Synthesis of brassinosteroids with a keto group in the side chain

Steroids 101 (2015) 90–95

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Synthesis of brassinosteroids with a keto group in the side chain Aliona G. Baradzenka, Barys M. Barysau, Alaksiej L. Hurski, Vladimir N. Zhabinskii ⇑, Vladimir A. Khripach Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevich Str., 5/2, 220141 Minsk, Belarus

a r t i c l e

i n f o

Article history: Received 4 February 2015 Received in revised form 1 April 2015 Accepted 5 June 2015 Available online 13 June 2015 Keywords: Brassinosteroids 24-Epibrassinolide Boric acid ester 24-Epicryptolide 22-Dehydro-24-epibrassinolide

a b s t r a c t The aim of this work was to prepare 24-epicryptolide and 22-dehydro-24-epibrassinolide as possible metabolites of 24-epibrassinolide. The main synthetic problem to be solved was the differentiation of functional groups in brassinosteroids. Distinguishing 2a,3a-diol function from another diol group in 24-epibrassinolide was achieved via selective hydrolysis of 2a,3a-cyclic carbonate or via regioselective reaction of boric acid with the functional groups in the side chain. The hydroxyl at C-23 was more reactive than the 22-OH in the oxidation with bromine in the presence of bis(tributyltin) oxide and in the benzylation reaction that resulted in the predominant formation of the corresponding a-hydroxy ketone derivatives with the ratio ranging from 4:1 to 1.5:1. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction The metabolic transformations of brassinosteroids (BS) proceed via various pathways, including dehydrogenation of one of the hydroxyl groups [1]. The evidence for the presence of this deactivation process for BS in plants is the identification of cryptolide 3 [2,3] in Japanese cedar pollen and anthers and of 23-dehydro-2epicastasterone 4 from immature seeds of Phaseolus vulgaris [4] as metabolites of brassinolide 1 and castasterone 2, respectively (Scheme 1). However, this process is still poorly studied, especially with respect to metabolic transformations of the side chain. Similar 22-dehydrogenation has not been described at all so far for BS, although steroidal 23-hydroxy-22-ketones are known as natural compounds [5–7]. In spite of various approaches are available for the synthesis of these compounds, none of them can be considered as practical or fully reliable. Thus, 23-dehydro-24-epibrassinolide was isolated as an impurity (0.11%) from commercially available 24-epibrassinolide [8]. An efficient method was proposed to build cryptolide side chain [9]. It is expedient for the preparation of steroids with a campestane carbon skeleton, but too cumbersome for making ergostane derivatives. Perhaps the easiest way to solve the problem is to use 22R,23R-diols as starting compounds. Such an approach implies selective protection of one of the two hydroxyls

Abbreviations: BS, brassinosteroids; TBAB, tetrabutylammonium bromide; DIPEA, diisopropylethylamine; DMP, Dess–Martin periodinane. ⇑ Corresponding author. Tel./fax: +375 172 678 647. E-mail address: [email protected] (V.N. Zhabinskii). http://dx.doi.org/10.1016/j.steroids.2015.06.004 0039-128X/Ó 2015 Elsevier Inc. All rights reserved.

followed by oxidation of the remaining free hydroxyl group. This strategy was applied for the preparation of cryptolide [3] and 22- and 23-oxo derivatives of 28-homocastasterone [10]. In both cases, partial acetylation of 22R,23R-diols was used to distinguish the hydroxyl groups. Apart from the lack of regioselectivity at the acetylation step, possible epimerization in the course of deacetylation of intermediate acetoxyketones is another disadvantage of this approach. The aim of the present work was therefore to develop a simple and reliable methodology for the preparation of steroids with an aketol in the side chain. 2. Experimental 2.1. General Melting points were recorded on a Boetius micro-melting point apparatus and are uncorrected. 1H and 13C NMR spectra were obtained using a Bruker AVANCE 500 (Bruker Biospin, Rheinstetten, Germany) spectrometer operating at 500 MHz for 1 H and 125 MHz for 13C. Chemical shift values are given in d (ppm) relative to the residual solvent peaks: dH 7.58 and dC 135.91 for C5D5N; dH 7.26 and dC 77.00 for CDCl3, and coupling constants are reported in Hz. Mass spectra were performed on an LCQ Fleet mass spectrometer (Thermo Electron Corporation, USA) with an APCI source. HRMS/MS-spectra were acquired in positive electrospray ionisation mode with an Agilent 6550 iFunnel QTOF (Agilent Technologies, USA). Chemicals were purchased from Aldrich and Fluka and used as received. 24-Epibrassinolide (1)

A.G. Baradzenka et al. / Steroids 101 (2015) 90–95

91

Scheme 1. 23-Dehydrogenation of brassinolide 1 and castasterone 2.

was prepared according to the procedure described in [11]. All solvents were purified according to standard methods [12]. Reactions were monitored by TLC using aluminium sheets, silica gel 60 F254 precoated (Merck Art. 5715). Column chromatography was carried out on Kieselgel 60 (Merck Art. 7734) (see Table 1). 2.2. Synthesis of the compounds 2.2.1. (22R,23R,24R)-2a,3a:22,23-Bis[carbonylbis(oxy)]-B-homo-7oxa-24-methyl-5a-cholestan-6-one (6) A solution of triphosgene (124 mg, 0.42 mmol) in CH2Cl2 (1.7 mL) was added to a stirred solution of epibrassinolide (5) Table 1 H and 13C NMR spectroscopic data for compounds 17 and 18a,b.

1

Position

17

18

d, C

d, H (a/b)

d, C

d, H (a/b)

1

33.5

33.5

2 3 4 5 6 7

73.1 72.4 27.6 40.2 176.6 71.1

2.32 dd (15.7, 3.2)/1.11 4.35 4.37 1.78 3.28

2.31 dd (15.6, 3.6)/1.11 4.35 4.37 1.80 3.28 dd (10.5, 4.1)

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 2,3-MeC<

39.3 54.6 35.9 22.9 39.7 43.2 51.7 24.8 28.6 52.9 11.7 19.6 41.4 13.6 81.2 75.0 43.2 26.6 21.7 16.1 10.2 107.6

2,3-MeC<

23.6

1.32 s

23.6

1.31 s

2,3-MeC< 1°

26.6

1.54 s

26.5

1.51 s

74.3

4.64 s

73.3

4.50 d (11.1)/4.69 d (11.1)

2° 3°/7° 4°/6° 5°

138.4 128.4 127.5 127.7

7.31 7.31 7.31

138.3 128.4 127.7 127.7

4.08

1.80 1.73 1.33c/1.83c 1.28/2.01 1.15 1.21c/1.59c 1.35c/1.90c 0.71 0.88 1.54 1.02 3.53 3.26 1.42 1.92 0.90 0.83 0.74

s s d (5.9) d (2.8)

d (7.0) d (6.8) d (6.9)

73.0 72.4 27.7 40.2 176.6 71.2

39.4 54.6 35.9 22.9 39.7 42.9 51.7 24.5 27.5 52.4 12.0 19.6 40.8 12.3 71.5 83.7 39.9 28.3 22.2 19.6 12.1 107.6

4.07 dd (12.7, 9.6)/4.11 dd (12.9, 2.6) 1.80 1.74 1.33c/1.82c 1.32/1.97 1.21 1.21c/1.61c 1.30c/2.00c 0.70 0.87 1.60 0.94 3.69 3.37 1.40 1.68 0.93 0.94 0.97

s s d (6.7) t (4.0) dd (5.5, 3.4)

d (6.5) d (6.2) d (7.0)

7.31 7.31 7.31

a NMR chemical shifts (d) and coupling constants (Hz) are from spectra obtained in CHCl3 solution. b Assigned by DEPT, COSY, HSQC, and HMBC experiments. c May be reversed.

(200 mg, 0.42 mmol) and pyridine (0.81 mL, 10 mmol) in CH2Cl2 (1.3 mL) at 80 °C. The cooling bath was removed, and the mixture was stirred at room temperature for 12 h. The excess of triphosgene was decomposed with a few drops of saturated NH4Cl, and the water phase was extracted with CH2Cl2 (3  3 mL). The combined organic phases were consecutively washed with 1 N HCl and saturated NaHCO3, dried (Na2SO4), and concentrated. The residue was chromatographed on SiO2 (CHCl3–EtOAc = 9:1 ? 4:1) to give dicarbonate 6 (194 mg, 89%) as white crystals. Mp 266– 1 267 °C (acetone). [a]20 D +140 (c 0.5, CHCl3). H NMR (CDCl3) d: 0.71 (s, 3H, C18-H), 0.76 (d, J = 6.9 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H), 0.93 (d, J = 6.6 Hz, 3H), 0.93 (s, 3H, C19-H), 1.02 (d, J = 6.6 Hz, 3H), 2.35 (dd, J = 15.8, 5.1 Hz, 1H), 3.03 (dd, J = 9.5, 5.4 Hz, 1H, C5-H), 4.04–4.17 (m, 3H, C7- and C23-H), 4.42 (d, J = 4.8 Hz, 1H, C22-H), 4.85–4.90 (m, 1H, C2- or C3-H), 4.94–4.98 (m, 1H, C3- or C2-H). 13C NMR (CDCl3) d: 8.6, 11.3, 11.8, 16.3, 18.9, 20.6, 22.9, 24.4, 26.5, 27.1, 27.7, 33.5, 36.0, 39.0, 39.1, 40.5, 40.6, 42.8, 42.9, 51.0, 51.7, 55.0, 71.0, 73.9, 74.8, 81.1, 82.2, 154.4, 154.8, 174.5. MS (APCI+) m/z (%): 532.9 ([M+H]+, 100). HRMS (ESI+): calcd. for C30H44NaO8 [M+Na]+ 555.2928, found 555.2917. 2.2.2. (22R,23R,24R)-22,23-Carbonylbis(oxy)-2a,3a-dihydroxy-Bhomo-7-oxa-24-methyl-5a-cholestan-6-one (7) A solution of LiOH (15.8 mg, 0.66 mmol) in water (0.4 mL) was added with stirring to a solution of dicarbonate 6 (175 mg, 0.33 mmol) in THF (4 mL). The mixture was stirred at room temperature for 40 min, then 0.1 N H2SO4 was added till pH 3 was achieved followed by extraction with CHCl3 (3  5 mL). The CHCl3 extract was dried (Na2SO4) and evaporated to give carbonate 7 (130 mg, 78%) as white crystals. Mp 259–260 °C (EtOAc). [a]20 D 4.1 (c 0.49, CHCl3). 1H NMR d: 0.70 (s, 3H, C18-H), 0.77 (d, J = 6.9 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H), 0.91 (s, 3H, C19-H), 0.94 (d, J = 6.9 Hz, 3H), 1.03 (d, J = 6.6 Hz, 3H), 3.12 (dd, J = 12.1, 4.5 Hz, 1H, C5-H), 3.71 (d, J = 10.1 Hz, 1H, C2-H), 4.02 (s, 1H, C3H), 4.06–4.10 (m, 2H, C7-H), 4.12 (dd, J = 9.1, 5.0 Hz, 1H, C23-H), 4.43 (d, J = 5.0 Hz, 1H, C22-H). 13C NMR d: 8.6, 11.3, 11.5, 15.4, 16.3, 20.6, 22.1, 24.7, 27.2, 27.6, 31.0, 38.3, 39.2, 40.6, 40.8, 41.4, 42.5, 42.8, 51.0, 51.8, 57.9, 68.0, 68.0, 70.2, 81.2, 82.3, 154.9, 176.2. HRMS (ESI+): calcd. for C29H47O7 [M+H]+ 507.3316, found 507.3309. 2.2.3. (22R,23R,24R)-2a,3a,22,23-Tetrahydroxy-B-homo-7-oxa-24methyl-5a-cholestan-6-one 2,3-acetonide (9) Variant A: A mixture of carbonate 7 (250 mg, 0.49 mmol), 2,2dimethoxypropane (0.31 mL, 2.5 mmol), TsOH (5 mg) and THF (2 mL) was stirred at room temperature for 45 min. Then Et3N (0.3 mL) was added, and solvents were evaporated under reduced pressure. The residue containing 8 was dissolved in MeOH (1.7 mL) and treated with K2CO3 (340 mg, 2.47 mmol). The mixture was stirred at room temperature for 5 days, diluted with water (3 mL) and extracted with CHCl3 (3  5 mL). The organic phase was dried (Na2SO4), filtered, and concentrated. The residue was chromatographed on silica gel (petroleum ether– EtOAc = 7:3 ? 11:9) to give the acetonide 9 (150 mg, 62%) as an

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oil. The physical and spectral data were identical to those previously reported for this compound [13]. Variant B: A solution of epibrassinolide (5) (173 mg, 0.36 mmol) and boric acid (25 mg, 0.40 mmol) in THF (2 mL) was stirred at ambient temperature for 1.5 h. The 2,2-dimethoxypropane (0.646 mL, 5.3 mmol) and TsOH
H2O (41 mg, 0.22 mmol) were added and stirring was continued for 1 h. The reaction mixture was treated with pyridine (0.1 mL), and then solvents were evaporated in vacuo. The residue containing 13 was dissolved in water (4 mL) and diethyl ether (2 mL), and treated with NaOH (158 mg, 3.95 mmol) and pentaerythritol (480 mg, 3.5 mmol). After stirring at room temperature for 2.5 h, the reaction mixture was extracted with CHCl3 (3  5 mL), and the organic layer was dried (Na2SO4) and evaporated. The residue was chromatographed on silica gel (petroleum ether–EtOAc = 65:35 ? 50:50) to give the acetonide 9 (149 mg, 80%) as an oil. 2.2.4. (22S,23S)-22,23-Dihydroxy-3a,5-cyclo-5a-stigmastan-6-one 22,23-cyclic boric ester (11) A mixture of (22S,23S)-22,23-dihydroxy-3a,5-cyclo-5a-stigmastan-6-one (10) (200 mg, 0.45 mmol, prepared according to [14]), boric acid (30 mg, 0.48 mmol) and MeOH (7 mL) was stirred at room temperature for 24 h. Then water (20 mL) was added, the precipitate was filtered off and dried by oil pump vacuum for 3 h to give ester 11 (0.15 g, 70%). 1H NMR (CDCl3) d: 0.71 (t, J = 4.7 Hz, 1H, C4-H), 0.74 (s, 3H, C18-H), 0.89 (d, J = 6.8 Hz, 3H), 0.91–1.02 (m, 15H), 2.42 (d, J = 12.5 Hz, 1H, C7-H), 4.10–4.17 (m, 1H, C22- or C23-H), 4.20–4.29 (m, 1H, C23- or C22-H). 13C NMR (CDCl3) d: 11.6, 11.8, 13.4, 18.6, 19.6, 22.7, 24.2, 25.8, 26.1, 28.0, 33.4, 34.7, 35.4, 39.6, 40.8, 40.8, 43.2, 44.6, 45.9, 46.2, 46.6, 50.5, 51.1, 51.2, 56.5, 60.8, 80.6, 80.7, 209.6. 2.2.5. Hydrolysis of (22S,23S)-22,23-dihydroxy-3a,5-cyclo-5astigmastan-6-one 22,23-cyclic boric ester (11) A mixture of ester 11 (20 mg, 0.043 mmol), pentaerythritol (60 mg, 0.44 mmol), 1 M NaOH (1.6 mL) and Et2O (1.6 mL) was stirred at ambient temperature for 24 h, and then it was extracted with Et2O (3  3 mL). The organic layer was dried (MgSO4) and evaporated to give diol 10 (18 mg, 95%) as an oil. 2.2.6. (22R,24R)-2a,3a,22-Trihydroxy-B-homo-7-oxa-24-methyl-5acholestan-6,23-dione 2,3-acetonide (14) A solution of Br2 (72 lL, 2.8 mmol) in CH2Cl2 (1.3 mL) was added dropwise to a solution of acetonide 9 (357 mg, 0.69 mmol) and (Bu3Sn)2O (0.46 mL, 0.89 mmol) in CH2Cl2 (2.6 mL). After stirring at room temperature for 20 min, the reaction solution was successively treated with saturated Na2SO3 and NaHCO3. The mixture was extracted with EtOAc (3  2 mL), the organic phase was dried (Na2SO4), filtered, and evaporated. The residue was

chromatographed on silica gel (petroleum ether– EtOAc = 4:1 ? 3:1) to give a mixture of 23- and 22-ketones 14 and 15 (4:1, 189 mg, 53%). It was rechromatographed over the same sorbent using petroleum ether–EtOAc as eluent in a gradient regime 4:1 ? 3:1 to afford the ketone 14 (151 mg, 42%) as an oil. [a]20 66 (c 1.22, CHCl3).). 1H NMR d: 0.71 (d, J = 6.7 Hz, 3H, D C21-H), 0.77 (s, 3H, C18-H), 0.88 (d, J = 7.0 Hz, 3H, C26-H), 0.88 (s, 3H, C19-H), 0.91 (d, J = 6.7 Hz, 3H, C27-H), 1.02 (d, J = 6.8 Hz, 3H, C28-H), 1.31 (s, 3H, acetonide-H), 1.51 (s, 3H, acetonide-H), 2.09 (ddd, J = 14.2, 11.8, 5.3 Hz, 1H), 2.31 (dd, J = 15.7, 3.6 Hz, 1H, C1a-H), 2.53 (p, J = 6.7 Hz, 1H, C24-H), 3.30 (dd, J = 10.9, 3.8 Hz, 1H, C5-H), 3.47 (d, J = 5.2 Hz, 1H, OH), 4.05–4.16 (m, 2H, C7-H), 4.22 (dd, J = 5.3, 1.4 Hz, 1H, C22-H), 4.32–4.41 (m, 2H, C2- and C3-H). 13C NMR d: 12.1, 12.2, 12.7, 18.9, 19.6, 20.9, 22.9, 23.6, 24.4, 26.4, 27.5, 28.2, 31.7, 33.4, 35.9, 38.4, 39.4, 39.4, 40.1, 42.9, 47.3, 51.8, 52.1, 54.5, 71.1, 72.4, 73.0, 79.5, 107.6, 176.6, 216.3. HRMS (ESI+): calcd. for C38H56NaO6 [M+Na]+ 631.3969, found 631.3954. 2.2.7. (22R,24R)-2a,3a,22-Trihydroxy-B-homo-7-oxa-24-methyl-5acholestan-6,23-dione (16) A mixture of acetonide 14 (80 mg), AcOH (8 mL) and water (2 mL) was stirred at 60 °C for 1 h, then the solvents were evaporated in vacuo and the residue was purified by flash chromatography on silica gel (CHCl3–EtOAc = 95:5 ? 85:15) to give compound 16 (72 mg, 97%) as white crystalls. Mp 232–233 °C (MeOH). [a]20 D 44 (c 0.25, CHCl3). 1H NMR (CDCl3) d: 0.71 (d, J = 6.7 Hz, 3H, C21-H), 0.76 (s, 3H, C18-H), 0.88 (d, J = 6.8 Hz, 3H, C26-H), 0.91 (d, J = 6.7 Hz, 3H, C27-H), 0.93 (s, 3H, C19-H), 1.03 (d, J = 6.8 Hz, 3H, C28-H), 2.53 (p, J = 6.7 Hz, 1H, C24-H), 3.12 (dd, J = 12.3, 4.5 Hz, 1H, C5-H), 3.49 (dd, J = 5.0, 3.3 Hz, 1H, OH), 3.68–3.76 (m, 1H), 4.03 (s, 1H, C3-H), 4.05–4.13 (m, 2H, C7-H), 4.22 (dd, J = 5.3, 1.5 Hz, 1H, C22-H). 13C NMR (CDCl3) d: 11.8, 12.2, 12.8, 15.5, 18.9, 21.0, 22.2, 24.7, 28.1, 31.0, 31.8, 38.3, 38.5, 39.2, 39.3, 40.9, 41.5, 42.4, 47.4, 51.3, 52.2, 58.1, 68.0, 68.1, 70.4, 79.6, 176.2, 216.4. HRMS (ESI+): calcd. for C28H47O6 [M+H]+ 479.3367, found 479.3360. 2.2.8. Benzylation of (22R,23R,24R)-2a,3a,22,23-tetrahydroxy-Bhomo-7-oxa-24-methyl-5a-cholestan-6-one 2,3-acetonide (9) A mixture of acetonide 9 (94 mg, 0.18 mmol), Bu2SnO (45 mg, 0.18 mmol), TBAB (17.5 mg, 0.054 mmol), DIPEA (63 lL, 0.36 mmol), BnBr (43 lL, 0.36 mmol) was stirred at 90 °C for 20 h, then another portions of BnBr (22 lL, 0.18 mmol) and DIPEA (32 lL, 0.18 mmol) were added and stirring was continued at the same temperature for 16 h. The volatile solvents were evaporated in vacuo, and the residue was chromatographed on silica gel (petroleum ether–EtOAc = 80:20 ? 70:30) to give:

Scheme 2. Synthesis of monoacetonide 9 via cyclic carbonate 6.

A.G. Baradzenka et al. / Steroids 101 (2015) 90–95

Scheme 3. Synthesis and cleavage of cyclic boric ester 11.

(a) (22R,23R,24R)-22-benzyl-2a,3a,23-trihydroxy-B-homo-7oxa-24-methyl-5a-cholestan-6-one 2,3-acetonide (17) as an 1 13 oil (28 mg, 26%). [a]20 C D +15 (c 0.59, CHCl3). Its H and NMR data are summarized in Table 1. HRMS (ESI+): calcd. for C38H58NaO6 [M+H]+ 633.4126, found 633.4114. (b) (22R,23R,24R)-23-benzyl-2a,3a,22-trihydroxy-B-homo-7oxa-24-methyl-5a-cholestan-6-one 2,3-acetonide (18) as an 1 13 oil (38 mg, 35%). [a]20 C D +11 (c 0.57, CHCl3). Its H and + NMR data are summarized in Table 1. HRMS (ESI ): calcd. for C38H58NaO6 [M+Na]+ 633.4126, found 633.4119. 2.2.9. (23R,24R)-23-Benzyl-2a,3a-dihydroxy-B-homo-7-oxa-24methyl-5a-cholestan-6,22-dione 2,3-acetonide (19) A mixture of compound 18 (20.6 mg, 0.034 mmol), DMP (15.7 mg, 0.037 mmol) and CH2Cl2 (4 mL) was stirred at room temperature for 1 h, and then was successively treated with saturated Na2S2O3 (1 mL) and NaHCO3 (3 mL). The water phase was extracted with EtOAc (3  5 mL). The combined organic layers were washed with saturated NaHCO3 (3  5 mL), dried (Na2SO4) and concentrated. The residue was purified on silica gel (petroleum ether–EtOAc = 80:20 ? 70:30) to afford ketone 19 (16.4 mg, 80%) 1 as an oil. [a]20 D +24 (c 0.5, CHCl3). H NMR (CDCl3) d: 0.70 (s, 3H, C18-H), 0.78 (d, J = 6.8 Hz, 3H), 0.81 (d, J = 7.0 Hz, 3H), 0.87 (s, 3H, C19-H), 0.90 (d, J = 6.9 Hz, 3H), 1.08 (d, J = 6.9 Hz, 3H), 1.32 (s, 3H, acetonide-H), 1.52 (s, 3H, acetonide-H), 3.00 (dt, J = 13.7, 6.9 Hz, 1H, C20-H), 3.29 (dd, J = 10.8, 3.8 Hz, 1H, C5-H), 3.69 (d, J = 7.2 Hz, 1H, C23-H), 4.04–4.10 (m, 2H, C7-H), 4.37 (m, 3H, C2-, C3-H and -OCH2Ph), 4.56 (d, J = 11.4 Hz, 1H, -OCH2Ph), 7.28– 7.38 (m, 5H, -OCH2Ph). 13C NMR (CDCl3) d: 10.7, 12.4, 16.8, 19.6, 21.8, 22.9, 23.6, 24.7, 26.5, 27.0, 27.6, 29.7, 33.5, 35.9, 39.3, 39.7, 40.2, 40.3, 43.2, 44.0, 51.3, 51.6, 54.7, 71.1, 72.4, 72.8, 73.0, 87.8, 107.6, 127.9, 128.4, 137.7, 176.6, 214.6. MS (APCI+) m/z (%): 609.3 ([M+H]+, 100), 551.4 ([M+H-acetone]+, 45). HRMS (ESI+): calcd. for C38H56NaO6 [M+Na]+ 631.3969, found 631.3976. 2.2.10. (23R,24R)-2a,3a,23-Trihydroxy-B-homo-7-oxa-24-methyl-5acholestan-6,22-dione (20) A mixture of benzyl ether 19 (17 mg, 0.028 mmol), MeOH (3 mL), THF (1 mL) and 10% Pd/C (34 mg) was stirred under hydrogen (1 atm) at 0 °C for 3 h. The catalyst was filtered off, and the organic layer was evaporated. The residue was dissolved in AcOH (1 mL) and water (0.2 mL) and kept at ambient temperature for 30 min. Solvents were evaporated under reduced pressure and the residue was purified by column chromatography on silica gel

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(CHCl3–iPrOH) to give ketone 20 (10.3 mg, 76%). Mp 216–218 °C (EtOAc). [a]20 6 (c 0.54, CHCl3). 1H NMR (C5D5N) d: 0.66 (s, 3H, D C18-H), 0.99–0.94 (m, 9H), 1.05 (s, 3H, C19-H), 1.26 (d, J = 6.9 Hz, 3H), 3.37–3. 45 (m, 1H), 3.62 (dd, J = 12.0, 4.2 Hz, 1H, C5-H), 3.99–4.15 (m, 3H, C2- and C7-H), 4.22 (d, J = 7.8 Hz, 1H, C23-H), 4.44 (s, 1H, C3-H). 13C NMR (C5D5N) d: 11.3, 12.5, 16.3, 17.0, 17.8, 22.3, 22.8, 25.5, 27.5, 28.0, 33.5, 38.9, 40.0, 40.1, 41.5, 42.1, 43.2, 43.3, 44.6, 51.2, 53.3, 58.7, 68.8, 69.1, 70.6, 80.7, 177.0, 216.7. MS (APCI+) m/z (%): 479.4 ([M+H]+, 100). HRMS (ESI+): calcd. for C28H47NaO6 [M+Na]+ 479.3367, found 479.3358.

3. Results and discussion Prior to any reaction, the 22R,23R-diol function in epibrassinolide (5) had to be differentiated from the similar functionality in cycle A. In view of the planned transformations in the side chain, the acetonide protection of 2a,3a-diol seemed to constitute a suitable solution. Two variants for the preparation of monoacetonide 9 were tested. The first one made use of cyclic dicarbonate 6 available from the reaction of epibrassinolide (5) with triphosgene (Scheme 2). LiOH mediated regioselective saponification of 6 in aqueous THF provided monocarbonate 7. Its treatment with 2,2dimethoxypropane followed by 22R,23R-diol deprotection in 8 afforded monoacetonide 9. The low overall yield (43% from 5) prompted us to search for an alternative route to this compound. A potentially useful reaction for the differentiation between 2a,3a- and 22,23-diol groups in BS is the formation of cyclic boronic or boric esters [13,15–17]. The reaction proceeds regioselectively at the side chain diol group in a high yield, but the removal of such protecting groups is not an easy task. The interaction between boronic or boric acids and diols is a reversible process [18] that in the case of steroidal 22,23-diols is shifted towards the formation of the corresponding cyclic esters. The only reliable, but time-consuming, method to regenerate such diols was based on the use of ion exchange resin [19]. In an attempt to find a solution to the problem, we had prepared the model boric ester 11 starting from the known diol 10 [14] and studied its hydrolysis (Scheme 3). No reaction was observed on heating the ester 11 in MeOH in the presence of TsOH. Treatment of the boronate 11 with KOH in MeOH led to an equilibrium mixture of 10:11 = 4:1 (NMR control). The shift of the equilibrium was achieved by the use of NaOH and pentaerythritol in a mixture of H2O and Et2O [20]. The desired diol 10 was obtained in a nearly quantitative yield. The developed methodology proved to be applicable to the preparation of monoacetonide 9. The desired compound was obtained from epibrassinolide (5) through a three step one-pot procedure in 80% overall yield (Scheme 4). A relatively simple procedure to achieve partial oxidation of 1,2-diols involves formation of intermediate alkyltin alkoxides followed by their treatment with bromine to afford a-ketols [21,22]. Alternatively, the same transformation can be effected by subjecting a solution of diol and bis(tributyltin) oxide to the action of bromine instead of oxidation of pre-formed tin alkoxides [23]. The latter variant was chosen for the partial oxidation of diol

Scheme 4. Synthesis of monoacetonide 9 via cyclic boric esters 12 and 13.

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Scheme 5. Synthesis of 24-epicryptolide 16.

Scheme 6. Synthesis of 22-dehydro-24-epicastasterone 20.

9 (Scheme 5). Although this transformation was quite regioselective, it proceeded in rather low yield. The main regioisomer 14 was further converted into the desired 24-epicryptolide 16, but attempts to achieve a more efficient differentiation of the functional groups in the side chain of 9 were continued by using benzyl ethers. A typical procedure for the monobenzylation of 1,n-diols consists of the formation of O-stannylene acetals by treatment with dibutyltin oxide in toluene at reflux with azeotropic removal of water followed by adding benzyl bromide and tetra-butylammonium bromide [24,25]. However, no reaction was observed under these conditions with the diol 9 (Scheme 6). Recently, a simple solvent-free procedure of the stannylene-mediated benzylation of polyols was reported [26]. The same protocol, when applied to the diol 8, gave a 1:1.5 mixture of benzyl ethers 17 and 18 in a good yield. After chromatographic separation, the major regioisomer 18 was subjected to Dess–Martin periodinane oxidation to afford 22-ketone 19. Removal of the protecting groups proceeded smoothly upon successive hydrogenolysis and exposure to aqueous acetic acid, yielding 22-dehydro-24-epicastasterone 20. 4. Conclusion In summary, we have prepared 24-epicryptolide and 22-dehydro-24-epibrassinolide as reference compounds for studying the metabolic and other processes occurring with epibrassinolide. The main chemical problem investigated included differentiation of functional groups of brassinosteroids. It was successfully solved with respect to vicinal 2a,3a- and 22,23-diol functions. Attempts to distinguish the hydroxyl groups in the side chain from one another gave less favourable results. The hydroxyl group at C-23 was more reactive than the 22-OH in the oxidation with bromine in the presence of bis(tributyltin) oxide and in the benzylation reaction. This resulted in the predominant formation of the corresponding a-hydroxy ketone derivatives with the ratio ranging from 4:1 to 1.5:1.

Acknowledgement The authors are grateful to the Belarusian Foundation for Fundamental Research for financial support (Project X14P-139).

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