Synthesis and anti-fibrotic effects of santamarin derivatives as cytotoxic agents against hepatic stellate cell line LX2

Article abstract of DOI:10.1016/j.bmcl.2021.127994

Liver fibrosis is a final result of extensive deposition of extracellular matrix (ECM) and starts with the activation and proliferation of hepatic stellate cells (HSCs). Our previous study showed that eudesmane sesquiterpenoid santamarin had cytotoxicity

Full text of DOI:10.1016/j.bmcl.2021.127994

Bioorg. Med. Chem. Lett. 41 (2021) 127994
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-fibrotic effects of santamarin derivatives as cytotoxic
agents against hepatic stellate cell line LX2
,
Jin-Ping Wanga b, Tian-Ze Lia, Xiao-Yan Huanga, Chang-An Genga, Cheng Shena,
,
,
,c,*
Jin-Jin Suna b, Dong Xueb *, Ji-Jun Chena
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Yunnan Key Laboratory of
Natural Medicinal Chemistry, Kunming 650201, People’s Republic of China
b Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education and School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an
710062, People’s Republic of China
c University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Liver fibrosis is a final result of extensive deposition of extracellular matrix (ECM) and starts with the activation
and proliferation of hepatic stellate cells (HSCs). Our previous study showed that eudesmane sesquiterpenoid
Santamarin derivatives
Anti-fibrotic effects
HSC-LX2
santamarin had cytotoxicity against hepatic stellate cell line LX2 (HSC-LX2) with IC50 values of 16.5 ± 0.7 μM.
To explore the structure–activity relationships, twenty-six derivatives were synthesized by modifying the hy-
droxyl group, double-bond and unsaturated lactone. Cytotoxicity evaluation suggested that eight derivatives (6,
9, 13, 17, 20 and 25–27) increased activity against HSC-LX2. Especially, derivatives 17, 20 and 25 displayed
Extracellular matrix
obvious cytotoxicity with IC50 values of 6.4 ± 0.4, 4.6 ± 0.1, and 3.5 ± 0.1 μM, which were 3 to 5-fold higher
than santamarin. Preliminary mechanisms study revealed that the active compound 20 exhibited more than 8-
fold and 6-fold enhancement of inhibitory effect on the deposition of human hyaluronic acid (HA) and human
laminin (HL) with IC50 values of 7.6 ± 0.6 and 3.3 ± 1.2 μM.
Introduction
bioactivities.10,11 Many natural products such as glycyrrhetinic acid,12
saikosaponin D,13 tetrandrine,14 resveratrol,15 silybin16 and cucurbita-
cin B17 have been reported as active ingredients against liver fibrosis. It
is reported that the inhibition of HSCs proliferation is one of the effective
anti-fibrotic strategies.2,18,19 Our previous study demonstrated that
santamarin (STM, 1), a eudesmane sesquiterpenoid from Artemisia lav-
andulaefolia, showed obvious cytotoxicity against HSC-LX2 with IC50
Liver fibrosis is the progressive pathological result of chronic liver
injury that causes by many liver diseases like chronic viral hepatitis,
alcoholic or nonalcoholic steatohepatitis, etc.1,2 The pathological pro-
cess originates from the activation and proliferation of hepatic stellate
cells (HSCs), which trans-differentiate into myofibroblasts as a repair
response to chronic injury.3,4 The extensive deposition of collagen-based
extracellular matrix (ECM) produced by myofibroblasts leads to liver
fibrosis.5 Without effective treatment, liver fibrosis will generally
progress to cirrhosis or hepatocellular carcinoma (HCC) which causes
two million deaths annually.6 In recent years, significant therapeutic
progress has been achieved, and more than 20 candidate drugs are in
clinical trials, but none of them have been approved for the treatment of
liver fibrosis.7–9 Therefore, there is still an urgent need to develop highly
effective and specific anti-fibrotic medicants.
value of 16.5 ± 0.7 μM, and inhibited the deposition of human collagen
type I (Col I) and human laminin (HL) in HSC-LX2 with IC50 values of
7.3 ± 1.7
μ
M and 18.6 ± 1.4
μ
M.20 Furthermore, it was also reported
that STM had various biological activities including anti-tumor, anti-
inflammatory and anti-bacterial and anti-ethanol activities.21–26 For
example, STM inhibited the growth of L1210, CCRF-CEM, KB, LS174T
and MCF-7 cells in vitro with IC50 in the range of 0.16–0.92 μg/mL.
Mechanism study revealed its cytotoxicity might be related to suppres-
sion of microtubular proteins, induction of cell cycle blockage in the G2/
M phase, and induction of apoptosis via activation of caspase 3.
Although various pharmacological properties of STM had been revealed,
Natural products and their derivatives played an important role in
the drug discovery due to their diverse structural skeletons and various
* Corresponding authors at: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sci-
ences, Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming 650201, People’s Republic of China (J.-J. Chen).
E-mail addresses: xuedong_welcome@snnu.edu.cn (D. Xue), chenjj@mail.kib.ac.cn (J.-J. Chen).
https://doi.org/10.1016/j.bmcl.2021.127994
Received 11 February 2021; Received in revised form 17 March 2021; Accepted 18 March 2021
Available online 26 March 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

J.-P. Wang et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127994
no investigation of chemical modified STM derivatives had been con-
ducted for their anti-fibrotic effects.
3. CuI-catalyzed coupling reaction of pyridin-2-ones with aromatic ha-
lides afforded 1,5-disubstituted-pyridin-2(1H)-ones S1 and S2 in good
yields. Hydrolysis of S1, followed by esterification of resulting carbox-
ylic acid with STM, yielded compound 23. Hydronidone S4 was pre-
pared from S3 via BBr3-mediated demethylation and was then
condensed with compound 8 to provide the desired hybrid 24, in which
two pharmacophores were hybridized via ester-ester bonds (Scheme 4).
Inspired by the interesting result that a lot of dimeric sesquiterpene
lactones are more potent than their monomers in the bioactivity,31
santamarin-derived dimers 25–27 containing different ester-linker to
connect the two monomers were prepared by reacting STM with cor-
responding diacids in the precense of DMAP and DIC.
Structurally, STM possesses a 6,6,5-tricyclic ring system bearing
functional groups of exomethylene group conjugated with carbonyl
–
γ-lactone, 1β-hydroxyl group, olefinic double bond C3 C4, which made
–
it to be perspective for chemical modification. In order to explore the
structure–activity relationships (SARs) and possibly develop more
potent anti-fibrotic agents, 26 analogues of STM were synthesized and
evaluated for cytotoxicity against HSC-LX2.
Investigations have shown that the presence of
α-methylene-
γ-lactone moiety is important for sesquiterpene lactones to achieve good
cytotoxicity. It is validated that the terminal double-bond between C-11
and C-13 is essential. Thus, changing of the
α
, β -unsaturated lactone
All synthesized compounds were characterized by spectral analyses
of 1H NMR, 13C NMR and HRESIMS. The cytotoxicity of all STM de-
rivatives against HSC-LX2 were tested in vitro using the MTT method
with silybin as the positive control. Twenty-six derivatives were tested at
system via an aza-Michael addition was conducted (Scheme 1). Reaction
of compound 1 with dimethylamine yielded nitrogen-containing com-
pounds 2 as a single diastereomer.27 To clarify the role of C3-C4 double
bond, epoxide 3 was obtained in 81% yield by m-CPBA oxidation, sub-
sequent HClO4 mediated epoxy isomerization delivered allylic alcohol 4
in moderate yield.28 In order to evaluate the function of hydroxy group
at C-1 for cytotoxicity, the 1β-hydroxyl group was oxidized to ketone via
Dess-Martin periodinane to deliver compound 5, acetylation of hydroxyl
group yielded compound 6. As shown in Table 1, acetylation product 6
showed an increase in the cytotoxicity with an IC50 value of 12.3 ± 0.4
concentrations of 100.0 and 50.0 μM (Table S1, Supporting Informa-
tion), of which 25 derivatives showed inhibitory ratios higher than 50%
at 100.0 μM and their IC50 values were further measured for their dose-
dependent effects (Table 1).
As expected, compound 2 showed almost no cytotoxicity against
HSC-LX2 at the concentration of 100 μM, which suggests that α-meth-
ylene-γ-lactone moiety is an essential pharmacophore. Modification on
the double bond (Δ3,4) led to significantly decrease in the cytotoxicity,
indicating that the double bond was important for maintaining activity.
Oxidation of the 1β-hydroxyl group at C-3 of STM delivered the ketone
5, which exhibited low cytotoxic activity relative to that of STM.
Compounds 6–24 with diverse acyloxy group at C-1 showed different
μ
M. This result prompted us to conduct the further modification focused
on introducing different substituents to the hydroxy group (Scheme 2).
In order to clarify the influence of ester side chain on their cyto-
toxicity against HSC-LX2, the esterification products 7–22 were syn-
thesized through the condensation of STM with different carboxylic
acids, including aliphatic, alicyclic, aromatic and cinnamic acids.
Treatment of STM with acids (two equivalents) in CH2Cl2 in the presence
of EDCI and DMAP for 2 to 24 h at room temperature afforded target
compounds in yield ranging from 43% to 87%.
activities with IC50 values ranging from 4.6 to 92.0
ester side chains had significant influence on the cytotoxicity. Com-
pound 6 (IC50, 12.3 ± 0.4 M) with an acetyl at C-1 displayed higher
activity than compound 7 (IC50, 32.4 ± 3.2 M) with an octanoyl group,
μM, suggesting that
μ
μ
Pirfenidone is an anti-fibrotic agent approved for the treatment of
idiopathic pulmonary fibrosis.29–30 It also has demonstrated activity in
reduce liver fibrosis, which is able to inhibit proliferation of HSCs in-
duced by numerous growth factors, and can decrease collagen deposi-
tion in a variety of animal models in vivo. With the aim of enhancing the
activity, it was designed to synthesize two STM-drug hybrids as novel
anti-fibrotic agents by incorporating the pharmacophores of pirfenidone
into the core skeleton of STM with different linkers. The target com-
pounds 23 and 24 were synthesized in three steps as depicted in Scheme
which indicated that extending the length of ester side chain was un-
favorable. Compound 8 containing an additional carboxyl in the acyloxy
was the weakest compound in this series, suggesting the incorporating
an additional carboxyl group was unfavorable toward cytotoxicity. Of
the aromatic derivatives, 3-O-benzoyl and 3-O-cinnamoyl analogues 10
and 14 showed similar cytotoxicity with STM. Derivatives 11, 12, 15
with nitrogen or sulfur heteroatomic rings did not improve the activity.
Comparison of compounds 13 and 16–18 with trifluoromethyl substit-
uent at the aromatic ring, the meta-substituted compounds 13 and 17
showed better activity than those of ortho- and para-substituted (16 and
18). Compound 17 exhibited cytotoxicity of nearly 3-fold more potent
than STM with IC50 value of 6.4 ± 0.4
rocinnamoyl analogue 19, the pentafluorocinnamyl analogue 20
enhanced 4-fold cytotoxicity with IC50 value of 4.6 ± 0.1 M, indicating
μ
M. In contrast to 4′-fluo-
μ
that polyfluorinated substituent was favorable. When the 4-formylcin-
namyl and acetoxyl cinnamyl groups were introduced into the sub-
strate, the obtained compounds 21 and 22 exhibited weaker
cytotoxicity. A dramatic reduction in cytotoxicity in STM-hydronidone
hybrids 23 and 24 was observed, it means that the STM and hydro-
nidone are not compatible towards cytotoxicity against HSC-LX2.
Among dimeric products, compound 25 with a two carbons bridge
displayed the most potent activity against HSC-LX2 with an IC50 value of
3.5 ± 0.1 μM, which is 5-fold higher than STM. Compound 26 with a
three carbons bridge showed cytotoxicity of nearly 3-fold less potent
than compound 25. Replacing its linker with aromatic fragment (27)
also resulted in an obvious decrease in cytotoxicity, suggesting a
different effect of the linker on the toxicity of the dimers.
Derivatives 17, 20 and 25 showed potent cytotoxicity against HSC-
LX2 with IC50 values of 6.4 ± 0.4, 4.6 ± 0.1, and 3.5 ± 0.1 μM, indi-
cating 3–5 fold higher than STM (IC50, 16.5 ± 0.7 μM). Furthermore,
inhibitory effects of the most active compounds 17, 20 and 25 on the
deposition of human hyaluronic acid (HA), HL and Col I in HSC-LX2
were performed to explore the possible mechanisms. As depicted in
Table 2, compounds 17, 20 and 25 all showed better inhibitory effects
Scheme 1. Reagents and conditions: (a) Me2NH, EtOH, r.t., 73%; (b) m-CPBA,
CH2Cl2, r.t., 81%; (c) 6% HClO4 aqueous, DME, r.t., 66%; (d) Dess-Martin
periodinane, CH2Cl2, r.t., 71%; (e) Ac2O, DMAP, dry CH2Cl2, r.t., 69%.
2

J.-P. Wang et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127994
Table 1
Cytotoxicity of compounds 3–27 and against HSC-LX2.
Compd.
R
IC50
(μ
M)a
Compd.
R
IC50
(
μ
M)a
STM
–
16.5 ± 0.7
16
16.5 ± 1.7
3
–
47.4 ± 5.9
17
6.4 ± 0.4
4
5
–
–
43.3 ± 2.0
44.4 ± 3.3
18
19
16.3 ± 0.5
21.0 ± 0.5
6
12.3 ± 0.4
20
4.6 ± 0.1
7
8
32.4 ± 3.2
21
22
45.5 ± 2.0
21.5 ± 1.1
92.0 ± 11.4
9
11.9 ± 1.2
16.9 ± 0.5
23
24
55.9 ± 0.1
55.9 ± 3.3
10
11
12
13
27.6 ± 1.6
31.3 ± 0.2
11.5 ± 0.5
25
26
27
3.5 ± 0.1
11.1 ± 0.7
9.0 ± 0.4
14
v
17.6 ± 0.3
16.1 ± 1.8
Silybinb
–
151.1 ± 3.3
a
Data were expressed as means ± SD (n = 3).
b
Silybin was used as the positive control.
on the deposition of ECM by comparing with silybin, and significantly
increased inhibitory effect on the deposition of HA and HL, which were 3
to 8-fold more potent than STM. Especially, compound 20 not only
showed similar efficacy on Col I deposition as STM, but also manifested
more than 8-fold and 6-fold enhancement of inhibitory effect on the
concluded as: (a) the α, β-unsaturated lactone is crucial pharmacophore
to maintain the activity; (b) change of the double-bond leads to decrease
activity; (c) oxidation of the hydroxyl group is unfavorable; (d) the
acyloxys at C-1 position play a crucial role in the activity. These results
demonstrated that compounds 20 and 25 as promising anti-fibrotic
candidates showed obvious inhibitory activity on the proliferation of
HSC-LX2 and secretion of Col I, HA and HL.
deposition of HA and HL with IC50 values of 7.6 ± 0.6 and 3.3 ± 1.2 μM.
In summary, 26 derivatives of STM were synthesized and evaluated
for their cytotoxic activity against HSC-LX2. Eight derivatives showed
higher activity, and compound 25 was approximately 5-fold stronger
than that of STM. Compound 20 exhibited more than 8-fold increase in
inhibitory activity on the deposition of HA and 6-fold increase in HL
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
with IC50 values of 7.6 ± 0.6 and 3.3 ± 1.2 μM. The SARs can be
3

J.-P. Wang et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127994
Scheme 2. Reagents and conditions: EDCI, DMAP, appropriate carboxylic acid, dry CH2Cl2, r.t., 7 (84%), 9 (80%), 10 (66%), 11 (74%), 12 (87%), 13 (60%), 14
(73%), 15 (43%), 16 (74%), 17 (74%), 18 (67%), 19 (82%), 20 (83%), 21 (81%), 22 (71%) or succinic anhydride, DMAP, dry CH2Cl2, r.t., 8 (63%).
Scheme 3. Reagents and conditions: (a) CuI, dry DMF, Ar, 160 ◦C, S1 (77%), S3 (79%); (b) 1 M LiOH aqueous solution, 1,4-dioxane, r.t., 91%; (c) EDCI, DMAP, dry
CH2Cl2, r.t., 23 (81%), 24 (75%); (d) BBr3, dry CH2Cl2, Ar, ꢀ 78 ◦C, 79%.
Scheme 4. Reagents and conditions: EDCI, DMAP, appropriate carboxylic diacid, dry CH2Cl2, sealed tube, 50 ◦C, 25 (60%), 26 (40%), 27 (32%).
the work reported in this paper.
Table 2
Inhibitory effect of compounds 17, 20 and 25 on HA, HL and Col I deposition in
Acknowledgements
HSC-LX2.
Compd.
IC50
HA
(μ
M)a
This work was supported by the Yunnan Wanren Project
(YNWR–KJLJ–2019-002), and the Program of Yunling Scholarship, the
Youth Innovation Promotion Association, CAS (2020386), and the
Reserve Talents of Young and Middle-aged Academic and Technical
Leaders in Yunnan Province (T.Z. Li).
HL
Col I
STM
17
>60.0
18.6 ± 1.4
7.3 ± 2.1
3.3 ± 1.2
6.6 ± 2.9
83.7 ± 2.5
7.3 ± 1.7
21.2 ± 1.3
7.6 ± 0.6
14.7 ± 1.8
6.1 ± 1.9
20
25
17.9 ± 1.8
164.4 ± 1.4
16.2 ± 4.1
51.5 ± 11.1
Silybinb
Appendix A. Supplementary data
a
Data were expressed as means ± SD (n = 3).
b
Silybin was used as the positive control.
Supplementary data to this article can be found online at https://doi.
4

J.-P. Wang et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127994
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