CN119698421A

CN119698421A - Preparation method of tetrodotoxin and its analogs

Info

Publication number: CN119698421A
Application number: CN202380058984.6A
Authority: CN
Inventors: 齐湘兵; 陈珮豪; 王静; 王岩; 孙毓泽; 吴青翠
Original assignee: National Institute of Biological Sciences Beijin
Current assignee: National Institute of Biological Sciences Beijin
Priority date: 2022-08-11
Filing date: 2023-08-11
Publication date: 2025-03-25
Also published as: WO2024032799A1

Abstract

本公开涉及TTX或其类似物的制备方法，还涉及可用于TTX或其类似物的制备方法的化合物。The present disclosure relates to a method for preparing TTX or an analog thereof, and also relates to a compound that can be used in the method for preparing TTX or an analog thereof.

Description

Process for preparing tetrodotoxin and analogues thereof

Cross Reference to Related Applications

The present disclosure claims priority from PCT patent application number PCT/CN2022/111861 filed 8/11 2022, the contents of which are incorporated herein by reference in their entirety as part of the present disclosure.

Technical Field

The present disclosure relates to methods of preparing tetrodotoxin or its analogues. The present disclosure also relates to various compounds useful for preparing tetrodotoxin or its analogs.

Background

Tetrodotoxin (TTX) is a well-known marine toxin that was originally isolated from puffer fish and later found in many other species such as salamander and octopus. The biological preparation of TTX remains a puzzle, although there are various lines of evidence that TTX is produced by microorganisms and several biosynthetic pathways are proposed. After isolation of analytically pure TTX in 1952, woodward, tsuda, goto and Mosher successfully broken down its complex structure in 1964. This unique structure consists of a densely functionalized, stereochemically complex framework with unprecedented dioxaadamantane containing an orthoacid, a cyclic guanidine hemiaminal moiety (cyclic guanidinium hemiaminal moiety), and nine consecutive stereocenters, including a bridgehead nitrogen-containing quaternary carbon center. There are three compounds in equilibrium-orthoesters, 4,9-anhydro compounds (4, 9-anhydro) and lactones, which are interconvertible under acidic conditions, and therefore it is difficult to obtain pure TTX by separation from natural mixtures.

Regarding the biological activity of TTX, the idea of disrupting voltage-gated sodium ion channels (Na _v) was proposed as early as the first 60 th century, while recent crystallographic studies revealed the binding motif of TTX to human Na _v 1.7.7. Extensive pharmacological studies have shown that TTX is largely promising for pain treatment, but its serious systemic toxicity limits its clinical use. To overcome the limitations of systemic toxicity and low bioavailability, structural modifications and suggestions for using innovative delivery systems have been proposed, but clinical evaluations have a need for reliable sources of pure TTX.

Such attractive molecules have also attracted considerable attention from the organic synthesis community due to the very dense array of heteroatoms surrounding its complex sp ³ hybridized carbon-rich core. To date, several groups have reported a full process for TTX, with Kishi achieving the first milestone in 1972 by racemization of the preparation. Subsequently, isobe, du Bois, sato, fukuyama and Yokoshima achieved asymmetric synthesis. Recently Trauner et al (2022) disclose a process for asymmetric preparation of TTX based on glucose derivatives which is simple and easy to implement. Furthermore Alonso, ciufolini and Hudlicky report innovative syntheses of key intermediates for the construction of TTX.

Despite the recent impressive advances in developing various strategies for assembling such complex molecules, chemoselective and stereoselective functionalization of sterically hindered carbocyclic systems with successive sp ³ hybridized and highly heteroatom substituents in a large scale and step-economical manner, starting from readily available simple starting materials, remains a considerable challenge.

Disclosure of Invention

The present disclosure presents a method for stereoselective asymmetric synthesis of TTX and its analogs from a large number of available furfuryl alcohol. A highly heteroatom-substituted pseudo-symmetrical cyclohexane skeleton is assembled through a stereoselective Diels-Alder reaction and a chemoselective cyclic anhydride ring opening strategy. Upjohn dihydroxylation has oxygen functionalities attached at C-6, 7, 8 and 11, and decarboxylation hydroxylation supports oxygen attachment at C-5. The scalable total synthesis of TTX is achieved by innovative Smi ₂ -mediated simultaneous free radical elimination, oxygen bridge ring opening and ester reduction sequence, followed by ruthenium catalyzed oxyalkynyl cleavage, and one pot formation of hemi-aminoacetals and orthoesters. The method should be readily adaptable to other natural (and non-natural) TTX homologs to further support research into biosynthesis, physiology, and pharmacology.

In one aspect, the present disclosure provides a method of preparation comprising any one or more of the following steps:

step (a) of converting compound 11 to compound 12,

Step (b) of converting compound 12 to compound 13,

Step (c) of converting compound 13 to compound 14,

Step (d) of converting compound 14 to compound 15,

Step (e) of converting compound 15 to compound 16,

Step (f) of converting compound 16 to compound 17,

Step (g) of converting compound 17 into compound A18,

Preferably, the method comprises the steps of,

Step (h) of converting compound A18 to compound A19,

Preferably, the method comprises the steps of,

Step (i) of converting compound A19 to compound A20,

Preferably, the method comprises the steps of,

Step (j) of converting the compound A20 into the compound A21,

Preferably, the method comprises the steps of,

Step (k) of converting the compound A21 into the compound A22,

Preferably, the method comprises the steps of,

Step (l) of converting the compound A22 into the compound A23,

Preferably, the method comprises the steps of,

Step (m) of converting the compound A23 into the compound A24,

Preferably, the method comprises the steps of,

Step (n) of converting the compound A24 into the compound A25 and/or A25a,

Preferably, the method comprises the steps of,

Step (o) of converting compound A25 into compound A26,

Preferably, the method comprises the steps of,

Step (p) of converting compound A26 to compound A27,

Preferably, the method comprises the steps of,

Step (q) of converting compound A27 into compound A28,

Preferably, the method comprises the steps of,

Step (r) of converting the compound A28 into tetrodotoxin 1,

Preferably, the method comprises the steps of,

Step(s) of converting the compound A27 and/or A27a into the compound A33,

Preferably, the method comprises the steps of,

Wherein,

Each R ₁ independently represents a protecting group, preferably a silicon-containing protecting group, preferably TBDPS, each R ₂ independently represents a protecting group, preferably a benzyl-containing protecting group, preferably p-methoxybenzyl (i.e., PMB), each R ₃ independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, and the like,

Each R ₄ independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, and the like, and

Each X independently represents halogen, preferably selected from Cl or Br.

According to some embodiments of the invention, the method of preparation comprises step (a).

According to some embodiments of the invention, the method of preparation comprises step (b).

According to some embodiments of the invention, the method of preparation comprises step (c).

According to some embodiments of the invention, the method of preparation comprises step (d).

According to some embodiments of the invention, the method of preparation comprises step (e).

According to some embodiments of the invention, the method of preparation comprises step (f).

According to some embodiments of the invention, the method of preparation comprises step (g).

According to some embodiments of the invention, the method of preparation comprises step (h).

According to some embodiments of the invention, the method of preparation comprises step (i).

According to some embodiments of the invention, the method of preparation comprises step (j).

According to some embodiments of the invention, the method of preparation comprises step (k).

According to some embodiments of the invention, the method of preparation comprises step (m).

According to some embodiments of the invention, the method of preparation comprises step (n).

According to some embodiments of the invention, the method of preparation comprises step (o).

According to some embodiments of the invention, the method of preparation comprises step (p).

According to some embodiments of the invention, the method of preparation comprises step (q).

According to some embodiments of the invention, the method of preparation comprises step (r).

According to some embodiments of the invention, the method of preparation comprises step(s).

According to some embodiments of the invention, the method of preparation further comprises step (x) and/or step (y):

Step (x) of converting compound A33 into compound 34,

Preferably, the method comprises the steps of,

Step (y) of converting compound 34 to tetrodotoxin 1, preferably,

According to some embodiments of the invention, the method further comprises any one or more of steps (z 1) to (z 4),

Step (z 1) of converting the compound A25a into the compound A35,

Step (z 2) of converting the compound A35 into the compound A36,

Step (z 3) of converting the compound A36 into the compound A37,

Step (z 4) of converting compound A37 to compound 1a,

Wherein,

Each R ₂ independently represents a protecting group, preferably containing a benzyl protecting group, preferably p-methoxybenzyl (i.e., PMB),

Each R ₃ independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, and the like,

Each X independently represents halogen, preferably selected from Cl or Br.

In some examples, R ₂ is PMB, R ₃ is methyl, and R ₄ is methyl.

According to some embodiments of the invention, step (q) comprises one or both of the following steps (q 1) and (q 2),

Step (q 1) of converting compound A27 into compound A27a,

Preferably, the method comprises the steps of,

Step (q 2) of converting compound A27a into compound A28,

Preferably, the method comprises the steps of,

Wherein each R ₁ independently represents a protecting group, preferably a silicon-containing protecting group, such as TBDPS,

Each R ₂ independently represents a protecting group, preferably a benzyl-containing protecting group, such as p-methoxybenzyl (i.e., PMB),

Each X independently represents halogen, preferably selected from Cl or Br.

According to some embodiments of the invention, step (z 3) comprises one or both of step (z 3-1) and step (z 3-2),

Step (z 3-1) of converting compound A36 into compound A36a,

Step (z 3-2) of converting compound A36a into compound A37,

According to some embodiments of the invention, step (c) comprises one or both of step (c 1) and step (c 2):

step (c 1) of converting compound 13 into compound 13a,

Step (c 2) of converting compound 13a to compound 14,

According to some embodiments of the invention, step (b) comprises step (b 1) and step (b 2):

step (b 1) of converting compound 12 into compound 12a,

Step (b 2) of converting compound 12a to compound 13,

According to some embodiments of the invention, step (f) comprises step (f 1) and step (f 2), step (f 1) converting compound 16 to compound 16a,

Step (f 2) of converting compound 16a to compound 17,

According to some embodiments of the invention, step (j) comprises step (j 1) and step (j 2), step (j 1) converting compound A20 to compound A20a,

Preferably, the method comprises the steps of,

Step (j 2) of converting the compound A20a into the compound A21,

Preferably, the method comprises the steps of,

According to some embodiments of the invention, step (l) comprises a step (l 1) and a step (l 2), step (l 1) of converting compound A22 into compound A22a,

Preferably, the method comprises the steps of,

Step (l 2) of converting the compound A22a into the compound A23,

Preferably, the method comprises the steps of,

According to some embodiments of the invention, step (n) comprises a step (n 1) and a step (n 2), step (n 1) of converting compound A24 to compound A25a,

Preferably, the method comprises the steps of,

Step (n 2) of converting the compound A25a into the compound A25,

Preferably, the method comprises the steps of,

In the above structure of the present application, each R ₁ independently represents a protecting group, preferably a silicon-containing protecting group, such as TBDPS,

Each R ₄ independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, and the like,

Each X independently represents halogen, preferably selected from Cl or Br.

According to some embodiments of the invention, R ₁ is TBDPS.

According to some embodiments of the invention, R ₂ is PMB.

According to some embodiments of the invention, R ₃ is methyl.

According to some embodiments of the invention, R4 is methyl.

According to some embodiments of the invention, X is Cl.

According to some embodiments of the invention, the method comprises any one or more of step (r) and steps of step (a), step (b), step (c), step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q).

According to some embodiments of the invention, the method comprises any two or three or more of the following steps, step (a), step (b), step (c), step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p) and step (q).

According to some embodiments of the invention, the method comprises any one or two or three or more of the following steps, step (b), step (c), step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p) and step (q).

According to some embodiments of the invention, the method comprises the steps of step (b), step (c), step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p) and step (q).

According to some embodiments of the invention, the method comprises step (o), step (p) and/or step (q).

According to some embodiments of the invention, the method comprises step (n), step (o), step (p) and/or step (q).

According to some embodiments of the invention, the method comprises step (m), step (n), step (o), step (p) and/or step (q).

According to some embodiments of the invention, the method comprises step (l), step (m), step (n), step (o), step (p) and/or step (q).

According to some embodiments of the invention, the method comprises step (k), step (l), step (m), step (n), step (o), step (p) and/or step (q).

According to some embodiments of the invention, the method comprises step (j), step (k), step (l), step (m), step (n), step (o), step (p) and/or step (q).

According to some embodiments of the invention, the method comprises step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p) and/or step (q).

According to some embodiments of the invention, the method comprises step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (q).

According to some embodiments of the invention, the method comprises step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p) and/or step (q).

According to some embodiments of the invention, the method comprises step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p) and/or step (q).

According to some embodiments of the invention, the method comprises step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p) and/or step (q).

According to some embodiments of the invention, the method comprises step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p) and/or step (q).

According to some embodiments of the invention, the method comprises step (c), step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p) and/or step (q).

According to some embodiments of the invention, the method comprises step (b), step (c), step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p) and/or step (q).

According to some embodiments of the invention, the method comprises step (a), step (b), step (c), step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p) and/or step (q).

According to some embodiments of the invention, the method comprises:

step (q) and/or step (r), or

Step (o), step (p), step (q) and/or step (r), or

Step (n), step (o), step (p), step (q) and/or step (r), or

Step (m), step (n), step (o), step (p), step (q) and/or step (r), or

Step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r), or

Step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r), or

Step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r), or

Step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r), or

Step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r), or

Step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r), or

Step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r), or

Step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r), or

Step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r), or

Step (c), step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r), or

Step (b), step (c), step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r), or

Step (a), step (b), step (c), step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r).

According to some embodiments of the invention, in step (a) the reagents used comprise quinine and MeOH, and the solvent used comprises CCl ₄ and/or toluene (preferably a mixture of CCl ₄ and/or toluene, preferably in a (1-2) to (1-2) volume ratio).

According to some embodiments of the invention, in step (b 1), the reagent used comprises 4-methylmorpholine N-oxide (NMO), 4-methylmorpholine (NMM) and/or an oxidizing agent, preferably OsO ₄, and the solvent used comprises acetone, preferably a mixture of acetone and water.

According to some embodiments of the invention, in step (b 2), the reagents used include 2, 2-dimethoxypropane and p-toluenesulfonic acid, and the solvents used include acetone.

According to some embodiments of the invention, in step (c 1), the reagent used comprises N-hydroxyphthalimide and a base, preferably an organic base, preferably DMAP, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCI).

According to some embodiments of the invention, in step (c 2) the reagents used comprise Ru (bpy) ₃Cl₂, TEMPO and Hantzsch ester (Hantzsch ester), and/or the solvents used comprise DMF, and/or in step (c 2) the conversion is carried out under irradiation of a blue LED.

According to some embodiments of the invention, in step (d) the reagent used comprises a base, preferably an inorganic base, preferably K ₂CO₃, and/or the solvent used comprises an alcohol, preferably a C1-C4 alcohol.

According to some embodiments of the invention, in step (e), the reagents used comprise I ₂, imidazole and PPh ₃, and/or the solvent used comprises toluene.

According to some embodiments of the invention, in step (f) the reagent used comprises SmI ₂, and preferably also a base, preferably an organic base such as Et ₃ N, and/or the solvent used comprises an ether (preferably THF).

According to some embodiments of the invention, in step (f 1), the reagent used comprises SmI ₂ and/or the solvent used comprises an ether (preferably THF).

According to some embodiments of the invention, in step (f 2), the reagent used comprises LiAlH ₄ and/or the solvent used comprises an ether (preferably THF).

According to some embodiments of the invention, in step (g), the reagent used comprises TBDPSCl, preferably imidazole and/or DMAP.

According to some embodiments of the invention, in step (h), the reagent used comprises zinc powder, and/or the solvent used comprises THF and AcOH.

According to some embodiments of the invention, in step (i), the reagents used include 2-methoxyacetic acid, PPh ₃ and diethyl diazenedicarboxylate (diethyl diazenecarboxylate), and/or the solvents used include THF.

According to some embodiments of the invention, in step (j 1), the reagent used comprises 4-methylmorpholine N-oxide and/or an oxidizing agent, preferably OsO ₄, and/or the solvent used comprises acetone.

According to some embodiments of the invention, in step (j 2), the reagent used comprises 2, 2-dimethoxypropane and camphorsulfonic acid, and/or the solvent used comprises CH ₂Cl₂.

According to some embodiments of the invention, in step (k), the reagent used comprises a Dess-Martin reagent, preferably also an inorganic base, preferably NaHCO ₃.

According to some embodiments of the invention, in step (l 1), the reagents used include N, N-diisopropylamine and N-butyllithium.

According to some embodiments of the invention, in step (l 2), the reagents used include NaHMDS and PMBBr.

According to some embodiments of the invention, in step (m) the reagent used comprises sodium azide, and preferably also crown ether, preferably 15-crown ether-5.

According to some embodiments of the invention, in step (n 1), the reagent used comprises lithium acetylide ethylenediamine complex (lithium ACETYLIDE ETHYLENEDIAMINE complex).

According to some embodiments of the invention, in step (n 2), the reagents used include an oxidizing agent, preferably MnO ₂, and a reducing agent, preferably NaBH ₄.

According to some embodiments of the invention, in step (o), the reagent used comprises an oxidizing agent, preferably 2-iodobenzoic acid, pyridinium p-toluenesulfonate and trimethyl orthoacetate, and/or the solvent used is DMSO.

According to some embodiments of the invention, in step (p), the reagents used comprise a catalyst, preferably RuCl ₃, an oxidizing agent, preferably NaIO ₄, preferably also EDCI and MeOH.

According to some embodiments of the invention, in step (q 1), the reagents used comprise a hydrogenation catalyst (preferably Pd/C) and hydrogen.

According to some embodiments of the invention, in step (q 2), the reagent used comprises 1, 3-bis (t-butoxycarbonyl) -2-methyl-2-thiopseudourea and mercury (II) chloride, preferably also an organic base, preferably Et ₃ N.

According to some embodiments of the invention, in step (r), the reagent used comprises trifluoroacetic acid.

According to some embodiments of the invention, in step(s), the reagents used include 1, 3-bis (benzyloxycarbonyl) -2-methyl-2-thiopseudourea and mercury (II) chloride.

According to some embodiments of the invention, in step (x), the reagent used comprises trifluoroacetic acid.

According to some embodiments of the invention, in step (y), the reagents used comprise a hydrogenation catalyst, preferably Pd/C, and hydrogen.

According to some embodiments of the invention, in step (z 1), the reagent used comprises an oxidizing agent, preferably 2-iodobenzoic acid, pyridinium p-toluenesulfonate and trimethyl orthoacetate, and/or the solvent used is DMSO.

According to some embodiments of the invention, in step (z 2), the reagents used comprise a catalyst, preferably RuCl ₃, an oxidizing agent, preferably NaIO ₄, preferably also EDCI and MeOH.

According to some embodiments of the invention, in step (z 3-1), the reagents used comprise a hydrogenation catalyst (preferably Pd/C) and hydrogen.

According to some embodiments of the invention, in step (z 3-2), the reagent used comprises 1, 3-bis (t-butoxycarbonyl) -2-methyl-2-thiopseudourea and mercury (II) chloride, preferably further comprising an organic base, preferably Et ₃ N.

According to some embodiments of the invention, in step (z 4), the reagent used comprises trifluoroacetic acid.

According to some embodiments of the invention, the method of preparation comprises the steps of:

According to some embodiments of the invention, the method of preparation comprises any one or more of the following steps:

in a second aspect, there is provided a compound selected from the group consisting of:

wherein,

Each R ₁ independently represents a protecting group, preferably a silicon-containing protecting group, such as TBDPS,

Each R ₂ independently represents a protecting group, preferably a benzyl-containing protecting group, such as p-methoxybenzyl (i.e., PMB), each R ₃ independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, and the like,

Each X independently represents halogen, preferably selected from Cl or Br.

Specifically, the compound is selected from any one of the following:

In a third aspect, there is provided the use of a compound according to the second aspect of the disclosure for the preparation of tetrodotoxin or its analogues.

In a fourth aspect, there is provided a method of preparation comprising the use of one or more of the compounds described in the second aspect of the present disclosure. In the preparation process, the compounds may be used as starting materials or as intermediate compounds.

In a fifth aspect, there is provided a method of preparing tetrodotoxin or its analogue, comprising the use of one or more of the compounds described in the second aspect of the disclosure. In the preparation process, the compounds may be used as starting materials or as intermediate compounds.

The scope of the present application includes intermediate compounds used in the preparation methods of the present application.

Detailed Description

Tetrodotoxin and its homologs are specific voltage-gated sodium ion channel blockers with remarkable anesthetic and analgesic effects. Extensive pharmacological studies, including clinical trials, have shown that TTX has potential in pain treatment and heroin addiction withdrawal.

In the present disclosure, a new scalable synthetic method for the neurotoxin family is provided, providing for rapid availability of highly oxidized natural products and scalable preparation of TTX and its analogs.

1. General information

Unless otherwise indicated, all reactions were performed in a flame-dried glassware under nitrogen atmosphere with magnetic stirring. Reagents were purchased from ALDRICH CHEMICAL, ALFA AESAR, TCI, adamas, ENERGY CHEMICAL or J & K with the highest commercial quality and used without further purification. The solvent was dried under argon over an activated alumina column. The liquid and solution are transferred through a syringe. The reaction was monitored by GC/MS, UPLC/MS and Thin Layer Chromatography (TLC) and visualized using 254nm uv light and heated phosphomolybdic acid solution staining. All flash column chromatography using Qingdao sea-yang chemical silica gel (300-400 mesh size). ¹ H and ¹³ C NMR spectra were recorded by a Varian Inova-400 spectrometer. ¹ H NMR spectral data were reported relative to CDCl ₃(7.26ppm)、CD₃ OD (3.31 ppm) or CD ₃CO₂ D (2.06 ppm) as internal standard and reported as chemical shift (delta ppm), multiplet (s=singlet, d=doublet, t=triplet, q=quartet, sept =heptadoublet, m=multiplet, br=broad), coupling constant J (Hz) and integral. Data for ¹³ C NMR spectra were reported relative to CDCl ₃(77.00ppm)、CD₃OD(49.00ppm)、CD₃CO₂ D (22.4 ppm) as an internal standard and reported in chemical shifts (δppm). UPLC-MS analysis was performed on a Waters system (column: BEH C18,1.7 μm, 2.1. Times.50 mm) equipped with a photodiode array (PDA) detector and a Single Quadrupole (SQ) detector. High resolution mass spectra were obtained from an Agilent 1290LC-6540QTOF mass spectrometer or Agilent Technologies 7250 GCQTOF. HPLC analysis on Waters (ColumnHILIC SILICA,5 μm, 4.6X105 mm and 19X 150 mm) equipped with 2998PDA and 3100MS detectors.

Scheme 1. Total synthesis of tetrodotoxin.

Scheme 2.9-Synthesis of epi tetrodotoxin.

In view of the fact that the core structure of TTX is a pseudo-C2-symmetrical structure, we contemplate that the symmetry of highly substituted functional groups can be exploited to rapidly assemble the scaffold. Subsequent chemoselective asymmetric cyclic anhydride ring opening and selective functional group interconversion strategies lay the foundation for gram-scale synthesis of the highly oxidized carbocyclic structural unit (-) -24 as a higher intermediate in only 15 steps and rapidly transfer it to the final TTX (1) in a scalable manner.

Synthesis of TTX 1 begins with stereoselective construction of the cyclohexane skeleton. Esterification of furfuryl alcohol 9 with the chiral auxiliary (-) - (1S) -camphoric acid 32 gives ester 10. To obtain enantiomerically pure 7-oxabicyclo [2.2.1] hept-2-ene derivative 11, we developed a highly efficient stereoselective Diels-Alder scheme, i.e. using isopropyl ether as solvent, heating 10 with maleic anhydride. The pseudo-asymmetry ³⁴ of anhydride 11 is promoted by quinine-mediated chemoselective methanolysis, yielding a monoacid 12 with high regioselectivity. Subsequently, stereospecific Upjohn racemic dihydroxylation of the alkene and simultaneous 1, 2-diol protection were carried out in one pot to give acid 13, the structure of which was confirmed by single crystal X-ray crystallographic analysis (CCDC#: 2184304).

Decarboxylation hydroxylation is performed, placing the TTX hydroxyl group in the C5 position. Initially, classical high valence metal reagents were studied as oxidants, but this resulted in substrate decomposition, and therefore we turned to milder free radical reaction conditions, including photocatalytic directed decarboxylation hydroxylation. Other decarboxylation methods, such as Barton decarboxylation or organic photoredox-promoted decarboxylation in the presence of a free radical initiator and oxygen, ultraviolet radiation, cannot produce a product. Finally, we performed Ru-catalyzed photo-redox decarboxylation hydroxylation of NHPI ester 13 using the method developed by the Liang group and successfully obtained 14, despite the inversion of C5 configuration compared to TTX. Previous syntheses found that direct construction of the correct stereochemical structure of C5 is challenging and that the steric effect of the substitution at the C5 position is troublesome for the subsequent installation of functional groups, so we decided to reverse the configuration of C5 at a later stage. Notably, this photo-redox decarboxylation hydroxylation can be extended to 1.5g by using a recycle stream photochemistry technique without affecting yield.

With compound 14, we studied the functional group interconversions of this oxygen bridged ring system and developed an innovative cascade to build up oxygen functionality at the C8a, C6 and C11 positions. Firstly, methanol is used for transesterification reaction, and auxiliary (-) -camphoric acid is removed to obtain primary alcohol 15. The chiral auxiliary can be used as methyl camphoride for recycling. Primary alcohol 15 undergoes an Appel reaction to yield alkyl iodide 16, however, various reduction conditions imposed on the alkyl iodide do not produce the desired product due to substrate decomposition. Through extensive exploration of the reductive ring opening conditions, we have now found that the cascade of initial Kagan reagent (SmI ₂) mediated single electron transfer and iodide loss produces primary carbon radicals which undergo radical elimination to form terminal olefins, while SmI ₂ simultaneously reduces the unactivated methyl esters to primary alcohols. In the presence of Hexamethylphosphoramide (HMPA), only eliminated product 17 (i.e. methyl ester was not reduced to diol 17 a) was obtained (entry 1). Activation of SmI ₂ with H ₂ O and Et ₃ N in a 1:2:2 ratio produced a thermodynamically stronger reductant ³⁸, reducing methyl ester (entry 2) in 77% ¹ HNMR yield. Either increasing the ratio of H ₂ O to Et ₃ N or replacing Et ₃ N with pyrrolidine resulted in a complex product mixture (entries 3 and 4). For scalability issues, the procedure described above can also be modified to a two-step process involving 17a with a small equivalent of SmI ₂, followed by LiAlH ₄ reduction to give 17 in the decimal order of 58% yield (entry 5). The absolute configuration of 17 was verified by single crystal X-ray crystallography (CCDC#: 2182018).

With 17, we focused on building the quaternary stereocenter of C8a and the inversion of the hydroxyl configuration of C5. The construction of azidoaldehyde 24 starts with the selective protection of primary alcohols in 17 using sterically hindered TBDPSCl. The N-O bond of TEMPO in the obtained olefin 18 is reduced by Zn powder to obtain allyl alcohol 19. The wrong configuration of the C5-OH was then reversed using Mitsunobu reaction with 2-methoxyacetic acid 29 to give secondary alcohol 20. 20 was subjected to a fine diastereoselective Upjohn dihydroxylation followed by protection with acetonide to give 21, the absolute configuration of which was verified by X-ray crystallography of derivative structure 31 (CCDC#: 2184298), which derivative 31 was obtained by hydrolysis of NH ₃/MeOH of dihydroxylated 20 (see supplementary materials).

Intermediate 21 was oxidized by Dess-Martin (DMP) to give ketone 22 in excellent yields. From ketone 22, our goal was to build a quaternary stereocenter at C8a, which was found to be challenging. Nucleophilic addition with imine derived from ketone 22 yields only diastereomers with undesired configuration at C8 a. Although Darzens condensation of 22 with alpha-haloesters successfully produced glycidyl esters, the corresponding epoxides were inert to stereoselective ammonolysis. It is appreciated that nucleophilic addition of the dichloromethyl anion successfully converts ketone 22 to the single isomer of spiro α -chloroepoxide 23, and simultaneously interconverts the ester protecting group at C5 to p-methoxybenzyl (PMB). After treatment of the resulting chloroepoxide 23 with NaN ₃, gram-grade alpha-azidoaldehyde 24 was obtained with the correct configuration at C8 a.

The construction of highly heteroatom substituted carbocyclic cores was completed in only 15 steps, and we have prepared to address the synthetic challenges of the key dioxaadamantane and guanamine acetal moieties. The alpha-azidoaldehyde 24 was 1, 2-added to lithium acetylide, followed by simultaneous removal of the TBDPS protecting group, yielding two diastereomers (25 and 25 a) in a 1:15 ratio with the undesired product 25a predominately. Extensive studies of the reaction conditions indicate that 25a can be converted to the desired propynyl alcohol 25 in a ratio of 2:1 (25/25 a = 2:1) by a sequence comprising MnO ₂ mediated selective oxidation followed by NaBH ₄ reduction. Primary alcohol 25 undergoes IBX oxidation to the corresponding hemiacetal, which is then converted to acetal 26 by reaction with trimethyl orthoacetate in the presence of pyridinium p-toluenesulfonate (PPTS). 26 was confirmed by single crystal X-ray crystallography (CCDC#: 2184305). Oxidative cleavage of alkyne 26 with RuCl ₃/NaIO₄ followed by esterification with methanol gives methyl carboxylate 27. The simultaneous deprotection of the PMB and reduction of the azide by hydrogenation effectively yields a tertiary amine which is guanidinated in situ with bis-Boc protected isothiourea 30 to afford the penultimate intermediate 28.

2. Detailed experimental procedure

Example 1

To a stirred solution of furfuryl alcohol 9 (39.6 g,404mmol,1.00 eq), (1S) - (-) -camphoric acid (80.0 g,404mmol,1.0 eq) and DMAP (4.93 g,40.4mmol,0.10 eq) in CH ₂Cl₂ (1400 mL) at 0℃was added 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCI) (116.6 g,606mmol,1.50 eq) and then stirred slowly under an argon atmosphere from 0℃to room temperature for 27 hours. The mixture was quenched with 0.5N HCl solution (800 mL) and then extracted three times with CH ₂Cl₂ (3X 800 mL). The combined organic extracts were dried over anhydrous Na ₂SO₄, filtered and concentrated under reduced pressure. The residue was recrystallized from EtOH and petroleum ether to give compound 10 (101.0 g, 90%) as a white solid.

R_f=0.33(PE/EtOAc=4:1)

(c=0.21,CHCl₃)

HRMS-ESI (m/z) C ₁₅H₁₈O₅Na[M+Na]⁺ calculated 301.1052, measured 301.1043.

¹H NMR(400MHz,CDCl₃)δ7.42(dd,J=1.8,0.8Hz,1H),6.45(d,J=3.3

Hz,1H),6.40–6.33(m,1H),5.22(q,J＝13.0Hz,2H),2.43(ddd,J＝14.2,10.7,4.2Hz,1H),2.03(ddd,J＝13.7,9.5,4.6Hz,1H),1.96–1.85(m,1H),1.68(ddd,J＝13.4,9.3,4.1Hz,1H),1.10(s,3H),1.00(s,3H),0.88(s,3H).

¹³C NMR(101MHz,CDCl₃)δ177.96,167.14,148.64,143.44,111.33,110.62,90.96,58.70,54.71,54.29,30.48,28.93,16.67,16.54,9.67.

Example 2

To a stirred solution of compound 10 (100.0 g,360mmol,1.00 eq) in 290mL of isopropyl ether was added maleic anhydride (35.3 g,360mmol,1.00 eq) in the dark, and the mixture was then heated to 55 ℃ and stirred overnight under an argon atmosphere. Then, 1.0eq of maleic anhydride was added to continue the reaction. The crude ¹ H NMR was examined until no compound 10 remained, and after filtration, crude compound 11 (131.0 g, containing a small amount of maleic anhydride) was obtained as a white solid without further purification.

Note that compound 11 is sensitive to protic solvents and should be protected from water or methanol during the whole process.

R_f=0.33(PE/EtOAc=1:1)

(c=0.16,CHCl₃)

HRMS-ESI (m/z) C ₁₉H₂₄NO₈[M+NH₄]⁺ calculated 394.1502, measured 394.1495.

¹H NMR(400MHz,CDCl₃)δ6.65(d,J＝5.7Hz,1H),6.54(d,J＝5.8Hz,1H),5.46(s,1H),4.97(d,J＝12.4Hz,1H),4.69(d,J＝12.4Hz,1H),3.31(dd,J＝26.4,6.9Hz,2H),2.44(ddd,J＝14.4,10.5,3.8Hz,1H),2.04(ddd,J＝13.7,9.3,4.5Hz,1H),1.93(ddd,J＝12.4,11.0,4.4Hz,1H),1.69(ddd,J＝13.3,9.3,4.1Hz,1H),1.12(s,3H),1.06(s,3H),1.00(s,3H).

¹³C NMR(101MHz,CDCl₃)δ178.06,169.11,167.81,166.85,138.05,137.34,90.94,90.11,82.34,61.48,54.86,54.50,51.22,49.66,30.75,28.84,16.67,16.62,9.70.

Example 3

Quinine (112.0 g,348mmol,1.00 eq) was added to a stirred solution of compound 11 (131.0 g,348mmol,1.00 eq) in toluene/CCl ₄ (750 mL/750 mL) at 0 ℃, followed by MeOH (55.7 g,1740mmol,5.00 eq) at 0 ℃. Then stirring was slow at 0 ℃ to room temperature under argon atmosphere for 22 hours. After concentration under reduced pressure, the residue was dissolved in CH ₂Cl₂ and acidified with 1.0N HCl solution to ph=2, extracted by CH ₂Cl₂ (3×800 mL) until no quinine was present in the organic layer. The combined organic extracts were dried over anhydrous Na ₂SO₄, filtered and concentrated under reduced pressure. The residue was recrystallized from CH ₂Cl₂ and petroleum ether to give compound 12 (127.5 g, 87% in two steps) as a white solid.

R_f=0.4(DCM/MeOH=10:1)

(c=0.32,CHCl₃)

HRMS-ESI (m/z) C ₂₀H₂₈NO₉[M+NH₄]⁺ calculated 426.1764, measured 426.1754.

¹H NMR(400MHz,CDCl₃)δ6.56(dd,J＝5.6,1.6Hz,1H),6.39(d,J＝5.7Hz,1H),5.46(d,J＝1.7Hz,1H),4.86(d,J＝12.2Hz,1H),4.70(d,J＝12.1Hz,1H),3.71(s,3H),3.07(d,J＝8.9Hz,1H),2.95(d,J＝9.0Hz,1H),2.48–2.34(m,2H),2.08–1.98(m,1H),1.98–1.84(m,2H),1.73–1.62(m,1H),1.11(s,3H),1.03(s,3H),0.96(s,3H).

¹³C NMR(101MHz,CDCl₃)δ178.09,175.59,171.01,166.85,137.80,136.59,91.07,89.02,79.96,62.30,54.81,54.32,52.46,49.94,48.28,30.65,28.88,16.72,16.61,9.66.

Example 4

To a stirred solution of compound 12 (70.0 g,171mmol,1.00 eq) in acetone (900 mL) and H ₂ O (36 mL) were added 4-methylmorpholine N-oxide (NMO) (30.0 g,256mmol,1.50 eq) and 4-methylmorpholine (NMM) (19.0 g,188mmol,1.10 eq) followed by slow addition of 0.05M aqueous OsO ₄ (34 mL,1.7mmol,0.01 eq) in the dark and then stirring of the mixture at room temperature for 3 hours. Quenched with saturated aqueous Na ₂SO₃ and then concentrated under reduced pressure. The residue was acidified to ph=2 with 1.0N HCl solution and extracted three times with ethyl acetate (3×500 mL), the remaining aqueous layer was extracted with ^n- BuOH until no product was present. After concentration under reduced pressure, pale yellow foam crude compound 12a was obtained for subsequent reaction without further purification.

To a stirred solution of compound 12a in 800mL of acetone were added 2, 2-dimethoxypropane (26.6 g,256mmol,1.50 eq) and PTSA.H ₂ O (3.3 g,17mmol,0.10 eq) and then stirred at room temperature for 3.5 hours. The reaction was quenched by addition of 4.0gNaHCO ₃ solids and concentrated under reduced pressure. The residue was directly purified by flash chromatography on silica gel (CH ₂Cl₂/MeOH, 100:1 to 20:1) to give compound 13 (63.7 g, 77%) as a white foam.

R_f=0.5(DCM/MeOH=10:1)

(c=0.36,CHCl₃)

HRMS-ESI (m/z) C ₂₃H₃₄NO₁₁[M+NH₄]⁺ calculated 500.2132, measured 500.2123.

¹H NMR(400MHz,CDCl₃)δ9.67(brs,1H),4.94(s,1H),4.65(d,J＝10.9Hz,1H),4.55(d,J＝10.9Hz,1H),4.34(dd,J＝20.7,5.5Hz,2H),3.66(s,3H),3.07(d,J＝9.6Hz,1H),2.95(d,J＝9.7Hz,1H),2.42(ddd,J＝14.7,9.0,4.1Hz,1H),2.06(ddd,J＝9.3,6.9,2.8Hz,1H),1.98–1.85(m,1H),1.69(ddd,J＝17.5,8.9,4.3Hz,2H),1.43(s,3H),1.28(s,3H),1.10(s,3H),1.05(s,3H),0.94(s,3H).

¹³C NMR(101MHz,CDCl₃)δ178.74,174.08,169.88,166.47,113.27,91.26,86.69,81.78,81.73,80.46,60.63,54.88,54.51,52.47,48.15,47.43,30.69,28.93,25.90,25.61,16.65,16.52,9.62.

Example 5

Method A:

To a stirred solution of compound 13 (30.0 g,62.2mmol,1.0 eq) in CH ₂Cl₂ (600 mL) was added N-hydroxyphthalimide (15.2 g,93.3mmol,1.5 eq) and DMAP (7516 mg,6.2mmol,0.1 eq) followed by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCI) (17.9 g,93.3mmol,1.5 eq) at 0℃and stirring slowly in the dark under an argon atmosphere for 24 hours from 0℃to room temperature. The resulting mixture was quenched with saturated aqueous NH ₄ Cl (800 mL) and then extracted three times with CH ₂Cl₂ (3X 500 mL). Dried over Na ₂SO₄, filtered, and concentrated under reduced pressure. The crude NHPI ester 13a was used directly in the next step without further purification.

A solution of the above crude NHPI ester 13a, ru (bpy) ₃Cl₂ (1.91 g,3.2mmol,0.05 eq), TEMPO (14.9 g,95.8mmol,1.54 eq) and Hans ester (15.7 g,62.2mmol,1.0 eq) in DMF (degassed, 500 mL) was vigorously stirred at room temperature under a 36W blue LED for 24 hours. The reaction mixture was then concentrated under reduced pressure. The residue obtained was subjected to silica gel column chromatography (petroleum ether/CH ₂Cl₂, 3:1 to 1:1) to give compound 14 (22.7 g,62%, dr > 95:5) as a pale yellow solid.

R_f=0.4(PE/EA=4:1)

(c=0.23,CHCl₃)

HRMS-ESI (m/z) C ₃₁H₄₈NO₁₀[M+H]⁺ calculated 594.3278, measured 594.3265.

¹H NMR(400MHz,CDCl₃)δ5.06–5.01(m,2H),4.80(d,J＝5.6Hz,1H),4.52–4.48(m,3H),3.71(s,3H),2.84(d,J＝3.2Hz,1H),2.54–2.45(m,1H),2.03(ddd,J＝13.7,9.3,4.5Hz,1H),1.92(ddd,J＝13.1,10.8,4.6Hz,1H),1.67(ddd,J＝13.4,9.4,4.2Hz,1H),1.59–1.37(m,9H),1.33(s,7H),1.21–1.05(m,14H),0.99(s,3H).

¹³C NMR(101MHz,CDCl₃)δ178.03,171.84,166.95,112.84,91.25,86.62,83.10,82.70,81.97,78.62,60.56,54.82,54.10,52.31,48.96,40.09,39.75,34.34,33.93,30.62,28.92,26.03,25.59,20.52,20.44,16.87,16.75,9.71.

Method B (flow chemistry procedure):

To a stirred solution of compound 13 (1.5 g,3.1mmol,1.0 eq) in CH ₂Cl₂ (25 mL) was added N-hydroxyphthalimide (760 mg,4.65mmol,1.5 eq) and DMAP (37 mg,0.3mmol,0.1 eq) followed by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCI) (892 mg,4.65mmol,1.5 eq) at 0℃and stirring slowly in the dark under an argon atmosphere for 24 hours from 0℃to room temperature. The resulting mixture was quenched with saturated aqueous NH ₄ Cl (20 mL) and then extracted three times with CH ₂Cl₂ (3X 20 mL). Dried over Na ₂SO₄, filtered, and concentrated under reduced pressure. The crude NHPI ester was used directly in the next step without further purification.

The freshly prepared NHPI activated ester 13a was added to a 100mL round bottom flask, followed by Ru (bpy) ₃Cl₂ (96 mg,0.16mmol,0.05 eq), TEMPO (750 mg,4.8mmol,1.54 eq) and hans ester (785 mg,3.1mmol,1.0 eq), DMF (degassed, 40 mL). A flow device is inserted. Argon was introduced into the system for 10 minutes, and the reaction was carried out under irradiation of a 36W blue LED at a flow rate of 14rpm for 24 hours. The reaction mixture was then concentrated under reduced pressure. The residue obtained was subjected to silica gel column chromatography (petroleum ether/CH ₂Cl₂, 3:1 to 1:1) to give compound 14 (1.22 g,66%, dr > 95:5) as a pale yellow solid.

Materials used in the flow procedure were peristaltic pumps (BT 02-YZ 1515), PFA tubing, silicone tubing, 36W 450nm LEDs, and electric fans (all materials purchased on Taobao mesh).

Example 6

To a stirred solution of compound 14 (20.0 g,33.7mmol,1.00 eq) in 350mL MeOH was added K ₂CO₃ (6.9 g,50.5mmol,1.50 eq) and then stirred at room temperature for 1 hour. Quench with 10mL of AcOH, then concentrate under reduced pressure and conduct silica gel column chromatography (CH ₂Cl₂/MeOH, 100:1 to 40:1) to give compound 15 (13.2 g, 95%) as a colourless oil.

R_f=0.5(DCM/MeOH=20:1)

(c=0.24,CHCl₃)

HRMS-ESI (m/z) C ₂₁H₃₆NO₇[M+H]⁺ calculated 414.2492, measured 414.2477.

¹H NMR(400MHz,CDCl₃)δ4.94(s,1H),4.79(d,J=6.3Hz,1H),4.51–

4.43(m,2H),4.21(d,J＝12.5Hz,1H),4.01(dd,J＝12.1,9.9Hz,1H),3.72(s,3H),2.80(d,J＝3.4Hz,1H),2.33(br s,1H),1.47–1.36(m,8H),1.35–1.20(m,7H),1.15–0.99(m,9H).

¹³C NMR(101MHz,CDCl₃)δ172.40,112.29,87.90,82.89,82.69,82.39,78.86,61.07,59.64,59.22,52.22,48.63,40.45,40.05,35.20,34.19,25.90,25.28,20.39,20.22,16.99.

Example 7

To a stirred solution of compound 15 (13.0 g,31.5mmol,1.00 eq), PPh ₃ (30.6 g,117mmol,3.74 eq) and imidazole (6.7 g,99.5mmol,3.16 eq) in 300mL toluene was added I ₂ (21.2 g,83.8mmol,2.66 eq) dissolved in 100mL toluene and then stirred at 110℃for 2 hours. The resulting mixture was concentrated under reduced pressure, and subjected to silica gel column chromatography (petroleum ether/ethyl acetate, 40:1 to 25:1) to give compound 16 (14.6 g, 89%) as a colorless oil.

R_f=0.4(PE/EA=10:1)

(c=0.26,CHCl₃)

HRMS-ESI (m/z) C ₂₁H₃₅INO₆[M+H]⁺ calculated 524.1509, measured 524.1503.

¹H NMR(400MHz,CDCl₃)δ4.98(d,J＝3.3Hz,1H),4.78(d,J＝5.5Hz,1H),4.63(d,J＝5.5Hz,1H),4.48(s,1H),3.79(d,J＝11.3Hz,1H),3.75(s,3H),3.56(d,J＝11.5Hz,1H),2.92(d,J＝3.3Hz,1H),1.54(s,3H),1.50–1.30(m,12H),1.22–1.00(m,9H).

¹³C NMR(101MHz,CDCl₃)δ171.92,112.51,86.51,84.36,83.98,82.28,77.92,60.93,59.33,52.23,48.39,40.54,40.03,35.08,26.01,25.64,20.45,20.39,16.96,1.18.

Example 8

1. Preparation of a 0.1M solution of Smi ₂ in THF

A flame-dried 1L round bottom flask was charged with samarium metal (12.70 g,86mmol,1.00 eq.) and a stirring bar, then thoroughly degassed THF (860 mL) and iodine crystals (22.68 g,86mmol,1.00 eq.) were added sequentially at room temperature under argon atmosphere. The reaction mixture was vigorously stirred at room temperature for more than 2 hours. As SmI2 was generated, the color of the solution changed from orange to yellow and green, and finally to deep blue.

Note that to ensure adequate conversion, the solution should be stirred for at least 2 hours before use.

Smi ₂ induced reduction reaction

A solution of 0.1M Smi ₂ (115 mL,11.5mmol,12.0 eq) in THF freshly prepared as described above was transferred using a double ended needle to a new flame-dried 500mL round bottom flask in which compound 16 (500 mg,0.95mmol,1.00 eq), et ₃ N (318. Mu.L, 23.0mmol,24.0 eq) and H ₂ O (413. Mu.L, 23.0mmol,24.0 eq) were dissolved in 2mL THF. The whole process needs to be protected from air and carried out under an argon atmosphere. The mixture was then stirred at room temperature for 1.5 hours. After completion of the reaction, the color turned yellow, which was then quenched with saturated aqueous NH ₄ Cl (10 mL), washed with 0.1N aqueous HCl (2 mL), and extracted with ethyl acetate (3X 10 mL). Dried over anhydrous MgSO ₄, filtered, and concentrated under reduced pressure. The crude product compound was purified by silica gel column chromatography (CH ₂Cl₂/MeOH, 80:1) to give compound 17 (229 mg, 65%) as a white solid.

Scalable preparation of Compound 17

A solution of 0.1M Smi ₂ in THF was freshly prepared in the same manner as described above. The above solution of Smi ₂ in freshly prepared THF (860 mL) was transferred using a double-ended needle to a fresh flame-dried 1L round bottom flask and compound 16 (15.0 g,28.7mmol,1.00 eq) was dissolved in a solution of 20mL THF. The whole process needs to be protected from air and carried out under an argon atmosphere. The mixture was then stirred at 55 ℃ for 2 hours. After completion of the reaction, the color turned yellow, and then quenched with saturated aqueous NH ₄ Cl (500 mL), the mixture was filtered to remove samarium salt, and then extracted with ethyl acetate (3×800 mL). Dried over Na ₂SO₄, filtered, and concentrated under reduced pressure. The crude compound 16a was used directly in the next step without further purification.

To a stirred solution of compound 16a in 150mL of THF at 0deg.C was slowly added LiAlH ₄ (1.09 g,28.7mmol,1.00 eq). The mixture was stirred slowly under an argon atmosphere, from 0 ℃ to room temperature for 12 hours. Excess Na ₂SO₄·10H₂ O was added to quench the reaction, which was then filtered and concentrated under reduced pressure. The crude product compound was purified by silica gel column chromatography (CH ₂Cl₂/MeOH, 80:1) to give compound 17 (6.1 g, 58% in two steps) as a white solid.

R_f=0.2(PE/EA=2:1)

(c=0.30,CHCl₃)

HRMS-ESI (m/z) C ₂₀H₃₆NO₅[M+H]⁺ calculated 370.2593, measured 370.2585.

¹H NMR(400MHz,CDCl₃)δ5.40(s,1H),5.23(s,1H),4.75(d,J=6.5Hz,

1H),4.51(s,1H),4.45–4.38(m,1H),4.30(d,J＝3.9Hz,1H),3.95(dd,J＝10.7,6.5Hz,1H),3.76(dd,J＝10.6,6.8Hz,1H),2.92(d,J＝4.3Hz,1H),2.56–2.46(m,1H),1.57–0.96(m,24H).

¹³C NMR(101MHz,CDCl₃)δ142.43,115.10,109.62,84.93,76.78,74.39,67.62,62.88,60.48,59.94,45.20,39.73,33.82,33.51,27.09,25.32,20.17,20.08,16.61.

Example 9

To a stirred solution of compound 17 (7.2 g,19.5mmol,1.00 eq) in 100mL CH ₂Cl₂ was added imidazole (2.6 g,39.0mmol,2.00 eq) and DMAP (470 mg,3.9mmol,0.20 eq) followed by slow addition of TBDPSCl (6.9 g,25.3mmol,1.30 eq) at 0deg.C. The mixture was stirred at room temperature for 2 hours under an argon atmosphere. The reaction was quenched by the addition of MeOH (20 mL), the resulting mixture was concentrated under reduced pressure and subjected to silica gel column chromatography (petroleum ether/ethyl acetate, 20:1 to 12:1) to give compound 18 (10.0 g, 85%) as a white foam.

R_f=0.4(PE/EA=10:1)

(c=0.22,CHCl₃)

HRMS-ESI (m/z) C ₃₆H₅₄NO₅Si[M+H]⁺ calculated 608.3771, measured 608.3748.

¹H NMR(400MHz,CDCl₃)δ7.70–7.63(m,4H),7.43–7.30(m,6H),5.36(t,J＝2.1Hz,1H),5.09(t,J＝2.0Hz,1H),4.76–4.69(m,1H),4.51–4.41(m,2H),4.33(d,J＝3.8Hz,1H),4.00(dd,J＝10.1,7.1Hz,1H),3.62(dd,J＝10.1,7.7Hz,1H),2.87(d,J＝7.5Hz,1H),2.82–2.75(m,1H),1.58–0.91(m,33H).

¹³C NMR(101MHz,CDCl₃)δ142.58,135.47,133.15,129.58,129.55,127.60,116.38,109.88,86.31,75.25,66.86,62.51,60.26,58.71,46.55,40.31,34.59,34.12,27.69,26.67,26.19,20.15,20.08,19.03,17.00.

Example 10

To a stirred solution of compound 18 (9.5 g,15.6mmol,1.00 eq) in 100mL THF and 130mL AcOH was added zinc powder (41 g, 264 mmol,40.00 eq). The mixture was stirred under argon atmosphere at 55 ℃ for 4.5 hours. The resulting mixture was concentrated under reduced pressure and subjected to silica gel column chromatography (petroleum ether/ethyl acetate, 4:1) to give compound 19 (6.5 g, 90%) as a white foam.

R_f=0.2(PE/EA=4:1)

(c=1.4,CHCl₃)

HRMS-ESI (m/z) C ₂₇H₃₆O₅SiNa[M+Na]⁺ calculated 491.2230, measured 491.2216.

¹H NMR(400MHz,CDCl₃)δ7.76–7.61(m,4H),7.49–7.34(m,6H),5.42–5.27(m,2H),4.80(d,J＝6.9Hz,1H),4.51(d,J＝7.6Hz,1H),4.31(dd,J＝6.9,4.3Hz,1H),4.13(t,J＝3.9Hz,1H),4.06–3.94(m,2H),2.66(s,2H),2.08–1.95(m,1H),1.47(s,3H),1.41(s,3H),1.07(s,9H).

¹³C NMR(101MHz,CDCl₃)δ145.09,135.54,132.79,129.82,127.79,127.76,115.07,109.81,76.76,75.66,70.10,66.82,65.13,47.43,26.79,26.69,25.17,19.08.

Example 11

To a stirred solution of compound 19 (6.4 g,13.7mmol,1.00 eq) in 137mL of dry THF were added 2-methoxyacetic acid (1.8 g,20.5mmol,1.50 eq) and PPh ₃ (7.2 g,27.4mmol,2.00 eq). Diazodicarbonic acid Diethyl Ester (DEAD) (3.7 g,20.5mmol,1.50 eq) was then added dropwise at-10 ℃. The mixture was stirred at-10 ℃ under an argon atmosphere for 12 hours. The resulting mixture was concentrated under reduced pressure, and subjected to silica gel column chromatography (petroleum ether/ethyl acetate, 8:1 to 5:1) to give compound 20 (6.6 g, 89%) as a colorless oil.

R_f=0.4(PE/EA=4:1)

(c=0.35,CHCl₃)

HRMS-ESI (m/z) C ₃₀H₄₀O₇SiNa[M+Na]⁺ calculated 563.2441, measured 563.2430.

¹H NMR(400MHz,CDCl₃)δ7.71–7.63(m,4H),7.47–7.34(m,6H),5.43(d,J＝4.1Hz,1H),5.37(d,J＝1.4Hz,1H),5.10(d,J＝1.4Hz,1H),4.54(d,J＝5.2Hz,1H),4.44–4.38(m,1H),4.35–4.25(m,1H),4.17(t,J＝3.9Hz,1H),3.93–3.75(m,3H),3.33(s,3H),2.55(s,1H),1.48(s,3H),1.44(s,3H),1.03(s,9H).

¹³C NMR(101MHz,CDCl₃)δ168.80,140.30,135.43,132.72,132.59,129.85,129.76,127.72,127.69,113.10,110.19,77.58,74.67,70.36,69.36,68.26,62.41,59.27,43.63,27.49,26.63,26.00,18.94.

Example 12

To a stirred solution of compound 20 (6.60 g,12.2mmol,1.00 eq) in acetone (70 mL) was added 4-methylmorpholine N-oxide (2.85 g,24.4mmol,2.00 eq) followed by slow addition of 0.05M aqueous OsO ₄ (35 mL,0.7mmol,0.06 eq) in the dark and then stirring of the mixture at room temperature for 12 hours. Quench with saturated aqueous NH ₄ Cl (50 mL), then extract three times with CH ₂Cl₂ (3×200 mL), then dry over Na ₂SO₄, filter and concentrate under reduced pressure to give crude compound 20a as a pale yellow solid, which is used in the subsequent reaction without further purification.

To a stirred solution of compound 20a in 80mL of CH ₂Cl₂ were added 2, 2-dimethoxypropane (2.54 g,24.4mmol,2.00 eq) and camphorsulfonic acid (CSA) (566 mg,2.44mmol,0.20 eq) and then stirred at room temperature for 0.5 hours. Quench with saturated aqueous NH ₄ Cl (50 mL) and extract with CH ₂Cl₂ (3X 200 mL). Dried over Na ₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (petroleum ether/ethyl acetate, 5:1) to give compound 21 (4.90 g, 66%) as a white foam.

R_f=0.4(PE/EA=4:1)

(c=0.43,CHCl₃)

HRMS-ESI (m/z) C ₃₃H₄₆O₉SiNa[M+Na]⁺ calculated 637.2809, measured 637.2799.

¹H NMR(400MHz,CDCl₃)δ7.70–7.59(m,4H),7.48–7.29(m,6H),5.52(s,1H),4.26–4.19(m,2H),4.15(d,J＝5.5Hz,1H),4.06(dd,J＝9.4,1.4Hz,1H),3.90–3.95(m,3H),3.84–3.72(m,2H),3.33(s,3H),2.52(brs,1H),2.28(t,J＝6.5Hz,1H),1.56(s,3H),1.44(s,6H),1.41(s,3H),1.05(s,9H).

¹³C NMR(101MHz,CDCl₃)δ168.86,135.58,133.25,129.63,129.59,127.62,127.60,110.96,109.65,79.73,77.81,74.81,71.89,69.60,69.10,66.34,61.50,59.37,40.45,26.74,26.68,26.50,25.74,25.52,19.10.

Example 13

To a stirred solution of compound 20 (54.0 mg,0.1mmol,1.00 eq) in acetone (1 mL) was added 4-methylmorpholine N-oxide (23.4 mg,0.2mmol,2.00 eq) followed by slow addition of 0.05M aqueous OsO ₄ (0.12 mL,0.006mmol,0.06 eq) in the dark and the mixture stirred overnight at room temperature. Quench with saturated aqueous NH ₄ Cl (2 mL), then extract three times with CH ₂Cl₂ (3 x 10 mL), then dry over Na ₂SO₄, filter and concentrate under reduced pressure to give the crude compound as a pale yellow solid, which is used in the subsequent reaction without further purification.

To a stirred solution of the crude product in 1mL MeOH was added 7N NH ₃ in 0.2mL MeOH and stirred at room temperature for 2 hours. Then extracted with H ₂ O (10 mL) and CH ₂Cl₂ (3X 10 mL). Dried over Na ₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (petroleum ether/ethyl acetate, 1:1) to give compound 31 (37.5 mg, 75%) as a white solid.

R_f=0.2(PE/EA=1:1)

(c=0.22,CHCl₃)

HRMS-ESI (m/z) C ₂₇H₃₈O₇SiNa[M+Na]⁺ calculated 525.2285, measured 525.2278.

¹H NMR(400MHz,CDCl₃)δ7.69(m,4H),7.45–7.34(m,6H),4.28(dd,J＝6.1,5.0Hz,1H),4.20(d,J＝5.7Hz,2H),4.13–3.99(m,3H),3.97(d,J＝10.7Hz,1H),3.88(d,J＝11.5Hz,1H),3.70(d,J＝10.6Hz,1H),2.93(s,1H),2.73(s,1H),2.51(s,1H),2.17(t,J＝7.3Hz,1H),1.50(s,3H),1.39(s,3H),1.06(s,9H).

¹³C NMR(101MHz,CDCl₃)δ135.57,133.63,129.60,127.64,109.24,74.53,73.33,70.98,67.25,64.83,62.54,38.60,26.85,26.18,24.72,19.26.

Example 14

To a stirred solution of compound 21 (4.89 g,7.96mmol,1.00 eq) in 22mL CH ₂Cl₂ at 0deg.C was added NaHCO ₃ (2.69 g,32.0mmol,4.00 eq) and Dess-Martin reagent (5.38 g,12.7mmol,1.60 eq). The solution was stirred at room temperature for 3 hours. The resulting mixture was directly purified by flash chromatography on silica gel (petroleum ether/ethyl acetate, 6:1) to give compound 22 (4.78 g, 98%) as a white foam.

R_f=0.5(PE/EA=4:1)

(c=0.24,CHCl₃)

HRMS-ESI (m/z) C ₃₃H₄₄O₉SiNa[M+H]⁺ calculated 635.2653, measured 635.2632.

¹H NMR(400MHz,CDCl₃)δ7.67–7.57(m,4H),7.46–7.31(m,6H),5.91(t,J＝2.3Hz,1H),4.47(d,J＝5.9Hz,1H),4.41(dd,J＝5.8,2.1Hz,1H),4.23(s,2H),3.96(dd,J＝10.9,4.8Hz,1H),3.91–3.76(m,2H),3.62(dd,J＝10.9,10.0Hz,1H),3.45–3.39(m,1H),3.37(s,3H),1.55(s,3H),1.51(s,3H),1.36(s,3H),1.35(s,3H),1.04(s,9H).

¹³C NMR(101MHz,CDCl₃)δ205.28,169.14,135.62,135.52,133.04,132.81,129.75,129.71,127.69,111.47,111.00,82.03,78.77,78.52,74.47,69.31,69.01,59.28,57.62,50.18,27.02,26.72,26.53,26.09,19.07.

Example 15

N-butyllithium (2.5M solution in hexane) (16.6 mL,41.6mmol,6.40 eq.) was added dropwise to a stirred solution of N, N-diisopropylamine (4.2 g,41.6mmol,6.40 eq.) in 42mL dry THF at-78℃under argon. After 1 hour, dry CH ₂Cl₂ (11.6 g,136.5mmol,21.00 eq) was added dropwise followed by a solution of compound 22 (4.0 g,6.5mmol,1.00 eq) in dry THF (50 mL) and stirred under argon atmosphere at-78 ℃. TLC was performed on 4:1 petroleum ether in ethyl acetate, and after disappearance of 22, the resulting mixture was quenched with saturated aqueous NH ₄ Cl (20 mL), extracted with EtOAc (3X 50 mL), dried over anhydrous MgSO ₄, and concentrated under reduced pressure to give crude compound 22a as a pale yellow oil, which was used in the subsequent reaction without further purification.

To a stirred solution of compound 22a in 15mL of dry THF was added NaHMDS (2.0 m,8.1mL,16.2mmol,2.50eq in THF), the reaction mixture was stirred at-78 ℃ for 1 hour, warmed to 0 ℃ and stirred at 0 ℃ for 1 hour, then the mixture was cooled again to-78 ℃ and PMBBr (3.3 g,16.2mmol,2.50 eq) was added dropwise, stirring continued at-78 ℃ for 1 hour, then warmed to 0 ℃, stirred at 0 ℃ for 1 hour, warmed to room temperature and stirred at room temperature for 12 hours. The resulting mixture was poured into saturated aqueous NH ₄ Cl (50 mL), extracted with EtOAc (3×100 mL), dried over anhydrous MgSO ₄, and then evaporated to give compound 23 (2.76 g,60% yield) which was purified on a silica gel column with petroleum ether: ethyl acetate (10:1) to give a colorless oil.

R_f=0.6(PE/EA=2:1)

(c=0.50,CHCl₃)

HRMS-ESI (m/z) C ₃₉H₄₉ClO₈SiNa[M+Na]⁺ calculated 731.2783, measured 731.2773.

¹H NMR(400MHz,CDCl₃)δ7.65–7.57(m,4H),7.39(dddd,J＝9.3,8.1,4.2,1.8Hz,6H),7.20(d,J＝8.7Hz,2H),6.80(d,J＝8.7Hz,2H),4.91(s,1H),4.62(d,J＝11.1Hz,1H),4.50(d,J＝11.1Hz,1H),4.44(d,J＝6.4Hz,1H),4.27–4.23(m,1H),4.22(s,1H),4.11(d,J＝9.5Hz,1H),3.89(dd,J＝10.4,7.7Hz,1H),3.80(dd,J＝4.2,1.5Hz,1H),3.78(s,3H),3.69(dd,J＝10.4,5.8Hz,1H),2.41(ddd,J＝7.6,5.7,4.2Hz,1H),1.42(s,3H),1.37(s,3H),1.35(s,3H),1.30(s,3H),1.05(s,9H).

¹³C NMR(101MHz,CDCl₃)δ158.89,135.51,135.46,133.17,130.55,129.85,129.78,128.73,127.78,127.75,113.49,110.54,110.40,79.84,78.85,78.72,74.59,72.41,70.55,70.06,60.74,59.81,55.22,41.65,29.70,27.07,26.87,26.63,25.52,19.11.

Example 16

To a solution of compound 23 (2.7 g,3.8mmol,1.00 eq) in dry dimethyl sulfoxide (38 mL) was added sodium azide (1.48 g,22.8mmol,6.00 eq) and 15-crown-5 (2.5 g,11.4mmol,3.00 eq) and stirred under argon at 70 ℃ for 21 hours. TLC was performed on 4:1 petroleum ether/ethyl acetate, after the disappearance of the starting compound, the reaction mixture was poured into saturated aqueous NH ₄ Cl (30 mL), extracted with CH ₂Cl₂ (3X 100 mL), dried over anhydrous MgSO ₄, evaporated to give compound 24 as a white foam (2.13 g,77% yield) which was purified on a silica gel column with petroleum ether/ethyl acetate (20:1).

R_f=0.5(PE/EA=2:1)

(c=0.13,CHCl₃)

HRMS-ESI (m/z) C ₃₉H₄₉N₃O₈SiNa[M+Na]⁺ calculated 738.3187, measured 738.3173.

¹H NMR(400MHz,CDCl₃)δ9.67(s,1H),7.65–7.58(m,4H),7.48–7.30(m,6H),7.10(d,J＝8.3Hz,2H),6.83(d,J＝7.9Hz,2H),4.61(q,J＝11.2Hz,2H),4.50(d,J＝6.4Hz,1H),4.38(d,J＝6.4Hz,1H),4.29–4.14(m,3H),3.95(dd,J＝10.4,3.9Hz,1H),3.80(s,3H),3.52(t,J＝10.8Hz,1H),2.34(d,J＝10.3Hz,1H),1.47(s,3H),1.46(s,3H),1.38(s,3H),1.36(s,3H),1.08(s,9H).

¹³C NMR(101MHz,CDCl₃)δ197.96,159.21,135.54,135.46,133.12,132.82,129.83,128.72,127.79,127.75,113.77,111.13,110.26,80.18,79.72,79.06,76.53,75.89,74.35,69.41,58.77,55.25,45.80,26.99,26.93,26.57,25.51,24.63,19.22.

Example 17

To a solution of lithium acetylide ethylenediamine complex (180 mg,1.96mmol,10.00 eq) in 2.5mL THF was added dropwise compound 24 (140 mg,0.20mmol,1.00 eq) dissolved in 2.5mL THF at 0 ℃, followed by stirring at 0 ℃ for 1 hour, quenching with saturated aqueous NH ₄ Cl (2 mL), extraction with EtOAc (3×10 mL), drying over anhydrous MgSO ₄, filtration and concentration under reduced pressure, purification on a silica gel column with 2:1 petroleum ether/ethyl acetate, affording compound 25a as a colourless oil (65 mg,66% yield) and its diastereoisomer 25 (25 a: 25=15:1, ratio detected by crude ¹ H NMR).

R_f=0.2(PE/EA=2:1)

(c=0.03,CHCl₃)

HRMS-ESI (m/z) C ₂₅H₃₃N₃O₈Na[M+Na]⁺ calculated 526.2166, measured 526.2154.

¹H NMR(400MHz,CDCl₃)δ7.31(d,J＝8.4Hz,2H),6.89(d,J＝8.0Hz,2H),5.00(d,J＝1.3Hz,1H),4.76(d,J＝11.1Hz,1H),4.62(d,J＝11.2Hz,1H),4.26(d,J＝6.3Hz,1H),4.13(d,J＝6.4Hz,1H),4.01–3.90(m,5H),3.82–3.75(m,4H),2.68–2.60(m,1H),2.53(d,J＝2.2Hz,1H),1.56(s,3H),1.47(s,3H),1.39(s,3H),1.33(s,3H).

¹³C NMR(101MHz,CDCl₃)δ159.40,129.80,129.56,113.90,109.23,108.64,85.91,81.59,79.81,75.11,74.62,74.27,66.75,66.30,64.89,58.13,55.27,43.29,29.69,27.04,26.26,26.07,24.43.

Example 18

25A to 25

To a stirred solution of compound 25a (148 mg,0.29mmol,1.00 eq) in 5mL CH ₂Cl₂ was added MnO ₂ (121 mg,1.4mmol,10.00 eq) and then stirred at room temperature for 2 hours. After filtration and concentration under reduced pressure, it was used in the subsequent reaction without further purification.

NaBH ₄ (426 mg,1.11mmol,4.00 eq) was added to a solution of the above-obtained oxidation product in 2ml dioxane and 200uL H ₂ O at room temperature, followed by stirring at 60℃for 0.5 hours. The mixture was quenched with saturated aqueous NH ₄ Cl (2 mL), extracted with CH ₂Cl₂ (3×5 mL), dried over anhydrous MgSO ₄, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (petroleum ether/ethyl acetate, 2:1) to give 25 as a colourless oil (76 mg,51%,70% brsm) and 25a as a white foam (39 mg, 26%). (25: 25a = 2:1, this ratio was detected by crude ¹ H NMR).

R_f=0.25(PE/EA=2:1)

(c=0.18,CHCl₃)

HRMS-ESI (m/z) C ₂₅H₃₃N₃O₈Na[M+Na]⁺ calculated 526.2166, measured 526.2156.

¹H NMR(400MHz,CDCl₃)δ7.30(d,J＝8.5Hz,2H),6.88(d,J＝8.4Hz,2H),4.83(d,J＝1.9Hz,1H),4.73(d,J＝10.7Hz,1H),4.64(d,J＝10.7Hz,1H),4.36(dd,J＝16.6,6.8Hz,2H),4.20(dd,J＝21.0,8.2Hz,2H),4.13(d,J＝4.4Hz,1H),3.80(s,3H),3.78–3.74(m,1H),3.69(dd,J＝11.8,5.7Hz,1H),2.65–2.63(m,1H),2.55(dd,J＝12.3,5.5Hz,2H),1.56(s,3H),1.48(s,3H),1.40(s,3H),1.37(s,3H).

¹³C NMR(101MHz,CDCl₃)δ159.49,129.78,129.30,113.91,109.56,108.50,85.45,81.54,79.63,78.40,76.76,74.89,74.75,65.85,65.74,65.51,59.22,55.25,43.89,29.68,26.87,26.26,26.09,24.76.

Example 19

To a stirred solution of compound 25 (120 mg,238mmol,1.00 eq) in 5.0mL of DMSO was added IBX (70 mg,250mmol,1.05 eq) and stirred at room temperature for 2 hours. TLC was performed with 4:1 petroleum ether/EtOAc, after which 25 had disappeared, meOH (5 mL), pyridinium p-toluenesulfonate (PPTS) (90.0 mg,356mmol,1.50 eq) and trimethyl orthoacetate (1.4 mL) were added and then stirred at room temperature for 12 hours. The mixture was quenched with saturated aqueous NaHCO ₃ (10 mL), extracted with CH ₂Cl₂ (3×20 mL), dried over anhydrous MgSO ₄, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (petroleum ether: ethyl acetate, 8/1) to give compound 26 (108 mg,88% yield) as a white foam.

R_f=0.4(PE/EA =4:1)

(c=0.05,CHCl₃)

HRMS-ESI (m/z) C ₂₆H₃₃N₃O₈Na[M+Na]⁺ calculated 538.2166, measured 538.2158.

¹H NMR(400MHz,CDCl₃)δ7.23(d,J＝8.7Hz,2H),6.84(d,J＝8.7Hz,2H),5.37(d,J＝4.2Hz,1H),4.85(d,J＝2.2Hz,1H),4.51–4.40(m,3H),4.23–4.09(m,3H),3.88(dd,J＝8.6,1.4Hz,1H),3.79(s,3H),3.40(s,3H),2.80(ddd,J＝8.5,4.2,0.9Hz,1H),2.61(d,J＝2.3Hz,1H),1.39(s,3H),1.37(s,3H),1.26(s,3H),1.26(s,3H).

¹³C NMR(101MHz,CDCl₃)δ159.52,130.29,129.10,113.72,111.08,110.07,107.08,79.55,77.38,76.81,76.62,75.79,75.17,74.95,73.62,69.17,67.74,56.03,55.23,47.35,27.13,26.34,24.73,24.18.

Example 20

To a stirred solution of compound 26 (27.0 mg,0.05mmol,1.00 eq) in CCl ₄ (1.0 mL), meCN (1.0 mL) and H ₂ O (1.5 mL) was added NaIO ₄ (45 mg,0.21mmol,4.00 eq) and RuCl ₃·xH₂ O (2.2 mg,0.01 mmol,0.20 eq) followed by stirring at room temperature for 2 hours. The mixture was quenched with H ₂ O (5 mL), extracted with CH ₂Cl₂ (3×100 mL), dried over anhydrous MgSO ₄, filtered and concentrated under reduced pressure, then used in the subsequent reaction without further purification.

EDCI (20 mg,0.10mmol,2.00 eq), DMAP (1.2 mg, 0.09 mmol,0.20 eq) and MeOH (16.77 mg,0.52mmol,10.00 eq) were added to a stirred solution of the crude product in 1mL CH ₂Cl₂, then stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure and purified by flash chromatography on silica gel (petroleum ether: ethyl acetate, 8/1 to 4/1) to give compound 27 (13.0 mg,48% yield) as a white foam.

R_f=0.25(PE/EA=4:1)

(c=0.05,CHCl₃)

HRMS-ESI (m/z) C ₂₆H₃₉N₄O₁₀[M+NH₄]⁺ calculated 567.2666, measured 567.2658.

¹H NMR(400MHz,CDCl₃)δ7.23(d,J＝8.4Hz,2H),6.86(d,J＝8.6Hz,2H),5.31(d,J＝3.3Hz,1H),4.72(s,1H),4.49(d,J＝7.2Hz,3H),4.20(dd,J＝18.2,7.9Hz,2H),4.04(d,J＝9.6Hz,1H),3.89(d,J＝6.4Hz,1H),3.80(d,J＝1.8Hz,6H),3.43(s,3H),2.83(dd,J＝7.5,3.3Hz,1H),1.39(s,3H),1.37(s,3H),1.22(s,3H),1.19(s,3H).

¹³C NMR(101MHz,CDCl₃)δ168.60,159.46,130.05,129.29,113.78,110.76,109.19,106.14,83.08,79.15,76.19,75.30,73.57,69.24,67.71,55.99,55.26,52.12,50.72,27.02,26.47,24.73,24.08.

Example 21

Compound 27 (13.0 mg,0.024mmol,1.00 eq) was hydrogenated in 2.0mL MeOH in the presence of 20% Pd-C in an atmosphere of H ₂ for 24 hours. The catalyst was then filtered off and evaporated to give compound 27a, which was used in the subsequent reaction without further purification.

To a stirred solution of the crude product in 2.5mL of dry dichloromethane at room temperature were added triethylamine (12.12 mg,0.12mmol,5.00 eq), 1, 3-bis (t-butoxycarbonyl) -2-methyl-2-thiopseudourea (13.9 mg,0.048mmol,2.00 eq) and mercury (II) chloride (17.0 mg,0.048mmol,2.00 eq). After stirring for 2 hours, the mixture was quenched with water (10 mL) and extracted three times with CH ₂Cl₂ (3X 10 mL). The combined organic layers were dried over anhydrous MgSO ₄. After filtration, the solvent was removed under reduced pressure to give a crude material which was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 1:1) to give the title compound 28 (11.2 mg,88% yield, 2 steps) as a white foam.

R_f=0.2(PE/EtOAc=2:1)

(c=0.16,CHCl₃)

HRMS-ESI (m/z) C ₂₄H₄₀N₃O₁₁[M+H]⁺ calculated 546.2663, measured 546.2650.

¹H NMR(400MHz,CDCl₃)δ11.35(brs,1H),9.02(brs,1H),5.46(s,1H),5.28(d,J＝5.8Hz,1H),5.24(s,1H),4.29(dd,J＝16.4,7.7Hz,2H),4.19(d,J＝9.6Hz,1H),3.92(dd,J＝12.0,6.8Hz,1H),3.80(s,3H),3.48(s,3H),3.35(d,J＝12.3Hz,1H),2.99–2.90(m,1H),1.47(s,12H),1.43(s,3H),1.39(s,3H),1.31(s,3H).

¹³C NMR(101MHz,CDCl₃)δ168.44,153.94,152.21,111.21,109.59,104.50,83.07,80.70,79.47,73.13,68.49,68.03,62.48,55.82,54.21,52.14,29.69,28.31,28.07,27.27,25.29.

Example 22

Compound 27 (13.0 mg,0.024mmol,1.00 eq) was hydrogenated in 2.0mL MeOH in the presence of 20% Pd-C in an atmosphere of H ₂ for 19 hours. The catalyst was then filtered off and evaporated to give compound 27a, which was used in the subsequent reaction without further purification.

To a stirred solution of the crude material in 2.5mL of dry dichloromethane at room temperature were added triethylamine (12.12 mg,0.12mmol,5.00 eq), 1, 3-bis (benzyloxycarbonyl) -2-methyl-2-thiopseudourea (17.2 mg,0.048mmol,2.00 eq) and mercury (II) chloride (17.0 mg,0.048mmol,2.00 eq). After stirring for 3 hours, the mixture was quenched with water (10 mL) and extracted three times with CH ₂Cl₂ (3X 10 mL). The combined organic layers were dried over anhydrous MgSO ₄. After filtration, the solvent was removed under reduced pressure to give a crude material which was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 2:1) to give the title compound 33 (13.4 mg,80% yield, 2 steps) as a white foam.

R_f=0.65(PE/EtOAc=2:1)

(c=0.22,CHCl₃)

HRMS-ESI (m/z) C ₃₅H₄₄N₃O₁₃[M+H]⁺ calculated 714.2874, measured 714.2863.

¹H NMR(400MHz,CDCl₃)δ11.64(brs,1H),9.03(brs,1H),7.41–7.28(m,10H),5.45(d,J＝2.8Hz,1H),5.26–5.21(m,1H),5.18–5.05(m,5H),4.31(d,J＝9.7Hz,1H),4.22–4.16(m,2H),3.93(dd,J＝12.0,6.7Hz,1H),3.61(s,3H),3.45(s,3H),3.36(d,J＝12.5Hz,1H),2.98(dd,J＝6.8,2.9Hz,1H),1.48(s,3H),1.42(s,3H),1.40(s,3H),1.20(s,3H).

¹³C NMR(101MHz,CDCl₃)δ168.07,163.03,154.15,152.89,136.58,134.61,128.80,128.67,128.40,127.90,127.75,111.35,109.80,104.45,80.58,79.47,72.96,68.51,68.14,67.90,67.01,62.67,55.81,54.15,52.01,29.69,27.22,25.36,25.29,24.11.

Example 23

Method A one-step synthesis of TTX

Compound 28 (11.0 mg,0.018mmol,1.00 eq) was dissolved in trifluoroacetic acid (500. Mu.L) and water (500. Mu.L) at room temperature. The resulting solution was heated to 60 ℃. After stirring for 24 hours, the mixture was concentrated in vacuo and the residue was examined by ¹ HNMR to give a mixture of tetrodotoxin and 4, 9-anhydrotetrodotoxin (1:1). The mixture was then redissolved in TFA-d (20. Mu.L) and deuterium oxide (1000. Mu.L) at room temperature and stirred at room temperature for 5 days. The mixture was concentrated in vacuo and the residue was purified by HPLC (sample preparation and purity analysis was performed on a sample equipped with a 2998PDA and 3100MS detector WATERS HPLC (ColumnHILIC SILICA,5 μm,4.6 x 150mm and 19 x 150 mm), mobile phase H ₂ O (0.1% AcOH)/acetonitrile (0.1% AcOH) (65% -50% within 10 min). The residual solution was lyophilized to give tetrodotoxin 1 and 4, 9-anhydrotetrodotoxin 2 (4:1) as white solids (detected by ¹ H NMR) (4.2 mg,65% total yield).

(c=0.06,0.05M AcOH)

HRMS-ESI (m/z) C ₁₁H₁₈N₃O₈[M+H]⁺ calculated 320.1094, measured 320.1085.

¹H NMR(600MHz,5％ CD₃CO₂D/D₂O)δ5.49(d,J=9.4Hz,1H),4.28

(brs,1H),4.24(brs,1H),4.07(brs,1H),4.04(d,J＝12.6Hz,1H),4.00(d,J＝12.6Hz,1H),3.95(s,1H),2.34(d,J＝9.4Hz,1H).

¹³C NMR(151MHz,5％ CD₃CO₂D/D₂O)δ156.52,110.76,79.59,75.06,73.77,72.72,71.39,70.80,65.45,59.65,40.62.

Example 24

Method B two-step Synthesis of TTX

Compound 33 (13.0 mg,0.018mmol,1.00 eq) was dissolved in trifluoroacetic acid (500. Mu.L) and water (500. Mu.L) at room temperature. The resulting solution was heated to 60 ℃. After stirring for 24 hours, the mixture was concentrated in vacuo and the residue was purified by prep. TLC to give hemiaminal 34 (4.1 mg,49.7% yield) as a white foam.

Note that the hemiaminal 34 and its anhydrous form can be isolated by PTLC (DCM: meoh=4:1) as described by Fukuyama.

R_f=0.15(DCM/MeOH=4:1)

(c=0.08,MeOH)

HRMS-ESI (m/z) C ₁₉H₂₄N₃O₁₀[M+H]⁺ calculated 454.1461, measured 454.1455.

¹H NMR(400MHz,CD₃OD)δ7.51–7.17(m,5H),5.63(d,J＝8.8Hz,1H),5.18(s,2H),4.19(d,J＝14.8Hz,2H),3.88–4.10(m,4H),2.31(d,J＝9.0Hz,1H).

¹³C NMR(151MHz,CD₃OD)δ169.65,156.11,137.49,129.54,129.31,129.25,109.77,78.97,74.80,73.03,71.91,70.03,68.61,64.79,58.81,39.96.

Compound 34 (4.0 mg, 0.09 mmol,1.00 eq) was hydrogenated in MeOH (0.5 mL) in the presence of 50% Pd-C in an atmosphere of H ₂ for 6 hours. The catalyst was then filtered off and evaporated to give a crude material which was purified by HPLC (sample preparation and purity analysis was performed on a solution of WATERS HPLC (ColumnHILIC SILICA,5 μm,4.6 x 150mm and 19 x 150 mm), mobile phase H ₂ O (0.1% AcOH)/acetonitrile (0.1% AcOH) (65% -50% within 10 min). The residual solution was lyophilized to give tetrodotoxin 1 (containing trace amounts of 4, 9-anhydrotetrodotoxin 2) as a white solid (2.5 mg,91% yield).

Synthesis of 9-epiTTX

Example 25

To a stirred solution of compound 25a (50.0 mg,0.10mmol,1.00 eq.) in DMSO (2.0 mL) was added IBX (29.0 mg,0.10mmol,1.00 eq.) at room temperature under argon atmosphere. The reaction mixture was then stirred at room temperature for 2 hours. TLC was performed with 4:1 petroleum ether/EtOAc, after which 25a disappeared, meOH (2 mL), pyridinium p-toluenesulfonate (PPTS) (37.8 mg,0.15mmol,1.50 eq.) and trimethyl orthoacetate (0.55 mL) were added to the above solution, and the solution was stirred at room temperature for 12 hours. The mixture was quenched with saturated aqueous NaHCO ₃ (2 mL) and extracted with CH ₂Cl₂ (3X 10 mL). The combined organic layers were dried over anhydrous MgSO ₄, filtered and concentrated under reduced pressure to give a residue which was purified by flash chromatography on silica gel (petroleum ether/ethyl acetate, 8:1) to give compound 35 (33.3 mg,65% yield) as a white foam.

R_f=0.35(PE/EA=4:1)

(c=0.108,CHCl₃)

HRMS-ESI (m/z) C ₂₆H₃₃N₃O₈Na[M+Na]⁺ calculated 538.2166, measured 538.2150.

¹H NMR(400MHz,CDCl₃)δ7.23(d,J=8.7Hz,2H),6.87(d,J=8.7

Hz,2H),5.09(d,J＝3.2Hz,1H),4.99(d,J＝2.2Hz,1H),4.53(dd,J＝30.7,11.1Hz,2H),4.39(d,J＝7.7Hz,1H),4.30–4.25(m,2H),4.09(d,J＝9.5Hz,1H),3.80(s,3H),3.79(d,J＝0.9Hz,1H),3.47(s,3H),2.78(dd,J＝5.9,3.2Hz,1H),2.68(d,J＝2.2Hz,1H),1.42(s,3H),1.39(s,3H),1.31(s,3H),1.29(s,3H).

¹³C NMR(101MHz,CDCl₃)δ159.36,129.39,113.80,110.23,109.55,106.48,80.49,78.71,78.10,76.47,76.33,76.00,75.00,74.64,69.76,68.46,56.33,55.25,51.23,29.69,26.81,26.60,25.25,23.97.

Example 26

To a stirred solution of compound 35 (20.0 mg,0.04mmol,1.00 eq.) and NaIO ₄ (34.2 mg,0.16mmol,4.00 eq.) in CCl ₄ (0.7 mL), meCN (0.7 mL) and H ₂ O (1.1 mL) under an argon atmosphere was added RuCl ₃·xH₂ O (1.7 mg,0.008mmol,0.20 eq.) at room temperature. The reaction mixture was then stirred at room temperature for 0.5 hours. H ₂ O (2.0 mL) and CH ₂Cl₂ (2.0 mL) were then added to the solution. The residue was partitioned and the separated organic layer was used directly. EDCI HCl (15.4 mg,0.08mmol,2.00 eq.) DMAP (1.0 mg,0.008mmol,0.20 eq.) and MeOH (12.8 mg,0.40mmol,10.00 eq.) were added to the organic solution at room temperature under an argon atmosphere. The reaction mixture was stirred at room temperature for 2 hours, then concentrated under reduced pressure to give a residue, which was purified by silica gel flash chromatography (PE/EtOAc, 8/1 to 4/1) to give compound 36 (10.4 mg,49% yield) as a white foam.

R_f=0.35(PE/EA=4:1)

(c=0.042,CHCl₃)

HRMS-ESI (m/z) C ₂₆H₃₅N₃O₁₀Na[M+Na]⁺ calculated 572.2220;

measured value 572.2214.

¹H NMR(400MHz,CDCl₃)δ7.23(d,J＝8.6Hz,2H),6.87(d,J＝8.7Hz,2H),5.14(d,J＝6.1Hz,1H),4.87(s,1H),4.50(q,J＝11.0Hz,2H),4.37(d,J＝7.4Hz,1H),4.26–4.17(m,2H),4.01(d,J＝9.6Hz,1H),3.87–3.84(m,1H),3.84(s,3H),3.80(s,3H),3.58(s,3H),2.72(t,J＝6.4Hz,1H),1.37(s,6H),1.25(s,6H).

¹³C NMR(101MHz,CDCl₃)δ168.61,159.43,129.88,129.48,113.75,110.61,109.11,106.30,80.97,79.99,75.26,75.14,75.07,68.67,68.29,57.41,55.25,52.28,49.03,29.70,26.94,26.41,24.83,24.17.

Example 27

A suspension of compound 36 (8.0 mg,0.015mmol,1.00 eq.) and Pd/C (1.6 mg,20 wt%) in MeOH (1.0 mL) was stirred at room temperature under an atmosphere of H ₂ for 24 hours. Passing the mixture throughAnd (3) a pad. The filtrate was evaporated to give crude product 36a, which was carried forward without further purification.

To a stirred solution of crude product 36a, 1, 3-bis (t-butoxycarbonyl) -2-methyl-2-thiopseudourea (8.7 mg,0.030mmol,2.00 eq.) and mercury (II) chloride (8.2 mg,0.030mmol,2.00 eq.) in dry dichloromethane (1.5 mL) was added Et ₃ N (7.6 mg,0.075mmol,5.00 eq.) under argon atmosphere at room temperature. The reaction mixture was stirred at room temperature for 2h, then quenched with water (10.0 mL) and extracted with CH ₂Cl₂ (3X 10 mL). The combined organic layers were dried over anhydrous MgSO ₄, filtered and concentrated under reduced pressure to give a residue which was purified by flash chromatography on silica gel (PE/EtOAc, 2:1) to give compound 37 (7.5 mg,80% yield, 2 steps) as a white foam.

R_f=0.4(PE/EtOAc=2:1)

(c=0.120,CHCl₃)

HRMS-ESI (m/z) C ₃₅H₄₄N₃O₁₃[M+H]⁺ calculated 646.3187, measured 646.3174.

¹H NMR(400MHz,CDCl₃)δ11.08(brs,1H),9.14(brs,1H),5.44(d,

J=2.1Hz,1H),5.27(s,1H),5.13(d,J=6.8Hz,1H),4.35(d,J=9.6

Hz,1H),4.24(d,J＝6.5Hz,1H),4.15(d,J＝9.6Hz,1H),3.97(dd,J＝11.7,7.0Hz,1H),3.65(s,3H),3.32(s,3H),3.10(d,J＝5.0Hz,1H),3.04(d,J＝12.0Hz,1H),1.50–1.40(m,24H),1.37(s,3H),1.31(s,3H).

¹³C NMR(101MHz,CDCl₃)δ169.12,162.69,154.29,151.96,111.28,109.32,106.71,82.62,82.15,78.78,78.47,77.10,74.03,68.89,68.03,61.79,55.15,54.23,51.54,28.26,28.07,27.24,25.46,25.37,23.67.

Example 28

Compound 37 (7.0 mg,0.01 mmol,1.00 eq.) was dissolved in trifluoroacetic acid (500. Mu.L) and water (500. Mu.L) under an argon atmosphere at room temperature. The reaction mixture was then heated to 60 ℃ and stirred at 60 ℃ for 12 hours. The resulting mixture was concentrated in vacuo to give a residue which was purified by HPLC (sample preparation and purity analysis was performed on a sample equipped with a 2998PDA and 3100MS detector WATERS HPLC (ColumnHILIC SILICA,5 μm,4.6 x 150mm and 19 x 150 mm), mobile phase H ₂ O (0.1% AcOH)/acetonitrile (0.1% AcOH) (65% -50% within 10 min). The residual solution was lyophilized to give 9-epiTTX-hemiacetal (hemilactal) and 9-epiTTX-10, 8-lactone (1:1, detected by ¹ H NMR) 1a (1.9 mg,55% yield) as white solids.

HRMS-ESI (m/z) C ₁₁H₁₈N₃O₈[M+H]⁺ calculated 320.1094, measured 320.1086.

9-EpiTTX (hemiacetal) ¹H NMR(400MHz,5％ CD₃CO₂D/D₂ O

δ5.24(d,J=9.2Hz,2H),4.24(s,1H),4.22(s,1H),4.15(brs,1H),

4.05(d,J=11.6Hz,1H),3.99(d,J=11.6Hz,1H),3.85(s,1H),2.45(d,J=9.2Hz,1H)。

9-EpiTTX (10, 8-lactone) ¹H NMR(400MHz,5％ CD₃CO₂D/D₂ O

δ5.44(s,1H),5.34(d,J＝10.4Hz,1H),4.18(d,J＝5.2Hz,1H),4.08(brs,1H),3.98(d,J＝12.0Hz,1H),3.85(d,J＝12.0Hz,1H),2.53(dd,J＝10.2,2.2Hz,1H).

9-EpiTTX (hemiacetals and 10, 8-lactones) )¹³C NMR(151MHz,5％ CD₃CO₂D/D₂O)δ180.03,156.80,155.14,110.74,81.06,79.64,76.78,75.33,74.25,73.68,73.27,72.99,71.03,70.14,69.42,68.50,66.00,65.30,63.40,56.18,43.90,43.34.

Biological assessment

To investigate the biological activity of pure TTX, we synthesized and purified a single form of TTX (S) from methyl carboxylate 24 according to the strategy of Fukuyama. Another sample (purchased from Tocris bioscience, analyzed by ¹ H NMR, ratio of TTX to 4,9-anhydroTTX 10:3, purity > 99%) designated TTX (C) was used for comparison. We compared the blocking ability of TTX from two different sources to a single subtype of sodium ion channel on human HEK-Na _v 1.7.7 cells and HEK-Na _v 1.5.5 cells. We measured the normal HEK-Na _v 1.7.7 current in the absence of TTX by a voltage clamp from-80 mV to 80 mV. HEK-Na _v 1.7.7 current was successfully induced and was completely blocked by 1. Mu.M TTX (C). The results indicate that HEK-Na _v 1.7.7 cells can be used to measure the blocking ability of TTX and TTX analogs from different sources. Then, we measured HEK-Na _v 1.7.7 currents of HEK-Na _v 1.7.7 cells treated with TTX (S) and TTX (C) under the same conditions. Our results indicate that TTX (S) has better blocking efficiency than TTX (C). In mice, we synthesized pure TTX (S) also showed a stronger effect in blocking sodium current amplitude of wild-type div (days of in vitro experiments) hippocampal neurons.

The biological activity of 9-epiTTX was also assessed based on blocking the sodium ion current amplitude of wild-type div hippocampal neurons, but it did not show any inhibitory activity.

In summary, we achieved one of the first asymmetric syntheses of 9-epiTTX (1 a) (22 steps), and the shortest one of TTX (1) syntheses starting from readily available furfuryl alcohol (24 steps, according to step count calculation rules 68, 69). The heck-scale asymmetric preparation of cyclohexane (+) -12 demonstrates the power of the stereoselective Diels-Alder reaction in the amplified synthesis of carbocycles with dense functionalities. 70 by photochemical decarboxylation hydroxylation, precise introduction of oxygen functions at the C-5 position, highlights the advances in radical conversion on the sterically demanding carbocyclic backbone. The sequential reactions of Smi 2-mediated reduction fragmentation, oxygen bridge ring opening and ester reduction followed by diastereoselective Upjohn dihydroxylation enabled gram-scale synthesis of highly oxidized intermediate (+) -19. The bridged tetrahydrofuran acetal arrangement simplifies the final stage and facilitates the rapid formation of cyclic guanidine hemiaminal and orthoesters in a one-pot process. It is notable that the synthesis method is used as a test platform for precisely manipulating functional groups on densely functionalized and stereochemically complex frameworks, and can be easily adapted to the synthesis of other highly oxygen-containing polycyclic natural products. This compact synthetic strategy is applicable to the production of TTX homologs or derivatives to support further pharmacological studies and can be used for large scale synthesis of TTX to develop analgesic drugs, particularly for non-opioid cancer pain treatment.

Claims

1. A preparation method comprising any one or more of the following steps: Step (a): converting compound 11 into compound 12,

Step (b): converting compound 12 into compound 13,

Step (c): converting compound 13 into compound 14,

Step (d): converting compound 14 into compound 15,

Step (e): converting compound 15 into compound 16,

Step (f): converting compound 16 into compound 17,

Step (g): converting compound 17 into compound A18,

Preferably,

Step (h): converting compound A18 into compound A19,

Preferably,

Step (i): converting compound A19 into compound A20,

Preferably,

Step (j): converting compound A20 into compound A21,

Preferably,

Step (k): converting compound A21 into compound A22,

Preferably,

Step (1): converting compound A22 into compound A23,

Preferably,

Step (m): converting compound A23 into compound A24,

Preferably,

Step (n): converting compound A24 into compound A25 and/or A25a,

and/or Preferably,

and/or Step (o): converting compound A25 into compound A26,

Preferably,

Step (p): converting compound A26 into compound A27,

Preferably,

Step (q): converting compound A27 into compound A28,

Preferably,

Step (r): converting compound A28 into tetrodotoxin 1,

Preferably,

Step (s): converting compound A27 and/or A27a into compound A33,

Preferably,

in,

Each R ₁ independently represents a protecting group, preferably a silicon-containing protecting group such as TBDPS,

Each R ₂ independently represents a protecting group, preferably a benzyl protecting group, such as p-methoxybenzyl (ie, PMB), each R ₃ independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc.,

Each R ₄ independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc.,

Each X independently represents a halogen, preferably selected from Cl or Br.

2. The method according to claim 1, further comprising step (x) and/or step (y):

Step (x): converting compound A33 into compound 34,

Preferably,

Step (y): converting compound 34 into tetrodotoxin 1,

Preferably,

3. The method according to claim 1 or 2, wherein step (q) comprises the following steps (q1) and (q2),

Step (q1): converting compound A27 into compound A27a,

Preferably,

Step (q2): converting compound A27a into compound A28,

Preferably,

in,

Each X independently represents a halogen, preferably selected from Cl or Br.

4. The method according to any one of claims 1 to 3, wherein step (c) comprises step (c1) and step (c2): step (c1): converting compound 13 into compound 13a,

Step (c2): converting compound 13a into compound 14,

5. The method according to any one of claims 1 to 4, wherein step (b) comprises step (b1) and step (b2): step (b1): converting compound 12 into compound 12a,

Step (b2): converting compound 12a into compound 13,

6. The method according to any one of claims 1 to 5, wherein the method further comprises steps (f1) and (f2),

Step (f1): converting compound 16 into compound 16a,

Step (f2): converting compound 16a into compound 17,

7. The method according to any one of claims 1 to 6, wherein step (j) comprises step (j1) and step (j2),

Step (j1): converting compound A20 into compound A20a,

Preferably,

Step (j2): converting compound A20a into compound A21,

Preferably,

8. The method according to any one of claims 1 to 7, wherein step (1) comprises step (11) and step (12), step (11): converting compound A22 into compound A22a,

Preferably,

Step (12): converting compound A22a into compound A23,

Preferably,

9. The method according to any one of claims 1 to 8, wherein step (n) comprises step (n1) and step (n2),

Step (n1): converting compound A24 into compound A25a,

and/or

Preferably

Step (n2): converting compound A25a into compound A25,

Preferably,

in,

Each R ₂ independently represents a protecting group, preferably a benzyl protecting group, such as p-methoxybenzyl (ie, PMB),

Each R ₃ independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc.,

Each X independently represents a halogen, preferably selected from Cl or Br.

10. The method according to any one of claims 1 to 9, wherein the method comprises step (r).

11. A method according to any one of claims 1 to 10, wherein the method comprises step (r) and any one or more of the following steps: step (a), step (b), step (c), step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), and step (q).

12. The method according to any one of claims 1 to 11, wherein the method comprises any two or three or more of the following steps: step (a), step (b), step (c), step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p) and step (q).

13. The method according to any one of claims 1 to 12, wherein the method comprises any one or two or three or more of the following steps: step (b), step (c), step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p) and step (q).

14. The method of any one of claims 1-13, wherein the method comprises:

step (q) and/or step (r); or

step (o), step (p), step (q) and/or step (r); or

step (n), step (o), step (p), step (q) and/or step (r); or

step (m), step (n), step (o), step (p), step (q) and/or step (r); or

step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r); or

step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r); or

step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r); or

step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r); or

step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r); or

step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r); or

step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r); or

step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r); or

step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r); or

step (c), step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r); or

step (b), step (c), step (d), step (e), step (f), step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and/or step (r); or

15. The method according to any one of claims 1 to 14, wherein

In step (a), the reagents used include quinine and MeOH, and the solvent used includes CCl ₄ and/or toluene (preferably a mixture of CCl ₄ and/or toluene, preferably, by volume (1-2): (1-2)); and/or

In step (b1), the reagent used comprises 4-methylmorpholine N-oxide (NMO), 4-methylmorpholine (NMM) and/or an oxidant, preferably OsO ₄ , and the solvent used comprises acetone, preferably a mixture of acetone and water; and/or

In step (b2), the reagents used include 2,2-dimethoxypropane and p-toluenesulfonic acid, and the solvent used includes acetone; and/or

In step (c1), the reagent used includes N-hydroxyphthalimide and a base, preferably an organic base, preferably DMAP, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI); and/or

In step (c2), the reagents used include Ru(bpy) ₃ Cl ₂ , TEMPO and Hans ester, and/or the solvent used includes DMF; and/or in step (c2), the conversion is carried out under blue LED irradiation; and/or

In step (d), the reagent used comprises a base, preferably an inorganic base, preferably K ₂ CO ₃ , and/or the solvent used comprises an alcohol, preferably a C1-C4 alcohol; and/or

In step (e), the reagents used include I ₂ , imidazole and PPh ₃ , and/or the solvent used includes toluene; and/or

In step (f), the reagent used comprises SmI ₂ , and preferably further comprises a base, preferably an organic base such as Et ₃ N, and/or the solvent used comprises an ether (preferably THF); and/or

In step (f1), the reagent used comprises SmI ₂ , and/or the solvent used comprises ether (preferably THF);

In step (f2), the reagent used comprises LiAlH ₄ , and/or the solvent used comprises ether (preferably THF);

In step (g), the reagent used comprises TBDPSCl, preferably comprises imidazole and/or DMAP; and/or

In step (h), the reagent used includes zinc powder; and/or the solvent used includes THF and AcOH; and/or

In step (i), the reagents used include 2-methoxyacetic acid, PPh ₃ and diethyl diazene dicarboxylate; and/or the solvent used includes THF; and/or

In step (j1), the reagent used includes 4-methylmorpholine N-oxide, and/or an oxidizing agent, preferably OsO ₄ , and/or the solvent used includes acetone; and/or

In step (j2), the reagent used includes 2,2-dimethoxypropane and camphorsulfonic acid, and/or the solvent used includes CH ₂ Cl ₂ ; and/or

In step (k), the reagent used includes Dess-Martin reagent, preferably also includes an inorganic base, preferably NaHCO ₃ ; and/or

In step (11), the reagents used include N,N-diisopropylamine and n-butyllithium; and/or

In step (12), the reagents used include NaHMDS and PMBBr; and/or

In step (m), the reagent used includes sodium azide, preferably also includes a crown ether, preferably 15-crown-5; and/or

In step (n1), the reagent used includes lithium ethylenediamine complex; and/or

In step (n2), the reagents used include an oxidizing agent, preferably MnO ₂ , and a reducing agent, preferably NaBH ₄ ; and/or

In step (o), the reagent used includes an oxidizing agent, preferably 2-iodobenzoic acid, pyridinium p-toluenesulfonate and trimethyl orthoacetate; and/or the solvent used is DMSO; and/or

In step (p), the reagents used include a catalyst, preferably RuCl ₃ , an oxidant, preferably NaIO ₄ , and preferably also include EDCI and MeOH; and/or

In step (q1), the reagents used include a hydrogenation catalyst, preferably Pd/C, and hydrogen; and/or

In step (q2), the reagents used include 1,3-bis(tert-butyloxycarbonyl)-2-methyl-2-thiopseudurea and mercuric (II) chloride, preferably also including an organic base, preferably Et ₃ N; and/or

In step (r), the reagent used includes trifluoroacetic acid; and/or

In step (s), the reagents used include 1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudurea and mercuric (II) chloride; and/or

In step (x), the reagent used includes trifluoroacetic acid; and/or

In step (y), the reagents used include a hydrogenation catalyst (preferably Pd/C) and hydrogen.

16. The method according to any one of claims 1 to 15, further comprising any one or more of steps (z1) to (z4),

Step (z1): converting compound A25a into compound A35,

Step (z2): converting compound A35 into compound A36,

Step (z3): converting compound A36 into compound A37,

Step (z4): converting compound A37 into compound 1a,

in,

Each R ₂ independently represents a protecting group, preferably a benzyl protecting group, preferably p-methoxybenzyl (ie, PMB), each R ₃ independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc., and

Each R ₄ independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc.;

Preferably, step (z3) includes one or both of step (z3-1) and step (z3-2),

Step (z3-1): converting compound A36 into compound A36a,

Step (z3-2): converting compound A36a into compound A37,

17. A compound selected from the group consisting of:

in,

Each X independently represents a halogen, preferably selected from Cl or Br;

18. A preparation method comprising using one or more of the compounds according to claim 17 as starting materials or intermediate compounds.

19. A method for preparing tetrodotoxin or an analog thereof, comprising using one or more of the compounds of claim 17 as starting materials or intermediate compounds.