Method for large-scale synthesis of tetrodotoxin

[0080] The technical problem to be solved by the present invention is to provide a method for artificially large-scale synthesis of tetrodotoxin that does not rely on natural sources such as puffer fish, that is, a chemical method for gram-scale synthesis of tetrodotoxin. Compared with the method reported in the prior art, the advantage of the chemical synthesis method of the present invention is that the improvement of the synthesis method and the purification method enables the present invention to have the ability to produce tetrodotoxin and its derivatives on a large scale according to the c-GMP standard above the gram level, It is possible to carry out clinical trials of tetrodotoxin as a candidate drug that meets regulatory specifications, and it has laid a solid API foundation for tetrodotoxin to become an important member of the field of human analgesia and detoxification drugs, including:

[0081] (1) Only one step of intramolecular transesterification reaction is needed from II to III, all reagents have only one cheap and easy-to-obtain base, the reaction yield is equivalent to the original route, replacing the 2-step reaction of the original route, and some expensive ones Reagents, operability and scale-up reproducibility of reactions are improved.

[0082] (2) IV to V use non-toxic metal Lewis acid (such as CuCl 2 etc.) to replace the highly toxic HgCl in the original route 2 , Safer operation, in line with API production requirements.

[0083] (3) Chiral preparative HPLC is used for the separation of VI-a and by-product VI-b, which has a good separation effect and can be prepared in large quantities, replacing the silica gel thin plate chromatography that is difficult to scale up in the original route.

[0084] (4) by-product VI-b can be converted into VI-a under dilute acid effect, through once recycling, the total yield of V to VI-a is 68%, compared with the yield of this step reaction 41% of original route. Greater improvement.

[0085] (5) From VI-a to TTX, use a water-based mixed fiber microporous filter membrane to remove palladium carbon, and the filtrate can be evaporated to dryness without further purification to obtain TTX with a purity of more than 95%, which avoids the difficult amplification of the original route. High pressure liquid chromatography separation.

[0086] (6) The present invention is the first to complete the synthesis of tetrodotoxin on a gram-level scale or above in the world, and the characterization data is complete and reliable, which can be used for large-scale preparation of tetrodotoxin API (API) that meets the c-GMP standard, making tetrodotoxin as a regulatory standard It is possible to carry out clinical trials of drug candidates.

[0095] Example 1 : the preparation of formula III compound

[0096]

[0097] At room temperature, 12 g of compound II and 6 g of potassium carbonate were dispersed in 300 ml of methanol, stirred at room temperature for 18 hours, HPLC detected that the raw materials disappeared, and the precipitate was removed by filtration, and most of the methanol was removed by rotary evaporation of the filtrate at room temperature. Then add 300 ml of water, extract the reaction solution three times with 300 ml of ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, filter and spin dry, and use ethyl acetate-petroleum ether (1:3) silica gel column chromatography to obtain white Foamy solid compound III (7.3 g, yield 75%). 1H NMR (CDCl 3 ,400MHz):δ5.16(s,1H),5.02(d,J=6.9Hz,1H),5.01(s,1H),4.42(s,1H),4.41(d,J=6.9Hz,1H) ,4.37(d,J=10.1Hz,1H),4.32(s,1H),4.25(d,J=10.1Hz,1H),3.48(br s,1H),3.44(s,3H),1.46(s ,9H), 1.43(s,3H), 1.38(s,3H), 1.29(s,3H).

[0098] Example 2 : the preparation of formula IV compound

[0099]

[0100] Under ice bath, add 180 ml of freshly prepared TMSI solution (0.32M) to 6.9 g of compound III in 240 ml of anhydrous acetonitrile solution, react at room temperature for 15 minutes, add 450 ml of sodium thiosulfate solution to quench the reaction, and use chloroform- Methanol (9:1) was extracted three times with 450 ml of mixed solvent, the combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated to obtain crude compound IV (5.4 g), which was directly put into the next reaction without purification. 1 H NMR (CDCl 3 ,400MHz):δ6.34(br,2H),5.32(d,J=7.2Hz,1H),5.30(s,1H),4.79(d,J=1.2Hz,1H),4.42(d,J= 7.6Hz, 1H), 4.39(s, 1H), 4.38(d, J=10.0Hz, 1H), 4.28(d, J=10.4Hz, 1H), 3.57(s, 3H), 3.36(t, J= 2.0Hz, 1H), 2.03(d, J=8.0Hz, 1H), 1.46(s, 3H), 1.44(s, 6H), 1.38(s, 3H), 1.36(s, 3H).