A method for large-scale synthesis of tetrodotoxin

Abstract

The application provides a method for synthesizing tetrodotoxin by combining biology and chemistry, and specifically comprises the following steps: using cheap and readily available benzyl acetate as a raw material, obtaining an optically pure intermediate ((5S, 6R)-5, 6-dihydroxycyclohexa-1, 3-dienyl) methyl acetate (formula I) through biological fermentation; the intermediate is further subjected to a series of chemical conversions, and the final product can obtain tetrodotoxin with a purity of greater than 95% without purification. The application has the potential for large-scale production, and is an excellent alternative to the existing extraction method from puffer fish.

Description

Technical Field

[0001] This invention belongs to the fields of medicine and organic synthesis, and specifically relates to a method for the artificial synthesis of tetrodotoxin (TTX). Background Technology

[0002] Tetrodotoxin (TTX) is an amino-perhydroquinazoline compound with a cage-like orthoester inner salt structure, as shown below:

[0003]

[0004] Tetrodotoxin is a white crystalline solid, odorless and tasteless, slightly soluble in water, soluble in water-soluble acetic acid solutions, and insoluble in organic solvents. Its molecular structure is very unique, existing as an inner salt. Tetrodotoxin is a typical sodium channel blocker, selectively binding to sodium channel receptors on the cell membrane surface of muscle and nerve cells. It has detoxification and analgesic effects without addiction, and also has hypotensive, local anesthetic, and tumor-inhibiting effects. In clinical applications, the single dose of tetrodotoxin is extremely low, in the microgram range; therefore, large-scale preparation at the gram level or above is of great significance for developing the medicinal value of tetrodotoxin.

[0005] The current source of tetrodotoxin is extraction from wild organisms, but its biological content is very low, generally on the order of 0.1-5 parts per million (ppm). Therefore, extraction is difficult, consumes a large number of wild pufferfish, is detrimental to ecological protection, and the quality of the extracted product is unstable. Furthermore, extraction of tetrodotoxin from artificially bred organisms has not been successful. It is currently believed that wild organisms achieve their 0.1-5 ppm tetrodotoxin levels through the enrichment of toxins found in nature. This results in the high price of tetrodotoxin, limiting its application in medicine and other fields.

[0006] The artificial total synthesis of tetrodotoxin began with the report of racemic compounds by Kishi's research group in 1972 (J. Am. Chem. Soc. 1972, 94, 9217-9219; 9219-9221). By 2018, when this invention was initiated, more than ten research teams worldwide had conducted artificial synthesis work on racemic compounds, chiral pure compounds, and derivatives of tetrodotoxin, achieving a series of research advances and results. However, the reported synthetic methods are lengthy (generally more than 30 steps), with very low synthesis efficiency, typically yielding only 1-5 mg of sample. The reported yields are unreliable due to systematic errors in measurement methods and equipment. The academic exploration value of these works outweighs their practical application value, and they lack the potential for large-scale tetrodotoxin synthesis and its use as a pharmaceutical raw material. For example, the synthetic route reported by Fukuyama's research group (Angew.Chem.Int.Ed.2017,56,1549–1552) is as long as 31 steps. In particular, the last six steps use a lot of highly toxic chemicals and expensive chemical reagents. The purification process of the related reactions is complicated and cannot prepare tetrodotoxin at the gram scale according to c-GMP standards. Summary of the Invention

[0007] To overcome the above-mentioned technical problems, the present invention provides a method for preparing tetrodotoxin at the gram scale, characterized in that: a compound of formula VI-a is hydrogenated to remove the protecting group Cbz, and after filtration and concentration, tetrodotoxin represented by TTX is prepared.

[0008]

[0009] Cbz represents benzyloxycarbonyl, and the solvents used in the reaction include, but are not limited to, methanol, ethanol, ethyl acetate or isopropanol; an aqueous mixed fiber microporous filter membrane is used for filtration.

[0010] In the above method for preparing tetrodotoxin, the compound of formula VI-a can be obtained from the compound of formula V under the action of acid:

[0011]

[0012] Among them, chiral preparation was used to separate compounds of formula VI-a and formula VI-b by HPLC.

[0013] The aforementioned formula VI-b can be further transformed into formula VI-a under the action of acid:

[0014]

[0015] Among them, chiral preparation was used to separate compounds of formula VI-a and formula VI-b by HPLC.

[0016] The compound of formula V can be obtained by reacting the compound of formula IV with Cbz-methylthiourea:

[0017]

[0018] The reaction uses metallic Lewis acids, including lead chloride, lead acetate, silver nitrate, copper chloride, and cuprous chloride.

[0019] The compound of formula IV can be obtained by removing the Boc protecting group from the compound of formula III:

[0020]

[0021] In this reaction, Boc represents tert-butyloxycarbonyl, acetonitrile is used as the solvent, trimethyliodosilane is used as the reagent, and the reaction temperature is from -10℃ to 50℃.

[0022] The compound of formula III can be obtained from the compound of formula II via intramolecular cyclization under the action of a base:

[0023]

[0024] Where R is C 1-6 Alkyl or substituted alkyl groups, and the bases used include potassium carbonate, sodium carbonate, potassium bicarbonate, and sodium bicarbonate; the solvents used include methanol, ethanol, and toluene.

[0025] This invention further provides a method for preparing tetrodotoxin (TTX) at the gram level, the method comprising steps (A)-(H):

[0026] (A) Biotransformation of benzyl acetate to prepare optically pure compound I.

[0027]

[0028] (B) Prepare compound II by subjecting compound I to 19 chemical transformations.

[0029]

[0030] Where R is C 1-6 Alkyl or substituted alkyl.

[0031] (C) To prepare compound III by subjecting compound II to an intramolecular transesterification reaction.

[0032]

[0033] (D) To prepare compound IV by removing the Boc protecting group from compound III.

[0034]

[0035] (E) Reacting compound IV with methylthiourea to prepare compound V.

[0036] (F) Intramolecular cyclization of compound V to prepare compounds of formula VI-a and VI-b.

[0037]

[0038] (G) Hydrolyzing compound VI-b to prepare compound VI-a

[0039]

[0040] (H) Hydrogenation of compound VI-a to remove the Cbz protecting group to prepare compound TTX.

[0041] Detailed Implementation

[0042] The technical content of this invention is described below through specific embodiments. Those skilled in the art can easily understand other advantages and effects of this invention from the content disclosed in this specification. This invention can also be implemented or applied through other different embodiments. Those skilled in the art can make various modifications and changes without departing from the spirit of this invention.

[0043] General terms and definitions

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of any conflict, the definitions provided herein shall prevail. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient. All patents, published patent applications, and publications cited herein are incorporated herein by reference.

[0045] When used with a numerical variable, the terms "about" or "approximately" usually mean that the value of the variable and all values ​​of the variable are within the experimental error (e.g., within a 95% confidence interval for the mean) or within ±10% of the specified value, or a wider range.

[0046] The term “optional” or “optional existence” means that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0047] The expression "comprising," or similar expressions such as "including," "containing," and "having," is open-ended and does not exclude additional unlisted elements, steps, or components. The expression "consisting of," excludes any unspecified elements, steps, or components. The expression "substantially consisting of," limits the scope to the specified elements, steps, or components, plus optional elements, steps, or components that do not materially affect the essential and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses both the expressions "substantially consisting of" and "consisting of."

[0048] The terms “one or more” or “at least one” can mean one, two, three, four, five, six, seven, eight, nine or more.

[0049] The ranges (e.g., numerical ranges) listed in this article can encompass every value within that range, as well as the subranges formed by those values. For example, the statement "reaction temperature is -20°C to 25°C" encompasses every point value and subrange within the range of -20°C to 25°C, such as -20°C to 0°C, 0°C to 25°C, -10°C to 10°C, and -20°C, -10°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, etc. Other similar statements such as "at -20°C to 40°C" or "at 0°C to 100°C" should also be understood in a similar manner. For example, the expression "molar equivalent between 0.01 and 1.5" includes 0.01-0.1, 0.02-0.05, 0.03-0.05, 0.04-0.06, 0.1-0.5, 0.5-1.0, as well as 0.01, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, etc.

[0050] Unless otherwise stated in the text, singular forms such as "a," "an," or "the" include plural references. Unless otherwise stated, concentrations are by weight, liquid proportions in mixed solutions are by volume, and the ratios (including percentages) of reagents to compounds are by moles.

[0051] Protecting derivatives of the compounds described herein can be prepared using methods well known to those skilled in the art. Protecting groups in protecting derivatives can be removed using methods well known to those skilled in the art. For detailed technical descriptions of methods for selecting protecting groups, as well as their addition and removal, please refer to: TW Greene, Protecting Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, Inc. 1999.

[0052] The term "alkyl," as used herein alone or in combination with other groups, refers to a saturated straight-chain, branched, or cyclic hydrocarbon group. As used herein, the term "C" refers to... 1-6 "Alkyl" refers to a saturated straight-chain, branched, or cyclic hydrocarbon group having 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5, or 6). For example, "C 1-6 "alkyl" can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 3-methylpentan-3-yl, hexyl (e.g., n-hexyl, cyclohexyl, etc.). "C" 1-6 "alkyl" encompasses its subrange, such as "C 1-3 Alkyl", C 2-3 Alkyl", C 4-6 Alkyl groups, etc.

[0053] Synthetic routes and intermediate compounds

[0054] This invention relates to a method for preparing tetrodotoxin (TTX) at the gram level, the method comprising the following steps (A)-(H):

[0055] Step (A): Biotransformation of benzyl acetate to prepare optically pure compound I.

[0056]

[0057] This biotransformation step mainly references Hudlicky's work (Angew. Chem. Int. Ed. 2018, 57, 10994-10998), and the improvements made will be detailed in a separate patent.

[0058] Step (B): Prepare compound II by subjecting compound I to 19 chemical transformations.

[0059]

[0060] This section primarily references the synthetic routes combining those of Hudlicky (Angew. Chem. Int. Ed. 2018, 57, 10994-10998) and Fukuyama (Angew. Chem. Int. Ed. 2017, 56, 1549–1552). Where R represents C 1-6 Alkyl or substituted alkyl.

[0061] Step (C): Intramolecular transesterification of compound II to prepare compound III.

[0062]

[0063] Formula II is obtained by intramolecular cyclization under the action of a base, and the base used includes, but is not limited to, potassium carbonate, sodium carbonate, potassium bicarbonate and sodium bicarbonate, among which potassium carbonate is the best; the solvent used includes, but is not limited to, methanol, ethanol and toluene, among which methanol is the best.

[0064] Step (D): Remove the Boc protecting group from compound III to prepare compound IV.

[0065]

[0066] Formula III is obtained by removing the Boc protecting group. Acetonitrile is used as the solvent and trimethyliodosilane is used as the reagent in the reaction. The reaction temperature is from -10℃ to 50℃, with 0℃ being the optimal temperature.

[0067] Step (E): React compound IV with methylthiourea to prepare compound V.

[0068]

[0069] Formula IV reacts with Cbz-methylthiourea to yield Formula V. The reaction uses a metallic Lewis acid, including but not limited to lead chloride, lead acetate, silver nitrate, copper chloride, and cuprous chloride, avoiding the use of highly toxic mercuric chloride, among which copper chloride is the best.

[0070] Step (F): Intramolecular cyclization of compound V to prepare compounds of formula VI-a and VI-b.

[0071]

[0072] Formula V was subjected to acid to give a mixture of formula VI-a and 4,9-dehydrated product formula VI-b. Pure formula VI-a and formula VI-b were separated by chiral preparative HPLC.

[0073] Step (G): Hydrolyze compound VI-b to prepare compound VI-a.

[0074]

[0075] The 4,9-dehydrated product VI-b can be converted into the intermediate VI-a in a certain ratio (1:8) under acidic conditions. Chiral preparative HPLC can then yield partially pure VI-a. After 2 to 3 cycles, the product is converted back to VI-b, and VI-a is obtained in a yield greater than 80%.

[0076] Step (H): Hydrogenate compound VI-a to remove the Cbz protecting group to prepare compound TTX.

[0077]

[0078] The VI-a hydrogenation process removes the protecting group Cbz, and after simple filtration and concentration, tetrodotoxin (TTX) can be obtained in high purity. Solvents used in the reaction include, but are not limited to, methanol, ethanol, ethyl acetate, or isopropanol, with methanol being the optimal solvent. A water-based mixed fiber microporous membrane is used for filtration to improve the filtration speed and effectively remove palladium on carbon.

[0079] Beneficial effects

[0080] The technical problem this invention aims to solve is to provide a method for the large-scale artificial synthesis of tetrodotoxin that does not rely on natural sources such as pufferfish, specifically a gram-scale chemical synthesis of tetrodotoxin. Compared to methods reported in the prior art, the advantages of this chemical synthesis method lie in the improvements in the synthesis and purification methods, enabling the production of tetrodotoxin and its derivatives at gram-scale or higher according to c-GMP standards. This makes it possible for tetrodotoxin to be used as a regulatory-compliant candidate drug in clinical trials, laying a solid foundation for tetrodotoxin to become an important active pharmaceutical ingredient in the field of human analgesia and addiction treatment. Specifically, this includes:

[0081] (1) Only one intramolecular transesterification reaction is needed from II to III. All reagents are only one cheap and readily available base. The reaction yield is comparable to the original route. The two-step reaction of the original route and some expensive reagents are replaced, and the operability and scalability of the reaction are improved.

[0082] (2) The IV to V route uses non-toxic metallic Lewis acid (such as CuCl2) to replace the highly toxic HgCl2 in the original route, making the operation safer and meeting API production requirements.

[0083] (3) The separation of VI-a and byproduct VI-b was carried out by chiral preparative HPLC, 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) Byproduct VI-b can be converted into VI-a under the action of dilute acid. After one cycle, the total yield from V to VI-a is 68%, which is significantly higher than the 41% yield of this step in the original route.

[0085] (5) VI-a to TTX is filtered using an aqueous mixed fiber microporous membrane, which has a good effect on removing palladium on carbon. The filtrate can be evaporated to dryness without further purification to obtain TTX with a purity of more than 95%, avoiding the high-performance liquid chromatography separation that is difficult to scale up in the original route.

[0086] (6) This invention is the first in the world to complete the synthesis of tetrodotoxin at a scale of gram or above. The characterization data is complete and reliable and can be used to prepare tetrodotoxin API that meets c-GMP standards on a large scale, making it possible for tetrodotoxin to be used as a candidate drug for clinical trials in compliance with regulatory requirements.

[0087] Example

[0088] The following embodiments are provided for illustrative purposes only and should not be construed as limiting the invention.

[0089] NMR spectroscopy recording was performed using an Aspect 3000 computer and a 5mm... 1 H / 13 On a BRUKER AC 250 Fourier transform NMR spectrometer with dual C probes. (Diagram showing DMSO-d) 6 The compound was studied in a (or CDCl3) solution at a probe temperature of 313 K. The instrument was locked to DMSO-d. 6 (or CDCl3) on the deuterium signal. Chemical shift is expressed in ppm for the low field of TMS with distance as internal standard.

[0090] HPLC conditions

[0091] Analysis was performed using an Agilent Technologies HPLC system equipped with an Agilent Eclipse PLUS C18 4.6 x 50 mm, 3.5 μm column. Elution was performed using a gradient of 95% 0.1% H3PO4 aqueous solution and 5% acetonitrile over 3.5 min, followed by elution with 5% 0.1% H3PO4 aqueous solution and 95% acetonitrile over another 1.5 min. The flow rate was set at 2.0 mL / min. The column temperature was set at 35 °C. The detection wavelength was 210 nm.

[0092] Chiral HPLC conditions (compounds VI-a and VI-b)

[0093] Analysis was performed using an Agilent Technologies HPLC system equipped with an AD-H, 4.6 x 250 mm, 5 μm column. Elution was performed over 25 minutes with 85% 1% trifluoroacetic acid in hexane and 15% 1% trifluoroacetic acid in ethanol. The flow rate was set at 0.5 mL / min. The detection wavelength was 205 nm.

[0094] The raw materials, reagents, and equipment used in the specific implementation of this invention are all commercially available products. Reagents can be obtained from WuXi AppTec (Wuhan) Chemical Technology Co., Ltd., Shanghai Titan Technology Co., Ltd., Saen Chemical Technology (Shanghai) Co., Ltd., and Shanghai Aladdin Biochemical Technology Co., Ltd.

[0095] Example 1 Preparation of Compounds of Formula III

[0096]

[0097] At room temperature, 12 g of compound II and 6 g of potassium carbonate were dispersed in 300 mL of methanol and stirred at room temperature for 18 hours. The starting material disappeared as detected by HPLC. The precipitate was removed by filtration, and most of the methanol was removed by rotary evaporation of the filtrate at room temperature. Then, 300 mL of water was added, and the reaction solution was extracted three times with 300 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The solution was then subjected to silica gel column chromatography with ethyl acetate-petroleum ether (1:3) to obtain a white foamy solid compound III (7.3 g, yield 75%). 1 H NMR (CDCl3, 400MHz): δ5.16 (s, 1H), 5.02 (d, J = 6.9Hz, 1H), 5.01 (s, 1H), 4.42 (s, 1H), 4.4 1(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 Preparation of Formula IV Compounds

[0099]

[0100] Under ice bath conditions, 180 mL of freshly prepared TMSI solution (0.32 M) was added to 240 mL of anhydrous acetonitrile solution containing 6.9 g of compound III. The reaction was allowed to proceed at room temperature for 15 minutes. The reaction was quenched by adding 450 mL of sodium thiosulfate solution. The mixture was extracted three times with 450 mL of a chloroform-methanol (9:1) mixture. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain crude compound IV (5.4 g). No purification was required before proceeding to the next step of the reaction. 1H NMR (CDCl3, 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).

[0101] Example 3 Preparation of compound V

[0102]

[0103] At room temperature, 15 g of copper chloride was added to 240 mL of a solution of 5.4 g crude compound IV, 15 mL of triethylamine, and 10 g of Cbz-methylthiourea in dichloromethane. The reaction was carried out at room temperature for 5 hours, and the reaction was quenched with 450 mL of water. The mixture was then extracted three times with 200 mL of dichloromethane, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography in petroleum ether-ethyl acetate (3:1) to give compound V (8.58 g, 86% yield in two steps). 1 H NMR (CDCl3, 400MHz): δ11.7(s,1H),8.8(s,1H),7.38-7.30(m,10H),5.24(d ,J=7.8Hz,1H),5.21(s,1H),5.18(d,J=13.0Hz,1H),5.13(s,1H),5.04(d,J= 13.0Hz,1H),4.58(s,1H),4.47(d,J=7.8Hz,1H),4.31(s,1H),4.28(s,2H),3 .91(s,1H),3.25(s,3H),1.36(s,3H),1.31(s,3H),1.24(s,3H),1.21(s,3H)

[0104] Example 4 Preparation of compounds of formula VI-a and formula VI-b

[0105]

[0106] At room temperature, 7.48 g of compound V was dissolved in a mixed solvent of 70 mL trifluoroacetic acid and 70 mL water, and then heated to 60 °C for 18 hours. HPLC analysis confirmed complete reaction of the starting material. The reaction solution was concentrated to dryness at 50 °C and then prepared by chiral HPLC (Chiralpak AD-H, n-Hexane:ETOH = 75:25) to give white solid compound VI-a (2.28 g, yield: 46%) and compound VI-b (1.28 g, yield: 27%). Compound VI-a: 1 ¹H NMR (CD₃OD, 400MHz): δ 7.39–7.29 (m, 5H), 5.56 (s, 1H), 5.09 (m, 2H), 4.48 (d, J = 1.8 Hz, 1H), 4.17 (m, 2H), 4.02 (m, 2H), 3.93 (m, 2H), 2.26 (d, J = 9.2 Hz, 1H); Compound VI-b: 1 H NMR (CD3OD, 400MHz): δ7.38-7.26 (m, 5H), 5.28 (s, 1H), 5.07 (s, 2H), 4.48 (d, J = 1.8Hz, 1H), 4.42 (s, 1H), 4.16 (br s, 1H), 4.02 (d, J = 1.8Hz, 1H), 3.97 (d, J = 11.9Hz, 1H), 3.90 (d, J = 11.9Hz, 1H), 2.70 (d, J = 2.8Hz, 1H).

[0107] Example 5 Recycling of Compound VI-b

[0108]

[0109] At room temperature, 1.28 g of compound V was dissolved in a mixed solvent of 7 mL trifluoroacetic acid and 70 mL water, and then heated to 60 °C for 24 h. HPLC was used to detect the constant ratio of compounds VI-a and VI-b. The reaction solution was concentrated to dryness at 50 °C and then prepared by chiral HPLC (Chiralpak AD-H, n-Hexane:ETOH = 75:25) to give white solid compounds VI-a (1.13 g, yield: 88%) and VI-b (0.14 g, yield: 12%).

[0110] Example 6 Preparation of TTX compounds

[0111]

[0112] At room temperature, 500 mg of 10% wet palladium on carbon was added to 150 mL of a methanol solution containing 2.39 g of compound VI-a. The reaction was hydrogenated for 3 hours. HPLC analysis confirmed complete reaction of the starting material. The catalyst was removed by filtration through a microporous membrane. The filter cake was washed with 500 mL of 0.05 M acetic acid aqueous solution. The filtrate was concentrated under reduced pressure at 45 °C to obtain compound TTX (1.65 g, 98%). [α] D 23 -10.4°(c 0.25,0.05M AcOH); IR(neat,cm-1):3211,1658,1608,1186,1126,1076,979; 1 HNMR (1% CF3COOD / 4% CD3COOD / 95% D2O, 400MHz): δ5.49 (d, J = 9.6Hz, 1H), 4.27 (br s, 1H), 4.23 (br s,1H),4.06(s,1H),4.02(d,J=7.0Hz,1H),4.00(d,J=7.0Hz,1H),3.94(s,1H),2.33(d,J=9.6Hz,1H); 13 CNMR(1%CF3COOD / 4%CD3COOD / 95%D2O,100MHz): δ156.7(C),110.9(C),79.8(CH),75.2( CH),74.0(CH),72.9(CH),71.6(C),71.0(CH),65.6(CH),59.8(C),40.8(CH); HRMS(ESI + ):Calcd for C 11 H 18 N3O8([M+H)) + ):320.1094,Found:320.1094. All analytical data are consistent with those reported in the literature.

[0113] While typical embodiments of the invention have been set forth and described, the invention is not limited to the details described. Since various possible modifications and substitutions do not depart from the spirit of the invention, variations and equivalents of the invention conceived by those skilled in the art through conventional experimentation fall within the spirit and scope of the invention as defined by the following claims.

Claims

1. A method of preparing tetrodotoxin, characterized by, The method comprises the following steps: (1) intramolecular cyclization of the compound of formula II under the action of a base to prepare a compound of formula III wherein R is C 1-6 alkyl or substituted alkyl, the base used is selected from potassium carbonate, sodium carbonate, potassium bicarbonate and sodium bicarbonate; the solvent used is selected from methanol, ethanol and toluene; (2) removing the Boc protecting group from the compound of formula III to prepare a compound of formula IV (3) reacting the compound of formula IV with methyl thiourea to prepare a compound of formula V wherein the reaction uses a metal Lewis acid selected from the group consisting of lead chloride, lead acetate, silver nitrate, copper chloride and cuprous chloride; (4) intramolecular cyclization of the compound of formula V to prepare a compound of formula VI-a and a compound of formula VI-b wherein the compound of formula VI-a and the compound of formula VI-b are separated by chiral preparative HPLC; (5) hydrolysis of the compound of formula VI-b to prepare a compound of formula VI-a wherein the compound of formula VI-a and the compound of formula VI-b are separated by chiral preparative HPLC; (6) hydrogenation of the compound of formula VI-a to remove the Cbz protecting group to prepare tetrodotoxin represented by TTX wherein Cbz represents a benzyloxy carbonyl group, the reaction uses a solvent selected from the group consisting of methanol, ethanol, ethyl acetate and isopropanol, and a water-based mixed fiber microporous filter membrane is used during filtration.

2. The method of preparing tetrodotoxin according to claim 1, wherein The compound of formula VI-a is obtained from a compound of formula V under the action of an acid: wherein the compound of formula VI-a and the compound of formula VI-b are separated by chiral preparative HPLC.

3. The method of preparing tetrodotoxin according to claim 2, wherein The compound of formula VI-b is converted into the compound of formula VI-a under the action of an acid: wherein the compound of formula VI-a and the compound of formula VI-b are separated by chiral preparative HPLC.

4. The method of preparing tetrodotoxin according to claim 1, wherein The metal Lewis acid is copper chloride.

5. The method of preparing tetrodotoxin according to claim 4, wherein The compound of formula IV is obtained from a compound of formula III by removing the Boc protecting group: wherein acetonitrile is used as the solvent and trimethylsilyl iodide is used as the reagent, and the reaction temperature is -10°C to 50°C.

6. The method of preparing tetrodotoxin according to claim 5, wherein The reaction temperature is 0°C.

7. The method of preparing tetrodotoxin according to claim 1, wherein The base is potassium carbonate, and the solvent used is methanol.

8. A method of preparing tetrodotoxin, characterized by, The method comprises the following steps: (1) intramolecular cyclization of the compound of formula II to prepare a compound of formula III wherein R is C 1-6 alkyl or substituted alkyl, the base used is selected from potassium carbonate, sodium carbonate, potassium bicarbonate and sodium bicarbonate; the solvent used is selected from methanol, ethanol and toluene; (2) removing the Boc protecting group from the compound of formula III to prepare a compound of formula IV wherein acetonitrile is used as the solvent and trimethylsilyl iodide is used as the reagent, and the reaction temperature is -10°C to 50°C; (3) reacting the compound of formula IV with methyl thiourea to prepare a compound of formula V wherein the reaction uses a metal Lewis acid selected from the group consisting of lead chloride, lead acetate, silver nitrate, copper chloride and cuprous chloride; (4) intramolecular cyclization of the compound of formula V to prepare a compound of formula VI-a and a compound of formula VI-b wherein the compound of formula VI-a and the compound of formula VI-b are separated by chiral preparative HPLC; (5) hydrolysis of the compound of formula VI-b to prepare a compound of formula VI-a wherein the compound of formula VI-a and the compound of formula VI-b are separated by chiral preparative HPLC; (6) hydrogenation of the compound of formula VI-a to remove the Cbz protecting group to prepare a compound of formula TTX wherein Cbz represents a benzyloxy carbonyl group, the reaction uses a solvent selected from the group consisting of methanol, ethanol, ethyl acetate and isopropanol, and a water-based mixed fiber microporous filter membrane is used during filtration.

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