Hydrogen sulfide donating ent-kaurane and spirolactone-type 6,7-seco-ent-kaurane derivatives: Design, synthesis and antiproliferative properties

Article abstract of DOI:10.1016/j.ejmech.2019.06.016

Motivated by our interest in hydrogen sulfide bio-chemistry and ent-kaurane diterpenoid chemistry, 14 hydrogen sulfide donating derivatives (9, 11a-c, 12a-c, 13, 14, 16a-c and 17a-b) of ent-kaurane and spirolactone-type 6,7-seco-ent-kaurane were designed and synthesized. Four human cancer cell lines (K562, Bel-7402, SGC-7901 and A549) and two normal cell lines (L-02 and PBMC) were selected for antiproliferative assay. Most derivatives showed more potent activities than the lead ent-kaurane oridonin. Among them, compound 12b exhibited the most potent antiproliferative activities, with IC₅₀ values of 1.01, 0.88, 4.36 and 5.21 μM against above human cancer cell lines, respectively. Further apoptosis-related mechanism study indicated that 12b could arrest Bel-7402 cell cycle at G1 phase and induce apoptosis through mitochondria related pathway. Through Western blot assay, 12b was shown to influence the intrinsic pathway by increasing the expression of Bax, cleaved caspase-3, cytochrome c and cleaved PARP, meanwhile suppressing procaspase-3, Bcl-2, Bcl-xL and PARP.

Full text of DOI:10.1016/j.ejmech.2019.06.016

Accepted Manuscript
Hydrogen sulfide donating ent-kaurane and spirolactone-type 6,7-seco-ent-kaurane
derivatives: Design, synthesis and antiproliferative properties
Haonan Li, Xiang Gao, Xiaofang Huang, Xianhua Wang, Shengtao Xu, Takahiro
Uchita, Ming Gao, Jinyi Xu, Huiming Hua, Dahong Li
PII:
S0223-5234(19)30535-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.016
EJMECH 11419
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 January 2019
Revised Date: 3 June 2019
Accepted Date: 4 June 2019
Please cite this article as: H. Li, X. Gao, X. Huang, X. Wang, S. Xu, T. Uchita, M. Gao, J. Xu, H. Hua,
D. Li, Hydrogen sulfide donating ent-kaurane and spirolactone-type 6,7-seco-ent-kaurane derivatives:
Design, synthesis and antiproliferative properties, European Journal of Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.ejmech.2019.06.016.
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ACCEPTED MANUSCRIPT
HO
O
H
IC50 ( M)
S
IC50 ( M)
H
OH
O
S
S
Bel-7402 4.67
Bel-7402 >50
CHO O
O
spirolactone-type diterpenoid
H2S-donating compound
O
H
IC50 ( M)
H
O
O
O
Bel-7402 0.88
CHO O
O
O
O
S
L-02
PBMC
20.82
19.85
S
S
Compound 12b
G1
phase
arrest
m
apoptosis

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Hydrogen sulfide donating ent-kaurane and spirolactone-type
6,7-seco-ent-kaurane derivatives: Design, synthesis and antiproliferative
properties
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Haonan Li , Xiang Gao , Xiaofang Huang , Xianhua Wang , Shengtao Xu , Takahiro Uchita ,
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Ming Gao , Jinyi Xu , Huiming Hua , Dahong Li
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Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, and School of Traditional
Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, P. R. China
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School of Public Health, Qingdao University, 38 Dengzhou Road, Qingdao 266021, P. R. China
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State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing
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210009, P. R. China
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School of Pharmaceutical Sciences, Mukogawa Women's University, 11-68 Koshien, Nishinomiya 663-8179,
Japan
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Corresponding author. E-mail address: lidahong0203@163.com.
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These authors contributed equally to this work.
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Abstract:
Motivated by our interest in hydrogen sulfide bio-chemistry and ent-kaurane diterpenoid
chemistry, 14 hydrogen sulfide donating derivatives (9, 11a-c, 12a-c, 13, 14, 16a-c and 17a-b) of
ent-kaurane and spirolactone-type 6,7-seco-ent-kaurane were designed and synthesized. Four
human cancer cell lines (K562, Bel-7402, SGC-7901 and A549) and two normal cell lines (L-02
and PBMC) were selected for antiproliferative assay. Most derivatives showed more potent
activities than the lead ent-kaurane oridonin. Among them, compound 12b exhibited the most
potent antiproliferative activities, with IC50 values of 1.01, 0.88, 4.36 and 5.21 µM against above
human cancer cell lines, respectively. Further apoptosis-related mechanism study indicated that
12b could arrest Bel-7402 cell cycle at G1 phase and induce apoptosis through mitochondria
related pathway. Through western blot assay, 12b was shown to influence the intrinsic pathway by
increasing the expression of Bax, cleaved caspase-3, cytochrome c and cleaved PARP, meanwhile
suppressing procaspase-3, Bcl-2, Bcl-xL and PARP.
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Key words: diterpenoid, hydrogen sulfide, antiproliferative activity, apoptosis
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1. Introduction
Nature has long been an invaluable source, providing molecular skeletons for drug
discovery [1]. During the 1940s to the end of 2014, almost 50% of approved anticancer small
molecules were nature derived molecules [2]. The Rabdosia, an important and cosmopolitan genus
of the Labiatae family, has aroused considerable interests due to the aboundance of ent-kaurane
diterpenoids [3-10]. Oridonin (1, Fig. 1), the main active component of Rabdosia rubescens, has
been the most extensively studied natural product of this genus [11-20]. Its structure and absolute
configuration were determined in 1970 [21]. It exhibited anticancer effects on leukemia [22],
pancreatic cancer [23], osteosarcoma [24], colorectal cancer [25], renal cell carcinoma [26] and
diffuse large B cell lymphoma [27]. One sub-type of 6,7-seco-ent-kauranes, spirolactone-type
(7,20-lactone) diterpenoids, contains multiple stereogenic centers and complex ring systems, such
as trichorabdal A, maoecrystal Z, longikaurin E, sculponeatin N, maoecrystal V, sculponeatin C,
laxiflorolide C and laxiflorin B (Fig. 1) [28-34]. In particular, some of them exhibited promising
antiproliferative effects [35-37] with IC50 values of 0.21, 0.29, 0.60 and 0.80 against K562,
HepG2, SW-480 and HL-60 human cancer cell lines, respectively.
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1
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Although spirolactone-type diterpenoids demonstrated sub-micromolar level
antiproliferative activities, their low isolation yields from Isodon plants can not meet the need for
further studies, which hindered their wide application as potential candidates in drug development.
Moreover, the complex stereogenic centers make it quite challenging to achieve via total synthesis
[38,39]. In general, through the analysis of active products [40-42], structural modification [43,44],
biological evaluation [45,46] and mechanism study [47-49], the deigned derivatives would show
better properties than the original forms and some of them would serve as drug candidates. In our
previous work, a convenient method for the conversion from oridonin to spirolactone-type
skeleton in quantitative yield was achieved by oxidation with lead tetraacetate, which could
provide enough starting materials for the medicinal study of spirolactone-type derivatives [50-53].
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Small molecule donors have been widely used in drug discovery [54,55]. Hydrogen
sulfide (H S), like nitric oxide (NO) and carbon monoxide (CO), has been recognized as a
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gasotransmitter related to health and disease [56,57]. The generation of endogenous H S is caused
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by three important enzymes, cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS) and
3-mercaptopyruvate sulfurtransferase (MST), with cysteine, homocysteine and cystathionine as
substrate [58,59]. Besides bioactivities in vascular tone, blood pressure, ischemia-reperfusion
injury, atherosclerosis, angiogenesis and neuroinflammation [60-65], recent studies elucidated that
H S showed moderate anticancer activity by inducing cell apoptosis via EGFR/ERK/MMP-2,
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PI3K/Akt/mTOR and p38 MAPK/ERK1/2-COX-2 pathways [66-70]. Although H S plays
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important roles in physiology and pathology, it can not be directly used in clinical treatment due to
uncontrollable dose and high toxicity. While the H S donors (including ADT-OH, thiobenzamide,
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α-thioctic acid, GYY4137, persulfides etc. Fig. 2A) could partially solve the problems which
release H S at relative slow rates [71-74]. H S-releasing compounds also showed promising
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properties and were instrumental for the exploration of drug candidates [75-79, Fig.2B].
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Based on the above, we used two ent-kaurane derivatives (2, 6) linked with two kinds of
H S donors (α-thioctic acid and ADT-OH) by ether bond and then converted them into
2
corresponding spirolactone-type derivatives. The antiproliferative effects of target compounds
were evaluated against chronic myelogenous leukemia K562, liver cancer Bel-7402, gastric cancer
SGC-7901, lung cancer A549, human normal liver L-02 and normal peripheral blood mononuclear
PBMC cell lines. Furthermore, apoptosis related mechanism of the most potent compound 12b
was also explored, including cell cycle progression, apoptotic induction, mitochondria membrane
potentials decline and the expression of apoptosis-related proteins.
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OH 211
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H14
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OH
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OH
H
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19 OH
Oridonin
AcO
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HO
AcO
H
H
H
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H
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O
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O
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OH
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CHO O
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HO
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longikaurin E
trichorabdal A
maoecrystal Z
sculponeatin N
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HO
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CHO
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maoecrystal V
sculponeatin C
laxiflorolide C
laxiflorin B
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Fig. 1. The structures of oridonin and natural spirolactone-type 6,7-seco-ent-kaurane diterpenoids.
S
S
S
A
O
P
N
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H N
O
S
S
2
HO
S
NH2
S
S
O
S
HO
HO
thiobenzamide
O
persulfide
RSSH)
ADT-OH
-thioctic acid
GYY4137
(
S
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B
S
H3CO
O
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S
S
S
S
O
NH2
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O
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ONO2
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ATB-346
NBS-1120
SG-1002
O
S
S
H3CO
H3CO
O
O
NH
O
S
OCH3
P
O
O
Br
NH2
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AP39
GIC-1001
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Fig. 2. The structures of selected H S donating molecules (A) and H S releasing drug candidates
2 2
(B).
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2. Result and discussion
2.1. Chemistry
The whole synthetic route of H S-releasing derivatives is outlined in Scheme 1 and Scheme 2.
2
Intermediate 2 was obtained through addition of Jones reagent to selectively oxidize the 1-OH of 1.
Treatment of 1 with 2,2-dimethoxypropane and TsOH in anhydrous acetone afforded derivative 4,
then reaction with Ac O/Et N in DCM acquired derivative 5. And intermediate 6 was afforded via
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3
deprotection in the presence of 10% HCl. The derivatives 3 and 7 were prepared by lead
tetraacetate and sodium carbonate (Na CO ) in THF. The generated 2 and 5 were reacted with
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3
corresponding anhydride under the condition of 4-dimethylaminopyridine (DMAP) and
1
1
1
1
1
triethylamine (TEA) in DCM at room temperature, which gave derivatives 10a-c and 15a-c in
quantitative yields. Then H S-releasing derivatives 8, 11a-c, 13 and 16a-c were obtained via
2
reaction of corresponding diterpenoid derivatives with ADT-OH or α-thioctic acid in the presence
of DMAP and carbodiimide hydrochloride (EDCI). Finally, spirolactone-type derivatives 9, 12a-c,
14 and 17a-b were achieved by oxidation using lead tetraacetate and Na CO in THF.
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Scheme 1. The synthetic route of intermediates. Reagents and conditions: (a) Jones reagent,
acetone, 0 °C, 0.5 h; (b) Pb(OAc) , Na CO , THF, rt, 2 h; (c) 2,2-dimethoxypropane, acetone,
4
2
3
TsOH, 56 °C, 1.5 h; (d) Ac O, TEA, DMAP, DCM, rt, 5 h; (e) 10% HCl, THF, rt, 4 h.
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Scheme 2. Synthesis of H S-releasing derivatives 9, 11a-c, 12a-c, 13, 14, 16a-c and 17a-b.
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Reagents and conditions: (a) α-thioctic acid, EDCI, DMAP, DCM, rt, 12 h; (b) Pb(OAc) , Na CO ,
4
2
3
THF, rt, 2 h; (c) corresponding anhydride, TEA, DMAP, DCM, rt, 5 h; (d) ADT-OH, EDCI,
DMAP, DCM, rt, 8-12 h.
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2.2. Biological evaluation
2.2.1 Antiproliferative activities and preliminary SAR
The antiproliferative activities of 14 target derivatives (9, 11a-c, 12a-c, 13, 14, 16a-c and
17a-b), lead compound oridonin (1), parental diterpenoids (2, 3, 6 and 7) and H S donors
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1
1
1
(ADT-OH and α-thioctic acid) were tested by MTT assay (5-Fu was used as positive control).
Four human cancer and two normal cell lines were selected to test their cytotoxicity. As shown in
Table 1, all derivatives displayed inhibitory activities against selected human cancer cell lines.
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Most derivatives exhibited more potent antiproliferative activities than oridonin (IC = 4.79-18.86
5
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µM), and even than positive control (IC50 = 2.33-17.59 µM). Besides promising antiproliferative
activities, low cytotoxicity to normal cells was also observed, which is a key character in
anticancer drug exploration. When the substitution R were alkyl groups (11a-b, 12a-b and 16a-b),
their antiproliferative activities were more potent than corresponding ones with aromatic groups
(11c, 12c and 16c) and improved IC50 values were observed with the extension of R group in
Bel-7402 cells. It demonstrated that linear linker rather than benzene ring was advantageous for
potency, and longer carbon bridge might contribute to superior inhibitory effects. Compounds 9,
13 and 14 whose 14-OH directly incorporated with α-thioctic acid showed similarly
antiproliferative activities to corresponding derivatives (11a-b, 12a-b, 16a-b and 17a-b) which
coupled with ADT-OH through a carbon bridge. Comparing 1-oxo ent-kaurane derivatives (11a-c)
with corresponding spirolactone-type derivatives (12a-c), stronger antiproliferative activities were
observed. But this phenomenon did not work in 1-acetylated derivatives (16a-c and 17a-b).
Furthermore, among spirolactone-type derivatives, 1-oxo derivatives (12a-c) showed more potent
activities than 1-acetylated derivatives (17a-b). Among them, compound 12b showed the
strongest antiproliferative activities, with IC50 values of 1.01, 0.88, 4.36 and 5.21 µM against
K562, Bel-7402, SGC-7901 and A549 human cancer cell lines, respectively. Furthermore,
compound 12b exhibited almost 24-fold less potent antiproliferative activities against L-02 (IC =
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20.82 µM) and PBMC (IC50 = 19.85 µM) normal cell lines, demonstrating selectivity between
cancer and normal cell lines. Hence, compound 12b was selected for further mechanism study.
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Table 1
a
Antiproliferative activities (IC50 µM) of 14 target derivatives, parental diterpenoids and H2S
donors against four human cancer and two normal cell lines.
Compound
K562
Bel-7402 SGC-7901
A549
L-02
PBMC
>50
1
2
3
4.79±0.12 8.31±0.29 7.87±0.36 17.80±0.75 18.68±1.28
4.02±0.18 3.88±0.18 16.54±0.30 9.86±0.45 23.41±1.39
3.26±0.16 4.67±0.25 19.48±0.94 5.31±0.40 29.70±1.07
>50
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4.92±0.24 3.59±0.18 6.45±0.37 13.47±0.53 22.36±1.25
>50
>50
7
8.96±0.49
2.59±0.22 2.56±0.19 6.43±0.37 7.92±0.77 19.85±1.37
3.66±0.30 3.52±0.31 12.50±0.44 9.78±0.75 >50
>50
49.84±2.51 27.44±1.52
>50
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>50
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1b
29.62±1.31
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3.84±0.28 3.04±0.23 6.59±0.26 4.38±0.17 21.02±1.36 17.23±1.05
4.80±0.10 5.34±0.27 14.87±0.53 7.69±0.40 42.64±2.63 >50
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1c
1
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2b
1.82±0.07 1.38±0.12 4.24±0.25 5.54±0.42 18.19±1.44 16.58±1.26
1.01±0.07 0.88±0.05 4.36±0.20 5.21±0.25 20.82±1.28 19.85±1.37
4.25±0.23 3.26±0.14 5.93±0.33 7.45±0.38 26.21±1.55 31.45±1.61
2.84±0.17 2.25±0.16 6.02±0.20 8.65±0.36 41.10±2.94 28.83±1.45
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2c
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14
6.75±0.31 4.58±0.24 10.04±0.39 19.89±0.81 36.47±1.32
>50
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6a
6b
2.54±0.18 2.87±0.08 5.49±0.14 6.34±0.41 23.24±1.54 44.15±2.26
1
2.02±0.06 1.94±0.12 5.36±0.28 9.76±0.48 14.70±1.14
>50
39.53±1.69
25.86±1.35
30.65±1.41
>50
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6c
11.43±0.74 7.68±0.35 12.37±0.85 16.66±0.70
15.26±1.08 9.73±0.52 17.95±0.76 27.53±1.21
3.27±0.21 4.59±0.37 7.66±0.37 14.82±1.14
>50
>50
>50
>50
>50
>50
1
7a
7b
1
ADT-OH
>50
>50
>50
>50
>50
>50
>50
>50
α-thioctic acid
>50
5
-Fu
4.02±0.22 17.59±0.86 6.38±0.32 2.33±0.12
>50
a
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IC : Half inhibitory concentrations measured by the MTT assay, the cells were incubated
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for 72 h. The values are expressed as averages ± standard deviations of three independent
experiments.
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2.2.2. H S release ability
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H S-releasing capability of all derivatives was tested by the methylene blue (MB ) method.
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As shown in Fig. 3, all derivatives could produce H S with a peak time around 15-20 min. It was
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obvious that those derivatives with α-thioctic acid (9, 13 and 14) showed the highest H2S
generating ability, while releasing characteristics of the derivatives with ADT moisties were not
varied significantly from each other, except for 17a and 17b. Interestingly, 1-oxo derivatives
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usually produced more H S (0.2-1 µM) than 1-acetylated derivatives. Two spirolactone-type
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derivatives with acetyl group at C-1 (17a-b) were found to release the lowest H S, which were
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consistent with their tempered antiproliferative activities. Among other derivatives with ADT-OH,
spirolactone-type derivatives 12a-c showed moderate H S releasing ability while exhibiting
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superior antiproliferative activities. Furthermore, in contrast to the low antiproliferative effects,
those with phthalic anhydride linker (11c, 12c and 16c) generated more H S. Moreover, with the
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increased length of R groups, stronger antiproliferative effects could be found but with a small
reduction in H S generation.
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Fig. 3. H S-releasing ability of target compounds. The values are expressed as averages of three
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independent experiments.
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2.2.3. Cell cycle analysis
Cell cycle is the basic process of cell life activities. An interruption of certain phase of cell
cycle leads to the inhibition of cell division and suppresses tumor growth. In order to demonstrate
the antiproliferative mechanism, the influence on cell cycle of 12b was tested and DNA content of
cell nuclei was measured via flow cytometry. In this assay, Bel-7402 cells were treated with
different concentrations of 12b (0, 0.45, 0.90 and 1.80 µM) for 48 h. Then stained with propidium
iodide (PI) for subsequent cytometry analysis. Cells treated with DMSO were used as control. As
shown in Fig. 4, after treatment with 0, 0.45, 0.90 and 1.80 µM 12b, the percentage of Bel-7402
cells at G1 phase achieved a substantial increase from 30.95% to 37.37%, 42.30% and 49.56%,
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respectively. According to the results, 12b led to a dose-dependent accumulation of Bel-7402 cells
at G1 phase.
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Fig. 4. Influence on Bel-7402 cell cycle of 12b. After incubated with 12b (0.45, 0.90 and 1.80 µM)
or DMSO (control) for 48 h, Bel-7402 cells were stained with PI and then cell cycle distribution
was analyzed by a flow cytometer.
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2.2.4. The morphological analysis by Hoechst 33258 staining
The deviant morphological changes in chromatin, including cell shrinkage, chromatin
condensation, the rupture of cell membrane and the nuclear fragmentation, indicate the occurrence
of apoptosis. Hoechst 33258 staining assay was used to indicate apoptosis by fluorescence
microscope [80]. As shown in Fig. 5, contrast to vehicle control, Bel-7402 cells presented bright
blue chromatin shrink and nuclear fragmentation after treatment with 12b (0.45, 0.90 and 1.80
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µM). While control cells remained homogeneous nuclei with deep blue color. The Hoechst 33258
staining assay revealed that 12b could induce apoptosis in Bel-7402 cells.
Control
0.45
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1.80
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Fig. 5. Hoechst staining of 12b treated Bel-7402 cells. After treatment with 12b (0.45, 0.90 and
1.80 µM) or vehicle for 48 h, Bel-7402 cells were stained in blue color and probed by a
fluorescent microscope.
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2.2.5. Cell apoptosis assay
Apoptosis is an important biological process of programmed cell death, which maintain
regular functions and activities of cells. But cancer cells could grow incessantly by suppressing
apoptosis. Thus, utilizing chemical agents induced apoptosis in cancer cells is a potent therapeutic
method. In order to further confirm 12b induced apoptosis in Bel-7402 cells, an annexin
V-FITC/PI binding assay was performed. Bel-7402 cells were treated with 12b at indicated
concentrations (0, 0.45, 0.90 and 1.80 µM), then the percentage of apoptotic cells was measured
by a flow cytometry. As shown in Fig. 6, after incubation with 12b for 48 h, apoptotic ratios
substantially rose from 2.77% of vehicle control to 17.65%, 27.47% and 55.32% for different
concentrations applied, respectively, in a dose-dependent manner. The results indicated that 12b
possessed the ability to induce apoptosis in Bel-7402 cells.
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Fig. 6. Apoptosis effects of 12b on Bel-7402 cells. Bel-7402 cells were treated with 12b for 48 h
and analyzed via an annexin V-FITC/PI binding assay.
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2.2.6. Mitochondria membrane potential analysis
The maintenance of mitochondrial membrane potentials plays an important role in
mitochondrial integrity and biological function. Mitochondrial changes, especially losing
mitochondrial membrane potentials, were significant events taking place during drug-induced
apoptosis. Additionally, endogenous H S was recognized to induce apoptosis by activating the
2
intrinsic mitochondrion-mediated pathways according to recent literatures [81]. The changes of
mitochondrial membrane potentials were measured via flow cytometry, in order to demonstrate
the influence of 12b on mitochondria. Bel-7402 cells were incubated for 48 h with different
concentrations of 12b (0.45, 0.90 and 1.80 µM) and vehicle control DMSO. Then cells were
1
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1
treated
with
the
lipophilic
cationic
fluorescent
probe
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1). As shown in Fig.
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7, the percentage of cells with depolarized mitochondria ranged from 1.28% of control to 18.48%,
30.96% and 58.59% in a dose-dependent manner. This indicated that 12b induced Bel-7402 cells
apoptosis via mitochondria related pathway.
4
5
6
7
Fig. 7. Influence on mitochondrial membrane potentials of 12b. Bel-7402 cells were treated with
12b for 48 h and then stained with JC-1. The changes of mitochondrial membrane potentials were
analyzed by a flow cytometry.
8
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2.2.7. Effects on apoptosis-related proteins
In general, apoptosis can be divided into two main pathways, intrinsic (the mitochondrial)
pathway and extrinsic (the death receptor) pathway. Apoptosis through intrinsic pathway is
usually mediated by a group of apoptosis related proteins. Bcl-2 family, an important protein
family including Bcl-2, Bcl-xL and Bax, adjusts the mitochondrial membrane permeabization and
eventually leads to apoptosis. Bcl-2 protein is an antiapoptotic protein and exerts inhibitory effects
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on proapoptotic proteins through mediating permeability transition pore. Bcl-xL protein exhibits
antiapoptotic effect by blocking the destruction of mitochondrial outer membrane caused by Bax.
In addition, it also plays an important role in cell necrosis. While Bax protein (proapoptotic
protein) can insert itself into mitochondrial outer membrane, uplift the permeabilization and
induce the mitochondria to release cytochrome c. Caspase activation is also a key event in the
initiation and execution of apoptosis in cells. With the releasing of cytochrome c, downstream
apoptotic protein caspase-9 is activated. Upon activation, caspase-9 cleaves procaspase-3 and
generates activated caspase-3 (cleaved caspase-3) directly, and then cleaves poly(ADP-ribose)
polymerase (PARP), eventually causing the execution of apoptosis. In order to find out the
mediative mechanism of aforementioned apoptosis related proteins behind 12b induced apoptosis
in Bel-7402 cells, corresponding Western blot analysis was conducted. As shown in Fig. 8,
treatment with 12b caused the increased expression of proapoptotic Bax, cleaved caspase-3,
cytochrome c and cleaved PARP and suppressed procaspase-3, PARP, Bcl-2 and Bcl-xL. The
results revealed that 12b induced Bel-7402 cells apoptosis via mitochondria mechanism mediated
by apoptotic related proteins.
1
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1
1
1
1
6
7
1
Fig. 8. Effects of 12b on apoptosis-related proteins in Bel-7402 cells.
18
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3. Conclusion
In this study, we designed and synthesized 14 H S-releasing derivatives. The antiproliferative
2
activities of all derivatives were evaluated against four tumor and two normal cell lines. Most
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derivatives displayed more potent antiproliferative activities than oridonin. Among them,
compound 12b showed the strongest antiproliferative effects, with IC50 values of 1.01, 0.88, 4.36
and 5.21 µM against K562, Bel-7402, SGC-7901 and A549 human cancer cell lines, respectively.
Additionally, 12b exhibited almost 24-fold less cytotoxicity against L-02 and PBMC cell lines,
indicating good selectivity between cancer and normal cells. Hence, compound 12b was selected
for further mechanism study. The results demonstrated that the strong apoptotic effects of 12b
were attributed to G1 phase cell cycle arrest, collapse of mitochondrial membrane potential and
mediation between proapoptotic and antiapoptotic proteins through mitochondrial pathway. In
conclusion, 12b merits further investigation as a potential candidate for anticancer therapeutics
due to high efficiency and selectivity.
1
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4. Experimental
4.1. Chemistry
Chemicals and solvents were commercially available. If required, further purification was
abided by standard methods. Oridonin was purchased from Nanjing Zelang Biology Technology
1
13
Co., Ltd with HPLC purity>98%. H NMR and C NMR spectra were measured on a Bruker 400
or 600 MHz spectrometer in the indicated solvents (TMS as internal standard): the values of the
chemical shifts were expressed in δ (ppm) and the coupling constants (J) in Hz. Mass spectra were
recorded on an Agilent 1100 LC-MSD-Trap-SL. High resolution mass spectra (HR-MS) were
analyzed on Agilent Q-TOF B.05.01 (B5125.2).
4.1.1 General procedures to synthesize target derivatives
The preparation of ADT-OH was according to previous literature [82]. Oridonin (1 g, 2.76
mmol) was added in 20 mL of acetone and cooled to 0 °C. Jones reagent (3.2 mL, 8.28 mmol) was
added dropwise and stirred at 0 °C for 0.5 h. The mixture was quenched by dropwise addition of
isopropyl alcohol (1 mL) and extracted with DCM (3×30 mL). The combined organic layer was
washed with brine, dried over anhydrous Na SO and evaporated under reduced pressure to
2
4
produce the crude compound 2. Oridonin (182 mg, 0.50 mmol) was added into 10 mL acetone,
then mixed with TsOH (43.1 mg, 0.25mmol), DMAP (30.5 mg, 0.25 mmol) and
2,2-dimethoxypropane (1.5 mL). The mixture was refluxed for 1.5 h and then concentrated in
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7
vacuo to get compound 4. 4 was dissolved in 5 mL DCM and mixed with 0.5 mL TEA, DMAP
(catalytic amount) and 0.75 mL Ac O. After stirring at room temperature for 6h, extracted with
2
DCM (3×30 mL), washed with brine, dried over anhydrous Na SO , and evaporated to give
2
4
compound 5. 5 was added to 10 mL of 10% HCl/THF (1:1) and the solution was stirred at room
temperature for 4 h. Through post-processed as described above, compound 6 was got.
2 (108.6 mg, 0.3 mmol) was added in 5 mL anhydrous DCM, then reacted with α-thioctic
acid (92.8 mg, 0.45 mmol) in the presence of EDCI (172.5 mg, 0.9mmol) and DMAP (7.4 mg,
0.06 mmol). The mixture was stirred at room temperature for 6 h and then extracted with DCM
(3×30 mL), washed with brine, dried over anhydrous Na SO and evaporated in vacuo. Compound
2
4
1
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1
1
1
1
1
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1
1
2
2
2
2
2
2
2
2
8 was obtained by purification with flash column chromatography (MeOH/DCM 1:200, v/v).
Following the procedures described for 8, compound 13 was prepared from 5.
Different ent-kaurane diterpenoid derivatives (1.5 mmol) were dissolved in 5 mL anhydrous
DCM and reacted with the corresponding anhydride (3.0 mmol), under the condition of TEA (1.04
mL, 7.5 mmol) and DMAP (91.6 mg, 0.75 mmol) for 5 h at room temperature. Then extracted
with DCM (3×30 mL), washed with brine, dried over anhydrous Na SO and evaporated in vacuo
2
4
to get intermediates 10a-c and 15a-c without further purification. Intermediates 10a-c and 15a-c
(0.6 mmol) were dissolved in 5 mL anhydrous DCM, reacted with ADT-OH (135.6 mg, 0.6 mmol)
in the presence of EDCI (345.1 mg, 1.8 mmol) and DMAP (36.6 mg, 0.3 mmol). The mixture was
stirred at room temperature for 6 h and then extracted with DCM (3×30 mL), washed with brine,
dried over anhydrous Na SO , and evaporated in vacuo. Compounds 11a-c and 16a-c were
2
4
acquired by purification with flash column chromatography (MeOH/DCM 1:300, v/v).
H S-releasing ent-kaurane diterpenoid derivatives 8, 11a-c, 13 and 16a-b (0.2 mmol) were
2
dissolved in 5 mL anhydrous THF, reacted with Pb(OAc) (0.7 mmol) and Na CO (1.6 mmol) at
4
2
3
room temperature for 3 h. The mixture was extracted with DCM (3×30 mL), washed with brine,
dried over anhydrous Na SO , and evaporated in vacuo to give corresponding crude products.
2
4
Spirolactone-type 6,7-seco-ent-kaurane derivatives 9, 12a-c, 14 and 16a-b were attained by
purification with flash column chromatography (MeOH/DCM 1:400, v/v).
1
28
4.1.1.1. Compound 9. Pale yellow solid, yield: 42.1%. H NMR (CDCl , 400 MHz), δ (ppm): 9.84
3
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(1H, d, J = 3.4 Hz, -CHO), 6.23 (1H, s, 17-CH ), 5.95 (1H, s, 14-CH), 5.58 (1H, s, 17-CH ), 4.82,
2
2
4.44 (each 1H, d, J = J = 12.5 Hz, 20-CH ), 3.14 (1H, s, 13-CH), 2.56 (2H, m, 8'-CH ), 2.44
A
B
2
2
(2H, m, 2-CH ), 2.26 (2H, m, 2'-CH ), 1.82 (1H, m, -CH ), 1.72-1.52 (7H, m, -CH ), 1.48-1.36
2
2
2
2
1
3
(4H, m, -CH ), 1.22 (3H, s, 18-CH ),1.20 (3H, s, 19-CH ); C NMR (CDCl , 100 MHz), δ(ppm):
2
3
3
3
209.10, 201.91, 197.82, 172.87, 166.49, 146.64, 120.85, 73.71, 67.80, 61.50, 59.57, 52.43, 44.79,
41.63, 37.16, 36.02, 34.11, 33.54, 30.48, 29.68, 29.47, 29.30, 28.60, 27.73, 26.37, 25.81, 23.99,
+
18.83; C H O S MS (ESI) m/z calcd for [M+K] 587.2, found 587.3.
28
38
7 2
1
8
9
0
1
2
3
4
5
6
7
8
4.1.1.2. Compound 11a. Orange red solid, yield: 26.2%. H NMR (CDCl , 400 MHz), δ (ppm):
3
7.67, 7.24 (each 2H, d, J = J = 8.7 Hz, Ar-H), 7.41 (1H, s, 8"-CH), 6.24 (1H, s, 17-CH ), 6.00
A
B
2
1
1
1
1
1
1
1
1
1
(1H, s, 17-CH ), 5.61 (1H, s, 7-OH), 5.42 (1H, d, J = 11.8 Hz, 6-OH), 4.31, 4.02 (each 1H, d, J =
2
10.7 Hz, 20-CH ), 3.76 (1H, dd, J = 11.7, 8.9 Hz, 6-CH), 3.10 (1H, d, J = 9.2 Hz, 13-CH), 2.88
2
(2H, m, 2'-CH ), 2.69 (2H, m, 3'-CH ), 2.59-2.42 (2H, m, -CH ), 2.38-2.29 (1H, m, -CH ), 2.21
2
2
2
2
(1H, dd, J = 13.7, 4.7 Hz, -CH), 1.94 (2H, m, -CH ), 1.77-1.58 (2H, m, -CH ), 1.31 (1H, m, -CH ),
2
2
2
1
3
1.19 (3H, s, 18-CH ), 0.99 (3H, s, 19-CH ); C NMR (CDCl , 100 MHz), δ (ppm): 215.46,
3
3
3
211.51, 204.87, 170.71, 170.37, 153.49, 149.44, 136.01, 129.21, 128.18 (×2), 122.88 (×2), 122.20,
96.85, 74.25, 73.46, 64.93, 61.38, 59.61, 50.69, 48.51, 41.50, 38.31, 35.71, 33.37, 32.84, 30.50,
+
30.06, 29.37, 29.07, 23.16, 19.19; HRMS (ESI) m/z calcd for C H O S [M+Na] 693.1257,
3
3
34
9 3
found 693.1289.
1
19
20
21
22
23
24
25
26
27
4.1.1.3. Compound 11b. Orange red solid, yield: 29.5%. H NMR (CDCl , 400 MHz), δ (ppm):
3
7.68, 7.22 (each 2H, d, J = J = 8.68 Hz, Ar-H), 7.41 (1H, s, 8"-H), 6.26 (1H, s, 17-CH ), 5.97
A
B
2
(1H, s, 17-CH ), 5.64 (1H, s, 7-OH), 5.41 (1H, d, J = 11.76 Hz, 6-OH), 4.31, 4.03 (each 1H, d, J =
2
10.64 Hz, 20-CH ), 3.77 (1H, dd, J = 11.5, 8.8 Hz, 6-CH), 3.10 (1H, d, J = 9.3 Hz, 13-CH), 2.65
2
(2H, td, J = 3.56, 7.4 Hz, 3'-CH ), 2.43 (2H, t, J = 7.16 Hz, 4'-CH ), 2.22 (1H, dd, J = 13.5, 4.4 Hz,
2
2
-CH), 2.03 (2H, t, J = 6.84 Hz, 2'-CH ), 1.94 (2H, m, -CH ), 1.78-1.60 (2H, m, -CH ), 1.32 (1H, m,
2
2
2
1
3
-CH ), 1.19 (3H, s, 18-CH ), 1.00 (3H, s, 19-CH ); C NMR (CDCl , 100 MHz), δ (ppm): 215.47,
2
3
3
3
211.54, 204.85, 171.33, 170.89, 153.49, 149.48, 136.01, 129.17, 128.20 (×2), 122.92 (×2), 122.14,
96.90, 74.15, 73.38, 64.95, 61.35, 59.68, 53.47, 50.68, 48.53, 41.55, 38.34, 35.73, 33.28, 33.14,
1
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ACCEPTED MANUSCRIPT
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1
2
32.85, 30.50, 30.05, 23.18, 19.55, 19.18; HRMS (ESI) m/z calcd for C H O S [M+Na]
3
4
36
9 3
707.1414, found 707.1451.
1
3
4
5
6
7
8
9
0
1
2
4.1.1.4. Compound 11c. Orange red solid, yield: 31.6%. H NMR (CDCl , 400 MHz), δ (ppm):
3
7.98 (1H, dd, J = 7.4, 1.7 Hz, 6'-H), 7.79 (1H, dd, J = 7.4, 1.7 Hz, 3'-H), 7.75 (2H, d, J = 8.7 Hz,
Ar-H), 7.58 (2H, m, 4', 5'-H), 7.45 (2H, d, J = 8.7 Hz, Ar-H), 7.44 (1H, s, 8"-H), 6.08 (1H, s,
17-CH ), 6.08 (1H, s, 14-CH), 5.42 (1H, s, 17-CH ), 4.70 (1H, d, J = 10.8 Hz, 6-OH), 4.63 (1H, s,
2
2
14-CH), 3.85, 3.27 (each 1H, d, J = 9.6 Hz, 20-CH ), 2.62 (1H, m, -CH ), 2.50 (1H, m, -CH ),
2
2
2
1
3
2.28 (1H, m, -CH ), 1.95-1.59 (5H, m, -CH ), 1.51 (3H, s, 18-CH ), 1.28 (3H, s, 19-CH );
C
2
2
3
3
NMR (CDCl , 100 MHz), δ (ppm): 215.51, 208.20, 202.90, 200.66, 171.77, 165.44, 153.90,
3
1
1
1
148.55, 136.06, 131.76, 131.72, 131.68, 131.01, 130.45, 129.74, 129.40, 128.62, 128.28 (×2),
123.05 (×2), 120.08, 93.20, 74.23, 67.62, 60.97, 58.46, 49.52, 41.04, 40.86, 36.22, 33.85, 33.40,
+
29.64, 22.70, 18.19; HRMS (ESI) m/z calcd for C H O S [M+H] 717.1286, found 717.1288.
3
7
34
9
3
1
13
14
15
16
17
18
19
20
21
22
4.1.1.5. Compound 12a. Orange red solid, yield: 21.8%. H NMR (CDCl , 600 MHz), δ (ppm):
3
9.85 (1H, d, J = 3.4 Hz, -CHO), 7.66, 7.23 (each 2H, d, J = J = 8.6 Hz, Ar-H), 7.40 (1H, s,
A
B
8''-CH), 6.24 (1H, s, 17-CH ), 6.04 (1H, s, 14-CH), 5.59 (1H, s, 17-CH ), 4.82, 4.48 (each 1H, d,
2
2
J = J = 12.4 Hz, 20-CH ), 3.16 (1H, d, J = 7.6 Hz, 13-CH), 2.94 (1H, m, 2'-CH ), 2.81 (1H, m,
A
B
2
2
2'-CH ), 2.72 (2H, m, 3'-CH ), 2.60 (2H, m, 2-CH ), 2.48-2.35 (4H, m, -CH ), 1.86 (2H, m, -CH ),
2
2
2
2
2
1
3
1.73-1.62 (4H, m, -CH ), 1.42 (2H, m, -CH ), 1.25 (3H, s, 18-CH ), 1.22 (3H, s, 19-CH );
C
2
2
3
3
NMR (CDCl , 150 MHz), δ (ppm): 215.53, 208.90, 201.89, 197.81, 171.81, 171.63, 170.41,
3
166.62, 153.55, 146.63, 136.03, 129.19, 128.86, 128.18 (×2), 122.95 (×2), 121.31, 74.13, 67.86,
61.26, 59.59, 52.52, 44.88, 41.53, 37.05, 36.25, 34.18, 30.63, 29.71, 29.53, 29.04, 28.99, 25.78,
+
18.81; HRMS (ESI) m/z calcd for C H O S [M+H] 669.1286, found 669.1299.
3
3
32
9 3
1
23
24
25
26
27
4.1.1.6. Compound 12b. Orange red solid, yield: 24.6%. H NMR (CDCl , 600 MHz), δ (ppm):
3
9.86 (1H, d, J = 3.4 Hz, -CHO), 7.67, 7.23 (each 2H, d, J = J = 8.6 Hz, Ar-H), 7.41 (1H, s,
A
B
8''-CH), 6.25 (1H, s, 17-CH ), 6.03 (1H, s, 14-CH), 5.59 (1H, s, 17-CH ), 4.86, 4.48 (each 1H, d,
2
2
J = J = 12.4 Hz, 20-CH ), 3.16 (1H, d, J = 7.3 Hz, 13-CH), 2.65 (2H, m, 3'-CH ), 2.60 (1H, m,
A
B
2
2
2'-CH ), 2.45 (2H, m, 4'-CH ), 2.38 (1H, m, 2'-CH ), 2.01 (2H, m, -CH ), 1.85 (1H, m, -CH ),
2
2
2
2
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5
1.69 (4H, m, -CH ), 1.43 (1H, m, -CH ), 1.25 (3H, s, 18-CH ), 1.22 (3H, s, 19-CH ); C NMR
2 2 3 3
(CDCl , 150 MHz), δ (ppm): 215.51, 209.17, 201.95, 197.81, 172.48, 171.91, 171.07, 166.64,
3
153.62, 146.60, 136.00, 129.09, 128.86, 128.20 (×2), 123.03 (×2), 121.07, 73.93, 67.85, 61.46,
59.65, 52.46, 44.85, 41.61, 37.15, 36.13, 34.14, 32.98, 32.73, 30.55, 29.49, 19.40, 18.83; HRMS
+
(ESI) m/z calcd for C H O S [M+H] 683.1443, found 683.1436.
3
4
34
9 3
1
6
7
8
9
0
1
2
3
4
5
4.1.1.7. Compound 12c. Orange red solid, yield: 14.8%. H NMR (CDCl , 600 MHz), δ (ppm):
3
9.86 (1H, d, J = 3.4 Hz, -CHO), 7.67, 7.23 (each 2H, d, J = J = 8.6 Hz, Ar-H), 7.41 (1H, s,
A
B
8''-CH), 6.25 (1H, s, 17-CH ), 6.03 (1H, s, 14-CH), 5.59 (1H, s, 17-CH ), 4.86, 4.48 (each 1H, d,
2
2
J = J = 12.4 Hz, 20-CH ), 3.16 (1H, d, J = 7.3 Hz, 13-CH), 2.65 (2H, m, 3'-CH ), 2.60 (1H, m,
A
B
2
2
1
1
1
1
1
1
2'-CH ), 2.45 (2H, m, 4'-CH ), 2.38 (1H, m, 2'-CH ), 1.86 (2H, m, -CH ), 1.80-1.60 (5H, m, -CH ),
2 2 2 2 2
1
3
1.44 (1H, m, -CH ), 1.25 (3H, s, 18-CH ), 1.22 (3H, s, 19-CH ); C NMR (CDCl , 150 MHz), δ
2
3
3
3
(ppm): 215.54, 208.89, 201.81, 197.76, 171.74, 166.42, 166.16, 153.81, 146.70, 136.09, 132.37,
131.80, 131.49, 130.28, 129.97, 129.74, 129.43, 129.16, 128.28 (×2), 123.02 (×2), 121.37, 74.87,
67.86, 61.24, 59.64, 52.69, 45.16, 41.50, 37.02, 36.36, 34.18, 30.69, 29.61, 25.73, 18.80; HRMS
+
(ESI) m/z calcd for C H O S [M+Na] 739.1106, found 739.1110.
3
7
32
9 3
1
16
17
18
19
20
21
22
23
24
4.1.1.8. Compound 13. Pale yellow solid, yield: 19.8%. H NMR (CDCl , 400 MHz), δ (ppm):
3
6.15 (1H, s, 17-CH ), 5.80 (1H, s, 14-CH), 5.51 (1H, s, 17-CH ), 4.63 (1H, q, J = 10.6 Hz, 1-CH),
2
2
4.26, 4.18 (each 1H, d, J = 10.6 Hz, 20-CH ), 3.78 (1H, brs, -OH), 3.17 (1H, d, J = 9.7 Hz,
2
13-CH), 2.53 (1H, dq, J = 6.4, 12.6 Hz, 8'-CH), 2.44 (1H, dt, J = 6.4, 12.6 Hz, 8'-CH), 2.30 (3H, t,
J = 7.5 Hz, 2', 7'-CH, -CH ), 1.99 (3H, s, -CH ), 1.93-1.23 (20H, m, -CH ), 1.12 (6H, s, 18,
2
3
2
1
3
19-CH3); C NMR (CDCl , 100 MHz), δ (ppm): 206.21, 171.81, 169.87, 149.37, 120.47, 96.10,
3
76.40, 75.37, 73.77, 63.53, 61.48, 60.24, 56.21, 53.68, 41.33, 41.04, 40.21, 39.75, 38.47, 38.13,
34.45, 34.35, 33.58, 32.36, 30.20, 28.54, 25.17, 24.39, 21.54, 17.96; HRMS (ESI) m/z calcd for
+
C H O S [M+Na] 617.2219, found 617.2204.
3
0
42
8 2
1
25
26
27
4.1.1.9. Compound 14. Pale yellow solid, yield: 39.1%. H NMR (CDCl , 400 MHz), δ (ppm):
3
9.79 (1H, d, J = 4.7 Hz, -CHO), 6.23 (1H, s, 17-CH ), 5.79 (1H, s, 14-CH), 5.59 (1H, s, 17-CH ),
2
2
5.10, 4.76 (each 1H, d, J = J = 12.5 Hz, 20-CH ), 4.63 (1H, m, 1-CH), 2.98 (1H, d, J = 7.4 Hz,
A
B
2
2
0

ACCEPTED MANUSCRIPT
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2
3
4
5
6
13-CH), 2.31-2.24 (2H, m, -CH ), 2.23 (2H, t, J = 4.4 Hz, 2'-CH ), 2.10 (3H, s, -CH ), 2.06 (2H,
2
2
3
m, -CH ), 1.96-1.76 (4H, m, -CH ), 1.71-1.31 (10H, m, -CH ), 1.21 (3H, s, 18-CH ), 0.99 (3H, s,
2
2
2
3
1
3
19-CH3); C NMR (CDCl , 100 MHz), δ (ppm): 202.41, 196.99, 172.60, 170.37, 166.59, 146.77,
3
121.45, 74.44, 72.23, 66.34, 62.68, 62.60, 61.62, 59.89, 55.42, 53.44, 45.63, 43.57, 42.16, 39.31,
35.46, 34.01, 32.86, 29.96, 28.69, 27.75, 23.93, 23.87, 21.44, 17.45; C H O S MS (ESI) m/z
30
40
8 2
+
calcd for [M+K] 631.2, found 631.3.
1
7
8
9
0
1
2
3
4
5
6
7
4.1.1.10. Compound 16a. Orange red solid, yield: 18.8%. H NMR (CDCl , 400 MHz), δ (ppm):
3
7.68, 7.24 (each 2H, d, J = J = 8.6 Hz, Ar-H), 7.40 (1H, s, 8''-CH), 6.14 (1H, s, 17-CH ), 6.08
A
B
2
(1H, d, J = 10.8 Hz, 6-OH), 5.93 (1H, s, 14-CH), 5.50 (1H, s, 17-CH ), 4.63 (1H, dd, J = 11.1, 5.5
2
1
1
1
1
1
1
1
1
Hz, 1-CH), 4.30, 4.20 (each 1H, d, J = J = 10.6 Hz, 20-CH ), 3.91 (1H, brs, -OH), 3.77 (1H, dd,
A
B
2
J = 10.3, 6.9 Hz, 6-CH), 3.14 (1H, d, J = 10.0 Hz, 13-CH), 2.90 (2H, m, 3'-CH ), 2.72 (2H, m,
2
2'-CH ), 2.52 (1H, m, -CH ), 1.78 (1H, m, -CH ), 1.48 (2H, m, -CH ), 1.37-1.29 (4H, m, -CH ),
2
2
2
2
2
1
3
1.99 (3H, s, -CH ), 1.13 (6H, s, 18,19-CH ); C NMR (CDCl , 100 MHz), δ (ppm): 215.53,
3
3
3
206.10, 171.57, 170.72, 170.19, 169.84, 153.45, 149.42, 136.06, 129.30, 128.17(×2), 122.84(×2),
120.74, 95.91, 75.78, 75.36, 74.37, 63.59, 61.71, 59.89, 53.87, 41.18, 39.79, 38.16, 33.57, 32.46,
30.27, 29.70, 29.41, 29.07, 25.15, 21.65, 21.52, 18.13; HRMS (ESI) m/z calcd for C H O S
3
5
38 10 3
+
[M+Na] 737.1525, found 737.1534.
1
18
19
20
21
22
23
24
25
26
27
4.1.1.11. Compound 16b. Orange red solid, yield: 23.4%. H NMR (CDCl , 400 MHz), δ (ppm):
3
7.68, 7.22 (each 2H, d, J = J = 8.7 Hz, Ar-H), 7.40 (1H, s, 8''-CH), 6.17 (1H, s, 17-CH ), 6.08
A
B
2
(1H, d, J = 10.6 Hz, 6-OH), 5.89 (1H, s, 14-CH), 5.52 (1H, s, 17-CH ), 4.63 (1H, dd, J = 11.2, 5.6
2
Hz, 1-CH), 4.30, 4.20 (each 1H, d, J = J = 10.6 Hz, 20-CH ), 4.09 (1H, brs, -OH), 3.78 (1H, t, J
A
B
2
= 7.7 Hz, 6-CH), 3.16 (1H, d, J = 9.9 Hz, 13-CH), 2.65 (2H, td, J = 7.3, 2.5 Hz, 3'-CH ), 2.44 (2H,
2
t, 2'-CH ), 2.01 (3H, s, -CH ), 1.78 (1H, m, -CH ), 1.48 (2H, m, -CH ), 1.38-1.28 (4H, m, -CH ),
2
3
2
2
2
13
1.13 (6H, s, 18,19-CH3); C NMR (CDCl , 100 MHz), δ (ppm): 215.52, 206.11, 171.64, 171.32,
3
170.72, 169.86, 153.44, 149.43, 136.04, 129.23, 128.20(×2), 122.89(×2), 120.64, 95.98, 75.85,
75.36, 74.13, 63.58, 61.62, 59.97, 53.78, 41.18, 39.76, 38.13, 32.43, 30.25, 25.16, 21.61, 21.55,
+
19.59, 18.08; HRMS (ESI) m/z calcd for C H O S [M+Na] 751.1682, found 751.1649.
3
6
40 10 3
2
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9
0
4.1.1.12. Compound 16c. Orange red solid, yield: 13.1%. H NMR (CDCl , 400 MHz), δ (ppm):
3
7.95 (1H, m, 6'-H), 7.75, 7.43 (each 2H, d, J = J = 8.6 Hz, Ar-H), 7.72 (1H, m, 3'-H), 7.62 (2H,
A
B
m, 4'-H,5'-H), 7.40 (1H, s, 8"-H), 6.07 (1H, s, 7-OH), 6.07, 5.39 (2H, s, 17-CH ), 4.65 (1H, m,
2
1-CH), 4.32, 4.21 (each 1H, d, J = 10.6 Hz, 20-H), 4.00 (1H, brs, -OH), 3.78 (1H, m, 6-CH), 3.37
(1H, d, J = 9.7 Hz, 13-CH), 2.01 (3H, s, -CH ), 1.78 (1H, m, -CH ), 1.49 (2H, m, -CH ), 1.39-1.30
3
2
2
1
3
(4H, m, -CH ), 1.13 (6H, s, 18,19-CH ); C NMR (CDCl , 100 MHz), δ (ppm): 215.52, 206.12,
2
3
3
171.51, 169.85, 166.02, 164.77, 153.65, 149.41, 136.13, 132.60, 132.45, 131.53, 129.82, 129.60,
129.54, 129.04, 128.22 (×2), 123.03 (×2), 120.76, 96.02, 75.36, 74.22, 63.62, 61.74, 60.04, 53.94,
40.69, 39.82, 38.16, 33.58, 32.44, 30.26, 29.70, 25.17, 21.63, 21.57, 18.10; HRMS (ESI) m/z calcd
+
1
for C H O S [M+Na] 785.1525, found 785.1526.
39
38 10 3
1
11
12
13
14
15
16
17
18
19
20
4.1.1.13. Compound 17a. Orange red solid, yield: 7.5%. H NMR (CDCl , 400 MHz), δ (ppm):
3
9.85 (1H, d, J = 3.4 Hz, -CHO), 9.79 (1H, d, J = 4.6 Hz, -CHO), 7.66, 7.23 (each 2H, d, J = J =
A
B
8.7 Hz, Ar-H), 7.39 (1H, s, 8''-CH), 6.23 (1H, s, 17-CH ), 5.81 (1H, s, 14-CH), 5.59 (1H, s,
2
17-CH ), 5.11, 4.76 (each 1H, d, J = J = 12.4 Hz, 20-CH ), 4.63 (1H, m, 1-CH), 3.02 (1H, d, J =
2
A
B
2
7.7 Hz, 13-CH), 2.93 (1H, m, 2'-CH ), 2.82 (1H, m, 2'-CH ), 2.73 (2H, m, 3'-CH ), 2.33-2.23 (2H,
2
2
2
m, -CH ), 2.10 (3H, s, -CH ), 1.90 (2H, m, -CH ), 1.71-1.49 (6H, m, -CH ), 1.21 (3H, s, 18-CH ),
2
3
2
2
3
1
3
1.01 (3H, s, 19-CH3); C NMR (CDCl , 100 MHz), δ (ppm): 215.50, 202.31, 196.82, 171.71,
3
171.22, 170.40, 166.68, 153.57, 146.77, 136.01, 129.18, 128.15 (×2), 122.87 (×2), 121.87, 74.55,
72.59, 66.44, 62.35, 59.80, 45.69, 43.58, 41.99, 39.44, 34.04, 32.90, 29.94, 29.21 (×2), 24.09 (×2),
+
21.41, 17.53; HRMS (ESI) m/z calcd for C H O S [M+H] 713.1549, found 713.1546.
3
5
36 10 3
1
21
22
23
24
25
26
27
4.1.1.14. Compound 17b. Orange red solid, yield: 10.1%. H NMR (CDCl , 400 MHz), δ (ppm):
3
9.80 (1H, d, J = 4.7 Hz, -CHO), 7.67, 7.22 (each 2H, d, J = J = 8.6 Hz, Ar-H), 7.41 (1H, s,
A
B
8''-CH), 6.26 (1H, s, 17-CH ), 5.83 (1H, s, 14-CH), 5.60 (1H, s, 17-CH ), 5.12, 4.77 (each 1H, d,
2
2
J = J = 12.4 Hz, 20-CH ), 4.64 (1H, m, 1-CH), 3.01 (1H, d, J = 7.7 Hz, 13-CH), 2.65 (2H, m,
A
B
2
4'-CH ), 2.45 (2H, m, 2'-CH ), 2.28-2.22 (2H, m, -CH ), 2.13 (1H, s, -CH ), 2.10 (1H, d, J = 4.8
2
2
2
3
1
3
Hz, -CH), 2.03 (2H, m, 3'-CH ), 1.22 (3H, s, 18-CH ), 1.01 (3H, s, 19-CH ); C NMR (CDCl3,
2
3
3
100 MHz), δ (ppm): 215.54, 202.34, 172.13, 171.04, 170.46, 166.73, 153.62, 146.80, 136.01,
2
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128.17 (×2), 123.00 (×2), 121.70, 74.53, 72.26, 66.40, 62.49, 59.89, 45.69, 43.59, 42.09, 39.43,
34.04, 32.97 (×2), 29.96, 29.71, 23.93 (×2), 21.44, 19.45, 17.52; HRMS (ESI) m/z calcd for
+
C H O S [M+H] 727.1705, found 727.1703.
3
6
38 10 3
4
5
6
7
8
9
0
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5
6
7
8
9
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8
4.2. MTT assay
The antiproliferative activities were examined by the MTT assay following the method
described previously. All selected cell lines were incubated with target compounds for different
concentrations (64, 16, 4, 1, 0.25, 0.0625 and 0.015625 µM) through a standard 96-well plates.
After 72 h, MTT solution (20 mL, 5 mg/mL) and DMSO (150 mL/well) were added, then
incubated for 10 min at room temperature. After that, the absorbance (OD) data of each well at
490 nm wavelength were measured by a Microplate Reader (BIO-RAD). Four human cancer and
two normal cell lines were selected for the assay, and half inhibition rates (IC ) were calculated.
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0
4.3. H S release experiment
2
Sodium phosphate buffer was used to prepare the stock solution of Na S (20 mM) in 100 mL
2
volumetric flask. Aliquots of Na S stock solution were transferred into a 50 mL volumetric flask
2
to obtain standard solutions of 5, 10, 20, 40, 60, 80, 100 and 150 µM, respectively. 1 mL of each
+
standard solutions were added to react with the methylene blue (MB ) cocktail (200 µL of 30 mM
FeCl in 1.2M HCl, 200 µL of 20 mM N,N-dimethyl-1,4-phenylenediamine sulfate in 7.2M HCl
3
and 100 µL of 1%w/v of Zn(OAc) in H O) at room temperature for 20 min in a triplicate manner
2
2
[83]. The mixture was measured at 670 nm in UV-Vis spectrophotometer and then the Na2S
calibration curve was obtained. In order to promote compounds to release H S, TCEP or
2
L-cysteine was used as an accelerator. All compounds were dissolved in THF solution (40 mM)
and added into phosphate buffer in the presence of TCEP or L-cysteine (1 mM). Then 2 mL
+
mixture was transferred to colorimetric cuvette containing methylene blue (MB ) cocktail in
designated time. After 20 min incubation, the absorbance of each compound was analyzed by
UV-Vis spectrophotometer at 670 nm. The H S concentration of each derivative was calculated
2
through standard curve.
4.4. Cell cycle analysis
Effects of 12b on cell cycle arrest in Bel-7402 cells were tested and DNA content of cell
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nuclei was measured via flow cytometry [84]. Bel-7402 cells were incubated in 6-well plates at
37 °C for 24 h. Then 12b was dissolved in DMSO and joined cell culture with designated
concentrations in triplicate manner. While DMSO was used as control. After 48 h incubation, all
cells were centrifuged and fixed with 70% ethanol at 4 °C overnight and suspended again in PBS
mixed with 100 mL RNase A and 400 mL PI. Cell cycle distribution of DNA content was
evaluated via flow cytometer (FACS Calibur Becton-Dickinson, Franklin Lake, NJ, USA).
4.5. Hoechst 333258 staining
Hoechst 33258 staining assay was used to observe the morphology change of nuclei. Bel-7402
cells were cultured in 6-well plates at 37 °C with 2 mL medium. After 24 h of incubation, 12b of
designated concentrations (0, 0.45, 0.90 and 1.80 µM) was added for another 48 h incubation.
Cells were harvested by mild trypsinization, collected by centrifugation and washed twice with
PBS. After that, 500 µL of Hoechst mixture (2 mg/mL) in PBS was added for 15 min at room
temperature in darkness in order to stain aforementioned cells. Finally, cells were washed by PBS
and assessed via a fluorescence microscope.
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2
4.6. Cell apoptosis assay
Bel-7402 cells were treated with designated concentrations of 12b (0, 0.45, 0.90 and 1.80 µM)
for 24 h incubation in 6-well plates. Then, cells were washed twice with PBS and suspended in
annexin V binding buffer. The mixture was incubated with V-FITC and PI at room temperature
for 15 min in dark. After that, cells with double-staining were analyzed by flow cytometry to
detect apoptotic cells.
4.7. Mitochondrial membrane potential assay
Bel-7402 cells were cultured in 6-well plates with different concentrations of 12b (0, 0.45,
0.90 and 1.80 µM) for 48 h, then washed with PBS and stained with JC-1 at room temperature in
darkness. A flow cytometry was used to measure the number of cells with collapsed mitochondrial
membrane potentials.
4.8. Western blot assay
Bel-7402 cells were treated with 12b (0, 0.45, 0.90 and 1.80 µM) in triplicate for 48 h.
Following the measurement of protein level, sodium dodecyl sulfate polyacrylamide gel
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electrophoresis (10% gel, SDS-PAGE) was applied to split each cell lysates, and then transferred
onto nitrocellulose membranes. Afterwards membranes were blocked with 5% of nonfat milk,
incubated with monoclonal antibodies for 12 h at 4 °C. Then washed with TBST, incubated with
appropriate second antibodies and followed by chemiluminescence detection [85]. Protein
visualization was analyzed by Keygen ECL system (KeyGEN Biotech, Nanjing, China) while
resulting images were obtained by Clinx ChemiScope chemiluminescence imaging system.
ChemiScope analysis program was used to gain relative optical densities for individual protein.
Acknowledgment
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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 University.
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