Synthesis and in vitro antiproliferative evaluation of d -secooxime derivatives of 13β- and 13α-estrone

Steroids 89 (2014) 47–55

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Synthesis and in vitro antiproliferative evaluation of D-secooxime derivatives of 13b- and 13a-estrone Erzsébet Mernyák a,⇑, Gabriella Fiser a, Johanna Szabó a, Brigitta Bodnár a, Gyula Schneider a, Ida Kovács b, Imre Ocsovszki c, István Zupkó b, János Wölfling a a b c

Department of Organic Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary Department of Biochemistry, University of Szeged, Dóm tér 9, H-6720 Szeged, Hungary

a r t i c l e

i n f o

Article history: Received 22 May 2014 Received in revised form 8 July 2014 Accepted 6 August 2014 Available online 20 August 2014 Keywords: Secoestrone 13a-Estrone Oxime Antitumor activity Cell cycle blockade

a b s t r a c t D-Secooximes were synthesized from the D-secoaldehydes in the 13b- and 13a-estrone series. The oximes were modified at three sites in the molecule: the oxime function was transformed into an oxime ether, oxime ester or nitrile group, the propenyl side-chain was saturated and the 3-benzyl ether was removed in order to obtain a phenolic hydroxy function. Triazoles were formed via Cu(I)-catalysed azide–alkyne cycloaddition (CuAAC) from 3-(prop-2-yniloxy)-D-secooximes and benzyl azides. All the products were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7, A2780 and A431). Some of them exhibited activities with submicromolar IC50 values, better than that of the reference agent cisplatin. The structural modifications led to significant differences in the cytostatic properties. Flow cytometry indicated that one of the most potent agents resulted in a cell cycle blockade. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Various types of substituted estrone analogs or homoestrones have been synthesized as cytotoxic or cytostatic (antiproliferative) anticancer agents [1–4]. One of the major requirements for the pharmacological use of antitumor estrone derivatives is that they must be devoid of estrogenic activity [5]. Natural 13b-estra1,3,5(10)-trienes possess a rigid steroidal framework with an aromatic A ring, while the B ring has a half-chair or sofa conformation, and the C ring possesses a chair conformation. The well-defined distances of the oxygen functionalities in estrone or estradiol are important for their estrogen receptor-binding capabilities and their biological activities. In contrast with the natural derivatives, the epimeric 13a-estra-1,3,5(10)-trienes have a relatively flexible molecular framework and different distances between the oxygen functions, which influence their receptor binding affinities [6]. Poirier et al. recently reported the impact of estradiol structural modifications (inversion of the configuration at C-13 and/or C-17) on the in vitro and in vivo estrogenic activity [7]. It has been found that the 13-epimers have a relative binding affinity for estrogen receptor alpha that is 1–2% of that of 17b-estradiol and they ⇑ Corresponding author. Fax: +36 (62)544199. E-mail address: [email protected] (E. Mernyák). http://dx.doi.org/10.1016/j.steroids.2014.08.015 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

have no significant uterotropic activity. These results suggest that inversion at C-13 in the estrane skeleton could be a powerful strategy in the design of non-estrogenic enzyme inhibitors. Additionally, D-homologization or substitution at C-2 of the estrane skeleton usually leads to the complete loss of hormonal behaviour [8]. 2-Ethoxyestradiol is potent both as a cytotoxic agent and as a tubulin polymerization inhibitor [9]. Its oximation at C-6 increased its cytotoxicity from micromolar to nanomolar IC50 values, but without an antitubulin effect. We recently synthesized 16-oxime derivatives of 13b- and 13a-estrone, determined their cytotoxic effects on human cancer cell lines and suggested their possible mechanism of action [10]. The 16-oxime of estrone selectively inhibits the proliferation of HeLa cells (IC50 = 4.41 lM), results in cell cycle arrest in the S phase and significantly increases caspase-3 activity, confirming the induction of programmed cell death. Derivatization of the oxime function, etherification of the 3-phenolic group and inversion at C-13 led to steroids with different antiproliferative properties. One 13a derivative, the 16-oxime propionate in the 3-benzyl ether series, proved to be as potent as the reference agent cisplatin, but with much better tumor selectivity. These promising results encouraged us to continue our investigations in the field of tumor-selective estrone oxime derivatives.

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E. Mernyák et al. / Steroids 89 (2014) 47–55

Cu(I)-catalysed azide–alkyne cycloaddition (CuAAC) is a widely applicable and facile method for the introduction of a triazole moiety into the molecules [11]. Incorporation of a triazole ring into the estrane skeleton has an additional advantage: it may enhance the solubility and bioavailability [12]. Triazoles are attractive units because of their stability against metabolic degradation and their ability to form hydrogen-bonds. We have recently reported the synthesis of various steroidal triazoles via the CuAAC reaction [13–17]. The aim of the present study was to synthetize D-secooximes in the 13b- and 13a-estrone series. Structural modifications of the secooximes were additionally planned in order to investigate the structure–activity relationships: etherification or esterification of the oxime function, removal of the 3-benzyl group and saturation of the propenyl side-chain. A propargyl function was incorporated into the D-secooxime and triazoles were formed from the secoestrone alkyne with benzyl azides via Cu(I)-catalysed azide–alkyne cycloaddition (CuAAC). All the synthesized compounds were tested in vitro by means of MTT assays for their antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7, A2780 and A431). 2. Experimental 2.1. Chemistry Melting points (mp) were determined with a Kofler hot-stage apparatus and are uncorrected. Elemental analyses were performed with a Perkin–Elmer CHN analyzer model 2400. Thin-layer chromatography: silica gel 60 F254; layer thickness 0.2 mm (Merck); detection with iodine or UV (365 nm) after spraying with 5% phosphomolybdic acid in 50% aqueous phosphoric acid and heating at 100–120 °C for 10 min. Flash chromatography: silica gel 60, 40–63 lm (Merck). The reactions under microwave irradiation were carried out with a CEM Corporation Focused Microwave System, Model Discover SP. 1H NMR spectra were recorded in CDCl3 solution (if not otherwise stated) with a Bruker DRX-500 instrument at 500 MHz, with Me4Si as internal standard. 13C NMR spectra were recorded with the same instrument at 125 MHz under the same conditions. 2.1.1. General procedure for the synthesis of D-secooximes (5–8) from 3-methoxy- or 3-benzyloxy-14b-(prop-2-en-yl)-des-D-estra-1,3,5(10) -trien-13a-carbaldehyde (1, 2) or from 3-methoxy- or 3-benzyloxy14b-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien-13b-carbaldehyde (3, 4) Compound 1 or 3 (298 mg, 1.00 mmol) or 2 or 4 (374 mg, 1.00 mmol) was dissolved in acetonitrile (10 mL), and water (4 mL), hydroxylamine hydrochloride (85 mg, 1.2 mmol) and sodium acetate (125 mg, 1.5 mmol) were added. The mixture was stirred at room temperature for 1 h, then evaporated to half volume and diluted with water (50 mL). The precipitate that formed was filtered off, washed with water until neutral and dried in the air. The crude product was subjected to flash chromatography over silica gel with dichloromethane as eluent. 2.1.1.1. 3-Methoxy-14b-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien13a-carbaldehyde oxime (5). From compound 1 (298 mg, 1.00 mmol). Yield: 301 mg (96%); mp 130–132 °C. 5 is identical with the compound described in the literature, mp 131–133 °C [18]. Anal. Calcd. for C20H27NO2: C, 76.64; H, 8.68. Found: C, 76.72; H, 8.55%. 2.1.1.2. 3-Benzyloxy-14b-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien13a-carbaldehyde oxime (6). From compound 2 (374 mg,

1.00 mmol). Yield: 362 mg (93%), mp 124–126 °C, Rf = 0.42 (2% ethyl acetate/98% dichloromethane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.12 (s, 3H, 18-H3), 2.86 (m, 2H, 6-H2), 5.00 (m, 2H, 16aH2), 5.05 (s, 2H, OCH2), 5.85 (m, 1H, 16-H), 6.74 (d, 1H, J = 2.2 Hz, 4-H), 6.81 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.21 (d, 1H, J = 8.6 Hz, 1-H), 7.32 (s, 1H, 17-H), 7.33 (t, 1H, J = 7.2 Hz, 40 -H), 7.40 (t, 2H, J = 7.2 Hz, 30 -H and 50 -H), 7.45 (d, 2H, J = 7.2 Hz, 20 -H and 60 -H), 7.85 (s, 1H, NOH). 13C NMR d [ppm] = 15.6 (C-18), 25.8, 27.3, 30.2, 34.1, 37.7, 40.6, 41.5 (C-13), 43.2, 47.5, 69.9 (OCH2), 112.5 (C-2), 114.5 (C-4), 115.0 (C-16a), 126.4 (C-1), 127.4 (2C, C-20 and C-60 ), 127.8 (C-40 ), 128.5 (2C: C-30 and C-50 ), 132.4 (C-10), 137.2 (C-10 ), 137.9 (C-5), 139.3 (C-16), 156.8 (C-3), 160.4 (C-17). Anal. Calcd. for C26H31NO2: C, 80.17; H, 8.02. Found: C, 79.97; H, 8.16%. 2.1.1.3. 3-Methoxy-14b-(prop-2-en-yl)-des-D-13a-estra-1,3,5(10)trien-13b-carbaldehyde oxime (7). From compound 3 (298 mg, 1.00 mmol). Yield: 295 mg (94%); mp 89–90 °C. 7 is identical with the compound described in the literature, mp 90–92 °C [19]. Anal. Calcd. for C20H27NO2: C, 76.64; H, 8.68. Found: C, 76.51; H, 8.76%. 2.1.1.4. 3-Benzyloxy-14b-(prop-2-en-yl)-des-D-13a-estra-1,3,5(10)trien-13b-carbaldehyde oxime (8). From compound 4 (374 mg, 1.00 mmol). Yield: 358 mg (92%), mp 105–107 °C, Rf = 0.52 (dichloromethane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.21 (s, 3H, 18H3), 2.83 (m, 2H, 6-H2), 4.98–5.06 (m, 2H, 16a-H2), 5.03 (s, 2H, OCH2), 5.84 (m, 1H, 16-H), 6.71 (d, 1H, J = 2.2 Hz, 4-H), 6.78 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.00 (s, 1H, 17-H), 7.19 (d, 1H, J = 8.6 Hz, 1-H), 7.32 (t, 1H, J = 7.2 Hz, 40 -H), 7.38 (t, 2H, J = 7.2 Hz, 30 -H and 50 -H), 7.42 (d, 2H, J = 7.2 Hz, 20 -H and 60 -H), 7.57 (s, 1H, NOH). 13C NMR d [ppm] = 26.4 (C-18), 27.0, 27.4, 30.3, 33.3, 39.2, 40.9 (C-13), 42.3, 43.5, 50.9, 70.0 (OCH2), 112.5 (C-2), 114.5 (C-4), 114.9 (C-16a), 126.4 (C-1), 127.4 (2C, C-20 and C-60 ), 127.8 (C-40 ), 128.5 (2C: C-30 and C-50 ), 132.5 (C-10), 137.3 (C-10 ), 137.8 (C-5), 139.4 (C-16), 156.0 (C-17), 156.8 (C-3). Anal. Calcd. for C26H31NO2: C, 80.17; H, 8.02. Found: C, 79.98; H, 8.17%. 2.1.2. General procedure for the hydrogenolysis and the saturation of the alkenyl side-chain A suspension of oxime 5 or 7 (314 mg, 1.00 mmol) or 6 or 8 (390 mg, 1.00 mmol) and Pd/C (0.60 g, 10%) in ethyl acetate (20 mL) was subjected to 30 bar of H2 pressure at room temperature for 2 h. The catalyst was then removed by filtration through a short pad of silica gel. After evaporation of the solvent, the crude product was subjected to flash chromatography. 2.1.2.1. 3-Methoxy-14b-propyl-des-D-estra-1,3,5(10)-trien-13a-carbaldehyde oxime (9). From compound 5 (314 mg, 1.00 mmol). Column chromatography over silica gel with dichloromethane yielded 268 mg (85%), mp 129–131 °C, Rf = 0.48 (2% ethyl acetate/ 98% dichloromethane). 1H NMR (500 MHz, DMSO-d6): d [ppm] = 0.83 (t, 3H, J = 6.8 Hz, 16a-H3), 0.97 (s, 3H, 18-H3), 2.78 (m, 2H, 6-H2), 3.69 (s, 3H, 3-OCH3), 6.60 (d, 1H, J = 2.2 Hz, 4-H), 6.67 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.17 (s, 1H, 17-H), 7.18 (d, 1H, J = 8.6 Hz, 1-H), 10.38 (s, 1H, NOH). 13C NMR d [ppm] = 14.4 and 15.4 (C-16a and C-18), 23.8, 25.7, 26.8, 29.9, 31.5, 37.1, 40.5, 40.6 (C-13), 42.7, 47.0, 54.8 (3-OCH3), 111.6 (C2), 113.0 (C-4), 126.3 (C-1), 131.8 (C-10), 137.3 (C-5), 157.0 (C-3), 157.8 (C-17). Anal. Calcd. for C20H29NO2: C, 76.15; H, 9.27. Found: C, 76.32; H, 9.05%. 2.1.2.2. 3-Methoxy-14b-propyl-des-D-13a-estra-1,3,5(10)-trien-13bcarbaldehyde oxime (10). From compound 7 (314 mg, 1.00 mmol). Column chromatography over silica gel with dichloromethane yielded 262 mg (83%), mp 127–130 °C, Rf = 0.47 (2% ethyl acetate/98% dichloromethane). 1H NMR (500 MHz, DMSO-d6):

E. Mernyák et al. / Steroids 89 (2014) 47–55

d [ppm] = 0.86 (t, 3H, J = 6.8 Hz, 16a-H3), 1.18 (s, 3H, 18-H3), 2.84 (m, 2H, 6-H2), 3.76 (s, 3H, 3-OCH3), 6.63 (d, 1H, J = 2.2 Hz, 4-H), 6.72 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.19 (d, 1H, J = 8.6 Hz, 1H), 7.23 (s, 1H, 17-H), 10.39 (s, 1H, NOH). 13C NMR d [ppm] = 23.6 and 25.9 (C-16a and C-18), 26.7, 27.6, 30.2, 34.3, 38.9, 40.3, 39.7 (C-13), 42.8, 42.9, 49.6, 55.3 (3-OCH3), 112.6 (C2), 114.6 (C-4), 126.5 (C-1), 131.8 (C-10), 137.7 (C-5), 156.9 (C-3), 157.6 (C-17). Anal. Calcd. for C20H29NO2: C, 76.15; H, 9.27. Found: C, 76.28; H, 9.32%. 2.1.2.3. 3-Hydroxy-14b-propyl-des-D-estra-1,3,5(10)-trien-13a-carbaldehyde oxime (11). From compound 6 (390 mg, 1.00 mmol). Column chromatography over silica gel with 30% tert-butyl methyl ether/60% n-hexane yielded 238 mg (79%), mp 133–135 °C, Rf = 0.48 (10% ethyl acetate/90% dichloromethane). 1H NMR (500 MHz, DMSO-d6): d [ppm] = 0.82 (t, 3H, J = 6.8 Hz, 16a-H3), 0.97 (s, 3H, 18-H3), 2.71 (m, 2H, 6-H2), 6.42 (d, 1H, J = 2.2 Hz, 4H), 6.51 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.05 (d, 1H, J = 8.6 Hz, 1-H), 7.17 (s, 1H, 17-H), 9.00 (s, 1H, 3-OH), 10.38 (s, 1H, NOH). 13 C NMR d [ppm] = 14.4 and 15.4 (C-16a and C-18), 23.8, 25.7, 26.9, 29.7, 31.5, 37.2, 40.6 (C-13), 40.7, 42.7, 47.0, 112.8 (C-2), 114.5 (C-4), 126.2 (C-1), 130.0 (C-10), 137.0 (C-5), 154.9 (C-3), 157.8 (C-17). Anal. Calcd. for C19H27NO2: C, 75.71; H, 9.03. Found: C, 75.83; H, 8.98%. 2.1.2.4. 3-Hydroxy-14b-propyl-des-D-13a-estra-1,3,5(10)-trien-13bcarbaldehyde oxime (12). From compound 8 (390 mg, 1.00 mmol). Column chromatography over silica gel with 30% tert-butyl methyl ether/60% n-hexane yielded 232 mg (77%), mp 163–167 °C, Rf = 0.39 (2% ethyl acetate/98% dichloromethane). 1H NMR (500 MHz, DMSO-d6): d [ppm] = 0.88 (t, 3H, J = 6.8 Hz, 16a-H3), 1.09 (s, 3H, 18-H3), 2.70 (m, 2H, 6-H2), 6.42 (d, 1H, J = 2.2 Hz, 4H), 6.51 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.03 (d, 1H, J = 8.6 Hz, 1-H), 7.38 (s, 1H, 17-H), 9.00 (s, 1H, 3-OH), 10.47 (s, 1H, NOH). 13 C NMR d [ppm] = 14.3 and 26.6 (C-18 and C-16a), 24.6, 26.8, 26.9, 29.9, 31.4, 38.7, 40.2, 42.3, 42.9, 50.7, 112.8 (C-2), 114.5 (C4), 126.2 (C-1), 130.2 (C-10), 137.0 (C-5), 153.5 (C-17), 154.8 (C3). Anal. Calcd. for C19H27NO2: C, 75.71; H, 9.03. Found: C, 75.79; H, 9.12%. 2.1.3. General procedure for the synthesis of oxime ethers (13–16) To a solution of oxime 5 or 7 (314 mg, 1.00 mmol) in acetonitrile (10 mL), water (4 mL), O-benzylhydroxylamine hydrochloride (160 mg, 1.00 mmol) or O-allylhydroxylamine hydrochloride (112 mg, 1.00 mmol) and sodium acetate (105 mg, 1.25 mmol) were added. The mixture was stirred at room temperature for 6 h, then evaporated to half volume and extracted with dichloromethane. The organic phase was washed with water until neutral, dried over sodium sulfate and evaporated to dryness. The residue obtained was subjected to flash chromatography on a silica gel column with dichloromethane as eluent. 2.1.3.1. 3-Methoxy-14b-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien13a-carbaldehyde oxime benzyl ether (13). From compound 5 (314 mg, 1.00 mmol). Yield: 347 mg (86%), oil, Rf = 0.44 (60% dichloromethane/40% n-hexane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.08 (s, 3H, 18-H3), 2.84 (m, 2H, 6-H2), 3.78 (s, 3H, OCH3), 4.90–4.95 (m, 2H, 16a-H2), 5.08 (s, 2H, OCH2), 5.79 (m, 1H, 16-H), 6.63 (d, 1H, J = 2.2 Hz, 4-H), 6.72 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.19 (d, 1H, J = 8.6 Hz, 1-H), 7.30 (t, 1H, J = 7.2 Hz, 40 -H), 7.32 (s, 1H, 17-H), 7.34–7.39 (overlapping multiplets, 4H, 20 -H, 30 -H, 50 -H, 60 -H). 13C NMR d [ppm] = 15.7 (C-18), 25.8, 27.4, 30.3, 34.1, 37.9, 40.8, 42.0 (C-13), 43.2, 47.4, 55.2 (OCH3), 75.6 (OCH2), 111.7 (C-2), 113.5 (C-4), 114.7 (C-16a), 126.3 (C-1), 127.2 (2C, C-20 and C-60 ), 127.7 (C-40 ), 128.3 (2C: C-30 and C-50 ), 132.2 (C-10), 137.3 (C-10 ), 137.8 (C-5), 139.6 (C-16), 157.5 (C-3), 159.5

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(C-17). Anal. Calcd. for C27H33NO2: C, 80.36; H, 8.24. Found: C, 80.15; H, 8.17%. 2.1.3.2. 3-Methoxy-14b-(prop-2-en-yl)-des-D-13a-estra-1,3,5(10)trien-13b-carbaldehyde oxime benzyl ether (14). From compound 7 (314 mg, 1.00 mmol). Yield: 331 mg (82%), oil, Rf = 0.45 (60% dichloromethane/40% n-hexane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.21 (s, 3H, 18-H3), 2.82 (m, 2H, 6-H2), 3.79 (s, 3H, OCH3), 4.98 (m, 2H, 16a-H2), 5.05 (s, 2H, OCH2), 5.83 (m, 1H, 16H), 6.64 (d, 1H, J = 2.2 Hz, 4-H), 6.73 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.19 (d, 1H, J = 8.6 Hz, 1-H), 7.24 (t, 1H, J = 7.2 Hz, 40 -H), 7.28 (t, 2H, J = 7.2 Hz, 30 -H and 50 -H), 7.43 (d, 2H, J = 7.2 Hz, 20 -H and 60 -H), 7.57 (s, 1H, 17-H). 13C NMR d [ppm] = 26.6 (C-18), 27.0, 27.4, 30.4, 33.3, 39.1, 41.0 (C-13), 42.3, 43.5, 50.9, 55.2 (OCH3), 75.6 (OCH2), 111.6 (C-2), 113.4 (C-4), 114.6 (C-16a), 126.4 (C-1), 127.6 and 128.2 and 128.3 (5C, C-20 , C-30 , C-40 , C-50 and C-60 ), 132.4 (C-10), 137.8 (2C, C-10 and C-5), 139.7 (C-16), 155.3 (C-17), 157.5 (C-3). Anal. Calcd. for C27H33NO2: C, 80.36; H, 8.24. Found: C, 80.25; H, 8.32%. 2.1.3.3. 3-Methoxy-14b-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien13a-carbaldehyde oxime allyl ether (15). From compound 5 (314 mg, 1.00 mmol). Yield: 276 mg (78%), oil, Rf = 0.42 (60% dichloromethane/40% n-hexane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.06 (s, 3H, 18-H3), 2.81 (m, 2H, 6-H2), 3.75 (s, 3H, OCH3), 4.52 (m, 2H, OCH2), 4.95 (m, 2H, 16a-H2), 5.26 (m, 2H, – CH = CH2), 5.82 (m, 1H, 16-H), 5.96 (m, 1H, –CH@CH2), 6.60 (d, 1H, J = 2.2 Hz, 4-H), 6.70 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.17 (d, 1H, J = 8.6 Hz, 1-H). 13C NMR d [ppm] = 15.7 (C-18), 25.8, 27.4, 30.3, 34.2, 37.9, 40.9, 41.5 (C-13), 43.2, 47.4, 55.2 (OCH3), 74.4 (OCH2), 111.7 (C-2), 113.4 (C-4), 114.7 (C-16a), 117.5 (–CH@CH2), 126.4 (C-1), 132.2 (C-10), 134.3 (–CH@CH2), 137.9 (C-5), 139.7 (C-16), 157.5 (C-3), 159.3 (C-17). Anal. Calcd. for C23H31NO2: C, 78.15; H, 8.84. Found: C, 78.23; H, 8.98%. 2.1.3.4. 3-Methoxy-14b-(prop-2-en-yl)-des-D-13a-estra-1,3,5(10)trien-13b-carbaldehyde oxime allyl ether (16). From compound 7 (314 mg, 1.00 mmol). Yield: 286 mg (81%), oil, Rf = 0.43 (60% dichloromethane/40% n-hexane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.20 (s, 3H, 18-H3), 2.83 (m, 2H, 6-H2), 3.78 (s, 3H, OCH3), 4.50 (m, 2H, OCH2), 4.96–5.06 (overlapping multiplets, 2H, 16a-H2), 5.15–5.28 (overlapping multiplets, 2H, –CH@CH2), 5.85 (m, 1H, 16-H), 5.95 (m, 1H, –CH@CH2), 6.62 (d, 1H, J = 2.2 Hz, 4-H), 6.71 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.19 (d, 1H, J = 8.6 Hz, 1-H), 7.56 (s, 1H, 17-H). 13C NMR d [ppm] = 26.6 (C-18), 27.1, 27.4, 30.4, 33.4, 39.2, 40.9 (C-13), 42.4, 43.6, 50.9, 55.1 (3-OCH3), 74.3 (OCH2), 111.7 (C-2), 113.4 (C-4), 114.7 (C-16a), 117.4 (–CH@CH2), 126.4 (C-1), 132.4 (C-10), 134.2 (–CH@CH2), 137.8 (C-5), 139.7 (C-16), 154.8 (C-17), 157.5 (C-3). Anal. Calcd. for C23H31NO2: C, 78.15; H, 8.84. Found: C, 78.06; H, 8.92%. 2.1.4. General procedure for the synthesis of oxime esters (17–20) To a solution of oxime 5 or 7 (314 mg, 1.00 mmol) or 6 or 8 (390 mg, 1.00 mmol) in pyridine (5 mL), acetic anhydride (2 mL, 21.2 mmol) was added. The mixture was stirred at room temperature for 1 h, then poured into water (50 mL) and extracted with dichloromethane. The organic phase was washed with water until neutral, dried over sodium sulfate and evaporated to dryness. The residue obtained was subjected to flash chromatography on a silica gel column with dichloromethane as eluent. 2.1.4.1. 3-Methoxy-14b-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien13a-carbaldehyde oxime acetate (17). From compound 5 (314 mg, 1.00 mmol). Yield: 263 mg (74%), oil, Rf = 0.63 (2% ethyl acetate/

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98% dichloromethane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.21 (s, 3H, 18-H3), 2.16 (s, 3H, Ac-CH3), 2.85 (m, 2H, 6-H2), 3.78 (s, 3H, 3-OCH3), 4.96–5.03 (m, 2H, 16a-H2), 5.83 (m, 1H, 16-H), 6.63 (d, 1H, J = 2.2 Hz, 4-H), 6.73 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.19 (d, 1H, J = 8.6 Hz, 1-H), 7.55 (s, 1H, 17-H). 13C NMR d [ppm] = 15.3 and 19.7 (C-18 and Ac-CH3), 25.6, 27.3, 30.2, 34.2, 37.3, 40.5, 42.3 (C-13), 43.1, 47.4, 55.2 (3-OCH3), 111.7 (C-2), 113.5 (C-4), 115.3 (C-16a), 126.3 (C-1), 132.0 (C-10), 137.8 (C-5), 139.0 (C-16), 157.6 (C-3), 167.0 (C-17), 168.9 (Ac-CO). Anal. Calcd. for C22H29NO3: C, 74.33; H, 8.22. Found: C, 74.42; H, 8.37%. 2.1.4.2. 3-Benzyloxy-14b-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien13a-carbaldehyde oxime acetate (18). From compound 6 (390 mg, 1.00 mmol). Yield: 354 mg (82%), oil, Rf = 0.67 (2% ethyl acetate/ 98% dichloromethane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.21 (s, 3H, 18-H3), 2.16 (s, 3H, Ac-CH3), 2.84 (m, 2H, 6-H2), 4.98 (m, 2H, 16a-H2), 5.04 (s, 2H, OCH2), 5.83 (m, 1H, 16-H), 6.72 (d, 1H, J = 2.2 Hz, 4-H), 6.79 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.19 (d, 1H, J = 8.6 Hz, 1-H), 7.32 (t, 1H, J = 7.2 Hz, 40 -H), 7.38 (t, 2H, J = 7.2 Hz, 30 -H and 50 -H), 7.42 (d, 2H, J = 7.2 Hz, 20 -H and 60 -H), 7.56 (s, 1H, 17-H). 13C NMR d [ppm] = 15.4 and 19.7 (C-18 and Ac-CH3), 25.6, 27.3, 30.2, 34.2, 37.3, 40.5, 42.3 (C-13), 43.2, 47.4, 70.0 (OCH2), 112.5 (C-2), 114.5 (C-4), 115.3 (C-16a), 126.4 (C-1), 127.4 (2C: C-20 and C-60 ), 127.9 (C-40 ), 128.5 (2C: C-30 and C-50 ), 132.2 (C-10), 137.2 (C-10 ), 137.8 (C-5), 139.0 (C-16), 156.9 (C-3), 167.0 (Ac-CO), 167.1 (C-17). Anal. Calcd. for C28H33NO3: C, 77.93; H, 7.71. Found: C, 78.03; H, 7.85%. 2.1.4.3. 3-Methoxy-14b-(prop-2-en-yl)-des-D-13a-estra-1,3,5(10)trien-13b-carbaldehyde oxime acetate (19). From compound 7 (314 mg, 1.00 mmol). Yield: 270 mg (76%), mp 53–55 °C, Rf = 0.37 (dichloromethane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.32 (s, 3H, 18-H3), 2.11 (s, 3H, Ac-H3), 2.85 (m, 2H, 6-H2), 3.78 (s, 3H, 3-OCH3), 4.99–5.07 (m, 2H, 16a-H2), 5.81 (m, 1H, 16-H), 6.62 (d, 1H, J = 2.2 Hz, 4-H), 6.72 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.18 (d, 1H, J = 8.6 Hz, 1-H), 7.87 (s, 1H, 17-H). 13C NMR d [ppm] = 19.6 and 26.0 (Ac-CH3 and C-18),, 27.0, 27.4, 30.2, 33.1, 39.0, 41.8 (C-13), 42.2, 43.4, 50.7, 55.2 (3-OCH3), 111.7 (C-2), 113.5 (C-4), 115.6 (C-16a), 126.4 (C-1), 132.0 (C-10), 137.7 (C-5), 138.6 (C-16), 157.6 (C-3), 163.2 (C-17), 168.9 (Ac-CO). Anal. Calcd. for C22H29NO3: C, 74.33; H, 8.22. Found: C, 74.45; H, 8.16%.

reactor. The mixture was heated for 6 min at 100 °C and then transferred into water and extracted with dichloromethane. The organic phase was washed with water until neutral, dried over sodium sulfate and evaporated to dryness. The residue obtained was subjected to flash chromatography on a silica gel column with 80% dichloromethane/20% n-hexane as eluent. 2.1.5.1. 3-Benzyloxy-14b-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien13a-carbonitrile (21). From compound 6 (390 mg, 1.00 mmol). Yield: 286 mg (77%), mp 78–82 °C, Rf = 0.54 (70% dichloromethane/30% n-hexane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.32 (s, 3H, 18-H3), 2.84 (m, 2H, 6-H2), 5.04 (s, 2H, OCH2), 5.06–5.20 (2 m, 2 1H, 16a-H2), 5.95 (m, 1H, 16-H), 6.72 (d, 1H, J = 2.2 Hz, 4-H), 6.80 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.16 (d, 1H, J = 8.6 Hz, 1-H), 7.32 (t, 1H, J = 7.2 Hz, 40 -H), 7.39 (t, 2H, J = 7.2 Hz, 30 -H and 50 -H), 7.43 (d, 2H, J = 7.2 Hz, 20 -H, 60 -H). 13C NMR d [ppm] = 17.3 (C-18), 25.1, 26.9, 30.0, 35.5, 37.4 (C-13), 37.7, 39.8, 42.8, 47.2, 69.9 (OCH2), 112.6 (C-2), 114.6 (C-4), 116.2 (C-16a), 126.1 (C-17), 126.3 (C-1), 127.4 (2C, C-20 and C-60 ), 127.9 (C-40 ), 128.5 (2C, C-30 and C-50 ), 131.4 (C-10), 137.1 (C-10 ), 137.6 (C-5), 137.8 (C-16), 157.0 (C-3). Anal. Calcd. for C26H29NO: C, 84.06; H, 7.87. Found: C, 83.92; H, 7.96%. 2.1.5.2. 3-Benzyloxy-14b-(prop-2-en-yl)-des-D-13a-estra-1,3,5(10)trien-13b-carbonitrile (22). From compound 8 (390 mg, 1.00 mmol). Yield: 275 mg (74%), mp 61–62 °C, Rf = 0.56 (70% dichloromethane/30% n-hexane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.45 (s, 3H, 18-H3), 2.85 (m, 2H, 6-H2), 5.05 (s, 2H, OCH2), 5.11 (m, 2H, 16a-H2), 5.95 (m, 1H, 16-H), 6.72 (d, 1H, J = 2.2 Hz, 4-H), 6.79 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.18 (d, 1H, J = 8.6 Hz, 1-H), 7.32 (t, 1H, J = 7.2 Hz, 40 -H), 7.39 (t, 2H, J = 7.2 Hz, 30 -H and 50 -H), 7.43 (d, 2H, J = 7.2 Hz, 20 -H and 60 -H). 13C NMR d [ppm] = 25.9 (C-18), 26.8, 27.8, 30.1, 34.1, 38.9, 39.4 (C-13), 42.8, 42.9, 49.6, 69.9 (OCH2), 112.6 (C-2), 114.5 (C-4), 115.9 (C-16a), 123.5 (C-17), 126.4 (C-1), 127.4 (2C, C-20 and C-60 ), 127.8 (C-40 ), 128.5 (2C: C30 and C-50 ), 131.6 (C-10), 137.2 (C-10 ), 137.5 (C-160 ), 137.7 (C-5), 156.9 (C-3). Anal. Calcd. for C26H29NO: C, 84.06; H, 7.87. Found: C, 84.22; H, 7.65%.

2.1.4.4. 3-Benzyloxy-14b-(prop-2-en-yl)-des-D-13a-estra-1,3,5(10)trien-13b-carbaldehyde oxime acetate (20). From compound 8 (390 mg, 1.00 mmol). Yield: 367 mg (85%), mp 60–62 °C, Rf = 0.45 (dichloromethane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.32 (s, 3H, 18-H3), 2.12 (s, 3H, Ac-H3), 2.85 (m, 2H, 6-H2), 5.01 (m, 2H, 16a-H2), 5.03 (s, 2H, OCH2), 5.83 (m, 1H, 16-H), 6.71 (d, 1H, J = 2.2 Hz, 4-H), 6.79 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.19 (d, 1H, J = 8.6 Hz, 1-H), 7.32 (t, 1H, J = 7.2 Hz, 40 -H), 7.38 (t, 2H, J = 7.2 Hz, 30 - H and 50 -H), 7.43 (d, 2H, J = 7.2 Hz, 20 -H and 60 -H), 7.87 (s, 1H, 17-H). 13C NMR d [ppm] = 19.6 and 26.0 (Ac-CH3 and C-18), 27.0, 27.3, 30.2, 33.1, 39.0, 41.7 (C-13), 42.1, 43.4, 50.7, 70.0 (OCH2), 112.5 (C-2), 114.5 (C-4), 115.5 (C-16a), 126.4 (C-1), 127.4 (2C: C-20 and C-60 ), 127.8 (C-40 ), 128.5 (2C: C-30 and C-50 ), 132.3 (C-10), 137.2 (C-10 ), 137.7 (C-5), 138.6 (C-16), 156.9 (C-3), 163.2 (C-17), 168.8 (Ac-CO). Anal. Calcd. for C28H33NO3: C, 77.93; H, 7.71. Found: C, 77.85; H, 7.89%.

2.1.6. Synthesis of the 3-(prop-2-inyloxy)-14b-propyl-des-D-estra1,3,5(10)-trien-13a-carbaldehyde oxime (23) Compound 11 (302 mg, 1.00 mmol) was dissolved in acetone (10 mL), propargyl-bromide (0.17 mL (80 wt.% in toluene), 1.5 mmol) and potassium carbonate (968 mg, 7 mmol) were added. The reaction mixture was stirred at 70 °C for 24 h, the solvent was evaporated off, and the residue was subjected to flash chromatography over silica gel with 2% ethyl acetate/98% dichloromethane as eluent. Yield: 302 mg (89%), oil, Rf = 0.46 (2% ethyl acetate/98% dichloromethane). 1H NMR (500 MHz, CDCl3): d [ppm] = 0.89 (t, 3H, J = 6.8 Hz, 16a-H3), 1.07 (s, 3H, 18-H3), 2.51 (s, 1H, C„CH), 2.86 (m, 2H, 6-H2), 4.66 (s, 2H, OCH2), 6.70 (d, 1H, J = 2.2 Hz, 4-H), 6.79 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.21 (d, 1H, J = 8.6 Hz, 1-H), 7.30 (s, 1H, 17-H). 13C NMR d [ppm] = 14.5 and 15.4 (C-16a and C-18), 24.3, 25.9, 27.2, 30.5, 32.2, 37.4, 40.9, 41.3 (C-13), 43.3, 47.9, 55.7 (OCH2), 75.3 (C„CH), 79.9 (C„CH), 112.5 (C-2), 114.5 (C-4), 126.5 (C-1), 133.2 (C-10), 137.9 (C-5), 155.5 (C-3), 160.5 (C-17). Anal. Calcd. for C22H29NO2: C, 77.84; H, 8.64. Found: C, 77.65; H, 8.82%.

2.1.5. General procedure for the synthesis of seconitriles (21, 22) Compound 6 or 8 (390 mg, 1.00 mmol) was dissolved in dichloromethane (30 mL), and acetic anhydride (1 mL, 10.6 mmol), 4(dimethylamino)pyridine (183 mg, 1.50 mmol) and silica gel (1 g) were added. The mixture was homogenized, the solvent was evaporated and the residue was placed into a pressure tube equipped with a stirrer bar and was inserted into the cavity of the microwave

2.1.7. General procedure for the synthesis of the triazoles 25 To a stirred solution of the appropriate terminal alkyne (23, 1.0 mmol) in toluene (10 mL), benzyl azide (24, 1.0 mmol), triphenylphosphine (52 mg, 0.2 mmol), copper(I) iodide (19 mg, 0.1 mmol) and N,N-diisopropylethylamine (0.52 mL, 3 mmol) were added. The reaction mixture was refluxed for 2 h, the solvent was evaporated off, and the residue was subjected to flash

E. Mernyák et al. / Steroids 89 (2014) 47–55

chromatography over silica gel with 10% ethyl acetate/90% dichloromethane as eluent. 2.1.7.1. 3-(1-Benzyl-1H-1,2,3-triazol-4-yl-methyloxy)-14b-propyldes-D-estra-1,3,5(10)-trien-carbaldehyde oxime (25a). From compound 23 (340 mg, 1.00 mmol) and benzyl azide (24a, 133 mg, 1.0 mmol). Yield: 435 mg (92%), mp 67–69 °C, Rf = 0.40 (10% ethyl acetate/90% dichloromethane). 1H NMR (500 MHz, CDCl3): d [ppm] = 0.88 (t, 3H, J = 6.8 Hz, 16a-H3), 1.06 (s, 3H, 18-H3), 2.83 (m, 2H, 6-H2), 5.16 (s, 2H, OCH2), 5.53 (s, 2H, NCH2), 6.69 (d, 1H, J = 2.2 Hz, 4-H), 6.77 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.18 (d, 1H, J = 8.6 Hz, 1-H), 7.26–7.29 (overlapping multiplets, 3H: 30 ,40 ,50 -H), 7.37 (s, 1H, 17-H), 7.38 (d, 2H, J = 7.3 Hz, 20 ,60 -H), 7.52 (s, 1H, C@CH). 13C NMR d [ppm] = 14.5 and 15.4 (C-16a and C18), 24.3, 25.9, 27.2, 30.4, 32.2, 37.4, 40.9, 41.3 (C-13), 43.3, 47.9, 54.3 (NCH2), 62.2 (OCH2), 112.5 (C-2), 114.4 (C-4), 122.5 (C@CH), 126.5 (C-1), 128.1 (2C) and 129.1 (2C): C-20 ,30 ,50 ,60 , 128.8 (C-40 ), 132.9 (C-10), 134.4 (C-10 ), 137.9 (C-5), 149.0 (C@CH), 156.2 (C-3), 160.5 (C-17). Anal. Calcd. for C29H36N4O2: C, 73.70; H, 7.68. Found: C, 73.86; H, 7.54%. 2.1.7.2. 3-(1-(4-Prop-2-ylbenzyl-1H-1,2,3-triazol-4-yl-methyloxy)14b-propyl-des-D-estra-1,3,5(10)-trien-carbaldehyde oxime (25b). From compound 23 (340 mg, 1.00 mmol) and 4-prop-2-ylbenzyl azide (24b, 176 mg, 1.0 mmol). Yield: 464 mg (90%), mp 59– 62 °C, Rf = 0.44 (10% ethyl acetate/90% dichloromethane). 1H NMR (500 MHz, CDCl3): d [ppm] = 0.88 (t, 3H, J = 6.8 Hz, 16a-H3), 1.06 (s, 3H, 18-H3), 1.25 (overlapping multiplets, 6H, 2 prop-2-ylCH3), 2.83 (m, 2H, 6-H2), 5.17 (s, 2H, OCH2), 5.49 (s, 2H, NCH2), 6.69 (d, 1H, J = 2.2 Hz, 4-H), 6.77 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2H), 7.18 (d, 1H, J = 8.6 Hz, 1-H), 7.20–7.24 (overlapping multiplets, 4H, 20 ,30 ,50 ,60 -H), 7.29 (s, 1H, (17-H), 7.53 (s, 1H, C@CH). 13C NMR d [ppm] = 14.5 and 15.4 (C-16a and C-18), 23.9 (2C: 2 prop-2-ylCH3), 24.3, 25.9, 27.2, 30.4, 32.1, 33.8, 37.4, 40.9, 41.3 (C-13), 43.3, 47.9, 54.1 (NCH2), 62.1 (OCH2), 112.5 (C-2), 114.4 (C-4), 122.4 (C@CH), 126.5 (C-1), 127.2 (2C) and 128.2 (2C): C-20 ,30 ,50 ,60 , 131.6 (C-10 ), 132.9 (C-10), 137.9 (C-5), 145.3 (C@CH), 149.7 (C40 ), 156.0 (C-3), 160.4 (C-17). Anal. Calcd. for C32H42N4O2: C, 74.67; H, 8.22. Found: C, 74.75; H, 8.36%. 2.1.7.3. 3-(1-(4-Nitrobenzyl-1H-1,2,3-triazol-4-yl-methyloxy)-14bpropyl-des-D-estra-1,3,5(10)-trien-carbaldehyde oxime (25c). From compound 23 (340 mg, 1.00 mmol) and 4-nitrobenzyl azide (24c, 178 mg, 1.0 mmol). Yield: 481 mg (93%), mp 76–79 °C, Rf = 0.33 (10% ethyl acetate/90% dichloromethane). 1H NMR (500 MHz, CDCl3): d [ppm] = 0.88 (t, 3H, J = 6.8 Hz, 16a-H3), 1.05 (s, 3H, 18H3), 2.83 (m, 2H, 6-H2), 5.19 (s, 2H, OCH2), 5.65 (s, 2H, NCH2), 6.69 (d, 1H, J = 2.2 Hz, 4-H), 6.77 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2H), 7.19 (d, 1H, J = 8.6 Hz, 1-H), 7.29 (s, 1H, (17-H), 7.40 (d, 2H, J = 8.6 Hz, 20 -H and 60 -H), 7.61 (s, 1H, C@CH), 8.22 (d, 2H, J = 8.6 Hz, 30 -H and 50 -H). 13C NMR d [ppm] = 14.5 and 15.4 (C16a and C-18), 24.3, 26.0, 27.2, 30.4, 32.1, 37.4, 40.9, 41.3 (C-13), 43.3, 47.9, 53.1 (NCH2), 62.0 (OCH2), 112.4 (C-2), 114.4 (C-4), 122.7 (C@CH), 124.3 (2C) and 128.6 (2C): C-20 ,30 ,50 ,60 , 126.6 (C-1), 133.0 (C-10), 138.0 (C-5), 141.5 and 145.6 and 148.1 (C-10 and C-40 and C@CH), 156.0 (C-3), 160.4 (C-17). Anal. Calcd. for C29H35N5O4: C, 67.29; H, 6.82. Found: C, 67.41; H, 6.95%. 2.2. Cell cultures and antiproliferative assays Human cancer cell lines (HeLa, MCF-7 and A431, isolated from cervical adenocarcinoma, breast adenocarcinoma and skin epidermoid carcinoma, respectively) and noncancerous human foreskin fibroblasts were maintained in minimal essential medium supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino-acids and an antibiotic–antimycotic mixture (AAM).

51

A2780 cells (isolated from ovarial cancer) were maintained in RPMI medium supplemented with 10% FBS, 1% AAM and 1% L-glutamine. All cell lines were purchased from the European Collection of Cell Cultures (Salisbury, U.K.). For pharmacological investigations, 10 mM stock solutions of the tested compounds were prepared with dimethyl sulfoxide (DMSO). The highest applied DMSO concentration of the medium (0.3%) did not have any substantial effect on the determined cellular functions. All the chemicals, if otherwise not specified, were purchased from Sigma–Aldrich Ltd. (Budapest, Hungary). The antiproliferative effects were determined in vitro on the four cell lines: HeLa, A431, MCF-7 and A2780. The cells were grown in a humidified atmosphere of 5% CO2 at 37 °C. Cells were seeded onto 96-well plates at a density of 5000 cells/well and allowed to stand overnight, after which the medium containing the tested compound was added. After a 72-h incubation, viability was determined by the addition of 20 lL of [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution (5 mg/mL). The precipitated formazan crystals were solubilized in DMSO and the absorbance was determined at 545 nm with an ELISA reader [20]. Two independent experiments were performed with 5 parallel wells; cisplatin, an agent administered clinically in the treatment of certain gynecological malignancies, was used as a positive control. Sigmoidal dose–response curves were fitted to the measured data. Calculations of IC50 values and statistical analyses (ANOVA) were performed by means of GraphPad Prism 4.0 (GraphPad Software; San Diego, CA, USA). 2.3. Cell cycle analysis by flow cytometry Cell cycle analysis by means of flow cytometry was performed in order to characterize the cellular DNA content of treated A2780 cells. After treatment for 24 or 48 h, cells (200,000/condition) were trypsinized (Gibco BRL, Paisley, U.K.), washed with phosphate-buffered saline (PBS) and fixed in 1.0 mL of cold 70% ethanol for 30 min on ice. After two washing steps in cold PBS, DNA was stained with propidium iodide (10 lg/mL) in the presence of RNA-ase (50 lg/mL). The samples were then analyzed with CyFlow (Partec GmbH, Münster, Germany). In each analysis, 20,000 events were recorded, and the percentages of the cells in the different cell-cycle phases (subG1, G1, S and G2/M) were calculated by using ModFit LT (Verity Software House, Topsham, ME, USA) [21]. 3. Results and discussion We earlier developed an efficient route for the synthesis of a of estrone 3-methyl (1) or 3-benzyl ether (2) [22,23]. This key compound, containing an aldehyde function and a propenyl side-chain in trans stereochemistry, proved to be a useful intermediate in the synthesis of D-homoestrone derivatives. The similar secoaldehydes in the 13a-estrone series (3, 4) with cis functional groups were later synthesized and used in cyclization reactions [23,24]. The additional structural modifications of the epimeric secoaldehydes (1–4) may lead to structurally diverse secoestrones. In the present work we first set out to synthetize oximes (5–8) from the secoaldehydes of 13b- and 13a-estrone 3-methyl- or 3-benzyl ether (1–4). The latter protecting group was used because of the possibility of its easy removal. We earlier reported the synthesis and Lewis aciD-induced intramolecular 1,3-dipolar cycloadditions of D-secooximes of estrone and 13a-estrone 3-methyl ether, furnishing isoxazolidines [18,19]. Electrophile-induced nitrone formation and the subsequent 1,3-dipolar cycloadditions of the cyclic nitrones with C@C or C@N dipolarophiles were additionally presented [19,25,26]. Here, we synthesized the oximes (5–8) D-secoaldehyde

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E. Mernyák et al. / Steroids 89 (2014) 47–55

Table 1 Synthesis of D-secooximes (5–8) and their derivatives (9–23, 25). Entry

Starting compound

Reagent

Time (h)

Product

Yield (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 7 6 8 5 7 5 7 5 6 7 8 6 8 11 23 23 23

NH2OHHCl/NaOAc NH2OHHCl/NaOAc NH2OHHCl/NaOAc NH2OHHCl/NaOAc Pd/C, 30 bar H2 Pd/C, 30 bar H2 Pd/C, 30 bar H2 Pd/C, 30 bar H2 O-Benzylhydroxylamine hydrochloride/NaOAc O-Benzylhydroxylamine hydrochloride/NaOAc O-Allylhydroxylamine hydrochloride/NaOAc O-Allylhydroxylamine hydrochloride/NaOAc Ac2O/py Ac2O/py Ac2O/py Ac2O/py Ac2O/DMAP silica gel, mw, 100 °C Ac2O/DMAP silica gel, mw, 100 °C Propargyl bromide/K2CO3 Benzyl azide/CuI/PPh3/DIPEA 4-(Prop-2-yl)benzyl azide/CuI/PPh3/DIPEA 4-Nitrobenzyl azide CuI/PPh3/DIPEA

1 1 1 1 2 2 2 2 6 6 6 6 1 1 1 1 6 min 6 min 2h 2h 2h 2h

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 25a 25b 25c

96 93 94 92 85 83 79 77 86 82 78 81 74 82 76 85 77 74 89 92 90 93

O N OH

17 13

H 3

RO

H

H

NH2OH.HCl/ NaOAc

16 16a

H

H 5, 6 13β-Me 7, 8 13α-Me

1, 2 13β-Me 3, 4 13α-Me H2, Pd/C

R = Me 1, 3, 5, 7 R = Bn 2, 4, 6, 8

H2, Pd/C

N OH

N OH

H H MeO

H H

9 13β-Me 10 13α-Me

H HO

H

11 13β-Me 12 13α-Me

Scheme 1. Oximation of D-secoaldehydes (1–4) and saturation of the alkenyl side-chain of the D-secooximes (5–8).

from the D-secoaldehydes (1–4) by using hydroxylamine hydrochloride and sodium acetate (Table 1, entries 1–4, Scheme 1). The first structural modification of the oximes (5–8) comprised the saturation of the propenyl side-chain and the simultaneous removal of the benzyl protecting group (Table 1, entries 5–8, Scheme 1). In the case of the 3-methyl ethers (5, 7), only the saturation of the alkenyl side-chain occurred (Table 1, entries 5 and 6). Compounds 5–12 and the starting D-secoaldehydes (1–4) were tested by means of MTT assays on a panel of human adherent cancer cell lines: HeLa, MCF-7, A2780 and A431 (Table 2). The oximes in the 13b-estrone series were active against all these cell lines, but the protecting group at C-3 moderately influenced the cytostatic behavior. Secooximes 5 and 6 displayed submicromolar IC50 values against A2780 cells. Compound 5 gave similar low IC50 values on all these cell lines, but compound 6, bearing a benzyl ether protecting group exhibited higher cancer selectivity. The epimeric derivatives of the secooximes (7, 8) had no impact on the proliferation of the tested cells. It can be concluded that the orientation of the angular methyl group greatly affects the antiproliferative properties of the D-secooximes (5–8). The secoaldehydes (1–4) appeared to be totally inactive. In order to examine the influence of saturation of the side-chain on the cytostatic behavior, 3-methyl ethers of secooximes with a propyl side-chain (9, 10) were additionally

tested. The antiproliferative effects of these latter compounds (9, 10) were similar to those of their unsaturated counterparts 5 and 7, and it may therefore be stated that the saturation of the alkenyl side-chain did not modulate the biological activity of these secoestrones. In the 13b-estrone series, removal of the benzyl ether protecting group resulted in compound 11, with a complete loss of the antiproliferative properties. In contrast, the presence of the phenolic OH group (in compound 12) in the 13a-estrone series, improved the growth-inhibitory effect, especially on HeLa cells. Etherifications of the oximes were carried out in order to examine whether the oxime OH function is necessary for the cytostatic potential. Epimeric oximes 5 and 7 bearing a 3-methyl ether group were chosen as model compounds, and were treated with O-benzyl- or O-allylhydroxylamine hydrochloride in the presence of sodium acetate (Table 1, entries 9–12, Scheme 2). Oxime ethers 13–16 were obtained in excellent yields and were subjected to MTT assays. None of these derivatives inhibited the proliferation of the tested cancer cells (Table 2). These results suggest that the free oxime OH group is essential for the antiproliferative effect. In view of our recently published results [10] that the oxime propionate in the 13a-estrone series results in cell cycle blockade and induces apoptosis, we now set out to synthetize oxime esters as well. Secooximes (5–8) were treated with acetic anhydride in

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E. Mernyák et al. / Steroids 89 (2014) 47–55 Table 2 Experimentally determined IC50 values of the synthesized D-secoestrone derivatives 1–22. Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 25a 25b 25c Cisplatin

N OH H H

Calculated IC50 valuesa (lM) HeLa

A2780

A431

MCF-7

>30 >30 >30 >30 1.2 7.1 >30 >30 1.7 >30 >30 12.1 >30 >30 >30 >30 0.8 13.3 >30 >30 >30 >30 1.7 12.7 5.3 5.7

>30 >30 >30 >30 0.9 1.4 >30 >30 0.7 >30 >30 21.1 >30 >30 >30 >30 0.4 3.8 >30 >30 >30 >30 1.9 10.1 7.7 8.8

>30 >30 >30 >30 0.8 0.9 >30 >30 0.9 >30 >30 28.4 >30 >30 >30 >30 0.9 4.7 >30 >30 >30 >30 1.5 8.1 4.4 0.9

>30 >30 >30 >30 2.6 >30 >30 >30 2.1 >30 >30 28.0 >30 >30 >30 >30 0.6 9.8 >30 >30 >30 >30 1.0 4.5 2.1 8.0

RO

H

5, 6 13β-Me 7, 8 13α-Me

Scheme 3. Synthesis of D-secooxime acetates (17–20) from the D-secooximes (5–8).

H H BnO

H

N OH Ac2O/DMAP silica gel, 100 oC, mw

6 13β-Me 8 13α-Me

Scheme 4. Synthesis of D-seconitriles (21, 22) from the microwave (mw) irradiation.

pyridine, leading to oxime acetates (17–20, Table 1, entries 13–16, Scheme 3). Compounds 17 and 18 greatly inhibited the proliferation of the tested cancer cells, while their epimeric counterparts 19 and 20 surprisingly appeared to be inactive (Table 2). Secooxime acetate 3-methyl ether (17) was the most potent, with submicromolar IC50 values (IC50 = 0.4–0.9 lM), and did not exhibit selectivity toward the different tumor cells. Cancer selectivity of this analog was additionally tested by determining its action on growth of skin fibroblast. Compound 17 exerted less than 30% inhibition of proliferation at 10 lM (data not presented) which indicates considerable selectivity towards cancer cells. Compound 18 was slightly less potent than its 3-methoxy derivative (17), but displayed greater selectivity: its activity against A2780 and on A431 was one order of magnitude better than that against HeLa and MCF-7 cells. It can be concluded that the determining structural moiety in the case of the oxime esters is the b-orientation of the angular methyl group. Additionally we set out to synthesize seconitriles from the oximes and to investigate the impact of this structural modification

BnONH2.HCl, NaOAc N OH

MeO

H 5 13β-Me 7 13α-Me

C N

H 21 13β-Me 22 13α-Me D-secooximes

(6, 8) by

on the biological activity. There are already literature examples of the synthesis and biological screening of secoestrone 16-nitriles with different functions at C-17 and protecting groups at C-3 [27,28]. It was reported that none of the seconitriles exerted estrogenic activity, but some of them expressed moderate antiestrogenic effects. This potential was regularly higher for the 3-benzyl ethers than for the seconitriles bearing a 3-OH group. In the light of these literature data, compounds 6 and 8 served as starting steroids for the formation of 17-nitriles. Water was eliminated from the oxime function of compounds 6 and 8 under microwave irradiation on silica gel as a solid support, with acetic anhydride and 4-(dimethylamino)pyridine as reagents (Table 1, entries 17 and 18, Scheme 4). The nitriles (21, 22) were formed in excellent yields and were subjected to MTT assays, which revealed the lack of inhibition of cell growth (Table 2). Based on our earlier promising results in the field of steroidal triazoles [13–17], finally, we planned to introduce a triazole moiety into the D-secooximes in the 13b-estrone series. Incorporation of an alkyne function into the steroid was achieved by the reaction of 11 with propargyl bromide and potassium carbonate (Table 1, entry 19, Scheme 5). The resulting secoestrone alkyne (23) was then reacted with benzyl azides (24a–c) under ‘click’ reaction conditions (CuI as a catalyst, triphenylphosphane as a stabilizing ligand, N,N-diisopropylethylamine as a base, toluene as a solvent, Table 1, entries 20–22, Scheme 5). All the reactions proceeded with full conversion of the starting compounds 23 and 24, no side products were formed. The triazoles (25) were subjected to MTT assays, which revealed that all the compounds (25a–c) greatly inhibited the cell proliferation with no selectivity toward the different tumor

N O CH2

H

H 17, 18 13β-Me 19, 20 13α-Me

R = Me 5, 7, 17, 19 R = Bn 6, 8, 18, 20

a Mean values from two independent determinations with 5 parallel wells; standard deviation <15%.

H

N OAc

Ac2O/py

AllylONH2.HCl, NaOAc

H 13 13β-Me 14 13α-Me

N O CH2CH CH2 H 15 13β-Me 16 13α-Me

Scheme 2. Synthesis of D-secooxime O-allyl or O-benzyl ethers (13–16) from the D-secooximes (5, 7).

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E. Mernyák et al. / Steroids 89 (2014) 47–55

N OH HC

C K2CO3

H H HO

CH2 Br

H C

11

HC

O 23

CH2 N3

R

24

N

C

N

O

CH

N H2C 1'

2'

6' 5'

3' 4'

R

25 24, 25 a b c

R H prop-2-yl NO2

Scheme 5. Synthesis of triazoles (25) from the 3-(prop-2-yniloxy)-D-secooxime (23) by azide-alkyne click reaction.

cells. The growth-inhibitory activity was influenced by the nature of the p-substituent of the N-benzyl ring. The unsubstituted compound (25a) was the most potent with IC50 = 1.0–1.9 lM values. It can be concluded that the difference in the antiproliferative potential of compounds 25 was the most outstanding on HeLa and A2780 cell lines. In order to acquire experimental data concerning the possible mechanism of the active substances, compound 6 (as one of the most potent and most cancer-selective) was chosen for additional cell cycle analyses. A2780 cells were exposed to 1 or 3 lM of the test compound for 24 or 48 h and the cell cycle distribution of the treated cells was determined by flow cytometry. No substantial

action was detected at 1 lM after 24 h of treatment, but 3 lM resulted in an increased population in the S phase at the expense of the cells in the G2/M phase (Fig. 1). Since DNA duplication is the most prominent feature of the S phase, it seems reasonable that the treated A2780 cells cannot achieve the synthesis of DNA, leading to a decreased G2/M population. Potent antiproliferative compounds selected from a set of estrone-16-oximes disturbed the DNA synthesis too [10]. After the longer incubation, the accumulation of hypodiploid (subG1) cells was observed, which is a generally acknowledged morphological marker of apoptosis. Our results therefore indicate that compound 6 induces a cell cycle disturbance in the S phase, which may initiate the apoptotic machinery. It can be concluded that the inversion at C-13 has a great impact on the cytostatic properties: the 13b derivatives proved to be the more potent in most cases. Oxime ethers or nitriles were less active than oxime esters. The functional group at C-3 also affected the antiproliferative behavior. 3-Methyl ethers displayed lower cancer selectivity, but a higher growth-inhibitory effect than those of their 3-benzyl ether counterparts. Besides its potency, compound 6 exhibited considerable tumor selectivity, which could be attributed to the cell cycle disturbance and apoptosis induction. The presented results may be regarded as evidence that these are novel D-secoestrone-based antiproliferative compounds suitable for further development.

Acknowledgments The authors thank the Hungarian Scientific Research Fund (OTKA K101659 and K109293) and the New Hungary Development Plan (TÁMOP-4.2.2.A-11/1/KONV-2012-0047) for financial support. This research was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP-4.2.4.A/2-11/1-2012-0001 ‘National Excellence Program’.

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Fig. 1. Effects of compound 6 on the A2780 cell cycle distribution after incubation for 24 h (upper panel) or 48 h (lower panel). ⁄, ⁄⁄ and ⁄⁄⁄ indicates p < 0.05, p < 0.01 and p < 0.001, respectively, as compared with the control cells.

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