Novel diosgenin derivatives containing 1,3,4-oxadiazole/thiadiazole moieties as potential antitumor agents: Design, synthesis and cytotoxic evaluation

Article abstract of DOI:10.1016/j.ejmech.2019.111897

Diosgenin, a naturally occurring steroidal saponin, has been confirmed to possess potent anticancer properties. In the current work, two series of novel diosgenin derivatives bearing 1,3,4-oxadiazole (6a?6e and 7a?7e) or 1,3,4-thiadiazole (8a?8e and 9a?9e) moieties were designed, synthesized and evaluated for their cytotoxicities in four human cancer cell lines (HepG2, A549, MCF-7 and HCT-116) and normal human gastric epithelial cells (GES-1) using the MTT assay in vitro. The results showed that compounds 8d and 9d exhibited significant cytotoxic activities against the HepG2 and A549 cells, being more potent than their parent compound diosgenin. Furthermore, the 1,3,4-thiadiazole series of compounds generally exhibited stronger cytotoxicity compared with the 1,3,4-oxadiazole series against HepG2 and A549 cells, and the substitution of 3-pyridyl group at the C5 position of the 1,3,4-thiadiazole ring was the preferred option for these compounds to display significant cytotoxic activities. Compound 8d showed potent cytotoxic activity against A549 cell line (IC₅₀ = 3.93 μM) and was 6.7-fold more potent than diosgenin (IC₅₀ = 26.41 μM). Moreover, compound 8d displayed low toxicity against GES-1 cells (IC₅₀ = 420.4 μM), showing specificity between normal and tumor cells. Further cellular mechanism studies in A549 cells indicated that compound 8d triggered the mitochondrial-mediated apoptosis by decreasing mitochondrial membrane potential, which was associated with up-regulation of Bax, down-regulation of Bcl-2 and activation levels of the caspase cascade. The above results indicated that compound 8d may be used as a promising skeleton for antitumor agents with improved efficacy.

Full text of DOI:10.1016/j.ejmech.2019.111897

Journal Pre-proof
Novel diosgenin derivatives containing 1,3,4-oxadiazole/thiadiazole moieties as
potential antitumor agents: Design, synthesis and cytotoxic evaluation
Jinling Zhang, Xuemei Wang, Jifang Yang, Lina Guo, Xiaoli Wang, Bo Song, Wei
Dong, Wenbao Wang
PII:
S0223-5234(19)31049-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.111897
EJMECH 111897
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 October 2019
Revised Date: 16 November 2019
Accepted Date: 16 November 2019
Please cite this article as: J. Zhang, X. Wang, J. Yang, L. Guo, X. Wang, B. Song, W. Dong, W. Wang,
Novel diosgenin derivatives containing 1,3,4-oxadiazole/thiadiazole moieties as potential antitumor
agents: Design, synthesis and cytotoxic evaluation, European Journal of Medicinal Chemistry (2019),
doi: https://doi.org/10.1016/j.ejmech.2019.111897.
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Graphical Abstract

Novel diosgenin derivatives containing 1,3,4-oxadiazole/thiadiazole moieties as
potential antitumor agents: Design, synthesis and cytotoxic evaluation
Jinling Zhanga, Xuemei Wangb, Jifang Yanga, Lina Guoa, Xiaoli Wanga, Bo Songa, Wei Donga, Wenbao Wanga*
aCollege of Pharmacy, Qiqihar Medical University, Qiqihar 161006, Heilongjiang, PR China
bThe second affiliated hospital of Qiqihar Medical University, Qiqihar 161006, Heilongjiang, PR China
E-mail address: wangwenbao0824@163.com (W. B. Wang).
1

Abstract
Diosgenin, a naturally occurring steroidal saponin, has been confirmed to possess potent anticancer properties. In the current
work, two series of novel diosgenin derivatives bearing 1,3,4-oxadiazole (6a−6e and 7a−7e) or 1,3,4-thiadiazole (8a−8e and 9a−9e)
moieties were designed, synthesized and evaluated for their cytotoxicities in four human cancer cell lines (HepG2, A549, MCF-7
and HCT-116) and normal human gastric epithelial cells (GES-1) using the MTT assay in vitro. The results showed that compounds
8d and 9d exhibited significant cytotoxic activities against the HepG2 and A549 cells, being more potent than their parent
compound diosgenin. Furthermore, the 1,3,4-thiadiazole series of compounds generally exhibited stronger cytotoxicity compared
with the 1,3,4-oxadiazole series against HepG2 and A549 cells, and the substitution of 3-pyridyl group at the C5 position of the
1,3,4-thiadiazole ring was the preferred option for these compounds to display significant cytotoxic activities. Compound 8d
showed potent cytotoxic activity against A549 cell line (IC50 = 3.93 µM) and was 6.7-fold more potent than diosgenin (IC50 = 26.41
µM). Moreover, compound 8d displayed low toxicity against GES-1 cells (IC50 = 420.4 µM), showing specificity between normal
and tumor cells. Further cellular mechanism studies in A549 cells indicated that compound 8d triggered the
mitochondrial-mediated apoptosis by decreasing mitochondrial membrane potential, which was associated with up-regulation of
Bax, down-regulation of Bcl-2 and activation levels of the caspase cascade. The above results indicated that compound 8d may be
used as a promising skeleton for antitumor agents with improved efficacy.
Key words: Diosgenin derivatives; 1,3,4-Oxadiazole; 1,3,4-Thiadiazole; Antitumor; Apoptosis.
2

1. Introduction
The incidence and mortality of cancer are rapidly growing worldwide. According to the latest Annual World
Cancer Report, there were approximately 18 million new cases and 9.6 million cancer deaths worldwide in 2018
[1]. Current studies showed that chemotherapy was still one of the most effective methods for cancer treatment.
However, undesirable side effects seriously impede its clinical applications [2, 3]. Therefore, new drugs killing
cancer cells without obvious side effects are of great importance for cancer chemotherapy. Natural products have
been a rich source of compounds for drug discovery and the majority of anticancer drugs currently used in clinic
are derived from natural product scaffolds [4].
Naturally occurring steroids have been shown to possess potent antitumor activity and provide privileged
structures for further modifications [5]. During the last few years, our research groups have been working on
structural modifications of steroids to obtain more active compounds as potential antitumor agents, and some
derivatives with highly promising cytotoxic activities against a number of cancer cell lines were found [6-8].
Diosgenin (DG, Fig. 1), a steroidal saponin abundantly present in the roots of wild yam (Dioscorea villosa),
is used as a traditional medicine because of its activities of antitumor [9-13], antidiabetes [14],
anti-inflammatory [15] and anti-Parkinson’s Disease [16]. More recently, it has been reported that DG inhibited
cancer cell proliferation and induced apoptosis in multiple cancer cell lines including colorectal cancer [9],
hepatocellular carcinoma [10], breast cancer [11], lung cancer [12] and prostate cancer [13]. Although DG has
extensive cytotoxic activity, the use of DG for cancer therapy was hampered due to its moderate potency.
Therefore, it is important to carry out structural modification of DG to obtain promising anticancer compounds.
Heterocycles are considered as privileged scaffolds in drug discovery and widely used in medicinal
chemistry [17, 18]. Among them, 1,3,4-oxadiazole and 1,3,4-thiadiazole are two well-known pharmacophores
and have drawn much attention due to their diverse pharmacological activities. Multiple studies have
demonstrated that 1,3,4-oxadiazole/thiadiazole containing Zibotentan (an anticancer drug candidate in late stage
clinical trials) and compounds A-D exhibited significant cytotoxic activity against various cancer cell lines
[19-23] (Fig. 2). Hence, the incorporation of these two moieties in the designed compounds may give new
effective antitumor agents.
Aiming at finding promising new leads with potential antitumor activity, it was considered worthwhile to
design and synthesize novel diosgenin derivatives that contain the 1,3,4-oxadiazole or 1,3,4-thiadiazole ring
systems. Accordingly, twenty novel diosgenin 1,3,4-oxadiazolyl/thiadiazolyl derivatives were designed,
3

synthesized, characterized and evaluated for their cytotoxicity against four tumor cell lines and one normal cell
line.
2. Results and discussion
2.1 Chemistry
The synthetic route for the target compounds is shown in Scheme 1. The acetylation of DG (1), using dry
dichloromethane as a co-solvent, afforded intermediate 2 according to the published method [24] with some
modifications. Next, intermediate 2 was converted into intermediate 3 by reductive opening of the cyclic
spiroketal using NaBH3CN in AcOH at room temperature in 81.0% yield [6]. Intermediate 3 was oxidized using
Jones reagent to give the key intermediate 4 in 76.0% yield. Subsequently, the intermediate 4 was coupled with
the appropriate acylhydrazines in the presence of O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium-
tetrafluoroboratetetrabutyl (TBTU) as a coupling catalyst to afford intermediates 5a−5e [25]. The
cyclodehydration of intermediates 5a−5e with POCl3 at 80 oC furnished the corresponding compounds 6a−6e in
good yields according to the method described in the literature [26], and subsequent deacetylation led to the
3β-hydroxy derivatives 7a−7e. On the other hand, the Lawesson reagent (LR) induced dehydrosulfurization of
intermediates 5a−5e in refluxing toluene for 1 h furnished compounds 8a−8e in good yields [26]. Finally,
deacetylation of compounds 8a−8e afforded the 3β-hydroxy analogs 9a−9e. All the synthesized compounds
were well characterized by their ESI-HRMS, 1H NMR, and 13C NMR spectral data.
2.2 Biological evaluation
2.2.1 In vitro cytotoxic activity
The in vitro cytotoxicities of the synthesized diosgenin derivatives against human hepatoma (HepG2), lung
carcinoma (A549), breast cancer (MCF-7), colorectal cancer (HCT-116) and normal gastric (GES-1) cell lines
were evaluated by the MTT assay, and Mitomycin C was used as the positive control. The IC50 values are
presented in Table 1. The results showed that compounds 8d and 9d exhibited marked cytotoxicities against the
HepG2 and A549 cell lines, and were more potent than their parent compound DG and Mitomycin C.
4

For HepG2 cell line, the 1,3,4-oxadiazolyl series of compounds 6a−6e (R2 = Ac) and 7a−7d (R2 = H)
displayed lower cytotoxic activities than DG (IC50 = 33.87 µM). Only compound 7e (IC50 = 29.24 µM) showed
similar cytotoxicity to DG. The substitutions (CH3, phenyl, p-methoxyphenyl and 1-naphthyl) on the
1,3,4-thiadiazole moiety led to compounds 8a−8e and 9a−9e without significant improvement in cytotoxicity
except for compounds 8d and 9d. These two compounds possessing 3-pyridyl group displayed significant
cytotoxic activities with IC50 values of 11.73 and 8.83 µM, respectively. Specifically, the cytotoxicity of
compound 8d was about 2.9-fold more potent than those of compounds 8a (IC50 = 34.25 µM) and DG. For
compound 9d, the cytotoxicity was 2.6−3.8-fold higher than those of compounds 9a (IC50 = 33.16 µM), 9b (IC50
= 32.14 µM), 9c (IC50 = 23.05 µM) and 9e (IC50 = 32.90 µM), and was about 3.8-fold more potent than that of
DG. Furthermore, compounds 8d and 9d were less toxic to non-cancer cells GES-1 with IC50 values of 420.4
and 354.4 µM, respectively. These data showed that the incorporation of a 3-pyridine ring at the C5 position of
the 1,3,4-thiadiazole ring led to significant improvement in cytotoxic activity when compared with methyl and
aryl rings.
For A549 cell line, compounds 8d (IC50 = 3.93 µM) and 9d (IC50 = 7.68 µM) also possessed stronger
cytotoxicity than that of DG (IC50 = 26.41 µM). Among these compounds, compound 8d was the most active
compound, which was 6.7-fold more potent than DG. Moreover, compound 8d (IC50 = 420.4 µM) showed lower
cytotoxicity against normal cells GES-1 than DG (IC50 = 100.7 µM). Compound 9d was 2.0−4.8-fold more
potent than compounds 9a (IC50 = 36.71 µM), 9b (IC50 = 15.04 µM), 9c (IC50 = 16.4 µM) and 9e (IC50 = 18.26
µM). The results also suggested that the substitution of 3-pyridyl group at the C5 position of the
1,3,4-thiadiazole ring was beneficial for compounds to display potent cytotoxicity against A549 cell line.
For MCF-7 cell line, the 20 synthesized derivatives exhibited lower cytotoxicity compared with DG (IC50
23.91 µM) with the exception of compound 7a (IC50 = 20.37 µM). For HCT-116 cell line, compound 8d (IC50
29.56 µM) showed higher cytotoxicity than DG (IC50 = 49.11 µM).
=
=
Taken together, the 1,3,4-thiadiazole series of compounds generally exhibited stronger cytotoxicity
compared with the 1,3,4-oxadiazole series against the HepG2 and A549 cell lines. The above results suggested
that the group with 3-pyridyl substitution at the C5 position of the 1,3,4-thiadiazole ring was the preferred
substitution pattern for compounds with potent cytotoxic activities.
The partition coefficient (log P) between two solvents n-octanol and water has been used as a classical
descriptor for lipophilicity. Compound 8d exhibited potent cytotoxic activity against A549 cell line. Therefore,
5

the calculated log P (clog P) values of compound 8d and DG were calculated using SwissADME web tool [27,
28]. The results showed that the clog P value of compound 8d (6.35) was relatively higher than that of DG
(5.03). Compound 8d with the medium clog P value showed stronger cytotoxicity, which may attribute to the
increase of lipophilicity.
To gain insight into the cellular mechanism of our synthesized compounds, the most potent compound 8d
was selected for further studies in A549 cells by performing DAPI, Annexin V/propidium iodide (PI) staining,
mitochondrial membrane potential (△Ψm) and Western blot assays.
2.2.2 Apoptosis study
Previous studies have reported that DG exhibited cytotoxic activity against DU145 and K562 cells by
inducing apoptosis in vitro [29, 30]. Compared with the control group, some of the 8d-treated cells exhibited
rounding, shrinkage, membrane blebbing and the formation of apoptotic bodies, which were hallmarks of
apoptotic cells (Fig. 3A). However, those changes were not obviously observed in the DG group. Changes of the
morphological characters in A549 cells were further studied by DAPI staining under fluorescence microscopy to
confirm that the growth inhibitory activity of 8d was related to the inducing of apoptosis. As shown in Fig. 3B,
cells of the untreated group were stained homogeneously, while some 8d-treated cells exhibited bright chromatin
condensation and nuclear fragmentation, a hallmark of apoptosis. The above observation indicated that the
treatment of 8d induced the apoptosis of A549 cells.
To further verify whether 8d could induce apoptosis in A549 cells, 8d-incubated A549 cells were stained
with Annexin V (AV) and propidium iodide (PI). As shown in Fig. 4, the percentage of total died cells (apoptotic
and necrotic cells) increased to 36.1% and 81.3% after treatment with 8d at the concentrations of 15 and 30 µM,
respectively, in comparison with the control (5.4%). Moreover, the apoptotic ratios of 8d-treated cells were
significantly increased than those of DG-treated cells. These findings suggested that 8d exerted its
antiproliferative effects by inducing apoptosis.
Subsequently, the expression levels of cleaved caspase-9, 3 and cleaved PARP were evaluated by Western
blot analysis in A549 cells. Fig. 5 showed that the expression levels of cleaved caspase-9, cleaved caspase-3 and
cleaved PARP were activated after treated with 8d. However, the expression levels of those proteins did not
change after treated with DG in A549 cells.
6

2.2.3 Measurement of mitochondrial membrane potential (MMP)
Mitochondrion plays an important role in regulating cellular functions, and mitochondrial dysfunction has
been identified as one of the characteristic events of apoptosis [8]. In order to determine whether 8d-induced
apoptosis was involved in a disruption of mitochondrial membrane integrity, we analyzed MMP changes of the
A549 cells by staining with the dye JC-1 and analyzing the cells by flow cytometry. As shown in Fig. 6, 8d
induced a concentration-dependent increase in depolarized cell population from 0.4% of control to 7.5% and
26.0%, respectively. The results indicated that apoptosis in A549 cells induced by 8d was associated with the
mitochondrial (intrinsic) pathways.
2.2.4 8d induces apoptosis through mitochondrial pathway
The mitochondria-dependent apoptotic pathway, also called the intrinsic pathway, is modulated by the Bcl-2
family of proteins, including Bax (pro-apoptotic protein) and Bcl-2 (anti-apoptotic protein), which could induce
cytochrome c (cyto-c) released into the cytosol, resulting in the activation of the caspase-9, -3, and PARP
cleavage, finally triggering the execution of apoptosis [31]. Therefore, to reveal the molecular mechanism of 8d,
we examined the expression of Bcl-2, Bax, and cyto-c by Western blot analysis. As shown in Fig. 7, in
comparison with untreated cells, 8d could increase the level of Bax and decrease the level of Bcl-2 in a
concentration-dependent manner. Moreover, the expression of cyto-c remarkably increased in a dose-related
fashion (Fig. 7). These results indicated that 8d could induce the apoptosis of A549 cells via increasing the level
of Bax and decreasing the level of Bcl-2, leading to the release of cyto-c and activation of caspase-9, -3, and
PARP cleavage, which triggered the execution of apoptosis.
The above results indicated that compound 8d showed low cytotoxicity to normal human cells and potential
anti-cancer activity against A549 cells by inducing apoptosis through mitochondrial pathway.
3. Conclusion
In the present work, twenty novel diosgenin 1,3,4-oxadiazolyl/thiadiazolyl derivatives were synthesized and
tested for their cytotoxic activities in vitro. Derivatives 8d and 9d showed stronger cytotoxic effects than their
parent compound diosgenin in the HepG2 and A549 cell lines. The 1,3,4-thiadiazole series of compounds
generally exhibited stronger cytotoxicity compared with the 1,3,4-oxadiazole series against the HepG2 and A549
cell lines, and the substitution of 3-pyridyl group at the C5 position of the 1,3,4-thiadiazole ring was the
7

preferred option for these compounds to display significant cytotoxic activities. Compound 8d displayed the
most potent cytotoxicity against A549 cells (IC50 = 3.93 µM) and low cytotoxicity against normal GES-1 cells
(IC50 = 420.4 µM). Further studies on the cellular mechanism of compound 8d showed that it caused
morphological changes, decreased mitochondrial membrane potential, and induced apoptosis through
mitochondrial pathway in A549 cells. The results of the current study imply that compound 8d is a lead
compound for further research.
4. Experimental protocols
4.1 Chemistry
Diosgenin was purchased from Xi’an Xiaocao Plant Technology Co., Ltd. Reagents and solvents were
purchased from commercial sources and used as received. Melting points (mp) were determined on a WRS-2
1
melting apparatus and were uncorrected. H NMR and 13C NMR spectra were recorded in CDCl3 with Bruker
AVANCE NEO 600 spectrometer (TMS as internal standard). The values of the chemical shifts were expressed
in δ values (ppm) and the coupling constants (J) in Hz. ESI-HRMS data were obtained using an AB SCIEX
TripleTOF 4600 instrument. Reaction progress was monitored by analytical thin layer chromatography (TLC) on
silica gel HSGF254 plates (Yantai Zifu Chemical Group Co. China). Silica gel (100−200 or 200−300 mesh,
Qingdao city, China) was used for column chromatography.
4.1.1 Synthesis of (25R)-3β-acetoxy-5-en-spirostan (2).
Ac2O (14.8 g, 144.9 mol) was added to a magnetically stirred solution of diosgenin (30 g, 72.4 mmol) in
CH2Cl2 (210 mL) and pyridine (60 mL). After the mixture was stirred at room temperature for 6 h, water (300
mL) was added, and then extracted with CH2Cl2 (100 mL). The combined organic layers were washed with
saturated NaHCO3, brine and dried over anhydrous Na2SO4, then the solids were removed by filtration. The
solvent was evaporated in vacuo, and the crude material was purified by reflux with anhydrous ethanol (240 mL)
o
1
to provide compound 2 (32.1 g, 97.0%). White amorphous powder, mp 194.8−195.6 C. H NMR (600 MHz,
CDCl3, δ): 5.37 (d, J = 4.9 Hz, 1H, H-6), 4.60 (m, 1H, H-3), 4.41 (dd, J = 14.8, 7.2 Hz, 1H, H-16), 3.47 (dd, J =
10.1, 4.1 Hz, 1H, H-26), 3.37 (t, J = 11.0 Hz, 1H, H-26), 2.03 (s, 3H, Ac-CH3), 1.03 (s, 3H, 19-CH3), 0.97 (d, J
= 7.0 Hz, 3H, 21-CH3), 0.78 (s, 3H, 18-CH3), 0.78 (d, J = 5.6 Hz, 3H, 27-CH3). 13C NMR (150 MHz, CDCl3, δ):
170.7 (Ac-CO), 139.8 (C-5), 122.5 (C-6), 109.4 (C-22), 80.9 (C-16), 74.0 (C-3), 67.0 (C-26), 62.2 (C-17), 56.6
8

(C-14), 50.1 (C-9), 41.8 (C-20), 40.4 (C-12), 39.9 (C-13), 38.2 (C-4), 37.1 (C-1), 36.9 (C-10), 32.2 (C-7), 32.0
(C-15), 31.8 (C-8), 30.4 (C-23), 29.8 (C-25), 29.0 (C-2), 27.9 (C-24), 21.6 (Ac-CH3), 21.0 (C-11), 19.5 (C-19),
17.3 (C-27), 16.4 (C-18), 14.7 (C-21). ESI-HRMS: m/z 457.3330 [M+H]+ (Calcd for C29H45O4, 457.3312).
4.1.2 Synthesis of (22R,25R)-3β-acetoxy-5-en-furostan-26-ol (3).
To a magnetically stirred solution of appropriate compound 2 (20 g, 43.9 mmol) in CH2Cl2 (80 mL) was
added acetic acid (20 mL) and NaBH3CN (5.53 g, 87.8 mmol). After 8 h, the mixture was alkalified by saturated
Na2CO3 solution. The organic layer was washed with water and brine and dried over anhydrous Na2SO4, then the
solids were removed by filtration. The solvent was evaporated in vacuo, and the crude material was purified by
column chromatography over the silica gel with petroleum ether–ethyl acetate (20:1) to get intermediate 3 (16.3
g, 81.0%). White amorphous powder, mp 107.2−108.6 oC. 1H NMR (600 MHz, CDCl3, δ): 5.36 (d, J = 5.0 Hz,
1H, H-6), 4.59 (m, 1H, H-3), 4.30 (m, 1H, H-16), 3.49 (dd, J = 10.6, 6.0 Hz, 1H, H-26), 3.43 (dd, J = 10.6, 6.0
Hz, 1H, H-26), 3.32 (td, J = 8.3, 3.7 Hz, 1H, H-22), 2.02 (s, 3H, Ac-CH3), 1.03 (s, 3H, 19-CH3), 1.00 (d, J = 6.7
Hz, 3H, 21-CH3), 0.91 (d, J = 6.8 Hz, 3H, 27-CH3), 0.80 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ):
170.7 (Ac-CO), 139.8 (C-5), 122.5 (C-6), 90.5 (C-22), 83.3 (C-16), 74.0 (C-3), 68.2 (C-26), 65.2 (C-17), 57.0
(C-14), 50.1 (C-9), 40.8 (C-12), 39.5 (C-13), 38.2 (C-4), 38.1 (C-20), 37.1 (C-1), 36.8 (C-10), 35.9 (C-25), 32.4
(C-7), 32.1 (C-15), 31.7 (C-8), 30.6 (C-24), 30.3 (C-23), 27.9 (C-2), 21.6 (Ac-CH3), 20.8 (C-11), 19.5 (C-19),
19.1 (C-21), 16.8 (C-27), 16.6 (C-18). ESI-HRMS: m/z 459.3472 [M+H]+ (Calcd for C29H47O4, 459.3469).
4.1.3 Synthesis of (22R,25R)-3β-acetoxy-5-en-furostan-26-oic acid (4).
To a stirred solution of compound 3 (10.0 g, 21.8 mmol) in a mixture of THF (50 mL) and acetone (50 mL),
Jones reagent was added dropwise at room temperature until the solution remained orange. Then the reaction
mixture was stirred for 2 h at room temperature. Upon completion, the precipitate was removed by filtration and
the solvent was evaporated under reduced pressure. The residue was dissolved in CH2Cl2 (50 mL) and washed
with brine (3×50 mL) and dried over anhydrous Na2SO4, then concentrated under reduced pressure. The residue
was purified on silica gel chromatography with petroleum ether-ethyl acetate (10:1) to give intermediate 4 (7.63
g, 76.0%). White amorphous powder, mp 124.5−125.2 oC. 1H NMR (600 MHz, CDCl3, δ): 5.37 (d, J = 4.8 Hz,
1H, H-6), 4.60 (m, 1H, H-3), 4.33 (m, 1H, H-16), 3.35 (td, J = 8.6, 2.4 Hz, 1H, H-22), 2.52 (m, 1H, H-25), 2.03
(s, 3H, Ac-CH3), 1.18 (d, J = 6.9 Hz, 3H, 27-CH3), 1.03 (s, 3H, 19-CH3), 1.00 (d, J = 6.7 Hz, 3H, 21-CH3), 0.79
9

(s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 181.2 (C-26), 170.7 (Ac-CO), 139.8 (C-5), 122.5 (C-6), 90.4
(C-22), 83.7 (C-16), 74.0 (C-3), 65.0 (C-17), 57.0 (C-14), 50.1 (C-9), 40.8 (C-12), 39.5 (C-13), 39.5 (C-25),
38.2 (C-4), 38.2 (C-20), 37.1 (C-1), 36.8 (C-10), 32.3 (C-7), 32.1 (C-15), 31.7 (C-8), 31.6 (C-24), 31.1 (C-23),
27.9 (C-2), 21.6 (Ac-CH3), 20.8 (C-11), 19.5 (C-19), 19.0 (C-21), 17.4 (C-27), 16.5 (C-18). ESI-HRMS: m/z
473.3220 [M+H]+ (Calcd for C29H45O5, 473.3262).
4.1.4 General procedure for synthesis of compounds 5a−5e.
To a solution of compound 4 (0.6 g, 1.28 mmol) and the appropriate acylhydrazines (2.56 mmol) in CH2Cl2
(5 mL) was added TBTU (0.82 g, 2.56 mmol) and DIPEA (0.33 g, 2.56 mmol) and stirred for 2 h at room
temperature. When the substrates disappeared (as detected by TLC), the mixture was washed with water (50 mL)
and brine (50 mL) and dried over anhydrous Na2SO4. The solvent was concentrated under vacuum. The crude
product was purified by flash chromatography on silica gel using petroleum ether-ethyl acetate (2:1) as eluent to
yield compounds 5a−5e.
4.1.4.1 (22R,25R)-N-Acetyl-3β-acetoxy-5-en-furostan-26-carbohydrazide (5a).
White amorphous powder, yield 74.5%, mp 221.9−223.0 oC. 1H NMR (600 MHz, CDCl3, δ): 9.24 (brs, 1H,
NH), 9.15 (d, J = 4.7 Hz, 1H, NH), 5.36 (d, J = 3.3 Hz, 1H, H-6), 4.59 (m, 1H, H-3), 4.30 (m, 1H, H-16), 3.28 (t,
J = 6.4 Hz, 1H, H-22), 2.52 (m, 1H, H-25), 2.03 (s, 3H, 2′-CH3), 2.02 (s, 3H, Ac-CH3), 1.13 (d, J = 6.6 Hz, 3H,
27-CH3), 1.02 (s, 3H, 19-CH3), 0.96 (d, J = 6.1 Hz, 3H, 21-CH3), 0.76 (s, 3H, 18-CH3). 13C NMR (150 MHz,
CDCl3, δ): 173.6 (C-26), 170.7 (Ac-CO), 167.0 (C-1′), 139.8 (C-5), 122.5 (C-6), 91.0 (C-22), 83.7 (C-16), 74.0
(C-3), 64.7 (C-17), 57.1 (C-14), 50.1 (C-9), 40.8 (C-12), 39.6 (C-13), 38.4 (C-25), 38.3 (C-20), 38.2 (C-4), 37.1
(C-1), 36.8 (C-10), 32.2 (C-7), 32.1 (C-15), 31.6 (C-8), 29.8 (C-24), 29.8 (C-23), 27.9 (C-2), 21.6 (Ac-CH3),
20.8 (C-2′), 20.8 (C-11), 19.5 (C-19), 18.7 (C-21), 17.9 (C-27), 16.5 (C-18). ESI-HRMS: m/z 529.3650 [M+H]+
(Calcd for C31H49N2O5, 529.3636).
4.1.4.2 (22R,25R)-N-Benzoyl-3β-acetoxy-5-en-furostan-26-carbohydrazide (5b).
White amorphous powder, yield 63.1%, mp 106.3−107.8 oC. 1H NMR (600 MHz, CDCl3, δ): 9.75 (d, J = 3.5
Hz, 1H, NH), 9.54 (d, J = 3.5 Hz, 1H, NH), 7.79 (d, J = 7.7 Hz, 2H, H-2′ and H-6′), 7.44 (t, J = 7.7 Hz, 2H,
H-4′), 7.33 (d, J = 7.7 Hz, 2H, H-3′ and H-5′), 5.34 (d, J = 4.3 Hz, 1H, H-6), 4.57 (m, 1H, H-3), 4.29 (m, 1H,
10

H-16), 3.27 (t, J = 7.8 Hz, 1H, H-22), 2.59 (m, 1H, H-25), 2.00 (s, 3H, Ac-CH3), 1.12 (d, J = 6.6 Hz, 3H,
27-CH3), 0.98 (s, 3H, 19-CH3), 0.94 (d, J = 6.5 Hz, 3H, 21-CH3), 0.74 (s, 3H, 18-CH3). 13C NMR (150 MHz,
CDCl3, δ): 174.2 (C-26), 170.6 (Ac-CO), 164.4 (C-28), 139.7 (C-5), 132.1 (C-4′), 131.8 (C-1′), 128.6 (2C,
C-3′ and C-5′), 127.4 (2C, C-2′ and C-6′), 122.4 (C-6), 90.8 (C-22), 83.6 (C-16), 73.9 (C-3), 64.7 (C-17), 57.0
(C-14), 50.0 (C-9), 40.7 (C-12), 39.5 (C-13), 38.5 (C-25), 38.3 (C-20), 38.1 (C-4), 37.0 (C-1), 36.8 (C-10), 32.2
(C-7), 32.0 (C-15), 31.6 (C-8), 31.0 (C-24), 30.7 (C-23), 27.8 (C-2), 21.5 (Ac-CH3), 20.7 (C-11), 19.4 (C-19),
18.7 (C-21), 17.8 (C-27), 16.4 (C-18). ESI-HRMS: m/z 591.3784 [M+H]+ (Calcd for C36H51N2O5, 591.3792).
4.1.4.3 (22R,25R)-N-(p-Methoxybenzoyl)-3β-acetoxy-5-en-furostan-26-carbohydrazide (5c).
White amorphous powder, yield 48.7%, mp 108.8−111.0 oC. 1H NMR (600 MHz, CDCl3, δ): 9.26 (brs, 1H,
NH), 9.20 (brs, 1H, NH), 7.77 (d, J = 8.6 Hz, 2H, H-2′ and H-6′), 6.86 (d, J = 8.6 Hz, 2H, H-3′ and H-5′), 5.35
(d, J = 3.3 Hz, 1H, H-6), 4.58 (m, 1H, H-3), 4.31 (m, 1H, H-16), 3.82 (s, 3H, 4′-OCH3), 3.30 (t, J = 6.7 Hz, 1H,
H-22), 2.58 (m, 1H, H-25), 2.02 (s, 3H, Ac-CH3), 1.16 (d, J = 6.6 Hz, 3H, 27-CH3), 1.00 (s, 3H, 19-CH3), 0.96
(d, J = 6.6 Hz, 3H, 21-CH3), 0.75 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 173.9 (C-26), 170.7
(Ac-CO), 164.0 (C-28), 162.8 (C-4′), 139.8 (C-5), 129.3 (2C, C-2′ and C-6′), 124.1 (C-1′), 122.5 (C-6), 113.9
(2C, C-3′ and C-5′), 91.0 (C-22), 83.7 (C-16), 74.0 (C-3), 64.7 (C-17), 57.1 (C-14), 55.5 (C-4′-OCH3), 50.1
(C-9), 40.8 (C-12), 39.5 (C-13), 38.6 (C-25), 38.4 (C-20), 38.2 (C-4), 37.1 (C-1), 36.8 (C-10), 32.3 (C-7), 32.1
(C-15), 31.6 (C-8), 31.1 (C-24), 30.7 (C-23), 27.9 (C-2), 21.6 (Ac-CH3), 20.8 (C-11), 19.4 (C-19), 18.8 (C-21),
17.9 (C-27), 16.5 (C-18). ESI-HRMS: m/z 621.3910 [M+H]+ (Calcd for C37H53N2O6, 621.3898).
4.1.4.4 (22R,25R)-N-(Pyridyl-3-carbonyl)-3β-acetoxy-5-en-furostan-26-carbohydrazide (5d).
White amorphous powder, yield 67.8%, mp 124.9−126.4 oC. 1H NMR (600 MHz, CDCl3, δ): 9.81 (brs, 1H,
NH), 9.32 (brs, 1H, NH), 9.01 (s, 1H, H-2′), 8.69 (brs, 1H, H-6′), 8.12 (d, 1H, J = 7.9 Hz, H-4′), 7.33 (dd, J =
7.9, 4.5 Hz, 1H, H-5′), 5.34 (d, J = 4.0 Hz, 1H, H-6), 4.58 (m, 1H, H-3), 4.33 (m, 1H, H-16), 3.32 (m, 1H, H-22),
2.64 (m, 1H, H-25), 2.01 (s, 3H, Ac-CH3), 1.15 (d, J = 6.7 Hz, 3H, 27-CH3), 0.98 (s, 3H, 19-CH3), 0.97 (d, J =
6.9 Hz, 3H, 21-CH3), 0.75 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 174.8 (C-26), 170.7 (Ac-CO),
163.0 (C-28), 152.7 (C-2′), 148.7 (C-6′), 139.8 (C-5), 135.2 (C-4′), 127.8 (C-5′), 123.5 (C-3′), 122.4 (C-6), 91.2
(C-22), 83.8 (C-16), 74.0 (C-3), 64.5 (C-17), 57.0 (C-14), 50.1 (C-9), 40.8 (C-12), 39.5 (C-13), 38.5 (C-25),
38.2 (C-4), 38.2 (C-20), 37.1 (C-1), 36.8 (C-10), 32.2 (C-7), 32.1 (C-15), 31.6 (C-8), 31.0 (C-24), 30.4 (C-23),
11

27.8 (C-2), 21.5 (Ac-CH3), 20.7 (C-11), 19.4 (C-19), 18.6 (C-27), 17.8 (C-21), 16.5 (C-18). ESI-HRMS: m/z
592.3769 [M+H]+ (Calcd for C35H50N3O5, 592.3745).
4.1.4.5 (22R,25S)-N-(1′-Naphthoyl)-3β-acetoxy-5-en-furostan-26-carbohydrazide (5e).
o
1
White amorphous powder, yield 70.6%, mp 157.7−158.6 C. H NMR (600 MHz, CDCl3, δ): 8.36 (m, 1H,
Ar-H), 7.92 (d, J = 8.2 Hz, 1H, Ar-H), 7.84 (m, 1H, Ar-H), 7.70 (d, J = 8.2 Hz, 1H, Ar-H), 7.51 (m, 2H, Ar-H),
7.42 (t, J = 8.2 Hz, 1H, Ar-H), 5.37 (d, J = 4.9 Hz, 1H, H-6), 4.59 (m, 1H, H-3), 4.36 (m, 1H, H-16), 3.35 (t, J =
8.4 Hz, 1H, H-22), 2.68 (m, 1H, H-25), 2.02 (s, 3H, Ac-CH3), 1.19 (d, J = 6.7 Hz, 3H, 27-CH3), 1.00 (s, 3H,
19-CH3), 0.98 (d, J = 6.7 Hz, 3H, 21-CH3), 0.82 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 174.1 (C-2′),
170.7 (Ac-CO), 166.2 (C-5′), 139.8 (C-5), 133.8 (Ar-C), 131.6 (Ar-C), 131.2 (Ar-C), 130.5 (Ar-C), 128.4
(Ar-C), 127.5 (Ar-C), 126.7 (Ar-C), 126.0 (Ar-C), 125.4 (Ar-C), 124.7 (Ar-C), 122.5 (C-6), 91.4 (C-22), 84.0
(C-16), 74.0 (C-3), 64.5 (C-17), 57.1 (C-14), 50.1 (C-9), 40.8 (C-12), 39.5 (C-13), 38.7 (C-25), 38.6 (C-20),
38.2 (C-4), 37.1 (C-1), 36.8 (C-10), 32.3 (C-7), 32.1 (C-15), 31.7 (C-8), 31.0 (C-24), 30.5 (C-23), 27.9 (C-2),
21.5 (Ac-CH3), 20.7 (C-11), 19.4 (C-19), 18.5 (C-21), 17.9 (C-27), 16.5 (C-18). ESI-HRMS: m/z 641.3959
[M+H]+ (Calcd for C40H53N2O5, 641.3949).
4.1.5 General procedure for synthesis of compounds 6a−6e.
N,N′-Disubstituted hydrazine (5a−5e, 0.40 mmol) was heated in POCl3 (5 mL) at 80 oC for 1 h until the
starting material was not observed by TLC. The reaction mixture was cooled to room temperature, carefully
poured onto crushed ice and made basic with saturated NaHCO3 solution. The resulting precipitate was filtered,
dried and purified by silica gel chromatography to obtain compounds 6a−6e.
4.1.5.1 (22R,25R)-3β-Acetoxy-5-en-25-(2′-[5′-methyl]-1′,3′,4′-oxadiazolyl) furostan (6a).
White amorphous powder, yield 75.0%, mp 120.4−121.7 oC. 1H NMR (600 MHz, CDCl3, δ): 5.36 (d, J = 4.8
Hz, 1H, H-6), 4.58 (m, 1H, H-3), 4.28 (m, 1H, H-16), 3.28 (m, 1H, H-22), 3.07 (dd, J = 14.9, 7.1 Hz, 1H, H-25),
2.49 (s, 3H, 5′-CH3), 2.02 (s, 3H, Ac-CH3), 1.34 (d, J = 7.1 Hz, 3H, 27-CH3), 1.02 (s, 3H, 19-CH3), 0.96 (d, J =
7.1 Hz, 3H, 21-CH3), 0.77 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 170.7 (Ac-CO), 170.5 (C-2′), 163.6
(C-5′), 139.8 (C-5), 122.5 (C-6), 89.8 (C-22), 83.4 (C-16), 74.0 (C-3), 65.2 (C-17), 57.0 (C-14), 50.1 (C-9), 40.8
(C-12), 39.5 (C-13), 38.2 (C-20), 38.0 (C-4), 37.1 (C-1), 36.8 (C-10), 32.3 (C-7), 32.1 (C-15), 32.0 (C-25), 31.9
12

(C-24), 31.7 (C-8), 31.1 (C-23), 27.9 (C-2), 21.6 (Ac-CH3), 20.8 (C-11), 19.5 (C-19), 19.0 (C-27), 18.4 (C-21),
16.5 (C-18), 11.1 (5′-CH3). ESI-HRMS: m/z 511.3562 [M+H]+ (Calcd for C31H47N2O4, 511.3530).
4.1.5.2 (22R,25R)-3β-Acetoxy-5-en-25-(2′-[5′-phenyl]-1′,3′,4′-oxadiazolyl) furostan (6b).
White amorphous powder, yield 81.5%, mp 97.2−98.6 oC. 1H NMR (600 MHz, CDCl3, δ): 8.04 (dd, J = 7.9,
1.3 Hz, 2H, H-2′′ and H-6′′), 7.50 (m, 3H, H-3′′, H-4′′ and H-5′′), 5.36 (d, J = 3.5 Hz, 1H, H-6), 4.59 (m, 1H,
H-3), 4.28 (m, 1H, H-16), 3.30 (dd, J = 13.6, 7.3 Hz, 1H, H-22), 3.20 (m, 1H, H-25), 2.02 (s, 3H, Ac-CH3), 1.43
(d, J = 7.0 Hz, 3H, 27-CH3), 1.02 (s, 3H, 19-CH3), 0.96 (d, J = 6.5 Hz, 3H, 21-CH3), 0.77 (s, 3H, 18-CH3). 13
C
NMR (150 MHz, CDCl3, δ): 170.7 (Ac-CO), 170.3 (C-2′), 164.7 (C-5′), 139.8 (C-5), 131.6 (C-4′′), 129.1 (2C,
C-3′′ and C-5′′), 126.9 (2C, C-2′′ and C-6′′), 124.3 (C-1′′), 122.5 (C-6), 89.9 (C-22), 83.4 (C-16), 74.0 (C-3),
65.1 (C-17), 57.0 (C-14), 50.1 (C-9), 40.8 (C-12), 39.5 (C-13), 38.2 (C-4), 38.1 (C-20), 37.1 (C-1), 36.8 (C-10),
32.3 (C-7), 32.2 (C-15), 32.1 (C-25), 31.7 (C-8), 31.2 (C-24), 31.1 (C-23), 27.9 (C-2), 21.6 (Ac-CH3), 20.7
(C-11), 19.5 (C-19), 19.0 (C-27), 18.5 (C-21), 16.5 (C-18). ESI-HRMS: m/z 573.3714 [M+H]+ (Calcd for
C36H49N2O4, 573.3687).
4.1.5.3 (22R,25R)-3β-Acetoxy-5-en-25-(2′-[5′-p-methoxyphenyl]-1′,3′,4′-oxadiazolyl) furostan (6c).
White amorphous powder, yield 73.2%, mp 69.8−71.3 oC. 1H NMR (600 MHz, CDCl3, δ): 7.97 (dd, J = 8.7,
2.9 Hz, 2H, H-2′′ and H-6′′), 6.98 (dd, J = 8.7, 2.9 Hz, 2H, H-3′′ and H-5′′), 5.36 (d, J = 2.0 Hz, 1H, H-6), 4.59
(m, 1H, H-3), 4.28 (m, 1H, H-16), 3.86 (s, 3H, H-4′′-OCH3), 3.30 (m, 1H, H-22), 3.16 (m, 1H, H-25), 2.02 (s,
3H, Ac-CH3), 1.41 (d, J = 6.8 Hz, 3H, 27-CH3), 1.02 (s, 3H, 19-CH3), 0.96 (d, J = 6.8 Hz, 3H, 21-CH3), 0.76 (s,
3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 170.7 (Ac-CO), 169.8 (C-2′), 164.6 (C-5′), 162.3 (C-4′′), 139.8
(C-5), 128.7 (2C, C-3′′ and C-5′′), 122.5 (C-6), 116.8 (C-1′′), 114.5 (2C, C-2′′ and C-6′′), 89.9 (C-22), 83.4
(C-16), 74.0 (C-3), 65.1 (C-17), 57.0 (C-14), 55.6 (C-4′′-OCH3), 50.1 (C-9), 40.8 (C-12), 39.5 (C-13), 38.2
(C-4), 38.1 (C-20), 37.1 (C-1), 36.8 (C-10), 32.3 (C-7), 32.2 (C-15), 32.1 (C-25), 31.7 (C-8), 31.2 (C-24), 31.0
(C-23), 27.9 (C-2), 21.6 (Ac-CH3), 20.7 (C-11), 19.4 (C-19), 19.0 (C-27), 18.5 (C-21), 16.5 (C-18). ESI-HRMS:
m/z 603.3795 [M+H]+ (Calcd for C37H51N2O4, 603.3972).
4.1.5.4 (22R,25R)-3β-Acetoxy-5-en-25-(2′-[5′-3′′-pyridyl]-1′,3′,4′-oxadiazolyl) furostan (6d).
o
1
White amorphous powder, yield 82.4%, mp 76.6−78.0 C. H NMR (600 MHz, CDCl3, δ): 9.25 (s, 1H,
13

H-2′′), 8.76 (d, J = 3.5 Hz, 1H, H-6′′), 8.34 (d, 1H, J = 7.9 Hz, H-4′′), 7.46 (dd, J = 7.9, 3.5 Hz, 1H, H-5′′), 5.36
(d, J = 4.8 Hz, 1H, H-6), 4.58 (m, 1H, H-3), 4.28 (m, 1H, H-16), 3.31 (dd, J = 12.6, 7.5 Hz, 1H, H-22), 3.22 (dd,
J = 14.4, 7.2 Hz, 1H, H-25), 2.02 (s, 3H, Ac-CH3), 1.44 (d, J = 6.9 Hz, 3H, 27-CH3), 1.02 (s, 3H, 19-CH3), 0.96
(d, J = 6.9 Hz, 3H, 21-CH3), 0.77 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 170.9 (C-2′), 170.6 (Ac-CO),
162.6 (C-5′), 152.3 (C-2′′), 147.8 (C-6′′), 139.8 (C-5), 132.4 (C-4′′), 123.9 (C-5′′), 122.4 (C-6), 120.8 (C-3′′),
89.8 (C-22), 83.4 (C-16), 74.0 (C-3), 65.1 (C-17), 57.0 (C-14), 50.1 (C-9), 40.8 (C-12), 39.4 (C-13), 38.2 (C-4),
38.0 (C-20), 37.1 (C-1), 36.8 (C-10), 32.3 (C-7), 32.1 (C-15), 32.1 (C-25), 32.1 (C-24), 31.7 (C-8), 31.1 (C-23),
27.8 (C-2), 21.5 (Ac-CH3), 20.7 (C-11), 19.4 (C-19), 18.9 (C-27), 18.4 (C-21), 16.5 (C-18). ESI-HRMS: m/z
574.3615 [M+H]+ (Calcd for C35H48N3O4, 574.3639).
4.1.5.5 (22R,25S)-3β-Acetoxy-5-en-25-(2′-[5′-(naphthalen-1′′-yl)]-1′,3′,4′-oxadiazolyl) furostan (6e).
White amorphous powder, yield 82.0%, mp 146.6−147.9 oC. 1H NMR (600 MHz, CDCl3, δ): 9.20 (d, J = 8.5
Hz, 1H, Ar-H), 8.15 (d, J = 7.3 Hz, 1H, Ar-H), 8.02 (d, J = 8.3 Hz, 1H, Ar-H), 7.92 (d, J = 8.3 Hz, 1H, Ar-H),
7.67 (m, 1H, Ar-H), 7.58 (m, 2H, Ar-H), 5.36 (d, J = 4.9 Hz, 1H, H-6), 4.59 (m, 1H, H-3), 4.30 (m, 1H, H-16),
3.34 (m, 1H, H-22), 3.27 (m, 1H, H-25), 2.03 (s, 3H, Ac-CH3), 1.49 (d, J = 6.9 Hz, 3H, 27-CH3), 1.01 (s, 3H,
19-CH3), 0.97 (d, J = 6.8 Hz, 3H, 21-CH3), 0.77 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 170.7 (C-2′),
169.9 (Ac-CO), 164.7 (C-5′), 139.8 (C-5), 134.0 (Ar-C), 132.5 (Ar-C), 130.2 (Ar-C), 128.8 (Ar-C), 128.4
(Ar-C), 128.2 (Ar-C), 126.8 (Ar-C), 126.4 (Ar-C), 125.0 (Ar-C), 122.5 (C-6), 120.9 (Ar-C), 89.9 (C-22), 83.5
(C-16), 74.0 (C-3), 65.2 (C-17), 57.0 (C-14), 50.1 (C-9), 40.8 (C-12), 39.5 (C-13), 38.2 (C-4), 38.1 (C-20), 37.1
(C-1), 36.8 (C-10), 32.4 (C-25), 32.2 (C-7), 32.1 (C-15), 32.1 (C-23), 31.7 (C-8), 31.2 (C-24), 27.9 (C-2), 21.6
(Ac-CH3), 20.8 (C-11), 19.5 (C-19), 19.0 (C-27), 18.6 (C-21), 16.6 (C-18). ESI-HRMS: m/z 623.3829 [M+H]+
(Calcd for C40H51N2O4, 623.3843).
4.1.6 General procedure for synthesis of compounds 7a−7e.
Compounds 6a−6e (0.2 mmol) was dissolved in MeOH (6 mL), respectively, and KOH (33.6 mg, 0.6 mmol)
was added. The mixture was stirred at room temperature for 1 h. Upon completion, the mixture was diluted with
water and acidified with dilute HCl. The precipitate was filtered, washed with water and dried.
4.1.6.1 (22R,25R)-3β-Hydroxy-5-en-25-(2′-[5′-methyl]-1′,3′,4′-oxadiazolyl) furostan (7a).
14

White amorphous powder, yield 91.0%, mp 162.2−163.5 oC. 1H NMR (600 MHz, CDCl3, δ): 5.33 (d, J = 4.3
Hz, 1H, H-6), 4.28 (m, 1H, H-16), 3.51 (m, 1H, H-3), 3.28 (dd, J = 13.5, 7.8 Hz, 1H, H-22), 3.06 (dd, J = 14.1,
7.2 Hz, 1H, H-25), 2.49 (s, 3H, 5′-CH3), 1.34 (d, J = 6.9 Hz, 3H, 27-CH3), 1.01 (s, 3H, 19-CH3), 0.96 (d, J = 6.9
Hz, 3H, 21-CH3), 0.77 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 170.5 (C-2′), 163.6 (C-5′), 141.0 (C-5),
121.5 (C-6), 89.8 (C-22), 83.4 (C-16), 71.8 (C-3), 65.2 (C-17), 57.1 (C-14), 50.2 (C-9), 42.4 (C-4), 40.8 (C-12),
39.5 (C-13), 38.0 (C-20), 37.4 (C-1), 36.7 (C-10), 32.3 (C-7), 32.1 (C-15), 32.0 (C-25), 31.9 (C-24), 31.7 (C-8),
31.1 (C-23), 31.1 (C-2), 20.8 (C-11), 19.5 (C-19), 19.0 (C-27), 18.3 (C-21), 16.5 (C-18), 11.1 (5′-CH3).
ESI-HRMS: m/z 469.3427 [M+H]+ (Calcd for C29H45N2O3, 469.3425).
4.1.6.2 (22R,25R)-3β-Hydroxy-5-en-25-(2′-[5′-phenyl]-1′,3′,4′-oxadiazolyl) furostan (7b).
o
1
White amorphous powder, yield 93.6%, mp 127.4−128.5 C. H NMR (600 MHz, CDCl3, δ): 8.04 (dd, J =
8.0, 1.3 Hz, 2H, H-2′′ and H-6′′), 7.50 (m, 3H, H-3′′, H-4′′ and H-5′′), 5.34 (d, J = 5.2 Hz, 1H, H-6), 4.29 (m,
1H, H-16), 3.52 (m, 1H, H-3), 3.31 (m, 1H, H-22), 3.20 (m, 1H, H-25), 1.43 (d, J = 7.1 Hz, 3H, 27-CH3), 1.01 (s,
3H, 19-CH3), 0.96 (d, J = 6.9 Hz, 3H, 21-CH3), 0.77 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 170.3
(C-2′), 164.7 (C-5′), 141.0 (C-5), 131.6 (C-4′′), 129.1 (2C, C-3′′ and C-5′′), 126.9 (2C, C-2′′ and C-6′′), 124.3
(C-1′′), 121.5 (C-6), 89.9 (C-22), 83.5 (C-16), 71.9 (C-3), 65.2 (C-17), 57.1 (C-14), 50.2 (C-9), 42.4 (C-4), 40.8
(C-12), 39.5 (C-13), 38.1 (C-20), 37.4 (C-1), 36.8 (C-10), 32.4 (C-7), 32.2 (C-24), 32.1 (C-15), 32.1 (C-25),
31.8 (C-23), 31.7 (C-8), 31.2 (C-2), 20.8 (C-11), 19.6 (C-19), 19.0 (C-27), 18.5 (C-21), 16.6 (C-18). ESI-HRMS:
m/z 531.3589 [M+H]+ (Calcd for C34H47N2O3, 531.3581).
4.1.6.3 (22R,25R)-3β-Hydroxy-5-en-25-(2′-[5′-p-methoxyphenyl]-1′,3′,4′-oxadiazolyl) furostan (7c).
White amorphous powder, yield 92.5%, mp 150.3−151.6 oC. 1H NMR (600 MHz, CDCl3, δ): 7.97 (dd, J =
8.8, 1.9 Hz, 2H, H-2′′ and H-6′′), 6.98 (dd, J = 8.8, 1.9 Hz, 2H, H-3′′ and H-5′′), 5.34 (d, J = 5.0 Hz, 1H, H-6),
4.29 (m, 1H, H-16), 3.87 (s, 3H, H-4′′-OCH3), 3.52 (m, 1H, H-3), 3.31 (m, 1H, H-22), 3.18 (m, 1H, H-25), 1.42
(d, J = 7.0 Hz, 3H, 27-CH3), 1.01 (s, 3H, 19-CH3), 0.96 (d, J = 6.7 Hz, 3H, 21-CH3), 0.77 (s, 3H, 18-CH3). 13
C
NMR (150 MHz, CDCl3, δ): 169.8 (C-2′), 164.6 (C-5′), 162.3 (C-4′′), 141.0 (C-5), 128.7 (2C, C-3′′ and C-5′′),
121.6 (C-6), 116.8 (C-1′′), 114.5 (2C, C-2′′ and C-6′′), 89.9 (C-22), 83.5 (C-16), 71.9 (C-3), 65.2 (C-17), 57.1
(C-14), 55.6 (C-4′′-OCH3), 50.2 (C-9), 42.4 (C-4), 40.8 (C-12), 39.5 (C-13), 38.1 (C-20), 37.4 (C-1), 36.8
(C-10), 32.4 (C-7), 32.2 (C-15), 32.1 (C-25), 31.8 (C-8), 31.8 (C-24), 31.2 (C-23), 29.5 (C-2), 20.8 (C-11), 19.6
15

(C-19), 19.0 (C-27), 18.5 (C-21), 16.6 (C-18). ESI-HRMS: m/z 561.3684 [M+H]+ (Calcd for C35H49N2O4,
561.3687).
4.1.6.4 (22R,25R)-3β-Hydroxy-5-en-25-(2′-[5′-3′′-pyridyl]-1′,3′,4′-oxadiazolyl) furostan (7d).
o
1
White amorphous powder, yield 93.0%, mp 81.6−83.0 C. H NMR (600 MHz, CDCl3, δ): 9.25 (s, 1H,
H-2′′), 8.76 (d, J = 2.8 Hz, 1H, H-6′′), 8.36 (d, 1H, J = 7.9 Hz, H-4′′), 7.47 (dd, J = 7.9, 2.8 Hz, 1H, H-5′′), 5.34
(d, J = 4.8 Hz, 1H, H-6), 4.29 (m, 1H, H-16), 3.50 (m, 1H, H-3), 3.31 (dd, J = 13.5, 7.5 Hz, 1H, H-22), 3.22 (dd,
J = 14.3, 7.1 Hz, 1H, H-25), 1.44 (d, J = 7.0 Hz, 3H, 27-CH3), 1.01 (s, 3H, 19-CH3), 0.96 (d, J = 6.7 Hz, 3H,
21-CH3), 0.77 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 171.0 (C-2′), 162.5 (C-5′), 152.1 (C-2′′), 147.7
(C-6′′), 141.0 (C-5), 134.5 (C-4′′), 124.0 (C-5′′), 121.5 (C-6), 120.7 (C-3′′), 89.8 (C-22), 83.5 (C-16), 71.8 (C-3),
65.1 (C-17), 57.1 (C-14), 50.2 (C-9), 42.4 (C-4), 40.8 (C-12), 39.5 (C-13), 38.1 (C-20), 37.4 (C-1), 36.8 (C-10),
32.4 (C-7), 32.1 (C-15), 32.1 (C-25), 32.1 (C-24), 31.8 (C-23), 31.7 (C-8), 31.1 (C-2), 20.8 (C-11), 19.6 (C-19),
19.0 (C-27), 18.5 (C-21), 16.6 (C-18). ESI-HRMS: m/z 532.3534 [M+H]+ (Calcd for C33H46N3O3, 532.3534).
4.1.6.5 (22R,25S)-3β-Hydroxy-5-en-25-(2′-[5′-(naphthalen-1′′-yl)]-1′,3′,4′-oxadiazolyl) furostan (7e).
White amorphous powder, yield 91.8%, mp 153.6−154.7 oC. 1H NMR (600 MHz, CDCl3, δ): 9.20 (d, J = 8.5
Hz, 1H, Ar-H), 8.15 (d, J = 7.3 Hz, 1H, Ar-H), 8.02 (d, J = 8.3 Hz, 1H, Ar-H), 7.92 (d, J = 8.3 Hz, 1H, Ar-H),
7.67 (m, 1H, Ar-H), 7.58 (m, 2H, Ar-H), 5.34 (d, J = 4.9 Hz, 1H, H-6), 4.30 (m, 1H, H-16), 3.52 (m, 1H, H-3),
3.34 (m, 1H, H-22), 3.27 (m, 1H, H-25), 1.49 (d, J = 6.9 Hz, 3H, 27-CH3), 1.00 (s, 3H, 19-CH3), 0.97 (d, J = 6.7
Hz, 3H, 21-CH3), 0.78 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 169.9 (C-2′), 164.7 (C-5′), 141.0 (C-5),
134.0 (Ar-C), 132.5 (Ar-C), 130.2 (Ar-C), 128.8 (Ar-C), 128.4 (Ar-C), 128.2 (Ar-C), 126.8 (Ar-C), 126.4
(Ar-C), 125.0 (Ar-C), 121.6 (C-6), 121.0 (Ar-C), 89.9 (C-22), 83.5 (C-16), 71.9 (C-3), 65.2 (C-17), 57.1 (C-14),
50.2 (C-9), 42.4 (C-4), 40.8 (C-12), 39.5 (C-13), 38.1 (C-20), 37.4 (C-1), 36.8 (C-10), 32.4 (C-7), 32.2 (C-15),
32.1 (C-25), 32.1 (C-24), 31.8 (C-23), 31.2 (C-8), 30.5 (C-2), 20.8 (C-11), 19.6 (C-19), 19.0 (C-27), 18.6 (C-21),
16.6 (C-18). ESI-HRMS: m/z 581.3777 [M+H]+ (Calcd for C38H49N2O3, 581.3738).
4.1.7 General procedure for synthesis of compounds 8a−8e.
To a solution of the appropriate compounds 5a−5e (0.23 mmol) in dry toluene (6 mL) was added Lawesson
reagent (186 mg, 0.46 mmol) and the reaction mixture was refluxed for 1 h. When TLC showed the completion
16

of the reaction, the solvent was removed in vacuo. The crude product was purified by flash chromatography on
silica gel using petroleum ether-ethyl acetate (10:1) as eluent.
4.1.7.1 (22R,25R)-3β-Acetoxy-5-en-25-(2′-[5′-methyl]-1′,3′,4′-thiadiazolyl) furostan (8a).
o
1
White amorphous powder, yield 75.6%, mp 92.2−93.4 C. H NMR (600 MHz, CDCl3, δ): 5.35 (d, J = 3.8
Hz, 1H, H-6), 4.57 (m, 1H, H-3), 4.27 (dd, J = 13.1, 7.5 Hz, 1H, H-16), 3.32 (m, 1H, H-22), 3.26 (m, 1H, H-25),
2.72 (s, 3H, 5′-CH3), 2.01 (s, 3H, Ac-CH3), 1.37 (d, J = 6.9 Hz, 3H, 27-CH3), 1.01 (s, 3H, 19-CH3), 0.94 (d, J =
7.2 Hz, 3H, 21-CH3), 0.76 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 176.7 (C-2′), 170.7 (Ac-CO), 164.7
(C-5′), 139.8 (C-5), 122.4 (C-6), 90.0 (C-22), 83.4 (C-16), 74.0 (C-3), 65.1 (C-17), 57.0 (C-14), 50.1 (C-9), 40.8
(C-12), 39.5 (C-13), 38.2 (C-4), 37.9 (C-20), 37.1 (C-1), 36.8 (C-10), 36.5 (C-25), 32.3 (C-7), 32.1 (C-15), 31.7
(C-8), 31.3 (C-24), 30.8 (C-23), 27.8 (C-2), 21.5 (Ac-CH3), 20.7 (C-11), 19.4 (C-19), 18.9 (C-27), 18.5 (C-21),
16.5 (C-18), 15.8 (5′-CH3). ESI-HRMS: m/z 527.3300 [M+H]+ (Calcd for C31H47N2O3S, 527.3302).
4.1.7.2 (22R,25R)-3β-Acetoxy-5-en-25-(2′-[5′-phenyl]-1′,3′,4′-thiadiazolyl) furostan (8b).
White amorphous powder, yield 72.0%, mp 66.4−67.8 oC. 1H NMR (600 MHz, CDCl3, δ): 7.94 (dd, J = 5.1,
1.7 Hz, 2H, H-2′′ and H-6′′), 7.46 (m, 3H, H-3′′, H-4′′ and H-5′′), 5.36 (d, J = 3.5 Hz, 1H, H-6), 4.60 (m, 1H,
H-3), 4.30 (dd, J = 13.0, 7.1 Hz, 1H, H-16), 3.40 (m, 1H, H-22), 3.30 (m, 1H, H-25), 2.02 (s, 3H, Ac-CH3), 1.45
(d, J = 6.6 Hz, 3H, 27-CH3), 1.02 (s, 3H, 19-CH3), 0.94 (d, J = 6.7 Hz, 3H, 21-CH3), 0.78 (s, 3H, 18-CH3). 13
C
NMR (150 MHz, CDCl3, δ): 176.2 (C-2′), 170.7 (Ac-CO), 168.2 (C-5′), 139.8 (C-5), 131.0 (C-4′′), 130.5 (C-1′′),
129.2 (2C, C-3′′ and C-5′′), 128.0 (2C, C-2′′ and C-6′′), 122.5 (C-6), 90.0 (C-22), 83.4 (C-16), 74.0 (C-3), 65.1
(C-17), 57.0 (C-14), 50.1 (C-9), 40.8 (C-12), 39.5 (C-13), 38.2 (C-4), 38.0 (C-20), 37.1 (C-1), 36.8 (C-10), 36.6
(C-25), 32.3 (C-7), 32.1 (C-15), 31.7 (C-8), 31.3 (C-24), 30.9 (C-23), 27.9 (C-2), 21.6 (Ac-CH3), 20.7 (C-11),
19.5 (C-19), 19.0 (C-27), 18.6 (C-21), 16.6 (C-18). ESI-HRMS: m/z 589.3434 [M+H]+ (Calcd for C36H49N2O3S,
589.3458).
4.1.7.3 (22R,25R)-3β-Acetoxy-5-en-25-(2′-[5′-p-methoxyphenyl]-1′,3′,4′-thiadiazolyl) furostan (8c).
o
1
White amorphous powder, yield 74.0%, mp 136.9−138.2 C. H NMR (600 MHz, CDCl3, δ): 7.87 (dd, J =
8.7 Hz, 2H, H-2′′ and H-6′′), 6.96 (d, J = 8.7 Hz, 2H, H-3′′ and H-5′′), 5.36 (br.s, 1H, H-6), 4.59 (m, 1H, H-3),
4.29 (m, 1H, H-16), 3.86 (s, 3H, H-4′′-OCH3), 3.39-3.30 (m, 2H, H-22, H-25), 2.02 (s, 3H, Ac-CH3), 1.44 (d, J
17

= 6.9 Hz, 3H, 27-CH3), 1.02 (s, 3H, 19-CH3), 0.95 (d, J = 6.8 Hz, 3H, 21-CH3), 0.77 (s, 3H, 18-CH3). 13C NMR
(150 MHz, CDCl3, δ): 175.4 (C-2′), 170.7 (Ac-CO), 167.9 (C-5′), 161.9 (C-4′′), 139.8 (C-5), 129.5 (2C,
C-3′′ and C-5′′), 123.2 (C-1′′), 122.5 (C-6), 114.6 (2C, C-2′′ and C-6′′), 89.7 (C-22), 83.4 (C-16), 74.0 (C-3),
65.2 (C-17), 57.0 (C-14), 55.6 (C-4′′-OCH3), 50.1 (C-9), 40.8 (C-12), 39.5 (C-13), 38.2 (C-4), 38.0 (C-20), 37.1
(C-1), 36.8 (C-10), 32.4 (C-7), 32.1 (C-15), 31.7 (C-25), 31.7 (C-8), 31.3 (C-24), 30.9 (C-23), 27.9 (C-2), 21.6
(Ac-CH3), 20.8 (C-11), 19.5 (C-19), 19.5 (C-27), 19.0 (C-21), 16.6 (C-18). ESI-HRMS: m/z 619.3524 [M+H]+
(Calcd for C37H51N2O4S, 619.3564).
4.1.7.4 (22R,25R)-3β-Acetoxy-5-en-25-(2′-[5′-3′′-pyridyl]-1′,3′,4′-thiadiazolyl) furostan (8d).
o
1
White amorphous powder, yield 68.9%, mp 72.0−73.1 C. H NMR (600 MHz, CDCl3, δ): 9.11 (brs, 1H,
H-2′′), 8.71 (brs, 1H, H-6′′), 8.32 (d, J = 7.6 Hz, 1H, H-4′′), 7.44 (dd, J = 7.6, 4.5 Hz, 1H, H-5′′), 5.36 (brs, 1H,
H-6), 4.59 (m, 1H, H-3), 4.29 (dd, J = 12.6, 6.9 Hz, 1H, H-16), 3.45 (m, 1H, H-22), 3.33 (m, 1H, H-25), 2.02 (s,
3H, Ac-CH3), 1.47 (d, J = 6.9 Hz, 3H, 27-CH3), 1.02 (s, 3H, 19-CH3), 0.95 (d, J = 6.5 Hz, 3H, 21-CH3), 0.78 (s,
3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 176.9 (C-2′), 170.7 (Ac-CO), 164.7 (C-5′), 151.5 (C-2′′), 148.7
(C-6′′), 139.8 (C-5), 135.0 (C-4′′), 127.0 (C-3′′), 124.1 (C-5′′), 122.5 (C-6), 90.0 (C-22), 83.5 (C-16), 74.0 (C-3),
65.1 (C-17), 57.0 (C-14), 50.1 (C-9), 40.8 (C-12), 39.5 (C-13), 38.2 (C-4), 38.0 (C-20), 37.1 (C-1), 36.8 (C-10),
36.6 (C-25), 32.3 (C-7), 32.1 (C-15), 31.7 (C-8), 31.3 (C-24), 30.9 (C-23), 27.9 (C-2), 21.6 (Ac-CH3), 20.7
(C-11), 19.5 (C-19), 19.0 (C-27), 18.4 (C-21), 16.6 (C-18). ESI-HRMS: m/z 590.3415 [M+H]+ (Calcd for
C35H48N3O3S, 590.3411).
4.1.7.5 (22R,25S)-3β-Acetoxy-5-en-25-(2′-[5′-(naphthalen-1′′-yl)]-1′,3′,4′-thiadiazolyl) furostan (8e).
White amorphous powder, yield 75.5%, mp 73.5−74.2 oC. 1H NMR (600 MHz, CDCl3, δ): 8.68 (dd, J = 8.3,
5.0 Hz, 1H, Ar-H), 7.97 (d, J = 8.3 Hz, 1H, Ar-H), 7.91 (d, J = 8.3 Hz, 1H, Ar-H), 7.78 (d, J = 8.3 Hz, 1H, Ar-H),
7.55 (m, 3H, Ar-H), 5.36 (d, J = 4.9 Hz, 1H, H-6), 4.59 (m, 1H, H-3), 4.31 (m, 1H, H-16), 3.50 (m, 1H, H-22),
3.35 (m, 1H, H-25), 2.02 (s, 3H, Ac-CH3), 1.51 (d, J = 6.8 Hz, 3H, 27-CH3), 1.01 (s, 3H, 19-CH3), 0.98 (d, J =
6.8 Hz, 3H, 21-CH3), 0.78 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 177.0 (C-2′), 170.7 (Ac-CO), 167.2
(C-5′), 139.8 (C-5), 134.0 (Ar-C), 131.3 (Ar-C), 130.7 (Ar-C), 129.7 (Ar-C), 128.6 (Ar-C), 127.8 (Ar-C), 127.3
(Ar-C), 126.7 (Ar-C), 125.8 (Ar-C), 125.1 (Ar-C), 122.5 (C-6), 90.0 (C-22), 83.5 (C-16), 74.0 (C-3), 65.1 (C-17),
57.0 (C-14), 50.1 (C-9), 40.8 (C-12), 39.5 (C-13), 38.2 (C-4), 38.0 (C-20), 37.1 (C-1), 36.8 (C-10), 36.5 (C-25),
18

32.3 (C-7), 32.1 (C-15), 31.7 (C-8), 31.3 (C-24), 31.0 (C-23), 27.9 (C-2), 21.5 (Ac-CH3), 20.7 (C-11), 19.4
(C-19), 19.0 (C-27), 18.5 (C-21), 16.5 (C-18). ESI-HRMS: m/z 639.3617 [M+H]+ (Calcd for C40H51N2O3S,
639.3615).
4.1.8 General procedure for synthesis of compounds 9a−9e.
Compounds 8a−8e (0.15 mmol) were used for the synthesis as described in Section 4.1.6.
4.1.8.1 (22R,25R)-3β-Hydroxy-5-en-25-(2′-[5′-methyl]-1′,3′,4′-thiadiazolyl) furostan (9a).
White amorphous powder, yield 92.0%, mp 135.5−136.4 oC. 1H NMR (600 MHz, CDCl3, δ): 5.32 (d, J = 3.7
Hz, 1H, H-6), 4.27 (dd, J = 13.1, 7.4 Hz, 1H, H-16), 3.50 (m, 1H, H-3), 3.32 (m, 1H, H-22), 3.26 (m, 1H, H-25),
2.72 (s, 3H, 5′-CH3), 1.37 (d, J = 7.0 Hz, 3H, 27-CH3), 1.01 (s, 3H, 19-CH3), 0.94 (d, J = 7.0 Hz, 3H, 21-CH3),
0.77 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 176.8 (C-2′), 164.7 (C-5′), 141.0 (C-5), 121.5 (C-6), 90.0
(C-22), 83.4 (C-16), 71.8 (C-3), 65.2 (C-17), 57.1 (C-14), 50.2 (C-9), 42.4 (C-4), 40.8 (C-12), 39.5 (C-13), 37.9
(C-20), 37.4 (C-1), 36.7 (C-10), 36.5 (C-25), 32.3 (C-7), 32.1 (C-15), 31.7 (C-8), 31.3 (C-24), 31.0 (C-2), 30.8
(C-23), 20.8 (C-11), 19.5 (C-19), 19.0 (C-27), 18.5 (C-21), 16.5 (C-18), 15.8 (5′-CH3). ESI-HRMS: m/z
485.3185 [M+H]+ (Calcd for C29H45N2O2S, 485.3196).
4.1.8.2 (22R,25R)-3β-Hydroxy-5-en-25-(2′-[5′-phenyl]-1′,3′,4′-thiadiazolyl) furostan (9b).
o
1
White amorphous powder, yield 93.6%, mp 134.0−135.4 C. H NMR (600 MHz, CDCl3, δ): 7.94 (dd, J =
5.1, 1.6 Hz, 2H, H-2′′ and H-6′′), 7.47 (m, 3H, H-3′′, H-4′′ and H-5′′), 5.34 (d, J = 4.5 Hz, 1H, H-6), 4.29 (m,
1H, H-16), 3.51 (m, 1H, H-3), 3.42 (m, 1H, H-22), 3.30 (m, 1H, H-25), 1.46 (d, J = 6.9 Hz, 3H, 27-CH3), 1.01 (s,
3H, 19-CH3), 0.95 (d, J = 6.5 Hz, 3H, 21-CH3), 0.79 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 176.3
(C-2′), 168.2 (C-5′), 141.0 (C-5), 131.0 (C-4′′), 130.5 (C-1′′), 129.2 (2C, C-3′′ and C-5′′), 128.0 (2C, C-2′′ and
C-6′′), 121.5 (C-6), 90.0 (C-22), 83.4 (C-16), 71.8 (C-3), 65.2 (C-17), 57.1 (C-14), 50.2 (C-9), 42.2 (C-4), 40.8
(C-12), 39.5 (C-13), 38.0 (C-20), 37.4 (C-1), 36.8 (C-10), 36.6 (C-25), 32.4 (C-7), 32.1 (C-15), 31.7 (C-8), 31.3
(C-24), 31.2 (C-2), 30.9 (C-23), 20.8 (C-11), 19.5 (C-19), 19.0 (C-27), 18.5 (C-21), 16.6 (C-18). ESI-HRMS:
m/z 547.3324 [M+H]+ (Calcd for C34H47N2O2S, 547.3353).
4.1.8.3 (22R,25R)-3β-Hydroxy-5-en-25-(2′-[5′-p-methoxyphenyl]-1′,3′,4′-thiadiazolyl) furostan (9c).
19

o
1
White amorphous powder, yield 92.5%, mp 193.6−194.2 C. H NMR (600 MHz, CDCl3, δ): 7.87 (dd, J =
8.7 Hz, 2H, H-2′′ and H-6′′), 6.97 (d, J = 8.7 Hz, 2H, H-3′′ and H-5′′), 5.34 (d, J = 2.0 Hz, 1H, H-6), 4.59 (m,
1H, H-3), 4.29 (m, 1H, H-16), 3.86 (s, 3H, H-4′′-OCH3), 3.51 (m, 1H, H-3), 3.39-3.30 (m, 2H, H-22, H-25),
1.44 (d, J = 6.9 Hz, 3H, 27-CH3), 1.01 (s, 3H, 19-CH3), 0.96 (d, J = 6.8 Hz, 3H, 21-CH3), 0.77 (s, 3H, 18-CH3).
13C NMR (150 MHz, CDCl3, δ): 175.3 (C-2′), 167.9 (C-5′), 161.8 (C-4′′), 141.0 (C-5), 129.5 (2C, C-3′′ and
C-5′′), 123.3 (C-1′′), 121.6 (C-6), 114.6 (2C, C-2′′ and C-6′′), 89.7 (C-22), 83.4 (C-16), 71.9 (C-3), 65.2 (C-17),
57.1 (C-14), 50.2 (C-9), 42.4 (C-4), 40.8 (C-12), 39.6 (C-13), 38.0 (C-20), 37.4 (C-1), 36.8 (C-10), 36.6 (C-25),
32.4 (C-7), 32.1 (C-15), 31.8 (C-8), 31.7 (C-2), 31.4 (C-24), 30.9 (C-23), 20.8 (C-11), 19.6 (C-19), 19.5 (C-21),
19.0 (C-27), 16.6 (C-18). ESI-HRMS: m/z 577.3559 [M+H]+ (Calcd for C35H48N3O3S, 577.3558).
4.1.8.4 (22R,25R)-3β-Hydroxy-5-en-25-(2′-[5′-3′′-pyridyl]-1′,3′,4′-thiadiazolyl) furostan (9d).
White amorphous powder, yield 91.4%, mp 135.9−137.2 oC. 1H NMR (600 MHz, CDCl3, δ): 9.12 (brs, 1H,
H-2′′), 8.72 (brs, 1H, H-6′′), 8.33 (d, J = 7.7 Hz, 1H, H-4′′), 7.45 (m, 1H, H-5′′), 5.33 (brs, 1H, H-6), 4.30 (m,
1H, H-16), 3.51 (m, 1H, H-3), 3.45 (m, 1H, H-22), 3.34 (m, 1H, H-25), 1.47 (d, J = 6.8 Hz, 3H, 27-CH3), 1.01 (s,
3H, 19-CH3), 0.96 (d, J = 6.7 Hz, 3H, 21-CH3), 0.79 (s, 3H, 18-CH3). 13C NMR (150 MHz, CDCl3, δ): 177.0
(C-2′), 164.7 (C-5′), 151.5 (C-2′′), 148.7 (C-6′′), 141.0 (C-5), 135.0 (C-4′′), 127.1 (C-3′′), 124.2 (C-5′′), 121.5
(C-6), 89.9 (C-22), 83.5 (C-16), 71.8 (C-3), 65.1 (C-17), 57.1 (C-14), 50.2 (C-9), 42.4 (C-4), 40.8 (C-12), 39.5
(C-13), 38.0 (C-20), 37.4 (C-1), 36.8 (C-10), 36.6 (C-25), 32.4 (C-7), 32.1 (C-15), 31.8 (C-8), 31.7 (C-2), 31.3
(C-24), 30.9 (C-23), 20.8 (C-11), 19.6 (C-19), 19.0 (C-27), 18.5 (C-21), 16.6 (C-18). ESI-HRMS: m/z 548.3304
[M+H]+ (Calcd for C33H46N3O2S, 584.3305).
4.1.8.5 (22R,25S)-3β-Hydroxy-5-en-25-(2′-[5′-(naphthalen-1′′-yl)]-1′,3′,4′-thiadiazolyl) furostan (9e).
White amorphous powder, yield 92.5%, mp 86.5−87.8 oC. 1H NMR (600 MHz, CDCl3, δ): 8.68 (dd, J = 8.3,
5.4 Hz, 1H, Ar-H), 7.97 (d, J = 8.3 Hz, 1H, Ar-H), 7.91 (d, J = 8.3 Hz, 1H, Ar-H), 7.78 (d, J = 8.3 Hz, 1H, Ar-H),
7.55 (m, 3H, Ar-H), 5.32 (br.s, 1H, H-6), 4.30 (m, 1H, H-16), 3.50 (m, 1H, H-3), 3.48 (m, 1H, H-22), 3.34 (m,
1H, H-25), 1.50 (d, J = 6.9 Hz, 3H, 27-CH3), 1.00 (s, 3H, 19-CH3), 0.97 (d, J = 6.9 Hz, 3H, 21-CH3), 0.78 (s, 3H,
18-CH3). 13C NMR (150 MHz, CDCl3, δ): 177.0 (C-2′), 167.2 (C-5′), 141.0 (C-5), 134.0 (Ar-C), 131.3 (Ar-C),
130.7 (Ar-C), 129.7 (Ar-C), 128.6 (Ar-C), 127.8 (Ar-C), 127.3 (Ar-C), 126.7 (Ar-C), 125.8 (Ar-C), 125.1
(Ar-C), 121.5 (C-6), 90.0 (C-22), 83.5 (C-16), 71.8 (C-3), 65.2 (C-17), 57.1 (C-14), 50.2 (C-9), 42.4 (C-4), 40.8
20

(C-12), 39.5 (C-13), 38.0 (C-20), 37.4 (C-1), 36.7 (C-10), 36.5 (C-25), 32.4 (C-7), 32.1 (C-15), 31.7 (C-8), 31.3
(C-24), 31.0 (C-2), 31.0 (C-23), 20.8 (C-11), 19.5 (C-19), 19.0 (C-27), 18.5 (C-21), 16.6 (C-18). ESI-HRMS:
m/z 597.3497 [M+H]+ (Calcd for C38H49N2O2S, 597.3509).
4.2 Cell Culture
Human hepatoma (HepG2), lung carcinoma (A549), breast cancer (MCF-7), colorectal cancer (HCT-116)
and gastric epithelial (GES-1) cells were obtained from the Cell Bank of Chinese Academy of Sciences
(Shanghai, China). Cells were cultured in DMEM medium (HyClone, Utah, USA) supplemented with 10% FBS
(Gibco BRL, Grand Island, USA), 100 units/mL penicillin, and 100 µg/mL streptomycin (HyClone, Utah, USA)
in a humidified atmosphere of 5% CO2 at 37 oC.
4.3 In vitro cytotoxic activity
Cytotoxic activities of all compounds against HepG2, A549, MCF-7, HCT-116 and GES-1 cell lines were
evaluated by the MTT method. The cells (5×103/well) were seeded into the 96-well plates filled with culture
medium containing various concentrations of test samples. They were incubated at 37 oC, 5% CO2 for 48 h.
After incubation, the cells were incubated with 10 µL of MTT (Sigma Chemical Co., Ltd., USA) solution (5
mg/mL in PBS) for 4 h. Subsequently, the growth medium was replaced with 100 µL of DMSO and the
absorbance values were measured at 492 nm using microplate reader (Bio-Rad iMARK, USA). The IC50 was
defined as the concentration of the compound that inhibited cell proliferation by 50%. Three replicates were
performed.
4.4 Determination of morphological changes of cells
For cell morphological analysis, A549 cells were grown in 6-well plates (2×105 cells/well). After
stabilization for 24 h, compound 8d (15 or 30 µM) and DG (30 µM) were cultured together with the cells for 48
h. Control cells were incubated with 0.1% DMSO. A phase contrast microscopy (Olympus, Tokyo, Japan) was
used to observe the cellular morphology.
For DAPI staining experiments, A549 cells were grown in 6-well plates (2×105 cells/well). After 24 h of
21

incubation, the cells were then treated with compound 8d (15 or 30 µM) and DG (30 µM) for 48 h. Control cells
were exposed to 0.1% DMSO. The cells were fixed with 4% formaldehyde for 1 h at 4 oC. After fixation, the
cells were treated with 5 µM DAPI (4’,6-diamidino-2-phenylindole) at 37 oC in the dark for 10 min. Then the
cells were observed using a fluorescence microscope [32].
4.5 Apoptosis analysis by flow cytometry
A549 cells were incubated with compound 8d and DG at different concentrations (0, 15 and 30 µM). After
48 h, the cells were washed twice in PBS and resuspended in the Annexin V-FITC/PI staining solution according
to the manufacturer’s instruction (BD Pharmingen Co., Ltd., USA). The cells were detected to analyze apoptosis
by a flow cytometry (Beckman Coulter, USA).
4.6 Analysis of mitochondrial membrane potential
A549 cells were incubated with compound 8d for 48 h, then washed with PBS and stained with the
lipophilic cationic dye JC-1 according to the manufacturer’s instruction (KGA601, KeyGEN Biotech). The
percentage of cells with healthy or collapsed mitochondrial membrane potentials was monitored by flow
cytometry analysis.
4.7 Western Blot Analysis
After treatment with compound 8d (0, 7.5, 15 and 30 µM) and DG (0, 15 and 30 µM), A549 cells were
o
harvested and lysed in RIPA buffer and boiled for 10 min at 100 C. Equal amount of protein (30 µg) were
separated on a 10% SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were blocked
with 5% BSA and probed with a 1:1000 dilution of primary antibody against Cl-caspase-3 (Cell Singaling
Technology, Inc., Danvers, MA, USA), Cl-caspase-9 (Cell Singaling Technology Inc), PARP (Cell Singaling
Technology, Inc), Bcl-2 (Santa Cruz Biotechnology, Inc), Cytochrome c (Proteintech Group, Inc), Bax
(Proteintech Group, Inc), β-actin (Ding-Guo Biotech Ltd., Beijing). Then the membranes were incubated with a
1:5000 dilution of horseradish peroxidase-conjugated secondary antibody for 2 h. Positive bands were visualized
on a X-ray film using an enhanced chemiluminescence system (Kodak).
Acknowledgments
22

The authors are grateful to financial supports from the Fundamental Research Funds for Education
Department of Heilongjiang Province (Grant No. 2018-KYYWF-0092). We kindly thank Mrs. Sun, Y., Mr. Liu,
Q., and Mr. Liu, L. of Qiqihar Medical University for recording ESI-HRMS and NMR spectra.
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24

Table 1
Cytotoxic activities of compounds 6a-6e
,
7a-7e
,
8a-8e
,
and 9a-9e in human cells.
IC50a (µM)
compound
R1
R2
HepG2
A549
MCF-7
HCT-116
GES-1
6a
6b
6c
CH3
Ac
Ac
Ac
57.64±1.17
63.24±1.34
-
34.38±1.22
84.49±2.57
-
44.81±2.17
78.82±2.81
98.24±1.17
82.34±3.56
151.0±6.31
>100
-
>100
-
6d
6e
Ac
Ac
82.56±2.07
-
33.55±1.34
-
>100
-
72.53±2.51
-
62.40±2.14
70.08±4.37
7a
7b
7c
CH3
CH3
CH3
H
H
H
34.23±0.87
39.51±1.01
-
30.36±1.09
64.16±2.77
-
20.37±1.03
52.97±2.47
-
51.63±2.01
>100
72.77±4.16
119.7±5.43
-
>100
7d
7e
H
H
>100
98.13±3.07
31.26±1.11
>100
>100
>100
198.9±6.43
29.24±1.09
69.66±2.44
298.07±7.34
8a
8b
8c
Ac
Ac
Ac
34.25±1.12
40.27±1.36
47.72±1.39
>100
75.78±4.35
-
-
-
-
-
-
-
-
-
-
8d
8e
Ac
Ac
11.73±0.67
-
3.93±0.37
>100
>100
-
29.56±1.02
>100
420.4±5.37
-
9a
9b
9c
H
H
H
33.16±1.14
32.14±1.03
23.05±1.02
36.71±1.35
15.04±0.87
16.40±0.97
33.75±1.25
27.37±1.05
>100
48.89±1.38
48.91±1.43
>100
107.4±2.36
527.0±5.57
208.7±5.38
9d
9e
H
H
8.83±0.67
7.677±0.57
18.26±0.95
>100
51.65±2.87
46.61±1.58
354.4±7.17
200.7±5.62
32.90±1.47
63.77±2.43
Diosgenin
−
−
−
−
33.87±1.37
32.63±1.22
26.41±1.42
11.03±1.04
23.91±1.34
16.71±1.02
49.11±2.11
11.03±1.01
100.7±4.53
21.12±1.01
Mitomycin C
a
IC50: concentration of the tested compound that inhibits 50% of cell growth. All data are presented as means ± SD of three
independent experiments.
“-” not active.

1. Legends for Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5 and Scheme 1
Fig. 1. Structure of diosgenin.
Fig. 2. Examples of 1,3,4-oxadiazole/thiadiazole-based antitumor compounds and designed diosgenin
1,3,4-oxadiazolyl/thiadiazolyl derivatives.
Fig. 3. Compound 8d and DG induced apoptosis in A549 cells. (A) Morphological changes of A549 cells were
observed with a phase contrast microscope (Scale bar: 50 µm). (B) The nuclear morphology changes of A549 cells
were detected by DAPI staining (Scale bar: 50 µm).
Fig. 4. A549 cells were treated with compound 8d and DG (0, 15, and 30 µM) for 48 h, and apoptosis was
measured by flow cytometry after staining with Annexin V/PI, ***P < .001 versus control.
Fig. 5. Effect of compound 8d and DG on the expression of apoptosis-related proteins in A549 cells was assayed
by Western blot analysis. β-actin was employed as loading control, ***P< .001 versus control. A representative
result from three separate experiments is shown.
Fig. 6. Compound 8d induced mitochondrial depolarization in A549 cells, **P< .01; ***P< .001 versus control.
Fig. 7. The expression levels of apoptosis-related proteins, including Bax, Bcl-2, and cyto-c were assayed by
Western blot analysis. β-actin was employed as loading control, **P< .01; ***P< .001 versus control.
Scheme 1. Synthesis of diosgenin 1,3,4-oxadiazolyl/thiadiazolyl derivatives. Reagents and conditions: (a) Ac2O,
dry pyridine, dry CH2Cl2, rt, 6 h; (b) NaBH3CN, AcOH, CH2Cl2, 8 h; (c) Jones reagent, THF/acetone (1/1), rt; (d)
appropriate acylhydrazines, TBTU, DIPEA, CH2Cl2, rt, 2 h; (e) POCl3, 80 oC, 1 h; (f) KOH, MeOH, rt, 1 h; (g) LR,
toluene, 110 oC, 1 h; (h) KOH, MeOH, rt, 1 h.
2. Graphics for Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5 and Scheme 1
Fig. 1

Fig. 2

Fig. 3