Preparation method of tetrodotoxin and analogues thereof

Abstract

The present disclosure relates to a process for the preparation of TTX or analogues thereof, and to compounds useful in the process for the preparation of TTX or analogues thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to PCT patent application No. PCT / CN2022 / 111861 filed on August 11, 2022, the contents of which are incorporated herein by reference in their entirety as a part of this application. Technical Field

[0003] The present disclosure relates to a method for preparing tetrodotoxin or its analogs. The present disclosure also relates to various compounds that can be used to prepare tetrodotoxin or its analogs. Background Art

[0004] Tetrodotoxin (TTX) is a well-known marine toxin that was originally isolated from pufferfish and later found in many other species such as salamanders and octopuses. The biological production of TTX remains a mystery, although various lines of evidence indicate that it is produced by microorganisms and several biosynthetic pathways have been proposed. After the isolation of analytically pure TTX in 1952, Woodward, Tsuda, Goto, and Mosher succeeded in deciphering its complex structure in 1964. This unique structure consists of a densely functionalized, stereochemically complex framework with an unprecedented dioxadamantane containing an orthoacid, a cyclic guanidinium hemiaminal moiety, and nine consecutive stereocenters, including a bridgehead nitrogen-containing quaternary carbon center. There are three compounds in equilibrium - the orthoester, the 4,9-anhydro, and the lactone, which interconvert under acidic conditions, making it difficult to obtain pure TTX by isolation from natural mixtures.

[0005] Regarding the biological activity of TTX, as early as the early 1960s, it was proposed that it could disrupt voltage-gated sodium channels (Na v ) view, and recent crystallographic studies have revealed that TTX binds to human Na v 1.7 binding motif. Extensive pharmacological studies have shown that TTX has great potential in pain treatment, but its severe systemic toxicity limits its clinical application. In order to overcome the limitations of systemic toxicity and low bioavailability, structural modifications and the use of innovative delivery systems have been proposed, but clinical evaluation urgently requires a reliable source of pure TTX.

[0006] Due to its complex sp 3This fascinating molecule, which has a very dense array of heteroatoms surrounding the hybrid carbon-rich core, has also attracted widespread attention in the organic synthesis community. To date, several research groups have reported the full preparation method of TTX, among which Kishi achieved the first milestone through racemic preparation in 1972. Subsequently, Isobe, Du Bois, Sato, Fukuyama, and Yokoshima achieved asymmetric synthesis. Recently, Trauner et al. (2022) disclosed a simple and easy asymmetric preparation method for TTX based on glucose derivatives. In addition, Alonso, Ciufolini, and Hudlicky also reported an innovative synthesis of key intermediates for the construction of TTX.

[0007] Despite recent impressive progress in the development of various strategies to assemble such complex molecules, the synthesis of stereohindered carbon ring systems with continuous sp 3 Chemoselective and stereoselective functionalization of heteroatomic substituents remains a considerable challenge. Summary of the invention

[0008] The present disclosure proposes a method for stereoselective asymmetric synthesis of TTX and its analogs from abundantly available furfuryl alcohol. A highly heteroatom-substituted pseudosymmetric cyclohexane skeleton was assembled through stereoselective Diels-Alder reaction and chemoselective cyclic anhydride opening strategy. Upjohn dihydroxylation installed oxygen functional groups at C-6, 7, 8 and 11, and decarboxylation hydroxylation supported the installation of oxygen at C-5. A scalable total synthesis of TTX was achieved through an innovative SmI2-mediated simultaneous free radical elimination, oxygen bridge opening and ester reduction sequence, followed by ruthenium-catalyzed oxidative alkynyl cleavage, and a one-pot formation of hemiaminoacetals and orthoesters. This method should be easily applicable to other natural (and non-natural) TTX homologues to further support studies on biosynthesis, physiology and pharmacology.

[0009] In one aspect, the present disclosure provides a preparation method, the method comprising any one or more of the following steps:

[0010] Step (a): converting compound 11 into compound 12,

[0011]

[0012] Step (b): converting compound 12 into compound 13,

[0013]

[0014] Step (c): converting compound 13 into compound 14,

[0015]

[0016] Step (d): converting compound 14 into compound 15,

[0017] Step (e): converting compound 15 into compound 16,

[0018]

[0019] Step (f): converting compound 16 into compound 17,

[0020]

[0021] Step (g): converting compound 17 into compound A18,

[0022] Preferably,

[0023]

[0024] Step (h): converting compound A18 into compound A19,

[0025] Preferably,

[0026]

[0027] Step (i): converting compound A19 into compound A20,

[0028] Preferably,

[0029]

[0030] Step (j): converting compound A20 into compound A21,

[0031] Preferably,

[0032]

[0033] Step (k): converting compound A21 into compound A22,

[0034]

[0035] Preferably,

[0036]

[0037] Step (1): converting compound A22 into compound A23,

[0038]

[0039] Preferably,

[0040]

[0041] Step (m): converting compound A23 into compound A24,

[0042] Preferably,

[0043]

[0044] Step (n): converting compound A24 into compound A25 and / or A25a,

[0045] Preferably,

[0046]

[0047] Step (o): converting compound A25 into compound A26,

[0048]

[0049] Preferably,

[0050]

[0051] Step (p): converting compound A26 into compound A27,

[0052] Preferably,

[0053]

[0054] Step (q): converting compound A27 into compound A28,

[0055] Preferably,

[0056]

[0057] Step (r): converting compound A28 into tetrodotoxin 1,

[0058] Preferably,

[0059]

[0060] Step (s): converting compound A27 and / or A27a into compound A33,

[0061]

[0062] Preferably,

[0063]

[0064] in,

[0065] Each R1 independently represents a protecting group, preferably a silicon-containing protecting group, preferably TBDPS, each R2 independently represents a protecting group, preferably a benzyl-containing protecting group, preferably p-methoxybenzyl (ie, PMB), each R3 independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc.,

[0066] Each R4 independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc., and

[0067] Each X independently represents a halogen, preferably selected from Cl or Br.

[0068] According to some embodiments of the present invention, the preparation method comprises step (a).

[0069] According to some embodiments of the present invention, the preparation method comprises step (b).

[0070] According to some embodiments of the present invention, the preparation method comprises step (c).

[0071] According to some embodiments of the present invention, the preparation method comprises step (d).

[0072] According to some embodiments of the present invention, the preparation method comprises step (e).

[0073] According to some embodiments of the present invention, the preparation method comprises step (f).

[0074] According to some embodiments of the present invention, the preparation method comprises step (g).

[0075] According to some embodiments of the invention, the preparation method comprises step (h).

[0076] According to some embodiments of the present invention, the preparation method comprises step (i).

[0077] According to some embodiments of the present invention, the preparation method comprises step (j).

[0078] According to some embodiments of the invention, the preparation method comprises step (k).

[0079] According to some embodiments of the present invention, the preparation method comprises step (1).

[0080] According to some embodiments of the present invention, the preparation method comprises step (m).

[0081] According to some embodiments of the present invention, the preparation method comprises step (n).

[0082] According to some embodiments of the present invention, the preparation method comprises step (o).

[0083] According to some embodiments of the present invention, the preparation method comprises step (p).

[0084] According to some embodiments of the invention, the preparation method comprises step (q).

[0085] According to some embodiments of the present invention, the preparation method comprises step (r).

[0086] According to some embodiments of the present invention, the preparation method comprises step (s).

[0087] According to some embodiments of the present invention, the preparation method further comprises step (x) and / or step (y):

[0088] Step (x): converting compound A33 into compound 34,

[0089]

[0090] Preferably,

[0091]

[0092] Step (y): converting compound 34 into tetrodotoxin 1, preferably,

[0093]

[0094] According to some embodiments of the present invention, the method further comprises any one or more of steps (z1) to (z4),

[0095] Step (z1): converting compound A25a into compound A35,

[0096]

[0097] Step (z2): converting compound A35 into compound A36,

[0098]

[0099] Step (z3): converting compound A36 into compound A37,

[0100]

[0101] Step (z4): converting compound A37 into compound 1a,

[0102]

[0103] in,

[0104] Each R2 independently represents a protecting group, preferably a benzyl protecting group, preferably p-methoxybenzyl (ie, PMB),

[0105] Each R3 independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc.,

[0106] Each R4 independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc., and

[0107] Each X independently represents a halogen, preferably selected from Cl or Br.

[0108] In some examples, R2 is PMB, R3 is methyl, and R4 is methyl.

[0109] According to some embodiments of the present invention, step (q) comprises one or both of the following steps (q1) and (q2),

[0110] Step (q1): converting compound A27 into compound A27a,

[0111]

[0112] Preferably,

[0113]

[0114] Step (q2): converting compound A27a into compound A28,

[0115]

[0116] Preferably,

[0117]

[0118] wherein each R1 independently represents a protecting group, preferably a silicon-containing protecting group, such as TBDPS,

[0119] Each R2 independently represents a protecting group, preferably a benzyl protecting group, such as p-methoxybenzyl (ie, PMB),

[0120] Each R3 independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc.,

[0121] Each R4 independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc., and

[0122] Each X independently represents a halogen, preferably selected from Cl or Br.

[0123] According to some embodiments of the present invention, step (z3) comprises one or both of step (z3-1) and step (z3-2),

[0124] Step (z3-1): converting compound A36 into compound A36a,

[0125]

[0126] Step (z3-2): converting compound A36a into compound A37,

[0127]

[0128] According to some embodiments of the present invention, step (c) comprises one or both of step (c1) and step (c2):

[0129] Step (c1): converting compound 13 into compound 13a,

[0130]

[0131] Step (c2): converting compound 13a into compound 14,

[0132]

[0133] According to some embodiments of the present invention, step (b) comprises step (b1) and step (b2):

[0134] Step (b1): converting compound 12 into compound 12a,

[0135]

[0136] Step (b2): converting compound 12a into compound 13,

[0137]

[0138] According to some embodiments of the present invention, step (f) comprises step (f1) and step (f2), step (f1): converting compound 16 into compound 16a,

[0139]

[0140] Step (f2): converting compound 16a into compound 17,

[0141]

[0142] According to some embodiments of the present invention, step (j) comprises step (j1) and step (j2), step (j1): converting compound A20 into compound A20a,

[0143]

[0144] Preferably,

[0145]

[0146] Step (j2): converting compound A20a into compound A21,

[0147]

[0148] Preferably,

[0149]

[0150] According to some embodiments of the present invention, step (1) comprises step (11) and step (12), step (11): converting compound A22 into compound A22a,

[0151]

[0152] Preferably,

[0153]

[0154] Step (12): converting compound A22a into compound A23,

[0155]

[0156] Preferably,

[0157]

[0158] According to some embodiments of the present invention, step (n) comprises step (n1) and step (n2), step (n1): converting compound A24 into compound A25a,

[0159]

[0160] Preferably,

[0161]

[0162] Step (n2): converting compound A25a into compound A25,

[0163]

[0164] Preferably,

[0165]

[0166] In the above structure of the present application, each R1 independently represents a protecting group, preferably a silicon-containing protecting group, such as TBDPS,

[0167] Each R2 independently represents a protecting group, preferably a benzyl protecting group, such as p-methoxybenzyl (ie, PMB),

[0168] Each R3 independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc.,

[0169] Each R4 independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc.,

[0170] Each X independently represents a halogen, preferably selected from Cl or Br.

[0171] According to some embodiments of the invention, R1 is TBDPS.

[0172] According to some embodiments of the invention, R2 is PMB.

[0173] According to some embodiments of the invention, R3 is methyl.

[0174] According to some embodiments of the invention, R4 is methyl.

[0175] According to some embodiments of the invention, X is Cl.

[0176] According to some embodiments of the present invention, the method includes 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).

[0177] According to some embodiments of the present invention, the method includes 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).

[0178] According to some embodiments of the present invention, the method includes 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).

[0179] According to some embodiments of the present invention, the method includes 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).

[0180] According to some embodiments of the invention, the method comprises: step (o), step (p) and / or step (q).

[0181] According to some embodiments of the invention, the method comprises: step (n), step (o), step (p) and / or step (q).

[0182] According to some embodiments of the invention, the method comprises: step (m), step (n), step (o), step (p) and / or step (q).

[0183] According to some embodiments of the present invention, the method comprises: step (l), step (m), step (n), step (o), step (p) and / or step (q).

[0184] 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).

[0185] 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).

[0186] According to some embodiments of the present invention, the method comprises: step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p) and / or step (q).

[0187] According to some embodiments of the present invention, the method includes: step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and / or step (q).

[0188] According to some embodiments of the present invention, the method includes: step (g), step (h), step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p) and / or step (q).

[0189] According to some embodiments of the present invention, the method includes: 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).

[0190] According to some embodiments of the present invention, the method includes: 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).

[0191] According to some embodiments of the present invention, the method includes: 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).

[0192] According to some embodiments of the present invention, the method includes: 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).

[0193] According to some embodiments of the present invention, the method includes: 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).

[0194] According to some embodiments of the present invention, the method includes: 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).

[0195] According to some embodiments of the invention, the method comprises:

[0196] step (q) and / or step (r); or

[0197] step (o), step (p), step (q) and / or step (r); or

[0198] step (n), step (o), step (p), step (q) and / or step (r); or

[0199] step (m), step (n), step (o), step (p), step (q) and / or step (r); or

[0200] step (l), step (m), step (n), step (o), step (p), step (q) and / or step (r); or

[0201] step (k), step (l), step (m), step (n), step (o), step (p), step (q) and / or step (r); or

[0202] step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and / or step (r); or

[0203] step (i), step (j), step (k), step (l), step (m), step (n), step (o), step (p), step (q) and / or step (r); or

[0204] 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

[0205] 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

[0206] 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

[0207] 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

[0208] 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

[0209] 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

[0210] 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

[0211] 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).

[0212] According to some embodiments of the present invention, in step (a), the reagents used include quinine and MeOH, and the solvent used includes CCl4 and / or toluene (preferably a mixture of CCl4 and / or toluene, preferably in a (1-2):(1-2) volume ratio by volume).

[0213] According to some embodiments of the present invention, in step (b1), the reagent used includes 4-methylmorpholine N-oxide (NMO), 4-methylmorpholine (NMM) and / or an oxidant, preferably OsO4, and the solvent used includes acetone, preferably a mixture of acetone and water.

[0214] According to some embodiments of the present invention, in step (b2), the reagent used includes 2,2-dimethoxypropane and p-toluenesulfonic acid, and the solvent used includes acetone.

[0215] According to some embodiments of the present invention, 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).

[0216] According to some embodiments of the present invention, in step (c2), the reagents used include Ru(bpy)3Cl2, TEMPO and Hantzsch ester, and / or the solvent used includes DMF; and / or in step (c2), the conversion is carried out under radiation of a blue LED.

[0217] According to some embodiments of the present invention, in step (d), the reagent used comprises a base, preferably an inorganic base, preferably K2CO3, and / or the solvent used comprises an alcohol, preferably a C1-C4 alcohol.

[0218] According to some embodiments of the present invention, in step (e), the reagent used comprises I2, imidazole and PPh3, and / or the solvent used comprises toluene.

[0219] According to some embodiments of the present invention, in step (f), the reagent used comprises SmI2, and preferably also comprises a base, preferably an organic base, such as Et3N, and / or the solvent used comprises an ether (preferably THF).

[0220] According to some embodiments of the present invention, in step (f1), the reagent used comprises SmI2, and / or the solvent used comprises ether (preferably THF).

[0221] According to some embodiments of the present invention, in step (f2), the reagent used comprises LiAlH4, and / or the solvent used comprises ether (preferably THF).

[0222] According to some embodiments of the present invention, in step (g), the reagent used comprises TBDPSCl, preferably comprises imidazole and / or DMAP.

[0223] According to some embodiments of the present invention, in step (h), the reagent used comprises zinc powder; and / or the solvent used comprises THF and AcOH.

[0224] According to some embodiments of the present invention, in step (i), the reagent used includes 2-methoxyacetic acid, PPh3 and diethyl diazenecarboxylate; and / or the solvent used includes THF.

[0225] According to some embodiments of the present invention, in step (j1), the reagent used comprises 4-methylmorpholine N-oxide and / or an oxidizing agent, preferably OsO4, and / or the solvent used comprises acetone.

[0226] According to some embodiments of the present invention, in step (j2), the reagent used includes 2,2-dimethoxypropane and camphorsulfonic acid, and / or the solvent used includes CH2Cl2.

[0227] According to some embodiments of the present invention, in step (k), the reagent used comprises Dess-Martin reagent, preferably further comprising an inorganic base, preferably NaHCO3.

[0228] According to some embodiments of the present invention, in step (l1), the reagents used include N,N-diisopropylamine and n-butyllithium.

[0229] According to some embodiments of the present invention, in step (12), the reagents used include NaHMDS and PMBBr.

[0230] According to some embodiments of the present invention, in step (m), the reagent used comprises sodium azide, and preferably further comprises a crown ether, preferably 15-crown-5.

[0231] According to some embodiments of the present invention, in step (n1), the reagent used includes lithium acetylide ethylenediamine complex.

[0232] According to some embodiments of the present invention, in step (n2), the reagents used include an oxidizing agent, preferably MnO2, and a reducing agent, preferably NaBH4.

[0233] According to some embodiments of the present invention, 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.

[0234] According to some embodiments of the present invention, in step (p), the reagents used include a catalyst, preferably RuCl3, an oxidant, preferably NaIO4, and preferably also include EDCI and MeOH.

[0235] According to some embodiments of the present invention, in step (q1), the reagents used include a hydrogenation catalyst (preferably Pd / C) and hydrogen.

[0236] According to some embodiments of the present invention, in step (q2), the reagent used includes 1,3-bis(tert-butyloxycarbonyl)-2-methyl-2-thiopseudurea and mercuric (II) chloride, preferably also includes an organic base, preferably Et3N.

[0237] According to some embodiments of the invention, in step (r), the reagent used comprises trifluoroacetic acid.

[0238] According to some embodiments of the present invention, in step (s), the reagent used includes 1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudurea and mercuric (II) chloride.

[0239] According to some embodiments of the invention, in step (x), the reagent used comprises trifluoroacetic acid.

[0240] According to some embodiments of the present invention, in step (y), the reagents used include a hydrogenation catalyst, preferably Pd / C, and hydrogen.

[0241] According to some embodiments of the present invention, in step (z1), the reagent used includes an oxidizing agent, preferably 2-iodobenzoic acid, pyridinium p-toluenesulfonate and trimethyl orthoacetate; and / or the solvent used is DMSO.

[0242] According to some embodiments of the present invention, in step (z2), the reagents used include a catalyst, preferably RuCl3, an oxidant, preferably NaIO4, and preferably also include EDCI and MeOH.

[0243] According to some embodiments of the present invention, in step (z3-1), the reagents used include a hydrogenation catalyst (preferably Pd / C) and hydrogen.

[0244] According to some embodiments of the present invention, in step (z3-2), the reagents used include 1,3-bis(tert-butyloxycarbonyl)-2-methyl-2-thiopseudurea and mercuric (II) chloride, and preferably also include an organic base, preferably Et3N.

[0245] According to some embodiments of the invention, in step (z4), the reagent used comprises trifluoroacetic acid.

[0246] According to some embodiments of the present invention, the preparation method comprises the following steps:

[0247]

[0248] According to some embodiments of the present invention, the preparation method comprises any one or more of the following steps:

[0249]

[0250]

[0251]

[0252]

[0253] According to some embodiments of the present invention, the preparation method comprises the following steps:

[0254] According to some embodiments of the present invention, the preparation method comprises any one or more of the following steps:

[0255] According to some embodiments of the present invention, the preparation method comprises any one or more of the following steps:

[0256]

[0257]

[0258] In a second aspect, a compound selected from the group consisting of:

[0259]

[0260]

[0261] in,

[0262] Each R1 independently represents a protecting group, preferably a silicon-containing protecting group such as TBDPS,

[0263] Each R2 independently represents a protecting group, preferably a benzyl protecting group, such as p-methoxybenzyl (ie, PMB), each R3 independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc.,

[0264] Each R4 independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc.,

[0265] Each X independently represents a halogen, preferably selected from Cl or Br.

[0266] Specifically, the compound is selected from any one of the following:

[0267]

[0268]

[0269] In a third aspect, provided is a use of the compound according to the second aspect of the present disclosure in the preparation of tetrodotoxin or an analog thereof.

[0270] In a fourth aspect, a preparation method is provided, comprising using one or more of the compounds described in the second aspect of the present disclosure. In the preparation method, the compound can be used as a starting material or an intermediate compound.

[0271] In a fifth aspect, a method for preparing tetrodotoxin or an analog thereof is provided, comprising using one or more of the compounds described in the second aspect of the present disclosure. In the preparation method, the compound can be used as a starting material or an intermediate compound.

[0272] The scope of the present invention includes the intermediate compounds used in the preparation method of the present application. DETAILED DESCRIPTION

[0273] Tetrodotoxin and its homologues are specific voltage-gated sodium channel blockers with significant anesthetic and analgesic effects. Extensive pharmacological studies (including clinical trials) have shown that TTX has potential in pain treatment and heroin addiction treatment.

[0274] In the present disclosure, a new scalable synthetic method for the neurotoxin family is provided, which provides conditions for rapid access to highly oxidized natural products and scalable preparation of TTX and its analogs.

[0275] 1. General Information

[0276] Unless otherwise noted, all reactions were performed in flame-dried glassware under nitrogen with magnetic stirring. Reagents were purchased from Aldrich Chemical, Alfa Aesar, TCI, Adamas, Energy Chemical, or J&K of the highest commercial quality and used without further purification. Solvents were dried by passing through an activated alumina column under argon. Liquids and solutions were transferred via syringe. Reactions were monitored by GC / MS, UPLC / MS, and thin layer chromatography (TLC), and visualization was achieved using 254 nm UV light and heated phosphomolybdic acid solution staining. Qingdao Haiyang Chemical Silica Gel (particle size 300-400 mesh) was used for all flash silica gel column chromatography. 1 H and 13 C NMR spectra were recorded on a Varian Inova-400 spectrometer. Reported relative to CDCl3 (7.26 ppm), CD3OD (3.31 ppm), or CD3CO2D (2.06 ppm) as internal standards 1H NMR spectral data are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, sept = septet, m = multiplet, br = broad), coupling constant J (Hz), and integration. Reported relative to CDCl3 (77.00 ppm), CD3OD (49.00 ppm), CD3CO2D (22.4 ppm) as internal standards 13 The data of C NMR spectra are reported in chemical shift (δppm). UPLC-MS analysis was performed on a Waters system (column: BEH C18, 1.7 μm, 2.1*50 mm) equipped with a photodiode array (PDA) detector and a single quadrupole (SQ) detector. High-resolution mass spectra were obtained by an Agilent 1290LC-6540QTOF mass spectrometer or an Agilent Technologies 7250GCQTOF. HPLC analysis was performed on a Waters (Column The analysis was performed on a HILIC Silica, 5 μm, 4.6*150 mm and 19*150 mm) equipped with a 2998 PDA and a 3100 MS detector.

[0277] Scheme 1. Total synthesis of tetrodotoxin.

[0278]

[0279] Scheme 2.9-Epi Synthesis of tetrodotoxin.

[0280]

[0281] Given the pseudo-C2-symmetric core structure of TTX, we envisioned that the symmetry of highly substituted functional groups could be exploited to rapidly assemble the backbone. The subsequent chemoselective asymmetric cyclic anhydride opening and selective functional group interconversion strategy provided the basis for a gram-scale synthesis of the highly oxidized carbocyclic building block (-)-24 as an advanced intermediate in only 15 steps, which was rapidly transferred to the final TTX (1) in a scalable manner.

[0282] The synthesis of TTX 1 began with the stereoselective construction of the cyclohexane skeleton. Furfuryl alcohol 9 was esterified with the chiral auxiliary (-)-(1S)-camphoric acid 32 to afford the ester 10. To obtain the enantiomerically pure 7-oxabicyclo[2.2.1]hept-2-ene derivative 11, we developed an efficient stereoselective Diels-Alder protocol, which involved heating 10 with maleic anhydride using isopropyl ether as solvent. The pseudo-asymmetric reaction of anhydride 11 was promoted by quinine-mediated chemoselective methanolysis. 34, affording the monoacid 12 with high regioselectivity. Subsequently, stereospecific Upjohn racemic dihydroxylation of the olefin and simultaneous 1,2-diol protection proceeded in one pot to afford the acid 13, whose structure was confirmed by X-ray crystallographic analysis of a single crystal (CCDC#:2184304).

[0283] Decarboxylative hydroxylation was performed to place the TTX hydroxyl group at the C5 position. Initially, classical high-valent metal reagents were investigated as oxidants, but this resulted in substrate decomposition; therefore, we turned to milder free radical reaction conditions, including photocatalytic directed decarboxylative hydroxylation. Other decarboxylation methods, such as Barton decarboxylation or organic photoredox-promoted decarboxylation in the presence of a free radical initiator and oxygen under UV irradiation, failed to generate products. Finally, we utilized the method developed by Liang’s group to perform Ru-catalyzed photoredox decarboxylative hydroxylation of the NHPI ester of 13 and successfully obtained 14, albeit with an inverted configuration at C5 compared to TTX. Previous syntheses found it challenging to directly construct the correct stereochemistry at C5, and the steric effects of substitution at C5 posed a problem for the installation of subsequent functional groups, so we decided to invert the configuration at C5 at a later stage. Remarkably, this photoredox decarboxylative hydroxylation could be scaled up to 1.5 g without compromising the yield using a cyclic flow photochemical technique.

[0284] With compound 14, we investigated the functional group interconversion of this oxygen-bridged ring system and developed an innovative cascade reaction to establish oxygen functional groups at C8a, C6, and C11 positions. The auxiliary (-)-camphoric acid was first removed by transesterification with methanol to afford primary alcohol 15. The chiral auxiliary can be recycled as methyl camphorate. Primary alcohol 15 underwent Appel reaction to afford alkyl iodide 16; however, various reduction conditions applied to the alkyl iodide failed to generate the desired product due to substrate decomposition. After further exploration of the reductive ring-opening conditions, we were delighted to observe a cascade reaction: the initial Kagan reagent (SmI2)-mediated single electron transfer and iodide loss generated a primary carbon radical, which underwent radical elimination to form the terminal olefin, while SmI2 simultaneously reduced the unactivated methyl ester to the primary alcohol. In the presence of hexamethylphosphoramide (HMPA), only the eliminated product 17 was obtained (i.e., the methyl ester was not reduced to the diol 17a) (entry 1). SmI2 was activated with H2O and Et3N in a 1:2:2 ratio, yielding a thermodynamically stronger reducing agent. 38 , making the methyl ester (entry 2) at 77% 1HNMR yield reduction. Increasing the ratio of H2O to Et3N or replacing Et3N with pyrrolidine resulted in a complex mixture of products (entries 3 and 4). For scalability issues, the above procedure can also be modified to a two-step process involving the use of a small amount of SmI2 to obtain 17a, followed by LiAlH4 reduction to afford decagram quantities of 17 in 58% yield (entry 5). The absolute configuration of 17 was confirmed by single crystal X-ray crystallography (CCDC#:2182018).

[0285] With 17 in hand, we focused on the construction of the quaternary stereocenter at C8a and the inversion of the hydroxyl configuration at C5. The construction of the azidoaldehyde 24 started with the selective protection of the primary alcohol in 17 using sterically hindered TBDPSCl. Reductive cleavage of the TEMPO-NO bond in the resulting olefin 18 by Zn powder gave the allylic alcohol 19. The misconfiguration of the C5-OH was then inverted using a Mitsunobu reaction with 2-methoxyacetic acid 29 to afford the secondary alcohol 20. Elaborate diastereoselective Upjohn dihydroxylation of 20 followed by protection with acetonide afforded 21, the absolute configuration of which was verified by X-ray crystallography of the derivative structure 31 (CCDC#:2184298), obtained by NH3 / MeOH hydrolysis of the dihydroxylated 20 (see Supplementary Materials).

[0286] Intermediate 21 underwent Dess-Martin oxidation (DMP) to afford ketone 22 in excellent yield. From ketone 22, we aimed to construct a quaternary stereocenter at C8a, which turned out to be challenging. Nucleophilic addition to the imine derived from ketone 22 produced only diastereoisomers with the undesired configuration at C8a. While Darzens condensation of 22 with α-haloesters successfully gave the glycidyl ester, the corresponding epoxide was inert to stereoselective aminolysis. Reassuringly, nucleophilic addition of the dichloromethyl anion successfully converted ketone 22 into a single isomer of the spiro α-chloro epoxide 23 with simultaneous interconversion of the ester protecting group at C5 to p-methoxybenzyl (PMB). The resulting chloro epoxide 23, upon treatment with NaN3, afforded gram quantities of α-azidoaldehyde 24 with the correct configuration at C8a.

[0287] With the construction of a highly heteroatom-substituted carbocyclic core completed in just 15 steps, we were ready to tackle the synthetic challenges of the key dioxadamantane and guanidine hemiamine acetal moieties. 1,2-Addition of α-azidoaldehyde 24 to lithium acetylide followed by simultaneous removal of the TBDPS protecting group afforded two diastereoisomers (25 and 25a) in a 1:15 ratio, with the undesired product 25a being the majority. Extensive investigation of the reaction conditions revealed that 25a could be converted to the desired propargyl alcohol 25 in a 2:1 ratio (25 / 25a=2:1) ​​via a sequence that included MnO2-mediated selective oxidation followed by NaBH4 reduction. IBX oxidation of primary alcohol 25 gave the corresponding hemiacetal, which was then converted to acetal 26 by reaction with trimethyl orthoacetate in the presence of pyridinium p-toluenesulfonate (PPTS). The structure of 26 was confirmed by single crystal X-ray crystallography (CCDC#:2184305). Oxidative cleavage of the alkyne 26 with RuCl3 / NaIO4 followed by esterification with methanol afforded the carboxylic acid methyl ester 27. Simultaneous PMB deprotection and azide reduction by hydrogenation efficiently afforded the tertiary amine, which was guanidinated in situ with bis-Boc protected isothiourea 30 to afford the penultimate intermediate 28.

[0288] 2. Detailed Experimental Procedure

[0289] Example 1

[0290]

[0291] To a stirred solution of furfuryl alcohol 9 (39.6 g, 404 mmol, 1.00 eq), (1S)-(-)-camphoric acid (80.0 g, 404 mmol, 1.0 eq) and DMAP (4.93 g, 40.4 mmol, 0.10 eq) in CH2Cl2 (1400 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (116.6 g, 606 mmol, 1.50 eq) at 0°C, followed by slow stirring from 0°C to room temperature under an argon atmosphere for 27 hours. The mixture was quenched with 0.5N HCl solution (800 mL) and then extracted three times with CH2Cl2 (3×800 mL). The combined organic extracts were dried over anhydrous Na2SO4, 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.

[0292] R f =0.33 (PE / EtOAc=4:1)

[0293] (c=0.21,CHCl3)

[0294] HRMS-ESI(m / z)C 15 H 18 O5Na[M+Na] + Calculated value: 301.1052; measured value: 301.1043.

[0295] 1 H NMR (400MHz, CDCl3) δ7.42 (dd, J=1.8, 0.8Hz, 1H), 6.45 (d, J=3.3

[0296] 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).

[0297] 13 C 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.

[0298] Example 2

[0299]

[0300] In the dark, maleic anhydride (35.3 g, 360 mmol, 1.00 eq) was added to a stirred solution of compound 10 (100.0 g, 360 mmol, 1.00 eq) in 290 mL of isopropyl ether, and the mixture was heated to 55 ° C and stirred overnight in an argon atmosphere. Another 1.0 eq of maleic anhydride was added and the reaction was continued. 1 H NMR detection was performed until no compound 10 remained. After filtration, crude compound 11 (131.0 g, containing a small amount of maleic anhydride) was obtained as a white solid without further purification.

[0301] Please note: Compound 11 is sensitive to protic solvents and should be avoided from contact with protic solvents such as water or methanol during the entire process.

[0302] R f =0.33 (PE / EtOAc=1:1)

[0303] (c=0.16,CHCl3)

[0304] HRMS-ESI(m / z)C 19 H 24 NO8[M+NH4] + Calculated value: 394.1502; measured value: 394.1495.

[0306] 1 H 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).

[0307] 13 C 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.

[0308] Example 3

[0309]

[0310] At 0°C, to a stirred solution of compound 11 (131.0 g, 348 mmol, 1.00 eq) in toluene / CCl4 (750 mL / 750 mL) was added quinine (112.0 g, 348 mmol, 1.00 eq), followed by MeOH (55.7 g, 1740 mmol, 5.00 eq) at 0°C. The mixture was then slowly stirred from 0°C to room temperature for 22 hours under an argon atmosphere. After concentration under reduced pressure, the residue was dissolved in CH2Cl2 and acidified to pH = 2 with 1.0 N HCl solution, extracted with CH2Cl2 (3 × 800 mL) until there was no quinine in the organic layer. The combined organic extracts were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was recrystallized from CH2Cl2 and petroleum ether to give compound 12 (127.5 g, 87% for two steps) as a white solid.

[0311] R f =0.4 (DCM / MeOH=10:1)

[0312] (c=0.32,CHCl3)

[0313] HRMS-ESI(m / z)C 20 H 28 NO9[M+NH4] + Calculated value: 426.1764; measured value: 426.1754.

[0315] 1 H 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).

[0316] 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.

[0317] Example 4

[0318]

[0319] To a stirred solution of compound 12 (70.0 g, 171 mmol, 1.00 eq) in acetone (900 mL) and H2O (36 mL) was added 4-methylmorpholine N-oxide (NMO) (30.0 g, 256 mmol, 1.50 eq) and 4-methylmorpholine (NMM) (19.0 g, 188 mmol, 1.10 eq), and then 0.05 M OsO4 aqueous solution (34 mL, 1.7 mmol, 0.01 eq) was slowly added in the dark, and then the mixture was stirred at room temperature for 3 hours. It was quenched with saturated Na2SO3 aqueous solution and then concentrated under reduced pressure. The residue was acidified to pH = 2 with 1.0 N HCl solution and extracted three times with ethyl acetate (3 × 500 mL), and the remaining aqueous layer was washed with water. n- The mixture was extracted with BuOH until no product was found. After concentration under reduced pressure, crude compound 12a was obtained as a pale yellow foam and used in subsequent reactions without further purification.

[0320] 2,2-dimethoxypropane (26.6 g, 256 mmol, 1.50 eq) and PTSA·H2O (3.3 g, 17 mmol, 0.10 eq) were added to a stirred solution of compound 12a in 800 mL of acetone, and then stirred at room temperature for 3.5 hours. 4.0 g of NaHCO3 solid was added to quench the reaction and concentrated under reduced pressure. The residue was directly purified by silica gel flash chromatography (CH2Cl2 / MeOH, 100:1 to 20:1) to obtain white foam compound 13 (63.7 g, 77%).

[0321] R f =0.5 (DCM / MeOH=10:1)

[0322] (c=0.36,CHCl3)

[0323] HRMS-ESI(m / z)C 23 H 34 NO 11[M+NH4] + Calculated value: 500.2132; measured value: 500.2123.

[0325] 1 H NMR (400MHz, CDCl3) δ9.67(brs,1H),4.94(s,1H),4.65(d,J=10.9Hz,1H),4.55(d,J=10.9Hz,1 H),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(d dd,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).

[0326] 13 C 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.

[0327] Example 5

[0328]

[0329] Method A:

[0330] To a stirred solution of compound 13 (30.0 g, 62.2 mmol, 1.0 eq) in CH2Cl2 (600 mL) was added N-hydroxyphthalimide (15.2 g, 93.3 mmol, 1.5 eq) and DMAP (756 mg, 6.2 mmol, 0.1 equiv), followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (17.9 g, 93.3 mmol, 1.5 eq) at 0°C and slowly stirred in the dark under argon atmosphere from 0°C to room temperature for 24 hours. The resulting mixture was quenched with saturated aqueous NH4Cl solution (800 mL) and then extracted three times with CH2Cl2 (3×500 mL). It was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude NHPI ester 13a was used directly in the next step without further purification.

[0331] At room temperature, under 36W blue LED, the above crude NHPI ester 13a, Ru (bpy) 3 Cl 2 (1.91g, 3.2mmol, 0.05eq), TEMPO (14.9g, 95.8mmol, 1.54eq) and Hans ester (15.7g, 62.2mmol, 1.0eq) in DMF (degassed, 500mL) were vigorously stirred for 24 hours. The reaction mixture was then concentrated under reduced pressure. The resulting residue was subjected to silica gel column chromatography (petroleum ether / CH 2 Cl 2, 3: 1 to 1: 1) to give a light yellow solid compound 14 (22.7g, 62%, dr> 95: 5).

[0332] R f =0.4(PE / EA=4:1)

[0333] (c=0.23,CHCl3)

[0334] HRMS-ESI(m / z)C 31 H 48 NO 10 [M+H] + Calculated value: 594.3278; measured value: 594.3265.

[0336] 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).

[0337] 13 C 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.

[0338] Method B (Flow Chemistry Procedure):

[0339] To a stirred solution of compound 13 (1.5 g, 3.1 mmol, 1.0 eq) in CH2Cl2 (25 mL) was added N-hydroxyphthalimide (760 mg, 4.65 mmol, 1.5 eq) and DMAP (37 mg, 0.3 mmol, 0.1 equiv), followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (892 mg, 4.65 mmol, 1.5 eq) at 0°C and slowly stirred in the dark under an argon atmosphere from 0°C to room temperature for 24 hours. The resulting mixture was quenched with saturated aqueous NH4Cl solution (20 mL) and then extracted three times with CH2Cl2 (3×20 mL). It was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude NHPI ester was used directly in the next step without further purification.

[0340] The newly prepared NHPI activated ester 13a was added to a 100 mL round-bottom flask, followed by Ru(bpy)3Cl2 (96 mg, 0.16 mmol, 0.05 eq), TEMPO (750 mg, 4.8 mmol, 1.54 eq) and Hans ester (785 mg, 3.1 mmol, 1.0 eq), DMF (degassed, 40 mL). A flow device was inserted. Argon was filled into the system for 10 minutes, and the reaction was carried out at a flow rate of 14 rpm under the irradiation of a 36 W blue LED for 24 hours. The reaction mixture was then concentrated under reduced pressure. The resulting residue was subjected to silica gel column chromatography (petroleum ether / CH2Cl2, 3:1 to 1:1) to give a light yellow solid compound 14 (1.22 g, 66%, dr>95:5).

[0341] Materials used in the flow procedure: peristaltic pump (BT02-YZ1515), PFA tube, silicone tube, 36W 450nm LED, and electric fan (all materials were purchased on Taobao).

[0342] Example 6

[0343]

[0344] To a stirred solution of compound 14 (20.0 g, 33.7 mmol, 1.00 eq) in 350 mL of MeOH was added KCO (6.9 g, 50.5 mmol, 1.50 eq), followed by stirring at room temperature for 1 hour. The mixture was quenched with 10 mL of AcOH, then concentrated under reduced pressure, and subjected to silica gel column chromatography (CHCl / MeOH, 100:1 to 40:1) to give compound 15 (13.2 g, 95%) as a colorless oil.

[0345] R f =0.5 (DCM / MeOH=20:1)

[0346] (c=0.24,CHCl3)

[0347] HRMS-ESI(m / z)C 21 H 36 NO7[M+H] + Calculated value: 414.2492; measured value: 414.2477.

[0348] 1 H NMR (400MHz, CDCl3) δ4.94 (s, 1H), 4.79 (d, J = 6.3Hz, 1H), 4.51–

[0349] 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).

[0350] 13 C 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.

[0351] Example 7

[0352]

[0353] To a stirred solution of compound 15 (13.0 g, 31.5 mmol, 1.00 eq), PPh3 (30.6 g, 117 mmol, 3.74 eq) and imidazole (6.7 g, 99.5 mmol, 3.16 eq) in 300 mL of toluene was added I2 (21.2 g, 83.8 mmol, 2.66 eq) dissolved in 100 mL of toluene, and then stirred at 110° C. 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.

[0354] R f =0.4(PE / EA=10:1)

[0355] (c=0.26,CHCl3)

[0356] HRMS-ESI(m / z)C 21 H 35 INO6[M+H] + Calculated value: 524.1509; measured value: 524.1503.

[0358] 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=11.3H z,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).

[0359] 13 C NMR (101MHz, CDCl3) δ171.92,112.51,86.51,84.36,83.98,82.28,77.92,60.93,5 9.33,52.23,48.39,40.54,40.03,35.08,26.01,25.64,20.45,20.39,16.96,1.18.

[0360] Example 8

[0361]

[0362] 1. Prepare 0.1M SmI2 solution in THF

[0363] A flame-dried 1L round-bottom flask was charged with samarium metal (12.70 g, 86 mmol, 1.00 eq.) and a stirring rod, and then thoroughly degassed THF (860 mL) and iodine crystals (22.68 g, 86 mmol, 1.00 eq.) were added in sequence at room temperature and argon atmosphere. The reaction mixture was stirred vigorously 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 dark blue.

[0364] Please note: To ensure adequate conversion, the solution should be stirred for at least 2 hours before use.

[0365] 2. SmI2-induced reduction reaction

[0366] The freshly prepared 0.1M SmI2 (115 mL, 11.5 mmol, 12.0 eq) solution in THF was transferred to a new flame-dried 500 mL round-bottom flask using a double-ended needle. Compound 16 (500 mg, 0.95 mmol, 1.00 eq), Et3N (318 μL, 23.0 mmol, 24.0 eq) and H2O (413 μL, 23.0 mmol, 24.0 eq) were dissolved in 2 mL of THF. The whole process needed to be protected from air and was carried out under an argon atmosphere. The mixture was then stirred at room temperature for 1.5 hours. After the reaction was complete, the color turned yellow and was then quenched with saturated NH4Cl aqueous solution (10 mL), washed with 0.1N HCl aqueous solution (2 mL), and extracted with ethyl acetate (3×10 mL). It was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (CH2Cl2 / MeOH, 80:1) to give white solid compound 17 (229 mg, 65%).

[0367] Scalable preparation of compound 17

[0368]

[0369] A 0.1M solution of SmI2 in THF was freshly prepared in the same manner as described above. A double-ended needle was used to transfer the freshly prepared solution of SmI2 in THF (860 mL) to a solution of compound 16 (15.0 g, 28.7 mmol, 1.00 eq) dissolved in 20 mL of THF in a new flame-dried 1 L round-bottom flask. The entire process needed to be air-free and was carried out under an argon atmosphere. The mixture was then stirred at 55 °C for 2 hours. After the reaction was complete, the color turned yellow and was then quenched with saturated aqueous NH4Cl solution (500 mL), the mixture was filtered to remove the samarium salt, and then extracted with ethyl acetate (3×800 mL). It was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product, compound 16a, was used directly in the next step without further purification.

[0370]

[0371] At 0°C, LiAlH4 (1.09 g, 28.7 mmol, 1.00 eq) was slowly added to a stirred solution of compound 16a in 150 mL THF. Under an argon atmosphere, the mixture was slowly stirred from 0°C to room temperature for 12 hours. Excess Na2SO4·10H2O was added to quench the reaction, then filtered and concentrated under reduced pressure. The crude product compound was purified by silica gel column chromatography (CH2Cl2 / MeOH, 80:1) to give a white solid compound 17 (6.1 g, 58% for two steps).

[0372] R f =0.2(PE / EA=2:1)

[0373] (c=0.30,CHCl3)

[0374] HRMS-ESI(m / z)C 20 H 36 NO5[M+H] + Calculated value: 370.2593; measured value: 370.2585.

[0375] 1 H NMR (400MHz, CDCl3) δ5.40 (s, 1H), 5.23 (s, 1H), 4.75 (d, J = 6.5Hz,

[0376] 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).

[0377] 13 C NMR (101MHz, CDCl3) δ142.43,115.10,109.62,84.93,76.78,74.39,67.62,62.8 8,60.48,59.94,45.20,39.73,33.82,33.51,27.09,25.32,20.17,20.08,16.61.

[0378] Example 9

[0379]

[0380] To a stirred solution of compound 17 (7.2 g, 19.5 mmol, 1.00 eq) in 100 mL of CH2Cl2, imidazole (2.6 g, 39.0 mmol, 2.00 eq) and DMAP (475 mg, 3.9 mmol, 0.20 eq) were added, and then TBDPSCl (6.9 g, 25.3 mmol, 1.30 eq) was slowly added at 0 ° C. Under an argon atmosphere, the mixture was stirred at room temperature for 2 hours. MeOH (20 mL) was added to quench the reaction, and the resulting mixture was concentrated under reduced pressure, and silica gel column chromatography (petroleum ether / ethyl acetate, 20: 1 to 12: 1) was performed to obtain white foam compound 18 (10.0 g, 85%).

[0381] R f =0.4(PE / EA=10:1)

[0382] (c=0.22,CHCl3)

[0383] HRMS-ESI(m / z)C 36 H 54 NO5Si[M+H] + Calculated value: 608.3771; measured value: 608.3748.

[0385] 1 H 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).

[0386] 13 C 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.

[0387] Example 10

[0388]

[0389] Zinc powder (41 g, 624 mmol, 40.00 eq) was added to a stirred solution of compound 18 (9.5 g, 15.6 mmol, 1.00 eq) in 100 mL THF and 130 mL AcOH. The mixture was stirred at 55 ° C for 4.5 hours under argon atmosphere. 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.

[0390] R f =0.2(PE / EA=4:1)

[0391] (c=1.4,CHCl3)

[0392] HRMS-ESI(m / z)C 27 H 36 O5SiNa[M+Na] + Calculated value: 491.2230; measured value: 491.2216.

[0393] 1 H 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).

[0394] 13 C 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.

[0395] Embodiment 11

[0396]

[0397] 2-Methoxyacetic acid (1.8 g, 20.5 mmol, 1.50 eq) and PPh3 (7.2 g, 27.4 mmol, 2.00 eq) were added to a stirred solution of compound 19 (6.4 g, 13.7 mmol, 1.00 eq) in 137 mL of dry THF. Diethyl diazine dicarboxylate (DEAD) (3.7 g, 20.5 mmol, 1.50 eq) was then added dropwise at -10 ° C. The mixture was stirred for 12 hours at -10 ° C under an argon atmosphere. 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.

[0398] R f =0.4(PE / EA=4:1)

[0399] (c=0.35,CHCl3)

[0400] HRMS-ESI(m / z)C 30 H 40 O7SiNa[M+Na] + Calculated value: 563.2441; measured value: 563.2430.

[0402] 1 H 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).

[0403] 13C NMR (101MHz, CDCl3) δ168.80,140.30,135.43,132.72,132.59,129.85,129.76,127.72,127.69,11 3.10,110.19,77.58,74.67,70.36,69.36,68.26,62.41,59.27,43.63,27.49,26.63,26.00,18.94.

[0404] Example 12

[0405]

[0406] To a stirred solution of compound 20 (6.60 g, 12.2 mmol, 1.00 eq) in acetone (70 mL) was added 4-methylmorpholine N-oxide (2.85 g, 24.4 mmol, 2.00 eq), then 0.05 M OsO4 aqueous solution (35 mL, 0.7 mmol, 0.06 eq) was slowly added in the dark, and the mixture was stirred at room temperature for 12 hours. It was quenched with saturated NH4Cl aqueous solution (50 mL), then extracted three times with CH2Cl2 (3 × 200 mL), then dried over Na2SO4, filtered and concentrated under reduced pressure to give a pale yellow solid crude compound 20a, which was used for subsequent reactions without further purification.

[0407] To a stirred solution of compound 20a in 80 mL of CH2Cl2 was added 2,2-dimethoxypropane (2.54 g, 24.4 mmol, 2.00 eq) and camphorsulfonic acid (CSA) (566 mg, 2.44 mmol, 0.20 eq), followed by stirring at room temperature for 0.5 hours. Quenched with saturated NH4Cl aqueous solution (50 mL), then extracted with CH2Cl2 (3 × 200 mL). Dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (petroleum ether / ethyl acetate, 5:1) to give compound 21 (4.90 g, 66%) as a white foam.

[0408] R f =0.4(PE / EA=4:1)

[0409] (c=0.43,CHCl3)

[0410] HRMS-ESI(m / z)C 33 H 46 O9SiNa[M+Na] + Calculated value: 637.2809; measured value: 637.2799.

[0411] 1 H 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),2.2 8(t,J=6.5Hz,1H),1.56(s,3H),1.44(s,6H),1.41(s,3H),1.05(s,9H).

[0412] 13 C NMR (101MHz, CDCl3) δ168.86,135.58,133.25,129.63,129.59,127.62,127.60,110.96,109.65,79.73,7 7.81,74.81,71.89,69.60,69.10,66.34,61.50,59.37,40.45,26.74,26.68,26.50,25.74,25.52,19.10.

[0413] Embodiment 13

[0414]

[0415] To a stirred solution of compound 20 (54.0 mg, 0.1 mmol, 1.00 eq) in acetone (1 mL) was added 4-methylmorpholine N-oxide (23.4 mg, 0.2 mmol, 2.00 eq), then 0.05 M OsO4 aqueous solution (0.12 mL, 0.006 mmol, 0.06 eq) was slowly added in the dark, and the mixture was stirred at room temperature overnight. It was quenched with saturated NH4Cl aqueous solution (2 mL), then extracted three times with CH2Cl2 (3 × 10 mL), then dried over Na2SO4, filtered and concentrated under reduced pressure to give a pale yellow solid crude compound, which was used in subsequent reactions without further purification.

[0416] To a stirred solution of the crude product in 1 mL of MeOH was added 0.2 mL of 7N NH3 in MeOH and stirred at room temperature for 2 hours. It was then extracted with H2O (10 mL) and CH2Cl2 (3×10 mL). It was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (petroleum ether / ethyl acetate, 1:1) to give compound 31 (37.5 mg, 75%) as a white solid.

[0417] R f =0.2(PE / EA=1:1)

[0418] (c=0.22,CHCl3)

[0419] HRMS-ESI(m / z)C 27 H 38 O7SiNa[M+Na] + Calculated value: 525.2285; measured value: 525.2278.

[0420] 1 H 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).

[0421] 13 C 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.

[0422] Embodiment 14

[0423]

[0424] To a stirred solution of compound 21 (4.89 g, 7.96 mmol, 1.00 eq) in 22 mL of CH2Cl2 was added NaHCO3 (2.69 g, 32.0 mmol, 4.00 eq) and Dess-Martin reagent (5.38 g, 12.7 mmol, 1.60 eq) at 0°C. The solution was stirred at room temperature for 3 hours. The resulting mixture was directly purified by silica gel flash chromatography (petroleum ether / ethyl acetate, 6:1) to give compound 22 (4.78 g, 98%) as a white foam.

[0425] R f =0.5(PE / EA=4:1)

[0426] (c=0.24,CHCl3)

[0427] HRMS-ESI(m / z)C 33 H 44 O9SiNa[M+H] + Calculated value: 635.2653; measured value: 635.2632.

[0429] 1 H 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).

[0430] 13 C NMR (101MHz, CDCl3) δ205.28,169.14,135.62,135.52,133.04,132.81,129.75,129.71,127.69,111.47,1 11.00,82.03,78.77,78.52,74.47,69.31,69.01,59.28,57.62,50.18,27.02,26.72,26.53,26.09,19.07.

[0431] Embodiment 15

[0432]

[0433] To a stirred solution of N,N-diisopropylamine (4.2 g, 41.6 mmol, 6.40 eq) in 42 mL of dry THF was added n-butyl lithium (2.5 M solution in hexane) (16.6 ml, 41.6 mmol, 6.40 eq) dropwise at -78 °C under argon atmosphere. After 1 hour, dry CH2Cl2 (11.6 g, 136.5 mmol, 21.00 eq) was added dropwise, followed by a solution of compound 22 (4.0 g, 6.5 mmol, 1.00 eq) in dry THF (50 mL) and stirred at -78 °C under argon atmosphere. TLC was performed with 4:1 petroleum ether:ethyl acetate. After the disappearance of 22, the resulting mixture was quenched with saturated NH4Cl aqueous solution (20 mL), extracted with EtOAc (3×50 mL), dried over anhydrous MgSO4, and concentrated under reduced pressure to give crude compound 22a as a light yellow oil, which was used in subsequent reactions without further purification.

[0434] To a stirred solution of compound 22a in 15 mL of dry THF was added NaHMDS (2.0 M in THF, 8.1 mL, 16.2 mmol, 2.50 eq), the reaction mixture was stirred at -78 ° C for 1 hour, warmed to 0 ° C and stirred at 0 ° C for 1 hour, then the mixture was cooled to -78 ° C again, and PMBBr (3.3 g, 16.2 mmol, 2.50 eq) was added dropwise, continued to stir at -78 ° C for 1 hour, then warmed to 0 ° C, stirred at 0 ° C for 1 hour, warmed to room temperature, and stirred at room temperature for 12 hours. The resulting mixture was poured into a saturated NH4Cl aqueous solution (50 mL), extracted with EtOAc (3 × 100 mL), dried over anhydrous MgSO4, 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.

[0435] R f =0.6(PE / EA=2:1)

[0436] (c=0.50,CHCl3)

[0437] HRMS-ESI(m / z)C 39 H 49 ClO8SiNa[M+Na] + Calculated value: 731.2783; measured value: 731.2773.

[0438] 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),4.2 2(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=1 0.4, 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).

[0439] 13 C 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.

[0440] Example 16

[0441]

[0442] To a solution of compound 23 (2.7 g, 3.8 mmol, 1.00 eq) in dry dimethyl sulfoxide (38 mL) was added sodium azide (1.48 g, 22.8 mmol, 6.00 eq) and 15-crown-5 (2.5 g, 11.4 mmol, 3.00 eq) and stirred at 70° C. under argon for 21 hours. TLC was performed with 4:1 petroleum ether:ethyl acetate. After the disappearance of the starting compound, the reaction mixture was poured into a saturated NH4Cl aqueous solution (30 mL), extracted with CH2Cl2 (3×100 mL), dried over anhydrous MgSO4, and evaporated to give a white foam compound 24 (2.13 g, 77% yield), which was purified on a silica gel column with petroleum ether / ethyl acetate (20:1).

[0443] R f =0.5(PE / EA=2:1)

[0444] (c=0.13,CHCl3)

[0445] HRMS-ESI(m / z)C 39 H 49 N3O8SiNa[M+Na] + Calculated value: 738.3187; measured value: 738.3173.

[0446] 1 H 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).

[0447] 13 C 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.

[0448] Embodiment 17

[0449]

[0450] To a solution of lithium acetylide ethylenediamine complex (180 mg, 1.96 mmol, 10.00 eq) in 2.5 mL THF was added dropwise compound 24 (140 mg, 0.20 mmol, 1.00 eq) dissolved in 2.5 mL THF at 0°C, followed by stirring at 0°C for 1 hour, quenching with saturated aqueous NH4Cl solution (2 mL), extracting with EtOAc (3×10 mL), drying over anhydrous MgSO4, filtering and concentrating under reduced pressure, and purified on a silica gel column with 2:1 petroleum ether / ethyl acetate to give a colorless oily compound 25a (65 mg, 66% yield) and its diastereomer 25 (25a:25=15:1, the ratio was determined by crude 1 H NMR detection).

[0451] R f =0.2(PE / EA=2:1)

[0452] (c=0.03,CHCl3)

[0453] HRMS-ESI(m / z)C 25 H 33 N3O8Na[M+Na] + Calculated value: 526.2166; measured value: 526.2154.

[0454] 1 H 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,1 H), 2.53 (d, J = 2.2Hz, 1H), 1.56 (s, 3H), 1.47 (s, 3H), 1.39 (s, 3H), 1.33 (s, 3H).

[0455] 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.

[0456] Embodiment 18

[0457] Epimerization of 25a to 25

[0458]

[0459] To a stirred solution of compound 25a (148 mg, 0.29 mmol, 1.00 eq) in 5 mL CH2Cl2 was added MnO2 (121 mg, 1.4 mmol, 10.00 eq), followed by stirring at room temperature for 2 hours. After filtration and concentration under reduced pressure, it was used in the subsequent reaction without further purification.

[0460] To a solution of the oxidation product obtained above in 2 ml of dioxane and 200 uL of H2O was added NaBH4 (421 mg, 1.11 mmol, 4.00 eq) at room temperature, and then stirred at 60°C for 0.5 hours. The mixture was quenched with saturated aqueous NH4Cl solution (2 mL), extracted with CH2Cl2 (3×5 mL), dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (petroleum ether / ethyl acetate, 2:1) to give a colorless oily compound 25 (76 mg, 51%, 70% brsm) and a white foamy compound 25a (39 mg, 26%). (25:25a=2:1, the ratio was determined by crude 1 H NMR detection).

[0461] R f =0.25(PE / EA=2:1)

[0462] (c=0.18,CHCl3)

[0463] HRMS-ESI(m / z)C 25 H 33 N3O8Na[M+Na] + Calculated value: 526.2166; measured value: 526.2156.

[0464] 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).

[0465] 13 C 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.

[0466] Embodiment 19

[0467]

[0468] IBX (70 mg, 250 mmol, 1.05 eq) was added to a stirred solution of compound 25 (120 mg, 238 mmol, 1.00 eq) in 5.0 mL DMSO, and stirred at room temperature for 2 hours. TLC was performed with 4:1 petroleum ether / EtOAc. After 25 disappeared, MeOH (5 mL), pyridinium p-toluenesulfonate (PPTS) (90.0 mg, 356 mmol, 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 NaHCO3 aqueous solution (10 mL), extracted with CH2Cl2 (3×20 mL), dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (petroleum ether: ethyl acetate, 8 / 1) to obtain white foam compound 26 (108 mg, 88% yield).

[0469] R f =0.4(PE / EA =4:1)

[0470] (c=0.05,CHCl3)

[0471] HRMS-ESI(m / z)C 26 H 33 N3O8Na[M+Na] + Calculated value: 538.2166; measured value: 538.2158.

[0472] 1 H 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(d d,J=8.6,1.4Hz,1H),3.79(s,3H),3.40(s,3H),2.80(ddd,J=8.5,4.2,0.9Hz,1 H), 2.61 (d, J = 2.3Hz, 1H), 1.39 (s, 3H), 1.37 (s, 3H), 1.26 (s, 3H), 1.26 (s, 3H).

[0473] 13 C 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.

[0474] Embodiment 20

[0475]

[0476]

[0477] To a stirred solution of compound 26 (27.0 mg, 0.05 mmol, 1.00 eq) in CCl4 (1.0 mL), MeCN (1.0 mL) and H2O (1.5 mL) was added NaIO4 (45 mg, 0.21 mmol, 4.00 eq) and RuCl3.xH2O (2.2 mg, 0.011 mmol, 0.20 eq), followed by stirring at room temperature for 2 hours. The mixture was quenched with H2O (5 mL), extracted with CH2Cl2 (3×100 mL), dried over anhydrous MgSO4, filtered and concentrated under reduced pressure, and then used in subsequent reactions without further purification.

[0478] To the stirring solution of crude product in 1mL CH Cl was added EDCI (20 mg, 0.10 mmol, 2.00 eq), DMAP (1.2 mg, 0.009 mmol, 0.20 eq) and MeOH (16.77 mg, 0.52 mmol, 10.00 eq), then stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure and purified by silica gel flash chromatography (petroleum ether: ethyl acetate, 8 / 1 to 4 / 1) to give white foam compound 27 (13.0 mg, 48% yield).

[0479] R f =0.25(PE / EA=4:1)

[0480] (c=0.05,CHCl3)

[0481] HRMS-ESI(m / z)C 26 H 39 N4O 10 [M+NH4] + Calculated value: 567.2666; measured value: 567.2658.

[0482] 1 H 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),4.0 4(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).

[0483] 13 C 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.

[0484] Embodiment 21

[0485]

[0486] Compound 27 (13.0 mg, 0.024 mmol, 1.00 eq) was hydrogenated in 2.0 mL MeOH in the presence of 20% Pd-C under H2 atmosphere for 24 h. The catalyst was then filtered off and evaporated to give compound 27a, which was used in subsequent reactions without further purification.

[0487] To a stirred solution of the crude product in 2.5 mL of dry dichloromethane was added triethylamine (12.12 mg, 0.12 mmol, 5.00 eq), 1,3-bis(tert-butyloxycarbonyl)-2-methyl-2-thiopseudurea (13.9 mg, 0.048 mmol, 2.00 eq) and mercuric (II) chloride (17.0 mg, 0.048 mmol, 2.00 eq) at room temperature. After stirring for 2 hours, the mixture was quenched with water (10 mL) and extracted three times with CH2Cl2 (3×10 mL). The combined organic layers were dried over anhydrous MgSO4. After filtration, the solvent was removed under reduced pressure to obtain the crude material, which was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 1:1) to obtain the title compound 28 (11.2 mg, 88% yield, 2 steps) as a white foam.

[0488] R f =0.2 (PE / EtOAc=2:1)

[0489] (c=0.16,CHCl3)

[0490] HRMS-ESI(m / z)C 24 H 40 N3O 11 [M+H] + Calculated value: 546.2663; measured value: 546.2650.

[0492] 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).

[0493] 13 C 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.

[0494] Embodiment 22

[0495]

[0496] Compound 27 (13.0 mg, 0.024 mmol, 1.00 eq) was hydrogenated in 2.0 mL MeOH in the presence of 20% Pd-C under H2 atmosphere for 19 h. The catalyst was then filtered off and evaporated to give compound 27a, which was used in subsequent reactions without further purification.

[0497] To a stirred solution of the crude material in 2.5 mL of dry dichloromethane was added triethylamine (12.12 mg, 0.12 mmol, 5.00 eq), 1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudurea (17.2 mg, 0.048 mmol, 2.00 eq) and mercuric (II) chloride (17.0 mg, 0.048 mmol, 2.00 eq) at room temperature. After stirring for 3 hours, the mixture was quenched with water (10 mL) and extracted three times with CH2Cl2 (3×10 mL). The combined organic layers were dried over anhydrous MgSO4. After filtration, the solvent was removed under reduced pressure to obtain the crude material, which was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 2:1) to obtain the title compound 33 as a white foam (13.4 mg, 80% yield, 2 steps).

[0498] Rf =0.65 (PE / EtOAc=2:1)

[0499] (c=0.22,CHCl3)

[0500] HRMS-ESI(m / z)C 35 H 44 N3O 13 [M+H] + Calculated value: 714.2874; measured value: 714.2863.

[0502] 1 H 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,1 H), 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).

[0503] 13 C 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.

[0504] Embodiment 23

[0505] Method A: One-step synthesis of TTX

[0506]

[0507] Compound 28 (11.0 mg, 0.018 mmol, 1.00 eq) was dissolved in trifluoroacetic acid (500 μL) and water (500 μL) at room temperature. The resulting solution was heated to 60°C. After stirring for 24 hours, the mixture was concentrated in vacuo and the residue was purified by 1 HNMR detection gave a mixture of tetrodotoxin and 4,9-dehydrotetrodotoxin (1:1). The mixture was then redissolved in TFA-d (20 μL) and deuterium oxide (1000 μL) at room temperature and stirred for 5 days at room temperature. The mixture was concentrated in vacuo and the residue was purified by HPLC (sample preparation and purity analysis were performed on a Waters HPLC (Column 2000) equipped with a 2998 PDA and 3100 MS detector). HILIC Silica, 5 μm, 4.6*150 mm and 19*150 mm), mobile phase: H2O (0.1% AcOH) / acetonitrile (0.1% AcOH) (65%-50% in 10 minutes). The residual solution was lyophilized to obtain white solid tetrodotoxin 1 and 4,9-dehydrotetrodotoxin 2 (4:1) (by 1 H NMR) (4.2 mg, 65% overall yield).

[0508] (c=0.06,0.05M AcOH)

[0509] HRMS-ESI(m / z)C 11 H 18 N3O8[M+H] + Calculated value: 320.1094; measured value: 320.1085.

[0511] 1 H NMR (600MHz, 5% CD3CO2D / D2O) δ5.49 (d, J=9.4Hz, 1H), 4.28

[0512] (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).

[0513] 13 C NMR (151MHz, 5% CD3CO2D / D2O) δ 156.52, 110.76, 79.59, 75.06, 73.77, 72.72, 71.39, 70.80, 65.45, 59.65, 40.62.

[0514] Embodiment 24

[0515] Method B: Two-step synthesis of TTX

[0516]

[0517] Compound 33 (13.0 mg, 0.018 mmol, 1.00 eq) was dissolved in trifluoroacetic acid (500 μL) and water (500 μL) at room temperature. The resulting solution was heated to 60°C. After stirring for 24 hours, the mixture was concentrated in vacuo and the residue was purified by preparative TLC to give hemiamine acetal 34 (4.1 mg, 49.7% yield) as a white foam.

[0518] Note: Hemiamine acetal 34 and its anhydrous form can be separated by PTLC (DCM:MeOH=4:1) as described by Fukuyama.

[0519] R f =0.15 (DCM / MeOH=4:1)

[0520] (c=0.08,MeOH)

[0521] HRMS-ESI(m / z)C 19 H 24 N3O 10 [M+H] + Calculated value: 454.1461; measured value: 454.1455.

[0523] 1 H 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).

[0524] 13 C 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.

[0525]

[0526] Compound 34 (4.0 mg, 0.009 mmol, 1.00 eq) was hydrogenated in MeOH (0.5 mL) in the presence of 50% Pd-C under H2 atmosphere for 6 h. The catalyst was then filtered off and evaporated to give the crude material which was purified by HPLC (sample preparation and purity analysis were performed on a Waters HPLC (Column 1000) equipped with a 2998 PDA and 3100 MS detector). HILIC Silica, 5 μm, 4.6*150 mm and 19*150 mm), mobile phase: H2O (0.1% AcOH) / acetonitrile (0.1% AcOH) (65%-50% in 10 minutes). The residual solution was freeze-dried to obtain white solid tetrodotoxin 1 (containing trace amounts of 4,9-dehydrated tetrodotoxin 2) (2.5 mg, 91% yield).

[0527] Synthesis of 9-epiTTX

[0528] Embodiment 25

[0529]

[0530] To a stirred solution of compound 25a (50.0 mg, 0.10 mmol, 1.00 eq.) in DMSO (2.0 mL) was added IBX (29.0 mg, 0.10 mmol, 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 the disappearance of 25a, MeOH (2 mL), pyridinium p-toluenesulfonate (PPTS) (37.8 mg, 0.15 mmol, 1.50 eq.) and trimethyl orthoacetate (0.55 mL) were added to the above solution, and the solution was then stirred at room temperature for 12 hours. The mixture was quenched with saturated aqueous NaHCO3 (2 mL) and extracted with CH2Cl2 (3×10 mL). The combined organic layers were dried over anhydrous MgSO 4 , filtered and concentrated under reduced pressure to obtain a residue, which was purified by silica gel flash chromatography (petroleum ether / ethyl acetate, 8:1) to afford compound 35 (33.3 mg, 65% yield) as a white foam.

[0531] R f =0.35(PE / EA=4:1)

[0532] (c=0.108,CHCl3)

[0533] HRMS-ESI(m / z)C 26 H 33 N3O8Na[M+Na] +Calculated value: 538.2166; measured value: 538.2150.

[0534] 1 H NMR (400MHz, CDCl3) δ7.23 (d, J = 8.7Hz, 2H), 6.87 (d, J = 8.7

[0535] 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).

[0536] 13 C 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.

[0537] Embodiment 26

[0538]

[0539] To a stirred solution of compound 35 (20.0 mg, 0.04 mmol, 1.00 eq.), NaIO4 (34.2 mg, 0.16 mmol, 4.00 eq.) in CCl4 (0.7 mL), MeCN (0.7 mL) and H2O (1.1 mL) was added RuCl3·xH2O (1.7 mg, 0.008 mmol, 0.20 eq.) at room temperature under argon atmosphere. The reaction mixture was then stirred at room temperature for 0.5 h. H2O (2.0 mL) and CH2Cl2 (2.0 mL) were then added to the above solution. The residue was distributed and the separated organic layer was used directly. At room temperature, under argon atmosphere, EDCI·HCl (15.4 mg, 0.08 mmol, 2.00 eq.), DMAP (1.0 mg, 0.008 mmol, 0.20 eq.), MeOH (12.8 mg, 0.40 mmol, 10.00 eq.) were added to the above organic solution. The reaction mixture was stirred at room temperature for 2 hours, then concentrated under reduced pressure to obtain a residue, which was purified by silica gel flash chromatography (PE / EtOAc, 8 / 1 to 4 / 1) to obtain white foam compound 36 (10.4 mg, 49% yield).

[0540] R f =0.35(PE / EA=4:1)

[0541] (c=0.042,CHCl3)

[0542] HRMS-ESI(m / z)C 26 H 35 N3O 10 Na[M+Na] + Calculated value: 572.2220;

[0543] Measurement value: 572.2214.

[0544] 1 H 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).

[0545] 13 C 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.

[0546] Embodiment 27

[0547]

[0548] A suspension of compound 36 (8.0 mg, 0.015 mmol, 1.00 eq.) and Pd / C (1.6 mg, 20 wt%) in MeOH (1.0 mL) was stirred at room temperature under H2 atmosphere for 24 h. The mixture was passed through The filtrate was evaporated to give crude product 36a which was carried on to the next step without further purification.

[0549] To a stirred solution of crude product 36a, 1,3-bis(tert-butyloxycarbonyl)-2-methyl-2-thiopseudurea (8.7 mg, 0.030 mmol, 2.00 eq.) and mercuric(II) chloride (8.2 mg, 0.030 mmol, 2.00 eq.) in dry dichloromethane (1.5 mL) was added Et3N (7.6 mg, 0.075 mmol, 5.00 eq.) at room temperature under argon atmosphere. The reaction mixture was stirred at room temperature for 2 h, then quenched with water (10.0 mL) and extracted with CH2Cl2 (3×10 mL). The combined organic layers were dried over anhydrous MgSO4, filtered and concentrated under reduced pressure to give a residue, which was purified by silica gel flash chromatography (PE / EtOAc, 2:1) to give compound 37 (7.5 mg, 80% yield, 2 steps) as a white foam.

[0550] R f =0.4 (PE / EtOAc=2:1)

[0551] (c=0.120,CHCl3)

[0552] HRMS-ESI(m / z)C 35 H 44 N3O 13 [M+H] +Calculated value: 646.3187; measured value: 646.3174.

[0553] 1 H NMR(400MHz, CDCl3)δ11.08(brs,1H),9.14(brs,1H),5.44(d,

[0554] J=2.1Hz,1H),5.27(s,1H),5.13(d,J=6.8Hz,1H),4.35(d,J=9.6

[0555] 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).

[0556] 13 C 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.

[0557] Embodiment 28

[0558]

[0559] Compound 37 (7.0 mg, 0.011 mmol, 1.00 eq.) was dissolved in trifluoroacetic acid (500 μL) and water (500 μL) at room temperature under argon atmosphere. The reaction mixture was then heated to 60°C and stirred at 60°C for 12 hours. The resulting mixture was concentrated in vacuo to give a residue, which was purified by HPLC (sample preparation and purity analysis were performed on a Waters HPLC (Column 1000) equipped with a 2998 PDA and 3100 MS detector). HILIC Silica, 5 μm, 4.6*150 mm and 19*150 mm), mobile phase: H2O (0.1% AcOH) / acetonitrile (0.1% AcOH) (65%-50% in 10 minutes). The residual solution was lyophilized to obtain white solid 9-epiTTX-hemiacetal and 9-epiTTX-10,8-lactone (1:1, by 1 H NMR) 1a (1.9 mg, 55% yield).

[0560] HRMS-ESI(m / z)C 11 H 18 N3O8[M+H] + Calculated value: 320.1094; measured value: 320.1086.

[0561] 9-epiTTX(hemiacetal) 1 H NMR(400MHz,5% CD3CO2D / D2O)

[0562] δ5.24(d,J=9.2Hz,2H),4.24(s,1H),4.22(s,1H),4.15(brs,1H),

[0563] 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).

[0564] 9-epiTTX(10,8-lactone) 1 H NMR(400MHz,5% CD3CO2D / D2O)

[0565] 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).

[0566] 9-epiTTX (hemiacetal and 10,8-lactone) 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.

[0567] Biological Assessment

[0568] To investigate the biological activity of pure TTX, we synthesized and purified a single form of TTX(S) from the carboxylic acid methyl ester 24 following the strategy of Fukuyama. Another sample, designated TTX(C) (purchased from Tocris bioscience, 1 H NMR analysis, TTX and 4,9-anhydroTTX ratio of 10:3, purity >99%) were used for comparison. We compared the effects of two different sources of TTX on human HEK-Na v 1.7 Cells and HEK-Na v We measured the blocking capacity of single subtype sodium channels on HEK-1.5 cells by voltage clamping from -80 mV to 80 mV in normal HEK-Na cells without TTX. v 1.7 current. HEK-Na v 1.7 current could be successfully induced and completely blocked by 1μM TTX(C). v 1.7 cells can be used to measure the blocking ability of TTX and TTX analogs from different sources. We then measured the blocking ability of HEK-Na treated with TTX(S) and TTX(C) under the same conditions. v 1.7 HEK-Na cells v 1.7 current. Our results show that TTX(S) has a better blocking efficiency than TTX(C). In mice, our synthesized pure TTX(S) also showed a stronger effect in blocking the amplitude of sodium ion current in wild-type div (in vitro experimental days) hippocampal neurons.

[0569] The biological activity of 9-epiTTX was also evaluated based on the blockade of the amplitude of sodium currents in wild-type div hippocampal neurons, but it did not show any inhibitory activity.

[0570] In summary, we achieved the first asymmetric synthesis of 9-epiTTX(1a) (22 steps) and one of the shortest syntheses of TTX(1) starting from readily available furfuryl alcohol (24 steps, following the step counting rule68,69). The 100-gram-scale asymmetric preparation of cyclohexane (+)-12 demonstrates the power of stereoselective Diels–Alder reactions in the scale-up of functionally dense carbocycles. The precise introduction of an oxygen functional group at the C-5 position by photochemical decarboxylative hydroxylation 70 highlights the advanced nature of radical transformations on stereo-demanding carbocyclic backbones. The sequential reactions of SmI2-mediated reductive fragmentation, oxygen bridge opening, and ester reduction followed by a diastereoselective Upjohn dihydroxylation enabled the gram-scale synthesis of the highly oxidized intermediate (+)-19. The bridged tetrahydrofuran acetal setup simplified the final stage and facilitated the rapid one-pot formation of the cyclic guanidine hemiamine acetal and orthoester. Notably, the present synthetic approach serves as a testbed for precise manipulation of functional groups on densely functionalized and stereochemically complex frameworks and can be readily adapted to synthesize other polycyclic natural products with high oxygen content. This concise synthetic strategy is suitable for the production of TTX homologues or derivatives to support further pharmacological studies and can be used for large-scale synthesis of TTX for the development of analgesic drugs, especially 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 R1 independently represents a protecting group, preferably a silicon-containing protecting group such as TBDPS, Each R2 independently represents a protecting group, preferably a benzyl protecting group, such as p-methoxybenzyl (ie, PMB), each R3 independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc., Each R4 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 R1 independently represents a protecting group, preferably a silicon-containing protecting group such as TBDPS, Each R2 independently represents a protecting group, preferably a benzyl protecting group, such as p-methoxybenzyl (ie, PMB), each R3 independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc., Each R4 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.

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 R1 independently represents a protecting group, preferably a silicon-containing protecting group such as TBDPS, Each R2 independently represents a protecting group, preferably a benzyl protecting group, such as p-methoxybenzyl (ie, PMB), Each R3 independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc., Each R4 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 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).

15. The method according to any one of claims 1 to 14, wherein In step (a), the reagent used includes quinine and MeOH, and the solvent used includes CCl4 and / or toluene (preferably a mixture of CCl4 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 OsO4, 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)3Cl2, 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 K2CO3, and / or the solvent used comprises an alcohol, preferably a C1-C4 alcohol; and / or In step (e), the reagent used includes I2, imidazole and PPh3, and / or the solvent used includes toluene; and / or In step (f), the reagent used comprises SmI2, and preferably also comprises a base, preferably an organic base, such as Et3N, and / or the solvent used comprises an ether (preferably THF); and / or In step (f1), the reagent used comprises SmI2, and / or the solvent used comprises ether (preferably THF); In step (f2), the reagent used comprises LiAlH4, 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, PPh3 and diethyl diazene dicarboxylate; and / or the solvent used includes THF; and / or In step (j1), the reagent used comprises 4-methylmorpholine N-oxide, and / or an oxidizing agent, preferably OsO4, and / or the solvent used comprises acetone; and / or In step (j2), the reagent used includes 2,2-dimethoxypropane and camphorsulfonic acid, and / or the solvent used includes CH2Cl2; and / or In step (k), the reagent used includes Dess-Martin reagent, preferably also includes an inorganic base, preferably NaHCO3; 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 MnO2, and a reducing agent, preferably NaBH4; 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 RuCl3, an oxidant, preferably NaIO4, 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 Et3N; 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 R2 independently represents a protecting group, preferably a benzyl protecting group, preferably p-methoxybenzyl (ie, PMB), each R3 independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc., and Each R4 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 R1 independently represents a protecting group, preferably a silicon-containing protecting group such as TBDPS, Each R2 independently represents a protecting group, preferably a benzyl protecting group, such as p-methoxybenzyl (ie, PMB), each R3 independently represents a C1-C5 alkyl group, preferably selected from methyl, ethyl, propyl, etc., Each R4 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; 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.

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