Preparation of 2'-deoxy-2',2'-difluorocytidine protides and antibody drug conjugates and uses thereof

Abstract

The present application relates to a series of 2'-deoxy-2',2'-difluoro cytidine (2',2'-difluoro 2'-deoxycytidine, dFdC, gemcitabine) Protide and antibody specific to tumor antigen form conjugates and its preparation method and application in the field of treatment. Further, the present application provides a series of antibody-linker-2'-deoxy-2',2'-difluoro cytidine Protide conjugate to form antibody drug conjugate (ADC). The structural features are mainly in the 5'-phosphate phenylamide amino acid ester corresponding group in the structure of 2'-deoxy-2',2'-difluoro cytidine and the linker for linkage, and then coupled with the corresponding tumor specific antibody to form ADC. These ADCs target antigens on tumor cells, endocytose into tumor cells, release the drug 2'-deoxy-2',2'-difluoro cytidine monophosphate, kill tumor cells, and cause tumor cell apoptosis.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field. Specifically, this invention provides a series of preparations and applications of 2'-deoxy-2',2'-difluorocytidine monophosphate amides and antibody-drug conjugates (ADCs). Background Technology

[0002] 2'-Deoxy-2',2'-Difluorocytidine (gemcitabine) is a cytosine nucleoside derivative with potent antitumor activity, primarily acting in the G1 / S phase and effective against various solid tumors. 2'-Deoxy-2',2'-Difluorocytidine enters the cell via nucleoside transporters (NTs) and is activated by deoxycytidine kinase (dCK) to form 2'-Deoxy-2',2'-Difluorocytidine monophosphate (dFdCMP), which is then converted to 2'-Deoxy-2',2'-Difluorocytidine diphosphate (dFdCDP) and 2'-Deoxy-2',2'-Difluorocytidine triphosphate (dFdCTP). Deoxycytidine kinase (dCK) is a key step in the activation of 2'-Deoxy-2',2'-Difluorocytidine. 2'-Deoxy-2',2'-Difluorocytidine triphosphate (2'-DETP) inhibits DNA polymerase and competitively incorporates into the DNA strand, extending it by one nucleotide and causing DNA strand extension to terminate and preventing recognition by DNA repair enzymes. 2'-Deoxy-2',2'-Difluorocytidine diphosphate (2'-DETP) inhibits ribonucleic acid reductase, reducing the production of deoxyribonucleoside triphosphate (DRPT). 2'-Deoxy-2',2'-Difluorocytidine itself inhibits Top1 inhibitors and thymidine nucleotide synthase. 2'-Deoxy-2',2'-Difluorocytidine triphosphate (2'-DETP) can also be incorporated into RNA synthesis, leading to ineffective RNA synthesis. All these effects of 2'-Deoxy-2',2'-Difluorocytidine contribute to apoptosis.

[0003] However, drug resistance often occurs during the use of 2'-deoxy-2',2'-difluorocytidine. The main resistance mechanisms include: the lack of nucleoside transporters (NTs) prevents 2'-deoxy-2',2'-difluorocytidine from entering cells; the lack of deoxycytidine kinase (dCK) prevents 2'-deoxy-2',2'-difluorocytidine from forming the key dFdCMP; and deoxycytidine deaminase (dCDA) deaminates 2'-deoxy-2',2'-difluorocytidine to form ineffective dFdU.

[0004] This invention involves derivatization at the 5'-OH of 2'-deoxy-2',2'-difluorocytidine to form a series of 2'-deoxy-2',2'-difluorocytidine-5-phosphate amide derivatives. These derivatives are then conjugated with antibodies specific to tumor cell membrane antigens to form ADCs. The ADCs bind to the antigens on the tumor cell membrane, and endocytosis carries the 2'-deoxy-2',2'-difluorocytidine-5-phosphate amide into the tumor cells. Intracellular enzymatic release of 2'-deoxy-2',2'-difluorocytidine-5-phosphate inhibits cell growth. Therefore, these ADCs can circumvent deoxycytosine kinase resistance and evade the action of deoxycytidine deaminase, exhibiting high antitumor activity and potential applications in cancer treatment. Summary of the Invention

[0005] In a first aspect of the invention, an intermediate for an antibody-drug conjugate (ADC), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is provided, characterized in that the structure of said intermediate is as shown in Formula I:

[0006] L-L1-D I

[0007] in,

[0008] D is

[0009] L is a connector that links the antibody and D;

[0010] "-" indicates a key or connector;

[0011] L1 has a structure selected from the following group:

[0012]

[0013] Where n = 0, 1, 2, 3 or 4;

[0014] R1 and R2 are each an amino acid side chain group;

[0015] R3 is selected from the following group: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 saturated or partially unsaturated carbocyclic group, 3-10 member saturated or partially unsaturated heterocyclic group, C6-C 10 Aryl, 5-12 membered heteroaryl; wherein the alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl groups are each optionally and independently substituted by one or more substituents selected from the group consisting of: halogen, hydroxyl, amino, and C1-C4 alkyl.

[0016] R4 is a side chain group of a natural amino acid or a side chain group of a non-natural amino acid that can be cleaved by intracellular enzymes.

[0017] In another preferred embodiment, the amino acid side chain group is selected from the group consisting of:

[0018] In another preferred embodiment, the natural amino acid side chain group is selected from the group consisting of:

[0019] In another preferred embodiment, the side chain groups of the non-natural amino acids that can be cleaved by intracellular enzymes are selected from the group consisting of:

[0020] In some embodiments, the L has a structure selected from the group consisting of:

[0021]

[0022] Each m is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0023] In a second aspect of the invention, there is provided the use of an intermediate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as described in the first aspect of the invention, characterized in that it is used for the preparation of antibody-drug conjugates.

[0024] In a third aspect of the invention, an antibody-drug conjugate (ADC), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is provided, characterized in that the structure of the antibody-drug conjugate is as shown in Formula II:

[0025] Ab-L-(L1-D) p II

[0026] in,

[0027] Ab represents antibodies;

[0028] D is

[0029] L is a connector that links the antibody and D;

[0030] p is the average number of drugs conjugated to the antibody, which can be an integer or a non-integer positive number, and 0.8≤p≤8;

[0031] "-" indicates a key or connector;

[0032] L1 has a structure selected from the following group:

[0033]

[0034] Where n = 0, 1, 2, 3 or 4;

[0035] R1 and R2 are each an amino acid side chain group;

[0036] R3 is selected from the following group: hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 saturated or partially unsaturated carbocyclic group, 3-10 member saturated or partially unsaturated heterocyclic group, C6-C 10 Aryl, 5-12 membered heteroaryl; wherein the alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl groups are each optionally and independently substituted by one or more substituents selected from the group consisting of: halogen, hydroxyl, amino, and C1-C4 alkyl.

[0037] R4 is a side chain group of a natural amino acid or a non-natural amino acid that can be cleaved by intracellular enzymes.

[0038] In another preferred embodiment, the antibody is an HS627 antibody.

[0039] In another preferred embodiment, the antibody is an antibody targeting Her2, Trop2, 5T4, ROR1, or B7-H3.

[0040] In another preferred embodiment, the amino acid side chain group is selected from the group consisting of:

[0041] In another preferred embodiment, the natural amino acid side chain group is selected from the group consisting of:

[0042] In another preferred embodiment, the side chain groups of the non-natural amino acids that can be cleaved by intracellular enzymes are selected from the group consisting of:

[0043] In some implementations, n is a positive integer or non-integer number, and 4≤p≤8.

[0044] In some embodiments, the L has a structure selected from the group consisting of:

[0045]

[0046] Each m is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0047] In some embodiments, the antibody-drug conjugate has a structure selected from the group consisting of:

[0048]

[0049]

[0050] In a fourth aspect of the invention, a pharmaceutical composition is provided, characterized in that the pharmaceutical composition comprises (1) an antibody-drug conjugate as described in the third aspect of the invention, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; optionally (2) a pharmaceutically acceptable carrier.

[0051] In a fifth aspect of the invention, the use of an antibody-drug conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof as described in the third aspect of the invention, or a pharmaceutical composition as described in the fourth aspect of the invention, is characterized in that it is used for the preparation of an antitumor drug.

[0052] In a sixth aspect of the invention, a method for preparing the intermediate described in the first aspect of the invention is provided, characterized in that the method comprises the steps of:

[0053] (1) 2'-deoxy-2',2'-difluorocytidine was reacted with Boc-anhydride to obtain the corresponding 3',4-ditert-butoxycarbonyl-2'-deoxy-2',2'-difluorocytidine.

[0054] (2) 3',4-di-tert-butoxycarbonyl-2'-deoxy-2',2'-difluorocytidine and phenyl dichlorophosphate were reacted together, and then reacted with L-amino acid esters to obtain the corresponding 3',4-di-tert-butoxycarbonyl-2'-deoxy-2',2'-difluorocytidine-5'-phosphate (phenyl ester) acyl amino acid esters.

[0055] (3) 3',4-Di-tert-butoxycarbonyl-2'-deoxy-2',2'-difluorocytidine-5'-phosphate (phenyl ester) acyl amino acid ester was reacted with trifluoroacetic acid to obtain 2'-deoxy-2',2'-difluorocytidine-5'-phosphate (phenyl ester) acyl amino acid ester;

[0056] (4) The 2'-deoxy-2',2'-difluorocytidine-5'-phosphate (phenyl ester) amino acid ester is reacted with the corresponding linker to obtain the corresponding intermediate as described in claim 1.

[0057] In a seventh aspect of the invention, a method for preparing the antibody-drug conjugate described in the third aspect of the invention is provided, characterized in that the method comprises the steps of:

[0058] (1) A reaction system is provided, the reaction system comprising an antibody and an intermediate as described in the first aspect of the present invention;

[0059] (2) In the reaction system, the antibody and the intermediate are coupled to obtain the antibody-drug conjugate of claim 1.

[0060] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation

[0061] Through extensive and in-depth research, the inventors unexpectedly discovered a series of conjugates formed by antibodies against gemcitabine and specific tumor antigens. Bioactivity tests revealed that these conjugates exhibited exceptionally significant anti-tumor effects. Based on this, the present invention was completed.

[0062] definition

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0064] As used herein, the terms “antibody-drug conjugate of the present invention,” “antibody-drug conjugate of the present invention,” “conjugate of the present invention,” or “ADC of the present invention” are used interchangeably to refer to an antibody-drug conjugate having the structure shown in Formula I.

[0065] Furthermore, in this invention, the same type or class of ADC molecules can be represented in multiple forms.

[0066] As used in this article, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0067] In this application, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0068] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, gluconates, lactates, sebates, adipates, glutarate, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbic acid salts, salicylates, 4-aminosalicylic acid salts, and naphthalene disulfonates. These salts can be prepared using methods known in this field.

[0069] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0070] Antibody

[0071] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0072] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0073] Vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (denoted as κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be further classified into different types based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0074] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). These regions are divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0075] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0076] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form single-chain antibodies.

[0077] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0078] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0079] Currently, the DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0080] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0081] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.

[0082] Typically, host cells transformed with the antibody are cultured under conditions suitable for antibody expression according to the present invention. The antibody of the present invention is then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are well known to those skilled in the art.

[0083] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis described by Munson et al., Anal. Biochem., 107:220 (1980).

[0084] The antibodies of this invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0085] In another preferred embodiment, the antibody is antibody hRS7.

[0086] Pharmaceutical Compositions and Administration

[0087] The present invention also provides pharmaceutical compositions containing the ADC of the present invention, and methods for treating mammalian diseases using the ADC of the present invention. Preferably, the disease is, for example, a tumor.

[0088] The present invention also provides the application of the antibody-drug conjugate in the preparation of antitumor drugs.

[0089] In this invention, the pharmaceutical composition comprises an effective amount of the ADC (as the active ingredient) according to the invention, and at least one pharmaceutically acceptable carrier, diluent, or excipient. In preparation, the active ingredient is typically mixed with, diluted with, or encapsulated in a carrier that may be in capsule or pouch form. When the excipient acts as a diluent, it may be a solid, semi-solid, or liquid material as the medium for the excipient, carrier, or active ingredient. Therefore, the composition may be a solution, a sterile injectable solution, etc.

[0090] Suitable excipients include lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, etc.; the formulation may also include wetting agents, emulsifiers, preservatives (such as methylparaben and propylparaben), etc. The antitumor drug can be formulated into single or multiple dosage forms, each containing a predetermined amount of the ADC of the present invention calculated to produce the desired therapeutic effect, and suitable pharmaceutical excipients.

[0091] The antitumor drugs mentioned above can be administered via conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, and local administration.

[0092] When using this drug, a safe and effective amount of the antibody-drug conjugate is administered to a human, preferably in the range of 0.5–50 mg / kg body weight, more preferably 1–10 mg / kg body weight. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health condition, all of which are within the scope of a skilled physician's expertise.

[0093] Furthermore, the conjugate of the present invention can also be used in combination with other therapeutic agents, including (but not limited to): various cytokines, such as TNF, IFN, IL-2, etc.; various tumor chemotherapy drugs, such as 5-FU, methotrexate, etc., which affect nucleic acid biosynthesis; alkylating agents such as nitrogen mustard, cyclophosphamide, etc.; drugs that interfere with transcription and prevent RNA synthesis such as doxorubicin D, etc.; drugs that affect protein synthesis such as vincristine, camptothecin, etc.; and certain hormone drugs, etc.

[0094] The main advantages of this invention are:

[0095] (1) The antibody-drug conjugate of the present invention can circumvent deoxycytosine kinase resistance.

[0096] (2) The antibody-drug conjugate of the present invention can evade the action of deoxycytidine deaminase.

[0097] (3) The antibody-drug conjugate of the present invention has high antitumor activity.

[0098] Example

[0099] In this embodiment, the ADC was prepared using, but not limited to, the HS627 antibody (Ab), which is a biosimilar of pertuzumab (perjeta) and is derived from Borui Biotechnology.

[0100] Example 1: Tras-mc-VC-PABC-Pip-4-O-Ala Gem Protide (ADC1)

[0101]

[0102] In a 10 ml single-necked flask, 1 ml of NMP, PT-1A (50 mg, 0.087 mmol), DIPEA (33 mg, 0.261 mmol), mc-VC-PAB-PNP (65 mg, 0.087 mmol), and HOBt (12 mg, 0.087 mmol) were added sequentially. The mixture was stirred at room temperature for 1.5 h. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water; the aqueous phase contained 0.1% TFA). The solution was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 15% to 40% acetonitrile in water, time 30 min), and lyophilized to obtain PT-1 (30 mg, 30%) as a white solid; LCMS: (M+1). + 1172.32 (Calculated value: 1171.46).

[0103] Preparation of ADC1:

[0104] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature (25 °C) by rotating the disc for 90 min.

[0105] Compound PT-1 (0.94 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain ADC-1 (3.1 mg / mL, 2 mL).

[0106] UV-HPLC calculated average: n = 7.30

[0107] Example 2: Tras-mc-GVAG-ha-pip-4-O-Ala Gem Protide (ADC2)

[0108]

[0109] In a 10ml single-necked flask, add DMF (2ml), and while stirring, add Fmoc-GVAG-ha (200mg, 0.3mmol); PT-1A (173mg, 0.3mmol); DIPEA (58mg, 0.45mmol); HOBT (61mg, 0.45mmol); and EDCI (87mg, 0.45mmol). After stirring until homogeneous, react overnight at room temperature to obtain crude PT-2C, which can be directly added to the next step.

[0110] Add 0.2 ml of piperidine (V / V = 10%) to the PT-2C reaction solution obtained above, react at room temperature for 0.5 h, add 30 ml of methyl tert-butyl ether to the reaction solution, centrifuge (5 min, 10000 r / min), remove the supernatant, add 3 ml of methanol again, sonicate to dissolve, add 30 ml of methyl tert-butyl ether again, centrifuge (5 min, 10000 r / min), remove the supernatant, and then rotary evaporate to remove excess solvent to obtain solid PT-2D (201 mg, yield 60%), which is directly added to the next reaction without further purification.

[0111] In a 10 mL single-necked flask, 0.5 mL of DMF, PT-2D (201 mg, 0.213 mmol), and mc-OSu (65 mg, 0.213 mmol) were added sequentially. The mixture was stirred at room temperature for 15 min. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water; the aqueous phase contained 0.1% TFA). The solution was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 15% to 50% acetonitrile in water, time 30 min). The eluted solution was lyophilized to obtain a white solid of PT-2 (85 mg, yield 40%). LCMS: (M+1) + 1138.10 (Calculated value: 1137.43).

[0112] Preparation of ADC2:

[0113] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature (25 °C) by rotating the disc for 90 min.

[0114] Compound PT-2 (0.91 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain ADC-2 (3.2 mg / mL, 2 mL).

[0115] UV-HPLC calculated average: n = 7.70

[0116] Example 3: Tras-mc-VC-PABC-Pip-4-O-Phe Gem Protide (ADC3)

[0117]

[0118] In a 10 ml single-necked flask, 1 ml of NMP, PT-12B (26.4 mg, 0.041 mmol), DIPEA (15.77 mg, 0.122 mmol), MC-VC-PAB-PNP (30 mg, 0.041 mmol), and HOBt (5.49 mg, 0.041 mmol) were added sequentially. The mixture was stirred at room temperature for 0.5 h. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water; the aqueous phase contained 0.1% TFA). The solution was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 10% to 45% acetonitrile in water, time 40 min), and lyophilized to obtain PT-12 (9.8 mg, 19.3%) as a white solid; LCMS: (M+1) + 1248.2 (Calculated value: 1247.4).

[0119] Preparation of ADC3:

[0120] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature (25 °C) by rotating the disc for 90 min.

[0121] Compound PT-12 (1 mg, 0.8 mmol) was dissolved in 0.1 ml of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain ADC-3 (3.0 mg / ml, 2 ml).

[0122] UV-HPLC calculated average: n = 7.60

[0123] Example 4: Tras-m(2O)c-GVAG-ha-Pip-4-O-Phe Gem Protide (ADC4)

[0124]

[0125] In a 10ml single-necked flask, add 2ml of DMF. While stirring, add Fmoc-GVAG-ha (50mg, 0.082mmol), PT-12B (53mg, 0.082mmol), DIPEA (22mg, 0.163mmol), HOBT (17mg, 0.122mmol), and EDCI (24mg, 0.122mmol) in sequence. After stirring evenly, react overnight at room temperature to obtain PT-13C reaction solution, which is then used in the next step without purification.

[0126] Add 0.2 ml of piperidine (V:V = 10%) to the PT-13C reaction solution and react at room temperature for 1 h. Add 30 ml of methyl tert-butyl ether to the reaction solution, centrifuge (5 min, 10000 r / min), remove the supernatant, add 3 ml of methanol again, sonicate to dissolve, add 30 ml of methyl tert-butyl ether again, centrifuge (5 min, 10000 r / min), remove the supernatant, remove the solvent under reduced pressure to obtain PT-13D (68 mg, 79%) solid product, which is used in the next step without purification.

[0127] In a 10 ml single-necked flask, DMF (2 ml), PT-13D (68 mg, 0.066 mmol), PT-13E (23.6 mg, 0.066 mmol), and TEA (6.7 mg, 0.066 mmol) were added sequentially. The mixture was stirred at room temperature for 15 min. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water; the aqueous phase contained 0.1% TFA). The solution was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 15% to 40% acetonitrile in water, time 30 min), and lyophilized to obtain PT-13 (28.5 mg, 34%) as a white solid; LCMS: (M+1) + 1260.2 (Calculated value: 1259.1).

[0128] Preparation of ADC4:

[0129] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature (25 °C) by rotating the disc for 90 min.

[0130] Compound PT-13 (1 mg, 0.8 mmol) was dissolved in 0.1 ml of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain ADC-4 (3.2 mg / ml, 2 ml).

[0131] UV-HPLC calculated average: n = 7.60

[0132] Example 5: Tras-m(2O)c-GK-PABC-Pip-4-O-Phe Gem Protide (ADC5)

[0133]

[0134] In a 10 ml single-necked flask, PT-12B (50 mg, 0.103 mmol), 2 ml NMP, DIPEA (40 mg, 0.310 mmol), Fmoc-GK(Trt)-PAB-PNP (72 mg, 0.103 mmol), and HOBt (14 mg, 0.103 mmol) were added sequentially. The mixture was stirred at room temperature for 2 hours to obtain PT-14B. The reaction solution was used directly in the next step.

[0135] Add 0.2 ml of piperidine (V:V = 10%) to the PT-14B reaction solution and react at room temperature for 0.5 h. Add 30 ml of methyl tert-butyl ether to the reaction solution, centrifuge (5 min, 10000 r / min), remove the supernatant, add 3 ml of methanol again, sonicate to dissolve, add 30 ml of methyl tert-butyl ether again, centrifuge (5 min, 10000 r / min), remove the supernatant, and remove the solvent by rotary evaporation under reduced pressure to obtain PT-14C (63 mg, 67%) solid, which is used in the next step without purification.

[0136] In a 10 ml single-necked flask, PT-14C (63 mg, 0.051 mmol) and PT-13E (19 mg, 0.051 mmol) were added sequentially. The mixture was stirred at room temperature for 30 min. 3 g of silica gel was added to the reaction solution. Column chromatography was performed using DCM and MeOH as the mobile phase. The product eluted at a DCM:MeOH ratio of 94%:6%. After concentration, PT-14E (52 mg, 69%) was obtained.

[0137] In a 10 mL single-necked flask, 1 mL of DCM (1 mL), PT-14E (52 mg, 0.035 mmol), and dichloroacetic acid (0.2 mL) were added sequentially. The mixture was stirred at room temperature for 60 min. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water; the aqueous phase contained 0.1% TFA). The solution was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 15% to 40% acetonitrile in water, time 30 min). The eluted solution was lyophilized to obtain PT-14 (14 mg, 33%) as a white solid; LCMS: (M+1). + 1223.1 (Calculated value: 1222.4).

[0138] Preparation of ADC5:

[0139] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature (25 °C) by rotating the disc for 90 min.

[0140] Compound PT-14 (1 mg, 0.8 mmol) was dissolved in 0.1 ml of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain ADC-5 (3.2 mg / ml, 2 ml).

[0141] UV-HPLC calculated average: n = 7.30

[0142] Example 6: Tras-m(2O)c-GK-PABC-ε-amino-Lys(2-ehtylbutyl ester)Gem Protide(ADC6)

[0143]

[0144] In a 10ml single-necked flask, PT-15A (60mg, 0.095mmol), 2ml NMP, DIPEA (36mg, 0.285mmol), Fmoc-GK(Trt)-PAB-PNP (70mg, 0.1mmol), and HOBt (15mg, 0.1mmol) were added sequentially. The mixture was stirred at room temperature for 0.5h to obtain PT-15B reaction solution, which was then directly added to the next step.

[0145] Add 0.2 ml of piperidine (V:V = 10%) to the PT-15B reaction solution and react at room temperature for 0.5 h. HPLC detection showed that the raw materials had reacted completely. Add 30 ml of methyl tert-butyl ether to the reaction solution, centrifuge (5 min, 10000 r / min), remove the supernatant, add 3 ml of methanol again, sonicate to dissolve, add 30 ml of methyl tert-butyl ether again, centrifuge (5 min, 10000 r / min), remove the supernatant, and then rotary evaporate to remove excess solvent to obtain PT-15C (79 mg, 71%) solid, which is used in the next step without purification.

[0146] In a 10 ml single-necked flask, PT-15C (79 mg, 0.065 mmol) and m(2O)c-OSu (23 mg, 0.065 mmol) were added sequentially. The mixture was stirred at room temperature for 30 min. 3 g of silica gel was added to the reaction solution. Column chromatography was performed using DCM and MeOH as the mobile phase. The product eluted at a DCM:MeOH ratio of 94%:6%. After concentration, PT-15E (83 mg, 87%) was obtained.

[0147] In a 10 mL single-necked flask, 1 mL of DCM, PT-15E (83 mg, 0.057 mmol), and 0.2 mL of dichloroacetic acid were added sequentially. The mixture was stirred at room temperature for 60 min. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water; the aqueous phase contained 0.1% TFA). The solution was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 15% to 40% acetonitrile in water, time 30 min). The eluted solution was lyophilized to obtain PT-15 (24 mg, 33%) as a white solid; LCMS: (M+1). + 1205.2 (Calculated value: 1204.5).

[0148] Preparation of ADC6:

[0149] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature (25 °C) by rotating the disc for 90 min.

[0150] Compound PT-15 (0.96 mg, 0.8 mmol) was dissolved in 0.096 mL of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain ADC-6 (3.0 mg / mL, 2 mL).

[0151] UV-HPLC calculated average: n = 7.70

[0152] Example 7: Tras-m(2O)c-GVAG-ha-ε-amino-Lys(2-ehtylbutyl ester)Gem Protide(ADC7)

[0153]

[0154] In a 10ml single-necked flask, add DMF (2ml), and while stirring, add Fmoc-GVAG-ha (50mg, 0.082mmol), PT-16B (52mg, 0.082mmol), DIPEA (22mg, 0.163mmol), and HATU (50mg, 0.163mmol) in sequence. After stirring evenly, let it react overnight at room temperature to obtain PT-16C reaction solution, which can be directly added to the next step.

[0155] Add 0.2 ml of piperidine (V:V = 10%) to the PT-16C reaction solution and react at room temperature for 1 h. Add 30 ml of methyl tert-butyl ether to the reaction solution, centrifuge (5 min, 10000 r / min), remove the supernatant, add 3 ml of methanol again, sonicate to dissolve, add 30 ml of methyl tert-butyl ether again, centrifuge (5 min, 10000 r / min), remove the supernatant, remove the solvent under reduced pressure, and obtain PT-16D (67 mg, 84%) solid, which can be directly added to the next step.

[0156] To the PT-16D solid obtained in the previous step, add m(2O)c-Osu (24 mg, 0.066 mmol), stir at room temperature for 15 min, and inject the reaction solution into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water, the aqueous phase containing 0.1% TFA). Elute by medium-pressure reversed-phase C18 chromatography (gradient: 15% to 40% acetonitrile in water, time 30 min), lyophilize, and obtain PT-16 (28.5 mg, 34%) as a white solid; LCMS: (M+1) + 1242.4 (Calculated value: 1241.5).

[0157] Preparation of ADC7:

[0158] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature (25 °C) by rotating the disc for 90 min.

[0159] Compound PT-16 (0.99 mg, 0.8 mmol) was dissolved in 0.099 mL of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain ADC-7 (3.2 mg / mL, 2 mL).

[0160] UV-HPLC calculated average: n = 7.60

[0161] Example 8: Tras-mc-VC-PABC-1,4-butanediamine Gem Protide (ADC8)

[0162]

[0163] In a 10 ml single-necked flask, 1 ml of NMP, PT-18A (28 mg, 0.067 mmol), DIPEA (26 mg, 0.200 mmol), MC-VC-PAB-PNP (50 mg, 0.067 mmol), and HOBt (9.2 mg, 0.067 mmol) were added sequentially. The mixture was stirred at room temperature for 2 h. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water; the aqueous phase contained 0.1% TFA). The solution was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 10% to 45% acetonitrile in water, time 40 min). The eluted solution was lyophilized to obtain PT-18 (12.5 mg, 20.8%) as a white solid; LCMS: (M+1) + 1012 (Calculated value: 1011.40).

[0164] Preparation of ADC8:

[0165] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature (25 °C) by rotating the disc for 90 min.

[0166] Compound PT-18 (0.81 mg, 0.8 mmol) was dissolved in 0.081 ml of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain ADC-8 (2.9 mg / ml, 2 ml).

[0167] UV-HPLC calculated average: n = 4.3

[0168] Example 9: ADC Inhibition of Tumor Cell Growth Activity

[0169] In vitro inhibitory activity assay method for ADC:

[0170] Human esophageal cancer cells OE33, lung cancer cells NCI-H1975, and breast cancer cells MDA-MB-231, used as activity assays, were cultured in RPMI1640 (Cellmax), RPMI1640 (Cellmax), and DMEM (Cellmax) media containing 10% fetal bovine serum (Cellmax), respectively, until the exponential growth phase. After trypsin digestion, the cells were centrifuged and the supernatant was discarded. The cells were then diluted with culture medium to 3×10⁴ cells / mL, 0.5×10⁴ cells / mL, and 1.5×10⁴ cells / mL, respectively. 100 μL of each cell was added to a 96-well cell culture plate and incubated overnight at 37°C with 5% CO₂. On the second day, the ADCs to be tested were diluted to 2000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.128 nM, and 0.026 nM using culture medium. 100 μL of the diluted ADC was added to each well of a 96-well cell culture plate, with three replicates for each concentration. 100 μL of culture medium was added to each well for the negative control and blank control groups (no ADC added). After sample addition, the plates were returned to a 37°C, 5% CO2 incubator for 6 days. After incubation, the cell culture plates were removed, the culture medium was aspirated from the plates, and 100 μL of medium containing 10% CCK-8 was added to each well. The plates were incubated at 37°C for 3 hours. After incubation, the plates were removed, protected from light, and placed in an ELISA plate. The absorbance was measured at 630 nm as the reference wavelength and 450 nm as the measurement wavelength. Based on the absorbance values, the IC50 was calculated using four-parameter regression in GraphPad (Table 1). The ADC drug DS-8201a was used as a positive control. For IC50 values, “++++” indicates IC50 < 10 nM; “+++” indicates IC50 between 10 nM and 100 nM; “++” indicates IC50 between 100 and 500 nM; and “+” indicates IC50 > 500 μM.

[0171] Table 1. Inhibitory activity (IC50, nM) of ADC drugs against NCI-H1975, MDA-MB-231, and OE-33 cells.

[0172]

[0173]

[0174] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, characterized in that, The antibody-drug conjugate has a structure selected from the group consisting of:

2. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises (1) the antibody-drug conjugate as claimed in claim 1 or a pharmaceutically acceptable salt thereof; optionally (2) a pharmaceutically acceptable carrier.

3. The use of the antibody-drug conjugate as claimed in claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as claimed in claim 2, characterized in that, The tumor is selected from the group consisting of esophageal cancer, lung cancer, and breast cancer. This is used to prepare an anti-tumor drug.

4. A method for preparing an intermediate of the antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The method includes the following steps: (1) 2'-deoxy-2',2'-difluorocytidine was reacted with Boc-anhydride to obtain the corresponding 3',4-ditert-butoxycarbonyl-2'-deoxy-2',2'-difluorocytidine. (2) 3',4-di-tert-butoxycarbonyl-2'-deoxy-2',2'-difluorocytidine and phenyl dichlorophosphate were reacted together, and then reacted with L-amino acid esters to obtain the corresponding 3',4-di-tert-butoxycarbonyl-2'-deoxy-2',2'-difluorocytidine-5'-phosphate (phenyl ester) acyl amino acid esters. (3) 3',4-Di-tert-butoxycarbonyl-2'-deoxy-2',2'-difluorocytidine-5'-phosphate (phenyl ester) acyl amino acid ester was reacted with trifluoroacetic acid to obtain 2'-deoxy-2',2'-difluorocytidine-5'-phosphate (phenyl ester) acyl amino acid ester; (4) React 2'-deoxy-2',2'-difluorocytidine-5'-phosphate (phenyl ester) acyl amino acid ester with the corresponding linker to obtain the corresponding intermediate.

5. A method for preparing the antibody-drug conjugate of claim 1, characterized in that, The method includes the following steps: (1) A reaction system is provided, the reaction system comprising an antibody and an intermediate as described in claim 4; (2) In the reaction system, the antibody and the intermediate are coupled to obtain the antibody-drug conjugate of claim 1.

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