Synthesis of a dual payload linker and its use in conjugated drugs

Abstract

The application relates to the field of medicines, and particularly discloses a preparation method and application of a double-load linker. The double-load linker takes glycerol as a mother nucleus, one hydroxyl group of the glycerol is connected with N-long-chain maleimide, and the other two hydroxyl groups are connected with two loads, so as to construct a novel double-load linker. The linker can realize the connection of two effect molecules through click chemistry or biological orthogonal reaction and the like; the maleimide end is connected with a targeting ligand, so as to construct a coupling drug of the double-load effect molecule.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, specifically to a method for preparing a dual-load linker with glycerol as the core and maleimide as the linker, and its application in drug coupling. Background Technology

[0002] Drug conjugates typically achieve their therapeutic or clinical purpose through a targeting ligand and effector molecules with different functions. The product mainly consists of three components: the targeting molecule (targeting ligand), the linker, and the effector molecule (cytotoxic, E3 ligase, oligonucleotide, etc.). Drug conjugate technology encompasses a variety of therapies, providing more innovative options for the treatment of diseases such as cancer, rare diseases, and hematological disorders.

[0003] Drug conjugates have formed many sub-sectors, such as antibody-drug conjugates (ADCs), radionuclide conjugates (RDCs), small molecule conjugates (SMDCs), peptide conjugates (PDCs), antibody-immunostimulatory conjugates (ISACs), antibody fragment conjugates (FDCs), antibody-cell conjugates (ACCs), virus-like drug conjugates (VDCs), antibody-oligonucleotide conjugates (AOCs), nucleic acid aptamer conjugates (ApDCs), antibody-biopolymer conjugates (ABCs), antibody degradation conjugates (ADeCs), prodrug conjugates (Pro-DCs), and protein degradation-targeting chimeras (PROTACs). In particular, antibody-drug conjugates (ADCs) have attracted widespread attention due to their clinical results and potential commercial value.

[0004] In drug-coupled compounds (ADCs), the linker is the molecular part that forms the connection between the target ligand and the effector molecule. It plays a crucial role in regulating the stability of the ADC in systemic circulation and the effective payload release efficiency at the target site, thus affecting pharmacokinetics (PK), efficacy, and toxicity characteristics. From a structural design perspective, the linker can be divided into three parts: the linker head at the target ligand end, the linker head at the effector molecule end, and the main body of the linker. The connection between maleimide and the target ligand is a commonly used linker, especially in ADCs where nucleophilic groups, such as the thiol group on cysteine ​​released during reduction reactions, form stable thioether bonds, achieving covalent bonding between the target ligand and the linker.

[0005] To improve the pharmaceutical properties of conjugated drugs, various approaches have been developed to enhance and control the loading capacity of linked drugs. Linkers carry sites for multiple effector molecules, providing a way to directionally and quantitatively control the effective loading capacity of conjugated drugs.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a novel dual-load linker. The maleimide linker can undergo a Michael addition reaction with nucleophilic groups (such as thiol groups) of a target ligand (such as an antibody), achieving coupling with the target ligand. Using glycerol as the linker's core, a multi-hydroxyl structure is combined with a linker at the effector molecule end to construct a dual-load linker, as shown in the following structural formula: , ; In the formula: W is the linker at the end of the effect molecule, including but not limited to dibenzocyclooctyne (DBCO), bicyclo[6,1,0]nonyne (BCN), azide, 1,2,4,5-tetraazine, transcyclooctene (TCO) and other cyclic alkenes; Y and Z are alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkoxy, alkoxyalkyl, polydiol, aminoacyl, ester, aromatic, acyl, diacyl, amino, peptide, phosphate ester, etc.

[0008] Preferably, the linker at the effector molecule end is dibenzocyclooctyne (DBCO), constructing a dual-loaded linker with the following structural formula: , ; In the formula: X and Y are alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkoxy, alkoxyalkyl, polydiol, aminoacyl, ester, aromatic, carbonyl, amino acid, peptide, phosphate ester, etc.

[0009] Preferably, DBCO is bonded to glycerol using succinyl, as shown in the following structural formula: , ; In the formula: Y represents alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkoxy, alkoxyalkyl, polydiol, aminoacyl, ester, aromatic, carbonyl, amino acid, peptide, phosphate ester, etc.

[0010] Preferably, maleimide and glycerol are linked via acyl chains of polyethylene glycol, as shown in the following structural formula: , ; In the formula, the degree of polymerization n is 1-30, and the water solubility of the coupled drug can be adjusted by changing the degree of polymerization n.

[0011] The technical solution for achieving the objective of this invention is as follows.

[0012] This invention provides a dual-load linker with a maleimide linker, using glycerol as the parent core. The three hydroxyl groups of the parent core are respectively linked to the maleimide and the linker of the load effect molecule. The linker at the effect molecule end can be selected from commonly used biological orthogonal reaction reagents such as dibenzocyclooctyne (DBCO), bicyclo[6,1,0]nonyne (BCN), azide, 1,2,4,5-tetraazine, and transcyclooctene (TCO).

[0013] The present invention also provides a method for synthesizing a dual-loaded linker with a maleimide linker, the method comprising linking glycerol with two orthogonal reaction reagents such as DBCO through an esterification reaction, and linking another hydroxyl group with an N-long chain of maleimide through a substitution reaction or an esterification reaction, thereby synthesizing a dual-loaded linker.

[0014] Preferably, the linker for the dual-loaded DBCO is synthesized by means of an esterification reaction in which one hydroxyl group of glycerol is linked to maleimide, and glycerol and maleimide are linked by a polyethylene glycol acyl group. The degree of polymerization n of polyethylene glycol is used to adjust the hydrophilicity of the linker.

[0015] This invention also provides a maleimide-based dual-load DBCO linker and an effect molecule containing an azide group, including but not limited to cytotoxic drugs, radioactive elements, peptides, ubiquitin, fluorescent molecules, and nucleic acid molecules, which are coupled by click chemistry. The analysis results show that the two effect molecules are successfully bonded to the linker proposed in this invention.

[0016] This invention also provides a dual-load linker with a maleimide linker, which links a maleimide linker to a nucleophilic group of a target ligand, such as an antibody. Subsequently, the dual-load DBCO is coupled to an effector molecule containing an azide group, including but not limited to cytotoxic drugs, radioactive elements, peptides, ubiquitin, fluorescent molecules, and nucleic acid molecules, via click chemistry. Analytical results show that one target ligand is linked to two effector molecules through the linker designed in this invention. Detailed Implementation

[0017] As used in this invention, commonly used abbreviations have their conventional meanings in the art.

[0018] The presence of a maleimide linker indicates the presence of a maleimide group.

[0019] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0020] In this invention, the substances corresponding to the abbreviations are as follows: Ab: Antibody; ADC: Antibody-drug conjugate; RDC: Radionuclide conjugate; PDC: Peptide conjugate; AOC: Antibody oligonucleotide conjugate; ApDC: Nucleic acid aptamer conjugate; PROTAC: Protein degradation-targeting chimera; PS: Cross-linked polystyrene; DBCO: Dibenzocyclooctylene; DBCO acid: Diphenylcyclooctylene-carboxylic acid; DCC: Dicyclohexylcarbodiimide; EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; DMAP : 4-Dimethylaminopyridine; DCM: dichloromethane; DMF: N,N-dimethylformamide; DMSO: dimethyl sulfoxide; DMTr: 4,4'-dimethoxytribenzoyl; OCE: phosphorous amide; U: uracil or uracil nucleotide; Ume: O2'-methyluracil nucleotide; THF: tetrahydrofuran; Tris: tris(hydroxymethyl)aminomethane hydrochloride; EDTA: ethylenediaminetetraacetic acid; TCEP: tris(2-carboxyethyl)phosphine; HPLC: high performance liquid chromatography. Attached Figure Description

[0021] Figure 1 The reaction equations for the linker and effector molecules; Figure 2 The structure of the nucleic acid molecule is UmeUmeUmeUme-(OCH2CH2)3-N3; Figure 3 This is the reaction equation between the linker and the nucleic acid molecule.

[0022] Example 1:

[0023] 0.553 g glycerol (6 mmol, 1.0 eq.), 2.600 g DCC (12.6 mmol, 2.1 eq.), and 0.220 g DMAP (1.8 mmol, 0.3 eq.) were dissolved in 60 mL of dry dichloromethane. Under nitrogen protection and at room temperature, a solution of 3.664 g DBCO acid (12 mmol, 2.0 eq.) dissolved in 60 mL of dry dichloromethane was slowly added dropwise. After the addition was complete, the reaction was continued for more than 12 hours. 100 mL of saturated sodium bicarbonate aqueous solution was added, and the organic phase was separated. The crude product was extracted twice with 50 mL of dichloromethane from the aqueous phase. The organic phases were combined, dried over sodium sulfate, concentrated, and purified by silica gel chromatography to obtain 1.161 g of target product A-1 (yield 29%, purity 98.4%) and 1.483 g of B-1 (yield 37%, purity 98.1%).

[0024] Example 2:

[0025] 0.667 g A-1 (1.0 mmol, 1.0 eq.), 0.380 g maleimide-tetraethylene glycol-carboxylic acid (1.1 mmol, 1.1 eq.), 0.227 g DCC (1.1 mmol, 1.1 eq.), and 0.134 g DMAP (1.1 mmol, 1.1 eq.) were dissolved in 30 mL of dry dichloromethane and reacted under nitrogen protection at room temperature for at least 12 hours. 50 mL of saturated sodium bicarbonate aqueous solution was added, and the organic phase was separated. The aqueous phase was extracted twice with 50 mL of dichloromethane to obtain the crude product. The combined organic phases were dried over sodium sulfate and concentrated. The product was then purified by silica gel chromatography to obtain 0.756 g of the target product A-2 (76% yield, 98.2% purity).

[0026] Example 3:

[0027] 0.667 g B-1 (1.0 mmol, 1.0 eq.), 0.380 g maleimide-tetraethylene glycol-carboxylic acid (1.1 mmol, 1.1 eq.), 0.227 g DCC (1.1 mmol, 1.1 eq.), and 0.134 g DMAP (1.1 mmol, 1.1 eq.) were dissolved in 30 mL of dry dichloromethane and reacted under nitrogen protection at room temperature for at least 12 hours. 50 mL of saturated sodium bicarbonate aqueous solution was added, and the organic phase was separated. The aqueous phase was extracted twice with 50 mL of dichloromethane to obtain the crude product. The combined organic phases were dried over sodium sulfate and concentrated. The product was then purified by silica gel chromatography to obtain 0.677 g of the target product B-2 (68% yield, 98.2% purity).

[0028] Furthermore, similar synthetic procedures are applicable to the preparation of the following linkers: , ; Wherein: W is the linker at the end of the effect molecule, including but not limited to dibenzocyclooctyne (DBCO), bicyclo[6,1,0]nonyne (BCN), azide, 1,2,4,5-tetraazine, transcyclooctene (TCO) and other cyclic alkenes; Y and Z are alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkoxy, alkoxyalkyl, polydiol, aminoacyl, ester, aromatic, acyl, diacyl, amino, peptide, phosphate ester, etc.

[0029] Example 4:

[0030] Please refer to the appendix for the reaction of linker A-2 with effector molecules. Figure 1 .

[0031] Under nitrogen protection, 20 mg A-2 (0.02 mmol, 1.0 eq.) and 23 mg tetramethylrhodamine-azide (CAS No. 1192590-89-8, 0.044 mmol, 2.2 eq.) were dissolved in 1 mL THF and reacted for more than 10 hours. The solvent was removed under reduced pressure, and the main fraction obtained was separated by reversed-phase liquid chromatography and freeze-dried to obtain 25 mg of solid (yield 62%, HPLC purity 97.1%). Mass spectrometry analysis confirmed that it was the target product coupled with two tetramethylrhodamine molecules.

[0032] Please refer to the following instructions for the process of linking linkers to oligonucleotide molecules.

[0033] Example 5:

[0034] This embodiment describes an oligonucleotide molecule synthesized in a solid-phase manner. The molecular structural formulas of the nucleotide monomers and azide reagents used in the solid-phase synthesis are as follows: , , ; Monomer Ume was loaded onto cross-linked polystyrene (PS). O5'-DMTr was removed using a 3% dichloroacetic acid-toluene solution. After washing with acetonitrile, monomer O3'-OCE-Ume was pumped in for coupling. P(III) was oxidized to P(V) using a 0.05 mol / L iodine-water / pyridine solution, and the system was capped. The process of coupling, oxidation, and capping with monomer O3'-OCE-Ume was repeated twice. Finally, N3-PEG3-POCE was pumped in for coupling and oxidation.

[0035] After ammonolysis and deprotection, the loaded nucleic acid molecules were concentrated under reduced pressure and purified by HPLC. The main fraction was collected, concentrated, and freeze-dried to obtain the final product. Molecular analysis confirmed the target sequence as UmeUmeUmeUme-(OCH2CH2)3-N3 nucleic acid molecule (labeled Ume4). Please refer to the appendix for the structural formula. Figure 2 .

[0036] Example 6:

[0037] Please refer to the appendix for the reaction of linkers with nucleic acid molecules. Figure 3 .

[0038] Under nitrogen protection, 6 mg A-2 (0.006 mmol, 1.0 eq.) and 0.015 mmol Ume4 (2.5 eq.) were dissolved in 1 mL of water (buffer solution pH 6.8–7.6). The reaction was carried out at room temperature and in the dark for more than 12 hours. After dialysis, the solvent was removed under reduced pressure. The main fraction obtained was separated by reversed-phase liquid chromatography and freeze-dried to obtain 17 mg of solid (yield 53%, HPLC purity 93.8%). Mass spectrometry analysis confirmed that it was the target product conjugated with two nucleic acids, Ume4.

[0039] For the procedure of linking the target ligand and effector molecule with a linker, please refer to the following steps.

[0040] Example 7:

[0041] Take antibody Ab1, adjust the pH to approximately 7.5 with Tris-EDTA solution, and analyze the protein concentration to calculate the required antibody dosage. Add an appropriate amount of TCEP solution and react in a shaker at room temperature for at least 2 hours. Add at least 2 equivalents of DMSO solution containing the A-2 linker to the reduced antibody solution and react in a shaker at room temperature for at least 1 hour. After the reaction, add excess N-acetylcysteine ​​solution and mix in a shaker at room temperature for at least 1 hour. Purify the reaction mixture by ultrafiltration and centrifugation, and separate the fractions using reversed-phase HPLC to obtain the conjugate Ab1-A2, which consists of antibody Ab1 linked to one A2 linker. After replacing the storage solution, sterilize and remove enzymes, and store at 4°C.

[0042] Example 8:

[0043] An appropriate amount of antibody Ab1-A2 conjugate was dissolved in a buffer solution (pH 6.8-7.6), and an excess of tetramethylrhodamine-azide compound was added. The mixture was shaken at room temperature for at least 4 hours. After dialysis and desalting, the main fraction obtained by reversed-phase HPLC was analyzed and confirmed to be a conjugate of Ab1-A2 and two tetramethylrhodamine-azides.

[0044] Example 9:

[0045] An appropriate amount of antibody Ab1-A2 conjugate and excess Ume4 were dissolved in a buffer solution (pH 6.8-7.6) and incubated on a shaker at room temperature for at least 6 hours. After dialysis and desalting, the main fraction obtained by reversed-phase HPLC was analyzed and confirmed to be a conjugate of Ab1-A2 and two nucleic acid molecules.

[0046] Furthermore, similar reaction procedures to those between effector molecules, antibodies, etc., are applicable to linker B-2.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. The synthesis of a dual payload linker and its use in coupling drugs, characterized in that, The structure of the dual-load connector includes the structures shown in Formula I and Formula II: … Formula I; … Formula II; Wherein: W is the linker at the end of the effect molecule, including but not limited to dibenzocyclooctyne (DBCO), bicyclo[6,1,0]nonyne (BCN), azide, 1,2,4,5-tetraazine, transcyclooctene (TCO) and other cyclic alkenes; Y and Z are alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkoxy, alkoxyalkyl, polydiol, aminoacyl, ester, aromatic, acyl, diacyl, amino, peptide, phosphate ester, etc.

2. The synthesis of a dual payload linker according to claim 1 and its use in coupling drugs, characterized by, The linker uses glycerol as the parent core, with two hydroxyl groups of glycerol bonded to the linker heads of the two loads, and the other hydroxyl group linked to the N-long chain of maleimide, thus constructing the dual-load linkers shown in Formula I and Formula II.

3. The synthesis of a dual-load linker according to claim 1 and its application in drug coupling, characterized in that, The nucleophilic group in the targeting ligand undergoes nucleophilic addition with the unsaturated double bond of the maleimide in the linker, thereby achieving the binding of the linker and the targeting ligand proposed in this invention.

4. The synthesis of a dual-load linker according to any one of claims 1 to 3 and its application in drug coupling, characterized in that, The maleimide in the linker binds to the targeting ligand, and the reaction between the two hydroxyl-bound linker and the two effector molecules is achieved through the linker proposed in this invention, thus realizing the dual loading of the targeting ligand.

5. The synthesis of a dual-load linker according to claim 1 and its application in drug coupling, characterized in that, The two hydroxyl groups in the glycerol of the linker are bonded to the two linkers through esterification or substitution reactions via appropriate spacer units, including but not limited to alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkoxy, alkoxyalkyl, polydiol, aminoacyl, ester, aromatic, acyl, amino, peptide, phosphate ester, etc.

6. The synthesis of a dual payload linker according to claim 1 and its use in coupling drugs, characterized by, The two hydroxyl groups in the glycerol of the linker are combined with linkers such as dibenzocyclooctyne (DBCO), bicyclic [6,1,0]nonyne (BCN), azide, 1,2,4,5-tetraazine, and transcyclooctene (TCO) to construct linkers that can carry two effect molecules.

7. The synthesis of a dual payload linker according to claim 6 and its use in coupling drugs, characterized in that, The two linkers, which are bound by two hydroxyl groups in the glycerol of the linker, can be connected to two effector molecules through reactions such as click chemistry or biological orthogonal reactions, thus achieving the binding of two effector molecules by one linker.

8. The synthesis of a dual payload linker according to any one of claims 1 to 7 and its use in the coupling of drugs, characterized in that, The effector molecules linked by the dual-load linker include, but are not limited to, cytotoxic drugs, radioactive elements, peptides, ubiquitin, fluorescent molecules, and nucleic acid molecules.

9. The synthesis of a dual payload linker according to claim 1 and its use in coupling drugs, characterized by, The linker is applicable to any one or a combination of at least two of the following conjugate drugs: antibody-drug conjugate (ADC), radionuclide conjugate (RDC), small molecule conjugate (SMDC), peptide conjugate (PDC), antibody immunostimulatory conjugate (ISAC), antibody fragment conjugate (FDC), antibody cell conjugate (ACC), virus-like drug conjugate (VDC), antibody oligonucleotide conjugate (AOC), nucleic acid aptamer conjugate (ApDC), antibody biopolymer conjugate (ABC), antibody degradation conjugate (ADeC), prodrug conjugate (Pro-DC), and protein degradation targeting chimera (PROTAC).

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