A method for preparing an intermediate of a linker-drug conjugate

Abstract

The present invention discloses a method for preparing an intermediate of a linker-drug conjugate, and a method for preparing compound LE using the intermediate. The preparation method of the present invention comprises the following steps: subjecting a compound of formula II to a deprotection reaction in the presence of a deprotecting agent in a solvent to obtain a compound of formula I; and subjecting the compound of formula I to an amide condensation reaction with a compound of formula VIII or its mesylate in the presence of a condensing agent, a base, and a solvent to obtain compound LE. The preparation method of the present invention has one or more of the following advantages: the compound of formula I is stable and does not decompose during the purification process; a high-purity compound of formula I can be obtained in a good yield; the compound of formula I acts as a linker and directly condenses with isotecan, which not only greatly reduces the exposure time of cytotoxic substances, but also improves the utilization rate of isotecan and reduces production costs. Therefore, the process route of the present invention is reasonable and suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to a method for preparing an intermediate of a linker-drug conjugate. Background Art

[0002] Antibody-drug conjugates (ADCs) have become a drug pipeline that domestic and foreign pharmaceutical companies have been competing to develop in recent years, and have become one of the hot topics of concern in the pharmaceutical industry in recent years. ADC drugs are composed of three basic modules, including antibodies, linkers, and effector molecules (drugs). ADC drugs utilize the specific targeting effect of antibodies to transport effector molecules to the tumor site for enrichment, thereby achieving the purpose of killing tumor cells. The linker serves as a bridge for ADC drugs, connecting the drug to the antibody through a cleavable or non-cleavable structure, and utilizing the specific targeting effect of the antibody to transport the effector molecules to the tumor site for enrichment, thereby achieving the purpose of killing tumor cells. Therefore, the linker requires a sophisticated design. It must not only be stable under physiological conditions and not be degraded before reaching the targeted tumor site, but also be able to be effectively degraded after reaching the targeted tumor site to release the effector molecules.

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

[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 exatecan 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 LE.

[0007] In this route, in the step of preparing intermediate 1-4 using intermediate 1-3 as raw material, since both the raw material and the product are unstable to acidity and alkalinity, 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, which will cause obvious impurities in the crude product 1-5. The polarity of this impurity is close to that of the product, making purification difficult, and thus affecting the product quality of LE.

[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 exatecan 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 LE.

[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 acidity and alkalinity. 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 for the next reaction, significant impurities will be produced 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 LE.

[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 5-1 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]

[0052] In the improved synthetic route for compound LE14, we first synthesized the compound of formula I. As the linker portion of the ADC molecule, it has excellent stability and is a key intermediate in the production process of the entire compound LE14 linker drug conjugate. Its purity has an extremely important impact on the quality control of the final product. Using the compound of formula I as the starting material for subsequent industrial production not only avoids the use of expensive DXd or DXd derivatives, but also allows the cytotoxic payload to be placed at the end, thereby avoiding excessive exposure to cytotoxic substances, greatly reducing the protection level of production personnel and environmental pressure. In addition, the compound LE14 obtained in the subsequent process needs to be conjugated with the corresponding antibody. Therefore, obtaining a high-purity compound of formula I has positive significance for reducing the potential safety toxicity of the entire antibody-drug conjugate and improving the conversion efficiency of the cytotoxic payload.

[0053] The compound of Formula I is a carboxylic acid structure with high polarity and water solubility. In the improved route, the synthesis of the compound of Formula I generates impurities of similar polarity, which are difficult to remove. If the compound is directly subjected to subsequent reactions to produce Compound LE14 without further purification, impurities with polarity very similar to that of Compound LE14 will also be generated, making them difficult to remove. Furthermore, if the compound is conjugated to an antibody, these impurities become even more difficult to remove. Therefore, further purification of the compound of Formula I to obtain a higher-purity intermediate is essential for controlling the quality and impurity content of the linker-drug and ADC molecules.

[0054] In the early stages of exploring and discovering improved routes, conventional column chromatography was used to purify the compound of Formula I, but the following problems persisted: 1) the crude compound of Formula I was a dark brown, viscous, solid oil that was difficult to dissolve and not suitable for wet or dry loading; 2) the sample was extremely difficult to completely elute using conventional elution systems, affecting the yield of the compound of Formula I; and 3) impurities of similar polarity were difficult to separate and remove, thereby affecting the difficulty of post-processing and the overall yield of subsequent reactions.

[0055] Conventional column chromatography purification steps are as follows:

[0056] Step 1: The reaction solution of the compound of formula I obtained by the improved route is vacuum concentrated to obtain a crude oil, and the crude oil is ultrasonically dissolved with dichloromethane at room temperature (the volume mass ratio of dichloromethane to the crude oil is 10 mL / g), and then methyl tert-butyl ether is added thereto (the volume mass ratio of methyl tert-butyl ether to the crude oil is 30-50 mL / g), and stirred at room temperature for 2 to 5 hours. After standing, the supernatant is poured off, and the lower oil is vacuum concentrated to obtain a pretreated crude product, which is used for subsequent chromatographic purification.

[0057] Step 2: Purify the crude product of Formula I obtained in Step 1 by column chromatography using conventional normal-phase silica gel as the stationary phase (see Table 2 for specific parameters of the normal-phase silica gel). Specifically, dissolve the crude product of Formula I in a 10:1 (volume ratio) dichloromethane:methanol mixture and wet-load the mixture onto the chromatography column. (This process does not allow for complete dissolution and clarification of the sample due to poor sample condition, resulting in partially non-uniform sample.) Elution is initiated at room temperature using dichloromethane and methanol as the eluents, with an elution gradient of 60:1 to 10:1. The intermediate of Formula I is eluted by adjusting the eluent gradient, and fractions with a purity greater than 80.0% are collected.

[0058] Step 3: Concentrate the fraction obtained in step 2 under reduced pressure to obtain a purified sample of the compound of formula I.

[0059] After verification and evaluation, it was found that the above purification method has the following defects: the sample recovery rate is not high (the recovery rate is only 44.35%, where the recovery rate = the weight of the purified sample / the weight of the crude product before purification × 100%), and there are still many impurities in the purified sample of Formula I (the purity is only 83.06%), which increases the difficulty of subsequent reaction post-processing and reduces the yield. Summary of the Invention

[0060] To overcome the instability, high impurity content, inability to effectively purify, and prolonged exposure time of cytotoxic substances of the carboxylic acid intermediates of linker-drug conjugates in existing technology routes, the present invention provides a new carboxylic acid intermediate compound of formula I for linker-drug conjugates and a method for preparing the same. The intermediate of the present invention is stable and does not decompose during the purification process. The preparation method of the present invention, based on obtaining a high-purity intermediate compound of formula I with a good yield, condenses the intermediate with ixetine to produce a high-purity compound LE. This significantly reduces the exposure time of cytotoxic substances and improves the utilization rate of ixetine, reducing production costs. The process route of the present invention is reasonable and more suitable for large-scale industrial production.

[0061] The present invention mainly solves the above technical problems through the following technical solutions.

[0062] The present invention provides a compound of formula I;

[0063]

[0064] The present invention also provides a method for preparing compound LE, comprising the following steps: performing an amide condensation reaction of a compound of formula I with a compound of formula VIII or its mesylate in the presence of a condensing agent, a base and a solvent to obtain compound LE,

[0065]

[0066] Among them, R 2 C 1-6 alkyl;

[0067] R 3 It is a halogen.

[0068] In some embodiments, in the method for preparing compound LE, the reaction materials are the compound of formula I, the compound of formula VIII or its mesylate, the condensing agent, the base and the solvent.

[0069] In some embodiments, the R 2 It is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, or hexyl, preferably methyl or ethyl, more preferably methyl.

[0070] In some embodiments, the R 3 It is fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine, more preferably fluorine.

[0071] In some embodiments, the reaction conditions (e.g., solvent dosage, order and method of adding materials, and time of addition) in the preparation method of compound LE can be conventional conditions for such reactions in the art and can be adjusted according to the type of condensing agent. For example, the method can include the following steps: dissolving the compound of formula I in a solvent, adding a condensing agent and a base, and then adding the compound of formula VIII immediately or after a period of reaction. Preferably, the amide condensation reaction is performed in the dark throughout.

[0072] In some embodiments, in the method for preparing compound LE, the molar ratio of the compound of formula VIII to the compound of formula I can be 0.8-1.5, preferably 0.9-1.2, and more preferably 1.0.

[0073] In some embodiments, in the preparation method of the compound LE, the condensing agent can be 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (DMTMM), diethyl cyanide phosphorothioate (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 One of triazole-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 (DMTMM).

[0074] In some embodiments, in the method for preparing compound LE, the molar ratio of the condensing agent to the compound of formula I can be 1-3, preferably 1-1.5, and more preferably 1.5.

[0075] In some embodiments, in the preparation method of the compound LE, the base can be a conventional base for this type of reaction 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.

[0076] In some embodiments, in the method for preparing compound LE, the molar ratio of the base to the compound of formula I can be 1-10, preferably 1-5, more preferably 1.2-3, and most preferably 1.5.

[0077] In some embodiments, in the preparation method of the compound LE, 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.

[0078] In some embodiments, in the method for preparing compound LE, the temperature of the amide condensation reaction can be 20°C-50°C, preferably 20°C-30°C.

[0079] In some embodiments, in the method for preparing compound LE, the amide condensation reaction is preferably carried out under the protection of an inert gas, such as in a nitrogen or helium environment.

[0080] In some embodiments, in the preparation method of the compound LE, the progress of the amide 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 VIII is no longer detected.

[0081] In some embodiments, in the method for preparing compound LE, the amide condensation reaction time can be 1-8 hours, preferably 1-5 hours, and more preferably 1.5-3 hours.

[0082] In some embodiments, in the preparation method of the compound LE, after the amide condensation reaction is completed, the following post-processing step may be further included: concentrating the reaction solution to obtain a crude compound LE; preferably, the crude compound LE is purified by silica gel column chromatography to obtain a compound LE product. Further preferably, the eluent of the silica gel column chromatography is a mixture of chloroform and methanol, and the volume ratio of chloroform to methanol is (100-10):1, further preferably 10:1.

[0083] The preparation method of the compound LE may further include a preparation method of the compound of formula I, which may include the following steps: in a solvent, subjecting the compound of formula II to a deprotection reaction in the presence of a deprotecting agent to obtain the compound of formula I;

[0084]

[0085] Wherein, R is a C1-C6 alkyl group substituted with -Si(C1-C6)3.

[0086] In some embodiments, in the preparation method of the compound of formula I, the R can be trimethylsilylethyl or tert-butyldimethylsilylethyl; preferably, the R is trimethylsilylethyl.

[0087] In some embodiments, in the method for preparing the compound of formula I, the reaction materials are the compound of formula II, the deprotecting agent and the solvent.

[0088] In some embodiments, the reaction conditions in the method for preparing the compound of formula I (such as the amount of solvent, order of feeding, method of adding materials and 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 deprotecting agents; for example, the method can include the following steps: dissolving the compound of formula II in a solvent, adding the deprotecting agent, and then heating to start the reaction; or dissolving the deprotecting agent in a solvent, adding the compound of formula II, and then heating to start the reaction.

[0089] In some embodiments, in the preparation method of the compound of formula I, 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 tetramethylammonium fluoride / tetrahydrofuran solution, tetraethylammonium fluoride / tetrahydrofuran solution or tetrabutylammonium fluoride / tetrahydrofuran solution, or one of potassium fluoride, sodium fluoride, lithium fluoride and cesium fluoride, or a mixture of any two or more thereof, preferably 1M tetrabutylammonium fluoride / tetrahydrofuran solution or potassium fluoride.

[0090] In some embodiments, in the method for preparing the compound of formula I, the molar ratio of the deprotecting agent to the compound of formula II can be 1-5, preferably 1-3, more preferably 1.1-2.0, and most preferably 1.5.

[0091] 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 or tetrahydrofuran, and more preferably N,N-dimethylformamide.

[0092] In some embodiments, in the method for preparing the compound of formula I, the temperature of the deprotection reaction is 20°C-80°C, preferably 30°C-70°C, more preferably 50°C-70°C, and most preferably 60°C.

[0093] In some embodiments, in the method for preparing the compound of formula I, the deprotection reaction is preferably carried out under the protection of an inert gas, such as in a nitrogen or helium environment.

[0094] In some embodiments, in the method for preparing the compound of formula I, 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 II is no longer detected.

[0095] In some embodiments, in the method for preparing the compound of formula I, the deprotection reaction time can be 4-24 hours, preferably 5-16 hours, and more preferably 5-8 hours.

[0096] In some embodiments, the method for preparing the compound of formula I may further comprise a post-treatment step after the deprotection reaction is completed: separating the crude compound of formula I under high pressure preparative chromatography conditions to obtain the compound of formula I. The high pressure preparative chromatography conditions are as follows:

[0097] The chromatographic column is a dynamic axial compression column;

[0098] The inner diameter of the chromatographic column is 50 mm;

[0099] The column length is 650 mm;

[0100] The stationary phase is unbonded silanol;

[0101] The particle size of the stationary phase is 8-20 μm;

[0102] The pore size of the stationary phase is

[0103] The mobile phase is a mixed solvent A of a halogenated alkane solvent and an alcohol solvent; the volume ratio of the halogenated alkane solvent to the alcohol solvent is 96:4 to 90:10;

[0104] The purity of the crude compound of formula I is 70%-80%.

[0105] In some embodiments, in the method for preparing the compound of formula I, the particle size of the stationary phase is 8 μm, 10 μm or 20 μm.

[0106] In some embodiments, in the preparation method of the compound of formula I, the high-pressure preparation further specifically comprises the following steps: dissolving the crude compound of formula I in a mixed solvent B, performing gradient elution under high-pressure preparative chromatography conditions, and collecting fractions with a chromatographic content greater than 95.0% to obtain the compound of formula I; the mixed solvent B is a mixed solvent of dichloromethane and methanol; the volume ratio of the methanol to the mixed solvent B can be 10%-30%, preferably 20%-30%, and most preferably 30%.

[0107] In some embodiments, in the method for preparing the compound of formula I, the stationary phase is 8-120 or 10-120, preferably 10-120.

[0108] In some embodiments, in the method for preparing the compound of formula I, the gradient elution can adopt any elution gradient from 4-9 to 4-13 in Table 1 below, preferably any elution gradient from 4-10 to 4-13, further preferably an elution gradient of 4-10 or 4-13, and most preferably an elution gradient of 4-10;

[0109] Table 1. Elution gradient of high pressure preparation system

[0110]

[0111]

[0112] In some embodiments, in the method for preparing the compound of formula I, the gradient elution rate can be 25 mL / min-45 mL / min, preferably 35 mL / min to 45 mL / min, and more preferably 40 mL / min to 45 mL / min.

[0113] In some embodiments, in the method for preparing the compound of formula I, the column temperature for gradient elution can be 20°C-50°C, preferably 25°C-40°C.

[0114] In some embodiments, in the method for preparing the compound of formula I, the detection wavelength of the high-pressure preparation can be 254 nm or 220 nm.

[0115] In some embodiments, the method for preparing the compound of formula I further comprises a method for preparing a crude product of the compound of formula I, comprising the following steps:

[0116] ① The reaction solution was concentrated to obtain a concentrate, which was ultrasonically dissolved with dichloromethane, and then methyl tert-butyl ether was added. The mixture was stirred at room temperature, allowed to stand, and the supernatant was removed. The remaining portion was concentrated to obtain a pretreated crude product.

[0117] ② Purify the pretreated crude product by rapid silica gel column chromatography. Dissolve the pretreated crude product in a small amount of mixed solvent C, apply the sample by wet method, elute with mobile phase, collect and combine the fractions, and concentrate to obtain a crude compound of formula I.

[0118] In some embodiments, in step ①, the volume mass ratio of the dichloromethane to the concentrate can be 8-12 mL / g, preferably 9-11 mL / g, and more preferably 10 mL / g.

[0119] In some embodiments, in step ①, the volume mass ratio of the methyl tert-butyl ether to the concentrate can be 30-70 mL / g, preferably 40-70 mL / g, more preferably 40-60 mL / g, further preferably 40-50 mL / g, and most preferably 50 mL / g.

[0120] In some embodiments, in step ①, the stirring time may be 2-10 hours, preferably 2-8 hours, and more preferably 2-5 hours.

[0121] In some embodiments, in step ②, the mixed solvent C is a mixed solvent of dichloromethane and methanol; the volume ratio of dichloromethane to methanol is 10:1-5:1, preferably 5:1.

[0122] In some embodiments, in step ②, the stationary phase used in the rapid silica gel column chromatography is 200-400 mesh normal phase chromatography silica gel.

[0123] In some embodiments, in step ②, the mobile phase is a mixture of dichloromethane and methanol; the volume ratio of dichloromethane to methanol can be 10:1-5:1, preferably 5:1.

[0124] In some embodiments, in the high pressure preparation,

[0125] The stationary phase is 8-120 or 10-120;

[0126] The volume ratio of the methanol to the mixed solvent B can be 20%-30%, preferably 30%;

[0127] The gradient elution rate may be 25 mL / min-45 mL / min, for example 35 mL / min, 40 mL / min or 45 mL / min;

[0128] The column temperature of the gradient elution can be 25°C-40°C;

[0129] The detection wavelength of the high pressure preparation can be 254nm or 220nm;

[0130] The elution gradient may be 4-9, 4-10, 4-11, 4-12 or 4-13 in Table 1.

[0131] In some embodiments, in the high pressure preparation,

[0132] The stationary phase is 10-120;

[0133] The volume ratio of the methanol to the mixed solvent B is 30%;

[0134] The gradient elution rate can be 40 mL / min to 45 mL / min;

[0135] The column temperature of the gradient elution can be 25°C;

[0136] The detection wavelength of the high pressure preparation can be 254nm or 220nm;

[0137] The elution gradient may be 4-10 in Table 1.

[0138] In some embodiments, the method for preparing the compound of formula I also includes a method for preparing the compound of formula II, which comprises the following steps: coupling the compound of formula III and 6-(maleimido)hexanoic acid succinimidyl ester in a solvent to obtain the corresponding compound of formula II;

[0139]

[0140] Wherein, R is a C1~C6 alkyl group substituted with -Si(C1~C6)3;

[0141] Furthermore, the preparation method of the compound of formula II also includes a preparation method of the compound of formula III, which comprises the following steps: performing a reduction reaction on the compound of formula IV and a reducing agent in a solvent and in the presence of an acid buffer to obtain the corresponding compound of formula III;

[0142]

[0143] Wherein, R is a C1~C6 alkyl group substituted with -Si(C1~C6)3;

[0144] Furthermore, the preparation method of the compound of formula III 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 the corresponding compound of formula IV;

[0145]

[0146] Wherein, R is a C1~C6 alkyl group substituted with -Si(C1~C6)3;

[0147] Furthermore, the preparation method of the compound of formula IV 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 the corresponding compound of formula VI;

[0148]

[0149] In some embodiments, in the preparation method of the compound of formula II, the R can be trimethylsilylethyl or tert-butyldimethylsilylethyl; preferably, the R is trimethylsilylethyl.

[0150] In some embodiments, in the method for preparing the compound of formula II, the reaction materials are the compound of formula III, the succinimide 6-(maleimido)hexanoate and the solvent.

[0151] In some embodiments, in the method for preparing the compound of formula II, the molar ratio of 6-(maleimido)hexanoic acid succinimide ester to the compound of formula III is 1-5, preferably 1-3, more preferably 1.2-2, and most preferably 1.5.

[0152] In some embodiments, in the method for preparing the compound of formula II, 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.

[0153] In some embodiments, in the method for preparing the compound of formula II, the coupling reaction temperature is 0°C-45°C, preferably 25°C-40°C, and more preferably 30°C-35°C.

[0154] In some embodiments, in the method for preparing the compound of formula II, the coupling reaction is carried out under the protection of an inert gas, for example, in a nitrogen or helium environment.

[0155] In some embodiments, in the method for preparing the compound of formula II, the progress of the coupling 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.

[0156] In some embodiments, in the method for preparing the compound of formula II, the coupling reaction time can be 3-24 hours, preferably 3-10 hours, and more preferably 3-6 hours.

[0157] In some embodiments, in the preparation method of the compound of formula II, after the coupling reaction is completed, the following post-treatment step may be further included: concentrating the obtained reaction solution under reduced pressure, extracting with dichloromethane and water, combining the dichloromethane organic phase, and concentrating to obtain a crude product of the compound of formula II; preferably, the crude product of the compound of formula II is purified by silica gel column chromatography to obtain a product of the compound of formula II; further preferably, the eluent for the silica gel column chromatography is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is (10-1):1.

[0158] In some embodiments, in the preparation method of the compound of formula III, the R can be trimethylsilylethyl or tert-butyldimethylsilylethyl; preferably, the R is trimethylsilylethyl.

[0159] In some embodiments, in the method for preparing the compound of formula III, the reaction conditions in the method for preparing the compound of formula III (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 reducing agents; for example, the following steps may be included: dissolving the reducing agent in a solvent, and then adding it dropwise to an acid buffer solution, placing the resulting reaction system under the protection of an inert gas, stirring and cooling, and then adding the compound of formula IV dissolved in the solvent dropwise to the above reaction system to carry out the reaction.

[0160] In some embodiments, in the method for preparing the compound of formula III, the reaction materials are the compound of formula IV, the reducing agent, the acid buffer and the solvent.

[0161] In some embodiments, in the method for preparing the compound of formula III, the reducing agent is one of triphenylphosphine, tri-tert-butylphosphine and trimethylphosphine, or a mixture of any two or more thereof, or a commercially available triphenylphosphine / tetrahydrofuran solution, tri-tert-butylphosphine / tetrahydrofuran solution or trimethylphosphine / tetrahydrofuran solution, preferably a trimethylphosphine / tetrahydrofuran solution, more preferably a 1 M trimethylphosphine / tetrahydrofuran solution.

[0162] In some embodiments, in the method for preparing the compound of formula III, the molar ratio of the reducing agent to the compound of formula IV is 1.0-3.0, preferably 1.2-1.8, and more preferably 1.5.

[0163] In some embodiments, in the method for preparing the compound of formula III, the volume mass ratio of the acid buffer to the compound of formula IV can be 2-10 mL / g, preferably 3-5 mL / g, and most preferably 4 mL / g.

[0164] In some embodiments, in the method for preparing the compound of formula III, the solvent is an ether solvent, preferably 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.

[0165] In some embodiments, in the method for preparing the compound of formula III, the acid buffer is an acetate buffer or a formic acid buffer, preferably an acetate buffer; and the acetate buffer is a sodium acetate buffer.

[0166] In some embodiments, in the method for preparing the compound of formula III, the pH of the acid buffer is 4.0-6.0, preferably 4.5-5.5, and more preferably 5.0.

[0167] In some embodiments, in the method for preparing the compound of formula III, the temperature of the reduction reaction is 0°C-20°C, preferably 0°C-10°C, and more preferably 0°C-5°C.

[0168] In some embodiments, in the method for preparing the compound of formula III, the progress of the reduction 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.

[0169] In some embodiments, in the method for preparing the compound of formula III, the reduction reaction time can be 0.5-8 hours, preferably 0.5-2 hours, and more preferably 0.5-1 hour.

[0170] In some embodiments, in the preparation method of the compound of formula III, after the reduction reaction is completed, the following post-treatment step may be further included: concentrating the reaction solution to remove the reaction solvent, adding dichloromethane or ethyl acetate for extraction, combining the organic phases, and concentrating to obtain a crude product of the compound of formula III. Preferably, the crude product of the compound of formula III is purified by silica gel column chromatography to obtain the compound of formula III product; further preferably, the eluent for the silica gel column chromatography is a mixed solvent of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is (10-1):1.

[0171] In some embodiments, in the preparation method of the compound of formula IV, the R can be trimethylsilylethyl or tert-butyldimethylsilylethyl; preferably, the R is trimethylsilylethyl.

[0172] 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.

[0173] 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, and more preferably 1.1-1.6.

[0174] In some embodiments, in the method for preparing the compound of formula IV, the base is 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, DMAP, pyridine and panpiperidin, or a mixture of any two or more thereof, and 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, and further preferably one of potassium phosphate, potassium carbonate, potassium hydroxide and cesium carbonate, or a mixture of any two or more thereof.

[0175] 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 is 1-5, preferably 1.2-4, and more preferably 1.5-3.

[0176] In some embodiments, in the method for preparing the compound of formula IV, the solvent is one of DMF, DMSO, tetrahydrofuran and 1,4-dioxane, or a mixture of any two or more thereof, preferably 1,4-dioxane or tetrahydrofuran, and more preferably tetrahydrofuran.

[0177] In some embodiments, in the method for preparing the compound of formula IV, the temperature of the etherification reaction is 0°C-80°C, preferably 40°C-80°C, and more preferably 50°C-70°C.

[0178] In some embodiments, in the method for preparing the compound of formula IV, the etherification reaction is carried out under the protection of an inert gas, for example, in a nitrogen or helium environment.

[0179] In some embodiments, in the method for preparing the compound of formula IV, 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 VI is no longer detected.

[0180] In some embodiments, in the method for preparing the compound of formula IV, the reaction time can be 2-48 hours, preferably 3-12 hours, and more preferably 4-8 hours.

[0181] In some embodiments, in the preparation method of the compound of formula IV, after the etherification reaction is completed, the following post-treatment step may be further included: concentrating the reaction solution, dissolving the obtained concentrate with a solvent, washing the organic phase, concentrating the organic phase, and purifying the obtained residue; wherein the solvent can be ethyl acetate or dichloromethane, preferably ethyl acetate; the aqueous phase used for the washing can be an acid aqueous solution, water and / or saturated brine; preferably, the organic phase is washed with saturated brine; the purification can adopt conventional purification methods in the art, such as beating, crystallization, preparative chromatography or silica gel column chromatography, etc., preferably silica gel column chromatography is selected, and the eluent for the silica gel column chromatography is preferably a mixture 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.

[0182] 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.

[0183] 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 may be 1-10, preferably 1-5, more preferably 1.3-3.0, and most preferably 1.3;

[0184] 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 3.

[0185] 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.

[0186] In some embodiments, in the method for preparing the compound of formula VI, the substitution reaction temperature may be -10°C to 50°C, preferably 15°C to 35°C, and more preferably 18-25°C;

[0187] 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.

[0188] 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 reaction endpoint is generally when the compound of formula VII is no longer detected (using methanol derivatization).

[0189] In some embodiments, the substitution reaction time may be 3-24 hours, preferably 6-18 hours, and more preferably 8-16 hours.

[0190] In some embodiments, the method for preparing the compound of formula VI may further include a post-treatment step after the substitution 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 the reagent V.

[0191] The present invention also provides a method for preparing a compound of formula I, comprising the following steps: subjecting a compound of formula II to a deprotection reaction in a solvent in the presence of a deprotecting agent to obtain a compound of formula I;

[0192]

[0193] Wherein, R is a C1-C6 alkyl group substituted with -Si(C1-C6)3.

[0194] The preparation method, reaction conditions and operation of the compound of formula I can be the same as described above.

[0195] The method for preparing the compound of formula I may further include the method for preparing the compound of formula II described herein.

[0196] 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.

[0197] The reagents and raw materials used in the present invention are commercially available.

[0198] The positive progress effect of the present invention is:

[0199] 1) The compound of formula I of the present invention is stable and does not decompose during the purification process. Using the compound of formula I to prepare the linker-drug molecule (Compound LE) not only greatly reduces the exposure time of cytotoxic substances and reduces the harm to the human body during the preparation process, but also improves the utilization rate of exitecan and reduces production costs;

[0200] 2) This method can obtain a linker-drug conjugate intermediate LE14, Formula I, with a content greater than 95.0%. Compared with low-pressure column chromatography purification methods, this method not only significantly improves purity but also increases yield and significantly reduces impurity content, facilitating subsequent reaction processing and improving yield.

[0201] 3) This method uses vacuum concentration and extractive drying to concentrate the fractions, avoiding the tedious freeze-drying process of reverse-phase separation. Sample powder can be obtained through simple operations, the product is in good condition, and the post-processing operation is simple and easy;

[0202] 4) The high-pressure preparation system filler stationary phase used in this method is simple, easy to obtain, economical and applicable, and can be reused, thereby improving utilization efficiency and reducing production costs. DETAILED DESCRIPTION

[0203] 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.

[0204] Unless otherwise specified, the ratios of various substances involved in the following examples are all volume ratios.

[0205] The stationary phase parameters of the column chromatography packing used in the following examples are shown in Table 2.

[0206] Table 2. Packing stationary phase parameters

[0207]

[0208] The parameters of the preparation and separation equipment used in the following examples are shown in Table 3.

[0209] Table 3. Preparative separation equipment parameters

[0210]

[0211] In the following examples, the HPLC analysis conditions for analyzing the content of the compound of formula I are as follows:

[0212] The instrument was Agilent 187260, the chromatographic column was Agilent AdvanceBio Peptide Map, 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, where phase A was 0.01 mol / L potassium dihydrogen phosphate aqueous solution (pH=5.0), phase B was 10% methanol acetonitrile solution, and the flow rate was 1.0 mL / min.

[0213] Table 4. HPLC analysis conditions

[0214]

[0215]

[0216] (Column temperature: 40°C, flow rate: 1.0 mL / min, detection wavelength: 254 nm.)

[0217] Example 1: Preparation of compound LE14

[0218] The synthetic route is as follows:

[0219]

[0220] Step 1: Synthesis of compound of formula VI

[0221]

[0222] The compound of formula VII (25.00 g, 53.36 mmol) and paraformaldehyde (2.09 g, 69.37 mmol) were mixed and dissolved in 250.0 mL of anhydrous 1,4-dioxane. Trimethylsilyl chloride (17.39 g, 160.08 mmol) was slowly added under stirring. The resulting mixed solution was kept at a temperature within the range of 18-25°C and stirred for 15 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a crude compound of formula VI (28.95 g) (directly used in the next reaction).

[0223] Step 2: Synthesis of compound of formula IVa

[0224]

[0225] The crude compound of Formula VI (28.95 g, 53.37 mmol, calculated based on theoretical yield) obtained in Step 1 was dissolved in 250.0 mL of anhydrous tetrahydrofuran. Panpiperidin (12.43 g, 80.05 mmol) and Va (14.12 g, 80.05 mmol) were added with stirring. The resulting mixture was heated to 60°C under nitrogen and allowed to react for 6 h. The reaction was monitored by TLC. After the reaction of the starting materials was complete, the tetrahydrofuran was removed by concentration under reduced pressure. The concentrate was then extracted with ethyl acetate and saturated brine. The resulting organic phase was dried and then concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (n-heptane:ethyl acetate = 10:1 to 1:1 (v / v)) to afford the compound of Formula IVa (18.12 g, 93.3% purity, 51.7% combined yield from Steps 1 and 2).

[0226] ESI-MS m / z: 657.4 (M+H).

[0227] 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).

[0228] Step 3: Synthesis of compound of formula IIIa

[0229]

[0230] A mixture of 32.0 mL of a 1 M solution of trimethylphosphine in tetrahydrofuran and 15.0 mL of THF was added dropwise to 60.0 mL of sodium acetate buffer (pH = 5.0). The resulting reaction system was stirred and cooled to 0°C-5°C under nitrogen. Subsequently, 45.0 mL of tetrahydrofuran containing the compound of Formula IVa (15.00 g, 21.20 mmol) was slowly added dropwise to the reaction system. The reaction system was maintained at 0-5°C and stirred for 0.5-1.0 h. The reaction was monitored by TLC. After the reaction of the starting materials was complete, the tetrahydrofuran was removed from the reaction solution by concentration under reduced pressure. The concentrated reaction solution was then extracted with dichloromethane. The resulting organic phase was dried and concentrated 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 IIIa (10.30 g, purity 96.5%, yield 76.9%).

[0231] ESI-MS m / z: 631.4 (M+H).

[0232] 1H-NMR (400MHz, DMSO-d6) δ10.10(s,1H),8.47(d,J=7.2Hz,1H),7.56(d,J=8.6Hz,2H),7.41–7.20(m, 2H), 5.16–4.99 (m, 2H), 5.16 (m, 2H), 4.86 (s, 2H), 4.45 (p, J=7.4Hz, 1H), 4.10 (dd, J=29.3, 10.5Hz, 4H ), 3.66 (dd, J=8.5, 6.0Hz, 2H), 3.47 (d, J=8.2Hz, 1H), 3.42 (t, J=7.5Hz, 2H), 2.97 (d, J=26.2Hz, 3H), 1 .35(d,J=7.1Hz,3H),1.27–1.16(m,2H),0.95(dd,J=9.6,6.6Hz,6H),0.85–0.80(m,1H),0.13(s,9H).

[0233] Step 4: Synthesis of compound of formula IIa

[0234]

[0235] The compound of formula IIIa (10.00 g, 15.30 mmol) was dissolved in 300.0 mL of anhydrous dichloromethane, and succinimidyl 6-(maleimido)hexanoate (7.08 g, 22.95 mmol) was added with stirring. The resulting reaction system was heated to 35°C under nitrogen and stirred for 3-6 hours. The reaction was monitored by TLC. After the reaction of the raw materials was complete, the reaction solution was concentrated under reduced pressure. The crude product was washed with dichloromethane and water. The organic phase was dried and concentrated under reduced pressure. The concentrate was then purified by silica gel column chromatography (dichloromethane:methanol = 10:1 to 1:1 (v / v)) to obtain the compound of formula IIa (9.80 g, purity 97.31%, yield 75.4%).

[0236] ESI-MS m / z: 824.5 (M+H).

[0237] 1H-NMR(400MHz, DMSO-d6)δ9.95(s,1H),8.14(d,J=6.9Hz,1H),7.79(d,J=8.6Hz,1H),7.59(d,J=8.2Hz,2H),7.39–7.22(m,2H), 6.99(s,2H),5.07–5.01(m,2H),4.85(s,2H),4.38(p,J=7.0Hz,1H),4.16(dd,J=8.6,6.9Hz,2H),4.08(d,J=12.3Hz,2H),3.68(d d,J=8.8,6.0Hz,2H),3.44–3.35(m,4H),3.07(s,1H),2.97(d,J=27.4Hz,3H),2.14(tt,J=14.1,6.9Hz,2H),1.95(h,J=6.7Hz,1H ),1.54–1.42(m,4H),1.29(d,J=7.1Hz,3H),1.18(q,J=7.7Hz,2H),0.98–0.91(m,2H),0.84(dd,J=15.4,6.8Hz,6H),0.06(s,9H).

[0238] Step 5: Synthesis of compound of formula I

[0239]

[0240] Method 1: Deprotection using potassium fluoride reagent

[0241] The compound of Formula IIa (5.00 g, 6.07 mmol) was dissolved in 30.0 mL of anhydrous DMF. Powdered potassium fluoride (0.53 g, 9.11 mmol) was added at room temperature. The resulting reaction system was heated to 60°C under nitrogen and stirred for 6 hours. The reaction was monitored by TLC. After the reaction of the starting materials was complete, the resulting solution was concentrated under reduced pressure to yield 6.75 g of a concentrate (50.06% purity). This concentrate (Batch 1) was purified using Purification Method 1 described below.

[0242] Another batch of materials was added according to this method. The amounts of materials used were: compound of formula IIa (8.50 g, 10.04 mmol), potassium fluoride (0.88 g, 15.06 mmol) and DMF (45.0 mL). The addition and reaction procedures were the same as those of batch 1 to obtain another batch of concentrate (batch 2). This batch of concentrate was purified according to the following purification method 2:

[0243] Purification method 1: Purification of the concentrate by high pressure preparative chromatography

[0244] The obtained concentrate (batch 1) was pretreated and then subjected to high pressure preparative chromatography (stationary phase: The obtained product was purified by HPLC (10-120, dichloromethane:methanol=96:4-90:10 (v / v), flow rate 40.0 mL / min) to obtain the compound of formula I (2.90 g, purity 96.5%, yield 66.0%).

[0245] Purification method 2: Purification of the concentrate by conventional column chromatography

[0246] The obtained concentrate (Batch 2) was pretreated and purified by conventional silica gel column chromatography (dichloromethane:methanol = 10:1 to 1:1 (v / v)) according to Example 3 to obtain the compound of Formula I (4.72 g, purity 83.06%, yield 65.0%; the actual amount of pure compound of Formula I contained was 3.92 g, and the yield calculated based on the amount of pure compound of Formula I obtained was 54.0%).

[0247] ESI-MS m / z: 722.3 (MH);

[0248] 1 H-NMR (400MHz, DMSO-d6) δ10.57(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,1 H),4.17(s,1H),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).

[0249] Method 2: Deprotection using sodium fluoride reagent

[0250] The operation steps were the same as those of Method 1, except that potassium fluoride was replaced by sodium fluoride. Purification was performed according to Purification Method 2 to obtain the compound of Formula I (3.44 g, yield 47.3%).

[0251] Method 3: Deprotection using cesium fluoride reagent

[0252] The operation steps were the same as those of Method 1, except that potassium fluoride was replaced by cesium fluoride. Purification was carried out according to Purification Method 2 to obtain the compound of Formula I (3.02 g, yield 41.50%).

[0253] Method 4: Deprotection using tetrabutylammonium fluoride reagent

[0254] The compound of Formula IIa (8.50 g, 10.04 mmol) was dissolved in tetrahydrofuran (50.0 mL). A 1.0 M solution of tetrabutylammonium fluoride in tetrahydrofuran (15.2 mL, 15.06 mmol) was added at room temperature. The temperature was slowly raised to 40°C under nitrogen and the reaction was continued for 8.0 h. The solvent was removed by concentration under reduced pressure, and the resulting concentrate was purified using Purification Method 2 in Method 1 to obtain the compound of Formula I (3.66 g, 50.3% yield).

[0255] Step 6: Synthesis of compound LE14

[0256]

[0257] Method 1: Using the compound of formula I obtained by purification method 1 in step 5 as a raw material

[0258] The compound of Formula I (4.80 g, 96.5% purity, 6.40 mmol) was dissolved in 15.0 mL of anhydrous DMF, and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) (2.65 g, 9.60 mmol) was added. The reaction was allowed to proceed at room temperature for 1.0 h. N,N-diisopropylethylamine (1.23 g, 9.60 mmol) and the compound of Formula VIII, exatecan mesylate (3.42 g, 6.40 mmol), were then added and the reaction continued for 1.5-2.0 h. After the reaction was complete, the solvent was evaporated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (chloroform:methanol = 10:1 (v / v)) to afford the compound of Formula LE14 (5.24 g, 99.17% purity, 71.8% yield).

[0259] Method 2. Using the compound of formula I obtained by purification method 2 in step 5 as a raw material

[0260] The compound of Formula I (4.72 g, 83.06% purity, 5.42 mmol) was dissolved in 15 mL of anhydrous DMF, and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) (2.24 g, 8.13 mmol) was added. The reaction was allowed to proceed at room temperature for 1.0 h. N,N-diisopropylethylamine (1.04 g, 8.13 mmol) and the compound of Formula VIII, exatecan mesylate (2.89 g, 5.42 mmol), were then added and the reaction continued for 1.5-2.0 h. The reaction was monitored by TLC. Upon completion of the reaction, the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (chloroform:methanol = 10:1 (v / v)) to afford compound LE14 (3.69 g, 98.77% purity, 59.6% yield).

[0261] ESI-MS m / z: 1141.7 (M+H);

[0262] 1 H-NMR(500MHz,DMSO-d6)δ9.92(s,1H),8.47(s,1H),8.13(d,J=6.9Hz,1H),7.75(d,J=28.5Hz,2H),7.55(s,2H),7.29(s,3H),6 .99(s,2H),6.47(s,1H),5.60(s,1H),5.49–5.33(m,2H),5.19(s,2H),5.05–4.80(m,4H),4.39(d,J=7.5Hz,1H),4.22–4.15(m, 1H),4.04(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. 70Hz,3H),2.23–2.08(m,4H),2.03–1.75(m,3H),1.53–1.43(m,4H),1.30(d,J=7.2Hz,3H),1.15(d,J=7.7Hz,2H),0.86(m,9H).

[0263] Example 2

[0264] After the reaction solution prepared in step 5 of Example 1 was concentrated, the resulting concentrate was pretreated by the following method: the concentrate was ultrasonically dissolved with dichloromethane at room temperature (the volume ratio of dichloromethane used to the mass of the concentrate was 10 mL / g), and then methyl tert-butyl ether was added thereto (the volume ratio of methyl tert-butyl ether used to the mass ratio of the concentrate was 20-70 mL / g), and stirred at room temperature for 2-5 hours. After standing, the supernatant was discarded, and the lower oil was vacuum concentrated to obtain a pretreated crude product, which was used for subsequent chromatographic purification.

[0265] The solvent ratios used in the pretreatment of this embodiment, the purity of the pretreated sample and the recovery rate are shown in Table 5. From the results given in Table 5, it can be seen that when the volume mass ratio of dichloromethane to the concentrate is 10 mL / g and the volume mass ratio of methyl tert-butyl ether to the concentrate is 30-70 mL / g, a crude pretreated product of the compound of formula I with a purity greater than 56% can be obtained with a recovery rate greater than 65%; when the volume mass ratio of dichloromethane to the concentrate is 10 mL / g and the volume mass ratio of methyl tert-butyl ether to the concentrate is 40-70 mL / g, a crude pretreated product of the compound of formula I with a purity greater than 56% can be obtained with a recovery rate greater than 70%; when the volume mass ratio of dichloromethane to the concentrate is 10 mL / g and the volume mass ratio of methyl tert-butyl ether to the concentrate is 40-70 mL / g, a crude pretreated product of the compound of formula I with a purity greater than 56% can be obtained with a recovery rate greater than 70%; when the volume mass ratio of dichloromethane to the concentrate is 10 mL / g and the volume mass ratio of methyl tert-butyl ether to the concentrate is When the volume mass ratio of dichloromethane to concentrate is 40-60 mL / g, a crude pretreatment product of the compound of formula I with a purity greater than 58% can be obtained with a recovery rate greater than 70%; when the volume mass ratio of dichloromethane to concentrate is 10 mL / g and the volume mass ratio of methyl tert-butyl ether to concentrate is 40-50 mL / g, a crude pretreatment product of the compound of formula I with a purity greater than 60% can be obtained with a recovery rate greater than 70%; when the volume mass ratio of dichloromethane to concentrate is 10 mL / g and the volume mass ratio of methyl tert-butyl ether to concentrate is 50 mL / g, a crude pretreatment product of the compound of formula I with a purity greater than 60% can be obtained with a recovery rate greater than 75%.

[0266] Table 5. Effect of solvent ratio in pretreatment step on the purity and recovery of crude product

[0267]

[0268] Note: Recovery rate = sample weight after treatment / sample weight before treatment × 100%.

[0269] Example 3

[0270] In this example, the crude product of the compound of formula I pretreated in Example 2 was purified by conventional column chromatography to obtain a crude product of the compound of formula I with a purity (greater than 80.0%). Specifically, in this example, the crude product of the compound of formula I pretreated was purified according to the following steps:

[0271] Step 1. Using conventional normal phase chromatography silica gel as the stationary phase, a normal phase chromatography method was used to prepare the compound of Formula I. The specific implementation method is as follows: the crude compound of Formula I was dissolved in dichloromethane:methanol = 10:1 (volume ratio), and then wet-loaded onto a conventional glass chromatography-type normal phase chromatography column. Specific column parameters are shown in Tables 2 and 3. Elution was performed at 25°C with a mixture of dichloromethane and methanol in a dichloromethane:methanol volume ratio of 60:1 to 10:1. The compound of Formula I was eluted by adjusting the eluent gradient, and the target fraction was collected;

[0272] Step 2. Combine the fractions with chromatographic contents greater than 80.0%, and vacuum concentrate the combined fractions or extract the concentrate after concentration or freeze-dry to obtain the finished product of the compound of formula I.

[0273] The chromatographic separation method, stationary phase type, elution system, and purity and recovery of the purified samples used in this example are shown in Table 6. From the results in Table 6, it can be seen that the normal phase chromatographic separation method can obtain a product with a purity of more than 80.0%, but has the disadvantage of low sample recovery.

[0274] Table 6. Chromatographic purification methods and purity and recovery of purified samples

[0275]

[0276] Note: Recovery rate = sample weight after treatment / sample weight before treatment × 100%.

[0277] Example 4

[0278] Step 1. Pretreat the crude compound of formula I according to the method of Example 2.

[0279] Step 2. Purification of the crude pretreated compound of Formula I by conventional flash column chromatography

[0280] In this step, the crude pre-treated compound of Formula I is preliminarily purified by conventional flash column chromatography to obtain a preliminarily purified sample of the compound of Formula I. Specifically, the purification is carried out according to the following steps: using conventional normal phase chromatography silica gel as the packing stationary phase, the compound of Formula I is prepared by normal phase chromatography. The specific implementation method is: the crude pre-treated compound of Formula I after the pre-treatment in step 1 is ultrasonically dissolved in a mixed solution of dichloromethane and methanol (the volume ratio and eluent ratio of the two are the same, and the smallest amount is used for dissolution) and injected into a conventional glass chromatography-type normal phase chromatography column. The specific column parameters are shown in Tables 2 and 3. The eluent is a mixed solvent of dichloromethane and methanol. Elution is carried out at 25°C. The crude pre-treated compound of Formula I is eluted in a single isocratic manner. As many target fractions as possible are collected. The combined fractions are vacuum concentrated or concentrated and then extracted or lyophilized to obtain a preliminarily purified crude compound of Formula I.

[0281] The chromatographic separation method, stationary phase type, elution system, and purity and recovery rate of the initially purified sample of the compound of Formula I obtained in this example are shown in Table 7. The results in Table 7 indicate that, using a mixed solvent of dichloromethane:methanol = 5:1 as the eluent, the conventional rapid column chromatography separation method of this example can obtain a sample of the compound of Formula I with a purity of 70-80% at a high recovery rate exceeding 90%, facilitating subsequent further purification.

[0282] Table 7. Chromatographic purification methods and purity and recovery of purified samples

[0283]

[0284]

[0285] Note: Recovery rate = sample weight after treatment / sample weight before treatment × 100%.

[0286] Step 3. Purification by high pressure preparative chromatography to prepare a higher purity (95.0% or more) compound of formula I

[0287] The sample of the compound of formula I (with a purity of 70.0% to 80.0%) initially purified by silica gel flash column in step 2 is dissolved in a mixed solvent of eluents A and B in a volume ratio of 7:3. The dissolved sample is loaded onto a well-equilibrated high-pressure preparative chromatographic column for elution. The compound of formula I is eluted by adjusting the eluent gradient using an isocratic or gradient elution method, and fractions with a purity greater than 95.0% are collected; the fractions with a chromatographic content greater than 95.0% are combined, and the combined fractions are concentrated under reduced pressure to obtain a finished compound of formula I.

[0288] In this example, the elution gradient was optimized to study the effect of the change in elution gradient on the purity and recovery of the purified product. The specific results are shown in Table 8, where the elution rate was 40 mL / min, the solvent A in the eluent was dichloromethane, the solvent B was methanol, and the stationary phase was selected. 8-120, column temperature is 25° C. As can be seen from the results in Table 8: According to the purification method described in this Example, when the elution gradients 4-9 to 4-13 in Table 8 are selected, the compound of Formula I with a purity higher than 91% can be obtained with a recovery rate higher than 74%, when the elution gradients 4-10 to 4-13 in Table 8 are selected, the compound of Formula I with a purity higher than 93% can be obtained with a recovery rate higher than 74%, when 4-10 or 4-13 in Table 8 are selected, the compound of Formula I with a purity higher than 94% can be obtained with a recovery rate higher than 74%, and when the elution gradient 4-10 in Table 8 is selected, the compound of Formula I with a purity higher than 95% can be obtained with a recovery rate higher than 74%.

[0289] Table 8. Elution gradient of high pressure preparation system

[0290]

[0291]

[0292] Note: Recovery rate = sample weight after treatment / sample weight before treatment × 100%.

[0293] In this example, the effects of the type of stationary phase and the type of eluent on the purity and recovery of the purified product were studied under the preferred elution gradients shown in 4-10 in Table 8 and an elution rate of 40 mL / min. The specific results are shown in Table 9 (the eluents No. 4-22 used isocratic elution), where the stationary phase is 8-120, 10-120 or 20-120, the eluent A is ethyl acetate or dichloromethane, B is methanol or ethanol. From the results in Table 9, it can be seen that according to the purification method described in this embodiment, the stationary phase is selected 8-120 or 10-120, when the eluent system (A:B) is dichloromethane:methanol, the compound of formula I with a purity higher than 95% can be obtained with a recovery rate higher than 74%. 10-120, when the eluent system (A:B) is dichloromethane:methanol, the compound of formula I with a purity higher than 95% can be obtained with a yield higher than 76%.

[0294] Among them, column temperature: 25°C, flow rate: 40.0 mL / min, detection wavelength 1: 254 nm, detection wavelength 2: 220 nm.

[0295] Table 9. Effects of stationary phase and elution system on the purity and recovery of purified products

[0296]

[0297] Note: Recovery rate = sample weight after treatment / sample weight before treatment × 100%.

[0298] This example studies the effect of elution rate on the purity and recovery of the purified product. The specific results are shown in Table 10. The stationary phase in Table 10 is 10-120, the eluent system is dichloromethane:methanol, and the elution gradient shown in 4-10 in Table 8 is selected. From the data in Table 10, it can be concluded that the compound of formula I with a purity greater than 95% can be obtained at an elution rate of 25 mL / min-45 mL / min, the compound of formula I with a purity greater than 95% can be obtained with a yield of greater than 70% at an elution rate of 35 mL / min to 45 mL / min, and the compound of formula I with a purity greater than 95% can be obtained with a yield of greater than 75% at an elution rate of 40 mL / min to 45 mL / min.

[0299] Table 10. Effect of elution rate on the purity and recovery of purified products

[0300] serial number Elution speed purity(%) Recovery rate (%) 4-23 25.0mL / min 96.30％ 58.41％ 4-24 30.0mL / min 96.18％ 59.29％ 4-25 35.0mL / min 96.24％ 70.66％ 4-26 40.0mL / min 96.29％ 76.42％ 4-27 45.0mL / min 95.79％ 79.25％ 4-28 50.0mL / min 93.61％ 81.63％ 4-29 55.0mL / min 92.49％ 82.47％

[0301] Note: Recovery rate = sample weight after treatment / sample weight before treatment × 100%.

[0302] The purification method of this example yields a higher purity (95.0% or higher) compound of Formula I with good sample recovery, making it suitable for commercial-scale production. Using the high-purity compound of Formula I as a key intermediate allows for controlled quality and impurity levels of the linger-drug molecule (compound LE14) and ADC, minimizing product quality while producing an ADC product that meets pharmaceutical quality standards. See Table 11 for details.

[0303] Table 11. Comparative data of purity of final product LE14 compound

[0304]

[0305] Compared with conventional chromatographic purification methods, this method can not only improve sample purity and yield, but also has the advantages of simple operation and good stability. Moreover, the solid phase filler used is easy to obtain and can be recycled repeatedly, saving production costs and reducing environmental pollution, making it suitable for industrial production.

[0306] Example 5 Comprehensive Comparison of Process Routes

[0307] 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 the purified intermediate 1-4 was not 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 the purified intermediate 2-2a was not obtained. However, the method of the present invention can obtain the carboxylic acid intermediate I with a good yield and good purity. The purification status of the carboxylic acid intermediates prepared by the three synthetic routes is summarized and compared in Table 12 below.

[0308] The purity of the LE14 final product synthesized according to Example 1 was compared with that of the LE14 final products obtained by Route 1, Route 2, Route 3, Route 4 and Route 5 by HPLC. The results are shown in Table 13 below.

[0309] Table 12 Comparison of carboxylic acid intermediates (1-4, 2-2a and I) obtained from several process routes

[0310]

[0311] Table 13. Comparative data of purity of final product LE14 from different process routes

[0312]

[0313] 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 four routes, and the total yield of the routes and the purity of the final product are summarized in Table 14.

[0314] Table 14. 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

[0315]

[0316]

[0317] In the prior art synthesis routes of CN 115215921 A and CN115385926A, using ixetemcan as the starting material, the total yields of routes 4 and 5 are lower than that of the present invention, as shown in Table 15.

[0318] Table 15. Comparison of total yields of different process routes based on the input of ixetem

[0319]

[0320] As can be seen from Tables 13, 14, and 15, the purity of the products prepared by Route 1 and Route 2 is far inferior to that of the present invention and does not meet the quality requirements of the products of the present invention for IND application (total impurities not exceeding 3%, single impurities not exceeding 1%). Route 3 uses DXd, which is more expensive than isotecan, as a payload source. Although it saves reaction steps, the yield and utilization rate of DXd in Route 3 are not high. In both Route 4 and Route 5, isotecan needs to be derivatized. On the one hand, the process reaction steps are more complicated and the production operation is cumbersome. On the other hand, there are also large losses during the preparation process, resulting in low utilization rate of the expensive material isotecan, which is not suitable for large-scale industrial production.

[0321] Specifically, in the synthetic route of the present invention, in the final step of reacting the compound of formula I with isotecan to prepare LE14, both method 1 and method 2 can obtain products that meet product quality standards. In the two process methods, isotecan is the key material with the largest cost share in the entire process route and is also the main cost control point. In method 1, the compound of formula I is purified by high-pressure column chromatography. On the one hand, it can indeed improve the product quality of the final product LE14. On the other hand, it can also reduce the consumption of isotecan by precise feeding, thereby achieving cost control. The utilization rate of isotecan in method 2 is much lower than that in method 1, which can be seen from the data comparison in Table 15 (yield calculated based on isotecan, method 1: 71.8%, method 2: 59.6%). After comprehensive comparison, of the two process routes provided in this application, method 1 can provide higher quality LE14 products at a lower cost, making it a more preferred production process and more suitable for large-scale industrial production.

[0322] While the specific embodiments of the present invention have been described in detail above, these are intended to be exemplary only, and the present invention is not limited thereto. Any equivalent modifications and substitutions to the present invention that would be apparent to those skilled in the art are also within the scope of the present invention. Therefore, any equivalent modifications and substitutions made without departing from the spirit and scope of the present invention are intended to be encompassed within the scope of the present invention.

Claims

1. A compound of formula I; 2. A method for preparing compound LE, characterized in that: The method comprises the following steps: performing an amide condensation reaction between a compound of formula I and a compound of formula VIII or its mesylate in the presence of a condensing agent, a base and a solvent to obtain a compound LE. Among them, R 2 C 1-6 alkyl; R 3 It is a halogen.

3. The preparation method according to claim 2, wherein The preparation method meets one or more of the following conditions: (1) R 2 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl; (2) The R 3 is fluorine, chlorine, bromine or iodine; (3) In the preparation method of the compound LE, the amide condensation reaction further specifically comprises the following steps: dissolving the compound of formula I in a solvent, adding a condensing agent and a base, and adding the compound of formula VIII immediately or after a period of reaction; (4) In the preparation method of the compound LE, the molar ratio of the compound of formula VIII to the compound of formula I is 0.8-1.5; (5) In the preparation method of the compound LE, 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; (6) In the preparation method of the compound LE, the molar ratio of the condensing agent to the compound of formula I is 1-3; (7) In the method for preparing compound LE, the base is an organic base, an inorganic base or a mixture thereof; (8) In the method for preparing compound LE, the molar ratio of the base to the compound of formula I is 1-10; (9) In the method for preparing compound LE, the solvent is one of N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran and 1,4-dioxane, or a mixture of any two or more thereof; (10) In the method for preparing compound LE, the temperature of the amide condensation reaction is 20°C-50°C; (11) In the method for preparing compound LE, the amide condensation reaction is carried out under the protection of an inert gas; The preparation method of compound LE described in (12) further includes the following post-treatment step: concentrating the reaction solution to obtain a crude product of compound LE.

4. The preparation method according to claim 3, wherein The preparation method meets one or more of the following conditions: (1) R 2 is methyl or ethyl; (2) The R 3 is fluorine or chlorine; (3) The amide condensation reaction is kept away from light throughout the entire process; (4) the molar ratio of the compound of formula VIII to the compound of formula I is 0.9-1.2; (5) The condensing agent is 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride; (6) The molar ratio of the condensing agent to the compound of formula I is 1-1.5; (7) The organic base is one of N,N-diisopropylethylamine, triethylamine and pyridine, or a mixture of any two or more thereof; (8) 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; (9) The molar ratio of the base to the compound of formula I is 1-5; (10) The solvent is N,N-dimethylformamide; (11) The temperature of the amide condensation reaction is 20°C-30°C; (12) The amide condensation reaction is carried out under the protection of nitrogen or helium; (13) The preparation method of compound LE further includes the following post-treatment step: concentrating the reaction solution to obtain a crude compound LE product, and purifying the crude compound LE product by silica gel column chromatography to obtain a compound LE product.

5. The preparation method according to claim 4, wherein The preparation method meets one or more of the following conditions: (1) R 2 is methyl; (2) The R 3 For fluorine; (3) the molar ratio of the compound of formula VIII to the compound of formula I is 1.0; (4) The organic base is N,N-diisopropylethylamine; (5) 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; (6) The molar ratio of the base to the compound of formula I is 1.2-3; (7) The preparation method of the compound LE further includes the following post-processing step: concentrating the reaction solution to obtain a crude compound LE, and purifying the crude compound LE by silica gel column chromatography to obtain a compound LE product; the eluent of the silica gel column chromatography is a mixture of chloroform and methanol, and the volume ratio of chloroform to methanol is (100-10):

1.

6. The preparation method according to claim 5, wherein The preparation method meets one or more of the following conditions: (1) The base is an organic base; (2) the molar ratio of the base to the compound of formula I is 1.5; (3) The preparation method of the compound LE further includes the following post-processing step: concentrating the reaction solution to obtain a crude compound LE, and purifying the crude compound LE by silica gel column chromatography to obtain a compound LE product; the eluent for the silica gel column chromatography is a mixture of chloroform and methanol, and the volume ratio of the chloroform to methanol is 10:

1.

7. The preparation method according to any one of claims 3 to 6, wherein The preparation method of the compound LE further includes a preparation method of the compound of formula I, which comprises the following steps: in a solvent, subjecting the compound of formula II to a deprotection reaction in the presence of a deprotecting agent to obtain the compound of formula I; Wherein, R is a C1-C6 alkyl group substituted with -Si(C1-C6)3.

8. The preparation method according to claim 7, wherein The preparation method meets one or more of the following conditions: (1) In the preparation method of the compound of formula I, R is trimethylsilylethyl or tert-butyldimethylsilylethyl; (2) In the method for preparing the compound of formula I, the deprotection reaction further comprises the following steps: dissolving the compound of formula II in a solvent, adding the deprotecting agent, and then heating to initiate the reaction; or dissolving the deprotecting agent in a solvent, adding the compound of formula II, and then heating to initiate the reaction; (3) In the preparation method of the compound of formula I, 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 tetramethylammonium fluoride / tetrahydrofuran solution, tetraethylammonium fluoride / tetrahydrofuran solution or tetrabutylammonium fluoride / tetrahydrofuran solution, or is one of potassium fluoride, sodium fluoride, lithium fluoride and cesium fluoride, or a mixture of any two or more thereof; (4) In the method for preparing the compound of formula I, the molar ratio of the deprotecting agent to the compound of formula II is 1-5; (5) 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; (6) In the method for preparing the compound of formula I, the temperature of the deprotection reaction is 20°C-80°C; (7) In the method for preparing the compound of formula I, the deprotection reaction is carried out under the protection of an inert gas; (8) In the method for preparing the compound of formula I, after the deprotection reaction is completed, a post-treatment step is further included: separating the crude compound of formula I under high pressure preparative chromatography conditions to obtain the compound of formula I. The high pressure preparative chromatography conditions are as follows: The chromatographic column is a dynamic axial compression column; The inner diameter of the chromatographic column is 50 mm; The column length is 650 mm; The stationary phase is unbonded silanol; The particle size of the stationary phase is 8-20 μm; The pore size of the stationary phase is The mobile phase is a mixed solvent A of a halogenated alkane solvent and an alcohol solvent; the volume ratio of the halogenated alkane solvent to the alcohol solvent is 96:4 to 90:10; The purity of the crude compound of formula I is 70%-80%.

9. The preparation method according to claim 8, wherein The preparation method meets one or more of the following conditions: (1) In the preparation method of the compound of formula I, R is trimethylsilylethyl; (2) In the method for preparing the compound of formula I, the fluorine reagent is 1M tetrabutylammonium fluoride / tetrahydrofuran solution or potassium fluoride; (3) In the method for preparing the compound of formula I, the molar ratio of the deprotecting agent to the compound of formula II is 1-3; (4) In the method for preparing the compound of formula I, the solvent is N,N-dimethylformamide or tetrahydrofuran; (5) In the method for preparing the compound of formula I, the temperature of the deprotection reaction is 30°C-70°C; (6) In the method for preparing the compound of formula I, the deprotection reaction is carried out under nitrogen or helium.

10. The preparation method according to claim 9, characterized in that 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 deprotecting agent to the compound of formula II is 1.1-2.0; (2) In the method for preparing the compound of formula I, the solvent is N,N-dimethylformamide; (3) In the method for preparing the compound of formula I, the temperature of the deprotection reaction is 50°C-70°C.

11. The preparation method according to claim 10, characterized in that 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 deprotecting agent to the compound of formula II is 1.5; (2) In the method for preparing the compound of formula I, the temperature of the deprotection reaction is 60°C.

12. The preparation method according to claim 8, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula I, the particle size of the stationary phase is 8 μm, 10 μm or 20 μm; (2) In the method for preparing the compound of formula I, the high-pressure preparation further comprises the following steps: dissolving the crude compound of formula I in a mixed solvent B, performing gradient elution under high-pressure preparative chromatography conditions, and collecting fractions with a chromatographic content greater than 95.0% to obtain the compound of formula I; the mixed solvent B is a mixed solvent of dichloromethane and methanol; the volume ratio of the methanol to the mixed solvent B is 10%-30%; (3) In the method for preparing the compound of formula I, the stationary phase is 8-120 or 10-120; (4) In the method for preparing the compound of formula I, the separation under high pressure preparative chromatography conditions is gradient elution separation, and the gradient elution adopts any elution gradient from 4-9 to 4-13 in Table 1 below; Table 1. Elution gradient of high pressure preparation system (5) In the method for preparing the compound of formula I, the separation under high pressure preparative chromatography conditions is gradient elution separation, and the gradient elution rate is 25 mL / min-45 mL / min; (6) In the method for preparing the compound of formula I, the separation under high pressure preparative chromatography conditions is gradient elution separation, and the column temperature of the gradient elution is 20°C-50°C; (7) In the method for preparing the compound of formula I, the detection wavelength of the high-pressure preparation is 254 nm or 220 nm; (8) In the method for preparing the compound of formula I, the method also includes a method for preparing a crude product of the compound of formula I, which comprises the following steps: ① The reaction solution was concentrated to obtain a concentrate, which was ultrasonically dissolved in dichloromethane, and then methyl tert-butyl ether was added. The mixture was stirred at room temperature, allowed to stand, the supernatant was removed, and the remaining portion was concentrated to obtain a pretreated crude product; ② Purify the pretreated crude product by rapid silica gel column chromatography, dissolve the pretreated crude product in a small amount of mixed solvent C, wet load, elute with mobile phase, collect and combine fractions, and concentrate to obtain a crude compound of formula I; the mixed solvent C is a mixed solvent of dichloromethane and methanol.

13. The preparation method according to claim 12, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula I, the high-pressure preparation further comprises the following steps: dissolving a crude compound of formula I in a mixed solvent B, performing gradient elution under high-pressure preparative chromatography conditions, and collecting fractions with a chromatographic content greater than 95.0% to obtain the compound of formula I; the mixed solvent B is a mixed solvent of dichloromethane and methanol; the volume ratio of the methanol to the mixed solvent B is 20%-30%; (2) In the method for preparing the compound of formula I, the stationary phase is 10-120; (3) In the method for preparing the compound of formula I, the gradient elution adopts any one of 4-10 to 4-13 in Table 1; (4) In the method for preparing the compound of formula I, the gradient elution rate is 35 mL / min to 45 mL / min; (5) In the method for preparing the compound of formula I, the column temperature for gradient elution is 25°C-40°C.

14. The preparation method according to claim 13, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula I, the high-pressure preparation further comprises the following steps: dissolving a crude compound of formula I in a mixed solvent B, performing gradient elution under high-pressure preparative chromatography conditions, and collecting fractions with a chromatographic content greater than 95.0% to obtain the compound of formula I; the mixed solvent B is a mixed solvent of dichloromethane and methanol; the volume ratio of the methanol to the mixed solvent B is 30%; (2) In the method for preparing the compound of formula I, the gradient elution adopts the elution gradient 4-10 or 4-13 in Table 1; (3) In the method for preparing the compound of formula I, the gradient elution rate is 40 mL / min to 45 mL / min.

15. The preparation method according to claim 14, wherein In the method for preparing the compound of formula I, the gradient elution adopts the elution gradient 4-10 in Table 1.

16. The preparation method according to claim 12, wherein The preparation method meets one or more of the following conditions: (1) In step ①, the volume mass ratio of the dichloromethane to the concentrate is 8-12 mL / g; (2) In step ①, the volume mass ratio of the methyl tert-butyl ether to the concentrate is 30-70 mL / g; (3) In step ①, the stirring time is 2-10 hours; (4) In step ②, the mixed solvent C is a mixed solvent of dichloromethane and methanol; (5) In step ②, the stationary phase used in the rapid silica gel column chromatography is 200-400 mesh normal phase chromatography silica gel; (6) In step ②, the mobile phase is a mixture of dichloromethane and methanol; the volume ratio of dichloromethane to methanol is 10:1-5:1; (7) In the high pressure preparation, the stationary phase is 8-120 or 10-120; the volume ratio of the methanol to the mixed solvent B is 20%-30%; the gradient elution rate is 25mL / min-45mL / min; the column temperature of the gradient elution is 25°C-40°C; the detection wavelength of the high pressure preparation is 254nm or 220nm; The elution gradient adopts 4-9, 4-10, 4-11, 4-12 or 4-13 in Table 1.

17. The preparation method according to claim 16, wherein The preparation method meets one or more of the following conditions: (1) In step ①, the volume mass ratio of the dichloromethane to the concentrate is 9-11 mL / g; (2) In step ①, the volume mass ratio of the methyl tert-butyl ether to the concentrate is 40-70 mL / g; (3) In step ①, the stirring time is 2-8 hours; (4) In step ②, the mixed solvent C is a mixed solvent of dichloromethane and methanol; the volume ratio of dichloromethane to methanol is 10:1-5:1; (5) In step ②, the mobile phase is a mixture of dichloromethane and methanol; the volume ratio of dichloromethane to methanol is 5:1; (6) In the high-pressure preparation, the volume ratio of the methanol to the mixed solvent B is 30%; (7) In the high-pressure preparation, the gradient elution rate is 35 mL / min, 40 mL / min or 45 mL / min.

18. The preparation method according to claim 17, wherein The preparation method meets one or more of the following conditions: (1) In step ①, the volume mass ratio of the dichloromethane to the concentrate is 10 mL / g; (2) In step ①, the volume mass ratio of the methyl tert-butyl ether to the concentrate is 40-60 mL / g; (3) In step ①, the stirring time is 2-5 hours; (4) In step ②, the mixed solvent C is a mixed solvent of dichloromethane and methanol; the volume ratio of dichloromethane to methanol is 5:

1.

19. The preparation method according to claim 18, characterized in that In step ①, the volume mass ratio of the methyl tert-butyl ether to the concentrate is 40-50 mL / g.

20. The preparation method according to claim 19, wherein In step ①, the volume mass ratio of the methyl tert-butyl ether to the concentrate is 50 mL / g.

21. The preparation method according to claim 16, wherein In the high pressure preparation, the stationary phase is 10-120; the volume ratio of the methanol to the mixed solvent B is 30%; the gradient elution rate is 40 mL / min to 45 mL / min; the column temperature of the gradient elution is 25°C; the detection wavelength of the high pressure preparation is 254 nm or 220 nm; the elution gradient adopts 4-10 in Table 1.

22. The preparation method according to claim 8, characterized in that The preparation method of the compound of formula I also includes the preparation method of the compound of formula II, which comprises the following steps: coupling the compound of formula III and 6-(maleimido)hexanoic acid succinimide ester in a solvent to obtain the corresponding compound of formula II; Wherein, R is a C1~C6 alkyl group substituted by -Si(C1~C6)3.

23. The preparation method according to claim 22, characterized in that The preparation method of the compound of formula II also includes a preparation method of the compound of formula III, which comprises the following steps: performing a reduction reaction on the compound of formula IV and a reducing agent in a solvent and in the presence of an acid buffer to obtain the corresponding compound of formula III; Wherein, R is a C1~C6 alkyl group substituted by -Si(C1~C6)3.

24. The preparation method according to claim 23, wherein The preparation method of the compound of formula III 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 the corresponding compound of formula IV; Wherein, R is a C1~C6 alkyl group substituted by -Si(C1~C6)3.

25. The preparation method according to claim 24, characterized in that The preparation method of the compound of formula IV also includes a preparation method of the compound of formula VI, which comprises the following steps: performing a substitution reaction on the compound of formula VII with paraformaldehyde and trimethylsilyl chloride in a solvent to obtain the corresponding compound of formula VI; 26. The preparation method according to claim 25, characterized in that The preparation method meets one or more of the following conditions: (1) In the preparation method of the compound of formula II, R is trimethylsilylethyl or tert-butyldimethylsilylethyl; (2) In the preparation method of the compound of formula II, the molar ratio of 6-(maleimido)hexanoic acid succinimide ester to the compound of formula III is 1-5; (3) In the method for preparing the compound of formula II, the solvent is one of an amide solvent, a chloroalkane solvent, an ether solvent, and a nitrile solvent, or a mixture of any two or more thereof; (4) In the method for preparing the compound of formula II, the coupling reaction temperature is 0°C-45°C; (5) In the method for preparing the compound of formula II, the coupling reaction is carried out under the protection of an inert gas; (6) In the method for preparing the compound of formula II, after the coupling reaction is completed, the following post-treatment step is further included: concentrating the reaction solution, extracting with dichloromethane and water, combining the dichloromethane organic phase, and concentrating to obtain a crude product of the compound of formula II; (7) In the preparation method of the compound of formula III, R is trimethylsilylethyl or tert-butyldimethylsilylethyl; (8) In the method for preparing the compound of formula III, the reduction reaction further comprises the following steps: the reducing agent is dissolved in a solvent and then added dropwise to an acid buffer solution; the resulting reaction system is placed under inert gas protection, stirred and cooled; and then the compound of formula IV dissolved in the solvent is added dropwise to the above reaction system to carry out the reaction; (9) In the method for preparing the compound of formula III, the reducing agent is one of triphenylphosphine, tri-tert-butylphosphine and trimethylphosphine, or a mixture of any two or more thereof, or a triphenylphosphine / tetrahydrofuran solution, a tri-tert-butylphosphine / tetrahydrofuran solution or a trimethylphosphine / tetrahydrofuran solution; (10) In the method for preparing the compound of formula III, the molar ratio of the reducing agent to the compound of formula IV is 1.0-3.0; (11) In the method for preparing the compound of formula III, the volume mass ratio of the acid buffer solution to the compound of formula IV is 2-10 mL / g; (12) In the method for preparing the compound of formula III, the solvent is an ether solvent; (13) In the method for preparing the compound of formula III, the acid buffer is an acetate buffer or a formic acid buffer; (14) In the method for preparing the compound of formula III, the pH of the acid buffer solution is 4.0-6.0; (15) In the method for preparing the compound of formula III, the temperature of the reduction reaction is 0°C-20°C; In the method for preparing the compound of formula III described in (16), after the reduction reaction is completed, the following post-treatment step is further included: concentrating the reaction solution to remove the solvent, adding dichloromethane or ethyl acetate for extraction, combining the organic phases, and concentrating to obtain a crude compound of formula III; (17) In the preparation method of the compound of formula IV, R is trimethylsilylethyl or tert-butyldimethylsilylethyl; (18) 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; (19) In the method for preparing the compound of formula IV, the base is an organic base, an inorganic base or a mixture thereof; (20) 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; (21) In the method for preparing the compound of formula IV, the solvent is DMF, DMSO, tetrahydrofuran, 1,4-dioxane, or a mixture of any two or more thereof; (22) In the method for preparing the compound of formula IV, the temperature of the etherification reaction is 0°C-80°C; (23) In the method for preparing the compound of formula IV, the etherification reaction is carried out under the protection of an inert gas; (24) In the method for preparing the compound of formula IV, after the etherification reaction is completed, the following post-treatment step is further included: concentrating the reaction solution, dissolving the obtained concentrate in an organic solvent, washing the organic phase, concentrating the organic phase, and purifying the obtained residue; wherein the organic solvent is ethyl acetate or dichloromethane; the aqueous phase used for the washing is an aqueous acid solution, water and / or saturated saline; (25) 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; (26) 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; (27) 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; (28) In the method for preparing the compound of formula VI, the substitution reaction temperature is -10°C to 50°C; (29) In the method for preparing the compound of formula VI, the substitution reaction is carried out under the protection of an inert gas; In the preparation method of the compound of formula VI described in (30), after the substitution reaction is completed, the following post-treatment step is further included: the reaction liquid is subjected to solid-liquid separation or not, and the organic phase is concentrated to obtain the compound of formula VI.

27. The preparation method according to claim 26, wherein The preparation method meets one or more of the following conditions: (1) In the preparation method of the compound of formula II, R is trimethylsilylethyl; (2) In the preparation method of the compound of formula II, the molar ratio of the 6-(maleimido)hexanoic acid succinimide ester to the compound of formula III is 1-3; (3) In the method for preparing the compound of formula II, the solvent is a chlorinated alkane solvent; (4) In the method for preparing the compound of formula II, the coupling reaction temperature is 25°C-40°C; (5) In the method for preparing the compound of formula II, the coupling reaction is carried out under nitrogen or helium; (6) In the method for preparing the compound of formula II, after the coupling reaction is completed, the following post-treatment steps are further included: concentrating the reaction solution, extracting with dichloromethane and water, combining the dichloromethane organic phase, and concentrating to obtain a crude product of the compound of formula II; purifying the crude product of the compound of formula II by silica gel column chromatography to obtain a product of the compound of formula II; (7) In the preparation method of the compound of formula III, R is trimethylsilylethyl; (8) In the method for preparing the compound of formula III, the reducing agent is trimethylphosphine / tetrahydrofuran solution; (9) In the method for preparing the compound of formula III, the molar ratio of the reducing agent to the compound of formula IV is 1.2-1.8; (10) In the method for preparing the compound of formula III, the volume mass ratio of the acid buffer solution to the compound of formula IV is 3-5 mL / g; (11) In the method for preparing the compound of formula III, the solvent is an ether solvent, and the ether 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; (12) In the method for preparing the compound of formula III, the acid buffer is an acetate buffer; (13) In the method for preparing the compound of formula III, the pH of the acid buffer solution is 4.5-5.5; (14) In the method for preparing the compound of formula III, the temperature of the reduction reaction is 0°C-10°C; (15) In the preparation method of the compound of formula III, after the reduction reaction is completed, the following post-treatment steps are further included: concentrating the reaction solution to remove the solvent, adding dichloromethane or ethyl acetate for extraction, combining the organic phases, and concentrating to obtain a crude product of the compound of formula III; purifying the crude product of the compound of formula III by silica gel column chromatography to obtain a product of the compound of formula III; (16) In the preparation method of the compound of formula IV, R is trimethylsilylethyl; (17) 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; (18) In the method for preparing the compound of formula IV, the organic base is one of potassium tert-butoxide, triethylamine, DMAP, pyridine and panpiperidin, or a mixture of any two or more thereof; (19) In the method for preparing 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; (20) 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; (21) In the method for preparing the compound of formula IV, the solvent is 1,4-dioxane or tetrahydrofuran; (22) In the method for preparing the compound of formula IV, the temperature of the etherification reaction is 40°C-80°C; (23) In the method for preparing the compound of formula IV, the etherification reaction is carried out under nitrogen or helium protection; (24) In the method for preparing the compound of formula IV, in the post-treatment step of the etherification reaction, the organic solvent is ethyl acetate; (25) In the method for preparing the compound of formula IV, in the post-treatment step of the etherification reaction, the aqueous phase used for washing is saturated brine; (26) In the method for preparing the compound of formula IV, in the post-treatment step of the etherification reaction, the purification is performed by beating, crystallization, preparative chromatography or silica gel column chromatography; (27) 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; (28) In the method for preparing the compound of formula VI, the molar ratio of trimethylsilyl chloride to the compound of formula VII is 2-4; (29) In the method for preparing the compound of formula VI, the solvent is tetrahydrofuran or 1,4-dioxane; (30) In the method for preparing the compound of formula VI, the substitution reaction temperature is 15°C-35°C; (31) In the method for preparing the compound of formula VI, the substitution reaction is carried out under nitrogen or helium protection; (32) In the preparation method of the compound of formula VI, after the substitution reaction is completed, the following post-treatment step is further included: the reaction liquid is subjected to solid-liquid separation or not, the organic phase is concentrated to obtain the compound of formula VI, and the crude compound of formula VI obtained after concentration is directly reacted with the reagent V.

28. The preparation method according to claim 27, wherein The preparation method meets one or more of the following conditions: (1) In the preparation method of the compound of formula II, the molar ratio of the 6-(maleimido)hexanoic acid succinimide ester to the compound of formula III is 1.2-2; (2) In the method for preparing the compound of formula II, 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 the compound of formula II, the coupling reaction temperature is 30°C-35°C; (4) In the method for preparing the compound of formula II, after the coupling reaction is completed, in the post-treatment step, the eluent for the silica gel column chromatography is a mixed solvent of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is (10-1):1; (5) In the method for preparing the compound of formula III, the reducing agent is a 1M trimethylphosphine / tetrahydrofuran solution; (6) In the method for preparing the compound of formula III, the molar ratio of the reducing agent to the compound of formula IV is 1.5; (7) In the preparation method of the compound of formula III, the volume mass ratio of the acid buffer solution to the compound of formula IV is 4 mL / g; (8) In the method for preparing the compound of formula III, the ether solvent is tetrahydrofuran; (9) In the method for preparing the compound of formula III, the acetate buffer is sodium acetate buffer; (10) In the method for preparing the compound of formula III, the pH of the acid buffer solution is 5.0; (11) In the method for preparing the compound of formula III, the temperature of the reduction reaction is 0°C-5°C; (12) In the method for preparing the compound of formula III, after the etherification reaction is completed, in the post-treatment step, the eluent for the silica gel column chromatography is a mixed solvent of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is (10-1):1; (13) 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; (14) In the method for preparing the compound of formula IV, the organic base is panpiperidin; (15) In the method for preparing the compound of formula IV, the inorganic base is one of potassium phosphate, potassium carbonate, potassium hydroxide and cesium carbonate, or a mixture of any two or more thereof; (16) 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; (17) In the method for preparing the compound of formula IV, the solvent is tetrahydrofuran; (18) In the method for preparing the compound of formula IV, the temperature of the etherification reaction is 50°C-70°C; (19) In the method for preparing the compound of formula IV, after the etherification reaction is completed, in the post-treatment step, the eluent for the silica gel column chromatography is a mixed solvent of n-heptane and ethyl acetate, and the volume ratio of the n-heptane to the ethyl acetate is (20-1):1; (20) 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; (21) In the method for preparing the compound of formula VI, the molar ratio of trimethylsilyl chloride to the compound of formula VII is 2-3; (22) In the method for preparing the compound of formula VI, the solvent is 1,4-dioxane; (23) In the method for preparing the compound of formula VI, the substitution reaction temperature is 18°C-25°C.

29. The preparation method according to claim 28, characterized in that The preparation method meets one or more of the following conditions: (1) In the preparation method of the compound of formula II, the molar ratio of 6-(maleimido)hexanoic acid succinimide ester to the compound of formula III is 1.5; (2) In the method for preparing the compound of formula II, the chlorinated alkane solvent is dichloromethane; (3) In the preparation method of the compound of formula IV, in the post-etherification reaction treatment, the volume ratio of the n-heptane to the ethyl acetate is (10-1):

1.

30. The preparation method according to claim 26, wherein In the preparation method of the compound of formula IV, the base is an organic base.

31. A method for preparing a compound of formula I, characterized in that: The method comprises the following steps: in a solvent, subjecting the compound of formula II to a deprotection reaction in the presence of a deprotecting agent to obtain a compound of formula I, wherein the reaction conditions and operation steps are as described in any one of claims 7 to 30; Wherein, R is a C1-C6 alkyl group substituted with -Si(C1-C6)3.

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