CN115873066A - Synthetic method of antibody-conjugated drug linker - Google Patents

Abstract

The invention provides a preparation method of an antibody conjugated drug linker, which comprises the following steps: the method takes Fmoc-valine-succinimide ester (Fmoc-Val-OSu) as an initial raw material, and Mc-Val-Cit-PABC-PNP is synthesized through ammonolysis reaction, amidation, deprotection, ammonolysis reaction and ester exchange reaction. The optimized synthesis process has the advantages of easily obtained raw materials, improved yield, improved defect that the original research route uses a large amount of ether and is not suitable for large-scale production, and is more suitable for industrial large-scale production.

Description

Synthetic method of antibody-conjugated drug linker

Technical Field

The invention relates to an industrial method for preparing an antibody-conjugated drug linker, in particular to a preparation method of an Mc-Val-Cit-PABC-PNP linker, belonging to the technical field of chemical synthesis of drugs.

Background

Antibody-conjugated drugs (ADCs) are a new class of targeted biopharmaceuticals that link biologically active cytotoxic drugs to monoclonal antibodies through linkers. On one hand, the action mechanism is that the antibody can identify the tumor antigen in a high-specificity targeting manner, after intravenous injection administration, the medicine is distributed to tumor tissues and combined with the tumor surface antigen through blood circulation, the compound of the ADC and the antigen undergoes endocytosis, small molecular cytotoxic load carried by the compound is internalized into tumor cells and is transported to lysosomes to be released in a high-efficiency active form, and the aim of tumor treatment is achieved by DNA damage or microtubule synthesis inhibition to further induce cancer cell apoptosis. On the other hand, the cell toxin releases permeable free small molecule cell toxin, and enters the tumor environment after passive diffusion or cell death to cause killing effect, namely bystander effect, on adjacent tumor cells.

The ADC has the advantages of high activity, small toxic and side effects, long action time and the like besides the advantages of accurate targeting of antibody drugs and high-efficiency killing of small-molecule cytotoxic drugs, and has become a popular research and development variety in the field of biological medicines, and the ADC has been developed in the last 10 years.

The ADC is composed of three modules, namely a monoclonal Antibody (Antibody), a Linker (Linker) and a cytotoxic drug (Cytotoxin). The monoclonal antibody provides targeting positioning, the linker ensures that the ADC can efficiently release cytotoxic drugs in tumor cells, and the ADC can be kept stable in blood circulation and tissue cells outside targets, and after the monoclonal antibody reaches the target, the small molecular cytotoxic drugs provide efficient killing effects on the tumor cells. Linkers are not only the moiety that forms the covalent link between the antibody and the small molecule cytotoxic, but are also key elements with design properties in targeted drug therapy.

Linkers affect many important properties of ADCs such as drug-antibody ratio (DAR), payload release time, therapeutic Index (TI), and pharmacokinetics/pharmacodynamics. Depending on the cleavage properties of the linker within the tumor cell, the linker can be divided into two types, cleavable and non-cleavable. The cleavable linker utilizes the specificity of the tumor microenvironment to release small molecule toxins, and comprises a hydrazone linker, a disulfide linker and an enzyme cleavable linker. The uncleavable linker breaks its linkage to the antibody through intracellular lysosomes, relying on complete degradation of the antibody for release. Both types of linkers have their own advantages and disadvantages: the metabolite generated by the cleavable linker is passively diffused and easily enters cells to generate bystander killing effect, has important significance for tumors with target antigen expression heterogeneity, can kill normal cells or immune cells near target tumor cells, and can be metabolized and degraded to a certain extent during in vivo circulation to further cause off-target toxicity; non-cleavable linkers are more stable than cleavable linkers and reduce off-target toxicity, but they are more dependent on the biological characteristics of the target cell, require good internalization to be activated by intracellular degradation, and have reduced bystander effects.

The linker Mc-Val-Cit-PABC-PNP is an enzyme-cleavable peptide linker, the linker has high blood circulation stability and release efficiency, and maleimide and carbonate structures at the chain ends in the linker structure can be easily combined with sulfydryl in antibody molecules and amino in cytotoxic drug molecules to form ADC molecules, so that the synthesis and structure screening research work of ADC is facilitated. The structural formula is as follows:

since the first ADC was marketed in 2000, a total of 12 ADCs were approved for marketing until now, with 4 of the Mc-Val-Cit-PABC-PNP linkers used, including Adcetris (benitumumab), polivy (pertuzumab), padcv (enrobiab), and Tivdak (Tivdak). Currently, up to 25 molecules using this structural linker are in clinical stage. And in recent related patent research reports, there are a number of ADC active molecules using this linker structure (PCT int.appl.,2021011834, 2020256721, 20210015941, 2020132658, 2020185069. The linker can be seen to be quite versatile.

The document Bioconjugate chem.2002,13,855-869, the synthetic route of which is included therein. The compound 2 is substituted, condensed, deprotected and subjected to a two-step substitution reaction to obtain the linker 1, which is carried out in small quantities, in the order of milligrams, with a final yield of only 8.6% and a large amount of ether for post-treatment, and the routes reported in the literature are as follows:

patent CN106380508 reports compound 3 as starting material to obtain compound 1 through four-step reaction. The purity of the final product can reach 99 percent, the yield is 55 percent, but a large amount of petroleum ether is used for post-treatment in the synthesis process. The routes reported in the patents are as follows:

in the above synthetic route, a large amount of controlled reagent ethyl ether is used for pulping treatment or the highly toxic and highly flammable reagent ethyl chloroformate is not suitable for large-scale production. In order to solve the defects of the synthesis process, the application aims to provide the synthesis method of the linker Mc-Val-Cit-PABC-PNP, which is applicable to industrial production, has good stability of process parameters, can reach the pharmaceutical industry standard in product purity and has high yield.

Disclosure of Invention

The invention aims to provide a preparation process of an antibody-conjugated drug linker Mc-Val-Cit-PABC-PNP, which aims to solve the problems mentioned in the background technology.

The technical scheme for realizing the purpose is as follows:

a synthetic method of an antibody coupling drug linker comprises the following steps of reacting a compound 6 with bis (4-nitrophenyl) carbonate, and purifying to obtain a compound 1:

pulping, performing silica gel column chromatography, recrystallizing, and pulping again;

it is preferable that: the solvent selected by pulping and re-pulping is methyl tert-butyl ether, and the mobile phase selected by silica gel column chromatography is dichloromethane/methanol =15:1, recrystallizing with DMF and methyl tert-butyl ether as selected solvents;

wherein,

the structural formula of compound 6 is:

the structural formula of compound 1 is:

the further synthesis method of the antibody coupling drug linker comprises the following purification steps after the compound 5 reacts with 6- (maleimide) hexanoic acid succinimidyl ester to obtain a compound 6:

pulping, recrystallizing and re-pulping;

it is preferable that: the solvent selected in pulping and re-pulping is methyl tert-butyl ether, and the solvent selected in recrystallization is DMF and methyl tert-butyl ether;

wherein,

the structural formula of the compound 5 is as follows:

the further synthesis method of the antibody-conjugated drug linker comprises the following purification steps after the compound 4 reacts with diethylamine to obtain a compound 5:

pulping;

it is preferable that: the solvent selected by pulping is dichloromethane;

wherein,

the structural formula of compound 4 is:

the further synthesis method of the antibody coupling drug linker comprises the following steps of reacting the compound 3 with p-aminobenzyl alcohol and EEDQ, and purifying to obtain a compound 4:

pulping, recrystallizing and re-pulping;

it is preferable that: the solvent selected in pulping and re-pulping is methyl tert-butyl ether, and the solvent selected in recrystallization is DMF and methyl tert-butyl ether;

wherein,

the structural formula of compound 3 is:

the further synthesis method of the antibody-conjugated drug linker comprises the following purification steps after the compound 2 reacts with citrulline to obtain a compound 3:

pulping;

it is preferable that: the solvent selected by pulping is methyl tert-butyl ether;

wherein,

the structural formula of compound 2 is:

the further synthesis method of the antibody coupling drug linker comprises the following steps of reacting the compound 6 with bis (4-nitrophenyl) carbonate:

adding the compound 6 and bis (4-nitrophenyl) carbonate into an organic solvent under nitrogen, stirring to react completely, evaporating under reduced pressure to remove the solvent, and washing.

The further synthesis method of the antibody coupling drug linker comprises the following steps of reacting the compound 5 with 6- (maleimide) hexanoic acid succinimidyl ester:

adding the compound 5 and Mc-OSu into an organic solvent at room temperature, stirring under the protection of nitrogen to react completely, evaporating under reduced pressure to remove the solvent, and washing.

The further synthesis method of the antibody coupling drug linker comprises the following steps of reacting the compound 4 with diethylamine:

adding the compound 4 into an organic solvent at room temperature, replacing nitrogen, adding diethylamine, stirring to react completely, and evaporating the solvent under reduced pressure.

The further synthesis method of the antibody coupling drug linker comprises the following steps of reacting the compound 3 with p-aminobenzyl alcohol and EEDQ:

adding the compound 3, p-aminobenzyl alcohol and EEDQ into an organic solvent under the protection of nitrogen, stirring at room temperature for complete reaction, concentrating under reduced pressure, and removing the solvent.

The further synthesis method of the antibody coupling drug linker comprises the following steps of reacting the compound 2 with citrulline:

under the cooling of ice water bath, dissolving citrulline in water, adding a compound 2 dissolved in an organic solvent, reacting at room temperature under the protection of nitrogen, stirring for completely reacting, and extracting an organic layer by using isopropanol/ethyl acetate; the reaction is stopped, washed, concentrated and dried in vacuum.

The beneficial effects of the invention include:

1) The synthesis method has mild conditions, easily obtained raw materials and no involvement of control reagents such as virulent reagents and the like.

2) The post-treatment method is simple and easy to operate, and the final product linker compound 1 with the purity of 98% is obtained in multiple batches by recrystallization, pulping and other methods.

3) Compared with the existing synthetic route, the synthetic method improves the total yield of the finished product.

4) The invention has developed preliminary gram-scale amplification experimental research, the material input in the front end step of the process route part reaches 50-100g level, and the method is not only suitable for small-amount preparation in a laboratory, but also suitable for industrial large-scale production.

Drawings

FIG. 1 is a 1H NMR chart of Compound 3 prepared by an example of the present invention;

FIG. 2 is a MS diagram of Compound 3 prepared by an example of the present invention;

FIG. 3 is a 1H NMR chart of Compound 4 prepared by the example of the present invention;

FIG. 4 is a MS picture of Compound 4 prepared by an example of the present invention;

FIG. 5 is a 1H NMR chart of Compound 5 prepared by an example of the present invention;

FIG. 6 is a MS picture of Compound 5 prepared by an example of the present invention;

FIG. 7 is a 1H NMR chart of Compound 6 prepared by the example of the present invention;

FIG. 8 is a MS plot of Compound 6 prepared according to the examples of the present invention;

FIG. 9 is a 1H NMR chart of Compound 1 prepared by an example of the present invention;

FIG. 10 is a 13C NMR chart of Compound 1 prepared by the example of the present invention;

FIG. 11 is an IR diagram of Compound 1 prepared according to an example of the present invention;

FIG. 12 is a MS picture of Compound 1 prepared by an example of the present invention;

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

The invention provides a preparation process of an antibody-conjugated drug linker Mc-Val-Cit-PABC-PNP, which comprises the following steps:

more specifically, the preparation method of the invention comprises the following steps:

step 1: carrying out ammonolysis reaction and purification on fluorenylmethyloxycarbonyl-L-valine (Fmoc-Val-OSu) (compound 2) serving as a raw material and citrulline in an organic solvent under nitrogen to obtain a compound 3; the structural formula of the compound 3 is as follows:

the preparation method comprises the following steps: adding the compound 2 into an organic solvent under the protection of nitrogen, and stirring and reacting with citrulline dissolved in water after being cooled by an ice bath, wherein the reaction temperature is room temperature. After stirring, the organic layer was extracted using isopropanol/ethyl acetate. After the reaction is stopped, the compound 3 is obtained by washing, concentrating, vacuum drying and purifying.

The purification method is pulping, and the selected reagent is methyl tert-butyl ether.

Step 2: carrying out amidation reaction on the raw material p-aminobenzyl alcohol and the compound 3 in the step 1 under the action of a catalyst 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), and purifying to obtain a compound 4; the structural formula of the compound 4 is as follows:

the preparation method comprises the following steps: adding the compound 3, p-aminobenzyl alcohol and EEDQ into an organic solvent under the protection of nitrogen, stirring at room temperature for complete reaction, concentrating under reduced pressure to remove the solvent, and purifying to obtain a compound 4.

The purification steps comprise pulping, recrystallization and pulping. Wherein, the solvent selected by pulping is methyl tert-butyl ether, and the solvent selected by recrystallization is DMF and methyl tert-butyl ether.

And step 3: carrying out amino deprotection and purification on the compound 4 obtained in the step 2 under the catalysis of strong base in an organic solvent under nitrogen to obtain a compound 5; the structural formula of the compound 5 is as follows:

the preparation method comprises the following steps: adding the compound 4 into an organic solvent at room temperature, replacing nitrogen, adding diethylamine, stirring to react completely, evaporating the solvent under reduced pressure, and purifying to obtain a compound 5.

The purification method is pulping, and the selected solvent is dichloromethane.

And 4, step 4: carrying out ammonolysis reaction on the compound 5 obtained in the step 3 and 6- (maleimide) caproic acid succinimidyl ester in an organic solvent under nitrogen, and purifying to obtain a compound 6; the structural formula of the compound 6 is as follows:

the preparation method comprises the following steps: and adding the compound 5 and MC-OSu into an organic solvent at room temperature, stirring under the nitrogen atmosphere to completely react, removing the solvent by reduced pressure evaporation, washing and purifying to obtain a compound 6.

The purification steps comprise pulping, recrystallization and pulping. Wherein, the solvent selected by pulping is methyl tert-butyl ether, and the solvent selected by recrystallization is DMF and methyl tert-butyl ether.

And 5: and (3) carrying out ester exchange reaction on the raw material bis (4-nitrophenyl) carbonate and the compound 6 obtained in the step (4) under the catalysis of alkali, and purifying to obtain a compound 1.

The preparation method comprises the following steps: adding the compound 6 and bis (4-nitrophenyl) carbonate into an organic solvent under nitrogen, stirring for complete reaction, evaporating the solvent under reduced pressure, washing and purifying to obtain a compound 1.

The purification steps comprise pulping, silica gel column chromatography, recrystallization and repulping. Wherein, the solvent selected by pulping is methyl tert-butyl ether, and the mobile phase selected by silica gel column chromatography is dichloromethane/methanol =15: the solvents chosen for recrystallization were DMF and methyl tert-butyl ether.

The following examples further illustrate the technical solution of the present application in non-limiting detail. The solvents, reagents, raw materials and the like used in the present application are all commercially available chemically pure or analytically pure products.

Example 1: synthesis of Compound 3

31.3g (178.7mmol, 1.3 equiv.) of L-citrulline, 15.0g (178.6mmol, 1.3 equiv.) of NaHCO3 and 360mL of water are put into a 3L reaction bottle and stirred to dissolve, and then cooled for 10min in an ice-water bath. 60g (137.5mmol, 1 eq) of Fmoc-Val-OSu were dissolved in 480mL DME and added to the flask with stirring. Then 480mL THF was added until the system was clear and stirred at room temperature for 100h under nitrogen (TLC monitoring progress). After the reaction, 672mL of 15% citric acid was added, and the mixture was extracted with ethyl acetate/isopropanol (9. The powder was transferred to a 1L flask, and 600mL of methyl t-butyl ether was added thereto, followed by stirring thoroughly for 30min, sonication for 5min, filtration, and repeated treatment 3 times to obtain 53.4g of Compound 3 as a white solid with a yield of 78.3%.1H NMR (400MHz, DMSO-d 6), delta: 12.48 (s, 1H); 8.20 (d, 1h, j = 7.2hz); 7.90 (d, 2h, j =7.6 hz); 7.76 (t, 2H, J= 7.2Hz); 7.43 (t, 2h, j =7.6 hz); 7.33 (t, 2h, j = 7.2hz); 5.96 (brs, 1H); 5.40 (brs, 2H); 4.29 to 4.26 (m, 1H); 4.25-4.22 (m, 2H); 4.15 (t, 1h, j = 5.2hz); 3.93 (t, 1h, j =16.4 hz); 2.98-2.94 (m, 2H); 2.01 to 1.96 (m, 1H); 1.71-1.56 (m, 2H); 1.42 to 1.40 (m, 2H); 0.87 (dd, 6H, J=13.2, 6.8Hz). HR-MS (ESI), C26H32N4O6, m/z:497.2404[ 2 ] M + H ] +.

Example 2: synthesis of Compound 4

A 3L reaction flask was charged with 48.0g (96.7mmol, 1 eq) of compound 3, 24.0g (194.9mmol, 2 eq) of p-aminobenzyl alcohol and 48.0g (194.1mmol, 2 eq) of EEDQ, 1500mL of dried dichloromethane/methanol (2, v). DMF was added until it was just dissolved, 20 volumes of methyl t-butyl ether was slowly added with stirring to precipitate the product, which was filtered, the filter cake was slurried with methyl t-butyl ether 3 times (800 mL. Times.3 times), filtered and dried to give 48.1g of Compound 4 as a white solid in 82.7% yield with 98.2% purity by HPLC.

1H NMR(400MHz,DMSO-d6),δ:9.99(brs,1H)；8.13(d,1H,J＝7.2Hz)；7.89(d,2H,J＝7.2Hz)；7.75(t,2H,J＝8.0Hz)；7.54(d,2H,J＝8.4Hz)；7.47～7.40(m,2H)；7.31(t,2H,J＝7.2Hz)；7.23(d,2H,J＝8.4Hz)；5.99(t,1H,J＝5.2Hz)；5.42(brs,2H)；5.12(t,1H,J＝5.6Hz)；4.44～4.42(m,3H)；4.31～4.26(m,1H)；4.25～4.22(m,2H)；3.94(t,1H,J＝7.2Hz)；3.04～2.93(m,2H)；2.02～1.96(m,1H)；1.69～1.59(m,2H)；1.47～1.35(m,2H)；0.87(dd,6H,J＝11.2,6.8Hz).HR-MS(ESI),C33H39N5O6,m/z:602.2953[M+H]+.

Example 3: synthesis of Compound 5

A1L reaction flask was charged with 42.0g (69.8mmol, 1 eq) of Compound 4 dissolved in 490mL of DMF, nitrogen was replaced, 144.5mL (1397.3mmol, 20 eq) of diethylamine was added, and the mixture was stirred at room temperature overnight. After TLC (dichloro/methanol =5. Adding 350mL of dichloromethane into the residue, slightly scraping the wall of the bottle to solidify the oily substance, grinding the block into powder, stirring for 30min, performing suction filtration, transferring the filter cake into a 500mL flask, adding 350mL of dichloromethane, stirring for 30min, performing ultrasonic treatment for 5min, performing suction filtration, repeating the treatment for 3 times, and performing vacuum drying to obtain 25.3g of compound 5 which is white or light gray powder, wherein the yield is 95.4%, and the purity is 95.5% by HPLC (high performance liquid chromatography).

1H NMR(400MHz,DMSO-d6),δ:10.08(brs,1H)；8.19(d,1H,J＝7.2Hz)；7.54(d,2H,J＝8.4Hz)；7.23(d,2H,J＝8.4Hz)；6.04(t,1H,J＝5.3Hz)；5.45(brs,2H)；5.12(brs,1H)；4.55～4.46(m,3H)；4.43(s,2H)；3.05(d,2H,J＝6.7Hz)；2.98～2.85(m,1H)；1.97～1.94(m,1H)；1.68～1.57(m,2H)；1.43～1.34(m,2H)；0.84(dd,6H,J＝39.6,7.8Hz).HR-MS(ESI),C18H29N5O4,m/z:380.2304[M+H]+.

Example 4: synthesis of Compound 6

To a 500mL reaction flask was added 19.0g (50.0 mmol,1 equivalent) of compound 5 and 18.5g (60.0 mmol,1.2 equivalents) of Mc-OSu dissolved in 280mL of DMF, the nitrogen was replaced, the reaction was carried out at room temperature for 24h (TLC monitoring, dichloro/methanol =10, v), DMF was distilled off under reduced pressure at 40 ℃. The residual oil was stirred with 350mL of methyl tert-butyl ether for 1h, filtered, the filter cake was ground to a powder, washed 3 times with methyl tert-butyl ether, recrystallized with DMF and methyl tert-butyl ether, the resulting solid was repeatedly slurried 3 times with methyl tert-butyl ether (350 mL × 3 times), dried by suction to give 26.6g of compound 6 as a white powder in 92.7% yield.

1H NMR(400MHz,DMSO-d6),δ:9.92(brs,1H)；8.08(d,1H,J＝7.6Hz)；7.83(d,1H,J＝8.4Hz)；7.53(d,2H,J＝8.4Hz)；7.23(d,2H,J＝8.4Hz)；7.01(brs,2H)；5.98(t,1H,J＝5.6Hz)；5.42(brs,2H)；5.11(t,1H,J＝5.6Hz)；4.42(d,2H,J＝5.2Hz)；4.40～4.35(m,1H)；4.19(t,1H,J＝8.0Hz)；3.38～3.33(m,2H)；3.03～3.00(m,2H)；2.20～2.09(m,2H)；1.99～1.94(m,1H)；1.68～1.33(m,8H)；1.22～1.16(m,2H)；0.84(dd,6H,J＝12.8,6.8Hz).HR-MS(ESI),C28H40N6O7,m/z:573.3038[M+H]+.

Example 5: synthesis of target product 1

A2L reaction flask was charged with 20.0g (34.9mmol, 1 equiv) of Compound 6, 13.4g (44.0mmol, 1.26 equiv) of bis (4-nitrophenyl) carbonate and 7.7mL (5.69g, 44.0mmol,1.26 equiv) of DIPEA in 1000mL of dry DMF. The reaction was stirred at room temperature under nitrogen for 40h and monitored by tlc (dichloro/methanol =10, 1,v. After the solvent was distilled off under reduced pressure at 40 ℃, the residue was powdered by adding 800mL of methyl tert-butyl ether, stirred for 30min, sonicated for 5min, filtered, the filter cake was slurried 3 times with methyl tert-ether, and dried to give a white solid powder, which was subjected to silica gel column chromatography (dichloro/methanol =15, v). Recrystallizing the crude product with DMF and methyl tert-butyl ether, transferring the precipitated crystal into a 1L flask, adding 800mL of methyl tert-butyl ether, stirring for 30min, performing ultrasonic treatment for 5min, performing suction filtration, repeatedly treating with methyl tert-butyl ether for 3 times, and performing suction drying to obtain 5.2g of compound 1 as white powder with yield of 20.2% and purity of 98.6% by HPLC detection.

1H NMR(400MHz,CDCl3/CD3OD)：δ:9.52(s,1H)；8.28(d,2H,J＝8.8Hz)；8.02(d,1H,J＝8.0Hz)；7.64(d,2H,J＝3.2Hz)；7.55(d,1H,J＝3.2Hz)；7.42(d,4H,J＝8.8Hz)；6.75(s,2H)；5.27(s,2H)；4.57～4.54(m,1H)；4.18(d,1H,J＝7.2Hz)；3.51(t,2H,J＝6.8Hz)；3.24～3.11(m,2H)；2.28(t,2H,J＝7.2Hz)；2.10～2.05(m,1H)；1.92～1.73(m,2H)；1.70～1.57(m,6H)；1.36～1.31(m,2H)；0.97(d,6H,J＝10.4Hz).13C NMR(101MHz,CDCl3/CD3OD),δ:174.9,172.8,171.5,171.0,160.9,155.9,152.9,145.7,139.1,134.5,131.0,129.9,125.6,122.2,120.4,71.0,59.3,53.6,39.2,37.9,36.1,30.9,29.5,28.5,26.6,25.5,19.4,18.5.IR(KBr,cm-1)ν3280,2938,1766,1706,1637,1529,1348,1215.HR-MS(ESI),C35H44N7O11,m/z 738.3091[M+H]+.

Comparative example 1: synthesis of target product 1

To a 500mL reaction flask was added 3.5g (6.11mmol, 1 eq) of compound 6 dissolved in 21mL DMF, 2.83mL (30.56mmol, 5 eq) pyridine was added and mixed well, the solution was cooled to 0 ℃, a mixed solution of 3.7g (18.34mmol, 3 eq) p-nitrophenyl chloroformate in 70mL dichloromethane was added, the reaction was stirred at room temperature under nitrogen for 24h, tlc monitored (dichloro/methanol =10, 1, v). After the layers were separated by adding 250mL of ethyl acetate and 375mL of 15% citric acid, the organic phase was washed with citric acid, water and saturated sodium chloride and then the solvent was distilled off under reduced pressure at 40 ℃, the obtained oil was separated by silica gel column chromatography (dichloro/methanol =15:1,v) to obtain 0.4g of a white powder in 8.9% yield, and thus the yield of comparative example 1 was significantly lower than that of the foregoing example.

Comparative example 2: synthesis of Compound 4

After 5.0g (10.1mmol, 1 eq) of compound 3,2.5g (20.1mmol, 2 eq) of p-aminobenzyl alcohol and 5.0g (21.1mmol, 2 eq) of EEDQ were charged to a 500mL reaction flask, 175mL of a dry dichloromethane/methanol (2.

1H NMR(400MHz,DMSO-d6),δ:9.99(brs,1H)；8.13(d,1H,J＝7.2Hz)；7.89(d,2H,J＝7.2Hz)；7.75(t,2H,J＝8.0Hz)；7.54(d,2H,J＝8.4Hz)；7.47～7.40(m,2H)；7.31(t,2H,J＝7.2Hz)；7.23(d,2H,J＝8.4Hz)；5.99(t,1H,J＝5.2Hz)；5.42(brs,2H)；5.12(t,1H,J＝5.6Hz)；4.44～4.42(m,3H)；4.31～4.26(m,1H)；4.25～4.22(m,2H)；3.94(t,1H,J＝7.2Hz)；3.04～2.93(m,2H)；2.02～1.96(m,1H)；1.69～1.59(m,2H)；1.47～1.35(m,2H)；0.87(dd,6H,J＝11.2,6.8Hz).

Comparative example 3: synthesis of target product 1

1.0g (1.75mmol, 1 eq) of compound 6,0.7g (2.21mmol, 1.26 eq) of bis (4-nitrophenyl) carbonate and 0.4mL (0.3g, 2.21mmol,1.26 eq) of DIPEA were dissolved in 50mL of dry DMF. The reaction was stirred at room temperature under nitrogen for 40h and monitored by tlc (dichloro/methanol =10, 1,v. After the solvent was evaporated under reduced pressure at 40 ℃, the residue was powdered by adding 40mL of methyl tert-butyl ether, stirred for 30min, sonicated for 5min, filtered, the filter cake was slurried 3 times with methyl tert-ether, and dried by suction to give a white solid powder, which was subjected to silica gel column chromatography (dichloro/methanol =15, v). The crude product was stirred with 400mL of methyl tert-butyl ether for 30min, sonicated for 5min, suction filtered, and treated with methyl tert-butyl ether repeatedly for 3 times in this manner to give 0.36g of Compound 1 as a white powder in 27.9% yield with 89.2% purity by HPLC, as seen in comparative example 3, which is significantly less pure than the previous examples.

1H NMR(400MHz,CDCl3/CD3OD)：δ:9.52(s,1H)；8.28(d,2H,J＝8.8Hz)；8.02(d,1H,J＝8.0Hz)；7.64(d,2H,J＝3.2Hz)；7.55(d,1H,J＝3.2Hz)；7.42(d,4H,J＝8.8Hz)；6.75(s,2H)；5.27(s,2H)；4.57～4.54(m,1H)；4.18(d,1H,J＝7.2Hz)；3.51(t,2H,J＝6.8Hz)；3.24～3.11(m,2H)；2.28(t,2H,J＝7.2Hz)；2.10～2.05(m,1H)；1.92～1.73(m,2H)；1.70～1.57(m,6H)；1.36～1.31(m,2H)；0.97(d,6H,J＝10.4Hz)。

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. The synthesis method of the antibody-conjugated drug linker is characterized in that after the compound 6 reacts with bis (4-nitrophenyl) carbonate, the compound 1 is obtained through the following purification steps:

pulping, performing silica gel column chromatography, recrystallizing, and pulping again;

it is preferable that: the solvent selected by pulping and re-pulping is methyl tert-butyl ether, and the mobile phase selected by silica gel column chromatography is dichloromethane/methanol =15:1, recrystallizing with DMF and methyl tert-butyl ether;

wherein,

the structural formula of compound 6 is:

the structural formula of compound 1 is:

2. the method of claim 1, wherein compound 5 is reacted with 6- (maleimido) hexanoic acid succinimidyl ester and then purified as follows to give compound 6:

pulping, recrystallizing and re-pulping;

it is preferable that: the solvent selected for pulping and repulping is methyl tert-butyl ether, and the solvent selected for recrystallization is DMF and methyl tert-butyl ether;

wherein,

the structural formula of the compound 5 is as follows:

3. the method of claim 2, wherein compound 4 is reacted with diethylamine to provide compound 5 by the following purification steps:

pulping;

it is preferable that: pulping the selected solvent to obtain dichloromethane;

wherein,

the structural formula of compound 4 is:

4. the method of claim 3, wherein compound 3 is reacted with p-aminobenzyl alcohol, EEDQ, and then purified to give compound 4 as follows:

pulping, recrystallizing and re-pulping;

it is preferable that: the solvent selected in pulping and re-pulping is methyl tert-butyl ether, and the solvent selected in recrystallization is DMF and methyl tert-butyl ether;

wherein,

the structural formula of compound 3 is:

5. the method according to claim 4, wherein compound 2 is reacted with citrulline and then purified as follows to give compound 3:

pulping;

it is preferable that: the solvent selected by pulping is methyl tert-butyl ether;

wherein,

the structural formula of the compound 2 is as follows:

6. the process of any one of claims 1 to 5, wherein compound 6 is reacted with bis (4-nitrophenyl) carbonate by:

adding the compound 6 and bis (4-nitrophenyl) carbonate into an organic solvent under nitrogen, stirring for complete reaction, evaporating the solvent under reduced pressure, and washing.

7. The method of any one of claims 1-6, wherein compound 5 is reacted with 6- (maleimido) hexanoic acid succinimidyl ester by:

adding the compound 5 and Mc-OSu into an organic solvent at room temperature, stirring under the protection of nitrogen to react completely, evaporating under reduced pressure to remove the solvent, and washing.

8. The method of any one of claims 1-7, wherein compound 4 is reacted with diethylamine by:

adding the compound 4 into an organic solvent at room temperature, replacing nitrogen, adding diethylamine, stirring to react completely, and evaporating the solvent under reduced pressure.

9. The process of any one of claims 1 to 8, wherein compound 3 is reacted with p-aminobenzyl alcohol, EEDQ, by:

adding the compound 3, p-aminobenzyl alcohol and EEDQ into an organic solvent under the protection of nitrogen, stirring at room temperature for complete reaction, concentrating under reduced pressure, and removing the solvent.

10. A method according to any one of claims 1 to 9, wherein compound 2 is reacted with citrulline by:

under the cooling of ice water bath, dissolving citrulline in water, adding a compound 2 dissolved in an organic solvent, reacting at room temperature under the protection of nitrogen, stirring for completely reacting, and extracting an organic layer by using isopropanol/ethyl acetate; the reaction is stopped, washed, concentrated and dried in vacuum.

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