Synthesis and biological evaluation of novel steroidal[17,16-d][1,2,4] triazolo[1,5-a]pyrimidines

Article abstract of DOI:10.1016/j.steroids.2012.03.002

The preparation of steroidal[17,16-d][1,2,4]triazolo[1,5-a]pyrimidines and their biological evaluation as potential anticancer agents are herein reported. These novel heterosteroids (2, 4) were prepared through the condensation reaction of 3-amino-1,2,4-t

Full text of DOI:10.1016/j.steroids.2012.03.002

Steroids 77 (2012) 710–715
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and biological evaluation of novel steroidal[17,16-d][1,2,4]triazolo
[1,5-a]pyrimidines
Li-Hua Huang a,b,c, Yong-Fei Zheng a,b, Yong-Zheng Lu c, Chuan-Jun Song c, Yan-Guang Wang a,b
,
Bin Yu a,b, Hong-Min Liu a,b,
⇑
a School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
b New Drug Research & Development Center, Zhengzhou University, Zhengzhou 450001, China
c Department of Chemistry, Zhengzhou University, Zhengzhou 450001, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The preparation of steroidal[17,16-d][1,2,4]triazolo[1,5-a]pyrimidines and their biological evaluation as
potential anticancer agents are herein reported. These novel heterosteroids (2, 4) were prepared through
the condensation reaction of 3-amino-1,2,4-triazole with 16-arylidene-17-ketosteroids (1, 3). All the syn-
thesized compounds were evaluated for their anticancer activity in vitro against PC-3 (human prostatic
carcinoma), MCF-7 (human breast carcinoma) and EC9706 (human esophageal carcinoma) cell lines.
Among the screened compounds, 2i, 2n and 4f showed significant inhibitory activity against all the three
human cell lines.
Received 14 January 2012
Received in revised form 25 February 2012
Accepted 5 March 2012
Available online 14 March 2012
Keywords:
Heterosteroids
Steroidal[17,16-d]triazolopyrimidines
1,2,4-Triazolo[1,5-a]pyrimidines
Anticancer activity
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
widely known triazolopyrimidine derivative is the simple molecule
of Trapidil, which acts as a platelet-derived growth factor antago-
During the last decades, steroids bearing heterocycles fused to
the A- or D-ring of the steroid skeleton have been of pharmaceutical
interest as many of these heterosteroids possess widespread bio-
logical activities [1–8]. For example, Cortivazol and similar arylpy-
razolo steroids exhibited powerful glucocorticoids and have been
extensively investigated as anti-inflammatory agents [2]. Potter
et al. reported that N-substituted 1,3,5(10)-estratrien[17,16-c]pyr-
azole showed potent inhibitory activity of 17b-hydroxysteroid
dehydrogenases (17b-HSD) in T47-D human breast cancer cells
[8]. Considering the remarkable importance from the pharmacolog-
ical and synthetic viewpoints, great efforts are being made to anne-
late steroidal moiety with pyrazole, pyridine, pyran, pyrrole or
pyrimidine rings using various synthetic strategies [9–13].
On the other hand, 1,2,4-triazolo[1,5-a]pyrimidines (TPs), a sub-
type of purine analogs, have been widely investigated and identi-
fied to possess multifaceted pharmacological properties, including
antihypertensive, cardiac stimulant, antimalarial, antifungal, anti-
HBV, antimicrobial and anticancer activities [14–21]. In addition,
triazolopyrimidines are versatile ligands and their coordination
compounds can be considered as model systems for metal–ligand
interactions observed in biological systems [20a,22]. The most
nist and as a phosphodiesterase inhibitor [15b]. Cevipabulin and
its analogs, a class of triazolo[1,5-a]pyrimidines, were proved to
be potent anticancer agents with a unique mechanism of action
in promoting tubulin polymerization reported by Beyer et al. [21b].
In view of the pharmacological importance of heterosteroids as
well as triazolopyrimidines and in continuation of our previous
work in developing new bioactive modified steroids [23], recently
we reported the synthesis of novel 70-aryl-androstano[17,16-
d][1,2,4]triazolo[1,5-a]pyrimidines derivatives [24]. In order to
study the effect on the bioactivity of the aryl at C-70 of these het-
erosteroids, several new substituted aryl derivatives were synthe-
sized. Herein, we present the synthesis of these heterosteroids and
their biological evaluation for anticancer activity against PC-3,
MCF-7 and EC9706 cell lines in vitro.
2. Experimental
2.1. General remarks
All reagents and solvents used were of analytical grade pur-
chased from commercial sources. Thin-layer chromatography
(TLC) was carried out on glass plates coated with silica gel
(Qingdao Haiyang Chemical Co., G60F-254) and visualized by UV
light (254 nm). The products were purified by column chromatog-
raphy over silica gel (Qingdao Haiyang Chemical Co., 200–300
⇑
Corresponding author at: New Drug Research & Development Center, Zhengz-
hou University, Zhengzhou 450001, China. Tel./fax: +86 371 67781739.
E-mail address: liuhm@zzu.edu.cn (H.-M. Liu).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.03.002

L.-H. Huang et al. / Steroids 77 (2012) 710–715
711
mesh). Melting points were determined on a Beijing Keyi XT4A
apparatus and are uncorrected. All NMR spectra were recorded
with a Bruker DPX 400 MHz spectrometer with TMS as internal
standard in CDCl3. Chemical shifts are given as d ppm values rela-
tive to TMS. Mass spectra (MS) were recorded on Q-Tof (Waters)
mass spectrometer by electrospray ionization (ESI).
3H, 18-H), 1.00 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 180.4,
172.3, 155.9, 154.8, 141.7, 137.3, 131.0, 129.1, 127.5, 122.4, 60.6,
55.3, 51.5, 46.8, 35.9, 34.3, 33.2, 32.8, 29.0, 29.0, 28.6, 27.0, 20.7,
17.4, 11.4. HRMS (ESI): m/z cacld. for
476.2217; found, 476.2223.
C
27H31ClN5O (M+H)+,
2.2.3. 3-Oxo-4-aza-70-(3-nitrophenyl)-5
a-androstano[17,16-
2.2. General procedure for the synthesis of steroidal[17,16-
d]triazolopyrimidines 2 and 4
d][1,2,4]triazolo[1,5-a]pyrimidine (2c)
White solid, yield 64%, mp > 300 °C. 1H NMR (400 MHz, CDCl3):
d 8.68 (t, J = 1.7 Hz, 1H, Ar–H), 8.44 (dd, J = 8.3, 1.3 Hz, 1H, Ar–H),
8.38 (s, 1H, 20-H), 8.21 (d, J = 7.8 Hz, 1H, Ar–H), 7.82 (t, J = 8.0 Hz,
1H, Ar–H), 6.49 (s, 1H, 4-NH), 3.09 (dd, J = 12.2, 3.7 Hz, 1H,
The 16-arylidene-17-ketosteroids
1 or 3 (1.0 mmol) was
dissolved in n-BuOH (10 mL). To the solution was added 3-
amino-1,2,4-triazole (2.0 mmol) and t-BuOK (2.0 mmol). The
resulting mixture was refluxed for 30 h. The solvent was removed
and CH2Cl2 was added. The insoluble t-BuOK was filtered and
washed thoroughly with CH2Cl2. After removal of the solvent, the
residue was purified by silica gel chromatography with ethyl ace-
tate/petroleum ether/acetone (4:2:1) and petroleum ether/ethyl
acetate (3:2) to give the corresponding steroidal[17,16-d]triazolo-
pyrimidines 2 and 4, respectively (Scheme 1).
5a
-H), 1.21 (s, 3H, 18-H), 1.00 (s, 3H, 19-H). 13C NMR (100 MHz,
CDCl3): d 180.7, 172.3, 155.8, 155.0, 148.3, 140.1, 135.5, 130.7,
129.9, 125.7, 124.8, 123.0, 60.5, 55.3, 51.5, 46.9, 36.0, 34.3, 33.2,
32.8, 29.0, 28.8, 28.5, 26.9, 20.6, 17.5, 11.4. HRMS (ESI): m/z cacld.
for C27H31N6O3 (M+H)+, 487.2458; found, 487.2459.
2.2.4. 3-Oxo-4-aza-70-(3,4,5-trimethoxyphenyl)-5
a-
androstano[17,16-d][1,2,4]triazolo[1,5-a]pyrimidine (2d)
White solid, yield 52%, mp 197–199 °C. 1H NMR (400 MHz,
CDCl3): d 8.36 (s, 1H, 20-H), 7.01 (s, 2H, Ar–H), 6.58 (s, 1H, 4-NH),
3.92 (d, J = 17.1 Hz, 9H, Ar–(OCH3)3), 3.08 (dd, J = 12.1, 3.3 Hz, 1H,
2.2.1. 3-Oxo-4-aza-70-phenyl-5
a-androstano[17,16-d][1,2,4]
triazolo[1,5-a]pyrimidine (2a)
White solid, yield 58%, mp > 300 °C. 1H NMR (400 MHz, CDCl3):
d 8.38 (s, 1H, 20-H), 7.81 (dd, J = 6.7, 3.0 Hz, 2H, Ar–H), 7.64–7.56
(m, 3H, Ar–H), 6.40 (s, 1H, 4-NH), 3.10 (dd, J = 12.2, 3.8 Hz, 1H,
5a
-H), 1.18 (s, 3H, 18-H), 0.98 (s, 3H, 19-H). 13C NMR (100 MHz,
CDCl3): d 180.3, 172.3, 156.0, 154.8, 153.3, 142.9, 140.4, 124.0,
122.2, 107.3, 61.0, 60.6, 56.4, 55.3, 51.5, 46.7, 35.9, 34.2, 33.2,
32.8, 29.2, 29.1, 28.5, 26.9, 20.7, 17.4, 11.4. HRMS (ESI): m/z cacld.
for C30H38N5O4 (M+H)+, 532.2924; found, 532.2921.
5a
-H), 1.20 (s, 3H, 18-H), 1.00 (s, 3H, 19-H). 13C NMR (100 MHz,
CDCl3): d 180.4, 172.4, 155.8, 154.7, 143.0, 131.1, 129.6, 129.1,
128.7, 122.4, 60.6, 55.3, 51.4, 46.7, 36.0, 34.2, 33.2, 32.8, 29.0,
29.0, 28.5, 27.0, 20.7, 17.4, 11.4. HRMS (ESI): m/z cacld. for
2.2.5. 3-Oxo-4-aza-70-(4-bromophenyl)-5
a-androstano[17,16-
C
27H32N5O (M+H)+, 442.2607; found, 442.2610.
d][1,2,4]triazolo[1,5-a]pyrimidine (2e)
White solid, yield 65%, mp > 300 °C. 1H NMR (400 MHz, CDCl3):
d 8.39 (s, 1H, 20-H), 7.73 (q, J = 8.6 Hz, 4H, Ar–H), 5.98 (s, 1H, 4-NH),
2.2.2. 3-Oxo-4-aza-70-(4-chlorophenyl)-5
a-androstano[17,16-
d][1,2,4]triazolo[1,5-a]pyrimidine (2b)
3.12 (dd, J = 11.9, 3.6 Hz, 1H, 5a-H), 1.20 (s, 3H, 18-H), 1.02 (s, 3H,
White solid, yield 67%, mp > 300 °C. 1H NMR (400 MHz, CDCl3):
d 8.37 (s, 1H, 20-H), 7.78 (d, J = 8.2 Hz, 2H, Ar–H), 7.57 (d, J = 8.2 Hz,
19-H). 13C NMR (100 MHz, CDCl3): d 180.4, 172.2, 155.9, 154.8,
141.8, 132.0, 131.2, 127.9, 125.8, 122.3, 60.6, 55.3, 51.5, 46.8,
36.0, 34.3, 33.2, 32.8, 29.7, 29.0, 28.5, 27.1, 20.7, 17.4, 11.4. HRMS
2H, Ar–H), 6.56 (s, 1H, 4-NH), 3.09 (d, J = 9.5 Hz, 1H, 5
a
-H), 1.19 (s,
N
O
N
N
N
NH
N
N
NH2
Ar
Ar
t-BuOK, n-BuOH, reflux
O
N
H
O
N
H
H
2
1
H
N
N
O
N
N
NH
N
Ar
NH2
Ar
N
t-BuOK, n-BuOH, reflux
HO
HO
4
3
O
NO2
2a
2g
Ar =
2c
2i
2f
2l
4c
2b
2h
2n
4e
2d
Cl
O
2e
Br
F
O
O
2k
2j
O
N
N
O
N
O
S
2m
4d
2o
4f
S
4a
4b
O
Cl
O
O
O
N
S
4g
4h
4i
N
O
O
O
N
Scheme 1. Synthesis of 70-aryl-androstano[17,16-d][1,2,4]triazolo[1,5-a]pyrimidines.

712
L.-H. Huang et al. / Steroids 77 (2012) 710–715
(ESI): m/z cacld. for C27H31BrN5O (M+H)+, 520.1712; found,
520.1710.
2.2.11. 3-Oxo-4-aza-70-(2-pyridyl)-5
d][1,2,4]triazolo[1,5-a]pyrimidine (2k)
a-androstano[17,16-
White solid, yield 79%, mp > 300 °C. 1H NMR (400 MHz, CDCl3):
d 8.84 (d, J = 4.1 Hz, 1H, proton of pyridine), 8.40 (s, 1H, 20-H), 8.34
(d, J = 7.9 Hz, 1H, proton of pyridine), 7.95 (td, J = 7.8, 1.2 Hz, 1H,
proton of pyridine), 7.48 (dd, J = 7.1, 5.1 Hz, 1H, proton of pyridine),
2.2.6. 3-Oxo-4-aza-70-(4-fluorophenyl)-5
a-androstano[17,16-
d][1,2,4]triazolo[1,5-a]pyrimidine (2f)
White solid, yield 68%, mp > 300 °C. 1H NMR (400 MHz, CDCl3):
6.47 (s, 1H, 4-NH), 3.09 (dd, J = 12.4, 3.7 Hz, 1H, 5a-H), 1.17 (s, 3H,
d 8.38 (s, 1H, 20-H), 7.84 (dd, J = 8.5, 5.3 Hz, 2H, Ar–H), 7.29 (t,
18-H), 0.99 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 181.1,
172.3, 156.0, 154.7, 149.9, 148.6, 140.5, 136.5, 126.4, 125.1,
124.3, 60.6, 54.8, 51.6, 46.5, 36.0, 34.3, 33.2, 32.8, 29.2, 29.0,
28.6, 27.0, 20.7, 17.4, 11.4. HRMS (ESI): m/z cacld. for C26H30N6ONa
(M+Na)+, 465.2379; found, 465.2383.
J = 8.5 Hz, 2H, Ar–H), 6.34 (s, 1H, 4-NH), 3.14– 3.06 (m, 1H, 5a-
H), 1.19 (s, 3H, 18-H), 1.00 (s, 3H, 19-H). 13C NMR (100 MHz,
CDCl3): d 180.4, 172.4, 155.9, 154.7, 143.0, 131.1, 129.6, 129.1,
128.7, 122.4, 60.6, 55.3, 51.5, 46.7, 36.0, 34.3, 33.2, 32.8, 29.0,
29.0, 28.5, 27.0, 20.7, 17.4, 11.4. HRMS (ESI): m/z cacld. for
C
27H31FN5O (M+H)+, 460.2513; found, 460.2518.
2.2.12. 3-Oxo-4-aza-70-(5-methylfuryl)-5
a-androstano[17,16-
d][1,2,4]triazolo[1,5-a]pyrimidine (2l)
2.2.7. 3-Oxo-4-aza-70-(4-methoxyphenyl)-5
a-androstano[17,16-
Yellow solid, yield 43%, mp 289–291 °C. 1H NMR (400 MHz,
CDCl3): d 8.44 (s, 1H, 20-H), 8.16 (d, J = 3.5 Hz, 1H, proton of furan),
6.36 (d, J = 3.4 Hz, 1H, proton of furan), 6.24 (s, 1H, 4-NH), 3.15 (dd,
d][1,2,4]triazolo[1,5-a]pyrimidine (2g)
White solid, yield 54%, mp 268–270 °C. 1H NMR (400 MHz,
CDCl3): d 8.37 (s, 1H, 20-H), 7.81 (d, J = 8.8 Hz, 2H, Ar–H), 7.10 (d,
J = 8.8 Hz, 2H, Ar–H), 6.36 (s, 1H, 4-NH), 3.92 (s, 3H, Ar–OCH3),
J = 12.1, 3.8 Hz, 1H, 5a-H), 2.50 (s, 3H, protons of furan), 1.12 (s, 3H,
18-H), 1.02 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 180.0,
172.3, 157.1, 155.7, 154.5, 142.8, 132.8, 122.5, 118.1, 109.6, 60.7,
54.5, 51.6, 46.1, 36.1, 34.2, 33.3, 32.8, 29.7, 29.1, 28.6, 27.2, 20.8,
3.10 (dd, J = 12.1, 3.7 Hz, 1H, 5a-H), 1.19 (s, 3H, 18-H), 1.00 (s,
3H, 19-H). 13C NMR (100 MHz, CDCl3): d 180.2, 172.3, 161.6,
156.0, 154.6, 142.9, 131.4, 121.6, 121.2, 114.1, 60.6, 55.5, 55.4,
51.5, 46.7, 36.0, 34.3, 33.2, 32.8, 29.3, 29.0, 28.6, 27.0, 20.7, 17.4,
11.4. HRMS (ESI): m/z cacld. for C28H34N5O (M+H)+, 472.2713;
found, 472.2702.
17.6, 14.2, 11.4. HRMS (ESI): m/z cacld. for
C26H31N5O2Na
(M+Na)+, 468.2375; found, 468.2379.
2.2.13. 3-Oxo-4-aza-70-(2-thienyl)-5
d][1,2,4]triazolo[1,5-a]pyrimidine (2m)
a-androstano[17,16-
2.2.8. 3-Oxo-4-aza-70-(4-dimethylaminophenyl)-5
a-
White solid, yield 51%, mp > 300 °C. 1H NMR (400 MHz, CDCl3):
d 8.48 (s, 1H, 20-H), 8.25 (d, J = 3.2 Hz, 1H, proton of thiophene),
7.80 (dd, J = 5.1, 0.8 Hz, 1H, proton of thiophene), 7.33 (dd, J = 5.0,
4.0 Hz, 1H, proton of thiophene), 6.29 (s, 1H, 4-NH), 3.15 (dd,
androstano[17,16-d][1,2,4]triazolo[1,5-a]pyrimidine (2h)
Yellow solid, yield 55%, mp 257-259 °C. 1H NMR (400 MHz,
CDCl3): d 8.35 (s, 1H, 20-H), 7.79 (d, J = 8.9 Hz, 2H, Ar–H), 6.83 (d,
J = 8.9 Hz, 2H, Ar–H), 6.52 (s, 1H, 4-NH), 3.09 (s, 7H, 5a-H and
J = 9.6, 5.3 Hz, 1H, 5a-H), 1.18 (s, 3H, 18-H), 1.02 (s, 3H, 19-H).
Ar–N(CH3)2), 1.18 (s, 3H, 18-H), 1.00 (s, 3H, 19-H). 13C NMR
(100 MHz, CDCl3): d 179.9, 172.4, 156.1, 154.4, 151.8, 143.7,
131.2, 125.0, 120.6, 115.6, 111.1, 60.6, 55.5, 51.5, 46.6, 40.1, 35.9,
34.3, 33.2, 32.9, 29.7, 29.1, 28.6, 27.0, 20.7, 17.3, 11.4. HRMS
(ESI): m/z cacld. for C29H37N6O (M+H)+, 485.3029; found, 485.3033.
13C NMR (100 MHz, CDCl3): d 180.0, 172.3, 155.8, 154.3, 137.3,
133.8, 132.3, 130.4, 127.6, 119.9, 60.6, 55.0, 51.5, 46.6, 36.0, 34.3,
33.2, 32.9, 30.9, 29.1, 28.6, 27.1, 20.7, 17.6, 14.2, 11.5. HRMS
(ESI): m/z cacld. for
C
25H30N5OS (M+H)+, 448.2171; found,
448.2172.
2.2.9. 3-Oxo-4-aza-70-(4-isopropylphenyl)-5
a-androstano[17,16-
2.2.14. 3-Oxo-4-aza-70-(3-phenoxyphenyl)-5
a-androstano[17,16-
d][1,2,4]triazolo[1,5-a]pyrimidine (2i)
White solid, yield 60%, mp 234–236 °C. 1H NMR (400 MHz,
CDCl3): d 8.37 (s, 1H, 20-H), 7.75 (d, J = 8.0 Hz, 2H, Ar–H), 7.45 (d,
J = 8.0 Hz, 2H, Ar–H), 6.33 (s, 1H, 4-NH), 3.10 (dd, J = 12.0, 3.4 Hz,
d][1,2,4]triazolo[1,5-a]pyrimidine (2n)
White solid, yield 72%, mp 193–195 °C. 1H NMR (400 MHz,
CDCl3): d 8.38 (s, 1H, 20-H), 7.60–7.53 (m, 2H, Ar–H), 7.45–7.35
(m, 3H, Ar–H), 7.25–7.15 (m, 2H, Ar–H), 7.15–7.07 (m, 2H, Ar–H),
1H, 5a-H), 3.08–2.97 (m, 1H, Ar-CH(CH3)2), 1.33 (d, J = 6.8 Hz, 6H,
Ar-CH(CH3)2), 1.19 (s, 3H, 18-H), 1.00 (s, 3H, 19-H). 13C NMR
(100 MHz, CDCl3): d 180.3, 172.2, 156.0, 154.7, 152.3, 143.1,
129.6, 126.8, 126.5, 122.0, 60.6, 55.4, 51.5, 46.7, 36.0, 34.3, 34.2,
33.3, 32.9, 29.1, 29.1, 28.6, 27.0, 23.7, 23.7, 20.7, 17.4, 11.4. HRMS
(ESI): m/z cacld. for C30H37N5ONa (M+Na)+, 506.2896; found,
506.2897.
6.25 (s, 1H, 4-NH), 3.11 (dd, J = 12.1, 3.7 Hz, 1H, 5a-H) 1.17 (s,
3H, 18-H), 1.01 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d
180.3, 172.2, 157.7, 156.3, 155.9, 154.8, 142.2, 130.6, 130.1,
130.0, 124.1, 124.1, 122.4, 120.9, 119.7, 119.4, 60.6, 55.2, 51.5,
46.7, 36.0, 34.3, 33.2, 32.8, 29.1, 29.0, 28.6, 27.1, 20.7, 17.4, 11.4.
HRMS (ESI): m/z cacld. for C33H36N5O2 (M+H)+, 534.2869; found,
534.2867.
2.2.10. 3-Oxo-4-aza-70-(4-morpholinylphenyl)-5
a-androstano[17,16-
d][1,2,4]triazolo[1,5-a]pyrimidine (2j)
2.2.15. 3-Oxo-4-aza-70-(4-methylsulfonylphenyl)-5
a-
Yellow solid, yield 51%, mp 298–300 °C. 1H NMR (400 MHz,
CDCl3): d 8.37 (s, 1H, 20-H), 7.81 (d, J = 8.7 Hz, 2H, Ar–H), 7.04 (d,
J = 8.7 Hz, 2H, Ar–H), 6.20 (s, 1H, 4-NH), 3.98–3.82 (m, 4H, protons
of morpholine), 3.43–3.27 (m, 4H, protons of morpholine), 3.10
androstano[17,16-d][1,2,4]triazolo[1,5-a]pyrimidine (2o)
White solid, yield 65%, mp > 300 °C. 1H NMR (400 MHz, CDCl3):
d 8.39 (s, 1H, 20-H), 8.18 (d, J = 8.5 Hz, 2H, Ar–H), 8.03 (d, J = 8.5 Hz,
2H, Ar–H), 6.32 (s, 1H, 4-NH), 3.16 (s, 3H, Ar–SO2CH3), 3.10 (dd,
(dd, J = 12.0, 3.7 Hz, 1H, 5
a
-H), 1.19 (s, 3H, 18-H), 1.01 (s, 3H, 19-
J = 12.2, 3.7 Hz, 1H, 5a-H), 1.21 (s, 3H, 18-H), 1.00 (s, 3H, 19-H).
H). 13C NMR (100 MHz, CDCl3): d 180.0, 172.2, 156.1, 154.6,
152.7, 143.1, 131.2, 121.1, 119.1, 114.0, 66.6, 60.6, 55.5, 51.5,
47.9, 46.6, 36.0, 34.3, 33.3, 32.9, 29.4, 29.1, 28.6, 27.1, 20.7, 17.3,
13C NMR (100 MHz, CDCl3): d 180.7, 172.2, 155.8, 155.0, 142.7,
140.6, 134.3, 130.7, 127.8, 123.2, 60.5, 55.3, 51.5, 46.9, 44.4, 36.0,
34.3, 33.2, 32.8, 29.0, 28.8, 28.5, 27.0, 20.7, 17.5, 11.4. HRMS
11.4. HRMS (ESI): m/z cacld. for
C
31H38N6O2Na (M+Na)+,
(ESI): m/z cacld. for C
28H34N5O3S (M+H)+, 520.2382; found,
549.2954; found, 549.2949.
520.2386.

L.-H. Huang et al. / Steroids 77 (2012) 710–715
713
2.2.16. 3b-Hydroxy-5-en-70-phenyl-androstano[17,16-
d][1,2,4]triazolo[1,5-a]pyrimidine (4a)
180.7, 156.0, 154.6, 152.2, 143.0, 141.4, 129.7, 126.8, 126.6,
122.4, 120.6, 71.5, 56.1, 50.3, 46.4, 42.2, 37.1, 36.8, 34.3, 33.0,
31.6, 31.2, 31.1, 29.3, 23.8, 23.7, 20.5, 19.5, 17.1. HRMS (ESI): m/z
cacld. for C31H39N4O (M+H)+, 483.3124; found, 483.3122.
White solid, yield 67%, mp > 300 °C. 1H NMR (400 MHz, CDCl3):
d 8.38 (s, 1H, 20-H), 7.82 (m, 2H, Ar–H), 7.64–7.57 (m, 3H, Ar–H),
5.37 (d, J = 5.0 Hz, 1H, 6-H), 3.63–3.46 (m, 1H, 3a-H), 1.21 (s, 3H,
18-H), 1.19–1.08 (m, 5H). 13C NMR (100 MHz, CDCl3): d 180.7,
156.0, 154.7, 142.8, 141.4, 131.0, 129.6, 129.2, 128.7, 122.6,
120.6, 71.5, 56.0, 50.3, 46.4, 42.2, 37.1, 36.8, 33.0, 31.6, 31.2,
31.1, 29.2, 20.5, 19.5, 17.1. HRMS (ESI): m/z cacld. for C28H32N4ONa
(M+Na)+, 463.2474; found, 463.2478.
2.2.22. 3b-Hydroxy-5-en-70-(4-morpholinylphenyl)-
androstano[17,16-d][1,2,4]triazolo[1,5-a]pyrimidine (4g)
Yellow solid, yield 63%, mp > 300 °C. 1H NMR (400 MHz, CDCl3):
d 8.37 (s, 1H, 20-H), 7.82 (d, J = 8.9 Hz, 2H, Ar–H), 7.05 (d, J = 8.9 Hz,
2H, Ar–H), 5.38 (d, J = 4.9 Hz, 1H, 6-H), 3.97–3.83 (m, 4H, protons of
morpholine), 3.61–3.49 (m, 1H, 3a-H), 3.42–3.26 (m, 4H, protons
2.2.17. 3b-Hydroxy-5-en-70-(4-methoxyphenyl)-androstano[17,16-
d][1,2,4]triazolo[1,5-a]pyrimidine (4b)
of morpholine), 1.21 (s, 3H, 18-H), 1.19–1.07 (m, 5H). 13C NMR
(100 MHz, CDCl3): d 180.4, 156.1, 154.5, 152.6, 143.0, 141.4,
131.2, 121.5, 120.6, 119.2, 114.0, 76.7, 71.6, 66.7, 56.2, 50.3, 47.9,
46.3, 42.2, 37.1, 36.8, 33.0, 31.6, 31.2, 31.1, 29.6, 20.5, 19.5, 17.1.
HRMS (ESI): m/z cacld. for C32H40N5O2 (M+H)+, 526.3182; found,
526.3185.
White solid, yield 63%, mp 275–277 °C. 1H NMR (400 MHz,
CDCl3): d 8.37 (s, 1H, 20-H), 7.83 (d, J = 8.7 Hz, 2H, Ar–H), 7.10 (d,
J = 8.6 Hz, 2H, Ar–H), 5.37 (d, J = 4.5 Hz, 1H, 6-H), 3.92 (s, 3H,
Ar–OCH3), 3.61–3.48 (m, 1H, 3a-H), 1.20 (s, 3H, 18-H), 1.18–1.08
(m, 5H). 13C NMR (100 MHz, CDCl3): d 180.6, 161.6, 156.0, 154.5,
142.8, 141.4, 131.5, 122.0, 121.3, 120.6, 114.8, 71.5, 56.1, 55.5,
50.3, 46.4, 42.2, 37.1, 36.8, 33.0, 31.6, 31.2, 31.1, 29.4, 20.5, 19.5,
2.2.23. 3b-Hydroxy-5-en-70-(4-methylsulfonylphenyl)-
androstano[17,16-d][1,2,4]triazolo[1,5-a]pyrimidine (4h)
White solid, yield 70%, mp > 300 °C. 1H NMR (400 MHz, CDCl3):
d 8.39 (s, 1H, 20-H), 8.18 (d, J = 8.4 Hz, 2H, Ar–H), 8.04 (d, J = 8.4 Hz,
17.1. HRMS (ESI): m/z cacld. for
C
29H34N4O2Na (M+Na)+,
493.2579; found, 493.2581.
2H, Ar–H), 5.38 (d, J = 4.9 Hz, 1H, 6-H), 3.62–3.48 (m, 1H, 3a-H),
2.2.18. 3b-Hydroxy-5-en-70-(4-chlorophenyl)-androstano[17,16-
d][1,2,4]triazolo[1,5-a]pyrimidine (4c)
3.16 (s, 3H, Ar–SO2CH3), 1.22 (s, 3H, 18-H), 1.20–1.07 (m, 5H).
13C NMR (100 MHz, CDCl3): d 181.1, 155.8, 154.9, 142.6, 141.4,
140.5, 134.5, 130.7, 127.8, 123.5, 120.4, 71.5, 56.0, 50.3, 46.6,
44.4, 42.2, 37.1, 36.8, 33.0, 31.6, 31.2, 31.1, 29.0, 20.5, 19.5, 17.1.
HRMS (ESI): m/z cacld. for C29H35N4O3S (M+H)+, 519.2430; found,
519.2429.
White solid, yield 65%, mp 291–293 °C. 1H NMR (400 MHz,
CDCl3): d 8.37 (s, 1H, 20-H), 7.79 (d, J = 8.4 Hz, 2H, Ar–H), 7.58 (d,
J = 8.4 Hz, 2H, Ar–H), 5.38 (d, J = 4.8 Hz, 1H, 6-H), 3.61–3.48 (m,
1H, 3a
-H), 1.21 (s, 3H, 18-H), 1.19–1.08 (m, 5H). 13C NMR
(100 MHz, CDCl3) d 180.8, 155.9, 154.7, 141.6, 141.4, 137.3,
131.1, 129.0, 127.6, 122.7, 120.5, 71.5, 56.0, 50.3, 46.5, 42.2, 37.1,
36.8, 33.0, 31.6, 31.2, 31.1, 29.2, 20.5, 19.5, 17.1. HRMS (ESI): m/z
cacld. for C28H32ClN4O (M+H)+, 475.2265; found, 475.2260.
2.2.24. 3b-Hydroxy-5-en-70-(2-pyridyl)-androstano[17,16-
d][1,2,4]triazolo[1,5-a]pyrimidine (4i)
White solid, yield 83%, mp 300–301 °C. 1H NMR (400 MHz,
CDCl3): d 8.86 (dd, J = 4.8, 0.7 Hz, 1H, proton of pyridine), 8.41 (s,
1H, 20-H), 8.34 (d, J = 8.0 Hz, 1H, proton of pyridine), 7.96 (td,
J = 7.8, 1.8 Hz, 1H, proton of pyridine), 7.49 (ddd, J = 7.6, 4.8,
1.0 Hz, 1H, proton of pyridine), 5.38 (d, J = 5.1 Hz, 1H, 6-H),
2.2.19. 3b-Hydroxy-5-en-70-(4-dimethylaminophenyl)-
androstano[17,16-d][1,2,4]triazolo [1,5-a]pyrimidine (4d)
Yellow solid, yield 61%, mp > 300 °C. 1H NMR (400 MHz, CDCl3):
d 8.36 (s, 1H, 20-H), 7.81 (d, J = 8.8 Hz, 2H, Ar–H), 6.84 (d, J = 8.8 Hz,
3.61–3.48 (m, 1H, 3a-H), 1.19 (s, 3H, 18-H), 1.17–1.09 (m, 5H).
2H, Ar–H), 5.37 (d, J = 4.1 Hz, 1H, 6-H), 3.60–3.49 (m, 1H, 3
a-H),
13C NMR (100 MHz, CDCl3): d 181.4, 156.0, 154.7, 149.9, 148.7,
141.3, 140.4, 136.5, 126.4, 125.0, 124.6, 120.7, 71.5, 55.5, 50.4,
46.3, 42.2, 37.1, 36.8, 33.0, 31.6, 31.2, 31.1, 29.4, 20.5, 19.5, 17.1.
HRMS (ESI): m/z cacld. for C27H32N5O (M+H)+, 442.2607; found,
442.2603.
3.09 (s, 6H, Ar–N(CH3)2), 1.20 (s, 3H, 18-H), 1.14 (m, 5H). 13C
NMR (100 MHz, CDCl3): d 180.3, 154.3, 151.8, 143.6, 141.4, 131.2,
125.0, 120.9, 120.7, 115.8, 111.1, 71.5, 56.2, 50.4, 46.3, 42.2, 40.1,
37.1, 36.8, 33.1, 31.6, 31.2, 31.1, 29.8, 20.5, 19.5, 17.1. HRMS
(ESI): m/z cacld. for C30H38N5O (M+H)+, 484.3076; found, 484.3079.
2.2.25. 3b-Hydroxy-5-en-70-(4-aminophenyl)-androstano[17,16-
d][1,2,4]triazolo[1,5-a]pyrimidine (4j)
2.2.20. 3b-Hydroxy-5-en-70-(3-phenoxyphenyl)-androstano[17,16-
d][1,2,4]triazolo[1,5-a]pyrimidine (4e)
Brown solid, yield 41%, mp > 300 °C. 1H NMR (400 MHz, CDCl3):
d 8.37 (s, 1H, 20-H), 7.72 (d, J = 8.5 Hz, 2H, Ar–H), 6.83 (d, J = 8.5 Hz,
White solid, yield 71%, mp 167–169 °C. 1H NMR (400 MHz,
CDCl3): d 8.37 (s, 1H, 20-H), 7.65–7.51 (m, 2H, Ar–H), 7.48–7.35
(m, 3H, Ar–H), 7.26–7.15 (m, 2H, Ar–H), 7.12 (d, J = 7.7 Hz, 2H,
2H, Ar–H), 5.38 (d, J = 4.9 Hz, 1H, 6-H), 3.60–3.51 (m, 1H, 3a-H),
1.20 (s, 3H, 18-H), 1.18–1.09 (m, 5H). 13C NMR (100 MHz, CDCl3):
d 180.4, 156.1, 154.4, 149.1, 143.3, 141.4, 131.5, 121.3, 120.7,
118.4, 114.2, 71.6, 56.2, 50.3, 46.3, 42.2, 37.1, 36.8, 33.0, 31.6,
31.2, 31.1, 29.7, 20.5, 19.5, 17.1. HRMS (ESI): m/z cacld. for
Ar–H), 5.39 (d, J = 4.9 Hz, 1H, 6-H), 3.60–3.52 (m, 1H, 3a-H), 1.18
(s, 3H, 18-H), 1.17–1.05 (m, 5H). 13C NMR (100 MHz, CDCl3): d
180.7, 157.7, 156.4, 155.9, 154.7, 142.1, 141.5, 130.7, 130.1,
130.0, 124.2, 124.1, 122.7, 120.8, 120.5, 119.7, 119.4, 71.5, 55.9,
50.3, 46.4, 42.2, 37.1, 36.8, 33.0, 31.6, 31.2, 31.1, 29.1, 20.5, 19.5,
17.1. HRMS (ESI): m/z cacld. for C34H37N4O2 (M+H)+, 533.2917;
found, 533.2915.
C
28H34N5O (M+H)+, 456.2763; found, 456.2761.
2.3. Bioactivity
All the synthesized heterosteroids were subjected to in vitro
cytotoxic evaluation against PC-3 (human prostatic carcinoma),
MCF-7 (human breast carcinoma) and EC9706 (human esophageal
carcinoma) cell lines. The cell lines were cultured in RPMI 1640
medium with 10% fetal bovine serum, 10 U penicillin and 100 lg/
mL streptomycin at 37 °C with 5% CO2 in a humidified incubator.
2.2.21. 3b-Hydroxy-5-en-70-(4-isopropylphenylphenyl)-
androstano[17,16-d][1,2,4]triazolo[1,5-a]pyrimidine (4f)
White solid, yield 66%, mp 248–250 °C. 1H NMR (400 MHz,
CDCl3): d 8.38 (s, 1H, 20-H), 7.77 (d, J = 8.0 Hz, 2H, Ar–H), 7.46 (d,
J = 8.1 Hz, 2H, Ar–H), 5.37 (d, J = 4.2 Hz, 1H, 6-H), 3.62–3.49 (m,
1H, 3
a
-H), 3.08–2.98 (m, 1H, Ar–CH(CH3)2), 1.34 (d, J = 6.8 Hz,
The anticancer potency of test compounds was measured using
3H, Ar–CH(CH3)2), 1.33 (d, J = 6.8 Hz, 3H, Ar–CH(CH3)2), 1.21 (s,
the
3-(4,5-dimethylthizao1-2-y1)-2,5-diphenyltetrazolium
3H, 18-H), 1.19–1.08 (m, 5H). 13C NMR (100 MHz, CDCl3): d
bromide (MTT) assay.

714
L.-H. Huang et al. / Steroids 77 (2012) 710–715
For the test procedure, cells were harvested by trypsinization,
washed, counted and distributed to wells of 96-well plates (about
6000 cells per well) in 200 L of RPMI 1640 culture medium. For
2l, 2m and 4i). All the synthesized hetero- steroids were character-
ized by 1H, 13C NMR and mass spectra, and the structure of 2b was
further established by X-ray analysis (Fig. 1).
l
each sample, eight serial twofold dilutions were prepared in DMSO.
After 24 h of incubation at 37 °C and 5% CO2 to allow cell attach-
ment, the cells were treated with various concentrations of test
samples. Six replicate wells were set up for each experimental con-
dition. The negative and blank control groups were set up at the
same time. Plates were returned to the incubator for 72 h under
Surprisingly, when the reaction of 3j and 3-AT was performed
under the same reaction conditions, the mass spectrum of the iso-
lated product 4j (m/z 455) was not consistent with the expected
product 4j0 (m/z 485). This indicated that the 70-substituent was
p-aminophenyl group instead of p-nitrophenyl group in the prod-
uct, which was also corroborated by the 1H and 13C NMR spectra.
A plausible mechanism for the formation of 4j is shown in Scheme
2. Under basic conditions, 3j reacted with 3-AT to afford the inter-
mediate A via aza-Michael addition and intramolecular cyclization
reaction followed by elimination of H2O. Subsequently, the
dihydrotriazolopyrimidine A dehydrogenated under the reaction
conditions and in presence of the oxidizing agent p-nitrophenyl
in the molecular structure. Simultaneouly, the nitro group was re-
duced to amino group. This intramolecular oxidation-reduction
was similar to Skraup reaction, in which nitrobenzene served as
the oxidizing agent to remove the hydrogen of dihydroquinoline
and itself was reduced to aniline [25]. However, the reaction of
1c and 3-AT afforded the normal product 2c, which is of interest
for further investigation.
same conditions. Thereafter, 20
was added to each well, and the cells were incubated for 4 h. The
medium was removed, 150 L of DMSO per well was added to dis-
lL of the MTT (0.5 mg/mL) solution
l
solve the purple formazan crystals formed and plates were gently
shaken for 10 min on a mechanical shaker. The optical density (OD)
of solubilized formazan was measured at 570 nm with an auto-
matic microplate reader. All the data of the experiment were calcu-
lated using the SPSS 16.0 software and were expressed as the IC50
(lM) values (Table 1).
3. Results and discussion
3.1. Chemistry
In our previous work, we have described in detail the protocol
for the synthesis of 70-aryl-androstano[17,16-d][1,2,4]triazol-
o[1,5-a]pyrimidines through the condensation reaction of the
intermediates 16-arylidene-17-ketosteroids with 3-amino-1,2,4-
triazole (3-AT) in presence of t-BuOK [24] (Scheme 1). These inter-
3.2. Biology
All the synthesized novel steroidal triazolopyrimidines deriva-
tives were evaluated for their cytotoxic activity in vitro against
PC-3 (human prostatic carcinoma), MCF-7 (human breast carci-
noma) and EC9706 (human esophageal carcinoma) cell lines. The
inhibition of test compounds was determined using the MTT assay.
The anticancer activity was indicated in terms of IC50 (lM) value
calculated by the SPSS 16.0 software and the results were
presented in Table 1.
mediates (1, 3) containing the aromatic
a,b-unsaturated ketone
moiety were prepared by Claisen-Schmidt condensation of
4-aza-androstane-3,17-dione or dehydroepiandrosterone (DHEA)
with various aromatic aldehydes catalyzed by KF/Al2O3. We then
applied the protocol to the preparation of several new substituted
aryl derivatives, including four 70-heteroaryl-substituted ones (2k,
As shown in Table 1, some of the test compounds showed
promising anticancer activity for certain cancer cell lines in vitro.
It was evident from the data that the changes of substituents on
the phenyl ring had a significant influence on the cytotoxicity.
When the phenyl rings were replaced by heteroaromatic rings,
compound 2k, 2l, 2m and 4i did not improve the inhibitory activ-
ity. For PC-3 cells, the most highly active compound was 4f with a
p-isopropyl group on the phenyl ring, which had an IC50 value of
Table 1
The in vitro anticancer activity of synthesized compounds 2 and 4.a
Compound
IC50 (l
M)b
PC-3
MCF-7
EC9706
2a
2b
2c
2d
2e
2f
2g
2h
2i
>100
>100
30.16
>100
>100
>100
>100
>100
87.51
19.80
>100
>100
>100
32.60
8.84
>100
>100
31.99
>100
20.87
>100
19.67
>100
>100
>100
29.45
>100
75.72
>100
>100
>100
>100
>100
>100
60.84
>100
>100
95.44
>100
14.95
>100
>100
>100
25.50
>100
62.22
22.24
>100
>100
>100
>100
35.15
>100
>100
>100
>100
>100
>100
18.02
>100
>100
>100
49.22
13.95
>100
>100
32.59
17.57
10.14
12.01
7.29
7.29
inhibition of PC-3 cells with the IC50 of 13.95, 10.14 and
12.01 M, respectively. Compound 2n with an o-phenoxyphenyl
group exhibited significant cytotoxic activity (8.84 M) in compar-
ison with the other test compounds against MCF-7 cells, which was
also the most effective one against EC9706 cells (14.95 M) among
lM. Compounds 2n, 4d and 4e also displayed remarkable
l
l
l
2j
2k
2l
these compounds. It was noteworthy that the compounds contain-
ing the same substituents in two different series exhibited similar
anticancer activity. For example, both of compounds 2i and 4f
showed potent anticancer activity against the three human cell
lines indicating the high degree of selectivity of these p-isopropyl-
phenyl compounds. Similar activity was also found for the com-
pounds 2n and 4e, both of which contained the o-phenoxyphenyl
2m
2n
2o
4a
4b
4c
4d
4e
4f
4g
4h
4i
>100
>100
>100
25.96
4j
PC-3: human prostatic carcinoma, MCF-7: human breast carcinoma, EC9706:
human esophageal carcinoma.
a
The results are the mean value of six replicate determinations.
IC50 represents the concentration of compound that is required for 50% inhi-
b
bition using the MTT assay.
Fig. 1. X-ray crystal structure of 2b.

L.-H. Huang et al. / Steroids 77 (2012) 710–715
715
N
N
H2N
H2N
N
N
O
O
- N
N
HO
HO
N
NO2
NO2
3j
- H2O
N
N
N
N
N
N
HN
N
N
N
N
[O]
Intramolecular
Oxidation-Reduction
2H
HO
HO
HO
NO2
NO2
NH2
4j'
4j
A
Scheme 2. Proposed mechanism for the formation of unexpected product 4j.
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groups. The preliminary results revealed that the p-isopropyl and
o-phenoxy on the phenyl rings were favorable structural moiety
to retain the anticancer activity.
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fused pyridines from steroidal enamides. Steroids 2008;73:1137–42.
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4. Conclusion
In summary, we have developed a facile approach for the prep-
aration of novel 70-aryl-androstano[17,16-d][1,2,4]triazolo[1,5-
a]pyrimidines derivatives. All the synthesized novel compounds
were evaluated for their anticancer activity in vitro against PC-3,
MCF-7 and EC9706 cell lines. The preliminary results exhibited
that the substituents on the phenyl ring remarkably influenced
the cytotoxicity. The three compounds namely 2i, 2n and 4f were
found to possess potent activity against the three human cell lines.
Guided by these initial findings, further modifications of these het-
erosteroids and research on their possible mechanism of inhibiting
proliferation of cancer cell lines are underway.
(b) Ohnishi H, Yamaguchi K, Shimada S, Suzuki Y, Kumagai A. A new approach
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Acknowledgment
(b) Gujjar R, Marwaha A, El Mazouni F, White J, White KL, Creason S, et al.
Identification
of
a
metabolically
stable
triazolopyrimidine-based
We are grateful to the National Natural Sciences Foundation of
China (No. 81172937) for financial support.
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