OH OH HO HO H O Castasterone 1 OH OH HO HO H O Brassinolide 2 O 360 J. CHEM. RESEARCH (S), 1997 J. Chem. Research (S), 1997, 360–361† Improved Synthesis of Castasterone and Brassinolide† Tsuyoshi Watanabe,a Suguru Takatsuto,*b Shozo Fujiokac and Akira Sakuraic aTama Biochemical Co. Ltd., 2-7-1 Nishishinjuku Shinjuku-ku, Toyko 163, Japan bDepartment of Chemistry, Joetsu University of Education, Joetsu-shi, Niigata 943, Japan cThe Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-01, Japan Castasterone 1 is synthesized in 32% overall yield in eight steps from the known (20S)-6,6-ethylenedioxy-20-formyl- 3a,5-cyclo-5a-pregnane 4.Brassinosteroids (BRs) constitute a new class of plant hormones with plant growth-promoting activity. It has been clarified that they occur widely in the plant kingdom and that more than 40 BRs have been chemically characterized from plant sources.1 Among the natural BRs, castasterone 1 occurs most frequently, followed by brassinolide 2, and they are assumed to play an important role in plant growth and development.1 The synthesis of castasterone 1 and brassinolide 2 has been reviewed.2 We have recently reported a method for constructing the side chain of C28 BRs, using the reaction of (20S)-20-formyl-6b-methoxy-3a,5-cyclo-5a-pregnane with (Z)-prop-1-enylmagnesium bromide, the orthoester Claisen rearrangement of the resulting (22S,23Z)-allylic alcohol, and the asymmetric dihydroxylation (AD) of the side chain of crinosterol, and we have synthesized the new 6-deoxo C28 BRs, 6-deoxoteasterone, 3-dehydro-6-deoxoteasterone and 6-deoxytyphasterol.3 In order to increase the overall yield of castasterone 1 and brassinolide 2, we investigated a convenient synthesis of 1 and 2 by employing our method to construct the side chain of C28 BRs.The starting material is a known 22-aldehyde 4, which was obtained in 70% yield in five steps from an abundant stigmasterol 3, according to the reported method.4 The reaction of the 22-aldehyde 4 with (Z)-prop-1-enylmagnesium bromide provided the (22S,23Z)-23-en-22-ol 5 in 57% yield.The orthoester Claisen rearrangement of 5 gave the ester 6 in 98% yield. The ethoxycarbonyl group of 6 was transformed in 89% yield into the methyl group by reduction with LiAlH4, methanesulfonation and then reduction with LiAlH4, constructing the side chain of crinosterol. Deprotection of 7 with acid gave the known cycloketone 8,5 which was subjected to acid catalysed isomerization6 to provide the known 2,22-dien- 6-one 95 in 82% yield from 7.With respect to the AD of the side chain of the crinosterol, Sun et al. have reported a high (8:1) stereoselectivity of (22R,23R)- and (22S,23S)-22,23-diols using dihydroquinidine p-chlorobenzoate as a chiral ligand.7 Recently, Marino et al. have reported the complete stereoselectivity for the (22R,23R)-22,23-diol by the AD using 1,4-bis(9-O-dihydroquinidinyl) phthalazine as a chiral ligand.8 We have previously reported a 57:43 ratio of (22R,23R)- and (22S,23S)-22,23-diols by the AD of (22E,24S)-3a,5-cyclo- 6b-methoxy-5a-ergost-22-ene by employing dihydroquinidine p-chlorobenzoate as a chiral ligand.3 The result of Marino et al.has prompted us to investigate the stereoselectivity of the AD of the same 22E-olefinic steroid using 1,4-bis(9- O-dihydroquinidinyl)phthalazine9 as a chiral ligand. Although we have not attained complete stereoselectivity, we have found a good ratio of 90:10 for the desired (22R,23R)-22,23-diol.Thus, the chiral ligand has been employed in the present synthesis. Castasterone 1 was synthesized in 80% yield by the AD of the 2,22-dien-6-one 9. As the transformation of 1 into brassinolide 2 is known,10 the formal synthesis of 2 was achieved. In conclusion, we have developed a convenient method for synthesizing castasterone 1 and brassinolide 2. The present synthesis provided castasterone 1 in 22% yield by 13 steps from stigmasterol 3, which is superior to the method recently reported by McMorris and co-workers.10d After submitting our paper, a concise and improved synthesis of brassinolide in 8% overall yield from stigmasterol by 12 steps has been published by Back et al.11 Although our route is the longer, our synthesis has merits of easier experimental manipulation and the expected better overall yield of brassinolide, inferred from the previous preparations of brassinolide from castasterone or its derivatives.2,10 Experimental Mps were determined under a hot-stage microscope (Yanaco micro melting point apparatus) and are uncorrected. 1H and 13C NMR spectra were recorded on a Varian XL-VXR 300 or JEOL a-400 spectrometer in a CDCl3 solution with tetramethylsilane as internal standard. HR-MS were recorded on a JEOL HX-110 mass spectrometer. Silica gel (Kieselgel 60, 70–230 mesh, Merck) was used for column chromatography.Reactions were monitored by TLC on silica gel plates (Kiesel gel 60F254, 0.25 mm thickness, Merck). Spots were visualized with 10% H2SO4, followed by heating. All purified compounds showed a single spot by TLC analysis. ( 2 2 S , 2 3 Z ) - 6 , 6 - E t h y l e n e d i o x y - 3 a, 5 - c y c l o- 2 6 , 2 7 - d i n o r - 5 a- c h o l e s t - 23-en-22-ol 5.·As described in our previous paper,3 (Z)-prop- 1-enylmagnesium bromide was prepared from (Z)-1-bromoprop- 1-ene (9.23 g, 76.3 mmol) and Mg (11.20 g, 0.461 mol).A solution of the 22-aldehyde 44 (24.30 g, 65.3 mmol) in THF (250 cm3) was added to the Grignard reagent at 0 °C under an argon atmosphere. The mixture was stirred at room temperature for 1 h. Work-up (hexane) gave a crude product, which was analysed by TLC, showing three spots with RF=0.33, 0.29 and 0.21 (hexane–EtOAc, 5:1), as described in the Grignard reaction of (20S)-20-formyl- 6b-methoxy-3a,5-cyclo-5a-pregnane.3 The product was applied to a column of silica gel (7.0 cm i.d.Å100 cm).Elution with hexane– EtOAc (100:5) separated the three products. The major product (15.35 g, 57%) with RF=0.29 was identified as the title compound 5, oil; dH (300 MHz): 0.73 (3 H, s, 18-H3), 0.96 (3 H, d, J 6.5 Hz, 21-H3), 1.01 (3 H, s, 19-H3), 1.66 (3 H, d, J 5 Hz, 25-H3), 3.75 (1 H, m, ethylene ketal), 3.87 (2 H, m, ethylene ketal), 4.02 (1 H, m, ethylene ketal), 4.57 (1 H, br d, J 6 Hz, 22-H) and 5.48–5.69 (2 H, m, 23-H and 24-H); dC (75 MHz): 7.3, 12.1, 12.3, 13.4, 19.0, 22.6, 23.1, 24.2, 24.9, 27.8, 33.3, 34.2, 39.3, 40.1, 40.2, 41.9, 42.8, 45.6, 47.4, 52.6, 56.2, 64.6, 64.9, 69.7, 109.9, 125.1 and 133.2.HR-MS (EI) (Found: M+, 414.3143. C27H42O3 requires Mr 414.3134). Ethyl (22E,24R)-6,6-Ethylenedioxy-3a,5-cyclo-5a-ergost-22-en- 26-oate 6.·A mixture of the allylic alcohol 5 (4.5 g, 10.86 mmol), triethyl orthopropionate (20 cm3, 0.160 mol), propionic acid (10 drops) and xylene (30 cm3) was refluxed under argon atmosphere for 2 h.MeOH was added and the solvent was removed in vacuo to give a residue which was applied to a column of silica gel (4.0 cm i.d.Å35 cm). Elution with hexane–EtOAc (30:1) afforded the title compound 6 (5.30 g, 98%) as an oil; TLC: hexane–EtOAc (10:1) RF=0.65; dH (300 MHz): 0.72 (3 H, s, 18-H3), 0.96 (3 H, d, J 6.5 Hz, *To receive any correspondence. †This is a Short Paper as defined in the Instructions for Authors, Section 5.0 [see J. Chem.Research (S), 1997, Issue 1]; there is therefore no corresponding material in J. Chem. Research (M). Fig. 1 Structures of castasterone and brassinolideHO 3 CHO 4 O O 5 O O OH 6 O O CO2Et i ii iii iv, v, vi 7 O O 8 O 9 O 1 2 vii viii ix x H J. CHEM. RESEARCH (S), 1997 361 28-H3), 0.99 (3 H, d, J 6.5 Hz, 21-H3), 1.00 (3 H, s, 19-H3), 1.07 (3 H, d, J 7 Hz, 27-H3), 1.26 (3 H, t, J 7 Hz, OCH2CH3), 3.74 (1 H, m, ethylene ketal), 3.86 (2 H, m, ethylene ketal), 4.01 (1 H, m, ethylene ketal), 4.12 (2 H, q, J 7 Hz, OCH2CH3) and 5.05–5.30 (2 H, m, 22-H and 23-H).HR-MS (FAB) (Found: M++H, 499.3785. C21H51O4 requires Mr 499.3787). ( 2 2 E , 2 4 S ) - 6 , 6 - E t h y l e n e d i o x y - 3 a, 5 - c y c l o - 5 a- e r g o s t - 2 2 - e n e 7.— The ester 6 (2.24 g, 4.50 mmol) in THF (100 cm3) was treated with LiAlH4 (1.0 g, 26.35 mmol) at reflux temperature under argon for 2 h. Work-up (hexane) gave a corresponding 26-ol, which was dissolved in toluene (50 cm3) and Et3N (5 cm3) and then treated with methanesulfonyl chloride (0.5 cm3, 6.46 mmol) at 0 °C for 2 h.Work-up (toluene) gave a corresponding sulfonate (2.25 g), which in THF (50 cm3) was treated with LiAlH4 (1.0 g, 26.35 mmol) at room temperature overnight. Work-up (hexane) followed by chromatography on silica gel (3.0 cm. i.d.Å30 cm) eluting with toluene afforded the title compound 7 (1.76 g, 89% in three steps) as an oil; TLC: toluene RF=0.70; dH (300 MHz): 0.73 (3 H, s, 18-H3), 0.83 (3 H, d, J 6.5 Hz, 28-H3), 0.84 (3 H, d, J 6.5 Hz, 26-H3), 0.91 (3 H, d, J 7 Hz, 27-H3), 1.00 (3 H, d, J 6.5 Hz, 21-H3), 1.01 (3 H, s, 19-H3), 3.75 (1 H, m, ethylene ketal), 3.87 (2 H, m, ethylene ketal), 4.02 (1 H, m, ethylene ketal) and 5.16 (2 H, m, 22-H and 23-H).HR-MS (FAB) (Found: M++H, 441.3736. C30H49O2 requires Mr 441.3733). (22E,24S)-3a,5-Cyclo-5a-ergost-22-en-6-one 8.·A solution of the compound 7 (1.76 g, 4.00 mmol) in acetone (75 cm3) was treated with 1 M H2SO4 (1 cm3) at room temperature for 2 h. Workup (hexane) and chromatography of a crude product on silica gel (3.0 cm i.d.Å30 cm) eluting with hexane–EtOAc (20:1) afforded the known compound 8 (1.57 g, 99%), mp 105–106 °C (from MeOH) (lit.,5 mp 105–108 °C).Its spectral data are in agreement with the reported data.5 (22E,24S)-5a-Ergosta-2,22-dien-6-one 9.·A mixture of compound 8 (505.6 mg, 1.277 mmol), pyridinium toluene-p-sulfonate (65 mg, 0.259 mmol), LiBr (52.6 mg, 0.606 mmol) and N,N-dimethylacetamide (30 cm3) was heated at 160 °C under argon for 5 h.Work-up (hexane) followed by chromatography on silica gel (1.6 cm i.d.Å25 cm) eluting with hexane–toluene–EtOAc (120:2:1) afforded the known compound 9 (416.3 mg, 82%), mp 110-111 °C (from acetone) (lit.,5 mp 111–112 °C). Its spectral data are in agreement with the reported data.5 ( 2 2 R , 2 3 R , 2 4 S ) - 2 a , 3 a, 2 2 , 2 3 - T e t r a h y d r o x y - 5 a- e r g o s t a n - 6 - o n e , Castasterone 1.·A mixture of the compound 8 (319.3 mg, 0.806 mmol), K3[Fe(CN)6] (2.0 g, 6.07 mmol), K2CO3 (0.84 g, 6.08 mmol), methanesulfonamide (1.15 g, 12.09 mmol), 1,4-bis(9-O-dihydroquinidinyl) phthalazine (400.3 mg, 0.514 mmol) and OsO4 (2.83 mg, 0.011 mmol) in ButOH–H2O (50 cm3, 1:1) was stirred at room temperature in the dark for 15 d.NaHSO3 (1.0 g, powder) was added and the mixture was stirred further for 1 h. Work-up (EtOAc) gave a crude product, which was analysed by TLC, showing two spots with RF=0.53 and 0.42 (CHCl3–EtOH, 9:1).The product was applied to a column of silica gel (1.8 cm i.d.Å40 cm). Elution with CH2Cl2–EtOH (30:1) separated the two products and castasterone 1 (300.4 mg, 80%) with RF=0.42 was obtained, mp 253–254 °C (from EtOAc) (lit.,5 mp 252–255 °C, lit.,10d mp 251–253 °C). Its spectral data are in agreement with the reported data.5,10 We thank Professor T. Yoshikawa and Dr Y. Orihara of Kitasato University for the measurements of 1H and 13C NMR and mass spectra.Received, 24th April 1997; Accepted, 3rd June 1997 Paper E/7/02805I References 1 (a) S. Fujioka and A. Sakurai, Nat. Prod. Rep., 1997, 1; (b) S. Takatsuto, J. Chromatogr., 1994, 658, 3; (c) Brassinosteroids: Chemistry, Bioactivity and Applications, ed. H. G. Cutler, T. Yokota and G. Adam, ACS Symposium Series 474, American Chemical Society, Washington, D.C., 1991. 2 T. G. Back, in Studies in Natural Products Chemistry, ed. Attaur- Rahman, Elsevier, Amsterdam, The Netherlands, 1995, vol. 16, pp. 321–364. 3 S. Takatsuto, T. Watanabe, S. Fujioka and A. Sakurai, J. Chem. Res., 1997, (S) 134; (M) 0901. 4 K. Okada and K. Mori, Agric. Biol. Chem., 1983, 47, 89. 5 M. Anastasia, P. Ciuffreda, M. D. Puppo and A. Fiecchi, J. Chem. Soc., Perkin Trans. 1, 1983, 383. 6 T. Watanabe, H. Kuriyama, T. Furuse, K. Kobayashi and S. Takatsuto, Agric. Biol. Chem., 1988, 52, 2117. 7 L.-Q. Sun, W.-S. Zhou and X.-F. Pan, Tetrahedron: Asymmetry, 1991, 2, 973. 8 J. P. Marino, A. Dedios, L. J. Anna and R. F. Delapradilla, J. Org. Chem., 1996, 61, 109. 9 W. Amberg, Y. L. Bennani, R. K. Chadha, G. A. Crispino, W. D. Davis, J. Hartung, K.-S. Jeong, Y. Ogino, T. Shibata and K. B. Sharpless, J. Org. Chem., 1993, 58, 844. 10 (a) S. Fung and J. B. Siddall, J. Am. Chem. Soc., 1980, 102, 6580; (b) S. Takatsuto, N. Yazawa, M. Ishiguro, M. Morisaki and N. Ikekawa, J. Chem. Soc., Perkin Trans. 1, 1984, 139; (c) K. Mori, M. Sakakibara and K. Okada, Tetrahedron, 1984, 40, 1767; (d) T. C. McMorris, R. G. Chavez and P. A. Patil, J. Chem. Soc., Perkin Trans. 1, 1996, 295. 11 T. G. Back, D. L. Baron, W. Luo and S. K. Nakajima, J. Org. Chem., 1997, 62, 1179. Scheme 1 Reagents and conditions: i, ref.4; ii, BrMgCH�CHCH3, THF, 0 °C to room temp., 1 h; iii, Et(OEt)3, propionic acid, xylene, reflux, 2 h; iv, LiAlH4, THF, reflux, 2 h; v, MeSO2Cl, Et3N, toluene, room temp., 2 h; vii, LiAlH4, THF, room temp., overnight; vii, 1 M H2SO4, acetone, room temp., 2 h; viii, PPTS, LiBr, DMA, 160 °C, 5 h; ix, OsO4, K3[Fe(CN)6], 1,4-bis(9-O-dihydroquinidinyl)phthalazine, K2CO3, methanesulfonamide, ButOH–H2O, room temp., 15 d; x, ref. 1