Synthesis and Antitumor Activity of Dehydroepiandrosterone Derivatives on Es-2, A549, and HepG2 Cells in vitro

Article abstract of DOI:10.1111/j.1747-0285.2011.01311.x

A series of dehydroepiandrosterone derivatives containing an acid ester was synthesized and evaluated for their antitumor activity on ES-2, A549, and HepG2 cells by the MTT assay. Most compounds showed antitumor activity, while compounds 1c, 2i, and 2o exhibited more potential inhibitory effects compared with dehydroepiandrosterone on ES-2 cells, A549 cells, and HepG2 cells, respectively.

Full text of DOI:10.1111/j.1747-0285.2011.01311.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01311.x
Chem Biol Drug Des 2012; 79: 523–529
Research Article
Synthesis and Antitumor Activity of
Dehydroepiandrosterone Derivatives on Es-2,
A549, and HepG2 Cells in vitro
Xue-Kun Liu1,2, Bai-Jun Ye1, Yan Wu1,
series of derivatives were synthesized through acylation of the
hydroxyl group of DHEA and compound 2 that was obtained by
reducing the ketone group. The antitumor activities of the deriva-
tives were evaluated against three different human tumor cell
Ji-Xing Nan1, Zhen-Hua Lin3,* and Hu-Ri
Piao1,*
1Key Laboratory of Natural Resources of Changbai Mountain &
lines.
Functional Molecules, Ministry of Education, Yanbian University
College of Pharmacy, Yanji 133002, China
2Department of Pharmaceutics, Tonghua Health School, Tonghua
Experimental Section
134000, China
3Yanbian University College of Preclinical Medicine, Yanji 133002,
Material and methods
China
Melting points were determined in open capillary tubes and are
uncorrected. Reaction courses were monitored by TLC on silica
gel precoated F254 Merck plates and developed plates were
examined under UV light (254 nm). Column chromatography was
performed using Merck 200 mesh silica gel. IR spectra were
recorded (in KBr) on a FT-IR1730. 1H NMR spectra were mea-
sured on a Bruker AV-300 spectrometer using TMS as an inter-
nal standard. Mass spectra were measured on an AXIMA CFR
Plus MALDI-TOF (Shimadzu Biotech, Columbia, MD, USA). Elemen-
tal analyses for C, H, and N were within €0.4% of the theoreti-
cal values and were performed on a 204Q CHN Rapid Analyzer
(Perkin-Elmer, Waltham, MA, USA). The major chemicals were
purchased from Aldrich Co. (St. Louis, MO, USA).
*Corresponding authors: Hu-Ri Piao, piaohuri@yahoo.com.cn
Zhen-Hua Lin, zhlin720@ybu.edu.cn
A
series of dehydroepiandrosterone derivatives
containing an acid ester was synthesized and eval-
uated for their antitumor activity on ES-2, A549,
and HepG2 cells by the MTT assay. Most com-
pounds showed antitumor activity, while com-
pounds 1c, 2i, and 2o exhibited more potential
inhibitory effects compared with dehydroepiand-
rosterone on ES-2 cells, A549 cells, and HepG2
cells, respectively.
Key words: antitumor activity, dehydroepiandrosterone, synthesis
Received 14 January 2011, revised 9 December 2011 and accepted for
publication 11 December 2011
DHEA-(3,17)-diol (2)
To a stirred solution of DHEA (2.9 g, 10 mmol) in methanol (50 mL),
NaBH4 (0.38 g, 10 mmol) was added in portions. After 2 h, the
resulting white solid was collected by filtration under reduced pres-
sure to afford 2 in 90% yield.
Dehydroepiandrosterone (DHEA) a major steroid secreted by the
adrenal gland, which decreases with age after adolescence, is
available as a nutritional supplement (1) and is the most abundant
steroid in humans (2). In addition to serving as a precursor of both
androgens and estrogens, DHEA has various other beneficial biolog-
ical effects. In experimental animals, it decreases body fat without
altering food intake, enhances the immune system, suppresses
spontaneous decreases in blood glucose in diabetic mice and
enhances the memory of old mice (3). In recent years, it has been
reported that DHEA possesses an antiproliferative effect on animal
tumor models and malignant cell lines (4). Several derivatives of
DHEA (3b, 7a, 17b-AET and 3b, 7b, 17b-AET) were evaluated for
their antitumor activities, although the mechanisms responsible for
the altered tumor growth are not yet established (5).
General procedure for the synthesis of
compounds 1a–1o and 2a–2o
A suspension of DHEA (0.29 g, 1 mmol), acyl acid (1 mmol), DCC
(0.41 g, 2 mmol), DMAP (0.12 g, 1 mmol), and KI (0.12 g,
0.72 mmol) in dry dichloromethane (25 mL) was stirred at room
temperature for 24 h and filtered (6). The filtrate was washed
with 5% NaHCO3 liquor (3 · 15 mL), dried over MgSO4, filtered,
and concentrated in vacuo. The residue was purified by silica
gel column chromatography (petroleum ether ⁄ ethyl acetate, 5:1)
to afford the corresponding ester. Compounds 2a–2o were pre-
pared by the same method using compound 2 as a reagent. The
yield, melting point, and spectra data of each compound are
given below.
With an aim to increase the antitumor effects of DHEA, structural
modifications were carried out at positions 3 and 17 of DHEA. Two
523

Liu et al.
DHEA-3-yl benzoate (1a)
DHEA-3-yl hexa-2,4-dienoate (1g)
Yield, 52%; mp 180–182 ꢀC; IR (KBr) per cm: 3267, 2958, 1735,
1712, 1632, 1450, 1378; H-NMR (300 MHz, CDCl3) d: 0.86–0.2.51
Yield, 52%; mp 175–177 ꢀC; IR (KBr) per cm: 2962, 1741, 1712,
1647, 1620, 1375; H-NMR (300 MHz, CDCl3) d: 0.88–2.44 (m, 30H,
1
1
(m, 25H, protons in dehydroepiandrosterone skeleton), 4.85–4.88 (m,
1H, CH–3), 5.45 (d, J = 3.0 Hz, 1H, CH–6), 7.44 (t, J = 15 Hz, 2H,
Ar–H), 7.54 (t, J = 9.0 Hz, 1H, Ar–H), 8.04 (d, J = 6.0 Hz, 2H, Ar–
H); MALDI-TOF MS: 393 (M+1); Anal. Calcd for C26H32O3: C, 79.56;
H, 8.22. Found: C, 79.44; H, 8.32.
protons in dehydroepiandrosterone skeleton and CH3–25 and CH2),
4.64–4.72 (m, 1H, CH–3), 5.41 (d, J = 4.8 Hz, 1H, CH–6), 5.75 (m,
1H, C=CH), 6.15 (m, 2H, CH=CH), 7.24 (m, 1H, C=CH); MALDI-TOF
MS: 383 (M+1); Anal. Calcd for C25H34O3: C, 78.49; H, 8.96. Found:
C, 78.51; H, 8.86.
DHEA-3-yl 4-fluorobenzoate (1b)
DHEA-3-yl pent-4-enoate (1h)
Yield, 50%; mp 173–175 ꢀC; IR (KBr) per cm: 2947, 1737, 1703,
1649, 1451, 1373;1H-NMR (300 MHz, CDCl3) d: 0.90–2.49 (m, 25H,
protons in dehydroepiandrosterone skeleton), 4.85–4.87 (m, 1H, CH–
3), 5.46 (d, J = 6.0 Hz, 1H, CH–6), 7.11 (t, J = 7.5 Hz, 2H, Ar–H),
8.05 (t, J = 7.5 Hz, 2H, Ar-H); MALDI-TOF MS: 411 (M+1); Anal.
Calcd for C26H31FO3: C, 76.07; H, 7.61. Found: C, 76.21; H, 7.55.
Yield, 55%; mp 168–170 ꢀC; IR (KBr) per cm: 2947, 1737, 1703,
1649, 1375; 1H-NMR (300 MHz, CDCl3) d: 0.89–2.48 (m, 29H,
protons in dehydroepiandrosterone skeleton and 4H,CH2–21, 22),
4.61–4.64 (m, 1H, CH–3), 4.98–5.89 (m, 2H, C=CH2), 5.18 (d,
J = 3.2 Hz, 1H, CH–6), 5.83 (m, 1H,CH=C); MALDI-TOF MS: 371
(M+1); Anal. Calcd for C24H34O3: C, 77.80; H, 9.25. Found: C,
77.85; H, 9.15.
DHEA-3-yl cinnamate (1c)
Yield, 56%; mp 150–150 ꢀC; IR (KBr) per cm: 2941, 1739, 1707,
1635, 1452, 1374; H-NMR (300 MHz, CDCl3) d: 0.90–2.45 (m, 25H,
DHEA-3-yl 2-methylbut-2-enoate (1i)
1
Yield, 52%; mp 165–167 ꢀC; IR (KBr) per cm: 2962, 2945, 1739,
1
protons in dehydroepiandrosterone skeleton), 4.75–4.77 (m, 1H, CH–
3), 5.44 (d, J = 3.0 Hz, 1H, CH–6), 6.44 (d, J = 15 Hz, 1H, CH=C),
7.38–7.54 (m, 5H, Ar–H), 7.69 (d, J = 15 Hz, 1H, C=CH); MALDI-TOF
MS: 393 (M+1); Anal. Calcd for C28H34O3: C, 80.35; H, 8.19. Found:
C, 80.23; H, 8.22.
1707, 1649, 1375; H-NMR (300 MHz, CDCl3) d: 0.89–2.50 (m, 31H,
protons in dehydroepiandrosterone skeleton and 6H, CH3–23, 24),
4.66 (m, 1H, CH–3), 5.40 (d, J = 3.0 Hz, 1H, CH–6), 6.83–6.84 (m,
CH=C); MALDI-TOF MS: 371 (M+1); Anal. Calcd for C24H34O3: C,
77.80; H, 9.25. Found: C, 77.88; H, 9.10.
DHEA-3-yl 3-(2-chlorophenyl)acrylate (1d)
DHEA-3-yl 2-methylpent-2-enoate (1j)
Yield, 52%; mp 198–200 ꢀC; IR (KBr) per cm: 2946, 1738, 1717,
1590, 1455, 1376; H-NMR (300 MHz, CDCl3) d: 0.89–2.51 (m, 25H,
Yield, 50%; mp 161–162 ꢀC; IR (KBr) per cm: 2959, 2935, 1736,
1710, 1634, 1377; 1H-NMR (300 MHz, CDCl3) d: 0.89–2.44 (m,
33H, protons in dehydroepiandrosterone skeleton and 6H, CH3–
24, 25 and 2H, CH2–23), 4.65–4.68 (m, 1H, CH–3), 5.40 (d,
J = 1.5 Hz, 1H, CH–6), 6.70–6.75 (m, CH=C); MALDI-TOF MS: 385
(M+1); Anal. Calcd for C25H36O3: C, 78.08; H, 9.44. Found: C,
78.28; H, 9.50.
1
protons in dehydroepiandrosterone skeleton), 4.71–4.78 (m, 1H, CH–
3), 5.44 (d, J = 3.0 Hz, 1H, CH–6), 6.72 (d, J = 15 Hz, 1H, CH=C),
7.28–7.50 (m, 4H, Ar–H), 7.59 (d, J = 15 Hz, 1H, C=CH); MALDI-TOF
MS: 453 (M+1); Anal. Calcd for C28H33ClO3: C, 74.24; H, 7.34.
Found: C, 74.44; H, 7.22.
DHEA-3-yl acrylate (1e)
DHEA-3-yl hex-2-enoate (1k)
Yield, 54%; mp 151–153 ꢀC; IR (KBr) per cm: 2948, 1730, 1717,
1380; H-NMR (300 MHz, CDCl3) d: 1.06–2.51 (m, 25H, protons in
Yield, 56%; mp 143–145 ꢀC; IR (KBr) per cm: 2961, 2955, 1735,
1717, 1639, 1381; H-NMR (300 MHz, CDCl3) d: 0.88–2.41 (m, 32H,
1
1
dehydroepiandrosterone skeleton), 4.68–4.71 (m, 1H, CH–3), 5.42 (d,
J = 4.2 Hz, 1H, CH–6), 5.81 (d, J = 10.5 Hz, 1H, C=CH2a), 6.10 (dd,
J = 10.5, 10.2 Hz, 1H, C=CH), 6.39 (d, J = 17.4 Hz, 1H, C=CH2b);
MALDI-TOF MS: 343 (M+1); Anal. Calcd for C22H30O3: C, 77.16; H,
8.83. Found: C, 77.20; H, 8.81.
protons in dehydroepiandrosterone skeleton and 3H, CH3–25 and
4H, CH2–23, 24), 4.61–4.65 (m, 1H, CH–3), 5.40 (d, J = 2.4 Hz, 1H,
CH–6), 5.78–5.82 (m, C=CH–22), 6.92–6.97 (m, C=CH–21); MALDI-
TOF MS: 385 (M+1); Anal. Calcd for C25H36O3: C, 78.08; H, 9.44.
Found: C, 78.05; H, 9.40.
DHEA-3-yl but-2-enoate (1f)
DHEA-3-yl undec-10-enoate (1l)
Yield, 54%; mp 218–220 ꢀC; IR (KBr) per cm: 2947, 1731, 1714,
1383; 1H-NMR (300 MHz, CDCl3) d: 0.88–2.41 (m, 28H, protons
in dehydroepiandrosterone skeleton and CH3–23), 4.61–4.70 (m,
1H, CH–3), 5.41 (d, J = 4.8 Hz, 1H, CH–6), 5.82 (dd, J = 1.5,
3 Hz, 1H, C=CH), 6.94 (m, 1H, CH=C); MALDI-TOF MS: 357
(M+1); Anal. Calcd for C23H32O3: C, 77.49; H, 9.05. Found: C,
77.40; H, 9.15.
Yield, 54%; mp oil; IR (KBr) per cm: 2964, 2945, 1737, 1718,
1635, 1380; 1H-NMR (300 MHz, CDCl3) d: 0.88–2.34 (m, 41H,
protons in dehydroepiandrosterone skeleton and 16H, CH2), 4.58–
4.63 (m, 1H, CH–3), 4.91–4.95 (m, 2H, C=CH2), 5.40 (d, J = 3 Hz,
1H, CH–6), 5.76–5.85 (m, 1H, CH=C); MALDI-TOF MS: 455 (M+1);
Anal. Calcd for C30H46O3: C, 79.25; H, 10.20. Found: C, 79.15; H,
10.40.
524
Chem Biol Drug Des 2012; 79: 523–529

Synthesis and Antitumor Activity
DHEA-3-yl octadeca-9,12-dienoate (1m)
protons in dehydroepiandrosterone skeleton), 4.74–4.79 (m, 2H, CH–
3, 17), 5.43 (d, J = 3.0 Hz, 1H, CH–6), 6.40–6.48 (dd, J = 6.0, 9 Hz,
2H, CH–21, 30), 7.38–7.70 (m, 10H, Ar–H); MALDI-TOF MS: 551
(M+1); Anal. Calcd for C37H42O4: C, 80.69; H, 7.69. Found: C, 80.72;
H, 7.65.
Yield, 58%; mp oil; IR (KBr) per cm: 2962, 2943, 1735, 1716, 1632,
1
1381; H-NMR (300 MHz, CDCl3) d: 0.86 (s, 3H, CH3–37), 0.87–2.40
(m, 47H, protons in dehydroepiandrosterone skeleton and 22H, CH2),
2.74–2.75 (m, 2H, CH2–30), 4.58–4.64 (m, 1H, CH–3), 5.27–5.57 (m,
5H, CH=C, C–6, 28, 29, 31, 32); MALDI-TOF MS: 551 (M+1); Anal.
Calcd for C37H58O3: C, 80.67; H, 10.61. Found: C, 79.89; H, 10.80.
(2E,2¢E)-(3S,8R,9S,10R,13S,14S,17S)-10,13-
dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-
3,17-diyl bis(3-(2-chlorophenyl)acrylate) (2d)
Yield, 52%; mp 198–200 ꢀC; IR (KBr) per cm: 2959, 1732, 1721,
DHEA-3-yl octadec-9-enoate (1n)
Yield, 51%; mp oil; IR (KBr) per cm: 2959, 2941, 1732, 1713, 1630,
1
1379; H-NMR (300 MHz, CDCl3) d: 0.85 (s, 3H, CH3–37), 0.88–2.77
1
(m, 53H, protons in dehydroepiandrosterone skeleton and 28H, CH2),
4.59–4.62 (m, 1H, CH–3), 5.33–5.41 (m, 3H, CH=C, C–6, 28, 29);
MALDI-TOF MS: 553 (M+1); Anal. Calcd for C37H58O3: C, 80.38; H,
10.95. Found: C, 80.22; H, 10.96.
1654, 1451, 1337; H-NMR (300 MHz, CDCl3) d: 0.85–2.42 (m, 25H,
protons in dehydroepiandrosterone skeleton), 4.72–4.78 (m, 2H, CH–
3, 17), 5.41 (d, J = 2.1 Hz, 1H, CH–6), 6.39–6.48 (dd, J = 9.0, 6 Hz,
2H, CH–21, 30), 7.28–7.41 (m, 8H, Ar–H); 7.57–7.62 (m, 2H, CH–22,
31); MALDI-TOF MS: 619 (M+1); Anal. Calcd for C37H40Cl2O4: C,
71.72; H, 6.51. Found: C, 71.61; H, 6.55.
DHEA-3-yl henicos-13-enoate (1o)
Yield, 60%; mp iol; IR (KBr) per cm: 2954, 2945, 1729, 1708, 1630,
1
1378; H-NMR (300 MHz, CDCl3) d: 0.86 (s, 3H, CH3–37), 0.89–2.51
(3S,8R,9S,10R,13S,14S,17S)-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-
3,17-diyl diacrylate (2e)
(m, 61H, protons in dehydroepiandrosterone skeleton and 36H, CH2),
4.60–4.63 (m, 1H, CH–3), 5.33–5.40 (m, 3H, CH=C, C–6, 32, 33);
MALDI-TOF MS: 609 (M+1); Anal. Calcd for C41H68O3: C, 80.86; H,
11.25. Found: C, 80.92; H, 11.05.
Yield, 49%; mp 113–115 ꢀC; IR (KBr) per cm: 2945, 1732, 1721,
1
1383; H-NMR (300 MHz, CDCl3) d: 0.84–2.39 (m, 25H, protons in
dehydroepiandrosterone skeleton), 4.67–4.72 (m, 2H, CH–3, 17),
5.40 (d, J = 2.4 Hz, 1H, CH–6), 5.80 (d, J = 12 Hz, 2H, C=CH–22a,
25b), 6.07–6.16 (m, 2H, C=CH–21, 24), 6.38 (d, J = 15 Hz, 2H,
C=CH–22b, 25b); MALDI-TOF MS: 399 (M+1); Anal. Calcd for
C37H40O4: C, 75.34; H, 8.60. Found: C, 75.14; H, 8.55.
(3S,8R,9S,10R,13S,14S,17S)-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-
3,17-diyl dibenzoate (2a)
Yield, 52%; mp 135–137 ꢀC; IR (KBr) per cm: 3266, 2959, 1733,
1
1710, 1631, 1451, 1381; H-NMR (300 MHz, CDCl3) d: 0.96–3.02 (m,
25H, protons in dehydroepiandrosterone skeleton), 4.83–4.89 (m,
2H, CH–3, 17), 5.45 (d, J = 3.0 Hz, 1H, CH–6), 7.44 (m, 4H, Ar–H),
7.54 (m, Hz, 2H, Ar–H), 8.05 (m, 4H, Ar–H). MALDI-TOF MS: 499
(M+1); Anal. Calcd for C33H38O4: C, 79.48; H, 7.68. Found: C, 79.21;
H, 7.55.
(2E,2¢E)-(3S,8R,9S,10R,13S,14S,17S)-10,13-
dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-
3,17-diyl bis(but-2-enoate) (2f)
Yield, 57%; mp 132–134 ꢀC; IR (KBr) per cm: 2962, 1735, 1710,
1
1385; H-NMR (300 MHz, CDCl3) d: 0.83–2.36 (m, 31H, protons in
dehydroepiandrosterone skeleton and 6H, CH3–23, 27), 4.63–4.69
(m, 2H, CH–3, 17), 5.38 (d, J = 6 Hz, 1H, CH–6), 5.78–5.87 (m, 2H,
C=CH–21, 25), 6.91–6.99 (m, 2H, C=CH–23, 26); MALDI-TOF MS:
427 (M+1); Anal. Calcd for C27H38O4: C, 76.02; H, 8.98. Found: C,
76.14; H, 8.95.
(3S,8R,9S,10R,13S,14S,17S)-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-
3,17-diyl bis(4-fluorobenzoate) (2b)
Yield, 50%; mp 161–163 ꢀC; IR (KBr) per cm: 2950, 1737, 1703,
1
1649, 1451, 1373; H-NMR (300 MHz, CDCl3) d: 0.96–2.48 (m, 25H,
protons in dehydroepiandrosterone skeleton), 4.85–4.87 (m, 2H, CH–
3, 17), 5.44 (d, J = 2.7 Hz, 1H, CH–6), 7.11 (m, 4H, Ar–H), 8.05 (m,
4H, Ar–H); MALDI-TOF MS: 535 (M+1); Anal. Calcd for C33H38O4: C,
74.14; H, 6.79. Found: C, 74.22; H, 6.65.
(2E,2¢E,4E,4¢E)-(3S,8R,9S,10R,13S,14S,17S)-
10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-
3,17-diyl bis(hexa-2,4-dienoate) (2g)
Yield, 53%; mp 181–183 ꢀC; IR (KBr) per cm: 2964, 1737, 1721,
1
(2E,2¢E)-(3S,8R,9S,10R,13S,14S,17S)-10,13-
dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-
3,17-diyl bis(3-phenylacrylate) (2c)
1647, 1622, 1381; H-NMR (300 MHz, CDCl3) d: 0.83–2.42 (m, 31H,
protons in dehydroepiandrosterone skeleton and 6H, CH3–23, 27),
4.73–4.76 (m, 2H, CH–3, 17), 5.43 (d, J = 3 Hz, 1H, CH–6), 6.39–
6.48 (m, 2H, C=CH–24, 30), 7.26–7.38 (m, 4H, C=CH–21, 23, 27, 29),
7.52–7.63 (m, 2H, C=CH–24, 28); MALDI-TOF MS: 479 (M+1); Anal.
Calcd for C31H42O4: C, 77.79; H, 8.84. Found: C, 78.04; H, 8.45.
Yield, 53%; mp 150–152 ꢀC; IR (KBr) per cm: 2964, 1730, 1713,
1
1645, 1450, 1380; H-NMR (300 MHz, CDCl3) d: 0.89–2.44 (m, 25H,
Chem Biol Drug Des 2012; 79: 523–529
525

Liu et al.
(3S,8R,9S,10R,13S,14S,17S)-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-
3,17-diyl bis(pent-4-enoate) (2h)
protons in dehydroepiandrosterone skeleton and 12H, CH3–24, 25,
30, 31), 2.35–2.37 (m, 4H, CH2–23, 29), 4.61–4.67 (m, 2H, CH–3,17),
5.38 (d, J = 3.0 Hz, 1H, CH–6), 6.72–6.96 (m, 2H, C=CH–22, 28);
MALDI-TOF MS: 483 (M+1); Anal. Calcd for C31H46O4: C, 77.14; H,
9.61. Found: C, 77.98; H, 9.23.
Yield, 50%; mp 143–145 ꢀC; IR (KBr) per cm: 2946, 1735, 1713,
1
1639, 1382; H-NMR (300 MHz, CDCl3) d: 0.80–2.39 (m, 33H, pro-
tons in dehydroepiandrosterone skeleton and 8H, CH2–21, 22, 26,
27), 4.58–4.64 (m, 2H, CH–3, 17), 4.98–5.08 (m, 4H, C=CH–24, 29),
5.37 (d, J = 3.6 Hz, 1H, CH–6), 5.78–5.86 (m, 2H, C=CH–23, 28);
MALDI-TOF MS: 455 (M+1); Anal. Calcd for C29H42O4: C, 76.61; H,
9.31. Found: C, 75.98; H, 9.54.
(2E,2¢E)-(3S,8R,9S,10R,13S,14S,17S)-10,13-
dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-
3,17-diyl hex-2-enoate (2k)
Yield, 52%; mp 154–156 ꢀC; IR (KBr) per cm: 2965, 2945, 1735,
1718, 1642, 1379; 1H-NMR (300 MHz, CDCl3) d: 0.83–2.37 (m,
39H, protons in dehydroepiandrosterone skeleton and 6H, CH3–
25, 31 and 8H, CH2–23, 24, 28, 29), 4.62–4.67 (m, 2H, CH–3,
17), 5.39 (d, J = 2.1 Hz, 1H, CH–6), 5.76–5.84 (m, 2H, C=CH–21,
27), 6.92–6.97 (m, 2H, C=CH–22, 28); MALDI-TOF MS: 483 (M+1);
Anal. Calcd for C31H46O4: C, 77.14; H, 9.61. Found: C, 76.98; H,
9.65.
(2E,2¢E)-(3S,8R,9S,10R,13S,14S,17S)-10,13-
dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-3,
17-diyl bis(2-methylbut-2-enoate) (2i)
Yield, 52%; mp 198–200 ꢀC; IR (KBr) per cm: 2965, 2943, 1738,
1
1717, 1645, 1380; H-NMR (300 MHz, CDCl3) d: 0.85–2.37 (m, 37H,
protons in dehydroepiandrosterone skeleton and 12H, CH3–23, 24,
28, 29), 4.61–4.67 (m, 2H, CH–3, 17), 5.37 (d, J = 3.0 Hz, 1H, CH–
6), 6.82–6.83 (m, 2H, C=CH–22, 27); MALDI-TOF MS: 455 (M+1);
Anal. Calcd for C29H42O4: C, 76.61; H, 9.31. Found: C, 76.54; H,
9.32.
(3S,8R,9S,10R,13S,14S,17S)-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-
3,17-diyl bis(undec-10-enoate) (2l)
Yield, 52%; mp oi; IR (KBr) per cm: 2961, 2947, 1738, 1719, 1638,
1
(2E,2¢E)-(3S,8R,9S,10R,13S,14S,17S)-10,
13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,
17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3,
17-diyl bis(2-methylpent-2-enoate) (2j)
1382; H-NMR (300 MHz, CDCl3) d: 0.81–2.32 (m, 57H, protons in
dehydroepiandrosterone skeleton and 16H, CH2), 4.58–4.63 (m, 2H,
CH–3, 17); 4.91–4.95 (m, 4H, C=CH–30, 41), 5.37 (d, J = 4.5 Hz, 1H,
CH–6), 5.74–5.85 (m, 2H, C=CH–29, 40). MALDI-TOF MS: 623 (M+1);
Anal. Calcd for C41H66O4: C, 79.05; H, 10.68. Found: C, 79.15; H,
10.54.
Yield, 57%; mp 123–125 ꢀC; IR (KBr) per cm: 2952, 2939, 1736,
1
1715, 1630, 1379; H-NMR (300 MHz, CDCl3) d: 0.83–2.37 (m, 37H,
O
O
a
O
RCOOH
HO
R
O
1a-1o
O
b
O
OH
R
a
O
RCOOH
R
O
HO
2
2a-2o
a: C6H5-
b: p-F-C6H4-
R
c: C6H5-CH=CH-
d: m-Cl-C6H4-CH=CH-
f: CH3CH=CH-
e: CH2=CH-
g: CH3CH=CHCH=CH-
i: CH3CH=C(CH3)-
k: CH3CH2CH2CH=CH-
h: CH2=CHCH2CH2-
j: CH3CH2CH=C(CH3)-
l: CH2=CH(CH2)7CH2-
m: CH3(CH2)4CH=CHCH2CH=CH(CH2)6CH2-
o: CH3(CH2)7CH=CH(CH2)10CH2-
n: CH3(CH2)7CH=CH(CH2)6CH2-
Scheme 1: Synthetic scheme for the synthesis of compounds 1a–1o, 2a–2o. Reagents and conditions: (a) DCC, DMAP, CH2Cl2;
(b) NaBH4, MeOH.
526
Chem Biol Drug Des 2012; 79: 523–529

Synthesis and Antitumor Activity
(9Z,9¢Z,12Z,12¢Z)-(3S,8R,9S,10R,13S,14S,17S)-
10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,
17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3,
17-diyl bis(octadeca-9,12-dienoate) (2m)
Table 1: The growth inhibitory (IC50 € SD, lM, 48 h) of DHEA,
1a–1o and 2a–2o in ES-2, A549 and HepG2 cells. p < 0.05
IC50 (lM)
Yield, 49%; mp oil; IR (KBr) per cm: 2966, 2948, 1735, 1720, 1636,
1380; H-NMR (300 MHz, CDCl3) d: 0.79–2.32 (m, 75H, protons in
Compound
ES-2
A549
Hep G2
1
1a
1b
1c
1d
1e
1f
22.5 € 0.7
43.2 € 0.2
23.6 € 0.3
31.8 € 0.1
11.6 € 0.4
10.7 € 0.5
19.4 € 0.3
21.8 € 0.5
12.2 € 0.4
34.6 € 0.3
14.7 € 0.4
26.8 € 0.6
22.8 € 0.8
22.9 € 0.7
>100
18.3 € 0.4
17.0 € 0.3
12.3 € 0.4
14.5 € 0.7
22.6 € 0.1
16.1 € 0.5
26.5 € 0.2
15.9 € 0.1
6.9 € 0.2
24.6 € 0.4
21.3 € 0.3
8.6 € 0.6
27.1 € 0.3
31.1 € 0.9
18.5 € 0.4
25.5 € 0.7
51.2 € 0.5
37.8 € 0.4
>100
12.1 € 0.3
20.2 € 0.2
13.9 € 0.1
17.1 € 0.2
27.2 € 0.3
22.1 € 0.4
21.6 € 0.5
>100
>100
>100
21.2 € 0.1
>100
>100
>100
>100
>100
25.5 € 0.4
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
15.4 € 0.4
24.5 € 0.3
21.2 € 0.2
11.1 € 0.9
27.2 € 0.3
10.9 € 0.1
15.5 € 0.3
17.6 € 0.8
11.7 € 0.1
19.2 € 0.5
10.6 € 0.4
18.0 € 0.6
13.2 € 0.5
11.2 € 0.6
9.1 € 0.3
18.3 € 0.5
dehydroepiandrosterone skeleton and 44H, CH2 and 6H, CH3–37,
55), 2.74–2.78 (m, 4H, CH2–30, 48), 4.58–4.64 (m, 2H, CH–3, 17),
5.28–5.36 (m, 9H, C=CH–6, 28, 29, 31, 32, 46, 47, 49, 50); MALDI-
TOF MS: 815 (M+1); Anal. Calcd for C55H90O4: C, 81.02; H, 11.13
Found: C, 81.05; H, 11.24.
1g
1h
1i
(9Z,9¢Z)-(3S,8R,9S,10R,13S,14S,17S)-10,13-
dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-
3,17-diyl bis(octadec-9-enoate) (2n)
1j
1k
1l
1m
1n
1o
2a
2b
2c
2d
2e
2f
Yield, 55%; mp oil; IR (KBr) per cm: 2963, 2938, 1731, 1715, 1630,
1
1378; H-NMR (300 MHz, CDCl3) d: 0.80–2.29 (m, 87H, protons in
dehydroepiandrosterone skeleton and 56H, CH2 and 6H, CH3–37,
55), 4.59–4.63 (m, 2H, CH–3,17), 5.30–5.34 (m, 5H, C=CH–6, 28, 29,
46, 47); MALDI-TOF MS: 819 (M+1); Anal. Calcd for C55H94O4: C,
81.63; H, 11.56 Found: C, 81.75; H, 11.55.
2g
2h
2i
(13Z,13¢Z)-(3S,8R,9S,10R,13S,14S,17S)-10,13-
dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthrene-
3,17-diyl bis(docos-13-enoate) (2o)
2j
16.4 € 0.3
23.8 € 0.4
26.7 € 0.2
18.2 € 0.8
18.9 € 0.1
17.7 € 0.6
17.5 € 0.3
2k
2l
Yield, 56%; mp oil; IR (KBr) per cm: 2965, 2945, 1734, 1710, 1632,
2m
2n
2o
DHEA
1
1378; H-NMR (300 MHz, CDCl3) d: 0.81–2.31 (m, 103H, protons in
17.2 € 0.2
>100
18.8 € 0.4
dehydroepiandrosterone skeleton and 72H, CH2 and 6H, CH3–37,
55), 4.58–4.62 (m, 2H, CH–3, 17), 5.32–5.35 (m, 5H, C=CH–6, 32,
33, 54, 55); MALDI-TOF MS: 932 (M+1); Anal. Calcd for C63H110O4:
C, 81.23; H, 11.90 Found: C, 81.35; H, 11.73.
DHEA, Dehydroepiandrosterone.
Cell culture
The ovarian cancer ES-2 cells, human hepatocellular carcinoma
HepG2 cells, and human lung tumor A-549 cells were routinely
maintained in Dulbecco's modified Eagle's minimal essential med-
ium (DMEM) supplemented with 10% heat-inactivated fetal bovine
serum, 100 units ⁄ mL penicillin and streptomycin antibiotics in a
humidified atmosphere containing 5% CO2 at 37 ꢀC. Cell culture
plates were washed to remove non-adherent cells, and adherent
cells were over 95% viable as determined with a trypan blue dye
exclusion test. The medium was changed every 2 days, and cells
were passaged after treatment with a solution of 0.05% tryp-
sin ⁄ 0.02% EDTA.
Table 2: Apoptosis induction was determined by FACS. Es-2
cells were treated with 2i (6.99 lM), A549 cells were treated with
1c (8.65 lM) and 2 cells were treated with 2o (9.17 lM).The com-
pounds were administrated with the indicated concentrations for
48 h. Apoptotic cells were determined after propidium iodide stain-
ing
Ap (%)
Compounds
Es-2
2.36
11.44
–
–
A549
HepG2
Normal
2i
1c
0.33
–
17.70
–
2.47
–
–
2o
28.81
Evaluation of antitumor activities in vitro
Ap, apoptotic cells. (–) The experiment did not test.
The antitumor activities of the thirty synthesized compounds were
evaluated against the three different human tumor cell lines, ovar-
ian cancer cells (ES-2), human lung tumor cells (A549), and human
hepatocellular liver carcinoma cells (HepG2), using the 3-(4,5-dim-
ethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (7),
which is based on the ability of glycolytic pathway enzymes to
cleave MTT to the blue compound formazan. MTT was added during
the last 3 h of incubation. The reduction of MTT to formazan was
measured using a precision microplate reader at 492 nm. The
Chem Biol Drug Des 2012; 79: 523–529
527

Liu et al.
Figure 1: Dose-dependent growth inhibitory of compounds Dehydroepiandrosterone (DHEA), 2i, 1c and 2o on different human tumor
cells. Cells were treated with indicated compounds for 48 h. Cell viability was measured by MTT assay and calculated as percent of control
untreated cultures. p < 0.05. a: Dose-dependent growth inhibitory of compound DHEA on Es-2 cells. b: Dose-dependent growth inhibitory of
compound 2i on Es-2 cells. c: Dose-dependent growth inhibitory of compound DHEA on A549 cells. d: Dose-dependent growth inhibitory of
compound 1c on A549 cells. e: Dose-dependent growth inhibitory of compound DHEA on HepG2 cells. f: Dose-dependent growth inhibitory of
compound 2o on HepG2 cells.
compounds were solubilized in DMSO, and a sample of each solu-
tion was added to the medium. The cancer cells were cultured and
incubated with test compounds at concentrations ranging from 0 to
100 lM for 48 h. The MTT assay was performed in triplicate for
each compound. Data were expressed as mean € SD. Statistical
significance was determined using one-way analysis of variance
(ANOVA) followed by Dunnett's contrast. Values of p < 0.05 were
considered statistically significant.
For the A549 cells, all of the mono-acylated compounds displayed
antitumor effects. Compound 1c, a cinnamic acid ester, showed the
most potent growth inhibitory effects (IC50 = 8.65 lM). For the bis-
acids derivatives, five compounds showed activity and only 2n
exhibited more potent than DHEA (IC50 = 18.86 lM).
For the HepG2 cells, most bis-acids derivatives showed more potent
growth inhibitory activity than DHEA, while the mono-acylated
esters showed no potency. Compound 2o, bearing the longest car-
bon chain acyl group, showed the most effective cytotoxic effect
(IC50 = 9.17 lM).
Results and Discussion
Chemistry
The apoptotic effects of these compounds were further confirmed
by subG1 induction, which was determined by flow cytometry (8).
As shown in Table 2, the subG1 phase (apoptotic cells) induced by
compound 2i (6.99 lM), 1c (8.65 lM), and 2o (9.17 lM) were
11.44%, 17.70% and 28.81%, respectively, after 48 h treatment.
The dose-dependent growth inhibition of the most effective com-
pounds 2i, 1c, and 2o are shown in Figure 1. These compounds
were more potent inhibitors than DHEA at the same concentration.
The synthesis of compounds 1a–1o and 2a–2o were presented in
Scheme 1. Dehydroepiandrosterone was treated with various unsat-
urated fatty acids (or arylcarboxylic acids) in dry dichloromethane in
the presence of DCC and DMAP at room temperature (24 h) to
afford a moderate yields of the corresponding esters 1a–1o. Com-
pound 2 was obtained by reducing compound 1 with NaBH4 in
methanol, followed by acylation with unsaturated fatty acids to give
the DHEA esters 2a–2o.
Conclusions
Biological results and discussion
As shown in Table 1, most tested compounds showed antitumor
effects on the three cell lines. For the ES-2 cells, all the com-
pounds, except 1o (IC50 > 100 lM) which has the longest carbon
chain acyl group at the 3-position, showed activity and eleven
compounds exhibited more potent antitumor effects than DHEA
(IC50, 17.56 lM). Compound 2i showed the most potent activity
(IC50, 6.99 lM), near 2.5 times better than DHEA. Among the
compounds, which contained a phenyl ring, the bis-acids esters
(2a–2d) exhibited more efficacy than mono-acylated esters
(1a–1d). For the unsaturated fatty acid esters, no significant dif-
ferences were found between the mono and bis-acids deriva-
tives.
In conclusion, we have synthesized two series of DHEA derivatives
and investigated their growth inhibitory activity against three differ-
ent human tumor cell lines. Several derivatives demonstrated stron-
ger potency than DHEA. The most promising compounds in this
series were 2i, 1c, and 2o. Further investigations into optimizing
the inhibitory activity of these three compounds are in progress.
Acknowledgment
This work was supported by the National Natural Science Founda-
tion of China (No. 20962021).
528
Chem Biol Drug Des 2012; 79: 523–529

Synthesis and Antitumor Activity
5. Graf M.R., Jia W., Lewbart M.L., Loria R.M. (2009) The anti-
tumor effects of androstene steroids exhibit a strict structure-
activity relationship dependent upon the orientation of the
hydroxyl group on carbon-17. Chem Biol Drug Des;74:625–629.
6. Banday M.R., Farshori N.N., Ahmad A., Khan A.U, Rauf A. (2010)
Synthesis and characterization of novel fatty acid analogs of cho-
lesterol: in vitro antimicrobial activity. Eur J Med Chem;45:1459–
1464.
7. Lian L.H., Park E.J., Piao H.S, Zhao Y.Z., Sohn D.H. (2005) Aloe
emodin-induced apoptosis in t-HSC ⁄ Cl-6 cells involves a mito-
chondria-mediated pathway. Basic Clin Pharmacol Toxicol;96:495–
502.
References
1. Yang N.C., Jeng K.C., Ho W.M., Chou S.J., Hu M.L. (2000) DHEA
inhibits cell growth and induces apoptosis in BV-2 cells and the
effects are inversely associated with glucose concentration in
the medium. J Steroid Biochem Mol Biol;75:159–166.
2. Mo Q., Lu S., Garippa C., Brownstein M.J., Simon N.G. (2009)
Genome-wide analysis of DHEA- and DHT-induced gene expres-
sion in mouse hypothalamus and hippocampus. J Steroid Bio-
chem Mol Biol;114:135–143.
3. Kroboth P.D., Salek F.S., Pittenger A.L., Fabian T.J., Frye R.F.
(1999) DHEA and DHEA-S: a review. J Clin Pharmacol;39:327–
348.
4. Shi B., Tang P., Hu X., Liu J.O., Yu B. (2005) OSW saponins: facile
synthesis toward a new type of structures with potent antitumor
activities. J Org Chem;70:10354–10367.
8. Liu D., Song D., Guo G., Wang R., Lv J., Jing Y., Zhao L. (2007)
The synthesis of 18beta-glycyrrhetinic acid derivatives which
have increased antiproliferative and apoptotic effects in leukemia
cells. Bioorg Med Chem;15:5432–5439.
Chem Biol Drug Des 2012; 79: 523–529
529