CN111620927B - One-pot preparation process of antibody drug conjugate intermediate - Google Patents
One-pot preparation process of antibody drug conjugate intermediate Download PDFInfo
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Abstract
A 'one-pot' method for preparing an antibody drug conjugate intermediate is characterized in that MC-Val-Cit-PAB-OH and di (p-nitrophenyl) carbonate are subjected to contact reaction in the presence of organic base, after the reaction is finished, organic base is added into a reaction system, and 1-hydroxybenzotriazole and a drug part D are subjected to reaction to obtain the antibody drug conjugate intermediate. The whole reaction system is only subjected to one-time separation and purification treatment, and the steps of concentration and washing of intermediate reaction liquid, treatment of organic waste liquid, packaging and storage of intermediates and the like are not required, so that the yield is higher.
Description
Technical Field
The invention relates to the field of antibody drug conjugates, in particular to a one-pot preparation process of an antibody drug conjugate intermediate (namely a linker part-drug part conjugate).
Background
Antibody Drug Conjugates (ADC) are a class of anti-tumor drugs that include three components: the Antibody part (Antibody), the Linker part (Linker) and the Drug part (Drug), wherein the Antibody part and the Drug part are connected through the Linker part, and the action mechanism is that the targeting of the Antibody is used to deliver the Drug to target cells (such as tumor cells) in a targeted manner and then release the Drug to kill the tumor cells.
Currently, the most common method for synthesizing antibody drug conjugates is to covalently link a linker moiety and a drug moiety in a liquid phase to form a linker-drug conjugate, and then to perform thiol or amino conjugation with an antibody to form an antibody drug conjugate. Chinese patent publication No. CN107427591A discloses a general synthetic method of linker-drug conjugates (page 34 and pages 47-48 of the specification) (as shown in fig. 1), which is divided into: the first step is that a linker containing free benzyl alcohol group is dissolved in a proper solvent, bis (p-nitrophenyl) carbonate and diisopropylethylamine are added into a reaction system, and an intermediate product is extracted and purified after reacting for several hours; and the second step is to dissolve the intermediate product and the medicine part containing free amino group in proper solvent, add 1-hydroxybenzotriazole and pyridine, react for several hours, and remove the solvent under reduced pressure to obtain the linker-medicine conjugate.
Because the intermediate needs to be extracted and purified in the two-step reaction system, the final reaction yield is affected. In addition, the above preparation process has inevitable defects in multi-system reaction during production, i.e. concentration, washing, filtration, treatment of organic waste liquid of reaction liquid in multiple steps, packaging and storage of intermediates in the first step, and the like are required, so that the costs of production consumables, labor, equipment, fields and the like are increased, more production waste liquid is generated, and the overall production cost and production time are increased.
A variety of linkers capable of covalent attachment to antibodies in a bridge coupling manner are also disclosed in Chinese patent application publication No. CN107921030A, which discloses on page 42 of the specification an antibody drug conjugate intermediate (Py-MAA-Val-Cit-PAB-MMAE) (where Py is 1,3,5-triacryloylhexahydro-1,3,5-triazine with CAS of 959-52-4, available from Bailingwei science and technology Co., ltd., nanjing Congralin chemical industry Co., ltd.) as shown below.
In addition, on page 32 of this patent application, a process for the preparation of an intermediate (Py-MAA-Val-Cit-PAB-MMAD) of an antibody drug conjugate wherein the drug moiety is MMAD (Demethyl olastatin 10) is disclosed:
the process comprises the steps of coupling Val-Cit-PAB with a drug part (MMAD) to form a Val-Cit-PAB-MMAD conjugate, purifying to obtain a target product, and reacting with Py-MAA to generate an antibody drug conjugate intermediate Py-MAA-Val-Cit-PAB-MMAD. Also, the multi-system synthesis is adopted, and since the linked drug moiety (e.g., MMAD/MMAE or MMAF) in the antibody drug conjugate is involved in the linking reaction in the early stage of the reaction (MMAD is added as a reactant to the reaction system when Val-Cit-PAB-MMAD is produced) rather than in the last step, the loss of the drug moiety (e.g., MMAD/MMAE or MMAF) produced by the above process is high, and since the price of the linked drug moiety (e.g., MMAD/MMAE or MMAF) in the antibody drug conjugate is generally expensive, the production cost is also greatly increased.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for preparing an antibody drug conjugate intermediate (i.e., linker-drug conjugate) by a "one-pot method".
Specifically, the invention provides a preparation process of an antibody drug conjugate intermediate comprising a linker moiety and a drug moiety, wherein the antibody drug conjugate intermediate is Py-MAA-Val-Cit-PAB-D or MC-Val-Cit-PAB-D, the intermediate is Py-MAA-Val-Cit-PAB or MC-Val-Cit-PAB is the linker moiety, the intermediate is D represents a linked drug moiety, and the linked drug moiety contains a free amino group, and the preparation process is characterized by comprising the following reaction routes:
the preparation process is a one-pot method, and the two reactions are carried out in one system.
Further, the preparation process comprises the following steps: py-MAA-Val-Cit-PAB-OH or MC-Val-Cit-PAB-OH and di (p-nitrophenyl) carbonate (NPC) are subjected to contact reaction in the presence of organic base, after the reaction is finished, the organic base is directly added into the same reaction system, and 1-hydroxybenzotriazole and the medicine part D are added for reaction.
Further, the drug moiety D is an auristatin (auristatin) cytotoxic agent, an antramycin (Anthramycin) cytotoxic agent, an anthracycline (anthracycline) cytotoxic agent, a puromycin (puromycin) cytotoxic agent, or a camptothecin analog.
Further, the auristatin cytotoxic agent is MMAE, MMAF, MMAD or its derivative; the Anthramycin (Anthramycin) cytotoxic agent is Anthramycin or a derivative thereof; the anthracycline cytotoxic agent is daunorubicin, adriamycin, epirubicin, idarubicin, mitoxantrone or derivatives thereof; the puromycin (puromycin) cytotoxic agent is puromycin toxin or a derivative thereof; the camptothecin analogue is DX8951 (irinotecan) or derivatives thereof.
Further, the structure of the Py-MAA-Val-Cit-PAB-D or MC-Val-Cit-PAB-D is shown as follows:
further, the organic base 1 and the organic base 2 are respectively and independently one or more of N, N diisopropylethylamine, triethylamine and pyridine; preferably, the organic base 1 and the organic base 2 are respectively and independently one or two of N, N diisopropylethylamine and pyridine.
Further, the triazole catalyst is one or more of 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazol, and 1-hydroxy-1H-1, 2, 3-triazole-4-carboxylic acid ethyl ester, preferably 1-hydroxybenzotriazole.
Further, the reaction temperature of the above-described reaction is about 15 to 35 ℃, and further, the reaction temperature may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 ℃.
The use of any one of the methods described above in the preparation of an anti-tumor medicament.
Use of a method as described in any of the preceding claims for the preparation of an antibody drug conjugate.
Compared with the traditional two-step reaction system, the preparation process for preparing the antibody drug conjugate intermediate by the one-pot method is simple to operate, the steps of concentration, washing, filtering, treatment of organic waste liquid, packaging and storage of the intermediate and the like of intermediate reaction liquid are not needed in the intermediate process, the next reaction operation is directly carried out in the same system after the first reaction step is finished, the whole reaction system is only subjected to separation and purification once, the production material consumption, labor, equipment, field, raw materials and other costs are saved, the produced production waste liquid is greatly reduced, the production cost is reduced, and the production efficiency is improved. In addition, in the reaction process, the connected medicine part is added in the last reaction link, so that the feeding loss of the medicine part (such as MMAD, MMAE or MMAF, DX8951 and the like) is effectively reduced. Therefore, the preparation process for preparing the antibody drug conjugate intermediate by the one-pot method is more suitable for large-scale production.
Drawings
FIG. 1 shows a method for preparing linker-drug conjugates disclosed on page 34 and pages 47-48 of the Chinese patent specification with publication No. CN 107427591A.
Detailed Description
Abbreviations
Unless otherwise defined, all abbreviations used in the present invention have the same meaning as understood by one of ordinary skill in the art. As used herein, the abbreviations used and their definitions are as follows:
definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term "antibody drug conjugate" as used herein means a compound in which antibody/antibody functional fragments, linkers, drug moieties are chemically linked together, the structure of which is generally composed of three parts: an antibody or antibody-like ligand, a drug moiety, and a linker (linker) coupling the antibody or antibody-like ligand and the drug. The preparation of antibody drug conjugates is currently generally divided into two steps: the first step is to chemically react the linker with the drug moiety to form a "linker-drug" conjugate, and the second step is to covalently couple the linker moiety of the "linker-drug" conjugate to the antibody/antibody functional fragment via a thiol or amino group. The term "antibody drug conjugate intermediate" as used herein refers to the above-mentioned "linker-drug" conjugate, and further, the term "antibody drug conjugate intermediate" as used herein refers to "linker-drug" conjugates in general, which are coupled together by amine transesterification to form a "-CO-NH-" bond between the linker and the drug.
The terms "linker" and "linker moiety" as used herein refer to the moiety that links an antibody to a drug in an antibody-drug conjugate, which may or may not be cleavable. The cleavable linker (i.e., cleavable linker or biodegradable linker) may be cleaved within or on the target cell, thereby releasing the drug. In some embodiments, the linker of the invention is selected from cleavable linkers, such as disulfide-based linkers (which selectively cleave in tumor cells with higher thiol concentrations), peptide linkers (which are cleaved by enzymes in tumor cells), hydrazone linkers. In other embodiments, the linkers of the present invention are selected from non-cleavable linkers (i.e., non-cleavable linkers), such as thioether linkers. In still other embodiments, the linkers of the present invention are a combination of cleavable linkers and non-cleavable linkers.
The terms "drug" and "drug moiety" as used herein generally refer to any compound having a desired biological activity and having reactive functional groups for the preparation of the conjugates of the invention. Desirable biological activities include diagnosing, curing, alleviating, treating, preventing diseases in humans or other animals. As new drugs continue to be discovered and developed, these new drugs should also be incorporated into the drugs described herein. Specifically, the drug includes, but is not limited to, cytotoxic drugs, cell differentiation factors, stem cell trophic factors, steroid drugs, drugs for treating autoimmune diseases, anti-inflammatory drugs, or drugs for infectious diseases. More specifically, the drugs include, but are not limited to, tubulin inhibitors or DNA, RNA damaging agents.
Examples
The technical solution of the present invention will be further described in non-limiting detail with reference to the following embodiments. It should be noted that the following examples are only for illustrating the technical idea and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
The general preparation method comprises the following steps: preparation of Py-MAA-Val-Cit-PAB-OH:
(1) Preparation of Py-MAA
The compound Py (1.87g, 7.51mmol) and Et 3 N (104. Mu.L, 0.75 mmol) in anhydrous CH 2 Cl 2 (40 mL), mercaptoacetic acid (103.9. Mu.L, 1.50 mmol) in CH was added dropwise 2 Cl 2 (40 mL) of the solution; after the addition was complete, the system was allowed to warm to room temperature and stirred overnight. After the reaction was completed, the solvent was removed in vacuo, and the crude product was purified by column chromatography to give Py-MAA (1.87 g) as a white solid.
(2) Preparation of Py-MAA-Val-Cit-PAB-OH
The compound Py-MAA (10.00g, 29.33mmol) was placed in tetrahydrofuran (200 mL), N' N-carbonyldiimidazole (7.13g, 44.00mmol), val-Cit-PAB-OH (13.34g, 35.20mmol) was added, and the mixture was stirred at room temperature for 24 hours. Petroleum ether (200 mL) was added, stirred for 0.5 h, and filtered to give a white solid. The white solid was purified by preparative high performance liquid chromatography, and the preparative liquid was rotary evaporated under reduced pressure to give Py-MAA-Val-Cit-PAB-OH (6.67 g, white solid powder).
EXAMPLE 1 preparation of Py-MAA-Val-Cit-PAB-MMAE
(1) Preparation by one-pot method
After dissolving Py-MAA-Val-Cit-PAB-OH (1.8g, 1.0eq) and DMF (40 mL) in this order in a three-necked round-bottomed flask, NPC (882mg, 1.1eq) and DIPEA (336mg, 1.0eq) were added thereto and stirred at 24. + -. 2 ℃ for 24 hours. Then DIPEA (672mg, 2.0eq), pyridine (2.3 mL), hoBt (351mg, 1.0eq) and MMAE (1.7g, 0.9eq) are sequentially added into the reaction solution, the reaction is continued for 48 hours at the temperature of 24 +/-2 ℃, and a product Py-MAA-Val-Cit-PAB-MMAE (1.9 g) is prepared by liquid phase purification, wherein the purity is 99.84%, and the yield is 51.3% [ a calculation formula ]: yield = Py-MAA-Val-Cit-PAB-MMAE yield ÷ (Py-MAA-Val-Cit-PAB-OH usage ÷ 702.8 × 1446.8) × 100%).
(2) "two step" preparation
The first step is as follows: preparation of Py-MAA-Val-Cit-PAB- (4-nitrophenyl) carbonate
DMF (40 mL) and Py-MAA-Val-Cit-PAB-OH (1.8g, 1.0eq.) were added to a reaction flask, and after stirring and dissolution, bis (p-nitrophenyl) carbonate (NPC, 882mg, 1.1eq.) and DIPEA (336mg, 1.0eq.) were added to react at 24. + -. 2 ℃ for 24 hours. Adding ethyl acetate (mL) into the reaction solution, dropwise adding petroleum ether (mL) for 20 minutes, continuously stirring for 10min, and filtering; then washing the mixture by ethyl acetate and petroleum ether for three times respectively; and (3) performing spin drying to obtain Py-MAA-Val-Cit-PAB- (4-nitrophenyl) carbonate (1.6 g), wherein the yield is 72.1%, and the purity is as follows: 86 percent.
The second step is that: preparation of Py-MAA-Val-Cit-PAB-MMAE
To a reaction flask, py-MAA-Val-Cit-PAB- (4-nitrophenyl) carbonate (1.5 g), 1-hydroxybenzotriazole (HoBt, 234mg, 1.0eq.), DMF (30 mL), MMAE (1.1g, 0.9eq.), pyridine (1.5 mL) and DIPEA (447mg, 2.0eq.) were added, reacted at 24 ± 2 ℃ for 48 hours, spin-dried, and prepared by HPLC to give Py-MAA-Val-Cit-PAB-MMAE (1.3 g), yield 52%, purity: 99 percent.
As can be seen by comparison, under the condition of the same feeding amount of the main raw materials, the yield of the final product of Py-MAA-Val-Cit-PAB-MMAE prepared by the one-pot method is 51.3 percent; the final product yield of the Py-MAA-Val-Cit-PAB-MMAE prepared by the two-step method is 37.49%, and by comparison, the final product yield of the Py-MAA-Val-Cit-PAB-MMAE prepared by the one-pot method is far higher than that of the Py-MAA-Val-Cit-PAB-MMAE prepared by the two-step method.
EXAMPLE 2 preparation of Py-MAA-Val-Cit-PAB-MMAD
(1) Preparation by one-pot method
DMF (4 mL), py-MAA-Val-Cit-PAB-OH (200mg, 1.0eq.) were added to a reaction flask, and after stirring and dissolution, bis (p-nitrophenyl) carbonate (NPC, 95mg, 1.1eq.) and DIPEA (36mg, 1.0eq.) were added and reacted at 24. + -. 2 ℃ for 24 hours, 1-hydroxybenzotriazole (Ho0Bt, 38mg, 1.eq.) and MMAD (197mg, 0.9eq.) were added to a reaction flask, and reacted at 24. + -. 2 ℃ for 48 hours, followed by spin-drying, and then prepared by HPLC to give Py-MAA-Val-Cit-PAB-MMAD (208 mg), with a yield: 48.7%, purity: 99 percent.
(2) "two step" preparation
The first step is as follows: preparation of Py-MAA-Val-Cit-PAB- (4-nitrophenyl) carbonate
DMF (4 mL) and Py-MAA-Val-Cit-PAB-OH (200mg, 1.0 eq.) were added to a reaction flask, and after stirring and dissolution, bis (p-nitrophenyl) carbonate (NPC, 95mg,1.1 eq.) and DIPEA (36mg, 1.0 eq.) were added and reacted at 24. + -. 2 ℃ for 24 hours. Adding ethyl acetate (6 mL) into the reaction solution, dropwise adding petroleum ether (12 mL) for 20 minutes, continuously stirring for 10min, and filtering; then washed three times with each of ethyl acetate and petroleum ether. And (3) performing spin drying to obtain Py-MAA-Val-Cit-PAB- (4-nitrophenyl) carbonate (170 mg), wherein the yield is as follows: 68.8%, purity: 86 percent.
The second step is that: preparation of Py-MAA-Val-Cit-PAB-MMAD
Py-MAA-Val-Cit-PAB- (4-nitrophenyl) carbonate (170 mg), 1-hydroxybenzotriazole (HoBt, 27mg, 1.0eq.), DMF (4 mL), MMAD (139mg, 0.9eq.), pyridine (174. Mu.L) and DIPEA (52mg, 2.0eq) were added to a reaction flask, reacted at 24 + -2 ℃ for 48 hours, spin-dried, and prepared by HPLC to obtain Py-MAA-Val-Cit-PAB-MMAD (139 mg), yield: 47.3%, purity: 99 percent.
As can be seen by comparison, under the condition of the same feeding amount of the main raw materials, the yield of the final product of Py-MAA-Val-Cit-PAB-MMAD prepared by the one-pot method is 48.7%; the final product yield of the Py-MAA-Val-Cit-PAB-MMAD prepared by the two-step method is 32.54 percent, and by comparison, the final product yield of the Py-MAA-Val-Cit-PAB-MMAD prepared by the one-pot method is far higher than that of the Py-MAA-Val-Cit-PAB-MMAD prepared by the two-step method.
EXAMPLE 3 preparation of Py-MAA-Val-Cit-PAB-DX8951
(1) Preparation by one-pot method
DMF (4 mL), py-MAA-Val-Cit-PAB-OH (200mg, 1.0 eq.) were added to the reaction flask, and after stirring and dissolution, di (p-nitrophenyl) carbonate (NPC, 95mg, 1.1eq.) and DIPEA (36mg, 1.0 eq.) were added. After 24 + -2 ℃ reaction for 24 hours, 1-hydroxybenzotriazole (HoBt, 38mg, 1.0eq.), DX8951 (136mg, 0.9eq.), pyridine (248 mu L) and DIPEA (110mg, 3.0eq.) were added, and the reaction was continued at 24 + -2 ℃ for 48 hours, followed by spin-drying to prepare Py-MAA-Val-Cit-PAB-DX8951 (123 mg) by HPLC, with the yield: 37.1%, purity: 97 percent.
(2) "two step" preparation
The first step is as follows: preparation of Py-MAA-Val-Cit-PAB- (4-nitrophenyl)) Carbonic acid esters
DMF (4 mL) and Py-MAA-Val-Cit-PAB-OH (200mg, 1.0 eq.) were added to a reaction flask, and after stirring and dissolution, di (p-nitrophenyl) carbonate (NPC, 95mg, 1.1eq.) and DIPEA (36mg, 1.0 eq.) were added and reacted at 24. + -. 2 ℃ for 24 hours. Adding ethyl acetate (6 mL) into the reaction solution, dropwise adding petroleum ether (12 mL) for 20 minutes, continuously stirring for 10min, and filtering; then washing the mixture by ethyl acetate and petroleum ether for three times respectively; and (3) performing spin drying to obtain Py-MAA-Val-Cit-PAB- (4-nitrophenyl) carbonate (176 mg), wherein the yield is as follows: 71.3%, purity: 86 percent.
The second step: preparation of Py-MAA-Val-Cit-PAB-DX8951
To a reaction flask, py-MAA-Val-Cit-PAB- (4-nitrophenyl) carbonate (170 mg), 1-hydroxybenzotriazole (HoBt, 27mg, 1.0eq.), DMF (4 mL), DX8951 (96mg, 0.9eq.), pyridine (174. Mu.L) and DIPEA (78mg, 3.0eq.) were added, reacted at 24. + -. 2 ℃ for 48 hours, spin-dried, and prepared by HPLC to give Py-MAA-Val-Cit-PAB-DX8951 (76 mg), yield: 33.3%, purity: 97 percent.
As can be seen by comparison, under the condition of the same feeding amount of main raw materials, the yield of the final product of Py-MAA-Val-Cit-PAB-DX8951 prepared by the one-pot method is 37.1%; the final product yield of the Py-MAA-Val-Cit-PAB-DX8951 prepared by the two-step method is 23.74%, and by comparison, the final product yield of the Py-MAA-Val-Cit-PAB-DX8951 prepared by the one-pot method is far higher than that of the Py-MAA-Val-Cit-PAB-DX8951 prepared by the two-step method.
Example 4 preparation of Mc-Val-Cit-PAB-MMAD
(1) Preparation by one-pot method
DMF (4 mL), MC-Val-Cit-PAB-OH (200mg, 1.0eq.) were added to a reaction flask, and after stirring and dissolution, bis (p-nitrophenyl) carbonate (NPC, 116mg, 1.1eq.) and DIPEA (45mg, 1.0eq.) were added, and after 24. + -. 2 ℃ reaction for 18 hours, 1-hydroxybenzotriazole (HoBt, 47mg, 1.0eq.) was added, MMAD (242mg, 0.9eq.), pyridine (304. Mu.L) and DIPEA (90mg, 2.0eq.) were added, and after 24. + -. 2 ℃ reaction was continued for 48 hours, spin-dried, and prepared by HPLC to obtain MC-Val-Cit-PAB-MMAD (228 mg), yield: 47.7%, purity: 99 percent.
(2) "two step" preparation
The first step is as follows: preparation of MC-Val-Cit-PAB- (4-nitrophenyl) carbonate
DMF (4 mL), MC-Val-Cit-PAB-OH (200mg, 1.0 eq.) were added to a reaction flask, and after stirring and dissolution, bis (p-nitrophenyl) carbonate (NPC, 116mg, 1.1eq.) and DIPEA (45mg, 1.0 eq.) were added. Reacting for 18 hours at the temperature of 24 +/-2 ℃, adding ethyl acetate (6 mL), dropwise adding petroleum ether (12 mL) after 20 minutes, continuously stirring for 10min, and filtering; then washed three times each with ethyl acetate and petroleum ether. Spin-drying to obtain MC-Val-Cit-PAB- (4-nitrophenyl) carbonate (177 mg), yield: 68.6%, purity: 90 percent.
The second step is that: preparation of MC-Val-Cit-PAB-MMAD
The MC-Val-Cit-PAB- (4-nitrophenyl) carbonate (170 mg), 1-hydroxybenzotriazole (HoBt, 31mg, 1.0eq.), DMF (4 mL), MMAD (160mg, 0.9eq.), pyridine (200. Mu.L) and DIPEA (59mg, 2.0eq) were added to the reaction flask, reacted at 24 + -2 ℃ for 48 hours, spin-dried, and prepared by HPLC to obtain MC-Val-Cit-PAB-MMAD (156 mg), yield: 49.4%, purity: 99 percent.
As can be seen by comparison, under the condition of the same feeding amount of the main raw materials, the yield of the final product of the MC-Val-Cit-PAB-MMAD prepared by the one-pot method is 47.7%; the final product yield of the MC-Val-Cit-PAB-MMAD prepared by the two-step method is 33.88 percent, and compared with the one-pot method, the final product yield of the MC-Val-Cit-PAB-MMAD prepared by the one-pot method is far higher than the final product yield of the MC-Val-Cit-PAB-MMAD prepared by the two-step method.
Example 5
Preparation of Mc-Val-Cit-PAB-DX8951
(1) Preparation by one-pot method
DMF (4 mL), MC-Val-Cit-PAB-OH (200mg, 1.0 eq.) were added to a reaction flask, and after stirring and dissolution, bis (p-nitrophenyl) carbonate (NPC, 116mg, 1.1eq.) and DIPEA (45mg, 1.0 eq.) were added. After reacting at 24. + -. 2 ℃ for 18 hours, 1-hydroxybenzotriazole (HoBt, 47mg, 1.0eq.), DMF (4 mL), DX8951 (167mg, 0.9eq.), pyridine (304. Mu.L) and DIPEA (135mg, 3.0eq.) were added, and reacting at 24. + -. 2 ℃ for 48 hours, followed by spin-drying, and HPLC was used to prepare MC-Val-Cit-PAB-DX8951 (139 mg), yielding: 38.4%, purity: 97 percent.
(2) "two step" preparation
The first step is as follows: preparation of MC-Val-Cit-PAB- (4-nitrophenyl) carbonate
DMF (4 mL), MC-Val-Cit-PAB-OH (200mg, 1.0 eq.) were added to a reaction flask, and after stirring and dissolution, bis (p-nitrophenyl) carbonate (NPC, 116mg, 1.1eq.) and DIPEA (45mg, 1.0 eq.) were added. Reacting for 18 hours at the temperature of 24 +/-2 ℃, adding ethyl acetate (6 mL), dropwise adding petroleum ether (12 mL) after 20 minutes, continuously stirring for 10min, and filtering; then washed three times with ethyl acetate and petroleum ether respectively and spin-dried to obtain MC-Val-Cit-PAB- (4-nitrophenyl) carbonate (174 mg), yield: 67.4%, purity: 90 percent.
The second step: preparation of MC-Val-Cit-PAB-DX8951
MC-Val-Cit-PAB- (4-nitrophenyl) carbonate (170 mg), 1-hydroxybenzotriazole (HoBt, 31mg, 1.0eq.), DMF (4 mL), DX8951 (110mg, 0.9eq.), pyridine (200. Mu.L) and DIPEA (89mg, 3.0eq.) were added into a reaction flask, reacted at 24 + -2 ℃ for 48 hours, spin-dried, and prepared by HPLC to obtain MC-Val-Cit-PAB-DX8951 (85 mg), yield: 35.6%, purity: 97 percent.
Compared with the prior art, the MC-Val-Cit-PAB-DX8951 prepared by the one-pot method has the final product yield of 38.4 percent under the condition of the same feeding amount of the main raw materials; the yield of the final product of the MC-Val-Cit-PAB-DX8951 prepared by the two-step method is 23.99 percent, and compared with the yield of the final product of the MC-Val-Cit-PAB-DX8951 prepared by the one-pot method, the yield of the final product of the MC-Val-Cit-PAB-DX8951 prepared by the two-step method is far higher than the yield of the final product of the MC-Val-Cit-PAB-DX8951 prepared by the two-step method.
Example 6
Preparation of Mc-Val-Cit-PAB-MMAE
(1) Preparation by one-pot method
MC-Val-Cit-PAB-OH (CAS No.: 159857-80-4) (200mg, 1.0 eq) and DMF (4 mL) were added in this order to a three-necked round-bottomed flask, and after stirring at room temperature until dissolved, NPC (117mg, 1.1 eq) and DIPEA (45mg, 1.0 eq) were added and stirred at 24. + -. 2 ℃ for 18 hours. Then DIPEA (90mg, 2.0eq), pyridine (0.3mL, V pyridine/VDIPEA = 2.5), hoBt (47mg, 1.0eq) and MMAE (251mg, 1.0eq) are sequentially added into the reaction liquid, the reaction is continuously carried out for 48 hours at the temperature of 24 +/-2 ℃, and a product MC-Val-Cit-PAB-MMAE (214 mg) is obtained through liquid phase purification, wherein the purity is 99.45%, and the yield is 46.5% [ a calculation formula ]: yield = MC-Val-Cit-PAB-MMAE yield ÷ (MC-Val-Cit-PAB-OH dosage ÷ 572.7 × 1316.6) × 100%).
(2) "two step" preparation
The first step is as follows: preparation of MC-Val-Cit-PAB- (4-nitrophenyl) carbonate
MC-Val-Cit-PAB-OH (200mg, 1.0eq) and DMF (4 mL) were added in this order to a three-necked round-bottomed flask, and after stirring at room temperature until dissolved, NPC (117mg, 1.1eq), DIPEA (45mg, 1.0eq) and stirring at 24. + -. 2 ℃ for 18 hours were added. Ethyl acetate (12 mL) and petroleum ether (18 mL) were added, stirred, filtered, and spun dry to give the crude product. The crude product was added to acetic acid (2 mL) and methanol (0.3 mL), dissolved with stirring, and then purified water (6 mL) was added dropwise and filtered. The solid obtained was spin-dried on a rotary evaporator to give MC-Val-Cit-PAB- (4-nitrophenyl) carbonate (185 mg), yield: 71.8%, purity: 97 percent.
The second step is that: preparation of MC-Val-Cit-PAB-MMAE
MC-Val-Cit-PAB- (4-nitrophenyl) carbonate (185 mg), DIPEA (64mg, 2.0eq), pyridine (0.2mL, V pyridine/VDIPEA = 2.5), hoBt (34mg, 1.0eq) and MMAE (180mg, 1.0eq) are sequentially added into a reaction bottle, and the reaction is continued for 48 hours at 24 +/-2 ℃, so that a product MC-Val-Cit-PAB-MMAE (115 mg) is obtained by liquid phase purification, wherein the yield is 34.8% and the purity is 99%.
As can be seen by comparison, under the condition of the same feeding amount of the main raw materials, the yield of the final product of the MC-Val-Cit-PAB-MMAE prepared by the one-pot method is 46.5 percent; the yield of the final product of the MC-Val-Cit-PAB-MMAE prepared by the two-step method is 24.99 percent, and through comparison, the yield of the final product of the MC-Val-Cit-PAB-MMAE prepared by the one-pot method is far higher than that of the MC-Val-Cit-PAB-MMAE prepared by the two-step method.
TABLE 1 comparison of the yields of the final products in the "one-pot" and "two-stage" processes
Note: the absolute value of the yield increase is = 'one-pot method' total yield- 'two-step method' total yield;
yield increase relative value = ("one-pot" total yield- "two-step" total yield)/"two-step" total yield;
compared with the 'one-pot' and 'two-step' preparation processes of Py-MAA-Val-Cib-MMAE, py-MAA-Val-Cit-PAB-MMAD, py-MAA-Val-Cit-PAB-DX8951, mc-Val-Cit-Cib-PAB-MMAD, mc-Val-Cit-PAB-MMAE, the yield of the final product is obviously improved, the product cost is greatly reduced based on the fact that the price of materials such as MMAE and DX8951 is very expensive, and therefore the 'one-pot' preparation process has obvious progress and unexpected technical effects.
In addition, the one-step preparation process provided by the invention only needs to be carried out in the same reaction system, while the traditional preparation process needs to be carried out in two reaction systems (namely, py-MAA-Val-Cit-PAB-PNP is extracted and then put into reaction), so that organic reagents (ethyl acetate, petroleum ether and the like), equipment (for concentration) and a new reaction container (for the next reaction) are needed, a large amount of work is undoubtedly brought to the synthesis process, and in addition, the use and recovery of the organic reagents need more manpower and also bring certain influence on the environment, so the one-step preparation process provided by the invention is more suitable for large-scale production and use.
The invention has been illustrated by various specific embodiments. However, it is understood by those skilled in the art that the present invention is not limited to the respective embodiments, and that various changes or modifications may be made by those skilled in the art within the scope of the present invention, and various technical features mentioned throughout the present specification may be combined with each other without departing from the spirit and scope of the present invention. Such modifications and variations are intended to be within the scope of the present invention.
Claims (9)
1. A process for the preparation of an antibody drug conjugate intermediate comprising a linker moiety and a drug moiety, said antibody drug conjugate intermediate being MC-Val-Cit-PAB-D, wherein MC-Val-Cit-PAB is the linker moiety, and wherein D represents the linked drug moiety, said linked drug moiety comprising a free amino group, comprising the following reaction scheme:
the preparation process is a one-pot method, the two reactions are carried out in one system, and the process comprises the following steps: the MC-Val-Cit-PAB-OH and di (p-nitrophenyl) carbonate are in contact reaction in the presence of organic base, after the reaction is finished, the organic base is added into a reaction system, and a triazole catalyst and the medicine part D are added for reaction, wherein the triazole catalyst is selected from 1-hydroxy benzotriazole, 1-hydroxy-7-azo benzotriazole or 1-hydroxy-1H-1, 2, 3-triazole-4-ethyl carboxylate.
2. The process of claim 1, wherein: and (2) carrying out contact reaction on MC-Val-Cit-PAB-OH and di (p-nitrophenyl) carbonate in the presence of organic base, after the reaction is finished, additionally adding the organic base into a reaction system, and adding 1-hydroxybenzotriazole and the medicine part D for reaction.
3. The process according to claim 1 or 2, wherein: the drug part D is an auristatin cytotoxic agent, an amphenicol cytotoxic agent, an anthracycline cytotoxic agent or a puromycin cytotoxic agent and a camptothecin analogue.
4. The process of claim 3, wherein: the auristatin cytotoxic agent is MMAE, MMAF and MMAD; the amrinomycin cytotoxic agent is amrinomycin; the anthracycline cytotoxic agent is daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone; the puromycin cytotoxic agent is puromycin toxin; the camptothecin analogue is DX8951.
5. The process of claim 4, wherein: the structure of the MC-Val-Cit-PAB-D is shown as (1-11):
6. the preparation process of any one of the preceding claims, wherein the organic base 1 and the organic base 2 are each independently one or more of N, N diisopropylethylamine, triethylamine and pyridine.
7. The preparation process of claim 6, wherein the organic base 1 and the organic base 2 are independently one or two of N, N diisopropylethylamine and pyridine.
8. Use of the process according to any one of claims 1 to 7 for the preparation of an antitumor medicament.
9. Use of the process of any one of claims 1 to 7 for the preparation of an antibody drug conjugate.
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