Synthesis and antitumor evaluation of novel hybrids of phenylsulfonylfuroxan and epiandrosterone/dehydroepiandrosterone derivatives

Article abstract of DOI:10.1016/j.steroids.2015.05.003

Abstract Thirteen novel furoxan-based nitric oxide (NO) releasing hybrids (14a-e, 15a-e, 17b-d) of 16,17-pyrazo-annulated steroidal derivatives were synthesized and evaluated against the MDA-MB-231, HCC1806, SKOV-3, DU145, and HUVEC cell lines for their in vitro anti-proliferative activity. Most of the compounds displayed potent anti-proliferative effects. Among them, 17c exhibited the best activity with IC₅₀ values of 20-1.4 nM against four cell lines (MDA-MB-231, SKOV-3, DU145, and HUVEC), and 1.03 μM against a tamoxifen resistant breast cancer cell line (HCC1806). Furthermore, five compounds (14a, 15a, 17b-d) were selected to screen for VEGF inhibitory activity. Compounds 15a, 17b,c showed obviously better activity than 2-Methoxyestradiol (2-ME) on reducing levels of VEGF secreted by MDA-MB-231 cell line. In a Capillary-like Tube Formation Assay, compounds 17b,c exhibited a significant suppression of the tubule formation in the concentration of 1.75 nM and 58 nM, respectively. The preliminary SAR showed that steroidal scaffolds with a linker in 3-position were favorable moieties to evidently increase the bioactivities of these hybrids. Overall, these results implied that 17c merited to be further investigated as a promising anti-cancer candidate.

Full text of DOI:10.1016/j.steroids.2015.05.003

Steroids 101 (2015) 7–14
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and antitumor evaluation of novel hybrids of
phenylsulfonylfuroxan and epiandrosterone/dehydroepiandrosterone
derivatives
Yaoqing Huang a,1, Mingming Liu a,1, Lanfang Meng b, Pan Feng a, Yalan Guo a,
Minghua Ying c, Xiuyan Zhu c, Ying Chen a,
⇑
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, China
b Department of Nuclear Medicine, ZhongShan Hospital, Fudan University, Shanghai, China
c Zhejiang Xianju Pharmaceutical Co. Ltd., Zhejiang, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirteen novel furoxan-based nitric oxide (NO) releasing hybrids (14a–e, 15a–e, 17b–d) of 16,17-pyrazo-
annulated steroidal derivatives were synthesized and evaluated against the MDA-MB-231, HCC1806,
SKOV-3, DU145, and HUVEC cell lines for their in vitro anti-proliferative activity. Most of the compounds
displayed potent anti-proliferative effects. Among them, 17c exhibited the best activity with IC50 values
Received 20 March 2015
Received in revised form 24 April 2015
Accepted 13 May 2015
Available online 22 May 2015
of 20–1.4 nM against four cell lines (MDA-MB-231, SKOV-3, DU145, and HUVEC), and 1.03 lM against a
tamoxifen resistant breast cancer cell line (HCC1806). Furthermore, five compounds (14a, 15a, 17b–d)
were selected to screen for VEGF inhibitory activity. Compounds 15a, 17b,c showed obviously better
activity than 2-Methoxyestradiol (2-ME) on reducing levels of VEGF secreted by MDA-MB-231 cell line.
In a Capillary-like Tube Formation Assay, compounds 17b,c exhibited a significant suppression of the
tubule formation in the concentration of 1.75 nM and 58 nM, respectively. The preliminary SAR showed
that steroidal scaffolds with a linker in 3-position were favorable moieties to evidently increase the
bioactivities of these hybrids. Overall, these results implied that 17c merited to be further investigated
as a promising anti-cancer candidate.
Keywords:
16,17-Pyrazo-annulated steroids
Furoxan
Cytotoxicity
VEGF inhibitor
Anti-cancer
Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
reported to reverse the resistance to doxorubicin in human colon
cancer cells [7,8]. Furoxan is an important class of NO donors,
Nitric oxide (NO), which is naturally synthesized from
-arginine by the action of NO synthase (NOS), is a key signaling
which can release high levels of NO in vitro [9]. And their hybrids
with other active compounds such as oleanolic acid, glycyrrhetinic
acid, farnesylthiosalicylic acid, displayed potent antitumor activity
both in vitro and in vivo [10–12]. In our prior research, several fur-
oxan-based NO releasing hybrids with the coumarin core were
synthesized to exhibit excellent biological activities including
antiproliferation of tumor cells, inducing apoptosis of tumor cells
and inhibition of angiogenesis [13].
Angiogenesis is dysregulated in many pathological conditions
such as cancers. It plays significant roles in tumor growth, invasion
and metastasis because of its function for supplying of oxygen,
nutrients, growth factors and dissemination of tumor cells to distal
sites [14]. Preventing the expansion of new blood vessel networks
may result in reduced tumor size and metastases. Vascular endothe-
lial growth factor (VEGF) is the major driver of tumor angiogenesis
by triggering endothelial cells to form new capillaries under hypoxic
conditions, and high expression of VEGF has been observed in many
cancers, including colorectal carcinoma, gastric carcinoma,
L
molecule which plays very important roles in diverse physiological
and pathophysiological processes including vascular homeostasis
maintenance, neurotransmission, and immune response. Nitric
oxide diffuses outward and into nearby target cells after being gen-
erated, and then all the effects mentioned above are triggered by
activating cytosolic guanylate cyclase, thereby stimulating intra-
cellular accumulation of cyclic guanosine monophosphate
(cGMP), which acts as an intracellular messenger to initiate a series
of reactions [1,2]. It is well-known that high levels of NO can act as
a potent anti-cancer agent by promoting apoptosis, inhibiting
angiogenesis, and sensitizing tumor cells to chemotherapy, radia-
tion, and immunotherapy [3–6]. High concentration of NO was also
⇑
Corresponding author. Tel./fax: +86 21 51980116.
E-mail address: yingchen71@fudan.edu.cn (Y. Chen).
Yaoqing Huang and Mingming Liu are co-first-authors.
1
http://dx.doi.org/10.1016/j.steroids.2015.05.003
0039-128X/Ó 2015 Elsevier Inc. All rights reserved.

8
Y. Huang et al. / Steroids 101 (2015) 7–14
pancreatic carcinoma, breast cancer, prostate cancer, lung cancer,
and melanoma [14–16]. All these results make VEGF a target for
many angiogenesis inhibitors and of keen interest in the field of
cancer research [17]. In our prior research, several 16,17-pyrazo-
annulated steroids were synthesized and proved to exhibit potent
VEGF inhibitory activity. Among them, compound 1 (3b-O-(3,3-
dimethylsuccinyl)-20-methyl-20H-androst-16-eno[17,16-c]pyrazole)
and 2 (3b-O-(3,3-dimethylsuccinyl)-20-phenyl-20H-androsta-5,16-
dieno[17,16-c]pyrazole) exhibited similar EC50 values and
obviously better TI values compared with 2-ME [18] (Fig. 1).
It is therefore of interest to develop more desirable leading
compounds with steroidal scaffold and explore whether introduc-
tion of phenylsulfonylfuroxan moiety to potent antiangiogenesis
compounds mentioned above would provide a novel class of NO
donating hybrids which may exert positive synergistic antitumor
effects. Herein, a series of novel hybrids of phenylsulfonylfuroxan
group and 16,17-pyrazo-annulated steroids were designed, syn-
thesized and biologically evaluated for antiproliferation of cancer
cell lines, inhibitory activity of VEGF and capillary-like tube
formation.
2.5. 20H-Androst-16-eno[17,16-c]pyrazole-3b-ol (12a)
Compound 10 (2.00 g, 6.3 mmol) was dissolved in anhydrous
ethanol (30 mL), and 85% hydrazine hydrate (1.10 g, 18.8 mmol)
was added dropwise to the solution. After being stirred for 0.5 h
at room temperature, the mixture was poured into ice water
(100 mL). Then the mixture was adjusted to pH 7 by 2 M HCl to
afford a white solid. Recrystallization from acetone gave 12a
(1.7 g, 86%). Mp 260–261 °C; ESI-MS m/z (%) 315.2 [M+H]+; 1H
NMR (400 MHz, CDCl3) d 0.87 (s, 3H, H-18), 0.97 (s, 3H, H-19),
3.60 (m, 1H, H-3), 7.14 (s, 1H, AN@CHA), 0.78–2.52 (m, 22H).
2.6. 20H-Androsta-5,16-dieno[17,16-c]pyrazole-3b-ol (13a)
13a (1.8 g, 92%) was obtained starting from 11 (2.0 g,
6.3 mmol). Mp 269–272 °C; ESI-MS m/z (%) 313.2 [M+H]+; 1H
NMR (400 MHz, CDCl3) d 1.02 (s, 3H, H-18), 1.08 (s, 3H, H-19),
3.54 (m, 1H, H-3), 5.39 (d, 1H, J = 5.16 Hz, H-6), 7.15 (s, 1H,
AN@CHA), 1.10–2.56 (m, 19H).
2.7. 20-methyl-20H-Androst-16-eno[17,16-c]pyrazole-3b-ol (12b)
2. Experimental
Compound 10 (2.00 g, 6.3 mmol) was dissolved in anhydrous
ethanol (30 mL), and 40% methylhydrazine (1.45 g, 12.6 mmol)
was added dropwise to the solution. After being stirred for 1 h at
room temperature, the mixture was poured into ice water
(100 mL). Then the mixture was adjusted to pH 7 by 2 M HCl to
afford a white solid. Recrystallization from methanol gave 12b
(1.72 g, 83%). Mp 222–223 °C; ESI-MS m/z (%) 329.1 [M+H]+; 1H
NMR (400 MHz, CDCl3) d 0.86 (s, 3H, H-18), 0.97 (s, 3H, H-19),
3.60 (m, 1H, H-3), 3.81 (s, 3H, ANACH3), 6.93 (s, 1H, AN@CHA),
0.79–2.48 (m, 21H).
2.1. General
Melting points were measured on a SGW X-4 microscopy melt-
ing point apparatus without correction. 1H and 13C NMR spectral
data were recorded with a Varian 400 MHz spectrometer at
303 K using TMS as an internal standard. Mass spectra were
recorded on Agilent Technologies 1260 infinity LC/MS instrument,
and HRMS spectra were recorded on an Agilent Technologies
LC/MSD TOF instrument. Analytical and preparative TLC was per-
formed on silica gel HSGF/UV 254. The chromatograms were con-
ducted on silica gel (100–200 mesh) and visualized under UV
light at 254 and 365 nm.
2.8. 20-methyl-20H-Androsta-5,16-dieno[17,16-c]pyrazole-3b-ol (13b)
13b (1.80 g, 87%) was obtained starting from 11 (2.00 g,
6.3 mmol). Mp 208–210 °C; ESI-MS m/z (%) 327.2 [M+H]+; 1H
NMR (400 MHz, CDCl3) d 1.00 (s, 3H, H-18), 1.07 (s, 3H, H-19),
3.55 (m, 1H, H-3), 3.78 (s, 3H, ANACH3), 5.38 (d, 1H, J = 4.64 Hz,
H-6), 7.12 (s, 1H, AN@CHA), 1.11–2.47 (m, 18H).
2.2. 3-Phenylsulfonyl-4-hydroxylethoxyl-1,2,5-oxadiazole 2-Oxide (7)
The synthesis of the intermediate 7 from benzenethiol was
reported previously in ref [19].
2.9. 20-Phenyl-20H-androst-16-eno[17,16-c]pyrazole-3b-ol (12c)
2.3. 3b-Hydroxy-16-(hydroxymethylene)-5a-androstan-17-one (10)
Compound 10 (0.80 g, 2.5 mmol) was dissolved in anhydrous
ethanol (20 mL) and phenylhydrazine (0.54 g, 5.0 mmol) was
added dropwise to the solution. After being refluxed for 2 h, the
mixture was poured into ice water (100 mL). Then the mixture
was adjusted to pH 7 by 2 M HCl to afford a light yellow solid.
Recrystallization from methanol gave 12c (0.70 g, 72%). Mp 217–
219 °C; ESI-MS m/z (%) 391.2 [M+H]+; 1H NMR (400 MHz, CDCl3)
d 0.84 (s, 3H, H-18), 0.97 (s, 3H, H-19), 3.59 (m, 1H, H-3), 7.32 (t,
1H, J = 7.48 Hz, 7.32 Hz, ANAC6H5), 7.37 (s, 1H, AN@CHA), 7.42
(t, 2H, J = 7.56 Hz, 8.04 Hz, ANAC6H5), 7.49 (d, 2H, J = 8.0 Hz,
ANAC6H5), 0.76–2.56 (m, 21H).
Sodium ethoxide (2.12 g, 31.2 mmol) was added to a round-bot-
tomed flask and dissolved in CH2Cl2 (40 mL). Then epiandrosterone
8 (0.91 g, 3.12 mmol) and ethyl formate (3.8 mL, 46.8 mmol) were
added. The reaction mixture was stirred under nitrogen atmo-
sphere for 5 h at room temperature. Then the solvent was evapo-
rated and water (50 mL) was added. The aqueous phase was
acidified with 2 M HCl. Then the precipitated solid was filtered
and washed with water. Evaporation of residual water under
reduced pressure afforded 10 (0.79 g, 80%) which was sufficiently
pure for the next stage. Mp 220–221 °C; ESI-MS m/z (%) 319.1
[M+H]+; 1H NMR (400 MHz, CDCl3) d 0.73 (s, 3H, H-18), 0.75 (s,
3H, H-19), 4.42 (d, 1H, J = 4.31 Hz, H-3), 7.32 (s, 1H, H-10), 0.61–
2.42 (m, 22H).
2.10. 20-Phenyl-20H-androsta-5,16-dieno[17,16-c]pyrazole-3b-ol
(13c)
2.4. 3b-Hydroxy-16-(hydroxymethylene)androst-5-en-17-one (11)
13c (0.42 g, 86%) was obtained starting from 11 (0.40 g,
1.3 mmol). Mp 232–235 °C; ESI-MS m/z (%) 389.0 [M+H]+; 1H
NMR (400 MHz, CDCl3) d 1.05 (s, 3H, H-18), 1.08 (s, 3H, H-19),
3.53 (m, 1H, H-3), 5.39 (d, 1H, J = 5.12 Hz, H-6), 7.32 (t, 1H,
J = 7.32 Hz, 7.24 Hz, ANAC6H5), 7.38 (s, 1H, AN@CHA),7.42 (t,
2H, J = 7.48 Hz, 8.04 Hz, ANAC6H5), 7.50 (d, 2H, J = 7.60 Hz,
ANAC6H5), 0.89–2.56 (m, 18H).
11 (0.85 g, 86%) was obtained starting from dehydroepiandros-
terone 9 (0.9 g, 3.12 mmol). Mp 238–241 °C; ESI-MS m/z (%) 317.2
[M+H]+; 1H NMR (400 MHz, DMSO-d6) d 1.03 (s, 3H, H-18), 1.09 (s,
3H, H-19), 3.53 (m, 1H, H-3), 5.40 (s, 1H, H-6), 7.16 (s, 1H, H-10),
1.12–2.59 (m, 19H).

Y. Huang et al. / Steroids 101 (2015) 7–14
9
Ph
N
N
N
N
O
O
HO
HO
O
O
1
2
O
O
Fig. 1. The structures of compounds 1 and 2.
2.11. 20-(1,1-Dimethylethyl)-20H-androst-16-eno[17,16-c]pyrazole-
28.40, 24.03, 20.74, 18.10, 12.33; ESI-HRMS (m/z) [M+H]+ Calcd
3b-ol (12d)
for C28H34N4O5S: 539.2323, Found: 539.2327.
Compound 10 (2.00 g, 6.3 mmol) was dissolved in anhydrous
ethanol (30 mL) and tert-Butylhydrazine hydrochloride (1.56 g,
12.6 mmol) was added to the solution. After being refluxed for
1 h, the mixture was poured into ice water (100 mL). Then the mix-
ture was adjusted to pH 7 by 2 M NaOH aqueous solution to afford
a white solid. Recrystallization from ethyl acetate gave 12d (1.50 g,
64%). Mp 242–245 °C; ESI-MS m/z (%) 371.2 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 0.85 (s, 3H, H-18), 1.02 (s, 3H, H-19), 1.60 (s,
9H, A(CH3)3), 3.61 (m, 1H, H-3), 7.13 (s, 1H, AN@CHA), 0.76–
2.47 (m, 21H).
2.14. 3b-{[5-Oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yl]oxy}-20H-
androsta-5,16-dieno[17,16-c]pyrazole (15a) and 20-{[5-Oxido-4-
(phenylsulfonyl)-1,2,5-oxadiazol-3-yl]oxy}-20H-androsta-5,16-
dieno[17,16-c]pyrazole-3b-ol (15e)
15a (120 mg, 14%) and 15e (40 mg, 5%) were obtained starting
from 13a (500 mg, 1.6 mmol).
15a: Mp 118–121 °C; ESI-MS m/z (%) 537.2 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 1.04 (s, 3H, H-18), 1.16 (s, 3H, H-19), 4.71
(m, 1H, H-3), 5.48 (s, 1H, H-6), 7.16 (s, 1H, AN@CHA),7.63 (t, 2H,
J = 7.72 Hz, 7.68 Hz, ASO2C6H5), 7.76 (t, 1H, J = 7.16 Hz, 7.20 Hz,
ASO2C6H5), 8.06 (d, 2H, J = 8.0 Hz, ASO2C6H5), 0.86–2.60 (m,
2.12. 20-(1,1-Dimethylethyl)-20H-androsta-5,16-dieno[17,16-c]
pyrazole-3b-ol (13d)
18H); 13C NMR (101 MHz, CDCl3)
d 168.50, 158.52, 139.25,
138.34, 135.80, 129.84, 128.85, 123.50, 123.21, 121.91, 110.76,
82.14, 62.48, 50.61, 40.47, 38.03, 37.11, 36.88, 34.06, 32.16,
31.64, 30.40, 27.73, 24.24, 20.75, 19.57, 18.25, 14.38; ESI-HRMS
(m/z) [M+H]+ Calcd for C28H32N4O5S: 537.2166, Found: 537.2167.
15e: Mp 190–193 °C; ESI-MS m/z (%) 537.2 [M+H]+; 1H NMR
(400 MHz, CDCl3) d1.11 (s, 3H, H-18), 1.12 (s, 3H, H-19), 3.56 (m,
1H, H-3), 5.40 (s, 1H, H-6), 7.61 (t, 2H, J = 8.16 Hz, 7.52 Hz,
ASO2C6H5), 7.66 (s, 1H, AN@CHA), 7.75 (t, 1H, J = 7.56 Hz,
7.48 Hz, ASO2C6H5), 8.18 (d, 2H, J = 7.36 Hz, ASO2C6H5), 0.86–
2.71 (m, 18H); 13C NMR (101 MHz, CDCl3) d 174.29, 149.56,
141.34, 138.16, 135.90, 129.65, 126.99, 124.87, 121.21, 113.52,
71.86, 61.79, 50.61, 42.47, 40.91, 37.37, 36.99, 33.81, 32.16,
31.82, 31.55, 30.40, 24.36, 22.94, 20.73, 19.66, 18.10, 14.38; ESI-
HRMS (m/z) [M+H]+ Calcd for C28H32N4O5S: 537.2166, Found:
537.2163.
13d (1.85 g, 80%) was obtained starting from 11 (2.00 g,
6.3 mmol). Mp 254–255 °C; ESI-MS m/z (%) 369.2 [M+H]+; 1H
NMR (400 MHz, CDCl3) d 1.08 (s, 6H, H-18, 19), 1.58 (s, 9H,
A(CH3)3), 3.54 (m, 1H, H-3), 5.40 (d, 1H, J = 4.64 Hz, H-6), 7.15 (s,
1H, AN@CHA), 1.06–2.50 (m, 18H).
2.13. 3b-{[5-Oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yl]oxy}-20H-
androst-16-eno[17,16-c]pyrazole (14a) and 20-{[5-Oxido-4-(phenyl-
sulfonyl)-1,2,5-oxadiazol-3-yl]oxy}-20H-androst-16-eno[17,16-c]pyra-
zole-3b-ol (14e)
Compound 12a (0.20 g, 0.6 mmol) was added to a stirred
solution of compound 6 (0.23 g, 0.6 mmol) in the presence of
8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.19 g, 1.28 mmol) in
CH2Cl2 (15 mL). The reaction mixture was stirred at room temper-
ature for 3 h and then washed with water (3 ꢀ 30 mL). The organic
layer was dried with anhydrous Na2SO4. Then the solvent was
removed under reduced pressure, and the residue was purified
by column chromatography (PE/EtOAc = 10:1–5:1) to yield 14a
(44 mg, 13%) and 14e (35 mg, 10%) as white solid, respectively.
14a: Mp 128–131 °C; ESI-MS m/z (%) 539.2 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 0.94 (s, 3H, H-18), 0.99 (s, 3H, H-19), 4.79 (m,
1H, H-3), 7.14 (s, 1H, AN@CHA), 7.61 (t, 2H, J = 7.72 Hz, 7.48 Hz,
ASO2C6H5), 7.75 (t, 1H, J = 7.26 Hz, 7.48 Hz, ASO2C6H5), 8.05 (d,
2H, J = 8.02 Hz, ASO2C6H5), 0.83–2.55 (m, 21H); 13C NMR
2.15. 3b-{[5-Oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yl]oxy}-20H-
20-methyl-androst-16-eno[17,16-c]pyrazole (14b)
14b (30 mg, 9%) was obtained starting from 12b (200 mg,
0.6 mmol).
14b: Mp 177–180 °C; ESI-MS m/z (%) 553.2 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 0.95 (s, 3H, H-18), 0.99 (s, 3H, H-19), 3.80 (s,
3H, ANACH3), 4.79 (m, 1H, H-3), 7.14 (s, 1H, AN@CHA), 7.61 (t,
2H, J = 8.04 Hz, 7.72 Hz, ASO2C6H5), 7.76 (t, 1H, J = 7.48 Hz,
7.56 Hz, ASO2C6H5), 8.06 (d, 2H, J = 7.32 Hz, ASO2C6H5), 0.84–
2.49 (m, 20H); 13C NMR (101 MHz, CDCl3) d 158.61, 157.47,
138.35, 135.78, 133.94, 129.81, 128.84, 124.01, 110.77, 82.02,
62.73, 54.61, 44.90, 41.64, 36.99, 36.60, 36.00, 34.29, 34.11,
33.76, 31.53, 29.94, 28.52, 27.42, 24.53, 20.91, 17.60, 12.57; ESI-
HRMS (m/z) [M+Na]+ Calcd for C29H36N4O5S: 575.2299, Found:
575.2295.
(101 MHz, CDCl3)
d 168.42, 158.41, 138.12, 135.52, 129.56,
128.56, 122.83, 121.55, 110.51, 81.95, 62.17, 54.64, 53.44, 44.56,
40.43, 36.40, 35.75, 34.32, 33.94, 33.51, 31.45, 29.67, 28.38,
27.26, 23.91, 20.83, 18.26, 12.30; ESI-HRMS (m/z) [M+H]+ Calcd
for C28H34N4O5S: 539.2323, Found: 539.2327.
14e: Mp 181–184 °C; ESI-MS m/z (%) 539.2 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 0.90 (s, 3H, H-18), 1.09 (s, 3H, H-19), 3.62 (m,
1H, H-3), 7.60 (t, 2H, J = 7.92 Hz, 7.72 Hz, ASO2C6H5), 7.65 (s, 1H,
AN@CHA), 7.75 (t, 1H, J = 7.36 Hz, 7.48 Hz, ASO2C6H5), 8.18 (d,
2H, J = 7.64 Hz, ASO2C6H5), 0.85–2.68 (m, 21H); 13C NMR
2.16. 3b-{[5-Oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yl]oxy}-20H-
20-phenyl-androst-16-eno[17,16-c]pyrazole (14c)
(101 MHz, CDCl3)
d 174.17, 149.20, 137.91, 135.62, 129.37,
129.29, 126.77, 124.52, 113.24, 76.68, 71.13, 61.41, 54.65, 44.85,
40.88, 38.05, 36.79, 35.73, 34.48, 33.61, 31.56, 31.41, 29.67,
14c (40 mg, 12%) was obtained starting from 12c (200 mg,
0.5 mmol).

10
Y. Huang et al. / Steroids 101 (2015) 7–14
14c: Mp 247–248 °C; ESI-MS m/z (%) 615.2 [M+H]+; 1H NMR
2.20. 3b-{[5-Oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yl]oxy}-20H-
20-(1,1-dimethylethyl)-androsta-5,16-dieno[17,16-c]pyrazole (15d)
(400 MHz, CDCl3) d 0.93 (s, 3H, H-18), 1.05 (s, 3H, H-19), 4.78 (m,
1H, H-3), 7.32 (t, 1H, J = 7.24 Hz, 7.32 Hz, ANAC6H5), 7.37 (s, 1H,
AN@CHA), 7.42 (t, 2H, J = 7.48 Hz, 8.12 Hz, ANAC6H5), 7.49 (d,
2H, J = 7.36 Hz, ANAC6H5), 7.62 (t, 2H, J = 8.12 Hz, 7.60 Hz,
ASO2C6H5), 7.75 (t, 1H, J = 7.40 Hz, 7.52 Hz, ASO2C6H5), 8.06 (d,
2H, J = 7.28 Hz, ASO2C6H5), 0.82–2.57 (m, 20H); 13C NMR
15d (52 mg, 17%) was obtained starting from 13d (188 mg,
0.54 mmol).
15d: Mp 219–222 °C; ESI-MS m/z (%) 593.2 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 1.08 (s, 3H, H-18), 1.14 (s, 3H, H-19), 1.59 (s,
9H, -(CH3)3), 4.71 (m, 1H, H-3), 5.48 (s, 1H, H-6), 7.15 (s, 1H,
AN@CHA), 7.63 (t, 2H, J = 7.80 Hz, 7.96 Hz, ASO2C6H5), 7.76 (t,
1H, J = 7.36 Hz, 7.56 Hz, ASO2C6H5), 8.07 (d, 2H, J = 8.36 Hz,
ASO2C6H5), 0.87–2.60 (m, 17H); 13C NMR (101 MHz, CDCl3) d
158.51, 156.19, 139.02, 138.33, 135.81, 131.16, 129.84, 128.85,
126.37, 123.57, 110.76, 82.05, 63.10, 59.56, 49.94, 43.88, 37.96,
36.95, 36.80, 31.43, 31.12, 30.91, 27.66, 23.92, 21.10, 19.50,
17.45; ESI-HRMS (m/z) [M+H]+ Calcd for C32H40N4O5S: 593.2792,
Found: 593.2787.
(101 MHz, CDCl3)
d 158.34, 156.98, 140.47, 138.09, 135.49,
135.20, 129.54, 128.86, 128.56, 127.11, 126.09, 123.47, 110.50,
81.77, 62.84, 54.21, 44.60, 42.63, 36.28, 35.66, 34.90, 34.19,
33.49, 31.28, 29.67, 28.27, 27.13, 24.08, 20.69, 17.76, 12.27; ESI-
HRMS (m/z) [M+H]+ Calcd for C34H38N4O5S: 615.2636, Found:
615.2631.
2.17. 3b-{[5-Oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yl]oxy}-20H-
20-(1,1-dimethylethyl)-androst-16-eno[17,16-c]pyrazole (14d)
14d (100 mg, 13%) was obtained starting from 12d (0.5 g,
1.35 mmol).
2.21. 3b-O-succinyl-20H-20-methyl-androsta-5,16-dieno[17,16-
c]pyrazole (16b)
14d: Mp 222–224 °C; ESI-MS m/z (%) 595.2 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 0.95 (s, 3H, H-18), 1.04 (s, 3H, H-19), 1.58 (s,
9H, A(CH3)3), 4.80 (m, 1H, H-3), 7.14 (s, 1H, AN@CHA), 7.62 (t,
2H, J = 8.04 Hz, 7.76 Hz, ASO2C6H5), 7.76 (t, 1H, J = 7.52 Hz,
7.48 Hz, ASO2C6H5), 8.06 (d, 2H, J = 7.40 Hz, ASO2C6H5), 0.83–
2.48 (m, 20H); 13C NMR (101 MHz, CDCl3) d 158.35, 155.65,
138.10, 135.50, 131.13, 129.54, 128.57, 126.03, 110.50, 81.81,
62.81, 58.85, 53.92, 44.55, 43.72, 36.88, 36.31, 35.59, 34.38,
33.46, 31.25, 30.64, 28.32, 27.14, 23.60, 21.14, 17.36, 12.26; ESI-
HRMS (m/z) [M+H]+ Calcd for C32H42N4O5S: 595.2949, Found:
595.2950.
Compound 13b (0.35 g, 1.1 mmol), succinic anhydride (0.69 g,
5.4 mmol) and DMAP (0.13 g, 1.1 mmol) were dissolved in anhy-
drous Py (20 mL). After being refluxed for 4 h, the the mixture
was poured into ice water (100 mL). The mixture was adjusted to
pH 3 by 2 M HCl to afford a brown solid. The crude product was
purified by column chromatography (PE/EtOAc = 5:1) to yield
16b (0.35 g, 77%).
16b: Mp 225–231 °C; ESI-MS m/z (%) 427.2 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 1.05 (s, 3H, H-18), 1.08 (s, 3H, H-19), 2.62 (m,
4H, ACOCH2CH2COA), 3.92 (s, 3H, ANACH3), 4.63 (m, 1H, H-3),
5.39 (d, 1H, J = 4.20 Hz, H-6), 7.01 (s, 1H, AN@CHA), 1.58–2.39
(m, 18H).
2.18. 3b-{[5-Oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yl]oxy}-20H-
20-methyl-androsta-5,16-dieno[17,16-c]pyrazole (15b)
2.22. 3b-O-succinyl-20H-20-phenyl-androsta-5,16-dieno[17,16-c]
pyrazole (16c)
15b (50 mg, 7%) was obtained starting from 13b (400 mg,
1.2 mmol).
15b: Mp 192–193 °C; ESI-MS m/z (%) 551.2 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 1.02 (s, 3H, H-18), 1.14 (s, 3H, H-19), 3.79 (s,
3H, ANACH3), 4.71 (m, 1H, H-3), 5.46 (s, 1H, H-6), 7.12 (s, 1H,
AN@CHA), 7.62 (t, 2H, J = 8.0 Hz, 7.68 Hz, ASO2C6H5), 7.76 (t, 1H,
J = 7.56 Hz, 7.48 Hz, ASO2C6H5), 8.06 (d, 2H, J = 7.44 Hz,
ASO2C6H5), 0.85–2.64 (m, 17H); 13C NMR (101 MHz, CDCl3) d
158.25, 158.81, 138.83, 138.07, 135.54, 133.05, 129.56, 128.59,
123.26, 110.50, 81.78, 62.55, 50.16, 41.03, 37.73, 36.79, 36.58,
33.84, 31.90, 31.41, 31.15, 30.13, 27.41, 24.31, 22.67, 20.31,
19.26, 17.10, 14.12; ESI-HRMS (m/z) [M+H]+ Calcd for
16c (1.07 g, 85%) was obtained starting from 13c (1.00 g,
2.6 mmol).
16c: Mp 222–226 °C; ESI-MS m/z (%) 489.8 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 1.06 (s, 3H, H-18), 1.08 (s, 3H, H-19), 2.62 (m,
4H, ACOCH2CH2COA), 4.63 (m, 1H, H-3), 5.42 (s, 1H, H-6), 7.33
(t, 1H, J = 7.16 Hz, 7.40 Hz, ANAC6H5), 7.40 (s, 1H, AN@CHA),
7.43 (t, 2H, J = 7.44 Hz, 8.08 Hz, ANAC6H5), 7.50 (d, 2H,
J = 7.56 Hz, ANAC6H5), 0.83–2.67 (m, 18H).
2.23. 3b-O-succinyl-20H-20-(1,1-dimethylethyl)-androsta-5,16-dieno
[17,16-c]pyrazole (16d)
C29H34N4O5S: 551.2323, Found: 551.2317.
2.19. 3b-{[5-Oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yl]oxy}-20H-
20-phenyl-androsta-5,16-dieno[17,16-c]pyrazole (15c)
16d (0.40 g, 90%) was obtained starting from 13d (0.35 g,
0.95 mmol).
16d: Mp 253–256 °C; ESI-MS m/z (%) 469.2 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 1.07 (s, 3H, H-18), 1.12 (s, 3H, H-19), 1.72 (s,
9H, AC(CH3)3), 2.63 (m, 4H, ACOCH2CH2COA), 4.62 (m, 1H, H-3),
5.41 (s, 1H, H-6), 7.54 (s, 1H, AN@CHA), 1.56–2.42 (m, 18H).
15c (10 mg, 13%) was obtained starting from 13c (50 mg,
0.13 mmol).
15c: Mp 237–239 °C; ESI-MS m/z (%) 613.2 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 1.10 (s, 3H, H-18), 1.12 (s, 3H, H-19), 4.70 (m,
1H, H-3), 5.47 (s, 1H, H-6), 7.33 (t, 1H, J = 7.16 Hz, 7.36 Hz,
ANAC6H5), 7.39 (s, 1H, AN@CHA), 7.43 (t, 2H, J = 7.48 Hz,
8.04 Hz, ANAC6H5), 7.50 (d, 2H, J = 7.44 Hz, ANAC6H5), 7.62 (t,
2H, J = 7.88 Hz, 7.84 Hz, ASO2C6H5), 7.76 (t, 1H, J = 7.48 Hz,
7.44 Hz, ASO2C6H5), 8.06 (d, 2H, J = 7.36 Hz, ASO2C6H5), 0.88–
2.60 (m, 17H); 13C NMR (101 MHz, CDCl3) d 158.24, 156.86,
140.46, 138.86, 138.06, 135.63, 135.20, 129.56, 128.89, 128.59,
127.14, 126.09, 123.47, 123.21, 110.49, 81.77, 62.94, 49.95, 42.36,
37.72, 36.73, 36.52, 34.71, 31.17, 30.61, 29.67, 27.38, 24.13,
20.35, 19.23, 17.56; ESI-HRMS (m/z) [M+H]+ Calcd for
2.24. 3b-{[4-{[5-Oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yl]oxy}
ethanoxy-4-oxobutanoyl]oxy}-20H -20-methyl-androsta-5,16-dieno
[17,16-c]pyrazole (17b)
DMAP (50 mg, 0.42 mmol) and compound
7
(100 mg,
0.35 mmol) were added to a mixture solution of compound 16b
(150 mg, 0.35 mmol) and DCC (90 mg, 0.42 mmol) in CH2Cl2
(15 mL). The reaction mixture was stirred at room temperature
for 24 h. Filtration and removal of the solvent in vacuo afforded
the crude product, which was subsequently purified by column
chromatography using (PE/EtOAc = 5:1) to give 17b (110 mg, 45%).
C34H36N4O5S: 613.2479, Found: 613.2476.

Y. Huang et al. / Steroids 101 (2015) 7–14
11
17b: Mp 62–64 °C; ESI-MS m/z (%) 695.2 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 1.01 (s, 3H, H-18), 1.08 (s, 3H, H-19), 2.67 (m,
4H, ACOCH2CH2COA), 3.83 (s, 3H, ANACH3), 4.53–4.64 (m, 5H,
H-3, AfuroxanAOCH2CH2), 5.40 (s, 1H, H-6), 6.95 (s, 1H,
AN@CHA), 7.63 (t, 2H, J = 7.52 Hz, 7.80 Hz, ASO2C6H5), 7.76 (t,
1H, J = 7.24 Hz, 7.40 Hz, ASO2C6H5), 8.07 (d, 2H, J = 7.84 Hz,
ASO2C6H5), 0.86–2.55 (m, 17H); 13C NMR (101 MHz, CDCl3) d
172.13, 171.53, 168.93, 158.69, 139.93, 138.06, 135.66, 129.69,
128.65, 124.28, 122.26, 121.95, 110.41, 74.40, 68.87, 62.12, 61.39,
50.51, 40.47, 38.61, 38.09, 36.88, 36.86, 34.01, 31.46, 30.79,
29.70, 29.31, 29.02, 27.73, 24.29, 20.56, 19.29, 18.17; ESI-HRMS
(m/z) [M+Na]+ Calcd for C35H42N4O9S: 717.2565, Found: 717.2580.
The solvent was removed under reduced pressure and the residue
was purified by column chromatography (CH2Cl2/MeOH = 50:1) to
yield 18c (0.5 g, 13%) as a white solid.
18c: Mp 195–198 °C; ESI-MS m/z (%) 533.4 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 1.06 (s, 3H, H-18), 1.08 (s, 3H, H-19), 2.65 (m,
4H, ACOCH2CH2COA), 3.68 (t, 2H, J = 5.84 Hz, 5.64 Hz, HOCH2A),
4.35 (t, 2H, J = 5.60 Hz, 5.84 Hz, HOCH2CH2COOA), 4.63 (m, 1H,
H-3), 5.42 (d, 1H, J = 4.84 Hz, H-6), 7.32 (t, 1H, J = 7.40 Hz,
7.24 Hz, ANAC6H5), 7.38 (s, 1H, AN@CHA), 7.42 (t, 2H,
J = 7.52 Hz, 8.0 Hz, ANAC6H5), 7.50 (d, 2H, J = 7.84 Hz, ANAC6H5),
1.06–2.59 (m, 18H); 13C NMR (101 MHz, CDCl3) d 172.58, 171.91,
157.03, 140.53, 139.79, 135.19, 128.89, 127.17, 126.16, 123.57,
122.23, 74.35, 66.33, 63.04, 61.11, 50.10, 42.43, 38.02, 36.77,
34.83, 31.21, 30.74, 29.61, 27.66, 24.18, 20.38, 19.22, 17.57; ESI-
HRMS (m/z) [M+H]+ Calcd for C32H40N2O5: 533.3010, Found:
533.3014.
2.25. 3b-{[4-{[5-Oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-yl]oxy}
ethanoxy-4-oxobutanoyl]oxy}-20H-20-phenyl-androsta-5,16-dieno
[17,16-c]pyrazole (17c)
17c (124 mg, 53%) was obtained starting from 16c (150 mg,
0.31 mmol) and 7 (80 mg, 0.30 mmol).
2.28. In vitro antiproliferative assay
17c: Mp 103–106 °C; ESI-MS m/z (%) 757.4 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 1.05 (s, 3H, H-18), 1.08 (s, 3H, H-19), 2.66 (m,
The in vitro antiproliferation of the chemical compounds was
measured by the MTT reagent. Briefly, 5 ꢀ 103 cells in 100
lL of
4H,
ACOCH2CH2COA),
4.51–4.64
(m,
5H,
H-3,
medium per well were plated in 96-well plates. After being incu-
bated for 24 h, the cells were treated with different concentration
of tested compound or DMSO (as negative control) for 48 h. Then
AfuroxanAOCH2CH2), 5.41 (s, 1H, H-6), 7.33 (t, 1H, J = 7.32 Hz,
7.28 Hz, ANAC6H5), 7.38 (s, 1H, AN@CHA), 7.43 (t, 2H,
J = 7.52 Hz, 8.12 Hz, ANAC6H5), 7.50 (d, 2H, J = 7.44 Hz,
ANAC6H5), 7.63 (t, 2H, J = 8.04 Hz, 7.68 Hz, ASO2C6H5), 7.76 (t,
1H, J = 7.52 Hz, 7.48 Hz, ASO2C6H5), 8.07 (d, 2H, J = 7.36 Hz,
ASO2C6H5), 0.86–2.58 (m, 17H); 13C NMR (101 MHz, CDCl3) d
172.39, 171.79, 158.90, 157.62, 140.18, 140.00, 138.21, 135.92,
135.02, 129.94, 129.23, 128.88, 127.73, 126.40, 123.89, 122.39,
110.64, 74.50, 69.09, 63.19, 61.62, 50.23, 42.71, 38.23, 36.96,
34.90, 31.40, 30.91, 29.94, 29.50, 29.22, 27.87, 24.42, 20.55,
19.46, 17.82; ESI-HRMS (m/z) [M+H]+ Calcd for C40H44N4O9S:
757.2902, Found: 757.2919.
the medium with compound or DMSO was replaced with 200 lL
of fresh medium containing 10% MTT (5 mg/mL in PBS) in each
well and incubated at 37 °C for 4 h. Last, the MTT-containing med-
ium was discarded and150 lL of DMSO per well was added to dis-
solve the formazan crystals newly formed. Absorbance of each well
was determined by a microplate reader (Synergy H4, Bio-Tek) at a
570 nm wavelength. The inhibition rates of proliferation were cal-
culated with the following equation:
À
Á
Inhibition ratio ð%Þ¼ ODDMSO ꢁODcompd =ðODDMSO ꢁODblankÞꢀ100
2.26. 3b-{[4-{[5-Oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-
yl]oxy}ethanoxy-4-oxobutanoyl]oxy}-20H-20-(1,1-dimethylethyl)-
androsta-5,16-dieno[17,16-c]pyrazole (17d)
The concentrations of the compounds that inhibited cell growth
by 50% (IC50) were calculated using GraphPad Prism, version 6.0.
17d (117 mg, 50%) was obtained starting from 16d (150 mg,
0.32 mmol) and 7 (920 mg, 0.32 mmol).
2.29. VEGF bioactivity detection
17d: Mp 154–156 °C; ESI-MS m/z (%) 737.2 [M+H]+; 1H NMR
(400 MHz, CDCl3) d 1.06 (s, 6H, H-18,19), 1.59 (s, 9H, AC(CH3)3),
2.67 (m, 4H, ACOCH2CH2COA), 4.52–4.64 (m, 5H, H-3,
AfuroxanAOCH2CH2), 5.41 (s, 1H, H-6), 7.17 (s, 1H, AN@CHA),
7.63 (t, 2H, J = 8.0 Hz, 7.64 Hz, ASO2C6H5), 7.76 (t, 1H, J = 7.56 Hz,
7.44 Hz, ASO2C6H5), 8.07 (d, 2H, J = 7.48 Hz, ASO2C6H5), 1.06–
2.50 (m, 17H); 13C NMR (101 MHz, CDCl3) d 172.13, 171.53,
158.69, 155.71, 139.68, 138.06, 135.66, 131.16, 129.70, 128.65,
126.12, 122.35, 110.42, 74.33, 68.87, 63.00, 61.39, 58.93, 49.83,
49.13, 43.54, 38.00, 36.83, 36.78, 36.72, 33.95, 31.93, 31.23,
30.95, 30.68, 29.70, 29.31, 29.02, 27.68, 25.63, 24.95, 23.69,
20.86, 19.22, 17.32; ESI-HRMS (m/z) [M+Na]+ Calcd for
The single human breast cancer cell MDA-MB-231 suspension
in 1640 medium with 10% foetal bovine serum at a concentration
of 5 ꢀ 103 cells in 100
lL of medium per well were plated in 96-
well plates. After incubated for 24 h, the cells were treated with
different concentration of tested compound or DMSO (as negative
control) at 37 °C with 5% CO2 for 48 h. The IC50 was obtained by the
MTT assay as described before. Conditioned medium from each
sample was then collected and stored at ꢁ20 °C. The VEFG inhibi-
tory rate was detected using VEGF ELISA kit (R & D Systems,
Minneapolis, MN). The EC50 was obtained by calculation from the
diagram of inhibitory rate vs. sample concentration.
C38H48N4O9S: 759.3034, Found: 759.3053.
2.30. Capillary-like tube formation assay
2.27. 3b-{[4-(2-Hydroxyl-ethanoxy)-4-oxobutanoyl]oxy}-20H-20-
phenyl-androsta-5,16-dieno[17,16-c]pyrazole (18c)
Capillary-like tube formation assay was performed by following
the procedure published previously. Briefly, 70 lL per well
Compound 16c (3.50 g, 7.2 mmol) was dissolved in anhydrous
CH2Cl2 (40 mL). Oxalyl chloride (2.45 mL, 25.2 mmol) was added
dropwise at 0 °C to a stirred solution. After being stirred for 3 h,
ethylene glycol (7.5 mL, 132.2 mmol) was added dropwise to the
solution. After being stirred for 3 h, water (5 mL) was added to
quench the reaction. Then the mixture was washed with water
(3 ꢀ 20 mL). The organic layer was dried with anhydrous Na2SO4.
Matrigel (Corning, NY) was added to 96-well plates and incubated
at 37 °C for 30 min to allow gelation to occur. HUVECs were added
to the top of the gel at a density of 3 ꢀ 104 cells/well in the pres-
ence of tested compound or vehicle control (DMSO). Cells were
incubated at 37 °C with 5% CO2 overnight, and pictures were cap-
tured with a CCD Sensicam camera mounted on a Olympus
inverted microscope.

12
Y. Huang et al. / Steroids 101 (2015) 7–14
3.2. Biological evaluations
3. Results
3.1. Chemistry
The thirteen newly synthesized hybrids (14a–e, 15a–e, 17b–d)
were evaluated for their cytotoxic effects against the following five
cell lines: MDA-MB-231 (human breast cancer cell line), HCC1806
(tamoxifen resistant human breast cancer cell line), SKOV-3
(human ovary cancer cell line), DU145 (human prostate cancer cell
line), and HUVEC (umbilical vein endothelium cell line). As shown
in Table 1, most target compounds displayed better anti-prolifera-
tive activity than that of control compound phenylsulfonylfuroxan
Using epiandrosterone 8 and dehydroepiandrosterone 9 as the
starting materials, via C-16 formylation, condensation with mono-
substituted hydrazines, esterification of C-3 hydroxyl group with
succinic anhydride, and coupling with phenylsulfonyl-substituted
furoxan, thirteen different 16,17-pyrazo-annulated steroidal
derivatives were synthesized.
6 (IC50 values of 0.65–2.97
d) had significant anti-proliferative effects with the IC50 values of
0.0014–0.34 M against MDA-MB-231, 0.0034–0.70 M against
SKOV-3, 0.02–0.36 M against DU145, and had considerable cyto-
toxicity for the tamoxifen resistant cancer cell line HCC1806 in
1.03–2.22 M values. Among them, compounds 17b–d exhibited
activities in the nanomolar range. In particular, compound 17c
was the most potent molecule. It not only inhibited the prolifera-
tion of MDA-MB-231, SKOV-3 and DU145 with IC50 values of 1.4,
3.4, and 20.0 nM, respectively, but also had a strong cytotoxicity
lM). Seven of them (14a,b, 15a,b, 17b–
As Scheme 1 depicted, phenylsulfonyl-substituted furoxans 6
and 7 were synthesized as described previously. Commercial avail-
able compounds 8 and 9 were reacted with ethyl formate to give
16-formylepiandrosterone (10) and 16-formyldehydroepiandros-
terone (11), respectively. Compound 10 was treated with different
monosubstituted hydrazines to give compounds 12a-d in 64–86%
yield. Compounds 13a–d were prepared in the same way from
compound 11 in 80–92% yield. Then phenylsulfonyl-substituted
furoxan 6 was introduced into compounds 12a–d and 13a–d in
the presence of 8-diazabicyclo[5.4.0]undec-7-ene (DBU) to give
target compounds 14a–d, 15a–d and two byproducts (14e, 15e),
respectively. Furthermore, the mixture of the compounds 13b–d,
succinic anhydride and catalytic amount 4-N,N-dimethylaminopy-
ridine (DMAP) were refluxed in dry pyridine to give compounds
16b–d in 77–90% yield, which esterified with 7 in the presence
of dicyclohexylcarbodiimide (DCC)/DMAP to generate compounds
17b–d in 45–53% yield. The compound 18c was synthesized via
the esterification of 16c with ethylene glycol in the CH2Cl2 solution
containing oxalyl chloride.
l
l
l
l
for the drug resistance cell line HCC1806 with IC50 value of 1.03 lM.
Moreover, angiogenesis is necessary for tumor growth and
metastasis, which makes it a promising target for cancer treatment.
Human umbilical vein endothelial cells (HUVECs) are normal cells
useful for assessing antiangiogenic potential [14]. HUVEC was also
screened in company with the previous four cancer cell lines. Ten
of the thirteen compounds showed potent activity with IC50 values
less than 1
lM. Compounds 17b,d demonstrated activity against
HUVEC with IC50 of 0.12 and 0.05
l
M, respectively. Compound 17c
SH
O
O
SO2Ph
SO2Ph
O
O
O
O
S
S
a
b
c
d
N
O
N
O
HO
O
HO
SO2Ph
O
N
N
3
5
6
4
7
OH
O
O
N
N
R
N
SO2Ph
R
N
O
O
N
N
N
N
N
OH
SO2Ph
O
g
f
e
e
O
N
O
HO
HO
HO
HO
N
O
8
9
10
12a-12d
14a-14d
15a-15d
14e
15e
O
N
N
R
N
SO2Ph
R
N
O
N
N
N
OH
SO2Ph
O
g
f
O
N
O
HO
HO
HO
N
HO
11
13a-13d
h
Ph
R
R
N
N
N
N
N
N
O
N
SO2Ph
j
i
OH
O
O
O
7
O
O
HO
O
O
O
O
O
N
18c
16b-16d
17b-17d
O
O
O
12a, 13a, 14a, 15a:
R=H ;
12b, 13b, 14b, 15b, 16b, 17b:
R=CH3
;
12c, 13c, 14c, 15c, 16c, 17c: R=Ph ;
12d, 13d, 14d, 15d, 16d, 17d: R=C(CH3)3
Scheme 1. Synthetic routes of target compoundsa. aReagents and conditions: (a) ClCH2COOH, NaOH (aq.), 140 °C, 2 h; (b) 30% H2O2, AcOH, r.t., 3.5 h; (c) fuming HNO3, 90 °C,
4 h; (d) Ethanediol, 25% NaOH (aq.), r.t., 1 h; (e) ethyl formate, C2H5ONa, CH2Cl2, r.t.; (f) NH2NHR, C2H5OH, r.t. or reflux; (g) furoxan 6, DBU, CH2Cl2, r.t.; (h) succinic anhydride,
DMAP, Py, reflux; (i) DCC, DMAP, CH2Cl2, r.t.; (j) (COCl)2, 0 °C, ethanediol, pyridine, r.t.

Y. Huang et al. / Steroids 101 (2015) 7–14
13
Table 1
Therefore, five compounds (14a, 15a, 17b–d) possessing better
activity were selected to test for VEGF inhibitory activity in MDA-
MB-231 using 2-Methoxyestradiol (2-ME) and furoxan 6 as refer-
ences. As shown in Table 2, three compounds (15a, 17b,c) effec-
tively reduced the secretion of VEGF in MDA-MB-231 with the
EC50 values of 40, 10 and 0.3 nM, respectively, and also exhibited
obviously better TI (IC50/EC50) values of 7.00, 9.00 and 4.67 com-
pared with 2-ME (TI = 3.57) and furoxan (TI = 1.10). Meanwhile,
in order to confirm whether phenylsulfonylfuroxan moiety played
an important role in these novel compounds on VEGF inhibitory
activity, compound 18c was also synthesized as a control which
Antiproliferation activities of 14a–e, 15a–e, 17b–d.
Compound MDA-MB-
231 (IC50
M)a
HCC1806
(IC50
M)a
SKOV-3
(IC50
M)a
DU145
(IC50
M)a
HUVEC
(IC50
M)a
,
,
,
,
,
l
l
l
l
l
14a
14b
14c
14d
14e
15a
15b
15c
15d
15e
17b
17c
17d
Furoxan
(6)
0.16
0.34
12.36
2.39
1.04
0.28
0.26
2.43
1.03
1.32
0.09
0.0014
0.04
1.62
1.82
2.04
>60
20.11
1.97
2.22
1.71
>60
19.55
2.46
1.80
1.03
1.34
2.97
0.40
0.70
20.35
3.88
0.89
0.61
0.50
6.72
2.31
1.58
0.19
0.0034
0.15
2.25
0.11
0.36
15.73
2.46
1.08
0.22
0.25
2.47
1.22
1.37
0.18
0.02
0.10
0.65
0.03
0.26
6.79
1.60
0.42
0.17
0.17
1.47
0.66
0.49
0.12
indeed showed worse activity with the EC50 value of 2.71 lM
and the TI value of 2.50 than that of the hybrids. These results elu-
cidated that the hybrids of 16,17-pyrazo-annulated steroidal
derivatives and furoxan were more favorable than the individuals
in inhibiting the secretion of VEGF.
0.0035
0.05
1.42
Furthermore, based on the above preliminary results of VEGF
inhibitory activity, compounds 17b,c were selected to perform cap-
illary-like tube formation assay to investigate their inhibition of
angiogenesis in vitro with Subitinib as a positive control. As dis-
played in Fig. 2, HUVEC cells formed a complete network structure
overnight with serum stimulation. Then the tubule formation was
slightly suppressed in a concentration of 23 nM (1/5 IC50) of 17b
and 0.7 nM (1/5 IC50) of 17c. And 17b with the concentration of
58 nM (1/2 IC50) and 17c with 1.75 nM (1/2 IC50) almost disrupted
the formation of tube structures. All above phenomena suggested
that the anti-angiogenesis mechanism of these derivatives of ster-
oidal scaffold coupling with furoxan might be related to the inhibi-
tion of secretion of VEGF.
According to the results mentioned above, the preliminary
structure–activity relationship (SAR) of the hybrids can be inferred.
First, the modification at the 20-position with H, methyl, phenyl
and tertiary butyl showed that the smaller group such as hydrogen
atom and methyl substitution (14a,b, 15a,b) were more favorable
for anti-proliferation activity. Meanwhile, the coupling site of
16,17-pyrazo-annulated steroidal skeleton with furoxan had a
slight impact on the cytotoxic potency of the derivatives. The com-
pounds 14a and 15a obtained by coupling phenylsulfonylfuroxan
to the 3-OH of the steroidal skeleton were more active than the
two byproducts 14e and 15e obtained by coupling furoxan to the
20-position. Second, the linker between steroidal skeleton and
a
The data are the mean of triplicate determinations; IC50 is the concentration of
sample for 50% cell growth inhibitory rate.
Table 2
VEGF inhibitory activities of 14a, 15a, 17b–d.
b
Compound
IC50
(l
M)a
VEGF EC50
(l
M)a
TI (IC50/EC50)
14a
15a
17b
17c
17d
18c
Furoxan (6)
2-ME
0.16
0.28
0.09
0.0014
0.04
6.78
1.62
0.25
0.06
0.04
0.01
0.0003
0.01
2.71
1.47
0.07
2.67
7.00
9.00
4.67
4.00
2.50
1.10
3.57
a
The data are the mean of triplicate determinations.
EC50 is the concentration of sample for 50% VEGF inhibitory rate; IC50 is the
b
concentration of sample for 50% cell growth inhibitory rate; TI (therapeutic
index) = IC50/EC50
.
exhibited the highest potency with an IC50 value of 3.5 nM in the
assay. The results implied that these new compounds might also
be active on angiogenesis targets like vascular endothelial growth
factor (VEGF) or its receptor (VEGFR) and encouraged us to further
explore their pharmacological mechanism in the following work.
Fig. 2. Capillary-like tube formation assay. (A) Tube formation for the vehicle control. HUVECs formed robust tube structures when they were only treated with vehicle
(DMSO). (B) Tubule formation was slightly suppressed with 0.70 nM 17c treatment. (C) Tubule formation was significantly suppressed with 1.75 nM 17c treatment. (D)
Tubule formation was slightly suppressed with 23 nM 17b treatment. (E) Tubule formation was significantly suppressed with 58 nM 17b treatment. (F) Sunitinib was used as
a positive control.

14
Y. Huang et al. / Steroids 101 (2015) 7–14
furoxan was crucial to preserve the strong anticancer activity. For
example, 17b–d with a linker in 3-position showed stronger anti-
proliferation activities against all four tested cancer cell lines than
15b–d, respectively. The activities of compound 17c bearing the
phenyl in the 20-position were enhanced from 60 to 1900 fold than
that of 15c without a linker at 3-position. The results also implied
that with or without double bond in the 5-position of androsterone
core had little influence on their activities. However, the structure–
activity relationship of the substituent group in the 20-position
seemed uncertain: in those target compounds 14a–d and 15a–d
without the linker, hydrogen atom and smaller size group like
methyl located at the 20-position were more beneficial to keep
the better potent activities, whereas in the hydrids 17b–d contain-
ing the linker, larger group size like phenyl substituted at 20-posi-
tion was observed to have the better anti-cancer activity. However,
the precise SAR of these hybrids and the optimal compound 17c
with potent anti-tumor activity remain to be further investigated.
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In summary, thirteen novel furoxan-based nitric oxide (NO)
releasing hybrids (14a–e, 15a–e, 17b–d) of 16, 17-pyrazo-annu-
lated steroidal derivatives were prepared and tested for their
in vitro anti-cancer activity. Most of the compounds showed strong
anti-proliferative effects better than that of furoxan toward MDA-
MB-231, SKOV-3, DU145 and HUVEC cell lines, and even nine of
them exhibited considerable cytotoxicity against a tamoxifen resis-
tant human breast cancer cell line HCC1806. Compound 17c had the
best activity against the five cell lines mentioned above. In the pre-
liminary pharmacological study, compounds 15a, 17b,c showed
obviously better activities compared with 2-ME on reducing levels
of VEGF secreted by MDA-MB-231. Besides, 17b,c significantly sup-
pressed tubule formation at the concentration of nanomolar level.
The initial SAR indicated that the linker between furoxan group
and steroidal scaffold was very important to notably improve their
anti-tumor potency. Further investigation of structure–activity
relationship and structure optimization should be conducted in
the future. In summary, all above results prompted that compound
17c might be a desirable candidate for further development.
Acknowledgements
This study was supported by grants from the Shanghai Natural
Science Foundation (Grant 14ZR1401900 for Y.C.), China
Postdoctoral Science Foundation (Grant 2013M531126 for M.-M.L.).