Improved synthesis of key intermediate of grayanotoxin III 
Weihao Ma, Zhi Huang, Yanxing Jia*   
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China 
Abstract:A concise improved synthesis of the key intermediate for the synthesis of grayanotoxin III was realized in the present study, featuring a tandem reaction of Michael addition-esterification, Mukaiyama hydration and Mukaiyama dehydrogenaiton.           
Keywords: Grayanotoxins; Improved synthesis; Tandem reaction; Hydration   
CLC number: R914                Document code: A                 Article ID: 10031057(2019)640206
1. Introduction
Grayantoxins, such as grayanotoxin I-IV (1?4, Fig. 1), are a well-known class of polyhydroxyl toxic diterpenoidsoccurring exclusively in the Ericaceae plants[1–3]. The grayanane diterpenoids exhibit various biological activities, including sodium channel modulating activity[4], insecticide activity[5], analgesic and sedative activity[6]. Structurally, grayanotoxins are characterized by the A-nor-B-homo-kaurane skeleton, a unique 5,7,6,5-tetracyclic A/B/C/D framework with a bicyclo[3.2.1]octane core, and by the dense arrangement of stereocenters and hydroxyl groups. The significant bioactivities and complex structure make grayanotoxins attractive but challenging synthetic targets.
To date, there are several articles describing the syntheticefforts toward grayanoids, including grayanotoxin II 2[7,8], grayanotoxin III 3[9], 14-deoxygrayanotoxin III 5[10] and pierisformaside C 6 (Fig. 1)[11]. However, there is only one total synthesis of grayanotoxin III 3, which is accomplished by Shirahama and co-workers in 1994[9]. Although their key intramolecular cyclization reaction induced by SmI2 has been proved to be efficient, the preparation of precursor 8 is lengthy, requiring 14 linear steps from the commercially available compound 7 and using protecting groups frequently.
As part of our ongoing studies towards the concise and efficient synthesis of bioactive natural products[12,13], and impressed by the significant bioactivities and original structure of grayanotoxins, we are interested in developing a more efficient synthetic strategy toward the total synthesis of grayanotoxins. Herein, we reportedthe concise synthesis of key intermediate 8 for the synthesis of grayanotoxin III 3 without using protecting groups (Scheme 1).  
Figure 1. Natural products containing grayanane core. 
Scheme 1. Shirahama’s key intermediate 8 for C/D ring formation. 
2. Results and discussion
As shown in Scheme 2, our synthesis of the key intermediate8 commenced with the commercially available 2-cyclohexen-1-one 10. The 1,4-addition of vinylmagnesium bromide to 10 in the presence of CuI gave the corresponding enolate intermediate that was intercepted with Mander’s reagent in THF to furnish the mixture of β-ketoester 11 and enolate 12, which could not be separated via column chromatography.However, the high cost of methyl cyanoformate limited the scalable preparation of 11 and 12 for further investigations. To our delight, using methyl chloroformateas the electrophilic reagent in the presence of CuCN gave the desired esteron a scale of 16 g. Subsequent alkylation of the mixture of 11 and 12 with (3-bromoprop-1-yn-1-yl) trimethsilane 13 in the presence of K2CO3 provided 14 in 70% yield over two steps.  
Scheme 2. Synthesis route of the key intermediate 8. 
With the alkylated compound 14 in hand, we investigated the hydration of vinyl group to form the γ-lactone ring using Mukaiyama’s procedure[1416]. Table 1 summarizes the results of reaction conditions for the hydration. We initially used Co(acac)2 as the catalyst and iPrOH as the solvent (entry 1), successfully obtaining the intermediateperoxide, which was reduced by PPh3 to provide 15 and 16 in a 25% combined yield as a pair of C10 diastereoisomersin a ratio of 1:1. The relative configurations of 15 and 16were determined by extensive NMR spectral analysis (see Supporting Information). The stereocenter at C10 generated here will be disappeared by oxidation according to Haruhisa Shirahama group’s procedure, therefore theoretically both 15 and 16 can be transformed into the advanced intermediate[9]. Encouraged by this preliminaryresult, we screened several catalysts and identified Mn(dpm)3 as the most promising catalyst for the hydration, which provided 15 and 16 in 72% with a ratio of 1:1. To further optimize this transformation, we next examined the solvent effects. However, when the reaction was carried out in EtOH, tBuOH, or DCE (entries 5?7, Table 1), the yield of the desired product was dramatically decreased. Therefore, it was found that Mn(dpm)3/iPrOH was the best condition for the hydration of 14.
Table 1. Hydration of 14 to 15/16.
We then turned our attention to the dehydrogenation of ketone 15. Initial attempts to transform 15 to 18 by using selenoxide elimination method reported proved unsuccessful[17]. Then we turned to the procedureof Mukaiyama dehydrogenation, which involvedsulfenylation of the lithium enolate of 15 with 17 and subsequent elimination to deliver 18 in 87% yield in a single step[18]. Subsequent subjection of 18 to NaBH4 in the presence of CeCl3·7H2O led to the regioselective and stereoselective reduction, providing 19 in 78% yield[19]. Finally, the regioselective hydration of internal alkyne 19 with NaAuCl4·2H2O afforded Shirahama’s key intermediate 8 in 86%[20].
In summary, we achieved a concise synthesis of the key intermediate 8 for the total synthesis of grayantoxinIII 3. The protecting-group-free synthesis proceeded in six steps featuring a tandem Michael addition-esterification, a Mukaiyama hydration and a Mukaiyama dehydrogenation. Further investigation in the concise synthesis of grayanotoxins and analogs is currently underway.
3. Experimental
All solvents were distilled prior to use unless otherwisenoted. Silica gel from Qingdao Mar. Chem. Ind. Co. Ltd. (200?300 mesh) was used for flash chromatography. Thin layer chromatography was performed with TLC plates from Merck (60 F254) using phosphomolybdic acid solution for visualization. High resolution mass spectra were recorded on a Bruker APEX IV FT-MS (ESI)spectrometer. 1H and 13C NMR spectra were recorded on Bruker Avance III 400-MHz and 600-MHz spectrometer. Chemical shifts δ were reported in ppm with the undeuterated solvent as internal standard.
The following abbreviations were used: PEpetroleum ether; EtOAcethyl acetate; CuCNcopper cyanide;THFtetrahydrofuran; EtOHethanol; iPrOHisopropanol; dpmdipivaloylmethanato.
3.1. Synthesis of compound 14
To a suspension of CuCN (19.5 g, 216.7 mmol) in anhydrous THF (350 mL), vinylmagnesium bromide (220 mL, 1 M in THF, 220 mmol) was added dropwise under an argon atmosphere at –78 °C. The reaction mixture was stirred at –78 °C for 30 min, and then a solution of 2-cyclohexen-1-one 10 (16.0 g, 166.7 mmol) in THF (100 mL) was added dropwise. The reaction was stirred at –78 °C for another 30 min. The neat methyl chloroformate (25.8 mL, 333.9 mmol) was added dropwise. The reaction temperature was gradually raised to room temperature. The reaction mixture wasstirred at room temperature for 8 h. Aqueous ammonium chloride was added to the mixture, and then the aqueouslayer was extracted with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, concentrated and filtered by a short column of silica gal to remove copper salts. The filtrate was evaporated to afford the crude product as a dark brown oil, which was used without further purification.
To a solution of crude ester in 300 mL acetone, K2CO3 (46.0 g, 333.4 mmol) and (3-bromoprop-1-yn-1-yl)trimethylsilane 13 (32.3 mL, 200.0 mmol) were added. The stirred mixture was refluxed overnight. When the ester was consumed, the mixture was filtered through a pad of Celite, and the Celite was washed with EtOAc. The filtrate was concentrated under reduced pressure to afford the crude product, which was purified by flash chromatography (PE–EtOAc, 100:1, v/v) to provide compound 14 (34.1 g, 116.7 mmol, 70% over 2 steps) as a yellow liquid. 1H NMR (400 MHz, CDCl3) δ: 5.81 (m, 1H), 5.18 (d, J = 17.0 Hz, 1H), 5.09 (d, J = 9.8 Hz, 1H), 3.68 (s, 3H), 2.98 (d, J = 17.0 Hz, 1H), 2.89 (m, 1H), 2.70 (m, 1H), 2.63 (d, J = 17.0 Hz, 1H), 2.50 (d, J = 14.9 Hz, 1H), 2.05–2.16 (m, 2H), 1.67–1.81 (m, 2H), 0.11 (s, 9H); 13C NMR (100 MHz, CDCl3) δ: 204.9, 169.7, 137.0, 117.3, 103.4, 87.7, 63.3, 52.3, 48.5, 39.7, 27.5, 23.7, 23.4, 0.0; HRMS (ESI) m/z calcd for C16H25O3Si [M+H]+ 293.1573, found 293.1575.
3.2. Synthesis of diastereoisomers 15/16
To a solution of 14 (2.0 g, 6.8 mmol) in iPrOH (68 mL), Mn(dpm)3 (206.8 mg, 0.34 mmol) and PhSiH3 (1.7 mL, 13.7 mmol) were added. The mixture was stirred at room temperature under an oxygen atmosphere. When 14 was almost consumed, the O2 balloon was removed, and triphenylphosphine (3.6 g, 13.7 mmol) was added. The mixture was stirred at 60 °C for 4 h. Then the mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, concentrated under reduced pressure to give the crude product, which was purified by flash chromatography (PEEtOAc, 10:1, v/v) to provide 15 (685.5 mg, 2.4 mmol, 36%) and 16 (684.0 mg, 2.4 mmol, 36%) as white solids.
Compound 15: 1H NMR (400 MHz, CDCl3) δ: 4.84 (m, 1H), 3.22 (d, J = 16.9 Hz, 1H), 2.95 (m, 1H), 2.52–2.57 (m, 2H), 2.27 (m, 1H), 2.07 (m, 1H), 1.94 (m, 1H), 1.56–1.69 (m, 2H), 1.44 (d, J = 6.6 Hz, 1H), 0.12 (s, 9H); 13C NMR (100 MHz, CDCl3) δ: 201.6, 172.4, 101.5, 88.3, 77.2, 61.3, 46.5, 39.7, 22.7(2C), 21.7, 14.8, –0.2; HRMS (ESI) m/z calcd for C15H23O3Si [M+H]+ 279.1416, found 279.1418.
Compound 16: 1H NMR (400 MHz, CDCl3) δ: 4.32 (m, 1H), 2.85 (d, J = 17.2 Hz, 1H), 2.77 (d, J = 17.2 Hz, 1H), 2.61–2.73 (m, 2H), 2.50 (m, 1H), 2.19 (m, 1H), 2.08 (m, 1H), 1.93 (m, 1H), 1.78 (m, 1H), 1.43 (d, J = 6.1 Hz, 3H), 0.15 (s, 9H); 13C NMR (100 MHz, CDCl3) δ: 204.7, 172.2, 100.7, 90.2, 75.9, 60.0, 49.1, 39.1, 25.6, 21.7, 21.3, 19.1, –0.2; HRMS (ESI) m/z calcd for C15H23O3Si [M+H]+ 279.1416, found 279.1420.
3.3.Synthesis of compound 18
Under an argon atmosphere, to a stirred solution of 15 (200 mg, 0.72 mmol) in anhydrous THF (7 mL), LiHMDS (1.44 mL, 1 M in THF, 1.43 mmol) was added at –78 °C. After stirred for 1 h at –78 °C, a solution of 17 (309.4 mg, 1.43 mmol) in THF (2.0 mL) was added, and the mixture was then stirred for another 1 h at the same temperature. Then the mixture was quenched by adding aqueous ammonium chloride, extracted with EtOAc, washed with brine, dried over Na2SO4, concentrated under reduced pressure to give the crude product, whichwas purified by flash chromatography (PE–EtOAc, 5:1, v/v) to provide 18 (172.7 mg, 0.63 mmol, 87%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ: 7.02 (m, 1H), 6.19 (d, J = 10.2 Hz, 1H), 4.80 (m, 1H), 3.31 (dd, J = 11.2, 6.3 Hz, 1H), 3.08 (d, J = 17.0 Hz, 1H), 2.77 (m, 1H), 2.70 (d, J = 17.0 Hz, 1H), 2.43 (d, J = 19.9 Hz, 1H), 1.31 (d, J = 6.6 Hz, 1H), 0.10 (s, 9H); 13C NMR (100 MHz, CDCl3) δ: 190.2, 171.8, 148.3, 129.6, 100.0,89.1, 76.7, 55.0, 41.1, 26.5, 22.9, 16.7, –0.2; HRMS (ESI) m/z calcd for C15H21O3Si [M+H]+ 277.1254, found 277.1255.
3.4. Synthesis of compound 19
To a solution of 18 (40 mg, 0.14 mmol) in CH2Cl2 (1.0 mL) and EtOH (1.0 mL), CeCl3·7H2O (53.9 mg, 0.14 mmol) and NaBH4 (2.8 mg, 0.072 mmol) were added at –78 °C. The reaction mixture was stirred for 5 h at –78 °C. The reaction mixture was quenched by aqueous ammonium chloride and diluted with CH2Cl2. The aqueous layer was extracted three times with CH2Cl2. The organic layers were combined, washed with brine, dried over Na2SO4, concentrated under reducedpressure to afford crude product. Flash chromatography (PEEtOAc, 20:1, v/v) furnished compound 19 (31.4 mg, 0.11 mmol, 78%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ: 5.86–5.92 (m, 2H), 4.89 (m, 1H), 4.35 (s, 1H), 2.92 (d, J = 17.2 Hz, 1H), 2.81 (q, J = 7.5 Hz, 1H), 2.51 (br s, 1H), 2.45 (d, J = 17.2 Hz, 1H), 2.32 (m, 1H), 2.00 (m, 1H), 1.35 (d, J = 6.6 Hz, 1H), 0.13 (s, 9H);13C NMR (100 MHz, CDCl3) δ: 179.9, 130.5, 127.9, 102.5, 88.0, 78.3, 69.0, 53.2, 40.2, 23.5, 21.6, 16.6, 0.1; HRMS (ESI) m/z calcd for C15H23O3Si [M+H]+ 279.1411, found 279.1413.
3.5. Synthesis of compound 8
To a stirred solution of 19 (30 mg, 0.11 mmol) in THF (1.0 mL) and H2O (0.1 mL), NaAuCl4·2H2O (0.9 mg, 0.0022 mmol) was added. Then the reaction mixture was stirred for 2 h at 60 °C. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, concentrated under reduced pressure to give the crude product, which was purified by flash chromatography (PE–EtOAc, 5:1, v/v) to provide 8 (20.8 mg, 0.092 mmol, 86%, d/r = 4:1) as a colorless oil. 1H NMR (400 MHz,CDCl3) δ: 5.86–6.05 (m, 2H), 4.72–4.81 (m, 2.2H), 4.63 (s, 0.8H), 2.48 (d, J = 13.1 Hz, 0.8H), 2.43 (d, J = 12.6 Hz, 0.2H), 2.09–2.24 (m, 2H), 2.04–2.08 (m, 1H), 1.63–1.80 (m, 1H), 1.56 (s, 0.6H), 1.55 (s, 2.4H), 1.38–1.40 (m, 3H); 13C NMR (100 MHz, CDCl3) δ:182.0, 179.2, 129.5, 127.7, 125.8, 124.7, 106.0, 104.1, 78.6, 77.4, 76.3, 74.2,54.9, 53.2, 48.2, 47.3, 42.2, 40.4, 27.1, 25.7, 20.9,20.6, 15.3, 14.8; HRMS (ESI) m/z calcd for C12H16O4Na[M+Na]+ 247.0941, found 247.0950.
This work was supported by the Drug Innovation Major Project (Grant No. 2018ZX09711-001-005-005).
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马伟豪, 黄智, 贾彦兴*
北京大学医学部 药学院 天然产物及仿生药物国家重点实验室, 北京 100191    
摘要: 本文报道了木藜芦毒素III关键中间体的简易合成的改进方法,合成路线中使用了Michael加成串联酯化反应、Mukaiyama水和反应以及Mukaiyama脱氢反应等关键反应。 
关键词: 木藜芦毒素;合成改进;串联反应;水合反应
Received: 2019-04-15; Revised: 2019-04-30; Accepted: 2019-05-13.
Foundation item: Drug Innovation Major Project (Grant No. 2018ZX09711-001-005-005).
*Corresponding author. Tel.: +86-010-82805166, E-mail:  

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