Steroselective synthesis of some steroidal oxazolines, as novel potential inhibitors of 17α-hydroxylase-C17,20-lyase

Article abstract of DOI:10.1016/j.steroids.2009.08.001

17β-Oxazolinyl steroids 7a-g and 8a-g were synthesized. The Lewis acid-catalysed reactions of (20R)-3β-acetoxy-21-azidomethyl-20-hydroxypregn-5-ene with substituted aromatic aldehydes led to the formation of 3β-acetoxyandrost-5-enes substituted in positio

Full text of DOI:10.1016/j.steroids.2009.08.001

Steroids 74 (2009) 1025–1032
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Steroselective synthesis of some steroidal oxazolines, as novel potential
inhibitors of 17␣-hydroxylase-C17,20-lyase
Dóra Ondréa, János Wölflinga, István Tóthb, Mihály Szécsib, János Juleszb, Gyula Schneidera,∗
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, 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:
Received 30 April 2009
Received in revised form 7 July 2009
Accepted 4 August 2009
Available online 8 August 2009
17␤-Oxazolinyl steroids 7a–g and 8a–g were synthesized. The Lewis acid-catalysed reactions of (20R)-
3␤-acetoxy-21-azidomethyl-20-hydroxypregn-5-ene with substituted aromatic aldehydes led to the
formation of 3␤-acetoxyandrost-5-enes substituted in position 17␤ with oxazolinyl residues (7a–g).
Oppenauer oxidation of the 3␤-hydroxy-exo-heterocyclic steroids yielded the corresponding ꢀ4-3-
ketosteroids. The inhibitory effects (IC50) of both 3-hydroxy compounds 7a–g and their ꢀ4-3-keto
counterparts 8a–g on rat testicular C17,20-lyase were investigated with an in vitro radioligand incubation
technique. The 3-chlorophenyl- (8d), and the 4-bromophenyl-17␤-(2-oxazolin-5-yl)androst-4-en-3-one
derivatives (8f) were found to be modest inhibitors (IC50 = 4.8 and 5.0 ␮M, respectively).
© 2009 Elsevier Inc. All rights reserved.
Keywords:
␣,␤-Azidoalcohols
Schmidt reaction
Steroid oxazolines
In vitro inhibition
Antiandrogenic effect
1. Introduction
number of inhibitors of P45017␣, of which 17-imidazolyl, pyra-
zolyl, isoxazoyl, oxazolyl, thiazolyl and indazol androstene are very
Prostatic cancer is the second most common cancer worldwide,
and this disease display androgen-dependency in about 80% of the
patients. The lack of available for the treatment of prostatic cancer
presents a significant challenge to researchers. 17␣-Hydroxylase-
potent [4–7]. We recently set out to synthesize a novel series of
steroidal 17␤-oxazolines, 17␤-oxazolidones, 17␤-dihydrooxazines
and 17␤-tetrahydrooxazin-2ꢀ-ones [8–11]. We report here the
syntheses of a variety of steroidal compounds with the com-
mon structural feature of a C-17␤ substituted phenyloxazoline on
C17,20-lyase plays an important role in the pathways of steroid
hormone biosynthesis. This enzyme catalyzes 17␣-hydroxylation
and cleavage of the C17–C20 bond in the side chain of the steroid
skeleton in both testes and adrenals. A complete inhibition of
this enzyme can block androgen synthesis, and may be suitable
in androgen dependent disorders [1]. In the past decade, num-
ber of different non-steroid compounds were developed, as very
potent P45017␣ inhibitors. Recently in the treatment of prostatic
cancer ketoconazole was employed, which was neither selec-
tive nor potent and side effects it showed were the reason why
it was not generally accepted [2]. Many steroidal compounds
bearing different heteroaromatic ring on the sterane skeleton
were investigated, of which abirateron [17-(3-pyridyl)androsta-
5,16-dien-3␤-ol] and its ꢀ4-3-keto analog exhibited prominent
inhibition of this enzyme. It has been suggested that such activ-
ity is related to the presence of the heterocyclic moiety in ring D,
with the nitrogen lone pair coordinating to the heme iron atom
at the active site of the enzyme [3]. Recently it was described a
steroid skeleton, as presumed inhibitors of P45017␣.
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 in units of 10−1 deg cm2 g−1. Elementary analysis data were
determined with a PerkinElmer 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) hexane/CH2Cl2
(30:70, v/v), (B) CH2Cl2, (C) ethyl acetate/CH2Cl2 (5:95, v/v), (D)
ethyl acetate/CH2Cl2 (10:90, v/v) and (E) ethyl acetate/CH2Cl2
(50:50, v/v). The spots were detected by spraying with 5% phos-
phomolybdic acid in 50% aqueous phosphoric acid. The Rf values
were determined for the spots observed by illumination at 254
and 365 nm. Flash chromatography: silica gel 60, 40–63 ␮m. All
solvents were distilled prior to use. NMR spectra were recorded
∗
Corresponding author. Tel.: +36 62 544276; fax: +36 62 544200.
E-mail address: schneider@chem.u-szeged.hu (G. Schneider).
on Bruker DRX 500 instruments at 500 (1H NMR) or 125 MHz (13
C
0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.08.001

1026
D. Ondré et al. / Steroids 74 (2009) 1025–1032
NMR). Chemical shifts are reported in ppm (ı scale), and coupling
constants (J) in Hertz. For the determination of multiplicities, the
J-MOD pulse sequence was used.
6), (128.6 and 129.4): (C-2ꢀꢀ, C-3ꢀꢀ, C-5ꢀꢀ, C-6ꢀꢀ), 126.7 (C-1ꢀꢀ), 137.3
(C-4ꢀꢀ), 139.8 (C-5), 163.2 (C-2ꢀ), 170.4 (Ac-CO).
2.2.4. (5ꢀR)-3ˇ-acetoxy-17ˇ-[2-(3-chlorophenyl)-4,5-
dihydrooxazol-5-yl]androst-5-ene
2.2. General procedure for preparation of compounds 6a–g
(6d)
6d (1.69 g, 85%), mp 170–174 ◦C, Rf = 0.60 (ss D); [␣]D − 38 (c 1
20
Compound 4b [8] (1.6 g, 4 mmol) was dissolved in dry CH2Cl2
(50 ml) and benzaldehyde (5a) and substituted benzaldehyde
(5b–g) (1.1 equivalent) was cooled to 0 ◦C, followed by the drop-
wise addition of BF3·OEt2 (50%) (8 mmol, 1.1 ml); the addition of
Lewis acid was accompanied by gas evolution. The reaction mixture
was stirred at room temperature for 6 h. After the disappearance
of starting material (TLC monitoring), saturated NaHCO3 solution
was added and the mixture was stirred until bubbling ceased. The
organic layer was washed with water, dried over anhydrous Na2SO4
and concentrated in vacuo. The product was purified by chromatog-
raphy on silica gel with hexane/CH2Cl2 (30:70, v/v).
in CHCl3) (found C, 72.55; H, 7.89. C30H38ClNO3 requires C, 72.63;
H, 7.72%). 1H NMR (ı, ppm, CDCl3): 0.86 (s, 3H, 18-H3), 1.05 (s, 3H,
19-H3), 2.02 (s, 3H, Ac-CH3), 3.63 (dd, 1H, J = 14.5 Hz and J = 8.0 Hz)
and 4.05 (dd, 1H, J = 14.5 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.62 (overlapping
multiplets, 2H, 3- and 5ꢀ-H), 5.37 (d, 1H, J = 4.5 Hz, 6-H), 7.33 (t, 1H,
J = 8.0 Hz, 5ꢀꢀ-H), 7.43 (d, 1H, J = 8.0 Hz, 4ꢀꢀ-H), 7.80 (d, 1H, J = 8.0 Hz,
6ꢀꢀ-H), 7.90 (s, 1H, 2ꢀꢀ-H). 13C NMR (ı, ppm, CDCl3): 12.7 (C-18), 19.3
(C-19), 20.8, 21.4 (Ac-CH3), 23.7, 24.7, 27.8, 31.7 (C-8), 31.9, 36.7 (C-
10), 37.0, 38.1, 39.0, 42.7 (C-13), 50.1, 55.0, 55.9, 59.8 (C-4ꢀ), 73.9
(C-3), 82.1 (C-5ꢀ), 122.3 (C-6), (126.2, 128.2, 129.6, and 131.1): (C-
2ꢀꢀ, C-4ꢀꢀ, C-5ꢀꢀ, C-6ꢀꢀ), 130.0 (C-3ꢀꢀ), 134.3 (C-1ꢀꢀ), 139.8 (C-5), 163.0
(C-2ꢀ), 170.4 (Ac-CO).
2.2.1. (5ꢀR)-3ˇ-acetoxy-17ˇ-[2-phenyl-4,5-dihydrooxazol-5-
yl]androst-5-ene
2.2.5. (5ꢀR)-3ˇ-acetoxy-17ˇ-[2-(2-chlorophenyl)-4,5-
dihydrooxazol-5-yl]androst-5-ene
(6a)
6a (1.64 g, 89%), mp 184–188 ◦C, Rf = 0.49 (ss D); [␣]D − 60 (c
20
(6e)
1 in CHCl3) (found C, 78.06; H, 8.95. C31H43NO3 requires C, 77.95;
H, 9.07%). 1H NMR (ı, ppm, CDCl3): 0.87 (s, 3H, 18-H3), 1.06 (s, 3H,
19-H3), 2.03 (s, 3H, Ac-CH3), 3.64 (dd, 1H, J = 14.5 Hz and J = 8.0 Hz)
and 4.05 (dd, 1H, J = 14.5 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.61 (overlapping
multiplets, 2H, 3- and 5ꢀ-H), 5.38 (d, 1H, J = 4.0 Hz, 5-H), 7.40 (t, 2H,
J = 7.5 Hz, 3ꢀꢀ- and 5ꢀꢀ-H), 7.46 (t, 1H, J = 7.5 Hz, 4ꢀꢀ-H), 7.92 (d, 2H,
J = 7.5 Hz, 2ꢀꢀ- and 6ꢀꢀ-H). 13C NMR (ı, ppm, CDCl3): 12.7 (C-18), 19.4
(C-19), 20.8, 21.4 (Ac-CH3), 23.8, 24.7, 27.8, 31.7 (C-8), 31.9, 36.7 (C-
10), 37.0, 38.1, 40.0, 42.7 (C-13), 50.2, 55.0, 55.9, 59.8 (C-4ꢀ), 73.9
(C-3), 81.7 (C-5ꢀ), 122.4 (C-6), (128.1 and 128.2): (C-2ꢀꢀ, C-3ꢀꢀ, C-5ꢀꢀ,
C-6ꢀꢀ), 128.2 (C-1ꢀꢀ), 131.1 (C-4ꢀꢀ), 128.2 (C-1ꢀꢀ), 139.8 (C-5), 164.1
(C-2ꢀ), 170.4 (Ac-CO).
6e (1.65 g, 83%), mp 162–164 ◦C, Rf = 0.60 (ss D); [␣]D − 59 (c 1
20
in CHCl3) (found C, 72.55; H, 7.82. C30H38ClNO3 requires C, 72.63;
H, 7.72%). 1H NMR (ı, ppm, CDCl3): 0.83 (s, 3H, 18-H3), 1.03 (s, 3H,
19-H3), 2.02 (s, 3H, Ac-CH3), 3.68 (dd, 1H, J = 14.5 Hz and J = 8.5 Hz)
and 4.11 (dd, 1H, J = 14.5 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.62 (overlapping
multiplets, 2H, 3- and 5ꢀ-H), 5.37 (s, 1H, 6-H), 7.28 (t, 1H, J = 7.5 Hz,
5ꢀꢀ-H), 7.34 (t, 1H, J = 7.5 Hz, 4ꢀꢀ-H), 7.43 (d, 1H, J = 7.5 Hz, 3ꢀꢀ-H), 7.76
(d, 1H, J = 7.5 Hz, 6ꢀꢀ-H). 13C NMR (ı, ppm, CDCl3): 12.6 (C-18), 19.3
(C-19), 20.8, 21.4 (Ac-CH3), 23.8, 24.7, 27.8, 31.7 (C-8), 31.9, 36.7 (C-
10), 36.9, 37.0, 38.1, 38.8, 42.6 (C-13), 50.1, 54.9, 55.9, 60.3 (C-4ꢀ),
73.9 (C-3), 81.5 (C-5ꢀ), 122.3 (C-6), (126.4, 130.7, 131.2 and 131.3):
(C-3ꢀꢀ, C-4ꢀꢀ, C-5ꢀꢀ, C-6ꢀꢀ), 127.7 (C-2ꢀꢀ), 133.4 (C-1ꢀꢀ), 139.8 (C-5), 162.6
(C-2ꢀ), 170.4 (Ac-CO).
2.2.2. (5ꢀR)-3ˇ-acetoxy-17ˇ-[2-(4-fluorophenyl)-4,5-
2.2.6. (5ꢀR)-3ˇ-acetoxy-17ˇ-[2-(4-bromophenyl)-4,5-
dihydrooxazol-5-yl]androst-5-ene
(6f)
dihydrooxazol-5-yl]androst-5-ene
(6b)
6b (1.33 g, 70%), mp 166–168 ◦C, Rf = 0.47 (ss D); [␣]D − 57 (c
20
6f (2.07 g, 92%). (Ref. [8] mp 103–105 ◦C).
1 in CHCl3) (found C, 75.24; H, 8.05. C30H38FNO3 requires C, 75.13;
H, 7.99%). 1H NMR (ı, ppm, CDCl3): 1.05 (s, 3H, 18-H3), 1.24 (s, 3H,
19-H3), 2.21 (s, 3H, Ac-CH3), 3.81 (dd, 1H, J = 14.5 Hz and J = 8.0 Hz)
and 4.22 (dd, 1H, J = 14.5 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.80 (overlapping
multiplets, 2H, 3- and 5ꢀ-H), 5.56 (d, 1H, J = 3.5 Hz, 6-H), 7.26 (t, 2H,
J = 8.5 Hz, 3ꢀꢀ- and 5ꢀꢀ-H), 8.10 (dd, 2H, J = 8.0 Hz, 2ꢀꢀ- and 6ꢀꢀ-H). 13C
NMR (ı, ppm, CDCl3): 12.7 (C-18), 18.9 (C-19), 20.4, 21.0 (Ac-CH3),
23.3, 24.3, 27.4, 31.3 (C-8), 31.5, 36.3 (C-10), 36.6, 37.7, 38.6, 42.3
(C-13), 49.8, 54.6, 55.5, 59.4 (C-4ꢀ), 73.5 (C-3), 81.5 (C-5ꢀ), 115.0
(2C, J = 21.7 Hz, C-3ꢀꢀ and C-5ꢀꢀ), 121.9 (C-6), 124.0 (C-1ꢀꢀ), 129.9 (2C,
J = 8.6 Hz, C-2ꢀꢀ and C-6ꢀꢀ), 139.4 (C-5), 163.0 (J = 47.8 Hz, C-4ꢀꢀ), 163.2,
165.2 (C-2ꢀ), 170.0 (Ac-CO).
2.2.7. (5ꢀR)-3ˇ-acetoxy-17ˇ-[2-(4-nitrophenyl)-4,5-
dihydrooxazol-5-yl]androst-5-ene
(6g)
6g (1.01 g, 90%). (Ref. [8] mp 111–113 ◦C).
2.3. General procedure for preparation of compounds 7a–g
The individual compounds 6a–g (2.5 mmol) were suspended in
methanol (50 ml), and 108 mg (2 mmol) NaOCH3 in 5 ml methanol
was added. The mixture was stirred for 8 h at room temperature
and than was poured into 300 ml of water. The precipitate that
formed was filtered off and chromatographed on silica starting
with CH2Cl2, followed by ethyl acetate/CH2Cl2 (5:95, v/v) and ethyl
acetate/CH2Cl2 (10:90, v/v) to afford 7a–g.
2.2.3. (5ꢀR)-3ˇ-acetoxy-17ˇ-[2-(4-chlorophenyl)-4,5-
dihydrooxazol-5-yl]androst-5-ene
(6c)
6c (1.78 g, 90%), mp 149–152 ◦C, Rf = 0.53 (ss D); [␣]D − 41 (c 1
20
in CHCl3) (found C, 72.74; H, 7.85. C30H38ClNO3 requires C, 72.63;
H, 7.72%). 1H NMR (ı, ppm, CDCl3): 0.86 (s, 3H, 18-H3), 1.05 (s, 3H,
19-H3), 2.02 (s, 3H, Ac-CH3), 3.63 (dd, 1H, J = 14.5 Hz and J = 8.0 Hz)
and 4.04 (dd, 1H, J = 14.5 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.62 (overlapping
multiplets, 2H, 3- and 5ꢀ-H), 5.37 (d, 1H, J = 4.5 Hz, 6-H), 7.37 (d, 2H,
J = 8.5 Hz, 3ꢀꢀ- and 5ꢀꢀ-H), 7.85 (d, 2H, J = 8.5 Hz, 2ꢀꢀ- and 6ꢀꢀ-H). 13C
NMR (ı, ppm, CDCl3): 12.7 (C-18), 19.3 (C-19), 20.8, 21.4 (Ac-CH3),
23.7, 24.7, 27.8, 31.7 (C-8), 31.9, 36.7 (C-10), 37.0, 38.1, 39.0, 42.7
(C-13), 50.2, 54.9, 55.9, 59.8 (C-4ꢀ), 73.9 (C-3), 82.0 (C-5ꢀ), 122.3 (C-
2.3.1.
(5ꢀR)-17ˇ-[2-phenyl)-4,5-dihydrooxazol-5-yl]androst-5-en-3ˇ-ol
(7a)
7a (890 mg, 81%), mp 215–216 ◦C, Rf = 0.27 (ss D); [␣]D − 55 (c
20
1 in CHCl3) (found C, 79.82; H, 9.36. C29H41NO2 requires C, 79.95;
H, 9.49%. 1H NMR (ı, ppm, CDCl3): 0.87 (s, 3H, 18-H3), 1.04 (s, 3H,
19-H3), 3.51 (m, 1-H, 3-H), 3.63 (dd, 1H, J = 14.5 Hz and J = 8.0 Hz)
and 4.05 (dd, 1H, J = 14.5 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.65 (m, 1H, 5ꢀ-H),
5.34 (d, 1H, J = 5.0 Hz, 6-H), 7.39 (t, 2H, J = 7.5 Hz, 3ꢀꢀ- and 5ꢀꢀ-H), 7.46

D. Ondré et al. / Steroids 74 (2009) 1025–1032
1027
and 131.3): (C-3ꢀꢀ, C-4ꢀꢀ, C-5ꢀꢀ, C-6ꢀꢀ), 127.7 (C-2ꢀꢀ), 133.4 (C-1ꢀꢀ), 140.9
(C-5), 1627 (C-2ꢀ).
(t, 1H, J = 7.5 Hz, 4ꢀꢀ-H), 7.92 (d, 2H, J = 7.5 Hz, 2ꢀꢀ- and 6ꢀꢀ-H). 13C NMR
(ı, ppm, CDCl3): 12.7 (C-18), 19.4 (C-19), 20.9, 23.8, 24.7, 31.7 (C-8),
31.7, 31.9, 36.6 (C-10), 37.3, 39.0, 42.3, 42.7 (C-13), 50.3, 55.0, 56.0,
59.7 (C-4ꢀ), 71.6, 81.7, 121.4 (C-6), (128.1 and 128.3): (C-2ꢀꢀ, C-3ꢀꢀ,
C-5ꢀꢀ, C-6ꢀꢀ), 128.1 (C-1ꢀꢀ), 131.1 (C-4ꢀꢀ), 140.9 (C-5), 164.1 (C-2ꢀ).
2.3.6. (5ꢀR)-17ˇ-[2-(4-bromophenyl)-4,5-dihydrooxazol-5-
yl]androst-5-en-3ˇ-ol
(7f)
7f (1.05 g, 84%). (Ref. [8] mp 202–203 ◦C).
2.3.2. (5ꢀR)-17ˇ-[2-(4-fluorophenyl)-4,5-dihydrooxazol-5-
yl]androst-5-en-3ˇ-ol
(7b)
2.3.7. (5ꢀR)-17ˇ-[2-(4-nitrophenyl)-4,5-dihydrooxazol-5-
yl]androst-5-en-3ˇ-ol
7b (950 mg, 87%), mp 232–235 ◦C, Rf = 0.29 (ss D); [␣]D − 61 (c
20
(7g)
1 in CHCl3) (found C, 76.72; H, 8.37. C28H36FNO2 requires C, 76.85;
H, 8.29%). 1H NMR (ı, ppm, CDCl3): 0.86 (s, 3H, 18-H3), 1.04 (s, 3H,
19-H3), 3.51 (m, 1H, 3-H), 3.62 (dd, 1H, J = 14.0 Hz and J = 8.0 Hz)
and 4.03 (dd, 1H, J = 14.0 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.67 (m, 1H, 5ꢀ-
H), 5.35 (s, 1H, 6-H), 7.07 (t, 2H, J = 8.5 Hz, 3ꢀꢀ- and 5ꢀꢀ-H), 7.92 (dd,
2H, J = 8.0 Hz, 2ꢀꢀ- and 6ꢀꢀ-H). 13C NMR (ı, ppm, CDCl3): 12.7 (C-18),
19.4 (C-19), 20.9, 23.8, 24.7, 31.7 (C-8), 31.7, 31.9, 36.6 (C-10), 37.3,
39.1, 42.3, 42.7 (C-13), 50.3, ꢀ5ꢀ 5.0, 56.0, 59.8 (C-4ꢀ), 71.7 (C-3), 81.9
(C-5ꢀ), 115.3 (J = 21.7 Hz, C-3 and C-5ꢀꢀ), 121.4 (C-6), 124.4 (C-1ꢀꢀ),
130.3 (J = 8.6 Hz, C-2ꢀꢀ and C-6ꢀꢀ), 140.9 (C-5), 163.4 (J = 42.4 Hz, C-4ꢀꢀ),
165.6 (C-2ꢀ).
7g (0.920 mg, 79%). (Ref. [8] mp 236–238 ◦C).
2.4. General procedure for preparation of compounds 8a–g
Compound 7a–g (2.0 mmol) was dissolved in toluene (50 ml),
Al(OiPr)3 (1.2 g, 6 mmol) and cyclohexanone (25 ml) were added to
it, and was stirred at 100 ◦C. The decrease of the starting material
was followed by TLC. When the reaction was complete, the mixture
was poured into water (200 ml) in which K/Na-tartarate (5 g) was
solved. Most of the organic solvent was removed in vacuo and the
residual emulsion was extracted with CH2Cl2. The organic phase
was evaporated to dryness and the residual crude product was
chromatographed on silica gel with ethyl acetate/CH2Cl2 (5:95).
2.3.3. (5ꢀR)-17ˇ-[2-(4-chlorophenyl)-4,5-dihydrooxazol-5-
yl]androst-5-en-3ˇ-ol
(7c)
7c (1.14 g, 70%), mp 215–218 ◦C, Rf = 0.29 (ss D); [␣]D − 48 (c 1
20
2.4.1.
(5ꢀR)-17ˇ-[2-phenyl-4,5-dihydrooxazol-5-yl]androst-5-en-3-one
(8a)
in CHCl3) (found C, 73.95; H, 8.03. C28H36ClNO2 requires C, 74.07;
H, 7.99%). 1H NMR (ı, ppm, CDCl3): 0.87 (s, 3H, 18-H3), 1.05 (s, 3H,
19-H3), 3.52 (m, 1H, 3-H), 3.63 (d, 1H, J = 14.5 Hz and J = 8.0 Hz) and
4.05 (d, 1H, J = 14.5 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.66 (m, 1H, 5ꢀ-H), 5.35
(d, 1H, J = 5.0 Hz, 6-H), 7.37 (d, 2H, J = 8.5 Hz, 3ꢀꢀ- and 5ꢀꢀ-H), 7.85 (d,
2H, J = 8.5 Hz, 2ꢀꢀ- and 6ꢀꢀ-H). 13C NMR (ı, ppm, CDCl3): 12.7 (C-18),
19.4 (C-19), 20.9, 23.8, 24.7, 31.7 (C-8), 31.7, 31.9, 36.6 (C-10), 37.3,
39.1ꢀ, 42.3, 42.7 (C-13), 50.3, 55.0, 56.0, 59.8 (C-4ꢀ), 71.7 (C-3), 82.0
(C-5 ), 121.4 (C-6), (128.6 and 129.4): (C-2ꢀꢀ, C-3ꢀꢀ, C-5ꢀꢀ, C-6ꢀꢀ), 126.7
(C-1ꢀꢀ), 137.3 (C-4ꢀꢀ), 140.9 (C-5), 163.3 (C-2ꢀ).
8a (350 mg, 40%), mp 89–93 ◦C, Rf = 0.29 (ss D); [␣]D20 + 94 (c 1
in CHCl3) (found C, 80.17; H, 9.25. C29H39NO2 requires C, 80.33; H,
9.07%). 1H NMR (ı, ppm, CDCl3): 0.90 (s, 3H, 18-H3), 1.21 (s, 3H,
19-H3), 3.63 (dd, 1H, J = 14.5 Hz and J = 8.0 Hz) and 4.05 (dd, 1H,
J = 14.5 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.64 (m, 1H, 5ꢀ-H), 5.72 (s, 1H, 4-
H), 7.39 (t, 2H, J = 7.5 Hz, 3ꢀꢀ- and 5ꢀꢀ-H), 7.45 (t, 1H, J = 7.5 Hz, 4ꢀꢀ-H),
7.91 (d, 2H, J = 7.5 Hz, 2ꢀꢀ- and 6ꢀꢀ-H). 13C NMR (ı, ppm, CDCl3): 17.4
(C-18), 20.8 (C-19), 23.7, 24.6, 32.0, 32.8, 33.9, 35.4 (C-8), 35.6, 35.7,
38.6 (C-10), 38.9, 39.0, 42.8 (C-13), 53.9, 54.9, 55.1, 55.2, 59.8 (C-4ꢀ),
81.5 (5ꢀ-H), 123.8 (C-4), (128.0 and 128.3): (C-2ꢀꢀ, C-3ꢀꢀ, C-5ꢀꢀ, C-6ꢀꢀ),
128.1 (C-1ꢀꢀ), 131.1 (C-4ꢀꢀ), 164.0 (C-2ꢀ), 171.1 (C-5), 199.3 (C-3).
2.3.4. (5ꢀR)-17ˇ-[2-(3-chlorophenyl)-4,5-dihydrooxazol-5-
yl]androst-5-en-3ˇ-ol
(7d)
7d (930 mg, 82%), mp 231–234 ◦C, Rf = 0.32 (ss D); [␣]D − 52 (c
2.4.2. (5ꢀR)-17ˇ-[2-(4-Fluorophenyl)-4,5-dihydrooxazol-5-
yl]androst-5-en-3-one
(8b)
20
1 in CHCl3) (found C, 74.12; H, 7.85. C28H36ClNO2 requires C, 74.07;
H, 7.99%). 1H NMR (ı, ppm, CDCl3): 0.86 (s, 3H, 18-H3), 1.04 (s, 3H,
19-H3), 3.51 (m, 1H, 3-H), 3.63 (dd, 1H, J = 14.5 Hz and J = 8.0 Hz)
and 4.05 (dd, 1H, J = 14.5 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.66 (m, 1H, 5ꢀ-
H), 5.35 (d, 1H, J = 5.0 Hz, 6-H), 7.33 (t, 1H, J = 8.0 Hz, 5ꢀꢀ-H), 7.43 (d,
1H, J = 8.0 Hz, 4ꢀꢀ-H), 7.80 (d, 1H, J = 8.0 Hz, 6ꢀꢀ-H), 7.9 (s, 1H, 2ꢀꢀ-H).
13C NMR (ı, ppm, CDCl3): 12.7 (C-18), 19.4 (C-19), 20.9, 23.7, 24.7,
31.7 (C-8), 31.7, 31.9, 36.6 (C-10), 37.3, 39.0, 42.3, 42.7 (C-13), 50.3,
55.0, 56.0, 59.8 (C-4ꢀ), 71.7 (C-3), 82.1 (C-5ꢀ), 121.4 (C-6), (126.2,
128.2, 129.6 and 131.3): (C-2ꢀꢀ, C-4ꢀꢀ, C-5ꢀꢀ, C-6ꢀꢀ), 129.9 (C-3ꢀꢀ), 134.3
(C-1ꢀꢀ), 140.9 (C-5), 163.0 (C-2ꢀ).
8b (425 mg, 48%), mp 164–167 ◦C, Rf = 0.35 (ss D); [␣]D20 + 94 (c
1 in CHCl3) (found C, 77.34; H, 7.65. C28H34FNO2 requires C, 77.21;
H, 7.87%). 1H NMR (ı, ppm, CDCl3): 0.89 (s, 3H, 18-H3), 1.20 (s,
3H, 19-H3), 3.61 (dd, 1H, J = 14.0 Hz and J = 8.0 Hz) and 4.04 (dd, 1H,
J = 14.0 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.64 (m, 1H, 5ꢀ-H), 5.72 (s, 1H, 4-
H), 7.07 (t, 2H, J = 8.5 Hz, 3ꢀꢀ- and 5ꢀꢀ-H), 7.90 (dd, 2H, J = 8.0 Hz, 2ꢀꢀ-
and 6ꢀꢀ-H). 13C NMR (ı, ppm, CDCl3): 12.8 (C-18), 17.4 (C-19), 20.8,
23.7, 24.6, 32.0, 32.8, 33.9, 35.4 (C-8), 35.7, 38.6 (C-10), 38.9, 42.8
(C-13), 53.9, 54.9, 55.1, 59.8 (C-4ꢀ), 81.8 (5ꢀ-C), 115.4 (J = 21.8 Hz,
C-3ꢀꢀ and C-5ꢀꢀ), 123.7 (C-4), 124.4 (C-1ꢀꢀ), 130.2 (J = 8.6 Hz, C-2ꢀꢀ and
C-6ꢀꢀ), 163.4 (J = 56.4 Hz, C-4ꢀꢀ), 165.6 (C-2ꢀꢀ), 171.0 (C-5), 199.3 (C-3).
2.3.5. (5ꢀR)-17ˇ-[2-(2-chlorophenyl)-4,5-dihydrooxazol-5-
yl]androst-5-en-3ˇ-ol
(7e)
2.4.3. (5ꢀR)-17ˇ-[2-(4-chlorophenyl)-4,5-dihydrooxazol-5-
yl]androst-5-en-3-one
(8c)
7e (970 mg, 85%), mp 165–169 ◦C, Rf = 0.35 (ss D); [␣]D − 58 (c
20
1 in CHCl3) (found C, 74.21; H, 8.12. C28H36ClNO2 requires C, 74.07;
H, 7.99%). 1H NMR (ı, ppm, CDCl3): 0.82 (s, 3H, 18-H3), 1.01 (s, 3H,
19-H3), 3.50 (m, 1-H, 3-H), 3.68 (dd, 1H, J = 14.5 Hz and J = 8.5 Hz)
and 4.12 (dd, 1H, J = 14.5 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.64 (m, 1H, 5ꢀ-
H), 5.34 (s, 1H, 6-H), 7.28 (t, 1H, 5ꢀꢀ-H), 7.34 (t, 1H, J = 7.5 Hz, 4ꢀꢀ-H),
7.43 (d, 1H, J = 7.5 Hz, 3ꢀꢀ-H), 7.75 (d, 1H, J = 7.5 Hz, 6ꢀꢀ-H). 13C NMR
(ı, ppm, CDCl3): 12.6 (C-18), 19.4 (C-19), 20.8, 23.8, 24.7, 31.6 (C-8),
31.7, 31.9, 36.6 (C-10), 37.3, 38.9, 42.3, 42.6 (C-13), 50.2, 54.9, 56.0,
60.2 (C-4ꢀ), 71.7 (C-3), 81.6 (C-5ꢀ), 121.4 (C-6), (126.4, 130.7, 131.2
8c (410 mg, 45%), mp 167–169 ◦C, Rf = 0.38 (ss D); [␣]D20 + 117
(c 1 in CHCl3) (found C, 74.26; H, 7.75. C28H34ClNO2 requires C,
74.40; H, 7.58%). 1H NMR (ı, ppm, CDCl3): 0.89 (s, 3H, 18-H3), 1.21
(s, 3H, 19-H3), 3.62 (dd, 1H, J = 14.5 Hz and J = 8.0 Hz) and 4.05 (dd,
1H, J = 14.5 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.65 (m, 1H, 5ꢀ-H), 5.72 (s, 1H,
4-H), 7.37 (d, 2H, J = 8.5 Hz, 3ꢀꢀ- and 5ꢀꢀ-H), 7.84 (d, 2H, J = 8.5 Hz, 2ꢀꢀ-
and 6ꢀꢀ-H). 13C NMR (ı, ppm, CDCl3): 12.8 (C-18), 17.4 (C-19), 20.8,
23.7, 24.6, 32.1, 32.8, 34.0, 35.4 (C-8), 35.7, 38.6 (C-10), 38.9, 42.8

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D. Ondré et al. / Steroids 74 (2009) 1025–1032
(C-13), 53.9, 54.9, 55.1, 59.8 (C-4ꢀ), 81.8 (C-5ꢀ), 123.9 (C-4), 126.6 (C-
1ꢀꢀ), (128.6 and 129.4): (C-2ꢀꢀ, C-3ꢀꢀ, C-5ꢀꢀ, C-6ꢀꢀ), 137.3 (C-4ꢀꢀ), 163.2
(C-2ꢀ), 171.0 (C-5), 199.4 (C-3).
2.5. Determination of C17,20-lyase activity and its inhibition in
the rat testis
Inhibition effects exerted on the C17,20-lyase activity by the
newly synthesized 17␤-(2-oxazolin-5-yl) steroids were deter-
mined by an in vitro radiosubstrate incubation method described
in our earlier publications [9,10] with some modifications.
2.4.4. (5ꢀR)-17ˇ-[2-(3-chlorophenyl)-4,5-dihydrooxazol-5-
yl]androst-5-en-3-one
(8d)
8d (375 mg, 41%), mp 78–81 ◦C, Rf = 0.44 (ss D); [␣]D20 + 101 (c 1
In brief, testicular tissue of adult Wistar rats was homogenized
with Ultra-Turrax in 0.1 M HEPES buffer (pH = 7.3) containing 1 mM
EDTA and 1 mM dithiotreitol. Aliquots of this homogenate were
incubated with 1 ␮M [3H]17-hydroxyprogesterone in the presence
of 1 mM NADPH at 37 ◦C for 20 min. The enzymatic reaction was
stopped by addition of diethyl ether and freezing. After extraction,
unlabelled carriers of the 17-hydroxyprogesterone and the prod-
uct androst-4-ene-3,17-dione were added to the samples. The two
steroids were separated by TLC on Kieselgel-G (Merck Si 254 F) lay-
ers (0.25 mm thick) with solvent system diisopropyl ether/CH2Cl2
(50:50, v/v) and UV spots were used to trace the separated steroids.
Spots were cut and radioactivity of the androst-4-ene-3,17-dione
formed and the 17␣-hydroxyprogesterone remaining was mea-
sured with liquid scintillation counting. C17,20-lyase activity was
calculated in picomoles of the androst-4-ene-3,17-dione formed.
Test compounds were applied at 50 ␮M. Control incubates with-
out test substances and incubates with the reference compound
ketoconazole were also prepared in every series. At least two exper-
iments were performed with each test compounds and standard
deviations of the mean enzyme activity results were within 10%.
IC50 values were determined for more potential inhibitors. In this
case, conversion was measured at five to six different concentra-
tions of the test compound. IC50 results were calculated by linear
regression analysis following a logit-log transformation of the data,
and the standard deviations were determined from the fitted lines.
in CHCl3) (found C, 74.32; H, 7.66; C28H34ClNO2 requires C, 74.40;
H, 7.58%). 1H NMR (ı, ppm, CDCl3): 0.89 (s, 3H, 18-H3), 1.20 (s,
3H, 19-H3), 3.62 (dd, 1H, J = 14.5 Hz and J = 8.0 Hz) and 4.05 (dd, 1H,
J = 14.5 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.65 (m, 1H, 5ꢀ-H), 5.71 (s, 1H, 4-H),
7.32 (t, 1H, J = 8.0 Hz, 5ꢀꢀ-H), 7.42 (d, 1H, J = 8.0 Hz, 4ꢀꢀ-H), 7.78 (d, 1H,
J = 8.0 Hz, 6ꢀꢀ-H), 7.88 (s, 1H, 2ꢀꢀ-H). 13C NMR (ı, ppm, CDCl3): 12.7
(C-18), 17.4 (C-19), 20.8, 23.7, 24.5, 32.0, 32.8, 33.9, 35.4 (C-8), 35.7,
38.6 (C-10), 38.9, 42.8 (C-13), 53.9, 54.9, 55.1, 59.8 (C-4ꢀ), 76.7, 77.0,
77.3, 81.9 (5ꢀ-C), 123.9 (C-4), (126.1, 128.1, 129.7, and 131.1): (C-
2ꢀꢀ, C-4ꢀꢀ, C-5ꢀꢀ, C-6ꢀꢀ), 129.9 (C-3ꢀꢀ), 134.3 (C-1ꢀꢀ), 162.9 (C-2ꢀ), 171.0
(C-5), 199.3 (C-3).
2.4.5. (5ꢀR)-17ˇ-[2-(2-chlorophenyl)-4,5-dihydrooxazol-5-
yl]androst-5-en-3-one
(8e)
8e (440 mg, 48%), mp 132–135 ◦C, Rf = 0.42 (ss D); [␣]D20 + 77 (c
1 in CHCl3) (found C, 74.26; H, 7.35. C26H35ClNO2 requires C, 74.40;
H, 7.58%). 1H NMR (ı, ppm, CDCl3): 0.86 (s, 3H, 18-H3), 1.18 (s, 3H,
19-H3), 3.67 (dd, 1H, J = 14.5 Hz and J = 8.5 Hz) and 4.11 (dd, 1H,
J = 14.5 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.64 (m, 1H, 5ꢀ-H), 5.71 (s, 1H, 4-
H), 7.28 (t, 1H, J = 7.5 Hz, 5ꢀꢀ-H), 7.34 (t, 1H, J = 7.5 Hz, 4ꢀꢀ-H), 7.43 (d,
1H, J = 7.5 Hz, 3ꢀꢀ-H), 7.74 (d, 1H, J = 7.5 Hz, 6ꢀꢀ-H). 13C NMR (ı, ppm,
CDCl3): 12.7 (C-18), 17.4 (C-19), 20.8, 23.7, 24.5, 32.1, 32.8, 33.9,
35.4 (C-8), 35.7, 38.6 (C-10), 38.7, 42.7 (C-13), 53.9, 54.8, 55.1, 60.2
(C-4ꢀ), 81.4 (C-5ꢀ), 123.8 (C-4), (126.4, 130.7, 131.2 and 131.4): (C-
3ꢀꢀ, C-4ꢀꢀ, C-5ꢀꢀ, C-6ꢀꢀ), 127.6 (C-2ꢀꢀ), 133.4 (C-1ꢀꢀ), 162.6 (C-2ꢀ), 171.1
(C-5), 199.4 (C-3).
3. Results and discussion
3.1. Synthetic studies
2.4.6. (5ꢀR)-17ˇ-[2-(4-bromophenyl)-4,5-dihydrooxazol-5-
yl]androst-5-en-3-one
Oxazolines have played important role in many areas of chem-
istry. Starting from ␣,␤-azidoalcohol several possibilities can be
given to prepare oxazoline moieties. Catalytic hydrogenation of azi-
doalcohols provides the corresponding aminoalcohols followed by
a ring closure with imidates furnishes the oxazolines [12]. Tsuboi
et al. from ␣,␤-benzamidoalcohol under the Mitsunobu reaction
conditions produced oxazolines directly in good yield [13]. From
␣,␤-aminoalcohol substituted oxazoline can be prepared in the
presence of aromatic orthoester [14]. Moreover, one-pot synthesis
was worked out from carboxylic acids with aromatic aminoalcohols
to oxazoles using Deoxo-fluor reagent [15]. New methodology for
the synthesis of variously substituted 2-oxazolines using aldehy-
des, aminoalcohol, and N-bromosuccinimide as an oxidizing agent
were described [16]. On the other hand, oxazolines can also be pro-
duced in the presence of molecular iodine using aldehydes, too
[17]. Not only the aminoalcohols can be starting material, but in
an extension of classic Schmidt reaction of ␣,␤-azidoalcohols with
aldehydes also can be suitable components in these cyclisations.
Boyer and Hammer found that the reactions of alkyl azides with
aromatic aldehydes could be carried out with protic or Lewis acids
in benzene to afford amides [18]. In contrast, to use ␣,␤- or ␣,␥-
azidoalcohols under similar conditions afforded oxazolines and
dihydrooxazines, respectively [19].
(8f)
8f (460 mg, 46%), mp 184–189 ◦C, Rf = 0.42 (ss D); [␣]D20 + 114
(c 1 in CHCl3) (found C, 67.55; H, 7.05. C28H34BrNO2 requires C,
67.74; H, 6.90%). 1H NMR (ı, ppm, CDCl3): 0.89 (s, 3H, 18-H3), 1.20
(s, 3H, 19-H3), 3.62 (dd, 1H, J = 14.5 Hz and J = 8.0 Hz) and 4.04 (dd,
1H, J = 14.5 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.65 (m, 1H, 5ꢀ-H), 5.72 (4-H),
7.53 (d, 2H, J = 8.5 Hz, 3ꢀꢀ- and 5ꢀꢀ-H), 7.77 (d, 2H, J = 8.5 Hz, 2ꢀꢀ- and
6ꢀꢀ-H). 13C NMR (ı, ppm, CDCl3): 12.8 (C-18), 17.4 (C-19), 20.8, 23.7,
24.6, 32.0, 32.8, 34.0, 35.4 (C-8), 35.7, 38.6 (C-10), 38.9, 42.8 (C-
13), 53.8, 54.8, 55.1, 59.8 (C-4ꢀ), 81.8 (C-5ꢀ), 123.9 (C-4), (125.8 and
127.0): (C-1ꢀꢀ, C-4ꢀꢀ), (129.6 and 131.6): (C-2ꢀꢀ, C-3ꢀꢀ, C-5ꢀꢀ, C-6ꢀꢀ), 163.3
(C-2ꢀ), 171.1 (C-5), 199.4 (C-3).
2.4.7. (5ꢀR)-17ˇ-[2-(4-nitrophenyl)-4,5-dihydrooxazol-5-
yl]androst-5-en-3-one
(8g)
8g (350 mg, 37%), mp 216–218 ◦C, Rf = 0.40 (ss D); [␣]D20 + 131 (c
1 in CHCl3) (found C, 72.87; H, 7.65. C28H34N2O2 requires C, 72.70;
H, 7.41%). 1H NMR (ı, ppm, CDCl3): 0.89 (s, 3H, 18-H3), 1.21 (s,
3H, 19-H3), 3.68 (dd, 1H, J = 15.0 Hz and J = 8.0 Hz) and 4.10 (dd, 1H,
J = 15.0 Hz and J = 9.5 Hz): 4ꢀ-H2, 4.71 (m, 1H, 5ꢀ-H), 5.72 (s, 1H, 4-H),
8.07 (d, 2H, J = 8.5 Hz, 2ꢀꢀ- and 6ꢀꢀ-H), 8.24 (d, 2H, J = 8.5 Hz, 3ꢀꢀ- and
5ꢀꢀ-H). 13C NMR (ı, ppm, CDCl3): 12.8 (C-18), 17.4 (C-19), 20.8, 23.6,
24.5, 32.0, 32.8, 33.9, 35.4 (C-8), 35.7, 38.6 (C-10), 38.9, 42.8 (C-13),
53.9, 54.9, 55.1, 60.0 (C-4ꢀ), 82.3 (C-5ꢀ), (123.5 and 129.0): (C-2ꢀꢀ, C-
3ꢀꢀ, C-5ꢀꢀ, C-6ꢀꢀ), 123.9 (C-4), 133.9 (C-1ꢀꢀ), 149.4 (C-4ꢀꢀ), 162.3 (C-2ꢀ),
170.9 (C-5), 199.3 (C-3).
This observation provides a possibility for the preparation
of compounds containing various substituted oxazolines con-
densed to ring D of the androstane skeleton. To prepare the
␣,␤-dihydroxy system on the side-chain of steroid skeleton we
chose 3␤-acetoxypregn-5-en-20-one (1) as starting material. Oxi-

D. Ondré et al. / Steroids 74 (2009) 1025–1032
1029
Scheme 1. Reagents and conditions: (i) Pb(OAc)4, BF3·OEt2, MeOH; (ii) KBH4, MeOH, rt; (iii) Al2O3, Mw; (iv) P(Ph)3, CCl4, reflux; (v) NaN3, DMF, 80 ◦C; (vi) benzaldehyde
(5a–g), CH2Cl2, BF3·OEt2, rt; (vii) KOH, MeOH, rt; (viii) Al(OiPr)3, cyclohexanone, toluene, reflux.
dation with Pb(OAc)4 in the presence of BF3·OEt2 furnished
3␤,21-diacetoxypregn-5-en-20-one (2). Reduction with KBH4 gave
two compounds, (20R)-3␤,21-diacetoxypregn-5-en-20-ol (3a) and
its 20S epimer, latter in a very small quantity. The required pure
epimer 3a was obtained by flash chromatography. Their selec-
tive deacetylation on alkaline alumina was carried out by an
earlier developed method to obtain 3b [8]. Chlorination of 3b
in the Appel reaction [20] produced the (20R)-3␤-acetoxy-21-
chloropregn-5-en-20-ol (4a). Nucleophilic exchange with NaN3
in dimethylformamide led to the required (20R)-3␤-acetoxy-21-
azidopregn-5-en-20-ol (4b) [8]. Reaction of the ␣,␤-azidoalcohol
4b and appropriately substituted aromatic aldehydes 5a–g acti-
vated by BF3·OEt2 as Lewis acid catalyst, proceeded cleanly to
give the corresponding steroid oxazolines 6a–g. Their deacetyla-
tion in methanol in the presence of NaOCH3 was carried out by
Zemplén method furnished 7a–g. Oppenauer oxidation of the 3␤-
hydroxy-exo-heterocyclic steroids 7a–g yielded the corresponding
ꢀ4-3-ketosteriods 8a–g (Scheme 1).
Mechanistically, it can be presumed [18] that the first step
in the Schmidt reaction involves hemiacetal formation between
the aromatic aldehyde and the steroid ␣,␤-azidoalcohol 9,
which undergoes elimination to afford a benzyl carbocation 10.
Intramolecular attack of the azide group on the carbocation fur-
nishes intermediate 11, and subsequent proton elimination and N2
detachment give the product 12. Since the hemiacetal formation
did not involve any chiral centre, the configurations of the com-
pounds obtained, agreed with those of the starting materials (of
confirmed configurations) (Scheme 2).
3.2. Effects of steroidal oxazolines on C17,20-lyase activity
The inhibitory effects of compounds 7a–g and 8a–g on rat testic-
ular C17,20-lyase were investigated with an in vitro radiosubstrate
incubation technique. Relative conversion percents measured at
Scheme 2.

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D. Ondré et al. / Steroids 74 (2009) 1025–1032
Table 1
Inhibition of C17,20-lyase activity.
Compound
Formula
Relative conversion, mean S.D. (%)
IC50 S.D. (␮M)
7a
NI
7b
NI
7c
80
4
7d
11 0.8
7e
83
4
7f
85
1
7g
7.9 1.5
8a
37
3
30 3.0

D. Ondré et al. / Steroids 74 (2009) 1025–1032
1031
Table 1 (Continued )
Compound
Formula
Relative conversion, mean S.D. (%)
IC50 S.D. (␮M)
8b
76
5
8c
50
1
52 6.6
8d
4.8 0.3
8e
14 3.0
8f
5.0 0.3
8g
7.7 0.4
Ketoconazole (reference)
0.35 0.02
NI: no inhibition.
S.D.: standard deviation.
Relative conversions (control incubation with no inhibition is taken as 100%) measured in the presence of 50 ␮M concentration of the compounds tested, and IC50 results for
more potent inhibitors.
the 50 ␮M inhibitor concentration applied and the IC50 values are
listed in Table 1 .
derivatives 7d and 7g of the 3-hydroxy compounds proved to be
more potent inhibitors, IC50 values of these compounds were found
11 ␮M and 7.9 ␮M, respectively.
Three derivatives of the 3-hydroxy compounds 7c, 7e and 7f
exerted weak inhibitory effect, when applied at 50 ␮M concentra-
tion in the incubates, these compounds decreased enzyme activity
by 80, 83 and 85%, respectively. Incubations with the 7a and 7b
resulted in conversions identical to those in the control exper-
iments; hence, these compounds did not inhibit rat C17,20-lyase
under the test conditions. The 3-chlorophenyl-, and 4-nitrophenyl
At the ꢀ4-3-ketosteroids the 4-fluorophenyl-derivatives 8b was
found to be weak inhibitor. This compound decreased enzyme
activity by 76% at the 50 ␮M test concentration. The phenyl-, and
4-chlorophenyl substituted derivatives 8a and 8c exhibited mod-
erate inhibitory action. 8a reduced enzyme activity by 37% and 8c
reduced enzyme activity by 50% at the 50 ␮M test concentration,

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D. Ondré et al. / Steroids 74 (2009) 1025–1032
and the IC50 values for these compounds were determined 30 ␮M
and 52 ␮M, respectively. Four ꢀ4-3-keto compounds proved to
be more modest inhibitors. IC50 results were found to be as fol-
lows: the 3-chlorophenyl 8d 4.8 ␮M, 2-chlorophenyl 8e 14 ␮M,
4-bromophenyl 8f 5.0 ␮M and 4-nitrophenyl 8g 7.7 ␮M.
[4] Ling YZ, Li JS, Liu Y, Kato K, Klus GT, Brodie AMH. 17-Imidazolyl, pyrazolyl,
and isoxazolyl androstene derivatives. Novel steroidal inhibitors of human
cytochrome C17,20-lyase (P45017␣). J Med Chem 1997;40:3297–304.
[5] Njar VCO, Kato K, Nnane IP, Grigoryev DM, Long BJ, Brodie AMH. Novel 17-azolyl
steroids, potent inhibitors of human cytochrome 17␣-hydroxylase-C17,20-lyase
(P45017␣): potent agents for the treatment of prostate cancer. J Med Chem
1998;41:902–12.
Numerous steroidal compounds bearing heterocyclic sub-
stituents at C-17 have been investigated as concerns their P45017␣
inhibitory effects [21,22]. Abiraterone and its derivatives (17-
pyridyl 16,17-unsaturated compounds) have IC50 values in the
interval 2–5 ␮M [4,23]. Other C-17 heteroaryl (imidazolyl, benz-
imidazolyl, pyrazolyl and isoxazolyl) or unsaturated heterocyclic
(aminothiazolyl and furanoyl) derivatives also with 16,17 double
bond are among the most effective inhibitors, with IC50 values
<0.01 ␮M [24,25]. Some compounds with a saturated heterocyclic
C-17 substituent have also been investigated. Compounds possess-
ing oxygen, sulfur or four-membered nitrogen heterocycles were
less effective inhibitors [26].
[6] Zhu N, Ling Y, Lei X, Handratta V, Brodie AMH. Novel P45017␣ inhibitors:
17-(2ꢀ-oxazolyl)- and 17-(2ꢀ-thiazolyl)-androstene derivatives. Steroids
2003;68:603–11.
[7] Moreira VMA, Vasaitis TS, Njar VCO, Salvador JAR. Synthesis and evaluation
of novel 17-indazole androstene derivatives designed as CYP17 inhibitors.
Steroids 2007;72:939–48.
[8] Wölfling J, Mernyák E, Sebo˝ k M, Schneider G. Synthesis of some steroidal oxa-
zolines. Collect Czech Chem Commun 2001;66:1831–40.
[9] Ondré D, Wölfling J, Iványi Z, Schneider G, Tóth I, Szécsi M, et al. Neighbor-
ing group participation. Part 17. Stereoselective synthesis of some steroidal
2-oxazolidones, as novel potential inhibitors of 17␣-hydroxylase-C17,20-lyase.
Steroids 2008;73:1375–84.
[10] Wölfling J, Oravecz EA, Ondré D, Mernyák E, Schneider G, Tóth I, et al. Stereos-
elective synthesis of some 17␤-dihydrooxazinyl steroids, as novel presumed
inhibitors of 17␣-hydroxylase-C17,20-lyase. Steroids 2006;71:809–16.
[11] Wölfling J, Hackler L, Mernyák E, Schneider G, Tóth I, Szécsi M, et al.
Neighboring group participation. Part 15. Stereoselective synthesis of some
steroidal tetrahydro-oxazin-2-ones, as novel presumed inhibitors of human
5␣-reductase. Steroids 2004;69:451–60.
[12] Gant TG, Meyers AI. The chemistry of 2-oxazolines (1985–present). Tetrahe-
dron 1994;50:2297–360.
[13] Hamamoto H, Mamedov VA, Kitamoto M, Hayashi N, Tsuboi S. Chemoenzymatic
synthesis of the C-13 side chain of paclitaxel (Taxol) and docetaxel (Taxotere).
Tetrahedron: Asymmetry 2000;11:4485–97.
[14] Saravanan P, Corey EJ. A short, stereocontrolled, and practical synthesis of
␣-methylomuralide, a potent inhibitor of proteasome function. J Org Chem
2003;68:2760–4.
[15] Kangani CO, Kelley DE, Day BW. One pot direct synthesis of oxazolines, benzox-
azoles, and oxadiazoles from carboxylic acids using the Deoxo-Fluor reagent.
Tetrahedron Lett 2006;47:6497–9.
[16] Schwekendiek K, Glorius F. Efficient oxidative synthesis of 2-oxazolines. Syn-
thesis 2006;18:2996–3002.
[17] Ishihara M, Togo H. Direct oxidative conversion of aldehydes and alco-
hols to 2-imidazolines and 2-oxazolines using molecular iodine. Tetrahedron
2007;63:1474–80.
We investigated the presumed
C17,20-lyase inhibition of
androstene derivatives with aryl substituted 5ꢀ-oxazoline ring at
the position 17␤. Compounds were synthesized and tested both in
the 3␤-hydroxy-, and ꢀ4-3-ketosteroids and in most of the cases,
the ꢀ4-3-keto compounds exerted a higher inhibition than their
3␤-hydroxy counterparts. Chlorine substitution in the phenyl ring
enhanced of the inhibition potential in both series, and the effect
was the most pronounced in the case of 3-chloro derivatives. The
two 4-bromophenyl derivatives 7f and 8f differed markedly from
each other in inhibitory action: the ꢀ4-en-3-one compound 8f was
found to be a potent inhibitor, but its 3␤-hydroxy 7f counterpart
exerted only a weak inhibition. Whereas both of the compounds
bearing 4-nitrophenyl structure 7g and 8g, were found equally
effective in inhibition. The 3-chlorophenyl-4-en-3-one 8d and the
4-bromophenyl-4-en-3-one 8f derivatives were proved to be the
most effective C17,20-lyase inhibitors among our compounds inves-
tigated, the IC50 value of this compounds are close to that of
ketoconazole and abiraterone (Table 1).
[18] Milligan GL, Mossman CJ, Aubé J. Intramolekular Schmidt reactions of alkyl
azides with ketones: scope and stereochemical studies.
1995;117:10449–59.
J Am Chem Soc
[19] Badiang JG, Aubé J. One-step conversion of aldehydes to oxazolines and
5,6-dihydro-4H-1,3-oxazines using 1,2- and 1,3-azido alcohols. J Org Chem
1996;61:2484–7.
[20] Appel R, Halstenberg M. In: Cadogan JIG, editor. Organophosphorus reagents
in organic synthesis. New York: Academic Press; 1979. p. 378–424.
[21] Jarman M, Smith HJ, Nicholls PJ, Simons C. Inhibitors of enzymes of andro-
gen biosynthesis: cytochrome P45017␣ and 5␣-steroid reductase. Nat Prod Rep
1998;15:495–512.
[22] Baston E, Leroux FR. Inhibitors of steroidal cytochrome P450 enzymes as targets
for drug development. Recent Pat Anti-Cancer Drug Discov 2007;2:31–58.
[23] Barrie SE, Potter GA, Goddard PM, Haynes BP, Dowsett M, Jarman M.
Pharmacology of novel steroidal inhibitors of cytochrome P45017␣ (17␣-
hydroxylase/C17-20 lyase). J Steroid Biochem Mol Biol 1994;50:267–73.
[24] Burkhart JP, Gates CA, Laughlin ME, Resvick RJ, Peet NP. Inhibition of
steroid C17(20) lyase with C-17-heteroaryl steroids. Bioorg Med Chem
1996;4:1411–20.
Acknowledgements
This work was supported by the Hungarian Scientific Research
Fund (OTKA K72309). Mihály Szécsi’s work was supported by the
award of a Bolyai János Research Fellowship.
References
[1] Njar VCO, Brodie AMH. Inhibitors of 17␣-hydroxylase/17,20-lyase (CYP17)
potential agents for the treatment of prostate cancer. Curr Pharm Des
1999;5:163–80.
[25] Handratta VD, Vasaitis TS, Njar VC, Gediya LK, Kataria R, Chopra P, et al.
Novel C-17-heteroaryl steroidal CYP17 inhibitors/antiandrogens: synthesis, in
vitro biological activity, pharmacokinetics, and antitumor activity in the LAPC4
human prostate cancer xenograft model. J Med Chem 2005;48:2972–84.
[26] Hartmann RW, Hector M, Wachall BG, Palusczak A, Palzer M, Huch
V, et al. Synthesis and evaluation of 17-aliphatic heterocycle-substituted
steroidal inhibitors of 17␣-hydroxylase/C17-20-lyase (P450 17). J Med Chem
2000;43:4437–45.
[2] Labrie F, Dupont A, Belanger A, Lefebvre FA, Cusan L, Monfette G, et al.
New hormonal treatment in cancer of the prostate: combined administra-
tion of an LHRH agonist and an antiandrogen. J Steroid Biochem 1983;19:999–
1007.
[3] Jarman M, Barrie SE, Llera JM. The 16,17-double bond is needed for
irreversible inhibition of human cytochrome p45017␣ by abirateron (17-(3␤-
pyridyl)androsta-5,16-diene-3␤-ol) and related steroidal inhibitors. J Med
Chem 1998;41:5375–81.