Novel isomeric compounds comprising a ring-opened thiosuccinimide group, an oligopeptide fragment and a chiral moiety

Abstract

The present disclosure relates to the field of medicinal chemistry, in particular to a method for separating isomeric compounds comprising a ring-opened thiosuccinimidyl group and a chiral moiety.

Description

Technical Field

[0001] This disclosure relates to the field of medicinal chemistry, and in particular to isomers of compounds with anticancer activity having an open-ring thiosuccinimide group, an oligopeptide fragment, and a chiral moiety. The invention also relates to methods for isolating isomers. Background Technology

[0002] The increasing recurrence of tumors in mammals and the severe side effects of chemotherapy drugs have reduced the clinical efficacy of many currently used anticancer drugs. Therefore, there is a constant need to develop alternative or synergistic anticancer drugs with minimal side effects. An important strategy for developing effective anticancer agents is to study anticancer agents derived from drug "combinations," and hundreds of trials are currently underway to evaluate novel combinations of targeted drugs. These targeted agents include antibody-drug conjugates.

[0003] Antibody-drug conjugates (ADCs) are not a new concept. As potent anti-tumor drugs based on monoclonal antibodies, they combine the advantages of antibodies (as the targeting and / or therapeutic components) with traditional cytotoxic drugs (which have strong cytotoxicity).

[0004] An ideal ADC linker must meet the following requirements: it should be sufficiently stable extracellularly to ensure the binding of the small molecule drug to the ligand; upon entry into the cell, the cleavable linker should break under appropriate conditions, releasing the active small molecule drug; for non-cleavable linkers, the active ingredient typically consists of amino acid residues generated from the enzymatic hydrolysis of the small molecule, linker, and ligand. Therefore, linker design and related coupling strategies are crucial in ADC development. These not only play a key role in the stability of the ADC but also directly affect the biological activity, aggregation state, in vivo bioavailability, distribution, and metabolism of the conjugate.

[0005] Currently, the mainstream conjugation technology is the chemical conjugation strategy, primarily based on lysine or cysteine ​​residues in antibodies. Due to the diversity in the number and position of these amino acids that can react with linkers in antibodies, the number and position of cytotoxins in ADCs are variable, resulting in heterogeneous ADCs. This heterogeneity affects the quality, stability, efficacy, metabolism, and toxicity of ADCs. For example, the drug information for the ADC Kadcyla, launched in 2013, explicitly states that the number of cytotoxins per antibody ranges from 0 to 8, with an average n of approximately 3.5. The heterogeneity of ADCs is a major challenge in developing next-generation ADCs.

[0006] One drawback of bioconjugates is off-target release, which can lead to toxicity to normal tissues and reduce the number of effective bioconjugates at the target site, resulting in decreased potency. More than half of commercially available or clinically tested antibody-drug conjugates (ADCs) use a thiosuccinimide structure (thiosuccinimide linker) to conjugate small molecule drugs to targeting antibodies or proteins. However, the thiosuccinimide linker is not stable. In vivo, reverse Michael addition or exchange with other thiol groups can occur, directly causing cytotoxicity to detach from the ADC and resulting in off-target toxicity.

[0007] Ring-opening of succinimide can increase the stability of bioconjugates by eliminating potential sites for thiol exchange via reverse Michael addition or through the reverse Michael addition mechanism.

[0008] The thiosuccinimide structure is formed by the reaction of a thiol group with a maleimide structure. Under conditions of low or incomplete regioselectivity, the ring-opening reaction yields a pair of positional isomers. Furthermore, the covalent linkage of the thiol group with the succinimide generates a chiral center. The presence of diastereomers and enantiomers poses a significant challenge to the post-processing of the ring-opening reaction. Therefore, further research on the isomers is needed, and new purification methods are urgently required. Summary of the Invention

[0009] This disclosure relates to compounds having the structure of any one of the following formulas (XI) to (XIV):

[0010]

[0011] in

[0012] Load, L 1 L 2 M is defined below.

[0013] This disclosure also provides an antibody-drug conjugate (ADC) prepared by using a compound of formula (XI), (XII), (XIII) or (XIV) with an antibody or an antigen-binding fragment thereof.

[0014] In one particular embodiment, the compounds of formula (XI) to (XIV) each have the structures of formula (i) to (iv):

[0015]

[0016] Where x is -OH or -NH2.

[0017] A mixture of ADCs obtained by conjugating a compound of formula (iii) or formula (iv) with a therapeutic antibody is referred to as "α-ADC".

[0018] α-ADCs comprising an open-ring thiosuccinimide group, an oligopeptide fragment, and a chiral moiety have been prepared, and their biological activity has been demonstrated by in vitro and in vivo assays. Unexpected anticancer results are as follows:

[0019] α-ADC has a significant inhibitory effect on the proliferation of HER2-positive cells, and its effect is dozens of times that of DM1.

[0020] α-ADCs can accumulate in tumors within 24 hours and maintain in situ stability.

[0021] In summary, under the current research conditions, repeated intravenous infusions of α-ADC at doses of 10, 30, and 45 mg / kg (3 times every 3 weeks) were tolerated in cynomolgus monkeys, with a single dose up to 45 mg / kg. The highest non-serious toxic dose (HNSTD) was determined to be 45 mg / kg, compared to a toxic dose of 10 mg / kg observed in Kadcyla.

[0022] The optimized molecular structure improved metabolism. Compared to Kadcyla, α-ADCs produced similar potency at lower doses.

[0023] This disclosure also provides a method for separating one or more target compounds from a mixture 1, the mixture 1 comprising four compounds, each of the four compounds comprising part 1, part 2 and part 3;

[0024] Part 1 has an open-ring thiosuccinimide structure, selected from formulas (I) to (IV):

[0025]

[0026] in

[0027] The thiol and amide groups in part 1 form two linkage sites, part 2 is linked to part 1 through one of them, and part 3 is linked to part 1 through the other of them;

[0028] Part 2 contains one or more chiral centers;

[0029] Part 1 of the four compounds is different;

[0030] Part 3 is the remaining portion of the molecule;

[0031] The molecular weight of part 3 does not exceed 1900;

[0032] The one or more target compounds are selected from the four compounds contained in mixture 1;

[0033] The method includes the following steps (1) and (2)

[0034] (1) Provide mixture 1;

[0035] (2) Mixture 1 was subjected to chromatography to obtain the target compound.

[0036] in

[0037] a. Each of the target compounds was obtained as an isolated product, or

[0038] b. The two target compounds were obtained as a mixture, which is defined as mixture 2.

[0039] In one embodiment, the method further includes steps (3) and (4).

[0040] (3) Recover the eluent collected in step (2), which contains mixture 3, wherein mixture 3 contains one or more additional target compounds that are different from the target compounds separated in step (2);

[0041] (4) Perform chromatography on the eluent recovered in step (3) to separate the additional target compound.

[0042] Based on the position of the thiol group, formulas (I) and (II) are referred to as β, and the configurations of formulas (III) and (IV) are referred to as α.

[0043] This disclosure also provides a method for analyzing one or more compounds in a mixture 1, the mixture 1 comprising four compounds, each of the four compounds comprising part 1, part 2 and part 3.

[0044] This disclosure also provides a method for analyzing one or more compounds in a mixture 2, the mixture comprising two compounds, each of the two compounds comprising part 1, part 2 and part 3. Attached Figure Description

[0045] Figure 1 HPLC chromatograms of the separation of β1, β2 and α isomers; Daisopak C4 SP-120-8-C4-P was used.

[0046] Figure 2 HPLC chromatograms of the separation of β1, β2 and α isomers; Kromasil C18 100-10C18 was used.

[0047] Figure 3 HPLC chromatograms showing the separation of β1, β2, and α isomers; using an Acchrom C18-ME 10µm microscope.

[0048] Figure 4HPLC chromatograms of the separation of β1, β2 and α isomers; Daisopak C18 100-10-ODS-P was used.

[0049] Figure 5 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 0.1% TFA aqueous solution, eluent B: MeOH.

[0050] Figure 6 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 0.1% H3PO4 aqueous solution, eluent B: MeOH.

[0051] Figure 7 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 0.2% TFA aqueous solution, eluent B: MeOH.

[0052] Figure 8 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 0.1% CH2O2 aqueous solution, eluent B: MeOH.

[0053] Figure 9 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 0.2% CH2O2, eluent B: MeOH.

[0054] Figure 10 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 0.5% AA aqueous solution, eluent B: MeOH.

[0055] Figure 11 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 0.1% CH3COOH aqueous solution, eluent B: MeOH.

[0056] Figure 12 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 0.3% CH3COOH aqueous solution, eluent B: MeOH.

[0057] Figure 13 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 0.3% CH3COOH + 0.05% L-TA aqueous solution, eluent B: MeOH.

[0058] Figure 14 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 10 mM NaH2PO4 pH=2.0, eluent B: MeOH.

[0059] Figure 15HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 10 mM NaH2PO4 pH=4.0, eluent B: MeOH.

[0060] Figure 16 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 10 mM NaH2PO4 pH = 6.0, eluent B: MeOH.

[0061] Figure 17 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 10 mM K2HPO4 pH=4.0, eluent B: MeOH.

[0062] Figure 18 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 10 mM TEAP pH = 4.0, eluent B: MeOH.

[0063] Figure 19 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 10 mM NaClO4 pH = 2.0, eluent B: MeOH.

[0064] Figure 20 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 0.3% CH3COOH aqueous solution, eluent B: MeOH; gradient: 46%-66% B, 30 min.

[0065] Figure 21 HPLC chromatograms showing the separation of β1, β2 and α isomers; eluent A: 0.3% CH3COOH aqueous solution, eluent B: MeOH; gradient: 46%-56% B, 80 min.

[0066] Figure 22 HPLC chromatograms of β1, β2, and α isomers were analyzed using Phenomenex Gemini. 5um 250*4.6mm.

[0067] Figure 23 HPLC chromatograms of the separation of α1 and α2 isomers; using Kromasil 100-10C18, 21.2*250mm. 10μm); Eluent C: 0.1% TFA aqueous solution, Eluent D: ACN, containing 0.1% TFA.

[0068] Figure 24 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 0.1% TFA aqueous solution, eluent D: ACN.

[0069] Figure 25 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 0.1% AcOH aqueous solution, eluent D: ACN.

[0070] Figure 26 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 0.3% AcOH aqueous solution, eluent D: ACN.

[0071] Figure 27 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 0.1% CH2O2 aqueous solution, eluent D: ACN.

[0072] Figure 28 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 0.3% CH2O2 aqueous solution, eluent D: ACN.

[0073] Figure 29 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 0.5% AA aqueous solution, eluent D: ACN.

[0074] Figure 30 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 0.1% H3PO4 aqueous solution, eluent D: ACN.

[0075] Figure 31 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 0.2% H3PO4 aqueous solution, eluent D: ACN.

[0076] Figure 32 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 10 mM NaH2PO4 pH=2.0, eluent D: ACN.

[0077] Figure 33 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 10 mM NaH2PO4 pH=4.0, eluent D: ACN.

[0078] Figure 34 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 10mM NaH2PO4 pH=6.0, eluent D: ACN.

[0079] Figure 35 HPLC chromatogram of separation of α1 and α isomers; eluent C: 10 mM K2HPO4 pH=2.0, eluent D: ACN.

[0080] Figure 36HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 10 mM K2HPO4 pH=4.0, eluent D: ACN.

[0081] Figure 37 HPLC chromatogram of separation of α1 and α2 isomers; eluent C: 10 mM K2HPO4 pH=6.0, eluent D: ACN.

[0082] Figure 38 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 10 mM K2HPO4 pH=8.0, eluent D: ACN.

[0083] Figure 39 HPLC chromatogram of separation of α1 and α isomers; eluent C: 30mM NaH2PO4 pH=2.0, eluent D: ACN.

[0084] Figure 40 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 30 mM NaH2PO4 pH = 6.0, eluent D: ACN.

[0085] Figure 41 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 30mM NaH2PO4 pH=4.0, eluent D: ACN.

[0086] Figure 42 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 10 mM TEAP pH = 6.0, eluent D: ACN.

[0087] Figure 43 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 10 mM TEAP pH = 2.0, eluent D: ACN.

[0088] Figure 44 HPLC chromatogram of the separation of α1 and α2 isomers; eluent C: 10 mM TEAP pH = 4.0, eluent D: ACN.

[0089] Figure 45 HPLC chromatograms used for analyzing α1 and α2 isomers; Waters X-select, CSH Phenyl-Hexyl 3.5um 150*4.6mm was used.

[0090] Figure 46 The effect of isoforms on cancer cell proliferation.

[0091] Figure 47 The effect of α-ADC on cancer cell proliferation.

[0092] Figure 48 Biodistribution characteristics of α-ADCs (single intravenous infusion) 89 (PET / CT scan results after Zr-α-ADC).

[0093] Figure 49 : In vivo antitumor efficacy of α-ADC. The x-axis represents time after a single dose. The y-axis represents tumor size.

[0094] Figure 50 Pharmacokinetic characteristics of α-ADCs. The x-axis represents the time following a single intravenous infusion. The y-axis represents serum concentration.

[0095] Figure 51 Summary of toxicokinetic data for α-ADCs. The x-axis represents time after the first dose. Hours 0, 504, and 1008 represent the time points of repeated intravenous infusion. Hours 1008 to 2016 represent the 6-week recovery period. The y-axis represents serum concentration.

[0096] exist Figure 1-45 In the diagram, the x-axis represents time (minutes). Figure 1-21 In 45, the y-axis represents UV absorbance (mAU). Figure 22-44 In the diagram, the y-axis represents UV absorbance (AU). Detailed Implementation

[0097] General definition

[0098] Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The term "technique" as used herein refers to techniques commonly understood in the art, including variations and equivalent substitutions that are obvious to one of ordinary skill in the art. While the following terms are thought to be readily understood by one of ordinary skill in the art, the following definitions are provided to better illustrate this disclosure. When a trade name appears herein, it refers to the corresponding product or its active ingredient. All patents, published patent applications, and publications cited herein are incorporated herein by reference.

[0099] When a quantity, concentration, or other numerical value or parameter is described as a range, preferred range, or preferred upper or lower limit, it should be understood as equivalent to specifically disclosing any range formed by combining any upper or preferred value with any lower or preferred value, whether or not the range is explicitly stated. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range as well as all integers and fractions (decimals) within the range. For example, the expression "about 0.01% to about 1%" means any value between 0.01% and 1%, such as 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, and 1%. Other similar expressions, such as "40%-50% to approximately 50%-70%", should also be understood in a similar way.

[0100] Unless otherwise stated herein, the singular forms such as “one” and “this” include the plural forms. The expressions “one or more” or “at least one” can mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0101] When used with numerical variables, the terms “about” and “approximately” generally indicate that the value of the variable and all values ​​of the variable are within the experimental error range (e.g., within the 95% confidence interval of the mean) or within ±10% of the specified value, or a wider range.

[0102] The term "mixture" refers to a mixture containing more than one compound, where one or more compounds may be the target compound. The term "target compound" refers to the compound to be separated or purified. When defining a separation process, the target compound is determined prior to the separation operation. It should be understood that the product containing the target compound can be in any desired form, such as a product containing pure isomers of the compound or a mixture containing multiple predetermined target compounds.

[0103] The term "stoichiometry" refers to the proportion of various substances by weight.

[0104] The terms “optional” or “optionally” mean that the events described below may occur but not necessarily occur, and the description includes the possibility that the events or situations described therein may or may not occur.

[0105] The expression "comprising," or similar expressions such as "including," "containing," and "having," is open-ended and does not exclude additional unlisted elements, steps, or components. The expression "consisting of," excludes any unspecified elements, steps, or components. The expression "substantially consisting of," limits the scope to the specified elements, steps, or components, plus optional elements, steps, or components that do not materially affect the essential and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses both the expressions "substantially consisting of" and "consisting of."

[0106] The chemical bonds in the compounds disclosed herein can be represented by solid lines. wavy lines solid wedge Or dashed wedge shape To describe it. Bonds to asymmetric atoms depicted with solid lines are intended to show that all possible stereoisomers of that atom (e.g., specific enantiomers, racemic mixtures, etc.) are taken into account. Bonds to asymmetric atoms depicted with wavy lines are intended to show that the bond is a solid wedge. Key or dashed wedge Bonds. Bonds with asymmetric atoms, indicated by solid or dashed wedges, indicate the presence of the stereoisomers shown. When present in racemic mixtures, solid or dashed wedges are used to define relative stereochemistry rather than absolute stereochemistry. Unless otherwise stated, the compounds of this disclosure may exist as stereoisomers (including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, trans-blocking isomers, and mixtures thereof). The compounds of this disclosure may exhibit one or more of the above-described isomer forms and may consist of mixtures thereof (e.g., racemic mixtures and / or diastereomer pairs).

[0107] The term "hydrocarbon group" refers to a monovalent group derived from a hydrocarbon. Examples of hydrocarbon groups include, but are not limited to, alkyl, alkenyl, ynyl, cycloalkyl, and aryl groups.

[0108] The term "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group composed of carbon and hydrogen atoms, connected to the rest of the molecule by single bonds. Alkyl groups can contain 1-20 carbon atoms, referring to C1-C2. 20Alkyl groups, such as C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C3 alkyl, C4 alkyl, and C3-C6 alkyl. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or isomers thereof.

[0109] The term "valence state" refers to the property of a group to be monovalent, divalent, or trivalent. A divalent group is a group obtained by removing a hydrogen atom from a carbon atom with free valence electrons in the corresponding monovalent group, and a trivalent group is a group obtained by removing a hydrogen atom from a carbon atom with free valence electrons in the corresponding divalent group. For example, "alkylene" refers to a straight-chain or branched saturated divalent hydrocarbon group. Examples of alkylene include, but are not limited to, methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), and pentylene (-C5H6-). 10 -), Hexyl (-C6H) 12 -), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropene, ethylpropene. Examples of cycloalkylene groups include, but are not limited to, divalent monocycloalkylene groups, such as cyclopropylene, cyclobutene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene, as well as divalent polycycloalkylene groups containing fused rings, spiro rings, or bridged rings. Examples of arylene groups include, but are not limited to, phenylene. Examples of heterocyclic groups include, but are not limited to, pyrrolidine, imidazoline, pyrazolidine, piperidinium, and morpholino. Examples of heteroarylene groups include, but are not limited to, pyrrolidine, furanyl, thiopheneyl, imidazolyl, oxazolyl, diazolyl, oxtriazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, pyrrolidine, triazolyl, and tetrazolyl.

[0110] The term "ring-opening thiosuccinimide group" refers to the product obtained by ring-opening the succinimide ring in the thiosuccinimide group. The ring-opening reaction of the thiosuccinimide group can occur by breaking either of the two amide bonds in the thiosuccinimide group, yielding two isomers. Specifically, the ring-opening thiosuccinimide group is selected from... Preferred

[0111] The term "position selectivity" refers to the phenomenon in a chemical reaction that may occur at multiple sites on a particular molecule, where a reagent preferentially reacts with one atom relative to another, or preferentially produces a certain positional isomer relative to another positional isomer. "Region specificity" refers to the selectivity where, for two or more possible sites on a particular molecule, a chemical reaction or reagent targets only one of those sites.

[0112] The term "diastereomer" refers to a stereoisomer of a molecule that has two or more chiral centers and is not a mirror image of the other molecules.

[0113] Small molecule compounds are molecules whose size is comparable to that of organic molecules commonly used in pharmaceuticals. This term excludes biological macromolecules (such as proteins and nucleic acids) but includes low molecular weight peptides or their derivatives, such as dipeptides, tripeptides, tetrapeptides, and pentapeptides. Typically, the molecular weight of small molecule compounds can be, for example, about 100–about 2000 Da, about 200–about 1000 Da, about 200–about 900 Da, about 200–about 800 Da, about 200–about 700 Da, about 200–about 600 Da, or about 200–about 500 Da. As used herein, small molecule compounds may also be referred to as pharmaceuticals.

[0114] As used herein, the term "antibody (Ab)" is an immunoglobulin (Ig) molecule or a derivative thereof that specifically binds to an antigen through at least one antigen-binding site. "Conventional" or "full-length" antibodies typically consist of four polypeptides: two heavy chains (HC) and two light chains (LC). As used herein, the definition of "antibody" includes, but is not limited to, conventional antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, biantibodies, or nanobodies (i.e., single-domain antibodies, VHH domains). Members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclasses (e.g., IgG2a and IgG2b), or any derivative thereof, are also considered.

[0115] As used herein, an "antibody fragment" refers to any portion of an antibody containing fewer amino acid residues than a full-length antibody, such as an antigen-binding fragment (e.g., one or more CDRs) containing at least a portion of a variable domain and specifically binding to the same homologous antigen as the full-length antibody, or an Fc fragment containing a constant region of the antibody heavy chain and binding to an Fc receptor on a cell surface. Antibody fragments can be obtained by a variety of methods, such as chemical or enzymatic treatment, chemical synthesis, or recombinant DNA technology. Examples of antibody fragments include, but are not limited to, Fv (fragment variable region), scFv (single-chain Fv fragment), dsFv (disulfide-stable variable fragment), scdsFv (single-chain disulfide-stable variable fragment), biantibodies, Fd (difficult fragment), Fab (antigen-binding fragment), scFab (single-chain Fab), Fab', F(ab')2, Fc (crystallizable region fragment), and any derivative thereof.

[0116] HER2 refers to human epidermal growth factor receptor 2, which belongs to the epidermal growth factor (EGFR) receptor tyrosine kinase family. In this application, the terms ErbB2 and HER2 have the same meaning and can be used interchangeably.

[0117] As used herein, the term "natural amino acid" refers to an amino acid that is a constituent amino acid of proteins, including the twenty common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), and the less common selenocysteine ​​and pyrrolidone. As used herein, the term "non-natural amino acid" refers to an amino acid that is not a constituent amino acid of proteins. Specifically, this term refers to an amino acid that is not a natural amino acid as defined above.

[0118] When specifying amino acid sequences in this document, standard single-letter amino acid codes are used unless otherwise stated. Thus, for example, LPXTG represents the sequence -Leu-Pro-X-Thr-Gly-, where X is any natural or non-natural single amino acid residue, LPETG represents Leu-Pro-Glu-Thr-Gly-, LPETGG represents -Leu-Pro-Glu-Thr-Gly-Gly-, and GGG represents -Gly-Gly-Gly-.

[0119] As used herein, the term "peptide mimic" refers to a compound that mimics the conformation and desired characteristics of a specific peptide.

[0120] As used herein, a "receptor" refers to a structure inside or on the surface of a cell that binds to specific substances and elicits specific effects within the cell. Receptors can include, as described herein, T-cell receptors, B-cell receptors, and receptors for signaling molecules, cell growth factors, and cytokines.

[0121] As described herein, interactions between chemical entities (e.g., ions, molecules, groups, or atomic groups) refer to non-covalent interactions, including but not limited to hydrogen bonds, π-π stacking, ionic interactions, ionic induced dipole forces, and ionic dipole forces.

[0122] As used herein, the term "ionization" refers to the dissociation of one or more acidic hydrogen atoms in an acidic group to form a group with one or more negative charges, or the combination of one or more protons with a basic group to form a group with one or more positive charges. For example, an "ionizable" carboxyl group can form a negatively charged group (-COO). - "Ionizable" amino groups can form positively charged groups (-NH3). + Molecules containing one or more ionizable acidic groups and one or more ionizable basic groups can form zwitterions.

[0123] The term “reversed-phase chromatography” (RPC) includes any chromatographic method that uses a hydrophobic stationary phase and a polar mobile phase, wherein the hydrophobic stationary phase has a stronger affinity for hydrophobic or less polar molecules.

[0124] Reversed-phase liquid chromatography (RPLC) is a widely used technique in biopharmaceutical research and manufacturing. A common stationary phase for RPLC is RP-modified silica gel, for example, silica gel grafted with hydrocarbon groups, such as alkyl chains and / or cycloalkyl or aryl groups (e.g., phenyl, pentafluorophenyl, cyclohexyl). "Monofunctional" alkyl silica gel stationary phases refer to those obtained by reacting a molecule of silane with a silanol group on a silica gel support. "Bidentate" bonded alkyl silica gel stationary phases refer to alkyl silica gel stationary phases obtained by reacting a molecule of bidentate silane containing two reactive groups with two silanol groups on a silica gel support. Bidentate stationary phases with two functional groups (e.g., alkyl groups, such as methyl, n-butyl, n-octyl, or n-octadecyl) on a bidentate silane are also called bifunctional stationary phases. Examples of alkyl silica gel stationary phases, such as alkyl-bonded silica gel, include, but are not limited to, silica gel bonded to C18, C8, C4, or C1 alkyl groups, especially C18 alkyl groups. The alkyl groups in alkyl-bonded silica gel can be straight-chain or branched. In one embodiment, the alkyl group in the alkyl-bonded silica gel is a straight-chain C18 alkyl group (n-octadecyl). In another embodiment, the C18 alkyl group in the alkyl-bonded silica gel is branched and contains one or more side-chain alkyl groups selected from C1-C6 alkyl groups (preferably isopropyl, isobutyl) and any combination thereof. Some alkyl silica gel stationary phases contain embedded functional groups, such as urethane groups (e.g., Symmetry Shield). TM Modified silica gels include RP C18 and amide groups (e.g., Zorbax Bonus-RP C18). Mixed modifiers are also used in RPLC, such as silica gel partially capped with hydrocarbon groups and containing a proportion of residual (uncapped) silica silanols, and silica gel partially capped with hydrocarbon groups and containing a proportion of modified polar groups such as NH2-. To ensure robust chromatographic separations can be achieved using suitable equipment, RP-modified silica gels can be particles of any suitable shape. For example, alkyl-bonded silica gels are typically spherical particles. In some cases, RP-modified silica gels are porous on the surface or completely porous (both surface and interior). In another embodiment, the RP-modified silica gel has a controlled surface porosity and a pore size of [missing information]. -1μm, preferred More For example In another embodiment, the RP-modified silica gel has a controlled surface porosity, and the pore size of the RP-modified silica gel is as shown above.

[0125] The mobile phase of RPLC can be a mixture of organic solvent and water. In some literature, the organic solvent is also referred to as an "organic modifier". Examples of aqueous binary mobile phases used in RPLC include, but are not limited to, ACN / H2O, MeOH / H2O, i-PrOH / H2O, or THF / H2O. Examples of aqueous ternary mobile phases used in RPLC include, but are not limited to, THF / MeOH / H2O.

[0126] The mechanism of RPLC generally involves the following aspects: the interaction between the stationary phase and the solute, which is controlled by changing the polarity of the mobile phase; solute-solvent and solvent-solvent dispersion interactions. Since the 1960s, the theory of solute retention in liquid chromatography has been developing. Efforts have been made to establish retention models based on molecular interactions, such as solute-solvent interactions (Scott-Kucera interaction model), solute-solvent interactions, and the localization of solute and / or solvent during adsorption (Snyder-Soczewinski model, also known as the linear solvent strength (LSS) model). However, considering the variability of many factors and parameters affecting chromatographic retention, the details of the LC retention mechanism remain unclear in general. In the development of high-pressure chromatography, the use of small-particle column packing materials and high column pressure has improved chromatographic accuracy and robustness, and some new retention modeling methods have been reported, such as lyochromic analysis (a method based on linear solvation-energy relationships, LSER). The RPLC mechanism of peptides is related to the size of the peptide, and the mechanism of oligopeptides is similar to that of other small molecules. Several semi-empirical models for predicting peptide retention have been proposed, and some calculated "coefficients" have been used to represent the interaction strength between amino acid residues and the stationary phase. These coefficients do not distinguish between hydrophobic, hydrophilic, ionic, and other interactions. Despite these developments, no predictive model has yet performed satisfactorily in real-world experimental settings. Successful predictions of chromatographic behavior for the identification of large numbers of unknown small or large molecules have not been reported.

[0127] "Hydrophobicity" refers to the association of nonpolar groups or molecules in an aqueous environment, arising from the tendency of water to repel nonpolar molecules, and is understood as a measure of the relative tendency of a solute to favor a non-aqueous environment relative to an aqueous environment, or as a measure of the tendency of two (or more) solute molecules to aggregate in an aqueous solution. "Lipophilicity" represents the affinity of a molecule or portion for a lipophilic environment. It is typically measured by its distribution behavior in a two-phase system, whether liquid-liquid (e.g., partition coefficient in 1-octanol / water) or solid-liquid (e.g., retention on TLC or RP HPLC). When used herein to describe a particular chemical entity, the terms "lipophilicity" and "hydrophobicity" represent the same characteristic of that chemical entity and are therefore used interchangeably.

[0128] The retention factor (k) of a component can be expressed by the formula k = (t)R -t M ) / t M =(V R -V M ) / V M Determined from the chromatogram, where t R and V R These are the retention time and retention volume of the components, t. M It is the retention time of non-retained components, such as air or non-retained solvent peaks or non-retained markers, V M This is the volume of mobile phase required to elute unretained components. The logarithmic form of the retention factor, log k, is used as a lipophilicity index. However, log k is not an equally important parameter as log P for quantifying lipophilicity. Although log k shows a good correlation with shake-flask partition data (log P) in some cases, significant differences are observed between slow equilibrium systems and chromatographic partitioning systems.

[0129] Chromatographic resolution (R) is calculated using the following formula: R = 2(t R2 -t R1 ) / (W1+W2), where t R2 and t R1 W1 represents the retention times of the two components; W2 and W1 are the corresponding widths of the peak bases obtained by extrapolating the relatively straight edges of the peaks to the baseline.

[0130] When used herein, the expressions “satisfactory chromatographic separation” and similar expressions mean a chromatographic resolution (R) > 0.8, preferably > 0.9, e.g., > 1.0, > 1.1, > 1.2, > 1.3, > 1.4, > 1.5, > 1.6, > 1.7, > 1.8, > 1.9, or > 2.0. In one particular embodiment, R > 1.0. In another particular embodiment, R > 1.5. Those skilled in the art can set and adjust the expected values ​​of the chromatographic results according to different needs and circumstances. It should be understood that in some cases, purification does not need to be thorough (100% purity), and a given purity (e.g., > 99%, > 98%, > 95%, > 90%, > 85%, > 80%, > 75%, > 70%, > 65%, or > 60%) prior to separation can be considered satisfactory.

[0131] In RPLC with ionizable solutes, the non-ionized form has the longest retention time, while the ionized form has the shortest. When the mobile phase pH is changed (e.g., in an elution series), the retention time of the ionizable solute will always fall between that of its ionized and non-ionized forms.

[0132] In RPLC, the elution gradient generally refers to the gradient of increasing organic solvent content or pH value over time. Peak compression is a characteristic of gradient separation. During gradient separation, the solute movement differs between the front (near the column outlet) and the rear (near the column inlet) of the peak. When the solute in the rear of the peak moves faster than the solute in the front, peak width compression is observed. Therefore, by adjusting the organic solvent content / mobile phase pH, not only retention time but also peak width can be adjusted. However, it is worth noting that the peak width change due to peak compression in pH gradient separation cannot be accurately predicted.

[0133] The pH gradient in the mobile phase is a result of parameters including, for example, the properties of the solute, the content of acidic / basic substances, temperature, and the proportion of organic solvents when the mobile phase is partially aqueous.

[0134] The specific pKa values ​​or pKa ranges mentioned in this disclosure refer to values ​​measured in aqueous solutions. For pKa values ​​not disclosed in this disclosure, refer to those published by IUPAC, such as Ionisation Constants of Organic Acids in Aqueous Solution, Serjeant, EP, Dempsey B., IUPAC Chemical Data Series No. 23, 1979. New York, New York: Pergamon Press, Inc., p. 989. Specific pH values ​​or pH ranges refer to the apparent pH values ​​when the liquid or solution in question contains only a portion of water.

[0135] Industrially, chromatographic methods for different pressure ranges have been developed, such as atmospheric pressure chromatography, medium-pressure chromatography, and high-pressure chromatography. The term "high-pressure chromatography" refers to a chromatographic method that applies pressures greater than ambient pressure. "High-pressure chromatography" refers to a chromatographic method in which the mobile phase and a liquid or suitably dissolved solid sample are forced through a column under high pressure, wherein the "high pressure" is sufficient to propel the mobile phase at a desired flow rate through particles of the stationary phase, which are typically smaller in size than those used in atmospheric or medium-pressure chromatography. In one embodiment, the stationary phase used in the RPLC method of this disclosure is RP-modified silica gel with a particle size of 1-300 μm, preferably 1-200 μm, more preferably 1-100 μm, even more preferably 1-80 μm, and more preferably 3-60 μm, such as 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 20 μm, 40 μm, 50 μm, and 60 μm.

[0136] Reversed-phase ion-pair chromatography (RPIPC) (also known as reversed-phase ion-pair chromatography (IPRP)) is a chromatographic method that adds a small amount of ion pairs to the mobile phase (e.g., by adding ion-pairing reagents), which leads to an increase in the retention of strongly polar compounds. The retention of the resulting ion pairs is controlled by pH, the concentration of the counter-charged ion, and the polarity of the mobile phase. In some cases, strongly polar compounds are charged molecules that are not retained in conventional reversed-phase chromatography. The mechanism of RPLC does not exclude the possible influence of ion pairs. The presence of acidic or basic substances in the RPLC mobile phase may affect the chromatographic behavior of the solute with respect to ambient pH, or it may affect the solute by forming ion pairs, such as with ionized forms of the solute.

[0137] The term "chromatographic process" is an abstract term that generally refers to a separation process using chromatographic methods. A separation process may include operations for separation and optionally operations for cleaning, equilibrating, or regenerating the chromatographic medium.

[0138] The "separation range" of an eluent refers to the eluent used for separation, and the "equilibrium range" of an eluent refers to the eluent used for equilibration. It should be understood that the separation range can represent the eluent composition that changes over time, and said change can be terminated by the end of a time period, for example due to a critical event such as the completion of elution of the target product.

[0139] The method disclosed herein

[0140] Separation methods

[0141] In a first aspect, this disclosure provides a method for separating one or more target compounds from a mixture 1, said mixture 1 comprising four compounds, each of said four compounds comprising part 1, part 2 and part 3;

[0142] Part 1 has an open-ring thiosuccinimide structure, selected from formulas (I) to (IV):

[0143]

[0144] in

[0145] The thiol and amide groups in part 1 form two linkage sites, part 2 is linked to part 1 through one of them, and part 3 is linked to part 1 through the other of them;

[0146] Part 2 contains one or more chiral centers;

[0147] Part 1 of the four compounds is different;

[0148] Part 3 is the remaining portion of the molecule;

[0149] The molecular weight of part 3 does not exceed 1900;

[0150] The one or more target compounds are selected from the four compounds contained in mixture 1;

[0151] The method includes the following steps (1) and (2)

[0152] (1) Provide mixture 1;

[0153] (2) Mixture 1 was subjected to chromatography to obtain the target compound.

[0154] in

[0155] a. Each of the target compounds was obtained as an isolated product, or

[0156] b. The two target compounds were obtained as a mixture, which is defined as mixture 2.

[0157] Based on the position of the thiol group, formulas (I) and (II) are referred to as β, and the configurations of formulas (III) and (IV) are referred to as α.

[0158] In one embodiment, the method further includes steps (3) and (4).

[0159] (3) Recover the eluent collected in step (2), which contains mixture 3, wherein mixture 3 contains one or more additional target compounds that are different from the target compounds separated in step (2);

[0160] (4) Perform chromatography on the eluent recovered in step (3) to separate the additional target compound.

[0161] In one embodiment, step (1) is optional. In another embodiment, mixture 1 is provided by a synthesis method known in the art.

[0162] In one embodiment, each of one or more target compounds is primarily, for example, a pure or substantially pure isomer form, for example, substantially free of other isomer forms, for example, having a purity of more than 90%, for example, more than 95%, for example, more than 98%, for example, at least 99%.

[0163] 1. Chromatographic Procedure

[0164] In one embodiment, part 1 is different in each target compound.

[0165] In one embodiment, in steps (2) and (4), four target compounds are obtained, two of which are obtained as isolated products in step (2) and the other two target compounds are obtained as isolated products in step (4).

[0166] In a preferred embodiment, in step (2), each of the two target compounds obtained from the separation product has a fraction 1 of formula (I) or formula (II); and in step (4), each of the two target compounds obtained from the separation product has a fraction 1 of formula (III) or formula (IV).

[0167] In another preferred embodiment, in step (2), the three target compounds contained in mixture 1 are obtained as a separation product, and the remaining one is obtained in step (4).

[0168] In another preferred embodiment, four target compounds are obtained in step (2), wherein two target compounds are obtained as isolated products, and the remaining two target compounds are obtained in mixture 2. In a more preferred embodiment, portions 1 of the two target compounds obtained as isolated products in step (2) each have formula (I) or formula (II); and portions 1 of the two target compounds obtained in mixture 2 each have formula (III) or formula (IV).

[0169] 2. Stationary phase

[0170] In one embodiment, the chromatography in steps (2) and (4) is reversed-phase chromatography. In another embodiment, the stationary phase used in the reversed-phase chromatography of steps (2) and (4) is independently selected from alkyl-bonded silica gel.

[0171] There are no particular limitations on the alkyl-bonded silica gels used in this disclosure. Any alkyl silica gel stationary phase can be used as long as acceptable chromatographic results can be obtained. In a preferred embodiment, the stationary phase used for the reversed-phase chromatography in steps (2) and (4) is C18-bonded silica gel (i.e., C18 alkyl-bonded silica gel). In a preferred embodiment, the alkyl group in the C18-bonded silica gel is a straight-chain C18 alkyl group (n-octadecyl). In one embodiment, the C18-bonded silica gel is generally in the form of spherical particles. In another embodiment, the C18-bonded silica gel has a controllable surface porosity and a pore size of [missing information]. Preferred More For example

[0172] 3. Mobile phase

[0173] In one embodiment, the mobile phase in step (2) is as follows:

[0174] Eluent composition:

[0175] Eluent A: Water, which optionally contains an acidic agent 1;

[0176] Eluent B: Organic solvent 1, which optionally contains an acidic agent 2;

[0177] The condition is that at least one of acidic agent 1 and acidic agent 2 is present;

[0178] Separation range: B is the gradient from approximately 0%-30% to approximately 30%-100%, and the remainder is A;

[0179] The acidic agent 1 and acidic agent 2 are independently inorganic or organic acids.

[0180] In one embodiment, organic solvent 1 is selected from methanol and ACN. In a preferred embodiment, organic solvent 1 is methanol.

[0181] In one embodiment, the separation range of the mobile phase in step (2) is as follows: B is a gradient of about 40%-50% to about 50%-70%, and the remainder is A.

[0182] In one embodiment, at least one of acidic agent 1 and acidic agent 2 is present. In a preferred embodiment, both acidic agent 1 and acidic agent 2 are present.

[0183] In one embodiment, the amounts of acidic agent 1 and acidic agent 2 (if present) are such that the gradients of eluent C and eluent D are associated with the gradients of acidic agent 1 and acidic agent 2.

[0184] In one embodiment, acid agent 1 has the structure R c (COOH) d Where d is 1 or 2, R c C 1-15 Hydrocarbon group or hydrocarbon subgroup, which is optionally composed of at least one selected from R y Substituents of R, wherein R y The derivative is selected from -OH, -OCH3, -OCH2CH3, -SH, and -SCH3, with -OH being preferred. In a preferred embodiment, R... c C in 1-15 The hydrocarbon group is C 1-15 Alkyl, alkylene, C 1-15 alkenyl or C 1-15 alkenyl groups, which may be selected from one, two, three or four R groups. y Substituents are substituted. In another preferred embodiment, d is 1, and R c C in 1-15 The hydrocarbon group is C 1-10 Alkyl, preferably C 1-4 Alkyl group. In a preferred embodiment, R c C in 1-15 The hydrocarbon group is C 1-2 Alkyl group. In one particular embodiment, R c C in 1-15 The hydrocarbon group is methyl. In a preferred embodiment, d is 2, and Rc C in 1-15 The hydrocarbon group is C 2-4 Alkylene, particularly ethylene, is optionally substituted with one or two -OH groups.

[0185] In one embodiment, the acidifying agent 1 is selected from organic acids. In one embodiment, the pKa value of the acidifying agent 1 is 0.1-6.5, for example 0.4-6.5, 0.6-6.5, 1.0-6.5, 2.0-6.5, or 2.0-5.5. In a preferred embodiment, the acidifying agent 1 is selected from organic acids, and the pKa value of the acidifying agent 1 is 4.0-5.5, for example about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4 or about 5.5, particularly about 4.7 or about 4.8, for example about 4.71, about 4.72, about 4.73, about 4.74, about 4.75, about 4.76, about 4.77, about 4.78 or about 4.79. In another preferred embodiment, the acidic agent 1 is selected from organic acids, and the pKa value of the acidic agent 1 is 2.3-3.8, for example about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7 or about 3.8, particularly about 3.0 or about 3.1, for example about 3.01, about 3.02, about 3.03, about 3.04, about 3.05, about 3.06, about 3.07, about 3.08 or about 3.09.

[0186] In another embodiment, acidifier 1 is selected from AcOH and L-tartaric acid (L-TA). In a preferred embodiment, acidifier 1 is AcOH. In one embodiment, the amount of acidifier 1 is about 0.01% to about 1% based on the total volume of eluent A. In a preferred embodiment, the amount of acidifier 1 is about 0.05% to about 0.5% based on the total volume of eluent A, preferably about 0.1% to about 0.5%, more preferably about 0.1% to about 0.3%, and particularly 0.3%.

[0187] In one embodiment, acidic agent 2 is absent. In another embodiment, the type of acidic agent 2 is the same as that of acidic agent 1.

[0188] In one embodiment, the amount of acidic agent 2 is 0.01% to about 1% based on the total volume of eluent B. In another embodiment, the amount of acidic agent 2 is about 0.05% to about 0.5% based on the total volume of eluent B, preferably about 0.1% to about 0.5%, more preferably about 0.1% to about 0.3%, and particularly 0.3%.

[0189] In one embodiment, when eluting the target compound, the total amount of acidic agent 1 and acidic agent 2, based on the total volume of eluent A and eluent B, does not exceed about 1%, preferably not more than about 0.8%, more preferably not more than about 0.6%, for example, not more than about 0.4%, and particularly not more than about 0.285%.

[0190] In one embodiment, the total content of acidic agent 1 and acidic agent 2 (if present) in the elution composition is about 0.005-0.06M.

[0191] In an alternative embodiment, the contents of acidifier 1 and acidifier 2 are dynamically set and / or adjusted to obtain a target pH profile, for example, to obtain a target isocratic pH value or a target pH gradient.

[0192] In one embodiment, the pH of the mobile phase is about 1.0 to about 4.0. In another embodiment, the pH of the mobile phase is about 1.0 to about 3.5. In one embodiment, the pH of the mobile phase is about 2.0 to about 3.5. In another embodiment, the pH of the mobile phase is about 2.4 to about 3.2.

[0193] In one embodiment, the mobile phase in step (4) is as follows:

[0194] Eluent composition:

[0195] Eluent C: Water, which optionally contains an acidic agent 3;

[0196] Eluent D: Organic solvent 2, which optionally contains an acidic agent 4;

[0197] Separation range: D is the gradient from approximately 0%-30% to approximately 30%-100%, and the remainder is C;

[0198] The acidic agent 3 and acidic agent 4 are independently inorganic or organic acids.

[0199] In one embodiment, organic solvent 2 is selected from methanol and ACN. In a preferred embodiment, organic solvent 2 is CAN.

[0200] In one embodiment, at least one of acidic agent 3 and acidic agent 4 is present. In a preferred embodiment, both acidic agent 3 and acidic agent 4 are present.

[0201] In one embodiment, the separation range of the mobile phase in step (4) is as follows: D is a gradient of about 10%-30% to about 30%-95%, and the remainder is C.

[0202] In one embodiment, the amounts of acidic agent 3 and acidic agent 4 (if present) are such that the gradients of eluent C and eluent D are associated with the gradients of acidic agent 3 and acidic agent 4.

[0203] The substances used for acidifying agents 3 and 4 are not particularly limited, as long as satisfactory chromatographic separation can be achieved. In one embodiment, acidifying agent 2 is selected from organic acids. In another embodiment, the pKa value of acidifying agent 2 is 0.1-6.5, for example, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3. 0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4 or about 6.5.

[0204] In one embodiment, acidifier 3 and acidifier 4 are independently selected from TFA, phosphate buffer, ammonium acetate (AA), AcOH, H3PO4, TEAP, and L-tartaric acid (L-TA). In a preferred embodiment, acidifier 3 is selected from TFA, phosphate buffer, AA, and TEAP. In a more preferred embodiment, acidifier 3 is selected from TFA, phosphate buffer, and TEAP, particularly TFA.

[0205] In one embodiment, acidic agent 4 is absent. In another embodiment, the type of acidic agent 4 is the same as that of acidic agent 3.

[0206] In one embodiment, the amount of acidic agent 3 is about 0.01% to about 1% based on the total volume of eluent C. In another embodiment, the amount of acidic agent 3 is about 0.05% to about 0.5% based on the total volume of eluent C, preferably about 0.05% to about 0.3%, particularly 0.1%.

[0207] In one embodiment, the amount of acidic agent 4 is about 0.01% to about 1% based on the total volume of eluent D. In another embodiment, the amount of acidic agent 4 is about 0.05% to about 0.5% based on the total volume of eluent D, preferably about 0.05% to about 0.3%, particularly 0.1%.

[0208] In one embodiment, the separation range of the mobile phase in step (4) is as follows: D is a gradient of about 10%-30% to about 30%-95%, and the remainder is C.

[0209] In one embodiment, the total content of acidic agent 3 and acidic agent 4 (if present) in the elution composition is about 0.01-0.25M.

[0210] In an alternative embodiment, the contents of acidifier 3 and acidifier 4 are dynamically set and / or adjusted to obtain a target pH spectrum, for example, to obtain a target isocratic pH value or a target pH gradient.

[0211] In one embodiment, the pH of the mobile phase is about 1.0 to about 6.0. In a specific embodiment, the pH of the mobile phase is about 2.0 to about 5.0, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0.

[0212] In one embodiment, an isocratic pH value is applied, and the isocratic pH value is in the range of about 2.0 to about 6.0. In another embodiment, the isocratic pH value of the mobile phase is about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 5.0, or about 6.0.

[0213] The method further includes an optional equilibration step prior to step (2), wherein the mobile phase is as defined above, and the equilibration range of the mobile phase is approximately 100%-95%A and approximately 0%-5%B, for example, approximately 95%A and approximately 5%B. In another optional embodiment, the method further includes an optional equilibration step prior to step (4), wherein the mobile phase is as defined above, and the equilibration range of the mobile phase is approximately 100%-95%C and approximately 0%-5%D, for example, approximately 95%C and approximately 5%D.

[0214] 4. Solute (target compound)

[0215] In one embodiment, the log P (octanol-water partition coefficient) value of part 2 is about 1 to about 5; preferably about 1.5 to about 4.5; about 2 to about 4.5; or about 2.5 to about 4.5, for example about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4 or about 4.5.

[0216] In one embodiment, part 2 has the following formula (V):

[0217]

[0218] Where Q is a group containing at least one chiral center; and

[0219] L 1 It does not exist, or it is a divalent group, wherein the divalent group is selected from one or more of the following: C 1-10 Alkylene, C 3-10 Cycloalkylene, C 6-10 Arylene, 4-10 heterocyclic alkylene, 5-10 heterocyclic alkylene, -NH-, -(CO)-, -NH(CO)- and -(CO)NH-.

[0220] In one implementation scheme, Q is C 5-50 hydrocarbon group, in which

[0221] One or more “CH2” structures in the hydrocarbon group are optionally replaced by -O-, -S-, -NH-, -C(=O)-, -S(=O)-, -S(=O)2-, -NH(C=O)-, -C(=O)NH-, -NH-, -S(=O)-, -S(=O)NH-, -NHS(=O)2- or -S(=O)2NH-, provided that a stable structure is formed;

[0222] One or more "CH" structures in the hydrocarbon group may be optionally replaced by N or P, provided that a stable structure is formed;

[0223] One or more carbon, sulfur, nitrogen, or phosphorus atoms in Q are independently and optionally substituted with oxygen (=O); Q is optionally substituted with at least one atom selected from R. q Substituents of R, wherein R q Each independently selected from R a1 -OR a1 -SR a1 -NR a1 R b1 -C(=O)OR a1 -C(=O)NR a1 R b1 -C(=O)R a1 -S(=O)2OR a1 -S(=O)2R a1 -S(=O)2NR a1 R b1 -S(=O)R a1 -C(=S)OR a1 -C(=S)NR a1 R b1 -C(=S)R a1 -P(=O)(OR) a1 OR b1-C(=NR) a1 )NR b1 R c1 ;and

[0224] R a1 R b1 and R c1 Each is independently selected from hydrogen and C. 1-6 alkyl.

[0225] In one embodiment, the cycloalkyl structures in Q are each optionally and independently replaced by heterocyclic structures with the same group valence state and the same number of ring atoms as the cycloalkyl structures.

[0226] In one embodiment, the aryl structures in Q are each optionally and independently replaced by heteroaryl structures having the same group valence state and the same number of ring atoms as the aryl structures.

[0227] In one implementation scheme, R q Each is independently selected from halogens, -C 1-3 Alkyl, -OH, -OC 1-3 Alkyl, -SH, -SC 1-3 Alkyl, -C(=O)-C 1-3 Alkyl groups and -S(=O)2-C 1-3 Alkyl group. In a very specific embodiment, R q Selected from halogens, -CH3, -OH and -OCH3.

[0228] In one implementation scheme, L 1 It does not exist.

[0229] In one embodiment, part 2 has the structure of the following formula (V-1):

[0230]

[0231] The loading material is selected from hydrogen and small molecule compounds containing at least one chiral center; and

[0232] L 1 As defined above.

[0233] In one embodiment, the small molecule is selected from enzyme inhibitors, enzyme agonists, receptor modulators (e.g., agonists or antagonists), toxins (e.g., cytotoxins), glycans, PEG moieties, and radionuclides (e.g., 225 Ac、 211 At、 212Bi , 213 Bi、 67 Ga、 123 I, 124 I, 125 I,131 I, 111 In、 177 Lu、 191m Os、 195m Pt, 186 Re、 188 Re、 119 Sb, 153 Sm、 99m Tc, 227 Th and 90 Y), nucleic acids and analogues (e.g., interfering RNA), tracer molecules (e.g., fluorophores and fluorescent molecules), low molecular weight peptides (e.g., protein tags, bioactive peptides, protein toxins and enzymes with molecular weights below 2000 Da, 1000 Da, 900 Da, 800 Da, 700 Da, 600 Da or 500 Da), low molecular weight peptide mimics, low molecular weight antibodies (e.g., nanobodies) and antibody fragments.

[0234] In one embodiment, fraction 2 does not contain ionizable acidic groups or ionizable basic groups. In another embodiment, fraction 2 contains a number of ionizable acidic groups and ionizable basic groups, and under the chromatographic conditions used herein, the total charge of fraction 2 is approximately zero. Ionizable acidic groups include, but are not limited to, carboxyl groups, sulfinic groups, sulfonic acid groups, phosphonic acid groups, and phosphonic acid groups, particularly carboxyl groups. Ionizable basic groups include, but are not limited to, amino groups, and particularly amino groups. In an alternative embodiment, the support does not contain ionizable carboxyl groups or ionizable amino groups. In another alternative embodiment, the support comprises an equal number of ionizable carboxyl groups and ionizable amino groups.

[0235] In one embodiment, part 2 has the structure of the following formula (V-1-1):

[0236]

[0237] The toxin is a cytotoxic moiety containing one or more chiral centers, and L 1 As defined above.

[0238] In one implementation scheme, L 1 Does not exist, or is selected from C 1-10 Alkylene, wherein one or more (-CH2-) structures of the alkyl group are optionally substituted with oxo (=O).

[0239] In a preferred embodiment, the cytotoxicant is selected from taxanes, maytansines, orlistatines, epothilones, comprehtidin A-4 phosphate, comprehtidin A-4 and its derivatives, indole-sulfonamides, vinblastines such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinlycinate, anhydrovinblastine, and dolastatin. 10) and its analogues, tebufenozide B, eribulin, indole-3-oxoacetamides, podophyllotoxins, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, and laulimalide. In another embodiment, the cytotoxicant is selected from the group consisting of DNA topoisomerase inhibitors such as camptothecins and their derivatives, mitoxantrone, and mitoxantrone hydrazone. In a preferred embodiment, the cytotoxicant is selected from the group consisting of nitrogen mustards such as chlorambucil, naphthalenemustine, cholophosphamide, estradiol, ifosfamide, nitrogen mustard, nitrogen oxychloride, melphalan, neonitrogen mustard, methionine mustard, benzylmustine, prednimustine, tromethamine, and uramustine. In another preferred embodiment, the cytotoxic agent is selected from the group consisting of nitrosoureas such as carmustine, flubenzuron, formoterol, lomustine, nimustine, and ramustine. In one embodiment, the cytotoxic agent is selected from the group consisting of azacyclopropanes. In a preferred embodiment, the cytotoxic agent is selected from the group consisting of benzodopa, carboquinone, methotrexate, and uretepa. In one embodiment, the cytotoxic agent is selected from the group consisting of antitumor antibiotics. In a preferred embodiment, the cytotoxic agent is selected from the group consisting of enedyne antibiotics. In a more preferred embodiment, the cytotoxic agent is selected from the group consisting of dynemicin, esperamicin, neomycin, and aclarubicin. In another preferred embodiment, the cytotoxicant is selected from the group consisting of actinomycin, atrazomycin, bleomycin derivatives, actinomycin C, carbapenem, erythromycin, anticancer cytotoxicin, erythromycin, actinomycin D, daunorubicin, detoxin, doxorubicin, epirubicin, esopycin, edabycin, metharubicin, mitomycin derivatives, nopramine, olivomycin, pepromycin, pofibromycin, puromycin, doxorubicin, rodorubicin, streptomycin, streptozotocin, fenestrated statin, and zolrubicin.In another preferred embodiment, the cytotoxic agent is selected from the group consisting of trichothecenes. In a more preferred embodiment, the cytotoxic agent is selected from the group consisting of T-2 toxin, verracurin A, baculosporin A, and anguidine. In one embodiment, the cytotoxic agent is selected from the group consisting of antitumor amino acid derivatives. In a preferred embodiment, the cytotoxic agent is selected from the group consisting of ubenimex, diazoserine, and 6-diazo-5-oxo-L-leucine. In another embodiment, the cytotoxic agent is selected from the group consisting of folic acid analogs. In a preferred embodiment, the cytotoxic agent is selected from the group consisting of folate, methotrexate, pteroxate, trimethoprim, and edaraxate. In one embodiment, the cytotoxic agent is selected from the group consisting of purine analogs. In a preferred embodiment, the cytotoxic agent is selected from the group consisting of fludarabine, 6-mercaptopurine, thioimidapurine, and thioguanine. In yet another embodiment, the cytotoxic agent is selected from the group consisting of pyrimidine analogs. In a preferred embodiment, the cytotoxic agent is selected from the group consisting of ancitabine, gemcitabine, enoxatabine, azacitidine, 6-azouridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, and fluorouridine. In one embodiment, the cytotoxic agent is selected from the group consisting of androgens. In a preferred embodiment, the cytotoxic agent is selected from the group consisting of capprotestone, drotalbuterone propionate, cyclothiosterol, meandrolone, and testrolide. In another embodiment, the cytotoxic agent is selected from the group consisting of anti-adrenergic agents. In a preferred embodiment, the cytotoxic agent is selected from the group consisting of aminoglutethimide, mitotane, and triplostertan. In one embodiment, the cytotoxic agent is selected from the group consisting of anti-androgens. In a preferred embodiment, the cytotoxic agent is selected from the group consisting of flutamide, niglutethimide, bicalutamide, leuprorelin acetate, and goserelin. In yet another embodiment, the cytotoxic agent is selected from the group consisting of protein kinase inhibitors and proteasome inhibitors. In one particular implementation, the cytotoxin is selected from the group consisting of vincristine, colchicine, taxane, orlistatine, and maytansine.

[0240] In one particular embodiment, the cytotoxin is maytansine, such as DM1. It should be noted that when using a cytotoxin containing a thiol moiety, the thiol moiety can react with the maleimide moiety to form a thiosuccinimide, such as maytansine, such as DM1, and the cytotoxin can be directly linked via the thiosuccinimide. In this case, it is understood that in some embodiments, the loading material and the thiol moiety together constitute the cytotoxin; therefore, in this case, the loading material represents the remainder of the cytotoxin molecule except for the thiol moiety.

[0241] In one embodiment, each of the four compounds in mixture 1 is identical in part 3, and the molecular weight of part 3 does not exceed 1900 Da.

[0242] In one embodiment, the molecular weight of part 3 is no more than 1800 Da or 1700 Da; preferably no more than 1600 Da, 1500 Da, 1400 Da, 1300 Da, 1200 Da, 1100 Da, 1000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, or 200 Da. In a particular embodiment, the molecular weight of part 3 is about 100 to about 2000 Da; preferably about 100 to about 1500 Da, about 100 to about 1000 Da, about 200 to about 1000 Da, or about 200 to about 600 Da.

[0243] In one particular embodiment, part 3 has the structure of the following formula (VI):

[0244]

[0245] Where L 2 It is a bond, or a divalent group, wherein the divalent group is selected from one or more of the following: C 1-10 Alkylene, C 3-10 Cycloalkylene, C 6-10 arylene, 4-10 membered heterocyclic, 5-10 membered heteroarylene, -NH-, -(CO)-, -NH(CO)- and -(CO)NH-;

[0246] M contains (1) an amino acid sequence 1 containing 1-20 amino acids, and (2) an optional polyethylene glycol (PEG) moiety containing "-(C2H4-O)". i -” structure, i is an integer from 1 to 100, and (3) optional group Y, which is divalent and selected from cleavable sequence 1, spacer Sp1 and combinations thereof;

[0247] The cleavable sequence comprises an amino acid sequence 2, which can be cleaved by an enzyme, and the cleavable sequence comprises 1-10 amino acids;

[0248] Sp1 is selected from spacer sequences containing 1-20 amino acids, PAB, and combinations thereof.

[0249] In one embodiment, amino acid sequence 1 includes a ligase recognition sequence, i.e., a ligase donor substrate recognition sequence or a ligase acceptor substrate recognition sequence. In one embodiment, the ligase is a transpeptidase. In a preferred embodiment, the ligase is a sortase. In one embodiment, the sortase is selected from sortase A (SrtA), sortase B (SrtB), sortase C (SrtC), sortase D (SrtD), sortase E (SrtE), sortase F (SrtF), and combinations thereof. In one embodiment, the ligase is SrtA. In another specific embodiment, the ligase recognition sequence is selected from LPTCG, where X can be any single amino acid, whether natural or non-natural. In a particular embodiment, the ligase recognition sequence is LPETG. In yet another specific embodiment, the ligase recognition sequence is G. n , where G is glycine (Gly) and n is an integer from 3 to 10.

[0250] In one implementation, i is 1-10.

[0251] In one embodiment, Y is a bond, or is selected from cleavable sequences, spacers Sp1, and combinations thereof. In a particular embodiment, Y is a bond. In one embodiment, amino acid sequence 2 can be recognized as an enzyme substrate and can be cleaved by an enzyme. In a particular embodiment, amino acid sequence 2 can be cleaved by an enzyme in lysosomes of a cell. In another specific embodiment, amino acid sequence 2 can be cleaved by a protease, particularly cathepsin. In yet another specific embodiment, amino acid sequence 2 can be cleaved by glutaminase. In one embodiment, amino acid sequence 2 is selected from cathepsin restriction sites, glutaminase restriction sites, and combinations thereof. In one embodiment, the cleavable sequence is selected from Phe-Lys, Val-Cit, Val-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, and combinations thereof.

[0252] In one embodiment, Y is a bond, or is selected from spacer Sp1. In another embodiment, Sp1 is a spacer sequence comprising 1-10, preferably 1-6, more preferably 1-4 amino acids. In a particular embodiment, Sp1 is Leu. In another specific embodiment, Sp1 is Gln. In one embodiment, Sp1 is PAB. In yet another embodiment, Y is selected from Phe-Lys-PAB, Val-Cit-PAB, and Val-Lys-PAB.

[0253] In one embodiment, the amino acid Y may be natural or non-natural. In a particular embodiment, Y is a bond or amino acid sequence 3. Amino acid sequence 3 contains 1-30 natural or non-natural amino acids, each of which may be independently identical or different. Amino acid sequence 3 is selected from: cleavable sequence 1 containing 1-10 amino acids, spacer Sp1 containing 1-20 amino acids, and combinations thereof.

[0254] In one embodiment, M is selected from lysine, oligoglycine, oligoalanine, oligoglycine / alanine mixtures with a degree of polymerization of 3-10, and combinations thereof.

[0255] In one embodiment, the pKa of portion 3 is 7 or more and 12 or less. In a preferred embodiment, the pKa of portion 3 is about 8 to about 12, preferably about 9 to about 11, for example about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10.0, about 10.1, about 10.2, about 10.3, about 10.4, about 10.5, about 10.6, about 10.7, about 10.8, about 10.9, about 11.0, and particularly about 10.0.

[0256] In one particular implementation, M is L(G). n Where G is glycine (Gly), and n is an integer from 3 to 10, specifically 3. In another particular embodiment, M is G n In one embodiment, the C-terminus of M is connected to L. 2 The linkage is formed, and M has an ionizable amino group. In a particular embodiment, M has only one ionizable group, which is an ionizable amino group. The ionizable amino group can be ionized into the group -NH3. + The ionized form of amino groups -NH3 + It may optionally interact with solvent molecules or chemical entities that carry a negative charge (+) or a partially negative charge (δ+).

[0257] In one alternative embodiment, M is LPXTGJ, where X can be any single amino acid, natural or non-natural; J is absent, or is an amino acid fragment containing 1-10 amino acids, optionally tagged. In one embodiment, J is absent. In another embodiment, J is an amino acid fragment containing 1-10 amino acids, where each amino acid is independently any natural or non-natural amino acid. In yet another embodiment, J is G. m Where m is an integer from 1 to 10. In another specific embodiment, M is LPETG. In yet another specific embodiment, M is LPETGG. In one embodiment, the N-terminus of M is connected to L 2M has a free C-terminal carboxyl group. In a particular embodiment, M has only one ionizable group, which is an ionizable carboxyl group. The ionizable carboxyl group can be ionized into the group -COO. - The ionized form of the carboxyl group -COO - It may optionally interact with solvent molecules or chemical entities that carry a positive or partially positive charge (δ+).

[0258] In one embodiment, the structure of formula (VI) has the structure of formula (VI-1).

[0259]

[0260] Where n is an integer from 3 to 10, and x is selected from hydrogen, OH, NH2, amino acid fragments containing 1 to 10 amino acids, and nucleotide fragments containing 1 to 10 nucleotides.

[0261] In one embodiment, n is 3. In a preferred embodiment, x is selected from OH, NH2, and amino acid fragments containing 1-10 amino acids. In a more preferred embodiment, x is selected from OH, NH2, and Gly. In a particular embodiment, x is NH2.

[0262] In one implementation scheme, L 1 and L 2 Each is independently selected from the key and C. 1-10 Alkyl group, wherein one or more (-CH2-) structures in the alkyl group are optionally substituted with oxo (=O).

[0263] In one implementation, L in part 2 1 Selected from And L in part 3 2 For key.

[0264] In another embodiment, L in part 2 1 For the key, and L in part 3 2 Selected from

[0265] In one embodiment, the mixture 1 comprises four compounds, each independently having a structure selected from the following formulas (1) and (1').

[0266]

[0267] Wherein, the load is defined as in equation (V-1), and M is defined as in equation (VI).

[0268] In one embodiment, the mixture 1 comprises four compounds, each independently having a structure selected from the following formulas (2) and (2').

[0269]

[0270] Wherein, respectively, the load is defined as in equation (V-1), Y is defined as in equation (VI), and n and x are defined as in equation (VI-1).

[0271] In one particular embodiment, the compound of formula (1) and the compound of formula (1') are ring-opening reaction products of the thiosuccinimide group in the following formula (3);

[0272]

[0273] Wherein, the load is defined as in equation (V-1), and M is defined as in equation (VI).

[0274] In one particular embodiment, the compound of formula (2) and the compound of formula (2') are ring-opening reaction products of the thiosuccinimide group in the following formula (4);

[0275]

[0276] Wherein, the load is defined as in equation (V-1), and n and x are defined as in equation (VI-1).

[0277] The ring-opening reaction of the thiosuccinimide group can be carried out by any method known in the art. For example, methods for ring-opening reactions can be found in WO2015165413A1.

[0278] In one particular embodiment, M has an ionizable amino group; acidic agent 1 is AcOH; the amount of acidic agent 1 is about 0.01% to about 1%, preferably about 0.05% to about 0.5%, more preferably about 0.1% to about 0.5%, more preferably about 0.1% to about 0.3%, particularly 0.3%, based on the total volume of eluent A; and acidic agent 2 is absent.

[0279] In another specific embodiment, M has an ionizable amino group; acidic agent 3 is TFA; the amount of acidic agent 3 is about 0.01% to about 1%, preferably about 0.05% to about 0.5%, more preferably about 0.05% to about 0.3%, particularly 0.1%, based on the total volume of eluent C; and acidic agent 4 is absent, or present in an amount of about 0.01% to about 1%, preferably about 0.05% to about 0.5%, more preferably about 0.05% to about 0.3%, particularly 0.1%, based on the total volume of eluent D.

[0280] In one embodiment, mixture 1 is a reaction mixture obtained from the ring-opening reaction of a thiosuccinimide group, which provides the ring-opening thiosuccinimide structure of part 1.

[0281] In one alternative embodiment, M comprises one or more primary amino groups; acidic agents 1-4 can each form ion pairs with one or more primary amino groups comprised in M. In another alternative embodiment, acidic agents 1-4 are independently selected from AcOH, L-TA, and TFA. In a particularly specific embodiment, acidic agent 1 is AcOH or L-TA, preferably AcOH. In another particularly specific embodiment, acidic agent 3 is TFA.

[0282] In an alternative implementation, part 1 is further R 1 Instead, part 1 has a structure selected from the following formulas (VII) to (X):

[0283]

[0284] Where R 1 Selected from hydrogen and C 1-10 Alkyl groups, and each R 1 The chiral configurations are the same in equations (VII) to (X).

[0285] In one embodiment, the chromatography in step (2) is performed using reversed-phase HPLC. In an alternative embodiment, the chromatography in step (4) is performed using a method selected from atmospheric pressure chromatography, medium pressure chromatography, and high pressure chromatography. In another embodiment, the reversed-phase chromatography in step (4) is performed using reversed-phase HPLC.

[0286] In one particular embodiment, the mixture 1 contains four compounds having the structures of formulas (i) to (iv).

[0287]

[0288] Where x is -OH or -NH2. In one specific embodiment, x is -NH2, and the compounds of formulas (i) to (iv) have the structures of formulas (i-1) to (iv-1).

[0289]

[0290] In one particular embodiment, compounds (i) and (ii) are obtained as isolated products in step (2); and compounds (iii) and (iv) are obtained as isolated products in step (4).

[0291] In another specific embodiment, in step (2), compounds (i) and (ii) are obtained as isolated products, and compounds (iii) and (iv) are obtained in mixture 2.

[0292] In one embodiment, mixture 1 is a reaction mixture obtained from the ring-opening reaction of the thiosuccinimide group in the following compounds.

[0293]

[0294] Where x is -OH or -NH2.

[0295] In one particular embodiment, the reversed-phase chromatographic separation range of step (2) is as follows:

[0296] B is a gradient of approximately 43% to approximately 53%, with the remainder being A, lasting 10-100 minutes, preferably 30-50 minutes.

[0297] In one particular embodiment, the reversed-phase chromatographic separation range of step (2) is as follows:

[0298] B is a gradient of approximately 43% to 48%, with the remainder being A, lasting 10-100 minutes, preferably 48 minutes.

[0299] In one particular embodiment, the column temperature of the reversed-phase chromatography in step (2) is about 10-40°C, preferably about 20-30°C. In another specific embodiment, the sample loading amount for reversed-phase column chromatography is no more than 20 g / kg, no more than 19 g / kg, no more than 18 g / kg, no more than 17 g / kg, no more than 16 g / kg, no more than 15 g / kg, no more than 14 g / kg, no more than 13 g / kg, no more than 12 g / kg, no more than 11 g / kg, no more than 10 g / kg, no more than 9 g / kg, no more than 8 g / kg, no more than 7 g / kg, and no more than 6 g / kg. In a preferred embodiment, the sample loading amount for reversed-phase column chromatography is no more than 14 g / kg. In a very particular embodiment, the loading amount of the reversed-phase column chromatography is about 0.1 g / Kg to 14 g / Kg, preferably 0.1 g / Kg to 14 g / Kg, and the flow rate of the mobile phase is constant and in the range of about 100 ± 50 mL / min to about 500 ± 50 mL / min, particularly about 300 ± 50 mL / min.

[0300] In another specific implementation, the reversed-phase chromatographic separation range of step (4) is as follows:

[0301] D is a gradient of approximately 22% to 42%, with the remainder being C, lasting 20-100 minutes.

[0302] In another specific implementation, the reversed-phase chromatographic separation range of step (4) is as follows:

[0303] D represents a gradient of approximately 22% to 28%, with the remainder being C, lasting 44 minutes.

[0304] In one particular embodiment, the column temperature of the reversed-phase chromatography in step (2) is about 10-40°C, preferably about 20-30°C. In another specific embodiment, the sample loading amount for reversed-phase column chromatography is no more than 20 g / kg, no more than 19 g / kg, no more than 18 g / kg, no more than 17 g / kg, no more than 16 g / kg, no more than 15 g / kg, no more than 14 g / kg, no more than 13 g / kg, no more than 12 g / kg, no more than 11 g / kg, no more than 10 g / kg, no more than 9 g / kg, no more than 8 g / kg, no more than 7 g / kg, and no more than 6 g / kg. In a preferred embodiment, the sample loading amount for reversed-phase column chromatography is no more than 14 g / kg. In a very particular embodiment, the loading amount of the reversed-phase column chromatography is about 0.1 g / Kg to 14 g / Kg, preferably 0.1 g / Kg to 14 g / Kg, and the flow rate of the mobile phase is constant and in the range of about 100 ± 50 mL / min to about 500 ± 50 mL / min, particularly about 300 ± 50 mL / min.

[0305] Analytical methods

[0306] The inventors unexpectedly discovered that the separation method provided in the first aspect of this disclosure can be effectively applied to the analysis of four compounds contained in mixture 1 or mixture 2. For example, the effectiveness of the separation process can be determined with satisfactory accuracy at the analytical scale under substantially the same chromatographic conditions.

[0307] Therefore, in a second aspect, this disclosure provides a method for analyzing one or more compounds in a mixture 1, the mixture 1 comprising four compounds, each of the four compounds comprising part 1, part 2 and part 3.

[0308] The method includes applying step (2) at the analytical scale; wherein part 1, part 2, part 3 and step (2) are as defined above.

[0309] In one embodiment, the method further includes applying step (4) at an analytical scale; wherein step (4) is as defined above.

[0310] In a third aspect, this disclosure provides a method for analyzing one or more compounds in a mixture 2, the mixture 2 comprising two compounds, each of the two compounds comprising part 1, part 2 and part 3.

[0311] The method includes applying step (4) at the analytical scale; wherein parts 1, 2, 3 and step (4) are as defined above.

[0312] In one embodiment, the robustness of the chromatographic conditions for the analytical process is optionally investigated. In one embodiment, the chromatographic conditions for the analytical process are adjusted based on the results of a system suitability determination. Each analytical process can be used independently as a process control method for one or more preparative processes. In one embodiment, the chromatographic conditions for the preparative and analytical processes are identical. In another embodiment, the only difference between the chromatographic conditions used in the preparative and analytical processes is the particle size and / or pore size of the stationary phase. In one embodiment, both the preparative and analytical processes use gradient elution. In another embodiment, the preparative process uses gradient elution, and the analytical process uses isocratic elution.

[0313] In one particular embodiment, the injection volume of the analytical process is less than 1 ml. In another particular embodiment, the injection volume of the analytical process is 1-300 μL, for example 5-100 μL. In a very specific embodiment, the flow rate of the mobile phase does not exceed 1 mL / min, for example, the flow rate can be 0.5-1 mL / min, particularly 0.7 mL / min, 0.8 mL / min, 0.9 mL / min or 1.0 mL / min.

[0314] The disclosed isomers

[0315] Load material - connector intermediate

[0316] In a fourth aspect, this disclosure provides compounds having the structure of any one of the following formulas (XI) to (XIV):

[0317]

[0318] in

[0319] Load, L 1 L 2 M is as defined above.

[0320] In one implementation scheme, L 1 L does not exist. 2 for M is Furthermore, equations (XI) to (XIV) each have the structure of equations (XI-1) to (XIV-1):

[0321]

[0322] in

[0323] The load, Y, n, and x are defined as above.

[0324] Based on the position of the thiol group, formulas (XI) and (XII) are referred to as β, and the configurations of formulas (XIII) and (XIV) are referred to as α.

[0325] In one embodiment, Y is a bond, n is 3, and equations (XI-1) to (XIV-1) have the structures of equations (XI-1-1) to (XIV-1-1), respectively.

[0326]

[0327] In one particular embodiment, the loading is a cytotoxic moiety containing one or more chiral centers, preferably maytansine, more preferably DM1. In one embodiment, the loading is DM1, and formulas (XI-1-1) to (XIV-1-1) represent the structures of isomers (i) to (iv), as defined above.

[0328] In a fifth aspect, this disclosure provides a mixture comprising two compounds, each having the structure of any one of formulas (XI) to (XIV), provided that the two compounds have different structures. In one embodiment, the mixture comprises two compounds having the structures of formulas (XI) and (XII), respectively. In another embodiment, the mixture comprises two compounds having the structures of formulas (XIII) and (XIV), respectively. In one embodiment, the mixture comprises two compounds having the structures of formulas (XI-1) and (XII-1), respectively. In another embodiment, the mixture comprises two compounds having the structures of formulas (XIII-1) and (XIV-1), respectively. In one embodiment, the mixture comprises two compounds having the structures of formulas (XI-1-1) and (XII-1-1), respectively. In another embodiment, the mixture comprises two compounds having the structures of formulas (XIII-1-1) and (XIV-1-1), respectively. In a particular embodiment, the mixture comprises two compounds having the structures of formulas (i) and (ii), respectively. In another embodiment, the mixture comprises two compounds having the structures of formula (iii) and formula (iv), respectively.

[0329] The mixture of compounds (iii) and (iv) is referred to below as the α intermediate or α isomer.

[0330] In some embodiments, the compounds provided in this disclosure may be described as follows:

[0331] <1> Compounds of formula (XI), (XII), (XIII) or (XIV):

[0332]

[0333]

[0334] in

[0335] The loading material is a cytotoxic moiety containing one or more chiral centers;

[0336] L 1 It does not exist, or it is a divalent group, wherein the divalent group is selected from one or more of the following: C 1-10 Alkylene, C 3-10 Cycloalkylene, C 6-10 arylene, 4-10 membered heterocyclic, 5-10 membered heteroarylene, -NH-, -(CO)-, -NH(CO)- and -(CO)NH-;

[0337] L 2 It is a bond, or a divalent group, wherein the divalent group is selected from one or more of the following: C 1-10 Alkylene, C 3-10 Cycloalkylene, C 6-10 arylene, 4-10 membered heterocyclic, 5-10 membered heteroarylene, -NH-, -(CO)-, -NH(CO)- and -(CO)NH-; and

[0338] M contains a ligase recognition sequence;

[0339] The compound is primarily, for example, a pure or substantially pure isomer, for example, substantially free of other isomers, for example, having a purity of more than 90%, for example, more than 95%, for example, more than 98%, for example, at least 99%.

[0340] <2> <1> Compounds of formula (XIII) or (XIV).

[0341] <3> <1> or <2> The compound wherein the ligase recognition sequence is selected from (a) LPXTG, where X is any amino acid residue, such as LPETG; and (b) G n , where G is glycine (Gly) and n is an integer from 3 to 10.

[0342] <4> <3> The compound wherein M is H-Gly-Gly-Gly-Lys-NH2, and M is linked to L via an ε-amino group on a lysine residue. 2

[0343] <5> <1> - <4> Compound of any one of the terms, where L 1 It does not exist, and L 2 Selected from

[0344] <6> <1> - <4> Compound of any one of the terms, where L 1 L does not exist. 2 for M is in

[0345] Y is a bond or a spacer sequence of 1-20 amino acids.

[0346] n is an integer between 3 and 10, for example, 3; and

[0347] x is selected from hydrogen, -OH, -NH2, amino acid fragments containing 1-10 amino acids, and nucleotide fragments containing 1-10 nucleotides; for example, selected from -OH, -NH2, and amino acid fragments containing 1-10 amino acids; for example, selected from -OH, -NH2, and Gly, such as -NH2.

[0348] <7> <1> - <6> The compound of any one of the following, wherein the cytotoxic agent comprises a thiol moiety capable of reacting with the maleimide moiety.

[0349] <8> <1> - <7> The compound of any one of the following, wherein the cytotoxin is maytansin, such as DM1:

[0350]

[0351] <9> <1> - <8> The compound of any one of the formulas is selected from compounds of formula (i), (ii), (iii) or (iv).

[0352]

[0353] Where x is -OH or -NH2.

[0354] <10> <9> Compounds of formula (iii) or (iv).

[0355] In one aspect, this disclosure provides a method for preparing compounds of formula (XI), (XII), (XIII) or (XIV), as follows:

[0356] <17> A method for preparing compounds of formula (XI), (XII), (XIII) or (XIV), comprising:

[0357] (i) A mixture of compounds of the synthetic formulas (XI), (XII), (XIII) and (XIV);

[0358] (ii) The mixture is subjected to chromatography to obtain the target compound.

[0359] Each of the target compounds is obtained as a separated product, or two target compounds are obtained in a second mixture; and

[0360] (iii) Optionally, the eluent collected in step (ii) is recovered, comprising a third mixture, wherein the third mixture contains one or more additional target compounds that are different from the target compounds separated in step (2), and the recovered eluent is subjected to chromatography to separate the additional target compounds.

[0361] <18> <17> The method wherein the chromatography is reversed-phase chromatography.

[0362] Antibody drug conjugates

[0363] Compounds of formula (XI), (XII), (XIII) or (XIV) can conjugate with biomolecules containing ligase recognition sequences to form bioconjugates, wherein the ligase recognition sequence contained in the biomolecule corresponds to the ligase recognition sequence contained in amino acid sequence 1 in structure M of the compound of formula (XI), (XII), (XIII) or (XIV).

[0364] In one embodiment, the compound of formula (XI), (XII), (XIII) or (XIV) contains the ligase acceptor substrate recognition sequence GGG, and the biomolecule contains the ligase donor substrate recognition sequence LPXTG.

[0365] In a sixth aspect, this disclosure provides antibody-drug conjugates (ADCs) prepared using compounds of formula (XI), (XII), (XIII), or (XIV) and antibodies or antigen-binding fragments thereof. The ADC can be prepared using any method known in the art, for example by coupling a ligase recognition sequence contained in structure M to a ligase recognition sequence contained in an antibody or antigen-binding fragment thereof. The resulting ADC also contains an isomerically open-ring thiosuccinimide group as in compounds of formula (XI), (XII), (XIII), or (XIV), and the resulting ADC has a structure of any one of formulas (XVII-1) to (XX-1):

[0366]

[0367] in

[0368] A is an antibody or its antigen-binding fragment, which is optionally modified to have a ligase donor substrate recognition sequence or a ligase acceptor substrate recognition sequence;

[0369] L 3 It does not exist, or contains: (1) amino acid sequence 4, which contains 1-20 amino acids, or (2) optionally present PEG fragment containing "-(C2H4-O"). j-” structure, j is an integer from 1 to 100, and (3) optional group U, which is divalent and selected from cleavable sequence 2, spacer Sp2 containing 2 to 100 amino acids and combinations thereof;

[0370] The cleavable sequence 2 contains an amino acid sequence 5 that can be cleaved by an enzyme, and the cleavable sequence 2 contains 1-10 amino acids;

[0371] The load, Y, n, and x are defined as above.

[0372] In one implementation, j is an integer from 1 to 10.

[0373] In one embodiment, Sp2 is a spacer sequence containing 2-20 amino acids. In a particular embodiment, Sp2 is a spacer sequence selected from GA, GGGS, and GGGGSGGGGS, especially GA.

[0374] In one embodiment, there is no limitation on the location of the introduction of the ligase substrate recognition sequence; for example, it may be introduced at, but is not limited to, the C-terminus or N-terminus of the antibody heavy or light chain.

[0375] The light chain of the antibody or its antigen-binding fragment includes three types: wild-type (LC); C-terminal modified light chain (LCCT) modified by directly introducing the ligase recognition sequence LPTG; and C-terminal modified light chain (LCCT) modified by introducing a short peptide spacer and the ligase donor substrate recognition sequence LPTG. L The heavy chain of an antibody or its antigen-binding fragment includes three types: wild-type (HC); C-terminal modified heavy chain (HCCT) modified by directly introducing the ligase recognition sequence LPTG; and C-terminal modified heavy chain (HCCT) modified by introducing a short peptide spacer and the ligase donor substrate recognition sequence LPTG. L X can be any natural or non-natural single amino acid. When z in compound (IV) is 1 or 2, the above combination of heavy and light chains can form 8 preferred antibody molecules. Sortase-mediated linkage results in the formation of a new amide bond between the C-terminal sorting sequence LPXTG and the N-terminal GGG, wherein the conjugation reaction proceeds by first cleaving the peptide bond between the threonine and glycine residues, and then linking LPXT to GGG.

[0376] In a preferred embodiment, the light chain of the antibody or its antigen-binding fragment comprises three types: wild-type (LC); N-terminal modified light chain (LCNT) modified by directly introducing the ligase recognition sequence GGG; and N-terminal modified light chain (LCNT) modified by introducing a short peptide spacer and the ligase receptor substrate recognition sequence GGG. LThe heavy chain of an antibody or its antigen-binding fragment includes three types: wild-type (HC); N-terminal modified heavy chain (HCNT) modified by directly introducing the ligase recognition sequence GGG; and N-terminal modified heavy chain (HCNT) modified by introducing a short peptide spacer and the ligase receptor substrate recognition sequence GGG. L ).

[0377] In one embodiment, Y is a bond, n is 3, and equations (XVII-1) to (XX-1) have the structures of equations (XVII-1-1) to (XX-1-1), respectively.

[0378]

[0379]

[0380] in

[0381] The load, A, x, and z are as defined above.

[0382] In one embodiment, the load is DM1, z is 2, and formulas (XI-1) to (XIV-1) have the structures of formulas (XI-1-1) to (XIV-1-1), respectively.

[0383] In one particular embodiment, the loading material is a cytotoxic moiety containing one or more chiral centers. The cytotoxic agent is preferably maytansine, more preferably DM1. In one embodiment, the loading material is DM1, L 3 No, formulas (XVII-1-1) to (XX-1-1) have the structures of compounds (v) to (viii), respectively.

[0384]

[0385]

[0386] in

[0387] A and x are as defined above. In one specific implementation, x is -NH2.

[0388] In one embodiment, A is trastuzumab, optionally modified to have one of a ligase donor substrate recognition sequence and a ligase acceptor substrate recognition sequence. In a preferred embodiment, A is a C-terminal modified light chain (LCCT) modified by introducing a short peptide spacer and the ligase donor substrate recognition sequence LPXTGJ. L The trastuzumab of the antibody, wherein J is as defined above. In one embodiment, the short peptide spacer is GA. In another embodiment, the heavy chain of the antibody or its antigen-binding fragment is a wild-type heavy chain (HC).

[0389] In a seventh aspect, this disclosure provides a mixture comprising two compounds, each having the structure of any one of formulas (XVII-1) to (XX-1), provided that the two compounds have different structures. In one embodiment, the mixture comprises two compounds having the structures of formulas (XVII-1) and (XVIII-1), respectively. In another embodiment, the mixture comprises two compounds having the structures of formulas (XIX-1) and (XX-1), respectively. In one embodiment, the mixture comprises two compounds having the structures of formulas (XVII-1-1) and (XVIII-1-1), respectively. In another embodiment, the mixture comprises two compounds having the structures of formulas (XIX-1-1) and (XX-1-1), respectively. In a particular embodiment, the mixture comprises two compounds having the structures of formulas (v) and (vi), respectively. In another embodiment, the mixture comprises two compounds having the structures of formulas (vii) and (viii), respectively. The mixture of compounds (vii) and (viii) is hereinafter referred to as αADC.

[0390] In some implementations, the antibody-drug conjugates provided in this disclosure may be described as follows:

[0391] <11> An antibody-drug conjugate comprising an open-ring thiosuccinimide structure of formula (I), (II), (III), or (IV):

[0392]

[0393] The compound is primarily, for example, a pure or substantially pure isomer, for example, substantially free of other isomers, for example, having a purity of more than 90%, for example, more than 95%, for example, more than 98%, for example, at least 99%.

[0394] <12> <11> Antibody drug conjugates, by making <1> - <11> Compounds from any one of the above react with antibodies in the presence of ligase to form

[0395] <13> <11> or <12> The antibody-drug conjugates have formulas (XVII-1-1) to (XX-1-1), respectively.

[0396]

[0397]

[0398] in

[0399] The loading and x are as defined above, for example, a cytotoxic moiety containing one or more chiral centers, such as the remainder of a maytansine molecule other than the thiol moiety, or the remainder of DM1 other than the thiol moiety;

[0400] A is an antibody or its antigen-binding fragment, which is optionally modified to have a ligase donor substrate recognition sequence or a ligase acceptor substrate recognition sequence;

[0401] For example, A is an antibody that binds to human epidermal growth factor receptor 2 (HER2), such as trastuzumab, which is modified to have a ligase donor substrate recognition sequence or a ligase receptor substrate recognition sequence.

[0402] z is an integer of 1 or 2.

[0403] <14> <13> The antibody-drug conjugate, wherein A is trastuzumab, has a C-terminal modified light chain (LCCT) modified by introducing a short peptide spacer, such as -GA-, and a ligase donor substrate recognition sequence LPXTGJ. L ), J in LPXTGJ is absent, or is an amino acid fragment containing 1-10 amino acids.

[0404] <15> <13> or <14> Antibody-drug conjugates, wherein the loading agent is DM1; L 3 It does not exist; and equations (XVII-1-1) to (XX-1-1) respectively have structures (v)-(viii).

[0405]

[0406]

[0407] Where x is -OH or -NH2.

[0408] <16> <14> Antibody drug conjugates, with <15> The structure shown is (vii).

[0409] Compound mixtures and antibody-drug conjugate mixtures

[0410] In some embodiments, the α-isomers of this disclosure are provided in the form of mixtures; therefore, in an eighth aspect, this disclosure provides a composition comprising the α-isomers of this disclosure. In some embodiments, the compositions of this disclosure may be described as follows:

[0411] <45> A composition comprising a mixture of a compound of formula (XIII) and a compound of formula (XIV), such as a racemic mixture:

[0412]

[0413] in

[0414] The loading material is a cytotoxic moiety containing one or more chiral centers;

[0415] L 1 It does not exist, or it is a divalent group, wherein the divalent group is selected from one or more of the following: C 1-10 Alkylene, C 3-10 Cycloalkylene, C 6-10 arylene, 4-10 membered heterocyclic, 5-10 membered heteroarylene, -NH-, -(CO)-, -NH(CO)- and -(CO)NH-;

[0416] L 2 It is a bond, or a divalent group, wherein the divalent group is selected from one or more of the following: C 1-10 Alkylene, C 3-10 Cycloalkylene, C 6-10 arylene, 4-10 membered heterocyclic, 5-10 membered heteroarylene, -NH-, -(CO)-, -NH(CO)- and -(CO)NH-; and

[0417] M contains a ligase recognition sequence;

[0418] The composition is substantially free of (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) compounds of formula XI or formula XII.

[0419]

[0420] <46> <45> The composition wherein the ligase recognition sequence is selected from (a) LPXTG, where X is any amino acid residue, such as LPETG; and (b) G n , where G is glycine (Gly) and n is an integer from 3 to 10.

[0421] <47> <45> or <46> The composition wherein M is H-Gly-Gly-Gly-Lys-NH2, and M is linked to L via an ε-amino group on a lysine residue. 2 .

[0422] <48> <45> - <47> The composition of any one of the following, wherein L 1 It does not exist, and L 2 Selected from

[0423] <49> <45> - <48> The composition of any one of the following, wherein L 1 L does not exist.2 for M is in

[0424] Y is a bond or a spacer sequence of 1-20 amino acids.

[0425] n is an integer between 3 and 10, for example, 3; and

[0426] x is selected from hydrogen, -OH, -NH2, amino acid fragments containing 1-10 amino acids, and nucleotide fragments containing 1-10 nucleotides; for example, selected from -OH, -NH2, and amino acid fragments containing 1-10 amino acids; for example, selected from -OH, -NH2, and Gly, such as -NH2.

[0427] <50> <45> - <49> The composition of any one of the following, wherein the cytotoxic agent comprises a thiol moiety capable of reacting with the maleimide moiety.

[0428] <51> <45> - <50> The composition of any one of the following, wherein the cytotoxin is maytansin, such as DM1:

[0429]

[0430] <52> <45> - <47> The composition of any one of the following is a mixture of compounds of formula (iii) and formula (iv) and substantially free of compounds of formula (i) or (ii).

[0431]

[0432] In a ninth aspect, this disclosure provides an antibody-drug conjugate composition comprising a mixture of the α-AADCs of this disclosure. In some embodiments, the antibody-drug conjugate compositions of this disclosure may be described as follows:

[0433] <53> An antibody drug conjugate composition comprising, for example, a racemic mixture, an antibody drug conjugate comprising an open-ring thiosuccinimide structure of formula (III), and an antibody drug conjugate comprising an open-ring thiosuccinimide structure of formula (IV):

[0434]

[0435] The composition thereof is substantially free of (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) compounds of formula (I) or formula (II):

[0436]

[0437] <54> <53> Antibody drug conjugate composition, by making <45> - <52> The composition of any one of the above is formed by reacting an antibody with an antibody in the presence of a ligase.

[0438] <55> <52> , <53> or <54> The antibody-drug conjugate composition is a mixture of a compound having the formula (XIX-1-1) and a compound having the formula (XX-1-1):

[0439]

[0440] in

[0441] The loading material is as defined above, for example, a cytotoxic moiety containing one or more chiral centers, such as the remainder of a maytansine molecule other than the thiol moiety, or the remainder of DM1 other than the thiol moiety;

[0442] A is an antibody or its antigen-binding fragment, which is optionally modified to have a ligase donor substrate recognition sequence or a ligase receptor substrate recognition sequence; for example, A is an antibody that binds to human epidermal growth factor receptor 2 (HER2), such as trastuzumab, which is modified to have a ligase donor substrate recognition sequence or a ligase receptor substrate recognition sequence.

[0443] z is an integer of 1 or 2.

[0444] <56> <55> The antibody-drug conjugate composition, wherein A is trastuzumab having a C-terminal modified light chain (LCCT) modified by introducing a short peptide spacer, such as -GA-, and a ligase donor substrate recognition sequence LPXTGJ. L ), J in LPXTGJ is absent, or is an amino acid fragment containing 1-10 amino acids.

[0445] <57> <55> or <56> An antibody-drug conjugate composition wherein the loading is DM1; L 3 It does not exist; and equations (XIX-1-1) to (XX-1-1) have structures (vii) and (viii) respectively.

[0446]

[0447] In the tenth aspect, the treatment method provided in this disclosure can be described as follows:

[0448] <58> A method for treating cancer in a patient in need, comprising administering an effective dose of medication to the patient. <53> - <57> An antibody-drug conjugate composition of any one of the following.

[0449] <59> <58> The method, wherein the cancer is a HER2-positive cancer, such as HER2-positive breast cancer.

[0450] <60> <58> or <59> The method wherein the antibody-drug conjugate composition is as follows: <57> As stated above.

[0451] Pharmaceutical compositions, formulations and kits

[0452] In another aspect, this disclosure also provides a pharmaceutical composition comprising (a) a compound of formula (XI), (XII), (XIII) or (XIV); or an antibody-pharmaceutical conjugate of the present invention; or a composition comprising a mixture of compounds of formula (XIII) and (XIV); or an antibody-pharmaceutical conjugate composition of the present disclosure; and (b) a pharmaceutically acceptable carrier.

[0453] Treatment and Uses

[0454] In another aspect, this disclosure also provides compounds of formula (XI), (XII), (XIII) or (XIV); or antibody-pharmaceutical conjugates of the present invention; or compositions comprising mixtures of compounds of formula (XIII) and (XIV); or antibody-pharmaceutical conjugate 1 compositions of the present disclosure; or the use of pharmaceutical compositions of the present disclosure in the preparation of medicaments for treating cancer.

[0455] In another aspect, this disclosure also provides a method of treating cancer, the method comprising administering to an individual in need an effective amount of a compound of formula (XI), (XII), (XIII) or (XIV); or an antibody-pharmaceutical conjugate of the present invention; or a composition comprising a mixture of compounds of formula (XIII) and (XIV); or an antibody-pharmaceutical conjugate composition of the present disclosure; or a pharmaceutical composition of the present disclosure.

[0456] In another aspect, the present invention provides compounds of formula (XI), (XII), (XIII) or (XIV); or antibody-pharmaceutical conjugates of the present invention; or compositions comprising mixtures of compounds of formula (XIII) and (XIV); or antibody-pharmaceutical conjugate compositions of the present invention; or pharmaceutical compositions disclosed herein for the treatment of cancer.

[0457] In one implementation scheme, the cancer is HER-2 positive.

[0458] Beneficial effects

[0459] The invention disclosed herein achieves at least one of the following technical effects:

[0460] (1) It can achieve complete separation and purification of the four isomers contained in mixture 1, and obtain pure isomers in high efficiency and high yield;

[0461] It is noteworthy that in the separation process (e.g., the process in Example 1) and analytical process (e.g., the process in Example 2) of the β1, β2, and α isomers, when the acidic agent used in the mobile phase is TFA, NaH2PO4, CH2O2, K2HPO4, AA, TEAP, and NaClO4 or other conventional reagents, almost no separation between the β1, β2, and α isomers was observed. Conversely, when CH3COOH and L-TA were selected as the acidic agents, the β1, β2, and α isomers could be successfully separated and purified. Therefore, the inventors unexpectedly discovered that specific acidic agents, namely CH3COOH or similar alkylcarboxylic acids, can effectively improve the separation degree between the β1, β2, and α isomers, and thus can be applied to separation and / or purification methods.

[0462] (2) The method is simple to operate, low in cost, and has been proven to be effective in pilot-scale production, thus it is conducive to industrial production.

[0463] (3) The separation efficiency of complex samples is usually monitored by complex and expensive post-column detection, such as LCMS methods which use expensive chromatographic columns for accurate analysis and in some cases require sample pretreatment to remove residual non-volatile salts in the product obtained by separation.

[0464] The analytical method disclosed herein employs chromatographic conditions similar to those used in the separation method, thus requiring fewer adjustments when designing the chromatographic method for either the separation or analysis process, facilitating the chromatographic processing of complex samples. Furthermore, volatile reagents are used in the separation method of this disclosure. Therefore, the separated product does not contain non-volatile salts, requires no desalting pretreatment, and can be directly analyzed by LCMS. This is particularly advantageous when mixture 1 is a reaction mixture from a ring-opening reaction. Impurities or byproducts can be detected promptly if needed.

[0465] (4) The method exhibits high efficiency, such as high resolution (parameter R in chromatography) and satisfactory accuracy.

[0466] (5) The isolated isomers exhibited differences in cytotoxicity. In the human breast cancer HCC1954 cell proliferation assay, the activity of isomer α2 was twice that of the β isomer (a mixture of β1 and β2 isomers).

[0467] The α-ADC disclosed herein achieves at least one of the following technical effects:

[0468] α-ADCs exhibit significant antitumor effects and can accumulate in tumors while maintaining in situ stability. Compared to Kadcyla, lower doses of α-ADCs produced similar potency, demonstrating the superior potency of the α-ADCs disclosed herein.

[0469] Example

[0470] To more clearly illustrate the purpose and technical solutions of this disclosure, the following detailed description is provided in conjunction with specific embodiments. It should be understood that these examples are not intended to limit the scope of the invention. Specific experimental methods not mentioned in the following embodiments are all performed using conventional experimental methods.

[0471] abbreviation

[0472]

[0473]

[0474] Instruments, materials and reagents

[0475] Unless otherwise stated, the instruments and reagents are commercially available or can be prepared according to conventional methods in the art. Reagents can be used directly without further purification.

[0476] 1 H NMR, 13 C NMR, 1 H- 1 H COSY and HMBC spectra were recorded on a Bruker AV-600 at 600 MHz. Chemical shifts are expressed in parts per million (ppm). Coupling constants are in Hertz (Hz). Splitting modes describe apparent multiplicity and are designated as s (single), d (double), t (triple), q (quadruple), m (multiple), and br (broad).

[0477] CellTiter read using a Biotek Cytation3 imaging reader The Luminescent Kit (Promega, Cat. #G7573) was used to test cell viability.

[0478] The HCC1954 human breast cancer xenograft model (HCC1954 model) and the NCI-N87 human gastric cancer xenograft model (NCI-N87 model) in female BALB / c nude mice were provided by ATCC. The cynomolgus monkeys were provided by GuangZhou XiangGuan, Ltd.

[0479] PLT, APTT, AST, ALT, GLOB, and A / G were tested on a blood analyzer (ADVIA2120i Siemens) and with auxiliary reagents.

[0480] Kadcyla is provided by Roche.

[0481] Preparation of mixtures containing β-isomers and α-isomers

[0482] Compounds having the following structure were prepared and the ring-opening reaction of the thiosuccinimide group was carried out using a method similar to that described in WO2015165413A1.

[0483]

[0484] The ring-opening reaction yielded four isomers (isomers (i-1), (ii-11), (iii-1), and (iv-1)). Based on the location of the thiol group at the α or β position of the unbroken amide bond, they were named the α isomer and β isomer, respectively. The structures of the four isomers are as follows:

[0485]

[0486] The reaction mixture obtained from the reaction is directly subjected to the separation process described below, wherein the reaction mixture contains the four isomers.

[0487] Example 1: Separation of β1, β2 and α isomers

[0488] 1.1 Separation Process

[0489] Gradient elution was used to separate the β1, β2, and α isomers. The elution gradient included a pH gradient and an organic solvent gradient. Chromatographic conditions are shown in Table 1.

[0490] Table 1

[0491]

[0492] The figure shows three main peaks, namely peak 1 (approximately t). R 27min to approximately t R The first elution peak within 39 min), peak 2 (approximately t) R 27min to approximately t R The second elution peak within the 39-minute range) and peak 3 (t R 42min to t R Peaks within a 51-minute range). The purity of each eluent fraction was determined by HPLC using the analytical method described in Example 2. Fractions were combined based on the analytical results, then lyophilized and subjected to NMR analysis. Based on 1D and 2D NMR data, peaks 1 and 2 each contained two β-isomers. The structures of the β-isomers are as follows:

[0493]

[0494] Where * denotes the asymmetric center (22-C).

[0495] The β isomer contained in peak 1 is denoted as β1. 1 H NMR,13 C NMR, 1 H- 1 The H COSY and HMBC data are shown in Table 2.

[0496] Table 2. 600MHz NMR assignment of isomer β1 in DMSO–d6

[0497]

[0498]

[0499] The β isomer contained in peak 2 is denoted as β2.

[0500] 1 H NMR, 13 C NMR, 1 H- 1 The H COSY and HMBC data are shown in Table 3.

[0501] Table 3. 600MHz NMR assignment of isomer β2 in DMSO–d6

[0502]

[0503]

[0504]

[0505] Based on 1D and 2D NMR data, peak 3 contains an α isomer. The content of peak 3 is inferred from the mechanism of the ring-opening reaction, and this peak is represented as a mixture of two α isomers, namely α1 and α2.

[0506] Separation under other chromatographic conditions was tested, and the results are listed in points 1.2–1.6 below.

[0507] 1.2 Stationary Phase

[0508] Packed columns from different manufacturers were tested. Chromatographic conditions are shown in Table 4.

[0509] Table 4. Tests using different columns

[0510]

[0511]

[0512] HPLC chromatogram as shown Figures 1-4 As shown:

[0513] The purity of each eluent fraction was determined by HPLC using the analytical method described in Example 2. Three main peaks are shown in the figure. Fractions were combined based on the analytical results, then lyophilized and subjected to NMR analysis. Based on the analytical results and 1D and 2D NMR data, the elution order of the isomers was the same as in Example 1 under the above chromatographic conditions. Figure 2-4 In the middle, the two β isomers eluted in the first two peaks, and the mixture of the two α isomers eluted in the third peak.

[0514] The calculation factors are listed in Table 5:

[0515] Table 5

[0516]

[0517] R greater than 1 was observed on all types of RP-C18 stationary phases. The method disclosed herein is applicable to various commercially available packed columns and is therefore robust.

[0518] 1.3 Acidic agent 1 as an acid

[0519] Different acids were tested as acidic agent 1 in eluent A. Chromatographic conditions are shown in Table 6.

[0520] Table 6

[0521]

[0522] The separated HPLC chromatogram is as follows Figure 5-13 As shown.

[0523] according to Figure 5 , 7 Moderate separation was observed in α, β, and β, when using optionally substituted alkyl carboxylic acids, with AcOH and L-TA exhibiting satisfactory separation. Inorganic acids (e.g., phosphoric acid) also showed some separation. Formic acid exhibited poor separation. The separating ability of the mobile phase may be affected by the anions of organic acids.

[0524] The purity of each eluent fraction was determined by HPLC using the analytical method described in Example 2. The fractions were combined based on the analytical results, then lyophilized and subjected to NMR analysis. Based on the analytical results and 1D and 2D NMR data, the elution order of the isomers was the same as in Example 1 under the above chromatographic conditions. Figure 11-13 In the middle, the two β isomers eluted in the first two peaks (partially fused with each other), and the mixture of the two α isomers eluted in the third peak.

[0525] 1.4 Buffer salts as acidifying agents

[0526] The use of phosphate buffer as acidic agent 1 in eluent A was tested.

[0527] The test results for eluent A are shown in Table 7. Except for eluent A, the other chromatographic conditions are the same as those in Table 6.

[0528] Table 7

[0529]

[0530] The separated HPLC chromatogram is as follows Figure 14-19 As shown.

[0531] The results from sections 1.3 and 1.4 show that pH is not the only factor affecting solute chromatographic behavior. The separation of isomers is significantly influenced by the acidic agent used. The applicant unexpectedly found that, among the acidic agents tested, AcOH and L-TA significantly improved resolution compared to other acidic agents.

[0532] 1.5 Organic solvents

[0533] By comparing with Example 3, the separation degree between the two α isomers was analyzed. A higher separation degree was obtained when MeOH was used as the organic solvent, indicating that the type of organic solvent may be a key factor.

[0534] 1.6 gradient

[0535] The use of different gradients in steps 1.1, 1.3, and 1.4 above, and the use of isocratic elution in step 1.2 above, demonstrate that the method of this disclosure can tolerate a range of elution compositions. This provides flexibility to the method and may be particularly beneficial in pilot-scale manufacturing, and helps alleviate pressure on robustness control of the elution composition.

[0536] A 0.3% CH3COOH aqueous solution was used as eluent A, and further tests were performed using the elution gradients in Tables 8-1 and 8-2. Except for eluent A and the elution gradients, the chromatographic conditions were the same as those in Table 6.

[0537] Table 8-1

[0538]

[0539] Table 8-2

[0540]

[0541] The separated HPLC chromatogram is as follows Figure 20-21 As shown

[0542] The purity of each eluent fraction was determined by HPLC using the analytical method described in Example 2. The fractions were combined based on the analytical results, then lyophilized and subjected to NMR analysis. Based on the analytical results and 1D and 2D NMR data, the elution order of the isomers was the same as in Example 1 under the above chromatographic conditions. Figure 20-21 In the middle, the two β isomers eluted in the first two peaks (where they are partially fused together), and the mixture of the two α isomers eluted in the third peak.

[0543] The calculation factors are listed in Table 9:

[0544] Table 9

[0545]

[0546] A B gradient of 46%–56% within 80 minutes achieves better separation than a B gradient of 46%–66% within 30 minutes. Considering the improved separation, the tailing factor of gradient 2 (increased) and the theoretical plate number (decreased) remain acceptable.

[0547] Further extending the elution time to over 80 minutes showed unsatisfactory results in system suitability testing.

[0548] Example 2: Process control for the separation of β1, β2 and α isomers

[0549] In Example 2, the method described in Example 1 was applied on an analytical scale, using a stationary phase with a smaller particle size (5 μm) than that used in Example 1 (10 μm). Chromatographic conditions are shown in Table 10.

[0550] Table 10

[0551]

[0552] The separated HPLC chromatogram is as follows Figure 22 As shown.

[0553] The calculated resolution was 1.28, and the theoretical plate number was 20020.6.

[0554] Example 3: Separation of α1 and α2 isomers

[0555] 3.1 Separation Method

[0556] The α isomer obtained in Example 1 was further separated to obtain isomers α1 and α2. The structures of the α isomers are as follows:

[0557]

[0558] Where * denotes the asymmetric center (22-C).

[0559] Gradient elution was used to separate the α1 and α2 isomers from the α isomers obtained in Example 1. The chromatographic conditions are shown in Table 11.

[0560] Table 11

[0561]

[0562] The separated HPLC chromatogram is as follows Figure 23 As shown in the figure. The figure displays two main peaks, namely peak 1'(t). R 40.075 min) and peak 2' (t R (42.497 min). The purity of each eluent fraction was determined by HPLC using the analytical method described in Example 4. The α isomer contained in peak 1 is designated as α1. 1 H NMR, 13 C NMR, 1 H- 1 The H COSY and HMBC data are shown in Table 12.

[0563] Table 12. 600MHz NMR assignment of isomer α1 in DMSO–d6

[0564]

[0565]

[0566] The α isomer contained in peak 2 is denoted as α2. 1 H NMR, 13 C NMR, 1 H- 1 The H COSY and HMBC data are shown in Table 13.

[0567] Table 13. 600MHz NMR assignment of isomer α2 in DMSO–d6

[0568]

[0569]

[0570]

[0571] Separation under other chromatographic conditions was tested, and the results are listed in points 3.2-3.6 below.

[0572] 3.2 Acids as acidic agents

[0573] Different acids were tested as acidic agents 3 in eluent C. Chromatographic conditions are shown in Table 14.

[0574] Table 14

[0575]

[0576] The separated HPLC chromatogram is as follows Figure 24-31 As shown.

[0577] 3.3 Buffer salts as acidifying agents

[0578] The use of phosphate buffer as acidic agent 1 in eluent A was tested.

[0579] The test results for eluent C are shown in Table 15. Except for eluent C, the other chromatographic conditions are the same as those in Table 14.

[0580] Table 15

[0581]

[0582] The separated HPLC chromatogram is as follows Figure 32-44 As shown.

[0583] 3.4 Summary of Acid Agent 3

[0584] The calculation factors are listed in Table 16:

[0585] Table 16

[0586] 0.1% TFA 1.1277 <![CDATA[10mM NaH2PO4 PH=2.0]]> 1.1493 0.1% AcOH 0.9400 <![CDATA[10mM NaH2PO4 PH=4.0]]> 1.0739 0.3% AcOH ND* <![CDATA[10mM NaH2PO4 PH=6.0]]> 1.0044 <![CDATA[0.1%CH2O2]]> 0.6984 <![CDATA[10mM K2HPO4 PH=2.0]]> 1.1803 <![CDATA[0.3%CH2O2]]> 0.6768 <![CDATA[10mM K2HPO4 PH=4.0]]> 1.1872 0.5% AA 1.0227 <![CDATA[10mM K2HPO4 PH=6.0]]> 1.0072 <![CDATA[0.1%H3PO4]]> 0.8875 <![CDATA[10mM K2HPO4 PH=8.0]]> 0.9989 <![CDATA[0.2%H3PO4]]> 0.9731 - - <![CDATA[30mM NaH2PO4 PH=2.0]]> 1.1929 10mM TEAP pH=6.0 0.9914 <![CDATA[30mM NaH2PO4 PH=6.0]]> 0.9612 10mM TEAP pH=2.0 1.1617 <![CDATA[30mM NaH2PO4 PH=4.0]]> 0.9958 10mM TEAP pH=4.0 1.1576

[0587] *: Not measured

[0588] When 0.1% TFA, 10mM K2HPO4 (pH=2.0), 10mM K2HPO4 (pH=4.0), 10mM TEAP (pH=2.0) or 10mM TEAP (pH=4.0) are used as acidic agent 3, a resolution greater than 1 is achieved.

[0589] Compared with the method in Example 1, in Figure 23-44 As shown in Table 16, the acidic agents exhibited a less significant effect, confirming the differences in chromatographic characteristics between the β and α isomers.

[0590] Example 4: Process control for separating α1 and α2 isomers

[0591] In Example 4, the method described in Example 3 was applied on an analytical scale, using a stationary phase with a smaller particle size (5 μm) than that used in Example 3 (10 μm). Chromatographic conditions are shown in Table 17.

[0592] Table 17

[0593]

[0594] The HPLC chromatogram of the analysis is as follows Figure 45 As shown.

[0595] The analytical methods disclosed herein can be performed using any of the parameters listed in Tables 17-19. The flexibility of these methods facilitates their application under different conditions and can meet the detection requirements at different stages of the target product preparation process, such as the preparation of bioconjugates.

[0596] Example 5: Cytotoxicity of the β-isomer, α1-isomer, and α2-isomer

[0597] The eluted peaks from Example 1 were collected, wherein: (1) the first and second peaks were collected as samples (β isomers), and (2) the third peak was collected as samples (α isomers), and the peaks were separated using the method described in Example 3 to obtain α1 isomers and α2 isomers.

[0598] The cytotoxicity of the β-isomer, α1-isomer, and α2-isomer was tested by tumor cell proliferation assay.

[0599] Detection method:

[0600] Human breast cancer HCC1954 cells with good growth conditions and approximately 80% cell confluence were digested with 0.25% trypsin, collected, and cultured overnight in 96-well plates. After cell attachment, test substances were diluted 3-fold from 10 nM, for a total of 10 gradients. After culturing at 37°C for 72 h, the results were analyzed using CellTiter... Cell viability was detected using a chemiluminescence assay kit. The cell viability formula is: Viability = (RLU(X) - RLU(Puro)) / (RLU(Control) - RLU(Puro)) * 100%. A logistic model (4 parameters) was used for data processing, IC50 calculation, and curve fitting using Prism 6 software. Results are as follows... Figure 46 As shown in Table 20.

[0601] Table 20. Cytotoxicity of isoforms in HER2-positive HCC1954 cells

[0602] β isomer 0.07399 100 0.9991 α1 isomer 0.09224 80.2 0.9976 α2 isomer 0.03601 205 0.9974

[0603] *: The biological activity of the β isoform in HCC1954 cells was set to 100%.

[0604] As shown in Table 20, the activity of the α1 isomer is within ±30% of that of the β isomer, therefore their activities are considered comparable. The relative activity of the α2 isomer is 205% (the biological activity of the β isomer is set at 100%), meaning that the activity of the α2 isomer is approximately twice that of the β isomer.

[0605] Example 6: Preparation of α-ADC

[0606] The pure product containing the α isomer (a mixture of α1 and α2) obtained in Example 1 was conjugated with a therapeutic antibody. The conjugation method was similar to that described in WO2015165413A1. The resulting product was a mixture of ADCs, denoted as "α-ADC". The α isomer was then conjugated with T-LCCT as described in WO2015165413A1 (T represents trastuzumab). L The α-ADC prepared by α-HC coupling is designated as "α-ADC composition 1".

[0607] Example 7: HER2-selective cytotoxicity targeting α-ADCs with HER2

[0608] Objective: To evaluate the selective cytotoxicity of α-ADCs, HER2-positive cells HCC1954 and HER2-negative cells MDA-MB-468 were treated with α-ADC composition 1, Kadcyla and DM1, respectively.

[0609] Research Design:

[0610] Table 21. Cytotoxicity design of HER2-positive HCC1954 cells

[0611]

[0612] Table 22. Cytotoxicity design of MDA-MB-468 in HER2-negative cells

[0613]

[0614] The study design is shown in Tables 21 and 22. Cells were incubated with α-ADC composition 1, Kadcyla, or DM1 for 72 h, and then analyzed by CellTiter- Cell viability was detected using a chemiluminescence assay kit. The cell viability formula is: Viability = (RLU(X) - RLU(Puro)) / (RLU(Control) - RLU(Puro)) * 100%. A logistic model (4 parameters) was used for data processing, IC50 calculation, and curve fitting using Prism 6 software.

[0615] Results: The results are as follows Figure 47 As shown in Table 23.

[0616] Table 23. Effects of α-ADC on cancer cell proliferation

[0617]

[0618] α-ADC significantly inhibited the proliferation of HER2-positive cells, and its effect was several times greater than that of DM1. T-DM1 (Kadcyla) was slightly more potent than α-ADC, because the toxin content of T-DM1 was about twice that of α-ADC (T-DM1 DAR≈3.5, α-ADC component 1 DAR 1.79).

[0619] α-ADC showed no inhibitory effect on HER2-negative cells at concentrations up to 100 nM, but exhibited significant inhibitory effects in HER2-positive cells. T-DM1 showed off-target cytotoxicity in HER2-negative cells at high concentrations, while α-ADC did not. This off-target cytotoxicity may be due to the decrease in DM1 via the reverse Michael reaction of MCC-DM1 in T-DM1. The MCC' linker of α-ADC (containing an open-ring MCC structure) is designed to reduce the reverse Michael reaction, which avoids or significantly reduces the shedding of DM1 before internalization. This suggests that α-ADC is safer than T-DM1 in normal cells.

[0620] Example 8: Biodistribution characteristics of α-ADC

[0621] Objective: To administer a single intravenous dose 89 The Zr-α-ADC composition was used to study different time points after application, using positron emission tomography / computed tomography (PET / CT) scans. 89 Biodistribution characteristics of Zr-α-ADC in tumor-bearing mice.

[0622] Research Design: [ 89 Zr isotope labeling was used to study the distribution of α-ADC composition 1 in BT-474 human breast cancer xenografts (referred to as the BT-474 xenograft model) in athymic mice after a single intravenous injection. Qualified ADC composition 1 was administered to the experimental animals. 89 Zr-α-ADC composition 1. Administration to 8 BT-474 xenograft tumor models. 89 Zr-α-ADC composition 1. PET / CT scans were performed at 1 h, 24 h, 48 h, 96 h, 168 h and 336 h after administration, with static scans lasting 10-30 min.

[0623] Results: The results are as follows Figure 48 As shown.

[0624] Single-dose intravenous injection in the BT-474 xenograft model 89 After Zr-α-ADC composition 1, the total radioactivity was mainly distributed within the tumor, followed by vascular organs (liver, heart, kidney, spleen, lung) and the knee, with less radioactivity in other tissues and organs (bones, brain, muscles). Based on radioactivity calculations, 89The AUC (0-336h) of Zr-α-ADC composition 1 in different tissues was in the following order: tumor > liver > knee > heart > kidney > spleen > lung > bone > muscle > brain. These results indicate that... 89 Zr-α-ADC composition 1 has difficulty crossing the blood-brain barrier, and 89 Zr-α-ADC composition 1 showed significant tumor targeting.

[0625] Single intravenous injection in the BT-474 xenograft model 89 After administration of Zr-α-ADC composition 1, the ratios (tumor to muscle and tumor to heart) gradually increased, reaching their highest values ​​of 34.78 and 15.54, respectively, at 336 hours post-administration.

[0626] Example 9: In vivo antitumor efficacy of α-ADC

[0627] Objective: The aim of this study was to evaluate the in vivo antitumor efficacy of α-ADC in female BALB / c nude mice following a single IV dose in the subcutaneous HCC1954 human breast cancer xenograft model (HCC1954 model) and NCI-N87 human gastric cancer xenograft model (NCI-N87 model).

[0628] Study design: The study design is shown in Tables 24 and 25.

[0629] Table 24. Research Designs in the HCC1954 Model

[0630]

[0631] Table 25. Research Design in the NCI-N87 Model

[0632]

[0633] The treatment effect was calculated as follows: T / C (%) = (mean RTV of the treatment group) / (mean RTV of the control group) × 100%; TGI = [1 - (mean tumor volume at the end of treatment - mean tumor volume at the beginning of treatment) / (mean tumor volume at the end of treatment in the control group - mean tumor volume at the beginning of treatment in the control group)] × 100%.

[0634] Results: In the HCC1954 human breast cancer xenograft model, the therapeutic efficacy of α-ADC composition 1 (DAR 1.79) alone was evaluated and compared with the positive control Kadcyla (DAR ~ 3.5). Tumor size at different time points after treatment initiation in different groups is shown in the table below. Figure 49Compared with the mediator group, treatment with test α-ADC composition 1 (T / C 18.83%, 9.12%, and 2.12%, respectively; TGI 86.61%, 96.97%, and 104.43%, respectively; p 0.002, 0.001, and 0.001, respectively) at 1.25 mg / kg, 2.5 mg / kg, and 5 mg / kg (T / C 6.47% and 1.57%, respectively; TGI 99.79% and 105.02%, respectively; p 0.001 and 0.001, respectively) and Kadcyla at 2.5 mg / kg and 5 mg / kg (T / C 6.47% and 1.57%, respectively; TGI 99.79% and 105.02%, respectively; p 0.001 and 0.001, respectively) produced significant antitumor activity; at the same time point, their mean tumor sizes were 418, 202, 47, 144, and 35 mm, respectively. 3 At the same dose levels, α-ADC compositions 1 and Kadcyla produced similar antitumor activity in the HCC1954 xenograft model, with p values ​​of 0.791 (5 mg / kg) and 0.701 (2.5 mg / kg), respectively.

[0635] In the NCI-N87 human gastric cancer xenograft model, the therapeutic efficacy of α-ADC composition 1 administered alone was evaluated. Tumor size results at different time points after tumor inoculation in different groups are shown in [Figure number missing]. Figure 49 Compared with the mediator group, treatment with the test α-ADC composition 1 at 5 mg / kg and 15 mg / kg (T / C 34.13% and 5.07%, respectively; TGI 76.62% and 110.38%, respectively; p 0.010 and 0.001, respectively) and Kadcyla at 5 mg / kg and 15 mg / kg (T / C 41.61% and 4.66%, respectively; TGI 67.93% and 110.87%, respectively; p 0.022 and 0.001, respectively) produced significant antitumor activity; at the same time point, their mean tumor sizes were 475, 71, 579, and 65 mm, respectively. 3 On day 35 post-treatment, the tumor volume of the patient treated with 1.67 mg / kg of α-ADC composition 1 was 1025 mm. 3 (T / C = 73.77%, TGI = 30.67%, p = 0.509). At the same dose level, α-ADC composition 1 and Kadcyla produced similar antitumor activity, with p values ​​of 0.791 (5 mg / kg) and 1.000 (15 mg / kg), respectively.

[0636] Example 10: Pharmacokinetic Characteristics of α-ADCs

[0637] Objective: The purpose of this study was to evaluate the pharmacokinetic characteristics of α-ADC after a single dose in cynomolgus monkeys.

[0638] Study Design: A total of 24 cynomolgus monkeys (3 animals / sex / group, 4 groups in total) were administered a single intravenous infusion of 0 mg / kg (20 minutes / dose / monkey) of the medium (α-ADC composition 1 preparation buffer), or 10, 30, and 45 mg / kg of α-ADC composition 1. Animals were necropsized on day 43 after a 6-week observation period. Details of animal numbers and dosage levels are given in Table 26.

[0639] Table 26. Groups and Dosage Levels

[0640]

[0641] Blood collection time points include 0h, 5min, 1h, 4h, 8h, 24h, 48h, 72h, 168h, 336h, 504h, 672h, 840h and 1008h.

[0642] Results: The aggregated pharmacokinetic data showed that... Figure 50 middle.

[0643] Following IV infusion, serum concentrations of α-ADC composition 1 and α-ADC composition 1TAb reached peak levels substantially rapidly and then declined in a roughly biphasic manner, with the rate of decline for α-ADC composition 1 being faster than that for TAb in the α-ADC composition 1 group. Exposure levels of α-ADC composition 1 and TAb (mean C) max AUC 0-t and AUC 0-∞ The dose was increased in an approximate dose-proportional manner. With increasing dose of α-ADC composition 1, little or no prolongation of the mean MRT was observed. For α-ADC composition 1 and α-ADC composition 1TAb, and α-ADC composition 1mAb, at C... max AUC 0-t or AUC 0-∞ No significant gender differences were found. The T1 of α-ADC composition 1 1 / 2 The TAb T of α-ADC composition 1 is 3.4–4.1 days. 1 / 2 It takes 5.2-8.6 days.

[0644] At 5 min post-dosing, DM1 was slightly higher than LLOQ in group 1 (≥30 mg / kg α-ADC composition) and at 1 h post-dosing, only in the 5 mg / kg α-ADC group. The mean C0 of DM1 was... max and AUC 0-t The dosage increases as the dose increases from 30 to 45 mg / kg.

[0645] Example 11: Toxicity and Potential Target Organs of α-ADCs

[0646] Objectives: This study aimed to: i) evaluate the toxicity and potential target organs of α-ADC following repeated intravenous infusions (3 times every 3 weeks) in cynomolgus monkeys during a 7-week main phase, and to assess the reversibility of observed toxicities or potential delayed toxicities after a 6-week recovery period, to support the study design for subsequent toxicity studies and clinical trials. ii) characterize the toxicokinetics of α-ADC in cynomolgus monkeys.

[0647] Study Design: Forty cynomolgus macaques (5 animals / sex / group, 4 groups in total) were randomly assigned to the study and administered α-ADC composition 1 (10, 30, and 45 mg / kg) or the carrier (0 mg / kg) every 3 weeks for a total of 3 administrations over a 7-week period. At the end of the administration period, necropsy was performed on 3 animals / sex / groups, and necropsy was performed on the remaining 2 animals / sex / groups after a 6-week recovery period. The results are shown in Table 27.

[0648] Table 27. Groups and Dosage Levels

[0649]

[0650] All dose levels in the table are nominal.

[0651] Results: The aggregated toxicokinetic data showed that... Figure 51 middle.

[0652] Following IV infusion, α-ADC composition 1 (10, 30, or 45 mg / kg), serum α-ADC composition 1, and TAb concentrations typically peaked rapidly and declined in a roughly biphasic manner, then decreased over time, with α-ADC composition 1 declining faster than TAb on both day 1 and day 43. Mean serum α-ADC composition 1 concentrations were close to but slightly lower than serum TAb concentrations. α-ADC composition 1 and TAb exposure (mean C...) max and AUC 0-t The dose-proportional increase was observed. No sex differences were found in α-ADC composition 1 and TAb exposure. Exposure was based on day 1 exposure (AUC). 0-t The average accumulation rate of all doses of α-ADC composition 1 and TAb on day 43 was approximately 1.0, indicating no significant drug accumulation after repeated administration.

[0653] On days 1 and 43, only in the 30 and 45 mg / kg α-ADC composition 1 treatment groups, the highest post-dose DM1 was detected at three time points (5 min, 1 h, and 4 h), slightly higher than LLOQ. The mean C of DM1 was... max and AUC 0-t The dosage increases as the dose increases from 30 to 45 mg / kg.

[0654] The maximum non-serious toxic dose (HNSTD) was determined to be 45 mg / kg.

[0655] Notable findings are shown below.

[0656] 45 mg / kg: Decreased PLT (male), prolonged APTT (male), and increased AST and / or ALT were observed periodically 7 days after administration in each dosing cycle, subsequently recovering. Decreased GLOB; decreased A / G ratio was observed during the dosing phase. Increased spleen weight (absolute and relative) was observed in both sexes. Histopathological changes included: hypertrophy of Kupffer cells in the liver; increased cell proliferation and single-cell necrosis in the splenic red pulp; epithelial cell necrosis in the cornea (1 male) and Brunner's gland in the duodenum; increased mitotic figures in Kupffer cells in the liver, spleen histiocytes, Brunner's gland epithelium in the duodenum (female), corneal epithelium (1 male), and esophageal squamous epithelium (male). Chronic perivascular inflammation and fibrosis were present at the administration site in one female. All of the above changes were not observed at the end of the recovery period, while a bilateral increase in mesangial matrix in the glomeruli was observed in one male, with ADA detected from day 43, suggesting the changes were related to immunogenicity.

[0657] In summary, under the current research conditions, repeated intravenous infusions of 10, 30, and 45 mg / kg of α-ADC composition 1 (3 times every 3 weeks) were tolerated in cynomolgus monkeys, with single doses up to 45 mg / kg. Potential target organs / tissues include the liver, spleen, epithelium (duodenum, esophagus, and cornea), and the administration site.

[0658] Compared to Kadcyla, α-ADC composition 1 releases significantly less DM1 during cycling, which can lead to fewer side effects. See Table 28 for a detailed comparison. DM1 C of α-ADC composition 1 max At a dose of 30 mg / kg, it is approximately 1% of T-DM1; the DM1 AUC of α-ADC composition 1 0-t At a dose of 30 mg / kg, it is approximately 0.002% of T-DM1.

[0659] Table 28. DM1 TK parameters (mean values) of α-ADC composition 1 and T-DM1 after repeated administration in cynomolgus monkeys.

[0660]

[0661] -: Not detected; Cycle 3 was α-ADC composition 1, Cycle 4 was Kadcyla

[0662] *:Kadcyla BLA PHARMACOLOGY REVIEW(S),APPLICATION NUMBER:125427Orig1s000

[0663] Representative pathological assessment items for α-ADC and Kadcyla are shown in Table 29.

[0664] Pathological evaluation identified the main DM1-related toxicities as hepatotoxicity and peripheral neuropathy. Compared to Kadcyla at 10 mg / kg, the HNSTD dose of α-ADC composition 1 (45 mg / kg) showed significantly less severe severity in histopathology. Since α-ADC composition 1 and Kadcyla produce similar antitumor activity at the same dose levels, and the HNSTD of α-ADC composition 1 is 4.5 times that of Kadcyla, the therapeutic window of α-ADC composition 1 should be much wider than that of Kadcyla.

[0665] Table 29. Representative Pathological Assessment Items for α-ADC and Kadcyla

[0666]

[0667]

[0668] *:Kadcyla BLA PHARMACOLOGY REVIEW(S),APPLICATION NUMBER:125427Orig1s000

[0669] Those skilled in the art will understand that many modifications and alterations can be made to this disclosure without departing from its spirit and scope. The embodiments described herein are provided by way of example only and should not be construed as limiting. The true scope and spirit of the invention are defined by the claims, and the description and examples are illustrative only.

Claims

1. Compounds of formula (iii) or (iv) ， in x is -NH2.

2. A method for separating one or more target compounds from a mixture 1, said mixture 1 comprising four compounds, said four compounds being isomers (i) to (iv): ， in x is -NH2; The one or more target compounds are selected from the four compounds contained in mixture 1; The method includes the steps (1) and (2), and further includes the steps (3) and (4): (1) Provide mixture 1; (2) Perform chromatography on mixture 1 to obtain the target compound. (3) Recover the eluent collected in step (2), which contains mixture 3, wherein mixture 3 contains one or more additional target compounds that are different from the target compounds separated in step (2); (4) The eluent recovered in step (3) is subjected to chromatography to obtain compounds (iii) and (iv) as separated products; in The chromatography in step (2) is reversed-phase chromatography; The stationary phase used in the reversed-phase chromatography in step (2) is selected from C18 alkyl-bonded silica gel; and The mobile phase in step (2) is as follows: Eluent A is water, containing acidic agent 1; Eluent B is: methanol; Separation range: B is the gradient from 0%-30% to 30%-100%, and the rest is A; in The acidifying agent 1 is selected from AcOH and L-tartaric acid (L-TA); the amount of acidifying agent 1 is 0.05%-0.5% based on the total volume of eluent A; and The chromatography in step (4) is reversed-phase chromatography; The stationary phase used in the reversed-phase chromatography in step (4) is selected from C18 alkyl-bonded silica gel; and The mobile phase in step (4) is as follows: Eluent C is water, containing acidic agent 3; Eluent D is ACN, which optionally contains acidic agent 4; Separation range: D is the gradient from 0%-30% to 30%-100%, and the rest is C; in The acidic agent 3 is selected from TFA, phosphate buffer, ammonium acetate, and TEAP; the amount of acidic agent 3 is 0.05%-0.5% based on the total volume of eluent D; and The acidic agent 4 is TFA; based on the total volume of eluent D, the amount of acidic agent 4 is 0.05%-0.5%.

3. The method of claim 2, wherein the amount of acidic agent 1 is 0.1%-0.5% based on the total volume of eluent A.

4. The method of claim 2, wherein the amount of acidic agent 1 is 0.1%-0.3% based on the total volume of eluent A.

5. The method of claim 2, wherein the amount of acidic agent 1 is 0.3% based on the total volume of eluent A.

6. The method of claim 2, wherein the acidic agent 1 is AcOH.

7. The method of claim 2, wherein the amount of acidic agent 3 is 0.05%-0.3% based on the total volume of eluent C.

8. The method of claim 2, wherein the amount of acidic agent 3 is 0.1% based on the total volume of eluent C.

9. The method of claim 2, wherein the acidic agent 3 is selected from TFA, phosphate buffer, and TEAP.

10. The method of claim 2, wherein the acidic agent 3 is TFA.

11. The method of claim 2, wherein the amount of acidic agent 4 is 0.05%-0.3% based on the total volume of eluent D.

12. The method of claim 2, wherein the amount of acidic agent 4 is 0.1% based on the total volume of eluent D.

13. The method of any one of claims 2-12, wherein Eluent C is: water containing 0.1% TFA; and Eluent D is ACN, which optionally contains 0.1% TFA.

14. The method of any one of claims 2-12, wherein Mixture 1 is a reaction mixture obtained from the ring-opening reaction of the thiosuccinimide group in the following compounds. 。 15. The compound of claim 1, wherein The compound is prepared by the method according to any one of claims 2-14.

16. An antibody-drug conjugate having the structure of formula (vii) or (viii): (vii) (viii) in x is -NH2; A is an antibody that binds to human epidermal growth factor receptor 2 (HER2). A is modified to have a ligase donor substrate recognition sequence; where X is any single amino acid, natural or non-natural.

17. The antibody-drug conjugate of claim 16, formed by reacting the compound of claim 1 or 15 with an antibody in the presence of a ligase.

18. The antibody-drug conjugate of claim 16 or 17, wherein A is trastuzumab having a C-terminal modified light chain modified by introducing -GA- and the ligase donor substrate recognition sequence LPXTGJ, wherein J in LPXTGJ is absent or is an amino acid fragment containing 1-10 amino acids.

19. A composition comprising a mixture of compounds of formula (iii) and formula (iv), and free from compounds of formula (i) or (ii). ， in x is -NH2.

20. An antibody-drug conjugate composition comprising a mixture of an antibody-drug conjugate having the structure of formula (vii) and an antibody-drug conjugate having the structure of formula (viii): (vii) (viii) in x is -NH2; A is an antibody that binds to human epidermal growth factor receptor 2 (HER2). A is modified to have a ligase donor substrate recognition sequence; where X is any single amino acid, natural or non-natural.

21. The antibody-drug conjugate composition of claim 20, formed by reacting the composition of claim 19 with an antibody in the presence of a ligase.

22. The antibody-drug conjugate composition of claim 20 or 21, wherein A is trastuzumab having a C-terminal modified light chain modified by introducing -GA- and the ligase donor substrate recognition sequence LPXTGJ, wherein J in LPXTGJ is absent or is an amino acid fragment comprising 1-10 amino acids.

23. Use of the antibody-drug conjugate of any one of claims 16-18 or the antibody-drug conjugate composition of any one of claims 20-22 in the preparation of a medicament for treating HER2-positive cancer; wherein the HER2-positive cancer is selected from HER2-positive breast cancer and HER2-positive gastric cancer.

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