Synthesis, characterization, and biological studies of diosgenyl analogs

Article abstract of DOI:10.1080/10286020.2016.1202240

A series of diosgenyl analogs were prepared from diosgenin to evaluate their anticancer activity and antithrombotic property. Analog 4, which had a spiroketal structure with a 6-aminohexanoic acid residue, exhibited the highest potency against all five tumor cell lines. It significantly blocked tumor growth, induced cell apoptosis and autophagy, and regulated cellular calcium concentration, mitochondrial membrane potential, adenosine triphosphate, and cell cycle. In addition, fluorescence-tagged compounds indicated that the analogs could rapidly accumulate in the cytoplasm, but no specific localization in the nucleus of cancer cells was observed. Furthermore, preliminary structure–activity relationship studies demonstrated that spiroketal analogs exhibit better antithrombotic activity than furostanic analogs, which exhibit the opposite effect by promoting thrombosis. Our study indicates that compound 4 may be a promising anticancer drug candidate for cancer patients with thromboembolism.

Full text of DOI:10.1080/10286020.2016.1202240

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Synthesis, characterization, and biological studies
of diosgenyl analogs
Bao-Zhan Huang, Guang Xin, Li-Mei Ma, Ze-Liang Wei, Yan Shen, Rui Zhang,
Hua-Jie Zheng, Xiang-Hua Zhang, Hai Niu & Wen Huang
To cite this article: Bao-Zhan Huang, Guang Xin, Li-Mei Ma, Ze-Liang Wei, Yan Shen, Rui Zhang,
Hua-Jie Zheng, Xiang-Hua Zhang, Hai Niu & Wen Huang (2016): Synthesis, characterization,
and biological studies of diosgenyl analogs, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2016.1202240
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Date: 07 September 2016, At: 21:24

Journal of asian natural Products research, 2016
http://dx.doi.org/10.1080/10286020.2016.1202240
Synthesis, characterization, and biological studies of
diosgenyl analogs
Bao-Zhan Huanga, Guang Xina, Li-Mei Maa,b, Ze-Liang Weia, Yan Shena,b, Rui Zhanga,b
Hua-Jie Zhenga,c, Xiang-Hua Zhanga,d, Hai Niua,e and Wen Huanga,b,c
,
alaboratory of ethnopharmacology, institute for nanobiomedical technology and Membrane Biology, West
china hospital, sichuan university, chengdu 610041, china; bregenerative Medicine research center, West china
hospital, sichuanuniversity, chengdu610041, china; cdepartmentofintegratedtraditionalchineseandWestern
d
Medicine, West china hospital, sichuan university, chengdu 610041, china; institute of sort science, sichuan
university, chengdu 610041, china; ecollege of Mathematics, sichuan university, chengdu 610041, china
ABSTRACT
ARTICLE HISTORY
received 4 March 2016
accepted 13 June 2016
A series of diosgenyl analogs were prepared from diosgenin to
evaluate their anticancer activity and antithrombotic property.
Analog 4, which had a spiroketal structure with a 6-aminohexanoic
acid residue, exhibited the highest potency against all five tumor cell
lines. It significantly blocked tumor growth, induced cell apoptosis
and autophagy, and regulated cellular calcium concentration,
mitochondrial membrane potential, adenosine triphosphate, and
cell cycle. In addition, fluorescence-tagged compounds indicated
that the analogs could rapidly accumulate in the cytoplasm, but
no specific localization in the nucleus of cancer cells was observed.
Furthermore, preliminary structure–activity relationship studies
demonstrated that spiroketal analogs exhibit better antithrombotic
activity than furostanic analogs, which exhibit the opposite effect by
promoting thrombosis. Our study indicates that compound 4 may
be a promising anticancer drug candidate for cancer patients with
thromboembolism.
KEYWORDS
diosgenin; diosgenyl analog;
antitumor; antithrombosis
1. Introduction
Because of its increasing incidence and mortality rate, cancer poses one of the most serious
challenges to public healthcare systems and places a huge burden on economic and social
development [1]. e World Health Organization attributed 8.2 million deaths to cancer in
2012. Within the next 20 years, the number of new cases of cancer is expected to increase
from 14 million in 2012 to as many as 22 million worldwide [2]. Symptomatic thrombosis
is a common complication of cancer. Patients with malignancies may have a seven-fold
increased risk of developing venous thromboembolism compared to those without cancer
[3]. Today, thromboembolism is the second most common cause of death in cancer patients;
it accounts for increased morbidity, mortality, and health care costs [4]. erefore, the
CONTACT Wen huang
baozhan2006@163.com
supplemental data for this article can be accessed http://dx.doi.org/10.1080/10.1080/10286020.2016.1202240.
© 2016 informa uK limited, trading as taylor & francis Group

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B.-Z. HuANG et AL.
treatment and prevention of tumors and thromboembolism in cancer patients have become
an important therapeutic aim and a major challenge for physicians [5].
Cancer is difficult to cure because of the successive mutations in oncogenes and suppres-
sor genes [6]. More than 100 types of cancers exist. Lung, stomach, liver, colon, and breast
cancers have been identified as the leading causes of cancer death each year [7]. Given that
current cancer therapy incurs expensive health care costs causes undesirable side effects, and
requires repeated treatments, the development of cancer therapies from naturally occurring
sterols and their semisynthetic analogs, which contain a 27-carbon atom skeleton with a
perhydro-1, 2-cyclopentenophenanthrene ring system, has become an attractive alternative
and many active molecules have been discovered [6,8].
Diosgenin (3β-hydroxy-5-spirostene), one of the most frequently encountered phy-
tochemical sterols, is a major steroidal sapogenin extracted from Dioscorea nipponoca,
Solanum incanum, and Trigonella foenum graecum [9]. Over the past few decades, diosgenin
and its analogs have been developed as chemopreventive/therapeutic agents against several
cancers. ey act by modulating multiple cell signaling events (e.g. inhibition of tumor
cell proliferation and invasion, causing cell cycle arrest, and induction of apoptosis and
autophagy) [9,10]. A series of preclinical and mechanistic studies indicate that they may
also contribute to a variety of biological activities, such as exerting antithrombotic [11,12]
and anti-inflammatory [13] effects, improving memory [14], enhancing immunity [15],
and attenuating oxidative stress [16].
In recent years, our group has been focusing on studying the synthesis and the prelimi-
nary structure–activity relationships of diosgenin and its derivatives for use in cancer therapy
[13,17–19]. Our previous work revealed that amino acid diosgenyl esters could significantly
inhibit tumor growth [13]. As an extension of this work, we herein designed and synthesized
a series of diosgenyl analogs with three different aglycon skeleton structures (S-A, S-B,
S-C, Figure 1) starting from diosgenin to evaluate anticancer activities and antithrombotic
properties of these compounds. e novel compounds were synthesized in good yields
through the esterification and opening of the F-spiroacetal ring of diosgenin. e analogs
were then investigated to elucidate the manner in which the amino acid side chains and
aglycon skeleton structures affect the cytotoxic activity of these compounds against murine
colon carcinoma C26 cells, mouse melanoma B16 cells, human hepatocellular carcinoma
HepG2 cells, human lung carcinoma A549 cells, and human breast cancer MDA-MB-231
cells. We further studied the antitumor effect of compound 4, the best analog of the series,
in C26 colon carcinoma cell lines in vitro and in the tumor xenograf model in vivo. en,
we designed a fluorescent probe, 4-chloro-7-nitrobenz-2-oxa-1, 3-diazole (NBD-Cl), to label
compound 4 for elucidating its effects on cellular processes and its subcellular distribution.
Finally, preliminary structure–activity relationships of several representative compounds
were analyzed via their antithrombotic activity using a mouse tail bleeding time assay.
Figure 1. skeleton structures of diosgenyl analogues (22β, 25r)-spirost-5-en-3β-yl 3β-esters (s-a), (25r)-
furost-5-en-26-ol-3β-yl 3β-esters (s-B) and (25r)-furost-5-en-3β-acetoxy-26-yl 3β-esters (s-c).

JOuRNAL OF ASIAN NAtuRAL PROductS ReSeARcH
3
2. Results and discussion
2.1. Preparation of diosgenyl analogs
In the present study, a series of straight-chain amino acid diosgenyl esters 2–6 (S-A-1)
with straight-chain amino acids containing 2–12 carbon atoms were synthesized in good
yields according to the previously reported methods with some modifications (Scheme 1)
[13]. Amino acid diosgenyl esters 2–6 were prepared by an esterification reaction involving
1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride/4-dimethylaminophenol
(EDC-HCl/DMAP), diosgenin, and the corresponding N-tert-butoxycarbonyl amino acids
in anhydrous dichloromethane. All N-protected amino acids 2b–6b used were synthesized
via the reaction of amino acids with di-tert-butyl pyrocarbonate ((Boc)2O) in acetone–
water mixed solvents in the presence of triethylamine (Et3N), except for the commercially
obtained compound 2b.
Scheme 2 shows the synthetic strategy of diosgenyl derivatives with furostanic skeleton
structures starting with diosgenin. Similar to the study by Hamid et al., the F-ring of the
spiroketal bond was opened under mild reductive cleavage conditions using sodium cyano-
borohydride in acetic acid at room temperature [7]. Compounds 2c–6c were converted
to 7a–11a, which possess furostanic skeleton structures. en, new straight-chain amino
acid diosgenyl derivatives 7, 8, and 11 (S-B) were obtained by deprotection of the N-tert-
butoxycarbonyl group using trifluoroacetic acid at room temperature.
As part of our ongoing research program, diosgenin was acetylated with acetic anhy-
dride–pyridine to yield diosgenyl acetate 12, and the F-ring of spiroketal linkage was reduced
according to the above method to yield a primary alcohol 13. e alcohol 13 was esterified
with four different straight-chain amino acids in the presence of EDC-HCl/DMAP in dry
dichloromethane. Finally, the corresponding straight-chain amino acid diosgenyl analogs
14–17 (S-C) were obtained by treating the compounds with trifluoroacetic acid in dichlo-
romethane (Scheme 3).
Scheme 1. synthesis of compounds 2–6. reagents and conditions: (a) (Boc)2o, et3n, ch3coch3-h2o (v:v
1:1), rt, 3–6 h, 93%–95%; (b) edc-hcl, dMaP, ch2cl2, 30–35 °c, 4–8 h, 77–82%; (c) cf3cooh, ch2cl2,
30–35 °c, 2–6 h, 80–87%.

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B.-Z. HuANG et AL.
Scheme 2. synthesis of compounds 7–11. reagents and conditions: (a) ch2cl2, ch3cooh, naBh3cn, rt,
16–20 h, 79–85%; (b) cf3cooh, ch2cl2, 30–35 °c, 2–6 h, 75–82%.
Scheme 3. synthesis of compounds 14–17. reagents and conditions: (a) ac2o, Py, 60 °c, 1 h, 93%;
(b) naBh3cn, ch2cl2, ch3cooh, rt, 18 h, 85%; (c) edc-hcl, dMaP, ch2cl2, 30–35 °c, 3–8 h, 79–88%;
(d) cf3cooh, ch2cl2, 30–35 °c, 2–6 h, 86–92%.
To further evaluate the antitumor effect of the primary amine of straight-chain amino
acid diosgenyl esters, another four straight-chain fatty acid diosgenyl esters 21–24 (S-A-
2) without primary amine residues were designed and synthesized with diosgenin as the
starting material. Compound 21 was obtained by acetylation with diosgenin, butyric anhy-
dride, and pyridine at room temperature (Scheme 4). Fatty acid diosgenyl esters 22–24

JOuRNAL OF ASIAN NAtuRAL PROductS ReSeARcH
5
Scheme 4. synthesis of compound 21. reagents and conditions: (a) butyric anhydride, pyridine, rt, 7 h,
89%.
Scheme 5. synthesis of compounds 22–24. reagents and conditions: (a) edc·hcl, dMaP, ch2cl2, 30–35 °c,
2–6 h, 79–84%.
were prepared by an esterification reaction involving EDC-HCl/DMAP, diosgenin, and
the corresponding straight-chain fatty acids in anhydrous dichloromethane (Scheme 5).
2.2. Structure–activity relationship of diosgenyl analogs against tumor cells
In this work, diosgenyl analogs, including five straight-chain amino acid (22β, 25R)-spirost-5-
en-3β-yl esters (2–6) (S-A-1), three straight-chain amino acid (25R)-furost-5-en-26-ol-3β-yl
esters (7, 8 and 11) (S-B), four straight-chain amino acid (25R)-furost-5-en-3β-acetoxy-
26-yl esters (14–17) (S-C), and five straight-chain fatty acid (22β, 25R)-spirost-5-en-3β-yl
esters (12, 21–24) (S-A-2) were synthesized and the cytotoxic activity of these compounds
was evaluated through the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bro-
mide (MTT) assay in the following tumor cell lines: murine colon carcinoma C26 cells,
mouse melanoma B16 cells, human hepatocellular carcinoma HepG2 cells, human lung
carcinoma A549 cells, and human breast cancer MDA-MB-231 cells. e IC50 values of
deltonin were also included in the study for comparison.
e data presented in Table 1 show that compounds 3–5 (S-A-1) and compound 8
(S-B), which have straight-chain amino acid side chains with a 4–8 carbon chain length,
demonstrate significantly higher inhibitory activity against all five cancer cell lines than
either diosgenin or other diosgenyl analogs that have straight-chain amino acid side chains
with a too short or long carbon chain length (e.g. 2 or 12 carbon atoms). Among them,
compound 4 containing a 6-aminocaproic acid residue with 6-carbon atom length shows

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B.-Z. HuANG et AL.
Table 1. In vitro antiproliferative activities of diosgenyl analogues.a
Ic50 (μM)
compound
c26
B16
HepG2
A549
MdA-MB-231
1
2
diosgenin
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
O
O
O
R
O
3
4
5
6
7
(s-a-1)
7.7 0.8
5.5 0.6
12.3 2.4 28.5 4.3 12.2 1.6
8.6 1.0 16.9 2.1 13.4 2.6
10.1 0.6
8.3 1.2
H2
N
n
R=
8.9 0.8
> 30
>30
10.6 1.2 20.8 2.7 17.8 2.9
13.0 1.5
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
OH
O
O
R
O
8
11
(s-B)
9.4 0.7
> 30
8.8 1.7
> 30
> 30
> 30
10.7 1.1
> 30
14.4 1.3
> 30
H2
N
n
R=
R
O
14
> 30
> 30
> 30
> 30
> 30
O
O
O
O
15
16
17
(s-c)
22.3 0.6
25.7 3.8
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
H2
N
n
R=
O
12
> 30
> 30
> 30
> 30
> 30
O
O
R
O
21
22
23
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
(s-a-2)
H3
C
n
R=
24
> 30
> 30
10.4 2.6
2.3 0.7
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
15.4 2.2
3.5 0.7
28
35
15.3 2.7 23.5 2.6 16.4 1.8
5.6 1.3 8.8 2.1 8.6 1.4
deltoninb
adata are obtained from a minimum of n = 3 independent Mtt assays for each compound, and represented as mean s.e.m.
bdeltonin was used in the study for comparison.
stronger activity than other diosgenyl analogs, especially compound 4 against C26 cell lines
(IC50 5.5 μM). It may be noted that straight-chain amino acid diosgenyl analogs with S-A-1
and S-B structures have better activity than those with S-C structures. Amino acid diosgenyl
analogs 2–6 (S-A-1) exhibited higher anticancer activity than highly lipophilic fatty acid
diosgenyl analogs 12, 21–24 (S-A-2), which do not have a primary amino group. ese
results suggest that the 4–8 carbon chain length of straight-chain amino acid residues and
S-A-1 and S-B aglycon structures are optimal for the antitumor effect of diosgenyl analogs.
2.3. Fluorescent cell imaging and cellular distribution of diosgenyl analogs
Increasing pre-clinical and mechanistic studies had been independently conducted and
showed that diosgenin and diosgenyl derivatives exhibited antiproliferative effects against
several types of cancer via multiple molecular mechanisms [9,10]. However, the mecha-
nisms of action of these molecules against tumors remained unclear, and there was little
information available on the localization and distribution of these molecules in live cells.
Fluorescence imaging of the living cells would be the most powerful means for the
real-time monitoring of cellular processes (including specific interactions or subcellular

JOuRNAL OF ASIAN NAtuRAL PROductS ReSeARcH
7
Scheme 6. synthesis of compound 28. reagents and conditions: (a) nBd-cl, et3n, thf, 50 °c, 5 h, 66%.
Scheme 7. synthesis of compound 32. reagents and conditions: (a) N-α-fmoc-N-ε-Boc-l-lysine, edc·hcl,
dMaP, ch2cl2, rt, 18 h; (b) piperidine, dMf, rt, 3 h; (c) nBd-cl, et3n, thf, 50 °c, 4 h; (d) cf3cooh, ch2cl2,
30 °c, 4 h.
localization) of bioactive molecules [20]. Reported protocols for the fluorescence-tagged
natural products are tedious and result in low yield because of laborious reaction steps
and protection–deprotection procedures. e molecular sizes of the fluorescent probes,
such as fluorescein and rhodamine, are usually too large and may induce alterations in
membrane organization [20]. It has been reported that 4-chloro-7-nitrobenz-2-oxa-1,3-
diazole (NBD-Cl) is nonfluorescent, small in size, has high permeability, and easily under-
goes nucleophilic substitution by the amine functional group to become a highly sensitive

8
B.-Z. HuANG et AL.
Figure 2. the cellular localization of diosgenyl analogues in live c26 cells.
notes: c26 cells were incubated with 10 μM compound 28 (a) and compound 35 (B) for 24 h, co-stained with hoechst
33,342, and then imaged using a laser scanning confocal microscopy. nucleus: blue (hoechst 33,342), cytoplasm: green
(nBd-labeled diosgenyl analogues).
fluorescent entity [20]. erefore, NBD-Cl is a good tag to label bioactive compounds, and
can be applied to track their mode of action through fluorescent cell imaging.
Here, NBD-Cl was successfully used to label diosgenyl analogs, and two NBD-tagged
diosgenyl derivatives 28 and 32 were synthesized in high yield (Schemes 6 and 7). C26
cells were treated with compounds 28 and 32 (10 μM) for 24 h, and analyzed for cellular
internalization and localization under a confocal microscope. Our data illustrated that
compounds 28 and 32 localized and played a role in the plasma membrane and cytoplasm
of C26 cells, but no specific localization in the nucleus was seen (Figure 2). en, the cyto-
toxic activity of these two compounds was evaluated through an MTT assay. Compounds
32 and 4, both containing a primary amino group, exhibited equal anticancer activity, and
showed higher inhibition against cancer cells than compound 28, which lacks a primary
amino group (Table 1).
e labeling of small molecules with fluorescent probes is an attractive technique to
elucidate cellular processes, and it has gradually become a trend in cell biology that this
technique was used to investigate the location of the bioactive molecules. Evaluation of
the cytotoxic activity of NBD-tagged diosgenyl derivative 32 and diosgenyl analog 4 indi-
cated that the labeling of NBD residues did not drastically alter the cytotoxic activity. e
internalization and localization of diosgenyl analogs were imaged successfully inside the
C26 cells. e analogs were found to be localized in the plasma membrane and cytoplasm
but not in the nucleus. In addition, NBD-based fluorescent labeling protocol works well
under mild conditions. erefore, this methodology can be applied for other diosgenyl

JOuRNAL OF ASIAN NAtuRAL PROductS ReSeARcH
9
Figure 3. diameter distribution of nanocompound 4 in ethanol–water solution. (a) schematic illustration
of compound 4 self-assembled into nanoparticles in 2% ethanol–water solution. (B) diameter distribution
of compound 4 in 2% ethanol–water solution, and the average size (Dh) was 163 nm. inset: digital
photographs of diosgenin (1 mg/ml) and nano-compound 4 (1 mg/ml) in 2% ethanol–water solution.
(c) teM image of compound 4 nanoparticles in 2% ethanol–water solution.
derivatives and even natural saponins to study cellular processes, especially cellular uptake,
distribution, or specific interactions.
2.4. The antitumor effect of analogue 4 in vivo and in vitro
Clinical application of diosgenin and its analogs in cancer therapy is limited due to their
undesirable pharmaceutical characteristics, such as poor solubility and low bioavailability
[21]. Nanotechnology for drug delivery can improve pharmacokinetics, prolong the sustain
release of drugs, and preferentially accumulate in tumor by the enhanced permeability and
retention effect [2,21]. Here, the amphiphilic analog 4 (5 mg) was dissolved in the ethanol
(100 μl), and then slowly added into deionized water (5 ml) under stirring. Compound 4
(1 mg/ml) was soluble in 2% ethanol–water solution, while diosgenin was insoluble at the
same concentration in 2% ethanol–water solution (Figure 3(A) and (B)). Surprisingly, we
found that compound 4 could successfully self-assemble into nanoparticles in water because
of its amphiphilic property (Figure 3(A)–(C)). Using nanoparticle size analyzer (DSA) and
transmission electron microscope (TEM), the mean hydrodynamic diameter size was about
163 nm with a unimodal size distribution (Figure 3 (B) and (C)). e nanodrug delivery
system of the diosgenyl analogue may attribute to its antitumor property.
In this study, compound 4, the most potent molecule of the series, was selected to further
elucidate the molecular mechanism of action of diosgenyl analogs against cancer in vitro
and in vivo. To determine the cytotoxicity of compound 4 in C26 cells, cells were treated

10
B.-Z. HuANG et AL.
Figure 4. the growth inhibition of c26 cells treated with compound 4 in vitro. (a) compound 4 blocked c26
cells growth in a dose-dependent manner. (B) compound 4 inhibited c26 cells proliferation and induced
cell morphology changes. (c) compound 4 suppressed c26 cell colonies formation in a dose-dependent
manner. (d) compound 4 blocked c26 cell cycle arrest in s phase.
note: *p < 0.05, p < 0.01 compared to control, error bars depict s.e.m.
with different concentrations of compound 4. Our data showed that compound 4 could
effectively inhibit cell proliferation (Figure 4(A)). Furthermore, results from microscopic
images of cell morphology showed that significant cell death was observed afer treatment
with compound 4 for 24 h (Figure 4(B)). In addition, compound 4 significantly blocked
C26 cell colony formation in a dose-dependent manner (Figure 4(C)). Cell cycle regulation
assures the fidelity of cell division and genomic replication. Induction of cell cycle arrest in
cancer cell lines leads to genomic instability, which is one of the most prevalent strategies
to stop cancer from spreading. Our data demonstrated that compound 4 arrested cancer
cells in S-phase to block DNA replication and induce cell death (Figure 4(D)).
In addition, compound 4 led to intracellular calcium concentration ([Ca2+]i) overload
in dose- and time-dependent manners (Figure 5(A)). [Ca2+]i overload in the cytoplasm
trigged the opening of the mitochondrial permeability transition pore, resulting in depolar-
ization of mitochondrial membrane potential (ΔΨ) (Figure 5(B)) and decrease in adenosine
triphosphate (ATP) (Figure 5(C)) [22]. With the presence of insufficient cellular ATP, AMP-
activated protein kinase (AMPK) was activated, leading to cell death [23].
Diosgenin and its derivatives are effective cytotoxic agents and induce apoptosis and
autophagy in various types of cancer cells [17–19,24]. Poly ADP-ribose polymerase (PARP)

JOuRNAL OF ASIAN NAtuRAL PROductS ReSeARcH
11
Figure 5. influence of compound 4 on cellular calcium concentration, mitochondrial membrane potential
and cellular atP level in c26 cells. (a) compound 4 increased cellular calcium concentration in c26 cells
in dose- and time-dependent manners. (B) compound 4 decreased mitochondrial membrane potential
in c26 cells in dose- and time-dependent manners. (c) compound 4 decreased cellular atP level in c26
cells in dose- and time-dependent manners.
note: *p < 0.05 compared to control, error bars depict s.e.m.
is a nuclear enzyme that is involved in DNA repair. During apoptosis, PARP is specifically
cleaved and inactivated by caspases. Accordingly, cleavage of PARP is a well-established
marker for the detection of apoptosis. In the present study, PARP was found to be cleaved
in C26 cells afer 24 h of treatment with compound 4, implying activation of caspases that
triggered apoptosis (Figure 6(A)). Morphological changes of C26 cells treated with com-
pound 4 were characterized by TEM. It can be noted that compound 4 led to an increase in
the number of autophagosomes (Figure 6(B)). e increase in endogenous PE-conjugated
LC3-II afer treatment with compound 4 further confirmed the activation of the autophagic
response induced by compound 4 (Figure 6(C)). ese results indicated that compound 4
inhibited tumor growth via apoptosis and autophagy. Consistently, dioscin, one of diosg-
enyl saponins, also induced autophagy and apoptosis in human lung cancer cell lines [24].

12
B.-Z. HuANG et AL.
Figure 6. compound 4 induced c26 cells death via apoptosis and autophagy. (a) c26 cells were treated
with compound 4 (0, 1.25, 2.5 and 5 μM) for 24 h, and compound 4 induced cleavage of ParP in c26 cells
to trigger cell apoptosis. (B) c26 cells were treated with compound 4 (0, 5 μM) for 24 h, and compound 4
increased autophagosome in cells and induced cells autophagy. a, the normal morphology of c26 cells by
transmission electron micrograph; b, the changed morphology of c26 cells with autophagic vesicles after
24 h treatment with compound 4 (5 μM); c and d, the representatively enlarged images of c26 cells with
typical autophagosomes and early autolysosomes (c) and late autolysosomes (d). (c) c26 cells were treated
with compound 4 (5 μM) for different time, and compound 4 promoted the conversion of lc3-i to lc3-ii.
To well evaluate the therapeutic potential of compound 4 on cancer in vivo, a tumor
xenograf model of C26 cells was successfully established in this study [19]. As shown in
Figure 7(A), the intraperitoneal administration of compound 4 at doses of 15 and 30 mg/kg
results in the reduction of tumor growth by 26 and 44%, respectively, compared with control
mice. During the experiment, no significant changes in the appetite, behavior, and weight of
the mice were observed (Figure 7(B)). Furthermore, no significant pathological changes in
the heart, liver, lung or kidney were detected by H&E staining (Figure 7(C)). Our previous
study indicated that diosgenyl analogs could inhibit tumor growth through suppressing
angiogenesis [18,19]. To further test whether compound 4 contributed to the inhibition of
angiogenesis in tumor, we used immunohistochemical staining assay by anti-CD31 antibody
to detect the microvessels in tumor tissues. It was noted that compound 4 significantly
decreased the vessel density (Figure 7(D)). Our data showed that compound 4 significantly
blocked C26 derived-tumor growth by suppressing angiogenesis without obvious toxicity
in vivo. In summary, compound 4 is a potential anticancer drug candidate.
2.5. Structure–activity relationship of diosgenyl analogs on thrombosis
The preliminary antithrombotic activity of the diosgenyl analogs was evaluated using
a mice tail bleeding time assay [11]. Diosgenin (1) and the representative diosgenyl
analogs (4, 16 and 22) with 6-carbon atom side chains were selected for testing. To
investigate the effect of these analogs on thrombosis, we measured tail bleeding times
1 h after the last administration. As shown in Figure 8, the bleeding times of the con-
trol group and diosgenin group were 342 and 532 s, respectively. Compounds 4 and
22, which had spiroketal structures (S-A), exhibited excellent efficacy in prolonging

JOuRNAL OF ASIAN NAtuRAL PROductS ReSeARcH
13
Figure 7. compound 4 inhibited tumor growth and angiogenesis in c26 implanted-tumor mouse model
in vivo. (a) compound 4 suppressed tumor growth in c26 tumor models. (B) toxicity-dependent weight
loss in tumor-bearing mice treated with compound 4. there are no significant differences in weight
between the three groups. (c) typical h&e staining of paraffin embedded sections of the heart, liver,
lung and kidney. compound 4 did not cause obvious pathologic abnormalities in normal tissues. (d)
frozen sections of tumor tissues were tested by immune histochemical analysis with anti-cd31 antibody.
compound 4 blocked blood vessel growth within tumors.
note: *p < 0.05 compared to control, error bars depict s.e.m.
bleeding time. Unexpectedly, compound 16, which had furostanic structure (S-C),
exhibited the opposite effect and reduced bleeding time compared to that observed
for the control group and the diosgenin group. Our data indicated that diosgenyl ana-
logs with spiroketal structures (S-A) blocked thrombosis while diosgenyl analogs with
furostanic structures (S-C) promoted thrombosis.

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B.-Z. HuANG et AL.
Figure 8. effect of diosgenyl analogues 1, 4, 16 and 22 on tail bleeding time in mice.
notes: the saline was used as the control, n = 10 per group. total bleeding times were recorded within 15 min. horizontal
bars represent means. *p < 0.05 and **p < 0.01 compared with control.
3. Conclusion
As an extension of our previous study, we synthesized 12 straight-chain amino acid diosgenyl
analogs, five straight-chain fatty acid diosgenyl derivatives, and two fluorescence-labeled
diosgenyl conjugates with different aglycon skeleton structures (S-A, S-B, S-C) with dios-
genin as the starting material to further evaluate the anticancer activity and antithrom-
botic properties of these compounds. Compound 4, which contained a spiroketal structure
with a 6-aminohexanoic acid residue, was the most effective molecule in blocking cancer
cell growth in vitro and in vivo. It rapidly accumulates in the cytoplasm (but not in the
nucleus), induces the accumulation of intracellular Ca2+, induces loss of ΔΨm and decrease
of cellular ATP, arrests the cell cycle in S-phase, and induces cell apoptosis and autophagy.
Structure–activity relationship studies indicated that spiroketal analogs exhibited higher
antithrombotic activity than diosgenin, and that furostanic analogs exhibited the opposite
effect of spiroketal analogs by promoting thrombosis. Our study also indicated that the
aglycon structure and the length of the carbon chain of straight-chain amino acid residues
in diosgenyl analogs were both important for the antitumor and antithrombotic activity
of the analogs.
Compound 4 effectively blocks tumor growth and inhibits thrombosis, suggesting that
it may be a promising anticancer drug candidate and may have translational potential
to complement currently used clinical drugs for cancer patients with thromboembolism.
Further investigation of the biological behavior of diosgenyl analogs is needed to elucidate
their anticancer effects and antithrombotic properties.
4. Experimental
4.1. Materials and methods
All air and moisture sensitive reactions were performed under nitrogen atmosphere.
Anhydrous pyridine was distilled over potassium hydroxide, dry dichloromethane over
calcium hydride. Unless otherwise stated, all commercially available reagents and solvents
were used as supplied without further purification. e structural characterization of com-
pounds was performed by NMR and high-resolution mass spectrometry. 1H NMR and 13C

JOuRNAL OF ASIAN NAtuRAL PROductS ReSeARcH
15
NMR spectra were recorded on Varian UNITY INOVA 400 or Bruker AV II-600 spectrom-
eter with tetramethylsilane as an internal standard, and chemical shifs are reported in δ
(ppm). HRESIMS were obtained with ESI-Q-TOF-MS (Bruker Daltonics, Billerica, USA).
Analytical thin-layer chromatography (TLC) was performed on Merck 60 F254 silica gel
plates, and visualization performed by UV or by spraying with sulfuric acid or ninhydrin.
Column chromatography was performed with silica gel H 60 (200–300 mesh, Qingdao,
China).
4.2. Chemical synthesis
4.2.1. Synthesis of (22β, 25R)-spirost-5-en-3β-yl amino acid esters (2–6)
4.2.1.1. General procedure for the synthesis of tert-butoxycarbonyl amino acid esters
3b–6b. Di-tert-butyl dicarbonate ((Boc)2O, 11.0 mmol) in a solution of CH3COCH3
(5 ml) was added dropwise to a stirred solution of amino acid (3a–6a, 10.0 mmol) and
triethylamine (Et3N, 20.0 mmol) in acetone-water (v:v 1:1, 20 ml), and the resulting solution
was stirred for 4–8 h at room temperature until absence of amino acid (checked by TLC).
e organic component of the solvent was removed under vacuum and the aqueous residue
was acidified with dilute HCl until pH 4–5. e solution was extracted with EtOAc, and the
combined organic fractions were washed with brine, dried over anhydrous Na2SO4, and
then concentrated in vacuo and freeze-dried in −80 °C to yield 3b–6b.
N-tert-Butoxycarbonyl-4-aminobutyric acid (3b): light yellow solid, (94%); Rf = 0.59
(CH2Cl2/CH3OH/CH3COOH 9:1:0.1); 1H NMR (400 MHz, CDCl3) δ 11.19 (s, 1H), 4.76
(s, 1H), 3.19 (d, J = 5.7 Hz, 2H), 2.39 (t, J = 7.2 Hz, 2H), 1.93–1.72 (m, 2H), 1.44 (s, 9H);
13C NMR (100 MHz, CDCl3) δ 177.4, 155.2, 78.5, 38.8, 30.3, 27.4, 24.1; HRESIMS: m/z
204.1226 [M + H]+ (calcd for C9H18NO4, 204.1236).
N-tert-Butoxycarbonyl-8-aminooctanoic acid (5b): light yellow solid, (93%); Rf = 0.58
(CH2Cl2/CH3OH/CH3COOH 9:1:0.1); 1H NMR (400 MHz, CDCl3) δ 10.99 (s, 1H), 4.51
(s, 1H), 3.03 (d, J = 5.9 Hz, 2H), 2.26 (t, J = 7.5 Hz, 2H), 1.63–1.49 (m, 2H), 1.37 (s, 11H),
1.25 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 178.3, 155.0, 78.1, 39.5, 33.0, 28.9, 27.9, 27.4,
25.5, 23.6; HRESIMS: m/z 260.1808 [M + H]+ (calcd for C13H26NO2, 260.1862).
N-tert-Butoxycarbonyl-12-aminolauric acid (6b): light yellow solid, (95%); Rf = 0.60
(CH2Cl2/CH3OH/CH3COOH 9:1:0.1); 1H NMR (400 MHz, CDCl3) δ 11.39 (s, 1H), 4.50 (s,
1H), 3.03 (d, J = 5.8 Hz, 2H), 2.27 (t, J = 7.5 Hz, 2H), 1.65–1.47 (m, 2H), 1.37 (s, 11H), 1.20
(s, 14H); 13C NMR (100 MHz, CDCl3) δ 178.4, 155.0, 78.1, 39.6, 33.1, 29.0, 28.4, 28.2, 28.0,
27.4, 25.8, 23.7; HRESIMS: m/z 338.2229 [M + Na]+ (calcd for C17H33NO4Na, 338.2307).
4.2.1.2. General procedure for the synthesis of (22β, 25R)-spirost-5-en-3β-yl N-tert-
butoxycarbonyl amino acid esters 2c–6c. Compounds 2c and 4c were synthesized as per
reported method [13].
To a solution of compound 1 (2.0 mmol), 2b–6b (2.5 mmol), DMAP (0.4 mmol) in
anhydrous CH2Cl2 (20 ml), EDC·HCl (2.5 mmol) in CH2Cl2 (6 ml) was added dropwise
under N2 atmosphere. e mixture was lef to stir at room temperature for 4–16 h under a
balloon of nitrogen (checked by TLC). en the reaction solution was washed with brine,
saturated NaHCO3 solution and brine, dried over anhydrous Na2SO4, and then filtered and
concentrated. e residue was purified by a silica gel column chromatography (petroleum
ether/EtOAc 12:1–8:1) to give 2c–6c.

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B.-Z. HuANG et AL.
(22β, 25R)-Spirost-5-en-3β-yl N-tert-butoxycarbonyl-4-aminobutyrate (3c): white solid
1
(81%); Rf = 0.30 (petroleum ether/EtOAc 4:1); H NMR (400 MHz, CDCl3) δ 5.30 (d,
J = 4.9 Hz, 1H), 4.64–4.41 (m, 2H), 4.34 (dd, J = 15.0, 7.5 Hz, 1H), 3.44–3.35 (m, 1H), 3.30
(t, J = 10.9 Hz, 1H), 3.09 (d, J = 5.8 Hz, 2H), 2.38–2.13 (m, 4H), 2.01–1.85 (m, 2H), 0.97
(s, 3H), 0.90 (d, J = 6.9 Hz, 3H), 0.72 (t, J = 3.1 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ
171.7, 154.9, 138.6, 121.4, 108.3, 79.8, 73.0, 65.8, 61.1, 55.4, 48.9, 40.6, 39.2, 38.7, 37.1, 35.9,
35.7, 31.0, 30.8, 30.4, 29.3, 27.8, 27.4, 26.7, 24.3, 19.8, 18.3, 16.1, 15.3, 13.5; HRESIMS: m/z
600.4294 [M + H]+ (calcd for C36H58NO6, 600.4264).
(22β, 25R)-Spirost-5-en-3β-yl N-tert-butoxycarbonyl-8-aminooctanoate (5c): white solid
1
(77%); Rf = 0.41 (petroleum ether/EtOAc 4:1); H NMR (400 MHz, CDCl3) δ 5.30 (d,
J = 5.0 Hz, 1H), 4.59–4.49 (m, 1H), 4.43 (s, 1H), 4.34 (dd, J = 14.9, 7.5 Hz, 1H), 3.45–3.35
(m, 1H), 3.30 (t, J = 10.9 Hz, 1H), 3.02 (t, J = 7.0 Hz, 2H), 2.31–2.13 (m, 4H), 1.98–1.86
(m, 2H), 1.84–1.74 (m, 3H), 0.97 (s, 3H), 0.90 (d, J = 6.9 Hz, 3H), 0.72 (t, J = 3.1 Hz, 6H);
13C NMR (100 MHz, CDCl3) δ 172.2, 155.0, 138.7, 121.3, 108.2, 79.8, 72.6, 65.8, 61.1, 55.4,
48.9, 40.6, 39.2, 38.7, 37.1, 36.0, 35.7, 33.6, 31.0, 30.8, 30.4, 29.3, 29.0, 28.0, 27.9, 27.8, 27.4,
26.8, 25.6, 23.9, 19.8, 18.3, 16.1, 15.3, 13.5; HRESIMS: m/z 656.4808 [M + H]+ (calcd for
C40H66NO6, 656.4890).
(22β, 25R)-Spirost-5-en-3β-yl N-tert-butoxycarbonyl-12-aminolaurate (6c): white solid
1
(82%); Rf = 0.48 (petroleum ether/EtOAc 4:1); H NMR (400 MHz, CDCl3) δ 5.30 (d,
J = 4.9 Hz, 1H), 4.65–4.48 (m, 1H), 4.42 (s, 1H), 4.34 (dd, J = 15.0, 7.5 Hz, 1H), 3.40 (dd,
J = 10.1, 3.4 Hz, 1H), 3.30 (t, J = 10.9 Hz, 1H), 3.02 (t, J = 7.1 Hz, 2H), 2.28–2.14 (m,
4H), 1.98–1.86 (m, 2H), 1.83–1.74 (m, 3H), 0.97 (s, 3H), 0.90 (d, J = 6.9 Hz, 3H), 0.72 (t,
J = 3.1 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 172.3, 155.0, 138.8, 121.3, 108.2, 79.8, 72.6,
65.8, 61.1, 55.4, 48.9, 40.6, 39.2, 38.7, 37.1, 36.0, 35.7, 33.7, 31.0, 30.8, 30.4, 29.3, 29.1, 28.5,
28.4, 28.3, 28.2, 28.1, 27.8, 27.4, 26.8, 25.8, 24.0, 19.8, 18.3, 16.1, 15.3, 13.5; HRESIMS: m/z
712.5445 [M + H]+ (calcd for C44H74NO6, 712.5516).
4.2.1.3. General procedure for the synthesis of (22β, 25R)-spirost-5-en-3β-yl amino acid
esters 2–6. CF3COOH (20.0 mmol) was added to a stirred solution of 2c–6c (1.5 mmol)
in CH2Cl2 (20 ml) at room temperature for 4–10 h. en the mixture solution was removed
in vacuo and purified by column chromatography (CH2Cl2/CH3COCH3 5:1–2:1) to yield
compounds 2–6.
Compounds 2 and 4 were synthesized as per reported method [13].
(22β, 25R)-Spirost-5-en-3β-yl 4-aminobutyrate (3): light yellow solid, (87%); Rf = 0.19
(CH2Cl2/CH3OH/CH3COOH 6:1:0.1); 1H NMR (400 MHz, CDCl3) δ 5.37 (d, J = 4.6 Hz,
1H), 4.67–4.53 (m, 1H), 4.41 (dd, J = 15.0, 7.4 Hz, 1H), 3.51–3.32 (m, 4H), 2.79 (t, J = 6.9 Hz,
2H), 2.38–2.28 (m, 4H), 2.05–1.92 (m, 2H), 1.03 (s, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.79 (t,
J = 3.1 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 171.1, 138.6, 121.4, 108.2, 79.8, 73.4, 65.8,
61.1, 55.5, 48.9, 40.6, 39.7, 39.2, 38.7, 38.4, 37.0, 35.9, 35.7, 31.0, 30.8, 30.5, 30.4, 29.3,
27.8, 26.7, 21.7, 19.8, 18.3, 16.1, 15.3, 13.5; HRESIMS: m/z 500.3780 [M + H]+ (calcd for
C31H50NO4, 500.3740).
(22β, 25R)-Spirost-5-en-3β-yl 8-aminooctanoate (5): light yellow solid, (84%); Rf = 0.31
(CH2Cl2/CH3OH/CH3COOH 6:1:0.1); 1H NMR (400 MHz, CDCl3) δ 5.37 (d, J = 4.7 Hz,
1H), 4.68–4.54 (m, 1H), 4.41 (dd, J = 15.0, 7.5 Hz, 1H), 3.47 (dd, J = 10.2, 3.3 Hz, 1H),
3.37 (t, J = 10.9 Hz, 1H), 2.68 (t, J = 7.0 Hz, 2H), 2.35–2.20 (m, 4H), 1.04 (s, 3H), 0.97 (d,
J = 6.9 Hz, 3H), 0.79 (t, J = 3.0 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 172.2, 138.7, 121.3,

JOuRNAL OF ASIAN NAtuRAL PROductS ReSeARcH
17
108.3, 79.8, 72.7, 65.8, 61.1, 55.4, 48.9, 44.9, 40.6, 39.2, 38.9, 38.7, 37.1, 36.0, 35.7, 33.5, 31.0,
30.8, 30.4, 29.3, 27.8, 27.6, 26.8, 26.5, 25.3, 23.8, 19.8, 18.3, 16.1, 15.3, 13.5, 7.7; HRESIMS:
m/z 556.4348 [M + H]+ (calcd for C35H58NO4, 556.4366).
(22β, 25R)-Spirost-5-en-3β-yl 12-aminolaurate (6): light yellow solid, (80%); Rf = 0.42
(CH2Cl2/CH3OH/CH3COOH 6:1:0.1); 1H NMR (400 MHz, CDCl3) δ 5.37 (d, J = 4.9 Hz, 1H),
4.67–4.53 (m, 1H), 4.41 (dd, J = 15.0, 7.5 Hz, 1H), 3.51–3.44 (m, 1H), 3.37 (t, J = 10.9 Hz,
1H), 2.72 (t, J = 7.2 Hz, 2H), 2.34–2.23 (m, 4H), 2.04–1.93 (m, 2H), 1.90–1.81 (m, 3H),
1.04 (s, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.79 (t, J = 3.1 Hz, 6H); 13C NMR (100 MHz, CDCl3)
δ 173.3, 139.8, 122.3, 109.3, 80.8, 73.6, 66.8, 62.1, 56.4, 49.9, 41.6, 40.3, 39.7, 38.1, 37.0, 36.7,
34.7, 32.0, 31.8, 31.4, 30.3, 29.5, 29.4, 29.3, 29.1, 28.8, 27.8, 26.8, 25.1, 20.8, 19.3, 17.1, 16.3,
14.5; HRESIMS: m/z 612.4990 [M + H]+ (calcd for C39H66NO4, 612.4992).
4.2.2. Synthesis of (25R)-furost-5-en-26-ol-3β-yl amino acid esters (7–11)
4.2.2.1. General procedure for the synthesis of (25R)-furost-5-en-26-ol-3β-yl N-tert-
butoxycarbonyl amino acid esters 7a–11a. Compounds 2c–6c were stirred with
CH3COOH (12 ml) in CH2Cl2 (6 ml) at room temperature for 30 min, and then sodium
cyanoborohydride (8 mmol) was added in portions over a period of 45 min. is mixture
was lef to stir at room temperature overnight. en brine (10 ml) was added and extracted
with CH2Cl2 (15 ml*3). e solution was washed with brine, saturated NaHCO3 solution
and brine, respectively. Afer drying the organic layer with Na2SO4 and filtering, the solvent
was removed in vacuo and purified by column chromatography (petroleum ether/EtOAc
5:1–3:1) to yield 7a-11a.
(25R)-Furost-5-en-26-ol-3β-yl N-tert-butoxycarbonyl-glycinate (7a): white solid (82%);
Rf = 0.44 (petroleum ether/EtOAc 1:1); 1H NMR (400 MHz, CDCl3) δ 5.37 (d, J = 4.3 Hz,
1H), 5.06 (s, 1H), 4.73–4.61 (m, 1H), 4.35–4.26 (m, 1H), 3.87 (s, 2H), 3.49 (dd, J = 10.6,
6.2 Hz, 1H), 3.43 (dd, J = 10.6, 6.2 Hz, 1H), 3.37–3.29 (m, 1H), 2.33 (d, J = 7.8 Hz, 2H),
2.06–1.93 (m, 3H), 1.87 (d, J = 10.4 Hz, 2H), 1.45 (s, 10H), 1.03 (s, 3H), 1.00 (d, J = 6.7 Hz,
3H), 0.91 (d, J = 6.7 Hz, 3H), 0.81 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 168.7, 154.7,
138.4, 121.6, 89.3, 82.2, 78.9, 74.0, 67.0, 64.1, 55.9, 49.0, 41.6, 39.7, 38.4, 37.0, 36.9, 35.9,
35.7, 34.7, 31.2, 31.0, 30.5, 29.5, 29.1, 28.7, 27.3, 26.7, 19.6, 18.3, 17.9, 15.6, 15.4; HRESIMS:
m/z 596.3937 [M + Na]+ (calcd for C34H55NO6Na, 596.3927).
(25R)-Furost-5-en-26-ol-3β-yl N-tert-butoxycarbonyl-4-aminobutyrate (8a): white solid
1
(85%); Rf = 0.41 (petroleum ether/EtOAc 1:1); H NMR (400 MHz, CDCl3) δ 5.37 (d,
J = 4.8 Hz, 1H), 4.71–4.54 (m, 2H), 4.34–4.27 (m, 1H), 3.50 (dd, J = 10.6, 6.2 Hz, 1H), 3.43
(dd, J = 10.6, 6.2 Hz, 1H), 3.33 (m, 1H), 3.16 (t, J = 6.8 Hz, 2H), 2.32 (t, J = 7.3 Hz, 4H),
2.09–1.91 (m, 3H), 1.44 (s, 9H), 1.03 (s, 3H), 1.00 (d, J = 6.7 Hz, 3H), 0.91 (d, J = 6.7 Hz,
3H), 0.81 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 171.7, 154.9, 138.6, 121.4, 89.3, 82.2, 78.3,
73.0, 67.0, 64.1, 55.9, 49.0, 39.7, 38.4, 37.1, 36.9, 36.0, 35.7, 34.7, 31.2, 31.0, 30.5, 29.5, 29.2,
27.4, 26.7, 24.3, 19.6, 18.3, 17.9, 15.6, 15.4; HRESIMS: m/z 624.4233 [M + Na]+ (calcd for
C36H59NO6Na, 624.4240).
(25R)-Furost-5-en-26-ol-3β-yl N-tert-butoxycarbonyl-12-aminolaurate (11a): white solid
1
(80%); Rf = 0.56 (petroleum ether/EtOAc 1:1); H NMR (400 MHz, CDCl3) δ 5.37 (d,
J = 4.8 Hz, 1H), 4.65–4.56 (m, 1H), 4.52 (s, 1H), 4.34–4.26 (m, 1H), 3.50 (dd, J = 10.6, 6.2 Hz,
1H), 3.43 (dd, J = 10.6, 6.2 Hz, 1H), 3.37–3.28 (m, 1H), 3.09 (t, J = 7.1 Hz, 2H), 2.35–2.21
(m, 4H), 2.06–1.94 (m, 3H), 1.89–1.81 (m, 2H), 1.44 (s, 9H), 1.04 (s, 3H), 1.00 (d, J = 6.7 Hz,
3H), 0.91 (d, J = 6.7 Hz, 3H), 0.81 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 172.3, 155.0, 138.7,

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B.-Z. HuANG et AL.
121.3, 89.4, 82.2, 78.0, 72.6, 67.0, 64.1, 55.9, 49.0, 39.7, 38.4, 37.1, 36.9, 36.0, 35.7, 34.7, 33.7,
31.2, 31.0, 30.6, 29.5, 29.2, 29.1, 28.5, 28.4, 28.3, 28.2, 28.1, 27.4, 26.8, 25.8, 24.0, 19.6, 18.3,
17.9, 15.6, 15.4; HRESIMS: m/z 736.5493 [M + Na]+ (calcd for C44H75NO6Na, 736.5492).
4.2.2.2. General procedure for the synthesis of (25R)-furost-5-en-26-ol-3β-yl amino acid
esters 7–11. CF3COOH (20.0–30.0 mmol) was added to a stirred solution of 7a–11a
(1.0–1.5 mmol) in CH2Cl2 (20 ml) at 30–35 °C and kept for 2–6 h. en the mixture
solution was removed in vacuo to give the crude product which was purified by column
chromatography (CH2Cl2/CH3OH 15:1) to yield compounds 7–11.
(25R)-Furost-5-en-26-ol-3β-yl glycinate (7): white solid (78%); Rf = 0.38 (CH2Cl2/
1
CH3OH/CH3COOH 6:1:0.1); H NMR (400 MHz, CDCl3) δ 5.37 (s, 1H), 4.73–4.55 (m,
1H), 4.30 (dd, J = 13.2, 7.3 Hz, 1H), 4.27–4.11 (m, 1H), 3.77 (s, 2H), 3.53–3.41 (m, 1H),
3.38–3.26 (m, 1H), 2.33 (d, J = 24.1 Hz, 2H), 0.81 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
167.0, 139.1, 122.8, 90.0, 83.2, 72.7, 68.0, 65.2, 57.0, 50.0, 40.7, 39.5, 37.9, 37.6, 36.9, 36.6,
35.7, 32.6, 32.0, 31.5, 30.6, 30.1, 27.3, 20.7, 19.3, 18.9, 16.6, 16.4; HRESIMS: m/z 474.3563
[M + H]+ (calcd for C29H48NO4, 474.3583).
(25R)-Furost-5-en-26-ol-3β-yl 4-aminobutyrate (8): light yellow solid (82%); Rf = 0.32
(CH2Cl2/CH3OH/CH3COOH 6:1:0.1); 1H NMR (400 MHz, CDCl3) δ 5.36 (d, J = 3.7 Hz,
1H), 4.65–4.51 (m, 1H), 4.31 (dd, J = 13.2, 7.5 Hz, 1H), 4.25 (dd, J = 10.6, 5.7 Hz, 1H),
4.16 (dd, J = 10.6, 6.9 Hz, 1H), 3.53–3.41 (m, 1H), 3.39–3.26 (m, 1H), 3.02 (s, 2H), 2.42
(t, J = 6.7 Hz, 2H), 2.30 (d, J = 7.8 Hz, 2H), 1.05–0.96 (m, 9H), 0.80 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 172.7, 139.5, 122.5, 89.9, 83.3, 74.9, 72.7, 65.1, 56.9, 50.0, 40.7, 39.5,
39.4, 37.9, 36.9, 36.7, 32.6, 32.2, 32.0, 31.6, 31.4, 30.6, 30.1, 27.5, 22.3, 20.7, 19.3, 18.9, 16.4;
HRESIMS: m/z 502.3893 [M + H]+ (calcd for C31H52NO4, 502.3896).
(25R)-Furost-5-en-26-ol-3β-yl 12-aminolaurate (11): light yellow viscous solid (75%);
1
Rf = 0.31 (CH2Cl2/CH3OH/CH3COOH 6:1:0.1); H NMR (400 MHz, MeOD) δ 5.29 (d,
J = 4.7 Hz, 1H), 4.50–4.37 (m, 1H), 4.27–4.17 (m, 1H), 3.32 (dd, J = 10.7, 5.9 Hz, 1H),
3.23–3.18 (m, 2H), 2.81 (m, 2H), 2.19 (dd, J = 15.7, 8.2 Hz, 4H), 1.97–1.86 (m, 2H), 1.81
(m, 1H), 1.77–1.63 (m, 3H), 0.97 (s, 3H), 0.92 (d, J = 6.7 Hz, 3H), 0.81 (d, J = 6.7 Hz, 3H),
0.74 (s, 3H); 13C NMR (100 MHz, MeOD) δ 175.1, 141.1, 123.4, 91.7, 84.6, 75.2, 68.4, 66.6,
58.2, 51.6, 41.9, 40.8, 40.5, 39.3, 39.2, 38.3, 37.9, 37.0, 35.4, 33.2, 33.1, 32.9, 32.0, 31.2, 30.6,
30.5, 30.4, 30.2, 30.1, 28.9, 28.6, 27.5, 26.1, 21.8, 19.8, 19.3, 17.0; HRESIMS: m/z 614.5142
[M + H]+ (calcd for C39H68NO4, 614.5148).
4.2.3. Synthesis of (25R)-furost-5-en-3β-acetoxy-26-yl amino acid esters (14–17)
4.2.3.1. Synthesis of (25R)-furost-5-en-3β-acetoxy-26-ol (13). Acetic anhydride (20 ml,
213 mmol) was added to a solution of diosgenin (8.29 g, 20 mmol) in pyridine (60 ml) at
room temperature. e solution was stirred at 60 °C for 1 h and then poured into ice-cool
water (2000 ml). e crude yellowish solid was filtered through the silica washing with brine,
dil. HCl and brine, the crude product was precipitated with methanol to yield compound 12.
(22β,25R)-Spirost-5-en-3β-acetate (12): white needle solid (93%); Rf = 0.54 (petroleum
ether/EtOAc 4:1); 1H NMR (400 MHz, CDCL3) δ 5.37 (d, J = 4.5 Hz, 1H), 4.78–4.49 (m,
1H), 4.41 (dd, J = 14.9, 7.5 Hz, 1H), 3.47 (dd, J = 10.0, 3.2 Hz, 1H), 3.37 (t, J = 10.9 Hz, 1H),
2.48–2.23 (m, 2H), 2.03 (s, 3H), 1.03 (s, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.79 (d, J = 3.9 Hz,
6H); 13C NMR (100 MHz, CDCl3) δ 169.5, 138.7, 121.4, 108.3, 79.8, 72.9, 65.8, 61.1, 55.4,

JOuRNAL OF ASIAN NAtuRAL PROductS ReSeARcH
19
48.9, 40.6, 39.2, 38.7, 37.1, 35.9, 35.7, 31.0, 30.8, 30.4, 29.3, 27.8, 26.7, 20.4, 19.8, 18.3, 16.1,
15.3, 13.5.
Compound 12 (2.28 g, 5 mmol) was dissolved in a mixture solution of CH3COOH/
CH2Cl2 (30 ml : 15 ml) at room temperature, and then, sodium cyanoborohydride (942 mg,
15 mmol) was added in portions over a period of 30 min. is mixture was stirred for 4 h
at room temperature (checked by TLC). en, brine (10 ml) was added and extracted with
CH2Cl2 for three times. e organic layer was dried over anhydrous Na2SO4 and concen-
trated in vacuo. e crude mass was purified through silica gel column (petroleum ether/
EtOAc 3:1) to yield 13.
Compound 13 was synthesized as previously reported method [7].
(25R)-Furost-5-en-3β-acetoxy-26-ol (13): white needle solid (85%); Rf = 0.51 (petroleum
ether/EtOAc 1:1); 1H NMR (600 MHz, CDCl3) δ 5.37 (d, J = 4.9 Hz, 1H), 4.60 (tt, J = 10.5,
5.1 Hz, 1H), 4.31 (dd, J = 13.1, 7.7 Hz, 1H), 3.49 (dd, J = 10.6, 6.2 Hz, 1H), 3.43 (dd, J = 10.6,
6.2 Hz, 1H), 3.33 (td, J = 8.3, 3.8 Hz, 1H), 2.36–2.27 (m, 2H), 2.03 (s, 3H), 2.02–1.96 (m,
3H), 1.86 (d, J = 10.6 Hz, 2H), 1.03 (s, 3H), 1.00 (d, J = 6.7 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H),
0.81 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 170.5, 139.7, 122.4, 90.4, 83.2, 73.9, 68.0, 65.1,
56.9, 50.0, 40.7, 39.4, 38.1, 37.9, 37.0, 36.7, 35.7, 32.2, 32.0, 31.6, 30.5, 30.1, 27.7, 21.4, 20.6,
19.3, 18.9, 16.6, 16.4; HRESIMS: m/z 481.3283 [M + Na]+ (calcd for C29H46O4Na, 481.3294).
4.2.3.2. General procedure for the synthesis of (25R)-furost-5-en-3β-acetoxy-26-yl N-tert-
butoxycarbonyl amino acid esters 14a–17a. To a solution of compound 13 (1.0 mmol),
2b–6b (1.2 mmol), DMAP (0.2 mmol) in anhydrous CH2Cl2 (15 ml), EDC·HCl (1.2 mmol)
in CH2Cl2 (5 ml) was added dropwise under N2 atmosphere. e mixture was lef to stir at
room temperature for 4–16 h under a balloon of nitrogen (checked by TLC). e reaction
solution was washed with brine, saturated NaHCO3 solution and brine, dried over anhydrous
Na2SO4, and then filtered and concentrated. e residue was purified by a silica gel column
chromatography (petroleum ether/EtOAc 12:1–8:1) to give 14a–17a.
(25R)-Furost-5-en-3β-acetoxy-26-yl N-tert-butoxycarbonyl-glycinate (14a): white viscous
1
solid (87%); Rf = 0.51 (petroleum ether/EtOAc 2:1); H NMR (400 MHz, CDCl3) δ 5.30
(d, J = 4.8 Hz, 1H), 4.97 (s, 1H), 4.60–4.46 (m, 1H), 4.29–4.16 (m, 1H), 3.98 (dd, J = 10.6,
5.8 Hz, 1H), 3.91–3.78 (m, 3H), 3.29–3.17 (m, 1H), 2.25 (d, J = 6.7 Hz, 2H), 1.96 (s, 3H),
1.38 (s, 9H), 0.97 (s, 3H), 0.93 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H), 0.73 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 169.5, 154.7, 138.7, 121.4, 89.1, 82.2, 78.9, 72.9, 69.1, 64.1,
55.9, 49.0, 41.4, 39.7, 38.4, 37.1, 36.9, 36.0, 35.7, 31.7, 31.2, 31.0, 30.6, 29.7, 29.3, 28.7,
27.3, 26.7, 20.4, 19.6, 18.3, 17.9, 15.7, 15.4; HRESIMS: m/z 638.4039 [M + Na]+ (calcd for
C36H57NO7Na, 638.4033).
(25R)-Furost-5-en-3β-acetoxy-26-yl N-tert-butoxycarbonyl-4-aminobutyrate (15a): white
viscous solid (84%); Rf = 0.46 (petroleum ether/EtOAc 2:1); 1H NMR (400 MHz, CDCl3)
δ 5.37 (d, J = 4.8 Hz, 1H), 4.75 (s, 1H), 4.65–4.53 (m, 1H), 4.35–4.25 (m, 1H), 3.97 (dd,
J = 10.7, 5.9 Hz, 1H), 3.87 (dd, J = 10.7, 6.8 Hz, 1H), 3.35–3.26 (m, 1H), 3.16 (dd, J = 12.5,
6.2 Hz, 2H), 2.40–2.29 (m, 4H), 2.03 (s, 3H), 1.44 (s, 9H), 1.04 (s, 3H), 1.00 (d, J = 6.7 Hz,
3H), 0.93 (d, J = 6.7 Hz, 3H), 0.81 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 173.3, 170.5,
155.9, 139.7, 122.4, 90.1, 83.2, 79.1, 73.9, 69.3, 65.1, 56.9, 50.0, 40.7, 40.0, 39.4, 38.1, 37.9,
37.0, 36.7, 32.7, 32.2, 32.0, 31.6, 30.7, 30.4, 29.7, 28.4, 27.7, 26.9, 25.3, 21.4, 20.6, 19.3, 18.9,
16.8, 16.4, 14.1; HRESIMS: m/z 666.4344 [M + Na]+ (calcd for C38H61NO7Na, 666.4346).

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B.-Z. HuANG et AL.
(25R)-Furost-5-en-3β-acetoxy-26-yl N-tert-butoxycarbonyl-6-aminohexanoate (16a):
1
white viscous solid (79%); Rf = 0.47 (petroleum ether/EtOAc 2:1); H NMR (400 MHz,
CDCl3) δ 5.30 (d, J = 4.8 Hz, 1H), 4.64–4.46 (m, 2H), 4.27–4.18 (m, 1H), 3.90 (dd, J = 10.7,
5.8 Hz, 1H), 3.79 (dd, J = 10.7, 6.8 Hz, 1H), 3.28–3.19 (m, 1H), 3.04 (dd, J = 12.8, 6.4 Hz,
2H), 2.24 (t, J = 7.5 Hz, 4H), 1.96 (s, 3H), 1.37 (s, 9H), 0.97 (s, 3H), 0.93 (d, J = 6.7 Hz, 3H),
0.86 (d, J = 6.7 Hz, 3H), 0.74 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 172.7, 169.5, 155.0,
138.7, 121.4, 89.1, 82.2, 77.9, 72.9, 68.1, 64.1, 55.9, 49.0, 39.7, 39.4, 38.4, 37.1, 36.9, 36.0,
35.7, 33.2, 31.8, 31.2, 31.0, 30.6, 29.8, 29.4, 28.8, 28.7, 27.4, 26.7, 25.3, 23.7, 20.4, 19.6, 18.3,
17.9, 15.8, 15.4; HRESIMS: m/z 672.4265 [M + H]+ (calcd for C40H66NO6, 672.4839).
(25R)-Furost-5-en-3β-acetoxy-26-yl N-tert-butoxycarbonyl-12-aminolaurate (17a): white
viscous solid (88%); Rf = 0.59 (petroleum ether/EtOAc 2:1); 1H NMR (400 MHz, CDCl3)
δ 5.37 (d, J = 4.9 Hz, 1H), 4.67–4.56 (m, 1H), 4.53 (s, 1H), 4.30 (td, J = 7.7, 5.4 Hz, 1H),
3.97 (dd, J = 10.7, 5.7 Hz, 1H), 3.86 (dd, J = 10.7, 6.8 Hz, 1H), 3.30 (td, J = 8.0, 4.3 Hz, 1H),
3.09 (s, 2H), 2.30 (dd, J = 14.9, 7.4 Hz, 4H), 1.04 (s, 3H), 1.00 (d, J = 6.7 Hz, 3H), 0.93 (d,
J = 6.7 Hz, 3H), 0.81 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 174.0, 170.5, 156.0, 139.7,
122.4, 90.2, 83.2, 79.0, 73.9, 69.1, 65.1, 56.9, 50.0, 40.7, 39.4, 38.1, 37.9, 37.0, 36.7, 34.4, 32.8,
32.2, 32.0, 31.6, 30.8, 30.5, 30.1, 29.5, 29.4, 29.3, 29.2, 28.4, 27.7, 26.8, 25.0, 21.4, 20.6, 19.3,
18.9, 16.8, 16.4; HRESIMS: m/z 778.5601 [M + Na]+ (calcd for C46H77NO7Na, 778.5598).
4.2.3.3. General procedure for the synthesis of (25R)-furost-5-en-3β-acetoxy-26-yl amino
acid esters 14–17. CF3COOH (10.0 mmol) was added to a stirred solution of 14a–17a
(0.5 mmol) in CH2Cl2 (10 ml) at room temperature for 4–10 h. en the mixture solution
was removed in vacuo and purified by column chromatography (CH2Cl2/CH3OH 15:1),
and then freeze-dried at −80 °C to yield 14–17.
(25R)-Furost-5-en-3β-acetoxy-26-yl glycinate (14): yellowish viscous solid (92%);
1
Rf = 0.40 (CH2Cl2/CH3OH/CH3COOH 6:1:0.1); H NMR (400 MHz, CDCl3) δ 5.37 (d,
J = 4.5 Hz, 1H), 4.66–4.52 (m, 1H), 4.30 (dd, J = 13.0, 7.6 Hz, 1H), 4.11–4.02 (m, 1H),
4.01–3.92 (m, 1H), 3.80 (s, 2H), 3.38–3.22 (m, 1H), 2.32 (d, J = 6.4 Hz, 2H), 2.03 (s, 3H), 1.03
(s, 3H), 0.99 (d, J = 6.6 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H), 0.80 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 170.6, 167.7, 139.7, 122.4, 90.0, 83.2, 73.9, 71.5, 65.0, 56.9, 50.0, 40.7, 39.4, 38.1,
37.9, 37.0, 36.7, 32.5, 32.2, 32.0, 31.6, 30.7, 30.2, 27.7, 21.4, 20.6, 19.3, 18.8, 16.4; HRESIMS:
m/z 516.3691 [M + H]+ (calcd for C31H50NO5, 516.3689).
(25R)-Furost-5-en-3β-acetoxy-26-yl 4-aminobutyrate (15): yellowish viscous solid (86%);
1
Rf = 0.32 (CH2Cl2/CH3OH/CH3COOH 6:1:0.1); H NMR (400 MHz, CDCl3) δ 5.37 (d,
J = 4.6 Hz, 1H), 4.68–4.52 (m, 1H), 4.30 (dd, J = 13.0, 7.6 Hz, 1H), 3.97 (dd, J = 11.2, 5.0 Hz,
1H), 3.85 (dd, J = 10.6, 7.0 Hz, 1H), 3.31 (td, J = 8.1, 4.0 Hz, 1H), 3.02 (s, 2H), 2.45 (t,
J = 6.5 Hz, 2H), 2.32 (d, J = 6.7 Hz, 2H), 2.03 (s, 3H), 2.01–1.93 (m, 3H), 1.86 (d, J = 10.7 Hz,
2H), 1.04 (s, 3H), 0.99 (d, J = 6.7 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H), 0.80 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 173.4, 170.6, 139.7, 122.4, 90.1, 83.2, 73.9, 70.0, 65.1, 56.9, 50.0, 40.7,
39.4, 38.1, 37.9, 37.0, 36.7, 32.7, 32.2, 32.0, 31.6, 31.1, 30.8, 30.4, 27.7, 22.3, 21.4, 20.6, 19.3,
18.9, 16.6, 16.4; HRESIMS: m/z 544.4007 [M + H]+ (calcd for C33H54NO5, 544.4002).
(25R)-Furost-5-en-3β-acetoxy-26-yl 6-aminohexanoate (16): yellowish viscous solid
(89%); Rf = 0.35 (CH2Cl2/CH3OH/CH3COOH 6:1:0.1); 1H NMR (400 MHz, CDCl3) δ 5.37
(d, J = 4.7 Hz, 1H), 4.60 (dt, J = 11.6, 6.7 Hz, 1H), 4.30 (dd, J = 13.0, 7.6 Hz, 1H), 3.96 (dd,
J = 10.5, 5.4 Hz, 1H), 3.84 (dd, J = 10.7, 7.0 Hz, 1H), 3.31 (td, J = 8.1, 4.1 Hz, 1H), 2.94 (s,
2H), 2.31 (t, J = 7.1 Hz, 4H), 2.03 (s, 3H), 2.02–1.95 (m, 2H), 1.86 (d, J = 10.6 Hz, 2H), 1.04

JOuRNAL OF ASIAN NAtuRAL PROductS ReSeARcH
21
(s, 3H), 0.99 (d, J = 6.7 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H), 0.80 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 174.0, 170.6, 139.7, 122.4, 90.2, 83.2, 73.9, 69.5, 65.1, 56.9, 50.0, 40.7, 39.8, 39.4,
38.1, 37.9, 37.0, 36.7, 33.7, 32.8, 32.2, 32.0, 31.6, 30.8, 30.4, 27.7, 26.9, 25.6, 24.0, 21.4, 20.6,
19.3, 18.9, 16.7, 16.4; HRESIMS: m/z 572.4327 [M + H]+ (calcd for C35H58NO5, 572.4315).
(25R)-Furost-5-en-3β-acetoxy-26-yl 12-aminolaurate (17): yellowish viscous solid (90%);
1
Rf = 0.42 (CH2Cl2/CH3OH/CH3COOH 6:1:0.1); H NMR (400 MHz, CDCl3) δ 5.37 (d,
J = 4.6 Hz, 1H), 4.65–4.54 (m, 1H), 4.31 (dd, J = 13.0, 7.6 Hz, 1H), 3.96 (dd, J = 10.6, 5.5 Hz,
1H), 3.85 (dd, J = 10.7, 6.9 Hz, 1H), 3.32 (td, J = 8.0, 4.2 Hz, 1H), 2.91 (s, 2H), 2.30 (dd,
J = 14.6, 7.2 Hz, 4H), 2.03 (s, 3H), 1.04 (s, 3H), 1.00 (d, J = 6.7 Hz, 3H), 0.93 (d, J = 6.7 Hz,
3H), 0.80 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 174.3, 170.7, 139.7, 122.4, 90.2, 83.2, 74.0,
69.2, 65.1, 56.9, 50.0, 40.7, 40.2, 39.4, 38.1, 37.9, 37.0, 36.7, 34.4, 32.8, 32.2, 32.0, 31.6, 30.8,
30.4, 29.3, 29.2, 28.9, 27.7, 27.4, 26.2, 25.0, 21.4, 20.7, 19.3, 18.9, 16.8, 16.4; HRESIMS: m/z
656.5258 [M + H]+ (calcd for C41H70NO5, 656.5254).
4.2.4. Synthesis of (22β, 25R)-spirost-5-en-3β-yl n-butyrate (21)
n-Butyric anhydride (3 ml, 18 mmol) was added to a solution of diosgenin (829 mg, 2 mmol)
in pyridine (5 mmol) and the resulting solution stirred at room temperature until absence of
diosgenin (checked by TLC). en the reaction mixture was poured into water and stirred
vigorously to get a white precipitate. e residue was washed with brine, recrystallized in
methane to afford a white needle solid 21.
(22β, 25R)-Spirost-5-en-3β-yl n-butyrate (21): white needle solid (89%); Rf = 0.55 (petro-
leum ether/EtOAc 8:1); 1H NMR (400 MHz, CDCl3) δ 5.37 (d, J = 5.0 Hz, 1H), 4.66–4.56
(m, 1H), 4.41 (dd, J = 14.9, 7.5 Hz, 1H), 3.51–3.43 (m, 1H), 3.37 (t, J = 10.9 Hz, 1H),
2.36–2.28 (m, 2H), 2.25 (t, J = 7.4 Hz, 2H), 2.06–1.93 (m, 2H), 1.90–1.82 (m, 3H), 1.04 (s,
3H), 0.95 (dd, J = 14.5, 7.2 Hz, 6H), 0.79 (t, J = 3.1 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ
172.1, 138.7, 121.3, 108.2, 79.8, 72.6, 65.8, 61.1, 55.4, 48.9, 40.6, 39.2, 38.7, 37.1, 36.0, 35.7,
35.6, 31.0, 30.8, 30.4, 29.3, 27.8, 26.8, 19.8, 18.3, 17.5, 16.1, 15.3, 13.5, 12.6; HRESIMS: m/z
507.3450 [M + Na]+ (calcd for C31H48O4Na, 507.3450).
4.2.5. Synthesis of (22β, 25R)-spirost-5-en-3β-yl aliphatic esters (22–24)
4.2.5.1. General procedure for the synthesis of (22β, 25R)-spirost-5-en-3β-yl aliphatic esters
22–24. Compound 1 (2.0 mmol), 22a–24a (3.0 mmol) and DMAP (0.5 mmol) were stirred
in anhydrous CH2Cl2 (20 ml) for 10 min. A solution of EDC·HCl (3.0 mmol) in CH2Cl2
(10 ml) was added dropwise under N2. e mixture was lef to stir for 2–6 h at 30–35 °C
(checked by TLC). e reaction solution was washed with brine, saturated NaHCO3 solution
and brine, dried over anhydrous Na2SO4, and then filtered and concentrated. e residue
was purified by a silica gel column chromatography (petroleum ether/EtOAc 25:1–15:1)
to give 22–24.
(22β, 25R)-Spirost-5-en-3β-yl n-hexanoate (22): white needle solid (79%); Rf = 0.59
1
(petroleum ether/EtOAc 8:1); H NMR (400 MHz, CDCl3) δ 5.37 (d, J = 5.0 Hz, 1H),
4.67–4.54 (m, 1H), 4.41 (dd, J = 14.9, 7.5 Hz, 1H), 3.52–3.43 (m, 1H), 3.37 (t, J = 10.9 Hz,
1H), 2.36–2.29 (m, 2H), 2.26 (t, J = 7.5 Hz, 2H), 2.08–1.91 (m, 1H), 1.90–1.81 (m, 1H),
1.04 (s, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.90 (t, J = 6.9 Hz, 3H), 0.79 (t, J = 3.1 Hz, 6H); 13
C
NMR (100 MHz, CDCl3) δ 172.3, 138.8, 121.3, 108.2, 79.8, 72.6, 65.8, 61.1, 55.4, 48.9, 40.6,
39.2, 38.7, 37.1, 36.0, 35.7, 33.7, 31.0, 30.8, 30.4, 30.3, 29.3, 27.8, 26.8, 23.7, 21.3, 19.8, 18.3,
16.1, 15.3, 13.5, 12.9; HRESIMS: m/z 513.3936 [M + H]+ (calcd for C33H53O4, 513.3944).

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(22β, 25R)-Spirost-5-en-3β-yl n-octanoate (23): white needle solid (84%); Rf = 0.62
(petroleum ether/EtOAc 8:1); 1H NMR (400 MHz, CDCl3) δ 5.37 (d, J = 5.0 Hz, 1H), 4.67–
4.54 (m, 1H), 4.41 (dd, J = 15.0, 7.5 Hz, 1H), 3.51–3.44 (m, 1H), 3.37 (t, J = 10.9 Hz, 1H),
2.37–2.29 (m, 2H), 2.27 (t, J = 7.5 Hz, 2H), 2.06–1.93 (m, 2H), 1.86 (m, 3H), 1.04 (s, 3H),
0.97 (d, J = 6.9 Hz, 3H), 0.88 (t, J = 6.8 Hz, 3H), 0.79 (t, J = 3.1 Hz, 6H); 13C NMR (100 MHz,
CDCl3) δ 172.3, 138.8, 121.3, 108.3, 79.8, 72.6, 65.8, 61.1, 55.4, 48.9, 40.6, 39.2, 38.7, 37.1,
36.0, 35.7, 33.7, 31.0, 30.8, 30.7, 30.4, 29.3, 28.1, 27.9, 27.8, 26.8, 24.1, 21.6, 19.8, 18.3, 16.1,
15.3, 13.5, 13.1; HRESIMS: m/z 563.4071 [M + Na]+ (calcd for C35H56O4Na, 563.4076).
(22β, 25R)-Spirost-5-en-3β-yl n-laurate (24): white needle solid (81%); Rf = 0.67 (petro-
leum ether/EtOAc 8:1); 1H NMR (400 MHz, CDCl3) δ 5.37 (d, J = 5.0 Hz, 1H), 4.67–4.54 (m,
1H), 4.41 (dd, J = 14.9, 7.5 Hz, 1H), 3.51–3.44 (m, 1H), 3.37 (t, J = 10.9 Hz, 1H), 2.35–2.29
(m, 2H), 2.26 (t, J = 7.5 Hz, 2H), 2.08–1.94 (m, 2H), 1.92–1.81 (m, 3H), 1.04 (s, 3H), 0.97
(d, J = 6.9 Hz, 3H), 0.88 (t, J = 6.8 Hz, 3H), 0.79 (t, J = 3.1 Hz, 6H); 13C NMR (100 MHz,
CDCl3) δ 173.3, 139.8, 122.3, 109.3, 80.8, 73.6, 66.8, 62.1, 56.4, 50.0, 41.6, 40.3, 39.7, 38.1,
37.0, 36.7, 34.7, 32.1, 31.9, 31.4, 30.3, 29.6, 29.5, 29.3, 29.1, 28.8, 27.8, 25.1, 22.7, 20.8, 19.3,
17.1, 16.3, 14.5, 14.1; HRESIMS: m/z 619.4697 [M + Na]+ (calcd for C39H64O4Na, 619.4702).
4.2.6. Synthesis of (22β, 25R)-spirost-5-en-3β-yl N-4-(7-nitrobenz-2-oxa-1,3- diazole)-
6-aminohexanoate (28)
4-Chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD-Cl, 200 mg, 1 mmol) and Et3N (140 μl,
1 mmol) was stirred in anhydrous THF (30 ml) for 10 min. A solution of compound 4
(528 mg, 1 mmol) in THF (20 ml) was added to the mixture solution in 30 min. en the
reaction was stirred at 50 °C for 5 h until absence of compound 4. en the reaction mixture
was filtered and the filtrate was concentrated in vacuo. e crude residue was purification
by column chromatography (petroleum ether/EtOAc 5:1) to yield 28.
(22β, 25R)-spirost-5-en-3β-yl N-4-(7-nitrobenz-2-oxa-1,3-diazole)-6- aminohexanoate
(28): orange powder solid (66%); Rf = 0.40 (petroleum ether/EtOAc 3:2); 1H NMR (400 MHz,
CDCl3) δ 8.49 (d, J = 8.6 Hz, 1H), 6.45 (t, J = 4.8 Hz,1H), 6.18 (d, J = 8.7 Hz, 1H), 5.36 (d,
J = 4.8 Hz, 1H), 4.67–4.55 (m, 1H), 4.41 (dd, J = 15.0, 7.5 Hz, 1H), 3.53 (dd, J = 12.8, 6.6 Hz,
2H), 3.48 (dd, J = 11.0, 4.1 Hz, 1H), 3.38 (t, J = 10.9 Hz, 1H), 2.39–2.27 (m, 4H), 1.03 (s,
3H), 0.97 (d, J = 6.9 Hz, 3H), 0.79 (t, J = 3.1 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 172.9,
144.3, 143.9, 139.6, 136.5, 123.9, 122.5, 109.3, 98.6, 80.8, 74.0, 66.8, 62.1, 56.4, 49.9, 43.7,
41.6, 40.3, 39.7, 38.1, 36.9, 36.7, 34.2, 32.0, 31.8, 31.4, 30.3, 28.8, 28.1, 27.8, 26.3, 24.3, 20.8,
19.3, 17.2, 16.3, 14.5; HRESIMS: m/z 691.4066 [M + H]+ (calcd for C39H55N4O6, 691.4071).
4.2.7. Synthesis of (22β, 25R)-spirost-5-en-3β-yl N-α-[4-(7-nitrobenz-2-oxa-1,3-
diazole)]-l-lysinate (32)
4.2.7.1. Synthesis of (22β, 25R)-spirost-5-en-3β-yl N-α-Fmoc-N-ε-Boc-l-lysinate (29). To
a solution of compound 1 (829 mg, 2.0 mmol), N-α-Fmoc-N-ε-Boc-l-lysine (1.12 g,
2.4 mmol), 4-dimethylaminopyridine (DMAP, 49 mg, 0.4 mmol) p-toluenesulfonic acid
(TsOH, 69 mg, 0.4 mmol) in dry CH2Cl2 (40 ml), and EDC·HCl (466 mg, 3.0 mmol) in
anhydrous CH2Cl2 (10 ml) were added dropwise under N2 atmosphere at room temperature.
e mixture was stirred vigorously for 18 h. e reaction mixture was washed with brine,
saturated NaHCO3 solution and brine, dried over anhydrous Na2SO4, and then filtered and
concentrated. e residue was purified by a silica gel column chromatography (petroleum
ether : EtOAc = 10:1) to give 29 (1.47 g, 85%) as a white powder. Rf 0.70 (petroleum ether

JOuRNAL OF ASIAN NAtuRAL PROductS ReSeARcH
23
1
: ethyl acetate = 1:1); H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.5 Hz, 2H), 7.63–7.57
(m, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 5.42 (d, J = 7.8 Hz, 1H), 5.38
(d, J = 3.9 Hz, 1H), 4.74–4.52 (m, 2H), 4.45–4.28 (m, 4H), 4.23 (t, J = 7.0 Hz, 1H), 3.47
(dd, J = 10.3, 3.4 Hz, 1H), 3.37 (t, J = 10.9 Hz, 1H), 3.11 (d, J = 5.1 Hz, 2H), 2.37–2.21 (m,
3H), 2.06–1.92 (m, 2H), 1.91–1.80 (m, 4H), 1.03 (s, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.79 (d,
J = 4.7 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 171.9, 156.1, 156.0, 144.0, 143.8, 141.3, 139.6,
139.3, 127.7, 127.1, 125.1, 122.8, 120.0, 109.3, 80.8, 75.2, 67.0, 66.9, 62.1, 56.4, 53.9, 49.9,
47.2, 41.6, 40.3, 39.7, 38.0, 36.9, 36.7, 32.4, 32.0, 31.8, 31.4, 30.3, 29.7, 28.8, 28.4, 27.7, 22.8,
22.4, 20.8, 19.3, 17.2, 16.3, 14.5; HRESIMS: m/z 865.5359 [M + H]+ (calcd for C53H73N2O8,
865.5367).
4.2.7.2. Synthesis of (22β, 25R)-spirost-5-en-3β-yl N-ε-Boc-l-lysinate (30). Compound 29
(643mg,1.0mmol)wasdissolvedinDMF(5ml),andpiperidine(1ml)wasaddedandstirredat
room temperature for 3 h. en the reaction mixture was concentrated in vacuo and the crude
residue was purified by column chromatography (CH2Cl2:CH3OH 25:1) to yield compound
1
30 (533 mg, 83%, light yellow solid). Rf 0.55 (CH2Cl2:CH3OH:CH3COOH = 6:1:0.1); H
NMR (400 MHz, CDCl3) δ 5.37 (d, J = 3.2 Hz, 1H), 4.79–4.57 (m, 1H), 4.41 (dd, J = 15.1,
7.2 Hz, 1H), 3.86 (s, 1H), 3.65 (s, 1H), 3.47 (dd, J = 9.8, 3.6 Hz, 1H), 3.37 (t, J = 10.9 Hz, 2H),
3.21 (s, 1H), 3.10 (dd, J = 12.6, 7.0 Hz, 2H), 2.71 (s, 1H), 2.47–2.23 (m, 5H), 2.07–1.92 (m,
2H), 1.03 (s, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.79 (d, J = 4.2 Hz, 6H); HRESIMS: m/z 643.4689
[M + H]+ (calcd for C38H63N2O6, 643.4686).
4.2.7.3. Synthesis of (22β, 25R)-spirost-5-en-3β-yl N-α-[4-(7-nitrobenz-2-oxa-1,3-
diazole)]-N-ε-Boc-l-lysinate (31). NBD-Cl (100 mg, 0.5 mmol) and Et3N (70 μl) were
stirred in anhydrous THF (10 ml) for 10 min. A solution of compound 30 (257 mg, 0.4 mmol)
in THF (10 ml) was added and the reaction was stirred at 50 °C for 4 h. e mixture solution
was concentrated in vacuo. Afer the crude residue was re-dissolved in CH2Cl2 (30 ml), the
organic solution was washed with brine, dried over anhydrous Na2SO4, and then purified
by flash chromatography. e purification by column chromatography (petroleum ether/
EtOAc 5:1) yielded 31 (238 mg, 74%, jacinth solid). Rf 0.29 (petroleum ether/EtOAc = 2:1);
HRESIMS: m/z 828.4566 [M + Na]+ (calcd for C44H63N5O9Na, 828.4523).
4.2.7.4. (22β, 25R)-Spirost-5-en-3β-yl N-α-[4-(7-nitrobenz-2-oxa-1,3-diazole)]-l-lysinate
(32). Trifluoroacetic acid (CF3COOH, 0.3 ml, 4 mmol) was added to a solution of
compound 31 (161 mg, 0.2 mmol) in CH2Cl2 (10 ml) at room temperature and reacted at
30 °C for 4 h. en the organic solution was concentrated and purified over silica gel column
(CH2Cl2:CH3OH = 15:1) to get compound 32 (124 mg, 88%, brownish red solid). Rf 0.35
(CH2Cl2:CH3OH:CH3COOH = 6:1:0.1); 1H NMR (400 MHz, CDCl3) δ 8.46 (d, J = 8.7 Hz,
1H), 6.14 (d, J = 8.7 Hz, 1H), 5.34 (d, J = 4.1 Hz, 1H), 5.31 (s, 1H), 4.71–4.58 (m, 1H), 4.41
(dd, J = 15.0, 7.4 Hz, 1H), 3.57–3.44 (m, 4H), 3.38 (t, J = 10.9 Hz, 1H), 2.29 (d, J = 7.8 Hz,
2H), 2.18 (d, J = 3.1 Hz, 1H), 2.06–1.94 (m, 3H), 1.92–1.82 (m, 6H), 1.02 (s, 3H), 0.97 (d,
J = 6.9 Hz, 3H), 0.79 (t, J = 3.0 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 175.1, 144.3, 144.0,
139.3, 136.6, 123.4, 122.7, 109.3, 80.8, 74.7, 66.8, 62.1, 56.4, 54.0, 49.9, 41.6, 40.2, 39.7, 38.1,
36.9, 36.7, 33.6, 32.0, 31.8, 31.4, 30.3, 29.7, 29.3, 28.8, 27.7, 27.5, 23.1, 22.7, 20.8, 19.3, 17.2,
16.3, 14.5, 14.1; HRESIMS: m/z 706.4281 [M + H]+ (calcd for C39H56N5O7, 706.4180).

24
B.-Z. HuANG et AL.
4.3. Biological evaluation
4.3.1. Cell culture
Mouse colon carcinoma C26 cells, mouse melanoma B16 cells, human HepG2 cells, human
lung carcinoma A549 cells, and human breast cancer MDA-MB-231 cells were maintained
in RPMI 1640 medium or DMEM medium (Hyclone, Logan, USA) supplemented with 10%
fetal bovine serum (Gibco, New York, USA). All experiments were performed in triplicate.
4.3.2. Cytotoxicity evaluation
Murine colon carcinoma C26 cells, mouse melanoma B16 cells, human hepatocellular
carcinoma HepG2 cells, human lung carcinoma A549 cells, and human breast cancer
MDA-MB-231 cells were plated at a density of 3–5 × 104 cells/ml in 96-well plates, followed
by stimulation of indicated concentrations of synthesized compounds, and carrier DMSO
(<0.05%) was used as the vehicle control. Afer 24 h, cell viability was measured using
MTT assay (Sigma-Aldrich, St. Louis., USA) according to the manufacturer’s protocol. e
absorbance at 490 nm was measured with VersaMax microplate reader (Molecular Devices,
San Francisco, USA).
4.3.3. Cellular localization imaging
e cellular localization of diosgenyl derivatives in C26 cells was illustrated by fluorescent
labeling assay. C26 cells were incubated with NBD-labeled compound 28 or 32 (10 μM).
Afer 24 h, cells were co-stained with 5 μg/ml Hoechst 33342 (Invitrogen, Carlsbad, USA),
and images were taken using a confocal laser scanning microscope (Nikon Eclipse Ti,
Tokyo, Japan).
4.3.4. Cell morphological analysis
C26 cells were treated with indicated concentrations of compound 4 for 24 h, and then
images were taken by inverted microscope Olympus IX71 (Olympus, Tokyo, Japan). To
obtain the changes of subcellular structures, TEM was used. Afer 24 h treatment with
vehicle or compound 4, cells were then fixed in glutaraldehyde, osmicated with osmium
tetroxide, dehydrated by graded ethanol, embedded in Epon–Araldite resin. Ultrathin sec-
tions were cut, double stained, and analyzed by H-600IV TEM (Hitachi, Tokyo, Japan).
4.3.5. Colony formation assay
C26 cells were plated at a density of 100 cells in culture dish (diameter 6 cm), followed by
stimulation of compound 4 (0.625, 1.25, 2.5 μM) for 7 days. en cells were fixed with 4%
paraformaldehyde, stained with 1% crystal violet and counted.
4.3.6. Cell cycle analysis
e effect of compound 4 on cell division cycle was assessed by flow cytometry with
PI-stained cellular DNA as described earlier. Briefly, cells (4 × 105 per well) were seeded
in 6-well culture plate and grown overnight. Afer exposure to compound 4 (0, 1.25, 2.5,
5 μM) for 24 h, cells were harvested by trypsinization and fixed with ice-cold 70% ethanol
overnight at 4 °C. e pellets were washed with PBS and resuspended in a solution contain-
ing PI (20 mg/ml), Triton X100 (0.1%) and RNase (1 mg/ml) in PBS. en cell cycle was
analyzed by Elite ESP flow cytometer (Beckman Coulter, High Wycombe, UK).

JOuRNAL OF ASIAN NAtuRAL PROductS ReSeARcH
25
4.3.7. Cytoplasmic Ca2+ concentration analysis
Cytoplasmic Ca2+ concentration was measured using Fluo-4 AM (ermoFisher Scientific,
South San Francisco, USA) by flow cytometry. C26 cells were treated with compound 4
(0, 2.5, 5 μM) for 6 and 12 h, and then incubated with 5 μM Fluo-4 AM in HBSS for 30 min
at 37 °C. en cells were subjected to flow cytometer, and 20,000 cells were collected for
the determination of fluorescence intensity of each sample.
4.3.8. Mitochondrial membrane potential analysis
Mitochondrial membrane potential was measured using JC-1 (ermoFisher Scientific,
South San Francisco, USA) by flow cytometry. C26 cells were treated with compound 4
(0, 2.5, 5 μM) for 6 and 12 h, and then incubated with 10 μg/ml JC-1 for 15 min at 37 °C.
en cells were subjected to flow cytometer, and 20,000 cells were collected for the deter-
mination of fluorescence intensity of each sample.
4.3.9. ATP measurement
Cellular ATP concentration was assessed as described previously. Cells were incubated with
compound 4 (0, 2.5, 5 μM) for 6 and 12 h at 37 °C. Subsequently, cells were lysed, centri-
fuged, and the supernatant was transferred to a black opaque 96-well microplate (Corning,
New York, USA). Cellular ATP levels were examined using an ATP Assay Kit (Invitrogen,
Carlsbad, USA), and the values were expressed using untreated wells at 100%.
4.3.10. Western blot analysis
For apoptosis testing, C26 cells were serum-starved overnight and then incubated with com-
pound 4 (0, 1.25, 2.5, 5 μM) for 24 h. For autophagy analysis, C26 cells were serum-starved
overnight and then incubated with compound 4 (5 μM) for 0, 1, 2, 4 h. Afer treatment with
compound 4, cell lysates were subjected to SDS-PAGE on 10–15% gels followed by transfer
to PVDF membrane. e membranes were incubated with antibodies against PARP and LC3
I/II (Cell Signaling Technology, Beverly, USA). GAPDH (Santa Cruz Biotechnology, Santa
Cruz, USA) was used as whole-cell extract loading control. Primary antibodies were detected
using either peroxidase-conjugated goat anti-rabbit IgG or peroxidase-conjugated goat anti-
mouse IgG secondary antibody (Proteintech, Chicago, USA). e proteins were visualized
with enhanced chemiluminescence detection (Amersham Biosciences, Sunnyvale, USA).
4.3.11. Experimental animals
All animal experiments were performed in accordance with the Guidelines for the Care and
Use of Laboratory Animals and the Association for Research in Vision and Ophthalmology.
All protocols were approved by the Institutional Animal Care and Use Committee of Sichuan
University and the approval number for the animal experiments is 2014008B. Male BALB/c
mice (4–6 weeks old) were obtained and supplied by Experimental Animal Research Center,
West China School of Pharmacy, Sichuan University. e animals had free access to stand-
ard laboratory food and water, and housed at 22 2 °C with a 12 h light/dark cycle. All
surgeries were performed under anesthesia, and supplemental anesthesia was administered
as needed. All efforts were made to minimize suffering and the number of animals used.

26
B.-Z. HuANG et AL.
4.3.12. Tumor xenograft model
Mice were injected subcutaneously with C26 cells (1 × 106 cells per mouse) as previously
described [19]. One week afer cells implantation, the animals were randomly divided into
three groups (n = 9). Compound 4 (0, 15, 30 mg/kg) was administrated intraperitoneally
every day for 28 days. Bodyweight and tumor growth were measured every 3 or 4 days. At
the time of killing, tumor and main visceral organs were removed, frozen tumor tissues
were used to detect the vessel density with an anti-CD31 antibody (BD Biosciences, San
Jose, USA); and the main visceral organs were fixed in 10% formalin immediately, and H&E
staining was performed.
4.3.13. Bleeding time assay
Mice tail bleeding time was measured as reported previously [11]. Male Balb/c mice were
randomly divided into six groups of ten each. Before the bleeding time was assessed, the
mice were given diosgenyl analogues 1, 4, 16, and 22, or control buffer orally (100 μl) twice
a day for five days. Afer 1 h for the last administration, mice were anesthetized intraperito-
neally with pentobarbital sodium (40 mg/kg). en an incision was made at the tip of the
tail with a scalpel, and the tail was immersed immediately in saline (37 °C). e time from
the incision to the cessation of bleeding was recorded, and a bleeding time of 900 s was used
as the cut-off time for the purpose of statistical analysis. e measurement of bleeding time
was performed in a blinded fashion.
4.3.14. Particle size analysis
Diosgenyl analogue 4 (5 mg) was dissolved in the ethanol (100 μl), and then slowly added
into deionized water (5 ml) under stirring. us some amphiphilic diosgenin analogues
could self-assemble into nanoparticles in water. e particle size distribution of compound 4
(1 mg/ml in 2% ethanol–water solution) was investigated on a Nano-Particle Size Analyzer
LB-550 (Horiba Ltd., Kyoto, Japan). Each sample was tested at least three times. e parti-
cle morphology of compound 4 in 2% ethanol–water solution was measured using TEM.
4.3.15. Statistical analysis
Statistical analysis was performed with unpaired two-tailed t-test using Graphpad Prism
5 (Graphpad Sofware, San Diego, USA) when two individual experimental groups were
analyzed. For multiple comparisons, a one-way ANOVA with Dunnett’s multiple compar-
ison test was used. Values of *p < 0.05 and **p < 0.01 were considered significant. All data
are represented as mean s.e.m.
Acknowledgments
We thank the study participants for their supports. e following are acknowledged for technical
assistance: Yanyan Zhang and Zhihui Zhong for live cell imaging; Jie Zhang and Ling Bai for confo-
cal microscope; Fang-Fang Wang and Fang-Fang Zhang for flow cytometer; Guang Yang for animal
care; Jing-Yi Zhang for fluorescence microplate reader; Bin Zhou for chemiluminescence detection.
Disclosure statement
No potential conflict of interest was reported by the authors.

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27
Funding
is work is supported in part by grants from China National “12.5” Research Foundation [grant
number 2011BAJ07B04], National Natural Science Foundation of China [grant number 20972105]
and Sichuan Province Research Foundation [grant number 2008SZ0024].
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