Synthesis and evaluation of 1H-pyrrole-2,5-dione derivatives as cholesterol absorption inhibitors for suppressing the formation of foam cells and inflammatory response

Article abstract of DOI:10.1016/j.bmc.2017.08.046

Excess lipid accumulation in the arterial intima and formation of macrophage-derived foam cells in the plaque could cause atherosclerotic lesion. Cholesterol absorption inhibitors could suppress the lipid accumulation of human macrophage, inflammatory res

Full text of DOI:10.1016/j.bmc.2017.08.046

Accepted Manuscript
Synthesis and evaluation of 1H-pyrrole-2,5-dione derivatives as cholesterol ab-
sorption inhibitors for suppressing the formation of foam cells and inflammatory
response
Xinrui Yuan, Yineng Xia, Peng Lu, Lijuan Zhu, Yuejiao Zhong, Yubin Wang
PII:
S0968-0896(17)31550-X
http://dx.doi.org/10.1016/j.bmc.2017.08.046
BMC 13950
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
31 July 2017
24 August 2017
27 August 2017
Please cite this article as: Yuan, X., Xia, Y., Lu, P., Zhu, L., Zhong, Y., Wang, Y., Synthesis and evaluation of
1H-pyrrole-2,5-dione derivatives as cholesterol absorption inhibitors for suppressing the formation of foam cells
and inflammatory response, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.
2017.08.046
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Synthesis and evaluation of 1H-pyrrole-2,5-dione derivatives as cholesterol absorption
inhibitors for suppressing the formation of foam cells and inflammatory response
Xinrui Yuan,a Yineng Xia,a Peng Lu,a Lijuan Zhu,a Yuejiao Zhongb and Yubin Wanga, *
a School of Pharmaceutical Sciences, Nanjing Tech University, No. 5 Xinmofan Road, Nanjing
210009, People’s Republic of China
b The Affiliated Cancer Hospital of Nanjing Medical University, No. 42 Baiziting, Nanjing 210009,
People’s Republic of China
* Corresponding author. E-mail address: wyb5393@njtech.edu.cn
Abstract
Excess lipid accumulation in the arterial intima and formation of macrophage-derived foam cells
in the plaque could cause atherosclerotic lesion. Cholesterol absorption inhibitors could suppress
the lipid accumulation of human macrophage, inflammatory response and the development of
atherosclerosis. In this research, a series of 1H-pyrrole-2,5-dione derivatives were synthesized as
cholesterol absorption inhibitor and tested in in vitro experiments. One of the most active
inhibitors, compound 20 exhibited stronger in vitro cholesterol absorption activity than ezetimibe,
no cytotoxicity in HEK293 and RAW264.7 cell lines and satisfied lipophilicity. The further study
indicated that 20 could inhibit lipid accumulation of macrophage and reduce the secretion of LDH,
MDA, TNF-α and ROS in a concentration-dependent manner. In conclusion, as a novel and potent
cholesterol absorption inhibitor, compound 20 could suppress the formation of foam cells and
inflammatory response.
Keywords: 1H-pyrrole-2,5-dione; cholesterol absorption inhibitors; macrophage-derived foam
cells; inflammatory response
1. Introduction
Atherosclerosis with excess lipid accumulation in the arterial intima is a chronic
inflammatory disease and the major underlying cause for cardiovascular events. The
formation of macrophage-derived foam cells in the plaque is the elementary feature of
atherosclerotic lesion initiation and one of characteristics for the development of
atherosclerosis[1]. On the other hand, lipid homeostasis, especially cholesterol homeostasis,

plays a vital role in the formation of macrophage-derived foam cells, indicating that
inhibition of lipid accumulation could be an effective anti-atherosclerotic strategy[2].
There are two main sources for cholesterol in the body: biosynthesis in liver and
cholesterol absorption from the small intestine[3]. Cholesterol absorption inhibitors can
suppress the exogenous cholesterol absorption by targeting a polytopic transmembrane
protein called the Niemann-Pick C1-like 1 (NPC1L1) protein. As the only approved
cholesterol absorption inhibitor, ezetimibe (Zetia, 1) widely used in the therapy of patients
with family hypercholesterolemia has been thoroughly studied in clinical trials to evaluate
its effects on lipid profiles, cardiovascular outcomes or other surrogate endpoints[4]. And in
the clinical administration, the combination of ezetimibe and statin have been shown to
lower LDL-C concentration and improve cardiovascular outcomes more effectively
compared with statin therapy alone[5,6]
.
Fig. 1. Structure of ezetimibe and its analogues
Recently, some studies demonstrated that cholesterol absorption inhibitors have the same
protective effect on the moderate atherosclerotic lesion as atorvastatin, and this function was
associated with lowering serum cholesterol, decreasing circulatory inflammation cytokines and
inhibiting macrophage accumulation in lesions[7,8]. In several animal models, cholesterol
absorption inhibitors were also confirmed to have the beneficial effect on atherosclerosis by its
reduction of atherosclerotic lesion[9]. Udo Seedorf et al. reported that ezetimibe could inhibit the
lipid accumulation of human macrophage and the formation of foam cells, which showed that
cholesterol absorption inhibitors had potential effect on the prevention and treatment of
atherosclerosis[10]. Though ezetimibe is safe in clinic so far undoubtedly, Alsheikh-Ali and Karas
reported that the combination of ezetimibe and simvastatin could increase the risk of cancer[11]. So,
there remains scant demand for developing new cholesterol absorption inhibitors that could

improve therapeutic potency and provide patients with more choices.
Earlier, we have reported the discovery and characterization of a series of
1H-pyrrole-2,5-dione derivatives
2 containing sulfonamide group as potent cholesterol
absorption inhibitors, which indeed exhibited certain inhibitory activity[12]. In this paper,
we retained the substituents (
p-fluoro, p-ethyl, p-trifluoromethyl and p-trifluoromethoxy)
on the benzene ring at the terminal of side chain with best potency, shorten the length of
side chain and completed the synthesis of eight 1
H-pyrrole-2,5-dione derivatives 7a-h.
Table 1. Structure of 1H-pyrrole-2, 5-dione derivatives 7a-h.
Compound
n
2
2
2
2
3
3
3
3
R
7a
7b
7c
7d
7e
7f
F
Et
CF3
CF3O
F
Et
CF3
CF3O
7g
7h
The phenolic hydroxyl group of ezetimibe tolerated various structural modifications
(disaccharide, carboxyalkyl and glucuronide) that retained inhibitory activity[13-15]. We
conjugated glycylglycine to phenolic hydroxyl group and synthesized ten
1H-pyrrole-2,5-dione derivatives 14a-j successfully. In the preceding work, we screened
out compound
3 which had significant effect on the inhibition of total cholesterol
absorption in an orally dosed, cholesterol-fed hamster model[16]. We maintained diphenyl
methylene moiety and replaced 2-azetidinone with 1
scaffold to obtain compound 20. At the same time, we introduced diethylamine or sodium
sulfonate to the phenolic hydroxyl group and synthesized other compounds 22a-f
H-pyrrole-2, 5-dione as new molecular
.

Table 2. Structure of 1H-pyrrole-2,5-dione derivatives 14a-j, 20 and 22a-f.
Compound
14a
Compound
20
n
0
2
3
4
2
3
4
R
Ar
-Ph
H
N(C2H5)2
N(C2H5)2
N(C2H5)2
SO3Na
SO3Na
SO3Na
14b
4-F-Ph
22a
4-CH3-Ph
4-CF3-Ph
3-Br-Ph
4-Et-Ph
14c
22b
14d
22c
14e
22d
14f
22e
14g
2-Thiophene
2-OCF3-Ph
4-OCF3-Ph
4-C(CH3)3-Ph
22f
14h
14i
14j
In this article, we perform a further structural modification of 1
derivatives to improve the cholesterol absorption inhibitory activity. And the
−activity relationship (SAR) will be discussed. The most desired compound will
H-pyrrole-2, 5-dione
structure
be evaluated for their effects on suppressing the lipid accumulation of macrophage and the
inflammatory response.
2. Results and discussion
2.1. Chemistry
A common intermediate was 1
The synthesis of 7a-h was accomplished using intermediate 4a whose phenolic hydroxyl
was protected by PMB ( -methoxybenzyl group)(Scheme 1). Treatment of 4a with
H .
-pyrrole-2,5-dione 4a-b, prepared as described formerly[12]
p
1,2-dibromoethane or 1,3-dibromopropane under K2CO3 produced compounds 5a and 5b
which reacted respectively with substituted aromatic sulfonamide to afford 6a-h. The
reaction of 6a-h with acetic acid afforded the desired products 7a-h with good yield.
,

Scheme 1. Synthesis of target compounds 7a-h. Reagents and conditions:(a) K2CO3, TBAB, CH3CN, r.t.; (b)
K2CO3, KI, CH3CN, reflux; (c) CH3COOH, reflux.
The general synthetic route toward 14a-j was depicted in Scheme 2
benzyl group reacted with 1,4-dibromobutane to afford compound
to compound by debenzylation reaction in the presence of BBr3. Subsequent reaction of
.
4b protected by
8, which was converted
9
9
with methyl bromoacetate prepared intermediate 10 with K2CO3. Compound 10 was
condensed with various substituted aromatic sulfonamide to give the corresponding
compounds 11a-j. The hydrolysis of 11a-j followed by reaction with glycylglycine methyl
ester hydrochloride yielded intermediate 13a-j. Subsequently, the target compounds 14a-j
were synthesized by hydrolysis reaction of 13a-j in acid condition.
Scheme 2. Synthesis of target compounds 14a-j. Reagents and conditions:(a) K2CO3, TBAB, CH3CN, r.t.; (b)
BBr3, CH2Cl2, -78 °C; (c) K2CO3, Acetone, reflux; (d) KI, K2CO3, CH3CN, reflux; (e) LiOH, THF/H2O, 0 °C; (f)
EDCl, HOBt, DIPEA, CH3CN, 50 °C; (g):(i) Et3N, LiCl, CH3CN /H2O; (ii) HCl.
Scheme 3 presents the synthetic routes for 20 and 22a-f. Commercially available benzophenone

15 reacted with ethyl bromoacetate by classical Reformatsky reaction gave intermediate 16.
Reduction of 16 in the presence of LiAlH4 following reaction with p-toluenesulfonyl chloride
produced compound 18. 18 was condensed with 4b to afford 19 in the presence of K2CO3 and
TBAB. Compound 20 preparing from deprotection reaction of 19 with 20% Pd(OH)2 was treated
with 1,2-dibromoethane, 1,3-dibromopropane and 1,4-dibromobutane to yield 21a-c. Intermediate
21a-c respectively reacted with diethylamine or sodium sulfite to produce other desired
compounds 22a-f.
Scheme 3. Synthesis of target compounds 20 and 22a-f. Reagents and conditions: (a) Zn, TMSCl, ethyl
bromoacetate, ether, reflux; (b) LiAlH4, THF, r.t.; (c) TsCl, Et3N, DMAP, DCM, r.t.; (d) K2CO3, TBAB, CH3CN,
r.t.; (e) Pd(OH)2, H2, THF, r.t.; (f) K2CO3, CH3CN, reflux; (g) K2CO3, (C2H5)2NH, CH3CN, reflux; (h) Na2SO3,
C2H5OH/H2O, reflux.
2.2. Inhibitory activity of cholesterol absorption in cellular assays
Previously, we combined the Caco-2 (a human colon adenocarcinoma cells) cell line, a reliable
human intestinal model with an HPLC method for the measurement of total cholesterol[12,17,18]
.
However, Caco-2 cell contains a large amount of cholesterol itself, which can affect the accuracy
of results to some degree. Considering stigmasterol was also the substrate of NPC1L1 protein and
it was absent in biological samples[19], we substituted cholesterol with stigmasterol to assessed in
vitro inhibitory activity of compounds 7a-h, 14a-j, 20 and 22a-f at 100 µM concentration. As

shown in Figure 2, most of target compounds maintained inhibitory potency, especially 7b, 7f-h,
14f, 20 and 22a-c. Surprisingly, though 22d-f containing sodium sulfonate group displayed a
significant loss of inhibitory activity, 22a-c containing tertiary amino group showed significant
inhibitory potency and inhibited cholesterol uptake by 69.6-72.6% (ezetimibe: 51.3%). On the
other hand, the length of carbon chain had no influence on compounds’ efficacy (e.g., 22a vs 22b
vs 22c). As can be seen in compounds 14a-j, when substituents at the p-position of phenyl ring
were ethyl or methyl, compounds has stronger potency. Of compounds 7a-h, when the length of
side chain was shortened from three carbons to two carbons, in vitro activity was decreased (e.g.,
7a vs 7e, 7b vs 7f), and when ethyl group was introduced into p-position of phenyl ring at the side
chain, compounds showed stronger efficacy (e.g., 7b vs 7a, 7c, 7d).
Fig. 2. Inhibitory potency of compounds 7a-h, 14a-j, 20 and 22a-f on cholesterol absorption in Caco-2 cell lines.
(mean SD, n=4)
2.3. Cytotoxicity and partition coefficients assays of candidate compounds
Based on the in vitro results, 7b, 7f-h, 14c, 14f, 20 and 22a-c were screened for further
evaluation. Firstly, MTT cell proliferation assay was adopted against the HEK293 (human
embryonic kidney cells) and RAW264.7 (mouse macrophage, Abelson murine leukemia
virus transformed) cell lines. If LC50 values were higher than 100 µM, compounds were
considered no cytotoxic[20]. As been shown in Table 3, LC50 values of ezetimibe were
57.58 µM against HEK293 and 44.20 µM against RAW264.7, respectively. Unfortunately,
compounds 22a-c with the best in vitro potency exhibited LC50 values of 31.5-47.3 µM
against HEK293 and RAW264.7. Except 14c, 14f and 20, the rest of compounds displayed
cytotoxicity in both the cell lines.

Table 3. Cytotoxicity assay of compounds 7b, 7f-h, 14c, 14f, 20 and 22a-c against HEK293 and RAW264.7.
LC50a (µM)
LC50 (µM)
Compound
Compound
HEK293 RAW264.7
HEK293 RAW264.7
80.4
11.5
19.7
24.8
>100
>100
>100
17.2
34.3
41.2
>100
>100
>100
31.5
32.7
47.3
57.6
>100
32.7
37.6
43.2
44.2
7b
7f
20
20a
20b
20c
Ez
7g
7h
14c
14f
a LC50 represented the concentration required to induce cell death by 50%.
As druglike physical property, lipophilicity is one of standards to estimate compounds and
represented by partition coefficients (P), and compounds with high lipophilicity could bind
to unwanted targets, causing cardiovascular and tissue toxicity[21]. Except LogP of 22a-c
exceeding 5.0, values of 7b
Figure 3). As the compromise in terms of potency and druglike properties, compound 20
turned out to be the best candidate.
, 7f-h, 14c, 14f and 20 ranged from 2 to 4, similar to ezetimibe
(
Fig. 3. Partition coefficients of compounds 7b, 7f-h, 14c, 14f and 20. (mean SD, n=3)
2.4. Cholesterol absorption inhibitory activity of compound 20 in RAW264.7 cell line treated
with Cholesterol-D7
Though previous studies demonstrated that ezetimibe and compound 20 could inhibit cholesterol
absorption in Caco-2 cell line significantly, we validated the cholesterol absorption inhibitory
potency of ezetimibe and compound 20 in RAW264.7 cell line treated with Cholesterol-D7. We
cultured RAW264.7 cells with 250 µg/mL Chol-D7: MβCD for 72 h, and intracellular
cholesterol-D7 was measured by LC-MS/MS. As shown in Figure 4, both of compounds showed
significant inhibition of Cholesterol–D7 absorption. 30 µM ezetimibe and compound 20 resulted
in reduction of Cholesterol–D7 in RAW264.7 cells by 80.3% and 67.9%, respectively.

Fig. 4. Cholesterol-D7 content of RAW264.7 treated with Chol-D7:MβCD. These values represented mean SD
(n=3).
2.5. Compound 20 suppresses lipid accumulation and formation of macrophage-derived
foam cell
It has been testified that ezetimibe could suppress macrophage-derived foam cell formation,
which is essential to atherosclerotic lesion development[7]. In this article,
macrophage-derived foam cell model was established by treating RAW264.7 with
Chol:MβCD, which is water-soluble and can induce foam cell formation[22]. The results of
preliminary experiment exhibited that the optimal concentration of Chol:MβCD was
determined as 250 µg/mL, and when the concentration of Chol:MβCD exceeded 300
µg/mL, the growth of RAW264.7 was inhibited. Considering that LC50 value of ezetimibe
was 44.20 µM against RAW264.7, the concentration of compounds was set as 7.5, 15 and
30 µM. Firstly, we treated RAW264.7 cell line with 250 µg/mL Chol:MβCD added with
7.5, 15 and 30 µM ezetimibe or compound 20 for 72 h, respectively. Interestingly, the
results of Oil red O staining exhibited that ezetimibe and compound 20 had suppression
effect in a concentration-dependent manner. And 30 µM ezetimibe and compound 20 could
greatly inhibit cellular lipid accumulation (Figure 5).
Fig. 5. The Oil red O staining results of RAW264.7 treated with Chol:MβCD.
The formation of foam cells can induce macrophages injury and increasing the release of LDH
(Lactate Dehydrogenase) and MDA (Malonic Dialdehyde). To further assess the effect of

ezetimibe or compound 20, we evaluated the amount of LDH and MDA in RAW264.7 cell line
treated with 250 µg/mL Chol: MβCD. The results showed that with the concentration of ezetimibe
or compound 20 increasing, the amount of LDH and MDA decreased (Figure 6). 30 µM
compound 20 decreased LDH and MDA by 33.3% and 79.6%, and 30 µM ezetimibe caused the
reduction by 36.4% and 76.3% respectively.
Fig. 6. LDH and MDA content of RAW264.7 treated with Chol:MβCD. These values represented mean SD
(n=3).
2.6. Compound 20 attenuates inflammatory response in macrophage-derived foam cell
During the lipid accumulation, macrophages are activated and can secrete various inflammatory
cytokines, such as TNF-α[23]. Redundant generation and accumulation of ROS (Reactive Oxygen
Species) can lead to oxidative stress and DNA damage[24]. Excessive ROS also activates several
injury-producing pathways, such as the nuclear factor-κB (NF-κB) inflammatory response[25]. To
further evaluate the anti-inflammatory effects of compound 20, we utilized RAW264.7 cell line
treated with Chol:MβCD, which could activate the inflammatory response in macrophages[22]. As
shown in Figure 7, ezetimibe treatment can reduced TNF-α and ROS by 30.9% and 46.5% at the
concentration of 30 µM. And 30 µM compound 20 also can decrease TNF-α and ROS by 35.1%
and 51% respectively.
Fig. 7. TNF-α and ROS content of RAW264.7 treated with Chol:MβCD. These values represented mean SD
(n=3).
3. Conclusions

The formation of macrophage-derived foam cells is considered to be the crucial aspect of
cardiovascular disease, especially in atherosclerosis. Macrophage is present in various
stages of atherosclerotic plaque and plays a major role during atherosclerotic lesion,
partially through inflammatory response[26]. Excessive accumulation of cholesterol in
various types of cells activates the inflammatory response and induces the development
and progression of atherosclerosis[22]
.
In this research, a series of 1H-pyrrole-2,5-dione derivatives were synthesized. In vitro
activity evaluation led to the discovery of a number of potent NPC1L1 inhibitors. One of
the most potent inhibitors, compound 20 was more active than ezetimibe in the in vitro
cholesterol absorption study and displayed the weakest cytotoxicity property in HEK293
and RAW264.7 cell lines and satisfied lipophilicity in a preliminary study. Furthermore,
cholesterol uptake inhibitory potency of compound 20 was verified by Cholesterol-D7.
Compound 20 also could inhibit macrophage lipid accumulation and reduce the secretion
of LDH, MDA, TNF-α and ROS comparable with ezetimibe. This in-depth study indicated
that compound 20 could suppress the formation of macrophage-derived foam cells and
inflammatory response. So, as a novel and potent cholesterol absorption inhibitor,
compound 20 showed the potential of relieving atherosclerosis.
4. Experimental section
4.1. Chemistry
All commercial reagents and solvents were used without further purification unless expressly
stated. Thin-layer chromatography (TLC) were performed on silica gel GF 254 (Tsingtao Haiyang
Chemicals, China) and observed under ultraviolet lamp at 254 nm. Column chromatography was
conducted on silica gel (200-300 mesh, Tsingtao Haiyang Chemicals, China). Melting points of
the synthesized compounds were determined with capillary apparatus and were not corrected.
1H-NMR and 13C-NMR spectra of the intermediates and final compounds were measured on
Bruker ACF-500Q apparatus at 500 MHz or Bruker AV-300 apparatus at 300 MHz using TMS as
the internal standard in CDCl3 or DMSO-d6 unless otherwise indicated. Mass spectrometry (MS)
were performed with Hewlett-Packard 1100 LC/MSD spectrometer. High-resolution-time-of-flight
(HR-TOF)-MS were recorded on a BioTOFTM-Q mass spectrometer (Bruker).
4.1.1. General procedure for the preparation of 7a-h

To a solution of 3,4-diphenyl-1H-pyrrole-2,5-dione 4a (2.11 mmol) in CH3CN (15 mL), K2CO3
(2.53 mmol), TBAB (0.2 mmol) and 1,2-dibromoethane or 1,3-dibromopropane (8.43 mmol) were
added and the mixture was stirred at 45 °C overnight. Solvent was evaporated to dryness, water
(30 mL) added and the resulting mixture extracted with ethyl acetate. Collected organic layers
were dried over anhydrous Na2SO4 and solvent evaporated to dryness. 5a-b was obtained by a
silica gel column chromatography. K2CO3 (0.784 mmol), KI (0.588 mmol), substituted aromatic
sulfonamide (0.588 mmol) and compound 5a-b (0.392 mmol) were added to 4 mL CH3CN. The
mixture was stirred reflux overnight, and solvent was concentrated, H2O added and the resulting
mixture extracted with ethyl acetate. Collected organic layers were dried over anhydrous Na2SO4
and evaporated. 6a-h was obtained by a silica gel column chromatography. A solution of 6a-h
(0.212 mmol) in 2.12 mL acetic acid proceeded at 90 °C overnight. H2O was added and the
resulting solution was extracted with ethyl acetate. The combined organic layers were dried over
anhydrous Na2SO4 and concentrated. Target compounds 7a-h were purified by the silica gel
column chromatography.
1-(2-bromoethyl)-3-(4-fluorophenyl)-4-(4-((4-methoxybenzyl)-oxy)phenyl)-1H-pyrrole-2,5-
dione (5a). Yield: 83%; mp: 81-85 °C; 1H-NMR (CDCl3, 300M): 3.60 (t, J = 6.6 Hz, 2H, -CH2-),
3.81 (s, 3H, CH3O-), 4.06 (t, J = 6.6 Hz, 2H, -CH2-), 5.01 (s, 2H, PhCH2O-), 6.95 (dd, J = 1.8, 6.9
Hz, 4H, Ar-H), 7.06 (t, J = 8.7 Hz, 2H, Ar-H), 7.34 (d, J = 8.7 Hz, 2H, Ar-H), 7.49 (m, 4H, Ar-H);
MS: found m/z [M+Na]+ 534.3, calcd. m/z [M] 511.06.
1-(3-bromopropyl)-3-(4-fluorophenyl)-4-(4-((4-methoxybenzyl)-oxy)phenyl)-1H-pyrrole-2,
5-dione (5b). Yield: 54.5%; mp: 90-94 °C; 1H-NMR (CDCl3, 300M): 2.27 (q, J = 6.6 Hz, 2H,
-CH2-), 3.43 (t, J = 6.6 Hz, 2H, -CH2-), 3.77 (d, J = 6.9 Hz, 2H, -CH2-), 3.82 (s, 3H, CH3O-), 5.01
(s, 2H, PhCH2O-), 6.93 (m, 4H, Ar-H), 7.06 (m, 2H, Ar-H), 7.34 (d, J = 8.7 Hz, -CH2-), 7.49 (m,
4H, Ar-H); MS: found m/z [M+Na]+ 546.2, calcd. m/z [M] 523.08.
N-(3-(3-(4-fluorophenyl)-4-(4-((4-methoxybenzyl)oxy)phenyl)-2,5-dioxo-2,5-dihydro-1H-p
yrrol-1-yl)propyl)-4-(trifluorometh-yl)benzenesulfonamide (6g). Yield: 95.3%; 1H-NMR
(CDCl3, 300M): 1.89 (m, 2H, -CH2-), 3.00 (m, 2H, -CH2-), 3.74 (t, J = 6 Hz, 2H, -CH2-), 3.84 (s,
3H, CH3O-), 5.01 (s, 2H, PhCH2O-), 5.32 (s, 1H, -NH-), 6.95 (m, 4H, Ar-H), 7.08 (t, J = 8.7 Hz,
2H, Ar-H), 7.38 (t, J = 9 Hz, 2H, Ar-H), 7.47 (m, 4H, Ar-H),7.71 (d, J = 8.4 Hz, 2H, Ar-H), 8.01

(d, J = 8.1 Hz, 2H, Ar-H); MS: found m/z [M-H]- 667.5, calcd. m/z [M] 668.16.
4-fluoro-N-(2-(3-(4-fluorophenyl)-4-(4-hydroxyphenyl)-2,5-di-oxo-2,5-dihydro-1H-pyrrol-
1-yl)ethyl)benzenesulfonamide (7a). Yield: 64.7%; mp: 174-176 °C; 1H-NMR (CDCl3, 300M):
3.31 (q, J = 5.7 Hz, 2H, -CH2-), 3.73 (t, J = 5.7 Hz, 2H, -CH2-), 4.96 (d, J = 5.4 Hz, 1H, -NHSO2-),
6.82 (d, J = 8.7 Hz, 2H, Ar-H), 7.08 (m, 4H, Ar-H), 7.38 (d, J = 8.7 Hz, 2H, Ar-H), 7.46 (m, 2H,
Ar-H), 7.84 (m, 2H, Ar-H); 13C-NMR (CDCl3+CH3OD, 300M): 37.69, 41.14, 115.54, 115.59,
115.71, 116.06, 116.24, 119.18, 124.86, 124.89, 129.43, 129.50, 131.50, 131.65, 131.71, 132.48,
136.17, 159.01, 162.22, 163.81, 164.22, 165.83, 171.03, 171.10; HRMS: found m/z [M+Na]+
507.08122, calcd. m/z [M] 484.09045.
4-ethyl-N-(2-(3-(4-fluorophenyl)-4-(4-hydroxyphenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)ethyl)benzenesulfonamide (7b). Yield: 82.4%; mp: 170-175 °C; 1H-NMR (CDCl3, 500M):
1.22 (t, J = 7.5 Hz, 3H, -CH2CH3), 2.65 (q, J = 5.5 Hz, 2H, -CH2CH3), 3.28 (m, J = 5.5 Hz, 2H,
-CH2-), 3.73 (t, J = 5.5 Hz, 2H, -CH2-), 4.92 (s, 1H, -NHSO2-), 6.81 (d, J = 8.5 Hz, 2H, Ar-H),
7.06 (t, J = 8.5 Hz, 2H, Ar-H), 7.27 (d, J = 8 Hz, 2H, Ar-H), 7.38 (d, J = 8.5 Hz, 2H, Ar-H), 7.46
(m, 2H, Ar-H), 7.74 (d, J = 8.5 Hz, 2H, Ar-H); 13C-NMR (CDCl3+CH3OD, 300M): 28.65, 29.58,
37.71, 41.42, 115.61, 115.65, 115.78, 119.33, 124.94, 126.97, 128.50, 131.57, 131.71, 131.78,
132.56, 136.21, 137.08, 149.47, 158.98, 162.27, 164.26, 171.09, 171.16; HRMS: found m/z
[M+NH4]+ 512.16734, calcd. m/z [M] 494.13117.
N-(2-(3-(4-fluorophenyl)-4-(4-hydroxyphenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)
-4-(trifluoromethyl)benzenesulfo-namide (7c). Yield: 61.8%; mp: 165-167 °C; 1H-NMR (CDCl3,
500M): 3.33 (m, 2H, -CH2-), 3.76 (t, J = 5.5 Hz, 2H, -CH2-), 5.18 (s, 1H, -OH), 5.20 (d, J = 5.5
Hz, 1H, -NHSO2-), 6.81 (d, J = 8.5 Hz, 2H, Ar-H), 7.06 (t, J = 8.5 Hz, 2H, Ar-H), 7.37 (d, J = 8.5
Hz, 2H, Ar-H), 7.45 (m, 2H, Ar-H), 7.73 (d, J = 8 Hz, 2H, Ar-H), 7.97 (d, J = 8 Hz, 2H, Ar-H);
13C-NMR (CDCl3+CH3OD, 300M): 37.77, 41.29, 115.62, 115.66, 115.80, 119.26, 122.07, 124.23,
124.90, 126.15, 126.18, 127.35, 131.54, 131.68, 131.75, 132.62, 133.98, 134.24, 136.27, 143.85,
159.02, 162.28, 164.28, 171.10, 171.18; 13C-NMR (CDCl3+CH3OD, 300M): HRMS: found m/z
[M+NH4]+ 552.12346, calcd. m/z [M] 534.08726.
N-(2-(3-(4-fluorophenyl)-4-(4-hydroxyphenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)
-4-(trifluoromethoxy)benzenesulfon-amide (7d). Yield: 77.7%; mp: 72-76 °C; 1H-NMR (CDCl3,
500M): 3.31 (m, J = 5.5 Hz, 2H, -CH2-), 3.75 (t, J = 5.5 Hz, 2H, -CH2-), 5.14 (t, J = 5.5 Hz, 1H,

-NHSO2-), 6.80 (d, J = 8.5 Hz, 2H, Ar-H), 7.06 (t, J = 8.5 Hz, 2H, Ar-H), 7.26 (d, J = 8 Hz, 2H,
Ar-H), 7.37 (d, J = 8.5 Hz, 2H, Ar-H), 7.46 (m, 2H, Ar-H), 7.89 (d, J = 8.5 Hz, 2H, Ar-H);
13C-NMR (CDCl3+CH3OD, 300M): 37.75, 41.21, 115.59, 115.64, 115.77, 119.24, 120.84, 124.89,
124.92, 128.97, 131.53, 131.67, 131.74, 132.59, 136.27, 138.58, 151.89, 159.02, 162.26, 164.26,
171.08, 171.16; HRMS: found m/z [M+NH4]+ 568.11804, calcd. m/z [M] 550.08217.
4-fluoro-N-(3-(3-(4-fluorophenyl)-4-(4-hydroxyphenyl)-2,5-di-oxo-2,5-dihydro-1H-pyrrol-
1-yl)propyl)benzenesulfonamide (7e). Yield: 54.8%; mp: 70-74 °C; 1H-NMR (CDCl3, 500M):
1.85 (m, J = 6 Hz, 2H, -CH2-), 2.96 (t, J = 6 Hz, 2H, -CH2-), 3.70 (t, J = 6 Hz, 2H, -CH2-), 5.26 (s,
1H, -NHSO2-), 6.81 (d, J = 9 Hz, 2H, Ar-H), 7.05 (d, J = 8.5 Hz, 2H, Ar-H), 7.11 (t, J = 8.5 Hz,
2H, Ar-H), 7.36 (d, J = 8.5 Hz, 2H, Ar-H), 7.44 (m, 2H, Ar-H), 7.87 (m, 2H, Ar-H); 13C-NMR
(CDCl3, 300M): 28.65, 34.85, 40.04, 115.86, 116.04, 116.20, 116.38, 120.33, 124.66, 124.69,
129.75, 129.83, 131.70, 131.79, 131.86, 133.13, 135.82, 135.88, 135.90, 157.72, 162.45, 163.99,
164.45, 166.02, 171.31, 171.44; MS: found m/z [M+NH4]+ 516.14229, calcd. m/z [M] 498.10610.
4-ethyl-N-(3-(3-(4-fluorophenyl)-4-(4-hydroxyphenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)propyl)benzenesulfonamide (7f). Yield: 81.8%; mp: 122-124 °C; 1H-NMR (CDCl3, 500M):
1.23 (t, J = 7.5 Hz, 3H, -CH2CH3), 1.83 (m, J = 6 Hz, 2H, -CH2-), 2.68 (q, J = 7.5 Hz, 2H,
-CH2CH3), 2.96 (d, J = 5.5 Hz, 2H, -CH2-), 3.69 (t, J = 6 Hz, 2H, -CH2-), 5.19 (s, 1H, -NHSO2-),
6.80 (d, J = 8.5 Hz, 2H, Ar-H), 7.04 (t, J = 8.5 Hz, 2H, Ar-H), 7.26 (d, J = 7.5 Hz, 2H, Ar-H), 7.36
(d, J = 8.5 Hz, 2H, Ar-H), 7.46 (m, 2H, Ar-H), 7.76 (d, J = 8.5 Hz, 2H, Ar-H); 13C-NMR (CDCl3,
300M): 28.71, 28.74, 29.66, 34.96, 40.08, 114.02, 115.74, 115.84, 116.03, 120.41, 124.76, 124.80,
127.15, 128.52, 129.73, 131.70, 131.78, 131.89, 133.11, 135.83, 137.11, 149.55, 157.70, 161.75,
165.09, 171.25, 171.36; HRMS: found m/z [M+NH4]+ 526.18311, calcd. m/z [M] 508.14682.
N-(3-(3-(4-fluorophenyl)-4-(4-hydroxyphenyl)-2,5-dioxo-2,5-di-hydro-1H-pyrrol-1-yl)prop
yl)-4-(trifluoromethyl)benzenesulfon-amide (7g). Yield: 77.6%; mp: 72-74 °C; 1H-NMR
(CDCl3, 500M): 1.86 (m, J = 6 Hz, 2H, -CH2-), 2.98 (q, J = 6 Hz, 2H, -CH2-), 3.71 (t, J = 6 Hz,
2H, -CH2-), 5.48 (t, J = 6.5 Hz, 1H, -NHSO2-), 6.80 (d, J = 8.5 Hz, 2H, Ar-H), 7.05 (t, J = 8.5 Hz,
2H, Ar-H), 7.36 (d, J = 8.5 Hz, 2H, Ar-H), 7.44 (m, 2H, Ar-H), 7.69 (d, J = 8.5 Hz, 2H, Ar-H),
7.98 (d, J = 8.5 Hz, 2H, Ar-H); 13C-NMR (CDCl3, 300M): 29.15, 30.07, 35.20, 40.50, 116.21,
116.27, 116.50, 120.77, 121.79, 125.04, 125.09, 125.40, 126.55, 126.60, 126.65, 126.69, 127.98,
130.14, 132.10, 132.16, 132.27, 133.59, 134.48, 134.92, 136.25, 144.00, 158.13, 162.22, 165.56,

171.74, 171.86; HRMS: found m/z [M+NH4]+ 566.13916, calcd. m/z [M] 548.10291.
N-(3-(3-(4-fluorophenyl)-4-(4-hydroxyphenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propy
l)-4-(trifluoromethoxy)benzene-sulfonamide (7h). Yield: 67.8%; mp: 68-72 °C; 1H-NMR
(CDCl3, 500M): 1.86 (m, J = 6 Hz, 2H, -CH2-), 2.98 (q, J = 6 Hz, 2H, -CH2-), 3.71 (t, J = 6 Hz,
2H, -CH2-), 5.35 (t, J = 6.5 Hz, 1H, -NHSO2-), 6.81 (d, J = 8.5 Hz, 2H, Ar-H), 7.06 (t, J = 8.5 Hz,
2H, Ar-H), 7.26 (d, J = 6 Hz, 2H, Ar-H), 7.37 (d, J = 8.5 Hz, 2H, Ar-H), 7.45 (m, 2H, Ar-H), 7.91
(d, J = 8.5 Hz, 2H, Ar-H); 13C-NMR (CDCl3, 300M): 29.15, 35.26, 40.49, 116.20, 116.26, 116.49,
120.79, 121.32, 125.07, 125.12, 129.61, 132.10, 132.17, 132.28, 133.60, 138.78, 158.12, 162.21,
165.55, 171.72, 171.84; HRMS: found m/z [M+NH4]+ 582.13424, calcd. m/z [M] 564.09782.
4.1.2. General procedure for the preparation of 14a-j
To a solution of 3,4-diphenyl-1H-pyrrole-2,5-dione 4b (4.96 mmol) in CH3CN (20 mL), K2CO3
(4.96 mmol), TBAB (0.5 mmol) and 1,4-dibromobutane (19.84 mmol) were added and the
mixture was stirred at 45 °C overnight. Solvent was evaporated to dryness, water (50 mL) added
and the resulting mixture extracted with ethyl acetate. Collected organic layers were dried over
anhydrous Na2SO4 and solvent evaporated to dryness. 8 was obtained by a silica gel column
chromatography. The solution of BBr3 (1 M, 9.47 mmol) in anhydrous CH2Cl2 was added to
compound 8 (3.94 mmol) in 20 mL anhydrous CH2Cl2 under nitrogen atmosphere at -78 °C. The
mixture was stirred at -78 °C for 1 h. The temperature rised to 0 °C, 10 mL H2O was added. and
the resulting solution was stirred for 0.5 h and extracted with ethyl acetate. Collected organic
layers were washed with brine and dried over anhydrous Na2SO4, and solvent was evaporated to
dryness to afford compound 9, a yellow solid with sufficient purity to be used without further
purification. To a solution of 9 (1.86 mmol) and K2CO3 (2.28 mmol) in 10 mL acetone, methyl
bromoacetate was added. The reaction proceeded reflux overnight and was added 30 mL water.
Then, the mixture was extracted with ethyl acetate and dried over anhydrous Na2SO4. The residue
was purified by a silica gel column chromatography to give 10. K2CO3 (1.76 mmol), KI (1.32
mmol), substituted aromatic sulfonamide (1.32 mmol) and compound 10 were added to 2 mL
CH3CN. The mixture was stirred reflux overnight, and solvent was concentrated, H2O (12 mL)
added and the resulting mixture extracted with ethyl acetate. Collected organic layers were dried
over anhydrous Na2SO4 and evaporated. 11 was obtained by a silica gel column chromatography.
1 M aqueous solution of LiOH (1.11 mmol) was added to the solution of compound 11 (0.742

mmol) in 4 mL THF at -5 °C. The mixture was stirred at -5 °C until complete by TLC and
adjusted to pH 5 by 1 M HCl. The resulting mixture was extracted with diethyl ether ,and the
organic layer was wash by brine and dried over anhydrous Na2SO4. The solution was evaporated
to afford compound 12. To a solution of glycylglycine methyl ester hydrochloride (0.81 mmol)
and DIPEA (1.62 mmol) in 5 mL CH3CN, 12 (0.67 mmol), HOBt (0.81 mmol) and EDCl (0.81
mmol) were added at 0 °C. The mixture proceeded at 50 °C overnight, diluted with CH2Cl2 and
washed with brine. The organic layer was dried over anhydrous Na2SO4 and evaporated.
Compound 13 was obtained by the silica gel column chromatography. For the synthesis of 14a-j,
Et3N (0.866 mmol) and LiCl (0.94 mmol) were added to the solution of compound 13 in 5 mL
CH3CN∶H2O (95∶5) at 0 °C. The solution was adjusted to pH 5 by 1 M HCl and evaporated by
CH2Cl2. Combined organic layer was washed by brine, dried over anhydrous Na2SO4 and
concentrated. The resulting residue was triturated with ethyl acetate and filtrated to give target
compound 14a-j.
3-(4-(benzyloxy)phenyl)-1-(4-bromobutyl)-4-(4-fluorophenyl)-1H-pyrrole-2,5-dione (8).
Yield: 60%; mp: 91-93 °C; 1H-NMR (CDCl3, 300 Hz): 1.867 (m, 2H, -CH2-), 1.92 (t, J = 7.2 Hz,
2H, -CH2-), 3.45 (t, J = 6.3 Hz, 2H, -CH2-), 3.67 (t, J = 6.8 Hz, 2H, -CH2N-), 3.82 (s, 3H, -OCH3),
5.00 (s, 2H, -PhCH2O-), 6.93 (t, J = 9.3 Hz, 4H, Ar-H), 7.05 (t, J = 8.5 Hz, 2H, Ar-H), 7.34 (d, J =
8.35 Hz, 2H, Ar-H), 7.45 (d, J = 8.65 Hz, 2H, Ar-H), 7.50 (dd, J = 5.75, 8.25 Hz, 2H, Ar-H); MS:
found m/z [M+Na]+ 530.10, 532.10, calcd. m/z [M] 507.08, 509.08.
1-(4-bromobutyl)-3-(4-fluorophenyl)-4-(4-hydroxyphenyl)-1H-pyrrole-2,5-dione (9). Yield:
94%; mp: 104-110 °C; 1H-NMR (CDCl3, 500M): 1.896 (m, 4H, -CH2-), 3.46 (t, J = 6.69 Hz, 2H,
-CH2-), 3.67 (t, J = 6.53 Hz, 2H, -CH2N-), 6.81 (d, J = 8.52 Hz, 2H, Ar-H), 7.06 (t, J = 8.58 Hz,
2H, Ar-H), 7.41 (d, J = 8.55 Hz, 2H, Ar-H), 7.49 (dd, J = 5.49, 8.46 Hz, 2H, Ar-H); MS: found
m/z [M-H]- 416.1, calcd. m/z [M] 417.04.
Methyl
2-(4-(1-(4-bromobutyl)-4-(4-fluorophenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)phenoxy)ac
etate (10). Yield: 94.7%; mp: 91-93 °C; 1H-NMR (CDCl3, 500M): 1.85 (dd, J = 6.8, 14.5 Hz, 2H,
-CH2-), 1.91 (m, 2H, -CH2-), 3.45 (t, J = 6.4 Hz, 2H, -CH2-), 3.67 (t, J = 6.775 Hz, 2H, -CH2N-),
3.80 (s, 3H, -OCH3), 4.66 (s, 2H, -OCH2CO-), 6.88 (d, J = 8.75 Hz, 2H, Ar-H), 7.06 (t, J = 8.6 Hz,

2H, Ar-H), 7.47 (m, 2H, Ar-H), 7.49 (d, J = 5.55 Hz, 2H, Ar-H); MS: found m/z [M+Na]+ 514.2,
calcd. m/z [M] 491.06.
Methyl
2-(4-(4-(4-fluorophenyl)-1-(4-(4-methylphenylsulfon-amido)butyl)-2,5-dioxo-2,5-dihydro-1H-
pyrrol-3-yl)phenoxy)ace-tate (11c). Yield: 68.4%; 1H-NMR (CDCl3, 500M): 1.53 (dd, J = 7.15,
14.75 Hz, 2H, -CH2-), 1.67 (dd, J = 7.15, 14.75 Hz, 2H, -CH2-), 2.41 (s, 3H, -CH3), 2.99 (d, J =
4.1 Hz, 2H, -CH2NH-), 3.60 (t, J = 6.85 Hz, 2H, -CH2N-), 3.81 (s, 3H, -OCH3), 4.52 (s, 1H,
-NH-), 4.66 (s, 2H, -OCH2CO-), 6.88 (d, J = 8.55 Hz, 2H, Ar-H), 7.05 (t, J = 8.55 Hz, 2H, Ar-H),
7.29 (d, J = 7.95 Hz, 2H, Ar-H), 7.47 (m, 4H, Ar-H), 7.73 (d, J = 8 Hz, 2H, Ar-H); MS: found m/z
[M-H]- 579.1, calcd. m/z [M] 580.17.
Methyl
2-(2-(2-(4-(4-(4-fluorophenyl)-1-(4-(4-methylphenyl-sulfonamido)butyl)-2,5-dioxo-2,5-dihydr
o-1H-pyrrol-3-yl)phenoxy)acetamido)acetamido)acetate (13c). Yield: 44.2%; mp: 155-158 °C;
1H-NMR (DMSO-d6, 300M): 1.39 (m, 2H, -CH2-), 1.56 (m, 2H, -CH2-), 2.36 (s, 3H, -CH3), 2.73
(dd, J = 6.36, 12.57 Hz, 2H, -CH2NH-), 3.47 (t, J = 6.36 Hz, 2H, -CH2N-), 3.62 (s, 3H, -OCH3),
3.81 (d, J = 5.88 Hz, 1H, -NCH2CO-), 3.86 (d, J = 5.85 Hz, 2H, -NCH2CO-), 4.58 (s, 2H,
-OCH2CO-), 7.02 (d, J = 8.85 Hz, 2H, Ar-H), 7.27 (t, J = 8.88 Hz, 2H, Ar-H), 7.34 (d, J = 7.14 Hz,
4H, Ar-H), 7.46 (m, 3H, Ar-H), 7.65 (d, J = 8.16 Hz, 2H, Ar-H); 8.34 (m, 2H, -NHCO-); MS:
found m/z [M+H]+ 695.3, calcd. m/z [M] 694.21.
(2-(4-(4-(4-fluorophenyl)-2,5-dioxo-1-(4-(phenylsulfonamido)-butyl)-2,5-dihydro-1H-pyrro
l-3-yl)phenoxy)acetyl)glycylglycine (14a). Yield: 80%; mp: 86-89 °C; 1H-NMR (DMSO-d6,
300M): 1.38 (m, 2H, -CH2-), 1.56 (m, 2H, -CH2-), 2.78 (t, J = 6 Hz, 2H, -CH2NH-), 3.47 (t, J = 6
Hz, 2H, -CH2N-), 3.75 (d, J = 5.1 Hz, 2H, -NCH2CO-), 3.81 (d, J = 5.7 Hz, 2H, -NCH2CO-), 4.58
(s, 2H, -OCH2CO-), 7.02 (d, J = 8.7 Hz, 2H, Ar-H), 7.27 (t, J = 9 Hz, 2H, Ar-H), 7.38 (d, J = 8.7
Hz, 2H, Ar-H), 7.46 (d, J = 8.4 Hz, 2H, Ar-H), 7.58 (m, 4H, Ar-H), 7.77 (d, J = 7.8 Hz, 2H, Ar-H),
8.16 (s, 1H, -NHCO-), 8.34 (d, J = 6.9 Hz, 1H, -NHCO-); 13C-NMR (DMSO-d6, 300M): 25.16,
26.33, 37.28, 40.83, 41.52, 42.00, 45.25, 66.72, 114.84, 115.53, 115.70, 121.19, 125.31, 126.34,
129.06, 131.20, 131.84, 131.91, 132.17, 133.30, 135.37, 140.55, 158.80, 161.52, 163.49, 167.62,
168.73, 170.30, 170.36, 171.08; HRMS: found m/z [M+NH4]+ 684.21673, calcd. m/z [M]
666.17958.

(2-(4-(4-(4-fluorophenyl)-1-(4-((4-fluorophenyl)sulfonamido)-butyl)-2,5-dioxo-2,5-dihydro-
1H-pyrrol-3-yl)phenoxy)acetyl)-glycylglycine (14b). Yield: 84%; mp: 97-101 °C; 1H-NMR
(DMSO-d6, 500M): 1.52 (m, 2H, -CH2-), 1.67 (m, 2H, -CH2-), 2.89 (t, J = 6.3 Hz, 2H, -CH2NH-),
3.58 (t, J = 6.75 Hz, 2H, -CH2N-), 3.96 (d, J = 5.15 Hz, 2H, -NCH2CO-), 4.06 (d, J = 5.15 Hz, 2H,
-NCH2CO-), 4.56 (s, 2H, -OCH2CO-), 6.95 (d, J = 8.75 Hz, 2H, Ar-H), 7.07 (t, J = 8.55 Hz, 2H,
Ar-H), 7.17 (t, J = 8.4 Hz, 2H, Ar-H), 7.47 (m, 4H, Ar-H), 7.72 (m, 2H, Ar-H), 7.87 (dd, J = 5.2,
8.5 Hz, 2H, 1H, -NHCO-); 13C-NMR (DMSO-d6, 300M): 25.15, 26.29, 37.26, 40.87, 41.51, 41.98,
66.71, 114.83, 115.52, 115.69, 116.10, 116.28, 121.18, 125.30, 129.31, 129.39, 131.19, 131.83,
131.90, 133.31, 135.37, 136.95, 158.79, 161.51, 162.96, 163.49, 164.95, 167.59, 168.68, 170.30,
170.36, 171.08; HRMS: found m/z [M+NH4]+ 702.20729, calcd. m/z [M] 684.17016.
(2-(4-(4-(4-fluorophenyl)-1-(4-((4-methylphenyl)sulfonamido)-butyl)-2,5-dioxo-2,5-dihydro
-1H-pyrrol-3-yl)phenoxy)acetyl)glycyl-glycine (14c). Yield: 87%; mp: 120-123 °C; 1H-NMR
(DMSO-d6, 300M): 1.39 (t, J= 7.41 Hz, 2H, -CH2-), 1.56 (t, J= 7.45 Hz, 2H, -CH2-), 2.36 (s, 3H,
-CH3), 2.73 (m, 2H, -CH2NH-), 3.47 (t, J= 6.45 Hz, 2H, -CH2N-), 3.56 (d, J= 4.95 Hz, 2H,
-NCH2CO-), 3.79 (d, J= 5.64 Hz, 2H, -NCH2CO-), 4.58 (s, 2H, -OCH2CO-), 7.02 (d, J= 8.61 Hz,
2H, Ar-H), 7.27 (t, J= 8.73 Hz, 2H, Ar-H), 7.37 (d, J= 6.48Hz, 4H, Ar-H), 7.47 (m, 3H, Ar-H),
7.66 (d, J= 8.04 Hz, 2H, Ar-H), 7.80 (s, 1H, -NHCO-), 8.37 (t, J= 5.46 Hz, 1H, -NHCO-);
13C-NMR (DMSO-d6, 300M): 20.85, 25.17, 26.32, 37.29, 41.69, 41.98, 42.19, 66.72, 114.85,
115.53, 115.71, 121.19, 125.29, 126.41, 128.36, 131.21, 131.85, 131.91, 133.33, 135.39, 137.65,
142.40, 158.79, 161.52, 163.49, 167.57, 168.09, 170.31, 170.37, 171.31; HRMS: found m/z
[M+H]+ 681.50514, [M+H2O]+ 698.24249, calcd. m/z [M] 680.19524.
(2-(4-(4-(4-fluorophenyl)-2,5-dioxo-1-(4-((4-(trifluoromethyl)-phenyl)sulfonamido)butyl)-2
,5-dihydro-1H-pyrrol-3-yl)phenoxy)-acetyl)glycylglycine (14d). Yield: 72%; mp: 125-129 °C;
1H-NMR (DMSO-d6, 300M): 1.42 (t, J = 7.29 Hz, 2H, -CH2-), 1.57 (t, J = 7.14 Hz, 2H, -CH2-),
2.82 (m, 2H, -CH2NH-), 3.49 (t, J = 6.18 Hz, 2H, -CH2N-), 3.68 (d, J = 5.4 Hz, 2H, -NCH2CO-),
3.80 (d, J = 5.7 Hz, 2H, -NCH2CO-), 4.58 (s, 2H, -OCH2CO-), 7.02 (d, J = 8.79 Hz, 2H, Ar-H),
7.26 (t, J = 8.88 Hz, 2H, Ar-H), 7.37 (d, J = 8.73 Hz, 2H, Ar-H), 7.45 (m, 2H, Ar-H), 7.87 (m, 1H,
-NHCO-), 7.98 (m, 5H, Ar-H), 8.35 (d, J= 5.37 Hz, 1H, -NHCO-); 13C-NMR (DMSO-d6, 300M):
25.18, 26.42, 37.29, 41.38, 41.63, 42.06, 66.75, 114.88, 115.52, 115.81, 121.22, 125.32, 126.39,
126.43, 127.42, 131.25, 131.86, 131.97, 133.36, 135.43, 144.49, 158.83, 160.90, 164.19, 167.65,

168.55, 170.37, 170.43, 171.31; HRMS: found m/z [M+NH4]+ 752.20423, calcd. m/z [M]
734.16696.
(2-(4-(1-(4-((3-bromophenyl)sulfonamido)butyl)-4-(4-fluoro-phenyl)-2,5-dioxo-2,5-dihydro
-1H-pyrrol-3-yl)phenoxy)acetyl)-glycylglycine (14e). Yield: 65%; mp: 104-107 °C; 1H-NMR
(DMSO-d6, 500M): 1.40 (t, J = 7.45 Hz, 2H, -CH2-), 1.55 (m, 2H, -CH2-), 2.78 (t, J = 6.65 Hz,
2H, -CH2NH-), 3.33 (m, 2H, -CH2N-), 3.47 (t, J = 6.4 Hz, 2H, -NCH2CO-), 3.75 (d, J = 5.6 Hz,
2H, -NCH2CO-), 4.59 (s, 2H, -OCH2CO-), 7.02 (d, J = 8.75 Hz, 2H, Ar-H), 7.28 (t, J = 8.8 Hz,
2H, Ar-H), 7.37 (d, J = 8.45 Hz, 3H, Ar-H), 7.44 (d, J = 5.6 Hz, 2H, Ar-H), 7.54 (t, J = 7.9 Hz,
1H, Ar-H), 7.78 (d, J = 7.8 Hz, 1H, Ar-H), 7.83 (d, J = 5.9 Hz, 1H, Ar-H), 7.91 (s, 1H, -NHCO-),
8.48 (t, J = 5.55Hz, 1H, -NHCO-); 13C-NMR (DMSO-d6, 300M): 25.16, 26.37, 37.28, 41.91,
42.05, 43.91, 66.70, 114.86, 115.54, 115.72, 121.19, 122.02, 125.29, 125.31, 125.45, 128.79,
131.23, 131.40, 131.86, 131.93, 133.32, 135.05, 135.40, 142.67, 158.81, 161.53, 163.50, 167.43,
167.59, 170.32, 170.38, 170.79; HRMS: found m/z [M+NH4]+ 762.12555, calcd. m/z [M]
744.09009.
(2-(4-(1-(4-((4-ethylphenyl)sulfonamido)butyl)-4-(4-fluorophen-yl)-2,5-dioxo-2,5-dihydro-1
H-pyrrol-3-yl)phenoxy)acetyl)glycyl-glycine (14f). Yield: 72%; mp: 95-98 °C; 1H-NMR
(DMSO-d6, 300M): 1.18 (t, J = 7.59 Hz, 3H, -CH3), 1.39 (t, J = 6.9 Hz, 2H, -CH2-), 1.56 (t, J =
5.94 Hz, 2H, -CH2-), 2.66 (m, 2H, -PhCH2-), 2.73 (m, 2H, -CH2NH-), 3.47 (t, J = 6.48 Hz,
-CH2N-), 3.60 (d, J = 5.13 Hz, 2H, -NCH2CO-), 3.79 (d, J = 5.82 Hz, 2H, -NCH2CO-), 4.58 (s,
2H, -OCH2CO-), 7.02 (d, J = 8.76 Hz, 2H, Ar-H), 7.27 (t, J = 8.82 Hz, 2H, Ar-H), 7.37 (d, J =
8.75 Hz, 2H, Ar-H), 7.39 (m, 4H, Ar-H), 7.48(m, 3H, Ar-H), 7.68 (d, J = 8.19 Hz, 2H, Ar-H),7.89
(s, 1H, -NHCO-), 8.44 (m, 1H, -NHCO-); 13C-NMR (DMSO-d6, 300M): 15.04, 25.19, 26.35,
27.90, 37.31, 41.66, 41.83, 42.00, 66.72, 114.85, 115.48, 115.77, 121.19, 125.29, 125.33, 126.52,
128.36, 131.21, 131.83, 131.94, 133.32, 135.39, 137.87, 148.43, 158.79, 160.86, 164.14, 167.60,
168.28, 170.32, 170.37, 171.30; HRMS: found m/z [M+Na]+ 717.20360, calcd. m/z [M]
694.21088.
(2-(4-(4-(4-fluorophenyl)-2,5-dioxo-1-(4-(thiophene-2-sulfon-amido)butyl)-2,5-dihydro-1H-
pyrrol-3-yl)phenoxy)acetyl)glycyl-glycine (14g). Yield: 78%; mp: 108-110 °C; 1H-NMR
(DMSO-d6, 300M): 1.44 (t, J = 7.32 Hz, 2H, -CH2-), 1.59 (m, 2H, -CH2-), 2.86 (m, 2H, -CH2NH-),
3.49 (t, J = 6.36 Hz, 2H, -CH2N-), 3.73 (d, J = 5.49 Hz, 2H, -NCH2CO-), 3.80 (d, J = 5.91 Hz,

2H, -NCH2CO-), 4.58 (s, 2H, -OCH2CO-), 7.02 (d, J = 8.67 Hz, 2H, Ar-H), 7.15 (m, 1H,
thiophene –H4), 7.27 (t, J = 8.79 Hz, 2H, Ar-H), 7.38 (d, J = 8.64 Hz, 2H, Ar-H), 7.45 (dd, J =
5.94, 8.28 Hz, 2H, Ar-H), 7.56 (d, J = 3.69 Hz, 1H, thiophene –H3), 7.71 (s, 1H, -COOH), 7.88 (d,
J = 4.98 Hz, 1H, -NHCO-); 8.12 (s,1H, thiophene –H5), 8.33 (s, 1H, -NHCO-); 13C-NMR
(DMSO-d6, 300M): 25.18, 26.17, 37.27, 41.02, 41.53, 42.21, 66.69, 114.82, 115.47, 115.76,
121.16, 125.27, 127.53, 131.19, 131.24, 131.81, 131.92, 132.13, 133.29, 135.36, 141.44, 158.76,
160.83, 164.12, 167.57, 168.58, 170.36, 170.98; HRMS: found m/z [M+Na]+ 695.12819, calcd.
m/z [M] 672.13600.
(2-(4-(4-(4-fluorophenyl)-2,5-dioxo-1-(4-((2-(trifluoromethoxy)-phenyl)sulfonamido)butyl)
-2,5-dihydro-1H-pyrrol-3-yl)phenoxy)-acetyl)glycylglycine (14h). Yield: 57%; mp: 85-88 °C;
1H-NMR (DMSO-d6, 300M): 1.42 (d, J = 7.2 Hz, 2H, -CH2-), 1.55 (d, J = 5.7 Hz, 2H, -CH2-),
2.92 (d, J = 5.4 Hz, 2H, -CH2NH-), 3.46 (d, J = 6.3 Hz, 2H, -CH2N-), 3.75 (d, J = 5.7 Hz, 2H,
-NCH2CO-), 3.81 (d, J = 5.7 Hz, 2H, -NCH2CO-), 4.58 (s, 2H, -OCH2CO-), 7.02 (d, J = 8.7 Hz,
2H, Ar-H), 7.27 (t, J = 8.7 Hz, 2H, Ar-H), 7.37 (d, J = 8.4 Hz, 2H, Ar-H), 7.43 (d, J = 5.7 Hz, 2H,
Ar-H), 7.54 (d, J = 6.9 Hz, 2H, Ar-H), 7.73 (t, J = 7.2 Hz, 1H, Ar-H), 7.82 (s, 1H, Ar-H), 7.91 (d,
J = 7.5 Hz, 1H, Ar-H), 8.15 (s, 1H, -NHCO-), 8.32 (s, 1H, -NHCO-); 13C-NMR (DMSO-d6,
300M): 25.08, 26.55, 37.22, 40.67, 41.47, 42.03, 66.70, 114.82, 115.51, 115.68, 118.79, 120.85,
120.94, 121.17, 125.27, 125.29, 127.38, 129.94, 131.17, 131.81, 131.88, 133.20, 133.28, 134.48,
135.34, 144.93, 158.78, 161.50, 163.47, 167.57, 168.74, 170.27, 170.33, 171.00; HRMS: found
m/z [M+NH4]+ 768.19931, calcd. m/z [M] 750.16188.
(2-(4-(4-(4-fluorophenyl)-2,5-dioxo-1-(4-((4-(trifluoromethoxy)-phenyl)sulfonamido)butyl)
-2,5-dihydro-1H-pyrrol-3-yl)phenoxy)-acetyl)glycylglycine (14i). Yield: 80%; mp: 138-142 °C;
1H-NMR (DMSO-d6, 300M): 1.42 (d, J = 5.7 Hz, 2H, -CH2-), 1.56 (d, J = 5.4 Hz, 2H, -CH2-),
2.80 (d, J = 5.4 Hz, 2H, -CH2NH-), 3.47 (s, 2H, -CH2N-), 3.76 (d, J = 5.4 Hz, 2H, -NCH2CO-),
3.81 (d, J = 5.4 Hz, 2H, -NCH2CO-), 4.57 (s, 2H, -OCH2CO-), 7.01 (d, J = 8.7 Hz, 2H, Ar-H),
7.26 (t, J = 8.7 Hz, 2H, Ar-H), 7.37 (d, J = 8.1 Hz, 2H, Ar-H), 7.44 (t, J = 6.9 Hz, 2H, Ar-H), 7.57
(d, J = 8.1 Hz, 2H, Ar-H), 7.72 (s, 1H, Ar-H), 7.91 (d, J = 8.4 Hz, 2H, Ar-H), 8.17 (s, 1H,
-NHCO-), 8.32 (s, 1H, -NHCO-); 13C-NMR (DMSO-d6, 300M): 25.11, 26.31, 37.23, 40.62, 41.46,
41.99, 66.70, 114.81, 115.50, 115.67, 116.73, 118.76, 120.81, 121.18, 121.35, 122.87, 125.27,
125.30, 128.93, 131.17, 131.81, 131.88, 133.30, 135.36, 139.53, 150.61, 158.77, 161.50, 163.47,

167.56, 168.76, 170.29, 170.35, 171.00; HRMS: found m/z [M+Na]+ 773.15473, calcd. m/z [M]
750.16188.
(2-(4-(1-(4-((4-(tert-butyl)phenyl)sulfonamido)butyl)-4-(4-fluor-ophenyl)-2,5-dioxo-2,5-dih
ydro-1H-pyrrol-3-yl)phenoxy)acetyl)-glycylglycine (14j). Yield: 67%; mp: 115-119 °C;
1H-NMR (DMSO-d6, 300M): 1.29 (s, 9H,-C(CH3)3), 1.41 (d, J = 6.96 Hz, 2H, -CH2-), 1.55 (d, J
= 7.26 Hz, 2H, -CH2-), 2.74 (d, J = 5.82 Hz, 2H, -CH2NH-), 3.46 (d, J = 6.48 Hz, 2H, -CH2N-),
3.65 (d, J = 5.04 Hz, 2H, -NCH2CO-), 3.79 (d, J = 5.64 Hz, 2H, -NCH2CO-), 4.58 (s, 2H,
-OCH2CO-), 7.02 (d, J = 8.61 Hz, 2H, Ar-H), 7.27 (t, J = 8.76 Hz, 2H, Ar-H), 7.37 (d, J = 8.67
Hz, 2H, Ar-H), 7.43 (d, J = 5.91 Hz, 2H, Ar-H), 7.50 (m, 1H, Ar-H), 7.59 (d, J = 8.58 Hz, 2H,
Ar-H), 7.70 (d, J = 8.16 Hz, 2H, Ar-H), 8.00 (s, 1H, -NHCO-), 8.36 (m, 1H, -NHCO-); 13C-NMR
(DMSO-d6, 300M): 25.22, 26.39, 30.78, 34.77, 37.34, 41.38, 41.61, 42.03, 66.69, 114.86, 115.54,
115.83, 121.19, 125.31, 125.35, 125.95, 126.32, 126.56, 128.43, 131.25, 131.87, 131.98, 133.33,
135.40, 137.59, 155.20, 158.79, 160.88, 164.17, 167.68, 168.57, 170.37, 170.43, 171.30; HRMS:
found m/z [M-H]- 721.5, calcd. m/z [M] 722.24218.
4.1.3. General procedure for the preparation of 20 and 22a-f
To activated zinc dust (16.455 mmol) was added a solution of benzophenone 15 (10.97 mmol) and
ethyl bromoacetate (13.16 mmol) in 50 mL dry ether dropwise followed by the addition of TMSCl
(1.1 mmol). The suspension was warmed to 40 °C for 3-4 h followed by cooling it to room
temperature. To it was added 10% sulphuric acid and extracted with ethyl acetate. The combined
organic layer was washed with 10% sulphuric acid, brine, dried and concentrated. Compound 16
was obtained by a silica gel column chromatography. To LiAlH4 (22.2 mmol) was added the
solution of compound 16 (11.1 mmol) in 20 mL dry THF dropwise at 0 °C. The reaction was
stirred at 0 °C for 1 h and then quenched with 20% NaOH. The mixture was extracted with ethyl
acetate, and the organic layer was washed with brine, dried and evaporated to afford compound
17.To a solution of compound 17 (10.5 mmol) in dry CH2Cl2 (20 mL) were added
p-toluenesulfonyl chloride (12.6 mmol), DMAP (1.05 mmol) and Et3N (21 mmol). The mixture
was stirred at room temperature for 2 h and washed with brine. The organic layer was dried over
anhydrous Na2SO4 and concentrated. The residue was purified by a silica gel column
chromatography to afford compound 18 without further purification. To a solution of
3,4-diphenyl-1H-pyrrole-2,5-dione 4b (7.84 mmol) in CH3CN (50 mL), K2CO3 (13.07 mmol),

TBAB (0.65 mmol) and compound 18 (6.54 mmol) were added and the mixture was stirred at
45 °C overnight. Solvent was evaporated to dryness, water (30 mL) added and the resulting
mixture extracted with ethyl acetate. Collected organic layers were dried over anhydrous Na2SO4
and solvent evaporated to dryness. Compound 19 was obtained by a silica gel column
chromatography. To a solution of compounds 19 (2.06 mmol) in 8 mL THF was added the
catalytic amount of 20% Pd(OH)2. The resulting mixture was stirred at room temperature under H2
at normal pressure for 1 h. Solvent was separated from 20% Pd(OH)2 by filtration through Celite
and removed. Compound 20 was obtained by a silica gel column chromatography. To a solution
of compound 20 (0.304 mmol) and K2CO3 (0.608 mmol) in 5 mL CH3CN was added
1,2-dibromoethane, 1,3-dibromopropane and 1,4-dibromobutane (1.22 mmol). The resulting
mixture was stirred reflux overnight and the solvent was concentrated. The residue was dissolved
with ethyl acetate and washed with brine, and the organic layer was dried over anhydrous Na2SO4
and evaporated. 21a-c was obtained by a silica gel column chromatography. A solution of 21a-c
(0.126 mmol ), diethylamine (0.252 mmol) and K2CO3 (0.252 mmol) in 5 mL CH3CN was
refluxed overnight. The reaction was evaporated and the residue was dissolved with CH2Cl2. The
solvent was washed with brine, dried and concentrated. Target compounds 22a-c were obtained by
a silica gel column chromatography. A mixed solution of 21a-c (0.218 mmol) and Na2SO3 (1.09
mmol) in 6 mL C2H5OH:H2O (2:1) was refluxed overnight. The reaction was evaporated and 2
mL CH3OH was added. The suspension was filtered and the organic layer was concentrated in
vacuum. The resulting solid was triturated with 4 mL hexane: ethyl acetate (3:1) and filtrated to
give target compounds 22d-f.
Ethyl 3-hydroxy-3,3-diphenylpropanoate (16). Yield: 64%; mp: 75-80 °C; 1H-NMR (CDCl3,
300M): 1.145 (t, J = 7.15 Hz, 3H, CH3CH2-), 3.259 (s, 2H, -CH2-), 4.087 (q, J = 7.10 Hz, 2H,
CH3CH2-), 7.212 (t, J = 7.30 Hz, 2H, Ar-H), 7.296 (m, 4H, Ar-H), 7.434 (m, 4H, Ar-H); MS:
found m/z [M+Na]+ 293.1, calcd. m/z [M] 270.13.
1,1-diphenylpropane-1,3-diol (17). Yield: 83%; mp: 85-90 °C; 1H-NMR (CDCl3, 300M):
2.564 (t, J = 5.45 Hz, 2H, -CH2-), 2.679 (s, 1H, -OH), 2.689 (s, 1H, -OH), 3.760 (t, J = 5.45 Hz,
2H, -CH2-), 7.236 (m, 2H, Ar-H), 7.318 (m, 4H, Ar-H), 7.426 (d, J = 7.7 Hz, 4H, Ar-H); MS:
found m/z [M+Na]+ 251.1, calcd. m/z [M] 228.12.

3-hydroxy-3,3-diphenylpropyl 4-methylbenzenesulfonate (18). Yield: 62.5%; 1H-NMR
(CDCl3, 300M): 2.438 (s, 3H, -PhCH3), 2.710 (t, J = 7.30 Hz, 2H, -CH2-), 4.092 ((t, J = 7.30 Hz,
2H, -CH2-), 7.27 (m, 12H, Ar-H), 7.679 (d, J = 8.20 Hz, 2H, Ar-H); MS: found m/z [M+Na]+
405.3, calcd. m/z [M] 382.12.
3-(4-(benzyloxy)phenyl)-4-(4-fluorophenyl)-1-(3-hydroxy-3,3-diphenylpropyl)-1H-pyrrole-
2,5-dione (19). Yield: 73.5%; 1H-NMR (CDCl3, 300M): 2.72 (t, 2H, J = 6.6 Hz, -CH2-), 3.87 (t,
2H, J = 6.6 Hz, -CH2-), 5.06 (s, 2H, PhCH2O-), 6.91 (d, 2H, J = 9 Hz, Ar-H), 7.03 (m, 2H, Ar-H),
7.10 (m, 2H, Ar-H), 7.27 (m, 8H, Ar-H), 7.40 (m, 8H, Ar-H); MS: found m/z [M+Na]+ 606.5,
calcd. m/z [M] 583.22.
3-(4-fluorophenyl)-1-(3-hydroxy-3,3-diphenylpropyl)-4-(4-hydroxyphenyl)-1H-pyrrole-2,5
-dione (20). Yield: 90%; mp: 63-65 °C; 1H-NMR (CDCl3, 300M): 2.74 (t, J = 6.48 Hz, 2H,
-CH2-), 3.88 (t, J = 6.48 Hz, 2H, -CH2-), 6.78 (m, 2H, Ar-H), 7.10 (m, 2H, Ar-H), 7.13 (m, 2H,
Ar-H), 7.24 (m, 4H, Ar-H), 7.30 (m, 2H, Ar-H), 7.44 (m, 4H, Ar-H); 13C-NMR (CDCl3, 300M):
38.63, 51.40, 70.10, 114.88, 115.36, 115.57, 116.14, 125.36, 125.65, 125.81, 126.72, 126.97,
127.41, 127.94, 128.05, 128.25, 128.40, 128.51, 128.97, 130.08, 130.70, 131.67, 131.85, 136.63,
146.57, 146.63, 154.23, 157.27, 157.91, 160.17, 177.82; HRMS: found m/z [M+Na]+ 516.16021,
calcd. m/z [M] 493.16894.
3-(4-(3-bromopropoxy)phenyl)-4-(4-fluorophenyl)-1-(3-hydroxy-3,3-diphenylpropyl)-1H-p
yrrole-2,5-dione (21b). Yield: 76.7%; 1H-NMR (CDCl3, 300M): 2.35 (m, J = 6.0 Hz, 2H, -CH2-),
2.76 (t, J = 6.3 Hz, 2H, -CH2-), 3.62 (t, J = 6.3 Hz, 2H, -CH2-), 3.91 (t, J = 6.3 Hz, 2H, -CH2-),
4.15 (t, J = 5.7 Hz, 2H, -CH2-), 6.87 (d, J = 9 Hz, 2H, Ar-H), 7.05 (t, J = 8.7 Hz, 2H, Ar-H), 7.14
(t, J = 7.20 Hz, 2H, Ar-H), 7.23 (d, J = 7.8 Hz, 3H, Ar-H), 7.33 (m, 3H, Ar-H), 7.47 (d, J = 7.20
Hz, 4H, Ar-H); MS: found m/z [M+Na]+ 636.4, calcd. m/z [M] 613.13.
3-(4-(2-(diethylamino)ethoxy)phenyl)-4-(4-fluorophenyl)-1-(3-hydroxy-3,3-diphenylpropyl
)-1H-pyrrole-2,5-dione (22a). Yield: 54.8%; mp: 99-103 °C; 1H-NMR (CDCl3, 300M): 1.33 (s,
6H, -CH2CH3), 2.73 (s, 4H, -CH2CH3), 2.74 (t, J = 6.5 Hz, 2H, -CH2-), 2.89 (s, 2H, -CH2-), 3.46
(s, 2H, -CH2-), 3.88 (t, J = 6.5 Hz, 2H, -CH2-), 4.16 (s, 2H, -CH2-), 6.84 (d, J = 9 Hz, 2H, Ar-H),
7.02 (t, J = 9 Hz, 2H, Ar-H), 7.12 (t, J = 7.5 Hz, 2H, Ar-H), 7.22 (t, J = 7.5 Hz, 4H, Ar-H), 7.26
(d, J = 8.5 Hz, 2H, Ar-H), 7.31 (m, 2H, Ar-H), 7.44 (d, J = 7.5 Hz, 2H, Ar-H); 13C-NMR (CDCl3,

300M): 11.40, 14.12, 22.69, 29.50, 33.76, 38.87, 47.75, 51.49, 66.24, 114.62, 115.64, 115.82,
120.49, 124.65, 124.68, 125.33, 126.71, 128.27, 131.47, 131.77, 131.84, 132.89, 135.45, 146.66,
160.05, 162.28, 164.28, 171.18, 171.20; HRMS: found m/z [M+H]+ 593.28385, calcd. m/z [M]
592.27374.
3-(4-(3-(diethylamino)propoxy)phenyl)-4-(4-fluorophenyl)-1-(3-hydroxy-3,3-diphenylprop
yl)-1H-pyrrole-2,5-dione (22b). Yield: 51.1%; mp: 94-99 °C; 1H-NMR (CDCl3, 300M): 1.37 (t, J
= 7 Hz, 6H, -CH2CH3), 2.32 (s, 2H, -CH2-), 2.74 (t, J = 6 Hz, 2H, -CH2-), 3.07 (d, J = 6 Hz, 2H,
-CH2-), 3.12 (s, 4H, -CH2CH3), 3.88 (t, J = 6.5 Hz, 2H, -CH2-), 4.10 (t, J = 5.5 Hz, 2H, -CH2-),
6.82 (d, J = 8.5 Hz, 2H, Ar-H), 7.02 (t, J = 8.5 Hz, 2H, Ar-H), 7.12 (t, J = 7.5 Hz, 2H, Ar-H), 7.22
(t, J = 7.5 Hz, 4H, Ar-H), 7.29 (m, 4H, Ar-H), 7.44 (d, J = 7.5 Hz, 2H, Ar-H); 13C-NMR (CDCl3,
300M): 25.05, 30.08, 30.60, 34.22, 39.33, 47.12, 49.62, 65.77, 114.92, 116.01, 116.30, 121.30,
125.05, 125.77, 127.12, 128.66, 131.95, 132.16, 132.27, 133.56, 135.75, 147.06, 160.16, 162.06,
165.39, 171.53; MS: found m/z [M+H]+ 607.5, calcd. m/z [M] 606.29.
3-(4-(4-(diethylamino)butoxy)phenyl)-4-(4-fluorophenyl)-1-(3-hydroxy-3,3-diphenylpropyl
)-1H-pyrrole-2,5-dione (22c). Yield: 54.8%; mp: 98-102 °C; 1H-NMR (CDCl3, 300M): 1.31 (t,
6H, J = 6.84 Hz, 6H, -CH2CH3), 1.88 (s, 4H, -CH2-), 2.73 (m, 2H, -CH2-), 3.25 (m, 2H, -CH2-),
3.44 (m, 4H, -CH2 CH2-), 3.76 (s, 2H, -CH2-), 4.07 (s, 2H, -CH2-), 6.90 (d, J = 8.22 Hz, 2H,
Ar-H), 7.09 (m, 4H, Ar-H), 7.22 (m, 4H, Ar-H), 7.33 (m, 4H, Ar-H), 7.43 (m, 4H, Ar-H);
13C-NMR (CDCl3, 300M): 19.64, 20.78, 26.05, 26.53, 33.81, 38.91, 46.44, 51.16, 58.71, 62.84,
66.93, 114.50, 115.66, 115.83, 120.73, 120.76, 124.62, 124.65, 125.36, 125.82, 126.72, 127.01,
128.25, 131.54, 131.78, 131.84, 133.06, 135.35, 146.66, 159.91, 162.31, 164.30, 171.15; HRMS:
found m/z [M+H]+ 621.31512, calcd. m/z [M] 620.30504.
Sodium 2-(4-(4-(4-fluorophenyl)-1-(3-hydroxy-3,3-diphenylprop-yl)-2,5-dioxo-2,5-dihydro
-1H-pyrrol-3-yl)phenoxy)ethane-1-sulfonate (22d). Yield: 61%; mp: >200 °C; 1H-NMR
(CDCl3, 300M): 2.76 (t, J = 7.2 Hz, 2H, -CH2-), 3.28 (t, J = 7.5 Hz, 2H, -CH2-), 3.79 (t, J = 6.9
Hz, 2H, -CH2-), 4.41 (t, J = 7.2 Hz, 2H, -CH2-), 6.96 (d, J = 9 Hz, 2H, Ar-H), 7.14 (m, 2H, Ar-H),
7.20 (m, 4H, Ar-H), 7.25 (m, 2H, Ar-H), 7.35 (m, 2H, Ar-H), 7.47 (m, 4H, Ar-H); 13C-NMR
(DMSO-d6, 300M): 34.04, 38.44, 50.32, 64.55, 75.38, 114.55, 115.48, 115.77, 120.61, 125.37,
125.42, 125.57, 125.78, 126.21, 127.85, 128.05, 131.32, 131.84, 131.95, 132.97, 135.39, 147.70,

159.45, 160.83, 164.11, 170.12, 170.18; HRMS: found m/z [M+H]+ 624.14916, calcd. m/z [M]
623.13900.
Sodium 3-(4-(4-(4-fluorophenyl)-1-(3-hydroxy-3,3-diphenylprop-yl)-2,5-dioxo-2,5-dihydro
-1H-pyrrol-3-yl)phenoxy)propane-1-sulfonate (22e). Yield: 57%; mp: >200 °C; 1H-NMR
(CDCl3, 300M): 2.26 (m, J = 6.9 Hz, 2H, -CH2-), 2.75 (t, J = 6.9 Hz, 2H, -CH2-), 2.97 (m, 2H,
-CH2-), 3.78 (t, J = 7.2 Hz, 2H, -CH2-), 4.16 (t, J = 6.3 Hz, 2H, -CH2-), 6.92 (d, J = 8.7 Hz, 2H,
Ar-H), 7.12 (m, 4H, Ar-H), 7.24 (d, J = 7.8 Hz, 4H, Ar-H), 7.35 (m, 2H, Ar-H), 7.40 (m, 2H,
Ar-H), 7.47 (m, 4H, Ar-H); 13C-NMR (DMSO-d6, 300M): 25.08, 34.01, 38.41, 47.74, 66.74,
75.35, 114.50, 115.46, 115.75, 120.38, 125.38, 125.42, 125.55, 126.18, 127.82, 128.01, 131.25,
131.80, 131.92, 132.83, 135.39, 147.68, 159.75, 160.79, 164.07, 170.10, 170.17; HRMS: found
m/z [M+Na]+ 660.14535, calcd. m/z [M] 637.15465.
Sodium 4-(4-(4-(4-fluorophenyl)-1-(3-hydroxy-3,3-diphenylprop-yl)-2,5-dioxo-2,5-dihydro
-1H-pyrrol-3-yl)phenoxy)butane-1-sulfonate (22f). Yield: 48%; mp: >200 °C; 1H-NMR (CDCl3,
300M): 1.83 (m, 2H, -CH2-), 1.94 (m, 2H, -CH2-), 2.71 (m, 2H, -CH2-), 2.84 (m, 2H, -CH2-), 3.76
(t, J = 6.9 Hz, 2H, -CH2-), 4.03 (t, J = 5.49 Hz, 2H, -CH2-), 6.83 (m, 2H, Ar-H), 7.08 (m, 4H,
Ar-H), 7.14 (m, 8H, Ar-H), 7.44 (m, 4H, Ar-H); 13C-NMR (DMSO-d6, 300M): 21.79, 27.84, 33.97,
38.40, 50.93, 67.43, 75.31, 114.42, 115.41, 115.70, 120.29, 125.37, 125.51, 125.71, 126.12,
127.76, 131.19, 131.76, 131.87, 135.35, 147.64, 159.80, 160.74, 164.02, 170.05, 170.13; HRMS:
found m/z [M+Na]+ 674.16286, calcd. m/z [M] 651.17030.
4.2. Biological materials
Chol:MβCD (Cholesterol–methyl-β-cyclodextrin) was water-soluble cholesterol. Caco-2 (a
human colon adenocarcinoma cells) and RAW264.7 (mouse macrophage, Abelson murine
leukemia virus transformed) cell lines were purchased from Shanghai Institute of
Biochemistry and Cell Biology (SIBCB). BCA protein assay kit (Cat. P0012) and
hematoxylin staining kit (Cat. C0105) were obtained from Beyotime (Shanghai, China).
Cholesterol-25,26,26,26,27,27,27-D7 (Cat. 677574) was purchased from Sigma-Aldrich.
LDH assay kit (Cat. A020-2), MDA assay kit (Cat. A003-4),TNF-α assay kit (Cat. H052)
and ROS assay kit (Cat. E004) were purchased from Nanjing Jiancheng Bioengineering

Institute (Nanjing, China). Oil red O staining kit (Cat. BIO1320) was purchased from J&K
Chemical.
4.3.In vitro cholesterol absorption inhibition assay
Caco-2 cell line was seeded to 6-well plates at a density of 1×106 cells/well in DMEM containing
20% fetal bovine serum (FBS), 1% penicillin-streptomycin, 1% non-essential-amino acids and
incubated in a 5 % CO2 incubator for 48 h before treated. Cells were washed twice with PBS and
incubated in medium (1.5 mL/well) containing tested compounds (100 µM). Following the
incubation for 24 h, cells were washed twice with PBS, added with stigmasterol micellar solutions
(1 mL/well) and incubated for another 24 h. Cells were washed twice with PBS again, and then
lysed with RIPA lysis buffer (200 µL/well). The lysate was transferred to centrifuge tubes for
centrifugation. BCA protein assay kit was used to quantify the protein concentration of lysate.
Stigmasterol was obtained by the hydrolysis of stigmasterol ester in the lysate with 8.9 mol/L
ethanol solution of KOH. 20 µL mixture of chromic acid (2 mol/L) and sulfuric acid (2 mol/L)
(Jones oxidation) was added to the mixture in sequence and the reaction was quenched with water
after 5 min. Stigmasterol oxidation product was extracted with n-hexane and measured by HPLC.
The results were expressed as mg stigmasterol /mg protein. An Inertsil ODS-SP C18 column (5
µm, 4.6 × 150 mm) (GL Science Inc., Japan) was used for the HPLC analysis. The mobile phase
was isopropanol-water-acetonitrile (1:1:8) and the flow-rate was 1 mL/min. The detection wave
length was 250nm.
4.4. Methods of cytotoxicity assay
Cytotoxicity assay was completed using the MTT cell proliferation assay. Human cell lines,
RAW264.7 (mouse macrophage, Abelson murine leukemia virus transformed, 2000 cells/well)
and HEK293 (human embryonic kidney cells, 8000 cells/well) were seeded in 96-well plates in a
100 µL culture medium. The cells were allowed to settle and attach for 12 h. Then, both of the cell
lines were exposed to different concentrations of tested compounds in 2-fold serial dilutions
ranging from 100 µM to 12.5 µM and incubated for further 48 h. Cell viability was measured by
MTT assay. The results were expressed as LC50. The absorption was measured on a microplate
reader at 490 nm.
4.5. Methods of partition coefficient

Lipophilic and hydrophilic properties were represented by the oil–water partition coefficient.
Partition coefficients were determined by dissolving compounds in octanol (o) and adding the
equal volume of water (w) and both of phases were presaturated with the other. Then, place the
tubes in a shaker for 4 h at room temperature and let them stand overnight to separate the octanol
and water phases. The concentrations of compounds in each phase were measured by HPLC
(Inertsil ODS-SP C18 column, 5 µm, 4.6 × 150 mm, GL Science Inc., Japan) and partition
coefficients were characterized by logPo/w = log (Co/Cw). The mobile phase A was water and the
mobile phase B was methanol. The flow rate was 1 mL/min.
4.6. LC-MS/MS analysis of cholesterol-D7
RAW264.7 cell line was seeded to 6-well plates at a density of 1×106 cells/well in DMEM
containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 1% non-essential-amino
acids and incubated in a 5 % CO2 incubator for 12 h. Cells were washed twice with PBS and
incubated with medium (1.5 mL/well) containg 250 µg/mL Chol-D7:MβCD and tested
compounds. After 72 h, cells were washed twice with PBS again, and then lysed with RIPA lysis
buffer (200 µL/well). The lysate was transferred to centrifuge tubes for centrifugation.
Cholesterol-D7 was obtained by the hydrolysis of cholesterol-D7 ester in the lysate with 8.9 mol/L
ethanol solution of KOH. Cholesterol-D7 product was extracted with n-hexane and measured by
LC-MS/MS.
The chromatographic separation of HPLC was performed using BEH C18 reversed-phase column
(2.0 mm×50 mm, 1.7µm). The mobile phase was water-methanol and the flow-rate was 0.4
mL/min. A triple quadrupole mass spectrometry equipped with an atmospheric pressure chemical
ionization (APCI) source was used for the mass analysis. The optimized operating parameters of
the APCI–MS interface were as follows: curtain gas pressure, 40 psi; collision gas pressure,
medium; spray gas pressure, 50 psi; corona pin current, 2.0 µA; vaporisation temperature, 400 °C.
4.7. Oil red O staining
RAW264.7 cell line was seeded to glass plates at a density of 1×106 cells/well in DMEM
containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 1% non-essential-amino
acids and incubated in a 5 % CO2 incubator for 12 h before treated. Cells were washed twice with
PBS and incubated with medium (1.5 mL/well) containg 250 µg/mL Chol:MβCD and tested

compounds. After 72 h, cells were washed twice with PBS and fixed with 50% isopropyl alcohol.
The fixed cells were stained with oil red O for 10 min and then stained with hematoxylin for 5 min.
The stained cells were observed and photographed with the microscope.
Acknowledgements
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, BK20141015) and the National Natural Science Foundation of
China (No. 81202393, 81600159).
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