Synthesis and biological evaluation of 3β-androsta-5,8(14),15-trien-17-one derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.steroids.2016.05.004

A novel and operationally simple method for highly efficient synthesis of promising anti-cancer 3β-hydroxy-16-arylandrosta-5,8(14),15-trien-17-ones was reported. Compounds were tested for their cytotoxic activities against A549, SKOV3, MKN-45 and MDA-MB-4

Full text of DOI:10.1016/j.steroids.2016.05.004

Steroids 112 (2016) 74–80
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and biological evaluation of 3b-androsta-5,8(14),15-trien-17-
one derivatives as potential anticancer agents
Yang Li a, Jinliang Liu a, Lizhong Wang b, Xushun Qing a, Cunde Wang a,
⇑
a School of Chemistry and Chemical Engineering, Yangzhou University, 180 Siwangting Street, Yangzhou 225002, PR China
b School of Pharmacy, Taizhou Polytechnic College, Taizhou 225300, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel and operationally simple method for highly efficient synthesis of promising anti-cancer 3b-
hydroxy-16-arylandrosta-5,8(14),15-trien-17-ones was reported. Compounds were tested for their cyto-
toxic activities against A549, SKOV3, MKN-45 and MDA-MB-435 cancer cell lines. The preliminary results
showed that compounds 5e, g were the most active especially against cancer cell lines tested.
Ó 2016 Elsevier Inc. All rights reserved.
Received 10 January 2016
Received in revised form 14 April 2016
Accepted 10 May 2016
Available online 15 May 2016
Keywords:
3b-Hydroxy-androsta-5,8(14),15-trien-17-
one
Dehydroepiandrosterone
Anticancer agent
Suzuki coupling reaction
a,b-Unsaturated ketone
1. Introduction
point, the development of new modified D-ring steroids promising
biological activity by new synthetic approaches using mild reac-
Steroids and their derivatives have been found to possess the
potential to be developed as drugs for the treatment of a large
number of diseases [1–3]. Among them, some modified steroids
have been extensively studied as their biological and clinical
importance is now well validated. And most recently abiraterone
acetate has been approved for use as anticancer drugs by the US
FDA [4]. However because of the drug resistance and drug toler-
ance problems, the development of new compounds to improve
the selectivity and to minimize side effects of steroidal drugs has
been a challenge for a long time [5]. For many years, the modifica-
tion of 3-hydroxysteroids has attracted considerable attention
from medicinal and synthetic organic chemists. Various derivatives
of steroids by modified at D ring with anticancer activity and cyto-
toxic activity have been reported [6–17]. Chemical modification of
the steroid D-ring provides a way to alter biologically important
properties of modified steroids. As an example on the modification
tion conditions remains an active research area. Many previous
studies proved that some steroidal compounds with ,b-unsatu-
rated ketone core gave the potency against human cancer cell lines
a
[20–24]. Recently, König and co-authors reported that three new
steroids with extended conjugated system via D4,5 D6,7 and D8,14
,
were isolated from the marine sponge Callyspongia cf. C. flammea,
which were found capable of preventing the enhanced production
of amyloid b-42 in Aftin-5 treated cells as candidates for the
treatment of neurodegenerative Alzheimer’s disease [25]. We envi-
sioned that the combination of 16-arylandroster-17-one with
extended conjugated system via D8,14 and D15,16 moieties should
also have cytotoxic activity. This encouraged us to further explore
the structural motif responsible for the biological properties of 16-
aryl multi doublet-bond androstenones. Thus in continuation of
our program, we herein present the synthesis of 3b-hydroxy-
androsta-5,8(14),15-trien-17-one derivatives and their biological
evaluation for anticancer activity against SKOV3, A549, MKN-45,
and MDA-MB-435 cell lines in vitro.
of steroidal D ring, 3
rosteroid antagonist [18].
antagonizes selectively the GABA-modulatory and GABA-mimetic
effects of -hydroxy-5 -pregnan-20-one (allopregnanolone,
,5 -THPROG) and related 5 -pregnane steroids [18,19]. In view
a,5a
-17-phenylandrost- 16-en-3-ol is a neu-
3a,5a-17-Phenylandrost-16-en-3-ol
3a
a
2. Experimental
3a
a
a
of the remarkable importance from pharmacological and synthetic
2.1. General remarks
⇑
All melting points were determined in a SGW X-4 melting point
apparatus and are uncorrected. IR spectra were recorded in a
Corresponding author.
E-mail address: wangcd@yzu.edu.cn (C. Wang).
http://dx.doi.org/10.1016/j.steroids.2016.05.004
0039-128X/Ó 2016 Elsevier Inc. All rights reserved.

Y. Li et al. / Steroids 112 (2016) 74–80
75
Nicolet FT-IR 5DX spectrometer. The 1H NMR (600 MHz) and 13C
NMR (150 MHz) spectra were recorded in a Bruker AV-600 spec-
trometer with TMS as internal reference in DMSO-d6 or CDCl3 solu-
tions. The J values are given in hertz. Only discrete or characteristic
signals for the 1H NMR are reported. The MS spectra were obtained
on a ZAB-HS mass spectrometer with 70 eV. High-resolution ESI
mass spectra were obtained on a UHR-TOF maXis (ESI) mass spec-
trometer. X-ray crystallographic analysis was performed with a
SMART APEX-II diffractometer. The elemental analyses were per-
formed in a Perkin-Elmer 240C instrument. Flash chromatography
was performed on silica gel (230–400 mesh) eluting with ethyl
acetate-hexanes mixture. All reactions were monitored by thin
layer chromatography (TLC). All reagents and solvents were pur-
chased from commercial sources and purified commonly before
used.
DCM:EA = 20:1) to give the desirable product (0.88 g, 31%) as a
white solid. Mp 194–195 °C (PE/AcOEt); IR (KBr, cmꢁ1): 3312,
2930, 2858, 1709, 1643, 1519, 1453, 1219, 1108, 1009, 940, 865,
700; 1H NMR (600 MHz, CDCl3) d (ppm): 7.81 (d, J = 5.7 Hz, 1H),
5.94 (d, J = 5.7 Hz, 1H), 5.28 (s, 1H), 3.53–3.51 (m, 1H), 3.47–3.22
(m, 2H, 4-H), 2.25–2.23 (m, 2H, 7-H), 1.12 (s, 3H), 0.95 (s, 3H);
13C NMR (150 MHz, CDCl3)
d (ppm): 212.2, 152.69, 141.65,
139.34, 133.78, 128.06, 118.87, 70.98, 48.28, 45.75, 41.59, 38.87,
36.34, 31.50, 28.97, 27.63, 22.95, 19.50, 18.82; MS(EI) (m/z):
285.43 [(M+H)+] (100%); Anal. Calcd for C19H24O2: C, 80.24; H,
8.51; Found: C, 80.19; H, 8.42.
2.4. Preparation of 3b-hydroxy-16-iodoandrosta-5,8(14),15-trien-17-
one (4)
The mixture of 3b-hydroxyandrosta-5,8(14),15-trien-17-one
(0.50 g, 1.74 mmol), iodine (0.89 g, 3.48 mmol), DMAP (0.01 g,
0.087 mmol) and pyridine (6 mL, 0.07 mmol) in CCl4 (15 mL) was
stirred at 0 °C for 12 h. After the completion of reaction was con-
firmed by TLC (Hexanes/EtOAc, 1:1), the solvent was removed by
reduced pressure. The residues were extracted with dichloro-
methane (20 mL) and the organic phase was washed with satu-
rated sodium thiosulfate (2 ꢀ 10 mL), 20% HCl (10 mL) and brine
(10 mL), and dried over anhydrate sodium sulfate. After removal
of dichloromethane, the crude product was purified by flash
chromatography (silica gel, DCM:EA = 20:1) to give the desirable
product (0.88 g, 80%) as a light brown solid. Mp 135–136 °C
(PE/AcOEt); IR(KBr): 3468, 2929, 2858, 1700, 1657, 1513, 1450,
1370, 1276, 1211, 1062, 1004, 937, 918, 846, 734; 1H NMR
(600 MHz, CDCl3) d (ppm): 8.16 (s, 1H), 5.28 (s, 1H), 3.68–3.39
(m, 1H), 1.15 (s, 3H), 0.94 (s, 3H); 13C NMR (150 MHz, CDCl3) d
(ppm): 204.62, 158.00, 140.59, 138.94, 133.13, 117.62, 97.47,
69.95, 47.06, 43.82, 40.52, 37.88, 35.24, 30.46, 28.06, 26.81,
22.11, 18.52, 17.70; HRMS (ESI) calcd for C19H23IO2 [M+H]+
412.0822; Found 411.0818; Anal. Calcd for C19H23IO2: C, 55.62;
H, 5.65; Found: C, 55.48; H, 5.60.
2.2. Preparation of 16
(Scheme 1)
a-bromo-3b-hydroxyandrost-5-en-17-one (2)
The mixture of dehydroepiandrosterone (1) (2.88 g, 0.01 mol)
and copper bromide (5.6 g, 0.025 mol) in methanol (30 mL) was
stirred under reflux for 8 h, and the completion of reaction was
confirmed by TLC (Hexanes/EtOAc, 1:1). After removal of methanol
by reduced pressure the residues was added with water (20 mL)
and was extracted with dichloromethane (50 mL ꢀ 2). The organic
phase was washed with water (20 mL) and brine (15 mL), and
dried over anhydrate sodium sulfate. After removal of dichloro-
methane, the crude product was purified via recrystallization with
PE/AcOEt to afford a white solid product (2) (3.5 g, 82%), mp 178–
179 °C (PE/AcOEt). IR (KBr, cmꢁ1): 3287, 2936, 2859, 1749, 1454,
1375, 1195, 1133, 1103, 910, 884, 608; 1H NMR (600 MHz, CDCl3)
d (ppm): 5.42 (d, 1H, J = 5.4 Hz, 6-H), 4.56 (t, 1H, J = 5.4 Hz, 16-H),
3.55–3.52 (m, 1H, 3
7-H), 1.04 (s, 3H, C18-Me), 0.93 (s, 3H, C19-Me); MS(EI) (m/z):
366.07 [M+] (82.0%), 368.10 [(M+2)+] (100%); Anal. Calcd for C19
27BrO2: C, 62.13; H, 7.41; Found: C, 62.02; H, 7.33.
a-H), 3.45–3.20 (m, 2H, 4-H), 2.25–2.22 (m, 2H,
-
H
2.5. General procedure for the preparation of 16-aryl-3b-
hydroxyandrosta-5,8(14),15-trien-17-ones (5a–h)
2.3. Preparation of 3b-hydroxyandrosta-5,8(14),15-trien-17-one (3)
The mixture of 16 -bromo-3b-hydroxyandrost-5-en-17-one
a
To the mixture of 3b-hydroxy-16-iodoandrosta-5,8(14),15-
trien-17-one (0.50 g, 1.2 mmol), arylboric acid (1.82 mmol), Pd
(PPh3)2Cl2 (17 mg, 0.024 mmol), CuI (2.4 mg, 0.024 mmol) in THF
(20 mL) and methanol (4 mL) was added 2 mol/L sodium carbonate
solution (4.8 mmol, 2.4 mL). The resultant mixture was stirred
under reflux for 10 h. After the completion of reaction was con-
firmed by TLC (Hexanes/EtOAc, 1:2), the solvent was removed by
reduced pressure. The residues were extracted with dichloro-
methane (20 mL) and the organic phase was washed with water
(2 ꢀ 10 mL) and brine (10 mL), and dried over anhydrate sodium
sulfate. After removal of dichloromethane, the crude product was
purified by flash chromatography (silica gel, DCM:EA = 10:1) to
give the desirable product 5a–h.
(3.67 g, 0.01 mol), LiBrꢂH2O (3.67 g, 0.035 mol) and Li2CO3 (2.96 g,
0.04 mol) in DMF (30 mL) was refluxed under nitrogen for 8 h,
and the completion of reaction was confirmed by TLC (Hexanes/
EtOAc, 1:1). After the mixture was cooled to room temperature,
to the mixture was added ice-water (15 g). A yellow solid was fil-
tered and dissolved with dichloromethane (20 mL). The solution
was washed with water (20 mL) and brine (15 mL), and dried over
anhydrate sodium sulfate. After removal of dichloromethane, the
crude product was purified by flash chromatography (silica gel,
O
O
O
Br
a
b
2.5.1. 3b-Hydroxy-16-phenylandrosta-5,8(14),15-trien-17-one (5a)
Light yellow solid, yield 91%, mp: 175–176 °C (PE/AcOEt); IR
(KBr, cmꢁ1): 3462, 2929, 2856, 1684, 1450,1339, 1292, 1176,
1059, 927, 795, 750, 654; 1H NMR (600 MHz, CDCl3) d (ppm):
7.96 (s, 1H), 7.77 (d, J = 7.7 Hz, 2H), 7.31 (dd, J = 7.6 Hz and
7.2 Hz, 2H), 7.24 (dd, J = 7.3 Hz and 7.2 Hz, 1H), 5.31 (s, 1H), 3.53
(m, 1H), 1.19 (s, 3H), 0.96 (s, 3H); 13C NMR (150 MHz, CDCl3) d
(ppm): 209.53, 146.57, 141.62, 137.77, 136.78, 133.70, 132.22,
128.49, 128.29, 127.12, 119.04, 71.05, 48.53, 47.50, 41.61, 38.98,
36.33, 31.52, 29.11, 27.87, 23.36, 19.58, 18.87; HRMS (ESI) calcd
for C25H28O2 [M+H]+ 361.2168; Found 361.2164; Anal. Calcd for
HO
HO
HO
3
1
2
O
O
d
c
I
Ar
HO
HO
4
5a-h
Scheme 1. Preparation of target compounds 5a–h. Reagents and conditions: (a)
CuBr2, CH3OH, reflux, 8 h, 82%; (b) LiBr, Li2CO3, DMF, reflux, 31%; (c) I2, DMAP,
pyridine, CCl4, 0 °C, 80%; (d) Pd(PPh3)2Cl2/CuI, ArB(OH)2, 2 mol/L Na2CO3, THF,
CH3OH, reflux, 70–91%.
C25H28O2: C, 83.29; H, 7.83; Found: C, 83.16; H, 7.89.

76
Y. Li et al. / Steroids 112 (2016) 74–80
2.5.2. 3b-Hydroxy-16-(pyridin-3-yl)androsta-5,8(14),15-trien-17-one
(5b)
2.5.6. 3b-Hydroxy-16-(pyrimidin-5-yl)androsta-5,8(14),15-trien-17-
one (5f)
Light yellow solid, yield 85%, mp: 167–168 °C (PE/AcOEt); IR
(KBr, cmꢁ1): 3472, 2928, 2857, 1697, 1647, 1458, 1415, 1376,
1299, 1066, 933, 808; 1H NMR (600 MHz, CDCl3) d (ppm): 8.91
(s, 1H), 8.46 (d, J = 4.4 Hz, 1H), 8.17 (d, J = 7.9 Hz, 1H), 8.06 (s,
1H), 7.28–7.23 (m, 1H), 5.32 (s, 1H), 3.58–3.51 (m, 1H), 1.98 (s,
1H), 1.20 (s, 3H), 0.98 (s, 3H); 13C NMR (150 MHz, CDCl3) d
(ppm): 208.10, 147.92, 147.00, 146.13, 140.75, 136.75, 134.59,
133.43, 132.83, 122.32, 119.83, 117.80, 69.99, 47.61, 46.25, 40.57,
38.05, 35.31, 30.49, 29.90, 26.80, 22.26, 18.57, 17.80; HRMS (ESI)
calcd for C24H27NO2 [M+H]+ 362.2121; Found 362.2112; Anal.
Calcd for C24H27NO2: C, 79.74; H, 7.53; N, 3.87; Found: C, 79.78;
H, 7.44; N, 3.82.
Light yellow solid, yield 87%, mp: 203–204 °C (PE/AcOEt); IR
(KBr, cmꢁ1): 3487, 3034, 2940, 2861, 2799, 1701, 1644, 1558,
1406, 1299, 1144, 1009, 932, 721; 1H NMR (600 MHz, CDCl3) d
(ppm): 9.11 (s, 2H), 9.07 (s, 1H), 8.13 (s, 1H), 5.32 (s, 1H), 3.59–
3.50 (m, 1H), 1.21 (s, 3H), 0.98 (s, 3H); 13C NMR (150 MHz, CDCl3)
d (ppm): 207.56, 156.63, 153.76, 146.98, 140.77, 136.67, 136.43,
129.91, 125.53, 117.52, 69.89, 47.69, 46.10, 40.54, 38.16, 35.32,
30.46, 28.26, 26.73, 22.19, 18.60, 17.75; HRMS (ESI) calcd for
C
C
23H26N2O2 [M+H]+ 363.2073; Found 363.2068; Anal. Calcd for
23H26N2O2: C, 76.21; H, 7.23; N, 7.73; Found: C, 76.14; H, 7.20;
N, 7.62.
2.5.7. 16-(3-Trifluoromethylphenyl)-3b-hydroxyandrosta-5,8(14),15-
trien-17-one (5g)
Light yellow solid, yield 93%, mp: 195–196 °C (PE/AcOEt); IR
(KBr, cmꢁ1): 3489, 3032, 2939, 2894, 2827, 1680, 1587, 1475,
1419, 1322, 1123, 1006, 805; 1H NMR (600 MHz, CDCl3) d (ppm):
8.04 (s, 1H), 8.02 (s, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.49 (d,
J = 7.7 Hz, 1H), 7.43 (dd, J = 7.2 and 7.8 Hz, 1H), 5.32 (s, 1H),
3.56–3.52 (m, 1H), 1.20 (s, 3H), 0.98 (s, 3H); 13C NMR (150 MHz,
2.5.3. 16-(2-Fluorophenyl)-3b-hydroxyandrosta-5,8(14),15-trien-17-
one (5c)
Light yellow solid, yield 86%, mp: 164–165 °C (PE/AcOEt); IR
(KBr, cmꢁ1): 3491, 3043, 2930, 2859, 1691, 1562, 1480, 1449,
1274, 1213, 1110, 1055, 930, 875, 800, 758, 722; 1H NMR
(600 MHz, CDCl3) d (ppm): 8.17 (s, 1H), 8.01 (dd, J = 7.2 Hz and
7.2 Hz, 1H), 7.22–7.17 (m, 2H), 7.10 (dd, J = 7.5 Hz and 6.1 Hz,
1H), 5.31 (s, 1H), 3.55–3.52 (m, 1H), 1.20 (s, 3H), 0.97 (s, 3H); 13C
NMR (150 MHz, CDCl3) d (ppm): 209.43, 159.53 (d, J = 249.6 Hz),
149.42 (d, J = 10.2 Hz), 140.49, 137.02, 133.70, 130.16, 129.91,
128.32 (d, J = 8.5 Hz), 123.03 (d, J = 3.3 Hz), 119.13 (d,
J = 12.3 Hz), 118.02, 114.62 (d, J = 22.3 Hz), 70.02, 47.58, 45.45,
40.58, 37.98, 35.32, 30.49, 28.05, 26.82, 22.31, 18.54, 17.84; HRMS
(ESI) calcd for C25H27FO2 [M+H]+ 379.2074; Found 379.2074; Anal.
Calcd for C25H27FO2: C, 79.34; H, 7.19; Found: C, 79.32; H, 7.22.
CDCl3)
d (ppm): 207.94, 146.41, 140.64, 136.56, 134.31 (d,
J = 8.5 Hz), 131.99, 129.87 (q, J = 32.6 Hz), 129.28, 127.91, 123.98,
123.69 (q, J = 3.7 Hz), 123.07 (q, J = 271.5 Hz), 122.74 (q,
J = 3.5 Hz), 117.83, 70.02, 47.63, 46.50, 40.58, 38.03, 35.31, 30.50,
28.17, 26.82, 22.28, 18.56, 17.81; HRMS (ESI) calcd for
C
C
26H27F3O2 [M+H]+ 429.2042; Found 429.2039; Anal. Calcd for
26H27F3O2: C, 72.88; H, 6.35; Found: C, 72.72; H, 6.32.
2.5.8. 3b-Hydroxy-16-(5-methoxypyridin-3-yl)androsta-5,8(14),15-
trien-17-one (5h)
Dark yellow solid, yield 74%, mp: 193–194 °C (PE/AcOEt); IR
(KBr, cmꢁ1): 3488, 2930, 2872, 1686, 1610, 1521, 1450, 1370,
1296, 1119, 1008, 940; 1H NMR (600 MHz, CDCl3) d (ppm): 8.49
(s, 1H), 8.16 (s, 1H), 8.07 (s, 1H), 7.79 (s, 1H), 5.32 (s, 1H), 3.82
(s, 3H), 3.57–3.50 (m, 1H), 1.20 (s, 3H), 0.97 (s, 3H); 13C NMR
2.5.4. 16-(3-Fluorophenyl)-3b-hydroxyandrosta-5,8(14),15-trien-17-
one (5d)
Light yellow solid, yield 84%, mp: 190–191 °C (PE/AcOEt); IR
(KBr, cmꢁ1): 3500, 3075, 2928, 2857, 1679, 1577, 1426, 1340,
1263, 1185, 1058, 886, 790, 681; 1H NMR (600 MHz, CDCl3) d
(ppm): 7.97 (s, 1H), 7.55 (dd, J = 7.7 and 7.2 Hz, 2H), 7.27 (dd,
J = 7.3 and 7.8 Hz, 1H), 6.93 (dd, J = 7.6 and 7.6 Hz, 1H), 5.32 (s,
1H), 3.55–3.52 (m, 1H), 1.19 (s, 3H), 0.97 (s, 3H); 13C NMR
(150 MHz, CDCl3)
d (ppm): 208.27, 154.50, 146.50, 140.67,
139.05, 136.60, 136.17, 134.84, 132.37, 127.83, 117.76, 117.45,
69.95, 54.57, 47.64, 46.32, 40.57, 38.07, 35.32, 30.48, 28.19,
26.80, 22.26, 18.57, 17.80; HRMS (ESI) calcd for C25H29NO3 [M
+H]+ 392.2226; Found 392.2229; Anal. Calcd for C25H29NO3: C,
76.70; H, 7.47; N, 3.58; Found: C, 76.62; H, 7.42; N, 3.52.
(150 MHz, CDCl3)
d (ppm): 207.90, 161.88 (d, J = 245.6 Hz),
146.03, 140.69, 136.62, 134.57 (d, J = 2.1 Hz), 133.75, 133.32,
128.89 (d, J = 8.1 Hz), 121.69 (d, J = 2.8 Hz), 117.90, 114.04 (d,
J = 21.3 Hz), 112.96 (d, J = 22.8 Hz), 70.05, 47.64, 46.57, 40.63,
38.02, 35.35, 30.56, 28.13, 26.88, 22.32, 18.56, 17.84; HRMS (ESI)
calcd for C25H27FO2 [M+H]+ 379.2074; Found 379.2074; Anal. Calcd
for C25H27FO2: C, 79.34; H, 7.19; Found: C, 79.28; H, 7.20.
2.6. Biology
2.6.1. Cell lines and culture conditions
Human lung carcinoma cell line A549, human ovarian carci-
noma cell line SKOV3, human gastric adenocarcinoma cell line
MKN-45 and human breast carcinoma cell line MDA-MB-435 used
in this work, were purchased from Shanghai Institute of Cell Biol-
ogy, Chinese Academy of Science. The cell lines were grown in
RPMI 1640 medium with 10% newborn calf serum. It was main-
tained in a humidified incubator with an atmosphere of 95% air
and 5% CO2 at 37 °C. The cells were continuously passaged once
every 3–4 days. Growing cells were collected on experiments.
2.5.5. 3b-Hydroxy-16-(6-fluoropyridin-3-yl)androsta-5,8(14),15-
trien-17-one (5e)
Light yellow solid, yield 90%, mp: 180–181 °C (PE/AcOEt); IR
(KBr, cmꢁ1): 3497, 2696, 2930, 2851, 2818, 1680, 1587, 1472,
1376, 1297, 1176, 1120, 1069, 1022, 928, 834, 744, 670; 1H NMR
(600 MHz, CDCl3) d (ppm): 8.56 (s, 1H), 8.26 (dd, J = 7.2 and
7.2 Hz, 1H), 8.02 (s, 1H), 6.89 (d, J = 6.6 Hz, 1H), 5.32 (s, 1H),
3.55–3.52 (m, 1H), 1.20 (s, 3H), 0.98 (s, 3H); 13C NMR (150 MHz,
CDCl3) d (ppm): 207.92, 162.11 (d, J = 238.5 Hz), 145.84, 145.04
(d, J = 14.7 Hz), 140.67, 138.64 (d, J = 8.0 Hz), 136.54, 134.65,
131.76 (d, J = 2.9 Hz), 125.34, 117.74, 108.24 (d, J = 37.3 Hz),
70.00, 47.61, 46.19, 40.57, 38.05, 35.31, 30.49, 28.17, 26.78,
22.25, 18.56, 17.79; HRMS (ESI) calcd for C24H26FNO2 [M+H]+
380.2027; Found 380.2012; Anal. Calcd for C24H26FNO2: C, 75.96;
H, 6.91; N, 3.69; Found: C, 76.08; H, 6.82; N, 3.76.
2.6.2. Cell viability assay (SRB)
The anticancer activity in vitro was measured using the SRB
assay. The assay was carried out according to previous study.
DMSO was used as latent solvent with the highest concentration
less than 0.1% in solution of the drug. The control groups of blank
(1640) and DMSO solvent were set up at the same time. Prolifera-
tive activity was evaluated by colorimetric sulforhodamine B (SRB)
assay. Briefly, cells were plated in 96-well plates. After cell
adhering, they were treated with different compounds in
a

Y. Li et al. / Steroids 112 (2016) 74–80
77
dose-dependent way for 44 h. Then the cells were fixed by 10% TDA
for 1 h and stained by SRB for 10 min. After washed with acetic
acid to remove the excess dye, protein bounding dye were
dissolved in 10 mM Tris and detected by a Model Elx 800 Autoplate
reader (Bio-Tek Instruments, U.S.A).
Compound 4 was synthesized by treating compound 3 with the
mixture of I2, DMAP and pyridine in CCl4 in 80% yield. IR spectrum
of compound 4, one sharp absorption band at 1700 cmꢁ1, three
bands at 1657, 1513 and 1450 cmꢁ1, could be related to CO and
C@C stretching frequencies. The high resolution mass spectrum
of compound
4 displayed the molecular ion peak at m/
z = 411.0818 (M+1)+, which is in good agreement with the pro-
posed structure. The 1H NMR spectrum of compound 4 showed
two singlet signals at 8.16 (s, 1H), 5.28 (s, 1H) for C15-H and C6-
H of the double bond, respectively, the characteristic 1H chemical
shift of C15-H unequivocally indicated the proton of C16 was
replaced by iodine. The important peaks of 13C NMR were related
to the CO and three C@C double bonds which appeared at
d = 204.62, 158.00, 140.59, 138.94, 133.13, 117.62, and 97.47 ppm.
2.6.3. Data analysis
Cell survival was calculated using the formula: Survival (%) =
[(absorbance of treated cells ꢁ absorbance of culture medium)/
(absorbance of untreated cells ꢁ absorbance of culture med-
ium)] ꢀ 100. The experiment was done in eight replicates and
the inhibitory concentration (IC) values were calculated from a
dose response curve. IC50 is the concentration in
lM required for
50% inhibition of cell growth as compared to that of untreated con-
trol. IC50 values were determined from the linear portion of the
curve by calculating the concentration of agent that reduced absor-
bance in treated cells, compared to control cells, by 50%. Evaluation
is based on mean values from three independent experiments,
each comprising at least six microcultures per concentration level.
All the data of the experiment were compiled and analyzed accord-
ing to SPSS 15.0 software. Measurement data were expressed as
the mean S.D.
a
-Iodo-a,b-unsaturated carbonyl compounds served as an acti-
vated iodoolefin having a great potential for the construction of
Csp2–Csp2 by Suzuki coupling reaction. The same was done for
the preparation of 16-aryl-3b-hydroxyandrosta-5,8(14),15-trien-
17-ones (Table 1, 5a–h) by employing the Suzuki coupling reaction
of intermediate 4 with appropriately substituted aromatic boric
acids in presence of the catalyst system: Pd(PPh3)2Cl2/CuI/Na2CO3.
The analytical and spectral data of compounds 5a–h were in agree-
ment with their structures. IR spectrum of compound 5a–h, one
sharp absorption band at 1680–1700 cmꢁ1 could be related to CO
stretching frequency. The 1H NMR spectrum of the 16-aryl-3b-
hydroxyandrosta-5,8(14),15-trien-17-ones in CDCl3 shows the
characteristic signals of the androsta-5,8(14),15-triene nucleus.
3. Results and discussion
D-ring modified steroids have attracted considerable attentions
from synthetic organic chemists and pharmaceuticals researchers.
Modifications at the D-ring of steroids are considerable important
and as per the accepted trend as such alterations result in effective
receptor binding or the increased bioavailability. In keeping with
our continuing interest for the modification of natural steroids as
effective cytotoxic agents, herein we report an efficient and simple
synthesis of 3b-hydroxy-16-arylandrosta-5,8(14),15-trien-17-
ones. The synthetic strategies are discussed below.
The multiplet of 3a-H is to be found at 3.60–3.50 ppm. Further-
more, two olefinic signals are observed at 5.30 and 8.10 ppm,
respectively, and can be assigned to C5-H and C15-H. However,
the chemical shift of the C5-H and C15-H singlets differs signifi-
cantly in two olefinic signals. This indicates that C5-H has a normal
chemical shift as an olefinic proton. The chemical shift of the C15-H
singlet moves to low field because it conjugates with a C@C double
bond, a carbonyl and an aromatic ring. The corresponding C16-aro-
matic ring shows low-field 1H NMR signals from 6.80 to 9.11 ppm.
The important peaks of 13C NMR were related to the C@O and HO-
3.1. 3b-Hydroxy-16-arylandrosta-5,8(14),15-trien-17-one
CH which appeared at d = ca 208 and 70 ppm, respectively. Three
C@C double bonds appeared at d = ca 146, 141, 137, 135, 125,
and 117 ppm. Taking compound 5e as an example, in the 13C
NMR spectrum compound 5e displayed signals of the carbonyl
group, hydroxyalkyl group and three double bonds at 207.92
(O@C17), 70.00 (HO-C3), 145.84 (C15), 140.67 (C14), 136.54 (C8),
134.65 (C5), 125.34 (16), and 117.74 (C6). Because the signals of
carbon atoms are spilt by coupling to 19F, the corresponding C16-
aromatic ring shows five distinctive doublets of aromatic ring car-
bons at 162.11 (d, 1JCF = 238.5 Hz), 145.04 (d, 3JCF = 14.7 Hz), 138.64
In this part, we design to synthesize a series of 3b-hydroxy-16-
arylandrosta-5,8(14),15-trien-17-ones with dehydroepiandros-
terone (1) as the starting material according to reported method
[26] (Scheme 1). Compound 2 was synthesized with the standard
procedure in our laboratory, by treating 1 with copper bromide
in methanol under reflux with a good yield. Compound 3 was pre-
pared by treating compound 2 with lithium bromide and lithium
carbonate in DMF and separated by column chromatography. Yield
was not very well which is only 31%. The molecular structure of
compound 3 was elucidated from its spectroscopic analyses as
described herein for compound 3. In the IR spectrum of compound
3, one sharp absorption band at 1709 cmꢁ1, three bands at 1643,
1519 and 1453 cmꢁ1, could be related to CO and C@C stretching
frequencies. The mass spectrum of compound 3 displayed the
molecular ion peak at m/z = 285.43 (M+1)+, which is in good agree-
ment with the proposed structure. The 1H NMR spectrum of com-
pound 3 exhibited two doublet signals at 7.81 (d, J = 5.7 Hz, 1H),
5.94 (d, J = 5.7 Hz, 1H) for C16-H and C15-H of the double bond,
respectively, one singlet signal at 5.28 (s, 1H) and one multipeak
(d, 3JCF = 8.0 Hz), 131.76 (d, JCF = 2.9 Hz), 108.24 (d, JCF = 37.3 Hz)
ppm. The structures of the compounds were additionally con-
firmed by X-ray crystal data. We obtained two X-ray crystal struc-
tures of 5a and 5b that were presented in Figs. 1 and 2.
Crystallographic data for 5a and 5b have been deposited with the
Cambridge Crystallographic Data Centre with the deposition num-
ber CCDC 935129 (5a), 937201(5b). These data can be obtained
free of charge on application to CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK [fax (+44) 1223 336033, e-mail: deposit@ccdc.cam.
ac.uk].
4
2
Both molecules consist of four six-membered rings and one
five-membered ring. The absolute configurations of both
compound 5a and 5b are C3S, C9S, C10R, and C13S, which is
inferred from the known stereochemistry of dehydroepiandros-
terone (3b-hydroxyandrost-5-en-17-one). The A, B, and C rings
for both molecules 5a and 5b are chair, twist-half-chair, and
twist-half-chair conformations. However, the chair conformation
of A ring is slightly disturbed by the presence of exocyclic double
at 3.53–3.51 (m, 1H) for C6-H and C3
a
-H respectively. Characteris-
tic 1H chemical shift of C16-H, C15-H, C6-H and C3
a-H unequivo-
cally indicated the exclusive chemical environment of compound
3 protons. The 1H-decoupled 13C NMR spectrum of compound 3
showed 19 distinct signals in agreement with the suggested struc-
ture. The important peaks were related to the CO and three C@C
double bonds which appeared at d = 212.20, 152.69, 141.65,
139.34, 133.78, 128.06, and 118.87 ppm.

78
Y. Li et al. / Steroids 112 (2016) 74–80
Fig. 1. Molecular structure of compound 5a (containing CH2Cl2).
Fig. 2. Molecular structure of compound 5b (containing CHCl3).
bond. The D ring and C16-aromatic ring for both molecules 5a and
5b are near-plane conformations. All of the bond lengths and
angles are normal. In the A, B, and C rings of compound 5a and
5b the average magnitude of the Csp3–Csp3 distances 1.53 Å, the
Csp2–Csp2 distances 1.33 Å, and the Csp3–Csp2 distances 1.50 Å
(Table 2). It is worth noting that the average magnitude of C
(15)–C(14)–C(13) angles 107.2°, the D ring with C16-aromatic ring
is leaning at a low angle from the A, B, and C rings of the steroid
structure.
our earlier communications [27] have shown their therapeutic
potential. As we are interested in thorough pharmacological stud-
ies of various steroidal analogs, we examined the anticancer poten-
tial of 3b-hydroxy-16-arylandrosta-5,8(14),15-trien-17-ones and
the results are described in Table 1.
The newly synthesized compounds 5a–h and 3 were evaluated
for their anticancer activity towards human tumor cell lines
derived from various human cancer types: A549 (human lung car-
cinoma), SKOV3 (human ovarian carcinoma), MKN-45 (human gas-
tric adenocarcinoma), MDA-MB-435 (human breast carcinoma)
cell lines. The results are summarized as IC50 values in l mol/L in
Table 1. From the data shown in Table 1, most of the newly synthe-
3.2. Biology
sized compounds showed
a measurable anti-cancer activity
a
,b-Unsaturated ketones of natural and synthetic steroids are
against A549 (human lung carcinoma), SKOV3 (human ovarian car-
cinoma), MKN-45 (human gastric adenocarcinoma), MDA-MB-435
(human breast carcinoma) cell lines tested. Although this is a
considered as resourceful molecules as for as their pharmacologi-
cal activities are concerned [23]. In reference to those natural
and synthetic steroids with
a,b-unsaturated ketone core, we in

Y. Li et al. / Steroids 112 (2016) 74–80
79
Table 1
Preparation and the in vitro cytotoxic activity (IC50, in l
mol Lꢁ1) of 5a–h.
Compd.
–Ar
Yield%a
Cancer cell linesb
A549
SKOV3
MKN-45
MDA-MB-435
5a
5b
91
85
32.1 3.0
12.5 1.8
70.2 2.2
20.2 1.6
22.5 1.6
14.3 2.6
10.3 1.5
9.6 1.3
21.2 0.6
17.2 1.6
18.2 2.2
12.6 2.4
N
5c
86
84
F
5d
12.1 1.6
8.9 1.2
15.3 2.2
9.7 2.6
F
5e
5f
90
87
11.5 2.0
20.2 0.8
7.7 0.4
18.4 1.2
21.6 2.6
8.8 1.5
F
N
N
10.1 1.0
34.6 1.9
N
5g
5h
93
74
10.9 1.8
20.9 2.8
7.5 2.5
16.6 3.2
23.6 2.2
8.3 2.8
CF3
14.5 1.5
32.3 1.6
N
OCH3
3
H
42.1 2.2
30.5 2.0
80.2 3.6
55.4 2.8
a
Isolated yields.
b
The results are the average mean of eight replicate determinations SD; Used as reference A549: human lung carcinoma, SKOV3: human ovarian carcinoma, MKN-45:
human gastric adenocarcinoma, MDA-MB-435: human breast carcinoma.
preliminary screening, compounds 5a–h exhibited different levels
of anticancer properties, the results showed all steroids tested
showed a higher cytotoxicity against SKOV3 cells than against
A549, MKN-45, and MDA-MB-435 cell lines. Compounds 5a–h with
same 3-hydroxyl structure and 5,8(14),15-trien-17-one motif, dif-
ferent types of substituted group of aromatic ring, showed a dis-
tinct difference in their cytotoxicity against these cancer cells.
However, the compound 3 without an aromatic ring at C16 posi-
Table 2
Selected bond lengths [Å] and angles [°] for compounds 5a and 5b.
Bond
Distance (Å) of 5a
Distance (Å) of 5b
C(5)–C(6)
C(5)–C(4)
1.312(6)
1.513(6)
1.517(6)
1.501(6)
1.510(6)
1.528(6)
1.356(6)
1.439(6)
1.508(6)
1.544(6)
1.545(6)
1.496(6)
1.546(6)
1.517(6)
1.337(5)
1.459(6)
1.483(6)
1.530(6)
1.322(9)
1.487(8)
1.534(8)
1.486(8)
1.537(10)
1.533(8)
1.344(8)
1.414(9)
1.543(9)
1.534(8)
1.543(9)
1.469(9)
1.547(9)
1.509(8)
1.337(8)
1.462(9)
1.503(9)
1.539(9)
C(5)–C(10)
C(13)–C(14)
C(13)–C(12)
C(13)–C(17)
C(15)–C(16)
C(15)–C(14)
C(9)–C(8)
C(9)–C(10)
C(9)–C(11)
C(6)–C(7)
C(10)–C(1)
C(7)–C(8)
C(14)–C(8)
C(16)–C(20)
C(16)–C(17)
C(2)–C(1)
tion decreased cytotoxicity. It was found that retaining the
a,b-
Unsaturated C17 carbonyl group in D-ring of androstene skeleton
lead to reduction in growth inhibition as observed for compound
of 5a–h and 3 which was also in accordance with previous reports
[10]. The reported results indicated that there were weak correla-
tion between molecular function of the proteins and the puckering
conformation of the steroid molecules. Both nuclear receptors and
G-protein coupled receptors prefer to bind steroid molecules in the
flat structure. Immunoglobulins also tend to bind steroid molecule
in flat, but enzyme tends to prefer steroid molecules in half-chair
[28]. The studies showed also clearly demonstrates that protein–
steroid interactions are hydrophobic in both flat and half-chair
steroid molecules. In both flat and half-chair cases, Asp, Glu, Arg
and Lys were less represented in the interface of steroid molecule
compared with the surface of proteins, and hydrophobic residues,
especially aromatic residues, were highly used in the interface
[28]. Thus, the near-plane conformations of D ring and C16-aro-
matic ring are helpful for the interface of steroid molecule com-
pared with the surface of proteins. It was also evident from the
data that the changes of substituents on the aromatic ring had a
significant influence on the cytotoxicity. Compound 5c, d, e and g
with a fluoro-substituted group of aromatic ring had a better cyto-
toxicity than compounds 5a, b, f, and h against SKOV3, A549, MKN-
45, and MDA-MB-435 cell lines. The analogs 5a with a phenyl
remarkably decreased its cytotoxic activity against A549, SKOV3,
MKN-45, and MDA-MB-435 cells in comparison with the analogs
5b, f, and h, which have a heterocycle containing nitrogen.
Compound 5e containing a fluoro-substituted group of heterocycle
containing nitrogen was found the best active to SKOV3, A549,
Angle
(°) of 5a
(°) of 5b
C(6)–C(5)–C(4)
120.6(4)
122.9(4)
116.5(3)
113.0(4)
125.8(4)
112.7(4)
128.9(4)
123.6(4)
107.2(4)
106.9(4)
112.1(4)
122.0(4)
123.0(4)
113.5(4)
109.7(4)
114.3(4)
107.9(4)
110.7(4)
121.7(6)
121.0(5)
117.3(5)
114.5(6)
127.0(6)
112.4(5)
128.4(6)
124.0(6)
107.3(5)
106.0(6)
113.4(6)
121.6(6)
123.9(6)
113.1(5)
109.5(6)
114.0(5)
106.7(5)
110.0(5)
C(6)–C(5)–C(10)
C(4)–C(5)–C(10)
C(16)–C(15)–C(14)
C(5)–C(6)–C(7)
C(6)–C(7)–C(8)
C(8)–C(14)–C(15)
C(8)–C(14)–C(13)
C(15)–C(14)–C(13)
C(15)–C(16)–C(17)
C(3)–C(4)–C(5)
C(14)–C(8)–C(9)
C(14)–C(8)–C(7)
C(9)–C(8)–C(7)
C(2)–C(3)–C(4)
C(12)–C(11)–C(9)
C(16)–C(17)–C(13)
C(3)–C(2)–C(1)

80
Y. Li et al. / Steroids 112 (2016) 74–80
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In summary, we have successfully developed a novel and oper-
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anti-cancer 3b-hydroxy-16-arylandrosta-5,8(14),15-trien-17-ones.
The preliminary results showed that those 3b-hydroxy-16-arylan-
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oro-substituted group of the heterocyclic ring containing
nitrogen at C-16 have significant impact on inhibiting human lung
carcinoma cell line A549, human ovarian carcinoma cell line
SKOV3, human gastric adenocarcinoma cell line MKN-45 and
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their possible mechanism of inhibiting proliferation of cancer cell
lines and the development of 3b-hydroxy-16-arylandrosta-5,8
(14),15-trien-17-ones as promising anticancer agents are ongoing.
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