CN113527418B

CN113527418B - Preparation method of ADC linker

Info

Publication number: CN113527418B
Application number: CN202110807191.2A
Authority: CN
Inventors: 罗前东; 谭永平
Original assignee: Chengdu Pukang Weixin Biotechnology Co ltd
Current assignee: Chengdu Pukang Weixin Biotechnology Co ltd
Priority date: 2021-07-16
Filing date: 2021-07-16
Publication date: 2022-05-03
Anticipated expiration: 2041-07-16
Also published as: CN113527418A

Abstract

The invention relates to a preparation method of an ADC linker, which comprises the following steps: 1) using A as raw material, using copper acetate as catalyst and passing through N₂After replacement, Pb (OAc) is added₄Heating and reacting to obtain B; 2) adding the compounds B, C and PPTS into DCM, and refluxing overnight to obtain D; 3) adding a compound D into DMF, stirring for dissolving, adding DBU, stirring for removing Fmoc, and obtaining a solution containing E; adding F, DIEA and HATU into DMF, stirring, adding the solution containing E, and continuing to react to obtain G; 4) adding DMF and Pd/C into the compound G, carrying out hydrogenolysis under hydrogen atmosphere, and carrying out operations such as crystallization on the product in DMF in sequence to obtain H; 5) and simultaneously adding DMF, H, I and DIEA, stirring to react, adjusting the pH value, and purifying to obtain the ADC linker. The preparation method is simple and easy to implement, the impurities are few, and the product can be finally crystallized to obtain a high-purity product.

Description

Preparation method of ADC linker

The technical field is as follows:

the invention belongs to the technical field of compound preparation, and particularly relates to a preparation method of an ADC linker.

Background art:

antibody Drug Conjugates (ADCs) are composed of three parts as novel targeted drugs, with a linker that links the cytotoxic payload to the mAb and maintains the stability of the ADC in the systemic circulation. The chemical properties and binding sites of linker play a crucial role in ADC performance in terms of stability, pharmacokinetic and pharmacodynamic properties, and therapeutic window.

At present, the technical route generated by ADC linker is mainly directed at the ADC medicine entirety, but the preparation method of ADC linker is rarely recorded. Therefore, the ADC linker is further researched to improve the yield, reduce the generation of impurities and reduce the cost, so that the method is more beneficial to expanding the production and is very important for increasing the product income.

The invention content is as follows:

the invention aims to provide a preparation method of ADC linker aiming at the defects in the prior art, and the method can improve the ADC linker yield, reduce the generation of impurities and reduce the cost, thereby being more beneficial to enlarging the production and increasing the product income.

Another object of the present invention is to provide a method for preparing the compound B involved in the process of preparing ADC linker.

The third invention of the present invention is to provide a method for preparing the compound D involved in the process of preparing ADC linker.

The fourth invention of the present invention is to provide a method for preparing the compound G involved in the process of preparing ADC-linker

The fifth invention of the present invention is to provide a method for preparing the compound H involved in the process of preparing ADC-linker.

In order to achieve the purpose, the specific technical scheme of the invention is as follows:

a preparation method of ADC linker comprises the following steps:

1) using A as raw material, using copper acetate as catalyst and passing through N₂After replacement, Pb (OAc) is added₄Then preparing a compound B through heating reaction;

2) adding the compounds B, C and PPTS into DCM, and refluxing overnight to prepare a compound D;

3) adding a compound D into DMF, stirring for dissolving, adding DBU, stirring for removing Fmoc, and obtaining a solution containing E; adding F, DIEA and HATU into DMF, stirring, adding the solution containing E, and continuously stirring for reaction; then quenching reaction, extraction, washing, drying and reduced pressure concentration are carried out to obtain a paste substance, and then methyl tert-butyl ether is added after heating and stirring washing, and cooling crystallization and drying are carried out to obtain G;

4) adding DMF and Pd/C into the compound G, carrying out hydrogenolysis in a hydrogen atmosphere, crystallizing a product in the DMF, filtering the DMF after the reaction is finished, dissolving a filter cake with water, filtering the Pd/C, concentrating the filtrate under reduced pressure to obtain a white solid, and drying to obtain H;

5) adding DMF, H, I and DIEA into the reaction vessel at the same time, stirring and reacting, adjusting the pH value, and purifying J, namely ADC linker.

As a preferred embodiment of the present application, the reaction formula of the substance B in the step 1) is,

the method specifically comprises the following steps:

1.1) adding A and Cu (OAc) to THF with stirring₂By N₂After replacement, Pb (OAc) is added₄Heating the reaction, and monitoring the completion of the reaction by TLC;

1.2) cooling to room temperature after the reaction is finished, then carrying out suction filtration, soaking and washing the solid with ethyl acetate, filtering, and concentrating the filtrate under reduced pressure to obtain a blue oily substance;

1.3) dissolving the substance obtained in the previous step with ethyl acetate and then with saturated NaHCO₃Washing with aqueous solution 2 times, then with saturated saline once, and Na₂SO₄Drying the mixture by using a drying agent;

1.4) carrying out reduced pressure concentration on the dried substance until the mass of A is reached, standing for crystallization, carrying out suction filtration and forced air drying to obtain a solid B.

As a preferred embodiment in this application, in step 1), A and Cu (OAc)₂In a molar ratio of 10: 1-5, preferably 5: 1; in terms of mole percent, Pb (OAc)₄The addition amount of (A) is 140% of that of A; the temperature for the heating reaction is 50 to 70 deg.C, preferably 65 deg.C.

As a preferred embodiment of the present application, the reaction formula of the substance D in the step 2) is,

the method specifically comprises the following steps:

2.1) adding B, C and PPTS into DCM, and refluxing at a certain temperature overnight;

2.2) Na after 3 washes with brine₂SO₄Drying, dissolving at 40-50 deg.C, and vacuum filtering;

2.3) decompressing and concentrating the filtered substance, adding ethyl acetate to dissolve at 50-60 ℃, crystallizing, filtering, cooling to normal temperature, crystallizing, repeating twice, and drying to obtain a compound D.

In a preferred embodiment of the present application, in step 2.1), the ratio of the addition amount mol of DCM, the addition amount of DCM L, B, the addition amount mol of C and the addition amount mol of PPTS is 10-20:10:20-50:1-10, preferably 15:10:30: 2; the temperature at reflux overnight is 40-45 deg.C, preferably 45 deg.C.

As a preferred embodiment of the present application, the reaction formula of the compound G in the step 3) is,

the method specifically comprises the following steps:

3.1) adding D into DMF, stirring and dissolving, adding DBU, stirring and removing Fmoc;

3.2) adding F and DIEA into DMF, cooling to T less than 0 ℃, adding HATU, stirring for 0.5-1h, and stirring the solution in the reaction 5.1) until the reaction is finished;

3.3) adding water into a reaction system to quench reaction, extracting twice by using ethyl acetate, washing the ethyl acetate twice by using acid water with the pH value of 3-5 and brine in sequence, drying the ethyl acetate by using anhydrous magnesium sulfate, concentrating at 45-60 ℃ under reduced pressure to obtain a paste-like substance, adding 100ml of ethyl acetate, heating to 45-60 ℃, stirring and washing for 1-3 hours, adding methyl tert-butyl ether with the same proportion of the ethyl acetate, cooling to normal temperature, crystallizing for 3-5 hours, filtering to obtain a white solid, and drying at 45-55 ℃ to obtain a product G.

In a preferred embodiment of the present invention, the ratio of the mol of DMF L, D to the mol of DBU is 1-5:0.5:0.5, preferably 1:0.5: 0.5.

As a preferred embodiment of the present application, the reaction formula of H in step 4) is,

the method specifically comprises the following steps:

adding G, DMF, Pd/C into a reaction vessel, carrying out hydrogenolysis in a hydrogen atmosphere, crystallizing a product in DMF, filtering DMF after TLC reaction is finished, dissolving a filter cake with water, filtering Pd/C, and concentrating the filtrate under reduced pressure to obtain a white solid; drying at 45-55 ℃ to obtain the product H.

In a preferred embodiment of the present application, the ratio of the amount of G added, the amount of DMF added, mL, to the amount of Pd/C added, G, is from 1:6 to 10:0.1 to 0.3, preferably 1:6: 0.1.

As a preferred embodiment of the present application, the reaction formula of J in step 5) is,

the method specifically comprises the following steps:

adding DMF, H, I and DIEA into the reaction vessel, stirring for 20 hours, adding glacial acetic acid to adjust the pH value to 4-5 after TLC shows that the reaction is finished, and performing column purification to obtain J.

In a preferred embodiment of the present invention, the ratio of the amount of DMF added in the form of L, H mol to the amount of DIEA added in the form of I added in the form of DIEA is 0.5-1.5:1:1:1-2.5, preferably 0.7:1:1: 1.5.

Compared with the prior art, the positive effects of the invention are as follows:

the method has high yield, can reduce the generation of impurities and reduce the cost, thereby being more beneficial to enlarging the production and increasing the product income.

And (II) the production method is simple and easy to implement and has strong operability.

And thirdly, in the step of preparing the substance B, copper acetate is used as a catalyst, so that the generation of impurities (the quality of the impurities is the same as the content of the main component) can be effectively avoided, and conditions are provided for obtaining high-purity products subsequently.

And (IV) the two-step reaction is adopted in the step of preparing the compound G, so that the side reaction of the compound F and the DBU is reduced, and the influence on the generation of the product due to the generation of impurities is avoided.

The specific implementation mode is as follows:

a preparation method of ADC linker comprises the following steps:

1) using A as raw material, using copper acetate as catalyst and passing through N₂After replacement, Pb (OAc) is added₄Then heating to react to prepare a compound B;

5) and simultaneously adding DMF, H, I and DIEA, stirring to react, adjusting the pH value, and purifying J, namely ADC linker.

the method specifically comprises the following steps:

1.4) concentrating the dried substance under reduced pressure until the mass of the substance A is reached, standing for crystallization, filtering, and drying by blowing to obtain a solid B.

Preferably, in the preparation, A and Cu (OAc)₂In a molar ratio of 10: 1-5, more preferably 5: 1; pb (OAc)₄In an amount of 140% of A (in mol); the temperature for the heating reaction is 50 to 70 deg.C, preferably 65 deg.C.

Preferably, the reaction formula of the substance D in step 2) is,

the method specifically comprises the following steps:

2.3) decompressing and concentrating the solvent, adding ethyl acetate to dissolve at the temperature of 50-60 ℃, then crystallizing and filtering, cooling to normal temperature for crystallization, repeating twice and drying to obtain a compound D.

Preferably, in step 2.1), the addition amount of DCM, i.e., L, B mol, and the ratio of the addition amount of C, i.e., mol, to the addition amount of PPTS, i.e., mol, are 10-20:10:20-50:1-10, preferably 15:10:30: 2. The temperature at reflux overnight is 40-45 deg.C, preferably 45 deg.C.

Preferably, the reaction formula of the compound G in the step 3) is,

the method specifically comprises the following steps:

3.1) adding D into DMF, stirring for dissolving, adding DBU, and stirring for removing Fmoc;

Preferably, the ratio of DMF, D and DBU is 1 to 5:0.5:0.5, and the most preferred ratio is 1:0.5:0.5, mol per L.

Preferably, the reaction formula of H in step 4) is,

the method specifically comprises the following steps:

the method specifically comprises the following steps:

adding DMF, H, I and DIEA into a reaction vessel, stirring for 20 hours, adding glacial acetic acid to adjust the pH value to 4-5 after TLC shows that the reaction vessel is finished, and performing column chromatography to obtain J.

In a preferred embodiment of the present invention, the ratio of DMF, H, I, DIEA is (L; mol: mol: mol: 0.5-1.5:1:1:1-2.5), preferably 0.7:1:1: 1.5.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It is to be noted that the features in the following embodiments and examples may be combined with each other without conflict. In the present application, the operations not described in detail are the ordinary operations in the art or do not have a specific influence on the effect of the present application.

In the present application, each letter is specifically represented as follows:

a: (Fmoc-glycyl-glycine);

B：(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate；

c: benzyl glycolate;

D：benzyl1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate；

E：benzyl 2-((2-aminoacetamido)methoxy)acetate；

f: cbz-glycyl-phenylalanine

G：benzyl(R)-11-benzyl-3,6,9,12,15-pentaoxo-1-phenyl-2,18-dioxa-4,7,10,13,16-pentaazaicosan-20-oate

H：(R)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahe xadecanoic acid

I: maleimidohexanoic acid succinimidyl ester

J：(R)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatricosanoic acid。

Each additive:

Cu(OAc)₂copper acetate

Pb(OAc)₄Lead tetra acetic acid

THF tetrahydrofuran

PPTS pyridine p-toluenesulfonate salt

DCM dichloromethane

DBU 1, 8-diazabicycloundec-7-enes

DMF N, N-dimethylformamide

HATU 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate

DIEA N, N-diisopropylethylamine

Pd/C palladium on carbon

Fmoc fluorenylmethyloxycarbonyl.

Example 1:

a preparation method of a compound B has a reaction formula as follows,

the method comprises the following steps:

1) 150gA and 20% (Cu (OAc))₂Mole percent of A, same below) Cu (OAc)₂，N₂After displacement 262.5g Pb (OAc)₄Heating to react at 65 ℃, and monitoring by TLC to finish the reaction;

2) cooling to room temperature, vacuum-filtering, soaking the solid with ethyl acetate, filtering, and concentrating the filtrate under reduced pressure to obtain blue oily substance;

3) dissolving the substance obtained in the previous step with ethyl acetate, and then using saturated NaHCO₃Washing with aqueous solution 2 times, and then with saturated brine once, Na₂SO₄Drying the mixture by using a drying agent;

4) concentrating the substance obtained after drying in the step 3) under reduced pressure to the mass of A, standing for crystallization, and performing suction filtration, air blowing and drying to obtain solid B77.8g; the calculated yield is 50%.

Comparative example 1: (different catalysts)

1) 150g (0.423mol) A and 40g (1.2eq) pyridine, N are stirred in 1.1L THF₂After the displacement, 262.5g (1.4eq) of Pb (OAc) were added₄The completion of the reaction was monitored by heating the reaction TLC at 65 ℃.

2) Cooling to room temperature, vacuum-filtering, soaking the solid with ethyl acetate, filtering, and concentrating the filtrate under reduced pressure to obtain reddish brown oily substance;

3) dissolving the obtained substance with ethyl acetate, and dissolving with saturated NaHCO₃Washing with aqueous solution 2 times, and then with saturated brine once, Na₂SO₄Drying the mixture by using a drying agent;

4) and (3) concentrating the substance obtained after drying in the step 3) under reduced pressure until the mass of the substance A is reached, standing for crystallization, and performing suction filtration, air blowing and drying to obtain 22.9g of solid, wherein the yield is 15% by calculation.

Test 1: investigating the influence of different amounts of catalyst on the yield of product B

The preparation method is basically the same as that of example 1, and the difference is only that the catalyst Cu (OAc)₂In different amounts (Cu (OAc))₂Calculated in mol), set to 1# 10% of the addition amount of a (mol ratio to compound a), 2# 20%, 3# 35%, 4# 50%), respectively, the remaining steps were in agreement, and the specific test results were as follows:

composition (I)	1#	2#	3#	4#
					A	150g	150g	150g	150g
Cu(OAc)₂	10％	20％	35％	50％
					B	43.5g	77.8g	76.2g	76.2g
Yield of	28％	50％	49％	49％

Example 2:

a process for the preparation of substance D having the formula,

1) to 1.4LDCM was added 121.5 g of B,219.1 g of C, 20% (molar percentage of PPTS to B, the same applies below) of PPTS, refluxed at 45 ℃ overnight, washed with brine 3 times, and Na₂SO₄Drying with desiccant, dissolving at 45 deg.C, and vacuum filtering.

2) Concentrating the dried substance under reduced pressure, adding ethyl acetate, dissolving at 55 deg.C, crystallizing, filtering, cooling to room temperature, repeating twice, and oven drying to obtain compound D81g with yield of 52%.

Comparative example 2-1 (different catalysts)

1) To 1.4LDCM was added 121.5 g of B (prepared in example 1), 219.1 g of C, 20% PTSA (20% is the molar percentage of PTSA to B, the same applies below), refluxed at 45 ℃ overnight, washed 3 times with brine and then washed with Na₂SO₄Drying with desiccant, dissolving at 45 deg.C, and vacuum filtering.

2) Concentrating the substance obtained in the previous step under reduced pressure, adding ethyl acetate, dissolving at 55 deg.C, crystallizing, filtering, cooling to room temperature, crystallizing, repeating twice, and oven drying to obtain compound D16.2g with yield of 10.4%.

Comparative examples 2 to 2: (different catalysts)

1) After addition of 121.5 g of B,219.1 g of C, 20% of PTSA (20% in mol% of PTSA to B) to 1.4LTHF, refluxing at 65 ℃ overnight, THF concentration at 50 ℃ under reduced pressure, dissolution of the oil in 1LDCM, washing with brine 3 times and Na₂SO₄Drying the mixture by using a drying agent.

2) Concentrating the obtained substance under reduced pressure, adding ethyl acetate, dissolving at 55 deg.C, crystallizing, vacuum filtering, cooling to room temperature, repeating twice, and oven drying to obtain compound D18.7g with yield of 12%.

Comparative examples 2 to 3: (different catalysts)

1) Reflux overnight at 45 ℃ after addition of 121.5 g of B,219.1 g of C, 20% PPTS to 1.4LTHF (PPTS catalysis is milder, less impurity generation); washed 3 times with brine and then Na₂SO₄Drying with desiccant, dissolving at 45 deg.C, and vacuum filtering.

2) Concentrating the substance obtained in the step 1) under reduced pressure, adding ethyl acetate, dissolving at 55 ℃, crystallizing, filtering, cooling to normal temperature, repeating twice, and drying to obtain compound D65.4g, wherein the yield is 42% by calculation.

Test 2: investigating the influence of different amounts of catalyst on the yield of product D

The preparation method is basically consistent with that of example 2, and only differs from the addition amount of the catalyst PPTS (the addition amount of PPTS is calculated by taking mol as a unit, and 1# 10% (mol ratio to the compound B), 2# 20% and 3# 30% are respectively set), the other steps are consistent, and the specific test results are as follows:

example 3:

a process for the preparation of substance G having the formula:

the method comprises the following steps:

1) d (prepared in example 2) is added into DMF, stirred and dissolved, DBU is added, and Fmoc is removed by stirring;

2) adding F and DIEA into DMF, cooling to T less than 0 ℃, adding HATU, stirring for 0.5-1h, and stirring the solution in the reaction 5.1 till the reaction is finished; and 5.3, adding water into the reaction system to quench the reaction, extracting twice by using ethyl acetate, washing the ethyl acetate twice by using hydrochloric acid with the pH value of about 4, water and saline water in sequence, drying the ethyl acetate by using anhydrous magnesium sulfate, concentrating under reduced pressure at about 55 ℃ to obtain a paste-like substance, adding 100ml of ethyl acetate, heating to about 55 ℃, stirring and washing for 2 hours, adding methyl tert-butyl ether with the same proportion as the ethyl acetate, cooling to normal temperature for crystallization for 4 hours, filtering to obtain a white solid, and drying at 50 ℃ to obtain a product G.

Example 4:

a process for the preparation of compound H having the reaction formula:

the method comprises the following steps:

adding G, DMF, Pd/C into a single-mouth bottle, carrying out hydrogenolysis in a hydrogen atmosphere, crystallizing a product in DMF, filtering DMF after TLC reaction is finished, dissolving a filter cake with water, filtering Pd/C, and concentrating the filtrate under reduced pressure to obtain a white solid; drying at 50 ℃ gave the product H9.1g, 93% yield.

Example 5:

a process for the preparation of compound J having the formula:

the method comprises the following steps:

adding DMF, H (prepared in example 4), I and DIEA into a reaction vessel, stirring for 20 hours, adding glacial acetic acid to adjust the pH value to 4.5 after TLC shows that the reaction is finished, and performing column purification to obtain 2.3g of J (namely ADC linker) with the yield of 81%; the ratio of DMF to H, I, DIEA is L: mol: mol: 0.7:1:1: 1.5.

Example 6:

a preparation method of ADC linker comprises the following steps:

1) using A as raw material, using copper acetate as catalyst and passing through N₂After replacement, Pb (OAc) is added₄And then heating to react to prepare the compound B.

The specific operation is as follows:

750gA and 20% (Cu (OAc))₂M. with AMole percent) Cu (OAc)₂From N to N₂After replacement 262.5Pb (OAc)₄The reaction was heated at 65 ℃ and monitored by TLC for completion. The treatment can avoid the generation of impurities and provide conditions for obtaining a product with higher purity through subsequent crystallization.

After the reaction is finished, cooling to room temperature, carrying out suction filtration, soaking and washing the solid with ethyl acetate, filtering, and concentrating the filtrate under reduced pressure to obtain a blue oily substance; the foregoing was dissolved in ethyl acetate followed by saturated NaHCO₃Washing with aqueous solution 2 times, then with saturated saline once, and Na₂SO₄Drying the mixture by using a drying agent; concentrating the dried substance under reduced pressure to the mass of A, standing for crystallization, performing suction filtration, air blowing and drying to obtain a solid B389g, wherein the yield is 50% by calculation.

2) Compound D was prepared after refluxing compound B, C and PPTS in DCM overnight.

Reflux overnight at 45 ℃ after addition of 121.5 g of B,219.1 g of C, 16.6 g of PPTS in DCM (PPTS catalysis milder, less impurity generation); washed with brine 3 times and then with Na₂SO₄Drying with desiccant, dissolving at 45 deg.C, and vacuum filtering. Concentrating the solvent under reduced pressure, adding ethyl acetate, dissolving at 55 deg.C, crystallizing, filtering, cooling to room temperature, crystallizing, repeating twice, and oven drying to obtain compound D81g with yield of 52%.

3) Adding a compound D into DMF, stirring for dissolving, adding DBU, stirring for removing Fmoc, and obtaining a solution containing E; adding F, DIEA and HATU into DMF, stirring, adding the solution containing E, and continuously stirring for reaction; then quenching reaction, extraction, washing, drying and decompression concentration are carried out to obtain a pasty substance, and then methyl tert-butyl ether is added after heating and stirring washing, and cooling, crystallization and drying are carried out to obtain G.

The concrete operation steps of G are as follows:

adding 24.3 g of D into 100ml of DMF, stirring and dissolving, adding 7.6 g of DBU, and stirring to remove Fmoc;

21.55 g of F and 9.7 g of DIEA are added into 100ml of DMF, the temperature is reduced to T less than 0 ℃, 22.8 g of HATU is added, the mixture is stirred for 0.7h, and the solution after Fmoc removal is added and stirred until the reaction is finished. (the side reaction of F and DBU is reduced and impurities are avoided by two-step reaction)

Adding water into a reaction system to quench reaction, extracting twice by using ethyl acetate, washing the ethyl acetate twice by using acid water with pH being 4 and brine in sequence, drying the ethyl acetate by using anhydrous magnesium sulfate, concentrating at 52 ℃ under reduced pressure to obtain a pasty substance, adding 100ml of ethyl acetate, heating at 52 ℃, stirring and washing for 2 hours, adding methyl tert-butyl ether with the same proportion of the ethyl acetate, cooling to normal temperature, crystallizing for 4 hours, filtering to obtain a white solid, and drying at 50 ℃. Product G20.9g is obtained, and the yield is 63% by well calculation.

4) Adding DMF and Pd/C into the compound G, carrying out hydrogenolysis under the hydrogen atmosphere, crystallizing the product in DMF, filtering DMF after the reaction is finished, dissolving a filter cake with water, filtering Pd/C, concentrating the filtrate under reduced pressure to obtain a white solid, and drying to obtain H.

The specific operation is as follows:

A1L single-neck flask was charged with 15G of G, 100ml of DMF, 2G of Pd/C, and subjected to hydrogenolysis under hydrogen atmosphere. And (3) crystallizing the product in DMF, filtering the DMF after TLC reaction is finished, dissolving a filter cake by using water, and filtering Pd/C. The filtrate was concentrated under reduced pressure to give a white solid which was dried at 50 ℃ to give the product, H9.1g, in a calculated yield of 93%.

The specific operation is as follows:

after a 100ml single-neck flask was charged with 30 ml DMF, 2g H, 1.5 g I, 1g DIEA and stirred for 20 hours, TLC showed completion, glacial acetic acid was added to adjust PH 4.5 and column purification was performed to obtain j2.3g, calculated as 81% yield.

¹H-NMR(400MHz,DMSO-d6)δ1.15-1.23(2H,m),δ1.43-1.52(4H,m),δ2.11(2H,t,7.3Hz),δ2.78-2.84(1H,m),δ3.04-3.09(1H,m),δ3.37(2H,t,7.0Hz)δ3.61-3.79(6H,m),δ3.94(2H,s),δ4.47-4.52(1H,m),δ4.61(2H,d,6.7Hz),δ6.99(2H,s),δ7.15-7.27(5H,m),δ8.11-8.15(2H,m),δ8.22(1H,d,8.5Hz),δ8.31(1H,t,5.8Hz),δ8.63(1H,t,6.4Hz).

¹³C-NMR(100MHz,DMSO-d6)δ24.6,25.8,27.8,34.9,37.0,37.2,41.9,42.1,42.1,54.2,65.1,69.2,126.2,128.1,129.1,134.4,137.9,168.9,169.5,169.8,171.1,171.4,171.9,172.6.

MS(ESI)(m/z):615([M-H]^-).

The foregoing basic embodiments of the invention and their various further alternatives can be freely combined to form multiple embodiments, all of which are contemplated and claimed herein. In the scheme of the invention, each selection example can be combined with any other basic example and selection example at will. Numerous combinations will be known to those skilled in the art.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A preparation method of ADC linker is characterized by comprising the following steps:

1) using A as raw material, using copper acetate as catalyst and passing through N₂After replacement, Pb (OAc) is added₄Then heating to react to prepare a compound B, wherein the reaction formula is as follows:

2) adding compounds B, C and PPTS into DCM, and refluxing overnight to obtain compound D, wherein the reaction formula is as follows:

3) adding a compound D into DMF, stirring for dissolving, adding DBU, stirring for removing Fmoc, and obtaining a solution containing E; adding F, DIEA and HATU into DMF, stirring, adding the solution containing E, and continuously stirring for reaction; then quenching reaction, extraction, washing, drying and reduced pressure concentration are carried out to obtain a pasty substance, then methyl tert-butyl ether is added after heating and stirring washing, and cooling, crystallization and drying are carried out to obtain G, wherein the reaction formula is as follows:

4) adding DMF and Pd/C into the compound G, carrying out hydrogenolysis in a hydrogen atmosphere, crystallizing a product in the DMF, filtering the DMF after the reaction is finished, dissolving a filter cake in water, filtering the Pd/C, concentrating the filtrate under reduced pressure to obtain a white solid, and drying to obtain H, wherein the reaction formula is as follows:

5) simultaneously adding DMF, H, I and DIEA, adjusting the pH value after stirring and reacting, and purifying J, namely ADC linker, wherein the reaction formula is as follows:

2. the method for preparing an ADC linker as set forth in claim 1, wherein the compound B of step 1) is prepared by the steps of:

1.1) adding A and Cu (OAc) to THF with stirring₂By N, of₂After replacement, Pb (OAc) is added₄Heating the reaction, and monitoring the completion of the reaction by TLC;

1.3) dissolving the substance obtained in the previous step by using ethyl acetate, and then using saturated NaHCO₃Washed 2 times with aqueous solution, then once with saturated brine, Na₂SO₄Drying the mixture by using a drying agent;

1.4) concentrating the substance obtained after drying in the previous step under reduced pressure until the mass of A is reached, standing for crystallization, and performing suction filtration and forced air drying to obtain a solid B.

3. The method of preparing ADC linker as set forth in claim 2, wherein A and Cu (OAc)₂In a molar ratio of 10: 1-5; in mol percent, Pb (OAc)₄The addition amount of (A) is 140% of the amount of A, and the heating reaction temperature is 50-70 ℃.

4. The method for preparing an ADC linker as set forth in any one of claims 1 to 3, wherein the step of D preparation in step 2) is:

2.2) brine 3 times followed by Na₂SO₄Drying with desiccant, dissolving at 40-50 deg.C, and vacuum filtering;

2.3) decompressing and concentrating the substance obtained by suction filtration, adding ethyl acetate to dissolve at 50-60 ℃, then crystallizing and suction filtering, cooling to normal temperature for crystallization, repeating twice and drying to obtain the compound D.

5. The method of preparing an ADC linker as set forth in claim 4, wherein: the proportion relation of the addition amount mol of the DCM L, B, the addition amount mol of the C and the addition amount mol of the PPTS is 10-20:10:20-50: 1-10; reflux overnight at 40-45 ℃.

6. The method for preparing ADC linker as described in claim 1, wherein the step of preparing compound G in step 3) is:

3.2) adding F and DIEA into DMF, cooling to T less than 0 ℃, adding HATU, stirring for 0.5-1h, adding the solution obtained in the reaction 3.1), and stirring until the reaction is finished;

7. The method of preparing an ADC linker as defined in claim 6, wherein: the ratio of the addition amount mol of DMF L, D to the addition amount mol of DBU is 1-5:0.5: 0.5.

8. The method for preparing ADC linker as described in claim 1, wherein the step of preparing H is: adding G, DMF, Pd/C into a reaction vessel, carrying out hydrogenolysis in a hydrogen atmosphere, crystallizing a product in DMF, filtering DMF after TLC reaction is finished, dissolving a filter cake with water, filtering Pd/C, and concentrating the filtrate under reduced pressure to obtain a white solid; drying at 45-55 ℃ to obtain the product H.

9. The method of preparing an ADC linker as set forth in claim 8, wherein: the proportion of the addition amount G of G, the addition amount mL of DMF and the addition amount G of Pd/C is 1:6-10: 0.1-0.3.

10. The method for preparing ADC linker as described in claim 1, wherein the step of preparing J comprises: adding DMF, H, I and DIEA into a reaction container, stirring for 20 hours, adding glacial acetic acid to adjust the pH value to 4-5 after TLC shows that the reaction is finished, and performing column chromatography purification to obtain J; the addition amount mol of the DMF L, H, the proportional relation of the addition amount mol of the I and the addition amount mol of the DIEA is 0.5-1.5:1:1: 1-2.5.