CN116217654A

CN116217654A - Preparation method of linker drug conjugate and intermediate thereof

Info

Publication number: CN116217654A
Application number: CN202211728485.7A
Authority: CN
Inventors: 李振东; 刘宁; 沈磊; 杨周; 王东生; 邱雪飞; 王宝霞
Original assignee: SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
Current assignee: SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2023-06-06

Abstract

The invention discloses a preparation method of a linker drug conjugate and an intermediate thereof, and particularly provides a preparation method of a compound shown in a formula II and a preparation method of a compound shown in a formula II I. The preparation method of the invention comprises the steps of carrying out reduction reaction on a compound of formula IV and a reducing agent in a solvent in the presence of an acid buffer solution to obtain a compound of formula III, and carrying out coupling reaction on the compound of formula III and 6- (maleimido) caproic acid succinimidyl ester to obtain a compound of formula II. The preparation method of the invention has one or more of the following advantages: the intermediate is stable, easy to purify and low in impurity content; the quality of LE14 is easy to control; the process is reasonable, expensive Dxd or Dxd derivatives are avoided, and the production cost is greatly reduced; excessive exposure of cytotoxic substances is avoided, and the production is safer; is suitable for large-scale industrial production.

Description

Preparation method of linker drug conjugate and intermediate thereof

Technical Field

The invention belongs to the field of drug synthesis, and particularly relates to a preparation method of a linker drug conjugate and an intermediate thereof.

Background

Antibody coupled drugs (ADCs) become drug lines which are in the arrangement of medical enterprises at home and abroad in recent years, and are one of the hot spots of attention in the pharmaceutical industry in recent years. The ADC medicine consists of three basic modules, including antibody, connector and effector molecule. The ADC medicine utilizes the specific targeting effect of the antibody to transmit effector molecules to tumor sites for enrichment, so that the aim of killing tumor cells is fulfilled. Among effector molecules, a class of camptothecin analogs is widely used in the ADC field. In recent years, the application of the camptothecin as an effector molecule in an ADC drug IMMU-132 (ZL 200980156218) of the compound by the company Immunometics shows better anti-tumor effect, and the first three uses another camptothecin analogue as an ADC drug DS-8201a (ZL 201380053256) of the effector molecule together also shows better anti-tumor effect.

Patent application WO2020259258A1 discloses an ADC compound with a camptothecin derivative DXd as effector molecule and provides a method for the preparation of a linker drug conjugate LE as shown in scheme 1 and scheme 2; patent applications WO2022204947A1, CN115215921a also provide improved route 3 and route 4.

Route 1:

the synthetic method of scheme 1 includes the steps of: the compound 1-1 reacts with 4-amino benzyl alcohol, the obtained compound reacts with di (p-nitrobenzene) carbonic ester and then reacts with substituted alkylamine to obtain a compound 1-2, the compound 1-2 reacts with paraformaldehyde and trimethylchlorosilane to obtain a compound 1-3, the compound 1-3 reacts with tert-butyl glycolate, the tert-butyl is removed under the action of trifluoroacetic acid to obtain a compound 1-4, the compound 1-4 reacts with Exatecan mesylate to obtain a compound 1-5, fmoc protection on deamination under the action of DBU is removed, and then the compound 1-4 undergoes coupling reaction with 6- (maleimide) caproic acid succinimidyl ester to obtain a target compound LE.

In the step of preparing the intermediate 1-4 by taking the intermediate 1-3 as a raw material in the route, as the raw material and the product are unstable to acid and alkali, certain decomposition risks exist in the deprotection and purification processes, the compound 1-4 is not purified, the crude product is directly used for the next reaction, obvious impurities are generated in the crude product 1-5, the polarity of the impurities is close to that of the product, the purification is difficult, and the quality of the LE product is further influenced.

Route 2:

the synthetic method of scheme 2 includes the steps of: reacting the compound 2-1 with paraformaldehyde and trimethylchlorosilane, reacting the obtained compound with tert-butyl glycolate to obtain a compound 2-2, removing tert-butyl groups from the compound 2-2 under the action of trifluoroacetic acid to obtain a compound 2-2a, reacting the compound 2-2 with Exatecan mesylate to obtain a compound 2-3, reducing azide into amino under the action of triethylphosphine to obtain a compound 2-4, and performing a coupling reaction on the compound 2-4 and MC-V to obtain a target compound LE.

In the route, the raw materials 2-2 and the carboxylic acid intermediate 2-2a generated by removing the tertiary butyl are unstable to acid and alkali, a certain decomposition risk exists in the deprotection and purification processes, the compound 2-2a is not purified, the crude product is directly used for the next reaction, obvious impurities can be generated in the 2-3 crude product, the polarity of the impurities is close to that of the product, the purification is difficult, and the quality of the LE product is further influenced.

Route 3:

the synthetic method of scheme 3 includes the steps of: the compound 3-1 reacts with p-nitrophenyl chloroformate to obtain a compound 3-2, the obtained compound reacts with corresponding amine to obtain a compound 3-3, deprotection is carried out to obtain a compound 3-4, the compound further reacts with amino acid active ester to obtain a compound 3-5, deprotection is carried out to obtain a compound 3-6, the compound 3-7 reacts with an acyl azide reagent to obtain a compound 3-8, the compound 3-8 reacts with DXd or DXd derivatives to obtain a compound 3-9, azide groups in the compound 3-9 are reduced to obtain a compound 3-10, and a maleimide joint is connected to obtain a final product LE14.

According to the method of scheme 3, the total yield of LE14 prepared from compound 3-1 as starting material was 3.5% and the total yield of LE14 prepared from compound 3-7 as starting material was 10.7%.

The use of DXd or DXd derivatives as a source of payload in this route is expensive and adds significantly to the cost of preparing LE14.

Route 4:

the synthetic method of scheme 4 includes the steps of: the compound 4-1 reacts with p-nitrophenyl chloroformate to obtain a compound 4-2, the obtained compound reacts with corresponding amine to obtain a compound 4-3, deprotection is carried out to obtain a compound 4-4, the compound further reacts with amino acid active ester to obtain a compound 4-5, deprotection is carried out to obtain a compound 4-6, the compound further reacts with an acyl azide reagent to obtain a compound 4-7, chloromethylation reaction is carried out to obtain a compound 4-8, the compound further reacts with DXd derivative to obtain a compound 4-9, azide groups in the compound 4-9 are reduced to obtain a compound 4-10, a maleimide joint is connected to obtain a compound 4-11, and finally hydroxy protecting groups are removed to obtain a final product LE14.

According to the method of scheme 4, using compound 4-1 as starting material and Dxd-a therein as the payload source, LE14 was prepared in an overall yield of 7.3% (4-1→ Dxd-a→le 14); the total yield of LE14 prepared from compound 4-1 as starting material and Dxd-b therefrom as payload source was 12.9% (4-1. Fwdarw. Dxd-b. Fwdarw.LE 14).

The DXd derivative Dxd-a (structure see scheme 4-a) or Dxd-b (structure see scheme 4-b) is used as a source of payload in this route, and is expensive, greatly increasing the cost of preparing LE 14.

To avoid the purchase of expensive DXd derivatives, this company developed synthetic routes 4-a and 4-b for Dxd-a and Dxd-b starting from irinotecan.

Irinotecan derivatization route 4-a: (irinotecan. Fwdarw. Dxd-a)

The synthetic procedure for preparing Dxd-a according to scheme 4-a includes: the method comprises the steps of reacting irinotecan or mesylate thereof with 4-methoxytriphenylmethane chloride in the presence of trimethylchlorosilane and N, N-diisopropylethylamine to obtain an amino-protected intermediate compound 17, reacting the compound 17 with acetic anhydride under alkaline conditions to obtain an acetylated intermediate 16a, and reacting the intermediate 15a obtained after deamination of the intermediate 16a under the action of triethylsilane with glycolic acid to obtain a compound Dxd-a, wherein the total yield of Dxd-a prepared from irinotecan as a starting material is 38.3% (yield of irinotecan- > Dxd-a; hereinafter referred to as yield of route 4-a).

The overall yield of LE14 prepared from compound 4-1 as starting material according to the synthesis method of scheme 4 and Dxd-a as payload source according to the synthesis method of scheme 4-a as starting material was 2.8% (the yield of 4-1→ Dxd-a→le14 multiplied by the yield of the derivative of irinotecan scheme 4-a, i.e. 7.3% ×38.3% = 2.8%).

The overall yield of LE14 prepared in the method of scheme 4 using compound 4-7 as starting material and using irinotecan as starting material according to the synthetic method of scheme 4-a as the payoad source was 8.2% (the yield of 4-7→ Dxd-a→le14 multiplied by the yield of irinotecan derivatisation scheme 4-a, i.e. 21.3% ×38.3% =8.2%).

Irinotecan derivatization route 4-b: (irinotecan. Fwdarw. Dxd-b)

The synthetic procedure for preparing Dxd-b according to scheme 4-b includes: the method comprises the steps of reacting irinotecan or mesylate thereof with 4-methoxytriphenylmethane chloride in the presence of trimethylchlorosilane and N, N-diisopropylethylamine to obtain an amino-protected intermediate compound 17, reacting the compound 17 with tert-butyldiphenylchlorosilane under alkaline conditions to obtain a tert-butyldiphenylsilanized intermediate 16b, and reacting the intermediate 15b obtained after deamination of the intermediate 16b under the action of triethylsilane with glycolic acid to obtain a compound Dxd-b, wherein the total yield of Dxd-b prepared from irinotecan as a starting material is 48.2% (yield of irinotecan- > Dxd-b; hereinafter referred to as yield of route 4-b).

The overall yield of LE14 prepared as per scheme 4 from compound 4-1 as starting material according to the synthesis of scheme 4 to give compound 4-8 and as per scheme 4-b as starting material according to the synthesis of scheme 4 to give Dxd-b as a payload source (the yield of 4-1→ Dxd-b→le14 multiplied by the yield of the derivative of irinotecan, scheme 4-b, i.e. 12.9% ×48.2% = 6.2%).

The overall yield of LE14 prepared according to the method of scheme 4 in using compound 4-7 as starting material and using Dxd-b prepared according to the synthetic method of scheme 4-b as starting material as a source of payload was 18.0% (the yield of 4-7→ Dxd-b→le14 multiplied by the yield of the derivative of irinotecan scheme 4-b, i.e. 37.5% ×48.2% = 18.0%).

If using irinotecan as starting material, dxd-a and Dxd-b prepared according to the methods of routes 4-a and 4-b are used as Dxd derivative sources for route 4, although the need of directly using Dxd which is relatively expensive is avoided, the number of 4 steps of reaction is increased, the experimental operation amount is increased, and meanwhile, the loss is huge in the process of derivatization of irinotecan, and the production cost is increased.

Route 5:

the synthetic method of scheme 5 includes the steps of: the compound 5-1 reacts with p-nitrophenyl chloroformate to obtain a compound 5-2, the obtained compound reacts with corresponding amine to obtain a compound 5-3, deprotection is carried out to obtain a compound 5-4, further reacts with amino acid active ester to obtain a compound 5-5, deprotection is carried out to obtain a compound 5-6, then reacts with an acyl azide reagent to obtain a compound 5-7, then a methylolation reaction is carried out to obtain a compound 5-8, then reacts with a DXd derivative to obtain a compound 5-9, azide groups in the compound 5-9 are reduced to obtain a compound 5-10, a maleimide joint is connected to obtain a compound 5-11, and finally a hydroxyl protecting group is removed to obtain a final product LE14.

The overall yield of LE14 prepared from compound 5-1 as starting material and Dxd-a '(numbered Dxd-a in the parent application, structure see scheme 5-a, renumbered Dxd-a' in this application for payload source as distinguished from Dxd-a in scheme 4-a) was 6.9% following the procedure of scheme 5; the overall yield of LE14 prepared from compound 5-1 as starting material, with Dxd-b '(numbered Dxd-b in the parent application, structure see scheme 5-b, for distinction from Dxd-b in scheme 4-b, renumbered Dxd-b' in the present application) as a payload source was 12.2%.

The DXd derivatives Dxd-a 'or Dxd-b' used as the sources of payload in this route are expensive to sell, greatly increasing the cost of preparing LE 14.

To avoid the purchase of expensive DXd derivatives, this company developed synthetic routes 5-a and 5-b for the preparation of Dxd-a 'and Dxd-b' from irinotecan.

Irinotecan derivatization route 5-a: (irinotecan. Fwdarw. Dxd-a')

The synthetic procedure for preparing Dxd-a' according to scheme 5-a includes: the method comprises the steps of reacting irinotecan or mesylate thereof with 4-methoxytriphenylmethane chloride in the presence of trimethylchlorosilane and N, N-diisopropylethylamine to obtain an amino-protected intermediate compound 17, reacting the compound 17 with acetic anhydride under alkaline conditions to obtain an acetylated intermediate 16a, and further reacting the intermediate 15a obtained after deamination of the intermediate 16a under the action of triethylsilane with 2-bromoacetic acid to obtain a compound Dxd-a ', wherein the total yield of Dxd-a ' prepared from irinotecan as a starting material is 27.6% (yield of irinotecan- > Dxd-a ', hereinafter referred to as scheme 5-a).

The overall yield of LE14 prepared from compound 5-1 as starting material according to the synthesis method of scheme 5 and Dxd-a 'prepared from irinotecan as starting material according to the synthesis method of scheme 5-a as a payload source according to the method of scheme 5 was 1.9% (the yield of 5-1→ Dxd-a' →le14 multiplied by the yield of the derivative of irinotecan, scheme 5-a, i.e., 6.9% ×27.6% = 1.9%).

The overall yield of LE14 prepared according to the method of scheme 5 using compound 5-7 as starting material and using as starting material irinotecan as starting material Dxd-a 'as the payload source was 6.5% (the yield of 5-7→ Dxd-a' →le14 multiplied by the yield of the irinotecan derivatisation scheme 5-a, i.e. 23.4% ×27.6% = 6.5%).

Irinotecan derivatization route 5-b: (irinotecan. Fwdarw. Dxd-b')

The synthetic procedure for preparing Dxd-b' according to scheme 5-b includes: the intermediate compound 17 with protection of amino is prepared by reacting irinotecan or mesylate thereof with 4-methoxytriphenylmethane chloride in the presence of trimethylchlorosilane and N, N-diisopropylethylamine, the intermediate compound 16b with protection of tert-butyldiphenylchlorosilane is prepared by reacting the compound 17 with tert-butyldiphenylchlorosilane under alkaline conditions, the intermediate 15b obtained after deamination of the intermediate 16b under the action of triethylsilane is further reacted with 2-bromoacetic acid to obtain the compound Dxd-b ', the total yield of Dxd-b ' prepared from irinotecan as a starting material is 44.5% (yield of irinotecan- > Dxd-b ', hereinafter referred to as route 5-b).

The overall yield of LE14 prepared from compound 5-1 as starting material according to the synthesis method of scheme 5 and Dxd-b 'as payload source according to the synthesis method of scheme 5-b as starting material was 5.4% (the yield of 5-1→ Dxd-b' →le14 multiplied by the yield of the derivative of irinotecan scheme 5-b, i.e. 12.2% ×44.5% = 5.4%).

The overall yield of LE14 prepared according to the method of scheme 5 using compound 5-7 as starting material and using as starting material irinotecan as starting material Dxd-b 'as a payload source was 18.3% (the yield of 5-7→ Dxd-b' →le14 multiplied by the yield of the irinotecan derivatisation scheme 5-b, i.e. 41.2% ×44.5% = 18.3%).

If the method for preparing the derivatives of Dxd-a 'and Dxd-b' by taking the irinotecan as the starting material according to the methods of the routes 5-1 and 5-2 is used as a Dxd derivative source for the route 5, the method avoids the use of relatively expensive Dxd directly, but increases the number of 4 steps of reactions and the experimental operation, and meanwhile, the loss of the irinotecan in the derivatization process is huge, so that the production cost is increased.

In summary, after experimental verification and evaluation, the above process synthetic route has the following disadvantages:

1. The intermediates 1-3 and 2-2 in the routes 1 and 2 need to undergo a tert-butyl removal reaction to continue the subsequent reaction, but 1-3 and 2-2 can react, but trifluoroacetic acid is used for deprotection, the conditions are severe, the trifluoroacetic acid is difficult to remove after-treatment, meanwhile, the generated 1-4 and 2-2a carboxylic acid intermediates are unstable in the purification process and almost completely lost in the purification process, and only crude products which are not purified can be directly used for the next reaction, so that a part of impurities with polarity similar to that of the impurities exist in the final product, and the purification difficulty is increased.

2. The need to use derivatives of DXd or DXd in route 3, route 4 and route 5 greatly increases the cost of production.

3. Furthermore, the five routes use the derivative intermediates of the irinotecan (Exatecan), DXd or DXd to participate in multi-step reactions in the process, so that the exposure degree of the cytotoxic substances is greatly increased, and great pressure is brought to production operators and environment protection.

The above drawbacks limit the commercial production of such ADC drugs, and therefore a new more efficient and rational route needs to be explored to solve the above problems.

Disclosure of Invention

The invention aims to overcome the defects that in the existing preparation method of the linker drug conjugate, the product and an intermediate thereof are not easy to purify, the cost is high and the damage to human bodies is large, and provides the preparation method of the linker drug conjugate and the intermediate thereof. The preparation method and the intermediate thereof provided by the invention have one or more of the following advantages: the intermediate is stable, easy to purify and low in impurity content, can be used for preparing a key carboxylic acid intermediate I in the subsequent process, and the carboxylic acid intermediate I is directly condensed with the Yi-xi-Kang Jia sulfonate, so that the quality of the linker drug conjugate is easy to control; the preparation route has reasonable process, avoids using expensive DXd or DXd derivatives, and greatly reduces the production cost; the cytotoxic payload irinotecan can be put into the last step to participate in the reaction, so that the overexposure of cytotoxic substances is avoided, and the production is safer.

The invention mainly solves the technical problems through the following technical scheme.

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

wherein R is-Si (C) ₁ ～C ₆ ) ₃ Substituted C ₁ ～C ₆ An alkyl group.

In some embodiments, R may be trimethylsilylethyl or t-butyldimethylsilylethyl; preferably, R is trimethylsilylethyl.

In some embodiments, the compound of formula II is

In some embodiments, the compound of formula III is

The invention also provides a preparation method of the compound shown in the formula II, which comprises the following steps: coupling the compound of formula III and 6- (maleimide) caproic acid succinimidyl ester in a solvent to obtain a compound of formula II;

In some embodiments, in the process for preparing the compound of formula II, R may be trimethylsilylethyl or t-butyldimethylsilylethyl; preferably, R is trimethylsilylethyl.

In some embodiments, the reaction materials in the preparation of the compound of formula II are the compound of formula III, the succinimidyl 6- (maleimido) caproate and the solvent.

In some embodiments, the molar ratio of the succinimidyl 6- (maleimido) hexanoate to the compound of formula III is 1-5, preferably 1-3, more preferably 1.2-2, and most preferably 1.5.

In some embodiments, the solvent may be one or a mixture of any two or more of amide solvents, chlorinated alkane solvents, ether solvents and nitrile solvents; preferably a chlorinated alkane solvent, wherein the chlorinated alkane solvent is preferably one or a mixture of any two or more of dichloromethane, 1, 2-dichloroethane and chloroform, and more preferably dichloromethane.

In some embodiments, the coupling reaction temperature is from 0 ℃ to 45 ℃, preferably from 25 ℃ to 40 ℃, and more preferably from 30 ℃ to 35 ℃.

In some embodiments, the method for preparing the compound of formula II, the coupling reaction is performed under inert gas, for example, under nitrogen or helium.

In some embodiments, the progress of the coupling reaction may be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC or NMR, etc.) in the preparation of the compound of formula II, typically with no more detection of the compound of formula III as the end point of the reaction.

In some embodiments, the coupling reaction time may be 3 to 24 hours, preferably 3 to 10 hours, and more preferably 3 to 6 hours in the preparation of the compound of formula II.

In some embodiments, the preparation method of the compound of formula II may further include the following post-treatment steps after the coupling reaction is completed: concentrating the obtained reaction liquid under reduced pressure, extracting with dichloromethane and water, combining dichloromethane organic phases, concentrating to obtain a crude compound of the formula II, and preferably, purifying the crude compound of the formula II by silica gel column chromatography to obtain a compound of the formula II; further preferably, the eluent of the silica gel column chromatography is a mixture of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is (10-1): 1.

Further, the preparation method of the compound of the formula II can further comprise a preparation method of the compound of the formula III, which can comprise the following steps: and (3) carrying out reduction reaction on the compound of the formula IV and a reducing agent in a solvent and in the presence of an acid buffer solution to obtain the compound of the formula III.

In some embodiments, in the process for preparing the compound of formula III, R may be trimethylsilylethyl or t-butyldimethylsilylethyl; preferably, R is trimethylsilylethyl.

In some embodiments, the reaction conditions (e.g., solvent amount, order of addition, mode of addition, time of addition, etc.) in the preparation process of the compound of formula III may be conventional conditions for such reactions in the art, which may be adjusted according to the type of reducing agent; for example, the method can comprise the following steps: dissolving a reducing agent in a solvent, then dropwise adding the solvent into an acid buffer solution, placing the obtained reaction system under the protection of inert gas, stirring and cooling, and then dropwise adding the solvent-dissolved compound of the formula IV into the reaction system to perform a reaction.

In some embodiments, the reaction materials in the preparation of the compound of formula III are the compound of formula IV, the reducing agent, the acid buffer, and the solvent.

In some embodiments, the reducing agent is one or a mixture of any two or more of triphenylphosphine, tri-tert-butylphosphine and trimethylphosphine, or a commercially available triphenylphosphine/tetrahydrofuran solution, tri-tert-butylphosphine/tetrahydrofuran solution or trimethylphosphine/tetrahydrofuran solution, preferably a trimethylphosphine/tetrahydrofuran solution, and more preferably a 1M trimethylphosphine/tetrahydrofuran solution.

In some embodiments, the molar ratio of the reducing agent to the compound of formula IV is 1.0 to 3.0, preferably 1.2 to 1.8, and more preferably 1.5.

In some embodiments, the volume to mass ratio of the acid buffer to the compound of formula III may be 2-10mL/g, preferably 3-5mL/g, and most preferably 4mL/g.

In some embodiments, the solvent is an ether solvent, preferably one or a mixture of any two or more of tetrahydrofuran, diethyl ether, 1, 4-dioxane, anisole and methyl tert-butyl ether, and more preferably tetrahydrofuran.

In some embodiments, the acid buffer is an acetate buffer or a formate buffer, preferably an acetate buffer; the acetic acid buffer solution is sodium acetate buffer solution.

In some embodiments, the pH of the acid buffer in the process for preparing the compound of formula III is from 4.0 to 6.0, preferably from 4.5 to 5.5, and more preferably 5.0.

In some embodiments, the temperature of the reduction reaction in the preparation of the compound of formula III is from 0 ℃ to 20 ℃, preferably from 0 ℃ to 10 ℃, and more preferably from 0 ℃ to 5 ℃.

In some embodiments, the monitoring of the progress of the reduction reaction in the preparation of the compound of formula III may be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC or NMR, etc.), typically with no more detection of the compound of formula IV as the end point of the reaction.

In some embodiments, the reduction reaction time may be from 0.5 to 8 hours, preferably from 0.5 to 2 hours, and more preferably from 0.5 to 1 hour.

In some embodiments, the method for preparing the compound of formula III may further include the following post-treatment steps after the reduction reaction is completed: concentrating the reaction solution to remove the reaction solvent, adding dichloromethane or ethyl acetate for extraction, combining organic phases, concentrating to obtain a crude compound of the formula III, and preferably, purifying the crude compound of the formula III by silica gel column chromatography to obtain a compound product of the formula III; further preferably, the eluent of 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.

Further, the preparation method of the compound of the formula II can further comprise a preparation method of the compound of the formula IV, which can comprise the following steps: etherification of a compound of formula VI with a reagent V in a solvent in the presence of a base gives a compound of formula IV.

In some embodiments, in the process for preparing the compound of formula IV, R may be trimethylsilylethyl or t-butyldimethylsilylethyl; preferably, R is trimethylsilylethyl.

In some embodiments, the reaction materials in the preparation of the compound of formula IV are the compound of formula VI, the reagent V, the base, and the solvent.

In some embodiments, the molar ratio of the reagent V to the compound of formula VI in the process for preparing the compound of formula IV may be 1-5, preferably 1-3, and more preferably 1.1-1.6.

In some embodiments, the base is an organic base, an inorganic base, or a mixture thereof in the process for preparing the compound of formula IV; preferably an organic base; wherein the organic base is preferably one or a mixture of any two or more of potassium tert-butoxide, triethylamine, DMAP, pyridine and Pan Bi pyridine, and is further preferably panpridine; the inorganic base is preferably one or a mixture of any two or more of alkali metal hydroxide, alkali metal carbonate and alkali metal phosphate, and more preferably one or a mixture of any two or more of potassium phosphate, potassium carbonate, potassium hydroxide and cesium carbonate.

In some embodiments, the molar ratio of the base to the compound of formula VI is 1 to 5, preferably 1.2 to 4, and more preferably 1.5 to 3.

In some embodiments, the solvent is one or a mixture of any two or more of DMF, DMSO, tetrahydrofuran and 1, 4-dioxane, preferably 1, 4-dioxane or tetrahydrofuran, more preferably tetrahydrofuran.

In some embodiments, the temperature of the etherification reaction is from 0 ℃ to 80 ℃, preferably from 40 ℃ to 80 ℃, and more preferably from 50 ℃ to 70 ℃.

In some embodiments, the etherification reaction is carried out under inert gas, for example, under nitrogen or helium atmosphere.

In some embodiments, the monitoring of the progress of the reaction in the preparation of the compound of formula IV may be monitored using conventional test methods in the art (e.g., TLC, GC, HPLC or NMR, etc.), typically with no more detection of the compound of formula VI as the end point of the reaction.

In some embodiments, the reaction time may be 2 to 48 hours, preferably 3 to 12 hours, and more preferably 4 to 8 hours in the process for preparing the compound of formula IV.

In some embodiments, the method for preparing the compound of formula IV may further include the following post-treatment steps after the etherification reaction is completed: concentrating the reaction solution, dissolving the obtained concentrate in a solvent, washing an 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 may be an aqueous acid solution, water and/or saturated saline; preferably, the organic phase is washed with saturated brine; the purification can be performed by conventional purification methods in the art, such as beating, crystallization, preparative chromatography or silica gel column chromatography, preferably by silica gel column chromatography, wherein the eluent of the silica gel column chromatography is preferably a mixture of n-heptane and ethyl acetate, and the volume ratio of the n-heptane to the ethyl acetate can be (20-1): 1, preferably (10-1): 1.

Further, the preparation method of the compound of the formula II can further comprise a preparation method of the compound of the formula VI, which can comprise the following steps: and carrying out substitution reaction on the compound of the formula VII, paraformaldehyde and trimethylchlorosilane in a solvent to obtain the compound VI.

Wherein the compound of the formula VII is an azide polypeptide compound, and the source is commercial or self-made.

In some embodiments, the reaction materials in the preparation of the compound of formula VI are the compound of formula VII, the paraformaldehyde, the trimethylchlorosilane, and the solvent.

In some embodiments, the molar ratio of paraformaldehyde to the compound of formula VII may be from 1 to 10, preferably from 1 to 5, more preferably from 1.3 to 3.0, most preferably 1.3.

In some embodiments, the molar ratio of the trimethylchlorosilane to the compound of formula VII in the process for the preparation of the compound of formula VI may be in the range of 1 to 5, preferably 2 to 4, further preferably 2 to 3, most preferably 3.

In some embodiments, the solvent may be one or a mixture of any two or more of N, N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran and 1, 4-dioxane, preferably tetrahydrofuran or 1, 4-dioxane, and more preferably 1, 4-dioxane.

In some embodiments, the substitution reaction temperature may be from-10 ℃ to 50 ℃, preferably from 15 ℃ to 35 ℃, and more preferably from 18 ℃ to 25 ℃.

In some embodiments, the substitution reaction is carried out under inert gas, such as in a nitrogen or helium atmosphere, in the process for preparing the compound of formula VI.

In some embodiments, the progress of the substitution reaction may be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC or NMR, etc.), typically by no longer detecting the compound of formula VII (using methanol derivatization) as the endpoint of the reaction.

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

In some embodiments, the preparation method of the compound of formula vi may further include a post-treatment step after the substitution reaction is completed: carrying out solid-liquid separation (under the condition of solid existence) or not, and concentrating an organic phase to obtain a compound of a formula VI; preferably, the crude compound of formula VI obtained after concentration is reacted directly with reagent V.

The invention also provides a preparation method of the compound shown in the formula III, which comprises the following steps: carrying out reduction reaction on a compound of formula IV and a reducing agent in a solvent and in the presence of an acid buffer solution to obtain a compound of formula III;

Wherein R is as defined above.

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

The methods of preparing the compounds of formula III may further include methods of preparing the compounds of formula IV described herein.

Definition of the definition

The table of the compounds of the present invention is shown in table 1.

TABLE 1

The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.

The reagents and raw materials used in the present invention are commercially available or self-made.

The invention has the positive progress effects that: a novel method for preparing a linker drug conjugate and intermediates thereof are provided. The intermediate provided by the invention is stable, easy to purify and low in impurity content; the method can be used for preparing a key carboxylic acid intermediate I in the subsequent process, and directly condenses with the Itothecitin Kang Jia sulfonate, so that the quality of LE14 is easy to control, and the prepared compound LE14 product meets the requirements of IND declaration on product quality; the preparation route has reasonable process, avoids using expensive Dxd or Dxd derivatives, and greatly reduces the production cost; the cytotoxic payload irinotecan can be put into the last step to participate in the reaction, so that the excessive exposure of cytotoxic substances is avoided, and the production is safer; is suitable for large-scale industrial production.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

In the following examples of the present invention,

the mass spectrum adopts a Waters Acquity Xevo G-XS QTof UPLC/MS ultra-high performance liquid chromatography high resolution mass spectrum combined system;

¹ H-NMR was performed using a Bruker AVANCE III MHz NMR apparatus or a Bruker AVANCE III HD MHz NMR apparatus;

HPLC detection conditions; the instrument was Agilent 187260, the column was Agilent AdvanceBio Peptide Map,3.5 μm, 2.1X1250 mm, and the detection wavelength was 254nm, using a mobile phase gradient setup as shown in Table 2, wherein phase A was 0.01mol/L aqueous potassium dihydrogen phosphate (pH=5.0), phase B was 10% methanolic acetonitrile solution, and the flow rate was 1.0mL/min.

TABLE 2 gradient setup of mobile phases

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

The conventional column chromatography conditions are as follows: the filler is 200-400 mesh amorphous silica gel, and column chromatography is carried out at room temperature.

In the examples below, room temperature refers to 20℃to 30 ℃.

Example 1 total synthetic route to LE14

Step 1: synthesis of Compound of formula VI

The compound of formula VII (25.00 g,53.36 mmol) and paraformaldehyde (2.09 g,69.37 mmol) were mixed and dissolved in 250.0mL of anhydrous 1, 4-dioxane, trimethylchlorosilane (17.39 g,160.08 mmol) was slowly added to the resultant mixture under stirring, the resultant reaction system was continuously stirred at 18℃to 25℃for 15.0 hours, the reaction was monitored by TLC, and after the completion of the reaction, 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)

Step 2: synthesis of Compound of formula IVa

The crude compound of formula VI from step 1 (28.95 g, calculated as theory yield, 53.37 mmol) was dissolved in 250.0mL of anhydrous tetrahydrofuran and panobidine (12.43 g,80.05 mmol) and Va (14.12 g,80.05 mmol) were added with stirring and the resulting mixture was heated to 60℃under nitrogen and reacted under these conditions for 6.0h. The reaction was monitored by TLC, after the starting material had reacted, tetrahydrofuran was removed by concentrating under reduced pressure, then ethyl acetate and saturated brine were added to the concentrate for extraction, the resulting organic phase was dried and concentrated under reduced pressure to remove the solvent, and the crude product obtained was purified by silica gel column chromatography (n-heptane: ethyl acetate=10:1 to 1:1 (v/v)) to give the compound of formula iv a (18.12 g, purity 93.3%, step 1 and step 2 combined yield 51.7%).

ESI-MS m/z：657.4(M+H)。

¹ H-NMR(400MHz,DMSO-d ₆ )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).

Step 3: synthesis of Compounds of formula IIIa

32.0mL of a 1M solution of trimethylphosphine in tetrahydrofuran and 15.0mL of THF are mixed and added dropwise to a sodium acetate buffer solution (pH=5.0) containing 60.0mL, the obtained reaction system is stirred and cooled to 0-5 ℃ under the protection of nitrogen, then 45.0mL of tetrahydrofuran in which a compound of formula IVa (15.00 g,21.20 mmol) is dissolved is slowly added dropwise to the reaction system, and the obtained reaction system is kept at 0-5 ℃ for continuous stirring for 0.5-1.0 h. Monitoring the reaction by TLC, after the reaction of the raw materials is finished, concentrating under reduced pressure to remove tetrahydrofuran in the reaction liquid, adding dichloromethane into the obtained concentrated reaction liquid for extraction, drying an obtained organic phase, concentrating under reduced pressure, and purifying the obtained crude product by silica gel column chromatography (dichloromethane: methanol=10:1-1:1 (v/v)) to obtain a compound shown in a formula IIIa (10.30 g, purity 96.5%, yield 76.9%);

ESI-MS m/z：631.4(M+H)。

¹ H-NMR(400MHz,DMSO-d ₆ )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).

step 4: synthesis of Compounds of formula IIa

The compound of formula IIIa (10.00 g,15.30 mmol) was dissolved in 300.0mL of anhydrous dichloromethane, succinimidyl 6- (maleimide) hexanoate (7.08 g,22.95 mmol) was added with stirring, the resulting reaction system was heated to 35℃under nitrogen protection, and stirring was continued under this condition for 3-6 h. The reaction was monitored by TLC, after the starting material had reacted, the resulting 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, and the resulting concentrate was purified by silica gel column chromatography (dichloromethane: methanol=10:1 to 1:1 (v/v)) to give the compound of formula ii a (9.80 g, purity 97.31%, yield 75.4%).

ESI-MS m/z：824.5(M+H)。

¹ H-NMR(400MHz,DMSO-d ₆ )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)。

Step 5: synthesis of Compound of formula I

Method 1: deprotection using potassium fluoride reagent

The compound of formula IIa (8.50 g,10.04 mmol) was dissolved in DMF (45.0 mL), potassium fluoride (0.88 g,15.06 mmol) was added at room temperature and the resulting reaction system was heated to 60℃under nitrogen with stirring and reacted under this condition for 6h. Monitoring the reaction by TLC, after the reaction of the raw materials is finished, concentrating under reduced pressure to remove the solvent, pretreating the crude product, purifying the crude product by silica gel column chromatography (dichloromethane: methanol=10:1-1:1 (v/v)) to obtain the compound of the formula I (4.72 g,83.06%, yield 65.0%); the amount of the pure compound of the formula I actually contained is 3.92g, so that the calculated yield of the amount of the pure compound of the formula I is 54.0%)

The pretreatment steps are as follows: the crude oil is dissolved by ultrasonic at room temperature with methylene dichloride (volume of methylene dichloride to weight of crude product is 10 mL/g) of which the volume is 10 times that of the crude product of the compound of the formula I, methyl tertiary butyl ether (volume of methyl tertiary butyl ether to weight of crude product is 50 mL/g) of which the volume is 50 times that of the crude product of the formula I is added, the mixture is stirred for 2 to 5 hours at normal temperature, the supernatant is poured off after standing, and the lower oily substance is concentrated in vacuum to obtain the pretreated crude product of the compound of the formula I.

ESI-MS m/z：722.3(M-H)；

¹ H-NMR(400MHz,DMSO-d ₆ )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,1H),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).

Method 2: deprotection using sodium fluoride reagent

The procedure was as in method 1, substituting potassium fluoride for sodium fluoride and purifying to give the compound of formula I (3.44 g, yield 47.3%).

Method 3: deprotection using cesium fluoride reagent

The procedure was as in method 1, substituting cesium fluoride for potassium fluoride and purifying to give the compound of formula I (3.02 g, yield 41.50%).

Method 4: deprotection using tetrabutylammonium fluoride reagent

The compound of formula IIa (8.50 g,10.04 mmol) was dissolved in tetrahydrofuran (50.0 mL), a 1.0M solution of tetrabutylammonium fluoride in tetrahydrofuran (15.2 mL,15.06 mmol) was added at room temperature, slowly warmed to 40℃under nitrogen, and reacted under this condition for 8.0h. The solvent was removed by concentration under reduced pressure, and the crude product obtained was purified by the same post-treatment method as in method 1 to give the compound of formula I (3.66 g, yield 50.3%).

Method 5: (the product, the compound of formula I, was used directly in the next reaction without purification):

the compound of formula IIa (8.50 g,10.04 mmol) was dissolved in DMF (45.0 mL), potassium fluoride (0.88 g,15.06 mmol) was added at room temperature and the reaction was heated to 60℃under nitrogen and allowed to react for 6h. After the reaction of the starting materials was completed, the solvent was removed by concentration under reduced pressure, and the crude product (9.85 g) of the obtained compound I was used for the next reaction.

Step 6: synthesis of LE14

Method one (reaction with purified Compound of formula I)

The compound of formula I (4.72 g, 83.06% purity, 5.42 mmol) was dissolved in 15.0mL anhydrous DMF and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMTMM) (2.24 g,8.13 mmol) was added and the reaction was allowed to proceed at room temperature for 1.0h. 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 thereto and the reaction continued for 1.5-2.0 h. The reaction was monitored by TLC, after the starting material had reacted, the solvent was removed under reduced pressure and the crude product obtained was purified by column chromatography on silica gel (chloroform: methanol=10:1 (v/v)) to give the target product LE14 (3.69 g, purity 98.77%, single step yield 59.6%, combined two step yield 32.2%).

Method II (crude unpurified compound of formula I prepared by method 5 above)

The crude compound of formula I (calculated as 100% conversion, 10.04 mmol) was dissolved in 15.0mL anhydrous DMF and 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 for 1.0h at room temperature. N, N-diisopropylethylamine (1.94 g,15.06 mmol) and Exatecan (mesylate) (5.37 g,10.04 mmol) were then added thereto and the reaction continued for 1.5-2.0 h. After the reaction of the raw materials was completed, the solvent was distilled off under reduced pressure, and the obtained crude product was purified by silica gel column chromatography (chloroform: methanol=10:1 (v/v)) to give the objective product LE14 (5.71 g, purity 98.06%, combined two-step yield 49.8%).

ESI-MS m/z：1141.7(M+H)；

¹ H-NMR(500MHz,DMSO-d ₆ )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).

Example 2 comprehensive comparison of Process routes

Purification of crude carboxylic acid intermediate 1-4 obtained according to the procedure of scheme 1 by column chromatography on silica gel (chloroform: methanol=10:1 (v/v)), without obtaining purified intermediate 1-4; crude carboxylic acid intermediate 2-2a obtained according to the procedure of scheme 2 was purified by silica gel column chromatography (chloroform: methanol=10:1 (v/v)), without obtaining purified intermediate 2-2a; the purification of the carboxylic acid intermediate I, which was obtained in good yields and in good purity by the method of example 1, was summarized in Table 3 below.

The LE14 end products synthesized according to example 1 were compared in purity with LE14 end products obtained in route 1, route 2, route 3, route 4 and route 5 by high performance liquid phase, and the results are shown in table 4 below.

TABLE 3 comparison of carboxylic acid intermediates (1-4, 2-2a and I) obtained by several process routes

TABLE 4 comparison data on the purity of the end products LE14 from different process routes

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In the prior art, the same intermediate (compound 5 has the same structure as compound VII) as the present invention is used in the synthesis routes 3, 4 and 5, and the final product LE14 can be prepared from the same intermediate compound 5, and the total yield and the purity of the final product in terms of 5 feeding are summarized in table 5.

TABLE 5 comparison of the final product purities of the different process routes and the overall yields starting from the same compound 5 (VII)

In the synthetic routes of the prior art CN 115215921A and CN115385926a, the final product LE14 was synthesized on the basis of the starting material of irinotecan, the overall yields of routes 4 and 5 were lower than in the present invention, see table 6.

TABLE 6 comparison of the total yields of the different process routes in terms of irinotecan inputs

As can be seen from tables 4, 5 and 6, the purity of the products prepared in routes 1 and 2 is far less than that of the present invention, and does not meet the quality requirements (total impurities are not more than 3%, single impurities are not more than 1%) of the IND declaration on the products in the present invention; in the route 3, DXd which is more expensive than the irinotecan is used as a payload source, although the reaction steps are saved, the yield of the route 3 and the Dxd utilization rate are not high, and in the route 4 and the route 5, the irinotecan needs to be derivatized, so that on one hand, the process has more reaction steps, the production operation is complicated, on the other hand, the loss in the preparation process is more, the utilization rate of the expensive material of the irinotecan is lower, and the method is not suitable for large-scale industrial production.

In the synthesis route of the invention, in the step of preparing LE14 by reacting the compound shown in the formula I with the irinotecan, both the method 1 and the method 2 can achieve products meeting the product quality standard, and in the two process methods, the irinotecan is a key material with the largest cost ratio in the whole process route and is also a main cost control point. The purification of the compound of formula I in method 1 can indeed improve the product quality of the final product LE14, and can also reduce the consumption of irinotecan by accurate feeding, thereby realizing cost control, while method 2 does not have the advantages of method 1, and the utilization rate of the irinotecan by method 2 is far lower than that of method 1, as can be seen from the comparison of the data in table 6 (calculated yield of irinotecan, method 1:59.6%, method 2:49.8%). In comparison to the overall tradeoff, of the two process routes provided herein, method 1 is a more preferred production process.

While particular embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are merely illustrative, and that many changes and modifications may be made to these embodiments without departing from the principles and spirit of the invention. Accordingly, the scope of the invention is defined by the appended claims.

Claims

1. A compound of formula II or III;

2. The compound of claim 1, wherein R is trimethylsilylethyl or t-butyldimethylsilylethyl.

3. The compound of claim 1, wherein the compound of formula II is

The compound of formula III is

4. A process for the preparation of a compound of formula II, comprising the steps of: coupling the compound of formula III and 6- (maleimide) caproic acid succinimidyl ester in a solvent to obtain a corresponding compound of formula II;

5. The method of claim 4, wherein the method of preparation satisfies one or more of the following conditions:

(1) In the preparation method of the compound of the formula II, R is trimethylsilylethyl or tert-butyldimethylsilylethyl;

(2) In the preparation method of the compound of the formula II, the molar ratio of the 6- (maleimide) caproic acid succinimidyl ester to the compound of the formula III is 1-5, preferably 1-3, and more preferably 1.2-2;

(3) In the preparation method of the compound shown in the formula II, the solvent is one or a mixture of any two or more of amide solvents, chlorinated alkane solvents, ether solvents and nitrile solvents, preferably chlorinated alkane solvents, wherein the chlorinated alkane solvents are preferably one or a mixture of any two or more of dichloromethane, 1, 2-dichloroethane and chloroform;

(4) In the preparation method of the compound of the formula II, the coupling reaction temperature is 0-45 ℃, preferably 25-40 ℃;

(5) In the preparation method of the compound of the formula II, the coupling reaction is carried out under the protection of inert gas;

(6) In the preparation method of the compound of the formula II, after the coupling reaction is finished, the preparation method further comprises the following post-treatment steps: the reaction solution is concentrated, dichloromethane and water are used for extraction, dichloromethane organic phases are combined, the concentration is carried out to obtain a crude product of the compound of the formula II, and preferably, the crude product of the compound of the formula II is subjected to silica gel column chromatography purification to obtain a compound of the formula II.

6. The method of claim 5, wherein the method of preparation satisfies one or more of the following conditions:

(1) In the preparation method of the compound of the formula II, R is trimethylsilylethyl;

(2) In the preparation method of the compound of the formula II, the molar ratio of the 6- (maleimide) caproic acid succinimidyl ester to the compound of the formula III is 1.5;

(3) In the preparation method of the compound of the formula II, the solvent is dichloromethane;

(4) In the preparation method of the compound of the formula II, the coupling reaction temperature is 30-35 ℃;

(5) In the preparation method of the compound of the formula II, the coupling reaction is carried out under the protection of nitrogen or helium;

(6) In the preparation method of the compound shown in the formula II, the eluent of 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.

7. The method of claim 6, wherein the method of preparing the compound of formula II further comprises a method of preparing the compound of formula III comprising the steps of: carrying out reduction reaction on a compound of the formula IV and a reducing agent in a solvent and in the presence of an acid buffer solution to obtain a corresponding compound of the formula III;

Wherein R is-Si (C) ₁ ～C ₆ ) ₃ Substituted C ₁ ～C ₆ An alkyl group;

further, the preparation method of the compound of the formula II also comprises a preparation method of the compound of the formula IV, and the preparation method comprises the following steps: etherification reaction is carried out on a compound in a formula VI and a reagent V in a solvent in the presence of alkali to obtain a corresponding compound in a formula IV;

further, the preparation method of the compound of the formula II also comprises a preparation method of the compound of the formula VI, which comprises the following steps: carrying out substitution reaction on the compound of the formula VII, paraformaldehyde and trimethylchlorosilane in a solvent to obtain a corresponding compound of the formula VI;

8. the method of claim 7, wherein the method of preparation satisfies one or more of the following conditions:

(1) In the preparation method of the compound of the formula III, R is trimethylsilylethyl or tert-butyldimethylsilylethyl;

(2) In the preparation method of the compound of the formula III, the reduction reaction further specifically comprises the following steps: the reducing agent is dissolved in a solvent and then is dripped into an acid buffer solution, the obtained reaction system is placed under the protection of inert gas, the stirring and the cooling are carried out, and then the compound of formula IVa dissolved in the solvent is dripped into the reaction system for reaction;

(3) In the preparation method of the compound shown in the formula III, the reducing agent is one or a mixture of any two or more of triphenylphosphine, tri-tert-butylphosphine and trimethylphosphine, or triphenylphosphine/tetrahydrofuran solution, tri-tert-butylphosphine/tetrahydrofuran solution or trimethylphosphine/tetrahydrofuran solution, preferably trimethylphosphine/tetrahydrofuran solution;

(4) In the preparation method of the compound of the formula III, the molar ratio of the reducing agent to the compound of the formula IV is 1.0-3.0, preferably 1.2-1.8;

(5) In the preparation method of the compound of the formula III, the volume-mass ratio of the acid buffer solution to the compound of the formula IV is 2-10mL/g, preferably 3-5mL/g;

(6) In the preparation method of the compound shown in the formula III, the solvent is an ether solvent, preferably one or a mixture of any two or more of tetrahydrofuran, diethyl ether, 1, 4-dioxane, anisole and methyl tertiary butyl ether;

(7) In the preparation method of the compound shown in the formula III, the acid buffer solution is acetic acid buffer solution or formic acid buffer solution;

(8) In the preparation method of the compound of the formula III, the pH value of the acid buffer solution is 4.0-6.0, preferably 4.5-5.5;

(9) In the preparation method of the compound of the formula III, the temperature of the reduction reaction is 0-20 ℃, preferably 0-10 ℃;

(10) In the preparation method of the compound of the formula III, after the reduction reaction is finished, the preparation method further comprises the following post-treatment steps: concentrating the reaction solution to remove the solvent, adding dichloromethane or ethyl acetate for extraction, combining organic phases, concentrating to obtain a crude compound of the formula III, and preferably, purifying the crude compound of the formula III by silica gel column chromatography to obtain a compound product of the formula III;

(11) In the preparation method of the compound shown in the formula IV, R is trimethylsilylethyl or tert-butyldimethylsilylethyl;

(12) In the preparation method of the compound of the formula IV, the mol ratio of the reagent V to the compound of the formula VI is 1-5, preferably 1-3;

(13) In the preparation method of the compound shown in the formula IV, the alkali is organic alkali, inorganic alkali or a mixture thereof; preferably an organic base; wherein the organic base is preferably one or a mixture of any two or more of potassium tert-butoxide, triethylamine, DMAP, pyridine and Pan Bi pyridine; the inorganic base is preferably one or a mixture of any two or more of alkali metal hydroxide, alkali metal carbonate and alkali metal phosphate, and more preferably one or a mixture of any two or more of potassium phosphate, potassium carbonate, potassium hydroxide and cesium carbonate;

(14) In the preparation method of the compound of the formula IV, the molar ratio of the alkali to the compound of the formula VI is 1-5, preferably 1.2-4;

(15) In the preparation method of the compound shown in the formula IV, the solvent is DMF, DMSO, tetrahydrofuran, 1, 4-dioxane or a mixture of any two or more of them, preferably 1, 4-dioxane or tetrahydrofuran;

(16) In the preparation method of the compound of the formula IV, the etherification reaction temperature is 0-80 ℃, preferably 40-80 ℃;

(17) In the preparation method of the compound of the formula IV, the etherification reaction is carried out under the protection of inert gas;

(18) In the preparation method of the compound of the formula IV, after the etherification reaction is finished, the preparation method further comprises the following post-treatment steps: concentrating the reaction solution, dissolving the obtained concentrate in an organic solvent, washing an organic phase, concentrating the organic phase, and purifying the obtained residue; wherein the organic solvent is ethyl acetate or dichloromethane, preferably ethyl acetate; the water phase used for washing is an aqueous acid solution, water and/or saturated saline water; preferably, the organic phase is washed with saturated brine; preferably, the purification adopts beating, crystallization, preparation chromatography or silica gel column chromatography;

(19) In the preparation method of the compound of the formula VI, the molar ratio of the paraformaldehyde to the compound of the formula VII is 1-10, preferably 1-5, and more preferably 1.3-3.0;

(20) In the preparation method of the compound of the formula VI, the molar ratio of the trimethylchlorosilane to the compound of the formula VII is 1-5, preferably 2-4, and more preferably 2-3;

(21) In the preparation method of the compound shown in the formula VI, the solvent is one or a mixture of any two or more of N, N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran and 1, 4-dioxane, and is preferably tetrahydrofuran or 1, 4-dioxane;

(22) In the preparation method of the compound of the formula VI, the substitution reaction temperature is-10 ℃ to 50 ℃, preferably 15 ℃ to 35 ℃;

(23) In the preparation method of the compound shown in the formula VI, the substitution reaction is carried out under the protection of inert gas;

(24) In the preparation method of the compound shown in the formula VI, after the substitution reaction is finished, the preparation method further comprises the following post-treatment steps: the reaction liquid is subjected to solid-liquid separation or not, the organic phase is concentrated to obtain the compound of the formula VI, and preferably, the crude product of the compound of the formula VI obtained after concentration is directly reacted with the reagent V.

9. The method of claim 8, wherein the method of preparation satisfies one or more of the following conditions:

(1) In the preparation method of the compound of the formula III, R is trimethylsilylethyl;

(2) In the preparation method of the compound of the formula III, the reducing agent is 1M trimethylphosphine/tetrahydrofuran solution;

(3) In the preparation method of the compound of the formula III, the molar ratio of the reducing agent to the compound of the formula IV is 1.5;

(4) In the preparation method of the compound of the formula III, the volume-mass ratio of the acid buffer solution to the compound of the formula IV is 4mL/g;

(5) In the preparation method of the compound of the formula III, the solvent is tetrahydrofuran;

(6) In the preparation method of the compound of the formula III, the acid buffer solution is acetic acid buffer solution; the acetic acid buffer solution is sodium acetate buffer solution;

(7) In the preparation method of the compound of the formula III, the pH value of the acid buffer solution is 5.0;

(8) In the preparation method of the compound of the formula III, the temperature of the reduction reaction is 0-5 ℃;

(9) In the preparation method of the compound shown in the formula III, the eluent of 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;

(10) In the preparation method of the compound shown in the formula IV, R is trimethylsilylethyl;

(11) In the preparation method of the compound of the formula IV, the mol ratio of the reagent V to the compound of the formula VI is 1.1-1.6;

(12) In the preparation method of the compound shown in the formula IV, the alkali is panpridine;

(13) In the preparation method of the compound of the formula IV, the molar ratio of the alkali to the compound of the formula VI is 1.5-3;

(14) In the preparation method of the compound shown in the formula IV, the solvent is tetrahydrofuran;

(15) In the preparation method of the compound of the formula IV, the temperature of the etherification reaction is 50-70 ℃;

(16) In the preparation method of the compound of the formula IV, the etherification reaction is carried out under the protection of nitrogen or helium;

(17) In the preparation method of the compound shown in the 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, preferably (10-1): 1;

(18) In the preparation method of the compound of the formula VI, the molar ratio of the paraformaldehyde to the compound of the formula VII is 1.3;

(19) In the preparation method of the compound of the formula VI, the molar ratio of the trimethylchlorosilane to the compound of the formula VII is 3;

(20) In the preparation method of the compound shown in the formula VI, the solvent is 1, 4-dioxane;

(21) In the preparation method of the compound shown in the formula VI, the substitution reaction temperature is 18-25 ℃;

(22) In the preparation method of the compound shown in the formula VI, the substitution reaction is carried out under the protection of nitrogen or helium.

10. A process for the preparation of a compound of formula III, comprising the steps of: carrying out reduction reaction on a compound of formula IV and a reducing agent in a solvent and in the presence of an acid buffer solution to obtain a compound of formula III; reaction conditions according to any one of claims 7 to 9;

wherein R is as claimed in any one of claims 7 to 9.