Discovery of 2-azetidinone and 1H-pyrrole-2,5-dione derivatives containing sulfonamide group at the side chain as potential cholesterol absorption inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.12.077

Cholesterol absorption inhibitor (CAI) targeting Niemann-Pick C1-like1 protein was developed for the treatment of hyperlipidaemia and only ezetimibe was approved so far. For developing novel CAIs, we synthesized sixteen 2-azetidinone derivatives and thirt

Full text of DOI:10.1016/j.bmcl.2015.12.077

Accepted Manuscript
Discovery of 2-azetidinone and 1H-pyrrole-2,5-dione derivatives containing
sulfonamide group at the side chain as potential cholesterol absorption inhibitors
Xinrui Yuan, Peng Lu, Xiaojian Xue, Hui Qin, Chen Fan, Yubin Wang, Qi
Zhang
PII:
S0960-894X(15)30390-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.12.077
BMCL 23438
Reference:
Bioorganic & Medicinal Chemistry Letters
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31 October 2015
8 December 2015
22 December 2015
Please cite this article as: Yuan, X., Lu, P., Xue, X., Qin, H., Fan, C., Wang, Y., Zhang, Q., Discovery of 2-azetidinone
and 1H-pyrrole-2,5-dione derivatives containing sulfonamide group at the side chain as potential cholesterol
absorption inhibitors, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.
2015.12.077
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Discovery of 2-azetidinone and 1H-pyrrole-
2,5-dione derivatives containing sulfonamide
group at the side chain as potential
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cholesterol absorption inhibitors
Xinrui Yuan, Peng Lu, Xiaojian Xue, Hui Qin, Chen Fan, Yubin Wang,* Qi Zhang

Bioorganic & Medicinal Chemistry Letters
Discovery of 2-azetidinone and 1H-pyrrole-2,5-dione derivatives containing
sulfonamide group at the side chain as potential cholesterol absorption inhibitors
Xinrui Yuan, Peng Lu, Xiaojian Xue, Hui Qin, Chen Fan, Yubin Wang,* Qi Zhang
School of Pharmaceutical Sciences, Nanjing Tech University, No. 5 Xinmofan Road, Nanjing 210009, People's Republic of China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Cholesterol absorption inhibitor (CAI) targeting Niemann-Pick C1-like1 protein was developed
for the treatment of hyperlipidaemia and only ezetimibe was approved so far. For developing
novel CAIs, we synthesized sixteen 2-azetidinone derivatives and thirteen 1H-pyrrole-2,5-dione
derivatives containing sulfonamide group at the side chain, and their inhibitory activity of
cholesterol absorption was evaluated in Caco-2 cell line in vitro. Furthermore, top six
compounds were measured by cytotoxicity and partition coefficient, and 2-azetidinone analogue
9e was selected for in vivo study. Finally, 9e considerably reduced total cholesterol, LDL-C,
FFA and triglyceride in the serum and increased the rate of HDL-C to total cholesterol,
suggesting it could regulate the lipid metabolism and act as a potent CAI.
Keywords:
2-azetidinone
1H-pyrrole-2,5-dione
Sulfonamide group
Cholesterol absorption inhibitor
Caco-2 cell line
2009 Elsevier Ltd. All rights reserved.
Atherosclerotic coronary artery disease (CAD) has become a
major cause of death and morbidity, especially in western
countries.1 Hyperlipidaemia with elevated serum cholesterol level
is one of the most significant risk factors for CAD. There are two
major sources of cholesterol in the body: de novo biosynthesis in
liver and absorption of dietary cholesterol in the small intestine.2
In most cases, inhibition of biosynthesis by 3-hydroxy-3-
methylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins) is
the first choice of patients with hyperlipidaemia.3 However, high-
dose statin treatment often leads to severe adverse effects, such
as hepatotoxicity and myopathy.4 As the new strategy of
managing hypercholesterolemia, cholesterol absorption inhibitor
(CAI) was developed to block exogenous cholesterol absorption.
Table 1. Structure of 2-azetidinone Derivatives 8a-f and 9a-j
Compound
Ar
R
—
Ph
8a
8b
8c
8d
8e
8f
4-C2H5-Ph
4-CF3O-Ph
2-CF3O-Ph
2-Thiophene
3-Br-Ph
—
—
—
—
—
Ph
CH3
9a
9b
9c
9d
9e
9f
Ph
4- CH3-Ph
CH3
4-C2H5-Ph
4-C2H5-Ph
4-CF3O-Ph
4-CF3O-Ph
2-CF3O-Ph
2-CF3O-Ph
2-Thiophene
2-Thiophene
4- CH3-Ph
CH3
4- CH3-Ph
CH3
9g
9h
9i
4- CH3-Ph
CH3
Figure 1. Structures of ezetimibe and its analogues.
4- CH3-Ph
9j
 Corresponding author. Tel.: +8625-83172108. E-mail: wyb5393@163.com.

displayed potent cholesterol absorption inhibitory activity.7,8
Given that sulfonamide has greater resistance to hydrolysis than
amide and ester in vivo, we adopted bioisosteric replacement and
introduced sulfonamide group into C-3 side chain of 2-
azetidinone, and six novel 2-azetidinone analogues 8a-f were
synthesized successfully (Table 1). To improve the
hydrophilicity and pharmacokinetics, we transformed nitro group
into sulfonamide and completed the synthesis of other ten 2-
azetidinone derivatives 9a-j including two sulfonamide groups
(Table 1). It was reported that the substituted γ-lactams,
imidazolidinones, oxazolidinones, pyrazolines and isoxazolines
still kept moderate inhibitory efficacy,9,10 revealing that 2-
azetidinone was a dispensable moiety. So, we selected 1H-
pyrrole-2,5-dione to replace 2-azetidinone as the new molecular
scaffold, retained the sulfonamide group at the terminal of side
chain and synthesized thirteen new compounds 20k-m and 21a-j
(Table 2). Last but not least, we combined the Caco-2 cell model
with an HPLC technique based on pre-column cholesterol
oxidation to evaluate the inhibitory activity of all twenty-nine
compounds in vitro for the first time.
Table 2. Structure of 1H-pyrrole-2,5-dione Derivatives 20k-m
and 21a-j
Compound
Ar
R1
NO2
NO2
NO2
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
R2
4-CF3O-Ph
4-CH3-Ph
4-NO2-Ph
Ph
OCH3
20k
20l
OCH3
OCH3
20m
21a
21b
21c
21d
21e
21f
F
F
F
F
F
F
F
F
F
F
4-F-Ph
4-CH3-Ph
4-CF3-Ph
3-Br-Ph
4-C2H5-Ph
2-Thiophene
2-CF3O-Ph
4-CF3O-Ph
4-t-Bu-Ph
21g
21h
21i
21j
Ezetimibe (1, Figure 1) (Zetia; approved in 2002) is the only
marketable cholesterol absorption inhibitor which was discovered
fortuitously in the program of acyl-CoA: cholesterol
acyltransferase (ACAT) inhibitors.5 In 2004, Niemann-Pick C1-
like1 (NPC1L1) protein was identified as the target of ezetimibe.6
It can be administered as monotherapy or combinatory treatment
with statins and has been confirmed to be safe and effective
among patients subjected to renal, cardiac and liver transplants,
as well as in HIV patients.3 Albeit such breakthrough, there
remains an unmet demand for developing new CAI that can
improve therapeutic efficacy and provide patients with more
choices.
Scheme 2. Synthesis of Compounds 20k-m and 21a-j: (i) Br2, CH3CN, r.t.,
overnight, 90%; (ii) NaN3, CH3OH/H2O, 0-5 °C, 4 h, 98%; (iii) Pd/C, H2,
HCl, CH3OH, r.t., 60%; (iv) EDCl, HOBT, 4-methylmorpholine, CH2Cl2, r.t.,
10 h, 80%; (v) Et3N, acetic anhydride, N2, 75 °C, 4 h, 67%; (vi) CH3ONa,
CH3OH, 0 °C, 1 h, 68%; (vii) NaH, DMF, O2, 0 °C, 30 min, 77%; (viii) 1,4-
dibromobutane, TBAB, K2CO3, CH3CN, 45 °C, overnight, 60%; (ix)
substituted aromatic sulfonamide, KI, K2CO3, CH3CN, reflux, overnight,
76%; (x) acetic acid, 90 °C, 24h, 64-83%.
The general synthetic route toward 8a-f and 9a-j was depicted
in Scheme 1. (3R,4S)-2-azetidinone 4 was prepared as previously
described.7 The hydrolysis and reduction of 4 followed by
reaction with p-toluenesulfonyl chloride gave intermediate 7.
Compounds 8a-f were prepared from the reaction of 7 with
various substituted aromatic sulfonamide. Subsequently, the
target compounds 9a-j were synthesized by reduction of 8a-e and
the following reaction with methanesulfonyl chloride or p-
toluenesulfonyl chloride.
The 1H-pyrrole-2,5-dione derivatives 20k-m and 21a-j were
synthesized as illustrated in Scheme 2. Substituted α-
bromoacetophenone 11 was prepared from bromination reaction
of 10. Compound 11 reacted with sodium azide to afford
corresponding substituted phenacyl azide 12. Phenylacetamide
hydrochloride 13 was obtained from reduction of 12. Then,
condensation between 13 and phenylacetic acid 14 yielded
intermediate 15. Ring closure of 15 in the presence of acetic
anhydride gave 16, which was deacetylated and oxidized to the
intermediate 18.11 Subsequent reaction of 18 with 1,4-
Scheme 1. Synthesis of Compounds 8a-f and 9a-j: (i) LiOH, THF, r.t., 2 h,
89%; (ii) BH3·Me2S, THF, r.t., 8 h, 45%; (iii) TsCl, Et3N, CH2Cl2, r.t., 6 h,
91%; (iv) substituted aromatic sulfonamide, K2CO3, CH3CN, reflux, 12 h,
78-84%; (v) Pd/C, H2, CH3OH, r.t., 1h, 90-93%; (vi) MsCl or TsCl, Et3N,
DMAP, CH2Cl2, r.t., 24 h, 60-78%.
Earlier, we reported several classes of 2-azetidinone
derivatives and shown that ezetimibe bioisosteres (2 and 3,
Figure 1) with amide group and ester group at C-3 side chain

dibromobutane under TBAB and K2CO3 prepared 19.
Intermediate 20 was obtained by reaction of 19 with various
substituted aromatic sulfonamide. Finally, deprotection reaction
of 20a-j with acetic acid afforded 21a-j.
Previously, in vitro inhibitory activity of cholesterol
absorption was evaluated in the brush border membrane vesicles
or hNPC1L1/MDCKⅡ cell line.12,13 In this article, we adopted
the Caco-2 cell line, a reliable human intestinal model for lipid
metabolism and lipoprotein assembly to initially assess the
inhibitory potency of compounds 8a-f, 9a-j, 20k-m and 21a-j at
100 μM concentration.14,15,16 An HPLC method based on a pre-
substituents and electron-donating substituents were introduced
to the p-position of phenyl ring at the side chain and most of
them were more potent than unsubstituted benzene sulfonamides,
especially ethyl (8b, 9c and 9d) and trifluoromethoxy (8c, 9e, 9f
and 21i). Interestingly, electron-withdrawing substituents were
more helpful to enhance inhibitory activity compared with
electron-donating substituents (e.g., 8b vs 8c, 9c vs 9e). On the
other hand, p-substituted benzene sulfonamides showed much
better inhibitory effect than o-substituted benzene sulfonamides
(e.g., 8c vs 8d, 21i vs 21h). Most of nitro-derived compounds
displayed better activity than those corresponding sulfonamide
derivatives (e.g., 8b vs 9c-d). Based on these results, 8b-c and
9c-f were screened for further evaluation.
Figure 4. Partition coefficients of compounds 8b-c and 9c-f. The
partition coefficient represents Po/w = Co/Cw of compound between the
two immiscible solvents octanol (o) and water (w). These values
represented mean ± SD (n=3).
Figure 2. Inhibitory effects of compounds 8a-f, 9a-j, 20k-m and 21a-j on
cholesterol absorption in Caco-2 cell line. These values represented mean ±
SD (n=4).
column Jones oxidation and ultraviolet detection for the
measurement of total cholesterol in Caco-2 cells was adopted and
stigmasterol was chosen as an internal standard because of its
absence in biological samples (Figure S1).17,18 As shown in
Figure 2, most of synthesized compounds containing
sulfonamide group at side chain retained the potency, especially
8b, 8c, 8e, 9c, 9d, 9e, 9f and 21d. To our delight, 8c and 9e
showed the significant inhibitory activity and decreased
cholesterol uptake by 47.9% and 41.4%, respectively. It was
noteworthy that most of 1H-pyrrole-2,5-dione derivatives
exhibited moderate efficacy except 21b, 21h and 21j, and only
21d was comparable with ezetimibe. These results suggested that
1H-pyrrole-2,5-dione scaffold could replace the 2-azetidinone
effectively, but its structure-activity relationship remained to be
further investigated. As can be seen, both electron-withdrawing
It was reported that ezetimibe had potential security risk in the
clinical trials and showed cytotoxicity against MDCKII and
HepG2 cell lines.2,19 To analyze the cytotoxicity of compounds
8b-c and 9c-f, MTT cell proliferation assay was adopted against
the human cell lines, GES1 (human gastric epithelial cells) and
HEK293 (human embryonic kidney cells). Compounds were
Figure 5. The mean body weight of five groups of hamsters in 7 days. These
values represented mean ± SD (n=6).
considered nontoxic when LC50 values were higher than 100
μM.13 The results of cytotoxicity assay were summarized in
Figure 3. LC50 values of ezetimibe were 61.3 μM against GES1
and 54.6 μM against HEK293. Unfortunately, Compound 8c
showed LC50 value of 56.8 μM against GES1, and 9d exhibited
cytotoxicity against GES1 (92.9 μM) and HEK293 (42.9 μM)
respectively. Compounds 8b, 9c, 9e and 9f displayed no toxicity
in both the cell lines.
Lipophilicity was one of the standards to estimate the druglike
physical properties of compounds and represented by the
partition coefficient (P) which was measured by the distribution
between the two immiscible phases, octanol and water. 20 High
lipophilicity could promote compounds binding to unwanted
targets and cause cardiovascular and tissue toxicity.21 LogP of
Figure 3. Cytotoxicity assay of compounds 8b-c and 9c-f against GES1 (a)
and HEK293 (b). LC50 represented the concentration required to induce cell
death by 50%.

compounds 8b and 8c reached or exceeded 4.4, and these values
of 9c-f ranged from 3.4 to 3.9, similar to ezetimibe (Figure 4).
Finally, we established an orally dosed, cholesterol-fed
hamster model as reported in literature to evaluate the in vivo
efficacy of ezetimibe and compound 9e (50 mg/kg/ day), which
displayed promising in vitro potency, nontoxicity and good
partition coefficient.22 The body weight of every group was
monitored throughout the observation period (Figure 5). The
model group gained weight greatly, but ezetimibe even decreased
weight slightly in the first three days. Interestingly, weight
change treated by compound 9e was similar to the one of control
group. At the same time, the ezetimibe treatment reduced total
cholesterol (TC), low-density lipoprotein cholesterol (LDL-C),
free fatty acid (FFA) and triglyceride (TG) by 30.2%, 24.8%,
29.5% and 36.0% respectively in the serum and significantly
increased the proportion of high-density lipoprotein cholesterol
(HDL-C) in TC (56.0%). Compound 9e lowered TC, LDL-C,
FFA and TG by 25.7%, 17.8%, 21.4% and 25.3% in the serum
and the rate of HDL-C to TC was increased to 42.9% (Figure 6
and Figure S4). These results suggested that compound 9e could
regulate the lipid metabolism and inhibit the cholesterol uptake in
vivo. Meanwhile, we measured the content of NO and NOS in the
serum, and these results indicated that compound 9e could
increase NO and (NO synthase) NOS obviously compared with
model group (Figure 6). Accumulating evidence suggested that
the synthesis and release of NO as the endothelium-derived
relaxing factor could maintain the vascular homeostasis.23
Besides these, we detected no obvious alteration of TC and TG in
the liver (Figure S4).
failed to achieve the level of ezetimibe in the in vivo study. The
probable reason for this result could be the rapid glucuronidation
of ezetimibe in the small intestine and liver. It was reported that
phenolic hydroxyl of ezetimibe served as a functional handle for
glucuronidation,24 and this active phenolic glucuronide can limit
systemic excretion by enterohepatic circulation and improve the
oral activity much better than ezetimibe.25 Synthesis and
evaluation of novel 2-azetidinone derivatives with p-
hydroxyphenyl replacing p-nitrophenyl group are underway in
our laboratory and will be reported in due course.
Acknowledgments
This work was supported by the Natural Science Foundation
for Colleges and Universities in Jiangsu Province (No.
11KJB350002), the Natural Science Foundation of Jiangsu
Province (No. BK2012422) and the National Natural Science
Foundation of China (NO. 81202393).
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Figure 6. TC (a), TG (b), LDL-C (c), FFA (d), NO (e) and NOS (f) content in
serum of hamsters. These values represented mean ± SD (n=6). #P<0.05 vs
control group; ###P<0.001 vs control group; *P<0.05 vs model group;
**P<0.01 vs model group; ***P<0.001 vs model group.
In summary, with the only marketable cholesterol absorption
inhibitor ezetimibe as the lead, we have synthesized sixteen 2-
azetidinone derivatives and thirteen 1H-pyrrole-2,5-dione
derivatives successfully and most of new compounds exhibited
significant inhibition of cholesterol uptake in Caco-2 cell line, an
in vitro model to mimic intestinal cholesterol absorption. This
was the first time that sulfonamide group was introduced to the
side chain and 1H-pyrrole-2,5-dione was selected as the scaffold.
Furthermore, top six compounds (8b-c and 9c-f) were evaluated
for cytotoxicity and partition coefficient, and 2-azetidinone
analogue 9e was screened out and tested in vivo. Taken together,
compound 9e was confirmed as a potent cholesterol absorption
inhibitor. Though 9e showed great inhibitory effect in vitro, it
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