A preparation method of a linker drug conjugate and its intermediate

Abstract

The present invention discloses a method for preparing a linker-drug conjugate and its intermediates, specifically providing methods for preparing compounds of Formula II and Formula III. The preparation method of the present invention comprises reducing a compound of Formula IV and a reducing agent in a solvent and in the presence of an acid buffer to obtain a compound of Formula III, and then coupling the compound of Formula III with 6-(maleimido)hexanoic acid succinimidyl ester to obtain a compound of Formula II. The preparation method of the present invention has one or more of the following advantages: stable intermediates, easy purification, and low impurity content; easy control of LE14 quality; a rational process that avoids the use of expensive Dxd or Dxd derivatives, significantly reducing production costs; avoiding excessive exposure to cytotoxic substances, making production safer; and suitability for large-scale industrial production.

Description

Technical Field

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

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

[0003] Patent application WO2020259258A1 discloses an ADC compound with a camptothecin derivative DXd as an effector molecule, and provides a preparation method 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] In summary, after experimental verification and evaluation, the above process synthesis route has the following disadvantages:

[0052] 1. Intermediates 1-3 and 2-2 in Route 1 and Route 2 require tert-butyl removal before proceeding to subsequent reactions. However, although 1-3 and 2-2 can react, trifluoroacetic acid is used for deprotection, which is harsh and difficult to remove after trifluoroacetic acid treatment. At the same time, the generated carboxylic acid intermediates 1-4 and 2-2a are unstable during the purification process and are almost completely lost during the purification process. The unpurified crude product can only be used directly for the next reaction, resulting in the presence of some impurities with similar polarity in the final product, which increases the difficulty of purification.

[0053] 2. Route 3, Route 4 and Route 5 require the use of DXd or DXd derivatives, which significantly increases production costs.

[0054] 3. Furthermore, the exatecan, DXd or DXd derivative intermediates used in these five routes participate in multi-step reactions during the process, which greatly increases the exposure level of this type of cytotoxic substances and puts great pressure on the protection of production operators and the environment.

[0055] The above defects limit the commercial production of this type of ADC drugs, so it is necessary to explore a more effective and reasonable new route to solve the above problems. Summary of the Invention

[0056] The technical problem to be solved by the present invention is to overcome the defects of existing methods for preparing linker-drug conjugates, such as the difficulty in purifying the products and intermediates thereof, the high cost, and the serious damage to the human body, and to provide a method for preparing a linker-drug conjugate and an intermediate thereof. The preparation method and intermediates provided by the present invention have one or more of the following advantages: the intermediates are stable, easy to purify, have low impurity content, and can be used for the subsequent preparation of the key carboxylic acid intermediate I, which is directly condensed with isotecan mesylate, making it easy to control the quality of the linker-drug conjugate; the preparation route is rational, avoids the use of expensive DXd or DXd derivatives, and significantly reduces production costs; and the cytotoxic payload isotecan can be placed in the last step of the reaction to avoid excessive exposure to cytotoxic substances, making production safer.

[0057] The present invention mainly solves the above technical problems through the following technical solutions.

[0058] The present invention provides a compound of formula II or formula III;

[0059]

[0060] Wherein, R is a C1~C6 alkyl group substituted by -Si(C1~C6)3.

[0061] In some embodiments, the R may be trimethylsilylethyl or tert-butyldimethylsilylethyl; preferably, the R is trimethylsilylethyl.

[0062] In some embodiments, the compound of formula II is

[0063]

[0064] In some embodiments, the compound of formula III is

[0065]

[0066] The present invention also provides a method for preparing a compound of formula II, comprising the following steps: subjecting a compound of formula III and 6-(maleimido)hexanoic acid succinimide ester to a coupling reaction in a solvent to obtain a compound of formula II;

[0067]

[0068] Wherein, R is a C1~C6 alkyl group substituted by -Si(C1~C6)3.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] Furthermore, the preparation method of the compound of formula II may further include a preparation method of the compound of formula III, which may include 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 compound of formula III.

[0079]

[0080] Wherein, R is a C1~C6 alkyl group substituted by -Si(C1~C6)3.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] Furthermore, the preparation method of the compound of formula II may further include a preparation method of the compound of formula IV, which may include the following steps: conducting an etherification reaction of the compound of formula VI with a reagent V in a solvent in the presence of a base to obtain the compound of formula IV.

[0095]

[0096] Wherein, R is a C1~C6 alkyl group substituted by -Si(C1~C6)3.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] Furthermore, the preparation method of the compound of formula II may further include a preparation method of the compound of formula VI, which may include the following steps: subjecting the compound of formula VII to a substitution reaction with paraformaldehyde and trimethylsilyl chloride in a solvent to obtain the compound of formula VI.

[0109]

[0110] The compound of formula VII is an azide polypeptide compound, which is commercially available or homemade.

[0111] 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.

[0112] In some embodiments, in the method for preparing the compound of formula VI, the molar ratio of the paraformaldehyde to the compound of formula VII can be 1-10, preferably 1-5, more preferably 1.3-3.0, and most preferably 1.3.

[0113] 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.

[0114] 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.

[0115] In some embodiments, in the method for preparing the compound of formula VI, the substitution reaction temperature can be -10°C-50°C, preferably 15°C-35°C, and more preferably 18°C-25°C.

[0116] 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.

[0117] 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).

[0118] In some embodiments, the substitution reaction time may be 3-24 hours, preferably 6-18 hours, and more preferably 8-16 hours.

[0119] 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.

[0120] The present invention also provides a method for preparing a compound of formula III, comprising the following steps: performing a reduction reaction on a compound of formula IV and a reducing agent in a solvent and in the presence of an acid buffer to obtain a compound of formula III;

[0121]

[0122] Wherein, R is defined as described above.

[0123] In the preparation method of the compound of formula III, the reaction conditions can be the same as described above.

[0124] The preparation method of the compound of formula III may further include the preparation method of the compound of formula IV described herein.

[0125] definition

[0126] The Chinese and English comparison table of the compounds involved in the present invention is shown in Table 1.

[0127] Table 1

[0128]

[0129] 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.

[0130] The reagents and raw materials used in the present invention are commercially available or homemade.

[0131] The present invention provides a novel method for preparing a linker-drug conjugate and its intermediate. The intermediate provided by the present invention is stable, easy to purify, and contains few impurities. It can be used to subsequently prepare the key carboxylic acid intermediate I, which is directly condensed with isotecan mesylate, making it easy to control the quality of LE14. The resulting compound LE14 meets the product quality requirements for IND filings. The preparation process is rational, avoiding the use of expensive Dxd or Dxd derivatives, significantly reducing production costs. The cytotoxic payload isotecan can be added to the final step of the reaction, avoiding excessive exposure to cytotoxic substances and making production safer. The intermediate is suitable for large-scale industrial production. DETAILED DESCRIPTION

[0132] 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.

[0133] In the following examples,

[0134] Mass spectrometry was performed using a Waters Acquity Xevo G2-XS QTof UPLC / MS ultra-high performance liquid chromatography-high resolution mass spectrometry system;

[0135] 1 H-NMR was performed using a Bruker AVANCE III 400 MHz NMR spectrometer or a Bruker AVANCE III HD 300 MHz NMR spectrometer;

[0136] HPLC detection conditions: The instrument was Agilent 187260, the chromatographic column was Agilent AdvanceBio PeptideMap, 3.5 μm, 2.1×250 mm, the detection wavelength was 254 nm, and the mobile phase gradient setting shown in Table 2 was adopted, wherein 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.

[0137] Table 2. Mobile phase gradient settings

[0138] Time (min) Mobile phase A% Mobile phase B% 0.00 80.0 20.0 5.00 60.0 40.0 35.00 60.0 40.0 40.00 30.0 70.0 46.00 30.0 70.0 46.10 80.0 20.0 60.00 80.0 20.0

[0139] Conventional column chromatography conditions are as follows: the filler is 200-400 mesh amorphous silica gel, and the column chromatography is performed at room temperature.

[0140] In the following examples, room temperature refers to 20°C-30°C.

[0141] Example 1 Overall Synthesis Route of LE14

[0142]

[0143] Step 1: Synthesis of compound of formula VI

[0144]

[0145] Compound 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 to the resulting mixture with stirring. The resulting reaction system was stirred at 18°C-25°C for 15.0 h. The reaction was monitored by TLC. After completion of the reaction, the reaction solution was concentrated under reduced pressure to obtain a crude compound of Formula VI (28.95 g). (Used directly in the next reaction)

[0146] Step 2: Synthesis of compound of formula IVa

[0147]

[0148] 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% yield from Steps 1 and 2 combined).

[0149] ESI-MS m / z: 657.4 (M+H).

[0150] 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).

[0151] Step 3: Synthesis of compound of formula IIIa

[0152]

[0153] A mixture of 32.0 mL of a 1 M trimethylphosphine solution 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 raw 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%).

[0154] ESI-MS m / z: 631.4 (M+H).

[0155] 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).

[0156] Step 4: Synthesis of compound of formula IIa

[0157]

[0158] 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%).

[0159] ESI-MS m / z: 824.5 (M+H).

[0160] 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(dd ,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).

[0161] Step 5: Synthesis of compound of formula I

[0162]

[0163] Method 1: Deprotection using potassium fluoride reagent

[0164] The compound of Formula IIa (8.50 g, 10.04 mmol) was dissolved in DMF (45.0 mL). Powdered potassium fluoride (0.88 g, 15.06 mmol) was added at room temperature. The resulting reaction system was heated to 60°C with stirring under nitrogen and allowed to react for 6 hours. The reaction was monitored by TLC. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The resulting crude product was pretreated and then purified by silica gel column chromatography (dichloromethane:methanol = 10:1 to 1:1 (v / v)) to obtain the compound of Formula I (4.72 g, 83.06%, yield 65.0%; the actual amount of pure compound of Formula I was 3.92 g, and the yield calculated based on the amount of pure compound of Formula I was 54.0%).

[0165] The above pretreatment step is: using dichloromethane with a volume (mL) 10 times the weight (g) of the crude compound of formula I (the ratio of the volume of dichloromethane to the weight of the crude product is 10 mL / g) to ultrasonically dissolve the crude oil at room temperature, and then adding methyl tert-butyl ether with a volume 50 times the weight of the crude product of formula I (the ratio of the volume of methyl tert-butyl ether to the weight of the crude product is 50 mL / g), and stirring at room temperature for 2-5 hours. After standing, the supernatant is poured off, and the lower oil is vacuum concentrated to obtain the pretreated crude compound of formula I.

[0166] ESI-MS m / z: 722.3 (MH);

[0167] 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).

[0168] Method 2: Deprotection using sodium fluoride reagent

[0169] The operation steps were the same as those of method 1, except that potassium fluoride was replaced by sodium fluoride, and the compound of formula I (3.44 g, yield 47.3%) was obtained by purification.

[0170] Method 3: Deprotection using cesium fluoride reagent

[0171] The operation steps were the same as those of method 1, except that potassium fluoride was replaced by cesium fluoride, and the compound of formula I (3.02 g, yield 41.50%) was obtained by purification.

[0172] Method 4: Deprotection using tetrabutylammonium fluoride reagent

[0173] 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 crude product was purified using the same post-treatment method as in Method 1 to obtain the compound of Formula I (3.66 g, 50.3% yield).

[0174] Method 5: (the product compound of formula I is not purified and is directly used in the next reaction):

[0175] Dissolve the compound of Formula IIa (8.50 g, 10.04 mmol) in DMF (45.0 mL). Add powdered potassium fluoride (0.88 g, 15.06 mmol) at room temperature. Heat to 60°C under nitrogen and allow to react for 6 hours. After the reaction is complete, concentrate under reduced pressure to remove the solvent. The resulting crude product, Compound I (9.85 g), is used in the next reaction.

[0176] Step 6: Synthesis of LE14

[0177]

[0178] Method 1 (using the purified compound of formula I for reaction)

[0179] The compound of Formula I (4.72 g, 83.06% purity, 5.42 mmol) was dissolved in 15.0 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 (methanesulfonate) (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 removed under reduced pressure, and the crude product was purified by silica gel column chromatography (chloroform:methanol = 10:1 (v / v)) to yield the desired product LE14 (3.69 g, 98.77% purity, single-step yield 59.6%, combined yield of two steps 32.2%).

[0180] Method 2 (using the unpurified crude compound of formula I prepared by the above method 5 to react)

[0181] The crude compound of Formula I (10.04 mmol, calculated based on 100% conversion) was dissolved in 15.0 mL of anhydrous DMF. 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) (4.16 g, 15.06 mmol) was added and the reaction was allowed to proceed at room temperature for 1.0 h. N,N-diisopropylethylamine (1.94 g, 15.06 mmol) and exatecan (methanesulfonate) (5.37 g, 10.04 mmol) were then added and the reaction continued for 1.5-2.0 h. After the reaction was complete, the solvent was removed by distillation under reduced pressure. The resulting crude product was purified by silica gel column chromatography (chloroform:methanol = 10:1 (v / v)) to yield the desired product LE14 (5.71 g, 98.06% purity, 49.8% yield for both steps).

[0182] ESI-MS m / z: 1141.7 (M+H);

[0183] 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).

[0184] Example 2 Comprehensive comparison of process routes

[0185] The crude carboxylic acid intermediate 1-4 obtained according to the method of Route 1 was purified by silica gel column chromatography (chloroform:methanol=10:1 (v / v)), but no purified intermediate 1-4 was obtained. The crude carboxylic acid intermediate 2-2a obtained according to the method of Route 2 was purified by silica gel column chromatography (chloroform:methanol=10:1 (v / v)), but no purified intermediate 2-2a was obtained. However, the carboxylic acid intermediate I of good purity was obtained in good yield according to the method of Example 1. The purification status of the carboxylic acid intermediates prepared by the three synthetic routes is summarized and compared in Table 3 below.

[0186] 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 4 below.

[0187] Table 3. Comparison of carboxylic acid intermediates (1-4, 2-2a and I) obtained from several process routes

[0188]

[0189]

[0190] Table 4. Comparative data of purity of final product LE14 from different process routes

[0191]

[0192] 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 compound 5. The total yield of each route based on 5 feeds and the purity of the final product are summarized in Table 5.

[0193] Table 5. Comparison of the purity of the final product of different process routes and the total yield of the same compound 5 (VII) as the starting material

[0194]

[0195] In the prior art synthesis routes of CN 115215921 A and CN115385926A, the total yields of Route 4 and Route 5 for synthesizing the final product LE14 using ixetemcan as the starting material are lower than that of the present invention, as shown in Table 6.

[0196] Table 6. Comparison of total yields of different process routes based on the input of ixetem

[0197]

[0198]

[0199] As can be seen from Tables 4, 5, and 6, 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 filing (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 numerous and the production operation is cumbersome. On the other hand, there are also significant losses during the preparation process, resulting in a low utilization rate of the expensive material isotecan, making it unsuitable for large-scale industrial production.

[0200] Specifically speaking of 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 a 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. 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. However, method 2 does not have these advantages of method 1. 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 6 (yield calculated based on isotecan, method 1: 59.6%, method 2: 49.8%). After comprehensive weighing and comparison, among the two process routes provided in this application, method 1 is a more preferred production process.

[0201] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A compound of formula II or III; in, R is trimethylsilylethyl.

2. A method for preparing a compound of formula II, characterized in that: The method comprises the following steps: carrying out a coupling reaction between a compound of formula III and 6-(maleimido)hexanoic acid succinimide ester in a solvent to obtain a corresponding compound of formula II; Wherein, R is trimethylsilylethyl.

3. The preparation method according to claim 2, 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 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 coupling reaction temperature is 0°C-45°C; (3) In the method for preparing the compound of formula II, the coupling reaction is carried out under the protection of an inert gas.

4. The preparation method according to claim 3, wherein The preparation method meets one or both 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 1-3; (2) In the method for preparing the compound of formula II, the coupling reaction temperature is 25°C-40°C.

5. The preparation method according to claim 4, wherein 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.

6. The preparation method according to claim 2, wherein In the preparation method of the compound of formula II, the solvent is one of amide solvents, chloroalkane solvents, ether solvents and nitrile solvents, or a mixture of any two or more thereof.

7. The preparation method according to claim 6, wherein In the preparation method of the compound of formula II, the solvent is a chlorinated alkane solvent.

8. The preparation method according to claim 7, wherein In the preparation method of 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.

9. The preparation method according to claim 2, wherein In the preparation method of 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.

10. The preparation method according to claim 9, characterized in that In the preparation method of the compound of formula II, the crude compound of formula II is purified by silica gel column chromatography to obtain the product compound of formula II.

11. The preparation method according to claim 3, 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 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 coupling reaction temperature is 30°C-35°C; (3) In the method for preparing the compound of formula II, the coupling reaction is carried out under the protection of nitrogen or helium.

12. The preparation method according to claim 7, wherein In the preparation method of the compound of formula II, the solvent is dichloromethane.

13. The preparation method according to claim 10, wherein In the preparation method of the compound of formula II, 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.

14. The preparation method according to claim 2, wherein The preparation method of the compound of formula II further 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 trimethylsilylethyl.

15. The preparation method according to claim 14, wherein The preparation method of the compound of formula II 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 trimethylsilylethyl.

16. The preparation method according to claim 15, characterized in that The preparation method of the compound of formula II 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; 17. The preparation method according to claim 14, wherein The preparation method meets one or more of the following conditions: (1) 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; (2) 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 tetrahydrofuran solution of triphenylphosphine, a tetrahydrofuran solution of tri-tert-butylphosphine or a tetrahydrofuran solution of trimethylphosphine; (3) 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; (4) In the method for preparing the compound of formula III, the volume mass ratio of the acid buffer to the compound of formula IV is 2-10 mL / g; (5) In the method for preparing the compound of formula III, the solvent is an ether solvent; (6) In the method for preparing the compound of formula III, the acid buffer is an acetate buffer or a formic acid buffer; (7) In the method for preparing the compound of formula III, the pH of the acid buffer is 4.0-6.0; (8) In the method for preparing the compound of formula III, the temperature of the reduction reaction is 0°C-20°C.

18. The preparation method according to claim 17, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula III, the reducing agent is a tetrahydrofuran solution of trimethylphosphine; (2) 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; (3) 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 3-5 mL / g; (4) In the method for preparing the compound of formula III, the solvent is one of tetrahydrofuran, diethyl ether, 1,4-dioxane, anisole and methyl tert-butyl ether, or a mixture of any two or more thereof; (5) In the method for preparing the compound of formula III, the pH of the acid buffer solution is 4.5-5.5; (6) In the method for preparing the compound of formula III, the temperature of the reduction reaction is 0°C-10°C.

19. The preparation method according to claim 14, wherein In the preparation method of the compound of formula III, 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 product of the compound of formula III.

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

21. The preparation method according to claim 15, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula IV, the molar ratio of the reagent V to the compound of formula VI is 1-5; (2) In the method for preparing the compound of formula IV, the molar ratio of the base to the compound of formula VI is 1-5; (3) 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; (4) In the method for preparing the compound of formula IV, the temperature of the etherification reaction is 0°C-80°C; (5) In the method for preparing the compound of formula IV, the etherification reaction is carried out under the protection of an inert gas.

22. The preparation method according to claim 21, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula IV, the molar ratio of the reagent V to the compound of formula VI is 1-3; (2) In the method for preparing the compound of formula IV, the molar ratio of the base to the compound of formula VI is 1.2-4; (3) In the method for preparing the compound of formula IV, the solvent is 1,4-dioxane or tetrahydrofuran; (4) In the method for preparing the compound of formula IV, the temperature of the etherification reaction is 40°C-80°C.

23. The preparation method according to claim 15, wherein In the preparation method of the compound of formula IV, the base is an organic base, an inorganic base or a mixture thereof.

24. The preparation method according to claim 23, wherein In the preparation method of the compound of formula IV, the base is an organic base.

25. The preparation method according to claim 24, wherein In the preparation method of 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.

26. The preparation method according to claim 23, wherein In the preparation method of the compound of formula IV, the inorganic base is one of alkali metal hydroxides, alkali metal carbonates and alkali metal phosphates, or a mixture of any two or more thereof.

27. The preparation method according to claim 26, wherein In the preparation method of 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.

28. The preparation method according to claim 15, wherein In the preparation method of the compound of formula IV, after the etherification reaction is completed, the following post-treatment steps are further included: concentrating the reaction solution, dissolving the obtained concentrate with an organic solvent, washing the organic phase, concentrating the organic phase, and purifying the obtained residue.

29. The preparation method according to claim 28, wherein In the preparation method of the compound of formula IV, the organic solvent is ethyl acetate or dichloromethane.

30. The preparation method according to claim 29, wherein In the preparation method of the compound of formula IV, the organic solvent is ethyl acetate.

31. The preparation method according to claim 28, wherein In the preparation method of the compound of formula IV, the aqueous phase used for washing is an acid aqueous solution, water and / or saturated saline.

32. The preparation method according to claim 31, wherein In the preparation method of the compound of formula IV, the organic phase is washed with saturated brine.

33. The preparation method according to claim 28, wherein In the preparation method of the compound of formula IV, the purification adopts beating, crystallization, preparative chromatography or silica gel column chromatography.

34. The preparation method according to claim 16, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula VI, the molar ratio of the paraformaldehyde to the compound of formula VII is 1-10; (2) In the preparation method of the compound of formula VI, the molar ratio of trimethylsilyl chloride to the compound of formula VII is 1-5; (3) In the method for preparing the compound of formula VI, the solvent is one of N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran and 1,4-dioxane, or a mixture of any two or more thereof; (4) In the method for preparing the compound of formula VI, the substitution reaction temperature is -10°C to 50°C; (5) In the method for preparing the compound of formula VI, the substitution reaction is carried out under the protection of an inert gas.

35. The preparation method according to claim 34, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula VI, the molar ratio of the paraformaldehyde to the compound of formula VII is 1-5; (2) In the preparation method of the compound of formula VI, the molar ratio of trimethylsilyl chloride to the compound of formula VII is 2-4; (3) In the method for preparing the compound of formula VI, the solvent is tetrahydrofuran or 1,4-dioxane; (4) In the method for preparing the compound of formula VI, the substitution reaction temperature is 15°C-35°C.

36. The preparation method according to claim 35, wherein The preparation method meets one or both of the following conditions: (1) In the method for preparing the compound of formula VI, the molar ratio of the paraformaldehyde to the compound of formula VII is 1.3-3.0; (2) In the preparation method of the compound of formula VI, the molar ratio of trimethylsilyl chloride to the compound of formula VII is 2-3.

37. The preparation method according to claim 16, wherein 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 solution is subjected to solid-liquid separation or not, and the organic phase is concentrated to obtain the compound of formula VI.

38. The preparation method according to claim 17, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula III, the reducing agent is a 1M trimethylphosphine solution in tetrahydrofuran; (2) In the preparation method of the compound of formula III, the molar ratio of the reducing agent to the compound of formula IV is 1.5; (3) 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; (4) In the method for preparing the compound of formula III, the solvent is tetrahydrofuran; (5) In the method for preparing the compound of formula III, the acid buffer solution is an acetate buffer solution; the acetate buffer solution is a sodium acetate buffer solution; (6) In the method for preparing the compound of formula III, the pH of the acid buffer solution is 5.0; (7) In the method for preparing the compound of formula III, the temperature of the reduction reaction is 0°C-5°C.

39. The preparation method according to claim 20, wherein In the preparation method of the compound of formula III, 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.

40. The preparation method according to claim 21, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula IV, the molar ratio of the reagent V to the compound of formula VI is 1.1-1.6; (2) In the method for preparing the compound of formula IV, the molar ratio of the base to the compound of formula VI is 1.5-3; (3) In the method for preparing the compound of formula IV, the solvent is tetrahydrofuran; (4) In the method for preparing the compound of formula IV, the temperature of the etherification reaction is 50°C-70°C; (5) In the method for preparing the compound of formula IV, the etherification reaction is carried out under the protection of nitrogen or helium.

41. The preparation method according to claim 23, wherein In the preparation method of the compound of formula IV, the base is panpiidine.

42. The preparation method according to claim 33, wherein In the preparation method of the compound of formula IV, the purification adopts a silica gel column chromatography method, the eluent of 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.

43. The preparation method according to claim 42, wherein In the preparation method of the compound of formula IV, the volume ratio of the n-heptane to the ethyl acetate is (10-1):

1.

44. The preparation method according to claim 34, wherein The preparation method meets one or more of the following conditions: (1) In the method for preparing the compound of formula VI, the molar ratio of the paraformaldehyde to the compound of formula VII is 1.3; (2) In the method for preparing the compound of formula VI, the molar ratio of trimethylsilyl chloride to the compound of formula VII is 3; (3) In the method for preparing the compound of formula VI, the solvent is 1,4-dioxane; (4) In the method for preparing the compound of formula VI, the substitution reaction temperature is 18°C-25°C; (5) In the method for preparing the compound of formula VI, the substitution reaction is carried out under the protection of nitrogen or helium.

45. A method for preparing a compound of formula III, characterized in that: The method comprises the following steps: performing a reduction reaction on a compound of formula IV and a reducing agent in a solvent and in the presence of an acid buffer to obtain a compound of formula III; the reaction conditions are as described in any one of claims 14, 17-20 or 38-39; Wherein, R is trimethylsilylethyl.

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