OH HO OH HO H OH HO OH HO H OH H O 1 2 3 4 OH OMe OMe OMe OMe O OH OR OH O RO H H OR RO 5 6 (22 R,23 R) 7 (22 S,23 S) 8 9 i ii + R = Et3Si R = H 10 3 vii R = Et3Si R = H 11 4 vii viii v,vi 1 iv 2 iii 418 J. CHEM. RESEARCH (S), 1997 J. Chem. Research (S), 1997, 418–419† Synthesis of Cathasterone and its Related Putative Intermediates in Brassinolide Biosynthesis† Suguru Takatsuto,*a Hiroki Kuriyama,b Takahiro Noguchi,b Hiroyuki Suganuma,b Shozo Fujiokac and Akira Sakuraic aDepartment of Chemistry, Joetsu University of Education, Joetsu-shi, Niigata 943, Japan bTama Biochemical Co.Ltd., 2-7-1 Nishishinjuku Shinjuku-ku, Tokyo 163, Japan cThe Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-01, Japan The putative intermediates in brassinolide biosynthesis, 22a-hydroxycampesterol 1, 6-deoxocathasterone and its 6a-hydroxylated compound 3, as well as cathasterone 4, are synthesized from a known (22E,24S)-6b-methoxy-3a,5-cyclo- 5a-ergost-22-ene 5.Using the cultured cells of Catharanthus roseus, we have previously demonstrated the occurrence of two biosynthetic pathways to brassinolide, namely the early C6-oxidation pathway (the sequence: campesterolh6a-hydroxycampestanolh6- oxocampestanol, and the sequence: cathasteroneh t e a s t e r o n e h3 - d e h y d r o t e a s t e r o n e ht y p h a s t e r o l hc a s t a s t e r - onehbrassinolide) and the late C6-oxidation pathway (the sequence: 6-deoxoteasteroneh3-dehydro-6-deoxoteastero n e h6 - d e o x o t y p h a s t e r o l h6 - d e o x o c a s t a s t e r o n e hc a s t a s t e r - onehbrassinolide.1,2 However, the step of campestanol to 6-deoxoteasterone and the step of 6-oxocampestanol to cathasterone 4 remain to be elucidated.In the former step, it is reasonably suggested that 6-deoxocathasterone 2 should be an intermediate, inferred from the natural occurrence of 4 and its conversion to teasterone.3 In addition, by analogy to the conversion of campesterol into 6-oxocampestanol via campestanol and 6a-hydroxycampestanol,4 there is the possibility that 4 would be biosynthesized via 2 and its 6a-hydroxylated compound 3 from 22a-hydroxycampesterol 1, which would be derived from campesterol by 22a-hydroxylation.In order to investigate these possibilities, we have now synthesized these putative intermediates 1–3. Our synthesis is described here along with our alternative synthesis of 4. We have previously synthesized 4 from (22R,23R,24S)- 3b-acetoxy-22,23-epoxy-5a-ergostan-6-one in four steps with epoxide opening by HBr as a key reaction.3 However, in this reaction, the desired 22-hydroxy-23-bromide and the undesired 23-hydroxy-22-bromide were obtained in a ratio of ca. 1:3. Thus, we have now taken an alternative short cut to the 22S-hydroxy-24R-methyl side chain of the target steroids. A known 22-ene compound 55 was epoxidized with m-chloroperbenzoic acid to provide the (22R,23R)-epoxide 6 (70%) and its (22S,23S)-isomer 7 (25%).The epoxide 6 was reduced with AlH3 to afford 22-ol 8 (42%) and 23-ol 9 (49%). When compared with the epoxide opening with HBr, the selectivity for 8 was increased and the desired side chain was completed in one step from 6. The AlH3 reduction of each (22R,23R)-epoxide derived from stigmasterol and brassicasterol has been reported to give a ratio of (26:74) and (66:34) of the corresponding 22-ol and 23-ol, respectively.6 Our present data and the reported data indicate that, possibly because of the steric hindrance, the hydride attacks the C-23 position in the descending order of the (22R,23R)- epoxides derived from brassicasterol, crinosterol and stigmasterol.The 22-ol 8 was subjected to acid treatment to give 1 in 96% yield. Catalytic hydrogenation of 1 provided 2 in 70% yield. After protecting two hydroxy groups as triethylsilyl ethers, the resulting compound was hydroborated and oxi- *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 cathasterone and its related compounds Scheme 1 Reagent and conditions: i, mCPBA, CH2Cl2, room temp., 5 h; ii, LiAlH4–AlCl3, THF, reflux, 2 h; iii, p-TsOH, 1,4-dioxane–H2O, reflux, 4 h; iv, H2, 10% Pd-C, EtOH, 40 °C, 3 h; v, Et3SiCl, pyridine, room temp., 2 h; vi, BH3–THF, THF, room temp., 5 h, then 2 M NaOH, 30% H2O2; vii, (Bu)4NH, THF, room temp., 6 h; viii, Collins reagent, CH2Cl2, room temp., 5 hJ.CHEM. RESEARCH (S), 1997 419 dized with alkaline H2O2 to give 10 in 86% yield. Deprotection of 10 provided 3 in 81% yield. Oxidation of 10 with Collins reagent and deprotection afforded 4 in 55% yield. Identification of the possible biosynthetic intermediates 1–3 from plants and their biological evaluation are now in progress. Experimental Mps are uncorrected. 1H NMR spectra were recorded at 400 MHz on a JEOL a-400 spectrometer in a CDCl3 solution with Me4Si as internal standard. HR-MS were recorded on a HITACHI M-80 or a JEOL HX-110 mass spectrometer. ( 2 2 R , 2 3 R , 2 4 S ) - 2 2 , 2 3 - E p o x y - 6 b- m e t h o x y - 3 a, 5 - c y c l o - 5 a- e r g o s t a n e 6 and its (22S,23S)-Isomer 7.·A known compound 55 (249 mg) in CH2Cl2 (15 cm3) was treated with m-chloroperbenzoic acid (296 mg) at room temp.in the dark for 5 h and then with Ca(OH)2 (380 mg) for 30 min. Work-up and chromatography on silica gel (100 g) with hexane–EtOAc (20:1) as eluent provided 6 (180 mg, 70%) as an oil, dH 1.05 (3 H, d, J 6.1 Hz, 21-H3), 2.50 (1 H, dd, J 7.0 and 2.4 Hz, 22-H), 2.72 (1 H, dd, J 6.4 and 2.4 Hz, 23-H). HR-MS (EI) (Found M+, 428.3645. C29H48O2 requires Mr 428.3651). Further elution gave 7 (64 mg, 25%), mp 81–82 °C (hexane–EtOAc), dH 0.97 (3 H, d, J 4.6 Hz, 21-H3), 2.40–2.50 (2 H, m, 22-H and 23-H).HR-MS (EI) (Found M+, 428.3658. C29H48O2 requires Mr 428.3651). (22S,24R)-6b-Methoxy-3a,5-cyclo-5a-ergostan-22-ol 8 and (23S, 24S)-6b-Methoxy-3a,5-cyclo-5a-ergostan-23-ol 9.·The epoxide 6 (152 mg) in THF (15 cm3) was treated with LiALH4 (160 mg) and AlCl3 (213 mg) at reflux under Ar for 2 h. Work-up (diethyl ether) and chromatography on silica gel (25 g) with toluene–EtOAc (50:1) afforded 8 (64 mg, 42%), mp 43.5–45 °C (toluene–EtOAc), dH 0.88 (3 H, d, J 6.8 Hz, 28-H3), 0.89 (3 H, d, J 6.3 Hz, 21-H3), 3.78 (1 H, t, J 6.4 Hz, 22-H).HR-MS (EI) (Found M+, 430.3818. C29H50O2 requires Mr 430.3808). Further elution gave 9 (75 mg, 49%), mp 90.5–91 °C (toluene–EtOAc), dH 0.96 (3 H, d, J 6.7 Hz, 28-H3), 0.97 (3 H, d, J 6.4 Hz, 21-H3), 3.89 (1 H, m, 23-H). HR-MS (EI) (Found M+, 430.3799. C29H50O2 requires Mr 430.3808). (22S,24R)-3b,22-Dihdyroxyergost-5-ene 1.·The 22-ol 8 (250 mg) in 1,4-dioxane (5 cm3) and H2O (0.75 cm3) was refluxed with p-TsOH (5 mg) for 4 h.Work-up (EtOAc) and chromatography on silica gel (20 g) with toluene–EtOAc (4:1) as eluent gave 1 (233 mg, 96%), mp 193–195 °C (toluene–EtOAc), dH 0.70 (3 H, s, 18-H3), 0.81 (3 H, d, J 6.7 Hz, 26-H3), 0.83 (3 H, d, J 6.7 Hz, 27-H3), 0.88 (3 H, d, J 6.7 Hz, 28-H3), 0.89 (3 H, d, J 6.7 Hz, 21-H3), 1.01 (3 H, s, 19-H3), 3.52 (1 H, m, 3a-H), 3.78 (1 H, t, J 6.6 Hz, 22-H), 5.35 (1 H, m, 6-H). HR-MS (EI) (Found M+, 416.3655. C28H48O2 requires Mr 416.3652).(22S,24R)-3b,22-Dihydroxy-5a-ergostane 2.·The 5-ene 1 (30 mg) in EtOH (3 cm3) was treated with 10% Pd–C (70 mg) under H2 at 40 °C overnight. Work-up and chromatography on silica gel (20 g) with toluene–EtOAc (5:1) as eluent gave 2 (21 mg, 70%), mp 200–202 °C (toluene–EtOAc), dH 0.67 (3 H, s, 18-H3), 0.81 (3 H, s, 19-H3), 0.81 (3 H, d, J 6.7 Hz, 26-H3), 0.83 (3 H, d, J 6.7 Hz, 27-H3), 0.88 (6 H, dÅ2, J 7.0 Hz, 21-H3 and 28-H3), 3.59 (1 H, ddd, J 15.9, 10.8 and 4.9 Hz, 3a-H), 3.77 ( H, t, J 6.4 Hz, 22-H).HR-MS (EI) (Found M+, 418.3812. C28H50O2 requires Mr 418.3808). (22S,24R)-3b-22Bis(triethylsiloxy)-5a-ergostan-6a-ol 11.·The 5-ene 1 (230 mg) in pyridine (5 cm3) was treated with Et3SiCl (0.58 cm3) at room temp. for 2 h. Work-up (diethyl ether) and chromatography on silica gel (25 g) with hexane–EtOAc (25:1) as eluent gave the corresponding silyl ether (322 mg, 90%), mp 126–127 °C (hexane–EtOAc), dH 0.96 (18 H, tÅ6, J 7.9 Hz, CH2CH3), 3.47 (1 H, ddd, J 15.6, 10.7 and 4.7 Hz, 3a-H), 3.76 (1 H, dd, J 9.8 and 4.7 Hz, 22-H), 5.32 (1 H, m, 6-H).HR-MS (EI) (Found M+, 644.5399. C40H76O2Si2 requires Mr 644.5380). This compound (236 mg) in THF (6 cm3) was reacted with 1 M BH3–THF complex in THF (1.2 cm3) at room temp. for 5 h. After treating with H2O (0.5 cm3), the mixture was treated with 2 M NaOH (0.4 cm3) and 30% H2O2 (0.4 cm3) for 30 min. Work-up (diethyl ether) and chromatography on silica gel (25 g) with toluene–EtOAc (100:1) provided 10 (231 mg, 95%), mp 64–65 °C (toluene–EtOAc), dH 0.96 (9 H, tÅ3, J 7.9 Hz, CH2CH3), 0.96 (9 H, tÅ3, J 7.9 Hz, CH2CH3), 3.40 (1 H, ddd, J 10.4, 10.4 and 4.3 Hz, 3a-H), 3.53 (1 H, ddd, J 15.6, 10.7 and 4.7 Hz, 6b-H), 3.76 (1 H, dd, J 9.5 and 4.3 Hz, 22-H).HR-MS (EI) (Found M+, 662.5511. C40H78O3Si2 requires Mr 662.5485). (22S,24R)-5a-Ergostan-3b,6a,22-triol 3.·Compound 10 (30 mg) in THF (2 cm3) was treated with 1 M (Bu)4NF in THF (0.3 cm3) at room temp.for 6 h. Work-up (EtOAc) and chromatography on silica gel (25 g) with hexane–EtOAc (1:3) as eluent gave 3 (16 mg, 81%), mp 225-226.5 °C (hexane–EtOAc), dH 0.67 (3 H, s, 18-H3), 0.81 (3 H, d, J 7.8 Hz, 26-H3), 0.82 (3 H, s, 18-H3), 0.83 (3 H, d, J 6.8 Hz, 27-H3), 0.88 (3 H, d, J 6.8 Hz, 28-H3), 0.88 (3 H, d, J 6.3 Hz, 21-H3), 3.42 (1 H, ddd, J 10.7, 10.7 and 4.4 Hz, 6b-H), 3.59 (1 H, ddd, J 16.1, 11.2 and 4.9 Hz, 3a-H), 3.77 (1 H, t, J 6.6 HJz, 22-H). HR-MS (negative-FAB) (Found [MµH]µ, 433.3698.C28H49O3 requires Mr 433.3684). (22S,24R)-3b,22-Dihydroxy-5a-ergostan-6-one 4.·The 6a-ol 10 (114 mg) in CH2Cl2 (5 cm3) was treated with Collins reagent (598 mg) at room temp. for 5 h. Work-up (diethyl ether) and chromatography on silica gel (25 g) with toluene–EtOAc (150:1) as eluent gave the cathasterone bis-triethylsilyl ether 11 (75 mg, 66%), mp 188–190 °C (toluene–EtAOc), dH 0.95 (9 H, t, J 7.9 Hz, CH2CH3), 0.97 (9 H, t, J 7.9 Hz, CH2CH3), 3.51 (1 H, m, 3a-H), 3.76 (1 H, dd, J 9.8 and 4.6 Hz, 22-H).HR-MS (EI) (Found M+, 660.5333. C40H76O3Si2 requires Mr 660.5329). The ether 11 (73 mg) was deprotected as described above and a crude product was purified by chromatography on silica gel (25 g) with hexane–EtOAc (1:2) as eluent to provide 4 (40 mg, 83%), mp 183.5–185 °C (MeOH) (lit.,3 mp 176–177 °C), dC (CDCl3) 11.18, 11.19, 13.13, 15.77, 17.81, 19.95, 21.52, 23.90, 27.64, 30.02, 30.68, 32.01, 35.32, 36.62, 37.94, 39.29, 39.44, 39.48, 40.93, 42.87, 46.68, 52.48, 53.85, 56.65, 56.73, 70.67, 71,59, 210.84. HR-MS (EI) (Found M+, 432.3610. C28H48O3 requires Mr 432.3601). Its 1H NMR and EI-MS spectral data are in good agreement with the reported data.3 We thank Mr Tetsu-ichiro Morita of The Institute of Physical and Chemical Research (RIKEN) for the measurement of NMR spectra. Received, 7th July 1997; Accepted, 31st July 1997 Paper E/7/04788F References 1 S. Fujioka and A. Sakurai, Nat. Prod. Rep., 1997, 1. 2 A. Sakurai and S. Fujioka, Biosci. Biotechnol. Biochem., 1997, 61, 757 3 S. Fujioka, T. Inoue, S. Takatsuto, T. Yanagisawa, T. Yokota and S. Sakurai, Biosci. Biotechnol. Biochem., 1995, 59, 1543. 4 H. Suzuki, T. Inoue, S. Fujioka, T. Saito, S. Takatsuto, T. Yokota, N. Murofushi, T. Yanagisawa and S. Sakurai, Phytochemistry, 1995, 40, 1391. 5 S. Takatsuto, T. Watanabe, S. Fujioka and A. Sakurai, J. Chem. Res., 1997, (S) 134; (M) 0901. 6 J.-L. Giner, C. Margot and C. Djerssai, J. Org. Chem., 1989, 54, 369.