Synthesis and biological evaluation of D-ring fused 1,2,3-thiadiazole dehydroepiandrosterone derivatives as antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2016.01.058

A series of D-ring fused 1,2,3-thiadiazole DHEA derivatives were synthesized and investigated for their activity against the growth of various tumor cell lines using the sulforhodamine B (SRB) assay. It is amazing that for these compounds, T47D cell line was much more sensitive than other tumor cell lines. The most potent saturated N-heterocyclic derivatives showed similar antitumor effect with the positive control compound ADM (adriamycin) on T47D cells, that was 44–60 folds more potent than the lead compound DHEA. Most compounds with potent antitumor activity displayed low toxicity on normal human fibroblasts (HAF). Especially compound 25 (CH33) showed an IC₅₀ of 0.058 μM on T47D cells and its selectivity index (SI) between HAF and T47D was 364, which was 214 folds better than ADM (SI = 1.7). The apoptosis, colony formation and transwell migration assays of 25 were performed on T47D cell line. The primary mechanism study showed that 25 caused a dose-dependent induction of apoptosis, and induced phosphorylation of EphA2 and EphB3 in T47D cells. The in vivo antitumor effect of 25 was also observed in T47D tumor-bearing mice without obvious toxicity.

Full text of DOI:10.1016/j.ejmech.2016.01.058

Accepted Manuscript
Synthesis and biological evaluation of D-ring fused 1,2,3-thiadiazole
dehydroepiandrosterone derivatives as antitumor agents
Hai-Wei Cui, Shihong Peng, Xiang-Zhong Gu, Huang Chen, Yuan He, Wei Gao, Fang
Lv, Jin-Hua Wang, Yan Wang, Jia Xie, Ming-Yao Liu, Zhengfang Yi, Wen-Wei Qiu
PII:
S0223-5234(16)30066-6
10.1016/j.ejmech.2016.01.058
EJMECH 8348
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 December 2015
Revised Date: 29 January 2016
Accepted Date: 30 January 2016
Please cite this article as: H.-W. Cui, S. Peng, X.-Z. Gu, H. Chen, Y. He, W. Gao, F. Lv, J.-H. Wang,
Y. Wang, J. Xie, M.-Y. Liu, Z. Yi, W.-W. Qiu, Synthesis and biological evaluation of D-ring fused 1,2,3-
thiadiazole dehydroepiandrosterone derivatives as antitumor agents, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.01.058.
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ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of D-ring fused 1,2,3-thiadiazole
dehydroepiandrosterone derivatives as antitumor agents
Hai-Wei Cui, Shihong Peng, Xiang-Zhong Gu, Huang Chen, Yuan He, Wei Gao , Fang Lv,
Jin-Hua Wang, Yan Wang, Jia Xie, Ming-Yao Liu, Zhengfang Yi, Wen-Wei Qiu
DMSO
25 (2.5 µM)
1
20
N
N
S
100
80
60
40
20
0
*
*
O
O
NH
***
2
5 (CH33)
IC 50 = 0.058 uM (T47D)
*
**
2
5 (µM)
0
0.1 0.5 2.5
SAR 14 compounds
1
20
00
O
1
*
**
8
6
4
2
0
0
0
0
0
HO
***
DHEA
***
IC 50 = 2.55 uM (T47D)
2
5 (µM)
0
0.1 0.5 2.5
A series of novel D-ring fused 1,2,3-thiadiazole dehydroepiandrosterone (DHEA) derivatives
were synthesized and investigated for their antitumor activity in vitro and in vivo.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of D-ring fused 1,2,3-thiadiazole
dehydroepiandrosterone derivatives as antitumor agents
a, 1
b, 1
c
b
b
a
Hai-Wei Cui , Shihong Peng , Xiang-Zhong Gu , Huang Chen , Yuan He , Wei Gao , Fang
b
b
a
b
b
b,
a,
Lv , Jin-Hua Wang , Yan Wang , Jia Xie , Ming-Yao Liu , Zhengfang Yi *, Wen-Wei Qiu *
a
Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal
University, 500 Dongchuan Road, Shanghai 200241, China
b
Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of
Life Sciences, East China Normal University, Shanghai 200241, China
c
Department of Research and Development, Jiangsu Jiaerke Pharmaceuticals Group Co., Ltd.,
Zhenglu Town, Changzhou 213111, China
Keywords: Antitumor, Dehydroepiandrosterone, 1,2,3-Thiadiazole, Apoptosis, Migration
*
Corresponding author.
E-mail address: zfyi@bio.ecnu.edu.cn (Z. Yi), wwqiu@chem.ecnu.edu.cn (W.-W. Qiu).
These authors contributed equally to this work.
1
ABSTRACT
A series of D-ring fused 1,2,3-thiadiazole DHEA derivatives were synthesized and investigated
for their activity against the growth of various tumor cell lines using the sulforhodamine B (SRB)
assay. It is amazing that for these compounds, T47D cell line was much more sensitive than other
tumor cell lines. The most potent saturated N-heterocyclic derivatives showed similar antitumor
effect with the positive control compound ADM (adriamycin) on T47D cells, that was 44-60 folds
more potent than the lead compound DHEA. Most compounds with potent antitumor activity
displayed low toxicity on normal human fibroblasts (HAF). Especially compound 25 (CH33)
showed an IC of 0.058 ꢀM on T47D cells and its selectivity index (SI) between HAF and T47D
5
0
was 364, which was 214 folds better than ADM (SI = 1.7). The apoptosis, colony formation and
Abbreviations: IC , half maximal inhibitory concentration; SAR, structure-activity relationship; SI, selectivity
50
index; SRB, sulforhodamine; RTKs, receptor tyrosine kinases; HE, hematoxylin and eosin; HAF, human
fibroblasts; ADM, adriamycin; DHEA, dehydroepiandrosterone.

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transwell migration assays of 25 were performed on T47D cell line. The primary mechanism study
showed that 25 caused a dose-dependent induction of apoptosis, and induced phosphorylation of
EphA2 and EphB3 in T47D cells. The in vivo antitumor effect of 25 was also observed in T47D
tumor-bearing mice without obvious toxicity.
1
. Introduction
Steroids are widely distributed in natural world and display a variety of biological activities,
especially antitumor activity [1,2]. Heterocycles are considered as privileged scaffolds in drug
discovery and widely used in medicinal chemistry [3,4]. The introduction of A- and D-ring fused
heterocycles into the steroid derivatives often result in amelioration of biological properties [5,6],
for example, enhancing the antitumor effect [7–10].
Dehydroepiandrosterone (DHEA) also known as androstenolone or prasterone, is the most
abundant circulating steroid hormone in humans [11]. DHEA and its sulfate ester also have a
variety of potential biological effects, such as anti-aging [12], anti-inflammatory [13],
immunomodulatory [14], antiviral [15,16], antidepressant [17] and anticancer effects [18,19]. As
heterocycles were fused with the D-ring of DHEA, the antitumor activity was greatly improved.
For instance (Fig. 1), compound A, that was obtained by introducing p-methoxyphenylpyrazoline
fragment displayed more cytotoxic activity than cisplatin on malignant human cell lines [20]; a
series of D-ring fused thiazole, thiazole imines, imidazo thiazoles and pyridine were synthesized
by Liu et al. showed good cytotoxicity against three tumor cell lines (EC109, EC9706 and MGC
8
03), especially compound B possessed the most potent antitumor activity [21,22]; compound C
was synthesized by fusing pyrazole-scaffold with D-ring exhibited the best antitumor activity with
IC values of 20-1.4 nM against four cell lines, and 1.03 ꢀM against a tamoxifen resistant breast
50
cancer cell line, and a preliminary structure-activity relationship (SAR) showed that with a linker
in 3-position was favorable to increase the bioactivity [23].
Thiadiazoles occur widely in nature in four isomeric forms, that is 1,2,3-thiadiazole,
1
,2,5-thiadiazole, 1,2,4-thiadiazole and 1,3,4-thiadiazole. Many drugs containing thiadiazole
nucleus are available in the market for instance acetazolamide, methazolamide, cefazoline,
sulfamethazole, etc. In recent decades, thiadiazole and their derivatives have attracted much
2

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attentation because of their wide range of biological activities [24]. Especially the
1
,3,4-thiadiazole derivatives have attracted widespread attention due to their diverse biological
activity such as antimicrobial, antifungal, antiviral, antitubercular, anti-inflammatory, analgesic,
antiepileptic, and antitumor activity, and been described in recent reviews [25–27]. However,
there are only a few researches performed on the 1,2,3-thiadiazole derivatives and their biological
activity [28–32]. In our laboratory, researches are focused on exploring the 1,2,3-thiadiazole
DHEA derivatives and their antitumor activity.
In order to obtain novel potent antitumor agents, a series of compounds with D-ring fused
1
,2,3-thiadiazole of DHEA were synthesized. Their inhibitory activity against the growth of T47D
cells and other 8 different tumor cell lines was evaluated using a SRB assay. DHEA and its
derivative were screened by flow cytometry to determine their apoptotic behaviors, and their
inhibitory effects on tumor cell migration and colony formation ability were also tested. The
primary anticancer mechanism was studied and the in vivo antitumor evaluation in T47D
tumor-bearing mice was also carried out.
Fig. 1. Chemical structures of D-ring fused heterocycles DHEA antitumor agents
2
. Chemistry
A series of D-ring fused 1,2,3-thiadiazole DHEA derivatives were synthesized according to the
pathways described in Scheme 1−2.
Compound 1 was prepared by protection of the 3-hydroxyl group of DHEA with acetyl group
under Ac O and DMAP in CH Cl . The intermediate 2 was afforded by reaction of 1 with
2
2
2
aminourea hydrochloride and sodium acetate in EtOH. Compound 4 was obtained by
condensation of 2 under thionyl chloride, then hydrolysis with K CO in MeOH. Reduction of the
2
3
double bond at B-ring of 1 under H and palladium in carbon in autoclave gave compound 5.
2
Compound 8 (CH21) was prepared in a manner similar to that of compound 4.
Compounds 9
−
13 were obtained by reaction of compound 8 with corresponding acyl chlorides
25 were obtained by condensation of 8 with
under DMAP in CH Cl . Compounds 20
−
2
2
corresponding N-Boc-amino-carboxylic acids under 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDCI), 1-hydroxybenzotriazole (HOBt) and DMAP in CH Cl , then deprotection of
2
2
3

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the Boc group under boron trifluoride etherate in Et O.
2
Scheme 1. Synthesis of derivatives 4 and 8. Reagents and conditions: (a) Ac O, DMAP, CH Cl , rt, 5 h, 98%; (b)
2
2
2
aminourea hydrochloride, CH COONa, EtOH, rt, 6 h, 92% for 2 and 95% for 6; (c) SOCl , CH Cl , rt, 5 h, 82%
3
2
2
2
o
for 3 and 78% for 7; (d) K CO , MeOH, rt, 4h, 93% for 4 and 95% for 8; (e) H (4.0 Mp), 5% Pd/C, MeOH, 40 C,
2
3
2
1
6 h, 87%.
Scheme 2. Synthesis of derivatives 9-13 and 20-25. Reagents and conditions: (a) acyl chlorides, DMAP, CH Cl , rt,
2
2
4
h, 83−90% for 9−13; (b) N-Boc-amino-carboxylic acids, EDCI, HOBt, DMAP, CH Cl , rt, 8 h, 86−98% for 14−
2 2
1
9; (c) boron trifluoride etherate, Et O, rt, 2 h, 72−82% for 20−25.
2
3
. Result and discussion
3
.1. Antiproliferative activity
The first round synthetic DHEA derivatives 4 and 8 were evaluated for their in vitro antitumor
activity in SRB assay against human breast cancer cell lines (T47D, MDA-MB-231 and MCF-7),
human prostate cancer cell lines (DU-145 and LNCaP), human colon carcinoma cell lines
(HCT-116 and HT-29), human promyelocytic leukemia cell line (HL-60) and human T lymphocyte
cell line (Jurkat). It is amazing that for these tested compounds (DHEAꢀEPIꢀ4 and 8), T47D was
the most sensitive tumor cell line. However for other various tumor cell lines, these compounds
just showed weak or even no inhibition activity (Table 1). All these tested compounds displayed
potent antiproliferative activity against T47D cells. Especially compound 8, which was about 2
and 4 folds higher activity than EPI and DHEA respectively. Thus, we considered the D-ring fused
1
,2,3-thiadiazole as an effective modification and the subsequent evaluation should be focused on
the most sensitive T47D cell line.
4

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Table 1
IC50 values of DHEA and its derivatives 4 and 8 against the growth of T47D cells and other various tumor cell
lines
a
From SRB assay after 96 h of treatment; IC data are an average of at least 3 independent experiments, variation
50
b
±
10%; Epiandrosterone (EPI) is a reduzate of DHEA.
The second round synthetic analogues were obtained by modification of the C3-hydroxyl group
of compound 8 and screened using the SRB assay on T47D cell line (Table 2). The results showed
that the alkyl esters (7, 9, 10, 20 and 21) and aromatic N-heterocyclic esters (11, 12 and 13) in
position C-3 were not favorable substituted groups for improving antitumor activity, which
resulted in a lower inhibition of cell growth compared with 8. Derivatives (22, 23, 24 and 25)
bearing a saturated N-heterocycle in position C-3, possessed more potent antiproliferative activity
than 8. It is interesting that for the five-membered saturated N-heterocycle, the unnatural
D-proline derivative 25 (IC = 0.058 ꢀM) possessed much higher antiproliferative activity than its
5
0
natural L-proline derivative 24 (IC50 = 0.24 ꢀM). The most potent saturated N-heterocyclic
derivatives 22, 23 and 25 showed similar antitumor effect with the positive control compound
ADM (adriamycin) on T47D cells, that was 44-60 folds more potent than DHEA (IC50 = 2.55
ꢀM).
In order to further investigate the selectivity between T47D tumor cell line and other tumor
cells, we tested the most potent compounds 22 and 25 and its L-proline isomer 24 against the
growth of these tumor cell lines (Table 3). The results revealed that selectivity was in accordance
with the former (Table 1), T47D was the most sensitive tumor cells in the all tested tumor cell
lines. Compound 25 (CH33) possessed the best selectivity, which was 43- to 793-fold for T47D
over other tumor cells.
Table 2
IC50 values of DHEA and its derivatives (7, 8 and 26-43) against the growth of T-47D tumor cell line and normal
human fibroblast (HAF) cells.
a
b
From SRB assay after 96 h of treatment; IC50 data are an average of at least 3 independent experiments; the
5

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selectivity indexes (SI) were calculated by IC50 values in HAF cells divided by IC50 values in T47D cancer cell
c
d
e
line; data from Table 1; ND = not determined; ADM is Adriamycin.
Table 3
IC50 values of compounds 22, 24 and 25 against the growth of T47D cells and other various tumor cell lines.
a
From SRB assay after 96 h of treatment; IC data are an average of at least 3 independent experiments, variation
50
b
c
±
10%; data from Table 2; Selectivity is the IC values of T47D cells divide IC50 values of other tumor cell lines.
50
3
.2. Selectivity
One of the major hindrances for druggability of compounds with effective antitumor activity is
their toxicity to normal cells. Thus, it is important to measure cytotoxicity on normal cells in
anticancer drug discovery.
Compound 8 and its derivatives were chosen for selectivity test on a normal human fibroblast
(
HAF) cell line using the SRB assay. The selectivity indexes (SI) were calculated by IC values in
50
HAF cells divided by IC values in T47D cell line. The results (Table 2) revealed that most of the
5
0
tested compounds were less toxic on HAF in comparison with the tumor cells. To our delight, the
SI of compounds 22, 24 and 25 were satisfactory. Especially compound 25 possessed the highest
selectivity (SI = 364), which was 214 folds better than the positive control compound ADM (SI =
1
.7), although the two compounds possessed almost equivalent antiproliferative activity against
T47D cells.
3
.3. Colony formation
Colony formation assay is the gold standard for measuring the effect of cytotoxic agents on
tumor cells in vitro [33]. It is closer to physiology and growth of tumor in vivo to mimic individual
tumor cell development into macroscopic cell clones [34]. So the colony formation assay on T47D
cell line was performed to confirm the antiproliferative activity of compound 25. The colony
formation ability of T47D cells was significantly inhibited by 25 (Fig. 2, IC50 = 0.35 ꢀM) in a
dose-dependent manner, which was much more effective than DHEA.
6

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Fig. 2. Compound 25 and DHEA inhibited the colony formation of T47D cell line. After treatment by various
doses of 25 and DHEA in 6-well plates for 7 days, cells were fixed and stained with crystal violet and the numbers
of cell colonies were counted. **P < 0.01, ***P < 0.001, versus the control group (0 ꢀM).
3
.4. Migration
Migration is an important step during the metastasis of cancer. The antimetastatic activity of
compound 25 was measured by in vitro transwell migration assay. The cell migration ability of
T47D cells was significantly inhibited by 25 (Fig. 3, IC = 0.42 ꢀM) in a dose-dependent manner,
5
0
which was much more effective than DHEA.
Fig. 3. Compound 25 and DHEA inhibited the cell migration of T47D cell line. After 12 h incubation with various
doses of 25 and DHEA, non-migrated cells were removed and migrated cells were fixed with cold 4%
paraformaldehyde and stained with 0.2% crystal violet. Images were taken and cells from three random areas per
filter were counted. **P < 0.01, ***P < 0.001, versus the control group (0 ꢀM).
3
.5. Apoptosis and cell cycle
Previous results of cellular cytotoxicity and colony formation assay indicated that compound 25
inhibited cancer cell proliferation. Many factors cause cell proliferation inhibition, including cell
cycle arrest and induction of apoptosis [35–37]. So we evaluated whether the antiproliferative
activity of the compound was resulted by cell apoptosis or cell cycle arrest using flow cytometry.
Results indicated that 25 did not induce cell-cycle arrest (Fig. 4A), but caused a dose-dependent
induction of apoptosis on T47D cells (Fig. 4B). The apoptotic cells rose from 5.74% to 30.14%
with doses of 25 increased from 0 to 12.5 ꢀM after 24 h treatment. These results suggested that
this compound inhibited cell proliferation through inducing apoptosis in the cell line mainly.
Fig. 4. (A) Compound 25 didn’t induce cell cycle arrest in T47D cell line. Cells were stained with PI and analyzed
using flow cytometry after treatment with 25 for 24 h. (B) Compound 25 mediated T47D cells apoptosis in
dose-dependent manner. Cells were treated with different dose of 25 for 24 h and then the percentages of apoptosis
7

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cells were analyzed using flow cytometry by staining with PI and Annexin V-FITC.
3
.6. Phospho-receptor tyrosine kinase array analysis
Receptor tyrosine kinases (RTKs) are a group of cell surface receptors that regulate a diverse
array of cellular functions including cellular proliferation, survival, differentiation, migration, and
metabolism [38]. We analyzed the activation of RTKs comprehensively of T47D cells using a
phospho-RTK array after compound 25 treated for 24 h. A remarkable increase in the
phosphorylation of EphA2 and EphB3 was detected (Fig. 5). According to previous studies, there
is accumulating evidence to suggest that EPH expression and function in the absence of kinase
activity is tumor promoting, whereas EPH forward signaling (phosphorylated Eph) is tumor
suppressive [39-42]. So, the phosphorylation of EphA2 and EphB3 may play pivotal role in the
proliferation suppression, migration inhibition and apoptosis-inducing of 25 in T47D cells and
further in-depth mechanism research is also required.
Fig. 5. Compound 25 increased the phosphorylation of EphA2 and EphB3 in T47D cells. After treated with 2.5 ꢀM
2
5 for 24 h, T47D cells were lysed and analyzed for the activation of RTKs comprehensively using a
phospho-RTK array.
3
.7. In vivo antitumor activity
In order to determine the efficacy of compound 25 in vivo, T47D subcutaneous xenograft
growth model and orthotopic xenograft growth and metastasis model were evaluated. Results of
subcutaneous xenograft growth model suggested that the tumor growth of T47D was significantly
inhibited by 25 (20mg/kg/day) compared with the control group (Fig. 6A, left), and there was
almost no obvious effect on body weight in the compound treated mice (Fig. 6A, right). The tumor
volume and body weight of 25 treated mice in orthotopic xenograft model displayed the same
results with subcutaneous xenograft model (Fig. 6B), moreover, the compound displayed
remarkable antimetastatic ability, the number of metastasized T47D nodules in the lungs of the
compound (20 mg/kg/day) treated mice reduced to about 40% compared with the control group
(
Fig. 6C). HE staining results of liver and kidney showed that 25 presented no obvious influence
8

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to the anatomical morphologies of mice (Fig. 6D).
Fig. 6. (A) T47D cells subcutaneous xenograft mice were intraperitoneally treated with compound 25 (20
mg/kg/day) or DMSO (served as control) for 19 days. Tumor volume and mice body weight were measured every
other day. N = 8; ns, no significant; ***P < 0.001. (B) T47D cells orthotopic xenograft mice intraperitoneally
treated with 25 (20 mg/kg/day) or DMSO (served as control) for 16 days. Tumor volume and mice body weight
were measured every three days. N = 5; ns, no significant; *P < 0.05. (C) Lungs were fixed, photographed, and
amount of metastasis nodules were manually counted using a dissecting microscope. *P < 0.05. (D) Liver and
kidney hematoxylin and eosin (HE) staining of control and 25 treatment groups.
4
. Conclusion
In this study we synthesized a novel class of D-ring fused 1,2,3-thiadiazole DHEA derivatives
and screened them for antiproliferative activity using the SRB assay on various cancer cell lines. It
is amazing that for these compounds, T47D cell line was much more sensitive than other tumor
cell lines. Compound 25 was an unnatural D-proline modified derivative, which possessed the
most potent antiproliferative activity on T47D cells and the best selectivity between the tumor cell
line and HAF cells. The colony formation and metastatic ability of T47D cells was significantly
inhibited by 25 in a dose-dependent manner, which was much more effective than lead compound
DHEA. The primary mechanism study showed that 25 did not induce cell-cycle arrest, but caused
a dose-dependent induction of apoptosis, and induced phosphorylation of EphA2 and EphB3 in
T47D cells. In vivo antitumor activity evaluation displayed that the tumor volume was
significantly inhibited and there was no obvious change in body weight of 25 treated mice in
subcutaneous xenograft model and orthotopic xenograft model. Moreover, the compound also
displayed remarkable antimetastatic ability, the number of metastasized T47D nodules in the lungs
reduced to about 40% compared with the control group, and the HE staining results showed that
the compound presented no obvious influence to the anatomical morphologies of mice.
In conclusion, we report D-ring fused 1,2,3-thiadiazole DHEA derivatives as a series of new
chemical entities for the first time. Especially 25, which exhibited potent antitumor and
9

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antimetastatic activity in vitro and in vivo, and could be used as a promising lead for the
development of a new class of antitumor and antimetastatic agents.
5
. Experimental section
5
.1. General
All reagents and chemicals were purchased from commercial suppliers and used without further
purification unless otherwise stated. When needed, the reactions were carried out in oven-dried
glassware under a positive pressure of dry N . Column chromatography was performed on silica
2
gel (QinDao, 200-300 mesh) using the indicated eluents. Thin-layer chromatography was carried
out on silica gel plates (QinDao) with a layer thickness of 0.25mm. Melting points were
1
13
determined using the MEL-TEMP 3.0 apparatus and uncorrected. H (400 MHz) and C (100
MHz) NMR spectra were recorded on Bruker AM-400 spectrometer with CDCl or DMSO-d as
3
6
solvent and tetramethylsilane (TMS) as the internal standard. All chemical shift values were
reported in units of δ (ppm). The following abbreviations were used to indicate the peak
multiplicity: s = singlet; d = doublet; t = triplet; m = multiplet; br = broad. High-resolution mass
data were obtained on a BrukermicroOTOF-Q II spectrometer.
5
.2. 3β-Acetoxy-5-androstene-17-one (1)
To a solution of DHEA (5 g, 17.2 mmol) in dry CH Cl (40 mL), Ac O (15.6 g, 103.3 mmol)
2
2
2
and DMAP (315 mg, 2.58 mmol) were added at room temperature. The reaction mixture was
stirred for 5 h under nitrogen atmosphere at room temperature and concentrated. The residue was
poured into H O (30 mL) and extracted with AcOEt (20 mL × 3). The organic layer was washed
2
with brine, dried with anhydrous Na SO and concentrated to give compound 1 (5.61 g, 98%) as a
2
4
1
white solid without purified. H NMR (400 MHz, CDCl ) δ 5.40 (d, J = 4.7 Hz, 1H), 4.63–4.56 (m,
3
1
3
1
1
2
5
H), 2.45 (dd, J = 19.2, 8.9 Hz, 1H), 2.33 (dd, J = 13.1, 2.7 Hz, 2H), 2.15–2.05 (m, 2H), 2.03 (s,
H), 1.96 (dd, J = 12.1, 5.8 Hz, 1H), 1.86 (s, 3H), 1.65 (d, J = 8.3 Hz, 3H), 1.33–1.24 (m, 2H),
1
3
.18–1.12(m, 1H), 1.05 (s, 3H), 0.88 (s, 3H). C NMR (100 MHz, CDCl ) δ 220.88, 170.41,
3
39.87, 121.84, 73.65, 51.64, 50.10, 47.46, 38.04, 36.90, 36.68, 35.79, 31.342, 37.37, 30.73, 27.66,
1.85, 21.38 , 20.28, 19.31, 13.51.
.3. 3β-Acetoxy-17-(2-carbamoylhydrazono)-5-androstene (2)
1
0

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To a solution of compound 1ꢁ400 mg, 1.2 mmolꢂin absolute EtOH (20 mL), aminourea
hydrochloride (675 mg, 6 mmol) and anhydrous CH COONa (492 mg, 6 mmol) were added at
3
room temperature. The reaction mixture was stirred for 6 h at room temperature and concentrated.
The residue was poured into H O (30 mL) and extracted with AcOEt (20 mL × 3). The organic
2
layer was washed with brine, dried with anhydrous Na SO and concentrated. The residue was
2
4
purified by silica gel column chromatography (CH Cl /MeOH, 20/1, v/v) to give compound 2
2
2
1
(
431 mg, 92%) as a white solid. H NMR (400 MHz, CDCl ) δ 7.82 (s, 1H), 5.43 (d, J = 5.1 Hz,
3
1
3
5
H),4.73–4.63 (m, 1H), 2.38 (dd, J = 13.1, 5.2 Hz, 2H), 2.27–2.14 (m, 1H), 2.02 (s, 3H), 0.86 (s,
H), 0.84 (s, 3H).
.4. 3β-Acetoxy-androsta-5,16-dieno[17,16-d][1,2,3]thiadiazole (3)
To a solution of compound 2 (435 mg, 1.12 mmol) in dry CH Cl (10 mL), thionyl chloride (2
2
2
o
mL) was added at 0 C. The reaction mixture was stirred for 5 h at room temperature and then
poured into H O (30mL) and extracted with CH Cl (10 mL × 3). The organic layer was washed
2
2
2
with brine, dried with anhydrous Na SO and concentrated. The residue was purified by silica gel
2
4
column chromatography (CH Cl /MeOH, 40/1, v/v) to give compound 3 (335 mg, 82%) as a
2
2
-1
1
white solid. IR (ATR) cm : 2930, 2840, 1719, 728. H NMR (400 MHz, CDCl ) δ 5.42 (d, J =
3
5
2
3
1
2
5
.1 Hz, 1H), 4.68–4.56 (m, 1H), 3.05 (dd, J = 15.5, 6.3 Hz, 1H), 2.63 (dd, J = 15.5, 11.9 Hz, 1H),
.48 (dd, J = 10.2, 2.4 Hz, 1H), 2.42–2.29 (m, 2H), 2.28–2.19 (m, 1H), 2.15–2.06 (m, 1H), 2.04 (s,
1
3
H), 1.13 (s, 3H), 1.12 (s, 3H). C NMR (100 MHz, CDCl ) δ 179.86, 170.48, 153.88, 140.30,
3
21.49, 73.65, 62.91, 50.41, 41.54, 38.09, 36.89, 36.82, 34.24, 30.96, 30.62, 27.67, 27.45, 21.41,
0.34, 19.29, 17.71.
.5. 3β-Hydroxy-androsta-5,16-dieno[17,16-d][1,2,3]thiadiazole (4)
To a solution of 3 (230 mg, 0.62 mmol) in MeOH (10 mL), K CO (428 mg, 3.1 mmol) was
2
3
added at room temperature. The reaction mixture was stirred for 4 h at room temperature and
concentrated. The residue was poured into H O (30mL) and acidified to pH 5-6 with hydrochloric
2
acid (1M), then extracted with AcOEt (20 mL × 3). The organic layer was washed with brine,
dried with anhydrous Na SO and concentrated. The residue was purified by silica gel column
2
4
chromatography (CH Cl /MeOH, 30/1, v/v) to give compound 4 (190mg, 93%) as a white solid;
2
2
o
-1
1
mp: 198–200 C. IR (ATR) cm : 3420, 2920, 2853, 720. H NMR (400 MHz, CDCl ) δ
3
1
1

ACCEPTED MANUSCRIPT
5
2
1
3
.42
–
–
5.35 (m, 1H), 3.61
–
3.49 (m, 1H), 3.07
–3.01 (m, 1H), 2.67–2.59 (m, 1H), 2.50–2.47 (m, 1H),
1
3
.36
2.19 (m, 3H), 2.12
–
2.06 (m, 1H), 1.13 (s, 3H), 1.10 (s, 3H). C NMR (100MHz, CDCl ) δ
3
80.06, 154.06, 141.57, 120.61, 77.48, 77.16, 76.84, 71.66, 63.15, 50.63, 42.31, 41.68, 37.20,
6.93, 34.39, 31.66, 31.10, 30.80, 27.57, 20.51, 19.48, 17.82. HRMS (ESI): calcd for C H N OS
1
9
27
2
+
[
M + H] ; 331.1839; found 331.1830.
5
.6. 3β-Acetoxy-5α-androstan-17-one (5)
To a solution of 1 (200 mg, 0.6 mmol) in MeOH (15 mL) in an autoclave, Pd/C (100 mg, 5%)
o
and the H were added and maintained at 4.0 Mp. The reaction mixture was stirred at 40 C for 16
2
h, then fitered with diatomite, and the filter cake was washed with CH Cl (15 mL × 3). The
2
2
filtrate was concentrated and the residue was purified by silica gel column chromatography
1
(
petroleum ether/AcOEt, 5/1, v/v) to give compound 5 (172 mg, 87%) as a white solid. H NMR
(
400 MHz, CDCl ) δ 4.68 (s, 1H), 2.42 (dd, J = 19.2, 8.9 Hz, 1H), 2.11–2.03 (m, 1H), 2.01 (d, J =
3
1
3
2
5
2
5
.3 Hz, 3H), 0.84 (d, J = 2.7 Hz, 6H). C NMR (100 MHz, CDCl ) δ 221.14, 170.60, 73.45, 54.27,
3
1.31, 47.73, 44.61, 36.67, 35.81, 35.61, 34.99, 33.91, 31.49, 30.77, 28.24, 27.37, 21.74, 21.42,
0.43, 13.79, 12.18.
.7. 3β-Acetoxy-17-(2-carbamoylhydrazono)-5α-androstan (6)
By a similar procedure described for compound 2, compound 6 was obtained as a white solid;
1
yield: 95%. H NMR (400 MHz, CDCl ) δ 7.82 (s, 1H), 4.73–4.63 (m, 1H), 2.38 (dd, J = 13.1, 5.2
3
Hz, 2H), 2.27–2.14 (m, 1H), 2.02 (s, 3H), 0.86 (s, 3H), 0.84 (s, 3H).
5
.8. 3β-Acetoxy-5α-androst-16-eno[17,16-d][1,2,3]thiadiazole (7)
By a similar procedure described for compound 3, compound 7 was obtained as a white solid;
o
-1
1
yield: 78%; mp: 184–186 C. IR (ATR) cm : 2917, 2853, 1716, 1245, 728. H NMR (400 MHz,
CDCl ) δ 4.75–4.65 (m, 1H), 3.02 (dd, J = 15.5, 6.3 Hz, 1H), 2.59 (dd, J = 15.5, 11.9 Hz, 1H),
3
1
3
2
.43 (dd, J = 12.0, 3.1 Hz, 1H), 2.25–2.18 (m, 1H), 2.02 (s, 3H), 1.09 (s, 3H), 0.91 (s, 3H).
C
NMR (100 MHz, CDCl ) δ 180.09, 170.76, 153.99, 73.54, 62.96, 54.83, 44.86, 41.91, 36.60,
3
3
5.92, 34.41, 34.33, 34.04, 31.32, 28.31, 27.50, 27.50, 21.54, 20.81, 18.06, 12.33. HRMS (ESI):
+
calcd for C H N NaO S [M + Na] ; 397.1920; found 397.1922.
2
1
30
2
2
5
.9. 3β-Hydroxy-5α-androst-16-eno[17,16-d][1,2,3]thiadiazole (8)
1
2

ACCEPTED MANUSCRIPT
By a similar procedure described for compound 4, compound 8 was obtained as a white solid;
o
-1
1
yield: 95%; mp: 208–210 C. IR (ATR) cm : 3410, 2923, 2847, 1720, 735. H NMR (400 MHz,
CDCl ) δ 3.66–3.56 (m, 1H), 3.02 (dd, J = 15.5, 6.3 Hz, 1H), 2.59 (dd, J = 15.5, 11.9 Hz, 1H),
3
1
3
2
.48–2.38 (m, 1H), 2.25–2.17 (m, 1H), 1.10 (s, 3H), 0.90 (s, 3H). C NMR (100 MHz, CDCl ) δ
3
1
80.15, 154.06, 71.17, 63.06, 54.96, 45.06, 41.92, 38.15, 36.84, 35.93, 34.44, 34.36, 31.53, 31.41,
+
2
8.44, 27.51, 20.86, 18.07, 12.43. HRMS (ESI): calcd for C H N NaOS [M + Na] ; 355.1815;
1
9
28
2
found 355.1818.
5
.10. General procedure for the preparation of compounds 9
–13
To a solution of compound 8ꢁ150 mg, 0.45 mmolꢂin dry CH Cl (20 mL), acyl chloride (9.0
2
2
mmol) and DMAP (55 mg, 0.45 mmol) were added at room temperature. The reaction mixture
was stirred for 4 h at room temperature under nitrogen atmosphere, then poured into H O (30 mL)
2
and extracted with CH Cl (20 mL × 3). The organic layer was washed with brine, dried with
2
2
anhydrous Na SO and concentrated. The residue was purified by silica gel column
2
4
chromatography (petroleum ether/AcOEt, 3/1, v/v) to give the desired product.
.10.1. 3β-Propionyloxy-5α-androst-16-eno[17,16-d][1,2,3]thiadiazole (9)
5
o
-1
1
White solid; yield: 88%; mp: 178–180 C. IR (ATR) cm : 2920, 2847, 1727, 1196. H NMR
(
400 MHz, CDCl ) δ 4.75–4.67 (m, 1H), 3.02 (dd, J = 15.5, 6.3 Hz, 1H), 2.59 (dd, J = 15.5, 12.0
3
Hz, 1H), 2.43 (dd, J = 11.9, 3.2 Hz, 1H), 2.29 (d, J = 7.6 Hz, 2H), 2.24–2.17 (m, 1H), 1.13 (t, J =
1
3
7
.6 Hz, 3H), 1.09 (s, 3H), 0.92 (s, 3H). C NMR (100 MHz, CDCl ) δ 180.11, 174.18, 154.01,
3
7
3.31, 62.97, 54.84, 44.87, 41.91, 36.62, 35.94, 34.42, 34.34, 34.08, 31.33, 28.33, 28.06, 27.53,
+
2
7.50, 20.82, 18.07, 12.35, 9.31. HRMS (ESI): calcd for C H N NaO S [M + Na] ; 411.2077;
2
2
32
2
2
found 411.2109.
5
.10.2. 3β-Methylsulfonyloxy-5α-androst-16-eno[17,16-d][1,2,3]thiadiazole (10)
o
-1
1
White solid; yield: 90%; mp: 166–167 C. IR (ATR) cm : 2919, 2855, 1728, 728. H NMR
(
400 MHz, CDCl ) δ 4.69–4.58 (m, 1H), 3.04 (d, J = 6.3 Hz, 1H), 3.00 (s, 3H), 2.60 (dd, J = 15.5,
3
1
1
4
1
1.9 Hz, 1H), 2.44 (dd, J = 11.4, 3.2 Hz, 1H), 2.21 (d, J = 29.5 Hz, 1H), 2.02 (d, J = 14.0 Hz, 1H),
1
3
.09 (s, 3H), 0.92 (s, 3H). C NMR (100 MHz, CDCl ) δ 179.98, 153.95, 81.78, 62.85, 54.65,
3
4.98, 41.87, 38.94, 36.62, 35.68, 35.19, 34.34, 34.24, 31.21, 28.69, 28.18, 27.47, 20.81, 18.05,
+
2.28. HRMS (ESI): calcd for C H N NaO S [M + Na] ; 433.1590; found 433.1612.
2
0
30
2
3 2
1
3

ACCEPTED MANUSCRIPT
5
.10.3. 3β-Nicotinoyloxy-5α-androst-16-eno[17,16-d][1,2,3]thiadiazole (11)
o
-1
1
White solid; yield: 83%; mp: 223–225 C. IR (ATR) cm : 2923, 2850, 1724, 1280, 730. H
NMR (400 MHz, CDCl ) δ 9.24 (s, 1H), 8.79 (d, J = 4.3 Hz, 1H), 8.41 (d, J = 7.7 Hz, 1H),
3
7
.54–7.45 (m, 1H), 5.06–4.95 (m, 1H), 3.03 (dd, J = 15.5, 6.4 Hz, 1H), 2.61 (dd, J = 15.5, 11.9 Hz,
H), 2.46 (dd, J = 11.7, 3.1 Hz, 1H), 2.28–2.20 (m, 1H), 2.00 (d, J = 12.5 Hz, 1H), 1.11 (s, 3H),
1
1
3
0
.98 (s, 3H). C NMR (100 MHz, CDCl ) δ 180.07, 164.88, 153.98, 153.35, 150.99, 137.11,
3
1
26.73, 123.32, 74.81, 62.94, 54.82, 44.90, 41.91, 36.60, 35.97, 34.41, 34.33, 34.07, 31.32, 28.32,
+
2
7.56, 27.50, 20.84, 18.07, 12.41. HRMS (ESI): calcd for C H N O S [M + H] ; 438.2210;
2
5
32
3
2
found 438.2232.
5
.10.4. 3β-Isonicotinoyloxy-5α-androst-16-eno[17,16-d][1,2,3]thiadiazole (12)
o
-1
1
White solid; yield: 89%; mp: 235–237 C. IR (ATR) cm : 2914, 2851, 1721, 1280, 727. H
NMR (400 MHz, CDCl ) δ 8.78 (d, J = 5.2 Hz, 2H), 7.90 (d, J = 5.5 Hz, 2H), 5.04–4.94 (m, 1H),
3
3
2
1
3
.03 (dd, J = 15.5, 6.3 Hz, 1H), 2.61 (dd, J = 15.5, 11.9 Hz, 1H), 2.45 (dd, J = 11.7, 3.2 Hz, 1H),
1
3
.27–2.20 (m, 1H), 2.04–1.98 (m, 1H), 1.11 (s, 3H), 0.97 (s, 3H). C NMR (100 MHz, CDCl ) δ
3
80.07, 164.71, 153.98, 150.62, 150.62,138.13, 122.97, 122.97, 75.25, 62.95, 54.82, 44.90, 41.92,
6.59, 35.98, 34.41, 34.35, 34.00, 31.32, 28.33, 27.51, 27.50, 20.86, 18.08, 12.41. HRMS (ESI):
+
calcd for C H N O S [M + H] ; 438.2210; found 438.2250.
2
5
32
3
2
5
.10.5. 3β-[(Pyrazine-2-carbonyl)oxy]-5α-androst-16-eno [17,16-d][1,2,3]thiadiazole (13)
o
-1
1
White solid; yield: 89%; mp: 222–224 C. IR (ATR) cm : 2916, 2853, 1712, 733. H NMR
(
400 MHz, CDCl ) δ 9.30 (s, 1H), 8.74 (d, J = 5.6 Hz, 2H), 5.15–5.04 (m, 1H), 3.03 (dd, J = 15.5,
3
6
2
1
3
.3 Hz, 1H), 2.60 (dd, J = 15.5, 12.0 Hz, 1H), 2.45 (dd, J = 11.8, 3.2 Hz, 1H), 2.28–2.20 (m, 1H),
1
3
.06–2.01 (m, 1H), 1.10 (s, 3H), 0.97 (s, 3H). C NMR (100 MHz, CDCl ) δ 180.04, 163.57,
3
53.95, 147.58, 146.38, 144.50, 143.97, 75.83, 62.92, 54.78, 44.94, 41.89, 36.61, 35.97, 34.39,
4.31, 33.93, 31.29, 28.28, 27.49, 27.43, 20.82, 18.05,12.40. HRMS (ESI): calcd for
+
C H N NaO S [M + Na] ; 461.1982; found 461.1986.
2
4
30
4
2
5
.11. General procedure for the preparation of compounds 14
–19
To a solution of compound 8 (250 mg, 0.75 mmol) in dry CH Cl2 (20 mL),
2
N-BOC-amino-carboxylic acid (3.0 mmol), HOBT (405 mg, 3.0 mmol), EDCI (575 mg, 3.0 mmol)
and DMA Pꢁ 366 mg, 3.0 mmolꢂwere added at room temperature. The reaction mixture was stirred
1
4

ACCEPTED MANUSCRIPT
for 8 h at room temperature under nitrogen atmosphere, then poured into water (30 mL) and
extracted with CH Cl (20 mL × 3). The organic layer was washed with brine, dried with
2
2
anhydrous Na SO and concentrated. The residue was purified by silica gel column
2
4
chromatography (petroleum ether/AcOEt, 3/1, v/v) to give the desired product.
.11.1. 3β-{2-[(tert-butoxycarbonyl)amino]acetoxy}-5α-androst-16-eno[17,16-d][1,2,3]
thiadiazole (14)
5
1
White solid; yield: 93%. H NMR (400 MHz, CDCl ) δ 5.01 (s, 1H), 4.83–4.72 (m, 1H), 3.89 (d,
3
J = 4.7 Hz, 2H), 3.04 (dd, J = 15.5, 6.3 Hz, 1H), 2.62 (dd, J = 15.4, 12.0 Hz, 1H), 2.46 (dd, J =
1
3
1
1.6, 3.4 Hz, 1H), 2.27–2.16 (m, 1H), 1.48 (s, 9H), 1.12 (s, 3H), 0.94 (s, 3H). C NMR (100 MHz,
CDCl ) δ 179.97, 169.87, 155.71, 153.91, 79.91, 74.64, 62.82, 54.66, 44.70, 42.67, 41.79, 36.41,
3
3
5.78, 34.27, 34.19, 33.82, 31.18, 28.33, 28.16, 27.40, 27.31, 20.70, 17.97, 12.22.
5
.11.2. 3β-{[4-((tert-butoxycarbonyl)amino)butanoyl]oxy}-5α-androst-16-eno[17,16-d][1,2,3]
thiadiazole (15)
1
White solid; yield: 96%. H NMR (400 MHz, CDCl ) δ 4.76–4.67 (m, 1H), 4.65 (d, J = 11.9 Hz,
3
1
2
3
6
2
6
H), 3.15 (d, J = 6.3 Hz, 2H), 3.01 (dd, J = 15.5, 6.3 Hz, 1H), 2.59 (dd, J = 15.5, 12.0 Hz, 1H),
.43 (dd, J = 11.8, 3.3 Hz, 1H), 2.31 (t, J = 7.3 Hz, 2H), 2.26–2.16 (m, 1H), 1.43 (s, 9H), 1.09 (s,
1
3
H), 0.91 (s, 3H). C NMR (100 MHz, CDCl ) δ 179.98, 172.82, 155.94, 153.92, 79.17, 73.51,
3
2.83, 54.67, 44.73, 41.78, 39.96, 36.46, 35.80, 34.27, 34.19, 33.92, 32.00, 31.20, 28.41, 28.19,
7.39, 25.33, 20.69, 17.97, 14.14, 12.23.
.11.3. 3β-{[1-(tert-butoxycarbonyl)piperidine-3-carbonyl]oxy}-5α-androst-16-eno
[
17,16-d][1,2,3]thiadiazole (16)
1
White solid; yield: 98%. H NMR (400 MHz, CDCl ) δ 4.78–4.65 (m, 1H), 3.99 (d, J = 13.4 Hz,
3
2
H), 3.01 (dd, J = 15.5, 6.3 Hz, 1H), 2.92–2.77 (m, 2H), 2.59 (dd, J = 15.4, 12.0 Hz, 1H),
1
3
2
.49–2.35 (m, 2H), 2.26–2.16 (m, 1H), 1.45 (s, 9H), 1.09 (s, 3H), 0.91 (s, 3H). C NMR (100
MHz, CDCl ) δ 180.00, 173.01, 154.71, 153.91, 79.66, 73.46, 62.84, 54.67, 44.71, 41.79,
3
4
2
5
1.59,41.57, 36.44, 35.81, 34.28, 34.20, 33.90, 33.87, 31.21, 28.43, 28.19, 27.37, 27.35, 27.33,
4.33, 20.70, 17.97, 12.24.
.11.4. 3β-{[1-(tert-butoxycarbonyl)piperidine-4-carbonyl]oxy}-5α-androst-16-eno
[
17,16-d][1,2,3]thiadiazole (17)
1
5

ACCEPTED MANUSCRIPT
1
White solid; yield: 96%. H NMR (400 MHz, CDCl ) δ 4.78–4.64 (m, 1H), 4.12 (d, J = 7.1 Hz,
3
1
H), 3.94–3.83 (m, 1H), 3.01 (dd, J = 15.5, 6.3 Hz, 1H), 2.86–2.76 (m, 1H), 2.59 (dd, J = 15.4,
2.0 Hz, 1H), 2.46–2.34 (m, 2H), 2.27–2.15 (m, 1H), 2.02 (d, J = 15.8 Hz, 1H), 1.46 (s, 9H), 1.09
1
1
3
(
s, 3H), 0.91 (s, 3H). C NMR (100 MHz, CDCl ) δ 179.98, 174.15, 154.72, 153.91, 79.55, 73.42,
3
6
2
5
2.83, 54.67, 44.71, 41.79, 41.29, 36.45, 35.81, 34.27, 34.20, 33.91, 31.20, 28.44, 28.19, 28.01,
7.98, 27.40, 27.37, 20.71, 17.97, 12.25.
.11.5. 3β-[N-tert-butoxycarbonyl-(L-prolyl)oxy]-5α-androst-16-eno[17,16-d][1,2,3]thiadiazole
(18)
1
White solid; yield: 98%. H NMR (400 MHz, CDCl ) δ 4.80–4.68 (m, 1H), 4.31–4.16 (m, 1H),
3
3
.59–3.34 (m, 2H), 3.02 (dd, J = 15.5, 6.3 Hz, 1H), 2.59 (dd, J = 15.1, 12.2 Hz, 1H), 2.44 (d, J =
1
3
9
.1 Hz, 1H), 2.28–2.11 (m, 2H), 1.46 (s, 3H), 1.42(s, 6H), 1.09 (s, 3H), 0.91 (s, 3H). C NMR
(
125 MHz, DMSO-d ) δ 179.81, 172.60, 155.17, 153.34, 79.13, 73.75, 62.50, 59.11, 54.25, 46.61,
6
4
2
5
4.44, 41.79, 36.25, 35.80, 34.49, 34.07, 31.07, 30.82, 29.88, 28.52, 28.38, 27.66, 24.28, 23.50,
0.77, 18.32, 12.30.
.11.6. 3β-[N-tert-butoxycarbonyl-(D-prolyl)oxy]-5α-androst-16-eno[17,16-d][1,2,3]thiadiazole
(19)
1
White solid; yield: 86%. H NMR (400 MHz, CDCl ) δ 4.80–4.67 (m, 1H), 4.33–4.14 (m, 1H),
3
3
.61–3.35 (m, 2H), 3.02 (dd, J = 15.5, 6.3 Hz, 1H), 2.65–2.53 (m, 1H), 2.43 (d, J = 11.4 Hz, 1H),
1
3
2
.30–2.11 (m, 2H), 1.46 (s, 3H), 1.42 (s, 6H), 1.09 (s, 3H), 0.91 (s, 3H). C NMR (125 MHz,
CDCl ) δ 179.96, 172.71, 154.36, 153.88, 79.77, 73.85, 62.82, 59.30, 54.68, 46.33, 44.72, 41.79,
3
3
1
5
6.43, 35.81, 34.28, 34.21, 34.01, 31.19, 31.00, 28.45, 28.39, 28.21, 27.39, 23.53, 20.71, 17.96,
2.25.
.12. General procedure for the preparation of compounds 20
–25
One of compounds 14–19 (0.4 mmol) was dissolved in anhydrous diethyl ether (20 mL) and
boron trifluoride etherate (5 mL) was added. The reaction mixture was stirred for 2 h at room
temperature and then quenched with saturated aqueous NaHCO (20 mL). The mixture was
3
extracted with AcOEt (20 mL × 3). The combined organic extract was washed with brine, dried
over anhydrous Na SO and concentrated. The residue was purified by silica gel chromatography
2
4
(
CH Cl /MeOH, 10/1, v/v) to give the desired product.
2 2
1
6

ACCEPTED MANUSCRIPT
5
.12.1. 3β-(2-Aminoacetoxy)-5α-androst-16-eno[17,16-d][1,2,3]thiadiazole (20)
o
-1
White solid; yield: 77%; mp: 163–165 C. IR (ATR) cm : 3378, 2918, 2853, 1718, 1280,
1
7
28. H NMR (400 MHz, CDCl ) δ 4.82–4.71 (m, 1H), 3.45 (s, 2H), 3.01 (dd, J = 15.5, 6.3 Hz,
3
1
H), 2.59 (dd, J = 15.5, 11.9 Hz, 1H), 2.43 (dd, J = 11.7, 3.2 Hz, 1H), 1.09 (s, 3H), 0.91 (s, 3H).
1
3
C NMR (100 MHz, CDCl ) δ 180.04, 173.69, 153.97, 74.19, 62.92, 54.79, 44.82, 44.19, 41.87,
3
3
6.54, 35.88, 34.37, 34.29, 34.01, 31.28, 28.27, 27.47, 27.47, 20.79, 18.04, 12.30. HRMS (ESI):
+
calcd for C H N O S [M + H] ; 390.2210; found 390.2211.
2
1
32
3
2
5
.12.2. 3β-[(4-Aminobutanoyl)oxy]-5α-androst-16-eno[17,16-d][1,2,3]thiadiazole (21)
o
-1
White solid; yield: 72%; mp: 170–172 C. IR (ATR) cm : 3342, 2918, 2855, 1719, 1685,
1
7
=
28. H NMR (400 MHz, CDCl ) δ 4.77–4.65 (m, 1H), 3.02 (dd, J = 15.5, 6.3 Hz, 1H), 2.73 (t, J
3
7.0 Hz, 1H), 2.59 (dd, J = 15.5, 11.9 Hz, 1H), 2.43 (dd, J = 12.0, 3.0 Hz, 1H), 2.39–2.26 (m,
1
3
2
H), 1.09 (s, 3H), 0.91 (s, 3H). C NMR (100 MHz, CDCl ) δ 180.06, 173.01, 154.01, 73.60,
3
7
1.07, 62.90, 54.75, 44.81, 41.86, 41.10, 38.13, 36.54, 35.89, 34.35, 34.26, 34.01, 32.09, 31.28,
+
2
8.27, 27.48, 20.78, 18.06, 12.32. HRMS (ESI): calcd for C H N O S [M + H] ; 418.2523;
2
3
36
3
2
found 418.2528.
5
.12.3. 3β-[(Piperidine-3-carbonyl)oxy]-5α-androst-16-eno[17,16-d][1,2,3]thiadiazole (22)
o
-1
1
White solid; yield: 75%; mp: 179–181 C. IR (ATR) cm : 3527, 2923, 2853, 1722, 728. H
NMR (400 MHz, CDCl ) δ 7.24 (s, 1H), 4.75 (s, 1H), 3.63–3.10 (m, 4H), 3.01 (dd, J = 15.5, 6.3
3
Hz, 1H), 2.90 (s, 1H), 2.59 (dd, J = 15.4, 12.0 Hz, 1H), 2.43 (dd, J = 11.9, 2.9 Hz, 1H), 1.09 (s,
1
3
3
H), 0.92 (s, 3H). C NMR (100 MHz, CDCl ) δ 180.02, 171.99, 154.06, 74.95, 62.96, 54.81,
3
4
5.42, 44.99, 44.82, 41.90, 38.08, 36.50, 35.90, 34.41, 34.29, 33.78, 31.31, 28.27, 27.51, 27.30,
+
2
5.30, 20.95, 20.84, 18.07, 12.34. HRMS (ESI): calcd for C H N O S [M + H] ; 444.2679;
2
5
38
3
2
found 444.2679.
5
.12.4. 3β-[(Piperidine-4-carbonyl)oxy]-5α-androst-16-eno[17,16-d][1,2,3]thiadiazole (23)
o
-1
1
White solid; yield: 82%; mp: 175–177 C. IR (ATR) cm : 3340, 2917, 2852, 1717, 728. H
NMR (400 MHz, CDCl ) δ 4.79–4.61 (m, 1H), 3.14–3.05 (m, 2H), 3.02 (dd, J = 15.5, 6.4 Hz, 1H),
3
1
3
2
.68–2.60 (m, 2H) , 2.48–2.33 (m, 2H), 2.25–2.18 (m, 1H), 1.09 (s, 3H), 0.91 (s, 3H). C NMR
(
100MHz, CDCl ) δ 180.07, 174.73, 153.98, 73.26, 62.93, 54.79, 45.87, 44.83, 41.88, 41.81,
3
3
6.56, 35.91, 34.38, 34.30, 34.02, 31.30, 29.32, 29.29, 28.29, 27.47, 20.79, 18.04, 12.33. HRMS
17

ACCEPTED MANUSCRIPT
+
(
ESI): calcd for C H N O S [M + H] ; 444.2679; found 444.2686.
2
5
38
3
2
5
.12.5. 3β-[(L-prolyl)oxy]-5α-androst-16-eno[17,16-d][1,2,3]thiadiazole (24)
o
-1
1
White solid; yield: 74%; mp: 82–84 C. IR (ATR) cm : 3391, 2917, 2853, 1719, 728. H NMR
(
400 MHz, CDCl ) δ 4.83–4.66 (m, 1H), 3.79–3.65 (m, 1H), 3.13–3.04 (m, 1H), 3.01 (dd, J = 15.5,
3
6
.3 Hz, 1H), 2.95–2.84 (m, 1H), 2.58 (dd, J = 15.3, 12.1 Hz, 1H), 2.45–2.40 (m, 1H), 2.25–2.09
1
3
(
m, 2H), 1.08 (s, 3H), 0.91 (s, 3H). C NMR (100 MHz, CDCl ) δ 180.10, 175.05, 153.99, 74.12,
3
6
2
4
5
2.96, 59.99, 54.82, 47.15, 44.85, 41.92, 36.58, 35.94, 34.41, 34.34, 33.99, 31.33, 30.54, 28.31,
+
7.51, 27.47, 25.58, 20.83, 18.07, 12.36. HRMS (ESI): calcd for C H N O S [M + H] ;
24
36
3
2
30.2523; found 430.2546.
.12.6. 3β-[(D-prolyl)oxy]-5α-androst-16-eno[17,16-d][1,2,3]thiadiazole (25)
o
-1
1
White solid; yield: 81%; mp: 148–150 C. IR (ATR) cm : 3389, 2931, 2853, 1719, 728. H
NMR (400 MHz, CDCl ) δ 4.80–4.68 (m, 1H), 3.71–3.68 (m, 1H), 3.10–3.06 (m, 1H), 3.04–2.99
3
(m, 1H), 2.91–2.85 (m, 1H), 2.59 (dd, J = 15.5, 11.9 Hz, 1H), 2.43 (dd, J = 11.8, 3.2 Hz, 1H),
1
3
2
1
3
.25–2.18 (m, 1H), 2.16–2.07 (m, 1H), 1.09 (s, 3H), 0.91 (s, 3H). C NMR (100 MHz, CDCl ) δ
3
80.07, 175.17, 153.97, 74.00, 62.94, 60.08, 54.80, 47.21, 44.85, 41.89, 36.55, 35.91, 34.39, 34.32,
3.98, 31.30, 30.53, 28.29, 27.49, 27.45, 25.61, 20.82, 18.06, 12.35. HRMS (ESI): calcd for
+
C H N O S [M + H] ; 430.2523; found 430.2543.
2
4
36
3
2
5
5
.13. Biological assay
.13.1. Cell lines and culture conditions
Tumor cell lines used in this study were obtained from the American Type Culture Collection
(ATCC). LNCaP, DU145, HCT116, HT29, T47D, MCF7, HL60 and Jurkat cell lines were cultured
in RPMI 1640 medium, MDA-MB-231 cell line was cultured in MEM medium, and HAF cell line
was cultured in DMEM medium with an additional final concentration of 2 mM L-glutamine.
o
Medium was supplemented with 10% FBS, and all tumor cells were incubated at 37 C and 5%
CO incubator.
2
5
.13.2. Cell Viability assay
Adherent cell viability was determined by SRB assay which was described previously [43]. In
brief, cells (including LNCaP, DU-145, HCT-116, HT-29, T47D, MDA-MB-231, MCF7 and HAF)
1
8

ACCEPTED MANUSCRIPT
were seeded into 96-well plates. After 24 h, the cells were treated with various doses of the
compounds for 96 h. Then fixed and washed after dyeing with SRB. After dried, 10 mM
Tris-based solution was added and absorbance was measured at 515 nm. Suspension cell viability
was determined by MTS assay which was described previously [44]. In brief, cells (including
HL60 and Jurkat) were seeded into 96-well plates. After 24 h, the cells were treated with various
doses of the compounds. MTS was added and incubated after 96 h treatment and absorbance was
measured at 490 nm. The IC was calculated using GraphPad software.
5
0
5
.13.3. In vitro transwell migration assay
Transwell migration assay was performed as previously reported [45]. The inhibition of tumor
cell migration was assessed by the Boyden chamber (Corning Falcon) migration assay with 8.0
ꢀm pore. Briefly, T47D cells were collected, centrifuged, and resuspended with serum-free
4
medium. The top chambers were seeded with 5 × 10 cells in serum-free 1640 medium containing
different dose of compounds. The bottom chambers were filled with complete medium
supplemented with different dose of compounds. After 12 h incubation, non-migrated cells were
removed, and migrated cells were fixed and stained. Images were taken and cells from three
random areas per filter were counted.
5
.13.4. Cell cycle analysis
Cell cycle analysis was conducted by PI staining as described previously [43]. T47D cells were
plated in 6 cm dishes and were treated with different dose of compound for 24 h. After ethanol
fixation, cells were washed in PBS once and suspended in PBS with RNAase and propidium
iodide (PI) in dark for 30 min. Then cells were analyzed by flow cytometry (FACS Calibur, BD
Biosciences).
5
.13.5. Cell apotosis analysis
Apoptotic cells were monitored with Annexin V-FITC Apoptosis Detection Kit I (BD
Biosciences) as described previously [44]. T47D cells were plated in 6 cm dishes and were treated
with different dose of compound for 24 h. Cells were washed with cold PBS, harvested and
re-suspended in 1×binding buffer, and incubated with Annexin V fluorescein isothiocyanate and
propidium iodide for 15 min in dark at room temperature. Then 1 × binding buffer was added and
analyzed immediately with flow cytometry (FACS Calibur, BD Biosciences).
1
9

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5
.13.6. Colony formation assay
T47D cells were trypsinized and seeded 2000 per well in 6-well dishes. Cells were allowed to
attach overnight and then exposed to different dose of compound for a week. After fixed for 20
min, cells were stained. The number of cell colonies was calculated and analyzed as the ratio of
the number of treated samples to untreated samples. Triplicate wells were set up for each
concentration.
5
.13.7. Subcutaneous and orthotopic xenograft animal model of T47D
These assays were performed as described previously [46,47]. In subcutaneous xenograft
6
animal model, 2 × 10 T47D cells were implanted subcutaneously on 8-week-old female nude
mice. On day 3, mice were divided into two groups (n=8) randomly. Compound (20 mg/kg) or
DMSO was injected intraperitoneally every day. The tumor length, width and body weight were
evaluated every other day and xenograft tumor growth rate was measured as the following
2
equation, volume = length × width × 0.52. Mice were continually observed until they were
sacrificed at the twenty-first day. The liver and kidney were fixed and prepared for H&E staining.
5
In orthotopic xenograft animal model, 1 × 10 T47D cells were injected subcutaneously into the
4
th abdominal mammary fat pad on 8-week-old female nude mice. On day 3, mice were divided
into two groups (n = 5) randomly. Compound (20 mg/kg/day) or DMSO was injected
intraperitoneally every day. The tumor length, width and body weight were evaluated every three
days and xenograft tumor growth rate was measured as the following equation, volume = length ×
2
width × 0.52. Mice were continually observed until they were sacrificed at the eighteenth day.
Lung metastases were manually counted using a dissecting microscope.
5
.13.8. Phospho-receptor tyrosine kinase array analysis
A Human Phospho-RTK Array Kit (R&D Systems) was used to measure the relative level of
tyrosine phosphorylation of 49 different receptor tyrosine kinases (RTKs). T47D cells were plated
in 10 cm dishes and treated with 2.5 ꢀM compound or DMSO for 24 h. Cells were lysed with
Lysis Buffer 17 and diluted to 1000ug/mL with Array Buffer 1. The arrays were blocked with
Array Buffer 1 for 1 h at room temperature on a rocking platform shaker and then incubated with
1
mL lysate overnight at 4°C on a rocking platform shaker. The arrays were washed, incubated
with Anti-Phospho-Tyrosine-HRP Detection Antibody, treated with Chemi Reagent Mix, and
2
0

ACCEPTED MANUSCRIPT
exposed to film.
Acknowledgment
This work was supported by Shanghai Science and Technology Council (Grant 12ZR1408500),
Major Research Plan of the National Natural Science Foundation of China (Grant 91413112), the
Open Project Program of Jiangsu Key Laboratory of Drug Screening (JKLDS2015KF-04) and
National Natural Science Foundation of China (81272463, 81472788).
Appendix A. Supplementary data
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Table 1
IC50 values of DHEA and its derivatives 4 and 8 against the growth of T47D cells and other various tumor cell
lines.
IC50 (ꢀM)a
Compound
T47D
2.55
1.23
1.36
0.67
MDA-MB-231 MCF-7
DU145
> 50
LNCaP
> 50
HCT116
> 50
HT29
> 50
> 50
> 50
> 50
HL-60
> 50
> 50
> 50
> 50
Jurkat
46.5
28.8
> 50
22.7
DHEA
> 50
> 50
>50
> 50
> 50
24.1
b
EPI
> 50
> 50
> 50
4
8
(CH12)
(CH21)
> 50
> 50
> 50
20.1
> 50
35.6
48.1
> 50
a
From SRB assay after 96 h of treatment; IC data are an average of at least 3 independent experiments, variation
50
b
±
10%; Epiandrosterone (EPI) is a reduzate of DHEA.
2
6

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Table 2
IC50 values of DHEA and its derivatives (7, 8 and 26-43) against the growth of T-47D tumor cell line and normal
human fibroblast (HAF) cells.
N
N
S
RO
H
IC50 (ꢀM)a
IC50 (ꢀM)a
SIb
32
SIb
16
70
Compound
R
Compound
R
T47D
HAF
T47D
HAF
O
c
8
(CH21)
H
0.67 ± 0.11
39.05 ± 4.15
21
22
1.15 ± 0.10
18.0 ± 0.22
NH
2
O
O
NDd
7
9
2.17 ± 0.09
> 50
0.042 ± 0.028
2.95 ± 0.28
N
H
O
O
2.66 ± 0.85
2.83 ± 0.49
2.25 ± 0.30
3.28 ± 0.51
3.04 ± 0.99
0.92 ± 0.05
> 50
> 50
> 50
> 50
> 50
ND
ND
ND
ND
ND
28
23
24
0.051 ± 0.010
0.24 ± 0.06
1.82 ± 0.03
36.0 ± 2.05
21.1 ± 5.06
> 50
36
NH
O
O
O
1
0
1
S
150
364
ND
1.7
HN
O
O
1
25 (CH33)
DHEA
ADMe
0.058 ± 0.016
2.55 ± 0.01
HN
N
O
O
1
1
2
2
3
0
N
N
N
0.040 ± 0.018 0.068 ± 0.064
O
NH
2
26.1 ± 0.77
a
b
From SRB assay after 96 h of treatment; IC50 data are an average of at least 3 independent experiments; the
selectivity indexes (SI) were calculated by IC50 values in HAF cells divided by IC50 values in T47D cancer cell
c
d
e
line; data from Table 1; ND = not determined; ADM is Adriamycin.
27

ACCEPTED MANUSCRIPT
Table 3
IC50 values of compounds 22, 24 and 25 against the growth of T47D cells and other various tumor cell lines.
IC50 (ꢀM)a
Compound
T47Db
0.042
0.24
c
MDA-MB-231 MCF-7
DU145
0.92
LNCaP
4.28
HCT116
2.01
HT29
2.12
13.6
16.5
HL-60
5.33
Jurkat
2.57
1.41
2.49
Selectivity
2
2
2
2
4
1.24
7.69
9.66
1.41
15.0
27.1
30-126
6-150
24.3
36.2
8.52
31.5
5 (CH33) 0.058
17.7
31.6
11.7
46.0
43-793
a
From SRB assay after 96 h of treatment; IC data are an average of at least 3 independent experiments, variation
50
b
c
±
10%; data from Table 2; Selectivity is the IC values of T47D cells divide IC50 values of other tumor cell lines.
50
Ph
OMe
N
N
N
N
N
CO Et
O
N
O
2
SO Ph
2
O
O
N
O
O
HO
AcO
O
A
B
C
Fig. 1. Chemical structures of D-ring fused heterocycles DHEA antitumor agents
0
µM
0.1 µM
0.5 µM
2.5 µM
1
1
20
00
8
6
4
2
0
0
0
0
0
ns
0.1 0.5 2.5
DHEA (µM)
0
1
1
20
00
*
*
8
6
4
2
0
0
0
0
0
*
**
*
**
2
5 (µM)
0
0.1 0.5 2.5
Fig. 2. Compound 25 and DHEA inhibited the colony formation of T47D cell line. After treatment by various
doses of 25 and DHEA in 6-well plates for 7 days, cells were fixed and stained with crystal violet and the numbers
of cell colonies were counted. **P < 0.01, ***P < 0.001, versus the control group (0 ꢀM).
2
8