J. Cent. South Univ. Technol. (2007)04-0524-04

DOI: 10.1007/s11771-007-0102-4

Synthesis and characterization of mibolerone

YANG Qing(杨  青), FAN Bo-lin(范柏林), TANG Rui-ren(唐瑞仁)

(School of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China)

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Abstract：A simple and effective route for the synthesis of mibolerone was described starting from the estr-5(10)-en-3,17-dione in four steps with the overall yield of 47.0 %. Thus, two methods for key intermediate methylnorandrost were investigated: one(method A) starting from estr-4-en-3,17-dione underwent 3-keto group protected with ethyl orthoformate to give 3-ethoxy-3,5-dien-estr-17-one, the other(method B) from estr-5(10)-en-3,17-dione and protected 3-keto group to give 3,3-dimethoxy-estr-5(10)-7-one in a mild acidic condition. Then, two intermediates were subsequently reacted with methyllithium followed by a mild hydrolytic procedure and gave methylnorandrost with total yield 25.0% and 86.0 %, respectively. In the preparation of 6-dehydrogenation product of methylnorandrost, two procedures(method C and method D) were investigated: one was the protected 17α-methyl-17β-hydroxy Δ^3,5-enol ethers estrendiene brominated and the resulting 6-bromo-19-methylnortestosterone was then immediately dehydrohaloenated to give 6-dehydro-19-methylnortestosterone, the total yield only reaches 36.0%; the other was directly dehydrogenated with chloranil and the yield reaches 75.6% under the optimum conditions: in refluxing tetrahydrofuran, the molar ratio of methylnorandrost to chloranil is 0.66 and reaction time of 5 h. The titled compound and intermediates were characterized by ¹H and ¹³C NMR, IRMS and elemental analysis.

Key words: mibolerone; methylnorandrost; steroids; synthesis; hepatic androgen receptor

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1 Introduction

The steroids belong to a group of substances of both plant and animal origin possessing a characteristic tetracylic backone, and is of very considerable medicinal importance. Some of them are reported to have anti-human immunodeficiency virus (anti-HIV) and anticancer activity^[1-2]. Related to these compounds are wide variety of analogues that have been introduced into therapy to correct hormone deficiencies as anabolic agents to alleviate rheumatoid in various conditions. In recent years, considerable interest has been focused on the total synthesis and partial synthesis of steroids^[3-7].

Mibolerone(7α,17α-dimethyl-17β-hydroxy-4-estren-3-one) is not only a key intermediate for modifying steroidal compounds, but also a high efficient and low side effect assimilation hormone. Mibolerone has been used widely as a ligand for the study of androgen receptor (AR) because of its high-affinity binding and its greater stability compared to methyltrienolone^[8]. When assayed orally in the rats, mibolerone was found to be approximately 18 times than methyltestosterone as an androgen agent^[8-10].

Although mibolerone is a known product of hepatic androgen receptor, there are no detailed published methods for its chemical synthesis and its spectral data. In this study, a new efficient and simple route for the synthesis of mibolerone starting from commercially available estr-5(10)-en-3,17-dione or estr-4-en-3,17-dione was reported.

2 Experimental

2.1 Reagents and instruments

Unless otherwise stated, all reagents were commercial analytical or chemical pure grades and were not additionally purified. Analytical thin-layer chromatography (TLC) was performed using 5 cm×  10 cm precoated silica gel F-254 from Merck with detection under ultraviolet (UV). Uncorrected melting points were determined on a XRC-1 apparatus. Infrared (IR) spectra were recorded on a Perkin-Elmer1420 spectrophotometer with KBr plates. All nuclear magnetic resonance (NMR) spectra were recorded on a INOVA-400MHz apparatus in CDCl₃ solution. Mass spectra (MS) were performed on an HP6890-HP5973 gas chromatography-mass spectra (GC-MS) united apparatus.

2.2 Synthetic route

The two synthetic routes of mibolerone are shown in Fig.1.

Fig.1 Synthetic route for mibolerone

Reagents: (a) Ethyl orthoformate/PTS; (b) CH₃OH/ CH₂(COOH)₂; (c)  CH₃Li/THF and then H₃O⁺; (d) Chloranil/THF; (e) (CH₃)₂CuLi/THF

2.3 Experimental procedures

2.3.1 Preparation of 3-ethoxy-3,5-dien-estr-17-one

To a round bottom flask with a thermometer and magnetic stirring bar was placed 25.00 g (91.9 mmol) estr-4-en-3,17-dione(compound 1) in 125 mL dry thiophene-free benzene, 12.5 mL of anhydrous ethanol, 15 mL of ethyl orthoformate and 12 mL of new prepared solution of p-toluene sulfonic acid (PTS) (250 mg PTS dissolving in 5 mL of anhydrous ethanol and 45 mL of dry benzene ), and the mixture was refluxed for 3 h, then, additional 10 mL of ethyl orthoformate was added and refluxed for another 2 h. After the reaction was completed as shown by TLC, the mixture was cooled to room temperature, and evaporated under vacuum to afford a yellow oil residue. The residue was dissolved in 30 mL of absolute ethanol and left in the refrigeratory overnight. The solid was crystallized and filtered to give 17.55 g crude product (yield: 64.00%, melting point(MP):  135-137 ℃) that was recrystallized from ether-hexane affording 7.00 g white crystal (compound 2) with MP of 141-143 ℃^[11]. The functional groups were identified by IR (cm^-1):1 656/1 630  (C==C), 1733 (C==O), 1177(C—O—C).

2.3.2 Preparation of 3,3-dimethoxy-estr-5(10)-en-17-one

20.00 g (73.5 mmol) of estr-5(10)-en-3,17-dione (compound 3) and 10.00 g (84.7 mmol) of methane dicarboxylic acid were dissolved in 300 mL of anhydrous methanol in a three-neck flask. The mixture was stirred at the room temperature for 5 h then cooled to 0 ℃ and treated with a cooled solution of sodium bicarbonate stirring for 0.5 h, and then the mixture was filtered to provide  20.20 g white solid of 3,3-dimethoxy-estr- 5(10)-en-17-one ( compound 4) with a 99.50% yield( MP: 115-116 ℃^[12]).  The functional groups were identified by IR (cm^-1): 1 739 (C==O), 2 959/2 831 (CH₃), 1 055   (C—O—C), 1 660 (C==C).

2.3.3 Preparation of 17β-hydroxy-17α-methyl-4- en-estr- 3-one(methylnorandrost)

1）Method A

A solution of 5.00 g (16.7 mmol) of 3-ethoxy-3,5-dien-estr-17-one (compound 2) in 20 mL anhydrous tetrahydrofuran (THF) was added to a freshly prepared solution of 40 mL (4.20 mol/L) of methyllithium in a round bottom flask. After refluxing for 3 h, the reaction mixture was poured into ice-water and acidified with 18% hydrochloric acid to make pH= 2-3. The 3-ethoxyl group was removed by allowing the acidic mixture to standing at room temperature for 1 h, whereupon the product was extracted with ether, dried and evaporated, giving 2.20 g product (compound 5) (yield: 40.30%, MP: 156-158 ℃). IR absorption of functional groups (cm^-1): 3 421 (—OH), 2 960/2 935   (—CH₃), 1 666 (C==O), 1 623 (C==C); MS: m/z=288 (M⁺, 100%), 270(M-H₂O, 25%).

2) Method B

A mixture of 5.00 g (15.7 mmol) of 3.3-dimethoxy-5(10)-en-estr-17-one (compound 4) in 20 mL anhydrous THF and a solution of 40 mL (4.2 mol/L) of methyllithium was refluxed for 4 h under stirring in nitrogen atmosphere. The following work-up procedure was used as method A.  Finally, 4.4 g of white solid methylnorandrost (compound 5) was obtained (yield: 86.7%, MP: 158-160 ℃).

2.3.4 Preparation of 17β-hydroxy-17α-methyl-4, 6-dien- estr-3-one

A solution of 5.00 g (17.4 mmol) of methylnorandrost (compound 6) and 6.40 g (26 mmol) of chloranil (QCl₄) in 80 mL THF was heated until the solid dissolved. Then, 1 mL 36% hydrochloric acid was added dropwise as catalyst. After the mixture was refluxed for 5 h, the reaction solution was concentrated under vacuum, affording a yellow solid. The resulting solid was washed with dilute sodium hydroxide and saturated sodium chloride for three times respectively. After removing the solvent by evaporation, the residue was recrystallized from acetone to give 4.28 g 17β-hydroxy-17α- methyl-4,6-dien-estr-3-one(compound 6) (yield: 75.6%; MP: 163-165 ℃);  IR (cm^-1): 3 425 (—OH), 1 713   (C==O), 1 642/1 658 (C==C).

2.3.5 Preparation of 7α,17α-dimethyl-4-en-estr-3-one (Mibolerone)

Under nitrogen atmosphere, to a THF solution of 30.0 mL freshly prepared 2.2 mol/L ethereal methyllithium which cooled in an ice-bath, was added 4.00 g (21 mmol) of cuprous iodide with stirring, followed by 3.00 g  (10.5 mmol) 6-dehydro product (compound 6). The reaction mixture was stirred at 25 ℃ for 4 h after the reaction completed as shown by TLC. After removing the cooled bath, the mixture was stirred for 30 min and poured into a cooled solution of saturated ammonium chloride, then, benzene was added under stirring. The reaction mixture was filtered through diatomite. The obtained solid was washed twice with benzene. The residue was isolated from the water phase, and the organic layer was washed consequently with saturated ammonium chloride and saturated sodium chloride to neutrality. The organic layer was dried with anhydrous sodium sulfate, evaporated under vacuum to give the oil residue. The resulting residue was purified by flash chromatography to afford 2.20 g titled compound 7α,17α-dimethyl-4-en-estr-3-one (compound 7)  with yield of 72.5% and MP: 193-196 ℃. The IR absorptions of the functional groups are(cm^-1) : 3 421   (—OH), 1 715 (C==O), 1 642 (C==C);  ¹³C-NMR/δ: 199.6(C-3), 123.8(C-4), 171.3(C-5), 81.5 (C-17); 55.7(C-13), 50.1(C-14), 45.3, 38.8, 36.6, 36.4, 35.7, 33.9, 32.8, 31.6, 31.4(C-2, C-6, C-7, C-8, C-9, C-10, C-11, C-12, C-16), 25.8(17′-αCH₃), 23.2(C-15), 20.6(C-1), 17.4(7′-αCH₃), 13.8(C-18);  ¹H-NMR: δ7.272 (C₄—H, 1H), 2.481/2.468 (C₆—H, 2H), 2.438/2.433/2.426   (C₁₀—H, 1H), 1.206/1.218 (C₇—H,3H), 5.734 (O—H,1H), 0.909 ( C_17/18—H, 6H). MS: m/z=302 (M, 100%), 284 (M—H₂O, 24%), 269 (M—H₂O—CH₃, 269, 36%). The elemental analysis results are shown in Table 1 and the mass spectrum in Fig.2.

Table 1 Elemental analysis data of (compound 7)    (mass fraction, %)

Fig.2 Mass spectrum of Mibolerone

3.1 Protection of 3-carbonyl and preparation of methylnorandrost

The search for suitable protecting group of carbonyl is of considerable importance in the steroids^[13-14]. In this work, two pathways (method A and method B in Fig.1) for methylnorandrost starting from estr-4-en-3,17-dione (compound 1) and estr-5(10)-en-3,17-dione(compound 3) were respectively investigated. The difference lies in the protection of the carbonyl group. In method A estr-4-en-3, 17-dione was treated with ethyl orthoformate to selectively protect 3-keto group, but the yield is only about 42%. In method B, however, methanol was employed to protect 3-keto group in estr-5 (10)-en-3, 17-dione. The result shows that the reaction is selective and quantitative with the yield of almost 100％. On the other hand, methanol is abundant and available in the industry and reaction happens at room temperature easily.

Protecting groups are generally formed by nucleophilic attack on the carbonyl group and the rate of this process is determined by steric and electronic factors associated with the ketone. In steroid ketones the steric effects are usually more important due to the rigid tetracyclic skeleton. Thus, the protection of 3-carbonyl proceeds selectively with good yield in the presence of 17-ketone. In estr-4-en-3, 17-dione(compound 1)  carbonyl group conjugates with the carbon-carbon double bond and diminishes the reaction propensity, and thus lowers the yield in method A. The result shows estr-5(10)-en-3,17-dione (compound 2) is the better starting material for methylnorandrost.

In the preparation of methylnorandrost(compound 2), the effects of various solvents were studied, such as anhydrous ether, THF and mixture of the benzene-anhydrous ether. The results show that THF is the best choice as a solvent for the nucleophilic addition of Grignard reagent on the 17-keto group with the yield improved from 40.3% (with anhydrous ether) to 86.7% in this reaction.

3.2 Introduction of double bonds in 6-position of methylnorandrost

The selection of a method for the introduction of unsaturation into the steroid molecule is highly dependent upon the presence of various functional groups in the molecule^[15-16].  Regarding the preparation of 6-dehydrogenation product (compound 6), two pathways were tried in this work as shown in Fig.3. The first route (f) employs 2,3-dichloro-5,6-dicyanoquinone (DDQ) or chloranil (QCl₄) to dehydrogenate the 7′-H to introduce the double bond in the 6-position under the catalysis of hydrogen chloride(method C).

Fig.3 Two methods (C: f and D: g→h→i) for 6-dehydrogenation of methylnorandrost

The other route is halogenation- dehydrohalogenation of methylnorandrost (g→h→i, method D) that is widely applied method for the preparation of unsaturated ketones, but needs 3 steps reaction. The overall yield is 44.30% in the present case.

The chloranil dehydrogenation of methylnorandrost (compound 5) offers a more convenient way with yield up to 75.6% to Δ^4,6-ketone (compound 6) than DDQ in which compound 5 is found to be converted into two by-product Δ^1,4,6 and Δ^1,4-ketone. In addition, different solvents are investigated in chloranil dehydrogenation reaction, for instance, butyl alcohol, ethyl acetate, tetrahydrofuran (THF), the reaction yield of giving 24.0%, 43.6%, 75.6%, respectively(Table 2). Clearly, THF is the best solvent for this reaction. The desired product of 4,6-dien-19-methylnortestosterone is obtained together with a little 1,4-dien-19-methylnortestosterone, which is easily separated from the former through silica gel column, what’s more, there is no product of 1,4,6-trien-methylnortestosterone in this condition.

Table 2 Yield of dehydrogenation of compound 5

The method to introduce the double bond of methylandrost in 6-position by bromogenation- dehydrohalogenation (with N-bromosuccinimide) was also investigated. It is found that the yield of the protection of 3-keto group is very low, only about 44%(Table 2). Several by-products are detected as shown by TLC in the brominating reaction, and furthermore, the work-up procedures are difficult.

The reaction condition was optimized and steps were decreased in this route, giving a high overall yield of 53.8%. The procedure is expected to be industrialized.

1) Mibolerone was synthesized in a simple pathway from estr-5 (10)-en-3,17-dione (compound 3) in 4-steps reactions with overall yield of 47.0%.

2) The intermediates and the title compound were fully characterized by NMR, IR and MS. 

3) The conditions for the protection of 3-carbonyl and preparation of methylnorandrost are optimized: the protection of 3-carbonyl with methanol starting from estr-5(10)-en-3,17-dione (compound 3) and then undergoing an addition reaction with Grignard reagent to give 86.7% methylnorandrost(compound 5).

4) The chloranil dehydrogenation of methyl- norandrost (compound 5) offers a more convenient way for introducing a double bond in methylnorandrost with yield of 75.6% in THF.

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Foundation item: Project(50573019) support by the National Natural Science Foundation of China

Received date: 2006-11-28; Accepted date: 2007-03-16

Corresponding author: TANG Rui-ren, Professor, PhD; Tel: +86-731-8836961; E-mail: trr@mail.csu.edu.cn

(Edited by CHEN Wei-ping)