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Steroselective synthesis of some steroidal oxazolines, as novel potential inhibitors of 17α-hydroxylase-C17,20-lyase
Steroids 74 (2009) 1025–1032
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Steroselective synthesis of some steroidal oxazolines, as novel potential inhibitors of 17␣-hydroxylase-C17,20 -lyase Dóra Ondré a , János Wölfling a , István Tóth b , Mihály Szécsi b , János Julesz b , Gyula Schneider a,∗ a b
Department of Organic Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary 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
Article history: Received 30 April 2009 Received in revised form 7 July 2009 Accepted 4 August 2009 Available online 8 August 2009 Keywords: ␣,-Azidoalcohols Schmidt reaction Steroid oxazolines In vitro inhibition Antiandrogenic effect
a b s t r a c t 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 -3ketosteroids. 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.
1. Introduction 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␣-HydroxylaseC17,20 -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, number 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 selective 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)androsta5,16-dien-3-ol] and its 4 -3-keto analog exhibited prominent inhibition of this enzyme. It has been suggested that such activity 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
∗ Corresponding author. Tel.: +36 62 544276; fax: +36 62 544200. E-mail address: [email protected] (G. Schneider). 0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2009.08.001
number of inhibitors of P45017␣ , of which 17-imidazolyl, pyrazolyl, isoxazoyl, oxazolyl, thiazolyl and indazol androstene are very potent [4–7]. We recently set out to synthesize a novel series of steroidal 17-oxazolines, 17-oxazolidones, 17-dihydrooxazines and 17-tetrahydrooxazin-2 -ones [8–11]. We report here the syntheses of a variety of steroidal compounds with the common structural feature of a C-17 substituted phenyloxazoline on 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/CH2 Cl2 (30:70, v/v), (B) CH2 Cl2 , (C) ethyl acetate/CH2 Cl2 (5:95, v/v), (D) ethyl acetate/CH2 Cl2 (10:90, v/v) and (E) ethyl acetate/CH2 Cl2 (50:50, v/v). The spots were detected by spraying with 5% phosphomolybdic 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 on Bruker DRX 500 instruments at 500 (1 H NMR) or 125 MHz (13 C
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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. 2.2. General procedure for preparation of compounds 6a–g Compound 4b [8] (1.6 g, 4 mmol) was dissolved in dry CH2 Cl2 (50 ml) and benzaldehyde (5a) and substituted benzaldehyde (5b–g) (1.1 equivalent) was cooled to 0 ◦ C, followed by the dropwise 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 Na2 SO4 and concentrated in vacuo. The product was purified by chromatography on silica gel with hexane/CH2 Cl2 (30:70, v/v). 2.2.1. (5 R)-3ˇ-acetoxy-17ˇ-[2-phenyl-4,5-dihydrooxazol-5yl]androst-5-ene (6a) 6a (1.64 g, 89%), mp 184–188 ◦ C, Rf = 0.49 (ss D); [␣]D 20 − 60 (c 1 in CHCl3 ) (found C, 78.06; H, 8.95. C31 H43 NO3 requires C, 77.95; H, 9.07%). 1 H 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): 4 -H2 , 4.61 (overlapping multiplets, 2H, 3- and 5 -H), 5.38 (d, 1H, J = 4.0 Hz, 5-H), 7.40 (t, 2H, J = 7.5 Hz, 3 - and 5 -H), 7.46 (t, 1H, J = 7.5 Hz, 4 -H), 7.92 (d, 2H, J = 7.5 Hz, 2 - and 6 -H). 13 C 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 (C10), 37.0, 38.1, 40.0, 42.7 (C-13), 50.2, 55.0, 55.9, 59.8 (C-4 ), 73.9 (C-3), 81.7 (C-5 ), 122.4 (C-6), (128.1 and 128.2): (C-2 , C-3 , C-5 , C-6 ), 128.2 (C-1 ), 131.1 (C-4 ), 128.2 (C-1 ), 139.8 (C-5), 164.1 (C-2 ), 170.4 (Ac-CO). 2.2.2. (5 R)-3ˇ-acetoxy-17ˇ-[2-(4-fluorophenyl)-4,5dihydrooxazol-5-yl]androst-5-ene (6b) 6b (1.33 g, 70%), mp 166–168 ◦ C, Rf = 0.47 (ss D); [␣]D 20 − 57 (c 1 in CHCl3 ) (found C, 75.24; H, 8.05. C30 H38 FNO3 requires C, 75.13; H, 7.99%). 1 H 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): 4 -H2 , 4.80 (overlapping multiplets, 2H, 3- and 5 -H), 5.56 (d, 1H, J = 3.5 Hz, 6-H), 7.26 (t, 2H, J = 8.5 Hz, 3 - and 5 -H), 8.10 (dd, 2H, J = 8.0 Hz, 2 - and 6 -H). 13 C 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-4 ), 73.5 (C-3), 81.5 (C-5 ), 115.0 (2C, J = 21.7 Hz, C-3 and C-5 ), 121.9 (C-6), 124.0 (C-1 ), 129.9 (2C, J = 8.6 Hz, C-2 and C-6 ), 139.4 (C-5), 163.0 (J = 47.8 Hz, C-4 ), 163.2, 165.2 (C-2 ), 170.0 (Ac-CO). 2.2.3. (5 R)-3ˇ-acetoxy-17ˇ-[2-(4-chlorophenyl)-4,5dihydrooxazol-5-yl]androst-5-ene (6c) 6c (1.78 g, 90%), mp 149–152 ◦ C, Rf = 0.53 (ss D); [␣]D 20 − 41 (c 1 in CHCl3 ) (found C, 72.74; H, 7.85. C30 H38 ClNO3 requires C, 72.63; H, 7.72%). 1 H 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): 4 -H2 , 4.62 (overlapping multiplets, 2H, 3- and 5 -H), 5.37 (d, 1H, J = 4.5 Hz, 6-H), 7.37 (d, 2H, J = 8.5 Hz, 3 - and 5 -H), 7.85 (d, 2H, J = 8.5 Hz, 2 - and 6 -H). 13 C 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-4 ), 73.9 (C-3), 82.0 (C-5 ), 122.3 (C-
6), (128.6 and 129.4): (C-2 , C-3 , C-5 , C-6 ), 126.7 (C-1 ), 137.3 (C-4 ), 139.8 (C-5), 163.2 (C-2 ), 170.4 (Ac-CO). 2.2.4. (5 R)-3ˇ-acetoxy-17ˇ-[2-(3-chlorophenyl)-4,5dihydrooxazol-5-yl]androst-5-ene (6d) 6d (1.69 g, 85%), mp 170–174 ◦ C, Rf = 0.60 (ss D); [␣]D 20 − 38 (c 1 in CHCl3 ) (found C, 72.55; H, 7.89. C30 H38 ClNO3 requires C, 72.63; H, 7.72%). 1 H 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): 4 -H2 , 4.62 (overlapping multiplets, 2H, 3- and 5 -H), 5.37 (d, 1H, J = 4.5 Hz, 6-H), 7.33 (t, 1H, J = 8.0 Hz, 5 -H), 7.43 (d, 1H, J = 8.0 Hz, 4 -H), 7.80 (d, 1H, J = 8.0 Hz, 6 -H), 7.90 (s, 1H, 2 -H). 13 C 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 (C10), 37.0, 38.1, 39.0, 42.7 (C-13), 50.1, 55.0, 55.9, 59.8 (C-4 ), 73.9 (C-3), 82.1 (C-5 ), 122.3 (C-6), (126.2, 128.2, 129.6, and 131.1): (C2 , C-4 , C-5 , C-6 ), 130.0 (C-3 ), 134.3 (C-1 ), 139.8 (C-5), 163.0 (C-2 ), 170.4 (Ac-CO). 2.2.5. (5 R)-3ˇ-acetoxy-17ˇ-[2-(2-chlorophenyl)-4,5dihydrooxazol-5-yl]androst-5-ene (6e) 6e (1.65 g, 83%), mp 162–164 ◦ C, Rf = 0.60 (ss D); [␣]D 20 − 59 (c 1 in CHCl3 ) (found C, 72.55; H, 7.82. C30 H38 ClNO3 requires C, 72.63; H, 7.72%). 1 H 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): 4 -H2 , 4.62 (overlapping multiplets, 2H, 3- and 5 -H), 5.37 (s, 1H, 6-H), 7.28 (t, 1H, J = 7.5 Hz, 5 -H), 7.34 (t, 1H, J = 7.5 Hz, 4 -H), 7.43 (d, 1H, J = 7.5 Hz, 3 -H), 7.76 (d, 1H, J = 7.5 Hz, 6 -H). 13 C 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 (C10), 36.9, 37.0, 38.1, 38.8, 42.6 (C-13), 50.1, 54.9, 55.9, 60.3 (C-4 ), 73.9 (C-3), 81.5 (C-5 ), 122.3 (C-6), (126.4, 130.7, 131.2 and 131.3): (C-3 , C-4 , C-5 , C-6 ), 127.7 (C-2 ), 133.4 (C-1 ), 139.8 (C-5), 162.6 (C-2 ), 170.4 (Ac-CO). 2.2.6. (5 R)-3ˇ-acetoxy-17ˇ-[2-(4-bromophenyl)-4,5dihydrooxazol-5-yl]androst-5-ene (6f) 6f (2.07 g, 92%). (Ref. [8] mp 103–105 ◦ C). 2.2.7. (5 R)-3ˇ-acetoxy-17ˇ-[2-(4-nitrophenyl)-4,5dihydrooxazol-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 CH2 Cl2 , followed by ethyl acetate/CH2 Cl2 (5:95, v/v) and ethyl acetate/CH2 Cl2 (10:90, v/v) to afford 7a–g. 2.3.1. (5 R)-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 20 − 55 (c 1 in CHCl3 ) (found C, 79.82; H, 9.36. C29 H41 NO2 requires C, 79.95; H, 9.49%. 1 H 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): 4 -H2 , 4.65 (m, 1H, 5 -H), 5.34 (d, 1H, J = 5.0 Hz, 6-H), 7.39 (t, 2H, J = 7.5 Hz, 3 - and 5 -H), 7.46
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(t, 1H, J = 7.5 Hz, 4 -H), 7.92 (d, 2H, J = 7.5 Hz, 2 - and 6 -H). 13 C 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-4 ), 71.6, 81.7, 121.4 (C-6), (128.1 and 128.3): (C-2 , C-3 , C-5 , C-6 ), 128.1 (C-1 ), 131.1 (C-4 ), 140.9 (C-5), 164.1 (C-2 ). 2.3.2. (5 R)-17ˇ-[2-(4-fluorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3ˇ-ol (7b) 7b (950 mg, 87%), mp 232–235 ◦ C, Rf = 0.29 (ss D); [␣]D 20 − 61 (c 1 in CHCl3 ) (found C, 76.72; H, 8.37. C28 H36 FNO2 requires C, 76.85; H, 8.29%). 1 H 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): 4 -H2 , 4.67 (m, 1H, 5 H), 5.35 (s, 1H, 6-H), 7.07 (t, 2H, J = 8.5 Hz, 3 - and 5 -H), 7.92 (dd, 2H, J = 8.0 Hz, 2 - and 6 -H). 13 C 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, 55.0, 56.0, 59.8 (C-4 ), 71.7 (C-3), 81.9 (C-5 ), 115.3 (J = 21.7 Hz, C-3 and C-5 ), 121.4 (C-6), 124.4 (C-1 ), 130.3 (J = 8.6 Hz, C-2 and C-6 ), 140.9 (C-5), 163.4 (J = 42.4 Hz, C-4 ), 165.6 (C-2 ). 2.3.3. (5 R)-17ˇ-[2-(4-chlorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3ˇ-ol (7c) 7c (1.14 g, 70%), mp 215–218 ◦ C, Rf = 0.29 (ss D); [␣]D 20 − 48 (c 1 in CHCl3 ) (found C, 73.95; H, 8.03. C28 H36 ClNO2 requires C, 74.07; H, 7.99%). 1 H 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): 4 -H2 , 4.66 (m, 1H, 5 -H), 5.35 (d, 1H, J = 5.0 Hz, 6-H), 7.37 (d, 2H, J = 8.5 Hz, 3 - and 5 -H), 7.85 (d, 2H, J = 8.5 Hz, 2 - and 6 -H). 13 C 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, 55.0, 56.0, 59.8 (C-4 ), 71.7 (C-3), 82.0 (C-5 ), 121.4 (C-6), (128.6 and 129.4): (C-2 , C-3 , C-5 , C-6 ), 126.7 (C-1 ), 137.3 (C-4 ), 140.9 (C-5), 163.3 (C-2 ). 2.3.4. (5 R)-17ˇ-[2-(3-chlorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3ˇ-ol (7d) 7d (930 mg, 82%), mp 231–234 ◦ C, Rf = 0.32 (ss D); [␣]D 20 − 52 (c 1 in CHCl3 ) (found C, 74.12; H, 7.85. C28 H36 ClNO2 requires C, 74.07; H, 7.99%). 1 H 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): 4 -H2 , 4.66 (m, 1H, 5 H), 5.35 (d, 1H, J = 5.0 Hz, 6-H), 7.33 (t, 1H, J = 8.0 Hz, 5 -H), 7.43 (d, 1H, J = 8.0 Hz, 4 -H), 7.80 (d, 1H, J = 8.0 Hz, 6 -H), 7.9 (s, 1H, 2 -H). 13 C NMR (ı, ppm, CDCl ): 12.7 (C-18), 19.4 (C-19), 20.9, 23.7, 24.7, 3 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-4 ), 71.7 (C-3), 82.1 (C-5 ), 121.4 (C-6), (126.2, 128.2, 129.6 and 131.3): (C-2 , C-4 , C-5 , C-6 ), 129.9 (C-3 ), 134.3 (C-1 ), 140.9 (C-5), 163.0 (C-2 ). 2.3.5. (5 R)-17ˇ-[2-(2-chlorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3ˇ-ol (7e) 7e (970 mg, 85%), mp 165–169 ◦ C, Rf = 0.35 (ss D); [␣]D 20 − 58 (c 1 in CHCl3 ) (found C, 74.21; H, 8.12. C28 H36 ClNO2 requires C, 74.07; H, 7.99%). 1 H 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): 4 -H2 , 4.64 (m, 1H, 5 H), 5.34 (s, 1H, 6-H), 7.28 (t, 1H, 5 -H), 7.34 (t, 1H, J = 7.5 Hz, 4 -H), 7.43 (d, 1H, J = 7.5 Hz, 3 -H), 7.75 (d, 1H, J = 7.5 Hz, 6 -H). 13 C 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-4 ), 71.7 (C-3), 81.6 (C-5 ), 121.4 (C-6), (126.4, 130.7, 131.2
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and 131.3): (C-3 , C-4 , C-5 , C-6 ), 127.7 (C-2 ), 133.4 (C-1 ), 140.9 (C-5), 1627 (C-2 ). 2.3.6. (5 R)-17ˇ-[2-(4-bromophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3ˇ-ol (7f) 7f (1.05 g, 84%). (Ref. [8] mp 202–203 ◦ C). 2.3.7. (5 R)-17ˇ-[2-(4-nitrophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3ˇ-ol (7g) 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 CH2 Cl2 . The organic phase was evaporated to dryness and the residual crude product was chromatographed on silica gel with ethyl acetate/CH2 Cl2 (5:95). 2.4.1. (5 R)-17ˇ-[2-phenyl-4,5-dihydrooxazol-5-yl]androst-5-en-3-one (8a) 8a (350 mg, 40%), mp 89–93 ◦ C, Rf = 0.29 (ss D); [␣]D 20 + 94 (c 1 in CHCl3 ) (found C, 80.17; H, 9.25. C29 H39 NO2 requires C, 80.33; H, 9.07%). 1 H 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): 4 -H2 , 4.64 (m, 1H, 5 -H), 5.72 (s, 1H, 4H), 7.39 (t, 2H, J = 7.5 Hz, 3 - and 5 -H), 7.45 (t, 1H, J = 7.5 Hz, 4 -H), 7.91 (d, 2H, J = 7.5 Hz, 2 - and 6 -H). 13 C 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-4 ), 81.5 (5 -H), 123.8 (C-4), (128.0 and 128.3): (C-2 , C-3 , C-5 , C-6 ), 128.1 (C-1 ), 131.1 (C-4 ), 164.0 (C-2 ), 171.1 (C-5), 199.3 (C-3). 2.4.2. (5 R)-17ˇ-[2-(4-Fluorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3-one (8b) 8b (425 mg, 48%), mp 164–167 ◦ C, Rf = 0.35 (ss D); [␣]D 20 + 94 (c 1 in CHCl3 ) (found C, 77.34; H, 7.65. C28 H34 FNO2 requires C, 77.21; H, 7.87%). 1 H 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): 4 -H2 , 4.64 (m, 1H, 5 -H), 5.72 (s, 1H, 4H), 7.07 (t, 2H, J = 8.5 Hz, 3 - and 5 -H), 7.90 (dd, 2H, J = 8.0 Hz, 2 and 6 -H). 13 C 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-4 ), 81.8 (5 -C), 115.4 (J = 21.8 Hz, C-3 and C-5 ), 123.7 (C-4), 124.4 (C-1 ), 130.2 (J = 8.6 Hz, C-2 and C-6 ), 163.4 (J = 56.4 Hz, C-4 ), 165.6 (C-2 ), 171.0 (C-5), 199.3 (C-3). 2.4.3. (5 R)-17ˇ-[2-(4-chlorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3-one (8c) 8c (410 mg, 45%), mp 167–169 ◦ C, Rf = 0.38 (ss D); [␣]D 20 + 117 (c 1 in CHCl3 ) (found C, 74.26; H, 7.75. C28 H34 ClNO2 requires C, 74.40; H, 7.58%). 1 H 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): 4 -H2 , 4.65 (m, 1H, 5 -H), 5.72 (s, 1H, 4-H), 7.37 (d, 2H, J = 8.5 Hz, 3 - and 5 -H), 7.84 (d, 2H, J = 8.5 Hz, 2 and 6 -H). 13 C 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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(C-13), 53.9, 54.9, 55.1, 59.8 (C-4 ), 81.8 (C-5 ), 123.9 (C-4), 126.6 (C1 ), (128.6 and 129.4): (C-2 , C-3 , C-5 , C-6 ), 137.3 (C-4 ), 163.2 (C-2 ), 171.0 (C-5), 199.4 (C-3). 2.4.4. (5 R)-17ˇ-[2-(3-chlorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3-one (8d) 8d (375 mg, 41%), mp 78–81 ◦ C, Rf = 0.44 (ss D); [␣]D 20 + 101 (c 1 in CHCl3 ) (found C, 74.32; H, 7.66; C28 H34 ClNO2 requires C, 74.40; H, 7.58%). 1 H 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): 4 -H2 , 4.65 (m, 1H, 5 -H), 5.71 (s, 1H, 4-H), 7.32 (t, 1H, J = 8.0 Hz, 5 -H), 7.42 (d, 1H, J = 8.0 Hz, 4 -H), 7.78 (d, 1H, J = 8.0 Hz, 6 -H), 7.88 (s, 1H, 2 -H). 13 C 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-4 ), 76.7, 77.0, 77.3, 81.9 (5 -C), 123.9 (C-4), (126.1, 128.1, 129.7, and 131.1): (C2 , C-4 , C-5 , C-6 ), 129.9 (C-3 ), 134.3 (C-1 ), 162.9 (C-2 ), 171.0 (C-5), 199.3 (C-3). 2.4.5. (5 R)-17ˇ-[2-(2-chlorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3-one (8e) 8e (440 mg, 48%), mp 132–135 ◦ C, Rf = 0.42 (ss D); [␣]D 20 + 77 (c 1 in CHCl3 ) (found C, 74.26; H, 7.35. C26 H35 ClNO2 requires C, 74.40; H, 7.58%). 1 H 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): 4 -H2 , 4.64 (m, 1H, 5 -H), 5.71 (s, 1H, 4H), 7.28 (t, 1H, J = 7.5 Hz, 5 -H), 7.34 (t, 1H, J = 7.5 Hz, 4 -H), 7.43 (d, 1H, J = 7.5 Hz, 3 -H), 7.74 (d, 1H, J = 7.5 Hz, 6 -H). 13 C 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-4 ), 81.4 (C-5 ), 123.8 (C-4), (126.4, 130.7, 131.2 and 131.4): (C3 , C-4 , C-5 , C-6 ), 127.6 (C-2 ), 133.4 (C-1 ), 162.6 (C-2 ), 171.1 (C-5), 199.4 (C-3). 2.4.6. (5 R)-17ˇ-[2-(4-bromophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3-one (8f) 8f (460 mg, 46%), mp 184–189 ◦ C, Rf = 0.42 (ss D); [␣]D 20 + 114 (c 1 in CHCl3 ) (found C, 67.55; H, 7.05. C28 H34 BrNO2 requires C, 67.74; H, 6.90%). 1 H 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): 4 -H2 , 4.65 (m, 1H, 5 -H), 5.72 (4-H), 7.53 (d, 2H, J = 8.5 Hz, 3 - and 5 -H), 7.77 (d, 2H, J = 8.5 Hz, 2 - and 6 -H). 13 C 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 (C13), 53.8, 54.8, 55.1, 59.8 (C-4 ), 81.8 (C-5 ), 123.9 (C-4), (125.8 and 127.0): (C-1 , C-4 ), (129.6 and 131.6): (C-2 , C-3 , C-5 , C-6 ), 163.3 (C-2 ), 171.1 (C-5), 199.4 (C-3). 2.4.7. (5 R)-17ˇ-[2-(4-nitrophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3-one (8g) 8g (350 mg, 37%), mp 216–218 ◦ C, Rf = 0.40 (ss D); [␣]D 20 + 131 (c 1 in CHCl3 ) (found C, 72.87; H, 7.65. C28 H34 N2 O2 requires C, 72.70; H, 7.41%). 1 H 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): 4 -H2 , 4.71 (m, 1H, 5 -H), 5.72 (s, 1H, 4-H), 8.07 (d, 2H, J = 8.5 Hz, 2 - and 6 -H), 8.24 (d, 2H, J = 8.5 Hz, 3 - and 5 -H). 13 C 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-4 ), 82.3 (C-5 ), (123.5 and 129.0): (C-2 , C3 , C-5 , C-6 ), 123.9 (C-4), 133.9 (C-1 ), 149.4 (C-4 ), 162.3 (C-2 ), 170.9 (C-5), 199.3 (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 determined by an in vitro radiosubstrate incubation method described in our earlier publications [9,10] with some modifications. 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 [3 H]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 product 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) layers (0.25 mm thick) with solvent system diisopropyl ether/CH2 Cl2 (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 measured 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 without test substances and incubates with the reference compound ketoconazole were also prepared in every series. At least two experiments 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 concentrations 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.
3. Results and discussion 3.1. Synthetic studies Oxazolines have played important role in many areas of chemistry. Starting from ␣,-azidoalcohol several possibilities can be given to prepare oxazoline moieties. Catalytic hydrogenation of azidoalcohols 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 aldehydes, aminoalcohol, and N-bromosuccinimide as an oxidizing agent were described [16]. On the other hand, oxazolines can also be produced 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]. This observation provides a possibility for the preparation of compounds containing various substituted oxazolines condensed 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
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Scheme 1. Reagents and conditions: (i) Pb(OAc)4 , BF3 ·OEt2 , MeOH; (ii) KBH4 , MeOH, rt; (iii) Al2 O3 , Mw; (iv) P(Ph)3 , CCl4 , reflux; (v) NaN3 , DMF, 80 ◦ C; (vi) benzaldehyde (5a–g), CH2 Cl2 , 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 selective 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-21chloropregn-5-en-20-ol (4a). Nucleophilic exchange with NaN3 in dimethylformamide led to the required (20R)-3-acetoxy-21azidopregn-5-en-20-ol (4b) [8]. Reaction of the ␣,-azidoalcohol 4b and appropriately substituted aromatic aldehydes 5a–g activated by BF3 ·OEt2 as Lewis acid catalyst, proceeded cleanly to give the corresponding steroid oxazolines 6a–g. Their deacetylation in methanol in the presence of NaOCH3 was carried out by Zemplén method furnished 7a–g. Oppenauer oxidation of the 3hydroxy-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 furnishes 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 compounds obtained, agreed with those of the starting materials (of confirmed configurations) (Scheme 2). 3.2. Effects of steroidal oxazolines on C17,20 -lyase activity
Scheme 2.
The inhibitory effects of compounds 7a–g and 8a–g on rat testicular C17,20 -lyase were investigated with an in vitro radiosubstrate incubation technique. Relative conversion percents measured at
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Table 1 Inhibition of C17,20 -lyase activity. Compound
Formula
Relative conversion, mean ± S.D. (%)
7a
NI
7b
NI
7c
80 ± 4
11 ± 0.8
7d
7e
83 ± 4
7f
85 ± 1
7.9 ± 1.5
7g
8a
IC50 ± S.D. (M)
37 ± 3
30 ± 3.0
D. Ondré et al. / Steroids 74 (2009) 1025–1032
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Table 1 (Continued ) Compound
Formula
Relative conversion, mean ± S.D. (%)
8b
76 ± 5
8c
50 ± 1
IC50 ± S.D. (M)
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 . Three derivatives of the 3-hydroxy compounds 7c, 7e and 7f exerted weak inhibitory effect, when applied at 50 M concentration 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 experiments; hence, these compounds did not inhibit rat C17,20 -lyase under the test conditions. The 3-chlorophenyl-, and 4-nitrophenyl
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. 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 moderate 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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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 follows: 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. Numerous steroidal compounds bearing heterocyclic substituents at C-17 have been investigated as concerns their P45017␣ inhibitory effects [21,22]. Abiraterone and its derivatives (17pyridyl 16,17-unsaturated compounds) have IC50 values in the interval 2–5 M [4,23]. Other C-17 heteroaryl (imidazolyl, benzimidazolyl, 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 possessing oxygen, sulfur or four-membered nitrogen heterocycles were less effective inhibitors [26]. We investigated the presumed C17,20 -lyase inhibition of androstene derivatives with aryl substituted 5 -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 investigated, the IC50 value of this compounds are close to that of ketoconazole and abiraterone (Table 1). 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. [2] Labrie F, Dupont A, Belanger A, Lefebvre FA, Cusan L, Monfette G, et al. New hormonal treatment in cancer of the prostate: combined administration 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-(3pyridyl)androsta-5,16-diene-3-ol) and related steroidal inhibitors. J Med Chem 1998;41:5375–81.
[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. [6] Zhu N, Ling Y, Lei X, Handratta V, Brodie AMH. Novel P45017␣ inhibitors: 17-(2 -oxazolyl)- and 17-(2 -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. ˝ M, Schneider G. Synthesis of some steroidal oxa[8] Wölfling J, Mernyák E, Sebok zolines. Collect Czech Chem Commun 2001;66:1831–40. [9] Ondré D, Wölfling J, Iványi Z, Schneider G, Tóth I, Szécsi M, et al. Neighboring 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. Stereoselective 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). Tetrahedron 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, benzoxazoles, 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. Synthesis 2006;18:2996–3002. [17] Ishihara M, Togo H. Direct oxidative conversion of aldehydes and alcohols to 2-imidazolines and 2-oxazolines using molecular iodine. Tetrahedron 2007;63:1474–80. [18] Milligan GL, Mossman CJ, Aubé J. Intramolekular Schmidt reactions of alkyl azides with ketones: scope and stereochemical studies. J Am Chem Soc 1995;117:10449–59. [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 androgen 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. [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.
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Steroselective synthesis of some steroidal oxazolines, as novel potential inhibitors of 17␣-hydroxylase-C17,20 -lyase Dóra Ondré a , János Wölfling a , István Tóth b , Mihály Szécsi b , János Julesz b , Gyula Schneider a,∗ a b
Department of Organic Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary 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
Article history: Received 30 April 2009 Received in revised form 7 July 2009 Accepted 4 August 2009 Available online 8 August 2009 Keywords: ␣,-Azidoalcohols Schmidt reaction Steroid oxazolines In vitro inhibition Antiandrogenic effect
a b s t r a c t 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 -3ketosteroids. 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.
1. Introduction 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␣-HydroxylaseC17,20 -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, number 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 selective 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)androsta5,16-dien-3-ol] and its 4 -3-keto analog exhibited prominent inhibition of this enzyme. It has been suggested that such activity 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
∗ Corresponding author. Tel.: +36 62 544276; fax: +36 62 544200. E-mail address: [email protected] (G. Schneider). 0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2009.08.001
number of inhibitors of P45017␣ , of which 17-imidazolyl, pyrazolyl, isoxazoyl, oxazolyl, thiazolyl and indazol androstene are very potent [4–7]. We recently set out to synthesize a novel series of steroidal 17-oxazolines, 17-oxazolidones, 17-dihydrooxazines and 17-tetrahydrooxazin-2 -ones [8–11]. We report here the syntheses of a variety of steroidal compounds with the common structural feature of a C-17 substituted phenyloxazoline on 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/CH2 Cl2 (30:70, v/v), (B) CH2 Cl2 , (C) ethyl acetate/CH2 Cl2 (5:95, v/v), (D) ethyl acetate/CH2 Cl2 (10:90, v/v) and (E) ethyl acetate/CH2 Cl2 (50:50, v/v). The spots were detected by spraying with 5% phosphomolybdic 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 on Bruker DRX 500 instruments at 500 (1 H NMR) or 125 MHz (13 C
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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. 2.2. General procedure for preparation of compounds 6a–g Compound 4b [8] (1.6 g, 4 mmol) was dissolved in dry CH2 Cl2 (50 ml) and benzaldehyde (5a) and substituted benzaldehyde (5b–g) (1.1 equivalent) was cooled to 0 ◦ C, followed by the dropwise 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 Na2 SO4 and concentrated in vacuo. The product was purified by chromatography on silica gel with hexane/CH2 Cl2 (30:70, v/v). 2.2.1. (5 R)-3ˇ-acetoxy-17ˇ-[2-phenyl-4,5-dihydrooxazol-5yl]androst-5-ene (6a) 6a (1.64 g, 89%), mp 184–188 ◦ C, Rf = 0.49 (ss D); [␣]D 20 − 60 (c 1 in CHCl3 ) (found C, 78.06; H, 8.95. C31 H43 NO3 requires C, 77.95; H, 9.07%). 1 H 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): 4 -H2 , 4.61 (overlapping multiplets, 2H, 3- and 5 -H), 5.38 (d, 1H, J = 4.0 Hz, 5-H), 7.40 (t, 2H, J = 7.5 Hz, 3 - and 5 -H), 7.46 (t, 1H, J = 7.5 Hz, 4 -H), 7.92 (d, 2H, J = 7.5 Hz, 2 - and 6 -H). 13 C 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 (C10), 37.0, 38.1, 40.0, 42.7 (C-13), 50.2, 55.0, 55.9, 59.8 (C-4 ), 73.9 (C-3), 81.7 (C-5 ), 122.4 (C-6), (128.1 and 128.2): (C-2 , C-3 , C-5 , C-6 ), 128.2 (C-1 ), 131.1 (C-4 ), 128.2 (C-1 ), 139.8 (C-5), 164.1 (C-2 ), 170.4 (Ac-CO). 2.2.2. (5 R)-3ˇ-acetoxy-17ˇ-[2-(4-fluorophenyl)-4,5dihydrooxazol-5-yl]androst-5-ene (6b) 6b (1.33 g, 70%), mp 166–168 ◦ C, Rf = 0.47 (ss D); [␣]D 20 − 57 (c 1 in CHCl3 ) (found C, 75.24; H, 8.05. C30 H38 FNO3 requires C, 75.13; H, 7.99%). 1 H 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): 4 -H2 , 4.80 (overlapping multiplets, 2H, 3- and 5 -H), 5.56 (d, 1H, J = 3.5 Hz, 6-H), 7.26 (t, 2H, J = 8.5 Hz, 3 - and 5 -H), 8.10 (dd, 2H, J = 8.0 Hz, 2 - and 6 -H). 13 C 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-4 ), 73.5 (C-3), 81.5 (C-5 ), 115.0 (2C, J = 21.7 Hz, C-3 and C-5 ), 121.9 (C-6), 124.0 (C-1 ), 129.9 (2C, J = 8.6 Hz, C-2 and C-6 ), 139.4 (C-5), 163.0 (J = 47.8 Hz, C-4 ), 163.2, 165.2 (C-2 ), 170.0 (Ac-CO). 2.2.3. (5 R)-3ˇ-acetoxy-17ˇ-[2-(4-chlorophenyl)-4,5dihydrooxazol-5-yl]androst-5-ene (6c) 6c (1.78 g, 90%), mp 149–152 ◦ C, Rf = 0.53 (ss D); [␣]D 20 − 41 (c 1 in CHCl3 ) (found C, 72.74; H, 7.85. C30 H38 ClNO3 requires C, 72.63; H, 7.72%). 1 H 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): 4 -H2 , 4.62 (overlapping multiplets, 2H, 3- and 5 -H), 5.37 (d, 1H, J = 4.5 Hz, 6-H), 7.37 (d, 2H, J = 8.5 Hz, 3 - and 5 -H), 7.85 (d, 2H, J = 8.5 Hz, 2 - and 6 -H). 13 C 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-4 ), 73.9 (C-3), 82.0 (C-5 ), 122.3 (C-
6), (128.6 and 129.4): (C-2 , C-3 , C-5 , C-6 ), 126.7 (C-1 ), 137.3 (C-4 ), 139.8 (C-5), 163.2 (C-2 ), 170.4 (Ac-CO). 2.2.4. (5 R)-3ˇ-acetoxy-17ˇ-[2-(3-chlorophenyl)-4,5dihydrooxazol-5-yl]androst-5-ene (6d) 6d (1.69 g, 85%), mp 170–174 ◦ C, Rf = 0.60 (ss D); [␣]D 20 − 38 (c 1 in CHCl3 ) (found C, 72.55; H, 7.89. C30 H38 ClNO3 requires C, 72.63; H, 7.72%). 1 H 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): 4 -H2 , 4.62 (overlapping multiplets, 2H, 3- and 5 -H), 5.37 (d, 1H, J = 4.5 Hz, 6-H), 7.33 (t, 1H, J = 8.0 Hz, 5 -H), 7.43 (d, 1H, J = 8.0 Hz, 4 -H), 7.80 (d, 1H, J = 8.0 Hz, 6 -H), 7.90 (s, 1H, 2 -H). 13 C 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 (C10), 37.0, 38.1, 39.0, 42.7 (C-13), 50.1, 55.0, 55.9, 59.8 (C-4 ), 73.9 (C-3), 82.1 (C-5 ), 122.3 (C-6), (126.2, 128.2, 129.6, and 131.1): (C2 , C-4 , C-5 , C-6 ), 130.0 (C-3 ), 134.3 (C-1 ), 139.8 (C-5), 163.0 (C-2 ), 170.4 (Ac-CO). 2.2.5. (5 R)-3ˇ-acetoxy-17ˇ-[2-(2-chlorophenyl)-4,5dihydrooxazol-5-yl]androst-5-ene (6e) 6e (1.65 g, 83%), mp 162–164 ◦ C, Rf = 0.60 (ss D); [␣]D 20 − 59 (c 1 in CHCl3 ) (found C, 72.55; H, 7.82. C30 H38 ClNO3 requires C, 72.63; H, 7.72%). 1 H 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): 4 -H2 , 4.62 (overlapping multiplets, 2H, 3- and 5 -H), 5.37 (s, 1H, 6-H), 7.28 (t, 1H, J = 7.5 Hz, 5 -H), 7.34 (t, 1H, J = 7.5 Hz, 4 -H), 7.43 (d, 1H, J = 7.5 Hz, 3 -H), 7.76 (d, 1H, J = 7.5 Hz, 6 -H). 13 C 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 (C10), 36.9, 37.0, 38.1, 38.8, 42.6 (C-13), 50.1, 54.9, 55.9, 60.3 (C-4 ), 73.9 (C-3), 81.5 (C-5 ), 122.3 (C-6), (126.4, 130.7, 131.2 and 131.3): (C-3 , C-4 , C-5 , C-6 ), 127.7 (C-2 ), 133.4 (C-1 ), 139.8 (C-5), 162.6 (C-2 ), 170.4 (Ac-CO). 2.2.6. (5 R)-3ˇ-acetoxy-17ˇ-[2-(4-bromophenyl)-4,5dihydrooxazol-5-yl]androst-5-ene (6f) 6f (2.07 g, 92%). (Ref. [8] mp 103–105 ◦ C). 2.2.7. (5 R)-3ˇ-acetoxy-17ˇ-[2-(4-nitrophenyl)-4,5dihydrooxazol-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 CH2 Cl2 , followed by ethyl acetate/CH2 Cl2 (5:95, v/v) and ethyl acetate/CH2 Cl2 (10:90, v/v) to afford 7a–g. 2.3.1. (5 R)-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 20 − 55 (c 1 in CHCl3 ) (found C, 79.82; H, 9.36. C29 H41 NO2 requires C, 79.95; H, 9.49%. 1 H 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): 4 -H2 , 4.65 (m, 1H, 5 -H), 5.34 (d, 1H, J = 5.0 Hz, 6-H), 7.39 (t, 2H, J = 7.5 Hz, 3 - and 5 -H), 7.46
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(t, 1H, J = 7.5 Hz, 4 -H), 7.92 (d, 2H, J = 7.5 Hz, 2 - and 6 -H). 13 C 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-4 ), 71.6, 81.7, 121.4 (C-6), (128.1 and 128.3): (C-2 , C-3 , C-5 , C-6 ), 128.1 (C-1 ), 131.1 (C-4 ), 140.9 (C-5), 164.1 (C-2 ). 2.3.2. (5 R)-17ˇ-[2-(4-fluorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3ˇ-ol (7b) 7b (950 mg, 87%), mp 232–235 ◦ C, Rf = 0.29 (ss D); [␣]D 20 − 61 (c 1 in CHCl3 ) (found C, 76.72; H, 8.37. C28 H36 FNO2 requires C, 76.85; H, 8.29%). 1 H 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): 4 -H2 , 4.67 (m, 1H, 5 H), 5.35 (s, 1H, 6-H), 7.07 (t, 2H, J = 8.5 Hz, 3 - and 5 -H), 7.92 (dd, 2H, J = 8.0 Hz, 2 - and 6 -H). 13 C 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, 55.0, 56.0, 59.8 (C-4 ), 71.7 (C-3), 81.9 (C-5 ), 115.3 (J = 21.7 Hz, C-3 and C-5 ), 121.4 (C-6), 124.4 (C-1 ), 130.3 (J = 8.6 Hz, C-2 and C-6 ), 140.9 (C-5), 163.4 (J = 42.4 Hz, C-4 ), 165.6 (C-2 ). 2.3.3. (5 R)-17ˇ-[2-(4-chlorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3ˇ-ol (7c) 7c (1.14 g, 70%), mp 215–218 ◦ C, Rf = 0.29 (ss D); [␣]D 20 − 48 (c 1 in CHCl3 ) (found C, 73.95; H, 8.03. C28 H36 ClNO2 requires C, 74.07; H, 7.99%). 1 H 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): 4 -H2 , 4.66 (m, 1H, 5 -H), 5.35 (d, 1H, J = 5.0 Hz, 6-H), 7.37 (d, 2H, J = 8.5 Hz, 3 - and 5 -H), 7.85 (d, 2H, J = 8.5 Hz, 2 - and 6 -H). 13 C 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, 55.0, 56.0, 59.8 (C-4 ), 71.7 (C-3), 82.0 (C-5 ), 121.4 (C-6), (128.6 and 129.4): (C-2 , C-3 , C-5 , C-6 ), 126.7 (C-1 ), 137.3 (C-4 ), 140.9 (C-5), 163.3 (C-2 ). 2.3.4. (5 R)-17ˇ-[2-(3-chlorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3ˇ-ol (7d) 7d (930 mg, 82%), mp 231–234 ◦ C, Rf = 0.32 (ss D); [␣]D 20 − 52 (c 1 in CHCl3 ) (found C, 74.12; H, 7.85. C28 H36 ClNO2 requires C, 74.07; H, 7.99%). 1 H 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): 4 -H2 , 4.66 (m, 1H, 5 H), 5.35 (d, 1H, J = 5.0 Hz, 6-H), 7.33 (t, 1H, J = 8.0 Hz, 5 -H), 7.43 (d, 1H, J = 8.0 Hz, 4 -H), 7.80 (d, 1H, J = 8.0 Hz, 6 -H), 7.9 (s, 1H, 2 -H). 13 C NMR (ı, ppm, CDCl ): 12.7 (C-18), 19.4 (C-19), 20.9, 23.7, 24.7, 3 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-4 ), 71.7 (C-3), 82.1 (C-5 ), 121.4 (C-6), (126.2, 128.2, 129.6 and 131.3): (C-2 , C-4 , C-5 , C-6 ), 129.9 (C-3 ), 134.3 (C-1 ), 140.9 (C-5), 163.0 (C-2 ). 2.3.5. (5 R)-17ˇ-[2-(2-chlorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3ˇ-ol (7e) 7e (970 mg, 85%), mp 165–169 ◦ C, Rf = 0.35 (ss D); [␣]D 20 − 58 (c 1 in CHCl3 ) (found C, 74.21; H, 8.12. C28 H36 ClNO2 requires C, 74.07; H, 7.99%). 1 H 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): 4 -H2 , 4.64 (m, 1H, 5 H), 5.34 (s, 1H, 6-H), 7.28 (t, 1H, 5 -H), 7.34 (t, 1H, J = 7.5 Hz, 4 -H), 7.43 (d, 1H, J = 7.5 Hz, 3 -H), 7.75 (d, 1H, J = 7.5 Hz, 6 -H). 13 C 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-4 ), 71.7 (C-3), 81.6 (C-5 ), 121.4 (C-6), (126.4, 130.7, 131.2
1027
and 131.3): (C-3 , C-4 , C-5 , C-6 ), 127.7 (C-2 ), 133.4 (C-1 ), 140.9 (C-5), 1627 (C-2 ). 2.3.6. (5 R)-17ˇ-[2-(4-bromophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3ˇ-ol (7f) 7f (1.05 g, 84%). (Ref. [8] mp 202–203 ◦ C). 2.3.7. (5 R)-17ˇ-[2-(4-nitrophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3ˇ-ol (7g) 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 CH2 Cl2 . The organic phase was evaporated to dryness and the residual crude product was chromatographed on silica gel with ethyl acetate/CH2 Cl2 (5:95). 2.4.1. (5 R)-17ˇ-[2-phenyl-4,5-dihydrooxazol-5-yl]androst-5-en-3-one (8a) 8a (350 mg, 40%), mp 89–93 ◦ C, Rf = 0.29 (ss D); [␣]D 20 + 94 (c 1 in CHCl3 ) (found C, 80.17; H, 9.25. C29 H39 NO2 requires C, 80.33; H, 9.07%). 1 H 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): 4 -H2 , 4.64 (m, 1H, 5 -H), 5.72 (s, 1H, 4H), 7.39 (t, 2H, J = 7.5 Hz, 3 - and 5 -H), 7.45 (t, 1H, J = 7.5 Hz, 4 -H), 7.91 (d, 2H, J = 7.5 Hz, 2 - and 6 -H). 13 C 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-4 ), 81.5 (5 -H), 123.8 (C-4), (128.0 and 128.3): (C-2 , C-3 , C-5 , C-6 ), 128.1 (C-1 ), 131.1 (C-4 ), 164.0 (C-2 ), 171.1 (C-5), 199.3 (C-3). 2.4.2. (5 R)-17ˇ-[2-(4-Fluorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3-one (8b) 8b (425 mg, 48%), mp 164–167 ◦ C, Rf = 0.35 (ss D); [␣]D 20 + 94 (c 1 in CHCl3 ) (found C, 77.34; H, 7.65. C28 H34 FNO2 requires C, 77.21; H, 7.87%). 1 H 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): 4 -H2 , 4.64 (m, 1H, 5 -H), 5.72 (s, 1H, 4H), 7.07 (t, 2H, J = 8.5 Hz, 3 - and 5 -H), 7.90 (dd, 2H, J = 8.0 Hz, 2 and 6 -H). 13 C 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-4 ), 81.8 (5 -C), 115.4 (J = 21.8 Hz, C-3 and C-5 ), 123.7 (C-4), 124.4 (C-1 ), 130.2 (J = 8.6 Hz, C-2 and C-6 ), 163.4 (J = 56.4 Hz, C-4 ), 165.6 (C-2 ), 171.0 (C-5), 199.3 (C-3). 2.4.3. (5 R)-17ˇ-[2-(4-chlorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3-one (8c) 8c (410 mg, 45%), mp 167–169 ◦ C, Rf = 0.38 (ss D); [␣]D 20 + 117 (c 1 in CHCl3 ) (found C, 74.26; H, 7.75. C28 H34 ClNO2 requires C, 74.40; H, 7.58%). 1 H 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): 4 -H2 , 4.65 (m, 1H, 5 -H), 5.72 (s, 1H, 4-H), 7.37 (d, 2H, J = 8.5 Hz, 3 - and 5 -H), 7.84 (d, 2H, J = 8.5 Hz, 2 and 6 -H). 13 C 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
1028
D. Ondré et al. / Steroids 74 (2009) 1025–1032
(C-13), 53.9, 54.9, 55.1, 59.8 (C-4 ), 81.8 (C-5 ), 123.9 (C-4), 126.6 (C1 ), (128.6 and 129.4): (C-2 , C-3 , C-5 , C-6 ), 137.3 (C-4 ), 163.2 (C-2 ), 171.0 (C-5), 199.4 (C-3). 2.4.4. (5 R)-17ˇ-[2-(3-chlorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3-one (8d) 8d (375 mg, 41%), mp 78–81 ◦ C, Rf = 0.44 (ss D); [␣]D 20 + 101 (c 1 in CHCl3 ) (found C, 74.32; H, 7.66; C28 H34 ClNO2 requires C, 74.40; H, 7.58%). 1 H 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): 4 -H2 , 4.65 (m, 1H, 5 -H), 5.71 (s, 1H, 4-H), 7.32 (t, 1H, J = 8.0 Hz, 5 -H), 7.42 (d, 1H, J = 8.0 Hz, 4 -H), 7.78 (d, 1H, J = 8.0 Hz, 6 -H), 7.88 (s, 1H, 2 -H). 13 C 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-4 ), 76.7, 77.0, 77.3, 81.9 (5 -C), 123.9 (C-4), (126.1, 128.1, 129.7, and 131.1): (C2 , C-4 , C-5 , C-6 ), 129.9 (C-3 ), 134.3 (C-1 ), 162.9 (C-2 ), 171.0 (C-5), 199.3 (C-3). 2.4.5. (5 R)-17ˇ-[2-(2-chlorophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3-one (8e) 8e (440 mg, 48%), mp 132–135 ◦ C, Rf = 0.42 (ss D); [␣]D 20 + 77 (c 1 in CHCl3 ) (found C, 74.26; H, 7.35. C26 H35 ClNO2 requires C, 74.40; H, 7.58%). 1 H 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): 4 -H2 , 4.64 (m, 1H, 5 -H), 5.71 (s, 1H, 4H), 7.28 (t, 1H, J = 7.5 Hz, 5 -H), 7.34 (t, 1H, J = 7.5 Hz, 4 -H), 7.43 (d, 1H, J = 7.5 Hz, 3 -H), 7.74 (d, 1H, J = 7.5 Hz, 6 -H). 13 C 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-4 ), 81.4 (C-5 ), 123.8 (C-4), (126.4, 130.7, 131.2 and 131.4): (C3 , C-4 , C-5 , C-6 ), 127.6 (C-2 ), 133.4 (C-1 ), 162.6 (C-2 ), 171.1 (C-5), 199.4 (C-3). 2.4.6. (5 R)-17ˇ-[2-(4-bromophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3-one (8f) 8f (460 mg, 46%), mp 184–189 ◦ C, Rf = 0.42 (ss D); [␣]D 20 + 114 (c 1 in CHCl3 ) (found C, 67.55; H, 7.05. C28 H34 BrNO2 requires C, 67.74; H, 6.90%). 1 H 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): 4 -H2 , 4.65 (m, 1H, 5 -H), 5.72 (4-H), 7.53 (d, 2H, J = 8.5 Hz, 3 - and 5 -H), 7.77 (d, 2H, J = 8.5 Hz, 2 - and 6 -H). 13 C 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 (C13), 53.8, 54.8, 55.1, 59.8 (C-4 ), 81.8 (C-5 ), 123.9 (C-4), (125.8 and 127.0): (C-1 , C-4 ), (129.6 and 131.6): (C-2 , C-3 , C-5 , C-6 ), 163.3 (C-2 ), 171.1 (C-5), 199.4 (C-3). 2.4.7. (5 R)-17ˇ-[2-(4-nitrophenyl)-4,5-dihydrooxazol-5yl]androst-5-en-3-one (8g) 8g (350 mg, 37%), mp 216–218 ◦ C, Rf = 0.40 (ss D); [␣]D 20 + 131 (c 1 in CHCl3 ) (found C, 72.87; H, 7.65. C28 H34 N2 O2 requires C, 72.70; H, 7.41%). 1 H 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): 4 -H2 , 4.71 (m, 1H, 5 -H), 5.72 (s, 1H, 4-H), 8.07 (d, 2H, J = 8.5 Hz, 2 - and 6 -H), 8.24 (d, 2H, J = 8.5 Hz, 3 - and 5 -H). 13 C 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-4 ), 82.3 (C-5 ), (123.5 and 129.0): (C-2 , C3 , C-5 , C-6 ), 123.9 (C-4), 133.9 (C-1 ), 149.4 (C-4 ), 162.3 (C-2 ), 170.9 (C-5), 199.3 (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 determined by an in vitro radiosubstrate incubation method described in our earlier publications [9,10] with some modifications. 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 [3 H]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 product 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) layers (0.25 mm thick) with solvent system diisopropyl ether/CH2 Cl2 (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 measured 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 without test substances and incubates with the reference compound ketoconazole were also prepared in every series. At least two experiments 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 concentrations 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.
3. Results and discussion 3.1. Synthetic studies Oxazolines have played important role in many areas of chemistry. Starting from ␣,-azidoalcohol several possibilities can be given to prepare oxazoline moieties. Catalytic hydrogenation of azidoalcohols 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 aldehydes, aminoalcohol, and N-bromosuccinimide as an oxidizing agent were described [16]. On the other hand, oxazolines can also be produced 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]. This observation provides a possibility for the preparation of compounds containing various substituted oxazolines condensed 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-
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Scheme 1. Reagents and conditions: (i) Pb(OAc)4 , BF3 ·OEt2 , MeOH; (ii) KBH4 , MeOH, rt; (iii) Al2 O3 , Mw; (iv) P(Ph)3 , CCl4 , reflux; (v) NaN3 , DMF, 80 ◦ C; (vi) benzaldehyde (5a–g), CH2 Cl2 , 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 selective 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-21chloropregn-5-en-20-ol (4a). Nucleophilic exchange with NaN3 in dimethylformamide led to the required (20R)-3-acetoxy-21azidopregn-5-en-20-ol (4b) [8]. Reaction of the ␣,-azidoalcohol 4b and appropriately substituted aromatic aldehydes 5a–g activated by BF3 ·OEt2 as Lewis acid catalyst, proceeded cleanly to give the corresponding steroid oxazolines 6a–g. Their deacetylation in methanol in the presence of NaOCH3 was carried out by Zemplén method furnished 7a–g. Oppenauer oxidation of the 3hydroxy-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 furnishes 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 compounds obtained, agreed with those of the starting materials (of confirmed configurations) (Scheme 2). 3.2. Effects of steroidal oxazolines on C17,20 -lyase activity
Scheme 2.
The inhibitory effects of compounds 7a–g and 8a–g on rat testicular C17,20 -lyase were investigated with an in vitro radiosubstrate incubation technique. Relative conversion percents measured at
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Table 1 Inhibition of C17,20 -lyase activity. Compound
Formula
Relative conversion, mean ± S.D. (%)
7a
NI
7b
NI
7c
80 ± 4
11 ± 0.8
7d
7e
83 ± 4
7f
85 ± 1
7.9 ± 1.5
7g
8a
IC50 ± S.D. (M)
37 ± 3
30 ± 3.0
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Table 1 (Continued ) Compound
Formula
Relative conversion, mean ± S.D. (%)
8b
76 ± 5
8c
50 ± 1
IC50 ± S.D. (M)
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 . Three derivatives of the 3-hydroxy compounds 7c, 7e and 7f exerted weak inhibitory effect, when applied at 50 M concentration 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 experiments; hence, these compounds did not inhibit rat C17,20 -lyase under the test conditions. The 3-chlorophenyl-, and 4-nitrophenyl
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. 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 moderate 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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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 follows: 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. Numerous steroidal compounds bearing heterocyclic substituents at C-17 have been investigated as concerns their P45017␣ inhibitory effects [21,22]. Abiraterone and its derivatives (17pyridyl 16,17-unsaturated compounds) have IC50 values in the interval 2–5 M [4,23]. Other C-17 heteroaryl (imidazolyl, benzimidazolyl, 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 possessing oxygen, sulfur or four-membered nitrogen heterocycles were less effective inhibitors [26]. We investigated the presumed C17,20 -lyase inhibition of androstene derivatives with aryl substituted 5 -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 investigated, the IC50 value of this compounds are close to that of ketoconazole and abiraterone (Table 1). 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. [2] Labrie F, Dupont A, Belanger A, Lefebvre FA, Cusan L, Monfette G, et al. New hormonal treatment in cancer of the prostate: combined administration 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-(3pyridyl)androsta-5,16-diene-3-ol) and related steroidal inhibitors. J Med Chem 1998;41:5375–81.
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