Synthesis of D-ring-substituted (5′R)- and (5′S)-17β- pyrazolinylandrostene epimers and comparison of their potential anticancer activities

Article abstract of DOI:10.1016/j.steroids.2012.02.001

Various steroidal benzylidenes were synthetized from pregnenolone with benzaldehyde and p-substituted benzaldehydes. The resulting 17β-chalconyl derivatives of pregnenolone were reacted with hydrazine hydrate in acetic acid solution. Regardless of the sta

Full text of DOI:10.1016/j.steroids.2012.02.001

Steroids 77 (2012) 566–574
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of D-ring-substituted (50R)- and (50S)-17b-pyrazolinylandrostene
epimers and comparison of their potential anticancer activities
Zoltán Iványi a, Nikoletta Szabó a,b, Judit Huber a, János Wölfling a, István Zupkó c, Mihály Szécsi b,
Tibor Wittmann b, Gyula Schneider a,
⇑
a Department of Organic Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary
b 1st Department of Medicine, University of Szeged, Korányi fasor 8-10, H-6720 Szeged, Hungary
c Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Various steroidal benzylidenes were synthetized from pregnenolone with benzaldehyde and p-substi-
tuted benzaldehydes. The resulting 17b-chalconyl derivatives of pregnenolone were reacted with hydra-
zine hydrate in acetic acid solution. Regardless of the starting material, the ring-closure reaction afforded
(in contrast with the literature data) a mixture of two steroidal pyrazoline epimers. The epimers were
critical isomer pairs, which could be separated only in their acetylated form; their structures were inves-
tigated by NMR techniques. The in vitro inhibition of rat testicular C17,20-lyase activity and the antiprolif-
erative effects on four human cancer cell lines were measured, and the results obtained from the two
epimer series were compared.
Received 12 October 2011
Received in revised form 11 January 2012
Accepted 2 February 2012
Available online 11 February 2012
Keywords:
Aldol condensation
Pregnenolone
Hydrazine
Ó 2012 Elsevier Inc. All rights reserved.
Pyrazoline
Epimers
Anticancer activities
1. Introduction
the reactions of pregnenolone with benzaldehyde and p-substi-
tuted benzaldehydes to give the corresponding benzylidene deriv-
Steroidal derivatives in which ring D is modified with exo-
heterocycles exhibit numerous forms of biological activity and
are attractive for medicine [1]. A large number of steroid deriva-
tives containing five- or six-membered 17b-exo-heterocycles are
atives, which were finally converted with hydrazine in acetic acid
to 17b-(1-acetyl-5-phenyl-3-pyrazolinyl)androst-5-en-3b-ol and
its p-substituted phenyl derivatives [12]. Only one isomer was
characterized. From a theoretical aspect, such a cyclization pro-
duces two epimers. Following the synthetic route described by
Banday et al., we concentrated on the formation of the two possible
epimers in the ring-closure step of the reaction. The epimers were
present as a critical isomer pair, which could be separated only in
acetylated form. The separation of steroidal epimers is essential
prior to the measurement of their biological effects. Stereoselective
known to cause the efficient inhibition of 17
a-hydroxylase/
C
17,20-lyase (P45017 ), which can block androgen synthesis at an
a
early stage, and may therefore be useful in the treatment of pros-
tatic carcinoma [2–7]. We recently reported the synthesis of regio-
isomeric 17b-N-phenylpyrazolyl steroids and the inhibitory effect
of these compounds on rat testicular C17,20-lyase activity in vitro
[8]. Some steroid compounds are known to exert hormone recep-
tor-independent antiproliferative activity via the inhibition of
angiogenesis, tubulin polymerization and the upregulation of
apoptotic pathways [9–11]; our novel steroidal pyrazole com-
pounds were therefore also designated for the in vitro screening
of their activities against a panel of three human cancer cell lines
(HeLa, MCF7 and A431). To continue this program, we now set
out to synthetize a novel series of phenypyrazolinyl steroid deriv-
atives in which a C-phenyl group is present instead of an N-phenyl
group. Such compounds were recently prepared by Banday et al. by
inhibition against the P45017 was observed among steroidal
a
dihydrooxazines and aziridines [5,13,14]. The antiproliferative
activities of the individual epimers may differ considerably, and
the epimeric mixture may exert only weak inhibitory effects [15].
2. Experimental
2.1. General
Melting points (mp) were determined on a Kofler block and are
uncorrected. Specific rotations were measured in CHCl3 (c 1) at
20 °C with a POLAMAT-A (Zeiss-Jena) polarimeter and are given
⇑
Corresponding author. Tel.: +36 62 544276; fax: +36 62 544200.
E-mail address: schneider@chem.u-szeged.hu (G. Schneider).
in units of 10–1 deg cm2 g–1
. Elementary analysis data were
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2012.02.001

Z. Iványi et al. / Steroids 77 (2012) 566–574
567
determined with a Perkin–Elmer CHN analyzer model 2400. The
reactions were monitored by TLC on Kieselgel-G (Merck Si 254 F)
layers (0.25 mm thick); solvent systems (ss): (A) methyl tert-butyl
ether/hexane (50:50 v/v), (B) methyl tert-butyl ether/hexane
(70:30 v/v), and (C) methyl tert-butyl ether. The spots were de-
tected by spraying with 5% phosphomolybdic acid in 50% aqueous
phosphoric acid. The Rf values were determined for the spots ob-
served by illumination at 254 and 365 nm. Flash chromatography:
and 129.4 (4C, C-20, C-30, C-50 and C-60), 133.3, 136.1, 140.0 (C-
22), 140.7 (C-5), 200.1 (C-20).
2.2.4. (21E)-3b-Hydroxy-21-(p-bromobenzylidene)pregn-5-en-20-one
(3d)
20
3d (4.42 g, 92%). mp 167–170 °C, Rf = 0.32 (ss A); [
a
]
D
ꢀ19 (c 1
in CHCl3). (Found C, 69.72; H, 7.18; C28H35BrO2 requires C, 69.56;
H, 7.30%). 1H NMR (d, ppm, CDCl3): 0.63 (s, 3H, 18-H3), 0.99 (s,
3H, 19-H3), 2.82 (t, 1H, J1 = 8.5 Hz, 17-H), 3.52 (m, 1H, 3-H), 5.35
(m, 1H, 6-H), 6.75 (d, 1H, J = 15.8 Hz, 21-H), 7.40 and 7.51 (d, 2H,
J = 8.3 Hz, and d, 2H, J = 8.3 Hz, 20-, 30-, 50-, 60-H), 7.46 (d, 1H,
J = 15.8 Hz, 22-H). 13C NMR (d, ppm, CDCl3): 13.4 (C-18), 19.4 (C-
19), 21.1, 22.7, 24.6, 31.6, 31.8, 32.0, 36.5, 37.2, 39.1, 42.2, 45.0,
50.0, 57.2, 62.2, 71.7 (C-3), 121.4 (C-6), 124.4, 127.2 (C-21), 129.6
and 132.1 (C-20, C-30, C-50 and C-60), 133.7, 140.0 (C-22), 140.7
(C-5), 200.1 (C-20).
silica gel 60, 40–63 lm. All solvents were distilled prior to use.
NMR spectra were recorded on a Bruker DRX 500 instrument at
500 (1H NMR) or 125 MHz (13C NMR). Chemical shifts are reported
in ppm (d scale), and coupling constants (J) in Hertz. For the deter-
mination of multiplicities, the J-MOD pulse sequence was used.
2.2. General procedure for the preparation of steroidal benzylidenes
(3a–f)
Three grams (75 mmol) NaOH was dissolved in 180 ml of etha-
nol. 3.16 g (10 mmol) of pregnenolone (1) was then added, and the
reaction mixture was stirred until a clear solution was obtained.
The aromatic aldehyde (2a–f, 15 mmol) was next added, and the
solution was stirred at room temperature for 12 h, and then poured
into a solution of 7 ml of cc HCl in ice-cold-water (700 ml). The
white precipitate that formed was filtered off, washed with water,
and crystallized from acetone/hexane.
2.2.5. (21E)-3b-Hydroxy-21-(p-cyanobenzylidene)pregn-5-en-20-one
(3e)
20
3e (3.71 g, 86%). mp 199–202 °C, Rf = 0.25 (ss A); [
a]
ꢀ30 (c 1
D
in CHCl3). (Found C, 80.96; H, 8.35; C29H35NO2 requires C, 81.08; H,
8.21%). 1H NMR (d, ppm, CDCl3): 0.64 (s, 3H, 18-H3), 0.99 (s, 3H, 19-
H3), 2.84 (t, 1H, J = 8.5 Hz, 17-H), 3.53 (m, 1H, 3-H), 5.35 (m, 1H, 6-
H), 6.82 (d, 1H, J = 16.0 Hz, 21-H), 7.50 (d, 1H, J = 16.0 Hz, 22-H),
7.62 and 7.67 (d, 2H, J = 8.0 Hz, and d, 2H, J = 8.0 Hz, 20-, 30-, 50-
and 60-H). 13C NMR (d, ppm, CDCl3): 13.5 (C-18), 19.4 (C-19),
21.1, 22.7, 24.6, 31.6, 31.8, 32.0, 36.5, 37.2, 39.2, 42.2, 45.1, 50.0,
57.2, 62.5, 71.7 (C-3), 113.3 (C-40), 118.4 (CN), 121.3 (C-6), 128.5
and 132.6 (4C, C-20, C-30, C-50 and C-60), 129.5 (C-21), 138.9 (C-
22), 139.2, 140.7 (C-5), 199.9 (C-20).
2.2.1. (21E)-3b-Hydroxy-21-benzylidenepregn-5-en-20-one (3a)
3a (3.54 g, 87%). mp 123–125 C (Ref. [16]: mp 128–131 °C),
20
Rf = 0.30 (ss A); [
a]
ꢀ18 (c 1 in CHCl3). (Found C, 82.98; H,
D
9.85; C28H36O2 requires C, 83.12; H, 8.97%). 1H NMR (d, ppm,
CDCl3): 0.64 (s, 3H, 18-H3), 0.99 (s, 3H, 19-H3), 2.85 (t, 1H,
J = 8.5 Hz, 17-H), 3.52 (m, 1H, 3-H), 5.36 (s, 1H, 6-H), 6.78 (d, 1H,
J = 16.0 Hz, 21-H), 7.38 (overlapping multiplets, 3H, 30-H, 40-H, 50-
H), 7.55 (overlapping multiplets, 3H, 20-H, 60-H, 22-H). 13C NMR
(d, ppm, CDCl3): 13.0 (C-18), 19.0 (C-19), 20.7, 22.3, 24.3, 31.2,
31.4, 31.6, 36.1, 36.8, 38.7, 41.8, 44.6, 49.7, 56.8, 61.7, 71.3 (C-3),
121.0 (C-6), 126.4 (C-21), 127.9 and 128.5 (4C, C-20, C-30, C-50
and C-60), 129.9 (C-40), 134.4 (C-10), 140.4 (C-5), 141.1 (C-22),
200.0 (C-20).
2.2.6. (21E)-3b-Hydroxy-21-(p-methoxybenzylidene)pregn-5-en-20-
one (3f)
3f (4.30 g, 99%). mp 180–183 °C (Ref. [16]: mp 200–203 °C),
20
Rf = 0.25 (ss A); [
a]
D
+1.5 (c 1 in CHCl3). (Found C, 80.22; H,
9.05; C29H38O3 requires C, 80.14; H, 8.81%). 1H NMR (d, ppm,
CDCl3): 0.63 (s, 3H, 18-H3), 0.99 (s, 3H, 19-H3), 3.52 (m, 1H, 3-H),
3.83 (s, 3H, OCH3), 5.35 (s, 1H, 6-H), 6.66 (d, 1H, J = 15.5 Hz, 21-
H), 6.90 (d, 2H, J = 8.0 Hz, 30- and 50-H), 7.49 (overlapping multi-
plets, 3H, 20-, 60-, 22-H). 13C NMR (d, ppm, CDCl3): 13.4 (C-18),
19.4 (C-19), 21.1, 22.7, 24.7, 31.5, 31.8, 32.0, 36.5, 37.2, 39.1,
42.2, 44.9, 50.0, 55.4, 57.1, 61.9, 71.7 (C-3), 114.3 (2C, C-3030, C-
50), 121.4 (C-6), 124.6, 127.4, 129.9 (2C, C-20, C-60), 140.7 (C-5),
141.3 (C-22), 161.4, 200.3 (C-20).
2.2.2. (21E)-3b-Hydroxy-21-(p-fluorobenzylidene)pregn-5-en-20-one
(3b)
3b (3.72 g, 88%). mp 153–156 °C (Ref. [16]: mp 196–198 °C),
20
Rf = 0.35 (ss A); [
a]
D
ꢀ20 (c 1 in CHCl3). (Found C, 79.65; H,
8.57; C28H35FO2 requires C, 79.58; H, 8.35%). 1H NMR (d, ppm,
CDCl3): 0.63 (s, 3H, 18-H3), 0.99 (s, 3H, 19-H3), 2.83 (t, 1H,
J = 8.5 Hz, 17-H), 3.52 (m, 1H, 3-H), 5.35 (s, 1H, 6-H), 6.69 (d, 1H,
J = 16.0 Hz, 21-H), 7.07 (t, 2H, J = 8.5 Hz, 300- and 500-H), 7.52 (over-
lapping multiplets, 3H, 20-, 60-, and 22-H). 13C NMR (d, ppm, CDCl3):
13.4 (C-18), 19.4 (C-19), 21.1, 22.7, 24.6, 31.6, 31.8, 32.0, 36.5, 37.2,
39.1, 42.2, 45.0, 50.1, 57.2, 62.2, 71.7 (C-3), 115.9 (d, 2C, J = 21.8 Hz,
C-30 and C-50), 121.4 (C-6), 126.5 (C-21), 130.1 (d, 2C, J = 8.3 Hz, C-
20 and C-60), 131.0, 140.2 (C-22), 140.7 (C-5), 163.9 (d, 1C,
J = 250 Hz, C-400), 200.1 (C-20).
2.3. General procedure for the preparation of 3b-acetylated
pyrazolinylandrost-5-ene derivatives (4a–f and 5a–f)
The individual compounds 3a–f (3 mmol) were dissolved in
60 ml of acetic acid, and 1.5 ml (30 mmol) of hydrazine hydrate
was added. The mixture was refluxed for 2 h and then poured into
water (600 ml). The precipitate that formed was filtered off,
washed with water, and dried. The crude product was dissolved
in a mixture of pyridine (5 ml) and acetic anhydride (5 ml) and
the solution was allowed to stand at room temperature for 8 h.
The mixture was then diluted with water and the precipitate that
separated out was filtered off.
2.2.3. (21E)-3b-Hydroxy-21-(p-chlorobenzylidene)pregn-5-en-20-one
(3c)
20
3c (3.83 g, 87%). mp 163–167 °C, Rf = 0.35 (ss A); [
a]
ꢀ17 (c 1
D
in CHCl3). (Found C, 76.85; H, 7.98; C28H35ClO2 requires C, 76.60; H,
8.04%). 1H NMR (d, ppm, CDCl3): 0.63 (s, 3H, 18-H3), 0.99 (s, 3H, 19-
H3), 2.83 (t, 1H, J = 8.5 Hz, 17-H), 3.52 (m, 1H, 3-H), 5.35 (t, 1H,
J = 2.5 Hz, 6-H), 6.73 (d, 1H, J = 16.0 Hz, 21-H), 7.35 and 7.47 (d,
2H, J = 8.0 Hz, and d, 2H, J = 8.0 Hz, 20-, 30-, 50-, and 60-H), 7.49 (d,
1H, J = 16.0 Hz, 22-H). 13C NMR (d, ppm, CDCl3): 13.4 (C-18), 19.4
(C-19), 21.1, 22.7, 24.6, 31.6, 31.8, 32.0, 36.5, 37.2, 39.1, 42.2,
45.0, 50.0, 57.2, 62.2, 71.7 (C-3), 121.4 (C-6), 127.1 (C-21), 129.1
2.3.1. (50R)- and (50S)-17b-(1-Acetyl-5-phenyl-3-pyrazolinyl)androst-
5-en-3b-yl acetate (4a and 5a)
The acetylated crude product was chromatographed on silica
gel with 50% methyl tert-butyl ether/hexane to yield pure 4a
20
(441 mg, 29%), mp 116–120 °C, Rf = 0.42 (ss B); [
a]
+24 (c 1 in
D
CHCl3). (Found C, 76.55; H, 8.34. C32H42N2O3 requires C, 76.46; H,
8.42%). 1H NMR (d, ppm, CDCl3): 0.66 (s, 3H, 18-H3), 1.03 (s, 3H,
19-H3), 2.03 (s, 3H, 3-Ac-CH3), 2.32 (s, 3H, N-Ac-CH3), 2.64 (dd,

568
Z. Iványi et al. / Steroids 77 (2012) 566–574
1H, J1 = 17.9 Hz, J2 = 4.0 Hz, 40-H), 3.37 (dd, 1H, J1 = 17.9 Hz,
J2 = 11.8 Hz, 40-H), 4.60 (m, 1H, 3-H), 5.41 (overlapping multiplets,
2H, 6-H and 50-H), 7.15 (d, 2H, J = 7.3 Hz, 200- and 600-H), 7.22 (t, 1H,
J = 7.3 Hz, 400-H), 7.30 (t, 2H, J = 7.3 Hz, 300- and 500-H). 13C NMR (d,
ppm, CDCl3): 13.2 (C-18), 19.3 (C-19), 20.9, 21.4 (3-Ac-CH3), 21.9,
24.4, 24.5, 27.7, 31.7, 32.0, 36.6, 37.0, 38.0, 38.3, 44.0, 46.0, 50.0
(C-40), 51.8, 56.3, 59.1 (C-50), 73.8 (C-3), 122.3 (C-6), 125.3 and
128.8 (4C, C-200, C-300, C-500 and C-600), 127.4 (C-400), 139.7 (C-5),
142.0 (C-100), 159.3 (C-30), 168.4 (N-Ac-CO), 170.5 (3-Ac-CO). Con-
2H, 6-H and 50-H), 7.09 and 7.27 (d, 2H, J = 8.5 Hz, 200- and 600-H, and
d, 2H, J = 8.5 Hz, 300- and 500-H). 13C NMR (d, ppm, CDCl3): 13.2 (C-
18), 19.3 (C-19), 20.8, 21.4 (3-Ac-CH3), 21.8, 24.4, 24.5, 27.7, 31.7,
31.9, 36.6, 37.0, 38.0, 38.3, 44.0, 45.8 (C-40), 49.9, 51.8, 56.2, 58.5
(C-50), 73.8 (C-3), 122.2 (C-6), 126.8 and 128.9 (4C, C-200, C-300, C-
500 and C-600), 133.1, 139.7 (C-5), 140.5, 159.2 (C-30), 168.5 (N-Ac-
CO), 170.5 (3-Ac-CO). Continued elution resulted in 5c (944 mg,
20
59%), mp 227–235 °C, Rf = 0.38 (ss B); [
a
]
D
ꢀ50 (c 1 in CHCl3).
(Found C, 71.60; H, 7.53. C32H41ClN2O3 requires C, 71.55; H,
7.69%). 1H NMR (d, ppm, CDCl3): 0.65 (s, 3H, 18-H3), 1.01 (s, 3H,
19-H3), 2.02 (s, 3H, 3-Ac-CH3), 2.31 (s, 3H, N-Ac-CH3), 2.70 (dd,
1H, J1 = 18.3 Hz, J2 = 4.8 Hz, 40-H), 3.26 (dd, 1H, J1 = 18.3 Hz,
J2 = 11.8 Hz, 40-H), 4.59 (m, 1H, 3-H), 5.37 (overlapping multiplets,
2H, 6-H and 50-H), 7.09 and 7.28 (d, 2H, J = 8.3 Hz, 200- and 600-H, and
d, 2H, J = 8.3 Hz, 300- and 500-H). 13C NMR (d, ppm, CDCl3): 13.4 (C-
18), 19.3 (C-19), 20.9, 21.4 (3-Ac-CH3), 21.8, 24.3, 24.5, 27.7, 31.7,
32.0, 36.6, 37.0, 38.0, 38.5, 43.9, 46.1 (C-40), 49.9, 51.6, 56.4, 58.5
(C-50), 73.8 (C-3), 122.2 (C-6), 126.7 and 129.0 (4C, C-200, C-300, C-
500 and C-600), 133.1, 139.7 (C-5), 140.8, 159.0 (C-30), 168.6 (N-Ac-
CO), 170.5 (3-Ac-CO).
tinued elution resulted in 5a (896 mg, 59%), mp 140–143 °C,
20
Rf = 0.38 (ss B); [
a]
D
ꢀ71 (c 1 in CHCl3). (Found C, 76.58; H,
8.64. C32H42N2O3 requires C, 76.46; H, 8.42%). 1H NMR (d, ppm,
CDCl3): 0.67 (s, 3H, 18-H3), 1.00 (s, 3H, 19-H3), 2.02 (s, 3H, 3-Ac-
CH3), 2.32 (s, 3H, N-Ac-CH3), 2.75 (dd, 1H, J1 = 18.0 Hz, J2 = 4.5 Hz,
40-H), 3.27 (dd, 1H, J1 = 18.0 Hz, J2 = 11.5 Hz, 40-H), 4.59 (m, 1H,
3-H), 5.40 (overlapping multiplets, 2H, 6-H and 50-H), 7.15 (d,
2H, J = 7.3 Hz, 200- and 600-H), 7.23 (t, 1H, J = 7.3 Hz, 400-H), 7.31 (t,
2H, J = 7.3 Hz, 300- and 500-H). 13C NMR (d, ppm, CDCl3): 13.4 (C-
18), 19.3 (C-19), 20.9, 21.4 (3-Ac-CH3), 21.8, 24.3, 24.6, 27.7, 31.7,
32.0, 36.6, 37.0, 38.0, 38.4, 43.8, 46.2 (C-40), 49.9, 51.7, 56.4, 59.1
(C-50), 73.8 (C-3), 122.3 (C-6), 125.3 and 128.8 (4C, C-200, C-300, C-
500 and C-600), 127.4 (C-400), 139.7 (C-5), 142.3 (C-100), 159.0 (C-30),
168.5 (N-Ac-CO), 170.5 (3-Ac-CO).
2.3.4. (50R)- and (50S)-17b-(1-Acetyl-5-p-bromophenyl-3-
pyrazolinyl)androst-5-en-3b-yl acetate (4d and 5d)
The acetylated crude product was chromatographed on silica
2.3.2. (50R)- and (50S)-17b-(1-Acetyl-5-p-fluorophenyl-3-
pyrazolinyl)androst-5-en-3b-yl acetate (4b and 5b)
gel with 50% methyl tert-butyl ether/hexane to yield pure 4d
20
(348 mg, 20%), mp 222–226 °C, Rf = 0.44 (ss B); [
a]
+17 (c 1 in
D
The acetylated crude product was chromatographed on silica
CHCl3). (Found C, 65.92; H, 7.21. C32H41BrN2O3 requires C, 66.09;
H, 7.11%). 1H NMR (d, ppm, CDCl3): 0.65 (s, 3H, 18-H3), 1.03 (s,
3H, 19-H3), 2.03 (s, 3H, 3-Ac-CH3), 2.31 (s, 3H, N-Ac-CH3), 2.59
(dd, 1H, J1 = 17.8 Hz, J2 = 4.3 Hz, 40-H), 3.37 (dd, 1H, J1 = 18.0 Hz,
J2 = 12.0 Hz, 40-H), 4.60 (m, 1H, 3-H), 5.36 (overlapping multiplets,
2H, 6-H and 50-H), 7.03 (d, 2H, J = 8.3 Hz, 200- and 600-H), 7.42 (d, 2H,
J = 8.3 Hz, 300- and 500-H). 13C NMR (d, ppm, CDCl3): 13.3 (C-18), 19.3
(C-19), 20.9, 21.4 (3-Ac-CH3), 21.8, 24.4, 24.5, 27.7, 31.7, 32.0, 36.6,
37.0, 38.0, 38.3, 44.0, 45.7 (C-40), 49.9, 51.8, 56.2, 58.6 (C-50), 73.8
(C-3), 121.2, 122.3 (C-6), 127.2 and 131.9 (4C, C-200, C-300, C-500
and C-600), 139.7 (C-5), 141.0, 159.2 (C-30), 168.5 (N-Ac-CO), 170.5
gel with 50% methyl tert-butyl ether/hexane to yield pure 4b
20
(276 mg, 18%), mp 180–184 °C, Rf = 0.40 (ss B); [
a]
+46 (c 1 in
D
CHCl3). (Found C, 73.65; H, 8.05. C32H41FN2O3 requires C, 73.82;
H, 7.94%). 1H NMR (d, ppm, CDCl3): 0.66 (s, 3H, 18-H3), 1.03 (s,
3H, 19-H3), 2.03 (s, 3H, 3-Ac-CH3), 2.30 (s, 3H, N-Ac-CH3), 2.61
(dd, 1H, J1 = 17.8 Hz, J2 = 3.8 Hz, 40-H), 3.36 (dd, 1H, J1 = 17.8 Hz,
J2 = 11.8 Hz, 40-H), 4.60 (m, 1H, 3-H), 5.38 (overlapping multiplets,
2H, 6-H and 50-H), 6.98 (t, 2H, J = 8.5 Hz, 300-, and 500-H), 7.12 (m, 2H,
200- and 600-H). 13C NMR (d, ppm, CDCl3): 13.2 (C-18), 19.3 (C-19),
20.9, 21.4 (3-Ac-CH3), 21.9, 24.4, 24.5, 27.7, 31.7, 32.0, 36.6, 37.0,
38.1, 38.3, 44.0, 45.9 (C-40), 50.0, 51.8, 56.3, 58.4 (C-50), 73.8 (C-
3), 115.6 (d, 2C, J = 21.5 Hz, C-300 and C-500), 122.3 (C-6), 127.1 (d,
2C, J = 8.0 Hz, C-200 and C-600), 137.8 (C-100), 139.7 (C-5), 159.3 (C-
30), 162.0 (d, 1C, J = 244 Hz, C-400), 168.5 (N-Ac-CO), 170.5 (3-Ac-
(3-Ac-CO). Continued elution resulted in 5d (705 mg, 40%), mp
20
230–234 °C, Rf = 0.38 (ss B); [
a]
D
ꢀ39 (c 1 in CHCl3). (Found C,
66.17; H, 7.28. C32H41BrN2O3 requires C, 66.09; H, 7.11%). 1H
NMR (d, ppm, CDCl3): 0.65 (s, 3H, 18-H3), 1.01 (s, 3H, 19-H3),
2.03 (s, 3H, 3-Ac-CH3), 2.31 (s, 3H, N-Ac-CH3), 2.70 (dd, 1H,
J1 = 18.3 Hz, J2 = 4.8 Hz, 40-H), 3.27 (dd, 1H, J1 = 18.3 Hz,
J2 = 11.8 Hz, 40-H), 4.59 (m, 1H, 3-H), 5.36 (overlapping multiplets,
2H, 6-H and 50-H), 7.03 (d, 2H, J = 8.3 Hz, 200- and 600-H), 7.43 (d, 2H,
J = 8.3 Hz, 300- and 500-H). 13C NMR (d, ppm, CDCl3): 13.5 (C-18), 19.3
(C-19), 20.9, 21.4 (3-Ac-CH3), 21.8, 24.3, 24.5, 27.7, 31.7, 32.0, 36.6,
37.0, 38.1, 38.5, 43.9, 46.0 (C-40), 49.9, 51.7, 56.4, 58.6 (C-50), 73.8
(C-3), 121.2, 122.3 (C-6), 127.1 and 132.0 (4C, C-200, C-300, C-500
and C-600), 139.7 (C-5), 141.4, 159.0 (C-30), 168.6 (N-Ac-CO), 170.5
(3-Ac-CO).
CO). Continued elution resulted in 5b (675 mg, 43%), mp 195–
20
200 °C, Rf = 0.35 (ss B); [
a]
ꢀ54 (c 1 in CHCl3). (Found C,
D
73.72; H, 7.98. C32H41FN2O3 requires C, 73.82; H, 7.94%). 1H NMR
(d, ppm, CDCl3): 0.66 (s, 3H, 18-H3), 1.01 (s, 3H, 19-H3), 2.03 (s,
3H, 3-Ac-CH3), 2.31 (s, 3H, N-Ac-CH3), 2.72 (dd, 1H, J1 = 18.0 Hz,
J2 = 5.0 Hz, 40-H), 3.27 (dd, 1H, J1 = 18.0 Hz, J2 = 12.0 Hz, 40-H),
4.60 (m, 1H, 3-H), 5.39 (overlapping multiplets, 2H, 6-H and 50-
H), 6.99 (t, 2H, J = 8.8 Hz, 300- and 500-H), 7.13 (m, 2H, 200- and 600-
H). 13C NMR (d, ppm, CDCl3): 13.4 (C-18), 19.3 (C-19), 20.9, 21.4
(3-Ac-CH3), 21.8, 24.3, 24.5, 27.7, 31.7, 32.0, 36.6, 37.0, 38.1, 38.5,
43.9, 46.2 (C-40), 50.0, 51.7, 56.4, 58.4 (C-50), 73.8 (C-3), 115.6 (d,
2C, J = 21.5 Hz, C-300 and C-500), 122.3 (C-6), 127.1 (d, 2C,
J = 8.0 Hz, C-200 and C-600), 138.1 (C-100), 139.7 (C-5), 159.0 (C-30),
162.0 (d, 1C, J = 244 Hz, C-400), 168.6 (N-Ac-CO), 170.5 (3-Ac-CO).
2.3.5. (50R)- and (50S)-17b-(1-Acetyl-5-p-cyanophenyl-3-
pyrazolinyl)androst-5-en-3b-yl acetate (4e and 5e)
The acetylated crude product was chromatographed on silica
gel with 5% CH2Cl2/diisopropyl ether to yield pure 4e (427 mg,
20
2.3.3. (50R)- and (50S)-17b-(1-Acetyl-5-p-chlorophenyl-3-
pyrazolinyl)androst-5-en-3b-yl acetate (4c and 5c)
27%), mp 232–236 °C, Rf = 0.25 (ss B); [
a
]
D
+24 (c 1 in CHCl3).
(Found C, 75.31; H, 7.58. C33H41N3O3 requires C, 75.11; H, 7.83%).
1H NMR (d, ppm, CDCl3): 0.66 (s, 3H, 18-H3), 1.03 (s, 3H, 19-H3),
2.03 (s, 3H, 3-Ac-CH3), 2.32 (s, 3H, N-Ac-CH3), 2.60 (dd, 1H,
J1 = 17.8 Hz, J2 = 4.0 Hz, 40-H), 3.42 (dd, 1H, J1 = 17.8 Hz,
J2 = 12.0 Hz, 40-H), 4.61 (m, 1H, 3-H), 5.41 (overlapping multiplets,
2H, 6-H and 50-H), 7.26 and 7.61 (d, 2H, J = 7.0 Hz, 200- and 600-H, and
d, 2H, J = 7.0 Hz, 300- and 500-H). 13C NMR (d, ppm, CDCl3): 13.3 (C-
18), 19.3 (C-19), 20.8, 21.4 (3-Ac-CH3), 21.8, 24.4, 24.5, 27.7, 31.7,
31.9, 36.6, 37.0, 38.0, 38.3, 44.0, 45.5 (C-40), 49.9, 51.7, 56.2, 58.8
The acetylated crude product was chromatographed on silica
gel with 5% CH2Cl2/diisopropyl ether to yield pure 4c (465 mg,
20
29%), mp 247–250 °C, Rf = 0.42 (ss B); [
a]
+22 (c 1 in CHCl3).
D
(Found C, 71.62; H, 7.81. C32H41ClN2O3 requires C, 71.55; H,
7.69%). 1H NMR (d, ppm, CDCl3): 0.66 (s, 3H, 18-H3), 1.03 (s, 3H,
19-H3), 2.03 (s, 3H, 3-Ac-CH3), 2.31 (s, 3H, N-Ac-CH3), 2.60 (dd,
1H, J1 = 17.9 Hz, J2 = 4.3 Hz, 40-H), 3.37 (dd, 1H, J1 = 17.9 Hz,
J2 = 11.5 Hz, 40-H), 4.61 (m, 1H, 3-H), 5.37 (overlapping multiplets,

Z. Iványi et al. / Steroids 77 (2012) 566–574
569
(C-50), 73.7 (C-3), 111.4, 118.6, 122.2 (C-6), 126.3 and 132.7 (4C, C-
J = 16.0 Hz, 40-H), 3.37 (dd, 1H, J1 = 16.0 Hz, J2 = 12.0 Hz, 40-H),
3.52 (s, 1H, 3-H), 5.39 (overlapping multiplets, 2H, 6-H és 50-H),
7.15 (d, 2H, J = 7.3 Hz, 200- és 600-H), 7.23 (t, 1H, J = 7.3 Hz, 400-H),
7.30 (t, 2H, J = 7.3 Hz, 300- és 500-H). 13C NMR (d, ppm, CDCl3): 13.2
(C-18), 19.4 (C-19), 20.9, 21.9 (N-Ac-CH3), 24.4, 24.5, 31.6, 31.7,
32.0, 36.5, 37.3, 38.4, 42.2, 44.0, 45.9, 50.1, 51.9, 56.4, 59.1, 71.7
(C-3), 121.3 (C-6), 125.3 and 128.8 (4C, C-200, C-300, C-500 and C-600),
127.4 (C-400), 140.8 (C-5), 142.0 (C-100), 159.3 (C-30), 168.5 (N-Ac-
CO).
200, C-300, C-500 and C-600), 139.7 (C-5), 147.1, 159.1 (C-30), 168.6 (N-
Ac-CO), 170.5 (3-Ac-CO). Continued elution resulted in 5e
20
(838 mg, 53%), mp 222–226 °C, Rf = 0.20 (ss B); [
a
]
D
ꢀ46 (c 1 in
CHCl3). (Found C, 75.25; H, 7.62. C33H41N3O3 requires C, 75.11; H,
7.83%). 1H NMR (d, ppm, CDCl3): 0.65 (s, 3H, 18-H3), 1.01 (s, 3H,
19-H3), 2.03 (s, 3H, 3-Ac-CH3), 2.32 (s, 3H, N-Ac-CH3), 2.70 (dd,
1H, J1 = 18.0 Hz, J2 = 4.5 Hz, 40-H), 3.32 (dd, 1H, J1 = 18.0 Hz,
J2 = 12.0 Hz, 40-H), 4.60 (m, 1H, 3-H), 5.41 (overlapping multiplets,
2H, 6-H and 50-H), 7.27 and 7.62 (d, 2H, J = 8.0 Hz, 200- and 600-H, and
d, 2H, J = 8.0 Hz, 300- and 500-H). 13C NMR (d, ppm, CDCl3): 13.5 (C-
18), 19.3 (C-19), 20.9, 21.4 (3-Ac-CH3), 21.7, 24.3, 24.5, 27.7, 31.7,
32.0, 36.6, 37.0, 38.0, 38.5, 43.9, 45.8 (C-40), 49.9, 51.6, 56.3, 58.8
(C-50), 73.7 (C-3), 111.4, 118.6, 122.2 (C-6), 126.2 and 132.8 (4C,
C-200, C-300, C-500 and C-600), 139.7 (C-5), 147.4, 158.9 (C-30), 168.7
(N-Ac-CO), 170.5 (3-Ac-CO).
2.4.2. (50S)-17b-(1-Acetyl-5-phenyl-3-pyrazolinyl)androst-5-en-3b-ol
(5g)
20
5g (80 mg, 87%). mp 194–197 °C, Rf = 0.62 (ss C); [
a]
ꢀ85 (c 1
D
in CHCl3). (Found C, 78.41; H, 8.78; C30H40N2O2 requires C, 78.22;H,
8.75%). 1H NMR (d, ppm, CDCl3): 0.67 (s, 3H, 18-H3), 0.99 (s, 3H, 19-
H3), 2.32 (s, 3H, N-Ac-CH3), 2.75 (d, 1H, J = 16.0 Hz, 40-H), 3.27 (dd,
1H, J1 = 16.0 Hz, J2 = 11.8 Hz, 40-H), 3.51 (s, 1H, 3-H), 5.39 (overlap-
ping multiplets, 2H, 6-H and 50-H), 7.15 (s, 2H, 200- and 600-H), 7.23
(t, 1H, J = 6.0 Hz, 400-H), 7.30 (s, 2H, 300- and 500-H). 13C NMR (d, ppm,
CDCl3): 13.0 (C-18), 19.0 (C-19), 20.6, 21.5 (N-Ac-CH3), 24.0, 24.2,
31.2, 31.3, 31.6, 36.1, 36.9, 38.1, 41.8, 43.5, 45.8, 49.7, 51.3, 56.1,
58.7, 71.2 (C-3), 120.9 (C-6), 124.9 and 128.4 (4C, C-200, C-300, C-500
and C-600), 127.0 (C-400), 140.4 (C-5), 141.9 (C-100), 158.7 (C-30),
168.2 (N-Ac-CO).
2.3.6. (50R)- and (50S)-17b-(1-Acetyl-5-p-methoxyphenyl-3-
pyrazolinyl)androst-5-en-3b-yl acetate (4f and 5f)
The acetylated crude product was chromatographed on silica
gel with 50% methyl tert-butyl ether/hexane to yield pure 4f
20
(387 mg, 24%), mp 159–161 °C, Rf = 0.33 (ss B); [
a]
+26 (c 1 in
D
CHCl3). (Found C, 74.55; H, 8.41. C33H44N2O4 requires C, 74.40; H,
8.33%). 1H NMR (d, ppm, CDCl3): 0.66 (s, 3H, 18-H3), 1.03 (s, 3H,
19-H3), 2.03 (s, 3H, 3-Ac-CH3), 2.29 (s, 3H, N-Ac-CH3), 2.62 (dd,
1H, J1 = 17.8 Hz, J2 = 4.0 Hz, 40-H), 3.34 (dd, 1H, J1 = 17.8 Hz,
J2 = 11.8 Hz, 40-H), 3.76 (s, 3H, OCH3), 4.60 (m, 1H, 3-H), 5.36 (over-
lapping multiplets, 2H, 6-H and 50-H), 6.83 (d, 2H, J = 8.5 Hz, 300-
and 500-H), 7.08 (d, 2H, J = 8.5 Hz, 200- and 600-H). 13C NMR (d, ppm,
CDCl3): 13.2 (C-18), 19.3 (C-19), 20.9, 21.4 (3-Ac-CH3), 21.9, 24.4,
24.5, 27.7, 31.7, 32.0, 36.6, 37.0, 38.1, 38.3, 43.9, 45.9 (C-40), 50.0,
51.9, 55.2, 56.3, 58.6 (C-50), 73.8 (C-3), 114.1 and 126.6 (4C, C-200,
C-300, C-500 and C-600), 122.3 (C-6), 134.2, 139.7 (C-5), 158.8, 159.2,
2.4.3. (50R)-17b-(1-Acetyl-5-p-fluorophenyl-3-pyrazolinyl)androst-5-
en-3b-ol (4h)
4h (70 mg, 76%). mp 269–272 °C, Rf = 0.62 (ss C); [a]
20 +8.5 (c 1
D
in CHCl3). (Found C, 75.37; H, 8.34; C30H39FN2O2 requires C,
75.28;H, 8.21%). 1H NMR (d, ppm, CDCl3): 0.66 (s, 3H, 18-H3),
1.02 (s, 3H, 19-H3), 2.30 (s, 3H, N-Ac-CH3), 2.61 (dd, 1H,
J1 = 18.0 Hz, J2 = 3.8 Hz, 40-H), 3.37 (dd, 1H, J1 = 18.0 Hz,
J2 = 12.0 Hz, 40-H), 3.51 (m, 1H, 3-H), 5.37 (overlapping multiplets,
2H, 6-H and 50-H), 6.98 (t, 2H, J = 8.5 Hz, 300-, and 500-H), 7.12 (m, 2H,
200- and 600-H). 13C NMR (d, ppm, CDCl3): 13.2 (C-18), 19.4 (C-19),
20.9, 21.9 (N-Ac-CH3), 24.4, 24.5, 31.6, 31.7, 32.0, 36.5, 37.3, 38.4,
42.2, 44.0, 45.9, 50.1, 51.9, 56.4, 58.5, 71.6 (C-3), 115.6 (d, 2C,
J = 21.5 Hz, C-300 and C-500), 127.1 (d, 2C, J = 7.9 Hz, C-200 and C-600),
121.3 (C-6), 137.8 (C-100), 140.8 (C-5), 159.3 (C-30), 162.0 (d, 1C,
J = 244 Hz, C-400), 168.5 (N-Ac-CO).
168.4 (N-Ac-CO), 170.5 (3-Ac-CO). Continued elution resulted in
20
5f (782 mg, 49%), mp 176–178 °C, Rf = 0.27 (ss B); [
a]
ꢀ45 (c 1
D
in CHCl3). (Found C, 74.32; H, 8.25. C33H44N2O4 requires C, 74.40;
H, 8.33%). 1H NMR (d, ppm, CDCl3): 0.68 (s, 3H, 18-H3), 1.01 (s,
3H, 19-H3), 2.03 (s, 3H, 3-Ac-CH3), 2.30 (s, 3H, N-Ac-CH3), 2.74
(dd, 1H, J1 = 18.0 Hz, J2 = 4.5 Hz, 40-H), 3.24 (dd, 1H, J1 = 18.0 Hz,
J2 = 11.5 Hz, 40-H), 3.77 (s, 3H, OCH3), 4.60 (m, 1H, 3-H), 5.36 (over-
lapping multiplets, 2H, 6-H and 50-H), 6.83 (d, 2H, J = 8.3 Hz, 300-
and 500-H), 7.08 (d, 2H, J = 8.3 Hz, 200- and 600-H). 13C NMR (d, ppm,
CDCl3): 13.4 (C-18), 19.3 (C-19), 20.9, 21.4 (3-Ac-CH3), 21.9, 24.3,
24.5, 27.7, 31.7, 32.0, 36.6, 37.0, 38.1, 38.4, 43.8, 46.2 (C-40), 50.0,
51.7, 55.2, 56.4, 58.5 (C-50), 73.8 (C-3), 114.2 and 126.6 (4C, C-200,
C-300, C-500 and C-600), 122.3 (C-6), 134.6, 139.7 (C-5), 158.8, 159.0,
168.5 (N-Ac-CO), 170.5 (3-Ac-CO).
2.4.4. (50S)-17b-(1-Acetyl-5-p-fluorophenyl-3-pyrazolinyl)androst-5-
en-3b-ol (5h)
20
5h (55 mg, 60%). mp 222–225 °C, Rf = 0.58 (ss C); [
a
]
D
ꢀ58 (c 1
in CHCl3). (Found C, 75.35; H, 8.30; C30H39FN2O2 requires C,
75.28;H, 8.21%). 1H NMR (d, ppm, CDCl3): 0.66 (s, 3H, 18-H3),
0.99 (s, 3H, 19-H3), 2.30 (s, 3H, N-Ac-CH3), 2.71 (dd, 1H,
J1 = 18.0 Hz, J2 = 3.5 Hz, 40-H), 3.26 (dd, 1H, J1 = 18.0 Hz,
J2 = 11.8 Hz, 40-H), 3.50 (m, 1H, 3-H), 5.38 (overlapping multiplets,
2H, 6-H and 50-H), 6.98 (t, 2H, J = 8.5 Hz, 300-, and 500-H), 7.12 (m, 2H,
200- and 600-H). 13C NMR (d, ppm, CDCl3): 13.4 (C-18), 19.4 (C-19),
21.0, 21.8 (N-Ac-CH3), 24.3, 24.6, 31.6, 31.7, 32.0, 36.5, 37.2, 38.5,
42.2, 43.9, 46.2, 50.0, 51.7, 56.5, 58.4, 71.6 (C-3), 115.6 (d, 2C,
J = 21.4 Hz, C-300 and C-500), 127.0 (d, 2C, J = 8.0 Hz, C-200 and C-600),
121.3 (C-6), 138.1 (C-100), 140.8 (C-5), 159.1 (C-30), 162.0 (d, 1C,
J = 244 Hz, C-400), 168.6 (N-Ac-CO).
2.4. General procedure for the preparation of 3b-
hydroxypyrazolinylandrostene derivatives (4g–l and 5g–l)
The individual compounds 4a–f or 5a–f (100 mg) were dis-
solved in methanol (15 ml), and NaOCH3 (18 mg, 0.33 mmol) was
added. The mixture was allowed to stand at room temperature,
and the progress of the reaction was monitored by TLC. After com-
pletion of the transformation, the reaction mixture was diluted
with water, and neutralized with dilute HCl. The resulting precip-
itate was filtered off, washed with water and dried at room
temperature.
2.4.5. (50R)-17b-(1-Acetyl-5-p-chlorophenyl-3-pyrazolinyl)androst-5-
en-3b-ol (4i)
4i (73 mg, 80%). mp 247–249 °C, Rf = 0.64 (ss C); [a]
20 +38 (c 1
D
2.4.1. (50R)-17b-(1-Acetyl-5-phenyl-3-pyrazolinyl)androst-5-en-3b-ol
(4g)
in CHCl3). (Found C, 72.94; H, 8.05; C30H39ClN2O2 requires C, 72.78;
H, 7.94%). 1H NMR (d, ppm, CDCl3): 0.66 (s, 3H, 18-H3), 1.02 (s, 3H,
19-H3), 2.31 (s, 3H, N-Ac-CH3), 2.60 (dd, 1H, J1 = 17.7 Hz, J2 = 2.8 Hz,
40-H), 3.37 (dd, 1H, J1 = 17.7 Hz, J2 = 12.0 Hz, 40-H), 3.51 (m, 1H, 3-
H), 5.36 (overlapping multiplets, 2H, 6-H and 50-H), 7.09 and 7.27
(d, 2H, J = 8.0 Hz, 200- and 600-H, and d, 2H, J = 8.0 Hz, 300- and
4g (65 mg, 71%). mp 205–209 ° °C, Rf = 0.64 (ss C); [a]
20 +26 (c
D
1 in CHCl3). (Found C, 78.34; H, 8.85; C30H40N2O2 requires C,
78.22;H, 8.75%). 1H NMR (d, ppm, CDCl3): 0.66 (s, 3H, 18-H3),
1.02 (s, 3H, 19-H3), 2.32 (s, 3H, N-Ac-CH3), 2.64 (d, 1H,

570
Z. Iványi et al. / Steroids 77 (2012) 566–574
500-H). 13C NMR (d, ppm, CDCl3): 13.2 (C-18), 19.4 (C-19), 20.9, 21.8
(N-Ac-CH3), 24.4, 24.5, 31.6, 31.7, 32.0, 36.5, 37.3, 38.4, 42.2, 44.0,
45.8, 50.1, 51.8, 56.3, 58.5, 71.6 (C-3), 121.3 (C-6), 126.8 and 128.9
(4C, C-200, C-300, C-500 and C-600), 133.1, 140.5, 140.8, 159.2 (C-30),
168.5 (N-Ac-CO).
2.4.10. (50S)-17b-(1-Acetyl-5-p-cyanophenyl-3-pyrazolinyl)androst-
5-en-3b-ol (5k)
20
5k (78 mg, 85%). mp 209–212 °C, Rf = 0.44 (ss C); [
a
]
D
ꢀ32 (c 1
in CHCl3). (Found C, 76.78; H, 8.15; C31H39N3O2 requires C, 76.67;H,
8.09%). 1H NMR (d, ppm, CDCl3): 0.65 (s, 3H, 18-H3), 0.99 (s, 3H, 19-
H3), 2.32 (s, 3H, N-Ac-CH3), 2.70 (dd, 1H, J1 = 18.3 Hz, J2 = 4.7 Hz, 40-
H), 3.32 (dd, 1H, J1 = 18.3 Hz, J2 = 11.8 Hz, 40-H), 3.50 (m, 1H, 3-H),
5.35 (m, 1H, 6-H), 5.42 (dd, 1H, J1 = 11.8 Hz, J2 = 4.7 Hz, 50-H), 7.27
and 7.62 (d, 2H, J = 8.0 Hz, 200- and 600-H, and d, 2H, J = 8.0 Hz, 300-
and 500-H). 13C NMR (d, ppm, CDCl3): 13.5 (C-18), 19.4 (C-19),
20.9, 21.7 (N-Ac-CH3), 24.3, 24.5, 31.5, 31.7, 32.0, 36.5, 37.2, 38.5,
42.2, 43.9, 45.8, 50.0, 51.6, 56.4, 58.8, 71.5 (C-3), 111.3, 118.6,
121.2 (C-6), 126.2 and 132.8 (4C, C-200, C-300, C-500 and C-600), 140.8
(C-5), 147.4, 159.0 (C-30), 168.8 (N-Ac-CO).
2.4.6. (50S)-17b-(1-Acetyl-5-p-chlorophenyl-3-pyrazolinyl)androst-5-
en-3b-ol (5i)
20
5i (69 mg, 75%). mp 227–229 °C, Rf = 0.58 (ss C); [
a]
ꢀ45 (c 1
D
in CHCl3). (Found C, 72.98; H, 7.95; C30H39ClN2O2 requires C, 72.78;
H, 7.94%). 1H NMR (d, ppm, CDCl3): 0.66 (s, 3H, 18-H3), 1.00 (s, 3H,
19-H3), 2.31 (s, 3H, N-Ac-CH3), 2.71 (d, 1H, J = 17.8 Hz, 40-H), 3.28
(dd, 1H, J1 = 17.8 Hz, J2 = 12.3 Hz, 40-H), 3.51 (m, 1H, 3-H), 5.37
(overlapping multiplets, 2H, 6-H and 50-H), 7.09 and 7.28 (d, 2H,
J = 7.5 Hz, 200- and 600-H, and d, 2H, J = 7.5 Hz, 300- and 500-H). 13C
NMR (d, ppm, CDCl3): 13.1 (C-18), 19.0 (C-19), 20.6, 21.4 (N-Ac-
CH3), 24.0, 24.2, 31.2, 31.3, 31.6, 36.1, 36.9, 38.1, 41.8, 43.5, 45.7,
49.7, 51.3, 56.1, 58.1, 71.2 (C-3), 120.9 (C-6), 126.4 and 128.6
(4C, C-200, C-300, C-500 and C-600), 132.7, 140.4 (2C), 158.7 (C-30),
168.2 (N-Ac-CO).
2.4.11. (50R)-17b-(1-Acetyl-5-p-methoxyphenyl-3-
pyrazolinyl)androst-5-en-3b-ol (4l)
4l (75 mg, 81%). mp 224–230 °C, Rf = 0.52 (ss C); [a]
20 +6.4 (c 1
D
in CHCl3). (Found C, 75.92; H, 8.51; C31H42N2O3 requires C, 75.88;
H, 8.63%). 1H NMR (d, ppm, CDCl3): 0.66 (s, 3H, 18-H3), 1.01 (s,
3H, 19-H3), 2.30 (s, 3H, N-Ac-CH3), 2.62 (dd, 1H, J1 = 18.0 Hz,
J2 = 3.5 Hz, 40-H), 3.34 (dd, 1H, J1 = 18.0 Hz, J2 = 11.8 Hz, 40-H),
3.51 (m, 1H, 3-H), 3.77 (s, 3H, OCH3), 5.36 (overlapping multiplets,
2H, 6-H and 50-H), 6.83 (d, 2H, J = 8.5 Hz, 300- and 500-H), 7.08 (d, 2H,
J = 8.5 Hz, 200- and 600-H). 13C NMR (d, ppm, CDCl3): 13.2 (C-18), 19.4
(C-19), 20.9, 21.9 (N-Ac-CH3), 24.4, 24.5, 31.6, 31.7, 32.0, 36.5, 37.3,
38.3, 42.2, 43.9, 45.9, 50.1, 51.9, 55.2, 56.4, 58.6, 71.6 (C-3), 114.1
and 126.6 (4C, C-200, C-300, C-500 and C-600), 121.3 (C-6), 134.2,
140.8 (C-5), 158.8, 159.3, 168.4 (N-Ac-CO).
2.4.7. (50R)-17b-(1-Acetyl-5-p-bromophenyl-3-pyrazolinyl)androst-5-
en-3b-ol (4j)
4j (68 mg, 73%). mp 212–215 °C, Rf = 0.64 (ss C); [a]
20 +9.4 (c 1
D
in CHCl3). (Found C, 66.54; H, 7.35; C30H39BrN2O2 requires C, 66.78;
H, 7.29%). 1H NMR (d, ppm, CDCl3): 0.65 (s, 3H, 18-H3), 1.01 (s, 3H,
19-H3), 2.30 (s, 3H, N-Ac-CH3), 2.59 (dd, 1H, J1 = 17.5 Hz, J2 = 3.8 Hz,
40-H), 3.37 (dd, 1H, J1 = 17.5 Hz, J2 = 12.0 Hz, 40-H), 3.51 (m, 1H, 3-
H), 5.35 (overlapping multiplets, 2H, 6-H and 50-H), 7.02 (d, 2H,
J = 8.0 Hz, 200- and 600-H), 7.42 (d, 2H, J = 8.0 Hz, 300- and 500-H). 13C
NMR (d, ppm, CDCl3): 13.3 (C-18), 19.4 (C-19), 20.9, 21.8 (N-Ac-
CH3), 24.4, 24.5, 31.6, 31.7, 32.0, 36.5, 37.3, 38.3, 42.2, 44.0, 45.7,
50.1, 51.8, 56.3, 58.6, 71.6 (C-3), 121.2, 121.3 (C-6), 127.2 and
131.9 (4C, C-200, C-300, C-500 and C-600), 140.8 (C-5), 141.0 (C-100),
159.3 (C-30), 168.5 (N-Ac-CO).
2.4.12. (50S)-17b-(1-Acetyl-5-p-methoxyphenyl-3-
pyrazolinyl)androst-5-en-3b-ol (5l)
20
5l (83 mg, 90%). mp 211–215 °C, Rf = 0.50 (ss C); [
a]
ꢀ48 (c 1
D
in CHCl3). (Found C, 75.73; H, 8.71; C31H42N2O3 requires C, 75.88;
H, 8.63%). 1H NMR (d, ppm, CDCl3): 0.68 (s, 3H, 18-H3), 0.99 (s,
3H, 19-H3), 2.30 (s, 3H, N-Ac-CH3), 2.74 (dd, 1H, J1 = 18.0 Hz,
J2 = 4.5 Hz, 40-H), 3.24 (dd, 1H, J1 = 18.0 Hz, J2 = 11.5 Hz, 40-H),
3.49 (m, 1H, 3-H), 3.77 (s, 3H, OCH3), 5.36 (overlapping multiplets,
2H, 6-H and 50-H), 6.83 (d, 2H, J = 8.5 Hz, 300- and 500-H), 7.08 (d, 2H,
J = 8.5 Hz, 200- and 600-H). 13C NMR (d, ppm, CDCl3): 13.4 (C-18), 19.4
(C-19), 21.0, 21.9 (N-Ac-CH3), 24.4, 24.6, 31.6, 31.7, 32.0, 36.5, 37.2,
38.5, 42.2, 43.9, 46.2, 50.1, 51.7, 55.2, 56.5, 58.5, 71.6 (C-3), 114.2
and 126.6 (4C, C-200, C-300, C-500 and C-600), 121.3 (C-6), 134.5,
140.8 (C-5), 158.8, 159.1, 168.5 (N-Ac-CO).
2.4.8. (50S)-17b-(1-Acetyl-5-p-bromophenyl-3-pyrazolinyl)androst-5-
en-3b-ol (5j)
20
5j (67 mg, 72%). mp 219–223 °C, Rf = 0.58 (ss C); [
a]
ꢀ46 (c 1
D
in CHCl3). (Found C, 66.62; H, 7.41; C30H39BrN2O2 requires C, 66.78;
H, 7.29%). 1H NMR (d, ppm, CDCl3): 0.65 (s, 3H, 18-H3), 0.99 (s, 3H,
19-H3), 2.31 (s, 3H, N-Ac-CH3), 2.70 (dd, 1H, J1 = 18.2 Hz, J2 = 4.8 Hz,
40-H), 3.26 (dd, 1H, J1 = 18.2 Hz, J2 = 12.0 Hz, 40-H), 3.52 (m, 1H, 3-
H), 5.36 (overlapping multiplets, 2H, 6-H and 50-H), 7.03 (d, 2H,
J = 8.0 Hz, 200- and 600-H), 7.43 (d, 2H, J = 8.0 Hz, 300- and 500-H). 13C
NMR (d, ppm, CDCl3): 13.5 (C-18), 19.4 (C-19), 21.0, 21.8 (N-Ac-
CH3), 24.4, 24.6, 31.6, 31.7, 32.0, 36.5, 37.3, 38.5, 42.2, 43.9, 46.0,
50.0, 51.7, 56.5, 58.6, 71.7 (C-3), 121.2 (C-400), 121.3 (C-6), 127.1
and 132.0 (4C, C-200, C-300, C-500 and C-600), 140.8 (C-5), 141.3 (C-
100), 159.1 (C-30), 168.6 (N-Ac-CO).
2.5. Determination of C17,20-lyase inhibition
Inhibitory effects exerted on the C17,20-lyase activity were
determined by an in vitro radiosubstrate incubation method de-
scribed in earlier publications [5–8], with some modifications.
In brief, adult Wistar rat testicular tissue was homogenized
with an Ultra-Turrax in 0.1 M HEPES buffer (pH = 7.3) containing
1 mM EDTA and 1 mM dithiotreitol. Aliquots of this homogenate
2.4.9. (50R)-17b-(1-Acetyl-5-p-cyanophenyl-3-pyrazolinyl)androst-5-
en-3b-ol (4k)
4k (80 mg, 87%). mp 193–200 °C, Rf = 0.50 (ss C); [
a
]
D
20 +77 (c 1
were incubated in 200
presence of 0.1 mM NADPH. 1
was added to the incubate in 20
solution. Test compounds were applied at 50
in 10 l of DMSO. (These organic solvent contents did not reduce
l
l final volume at 37 °C for 20 min in the
M [3H]17-hydroxyprogesterone
l of a 25 v/v% propylene glycol
M and introduced
in CHCl3). Found C, 76.82; H, 7.96; C31H39N3O2 requires C, 76.67;H,
8.09%). 1H NMR (d, ppm, CDCl3): 0.66 (s, 3H, 18-H3), 1.02 (s, 3H, 19-
H3), 2.32 (s, 3H, N-Ac-CH3), 2.60 (dd, 1H, J1 = 18.0 Hz, J2 = 4.5 Hz, 40-
H), 3.42 (dd, 1H, J1 = 18.0 Hz, J2 = 12.0 Hz, 40-H), 3.51 (m, 1H, 3-H),
5.35 (d, 1H, J = 5.0 Hz, 6-H), 5.42 (dd, 1H, J1 = 12.0 Hz, J2 = 4.5 Hz,
50-H), 7.26 and 7.61 (d, 2H, J = 8.0 Hz, 200- and 600-H, and d, 2H,
J = 8.0 Hz, 300- and 500-H). 13C NMR (d, ppm, CDCl3): 13.3 (C-18),
19.4 (C-19), 20.9, 21.8 (N-Ac-CH3), 24.4, 24.5, 31.5, 31.7, 32.0,
36.5, 37.2, 38.4, 42.2, 44.0, 45.5, 50.0, 51.7, 56.3, 58.8, 71.6 (C-3),
111.4, 118.6, 121.2 (C-6), 126.2 and 132.7 (4C, C-200, C-300, C-500
and C-600), 140.8 (C-5), 147.1, 159.2 (C-30), 168.6 (N-Ac-CO).
l
l
l
l
the enzyme activity substantially.) Control incubates without test
substances, and incubates with the reference compound ketocona-
zole were also prepared in every series. Following incubation, the
androst-4-ene-3,17-dione formed and the 17-hydroxyprogester-
one remaining were isolated through extraction and TLC. C17,20
lyase activity was calculated from the radioactivity of the and-
rost-4-ene-3,17-dione obtained. At least two experiments were
-

Z. Iványi et al. / Steroids 77 (2012) 566–574
571
performed with each test compound and the standard deviations
of the mean enzyme activity results were within 10%.
and no further purification was needed. The condensation formed
a new double bond at position 21. The 1H NMR spectra of the crys-
tallized products revealed that the reaction resulted only in the E
isomers, as described by Catsoulacos and Stassinopoulou for
(21E)-3b-acetoxy-21-benzylidenepregn-5-en-20-one [21]. It has
2.6. Determination of antiproliferative effects
also been reported that the aldol condensation of 3b,7a,11a-tri-
hydroxypregn-5-en-20-one with benzaldehyde gives the E isomer
as the sole product [22].
Human cancer cell lines were purchased from ECACC (Salisbury,
UK). HeLa (cervix adenocarcinoma), MCF7 (breast adenocarci-
noma), A2780 (ovarian carcinoma) and A431 (skin epidermoid car-
cinoma) cells were cultivated in minimal essential medium
supplemented with 10% fetal bovine serum, 1% non-essential ami-
no acids and an antibiotic–antimycotic mixture.
To obtain pyrazolinylandrostene derivatives and to study the
stereochemistry of their formation, we reacted (21E)-3b-hydrox-
ybenzylidenepregn-5-en-20-one (3a) with hydrazine hydrate in
acetic acid. The consumption of the starting material was moni-
tored by TLC. We found that the reaction took 2 h to complete un-
der reflux, and the TLC indicated two products. The spots were
distinguishable only when methyl tert-butyl ether was used as elu-
ent. The more polar of these two spots was the more intense, and
the 1H NMR spectra of the crude mixtures revealed that the prod-
uct ratio was 1:2. Two products appeared in the ring-closure reac-
tions of all the other benzylidene derivatives (3b–f) and their ratio
was independent of the substituent in the para position of the phe-
nyl group. For easier separation, the product mixture was acety-
lated. For all of the substituted pyrazolines, the two products
could be fully separated by column chromatography. After separa-
tion, 2D NMR demonstrated that the minor products were the 50R
epimers (4a–f), while the major products were the 50S epimers
(5a–f) of the corresponding phenylpyrazolinylandrostene deriva-
tives. After separation of the acetylated pyrazoline products (4a–f
and 5a–f), they were hydrolyzed by the method of Zemplen to ob-
tain the 3b-hydroxy derivatives (4g–l and 5g–l), which are pre-
sumably more effective in the C17,20-lyase inhibition [2,23,24]
and in antiproliferative measurements [25]. Their NMR spectra
were also taken, and it was proved that the pyrazoline ring with
the N-acetyl group remained unchanged under the mild conditions
of deacetylation.
Near-confluent cancer cells were seeded onto a 96-well micro-
plate (5000/well) and attached to the bottom of the well overnight.
On the second day, 200
compound (at 10 or 30
l
l of new medium containing the tested
lM) was added. After incubation for 72 h
at 37 °C in humidified air containing 5% CO2, the living cells were
assayed by the addition of 20 l of 5 mg/ml MTT solution. MTT
l
was converted by intact mitochondrial reductase and precipitated
as blue crystals during a 4-h contact period. The medium was then
removed and the precipitated crystals were dissolved in 100 ll of
DMSO during a 60-min period of shaking at 25 °C. Finally, the re-
duced MTT was assayed at 545 nm, using a microplate reader;
wells with untreated cells were utilized as controls [17]. All
in vitro experiments were carried out on two microplates with at
least five parallel wells. Cisplatin was used as positive control.
Stock solutions of the tested substances (10 mM) were prepared
with DMSO. The DMSO content of the medium (0.1% or 0.3%) did
not have any significant effect on the cell proliferation.
3. Results and discussion
3.1. Synthetic studies
The reactions between
are widely used for the preparation of substituted 2-pyrazolines
a
,b-unsaturated ketones and hydrazines
The optical rotations of the pyrazolinyl compounds (4a–l and
5a–l) in CHCl3 presented a characteristic picture. The [a]
20 values
D
[18].
of 4a–l were always positive (between +6.4 and +77), while those
of 5a–l were negative (between ꢀ85 and ꢀ32).
When an appropriately substituted starting material is used,
one or two new stereogenic centers are formed during the ring-
closure, and as a result the reaction can lead to a diastereomeric
mixture of pyrazoline products [18]. However, in some cases the
ring closure results in only a single diastereomer [19]. Amr et al.
found that the reaction of (16E)-3b-trifluoroacetoxy-16-benzyli-
dene-5
tic acid afforded a single D-ring fused pyrazoline diastereomer,
(16R,50S)-3b-trifluoroacetoxy-5 -androstano[17,16-c]-10H-50-(p-
methoxyphenyl)-N-acetylpyrazoline [19]. In this reaction, a fused
pyrazoline ring is formed from a hydrazine derivative and an ,b-
unsaturated ring ketone, and the stereochemistry of the ring
closure can therefore be influenced by the conformation of the
starting material. There are very few examples in the literature
where the stereochemistry of the reaction between an open-chain
unsaturated ketone and hydrazine was studied [20].
Banday et al. synthetized a series of steroidal pyrazolines by
reacting 21-benzylidenepreg-5-ene derivatives (3a, 3b and 3f)
[12] with hydrazine hydrate in glacial acetic acid. This reaction
leads to a formation of a new stereogenic center at position 50; in
principle, therefore a mixture of 50R and 50S epimers can be ob-
tained. However, the separation of these two possible isomers
and a comparison of their physical and antiproliferative properties
have not yet been described.
The structures of the newly synthetized steroids were deter-
mined by NMR spectroscopy. The 1H NMR spectra of the steroidal
benzylidenes (3a–f) contained two characteristic peaks at around
6.7 and 7.5 ppm, which can be assigned to the coupling protons
of the double bond at position 21. Their coupling constant was
16 Hz, indicating their trans position relative to each other, and
the geometry of the double bond is therefore E. One of these two
peaks overlaps with the signals of the phenyl group (d = 6.89–
7.68 ppm) in the spectra of 3a, 3b, and 3f. The 3-H multiplet is to
be found at ꢁ3.5 ppm in the spectra of all the 3b-hydroxy products
(3a–f, 4g–l and 5g–l). In the 13C NMR spectra of 3a–f, the signals of
the phenyl group (d = 114–164 ppm) and C21 (d = 124–130) and
C22 (d = 139–141) are separate from the aliphatic region, and the
C20 carbonyl group resonates at ꢁ200 ppm.
a-androstan-17-one with hydrazine hydrate in glacial ace-
a
a
In the 1H NMR spectra of the newly synthetized pyrazolines
(4a–f and 5a–f), the 22- and the 21-(sp2-)H signals have disap-
peared and there are three new signals. Two of them are located
between 2.59 and 3.42 ppm, while the third one overlaps with
the 6-H signal at ꢁ5.7 ppm. These signals forming an AMX spin
system can be assigned to the protons of the pyrazoline ring; their
chemical shifts are in good agreement with those of other, non-ste-
roidal 2-pyrazoline derivatives [26,27]. It can be concluded that the
two protons at higher fields (d = 2.59–3.37) are the 40-CH2 protons,
and their chemical shifts in the two epimer series differ in a char-
acteristic manner. One of these signals is situated at ꢁ2.61 in the
spectra of the 50R epimeric series, and at ꢁ2.72 in the 50S series,
while the other is at ꢁ3.37 in the 50R series and ꢁ3.27 in the 50S
series. Other than that, there are very few differences in the spectra
The benzylidene products (3a–f) were synthetized by the aldol
condensation of pregnenolone (1) with various benzaldehyde
derivatives (2a–f) (Scheme 1). After crystallization, the purity of
the condensed products was tested with TLC. The reaction led to
a single product, the starting material was completely consumed,

572
Z. Iványi et al. / Steroids 77 (2012) 566–574
2, 3
R
a
b
c
d
e
f
H
5'
R
4'
F
6'
1'
22
21
Cl
CH3
O
C
Br
3'
O
C O
2'
ii
CN
OCH3
C
R
H
2
i
HO
HO
1
3
R2
R2
O
O
N
H3C
H3C
1'
R1
R2
5'
4, 5
N
H
H
2'
a
b
c
d
e
f
H
Ac
Ac
Ac
Ac
Ac
Ac
H
N
4'
N
ii
F
3'
Cl
+
Br
CN
OCH3
H
g
h
i
R1O
R1O
F
H
4
5
Cl
H
Br
H
j
CN
OCH3
5a-f
5g-l
H
4a-f
4g-l
k
l
iii
iii
H
Scheme 1. Reagents and conditions: (i) ethanol, NaOH, rt; (ii) acetic acid, hydrazine hydrate, reflux/pyridine, acetic anhydride, rt; (iii) methanol, NaOCH3, rt.
of the two series; minor changes can be observed in the aliphatic
region. The third pyrazoline proton is located at ꢁ5.7 ppm in both
epimeric series. From the 1H NMR spectra of 4a–f and 5a–f, it can
be concluded that N-acetylation also takes place because of the
presence of the glacial acetic acid. In the spectra of 4a–f and 5a–
f, the singlet that appears at 2.3 ppm can be assigned to the methyl
group of the N-acetyl function. Acetylation with acetic anhydride/
pyridine resulted in a shift of the 3-H signal toward lower fields,
ꢁ4.5 ppm, and the 3-Ac-CH3 resonated at around d = 2.02. In the
13C NMR spectra of compounds 4a–f and 5a–f, the characteristic
signals of the pyrazoline ring were at ꢁ46, 59 and 159 ppm, while
the N-Ac-CH3 resonated at d = ꢁ168.6 ppm, and the 3-Ac-CH3 at
ꢁ170.5 ppm. The 1H and 13C NMR spectra of 4g–l and 5g–l pre-
sented a similar picture to that for 4a–f and 5a–f, except for the
lack of the signals of the 3-Ac-CH3 group.
Spectral data on the phenylpyrazolinylandrostene compound
and its p-fluoro and p-methoxy derivatives have already been pub-
lished [12]. A comparison of the spectra of the major products (5g,
5h and 5l) with those reported by Banday et al. reveals several dif-
ferences. Our measurements indicated that Ha, and Hm in 5g reso-
nate at 2.75 and 3.27, while those in 5h do so at 2.71 and 3.26, and
those in 5l at 2.74 and 3.24, respectively. In the literature data,
three multiplets are described in that region for each compound:
2.65, 2.79 and 3.26 for the phenylpyrazolinylandrostene com-
pound, and 2.76, 3.26 and 3.35 for its p-fluoro and 2.53–2.73,
3.20 and 3.29 for its p-methoxy derivative.
of the two 40-CH2 peaks, which can be found at lower fields. In
the case of 5c, 12b-H gives a cross-peak with the 40-H signal which
resonates at higher fields. It was previously established from the
assignment that in both epimers the 40-H that resonates at higher
fields is on the same side of the pyrazoline ring as the phenyl
group. By identifying the positions of the two 40-H protons relative
to the steroidal skeleton (cross-peak with 12b-H), the absolute
configuration at the neighboring 50 position was ascertained.
For the formation of the epimers 4g–l and 5g–l, three mechanis-
tic pathways may be suggested (Scheme 2). According to a widely
accepted interpretation (A–B–C) [18,19,21], the nucleophilic attack
of the hydrazine nitrogen takes place on the carbonyl group of the
enone, and intermediate A is formed containing a chiral carbon
atom (C-20). In the case of the present molecules, the direction
of the nucleophilic attack may be affected by the steric hindrance
of the angular 18-CH3, and one side of the carbonyl group therefore
becomes more favorable. Then with the elimination of one mole-
cule of water, a hydrazone B is formed, with the loss of the C-20
asymmetric center. Intramolecular Michael addition of the NH2
group of B to the double bond leads to pyrazoline C. This step is
probably not affected by the steric effect of the angular 18-CH3,
as it is several bonds away from it, and this should therefore result
in an approximately 1:1 ratio of the epimers. The acetylation of C
by the solvent leads to the formation of the N-acetyl products
4g–l and 5g–l.
Another possible mechanistic pathway (A–D–C) starts with the
formation of A, but without the elimination of water a ring-closure
step takes place next, furnishing a hydroxypyrazolidine intermedi-
ate D. The formation of a similar intermediate was reported in the
reaction between b-dicarbonyls and hydrazine derivatives, which
is isolable in some cases [28]. As the asymmetric center at C-20
is still present in the ring closure step, its configuration determines
The 1H NMR peaks of 4c and 5c were fully assigned with the aid
of the H,H COSY and HSQC spectra. NOESY techniques were then
used to determine the absolute configuration of C-50. The NOESY
spectra of the two isolated products are very similar, but do reveal
some characteristic differences. The main difference is that the
12b-H signal in the spectrum of 4c gives a cross-peak with one

Z. Iványi et al. / Steroids 77 (2012) 566–574
573
R
R
R
R
HN
N
NH2
HN
NH2
N
H
O
R + S
OH
H2N NH2
- H2O
C
3a-f
A
B
- H2O
CH3COOH
- H2O
H2N NH2
R
R
R
O
H
N
H3C
H2N
HN
HN
N
N
H
H
H
O
R + S
OH
4g-l + 5g-l
D
E
Scheme 2. Presumed mechanisms for the formation of epimers 4g–l, 5g–l.
the configuration of the new chiral carbon atom (C-50). As the two
epimers of intermediate A may not be present in 1:1 ratio because
of the steric effects mentioned above, the 50S and 50R epimers of C
(and therefore 4g–l and 5g–l) may not form in equal quantities,
which is in agreement with our experimental findings.
According to some statements in the literature [18], the Michael
addition of the hydrazine derivate can be regarded as the first step,
but this mechanism (E–D–C) needs special experimental condi-
tions (addition of piperidine), which were not present in our
reactions.
compound cisplatin, many of them do exhibit pronounced activity
in vitro (Table 1). Skin cancer cells (A431) seemed to be commonly
less sensitive than cell lines of gynecological origin.
Since substantial differences were found in the in vitro results
on the epimer pairs, the configuration at position 50 has a crucial
impact on the activities of the synthetized compounds. In general,
the 50S epimers were more potent than their 50R counterparts, the
difference proving most substantial in compounds h and l. 5h and
5l exhibited >90% inhibition of cancer cell growth in all four cell
lines used, whereas the growth inhibition by 4h did not exceed
50%, and 4l was also less effective. The differences between the epi-
mer pairs were not so pronounced in the case of analogs g and k,
As concerns the three potential mechanisms, path A–D–C ap-
pears to be the most probable, as it can explain the unequal
amounts of the two epimers (4g–l, 5g–l).
Table 1
3.2. C17,20-lyase inhibition tests
Antiproliferative effects of the synthetized compounds.
Product
l
M
Growth inhibition% ( SEM)
The inhibitory effects of compounds 4g–l and 5g–l on rat testic-
ular C17,20-lyase were investigated with an in vitro radioincubation
HeLa
MCF7
A2780
A431
technique. At the applied concentration of 50 lM, compound 4g
4g
4h
4i
10
30
10
30
10
30
10
30
10
30
10
30
10
30
10
30
10
30
10
30
10
30
10
30
>20
22.0 ( 1.1) 40.3 ( 0.9) >20
( 0.8) 75.3 ( 0.5) 92.9 ( 0.9) >20
( 0.8) 23.3 ( 1.6) 25.4 ( 0.6) 27.6 ( 0.6)
( 0.5) 42.4 ( 0.6) 45.2 ( 0.5) >20
( 2.1) 50.3 ( 0.4) 69.3 ( 1.9) >20
( 0.2) 95.4 ( 0.4) 96.1 ( 1.0) 93.9 ( 0.7)
47.2 ( 2.1) 54.8 ( 2.8) >20
( 0.2) 95.4 ( 0.5) 97.7 ( 0.1) 94.1 ( 0.5)
( 1.7) 31.9 ( 1.1) 53.0 ( 1.2) >20
( 2.0) 41.3 ( 1.2) 54.5 ( 2.7) >20
( 2.7) 30.8 ( 1.5) 46.6 ( 1.1) >20
( 1.5) 67.1 ( 2.2) 81.4 ( 1.0) 21.5 ( 1.2)
( 1.7) 54.1 ( 1.6) 53.2 ( 1.9) >20
( 0.3) 96.2 ( 0.2) 96.2 ( 0.2) 91.9 ( 0.4)
and 5g with the non-substituted phenyl group on the heterocycle
decreased the enzyme activity by 72 3% and 92 4%, respectively.
Enzyme incubations with the other test compounds resulted in
conversions identical to those in the control experiments; hence
these compounds did not inhibit rat C17,20-lyase in vitro even in
55.6
39.8
41.5
21.3
98.0
>20
98.5
27.1
31.9
21.4
48.9
22.3
97.8
>20
97.1
>20
97.8
>20
97.2
20.7
4j
the relatively high concentration of 50
lM.
4k
4l
The attachment of pyrazolyl groups to a steroid skeleton at C-17
may produce potent inhibitors of P45017 [2,3] and extension of
a
the 17b heterocyclic side-chain with various substituted aromatic
rings may improve the inhibitory effect [7]. Our tested steroidal
phenylpyrazolines, however, did not exert efficient C17,20-lyase
inhibition. Nevertheless, the stereoselectivity of the inhibition
could be demonstrated in our experiments too, as the phenyl-
substituted 50R isomer 4g exhibited a higher inhibitory effect than
5g
5h
5i
>20
38.7 ( 1.4) 21.9 ( 1.9)
( 0.5) 95.6 ( 0.5) 96.1 ( 0.2) 91.8 ( 1.0)
25.8 ( 1.6) 52.3 ( 1.1) >20
( 0.2) 96.6 ( 0.1) 96.0 ( 0.2) 92.1 ( 0.4)
37.9 ( 2.7) 54.0 ( 1.6) >20
( 0.2) 96.1 ( 0.3) 95.7 ( 0.3) 93.1 ( 0.2)
( 1.6) 46.2 ( 0.6) 60.4 ( 0.7) >20
that of the 50S isomer counterpart 5g in the rat testicular C17,20
-
5j
lyase test.
5k
5l
31.48 ( 2.3) 81.8 ( 1.7) 89.2 ( 0.4) >20
>20
97.7
42.6
99.9
42.1 ( 2.6) 61.5 ( 2.0) >20
3.3. Antiproliferative measurements
( 0.2) 96.6 ( 0.1) 96.0 ( 0.2) 91.2 ( 1.2
( 2.3) 53.0 ( 2.3) 83.6 ( 1.2) 88.6 ( 0.5)
( 0.3) 86.9 ( 1.3) 95.0 ( 0.3) 90.2 ( 1.8)
Cisplatin 10
30
Although the antiproliferative potencies of the currently
reported compounds are not comparable with that of the reference

574
Z. Iványi et al. / Steroids 77 (2012) 566–574
[13] Njar VCO, Hector M, Hartmann RW. 20-Amino and 20,21-aziridinyl pregnene
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while the efficacies of compounds i and j seemed to be indepen-
dent of the configuration at position 50.
a
a
A large body of evidence demonstrates that exo-heterocyclic
compounds with a steroidal skeleton possess valuable pharmaco-
logical properties, including antiproliferative activity [25,29–31].
The current results allow the conclusion that the configuration of
the exo ring should be considered in the design of drugs and in
the selection of agents for further development.
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a -hydroxylase/C17-20-lyase (P450 17). J Med Chem
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Immunol Methods
b-unsaturated
This work was supported by the Hungarian Scientific Research
Fund (OTKA K101659) and New Hungary Development Plan
(TÁMOP 4.2.2-08/01-2008-0002). Nikoletta Szabó’s work was sup-
ported by a PhD Fellowship of the Talentum Fund of Richter Gede-
on Plc.
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hydrazines. J. Heterocyclic Chem 2002;39:1–13.
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