Connexons for antibody drug conjugates and uses thereof

Abstract

The invention relates to a connexon for an antibody drug conjugate and application of the connexon. Specifically, the invention provides a connexon shown in the formula (I), and the antibody drug conjugate prepared from the connexon is more stable and more effective and can be effectively used for treating various diseases such as tumors. The invention also provides an antibody drug conjugate prepared from the connexon, and application of the antibody drug conjugate in treatment of tumors or other diseases. # imgabs0 #

Description

Linkers for antibody-drug conjugates and their applications Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a linker for an antibody-drug conjugate, an antibody-drug conjugate prepared using the linker, and use of the antibody-drug conjugate in treating tumors or other diseases. Background Art

[0002] Antibody-Drug Conjugates (ADCs) are a new class of targeted therapeutics primarily used in the treatment of cancer, autoimmune diseases, and infections. ADCs generally consist of three components: an antibody or antibody-like ligand, a small molecule drug, and a linker that connects the ligand to the drug. The mechanism of action of ADCs is as follows: the antibody or antibody-like ligand specifically recognizes and binds to cell surface antigens; the resulting conjugate enters the cell via endocytosis, simultaneously carrying the small molecule drug into the cell; the antibody is enzymatically hydrolyzed or the linker itself cleaves, releasing the small molecule drug as an appropriate active ingredient, which kills the target cell.

[0003] Linkers are a core part of ADC drug design and are key to achieving targeted drug release. When delivering hydrophobic cytotoxic drugs, hydrophobic linkers can increase conjugate aggregation or reduce the affinity of antibodies, especially at high drug loads. At the same time, drug-resistant tumor cells may limit the activity of ADCs, most of the time due to increased expression or activity of drug transporters, which accelerates the efflux of hydrophobic compounds. Therefore, one of the challenges faced in the design and development of ADCs is the generation of hydrophilic linkers suitable for antibody and drug conjugation. By using hydrophilic linkers, higher drug loads can be achieved, and local dense distribution of drugs (mostly hydrophobic) can be avoided, causing aggregation between hydrophobic groups and thus reducing drug efficacy.

[0004] In view of the above-mentioned defects in the prior art for preparing antibody drug conjugates, it is necessary to develop new linker technologies so that the obtained conjugates are more stable and more effective.

[0005] Summary of the Invention

[0006] The first aspect of the present invention provides a linker, the structure of which is shown in Formula A:

[0007] Wherein, L1 is a functional group capable of reacting with a thiol group; L2 and Y are each independently selected from -(CH2) p -、-(OCH2CH2) p -and-(CH2CH2O) p-; L3 is a hydrophilic group; L4 is an amino acid group; R' is -OR or -NR"-C1-C6 alkylene-NR"R"'; R" and R"' are each independently H and C1-C6 alkyl; R is selected from H and phenyl optionally substituted with one or more nitro groups; R6 is selected from H, C1-C6 alkoxy, -(OCH2CH2) p O-C1-C6 alkyl and -(CH2CH2O) p -C1-C6 alkyl; X is selected from -NH-, -NH(CH2) n C(=O)-, -C(=O)(CH2) n NH- and -(CH2) n C(═O)—; Z is selected from C and S; m is selected from 0 and 1; n is selected from 0, 1 and 2; and p is selected from an integer from 1 to 10.

[0008] In a second aspect, the present application provides a linker-drug conjugate, the structure of which is shown in Formula XIII':

[0009] Wherein, L1, L2, L3, L4, X, Y, Z, R6 and m are as described in the first aspect; L' is a bond, or is -NR"-C1-C6 alkylene-NR"-CO-*, or is -NH-CH2-O-; D is a drug moiety; R" are each independently H and C1-C6 alkyl, and * indicates the position where L' is connected to D.

[0010] The third aspect of the present application provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, the structure of which is shown in Formula XV: Ab-(LD) s (Formula XV)

[0011] Wherein, L is the linker described in the first aspect; D is the drug described in the second aspect; s represents the average number of LD units coupled to Ab, and s ranges from 1 to 8; Ab is the targeting portion.

[0012] In a fourth aspect, the present application provides a linker for connecting a drug to a targeting moiety to form a targeting moiety-drug conjugate, which has the following structure: -L1-L2-X-L3-YZ(=O)-L4-(L5) m -L6-

[0013] Wherein, L1 is selected from -(maleimide-3-yl-N)-, -C(=O)-, -(CH2) n C(=O)-, -S(=O)-, -(CH2) n S(=O)-、-CH2-(CH2) n - and -CH=CH-; L2 and Y are each independently selected from -(CH2) p-、-(OCH2CH2) p -and-(CH2CH2O) p -; L3 is a hydrophilic group; L4 is an amino acid group; L5 is:

[0014] R6 is selected from H, C1-C6 alkoxy, -(OCH2CH2) p O-C1-C6 alkyl and -(CH2CH2O) p -C1-C6 alkyl; L6 is a bond, or is -NR"-C1-C6 alkylene-NR"- or -NR"-C1-C6 alkylene-NR"-CO-; R" are each independently H or C1-C6 alkyl, preferably each independently H, methyl or ethyl, more preferably both are methyl; X is selected from -NH-, -NH(CH2) n C(=O)-, -C(=O)(CH2) n NH- and -(CH2) n C(=O)-; Z is selected from C and S; n is selected from 0, 1 and 2; p is selected from an integer from 1 to 10; m is selected from 0 and 1; wherein, L1 is used to connect to the targeting portion, and the other end of the linker is used to connect to the drug; wherein, when m is 0, the drug is connected to the carbonyl end (C(=O)) of L4; when m is 1, the drug is connected to the carbonyl end (C(=O)) of L5, and L4 is connected to the amino end (NH) of L5.

[0015] The present invention also provides a compound comprising a linker as described in any embodiment of the fourth aspect of the present invention. The compound can be a linker-drug conjugate or an antibody-linker-drug conjugate. In the compound, the linker is a portion of the compound that is covalently linked to the remainder of the compound. In some embodiments, the linker-drug conjugate or antibody-linker-drug conjugate is as described in any embodiment of the present application.

[0016] The present application also provides a pharmaceutical composition, which includes any antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described herein, and a pharmaceutically acceptable carrier or excipient.

[0017] The present application also provides use of any linker described herein in preparing a linker-drug conjugate or an antibody-drug conjugate.

[0018] The present application also provides the use of any antibody-drug conjugate or a pharmaceutically acceptable salt thereof described herein, or any pharmaceutical composition described herein, in the preparation of a medicament for treating and / or preventing cancer, autoimmune diseases, inflammatory diseases or infectious diseases.

[0019] The present application also provides any antibody-drug conjugate described herein or a pharmaceutically acceptable salt thereof, or any pharmaceutical composition described herein, for use in treating and / or preventing cancer, autoimmune diseases, inflammatory diseases, or infectious diseases.

[0020] The present application also provides a method for treating and / or preventing cancer, autoimmune diseases, inflammatory diseases or infectious diseases, comprising administering to a subject in need thereof any antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described herein or any pharmaceutical composition described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1: Antitumor effect of antibody-drug conjugates in a subcutaneous transplant model of human lung squamous cell carcinoma (EBC-1).

[0022] Figure 2: Body weight test results of antibody drug conjugates in a subcutaneous transplant tumor model of human lung squamous cell carcinoma cells (EBC-1).

[0023] Figure 3: Antitumor effect of the antibody drug conjugate in a subcutaneous transplant model of human lung adenocarcinoma cells (Calu-3).

[0024] Figure 4: Body weight test results of antibody drug conjugates in a subcutaneous transplant tumor model of human lung adenocarcinoma cells (Calu-3).

[0025] Figure 5: Comparison of the inhibitory effects of candidate molecules and negative control ADC on BxPC-3 cell proliferation in vitro.

[0026] Figure 6: Comparison of the inhibitory effects of candidate molecules and negative control ADC on BxPC-3 cell proliferation in vitro.

[0027] Figure 7: Comparison of the inhibitory effects of candidate molecules and negative control ADC on BxPC-3 cell proliferation in vitro.

[0028] Figure 8: Comparison of the inhibitory effects of candidate molecules and negative control ADC on MM.1S cell proliferation.

[0029] Figure 9: Comparison of the inhibitory effects of candidate molecules and negative control ADC on MM.1S cell proliferation. DETAILED DESCRIPTION

[0030] The present invention will be described in detail below. Before describing, it should be understood that the terms used in this specification and the appended claims should not be interpreted as limited to the general meaning and dictionary meaning, but should be interpreted according to the meaning and concept corresponding to the technical aspects of the present invention on the basis of the principle that allows the inventor to appropriately define the terms for the best interpretation. Therefore, the descriptions presented here are merely preferred examples for illustrative purposes and are not intended to limit the scope of the present invention. It should be understood that other equivalents or improvements can be obtained therefrom without departing from the spirit and scope of the present invention.

[0031] the term

[0032] In order to make the present invention easier to understand, certain technical terms are specifically defined below. Unless otherwise explicitly stated elsewhere in this document, the technical terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0033] As used herein, "a" or "an" may mean one or more. As used in one or more claims, when used in conjunction with the word "comprising," the word "a" or "an" may mean one or more than one. As used herein, "another" may mean at least a second or more.

[0034] As used herein, "about" when referring to a measurable value (e.g., an amount, a duration, etc.) is intended to encompass variations of +20% or ±10% relative to the particular value, including ±5%, ±1%, and ±0.1%, as such variations are suitable for performing the disclosed methods.

[0035] The term "and / or" should be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0036] As used herein, a "linker" has two reactive ends, one end for binding or associating with a biological moiety or fragment thereof, such as an antibody or fragment thereof, and the other end for coupling with an active agent, such as a cytotoxin.

[0037] As used herein, the term "alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 12 carbon atoms, such as 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl (e.g., 1-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and the like.

[0038] "Alkenyl" is a straight or branched unsaturated hydrocarbon group containing at least one carbon-carbon sp2 double bond (C=C). The number of carbon atoms in an alkenyl group is generally 2-8, for example 2-6 or 2-4. Exemplary alkenyl groups include vinyl and allyl.

[0039] "Alkynyl" is a straight or branched unsaturated hydrocarbon group containing at least one carbon-carbon sp triple bond (C≡C). The number of carbon atoms in an alkynyl group is generally 2-8, for example 2-6 or 2-4. Exemplary alkynyl groups include ethynyl and propargyl.

[0040] "Carbocyclyl" refers to a saturated or partially saturated cyclic hydrocarbon group, including saturated cycloalkyl, partially saturated cycloalkenyl, and cycloalkynyl. The number of carbon atoms in the ring of the carbocyclyl group may be 3-9, such as 3-8 or 3-6. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0041] "Aldehyde" refers to a RC(O)- group, where R can be an alkyl, alkenyl, or alkynyl group as described herein. Preferred aldehyde groups are C1-C6 aldehyde groups, i.e., C1-C5 alkyl-C(O)-.

[0042] "Alkanone" refers to a straight or branched chain group or compound, or a cyclic saturated or unsaturated group or compound containing -CH2COCH2-.

[0043] "Silane" can be represented by Si n H 2n+2 , wherein n is an integer. For example, a silyl group can be represented as -SiH3.

[0044] "Isocyanate" typically refers to -N=C=O.

[0045] "Sulfonyl" refers to a RS(O)2- group, where R can be alkyl, alkenyl, or alkynyl as described herein.

[0046] "Halogen" refers to F, Cl, Br or I.

[0047] "Alkoxy" refers to a RO- group, where R can be an alkyl group as described herein, such as a C1-C6 alkyl group or a C1-C3 alkyl group.

[0048] "Aryl" refers to a monovalent aromatic hydrocarbon radical of 6 to 14 carbon atoms derived by removing a hydrogen atom from a carbon atom of a parent aromatic ring system, such as phenyl, naphthyl, anthracenyl, biphenyl, and the like.

[0049] "Heteroaryl" refers to a group in which one or more carbon atoms in the "aryl" backbone are replaced by one or more heteroatoms selected from N, O, P, and S. Heteroaryl groups typically contain 5-14, preferably 5-10, ring atoms, and have 6, 10, or 14 electrons shared within the ring system. Exemplary heteroaryl groups include, but are not limited to, pyridine, thiophene, furan, pyrrolyl, pyrazolyl, thiazolyl, and the like.

[0050] "Heterocyclyl" refers to a group in which one or more carbon atoms in a parent aromatic or non-aromatic ring system are replaced by one or more heteroatoms selected from N, O, P, and S. Heterocyclyl groups can include saturated or partially saturated 3-7 membered monocyclic groups, 7-10 membered bicyclic groups, spirocyclic groups, or bridged ring groups. Exemplary heterocyclyl groups include tetrahydrofuranyl, pyranyl, piperidinyl, piperazinyl, 1,4-diazepanyl, azetidinyl, oxetanyl, pyrrolidinyl, imidazolidinyl, imidazolinyl, indolinyl, isoindolinyl, morpholinyl, and the like.

[0051] "Aralkyl" refers to an aliphatic alkyl group in which one hydrogen atom attached to a terminal or sp3 carbon atom is replaced by an aryl group, such as benzyl, 3-phenylpropyl, etc. The alkyl portion of the aralkyl group may also include an alkenyl or alkynyl group.

[0052] "Heteroaralkyl" refers to an aliphatic alkyl group in which one hydrogen atom attached to a terminal or sp3 carbon atom is replaced by a heteroaryl group, such as 2-pyridylethyl, 3-furylpropyl, etc. The alkyl portion of the heteroaralkyl group may also include an alkenyl or alkynyl group.

[0053] "Alkylene" is a saturated hydrocarbon group containing a straight chain, branched chain or carbon ring, which is formed by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkane to produce two monovalent radical centers, such as methylene (-CH2-), 1,2-ethylene (-CH2CH2-), 1,3-propylene (-CH2CH2CH2-), etc.

[0054] "Alkenylene" is an unsaturated hydrocarbon radical containing a straight chain, branched chain, or carbon ring, which is formed by removing two hydrogen atoms from the same or two different carbon atoms of the parent olefin to produce two monovalent radical centers, such as 1,2-vinylene (-CH=CH-), 1,3-propenylene (-CH2CH=CH-), etc.

[0055] "Alkynylidene" is an unsaturated hydrocarbon group containing a straight chain, branched chain, or carbon ring, which is formed by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkyne to produce two monovalent radical centers, such as ethynylene (-CH≡CH-), 1,3-propynylene (-CH2C≡CH-), etc.

[0056] "Arylene" refers to an aromatic hydrocarbon radical of 6 to 14 carbon atoms, formed by removing two hydrogen atoms from two different carbon atoms of a parent aromatic ring system to produce two monovalent radical centers, such as 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, and the like.

[0057] Whenever a group is described herein as "substituted," the group is substituted with one or more (eg, 1, 2, 3, 4, or 5 or more) of the specified substituents. If no substituents are specified, it is meant that the specified "substituted" group may be substituted by one or more (e.g., 1, 2, 3, 4, or 5 or more) groups individually and independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, aryl, heteroaryl, heteroalicyclic, aralkyl, heteroaralkyl, (heteroalicyclic)alkyl, hydroxy, alkoxy, aryloxy, acyl, thiol, alkylthio, arylthio, cyano, halogen, thiocarbonyl, carbamoyl, thiocarbamoyl, amido, sulfonylamino, sulfonylamino, carboxyl, isocyanate, thiocyanate, isothiocyanate, nitro, silyl, sulfinyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethylsulfonyl, trihalomethylsulfonylamino, amino, mono-substituted amino, and di-substituted amino groups, and protected derivatives thereof.

[0058] Where the number of substituents is not specified (e.g., haloalkyl), one or more substituents may be present. For example, "haloalkyl" may contain one or more of the same or different halogens. As another example, "C1-C3 alkoxyphenyl" may contain one or more of the same or different alkoxy groups containing one, two, or three atoms.

[0059] In some embodiments, "substituted alkyl," "substituted alkenyl," "substituted alkynyl," "substituted aryl," "substituted heteroaryl," "substituted heterocyclyl," "substituted aralkyl," and "substituted heteroaralkyl" refer to the corresponding "alkyl," "alkenyl," "alkynyl," "aryl," "heteroaryl," "heterocyclyl," "aralkyl," and "heteroaralkyl," respectively, in which one or more hydrogen atoms are independently replaced by a substituent. Substituents generally include, but are not limited to, -X, -OR, -NR2, -NO2, -CN, -S03R, -C02R, and the like, where X is a halogen atom and R is H, an alkyl, an aryl, a heterocyclyl, a protecting group, or a prodrug moiety. The aforementioned alkylene, alkenylene, and alkynylene groups may also be similarly substituted.

[0060] "Any combination" refers to a new substituent formed by combining two or more substituent groups in a certain connection manner, such as benzyl (phenyl + methylene), 3-phenylpropyl (phenyl + 1,3-propylene), 2-cyclohexylpropyl (cyclohexyl + 1,2-propylene), 3-(3-pyridyl)propyl (3-pyridyl + 1,3-propylene), etc.

[0061] When a group is defined as a "bond", it usually means that the group is a monovalent covalent bond. For example, when L6 is a bond, the linker -L1-L2-X-L3-YZ(=O)-L4-(L5) mThe structural formula of -L6- is actually -L1-L2-X-L3-YZ(=O)-L4-(L5) m -.

[0062] The term "Drug / Antibody Ratio (DAR)" as used herein refers to the number of drug-conjugated molecules per antibody molecule. Because antibody-drug conjugate samples contain components with varying DAR values, the concepts of "average DAR" and "DAR distribution" are more appropriate for describing the composition of antibody-drug conjugates. The average DAR is the ratio of the total number of drug molecules to the total number of antibodies in a sample, while the DAR distribution refers to the distribution of the concentrations of components with varying DAR values ​​in a sample.

[0063] The term "drug" as used herein generally refers to any compound having a desired biological activity and a reactive functional group for preparing the conjugates of the present invention. The desired biological activity includes the diagnosis, cure, alleviation, treatment, or prevention of human or other animal diseases. As new drugs are continuously discovered and developed, these new drugs should also be included in the drugs of the present invention. Specifically, the drugs include, but are not limited to, cytotoxic drugs, cell differentiation factors, stem cell trophic factors, steroid drugs, drugs for treating autoimmune diseases, anti-inflammatory drugs, or drugs for infectious diseases. More specifically, the drugs include, but are not limited to, microtubule inhibitors or DNA or RNA damaging agents.

[0064] The term "antibody" in the present invention is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments. In particular, "antibody" as used herein refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into multiple regions with high variability, known as complementarity determining regions (CDRs), interspersed with multiple regions that are more conserved, known as framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of heavy and light chains comprise a binding domain that interacts with an antigen. The constant region of an antibody can mediate the binding of an immunoglobulin to a tissue or factor of the host, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system. Chimeric or humanized antibodies are also encompassed in the antibodies according to the present invention.

[0065] The term "humanized antibody" refers to an antibody that comprises a CDR region derived from a non-human antibody and the rest of the antibody molecule is derived from one (or several) human antibodies. In addition, some residues in the framework (called FR) segment may be modified to retain binding affinity (Jones et al., Nature, 321: 522-525, 1986; Verhoeyen et al., Science, 239: 1534-1536, 1988; Riechmann et al., Nature, 332: 323-327, 1988). The humanized antibodies or fragments thereof according to the present invention can be prepared by techniques known to those skilled in the art (for example, as described in the documents Singer et al., J. Immun. 150: 2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10: 1-142, 1992; or Bebbington et al., Bio / Technology, 10: 169-175, 1992).

[0066] The term "chimeric antibody" refers to an antibody in which the variable region sequence is from one species and the constant region sequence is from another species, for example, an antibody in which the variable region sequence is from a mouse antibody and the constant region sequence is from a human antibody. Chimeric antibodies or fragments thereof according to the present invention can be prepared by using genetic recombination technology. For example, the chimeric antibody can be produced by cloning recombinant DNA, which comprises a promoter and a sequence encoding the variable region of a non-human, especially mouse, monoclonal antibody according to the present invention, and a sequence encoding the constant region of a human antibody. The chimeric antibody of the present invention encoded by such a recombinant gene will be, for example, a mouse-human chimera, the specificity of the antibody being determined by the variable region derived from the mouse DNA, and its isotype being determined by the constant region derived from the human DNA. For methods of preparing chimeric antibodies, for example, reference can be made to the document Verhoeyn et al. (BioEssays, 8:74, 1988).

[0067] The term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0068] The term "functional fragment" in the present invention refers to an antibody fragment consisting of or comprising a partial sequence of the heavy or light variable chain of the antibody from which it is derived, wherein the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least 1 / 100, and more preferably at least 1 / 10, of the affinity of the antibody from which it is derived. Such a functional fragment will contain a minimum of 5 amino acids, preferably 10, 15, 25, 50, or 100 consecutive amino acids of the antibody sequence from which it is derived.

[0069] The antibodies constituting the antibody drug conjugates of the present invention preferably retain their antigen binding ability in their original wild state. Therefore, the antibodies of the present invention are able to bind to antigens, preferably specifically. The antigens involved include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulatory factors, cell proliferation regulatory factors, molecules related to tissue growth and differentiation (such as those known or predicted to be functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (such as those known or predicted to be functional). Tumor-related factors can be cluster differentiation factors (such as CD proteins). The antigens bound to the antibodies of the present invention can be one or a subset of the above classifications, while other subsets contain other molecules / antigens with special properties (compared to the target antigen).

[0070] Antibodies used in antibody-drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the industry and can be prepared using methods and information for antibody preparation that are well known in the industry. In order to develop effective cellular targets that can be used for cancer diagnosis and treatment, researchers strive to find transmembrane or other tumor-associated polypeptides. These targets can be specifically expressed on the surface of one or more cancer cells, while being rarely or not expressed on the surface of one or more non-cancerous cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells than on the surface of non-cancerous cells. Identifying such tumor-associated factors can greatly improve the specific targeting properties of antibody-based cancer treatment.

[0071] Tumor-associated antigens include, but are not limited to, the tumor-associated antigens listed below (1)-(36). For convenience, information related to antigens well known in the art is indicated below, including name, other names, and gene bank accession number. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases such as Genbank. Antibodies targeting tumor-associated antigens include all amino acid sequence variants and isotypes that have at least 70%, 80%, 85%, 90%, or 95% homology with the sequences confirmed in the references, or have biological properties and characteristics that are completely consistent with the tumor-associated antigen sequences in the references.

[0072] Tumor-associated antigens (1)-(36) are:

[0073] (1) BMPR1B (bone morphogenetic protein receptor type IB, Genbank accession number NM_001203);

[0074] (2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486);

[0075] (3) STEAP1 (six-transmembrane prostate epithelial antigen, Genbank accession number NM_012449);

[0076] (4) 0772P (CA125, MUC16, Genbank accession number AF361486);

[0077] (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession number NM_005823);

[0078] (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate) member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM_006424);

[0079] (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5bHlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession number AB040878);

[0080] (8) PSCAhlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628);

[0081] (9) ETBR (endothelin type B receptor, Genbank accession number AY275463);

[0082] (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM_017763);

[0083] (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six-transmembrane prostate epithelial antigen 2, six-transmembrane prostate protein, Genbank accession number AF455138);

[0084] (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM_017636);

[0085] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor, Genbank accession number NP_003203 or NM_003212);

[0086] (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792, Genbank accession number M26004);

[0087] (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-related β), B29, Genbank accession number NM_000626);

[0088] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchoring protein 1a), SPAP1B, SPAP1C, Genbank accession number NM_030764);

[0089] (17) HER2 (ErbB2, Genbank accession number M11730);

[0090] (18) NCA (CEACAM6, Genbank accession number M18728);

[0091] (19) MDP (DPEP1, Genbank accession number BC017023);

[0092] (20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession number AF184971);

[0093] (21) Brevican (BCAN, BEHAB, Genbank accession number AF229053);

[0094] (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession number NM_004442);

[0095] (23) ASLG659 (B7h, Genbank accession number AX092328);

[0096] (24) PSCA (prostate stem cell antigen precursor, Genbank accession number AJ297436);

[0097] (25) GEDA (Genbank accession number AY260763);

[0098] (26) BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR3, Genbank accession number AF116456);

[0099] (27) CD22 (B cell receptor CD22-B isoform, Genbank accession number AK026467);

[0100] (28) CD79a (CD79A, CD79α, immunoglobulin-associated α, can covalently interact with Igβ (CD79B) and form a complex with Ig M molecules on the surface, transducing B cell-specific proteins involved in B cell differentiation signals, Genbank accession number NP_001774.1);

[0101] (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine, plays a role in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and possibly in AIDS, lymphoma, myeloma, and leukemia, Genbank accession number NP_001701.1);

[0102] (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen), which binds peptides and presents them to CD4 + T lymphocytes, Genbank accession number NP_002111.1);

[0103] (31) P2X5 (purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, its defect may lead to the pathophysiological condition of idiopathic detrusor instability, Genbank accession number NP_002552.2);

[0104] (32) CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP_001773.1);

[0105] (33) LY64 (lymphocyte antigen 64 (RP105), a member of the leucine-rich repeat-rich type I membrane protein (LRR) family, regulates B cell activation and apoptosis, and loss of function is associated with increased disease activity in patients with systemic lupus erythematosus, Genbank accession number NP_005573.1);

[0106] (34) FcRH1 (Fc receptor-like protein 1, a putative immunoglobulin Fc domain receptor containing C2-type Ig-like and ITAM domains, which may play a role in B lymphocyte differentiation, Genbank accession number NP_443170.1);

[0107] (35) IRTA2 (translocation-associated immunoglobulin superfamily receptor 2, a putative immune receptor that may play a role in B cell development and lymphomagenesis; gene dysregulation caused by translocation occurs in some B cell malignancies, Genbank accession number NP_112571.1);

[0108] (36) TENB2 (putative transmembrane proteoglycan related to the EGF / heregulin family of growth factors and follistatin, Genbank accession number AF179274).

[0109] As used herein, "drug" or the code "D" refers to any compound having a desired biological activity and a reactive functional group for preparing the conjugates of the present invention. The desired biological activity includes diagnosing, curing, alleviating, treating, and preventing diseases in humans or other animals. Therefore, as long as the necessary reactive functional groups are present, the compounds referred to by the term "drug" include drugs recognized in official national pharmacopeias, as well as, for example, the official U.S. Homeopathic Pharmacopoeia, the official National Formulary, or any supplements thereto. Typical drugs are listed in the Physician's Desk Reference (PDR) and the Orange Book of the U.S. Food and Drug Administration (FDA). As new drugs are continuously discovered and developed, this application stipulates that these drugs should also be included in the prodrugs of the conjugate drugs of the present invention.

[0110] As used herein, the term "prevent" or "prevent" refers to reducing the frequency of symptoms of a medical condition or delaying the onset of a compound or drug (e.g., a combination product claimed herein) in a subject when used for a disease or condition (e.g., cancer) as compared to a subject to which the compound or drug (e.g., a combination product claimed herein) is not administered.

[0111] As used herein, the term "treat," ...

[0112] As used herein, the term "effective amount" or "prophylactically and / or therapeutically effective amount" refers to a sufficient amount (e.g., dosage) of a drug or compound administered that will alleviate to some extent one or more symptoms of the disease or condition being treated. The result can be a reduction and / or alleviation of symptoms or the cause of the disease or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is an amount of a compound or drug (e.g., a combination product claimed herein) that provides a significant alleviation of the clinical symptoms of a disease or condition without producing excessive toxic side effects.

[0113] "Administering," "giving," and "administering" refer to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those skilled in the art.

[0114] The terms "subject," "individual," and "object" include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. The terms "subject" and "patient" are used interchangeably herein.

[0115] The term "pharmaceutically acceptable carrier or excipient" refers to a carrier or excipient that can be administered to a subject together with the compound of one aspect of the present invention and does not destroy its pharmacological activity and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the compound. Exemplary pharmaceutically acceptable carriers or excipients include inert diluents such as water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, EtOAc, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (including cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof; adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings and fragrances, etc. The terms "administration mode" and "dosage regimen" are used interchangeably and refer to the dosage and time of use of each therapeutic agent in the combination of the present invention.

[0116] "Cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included within this definition are both benign and malignant cancers, as well as dormant tumors or micrometastases. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, as well as B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, storage diseases (bulky disease) NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs syndrome.

[0117] Linker

[0118] In a first aspect, the present application provides a linker, the structure of which is shown in Formula A:

[0119] Wherein: L1 is a functional group capable of reacting with a thiol group; L2 and Y are each independently selected from -(CH2) p -、-(OCH2CH2) p -and-(CH2CH2O) p -; L3 is a hydrophilic group; L4 is an amino acid group; R' is -OR or -NR"-C1-C6 alkylene-NR"R"'; R" and R"' are each independently H and C1-C6 alkyl; R is selected from H and phenyl optionally substituted with one or more nitro groups; R6 is selected from H, C1-C6 alkoxy, -(OCH2CH2) p O-C1-C6 alkyl and -(CH2CH2O) p -C1-C6 alkyl; X is selected from -NH-, -NH(CH2) n C(=O)-, -C(=O)(CH2)n NH- and -(CH2) n C(═O)—; Z is selected from C and S; m is selected from 0 and 1; n is selected from 0, 1 and 2; and p is selected from an integer from 1 to 10.

[0120] In some embodiments, the present invention provides a linker having a structure as shown in Formula I:

[0121] Wherein: L1 is a functional group capable of reacting with a thiol group; L2 and Y are each independently selected from -(CH2) p -、-(OCH2CH2) p -and-(CH2CH2O) p -; L3 is a hydrophilic group; L4 is an amino acid group; R is selected from H and phenyl optionally substituted with one or more nitro groups; R6 is selected from H, C1-C6 alkoxy, -(OCH2CH2) p O-C1-C6 alkyl and -(CH2CH2O) p -C1-C6 alkyl; X is selected from -NH-, -NH(CH2) n C(=O)-, -C(=O)(CH2) n NH- and -(CH2) n C(=O)-; Z is selected from C and S; m is selected from 0 and 1; n is selected from 0, 1 and 2; p is selected from an integer from 1 to 10, such as 1-8 or 1-6.

[0122] In one or more embodiments, L1 is at least one group selected from maleimide, halogen, halogen-substituted functional groups (such as halogen-substituted aldehyde groups, halogen-substituted -S(O)2-), aldehyde groups, alkenyl groups, alkynyl groups, alkanone groups, sulfonyl groups, silane groups, isocyanate groups and norbornene groups.

[0123] In some embodiments, the norbornene group has the following structure:

[0124] wherein R7, R8 and R9 are each independently selected from H, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, heteroaryl, C3-C9 cycloalkyl, C3-C9 heterocyclyl, -(OCH2CH2) p OCH3, -NO2, -CN, -SCN, -OR 10 、-SR 10 、-N(R 10 )2, -C(=O)R 10 、-C(=O)OR 10 、-C(=O)N(R 10 )2、-C(=S)OR 10 、-C(=S)N(R10 )2、-C(=S)SR 10 、-NR 1010 (C=O)R 10 、-NR 10 (C=S)N(R 10 )2、-O(C=O)N(R 10 )2, -S(=O)2Ri0, -S(=O)2OR 10 ; where each R 10 Independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl.

[0125] In one or more embodiments, L1 is selected from:

[0126] and their derivatives; wherein: R7, R8 and R9 are each independently selected from H, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, heteroaryl, C3-C9 cycloalkyl, C3-C9 heterocyclyl, -(OCH2CH2) p OCH3, -NO2, -CN, -SCN, -OR 10 、-SR 10 、-N(R 10 )2, -C(=O)R 10 、-C(=O)OR 10 、-C(=O)N(R 10 )2、-C(=S)OR 10 、-C(=S)N(R 10 )2、-C(=S)SR 10 、-NR 10 (C=O)R 10 、-NR 10 (C=S)N(R 10 )2、-O(C=O)N(R 10 )2、-S(=O)2R 10 、-S(=O)2OR 10 ; where each R 10 Independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl; p is an integer of 1-6; A is halogen; the wavy line indicates the position where L1 and L2 are connected.

[0127] In one or more embodiments, L1 is:

[0128] In one or more embodiments, L2 is -(CH2) p -. In one or more embodiments, L2 is -(CH2)p -, and p is an integer from 1 to 4. In one or more embodiments, L2 is -CH2CH2- or -CH2CH2CH2-.

[0129] In one or more embodiments, X is -NH- or -NH(CH2) n C(=O)-. In some embodiments, X is -NH- or -NHC(=O)-.

[0130] In one or more embodiments, L3 is selected from monosaccharides, disaccharides, and five- or six-membered saturated heterocycles containing 1-2 nitrogen atoms, and derivatives thereof, which are divalent hydrophilic groups with two monovalent radical centers generated by removing two hydrogen atoms.

[0131] In one or more embodiments, the monosaccharide is selected from at least one of trisaccharide, tetrasaccharide, pentose, hexose and heptose, the disaccharide is selected from at least one of maltose, sucrose and lactose, and the five-membered or six-membered saturated heterocycle containing 1-2 nitrogen atoms can be piperazinyl, piperidinyl and pyrrolidinyl. The derivative can be a monosaccharide or disaccharide in which the H of one or more hydroxyl groups is replaced by, for example, a C1-C6 alkyl, -(CH2CH2O) t CH3, sulfonic acid and phosphoric acid groups, wherein t is selected from an integer from 1 to 20, or one or more ring carbon atoms of the five-membered or six-membered saturated heterocyclic ring containing 1-2 nitrogen atoms are replaced by one or more ring carbon atoms selected from -OR 20 and -CH2R 20 The group substituted, the R 20 Can be C1-C6 alkyl, -(CH2CH2O) t CH3, sulfonic acid group or phosphoric acid group, wherein t is selected from an integer from 1 to 20.

[0132] In one or more embodiments, L3 is a divalent hydrophilic group having two monovalent radical centers generated by removing two hydrogen atoms from glucose, galactose, mannose, glucuronic acid, galactonic acid, mannuronic acid, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine and N-acetylmuramic acid.

[0133] In one or more embodiments, L3 is selected from:

[0134] Wherein, R1 is selected from -O(CH2) p - and -C(=O)-, p is an integer from 1 to 6, preferably 1, 2, 3 or 4; R5 is a bond or a C1-C3 alkylene group which is unsubstituted or substituted by one or more substituents selected from the group consisting of: -OR 20 and -C(=O)OR 20 -; R2, R3, R4, R11 、R 12 、R 13 Independently selected from H, C1-C6 alkyl, -(CH2CH2O) t -C1-C6 alkyl, sulfonic acid and phosphoric acid groups; R 14 、R 15 Independently selected from H, -OR 20 , and -CH2R 20 ; R 16 、R 17 、R 18 、R 19 Independently selected from H and -OR 20 ; R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t -C1-C6 alkyl, sulfonic acid and phosphoric acid; t is an integer from 1 to 20; the wavy line indicates the position where L3 is connected to X and Y.

[0135] In one or more embodiments, R1 is selected from -OCH2- and -C(=O)-.

[0136] In one or more embodiments, R5 is unsubstituted C1-C3 alkylene, or is substituted by one or more selected from -OR 20 and -C(=O)OR 20 -substituted C1-C3 alkylene, wherein R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t CH3, sulfonic acid group and phosphoric acid group, wherein t is selected from an integer from 1 to 10.

[0137] In one or more embodiments, R 14 、R 15 Independently selected from H, -OR 20 , and -CH2R 20 , where R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t CH3, sulfonic acid group and phosphoric acid group, wherein t is selected from an integer from 1 to 10.

[0138] In one or more embodiments, R 16 、R 17 、R 18 、R 19 Independently selected from H and -OR 20 , where R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t CH3, sulfonic acid group and phosphoric acid group, wherein t is selected from an integer from 1 to 10.

[0139] In one or more embodiments, R2, R3, R4, R 11 、R 12 、R 13 Independently selected from H, C1-C6 alkyl, -(CH2CH2O) t CH3, sulfonic acid group and phosphoric acid group, wherein t is selected from an integer of 1 to 20, such as 1 to 10.

[0140] In one or more embodiments, L3 is:

[0141] Wherein, R1 is selected from -O(CH2) p - and -C(=O)-, p is 1, 2 or 3; R2, R3 and R4 are all H.

[0142] In one or more embodiments, Y is -(CH2) p -, p is 1, 2 or 3; preferably, Y is a methylene group.

[0143] In one or more embodiments, Z is C.

[0144] In one or more embodiments, L4 is selected from the following amino acid residues: valine, citrulline, alanine, glycine, phenylalanine, asparagine, glutamic acid, lysine, serine, threonine, cysteine ​​and tyrosine; said amino acid group is optionally substituted with one or more substituents selected from the group consisting of: -OR 20 , where R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t -C1-C6 alkyl, sulfonic acid and phosphate groups, t is an integer from 1 to 20. Preferably, the amino acid residue is linked to the rest of the compound via a peptide bond (-NH-CO-). Preferably, R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t CH3, sulfonic acid group and phosphoric acid group, wherein t is selected from an integer from 1 to 10.

[0145] In one or more embodiments, L4 is selected from the following peptide segments: valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), glycine-glycine-phenylalanine-glycine-glycine, glycine-glycine-phenylalanine-glycine-glycine, citrulline-valine, alanine-valine, alanine-alanine, citrulline-alanine, asparagine-citrulline, citrulline-asparagine, citrulline-citrulline, phenylalanine-lysine and lysine-phenylalanine, preferably selected from the following peptide segments: valine-citrulline (VC), valine-alanine (VA) and glycine-glycine-phenylalanine-glycine (GGFG); each peptide segment is optionally substituted by one or more substituents selected from the following group: -OR 20 and -(OCH2CH2) p O-C1-C6 alkyl; wherein R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t -C1-C6 alkyl, sulfonic acid and phosphate groups, t is an integer from 1 to 20, and p is an integer from 1 to 6. It should be understood that the peptide segment is a divalent group, and the two amino acid residues at the head and tail are connected to the other parts of the compound through the -NH- part or -CO- part in the peptide bond. Preferably, R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t CH3, sulfonic acid group and phosphoric acid group, wherein t is selected from an integer from 1 to 10. Preferably, the -(OCH2CH2) p O-C1-C6 alkyl is -(OCH2CH2) p O-C1-C3 alkyl, more preferably -(OCH2CH2) p OCH3, preferably, p is 1, 2 or 3.

[0146] In one or more embodiments, L4 is selected from:

[0147] In one or more embodiments, m is 0.

[0148] In one or more embodiments, m is 1.

[0149] In one or more embodiments, R6 is H, C1-C3 alkoxy or -(OCH2CH2) p O-C1-C3 alkyl, wherein p is an integer from 1 to 6. In one or more embodiments, R6 is H, methoxy or -(OCH2CH2) p OCH3, wherein p is an integer of 1-6 or 1-4.

[0150] In one or more embodiments, R is selected from H and phenyl optionally substituted with 1-3 nitro groups, preferably H or 4-nitrophenyl.

[0151] In one or more embodiments, the structure of the linker is shown in any one of Formula II to Formula XIV below:

[0152] In each type, R”, R”’, R, L2, X, L3, R2-R4, R 11 -R 13 and R 15 As described in any of the above embodiments.

[0153] In one or more embodiments, the present application provides linkers represented by the following formulae 1 to 46:

[0154] Linker-drug conjugates

[0155] In a second aspect, the present application provides a linker-drug conjugate, the structural formula of which is shown in Formula XIII':

[0156] In the formula, L1, L2, L3, L4, X, Y, Z, R6 and m are as described in the first aspect; L' is a bond, or is -NR"'-C1-C6 alkylene-NR"'-CO-*, or is -NH-CH2-O-; D is a drug moiety; wherein R" are each independently H and C1-C6 alkyl, and * indicates the position where L' is connected to D.

[0157] In some embodiments, the structure of the linker-drug conjugate is shown in Formula XIII:

[0158] Wherein, L1, L2, L3, L4, X, Y, Z, R6 and m are as described in the first aspect; and D is a drug moiety.

[0159] Herein, the drugs include but are not limited to cytotoxic drugs, cell differentiation factors, stem cell trophic factors, steroid drugs, drugs for treating autoimmune diseases, anti-inflammatory drugs and drugs for infectious diseases. Further, the drug part D includes but is not limited to microtubule inhibitors or DNA damaging agents. The microtubule inhibitors include but are not limited to dolastatin and auristatin, maytansine; the DNA damaging agents include but are not limited to calicheamicin, duocarmycin, anthramycin derivatives PBD (pyrrolobenzodiazepine, pyrrolobenzodiazepine), camptothecin derivatives SN-38, topoisomerase I inhibitors. The auristatin drugs include but are not limited to monomethyl auristatin E (monomethyl auristatinE; MMAE), monomethyl auristatin F (monomethyl auristatin F; MMAF), and auristatin F (auristatin F; AF) or their derivatives, the maytansine drugs include but are not limited to DM1, DM3, DM4 or their derivatives ("Progress in Research on Warhead Molecules of Antibody Drug Conjugates", Hu Xinyue et al., "China Pharmaceutical Biotechnology", December 2017, Vol. 12, No. 6; "Progress in Research on Maytansine Antibody Drug Conjugates", Zhou Lei et al., "China Journal of New Drugs", Vol. 25, No. 22, 2016, pp. 2521-2530), the topoisomerase I inhibitors include but are not limited to exitecan, topotecan, irinotecan, 9-nitrocamptothecin and the macromolecular camptothecin conjugate PNU-166148 (compound A1 in WO99 / 17804).The drug moiety D can also be amanitins, anthracyclines, baccatins, camptothecins, cemadotins, colchicines, colcimids, combretastatins, cryptophycins, discodermolides, docetaxel, doxorubicin, echinocin The drug moiety D can also be a vitamin A precursor, folic acid, or the like. The drug moiety D is not limited to the above categories, but also includes all drugs that can be used for ADC.

[0160] It should be understood that the position at which D is linked to the rest of the linker-drug conjugate generally does not affect the biological activity of the drug D itself. Those skilled in the art can readily determine the active site of drug D based on existing techniques and covalently link the rest of the linker-drug conjugate to the position of the active site. Covalent linkage methods are well known in the art and include acylamino groups (-NRCO-, where R is H or C1-C4 alkyl), ester bonds (-COO-), and -S-.

[0161] In one or more embodiments, the structure of the drug moiety (D) is shown in any one of the following formulas D-1 to D-9:

[0162] Wherein, the wavy line indicates the position where D is attached to the rest of the linker-drug conjugate.

[0163] In one or more embodiments, the present application provides linker-drug conjugates numbered herein as CPD1, CPD2, CPD3, CPD4, CPD5, CPD6, CPT004, CPT005, CPT006, CPT008, CPT009, CPT010, MMAE Derivate (MMAE derivative 1), MMAE Derivate (o-OMe-PAB) (MMAE derivative 2), Comp.5, Comp6, Comp7, Comp8, and Comp9.

[0164] Antibody Drug Conjugates

[0165] In a third aspect, the present application provides an antibody-drug conjugate, the structure of which is shown in Formula XV: Ab-(LD) s (Formula XV)

[0166] wherein L is the linker described in any embodiment of the first aspect; D is the drug described in any embodiment of the second aspect; s represents the average number of LD units coupled to Ab, and s ranges from 1 to 8; Ab is a targeting moiety. In some embodiments, s is 2-6.

[0167] In some embodiments, the structure of the antibody drug conjugate of the present invention is shown in the following formula:

[0168] wherein L2, X, L3, Y, Z, L4, R6, L', and m are as described in any embodiment herein; s represents the average number of LD units conjugated to Ab, and s ranges from 1 to 8; Ab is a targeting moiety; and S represents a sulfur atom in a cysteine ​​residue in the antibody. In some embodiments, s is 2-6.

[0169] In one or more embodiments, Ab is any antibody or functional fragment of an antibody. Furthermore, antibodies include derivatives or analogs of antibodies; a "functional" fragment means that the fragment, derivative, or analog can recognize the same antigen, recognizes an antibody derived from a fragment, derivative, or analog of the antigen, such as, but not limited to, F(ab')2, Fab, Fab', Fv fragments, and antibody heavy and light chain dimers, or any minimal fragment thereof such as Fvs or single-chain antibody fragments / single-chain variable fragments (scFv). Furthermore, the antibody may be an antibody fusion protein. Antibodies may also include analogs and derivatives that are modified or unmodified (i.e., covalently linked to any molecule), as long as such covalent linkage allows the antibody to retain its antigen-binding immunospecificity. Examples include, but are not limited to, analogs and derivatives of antibodies, including those that have been further modified, such as glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protease cleavage, attachment to cellular antibody units or other proteins, and the like. Any of a number of chemical modifications can be achieved using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, and the like. In addition, analogs or derivatives may include one or more unnatural amino acids. In some embodiments, the antibody may have modifications (e.g., substitutions, deletions, or additions) in amino acid residues that interact with Fc receptors; further, the antibody is selected from a murine antibody, a mammalian antibody, a chimeric antibody, a humanized antibody, a human antibody, and a multispecific (e.g., bispecific) antibody. Furthermore, the antibody or functional fragment of the antibody specifically binds to a cell surface receptor or tumor-associated antigen.

[0170] In some embodiments, the antibody is an anti-TROP2 antibody, such as hRS7 or Datopotamab. Exemplary anti-TROP2 antibodies have a light chain sequence as shown in SEQ ID NO: 1 and a heavy chain as shown in SEQ ID NO: 2, or have a light chain sequence as shown in SEQ ID NO: 3 and a heavy chain as shown in SEQ ID NO: 4. In some embodiments, the antibody is anti-KLH hIgG1.

[0171] In some embodiments, the antibody is an anti-BCMA antibody. Preferably, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO: 6.

[0172] In one or more embodiments, the LD is a linker-drug conjugate as described in any embodiment herein, in particular, the linker-drug conjugates numbered herein as CPD1, CPD2, CPD3, CPD4, CPD5, CPD6, CPT004, CPT005, CPT006, CPT008, CPT009, CPT010, MMAE Derivate (MMAE derivative 1), MMAE Derivate (o-OMe-PAB) (MMAE derivative 2), Comp.5, Comp6, Comp7, Comp8 and Comp9.

[0173] In one or more embodiments, the structure of the antibody drug conjugate (ADC) is:

[0174] Wherein, s is 1-8.

[0175] connector

[0176] In a fourth aspect, the present application provides a linker for connecting a drug to a targeting moiety to form a targeting moiety-drug conjugate: -L1-L2-X-L3-YZ(=O)-L4-(L5) m -L6-

[0177] Wherein, L1 is selected from -(maleimide-3-yl-N)-, -C(=O)-, -(CH2) n C(=O)-, -S(=O)-, -(CH2) n S(=O)-、-CH2-(CH2) n - and -CH=CH-; L2 and Y are each independently selected from -(CH2) p -、-(OCH2CH2) p -and-(CH2CH2O) p -; L3 is a hydrophilic group; L4 is an amino acid group; L5 is:

[0178] R6 is selected from H, C1-C6 alkoxy, -(OCH2CH2) p O-C1-C6 alkyl and -(CH2CH2O) p -C1-C6 alkyl; L6 is a bond, or is -NR"-C1-C6 alkylene-NR"- or -NR"-C1-C6 alkylene-NR"-CO-; R" are each independently H or C1-C6 alkyl, preferably each independently H, methyl or ethyl, more preferably both are methyl; X is selected from -NH-, -NH(CH2) nC(=O)-, -C(=O)(CH2) n NH- and -(CH2) n C(=O)-; Z is selected from C and S; n is selected from 0, 1 and 2; p is selected from an integer from 1 to 10; m is selected from 0 and 1; wherein, L1 is used to connect to the targeting portion, and the other end of the linker is used to connect to the drug; wherein, when m is 0, the drug is connected to the carbonyl end (C(=O)) of L4; when m is 1, the drug is connected to the carbonyl end (C(=O)) of L5, and L4 is connected to the amino end (NH) of L5.

[0179] In one or more embodiments, L1 is -(maleimide-3-yl-N)-, wherein -(maleimide-3-yl-N)- is a structure shown in the following formula, which is connected to the targeting moiety through its 3-position,

[0180] In one or more embodiments, L2 is -(CH2) p -, and p is an integer of 1-4; preferably, L2 is -CH2CH2- or -CH2CH2CH2-.

[0181] In one or more embodiments, X is -NH- or -NH(CH2) n C(=O)-, preferably, X is -NH- or -NHC(=O)-.

[0182] In one or more embodiments, Y is -(CH2) p -, p is 1, 2 or 3; preferably, Y is a methylene group.

[0183] In one or more embodiments, Z is C.

[0184] In one or more embodiments, L3 is selected from monosaccharides, disaccharides and five-membered or six-membered saturated heterocycles containing 1-2 nitrogen atoms and their derivatives, which are divalent hydrophilic groups with two monovalent radical centers produced by removing two hydrogen atoms; preferably, the monosaccharide is selected from at least one of trisaccharides, tetrasaccharides, pentoses, hexoses and heptoses, the disaccharide is selected from at least one of maltose, sucrose and lactose, and the five-membered or six-membered saturated heterocycles containing 1-2 nitrogen atoms are selected from piperazinyl, piperidinyl and pyrrolidinyl.

[0185] In one or more embodiments, L3 is a divalent hydrophilic group having two monovalent radical centers generated by removing two hydrogen atoms from glucose, galactose, mannose, glucuronic acid, galactonic acid, mannuronic acid, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine and N-acetylmuramic acid.

[0186] In one or more embodiments, L3 is selected from:

[0187] Wherein, R1 is selected from -O(CH2) p - and -C(=O)-, p is an integer from 1 to 6, preferably 1, 2, 3 or 4; R5 is a C1-C3 alkylene group which is unsubstituted or substituted by one or more substituents selected from the group consisting of: -OR 20 and -C(=O)OR 20 -; R2, R3, R4, R 11 、R 12 、R 13 Independently selected from H, C1-C6 alkyl, -(CH2CH2O) t -C1-C6 alkyl, sulfonic acid and phosphoric acid groups; R 14 、R 15 Independently selected from H, -OR 20 , and -CH2R 20 ; R 16 、R 17 、R 18 、R 19 Independently selected from H and -OR 20 ; R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t -C1-C6 alkyl, sulfonic acid and phosphoric acid; t is an integer from 1 to 20; the wavy line indicates the position where L3 is connected to X and Y.

[0188] In one or more embodiments, L4 is selected from the following amino acid residues: valine, citrulline, alanine, glycine, phenylalanine, asparagine, glutamic acid, lysine, serine, threonine, cysteine ​​and tyrosine; said amino acid group is optionally substituted with one or more substituents selected from the group consisting of: -OR 20 , where R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t -C1-C6 alkyl, sulfonic acid and phosphoric acid groups, t is an integer from 1 to 20.

[0189] In one or more embodiments, L4 is selected from the following peptide segments: valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), glycine-glycine-phenylalanine-glycine-glycine, glycine-glycine-phenylalanine-glycine-glycine, citrulline-valine, alanine-valine, alanine-alanine, citrulline-alanine, asparagine-citrulline, citrulline-asparagine, citrulline-citrulline, phenylalanine-lysine, and lysine-phenylalanine.

[0190] In one or more embodiments, L4 is selected from the following peptide segments: valine-citrulline (VC), valine-alanine (VA) and glycine-glycine-phenylalanine-glycine (GGFG); each of the peptide segments is optionally substituted with one or more substituents selected from the group consisting of: -OR 20 and -(OCH2CH2) p O-C1-C6 alkyl; wherein R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t -C1-C6 alkyl, sulfonic acid and phosphoric acid groups, t is an integer from 1 to 20, and p is an integer from 1 to 6;

[0191] In one or more embodiments, L4 is selected from:

[0192] In one or more embodiments, R6 is H, C1-C3 alkoxy or -(OCH2CH2) p O-C1-C3 alkyl, wherein p is an integer of 1-6.

[0193] In one or more embodiments, R6 is H, methoxy or -(OCH2CH2) p OCH3, wherein p is an integer of 1-6 or 1-4.

[0194] In one or more embodiments, the linker is selected from the linkers having the structures shown in the following formulae II-1 to XIV-1:

[0195] In each type, R, L2, X, L3, R2-R4, R 11 -R 13 and R 15 As described in any embodiment herein.

[0196] In one or more embodiments, the linker is selected from the linkers having the structures shown in the following formulae 1-1 to 46-1:

[0197] In some embodiments, the targeting moiety is an antibody, such as the various antibodies (Ab) described in the "Antibody Drug Conjugates" section of this application. In some embodiments, the drug includes various drug molecules well known in the art, including the various drugs (D) described in the "Linker-Drug Conjugates" section of this application.

[0198] In some embodiments, the present invention provides an antibody-drug conjugate comprising a linker as described in any embodiment herein. In some embodiments, the present invention also provides applications of the linker structure as described in any embodiment herein, specifically for increasing the drug loading of an antibody-drug conjugate and preventing the reduction of drug efficacy caused by hydrophobic aggregation due to localized dense distribution of the drug.

[0199] Pharmaceutical composition

[0200] In a fifth aspect, the present application provides a pharmaceutical composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any embodiment herein, and a pharmaceutically acceptable carrier or excipient.

[0201] As used herein, a pharmaceutically acceptable carrier or excipient may be any pharmaceutically acceptable carrier or excipient, which may vary depending on the dosage form and route of administration. Pharmaceutically acceptable carriers are generally safe and non-toxic and may comprise any known substance used in the pharmaceutical industry to formulate pharmaceutical compositions, including fillers, diluents, coagulants, binders, lubricants, glidants, stabilizers, colorants, wetting agents, and disintegrants, among others. Suitable pharmaceutically acceptable carriers include sugars, such as lactose or sucrose, mannitol or sorbitol; cellulose preparations and / or calcium phosphates, such as tricalcium phosphate or dibasic calcium phosphate; starch; silicon dioxide, talc, stearic acid or its salts, such as magnesium stearate or calcium stearate; and / or polyethylene glycol, among others. When selecting a pharmaceutically acceptable carrier, the primary consideration is the route of administration of the pharmaceutical dosage form.

[0202] The pharmaceutical composition may contain a therapeutically or prophylactically effective amount of the antibody drug conjugate. "Effective amount" means the amount of the component sufficient to produce the desired response. The specific effective amount depends on various factors, such as the specific disease to be treated, the patient's physical condition, such as weight, age and sex, the duration of treatment, co-administered therapies (if any), and the specific formulation used.

[0203] The pharmaceutical composition of the present invention can be formulated into various suitable dosage forms, including but not limited to tablets, capsules, injections, and the like, and can be administered by any suitable route to achieve the intended purpose. For example, it can be administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally, orally, intrathecally, intracranially, intranasally, or topically. The dosage of the drug can depend on the patient's age, health status, and weight, concurrent treatments, and the frequency of treatment. The pharmaceutical composition of the present invention can be administered to any subject in need thereof, such as a mammal, particularly a human.

[0204] Uses and methods

[0205] In a sixth aspect, the present application provides the use of the linker described in any embodiment of the first aspect herein in preparing a linker-drug conjugate or ADC. In some embodiments, the linker-drug conjugate is the linker-drug conjugate described in any embodiment of the second aspect herein. In some embodiments, the ADC is the ADC described in any embodiment of the third aspect herein.

[0206] In a seventh aspect, the present application provides the use of the antibody-drug conjugate described in any embodiment of the third aspect or the pharmaceutical composition described in any embodiment of the fourth aspect in the preparation of a drug, or the antibody-drug conjugate described in any embodiment of the third aspect or the pharmaceutical composition described in any embodiment of the fourth aspect for treating or preventing a disease. The disease that the drug can treat or prevent depends on the drug moiety contained in the antibody-drug conjugate. In some embodiments, the drug moiety contained in the antibody-drug conjugate is suitable for treating or preventing cancer, autoimmune diseases, inflammatory diseases or infectious diseases, and thus the diseases that the antibody-drug conjugate can be used to treat or prevent include cancer, autoimmune diseases, inflammatory diseases or infectious diseases.

[0207] In an eighth aspect, the present application provides a method for treating or preventing a disease, comprising administering to a subject in need thereof a therapeutically effective amount or a prophylactically effective amount of an antibody-drug conjugate or its pharmaceutical composition according to any embodiment of the third aspect of the present application. The antibody-drug conjugate or its pharmaceutical composition of the present invention can be administered in any suitable manner, including parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, intrathecal, intracranial, intranasal or external administration. The dosage administered may depend on the patient's age, health status and weight, concurrent treatments, and the frequency of treatment. The diseases include, but are not limited to, diseases that can be treated or prevented by the drug in the antibody-drug conjugate, including cancer, autoimmune diseases, inflammatory diseases, and infectious diseases.

[0208] Abbreviations

[0209] Throughout the description and examples of the present invention, the following abbreviations are used:

[0210] ADC: Antibody-drug conjugate;

[0211] PBS solution: phosphate buffered saline;

[0212] EDTA: ethylenediaminetetraacetic acid;

[0213] TCEP: tris(2-carboxyethyl)phosphine;

[0214] DMSO: dimethyl sulfoxide;

[0215] L-His solution: L-histidine solution;

[0216] DMF: N,N-dimethylformamide;

[0217] DIC: N,N′-diisopropylcarbodiimide;

[0218] HOBt: 1-hydroxybenzotriazole;

[0219] DIEA: N,N-diisopropylethylamine;

[0220] DCM: dichloromethane;

[0221] PE: petroleum ether;

[0222] 4A MS: 4A molecular sieve;

[0223] TMSOTf: trimethylsilyl trifluoromethanesulfonate;

[0224] THF: tetrahydrofuran;

[0225] TBTU: O-benzotriazole-N,N,N′,N′-tetramethyluronium tetrafluoroborate.

[0226] The present invention is further illustrated by the following examples, which should not be construed as limiting the present invention.The contents of all references cited throughout this application are expressly incorporated herein by reference.

[0227] Example

[0228] Example 1: Synthesis of drugs and linkers

[0229] Synthesis of CPD1 (whose structure is Formula D)

[0230] Synthesis of compound A1, solid phase synthesis of tetrapeptide

[0231] The GGFG tetrapeptide was synthesized using the standard Fmoc method.

[0232] Synthesis of compound A3

[0233] Compound A1 (10.0 g, 22.9 mmol) and commercially available compound A2 (10.9 g, 20.6 mmol) were dissolved in DMF (120 mL). DIC (7.1 mL, 45.8 mmol), HOBt (3.41 g, 25.2 mmol), and DIEA (7.98 mL, 45.8 mmol) were added to the resulting solution in sequence. The mixture was stirred and allowed to react at 25°C for three hours. After the reaction, MTBE was used for precipitation. The precipitate was removed by filtration, and the filtrate was concentrated and dried to obtain compound A3. Compound A3 was used directly in the synthesis of compound A4 without purification. Compound A3 (21.6 g, 22.7 mmol, 90% purity) was obtained as a brown solid with an MS of 854.4.

[0234] Synthesis of compound A4

[0235] Compound A3 (21.6 g, 22.7 mmol, purity 90%) was dissolved in HCl / dioxane (200 mL) and reacted at 25 degrees for 0.5 hour. After the reaction, the solvent was removed by distillation under reduced pressure. The crude product was then dissolved in DCM and PE, and the solution was evaporated to dryness under reduced pressure to obtain compound A4. Compound A4 was directly used in the synthesis of compound A9 without purification. Compound A4 was a yellow solid (17.3 g, 22.9 mmol, yield 90.7%), MS: 754.5.

[0236] Synthesis of compound A7

[0237] Commercially available compound A5 (50 g, 132 mmol), compound A6 (33.1 g, 199 mmol, 28.3 mL), 4A MS (50 g), and TMSOTf (26.58 g, 119.5 mmol, 21.6 mL) were dissolved in anhydrous DCM, replaced with nitrogen three times, and reacted at 25° C. for 3 hours under nitrogen protection. After the reaction, 4A MS (50 g) was removed by filtration, and the filtrate was diluted with DCM (500 mL) and washed with saturated sodium bicarbonate (1.50 L). The washings were extracted with DCM (1.50 L*3), the extract was washed with brine and dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The concentrate was purified using preparative HPLC [Phenomenex luna C18 (250*70mm, 10um, mobile phase [water (TFA)-ACN], B%: 36%-66%, 25min], to obtain compound A7 (24.7g, 51.2mmol, yield 38.5%) as a white solid.

[0238] 1H NMR: δppm 7.36 (br s, 5H), 5.22-5.32 (m, 2H), 5.18 (s, 2H), 5.07 (br t, J=7.82Hz, 1H), 4.74 (br d, J=7.25Hz, 1H), 4.35 (s, 2H), 4.04 (br d, J=9.13Hz, 1H), 3.74 (s, 3H), 2.03 (br d, J=4.00Hz, 9H).

[0239] Synthesis of compound A8

[0240] A 500ml round-bottom flask was replaced with Ar, and Pd / C (1.50g, 31.9mmol) was added to the Ar-replaced round-bottom flask, followed by addition of THF (10.0mL) to completely wet the Pd / C. Compound A7 (15.0g, 31.9mol) was dissolved in THF (150mL), and the solution was slowly added to the round-bottom flask under Ar protection. The reaction solution was replaced with H2 three times and reacted at 25°C under a hydrogen environment for two hours. After the reaction was completed, it was filtered under N2 protection, and the filtrate was concentrated to obtain a crude compound A8. Compound A8 can be used for the synthesis of compound A9 without purification. Compound A8 is a white solid.

[0241] 1 H NMR: δppm 8.30-9.05(m, 1H), 5.21-5.34(m, 2H), 5.03-5.12(m, 1H), 4.74(d,.J=7.38Hz, 1H), 4. 38 (d, J=1.25Hz, 2H), 4.10 (d,.J=9.51Hz, 1H), 3.73-3.78 (m, 3H), 2.01-2.09 (m, 9H).

[0242] Synthesis of compound A9

[0243] Compound A8 (3.00 g, 7.65 mmol) and compound A4 (5.76 g, 7.65 mmol) were dissolved in DMF (30.0 mL), and DIC (2.37 mL, 15.3 mmol), HOBt (1.14 g, 8.41 mmol), and DIEA (2.66 mL, 15.3 mmol) were added sequentially to the solution. The resulting mixture was reacted at 25° C. for 3 hours. After the reaction, the pH was adjusted to 5 with acetic acid and purified using preparative HPLC [preparative column: YMC Triart C18 250*50 mm*7 um, mobile phase: [water (TFA)-ACN]; B%: 21%-55%, 30 min]. The resulting solution was lyophilized to obtain compound A9, which was a yellow solid with an MS of 1129.1.

[0244] Synthesis of compound A10

[0245] To a solution of compound A9 (2.00 g, 1.77 mmol) was added an aqueous solution (30.0 mL) of lithium hydroxide (744 mg, 17.7 mmol), and the mixture was reacted at 0°C for 0.5 hour. After the reaction, the pH was adjusted to 5 with acetic acid and purified using preparative HPLC [preparative column: Phenomenex luna C18 (250*70 mm, 10 um, mobile phase: [water (TFA)-ACN]; B%: 17%-47%, 20 min]. The resulting solution was lyophilized to obtain compound A10, which was a white solid with an MS of 988.4.

[0246] Synthesis of CPD1

[0247] Compound A10 (1.18 g, 1.19 mmol) was dissolved in DMF (10.0 mL), and commercially available compound A11 (184 mg, 1.31 mmol), TBTU (767 mg, 2.39 mmol) and DIEA (416 uL, 2.39 mol) were added sequentially to the solution. The reaction solution was reacted at 25°C for 1 hour. After completion of the reaction, the insoluble material was removed by filtration and purified using preparative HPLC [preparative column: YMC Triart C18 250*50mm*7um, mobile phase: [water (TFA)-ACN]; B%: 13%-39%, 33 min]. The obtained solution was lyophilized to obtain CPD (820 mg, 738 umol, yield: 62.0%). CPD1 was a yellow solid with MS: 1110.2 and a purity of 100%.

[0248] 1H NMR (400MHz, DMSO) δ8.43 (d, J=8.4Hz, 1H), 8.31 (d, J=5.5Hz, 1H), 8.15-8.0 3(m, 4H), 7.80(d, J=10.9Hz, 1H), 7.32(s, 1H), 7.27-7.14(m, 5H), 6.97(s, 2 H), 5.61-5.53 (m, 1H), 5.40 (t, J=11.0Hz, 2H), 5.22 (d, J=21.6Hz, 2H), 4.49 -4.40 (m, 1H), 4.27 (t, J=11.8Hz, 2H), 4.02 (d, J=15.4Hz, 1H), 3.83 (dd, J=16 .5, 6.0Hz, 1H), 3.72 (dd, J=21.1, 6.2Hz, 4H), 3.61 (d, J=5.6Hz, 1H), 3.54 (d d, J=16.2, 7.7Hz, 3H), 3.48 (d, J=6.0Hz, 3H), 3.25 (s, 3H), 3.18 (d, J=9.0Hz, 4H), 3.12-3.07 (m, 1H), 2.99 (dd, J=14.0, 4.5Hz, 1H), 2.82-2.72 (m, 1H), 2. 41 (s, 3H), 2.16 (d, J = 31.0Hz, 2H), 1.92-1.77 (m, 2H), 0.87 (t, J = 7.2Hz, 3H).

[0249] Synthesis of CPD2

[0250] General steps for the preparation of compound 5-1

[0251] Compound 4-1 (10.0 g, 24.4 mmol) and p-aminobenzyl alcohol (6.00 g, 48.7 mmol) were dissolved in a mixture of methyl acetate (90 mL) and methanol (90 mL). EEDQ (12.1 g, 48.7 mmol) was then added under light-shielding conditions. The mixture was stirred at 45°C for 1.5 hours. LC-MS analysis showed complete consumption of reactant 4-1, with a major peak representing the product. The reaction mixture was distilled under reduced pressure to remove the solvent, and the product was crystallized with MTBE:ethyl acetate (10:1). The product was filtered and centrifuged to obtain the crude product. The crude product was used in the next reaction without further purification. The product was obtained as a gray solid (11.7 g, 99.3% yield).

[0252] MS(ES+)516.3[M+H]+

[0253] General steps for the preparation of compound 6-1

[0254] Compound 5-1 (11.7 g, 22.7 mmol) and PNP (13.8 g, 45.5 mmol) were dissolved in DMF (117 ml), and DIEA (7.92 ml, 45.5 mmol) was added to the mixture. The mixture was stirred at 25 ° C for 2 hours. LC-MS analysis showed that the reactant 5-1 had been completely consumed, and a main peak was detected as the product peak. The reaction mixture was precipitated with 1500 ml of ice isopropyl ether and repeated twice, filtered, and the precipitate was centrifuged to obtain a crude product. The crude product was used directly in the next reaction without further purification. The product was a brown solid (8.10 g, yield 52.3%).

[0255] MS(ES+)681.4[M+H]+

[0256] General steps for the preparation of compound 7-1

[0257] Compound 6-1 (8.10 g, 11.9 mmol) and Exatecan (5.06 g, 9.52 mmol) were dissolved in DMF (49 ml), and DIEA (4.15 ml, 23.8 mmol) and HOBt (1.93 g, 14.3 mmol) were added to the solution. The mixture was stirred and reacted at 25°C for 16 hours. LC-MS analysis showed that the reactants were completely consumed, and a major product peak was detected. 20% TEA was then added to the reaction solution, and the reaction was stirred for 5.5 hours. The reaction mixture was acidified by adding TFA at 0°C. The mixture was purified by preparative HPLC (TFA conditions) using a UniSil 10-120 C18 50x250 mm column; mobile phase: [water (TFA)-acetonitrile]; B%: 20%-50% over 20 minutes. Compound 7-1 (5.40 g, 60.1% yield) was obtained as a yellow solid.

[0258] MS(ES+)755.3[M+H]+

[0259] General steps for the preparation of compound 8-1

[0260] Compound 7-1 (2.70 g, 3.58 mmol) and compound 5A (2.11 g, 5.37 mmol) were dissolved in DMF (27 ml), and HOBt (532 mg, 3.93 mmol), DIC (1.11 ml, 7.15 mmol) and DIEA (1.25 ml, 7.15 mmol) were added to the solution. The mixture was stirred at 25°C for 2 hours. LC-MS analysis showed that the reactant 7-1 was completely consumed and a major product peak was detected. CH3COOH was added at 0°C to adjust the pH of the reaction mixture to acidic, and then precipitated twice with 1000 ml of ice isopropyl ether, filtered, and centrifuged to obtain a crude product. The crude product was used directly in the next reaction without further purification. The product was an off-white solid 8-1 (3.97 g, yield 98.3%).

[0261] MS(ES+)1129.4[M+H]+

[0262] General steps for the preparation of compound 9-1

[0263] Compound 8-1 (3.97 g, 3.52 mmol) was dissolved in a mixed solvent of H₂O (2 mL) and THF (22 mL). A solution of LiOH·H₂O (1.48 g, 35.2 mmol) in H₂O (5 mL) was then added to the solution. The mixture was stirred at 0°C for 5 minutes. LC-MS analysis showed complete consumption of reactant 8-1, with a major product peak detected. The pH of the reaction mixture was adjusted to 3-4 by adding CH₃COOH at 0°C. The reaction mixture was purified using preparative HPLC (TFA conditions) using a Phenomenex luna C₁₈ column (250*70 mm, 10 μm); mobile phase: [water (TFA)-acetonitrile]; B%: 15%-45% over 20 minutes. Compound 9-1 (1.82 g, 52.4% yield) was obtained as a yellow solid.

[0264] MS(ES+)989.2[M+H]+

[0265] General steps for CPD2 preparation

[0266] Compound 9-1 (1.82 g, 1.84 mmol) and compound 10-1 (387 mg, 2.76 mmol) were dissolved in DMF (18 ml), followed by the addition of TBTU (1.18 g, 3.68 mmol) and DIEA (1.28 ml, 7.36 mmol). The mixture was stirred at 25°C for 1 hour. LC-MS analysis showed complete consumption of the reactants, with a major product peak detected. The reactants were purified by preparative HPLC (TFA conditions) using a Phenomenex Luna C18 column (250*70 mm, 10 μm); mobile phase: [water (TFA)-acetonitrile]; B%: 26%-56% over 23 minutes. Compound CPD2 (1.15 g, 56.0% yield) was obtained as a yellow solid. MS (ES+) 1111.5 [M+H]+

[0267] Synthesis of CPD3

[0268] General steps for the preparation of compound 2

[0269] Compound 1 (2 g, 2.61 mmol) and Exatecan (970 mg, 1.83 mmol) were dissolved in DMF (10 ml), and HOBt (422 mg, 3.13 mmol) and DIEA (5.22 mmol, 908 μL) were added to the solution. The mixture was stirred at 25°C for 3 hours. LC-MS analysis showed that reactant 1 was completely consumed. The reaction mixture was distilled under reduced pressure to remove the solvent. The crude compound 2 (2.7 g) was used directly in the next reaction as a white oil without further purification. MS (ES+) 1063.3 [M+H] +

[0270] General steps for the preparation of compound 3

[0271] Compound 2 (2.70 g, 2.54 mmol) was dissolved in DMF (16 ml), and TEA (28.7 mmol, 4 ml) was added to the solution. The mixture was stirred at 25°C for 10 hours. LC-MS analysis showed that compound 2 was completely consumed. The reaction mixture was distilled under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography (TFA conditions). A yellow solid compound 3 (1.3 g, yield 60.8%) was obtained. MS (ES+) 841.2 [M+H]+

[0272] General steps for the preparation of compound 4

[0273] Compound 3 (1.2 g, 1.43 mmol) was dissolved in DMF (10 ml), and DIEA (2.85 mmol, 497 μl) was added to the solution. The mixture was stirred and reacted at 25°C for 10 minutes. Compound 6 (559 mg, 1.43 mmol), HOBt (212 mg, 1.57 mmol) and DIC (2.85 mmol, 442 μl) were then added to the mixture. The mixture was stirred and reacted at 25°C for 3 hours. LC-MS analysis showed that compound 3 was completely consumed and a major product peak was detected. The reaction mixture was distilled under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography (TFA conditions) to obtain yellow solid compound 4 (1.3 g, yield 74.9%). MS (ES+) 1215.3 [M+H] +

[0274] General steps for the preparation of compound 5

[0275] Compound 4 (500 mg, 411 μmol) was dissolved in THF (100 μL), and LiOH.H2O (172 mg, 4.11 mmol) was added to the solution. The mixture was stirred at 0°C for 5 minutes. LC-MS analysis showed that compound 4 was completely consumed and a major product peak was detected. The reaction mixture was distilled under reduced pressure to remove the solvent to obtain a crude product. The crude product was a green solid compound 5 (158 mg, yield 35.7%). Note: The reaction was carried out in parallel, and a total of 500 mg of the reaction was carried out to obtain 300 mg of compound 5. MS (ES+) 1075.2 [M+H] +

[0276] General steps for CPD3 preparation

[0277] Compound 5 (300 mg, 278 μmol) and compound 8a (30.8 mg, 418 μmol) were dissolved in DMF (600 μL), and TBTU (94.3 mg, 556 μmol) and DIEA (1.11 mmol, 102 μL) were added to the solution. The mixture was stirred at 25°C for 1 hour. LC-MS analysis showed that compound 5 was completely consumed and a major product peak was detected. The reaction mixture was distilled under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography (TFA conditions) to obtain a yellow solid compound CPD3 (106 mg, yield 31.8%). MS (ES+) 1197.2 [M+H] +

[0278] Synthesis of CPD4

[0279] General steps for the preparation of compound 5A-1

[0280] Compound 5A-1 (5 g, 25.8 mmol) was dissolved in Ac2O (80 mL). I2 (392 mg, 1.55 mmol) was slowly added to the solution, and the reaction mixture was maintained at 0°C. After the addition, the reaction was stirred at this temperature for 2 hours. LC-MS analysis showed complete consumption of the reactants, with several new peaks appearing on the spectrum, approximately 80% of which was product. The reaction mixture was maintained at 0°C, and 50 mL of dry MeOH was added dropwise. The reaction mixture was then allowed to react at 25°C for 18 hours. The reaction mixture was diluted with 150 mL of 1M Na2SO3 and extracted with 150 mL of DCM (50 mL x 3). The organic phase was dried over Na2SO4, filtered, and evaporated under reduced pressure to obtain the crude product. The crude product was slurried with 2-acetonylpropane at 25°C for 30 minutes. Compound 5A-2 (5 g, 53.6% yield) was obtained as a white solid. MS (ES+) 380.2 [M+H2O]+.

[0281] General steps for the preparation of compound 5A-3

[0282] Compound 5A-2 (5 g, 13.8 mmol), iodomethane (3.13 g, 22.1 mmol) and K2CO3 (5.72 g, 41.4 mmol) were dissolved in DMF (2 ml), replaced with nitrogen three times, and then stirred at 25 ° C under N2 protection for 16 hours. LC-MS analysis showed that the reactants were completely consumed and a major product peak was detected. 10 ml of water and 130 ml of ethyl acetate (10 ml * 3) were added to the reaction mixture, the organic phase was separated, washed with 20 ml of 1M NaCl (10 ml * 2), dried with Na2SO4, filtered and distilled under reduced pressure to obtain a crude product. The crude product was slurried with 2-acetonylpropane at 25 ° C for 30 minutes. Compound 5A-3 (4.4 g, yield 84.7%) was obtained as a yellow oily substance.

[0283] MS(ES+)394.3[M+Na]+

[0284] General steps for the preparation of compound 5A-3-1

[0285] Compound 5A-3 (1 g, 2.66 mmol) was dissolved in DCM (10 mL), and 35% HBrinAcOH (2 mL) was added to the solution. The mixture was stirred at 25°C for 16 hours. LC-MS analysis showed complete consumption of the reactants and the formation of several new peaks, with the main peak being the product. The reaction mixture was quenched by adding 10 mL of H2O at 25°C, then diluted with 10 mL of EtOAc and extracted with 30 mL of EtOAc (10 mL*3). The organic phase was washed with 30 mL of 1M NaCl (10 mL*3), dried over Na2SO4, filtered, and distilled under reduced pressure to obtain the crude product. The crude product was directly subjected to the next step without further purification, resulting in Compound 5A-3-1 (1.00 g, 94.8% yield) as a yellow oil.

[0286] MS(ES+)342.9

[0287] General steps for the preparation of compound 5A-4

[0288] Compound 5A-3-1 (2.00 g, 5.04 mmol) was dissolved in DCM (10 ml), and Ag2CO3 (1.39 g, 5.04 mmol) and benzyl alcohol 2-hydroxyacetate (1.26 g, 7.55 mmol) were added to the solution. The mixture was stirred at 25°C for 16 hours. LC-MS analysis showed that reactant 1 was completely consumed, and the main peak detected was the product peak. The reaction mixture was quenched by adding 10 ml of H2O at 25°C, then diluted with 5 ml of EtOAc and extracted with 15 ml of EtOAc (5 ml * 3). The combined organic phase was washed with 15 ml of NaCl (5 ml * 3), dried over Na2SO4, filtered, and distilled under reduced pressure to obtain a crude product. The crude product was purified using silica gel column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 3). The product was a yellow oily compound 5A-4 (800 mg, 1.66 mmol, yield 32.93%). MS (ES+) 500.2 [M+H2O]+.

[0289] General steps for the preparation of compound 5A

[0290] A 50 ml round-bottom flask was first replaced with Ar three times, and then 100 mg of Pd / C (50 mg, 1.04 mmol, 10% purity) (dried) was carefully added. Subsequently, THF (1 ml) was added to completely permeate the Pd / C, and then a THF (4 ml) solution of compound 5A-4 (500 mg, 1.04 mmol) was slowly added dropwise under Ar protection. The resulting mixture was replaced with H2 three times, and then stirred at 25°C under H2 for 2 hours. The reaction was monitored using LC-MS to complete the reaction. Under a N2 atmosphere, the reaction mixture was carefully filtered under reduced pressure. After filtration, water was immediately added to the filter membrane to keep the Pd / C wet. The organic phase was distilled under reduced pressure to obtain a crude product. The crude product was not further purified and was directly used in the next reaction. The crude product was a yellow solid compound 5A (400 mg, 98.4% yield). MS (ES+) 410.1 [M+H2O]+.

[0291] General steps for the preparation of compound 5

[0292] Compound 4 (1.00 g, 2.44 mmol) and p-aminobenzyl alcohol (600 mg, 4.87 mmol) were dissolved in a mixed solvent of DCM (5 ml) and MeOH (5 ml). EEDQ (1.20 g, 4.87 mmol) was added to the solution under light-shielding conditions. The mixture was stirred at 45°C for 1.5 hours. LC-MS monitoring showed that the reactants were completely consumed, and the main peak detected was the product peak. The reaction mixture was distilled under reduced pressure to remove the solvent to obtain a crude product. The crude product was slurried with MTBE:EtOAc (10:1), filtered, and the solid was dried. The solid was not further purified and was used directly in the next reaction. Compound 5 (1.26 g, yield 100%) was obtained as a white solid.

[0293] MS(ES+)516.0[M+H]+

[0294] General steps for the preparation of compound 6

[0295] Compound 5 (1.26 g, 2.44 mmol) and PNP (1.49 g, 4.89 mmol) were dissolved in DMF (12.6 ml), and DIEA (851 μL, 4.89 mmol) was added to the solution. The mixture was stirred at 25 ° C for 1.5 hours. LC-MS monitoring showed that compound 4 was completely consumed, and the main peak was detected as the product. The reaction mixture was precipitated with ice isopropyl ether (500 ml), filtered, and the solid was dried to obtain a crude product. The crude product was used directly in the next reaction without further purification. Compound 6 (1.23 g, yield 73.9%) was a light pink solid.

[0296] MS(ES+)681.1[M+H]+

[0297] General steps for the preparation of compound 7

[0298] Compound 6 (1.23 g, 1.81 mmol) and exatecan (866 mg, 1.63 mmol) were dissolved in DMF (12.3 ml). DIEA (631 μL, 3.62 mmol) and HOBt (293 mg, 2.17 mmol) were added to the solution. The mixture was stirred at 25°C for 16 hours. LC-MS monitoring showed complete consumption of the reactants, with the main peak being the product. 20% TEA was then added to the reaction solution, and stirring was continued for 4 hours. The reaction solution was purified using preparative HPLC (TFA conditions). Column: UniSil 10-120 C18 50x250 mm; Mobile phase: [water (TFA)-propionitrile]; B%: 15%-45% over 23 minutes. Compound 7 (1.69 g, yield 95.56%) was obtained as a yellow solid. MS (ES+) 755.2 [M+H]+.

[0299] General steps for the preparation of compound 8

[0300] Compound 7 (117 mg, 155 μmol) and compound 5A (60.8 mg, 155 μmol) were dissolved in DMF (1 ml), and HOBt (23.0 mg, 171 μmol), DIC (48.0 μl, 310 μmol), and DIEA (54.0 μl, 310 μmol) were added to the solution. The mixture was stirred at 25°C for 3 hours. LC-MS monitoring showed that the reaction was complete. The reaction mixture was slurried twice with isopropyl ether, filtered, and dried to a solid state. The crude product was not further purified and was used directly in the next reaction. Compound 8 (149 mg, yield 85.1%) was a green solid. MS (ES+) 1129.0 [M+H]+.

[0301] General steps for the preparation of compound 9

[0302] Compound 8 (149 mg, 132 μmol) was dissolved in a mixture of MeOH (5.96 mL) and water, followed by the addition of LiOH.H₂O (55.4 mg, 1.32 mmol). The mixture was stirred at 0°C for 4 minutes, followed by the addition of H₂O (1.49 mL) and stirring for 30 minutes. LC-MS monitoring confirmed the completion of the reaction, with the main peak being the product. TFA was added to the reaction mixture at 0°C, and the solvent was removed by distillation under reduced pressure. The crude product was purified by preparative HPLC (TFA conditions) using a Welch Ultimate C₁₈ column (150 × 25 mm × 5 μm) and a mobile phase of [water (TFA)-ACN] with a gradient elution increasing from 22% to 52% B% over 10 minutes. Compound 9 (47.0 mg, 36.0% yield) was obtained as a yellow solid. MS (ES+) 989.3 [M+H]⁺.

[0303] General steps for CPD4 preparation

[0304] Compound 9 (47.0 mg, 47.5 μmol) and compound 9-1 (9.99 mg, 71.3 μmol) were dissolved in DMF (0.5 mL), followed by the addition of TBTU (30.5 mg, 95.1 μmol) and DIEA (33.2 μL, 190.10 μmol). The mixture was stirred at 25°C for 1 hour. LC-MS analysis indicated the reaction was complete, with the main peak being the product. The reaction mixture was acidified by the addition of TFA at 0°C. The crude product was purified by preparative HPLC (TFA conditions) using a C18-1 column (150 × 30 mm × 5 μm) and a mobile phase of [water (TFA)-ACN], with a gradient elution increasing from 23% to 46% B% over 21 minutes. Compound CPD4 (29.0 mg, 54.9% yield) was obtained as a yellow solid. MS (ES+) 1111.2 [M+H]+.

[0305] Synthesis of CPD5

[0306] General steps for the preparation of compound 2

[0307] Compound 1 (2.00 g, 4.87 mmol) and compound 1-1 (1.49 g, 9.75 mmol) were dissolved in a mixed solvent of DCM (16 ml) and MeOH (16 ml), and EEDQ (2.41 g, 9.75 mmol) was added under light-shielding conditions. The mixture was stirred at 25°C for 2 hours. LC-MS analysis showed that the reaction was complete, and the main peak was the product peak. The reaction mixture was distilled under reduced pressure to remove the solvent. The crude product was slurried with MTBE: EtOAc (10: 1), filtered, and the solid was dried. The solid was used directly in the next reaction without further purification. Compound 2 (1.54 g, yield 57.9%) was obtained as a brown solid. MS (ES+) 546.2 [M+H]+.

[0308] General steps for the preparation of compound 3

[0309] Compound 2 (1.54 g, 2.82 mmol) and PNP (3.43 g, 11.3 mmol) were dissolved in DMF (15.5 ml), and DIEA (1.47 ml, 8.47 mmol) was added. The mixture was stirred at 25°C for 2 hours. LC-MS monitored the reaction completion. The reaction mixture was precipitated with icy isopropyl ether (500 ml * 3), the mixture was filtered, and the solid was dried. The solid was used in the next reaction without further purification. Compound 3 (1.14 g, yield 56.6%) was obtained as a brown solid. MS (ES+) 711.3 [M+H]+.

[0310] General steps for the preparation of compound 4

[0311] Compound 3 (500 mg, 704 μmol) and exatecan (337 mg, 633 μmol) were dissolved in DMF (5 mL), followed by the addition of HOBt (114 mg, 844 μmol) and DIEA (245 μL, 1.41 mmol). The mixture was stirred at 25°C for 1.5 hours. LC-MS monitored the reaction for completion. TEA (1.25 mL, 8.98 mmol) was then added to the reaction solution, and the mixture was stirred at 25°C for 16 hours. Upon completion of the reaction, TFA was added to the reaction solution at 0°C to adjust the acidity. The crude product was purified by preparative HPLC (TFA conditions) using a Phenomenex Luna C18 column (200 x 40 mm x 10 μm); mobile phase: [water (TFA)-acetonitrile]; B%: 18%-48% over 10 minutes. Compound 4 (331 mg, 59.9% yield) was obtained as a yellow solid. MS(ES+)785.3[M+H]+.

[0312] General steps for the preparation of compound 5

[0313] Compound 4 (331 mg, 422 μmol) and compound 5A (331 mg, 843 μmol) were dissolved in DMF (3.5 ml), followed by the addition of HOBt (125 mg, 928 μmol), DIC (261 μl, 1.69 mmol), and DIEA (367 μl, 2.11 mmol). The mixture was stirred at 25°C for 6 hours. LC-MS monitored the reaction completion. The reaction mixture was precipitated with 50 ml of isopropyl ether (50 ml * 2), and the turbid liquid was filtered and dried. The solid was used in the next reaction without further purification. Compound 5 (555 mg, crude product) was obtained as a gray solid. MS (ES+) 1159.1 [M+H]+.

[0314] General steps for the preparation of compound 6

[0315] Compound 5 (155 mg, 134 μmol) was dissolved in a mixed solvent of H₂O (1 mL) and THF (0.5 mL), followed by the addition of a solution of LiOH·H₂O (56.11 mg, 1.34 mmol) in H₂O (200 μL). The mixture was stirred at 0°C for 10 minutes. LC-MS analysis indicated complete consumption of the reactant, and the product peak was detected as the main peak. The reaction mixture was acidified by the addition of CH₃COOH at 0°C. The crude product was purified by preparative HPLC (TFA conditions) using a Welch Xtimate C₁₈ 150*25mm*5um column; mobile phase: [water (TFA)-acetonitrile]; B%: 20%-50% over 40 minutes. Compound 6 (50.0 mg, yield 36.7%) was obtained as a yellow solid. MS (ES+) 1019.4 [M+H]+.

[0316] General steps for CPD5 preparation

[0317] Compound 6 (50.0 mg, 49.1 μmol) and compound 7-1 (10.3 mg, 73.6 μmol) were dissolved in DMF (0.5 mL), followed by the addition of TBTU (31.5 mg, 98.1 μmol) and DIEA (34.2 μL, 196 μmol). The mixture was stirred at 25°C for 1 hour. LC-MS analysis confirmed the reaction was complete. The reaction solution was purified by preparative HPLC (TFA conditions) using a Welch Xtimate C18 150*25 mm*5 μm column; mobile phase: [water (TFA)-acetonitrile]; B%: 22%-52% over 10 minutes. Compound Target 1 (27.0 mg, 48.2% yield) was obtained as a yellow solid. MS (ES+) 1141.1 [M+H]+.

[0318] Synthesis of CPD6

[0319] General steps for the preparation of compound 2

[0320] Compound 1 (2.93 g, 8.08 mmol) and compound 1D (1.35 g, 8.08 mmol) were dissolved in DMF (40 ml), and K2CO3 (2.23 g, 16.2 mmol) was added. The mixture was stirred at 80°C for 3 hours. LC-MS monitoring showed that the reaction was complete, with the main peak being the product peak. The reaction mixture was filtered to remove K2CO3. The filtrate was purified using preparative HPLC (TFA conditions). Compound 2 (2.30 g, yield 79.6%) was obtained as a yellow liquid. MS (ES+) 358.1 [M+H]+.

[0321] General steps for the preparation of compound 2A

[0322] Compound 2 (1.93 g, 5.40 mmol) was dissolved in MeOH (40 ml), and then NaBH4 (306 mg, 8.10 mmol) was added at 0°C. The mixture was stirred at 0°C for 30 minutes. LC-MS monitoring showed that compound 2 was completely consumed, and the main peak was the product peak. 1NHCl (200 ml) was added to the reaction mixture at 0°C for quenching, followed by dilution with DCM (100 ml) and extraction (200 ml × 2). The organic phases were combined, washed with brine (200 ml), dried over Na2SO4, filtered, and then distilled under reduced pressure to obtain the product. Compound 2A (crude, 1.89 g) was obtained as a white oil. MS (ES+) 360.1 [M+H]+.

[0323] General steps for the preparation of compound 2B

[0324] Compound 2A (3.00 g, 8.35 mmol) was dissolved in DMF (30 ml), and 1H-imidazole (1.70 g, 25.0 mmol) and tert-butyldimethylsilyl chloride (2.52 g, 16.7 mmol) were added. The mixture was stirred at 25°C for 2 hours. LC-MS monitoring showed that compound 2A was completely consumed, and the main peak was the product peak. The reaction mixture was poured into ice water (250 ml). The mixture was extracted with ethyl acetate (200 ml × 3). The organic phases were combined and washed with brine (200 ml), and the organic phase was dried over Na2SO4 and the solvent was removed by distillation under reduced pressure. The crude product was used directly in the next reaction without purification. Compound 2B (3.50 g, yield 79.4%) was obtained as a yellow oil. MS (ES+) 474.3 [M+H] +.

[0325] General steps for the preparation of compound 2C

[0326] A 250 ml round-bottom flask was replaced with argon three times and 300 mg of dry Pd / C was added. Ethyl acetate (20 ml) was then slowly added to completely wet the Pd / C, followed by the slow addition of a solution of compound 2B (3.50 g, 7.39 mmol) in ethyl acetate (20 ml) under argon protection. The resulting mixture was replaced with hydrogen three times and then stirred at 25°C under H2 (15 psi) for 3 hours. The reaction was monitored by LC-MS. The reaction mixture was carefully filtered under reduced pressure under nitrogen protection. Water was then immediately added to the filter cake. The organic phase was concentrated under reduced pressure to obtain a crude product. The crude product was used in the next reaction without further purification. Compound 2C (3.18 g, yield 86.6%) was obtained as a yellow liquid. MS (ES+) 444.3 [M+H]+.

[0327] General steps for the preparation of compound 3

[0328] Compound 2C (2.90 g, 6.54 mmol) and compound 2D (3.22 g, 7.84 mmol) were dissolved in DMF (30 ml), and EEDQ (3.23 g, 13.0 mmol) was added. The mixture was stirred at 25°C for 2 hours. LC-MS analysis showed that compound 2C was completely consumed, with the main peak being the product. The reaction solution was purified by preparative HPLC (TFA conditions). Compound 3 (2.8 g) was obtained as a white solid. MS (ES+) 858.6 [M+Na]+.

[0329] General steps for the preparation of compound 4

[0330] Compound 3 (370 mg, 442.53 μmol) was dissolved in a mixed solvent of THF (1 ml) and water (1 ml), and AcOH (2 ml) was added. The mixture was stirred at 25°C for 1 hour. LC-MS detection showed that compound 3 was completely consumed and a main peak of the desired m / z was detected. The reaction mixture was cooled in an ice bath and diluted with a saturated NaHCO3 solution (10 ml). The mixture was extracted with ethyl acetate (10 ml*3), and the combined organic phases were washed with water (20 ml) and brine (10 ml), dried over sodium sulfate, and evaporated under reduced pressure. Compound 4 (290 mg, crude product) was obtained as a white solid. MS (ES+) 704.4 [M-H2O]+.

[0331] General steps for the preparation of compound 5

[0332] Compound 4 (290 mg, 401 μmol) and bis(4-nitrophenyl) carbonate (244 mg, 803 μmol) were dissolved in DMF (4 ml), and DIEA (103 mg, 803 μmol) was added. The mixture was stirred at 25°C for 2 hours. LC-MS detection revealed that compound 4 was completely consumed, and a main peak with the product m / z was detected. The mixture was diluted with 1N hydrochloric acid (20 ml) and then extracted with DCM (20 ml * 3). The combined organic phases were dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative HPLC (TFA conditions). Compound 5 (135 mg, yield 37.9%) was obtained as a white solid. MS (ES+) 887.5 [M+H]+.

[0333] General steps for the preparation of compound 6

[0334] Compound 5 (135 mg, 152 μmol) and isotecan mesylate (89.0 mg, 167 μmol) were dissolved in DMF (2 ml), and HOBt (24.6 mg, 182 μmol) and DIEA (39.3 mg, 304 μmol) were added. The mixture was stirred at 25°C for 2 hours. LC-MS monitoring showed that compound 5 was completely consumed, and the main peak of the product m / z was detected. The reaction mixture was added dropwise to MTBE (30 ml * 2) for precipitation to obtain a crude product. Compound 6 (190 mg, crude product) was obtained as a yellow solid. MS (ES+) 1184.7 [M+H]+.

[0335] General steps for the preparation of compound 7

[0336] Compound 6 (190 mg, 160 μmol) was dissolved in DMF (2 mL) and TEA (0.5 mL) was added. The reaction was stirred at 25°C for 4 hours. LC-MS monitoring of compound 6 revealed complete reaction, with a major peak of the product m / z detected. The reaction solution was purified by preparative HPLC (TFA). Compound 7 (100 mg, 64.8% yield) was obtained as a yellow solid. MS (ES+) 961.6 [M+H]+.

[0337] General steps for the preparation of compound 8

[0338] Compound 7 (100 mg, 104 μmol) and compound 5A (40.8 mg, 104 μmol) were dissolved in DMF (2 ml), and DIEA (26.9 mg, 208 μmol), HOBt (21.1 mg, 156 μmol), and DIC (26.4 mg, 208 μmol) were added. The mixture was stirred at 25°C for 2 hours. LC-MS monitoring showed that the reaction of compound 7 was complete, with the main peak being the product peak. The reaction mixture was added to MTBE (30 ml*2) for precipitation, filtered, and dried to obtain a crude product. The crude product was used directly in the next reaction without purification. Compound 8 (136 mg, crude product) was obtained as a yellow solid. MS (ES+) 1335.8 [M+H]+.

[0339] General steps for the preparation of compound 9

[0340] Compound 8 (136 mg, 102 μmol) was dissolved in a mixed solvent of THF (1 mL) and H₂O (1 mL), and LiOH.H₂O (42.7 mg, 1.02 mmol) was added. The mixture was stirred at 0°C for 0.5 h. LC-MS analysis confirmed the complete reaction of compound 8, with the main peak being the product. Acetic acid was added to the reaction mixture to adjust the pH to 5. The reaction mixture was purified by preparative HPLC (TFA conditions). Compound 9 (65.0 mg, 53.4% ​​yield) was obtained as a white solid. MS (ES+) 1195.7 [M+H]+.

[0341] General steps for CPD6 preparation

[0342] Compound 9 (65.0 mg, 54.4 μmol) was dissolved in DMF (1 ml), and compound 10-1 (9.15 mg, 65.2 μmol) was added. TBTU (34.9 mg, 108 μmol) and DIEA (28.1 mg, 217 μmol) were then added to the reaction mixture. The reaction was stirred at 25°C for 1 hour. LC-MS monitoring indicated complete reaction of compound 9, with the main peak being the product. The reaction solution was directly purified by preparative HPLC (TFA conditions). Compound CPD6 (35 mg, 48.8% yield) was obtained as a yellow solid. MS (ES+) 1317.3 [M+H]+.

[0343] Synthesis of CPT004

[0344] General steps for the preparation of compound 2

[0345] Compound 1 (7.0 g, 21.99 mmol) was dissolved in DMF (50 mL), the solution was diluted with 0.1 M pH 7.0 phosphate buffer (450 mL), and then lipase C (5.93 g) was added. The mixture was stirred at 25° C. for 24 hours. The reaction mixture was filtered, and the filtrate was extracted with 1500 mL of ethyl acetate (3*500 mL). The organic phase was washed with 500 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. The crude product was purified using a silica gel column to obtain compound 2 (3.0 g, 40% yield). Compound 2 is a colorless oil.

[0346] General steps for the preparation of compound 3

[0347] Compound 2 was dissolved in a mixed solvent of acetonitrile and water (30 mL, v / v = 1 / 1), TEMPO (424 mg, 2.72 mmol) and BAIB (7.0 g, 21.72 mmol) were added to the solution, and the mixture was stirred at 25 ° C. for 16 hours. The solvent was removed by evaporation under reduced pressure, and the crude product was diluted with water and DCM, extracted with DCM (3*50 mL), and the organic phase was washed with saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure to remove the solvent to obtain a crude product. The crude product (4.0 g) was used directly in the next reaction without purification.

[0348] General steps for the preparation of compound 4

[0349] Compound 3 (4.0 g, 13.78 mmol) was dissolved in methanol (50 mL), and EDCI (5.28 g, 27.56 mmol) and DMAP (0.34 mg, 2.76 mmol) were added to the solution. The mixture was stirred at 25 ° C. for 2 hours, and the methanol was removed by rotary evaporation. The crude product was diluted with DCM (50 mL), washed with water (50 mL * 3) and saturated brine (2 * 50 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and rotary evaporation to obtain a crude product. The crude product was purified using a silica gel column to obtain compound 4 (1.3 g, 31.0% yield), which was a colorless oily substance.

[0350] General steps for the preparation of compound 5

[0351] 4A molecular sieves (0.3 g) were added to a three-necked flask and heated at high temperature for 5 minutes. After cooling, anhydrous DCM (200 mL) was added. Compound 4 (0.3 g, 0.99 mmol), compound 6 (0.2 g, 1.18 mmol), and TMSOTf (0.26 g, 1.18 mmol) were added sequentially to the mixture. The atmosphere was purged with nitrogen three times. The mixture was stirred at 25°C under nitrogen for 3 hours. The reaction was monitored by TLC (developing solvent: PE / EA = 3 / 1). After completion of the reaction, the 4A molecular sieves were removed by filtration. The filtrate was diluted with 50 mL of DCM and washed with saturated sodium bicarbonate (30 mL) and saturated brine (30 mL x 3). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain a crude product. The crude product was purified using a silica gel column to obtain compound 5 (0.2 g, 49.4% yield) as a white solid. MS (ES+) 428.6 [M+H2O+H]+.

[0352] General steps for the preparation of compound 6

[0353] Compound 5 (0.2 g, 2.07 mmol) was dissolved in 4.4% formic acid in methanol (20 mL), and wet Pd / C (0.2 g) was added to the solution. The mixture was reacted at 25°C for 5 hours. The reaction of compound 5 was completed as monitored by TLC (developing solvent: PE / EA = 2 / 1). The mixture was filtered through celite to obtain a filtrate, and the filtrate was evaporated to remove the solvent to obtain a crude product. The crude product (0.15 mg, 96.1% yield) was used directly in the next step without purification.

[0354] General steps for the preparation of compound 7

[0355] Compound 6 (350 mg, 0.46 mmol) and GGFG-irinotecan (150 mg, 0.46 mmol) were dissolved in DMF (5 mL), and HOBt (94 mg, 0.70 mmol), EDCI (133 mg, 0.70 mmol) and DIEA (171 uL, 0.93 mmol) were added. The mixture was stirred at 25 ° C for 2 hours. HPLC detection showed that the reaction of compound 6 was complete. The reaction mixture was evaporated to remove the solvent, and the residue was purified by preparative HPLC to obtain compound 7 (115 mg, 23.5% yield). Compound 7 is a yellow solid.

[0356] General steps for the preparation of compound 8

[0357] Compound 7 (115 mg, 0.109 mmol) was dissolved in a mixed solvent of THF / MeOH / H2O (5 mL), and a solution of LiOH (13 mg, 0.54 mmol) in H2O (0.5 mL) was added at 0°C. The mixture was reacted at 0°C for 0.5 h. The reaction of compound 7 was complete as monitored by HPLC. The reaction solution was evaporated to remove the solvent, and the residue was purified by preparative HPLC to give compound 8 (50.0 mg, 47.9% yield) as a white solid.

[0358] General steps for the preparation of CPT004

[0359] Compound 8 (50.0 mg, 52.20 μmol) and 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (18.44 mg, 104.39 μmol) were dissolved in DMF (2.0 mL). TBTU (25.14 mg, 78.29 μmol) and DIEA (19.27 μL, 104.39 μmol) were added. The mixture was stirred at 25°C for 1 hour. The reaction was monitored for completion by HPLC. The reaction solution was diluted with MeOH (2 mL) and purified by preparative HPLC to obtain the product, CPT004 (14 mg, 24.8% yield), as a yellow solid. HRMS (ES+) revealed 1079.854 [M+H]+.

[0360] 1 H NMR (400MHz, DMSO-d6) δ8.42 (d, J=8.6Hz, 1H), 8.31 (s, 1H), 8.16 (t, J=5.6Hz, 2H), 8.09 (d, J=8.0Hz, 1H), 8.04 (t, J=5.5Hz, 1H), 7.80 (d, J=11.0Hz, 1H), 7.32 (s, 1H), 7.27-7.13 (m, 5H), 6.94 (s, 2H), 5.65-5.51 (m, 1H), 5.4 8-5.35 (m, 2H), 5.31-5.18 (m, 2H), 4.89 (d, J=2.0Hz, 1H), 4.48-4.40 (m, 1H), 4.18-4.02 (m, 4H), 3.83 (dd, J=4. 4, 2.1Hz, 1H), 3.73 (t, J=6.2Hz, 6H), 3.29-3.16 (m, 6H), 2.98 (dd, J=13.7, 4.7Hz, 1H), 2.77 (dd, J=13.5, 9.5Hz, 1H), 2.41 (s, 3H), 2.20 (s, 1H), 2.12 (s, 1H), 1.86 (tt, J=14.0, 7.1Hz, 2H), 0.87 (t, J=7.3Hz, 3H).

[0361] Synthesis of CPT005

[0362] General steps for the preparation of compound 3

[0363] Compound 1 (2.0 g, 16.94 mmol) and compound 2 (2.84 g, 15.24 mmol) were dissolved in DMF (20 ml), and DIEA (2.85 mmol, 9.4 ml) and HATU (9.66 g, 25.40 mmol) were added. The reaction was stirred at 25 ° C for 3 hours. TLC monitored the complete reaction of compound 2. The reaction mixture was diluted with H2O (50 ml) and extracted with ethyl acetate (50 ml * 3). The organic phases were combined and washed with saturated sodium chloride aqueous solution (50 ml). The organic phase was dried over anhydrous Na2SO4, filtered, and the solvent was removed by rotary evaporation to obtain a crude product. The crude product was purified by silica gel column chromatography. Compound 3 (4.0 g, yield 82.5%) was obtained as a yellow solid.

[0364] MS(ES+)309.2[M+Na]+.

[0365] General steps for the preparation of compound 4

[0366] Compound 3 (4.0 g, 13.97 mmol) was dissolved in a THF / MeOH / H2O (50 ml, 3 / 1 / 1) mixed solvent, and then LiOH (1.0 g, 41.91 mmol) was added. The mixture was stirred at 25°C for 3 hours. TLC monitoring showed that the reaction of compound 3 was complete. The reaction mixture was diluted with H2O (50 ml) and extracted with ethyl acetate (50 ml*3). The organic phases were combined and washed with saturated sodium chloride aqueous solution (20 ml), the organic phase was dried over Na2SO4, filtered, and the solvent was removed by rotary evaporation to obtain a crude product. The crude product (2.0 g, yield 52.6%) was used directly in the next reaction without further purification. MS (ES+) 295.1 [M+Na]+.

[0367] General steps for the preparation of compound 7

[0368] Compound 5 (1.0 g, 2.30 mmol) and compound 6 (1.28 g, 2.30 mmol) were dissolved in DMF (20 ml), and HOBt (0.46 g, 3.44 mmol), DIEA (1.27 ml, 6.89 mmol) and EDCI (0.66 g, 3.44 mmol) were added. The mixture was reacted at 25°C for 2 hours. LC-MS monitoring showed that reactant 5 was completely reacted, and the main peak was the product peak. The reaction mixture was diluted with H2O (50 ml) and extracted with ethyl acetate (20 ml * 3). The organic phases were combined and washed with saturated sodium chloride aqueous solution (50 ml), the organic phase was dried over Na2SO4, filtered, and the solvent was removed by rotary evaporation to obtain a crude product. The crude product was purified by silica gel column chromatography. Compound 7 (2.0 g, yield 89.2%) was obtained as a yellow solid.

[0369] General steps for the preparation of compound 8

[0370] Compound 7 (1.0 g, 1.02 mmol) was dissolved in DMF (10 ml) and TEA (2 ml) was added. The mixture was stirred at 25°C for 12 hours. HPLC monitoring showed that the reaction of compound 7 was complete. The reaction mixture was poured into ice water (50 ml) for precipitation, and the turbid liquid was filtered to obtain a solid. The solid was washed with ethyl acetate and dried to obtain compound 8 (0.5 g, yield 64.7%) as a yellow solid. MS (ES+) 754.4 [1 / 2M+H]+.

[0371] General steps for the preparation of compound 9

[0372] Compound 8 (0.4 g, 0.53 mmol) and compound 4 (0.22 g, 0.80 mmol) were dissolved in DMF (10 ml), and HOBt (0.11 g, 0.80 mmol), EDCI (0.15 g, 0.80 mmol) and DIEA (0.19 ml, 1.06 mmol) were added. The mixture was stirred at 25 ° C for 2 hours. TLC monitoring showed that the reaction of reactant 8 was complete. The reaction mixture was diluted with H2O (50 ml) and extracted with ethyl acetate (20 ml * 3). The organic phases were combined and washed with saturated sodium chloride aqueous solution (50 ml). The organic phase was dried over Na2SO4, filtered, and rotary evaporated to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain the product. Compound 9 (0.17 g, yield 31.8%) was obtained as a yellow solid.

[0373] General steps for the preparation of compound 10

[0374] Compound 9 (0.150 g, 0.15 mmol) was dissolved in DMF (3 mL), and TFA (13.39 mmol, 1 mL) was added. The mixture was stirred at 25°C for 5 hours. HPLC monitoring indicated that compound 9 had reacted completely. The solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by preparative HPLC. Compound 10 (0.1 g, 74% yield) was obtained as a yellow solid. MS (ES+) 454.8 [1 / 2 M+H]+.

[0375] General steps for the preparation of CPT005

[0376] Compound 10 (100 mg, 0.11 mmol) and GMBS (46.3 mg, 0.17 mmol) were dissolved in DMF (2.2 mL), and DIEA (40.7 mL, 0.22 mmol) was added. The mixture was stirred at 0°C for 1 hour. HPLC monitoring indicated complete reaction of compound 10. The solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by preparative HPLC to obtain CPT005 (30 mg, 25.4% yield) as a yellow solid. MS (ES+) 537.3 [1 / 2 M+H]+.

[0377] 1 H NMR (400MHz, DMSO) δ8.42 (s, 2H), 8.32 (s, 1H), 8.18 (s, 1H), 8.06 (s, 1H), 7.81 (d, J=11.1Hz, 1H), 7.32 (s, 1 H), 7.20 (dd, J=18.0, 10.1Hz, 5H), 6.99 (s, 2H), 5.57 (s, 1H), 5.42 (s, 2H), 5.25 (s, 2H), 4.43 (s, 1H), 3.71 (s , 6H), 3.42 (d, J=18.6Hz, 15H), 3.18 (s, 2H), 2.98 (d, J=10.3Hz, 1H), 2.78 (d, J=10.6Hz, 1H), 2.41 (s, 3H), 2. 32 (d, J=7.2Hz, 2H), 2.20 (s, 1H), 2.08 (s, 1H), 1.91-1.79 (m, 2H), 1.77-1.68 (m, 2H), 0.87 (t, J=7.2Hz, 3H).

[0378] Synthesis of CPT006

[0379] General steps for the synthesis of CPT006-2

[0380] Compound CPT006-1 (100.0 mg, 243.9 μmol, 1.0 eq.), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 60.0 mg, 246.7 μmol, 1.0 eq.), and p-aminobenzyl alcohol (30.0 mg, 243.6 μmol, 1.0 eq.) were added sequentially to a 50 ml flask. A methanol / dichloromethane (1:4) solvent mixture was added. The reaction mixture was stirred at 25°C for 6 hours and monitored by HPLC until completion. The solvent was removed by rotary evaporation, and 20 ml of petroleum ether was added. The mixture was stirred at room temperature for 2 hours and filtered to obtain a pale yellow solid, CPT006-2. LC-MS: 538.2 [M+Na].

[0381] General steps for the synthesis of CPT006-3

[0382] Compound CPT006-2 (100.0 mg, 194.2 μmol, 1.0 eq.) was dissolved in a mixture of 3 mL of dichloromethane and 1 mL of N,N-dimethylformamide. 40.0 μL of piperidine was added, and the mixture was stirred at 25°C for 1 hour. The reaction was monitored by thin-layer chromatography (TLC). After completion of the reaction, the solvent was removed by rotary evaporation to obtain a yellow solid, crude product CPT006-3. The crude product was used directly in the next reaction without purification. LC-MS: 294.3 [M+H].

[0383] General steps for the synthesis of CPT006-4

[0384] The crude compound CPT006-3 was dissolved in 5 ml of dichloromethane, and CPT006-9 (53.0 mg, 194.9 μmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 48.2 mg, 194.9 μmol, 1.0 eq.) were added. The reaction was monitored by thin-layer chromatography (TLC). After completion of the reaction, the solvent was removed by rotary evaporation to obtain a yellow semisolid, the crude product CPT006-4. Purification on a silica gel column with petroleum ether / ethyl acetate (10:1) afforded 60 mg of the desired product as a colorless oil.

[0385] Structural characterization: LC-MS: 570.3 [M+Na], 474.3 [M-tBuO], 448.3 [M-Boc].

[0386] General steps for the synthesis of CPT006-5

[0387] Compound CPT006-4 (120.0 mg, 219.3 μmol, 1.0 eq.), di(p-nitrobenzene) carbonate (132.0 mg, 434.2 μmol, 2.0 eq.), and N,N-diisopropylethylamine (72 μL, 434.2 μmol, 2.0 eq.) were dissolved in 10 mL of N,N-dimethylformamide and stirred overnight at 25°C. The mixture was diluted with 20 mL of pure water and stirred for 1 hour. The mixture was then extracted with ethyl acetate (50 mL x 3). The combined organic phases were washed with a semi-saturated aqueous solution of NaCO (50 mL x 2). The mixture was dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain 90 mg of a pale yellow solid, CPT006-5. The crude product was used directly in the next reaction without further purification.

[0388] General steps for the synthesis of CPT006-6

[0389] Compound CPT006-5 (20 mg, 30.0 μmol, 1.2 eq.) and isotecan mesylate (11.5 mg, 26.4 μmol, 1.0 eq.) were dissolved in 2 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine (8.3 μL, 50.0 μmol, 2.0 eq.) was added. The reaction was monitored for completion by HPLC, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was directly purified by high performance liquid chromatography and freeze-dried to obtain 13 mg of the trifluoroacetate salt of compound CPT006-6.

[0390] Mobile phase A: acetonitrile (0.1% trifluoroacetic acid), mobile phase B: water (0.1% trifluoroacetic acid)

[0391] Structural characterization: LC-MS: 1009.4 [M+H].

[0392] General steps for the synthesis of CPT006-7

[0393] Compound CPT006-6 (13.0 mg, 12.9 μmol, 1.0 eq.) was dissolved in 2 mL of dichloromethane containing 20% ​​trifluoroacetic acid. The reaction was monitored by thin layer chromatography (TLC) after 2 hours. After the reaction was completed, the solvent was removed by rotary evaporation to obtain crude product CPT006-7 as a light yellow solid (15 mg).

[0394] Structural characterization: LC-MS: 455.2 [M+2H] / 2, 909.4 [M+H].

[0395] General steps for the synthesis of CPT006

[0396] Compound CPT006-7 (15.0 mg, 14.7 μmol, 1.0 eq.) and 4-maleimidobutyric acid N-hydroxysuccinimide ester (10.0 mg, 35.7 μmol, 2.5 eq.) were dissolved in 2 mL of N,N-dimethylformamide. N,N-Diisopropylethylamine (4.8 μL, 29.4 μmol, 2.0 eq.) was added with stirring. The mixture was reacted at 25°C for 16 hours and monitored by HPLC until the starting material was completely consumed. The reaction solvent was removed under reduced pressure, and the concentrate was directly purified by HPLC and freeze-dried to obtain 5 mg of the pale yellow compound CPT006.

[0397] Mobile phase A: acetonitrile (0.1% trifluoroacetic acid), mobile phase B: water (0.1% trifluoroacetic acid)

[0398] Structural characterization: LC-MS: 1074.4 [M+H].

[0399] 1 H NMR (400MHz, DMSO) δ9.78 (s, 1H), 8.35 (dd, J=5.0, 1.9Hz, 1H), 8.21 (d, J=8.0Hz, 1H), 8.07 (dd, J=8.8, 1.6Hz, 1H), 7.79 (d , J=10.9Hz, 1H), 7.61 (d, J=8.3Hz, 2H), 7.37 (d, J=8.3Hz, 2H), 7.32 (s, 1H), 6.99 (s, 2H), 6.53 (m, 1H), 5.45 (s, 2H), 5.29 ( s, 3H), 5.08 (s, 2H), 4.36 (dd, J=10.5, 4.1Hz, 2H), 4.20 (dd, J=11.2, 3.6Hz, 2H), 2.38 (s, 2H), 2.32 (dd, J=11.8, 4.3Hz, 2H ), 2.18 (m, 4H), 2.00 (dd, J=14.7, 7.1Hz, 4H), 1.88 (m, 3H), 1.73 (m, 2H), 1.33 (d, J=7.1Hz, 2H), 1.24 (s, 6H), 0.89 (m, 9H).

[0400] Synthesis of CPT008

[0401] Synthesis of compound 2

[0402] Dissolve 1,4-dibromomaleimide and N-methylmorpholine in tetrahydrofuran, then add propyl chloroformate. Stir the reaction at room temperature for 20 minutes. After stirring, add an appropriate amount of dichloromethane, wash the organic phase with water, dry over anhydrous sodium sulfate, filter, and evaporate the solvent under reduced pressure. The crude product is used directly in the next reaction.

[0403] Synthesis of compound 3

[0404] Dissolve compound 2 and N-tert-butylcarbonyl-ethylenediamine separately in a dichloromethane solution. Slowly add the N-tert-butylcarbonyl-ethylenediamine solution to the dichloromethane solution containing compound 2 via an addition funnel. Stir for 2 hours. After the reaction is complete, wash with a saturated aqueous ammonium chloride solution and then with a saturated aqueous sodium chloride solution. The organic phase is dried over anhydrous sodium sulfate, filtered, and the solvent evaporated under reduced pressure. Further purification is performed by silica gel chromatography, and the solvent is evaporated under reduced pressure to obtain the pure product.

[0405] Synthesis of compound 4

[0406] Compound 3 was dissolved in a mixture of dichloromethane and trifluoroacetic acid (TFA) (1:1), stirred at room temperature for 1 hour, and the solvent was evaporated under reduced pressure to obtain a crude product.

[0407] Synthesis of compound CPT008

[0408] Compounds 4 and 5 were dissolved in N,N-dimethylformamide solution, followed by the addition of triethylamine and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate. The reaction was stirred at room temperature for 30 minutes. After stirring, methanol was added for dilution, and the mixture was purified by preparative liquid chromatography, and the solvent was evaporated under reduced pressure to obtain the pure product.

[0409] Synthesis of CPT009

[0410] General steps for the preparation of compound 2

[0411] Compound 1 (10 g, 26.57 mmol), compound 6 (6.62 g, 39.86 mmol), 4A MS (10 g), and trimethylsilyl trifluoromethanesulfonate (5.91 g, 26.57 mmol) were dissolved in anhydrous dichloromethane (200 ml). After three nitrogen replacements, the mixture was stirred at 25°C under N2 protection for 3 hours. The reaction was monitored for completion by TLC. The reaction mixture was filtered to remove 4A MS (50 g). The filtrate was diluted with dichloromethane (200 ml), washed with saturated sodium bicarbonate solution (100 ml), and the aqueous phase was extracted with dichloromethane (150 ml * 3). The combined organic phases were washed with saturated brine (150 ml), dried over Na2SO4, and rotary evaporated to obtain the crude product. The crude product was purified by silica gel column chromatography. Compound 2 (3.0 g, yield 23.4%) was obtained as a white solid.

[0412] General steps for the preparation of compound 3

[0413] Compound 2 (1.0 g, 2.07 mmol) was dissolved in 4.4% formic acid-methanol solution (20 ml), and then Pd / C (0.1 g) was added. The mixture was stirred at 25°C for 5 hours. TLC monitoring showed that compound 2 was completely reacted. The reaction mixture was filtered through silica filter paper. The filtrate was rotary evaporated to obtain a crude product (0.8 g, yield 98.4%), which was used in the next reaction without further purification.

[0414] General steps for the preparation of compound 5

[0415] Compound 4 (5.00 g, 12.18 mmol) and PAB (3.0 g, 24.36 mmol) were dissolved in a mixed solvent of dichloromethane (100 ml) and methanol (10 ml), and then EEDQ (6.02 g, 24.36 mmol) was added. The mixture was stirred at 25°C for 1.5 hours. TLC monitoring showed that compound 3 was completely reacted. The reaction solution was a suspension and filtered to obtain a solid. The solid was washed with dichloromethane (10 ml) and then dried under reduced pressure to obtain compound 5 (5.0 g, yield 79.6%) as a white solid.

[0416] General steps for the preparation of compound 6

[0417] Compound 5 (3.0 g, 5.82 mmol) was dissolved in DMF (10 ml) and TEA (11.64 mmol, 2 ml) was added. The mixture was stirred at 25°C for 12 hours. HPLC monitoring showed that compound 5 was completely reacted. The reaction mixture was diluted with H2O (50 ml) and extracted with ethyl acetate (20 ml * 3). The combined organic phase was washed with saturated sodium chloride solution (50 ml), dried over Na2SO4, filtered, and rotary evaporated to obtain a crude product. The crude product (2.7 g) was used in the next reaction without further purification.

[0418] General steps for the preparation of compound 7

[0419] Compound 6 (2.4 g, 8.18 mmol) and Fmoc-Gly-OH (2.68 g, 9.00 mmol) were dissolved in DMF (20 ml), and DIC (2.06 g, 16.36 mmol) was added. The mixture was stirred at 25°C for 2 hours. TLC monitoring showed that reactant 6 was completely reacted. The reaction mixture was poured into ice water (50 ml) for precipitation, and the suspension was filtered to obtain a solid. The filter cake was washed with ethyl acetate and dried under reduced pressure to obtain compound 7 (2.0 g, yield 42.7%) as a white solid. MS (ES+) 595.3 [M+Na]+.

[0420] General steps for the preparation of compound 8

[0421] Compound 7 (2.0 g, 3.49 mmol) and PNP (2.12 g, 6.98 mmol) were dissolved in DMF (20 ml), and DIEA (1.3 ml, 6.98 mmol) was then added. The mixture was stirred at 25°C for 1.5 hours. TLC monitoring showed that compound 7 had completely reacted. The reaction mixture was diluted with H2O (50 ml) and extracted with ethyl acetate (20 ml * 3). The combined organic phase was washed with saturated sodium chloride solution (50 ml), dried over Na2SO4, filtered, and rotary evaporated to obtain a crude product. The crude product was purified by silica gel chromatography. Compound 8 (1.5 g, yield 58.2%) was obtained as a yellow solid.

[0422] General steps for the preparation of compound 9

[0423] Compound 8 (400 mg, 0.54 mmol) and exatecan mesylate (236.09 mg, 0.54 mmol) were dissolved in DMF (10 ml), followed by the addition of HOBt (80.59 mg, 0.60 mmol) and DIEA (200 μL, 1.08 mmol). The mixture was stirred at 25°C for 16 hours. TLC monitoring showed complete reaction of exatecan. The reaction mixture was diluted with H2O (50 ml) and extracted with EA (20 ml*3). The combined organic phases were washed with saturated sodium chloride solution (50 ml), dried over Na2SO4, filtered, and rotary evaporated to obtain a crude product. The crude product was purified by silica gel chromatography. Compound 9 (0.38 g, 67.8% yield) was obtained as a yellow solid.

[0424] General steps for the preparation of compound 10

[0425] Compound 9 (380 mg, 0.47 mmol) was dissolved in DMF (5 mL), and then TEA (1.06 mL, 7.35 mmol) was added. The mixture was stirred at 25°C for 12 hours. HPLC monitoring showed that compound 9 was completely reacted. The reaction mixture was poured into ice water (50 mL) for precipitation, and the solid was filtered to obtain a solid. The solid was washed with ethyl acetate and dried under reduced pressure to obtain compound 10 (250 mg, yield 83.8%) as a white solid. MS (ES+) 406.8 [1 / 2M+H]+.

[0426] General steps for the preparation of compound 11

[0427] Compound 10 (250 mg, 0.31 mmol) and compound 3 (145 mg, 0.37 mmol) were dissolved in DMF (5 ml), followed by the addition of HOBt (62 mg, 0.46 mmol), EDCI (88 mg, 0.46 mmol), and DIEA (113 μL, 0.61 mmol). The mixture was stirred at 25°C for 2 hours. HPLC monitoring showed that reactant 10 was completely reacted. The reaction mixture was rotary evaporated to remove the solvent to obtain a crude product. The crude product was purified by preparative HPLC. Compound 11 (70 mg, 19.1% yield) was obtained as a yellow solid. MS (ES+) 1186.4 [M+H]+.

[0428] General steps for the preparation of compound 12

[0429] Compound 11 (70.0 mg, 0.059 mmol) was dissolved in a THF / MeOH / H2O (5 mL) mixed solvent, and a solution of LiOH (14.1 mg, 0.59 mmol) in H2O (0.5 mL) was added. The mixture was stirred at 0°C for 0.5 hours. HPLC monitoring showed that compound 11 was completely reacted. The reaction mixture was rotary evaporated to remove the solvent to obtain a crude product. The crude product was purified by preparative HPLC. Compound 12 (40.0 mg, 64.8% yield) was obtained as a white solid. MS (ES+) 1046.4 [M+H]+.

[0430] General steps for the preparation of CPT009

[0431] Compound 12 (13.0 mg, 12.43 μmol) was dissolved in DMF (2.0 mL), followed by the addition of compound 13 (2.61 mg, 18.64 μmol). EDCI (3.57 mg, 18.64 μmol), HOBt (2.52 mg, 18.64 μmol), and DIEA (4.59 μL, 24.86 μmol) were subsequently added to the reaction mixture. The mixture was stirred at 25°C for 1 hour. HPLC monitoring indicated the reaction was complete. The reaction mixture was diluted with MeOH (2 mL), directly purified by preparative HPLC, and freeze-dried to afford CPT009 (5 mg, 34.4% yield) as a yellow solid. MS (ES+) 1076.4 [M+H]+.

[0432] Synthesis of CPT010

[0433] Synthesis method of compound 2:

[0434] A solution of compound 1 in N,N-dimethylformamide was diluted with 0.1 M sodium phosphate buffer (pH 7.0). C. rugosa lipase was added to the mixture, and the mixture was stirred at 25°C for 24 hours. The mixture was filtered, and the filtrate was extracted with ethyl acetate. The organic phase was washed with brine, dried over sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography to yield compound 2.

[0435] Synthesis method of compound 3

[0436] Compound 2 was added to an acetonitrile / water solution with TEMPO and BAIB. The mixture was stirred at 25°C for 18 hours. The solvent was evaporated in vacuo. The residue was extracted with DCM. The organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure to obtain a residue. The crude product was used in the next reaction.

[0437] Synthesis method of compound 4

[0438] Compound 3 was added to a methanol solution with EDCI and DMAP. The mixture was stirred at room temperature for 2 hours. The methanol was evaporated, and the residue was diluted with dichloromethane, washed with water, and then washed with brine. The organic extract was dried over anhydrous water, filtered, and concentrated under reduced pressure to obtain a residue. The crude product was purified by silica gel column chromatography to obtain compound 4 as a colorless oil.

[0439] Preparation method of compound 5

[0440] Compound 4, benzyl glycolate, 4A MS, and trimethylsilyltrifluoromethanesulfonic acid were degassed in anhydrous DCM and purged with N2 three times. The mixture was then stirred at 25°C under an N2 atmosphere for 3 hours. The reaction was monitored by TLC (eluent: PE / EA = 3 / 1). The reaction mixture was filtered to remove 4A MS. The residue was diluted with DCM and washed with Sat. NaHCO3. The organic phase was dried over Na2SO4 and concentrated. Purification was performed by silica gel chromatography.

[0441] Preparation method of compound 6

[0442] Compound 5 was dissolved in 4.4% formic acid-methanol solution and Pd / C was added. The mixture was stirred at 25°C for 5 hours. TLC (eluent: PE / EA = 2 / 1) showed complete consumption of compound 5. The reaction mixture was filtered through a layer of celite. The filtrate was concentrated under reduced pressure. The crude product was carried on to the next reaction without further purification.

[0443] Preparation method of compound 8

[0444] HOBt, EDCI and DIEA were added to the DMF solution of compound 6 and compound 7. The mixture was stirred at 25°C for 2 hours. HPLC showed that reactant 6 was completely consumed. The reaction mixture was concentrated under reduced pressure to remove the solvent. Pre-HPLC purification was performed. Compound 8 was obtained as a yellow solid.

[0445] Preparation method of compound 9

[0446] Compound 8 was dissolved in a THF / MeOH / H2O solution, followed by the addition of LiOH in H2O. The mixture was stirred at 0°C for 0.5 h. HPLC indicated complete consumption of compound 8. The reaction mixture was concentrated under reduced pressure to remove the solvent. Pre-HPLC purification was performed. Compound 9 was obtained as a white solid.

[0447] CPT010 preparation method

[0448] To a DMF solution of compound 9 and 1-(2-aminoethyl)-1h-pyrrole-2,5-dione hydrochloride, TBTU and DIEA were added. The mixture was stirred at 25°C for 1 hour. HPLC indicated the reaction was complete. The reaction mixture was diluted with MeOH and directly purified by pre-HPLC. Lyophilization afforded CPT010 as a yellow solid.

[0449] Synthesis of MMAE derivative 1

[0450] General steps for the preparation of compound 2

[0451] Compound 1 (10.0 g, 24.4 mmol) and PAB (6.00 g, 48.7 mmol) were dissolved in a mixed solvent of DCM (80 ml) and MeOH (80 ml), and then EEDQ (12.0 g, 4.87 mmol) was added under light-shielding conditions. The mixture was stirred at 45°C for 1.5 hours. LC-MS monitoring showed that reactant 1 had reacted completely, and the main peak was the product peak. The reaction mixture was evaporated to remove the solvent to obtain a crude product. The crude product was slurried with MTBE: EtOAc (10: 1), filtered and centrifuged to dry to obtain the product. The product was used for the next reaction without further purification. Compound 2 (11.4 g, yield 90.7%) was obtained as a white solid. MS (ES+) 516.3 [M+H] +.

[0452] General steps for the preparation of compound 3

[0453] Compound 2 (11.4 g, 22.1 mmol) and PNP (13.4 g, 44.2 mmol) were dissolved in DMF (114 ml), and DIEA (7.69 ml, 44.2 mmol) was added. The mixture was stirred at 25°C for 1.5 hours. LC-MS monitoring showed that reactant 2 had reacted completely, with the main peak being the product. The reaction mixture was precipitated with icy isopropyl ether (1500 ml), filtered, and centrifuged to obtain a solid. The solid was used in the next reaction without further purification. Compound 3 (13.9 g, 92.3% yield) was obtained as a yellow solid. MS (ES+) 681.4 [M+H]+.

[0454] General steps for the preparation of compound 4

[0455] Compound 3 (1.00 g, 1.47 mmol) and MMAE (1.06 g, 1.47 mmol) were dissolved in DMF (10 ml), followed by the addition of HOBt (218 mg, 1.62 mmol) and DIEA (512 μL, 2.94 mmol). The mixture was stirred at 25°C for 16 hours. LC-MS monitoring indicated complete consumption of the reactants, with the product as the main peak. 20% triethylamine was then added to the reaction mixture, and the reaction was continued at 25°C with stirring for 2 hours. The reaction solution was purified by preparative HPLC (TFA conditions). Column: Phenomenex luna C18 250*50 mm*10 μm; mobile phase: [water (TFA)-acetonitrile]; B%: 30%-60% over 20 minutes. Compound 4 (910 mg, 59.7% yield) was obtained as a white solid. MS (ES+) 1037.6 [M+H]+.

[0456] General steps for the preparation of compound 5

[0457] Compound 4 (400 mg, 386 μmol) and compound 5-1 (151 mg, 386 μmol) were dissolved in DMF (4 ml), followed by the addition of HOBt (115 mg, 848 μmol), DIEA (269 μl, 1.54 mmol), and DIC (239 μl, 1.54 mmol). The mixture was stirred at 25°C for 6 hours. LC-MS monitoring showed that compound 4 had reacted completely, with the main peak being the product. The reaction mixture was precipitated with 100 ml of isopropyl ether (100 ml * 3), filtered, and centrifuged to obtain a solid. The solid was used in the next reaction without further purification. Compound 5 (429 mg, 78.8% yield) was obtained as a white solid. MS (ES+) 1411.5 [M+H]+.

[0458] General steps for the preparation of compound 6

[0459] Compound 5 (200 mg, 142 μmol) was dissolved in a mixed solvent of THF (1 mL) and H₂O (1 mL), followed by the addition of LiOH·H₂O (59.5 mg, 1.42 mmol). The mixture was stirred at 0°C for 5 minutes. LC-MS monitoring indicated complete consumption of compound 5, with the main peak being the product. The pH of the reaction mixture was adjusted to acidic by the addition of CH₃COOH at 0°C. The mixture was purified by preparative HPLC (TFA conditions). Column: Welch Ultimate C₁₈ 150*25mm*5um; Mobile phase: [water (TFA)-acetonitrile]; B%: 31%-61% over 10 minutes. Compound 6 (100 mg, 55.5% yield) was obtained as a white solid. MS (ES+) 1271.5 [M+H]+.

[0460] General procedure for the preparation of MMAE derivative 1

[0461] Compound 6 (50.0 mg, 39.3 μmol) and compound 7-1 (16.5 mg, 118 μmol) were dissolved in DMF (0.5 mL), followed by the addition of DIEA (27.4 μL, 157 μmol) and TBTU (25.3 mg, 78.7 μmol). The mixture was stirred at 25°C for 1 hour. LC-MS monitoring indicated complete reaction of compound 6, with the main peak being the product. The pH of the reaction mixture was adjusted to acidic by the addition of TFA at 0°C. The mixture was purified by preparative HPLC (TFA conditions). Column: Welch Ultimate C18 150*25 mm*5 μm; mobile phase: [water (TFA)-acetonitrile]; B%: 30%-60% over 10 minutes. MMAE derivative 1 (28.0 mg, 51.09% yield) was obtained as a white solid. MS (ES+) 1393.7 [M+H]+.

[0462] Synthesis of MMAE derivative 2

[0463] General steps for the preparation of compound 2

[0464] Compound 1 (1.00 g, 2.44 mmol) and compound 1-1 (746 mg, 4.87 mmol) were dissolved in a mixed solvent of DCM (8 ml) and MeOH (8 ml), and then EEDQ (1.20 g, 4.87 mmol) was added under light-shielding conditions. The mixture was stirred at 25°C for 2 hours. LC-MS monitoring showed that compound 1 had completely reacted, and the main peak was the product. The solvent was removed by rotary evaporation to obtain a crude product. The crude product was slurried with MTBE: EtOAc (10: 1), filtered, and the solid was dried by rotary evaporation. The product was used in the next step without further purification. Compound 2 (611 mg, yield 46%) was obtained as a brown solid. MS (ES+) 546.4 [M+H]+.

[0465] General steps for the preparation of compound 3

[0466] Compound 2 (0.611 g, 1.12 mmol) and PNP (1.36 g, 4.48 mmol) were dissolved in DMF (6.2 ml), followed by the addition of DIEA (585 μL, 3.36 mmol). The reaction mixture was stirred at 25°C for 4.5 hours. LC-MS monitoring indicated that compound 2 had reacted completely, with the main peak being the product. 150 ml of ice-cold isopropyl ether (150 ml * 3) was added to the reaction mixture for precipitation, which was then filtered and the solid was spin-dried. The product was used in the next step without further purification. Compound 3 (0.272 g, 34.2% yield) was obtained as a brown solid. MS (ES+) 711.4 [M+H]+.

[0467] General steps for the preparation of compound 4

[0468] Compound 3 (0.272 g, 383 μmol) and MMAE (275 mg, 382.71 μmol) were dissolved in DMF (3 mL), followed by the addition of HOBt (56.9 mg, 421 μmol) and DIEA (133 μL, 765 μmol). The mixture was stirred at 25°C for 16 hours. LC-MS monitoring indicated complete reaction, with the product as the main peak. 20% TEA was then added to the reaction mixture, and the reaction was continued at 25°C with stirring for 2 hours. The reaction solution was purified by preparative HPLC (TFA conditions). Column: Phenomenex Luna C18 200*40 mm*10 μm; mobile phase: [water (TFA)-acetonitrile]; B%: 30%-60% over 10 minutes. Compound 4 (0.212 g, 51.9% yield) was obtained as a brown solid. MS (ES+) 1067.5 [M+H]+.

[0469] General steps for the preparation of compound 5

[0470] Compound 4 (0.212 g, 199 μmol) and compound 5-1 (156 mg, 397 μmol) were dissolved in DMF (2.2 mL). HOBt (59.0 mg, 437 μmol), DIEA (138 μL, 794 μmol), and DIC (123 μL, 794 μmol) were then added. The mixture was stirred at 25°C for 2 hours. LC-MS monitoring indicated complete reaction of compound 4, with the main peak being the product. The pH of the reaction mixture was adjusted to acidic by adding CH3COOH. The mixture was purified by preparative HPLC (TFA conditions). Column: Phenomenex Luna C18 200*40 mm*10 μm; mobile phase: [water (TFA)-acetonitrile]; B%: 43%-73% over 10 minutes. Compound 5 (0.118 g, 41.2% yield) was obtained as a yellow solid. MS(ES+)1441.6[M+H]+.

[0471] General steps for the preparation of compound 6

[0472] Compound 5 (100 g, 69.4 μmol) was dissolved in a mixed solvent of H₂O (0.8 ml) and THF (0.2 ml), followed by the addition of LiOH·H₂O (29.1 mg, 694 μmol). The mixture was stirred at 0°C for 5 minutes. LC-MS monitoring indicated complete reaction of compound 5, with the main peak being the product. The pH of the reaction mixture was adjusted to acidic by the addition of CH₃COOH. The mixture was purified by preparative HPLC (TFA conditions). Column: Welch Ultimate C₁₈ 150*25mm*5um; Mobile phase: [water (TFA)-acetonitrile]; B%: 30%-60% over 10 minutes. Compound 6 (45.0 mg, 49.9% yield) was obtained as a white solid. MS (ES+) 1301.6 [M+H]+.

[0473] General steps for the synthesis of MMAE derivative 2

[0474] Compound 6 (45.0 mg, 34.6 μmol) and compound 7-1 (7.27 mg, 51.9 μmol) were dissolved in DMF (0.5 mL), followed by the addition of TBTU (22.2 mg, 69.2 μmol) and DIEA (24.1 μL, 138 μmol). The mixture was stirred at 25°C for 1 hour. LC-MS monitoring indicated complete reaction of compound 6, with the main peak being the product. The pH of the reaction mixture was adjusted to acidic by the addition of trifluoroacetic acid (TFA). The mixture was purified by preparative HPLC (TFA conditions). Column: Welch Xtimate C18 150*25 mm*5 μm; mobile phase: [water (TFA)-acetonitrile]; B%: 30%-60% over 10 minutes. The MMAE derivative 2 (o-OMe-PAB) (32.0 mg, 65.0% yield) was obtained as a white solid. MS(ES+)1424.6[M+H]+.

[0475] Synthesis of Comp.5

[0476] General steps for the synthesis of Comp.5-2:

[0477] Fmoc-VA-Pab-OH (1.0 g, 1.9 mmol, 1.0 eq) was dissolved in dichloromethane (50 ml, 20% v / v triethylamine) containing 20% ​​triethylamine. The mixture was stirred at 25°C for 16 hours. No starting material remained after TLC. The solvent was removed by rotary evaporation to obtain a crude product as a white solid. The crude product was used directly in the next reaction without purification. The crude product was dissolved in 50 mL of dichloromethane and comp.5-1 (0.91 g, 2.3 mmol, 1.2 eq) was added. After stirring, N,N′-diisopropylcarbodiimide (DIC, 0.29 g, 2.3 mmol, 1.2 eq) was added dropwise. The mixture was stirred at 25°C for 5 hours. No NH2-VA-Pab-OH remained after TLC. The reaction was terminated by adding 50 mL of water. After separation, the organic phase was washed twice with 50 mL of water, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10:1) and rotary evaporation to obtain 800 mg of an off-white solid product comp.5-2.

[0478] General steps for the synthesis of Comp.5-3:

[0479] Comp.5-2 (800 mg, 1.2 mmol, 1.0 eq) was dissolved in 50 mL of N,N-dimethylformamide, and di-p-nitrophenol carbonate (800 mg, 2.6 mmol, 2.2 eq) was added. The mixture was stirred until uniformly dissolved, and triethylamine (400 μL, 2.9 mmol, 2.4 eq) was added dropwise. The mixture was stirred at 25°C for 2 hours. TLC analysis revealed no residual comp.5-2. 100 mL of water and 100 mL of ethyl acetate were added, stirred uniformly, and the layers separated. The aqueous phase was extracted twice with 50 mL of ethyl acetate, and the organic phases were combined. The organic phases were washed twice with 50 mL of 3% (m / m) sodium carbonate solution and once with 50 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10:1) and rotary evaporation to yield 620 mg of comp.5-3 as a pale yellow solid.

[0480] General steps for the synthesis of Comp.5-4:

[0481] Comp. 5-3 (400 mg, 0.48 mmol, 1.0 eq) was dissolved in 4 mL of N,N-dimethylformamide, and a solution of N,N'-dimethylethylenediamine (600 mg, 4.8 mmol, 10.0 eq) in 4 mL of N,N-dimethylformamide was added dropwise. The mixture was stirred at 25°C for half an hour. The reaction was monitored by TLC. The solvent was removed by rotary evaporation to obtain the crude product, which was purified by silica gel column chromatography (dichloromethane / methanol = 10:1). 210 mg of the product, comp. 5-4, was obtained as a pale yellow solid by rotary evaporation.

[0482] General steps for the synthesis of Comp.5-5:

[0483] SN38 (5.0 mg, 12.7 μmol, 1.0 eq) and di-p-nitrophenol carbonate (3.8 mg, 12.7 μmol, 1.0 eq) were dissolved in 5 mL of dichloromethane. After stirring for three hours, the solvent was evaporated and 2 mL of N,N-dimethylformamide was added to prepare an SN38-PNP solution.

[0484] Comp.5-4 (9 mg, 11.5 μmol, 1.0 eq) was dissolved in 2 mL of tetrahydrofuran. 200 μL of aqueous lithium hydroxide solution (0.3 mol / mL, 60 μmol, 5.2 eq) was added at -5°C and the reaction was maintained for half an hour. TLC confirmed that no starting material remained. Dilute hydrochloric acid was added to adjust the pH to 3-4. The solvent was removed by rotary evaporation, and 2 mL of N,N-dimethylformamide was added and stirred until dissolved. A solution of SN38-PNP was added, and the mixture was stirred at 25°C for four hours. The reaction solution was purified by preparative liquid chromatography to obtain 10.4 mg of comp.5-5 as a yellow solid.

[0485] Mobile phase A: acetonitrile (0.1% trifluoroacetic acid), mobile phase B: water (0.1% trifluoroacetic acid)

[0486] Structural characterization: LC-MS: 1059.4[M]

[0487] General steps for the synthesis of Comp.5:

[0488] Comp.5-5 (10.4 mg, 9.8 μmol, 1.0 eq) was dissolved in 2 mL of N,N-dimethylformamide and stirred at 25°C. N-(2-aminoethyl)maleimide hydrochloride (4.0 mg, 22.6 μmol, 2.3 eq), benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (4.5 mg, 12.0 μmol, 1.2 eq), and diisopropylethylamine (5 μL) were added sequentially. The mixture was stirred at 25°C for 1.5 hours. HPLC analysis confirmed the absence of starting material comp.5-5. The reaction solution was purified by preparative liquid chromatography to yield 5.7 mg of comp.5 as a yellow solid.

[0489] Mobile phase A: acetonitrile (0.1% trifluoroacetic acid), mobile phase B: water (0.1% trifluoroacetic acid)

[0490] Structural characterization: LC-MS: 1081.4[M].

[0491] Synthesis of Comp6

[0492] Compound R1 (19 mg, 19.2 umol) was dissolved in DMF (3.0 mL), and commercially available compound R2 (5.5 mg, 28.8 umol), TBTU (12.3 mg, 38.4 umol) and DIEA (13.4 uL, 76.8 umol) were added to the solution in sequence. The reaction solution was reacted at 25°C for 1 hour. The reaction of R1 was completed as monitored by HPLC. The reaction solution was filtered to remove insoluble matter and purified using preparative HPLC. The resulting solution was lyophilized to obtain Comp6, which was a yellow solid with MS: 1125.1.

[0493] Comp7 Synthesis

[0494] Compound R1 (19 mg, 19.2 umol) was dissolved in DMF (3.5 mL), and commercially available compound R2 (5.9 mg, 28.8 umol), TBTU (12.3 mg, 38.4 umol) and DIEA (13.4 uL, 76.8 umol) were added to the solution in sequence. The reaction solution was reacted at 25°C for 1 hour. The reaction of R1 was completed by HPLC monitoring. The reaction solution was filtered to remove insoluble matter and purified by preparative HPLC. The obtained solution was lyophilized to obtain Comp7, which was a yellow solid with MS: 1139.2.

[0495] Comp8 Synthesis

[0496] Compound R1 (19 mg, 19.2 umol) was dissolved in DMF (4.0 mL), and commercially available compound R2 (8.5 mg, 28.8 umol), TBTU (12.3 mg, 38.4 umol) and DIEA (13.4 uL, 76.8 umol) were added to the solution in sequence. The reaction solution was reacted at 25 ° C for 1 hour. The reaction of R1 was monitored by HPLC. The reaction solution was filtered to remove insoluble matter and purified by preparative HPLC. The obtained solution was lyophilized to obtain Comp8, which was a yellow solid with MS: 1153.2.

[0497] Comp9 Synthesis

[0498] Compound R1 (19 mg, 19.2 umol) was dissolved in DMF (3.0 mL), and commercially available compound R2 (8.5 mg, 28.8 umol), TBTU (12.3 mg, 38.4 umol) and DIEA (13.4 uL, 76.8 umol) were added to the solution in sequence. The reaction solution was reacted at 25°C for 1 hour. The reaction of R1 was monitored by HPLC. The reaction solution was filtered to remove insoluble matter and purified by preparative HPLC. The obtained solution was lyophilized to obtain Comp9, which was a yellow solid with MS: 1155.2.

[0499] Example 2: Preparation and effect evaluation of ADC

[0500] CPD-ADC preparation

[0501] The anti-TROP2 antibody hRS7 (concentration 13.6 mg / mL) was replaced with a 40 mM Pb / 2.0 mM EDTA (pH 7.0) buffer using a tangential flow ultrafiltration system. 1.8-2.2 equivalents of TCEP were added and stirred at 25°C for 2-4 hours to partially open the disulfide bonds between the antibody chains. The linker-drug conjugates CPD1, CPD2, CPD3, and GGFG-Dxd prepared in Example 1 were dissolved in DMSO, and 5-8 equivalents of the linker-drug conjugate were slowly added to the reduced antibody solution. DMSO was added to a final concentration of 5-20% (v / v). The reaction was stirred at 25°C for 1-4 hours. After the reaction was complete, the sample was filtered using a 0.22 μm membrane. The unconjugated small molecules were purified using a tangential flow ultrafiltration system. The buffer was 20 mM L-His solution (pH 5.5) and stored in a -80°C refrigerator. The DAR value was determined using hydrophobic interaction chromatography. The results are shown in Table 1 below.

[0502] Table 1: DAR value results

[0503] The structure of GGFG-Dxd is as follows:

[0504] hRS7-light chain (SEQ ID NO: 1)

[0505] hRS7-heavy chain (SEQ ID NO: 2)

[0506] Datopotamab-light chain (SEQ ID NO: 3)

[0507] Datopotamab-heavy chain (SEQ ID NO: 4)

[0508] Effect Example 1: In vitro cell activity test

[0509] A431 cells with high expression of TROP2 were selected as the cell line for this in vitro activity test to investigate the dose-effect of ADC on cell killing. The initial seeding density of each cell type was 10 3 -10 5Cells / well were added, and cytotoxic activity was determined after 1-7 days. For sample concentration, the final concentration of the antibody-drug conjugate prepared in Example 2 was set to 50 μg / mL as the starting concentration after sample addition. A series of 8 concentrations (2-10 dilutions) from 50 to 0.00064 μg / mL was designed. The killing (or inhibition) was detected after 1-7 days using CellTiter-Glo@Luminescent Cell Viability Assay chemiluminescence staining, and the IC was calculated. 50 The results of the in vitro cell activity test are shown in Table 2 below.

[0510] Table 2: In vitro cell activity test results

[0511] Effect Example 2

[0512] In vivo evaluation 1

[0513] Female Balb / c nude mice aged 6-8 weeks were subcutaneously injected with 5×10 6 Human lung squamous cell carcinoma cells (EBC-1). The average tumor volume is about 100 mm 3 At 11 days after inoculation, the 48 nude mice were randomly divided into 8 groups according to tumor size, with 6 animals in each group, and the drugs were injected into the tail vein once:

[0514] Group 1 was the blank control group;

[0515] Group 2: ADC07 (10 mg / kg);

[0516] Group 3: ADC01 (2 mg / kg);

[0517] Group 4: ADC01 (10 mg / kg);

[0518] Group 5: ADC02 (10 mg / kg);

[0519] Group 6: ADC03 (2 mg / kg);

[0520] Group 7: ADC03 (10 mg / kg);

[0521] Group 8 was ADC04 (10 mg / kg).

[0522] Animal body weight and tumor volume were measured twice weekly, and animal survival was observed throughout the experiment. The results (Figures 1 and 2) show that ADC01, ADC02, ADC03, and ADC04 all exhibited anti-tumor activity compared to the control, with ADC03 and ADC04 showing superior in vivo anti-tumor activity. Furthermore, no mortality or weight loss was observed in any of the experimental mice, demonstrating the excellent safety of ADC03 and ADC04.

[0523] In vivo evaluation 2

[0524] Female Balb / c nude mice aged 6-8 weeks were subcutaneously injected with 5×10 6 Human lung adenocarcinoma cells (Calu-3). The average tumor volume is approximately 100 mm 3 At 4 days after inoculation, the 48 nude mice were randomly divided into 8 groups according to the tumor size, with 6 animals in each group, and the drugs were injected into the tail vein once:

[0525] Group 1 was the blank control group;

[0526] Group 2: ADC07 (8 mg / kg);

[0527] Group 3: ADC01 (2 mg / kg);

[0528] Group 4: ADC01 (8 mg / kg);

[0529] Group 5: ADC02 (8 mg / kg);

[0530] Group 6: ADC03 (2 mg / kg);

[0531] Group 7: ADC03 (8 mg / kg);

[0532] Group 8 was ADC04 (8 mg / kg).

[0533] Animal body weight and tumor volume were measured twice weekly, and animal survival was observed throughout the experiment. The results (Figures 3 and 4) show that ADC01, ADC02, ADC03, ADC04, and ADC07 all exhibited anti-tumor activity compared to the control, with ADC03 and ADC04 showing superior in vivo anti-tumor activity. Furthermore, no mortality or weight loss was observed in any of the experimental mice, demonstrating the excellent safety of ADC03 and ADC04.

[0534] Example 3: Preparation and efficacy evaluation of drug-conjugated anti-Trop2 antibody ADC

[0535] Drug sources: Deruxtecan (linker-cytotoxin, CAS No.: 1599440-13-7) and CL2A-SN-38 were purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd.

[0536] The anti-Trop2 antibody used was sacituzumab (hRS7, prepared by the applicant).

[0537] The preparation method of the conjugate prepared by coupling the anti-Trop2 antibody to the linker-cytotoxin is as follows:

[0538] Step 1: Add TCEP (tris(2-carbonylethyl)phosphine, 2.1 eq.) to the antibody in a buffer solution of pH 6.5, 50 mM PB / 2 mM EDTA. The reaction is stirred at 25°C for 4 hours to obtain a reduced antibody solution. Step 2: Add DMA (N,N-dimethylacetamide) and an appropriate amount of cytotoxin CPD2 (e.g., 6.0 eq., 5.5 eq.) (total DMA ratio is 10%) to the reduced antibody solution. The reaction is then maintained at 25°C for 1 hour. The reaction solution is then replaced with a buffer solution (e.g., 20 mM L-histidine / pH 5.5, 50 mM MES / pH 6.0, or 20 mM L-histidine / pH 5.5), filtered to obtain the antibody-drug conjugate ADC of the present invention, and stored at -60 to -90°C. Where m = 0-8. Free small molecules are detected by reverse-phase high-performance liquid chromatography, and DAR is detected by hydrophobic interaction chromatography. The purity was determined by size exclusion chromatography.

[0539] In vitro killing experiment of anti-Trop2 antibody ADC

[0540] Sacituzumab (hereinafter referred to as hRS7-DXD, synthesized by the applicant) coupled with DXD (Deruxtecan, #HY-13631E, MCE) after concentration gradient dilution, control IgG1 protein (antiKLH-DXD) coupled with DXD, hRS7-CPD1, hRS7-CPD2, hRS7-CPD3, hRS7-CPD4, hRS7-CPD5, hRS7-CPD6, and hRS7-CPD7 obtained after coupling the linker-drug conjugate of the present invention. PD6, hRS7-CPT004, hRS7-CPT005, hRS7-CPT006, hRS7-CPT009, hRS7-SN38 obtained by coupling comp.5 to SN38, hRS7-CL2A-SN38 obtained by coupling CL2A-SN38 (MCE), and coupled control IgG1 protein (antiKLH-CPD1) were co-cultured with Trop2-expressing cells BxPC-3 (ATCC#CRL-1555) for 6 days, and the number of viable cells was determined by cell counting lite (Vazyme#DD1102-01). The in vitro killing activity of the candidate molecules was determined by calculating the proliferation inhibition rate (proliferation inhibition rate (%) = 100*(the number of viable cells in the untreated group - the number of viable cells in the treated group) / (the number of viable cells in the untreated group - the number of viable cells in the solution control group)%).

[0541] The results are shown in Figures 5 and 6 and Tables 3 and 4.

[0542] Table 3: Inhibitory effect of candidate molecules and negative control ADC on BxPC-3 cell proliferation (EC 50 ) Note: NA means no killing was observed and cannot be calculated.

[0543] As shown in Figure 5 and Table 3, compared with anti-KLH-CPD1, hRS7-CPD1 / hRS7-CPD2 / hRS7-CPD3 / hRS7-CPD4 / hRS7-CPD5 / hRS7-CPD6 had specific inhibitory effects on the proliferation of BxPC-3 cells. hRS7-DXD also had a specific inhibitory effect on the proliferation of BxPC-3 cells. The proliferation inhibitory activities of the CPD1, CPD2, CPD3, CPD4, CPD5, and CPD6 conjugates were comparable to those of the DXD conjugate.

[0544] Table 4: Inhibitory effect of candidate molecules and negative control ADC on BxPC-3 cell proliferation (EC 50 ) Note: NA means no killing was observed and cannot be calculated.

[0545] As shown in Table 4 and Figure 6, hRS7-CPT004, hRS7-CPT005, hRS7-CPT006, and hRS7-CPT009 significantly inhibited the proliferation of BxPC-3 cells, with activity comparable to that of hRS7-DXD. hRS7-SN38 also had an inhibitory effect on the proliferation of BxPC-3 cells.

[0546] Example 4: Preparation and efficacy evaluation of comp6 and other conjugated anti-Trop2 antibody ADCs

[0547] Drug source: comp6, comp7, comp8 and comp9 are prepared by the applicant.

[0548] The anti-Trop2 antibody used was Sacituzumab (hRS7, prepared by the applicant).

[0549] Preparation of ADC

[0550] The preparation method of the conjugate prepared by coupling the anti-Trop2 antibody to the linker-cytotoxin is as follows:

[0551] Step 1: Add TCEP (tris(2-carbonylethyl)phosphine, 2.4 eq.) to the antibody in a buffer solution of 50 mM PBS / 2 mM EDTA, pH 6.5, and stir at 25°C for 3.5 h to obtain a reduced antibody solution.

[0552] Step 2: Add DMA (N,N-dimethylacetamide), comp6, comp7, comp8, or comp9 (7.0 eq.) (total DMA ratio 20%) to the reduced antibody solution, control the temperature at 25°C, and react for 1 hour. Replace the reaction solution with a buffer solution (20 mM Histidine / pH 5.5), filter, and obtain the antibody-drug conjugate ADC of the present invention. Store at -60 to -90°C. Where m = 0-8. Free small molecules are detected by reverse-phase HPLC, and DAR is detected by PLRP chromatography. Purity is determined by size-exclusion chromatography.

[0553] The DAR values ​​of ADC of comp6, comp7, comp8 and comp9 detected by PLRP chromatography were 3.49, 3.56, 3.52 and 3.54 respectively.

[0554] In vitro killing experiment

[0555] DXD (Deruxtecan, #HY-13631E, MCE)-conjugated saxotuzumab ADC (hRS7-DXD), DXD-conjugated control IgG1 protein (antiKLH-DXD), and Comp6, 7, 8, or 9-conjugated saxotuzumab ADCs, designated hRS7-C6, hRS7-C7, hRS7-C8, and hRS7-C9, respectively, were co-cultured with Trop2-expressing BxPC-3 cells (ATCC #CRL-1555) for 6 days. The number of viable cells was determined using cell counting lite (Vazyme #DD1102-01), and the in vitro cytotoxic activity of the candidate molecules was determined by calculating the proliferation inhibition rate (proliferation inhibition rate (%) = 100 * (number of viable cells in the untreated group - number of viable cells in the treated group) / (number of viable cells in the untreated group - number of viable cells in the solution control group) %).

[0556] As shown in Table 5 and Figure 7, hRS7-C6, hRS7-C7, hRS7-C8, and hRS7-C9 had significant inhibitory effects on the proliferation of BxPC-3 cells, which were comparable to the activity of hRS7-DXD.

[0557] Table 5: Inhibitory effect of candidate molecules and negative control ADC on BxPC-3 cell proliferation (EC 50 ) Note: NA means no killing was observed and cannot be calculated.

[0558] Example 5: Anti-BCMA Antibody ADC

[0559] The anti-BCMA antibody is coupled to a linker-cytotoxin vc-MMAE or CPD to prepare a conjugate.

[0560] Drug sources: vc-MMAE, vc-MMAF, and deruxtecan (DXD) were purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd.; Linker-MMAE derivative 1, Linker-MMAE derivative 2, and CPD (i.e., CPD1) were commissioned to Shanghai WuXi AppTec Co., Ltd. for preparation.

[0561] The anti-BCMA antibody anti-BCMA-005-Hu178 was prepared by the applicant, and the amino acid sequences of its heavy and light chains are shown below:

[0562] anti-BCMA-005-Hu178 heavy chain (SEQ ID NO: 5):

[0563] anti-BCMA-005-Hu178 light chain (SEQ ID NO: 6):

[0564] The control IgG1 protein was prepared by the applicant.

[0565] Preparation of ADC

[0566] Step 1: Add TCEP (tris(2-carbonylethyl)phosphine, 2.4 eq.) to the antibody in a pH 7.0 buffer solution containing 40 mM PB. Control the temperature at 37°C and stir the reaction for 4 h to obtain a reduced antibody solution.

[0567] Step 2: DMA (N,N-dimethylacetamide), vc-MMAE, vc-MMAF, deruxtecan (DXD), Linker-MMAE derivative 1, Linker-MMAE derivative 2, or CPD (i.e., CPD1) (8.0 eq.) were added to the reduced antibody solution (total DMA ratio was 10%), the temperature was controlled at 25°C, and the reaction was allowed to proceed for 1 hour. The reaction solution was replaced with a buffer solution (20 mM L-His / pH 5.5), filtered to obtain the antibody-drug conjugate ADC of the present invention, and stored at -60 to -90°C. Where m = 0-8. Free small molecules were detected by reverse-phase high-performance liquid chromatography, and DAR was detected by HIC chromatography. Purity was determined by size exclusion chromatography.

[0568] Results The HIC-DAR detection value of the antibody conjugated with vc-MMAE was 3.78, the HIC-DAR detection value of the antibody conjugated with vc-MMAF was 3.86, the HIC-DAR detection value of the antibody conjugated with Linker-MMAE derivative 1 was 3.73, the HIC-DAR detection value of the antibody conjugated with Linker-MMAE derivative 2 was 3.79, the HIC-DAR detection value of anti-BCMA-CPD1-D4 was 4.24, the HIC-DAR detection value of anti-BCMA-CPD1-D8 was 7.73, and the HIC-DAR detection value of the anti-KLH antibody conjugated with linker-cytotoxin DXD was 7.34.

[0569] In vitro killing experiment

[0570] vcMMAE (MCE)-conjugated anti-BCMA monoclonal antibody (abbreviated as anti-BCMA-vcMMAE), vcMMAF-conjugated anti-BCMA monoclonal antibody (abbreviated as anti-BCMA-vcMMAF), Linker-MMAE derivative 1, Linker-MMAE derivative 2, CPD-conjugated anti-BCMA (abbreviated as anti-BCMA-Linker-MMAE derivative 1, anti-BCMA-Linker-MMAE derivative 2, anti-BCMA-CPD-D4, anti-BCMA-CPD-D8, respectively), CPD-conjugated control IgG1 protein (abbreviated as anti-KLH-CPD-D4), MMAF or MMAE-conjugated control IgG1 protein (abbreviated as anti-KLH-MMAF-D4, anti-KLH-MMAE-D4, respectively) diluted in a concentration gradient were co-cultured with BCMA-expressing MM.1S cells for 4 days, and the cells were counted by cell counting. The number of viable cells was determined by the ELISA lite (Vazyme #DD1102-01) assay, and the in vitro cytotoxic activity of the candidate molecule was determined by calculating the proliferation inhibition rate (proliferation inhibition rate%, proliferation inhibition rate = 100*(number of viable cells in the untreated group - number of viable cells in the treated group) / (number of viable cells in the untreated group - number of viable cells in the solution control group)%).

[0571] The results are shown in Tables 6, 7 and Figures 8, 9.

[0572] Table 6: Inhibitory effect of candidate molecules and negative control ADC on MM.1S cell proliferation (EC 50 )

[0573] Table 7: Inhibitory effect of candidate molecules and negative control ADC on MM.1S cell proliferation (EC50) Note: NA means no killing was observed and cannot be calculated.

[0574] As shown in Tables 6, 7, Figures 8 and 9, anti-BCMA-vcMMAE, anti-BCMA-vcMMAF, anti-BCMA-Linker-MMAE derivative 1, anti-BCMA-Linker-MMAE derivative 2, anti-BCMA-CPD-D4, and anti-BCMA-CPD-D8 significantly inhibited the proliferation of MM.1S cells. Among them, anti-BCMA-Linker-MMAE derivative 1 is anti-BCMA-005-Hu178-glucoseMMAE-NC-D4, and anti-BCMA-Linker-MMAE derivative 2 is anti-BCMA-005-Hu178-OMeMMAE-D4; anti-BCMA-vcMMAE refers to anti-BCMA-005-Hu178-vcMMAE, and anti-BCMA-vcMMAF refers to anti-BCMA-005-Hu178-vcMMAF.

Claims

1. A linker, the structure of which is shown in Formula A: in: L1 is a functional group that can react with sulfhydryl groups; L2 and Y are each independently selected from -(CH2) p -、-(OCH2CH2) p -and-(CH2CH2O) p -; L3 is a hydrophilic group; L4 is an amino acid group; R' is -OR or -NR"-C1-C6 alkylene-NR"R"'; R" and R"' are each independently H and C1-C6 alkyl; R is selected from H and phenyl optionally substituted with one or more nitro groups; R6 is selected from H, C1-C6 alkoxy, -(OCH2CH2) p O-C1-C6 alkyl and -(CH2CH2O) p -C1-C6 alkyl; X is selected from -NH-, -NH(CH2) n C(=O)-, -C(=O)(CH2) n NH- and -(CH2) n C(=O)-; Z is selected from C and S; m is selected from 0 and 1; n is selected from 0, 1 and 2; p is selected from integers from 1 to 10.

2. The linker according to claim 1, wherein The structure of the linker is shown in Formula I: in: L1 is a functional group that can react with sulfhydryl groups; L2 and Y are each independently selected from -(CH2) p -、-(OCH2CH2) p -and-(CH2CH2O) p -; L3 is a hydrophilic group; L4 is an amino acid group; R is selected from H and phenyl optionally substituted with one or more nitro groups; R6 is selected from H, C1-C6 alkoxy, -(OCH2CH2) p O-C1-C6 alkyl and -(CH2CH2O) p -C1-C6 alkyl; X is selected from -NH-, -NH(CH2) n C(=O)-, -C(=O)(CH2) n NH- and -(CH2) n C(=O)-; Z is selected from C and S; m is selected from 0 and 1; n is selected from 0, 1 and 2; p is selected from integers from 1 to 10.

3. The linker according to claim 1 or 2, wherein L1 is selected from maleimide, halogen, halogen-substituted functional groups (such as halogen-substituted aldehyde groups, halogen-substituted -S(O)2-), aldehyde groups, alkenyl groups, alkynyl groups, alkanone groups, sulfonyl groups, silane groups, isocyanate groups and norbornene groups.

4. The linker according to claim 3, wherein L1 is selected from: and their derivatives; among which: R7, R8 and R9 are each independently selected from H, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, heteroaryl, C3-C9 cycloalkyl, C3-C9 heterocyclyl, -(OCH2CH2) p OCH3, -NO2, -CN, -SCN, -OR 10 、-SR 10 、-N(R 10 )2, -C(=O)R 10 、-C(=O)OR 10 、-C(=O)N(R 10 )2、-C(=S)OR 10 、-C(=S)N(R 10 )2、-C(=S)SR 10 、-NR 10 (C=O)R 10 、-NR 10 (C=S)N(R 10 )2、-O(C=O)N(R 10 )2、-S(=O)2R 10 、-S(=O)2OR 10 ; where each R 10 Independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl; p is an integer from 1 to 6; A is a halogen; The wavy line indicates where L1 joins L2; Preferably, L1 is:

5. The linker according to any one of claims 1 to 4, wherein L2 is -(CH2) p -, and p is an integer of 1-4; preferably, L2 is -CH2CH2- or -CH2CH2CH2-; and / or X is -NH- or -NH(CH2) n C(=O)-; preferably, X is -NH- or -NHC(=O)-; and / or Y is -(CH2) p -, p is 1, 2 or 3; preferably, Y is methylene; and / or Z is C.

6. The linker according to any one of claims 1 to 5, wherein L3 is a divalent hydrophilic group having two monovalent radical centers generated by removing two hydrogen atoms from a monosaccharide, a disaccharide, a five-membered or six-membered saturated heterocycle containing 1-2 nitrogen atoms, and a derivative thereof; Preferably, the monosaccharide is selected from the group consisting of triose, tetraose, pentose, hexose and heptose; Preferably, the disaccharide is selected from maltose, sucrose and lactose; Preferably, the five-membered or six-membered saturated heterocycle containing 1-2 nitrogen atoms or its derivatives are selected from piperazinyl, piperidinyl and pyrrolidinyl.

7. The linker according to any one of claims 1 to 6, wherein L3 is a divalent hydrophilic group having two monovalent radical centers generated by removing two hydrogen atoms from glucose, galactose, mannose, glucuronic acid, galactonic acid, mannuronic acid, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine and N-acetylmuramic acid.

8. The linker according to any one of claims 1 to 6, wherein L3 is selected from: Where, R1 is selected from -O(CH2) p - and -C(=O)-, p is an integer from 1 to 6, preferably 1, 2, 3 or 4; R5 is a C1-C3 alkylene group which is unsubstituted or substituted by one or more substituents selected from the group consisting of: -OR 20 and -C(=O)OR 20 -; R2, R3, R4, R 11 、R 12 、R 13 Independently selected from H, C1-C6 alkyl, -(CH2CH2O) t -C1-C6 alkyl, sulfonic acid and phosphoric acid groups; R 14 、R 15 Independently selected from H, -OR 20 , and -CH2R 20 ; R 16 、R 17 、R 18 、R 19 Independently selected from H and -OR 20 ; R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t -C1-C6 alkyl, sulfonic acid and phosphoric acid groups; t is an integer from 1 to 20; The wavy lines indicate where L3 connects to X and Y.

9. The linker according to any one of claims 1 to 8, wherein L4 is selected from the following amino acid residues: valine, citrulline, alanine, glycine, phenylalanine, asparagine, glutamic acid, lysine, serine, threonine, cysteine ​​and tyrosine; said amino acid group is optionally replaced by one or more -OR 20 Replaced by, where R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t - C1-C6 alkyl, sulfonic acid and phosphoric acid, wherein t is an integer from 1 to 20; or L4 is selected from the following peptide segments: valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), glycine-glycine-phenylalanine-glycine-glycine, glycine-glycine-phenylalanine-glycine-glycine, citrulline-valine, alanine-valine, alanine-alanine, citrulline-alanine, asparagine-citrulline, citrulline-asparagine, citrulline-citrulline, phenylalanine-lysine and lysine-phenylalanine, preferably selected from the following peptide segments: valine-citrulline (VC), valine-alanine (VA) and glycine-glycine-phenylalanine-glycine (GGFG); each peptide segment is optionally substituted by one or more substituents selected from the following group: -OR 20 and -(OCH2CH2) p O-C1-C6 alkyl, wherein R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t - C1-C6 alkyl, sulfonic acid and phosphoric acid groups, t is an integer from 1 to 20, and p is an integer from 1 to 6.

10. The linker according to claim 9, wherein L4 is selected from:

11. The linker according to any one of claims 1 to 10, wherein R6 is H, C1-C3 alkoxy or -(OCH2CH2) p O-C1-C3 alkyl, wherein p is an integer from 1 to 6; preferably, R6 is H, methoxy or -(OCH2CH2) p OCH3, wherein p is an integer from 1 to 6 or from 1 to 4; and / or R is selected from H and phenyl optionally substituted by 1 to 3 nitro groups, preferably H or 4-nitrophenyl.

12. The linker according to any one of claims 1 to 11, wherein The linker is selected from the linkers shown in Formula II to Formula XIV, wherein R", R"', R, L2, X, L3, R2-R4, R6, R 11 -R 13 and R 15 As claimed in any one of claims 1 to 11.

13. The linker according to any one of claims 1 to 12, wherein The linker is selected from the linkers shown in Formula 1 to Formula 46.

14. A linker-drug conjugate, the structural formula of which is shown in Formula XIII': In the formula, L1, L2, L3, L4, X, Y, Z, R6 and m are as described in any one of claims 1 to 11; L' is a bond, or is -NR"-C1-C6 alkylene-NR"-CO-*, or is -NH-CH2-O-; D is a drug moiety; wherein, R" are each independently H and C1-C6 alkyl, * represents the position where L' is connected to D; preferably, the structure of formula XIII' except for the D part is as shown in claim 12 or 13.

15. The linker-drug conjugate according to claim 14, wherein The structure of the linker-drug conjugate is shown in Formula XIII: wherein L1, L2, L3, L4, X, Y, Z, R6 and m are as described in any one of claims 1 to 11; D is a drug moiety; preferably, the structure of Formula XIII except for moiety D is as shown in claim 12 or 13.

16. The linker-drug conjugate according to claim 14 or 15, wherein The drug moiety D is selected from the drug moieties shown in formulas D-1 to D-9, wherein the wavy line represents the connection position between D and the rest of the linker-drug conjugate.

17. The linker-drug conjugate according to any one of claims 14 to 16, wherein The linker-drug conjugate is selected from CPD1, CPD2, CPD3, CPD4, CPD5, CPD6, CPT004, CPT005, CPT006, CPT008, CPT009, CPT010, MMAE Derivate, MMAE Derivate (o-OMe-PAB), Comp.5, Comp6, Comp7, Comp8 and Comp9.

18. An antibody-drug conjugate or a pharmaceutical composition thereof, characterized in that: The structure of the antibody drug conjugate is shown in Formula XIV: Ab-(LD) s (Formula XV) wherein L is a linker according to any one of claims 1 to 13; D is a drug; s represents the average number of LD units coupled to Ab, and s ranges from 1 to 8, preferably s is 2 to 6; Ab is a targeting moiety; preferably, LD is a linker-drug conjugate according to any one of claims 14 to 17.

19. The antibody-drug conjugate or pharmaceutical composition thereof according to claim 18, wherein: The structure of the antibody drug conjugate is shown below: wherein L2, X, L3, Y, Z, L4, R6 and m are as described in any one of claims 1 to 11; L' is as described in claim 14; s represents the average number of LD units coupled to Ab, and s ranges from 1 to 8, preferably s is 2 to 6; Ab is a target part; S represents the sulfur atom in the cysteine ​​residue in the antibody.

20. The antibody drug conjugate or pharmaceutical composition thereof according to claim 18, wherein: The structure of the antibody drug conjugate is: Wherein, s is 1-8.

21. A linker having a structure shown in the following formula, which is used to connect a drug to a targeting moiety to form a targeting moiety-drug conjugate: -L1-L2-X-L3-YZ(=O)-L4-(L5) m -L6- Where, L1 is selected from -(maleimide-3-yl-N)-, -C(=O)-, -(CH2) n C(=O)-, -S(=O)-, -(CH2) n S(=O)-、-CH2-(CH2) n - and -CH=CH-; L2 and Y are each independently selected from -(CH2) p -、-(OCH2CH2) p -and-(CH2CH2O) p -; L3 is a hydrophilic group; L4 is an amino acid group; L5 is: R6 is selected from H, C1-C6 alkoxy, -(OCH2CH2) p O-C1-C6 alkyl and -(CH2CH2O) p -C1-C6 alkyl; L6 is a bond, or is -NR"-C1-C6 alkylene-NR"- or -NR"-C1-C6 alkylene-NR"-CO-; R" are each independently H or C1-C6 alkyl, preferably each independently H, methyl or ethyl, more preferably both are methyl; X is selected from -NH-, -NH(CH2) n C(=O)-, -C(=O)(CH2) n NH- and -(CH2) n C(=O)-; Z is selected from C and S; n is selected from 0, 1 and 2; p is an integer selected from 1 to 10; m is selected from 0 and 1; in, The L1 is used to connect to the targeting moiety, and the other end of the linker is used to connect to the drug; When m is 0, the drug is connected to the carbonyl end (C(=O)) of L4; when m is 1, the drug is connected to the carbonyl end (C(=O)) of L5, and L4 is connected to the amino end (NH) of L5; Wherein, the -(maleimide-3-yl-N)- is a structure shown in the following formula, which is connected to the targeting moiety via the 3-position, 22. The connector according to claim 21, wherein L1 is -(maleimide-3-yl-N)-; and / or L2 is -(CH2) p -, and p is an integer of 1-4; preferably, L2 is -CH2CH2- or -CH2CH2CH2-; and / or X is -NH- or -NH(CH2)nC(=O)-, preferably, X is -NH- or -NHC(=O)-; and / or Y is -(CH2) p -, p is 1, 2 or 3; preferably, Y is methylene; and / or Z is C.

23. The joint according to claim 21 or 22, wherein: L3 is selected from monosaccharides, disaccharides and five-membered or six-membered saturated heterocycles containing 1-2 nitrogen atoms and their derivatives, which are divalent hydrophilic groups with two monovalent radical centers produced by removing two hydrogen atoms; preferably, the monosaccharide is selected from at least one of trisaccharides, tetrasaccharides, pentoses, hexoses and heptoses, the disaccharide is selected from at least one of maltose, sucrose and lactose, and the five-membered or six-membered saturated heterocycles containing 1-2 nitrogen atoms are selected from piperazinyl, piperidinyl and pyrrolidinyl.

24. The joint according to any one of claims 21 to 23, wherein L3 is a divalent hydrophilic group having two monovalent radical centers generated by removing two hydrogen atoms from glucose, galactose, mannose, glucuronic acid, galactonic acid, mannuronic acid, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine and N-acetylmuramic acid.

25. The joint according to any one of claims 21 to 24, characterized in that L3 is selected from: Where, R1 is selected from -O(CH2) p - and -C(=O)-, p is an integer from 1 to 6, preferably 1, 2, 3 or 4; R5 is a C1-C3 alkylene group which is unsubstituted or substituted by one or more substituents selected from the group consisting of: -OR 20 and -C(=O)OR 20 -; R2, R3, R4, R 11 、R 12 、R 13 Independently selected from H, C1-C6 alkyl, -(CH2CH2O) t -C1-C6 alkyl, sulfonic acid and phosphoric acid groups; R 14 、R 15 Independently selected from H, -OR 20 , and -CH2R 20 ; R 16 、R 17 、R 18 、R 19 Independently selected from H and -OR 20 ; R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t -C1-C6 alkyl, sulfonic acid and phosphoric acid groups; t is an integer from 1 to 20; The wavy lines indicate where L3 connects to X and Y.

26. The joint according to any one of claims 21 to 25, characterized in that L4 is selected from the following amino acid residues: valine, citrulline, alanine, glycine, phenylalanine, asparagine, glutamic acid, lysine, serine, threonine, cysteine ​​and tyrosine; said amino acid group is optionally replaced by one or more -OR 20 Substitute, where R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t - C1-C6 alkyl, sulfonic acid and phosphoric acid, wherein t is an integer from 1 to 20; or L4 is selected from the following peptide segments: valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), glycine-glycine-phenylalanine-glycine-glycine, glycine-glycine-phenylalanine-glycine-glycine-glycine, citrulline-valine, alanine-valine, alanine-alanine, citrulline-alanine, asparagine-citrulline, citrulline-asparagine, citrulline-citrulline, phenylalanine-lysine and lysine-phenylalanine, preferably Selected from the following peptide segments: valine-citrulline (VC), valine-alanine (VA) and glycine-glycine-phenylalanine-glycine (GGFG); each peptide segment is optionally substituted by one or more substituents selected from the following group: -OR 20 and -(OCH2CH2) p O-C1-C6 alkyl, wherein R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t - C1-C6 alkyl, sulfonic acid and phosphoric acid groups, t is an integer from 1 to 20, and p is an integer from 1 to 6.

27. The joint according to any one of claims 21 to 26, wherein L4 is selected from:

28. The joint according to any one of claims 21 to 27, wherein R6 is H, C1-C3 alkoxy or -(OCH2CH2) p O-C1-C3 alkyl, wherein p is an integer from 1 to 6; preferably, R6 is H, methoxy or -(OCH2CH2) p OCH3, wherein p is an integer of 1-6 or 1-4.

29. The joint according to any one of claims 21 to 28, wherein The linker is selected from the linkers shown in formula II-1 to XIV-1, wherein R, L2, X, L3, R2-R4, R 11 -R 13 and R 15 As described in any one of claims 1 to 11.

30. The joint according to any one of claims 21 to 29, wherein The linker is selected from the linkers shown in Formula 1-1 to Formula 46-1. 31.Selected from the following applications: Use of the linker according to any one of claims 1 to 13 in the preparation of a linker-drug conjugate or an antibody-drug conjugate; preferably, the linker-drug conjugate is the linker-drug conjugate according to any one of claims 14 to 17, and the antibody-drug conjugate is the antibody-drug conjugate according to any one of claims 18 to 20; Use of the antibody-drug conjugate or pharmaceutical composition thereof according to any one of claims 18 to 20 in the preparation of a medicament; preferably, the medicament is used to treat or prevent a disease selected from cancer, autoimmune diseases, inflammatory diseases and infectious diseases.

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