Hydrogen sulfide releasing oridonin derivatives induce apoptosis through extrinsic and intrinsic pathways

Article abstract of DOI:10.1016/j.ejmech.2019.111978

Hydrogen sulfide (H₂S) has been recognized as the third endogenous signaling gasotransmitter following nitric oxide (NO) and carbon monoxide (CO), and exhibits antiproliferative activity against several cancer cells. In order to stably and controllably release H₂S, H₂S donating compound (ADT-OH) was used in the present study and 18H₂S releasing natural ent-kaurane diterpenoid oridonin derivatives were designed and synthesized. Most derivatives showed more potent antiproliferative activities than oridonin against HepG2 and K562 cell lines, while they were lack of sensitivity to HCT-116 and B16 cells. In particular, 12b showed the most potent antiproliferative activities against HepG2, HCT-116 and K562 cells with IC₅₀ values of 2.57, 5.81 and 0.95 μM, respectively. Through cell cycle analysis, 12b caused cell cycle arrest at S phase in K562 cells and G1 phase in HepG2 cells. In Hoechst 33258 staining assay, cell shrinkage and fragmentation of cell nuclei indicated apoptotic morphological changes. Considering the decline of mitochondrial membrane potential and changes in the levels of apoptosis-related proteins, 12b was shown to induce apoptosis through extrinsic and intrinsic apoptosis pathways.

Full text of DOI:10.1016/j.ejmech.2019.111978

European Journal of Medicinal Chemistry 187 (2020) 111978
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Hydrogen sulfide releasing oridonin derivatives induce apoptosis
through extrinsic and intrinsic pathways
Haonan Li a, Jiahui Mu a, Jianan Sun a, Shengtao Xu b, Weiwei Liu c, Fanxing Xu c,
,
*
Zhanlin Li a, Jinyi Xu b, Huiming Hua a, Dahong Li a
a Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University, 103 Wenhua Road, Shenyang, 110016, PR China
b State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing, 210009, PR China
c Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, 110016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Hydrogen sulfide (H2S) has been recognized as the third endogenous signaling gasotransmitter following
nitric oxide (NO) and carbon monoxide (CO), and exhibits antiproliferative activity against several cancer
cells. In order to stably and controllably release H2S, H2S donating compound (ADT-OH) was used in the
present study and 18H2S releasing natural ent-kaurane diterpenoid oridonin derivatives were designed
and synthesized. Most derivatives showed more potent antiproliferative activities than oridonin against
HepG2 and K562 cell lines, while they were lack of sensitivity to HCT-116 and B16 cells. In particular, 12b
showed the most potent antiproliferative activities against HepG2, HCT-116 and K562 cells with IC50
Received 23 September 2019
Received in revised form
12 December 2019
Accepted 16 December 2019
Available online 19 December 2019
Keywords:
ent-kaurane
values of 2.57, 5.81 and 0.95 mM, respectively. Through cell cycle analysis, 12b caused cell cycle arrest at S
phase in K562 cells and G1 phase in HepG2 cells. In Hoechst 33258 staining assay, cell shrinkage and
fragmentation of cell nuclei indicated apoptotic morphological changes. Considering the decline of
mitochondrial membrane potential and changes in the levels of apoptosis-related proteins, 12b was
shown to induce apoptosis through extrinsic and intrinsic apoptosis pathways.
Oridonin
Hydrogen sulfide
Antiproliferative activity
Apoptosis
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
inflammatory, anticancer, antibacterial and antimutagenic activ-
ities [8e17]. Oridonin (1, Fig. 1), a natural ent-kaurane diterpenoid
Natural products have made great contributions to drug design.
With the development of analytical techniques, more and more
active ingredients were isolated and identified from herbal medi-
cines, natural plants and their metabolites. Many novel structural
skeletons were provided for drug discovery, and relatively com-
plete quality control systems were also established [1e4].
Compared with combinatorial chemistry, natural products remain
active in drug discovery due to their prominent biological activities,
complex and diverse structures and distinctive stereogenic centers
which could better combine with corresponding targets in vivo [5].
In the past three decades, the percentage of natural products or
nature derived molecules has increased to 74% of all approved
chemical entities in anticancer domain [6,7].
from Rabdosia, was first isolated and identified by Fujita et al., in
1970 [18]. Further in-depth studies on pharmacology showed
potent anticancer effects t(8; 21) acute myeloid leukemic (AML)
cells [19], human pancreatic cancer PANC-1 cells [20], human os-
teosarcoma cells [21], p53-mutated colorectal cancer cells [22],
drug-resistant renal cell carcinoma (RCC) [23] and non-germinal
center B cell-like subtype of diffuse large B cell lymphoma (non-
GCB DLBCL) [24]. Although oridonin can be obtained commercially
and shows cytotoxicity for many cancer cells, it deserves further
structural modification due to its poor selectivity and low aqueous
solubility [25]. SAR studies indicated that unsaturated cyclo-
pentanone conjugated with extracyclic methylene was a pivotal
structure for antiproliferative activity, while the activity slumped
when cyclopentanone was cleaved or methylene was saturated
[26]. Furthermore, 14-hydroxy of oridonin was a prominent struc-
ture modification site. Most 14-O-derivatives showed maintained
or better antiproliferative activities toward several human cancer
cells [27,28].
As a rich source of ent-kaurane diterpenoids (Fig. 1), the genus
Rabdosia exhibits many biological activities, including anti-
* Corresponding author.
E-mail address: lidahong0203@163.com (D. Li).
https://doi.org/10.1016/j.ejmech.2019.111978
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
H. Li et al. / European Journal of Medicinal Chemistry 187 (2020) 111978
Fig. 1. The structures of natural ent-kaurane diterpenoids.
Due to the endogenous pathophysiological functions, small
mononuclear PBMC). Moreover, in-depth apoptosis related mech-
anisms of the most promising compound 12b which included cell
cycle arrest, morphological change, apoptosis induction, mito-
chondria membrane potentials decline and the expression of
apoptosis-related proteins were studied.
molecule donors have comprised a key pillar of drug design [29,30].
Before nitric oxide (NO) and carbon monoxide (CO) were famous
for endogenous bioactivity, hydrogen sulfide (H2S) was considered
to be a colorless and toxic gas with a strong smell of rotten eggs. In
recent years, it has been recognized as the third endogenous
signaling gasotransmitter following NO and CO, acting as a neuro-
modulator and neuroprotective agent [31,32]. Endogenous H2S
commonly generates through two specific pyridoxal-50-phosphate
2. Result and discussion
2.1. Chemistry
(PLP)-dependent enzymes, cystathionine-
g-lyase (CSE) and cys-
tathionine- -synthase (CBS), or biosynthesized by the synergistic
b
The intermediates 2 and 5 were synthesized according to our
previous report [57]. The H2S donating derivatives 7a-c were ob-
tained by substitution reactions of ADT-OH 6 with several brom-
hydrins (2-bromoethanol, 3-bromo-1-propanol and 6-bromo-1-
hexanol). By treatment of 1, 2 and 5 with corresponding anhy-
dride in the presence of TEA/DMAP, derivatives 8a-c and 9a-c were
got. Derivatives 8a-c and 9a-c were directly reacted with H2S
releasing 7a-c via esterification reaction to gain the target com-
pounds 10a-c, 11a-c, 12a-c, 13a-c, 14a-c and 15a-c. Among them,
compounds 12c and 15c were dimeric diterpenoid compounds. The
dimerization phenomenon would be due to the residual 6-bromo-
1-hexanol after silica column chromatography (see Schemes 1 and
2).
effects of 3-mercaptopyruvate sulfurtransferase (3-MST) and
cysteine aminotransferase (CAT) [33e35]. Increasing evidences
confirm the pathophysiological functions of H2S in atherosclerosis,
cytoprotection against oxidative stress, angiogenesis, ischemia-
reperfusion injury and so on [36e40]. In addition, recent studies
demonstrate the importance of H2S in biological processes of can-
cer which shows antiproliferative activities through EGFR/ERK/
MMP-2, PTEN/AKT, PI3K/Akt/mTOR and p38 MAPK/ERK1/2-COX-2
pathways [41e44]. This reactivation of programmed cell death by
delivery of H2S could be suggested as an effective approach for
cancer therapy. However, H2S is not suitable for clinical applications
directly owing to uncontrollability, high toxicity and short half-life.
To overcome these disadvantages, a number of H2S releasing agents
(H2S donors) have been developed, such as ADT-OH, thio-
2.2. Biological evaluation
benzamide, Jks, L-propargyl cysteine,
a-thioctic acid, GYY4137,
DATS, -cysteine and so on (Fig. 2A). These H2S donating com-
D
2.2.1. Antiproliferative activity and preliminary SAR
pounds could deliver H2S in sustained manner and prolong the
term of treating times [45e49]. With the development of H2S do-
nors, more and more H2S releasing derivatives have been designed
and synthesized. Some of them have entered into Phase I or Phase II
clinical trials [ [50e55], Fig. 2C]. Among them, compounds with 3H-
1,2-dithiole-3-thione show prominent cytoprotective properties [
[56], Fig. 2B].
In the present study, we designed and synthesized three series
of H2S releasing oridonin derivatives in which oridonin was con-
jugated with a H2S generating moiety (5-(4-hydroxyphenyl)-3H-
1,2-dithiole-3-thione, ADT-OH) via different anhydride linkers at
14-hydroxyl group. The antiproliferative activities of all the de-
rivatives were tested against five human cancer cell lines (human
hepatoma HepG2, breast cancer MCF-7, colorectal cancer HCT-116,
melanoma B16 and chronic myelogenous leukemia K562) and two
normal cell lines (human liver L-02 and peripheral blood
MTT assay and trypan blue assay were performed to evaluate the
antiproliferative activities of 18 synthetic derivatives (10a-c, 11a-c,
12a-c, 13a-c, 14a-c and 15a-c), lead compound oridonin (1) and H2S
donor ADT-OH (6). The IC50 values against five human cancer cells
(HepG2, MCF-7, HCT-116, B16 and K562) and two human normal
cells (L-02 and PBMC) were calculated and listed in Table 1.
Although the antiproliferative activities of most derivatives were
more potent than oridonin in HepG2 and K562 cell lines, they were
lack of sensitivity to HCT-116 and B16 cells. Only 12b was stronger
than oridonin with IC50 values of 5.81 and 12.94
exhibited only appropriate effect in MCF-7 cell line (16.15
which was similar to that of oridonin (13.85 M). In HepG2 and
m
M, while
mM)
m
K562 cells, compared with compounds containing carbonyl or hy-
droxyl group at C-1, acetylated derivatives (10e15b) usually
possessed more potent cytotoxicity, their IC50 values were univer-
sally below 6
mM. Furthermore, acetylated derivatives which

H. Li et al. / European Journal of Medicinal Chemistry 187 (2020) 111978
3
Fig. 2. The structures of H2S donors (A), compounds with 3H-1,2-dithiole-3-thione group (B) and H2S releasing drug candidates (C).
conjugated with succinic anhydride as a linker (10e12b) were
2.2.2. H2S release ability
stronger than those with glutaric anhydride (13e15b). However, it
will no longer be in line with this trend when acetyl group changed
to carbonyl or hydroxyl group at C-1. Comprehensive analysis of
antiproliferative data in HepG2, MCF-7 and K562 cells indicated
that more efficacious inhibitory activities were not observed with
the extension of carbon chain. It seemed that compounds with
medium-length carbon chains (n2 ¼ 3) showed better anti-
proliferative activities in general, yet compound 12b with long
carbon chains (n2 ¼ 6) still exhibited the most potent anti-
proliferative activity in all derivatives against HepG2, HCT-116 and
In order to observe H2S generation capability of all hybrids, the
methylene blue (MBþ) method was carried out and H2S-releasing
curves were calculated and summarized in Fig. 3. The delivery of
H2S elevated as time increased and reached the peak value at 15 or
20 min, indicating all H2S-donating compounds could release H2S
in a relatively slow manner. Considering the cytotoxicity and po-
tential SAR of the derivatives, it did not seem that the more H2S
released, the better inhibitory activity exhibited. Compounds con-
tained a hydroxyl group at C-1 (10e12c, 14c and 15c) showed
higher H2S releasing capacity (15e20 mM) when compared with 1-
K562 cells with IC50 values of 2.57, 5.81 and 0.95
m
M, respectively.
acetylated and 1-oxo derivatives. Furthermore, the length of n1 and
n2 also seemed to be another decisive factor to the release of H2S.
With the increased length of carbon chains, higher H2S release was
observed. It seemed that a certain amount of H2S was sufficient for
antiproliferative treatments.
Cultivated with 18 derivatives, the cell viability of L-02 and
PBMC cells was not significantly affected which indicated good
selectivity of inhibitory activity between tumor and normal cells.
Besides efficacious antiproliferative activity, 12b also showed
nearly 20-fold lower inhibitory activities against L-02 and PBMC
cells than those of malignant cells.

4
H. Li et al. / European Journal of Medicinal Chemistry 187 (2020) 111978
responsible for the inhibition of tumor cell proliferation.
2.2.4. Hoechst 33258 staining indicated that 12b induced pyknosis
in K562 cells
Apoptosis is prescribed as the major killing way of tumor cells
after antitumor drugs treatment. Hence, we verified whether 12b
induces K562 cell apoptosis. The occurrence of apoptosis usually
concomitant of deviant morphological characteristics included cell
shrinkage, chromatin condensation, cell membrane rupture and
fragmentation of cell nuclei [58]. In order to preliminarily verify the
occurrence of apoptosis, Hoechst 33258 staining assay and fluo-
rescence microscope were used to observe the morphology of
aberrant. Three random microscopic fields per sample of approxi-
mately 200 nuclei were counted and expressed as the percentage of
apoptotic nuclei in histogram. As shown in Fig. 5, compared with
vehicle control (DMSO), some fragment could be observed in
K562 cells incubated with 12b (1.0, 2.0 and 4.0 mM) for 24 h and
48 h, demonstrating the occurrence of chromatin shrink and nu-
clear fragmentation, and showed obvious concentration
dependence.
2.2.5. Flow cytometric analysis suggested that 12b induced
apoptosis in K562 and HepG2 cells
Apoptosis is essential to multicellular organisms development,
it will be triggered when a cell commits suicide. Apoptosis is also
referred to programmed cell death and follows a controlled pre-
dictable routine, while cancer cells can suppress apoptosis and
proliferate incessantly. Hence, great efforts have been exerted to
induce cancer cells apoptosis by developing chemical entities. To
evaluate the effects of 12b on induction of apoptosis and quantify
the percentage of apoptotic cells, an annexin V-FITC/PI binding
Scheme 1. Synthetic routes of the intermediates. Reagents and conditions: (a) Jones
reagent, acetone, 0 ꢀC, 0.5 h; (b) 2,2-dimethoxypropane, acetone, TsOH, reflux, 2 h; (c)
Ac2O, TEA, DMAP, DCM, rt, 5 h; (d) 10% HCl, THF, rt, 4 h; (e) correspondent bro-
moalcohol, K2CO3, acetone, reflux, 6 h.
2.2.3. 12b caused cell cycle arrest at S phase in K562 cells and G1
phase in HepG2 cells
assay was carried out. Cultivated with 12b (0, 0.50, 1.0 and 2.0 mM),
Cell cycle plays an important role in cell life activities due to its
regulation of division and duplication of DNA. Dysregulation of cell
cycle may contribute to persistent tumor cell proliferation.
Considering many anticancer compounds showed cytotoxicity
which was associated with cell cycle arrest at a particular check-
point, the influence of 12b to cell cycle arrest was analyzed and
DNA content of cell nuclei which stained by propidium iodide (PI)
was monitored by flow cytometry. As shown in Fig. 4AeB, 12b
arrested K562 cell cycle at S phase and HepG2 cell at G1 phase. In
contrast, oridonin caused cell cycle arrest of K562 and HepG2 cells
both at G1 phase. In short, 12b-mediated cell cycle arrest could be
the percentage of K562 and HepG2 cells underwent apoptosis
which was tested via flow cytometry. As shown in Fig. 6, apoptotic
ratios of K562 and HepG2 cells (annexin Vþ) obviously increased in
a
concentration-dependent manner. Consequently, 12b was
capable of inducing apoptosis both in K562 and HepG2 cells.
2.2.6. 12b induced mitochondrial membrane potentials decline
The decline of mitochondrial membrane potentials is known as
the classic characteristics of early apoptosis process, which is also
critical to the apoptotic pathway. To ensure mitochondrial integrity
and biological function, the maintenance of mitochondrial
Scheme 2. Synthesis of H2S donating derivatives 10a-c, 11a-c, 12a-c, 13a-c, 14a-c and 15a-c. Reagents and conditions: (a) correspondent anhydride, TEA, DMAP, DCM, rt, 7 h; (b) 7a-
c, EDCI, DMAP, DCM, rt, overnight.

H. Li et al. / European Journal of Medicinal Chemistry 187 (2020) 111978
5
Table 1
Antiproliferative activities (IC50a
m
M) of 18 target derivatives, parental compounds and H2S donors against five human cancer and two normal cell lines.
Compd.
IC50 (mM)
HepG2
MCF-7
HCT-116
B16
K562
L-02
PBMC
1
2
5
6
8.95 0.68
10.51 0.73
11.94 1.02
>50
13.85 1.23
15.39 1.50
19.82 1.36
>50
25.44 1.23
19.52 1.30
34.80 2.56
27.51 1.64
12.47 1.03
>50
24.72 1.53
20.81 1.37
16.15 1.02
>50
15.75 1.23
>50
11.73 1.01
10.67 0.75
13.48 0.86
>50
>50
13.97 0.71
>50
34.88 1.59
23.70 1.51
>50
36.85 1.54
22.06 1.41
5.81 1.17
>50
37.47 1.66
>50
42.61 2.95
27.48 1.60
>50
24.89 1.32
25.46 1.08
32.57 1.33
>50
>50
>50
>50
>50
39.94 1.61
>50
>50
16.38 1.25
12.94 0.88
11.09 0.86
>50
>50
>50
27.50 0.92
>50
>50
>50
>50
4.57 0.20
4.17 0.17
5.21 0.19
>50
21.59 1.46
28.54 1.31
25.39 1.78
>50
39.64 1.20
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
10a
11a
12a
13a
14a
15a
10b
11b
12b
13b
14b
15b
10c
11c
12c
13c
14c
15c
5-Fu
7.52 0.72
5.01 0.26
10.47 0.73
7.65 0.57
2.58 0.20
9.33 0.65
5.89 0.36
6.30 0.58
2.57 0.12
4.78 0.40
3.57 0.31
15.60 1.52
5.36 0.28
4.45 0.34
12.26 1.04
6.60 0.53
5.93 0.47
10.05 0.55
32.57 1.98
8.79 0.21
7.12 0.33
5.87 0.43
5.75 0.12
3.59 0.09
8.89 0.25
2.85 0.10
2.44 0.13
0.95 0.06
1.86 0.15
5.93 0.18
13.88 0.46
4.57 0.21
2.17 0.15
14.86 0.94
4.89 0.34
4.19 0.22
13.47 0.68
3.94 0.17
>50
42.16 1.58
27.18 1.72
>50
29.93 1.38
22.31 1.72
17.59 0.93
26.84 1.40
33.51 1.85
>50
33.96 2.55
26.54 1.70
>50
39.11 1.42
40.92 1.77
>50
17.86 1.35
14.67 1.21
>50
18.30 1.42
16.63 1.35
>50
38.35 2.26
34.16 1.73
>50
26.65 1.92
6.86 0.37
12.62 1.06
>50
aIC50: Half inhibitory concentrations measured by the MTT assay and trypan blue assay. The values are expressed as averages standard deviations of three independent
experiments.
membrane potentials is crucial [59]. Cells pretreated with 12b
(0.50, 1.0 and 2.0 M) or DMSO (vehicle control) were stained with
binding to DR4 and DR5 death receptors [61]. The higher binding
level of DR4/DR5 to TRAIL, the more apoptosis would be observed.
To analyze the mediative mechanism of 12b on apoptosis, the
Human Apoptosis Array kit was used to detect the relative
expression of 35 apoptosis-related proteins. Each dot represents
which antibodies were shown in the Human Apoptosis Array co-
ordinates (Fig. S1), and the original image of Human Apoptosis
Array kit was shown in Fig. S2. As shown in Fig. 8, the down-
regulated level of procaspase-3 and the increase in cleaved
caspase-3 demonstrated that 12b could trigger caspase-dependent
apoptosis pathway. Furthermore, the level of pro-apoptotic protein
m
the lipophilic cationic fluorescent probe 5,50,6,60-tetrachloro-
1,10,3,30-tetraethylbenzimidazol-carbocyanine iodide (JC-1), and
then the effects of 12b on mitochondrial membrane potentials were
detected by flow cytometry. As shown in Fig. 7A and B, mito-
chondrial membrane potentials of K562 and HepG2 cells treated
with 12b was significantly decreased in a concentration-dependent
manner. These results indicated that 12b influenced the perme-
ability of mitochondrial membrane and eventually led to apoptosis.
Bax was elevated after the treatment with 12b (1 mM) for 24 h,
2.2.7. 12b treatment activated extrinsic and intrinsic apoptosis
pathways followed by regulation of apoptosis-related protein levels
Apoptosis is a process of autonomous cell death that is strictly
controlled by multiple genes, which plays an important role in the
evolution of organisms, the homeostasis of internal environment
and the development of multiple systems. Extrinsic and intrinsic
apoptosis pathways generally mediate by several apoptosis related
proteins. When the expression of Bax, Bcl-2 and other factors
changes, the opening of permeability transition pore on outer
mitochondrial membrane will be triggered which concomitantly
delivers cytochrome c. Once cytochrome c releases into outer
cytoplasm, it will form apoptotic bodies with Apaf-1 and then
activate downstream apoptotic protein caspase-9. Activated
caspase-9 then cleaves downstream procaspase-3 to cut it into
active for activated caspase-3 immediately. Finally, caspase-3 cau-
ses the execution of intrinsic apoptosis by cleaving targeted cellular
proteins. IAPs (Inhibitor of apoptosis proteins) are a group of
apoptotic regulatory proteins which mediate intracellular or
extracellular apoptosis pathways by inhibiting caspase activity
directly or indirectly, or preventing the release of mitochondrial
apoptotic precursors. Up to now, eight apoptotic inhibitory proteins
have been found: c-IAP1, c-IAP2, NAIP, Survivin, XIAP, Bruce, ILP-2
and Livin. The activation of caspase family can be inhibited by c-
IAPs, Survivin and XIAP through BIR structural domain. Hence,
inhibiting IAPs is one of the most effective method to prevent
tumorigenesis [60]. Moreover, TRAIL induces apoptosis mainly by
suggesting the participation of the endogenous apoptosis pathway.
Compared with the control group, the protein levels of c-IAP1, c-
IAP2, Survivin, XIAP and Livin were significantly decreased. Hence,
we reasoned that 12b could inhibit the expression of related pro-
teins in IAPs to ensure the activation of caspase pathway and ulti-
mately induce apoptosis through mitochondria-dependent
intrinsic pathway. Furthermore, the expressions of DR4 and DR5
were found to be elevated compared with the control group, indi-
cating the extrinsic apoptotic pathway would also be associated
with apoptosis induced by 12b (Fig. 9).
3. Conclusion
Overall, by using oridonin as lead compound, we designed and
synthesized 18H2S-delivering derivatives in the present study. Five
tumor and two normal cell lines were selected to test the anti-
proliferative activities of all derivatives. Most derivatives were
sensitive to HepG2 and K562 cells and several compounds showed
improved effects than parent compound oridonin. In particular, 12b
not only showed the most potent antiproliferative activities against
HepG2, HCT-116 and K562 cells with IC50 values of 2.57, 5.81 and
0.95 mM respectively, but also exhibited lower toxicity to two hu-
man normal cells. Further in-depth cellular mechanism exploration
showed that 12b caused cell cycle arrest at S phase in K562 cells
and G1 phase in HepG2 cells. 12b also reduced mitochondrial

6
H. Li et al. / European Journal of Medicinal Chemistry 187 (2020) 111978
4.1.1. General procedures to synthesize target derivatives
1-oxo intermediate 2, acetylated derivative 5 and ADT-OH 6
were synthesized according to our previous report [57]. Corre-
spondent bromoalcohol was added into anhydrous acetone in the
presence of sodium carbonate and ADT-OH 6. After reflux for 8 h,
the reaction mixture was filtered and washed with acetone, and
then the filtrate was evaporated in vacuo, the crude product was
purified by silica column chromatography (PE/EA 4:1, v/v) to yield
7a-c.
Compounds 1, 2 or 5 (0.50 mmol) were dissolved in 6 mL
anhydrous DCM in the presence of TEA (2.5 mmol), DMAP
(0.25 mmol) and succinic anhydride or glutaric anhydride
(1.0 mmol) and stirred at room temperature for 6e8 h. The mixture
was extracted with DCM (3 ꢁ 20 mL), washed with brine, dried over
anhydrous Na2SO4, and evaporated in vacuo to yield intermediates
8a-c and 9a-c. Intermediates 10a-c or 15a-c (0.20 mmol) were
dissolved in anhydrous DCM and directly reacted with 7a-c
(0.22 mmol) under the condition of EDCI (0.60 mmol) and DMAP
(0.10 mmol). After stirring overnight at room temperature, the re-
action was quenched and extracted with DCM (3 ꢁ 20 mL), washed
with brine, dried over anhydrous Na2SO4, and evaporated in vacuo.
Finally, H2S-releasing derivatives 10a-c, 11a-c, 12a-c, 13a-c, 14a-c
and 15a-c were obtained by flash column chromatography (DCM/
MeOH 100:1e200:1, v/v).
4.1.1.1. Compound 10a.
(4aR,5S,6S,6aR,9S,11aS,11bS,14R)-5,6-dihydroxy-4,4-dimethyl-8-
methylene-1,7-dioxododecahydro-1H-6,11b-(epoxymethano)-
6a,9-methanocyclohepta[a]naphthalen-14-yl(2-(4-(3-thioxo-3H-
1,2-dithiol-5-yl)phenoxy)ethyl) succinate. Orange red solid, yield:
37.6%, m.p. 57e59 ꢀC. 1H NMR (CDCl3, 400 MHz),
d (ppm): 7.63, 7.00
(each 2H, d, JA ¼ JB ¼ 8.8 Hz, AreH), 7.40 (1H, s, 800-CH), 6.25 (1H, s,
17-CH2), 5.93 (1H, s, 14-CH), 5.62 (1H, s, 17-CH2), 5.36 (1H, d,
J ¼ 11.9 Hz, 6-OH), 4.46 (2H, dd, J ¼ 9.6, 4.7 Hz, 50-CH2), 4.23 (2H, t,
J ¼ 4.7 Hz, 60-CH2), 4.29, 4.01 (each 1H, d, J ¼ 10.7 Hz, 20-CH2), 3.75
(1H, dd, J ¼ 11.7, 8.9 Hz, 6-CH), 3.10 (1H, d, J ¼ 9.4 Hz, 13-CH), 2.69
(2H, m, 30-CH2), 2.58 (2H, m, 20-CH2), 2.54e2.41 (2H, m, eCH2),
2.33, 2.20 (each 1H, m, eCH2), 1.93 (2H, m, eCH2), 1.77e1.57 (4H, m,
eCH2), 1.31 (2H, m, eCH2), 1.19 (3H, s, 18-CH3), 0.98 (3H, s, 19-CH3);
13C NMR (CDCl3, 100 MHz),
d (ppm): 215.48, 211.51, 204.79, 172.09,
Fig. 3. In vitro H2S release of 10e15a (A), 10e15b (B) and 10e15c (C) at a dose of
0.1 mM using TCEP as accelerator. The absorbance of each resulting mixture was
detected by UVeVis spectrometer (670 nm). The amount of H2S was calculated
through the Na2S standard curve. All of the data were the mean SD of three inde-
pendent tests.
170.82, 161.93, 149.50, 134.83, 128.62 ( ꢁ 2), 122.08, 115.61 ( ꢁ 2),
96.85, 74.50, 73.39, 66.13, 64.92, 62.73, 61.32, 59.75, 50.73, 48.54,
41.47, 38.38, 35.73, 32.85, 30.51, 30.05, 29.49, 28.83, 23.17, 19.17;
HRMS (ESI) m/z calcd for C35H38O10S3 [M ꢂ H]- 713.1549, found
713.1566.
membrane potentials in both K562 and HepG2 cells and affected
apoptosis-related proteins through extrinsic and intrinsic path-
ways. In conclusion, 12b with potent antiproliferative activities,
deserved further investigation as a potential anticancer candidate.
4.1.1.2. Compound 11a.
(4aR,5S,6S,6aR,9S,11aS,11bS,14R)-5,6-dihydroxy-4,4-dimethyl-8-
methylene-1,7-dioxododecahydro-1H-6,11b-(epoxymethano)-
6a,9-methanocyclohepta[a]naphthalen-14-yl(3-(4-(3-thioxo-3H-
1,2-dithiol-5-yl)phenoxy)propyl) succinate. Orange red solid, yield:
36.2%, m.p. 60e62 ꢀC. 1H NMR (CDCl3, 400 MHz),
d (ppm): 7.62, 6.98
(each 2H, d, JA ¼ JB ¼ 8.7 Hz, AreH), 7.39 (1H, s, 800-CH), 6.24 (1H, s,
17-CH2), 5.92 (1H, s, 14-CH), 5.62 (1H, s, 17-CH2), 5.37 (1H, d,
J ¼ 11.3 Hz, 6-OH), 4.31e4.25 (3H, m, 50-CH2, 20-CH2), 4.10 (2H, t,
J ¼ 6.1 Hz, 70-CH2), 4.01 (1H, d, J ¼ 10.7 Hz, 20-CH2), 3.76 (1H, m, 6-
CH), 3.09 (1H, d, J ¼ 9.4 Hz, 13-CH), 2.61 (2H, m, 30-CH2), 2.58 (2H,
m, 20-CH2), 2.53e2.40 (2H, m, eCH2), 2.33, 2.20 (each 1H, m, eCH2),
2.13 (2H, t, J ¼ 6.1 Hz, 20-CH2), 1.93 (2H, m, eCH2), 1.76e1.56 (4H, m,
eCH2), 1.31 (2H, m, eCH2), 1.19 (3H, s, 18-CH3), 0.98 (3H, s, 19-CH3);
4. Experimental
4.1. Chemistry
Chemical materials and reagents were obtained from commer-
cial suppliers, the preparation of anhydrous solvents was based on
standard methods. 1H and 13C NMR spectra were determined with
Bruker 400 MHz spectrometer in the solvent of CDCl3 (TMS as in-
ternal standard): the values of the chemical shifts were reported in
13C NMR (CDCl3, 100 MHz),
d (ppm): 215.17, 211.52, 204.79, 172.99,
172.14, 170.92, 162.13, 149.48, 134.68, 128.62 ( ꢁ 2), 124.31, 122.08,
115.49 ( ꢁ 2), 96.86, 74.51, 73.39, 64.92, 64.82, 61.42, 61.32, 59.78,
50.73, 48.54, 41.46, 38.39, 35.74, 32.86, 30.51, 30.04, 29.53, 28.87,
28.42, 23.18, 19.16; HRMS (ESI) m/z calcd for C36H40O10S3 [M ꢂ H]-
d
values (ppm) and the coupling constants (J) in Hz. High resolution
mass spectra (HR-MS) were analyzed on Agilent Q-TOF B.05.01
(B5125.2).

H. Li et al. / European Journal of Medicinal Chemistry 187 (2020) 111978
7
727.1706, found 727.1711.
4.1.1.6. Compound 15a.
(4aR,5S,6S,6aR,9S,11aS,11bS,14R)-5,6-dihydroxy-4,4-dimethyl-8-
methylene-1,7-dioxododecahydro-1H-6,11b-(epoxymethano)-
6a,9-methanocyclohepta[a]naphthalen-14-yl(6-(4-(3-thioxo-3H-
4.1.1.3. Compound 12a.
(4aR,5S,6S,6aR,9S,11aS,11bS,14R)-5,6-dihydroxy-4,4-dimethyl-8-
methylene-1,7-dioxododecahydro-1H-6,11b-(epoxymethano)-
6a,9-methanocyclohepta[a]naphthalen-14-yl(6-(4-(3-thioxo-3H-
1,2-dithiol-5-yl)phenoxy)hexyl) succinate. Orange red solid, yield:
1,2-dithiol-5-yl)phenoxy)hexyl) glutarate. Orange red solid, yield:
34.2%, m.p. 69e70 ꢀC. 1H NMR (CDCl3, 400 MHz),
d
(ppm): 7.61, 6.96
(each 2H, d, JA ¼ JB ¼ 8.7 Hz, AreH), 7.40 (1H, s, 800-H), 6.25 (1H, s,
17-CH2), 5.90 (1H, s, 14-CH), 5.62 (1H, s, 17-CH2), 5.37 (1H, d,
J ¼ 11.7 Hz, 6-OH), 4.30 (1H, d, J ¼ 10.6 Hz, 20-CH2), 4.08 (2H, t,
J ¼ 6.7 Hz, 60-CH2), 4.05e4.00 (3H, m, 110-CH2, 20-CH2), 3.77 (1H, t,
J ¼ 10.0 Hz, 6-CH), 3.10 (1H, d, J ¼ 9.4 Hz, 13-CH), 2.59 (2H, m, 20-
CH2), 2.58e2.40 (2H, m, eCH2), 2.21 (1H, dd, J ¼ 13.8, 4.2 Hz, eCH2),
1.99e1.78 (8H, m, eCH2), 1.31 (2H, m, eCH2), 1.19 (3H, s, 18-CH3),
31.2%, m.p. 65e67 ꢀC. 1H NMR (CDCl3, 400 MHz),
d (ppm): 7.61, 6.96
(each 2H, d, JA ¼ JB ¼ 8.7 Hz, AreH), 7.40 (1H, s, 800-CH), 6.25 (1H, s,
17-CH2), 5.92 (1H, s, 14-CH), 5.62 (1H, s, 17-CH2), 5.38 (1H, d,
J ¼ 11.1 Hz, 6-OH), 4.29 (1H, d, J ¼ 10.7 Hz, 20-CH2), 4.08 (2H, m, 50-
CH2), 4.05e3.99 (3H, m, 100-CH2, 20-CH2), 3.77 (1H, t, J ¼ 9.4 Hz, 6-
CH), 3.10 (1H, d, J ¼ 9.4 Hz,13-CH), 2.59 (2H, m, 20-CH2), 2.55 (2H, m,
30-CH2), 2.52e2.40 (2H, m, eCH2), 2.33, 2.21 (each 1H, m, eCH2),
1.94 (2H, m, eCH2), 1.89e1.56 (10H, m, eCH2), 1.31 (2H, m, eCH2),
1.19 (3H, s, 18-CH3), 0.99 (3H, s, 19-CH3); 13C NMR (CDCl3, 100 MHz),
1.00 (3H, s, 19-CH3); 13C NMR (CDCl3, 100 MHz),
d (ppm): 215.28,
211.51, 204.82, 172.89, 171.40, 162.51, 149.48, 134.57, 128.58 ( ꢁ 2),
124.00, 121.98, 115.46 ( ꢁ 2), 96.92, 74.47, 73.31, 68.26, 64.93, 64.38,
61.29, 59.89, 50.74, 48.57, 41.49, 38.44, 35.77, 33.53, 33.14, 32.86,
30.52, 30.05, 29.69, 28.93, 28.51, 25.64, 23.19, 22.67, 19.79, 19.13,
14.10; HRMS (ESI) m/z calcd for C40H48O10S3 [M ꢂ H]- 783.2332,
found 783.2305.
d
(ppm): 215.08, 211.57, 204.80, 173.15, 172.26, 170.99, 162.52,
149.49, 134.58, 129.21, 128.60 ( ꢁ 2), 124.03, 122.04, 115.47 ( ꢁ 2),
96.86, 74.56, 73.36, 66.24, 64.92, 64.78, 61.31, 59.83, 50.77, 48.56,
41.45, 38.42, 35.76, 32.86, 30.51, 30.04, 29.69, 29.57, 28.92, 28.46,
25.63, 23.20, 19.16; HRMS (ESI) m/z calcd for C39H46O10S3 [M ꢂ H]-
769.2175, found 769.2182.
4.1.1.7. Compound 10b.
(1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1-acetoxy-5,6-dihydroxy-4,4-
dimethyl-8-methylene-7-oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl(2-(4-(3-
thioxo-3H-1,2-dithiol-5-yl)phenoxy)ethyl) succinate. Orange red
solid, yield: 28.3%, m.p. 63e64 ꢀC. 1H NMR (CDCl3, 400 MHz),
4.1.1.4. Compound 13a.
(4aR,5S,6S,6aR,9S,11aS,11bS,14R)-5,6-dihydroxy-4,4-dimethyl-8-
methylene-1,7-dioxododecahydro-1H-6,11b-(epoxymethano)-
6a,9-methanocyclohepta[a]naphthalen-14-yl(2-(4-(3-thioxo-3H-
1,2-dithiol-5-yl)phenoxy)ethyl) glutarate. Orange red solid, yield:
d
(ppm): 7.63, 7.00 (each 2H, d, JA ¼ JB ¼ 8.6 Hz, AreH), 7.40 (1H, s,
800-CH), 6.16 (1H, s,17-CH2), 6.07 (1H, d, J ¼ 10.8 Hz, 6-OH), 5.88 (1H,
s, 14-CH), 5.53 (1H, s, 17-CH2), 4.62 (1H, dd, J ¼ 11.2, 5.5 Hz, 1-CH),
4.46 (2H, m, 50-CH2), 4.28, 4.18 (each 1H, d, JA ¼ JB ¼ 10.6 Hz, 20-
CH2), 4.23 (2H, t, J ¼ 4.6 Hz, 60-CH2), 3.77 (1H, m, 6-CH), 3.14 (1H,
d, J ¼ 9.2 Hz, 13-CH), 2.65 (2H, m, 20-CH2), 2.61 (4H, m, eCH2), 2.00
(3H, s, eCH3), 1.13 (6H, s, 18,19-CH3); 13C NMR (CDCl3, 100 MHz),
28.4%, m.p. 59e61 ꢀC. 1H NMR (CDCl3, 400 MHz),
d (ppm): 7.62, 6.98
(each 2H, d, JA ¼ JB ¼ 8.8 Hz, AreH), 7.39 (1H, s, 800-H), 6.23 (1H, s,
17-CH2), 5.90 (1H, s, 14-CH), 5.60 (1H, s, 17-CH2), 5.35 (1H, d,
J ¼ 11.6 Hz, 6-OH), 4.44 (2H, t, J ¼ 4.8 Hz, 60-CH2), 4.28, 4.01 (each
1H, d, J ¼ 10.6 Hz, 20-CH2), 4.22 (2H, t, J ¼ 4.8 Hz, 70-CH2), 4.06 (1H,
brs, eOH), 3.74 (1H, dd, J ¼ 10.8, 9.0 Hz, 6-CH), 3.08 (1H, d,
J ¼ 9.3 Hz, 13-CH), 2.59e2.42 (2H, m, 30-CH2), 2.39 (2H, t, J ¼ 7.2 Hz,
40-CH2), 2.32 (2H, t, J ¼ 7.2 Hz, 20-CH2), 2.20 (1H, dd, J ¼ 13.7, 4.6 Hz,
eCH2), 1.93 (2H, m, eCH2), 1.72, 1.61 (each 1H, m, eCH2), 1.31 (2H,
m, eCH2), 1.18 (3H, s, 18-CH3), 0.98 (3H, s, 19-CH3); 13C NMR (CDCl3,
d
(ppm): 215.42, 206.31, 171.93, 170.86, 169.84, 161.69, 149.44,
134.82, 128.63 ( ꢁ 2), 124.69, 120.64, 115.58 ( ꢁ 2), 95.92, 76.07,
75.94, 75.38, 74.19, 66.10, 63.53, 62.78, 61.63, 59.98, 53.79, 41.14,
39.75, 38.15, 33.57, 32.44, 30.24, 29.70, 29.51, 28.78, 25.16, 21.62,
21.53, 18.10; HRMS (ESI) m/z calcd for C37H42O11S3 [M ꢂ H]-
757.1811, found 757.1839.
100 MHz),
d (ppm): 215.20, 211.48, 204.81, 172.80, 172.70, 171.33,
161.74, 149.50, 134.80, 128.65 ( ꢁ 2), 124.66, 122.00, 115.58 ( ꢁ 2),
96.85, 74.37, 73.33, 66.17, 64.93, 62.33, 61.30, 59.81, 50.71, 48.55,
41.51, 38.40, 35.75, 33.38, 32.92, 32.84, 30.51, 30.05, 23.17, 19.67,
19.14; HRMS (ESI) m/z calcd for C36H40O10S3 [M ꢂ H]- 727.1706,
found 727.1691.
4.1.1.8. Compound 11b.
(1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1-acetoxy-5,6-dihydroxy-4,4-
dimethyl-8-methylene-7-oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl(3-(4-(3-
thioxo-3H-1,2-dithiol-5-yl)phenoxy)propyl) succinate. Orange red
solid, yield: 39.0%, m.p. 66e68 ꢀC. 1H NMR (CDCl3, 400 MHz),
4.1.1.5. Compound 14a.
(4aR,5S,6S,6aR,9S,11aS,11bS,14R)-5,6-dihydroxy-4,4-dimethyl-8-
methylene-1,7-dioxododecahydro-1H-6,11b-(epoxymethano)-
6a,9-methanocyclohepta[a]naphthalen-14-yl(3-(4-(3-thioxo-3H-
1,2-dithiol-5-yl)phenoxy)propyl) glutarate. Orange red solid, yield:
d
(ppm): 7.62, 6.98 (each 2H, d, JA ¼ JB ¼ 8.6 Hz, AreH), 7.40 (1H, s,
800-CH), 6.15 (1H, s,17-CH2), 6.08 (1H, d, J ¼ 10.8 Hz, 6-OH), 5.88 (1H,
s, 14-CH), 5.52 (1H, s, 17-CH2), 4.62 (1H, dd, J ¼ 11.2, 5.4 Hz, 1-CH),
4.28 (3H, m, 20-CH2, 50-CH2), 4.18 (1H, d, J ¼ 10.6 Hz, 20-CH2), 4.10
(2H, t, J ¼ 6.0 Hz, 70-CH2), 3.78 (1H, m, 6-CH), 3.14 (1H, d, J ¼ 9.9 Hz,
13-CH), 2.61 (2H, m, 20-CH2), 2.15 (2H, m, 30-CH2),1.99 (3H, s, eCH3),
1.47 (2H, m, eCH2), 1.13 (6H, s, 18,19-CH3); 13C NMR (CDCl3, 100 M
25.5%, m.p. 62e63 ꢀC. 1H NMR (CDCl3, 400 MHz),
d (ppm): 7.61, 6.97
(each 2H, d, JA ¼ JB ¼ 8.7 Hz, AreH), 7.39 (1H, s, 800-H), 6.24 (1H, s,
17-CH2), 5.90 (1H, s, 14-CH), 5.61 (1H, s, 17-CH2), 5.37 (1H, d,
J ¼ 11.6 Hz, 6-OH), 4.31e4.24 (3H, m, 60-CH2, 20-CH2), 4.10 (2H, t,
J ¼ 6.2 Hz, 80-CH2), 4.02 (1H, d, J ¼ 10.6 Hz, 20-CH2), 3.74 (1H, dd,
J ¼ 11.5, 9.0 Hz, 6-CH), 3.08 (1H, d, J ¼ 9.4 Hz,13-CH), 2.59e2.40 (2H,
m, eCH2), 2.37e2.29 (5H, m, eCH2), 2.13 (2H, t, J ¼ 6.1 Hz, 20-CH2),
1.90 (4H, m, eCH2), 1.31 (2H, m, eCH2), 1.18 (3H, s, 18-CH3), 0.99
Hz),
d (ppm): 215.15, 206.07, 172.96, 171.95, 170.92, 169.83, 162.09,
149.44, 134.67, 128.62 ( ꢁ 2), 124.32, 120.63, 115.47 ( ꢁ 2), 95.92,
75.96, 75.37, 74.16, 64.80, 63.53, 61.62, 61.48, 59.98, 53.78, 41.12,
39.75, 38.14, 33.56, 32.43, 30.24, 29.70, 29.54, 28.83, 28.40, 25.16,
21.61, 21.53, 18.08; HRMS (ESI) m/z calcd for C38H44O11S3 [M ꢂ H]-
771.1968, found 771.1999.
(3H, s, 19-CH3); 13C NMR (CDCl3, 100 MHz),
d (ppm): 215.14, 211.47,
204.80, 172.99, 172.76, 171.34, 162.12, 149.49, 134.67, 128.62 ( ꢁ 2),
124.31, 122.00, 115.48 ( ꢁ 2), 96.92, 74.39, 73.33, 64.94, 64.83, 61.30,
61.03, 59.83, 50.71, 48.55, 41.50, 38.41, 35.75, 33.48, 33.05, 32.86,
30.52, 30.05, 28.46, 23.18, 19.74, 19.14; HRMS (ESI) m/z calcd for
4.1.1.9. Compound 12b.
(1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1-acetoxy-5,6-dihydroxy-4,4-
dimethyl-8-methylene-7-oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl(6-(4-(3-
C
37H42O10S3 [M ꢂ H]- 741.1862, found 741.1845.

8
H. Li et al. / European Journal of Medicinal Chemistry 187 (2020) 111978
Fig. 4. 12b significantly induced cell cycle arrest. (A) Effect of 12b on cell cycle distribution of K562 cells. (B) Effect of 12b on cell cycle distribution of HepG2 cells. (C) Effect of
oridonin on cell cycle distribution of K562 cells. (D) Effect of oridonin on cell cycle distribution of HepG2 cells. After the treatment with 12b or oridonin (0, 0.50, 1.0 and 2.0 M) for
m
48 h, K562 or HepG2 cells were stained with PI and cell cycle distribution was investigated by flow cytometer. Data are represented as mean SD of three independent experiments.
*p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group.

H. Li et al. / European Journal of Medicinal Chemistry 187 (2020) 111978
9
Fig. 5. Effects of 12b on morphology of K562 cells. (A) Cell morphology after 24 h of 12b treatment. (B) Cell morphology after 48 h of 12b treatment. K562 cells were incubated with
12b (1.0, 2.0 and 4.0 M) for 24 h and 48 h and then stained with Hoechst 33258 to monitor apoptosis under the fluorescence microscope. The red arrows were pointed to K562 cells
m
with chromatin condensation and fragmentation of cell nuclei. Data are represented as mean SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001
compared with control group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
thioxo-3H-1,2-dithiol-5-yl)phenoxy)hexyl) succinate. Orange red
solid, yield: 29.6%, m.p. 71e73 ꢀC. 1H NMR (CDCl3, 400 MHz),
(epoxymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl(2-
(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)ethyl) glutarate. Orange
red solid, yield: 33.6%, m.p. 65e67 ꢀC. 1H NMR (CDCl3, 400 MHz),
d
(ppm): 7.61, 6.96 (each 2H, d, JA ¼ JB ¼ 8.9 Hz, AreH), 7.40 (1H, s,
800-CH), 6.16 (1H, s,17-CH2), 6.09 (1H, d, J ¼ 10.5 Hz, 6-OH), 5.88 (1H,
s, 14-CH), 5.52 (1H, s, 17-CH2), 4.62 (1H, dd, J ¼ 11.2, 5.4 Hz, 1-CH),
4.28, 4.19 (each 1H, d, JA ¼ JB ¼ 10.6 Hz, 20-CH2), 4.09 (2H, m, 50-
CH2), 4.02 (2H, t, J ¼ 6.3 Hz,100-CH2), 3.78 (1H, m, 6-CH), 3.16 (1H, d,
J ¼ 9.9 Hz, 13-CH), 2.60 (2H, m, 20-CH2), 1.99 (3H, s, eCH3), 1.12 (6H,
d
(ppm): 7.63, 7.00 (each 2H, d, JA ¼ JB ¼ 8.6 Hz, AreH), 7.40 (1H, s,
800-CH), 6.16 (1H, s,17-CH2), 6.05 (1H, d, J ¼ 10.5 Hz, 6-OH), 5.84 (1H,
s, 14-CH), 5.50 (1H, s, 17-CH2), 4.61 (1H, dd, J ¼ 11.1, 5.4 Hz, 1-CH),
4.44 (2H, t,
J
¼
4.7 Hz, 60-CH2), 4.27, 4.18 (each 1H, d,
JA ¼ JB ¼ 10.5 Hz, 20-CH2), 4.23 (2H, t, J ¼ 4.8 Hz, 70-CH2), 4.07 (1H, s,
eOH), 3.77 (1H, m, 6-CH), 3.15 (1H, d, J ¼ 9.2 Hz, 13-CH), 2.58e2.32
(8H, m, eCH2), 2.00 (3H, s, eCH3), 1.13 (3H, s, eCH3), 1.12 (3H, s,
s, 18,19-CH3); 13C NMR (CDCl3, 100 MHz),
d (ppm): 215.11, 206.11,
173.13, 172.06, 171.00, 169.84, 162.50, 149.44, 134.57, 128.59 ( ꢁ 2),
124.01, 120.58, 115.44 ( ꢁ 2), 95.94, 76.03, 75.38, 74.10, 68.23, 64.83,
63.52, 61.60, 60.04, 53.78, 41.10, 39.75, 38.15, 33.57, 32.42, 30.23,
29.70, 29.59, 28.92, 28.89, 28.46, 25.63, 25.17, 21.60, 21.53, 18.06;
HRMS (ESI) m/z calcd for C41H50O11S3 [M ꢂ H]- 813.2437, found
813.2459.
eCH3); 13C NMR (CDCl3, 100 MHz),
d (ppm): 215.22, 206.06, 172.52,
171.34, 169.86, 161.72, 149.42, 134.83, 128.66 ( ꢁ 2), 124.69, 120.53,
115.56 ( ꢁ 2), 96.03, 77.20, 76.06, 75.37, 74.03, 66.15, 63.55, 62.42,
61.56, 60.07, 53.76, 41.12, 39.77, 38.15, 33.57, 33.45, 32.87, 32.40,
30.24, 29.69, 25.17, 21.53, 19.74, 18.04; HRMS (ESI) m/z calcd for
C
38H44O11S3 [M ꢂ H]- 771.1968, found 771.2000.
4.1.1.10. Compound 13b.
(1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1-acetoxy-5,6-dihydroxy-4,4-
dimethyl-8-methylene-7-oxododecahydro-1H-6,11b-
4.1.1.11. Compound 14b.
(1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1-acetoxy-5,6-dihydroxy-4,4-

10
H. Li et al. / European Journal of Medicinal Chemistry 187 (2020) 111978
Fig. 6. Effects of 12b on induction of apoptosis. (A) 12b induced apoptosis in K562 cells. (B) 12b induced apoptosis in HepG2 cells. K562 cells were treated with 12b (0.50, 1.0 and
2.0 M) for 48 h followed by annexin V/PI staining for 15 min in darkness. Flow cytometric analysis was used to detect apoptosis in K562 cells. Apoptotic cells were on the right
panels of each image. Data are represented as mean SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group.
m
dimethyl-8-methylene-7-oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl(3-(4-(3-
thioxo-3H-1,2-dithiol-5-yl)phenoxy)propyl) glutarate. Orange red
solid, yield: 41.6%, m.p. 66e68 ꢀC. 1H NMR (CDCl3, 400 MHz),
s, 14-CH), 5.51 (1H, s, 17-CH2), 4.63 (1H, dd, J ¼ 11.2, 5.5 Hz, 1-CH),
4.28 (2H, t, J ¼ 6.3 Hz, 60-CH2), 4.26 (1H, m, 20-CH2), 4.19 (1H, d,
J ¼ 10.6 Hz, 20-CH2), 4.10 (2H, t, J ¼ 6.1 Hz, 80-CH2), 3.77 (1H, dd,
J ¼ 10.3, 6.6 Hz, 6-CH), 3.15 (1H, d, J ¼ 9.2 Hz, 13-CH), 2.35 (4H, m,
eCH2), 2.14, 1.91 (each 2H, m, 20-CH2, 40-CH2), 2.00 (3H, s, eCH3),
d
(ppm): 7.62, 6.98 (each 2H, d, JA ¼ JB ¼ 8.6 Hz, AreH), 7.40 (1H, s,
800-CH), 6.15 (1H, s,17-CH2), 6.06 (1H, d, J ¼ 10.5 Hz, 6-OH), 5.84 (1H,
1.12 (6H, s, 18, 19-CH3); 13C NMR (CDCl3, 100 MHz),
d (ppm): 215.17,

H. Li et al. / European Journal of Medicinal Chemistry 187 (2020) 111978
11
Fig. 7. Decrease of mitochondrial membrane potentials triggered by 12b. (A) Decrease of mitochondrial membrane potentials in K562 cells. (B) Decrease of mitochondrial
membrane potentials in HepG2 cells. After the treatment with 12b (0.50, 1.0 and 2.0 M) for 48 h, K562 and HepG2 cells were fixed and labeled with JC-1 for flow cytometry
m
analysis. K562 and HepG2 cells with decreased mitochondrial membrane potentials were on the bottom right panels of each image. Data are represented as mean SD of three
independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group.

12
H. Li et al. / European Journal of Medicinal Chemistry 187 (2020) 111978
Fig. 8. Effects of 12b on apoptosis-related protein expression of K562 cells. The total cell lysates were collected from K562 cells pretreated with 1
mM 12b for 24 h and then seeded in
Human Apoptosis Array kit. Anti-apoptotic proteins were labeled with “*“. Some dots of changed apoptosis-related proteins were locally amplified.
206.07, 172.98, 172.59, 171.34, 169.84, 162.10, 149.42, 134.69, 128.63
( ꢁ 2), 124.32, 120.53, 115.47 ( ꢁ 2), 96.03, 76.08, 75.36, 74.02, 64.79,
63.56, 61.56, 61.08, 60.07, 53.76, 41.12, 39.77, 38.14, 33.56, 32.99,
32.41, 30.24, 29.69, 28.45, 25.17, 21.58, 21.53, 19.78, 18.04; HRMS
(ESI) m/z calcd for C39H46O11S3 [M ꢂ H]- 785.2124, found 785.2172.
s, 14-CH), 5.51 (1H, s, 17-CH2), 4.63 (1H, dd, J ¼ 11.2, 5.5 Hz, 1-CH),
4.28, 4.18 (each 1H, d, JA ¼ JB ¼ 10.6 Hz, 20-CH2), 4.07 (2H, t,
J ¼ 6.7 Hz, 60-CH2), 4.02 (2H, t, J ¼ 6.4 Hz, 110-CH2), 3.78 (1H, dd,
J ¼ 10.2, 6.6 Hz, 6-CH), 3.16 (1H, d, J ¼ 9.9 Hz,13-CH), 2.39e2.30 (4H,
m, eCH2), 2.00 (3H, s, eCH3), 1.91 (2H, m, 20-CH2), 1.83 (2H, m, 40-
CH2), 1.13 (6H, s, 18, 19-CH3); 13C NMR (CDCl3, 100 M Hz),
d (ppm):
215.27, 206.13, 173.12, 172.73, 171.43, 169.85, 162.51, 149.43, 134.58,
128.58 ( ꢁ 2), 124.01, 120.49, 115.45 ( ꢁ 2), 96.03, 76.10, 75.37, 73.97,
68.24, 64.44, 63.54, 61.56, 60.12, 53.75, 41.12, 39.77, 38.15, 33.57
( ꢁ 2), 33.07, 32.39, 30.23, 29.69 ( ꢁ 2), 28.92, 28.51, 25.64 ( ꢁ 2),
25.17, 21.56, 19.84, 18.03; HRMS (ESI) m/z calcd for C42H52O11S3
[M ꢂ H]- 827.2594, found 827.2588.
4.1.1.12. Compound 15b.
(1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1-acetoxy-5,6-dihydroxy-4,4-
dimethyl-8-methylene-7-oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl(6-(4-(3-
thioxo-3H-1,2-dithiol-5-yl)phenoxy)hexyl) glutarate. Orange red
solid, yield: 33.4%, m.p. 72e74 ꢀC. 1H NMR (CDCl3, 400 MHz),
d
(ppm): 7.61, 6.96 (each 2H, d, JA ¼ JB ¼ 8.6 Hz, AreH), 7.40 (1H, s,
800-CH), 6.15 (1H, s, 17-CH2), 6.10 (1H, d, J ¼ 10.5 Hz, 6-OH), 5.84 (1H,

H. Li et al. / European Journal of Medicinal Chemistry 187 (2020) 111978
13
1.70e1.43 (6H, m, eCH2), 1.11 (6H, s, 18-CH3, 19-CH3); 13C NMR
(CDCl3, 100 M Hz), (ppm): 215.12, 206.37, 173.16, 172.04, 170.90,
d
162.23, 149.87, 134.63, 128.61 ( ꢁ 2), 124.21, 120.27, 115.55 ( ꢁ 2),
96.05, 76.12, 74.32, 73.43, 64.87, 63.42, 61.94, 61.31, 59.51, 54.67,
41.34, 41.30, 38.63, 33.73, 32.61, 30.51, 30.06, 29.54, 28.86, 28.49,
21.75, 19.93; HRMS (ESI) m/z calcd for C36H42O10S3 [M ꢂ H]-
729.1862, found 729.1890.
4.1.1.15. Compound 12c.
6-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)hex-
yl((1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7-oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl) succi-
nate. Orange red solid, yield: 40.2%, m.p. 64e66 ꢀC. 1H NMR (CDCl3,
400 MHz),
d
(ppm): 7.61, 6.97 (each 2H, d, JA ¼ JB ¼ 8.7 Hz, AreH),
7.40 (1H, s, 800-H), 6.15 (2H, s, 17-CH2), 6.09 (2H, d, J ¼ 10.2 Hz, 6-
OH), 5.91 (2H, s, 17-CH2), 5.52 (2H, s, 14-CH), 4.30 (2H, d,
J ¼ 10.2 Hz, 20-CH2), 4.08 (6H, m, 20-CH2, 50-CH2), 4.03, 3.41 (each
2H, t, J ¼ 6.6 Hz, 100-CH2), 3.76 (2H, m, 6-CH), 3.50 (2H, dd, J ¼ 11.2,
5.7 Hz, 1-CH), 3.16 (2H, d, J ¼ 9.7 Hz, 13-CH), 2.58 (8H, m, eCH2),
1.99e1.71 (10H, m, eCH2), 1.70e1.41 (18H, m, eCH2), 1.12 (12H, s,
18-CH3, 19-CH3); 13C NMR (CDCl3, 100 M Hz),
d (ppm): 215.17,
206.41, 173.15, 172.10, 170.93, 162.64, 149.90, 134.57, 128.60 ( ꢁ 2),
120.24, 115.48 ( ꢁ 2), 96.08, 76.24, 74.24, 73.52, 68.25, 64.76, 63.35,
61.91, 59.55, 54.67, 41.34, 38.67, 33.73, 32.59, 30.51, 30.07, 29.69,
29.57, 28.90, 28.44, 28.37, 27.77, 25.62, 25.09, 21.72, 19.87; HRMS
(ESI) m/z calcd for
1319.5395.
C
69H90O19S3 [MþH]þ 1319.5316, found
Fig. 9. The apoptosis-related pathways would involve in 12b treated K562 cells.
4.1.1.16. Compound 13c.
2-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)ethyl((1-
S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-
methylene-7-oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-
methanocyclohepta[a]naphthalen-14-yl) glutarate. Orange red
solid, yield: 31.8%, m.p. 70e72 ꢀC. 1H NMR (CDCl3, 400 MHz),
4.1.1.13. Compound 10c.
2-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)ethyl((1-
S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-dimethyl-8-
methylene-7-oxododecahydro-1H-6,11b-(epoxymethano)-6a,9-
methanocyclohepta[a]naphthalen-14-yl) succinate. Orange red
solid, yield: 27.4%, m.p. 68e70 ꢀC. 1H NMR (CDCl3, 400 MHz),
d
(ppm): 7.63, 6.99 (each 2H, d, JA ¼ JB ¼ 8.8 Hz, AreH), 7.40 (1H, s,
800-H), 6.13 (1H, s, 17-CH2), 6.05 (1H, d, J ¼ 8.6 Hz, 6-OH), 5.87 (1H, s,
17-CH2), 5.50 (1H, s, 14-CH), 4.30, 4.07 (each 1H, d, J ¼ 10.4 Hz, 20-
CH2), 4.44, 4.23 (each 2H, t, J ¼ 4.4 Hz, 60-CH2, 70-CH2), 3.74 (1H, m,
6-CH), 3.50 (1H, dd, J ¼ 11.2, 5.7 Hz,1-CH), 3.15 (1H, d, J ¼ 9.8 Hz,13-
CH), 2.60, 2.24 (each 1H, m, 30-CH2), 2.42e2.33 (4H, m, 20-CH2, 40-
CH2), 1.94 (4H, m, eCH2), 1.11 (3H, s, 18-CH3), 1.10 (3H, s, 19-CH3);
d
(ppm): 7.63, 7.01 (each 2H, d, JA ¼ JB ¼ 8.6 Hz, AreH), 7.40 (1H, s,
800-H), 6.15 (1H, s, 17-CH2), 6.05 (1H, d, J ¼ 10.7 Hz, 6-OH), 5.89 (1H,
s, 17-CH2), 5.51 (1H, s, 14-CH), 4.46 (2H, m, 50-CH2), 4.29, 4.06 (each
1H, d, J ¼ 10.5 Hz, 20-CH2), 4.24 (2H, t, J ¼ 4.6 Hz, 60-CH2), 3.75 (1H,
d, 10.0 Hz, 6-CH), 3.49 (1H, dd, J ¼ 11.1, 5.7 Hz, 1-CH), 3.14 (1H, d,
J ¼ 9.8 Hz, 13-CH), 2.65 (2H, m, 30-CH2), 2.59 (2H, m, 20-CH2),
2.54e2.41 (2H, m, eCH2), 2.22,1.97 (each 1H, m, eCH2), 1.93 (2H, m,
eCH2), 1.80e1.52 (2H, m, eCH2), 1.46 (2H, m, eCH2), 1.11 (3H, s, 18-
13C NMR (CDCl3, 100 M Hz),
d (ppm): 215.19, 206.38, 172.88, 172.57,
171.27, 161.75, 149.86, 134.80, 128.66 ( ꢁ 2), 124.63, 120.19, 115.58
( ꢁ 2), 96.15, 76.33, 74.16, 73.48, 66.15, 63.41, 62.38, 61.88, 59.60,
54.62, 41.34, 41.31, 38.64, 33.73, 33.44, 32.89, 32.58, 30.52, 30.07,
29.69, 21.70, 19.83; HRMS (ESI) m/z calcd for C36H42O10S3 [M ꢂ H]-
729.1862, found 729.1889.
CH3), 1.10 (3H, s, 19-CH3); 13C NMR (CDCl3, 100 M Hz),
d (ppm):
215.20, 206.36, 172.85, 171.96, 170.76, 161.77, 149.90, 134.82, 128.63
( ꢁ 2), 124.64, 120.28, 115.64 ( ꢁ 2), 96.05, 76.18, 74.33, 73.49, 66.15,
63.37, 62.73, 61.93, 59.49, 54.68, 41.33, 38.65, 33.73, 32.61, 30.53,
30.06, 29.69, 29.53, 28.83, 21.75, 19.89; HRMS (ESI) m/z calcd for
4.1.1.17. Compound 14c.
3-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)pro-
pyl((1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7-oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl) gluta-
rate. Orange red solid, yield: 30.2%, m.p. 74e75 ꢀC. 1H NMR (CDCl3,
C
35H40O10S3 [M ꢂ H]- 715.1706, found 715.1731.
4.1.1.14. Compound 11c.
3-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)pro-
pyl((1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7-oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl) succi-
nate. Orange red solid, yield: 32.6%, m.p. 71e73 ꢀC. 1H NMR (CDCl3,
400 MHz),
d
(ppm): 7.62, 6.98 (each 2H, d, JA ¼ JB ¼ 8.7 Hz, AreH),
7.40 (1H, s, 800-H), 6.13 (1H, s,17-CH2), 6.05 (1H, s, 6-OH), 5.87 (1H, s,
17-CH2), 5.50 (1H, s, 14-CH), 4.30, 4.07 (each 1H, d, J ¼ 10.8 Hz, 20-
CH2), 4.27, 4.10 (each 2H, t, J ¼ 6.4 Hz, eCH2), 3.75 (1H, brs, 6-CH),
3.50 (1H, dd, J ¼ 11.1, 5.7 Hz, 1-CH), 3.15 (1H, d, J ¼ 9.8 Hz, 13-CH),
2.60, 2.25 (each 1H, m, 30-CH2), 2.34 (4H, m, -20-CH2, 40-CH2), 2.15,
1.90 (each 2H, m, eCH2), 1.80e1.42 (8H, m, eCH2), 1.12 (3H, s, 18-
400 MHz),
d
(ppm): 7.63, 7.00 (each 2H, d, JA ¼ JB ¼ 8.0 Hz, AreH),
7.42 (1H, s, 800-H), 6.14 (1H, s, 17-CH2), 6.06 (1H, d, J ¼ 8.5 Hz, 6-OH),
5.90 (1H, s, 17-CH2), 5.51 (1H, s, 14-CH), 4.30 (3H, m, 50-CH2, 20-
CH2), 4.10 (3H, m, 70-CH2, 20-CH2), 3.75 (1H, m, 6-CH), 3.50 (1H,
dd, J ¼ 11.1, 5.5 Hz,1-CH), 3.12 (1H, d, J ¼ 9.7 Hz,13-CH), 2.58 (4H, m,
eCH2), 2.16 (2H, t, J ¼ 5.8 Hz, 20-CH2), 1.96, 1.76 (each 1H, m, eCH2),
CH3), 1.11 (3H, s, 19-CH3); 13C NMR (CDCl3, 100 M Hz),
215.13, 206.38, 173.10, 172.63, 171.28, 162.15, 149.86, 134.66, 128.64
( ꢁ 2), 124.27, 120.18, 115.49 ( ꢁ 2), 96.17, 76.34, 74.15, 73.47, 64.82,
d (ppm):

14
H. Li et al. / European Journal of Medicinal Chemistry 187 (2020) 111978
63.42, 61.88, 61.06, 59.61, 54.62, 41.34, 41.32, 38.64, 33.74, 33.53,
33.03, 32.58, 30.52, 30.07, 29.70, 28.44, 21.71, 19.84; HRMS (ESI) m/z
calcd for C37H44O10S3 [M ꢂ H]- 743.2019, found 743.2034.
4.4. Cell cycle analysis
K562 and HepG2 cells were seeded in 6-well plates and incu-
bated with 12b or oridonin for 48 h and then harvested by
centrifugation. The cells were washed with cold PBS and fixed with
70% ethanol at ꢂ20 ꢀC overnight. After re-centrifugation, all cells
were suspended again using PBS buffer and then incubated with
RNase A (1 mg/mL) and PI (0.1 mg/mL) for 30 min in darkness. All
samples were then analyzed by flow cytometer to determine the
distribution of DNA content [63].
4.1.1.18. Compound 15c.
6-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)hex-
yl((1S,4aR,5S,6S,6aR,9S,11aS,11bS,14R)-1,5,6-trihydroxy-4,4-
dimethyl-8-methylene-7-oxododecahydro-1H-6,11b-(epox-
ymethano)-6a,9-methanocyclohepta[a]naphthalen-14-yl) gluta-
rate. Orange red solid, yield: 37.1%, m.p. 67e69 ꢀC. 1H NMR (CDCl3,
400 MHz),
d
(ppm): 7.61, 6.97 (each 2H, d, JA ¼ JB ¼ 8.8 Hz, AreH),
7.40 (1H, s, 800-H), 6.14 (2H, s, 17-CH2), 6.06 (2H, d, J ¼ 10.2 Hz, 6-
OH), 5.87 (2H, s, 17-CH2), 5.50 (2H, s, 14-CH), 4.30 (2H, d,
J ¼ 10.2 Hz, 20-CH2), 4.10e4.01 (8H, m, 20-CH2, 50-CH2, 100-CH2),
3.76 (2H, t, J ¼ 7.4 Hz, 6-CH), 3.50 (2H, dd, J ¼ 11.1, 5.6 Hz,1-CH), 3.41
(2H, t, J ¼ 6.7 Hz, 100-CH2), 3.17 (2H, d, J ¼ 9.9 Hz, 13-CH), 2.60, 2.24
(each 2H, m, 30-CH2), 2.33 (8H, m, 20-CH2, 40-CH2), 1.90 (4H, t,
J ¼ 7.3 Hz, eCH2), 1.83 (4H, m, eCH2), 1.69e1.60 (8H, m, eCH2),
1.53e1.43 (8H, m, eCH2), 1.12 (12H, s, 18-CH3, 19-CH3); 13C NMR
4.5. Hoechst 333258 staining
K562 cells were grown on 6-well plates which contained 2 mL
medium and adhered for 24 h or 48 h. Later, 12b (0, 1.0, 2.0 and
4.0 mM) was added into each well and cultured for another 48 h.
After mild trypsinization and centrifugation, cells were harvested
and washed twice with PBS buffer. Then, using Hoechst mixture
(0.5 mg/mL) in PBS buffer to stain cells for 30 min in darkness. After
washed by PBS, cells were mounted on slides and analyzed through
a DAPI filtered fluorescence microscope.
(CDCl3, 100 M Hz),
d (ppm): 215.10, 206.42, 173.23, 172.78, 171.35,
162.55, 149.87, 134.54, 128.60 ( ꢁ 2), 123.97, 120.14, 115.48 ( ꢁ 2),
96.17, 76.40, 74.12, 73.47, 68.25, 64.43, 63.40, 61.87, 59.66, 54.62,
41.34, 38.65, 33.73, 33.56, 33.10, 32.57, 30.51, 30.06, 28.92, 28.50,
27.77, 25.63, 25.13, 21.69, 19.82; HRMS (ESI) m/z calcd for
4.6. Cell apoptosis assay
C
71H94O19S3 [MþH]þ 1347.5629, found 1347.5678.
To uncovered the influence of 12b on apoptosis, K562 and
HepG2 cells were incubated in 6-well plates for 72 h and then
4.2. MTT assay and trypan blue assay
different concentrations (0, 0.50, 1.0 and 2.0
mM) of 12b were
injected for another 48 h. After washed twice with PBS, cells were
suspended in annexin V binding buffer. Then AV-FITC and PI were
added to the mixture at room temperature and incubated for
15 min in darkness. Finally, double-staining cells were measured
via flow cytometry to detect apoptotic process.
MTT assay and trypan blue assay were carried out to investigate
the cytotoxicity of all derivatives against five cancer cell lines and
two normal cells. All cells were seeded in standard 96-well plates
under the condition of humidified 5% CO2 and kept at 37 ꢀC to
simulate human body temperature. After 24 h, gradient diluted
target derivatives (DMSO in each well <0.5%) were added and
incubated for 72 h. MTT solution (0.5 mg/mL) was added to each
well and cultivated for another 4 h, and then removed the media
4.7. Mitochondrial membrane potential assay
followed by dissolution formazan crystals in DMSO (200
mL).
K562 and HepG2 cells were grown on 6-well plates and incu-
Finally, the absorbance (OD) of each well was determined quanti-
tatively by Microplate Reader at 490 nm wavelength and half in-
hibition rates (IC50) were calculated by a data analysis software. In
trypan blue assay, after 72 h incubated with all derivatives, cells
were stained with 0.4% trypan blue for 3 min and then observed by
using optical microscopy.
bated with 12 b at 0, 0.50, 1.0 and 2.0 mM concentrations for 48 h.
After trypsinization, cells were collected and then stained with JC-1
(0.5 mg/mL) at 37 ꢀC in darkness. Washed out excess dye with PBS
after 30 min incubation, and then monitored the mitochondrial
membrane potentials by flow cytometry.
4.8. Human apoptosis protein array
4.3. H2S release experiment
According to the manufacturer instructions, the relative
expression levels of 35 apoptosis-related proteins were investi-
gated after the treatment with 12b (1 mM) [64]. Diluted cellular
extracts were seeded in Human Apoptosis Array kit for 24 h.
Washed to remove unbound proteins, and then incubated with a
biotinylated detection antibody for 1 h. After that, chemilumines-
cent signals were detected by digital imaging system.
Na2S was dissolved in sodium phosphate buffer (20 mM, pH 7.4)
in 100 mL volumetric flask, which was used as the stock solution
and then standard solutions of 5, 10, 20, 40, 60, 80, 100 and 150
in 50 mL volumetric flask were prepared. 1 mL of each standard
solution was added into methylene blue (MBþ) cocktail: 200
L of
30 mM FeCl3 in 1.2 M HCl, 200 L of 20 mM N,N-dimethyl-1,4-
phenylenediamine sulfate in 7.2 M HCl and 100 L of 1% w/v
mM
m
m
m
Zn(OAc)2 in H2O and stored for 20 min at room temperature (each
reaction was carried out in triplicate). Later, each of colorimetric
cuvette was placed in UVeVis spectrophotometer at 670 nm to
draft the Na2S calibration curve [62]. Target derivatives were added
into the mixture of THF and PBS in the presence of 1 mM TCEP (an
effective mercaptan reductant) which was used as an accelerator. At
different time points, the mixture (2 mL) was transferred to
colorimetric cuvette included methylene blue (MBþ) cocktail. Ul-
timately, each of colorimetric cuvette was placed in UVeVis spec-
trophotometer at 670 nm after 20 min at room temperature.
According to the absorbance of each compound, the H2S release
was calculated through standard curve.
4.9. Statistical analysis
All data were expressed as the means standard deviation (SD),
from at least three independent experiments. Statistical analysis
and figures were performed by GraphPad.Prism. v5.0.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.

H. Li et al. / European Journal of Medicinal Chemistry 187 (2020) 111978
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Acknowledgment
This paper was financially supported by the National Natural
Science Foundation of China (21772124, 21502121), Natural Science
Foundation of Liaoning Province (20170540858), General Scientific
Research Projects of Department of Education in Liaoning Province
(2017LQN05), Key Laboratory of Quality Control of TCM of Liaoning
Province (17-137-1-00) and Career Development Support Plan for
Young and Middle-aged Teachers in Shenyang Pharmaceutical
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