Enantioselective total synthesis of (–)-colchicine, (+)-demecolcinone and metacolchicine: determination of the absolute configurations of the latter two alkaloids

A highly concise, enantioselective synthesis of (–)-colchicine, the first syntheses of (+)-demecolcinone and metacolchicine, was reported.

To a solution of furfuryl alcohol 13 (2.54 g, 26.0 mmol) in THF (150 mL) was added BuLi (21.6 mL, 2.4 M, 52.0 mmol) at -78 o C. The reaction mixture was stirred at -78 o C for 10 minutes, then warmed to 0 o C, and stirred for another 30 minutes. Compound 12 (4.0 g, 13.0 mmol) dissolved in THF (20 mL) was added dropwise. The reaction mixture was stirred for 3h, then poured into saturated aqueous NH 4 Cl (100 mL) and dilute with ether (150 mL).
After separation, the aqueous phase was re-extracted with ether (2 х 50 mL). The combined organic phase was washed with brine (100 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. Purification of the crude product by flash column chromatography on silica gel (hexane/ethyl acetate = 1/1) afforded 18 (4.12 g, 92% yield) as a colorless oil.  A solution of compound 18 (3.0 g, 8.72 mmol) in anhydrous THF (50 mL) was cooled to 0 o C and R-tertbutanesulfinamide 11 (1.27 g, 10.5 mmol) was added, followed by Ti(OBu) 4 (5.8 g, 17.0 mmol). The reaction S7 mixture was stirred at 120 o C for 12 h, and then cooled to -78 o C. DIBAL-H (1 M, 17mL) was added dropwise, and the reaction mixture was allowed to warm to room temperature. Once the reduction was determined to be completed by TLC, the reaction mixture was cooled to 0 °C and MeOH (1 mL) was added dropwise until gas evolution was no longer observed. Aqueous solution of HCl (1M) was added until the pH = 1-2, and then the reaction mixture was stirred at room temperature for 1h. Saturated aqueous NaHCO 3 was added at 0 o C until pH of the reaction mixture was up to 10. MeOH (5 mL) was added to the reaction mixture, followed by slowly addition of Ac 2 O. The reaction mixture was evaporated under reduced pressure, and the aqueous phase was extracted with ethyl acetate (50 mL x 2). The combined organic phase was washed with brine (15 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. Purification of the crude product by flash column chromatography on silica gel (hexane/ethyl acetate = 1/2) afforded alcohol 10 (2.63 g, 78% yield) as colorless oil.  6 To a solution of 8 (0.60 g, 1.56 mmol) in DCM/pyridine (10 mL/10 mL), was added I 2 (0.47 g, 1.87 mmol) in one portion. The resulting reaction mixture was heated at 60 o C in a sealed tube for 10h. The reaction solution was poured into saturated Na 2 S 2 O 3 (50 mL), and extracted with DCM (20 mL X 2). The combined organic phase was washed successively with aqueous HCl (1 M, 100 mL), saturated aqueous NaHCO 3 (30 mL), brine (30 mL), then dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. Purification of the crude product by flash column chromatography on silica gel (hexane/ethyl acetate = 1/1) afforded 19 (0.71 g, 88% yield) as a white solid.
Purification of the crude product by flash column chromatography on silica gel (hexane/ethyl acetate = 2/1) afforded 21 (0.75 g, 78% yield) as a white solid.  Trimethylsilyl trifluoromethanesulfonate (0.8 mL) was added dropwise at 0 o C to a solution of 21 (280 mg, 0.68 mmol) and N,N-Dimethylethylamine (1.2 mL) in DCM (20 mL). After the reaction mixture was stirred at room temperature for 12 h, saturated aqueous NaHCO 3 (20 mL) was added. After separation, the aqueous phase was further extracted with DCM (20 mL x 2). The combined organic phases were washed with saturated NH 4 Cl (10 mL), brine (10 mL) and dried over Na 2 SO 4 . The solution was concentrated under reduced pressure and purified by chromatography (DCM/MeOH = 9/1) to afford 1 (220 mg, 81%) as a white solid.  Note: Using the described route, a total of 1.1 g of colchicine was prepared readily after five simple parallel operations. To a solution of AcONH 4 (4.9 g, 64.0 mmol) in methanol (30 mL) was added 18 (2.2 g, 6.4mmol), the resulting mixture was cooled to 0 o C. NaBH 3 CN (2.0 g, 32.0 mmol) was added with portions. The mixture was stirred for 24 h at 55 o C and cooled to 0 o C. Aqueous NaOH (1M, 10 mL) was added, followed by MeOH (5 mL). To the resulting S12 mixture was added Ac 2 O (3 mL). After the reaction mixture was stirred at room temperature for 10 minutes, the reaction mixture was evaporated under reduced pressure. The residue was extracted with DCM (50 mL x 2), and the combined organic phase was washed with saturated aqueous NH 4 Cl (20 mL), brine (20 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. Purification of the crude product by flash column chromatography on silica gel (hexane/ethyl acetate = 1/2) afforded (±)-10 (2.03 g, 82% yield) as colorless oil.

Racemic synthesis of (±)-10 for HPLC analysis
The synthesis of (±)-colchicine (1) from (±)-10 was using the same route as described above.    To a solution of N-Boc-N-deacetylcolchicine 30 (110 mg, 0.48 mmol) and NaH (19.7 mg, 0.49 mmol) in DMF (10 mL) was added CH 3 I (0.034 mL, 0.49 mmol) dropwise at 0 o C, and the mixture was stirred at the same temperature for 5 minutes, followed by stirring at room temperature for 10 minutes. The reaction was quenched by water (5 mL), followed by extraction with Et 2 O (3 x 15 mL). The combined organic extracts were dried over by  To a solution of 2-demethyldemecolcine 24 (42 mg, 0.12 mmol) in (CF 3 ) 2 CHOH (HFIP, 5 mL) was added PhI(OAc) 2 (57 mg, 0.18 mmol) at 25 o C, and then the mixture was stirred at the same temperature for 5 minutes.

MeO
The reaction was quenched by addition of saturated aqueous Na 2 S 2 O 3 (2.5 mL). The mixture was extracted with DCM (3 x 10 mL). The combined organic extracts were washed with brine (5 mL) and then were dried over by

S20
The process from 1 to 25 involved a series of sequential reactions as follow.     Compound 25 (20 mg, 0.05 mmol) was dissolved in a solution of acetone and water (1:1, 5 mL) at room temperature and then AgBF 4 (48 g, 0.25 mmol) was added at the same temperature. After stirred for 10 hours at room temperature, the reaction was diluted with DCM (3 mL) and washed with water (2 x 5mL). The layers were separated and the aqueous layer was extracted with DCM (2 x 5 mL). The combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM/MeOH = 50/1-20/1) to afford alkene 25 (15.5 mg, 78% yield) as a yellow solid.   Then the mixture was diluted with ether (3 mL) and washed with 1M HCl (0.4 mL), water (0.5 mL) and brine (0.5 mL) sequentially. Organic layer was dried over Na 2 SO 4 , filtered, concentrated and the residue was purified by flash column chromatography (DCM: MeOH = 40:1  30:1) to yield the triflates mixture as a yellow oil, which S24 was used in the next step.

Cell cycle analysis
DNA content analysis was performed as described previously 12 .

Cell Morphology
Tubulin cytoskeleton organization in MDA-MB-231 cells treated with 30 and colchicine were incubated with anti-β-tubulin antibody and Alexa Fluor 488 secondary antibody. Images were taken using a laser scanning confocal microscope (LSM 510, Zeiss).

S58
Tubulin polymerization assay was performed using tubulin polymerization assay kit according to the manufacturer's instruction.

Microscale thermophoresis binding assay
Binding were calculated for 30 or colchicine and tubulin protein, using microscale thermophoresis 13         a "The partial racemisation observed in the final stages of this synthesis is attributed to the substantial acidity of the C-7 proton associated with the intermediate phosphoimine involved in the azide reduction step. " 18