RU2793125C2 - Antibody-drug conjugate having an acid self-stabilizing binding site - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a drug conjugate having a hydrophilic acid stabilized binding site. Compared to a conjugate having a lower drug load due to the addition of an acidic stabilized compound site, the conjugate of the present invention can have a higher drug load (i.e., each given reagent has more hydrophilic attachment sites), has desirable pharmacokinetic properties.

EFFECT: conjugate with a high drug load.

15 cl, 13 dwg, 1 tbl, 56 ex

Description

Technical field

The present invention relates to the use of an antibody-drug conjugate for the treatment of cancer or other diseases and, in particular, to the use of a specific hydrophilic acidic stabilized compound site in the preparation of an antibody-drug conjugate to increase drug stability in plasma while significantly improving pharmacokinetics ( P.K.).

State of the art

Antibody-drug conjugates (ADCs) are a class of novel targeted drugs that typically consist of three components: an antibody or antibody-like ligand, a small molecule drug, and a linker that binds the antibody to the drug. Antibody-drug conjugates use the specific recognition of certain antigens by antibodies to transport drug molecules to a location near target cells and release them efficiently to provide a therapeutic effect. In August 2011, the US Food and Drug Administration (FDA) approved a new ADC from Seattle Genetics, Adecteis ^TM , for the treatment of Hodgkin's lymphoma as well as anaplastic large cell lymphoma (ALCL), and clinical use has demonstrated the safety and efficacy of this drug class.

For antibody-drug conjugates, in order to ensure efficient attachment of a drug molecule to an antibody, the conjugates currently in clinical trials are typically linked via linkers to lysine residues on the surface of the ligand or to cysteine residues (obtained by partial reduction of interstrand disulfide bonds) in the hinge region of the antibody. However, the presence of a large number of lysine residues (greater than 80) on the surface of the antibody and the non-selective nature of the attachment leads to uncertainty regarding the number of both attachments and sites, which in turn leads to a lack of uniformity in antibody-drug conjugates. Attachment to sulfhydryl can effectively reduce the problem of poor product quality, and maleimide acting as a linker can quickly and highly selectively form thioether products with sulfhydryl groups of antibodies under mild conditions.

However, a growing body of research has shown that the addition of succinimide to sulfhydryl is a reversible process (reverse Michael reaction), and when the addition product enters the plasma, the exchange of the addition product with the sulfhydryl group of albumin can be clearly observed, resulting in the release of the drug molecule, due to the presence of a large the amount of proteins containing free sulfhydryl groups in plasma. The release of the drug molecule on the one hand causes toxic side effects, while on the other hand reduces the effectiveness of the antibody-drug conjugate. (See Shen, et al . "Conjugation site modulate the vivo stability and therapeutic activity of antibody-drug conjugates" Nature Biotech (2012) 30:184-189; Baldwin & Kiick, Bioconj. Chem 2011, 22, 1946-1953; Alley, et al . Bioconjugate Chem. 2008, 19, 759-765.)

Many investigators have conducted published trials to improve the stability of antibody-drug conjugates in plasma and reduce the back reaction of Michael on the sulfhydryl group. It has been described that the cyclic thioether addition product formed by maleimide and sulfhydryl can undergo hydrolysis in an aqueous medium to form an open ring thioether product. Unlike cyclic sulfide addition products, open ring sulfide products remain stable in plasma and are no longer exchanged with other sulfhydryl groups. Thus, the exchange of sulfhydryl groups can be effectively avoided if the antibody-drug conjugate is converted to an open ring structure prior to its entry into the body.

The rate of the ring-opening addition reaction is influenced by many factors, including the pH and temperature of the reaction, as well as the structure undergoing the reaction, and due to the pH and temperature sensitivity of antibodies, rapid ring opening at the pH and temperature that the antibody can tolerate is the best choice. , feasible by optimizing the structure of the linker. WO2016025752 describes a method in which a strong electron attracting group is introduced on the outside of the succinimide ring, wherein said method uses electron absorption to reduce the exchange of thioether addition products with albumin; however, a consequence of the presence of said electron-attracting group is definitely that the bond between succinimide and the drug is difficult to maintain in vivo and the succinimide bearing a strong electron-attracting group is highly susceptible to ring hydrolysis, after which it is no longer able to undergo further attachment to a simple thioether.

Seattle Genetics (US) Patent No. US20130309256 describes a linker that introduces a main group and an electron attracting group near succinimide, where the rate of exchange of the adduct with albumin can be effectively reduced due to the effect of the main group, greatly increasing plasma stability. Human plasma pH is known to be weakly basic, and although the inclusion of basic groups may facilitate the opening of the succinimide ring, increasing the hydrophobicity of the entire molecule becomes more difficult. This phenomenon is convincingly confirmed in the subsequent Seattle Genetics patent WO2015057699. After incorporating a stabilized attachment site bearing a basic group into the target molecule, the present inventors discovered that the entire antibody-drug conjugate molecule was still rapidly degraded in mice with higher drug loading due to hydrophobicity (see Patent No. WO2015057699, page .225), and in order to improve the situation described above, the inventors were forced to introduce a complex structure of polyethylene glycol into the side chain of the molecule to improve the aggregation of the molecule (see patent No. WO2015057699).

In addition to reducing albumin turnover and improving plasma stability, another important factor to consider when designing an antibody-drug conjugate is the amount of drug that can be delivered to the targeting agent (i.e., the amount of cytotoxic agent attached to each targeting agent (for example, each antibody)), which is called the drug load. It has been suggested that higher drug loading provides better efficacy than lower drug loading (eg, 8 element loading should show better in vivo and in vitro efficacy than 4 element loading). The rationale for this theory is that conjugates with a higher drug load will deliver more drug (cytotoxic agent) to the target cells. The in vitro results also confirmed that the higher drug loading conjugates showed higher activity against in vitro cell lines. However, certain subsequent trials have shown that this hypothesis is likewise yet to be confirmed in vivo in animal models. The literature describes that the 4-unit and 8-unit drug loading conjugates of auristatin have unexpectedly similar activity in a mouse model, and no higher efficacy was observed with the 8-unit drug loading. See Hamblett, et al ., Clinical Cancer Res. 10:7063-70 (2004). Hamblett et al. identified the causes of the aforementioned experimental phenomenon and further described that in animal models, higher loading ADCs are cleared from the circulation more rapidly. This faster clearance indicates a tendency for higher loading conjugates to have a more unstable PK compared to lower loading conjugates. In addition, higher loading conjugates show lower MTD in mice and thus have a narrower therapeutic index. In contrast, ADCs with a loading of two in an artificially modified region on a monoclonal antibody have been described to have the same or better PK properties and therapeutic indices than some of the ADCs with a loading of four elements. See reports published by Junutula, et al ., Clinical Cancer Res. 16:4769 (2010).

Increasing the drug load theoretically increases the amount of drug that a single antibody carries to the target cell, however, due to the hydrophobicity of the drug, the hydrophobicity of the ADC will increase as the amount of drug increases, causing the ADC to aggregate in the body and reduce the therapeutic index. Alternative ways to overcome the tendency to show ADCs with a higher load of less desirable PK properties include adding solubilizing groups to the structure of the ADCs. For example, a polyethylene glycol polymer or other water-soluble polymer (eg, the region between the drug and antibody binding sites) can be added to the linker to overcome the tendency of the ADC to aggregate. For example, despite the fact that Seattle Genetics has developed the ability to stabilize attachment sites by incorporating major groups, there is still a need to improve the PK of drug molecules at high loadings by, for example, introducing a PEG side chain into the attachment site-drug conjugate. However, the addition of solubilizing groups increases the complexity of the preparation process used for such conjugates.

Thus, in the field of novel ADCs used to mitigate the deleterious effects of high drug-to-antibody ratio (DAR), a new approach is urgently needed that can simultaneously overcome the increase in plasma stability caused by succinimide exchange between sulfhydryls while also simultaneously improving the pharmacokinetic properties of antibody conjugates. -drug with high DAR. The acid stabilized attachment site of the present invention surprisingly satisfies both of the above requirements.

The essence of the invention

The present invention is directed to providing a specific hydrophilic acid stabilized site-drug conjugate. Through experiments, the present inventors unexpectedly found that, in contrast to the introduction of the main groups described in US20130309256, the introduction of an acidic amino acid or an oligopeptide element reduces the sulfhydryl exchange of the succinimide-antibody conjugate while maintaining a high degree of stability in blood plasma. It has also been experimentally demonstrated that the engineered conjugate exhibits a similar level of hydrophobicity to the unconjugated targeting reagent due to the incorporation of one or more amino acids, thus maintaining pharmacokinetic (PK) properties similar to those of the unconjugated targeting reagent in vivo . Thus, the presence of an acid stabilized attachment site contributes to drug efficacy through the two cascades described above, which together increase drug plasma stability and improve drug pharmacokinetics (PK).

The introduction of an acidic stabilized junction site also allows the conjugate to have a higher drug loading (i.e., a higher number of hydrophilic drug attachment sites per targeting reagent) compared to lower drug loading conjugates while maintaining the desired PK properties and exhibiting the same or better in vivo activity. (For example, a conjugate loaded with 4 elements or 8 elements may have the same or better PK properties than its counterparts loaded with 2 elements with 4 elements, respectively; such conjugates loaded with 4 elements or 8 elements may have the same or better PK properties, than their counterparts with a load of 2 elements or 4 elements, respectively).

In particular, the present invention relates to an antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, as shown in formula I:

Where:

L corresponds to an antibody, antibody fragment, or protein;

M is succinimide or hydrolysed succinimide;

Ac corresponds to a fragment consisting of an amino group or an acid group, or an oligopeptide consisting of many amino acids, where the amino part is attached to the ring shown;

D corresponds to the medicinal part;

A corresponds to the linker part;

The ring indicates a framework corresponding to a substituted or unsubstituted C _1-8 alkylidene, C _1-8 heteroalkylidene, C _6-10 arylidene, or C _4-10 heteroarylidene.

m corresponds to an integer from 1 to 20, and n corresponds to 1 or 2.

Preferably, said antibody corresponds to an antibody to a cell surface receptor or a tumor associated antigen.

Preferably, Ac corresponds to an amino acid element or an acid oligopeptide, the acid group Ac is selected from one or more of the group consisting of a carboxylic acid, phosphoric acid, phosphorous acid or sulfonic acid group.

More preferably, Ac preferably corresponds to a natural amino acid or a non-natural amino acid having an isoelectric point of 7.0 or less, or an oligopeptide consisting of them.

In some preferred examples, the oligopeptide element preferably consists of 2-5 amino acids.

In some preferred examples, A corresponds to a cleavable linker or a non-cleavable linker.

In some preferred examples, said drug corresponds to a cytotoxic drug, an autoimmune disease drug, and an anti-inflammatory drug.

More preferably, drug D is selected from the group consisting of maytansine drugs, australin drugs, benzodipyrrole drugs, pyrrolosodiazole drugs, amanitin and its derivatives, and camptothecin compounds.

In some preferred examples, A has the following formula:

where C corresponds to an optional extendable element at the end, E corresponds to an optional splitting element, F corresponds to a spacer element, and the subscripts e and f correspond to 0 or 1. The wavy line indicates the connection point between the acidic self-stabilized attachment site and the drug element.

In some preferred examples, the ring corresponds to a C _1-8 alkylene group or a C _1-8 heteroalkylene group.

More preferably, the ring corresponds to a C _1-3 alkylene group;

In some preferred examples, Ac is selected from the group consisting of (D/L) glycine, (D/L) alanine, (D/L) leucine, (D/L) isoleucine, (D/L) valine, (D/L) phenylalanine, (D/L) proline, (D/L) tryptophan, (D/L) serine, (D/L) tyrosine, (D/L) cysteine, (D/L) methionine, (D/L) asparagine , (D/L) glutamine, (D/L) threonine, (D/L) aspartic acid, (D/L) glutamic acid, or compounds of the following structural formulas:

where the wavy line indicates the site of attachment to the frame and R corresponds to an arbitrary linker fragment between the amino group and the phosphate group.

Another aspect of the present invention relates to a site of attachment-drug having the following structure:

Ac corresponds to an amino acid element with an isoelectric point of less than 7;

D corresponds to the drug part;

A corresponds to the linker part;

q corresponds to an integer in the range 1 to 8, and n corresponds to 1 or 2.

The present invention relates to an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, as well as to a pharmaceutical composition with a pharmaceutically acceptable diluent, carrier or excipient.

The antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, is used in the manufacture of medicaments for the treatment of cancer, immune disease, and inflammation.

Description of drawings

Figure 1 shows the results of the MS-TOF analysis obtained for compound 11.

On figa presents the results of the analysis of SEC-HPLC obtained for H-11.

Figure 2B presents the results of the SEC-HPLC analysis obtained for H-20.

Figure 2C presents the results of the SEC-HPLC analysis obtained for H-29.

Figure 2D presents the results of the SEC-HPLC analysis obtained for H-39.

Figure 2E presents the results of the SEC-HPLC analysis obtained for H-41.

Figure 2F presents the results of the SEC-HPLC analysis obtained for H-43.

Figure 2G shows the results of the SEC-HPLC analysis obtained for MC-VC-PAB-MMAE.

Figure 2H shows the results of the SEC-HPLC analysis obtained for DPR-VC-PAB-MMAE.

Figure 3 shows the results of in vivo stability testing for unconjugated antibodies, ADCs with acid-stabilized attachment sites, and control ADCs.

Figure 4 shows the results of HIC chromatography for an ADC with acid stabilized attachment sites and a control ADC.

FIG. 5 shows in vivo performance results for ADC with acid-stabilized attachment sites and control ADC.

Figure 6 is a graph showing the results of the experiment obtained for example 56.

Specific Embodiments

After extensive and careful research and development, the present inventors unexpectedly found that antibody-drug conjugates having an acidic stabilized attachment site exhibit better plasma stability and are more hydrophilic than conventional antibody-drug conjugates, and that the conjugate structures can withstand a higher load while maintaining the desired pharmacokinetic properties of the structures at low load. Higher drug loading results in better potency and therapeutic efficacy.

In particular, the antibody-drug conjugate having an acidic stabilized attachment site provided by the present invention has a maleimide element, which, when bound to the sulfhydryl group of the antibody and with the participation of the stabilizing acid group, hydrolyzes the ring, and the resulting structure does not undergo exchange with other sulfhydryl-containing macromolecules in plasma, thus preventing the release of the drug molecule.

At the same time, the presence of one or more hydrophilic acid groups results in an open ring structure that is more hydrophilic than the antibody-drug conjugate, thereby preventing problems such as decreased activity due to increased drug loading.

The resulting antibody-drug conjugate can be used to ensure that the drug reaches a target cell population, such as a group of tumor cells. The antibody-drug conjugate binds specifically to cell surface proteins and the resulting conjugates are randomly endocytosed. In the cell, the drug is released in the form of the active drug, producing one or more effects. Antibodies include chimeric antibodies, humanized antibodies, human antibodies; antibody fragments that can bind to antigens; antibody Fc fusion proteins; or squirrels. Suitable drugs are highly potent drugs including, but not limited to, maytansinoids, auristatins, calicheamicins, doxorubicins, benzodipyrrole antibiotics (duocarmycins and CC-1065), pyrrolobenzodiazepine dimers (PBDS), goitrogens, camptothecin, and derivatives such as SN38, irinotecan and ixitecan.

Detailed description of the patent.

Abbreviations and definitions

Unless otherwise indicated, the following terms and expressions used in the present description have the following meanings. When a trade name is used in this specification, unless the context indicates otherwise, the trade name includes the formulation of the product, the generic drug, and the active pharmaceutical ingredient of the trade name of said product.

The term "alkylene" refers to a divalent straight chain saturated hydrocarbon group with 1-20 carbon atoms, including groups in the range from 1 to 10 carbon atoms. Examples of alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2-CH2-), n-propylene, n-butylene, n-pentylene, n-hexylene, etc. Unless otherwise indicated, the term "aryl" refers to a polyunsaturated, typically aromatic, hydroxyl group, which may be monocyclic or fused, or a covalently linked polycyclic ring (up to three rings). The term "heteroaryl" refers to an aryl group (or ring) containing 1-5 heteroatoms selected from the group consisting of N, O, and S, where the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atoms may optionally undergo quaternary ammonization. The heteroaryl group may be attached to the rest of the molecule via a heteroatom. Non-limiting examples of aryl groups include: phenyl, naphthyl, and diphenyl, while non-limiting examples of heteroaryl groups include: pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl (pyrimidinyl), triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, misolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl , benzopyrazolyl, benzotriazolyl, benzotriazolyl, isobenzofuranyl, isoindolyl, indolyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrimidinyl, imidazopyridine, benzothiazolyl, benzofuranyl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, isothiazolyl, pyrazolyl, indolyl, trizolyl, thyridinyl, , isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furanyl, thienyl, etc. When described as "substituted", the substituents on the aromatic and heteroaromatic systems described above are selected from the acceptable substituents as indicated below.

As stated in this patent, the arylidene group refers to the presence of two covalently linked structures in the above aryl structure in the ortho, meta or para position.

Unless otherwise indicated herein, substituents consisting of hydrocarbon groups (including groups commonly referred to as alkylidene, alkenyl, alkynyl, and cycloalkyl) may correspond to a variety of different groups selected from the following group: -halogen, -OR', -NR 'R', -SR', -SiR'R "R"', -OC(O)R', -C(O)R', -CO ₂ R', -CONR'R", -OC(O) NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O) ₂ R', -NH-C(NH ₂ )=NH , -NR'C(NH ₂ )=NH, -NH-C(NH ₂ )=NR', -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R ", -NR'S(O) ₂ R", -CN and -NO ₂ , with a number of substituents in the range from 0 to (2m'+1), where m' represents the total number of carbon atoms in the group. Each of R', R" and R"' independently correspond to a hydrogen atom, an unsubstituted C _1-8 alkyl, an unsubstituted aryl, an aryl substituted with 1-3 halogen atoms, an unsubstituted C _1-8 alkyl, a C _1-8 alkoxy, or a C _1-8 thioalkoxy, or an unsubstituted aryl-C _1-4 alkyl group. When R' and R" are bonded to the same nitrogen atom, they can form rings of 3, 4, 5, 6 or 7 elements with said nitrogen atom. For example, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.

As used herein, a "derivative" of a compound refers to a substance that has a chemical structure similar to that of the corresponding compound, but which also contains at least one chemical group that is not present in the compound and/or lacks at least one chemical group that is present in the compound. The compound to which the derivative is compared is referred to as the "parent" compound. Typically, a "derivative" can be obtained from the parent compound through one or more chemical reaction steps.

Antibodies, antibody fragments and proteins

In embodiments of the present invention, the antibodies, antibody fragments, and protein elements correspond to targeting agents that specifically bind to the target element. Antibodies, as described in the context of the present invention, are capable of specifically binding to cellular components or other target molecules of interest. In some aspects, the antibody element delivers the drug element to a specific population of target cells with which the ligand element interacts. Ligands may include, but are not limited to, proteins, polypeptides, and peptides, as well as non-proteins such as sugars. Suitable ligand elements include, for example, antibodies, such as full-length (full) antibodies, as well as antigen-binding fragments thereof. In embodiments where the ligand element is a non-antibody targeting reagent, it may correspond to a peptide or polypeptide, or non-protein molecule. Examples of such targeting reagents include interferons, lymphokines, hormones, growth and colony stimulating factors, vitamins, nutrient transport molecules, or any other cell-binding molecule or substance. In some embodiments, the linker is covalently bonded to a sulfur atom on the ligand. In some aspects, the sulfur atom is a sulfur atom of a cysteine residue that forms an interchain disulfide bond of an antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine residue which is introduced into the ligand element and which forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine residue that has been introduced into the ligand element (eg, via site-directed mutagenesis or a chemical reaction). In other aspects, the sulfur atom to which the linker is bound is selected from the sulfur atom of a cysteine residue that forms an interchain disulfide bond of an antibody, or a frontal cysteine residue that is introduced into the ligand element (eg, via site-directed mutagenesis or a chemical reaction). In some embodiments, a numbering system according to the EU index is used, as defined by Kabat (Kabat EA, et al ., (1991) Sequences of proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242).

As used in this patent, "antibody" or "antibody element" includes, within its scope, any part of the antibody structure. This element can bind, reactively associate with, or complex with a receptor, antigen, or other target receptor element belonging to a cell population. An antibody can be any protein or protein-like molecule that binds, complexes with, or reacts with the part of the cell population that is being treated or biologically modified.

The antibodies used herein include polyclonal antibodies and monoclonal antibodies, wherein said polyclonal antibodies are heterogeneous groups of antibody molecules derived from the serum of an immune animal. Monoclonal antibodies include, but are not limited to, murine and human monoclonal antibodies, humanized monoclonal antibodies or chimeric monoclonal antibodies, and antibodies derived from other species. Human monoclonal antibodies can be prepared by any of the methods known in the art (e.g., Teng, et al . 1983, proc. Nat1. Acad. Sci. USA. 80: 7308-7312; Olsson, et al . 1982, Methan, Enzymol 92 : 3-16).

The antibody contained in the antibody-drug conjugate described in this patent should preferably retain the antigen-binding ability it had in its original wild state. Thus, an antibody, as described in the context of the present invention, is capable, preferably exclusively, of binding to the corresponding antigen. Antigens within the scope of the present invention include, for example, tumor associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules associated with tissue growth and differentiation (when known or predicted to be they are functional), lymphokines, cytokines, molecules involved in the regulation of cell circulation, molecules involved in angiogenesis, and molecules associated with angiogenesis (when known or predicted to be functional). Tumor-associated factors may correspond to factors of differentiation clusters (eg, CD proteins).

Antibodies described herein for use in an antibody-drug conjugate include, but are not limited to, antibodies that target cell surface receptors and antibodies that target tumor-associated antigens. Such tumor-associated antigens are well known to those skilled in the art and can be generated using antibody production methods and information that are well known to those of skill in the art. To develop effective cellular level targets that can be used to diagnose and treat cancer, researchers have attempted to identify transmembrane or other tumor-associated peptides. These targets are specifically expressed on the surface of one or more cancer cells with little or no expression on the surface of one or more non-cancerous cells. Typically, such tumor-associated polypeptides are overexpressed to a greater extent on the surface of malignant cells compared to the surface of non-malignant cells. The identification of such tumor-associated factors has the potential to significantly increase the specificity of antibody-based cancer therapy.

The proteins or peptides used in the context of this patent to prepare an antibody-drug conjugate can be any arbitrary peptide or protein that has affinity for an epitope or corresponding receptor, and they do not have to belong to the immunoglobulin family. These peptides can be isolated by methods similar to those used for antibody phage display (Szardenings, J Recept Signal Transduct Res. 2003: 23(4): 307-49). The use of peptides from such random peptide libraries may be similar to the use of antibodies and antibody fragments. Peptide or protein binding molecules may be linked or linked to a macromolecule or other material, including, but not limited to, albumin, polymers, liposomes, nanoparticles, or dendrimers, provided that said binding allows the peptide or protein to retain its antigen-binding specificity.

Tumor-associated antigens include antigens that are well known to those skilled in the art. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases such as Genbank. Tumor-associated antigens intended for antibody targeting include all amino acid sequence variants and homologues that share at least 70%, 80%, 85%, 90%, or 95% homology with a sequence reported in the available literature or have biological properties. and characteristics that are identical to the tumor-associated antigen sequence reported in the literature.

The term "inhibition" or "inhibition by" refers to the reduction or complete elimination of a detectable amount.

The term "cancer" refers to a physiological condition or disease characterized by unregulated cell growth. "Tumor" includes malignant cells.

The term "autoimmune disease" refers to a disease or disorder resulting from targeting an individual's own tissues or proteins.

As used herein, the expression "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound (eg, a drug, a drug-site conjugate of a compound, or a ligand-site of a compound-drug). Said compound may contain at least one amino or carboxyl group and thus may form an adduct salt with an appropriate acid or base. Illustrative examples of salts include, but are not limited to: sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, hydrogen sulfate, phosphate, acid phosphate, isonicotinic acid salts, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, hydrogen tartrate, ascorbate, salicylate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, potassium salts, sodium salts, etc. In addition, pharmaceutically acceptable salts have more than one charged atom in their structure. There are instances where a plurality of charged atoms that are part of a pharmaceutically acceptable salt can be balanced in many cases. For example, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more balancing atoms.

Preferred drugs include: cytotoxic drugs used in cancer therapy, including but not limited to maytansine or maytansinoids, dolastatin 10 analogs, cachymycins, doxorubicins, benzodipyrrole antibiotics (duocarmycins, CC-1065, etc. ), pyrrolo[2,1-c][1,4]benzodiazepines (PBD) or PBD dimers and their derivatives, amanitin or its derivatives and camptothecin compounds, including camptothecin, hydroxycamptothecin, SN-38, icticam, irinotecan, etc. .

On the other hand, suitable drugs are not limited to the categories mentioned above, but rather include all drugs that can be used in the antibody-drug conjugate.

In some embodiments, the invention also includes radioisotopes attached via an acidic self-stabilized attachment site. These include, but are not limited to: ³ H, ¹¹ C, ¹⁸ F, ³² P, ³⁵ S, ⁶⁴ Cu, ⁶⁸ Ga, ⁸⁶ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²³ I, ¹²⁵ I, ¹³¹ I, ¹³³ Xe, ^177Lu , ^211At and ^213Bi .

Based on the mechanism of drug release into the cell, as used herein, "linkers" or "antibody-drug conjugate linkers" can be divided into two categories: non-cleavable linkers and cleavable linkers.

For antibody-drug conjugates containing a non-cleavable linker, the drug release mechanism is as follows: after binding of the conjugate to the antigen and endocytosis, the antibody is enzymatically cleaved in the lysosome to release the active molecule, consisting of the small molecule drug, the linker, and the amino acid residue of the antibody. The final change in the molecular structure of the drug does not reduce its cytotoxicity, however, since the active molecule is charged (due to amino acid residues), it is not able to penetrate into neighboring cells. Thus, said active drug will not be able to kill cells adjacent to the tumor that do not express the target antigen (antigen-negative cells) (known as the "bystander effect") (Ducry, et al . 2010, Bioconjugate Chem. 21:5 -13).

A cleavable linker, as the name suggests, can break down and release the active drug (the small molecule drug itself) in the target cell. Cleavable linkers can be divided into two main classes: chemically unstable linkers and enzymatically unstable linkers.

Chemically unstable linkers can be selectively degraded due to differences in plasma and cytoplasmic properties. Such properties include pH, glutathione concentration, etc.

In some embodiments of the present patent, the linker is a pH sensitive linker, also commonly referred to as an acid sensitive linker. Such linkers are relatively stable in the neutral blood medium (pH 7.3-7.5), but are hydrolyzed in the slightly acidic environment of endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). Most first generation antibody-drug conjugates use these types of linkers, examples of which include hydrazones, carbonates, acetals, and ketals. Due to the limited plasma stability of acid-sensitive linkers, antibody-drug conjugates based on such linkers typically have a short half-life (2-3 days). This short half-life somewhat limits the use of pH-sensitive linkers in the latest generation of antibody-drug conjugates.

Glutathione sensitive linkers are also known as disulfide linkers. The corresponding drug release mechanism is based on the difference between high intracellular glutathione concentration (millimolar range) and relatively low glutathione concentration (micromolar range) found in the blood. This is especially true in tumor cells, where low oxygen leads to increased reductase activity, and thus higher levels of glutathione. The disulfide bonds are thermodynamically stable and thus exhibit high plasma stability.

Enzyme-unstable linkers, such as peptide linkers, allow better control of drug release. Peptide linkers can be efficiently cleaved by lysosomal proteases such as cathepsin B or fibronectin (elevated levels of these enzymes are found in some tumor tissues). This peptide bond is believed to be highly stable in plasma due to unsuitable extracellular pH as well as the presence of serum protease inhibitors which result in proteases being normally inactive. Given the high stability in plasma, as well as the favorable selectivity and efficiency of intracellular destruction, enzyme-unstable linkers are widely used as cleavable linkers for antibody-drug conjugates. Typical enzyme-unstable linkers include Val-Cit(vc), Phe-Lys, and others.

The spacer element is usually located between the cleavable linker and the active drug, or is itself part of the cleavable linker. The mechanism of action of the spacer element is as follows: when the cleavable linker is cleaved under suitable conditions, the spacer element undergoes structural rearrangement, thus releasing the active drug to which it is associated. Typically, such spacer elements include p-aminobenzyl alcohols (PAB) and β-glucuronides, substituted or unsubstituted ethylenediamines, and the like.

The following abbreviations may be used in this patent and should be understood as having the following fixed meanings: Boc: tert-butoxycarbonyl; DCC: dicyclohexylcarbodiimide; DCM: dichloromethane; DIPEA: diisopropylcarbodiimide; DMF: N , N -dimethylformamide; DMAP: 4-( N,N -dimethylamino)pyridine; HATU: (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HPLC: high performance liquid chromatography; PEG: polyethylene glycol; TFA: trifluoroacetic acid; THF: tetrahydrofuran PBS: phosphate buffer solution (pH 7.0-7.5).

Pharmaceutically acceptable excipients include any carrier, diluent, adjuvant or excipient such as preservatives and antioxidants, bulking agents, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersants, coating agents, antimicrobial and antifungal agents, retarding agents, etc. . The use of such media and substances with pharmaceutically active substances is well known in the art. Except in cases of incompatibility between any conventional medium or reagent with the active ingredient, their use in therapeutic compositions is also contemplated. As a suitable form of therapeutic combination, additional active ingredients may also be included in said composition.

The main advantages of the present invention are the following:

1. An antibody-drug conjugate having an acidic stabilized attachment site provided by the present invention can significantly reduce the rate of exchange with albumin sulfhydryl groups in vivo , significantly increasing plasma stability.

2. Due to the incorporation of an acidic group, the acidic stabilized attachment site provided by the present invention exhibits better water solubility at the molecular level, effectively improving the PK properties of the corresponding antibody-drug conjugate, which exhibits better in vivo efficacy.

3. The antibody-drug conjugate having an acid stabilized attachment site provided by the present invention not only provides increased plasma stability, it also improves the PK properties of the final conjugate, and these advantages can fully satisfy the need for higher drug loading by reducing conjugate aggregation antibody-drug at high drug loading, while also achieving better efficacy compared to lower drug loading.

In the following section, the invention is described in more detail with reference to specific examples, and it should be understood that these examples are used only to illustrate the invention and are not intended to limit the scope of the invention. Test methods for which specific conditions are not indicated in the examples below were generally carried out under normal conditions or under the conditions recommended by the manufacturer. Unless otherwise indicated, all percentages, parts, ratios or parts are by weight. Unless otherwise specified, all professional and scientific terms used in the text below are intended to have the meanings generally understood by those skilled in the art. In addition, any methods and materials similar or equivalent to the described methods and materials can be used in the methods of the present invention. The preferred methods and materials described in the text below are for demonstration purposes only.

The general techniques used in the following embodiments of the invention are as follows:

General procedure A

After preliminary purification, antibodies with a monomer fraction of more than 95% were replaced with a phosphate buffer containing EDTA at a concentration of 10 mg/ml using an ultrafiltration centrifuge tube. TCEP was added in a molar amount of 10 times the amount of antibody, and the resulting reaction was allowed to proceed for eight hours. Disulfide bonds between antibody chains were opened and free sulfhydryl groups were quantified using the Ellman method to determine if all disulfide bonds were opened. Next, a load was added in a molar amount of 10 times the amount of antibody, and the resulting reaction was allowed to proceed for eight hours. After completion of the reaction, an ultrafiltration centrifuge tube with a cut-off molecular weight of 30 kDa was used to replace the solution with PBS, and the unbound load was removed.

General Method B

Pharmacokinetic study

ELISA method for detecting antibodies in serum: application of antibody (2 μg/ml) was carried out at 4°C overnight, and then washed with PBST, which was repeated three times, and blocking with 1% BSA + PBST at 37°C for one hour; serum samples were incubated and washed with PBST three times; detection antibodies (anti-Fc monoclonal antibody or polyclonal antibody [HRP-labeled]) were incubated at 37°C for one hour, washed three times with PBST and developed with TMB, after which 2 MH ₂ SO ₄ was used to complete the reaction and measured using microplate readers.

General Method C

Analysis using hydrophobic interaction chromatography (HIC)

ADC analysis was performed using hydrophobic interaction chromatography (HIC). Elution was performed using 0-100% mobile phase B (MPB), where mobile phase A (MPA) consisted of 1.5 M ammonium sulfate and 0.025 M sodium phosphate, and MPB consisted of 0.025 M sodium phosphate and 25% isopropanol. The sample loading volume was approximately 20 μg and the gradient elution was carried out for 15 minutes. UV 280 nm was used for detection, and the stronger the water-transporting sample was, the later the peak was.

General procedure D

Plasma stability tests

A fixed amount of ADC sample was added to human plasma from which human IgG had already been removed, with each ADC tube obtained in triplicate; followed by incubation in a water bath at 37°C for 0 hours and 72 hours, after which the ADC sample was removed and 100 μl of protein A (MabSelect SuReTM LX batch: #10221479GE, washed with PBS) was added to each tube; then allowed to occur adsorption with shaking on a vertical mixer for two hours, and then carried out washing and elution to obtain incubated ADC; then the stability of each incubated ADC sample in plasma was determined by RP-HPLC.

General Method E: Site-Specific Attachment of ADC

After preliminary purification, for antibody molecules with a monomer fraction of more than 95%, the buffer was exchanged for phosphate buffer containing EDTA at a concentration of 10 mg/ml using an ultrafiltration centrifuge tube. TCEP was added in a molar amount of 10 times the amount of antibody, and the resulting reaction was allowed to proceed for two hours. Using an ultrafiltration centrifuge tube to replace the solution with phosphate buffer at pH 6.5, DHAA was added at a molar amount of 10 times the amount of antibody, and the final reaction was allowed to proceed for two hours. Next, a load was added in a molar amount of 3 times the amount of antibody, and the resulting reaction was allowed to proceed for four hours. After completion of the reaction, an ultrafiltration centrifuge tube with a cut-off molecular weight of 30 kDa was used to replace the solution with PBS and the unbound cargo was removed to give ADC with site-specific attachment (DAR=2).

Example 1

Get connection 1

50 g of ( S ) -N -benzyloxycarbonyl- N' -tert-butoxycarbonyl-2,3-diaminopropionic acid was dissolved in 500 ml of dichloromethane, after which 50 ml of trifluoroacetic acid was added, and the reaction solution was stirred at room temperature overnight. After completion of the reaction, the reaction solution was concentrated to dryness under reduced pressure, after which ethyl acetate was added to dissolve the resulting solid, followed by the addition of hexane and stirring to precipitate the solids, which were filtered and dried to give a solid product weighing 21 g. LC-MS m/ z(ES ⁺ ): 239.1(M+H) ⁺

Example 2

Get connection 2

200 ml of benzyl alcohol was added to the reaction flask and thionyl chloride (6.69 ml, 92.4 mmol) was slowly added dropwise in a water bath; after completion of the drop addition, stirring was carried out for one hour, followed by periodic addition of compound 1 (20 g, 84 mmol) and after completion of the addition, the mixture was stirred at room temperature overnight. After completion of the reaction, benzyl alcohol was distilled off by an oil pump under reduced pressure, and the resulting residue was subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol=150:1) to obtain 22 g of a product. LC-MS m/z (ES ⁺ ): 329.1 (M+H) ⁺ .

Example 3

Get Compound 3

Compound 2 (10 g, 30.4 mmol) was dissolved in 150 ml of acetonitrile, diisopropylethylamine (DIEA, 4.72 ml, 36.58 mmol) was added and tert-butyl bromoacetate (4.75 g, 24.4 mmol) was added dropwise ) in an ice bath; after the dropwise addition, the reaction was allowed to proceed for 30 minutes, and then the reaction mixture was placed under room temperature conditions for further reaction, after which it was monitored by TLC (developer: DCM: MeOH=10:1). After completion of the reaction, the solution was concentrated under reduced pressure, and the resulting residue was subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol=100:1) to obtain 5.9 g of a product. LC-MS m/z (ES ⁺ ): 443.3 (M+H) ⁺ .

Example 4

Get Compound 4

Compound 3 (5.9 g, 13.3 mmol), 30 ml of 1,4-dioxane and 30 ml of water were added to the reaction flask, and then DIEA (3.3 ml, 20 mmol) was added and Boc anhydride ( 14.5 g, 66.7 mmol) at room temperature; the resulting reaction solution was yellow, and the reaction was allowed to proceed at room temperature for about 3-4 hours after the dropwise addition, after which it was monitored by TLC (developer: DCM:MeOH=30:1). After completion of the reaction, dioxane was removed by concentration under reduced pressure, and DCM was added to carry out extraction, followed by concentration under reduced pressure to give a crude yellow oily product; it was then subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol=300:1) to obtain 6.6 g of a product. LC-MS m/z (ES ⁺ ): 443.3 (M+H) ⁺ -Boc, 543.2 (M+H) ⁺ .

Example 5

Get Compound 5

6.6 g of compound 4 was dissolved in 50 ml of methanol, 1.32 g of 5% Pd/C was added, and hydrogen gas was changed two to three times while the mixture was reacting at room temperature, and monitored by TLC (developer: DCM:MeOH =5:1). After completion of the reaction, the reaction solution was filtered and concentrated under reduced pressure at 40° C. to obtain 4.4 g of a product, which was used directly in the next reaction.

Example 6

Getting Compound 6

Compound 5 (4.4 g, 13.8 mmol) was dissolved in 40 ml of glacial acetic acid, maleic anhydride (2.71 g, 27.6 mmol) was added and the reaction was allowed to proceed with stirring at room temperature. The reaction was monitored by TLC (DCM:MeOH=3:1). After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the resulting residue was purified using HPLC liquid phase.

(Column: YMC-C18, 100 mm×450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 200 ml/min, detection at λ=215 nm)

Solvent A: 0.2% TFA in the input

Solvent B: Acetonitrile

Gradient: 0 to 10 min, 90%A, 10 to 25 min, 90 to 45%A, 25 to 55 min, 45 to 40%A

The 43 min retention time fraction was collected and lyophilized to give 1.51 g of a white solid. LC-MS m/z (ES ⁺ ): 317.9 (M+H) ⁺ -Boc, 417.9 (M+H) ⁺ .

Example 7

Obtaining Compound 7

Compound 6 (1.2 g, 2.88 mmol) was dissolved in 25 ml dry toluene and 2.5 ml DMA solution and triethylamine (1.2 ml, 8.65 mmol) was added (a few dry molecular sieves may be added), a nitrogen gas exchange was performed, and the mixture was heated to 120° C. while the reaction was monitored by TLC (developer: DCM:MeOH=3:1). After completion of the reaction, the solution was concentrated under reduced pressure using an oil pump, and the resulting residue was subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol 20:1 to 10:1) to obtain 700 mg of a product. LC-MS m/z (ES ⁺ ): 299.2 (M+H) ⁺ -Boc, 399.3 (M+H) ⁺ .

Example 8

Getting connection 8

700 mg (1.7 mmol) of compound 7 was dissolved in 3.5 ml of DMF, after which 531 mg (2.11 mmol) of EEDQ and 733 mg (1.93 mmol) of Val-Cit-PABOH were added and the reaction was allowed to proceed at room temperature. temperature. Monitored by TLC (developer: DCM: MeOH=5:1). After completion of the reaction, the solution was concentrated under reduced pressure using an oil pump at 45°C, and the resulting residue was subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol 50:1 to 20:1) to obtain 520 mg of product. LC-MS m/z (ES ⁺ ): 660.3 (M+H) ⁺ -Boc, 760.4 (M+H) ⁺ .

Example 9

Getting Compound 9

520 mg (0.685 mmol) of compound 8 and 1.04 g (3.42 mmol) of NCP were sequentially added to the reaction flask, 10 ml of DMF was added to dissolve the solids, and 0.33 ml (2.05 mmol) of DIEA was added, after which the reaction was allowed to proceed at room temperature. Monitored by TLC (developer: DCM:MeOH=5:1). After completion of the reaction, the solution was concentrated under reduced pressure using an oil pump at 45°C, and the resulting residue was subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol=30:1) to obtain 530 mg of a product. LC-MS m/z (ES ⁺ ): 825.3 (M+H) ⁺ -Boc, 925.2 (M+H) ⁺ .

Example 10

Get connection 10

530 mg (0.57 mmol) of compound 9 and 13.5 mg (0.1 mol) of HOBT were dissolved in 5 ml of DMF, 0.17 ml (1.04 mmol) of DIEA were added and after activation at room temperature for 1 h , 373 mg (0.52 mmol) MMAE was added and the solution was allowed to react overnight at room temperature. Monitoring by HPLC showed that the MMAE starting material was fully reacted. Purification was carried out using liquid phase HPLC.

(Column: YMC-C18, 50 mm × 450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 50 ml/min, detection at λ=205 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0-10 min, 90%A, 10-25 min, 90-45%A, 25-55 min, 45-40%A

The 34 min retention time fraction was collected and lyophilized to give 200 mg of product; LC-MS m/z (ES ⁺ ): 1503.2 (M+H) ⁺ .

Example 11

Getting Compound 11

70 mg of compound 10 was dissolved in 10 ml of dry dichloromethane, 4 ml of trifluoroacetic acid was added and the reaction was allowed to proceed at room temperature for one hour, and then purification was carried out by HPLC.

(Column: YMC-C18, 30 mm×450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 25 ml/min, detection at λ=205 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0-10 min, 95%A, 10-30 min, 95-80%A, 30-50 min, 80-50%A.

The 33 min retention time fraction was collected and lyophilized to give 34 mg of product; LC-MS m/z (ES ⁺ ): 1347.8 (M+H) ⁺ .

Example 12

Getting Compound 12

Di-tert - butyl -L -glutamate (8 g, 30.8 mmol) was dissolved in 150 ml of acetonitrile, DIEA (6 ml, 37 mmol) was added and 2-bromo- N- (benzyloxycarbonyl) -L benzyl ester was added dropwise -alanine (10.2 g, 26.1 mmol) in an ice bath; after dropwise addition, the reaction was allowed to proceed for 40 minutes, the reaction solution was placed at room temperature and allowed to react while monitoring by TLC (developer: DCM:MeOH=10:1). After completion of the reaction, the solution was concentrated under reduced pressure, and the resulting residue was subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol=100:1) to obtain 8.1 g of a product. LC-MS m/z (ES ⁺ ): 571.2 (M+H) ⁺ .

Example 13

Obtaining Compound 13

8.1 g of compound 12 (14.2 mmol), 50 ml of 1,4-dioxane and 50 ml of water were added to the reaction flask, and then 3.5 ml of DIEA (21.3 mmol) were added and 14.5 g of Boc anhydride (66.7 mmol) at room temperature; the resulting reaction solution was yellow, and the reaction was allowed to proceed to completion at room temperature, and at this time was monitored by TLC (developer: DCM: MeOH=30:1). After completion of the reaction, dioxane was removed by concentration under reduced pressure, and DCM was added to carry out extraction, followed by concentration under reduced pressure to give a crude yellow oily product; then, it was subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol=300:1) to obtain 8.55 g of a product. LC-MS m/z (ES ⁺ ): 471.1 (M+H) ⁺ -Boc, 571.3 (M+H) ⁺ .

Example 14

Getting Compound 14

8.5 g of compound 13 was dissolved in 70 ml of methanol, 1.7 g of 5% Pd/C was added and the hydrogen gas was replaced two to three times while the mixture was reacted at room temperature and monitored by TLC (developer : DCM:MeOH=5:1). After completion of the reaction, the reaction solution was filtered and concentrated under reduced pressure at 40°C to obtain 5 g of a product, which was directly used in the next reaction.

Example 15

Getting Compound 15

5 g (11.2 mmol) of compound 14 was dissolved in 40 ml of glacial acetic acid, 2.19 g (22.4 mmol) of maleic anhydride was added and the reaction was allowed to proceed with stirring at room temperature. The reaction was monitored by TLC (DCM: MeOH=3:1). After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the resulting residue was purified using HPLC liquid phase.

(Column: YMC-C18, 100 mm × 450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 200 ml/min, detection at λ=215 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0-10 min, 90%A, 10-25 min, 90-45%A, 25-55 min, 45-40%A.

The 48 min retention time fraction was collected and lyophilized to give 2.3 g of product. LC-MS m/z (ES ⁺ ): 445.2 (M+H) ⁺ -Boc, 545.3 (M+H) ⁺ .

Example 16

Obtaining Compound 16

2 g of compound 15 (2.88 mmol) was dissolved in 25 ml of dry toluene and 2.5 ml of DMA solution and 1.58 ml of triethylamine (11.4 mmol) was added, nitrogen gas was replaced and the mixture was heated to 120°C at that time. while the reaction was monitored by TLC (developer: DCM: MeOH=3:1). After completion of the reaction, the solution was concentrated under reduced pressure using an oil pump, and the resulting residue was subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol 20:1 to 10:1) to obtain 1.19 g of a product; LC-MS m/z (ES ⁺ ): 427.1 (M+H) ⁺ -Boc, 527.3 (M+H) ⁺ .

Example 17

Obtaining Compound 17

1.19 g (1.7 mmol) of compound 16 was dissolved in 3.5 ml of DMF, after which 531 mg (2.11 mmol) of EEDQ and 733 mg (1.93 mmol) of Val-Cit-PABOH were added and the reaction was allowed to proceed at room temperature. Monitored by TLC (developer: DCM:MeOH=5:1). After completion of the reaction, the solution was concentrated under reduced pressure using an oil pump at 45°C, and the resulting residue was subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol 50:1 to 25:1) to obtain 753 mg of product. LC-MS m/z (ES ⁺ ): 788.5 (M+H) ⁺ -Boc, 888.4 (M+H) ⁺ .

Example 18

Getting Compound 18

753 mg (0.849 mmol) of compound 17 and 1.28 g (4.24 mmol) of NCP were sequentially added to the reaction flask, 15 ml of DMF was added to dissolve the solids, and 0.35 ml (2.55 mmol) of DIEA was added, after which the reaction was allowed to proceed at room temperature. Monitored by TLC (developer: DCM:MeOH=5:1). After completion of the reaction, the solution was concentrated under reduced pressure using an oil pump at 45°C, and the resulting residue was subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol=25:1) to obtain 762 mg of a product. LC-MS m/z (ES ⁺ ): 1053.6 (M+H) ⁺ .

Example 19

Getting Compound 19

762 mg (0.71 mmol) of compound 18 and 13.5 mg (0.1 mol) of HOBT were dissolved in 8 ml of DMF, 0.23 ml (1.42 mmol) of DIEA were added and after activation at room temperature for 1 h 463 mg (0.645 mmol) MMAE was added and the solution was allowed to react overnight at room temperature. Monitoring by HPLC showed that the MMAE starting material was fully reacted. Purification was carried out using liquid phase HPLC.

(Column: YMC-C18, 100 mm×450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 200 ml/min, detection at λ=215 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0-10 min, 90%A, 10-25 min, 90-45%A, 25-55 min, 45-40%A

The 30 min retention time fraction was collected and lyophilized to give 300 mg of product. LC-MS m/z (ES ⁺ ): 1631.6 (M+H) ⁺ .

Example 20

Getting Compound 20

100 mg of compound 19 was dissolved in 10 ml of dry dichloromethane, 4 ml of trifluoroacetic acid was added and the reaction was allowed to proceed at room temperature for one hour, and then purification was carried out by HPLC.

(Column: YMC-C18, 30 mm × 450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 25 ml/min, detection at λ=215 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0-10 min, 95%A, 10-25 min, 95-80%A, 25-55 min, 80-55%A

The 30 min retention time fraction was collected and lyophilized to give 45 mg of product. LC-MS m/z (ES ⁺ ): 1419.1 (M+H) ⁺ .

Examples 21-29

Synthesis of compounds 21 - 29

Example 21

Getting connection 21

15 g of 3-nitro-4-aminobenzoic acid was dissolved in 100 ml of methanol, 3 g of 5% Pd/C was added, and hydrogenation was carried out at atmospheric pressure for five hours, and then filtration was carried out; the resulting filter residue was washed twice with methanol, then concentrated to dryness at room temperature under high vacuum, and directly used in the next reaction without further purification.

Example 22

Getting connection 22

3,4-diaminobenzoic acid (10 g, 65.7 mmol) was dissolved in 100 ml DMF, powdered potassium carbonate (13.6 g, 98.55 mol) and potassium iodide (2.2 g, 1.31 mmol) were added , and tert-bromoacetic acid in nitrogen gas was added dropwise, after which butyl ether (12.8 g, 65.7 mol) was added dropwise while the reaction was allowed to proceed at room temperature. Monitored by TLC (developer: DCM: MeOH=10:1). After completion of the reaction, water was added, extraction was carried out with ethyl acetate, and the extract was combined with the organic phase; thereafter, washing with saturated saline followed by drying with anhydrous sodium sulfate and filtration was carried out, and the solution was concentrated under reduced pressure, and the resulting residue was subjected to separation using purification by silica gel column chromatography (eluent: dichloromethane:methanol=150:1 to 100: 1) to obtain 3.46 g of product. LC-MS m/z (ES ⁺ ): 267.4 (M+H) ⁺ ; ¹ H-NMR: 7.51-7.53, dd, 1H: 7.44-7.45, d, 1H: 6.66-6.69, d, 1H; 4.69, s, 2H: 1.49, s, 9H.

Example 23

Getting Compound 23

3 g (11.2 mmol) of compound 22 , 15 ml of glacial acetic acid and maleic anhydride (0.55 g, 5.6 mmol) were added to the reaction flask, and the resulting reaction mixture was allowed to proceed at room temperature and monitored by HPLC. After completion of the reaction, concentration under reduced pressure was carried out to remove glacial acetic acid, and purification was carried out. (Column: YMC-C18, 100 mm×450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 200 ml/min, detection at λ=214 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0-5 min 80%A, 5-35 min, 80-65%A, 35-45 min, 65-60%A, the fraction showing a retention time of 37-42 min was collected and lyophilized to give 2 g of product . LC-MS m/z (ES ⁺ ): 365.4 (M+H) ⁺ , 309.3 (M+H) ⁺ -tBu. ¹ H-NMR: 10.48, s, 1H; 7.41-7.44, dd, 1H; 7.35-7.36, d, 1H; 6.92, s, 2H; 6.70-6.72, d, 1H; 4.68, s, 2H; 1.42, s, 9H.

Example 24

Getting connection 24

Compound 23 (2 g, 5.49 mmol) was dissolved in 30 ml THF and Boc anhydride (1.44 g, 6.59 mmol), DMAP (1.32 g, 10.9 mmol) and DIEA (1 .8 ml, 10.98 mmol) and the reaction was allowed to proceed at room temperature. Monitored by TLC (developer: DCM: MeOH=10:1). After completion of the reaction, the solution was subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol=100:1 to 20:1) to obtain 2.2 g of a product. LC-MS m/z (ES ⁺ ); 365.4(M+H) ⁺ -Boc ¹ H-NMR: 10.50, s, 1H; 7.46-7.48, dd, 1H; 7.41-7.42, d, 1H; 6.95, s, 2H; 6.73-6.75, d, 1H; 4.69, s, 2H; 1.48, s, 9H; 1.40, s, 9H.

Example 25

Getting Compound 25

2 g (4.31 mmol) of compound 24 was dissolved in 15 ml of toluene and 2 ml of DMA, triethylamine (1.2 ml, 8.62 mmol) was added and the resulting reaction was allowed to proceed at 120°C. The reaction was monitored by TLC (DCM:MeOH=10:1). After completion of the reaction, the solution was concentrated under reduced pressure, and the resulting residue was subjected to separation using purification by silica gel column chromatography (eluent: dichloromethane:methanol 100:1 to 50:1) to obtain 1.63 g of a product; LC-MS m/z (ES ⁺ ): 347.4 (M+H) ⁺ -Boc ¹ H-NMR: 7.50-7.51, dd, 1H; 7.43-7.44, d, 1H; 6.97, s, 2H; 6.75-6.77, d, 1H; 4.67, s, 2H; 1.48, s, 9H; 1.40, s, 9H.

Example 26

Getting Compound 26

1.6 g (3.58 mmol) of compound 25 was dissolved in 10 ml of DMF, after which 1.77 g (7.17 mmol) of EEDQ and 2.98 g (7.89 mmol) of Val-Cit-PABOH were added, and the reaction was allowed to proceed at room temperature. Monitored by TLC (developer: DCM:MeOH=10:1). After completion of the reaction, the solution was concentrated under reduced pressure using an oil pump at 45°C, and the resulting residue was subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol 100:1 to 25:1) to obtain 2.1 g of product . LC-MS m/z (ES ⁺ ): 709.8 (M+H) ⁺ -Boc, 809.7 (M+H) ⁺ .

Example 27

Getting Compound 27

2 g (2.47 mmol) of compound 26 and 3.71 g (12.3 mmol) of NCP were sequentially added to the reaction flask, 20 ml of DMF was added to dissolve the solids, and 1.2 ml (7.41 mmol) of DIEA was added, after which the reaction was allowed to proceed at room temperature. Monitored by TLC (developer: DCM: MeOH=15:1). After completion of the reaction, the solution was concentrated under reduced pressure using an oil pump at 45°C, and the resulting residue was subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol=100:1) to obtain 2.1 g of a product. LC-MS m/z (ES ⁺ ): 973.3 (M+H) ⁺ .

Example 28

Getting connection 28

1 g (1 mmol) of compound 27 and 13.5 mg (0.1 mol) of HOBT were dissolved in 8 ml of DMF, 0.33 ml (2 mmol) of DIEA was added, and after activation at room temperature for 1 h, 646 mg of (0.9 mmol) MMAE and the solution was allowed to react overnight at room temperature. Monitoring by HPLC showed that the MMAE starting material was fully reacted. Purification was carried out using liquid phase HPLC.

(Column: YMC-C18, 100 mm×450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 200 ml/min, detection at λ=214 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0-10 min, 90%A, 10-25 min, 90-45%A, 25-55 min, 45-40%A.

The 30 min retention time fraction was collected and lyophilized to give 560 mg of product. LC-MS m/z (ES ⁺ ): 1551.7 (M+H) ⁺ .

Example 29

Getting connection 29

100 mg of compound 28 was dissolved in 10 ml of dry dichloromethane, 4 ml of trifluoroacetic acid was added and the reaction was allowed to proceed at room temperature for two hours, and then purification was carried out by HPLC.

(Column: YMC-C18, 30 mm × 450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 25 ml/min, detection at λ=214 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0-10 min, 95%A, 10-25 min, 95-80%A, 25-55 min, 80-55%A

The 30 min retention time fraction was collected and lyophilized to give 45 mg of product. LC-MS m/z (ES ⁺ ): 1395.7 (M+H) ⁺ .

Examples 30-39

Synthesis of compounds 30-39

Example 30

Getting Compound 30

Using a 500 ml round bottom flask, potassium carbonate (24.7 g, 179.24 mmol) was dissolved in 220 ml of pure water, after which ( S )-3-amino-2-(benzyloxycarbonylamino) propionic acid (16 g, 67.16 mmol) and the mixture was stirred almost until complete dissolution; further, 2-bromoethyl diethyl phosphate (10.98 g, 44.81 mmol) was added and after completion of the addition, the mixture was placed in an oil bath at 80° C. and stirred for six hours until the reaction was complete. The reaction liquid was purified using HPLC liquid phase. (Column: YMC-C18, 100 mm×450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 200 ml/min, detection at λ=214 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0-10 min, 5-15%B, 10-15 min, 15%B, 15-25 min, 15-20%B, 25-50 min, 20-30%B, 50-55 min, 30 -50%B, 55-65 min, 50-90%B. The fraction with a retention time of 42-49 minutes was collected and lyophilized to obtain 8.38 g of a colorless oily product with a yield of 46.5%. ¹ H-NMR data (CDCl ₃ , 400 MHz): 7.39-7.28 (m, 5H), 6.54-6.41 (m, 1H), 5.12-5.01 (m, 2H ), 4.63-4.53 (m, 1H), 4.16-4.03 (m, 4H), 3.48-3.36 (m, 2H), 3.35-3.25 (m , 2H), 2.35-2.23 (m, 2H), 1.29 (t, J=6.8Hz, 6H); LCMS[M+H] ⁺ m/z 403.3 (calculated for C ₁₇ H ₂₇ N ₂ O ₇ P, 402.16).

Example 31

Getting connection 31

In a 100 ml three-necked flask, compound 30 (8.3 g, 20.85 mmol) was dissolved using 50 ml dichloromethane solvent and the solution was placed in a low temperature reactor set at 0° C. under nitrogen gas, after which DIEA base (6.7 g, 52.11 mmol) with stirring and Boc anhydride (5.0 g, 22.935 mmol) added dropwise over half an hour; after completion of the dropwise addition, the mixture was placed under room temperature conditions and stirred for 2 hours, and monitored by TLC until the reaction was completed (developer: DCM:MeOH=4:1). Post-treatment: Water was added to the reaction solution to wash it three times, the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the resulting residue was purified by HPLC. (Column: YMC-C18, 100 mm 450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 200 ml/min, detection at λ=214 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0 min, 40%B, 0-10 min, 40%B, 10-20 min, 40-45%B, 20-30 min, 45%B, 30-45 min, 45-80%B, 45 -50 min, 80-95%B. The fraction with a retention time of 26-29.5 minutes was collected and lyophilized to obtain 4.02 g of a colorless oily liquid with a yield of 38.3%. ¹ H-NMR data (CDCl ₃ , 400 MHz): 7.36-7.28 (m, 5H), 5.09 (s, 2H), 4.63-4.43 (m, 1H), 4. .14-4.01 (m, 4H), 3.92-3.65 (m, 2H), 3.61-3.29 (m, 2H), 2.34-1.95 (m, 2H) , 1.45 (s, 9H), 1.36-1.22 (m, 6H); LCMS[M+H] ⁺ m/z 503.4 (calculated for C ₂₂ H ₃₅ N ₂ O ₉ P, 502.21).

Example 32

Getting connection 32

In a 150 ml round bottom flask, 50 ml of methanol was used to dissolve compound 31 (4.0 g, 8.0 mmol), after which 800 mg of Pd/C (containing 5% Pd) was added and hydrogen gas was replaced, after which the reaction solution was stirred at room temperature for four hours; monitored by TLC (developer: DCM:MeOH=4:1) until the completion of the reaction, and the reaction was stopped. Filtration through an organic membrane was used to remove the palladium-carbon and concentrated under reduced pressure to give a residue which was directly used in the next step without the need for any purification. LCMS[M+H] ⁺ m/z 369.4 (calculated for C ₁₄ H ₂₉ N ₂ O ₇ P, 368.17).

Example 33

Getting Compound 33

To a 100 ml round bottom flask was added 30 ml of glacial acetic acid to dissolve the crude compound 32 , after which maleic anhydride (1.57 g, 16.0 mmol) was added with stirring and the reaction was allowed to proceed with stirring at room temperature overnight. The reaction was stopped the next day and glacial acetic acid was removed by concentration under reduced pressure using an oil pump at 45°C; then the resulting residue was purified by HPLC. (Column: YMC-C18, 100 mm × 450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 200 ml/min, detection at λ=214 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0 min, 5%B, 0-10 min, 5-15%B, 10-20 min, 15-30%B, 20-35 min, 30-55%B, 35-45 min, 55%B , 45-55 min, 55-80%B, 55-60 min, 80-98%B. A fraction with a retention time of 25-26.5 minutes was collected and lyophilized to give 3.35 g of an oily substance with a 90% yield over two steps. ¹ H-NMR data (CDCl ₃ , 400 MHz): 6.40 (d, J=12Hz, 1H), 6.35 (d, J=12Hz, 1H), 4.96 to 4.82 (m, 1H ), 4.19-3.99 (m, 4H), 3.98-3.61 (m, 2H), 3.60-3.36 (m, 2H), 2.22-2.00 (m , 2H), 1.45 (s, 9H), 1.33 (t, J=7.0Hz, 3H), 1.32 (t, J=7.0Hz, 3H); LCMS[M+H] ⁺ m/z 467.3 (calculated for C ₁₈ H ₃₁ N ₂ O ₁₀ P, 466.17).

Example 34

Getting connection 34

In a 100 ml round bottom flask, compound 33 (3.3 g, 7.07 mmol) was dissolved with 40 ml of toluene and 4 ml of DMA with stirring, after which triethylamine (2.1 g, 21.2 mmol) was added and a trap and reflux were installed. refrigerator, and the reaction was allowed to proceed under reflux and stirring at 120°C for 3 hours until TLC monitoring showed that the reaction was complete (developer: DCM:MeOH=4:1). Post-treatment: The reaction solution was concentrated under reduced pressure at 45° C. to remove toluene, and the resulting residue was purified by HPLC. (Column: YMC-C18, 100 mm×450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 200 ml/min, detection at λ=214 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0 min, 5%B, 0-10 min, 5-15%B, 10-20 min, 15-30%B, 20-35 min, 30-55%B, 35-45 min, 55%B , 45-55 min, 55-80%B, 55-60 min, 80-98%B. A fraction with a retention time of 27-29 minutes was collected and lyophilized to give 2.12 g of a white powdery solid (which rapidly absorbed moisture from the air and became a viscous oil) in 67% yield. LCMS[M+H] ⁺ m/z 449.3 (calculated for C ₁₈ H ₂₉ N ₂ O ₉ P, 448.16).

Example 35

Getting Compound 35

A 100 ml round bottom flask containing compound 34 (2.1 g, 4.69 mmol) was dried using an oil pump, 20 ml of dry DMF was added and the resulting mixture was stirred until complete dissolution; further DIEA (1.8 g, 14.07 mmol) was added followed by EDCI (1.81 g, 9.38 mmol) and HoAt (1.28 g, 9.38 mmol) and the activated compound was stirred at room temperature for five and a half hours, after which compound 6 (2.66 g, 7.03 mmol) was added and the reaction was allowed to proceed at room temperature overnight, and then it was stopped the next day. Post-treatment: The DMF solvent was removed by concentration under reduced pressure at 45°C using an oil pump, and the resulting residue was purified using column chromatography (eluent: dichloromethane:methanol=20:1 to 10:1 to 5:1) to obtain 1 .01 g of a white powdery solid in 26.6% yield. LCMS[M+H] ⁺ m/z 810.4 (calculated for C ₃₆ H ₅₆ N ₇ O ₁₂ P, 809.37).

Example 36

Getting Compound 36

In a 50 ml round bottom flask, 15 ml dry DMF was used to dissolve compound 35 (1.0 g, 1.24 mmol) with stirring, after which DIEA (961 mg, 7.44 mmol) and NPC (1.88 g, 6.2 mmol) and the reaction mixture was allowed to react at room temperature overnight with stirring; the next morning monitoring by TLC showed that the reaction was complete (developer: dichloromethane:methanol=5:1) and the reaction was stopped. Post-treatment: The reaction solution was concentrated under reduced pressure using an oil pump at 45°C, and the resulting residue was purified by thin layer chromatography (developer: dichloromethane:methanol=6:1) to obtain 1.02 g of a white powdery solid with a yield of 84, 5%. LCMS[M+H] ⁺ m/z 975.3 (calculated for C ₄₃ H ₅₉ N ₈ O ₁₆ P, 974.38).

Example 37

Getting Compound 37

In a 100 ml round bottom flask, 10 ml dry DMF was used to dissolve compound 36 (1.0 g, 1.03 mmol) followed by the addition of HoBt (28 mg, 0.21 mmol) and DIEA (266 mg, 2.06 mmol ), and the resulting reaction was allowed to proceed with stirring for half an hour; further MMAE (740 mg, 1.03 mmol) was added and the reaction was allowed to proceed overnight at room temperature. Post-treatment: The reaction solution was purified by preparative liquid phase chromatography. (Column: YMC-C18, 50 mm×450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 50 ml/min, detection at λ=214 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0 min, 5%B, 0-10 min, 5-15%B, 10-20 min, 15-30%B, 20-35 min, 30-55%B, 35-45 min, 55-70 %B, 45-55 min, 70%B, 55-60 min, 70-98%B.

The 38-40 min retention time fraction was collected and lyophilized to give 320 mg of a white powdery solid in 20% yield. LCMS[M+H] ⁺ m/z 1553.8 (calculated for C ₇₆ H ₁₂₁ N1 ₂ O ₂₀ P, 1552.85).

Example 38

Getting connection 38

Compound 37 (260 mg, 0.17 mmol) was dissolved in a 25 ml round bottom flask using 5 ml of distilled dichloromethane, followed by the addition of bromotrimethylsilane (78 mg, 0.51 mmol) in an ice bath under nitrogen gas; the reaction was allowed to proceed overnight at room temperature with stirring and the next morning 1 ml of methanol was added, after which the reaction was allowed to proceed with stirring at room temperature for one hour and liquid phase analysis was used to confirm that compound 37 had completely reacted. Post-treatment: The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by HPLC. (Column: YMC-C18, 30 mm × 450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 25 ml/min, detection at λ=214 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0 min, 5%B, 0-10 min, 5-15%B, 10-20 min, 15-30%B, 20-35 min, 30-55%B, 35-45 min, 55-70 %B, 45-55 min, 70%B, 55-60 min, 70-98%B.

The 20-23 min retention time fraction was collected and lyophilized to give 140 mg of a white powdery solid in 55% yield.

Example 39

Getting Compound 39

In a 25 ml round bottom flask, 2 ml of dichloromethane was used to dissolve compound 38 (120 mg, 0.08 mmol) with stirring, after which 800 microliters of trifluoroacetic acid were added dropwise in an ice bath; after the dropwise addition, the reaction solution was placed under room temperature conditions, and the reaction was allowed to proceed with stirring for one hour to complete the reaction. Post-treatment: The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by HPLC. (Column: YMC-C18, 30 mm×450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 25 ml/min, detection at λ=214 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0 min, 5%B, 0-10 min, 5-15%B, 10-20 min, 15-30%B, 20-35 min, 30-55%B, 35-45 min, 55-70 %B, 45-55 min, 70%B, 55-60 min, 70-98%B.

The 18-19.5 min retention time fraction was collected and lyophilized to give 55 mg of a white powdery solid in 49% yield. LC-MS m/z (ES ⁺ ): 1397.8 (M+H) ⁺ .

Example 40

Getting connection 40

Compound 7 (332 mg, 0.836 mmol) was dissolved in dry dichloromethane, pentafluorophenol (184 mg, 1 mmol) and dicyclohexylcarbodiimide (2 mmol) were added and stirring was carried out at room temperature for three hours until compound 7 was eliminated. MMAF (460 mg, 0.585 mmol) and diisopropylethylamine (DIEA, 0.27 ml, 1.67 mmol) were added and dissolved in 15 ml dry dichloromethane solution and the resulting reaction was allowed to proceed at room temperature for four hours under nitrogen gas. The solution was concentrated under reduced pressure, and the resulting residue was subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol=50:1) to obtain 290 mg of a product. LC-MS m/z (ES ⁺ ): 1168.8 (M+H) ⁺ .

Example 41

Getting Compound 41

100 mg of compound 39 was dissolved in 10 ml of dry dichloromethane, 4 ml of trifluoroacetic acid was added and the reaction was allowed to proceed at room temperature for two, and then purification was carried out by HPLC.

(Column: YMC-C18, 30 mm × 450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 25 ml/min, detection at λ=215 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0-10 min, 95%A, 10-25 min, 95-70%A, 25-55 min, 70-40%A.

The 33 min retention time fraction was collected and lyophilized to give 50 mg of product. LC-MS m/z (ES ⁺ ): 956.6 (M+H) ⁺ .

Example 42

Getting Compound 42

Compound 16 (400 mg, 0.76 mmol) was dissolved in dry dichloromethane, pentafluorophenol (184 mg, 1 mmol) and dicyclohexylcarbodiimide (2 mmol) were added and stirring was carried out at room temperature for four hours until compound 16 was eliminated. MMAF (389 mg, 0.49 mmol) and diisopropylethylamine (DIEA, 0.25 ml, 1.52 mmol) were added and dissolved in 15 ml dry dichloromethane and the final reaction was allowed to proceed at room temperature for four hours under nitrogen gas. The solution was concentrated under reduced pressure, and the resulting residue was subjected to separation using silica gel column chromatography (eluent: dichloromethane:methanol=50:1) to obtain 330 mg of a product. LC-MS m/z (ES ⁺ ): 1296.8 (M+H) ⁺ .

Example 43

Getting Compound 43

100 mg of compound 41 was dissolved in 10 ml of dry dichloromethane, 4 ml of trifluoroacetic acid was added and the reaction was allowed to proceed at room temperature for two hours, followed by purification by HPLC.

(Column: YMC-C18, 30 mm × 450 mm, 10 µm, acetonitrile/water (0.2% TFA), flow rate 25 ml/min, detection at λ=215 nm)

Solvent A: 0.2% TFA in water

Solvent B: Acetonitrile

Gradient: 0-10 min, 95%A, 10-25 min, 95-70%A, 25-55 min, 70-40%A

The 30 min retention time fraction was collected and lyophilized to give 48 mg of product. LC-MS m/z (ES ⁺ ): 1028.5 (M+H) ⁺ .

Example 44

The H-11 antibody-drug conjugate was prepared according to General Method B and reverse phase HPLC showed that the average drug/antibody ratio (DAR) was 7.2.

Example 45

The H-20 antibody-drug conjugate was prepared according to General Method B and reverse phase HPLC showed that the average drug/antibody ratio (DAR) was 7.2.

Example 46

The H-29 antibody-drug conjugate was prepared according to General Method B and reverse phase HPLC showed that the average drug/antibody ratio (DAR) was 6.8.

Example 47

The H-39 antibody-drug conjugate was prepared according to General Method B and reverse phase HPLC showed that the average drug/antibody ratio (DAR) was 7.0.

Example 48

The H-41 antibody-drug conjugate was prepared according to General Method B and reverse phase HPLC showed that the average drug/antibody ratio (DAR) was 7.5.

Example 49

The H-43 antibody-drug conjugate was prepared according to General Method B and reverse phase HPLC showed that the average drug/antibody ratio (DAR) was 7.7.

Example 50

The synthesis was continued to give the following drug-site linkers:

where Ac denotes a non-basic amino acid or oligopeptide, and the amino group in the amino acid is connected to an alkyl group;

n=1,2, 3...;

Table 1 below summarizes the synthesis and characterization of drug attachment sites containing various amino acids and oligopeptides. In the table, the first column on the left is the compound number, the second column is the amino acid class, the third column is the value of n, the fourth column is the general synthesis method, and the fifth and sixth columns show the calculated mass of the compound drug-site of attachment and the mass at determination when determined by mass spectrometry.

Table 1

Connection number Connection section Synthesis method Theoretical molecular weight True molecular weight Ac amino acid Value n 42 gly 2 General Formula I 1360.7 1360.3 43 1 General Formula II 969.5 969.2 44 Phe 1 General Formula II 1045.6 1045.8 45 trp 1 General Formula I 1452.7 1452.8 46 asp 2 General Formula I 1418.7 1418.5 47 Ser 2 General Formula II 999.5 999.4 48 Val 1 General Formula II 997.5 999.6 49 Pyrazole 2 General Formula I 1470.7 1470.5 50 Fur 1 General Formula II 1023.5 1023.3 51 Gly-Ala 1 General Formula I 1431.4 1431.4

Abbreviations:

Gly: L-glycine, Ala: L-alanine, Phe: L-phenylalanine, Trp: L-tyrosine, Asp: L-aspartic acid, Ser: L-serine, Val: L-Valine

Examples 52-56

To evaluate the stability, hydrophilicity and pharmacological activity of ADCs prepared using an acidic self-stabilizing attachment site, the present inventors prepared drug-attachment compounds with acidic stabilizing attachment sites according to the examples. All of these compounds had an acid stabilized attachment site that was linked to the cytotoxic drug MMAE or MMAF via a cleavable or non-cleavable linker. As a comparison, a typical non-self-stabilizing compound drug-attachment site (hereinafter referred to as MC-VC-PAB-MMAE) was prepared by the methods described in the literature, and a compound drug-attachment site having a basic self-stabilizing attachment site was obtained, as described in this patent (hereinafter referred to as DPR-VC-PAB-MMAE).

Example 52 In Vitro Plasma Stability

The results of plasma stability studies conducted according to the method described in General Procedure D were as shown in the table below: ADCs with acid-stabilized attachment sites show little to no drug loss during plasma incubation, while a typical ADC with an attachment site MC showed a very significant decrease in DAR after incubation for 72 hours. The results of the experiment demonstrate that the acid stabilized attachment site can significantly increase the stability of ADC in plasma.

Target conjugate DAR 0 h DAR 72 h H-11 7.2 6.8 H-20 7.2 7.1 H-29 6.8 6.4 H-39 7.0 6.8 H-41 7.5 6.9 H-43 7.7 6.4 MC-VC-PAB-MMAE 7.2 2.3

Example 53: Detection by SEC-HPLC

ADC samples obtained by conjugation were centrifuged at 14,000 rpm for five minutes, and the supernatant was collected for analysis.

Device: Waters e2695 (2489UV/Vis)

Chromatography column: TSKgel G3000SWXL (7.8×300 mm, 5 µm)

Mobile phase: A: 50 mM PB, 300 mM NaCl, 200 mM Arg, 5% IPA, pH 6.5

Mobile phase A was subjected to isocratic elution for 30 minutes; flow rate: 0.714 ml/min, column temperature: 25°C, detection wavelength: 280 nm.

The degree of aggregation of each ADC was determined by SEC-HPLC and SEC-HPLC peak plots are shown in FIGS. 2A-2H; the corresponding data are summarized in the table below, where the level of monomer increased significantly and the degree of aggregation decreased significantly in ADCs that included an acid stabilized attachment site.

Target conjugate Monomer share Aggregation level H-11 96.25% 3.75% H-20 98.85% 1.15% H-29 98.26% 1.74% H-39 98.86% 1.14% H-41 97.02% 2.98% H-43 96.54% 3.46% MC-VC-PAB-MMAE 67.51% 32.46% DPR-VC-PAB-MMAE 79.10% 20.90%

Example 54

PK experiments in mice in vivo

PK properties for ADC (load 8) were determined using a mouse model. The drug was administered by a single intravenous injection in the tail at a dose of 2 mg/kg, and the concentration of total antibodies (total Ab) in the blood was evaluated according to the General method B; the results are presented in Fig.3. The results of the experiment show that in a mouse model, ADC with an acid-stabilized attachment site showed significantly improved PK properties with total antibody concentration maintained for a longer period of time.

Example 55

Analysis using hydrophobic interaction chromatography (HIC)

After complete reduction of the IgG1 antibody to 8 thiols per antibody according to General Method A, the corresponding antibody-drug conjugates were obtained by non-specific binding. ADCs with acid stabilized attachment sites and ADCs with conventional attachment sites (i.e. MC-VC-PAB-MMAE) with eight drug units per antibody were further separated and purified by hydrophobic interaction chromatography, and hydrophobic interaction chromatography (HIC) was used to perform analysis of ADCs according to General Method C. ADCs with greater hydrophobicity and higher drug/molecule ratios eluted with longer retention times. The results are shown in Figure 4: ADCs with acidic attachment sites showed a relatively short retention time in the HIC, with the MC-VC-PAB-MMAE antibody conjugate showing the longest retention time. The experimental results demonstrate that ADC molecules having an acidic stabilized attachment site show better hydrophilicity.

Example 56

In vivo drug efficacy experiments (cell proliferation assay)

Human pancreatic adenocarcinoma cells (BxPc3) were cultured in vitro and inoculated subcutaneously into the flank of BALB/c mice with an inoculated cell number of 5×10 ⁶ and after tumor growth to 100-200 mm ³ , the animals were divided into groups and they were injected with a single dose of ADC 3 mg/kg (injected via the tail vein) while also receiving a vehicle control group; mice were subjected to regular weighing and measurement of tumor volume, and drug efficacy against the BxPc3 model was evaluated by examining the efficacy of tumor suppression by ADC as well as other indicators. The results of the experiment are presented in Figures 5 and 6. The results show that ADCs having an acid stabilized attachment site show similar performance to Dpr-MC-VC-PAB-MMAE at DAR=2, and significantly better than Mc-VC-PAB -MMAE, for that matter. At high drug loading DAR=8, the same ADCs showed high efficacy in a subcutaneous BxPc3 tumor graft model in mice.

Claims (36)

1. An antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, as shown in Formula I:

, I Where: the ring corresponds to a backbone, where the backbone contains a C _1-8 alkylidene group or a C _6-10 arylidene group; L is an antibody, antibody fragment, or functional targeting protein; M is a succinimide group or a hydrolyzed succinimide group; Ac is a fragment consisting of at least one amino group and at least one acid group, or an oligopeptide group consisting of many amino acid units; where Ac is connected to the framework through an amino group; D is a cytotoxic drug, a drug for the treatment of an autoimmune disease, or an anti-inflammatory drug; A is a cleavable linker or a non-cleavable linker; m is an integer selected from 1-20; And n is 1 or 2. 2. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein said antibody corresponds to an antibody to a cell surface receptor or a tumor associated antigen. 3. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the acidic element Ac is selected from the group consisting of carboxylic acid, phosphoric acid, phosphorous ester, and sulfonic acid groups. 4. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein Ac comprises an acidic amino acid group or an acidic oligopeptide group. 5. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 4, wherein said oligopeptide group comprises 2-5 natural amino acid elements, 2-5 non-natural amino acid elements, or 2-5 natural and non-natural amino acid elements. 6. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the arylidene group is selected from the group consisting of phenyl, naphthyl, or diphenyl. 7. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the drug element D is selected from the group consisting of maytansine-based drugs, auristatin-based drugs, benzodipyrrole-based drugs, pyrrolozodiazol, amanitin and camptothecin compounds, their pharmaceutically acceptable salts or derivatives thereof. 8. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein A has the structure as shown in the following formula:

, where C denotes an extension element at the end, E denotes a cleavable element, F denotes a spacer element, each of the subscripts e and f is independently 0 or 1, the wavy line on the left indicates the site of attachment to the frame, and the wavy line on the right indicates the site of attachment to the drug element facilities. 9. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 8, wherein E is designed to be cleaved from the drug element D or spacer element F by a tumor-associated protease or at acidic pH. 10. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 8, wherein F is selected from the group consisting of p-aminobenzyl alcohol, ethylenediamine elements, and derivatives thereof. 11. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 6, wherein the backbone comprises a C _1-8 alkylidene. 12. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 11, wherein the backbone comprises a C _1-3 alkylidene. 13. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein Ac is selected from the group consisting of (D/L) glycine, (D/L) alanine, (D/L) leucine, (D/L ) isoleucine, (D/L) valine, (D/L) phenylalanine, (D/L) proline, (D/L) tryptophan, (D/L) serine, (D/L) tyrosine, (D/L) cysteine, (D/L) methionine, (D/L) asparagine, (D/L) glutamine, (D/L) threonine, (D/L) aspartic acid, (D/L) glutamic acid, or compounds of the following structural formulas :

or

, where the wavy line denotes the site of binding to the framework and R denotes any structure between the amino group and the phosphate group. 14. A fragment of a drug compound having the following structure:

, where Ac is a fragment consisting of at least one amino group and at least one acid group, or an oligopeptide group consisting of many amino acid units; where Ac is connected to the hydrocarbyl group through an amino group; D is a cytotoxic drug, a drug for the treatment of an autoimmune disease, or an anti-inflammatory drug; A represents a cleavable linker or a non-cleavable linker; q is an integer in the range 1 to 8, and n is 1 or 2. 15. The use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1 for the preparation of a medicament for the treatment of cancer, immune disease or inflammation.

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