Preparation methods and applications of pyridine-derived neighboring thiol covalent bridging reagents and their couplings

Abstract

This invention discloses a pyridine-derived neighboring thiol covalent bridging reagent and its conjugates, along with their preparation methods and applications. The pyridine-derived neighboring thiol covalent bridging reagent (i.e., disulfide bridging reagent) of this invention contains three different electrophilic functional groups. By rationally designing and adjusting the substitution positions and substituent groups, the reactivity of two of the electrophilic groups with the thiol group can be modulated, achieving intramolecular or intermolecular covalent bridging of polypeptides, proteins, antibodies, and their fragments as disulfides. Furthermore, the third electrophilic functional group further reacts with reactive active functional molecules to generate stable bioconjugates. The antibody-drug conjugates prepared by this invention exhibit significant inhibitory activity against cell lines expressing corresponding antigens, demonstrating potential clinical application value. Compared with existing technologies, the conjugate reagent of this invention exhibits higher reaction selectivity and product stability in the field of bioconjugation, providing new tools and methods for the modification and functionalization of biomolecules.

Description

Technical Field

[0001] This invention relates to the field of chemistry, and in particular to a pyridine-derived adjacent thiol covalent bridging reagent and its conjugates, along with their preparation methods and applications. This invention designs and synthesizes a series of novel disulfide covalent bridging reagents with multiple active functional groups, enabling the efficient conjugation of various functional molecules (such as fluorescent molecules and cytotoxic drugs) with biomolecules (such as peptides, proteins, antibodies, and their fragments) to form conjugates with specific functions. This invention also provides methods for preparing these conjugates and their applications in drug development, including but not limited to drugs for drug delivery, bioimaging, and disease treatment. Background Technology

[0002] Antibody-drug conjugates (ADCs) combine the tumor-targeting ability of antibodies with the cell-killing ability of potent cytotoxic drugs. Therefore, ADCs need to maintain stability in the bloodstream, accurately reach the therapeutic target, and release the cytotoxic drug. A significant reason for clinical failures is safety concerns; the activity of ADCs at the maximum tolerated dose remains insufficient. Limiting factors include the stability of ADCs in the bloodstream and the internalization efficiency of ADCs (Nat Rev Chem, 2022, 6, 844-861; Nat. Rev. Drug Discov., 2023, 22, 641-661). Therefore, minimizing the impact of conjugation chemistry on the affinity between antibodies and antigens and maintaining high stability in the bloodstream is one of the development goals of this research field. However, a conjugation method for antibodies and drugs that combines both uniformity and stability is still lacking.

[0003] In recent years, the development of protein site-selective modification methods has improved the consistency and stability of next-generation ADCs. These methods include enzyme-catalyzed N- and C-terminal modification methods, the introduction of biological orthogonal handles through genetic code expansion technology, site-specific introduction of cysteine ​​residues through genetic engineering technology, glycochemical modification methods, chemical or regioselective modification, and neighboring ligand-guided site-directed modification technology (Chem. Soc. Rev., 2021, 50, 1305-1353). However, these methods require antibody modification, selection of optimal coupling sites, low yields, and difficulty in isolating, purifying, and removing catalytic enzymes, thus increasing the manufacturing cost of ADCs.

[0004] Site-specific modification of natural antibodies can maintain antibody integrity, improve production efficiency, and reduce production costs. Currently, most FDA-approved antibody-drug conjugates (ADCs) are produced by modifying the cysteine ​​residues after disulfide bond reduction between antibody chains. This not only compromises antibody stability but also, due to coupling efficiency and steric hindrance, prevents the homogeneous and complete coupling of the eight reactive thiols, resulting in heterogeneous conjugates. Furthermore, while using maleimide or sulfadiazine derivatives as thiol-based reactive linkers improves coupling efficiency, it also potentially introduces unstable linkers. The thioether bonds generated in this way can undergo thiol exchange reactions with free thiols, leading to premature decomposition of the conjugate (Drug Discov Today Technol, 2018, 30, 27-34). These issues result in low serum stability and off-target effects, poor pharmacokinetics, and a narrow therapeutic window for ADCs.

[0005] Using dual-linker re-bridging of antibody interchain disulfide bonds is a recently developed novel conjugation method. This method not only improves antibody stability but also enables site-controllable formation of homogeneous conjugates (Chem. Soc. Rev., 2021, 50, 1305-1353). This method requires no antibody modification, is low-cost, and produces ADCs with a controllable DAR (drug-to-antibody ratio). Currently, bridging reagents used for re-bridging antibody interchain disulfide bonds include mono / disulfone reagents, next-generation maleimides (dibromomaleimides), dibromopyridazine diones, divinylpyrimidine derivatives, and C-Lock. TN (Dibromomethyl heterocyclic compounds), diethylenesulfonamides, and 1,3-dihaloacetones, etc. (Chem. Soc. Rev., 2021, 50, 1305-1353). Conjugates constructed from disulfone conjugates are relatively stable (US2006 / 0210526), ​​but have low reaction efficiency and produce heterogeneous products; disubstituted maleimide conjugates (CA2866699A1), although having high conjugation efficiency, still undergo reverse Michael reaction and thiol exchange reaction; conjugates of dibromopyridazine dione compounds are not easily hydrolyzed (EP4065978B1; WO2023 / 076848A1), but under high concentrations of thiol, exchange reaction occurs, causing the linker and drug conjugate to dissociate prematurely from the antibody; divinylpyrimidine derivatives (US20210308275A1), C-Lock... TM (Dibromomethyl heterocyclic) (WO2013173391A1) and piperazine-based divinylsulfonamide linkers (CN110251680A) have low coupling efficiency, and the double bond groups and benzyl bromide groups can react with TCEP. Therefore, these reactions require a large amount of coupling reagents, resulting in partial antibody precipitation, low coupling efficiency, and difficulty in controlling the uniformity of the product.

[0006] To improve the uniformity and stability of antibody-conjugates, there is an urgent need to develop more robust and efficient conjugation reagents.

[0007] Given the dynamic characteristics of the hinge region structure, prolonged reactions can easily lead to misaligned rebridging and incomplete bridging of dithiols. Therefore, on the one hand, the reaction rate between the linker and the reduced thiol should be accelerated to avoid excessively long reaction times; on the other hand, to enhance the stability of the linker bond, the coupling reagent must be rationally designed to reduce the reversibility of the reaction between the rebridging reagent and the thiol. Furthermore, by adjusting the coupling strategy, such as using a stepwise coupling method, a thiol-inert reactive group can be introduced first using a disulfide bond to bridge the linker, and then reacted with the linker and drug to form an antibody-drug conjugate. Although this increases the coupling steps, it significantly improves the reaction efficiency between the coupling reagent and the reduced thiol, while the mild reaction conditions help maintain the integrity of the antibody structure and its functional stability. For example, Novartis uses 1,3-dihaloacetone to first introduce a ketone group onto the antibody before further forming an ADC (WO2014083505A1). However, 1,3-dihaloacetone is easily hydrolyzed and has poor stability. Furthermore, these site-specific disulfide bond rebridging techniques not only maintain the integrity of the antibody structure but also enable the incorporation of various bioorthogonal groups onto the antibody, including ultra-fast and quantitative reactive groups such as azide, alkynyl, cyclooctyne, and tetrazine. These can be applied to antibody refunctionalization and coupling with various fluorescent groups, polyethylene glycol, peptides, proteins, protein ligands, nucleic acid aptamers, and small molecule toxins. Therefore, an ideal rebridging reagent should possess the ability to achieve site-specific labeling of antibodies under mild reaction conditions, while simultaneously endowing the antibody with multifunctional properties and ensuring the high stability of the formed coupling bonds.

[0008] In summary, there is still a need in the field for effective disulfide bridging reagents and novel coupling strategies that can react rapidly with reduced thiol groups, while the resulting coupling bonds exhibit high stability, do not disrupt the three-dimensional structure of the antibody during the coupling reaction, achieve site-specific coupling, and maintain or further improve the efficacy, stability, and safety properties of antibody-drug conjugates. Summary of the Invention

[0009] This invention aims to provide a novel class of disulfide covalent bridging reagents, their preparation methods, and applications. This reagent, through the design and synthesis of a series of novel disulfide covalent bridging reagents with multiple active functional groups, can efficiently covalently bridge peptides, proteins, antibodies, or antibody fragments containing at least one disulfide bond to form disulfide-bridged modified biomolecules. Specifically, the two leaving groups (X...) on the pyridine derivative corresponding to the disulfide covalent bridging reagent... 1 X 2First, it reacts with two adjacent thiol groups between or within the chain of the biomolecule to form a homogeneous conjugate; subsequently, the disulfide bridges the third functional group (X) on the pyridine ring of the modified biomolecule. 3 (e.g., aldehyde, cyano, alkynyl groups) are further reacted with functional conjugates containing active groups (including peptides, proteins, linkers, fluorescent molecules, drugs, etc.) to generate homogeneous functional bioconjugates. The conjugates generated by this invention exhibit high stability, and the antibody-drug conjugates (ADCs) prepared by this invention show significant inhibitory activity against cell lines expressing corresponding antigens, possessing potential clinical application value. Compared with existing technologies, this invention, through the design and synthesis of a series of pyridine derivatives with multiple active functional groups, can efficiently crosslink various functional molecules (such as fluorescent molecules, cytotoxic drugs, etc.) with biomolecules (such as peptides, proteins, antibodies, and their fragments) to form conjugates with specific functions. Furthermore, the disulfide covalent bridging reagent of this invention exhibits higher reaction selectivity and product stability in the field of bioconjugation, providing new tools and methods for the modification and functionalization of biomolecules. This invention also provides methods for preparing these conjugates and their applications in biomedicine, including but not limited to drug delivery, bioimaging, and disease treatment.

[0010] The first aspect of the present invention is to provide a disulfide bridging agent having a general structural formula as shown in Formulas IA and IB:

[0011]

[0012] in,

[0013] X in equations IA and IB 1 Whether the groups are the same or different, they are each independently selected from -SO2R1; where R1 is selected from aryl and halogen-substituted aryl groups.

[0014] X in equations IA and IB 2 Whether the groups are the same or different, they are each independently selected from -SO2R2, halogens, and halogen-substituted C1-C10 alkyl groups; wherein R2 is selected from aryl and halogen-substituted aryl groups.

[0015] X in equations IA and IB 3 They may be the same or different, and are each independently selected from -CHO, -CN, and -COR3; where -COR3 is selected from C1-C10 alkyl groups.

[0016] The reagent comprises a functionalized pyridine derivative containing three distinct electrophilic functional groups, of which two electrophilic functional groups (X...) 1 and X 2It can undergo a cascade of nucleophilic aromatic substitution reactions with two neighboring thiols on a biomolecule, thereby covalently bridging the two thiols to form a disulfide-bridged biomolecule. The third electrophilic functional group (X) 3 It can react with functional molecules containing active groups to form conjugates (i.e., biomolecule-load conjugates covalently bridged by disulfide).

[0017] As a preferred embodiment, X in formulas IA and IB 1 Whether the two are the same or different, they are each independently selected from -SO2R1; wherein R1 is selected from phenyl or halogen-substituted phenyl.

[0018] X in equations IA and IB 2 Whether the groups are the same or different, each is independently selected from -SO2R2, halogens, and halogen-substituted C1-C5 alkyl groups; wherein R2 is selected from phenyl groups and halogen-substituted phenyl groups.

[0019] X in equations IA and IB 3 They may be the same or different, and are each independently selected from -CHO, -CN, and -COR3; where -COR3 is selected from C1-C5 alkyl groups;

[0020] The halogen is selected from F, Cl, and Br;

[0021] Preferably,

[0022] X in equations IA and IB 1 Same or different, each independently selected from -SO2Ph; and / or,

[0023] X in equations IA and IB 2 They may be the same or different, each independently selected from -SO2Ph, -F, -Cl, -Br, -I; and / or,

[0024] X in equations IA and IB 3 Whether they are the same or different, they are each independently selected from -CHO, -CN, and -COCH3;

[0025] More preferably,

[0026] The disulfide multibridge reagent is selected from at least one of the following compounds:

[0027]

[0028] A second aspect of the present invention is to provide a method for preparing a disulfide bridging reagent as described in the first aspect of the present invention, wherein the method for preparing the compound represented by formula IB is selected from method one or method two;

[0029] Method 1 or Method 2 has the following general reaction formula:

[0030] Method 1

[0031]

[0032] Method 2

[0033]

[0034] Method 1 includes the following steps:

[0035] Step (1): Compound IB-1 is dissolved in an organic solvent, and oxalyl chloride is added under ice bath conditions for a primary reaction. After concentration under reduced pressure, methanol and triethylamine are added for a secondary reaction to obtain compound IB-2. The structural formulas of compounds IB-1 and IB-2 are as follows:

[0036] Step (2): IB-2 is dissolved in an organic solvent, and a reducing agent is added under ice bath conditions to carry out a reduction reaction to obtain compound IB-3; wherein, the structural formula of compound IB-3 is as follows:

[0037] Step (3): IB-3 is dissolved in an organic solvent, and an oxidant is added under ice bath conditions, followed by heating to carry out an oxidation reaction, yielding compound IB-4; the structural formula of compound IB-4 is as follows:

[0038] In step (4), compound IB-4 undergoes a secondary oxidation reaction in a solvent with the addition of oxidant II to obtain compound with the general formula IB-5-0,X. 3 =CHO;

[0039] Optionally, in step (4-1), compound IB-5-0 is reacted with compound R3MgBr in a solvent, wherein R3 reacts with X in claims 1-2. 3 =The R3 in COR3 corresponds to the same value, resulting in compound IB-5-1; the structural formula of compound IB-5-1 is as follows: R3 and X in the first aspect of the present invention 3 =The R3 in COR3 corresponds to the same value;

[0040] or,

[0041] Optionally, in step (4-2), compound IB-5-0 is reacted in methanol solvent with the addition of hydroxylamine hydrochloride in a single reaction to give the compound a solution in acetonitrile solvent. Triethylamine and dimethyl sulfoxide are then added, and oxalyl chloride is added dropwise under ice bath conditions to generate compound IB-5-2 in a second reaction. The structural formula of compound IB-5-2 is as follows:

[0042]

[0043] Step (5): One of the compounds IB-5 is reacted with trifluoroacetic anhydride in an organic solvent to obtain compound IB-6; compound IB-5 is selected from at least one of compounds IB-5-0, IB-5-1, and IB-5-2; the structural formula of compound IB-5 is as follows: Among them, X 3 In the first aspect of the invention, X 3 The corresponding ones in the text are the same;

[0044] The structural formula of compound IB-6 is as follows: In compound IB-6, X 3 In the first aspect of the invention, X 3 The corresponding ones in the text are the same;

[0045] Step (6): Compound IB-6 reacts with sodium benzenesulfinate in an organic solvent to obtain compound IB-7-0; the structural formula of compound IB-7-0 is as follows: In compound IB-7-0, X 3 In the first aspect of the invention, X 3 The corresponding ones in the text are the same;

[0046] Step (7): Compound IB-7 and an organic solvent were added to phosphorus tribromide under ice bath conditions, and then the temperature was increased to react and obtain compound IB-8, X. 2 =CH2Br; Compound IB-7 is selected from at least one of compounds IB-7-0 and IB-7-1; The structural formula of compound IB-7 is as follows:

[0047] In compound IB-7, X 1 In the first aspect of the invention, X 1 The corresponding values ​​in X are the same; 3 In the first aspect of the invention, X 3 The corresponding ones in the text are the same;

[0048] In compound IB-8, X 1 In the first aspect of the invention, X 1 The corresponding values ​​in X are the same; 3 In the first aspect of the invention, X 3 The corresponding ones in the text are the same;

[0049] Method 2:

[0050] Using IB-9 as the starting material, R4 as a halogen, X 3 It has an aldehyde or acyl group, dissolves in an organic solvent, and then reacts with sodium benzenesulfinate to give compound IB-10, X. 1X 2 Both are PhSO2;

[0051] In IB-9, both R4 atoms are identical, both being halogenated. X 3 In the first aspect of the invention, X 3 The corresponding ones in the text are the same;

[0052] IB-10 X 3 In the first aspect of the invention, X 3 The corresponding ones in the text are the same;

[0053] Preferably, in method one, in step (1),

[0054] The organic solvents are dichloromethane and N,N-dimethylformamide; the molar ratio of IB-1 to oxaloyl chloride is 1:2-4; the molar ratio of IB-1 to dichloromethane is 1 g:10-15 mL; the molar ratio of IB-1 to N,N-dimethylformamide is 1 g:10-15 μL; the primary reaction temperature is 0–4 °C; the primary reaction time is 4–6 hours; the molar ratio of IB-1 to methanol is 1 g:10-15 mL; the molar ratio of IB-1 to triethylamine is 1:1.5-2; the secondary reaction temperature is 20–25 °C; the secondary reaction time is 4–6 hours; and / or,

[0055] In Method 1, in step (2), the organic solvent is tetrahydrofuran; the reducing agent is lithium aluminum hydride; the molar ratio of IB-2 to the reducing agent is 1:1-1.5; the volume ratio of IB-2 to the organic solvent is 1g:10-15mL; the temperature of the reduction reaction is 0–4℃; the time of the reduction reaction is 1-2 hours; and / or,

[0056] In Method 1, in step (3), the organic solvent is dichloromethane; the oxidant is m-chloroperoxybenzoic acid; the molar ratio of IB-3 to oxidant is 1:1.2-1.5; the volume ratio of IB-3 to organic solvent is 1g:9-12mL; the oxidation reaction temperature is 20–25℃; the oxidation reaction time is 1–1.5 hours; and / or,

[0057] In Method 1, in step (4), the organic solvent is at least one of dichloromethane, chloroform, or tetrahydrofuran; the oxidant 2 is active manganese dioxide, tin dioxide, or Desmartin oxidant; the molar ratio of IB-4 to oxidant 2 is 1:3-5; the volume ratio of IB-4 to organic solvent is 1g:20-30mL; the temperature of the secondary oxidation reaction is 60–70℃; the time of the secondary oxidation reaction is 8–10 hours; and / or,

[0058] In Method 1, in step (4-1), the organic solvent is at least one of tetrahydrofuran, pyrrolidone, dichloromethane, and chloroform; the molar ratio of IB-5-0 to RMgBr is 1:4-5; the volume ratio of IB-5-0 to the organic solvent is 1g:20-50mL; the reaction temperature is 0℃–4℃; and the reaction time is 5–10 hours.

[0059] or,

[0060] In Method 1, in step (4-2), the molar ratio of IB-5-0 to hydroxylamine hydrochloride is 1:10-15; the molar ratio of IB-5-0 to methanol is 1g:10-50mL; the temperature of the first reaction is 20℃–25℃; the reaction time is 5–8 hours; the molar ratio of IB-5-0 to triethylamine is 1:2-3; the molar ratio of IB-5-0 to dimethyl sulfoxide is 80-100:1; the molar ratio of IB-5-0 to oxalyl chloride is 1:1.0-1.5; the molar ratio of IB-5-0 to acetonitrile is 1g:12-18mL; the temperature of the second reaction is 20℃–25℃; the reaction time is 9–11 hours.

[0061] In Method 1, in step (5), the organic solvent is chloroform; the molar ratio of compound IB-5 to trifluoroacetic anhydride is 1:2-3; the volume ratio of compound IB-5 to organic solvent is 1g:8-12mL; the reaction temperature is 20℃–25℃; the reaction time is 10–25 hours; and / or,

[0062] In Method 1, in step (6), the organic solvent is dimethyl sulfoxide; the molar ratio of compound IB-6 to sodium benzenesulfinate is 1:2.5-3.5; the volume ratio of compound IB-6 to organic solvent is 1g:15-20mL; the reaction temperature is 45℃–55℃; and the reaction time is 2–4 hours.

[0063] And / or,

[0064] In Method 1, in step (7), the organic solvent is chloroform; the molar ratio of compound IB-7 to phosphorus tribromide is 1:2.0-4.0; the volume ratio of compound IB-7 to organic solvent is 1g:20-30mL; the reaction temperature is 20℃–25℃; the reaction time is 2–4 hours; and / or,

[0065] In Method 2, the organic solvent is dimethyl sulfoxide; the molar ratio of compound IB-9 to sodium benzenesulfinate is 1:4.5-5.5; the volume ratio of compound IB-9 to organic solvent is 1g:30-40mL; the reaction temperature is 45–55℃; and the reaction time is 1.5–3.0 hours.

[0066] A third aspect of the present invention is to provide a disulfide covalently bridged biomolecule having the following general formula IIA or general formula IIB or its salt form:

[0067] Among them, the The corresponding precursor is a biomolecule containing at least two cysteine ​​residues. Or biomolecules containing at least one pair of disulfide bonds

[0068] The X 3 X in the disulfide bridging agent according to any one of the first aspects of the present invention 3 The correspondences are the same; n1 in Equations IIA and IIB are each independently selected from integers from 1 to 8.

[0069] As a preferred embodiment, the The corresponding precursor is selected from polypeptides, proteins, antibodies or antibody fragments containing at least two cysteine ​​residues or polypeptides, proteins, antibodies or antibody fragments containing at least one disulfide bond.

[0070] Preferably,

[0071] The polypeptide is an active cyclic peptide containing at least one pair of disulfide bonds, a linear polypeptide containing at least one pair of disulfide bonds, or two polypeptide chains linked by a pair of disulfide bonds; or a linear polypeptide or cyclic peptide composed of 1-50 amino acids containing at least two cysteine ​​residues; these polypeptides have pharmaceutical activity, cell penetration or receptor protein binding ability; preferably at least one of octreotide, somatostatin, oxytocin, terlipressin, lysine vasopressin, RGD tripeptide and its homologs.

[0072] The protein is a native protein containing at least one pair of solvent-oriented disulfide bonds, a recombinant protein containing at least two adjacent cysteine ​​residues, or a protein complex containing at least one pair of covalently linked disulfide bonds; preferably at least one of the following: autolysin, thioredoxin, serum albumin, green fluorescent protein, small ubiquitin protein, ubiquitin ligase, heat shock chaperone protein, epidermal growth factor receptor protein, copper transporter, transferrin, calmodulin, murine sarcoma protein (Ras), and peptidyl proline isomerase;

[0073] The antibody or antibody fragment is an antibody or antibody fragment comprising at least one pair of accessible disulfide bonds, wherein the antibody fragment comprises a fragment of a single antibody or a combination of light chains or heavy chains of multiple antibodies; preferably selected from at least one of murine antibodies, rabbit antibodies, phage display-derived antibodies, yeast display-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies and multispecific antibodies, antigen-binding fragments of bispecific antibodies and multispecific antibodies, immunologically active portions, or mutants of the above antibodies; preferably, whole antibodies or antibody fragments of abciximab, cetuximab, trastuzumab, sacituzumab, and palivizumab, and nanobodies selected from at least one of nanobodies targeting membrane proteins EGFR, green fluorescent protein, PD-L1, and β-amyloid protein. More preferably, the antibody is selected from monoclonal antibodies, and is not limited to the following: abciximab, alemtuzumab, anetumab, atezolizumab, avelumab, basiliximab, bevacizumab, blinatomumab, brentuximab, catutoxomab, cetuximab, cirmtuzumab, coltuximab, daclizumab, daratumumab, denintuzumab, and denosumab. Depatuxizumab, Dinutuximab, Durvalumab, Elotuzumab, Enfortumab, Glembatumumab, Gemtuzumab, Ibritumomab, Indatuximab, Indusatumab, Inotuzumab, Ipilimumab, Labetuzumab, Ladiratuzumab, Laprituximab, Lifastuzumab, LorvotuzumabMilatuzumab, Mirvetuximab, Naratuximab, Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab, Omalizumab, Palivizumab, Panitumumab, Patritumab, Pembrolizumab, Pertuzumab Anti-CD4 antibody, anti-CD5 antibody, anti-CD13 antibody, and anti-CD30 antibody, and their antigen-binding fragments or immunologically active portions. (The text also mentions various anti-CD4 antibodies, anti-CD5 antibodies, anti-CD13 antibodies, and anti-CD30 antibodies, but these appear unrelated to the main topic and are likely separate entries.)

[0074] A fourth aspect of the present invention is to provide a method for preparing a disulfide-bridged modified biomolecule as described in the second aspect of the present invention, the method comprising method A or method B;

[0075] Method A includes the following steps: simultaneously adding a reducing agent and a disulfide bridging reagent to a biomolecule solution, adjusting the pH with acid or base, carrying out a bridging reaction, and obtaining a disulfide-bridged modified biomolecule.

[0076] Method B includes the following steps: first, a reducing agent is added to the biomolecule solution to carry out a reduction reaction, and then a disulfide bridging reagent is added directly. The pH is adjusted with acid or base to carry out a bridging reaction to obtain a disulfide-bridged modified biomolecule.

[0077] The disulfide bridging agent is the same as the disulfide bridging agent described in any of the first aspects of the present invention;

[0078] The biomolecule is related to the second aspect of the present invention. The corresponding precursors are the same.

[0079] The third functional group X in the disulfide heavy bridging reagent 3 It does not participate in the reaction, but its reactivity is still retained.

[0080] In a preferred embodiment, the molar ratio of the disulfide bridging reagent to the biomolecule is ≥1; for example, a molar ratio of 1-10:1; the solvent of the biomolecule solution is phosphate buffer, ammonium bicarbonate buffer, sodium acetate buffer, or 4-hydroxyethylpiperazine ethanesulfonic acid buffer; the reducing agent is at least one of tris(2-carboxyethyl)phosphine (TCEP) and tris(hydroxypropyl)phosphine (THPP); the molar ratio of the reducing agent to the disulfide bridging reagent is ≥1; for example, a molar ratio of 1-10:1; the pH is adjusted with hydrochloric acid or sodium hydroxide; the pH of the bridging reaction is 7.0-8.5; the temperature of the bridging reaction is 25-37°C; and the time of the bridging reaction is 0.5-16 hours.

[0081] A fifth aspect of the present invention is to provide a biomolecule-load conjugate constructed by disulfide covalent bridging, wherein the biomolecule-load conjugate constructed by disulfide covalent bridging has the following general structural formula IIIA, IIIB or salts thereof:

[0082]

[0083] Formula IIIA and Formula IIIB As described in the second aspect of the invention The corresponding ones are the same;

[0084] The n2 is selected from integers from 1 to 20;

[0085] The X 5 Functional group X in biomolecules constructed by disulfide covalent bridging for the second aspect of this invention 3 With general formula X 4 ...Active functional group X in L1...L2-D 4 The linking group generated by the reaction; the X 5 Selected from the following structural fragments:

[0086] R6, R7, and R8 are amino acid side chains excluding proline; R9, R... 10 The alkyl group is selected from C1-C10; m1 and m2 are each independent integers from 1 to 20;

[0087] The L1 contains at least one spacer selected from the following: C(O), S, S(O), S(O)2. n3-n 11 Each is an independent integer between 1 and 20;

[0088] The L2 is selected from alkylene groups, C(O), S, S(O), S(O)2, hydrazones, etc. Oligopeptides or polyethylene glycol-modified oligopeptides, monosaccharide groups, sulfate groups, aminobenzyloxycarbonyl self-degrading groups, diazonium salt photosensitive degradation cleavage groups, and combinations thereof; preferably.

[0089] n 12 n is an integer between 1 and 20; 13 n is an integer between 1 and 20; 14 Integers between 1 and 20;

[0090] The D is selected from compounds containing at least one active group selected from alkyl, cycloalkyl, aryl, azido, alkynyl, tetrazine, and cyclooctyne, proteins, polypeptides, nucleophilic tags, fluorescent molecules, cytotoxins, and drugs; preferably, the nucleophilic tag is selected from polypeptides with 6-10 histidine residues, glutathione thiotransferase (GST), maltose-binding protein (MBP), biotin, or biotin molecules containing polyethylene glycol; the cytotoxin includes microtubule inhibitors, topoisomerase inhibitors, and DNA binders; preferably, methylaurestatin E (MMAE), methylaurestatin F (MMAF), camptothecin (CPT), irinotecan (CPT-11), and pyrrolobenzodiazepines. (PBD);

[0091] The antibody is a monoclonal antibody, including but not limited to murine antibodies, rabbit antibodies, phage-display antibodies, yeast-display antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies and multispecific antibodies, their antigen-binding fragments, immunologically active portions, or mutants of the above antibodies, and is not limited to being selected from: cetuximab, ciromtuzumab, mirvetuximab, patritumab, sacituzumab, or trastuzumab. The antibody is selected from humanized antibodies.

[0092] The polypeptides mentioned include polypeptides with pharmaceutical properties such as multihistidine tags, polypeptide ligands, and oxytocin;

[0093] The drugs mentioned are microtubule inhibitors, RNA polymerase inhibitors, topoisomerase inhibitors, inserters, DNA-reactants, tunicaein and its derivatives, drugs for inflammatory diseases, immunosuppressants, drugs for nervous system diseases, or drugs for cardiovascular diseases. Microtubule inhibitors such as monomethylaurestatin... Auristatin (e.g., methylauritstatin E or methylauritstatin F, etc.), maytansinoid (e.g., maytansinol or DM1 maytansin, etc.); RNA polymerase inhibitors such as α-amaminine, etc.; topoisomerase inhibitors such as etoposide, teniposide, acridine, SN-38 or eczetidine, etc.; intercalating agents such as pyrrolobenzodiazepine (PBD), etc.; DNA-reactive agents such as chazim, tiancimycins or other enediynes, etc.; sea sucrose and its derivatives such as trabectedin or rupettedin, etc.; inflammatory drugs such as azithromycin, roxithromycin, etc.; immunosuppressants such as prednisone, hydrocortisone or dexamethasone, etc.; drugs for nervous system diseases such as chlorpromazine or amitriptyline, etc.; cardiovascular drugs such as moricizine hydrochloride or digoxin, etc.

[0094] In a further preferred embodiment, the general formula (IIIA) is a polypeptide-fluorescent molecule conjugate constructed by covalent bridging with disulfides, wherein the polypeptide includes, but is not limited to, octreotide, and the fluorescent molecule includes, but is not limited to, rhodamine B compounds containing 1,2-amino acids.

[0095] In a further preferred embodiment, the general formulas (IIIA) and (IIIB) are antibody fragment-peptide conjugates constructed by covalent bridging with disulfides, wherein the antibody fragment includes, but is not limited to, trastuzumab antigen-binding fragments, and the peptide includes, but is not limited to, a multihistidine nucleophilic tag with an N-terminal cysteine ​​residue.

[0096] In a further preferred embodiment, the general formula (IIIA) is an antibody-fluorescent molecule conjugate constructed by covalent bridging with disulfides; wherein the antibody fragment includes, but is not limited to, trastuzumab, and the fluorescent molecule includes, but is not limited to, rhodamine B compound containing 1,2-amino acids.

[0097] In a further preferred embodiment, the disulfide covalently bridged biomolecule-load coupling of the general formula (IIIA) is selected from at least one compound with the following structural formula:

[0098]

[0099] A sixth aspect of the present invention is to provide a method for preparing the disulfide covalently bridged biomolecule-load coupling described in the fifth aspect of the present invention, the method comprising the following steps:

[0100]

[0101] Add the payload conjugate to the solution of disulfide-bridged biomolecules, adjust the pH with acid or base, carry out the coupling reaction, and perform post-treatment to obtain the biomolecule-payload conjugate constructed by disulfide covalent bridging.

[0102] In further optimized examples,

[0103] The molar ratio of the payload conjugate to the disulfide-bridged biomolecule is ≥1; for example, the molar ratio is 1-10:1.

[0104] The solvent for the disulfide-bridged modified biomolecule solution is phosphate buffer, ammonium bicarbonate buffer, sodium acetate buffer, or 4-hydroxyethylpiperazine ethanesulfonic acid buffer; HCl or NaOH is added to adjust the pH to 5.0-7.5.

[0105] The reaction temperature is 25℃-37℃;

[0106] The reaction time is 0.5-24 hours.

[0107] The payload conjugate is X in a biomolecule that can be bridged with disulfide-modified molecules. 3 The reacting compounds;

[0108] Preferably, the payload coupling compound is selected from compounds containing 1,2-aminothiol, hydroxylamine, or hydrazide groups as linkers;

[0109] More preferably, the payload coupling has the following structural formula: X 4 ...L1...L 2 -D;

[0110] The X 4 Selected from R 11 R 12 It consists of amino acid side chains other than proline;

[0111] L1, L2, and D correspond to the same L1, L2, and D as described in claim 8;

[0112] More preferably, the X 4 ...L1...L 2 -D is selected from at least one of the compounds in the following table:

[0113]

[0114] A seventh aspect of the present invention is the application of a method for preparing a disulfide covalently bridged biomolecule-load conjugate as described in the fifth aspect of the present invention or a method for preparing a disulfide covalently bridged biomolecule-load conjugate as described in the sixth aspect of the present invention in the preparation of a pharmaceutical product; preferably, in the preparation of a targeted drug; more preferably, for the preparation of a polypeptide-drug conjugate or an antibody-drug conjugate; even more preferably, for the preparation of a drug for treating cancer, inflammatory diseases, autoimmune diseases, cardiovascular diseases, or neurological diseases; and even more preferably, for the preparation of a drug for treating breast cancer, gastric cancer, liver cancer, ovarian cancer, pancreatic cancer, bladder cancer, lung cancer, epithelial cancer, or malignant lymphoma.

[0115] The cancers included in the medicament for treating cancer described in the invention also include lymphoma, blastoma, sarcoma, leukemia or lymphoma, squamous cell carcinoma (such as epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, liver cancer, stomach cancer, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioma, cervical cancer, ovarian cancer, oral cancer, liver cancer, bladder cancer, urinary tract cancer, liver tumor, breast cancer, HER2-positive breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, thyroid cancer, liver cancer, melanoma, multiple myeloma, B-cell lymphoma, brain cancer, head and neck cancer, and related metastatic tumors.

[0116] The aforementioned disulfide covalently bridged biomolecule-payload conjugates can also be used to deliver other active agents for the treatment of a variety of other diseases, including but not limited to inflammatory diseases, autoimmune diseases, nervous system diseases, and cardiovascular diseases.

[0117] The innovation of this invention lies in:

[0118] This invention provides a novel class of multifunctional pyridine derivatives containing three heterogeneous electrophilic groups capable of reacting with spatially adjacent dithiol groups. This novel conjugation chemistry approach modulates the reactivity of the electrophilic groups in the heavy bridging reagent with thiols through substituents and utilizes the spatial proximity induction effect to enhance the reaction rate between the less reactive electrophilic groups and the thiol groups, thereby achieving covalent bridging between or within adjacent cysteine ​​residues in biomolecules.

[0119] The novel rebridging reagent of this invention is small in size and highly water-soluble, exhibiting significant advantages in the preparation of peptide conjugates, protein conjugates, antibody fragments, or antibody-drug conjugates (ADCs). It can highly stably link antibodies and drugs together, and the resulting conjugates possess high stability. This overcomes the limitation of poor stability in ADCs prepared by existing conjugation methods, providing broader application prospects for disease treatment drugs.

[0120] This invention provides a novel disulfide heavy bridging reagent that can prepare various biomolecular conjugates using a simple two-step chemical coupling strategy. This method has a wide range of applications.

[0121] The novel linker of this invention is suitable for preparing a variety of conjugates, including protein conjugates, polypeptide conjugates, antibody or antibody fragment conjugates. It is simple to operate, operates under mild reaction conditions, and requires no modification to the protein or antibody, offering significant economic advantages.

[0122] The heavy bridging reagent of the present invention performs well in the preparation of antibody-drug conjugates, and the resulting conjugates have a relatively uniform drug-antibody conjugate ratio (DAR), high product homogeneity, and a single distribution component (DAR 4) accounting for more than 80%.

[0123] The antibody-drug conjugates prepared by the novel linker of the present invention have high stability; after incubation in human plasma for 14 days, more than 95% of the conjugates remain intact.

[0124] The antibody-drug conjugate prepared by the novel linker of the present invention has a significant killing effect on tumor cells, with a half-maximal inhibitory concentration (IC50) reaching the sub-pM level. Attached Figure Description

[0125] Figure 1 This is a schematic diagram illustrating the application of pyridine derivative-based heavy-bridged disulfides in the preparation of various coupling compounds.

[0126] Figure 2 This is the LC-MS image of the lysozyme conjugate prepared using the partially re-bridging reagent of this invention in Example 10 of this invention. Figure 2 It can be seen that each lysozyme molecule reacts with only one molecule of the coupling agent on all the conjugates, indicating that only the solvent-exposed disulfide bonds (C6-C127) are covalently rebridged. The conjugate yield of FPPAc is about 50%, the conjugate yield of BPNA is about 60%, and the conjugate yields of FPPA, FPPN and PPNA are all about 85%.

[0127] Figure 3 This is an LC-MS image of the bacterial thioredoxin conjugate (Trx_FPPN) prepared in Example 10 of this invention. Figure 3 It can be seen that the obtained conjugate has a single component and a conjugation efficiency of nearly 100%. The cyano group can be used to further conjugate a polypeptide containing an N-terminal cysteine ​​(CGGSHHHHHH, CH6). The reaction product is single and the conjugation efficiency is nearly 100%.

[0128] Figure 4This is the LC-MS chromatogram of the polypeptide conjugate prepared by reacting FPPN, FPPA, FPPac with octreotide in Example 11 of this invention. In the figure, black represents the molecular ion peak of free octreotide, and red represents the molecular ion peak of the conjugate after the reaction of reduced octreotide with the heavy-bridge reagent. Figure 4 It can be seen that the coupling yields of the three heavy bridging reagents and octreotide to reduce disulfide bonds are all greater than 95%.

[0129] Figure 5 This is an LC-MS image of the polypeptide conjugate prepared by reacting FPPN with lypressin and oxytocin in Example 11 of this invention. After reduction, one cysteine ​​residue is located at the N-terminus of these two polypeptides, therefore... Figure 5 It can be seen that the coupling products mainly consist of two types: one is the product of the reaction between fluorine and sulfone groups with two cysteine ​​residues, and the other is the product of the reaction between fluorine and cyano groups with two cysteine ​​residues, in which the cyano group reacts with the N-terminal cysteine ​​residue to form a cyclized thiazoline-linked coupling product. From Figure 4 It can be seen that the coupling yield of FPPN with the polypeptide to reduce disulfide bonds is close to 100%.

[0130] Figure 6 The image shows the LC-MS chromatogram of the conjugate prepared by reacting FPPN with somatostatin and terlipressin in Example 11 of this invention. As can be seen from the image, the conjugation yield is nearly 100%, and the conjugation product is singular.

[0131] Figure 7 The image shows the LC-MS chromatogram of the conjugates of octreotide, FPPN, and FPPA from Example 11 of this invention, further obtained by reacting the cyano and aldehyde groups with rhodamine containing 1,2-aminothiol. The image shows that the conjugation yield is nearly 100%, and the conjugated product is singular.

[0132] Figure 8 This is an imaging map of A549 cells internalized with a polypeptide conjugate prepared by co-focusing imaging analysis in Example 11 of the present invention.

[0133] Figure 9 This is an LC-MS image of the antibody fragment Fab conjugate prepared in Example 12 of this invention. From... Figure 9 It can be seen that the coupling efficiency of the five re-bridging reagents in the examples is all above 95%.

[0134] Figure 10 This is an LC-MS image of the conjugate obtained by further conjugating the antibody fragment Fab conjugate with a polypeptide in Example 12 of this invention. The conjugate is generated by reacting Tmab-Fab-L1 (L2 / L4) with an N-terminal cysteine ​​polypeptide (CGGSHHHHHH, CH6). Figure 10 It can be seen that the heavy-bridged coupling compounds can further couple functional molecules, with the coupling efficiency of nitrile groups exceeding 70% and the coupling efficiency of aldehyde groups exceeding 95%.

[0135] Figure 11 This is an SDS-PAGE image of the antibody fragment Fab conjugate prepared in Example 12 of the present invention.

[0136] Figure 12 This is an LC-MS image of the interchain disulfide bonds in the FPPA-bridged full antibody (Trastuzumab) from Example 13 of this invention. From... Figure 12 As can be seen, FPPA can achieve a conjugation efficiency of 90%, mainly existing in the form of inter-chain conjugated full antibodies and intra-chain conjugated half antibodies.

[0137] Figure 13 This is an SDS-PAGE image of the interchain disulfide bonds of the FPPA-reinforced full antibody (Trastuzumab) in Example 13 of this invention. Lane 1: Marker, Lane 2: Trastuzumab (Tmab), Lane 3: Reduced Tmab, Lane 4: FPPA-Tmab conjugate (Tmab-PyA), Lane 5: Tmab-PyA conjugate incubated at 10 times the amount of TCEP for 1 hour (Tmab-PyA + TCEP). Figure 13 It can be seen that the ratio of full antibody to half antibody is about 10:7, with trace amounts of unbridged light and heavy chains.

[0138] Figure 14 This is an LC-MS image of the FPPA-Trastuzumab conjugate linked to the fluorescent molecule (Rhodamine B) in Example 14 of this invention. Figure 15 The conversion rate is greater than 95%.

[0139] Figure 15 This is an SDS-PAGE image of the FPPA-Trastuzumab-Rhodamine conjugate in human plasma for 1-14 days, as analyzed in Example 15 of this invention. Figure 15 This shows that the coupling compound has high stability.

[0140] Figure 16 This is an intracellular imaging image of the FPPA-Trastuzumab-Rhodamine conjugate in Example 16 of this invention. From... Figure 16As can be seen in Example 15, when the FPPA-Trastuzumab-Rhodamine conjugate was incubated with HER2-positive cells SK-Br-3 at 4°C for 1 hour, the antibody-fluorescent conjugate bound to the cell membrane receptor. After incubation at 37°C for 24 hours, it was internalized into the cells and obvious bright spots were observed. This indicates that the antibody conjugate can selectively internalize into positive tumor cells and can enter lysosomes.

[0141] Figure 17 This is an LC-MS image of the green fluorescent protein linked by the FPPA-Trastuzumab conjugate in Example 17 of the present invention.

[0142] Figure 18 This is an SDS-PAGE image of the stability of the FPPA-Trastuzumab-fluorescent protein conjugate in human plasma, as analyzed in Example 17 of this invention.

[0143] Figure 19 These are SDS-PAGE images of ADC-2, ADC-3, ADC-4, and ADC-5 prepared in Examples 18-22 of this invention. From... Figure 19 It can be seen that the obtained conjugate contains two main components: half-antibody and full-antibody, with a small amount of free light and heavy chains. The ratio of half-antibody to full-antibody is approximately 4:6, calculated by grayscale.

[0144] Figure 20 These are LC-MS images of ADC-2, ADC-3, ADC-4, ADC-5, and ADC-8 prepared in Examples 18-22 of this invention. From... Figure 20 It can be seen that the observed molecular weight is basically consistent with the theoretically calculated molecular weight, and the proportion of the single-distribution component (DAR4) in the obtained conjugate can exceed 60%.

[0145] Figure 21 These are SEC plots of ADC-2, ADC-3, ADC-4, ADC-5, and ADC-8 prepared in Examples 18-22 of this invention. From... Figure 21 It can be seen that the obtained conjugates retain the monomer form, with the aggregated component being less than 1%. Detailed Implementation

[0146] The present invention will be further illustrated below with reference to specific embodiments. The following are examples of the synthesis and analysis of the compounds (including linkers and linker-drug conjugates) and peptides, proteins, and antibody-drug conjugates described in this patent. It should be noted that these examples do not limit the scope of protection of the present invention.

[0147] Unless otherwise stated, all anhydrous reagents were purchased directly from the supplier and stored under nitrogen. All other reagents and solvents purchased were of high purity and were not further purified before use.

[0148] Example 1: Synthesis of L1 and L2

[0149]

[0150] Step 1: Synthesize 1-2

[0151] 10.0 g of 2-methyl-5-fluoropyridine was dissolved in 100 mL of chloroform, and 20.1 g of mCPBA was added in portions. The mixture was reacted at 40 °C for 12 h. The generated m-chlorobenzoic acid was filtered off, the solvent was removed by rotary evaporation, and the product was dried under vacuum to give 11.0 g of a white solid, with a yield of 96.0%. 1 HNMR (600MHz, CDCl3) δ8.22(dd,J=4.4,2.4Hz,1H),7.23(t,J=8.0Hz,1H),7.01(t,J=7.6Hz,1H),2.48(s,3H). 13 C NMR(151MHz,CDCl3)δ158.85(d,J FC =250.3Hz), 145.81, 129.46 (d, J) FC =35.7Hz), 125.81(d,J FC =8.9Hz), 113.51(d,J FC =19.3Hz), 17.12.

[0152] Step 2: Synthesize 1-3

[0153] 26 mL of fuming nitric acid and 52 mL of concentrated sulfuric acid were added to 11 g of 2-methyl-5-fluoropyridine nitrogen oxides in an ice-water bath, and the reaction was carried out at 100 °C for 8 h. After cooling to room temperature, the mixture was poured into ice water and extracted three times with dichloromethane. The organic phase was washed with sodium bicarbonate solution, recovered, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The product was then dried under vacuum to give 20.8 g of a yellow viscous liquid, with a yield of 95.0%. 1 H NMR (600MHz, CDCl3) δ8.33 (d, J = 5.9 Hz, 1H), 8.06 (d, J = 9.0 Hz, 1H), 2.52 (s, 3H). 13 CNMR(151MHz,CDCl3)δ151.82(d,J FC =269.2Hz),147.21,131.74,131.25(d,J FC =34.2Hz), 121.26, 17.08.

[0154] Step 3: Synthesize 1-4

[0155] 64 mL (89.0 g) of a 33% hydrobromic acid-acetic acid solution was added to 20.8 g of 2-methyl-4-nitro-5-fluoropyridine nitrogen oxides, and the reaction was carried out at 90 °C for 3 h. The pH was slowly adjusted to neutral with sodium hydroxide solution under ice-water bath conditions. The mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and the dichloromethane was removed by rotary evaporation. The solid was then dried under vacuum to give 16.4 g of a yellow solid, with a yield of 65.9%. 1 HNMR (600MHz, CDCl3) δ8.23 (d, J = 4.0Hz, 1H), 7.47 (s, 1H), 2.48 (s, 3H). 13 C NMR(151MHz,CDCl3)δ155.64(d,J FC =251.3Hz), 146.84, 129.85 (d, J) FC =35.2Hz), 128.85, 106.36 (d, J) FC =20.7Hz), 16.99.

[0156] Step 4: Synthesize 1-5

[0157] 16.4 g of 2-methyl-4-bromo-5-fluoropyridine nitride was dissolved in 100 mL of chloroform. 34 mL of trifluoroacetic anhydride was slowly added dropwise under an ice-water bath, and the reaction was carried out at 65 °C for 8 h. Trifluoroacetic acid and trifluoroacetic anhydride were removed by rotary evaporation, and saturated sodium carbonate solution was added with stirring for another 4 h. The aqueous phase was extracted three times with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, the solvent was removed by rotary evaporation, and the solution was dried under vacuum to give 12.6 g of a brownish-yellow liquid, with a yield of 76.8%. 1 HNMR (600MHz, CDCl3) δ8.39 (s, 1H), 7.60 (d, J = 5.2Hz, 1H), 4.75 (s, 2H), 3.38 (brs, 1H). 13 C NMR(151MHz,CDCl3)δ156.38(d,J FC =4.9Hz), 155.92(d,J FC =255.9Hz), 137.21(d,J FC =25.6Hz), 125.30, 120.14(d,J) FC =18.9Hz), 63.73.

[0158] Step 5: Synthesize 1-6

[0159] 12.6 g of 2-hydroxymethyl-4-bromo-5-fluoropyridine was dissolved in 100 mL of acetonitrile, and 1.8 g of acetic acid and 20.1 g of sodium benzenesulfinate were added. The mixture was refluxed at 90 °C for 24 h. The reaction solution was adjusted to neutral with sodium carbonate solution, and the aqueous phase was extracted three times with dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography to give 7.4 g of a brown solid, with a yield of 45.0%. 1 H NMR (400MHz, CDCl3) δppm: 8.50 (s, 1H), 8.08–8.03 (d, 2H), 8.02 (d, J = 5.4Hz, 1H), 7.73–7.66 (t, 1H), 7.60 (t, J = 7.7Hz, 2H), 4.86 (s, 2H). 13 C NMR (101MHz, CDCl3) δppm: 157.43, 154.92, 152.29, 141.28, 137.36, 134.67, 129.52, 128.61, 119.00, 64.18. 13 C NMR(101MHz,CDCl3)δ157.43(d,J FC =5.3Hz), 153.60(d,J FC =264.7Hz), 139.21, 139.17 (d, J) FC =24.9Hz), 137.36(d,J) FC =13.0Hz),134.67,129.52,128.60,118.99,64.18.

[0160] Step 6: Synthesize L2

[0161] 7.0 g of 2-hydroxymethyl-4-phenylsulfonyl-5-fluoropyridine was dissolved in 100 mL of chloroform, and 11.4 g of freshly prepared manganese dioxide was added. The mixture was reacted at 60 °C for 6 h. The manganese dioxide was filtered off, the filter cake was washed three times with methanol, the filtrate was recovered, and the solvent was removed by rotary evaporation to give 6.6 g of a brown solid, with a yield of 95.0%. 1 HNMR (600MHz, CDCl3) δ10.08(s,1H),8.72(s,1H),8.61(d,J=5.7Hz,1H),8.08(d,J=8.2Hz,2H),7.73(t,J=7.4Hz,1H),7.62(t,J=7.7Hz,2H). 13 CNMR(151MHz,CDCl3)δ190.16,156.61(d,J FC =275.1Hz), 150.12(d,J FC =5.7Hz), 141.02(d,J FC=25.4Hz), 138.78, 138.29 (d, J) FC =13.2Hz),134.99,129.68,128.77,121.06.

[0162] Step 7: Synthesize 1-7

[0163] 5.3 g of 4-phenylsulfonyl-5-bromo-2-pyridinecarboxaldehyde was dissolved in methanol, and 1.7 g of hydroxylamine hydrochloride was added. The mixture was stirred at room temperature for 6 h. A large amount of yellow solid was produced in the system. The solvent was evaporated and the solid was dried under vacuum to give 5.4 g of yellow solid, with a yield of 96.0%. 1 H NMR (600MHz, DMSO-d6) δ8.82(s,1H),8.26(d,J=5.4Hz,1H),8.19(s,1H),8.05(d,J=7.9Hz,2H),7.84(t,J=7.4Hz,1H),7.72(t,J=7.8Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ153.75(d,J FC =266.1Hz), 150.65(d,J FC =5.6Hz), 147.51, 140.85 (d, J) FC =24.8Hz), 138.89, 136.88 (d, J) FC =12.9Hz),135.76,130.52,128.67,118.34.

[0164] Step 8: Synthesize L1

[0165] E-4-phenylsulfonyl-5-fluoro-2-pyridinecarboxaldehyde oxime (500 mg) was dissolved in acetonitrile, and triethylamine (451 mg) and dimethyl sulfoxide (2 mg) were added. Oxaloyl chloride (272 mg) was added dropwise, and the reaction was carried out at room temperature for about 10 h. After the reaction was complete, the acetonitrile was removed by rotary evaporation, and the acid was removed by adding NaHCO3 solution. The aqueous phase was extracted three times with dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to give 350 mg of white solid, with a yield of 75%. 1 H NMR (400MHz, CDCl3) δ8.65 (s, 1H), 8.34 (d, J = 5.2Hz, 1H), 8.05 (d, J = 8.4Hz, 2H), 7.75 (t, J = 7.5Hz, 1H), 7.63 (t, J = 7.8Hz, 2H). 13 C NMR(101MHz,CDCl3)δ155.75(d,J FC =276.3Hz), 142.40(d,J FC=26.3Hz), 138.24(d,J FC =12.7Hz), 135.35, 130.87 (d, J) FC =6.4Hz), 129.85, 128.85 (d, J) FC =1.7Hz), 127.47, 115.33.

[0166] Example 2: Synthesis of L3

[0167]

[0168] Step 1: Synthesize 2-2

[0169] 10 g of 2-bromo-5-fluoropyridine was dissolved in 100 mL of chloroform, and 12.6 g of m-CPBA was added in portions. The mixture was reacted at 40 °C for 24 h. The generated m-chlorobenzoic acid was filtered off, and the solution was concentrated and separated by column chromatography to give 10 g of a white solid, with a yield of 94%. 1 H NMR (400MHz, CDCl3) δppm: 8.38 (dd, J=4.2, 2.6Hz, 1H), 7.66 (dd, J=9.1, 6.6Hz, 1H), 7.01 (ddd, J=9.1, 6.4, 2.6Hz, 1H).

[0170] Step 2: Synthesize 2-3

[0171] 15 mL of fuming nitric acid and 30 mL of concentrated sulfuric acid were added to 10 g of 2-bromo-5-fluoropyridine nitrogen oxides under ice-water bath conditions, and the reaction was carried out at 100 °C for 6 h. After cooling the system to room temperature, it was poured into an appropriate amount of ice water, extracted three times with dichloromethane, the organic layer was separated, the organic phase was washed with sodium bicarbonate solution, the organic phase was recovered, dried over anhydrous sodium sulfate, filtered, the solvent was removed by rotary evaporation, and dried under vacuum to give 6 g of brown oily liquid, with a yield of 50%. 1 H NMR (400MHz, CDCl3) δppm: 8.60 (s, 1H), 8.07 (d, J = 5.2Hz, 1H).

[0172] Step 3: Synthesize 2-4

[0173] 20 mL of a 33% hydrobromic acid-acetic acid solution was added to 6 g of 2-bromo-4-nitro-5-fluoropyridine nitrogen oxides, and the reaction was carried out at 90 °C for 6 h. The pH of the system was carefully adjusted to neutral with sodium hydroxide solution under ice-water bath conditions. The mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, the solvent was removed by rotary evaporation, and column chromatography was performed to obtain 3 g of a yellow solid, yield 46%. 1 H NMR (400MHz, CDCl3) δppm: 8.60 (d, J = 2.0 Hz, 1H), 8.07 (d, J = 5.2 Hz, 1H).

[0174] Step 4: Synthesize 2-5

[0175] 2,4-Dibromo-5-fluoropyridine (2.5 g) was dissolved in 30 mL of acetic acid, and sodium benzenesulfinate (3.2 g) was added. The reaction was carried out at 90 °C for 24 h. The pH of the system was adjusted to neutral with saturated sodium carbonate solution, and the mixture was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography to give 1.1 g of a pale yellow solid, with a yield of 38%. 1 H NMR (400MHz, CDCl3) δppm: 8.35 (s, 1H), 8.13 (d, J = 4.9Hz, 1H), 8.06 (d, J = 7.2Hz, 2H), 7.74 (t, J = 7.5Hz, 1H), 7.63 (t, J = 7.6Hz, 2H). 13 C NMR(151MHz,CDCl3)δ154.21(d,J F-C =267.0Hz), 140.56(d,J) FC =26.1Hz), 139.23(d,J FC =14.2Hz),138.69,136.83,135.00,129.66,128.78,126.58.

[0176] Step 5: Synthesize 2-6

[0177] Dissolve 2-bromo-4-phenylsulfonyl-5-fluoropyridine (4 g) in 100 mL of anhydrous acetonitrile, add cuprous iodide (360 mg), bis(triphenylphosphine)palladium dichloride (442.2 mg), and tributyl(1-ethoxyethylene)tin (5.0 g), react at 90 °C for 12 h, evaporate the solvent, and proceed directly to the next step. 1 HNMR (600MHz, CDCl3) δ8.48(s,1H),8.29(d,J=5.6Hz,1H),8.07(d,J=7.9Hz,2H),7.70(t,J=7.4Hz,1H),7.60 (t,J=7.7Hz,2H),5.43(d,J=2.2Hz,1H),4.46(d,J=2.2Hz,1H),4.03(q,J=7.0Hz,2H),1.51(t,J=7.0Hz,3H). 13 C NMR(151MHz,CDCl3)δ156.52,153.96(d,J FC =267.2Hz),151.37,139.53,139.24(d,J FC =24.9Hz), 136.84(d,J FC=11.9Hz),134.49,129.42,128.59,117.56,86.26,63.97,14.52.

[0178] Step 6: Synthesize L3

[0179] Add 80 mL of hydrochloric acid (1 M) to the black viscous system obtained in the previous step, and reflux for 2 h. Adjust the pH to neutral with potassium carbonate solution, and a black precipitate is formed. Filter, wash the filter cake several times with dichloromethane, recover the filtrate, extract the aqueous phase with dichloromethane, combine the organic layers, dry with anhydrous sodium sulfate, concentrate, and separate by column chromatography to give 1.1 g of yellow solid, yield 30%. 1 H NMR (600MHz, CDCl3) δ8.68(d,J=5.7Hz,1H),8.60(s,1H),8.07(d,J=7.9Hz,2H),7.72(t,J=7.5Hz,1H),7.61(t,J=7.7Hz,2H),2.72(s,3H). 13 C NMR(151MHz,CDCl3)δ197.04,156.37(d,J FC =273.1Hz), 151.00, 139.59 (d, J) FC =25.1Hz), 139.04, 137.91 (d, J) FC =13.2Hz),134.82,129.60,128.71,121.32,25.71.

[0180] Example 3: Synthesis of L4

[0181]

[0182] Step 1: Synthesize 3-2

[0183] Compound 3-1 (9.21 g), N,N-dimethylformamide (100 μL), and anhydrous dichloromethane (100 mL) were added sequentially to a 500 mL round-bottom flask. Oxaloyl chloride (15.2 g) was added dropwise under ice bath conditions. After the addition was complete, the mixture was brought to room temperature and stirred for 6 hours. The mixture was concentrated under reduced pressure, and anhydrous methanol (100 mL) and triethylamine (10.1 g) were slowly added sequentially under ice bath conditions. After the addition was complete, the mixture was brought to room temperature and stirred for 4 hours. The reaction was monitored by TLC until complete. The mixture was concentrated under reduced pressure, and the residue was separated by silica column chromatography (petroleum ether: ethyl acetate 5:1) to obtain the target product 3-2 (8.3 g, 75% yield, pale yellow solid). 1 HNMR (400MHz, CDCl3) δ8.95(s,1H),7.28(s,1H),3.96(s,3H),2.59(s,3H).

[0184] Step 2: Synthesize 3-3

[0185] Compound 3-2 (7.40 g) and anhydrous tetrahydrofuran (100 mL) were added to a 500 mL round-bottom flask. Lithium aluminum hydride (1.52 g) was added in portions under ice bath conditions, and the reaction was stirred for 1 hour while maintaining this temperature. Then, 1.52 mL of water and a 15% sodium hydroxide aqueous solution were added sequentially, and stirring was continued for 15 minutes. The mixture was filtered through diatomaceous earth and the filtrate was collected. The filtrate was dried over anhydrous sodium sulfate, filtered again, and concentrated under reduced pressure. The residue was separated by silica column chromatography (petroleum ether: ethyl acetate 1:1) to obtain the target product 3-3 (8.3 g, 86% yield, white solid). 1 H NMR (400MHz, CDCl3): δ8.65(s,1H),7.87(s,1H),4.05(s,2H),3.65(s,3H),2.01(s,3H).

[0186] Step 3: Synthesize 3-4

[0187] Compound 3-3 (5.2 g) and anhydrous dichloromethane (50 mL) were added to a 250 mL round-bottom flask. Under ice bath conditions, m-chloroperoxybenzoic acid (6.9 g) was added in portions, and the mixture was stirred at room temperature for 1 hour. 100 mL of a 10% potassium carbonate aqueous solution was added, and stirring continued for 15 minutes. The aqueous phase was washed with dichloromethane (3 × 100 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated by silica column chromatography (dichloromethane:methanol 100:1) to obtain the target product 3-4 (4.9 g, 94% yield, white solid). 1 HNMR (400MHz, CDCl3): δ8.85(s,1H),7.06(s,1H),4.25(s,2H),3.79(s,3H),2.65(s,3H). 13 C NMR(101MHz, CDCl3)δ169.23,163.21,158.21,153.29,150.98,58.23,30.27.HRMS(ESI)calcd for[C7H8ClNO2+H] + :174.0316,found:174.0319.

[0188] Step 4: Synthesize 3-5

[0189] Compound 3-4 (4.3 g), activated manganese dioxide (8.7 g), and chloroform (100 mL) were added sequentially to a 250 mL round-bottom flask. The mixture was heated to 65 °C and stirred for 8 hours. The reaction was monitored by TLC until complete. The mixture was filtered through diatomaceous earth and the filtrate was collected. The filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated by silica column chromatography (dichloromethane:methanol 100:1) to obtain the target product 3-5 (3.9 g, 91% yield, white solid). 1 H NMR (400MHz, CDCl3) δ9.87(s,1H),8.02(s,1H),7.61(s,1H),3.67(s,3H),2.03(s,3H).

[0190] Step 5: Synthesize 3-6

[0191] Compound 3-5 (3.4 g) and chloroform (30 mL) were added to a 250 mL round-bottom flask. Trifluoroacetic anhydride (10 mL) was added dropwise under ice bath conditions, and the mixture was stirred at room temperature for 10 hours. The mixture was concentrated under reduced pressure to obtain a brown oily liquid. 30 mL of dichloromethane was added and the mixture was concentrated under reduced pressure. Finally, 50 mL of dichloromethane and 50 mL of saturated sodium bicarbonate solution were added sequentially, and the mixture was stirred at room temperature for 10 hours. The organic phase was washed with dichloromethane (3 × 50 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated by silica column chromatography (petroleum:ethyl ether 1:1) to obtain the target product 3-6 (1.8 g, 10.4 mmol, yield 52%, pale yellow solid). 1 H NMR (400MHz, CDCl3) δ9.93(s,1H),8.01(s,1H),7.02(s,1H),4.24(s,2H),3.59(s,3H).

[0192] Step 6: Synthesize 3-7

[0193] Compound 3-6 (1.72 g), sodium benzenesulfinate (4.92 g), and dimethyl sulfoxide (30 mL) were added sequentially to a 100 mL round-bottom flask. The mixture was heated to 50 °C and stirred for 2 hours. The mixture was diluted with 100 mL of ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated by silica column chromatography (petroleum:ethyl ether 1:1) to obtain the target product 3-7 (2.6 g, 93% yield, pale yellow solid). 1 H NMR (400MHz, CDCl3) δ9.98(s,1H),8.69(s,1H),8.43(s,1H),8.12–8.01(m,2H),7.56–7.40(m,1H),7.34–7.19(m,2H).4.65(s,2H).

[0194] Step 7: Synthesize L4

[0195] Compound 3-7 (831 mg) and chloroform (20 mL) were added sequentially to a 100 mL round-bottom flask. Phosphorus tribromide (976 mg) was slowly added at 0 °C, and the reaction was continued at room temperature for 2 hours. The reaction was monitored by TLC until complete. The reaction system was neutralized with saturated sodium bicarbonate solution, diluted with 200 mL of ethyl acetate, and washed with saturated sodium chloride solution (3 × 20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated by silica column chromatography (petroleum ether: ethyl acetate 20:1) to obtain the target product L4 (918 mg, 90% yield, white solid). 1 H NMR (400MHz, CDCl3) δ10.88(s,0H),9.15(s,0H),8.09(s,0H),8.03–7.94(m,1H),7.78–7.69(m,0H),7.68–7.61(m,1H),4.66(s,1H).

[0196] Example 4: Synthesis of L5

[0197]

[0198] Synthesize L5

[0199] Compound 4-1 (528 mg), sodium benzenesulfinate (1.64 g), and dimethyl sulfoxide (20 mL) were added sequentially to a 100 mL round-bottom flask. The mixture was heated to 50 °C and stirred for 2 hours. The mixture was diluted with 100 mL of ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated by silica column chromatography (petroleum:ethyl ether 1:1) to obtain the target product L5 (658 mg, 85% yield, pale yellow solid). 1 H NMR (400MHz, CDCl3) δ10.94 (s, 1H), 9.15 (s, 1H), 8.70 (s, 1H), 8.10 (d, J = 7.2Hz, 2H) ,8.00(d,J=7.2Hz,2H),7.82–7.74(m,1H),7.74–7.65(m,3H),7.61(t,J=7.8Hz,2H).

[0200] Example 5: Synthesis of compound LP2

[0201]

[0202] Step 1: Synthesize LP2-3:

[0203] Ethyl 6-bromohexanoate (5.00 g) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 17.0 g) were added to a solution of tert-butyl hydroxycarbamate (7.50 g) in acetonitrile (75 mL). The mixture was stirred at 60 °C for 12 hours. LC-MS showed that the reaction was complete. The resulting solution was extracted with ethyl acetate (3 × 100 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (PE:EA = 1:1) to give a colorless oily 6-(((tert-butoxycarbonyl)amino)oxy)hexanoate (1.40 g, 23% yield). Characterization data: 1 HNMR(400MHz, CDCl3): δ7.09(s,1H),4.11–4.10(m,2H),3.46-3.35(m,2H),2.29-2. 25(m,2H),1.70-1.58(m,4H),1.46(s,9H),1.38-1.30(m,2H),1.24(t,J=7.2Hz,3H).

[0204] Step 2: Synthesize LP2-4:

[0205] To a solution of 1.40 g of 6-(((tert-butoxycarbonyl)amino)oxy)hexanoate in 20 mL of EtOH and 2 mL of H₂O, 0.570 g of NaOH was added. The mixture was stirred at 25 °C for 12 h. LC-MS showed that the reaction was complete. The pH of the mixture was adjusted to 3 with 1 M HCl, and the resulting solution was extracted with dichloromethane (25 mL × 3) and washed with saturated brine (10 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give a colorless oily 6-(((tert-butoxycarbonyl)amino)oxy)hexanoic acid (1.0 g, 80% yield), which was used directly for the next step without further purification. Characterization data: 1 H NMR (400MHz, CDCl3): δ3.48-3.40(m,2H),2.36-2.30(m,2H),1.72-1.59(m,4H),1.47(s,9H),1.41-1.33(m,2H).

[0206] Step 3: Synthesize LP2-6:

[0207] N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU, 66.8 mg) and 1-hydroxybenzotriazole (HOBt, 2.50 mg) were added to 10 mL of N,N-dimethylformamide (DMF) solution of LP2-5 (90 mg) and LP2-4 (20 mg). The mixture was stirred at 0 °C for 15 min, and then N,N-diisopropylethylamine (DIPEA, 42 mg) was added to the reaction mixture. The reaction mixture was stirred at 25 °C for 12 h. LC-MS showed that the reaction was complete. Water (10 mL) was added to the mixture, and the resulting mixture was extracted with ethyl acetate (25 mL × 3). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give LP2-6 (100 mg, 83% yield) as a yellow solid. LC-MS (ESI) m / z: 1374.7 [M+Na] +

[0208] Step 4: Synthesize LP2:

[0209] Trifluoroacetic acid (TFA, 1 mL) was added to a solution of LP2-6 (100 mg) in dichloromethane (5 mL). The reaction mixture was stirred at 0 °C for 2 hours. LC-MS showed the reaction was complete. The mixture was concentrated to give a crude product, which was further purified by reversed-phase column chromatography (0-45% ACN in H2O, 0.1% TFA) to give LP2 (41.8 mg, 35% yield, TFA salt) as a white solid. Characterization data: LC-MS (ESI) m / z: 1273.8 [M+Na] + .

[0210] 1H NMR (400MHz, DMSO-d6): δ11.02-10.95(m,1H),10.70(s,1H),10.00-9.90(m,1H),8.30-8.00(m,2H),7.95-7.75(m,2H),7.60-7.55(m, 2H),7.39-7.12(m,8H),6.00-5.95(m,1H),5.43-5.35(m,2H),5.02-4.95(m,2H),4.70-4.60(m,1H),4.45-4.35(m,3H),4.30-4.16(m, 2H),4.00-3.90(m,2H),3.81-3.51(m,2H),3.24-3.22(m,2H),3.19-3.16(m,2H),3.12-3.02(m,2H),2.97(s,1H),2.87-2.83(m,2H),2 .34-2.08(m,5H),1.97-1.90(m,2H),1.80-1.70(m,4H),1.65-1.45(m,8H),1.37-1.20(m,5H),1.08-0.96(m,7H),0.91-0.72(m,22H).

[0211] Example 6: Synthesis of compound LP6

[0212]

[0213] Step 1: Synthesize LP6-3:

[0214] LP6-1 (0.3 g) was added to a 50 mL three-necked flask, followed by 5 mL of N,N-dimethylformamide (DMF). Under nitrogen protection, LP6-2 (0.124 g) was added. At 25 °C, diisopropylethylamine (DIEA, 0.078 g) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU, 0.120 g) were added, and the reaction was carried out for 2 h. LC-MS was used to monitor the reaction progress. Post-treatment: The reaction solution was added to water (100 mL) and ethyl acetate (100 mL) and purified to obtain a yellow solid LP6-3 (0.19 g, yield 45%).

[0215] Characterization data: LC-MS (ESI) m / z: 1683.8 [M+H] +

[0216] Step 2: Synthesize LP6:

[0217] LP6-3 (60 mg, 1.0 eq) was added to a 10 mL three-necked flask, followed by the addition of dichloromethane (1 mL). Under nitrogen protection, triethylsilane (0.016 g, 4 eq) was added, and the mixture was reacted at 25 °C with trifluoroacetic acid (TFA, 0.078 g, 2.5 eq) at 25 °C for 2 h. The reaction was monitored by LC-MS to ensure complete reaction of the starting material. Post-treatment: The reaction was quenched by adding water (10 mL), and the mixture was purified by reverse phase to obtain yellow powder LP6 (0.02 g, yield 41.9%).

[0218] Characterization data: LC-MS (ESI) m / z: 1340.7 [M+Na] + 1363.7 [M+Na] + .

[0219] 1 H NMR (400MHz, DMSO) δ10.12–10.02(m,1H),8.48(t,J=5.4Hz,1H),8.44–8.24(m,3H),8.18(dd,J=7.9,4.0Hz,1H),8.10–7.96(m ,1H),7.94–7.86(m,1H),7.80–7.72(m,0.5H),7.68–7.58(m,2H),7.44–7.30(m,5.5H),7.29–7.17(m,1H),6.17–6.06(m,1H), 5.20–5.05(m,2H),4.85–4.63(m,2H),4.62–4.39(m,4H),4.37–4.16(m,4H),4.14–4.00(m,4H),3.99–3.88(m,4H),3.45–3.35 (m,2H),3.34–2.81(m,19H),2.55–1.97(m,8H),1.95–1.67(m,4H),1.67–1.26(m,11H),1.19–0.97(m,7H),0.97–0.75(m,21H).

[0220] Example 7: Synthesis of compound LP8

[0221]

[0222] Step 1: Synthesize LP8-3:

[0223] LP8-1 (1.98 g) and LP8-2 (950 mg) were dissolved in 40 mL of N,N-dimethylformamide (DMF) solution, and diisopropylethylamine (DIEA, 1.37 g) was added. The mixture was stirred at 15 °C for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography and lyophilized to obtain the target product LP8-3 (2 g, white viscous substance, yield: 80%). Characterization data: LC-MS (ESI) m / z: 733.3 [M+Na] +

[0224] Step 2: Synthesize LP8-5:

[0225] At 0°C, N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU, 88 mg) was added to a solution of LP8-4 (200 mg), LP8-3 (137 mg), and diisopropylethylamine (DIEA, 50 mg) in N,N-dimethylformamide (DMF) (4 mL). The reaction mixture was stirred at 15°C for 2 hours. The reaction mixture was directly separated by reverse-phase column chromatography and lyophilized to obtain the target product LP8-5 (320 mg, yield: 95%, white solid). LC-MS (ESI) m / z: 1729.8 [M+H] + .

[0226] Step 2: Synthesize LP8:

[0227] To a solution of LP8-5 (280 mg, 0.162 mmol) in dichloromethane (DCM, 2.8 mL), triisopropylsilane (0.14 mL) and trifluoroacetic acid (TFA, 0.56 mL) were added sequentially. The reaction mixture was stirred at 15 °C for 30 minutes. The reaction mixture was concentrated to remove DCM and TFA. The residue was dissolved in acetonitrile, directly separated by reverse-phase chromatography, and lyophilized to obtain the target product LP8 (65 mg, yield: 26.7%, white solid) with a purity of 98.7%. Characterization data: LC-MS (ESI) m / z: 1387.6 [M+H] +

[0228] Example 8: Synthesis of compound LP9

[0229]

[0230] Step 1: Synthesize LP9-3:

[0231] To a solution of LP9-1 (390 mg) in N,N-dimethylformamide (DMF) (8 mL), LP9-2 (430 mg), pyridine (Py, 2 mL), N-hydroxy-7-azabenzotriazole (HOAt, 80 mg), and diisopropylethylamine (DIEA, 76 mg) were added sequentially, and the mixture was stirred at 15 °C for 16 hours. The reaction solution was directly purified by reverse-phase column chromatography and lyophilized to obtain the target product LP9-3 (680 mg, white solid, yield: 92.2%). Characterization data: LC-MS (ESI) m / z: 1287.6 [M+H] + .

[0232] Step 2: Synthesize LP9-4:

[0233] At 0°C, a solution of lithium hydroxide (LiOH, 50 mg) in water (6 mL) was added to a MeOH / THF (6 mL / 6 mL) solution of LP9-3 (660 mg). The reaction mixture was stirred at 15°C for 2 hours. The reaction mixture was neutralized with acetic acid, separated by reverse-phase column chromatography, and lyophilized to obtain the target product LP9-4 (410 mg, yield: 72%, white solid, formate). Characterization data: LC-MS (ESI) m / z: 1051.7 [M+H] + .

[0234] Step 3: Synthesize LP9-5:

[0235] To a solution of LP8-3 (140 mg) in N,N-dimethylformamide (DMF) (3.5 mL), diisopropylethylamine (DIEA, 64 mg) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU, 75 mg) were added sequentially, and the reaction mixture was stirred at 5 °C for 1 hour. Under ice bath conditions (5 °C), a solution of LP9-4 (180 mg) in N,N-dimethylformamide (DMF) (1.5 mL) was added, and the reaction mixture was stirred at 15 °C for 1 hour. The reaction mixture was directly separated by reverse-phase column chromatography and lyophilized to obtain the target product LP9-5 (200 mg, yield: 70%, white solid). LC-MS (ESI) m / z: 1743.8 [M+H] + .

[0236] Step 4: Synthesize LP9-6:

[0237] Triisopropylsilane (0.09 mL) and trifluoroacetic acid (TFA, 0.36 mL) were added sequentially to a solution of LP9-5 (180 mg) in dichloromethane (DCM, 1.8 mL). The reaction mixture was stirred at 15 °C for 30 min. The DCM and TFA were concentrated to remove the DCM and TFA. The residue was dissolved in acetonitrile, separated directly by reverse-phase chromatography, and lyophilized to obtain the target product LP9-6 (45 mg, yield: 28.7%, white solid). Characterization data: LC-MS (ESI) m / z: 1401.6 [M+H] + .

[0238] Example 9: Synthesis of compound LP13

[0239]

[0240] Step 1: Synthesize LP13-2

[0241] Compound LP13-1 (958 mg), piperazine (860 mg), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU, 913 mg), diisopropylethylamine (DIEA, 310 mg), and acetonitrile (20 mL) were added sequentially to a 100 mL round-bottom flask. The reaction mixture was stirred at room temperature for 6 hours. The mixture was concentrated under reduced pressure, and the residue was separated by silica column chromatography to obtain the target product LP13-2 (985 mg, 90% yield, purple-red solid). Characterization data: LC-MS (ESI) m / z: 512.3074 [M+H] + .

[0242] Step 2: Synthesize LP13-4

[0243] Compounds LP13-2 (547 mg), LP13-3 (556 mg), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU, 456 mg), diisopropylethylamine (DIEA, 155 mg), and acetonitrile (20 mL) were added sequentially to a 100 mL round-bottom flask. The reaction mixture was stirred at room temperature for 4 hours. The mixture was concentrated under reduced pressure, and the residue was separated by silica column chromatography to obtain the target product LP13-4 (744 mg, 75% yield, purple-red solid). Characterization data: LC-MS (ESI) m / z: 957.4786 [M+H] + .

[0244] Step 3: Synthesize LP13

[0245] Compound LP13-4 (198 mg), triethylsilane (1 mL), dichloromethane (5 mL), and trifluoroacetic acid (5 mL) were added sequentially to a 100 mL round-bottom flask. The reaction mixture was stirred at room temperature for 6 hours. The product LP13 (78 mg, 60% yield, purple-red solid) was then concentrated under reduced pressure. Characterization data: LC-MS (ESI) m / z: 615.3216 [M+H] + .

[0246] Example 10: Preparation and characterization of natural protein conjugates containing disulfide bonds

[0247] The following are examples of methods for the preparation and analytical characterization of natural protein conjugates containing at least one disulfide bond pair. It should be noted that the following examples are only partial embodiments and are not limited to the embodiments described herein. For example, the protein is not limited to the protein currently shown, but can be any other protein containing at least one disulfide bond pair; the specific reaction process is as follows... Figure 1 As shown.

[0248] A general coupling process for preparing natural protein conjugates containing disulfide bonds

[0249] 200 μL of lysozyme (1.5 mg / mL, 0.1 mM, 1.0 eq.) and thioredoxin (Trx) were reacted with tris(2-carboxyethyl)phosphine (TCEP) in 1.5 mL centrifuge tubes. The protein concentration was diluted to 1.5 mg / mL (0.1 mM, 1.0 eq.) with 25 mM PBS buffer (25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl, pH 7.5). After vortexing and mixing, 0.01 mg / mL TCEP (0.04 mM, 4 eq.) aqueous solution was added, followed by the addition of heavy bridging reagents (i.e., disulfide bridging reagents) (FPPA, FPPN, FPPAc, BPNA, PPNA) (0.15 mM, 1.5 eq.). The mixture was then placed on a heated and cooled thermostatic mixer and reacted at 30 °C for 4 hours. The conjugates of the five re-bridged reagents (T1-T5) that re-bridged the disulfide bonds of lysozyme were named lysozyme-FPPA, lysozyme-FPPN, lysozyme-FPPAc, lysozyme-BPNA, and lysozyme-PPNA, respectively; the conjugate of FPPN that re-bridged the disulfide bonds of thioredoxin was named Trx_FPPN.

[0250] Coupling reactions of aldehyde groups and other functional molecules with different functional molecules

[0251] The reaction was carried out in 1.5 mL centrifuge tubes. 150 μL of thioredoxin disulfide heavy bridging molecules (1.5 mg / mL, 0.1 mM, 1.0 eq.) were added to 25 mM PBS buffer (25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl, pH 6.5). The N-terminal 1,2-aminothiol peptide (0.2 mM) was added first, and the mixture was vortexed and then placed on a thermostatic mixer at 30 °C for 5 hours. The resulting conjugate was named Trx_Py-Thz(2H)-H6.

[0252] After the connection is complete, use Zeba TM Desalination centrifuge column, 7KMWCO (Zeba TM Spin Desalting Columns (7KMWCO) removes free peptides.

[0253] General characterization methods for protein conjugates

[0254] (a) LC-MS (ESI-Q-TOF) analysis of the linker products

[0255] All samples were analyzed using an Agilent 1290 Series High Performance Liquid Chromatography (HPLC) system equipped with an Agilent 6545 Series Liquid Chromatography-Electrospray Ionization-Quadrupole Time-of-Flight (LC-ESI-Q-TOF) Mass Spectrometer.

[0256] Column: Agilent ZORBAX 300SB-C8 (2.1×150mm)

[0257] Mobile phase: Buffer A: double-distilled water containing 0.1% formic acid; Buffer B: acetonitrile containing 0.1% formic acid. Gradient elution was performed at a column temperature of 70°C, with the following linear gradient conditions: 0–2 min: 95% Buffer A, 5% Buffer B; 2–12 min: Buffer B increased from 10% to 60%; 12–14 min: Buffer B increased from 60% to 95%; 14–15 min: Buffer B decreased from 95% to 5%. The flow rate was maintained at 0.5 mL / min.

[0258] Mass spectrometry: The column effluent was continuously analyzed using a capillary electrospray ionization source connected to the Agilent 6545Q-TOF mass spectrometer. Mass spectra were acquired in positive electrospray ionization (ESI) mode, with the acquisition range set to 100–2500 Da, using profilometry mode to obtain the total ion chromatogram. The retention peaks of the target protein in the total ion chromatogram were selected using Bioconfirm software (Agilent Technologies Inc., V10.0), and the raw mass spectrometry data were converted to zero-charge mass spectra using a maximum entropy deconvolution algorithm.

[0259] The deconvolution settings are configured as follows: Mass spectrometry range: 100.0-2500.0 Daltons, Mass spectrometry step size: 1.0000 Daltons, Baseline factor: 7.00, Additives: Protons, Isotope width: Auto. This setting provides comprehensive analysis of protein samples, enabling accurate characterization of their quality and composition.

[0260] from Figure 2 and Figure 3 It can be seen that the solvent-exposed disulfide bonds (C6-C127) on lysozyme can be rebridged, with coupling yields ranging from 50% to 85%. The third functional group can further couple to peptides containing N-terminal cysteine ​​residues, with a coupling efficiency of nearly 100%.

[0261] Example 11: Preparation and Characterization of Peptide-Drug Conjugates

[0262] The following are examples of methods for the preparation and analytical characterization of peptide-functional molecule conjugates. It should be noted that the following examples are only partial embodiments and are not limited to the embodiments described herein. For example, the peptide is not limited to the peptide presented herein, but may be any other peptide; the functional molecule is not limited to the peptide presented herein, but may be any other biomolecule.

[0263] Coupling reaction of peptide-reduced dithiol with coupling reagent

[0264] Taking some commercially available cyclic peptides as examples, including but not limited to octreotide, somatostatin, catalytic peptides, and terlipressin, the reactions of these cyclic peptides with FPPA, FPPN, and FPPac are used as application examples. The reaction of 0.2 mM cyclic peptides with a heavy-bridge reagent is carried out in a 1.5 mL centrifuge tube. The pre-prepared cyclic peptide solution (0.2 mM, 1.0 eq.) is mixed with 25 mM PBS (25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl, pH 7.5) buffer. Then, TCEP (0.4 mM, 2 eq.) is added, followed immediately by the heavy-bridge reagent (0.25 mM, 1.25 eq.). After mixing, the mixture is placed in a heated-cooled thermostatic mixer and reacted continuously at 30°C for 3 hours to achieve a complete reaction with a coupling yield of over 95%. The resulting polypeptide disulfide rebridged conjugates were named octreotide-FPPA, octreotide-FPPN, octreotide-FPPAc, lypression-FPPN, oxytocin-FPPN, somatostatin-FPPN, and terlipressin-FPPN, respectively.

[0265] The coupling reaction between orthogonal groups (aldehyde and nitrile groups) and fluorescent molecules in disulfide bond-bridged polypeptides.

[0266] Taking rhodamine (Cys-Rhodamin B, LP13) containing 1,2-aminothiol as an example, it was reacted with the octreotide conjugate from the previous step. The reaction was carried out in a 1.5 mL centrifuge tube. 0.2 mM of the heavy-bridged conjugate (octreotide-FPPA, octreotide-FPPN) was added to 25 mM PBS buffer (25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl, pH 6.5), and 0.4 mM LP13 (2 eq., 25 mM stock solution dissolved in N,N-dimethylformamide (DMF)) was added. After mixing, the reaction was placed on a heated-cooled thermostatic mixer and reacted at 30 °C for 3 hours. The resulting conjugates were named octreotide-Py-Thz-RhB and octreotide-Py-Thz(2H)-RhB.

[0267] After connecting, first use BeyoDesalt. TM The G-10Mini desalting column removes free LP13, and the sample is then freeze-dried and preserved using a freeze dryer.

[0268] General characterization methods for peptide conjugates

[0269] (a) RP-HPLC analysis

[0270] High performance liquid chromatograph: High performance liquid chromatography system.

[0271] Column: BioResolve RP C8 (4.6×150mm, 3.2μm) (manufacturer: Phenomenex).

[0272] Mobile phase: Mobile phase A: 0.1% TFA-H2O; Mobile phase B: 0.1% TFA-ACN; Elution was performed according to the following program: 0-2 min, mobile phase A volume was 90%-90%, mobile phase B volume was 10%-10%; 2-12 min, mobile phase A volume was 90%-40%, mobile phase B volume was 10%-60%; 12-15 min, mobile phase A volume was 40%-10%, mobile phase B volume was 60%-90%.

[0273] Detection conditions: The mobile phase flow rate was set to 1 ml / min, the detection wavelength to 280 nm, and the column temperature to 70 ℃.

[0274] Experimental procedure: Take 5 μg of the coupled sample (converted to mass and volume according to concentration), add storage buffer to a final volume of 25 μL, and vortex to mix. Centrifuge at 12000 rpm for 5 min, inject 20 μL of the supernatant into the high-performance liquid chromatograph, elute using the above elution program, and record the chromatogram.

[0275] (b) LC-MS (ESI-Q-TOF) analysis of the linker products

[0276] All samples were analyzed using an Agilent 1290 Series High Performance Liquid Chromatography (HPLC) system equipped with an Agilent 6545 Series Liquid Chromatography-Electrospray Ionization-Quadrupole Time-of-Flight (LC-ESI-Q-TOF) Mass Spectrometer.

[0277] Column: Agilent ZORBAX 300SB-C8 (2.1×150mm)

[0278] Mobile phase: Buffer A: double-distilled water containing 0.1% formic acid; Buffer B: acetonitrile containing 0.1% formic acid. Gradient elution was performed at a column temperature of 70°C, with the following linear gradient conditions: 0–2 min: 95% Buffer A, 5% Buffer B; 2–12 min: Buffer B increased from 10% to 60%; 12–14 min: Buffer B increased from 60% to 95%; 14–15 min: Buffer B decreased from 95% to 5%. The flow rate was maintained at 0.5 mL / min.

[0279] Mass spectrometry: The column effluent was continuously analyzed using a capillary electrospray ionization source connected to the Agilent 6545Q-TOF mass spectrometer. Mass spectra were acquired in positive electrospray ionization (ESI) mode, with the acquisition range set to 100–2500 Da, using profilometry mode to obtain total ion chromatograms. Using Bioconfirm software (Agilent Technologies Inc., V10.0), the retention peaks of the target protein in the total ion chromatogram were selected to obtain raw mass spectrometry data with different charges.

[0280] from Figure 4 , Figure 5 and Figure 6 It can be seen that the disulfide bonds on octreotide, lysine vasopressin, oxytocin, somatostatin, and terlipressin can be nearly 100% coupled by the heavy bridging reagents FPPA, FPPN, and FPPAc. In lysine vasopressin and oxytocin, one cysteine ​​residue from the reduced disulfide bond is located at the N-terminus, resulting in a coupling product where the cyano group on FPPN is partially cyclized with a cysteine ​​residue. From... Figure 7 and Figure 8 It can be seen that the conjugate can be further conjugated to fluorescent molecules, which can be applied to imaging analysis of peptide internalization into cells.

[0281] Example 12: Preparation and Characterization of Antibody Fragment Conjugates

[0282] The following are examples of methods for the preparation and analytical characterization of antibody fragment conjugates. It should be noted that the following examples are only partial embodiments and are not limited to the embodiments described herein. For example, the antibody fragment is not limited to the antibody currently shown, but can be any other antibody fragment containing at least one disulfide bond pair.

[0283] Coupling reaction of reducing dithiol on antibody fragment with coupling reagent

[0284] Taking the reaction of trastuzumab Fab (Tmab-Fab) with five coupling reagents (i.e., disulfide bridging reagents) FPPA, FPPN, FPPAc, BPNA, and PPNA as an example, Tmab-Fab (100 μM, 200 μL) was added to 25 mM PBS (25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl, pH 7.5) buffer, followed by the addition of 0.5 mM TCEP (5 eq.) aqueous solution. After mixing, 125 μM of the coupling reagent (1.25 eq., 25 mM stock solution dissolved in N,N-dimethylformamide (DMF)) was added, and the mixture was stirred. The reaction was then placed on a heated and cooled thermostatic mixer and reacted at 30 °C for 5 hours. The resulting rebridged antibody fragments were named Tmab_Fab-L1, Tmab_Fab-L2, Tmab_Fab-L3, Tmab_Fab-L4, and Tmab_Fab-L5, respectively.

[0285] After the connection is complete, use Zeba first. TM Desalination centrifuge column, 7K MWCO (Zeba TM Spin Desalting Columns (7KMWCO) removes free coupling reagents.

[0286] from Figure 9 It can be seen that the pair of disulfide bonds at the Fab tail of the antibody fragment can be rebridged by five rebridge reagents, with a coupling ratio greater than 95%; from Figure 10 It can be seen that more than 95% of the disulfide bonds are rebridged, with a small amount of single-linked products (L and H chains).

[0287] The coupling reaction of Tmab-Fab conjugate with N-terminal cysteine-containing peptides

[0288] Taking the reaction of Tmab-Fab conjugates with FPPA, FPPN, and BPNA with peptides (CGGSHHHHHH) as an example, 100 μM Tmab-Fab conjugate was dissolved in 25 mM PBS buffer (25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl, pH 6.5), and 0.2 mM of a peptide containing an N-terminal cysteine ​​(2 eq., 10 mM stock solution dissolved in H2O) was added. Then, 0.1 mM TCEP was added, and the mixture was stirred. The reaction was then carried out at 30°C for 5 hours on a thermostatic mixer. The resulting conjugates were named Tmab_Fab-L1-Thz-H6, Tmab_Fab-L2-Thz(2H)-H6, and Tmab_Fab-L4-Thz-H6, respectively.

[0289] After the connection is complete, use Zeba first. TM Desalination centrifuge column, 7K MWCO (Zeba TM The free peptides were removed by Spin Desalting Columns (7K MWCO), and then repeatedly filtered and purified using an Amicron-Ultra centrifugal filter (10000MWCO, Merck Millipore) to remove free peptides that were not coupled with the aldehyde or cyano groups of the Tmab-Fab conjugate in the reaction solution.

[0290] from Figure 11 It can be seen that the third functional group on the pyridine ring of the Fab coupling can react efficiently with peptides containing N-terminal cysteine. The coupling efficiency of the nitrile group is over 70%, and the coupling efficiency of the aldehyde group is over 95%.

[0291] Example 13: A general conjugation process for preparing antibody-drug conjugates based on FPPA

[0292] Coupling reaction of antibody-chain reduced dithiols with FPPA

[0293] The reaction of monoclonal antibody with FPPA was carried out in 5 mL centrifuge tubes. The antibody concentration was diluted to 10 μM (1.5 mg / mL, 1.0 eq.) with 25 mM PBS (25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl) pH 7.5 buffer. After vortexing and mixing, 10 μM TCEP (0.02 mg / mL, 8 eq.) aqueous solution was added first, followed by FPPA (0.012 mg / mL, 4.5 eq., FPPA stock solution dissolved in N,N-dimethylformamide (DMF)) to a final concentration of 45 μM. The DMF content in the entire reaction solution was less than 1%. After vortexing and mixing, the mixture was placed in a heated and cooled thermostatic mixer and reacted continuously at 30 °C for 12 hours.

[0294] After ligation, the solution was repeatedly purified by filtration using an Amicron-Ultra centrifugal filter (50000MWCO, Merck Millipore) to remove unreacted FPPA from the reaction solution. The product from this step was named Tmab-PyA. Figure 12 and Figure 13 It can be seen that the coupling ratio of FPPA to the interchain disulfide bonds of the whole antibody is greater than 90%, mainly generating interchain coupled whole antibodies and intrachain coupled half antibodies; FPPA-modified L chain is named L-PPA, the two pairs of disulfide bonds on the two FPPA heavy-bridged half antibody (HL) are named HL-2PyA, and the four pairs of disulfide bonds on the four FPPA heavy-bridged whole antibody (LHHL) are named HL-4PyA.

[0295] Coupling reactions of aldehyde groups with different linker-drug conjugates in Tmab-PyA

[0296] The reaction was carried out in 5 mL centrifuge tubes. The Tmab-4PyA concentration was diluted to 20 μM (3.0 mg / mL, 1.0 eq.) with 25 mM PBS (25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl) pH 5.0 or pH 7.2 buffer. After vortexing, 80 μM of linker-drug conjugates (LP2, LP6, LP8, LP9, LP13) (4 eq., 10 mM stock solution dissolved in DMF) was added. After vortexing, the reaction was placed on a heated-cooled thermostatic mixer and reacted at 30 °C for 3 hours. Then, 80 μM of linker-conjugates (4 eq.) were added, vortexed, and the reaction was continued on a heated-cooled thermostatic mixer at 30 °C for another 9 hours. The DMF content in the entire reaction solution was less than 1%.

[0297] After the connection is complete, use Zeba first.TM Desalination centrifuge column, 7K MWCO (Zeba TM The free linker conjugate was removed by Spin Desalting Columns (7K MWCO), and then repeatedly filtered and purified using an Amicron-Ultra centrifugal filter (50000MWCO, Merck Millipore) to remove the free linker-drug conjugate in the reaction solution.

[0298] Next, use 300 mg / mL dextran-coated activated charcoal (manufacturer: Sigma) to remove residual toxin linkers-drug conjugates that are not conjugated with the antibody from the reaction solution. Add 10% (v / v) of activated charcoal solution to the reaction solution, mix well, and shake for 2 hours at 4°C. Repeat this process three times. After treatment, centrifuge the mixture, collect the supernatant using a syringe and a hydrophilic membrane filter, and filter out the activated charcoal. Replace the buffer solution for the desired antibody-drug conjugate with a suitable storage buffer through four ultrafiltrations and store at -80°C.

[0299] General characterization methods for antibody-drug conjugates

[0300] (a) LC-MS analysis

[0301] All samples were analyzed using an Agilent 1290 Series High Performance Liquid Chromatography (HPLC) system equipped with an Agilent 6545 Series Liquid Chromatography-Electrospray Ionization-Quadrupole Time-of-Flight (LC-ESI-Q-TOF) Mass Spectrometer.

[0302] Column: Agilent ZORBAX 300SB-C8 (2.1×150mm)

[0303] Mobile phase: Buffer A: double-distilled water containing 0.1% formic acid; Buffer B: acetonitrile containing 0.1% formic acid. Gradient elution was performed at a column temperature of 70°C, with the following linear gradient conditions: 0–2 min: 95% Buffer A, 5% Buffer B; 2–12 min: Buffer B increased from 10% to 60%; 12–14 min: Buffer B increased from 60% to 95%; 14–15 min: Buffer B decreased from 95% to 5%. The flow rate was maintained at 0.5 mL / min.

[0304] Mass spectrometry: The column effluent was continuously analyzed using a capillary electrospray ionization source connected to the Agilent 6545Q-TOF mass spectrometer. Mass spectra were acquired in positive electrospray ionization (ESI) mode, with the acquisition range set to 600–3200 Da, using profilometry mode to obtain the total ion chromatogram. The retention peaks of the target protein in the total ion chromatogram were selected using Bioconfirm software (Agilent Technologies Inc., V10.0), and the raw mass spectrometry data were converted to zero-charge mass spectra using a maximum entropy deconvolution algorithm.

[0305] The deconvolution settings are configured as follows: Mass spectrometry range: 600.0-3200.0 Daltons, Mass spectrometry step size: 1.0000 Daltons, Baseline factor: 7.00, Additives: Protons, Isotope width: Auto. This setting provides comprehensive analysis of protein samples, enabling accurate characterization of their quality and composition.

[0306] from Figure 12 The LC-MS results showed that the half antibody (HL) was covalently modified with two FPPA molecules (HL-2PyA), and the whole antibody (LHHL) was covalently modified with four FPPA molecules (LHHL-4PyA). There were only a small amount of free light chain (L-PPA). Due to the large difference in molecular weight between HL and LHHL, the intensity of the mass spectrum peak does not represent the actual intensity of the species.

[0307] (b) SEC-HPLC analysis of ADC aggregates

[0308] High performance liquid chromatograph: Agilent 1260 liquid chromatograph.

[0309] Column: Waters Xbridge BEH200 SEC (7.8×300mm, 3.5μm)

[0310] Mobile phase: 50mM PB, 100mM NaCl, pH 7.2. Elute according to the following procedure, with the volume of mobile phase A being 100% for 0-30 min.

[0311] Detection conditions: The mobile phase flow rate was set to 0.5 ml / min, the detection wavelength to 280 nm, and the column temperature to 30 ℃.

[0312] Experimental procedure: Take 20 μg of the coupled sample (volume depends on sample concentration), inject it into the high performance liquid chromatograph, elute using the above elution procedure, and record the chromatogram.

[0313] Calculation formulas: Monomer purity (%) = (A monomer / A total) × 100%; Polymer purity (%) = (A polymer / A total) × 100%

[0314] (c) Reductive SDS-PAGE analysis of the ADC connector coupling method and ADC components

[0315] Electrophoresis apparatus: Tianneng EPS-600

[0316] Acrylamide gel: GenScript M00656

[0317] Imaging System Analyzer: Tianneng 4600SF

[0318] Experimental Procedure: Mix 5 μg of sample with the corresponding loading buffer and heat in a boiling water bath for 5 minutes; sequentially add the sample and standard protein (10 μL / well) to the wells of a 12% stacking gel and electrophoresis at a constant voltage of 160 V for 60 minutes; remove the gel, rinse once with deionized water, then add an appropriate amount of Coomassie Brilliant Blue R250 staining solution to cover the gel and stain on a shaker for 0.3 hours; rinse the stained gel three times with deionized water, shaking on a horizontal shaker for 30 minutes each time; after destaining, remove the gel and transfer it to an imaging system to record gel images. For samples with Tmab-Py-Thz-RhB coupled fluorescent molecules, first photograph them using a gel fluorescence imaging system before subsequent staining and destaining.

[0319] from Figure 13 Analysis shows that the presence of bands ≥50 kDa indicates that thiol bridging coupling is effective (H, L). A 75 kDa band represents a portion consisting of a heavy chain and a light chain linked by a pair of bridging thiol groups (LH). A band formed by two heavy chains linked by one or two pairs of bridging thiol groups is 100 kDa (HH). A 125 kDa band represents only one heavy chain and light chain without bridging (LHH). Finally, a 150 kDa band represents all chains being bridged with the antibody (LHHL).

[0320] Example 14: Construction of antibody-fluorescent conjugates

[0321]

[0322] Following the general conjugation procedure for preparing FPPA-based antibody-drug conjugates, TCEP (0.028 mg / mL, 8.0 eq.) aqueous solution was added to a buffer solution of trastuzumab (1.5 mg / mL, 1.0 eq.) containing 25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl, and pH 7.5, and the mixture was reduced at 30°C for 2 hours. Then, FPPA (0.012 mg / mL, 4.5 eq.) was added, and the conjugation reaction was carried out at 30°C for 12 hours. After repeated filtration five times, Tmab-4PyA was obtained. Tmab-4PyA was then diluted to 4.5 mg / mL with 25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl, and pH 7.0. 0.11 mg / mL LP13 (6 eq.) was added, and the reaction was carried out at 30°C for 10 hours. Finally, the reaction solution was repeatedly filtered and washed three times, and then free LP13 was removed by BeyoDesalt™ G-10Mini to finally obtain Tmab-Py-Thz-RhB (C(mg / mL): 3.6, yield 75-80%).

[0323] from Figure 14 It can be seen that the proportion of coupling with 4 rhodamine molecules is greater than 80%.

[0324] Example 15: Application of antibody-fluorescent conjugates in plasma stability analysis

[0325] The Tmab_Py-Thz-RhB conjugate (Tmab-RhB for short) was added to 35% human plasma (female) in 50 mM PBS buffer (45.5 mM Na2HPO4, 4.5 mM KH2PO4, 150 mM NaCl, 5 mM EDTA, pH 7.4, final conjugate concentration 0.37 mg / mL (2.5 μM). At the initial time, a 20 μL sample of the mixture was taken and 6 μL of 6×SDS-PAGE loading buffer (without reducing agents) was added, and the mixture was rapidly frozen and stored at -20°C. The remaining mixture was gently shaken (200 rpm) at 37°C and incubated in the dark. Samples (20 μL) were taken on days 0, 1, 3, 5, 7, 9, 11, and 14, and added to 6 μL of the buffer. The 6×SDS-PAGE loading buffer was thoroughly mixed, rapidly frozen, and stored at -20°C. Finally, the sample was analyzed by 10% SDS-PAGE (samples were run at 160V for 70 minutes). Fluorescent bands were observed using a TITAN 100 followed by Coomassie Brilliant Blue staining.

[0326] from Figure 15 It can be seen that the antibody-fluorescent conjugate constructed using the heavy-bridge reagent FPPA has high stability in plasma.

[0327] Example 16: Application of antibody-fluorescent conjugates in intracellular imaging analysis

[0328] To test the endocytosis efficiency of the Tmab-PyA conjugate by tumor cells, SK-Br-3 cells (HRE2-positive cells) and MCF7 cells (HER2-negative cells) were seeded at a density of 20,000 cells / well on poly-D-lysine-coated coverslips and placed in 24-well culture plates. Cells were incubated at 37°C and 5% CO2 for 24 hours. Cells were washed three times with 1×PBS buffer (pH 7.2-7.4). Three experimental groups were set up. First, 300 μL of 25 nM Tmab-PyA-Thz-RhB conjugate and DMEM medium were added to the cells as a blank control, followed by incubation at 4°C in the dark for 1 hour. Cells were washed with PBS to remove unbound antibodies. Next, one group of cells was fixed with 4% paraformaldehyde and stained with Hoechst 33342 to observe antibody binding to the cell membrane surface. The other two groups were supplemented with 600 μL of complete DMEM medium containing 10% FBS and incubated at 37°C and 5% CO2 for 3.0 h or 24 h to assess the endocytosis efficiency of the antibody conjugates. At each time point (3 h and 24 h), the growth medium was removed, cells were washed three times with PBS, and then fixed with 4% paraformaldehyde for 10 min. After fixation, the cells were washed three more times with PBS and incubated with Hoechst 33342 for 5 min for nuclear staining. Finally, coverslips with fixed cells were mounted onto glass slides.

[0329] Cell imaging was performed using a confocal laser scanning microscope. Confocal imaging was performed using an Olympus IX83 inverted microscope equipped with a FluoView 3000 scanning system (Olympus, Center Valley, PA). Standard emission filters (DM405 / 488 / 561) were used. Kaede-red at 561 nm and Hoechst at 333-42405 nm were selected as dyes and detectors, respectively. All images were acquired using UPLSAPO ×40 0.95 numerical aperture dry objectives (Olympus).

[0330] from Figure 16 It can be seen that the antibody-fluorescent conjugate is effectively internalized into the cell. After 3 hours and 24 hours of incubation, it gradually enters the lysosome from the cell, indicating that the antibody conjugate with the FPPA heavy-bridged inter-chain disulfide bond conformation has a high cell internalization efficiency.

[0331] Example 17: Construction of Tmab-GFP conjugate

[0332]

[0333] 4.5 mg / mL Tmab-4PyA was added to 25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl, pH 7.0. The recombinant GFP protein was first added, and CGGSGGSGGS (3.5 mg / mL, 4 eq.) was fused to the N-terminus. The reaction was carried out at 30 °C for 8-10 hours. The reaction solution was then repeatedly filtered and washed three times. Uncoupled free GFP was not removed (it can be purified by SEC or ion exchange column analysis), ultimately yielding Tmab-Py-Thz-GFP in 80% yield.

[0334] from Figure 17 It can be seen that on average, two fluorescent proteins are coupled to each antibody. From... Figure 18 As shown in the figure, the antibody-fluorescent protein conjugate maintains high stability even after prolonged incubation in human plasma, proving that the thioether bond formed by the pyridine derivative has high stability.

[0335] Example 18: Preparation of antibody-drug conjugate ADC-2

[0336]

[0337] Following the general conjugation procedure for preparing FPPA-based antibody-drug conjugates, TCEP (0.028 mg / mL, 8.0 eq.) aqueous solution was added to a buffer solution of trastuzumab (1.5 mg / mL, 1.0 eq.) containing 25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl, and pH 7.5. Immediately afterward, FPPA (0.012 mg / mL, 4.5 eq.) was added, and the conjugation reaction was carried out at 30°C for 8 hours. After purification by repeated filtration five times, Tmab-PyA was obtained. Tmab-PyA was then diluted to 3.0 mg / mL with 25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl, and pH 5.0. 0.3 mg / mL LP2 (12 eq.) was added, and the reaction was carried out at 30°C for 10 hours. Finally, the reaction solution was repeatedly filtered and washed three times, and then passed through dextran-coated activated carbon to remove free LP2, ultimately yielding ADC-2 (C ADC (mg / mL): 2.43, yield 75-80%).

[0338] from Figure 19 and Figure 20 It can be seen that the single-distribution component (DAR4) accounts for 60-70% of the obtained ADC-2. Figure 21 It can be seen that the aggregate size is less than 1%, and in addition... Figure 19The ratio of the half antibody (DAR4) formed by bridging between light and heavy chains and bridging within heavy chains to the full antibody (DAR4) formed by bridging between heavy chains is approximately 7:10.

[0339] Example 19: Preparation of antibody-drug conjugate ADC-3

[0340]

[0341] 3.0 mg / mL Tmab-4PyA was reacted with 0.22 mg / mL LP6 (8 eq.) in a solution of 25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl, and pH 7.0 at 30 °C for 10 hours. The reaction solution was then repeatedly filtered and washed three times, followed by dextran-coated activated carbon to remove free LP6, ultimately yielding ADC-3 (C ADC (mg / mL): 2.43, yield 75-80%).

[0342] from Figure 19 and Figure 20 It can be seen that the proportion of the single-distributed component (DAR4) in the obtained ADC-3 is 85%-90%, which is an increase compared to the DAR4 proportion in ADC-2. Figure 21 It can be seen that there are no aggregated components. Figure 19 The ratio of the half antibody (DAR4) formed by bridging between light and heavy chains and bridging within heavy chains to the full antibody (DAR4) formed by bridging between heavy chains is approximately 7:10.

[0343] Example 20: Preparation of antibody-drug conjugate ADC-4

[0344]

[0345] 3.0 mg / mL Tmab-4PyA was reacted with 0.22 mg / mL LP8 (8 eq.) in a solution of 25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl, and pH 7.0 at 30°C for 10 hours. The reaction solution was then repeatedly filtered and washed three times, followed by dextran-coated activated carbon to remove free LP8, ultimately yielding ADC-4 (C ADC (mg / mL): 2.43, yield 75-80%).

[0346] from Figure 19 and Figure 20 It can be seen that the single-distribution component (DAR4) accounts for 85%-90% of the obtained ADC-4. Figure 21 It can be seen that the aggregated component of the antibody-drug conjugate is less than 5%.

[0347] Example 21: Preparation of antibody-drug conjugate ADC-5

[0348]

[0349] 3.0 mg / mL Tmab-4PyA was reacted with 0.22 mg / mL LP9 (8 eq.) in a solution of 25 mM Na2HPO4, 5 mM EDTA, 150 mM NaCl, and pH 7.0 at 30 °C for 10 hours. The reaction solution was then repeatedly filtered and washed three times, followed by dextran-coated activated carbon to remove free LP9, ultimately yielding ADC-5 (C ADC (mg / mL): 0.66, yield 75-80%).

[0350] from Figure 19 and Figure 20 It can be seen that the single-distribution component (DAR4) accounts for 85%-90% of the obtained ADC-5. Figure 21 It can be seen that the aggregated component of the antibody-drug conjugate is less than 5%.

[0351] Example 22: Preparation of antibody-drug conjugate ADC-8

[0352]

[0353] 3.0 mg / mL Tmab-4PyA was reacted in a solution of 25 mM Na₂HPO₄, 5 mM EDTA, 150 mM NaCl, pH 7.0. First, 0.11 mg / mL LP₈ (4 eq.) was added, and the reaction was carried out at 30 °C for 4 hours. Then, 0.11 mg / mL LP₈ (4 eq.) was added, and the reaction was carried out at 30 °C for 8 hours. Finally, the reaction solution was repeatedly filtered and washed three times, and then passed through dextran-coated activated carbon to remove free LP₈ and LP₈, ultimately yielding ADC-8 (C ADC (mg / mL): 2.4, yield 75-80%).

[0354] from Figure 19 and Figure 20 It can be seen that the proportion of the single-distributed component (DAR4) in the obtained ADC-8 is >95%, from Figure 21 It can be seen that the aggregated component of the antibody-drug conjugate is less than 5% after incubation at 37°C for 48 hours.

[0355] Example 23: Biological Evaluation

[0356] Tumor cell proliferation inhibition assay based on antibody-drug conjugates prepared by FPPA

[0357] 5×10 3Cells were seeded in 96-well plates (excluding wells filled with PBS) and cultured in 100 μL of suitable culture medium in a 37°C, 5% CO2 incubator. After overnight culture in the incubator (37°C, 5% CO2), the supernatant was removed, and 100 μL of antibody-drug conjugate solution diluted with DMEM was added to each well, bringing the final conjugate concentration range to 0.1 pM-50 nM (SK-Br-3 cells), 0.5 pM-250 nM (NCI-N87 cells), and 1.0 pM-250 nM (MCF7 cells). After co-incubating the cells and conjugate for 72 h, the 96-well plates were equilibrated at room temperature for 30 min. 40 μL of the conjugate solution was then added to each well. The (Promega, G7572) reagent was reacted in the dark for 10 min, and the fluorescence value was measured using a SpectraMax i3X microplate reader. A curve of fluorescence value versus conjugate concentration (nM) was fitted using Graphpad Prism software.

[0358] The in vitro cytotoxicity of the antibody-drug conjugate (++++: <1 nM; +++: <10 nM; ++: 10-200 nM; +: >200 nM) is shown in Table 1.

[0359] Table 1

[0360]

[0361] Table 1 shows the biological activities of ADC-2, ADC-3, ADC-4, ADC-5, and ADC-8 prepared in Examples 18-22 of this invention. As can be seen from the table, the IC50 values ​​of the prepared ADCs against HER2-positive cells SK-Br-3 are as low as 3 pM, and the IC50 values ​​against HER2-positive cells NCI-N87 with low expression of HER2 receptor are as low as 22 pM. However, they have almost no cytotoxicity against HER2-negative cells within the measured concentration range.

[0362] In summary, this invention provides a novel pyridine derivative bridging reagent that is simple to synthesize, highly water-soluble, and capable of reacting selectively with reduced dithiols, producing conjugates with high stability and uniformity. This bridging reagent enables efficient modification and functionalization of peptides, proteins, antibodies, and their fragments, broadening the application range of biomolecules. This invention also provides specific applications of the above-mentioned conjugates in cell imaging, disease diagnosis, and treatment, particularly the application of antibody-drug conjugates (ADCs) in cancer treatment, demonstrating significant biological activity and therapeutic effects.

[0363] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, or improvements within the spirit of the present invention are covered within the scope of the claims of the present invention. All patent and non-patent literature mentioned in this invention are incorporated herein by reference.

Claims

1. A disulfide bridging reagent, characterized in that, The bridging reagent has the general structural formula shown in Formula IB: ⅠB； in, X in Formula IB 1 Selected from -SO2R1; wherein, R1 is selected from phenyl or halogen-substituted phenyl; X in Formula IB 2 Selected from -SO2R2, halogen, halogen-substituted C1-C10 alkyl groups; wherein, R2 is selected from phenyl or halogen-substituted phenyl groups; X in Formula IB 3 Selected from -CHO and -CN.

2. The disulfide bridging reagent according to claim 1, characterized in that, X in Formula IB 2 Selected from -SO2R2, halogens, and halogen-substituted C1-C5 alkyl groups; X in Formula IB 3 Selected from -CHO, -CN; The halogen is selected from F, Cl, and Br.

3. The disulfide bridging reagent according to claim 2, characterized in that, X in Formula IB 1 Selected from -SO2Ph; and / or, X in Formula IB 2 Selected from -SO2Ph, -F, -Cl, -Br, -I; and / or, X in Formula IB 3 Selected from -CHO and -CN.

4. The disulfide bridging reagent according to claim 3, characterized in that, The disulfide multibridge reagent is selected from at least one of the following compounds: 。 5. A method for preparing a disulfide bridging reagent as described in any one of claims 1-4, characterized in that, The preparation method of the compound represented by formula IB is selected from method one or method two; Method 1 includes the following steps: Step (1): Compound IB-1 is dissolved in an organic solvent, and oxalyl chloride is added under ice bath conditions for a primary reaction. After concentration under reduced pressure, methanol and triethylamine are added for a secondary reaction to obtain compound IB-2. The structural formulas of compounds IB-1 and IB-2 are as follows: ; Step (2): IB-2 is dissolved in an organic solvent, and a reducing agent is added under ice bath conditions to carry out a reduction reaction to obtain compound IB-3; wherein, the structural formula of compound IB-3 is as follows: ; Step (3): IB-3 is dissolved in an organic solvent, and oxidant is added under ice bath conditions, followed by heating to carry out an oxidation reaction, yielding compound IB-4; the structural formula of compound IB-4 is as follows: ; Step (4): Compound IB-4 undergoes a secondary oxidation reaction in a solvent with the addition of oxidant II to obtain compound with the general formula IB-5-0,X. 3 =CHO; ; Optionally, in step (4-2), compound IB-5-0 is reacted in methanol solvent with the addition of hydroxylamine hydrochloride in a single reaction to give the compound a solution in acetonitrile solvent. Triethylamine and dimethyl sulfoxide are then added, and oxalyl chloride is added dropwise under ice bath conditions to generate compound IB-5-2 in a second reaction. The structural formula of compound IB-5-2 is as follows: ； Step (5): Compound IB-5 is reacted with trifluoroacetic anhydride in an organic solvent to obtain compound IB-6; compound IB-5 is selected from compound IB-5-0 or IB-5-2; The structural formula of compound IB-6 is as follows: In compound IB-6, X 3 X in claims 1-4 3 The corresponding ones in the text are the same; Step (6): Compound IB-6 reacts with sodium benzenesulfinate in an organic solvent to obtain compound IB-7-0; the structural formula of compound IB-7-0 is as follows: IB-7-0; In compound IB-7-0, X 3 X in claims 1-4 3 The corresponding ones in the text are the same; Step (7): Compound IB-7 and organic solvent were added to phosphorus tribromide under ice bath conditions, and the temperature was raised to react and obtain compound IB-8; compound IB-7 was selected from compound IB-7-0; In compound IB-8, X 1 -SO2Ph; X 3 X in claims 1-4 3 The corresponding ones in the text are the same; Method 2: Using IB-9 as the starting material, R4 as a halogen, X 3 It has an aldehyde group, is soluble in an organic solvent, and then reacts with sodium benzenesulfinate to give compound IB-10,X. 1 X 2 Both are -SO2Ph; In IB-9, both R4 atoms are identical, both being halogens, and X... 3 X in claims 1-4 3 The corresponding ones in the text are the same; ;IB-10 X 3 X in claims 1-4 3 The corresponding ones are the same.

6. The method for preparing the disulfide bridging reagent according to claim 5, characterized in that, In Method 1, in step (1), The organic solvents are dichloromethane and N,N-dimethylformamide; The molar ratio of IB-1 to oxalyl chloride is 1:2-4; The ratio of IB-1 to dichloromethane is 1 g : 10-15 mL; The ratio of IB-1 to N,N-dimethylformamide is 1 g : 10-15 μL; The primary reaction temperature is 0–4°C; The reaction time is 4–6 hours. The ratio of IB-1 to methanol is 1 g : 10-15 mL; The molar ratio of IB-1 to triethylamine is 1:1.5-2; The secondary reaction temperature is 20–25°C; The secondary reaction time is 4–6 hours; and / or, In method one, in step (2), The organic solvent is tetrahydrofuran; The reducing agent is lithium aluminum hydride; The molar ratio of IB-2 to the reducing agent is 1:1-1.5; The ratio of IB-2 to organic solvent is 1 g : 10-15 mL; The reduction reaction occurs at temperatures of 0–4°C. The reduction reaction takes 1-2 hours; and / or, In method one, in step (3), The organic solvent is dichloromethane; The oxidizing agent is m-chloroperoxybenzoic acid; The molar ratio of IB-3 to oxidant one is 1:1.2-1.5; The ratio of IB-3 to organic solvent is 1 g : 9-12 mL; The oxidation reaction takes place at a temperature of 20–25°C. The oxidation reaction takes 1–1.5 hours; and / or, In method one, in step (4), The organic solvent is at least one of dichloromethane, chloroform, or tetrahydrofuran; Oxidizing agent two consists of active manganese dioxide, tin dioxide, and Desmartin oxidizing agent; The molar ratio of IB-4 to oxidant II is 1:3-5; The ratio of IB-4 to organic solvent is 1g : 20-30 mL; The temperature for the secondary oxidation reaction is 60–70°C; The secondary oxidation reaction takes 8–10 hours; and / or, In Method 1, in step (4-2), The molar ratio of IB-5-0 to hydroxylamine hydrochloride is 1:10-15; The ratio of IB-5-0 to methanol is 1g : 10-50 mL; The temperature for one reaction is 20°C–25°C; The reaction time is 5–8 hours; The molar ratio of IB-5-0 to triethylamine is 1:2-3; The molar ratio of IB-5-0 to dimethyl sulfoxide is 80-100:1; The molar ratio of IB-5-0 to oxalyl chloride is 1:1.0-1.5; The ratio of IB-5-0 to acetonitrile is 1 g : 12-18 mL; The temperature for the secondary reaction is 20°C–25°C; The secondary reaction takes 9–11 hours; In method one, in step (5), The organic solvent is chloroform; The molar ratio of compound IB-5 to trifluoroacetic anhydride is 1:2-3; The ratio of compound IB-5 to organic solvent is 1 g : 8-12 mL; The reaction temperature is 20°C–25°C; The reaction time is 10–25 hours; and / or, In method one, in step (6), The organic solvent is dimethyl sulfoxide; The molar ratio of compound IB-6 to sodium benzenesulfinate is 1:2.5-3.5; The ratio of compound IB-6 to organic solvent is 1 g : 15-20 mL; The reaction temperature is 45°C–55°C; The reaction time is 2–4 hours; And / or, In method one, in step (7), The organic solvent is chloroform; The molar ratio of compound IB-7 to phosphorus tribromide is 1:2.0-4.0; The ratio of compound IB-7 to organic solvent is 1 g : 20-30 mL; The reaction temperature is 20°C–25°C; The reaction time is 2–4 hours; and / or, In method two, The organic solvent is dimethyl sulfoxide; The molar ratio of compound IB-9 to sodium benzenesulfinate is 1:4.5-5.5; The ratio of compound IB-9 to organic solvent is 1 g : 30-40 mL; The reaction temperature is 45–55℃; The reaction time is 1.5–3.0 hours.

7. A biomolecule covalently bridged by a disulfide, characterized in that, The disulfide-bridged biomolecules have the following general structural formula IIA or general formula IIB or their salt forms: Formula IIA; Formula IIB; Among them, the The corresponding precursor is a biomolecule containing at least two cysteine ​​residues. or biomolecules containing at least one pair of disulfide bonds. ; The X 3 X in the disulfide bridging agent according to any one of claims 1-4 3 The corresponding ones are the same; In Equations IIA and IIB, n1 is selected from integers from 1 to 8.

8. The disulfide-bridged biomolecule according to claim 7, characterized in that, The The corresponding precursor is selected from polypeptides, proteins, antibodies or antibody fragments containing at least two cysteine ​​residues or polypeptides, proteins, antibodies or antibody fragments containing at least one disulfide bond.

9. The disulfide-bridged modified biomolecule according to claim 8, characterized in that, The polypeptide is an active cyclic peptide containing at least one pair of disulfide bonds, a linear polypeptide containing at least one pair of disulfide bonds, two polypeptide chains linked by a pair of disulfide bonds; or a linear polypeptide or cyclic peptide composed of 1-50 amino acids containing at least two cysteine ​​residues. The protein is a native protein containing at least one pair of solvent-oriented disulfide bonds, a recombinant protein containing at least two adjacent cysteine ​​residues, or a protein complex containing at least one pair of disulfide bonds covalently linked; the antibody or antibody fragment is an antibody or antibody fragment comprising at least one pair of accessible disulfide bonds, the antibody fragment comprising one or more portions of a single antibody that retain the ability to specifically bind to an epitope.

10. The disulfide-bridged biomolecule according to claim 9, characterized in that, The polypeptide is selected from at least one of octreotide, somatostatin, oxytocin, terlipressin, lysine vasopressin, RGD tripeptide and its homologs; The protein is selected from at least one of lysozyme, thioredoxin, serum protein, green fluorescent protein, small ubiquitin protein, ubiquitin ligase, heat shock chaperone protein, epidermal growth factor receptor protein, copper transporter, transferrin, calmodulin, mouse sarcoma protein, and peptidyl proline isomerase. The antibody or antibody fragment is selected from at least one of the following: mouse antibody, rabbit antibody, phage display antibody, yeast display antibody, chimeric antibody, humanized antibody, fully human antibody, antibody fragment, bispecific antibody and multispecific antibody, antigen-binding fragment of bispecific antibody and multispecific antibody, immunologically active portion, or mutant of the above antibodies.

11. The disulfide-bridged biomolecule according to claim 10, characterized in that, The antibody or antibody fragment is selected from the whole antibody or antibody fragment of abciximab, cetuximab, trastuzumab, sacixituzumab, and pavirizumab, and the nanobody is selected from at least one of nanobodies targeting epidermal growth factor receptor protein, green fluorescent protein, programmed death ligand-1, and β-amyloid protein.

12. A method for preparing a disulfide-bridged modified biomolecule as described in any one of claims 7-11, characterized in that, The method includes method A or method B; Method A includes the following steps: A bridging reaction was carried out by simultaneously adding a reducing agent and a disulfide bridging reagent to a biomolecule solution, adjusting the pH with acid or base, and obtaining disulfide-bridged modified biomolecules. Method B includes the following steps: A reducing agent is first added to the biomolecule solution to carry out a reduction reaction, and then a disulfide bridging reagent is directly added. The pH is adjusted with acid or base to carry out a bridging reaction, resulting in a disulfide-bridged modified biomolecule. The disulfide bridging reagent is the same as the disulfide bridging reagent described in any one of claims 1-4; The biomolecule is consistent with any one of claims 4-5. The corresponding precursors are the same.

13. The preparation method according to claim 12, characterized in that, The molar ratio of the disulfide bridging agent to the biomolecule is ≥1; The solvents for the biomolecule solution are phosphate buffer, ammonium bicarbonate buffer, sodium acetate buffer, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer. The reducing agent is at least one of tris(2-carboxyethyl)phosphine and tris(hydroxypropyl)phosphine; The molar ratio of the reducing agent to the disulfide bridging agent is ≥1; Adjust the pH using hydrochloric acid or sodium hydroxide; The pH of the bridging reaction is 7.0-8.5; The bridging reaction is carried out at a temperature of 25-37°C. The bridging reaction takes 0.5-16 hours.

14. A biomolecule-payload conjugate constructed via disulfide covalent bridging, characterized in that, The biomolecule-load conjugate constructed by disulfide covalent bridging has the following general formula IIIA, general formula IIIB, or their salt forms: Formula IIIA; Formula IIIB; Formula IIIA and Formula IIIB With any one of claims 7-11 The corresponding ones are the same; The n2 is selected from integers from 1 to 20; The X 5 Selected from the following structural fragments: ； R6, R7, and R8 are amino acid side chains excluding proline; R9, R... 10 The alkyl group is selected from C1-C10; m1 and m2 are each independent integers from 1 to 20; The L1 contains at least one spacer selected from the following: C(O), S, S(O), S(O)2. n3-n 11 Each is an independent integer between 1 and 20; The L2 is selected from alkylene groups, C(O), S, S(O), S(O)2, hydrazones, etc. Oligopeptides or polyethylene glycol-modified oligopeptides, monosaccharide groups, sulfate groups, aminobenzyloxycarbonyl self-degrading groups, diazonium salt photosensitive degradation cleavage groups, and combinations thereof; The D is selected from compounds containing at least one active group selected from alkyl, cycloalkyl, phenyl, compounds with at least one active group selected from azide, alkynyl, tetrazine, and cyclooctyne, proteins, polypeptides, nucleophilic tags, fluorescent molecules, and cytotoxins.

15. The biomolecule-payload conjugate constructed via disulfide covalent bridging according to claim 14, characterized in that, The L2 is selected from the following groups: ； n 12 n is an integer between 1 and 20; 13 n is an integer between 1 and 20; 14 Integers between 1 and 20; The nucleophilic tag is selected from polypeptides with 6-10 histidine residues; the cytotoxin includes microtubule inhibitors, topoisomerase inhibitors, and DNA binders.

16. The biomolecule-payload conjugate constructed via disulfide covalent bridging according to claim 15, characterized in that, The cytotoxins mentioned are selected from methylaurestatin E, methylaurestatin F, camptothecin, irinotecan, and pyrrolobenzodiazepine.

17. The biomolecule-payload conjugate constructed via disulfide covalent bridging according to claim 16, characterized in that, The disulfide covalently bridged biomolecule-load coupling is selected from at least one compound with the following structural formula: ； ； ； ； ； ； 。 18. A method for preparing a disulfide covalently bridged biomolecule-load coupling according to any one of claims 14-17, characterized in that, The method includes the following steps: Add the payload conjugate to the solution of disulfide-bridged biomolecules, adjust the pH with acid or base, carry out the coupling reaction, and perform post-treatment to obtain the biomolecule-payload conjugate constructed by disulfide covalent bridging. The payload conjugate is X in a biomolecule that can be bridged with disulfide-modified molecules. 3 The reacting compounds.

19. The method for preparing the disulfide covalently bridged biomolecule-load coupling according to claim 18, characterized in that, The payload coupling compound is selected from compounds containing 1,2-aminothiol, hydroxylamine, or hydrazide groups as linkers.

20. The method for preparing the disulfide covalently bridged biomolecule-load coupling according to claim 19, characterized in that, The payload coupling has the following structural formula: ; The X 4 Selected from ;R 11 R 12 It consists of amino acid side chains other than proline; L1, L 2、 D and L1, L as described in claims 14-17 2、 D corresponds to the same.

21. The method for preparing the disulfide covalently bridged biomolecule-load coupling according to claim 20, characterized in that, The payload coupling material is selected from the following compounds: ； ； 。 22. The method for preparing the disulfide covalently bridged biomolecule-load coupling according to claim 21, characterized in that, The molar ratio of the payload conjugate to the disulfide-bridged biomolecule is ≥1. The solvent for the disulfide-bridged modified biomolecule solution is phosphate buffer, ammonium bicarbonate buffer, sodium acetate buffer, or 4-hydroxyethylpiperazine ethanesulfonic acid buffer; HCl or NaOH is added to adjust the pH to 5.0-7.

5. The reaction temperature is 25°C-37°C; The reaction time is 0.5-24 hours.

23. The application of a method for preparing a disulfide covalently bridged biomolecule-load conjugate as described in any one of claims 14-17 or as described in any one of claims 18-22 in the preparation of targeted drugs, wherein, The biomolecule-payload conjugate is selected from protein-drug conjugates, peptide-drug conjugates, or antibody-drug conjugates; the targeted drug is used to treat diseases, the diseases being selected from cancer, inflammatory diseases, autoimmune diseases, cardiovascular diseases, or nervous system diseases; the cancer is selected from breast cancer, gastric cancer, liver cancer, ovarian cancer, pancreatic cancer, bladder cancer, lung cancer, epithelial tumors, or malignant tumors of the lymphatic system.

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