the
liver for degradation to bile acids. ATP-binding cassette
Introduction
Coronary heart disease (CHD), stroke, and peripheral vascular
disease are the leading causes of death and represent a heavy
burden to healthcare systems around the world.1–4 High-
density lipoprotein (HDL) is an independent protective factor
against cardiovascular disease through many potential
mechanisms such as inhibiting inflammation, regulating
nitric oxide (NO) production, and anti-thrombotic and anti-
apoptotic activities.5–8 The most established cardioprotective
effect of HDL is its role in reverse cholesterol transport
(RCT),8 in which HDL removes excess cholesterol from
transporter A1 (ABCA1) mediates the rate-controlling step in
the initial stages of HDL formation and as much as 70% of
the total cellular cholesterol is effluxed to apolipoprotein A-I
(ApoA-I) from macrophages.9,10 Overexpression of ABCA1 in
macrophages promotes HDL formation and RCT, and reduces
atherosclerosis.11,12 However, humans with Tangier disease
(TD), who are deficient in ABCA1, exhibit severe HDL
deficiency and a higher incidence of cardiovascular disease.13
Thus, ABCA1 has been identified as a potential target for
novel pharmacological agents that are designed to increase
the cardiovascular protective potential of HDL.14 ABCA1
a Department of Pharmacy, Hebei General Hospital, Shijiazhuang 05005, China
b Hebei Key Laboratory of Organic Functional Molecules, College of Chemistry and
Materials Science, Hebei Normal University, Shijiazhuang, 050024, China.
E-mail: wyli8512@mail.hebtu.edu.cn
c NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal
Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical
College (CAMS&PUMC), Beijing 100050, China.
E-mail: xuyanniwendeng@hotmail.com
d Department of Cardiology, Tianjin Union Medical Center, Nankai University
Affiliated Hospital, 190 Jieyuan Road, Hongqiao, Tianjin 300121, P. R. China
e Department of Cardiology & National Clinical Research Center of Geriatrics
Disease, Chinese PLA General Hospital, Beijing 100853, China
† Electronic supplementary information (ESI) available: NMR and MS spectra of
compounds 6a–6n. See DOI: 10.1039/c9md00556k
‡ These authors contributed equally to this work.
Fig. 1 The structure of some ABCA1 expression upregulators.
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expression upregulators may be potentially used in
atherosclerosis treatment.15–17 In addition, ABCA1 is also
associated with the occurrence and development of other
diseases such as Alzheimer's disease, inflammation, ischemic
heart disease, and diabetes. Therefore, regulating ABCA1 may
play an important role for the treatment of these diseases.18–21
Currently, several natural or synthetic compounds such as
pratensein,22 rutaecarpine (Fig. 1),23,24 and CS6253 (ref. 25) (a
26-amino acid peptide) have been reported as ABCA1
expression upregulators, but none of these has been released
to the market to date. In our previous study, WY06 with a
benzothiazole scaffold was found to display potent ABCA1
upregulatory activity using a high-throughput assay.22 In this
study, the structure of WY06 was optimized by replacing the
substituents on the benzothiazole ring and sulfonamide
nitrogen atoms and by changing the position of the amine
group on the benzene ring. A series of WY06 analogues were
synthesized and evaluated. Our primary objective was to
identify more potent ABCA1 expression upregulators. The
structure and activity relationship (SAR) was summarized to
facilitate further design. Moreover, the primary mechanism
of action of the most potent compound was also investigated.
Pharmacology
Table 1 presents the series of WY06 derivatives that were
synthesized and evaluated for their ABCA1 upregulatory
activities. We first examined the influence of substituents on
the benzothiazole moiety. Removing or replacing the methoxy
group on the benzothiazole moiety with a fluorine or chlorine
atom, as in 6b–d, resulted in more potent activity, including
increased upregulating value and decreased EC50 value. In
particular, compound 6d with a chlorine atom showed a 17-
fold increase in potency (EC50, 0.37 0.08 μM) compared with
WY06 (EC50, 6.38 0.09 μM). Subsequently, the ester group at
the R2 position, which might be hydrolyzed in vivo, was
exchanged with a cyano group or vinyl group (6e–j). Generally,
most of these analogues displayed lower potency than the
corresponding ester targets. The contribution of R2 to the EC50
activity seemed to be as follows: ester > cyano > vinyl (6b vs.
6e vs. 6f; 6c vs. 6g vs. 6h; 6d vs. 6i vs. 6j). However, compound
6i with the cyano group not only displayed comparable EC50
activity to 6d, but also exhibited enhanced upregulating
potency. In addition, a series of compounds 6k–m with an
amino group at the meta-position of benzene were also
synthesized and evaluated, all of them exhibiting decreased
potency or even lost potency (6k and 6l, EC50 > 50 μM).
Results and discussion
Chemistry
Table
1 shows that compounds 6d and 6i exhibit
exceptional EC50 values, which are >15-fold higher than that
of lead compound WY06, and 6i showed slightly higher
ABCA1 upregulating activity than 6d. Subsequently, the
luciferase assay of compound 6i at 6 h, 12 h, 24 h, 48 h, and
The detailed synthetic pathway to target compounds 6a–n is
shown in Scheme 1. Anilines 1a–d were treated with
potassium thiocyanate in chlorobenzene at 100 °C to yield
thioureas 2a–d, which were cyclized in the presence of Br2 to
yield benzo[d]-2-amines 3a–d. Coupling of the amines 3a–d
72
h was examined to further understand the ABCA1
upregulatory process.
with
3-/4-nitrobenzenesulfonyl
chloride
afforded
The results (Fig. 2) show that compound 6i exhibited
dose-dependent upregulating ABCA1 activity at different time
points, and the upregulating ABCA1 activity peaked at 24 h
and 48 h.
To confirm the above luciferase assay results, compound
6i was further evaluated in terms of ABCA1 mRNA and
protein expression levels in RAW264.7 cells. Western blot and
real time quantitative PCR (RT-qPCR) analyses were
performed to investigate the effect of 6i on ABCA1 mRNA and
protein expression. RT-qPCR analysis showed that 6i
benzenesulfonamides 4a–d, and then the nitro group was
reduced with Zn powder and NH4Cl to yield 3-/4-
aminobenzenesulfonamides 5a–d. Targets 6a–n were obtained
by nucleophilic substitution of the resulting sulfonamides
5a–d with substituted haloalkanes in the presence of NaH in
DMF.
significantly increases ABCA1 mRNA expression, with
a
maximum upregulating rate of 210% (Fig. 3A). Western blot
analysis revealed that ABCA1 protein levels significantly
increased in a dose-dependent manner when incubated with
2.5, 5, or 10.0 μM 6i (Fig. 3B), with a maximum upregulating
rate of 240% compared to the control group (Fig. 3C).
In addition, compounds 6a, 6d, and 6n with an ABCA1
upregulating EC50 gradient were selected for ABCA1 mRNA
and protein analysis. Fig. 4 shows that compound 6d (EC50:
0.37 μM, maximum value: 1.74-fold higher) significantly
increased ABCA1 mRNA expression in RAW264.7 cells at a
concentration of 5.0 μM, and increased ABCA1 protein
expression in a dose-dependent manner. Compound 6n
(EC50: 5.84 μM, maximum value: 1.68-fold higher)
significantly increased ABCA1 mRNA expression in RAW264.7
Scheme 1 Synthesis of target compounds 6a–n.
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Table 1 Structures and activity of target compounds 6a–n
Compd.
R1
R2
Max.a (%)
EC50b (μM)
6a (WY06)
OMe
H
F
Cl
H
H
F
CO2Et
CO2Et
CO2Et
CO2Et
CN
CHCH2
CN
CHCH2
CN
CHCH2
CHCH2
CN
CHCH2
CN
138
209
167
174
157
115
163
135
206
135
136
124
152
168
6.38 0.09
1.24 0.33
0.97 0.03
0.37 0.08
8.51 0.12
33.42 1.21
2.57 0.15
7.4 0.32
0.41 0.05
10.57 0.16
>50
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
F
Cl
Cl
H
H
F
>50
25.98 0.45
5.84 0.57
Cl
a
Maximum upregulating value (%). b Data represent the average of multiple determinations (n ≥ 3) standard error of the mean (SEM).
cells at a concentration of 5.0 μM with a maximum value of
1.5-fold, and slightly increased ABCA1 protein expression in a
dose-dependent manner. Compound 6a (EC50: 6.38 μM,
maximum value: 1.38-fold higher) had no obvious effect on
ABCA1 mRNA and protein expression in RAW264.7 cells.
These results coincided with the above SAR.
ABCA1 is one of the crucial transporter proteins that
facilitate cholesterol efflux from macrophages. Considering
that compound 6i could increase the expression of ABCA1 in
RAW264.7 cells, a 22-NBD-cholesterol efflux assay to investigate
the cholesterol efflux effect of compound 6i in RAW264.7 cells
affect ApoA-I protein expression at the indicated
concentrations [0 (control), 2.5, 5, 10 μM]. Therefore,
cholesterol efflux due to compound 6i involved an increase
in ABCA1 expression.
To evaluate the potential therapeutic application of the hit
compounds, the cytotoxicity of six representative compounds
(6a, 6b, 6c, 6d, 6i, and 6n) were evaluated in HepG2 cells
using Cell Counting Kit 8 (Solarbio Life Sciences, Beijing,
China). The results (ESI† Fig. S2) showed that all the tested
compounds showed low cytotoxicity (CC50 > 100 μM).
was performed. Fig. 5 shows that the 22-NBD-cholesterol levels Experimental methods
(green color) of cells treated with compound 6i significantly
decreased compared to that of the model group (Fig. 5A).
General
1H NMR and 13C NMR spectra were measured using a Bruker
Quantitative analysis results showed that compound 6i
significantly decreased cellular 22-NBD-cholesterol content
(Fig. 5B), which agreed with the imaging results. In summary,
these results indicate that compound 6i significantly increases
cholesterol efflux from RAW264.7 macrophages.
To exclude the effect of ApoA-I on cholesterol efflux,
western blotting was performed to examine whether
compound 6i affects ApoA-I protein expression in RAW264.7
cells. As shown in the ESI† (Fig. S1), compound 6i did not
ARX-500 nuclear magnetic resonance instrument. Mass spectra
were recorded with an AB Sciex 3200 LC-MS. Elemental analysis
was carried out with an Elementar Vario ELIII. Commercially
produced solvents and reagents were used in this study. The
boiling range of petroleum ether used in column
chromatography was 60–90 °C. The silica gel (300–400 mesh)
for flash column chromatography was purchased from Qingdao
Marine Chemical Factory in China (Qingdao, China).
Representative example: synthesis of 1-(4-chlorophenyl)-
thiourea (2a)
4-Chlorobenzenamine (10.0 g, 78.4 mmol) was dissolved in
70 mL of chlorobenzene. Concentrated sulfuric acid (3.0 mL,
43.1 mmol) was then added at 0 °C. The mixture was heated
to 70 °C, followed by the addition of potassium thiocyanate
(8.4 g, 86.2 mmol). Then, the solution was reacted at 100 °C
for 6 h. After cooling to room temperature, the mixture was
filtered, and the filter cake was washed with diethyl ether
and water and dried in vacuo to obtain compound 2a as a
white solid (yield: 63.0%). M.p.: 180.1–181.3 °C; 1H-NMR
Fig. 2 The luciferase assay of compound 6i at different time points.
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Fig. 3 Compound 6i increases ABCA1 expression in RAW264.7 cells. (A) ABCA1 mRNA expression levels in RAW264.7 cells. (B) Representative
images of ABCA1 and β-actin. (C) Relative ABCA1 protein expression levels in RAW264.7 cells were normalized to β-actin. *P < 0.05, **P < 0.01 vs.
control group.
Fig. 4 The ABCA1 mRNA and protein analysis of compounds 6a, 6d, and 6n in RAW264.7 cells. RAW264.7 cells were treated with compounds 6a,
6d, or 6n [0 (control), 2.5, 5, 10 μM] as described in Experimental methods. ABCA1 mRNA and protein expression levels induced by compounds 6a
(A), 6d (B), and 6n (C) were analyzed by RT-qPCR and western blotting. Representative images of ABCA1 and β-actin. Relative ABCA1 protein levels
in RAW264.7 cells were normalized to β-actin. *P < 0.05 vs. control group.
Fig. 5 Compound 6i promotes cholesterol efflux in RAW264.7 cells. (A) Representative images of the effects of 6i on cholesterol efflux in
RAW264.7 cells that were treated as described in Experimental methods (400×). (B) Fluorescence intensity of 22-NBD-cholesterol per cell from
three independent experiments. *P < 0.05, **P < 0.01 vs. model group.
(DMSO-d6, 500 MHz) δ: 7.36 (d, J = 9.0 Hz, 2H), 7.46 (d, J =
9.0 Hz, 2H), 9.75 (s, 1H). ESI-MS, m/z: 187.2 (M + H)+. Anal.
calcd for C7H7ClN2S: C 45.04, H 3.78, N 15.01; found C 45.21,
H 3.84, N 15.23.
added slowly at 0 °C. The mixture was heated to 70 °C for 9 h
and cooled to room temperature. The reaction mixture was
filtered, and the filtrate was neutralized with ammonium
hydroxide, forming white solids. The precipitate was filtered,
washed with water, and dried in vacuo to obtain compound
3a as a white solid (yield: 43.1%). M.p. 175.3–179.2 °C; 1H-
NMR (DMSO-d6, 500 MHz) δ: 7.76 (d, J = 9.0 Hz, 2H), 7.45 (d,
J = 9.0 Hz, 2H), 9.75 (s, 1H). ESI-MS, m/z: 185.2 (M + H)+.
Anal. calcd for C7H5ClN2S: C 45.53, H 2.73, N 15.17; found C
45.62, H 2.84, N 15.09.
Representative example: synthesis of 6-chlorobenzo[d]thiazol-
2-amine (3a)
1-(4-Chlorophenyl)thiourea (8.5 g, 45.7 mmol) was dissolved
in 50 mL of chloroform, and Br2 (2.6 mL, 50.3 mmol) was
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Representative example: synthesis of N-(6-chlorobenzoijd]-
Ethyl
2-(4-amino-N-(6-methoxybenzoijd]thiazol-2-yl)-
thiazol-2-yl)-4-nitrobenzenesulfonamide (4a1)
phenylsulfonamido)acetate (6a). Yield 39.8%. M.p. 170.5–
172.0 °C; H-NMR (DMSO-d6, 500 MHz) δ: 1.11 (t, J = 7.3 Hz,
1
6-Chlorobenzo[d]thiazol-2-amine (5.0 g, 27.0 mmol) was
dissolved in 100 mL of THF. Pyridine (2.4 mL, 29.8 mmol)
was added slowly thrice, at intervals of 4 h. After stirring for
10 min each time pyridine was added, 4-nitrobenzenesulfonyl
chloride (8.9 g, 40.5 mmol) was added in three portions.
About 7 h later, the reaction was completed, and the mixture
was concentrated to obtain a flesh pink solid. The solid was
added to 100 mL of water and stirred for 1 h and then
filtered. The filter cake was washed with water and dried in
vacuo, and then purified by silica gel chromatography using a
solvent of petroleum ether/ethyl acetate (5 : 1) to provide the
desired product 4a1 as a flesh pink solid, with a yield of
3H), 3.78 (s, 3H), 4.06 (q, J = 7.0 Hz, 2H), 4.97 (s, 2H), 5.94 (s,
2H), 6.56 (d, J = 8.5 Hz, 2H), 7.02 (dd, J = 2.4, 8.5 Hz, 1H),
7.45 (d, J = 9.0 Hz, 2H), 7.48 (d, J = 9.0 Hz, 1H), 7.51 (s, 1H).
13C-NMR (DMSO-d6, 125 MHz) δ: 14.34, 46.37, 56.28, 61.84,
107.98, 112.91, 113.35, 114.69, 124.88, 127.22, 128.28, 131.32,
149.24, 153.21, 156.82, 164.67, 167.32, 175.43. ESI-MS, m/z:
422.3 (M + H)+, 444.1 (M + Na)+. Anal. calcd for C17H17N3O5S2:
C 50.11, H 4.21, N 10.31; found C 50.19, H 4.30, N 10.33.
Ethyl
2-(4-amino-N-(benzoijd]thiazol-2-yl)-
phenylsulfonamido)acetate (6b). Yield 41.4%. M.p. 167.5–
1
170.2 °C; H-NMR (DMSO-d6, 500 MHz) δ: 1.12 (t, J = 7.0 Hz,
1
3H), 4.05–4.09 (m, 2H), 5.01 (s, 2H), 5.96 (s, 2H), 6.56 (d, J =
9.0 Hz, 2H), 7.30 (t, J = 8.0 Hz, 1H), 7.45 (d, J = 8.5 Hz, 3H),
7.57 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 7.5 Hz, 1H). 13C-NMR
(DMSO-d6, 125 MHz) δ: 14.35, 46.28, 61.87, 112.62, 112.93,
123.35, 123.68, 124.50, 126.96, 127.65, 128.33, 128.46, 137.48,
153.32, 165.08, 167.32, 175.27. ESI-MS, m/z: 392.3 (M + H)+,
414.2 (M + Na)+. Anal. calcd for C16H15N3O4S2: C 50.91, H
4.01, N 11.13; found C 50.84, H 4.26, N 11.23.
63.5%. M.p. 169.3–171.5 °C; H-NMR (DMSO-d6, 500 MHz) δ:
7.26 (dd, J = 2.0, 7.5 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.40 (d,
J = 7.5 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 7.76 (s, 1H), 7.85 (s,
1H), 8.25 (s, 1H), 13.35 (s, 1H). ESI-MS, m/z: 370.2 (M + H)+,
392.4 (M + Na)+. Anal. calcd for C13H8ClN3O4S2: C 42.22, H
2.18, N 11.36; found C 42.31, H 2.97, N 11.41.
Representative example: synthesis of 4-amino-N-(6-
chlorobenzoijd]thiazol-2-yl)benzenesulfonamide (5a1)
Ethyl
2-(4-amino-N-(6-fluorobenzoijd]thiazol-2-yl)-
phenylsulfonamido)acetate (6c). Yield 40.2%. M.p. 193.6–
194.5 °C; H-NMR (DMSO-d6, 500 MHz) δ: 1.16 (t, J = 7.0 Hz,
1
N-(6-Chlorobenzo[d]thiazol-2-yl)-4-nitrobenzenesulfonamide
(2.0 g, 5.4 mmol) was dissolved in 15 mL of water–ethanol–
methanol (2 : 6 : 3) and heated to 60 °C for 20 min to obtain a
clear solution. The solution was cooled to room temperature
and then ammonium chloride was added. The zinc powder
was added in batches. The reaction was stirred for an
additional 2 h at room temperature and then filtered. The
filtrate was concentrated and quenched with water, then
extracted with ethyl acetate. The organic layer was washed
with water and brine and then dried over anhydrous sodium
sulfate. The solvent was removed under reduced pressure
and the obtained compound 5a1 as a yellow solid was used
in the next step without any purification (yield: 52.4%). M.p.
3H), 4.12 (q, J = 7.0 Hz, 2H), 5.06 (s, 2H), 6.04 (s, 2H), 6.62 (d,
J = 8.0 Hz, 2H), 7.38 (t, J = 7.5 Hz, 1H), 7.49 (d, J = 8.0 Hz,
2H), 7.67 (dd, J = 2.4, 8.0 Hz, 1H), 7.98 (dd, J = 2.0, 7.5 Hz,
1H). 13C-NMR (DMSO-d6, 125 MHz) δ: 14.12, 46.72, 61.03,
109.59, 112.98. 114.03, 123.26, 125.69, 127.63, 128.10, 128.61,
136.98, 151.22, 152.42, 164.16, 167.34, 175.66. ESI-MS, m/z:
410.1 (M
+ +
H)+, 432.3 (M Na)+. Anal. calcd for
C16H14FN3O4S2: C 48.60, H 3.57, N 10.63; found C 48.72, H
3.52, N 10.73.
Ethyl
2-(4-amino-N-(6-chlorobenzoijd]thiazol-2-yl)-
phenylsulfonamido)acetate (6d). Yield 42.3%. M.p. 185.0–
186.7 °C; H-NMR (DMSO-d6, 500 MHz) δ: 1.10 (t, J = 7.0 Hz,
1
1
3H), 4.06 (q, J = 7.00 Hz, 2H), 5.00 (s, 2H), 5.98 (s, 2H), 6.57
(d, J = 8.5 Hz, 2H), 7.44 (d, J = 8.5 Hz, 2H), 7.50 (dd, J = 2.4,
8.7 Hz, 1H), 7.60 (d, J = 9.0 Hz, 1H), 8.02 (s, 1H). 13C-NMR
(DMSO-d6, 125 MHz) δ: 14.12, 46.72, 61.03, 109.59, 112.98,
114.03, 123.26, 125.69, 127.63, 128.10, 128.61, 136.98, 151.22,
152.42, 164.16, 167.34, 175.66. ESI-MS, m/z: 426.3 (M + H)+,
448.1 (M + Na)+. Anal. calcd for C16H14ClN3O4S2: C 46.66, H
3.43, N 10.20; found C 46.59, H 3.49, N 10.34.
182.5–184.6 °C; H-NMR (DMSO-d6, 500 MHz) δ: 5.96 (s, 2H),
7.13 (dd, J = 2.0, 7.5 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.36 (d,
J = 7.5 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.68 (s, 1H), 7.98 (s,
1H), 8.23 (s, 1H), 13.26 (s, 1H). ESI-MS, m/z: 340.2 (M + H)+,
362.4 (M + Na)+. Anal. calcd for C13H10ClN3O2S2: C 45.95, H
2.97, N 12.37; found C 45.87, H 2.84, N 12.42.
General synthetic procedure for compounds 6a–6n
N - C y a n o m e t h y l - N - ( b e n z o ij d ] t h i a z o l - 2 - y l ) - 4 -
aminobenzenesulfonamide (6e). Yield 39.3%. M.p. 176.2–
177.5 °C; 1H-NMR (DMSO-d6, 500 MHz) δ: 5.41 (s, 2H), 5.58
(s, 2H), 6.76 (dd, J = 2.4, 8.3 Hz, 1H), 7.00 (d, J = 7.5 Hz, 1H),
7.10 (s, 1H), 7.17 (t, J = 8.0 Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H),
7.54 (t, J = 7.7 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 7.5
Hz, 1H). 13C-NMR (DMSO-d6, 125 MHz) δ: 32.79, 110.58,
112.33, 112.77, 114.85, 117.72, 123.26, 123.40, 124.94, 127.69,
129.53, 135.55, 141.59, 149.47, 165.30. ESI-MS, m/z: 345.4 (M
+ H)+, 367.1 (M + Na)+. Anal. calcd for C15H12N4O2S2: C 52.31,
H 3.51, N 16.27; found C 52.54, H 3.61, N 16.32.
Amino-N-(6-chlorobenzoijd]thiazol-2-yl)benzenesulfonamide (1
mmol) was dissolved in 1 mL of DMF. NaH (3 mmol) was
added at 0 °C. Chloroacetonitrile, ethyl bromoacetate, or allyl
chloride (2.5 mmol) was then added. The reaction was stirred
for an additional 8 h at room temperature and diluted with
water (30 mL). The mixture was extracted with ethyl acetate
(4 × 30 mL), washed with brine (30 mL), and dried over
anhydrous sodium sulfate. The target compounds were
purified by silica gel chromatography using petroleum ether
and ethyl acetate as a solvent as white to light yellow solids.
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1
N-Allyl-N-(benzo[d]thiazol-2-yl)-4-aminobenzenesulfonamide
(6f). Yield 40.0%. M.p. 199.7–200.6 °C; 1H-NMR (DMSO-d6,
500 MHz) δ: 3.59 (d, J = 6.5 Hz, 2H), 5.03 (d, J = 10.0 Hz, 1H),
5.13 (d, J = 17.0 Hz, 1H), 5.78–5.88 (m, 1H), 7.11 (t, J = 7.5 Hz,
1H), 7.16 (t, J = 7.5 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.28 (d, J
= 7.5 Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H),
7.60 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 7.5 Hz, 1H). 13C-NMR
(DMSO-d6, 125 MHz) δ: 35.93, 118.07, 118.94, 121.03, 122.21,
125.40, 125.55, 125.93, 127.06, 130.07, 130.83, 133.68, 137.34,
151.72, 155.10, 173.51. ESI-MS, m/z: 346.4 (M + H)+, 368.1
(M + Na)+. Anal. calcd for C16H15N3O2S2: C 55.63, H 4.38,
N 12.16; found C 55.72, H 4.28, N 12.37.
N-Cyanomethyl-N-(6-fluorobenzoijd]thiazol-2-yl)-4-
aminobenzenesulfonamide (6g). Yield 38.8%. M.p. 197.5–
198.0 °C; 1H-NMR (DMSO-d6, 125 MHz) δ: 5.37 (s, 2H), 6.03
(s, 2H), 6.59 (d, J = 9.00 Hz, 2H), 7.41 (t, J = 9.0 Hz, 1H), 7.52
(d, J = 8.5 Hz, 2H), 7.74 (dd, J = 3.0, 9.0 Hz, 1H), 7.86 (dd, J =
8.3 Hz, 1H). 13C-NMR (DMSO-d6, 125 MHz) δ: 33.29, 110.89,
111.12, 113.03, 113.75, 115.23, 115.39, 126.31, 128.58, 132.72,
153.61, 157.66, 158.47, 160.39, 164.66. ESI-MS, m/z: 363.2 (M
+ H)+, 385.1 (M + Na)+. Anal. calcd for C15H11FN4O2S2: C
49.71, H 3.06, N 15.46; found C 49.75, H 3.43, N 15.62.
176.0 °C; H-NMR (DMSO-d6, 500 MHz) δ: 4.83 (d, J = 4.5 Hz,
2H), 5.01 (d, J = 17.5 Hz, 1H), 5.15 (d, J = 10.5 Hz, 1H), 5.58 (s,
1H), 5.81–5.89 (m, 1H), 6.73 (dd, J = 2.4, 8.0 Hz, 1H), 6.94 (d, J =
7.5 Hz, 1H), 7.05 (s, 1H), 7.13 (t, J = 8.0 Hz, 1H), 7.31 (t, J = 7.7
Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.87 (d,
J = 8.0 Hz, 1H). 13C-NMR (DMSO-d6, 125 MHz) δ: 47.14, 110.88,
113.07, 117.84, 118.25, 121.30, 123.35, 123.98, 124.71, 127.76,
129.80, 130.95, 137.11, 142.55, 149.75, 165.75. ESI-MS, m/z:
346.3 (M + H)+, 368.2 (M + Na)+. Anal. calcd for C16H15N3O2S2:
C 55.63, H 4.38, N 12.16; found C 55.72, H 4.26, N 12.74.
N - C y a n o m e t h y l - N - ( b e n z o ij d ] t h i a z o l - 2 - y l ) - 3 -
aminobenzenesulfonamide (6l). Yield 35.3%. M.p. 210.0–
211.5 °C; 1H-NMR (DMSO-d6, 500 MHz) δ: 5.44 (s, 2H), 5.62
(s, 2H), 6.76 (dd, J = 2.4, 7.8 Hz, 1H), 6.99 (d, J = 8.0 Hz, 1H),
7.10 (s, 1H), 7.16 (t, J = 7.8 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H),
7.54 (t, J = 8.0 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.0
Hz, 1H). 13C-NMR (DMSO-d6, 125 MHz) δ: 32.79, 110.58,
112.33, 112.77, 114.85, 117.72, 123.26, 123.40, 124.94, 127.69,
129.53, 135.55, 141.59, 149.47, 165.30. ESI-MS, m/z: 345.1 (M
+ H)+, 367.3 (M + Na)+. Anal. calcd for C15H12N4O2S2: C 52.31,
H 3.51, N 16.27; found C 52.52, H 3.54, N 16.34.
N - A l l y l - N - ( 6 - f l u o r o b e n z o ij d ] t h i a z o l - 2 - y l ) - 3 -
aminobenzenesulfonamide (6m). Yield 39.6%. M.p. 153.3–
N - A l l y l - N - ( 6 - f l u o r o b e n z o ij d ] t h i a z o l - 2 - y l ) - 4 -
aminobenzenesulfonamide (6h). Yield 39.6%. M.p. 208.5–
1
155.0 °C; H-NMR (DMSO-d6, 500 MHz) δ: 3.66 (d, J = 6.5 Hz,
1
210.5 °C; H-NMR (DMSO-d6, 500 MHz) δ: 4.78 (d, J = 4.0 Hz,
2H), 5.09 (d, J = 10.0 Hz, 1H), 5.25 (d, J = 16.5 Hz, 1H), 5.79–
5.88 (m, 1H), 7.03 (t, J = 7.3 Hz, 1H), 7.09 (t, J = 8.0 Hz, 1H),
7.21 (d, J = 7.5 Hz, 1H), 7.35 (s, 1H), 7.47 (dd, J = 3.0, 8.0 Hz,
1H), 7.60 (dd, J = 2.4, 7.5 Hz, 1H), 8.05 (s, 1H), 9.28 (s, 1H).
13C-NMR (DMSO-d6, 125 MHz) δ: 35.06, 108.10, 108.56,
113.28, 113.31, 117.41, 117.79, 119.01, 119.85, 119.98, 134.21,
148.63, 157.41, 159.50, 174.20. ESI-MS, m/z: 364.1 (M + H)+,
386.2 (M + Na)+. Anal. calcd for C16H14FN3O2S2: C 52.88, H
3.88, N 11.56; found C 52.95, H 3.97, N 11.67.
2H), 4.98 (d, J = 17.0 Hz, 1H), 5.14 (d, J = 10.5 Hz, 1H), 5.78–
5.86 (m, 1H), 5.97 (s, 2H), 6.57 (d, J = 8.5 Hz, 2H), 7.31 (t, J =
9.0 Hz, 1H), 7.47 (d, J = 9.0 Hz, 3H), 7.82 (dd, J = 2.4, 8.5 Hz,
1H). 13C-NMR (DMSO-d6, 125 MHz) δ: 35.04, 108.01, 108.22,
113.37, 113.56, 117.53, 117.60, 118.83, 119.85, 119.91, 133.63,
148.80, 157.41, 159.30, 173.90. ESI-MS, m/z: 364.4 (M + H)+,
386.6 (M + Na)+. Anal. calcd for C16H14FN3O3S2: C 52.88, H
3.88, N 11.56; found C 52.91, H 3.74, N 11.62.
N-Cyanomethyl-N-(6-chlorobenzo[d]thiazol-2-yl)-4-
aminobenzenesulfonamide (6i). Yield 40.4%. M.p. 183.2–
185.3 °C; 1H-NMR (DMSO-d6, 500 MHz) δ: 5.35 (s, 2H), 6.03
(s, 2H), 6.59 (d, J = 8.5 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.58
(dd, J = 2.4, 8.5 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H). 13C-NMR
(DMSO-d6, 125 MHz) δ: 33.24, 112.95, 113.04, 113.76, 115.16,
123.40, 125.72, 126.25, 127.93, 128.40, 128.61, 129.10, 135.16,
153.62, 164.50. ESI-MS, m/z: 379.5 (M + H)+, 401.2 (M + Na)+.
Anal. calcd for C15H11ClN4O2S2: C 47.55, H 2.93, N 14.79;
found C 47.69, H 2.85, N 14.52.
N-Cyanomethyl-N-(6-chlorobenzoijd]thiazol-2-yl)-3-
aminobenzenesulfonamide (6n). Yield 39.6%. M.p. 157.0–
158.6 °C; 1H-NMR (DMSO-d6, 500 MHz) δ: 5.39 (s, 1H), 5.60
(s, 2H), 6.75 (dd, J = 7.3 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 7.08
(s, 1H), 7.12 (dd, J = 2.4, 8.0 Hz, 1H), 7.15 (t, J = 8.5 Hz,
1H,),7.58 (s, 1H), 7.68 (d, J = 8.5 Hz, 1H). 13C-NMR (DMSO-d6,
125 MHz) δ: 33.14, 110.96, 112.88, 113.59, 115.08, 115.23,
125.28, 126.15, 128.42, 132.56, 153.45, 157.51, 158.31, 160.23,
164.50. ESI-MS, m/z: 379.1 (M + H)+, 401.2 (M + Na)+. Anal.
calcd for C15H11ClN4O2S2: C 47.55, H 2.93, N 14.79; found C
47.69, H 3.84, N 14.82.
N - A l l y l - N - ( 6 - c h l o r o b e n z o ij d ] t h i a z o l - 2 - y l ) - 4 -
aminobenzenesulfonamide (6j). Yield 40.3%. M.p. 180.5–
1
182.1 °C; H-NMR (DMSO-d6, 500 MHz) δ: 4.77 (d, J = 5.0 Hz,
Cell culture
2H), 4.97 (d, J = 17.0 Hz, 1H), 5.13 (d, J = 10.5 Hz, 1H), 5.78–
5.86 (m, 1H), 5.98 (s, 2H), 6.57 (d, J = 8.5 Hz, 2H), 7.47 (s,
1H), 7.48 (s, 3H), 8.01 (s, 1H). 13C-NMR (DMSO-d6, 125 MHz)
δ: 15.44, 47.13, 49.05, 112.99, 114.28, 118.10, 122.97, 126.00,
127.01, 127.59, 128.35, 128.43, 130.89, 136.31, 153.30, 164.40,
173.42. ESI-MS, m/z: 380.2 (M + H)+, 402.1 (M + Na)+. Anal.
calcd for C16H14ClN3O2S2: C 50.59, H 3.71, N 11.06; found C
50.70, H 3.69, N 11.32.
HepG2 cells were cultured in DMEM (Corning Inc., Corning,
NY, USA) with 10% fetal bovine serum (FBS) (Gibco, Grand
Island, NY, USA). RAW264.7 (ATCC, Rockville, MD, USA) cells
were grown in RPMI (Corning, Inc.) with 10% FBS (medium
A). Human ABCA1p-LUC HepG2 cells were grown in medium
A supplemented with 500 μg mL−1 G418 (medium B). All cells
were cultured at 37 °C with 5% CO2 in a cell incubator and
maintained at the NHC Key Laboratory of Biotechnology of
Antibiotics (CAMS & PUMC).
N - A l l y l - N - ( b e n z o ij d ] t h i a z o l - 2 - y l ) - 3 -
aminobenzenesulfonamide (6k). Yield 36.2%. M.p. 175.3–
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Research Article
ABCA1 upregulating activity assay
(medium
A)
containing
22-NBD-cholesterol
(final
concentration: 50 μM) (Thermo Fisher Scientific) for 6 h.
Then, the cells were washed twice with phosphate-buffered
saline (PBS). Then, the cells were incubated with medium A
containing ApoA-I (final concentration: 10 μg mL−1) (Peking
The ABCA1p-LUC HepG2 cells were seeded into a 96-well
clear bottom white plate (Costar) at a density of 5 × 105 cells
per well in 100 μL medium B. The compounds were diluted
to concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625,
0.78125, 0.39, 0.039, 0.0039, and 0 μM with RPMI-1640 cell
culture medium containing 5% FBS (medium C). After 6–8 h
of incubation, the culture medium was discarded and
replaced with 200 μL of cell culture medium containing the
indicated concentrations of the compounds. After 24 h, the
cells were washed with PBS once, and then luciferase activity
was assessed using a Luciferase Assay System (Promega) and
a microplate reader.22
Union-Biology, Beijing, China) for another
6 h. DAPI
(Invitrogen) was used as a nuclear counterstain. Fluorescence
images were captured and the fluorescence was quantified
using a high content analysis system (PerkinElmer, Fremont,
CA, USA).
Conclusions
In this study, 14 benzothiazole derivatives were synthesized
and evaluated in terms of their upregulatory effects on
human ABCA1 expression. The results showed that
compounds 6b, 6c, 6d, 6g, and 6i had better upregulatory
activities than the lead compound 6a. Further RT-qPCR and
western blot analyses showed that 6i increases both ABCA1
mRNA expression and protein expression in RAW264.7 cells.
In addition, 22-NBD-cholesterol efflux assay showed that 6i
significantly increases cholesterol efflux from RAW264.7
macrophages. Currently, 6i is the leading anti-atherosclerosis
drug that requires further modification.
Western blotting
RAW264.7 cells were grown in a six-well plate (Costar) and
then treated with compound 6i (0, 2.5, 5, and 10 μM) for 24
h. Total proteins were extracted with RIPA buffer (Applygen
Technologies, Beijing, China). The cell lysate was centrifuged
at 12 000 rpm for 15 min at 4 °C. Protein concentrations of
the supernatants were determined with a BCA Protein Assay
kit (Thermo Fisher Scientific). Protein samples were
separated using SDS-PAGE gels and then transferred onto
polyvinylidene fluoride membranes (Merck Millipore,
Darmstadt, Germany). The membranes were blocked with 5%
skim milk for 1 h and then incubated with the corresponding
primary antibodies overnight at 4 °C. After washing with 1×
Tris buffered saline with 0.1% Tween-20 (TBST) thrice, the
membranes were incubated with the appropriate secondary
antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) for 1
h at room temperature. The primary antibodies used include
ABCA1 (Novus Biologicals, Littleton, CO, USA), ApoA-I
(Abcam) and β-actin (Sigma-Aldrich).
Conflicts of interest
The authors hereby declare no conflict of interest.
Acknowledgements
This work was supported by the Natural Science Foundation
of Hebei Province (grant number 19JCZDJC63900), the Drug
Innovation Major Project (grant number 2018ZX09711001-
003-006), the National Natural Science Foundation of China
(grant number 81573482), the Health and Medical Creation
Program of CAMS (grant number 2016-I2M-1-011), the Natural
Science Foundation of Hebei Province (grant number
H2018307055) and the Science Foundation of Hebei Normal
University (grant number L2017K06). We thank LetPub (www.
letpub.com) for its linguistic assistance during the
preparation of this manuscript.
Real-time quantitative PCR (RT-qPCR)
RAW264.7 cells were grown in a six-well plate and treated with
compound 6i (0, 2.5, 5 and 10 μM) for 24 h. Total RNA was
extracted with a QIAGEN RNeasy Mini kit (Qiagen, 40724
Hilden, Germany). Then, the total RNA was reverse
transcribed using a TransScript One-Step gDNA Removal and
cDNA Synthesis SuperMix (Transgen Biotech, Beijing, China).
RT-qPCR was performed using a FastStart Universal SYBR
Green PCR Master (Roche Diagnostics, Lewes, UK) and special
primers with an FTC-3000 real-time quantitative thermal
cycler (Funglyn Biotech Inc., Richmond Hill, Canada).
Notes and references
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