Method for preparing halichondrin b, and use thereof

Abstract

Provided are a method for preparing halichondrin B, and a use thereof. Specifically, the method comprises the steps of preparing a compound as shown in formula C-e, or a salt thereof. A corresponding process can effectively reduce production costs, and is applicable to industrial production.

Description

A method for preparing soft sponge B and its application Technical Field

[0001] This disclosure belongs to the field of medicinal chemistry and relates to a method for preparing soft sponge B and its application. Background Technology

[0002] Halichondrin B is a natural product with antitumor activity, originally isolated from the marine sponge, the black soft sponge. Eribulin, the first macrocyclic ketone analog derived from halichondrin B through structural optimization, is used to treat metastatic breast cancer.

[0003] Eribulin derivatives have also been reported; for example, compound E-305 is disclosed in Bioorganic & Medicinal Chemistry Letters 14 (2004) 5551–5554.

[0004] WO2021148003A reports a C-35 methylated eribulin derivative that exhibits superior cell-killing effects in vitro.

[0005] WO2023061466 discloses a method for preparing a C-35 methylated eribulin derivative, which obtains C-35 methyl eribulin by remethylating the protected amino group.

[0006] This process uses eribulin as a substrate, and the cost of eribulin is approximately 3.8 w / g. The cost of obtaining the target product using this process would not be lower than 3.8 w / g, which is extremely high for drugs entering clinical development. To effectively reduce costs, this disclosure provides a new synthetic process. Summary of the Invention

[0007] This disclosure provides a method for preparing a compound of the formula Ce or a salt thereof.

[0008] This includes the conversion of a compound of formula Ca or a salt thereof into a compound of formula Cb or a salt thereof, and the reaction of a compound of formula Cb or a salt thereof with a methylating agent to form a compound of formula Cc or a salt thereof.

[0009] Among them, R 1 Selected from C 1-3 Alkyl or benzyl, R 2 Selected from phenyl, wherein the phenyl group is optionally substituted with one or more halogens, methyl or nitro groups, P 1Selected from hydroxyl protecting groups, such as tert-butyldimethylsilyl or triisopropylsilyl.

[0010] In some embodiments, the methylating agent in the method is selected from iodomethane, dimethyl sulfate, or trimethyloxonium tetrafluoroborate.

[0011] In some embodiments, the methylating agent in the method is selected from trimethyloxonium tetrafluoroborate.

[0012] In some embodiments, the molar ratio of the compound represented by formula Cb or its salt to the methylating agent is 1:0.8 to 1:5, for example, 1:1 to 1:3, including but not limited to 1:0.8, 1:0.9, 1:1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, and 1:4.

[0013] In some embodiments, the molar ratio of the compound of formula Cb or its salt to a methylating agent such as trimethyloxonium tetrafluoroborate is 1:3.2.

[0014] In some embodiments, the compound represented by formula Cb or its salt reacts with the methylating agent at a temperature of -50 to 50°C, for example, 5°C to 25°C.

[0015] In some embodiments, the method for preparing the compound of formula Ce or a salt thereof further includes the step of oxidative cleavage of the compound of formula Cc or a salt thereof to an olefin to form the compound of formula Ce or a salt thereof.

[0016] In some implementations, the olefin oxidative cracking conditions include, but are not limited to, ozone oxidation reactions.

[0017] In some embodiments, the method for preparing the compound of formula Ce or a salt thereof includes the step of converting the compound of formula Cc or a salt thereof into the compound of formula Cd or a salt thereof.

[0018] Where R 1 R 2 and P 1 As defined above.

[0019] The olefin dihydroxylation reaction disclosed herein can be performed using the potassium permanganate method, osmium tetroxide oxidation or potassium osmium tetroxide method, Prévost reaction, Woodward reaction or Plietker dihydroxylation reaction. Specific operational procedures can be found in Strategic Applications of Named Reactions in Organic Synthesis, Elsevier Inc., 2005, and the relevant content is incorporated herein for illustration.

[0020] In some embodiments, the compound of formula Cc or a salt thereof is formed under osmium tetroxide conditions to form the compound of formula Cd or a salt thereof.

[0021] In some embodiments, the compound of formula Cc or its salt is formed under potassium permanganate conditions to form the compound of formula Cd or its salt.

[0022] In some embodiments, the compound of formula Cc or a salt thereof is formed under potassium osmium tetroxide conditions to form the compound of formula Cd or a salt thereof.

[0023] In some embodiments, the compound of formula Cc or a salt thereof is formed under potassium osmium tetroxide / N-methylmorpholine-N-oxide (NMO) conditions to form the compound of formula Cd or a salt thereof. In some embodiments, the compound of formula Cc or a salt thereof is formed under RuCl3 or RuO4 conditions to form the compound of formula Cd or a salt thereof.

[0024] Furthermore, the compound represented by formula Cd or its salt is oxidized to an aldehyde, for example, by oxidative cleavage using periodates such as NaIO4.

[0025] In some embodiments, the compound of formula Cd or its salt is reacted under NaIO4 conditions to form the compound of formula Ce or its salt.

[0026] On the other hand, the compound represented by formula Ce or its salt is the compound represented by formula C-e1 or its salt.

[0027] Where R 1 R 2 and P 1 As defined above.

[0028] In some embodiments, a method for preparing a compound of formula C-e1 or a salt thereof includes converting a compound of formula C-a1 or a salt thereof into a compound of formula C-b1 or a salt thereof, and reacting a compound of formula C-b1 or a salt thereof with a methylating agent to form a compound of formula C-c1 or a salt thereof, wherein the methylating agent is selected from iodomethane, dimethyl sulfate, or trimethyloxonium tetrafluoroborate.

[0029] Where R 1 R 2 and P 1 As defined above.

[0030] In some embodiments, the compound represented by formula Ce or a salt thereof is a compound represented by formula C-e1-a or a salt thereof. Where P 1 As defined above, for example, triisopropylsilyl.

[0031] In some embodiments, a method for preparing a compound of formula C-e1-a or a salt thereof includes converting a compound of formula C-a1-a or a salt thereof into a compound of formula C-b1-a or a salt thereof, and reacting a compound of formula C-b1-a or a salt thereof with a methylating agent to form a compound of formula C-c1-a or a salt thereof.

[0032] Where P 1 As defined above, for example, triisopropylsilyl.

[0033] In some embodiments, the compound represented by formula Ce or a salt thereof is the compound represented by formula C-e1-b or a salt thereof. Where P 1 As defined above, for example, triisopropylsilyl.

[0034] In some embodiments, a method for preparing a compound of formula C-e1-b or a salt thereof includes converting a compound of formula C-a1-b or a salt thereof into a compound of formula C-b1-b or a salt thereof, and reacting a compound of formula C-b1-b or a salt thereof with a methylating agent to form a compound of formula C-c1-b or a salt thereof.

[0035] Where P 1 As defined above, for example, triisopropylsilyl.

[0036] In some embodiments, a compound of formula Ca or a salt thereof reacts with a hydroxyl protecting agent to form a compound of formula Cb or a salt thereof, wherein the hydroxyl protecting group is selected from silane hydroxyl protecting groups, including but not limited to tert-butyldimethylchlorosilane and triisopropylchlorosilane.

[0037] In some embodiments, a compound of formula Ca or a salt thereof reacts with a hydroxyl protecting agent under basic conditions to form a compound of formula Cb or a salt thereof, wherein the base includes, but is not limited to, triethylamine, isopropylamine, imidazole, or pyridine. Specific procedures are described in Greene's Protective Groups in Organic Synthesis, Wiley-Interscience, 4th edition, 2006, and the relevant content is incorporated herein for illustrative purposes.

[0038] In other embodiments, the method for preparing the compound of formula Ce or a salt thereof includes converting the compound of formula Ca or a salt thereof into the compound of formula Cb or a salt thereof, reacting the compound of formula Cb or a salt thereof with a methylating agent to form the compound of formula Cc or a salt thereof, and subjecting the compound of formula Cc or a salt thereof to olefin oxidative cleavage to form the compound of formula Ce or a salt thereof.

[0039] In other embodiments, the method for preparing the compound of formula Ce or a salt thereof includes converting the compound of formula Ca or a salt thereof into the compound of formula Cb or a salt thereof, reacting the compound of formula Cb or a salt thereof with a methylating agent to form the compound of formula Cc or a salt thereof, subjecting the compound of formula Cc or a salt thereof to an olefin dihydroxylation reaction to form the compound of formula Cd or a salt thereof, and oxidizing the compound of formula Cd or a salt thereof to form the compound of formula Ce or a salt thereof.

[0040] In other embodiments, the aforementioned preparation method includes a) converting the compound of formula C-a1 or a salt thereof into the compound of formula C-b1 or a salt thereof; b) reacting the compound of formula C-b1 or a salt thereof with a methylating agent to form the compound of formula C-c1 or a salt thereof; and c) subjecting the compound of formula C-c1 or a salt thereof to olefin oxidative cleavage to form the compound of formula C-e1 or a salt thereof.

[0041] In some embodiments, the compound or its salt represented by formula C-c1 in step c) of the aforementioned preparation method reacts under ozone conditions to form the compound or its salt represented by formula C-e1.

[0042] In other embodiments, the preparation method includes a) converting the compound of formula C-a1 or a salt thereof into the compound of formula C-b1 or a salt thereof; b) reacting the compound of formula C-b1 or a salt thereof with a methylating agent to form the compound of formula C-c1 or a salt thereof; c) subjecting the compound of formula C-c1 or a salt thereof to an olefinic dihydroxylation reaction to form the compound of formula C-d1 or a salt thereof; and d) oxidizing the compound of formula C-d1 or a salt thereof to form the compound of formula C-e1 or a salt thereof.

[0043] In some embodiments, the compound or its salt represented by formula C-c1 in step c) of the aforementioned preparation method is subjected to osmium tetroxide conditions to form the compound or its salt represented by formula C-d1.

[0044] In some embodiments, the compound or its salt represented by formula C-c1 in step c) of the aforementioned preparation method is subjected to potassium osmium tetroxide conditions to form the compound or its salt represented by formula C-d1.

[0045] In some embodiments, the compound or its salt represented by formula C-c1 in step c) of the aforementioned preparation method is subjected to potassium permanganate conditions to form the compound or its salt represented by formula C-d1.

[0046] In some embodiments, the compound or its salt represented by formula C-c1 in step c) of the aforementioned preparation method is formed under RuCl3 or RuO4 conditions to form the compound or its salt represented by formula C-d1.

[0047] In some embodiments, the compound or its salt represented by formula C-d1 in step d) of the aforementioned preparation method is formed by reacting it with periodate such as NaIO4 to form the compound or its salt represented by formula C-e1.

[0048] In other embodiments, the preparation method includes converting a compound of formula C-a1-a or a salt thereof into a compound of formula C-b1-a or a salt thereof, reacting a compound of formula C-b1-a or a salt thereof with a methylating agent to form a compound of formula C-c1-a or a salt thereof, subjecting a compound of formula C-c1-a or a salt thereof to an olefinic dihydroxylation reaction to form a compound of formula C-d1-a or a salt thereof, and oxidizing a compound of formula C-d1-a or a salt thereof to form a compound of formula C-e1-a or a salt thereof.

[0049] In other embodiments, the preparation method includes converting the compound of formula C-a1-b or a salt thereof into the compound of formula C-b1-b or a salt thereof, reacting the compound of formula C-b1-b or a salt thereof with a methylating agent to form the compound of formula C-c1-b or a salt thereof, subjecting the compound of formula C-c1-b or a salt thereof to an olefin dihydroxylation reaction to form the compound of formula C-d1-b or a salt thereof, and oxidizing the compound of formula C-d1-b or a salt thereof to form the compound of formula C-e1-b or a salt thereof.

[0050] In some embodiments, the compound represented by formula Ce or a salt thereof is the compound represented by formula C-e1-b1 or a salt thereof.

[0051] In some embodiments, the compound represented by formula Ce or a salt thereof is the compound represented by formula C-e1-a1 or a salt thereof.

[0052] In some embodiments, a method for preparing the compound shown in C-e1-b1 or a salt thereof includes the following steps:

[0053] In step 1), compound C-a1-a1 or its salt reacts with triisopropylchlorosilane under imidazole conditions to form compound C-b1-a1 or its salt.

[0054] Step 2) Compound C-b1-a1 or its salt reacts with trimethyloxonium tetrafluoroborate to form compound C-c1-a1 or its salt.

[0055] Step 3) Compound C-c1-a1 or its salt reacts under potassium osmium tetroxide / NMO conditions to form compound C-d1-a1 or its salt.

[0056] Step 4) Compound C-d1-a1 or its salt reacts under sodium periodate conditions to form compound C-e1-a1 or its salt.

[0057] In some embodiments, a method for preparing the compound shown in C-e1-b1 or a salt thereof includes the following steps:

[0058] In step 1), compound C-a1-a1 or its salt reacts with triisopropylchlorosilane under imidazole conditions to form compound C-b1-a1 or its salt.

[0059] Step 2) Compound C-b1-a1 or its salt reacts with trimethyloxonium tetrafluoroborate to form compound C-c1-a1 or its salt.

[0060] Step 3) Compound C-c1-a1 or its salt reacts under potassium osmium tetroxide / NMO conditions to form compound C-d1-a1 or its salt.

[0061] Step 4) Compound C-d1-a1 or its salt reacts under sodium periodate conditions to form compound C-e1-a1 or its salt.

[0062] This disclosure also provides a method for preparing the compound of formula E3-23a or a salt thereof, comprising the steps of the aforementioned method for preparing the compound of formula Ce or a salt thereof.

[0063] Where R 2 Selected from phenyl, wherein the phenyl group is optionally substituted with one or more halogens, methyl or nitro groups, P 1 Selected from hydroxyl protecting groups, such as tert-butyldimethylsilyl or triisopropylsilyl.

[0064] In some embodiments, the method for preparing the compound shown in E3-23a or a salt thereof further includes the step of reacting the compound shown in C-e1 or a salt thereof with the compound shown in Ba or a salt thereof to form the compound shown in E3-21a or a salt thereof.

[0065] Where P 2 Selected from hydroxyl protecting groups, such as tert-butyldimethylsilyl or piv (trimethylacetyl).

[0066] In some embodiments, the compound represented by formula C-e1 or a salt thereof is coupled with the compound represented by formula Ba to form the compound represented by formula E3-21a or a salt thereof, and the coupling reaction is carried out using a Ni / Cr catalyst. The coupling reaction is described in US621486865, the relevant content of which is incorporated herein by reference.

[0067] In other embodiments, a method for preparing the compound shown in E3-23a or a salt thereof includes forming a compound of formula E3-22a or a salt thereof under alkaline conditions from E3-21a, and deprotecting the compound of formula E3-22a to form the compound of formula E3-23a or a salt thereof.

[0068] The preparation methods for compounds similar to those shown in Formula Ba are described in WO2005 / 118565, J.AM.Chem.Soc.,2009,131,15387, J.AM.Chem.Soc.,2009,131,15636, Org.Lett.,2002,4(25),4435, and the relevant content is incorporated into the text for illustration.

[0069] In some embodiments, the compound represented by formula Ba is the compound represented by formula B-a1.

[0070] In other embodiments, the compound of formula E3-23a or a salt thereof is the compound of formula E3-23a1 or a salt thereof.

[0071] Where P 1 It is a hydroxyl protecting group, such as tert-butyldimethylsilyl or triisopropylsilyl.

[0072] In some embodiments, the compound of formula E3-23a or a salt thereof is of formula E3-23b or the compound of formula E3-23 or a salt thereof.

[0073] In some embodiments, the method for preparing the compound shown in E3-23 or a salt thereof further includes the step of reacting the compound shown in C-e1 or a salt thereof with the compound shown in Ba or a salt thereof to form the compound shown in E3-21a or a salt thereof.

[0074] Where P 2 Selected from hydroxyl protecting groups, such as tert-butyldimethylsilyl or tert-valeryl.

[0075] In some implementations, the reaction conditions for step 5) are (s)-ligands. CrCl2, NET3, and NiCl2. The relevant reaction procedures can be found in CN105713031B, and the relevant content is incorporated into this document for illustrative purposes.

[0076] In some implementations, the reaction conditions for step 5) are: (S)-Me-Ligand, CrCl2, NET3, and NiCl2. Related reaction procedures can be found in CN105713031B, and the relevant content is incorporated herein for illustration. In some embodiments, the method for preparing the compound shown in E3-23 or a salt thereof includes reacting formula E3-21 to form the compound shown in E3-22 or a salt thereof, and deprotecting the compound shown in E3-22 to form the compound shown in E3-23 or a salt thereof.

[0077] In some embodiments, the reaction conditions for step 6) are potassium bis(trimethylsilyl)amino (KHMDS). In some embodiments, the solvent used for the reaction in step 6) includes, but is not limited to, benzene, toluene, or tetrahydrofuran.

[0078] In some implementations, the reaction conditions for step 6) are KHMDS / toluene.

[0079] In some embodiments, the reaction conditions for step 7) are DIBAL-H. In some embodiments, the solvent used for the reaction in step 7) includes, but is not limited to, benzene, toluene, or tetrahydrofuran.

[0080] In some implementations, the reaction conditions for step 6) are DIBAL-H / toluene.

[0081] On the other hand, this disclosure also provides a method for preparing the compound of formula I or a salt thereof, which includes the aforementioned steps for preparing the compound of formula Ce or a salt thereof, and / or the steps for preparing the compound of formula E3-23a or a salt thereof.

[0082] In some embodiments, the method for preparing the compound of formula I or a salt thereof includes the step of converting the compound of formula Ic or a salt thereof into the compound of formula I or a salt thereof.

[0083] For relevant reaction procedures, please refer to CN109694379, and the relevant content is included in the text for illustration.

[0084] In some embodiments, the compound of formula Ic or a salt thereof is amination reaction to generate compound of formula I, wherein the amination reagent is selected from ammonia or an organic solvent of ammonia.

[0085] In some embodiments, a method for preparing a compound of formula I or a salt thereof includes the step of reacting a compound of formula Ic or a salt thereof with an azide reagent to generate a compound of formula Id or a salt thereof, wherein the azide reagent includes, but is not limited to, sodium azide, potassium azide, lithium azide, and trimethylsilyl azide (TMSN3).

[0086] Where R 4 The sulfonyl group is preferably toluenesulfonyl, benzenesulfonyl, trifluoromethanesulfonyl, or methanesulfonyl.

[0087] Furthermore, the compound of formula Id undergoes a reduction reaction to form the compound of formula I or its salt, wherein the reduction reaction conditions are selected from Pd / C, H2.

[0088] In other embodiments, the compound of formula Id is reacted under organophosphorus conditions to form the compound of formula Id, wherein the organophosphorus reagent includes, but is not limited to, triphenylphosphine, tributylphosphine, and diethylphenylphosphine. In other embodiments, the solvent used in the aforementioned reduction reaction is selected from at least one of tetrahydrofuran, 2-methyltetrahydrofuran, acetonitrile, ethyl acetate, dichloromethane, chloroform, N,N-dimethylacetamide, and N,N-dimethylformamide.

[0089] In some embodiments, the compound of formula Ic or a salt thereof is a compound of formula I-c1 or a salt thereof.

[0090] In some embodiments, the compound represented by formula Ic or a salt thereof is a compound of formula I-c1-a or a salt thereof.

[0091] In some embodiments, the method for preparing the compound of formula I or a salt thereof includes the following steps:

[0092] In some embodiments, step 15) involves reacting the compound of formula ER-3005a or a salt thereof with sulfonyl anhydride or sulfonyl chloride to form the compound of formula Ic or a salt thereof.

[0093] In some embodiments, the aforementioned sulfonation reaction is carried out under organic or inorganic base conditions, wherein the base is selected from triethylamine. Related reaction procedures can be found in CN109694379, and the relevant content is incorporated herein by reference.

[0094] In some embodiments, the method for preparing the compound of formula I or a salt thereof includes the following steps:

[0095] In some embodiments, step 15) reacts the compound of formula ER-3005a or its salt with methanesulfonyl anhydride or methanesulfonyl chloride to form the compound of formula I-c1 or its salt; step 16) reacts the compound of formula I-c1 or its salt with tetrabutylammonium azide to form the compound of formula Id or its salt; and step 16) reacts the compound of formula Id or its salt under organophosphorus conditions such as triphenylphosphine to form the compound of formula I or its salt.

[0096] In other embodiments, the compound of formula I or a salt thereof is a compound of formula ER-300.

[0097] In some embodiments, the ER-300 compound is prepared via a corresponding chiral intermediate, for example using... The compound ER-300 was prepared by reduction reaction.

[0098] In other embodiments, the ER-300 compound is prepared via a non-chiral intermediate and then obtained by chiral separation. For example:

[0099] In some other embodiments, the method for preparing the compound of formula I or a salt thereof further includes the following steps:

[0100] In some other embodiments, the method for preparing the compound of formula I or a salt thereof further includes the following steps:

[0101] In some embodiments, step 8) is reacted under n-BuLi conditions; step 9) under Dess-Martin conditions; step 10) under SmI2 conditions; step 11) under CrCl2 or NiCl2 conditions; step 12) under Dess-Martin conditions; step 13) under imidazole hydrochloride or tetrabutylammonium fluoride conditions; and step 14) under p-toluenesulfonic acid pyridinium conditions. Related reaction procedures can be found in CN105713031B, and the relevant content is incorporated herein for illustrative purposes.

[0102] This disclosure also provides methods for preparing compounds of formula II or salts thereof, including the aforementioned methods for preparing compounds of formula Ce or salts thereof, and / or methods for preparing compounds of formula E3-23a or salts thereof, and / or methods for preparing compounds of formula I or salts thereof.

[0103] Where L is the connector.

[0104] In some embodiments, the cleavable portion in the linker is a cleavable peptide portion. In some embodiments, an ADC containing a cleavable peptide portion exhibits lower aggregation levels, improved antibody:drug ratios, increased targeted killing of cancer cells, reduced off-target killing of non-cancer cells, and / or higher drug loading compared to an ADC containing an alternative cleavable portion. In some embodiments, adding a cleavable portion increases cytotoxicity and / or potency compared to a non-cleavable linker. In some embodiments, the increased potency and / or cytotoxicity is in cancers expressing moderate levels of antigens targeted by the antibody portion of the ADC (e.g., moderate FRA expression). In some embodiments, the cleavable peptide portion is enzymatically cleavable, and the linker is an enzyme-cleavable linker. In some embodiments, the enzyme is a cathepsin, and the linker is a cathepsin-cleavable linker. In some embodiments, an enzyme-cleavable linker (e.g., a cathepsin-cleavable linker) exhibits one or more of the aforementioned improved properties compared to an alternative cleavage mechanism.

[0105] In some embodiments, the linker L in the compound of Formula II or its salt comprises an amino acid unit selected from peptide residues consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid.

[0106] In some embodiments, L in the compound of Formula II or its salt comprises valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lys (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe), or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0107] In some implementations, the connector L includes:

[0108] Furthermore, the linker L also includes a spacer unit, such as a p-aminobenzoxycarbonyl (PAB) group. In other embodiments, the spacing unit comprises a subset of:

[0109] -(CH2)3-C(O)-, -CH2-O-CH2-C(O)-, -(CH2)2-O-CH2-C(O)-,

[0110] In other embodiments, the connector L is

[0111] On the other hand, this disclosure also provides a method for preparing the antibody-drug conjugate of Formula III, including the aforementioned methods for preparing the compound of Formula Ce or its salt, and / or the methods for preparing the compound of Formula E3-23a or its salt, and / or the methods for preparing the compound of Formula I or its salt, and / or the methods for preparing the compound of Formula II or its salt.

[0112] Where L is the linker, Ab is the antibody, and k is 1 to 20.

[0113] In some embodiments, the antibody-drug conjugate is:

[0114] Where Ab is the antibody and k is 1 to 20.

[0115] In some embodiments, the antibody is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-CD79 antibody, anti-TROP-2 antibody, anti-CD79B antibody, anti-Mesothelin antibody, and anti-folate receptor α (FRA) antibody.

[0116] In some embodiments, the antibody in the antibody-drug conjugate (ADC) is a known antibody selected from, but not limited to, trastuzumab, pertuzumab, nimotuzumab, enoblituzumab, emibetuzumab, Inotuzumab, vitine-pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96, farletuzumab, and glematumamab.

[0117] This disclosure also provides compounds or salts thereof, selected from...

[0118] Where R 1 Selected from C 1-3 Alkyl or benzyl, R 2 Selected from phenyl, wherein the phenyl group is optionally substituted with one or more halogens, methyl or nitro groups, P 1 Selected from hydroxyl protecting groups, such as tert-butyldimethylsilyl, triisopropylsilyl; P 2 Selected from ester protecting groups, such as trimethylacetyl (Piv, pivaloyl).

[0119] On the other hand, this disclosure also provides the following compounds:

[0120] Where P 1 Selected from hydroxyl protecting groups, such as tert-butyldimethylsilyl, triisopropylsilyl; R 2 Selected from phenyl, wherein the phenyl group is optionally substituted with one or more halogens, methyl groups, or nitro groups.

[0121] This disclosure also provides the use of the aforementioned compounds or salts thereof in methods for preparing compounds or salts thereof of Formula I.

[0122] This disclosure also provides the use of the aforementioned compounds or salts thereof in methods for preparing compounds or salts thereof of Formula II.

[0123] Where L is the connector.

[0124] This disclosure also provides the use of the aforementioned compounds or salts thereof in methods for preparing compounds or salts thereof of Formula III.

[0125] Where L is the linker, Ab is the antibody, and k is 1 to 20.

[0126] the term:

[0127] Unless otherwise stated, the terms used in the specification and claims shall have the following meanings.

[0128] The term "transformation" in this disclosure does not specifically refer to a single-step transformation reaction between two substrates; it can be a single-step or multi-step reaction between two substrates.

[0129] The term "amino acid" refers to an organic compound whose molecular structure contains both an amino group and a carboxyl group, with both groups directly attached to the -CH- structure. The general formula is H₂NCHRCOOH, where R is H, a substituted or unsubstituted alkyl group, etc. Based on the position of the amino group attached to the carbon atom in the carboxylic acid, amino acids can be classified into α, β, γ, δ, ε…-amino acids. In the biological world, the amino acids that constitute natural proteins have specific structural characteristics, namely, their amino groups are directly attached to the α-carbon atom, i.e., α-amino acids, including glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, and proline. Non-natural amino acids, such as citrulline, are also present. As is known to those skilled in the art, non-natural amino acids do not constitute natural proteins and therefore do not participate in the synthesis of the antibodies disclosed herein.

[0130] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain and branched groups with 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, etc. Alkyl groups can be substituted or unsubstituted.

[0131] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0132] "Nitro" refers to -NO2.

[0133] The “hydroxyl protecting group” disclosed herein is a group known in the art that can be used to protect hydroxyl groups, see the hydroxyl protecting groups in the literature (Protective Groups in Organic Synthesis, 5th Ed. TW Greene & P. ​​GMWuts). As examples, including but not limited to tert-butyldimethylsilyl and triisopropylsilyl.

[0134] When the functional group disclosed herein is substituted, the substituent is preferably one or more of the following groups: halogen, methyl, or nitro.

[0135] "Sulfoyl" refers to -S(O)2R, where R is alkyl, alkenyl, aryl, arylalkyl, or silyl. In exemplary sulfonyl groups, R is C1-C12 alkyl (e.g., C1-C8, C1-C6, C1-C4, C2-C7, C3-C12, and C3-C6 alkyl), C2-C12 alkenyl (e.g., C2-C8, C2-C6, C2-C4, C3-C12, and C3-C6 alkenyl), or carbocyclic C6-C20 aryl (e.g., C6-C15, C6-C...). 10. C8-C20 and C8-C15 aryl groups, monocyclic C1-C6 heteroaryl groups (e.g., C1-C4 and C2-C6 heteroaryl groups), C4-C19 heteroaryl groups (e.g., C4-C10 heteroaryl groups), (C6-C15)aryl(C1-C6)alkyl, (C4-C19)heteroaryl(C1-C6)alkyl, or (C1-C6)heteroaryl(C1-C6)alkyl. As defined herein, any heteroaryl group present in the sulfonyl group has 1 to 4 heteroatoms independently selected from O, N, and S. Exemplary sulfonyl groups include toluenesulfonyl, trifluoromethanesulfonyl, and methanesulfonyl.

[0136] A "linker" is a chemical structural fragment that is linked at one end to a drug (toxin molecule) and at the other end to a ligand such as an antibody.

[0137] In the chemical structure of the compounds described in this disclosure, the bonds... This indicates that the configuration is not specified; that is, if chiral isomers exist in the chemical structure, the bond... It can be Or simultaneously include Two configurations. For example... include

[0138] "Optional" or "optional" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of the event or environment. For example, "optionally halogenated phenyl" means that a halogen may but does not have to be present, and the description includes cases where the phenyl is halogenated and cases where the phenyl is not halogenated.

[0139] The term "drug loading" refers to the average amount of drug loaded per antibody-drug conjugate molecule, or it can be expressed as the ratio of drug amount to antibody amount. The drug loading range can be 1-20, preferably 1-10, cytotoxic drugs (D) linked to each antibody (Ab). In embodiments of this disclosure, the drug loading is expressed as k, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or the average of any two values. Preferably 1-10, more preferably 1-8, or 2-8, or 2-7, or 3-8, or 3-7, or 3-6, or 4-7, or 4-6, or 4-5. The average amount of drug per ADC molecule after the conjugation reaction can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, monoclonal antibody molecular size variant assay (CE-SDS), and HPLC characterization.

[0140] The present invention discloses a method for determining the molecular size variant of monoclonal antibodies (CE-SDS), which uses sodium dodecyl sulfate capillary electrophoresis (CE-SDS) with ultraviolet detection. Under reducing and non-reducing conditions, the purity of recombinant monoclonal antibody products is quantitatively determined according to molecular weight using capillary electrophoresis (2015 edition of the Chinese Pharmacopoeia 0542).

[0141] The term "antibody" encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibodies can refer to immunoglobulins, which are tetrapeptide chains composed of two heavy chains and two light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, different subclasses can be distinguished; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into κ chains or λ chains based on differences in their constant regions. Each of the five types of Ig can have either a κ chain or a λ chain.

[0142] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this invention utilize genetic engineering methods to add one or more human FR regions to a non-human CDR region. Human FR germline sequences can be obtained by alignment with the IMGT Human Antibody Variable Region Germplasm Database and MOE software, such as from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr.

[0143] The values ​​in this disclosure are instrument measurements and are subject to a certain degree of error. Generally, ±10% is within the reasonable error range. Of course, the context in which the value is used must be considered. For example, in the case of reactant dosage, where the measured error variation does not exceed ±10%, the value can be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%, preferably ±5%.

[0144] Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents whose specific source is not specified are commercially available conventional reagents.

[0145] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) are given in units of 10⁻⁶ (ppm).

[0146] The NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer, with deuterated chloroform (CDCl3) as the solvent.

[0147] MS measurements were performed using a Waters Micromass Quattro micro API triple quadrupole mass spectrometer, in positive / negative ion mode, with a mass scan range of 120–1300.

[0148] HPLC column: YMC-Pack ODS-A (3μm, 4.6mm x 150mm)

[0149] The silica gel plates used for thin-layer chromatography are Yantai Huanghai HSGF254 silica gel plates. The silica gel plates used in thin-layer chromatography (TLC) have a size of 0.2mm ± 0.03mm, and the size used for thin-layer chromatography separation and purification of products is 0.4mm-0.5mm. Detailed Implementation

[0150] The present disclosure will be explained in more detail below with reference to the embodiments. The embodiments of the present disclosure are only used to illustrate the technical solutions of the present disclosure, and the nature and scope of the present disclosure are not limited thereto.

[0151] Example 1: Preparation of compound C-e1-a1

[0152] Step 1:

[0153] Compound C-a1-a1 (11.82 g, 30.78 mmol, prepared by a known method, “Tetrahedron Letters, 2013, vol. 54, #51, p. 7059–7061”) was dissolved in DMF (80 mL). Imidazole (6.3 g, 92.5 mmol) and triisopropylchlorosilane (7.12 g, 36.9 mmol) were added at room temperature, the mixture was purged with nitrogen three times, and then reacted at room temperature for 1 hour. After adding water (100 mL) and dichloromethane (100 mL), the mixture was separated. The aqueous phase was extracted with dichloromethane, the organic phases were combined, concentrated to dryness, and purified by column chromatography (PE:EA = 100:0-65:35) to give 15.05 g of compound C-b1-a1 (yield 91%).

[0154] MS(ESI): m / z 541.3 [M+1] + .

[0155] 1 H NMR (400MHz, CD3Cl): δ7.87-7.77(m,2H),7.39-7.37(m,2H),5.79-5.65(m,1H) ,5.02-4.96(m,2H),3.96-3.88(m,2H),3.81-3.78(m,1H),3.65-3.63(m,2H),3. 55-3.53(m,1H),3.41(s,3H),3.14-2.97(m,3H),2.49-2.47(m,4H),2.33-2.40 (m,2H),1.94-1.82(m,2H),1.73(s,1H),1.28-1.24(m,1H),1.15-1.04(m,19H).

[0156] Step 2:

[0157] Compound C-b1-a1 (15.0 g, 27.8 mmol) was dissolved in dichloromethane (200 mL), and 1,8-bis(dimethylaminonaphthalene) (21.7 g, 101.3 mmol) and trimethyloxonium tetrafluoroborate (13.6 g, 91.6 mmol) were added. The mixture was reacted at room temperature for 1 hour. The dichloromethane was removed by concentration below 30 °C, and methyl tert-butyl ether (200 mL) was added. The mixture was stirred at room temperature for 30 minutes, and the solid residue was removed by filtration. The filter cake was washed with methyl tert-butyl ether, and the filtrate was collected. 1 M dilute hydrochloric acid (100 mL) was added under ice-water bath. The mixture was separated, and the aqueous phase was extracted again with methyl tert-butyl ether. The organic phase was washed again with saturated brine, and the organic phase was collected. The organic phase was concentrated to dryness and purified by column chromatography (PE:EA = 100:0-75:25) to give 12.87 g of compound C-c1-a1 (yield 84%).

[0158] MS(ESI): m / z 555.2 [M+1] + .

[0159] 1 H NMR (400MHz, CD3Cl): δ7.81-7.79(m,2H),7.39-7.37(m,2H),5.71-5.65(m,1H),5.00-4.94(m,2H),3.85-3.69(m,4H),3.46 -3.36(m,8H),3.09-3.01(m,2H),2.47-2.29(m,6H),1.96-1.87(m,2H),1.66(s,1H),1.28-1.21(m,1H),1.09-1.05(m,19H).

[0160] Step 3:

[0161] Compound C-c1-a1 (12.75 g, 23.0 mmol) was dissolved in acetone (80 mL) and water (8 mL). A 50% (wt%) aqueous solution of N-methylmorpholine-N-oxide (10.8 g, 46.1 mmol) and potassium osmium tetroxide dihydrate (42.4 mg, 0.12 mmol) were slowly added, and the reaction was carried out at room temperature for 18 hours. The reaction was quenched by adding saturated sodium thiosulfate aqueous solution (80 mL) in an ice-water bath. The mixture was then heated to room temperature and stirred vigorously for 30 minutes. Ethyl acetate (200 mL) was added, the mixture was separated, the organic phase was collected, concentrated to dryness, and purified by column chromatography (PE:EA = 100:0-20:80) to give 12.06 g of compound C-d1-a1 (yield 89%).

[0162] MS(ESI): m / z 589.3 [M+1] + .

[0163] 1H NMR (400MHz, CD3Cl): δ7.81-7.79(m,2H),7.40-7.38(m,2H),3.94-3.74(m,5H),3.69-3.31(m,11H ),3.14-3.01(m,2H),2.59-2.35(m,5H),2.04-1.69(m,5H),1.28-1.24(m,1H),1.09-1.05(m,19H).

[0164] Step 4:

[0165] Compound C-d1-a1 (11.82 g, 20.1 mmol) was dissolved in dichloromethane (100 mL) and water (50 mL), followed by the addition of saturated sodium bicarbonate (5 mL) and sodium periodate (12.89 g, 60.3 mmol). The mixture was then reacted at room temperature for 18 hours. The reaction mixture was diluted with saturated sodium bicarbonate aqueous solution (50 mL) and ethyl acetate (100 mL) to separate the layers. The organic phase was collected, concentrated to dryness, and purified by column chromatography (PE:EA = 100:0-60:40) to give 11.12 g of compound C-e1-a1 (99% yield).

[0166] MS(ESI): m / z 557.3 [M+1] + .

[0167] 1 H NMR (400MHz, CD3Cl): δ9.70(s,1H),7.83-7.80(m,2H),7.40-7.38(m,2H),4.02-3.89(m,2H),3.83-3.66(m,3H),3.42-3.30 (m,8H),3.16-3.10(m,1H),2.91-2.74(m,2H),2.52-2.47(m,4H),2.04-1.75(m,3H),1.28-1.24(m,1H),1.13-1.02(m,19H).

[0168] Example 2 Preparation of Compound I

[0169] Step 1:

[0170] In a dry three-necked flask, compound (S)-Me-Ligand (13.32 g, 44.98 mmol, prepared by the well-known method "Organic Letters, 2002, vol. 4, #25, pp. 4431-4434"), anhydrous chromium dichloride (5.03 g, 40.9 mmol), anhydrous tetrahydrofuran (80 mL), and triethylamine (4.55 g, 44.98 mmol) were added. After the addition was complete, the mixture was heated to 32–35 °C and reacted for 2 h. Nickel dichloride (0.14 g, 1.06 mmol) was rapidly added to the reaction mixture under an ice-water bath. Then, a pre-prepared tetrahydrofuran solution (20 mL) of B-a1 (5.92 g, 10.63 mmol) and C-e1-a1 (4.55 g, 8.18 mmol) was added to the three-necked flask. After the addition was complete, the mixture was heated to room temperature and reacted for 18 h.

[0171] Ethylenediamine (7.4 g, 122.7 mmol) was added and stirred for 1 hour, precipitating a solid. Water (45 mL) and n-heptane (100 mL) were added, and the mixture was stirred and separated. The aqueous phase was then extracted with methyl tert-butyl ether. The combined organic phases were washed with an aqueous solution of sodium bicarbonate (7 g, sodium chloride 18.4 g, water 120 mL), and the aqueous phase was back-extracted with MEBT. The combined organic phases were dried over magnesium sulfate and concentrated to dryness. MTBE / PE was added at room temperature and the mixture was stirred (100 mL, V / V = 1 / 3). The mixture was filtered, and the filter cake was washed with MTBE / PE (V / V = 1 / 3). The filtrate was collected, concentrated, and dried to obtain 13.83 g of crude E3-21.

[0172] Step 2:

[0173] Compound E3-21 (13.83 g crude product, 8.18 mmol) was dissolved in anhydrous tetrahydrofuran (120 mL), cooled to -20 to -15 °C (internal temperature), and 0.5 M KHMDS in toluene solution (51 mL, 25.5 mmol) was slowly added dropwise. After the addition was complete, the mixture was kept warm and stirred for 10 minutes. The reaction was then reversed and added to a saturated ammonium chloride aqueous solution (50 mL), followed by washing and separation with methyl tert-butyl ether (150 mL). The organic phase was washed with 30 mL of saturated brine, and the aqueous phase was back-extracted with MTBE. The organic phases were combined, dried, filtered, and concentrated to obtain 13.5 g of crude product. The crude product was purified by column chromatography (PE:EA = 100:0-80:20) to obtain 4.66 g of the target compound. The 4.66 g product was then purified again by C18 column chromatography (water:methanol = 90:10-2:98) to obtain a total of 3.85 g of E3-22 (two-step yield 53%).

[0174] 1H NMR (400MHz, CDCl3): δ7.84(d,2H),7.40(d,2H),4.91-4.65(m,4H),4.25(br s,1H),4.09-4.04(m,2H),3.99-3.93(m,1H),3.84(d,1H),3.81-3.76(m,2H), 3.73-3.65(m,2H),3.55-3.52(m,1H),3.44(s,6H),3.40-3.34(m,2H),3.11-2 .99(m,2H),2.65-2.60(m,1H),2.54-2.50(m,1H),2.46(s,3H),2.24-2.11(m, 3H),2.04-1.83(m,3H),1.76-1.26(m,13H),1.19(s,9H),1.09-1.04(m,21H).

[0175] Step 3:

[0176] Compound E3-22 (5.96 g, 6.69 mmol) was dissolved in 40 mL of toluene dried with molecular sieves. The solution was stirred until dissolved, and the temperature was lowered to -65 °C (internal temperature). DIBAL-H (1.5 M in toluene, 9.4 mL, 14.1 mmol) was slowly added dropwise. After the addition was complete, the reaction was maintained at ≤-65 °C for 0.5 h. The reaction was quenched by adding methanol (0.4 mL). The temperature was raised to -20 to -30 °C, and 50 mL of saturated sodium potassium tartrate aqueous solution was added. The mixture was stirred until the organic phase was clear. Methyl tert-butyl ether (80 mL) was added, and the mixture was separated. The solution was washed with saturated brine, and the aqueous phase was back-extracted with MTBE. The organic phases were combined, dried, filtered, and concentrated to obtain 5.45 g of crude product. Column purification (PE:EA = 100:0-50:50) yielded 4.96 g of compound E3-23 (yield 91%).

[0177] 1H NMR (400MHz, CDCl3): δ7.84(d,2H),7.40(d,2H),4.91-4.65(m,4H),4.31(br s,1H),4.01-3.95(m,1H),3.84-3.83(m,1H),3.81-3.72(m,4H),3.70-3.62 (m,3H),3.54-3.51(m,1H),3.44(s,6H),3.40-3.34(m,2H),3.11-2.99(m,2H ),2.65-2.60(m,1H),2.54-2.50(m,2H),2.46(s,3H),2.27-2.13(m,3H),2. 04-1.83(m,3H),1.76-1.72(m,1H),1.68-1.26(m,11H),1.15-1.01(m,21H).

[0178] Step 4:

[0179] Compound E3-23 (2.08 g, 2.58 mmol) was dissolved in anhydrous THF (20 mL) and transferred to a pre-dried 250 mL three-necked flask. Under argon protection, the mixture was cooled to 0–5 °C, and n-butyllithium (3.39 mL, 5.42 mmol) was slowly added dropwise over 20 min. The mixture was kept at this temperature for half an hour, then cooled to -55–-65 °C. A solution of n-heptane (26 mL) of A-18 (2.62 g, 3.54 mmol, prepared by the known method “Synlett, 2013, vol. 24, #3, p. 333-337”) was slowly added dropwise over approximately 40 min. The mixture was kept at this temperature for 2 h. Add 10 mL of saturated ammonium chloride aqueous solution, 10 mL of water, and 20 mL of methyl tert-butyl ether to the reaction solution to quench the reaction. Stir and separate the liquids. Wash with saturated brine. After back-extracting the aqueous phase with MTBE, combine the organic phases, dry, filter, and concentrate to obtain an oily substance. Add 100 mL of n-heptane and 20 mL of acetonitrile to dissolve the oily substance, then cool to -30 to -20 °C (internal temperature). Stir and separate the liquids. Wash the n-heptane phase with 20 mL of acetonitrile. Combine the acetonitrile phases and back-extract with 20 mL of n-heptane. Combine the n-heptane phases and concentrate to remove the solvent, obtaining 4.80 g of crude E3-24 product. The crude product is directly added to the next reaction.

[0180] Step 5:

[0181] Compound E3-24 (4.79 g crude product, 2.58 mmol) was dissolved in 48 mL of toluene dried on molecular sieves. The solution was stirred until dissolved, cooled in an ice-water bath, and water (2.3 mg, 0.13 mmol) was added. Dess-Martin oxidant (3.28 g, 7.7 mmol) was added, and the mixture was allowed to react at room temperature for 1 h. Under ice-water bath conditions, 48 ​​mL each of 5% NaHCO3 and 20% Na2SO3 were added. After addition, the mixture was stirred at room temperature for 5 min to separate the phases. The aqueous phase was extracted with methyl tert-butyl ether (48 mL). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain crude E3-25. Column purification (PE:EA = 100:0-80:20) yielded 3.22 g of compound E3-25 (two-step yield 81%).

[0182] 1 H NMR (400MHz, CDCl3): δ9.78(s,1H),7.92-7.67(m,2H),7.37(d,2H),6.87-6.80(m,1H),6.27(d,1H), 5.01-4.65(m,5H),4.36-4.30(m,2H),4.20-4.02(m,3H),3.89-3.83(m,2H),3.78-3.60(m,5H),3.57- 3.51(m,2H),3.46-3.35(m,8H),2.92-2.79(m,2H),2.74-2.43(m,7H),2.36-2.16(m,2H),2.12-1.56( m,20H),1.12-1.02(m,24H),0.95-0.91(m,15H),0.89(s,9H),0.13-0.08(m,12H),0.02-0.00(m,6H).

[0183] Step 6:

[0184] Add 50 mL of SmI2 solution (0.1 M tetrahydrofuran solution, 5 mmol) to a pre-dried 1 L three-necked flask, under argon protection, and cool to below -65 °C. Dissolve E3-25 (3.1 g, 2.0 mmol) in 20 mL of pre-degassed THF and 14 mL of pre-degassed MeOH, and slowly add this solution dropwise to the 1 L three-necked flask. After the addition is complete, maintain the reaction temperature below -65 °C for 1 h. Maintain the internal temperature at -55 to -50 °C, and quench the reaction by adding a pre-prepared potassium carbonate-sodium tartrate solution (20 g potassium carbonate + 20 g sodium potassium tartrate + 200 g water) dropwise to the 1 L three-necked flask (the quenching process should be carried out with the flask open), while simultaneously slowly adding 100 mL of methyl tert-butyl ether. The mixture was heated to 10–20 °C and stirred for 1 h. After separation, the liquid was washed successively with potassium carbonate-sodium potassium tartrate solution (50 mL) and saturated saline solution (20 mL). The organic phases were combined, dried, filtered, and concentrated to obtain the crude product. Column purification (PE:EA = 100:0-83:17) yielded 2.43 g of compound E3-26 (yield 87%).

[0185] 1 H NMR (400MHz, CDCl3): δ9.68(s,1H),6.75(dd,1H),6.18(d,1H),4.88(d,1H),4.79-4.75(m,3H),4.68(s,1 H),4.26-4.20(m,1H),3.98-3.90(m,2H),3.79-3.55(m,8H),3.41-3.32(m,5H),3.28-3.23(m,4H),3.15(d ,1H),2.83(dd,1H),2.65-2.55(m,2H),2.52-2.28(m,6H),2.19-2.10(m,2H),2.03-1.97(m,1H),1.94-1. 15(m,16H),1.02-0.93(m,24H),0.84-0.82(m,15H),0.75(s,9H),0.04-0.00(m,12H),-0.09--0.1(m,6H).

[0186] Step 7:

[0187] In a pre-dried 500 mL three-necked flask, add (S)-Me-Ligand (6.22 g, 20.91 mmol). Under argon protection, add chromium chloride (2.58 g, 20.91 mmol), pre-degassed anhydrous acetonitrile (70 mL), and triethylamine (2.12 g, 20.91 mmol). After the addition is complete, raise the temperature to 32–35 °C (internal temperature) and stir for 1.5 h. Under ice bath conditions, rapidly add nickel chloride (0.23 g, 1.74 mmol). Dissolve E3-26 (2.42 g, 1.74 mmol) in a mixture of tetrahydrofuran (25 mL) and acetonitrile (12.5 mL) (tetrahydrofuran and acetonitrile need to be pre-degassed). Add this solution dropwise to the 500 mL three-necked flask over approximately 2 h. Maintain the temperature (20–25 °C) during the dropwise addition and continue the reaction for 18 h. Add 200 mL of n-heptane to the reaction solution, stir for 30 min, filter through a diatomaceous earth filter, wash the filter cake with n-heptane / acetonitrile = (50 mL / 50 mL) × 2, separate the liquids, combine the n-heptane phases, concentrate to remove solvent and obtain 2.5 g of crude E3-27.

[0188] Step 8:

[0189] Crude E3-27 (2.5 g, 1.74 mmol) was dissolved in 25 mL of dried DCM. Purified water (1.6 mg, 0.09 mmol) was added under an ice-water bath, maintaining an internal temperature <20 °C. Dess-Martin oxidant (0.96 g, 2.26 mmol) was added, and the mixture was brought to room temperature. After 45 minutes of reaction, 5% NaHCO3 (15 mL) and 20% Na2SO3 (15 mL) were added to the reaction solution under an ice bath to quench the reaction. The mixture was brought to room temperature, stirred, and separated into layers. The aqueous phase was extracted with MTBE (30 mL). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain 2.2 g of crude E3-28. Column purification (PE:EA = 100:0-80:20) yielded 1.5 g of compound E3-28 (two-step yield 68%).

[0190] 1H NMR (400MHz, CDCl3): δ7.14(dd,1H),6.22(d,1H),4.90-4.65(m,5H),4.13-4.09(m,1H),3.95-3.90(m,3H),3. 78-3.68(m,4H),3.66-3.56(m,3H),3.47-3.40(m,1H),3.33-3.23(m,6H),3.18(d,3H),2.81(dd,1H),2.73(dd, 1H),2.63-2.33(m,7H),2.19-2.07(m,2H),1.94-1.88(m,1H),1.84-1.78(m,2H),1.75-1.51(m,7H),1.33-1.1 3(m,6H),0.99-0.89(m,24H),0.85(s,9H),0.81(s,9H),0.75(s,9H),0.07-0.00(m,6H),-0.09--0.17(m,12H).

[0191] Step 9:

[0192] Under argon protection, E3-28 (1.47 g, 1.16 mmol) was dissolved in anhydrous tetrahydrofuran (22 mL) and anhydrous N,N-dimethylacetamide (7.4 mL) with stirring and cooled to 16 °C. Imidazole hydrochloride (0.364 g, 3.48 mmol) and a 1 MTBAF tetrahydrofuran mixture (7.4 mL) were added sequentially. After the addition was complete, the mixture was stirred at 16 °C for 16 h. 22 mL of water and 22 mL of toluene were added, and the mixture was stirred to separate the layers. The aqueous phase was then extracted with THF / toluene = 1 / 1 (22 mL × 3). The combined organic phases were dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain 1.6 g of crude E3-29. This crude product was dissolved in acetonitrile (8 mL) and DCM (8 mL) respectively, and evaporated to dryness before being used directly in the next step.

[0193] Step 10:

[0194] Under argon protection, crude E3-29 (1.6 g, 1.16 mmol) was dissolved in anhydrous DCM (20 mL) with stirring. Pyridinium 4-methylbenzenesulfonate (1.6 g, 6.38 mmol) was added, and the mixture was stirred at room temperature for 18 h. The solution was diluted with toluene (5 mL), and most of the DCM was evaporated to obtain the crude product. Column purification (PE:EA = 100:0-0:100) yielded 0.8 g of compound ER-3005. The 0.8 g product was further purified by C18 column purification (water:acetonitrile = 100:0-40:60) to obtain a total of 690 mg of ER-3005 (80% yield in both steps).

[0195] MS(ESI): m / z 745.3 [M+1]+ .

[0196] 1 H NMR (400MHz, CDCl3): δ5.02(d,2H),4.85(d,2H),4.65(dt,2H),4.37-4.27(m,3H),4.20-4.17(m, 1H),4.14-4.09(m,1H),4.05-4.02(m,1H),3.99-3.94(m,1H),3.90-3.87(m,1H),3.78-3.73(m,2H ),3.64-3.61(m,3H),3.52-3.47(m,1H),3.45(s,3H),3.40(s,3H),3.22(d,1H),2.91-2.84(m,2H) ,2.78-2.69(m,2H),2.55-1.88(m,15H),1.77-1.67(m,3H),1.49-1.26(m,7H),1.13-1.03(m,4H).

[0197] Step 11:

[0198] Add ER-3005 (668 mg, 0.897 mmol) to a pre-dried three-necked flask, under argon protection, add anhydrous tetrahydrofuran (7 mL) and stir to dissolve. Add triethylamine (182 mL, 1.79 mmol) under an ice-water bath, followed by slow dropwise addition of methanesulfonyl chloride (154 mL, 1.35 mmol). React for 0.5 hours under an ice-water bath after the addition is complete. Add 10 mL of saturated sodium bicarbonate aqueous solution and 20 mL of MTBE to the reaction mixture, stir to separate the layers, wash the organic phase with 8 mL of saturated brine, back-extract the aqueous phase with MTBE, combine the organic phases, dry, filter, and concentrate to dryness to obtain 747 mg of crude I-c1-a product, which can be directly added to the next step.

[0199] MS(ESI): m / z 822.92 [M+1] + .

[0200] Step 12:

[0201] Crude I-c1-a (747 mg, 0.897 mmol) was added to a pre-dried three-necked flask. Tetrabutylammonium azide (1.02 g, 3.59 mmol) was weighed in a glove box, and anhydrous N,N-dimethylformamide (7 mL) was added. The mixture was heated to 83 °C (oil bath temperature) and stirred for 2 h. The temperature was lowered to 20-30 °C, and 10 mL of purified water and 20 mL of MTBE were added. The mixture was stirred to separate the layers. The organic phase was washed with saturated brine, and the aqueous phase was back-extracted with MTBE until no product residue remained. The organic phases were combined and dried over magnesium sulfate. The mixture was filtered, concentrated to dryness, and the crude product was obtained. Column purification (PE:EA = 100:0-40:60) yielded 614 mg of compound Ida (two-step yield 89%).

[0202] MS(ESI): m / z 769.93 [M+1] + .

[0203] 1 H NMR (400MHz, CDCl3): δ5.02(d,2H),4.85(d,2H),4.65(dt,2H),4.37-4.27(m,3H),4.20 -4.17(m,1H),4.14-4.09(m,1H),4.05-4.02(m,1H),3.99-3.93(m,1H),3.83-3.80(m,1H ),3.78-3.75(m,1H),3.66-3.60(m,3H),3.56-3.53(m,1H),3.43(s,3H),3.41(s,3H),3. 38-3.36(m,2H),3.22(d,1H),2.93-2.85(m,3H),2.76-2.69(m,1H),2.55-0.82(m,27H).

[0204] Step 13:

[0205] Ida (601 mg, 0.78 mmol), tetrahydrofuran (10 mL), and purified water (2.5 mL) were added to a three-necked flask and stirred to dissolve. Then, triphenylphosphine (615 mg, 2.34 mmol) was added, and the mixture was reacted at room temperature for 18 hours. The solvent was removed by concentration below 30 °C to obtain the crude product. Column purification (MeOH / EA / ammonia water = 100:0:0-10:90:0-9:86:5) yielded 522 mg of compound I (90% yield).

[0206] MS(ESI): m / z 743.99 [M+1] + .

[0207] 1H NMR(400MHz,CDCl3):δ5.02(d,2H),4.85(d,2H),4.65(dt,2H),4.37-4.27(m,3H),4.20-4.17(m,1H),4.14-4.09(m,1H),4.05-4.02(m,1H),4.00-3.93(m,1H),3.84-3.80(m,1H),3.73-3.70(m,3H),3.68-3.61(m,3H),3.44(s,3H),3.37(s,3H),3.36-3.32(m,1H),3.21(d,1H),2.89-2.84(m,6H),2.76-2.69(m,2H),2.55-1.09(m,25H)。

Claims

1. A method for preparing a compound of the formula Ce or a salt thereof, This includes the conversion of a compound of formula Ca or a salt thereof into a compound of formula Cb or a salt thereof, and the reaction of a compound of formula Cb or a salt thereof with a methylating agent to form a compound of formula Cc or a salt thereof, wherein the methylating agent is preferably iodomethane, dimethyl sulfate, or trimethyloxonium tetrafluoroborate. in, R 1 Selected from C 1-3 Alkyl or benzyl, R 2 Selected from phenyl, wherein the phenyl group is optionally substituted with one or more halogens, methyl or nitro groups, P 1 Selected from hydroxyl protecting groups, such as tert-butyldimethylsilyl or triisopropylsilyl.

2. The method according to claim 1, further comprising the step of oxidative cleavage of the compound of formula Cc or its salt via olefin to form the compound of formula Ce or its salt.

3. The method according to claim 1 or 2, further comprising reacting a compound of formula Cc or a salt thereof to form a compound of formula Cd or a salt thereof. Where R 1 R 2 and P 1 As defined in claim 1.

4. The method according to claim 2, wherein the compound represented by formula Ce is the compound represented by formula C-e1. Where R 1 R 2 and P 1 As defined in claim 1; further, preferably Where P 1 As defined in claim 1.

5. The method according to claim 4, comprising: a) forming a compound of formula C-a1 or a salt thereof into a compound of formula C-b1 or a salt thereof; reacting a compound of formula C-b1 or a salt thereof with a methylating agent to form a compound of formula C-c1 or a salt thereof; subjecting a compound of formula C-c1 or a salt thereof to an olefinic dihydroxylation reaction to form a compound of formula C-d1 or a salt thereof; and oxidizing a compound of formula C-d1 or a salt thereof to form a compound of formula C-e1 or a salt thereof. Where R 1 R 2 and P 1 As defined in claim 1.

6. The method according to any one of claims 1-5, wherein P 1 Selected from triisopropylsilyl groups.

7. A method for preparing the compound of formula E3-23a or a salt thereof, comprising the steps of the method according to any one of claims 1-6. Where R 2 and P 1 As defined in claim 1.

8. The method according to claim 7, further comprising the step of reacting the compound of formula C-e1 or a salt thereof with the compound of formula Ba or a salt thereof to form the compound of formula E3-21a or a salt thereof. Where R 2 and P 1 As defined in claim 1; P 2 Selected from ester protecting groups, preferably trimethylacetyl.

9. A method for preparing the compound of formula I or a salt thereof, comprising the steps of the method according to any one of claims 1-8.

10. The method of claim 9, further comprising the step of converting the compound of formula Ic or a salt thereof into the compound of formula I or a salt thereof. Where R 4 The sulfonyl group is preferably toluenesulfonyl, benzenesulfonyl, trifluoromethanesulfonyl, and methanesulfonyl, and more preferably toluenesulfonyl.

11. The method according to claim 10, wherein the compound of formula Ic or a salt thereof is amination reaction to generate compound I, wherein the amination reagent is selected from ammonia or an ammonia-containing organic solvent.

12. The method of claim 10, further comprising the step of reacting the compound of formula Ic or a salt thereof with an azide reagent to generate the compound of formula Id or a salt thereof, wherein the azide reagent is preferably sodium azide, potassium azide, lithium azide, or trimethylsilyl azide (TMSN3). Where R 4 As defined in claim 10.

13. A method for preparing the compound of formula II or a salt thereof, comprising the steps of the method described in any one of claims 1-12. Wherein L is a linker, and the linker preferably comprises an amino acid unit, the amino acid unit being selected from peptide residues consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, preferably valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lys (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe), or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

14. A method for preparing the antibody-drug conjugate of Formula III, comprising the method step of any one of claims 1-13, and the step of conjugating the compound of Formula II or a salt thereof with an antibody or an antigen fragment thereof. Wherein L is defined as in claim 13, Ab is an antibody, and k is 1 to 20.

15. A compound or a salt thereof, said compound being selected from... Where R 1 Selected from C 1-3 Alkyl or benzyl, R 2 Selected from phenyl, wherein the phenyl group is optionally substituted with one or more halogens, methyl or nitro groups, P 1 Selected from hydroxyl protecting groups, such as tert-butyldimethylsilyl, triisopropylsilyl; P 2 Selected from ester protecting groups, such as trimethylacetyl.

16. Use of the compound of claim 15 or a salt thereof in a method for preparing the compound of formula I or a salt thereof.

17. Use of the compound of claim 15 or a salt thereof in a method for preparing the compound of formula II or a salt thereof. Where L is the connector.

18. The use of the compound of claim 15 or a salt thereof in the method for preparing the antibody-drug conjugate of formula III, Wherein L is defined as in claim 13, Ab is an antibody, and k is 1 to 20.

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