CN117964685A - Antibody drug conjugate, intermediate thereof, preparation method and application - Google Patents

Abstract

The invention relates to the technical field of biological medicines, in particular to an antibody drug conjugate intermediate with structures shown in formulas I, II and III, and a preparation method and application thereof. The invention provides a preparation method of an antibody drug conjugate intermediate and application thereof in preparing an anti-tumor drug. The preparation method has the advantages of mild reaction conditions, simple steps, convenient post-treatment and low cost, and is suitable for industrial production.

Description

Antibody drug conjugate, intermediate thereof, preparation method and application

Technical Field

The invention relates to the field of biological medicine, in particular to an antibody drug conjugate, an intermediate thereof, a preparation method and application.

Background

The antibody drug conjugate (Antibody drug conjugate, ADC) takes an antibody as a carrier, carries cytotoxic molecules into tumor cells in a covalent connection mode, and then kills the tumor cells by using small molecules dissociated from the conjugate in a special tumor environment.

Camptothecin (CPT) analogs and derivatives are small molecules with cytotoxicity that have an antitumor effect through inhibition of topoisomerase I (Topoisomerase, topo I), which shows remarkable activity against many tumor types. Irinotecan (Exatecan) is a water-soluble CPT derivative, has strong inhibitory activity on Topo I, and shows excellent anti-tumor effect on various tumor cells in vitro.

The linker design plays a key role in regulating the stability of the ADC in the systemic circulation and the efficiency of payload release in tumors, it has now been clearly demonstrated from at least two aspects that the linker is a key element of the overall ADC design: first, the linker may be structurally modified to optimize the therapeutic index (Therapeutic index, TI); second, the linker must ensure that the correct amount of cytotoxin is delivered to the correct cells. The presence of different structural elements of the linker, including the site of attachment, the release moiety, the solubilising moiety, and the appropriate design of these different units, can affect the pharmacokinetic, therapeutic and toxic profile of the ADC, and the ideal linker should remain stable in the circulatory system and release the cytotoxic payload in the tumour.

Therefore, the development of new cytotoxic small molecules and linkers is particularly important in ADC drug development.

Disclosure of Invention

The invention aims to provide an antibody drug conjugate intermediate;

a second object of the present invention is to provide a method for preparing an antibody drug conjugate intermediate;

A third object of the present invention is to provide a linker compound;

the fourth object of the invention is to provide the application of the antibody drug conjugate intermediate and the linker compound in preparing antitumor drugs.

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

An antibody drug conjugate intermediate has a structure shown in formulas I, II and III:

The compound of the formula II is used as linker in ADC drugs, the carboxylic end of the linker is connected with toxin drug molecules, for example, the carboxylic end of the compound of the formula II is connected with irinotecan to obtain the compound of the formula III. Of course, the carboxylic acid end of the compounds of formula II may also be linked to other toxin molecules.

The compound of the formula II is used as a linker in ADC drugs, and the maleimide end of the compound can be connected with an antibody, such as Herceptin.

The preparation method of the compound I comprises the following reaction route:

the specific technical scheme of the preparation method of the antibody drug conjugate intermediate compound I is as follows: mixing the Sha Tikang, 3-hydroxy propionic acid and the organic solvent, cooling to below 10 ℃, adding DEPC and DIEA, and heating to room temperature for reaction for 1-3 h to obtain the compound I. Wherein the mol ratio of the irinotecan to the 3-hydroxy propionic acid is 1:1-1:5; the molar ratio of DEPC to irinotecan was 1.5:1 and the molar ratio of DIEA to irinotecan was 3:1.

The compound of formula II is obtained by adopting a solid phase synthesis method, and the reaction route is as follows:

wherein the glycine analog is Fmoc-Gly-OH or Fmoc-Gly-Gly-OH, and Fmoc is fluorenylmethoxycarbonyl.

Fmoc solid phase synthesis is adopted, and the coupling reagent adopts carbodiimide condensing agent, phosphorus positive ion condensing agent, urea positive ion condensing agent and other condensing agents. The condensing agent is any one or more of HOBt、HOAt、HOOBt、HOPyU、TBTU、HBPyU、HBPipU、HBMDU、HATU、HAPyU、HAMDU、TAPipU、HDTU、HPyOPfp、HPySPfp、HAPyTU、TOTU、HAPipU、BOP-Cl、FDP、FDPP、DEPBT、EEDQ、EDC.HCl、DCC、DIC.

The preparation method of the compound 5 comprises the following reaction route:

the preparation method of the compound III comprises the following reaction route:

The specific technical scheme of the preparation method of the antibody drug conjugate intermediate compound III is as follows: and mixing the compound of the formula II, the I Sha Tikang and the organic solvent, cooling to below 10 ℃, adding TBTU and DIEA, and reacting for 0.5-3 h at room temperature to obtain the compound III.

The linker used in the antibody conjugate intermediate III is a cleavable hydrophilic polypeptide linker, and can be cleaved by lysosome protease after entering tumor cells, so that the systemic toxicity can be limited while the systemic circulation stability is ensured.

According to the method for synthesizing the compound in the formula II in the solid phase, the hydroxyl end of the compound 5 is coupled to the solid phase resin, fmoc-Phe-OH, glycine analogue, 6-maleimide caproic acid or succinimidyl ester thereof are spliced in sequence, a linker fragment is synthesized rapidly and efficiently by using the solid phase synthesis method, purification is not needed in each step of solid phase synthesis, only the rest reagent/raw material is needed to be washed after the condensation is finished in each step, and finally the linker is cut from branches, so that the linker with higher purity can be obtained.

The invention provides a preparation method of an antibody coupling intermediate, which comprises the steps of preparing a linker by adopting solid-phase peptide synthesis, splicing the linker with the isaatide Kang Suge by adopting TBTU and DIEA condensing agents, and reacting for 1-3 h at room temperature to obtain a compound III, thereby reducing the production cost and improving the production efficiency.

The antibody drug conjugate has a targeting effect, and when reaching a tumor part, the linker breaks down to effectively release toxin molecules, namely a compound I, to kill tumor cells.

Drawings

FIG. 1 is a mass spectrum of compound I;

FIG. 2 is a nuclear magnetic resonance diagram of compound I;

FIG. 3 is a mass spectrum of compound II;

FIG. 4 is a nuclear magnetic resonance diagram of compound II;

FIG. 5 is a scheme for the preparation of Compound II;

FIG. 6 is a mass spectrum of compound III;

FIG. 7 is a nuclear magnetic resonance diagram of compound III.

Detailed description of the preferred embodiments

Unless otherwise indicated, the terms used herein have the following meanings:

DCM: dichloromethane (dichloromethane)

DMF: n, N-dimethylformamide

DEPC: cyanophosphoric acid diethyl ester

DIEA: n, N-diisopropylethylamine

DIC: n, N' -diisopropylcarbodiimide

TFA: trifluoroacetic acid

TLC: thin layer chromatography

TBTU: benzotriazol-N, N, N ', N' -tetramethylurea hexafluorophosphate borate

MeOH: methanol

EXAMPLE 1 Synthesis of Compounds of formula I

3-Hydroxy propionic acid (38 mg,1.2 eq), irinotecan (200 mg,1 eq) and DEPC (80 mu L,1.5 eq) are added into a 5mL reaction bottle, the temperature is reduced to below 10 ℃ by using 2mLDMF as a solvent, DIEA (175 mu L,3 eq) is added, the reaction is carried out for 1h at room temperature, after TLC monitoring, the reaction is finished, the reaction is concentrated to dryness, so that a crude product is obtained, the crude product is purified by a reverse-phase high-efficiency liquid phase to obtain 150mg of an off-white solid compound, and the mass spectrum and the nuclear magnetic resonance chart of a compound I of yield 83.9％.LC/MS(m/z):calcd for C₂₇H₂₆FN₃O₆,507.18;found 508.20[M+H]⁺,506.20[M-H]-, are shown as figures 1 and 2.

EXAMPLE 2 Synthesis of Compound 5

Synthesis of Compound 4

Into 250mL three-necked flask, compound 2 (10.00 g,1.0 eq), compound 3 (14.70 g,3.0 eq), 200mL tetrahydrofuran, potassium tert-butoxide (9.15 g,2.0 eq) were added, water and DCM were added after stirring at room temperature for 0.5h, the organic phase was collected, 100mL of water was added after concentrating the organic phase, pH was adjusted to 7-8 with sodium bicarbonate, fmoc-OSU (7.30 g,0.8 eq) was added in 100mL of ethylene glycol dimethyl ether solution, 50mL of tetrahydrofuran was added after stirring at room temperature, after TLC monitoring reaction was completed, ethyl acetate extraction was added after concentrating, washing with dilute hydrochloric acid, washing with sodium bicarbonate and concentrating, column chromatography was performed to obtain white solid-like compound 4,3.90g, yield 29.5％.LC/MS(m/z):calcd for C₂₈H₂₈N₂O₆,488.19;found 511.10[M+Na]⁺.

Synthesis of Compound 5

Adding compound 4 (3.90 g,1.0 eq), 50mL MeOH,0.4g palladium carbon, introducing hydrogen after nitrogen replacement, maintaining 4 atmosphere, reacting at room temperature for 2h, filtering after TLC monitoring reaction, concentrating to obtain off-white solid compound 5,1.50g, yield 47.2％.LC/MS(m/z):calcd for C₂₁H₂₂N₂O₆,398.15;found 421.15[M+Na]⁺.

EXAMPLE 3 Synthesis of Compounds of formula II

2-CTC Resin (4.76 g,1.9 eq) with 0.75mmol/g substitution was weighed into a 200mL solid phase reaction column, 50mL DCM was added, compound 5 (0.75 g,1 eq), DIEA (1.21 g,5 eq) and nitrogen sparged for 2h; 5mL of MeOH was added and the mixture was reacted for 1 hour. The resin was washed three times with DMF, fmoc protecting groups were removed with 20% piperidine/DMF for 20min and washed 5 times with DMF. Fmoc-Phe-OH (1.46 g,2 eq), HOBt (0.63 g,2.2 eq), DIC (0.52 g,2.2 eq) were weighed, dissolved in DMF, added to the reaction column, after 2h of reaction the resin was washed 3 times with DMF, fmoc protecting group was removed with 20% piperidine/DMF for 20min, washed 6 times with DMF and 3 times with DCM. Repeating the coupling operation, and coupling Fmoc-Gly-OH, fmoc-Gly-OH and 6-maleimide caproic acid sequentially according to the peptide sequence. After the reaction, the resin was contracted with methanol and dried in vacuo to obtain a peptide resin. Adding the obtained peptide resin into 70mL of DCM solution prepared with 1% TFA in advance, reacting for 2.0h at room temperature, filtering off the resin, adding pyridine to neutralize TFA, concentrating to remove the solvent, adding 100mL of methyl tertiary butyl ether for pulping, filtering, collecting the filter cake, and drying in vacuum to obtain white compound of formula II, wherein the mass spectrum and nuclear magnetic resonance diagram of the compound of formula II with yield 70.0％.LC/MS(m/z):calcd for C₂₉H₃₈N₆O₁₀,630.26;found 653.25[M+Na]⁺,629.20[M-H]^-. of 0.83g are shown in figures 3and 4, and the preparation flow chart of the compound of formula II is shown in figure 5.

EXAMPLE 4 Synthesis of Compound III

To a 25mL three-necked flask, the compound of formula II (0.80 g,1.2 eq), the compound 7 (0.60 g,1.00 eq), 8mL DMF was added, the temperature was lowered to 10℃or lower, TBTU (0.41 g,1.2 eq), DIEA (0.53 mL,3.0 eq) was added, the reaction was allowed to react at room temperature for 1h, after completion of TLC monitoring, the reaction solution was poured into water, DCM/MeOH mixed solvent was extracted 2 times, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give a crude product, which was subjected to column chromatography (DCM: meOH=200:1 to 10:1) to give a pale yellow solid compound III, 0.79g, the mass spectrum and nuclear magnetic resonance chart of the compound III of which yield 71.4％.LC/MS(m/z):calcd for C₅₃H₅₈FN₉O₁₃,1047.41;found 1048.35[M+H]⁺,1046.30.25[M-H]-, were shown in FIG. 6, 7.

It will be apparent to those skilled in the art from this disclosure that various other changes and modifications can be made which are within the scope of the invention as defined in the appended claims.

Claims (10)

1. An antibody drug conjugate intermediate, which is characterized in that the compound, tautomer, meso, racemate, enantiomer, diastereoisomer or mixture form and pharmaceutically acceptable salt of the structure shown in formula I, formula II or formula III:

2. the method of preparing an antibody drug conjugate intermediate of claim 1, wherein the compound of formula I is synthesized according to the following route:

3. The method of preparing an antibody drug conjugate intermediate of claim 2, wherein: mixing Sha Tikang with 3-hydroxy propionic acid and organic solvent, cooling to below 10deg.C, adding DEPC and DIEA, and reacting at room temperature for 1-3 hr to obtain the final product

A compound of formula I.

4. The method for preparing an antibody drug conjugate intermediate according to claim 1, wherein the compound represented by formula ii is obtained by a solid phase synthesis method, and the reaction route is as follows:

wherein the glycine analog is Fmoc-Gly-OH or Fmoc-Gly-Gly-OH, and Fmoc is fluorenylmethoxycarbonyl.

5. The method for preparing an antibody drug conjugate intermediate according to claim 4, wherein the compound 5, fmoc-Phe-OH, glycine analog, 6-maleimide caproic acid or succinimidyl ester thereof is sequentially coupled to a solid phase resin by Fmoc solid phase synthesis method, and then washed and dried, and then added with a cleavage reagent for cleavage reaction to obtain the compound shown in formula II.

6. The method of preparing an antibody drug conjugate intermediate of claim 4, wherein the synthetic route of compound 5 is as follows:

7. The method of preparing an antibody drug conjugate intermediate of claim 1, wherein the method of preparing the compound of formula iii comprises: and mixing the compound shown in the formula II, the Sha Tikang and the organic solvent, cooling to below 10 ℃, adding TBTU and DIEA, and heating to room temperature to react for 0.5-3 h to obtain the compound shown in the formula III.

8. An antibody drug conjugate characterized by having the structure:

ab is an antibody.

9. Use of an antibody drug conjugate intermediate, tautomer, meso, racemate, enantiomer, diastereomer or a mixture thereof according to claim 1 or a pharmaceutically acceptable salt thereof or an antibody drug conjugate according to claim 8 for the preparation of a medicament for the prevention and/or treatment of tumors.

10. The use of claim 9, wherein the tumor comprises gastric cancer, pancreatic cancer, breast cancer, colon cancer.