Synthesis and Screening of Aromatase Inhibitory Activity of Substituted C19 Steroidal 17-Oxime Analogs

The synthesis and aromatase inhibitory activity of androst-4-en-, androst-5-en-, 1β,2β-epoxy- and/or androsta-4,6-dien-, 4β,5β-epoxyandrostane-, and 4-substituted androst-4-en-17-oxime derivatives are described. Inhibition activity of synthesized compounds was assessed using aromatase enzyme and [1β-3H]androstenedione as substrate. Most of the compounds displayed similar to or more aromatase inhibitory activity than formestane (74.2%). 4-Chloro-3β-hydroxy-4-androsten-17-one oxime (14, 93.8%) showed the highest activity, while 4-azido-3β-hydroxy-4-androsten-17-one oxime (17, 32.8%) showed the lowest inhibitory activity for aromatase.


Introduction
Breast cancer is the most common cause of death from cancer in women. Estrogens are involved in numerous physiological processes including the development and maintenance of the female sexual organs, the reproductive cycle, reproduction, and various neuroendocrine functions. On the other hand, estrogens enhance growth and proliferation of certain target cells, such as breast epithelial cells and estrogen-dependent mammary carcinoma cells [1]. Most cases (around 80%) of breast cancer occur in postmenopausal women, and the majority of the tumor is found to be hormone-dependent, where estrone (E1) and estradiol (E2) play an important role in the development and evolution of the OPEN ACCESS disease [2][3][4][5]. The conversion of E1 and E2 from androst-4-ene-3,17-dione (androstenedione, AD) and testosterone is catalyzed by aromatase. Compounds that inhibit enzyme aromatase have applications in the treatment of advanced estrogen-dependent breast cancer [6,7]. Over the past two decades, highly potent and specific aromatase inhibitors have been studied as a logical treatment strategy and some have already been approved for clinical use. These include two classes of compounds: steroids, exemestane (Aromasin®), formestane (Lentaron®) and nonsteroids, anastrozole (Arimidex®) and letrozole (Femara®) (Figure 1). Brodie and colleagues [8] showed that extra unsaturation in the A and/or B rings of 4-androstenedione leads to compounds that are effective inhibitors of aromatase. Numerous aromatase inhibitors, analogs of 4-androstenedione, have been described, including 4-hydroxy- [9], 4-amino- [10], 4-mercapto- [11], 4-(O-alkyl)-, 4-(O-aryl)-, 4-(alkyl)-and 4-(aryl) [12] derivatives, which have been evaluated clinically.
Hydroxyimino steroids represent a distinct class of antineoplastic agents [13,14] and varied placement of the hydroxyimino group on the parental steroid skeleton results in remarkable changes in the antineoplastic activity profile of the compounds [15,16].
Some steroidal 6-hydroxyimino-4-en-3-ones have shown a high affinity for human placental aromatase and function as competitive inhibitors of this enzyme [17]. 3-and 17-Hydroxyimino-2alkylaminoethyl steroid derivatives were synthesized and evaluated for antineoplastic activity and aromatase inhibitory activity [18].
In the present study, to explore the effect of extended linear conjugation in the rings A and/or B and the effect on the position of the epoxy ring and effect of substituent at 4-position of 17-hydroxyimino androstane skeleton structure on aromatase inhibition, we synthesized 17-hydroxyimino derivatives having additional double bonds at C-1-C-2, at C-4-C-5, at C-6-C-7, or both positions and 17-hydroxyimino derivatives of 1,2-or 4,5-epoxyandrostene and/or -diene and of 4-substituted 4-androstene. Synthesized compounds primarily evaluated their aromatase inhibitory activity by the radiometric method in vitro.
DHEA was reacted with hydroxylamine hydrochloride in the presence of pyridine to afford 3-hydroxy-5-androstene-17-oxime (6). The presence of one broad singlet oxime peak at δ 10.02 ppm in the 1 H-NMR spectrum and the disappearance of the 17-carbonyl carbon peak and the appearance of a new peak at δ 167.8 ppm (C=NOH) in the corresponding 13 C-NMR spectrum and the mass spectral peak at m/z 303 ([M] + ) confirmed the structure.
Compound 6 was subjected to Oppenauer oxidation in the presence of N-methylpiperidone and aluminium iso-propoxide to yield the 3-carbonyl compound, 4-androstene-3,17-dione-17-oxime (7). Oxidation of 7 with chloranil instead of DDQ afforded the diene analogue 4,6-androstadiene-3,17dione-17-oxime (8). The structure of 7 was identified by two peaks at δ 6.15 (H-7) and 6.14 (H-6) in the 1 H-NMR spectrum and new double bond carbon peaks at δ 139.6 ppm (C-7) and δ 128.7 ppm (C-6) in the 13 C-NMR spectrum. Further reduction of compound 7 with sodium borohydride in absolute ethanol afforded the 3α-hydroxy compound 9 whose structure was confirmed by 1D-NOESY as there was a correlation between 3β-H and 19β-CH 3 group. A new multiplet peak at δ 4.14 ppm (H-3) in the 1 H-NMR spectrum and the disappearance of the carbonyl peak from the 13 C-NMR and a new peak at δ 67.9 ppm (C-3) confirmed this structure.
Epoxidation of compound 9 with m-CPBA in chloroform afforded 4α,5α-epoxy compound 10, which was confirmed by a new epoxy peak at δ 3.21 ppm in the 1 H-NMR spectrum and disappearance of two double bond carbon peaks and two epoxy carbon peaks at δ 68.8 ppm (C-5) and δ 64.4 ppm (C-4) in the 13 C-NMR spectrum.
A correlation between 3β-H and 4β-H was observed in the 1 H-1 H COSY spectrum of 10 ( Figure 3). Furthermore, it was reported that the presence of the 3α-hydroxy group of the allylic alcohol directed the epoxidation exclusively to the α-face of the double bond of the compound [20,21]. The presence of one new singlet peak at δ 5.74 ppm (H-4) in the 1 H-NMR spectrum and the appearance of new 3-carbonyl peak (C-3) at δ 199.6 ppm in 13 C-NMR spectrum confirmed the structure. The syntheses of 4-substituted androst-4-en-17-oximes are shown in Scheme 2. Epoxidation of 7 was carried out with 30% hydrogen peroxide in the presence of base to afford 4β,5β-epoxyandrostane-3,17-dione-17-oxime (11). The presence of a new epoxy peak at δ 2.99 ppm (H-4) in the 1 H-NMR spectrum and the appearance of two new epoxy carbon peaks at δ 70.2 and 62.8 ppm in the 13 C-NMR spectrum confirmed the structure. The 4β,5β-epoxy stereochemistry was determined from the 1D-NOESY ( Figure 4) and 1 H-1 H COSY ( Figure 5) spectra. The β-configuration of the 4,5-epoxy ring was determined by irradiation of the H-4 proton, which did not result in a NOE at H-19 (δ 1.19 ppm) and by the fact that no correlation between any proton and 4α-H was observed in the 1 H-1 H COSY spectrum of 11.
Reduction of compound 11 with sodium borohydride in absolute ethanol afforded 3β-hydroxy compound 12, as confirmed by 1D-NOESY, the presence of a new peak at δ 3.69 ppm (H-3) in the 1 H-NMR spectrum and a new peak at δ 65.7 ppm (C-3) and absence of C-3 carbonyl peak in the 13 C-NMR spectrum.   Chlorination of 11 to cleave the epoxy group under acidic conditions (concentrated HCl in acetone) afforded 4-chloro-4-androstene-3,17-dione-17-oxime (13). Disappearance of the epoxy peak from the 1 H-NMR spectrum and appearance of two new quaternary carbon peaks of the C-4 and C-5 carbons at δ 164.2 and 127.2 ppm in the 13 C-NMR spectrum verified the structure. Reduction of compound 13 with sodium borohydride in absolute ethanol afforded 4-chloro-3β-hydroxy-4-androsten-17-one oxime (14), which was confirmed by 1D-NOESY. The β-configuration of the OH group at the 4 position was determined by irradiation of the H-3 proton which did not show a NOE at H-19. A new multiplet peak at δ 4.14 ppm (H-3) in the 1 H-NMR spectrum and disappearance of the carbonyl peak from the 13 C-NMR spectrum and a new peak for C-3 at δ 69.7 ppm confirmed the proposed structure of compound 14. Reaction of 11 with sodium cyanide in the presence of ammonium chloride in ethanol yielded the compound 3,4-dicyano-3-hydroxy-4-androsten-17-one oxime (15). Disappearance of the epoxy peak from the 1 H-NMR spectrum and the C-3 carbonyl peak and new peaks at δ 171.1 ppm (C-5), δ 122.2 ppm (C-3 CN and C-4 CN) and δ 116.3 ppm (C-4) in the 13 C-NMR spectrum and the peak at m/z 326 [M-HCN] + in its mass spectrum confirmed the structure. Reaction of compound 11 with sodium azide in ethanol and in the presence of ammonium chloride afforded 4-azido-4androstene-3,17-dione-17-oxime (16). Disappearance of the epoxy peak from the 1 H-NMR spectrum, two new peaks at δ 154.5 ppm (C-4) and δ 128.9 ppm (C-5) in the 13 C-NMR spectrum confirmed this structure. Further reduction of compound 16 with sodium borohydride in absolute ethanol afforded the 3β-hydroxy compound 17, which was confirmed by 1D-NOESY. A new multiplet peak at δ 4.13 ppm in the 1 H-NMR spectrum and disappearance of the carbonyl peak from the 13 C-NMR spectrum and a new peak at δ 67.5 ppm of C-3 and the MS peak at m/z 316 [M-N 2 ] + confirmed the structure.

General
All non-aqueous reactions were performed under a dry atmosphere of nitrogen. The commercial reagents were purchased from Aldrich, Fluka, or Sigma Chemical Company. Solvents were purified and dried prior to use. Melting points were measured on Thomas-Hoover melting point apparatus and not corrected. 1 H-, 13 C-NMR, HSQC, HMQC and NOESY spectra were taken on Varian 400 MHz spectrometer in CDCl 3 , and DMSO-d 6 . Chemical shifts (δ) are in parts per million (ppm) relative to tetramethylsilane, and coupling constants (J) are in Hertz. IR spectra were determined on a FT-IR JASCO 4100 spectrometer. GC/MS spectra were obtained on a Shimadzu QP 5050 and JEOL GC Mate 2 mass spectrometers. Elemental analysis was performed on a Yanaco CHN Corder MF-3 automatic elemental analyser. Analytical TLC was performed on pre-coated silica gel 60 F 254 plates (Merck). Solvent systems for TLC were ethyl acetate/n-hexane mixtures and 10% MeOH in dichloromethane. Column chromatography was carried out on Merck silica gel 9385 (230-400 mesh) eluting with ethyl acetate/n-hexane mixtures.

1,4,6-Androstatriene-3,17-dione 17-oxime (2)
To a solution of 1,4,6-androstatriene-3,17-dione [28] (1, 1 g, 3.54 mmol) in ethanol (10 mL) hydroxylamine hydrochloride (386 mg, 5.6 mmol) and pyridine (0.5 mL) were added and the mixture was allowed to reflux for 1 h. After cooling to room temperature, the ethanol was evaporated and water was added and the mixture extracted with ethyl acetate (3 × 15 mL). The organic solution was dried with anhydrous MgSO 4 , filtered and concentrated to yield a crude oily product which was further purified by silica gel column chromatography (ethyl acetate-n-hexane = 1:2) to afford compound 2 (716 mg, 70%,) as a whitish solid.  2. 1α,2α-Epoxy-4,6-androstadiene-3,17-dione 17-oxime (3) To a solution of 2 (761 mg, 2.5 mmol) in methanol (40 mL) was added 5% NaOH (1.5 mL) and 30% H 2 O 2 (8.5 mL) and the mixture was allowed to stir at room temperature for 5 h. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to remove the methanol and hydrogen peroxide, water was added to the residue and the mixture was extracted with dichloromethane (3× 20 mL). The organic solvent was dried with anhydrous MgSO 4 , filtered and concentrated to yield a crude oily product which was purified by silica gel column chromatography

2-Chloro-1,4,6-androstatriene-3,17-dione 17-oxime (4)
To a ice cooled solution of 4 (100 mg, 0.31 mmol) in acetone (4 mL) in an ice bath was added concentrated HCl (1 mL) and the mixture was allowed to stir for 12 h. Then it was neutralized with aqueous 10% NaOH. The solid that appeared was filtered and dried to give a yellowish-white solid which was further purified by recrystallization with methanol to afford compound 4 (50 mg, 32%).

1α,2α-Epoxy-3β-hydroxy-2,4-androstadien-17-one Oxime (5)
To a solution of 4 (200 mg, 0.63 mmol) in absolute ethanol (10 mL) was added sodium borohydride (48 mg, 1.27 mmol) and the mixture was allowed to stir at room temperature for 2 h. Then ethanol was removed by evaporation followed by addition of water and extraction with ethyl acetate (3 × 10 mL). The combined organic layers were dried with anhydrous MgSO 4 , and concentrated to give a crude oily product which was further purified by silica gel column chromatography (ethyl acetate-n-hexane = 1:1) to afford compound 5 (49%, 100 mg) (6) To a solution of DHEA (3.0 g, 10.41 mmol) in ethanol (50 mL) was added hydroxylamine hydrochloride (792 mg, 25.0 mmol) and pyridine (1.6 mL) and the mixture allowed to reflux for 3 h. Then, after cooling to room temperature, the ethanol was evaporated and the white precipitate formed upon addition of water was filtered to afford a crude precipitate which was recrystallized from methanol and H 2 O to give 6 (3 g, 95%) as a pure white product .  (7) To a solution of 6 (3 g, 9.9 mmol) in toluene (250 mL) was added N-methylpiperidone (14 mL, mmol) and aluminiun isopropoxide (1.10 g, 5.4 mmol) and the mixture was was allowed to reflux for 5 h under nitrogen. After cooling the toluene was evaporated and water (80 mL) and ethyl acetate (50 mL) were added and left to stir at room temperature overnight and the aqueous layer was then extracted with additional ethyl acetate (3 × 50 mL). The organic layer was further washed with 5% HCl followed by aqueous 10% NaOH and water and finally dried with anhydrous MgSO 4 and concentrated in vacuo to afford the desired product 7 (2.97 g, 93%) as a white solid. To a solution of 7 (100 mg, 0.33 mmol) in n-butyl alcohol was added chloranil (86 mg, 0.35 mmol) and the mixture allowed to reflux for 5 h. Then it was allowed to cool and concentrated to remove the n-butyl alcohol, water was added and the mixture extracted with ethyl acetate (3 × 10 mL). The organic solvent was dried with anhydrous MgSO 4 , filtered and concentrated to yield a crude oily product which was further purified by silica gel column chromatography (ethyl acetate-n-hexane = 1:3) to afford the desired product 8 ( (9) To a solution of 7 (1 g, 3.3 mmol) in absolute ethanol (50 mL) was added sodium borohydride (250 mg, 6.6 mmol) and the mixture was allowed to stir at room temperature for 3 h. Absolute ethanol was removed by evaporation followed by addition of water afterwards to obtain a white solid which was filtered to afford compound 9 (800 mg, 78%,) as a white solid. 3.2.9. 4α,5α-Epoxy-3α-hydroxyandrostan-17-one Oxime (10) To a solution of 9 (750 mg, 2.47 mmol) in chloroform was added m-CPBA (673 mg, 3.9 mmol) and the mixture was allowed to stir at room temperature for 3 h. Water was added water and the mixture was extracted with dichloromethane (3 × 50 mL). The organic solvent was dried with anhydrous MgSO 4 , filtered and concentrated to give a crude pale yellow precipitate which was further purified by silica gel column chromatography (ethyl acetate-n-hexane = 1:1) to afford compound 10 (300 mg, 38%) as a white solid.   (11) To a solution of 7 (1 g, 3.31 mmol) in methanol was added 5% NaOH in methanol (2.24 mL) and 30% H 2 O 2 (12 mL) and the mixture was allowed to stir at room temperature for 2 h. Then it was concentrated to remove the methanol and 30% H 2 O 2 and further purified by silica gel column chromatography (ethyl acetate-n-hexane = 1:3) to afford the desired product 11 (500 mg, 45%) as a white solid.   (12) To a solution of 11 (266 mg, 0.83 mmol) in absolute ethanol (7 mL) was added sodium borohydride (63 mg, 1.66 mmol) and the mixture stirred at room temperature for 2 h. Then ethanol was removed and water was added and the reaction mixture was extracted with dichloromethane (3 × 10 mL) to afford the crude compound which was purified by column chromatography (ethyl acetate-n-hexane = 1:1) to afford 12 (222 mg, 83%) as a white solid.  (13) To a solution of 11 (100 mg, 0.31 mmol) in acetone (2 mL) in an ice bath was added conc. HCl (0.46 mL) and the mixture was allowed to stir for 4 h. Then it was neutralized with aq. 10% NaOH. A solid appeared that was filtered off and further purified by silica gel column chromatography (ethyl acetate-n-hexane = 1:3) to afford the desired product 13 (80 mg, 74%) as a white solid.  (14) To a solution of 13 (50 mg, 0.14 mmol) in absolute ethanol (5 mL) was added NaBH 4 (10.5 mg, 0.28 mmol) and the mixture was stirred at room temperature for 1 h. After completion of the reaction the absolute ethanol was removed in vacuo and the product further purified by silica gel column chromatography (ethyl acetate-n-hexane = 1: 3) to afford compound 14 (20 mg, 35%) as a white solid.  (15) To a solution of 11 (100 mg, 0.52 mmol) in ethanol-water (8:1, 9 mL) was added sodium cyanide (120 mg, 2.45 mmol) and ammonium chloride (75 mg, 1.4 mmol) and the mixture was allowed to reflux for 12 h. Then it was cooled down and neutralized with 10% HCl, followed by removal of ethanol and the mixture was further extracted with ethyl acetate (3 × 15 mL) and dried with anhydrous MgSO 4 to afford the the desired product 15 (80 mg, 64%) as a creamy white solid. Mp 232-233 °C;