A preparation method of a linker drug conjugate and its intermediate

Abstract

The present invention discloses a method for preparing a linker-drug conjugate and its intermediates. Specifically, a method for preparing compound LE14 is provided, which comprises subjecting an intermediate compound of formula II to an amide condensation reaction with a compound of formula III or its mesylate in the presence of a condensing agent, a base, and a solvent to obtain a compound of formula I. This method can produce a high-purity intermediate II, which is directly condensed with isotecan mesylate to obtain compound I, and then subjected to subsequent reactions to obtain the final product LE14. The preparation method of the present invention has one or more of the following advantages: using intermediate II as the starting material for the production process reduces process steps, facilitates product quality control, significantly reduces impurities in the final product, and improves purity. Furthermore, using the more economical isotecan for the condensation reaction significantly reduces production costs, making it more suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of drug synthesis, and in particular relates to a preparation method of a linker-drug conjugate and an intermediate thereof. Background Art

[0002] Antibody-drug conjugates (ADCs) have become a hot topic in the pharmaceutical industry in recent years, with both domestic and international pharmaceutical companies vying to develop their pipelines. ADCs consist of three basic building blocks: an antibody, a linker, and an effector molecule. ADCs leverage the specific targeting properties of antibodies to deliver effector molecules to tumor sites for enrichment, thereby killing tumor cells. Camptothecin analogs, a class of effector molecules, are widely used in the ADC field. In recent years, Immunomedics has used them as effector molecules in its ADC drug IMMU-132 (ZL200980156218), demonstrating promising anti-tumor effects. Daiichi Sankyo's ADC drug DS-8201a (ZL201380053256), using another camptothecin analog as an effector molecule, has also demonstrated promising anti-tumor effects.

[0003] Patent application WO2020259258A1 discloses an ADC compound with a camptothecin derivative DXd as an effector molecule, and provides a preparation method for the linker-drug conjugate LE14 as shown in Route 1 and Route 2; patent applications WO2022204947A1, CN115215921A and CN115385926A provide improved Route 3, Route 4 and Route 5.

[0004] Route 1:

[0005]

[0006] The synthesis method of route 1 comprises the following steps: reacting compound 1-1 with 4-aminobenzyl alcohol, reacting the resulting compound with di(p-nitrobenzyl) carbonate and then reacting with a substituted alkylamine to obtain compound 1-2, reacting compound 1-2 with paraformaldehyde and trimethylsilyl chloride to obtain compound 1-3, reacting compound 1-3 with tert-butyl glycolate and then removing the tert-butyl group under the action of trifluoroacetic acid to obtain compound 1-4, reacting compound 1-4 with isotecan mesylate to obtain compound 1-5, removing the Fmoc protection on the amino group under the action of DBU, and then coupling reaction with 6-(maleimido)hexanoic acid succinimidyl ester to obtain the target compound LE14.

[0007] In this route, in the step of preparing intermediate 1-4 using intermediate 1-3 as a raw material, since both the raw material and the product are unstable to acids and bases, there is a certain risk of decomposition during the deprotection and purification process. Compound 1-4 is not purified and the crude product is used directly for the next reaction. This will cause obvious impurities in the crude product 1-5. The polarity of these impurities is close to that of the product, making purification difficult, and thus affecting the product quality of LE14.

[0008] Route 2:

[0009]

[0010] The synthesis method of Route 2 comprises the following steps: compound 2-1 reacts with paraformaldehyde and trimethylsilyl chloride, the resulting compound then reacts with tert-butyl glycolate to obtain compound 2-2, compound 2-2 is de-tert-butylated under the action of trifluoroacetic acid to obtain compound 2-2a, which is then reacted with isotecan mesylate to obtain compound 2-3, the azide of compound 2-3 is reduced to an amino group under the action of triethylphosphine to obtain compound 2-4, and compound 2-4 undergoes a coupling reaction with MC-V to obtain the target compound LE14.

[0011] In this route, the raw material 2-2 and the carboxylic acid intermediate 2-2a generated by removing the tert-butyl group are both unstable to acids and bases, and there is a certain risk of decomposition during the deprotection and purification process. If the compound 2-2a is not purified and the crude product is used directly in the next reaction, significant impurities will be generated in the crude product 2-3. The polarity of these impurities is close to that of the product, making purification difficult and thus affecting the product quality of LE14.

[0012] Route 3:

[0013]

[0014] The synthesis method of route 3 comprises the following steps: compound 3-1 is reacted with p-nitrophenyl chloroformate to obtain compound 3-2, the obtained compound is then reacted with the corresponding amine to obtain compound 3-3, which is then deprotected to obtain compound 3-4, which is further reacted with an amino acid active ester to obtain 3-5, which is then deprotected to obtain 3-6, which is then reacted with an acyl azide reagent to obtain compound 3-7, which is then chloromethylated to obtain compound 3-8, which is then reacted with DXd or a DXd derivative to obtain compound 3-9, and the azide group in 3-9 is then reduced to obtain compound 3-10, which is finally connected with a maleimide linker to obtain the final product LE14.

[0015] According to the method of route 3, the total yield of LE14 prepared from compound 3-1 as the starting material is 3.5%, and the total yield of LE14 prepared from compound 3-7 as the starting material is 10.7%.

[0016] This route uses DXd or DXd derivatives as the source of payload, which is relatively expensive and greatly increases the cost of preparing LE14.

[0017] Route 4:

[0018]

[0019] The synthesis method of route 4 comprises the following steps: compound 4-1 is reacted with p-nitrophenyl chloroformate to obtain compound 4-2, the obtained compound is then reacted with the corresponding amine to obtain compound 4-3, which is then deprotected to obtain compound 4-4, which is further reacted with an amino acid active ester to obtain 4-5, which is then deprotected to obtain 4-6, which is then reacted with an acyl azide reagent to obtain compound 4-7, which is then chloromethylated to obtain compound 4-8, which is then reacted with a DXd derivative to obtain compound 4-9, and the azide group in 4-9 is then reduced to obtain compound 4-10, which is then coupled with a maleimide linker to obtain 4-11, and finally the hydroxyl protecting group is removed to obtain the final product LE14.

[0020] According to the method of route 4, the total yield of LE14 prepared from compound 4-1 as the starting material and Dxd-a as the payload source was 7.3% (4-1→Dxd-a→LE14); the total yield of LE14 prepared from compound 4-1 as the starting material and Dxd-b as the payload source was 12.9% (4-1→Dxd-b→LE14).

[0021] In this route, DXd derivatives Dxd-a (structure shown in route 4-a) or Dxd-b (structure shown in route 4-b) are used as the source of payload, which is expensive and greatly increases the cost of preparing LE14.

[0022] In order to avoid the purchase of expensive DXd derivatives, our company developed synthetic routes 4-a and 4-b for preparing Dxd-a and Dxd-b from ixitecan.

[0023] Exotecan derivatization route 4-a: (Exotecan → Dxd-a)

[0024]

[0025] The synthetic method for preparing Dxd-a according to Route 4-a comprises: reacting isotecan or its mesylate with 4-methoxytriphenylmethane chloride in the presence of trimethylsilyl chloride and N,N-diisopropylethylamine to prepare an amino-protected intermediate compound 17; reacting compound 17 with acetic anhydride under alkaline conditions to obtain an acetylated intermediate 16a; and removing the amino protection of intermediate 16a under the action of triethylsilane to obtain intermediate 15a, which is further reacted with glycolic acid to obtain compound Dxd-a. The total yield of Dxd-a prepared using isotecan as the starting material is 38.3% (isotecan → Dxd-a; hereinafter referred to as the yield of Route 4-a).

[0026] Compound 4-8 was prepared using compound 4-1 as the starting material according to the synthetic method of route 4, and Dxd-a was prepared using isotecan as the starting material according to the synthetic method of route 4-a as the payload source and LE14 was prepared according to the method of route 4. The overall yield was 2.8% (the yield of 4-1→Dxd-a→LE14 multiplied by the yield of isotecan derivatization route 4-a, i.e., 7.3%×38.3%=2.8%).

[0027] The overall yield of LE14 prepared according to the method of Route 4 using compound 4-7 as the starting material and Dxd-a prepared according to the synthetic method of Route 4-a using isotecan as the starting material was 8.2% (the yield of 4-7→Dxd-a→LE14 multiplied by the yield of isotecan derivatization Route 4-a, i.e., 21.3%×38.3%=8.2%).

[0028] Exotecan derivatization route 4-b: (Exotecan → Dxd-b)

[0029]

[0030] The synthetic method for preparing Dxd-b according to Route 4-b includes: reacting isotecan or its mesylate with 4-methoxytriphenylmethane chloride in the presence of trimethylsilyl chloride and N,N-diisopropylethylamine to prepare an amino-protected intermediate compound 17; reacting compound 17 with tert-butyldiphenylsilyl chloride under alkaline conditions to obtain tert-butyldiphenylsilylated intermediate 16b; deprotecting the amino group of intermediate 16b under the action of triethylsilane to obtain intermediate 15b, which is further reacted with glycolic acid to obtain compound Dxd-b. The total yield of Dxd-b prepared using isotecan as the starting material is 48.2% (isotecan → Dxd-b; hereinafter referred to as the yield of Route 4-b).

[0031] The overall yield of compound 4-8 prepared from compound 4-1 according to the synthetic method of route 4 and LE14 prepared from isetec as the starting material according to the synthetic method of route 4-b using Dxd-b as the payload source according to the method of route 4 was 6.2% (the yield of 4-1→Dxd-b→LE14 multiplied by the yield of isetec derivatization route 4-b, i.e., 12.9%×48.2%=6.2%).

[0032] The overall yield of LE14 prepared according to the method of Route 4 using compound 4-7 as the starting material and Dxd-b prepared according to the synthetic method of Route 4-b using isotecan as the starting material was 18.0% (the yield of 4-7→Dxd-b→LE14 multiplied by the yield of isotecan derivatization Route 4-b, i.e., 37.5%×48.2%=18.0%).

[0033] If Dxd-a and Dxd-b prepared using isotecan as the starting material according to the methods of Routes 4-a and 4-b are used as the source of Dxd derivatives in Routes 4, although the direct use of the relatively expensive Dxd is avoided, four reaction steps are added, which increases the experimental operation volume. At the same time, the loss during the derivatization process of isotecan is also huge, which in turn increases the production cost.

[0034] Route 5:

[0035]

[0036] The synthesis method of Route 5 comprises the following steps: compound 5-1 is reacted with p-nitrophenyl chloroformate to obtain compound 5-2, the resulting compound is then reacted with the corresponding amine to obtain compound 5-3, which is then deprotected to obtain compound 5-4, which is further reacted with an amino acid active ester to obtain 5-5, which is then deprotected to obtain 5-6, which is then reacted with an acyl azide reagent to obtain compound 5-7, which is then subjected to a hydroxymethylation reaction to obtain compound 5-8, which is then reacted with a DXd derivative to obtain compound 5-9, and the azide group in 5-9 is then reduced to obtain compound 5-10, which is then coupled with a maleimide linker to obtain 5-11, and finally the hydroxy protecting group is removed to obtain the final product LE14.

[0037] According to the method of Scheme 5, LE14 was prepared with compound 5-1 as the starting material and Dxd-a' (numbered as Dxd-a in the original patent application, the structure of which is shown in Scheme 5-a, and renumbered as Dxd-a' in this application to distinguish it from Dxd-a in Scheme 4-a) as the payload source, with a total yield of 6.9%; LE14 was prepared with compound 5-1 as the starting material and Dxd-b' (numbered as Dxd-b in the original patent application, the structure of which is shown in Scheme 5-b, and renumbered as Dxd-b' in this application to distinguish it from Dxd-b in Scheme 4-b) as the payload source, with a total yield of 12.2%.

[0038] This route uses DXd derivatives Dxd-a' or Dxd-b' as the source of payload, which is expensive and greatly increases the cost of preparing LE14.

[0039] In order to avoid the purchase of expensive DXd derivatives, our company developed synthetic routes 5-a and 5-b for preparing Dxd-a' and Dxd-b' from ixitecan.

[0040] Exotecan derivatization route 5-a: (Exotecan → Dxd-a')

[0041]

[0042] The synthetic method for preparing Dxd-a' according to Route 5-a comprises: reacting isotecan or its mesylate with 4-methoxytriphenylmethane chloride in the presence of trimethylsilyl chloride and N,N-diisopropylethylamine to prepare an amino-protected intermediate compound 17; reacting compound 17 with acetic anhydride under alkaline conditions to obtain an acetylated intermediate 16a; and removing the amino protection of intermediate 16a under the action of triethylsilane to obtain intermediate 15a, which is further reacted with 2-bromoacetic acid to obtain compound Dxd-a'. The total yield of Dxd-a' prepared using isotecan as the starting material is 27.6% (isotecan → Dxd-a', hereinafter referred to as the yield of Route 5-a).

[0043] Compound 5-8 was prepared using compound 5-1 as the starting material according to the synthetic method of route 5, and Dxd-a' was prepared using isotecan as the starting material according to the synthetic method of route 5-a as the payload source and LE14 was prepared according to the method of route 5. The overall yield was 1.9% (the yield of 5-1→Dxd-a'→LE14 multiplied by the yield of isotecan derivatization route 5-a, i.e., 6.9%×27.6%=1.9%).

[0044] The overall yield of LE14 prepared according to the method of Route 5 using compound 5-7 as the starting material and Dxd-a' prepared according to the synthetic method of Route 5-a using isotecan as the starting material was 6.5% (the yield of 5-7→Dxd-a'→LE14 multiplied by the yield of isotecan derivatization Route 5-a, i.e., 23.4%×27.6%=6.5%).

[0045] Exotecan derivatization route 5-b: (Exotecan → Dxd-b')

[0046]

[0047] The synthetic method for preparing Dxd-b' according to route 5-b includes: reacting isotecan or its mesylate with 4-methoxytriphenylmethane chloride in the presence of trimethylsilyl chloride and N,N-diisopropylethylamine to prepare an amino-protected intermediate compound 17, reacting compound 17 with tert-butyldiphenylsilyl chloride under alkaline conditions to obtain tert-butyldiphenylsilylated intermediate 16b, removing amino protection from intermediate 16b under the action of triethylsilane to obtain intermediate 15b, which is further reacted with 2-bromoacetic acid to obtain compound Dxd-b'. The total yield of Dxd-b' prepared using isotecan as the starting material is 44.5% (isotecan → Dxd-b', hereinafter referred to as the yield of route 5-b).

[0048] Compound 5-8 was prepared using compound 5-1 as the starting material according to the synthetic method of route 5, and Dxd-b' was prepared using isotecan as the starting material according to the synthetic method of route 5-b as the payload source and LE14 was prepared according to the method of route 5. The overall yield was 5.4% (the yield of 5-1→Dxd-b'→LE14 multiplied by the yield of isotecan derivatization route 5-b, i.e., 12.2%×44.5%=5.4%).

[0049] The overall yield of LE14 prepared according to the method of Route 5 using compound 5-7 as the starting material and Dxd-b' prepared according to the synthetic method of Route 5-b using isotecan as the starting material was 18.3% (the yield of 5-7→Dxd-b'→LE14 multiplied by the yield of isotecan derivatization Route 5-b, i.e., 41.2%×44.5%=18.3%).

[0050] If Dxd-a' and Dxd-b' prepared using isotecan as the starting material according to the methods of Routes 5-a and 5-2 are used as the source of Dxd derivatives in Route 5, although the direct use of the relatively expensive Dxd is avoided, four reaction steps are added, which increases the experimental operation volume. At the same time, the loss during the derivatization process of isotecan is also huge, which in turn increases the production cost.

[0051] After verification and evaluation, it was found that the above route has the following defects:

[0052] Intermediates 1-3 and 2-2 in Routes 1 and 2 require removal of the tert-butyl group to prepare carboxylic acid intermediates. However, this reaction uses trifluoroacetic acid for deprotection, which is harsh and difficult to remove after post-treatment. Furthermore, the resulting carboxylic acid intermediates 1-4 and 2-2a are unstable and almost completely lost during the purification process. Consequently, the unpurified crude product can only be used directly for the next reaction, resulting in the presence of impurities of similar polarity in the final product, increasing the difficulty of purification. Furthermore, Routes 3, 4, and 5 require the use of DXd or a DXd derivative, significantly increasing production costs.

[0053] To obtain a purified, stable carboxylic acid intermediate, we also attempted to protect the carboxyl group with a methyl or ethyl group. For example, we reacted the methylsulfonylethylamine intermediate with methyl glycolate or ethyl glycolate, followed by ester hydrolysis under alkaline or acidic conditions to obtain the carboxylic acid. However, these ester structures and the resulting carboxylic acids after hydrolysis were not acid- and alkali-resistant, making it impossible to obtain a purified carboxylic acid product. We also attempted to use silicon-based protecting groups, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), to protect the carboxyl group. However, the corresponding products were also unstable, and we were unable to obtain a purified product.

[0054] The defects and failed attempts of the above synthetic routes have limited the commercial production of this type of ADC drugs. Therefore, it is necessary to find a more efficient and reasonable preparation route and an intermediate with stable and easy purification and separation to solve this problem. Summary of the Invention

[0055] The technical problem to be solved by the present invention is to overcome the drawbacks of existing methods for preparing linker-drug conjugates of formula LE14, such as the difficulty in purifying intermediates, resulting in low purity of the prepared LE14, and the high cost of DXd derivatives. Thus, a new method for preparing linker-drug conjugate LE14 and its intermediates are provided. The preparation method of the present invention directly utilizes isotecan or its mesylate salt as a payload source, which reacts with the novel intermediate II of the present invention to form linker-drug conjugate compound I, and ultimately, through subsequent process reactions, obtains the final product LE14. The preparation method of the present invention has one or more of the following advantages: the purified carboxylic acid intermediate II can be stably obtained and used as a starting material in the production process, significantly reducing the number of process steps and the impurity content of the final product LE14. Furthermore, the process utilizes the more economical isotecan or its mesylate salt as a key raw material, significantly reducing production costs.

[0056] The structure of the antibody-drug conjugate LE14 of the present invention is shown below:

[0057]

[0058] The present invention mainly solves the above technical problems through the following technical solutions.

[0059] The present invention provides a method for preparing a compound of formula I, comprising the following steps: subjecting an intermediate compound of formula II to an amide condensation reaction with a compound of formula III or its mesylate in the presence of a condensing agent, a base, and a solvent to obtain the compound of formula I;

[0060]

[0061] In the method for preparing the compound of formula I, the reaction conditions (such as the amount of solvent, the order and method of adding materials, the feeding time, etc.) can be conventional conditions for such reactions in the art. In the present invention, the following scheme is preferred:

[0062] In some embodiments, in the method for preparing the compound of formula I, the reaction materials are the compound of formula II, the compound of formula III or its mesylate, the condensing agent, the base and the solvent.

[0063] In some embodiments, in the method for preparing the compound of formula I, the amide condensation reaction further comprises the following specific steps: dissolving the compound of formula II in a solvent, adding the condensing agent, and immediately or after a period of reaction, adding the compound of formula III or its mesylate and a base.

[0064] In some embodiments, in the method for preparing the compound of formula I, the reaction is protected from light throughout the entire process.

[0065] In some embodiments, in the method for preparing the compound of formula I, the molar ratio of the compound of formula III to the compound of formula II can be 0.8-1.5, preferably 0.9-1.2, more preferably 0.9-1.0, and most preferably 0.9.

[0066] In some embodiments, in the preparation method of the compound of formula I, the condensing agent can be 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (DMTMM), diethyl phosphorocyanide (DECP), diphenyl phosphorazide (DPPA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 6 -chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), 2-(1H-benzotriazol L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) and 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate quaternary ammonium salt (TNTU) or a mixture of any two or more thereof. Preferably, the condensing agent can be 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride.

[0067] In some embodiments, in the method for preparing the compound of formula I, the molar ratio of the condensing agent to the compound of formula II can be 1-3, preferably 1-1.5, and more preferably 1.5.

[0068] In some embodiments, in the preparation method of the compound of formula I, the base can be a conventional base for such reactions in the art, and can be an organic base, an inorganic base, or a mixture thereof, preferably an organic base; wherein the organic base is preferably one of N,N-diisopropylethylamine, triethylamine, and pyridine, or a mixture of any two or more thereof, further preferably N,N-diisopropylethylamine; the inorganic base is preferably one of alkali metal hydroxides, alkali metal carbonates, and alkali metal phosphates, or a mixture of any two or more thereof, further preferably one of sodium bicarbonate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, and potassium hydroxide, or a mixture of any two or more thereof.

[0069] In some embodiments, in the method for preparing the compound of formula I, the molar ratio of the base to the compound of formula II can be 1-10, preferably 1-6, more preferably 1-3, and most preferably 1.5.

[0070] In some embodiments, in the method for preparing the compound of formula I, the solvent can be one of N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran and 1,4-dioxane, or a mixture of any two or more thereof, preferably N,N-dimethylformamide.

[0071] In some embodiments, in the method for preparing the compound of formula I, the temperature of the condensation reaction can be 20°C-50°C, preferably 20°C-30°C.

[0072] In some embodiments, in the method for preparing the compound of formula I, the condensation reaction is preferably carried out under the protection of an inert gas, for example, in a nitrogen or helium environment.

[0073] In some embodiments, in the method for preparing the compound of formula I, the reaction progress of the condensation reaction can be monitored using conventional testing methods in the art (such as TLC, GC, HPLC or NMR, etc.), and the reaction endpoint is generally when the compound of formula III is no longer detected.

[0074] In some embodiments, in the method for preparing the compound of formula I, the reaction time of the condensation reaction can be 2-8 hours, preferably 3-5 hours, and more preferably 2-4 hours.

[0075] In some embodiments, in the preparation method of the compound of formula I, after the condensation reaction is completed, the following post-treatment step may be further included: concentrating the reaction solution to obtain a crude compound of formula I; preferably, purifying the crude compound of formula I by silica gel column chromatography to obtain a product compound of formula I. More preferably, the eluent for the silica gel column chromatography can be a mixed solvent of chloroform and methanol, and the volume ratio of chloroform to methanol is (100-10):1, more preferably 10:1.

[0076] The preparation method of the compound of formula I may further include a preparation method of the compound of formula II, which may include the following steps: subjecting the compound of formula IV to a deprotection reaction in a solvent in the presence of a deprotecting agent to obtain the compound of formula II;

[0077]

[0078] Wherein, R is a C1~C6 alkyl group substituted by -Si(C1~C6)3.

[0079] In some embodiments, in the preparation method of the compound of formula II, the C1~C6 alkyl substituted by -Si(C1~C6)3 can be trimethylsilylethyl or tert-butyldimethylsilylethyl; preferably, the C1~C6 alkyl substituted by -Si(C1~C6)3 is trimethylsilylethyl.

[0080] In some embodiments, in the method for preparing the compound of formula II, the reaction materials are the compound of formula IV, the deprotecting agent and the solvent.

[0081] In some embodiments, in the preparation method of the compound of formula II, the deprotecting agent can be a fluorine reagent, and the fluorine reagent can be tetrabutylammonium fluoride, tetramethylammonium fluoride, tetrabutylammonium fluoride / acetic acid, pyridine hydrogen fluoride complex, tert-butylammonium fluoride, tert-butylammonium fluoride / acetic acid, tetraethylammonium fluoride, or commercially available tetramethylammonium fluoride / tetrahydrofuran solution, tetraethylammonium fluoride / tetrahydrofuran solution, tetrabutylammonium fluoride / tetrahydrofuran solution, or one or more of silicon tetrafluoride, potassium fluoride, sodium fluoride, lithium fluoride and cesium fluoride, preferably 1M tetrabutylammonium fluoride / tetrahydrofuran solution or potassium fluoride.

[0082] In some embodiments, in the method for preparing the compound of formula II, the molar ratio of the deprotecting agent to the compound of formula IV can be 1-5, preferably 1-3, more preferably 1.1-2.0, and most preferably 1.5.

[0083] In some embodiments, in the method for preparing the compound of formula II, the solvent can be one of purified water, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran and 1,4-dioxane, or a mixture of any two or more thereof, preferably N,N-dimethylformamide or tetrahydrofuran, and most preferably N,N-dimethylformamide.

[0084] In some embodiments, the reaction conditions (e.g., solvent amount, solvent type, order of addition, method of addition, and time of addition) in the method for preparing the compound of Formula II may be conventional conditions for such reactions in the art and may be adjusted according to the type of deprotecting group. For example, the method may include the following steps: dissolving the compound of Formula IV in a solvent, adding a deprotecting agent, and then heating to initiate the reaction; or dissolving the deprotecting agent in a solvent, adding the compound of Formula IV, and then heating to initiate the reaction.

[0085] In some embodiments, in the method for preparing the compound of formula II, the temperature of the deprotection reaction is 20°C-80°C, preferably 30°C-70°C, more preferably 40°C-70°C, even more preferably 40°C-60°C, and most preferably 60°C.

[0086] In some embodiments, in the method for preparing the compound of formula II, the deprotection reaction is preferably carried out under the protection of an inert gas, such as in a nitrogen or helium environment.

[0087] In some embodiments, in the method for preparing the compound of formula II, the progress of the deprotection reaction can be monitored using conventional testing methods in the art (such as TLC, GC, HPLC or NMR, etc.), and the reaction endpoint is generally when the compound of formula IV is no longer detected.

[0088] In some embodiments, in the method for preparing the compound of formula II, the reaction time of the deprotection reaction can be 4-24 hours, preferably 5-16 hours, and more preferably 5-8 hours.

[0089] In some embodiments, the method for synthesizing the compound of formula II may further include a post-treatment step after the reaction is completed: concentrating the reaction solution to obtain a crude compound of formula II; preferably, purifying the crude compound of formula II by silica gel column chromatography to obtain a product compound of formula II; more preferably, the eluent used for the silica gel column chromatography is a mixed solvent of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol may be (100-1):1, preferably (10-1):1.

[0090] The preparation method of the compound of formula I may further include a preparation method of the compound of formula IV, which may include the following steps: conducting an etherification reaction of the compound of formula VI with a reagent V in a solvent in the presence of a base to obtain a compound of formula IV;

[0091]

[0092] Wherein, R is as described above.

[0093] In some embodiments, in the method for preparing the compound of formula IV, the reaction materials are the compound of formula VI, the reagent V, the base and the solvent.

[0094] In some embodiments, in the method for preparing the compound of formula IV, the molar ratio of the reagent V to the compound of formula VI can be 1-5, preferably 1-3, more preferably 1.1-1.6, and most preferably 1.5.

[0095] In some embodiments, in the method for preparing the compound of formula IV, the alkaline reagent can be an organic base, an inorganic base or a mixture thereof, preferably an organic base; wherein the organic base is preferably one of potassium tert-butoxide, triethylamine, 4-dimethylaminopyridine, pyridine and panpiperidin, or a mixture of any two or more thereof, further preferably panpiperidin; the inorganic base is preferably one of alkali metal hydroxides, alkali metal carbonates and alkali metal phosphates, or a mixture of any two or more thereof.

[0096] In some embodiments, in the method for preparing the compound of formula IV, the molar ratio of the base to the compound of formula VI can be 1-5, preferably 1.2-4, more preferably 1.5-3, and most preferably 1.5.

[0097] In some embodiments, in the method for preparing the compound of formula IV, the solvent is one of N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran and 1,4-dioxane, or a mixture of any two or more thereof, preferably tetrahydrofuran or 1,4-dioxane, and more preferably tetrahydrofuran.

[0098] In some embodiments, in the method for preparing the compound of formula IV, the reaction conditions (such as the amount of solvent, the order of feeding, the method of adding materials and the time of feeding, etc.) can be conventional conditions for this type of reaction in the art, which can be adjusted according to the type of different alkaline reagents. For example, it can include the following steps: the compound of formula VI is dissolved in a solvent, and after adding a base, reagent V is added immediately or after a period of reaction.

[0099] In some embodiments, in the method for synthesizing the compound of formula IV, the temperature of the etherification reaction can be 0°C-80°C, preferably 40°C-70°C, and most preferably 60°C.

[0100] In some embodiments, in the method for preparing the compound of formula IV, the etherification reaction is preferably carried out under the protection of an inert gas, such as in a nitrogen or helium environment.

[0101] In some embodiments, in the method for preparing the compound of formula IV, the progress of the etherification reaction can be monitored using conventional testing methods in the art (such as TLC, GC, HPLC or NMR, etc.), and the reaction endpoint is generally when the compound of formula VI is no longer detected.

[0102] In some embodiments, in the method for preparing the compound of formula IV, the etherification reaction time can be 3-24 hours, preferably 3-12 hours, and more preferably 4-8 hours.

[0103] In some embodiments, the preparation method of the compound of formula IV may further include a post-treatment step after the reaction is completed: extracting the reaction solution and concentrating the organic phase to obtain a crude compound of formula IV, or directly concentrating the reaction solution to obtain a crude compound of formula IV; preferably, the crude compound of formula IV is purified by silica gel column chromatography to obtain the compound IV product; more preferably, the eluent used for the silica gel column chromatography is a mixed solution of n-heptane and ethyl acetate, and the volume ratio of n-heptane to ethyl acetate can be (20-1):1, preferably (10-1):1.

[0104] The preparation method of the compound of formula I may further include a preparation method of the compound of formula VI, which may include the following steps: subjecting the compound of formula VII to a substitution reaction with paraformaldehyde and trimethylsilyl chloride in a solvent to obtain the compound of formula VI;

[0105]

[0106] The compound of formula VII is an azide polypeptide compound, which is commercially available or homemade.

[0107] In some embodiments, in the method for preparing the compound of formula VI, the reaction materials are the compound of formula VII, the paraformaldehyde, the trimethylsilyl chloride and the solvent.

[0108] In some embodiments, in the method for preparing the compound of formula VI, the molar ratio of the paraformaldehyde to the compound of formula VII can be 1-10, preferably 1-5, more preferably 1.3-3.0, and most preferably 1.3.

[0109] In some embodiments, in the method for preparing the compound of formula VI, the molar ratio of trimethylsilyl chloride to the compound of formula VII can be 1-5, preferably 2-4, more preferably 2-3, and most preferably 2.5.

[0110] In some embodiments, in the method for preparing the compound of formula VI, the solvent can be one of N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran and 1,4-dioxane, or a mixture of any two or more thereof, preferably tetrahydrofuran or 1,4-dioxane, and more preferably 1,4-dioxane.

[0111] In some embodiments, in the method for preparing the compound of formula VI, the temperature of the substitution reaction is -10°C-50°C, preferably 15°C-35°C, and more preferably 18°C-25°C.

[0112] In some embodiments, in the method for preparing the compound of formula VI, the substitution reaction is carried out under the protection of an inert gas, for example, in a nitrogen or helium environment.

[0113] In some embodiments, in the preparation method of the compound VI, the progress of the substitution reaction can be monitored using conventional testing methods in the art (such as TLC, GC, HPLC or NMR, etc.), and the end point of the reaction is generally when the compound of formula VII is no longer detected (using methanol derivatization).

[0114] In some embodiments, in the method for preparing compound VI, the substitution reaction time may be 3-24 hours, preferably 6-18 hours, and more preferably 8-16 hours.

[0115] In some embodiments, the method for preparing the compound of formula VI may further include a post-treatment step after the reaction is completed: the reaction solution is subjected to solid-liquid separation (in the presence of solids) or no solid-liquid separation is performed, and the organic phase is concentrated to obtain the compound of formula VI; preferably, the crude compound of formula VI obtained after concentration is directly reacted with reagent V.

[0116] The present invention also provides a method for preparing a compound of formula VIII, characterized in that it comprises the following steps: performing a reduction reaction on a compound of formula I and a reducing agent in a solvent and in the presence of an acid buffer to obtain a compound of formula VIII;

[0117]

[0118] In some embodiments, in the method for preparing the compound of formula VIII, the reaction materials are the compound of formula I, the reducing agent, the acid buffer and the solvent.

[0119] In some embodiments, in the method for preparing the compound of formula VIII, the reducing agent can be triphenylphosphine, tri-tert-butylphosphine or trimethylphosphine, or commercially available triphenylphosphine / tetrahydrofuran solution, tri-tert-butylphosphine / tetrahydrofuran solution or trimethylphosphine / tetrahydrofuran solution, preferably trimethylphosphine / tetrahydrofuran solution, more preferably 1 M trimethylphosphine / tetrahydrofuran solution.

[0120] In some embodiments, in the method for preparing the compound of formula VIII, the acid buffer solution may be sodium acetate buffer solution or sodium formate buffer solution, preferably sodium acetate buffer solution; the pH of the acid buffer solution is preferably 4.0-6.0, more preferably 4.5-5.5, and even more preferably 5.0.

[0121] In some embodiments, in the method for preparing the compound of formula VIII, the molar ratio of the reducing agent to the compound of formula I can be 1.0-3.0, preferably 1.2-1.8, and more preferably 1.5.

[0122] In some embodiments, in the method for preparing the compound of formula VIII, the volume mass ratio of the acid buffer to the compound of formula I can be 2-10 mL / g, preferably 5-10 mL / g; further preferably 7-8 mL / g, and most preferably 7.7 mL / g.

[0123] In some embodiments, in the method for preparing the compound of formula VIII, the solvent is an ether solvent, such as one of tetrahydrofuran, diethyl ether, 1,4-dioxane, anisole, and methyl tert-butyl ether, or a mixture of any two or more thereof, more preferably tetrahydrofuran.

[0124] In some embodiments, in the method for preparing the compound of formula VIII, the temperature of the reduction reaction can be 0°C-20°C, preferably 0°C-10°C.

[0125] In some embodiments, in the method for preparing the compound of formula VIII, the reaction progress can be monitored using conventional testing methods in the art (such as TLC, GC, HPLC or NMR, etc.), and the reaction endpoint is generally when the compound of formula I is no longer detected.

[0126] In some embodiments, in the method for preparing the compound of formula VIII, the reaction time can be 0.5-5 hours, preferably 0.5-2.5 hours, and more preferably 0.5-1.0 hour.

[0127] In some embodiments, the method for preparing the compound of formula VIII may further include a post-treatment step after the reaction is completed: extracting the reaction solution and concentrating the organic phase to obtain a crude compound of formula VIII, or directly concentrating the reaction solution to obtain a crude compound of formula VIII; preferably, the crude compound of formula VIII is purified by silica gel column chromatography to obtain a product compound of formula VIII; more preferably, the eluent used for the silica gel column chromatography is a mixed solvent of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol may be (100-1):1, preferably (10-1):1.

[0128] The present invention also provides a method for preparing compound LE14, comprising the following steps: coupling the compound of formula VIII and 6-(maleimido)hexanoic acid succinimidyl ester in a solvent to obtain the compound LE14, i.e., the linker-drug conjugate LE14.

[0129]

[0130] In some embodiments, in the method for preparing compound LE14, the reaction materials are the compound of formula VIII, the succinimidyl 6-(maleimido)hexanoate, and the solvent.

[0131] In some embodiments, in the method for preparing compound LE14, the molar ratio of 6-(maleimido)hexanoic acid succinimidyl ester to the compound of formula VIII can be 1-5, preferably 1-3, more preferably 1.2-2.0, and most preferably 2.0.

[0132] In some embodiments, in the preparation method of the compound LE14, the solvent can be one of an amide solvent, a chlorinated alkane solvent, an ether solvent, and a nitrile solvent, or a mixture of any two or more thereof; preferably a chlorinated alkane solvent, wherein the chlorinated alkane solvent is preferably one of dichloromethane, 1,2-dichloroethane, and chloroform, or a mixture of any two or more thereof, further preferably dichloromethane.

[0133] In some embodiments, in the method for preparing compound LE14, the reaction temperature is 0°C-45°C, preferably 25°C-40°C, more preferably 30°C-35°C, and most preferably 35°C.

[0134] In some embodiments, in the preparation method of the compound LE14, the reaction progress can be monitored using conventional testing methods in the art (such as TLC, GC, HPLC or NMR, etc.), and the reaction endpoint is generally when the compound of formula VIII is no longer detected.

[0135] In some embodiments, in the method for preparing compound LE14, the reaction time can be 3-24 hours, preferably 3-10 hours, and more preferably 3-6 hours.

[0136] In some embodiments, the preparation method of the compound LE14 may further include a post-treatment step after the reaction is completed: concentrating the reaction solution to obtain a crude compound LE14; preferably, the crude compound LE14 is purified by silica gel column chromatography to obtain the compound LE14 product; more preferably, the eluent used for the silica gel column chromatography is a mixed solvent of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol may be (100-1):1, preferably (10-1):1.

[0137] In some embodiments, in the method for preparing compound LE14, the compound of formula VIII can be prepared by any of the methods for preparing the compound of formula VIII above.

[0138] The present invention also provides a compound of formula II:

[0139]

[0140] The present invention also provides a method for preparing a compound of formula II, comprising the following steps: subjecting a compound of formula IV to a deprotection reaction in a solvent in the presence of a deprotecting agent to obtain a compound of formula II;

[0141]

[0142] Wherein, R is a C1~C6 alkyl group substituted by -Si(C1~C6)3.

[0143] In the preparation method of the compound of formula II, the reaction conditions can be the same as described above.

[0144] The method for preparing the compound of formula II may further include the method for preparing the compound of formula IV described herein.

[0145] definition

[0146] In the present invention, the term "C1-C6 alkyl" refers to a saturated straight-chain or branched alkyl group containing 1-6, especially 1-4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc., especially methyl or ethyl.

[0147] In the present invention, room temperature refers to 20℃-30℃

[0148] The English abbreviations and Chinese names of the compounds involved in the present invention are shown in Table 1.

[0149] Table 1

[0150]

[0151]

[0152] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0153] The reagents and raw materials used in the present invention are commercially available or homemade.

[0154] The positive progress of the present invention is that: the present invention provides a novel method for preparing a linker-drug conjugate intermediate represented by Formula I. First, the preparation method can stably provide a pure intermediate II with a yield of up to 89.1% and a purity of up to 90.16%. Compared with the methods of removing the tert-butyl group to prepare the carboxylic acid intermediate in Routes 1 and 2, both the yield and purity are greatly improved;

[0155] Secondly, using the purified intermediate II for condensation reactions can greatly improve the purity of intermediate I and reduce the generation of impurities. Continuing with subsequent synthesis steps can also significantly improve the purity of the final product LE14, with the maximum single impurity in the final product LE14 reduced to less than 0.1%.

[0156] Finally, using intermediate II as the starting material for the commercial production process can simplify the production steps. At the same time, condensing intermediate II with commercially available Exatecan mesylate avoids the use of expensive DXd materials, which can significantly reduce production costs. DETAILED DESCRIPTION

[0157] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0158] In the following examples,

[0159] Mass spectrometry was performed using a Waters Acquity Xevo G2-XS QTof UPLC / MS ultra-high performance liquid chromatography-high resolution mass spectrometry system;

[0160] 1 H-NMR was performed using a Bruker AVANCE III 400 MHz NMR instrument or a Bruker AVANCE III HD300 MHz NMR instrument;

[0161] HPLC detection conditions: The instrument was Agilent 187260, the chromatographic column was Agilent AdvanceBio PeptideMap, 3.5 μm, 2.1×250 mm, the detection wavelength was 254 nm, and the mobile phase gradient setting shown in Table 2 was used, wherein phase A was 0.01 mol / L potassium dihydrogen phosphate aqueous solution (pH=5.0), phase B was 10% methanol in acetonitrile, and the flow rate was 1.0 mL / min.

[0162] Table 2. HPLC detection mobile phase gradient settings

[0163] Time (min) Mobile phase A% Mobile phase B% 0.00 80.0 20.0 5.00 60.0 40.0 35.00 60.0 40.0 40.00 30.0 70.0 46.00 30.0 70.0 46.10 80.0 20.0 60.00 80.0 20.0

[0164] Conventional column chromatography conditions are as follows: the filler is 200-400 mesh amorphous silica gel, and the column chromatography is performed at room temperature.

[0165] In the following examples, room temperature refers to 20-30°C.

[0166] Example 1 Synthesis of Compound LE14

[0167]

[0168] Step 1: Synthesis of compound of formula VI

[0169]

[0170] A mixture of the compound of formula VII (25.0 g, 53.36 mmol) and paraformaldehyde (2.09 g, 69.37 mmol) was dissolved in 250.0 mL of anhydrous 1,4-dioxane. Trimethylsilyl chloride (14.43 g, 133.40 mmol) was slowly added and the mixture was reacted at 18-25°C for 15.0 h to obtain a crude solution of the compound of formula VI. The reaction solution was sampled for central control testing. After the reaction was complete, the reaction solution was filtered and the filtrate was concentrated to obtain a crude solution of the compound of formula VI (28.40 g). (Used directly in the next reaction)

[0171] Step 2: Synthesis of compound of formula IVb

[0172]

[0173] The crude compound of Formula VI (28.40 g, 53.36 mmol, calculated based on theoretical yield) was dissolved in 250.0 mL of anhydrous tetrahydrofuran, and panpiperidin (12.43 g, 80.05 mmol) and the compound of Formula Vb (14.12 g, 80.05 mmol) were added. The reaction was heated to 60°C under nitrogen and allowed to react for 4.0 to 8.0 hours. After the reaction was complete, the tetrahydrofuran was removed by concentration under reduced pressure, and then the mixture was extracted with ethyl acetate and saturated brine. The resulting organic phase was dried and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (n-heptane:ethyl acetate = 10:1 to 1:1 (v / v)) to obtain the compound of Formula IVb (17.88 g, purity 92.81%, combined yield of Steps 1 and 2 51.0%).

[0174] ESI-MS m / z: 657.4 (M+H);

[0175] 1H NMR (400MHz, DMSO-d6) δ10.12(s,1H),8.49(d,J=7.0Hz,1H),7.59(d,J=8.6Hz,2H),7.42–7.21(m ,2H),5.15–4.97(m,2H),4.85(s,2H),4.46(p,J=7.0Hz,1H),4.12(dd,J=29.5,10.7Hz,4H),3.68( dd,J=8.7,6.0Hz,2H),3.49(d,J=8.2Hz,1H),3.40(t,J=7.3Hz,2H),2.97(d,J=26.2Hz,3H),1.33 (d,J=7.1Hz,3H),1.29–1.18(m,2H),0.93(dd,J=9.6,6.6Hz,6H),0.87–0.82(m,1H),0.15(s,9H).

[0176] Step 3: Synthesis of compound of formula II

[0177]

[0178] Method 1: Deprotection using potassium fluoride reagent

[0179] Dissolve the compound of Formula IVb (5.0 g, 7.62 mmol) in 50.0 mL of DMF. Add potassium fluoride (0.67 g, 11.42 mmol) at room temperature to form a mixed solution. Heat the reaction to 60°C under nitrogen and allow to react for 5.0 to 8.0 hours. After the reaction is complete, concentrate under reduced pressure to remove the solvent. The resulting crude product is purified by silica gel column chromatography (dichloromethane:methanol = 10:1 to 1:1 (v / v)) to yield the compound of Formula II (3.78 g, 89.1% yield).

[0180] ESI-MS m / z: 556.0 (MH);

[0181] 1H NMR (400 MHz, DMSO-d6) δ ppm 10.57(d,J=38.3Hz,1H),9.36(d,J=94.5Hz,1H),7.61(d,J=8.5Hz,2H),7.28 (d,J=7.2Hz,2H),5.01(d,J=10.8Hz,2H),4.83(s,2H),4.38(s,1H),4.17(s,1 H),3.71(s,2H),3.54(s,2H),3.48(d,J=8.3Hz,1H),3.44(s,1H),3.14(s,2H) ,2.95(d,J=22.9Hz,3H),1.32(d,J=7.2Hz,3H),0.89(dd,J=14.7,6.7Hz,6H).

[0182] Method 2: Deprotection using sodium fluoride reagent

[0183] The operation steps and reaction conditions were the same as those in method 1, except that potassium fluoride was replaced by an equivalent amount of sodium fluoride. The amounts of raw materials and solvents used were the same as those in method 1, and the compound of formula II (2.20 g, yield 52.0%) was obtained by purification.

[0184] Method 3: Deprotection using cesium fluoride reagent

[0185] The operation steps and reaction conditions were the same as those in method 1, except that potassium fluoride was replaced by an equivalent amount of cesium fluoride. The amounts of raw materials and solvents used were the same as those in method 1, and the compound of formula II (1.84 g, yield 43.5%) was obtained by purification.

[0186] Method 4: Deprotection using tetrabutylammonium fluoride reagent

[0187] Dissolve the compound of Formula IVb (5.0 g, 7.62 mmol) in tetrahydrofuran (50.0 mL). Add a 1.0 M solution of tetrabutylammonium fluoride in tetrahydrofuran (11.5 mL, 11.43 mmol) at room temperature. Slowly raise the temperature to 40°C under nitrogen and allow to react for 8.0 h. The solvent is removed by concentration under reduced pressure. The resulting crude product is purified by silica gel column chromatography (dichloromethane:methanol = 10:1 to 1:1 (v / v)) to afford the compound of Formula II (2.99 g, 70.3% yield).

[0188] ESI-MS m / z: 557.5 (M+H).

[0189] Step 4: Synthesis of compound of formula I

[0190]

[0191] Dissolve the compound of Formula II (2.79 g, 5.00 mmol) in 20.0 mL of anhydrous DMF, add 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) (2.08 g, 7.50 mmol), and allow to react at room temperature for 1.0 h. Then, add N,N-diisopropylethylamine (1.30 mL, 7.50 mmol) and the compound of Formula III (Exatecan methanesulfonate) (2.40 g, 4.50 mmol) and continue the reaction for 1.5-2.0 h. After the reaction is complete, concentrate under reduced pressure to remove the solvent. The resulting crude product is purified by silica gel column chromatography (chloroform:methanol = 10:1 (v / v)) to obtain the compound of Formula I (3.44 g, 91.30% purity, 70.5% yield).

[0192] ESI-MS m / z: 974.5 (M+H).

[0193] Step 5: Synthesis of compound of formula VIII

[0194]

[0195] A mixture of 2.1 mL of a 1.0 mol / L trimethylphosphine tetrahydrofuran solution (2.1 mL, 2.01 mmol) and 5.0 mL of THF was added dropwise to 10.0 mL of sodium acetate buffer (pH = 5.0). The reaction was cooled to 0-5°C under nitrogen. Subsequently, 5.0 mL of tetrahydrofuran containing the compound of Formula I (1.30 g, 1.34 mmol) was slowly added dropwise to the reaction system. The reaction was maintained at 0-10°C for 0.5-1.0 h. After the reaction was complete, the tetrahydrofuran solvent was distilled off under reduced pressure, and the mixture was extracted with dichloromethane. The resulting organic phase was dried and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 10:1 to 1:1 (v / v)) to obtain the compound of Formula VIII (980 mg, purity 89.75%, yield 77.5%).

[0196] ESI-MS m / z: 948.7 (M+H);

[0197] Step 6: Synthesis of compound LE14

[0198]

[0199] Compound VIII (0.98 g, 1.04 mmol) was dissolved in 30.0 mL of anhydrous dichloromethane, and succinimidyl 6-(maleimido)hexanoate (0.64 g, 2.08 mmol) was added. The reaction was heated to 35°C under nitrogen and allowed to react for 3-6 hours. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 10:1 to 1:1 (v / v)) to afford compound LE14 (861 mg, 99.66% purity, 72.5% yield).

[0200] ESI-MS m / z: 1141.6 (M+H);

[0201] 1 H NMR(500MHz,DMSO-d6)δppm 9.93(s,1H),8.49(s,1H),8.13(d,J=6.9Hz,1H),7.76(d,J=28.7Hz,2H),7.57(s,2H),7.28(s,3H),6.99(s,2H),6.48(s,1 H),5.59(s,1H),5.47–5.32(m,2H),5.17(s,2H),5.04–4.79(m,4H),4.38(d,J=7.1Hz,1H),4.21–4.14(m,1H),4.03(s,2H), 3.69(d,J=2.8Hz,2H),3.37(dt,J=14.2,7.7Hz,4H),3.24–3.09(m,2H),2.93(d,J=37.1Hz,3H),2.36(t,J=1.8Hz,3H),2.2 2–2.07(m,4H),2.01–1.77(m,3H),1.52–1.41(m,4H),1.30(d,J=7.1Hz,3H),1.19(d,J=7.7Hz,2H),0.84(d,J=12.0Hz,9H).

[0202] Example 2

[0203] The other feeds and reaction conditions remained unchanged, and the crude product of the compound of formula II was prepared according to the methods of steps 1 to 3 of Example 1 (the reaction solution in step 3 was directly concentrated without purification) and directly subjected to subsequent reactions. The operations of steps 4 to 6 of Example 1 were followed to finally obtain the purified compound LE14 (894 mg, purity 95.46%, yield 75.3%).

[0204] Example 3

[0205]

[0206] The synthesis steps of IVb-1 were similar to steps 1 and 2 in Example 1, except that Vb was replaced with Vb-1 (ethyl hydroxyacetate) to obtain compound IVb-1 (18.28 g, purity 94.10%, combined yield of steps 1 and 2 58.6%). The synthesis steps of compound II were as follows:

[0207] The compound of formula IVb-1 (5.9 g, 10.00 mmol) was dissolved in 50.0 mL of methanol, and an aqueous lithium hydroxide solution (360 mg of lithium hydroxide was dissolved in 10 mL of water) was added at 0-10°C. After the addition was completed, the reaction was carried out at 20-30°C for 3-4 hours. After the reaction of the raw materials was monitored, the methanol was removed by concentration under reduced pressure. A 1 mol / L aqueous ammonium chloride solution (15 mL) was added dropwise to the concentrate for neutralization. The resulting mixture was extracted with a dichloromethane / water system and the organic phase was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain a compound of formula II.

[0208] Example 4 Comprehensive Comparison of Process Routes

[0209] 1. Comparison of carboxylic acid intermediates prepared by different routes

[0210] The crude carboxylic acid intermediate 1-4 obtained according to the method of Route 1 was purified by silica gel column chromatography (chloroform:methanol=10:1 (v / v)), but no purified intermediate 1-4 was obtained. The crude carboxylic acid intermediate 2-2a obtained according to the method of Route 2 was purified by silica gel column chromatography (chloroform:methanol=10:1 (v / v)), but no purified intermediate 2-2a was obtained. However, the carboxylic acid intermediate II was obtained in a good yield according to the method of Example 1. The purification status of the carboxylic acid intermediates prepared by the three synthetic routes is summarized and compared in Table 3 below.

[0211] 2. Comparison of compound LE14 prepared by different routes

[0212] The purity of compound LE14 synthesized according to Example 1 and Example 2 was compared with that of compound LE14 obtained by Route 1, Route 2, Route 3, Route 4 and Route 5 by HPLC. The results are shown in Table 4 below.

[0213] The data in Table 4 show that compound LE14 was prepared according to the method of Example 1 of the present invention. Due to the replacement of the protecting group, the deprotection conditions were milder, and the resulting carboxylic acid intermediate was able to exist stably. This not only reduced the maximum single impurity content, but also increased the purity of the compound LE14 product to over 99.5%, which was superior to the methods of Routes 1 to 5 (the maximum single impurity content was greater than 0.3%, and the purity of the compound LE14 product was less than 98.7%). In Example 2, the compound of Formula II in Step 3 was used directly in the subsequent reaction without purification. The maximum single impurity content of the obtained compound LE14 product was greater than 1%, and the product purity was only 95.46%. This indicates that whether or not the crude compound of Formula II in Step 3 is purified has a significant impact on the purity and maximum single impurity content of the LE14 product.

[0214] In the present invention, the crude compound IVb obtained by protecting the carboxylic acid with a trimethylsilyl group in step 2 of Example 1 can stably exist in the compound IVb. The crude compound IVb can be purified by conventional column chromatography. During the purification process, the compound IVb is stable and almost never lost, and a purified compound of formula IVb is obtained, thereby avoiding the introduction of impurities into subsequent reaction steps.

[0215] In the present invention, the carboxylic acid intermediate compound II can withstand the conditions of deprotection in method 1 in step 3, and can also stably exist under the conditions of the crude compound II obtained by deprotection according to method 1 in step 3 and conventional column chromatography conditions. The crude compound II is purified by conventional column chromatography to obtain a carboxylic acid intermediate II with high purity and low impurity content, thereby avoiding the introduction of impurities into subsequent reaction steps, thereby improving the purity of the final product of compound LE14 while reducing the maximum single impurity, and obtaining a final product of compound LE14 with a purity greater than 99.5% and a maximum single impurity content less than 0.1%.

[0216] Liquid phase conditions used: Phase A: 0.01 mol / L potassium dihydrogen phosphate (pH 5.0), Phase B: 10% methanol in acetonitrile, Detection wavelength: 254 nm, Instrument: Agilent 187260, Column: Agilent AdvanceBio PeptideMap, 3.5 μm, 2.1 × 250 mm, Flow rate: 1.0 mL / min. Gradient settings are shown in Table 2.

[0217] Table 2. Mobile phase gradient settings

[0218] Time (min) Mobile phase A% Mobile phase B% 0.00 80.0 20.0 5.00 60.0 40.0 35.00 60.0 40.0 40.00 30.0 70.0 46.00 30.0 70.0 46.10 80.0 20.0 60.00 80.0 20.0

[0219] Table 3. Comparison of carboxylic acid intermediates (1-4, 2-2a, II) prepared by four different process routes

[0220]

[0221]

[0222] Table 4. Comparative data of purity of final product LE14 from different process routes

[0223]

[0224] In the prior art, synthetic routes 3, 4, and 5 all use the same intermediate as the present invention (compound 5 has the same structure as compound VII), and the final product LE14 can be prepared from the same intermediate. The final product LE14 was prepared by the three routes, and the total yield of the routes and the purity of the final product are summarized in Table 5.

[0225] Table 5. Comparison of the purity of the final product of different process routes and the total yield of the same compound 5 (VII) as the starting material

[0226]

[0227] In the prior art synthesis routes of CN115215921A and CN115385926A, the total yield of the routes for synthesizing the final product 1 using ixetec as the starting material is lower than that of the present invention, as shown in Table 6.

[0228] Table 6. Comparison of total yields of different process routes based on the input of ixetem

[0229]

[0230] As can be seen from Tables 5 and 6, the purity of the products prepared in Route 3, Route 4, and Route 5 is far inferior to that of the present invention; Route 3 uses DXd, which is more expensive than isotecan, as a payload source. Although it saves reaction steps, the yield of Route 3 is far lower than that of the present invention and the utilization rate of DXd is not high. In Route 4, Route 5, and the synthetic routes of the present invention, isotecan is the key material with the largest cost share in each process route and is also the main cost control point. Route 4 and 5 both derivatize isotecan. On the one hand, the process reaction steps are more and the production operation is cumbersome. On the other hand, isotecan loss is also relatively high during the preparation process. The total yield of the routes calculated using isotecan as the starting material is far lower than that of the routes of the present invention. Therefore, in Route 4 and Route 5, the utilization rate of the expensive material isotecan is low, resulting in high production costs and is not suitable for large-scale industrial production.

[0231] The synthetic route of the present invention can not only provide a high-purity LE14 compound, but also significantly reduce the production cost by improving the utilization rate of exotecan, and is more suitable for large-scale industrial production.

[0232] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for preparing a compound of formula I, characterized in that: The method comprises the following steps: subjecting a compound of formula II to an amide condensation reaction with a compound of formula III or its mesylate in the presence of a condensing agent, a base and a solvent to obtain a compound of formula I; The preparation method of the compound of formula I also includes a preparation method of the compound of formula II, which comprises the following steps: in a solvent, subjecting the compound of formula IV to a deprotection reaction in the presence of a deprotecting agent to obtain the compound of formula II; Wherein, R is a C1~C6 alkyl group substituted by -Si(C1~C6)3.

2. The preparation method according to claim 1, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula I, the amide condensation reaction further comprises the following specific steps: dissolving the compound of formula II in a solvent, adding the condensing agent, and immediately or after a period of reaction, adding the compound of formula III or its mesylate and the base; (2) In the method for preparing the compound of formula I, the condensation reaction is kept away from light throughout the entire process; (3) In the method for preparing the compound of formula I, the molar ratio of the compound of formula III to the compound of formula II is 0.8-1.5; (4) In the preparation method of the compound of formula I, the condensing agent is 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride, diethyl cyanophosphate, diphenyl phosphorazide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'- One of tetramethyluronium hexafluorophosphate, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate, 2-(1H-benzotriazol L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate quaternary ammonium salt, or a mixture of any two or more thereof; (5) In the method for preparing the compound of formula I, the molar ratio of the condensing agent to the compound of formula II is 1-3; (6) In the method for preparing the compound of formula I, the molar ratio of the base to the compound of formula II is 1-10; (7) In the method for preparing the compound of formula I, the solvent is one of N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran and 1,4-dioxane, or a mixture of any two or more thereof; (8) In the method for preparing the compound of formula I, the temperature of the condensation reaction is 20°C-50°C; (9) In the method for preparing the compound of formula I, the condensation reaction is carried out under the protection of an inert gas.

3. The preparation method according to claim 1, wherein In the preparation method of the compound of formula I, the base is an organic base, an inorganic base or a mixture thereof.

4. The preparation method according to claim 1, wherein In the method for preparing the compound of formula I, after the condensation reaction is completed, the following post-treatment step is further included: concentrating the organic phase to obtain a crude product of the compound of formula I.

5. The preparation method according to claim 2, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula I, the molar ratio of the compound of formula III to the compound of formula II is 0.9-1.2; (2) In the method for preparing the compound of formula I, the condensing agent is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride; (3) In the method for preparing the compound of formula I, the molar ratio of the condensing agent to the compound of formula II is 1-1.5; (4) In the method for preparing the compound of formula I, the molar ratio of the base to the compound of formula II is 1-6; (5) In the method for preparing the compound of formula I, the solvent is N,N-dimethylformamide; (6) In the method for preparing the compound of formula I, the temperature of the condensation reaction is 20°C-30°C; (7) In the method for preparing the compound of formula I, the condensation reaction is carried out in a nitrogen or helium environment.

6. The preparation method according to claim 3, wherein In the preparation method of the compound of formula I, the base is an organic base.

7. The preparation method according to claim 4, wherein In the preparation method of the compound of formula I, the crude compound of formula I is purified by silica gel column chromatography to obtain the product compound of formula I.

8. The preparation method according to claim 5, wherein The preparation method meets one or both of the following conditions: (1) In the method for preparing the compound of formula I, the molar ratio of the compound of formula III to the compound of formula II is 0.9-1.0; (2) In the method for preparing the compound of formula I, the molar ratio of the base to the compound of formula II is 1-3.

9. The preparation method according to claim 6, wherein In the preparation method of the compound of formula I, the organic base is one of N,N-diisopropylethylamine, triethylamine and pyridine, or a mixture of any two or more thereof.

10. The preparation method according to claim 7, wherein In the preparation method of the compound of formula I, the eluent for the silica gel column chromatography is a mixed solvent of chloroform and methanol, and the volume ratio of chloroform to methanol is (100-10):

1.

11. The preparation method according to claim 8, characterized in that In the preparation method of the compound of formula I, the molar ratio of the base to the compound of formula II is 1.

5.

12. The preparation method according to claim 9, wherein In the preparation method of the compound of formula I, the organic base is N,N-diisopropylethylamine.

13. The preparation method according to claim 10, wherein In the preparation method of the compound of formula I, the eluent for the silica gel column chromatography is a mixed solvent of chloroform and methanol, and the volume ratio of chloroform to methanol is 10:

1.

14. The preparation method according to claim 3, wherein In the preparation method of the compound of formula I, the inorganic base is one of alkali metal hydroxides, alkali metal carbonates and alkali metal phosphates, or a mixture of any two or more thereof.

15. The preparation method according to claim 14, wherein In the preparation method of the compound of formula I, the inorganic base is one of sodium bicarbonate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and potassium hydroxide, or a mixture of any two or more thereof.

16. The preparation method according to claim 1, wherein The preparation method of the compound of formula I also includes a preparation method of the compound of formula IV, which comprises the following steps: conducting an etherification reaction of the compound of formula VI with a reagent V in a solvent in the presence of a base to obtain a compound of formula IV; Wherein, R is a C1~C6 alkyl group substituted by -Si(C1~C6)3.

17. The preparation method according to claim 16, wherein The preparation method of the compound of formula I also includes a preparation method of the compound of formula VI, which comprises the following steps: subjecting the compound of formula VII to a substitution reaction with paraformaldehyde and trimethylsilyl chloride in a solvent to obtain a compound of formula VI; 18. The preparation method according to claim 1, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula II, the deprotection reaction further comprises the following specific steps: dissolving the compound of formula IV in a solvent, adding the deprotecting agent, and heating to initiate the reaction; or dissolving the deprotecting agent in a solvent, adding the compound of formula IV, and heating to initiate the reaction; (2) In the preparation method of the compound of formula II, the C1-C6 alkyl substituted by -Si(C1-C6)3 is trimethylsilylethyl or tert-butyldimethylsilylethyl; (3) In the preparation method of the compound of formula II, the deprotecting agent is a fluorine reagent, and the fluorine reagent is tetrabutylammonium fluoride, tetramethylammonium fluoride, tetrabutylammonium fluoride / acetic acid, pyridine hydrogen fluoride complex, tert-butylammonium fluoride, tert-butylammonium fluoride / acetic acid, tetraethylammonium fluoride, or commercially available tetramethylammonium fluoride / tetrahydrofuran solution, tetraethylammonium fluoride / tetrahydrofuran solution, tetrabutylammonium fluoride in tetrahydrofuran solution, or one or more of silicon tetrafluoride, potassium fluoride, sodium fluoride, lithium fluoride and cesium fluoride; (4) In the method for preparing the compound of formula II, the molar ratio of the deprotecting agent to the compound of formula IV is 1-5; (5) In the method for preparing the compound of formula II, the solvent is one of purified water, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran and 1,4-dioxane, or a mixture of any two or more thereof; (6) In the method for preparing the compound of formula II, the temperature of the deprotection reaction is 20°C-80°C; In the method for preparing the compound of formula II described in (7), the deprotection reaction is carried out under the protection of an inert gas.

19. The preparation method according to claim 1, wherein The method for preparing the compound of formula II further comprises a post-treatment step after the reaction is completed: concentrating the reaction solution to obtain a crude product of the compound of formula II.

20. The preparation method according to claim 16, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula IV, the etherification reaction further comprises the following specific steps: dissolving the compound of formula VI in a solvent, adding the base, and then immediately or after a period of reaction, adding the reagent V; (2) In the method for preparing the compound of formula IV, the molar ratio of the reagent V to the compound of formula VI is 1-5; (3) In the method for preparing the compound of formula IV, the molar ratio of the base to the compound of formula VI is 1-5; (4) In the method for preparing the compound of formula IV, the solvent is one of N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran and 1,4-dioxane, or a mixture of any two or more thereof; (5) In the method for preparing the compound of formula IV, the temperature of the etherification reaction is 0°C-80°C; (6) In the method for preparing the compound of formula IV, the etherification reaction is carried out under the protection of an inert gas.

21. The preparation method according to claim 16, wherein In the preparation method of the compound of formula IV, the base is an organic base, an inorganic base or a mixture thereof.

22. The preparation method according to claim 16, wherein In the preparation method of the compound of formula IV, after the reaction is completed, a post-treatment step is further included: extracting the reaction solution and concentrating the organic phase to obtain a crude product of the compound of formula IV, or directly concentrating the reaction solution to obtain a crude product of the compound of formula IV.

23. The preparation method according to claim 17, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula VI, the molar ratio of the paraformaldehyde to the compound of formula VII is 1-10; (2) In the method for preparing the compound of formula VI, the molar ratio of trimethylsilyl chloride to the compound of formula VII is 1-5; (3) In the method for preparing the compound of formula VI, the solvent is one of N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran and 1,4-dioxane, or a mixture of any two or more thereof; (4) In the method for preparing the compound of formula VI, the temperature of the substitution reaction is -10°C to 50°C; (5) In the method for preparing the compound of formula VI, the substitution reaction is carried out under the protection of an inert gas.

24. The preparation method according to claim 17, wherein The method for preparing the compound of formula VI further comprises a post-treatment step after the reaction is completed: performing solid-liquid separation on the reaction solution or not performing solid-liquid separation, and concentrating the organic phase to obtain the compound of formula VI.

25. The preparation method according to claim 18, wherein The preparation method meets one or more of the following conditions: (1) In the preparation method of the compound of formula II, the C1-C6 alkyl substituted by -Si(C1-C6)3 is trimethylsilylethyl; (2) In the preparation method of the compound of formula II, the deprotecting agent is a fluorine reagent, and the fluorine reagent is a 1M tetrabutylammonium fluoride tetrahydrofuran solution or potassium fluoride; (3) In the method for preparing the compound of formula II, the molar ratio of the deprotecting agent to the compound of formula IV is 1-3; (4) In the method for preparing the compound of formula II, the solvent is N,N-dimethylformamide or tetrahydrofuran; (5) In the method for preparing the compound of formula II, the temperature of the deprotection reaction is 30°C-70°C; (6) In the method for preparing the compound of formula II, the deprotection reaction is carried out in a nitrogen or helium environment.

26. The preparation method according to claim 19, wherein In the preparation method of the compound of formula II, the crude compound of formula II is purified by silica gel column chromatography to obtain the product compound of formula II.

27. The preparation method according to claim 20, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula IV, the molar ratio of the reagent V to the compound of formula VI is 1-3; (2) In the method for preparing the compound of formula IV, the molar ratio of the base to the compound of formula VI is 1.2-4; (3) In the method for preparing the compound of formula IV, the solvent is tetrahydrofuran or 1,4-dioxane; (4) In the method for preparing the compound of formula IV, the temperature of the etherification reaction is 40°C-70°C; (5) In the method for preparing the compound of formula IV, the etherification reaction is carried out in a nitrogen or helium environment.

28. The preparation method according to claim 21, wherein In the preparation method of the compound of formula IV, the base is an organic base.

29. The preparation method according to claim 22, wherein In the preparation method of the compound of formula IV, the crude compound of formula IV is purified by silica gel column chromatography to obtain the compound IV product.

30. The preparation method according to claim 23, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula VI, the molar ratio of the paraformaldehyde to the compound of formula VII is 1-5; (2) In the preparation method of the compound of formula VI, the molar ratio of trimethylsilyl chloride to the compound of formula VII is 2-4; (3) In the method for preparing the compound of formula VI, the solvent is tetrahydrofuran or 1,4-dioxane; (4) In the method for preparing the compound of formula VI, the temperature of the substitution reaction is 15°C-35°C; (5) In the method for preparing the compound of formula VI, the substitution reaction is carried out in a nitrogen or helium environment.

31. The preparation method according to claim 24, wherein In the preparation method of the compound of formula VI, the compound of formula VI obtained after concentration is directly subjected to subsequent reactions.

32. The preparation method according to claim 25, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula II, the molar ratio of the deprotecting agent to the compound of formula IV is 1.1-2.0; (2) In the method for preparing the compound of formula II, the solvent is N,N-dimethylformamide; (3) In the method for preparing the compound of formula II, the temperature of the deprotection reaction is 40°C-60°C.

33. The preparation method according to claim 26, wherein In the preparation method of the compound of formula II, the eluent used in the silica gel column chromatography is a mixed solvent of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is (100-1):

1.

34. The preparation method according to claim 27, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula IV, the molar ratio of the reagent V to the compound of formula VI is 1.1-1.6; (2) In the method for preparing the compound of formula IV, the molar ratio of the base to the compound of formula VI is 1.5-3; (3) In the method for preparing the compound of formula IV, the solvent is tetrahydrofuran; (4) In the method for preparing the compound of formula IV, the temperature of the etherification reaction is 60°C.

35. The preparation method according to claim 28, wherein In the preparation method of the compound of formula IV, the organic base is one of potassium tert-butoxide, triethylamine, 4-dimethylaminopyridine, pyridine and panpiperidin, or a mixture of any two or more thereof.

36. The preparation method according to claim 29, wherein In the preparation method of the compound of formula IV, the eluent used in the silica gel column chromatography is a mixed solution of n-heptane and ethyl acetate, and the volume ratio of n-heptane to ethyl acetate is (20-1):

1.

37. The preparation method according to claim 30, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula VI, the molar ratio of the paraformaldehyde to the compound of formula VII is 1.3-3.0; (2) In the preparation method of the compound of formula VI, the molar ratio of trimethylsilyl chloride to the compound of formula VII is 2-3; (3) In the method for preparing the compound of formula VI, the solvent is 1,4-dioxane; (4) In the method for preparing the compound of formula VI, the temperature of the substitution reaction is 18°C-25°C.

38. The preparation method according to claim 32, wherein In the preparation method of the compound of formula II, the molar ratio of the deprotecting agent to the compound of formula IV is 1.

5.

39. The preparation method according to claim 33, wherein In the preparation method of the compound of formula II, the eluent used in the silica gel column chromatography is a mixed solvent of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is (10-1):

1.

40. The preparation method according to claim 34, wherein In the preparation method of the compound of formula IV, the molar ratio of the reagent V to the compound of formula VI is 1.

5.

41. The preparation method according to claim 35, wherein In the preparation method of the compound of formula IV, the organic base is panpiidine.

42. The preparation method according to claim 36, wherein In the preparation method of the compound of formula IV, the eluent used in the silica gel column chromatography is a mixed solution of n-heptane and ethyl acetate, and the volume ratio of n-heptane to ethyl acetate is (10-1):

1.

43. The preparation method according to claim 37, wherein The preparation method meets one or both of the following conditions: (1) In the method for preparing the compound of formula VI, the molar ratio of the paraformaldehyde to the compound of formula VII is 1.3; (2) In the preparation method of the compound of formula VI, the molar ratio of trimethylsilyl chloride to the compound of formula VII is 2.

5.

44. The preparation method according to claim 21, wherein In the preparation method of the compound of formula IV, the inorganic base is one of alkali metal hydroxides, alkali metal carbonates and alkali metal phosphates, or a mixture of any two or more thereof.

45. A method for preparing a compound of formula VIII, characterized in that: The method comprises the following steps: performing a reduction reaction on a compound of formula I and a reducing agent in a solvent and in the presence of an acid buffer to obtain a compound of formula VIII; Wherein, the preparation method also includes the preparation method as described in any one of claims 1-44.

46. ​​The preparation method according to claim 45, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula VIII, the reducing agent is triphenylphosphine, tri-tert-butylphosphine or trimethylphosphine, or a triphenylphosphine / tetrahydrofuran solution, a tri-tert-butylphosphine / tetrahydrofuran solution or a trimethylphosphine / tetrahydrofuran solution; (2) In the method for preparing the compound of formula VIII, the acid buffer is sodium acetate buffer or sodium formate buffer; (3) In the method for preparing the compound of formula VIII, the molar ratio of the reducing agent to the compound of formula I is 1.0-3.0; (4) In the method for preparing the compound of formula VIII, the volume mass ratio of the acid buffer to the compound of formula I is 2-10 mL / g; (5) In the method for preparing the compound of formula VIII, the solvent is an ether solvent; (6) In the method for preparing the compound of formula VIII, the temperature of the reduction reaction is 0°C-20°C.

47. The preparation method according to claim 45, wherein The preparation method of the compound of formula VIII further includes a post-treatment step after the reaction is completed: extracting the reaction solution and concentrating the organic phase to obtain a crude compound of formula VIII, or directly concentrating the reaction solution to obtain a crude compound of formula VIII.

48. The preparation method according to claim 46, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula VIII, the reducing agent is a tetrahydrofuran solution of trimethylphosphine; (2) In the method for preparing the compound of formula VIII, the acid buffer is a sodium acetate buffer; (3) In the method for preparing the compound of formula VIII, the molar ratio of the reducing agent to the compound of formula I is 1.2-1.8; (4) In the preparation method of the compound of formula VIII, the volume mass ratio of the acid buffer to the compound of formula I is 5-10 mL / g; (5) In the method for preparing the compound of formula VIII, the solvent is one of tetrahydrofuran, diethyl ether, 1,4-dioxane, anisole, and methyl tert-butyl ether, or a mixture of any two or more thereof; (6) In the method for preparing the compound of formula VIII, the temperature of the reduction reaction is 0°C-10°C.

49. The preparation method according to claim 47, wherein In the preparation method of the compound of formula VIII, the crude compound of formula VIII is purified by silica gel column chromatography to obtain the product compound of formula VIII.

50. The preparation method according to claim 48, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula VIII, the reducing agent is a 1M trimethylphosphine solution in tetrahydrofuran; (2) In the method for preparing the compound of formula VIII, the pH of the acid buffer is 4.0-6.0; (3) In the method for preparing the compound of formula VIII, the molar ratio of the reducing agent to the compound of formula I is 1.5; (4) In the method for preparing the compound of formula VIII, the volume mass ratio of the acid buffer to the compound of formula I is 7-8 mL / g; (5) In the method for preparing the compound of formula VIII, the solvent is tetrahydrofuran.

51. The preparation method according to claim 49, wherein In the preparation method of the compound of formula VIII, the eluent used in the silica gel column chromatography is a mixed solvent of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is (100-1):

1.

52. The preparation method according to claim 50, wherein The preparation method meets one or both of the following conditions: (1) In the method for preparing the compound of formula VIII, the pH of the acid buffer solution is 4.5-5.5; (2) In the preparation method of the compound of formula VIII, the volume mass ratio of the acid buffer solution to the compound of formula I is 7.7 mL / g.

53. The preparation method according to claim 51, wherein In the preparation method of the compound of formula VIII, the eluent used in the silica gel column chromatography is a mixed solvent of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is (10-1):

1.

54. The preparation method according to claim 52, wherein In the preparation method of the compound of formula VIII, the pH of the acid buffer solution is 5.

0.

55. A method for preparing compound LE14, characterized in that: The method comprises the following steps: coupling a compound of formula VIII and 6-(maleimido)hexanoic acid succinimidyl ester in a solvent to obtain compound LE14; Wherein, the preparation method also includes the preparation method as described in any one of claims 45-54.

56. The preparation method according to claim 55, wherein The preparation method meets one or more of the following conditions: (1) In the preparation method of compound LE14, the molar ratio of 6-(maleimido)hexanoic acid succinimidyl ester to the compound of formula VIII is 1-5; (2) In the method for preparing compound LE14, the solvent is one of an amide solvent, a chlorinated alkane solvent, an ether solvent, and a nitrile solvent, or a mixture of any two or more thereof; (3) In the preparation method of compound LE14, the coupling reaction temperature is 0°C-45°C.

57. The preparation method according to claim 55, wherein The preparation method of compound LE14 further includes a post-treatment step after the reaction is completed: the reaction solution is concentrated to obtain a crude product of compound LE14.

58. The preparation method according to claim 56, wherein The preparation method meets one or more of the following conditions: (1) In the preparation method of compound LE14, the molar ratio of 6-(maleimido)hexanoic acid succinimidyl ester to the compound of formula VIII is 1-3; (2) In the preparation method of compound LE14, the solvent is a chlorinated alkane solvent; (3) In the preparation method of compound LE14, the coupling reaction temperature is 25°C-40°C.

59. The preparation method according to claim 57, wherein In the preparation method of the compound LE14, the crude compound LE14 is purified by silica gel column chromatography to obtain the compound LE14 product.

60. The preparation method according to claim 58, wherein The preparation method meets one or more of the following conditions: (1) In the preparation method of compound LE14, the molar ratio of 6-(maleimido)hexanoic acid succinimidyl ester to the compound of formula VIII is 1.2-2.0; (2) In the preparation method of compound LE14, the solvent is a chlorinated alkane solvent; the chlorinated alkane solvent is one of dichloromethane, 1,2-dichloroethane and chloroform, or a mixture of any two or more thereof; (3) In the preparation method of compound LE14, the coupling reaction temperature is 30°C-35°C.

61. The preparation method according to claim 59, wherein In the preparation method of the compound LE14, the eluent used in the silica gel column chromatography is a mixed solvent of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is (100-1):

1.

62. The preparation method according to claim 60, wherein In the preparation method of compound LE14, the solvent is a chlorinated alkane solvent; the chlorinated alkane solvent is dichloromethane.

63. The preparation method according to claim 61, wherein In the preparation method of the compound LE14, the eluent used in the silica gel column chromatography is a mixed solvent of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is (10-1):

1.

64. A method for preparing a compound of formula II, characterized in that: The method comprises the following steps: subjecting a compound of formula IV to a deprotection reaction in the presence of a deprotecting agent in a solvent to obtain a compound of formula II; the reaction conditions are as described in any one of claims 18 to 19, 25 to 26, 32 to 33 or 38 to 39; wherein R is defined as in any one of claims 1, 18 or 25.

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