Special Collection of Papers: Regular Articles
Chemical Conversion of Ryanodol to Ryanodine
2016 年 64 巻 7 号 p. 874-879
詳細
2016 年 64 巻 7 号 p. 874-879
Ryanodine (1) is a plant-derived natural product with powerful pharmacological and insecticidal action, and is a potent modulator of intracellular calcium release channels. Compound 1 possesses a 1H-pyrrole-2-carboxylate ester at the C3-position of heptahydroxylated terpenoid ryanodol (2). Whereas 2 was readily obtained from 1 by basic hydrolysis, 1 has never been synthesized from 2, due to the extreme difficulty in selectively introducing the bulky pyrrole moiety at the severely hindered C3-hydroxyl group of heptaol 2. Here we report chemical conversion of 2 to 1 for the first time. The derivatization was realized through the use of a new protective group strategy and the application of on-site construction of the pyrrole-2-carboxylate ester from the glycine ester and 1,3-bis(dimethylamino)allylium tetrafluoroborate.
Ryanodine (1, Chart 1) has been isolated from the wood of Ryania speciosa VAHL and shown to possess potent insecticidal and pharmacological actions.1) Compound 1 binds specifically to a high-conductance intracellular calcium channel known as the ryanodine receptor (RyR), altering its conductance.2,3) Since the RyR plays an important role in many biological processes and malfunctions in its action have been linked to skeletal and cardiac diseases,4–7) 1 has been used as a biological probe for investigations of RyR function, and is regarded as a strong candidate in the development of therapeutic agents for various diseases.8–10)
In 1968, the structure of ryanodine (1) was determined to be the ester between 1H-pyrrole-2-carboxylic acid (3) and the C3-hydroxyl group of ryanodol (2).11–14) Compound 2 is a diterpenoid containing the exceedingly complex five fused rings (ABCDE-rings) with eleven contiguous stereocenters. Interestingly, the affinity of 2 toward RyRs of vertebrate skeletal muscle is 1700 times weaker than that of 1 (KD=7 nM),15–17) demonstrating that the pyrrole group is a primary determinant for strong interactions of 1. Because of its important biological activities and intriguing structural features, many studies attempting the chemical construction of 1 has been carried out, which resulted in the total syntheses of ryanodol (2) by the Deslongchamps group in 197918,19) and by our laboratory in 2014.20–22)
In 1951, before the complete structure of 1 was known, Wiesner and colleagues found that basic hydrolysis of ryanodine (1) generated 2 and 3.11) However, the reverse reaction has not been realized even to date. The difficulty of the transformation arises from the need to control the regioselectivity of the four tertiary (C2, 4, 6, 12), two secondary (C3, 10) and one acetalic (C15) alcohols within heptaol 2 and to accommodate the bulky pyrrole moiety at the hindered C3-hydroxyl group. The computer-generated23) structure of 2 revealed that its axially oriented C3-OH was shielded by the proximal C14-hydrogen atom and by the C19-, 20-methyl groups (Fig. 1). In fact, direct esterification (2+3→1, Chart 1) was attempted by Ruest and Deslongchamps, but was not successful: Treatment of 2 and 3 with dicyclohexyl carbodiimide (DCC) and N,N-dimethylamino pyridine (DMAP) led to condensation at the more accessible equatorially oriented C10-OH to afford the regioisomer 4.24)
In 2016, we accomplished the first total synthesis of ryanodine (1) by developing a novel synthetic strategy for installation of the pyrrole-2-carboxylate ester at C3-OH.25,26) Pentaol 7, which was constructed from 2,5-dimethylbenzene-1,4-diol (8), was regioselectively protected as the two boronate esters to yield 6. The remaining C3-hydroxy group of 6 then was conjugated with the less sterically cumbersome glycine A,27) followed by construction of the pyrrole ring on-site via reaction with 1,3-bis(dimethylamino)allylium tetrafluoroborate B.28) The thus synthesized 5 was transformed to 1 by treating with KHF2 and subsequently with Pd/C under an H2 atmosphere. This stepwise protocol for attachment of the pyrrole moiety was expected to allow preparation of 1 from 2, although the heptaol structure of 2 would further complicate the protective group manipulations as compared to pentanol 7. The present report describes the chemical conversion of 2 to 1 for the first time. Problems in controlling the regioselectivity of heptaol 2 were overcome successfully by judicious selection of the protective groups and proper arrangement of the order of their introductions.
The regioselective protection of heptaol 2 started with formation of the boronate ester by taking advantage of the proximal relationship of the syn-oriented tertiary hydroxy groups at C2, 4, 6, and 1229) (Chart 2). Namely, 2 was treated with phenyl boronic acid30) to produce bis-boronate ester 9. Interestingly, upon purification with silica gel, 9 was partially deprotected to afford mono-boronate ester 10, the structure of which was determined by the nuclear Overhauser effect (NOE) correlation between the protons of the phenyl group and the C3/10-methine protons.31) As full protection of the tetraol on the top face from 2 was ineffective, 10 was employed as the intermediate for synthesis of targeted 1. Thus, the combination of reagents Ac2O and DMAP regioselectively transformed the least sterically hindered equatorial C10-hydroxy group of pentaol 10 to the corresponding acetate of 11. The remaining C3-secondary alcohol of tetraol 11 was condensed selectively over the three tertiary alcohols with N,N-Boc2-glycine (A), 2,4,6-trichlorobenzoyl chloride, DMAP, and Et3N,32,33) giving rise to 12.34) Next, an attempt was made to convert the amino methyl group in a pyrrole ring. After treatment of 12 with CF3CO2H to detach the two Boc groups, amine 13 was subjected to optimized conditions for preparation of 5 (see Chart 1) [B and Et3N at 150°C in MeCN].35,36) However, reaction of 13 resulted in a complex mixture of products, and did not furnish 14.
Reagents and conditions: (a) PhB(OH)2, benzene–MeOH, rt; SiO2, 91%; (b) Ac2O, N,N-dimethylamino pyridine (DMAP), pyridine, rt, 72%; (c) A, 2,4,6-trichlorobenzoyl chloride, DMAP, Et3N, toluene, rt, 34%; (d) CF3CO2H, CH2Cl2, rt; (e) B, Et3N, MeCN, 150°C (microwave); (f) NaH, BnCl, DMF, –15°C, 90%; (g) Ac2O, DMAP, pyridine, rt, 59%; (h) PhB(OH)2, benzene, rt, 63%; (i) A, 2,4,6-trichlorobenzoyl chloride, DMAP, Et3N, toluene, rt, 71% (calculated yield); (j) CF3CO2H, CH2Cl2, rt; (k) B, Et3N, MeCN, 150°C (microwave); (l) aqueous KHF2, MeOH, rt, 39% (four steps from 17); (m) K2CO3, MeOH, rt; (n) H2, Pd/C, MeOH/EtOAc, rt, 78% (two steps from 21).
The unsuccessful on-site pyrrole formation from 13 was attributable to the unique reactivity of the ryanodine skeletons: dehydrative Grob-type fragmentation readily occurs upon activation of the C2-OH37) (Chart 3). Therefore, the electrophilic reagent B was assumed to react with the C2-OH of 13 and trigger the scission of the C2-O and C1–C15 bonds through the reactive intermediate D to generate E, which would decompose further under the reaction conditions. Based on this consideration, we decided to protect the free C2- and C15-hydroxy groups prior to pyrrole construction to prevent the undesired fragmentation.
Protection tactics were developed for preparation of the alternative substrate 17 from 10, in which all the hydroxy groups were capped except for the C3-OH (Chart 2). By exploiting the lower pKa value of the hemiacetal compared to that of the secondary or tertiary alcohol, the C15-OH of pentaol 10 was regioselectively benzylated at −15°C using the reagent combination of BnCl and a strong base (NaH), leading to tetraol 15.38) Although the less acidic, yet most sterically exposed, C10-secondary hydroxyl group did not react in this benzylation, C10-acetylation of 15 proceeded smoothly in the presence of Ac2O and DMAP to furnish triol 16. At this stage, we found that bis-boronate ester 17 was formed from mono-boronate ester 16 by the action of phenyl boronic acid and was inert toward silica gel. The greater stability of 17 compared to that of 9 can be explained by the difference in the substitutions at the C10- and C15-hydroxy groups. While strong interaction of polar 9 with silica gel could facilitate the intermolecular nucleophilic addition of the silanol residue to the boron atom of 9, such undesired reaction from less polar 17 would be slower due to its weaker coordination to the silica gel surface.39) Hence, the chemically stable and fully protected C3-alcohol 17 was synthesized from ryanodol (2) in four steps.
Three-step installation of the pyrrole-2-carboxylic acid ester at the C3-position and three-step detachment of the protecting groups completed the synthesis of 1 from 17 (Chart 2). The glycine ester of 19 was appended at the C3-secondary hydroxy group by condensation between 17 and N,N-Boc2-glycine (A) under the Yamaguchi conditions, followed by CF3CO2H-promoted Boc-removal from 18. Most importantly, treatment of amine 19 with B and Et3N at 150°C under microwave irradiation indeed resulted in on-site formation of the pyrrole ring to afford 20 in high yield. During the reaction, the amino group of 19 first added to bis-electrophile B, and subsequent deprotonation, intramolecular Mannich-type reaction of C, and ejection of dimethylamine provided the requisite pyrrole-2-carboxylate ester. Next, all six hydroxyl groups were liberated. The two phenyl boronate moieties of 20 were removed with aqueous KHF2 in methanol40) to generate 21. Finally, the C13-acetate ester of 21 was chemoselectively saponified in the presence of the C3-ester using K2CO3 in MeOH, and the C15-benzyl ether of 22 was detached using H2 and Pd/C in MeOH, delivering ryanodine (1). All of the spectral and physical data of thus obtained 1 were identical to those of naturally occurring (+)-ryanodine.
In summary, the first synthesis of ryanodine (1) from ryanodol (2) was accomplished in 10 steps by regioselectively installing the pyrrole-2-carboxylic acid ester at the extremely hindered C3-secondary hydroxy group. In doing so, three types of protective groups (PhB, Ac, Bn) were judiciously applied to cap the six hydroxy groups of heptaol 2. Then, the remaining C3-OH was converted to the less sterically cumbersome glycine ester, which was transformed into the pyrrole ring via condensation with 1,3-bis(dimethylamino)allylium tetrafluoroborate B. Finally, three-step chemoselective deprotection furnished hexaol 1. The new methodology and protection tactics described here will enable generation of chemical derivatives of this exceedingly complex structure41–44) and have further application to total syntheses of biologically active natural products with pyrrole esters.45–47)
To prevent formation of the stable borate esters with B2O3 leached from borosilicate glassware, soda-lime and quartz glassware were used for all reactions and purification. All reactions sensitive to air or moisture were carried out under argon atmosphere in dry solvents under anhydrous conditions, unless otherwise noted. CH2Cl2, N,N-dimethylformamide (DMF) and toluene were purified by Glass Contour solvent dispensing system (Nikko Hansen & Co., Ltd., Japan). All other reagents were used as supplied. The microwave irradiation experiments were performed with Initiator System (Biotage Japan Co., Ltd.). Analytical TLC was performed using E. Merck Silica gel 60 F254 pre-coated plates (0.25 mm). Preparative thin-layer chromatography (PTLC) was performed using Merck silica gel 60 F254 pre-coated plates (0.5 mm). Flash chromatography was performed using 40–100 µm Silica Gel 60N (Kanto Chemical Co., Inc.), unless otherwise noted. Optical rotations were measured on JASCO P-2200 Digital Polarimeter at room temperature using the sodium D line. Melting points were measured on Yanaco MP-J3 micro melting point apparatus, and were uncorrected. Infrared (IR) spectra were recorded on JASCO FT/IR-4100 spectrometer. 1H- and 13C-NMR spectra were recorded on JNM-ECS-400 spectrometer. Chemical shifts were reported in ppm on the δ scale relative to CHCl3 (δ=7.26 for 1H-NMR), CDCl3 (δ=77.0 for 13C-NMR), C6D5H (δ=7.16 for 1H-NMR), C6D6 (δ=128.0 for 13C-NMR), CD2HOD (δ=3.31 for 1H-NMR), and CD3OD (δ=49.0 for 13C-NMR) as internal references. Signal patterns are indicated as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broaden peak. The numbering of compounds corresponds to that of ryanodine (1). High resolution (HR)-MS were measured JEOL JMS-T100LP.
Boronate 10A solution of (+)-ryanodol (2, 8.2 mg, 20 µmol) and PhB(OH)2 (7.5 mg, 61 µmol) in benzene (1 mL) and MeOH (1 mL) was stirred for 5 min at room temperature. The reaction mixture was concentrated. The residue was loaded on PTLC for 40 h at room temperature to complete the conversion to boronate 10. Then, the PTLC was developed (CHCl3–MeOH 10 : 1) to obtain boronate 10 (9.1 mg, 19 µmol) in 91% yield: colorless solid; mp >300°C; [α]D23 −25 (c=0.46, MeOH); IR (film) 3366, 2955, 2925, 2854, 1736, 1714, 1458, 1375, 1259 cm−1; 1H-NMR (400 MHz, CD3OD) δ: 1.045 (3H, d, J=6.3 Hz, H18) 1.049 (3H, d, J=6.3 Hz, H21), 1.08 (3H, s, H20), 1.12 (3H, d, J=6.8 Hz, H19), 1.33 (3H, s, H17), 1.41 (1H, ddd, J=12.7, 4.1, 2.3 Hz, H7a), 1.66 (1H, dddd, J=12.2, 5.0, 4.5, 2.3 Hz, H8a), 1.75 (1H, dddd, J=12.2, 12.2, 12.2, 4.1 Hz, H8b), 1.83 (1H, ddqd, J=12.2, 10.0, 6.3, 4.5 Hz, H9), 1.92 (1H, d, J=13.1 Hz, H14a), 2.03 (1H, ddd, J=12.7, 12.2, 5.0 Hz, H7b), 2.24 (1H, qq, J=6.8, 6.3 Hz, H13), 2.59 (1H, d, J=13.1 Hz, H14b), 3.65 (1H, d, J=10.0 Hz, H10), 4.42 (1H, s, H3), 7.32 (2H, ddd, J=7.3, 7.3, 0.9 Hz, m-H2C6H3B), 7.42 (1H, tt, J=7.3, 1.4 Hz, p-HC6H4B), 7.78 (2H, dd, J=7.3, 1.4 Hz, o-H2C6H3B); 13C-NMR (100 MHz, CD3OD) δ: 10.2, 13.6, 19.0, 19.4, 19.5, 25.7, 29.4, 30.9, 35.4, 40.9, 46.9, 64.6, 72.6, 85.5, 87.3, 88.2, 89.0, 92.5, 97.2, 103.7, 128.5 (2C), 131.9, 135.1 (2C) (ipso carbon peak of phenyl boronate was not observed); HR-MS (electrospray ionization (ESI)) Calcd for C26H35BNaO8 [M+Na]+ 509.2317. Found 509.2324.
Benzyl Ether 15NaH in mineral oil (purity 50–72%) was washed with n-hexane, and dried in vacuo. The purified NaH (18 mg, 0.76 mmol) was added to a solution of boronate 10 (7.4 mg, 15 µmol) in DMF (1.5 mL) at –40°C. After the resultant mixture was stirred for 10 min at the same temperature, BnCl (18 µL, 0.15 mmol) was added. The reaction mixture was stirred for 20 h at −15°C. The reaction was quenched with saturated aqueous NH4Cl (2 mL). The resultant solution was extracted with CHCl3–i-PrOH 3 : 1 (3 mL×7), and the combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by PTLC on silica gel (CHCl3–MeOH 10 : 1) to afford benzyl ether 15 (7.9 mg, 14 µmol) in 90% yield: colorless solid; mp 96–97°C; [α]D24 −0.43 (c=0.40, MeOH); IR (film) 3446, 2960, 2928, 2876, 1455, 1440, 1360, 1330 cm−1; 1H-NMR (400 MHz, CD3OD) δ: 1.05 (3H, d, J=6.3 Hz, H18), 1.06 (3H, d, J=6.3 Hz, H21), 1.09 (3H, d, J=6.8 Hz, H19), 1.11 (3H, s, H20), 1.39 (3H, s, H17), 1.46 (1H, ddd, J=12.7, 4.1, 2.3 Hz, H7a), 1.70 (1H, dddd, J=12.7, 5.0, 4.5, 2.3 Hz, H8a), 1.77 (1H, dddd, J=12.7, 12.7, 12.2, 4.1 Hz, H8b), 1.90 (1H, ddqd, J=12.7, 10.4, 6.3, 4.5 Hz, H9), 2.04 (1H, ddd, J=12.7, 12.2, 5.0 Hz, H7b), 2.25 (1H, qq, J=6.8, 6.3 Hz, H13), 2.34 (1H, d, J=13.1 Hz, H14a), 2.43 (1H, d, J=13.1 Hz, H14b), 3.68 (1H, d, J=10.4 Hz, H10), 4.44 (1H, s, H3), 4.75 (1H, d, J=11.8 Hz, PhCHAHB), 4.80 (1H, d, J=11.8 Hz, PhCHAHB), 7.25 (1H, tt, J=6.3, 1.4 Hz, aromatic), 7.30–7.40 (6H, m, aromatic), 7.42 (1H, tt, J=6.3, 1.4 Hz, p-HC6H4B), 7.78 (2H, dd, J=7.7, 1.4 Hz, o-H2C6H3B); 13C-NMR (100 MHz, CD3OD) δ: 10.5, 13.8, 19.0, 19.4 (2C), 25.8, 29.4, 31.1, 35.4, 36.4, 46.9, 66.3, 66.9, 72.6, 85.5, 88.0, 88.2, 88.9, 92.5, 97.2, 106.7, 128.3 (3C), 128.5 (2C), 129.2 (2C), 131.9, 135.1 (2C), 140.2 (ipso carbon peak of phenyl boronate was not observed); HR-MS (ESI) Calcd for C33H41BNaO8 [M+Na]+ 599.2787. Found 599.2794.
Acetate 16Ac2O (130 µL, 1.4 mmol) was added to a solution of benzyl ether 15 (7.9 mg, 14 µmol) and DMAP (5 mg, 40 µmol) in pyridine (1.4 mL) at room temperature. After being stirred for 1 h at room temperature, the reaction mixture was quenched with saturated aqueous NaHCO3 (5 mL) at 0°C. The resultant solution was extracted with CHCl3–i-PrOH 3 : 1 (5 mL×7). The combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by PTLC on silica gel (CHCl3–MeOH 50 : 1, ×2) to afford acetate 16 (5.0 mg, 8.1 µmol) in 59% yield: colorless solid; mp 91–92°C; [α]D24 +16 (c=0.25, MeOH); IR (film) 3502, 2962, 2931, 2876, 1718, 1331, 1254, 1127 cm−1; 1H-NMR (400 MHz, CD3OD) δ: 0.90 (3H, d, J=6.3 Hz, H21), 1.03 (3H, d, J=6.8 Hz, H18), 1.08 (3H, d, J=6.8 Hz, H19), 1.11 (3H, s, H20), 1.36 (3H, s, H17), 1.50 (1H, ddd, J=12.7, 4.1, 2.3 Hz, H7a), 1.74 (1H, dddd, J=13.1, 5.0, 4.5, 2.3 Hz, H8a), 1.86 (1H, dddd, J=13.1, 12.7, 12.7, 4.1 Hz, H8b), 2.04 (3H, s, COCH3), 2.05 (1H, ddqd, J=12.7, 10.9, 6.3, 4.5 Hz, H9), 2.07 (1H, ddd, J=12.7, 12.7, 5.0 Hz, H7b), 2.23 (1H, qq, J=6.8, 6.8 Hz, H13), 2.35 (1H, d, J=13.6 Hz, H14a), 2.44 (1H, d, J=13.6 Hz, H14b), 4.38 (1H, s, H3), 4.76 (1H, d, J=11.3 Hz, PhCHAHB), 4.80 (1H, d, J=11.3 Hz, PhCHAHB), 5.16 (1H, d, J=10.9 Hz, H10), 7.27 (1H, tt, J=6.3, 1.4 Hz, p-HC6H4CH2), 7.30–7.42 (6H, m, aromatic), 7.43 (1H, tt, J=6.3, 1.4 Hz, p-HC6H4B), 7.81 (2H, dd, J=8.2, 1.4 Hz, o-H2C6H3B); 13C-NMR (100 MHz, CD3OD) δ: 10.6, 13.9, 18.4, 19.36, 19.39, 21.4, 25.8, 29.1, 31.1, 34.4, 36.3, 46.8, 66.5, 67.0, 74.1, 85.6, 87.8, 88.1, 88.5, 92.7, 96.6, 106.8, 128.37, 128.44 (2C), 128.5 (2C), 129.2 (2C), 131.9, 135.2 (2C), 140.2, 172.8 (ipso carbon peak of phenyl boronate was not observed); HR-MS (ESI) Calcd for C35H43BNaO9 [M+Na]+ 641.2892. Found 641.2899.
Bis-boronate 17PhB(OH)2 (10 mg, 82 µmol) was added to a solution of acetate 16 (5.0 mg, 8.1 µmol) in benzene (2 mL). The reaction mixture was stirred for 5 min at room temperature, and then concentrated. The crude was dissolved in benzene (2 mL) and concentrated three times to complete the boronate formation. The residue was purified by PTLC on silica gel (CHCl3–MeOH 50 : 1) to afford bis-boronate 17 (3.6 mg, 5.1 µmol) in 63% yield: colorless solid; mp 93–94°C; [α]D27 −32 (c=0.18, CHCl3); IR (film) 3491, 2962, 2930, 2877, 1719, 1335, 1250, 1094 cm−1; 1H-NMR (400 MHz, CDCl3) δ: 0.96 (3H, d, J=6.8 Hz, H21), 1.13 (3H, s, H20), 1.28 (3H, d, J=6.8 Hz, H18), 1.29 (3H, d, J=6.8 Hz, H19), 1.34 (3H, s, H17), 1.57 (1H, m, H7a), 1.77 (1H, m, H8a), 1.84 (3H, s, COCH3), 1.93 (1H, dddd, J=12.7, 12.7, 12.7, 4.1 Hz, H8b), 2.08 (1H, m, H9), 2.08 (1H, ddd, J=12.7, 12.7, 4.5 Hz, H7b), 2.19 (1H, qq, J=6.8, 6.8 Hz, H13), 2.20 (1H, d, J=6.3 Hz, OH), 2.42 (1H, d, J=13.6 Hz, H14a), 2.85 (1H, d, J=13.6 Hz, H14b), 4.66 (1H, d, J=6.3 Hz, H3), 4.79 (1H, d, J=11.3 Hz, PhCHAHB), 4.89 (1H, d, J=11.3 Hz, PhCHAHB), 5.42 (1H, d, J=10.9 Hz, H10), 7.28–7.46 (11H, m, aromatic), 7.72–7.80 (4H, m, aromatic); 13C-NMR (100 MHz, CDCl3) δ: 10.3, 13.1, 17.0, 17.4, 17.8, 21.1, 24.7, 27.9, 31.6, 32.9, 35.3, 45.6, 62.8, 65.9, 71.7, 85.3, 85.6, 87.6, 90.9, 93.9, 96.3, 105.8, 127.0 (2C), 127.4, 127.5 (2C), 127.6 (2C), 128.3 (2C), 131.07, 131.14, 134.2 (2C), 134.4 (2C), 138.2, 170.0 (two ipso carbon peaks of phenyl boronate were not observed); HR-MS (ESI) Calcd for C41H46B2NaO9 [M+Na]+ 727.3220. Found 727.3236.
Glycine Ester 182,4,6-Trichlorobenzoyl chloride (24 µL, 0.15 mmol) was added to a suspension of bis-boronate 17 (3.6 mg, 5.1 µmol), N,N-Boc2-glycine (A, 28 mg, 0.10 mmol), DMAP (19 mg, 0.16 mmol) and Et3N (32 µL, 0.23 mmol) in toluene (1.7 mL) at room temperature. The reaction mixture was stirred for 20 h at the same temperature, and then treated with saturated aqueous NaHCO3 (3 drops). The resultant mixture was dried over Na2SO4, filtered through a short pad of silica gel (0.5 g, Et2O), and concentrated. The residue was purified by PTLC on silica gel (n-hexane–EtOAc 5 : 1) to afford 1.00 : 1.06 inseparable mixture (5.3 mg) of glycine ester 18 (3.6 µmol, 71% calculated yield) and the byproduct N,N,N′,N′-Boc4-1,3-diaminoacetone (3.8 µmol), which was used in the next reaction without further purification. The analytical data of 18 was collected by using the mixture: 1H-NMR (400 MHz, C6D6) δ: 1.08 (3H, s, H20), 1.10 (3H, d, J=6.3 Hz, H21), 1.37 (1H, m, H7a), 1.38 (3H, d, J=7.3 Hz, H18), 1.38 (3H, d, J=6.8 Hz, H19), 1.39 (18H, s, CH3×6 of Boc), 1.57 (1H, ddd, J=13.3, 13.3, 4.6 Hz, H7b), 1.60 (6H, s, H17 and COCH3), 1.62 (1H, m, H8a), 2.06 (1H, dddd, J=13.3, 13.2, 13.2, 4.1 Hz, H8b), 2.24 (1H, m, H9), 2.25 (1H, qq, J=7.3, 6.8 Hz, H13), 2.35 (1H, d, J=13.6 Hz, H14a), 2.69 (1H, d, J=13.6 Hz, H14b), 4.41 (1H, d, J=17.7 Hz, NCHAHBC), 4.51 (1H, d, J=17.7 Hz, NCHAHBC), 4.65 (1H, d, J=11.3 Hz, PhCHAHB), 4.85 (1H, d, J=11.3 Hz, PhCHAHB), 5.92 (1H, d, J=10.9 Hz, H10), 6.36 (1H, s, H3), 7.03–7.10 (4H, m, aromatic), 7.11–7.17 (2H, m, aromatic), 7.19–7.25 (3H, m, aromatic), 7.39–7.43 (2H, m, aromatic), 7.97–8.01 (2H, m, aromatic), 8.02–8.06 (2H, m, aromatic); 13C-NMR (100 MHz, C6D6) δ: 10.5, 12.7, 17.17, 17.24, 18.1, 20.7, 25.1, 27.9 (6C), 28.3, 32.4, 33.4, 35.8, 45.7, 47.6, 63.4, 66.4, 71.5, 83.0 (2C), 84.7, 85.9, 87.9, 91.2, 94.0, 97.3, 106.0, 128.7 (2C), 131.5, 131.7, 134.8 (2C), 135.1 (2C), 138.3, 152.5 (2C), 167.6, 169.5 (two ipso carbon peaks of phenyl boronate were not observed; other four aromatic peaks (9 carbons) overlapped with the solvent peaks); HR-MS (ESI) Calcd for C53H65B2NNaO14 [M+Na]+ 984.4483. Found 984.4493.
Analytical Data of N,N,N′,N′-Boc4-1,3-Diaminoacetone1H-NMR (400 MHz, C6D6) δ: 1.39 (36H, s), 4.32 (4H, s); 13C-NMR (100 MHz, C6D6) δ: 27.9 (12C), 52.5 (2C), 82.5 (4C), 152.3 (4C), 198.9; HR-MS (ESI) Calcd for C23H40N2O9Na [M+Na]+ 511.2626. Found 511.2623.
10-Acetyl-15-benzylryanodine (21)Trifluoroacetic acid (60 µL) was added to a solution of the 1.00 : 1.06 mixture (5.3 mg) of glycine ester 18 and the byproduct N,N,N′,N′-Boc4-1,3-diaminoacetone in CH2Cl2 (1.2 mL) at room temperature. The reaction mixture was stirred for 90 min at the same temperature. The resultant solution was diluted with toluene (3 mL), and concentrated to afford the crude amine trifluoroacetate salt 19 (5 mg), which was used in the next reaction without further purification.
A solution of the above crude amine trifluoroacetate salt 19 (5 mg), 1,3-bis(dimethylamino)allylium tetrafluoroborate B (15 mg, 70 µmol) and Et3N (50 µL, 0.36 mmol) in MeCN (0.7 mL) was stirred for 18 h at 150°C under microwave irradiation in a sealed tube. After being cooled to room temperature, the resultant mixture was concentrated. The resultant mixture was filtered through a short pad of silica gel (0.5 g) with EtOAc, and the filtrate was concentrated to afford the crude pyrrole-carboxylic acid ester 20 (5 mg), which was used in the next reaction without further purification.
Aqueous KHF2 (3.0 M, 60 µL, 0.18 mmol) was added to a solution of the crude pyrrole-carboxylic acid ester 20 (5 mg) in MeOH (1 mL) at room temperature. The reaction mixture was stirred for 4 h at the same temperature. After addition of EtOH (1 mL), the resultant solution was directly concentrated. The residue was purified by PTLC on silica gel (CHCl3–MeOH 20 : 1, ×2) to afford 10-acetyl-15-benzylryanodine (21, 1.24 mg, 1.98 µmol) in 39% yield over 4 steps from 17: colorless solid; mp 106–107°C; [α]D27 +71 (c=0.062, MeOH); IR (film) 3342, 2967, 2930, 2876, 1720, 1694, 1409, 1256, 1165, 1121, 1081, 1032 cm−1; 1H-NMR (400 MHz, CD3OD) δ: 0.74 (3H, d, J=6.3 Hz, H18), 0.87 (3H, d, J=6.8 Hz, H21), 0.92 (3H, s, H20), 1.07 (3H, d, J=6.3 Hz, H19), 1.36 (1H, ddd, J=12.7, 4.5, 2.3 Hz, H7a), 1.43 (3H, s, H17), 1.60 (1H, dddd, J=13.1, 12.7, 12.7, 4.5 Hz, H8b), 1.61 (1H, m, H8a), 2.08 (1H, m, H9), 2.09 (3H, s, COCH3), 2.11 (1H, ddd, J=13.1, 12.7, 5.4 Hz, H7b), 2.27 (1H, qq, J=6.3, 6.3 Hz, H13), 2.35 (1H, d, J=14.0 Hz, H14a), 2.42 (1H, d, J=14.0 Hz, H14b), 4.78 (1H, d, J=11.3 Hz, PhCHAHB), 4.82 (1H, d, J=11.3 Hz, PhCHAHB), 5.29 (1H, d, J=10.9 Hz, H10), 5.60 (1H, s, H3), 6.24 (1H, dd, J=3.6, 2.7 Hz, H25), 6.84 (1H, dd, J=3.6, 1.4 Hz, H24), 7.03 (1H, dd, J=2.7, 1.4 Hz, H26), 7.28 (1H, m, aromatic), 7.33–7.38 (2H, m, aromatic), 7.42 (2H, m, aromatic); 13C-NMR (100 MHz, CD3OD) δ: 10.5, 12.7, 18.4, 18.9, 19.5, 21.5, 26.8, 29.0, 31.0, 34.3, 37.1, 48.6 (deduced from heteronuclear multiple bond connectivity (HMBC) correlation), 67.0, 67.7, 74.4, 84.3, 86.5, 87.7, 90.7, 92.4, 96.1, 106.0, 111.0 (2C), 117.2, 125.6, 128.4, 128.6 (2C), 129.3 (2C), 140.2, 161.8, 172.9; HR-MS (ESI) Calcd for C34H43NNaO10 [M+Na]+ 648.2779. Found 648.2775.
Ryanodine (1)K2CO3 (6 mg, 40 µmol) was added to a solution of 10-acetyl-15-benzylryanodine (21, 1.24 mg, 1.98 µmol) in MeOH (0.7 mL) at room temperature. The reaction mixture was stirred for 5 h at the same temperature. The reaction was quenched with AcOH (15 µL), and the resultant solution was directly concentrated. The residue was filtered through a short pad of silica gel (0.3 g, CHCl3–MeOH 5 : 1), and the filtrate was concentrated to afford crude 15-benzylryanodine (22, 1.5 mg), which was used in the next reaction without further purification.
A suspension of the above crude 15-benzylryanodine (22, 1.5 mg) and Pd–C (10 wt% Pd on carbon, 4 mg) in MeOH (0.7 mL) and EtOAc (0.7 mL) was stirred for 30 min under H2 atmosphere (1 atm) at room temperature. After additional Pd–C (10 mg) was added, the reaction mixture was stirred under H2 atmosphere for 3 h at the same temperature. The resultant mixture was filtered through a membrane filter with MeOH, and the filtrate was concentrated. The residue was purified by PTLC on silica gel (CHCl3–MeOH 10 : 1) to afford ryanodine (1, 0.76 mg, 1.5 µmol) in 78% yield over 2 steps from 21: colorless solid; [α]D26 +14 (c=0.038, MeOH); 1H-NMR (400 MHz, CD3OD) δ: 0.76 (3H, d, J=6.3 Hz, H18), 0.90 (3H, s, H20), 1.02 (3H, d, J=6.3 Hz, H21), 1.12 (3H, d, J=6.8 Hz, H19), 1.26 (1H, ddd, J=12.7, 4.5, 2.3 Hz, H7a), 1.39 (3H, s, H17), 1.46 (1H, dddd, J=13.1, 13.1, 12.7, 4.5 Hz, H8a), 1.53 (1H, dddd, J=13.1, 5.9, 5.4, 2.3 Hz, H8b), 1.85 (1H, ddqd, J=13.1, 10.4, 6.3, 5.9 Hz, H9), 1.93 (1H, d, J=13.6 Hz, H14a), 2.10 (1H, ddd, J=12.7 12.7, 5.4 Hz, H7b), 2.27 (1H, qq, J=6.8, 6.3 Hz, H13), 2.57 (1H, d, J=13.6 Hz, H14b), 3.80 (1H, d, J=10.4 Hz, H10), 5.64 (1H, s, H3), 6.24 (1H, dd, J=3.6, 2.7 Hz, H25), 6.88 (1H, dd, J=3.6, 1.4 Hz, H24), 7.04 (1H, dd, J=2.7, 1.4 Hz, H26); 13C-NMR (100 MHz, CD3OD) δ: 10.2, 12.6, 18.9, 19.0, 19.5, 26.8, 29.3, 30.9, 35.4, 41.8, 65.9, 72.8, 84.4, 86.6, 87.4, 90.9, 92.4, 96.7, 102.9, 110.9, 117.0, 123.4, 125.6, 161.9 (one peak overlapped with the solvent peaks); HR-MS (ESI) Calcd for C25H35NO9Na [M+Na]+ 516.2204. Found 516.2207.
This research was financially supported by the Funding Program a Grant-in-Aid for Scientific Research (A) (JSPS) and Challenging Exploratory Research to M.I., and a Grant-in-Aid for Young Scientists (B) (JSPS) to M.N. A Fellowship from JSPS to K.M. is gratefully acknowledged.
The authors declare no conflict of interest.
The online version of this article contains supplementary materials; the NMR spectra of newly synthesized compounds.
