Discovery of triazolyl thalidomide derivatives as anti-fibrosis agents

Article abstract of DOI:10.1039/d0nj03139a

Fibrosis with excessive accumulation of extracellular matrix (ECM) often causes progressive organ dysfunction and results in many inflammatory and metabolic diseases, including systemic sclerosis, pulmonary fibrosis, advanced liver disease and advanced kidney disease. The store-operated calcium entry (SOCE) pathway and the related signaling pathway were both found to be the important routes for fibrogenesis. Our aim in this study was to discover novel compounds to inhibit fibrogenesis. A number of triazolyl thalidomide derivatives were synthesized and evaluated for their anti-fibrosis activities. Compounds 7b-e, 8c-d, 10a-b and 10e inhibited intracellular Ca2+ activation and showed no cytotoxicity. Among them, 6-{4-[(3-(1,3-dioxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}hexanoic acid (10e) with the most potent inhibitory effect was chosen for further examination. The results revealed that compound 10e, a SOCE inhibitor, reversed the migratory ability of TGF-β1-induced myofibroblasts, dedifferentiated myofibroblasts to fibroblasts due to cytoskeleton remodeling, and restrained myofibroblast activation by targeting Orai1 and TGF-β1/SMAD2/3 signaling pathways. The in silico study indicated that compound 10e, with the appropriate lipophilic carbon chain and carboxylic acid, showed a good drug-likeness model score. Conclusively, the SOCE inhibitor, compound 10e, is used as a promising lead compound for the development of a new treatment for fibrosis. This journal is

Full text of DOI:10.1039/d0nj03139a

NJC
View Article Online
View Journal | View Issue
PAPER
Discovery of triazolyl thalidomide derivatives as
anti-fibrosis agents†
Cite this: New J. Chem., 2021,
45, 3589
Kai-Wei Tang,‡a Wen-Li Hsu,‡bc Cheng-Ru Chen,d Ming-Hsien Tsai,e
Chia-Jung Yenc and Chih-Hua Tseng
*
adfgh
Fibrosis with excessive accumulation of extracellular matrix (ECM) often causes progressive organ
dysfunction and results in many inflammatory and metabolic diseases, including systemic sclerosis,
pulmonary fibrosis, advanced liver disease and advanced kidney disease. The store-operated calcium
entry (SOCE) pathway and the related signaling pathway were both found to be the important routes for
fibrogenesis. Our aim in this study was to discover novel compounds to inhibit fibrogenesis. A number
of triazolyl thalidomide derivatives were synthesized and evaluated for their anti-fibrosis activities.
Compounds 7b–e, 8c–d, 10a–b and 10e inhibited intracellular Ca2+ activation and showed no cytotoxicity.
Among them, 6-{4-[(3-(1,3-dioxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}hexanoic
acid (10e) with the most potent inhibitory effect was chosen for further examination. The results revealed
that compound 10e, a SOCE inhibitor, reversed the migratory ability of TGF-b1-induced myofibroblasts,
dedifferentiated myofibroblasts to fibroblasts due to cytoskeleton remodeling, and restrained myofibroblast
activation by targeting Orai1 and TGF-b1/SMAD2/3 signaling pathways. The in silico study indicated that
compound 10e, with the appropriate lipophilic carbon chain and carboxylic acid, showed a good drug-
likeness model score. Conclusively, the SOCE inhibitor, compound 10e, is used as a promising lead
compound for the development of a new treatment for fibrosis.
Received 29th June 2020,
Accepted 15th January 2021
DOI: 10.1039/d0nj03139a
rsc.li/njc
diseases, such as systemic sclerosis, pulmonary fibrosis,
advanced liver disease and advanced kidney disease.2 The
Introduction
Fibrosis leads to severe disorders for decreased quality of life fibrosis process is potentially triggered by tissue injury, and it
having both psychological and social impact, and even death.1 could be that autoimmunity in the pathogenesis of tissue repair
The fibrotic change that is attributed to abnormal or excessive drives fibrosis in injured tissues with an inflammatory
accumulation of extracellular matrix (ECM) causes progressive response.3,4 Supportive therapy5–7 and suppression immuno-
organ dysfunction and many inflammatory and metabolic therapeutic drugs such as methotrexate, mycophenolate mofetil,
and cyclophosphamide (Fig. S1, ESI†) have been currently used
a School of Pharmacy, College of Pharmacy, Kaohsiung Medical University,
Kaohsiung 807, Taiwan. E-mail: chihhua@kmu.edu.tw; Fax: +886-7-3125339;
Tel: +886-7-3121101 (ext. 2163)
for the treatment of fibrotic diseases. However, the therapeutic
benefit is limited due to low efficacy and significant cytotoxicity.8
Therefore, the discovery of less cytotoxic and more effective anti-
fibrosis agents is urgently needed.
b Department of Dermatology, Kaohsiung Municipal Ta-Tung Hospital,
Kaohsiung Medical University, Kaohsiung 801, Taiwan
c Regenerative Medicine and Cell Therapy Research Center, Kaohsiung Medical
University, Kaohsiung 807, Taiwan
Recent research has pointed out that transforming growth
factor (TGF)-b1 is the key driver of several fibrosis modes such
as skin, cardiac, pulmonary and renal fibrosis.4 TGF-b1 acti-
vates SMAD downstream-signaling pathways, which induce the
differentiation of dermal fibroblasts to generate superabundant
collagens, contributing to increased fibrosis in the ECM.9
Fibroblasts when stimulated by TGF-b1 will differentiate into
myofibroblasts, and this process is mediated by Ca2+-
dependent signaling pathways, causing an increased expres-
sion of fibrosis markers such as a-smooth muscle actin (a-SMA)
and fibronectin.10 Store-operated calcium entry (SOCE), asso-
ciated with inflammation and carcinogenesis, is the most crucial
Ca2+ signal in this process. Thus, blockage of the increased SOCE
d Department of Fragrance & Cosmetic Science, College of Pharmacy, Kaohsiung
Medical University, Kaohsiung 807, Taiwan
e Department of Child Care, College of Humanities and Social Sciences,
National Pingtung University of Science and Technology, Pingtung 91201, Taiwan
f Drug Development and Value Creation Research Center, Kaohsiung Medical
University, Kaohsiung 807, Taiwan
g Department of Medical Research, Kaohsiung Medical University Hospital,
Kaohsiung 807, Taiwan
h Department of Pharmacy, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung 801,
Taiwan
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0nj03139a
‡ These two authors contributed equally to this work.
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 3589ꢀ3599 | 3589

View Article Online
Paper
NJC
activity of the myofibroblasts limits a pathogenic fibrotic
response and regulates the ECM–protein production in the
fibroblasts.11 Targeting SOCE and its related signaling pathways
would be one of the practical strategies for treating fibrotic
diseases.12,13
Thalidomide, the derivative of glutamic acid, possessing the
properties of anti-inflammation and immunomodulation, is
extensively used in the clinical treatment of multiple myeloma,
acquired immune deficiency syndrome, carcinomas, and
inflammatory bowel disease.14–16 Moreover, the inhibitory
activity toward the calcium channel and the anti-fibrotic effects
involving the SMAD signaling pathways of thalidomide have
been reported by several studies.17,18 Our previous studies
indicated that the modification of thalidomide restrained the
expression of interleukin-6 (IL-6) and tumor necrosis factor-a
(TNF-a) via MAPK pathways.19 As a consequence, we antici-
pated that further modification of thalidomide could enhance
the inhibition of SOCE and its related signaling pathways to
develop a new treatment for fibrosis. 1,2,3-Triazole, a versatile
functional moiety with the features of structural stability and
less degradability, is extensively applied to small-molecule drug
development.20,21 Several 1,2,3-triazole-containing drugs have
been approved by the FDA, for instance, rufinamide, tazobactam,
and cefatrizine (Fig. S1, ESI†). Additionally, 1,2,3-triazole is prone
to generate interaction with the target in the organism and it can
also escalate hydrophilicity by forming hydrogen bonds and
increasing polarity.22 Many studies have shown that the
structure containing 1,2,3-triazole exhibits a wide range of
pharmacological activities such as having anti-carcinotic, anti-
viral, anti-tuberculid bacillus, anti-dengue, and anti-bacterial
infection properties.23,24 These advantages imply that 1,2,3-
triazole might serve as a potential pharmacophore and the introduc-
tion of 1,2,3-triazole to our drug design may well promote
interaction in the organism.
None of the approved drugs have been developed for fibrosis.
Although several agents are available for current treatment, an
undesired off-target toxicity has limited any therapeutic effect.
Inhibitors of SOCE serve as a potential development source
against fibrosis.25 A number of agents to inhibit SOCE have
been developed as shown in Fig. 1.26–29 Most of them, however,
cannot qualify for clinical trials due to their poor selectivity and
high toxicity. In this study, thalidomide was used as the main
scaffold for modification. Several SOCE inhibitors have been
reviewed and the results indicated that the common structural
feature phenylpyrazole on Pyr6 and BTP2 might be a potential
pharmacophore.30 GSK-7579A with a similar structural charac-
teristic benzylpyrazole also exhibited good inhibitory activity
toward calcium channels.31 Furthermore, carboxyamido-
triazole (CAI) was initially identified as a SOCE inhibitor. We
inferred that the application of triazole, the bioisostere of
pyrazole, might enhance the effect on SOCE.32 Therefore, our
design in this study substituted pyrazolyl to the triazolyl group to
build phenyltriazolyl, benzyltriazolyl, and phenacyltriazolyl as
pharmacophores, which are added on the glutarimide ring of
thalidomide. The importance of the benzene ring was also
investigated by replacing the selected alkanoic acid. By the
Fig. 1 Design and molecular hybrid strategy of anti-fibrosis agents.
hybridization of these functional structures, our aim is to
develop a novel therapy against fibrosis with high selectivity
and low toxicity, providing an alternative option for clinical
treatment (Fig. 1).
Results and discussion
Chemistry
Alkyne and azide derivatives were used as starting materials
and were initially prepared for the 1,2,3-triazole click reaction.
First, in Scheme 1, compound 2 was obtained by the substitution
reaction of thalidomide (1) and propargyl bromide under the
condition of potassium carbonate and acetone. The selected
azides, compounds 3–6 series, were then prepared under different
conditions according to the previous studies (Scheme 2).24 The
synthesis of compounds 7–10, depicted in Scheme 3, was accom-
plished by the click reaction of compound 2 and selected azide
(3–6) under the presence of copper sulfate and sodium ascorbate.
The stereochemical structure of the starting agent thalidomide
(1) was depicted in our previous study.19 The structures of the final
compounds were confirmed by 1H NMR and 13C NMR spectra
(spectral data can be found in the ESI†). The detailed synthesized
methods, and mass and purity analysis are as described in the
‘Experimental’ section.
Biology
Triazolyl thalidomide derivatives-attenuated intracellular
Ca2+ activation. SOCE plays a crucial role in determining the
progression of skin fibrosis.33 The inhibition of SOCE eliminated
the fibrogenesis through a changing microenvironment and
Scheme 1 Synthesis of alkynyl thalidomide analogue (2).
3590 | New J. Chem., 2021, 45, 3589ꢀ3599
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

View Article Online
NJC
Paper
Table 1 Preliminary inhibitory activity of intracellular Ca2+ activation and
cytotoxicities of triazolyl thalidomide derivatives at 5 mM
Compounds
% inhibition of intracellular calciuma
% viabilityb
7a
7b
7c
7d
7e
8a
8c
8d
8e
9a
9c
9d
9e
10a
10b
10c
49.7 ꢁ 8.4
53.0 ꢁ 7.4
90.8 ꢁ 0.8
98.3 ꢁ 2.0
101.9 ꢁ 0.9
99.8 ꢁ 3.4
97.7 ꢁ 1.9
N.D.c
102.7 ꢁ 7.6
97.7 ꢁ 3.4
N.D.
N.D.
N.D.
100.8 ꢁ 0.8
100.9 ꢁ 0.3
98.3 ꢁ 2.8
99.2 ꢁ 1.9
N.D.
45.3 ꢁ 11.4
44.6 ꢁ 11.7
42.6 ꢁ 6.7
ꢀ58.1 ꢁ 22.9
31.2 ꢁ 16.8
32.9 ꢁ 10.1
ꢀ40.3 ꢁ 31.8
ꢀ13.8 ꢁ 22.7
ꢀ102.0 ꢁ 18.1
26.8 ꢁ 18.5
32.9 ꢁ 7.0
40.9 ꢁ 9.1
75.8 ꢁ 4.4
ꢀ83.2 ꢁ 9.3
ꢀ39.9 ꢁ 32.6
27.9 ꢁ 3.0
Scheme 2 Preparation of the selected azide derivatives 3–6.
10d
10e
Thalidomide
N.D.
97.7 ꢁ 0.8
55 ꢁ 12.0
94.7 ꢁ 7.5
Results are presented as mean ꢁ standard deviation (n = 3). a The mean
area under the [Ca2+
] response curves compared with the TGF-b1-
i
b
induced myofibroblasts from 100 cells after the addition of TG. The
TGF-b1-induced myofibroblasts were treated with each compound in
c
5 mM for 24 h and cytotoxicity was assessed by MTT assays. N.D.
represents not determined.
We next identified the structure–activity relationship
(Fig. 2). Compound 7 series with the structure of phenyltriazolyl
moiety exhibited a strong inhibition of intracellular Ca2+ acti-
vation. The inhibition percentage of the intracellular Ca2+ level
was 42–53%. Compounds 8 and 9 series with benzyltriazolyl
and phenacyltriazolyl moiety showed a moderate inhibitory
activity (26–32%) except for compounds 8a, 8e, 9a and 9c.
The results indicated that compounds with the aryl group
connected directly to triazole, such as 7a–e, which caused a
strong inhibitory effect on the intracellular Ca2+ activation.
However, the insertion of CH2 or CH2CO between the triazole
and the aryl groups decreased the inhibitory activity (7c: 45.3 4
8c: 31.2; 7d: 44.6% 4 8d: 32.9% and 9d: 26.8%; 7e: 42.6 4 9e:
32.9). Interestingly, different substituents also contributed to
different inhibitory effects. Compounds 8a (ꢀ58.1%) and 9a
(ꢀ13.8%) with no substituent on the aryl group showed no
inhibitory effect on the intracellular Ca2+ activation, whereas
there appeared no different effects between the electron-
donating and the electron-withdrawing substituents. However,
Scheme 3 Synthesis of triazolyl-thalidomide-targeted compounds 7–10.
cytoskeleton remodeling.11 To develop an efficient strategy
against fibrosis, we first examined whether intracellular Ca2+
activation was inhibited by our synthesized compounds, triazolyl
thalidomide derivatives, in the TGF-b1-induced myofibroblasts.
The myofibroblast was treated with each compound (5 mM) for
30 min. Then, the intracellular Ca2+ activation was stimulated by
thapsigargin (TG) and was detected utilizing a Ca2+ imaging
system (Fig. S2, ESI†).34 The area under the curve of each group
was calculated as an intracellular Ca2+ elevation and the percen-
tage of inhibition is shown in Table 1. The results revealed that
compounds 7a–e, 8c–d, 9d–e, 10a–b, and 10e significantly facili-
tated the inhibition of intracellular Ca2+ activation in the myofi-
broblasts. Other compounds enhanced the intracellular Ca2+
activation with a higher area under the curve. The reason for
why these compounds enhanced the intracellular Ca2+ activation
remains unknown. The viability of each group shown in Table 1 is
over 97%, which means each compound exhibited no cytotoxicity
at 5 mM, except for compound 7a. Therefore, compounds 7b–e,
8c–d, 9d–e, 10a–b, and 10e were used for further investigations.
Fig. 2 Structure–activity relationship of compound 7–10 series and its
inhibitory effect of intracellular calcium.
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 3589ꢀ3599 | 3591

View Article Online
Paper
NJC
the carboxylic group, which was added on compounds 7e, 8e, Whether these two pathways are connected or not still needs
9e, 10b–e, seemingly affected the intracellular Ca2+ activation. further investigation. However, compound 10e presented a more
The carbon side chain, which was substituted instead of the potent inhibitory ability on a-SMA than compound 7e. In fibrosis,
aryl group in compound 10 series, displayed an irregular especially for myofibroblasts, a-SMA is a more well-known and
inhibitory effect. Thus, compounds 10c (ꢀ83.2%) and 10d typical marker compared with fibronectin. In consequence, com-
(ꢀ39.9%) with the moiety of propanoic acid and pentanoic pound 10e was used for further biological investigation.
acid promoted the intracellular Ca2+ activation, while com-
Compound 10e reversed the migratory ability of the TGF-b1-
pounds 10b (75.8%) and 10e (27.9%) with the moiety of acetic induced myofibroblasts via inhibiting SOCE. Because cell
acid and hexanoic acid showed an inhibitory effect. Taken migration is restrained in the process of TGF-b1-induced
together, the results implied that the carboxylic group was an fibrogenesis due to the cytoskeleton remodeling, we investi-
important pharmacophore to interfere with the intracellular gated whether blockage of SOCE by compound 10e improved
Ca2+ activation, and the substituent effect, no matter whether the migratory ability of myofibroblasts.10 As shown in Fig. 3A,
electron-donating or electron-withdrawing groups on the aryl compound 10e demonstrates low cytotoxicity even in high
group in thalidomide derivatives, was also necessary to restrain concentrations that achieve 100 mM. The SOCE inhibitory
the intracellular Ca2+ activation.
activity of compound 10e was examined in 5 and 10 mM and
Compounds 7e and 10e inhibited the expression of fibro- compared with that of thalidomide and the SOCE inhibitor,
nectin and a-SMA. To clarify the effects of compounds 7b–e, 2-aminoethoxydiphenyl borate (2-APB)37 (Fig. 3B). Treatment
8c–d, 9d–e, 10a–b, and 10e on TGF-b1-induced fibrogenesis, the with thalidomide showed a decreased tendency of SOCE activ-
expressions of fibrotic markers, fibronectin and a-SMA were ity, but treatment with 2-APB and compound 10e significantly
determined by western blot analysis. Fibronectin is a multi- inhibited the SOCE activity in the TGF-b1-induced myofibro-
functional glycoprotein and ECM component, which plays an blasts (Fig. 3C). Compound 10e possesses a stronger inhibitory
important role in cell adhesion, migration, growth, and ability of SOCE than that in thalidomide. Thus, compound 10e
differentiation.35 a-SMA is an actin isoform, which shows over- also recovered the migratory ability in a dose-dependent manner
expression in fibrogenesis.36 Both of them are correlated with in the TGF-b1-induced myofibroblasts (Fig. 3D), significantly
the activation of myofibroblasts. Herein, the inhibitory effects accelerating the number of migrated cells (Fig. 3E). Our results
of fibronectin and a-SMA were considered to screen the chosen suggest that compound 10e, a SOCE inhibitor, restored the
compounds 7b–e, 8c–d, 9d–e, 10a–b and 10e, testing whether
the activation of myofibroblasts was blocked by the application
of these compounds (Table 2). Most of the tested compounds
were able to decrease the expression of fibronectin, excluding
compounds 9d and 9e. Compounds 7b–e, 8c–d, 10a–b, and 10e
provided greater inhibition than thalidomide on fibronectin,
but only compounds 7e (32.5%) and 10e (38.4%) significantly
decreased the expression of a-SMA. The results revealed that
both compounds 7e and 10e simultaneously inhibited the
expressions of fibronectin and a-SMA to suppress fibrogenesis.
As we described earlier, the carboxylic group, the substituent of
thalidomide derivatives, was important in governing the inhi-
bition of SOCE activity, especially compounds 7e and 10e.
Table 2 The inhibitory effect toward fibronectin and a-SMA of selected
thalidomide derivatives
Fig. 3 The migratory ability of TGF-b1-induced myofibroblasts was
improved by treating with the SOCE inhibitor, compound 10e. (A) Effects
of treatment with each concentration of compound 10e (0 mM, 5 mM,
% inhibition at 5 mM
Compounds
Fibronectin
a-SMA
10 mM, 50 mM and 100 mM) on the percentage of cell survival. (B) Pre-
treatment with SOCE inhibitor 2-aminoethoxydiphenyl borate (2-APB),
compound 10e (5 mM and 10 mM) and thalidomide limited the TG-induced
SOCE activity in the TGF-b1-induced myofibroblasts. After the application of
thapsigargin (TG) (black arrow) to activate store-operated Ca2+ channels in a
Ca2+-free balanced salt solution (thick white bar), extracellular CaCl2 was
added (thick black bar) to induce intracellular Ca2+ entry. Figure B is a
schematic diagram which shows one of the experimental data. (C) Mean
area under the intracellular Ca2+ response curves after the addition of
extracellular CaCl2 in (B) (n 4 100 cells; mean ꢁ SD, ***, p o 0.001).
(D) The migratory ability of the TGF-b1-induced myofibroblasts with or
without treating with 2-APB, compound 10e (5 mM and 10 mM) and
thalidomide was analyzed using a transwell assay. (E) Quantification of
migratory cells as in (D) (mean ꢁ SD, *, p o 0.05; ***, p o 0.001).
7b
7c
7d
7e
8c
8d
9d
9e
39.8 ꢁ 3.1
33.1 ꢁ 6.2
46.0 ꢁ 4.1
28.6 ꢁ 3.1
38.9 ꢁ 5.8
36.9 ꢁ 6.2
0.6 ꢁ 1.0
ꢀ10.6 ꢁ 3.3
11.4 ꢁ 0.8
ꢀ27.2 ꢁ 1.3
32.5 ꢁ 4.0
ꢀ9.3 ꢁ 6.1
ꢀ14.3 ꢁ 5.0
ꢀ4.2 ꢁ 1.1
3.4 ꢁ 0.3
4.0 ꢁ 11.4
27.7 ꢁ 1.0
29.2 ꢁ 4.1
22.8 ꢁ 2.1
15.6 ꢁ 9.1
10a
10b
10e
Thalidomide
3.7 ꢁ 5.6
ꢀ5.6 ꢁ 2.4
38.4 ꢁ 3.2
ꢀ23.1 ꢁ 12.9
Results are presented as mean ꢁ standard deviation (n = 3).
3592 | New J. Chem., 2021, 45, 3589ꢀ3599
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

View Article Online
NJC
Paper
migratory ability via attenuating the SOCE activity in the TGF-b1- order to identify the effect of compound 10e on SOCE and its
induced myofibroblasts. related signaling pathways, several molecules involved in SOCE
Compound 10e-induced dedifferentiation of myofibroblasts and TGF-b1/SMAD signaling pathways were analyzed. Because
was due to the cytoskeleton remodeling. A previous study the most extensively tested molecules of SOCE are STIM1 and
illuminated that SOCE activity deficiency in myofibroblasts Orai1,25 we first confirmed the expressions of STIM1 and Orai1.
induces dedifferentiation and simultaneously changes the cytos- The results indicated that the expression of Orai1 was signifi-
keleton remodeling against fibrogenesis.11 Our study found that cantly upregulated in the TGF-b1-induced myofibroblasts,
compound 10e facilitated the recovery of the migratory ability via which was dramatically downregulated by treating with com-
inhibiting the SOCE activity in the TGF-b1-induced myofibro- pound 10e (Fig. 5A and B). Compound 10e suppressed the
blasts. As a result, we explored whether compound 10e affecting expression of Orai1 but not STIM1 (Fig. 5A and B), attenuating
the migratory ability was dependent on cytoskeleton remodeling. the increased level of SOCE against myofibroblast activation. In
To demonstrate the dedifferentiation of myofibroblasts, cell addition, the phosphorylated SMAD2/3 complex through TGF-
morphology was first determined. As shown in Fig. 4A, the b1 induction drives the expression of ECM-related genes, such
control group represented the normal type of fibroblasts with as collagens, fibronectin and a-SMA, regulating the differentia-
spindle-shaped elongated cells. The fibroblasts treated with tion from fibroblasts to myofibroblasts in the process of
TGF-b1 displayed the typical type of myofibroblasts with a fibrogenesis.38 Fig. 5C and D show that compound 10e signifi-
complex and aggregated shape. Compared to myofibroblasts, cantly inhibited the TGF-b1/SMAD2/3 signaling pathways in the
the group with the addition of compound 10e showed the shape myofibroblasts. Once the TGF-b1/SMAD2/3 signaling pathways
that was similar to the fibroblast. We have also calculated the were blocked, the microenvironment and cytoskeleton remodeling
ratio of differentiated fibroblasts by identifying the cell morphol- were affected by disrupting the expression of ECM-related genes.
ogy and found that the higher ratio of the differentiated fibro- The results gave strong evidence that compound 10e inhibited
blasts in the TGF-b1-induced myofibroblasts was significantly not only the expression of Orai1 but also the TGF-b1/SMAD2/3
decreased by treating with compound 10e (Fig. 4B). We further signaling pathways. As previously mentioned, these pathways
identified that compound 10e-induced dedifferentiation from play important roles in several kinds of fibrosis. Previous results
myofibroblasts to fibroblasts could attribute to cytoskeleton indicated that thalidomide reduced the expressions of IL-6, IL-8,
remodeling. Consequently, the variation of cytoskeleton was TNF-a and TGF-b. Among them, the decrease of IL-6, IL-8 and
detected by immunofluorescence staining. Fibronectin and TNF-a contributed to the anti-inflammatory effect while the
a-SMA in the TGF-b1-induced myofibroblasts revealed a crossed decrease of TGF-b1 contributed to the anti-fibrotic effect.39 Com-
and inattentive arrangement (Fig. 4C); in contrast, with the pared to our new triazolyl thalidomide derivatives, compound 10e
addition of compound 10e, the shape changed and rearranged inhibited not only SOCE but also TGF-b1 (Fig. 6C) and its related
into a straight and loosened type, which was similar to the control
group. Conclusively, the results validated that compound 10e
induced the rearrangement of fibers by altering the cytoskeleton
remodeling, leading to the dedifferentiation of myofibroblasts
and increasing the ability of cell migration.
Compound 10e restrained the myofibroblast activation by
targeting Orai1 and TGF-b1/SMAD2/3 signaling pathways. In
Fig. 4 SOCE inhibitor, compound 10e, affected the cell morphology
through cytoskeleton remodeling in the TGF-b1-induced myofibroblasts.
(A) Compound 10e (5 mM) dramatically affected the cell morphology of the
myofibroblasts, indicating the dedifferentiation of the myofibroblasts. The
percentage of the differentiated fibroblasts was microscopically quantified
in (B) (mean ꢁ SD, ***, p o 0.001). (C) Distribution of a-SMA and
fibronectin was analyzed using immunofluorescence staining; compound
10e influenced the cytoskeleton remodeling in the TGF-b1-induced
myofibroblasts.
Fig. 5 Treatment with compound 10e eliminated the activation of TGF-b1-
induced myofibroblasts due to the inhibition of Orai1 and TGF-b1/SMAD2/3
signaling pathways. (A) Analysis of the expression of Orai1 and STIM1 after
treating with compound 10e for 24 h. Quantification of (A) as for (B). (C) Analysis
of the expression of TGF-b1, phospho-SMAD2 (pSMAD2) and phospho-SMAD3
(pSMAD3). Quantification of (C) as for (D). Data were normalized to the protein
expression of the internal control and GAPDH, and all data are presented as
mean ꢁ SD (*, p o 0.05; **, p o 0.01; ***, p o 0.001).
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 3589ꢀ3599 | 3593

View Article Online
Paper
NJC
series. We could also infer that compound 10e may be involved in
other mechanisms to decrease the expression of Orai1 instead of
directly blocking the channels. However, the other pharmacoki-
netic properties also play an important role in affecting the
binding affinity and permeability. Hydrogen bond acceptor
(HBA) and hydrogen bond donor (HBD) groups optimized the
interaction between the compounds and the receptor. Among
these compounds, 7e, 8e, 9e and 10b–e with the carboxylic acid
group possessed the hydrogen bond donor (HBD). log P is an
essential factor in passive membrane partition. The carboxylic
group in the compound improved the dissolution and decreased
the log P values, while the appropriate lipophilic moiety may
balance the dissolution contributing to suitable log P values.
Compound 10e comprised the appropriate lipophilic carbon
chain and carboxylic acid. The combination of these structures
enhanced their absorption or permeability properties through
the biological membrane, which gave rise to a good drug-
Fig. 6 Schematic representation of the mechanism of compound 10e in
inhibiting the process of fibrogenesis.
signaling pathways. These effects make compound 10e a pro- likeness model score. The prediction of drug-likeness model
mising anti-fibrotic agent. We can infer from the results that score was performed with Molsoft’s chemical fingerprints,
one of our synthesized SOCE inhibitors, compound 10e, pro- which was set to consist of 5k of marketed drugs (positive)
vides us with a promising lead compound for a new therapy for and 10k of carefully selected non-drugs compound.41 The
fibrosis.
molecular weight of all the compounds lies between 397 and
The in silico study of compound 10e compared with other 501, indicating that all the compounds follow Lipinski’s rule of
synthesized compounds. To investigate the deep mechanism of five. Molecular polar surface area (PSA), a sum of surfaces of
the SOCE inhibitor, compound 10e, the in silico study was polar atoms, is a crucial parameter for the prediction of drug
carried out. In a molecular docking study, one of the dominant transport property. There is a little difference in each com-
SOCE channels, Orai1, was chosen to dock with compound 10e pound’s PSA due to the high similarity in their structures.
to see whether Orai1 would be blocked by our compound
leading to the decrease of SOCE.40 Other synthesized com-
pounds were also docked with Orai1 to compare with com-
pound 10e. The results are shown in Table 3. The docking score
Experimental section
Chemistry section
of all the synthesized compounds were in the range of ꢀ5.8 to
ꢀ6.8 kcal molꢀ1. Comparing the docking score of each com-
General information. Commercial reagents were used as
pound, we found that the score of each compound was similar. received without additional purification. Melting points were
The reason for the results could be due to the fact that there is determined with an Electrothermal IA9100 (Electrothermal,
only a limited variation between the structures of each compound Staffordshire, UK) micro-melting point apparatus and were
Table 3 Calculated absorption (%ABS), polar surface area (PSA), Lipinski’s parameters, drug-likeness model score and molecular docking score of
synthesized compounds 7–10
Drug-likeness Docking score
Compound
R
% ABS Volume PSA
NROTB HBA HBD log P, calculated Molecular weight model score (kcal molꢀ1
)
7a
7b
7c
7d
7e
8a
8c
8d
8e
9a
9c
9d
9e
10a
10b
10c
10d
10e
H
Cl
F
OMe
COOH
H
72.03 350.97 107.17
72.03 364.50 107.17
72.03 355.90 107.17
68.84 376.51 116.41
59.16 377.97 144.47
72.03 367.77 107.17
72.03 372.70 107.17
68.84 393.31 116.41
59.16 394.77 144.47
66.13 386.75 124.25
66.13 391.68 124.25
62.95 412.30 133.48
53.27 413.75 161.54
4
4
4
5
5
5
5
6
6
6
6
7
7
7
5
6
8
9
9
9
9
10
11
9
0
0
0
0
1
0
0
0
1
0
0
0
1
0
1
1
1
1
1.41
2.09
1.58
1.47
1.32
1.74
1.90
1.79
1.65
1.29
1.45
1.34
1.20
0.38
-0.61
-0.34
0.43
0.94
415.41
449.85
433.40
445.44
459.42
429.44
447.43
459.46
473.44
457.45
475.44
487.47
501.45
425.40
397.35
411.37
439.43
453.45
0.54
0.86
0.73
0.49
0.63
0.53
1.02
0.81
1.06
0.66
1.13
1.02
1.06
0.30
0.62
0.63
0.68
0.68
ꢀ6.8
ꢀ6.7
ꢀ6.6
ꢀ6.4
ꢀ6.7
ꢀ6.6
ꢀ6.7
ꢀ6.6
ꢀ6.6
ꢀ6.2
ꢀ6.6
ꢀ6.3
ꢀ6.8
ꢀ5.8
ꢀ6.4
ꢀ6.0
ꢀ6.4
ꢀ6.2
F
9
OMe
COOH
H
10
11
10
10
11
12
11
11
11
11
11
F
OMe
COOH
n = 1, Et 62.95 357.69 133.48
n = 1, OH 59.16 323.36 144.47
n = 2, OH 59.16 340.16 144.47
n = 4, OH 59.16 373.77 144.47
n = 5, OH 59.16 390.57 144.47
3594 | New J. Chem., 2021, 45, 3589ꢀ3599
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

View Article Online
NJC
Paper
uncorrected. NMR spectra were recorded with a Varian Unity- individually dissolved in 0.5 mL water. The solution of copper
400 MHz spectrometer (LabX, Midland, Canada) using DMSO-d6 sulfate pentahydrate and sodium ascorbate was then added
as the solvent and tetramethylsilane as the internal standard. dropwise into tert-butanol and stirred at room temperature for
Chemical shifts were expressed as d (ppm). Splitting patterns 24 h (monitored by TLC). The mixtures were then concentrated
have been described as follows: s = singlet; d = doublet; t = triplet; under reduced pressure and extracted twice by 80 mL dichlor-
q = quartet; dd = double doublet; m = multiplet. The integration omethane/water (1/1). The organic layers were dried with
of one proton in the NMR spectra was set to 10. Analytical thin magnesium sulfate and concentrated under reduced pressure.
layer chromatography (TLC) was performed on an Art. 5554 The samples were further purified by silica gel column chro-
Kieselgel 60 GF254 (Merck, Darmstadt, Germany) produced by matography, using dichloromethane/methanol (100/1 to 20/1)
E. Merck and the spots of compounds were detected with UV as the mobile phase, to produce compounds with variable
light indicator irradiated at 254 and 366 nm. Art. 7734 Kieselgel yields (27–98%).
60 GF254 (70–400 mesh, Merck, Darmstadt, Germany) made by
2-{2,6-Dioxo-1-[(1-phenyl-1H-1,2,3-triazol-4-yl)methyl]piperidin-
E. Merck was used for column chromatography. The purity of 3-yl}isoindoline-1,3-dione (7a). Compound 7a was obtained at
compounds was determined by HPLC and elemental analysis 82% as a yellow solid. Melting point: 212.1–213.1 1C. Purity:
1
(EA). High performance liquid chromatography (HPLC) analysis 97.7%. H NMR (400 MHz, DMSO-d6) d 8.52 (s, 1H, triazole-H),
was performed on a HITACHI Chromaster 5110 HPLC (Hitachi 7.94–7.88 (m, 4H, Ar-H), 7.88–7.84 (m, 2H, Ar-H), 7.63–7.58
High-Technologies, Tokyo, Japan), fitted with a UV detector and (m, 2H, Ar-H), 7.51–7.47 (m, 1H, Ar-H), 5.34 (dd, 1H, J = 5.6,
an auto sampler. The compounds were tested on a Mightysil 13.2 Hz, 30-H), 5.05, 4.99 (AB system, 2H, J = 24 Hz, N-CH2), 3.11–
RP-18 GP 250-4.6 (5 mm) (Kanto Chemical, Tokyo, Japan) 2.12 (4m, 4H, 40-H, 50-H). 13C NMR (100 MHz, DMSO-d6) d 171.28,
with a mobile phase of 10 mM KH2PO4 : acetonitrile (35: 65) 169.29, 167.14 (2C), 144.19, 136.57, 134.96 (2C), 131.20 (2C), 129.93
pH = 2.89 for compounds 2, 7a–d, 8a, 8c–d, 9a, 9c–d, and 10a and (2C), 128.68, 123.49 (2C), 121.25, 120.01 (2C), 49.63, 35.18, 31.21,
methanol : 0.1% formic acid (9 : 1) for compounds 7e, 8e, 9e, and 21.14. MS (ESI): m/z [M + H]+ 415.92.
10b–e. The flow rate was 1 mL minꢀ1 and the sample injection
2-{1-[(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl]-2,6-dioxo-
was 100 mL (5.00 mg dissolved in 0.5 mL DMSO, and then diluted piperidin-3-yl}isoindoline-1,3-dione (7b). Compound 7b was
with methanol to 5 mL). The wavelength was set at 295 nm. obtained at 87% as a white solid. Melting point: 237.5–238.4 1C.
Waters ZQ-4000 (Waters, Milford, MA, USA) liquid chromato- Purity: 96.3%. 1H NMR (400 MHz, DMSO-d6) d 8.56 (s, 1H,
graphy electrospray ionization mass spectrometry was used to triazole-H), 7.95–7.87 (2m, 6H, 4Ar-H, 2H of Ar-H), 7.67 (d, J =
record mass spectra.
8.8Hz, 2H, Ar-H), 5.34 (dd, 1H, J = 5.2, 13.2 Hz, 30-H), 5.04, 4.99
Procedure for the synthesis of 2-[2,6-dioxo-1-(prop-2-yn-1- (AB system, 2H, J = 23.4 Hz, N-CH2), 3.10–2.12 (4m, 4H, 40-H,
yl)piperidin-3-yl]isoindoline-1,3-dione (2). Thalidomide was 50-H). 13C NMR (100 MHz, DMSO-d6) d 171.25, 169.28, 167.13
prepared by the approach based on our previous study.17 The (2C), 144.34, 135.35, 134.95 (2C), 132.92, 131.19 (2C), 129.89 (2C),
mixture of thalidomide (1.29 g, 5.00 mmol), potassium carbo- 123.49 (2C), 121.67 (2C), 121.40, 49.62, 35.11, 31.19, 21.13. MS
nate (3.45 g, 25.0 mmol) and acetone (40.0 mL) was stirred at (ESI): m/z [M + H]+ 449.91.
room temperature for 10 minutes. Propargyl bromide 80 wt% in
2-{1-[(1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl]-2,6-dioxo-
toluene (1.20 g, 10.0 mmol) was then added into the mixture piperidin-3-yl}isoindoline-1,3-dione (7c). Compound 7c was
and stirred at room temperature for 2 days (monitored by TLC). obtained at 88% as a white solid. Melting point: 240.3–240.7 1C.
The mixtures were first concentrated under reduced pressure Purity: 97.8%. 1H NMR (400 MHz, DMSO-d6) d 8.52 (s, 1H,
and then extracted twice with 80 mL dichloromethane/water triazole-H), 7.96–7.88 (m, 6H, 4Ar-H, 2H of Ar-H), 7.50–7.44
(1/1). The organic layers were collected, dried with magnesium (m, 2H, Ar-H), 5.34 (dd, 1H, J = 5.2, 13.2 Hz, 30-H), 5.05, 4.99
sulfate and concentrated under reduced pressure. The crude (AB system, 2H, J = 22.8 Hz, N-CH2), 3.11–2.12 (4m, 4H, 40-H,
compound was further purified by silica gel column chromato- 50-H). 13C NMR (100 MHz, DMSO-d6) d 171.24, 169.26, 167.12
graphy, using hexane/dichloromethane (1/1) as the mobile (2C), 161.60 (1JCF = 243.3 Hz), 144.19, 134.95 (2C), 133.11, 131.19
3
phase, to produce compound 2 with a yield of 87% as a white (2C), 123.47 (2C), 122.36 (2C, JCF = 9.1 Hz), 121.55, 116.74 (2C,
solid. Melting point: 187.2–187.9 1C. Purity: 97.5%. 1H NMR 2JCF = 23.5 Hz), 49.62, 35.12, 31.18, 21.12. MS (ESI): m/z [M + H]+
(400 MHz, DMSO-d6) d 7.97–7.90 (m, 4H, Ar-H), 5.31 (dd, 1H, 433.91.
J = 5.2, 13.2 Hz, 30-H), 4.39 (d, J = 2.4 Hz, N-CH2), 3.11 (t, J =
2-{1-[((1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)]-2,6-
2.4 Hz, CH), 3.09–2.08 (4m, 4H, 40-H, 50-H). 13C NMR (100 MHz, dioxopiperidin-3-yl}isoindoline-1,3-dione (7d). Compound 7d
DMSO-d6) d 170.75, 168.75, 167.06 (2C), 134.92 (2C), 131.18, was obtained at 98% as a brown solid. Melting point: 106.4–
123.45 (2C), 78.97, 75.74, 72.98, 49.49, 30.96, 29.05, 20.98. MS 108.0 1C. Purity: 97.6%. 1H NMR (400 MHz, DMSO-d6) d 8.41
(ESI): m/z [M + H]+ 296.87.
(s, 1H, triazole-H), 7.96–7.88 (m, 4H, Ar-H), 7.79–7.75 (m, 2H, Ar-
General procedure for the synthesis of compounds 7–10 H), 7.16–7.12 (m, 2H, Ar-H), 5.35 (dd, 1H, J = 5.2, 13.2 Hz, 30-H),
series. The selected azide 3–6 was prepared under different 5.04, 4.98 (AB system, 2H, J = 23.6 Hz, N-CH2), 3.84 (s, 3H, OCH3),
conditions according to the published research.24 Compound 2 3.11–2.12 (4m, 4H, 40-H, 50-H). 13C NMR (100 MHz, DMSO-d6)
(0.29 g, 1.00 mmol) and the selected azide (1.20 mmol) were first d 171.23, 169.25, 167.12 (2C), 159.23, 143.91, 134.92 (2C), 131.18
dissolved in 8 mL tert-butanol. Copper sulfate pentahydrate (0.13 g, (2C), 130.00, 123.46 (2C), 121.65 (2C), 121.18, 114.88 (2C), 55.54,
0.50 mmol) and sodium ascorbate (0.10 g, 0.50 mmol) were 49.61, 35.17, 31.18, 21.12. MS (ESI): m/z [M + H]+ 445.91.
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 3589ꢀ3599 | 3595

View Article Online
Paper
4-{4-[(3-(1,3-Dioxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl]-
NJC
2-{2,6-Dioxo-1-[(1-(2-oxo-2-phenylethyl)-1H-1,2,3-triazol-4-yl)-
1H-1,2,3-triazol-1-yl}benzoic acid (7e). Compound 7e was obtained methyl]piperidin-3-yl}isoindoline-1,3-dione (9a). Compound 9a
at 33% as a white solid. Melting point: 219.3–221.0 1C. Purity: was obtained at 54% as a yellow solid. Melting point: 208.8–
1
1
98.6%. H NMR (400 MHz, DMSO-d6) d 8.62 (s, 1H, triazole-H), 210.2 1C. Purity: 96.1%. H NMR (400 MHz, DMSO-d6) d 8.07–
8.16–8.14 (m, 2H, Ar-H), 7.96–7.89 (m, 6H, Ar-H), 5.36 (dd, 1H, J = 8.05 (m, 2H, Ar-H), 7.95–7.89 (m, 4H, Ar-H), 7.88 (s, 1H, triazole-H),
5.2, 12.8 Hz, 30-H), 5.06, 5.00 (AB system, 2H, J = 22.8 Hz, N-CH2), 7.76–7.72 (m, 1H, Ar-H), 7.63–7.59 (m, 2H, Ar-H), 6.15 (s, 2H, CH2-
3.12–2.14 (4m, 4H, 40-H, 50-H). 13C NMR (100 MHz, DMSO-d6) CO), 5.33 (dd, 1H, J = 5.2, 13.2 Hz, 30-H), 4.99, 4.95 (AB system,
d 171.29, 169.31, 167.15 (2C), 144.40, 138.42, 134.98 (2C), 131.21 2H, J = 16 Hz, N-CH2), 3.12–2.10 (4m, 4H, 40-H, 50-H). 13C NMR
(2C), 130.91 (2C), 123.51 (2C), 121.35, 119.34 (2C), 49.61, 35.16, (100 MHz, DMSO-d6) d 192.09, 171.22, 169.19, 167.08 (2C),
31.21, 21.14. MS (ESI): m/z [M + H]+ 459.90.
142.59, 134.89 (2C), 134.14, 131.19 (2C), 128.92 (2C), 128.12
2-{1-[(1-Benzyl-1H-1,2,3-triazol-4-yl)methyl]-2,6-dioxopiperidin- (2C), 124.97, 123.43 (2C), 55.69, 49.61, 35.24, 31.15, 21.11. MS
3-yl}isoindoline-1,3-dione (8a). Compound 8a was obtained at (ESI): m/z [M + H]+ 457.92.
67% as a white solid. Melting point: 173.9–174.6 1C. Purity:
2-{1-[(1-(2-(4-Fluorophenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)-
1
97.7%. H NMR (400 MHz, DMSO-d6) d 7.95–7.89 (m, 5H, 4 of methyl]-2,6-dioxopiperidin-3-yl}isoindoline-1,3-dione (9c). Com-
Ar-H, 1 of triazole-H), 7.40–7.28 (m, 5H, Ar-H), 5.56 (s, 2H, CH2- pound 9c was obtained at 60% as an orange solid. Melting point:
1
Ar), 5.30 (dd, 1H, J = 5.2, 13.2 Hz, 30-H), 4.94, 4.88 (AB system, 2H, 169.6–170.3 1C. Purity: 95.6%. H NMR (400 MHz, DMSO-d6) d
J = 23.2 Hz, N-CH2), 3.08–2.08 (4m, 4H, 40-H, 50-H). 13C NMR 8.17–8.12 (m, 2H, Ar-H), 7.95–7.89 (m, 4H, Ar-H), 7.87 (s, 1H,
(100 MHz, DMSO-d6) d 171.19, 169.19, 167.05 (2C), 143.00, 135.93, triazole-H), 7.47–7.42 (m, 2H, Ar-H), 6.14 (s, 2H, CH2-CO), 5.33
134.90 (2C), 131.18 (2C), 128.69 (2C), 128.05, 127.90 (2C), 123.41 (dd, 1H, J = 5.2, 13.2 Hz, 30-H), 4.98, 4.94 (AB system, 2H, J =
(2C), 52.72, 49.54, 35.16, 31.09, 21.10. MS (ESI): m/z [M + H]+ 16 Hz, N-CH2), 3.12–2.10 (4m, 4H, 40-H, 50-H). 13C NMR (100 MHz,
429.91.
DMSO-d6) d 190.79, 171.22, 169.19, 167.08 (2C), 165.50 (1JCF
=
3
2-{1-[(1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl]-2,6-dioxo- 250.9 Hz), 142.61, 134.90 (2C), 131.24 (2C, JCF = 9.9 Hz), 131.19,
piperidin-3-yl}isoindoline-1,3-dione (8c). Compound 8c was 130.93 (4JCF = 3.0 Hz), 124.96 (2C), 123.43 (2C), 116.02 (2C, 2JCF
=
obtained at 53% as a yellow solid. Melting point: 80.7–81.8 1C. 22.0 Hz), 55.62, 49.60, 35.23, 31.14, 21.10. MS (ESI): m/z [M + H]+
Purity: 97.5%. 1H NMR (400 MHz, DMSO-d6) d 7.95–7.89 (m, 5H, 475.91.
4Ar-H, 1 of triazole-H), 7.40–7.36 (m, 2H, Ar-H), 7.24–7.18 (m, 2H,
2-{1-[(1-(2-(4-Methoxyphenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)-
Ar-H), 5.55 (s, 2H, CH2-Ar), 5.30 (dd, 1H, J = 5.2, 13.2 Hz, 30-H), methyl]-2,6-dioxopiperidin-3-yl}isoindoline-1,3-dione (9d). Com-
4.94, 4.87 (AB system, 2H, J = 24.8 Hz, N-CH2), 3.08–2.08 (4m, 4H, pound 9d was obtained at 62% as an orange solid. Melting point:
40-H, 50-H). 13C NMR (100 MHz, DMSO-d6) d 171.20, 169.19, 113.4–114.8 1C. Purity: 94.7%. H NMR (400 MHz, DMSO-d6) d
1
167.06 (2C), 161.84 (1JCF = 243.3 Hz), 143.04, 134.91 (2C), 132.19, 8.06–8.02 (m, 2H, Ar-H), 7.95–7.89 (m, 4H, Ar-H), 7.86 (s, 1H,
131.18 (2C), 130.25 (2C, 3JCF = 8.3 Hz), 123.41 (2C), 123.34, 115.52 triazole-H), 7.14–7.10 (m, 2H, Ar-H), 6.07 (s, 2H, CH2-CO), 5.33
2
(2C, JCF = 21.2 Hz), 51.92, 49.55, 35.15, 31.09, 21.10. MS (ESI): (dd, 1H, J = 5.2, 13.2 Hz, 30-H), 4.98, 4.94 (AB system, 2H, J = 16.4
m/z [M + H]+ 447.91.
Hz, N-CH2-triazole), 3.88 (s, 3H, OCH3), 3.12–2.09 (4m, 4H, 40-H,
2-{1-[(1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl]-2,6-dioxo- 50-H). 13C NMR (100 MHz, DMSO-d6) d 190.30, 171.21, 169.18,
piperidin-3-yl}isoindoline-1,3-dione (8d). Compound 8d was 167.07 (2C), 163.83, 142.53, 134.88 (2C), 131.19 (2C), 130.52 (2C),
obtained at 57% as a yellow solid. Melting point: 81.0–82.5 1C. 126.99, 124.95, 123.42 (2C), 114.16 (2C), 55.64, 55.31, 49.60,
Purity: 96.4%. 1H NMR (400 MHz, DMSO-d6) d 7.95–7.89 (m, 4H, 35.25, 31.14, 21.10. MS (ESI): m/z [M + H]+ 487.93.
Ar-H), 7.87 (s, 1H, triazole-H), 7.29–7.27 (m, 2H, Ar-H), 6.95–6.91
4-{2-[4-((3-(1,3-Dioxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)-
(m, 2H, Ar-H), 5.46 (s, 2H, CH2-Ar), 5.30 (dd, 1H, J = 5.2, 13 Hz, methyl)-1H-1,2,3-triazol-1-yl]acetyl}benzoic acid (9e). Compound
30-H), 4.92, 4.86 (AB system, 2H, J = 22.4 Hz, N-CH2), 3.74 (s, 3H, 9e was obtained at 60% as a white solid. Melting point: 230.7–
1
OCH3), 3.08–2.09 (4m, 4H, 40-H, 50-H). 13C NMR (100 MHz, 232.4 1C. Purity: 99.7%. H NMR (400 MHz, DMSO-d6) d 8.15–
DMSO-d6) d 171.20, 169.19, 167.06 (2C), 159.09, 142.96, 134.91 8.10 (m, 4H, Ar-H), 7.96–7.89 (m, 5H, 4 of Ar-H, 1 of triazole-H),
(2C), 131.19 (2C), 129.59 (2C), 127.81, 123.41 (2C), 123.00, 114.08 6.19 (s, 2H, CH2-CO), 5.34 (dd, 1H, J = 5.6, 13.2 Hz, 30-H), 5.01–
(2C), 55.09, 52.29, 49.54, 35.19, 31.08, 21.10. MS (ESI): m/z [M + H]+ 4.93 (m, 2H, N-CH2), 3.12–2.11 (4m, 4H, 40-H, 50-H). 13C NMR
459.96.
(100 MHz, DMSO-d6) d 192.05, 171.29, 169.25, 167.13 (2C),
4-{[4-((3-(1,3-Dioxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl)- 142.66, 134.95 (2C), 131.22 (2C), 129.60 (2C), 128.25 (2C),
1H-1,2,3-triazol-1-yl]methyl}benzoic acid (8e). Compound 8e was 125.02, 123.48 (2C), 55.97, 49.62, 35.27, 31.18, 21.13. MS (ESI):
obtained at 42% as a white solid. Melting point: 246.8–248.2 1C. m/z [M + H]+ 501.91.
Purity: 99.2%. 1H NMR (400 MHz, DMSO-d6) d 7.98–7.89 (m, 7H,
2-{4-[(3-(1,3-Dioxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl]-
6 of Ar-H, 1 of triazole-H), 7.38–7.36 (m, 2H, Ar-H), 6.95–6.91 1H-1,2,3-triazol-1-yl}acetate (10a). Compound 10a was obtained
(m, 2H, Ar-H), 5.66 (s, 2H, CH2-Ar), 5.31 (dd, 1H, J = 5.6, 13.2 Hz, at 52% as a yellow solid. Melting point: 71.1–72.3 1C. Purity:
1
30-H), 4.96, 4.88 (AB system, 2H, J = 35.2 Hz, N-CH2), 3.08–2.08 98.5%. H NMR (400 MHz, DMSO-d6) d 7.96–7.89 (m, 5H, 4 of
(4m, 4H, 40-H, 50-H). 13C NMR (100 MHz, DMSO-d6) d 171.26, Ar-H, 1 of triazole-H), 5.35 (s, 2H, CH2-CO), 5.32 (dd, 1H, J = 5.6,
169.24, 167.09 (2C), 143.14, 140.63, 134.93 (2C), 131.22 (2C), 13.2 Hz, 30-H), 4.97–4.89 (m, 2H, N-CH2), 4.17 (q, J = 7.2 Hz, 2H,
129.73 (2C), 127.89 (2C), 123.79, 123.45 (2C), 52.31, 49.57, 35.15, O-CH2), 3.10–2.09 (4m, 4H, 40-H, 50-H), 1.22 (t, J = 7.2 Hz, 3H,
31.12, 21.12. MS (ESI): m/z [M + H]+ 473.91.
CH3). 13C NMR (100 MHz, DMSO-d6) d 171.20, 169.16, 167.07
3596 | New J. Chem., 2021, 45, 3589ꢀ3599
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

View Article Online
NJC
Paper
(2C), 142.53, 134.90 (2C), 131.19, 124.73 (2C), 123.42 (2C), 61.39, and synthesized compounds 1 and 7–10 series (5 mM) for
50.29, 49.57, 35.13, 31.12, 21.09, 13.89. MS (ESI): m/z [M + H]+ 30 min. Then, the intracellular Ca2+ responses were induced
425.91.
by thapsigargin (20 nM, TG, Sigma-Aldrich) as previously
2-{4-[(3-(1,3-Dioxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl]- described.42 Cells were loaded with 1 mM Fluo-4-AM (molecular
1H-1,2,3-triazol-1-yl}acetic acid (10b). Compound 10b was probes) at 37 1C for 20 minutes before the experiments and
obtained at 46% as a brown solid. Melting point: 232.9– then washed with a balanced salt solution buffer (5.4 mM KCl,
1
233.6 1C. Purity: 96.0%. H NMR (400 MHz, DMSO-d6) d 7.95– 5.5 mM D-glucose, 1 mM MgSO4, 130 mM NaCl, 20 mM hepes
7.88 (m, 4H, Ar-H), 7.88 (s, 1H, triazole-H), 5.32 (dd, 1H, J = 5.2, pH 7.4, and 2 mM CaCl2). Intracellular Ca2+ concentrations
13.2 Hz, 30-H), 5.23 (s, 2H, CH2-CO), 4.95, 4.91 (AB system, 2H, ([Ca2+]i) were calculated from the ratio of fluorescence inten-
J = 16.4 Hz, N-CH2), 3.10–2.10 (4m, 4H, 40-H, 50-H). 13C NMR sities in the absence of Ca2+ and at saturation, emitted (509 nm)
(100 MHz, DMSO-d6) d 171.22, 169.18, 168.45, 167.09 (2C), upon excitation with consecutive 3 second pulses of 488 nm
142.63, 134.91 (2C), 131.19 (2C), 124.64, 123.45 (2C), 50.39, light at a resolution of 1376 ꢂ 1038 pixels using an Olympus
49.60, 35.19, 31.14, 21.11. MS (ESI): m/z [M + H]+ 397.91.
Cell^R IX81 fluorescence microscope (Olympus) equipped with
3-{4-[(3-(1,3-Dioxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl]- an MT 20 illumination system (Olympus) and UPLanApo
1H-1,2,3-triazol-1-yl}propanoic acid (10c). Compound 10c was 10ꢂ objective lens. [Ca2+]i was estimated based on a [Ca2+] calibra-
obtained at 43% as a yellow solid. Melting point: 91.1–93.3 1C. tion curve created using a Ca2+ calibration buffer kit (Thermo Fisher
Purity: 99.4%. 1H NMR (400 MHz, DMSO-d6) d 7.96–7.89 (m, 4H, Scientific). [Ca2+]i was calculated using Fluo-4 excited at 488 nm and
Ar-H), 7.87 (s, 1H, triazole-H), 5.31 (dd, 1H, J = 5.6, 13.2 Hz, 30-H), imaged at 20 1C using the same instrument. Fluo-4 signals were
4.90 (s, 2H, N-CH2), 4.50 (t, 2H, J = 7.2 Hz, CH2-CO), 3.09–2.10 calibrated by measuring the fluorescence intensity from microcuv-
(4m, 6H, 40-H, 50-H, CH2-CH2CO). 13C NMR (100 MHz, DMSO-d6) d ettes containing 10 mM K2-EGTA (pH 7.20) buffered to various Ca2+
ꢀ ꢀ
171.78, 171.22, 169.21, 167.12 (2C), 142.68, 134.95 (2C), 131.22 concentrations. [Ca2+]i was calculated using the following formula:
(2C), 123.48 (2C), 123.39, 49.58, 45.33, 35.19, 34.03, 31.14, 21.13. [Ca2+]free = Kd ꢂ (F ꢀ Fmin/Fmax ꢀ F). Plotting the fluorescence
MS (ESI): m/z [M + H]+ 411.91.
intensity versus [Ca2+]i yielded the calibration curve with the
5-{4-[(3-(1,3-Dioxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl]- following formula: [Ca2+]i = Kd ꢂ (F ꢀ Fmin/Fmax ꢀ F), where Kd =
1H-1,2,3-triazol-1-yl}pentanoic acid (10d). Compound 10d was 345 nM, F = Fluo-4 intensity, Fmax = 640, and Fmin = 21.7 for Fluo-4.
obtained at 64% as a yellow semi-solid. Melting point: N.D.
Western blot. Western blot analyses were performed to
Purity: 99.3%. 1H NMR (400 MHz, DMSO-d6) d 7.95–7.89 (m, 4H, measure the protein expression with antibodies against TGF-
Ar-H), 7.86 (s, 1H, triazole-H), 5.31 (dd, 1H, J = 5.2, 13.2 Hz, 30-H), b1 (GeneTex), fibronectin (Abcam), a-SMA (Abcam), Orai1
4.93, 4.88 (AB system, 2H, J = 20.8 Hz, N-CH2), 4.32 (t, 2H, J = (Millipore), STIM1 (OriGene), SMAD2 (enogene), SMAD3 (eno-
7.2 Hz, CH2-CO), 3.13–2.09 (4m, 4H, 40-H, 50-H), 2.25 (t, 2H, gene) and GAPDH (GeneTex), and the method was previously
J = 7.2 Hz, triazole-CH2), 1.84–1.42 (2m, 4H, CH2CH2). 13C NMR described elsewhere.43
ꢀ ꢀ
(100 MHz, DMSO-d6) d 174.25, 171.28, 169.26, 167.16 (2C),
Cell morphology. The cell morphology was observed with a
142.77, 134.99 (2C), 131.24 (2C), 123.51 (2C), 123.14, 49.61, microscope and the percentage of differentiated fibroblasts was
49.04, 35.24, 32.94, 31.17, 29.18, 21.45, 21.18. MS (ESI): m/z then quantified. The fibroblasts were treated with TGF-b1 to
[M + H]+ 439.91.
become myofibroblasts. When the myofibroblasts were avail-
6-{4-[(3-(1,3-Dioxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl]- able, the tested compound was added. The morphology was
1H-1,2,3-triazol-1-yl}hexanoic acid (10e). Compound 10e was observed after 3 days.
obtained at 75% as a yellow semi-solid. Melting point: N.D.
Cell migration assay. To detect the cell migration ability, a
Purity: 99.6%. 1H NMR (400 MHz, DMSO-d6) d 7.95–7.89 (m, 4H, transwell system (24-well insert; pore size: 8 mm; Corning
Ar-H), 7.86 (s, 1H, triazole-H), 5.31 (dd, 1H, J = 5.2, 13.2 Hz, 30-H), Costar) was utilized in this assay. 2.5 ꢂ 104 cells were plated
4.94, 4.84 (AB system, 2H, J = 24.4 Hz, N-CH2), 4.30 (t, 2H, J = 7.2 Hz, in the media of the top chamber without serum or growth
CH2-CO), 3.09–2.08 (4m, 4H, 40-H, 50-H), 2.20 (t, 2H, J = 7.2 Hz, factors, and the medium supplemented with serum in the lower
triazole-CH2), 1.82–1.20 (3m, 6H, CH2CH2CH2). 13C NMR (100 MHz, chamber was applied as a chemoattractant. After incubating the
ꢀ ꢀ
DMSO-d6) d 174.45, 171.29, 169.28, 167.18 (2C), 142.77, 135.01 (2C), cells for 24 h, the cells that had not penetrated the membrane
131.25 (2C), 123.52 (2C), 123.15, 49.62, 49.20, 35.25, 33.48, 31.19, pores were quenched with a cotton swab, and the cells on the
29.49, 25.44, 23.92, 21.18. MS (ESI): m/z [M + H]+ 453.91.
lower surface of the membrane corresponding to the migrated
cells were stained with Giemsa stain (Gibco) and counted.
Immunofluorescence staining. Fixed cells and primary anti-
Biology section
Cell culture. Human dermal fibroblasts were purchased bodies against fibronectin (Abcam) and a-SMA (Abcam) were
from Lonza. Fibroblasts were grown in Dulbecco’s modified incubated together at 4 1C overnight. Secondary antibody and
Eagle’s medium (DMEM; Gibco Invitrogen) supplemented with 40,6-diamidino-2-phenylindole (DAPI, ThermoFisher Scientific)
10% fetal bovine serum (FBS, Gibco, Thermos Fisher Scientific) were subsequently added into the cells for 1 h and 5 min
at 37 1C in a humidified incubator containing 5% CO2 in an air individually. Coverslips were inverted and fixed onto glass slides
atmosphere.
with an antifade mounting reagent (Biotium). The fluorescence
Calcium imaging. Myofibroblasts were first treated with imaging was performed with a confocal microscope (Olympus
2-aminoethoxydiphenyl borate (2-APB, 2 mM, Sigma-Aldrich) FV1000).
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 3589ꢀ3599 | 3597

View Article Online
Paper
NJC
Statistical analysis. Bar charts were generated with GraphPad the inhibition of SOCE. As a result, this study verified that our
Prism (La Jolla, CA, USA); error bars indicated the SDs, unless design successfully suppressed fibrogenesis by the inhibition of
otherwise noted. GraphPad-Prism Analysis software (GraphPad- SOCE and its related signaling pathways, and compound 10e can
Prism Software Inc., San Diego, CA. USA) was used to analyze all be used as a promising lead compound. The in-depth in vivo study
statistical analysis data. Comparation of the difference between should be further investigated.
groups was analyzed by variance and Student’s t-tests. P-values
less than 0.05 were considered statistically significant for the
differences between groups.
Abbreviations used
In silico study. The target protein structure, Orai1, and the
active binding site are available in ref. 40 and reproduced
online at https://zhanglab.ccmb.med.umich.edu. The 3D confor-
mations of all compounds were generated by ChemBio 3D Ultra
14.0 (PerkinElmer, Waltham, MA, USA). Calculations were per-
formed with Autodock Vina.44 Absorption percent (% ABS) was
calculated (%ABS = 109 ꢀ 0.345 ꢂ PSA) and the degree of
absorption was represented. Lipinski’s rule of five, including
molecular polar surface area (PSA), log P, number of rotatable
bonds, number of hydrogen bond donor and accepter atoms, was
computed with a Molinspiration online property calculation
toolkit (http://www.molinspiration.com/services/properties.html).
The drug-likeness model score (a collective property of physical-
chemical properties, pharmacokinetics and pharmacodynamics of
a compound is represented by a numerical value) was calculated
using MolSoft software (http://www.molsoft.com/mprop/).
ECM
SOCE
TGF
Extracellular matrix
Store-operated calcium entry
Transforming growth factor
a-SMA a-Smooth muscle actin
IL-6 Interleukin-6
TNF-a Tumor necrosis factor-a
MAPK Mitogen-activated protein kinase
TG,
2-APB 2-Aminoethoxydiphenyl borate
STIM1 stromal interaction molecule 1
Orai1
HBA
HBD
PSA
Thapsigargin
Calcium release-activated calcium modulator 1
hydrogen bond acceptor
Hydrogen bond donor
molecular polar surface area
% ABS Absorption percent
NROTB Number of rotatable bonds.
Author contributions
Conclusion
K.-W. T. participated in the synthesis, purification, characteriza-
tion of the chemical compounds, and molecular docking; C.-R. C.
participated in the synthesis and interpretation of the results;
W.-L. H., M.-H. T. and C.-J. Y. cultured primary fibroblasts and
performed the cellular imaging study of Ca2+ dynamics, western
blotting, cell migration assay and immunofluorescence staining;
C.-H. T. conceptualized the project, reviewed the literature, and
wrote the paper with the help of K.-W. T. and W.-L. H.
In this study, a number of triazolyl thalidomide derivatives
were designed and synthesized to develop novel anti-fibrosis
agents by the inhibition of SOCE and its related pathways. The
inhibitory effects of SOCE, fibronectin and a-SMA, and cyto-
toxicity were first considered for screening. None of these
derivatives showed cytotoxicity in 5 mM except for compound
7a. Compounds 7a–e, 8c–d, 10a–b and 10e exhibited the SOCE
inhibitory activity. Among them, compound 10e, which inhib-
ited SOCE, fibronectin, and a-SMA simultaneously, was chosen
for further examination. The results revealed that by inhibiting
SOCE, compound 10e, a SOCE inhibitor, reversed the migratory
ability of TGF-b1-induced myofibroblasts. Compound 10e also
dedifferentiated myofibroblasts to fibroblasts due to cytoskeleton
remodeling. The mechanism of investigation revealed that com-
pound 10e restrained the myofibroblast activation by targeting
Orai1 and TGF-b1/SMAD2/3 signaling pathways. The in silico study
indicated that compound 10e may involve in other mechanisms,
giving the decrease of Orai1 expression. Also, compound 10e
comprised the appropriate lipophilic carbon chain and carboxylic
acid. The combination of these structures enhanced their absorp-
tion or permeability properties through the biological membrane,
which gave rise to a good drug-likeness model score. The mecha-
nism of compound 10e in regulating the process of fibrogenesis is
shown in Fig. 6. Compound 10e restored fibrosis by not only
Funding
The financial support of this work by the Minister of Science and
Technology of the Republic of China (Grant No. MOST 107-2320-B-
037-015, MOST 108-2320-B-037-026-MY2, MOST 107-2320-B-020-
002 and MOST 108-2314-B-020-001) and Kaohsiung Medical
University (Grant No. KMU-TC108A03-2, KMU-TC108A03-10,
KMU-KI110003 and KMU-KI110004) are gratefully acknowledged.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
inhibiting SOCE initially but also by down-regulating Orai1 and The authors thank the Center for Research Resources and Develop-
TGF-b1/SMAD2/3 signaling pathways to inhibit SOCE over long ment at Kaohsiung Medical University for the instrumentation and
periods. We also determined from the structure–activity relation- equipment support, and they also thank Yi-Chun Hsieh for the
ship that carboxylic acid was the important pharmacophore for technical support.
3598 | New J. Chem., 2021, 45, 3589ꢀ3599
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

View Article Online
NJC
Paper
23 R. Kant, D. Kumar, D. Agarwal, R. D. Gupta, R. Tilak,
S. K. Awasthi and A. Agarwal, Eur. J. Med. Chem., 2016,
113, 34–49.
24 K. W. Tang, S. C. Yang and C. H. Tseng, Int. J. Mol. Sci., 2019,
20(18), 4564.
References
1 T. Kisseleva and D. A. Brenner, Proc. Am. Thorac. Soc., 2008,
5(3), 338–342.
2 C. B. Nanthakumar, R. J. Hatley, S. Lemma, J. Gauldie,
R. P. Marshall and S. J. Macdonald, Nat. Rev. Drug Discovery,
2015, 14(10), 693–720.
25 C. Tian, L. Du, Y. Zhou and M. Li, Future Med. Chem., 2016,
8(7), 817–832.
3 T. A. Wynn, J. Pathol., 2008, 214(2), 199–210.
4 D. C. Rockey, P. D. Bell and J. A. Hill, N. Engl. J. Med., 2015,
37, 1138–1149.
5 B. M. Klinkhammer, R. Goldschmeding, J. Floege and
P. Boor, Adv. Chronic Kidney Dis., 2017, 24(2), 117–129.
6 G. Raghu and L. Richeldi, Respir. Med., 2017, 129, 24–30.
¨
26 D. Franzius, M. Hoth and R. Penner, Pflugers Arch., 1994,
428(5), 433–438.
27 E. Clementi and J. Meldolesi, Cell Calcium, 1996, 19(4), 269–279.
28 J. W. Putney, Mol. Interv., 2001, 1(2), 84–94.
29 Z. K. Sweeney, A. Minatti, D. C. Button and S. Patrick,
ChemMedChem, 2009, 4(5), 706–718.
30 J. Ishikawa, K. Ohga, T. Yoshino, R. Takezawa, A. Ichikawa,
H. Kubota and T. Yamada, J. Immunol., 2003, 170(9),
4441–4449.
´
7 A. Fernandez-Codina, K. M. Walker, J. E. Pope and Scler-
oderma Algorithm Group, Arthritis Rheumatol., 2018, 70(11),
1820–1828.
8 H. R. Davies and L. Richeldi, Am. J. Respir. Med., 2002, 1(3),
211–224.
9 X. M. Meng, D. J. Nikolic-Paterson and H. Y. Lan, Nat. Rev.
Nephrol., 2016, 12(6), 325–338.
10 A. C. Midgley, M. Rogers, M. B. Hallett, A. Clayton,
T. Bowen, A. O. Phillips and R. Steadman, J. Biol. Chem.,
2013, 288(21), 14824–14838.
11 C. Y. Wu, W. L. Hsu, M. H. Tsai, C. Y. Chai, C. J. Yen and
C. H. Chen, PLoS One, 2019, 14(3), 0213400.
12 X. Mai, J. Shang, S. Liang, B. Yu, J. Yuan, Y. Lin, R. Luo,
F. Zhang, Y. Liu, X. Lv, C. Li, X. Liang, W. Wang and J. Zhou,
J. Am. Soc. Nephrol., 2016, 27(10), 3063–3078.
13 B. Zhang, J. Jiang, Z. Yue, S. Liu, Y. Ma, N. Yu, Y. Gao, S. Sun,
S. Chen and P. Liu, J. Pharmacol. Sci., 2016, 132(3), 171–180.
14 V. Eleutherakis-Papaiakovou, A. Bamias and M. A. Dimopoulos,
Ann. Oncol., 2004, 15, 1151–1160.
31 I. Derler, R. Schindl, R. Fritsch, P. Heftberger, M. C. Riedl,
M. Begg, D. House and C. Romanin, Cell Calcium, 2013,
53(2), 139–151.
32 K. D. Rodland, R. P. Wersto, S. Hobson and E. C. Kohn, Mol.
Endocrinol., 1997, 11(3), 281–291.
33 H. E. Collins, X. Zhu-Mauldin, R. B. Marchase and
J. C. Chatham, Am. J. Physiol.: Heart Circ. Physiol., 2013,
305(4), 446–458.
34 M. Kasabova, A. Joulin-Giet, F. Lecaille, B. F. Gilmore,
S. Marchand-Adam, A. Saidi and G. Lalmanach, J. Biol.
Chem., 2014, 289(23), 16239–16251.
35 X. Y. Liu, R. X. Liu, F. Hou, L. J. Cui, C. Y. Li, C. Chi, E. Yi,
Y. Wen and C. H. Yin, Mol. Med. Rep., 2016, 14(4),
3669–3675.
36 A. V. Shinde, C. Humeres and N. G. Frangogiannis, Biochim.
Biophys. Acta, Mol. Basis Dis., 2017, 1863(1), 298–309.
37 M. Wei, Y. Zhou, A. Sun, G. Ma, L. He, L. Zhou, S. Zhang,
15 S. V. Rajkumar, E. Blood, D. Vesole, R. Fonseca, P. R. Greipp
and Eastern Cooperative Oncology Group, J. Clin. Oncol.,
2006, 24, 431–436.
¨
J. Liu, S. L. Zhang, D. L. Gill and Y. Wang, Pflugers Arch.,
2016, 468(11–12), 2061–2074.
16 M. Bramuzzo, A. Ventura, S. Martelossi and M. Lazzerini,
Medicine, 2016, 95, 4239–4254.
38 K. L. Walton, K. E. Johnson and C. A. Harrison, Front.
Pharmacol., 2017, 8, 461.
17 S. W. Seto and J. R. Docherty, Br. J. Pharmacol., 2010, 159(6),
1211–1216.
39 X. Dong, X. Li, M. Li, M. Chen, Q. Fan and W. Wei, Am.
J. Transl. Res., 2017, 9(10), 4390–4401.
18 X. L. Zhou, P. Xu, H. H. Chen, Y. Zhao, J. Shen, C. Jiang,
S. Jiang, S. Z. Ni, B. Xu and L. Li, Sci. Rep., 2017, 7(1), 14727.
19 K. W. Tang, Z. C. Lin, Y. L. Chen, C. C. Tzeng, J. Y. Fang and
C. H. Tseng, Int. J. Mol. Sci., 2018, 19(10), 3061.
20 S. G. Agalave, S. R. Maujan and V. S. Pore, Chem. – Asian J.,
2011, 6, 2696–2718.
21 A. Massarotti, S. Aprile, V. Mercalli, E. D. Grosso, G. Grosa,
G. Sorba and G. C. Tron, ChemMedChem, 2014, 9,
2497–2508.
40 B. Sampath and K. Sankaranarayanan, J. Recept. Signal
Transduction Res., 2016, 36(6), 572–585.
41 Molinspiration Cheminformatics. Available online: http://
http://www.molsoft.com/mpropdesc.html, accessed on 9
December 2020.
42 W. L. Hsu, J. H. Lu, M. Noda, C. Y. Wu, J. D. Liu and
M. Sakakibara, et al., Molecules, 2015, 20(11), 20297–20311.
43 C. Y. Wu, W. L. Hsu, C. H. Wang, J. L. Liang, M. H. Tsai and
C. J. Yen, PLoS One, 2016, 11(7), 0159299.
22 H. C. Kolb and K. B. Sharpless, Drug Discovery Today, 2003,
8, 1128–1137.
44 O. Trott and A. J. Olson, J. Comput. Chem., 2009, 31(2),
455–461.
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 3589ꢀ3599 | 3599