Exatecan derivative and use thereof

Abstract

The present application relates to an exatecan derivative having a structure as represented by formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The present application further relates to an ADC of the exatecan derivative, a pharmaceutical composition, a preparation method therefor and the use thereof.

Description

Iciticon derivatives and their uses Technical Field

[0001] This application relates to the field of biomedicine, specifically to an ixotecan derivative, an ADC of the ixotecan derivative, a pharmaceutical composition, a preparation method, and its uses. Background Technology

[0002] Antibody-drug conjugates (ADCs), as novel targeted therapies, generally consist of three parts: an antibody, a toxin (payload, usually a small molecule drug with cytotoxicity), and a linker that conjugates the antibody and toxin. Through the specific binding of the antibody to the antigen, the toxin is carried to the target cell, where it is released to effectively kill tumor cells. These drugs not only fully utilize the specificity of antibodies in binding to antigens on the surface of normal and tumor cells and the high efficiency of toxins, but also avoid the drawbacks of the former (low efficacy) and the latter (excessive toxicity). Several classes of cytotoxic small molecules are used in antibody-drug conjugates, one of which is camptothecin derivatives, such as eczema and its derivatives.

[0003] Ecinotecan (CAS: 171335-80-1) and its derivatives are potent DNA topoisomerase I inhibitors, exhibiting strong antitumor activity in animal models of lung cancer, breast cancer, colorectal cancer, and ovarian cancer. However, their toxicity is also significant, particularly in the gastrointestinal and myelotoxic regions, limiting their clinical application. Therefore, it is necessary to provide an eccinotecan derivative with improved safety and antitumor activity, as well as to develop more effective ADC drugs. Summary of the Invention

[0004] The first aspect of this invention provides a compound of formula (I), its stereoisomers, or a pharmaceutically acceptable salt thereof:

[0005] Among them, X 1 For O, S, or NH;

[0006] X 2 For -NR 2 - or key, R 2 -H, -C 1-3 Alkyl or 3-6 membered cycloalkyl;

[0007] X 3 It can be -O- or -NH-;

[0008] R 1 -H, -C 1-3 Alkyl or 3-6 membered cycloalkyl;

[0009] M is -CR3a R 4a -(CR 3 R 4 ) m -or Among them, R 3a R 4a R 3 R 4 Each is independently -H, -C 1-5 Alkyl or 3-6 membered cycloalkyl, where m is 0, 1, 2 or 3, and ring A is a 3-6 membered cycloalkane;

[0010] Where X 1 For O or S and X 2 When R is -NH-, 1 R 3a R 4a At least one of them is not -H.

[0011] In some implementations, R 1 It is -H, methyl or cyclopropyl, preferably -H.

[0012] In some implementations, R 2 It is -H, methyl or cyclopropyl, preferably -H or methyl.

[0013] In some implementations, R 3a R 4a One is -H, and the other is -C 1-5 Alkyl or 3-6 membered cycloalkyl, preferably R 3a R 4a One of them is -H, and the other is methyl, isopropyl, or cyclopropyl.

[0014] In some implementations, R 3 R 4 Each is independently -H, -C 1-3 Alkyl or 3-5 membered cycloalkyl; preferably R 3 R 4 Each is independently -H, methyl, cyclopropyl, or isopropyl; more preferably, R 3 R 4 It is -H.

[0015] In some implementations, m is 0, 1, or 2, preferably 0 or 1.

[0016] In some embodiments, ring A is a 3-5 membered cycloalkyl group, preferably a cycloalkyl group.

[0017] In some implementations, M is -CR 3a R 4a -CR3 R 4 -or Preferably, it is -CH(CH3)CH2-. More preferably, M is -CH(CH3)CH2-.

[0018] In some implementations, X 1 For NH, X 2 For key, M is -CR 3a R 4a -(CR 3 R 4 ) m -or Among them, R 3a R 4a R 3 R 4 Each is independently -H, -C 1-5 Alkyl or 3-6 membered cycloalkyl, where m is 0, 1, 2 or 3, and ring A is 3-6 membered cycloalkane.

[0019] In some embodiments, the compound represented by formula (I), its stereoisomers, or pharmaceutically acceptable salts thereof have the structure shown in formula (Ia):

[0020] M is as described above;

[0021] Preferably, M is -CR 3a R 4a -(CR 3 R 4 ) m -, R 3a R 4a One is -H, and the other is -C 1-5 Alkyl or 3-6 membered cycloalkyl, R 3 R 4 Each is independently -H, -C 1-5 Alkyl or 3-6 membered cycloalkyl, m is 1, 2 or 3; more preferably, m is 1; even more preferably, M is -CR 3a R 4a CH2-, R 3a R 4a One of them is -H, and the other is methyl, isopropyl, or cyclopropyl.

[0022] In some embodiments, the compound represented by formula (I), its stereoisomers, or pharmaceutically acceptable salts thereof have the structure represented by formula (Ib):

[0023] Among them, R 2M and M are as described above, each independently.

[0024] In some embodiments, the compound represented by formula (I), its stereoisomers, or pharmaceutically acceptable salts thereof have the structure represented by formula (Ic):

[0025] M is as described above;

[0026] Preferably, M is -CR 3a R 4a -(CR 3 R 4 ) m -or R 3a R 4a R 3 R 4 Each is independently -H or a 3-6 membered cycloalkyl group, ring A is a 3-6 membered cycloalkane, and m is 0, 1, 2 or 3; more preferably, M is -CR. 3a R 4a -(CR 3 R 4 ) m -or R 3a R 4a R 3 R 4 Each is independently -H or a 3-5 membered cycloalkyl group, ring A is a 3-5 membered cycloalkane, and m is 0, 1, or 2; more preferably, M is -CR. 3a R 4a -(CR 3 R 4 ) m -or R 3a R 4a R 3 R 4 Each is independently -H or cyclopropyl, and m is 0 or 1.

[0027] A second aspect of the present invention provides a compound, its stereoisomer, or a pharmaceutically acceptable salt thereof, the structure of which is shown below:

[0028] In some embodiments, the compound, its stereoisomers, or pharmaceutically acceptable salts thereof have the following structures:

[0029] A third aspect of the present invention provides a compound of formula (II), its stereoisomers, or a pharmaceutically acceptable salt thereof:

[0030] Among them, R1 X 1 X 2 M, X 3 Each is independent as described in the first aspect of the invention;

[0031] L 1 For key, Among them, the -NH- end and L 2 Connected, the other end is connected to X 3 Connected, R a Or R b Each is independently -H or -C 1-3 Alkyl groups, preferably L 1 for

[0032] L 2 A peptide consisting of 2 to 7 amino acid residues, wherein each amino acid is independently phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, or glycine, and each amino acid residue is optionally independently divided by one or more halogens, hydroxyl groups, cyano groups, amino groups, or -C groups. 1-5 Alkyl, -C 1-5 Alkoxy or 3-6 membered cycloalkyl substitution, wherein the -NH- terminus is associated with an L-terminus. 3 Connected to L, the other end is connected to L. 1 Linked; preferably, each of the amino acids is independently glycine, phenylalanine, valine, or citrulline; more preferably, L 2 The residues are -glycine residues - phenylalanine residues - glycine residues - (-Gly-Phe-Gly-), -glycine residues - glycine residues - phenylalanine residues - glycine residues - (-Gly-Gly-Phe-Gly-), or -valine residues - citrulline residues - (-Val-Cit-); more preferably, L 2 for

[0033] L 3 -(CH2) n1 -C(O)-,-(CH2CH2O) n2 -C(O)- or -(CH2) n3 -W-(CH2) n4 -C(O)-, where the -C(O)- terminal is connected to L 2 Connected to L, the other end is connected to L. 4Connected, n1, n2, n3, and n4 are each independently an integer from 0 to 8, and W is selected from -O-, -C(O)-NH-, 6-10 aryl, 5-10 aryl heteroalkyl, 3-6 cycloalkyl, or 3-6 heterocyclic groups. The 6-10 aryl, 5-10 aryl heteroalkyl, 3-6 cycloalkyl, or 3-6 heterocyclic groups are optionally converted by one or more halogens, hydroxyl groups, cyano groups, amino groups, or -C groups. 1-5 Alkyl, -C 1-5 Alkoxy or 3-6 membered cycloalkyl substitution; preferably, L 3 for

[0034] L 4x for -C(=O)CH2Br or -C(=O)CH2I, preferably

[0035] In some embodiments, the compound represented by formula (II), its stereoisomers, or pharmaceutically acceptable salts thereof have the structure shown in formula (II-1):

[0036] Among them, X 1 R 2 M and M are each independently as described in the first aspect of the invention;

[0037] L 1 L 2 L 3 L 4x Each is independent as described above.

[0038] A fourth aspect of the present invention provides a compound, its stereoisomer, or a pharmaceutically acceptable salt thereof, the structure of which is shown below:

[0039] The fifth aspect of the present invention provides an antibody conjugate of formula (III) or a pharmaceutically acceptable salt or solvate thereof:

[0040] Among them, R 1 X 1 X 2 X 3 M, L 1 L 2 L 3 Each is independent as described in the third aspect of the invention;

[0041] mAb is an antibody or antigen-binding fragment;

[0042] L 4 for -C(=O)-、a-CH2C(=O)-b, where end a is connected to mAb and end b is connected to L. 3 Connected; preferably

[0043] n is a decimal or integer from 1 to 10, preferably n is a decimal or integer from 3 to 10, and more preferably n is 4, 6 or 8.

[0044] In some embodiments, the mAb is an antibody selected from the group consisting of monoclonal antibodies, single-chain antibodies, chimeric antibodies, multispecific antibodies, humanized antibodies, or fully human antibodies.

[0045] In some embodiments, mAb is an antigen-binding fragment selected from the group consisting of Fab, Fab', Fv, F(ab')2, scFv, di-scFv, VHH, or dAb fragments.

[0046] In some embodiments, the mAb is a Her2 antibody; preferably, the Her2 antibody is Trastuzumab.

[0047] In some embodiments, the antibody conjugate of formula (III) or its pharmaceutically acceptable salt or solvate has the structure shown in formula (III-1):

[0048] Among them, X 1 R 2 M, L 1 L 2 L 3 Each is independent as described in the third aspect of the invention;

[0049] mAb, n, L 4 Each is independent as described above.

[0050] A sixth aspect of the present invention provides an antibody conjugate or a pharmaceutically acceptable salt or solvate thereof having the following structure:

[0051] Wherein, mAb and n are each independently as described in the fifth aspect of the present invention;

[0052] Preferably, the mAb is a Her2 antibody; and / or

[0053] Preferably, n is a decimal or integer from 3 to 10; more preferably, n is 4, 6 or 8.

[0054] A seventh aspect of the present invention provides a pharmaceutical composition comprising a compound described in the first or second aspect of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or an antibody-drug conjugate described in the fifth or sixth aspect of the present invention or a pharmaceutically acceptable salt or solvate thereof, and optionally a pharmaceutically acceptable carrier.

[0055] The eighth aspect of the present invention provides the use of a compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as described in the first or second aspect of the present invention, or an antibody-drug conjugate thereof, or a pharmaceutically acceptable salt or solvate thereof, as described in the fifth or sixth aspect of the present invention, or a pharmaceutical composition thereof, in the preparation of a medicament for the prevention or treatment of tumors.

[0056] The ninth aspect of the present invention provides a method for preventing or treating tumors, the method comprising administering to a patient in need a therapeutic amount of a compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as described in the first or second aspect of the present invention, or an antibody-drug conjugate thereof, or a pharmaceutically acceptable salt or solvate thereof, as described in the fifth or sixth aspect of the present invention, or a pharmaceutical composition as described in the seventh aspect.

[0057] In some embodiments, the tumor described in the eighth or ninth aspect of the present invention is an advanced solid tumor; in some embodiments, the tumor is selected from lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.

[0058] In some embodiments, the tumor described in the eighth or ninth aspect of the present invention is selected from tumors associated with the expression of the following group: HER2.

[0059] The tenth aspect of the present invention provides a medicine box comprising a first therapeutic agent and a second therapeutic agent, the first therapeutic agent comprising an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as described in the fifth or sixth aspect of the present invention, or a pharmaceutical composition as described in the seventh aspect; the second therapeutic agent comprising at least one other therapeutic agent, or a pharmaceutical composition comprising other therapeutic agents as a pharmaceutical composition; and optionally, packaging and / or instructions.

[0060] The kit may contain 0.01 mg to 1000 mg of at least one antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as described in the fifth or sixth aspect of this invention.

[0061] The present invention also provides a method for preparing the above-mentioned medicine box, which includes combining at least one antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as described in the fifth or sixth aspect of the present invention, or a pharmaceutical composition as described in the seventh aspect of the present invention, with at least one other therapeutic agent optionally present, or a pharmaceutical composition containing other therapeutic agents, packaging and / or instructions.

[0062] This invention provides an ixotecan derivative, which has one or more of the following advantages: (1) its in vitro killing effect on various cell lines is superior to Dxd (CAS: 1599440-33-1) under the same experimental conditions, including but not limited to AGS, NCI-N87, SK-BR-3 and MCF-7 cell lines; (2) according to cLogP prediction, under the same conditions, its hydrophilicity is superior to SN-38 (CAS: 86639-52-3) and ixotecan; (3) compared with DS8201, an ADC drug using Dxd as a toxin, the ixotecan derivative has significantly better in vitro killing and anti-tumor proliferation effects in both NCI-N87 cell lines with high antigen expression and AGS cell lines with low antigen expression; (4) compared with Dxd, the ixotecan derivative has better safety. Attached Figure Description

[0063] Figure 1 shows the tumor volume change curves in a mouse model of gastric cancer NCI-N87 cells with high Her2 expression: a comparison between ADC-01 and DS8201. Detailed Implementation

[0064] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive.

[0065] Unless otherwise stated, conventional methods within the scope of the art, such as mass spectrometry, NMR, and pharmacological methods, are employed. Unless specifically defined, the terminology used herein in the relevant descriptions of analytical chemistry, organic synthetic chemistry, and pharmaceutical and medicinal chemistry is known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and in the treatment of patients. For example, reactions and purifications can be carried out using the manufacturer's instructions for use of reagent kits, or in accordance with methods known in the art or the descriptions of this invention. The techniques and methods described herein are generally carried out according to conventional methods well known in the art, based on the descriptions in the various summary and more specific literatures cited and discussed herein.

[0066] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0067] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of".

[0068] The terms “optional” or “optionally” mean that the event or situation described below may, but does not have to, occur, and the description includes the circumstances under which the event or situation may or may not occur.

[0069] The term "one or more" means one or more under reasonable conditions, such as two, three, four, five or ten.

[0070] As used in this article, This indicates the location where a structural segment connects to other parts of the molecule. For example... The N group is connected to other parts of the molecule. Unless otherwise specified, the direction of connection of the linking groups mentioned in this article is arbitrary.

[0071] The term "pharmaceutically acceptable salt" refers to a salt formed by the compound of the general formula with an acid or base that is suitable for use as a medicine. In some embodiments, a pharmaceutically acceptable salt may include an inorganic salt or an organic salt.

[0072] The term "isotope label" refers to different atoms of the same element with the same number of protons but different numbers of neutrons, and is not limited to radioactive isotopes. For example, the isotope label could be deuterium (D, 2 H), tritium (T), 3 H) 12 C 13 C or 14 C. The isotopic markers also include naturally occurring isotopes and artificially synthesized isotopes.

[0073] The compounds of this invention encompass, within their scope, compounds, stereoisomers, tautomers, isotope labels, pharmaceutically acceptable salts, or prodrugs.

[0074] The compounds of the present invention can exist in both non-solventized and solvated forms. The term "solvent" is used herein to describe a molecular complex comprising the compounds of the present invention and one or more pharmaceutically acceptable solvent molecules (e.g., ethanol).

[0075] The compounds of the present invention also cover all possible crystalline forms or polymorphs of the compounds of the present invention, which may be a single polymorph or a mixture of more than one polymorph in any proportion.

[0076] The term "stereoisomer" refers to a molecule in which atoms or groups have the same connection order but different spatial arrangements. A stereoisomer comprises an isomer formed by at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and single diastereomers can be produced. The compounds of the present invention can exist in the following forms: optically pure enantiomers, pure diastereomers, mixtures of enantiomers, mixtures of diastereomers, racemic mixtures of enantiomers, racemates, or mixtures of racemates. Certain individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers).

[0077] The term "tautomers" refers to functional group isomers that arise from the rapid movement of an atom between two positions in a molecule. Representative examples of tautomers include keto-enol tautomers, imine-enamine tautomers, etc. It should be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0078] Certain compounds of the present invention may be present in their free form for therapeutic purposes, or, where appropriate, in their pharmaceutically acceptable derivative forms. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, metabolites, or prodrugs, which, upon administration to a patient in need, can directly or indirectly provide the compounds of the present invention or their metabolites or residues. Therefore, when referred to herein as "compounds of the present invention," it is also intended to encompass the various derivative forms of the compounds described above.

[0079] The term "prodrug" refers to a prodrug with no or very low activity that is converted into an active drug through a chemical reaction or enzymatic action. In this application, the prodrug may be an inactive or very inactive ixotecan derivative.

[0080] The scope of this invention also includes metabolites of the compounds of this invention, i.e., substances formed in the body when the compounds of this invention are administered. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic hydrolysis, etc., of the administered compound. Therefore, this invention includes metabolites of the compounds of this invention, including compounds obtained by methods that expose the compounds of this invention to mammals for a time sufficient to produce their metabolites.

[0081] The term “substitution” means that any one or more atoms or groups (e.g., hydrogen) on a specified atom or group are replaced by a specified atom or group selected from the specified atom or group, such as hydroxyl, amino, halogen, alkoxy, alkylamine and three to six-membered rings or more.

[0082] The term "alkyl", either on its own or as part of another substituent, refers to an alkyl group having a specified number of carbon atoms (i.e., -C). 1-10 This refers to a saturated aliphatic hydrocarbon group (one to ten carbon atoms), which is an uncyclic straight-chain or branched carbon chain (or carbon), or a combination thereof. Examples of saturated hydrocarbon groups include, but are not limited to, homologues and isomers of groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, (e.g.) n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Preferably, it is an alkyl group containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, and even more preferably an alkyl group containing 1 to 3 carbon atoms. For example, -C 1-3 Alkyl refers to methyl, ethyl, n-propyl, and isopropyl.

[0083] The term "cycloalkane" refers to a saturated or partially unsaturated monocyclic or polycyclic ring, and the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 8 (e.g., 3, 4, 5, 6, 7, and 8) carbon atoms, and even more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, etc. Cycloalkyl can be optionally substituted or unsubstituted, and when substituted, the substituent can be substituted at any usable connection point.

[0084] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic group with a fully conjugated π-electron system, including but not limited to phenyl, naphthyl, anthracene, etc., with phenyl being preferred.

[0085] The term "halogen" refers to the six elements belonging to Group 17 (formerly Group VIIA) of the periodic table, including fluorine (F), chlorine (Cl), bromine (Br), iodine (I), astatine (At), and... (Ts).

[0086] The term "heterocyclic" refers to a saturated or partially unsaturated monocyclic, bicyclic, or polycyclic cyclic hydrocarbon with a non-aromatic structure containing 3 to 20 ring atoms, wherein one, two, three, or more ring atoms are selected from N, O, or S, and the remaining ring atoms are C. The term "heterocyclic group" refers to a substituent generated based on the foregoing definition of a heterocyclic group. Exemplary 3-membered heterocyclic groups containing one heteroatom include azirropropyl, oxacyclopropyl, and thioherropropyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include azirrobutyl, oxacyclobutyl, and thioherrobutyl.

[0087] The term "aromatic heterocycle" refers to an aromatic monocyclic, bicyclic, or polycyclic cyclic system containing a 5-16 member structure, preferably a 5-14, 5-12, 5-10, or 5-8 member structure, and more preferably a 5-6 member structure, wherein one, two, three, or more ring atoms are heteroatoms and the remaining atoms are carbon atoms, and the heteroatoms are independently selected from O, N, or S, and the number of heteroatoms is preferably one, two, or three. The term "aromatic heterolyl" refers to a substituent generated based on the aforementioned definition of aromatic heterocycles. Examples of heteroaryl groups include, but are not limited to, furanyl, thiophene, oxazolyl, thiazolyl, isoxazolyl, oxadiazolyl, thiazolyl, pyrroleyl, and pyrazolyl.

[0088] This invention also covers methods for preparing the compounds described herein. It should be understood that the compounds of this invention can be synthesized using the methods described below, as well as synthetic methods known in the field of synthetic organic chemistry or variations thereof understood by those skilled in the art. Preferred methods include (but are not limited to) those described below. Reactions can be carried out in solvents or solvent mixtures suitable for the reagents and materials used and suitable for achieving the conversion. The methods for preparing the compounds described herein, unless otherwise specified, are methods commonly used in the art, for example, referring to the methods for preparing the compounds in the embodiments of this invention, or the methods for preparing the compounds of patents WO2024125627A1 or CN116239601B.

[0089] The term "antibody," used in its broadest sense, refers to a unit containing sufficient sequence from the variable region of the immunoglobulin heavy chain and / or from the variable region of the immunoglobulin light chain to bind, reactively associate, or chelate a receptor, antigen, or other receptor units present in a target cell population. Antibodies can be any protein or protein-like molecule that can bind, chelate, or react with a portion of a cell population to be treated or bioengineered. In this document, "antibody" encompasses a wide range of forms and structures, as long as they exhibit the desired antigen-binding activity.

[0090] The “antibody” in this article can be derived from any animal, including but not limited to humans and non-human animals. The non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camels, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0091] In this application, the term "amino acid" includes both natural and non-natural amino acids, and the conventional designation of amino acids follows standard usage. See, for example, Immunology-A Synthesis (2nd Edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this application, the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this application, amino acids or residues thereof are generally represented by single-letter and three-letter abbreviations well known in the art. For example, alanine may be represented by A or Ala; arginine by R or Arg; glycine by G or Gly; glutamine by Q or Gln; and citrulline by Z or Cit.

[0092] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use that comprises a small molecule drug, a large molecule antibody (or antibody ligand), or a conjugate of both, as an active pharmaceutical ingredient (the eczemab derivative described in this application), and other components (e.g., pharmaceutically acceptable excipients). Pharmaceutical compositions can be prepared using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, and / or lyophilization processes.

[0093] The pharmaceutical compositions provided by this invention can be prepared in any form. Depending on the clinical indication, route of administration, and method of administration, the pharmaceutical compositions include, but are not limited to, oral formulations such as tablets, gels, soft / hard capsules, emulsions, dispersible powders, granules, and water / oil suspensions; injectable formulations including intravenous injections, intramuscular injections, intraperitoneal injections, rectal suppositories, and intracranial injections, which may be aqueous or oil solutions; topical formulations including creams, ointments, gels, water / oil solutions, and inclusion complex formulations; and inhaled formulations including fine powders, liquid aerosols, and various formulations suitable for implantation.

[0094] The pharmaceutical compositions of the present invention may further comprise one or more pharmaceutically acceptable carriers. The term "carrier" refers to a diluent, adjuvant, excipient, or carrier that can be administered to a patient together with the active ingredient. Such carriers can be sterile liquids, such as water and oils, including petroleum, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, glucose solutions, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers such as acetates, citrates, or phosphates. Antimicrobial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as EDTA, and tonic agents such as sodium chloride or dextran are also foreseeable. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition can be formulated into suppositories using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in EW Martin's Remington's Pharmaceutical Sciences, which are incorporated herein by reference. Such compositions will contain a clinically effective dose of an antibody or its antigen-binding unit or ADC, preferably in purified form, along with a suitable number of carriers to provide a dosage form suitable for the patient. The formulation can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0095] The term "pharmaceutically acceptable" generally means that, within the limits of reasonable medical judgment, it is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and has a reasonable benefit / risk ratio.

[0096] As used herein, “treatment” includes preventative treatment. Treatment methods include administering a therapeutically effective amount of the compound described herein to an individual. Administration may include a single dose or a series of doses. The duration of treatment depends on various factors, such as symptom severity, patient age, compound concentration, the activity of the composition used for treatment, and their combination. It should also be understood that the effective amount of the agent used for treatment or prevention may increase or decrease as a particular treatment or prevention regimen progresses. Dosage changes can be obtained and become apparent through standard diagnostic analyses known in the art.

[0097] The term "prevention" refers to reducing the occurrence of disease symptoms in a patient. As mentioned above, prevention can be complete (no detectable symptoms) or partial prevention, resulting in fewer observed symptoms than would be possible without treatment. In implementation, prevention means slowing the progression of a disease, symptom, or illness, or inhibiting its progression to a harmful or other undesirable state.

[0098] "Patient" or "patient in need" means a living organism or individual who suffers from or is susceptible to a disease or symptom that can be treated by administration of the pharmaceutical compositions provided herein. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, dairy cows, deer, and other non-mammalian animals. In some embodiments, the patient is a human.

[0099] Unless otherwise specified, all raw materials or reagents used in the embodiments of this invention are commercially available.

[0100] Some common abbreviations in this manual have the following meanings:

[0101] The embodiments described below are not intended to be limited by any theory; they are merely for illustrating the fusion protein, preparation method, and uses of this application, and are not intended to limit the scope of the invention. The invention is further illustrated by the following embodiments, which are illustrative and do not limit the invention in any way. Any modifications or alterations to the invention that are easily achievable by those skilled in the art will fall within the scope of the invention.

[0102] Example 1: Preparation and Synthesis of Compound 1

[0103] Ecinotecan mesylate (200 mg, 0.38 mmol) was dissolved in DMF (10 mL), and DIEA (73 mg, 0.56 mmol) and N,N'-carbonyldiimidazole (210 mg, 1.32 mmol) were added. The mixture was purged with nitrogen three times, and stirred at room temperature for 2 h after the addition was complete. The reaction mixture was monitored by LCMS to indicate the disappearance of the starting material. Then, compound SM1 (110 mg, 0.56 mmol) was dissolved in DMF (2 mL) and added to the reaction flask. The mixture was stirred at room temperature for 12 h, and the reaction was monitored by LCMS to indicate completion. The reaction solution was poured into distilled water (100 mL), extracted with ethyl acetate (125 mL × 3), and then extracted with DCM (125 mL × 1). The organic phases were combined, washed with saturated NaCl aqueous solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was separated by silica gel column chromatography (PE / EA = 1 / 5) to obtain compound Int1 (190 mg).

[0104] Compound Int1 (190 mg, 0.29 mmol) was added to a reaction flask and dissolved in DCM (10 mL) at room temperature. The reaction solution was cooled to 0 °C, and 0.2 M HCl / dioxane solution (10 mL) was added dropwise. The mixture was stirred at 0 °C for 30 min, and the reaction was monitored for completeness by LC-MS. The reaction solution was poured into ice water (50 mL), extracted with DCM (25 mL × 3), and then extracted with EA (25 mL × 1). The organic phases were combined, washed with saturated NaCl aqueous solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by pre-HPLC to obtain compound 1 (12 mg).

[0105] 1 H NMR (400Hz, DMSO-d6): δ7.758-7.731 (d, J=10.5Hz, 1H), 7.304 (s, 1H), 6.811-6.791 (d,J=8.0Hz,1H),6.515(s,1H),5.417(s,3H),5.237-5.109(m,2H),4.778(s,1H),3. 535-3.525(d,J=4.0Hz,2H),3.189-3.113(m,2H),2.915(s,3H),2.378(s,3H),2.160 -2.148(d,J=5.0Hz,2H),1.877-1.842(m,2H),1.233(s,1H),0.871(t,J=6.5Hz,3H).

[0106] MS(ESI) m / z 537.2 [M+1] + .

[0107] Example 2: Preparation and Synthesis of Compound 2

[0108] Ecinotecan mesylate (200 mg, 0.38 mmol) was added to the reaction flask and dissolved in DMF (10 mL). Under nitrogen protection, DIEA (73.7 mg, 0.57 mmol) and N,N'-carbonyldiimidazole (216 mg, 1.33 mmol) were added. The reaction was allowed to proceed at room temperature for 1 h, and LCMS monitoring showed complete reaction. Compound SM2 (360 mg, 1.9 mmol) was added, and the reaction was continued at room temperature for 2 h, with LCMS monitoring showing complete reaction. The reaction solution was poured into ice water (50 mL), extracted with EA (100 mL × 3), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE / EA = 1 / 8) to obtain compound Int2 (150 mg).

[0109] Compound Int2 (150 mg, 0.23 mmol) was added to a reaction flask, dissolved in DCM (10 mL), and cooled to 0 °C. Then, 0.2 M HCl / Dioxane (8 mL) solution was added, and the reaction was continued at 0 °C for 10 min. LC-MS showed the reaction was complete. The reaction solution was poured into ice water (50 mL), extracted with DCM (50 mL × 3), and the organic phases were combined. The mixture was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by pre-HPLC to obtain compound 2 (70 mg).

[0110] 1 H NMR (400Hz, DMSO-d6): δ7.743-7.716 (d, J=10.5Hz, 1H), 7.292-7.280 (d, J=4.5Hz, 1H), 6.628-6 .606(d,J=9.0Hz,1H),6.513-6.504(d,J=3.5Hz,1H),5.827-5.796(m,1H),5.415-5.130(m,5H) ,4.760-4.702(m,1H),3.712-3.681(m,1H),3.420-3.263(m,2H),3.150-3.138(m,2H),2.359(s ,3H),2.172-2.072(m,2H),1.911-1.802(m,2H),1.064(t,J=7.0Hz,3H),0.869(t,J=7.5Hz,3H).

[0111] MS(ESI) m / z 537.2 [M+1] + .

[0112] Example 3: Preparation and Synthesis of Compound 2A

[0113] Ecinotecan mesylate (1.2 g, 2.28 mmol) was added to the reaction flask and dissolved in DMF (60 mL). Under nitrogen protection, DIEA (442.2 mg, 3.42 mmol) and N,N'-carbonyldiimidazole (1.3 g, 7.98 mmol) were added. The reaction was allowed to proceed at room temperature for 1 h, and LCMS monitoring showed complete reaction. Compound SM3 (2.16 g, 11.40 mmol) was added, and the reaction was continued at room temperature for 2 h, with LCMS monitoring showing complete reaction. The reaction solution was poured into ice water (300 mL), extracted with EA (600 mL × 3), the organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE / EA = 1 / 8) to obtain compound Int3 (900 mg).

[0114] Compound Int3 (575 mg, 0.74 mmol) was added to a reaction flask, dissolved in DCM (15 mL), and cooled to 0 °C. Then, HCl / Dioxane (0.2 M, 12 mL) solution was added, and the reaction was continued at 0 °C for 10 min. LC-MS showed the reaction was complete. The reaction solution was poured into ice water (100 mL), extracted with DCM (100 mL × 3), and the organic phases were combined. The mixture was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by pre-HPLC to obtain compound 2A (155 mg).

[0115] 1 H NMR (400Hz, DMSO-d6): δ7.789-7.761(d,J=11.0Hz,1H),7.307(s,1H),6.640-6.617(d,J=9.0Hz,1H),6. 509(s,1H),5.801-5.781(d,J=8.0Hz,1H),5.425(s,2H),5.377-5.330(m,2H),5.252-5.204(m,1H),4.7 22-4.695(m,1H),3.678-3.662(m,1H),3.404-3.367(m,1H),3.298-3.284(m,1H),3.161(s,2H),2.392( s,3H),2.181-2.115(m,2H),1.881-1.841(m,2H),1.061-1.044(d,J=7.0Hz,3H),0.871(t,J=7.5Hz,3H).

[0116] MS(ESI) m / z 537.2 [M+1].

[0117] Example 4: Preparation and Synthesis of Compound 2B

[0118] Using the same experimental methods and routes as in Example 3, compound 2B was synthesized from the S-configuration intermediate of compound SM3 as the starting material.

[0119] 1H NMR (400Hz, DMSO-d6): δ7.761-7.734(d,J=11.0Hz,1H),7.288(s,1H),6.628-6.606(d,J=9.0Hz,1H) ,6.497(s,1H),5.817-5.797(d,J=8.0Hz,1H),5.416(s,2H),5.372-5.307(m,2H),5.208-5.160(m,1 H),3.710-3.693(m,1H),3.395-3.349(m,1H),3.308-3.268(m,2H),3.171-3.159(m,2H),2.371(s,3 H),2.176-2.077(m,2H),1.894-1.820(m,2H),1.080-1.063(d,J=7.0Hz,2H),0.870(t,J=7.5Hz,3H).

[0120] MS(ESI) m / z 537.2 [M+1].

[0121] Example 5: Preparation and Synthesis of Compound 5

[0122] Ecinotecan mesylate (100 mg, 229.65 μmol) was dissolved in DMF (2 mL), and DIEA (29.68 mg, 229.65 μmol, 40.00 μL) and CDI (52.13 mg, 321.51 μmol) were added. The mixture was stirred at 20 °C for 3 h. 2-Amino-2-cyclopropylethanol SM6 (27.87 mg, 275.58 μmol) was added, and the mixture was stirred at 20 °C for another 1.5 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by pre-HPLC to give compound 5 (98.74 mg).

[0123] 1 H NMR (400MHz, DMSO-d6): δ7.78(dd,J=5.6,10.8Hz,1H),7.68-7.26(m,1H),7.09-6.50(m,2H),5.94-5.78( m,1H),5.42(s,1H),5.39-5.04(m,3H),4.92-4.54(m,2H),3.59-3.40(m,2H),3.20-3.07(m,3H),2.39(br s,3H),2.24-1.99(m,3H),1.92-1.80(m,1H),1.00-0.74(m,4H),0.53-0.05(m,4H).

[0124] MS(ESI) m / z 563.2 [M+1] + .

[0125] Example 6: Preparation and Synthesis of Compound 6

[0126] The starting material, eczemacin mesylate (173 mg, 397.29 μmol), was dissolved in DMF (2 mL). DIEA (51.35 mg, 397.29 μmol, 69.20 μL) and CDI (90.19 mg, 556.21 μmol) were added, and the mixture was stirred at 20 °C for 3 h. Then, 2-(cyclopropylamino)ethanol SM7 (48.22 mg, 476.75 μmol) was added, and the mixture was stirred at 20 °C for another 1.5 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The crude product was milled with ACN / PE at 20 °C for 10 min to remove impurities, yielding compound 6 (49.08 mg).

[0127] 1 H NMR (400MHz, DMSO-d6): δ7.76 (d, J = 10.8 Hz, 1H), 7.32 (s, 1H), 6.89 (d, J = 8. 8Hz,1H),6.51(s,1H),5.49-5.37(m,3H),5.34-5.09(m,2H),4.72(t,J=5.2H z,1H),3.66-3.41(m,3H),3.28-3.05(m,3H),2.64-2.55(m,1H),2.39(s,3H) ,2.21(q,J=6.4Hz,2H),1.86(quind,J=7.2,14.4Hz,2H),0.92-0.65(m,7H).

[0128] MS(ESI) m / z 563.2 [M+1] + .

[0129] Example 7: Preparation and Synthesis of Compound 7

[0130] Ecinotecan mesylate (150 mg, 344.47 μmol) was dissolved in DMF (2 mL), and DIEA (44.52 mg, 344.47 μmol, 60.00 μL) and CDI (78.20 mg, 482.26 μmol) were added. The mixture was stirred at 20 °C for 3 h. Then, 2-amino-3-methyl-1-butanol SM8 (42.64 mg, 413.37 μmol, 45.56 μL) was added, and the mixture was stirred at 20 °C for another 1.5 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by pre-HPLC to give compound 7 (46.04 mg).

[0131] 1 H NMR (400MHz, DMSO-d6): δ7.78(dd,J=11.2Hz,1H),7.36-7.26(m,1H),6.73-6.63(m,1H),5.77(br d,J=7.6Hz,1H),5.53-5.28(m,4H),5.27-5.13(m,1H),3.48-3.43(m,3H),3.40-3.29(m,2H),3.17(br s,2H),2.39(s,3H),2.21-2.06(m,2H),1.95-1.77(m,3H),0.96-0.79(m,9H).

[0132] MS(ESI) m / z 565.3 [M+1] + .

[0133] Example 8: Preparation and Synthesis of Compound 9

[0134] A mixture of CHCl3 (8 mL), H2O (4 mL), and thiocarbonyl dichloro(158.43 mg, 1.38 mmol, 105.62 μL) was stirred, and a solution of eczemab mesylate (600 mg, 1.38 mmol) in H2O (4 mL) was added dropwise. The mixture was stirred at 40 °C for 6 h. After stirring, the CHCl3 layer was separated, and the aqueous solution was extracted with dichloromethane (10 mL). The composite organic layer was dried, filtered, and concentrated. The residue was purified by rapid silica gel column chromatography (0–70% petroleum ether / ethyl acetate gradient) to obtain compound Int5 (480 mg).

[0135] 2-(methylamino)ethanol SM10 (35.39 mg, 471.20 μmol, 37.85 μL) was added to a THF (1.5 mL) solution of compound Int5 (150 mg, 314.13 μmol). The mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The crude product was milled with PE at 20 °C for 10 min to remove impurities, giving compound 9 (36.65 mg).

[0136] 1H NMR (400MHz, DMSO-d6): δ7.93(d,J=8.4Hz,1H),7.77(d,J=11.2Hz,1H),7.32(s ,1H),6.51(s,1H),6.45-6.32(m,1H),5.42(s,2H),5.28-5.06(m,2H),4.94(br s,1H),3.92(br d,J=1.6Hz,1H),3.76-3.62(m,3H),3.25(s,3H),3.16-2.91(m,2H),2.39(s,3H),2.31-2.15(m,2H),1.95-1.79(m,2H),0.88(t,J=7.2Hz,3H).

[0137] MS(ESI) m / z 553.1 [M+1] + .

[0138] Example 9: Preparation and Synthesis of Compound 10

[0139] 2-Amino-1-propanol SM11 (35.39 mg, 471.20 μmol, 37.53 μL) was added to a THF (1.5 mL) solution of compound Int5 (150 mg, 314.13 μmol). The mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by pre-HPLC to obtain compound 10 (14.35 mg).

[0140] 1 H NMR (400MHz, DMSO-d6): δ8.09(br d,J=8.8Hz,1H),7.79(br d,J=11.2Hz,1H),7.50(br s,1H),7.31(d,J=4.4Hz,1H),6.50(d,J=7.6Hz,1H),6.23(br d,J=5.2Hz,1H),5.42(s,2H),5.25(s,2H),4.94-4.80(m,1H),4.51-4.25(m,1H),3.60-3.38(m,2H),3.24- 3.12(m,2H),2.39(s,3H),2.27-2.08(m,2H),1.93-1.77(m,2H),1.25-1.12(m,3H),0.87(t,J=7.2Hz,3H).

[0141] MS(ESI) m / z 553.1 [M+1] + .

[0142] Example 10: Preparation and Synthesis of Compound 11

[0143] Compound SM12 (5 g, 70.34 mmol) was added to a three-necked flask, followed by the addition of ethanol (15 mL) and diethyl ether (85 mL) to dissolve it. The mixture was cooled to 0 °C, and dried hydrochloric acid was bubbled through it for half an hour. After bubbling, the mixture was stirred at 0 °C for 12 hours. After concentration, diethyl ether (100 mL) was added, and the mixture was cooled to -20 °C, causing a solid to precipitate. The solid was then stirred at room temperature for 10 minutes and filtered. The filter cake was collected and concentrated under reduced pressure to obtain compound Int6 (2.5 g).

[0144] Compound Int6 (564 mg, 3.68 mmol) was added to a single-necked flask, followed by the addition of DMSO (5.0 mL) for dissolution and DIEA (237 mg, 1.84 mmol) and stirring for 1 h. Then, eczemacon mesylate (0.4 g, 0.92 mmol) and DIEA (0.95 g, 7.36 mmol) were added, and the reaction mixture was stirred at 40 °C for 12 h. The reaction mixture was cooled to room temperature and filtered. The filtrate was purified by pre-HPLC to obtain compound 11 (19 mg).

[0145] 1 H NMR (400MHz, DMSO-d6): δ9.786-9.765(d,J=8.5Hz,1H),9.496(s,1H),9.271(s,1H),7.895-7.8 68(d,J=11.0Hz,1H),7.346(s,1H),6.558(s,1H),5.472-5.440(m,3H),5.325-5.224(m,2H),5.0 95(m,1H),3.710(s,2H),3.271-3.228(m,1H),3.112-3.048(m,1H),2.578-2.548(m,1H),2.427 (s,3H),2.381-2.337(m,1H),2.179-2.115(m,1H),1.926-1.821(m,2H),0.877(t,J=7.5Hz,3H).

[0146] MS(ESI) m / z 507.2 [M+1] + .

[0147] Example 11: Preparation and Synthesis of Compound 12

[0148] Compound SM13 (3g, 30.89mmol) was added to a three-necked flask and dissolved in ethanol (12mL) and diethyl ether (120mL). The mixture was cooled to 0°C and dried hydrochloric acid was passed through it for half an hour. After the aeration was complete, the mixture was stirred at 0°C for 12 hours. The reaction solution was concentrated, and diethyl ether (100mL) was added. The mixture was cooled to -20°C, and a solid precipitated. The solid was then stirred at room temperature for 10 minutes and filtered. The filter cake was collected and concentrated under reduced pressure to obtain compound Int7 (2.0g).

[0149] Compound Int7 (825 mg, 4.6 mmol) was added to a single-necked flask, dissolved in 10 mL of DMSO, and then DIEA (1.19 g, 9.2 mmol) was added and stirred for 1 h. Then, eczemacon mesylate (0.50 g, 1.15 mmol) and DIEA (1.19 g, 9.2 mmol) were added, and the reaction mixture was stirred at 40 °C for 12 h. The reaction mixture was cooled to room temperature and filtered. The filtrate was purified by pre-HPLC to obtain compound 12 (18 mg).

[0150] 1 H NMR (400MHz, DMSO-d6): δ9.662-9.641 (d, J=8.5Hz, 1H), 9.360-9.321 (d, J=15.5Hz, 2H),7.881-7.854(d,J=10.5Hz,1H),7.341(s,1H),6.556(s,1H),5.494-5.144(m,6H ),4.046-4.010(m,1H),3.213-3.157(m,2H),2.878-2.788(m,1H),2.581-2.539(m,1 H),2.422(s,3H),2.327-2.109(m,4H),1.925-1.837(m,2H),0.877(t,J=7.5Hz,3H).

[0151] MS(ESI) m / z 533.5 [M+1] + .

[0152] Example 12: Preparation and Synthesis of Compound 13

[0153] Compound SM14 (1.0 g, 10 mmol) was added to a three-necked flask and dissolved in ethanol (5.0 mL) and diethyl ether (50 mL). The mixture was cooled to 0 °C and purged with dried hydrochloric acid for half an hour. After purging, the mixture was stirred at 0 °C for 12 h. The reaction solution was concentrated, and diethyl ether (100 mL) was added. The mixture was cooled to -20 °C, and a solid precipitated. The solid was then stirred at room temperature for 10 min and filtered. The filter cake was collected and concentrated under reduced pressure to obtain compound Int8 (0.60 g).

[0154] Compound Int8 (495 mg, 2.76 mmol) was added to a single-necked flask, dissolved in 10 mL of DMSO, and DIEA (178 mg, 1.38 mmol) was added. The mixture was stirred for 1 h. Ecinotecan (0.30 g, 0.69 mmol) and DIEA (713 mg, 5.52 mmol) were then added, and the reaction mixture was stirred at 40 °C for 12 h. The reaction mixture was cooled to room temperature and filtered. The filtrate was purified by pre-HPLC to obtain compound 13 (17 mg).

[0155] 1 H NMR (400MHz, DMSO-d6): δ9.871-9.758 (dd, J=36.5, 8.5Hz, 1H), 9.511-9.427 (d, J=33.5Hz, 2H), 7.8 84-7.857(d,J=10.5Hz,1H),7.345(s,1H),6.554(s,1H),6.348-6.323(m,1H),5.556-5.432(m,1H), 5.303-5.256(m,2H),5.209-5.144(m,2H),4.054-3.938(m,1H),3.209-3.196(m,1H),2.427(s,3H), 2.263(m,2H),1.906-1.837(m,2H),1.229-1.196(m,1H),0.902-0.857(m,3H),0.595-0.455(m,4H).

[0156] MS(ESI) m / z 533.2 [M+1] + .

[0157] Example 13: Preparation and Synthesis of Compound 14

[0158] Step 1: Compound 1.1 (5.0 g, 67 mmol) was dissolved in acetonitrile (120 mL), and AllocOSu (14 g, 70 mmol) and triethylamine (7.4 g, 73 mmol) were added. The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was concentrated, dissolved in ethyl acetate, and the organic phase was washed with water. The organic phase was concentrated and dried to obtain a crude product. The crude product was purified by silica gel column chromatography (DCM / EtOAc = 1 / 1) to obtain compound 1.2 (9.5 g).

[0159] Step 2: Compound 1.3 (10 g, 76 mmol) was dissolved in H2O / ACN (200 mL), and FmocOSu (26.8 g, 79.47 mmol) and triethylamine (8.04 g, 79.47 mmol) were added. The reaction mixture was stirred at room temperature for 8 h. After the reaction was completed, HCl (1 M) was added to the reaction mixture to adjust the pH to 4. The precipitated solid was filtered, washed three times with water, and lyophilized to obtain compound 1.4 (20 g).

[0160] Step 3: Compound 1.4 (10 g, 28.22 mmol) was dissolved in DMF (150 mL), and Pb(OAc)4 (18.7 g, 42.33 mmol) was added. The reaction mixture was stirred at 60 °C for 0.5 h under N2 protection. After the reaction was completed, water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was washed with water, dried, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (DCM / EtOAc = 4 / 1) to obtain compound 1.5 (10 g).

[0161] Step 4: Compound 1.5 (5 g, 13.57 mmol) was dissolved in THF (70 mL), and compound 1.2 (2.27 g, 14.25 mmol) and TsOH (2.57 g, 16.28 mmol) were added. The mixture was reacted at 0 °C for 1 h under N2 protection. Water was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (DCM / EtOAc = 4 / 1) to obtain compound 1.6 (2.9 g).

[0162] Step 5: Compound 1.6 (2.6 g, 5.56 mmol) was dissolved in DCM (30 mL), and diethylamine (6 mL) was added. The reaction mixture was stirred at room temperature for 2 h. LCMS showed that the reaction was complete, and the reaction mixture was directly concentrated to give crude compound 1.7 (1.1 g).

[0163] Step 6: Compound 1.7 (0.9 g, 3.67 mmol) and ((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (1.93 g, 3.85 mmol) were dissolved in DMF (30 mL), and HATU (2.09 g, 5.51 mmol) and triethylamine (0.742 g, 7.34 mmol) were added. The mixture was stirred at room temperature for 2 h. LCMS showed that the reaction was complete. The reaction solution was directly purified by pre-HPLC to obtain compound 1.8 (1.5 g).

[0164] Step 7: Compound 1.8 (1.5 g, 2.06 mmol) was dissolved in THF (20 mL), and 5,5-dimethylcyclohexane-1,3-dione (115.4 mg, 0.82 mmol) and tetraphenylphosphine palladium (713.5 mg, 0.62 mmol) were added. The reaction mixture was stirred for 30 min under N2 protection. LCMS showed that the reaction was complete, and the reaction solution was directly purified by pre-HPLC to obtain compound 1.9 (0.8 g).

[0165] Step 8: The nitrogen-indo[1,2-b]quinoline-10,13-dione (0.337 g, 0.78 mmol) of compound (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7] was dissolved in DMF (10 mL), and triethylamine (0.159 g, 1.56 mmol) and CDI (0.125 mg, 0.78 mmol) were added. The mixture was stirred at room temperature for 20 min. Compound 1.9 (0.5 g, 0.78 mmol) was dissolved in DMF (2 mL) and added to the reaction mixture. The mixture was stirred at room temperature for 1 h, and then directly purified by pre-HPLC to obtain compound 1.10 (0.8 g).

[0166] Step 9: Compound 1.10 (0.8 g, 0.723 mmol) was dissolved in DMF (10 mL), and diethylamine (4 mL) was added. The reaction mixture was stirred at room temperature for 1 h. LCMS showed that the reaction was complete. The reaction mixture was directly purified by pre-HPLC to obtain compound 1.11 (0.2 g).

[0167] Step 10: Compound 1.11 (0.125 g, 0.141 mmol) was dissolved in DMF (2 mL), and 2,5-dioxopyrrolidine-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid ester (45.78 mg, 0.148 mmol) and triethylamine (14.3 mg, 0.141 mmol) were added. The reaction mixture was stirred at room temperature for 1 h. LCMS showed that the reaction was complete, and the reaction mixture was directly purified by pre-HPLC to obtain compound 14 (23 mg).

[0168] 1H NMR (400MHz, DMSO-d6): δ8.50(m,1H),8.32(m,1H),8.20-8.00(m,3H),7.75(d,J=4.5Hz,1 H),7.29-7.11(m,6H),7.01(s,2H),6.85(d,J=4.5Hz,1H),7.45(s,1H),5.35(m,3H),5.15 (m,2H),4.51-4.40(m,3H),3.70-3.00(m,15H),2.85(s,3H),2.71(m,1H),2.32(s,3H),2. 15-2.01(m,4H),190-1.75(m,2H),1.40-1.30(m,4H),1.15(m,2H),0.84(t,J=8.5Hz,3H).

[0169] MS(ESI) m / z 1078.4 [M+1] + .

[0170] Example 14: Preparation and Synthesis of Compound 15

[0171] Step 1: Add an acetone (350 mL) solution of Alloc-OSU (11.622 g, 58.4 mmol) to a water (236 mL) solution of compound 2.1 (4.38 g, 58.4 mmol) and NaHCO3 (4.906 g, 58.4 mmol), stir at room temperature for 4 h, remove acetone by rotary evaporation, extract with aqueous phase EA (200 mL × 2), combine organic phases, wash with saturated brine, dry with anhydrous sodium sulfate, filter, and evaporate to dryness to obtain crude compound 2.2 (11.0 g).

[0172] Step 2: Under nitrogen protection at 0°C, a THF solution of compound 2.2 (4.3 g, 27.04 mmol) in 10 mL of THF was added to a THF solution of compound 1.5 (4.976 g, 13.52 mmol) and TsOH (1.163 g, 6.76 mmol) in 40 mL of THF. The mixture was stirred at this temperature for 2 h. After removing the solvent by evaporation, the sample was dissolved in a small amount of DCM and loaded onto a wet plate. The sample was then purified by silica gel column chromatography (MeOH / DCM = 0%-10%) to obtain compound 2.3 (6.3 g).

[0173] Step 3: At room temperature, DEA (20 mL) was added to a DCM (40 mL) solution of compound 2.3 (6.3 g, 13.48 mmol). After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction solution was then evaporated to dryness using an oil pump until constant weight was obtained, yielding crude compound 2.4 (5.2 g).

[0174] Step 4: At 0°C, DIEA (1.316 g, 10.2 mmol) and HATU (3.876 g, 10.2 mmol) were added to a DMF (25 mL) solution of compound 2.4 (2.5 g, 10.2 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (12.778 g, 25.5 mmol). After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction solution was then directly purified by pre-HPLC and lyophilized to obtain compound 2.5 (2.8 g).

[0175] Step 5: Under nitrogen protection, phenylsilane (0.327 g, 3.02 mmol) was added to a DCM / MeOH (1 / 1, 11 mL) solution of compound 2.5 (1.1 g, 1.51 mmol) and Pd(PPh3)4 (0.872 g, 0.76 mmol). After the addition was complete, the mixture was stirred at room temperature for 20 min. The reaction solution was filtered, purified by pre-HPLC, and lyophilized to obtain compound 2.6 (520 mg).

[0176] Step 6: Under nitrogen protection, CDI (302 mg, 1.86 mmol) was added to a DMF (16 mL) solution of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione (825 mg, 1.55 mmol) and DIEA (240 mg, 2.33 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 h. Compound 2.6 (400 mg, 0.62 mmol) was added to the above reaction solution and stirred at room temperature for 2 h. The reaction solution was purified by pre-HPLC and lyophilized to obtain compound 2.7 (150 mg).

[0177] Step 7: Add DEA (1 mL) to 2 mL of DMF solution containing compound 2.7 (150 mg, 0.23 mmol). After addition, stir at room temperature for 2 h. The reaction solution was purified by pre-HPLC and lyophilized to obtain compound 2.8 (80 mg).

[0178] Step 8: 2,5-Dioxopyrrolidone-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate (33 mg, 0.11 mmol) was added to a DMF (2 mL) solution of compound 2.8 (80 mg, 0.09 mmol) and DIEA (18 mg, 0.14 mmol). After addition, the mixture was stirred at room temperature for 1 h. The reaction solution was purified by pre-HPLC and lyophilized to obtain compound 15 (30 mg).

[0179] 1 H NMR (400MHz, DMSO-d6): δ8.50(m,1H),8.30(m,1H),8.10(m,3H),7.80(d,J=4.4Hz, 1H),7.21(m,6H),7.01(s,2H),6.55(s,2H),5.85(d,J=4.4Hz,1H),5.40-5.10(m,5 H),4.51(m,3H),3.80-3.00(m,11H),2.80-2.71(m,1H),2.31(s,3H),2.27-2.08(m ,4H),1.93-1.77(m,2H),1.42-1.30(m,4H),1.15(m,3H),1.05(m,3H),0.87(m,5H).

[0180] MS(ESI) m / z 1077.3 [M+1] + .

[0181] Example 15: Preparation and Synthesis of Compound 15A

[0182] Step 1: At 0°C, a solution of NaHCO3 (4.906 g, 58.4 mmol) and Alloc-OSU (11.622 g, 58.4 mmol) in THF (50 mL) was added to a solution of compound 3.1 (4.38 g, 58.4 mmol) in H2O (50 mL). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS until completion. The reaction solution was directly concentrated at low temperature. The aqueous phase was extracted with ethyl acetate (80 mL × 2). The organic phases were combined, washed with saturated brine (80 mL × 2), dried over anhydrous sodium sulfate, and concentrated to obtain compound 3.2 (8.4 g).

[0183] Step 2: Under nitrogen protection at 0°C, a THF solution of compound 3.2 (3.24 g, 20.38 mmol) in 10 mL of THF was added to a THF solution of compound 1.5 (5 g, 13.57 mmol) and TsOH (1.17 g, 6.79 mmol) in 30 mL. The mixture was stirred at this temperature for 0.5 h. The reaction was monitored by LCMS until completion. The sample was directly loaded onto a wet plate and purified by silica gel column chromatography (55-75% EA / PE) to obtain compound 3.3 (2.2 g).

[0184] Step 3: DEA (7 mL) was added to a DCM solution (14 mL) of compound 3.3 (2.1 g, 4.5 mmol) at room temperature, and the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS until it was complete. The reaction solution was then concentrated at low temperature and dried by pump to obtain compound 3.4 (2.4 g).

[0185] Step 4: At 0-5℃, 2,6-dimethylpyridine (1.44 g, 13.47 mmol) and HATU (2.05 g, 5.39 mmol) were added to a DMF (25 mL) solution of compound 3.4 (2.2 g, 8.98 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (2.25 g, 4.49 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS until completion. The reaction solution was directly purified by pre-HPLC to obtain compound 3.5 (1.7 g).

[0186] Step 5: At room temperature, phenylsilane (475 mg, 4.4 mmol) and Pd(PPh3)4 (507 mg, 0.44 mmol) were added to a THF / MeOH (35 mL, 2 / 1) solution of compound 3.5 (1.6 g, 2.2 mmol). The mixture was stirred at room temperature for 20 min after the addition was complete. The reaction was monitored by LCMS until it was complete. The reaction solution was filtered and purified by pre-HPLC to obtain compound 3.6 (1 g).

[0187] Step 6: At 0-5℃, DIEA (182 mg, 1.41 mmol) and CDI (183 mg, 1.13 mmol) were added to a DMF (10 mL) solution of eczemacon mesylate (500 mg, 0.942 mmol). The mixture was stirred at room temperature for 1.5 h after the addition was complete. After the reaction was completed as monitored by LCMS, compound 3.6 (364 mg, 0.565 mmol) was added to the reaction solution, and the reaction was continued for 2 h. After the reaction was completed as monitored by LCMS, the pH of the reaction solution was adjusted to 5-6 with acetic acid, and then purified and concentrated by pre-HPLC to obtain compound 3.7 (260 mg).

[0188] Step 7: At room temperature, DABCO (106 mg, 0.95 mmol) was added to a DMF (3 mL) solution of compound 3.7 (210 mg, 0.19 mmol), and the mixture was stirred at room temperature for 1.5 h. The reaction was monitored by LCMS until it was complete. The pH of the reaction solution was adjusted to 5-6 with acetic acid, and then purified by pre-HPLC, concentrated, and lyophilized to obtain compound 3.8 (126 mg).

[0189] Step 8: At room temperature, 2,6-dimethylpyridine (31 mg, 0.286 mmol) was added to a DMF (2 mL) solution of compound 3.8 (126 mg, 0.143 mmol) and 2,5-dioxopyrrolidone-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate (44 mg, 0.143 mmol). The mixture was stirred at room temperature for 1 h after the addition was complete. The reaction was monitored by LCMS until it was complete. The pH of the reaction solution was adjusted to 5-6 with acetic acid, and then purified by pre-HPLC, concentrated, and lyophilized to obtain compound 15A (115 mg).

[0190] 1 H NMR (400MHz, DMSO-d6): δ8.500(t,J=5.0,10.5Hz,1H),8.280-8.253(t,J=5.0,10.5Hz,1H),8.104-7.971(m,3H),7.776(d,J= 11.0Hz,1H),7.305(s,1H),7.216-7.140(m,5H),6.979(s,2H),6.625(d,J=9.0Hz,1H),6.512(s,1H),5.857(d,J=8.0Hz,1H),5 .427-5.205(m,5H),4.600-4.477(m,3H),3.816-3.407(m,9H),3.347-3.264(m,2H),3.141(m,2H),3.043-2.998(m,1H),2.79 8-2.739(m,1H),2.379(s,3H),2.137(m,4H),1.810(m,2H),1.400(m,4H),1.151(m,2H),1.095(m,3H),0.889(t,J=8.5Hz,3H).

[0191] MS(ESI) m / z 1077.4 [M+1] + .

[0192] Example 16: Preparation and Synthesis of Compound 15B

[0193] Following the method described in Example 15 above, compound 15B was prepared using S-configuration aminopropanol as the starting material.

[0194] 1H NMR (400MHz, DMSO-d6): δ8.528(t,J=5.0,10.5Hz,1H),8.325(t,J=5.0,10.5Hz,1H),8.156-8.031(m,3H),7.787(d,J=11.0Hz ,1H),7.310-7.192(m,6H),7.005(s,2H),6.641-6.619(d,J=8.5Hz,1H),6.521(s,1H),5.878(d,J=8.0Hz,1H),5.436-5.181(m ,5H),4.601-4.511(m,3H),3.794-3.625(m,7H),3.403-3.372(m,3H),3.344-3.322(m,1H),3.176-3.045(m,3H),2.850-2.81 5(m,1H),2.392(s,3H),2.135-2.098(m,4H),1.894(m,2H),1.412(m,4H),1.115(m,2H),1.095(m,3H),0.889(t,J=8.5Hz,3H).

[0195] MS(ESI) m / z 1077.4 [M+1] + .

[0196] Example 17: Preparation and Synthesis of Compound 16

[0197] Step 1: Compound 4.1 (3 g, 29.66 mmol) was added to a 250 mL single-necked flask and dissolved in THF (30 mL) and water (30 mL). NaHCO3 (7.48 g, 88.98 mmol) was then added, followed by allyl chloroformate (24.07 g, 35.59 mmol). The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was concentrated and extracted with EA (100 mL × 3). The combined organic phases were concentrated under vacuum and evaporated to dryness. The crude product was purified by silica gel column chromatography (PE / EA = 1 / 1) to obtain compound 4.2 (4 g).

[0198] Step 2: In a 500 mL round-bottom flask, (S)-11-benzyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentoxo-2-oxo-4,7,10,13,16-pentazaheptadecane-17-acetate (6.5 g, 10.3 mmol) was added and dissolved in THF (70 mL). Compound 4.2 (2.0 g, 10.8 mmol) and TsOH (2.1 g, 12.4 mmol) were added at 0 °C. The reaction mixture was stirred at 0 °C for 1 h under argon protection. The reaction mixture was purified by pre-HPLC to obtain compound Cpd 4.3 (2.4 g).

[0199] Step 3: Compound 4.3 (2.3 g, 3.05 mmol) was added to a 250 mL round-bottom flask and dissolved in 30 mL of THF. Morpholine (796 mg, 9.15 mmol) and Pd(PPh3)4 (1.06 g, 0.915 mmol) were added at 0 °C. The reaction solution was stirred at 0 °C for 30 min under argon protection. The reaction solution was purified by pre-HPLC to obtain compound 4.4 (1 g).

[0200] Step 4: Add eczema (792 mg, 1.5 mmol) to a 250 mL round-bottom flask and dissolve it in DMF (10 mL). Add DIEA (385 mg, 3.0 mmol) and CDI (242 mg, 1.5 mmol) at 0 °C. Stir the reaction mixture at room temperature for 30 min. Continue to add compound 4.4 (1 g, 1.5 mmol, dissolved in 10 mL DMF) to the reaction mixture at 0 °C, and stir the mixture at room temperature for 1 h. The reaction mixture is then purified by pre-HPLC to obtain compound 4.5 (1 g).

[0201] Step 5: Add compound 4.5 (1 g, 0.88 mmol) to a 100 mL flask, dissolve it in DCM (20 mL), and add DEA (10 mL) at 0 °C. Stir the reaction solution at room temperature for 2 h. Concentrate the reaction solution to dryness, and purify it by pre-HPLC to obtain compound 4.6 (250 mg).

[0202] Step 6: Compound 4.6 (250 mg, 0.27 mmol) was added to a 100 mL round-bottom flask and dissolved in 10 mL of DMF. Then, 6-(maleimide)hexanoic acid succinimide ester (85 mg, 0.27 mmol) and DIEA (71 mg, 0.55 mmol) were added at 0 °C. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was purified by pre-HPLC to obtain compound 16 (46 mg).

[0203] 1H NMR (400MHz, DMSO-d6): δ8.50(m,1H),8.25(m,1H),8.10-8.00(m,3H),7.77(d,J=11.0Hz,1H),7.31(m,1H),7.22 -7.14(m,5H),6.98(s,2H),6.90-6.88(m,2H),5.43(s,3H),5.31-5.15(m,2H),4.64-4.56(m,2H),4.51-4.46(m, 1H),3.76-3.52(m,10H),3.34-3.33(m,3H),3.23(m,1H),3.11-3.00(m,2H),2.81-2.76(m,1H),2.54(m,1H),2.3 8(s,3H),2.21-2.20(m,2H),2.11(m,2H),1.82(m,2H),1.35(m,4H),1.11(m,2H),0.69-0.79(m,5H),0.60(s,2H).

[0204] MS(ESI) m / z 1104.4 [M+1] + .

[0205] Example 18: Preparation and Synthesis of Compound 17

[0206] Step 1: Compound 5.1 (10 g, 133.14 mmol) was added to a 500 mL single-necked flask, dissolved in 100 mL THF and 100 mL water, followed by the addition of 33.6 g NaHCO3 (399.41 mmol) and allyl chloroformate (24.07 g, 199.70 mmol). The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was concentrated, extracted with ethyl acetate (200 mL × 3), and the combined organic phases were concentrated under vacuum and evaporated to dryness. The crude product was purified by silica gel column chromatography (PE / EA = 1 / 1) to obtain compound 5.2 (12 g).

[0207] Step 2: In a 500 mL round-bottom flask, (S)-11-benzyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentoxo-2-oxo-4,7,10,13,16-pentazaheptadecane-17-acetate (10 g, 15.9 mmol) was added and dissolved in 100 mL of THF. Compound 5.2 (2.65 g, 16.7 mmol) and TsOH (3.28 g, 19.1 mmol) were added at 0 °C. The reaction mixture was stirred at 0 °C for 1 h under argon protection. The reaction mixture was purified by pre-HPLC to obtain compound 5.3 (2.8 g).

[0208] Step 3: Compound 5.3 (2.7 g, 3.7 mmol) was added to a 50 mL round-bottom flask and dissolved in 30 mL of THF. Morpholine (968 mg, 11.1 mmol) and Pd(PPh3)4 (1.28 g, 1.11 mmol) were added at 0 °C. The reaction mixture was stirred at 0 °C for 30 min under argon protection. The reaction mixture was purified by pre-HPLC to obtain compound 5.4 (1.45 g).

[0209] Step 4: In a 50 mL round-bottom flask, add eczema (1.19 g, 2.25 mmol) dissolved in 20 mL of DMF. Add DIEA (581 mg, 4.50 mmol) and CDI (401 mg, 2.25 mmol) at 0 °C. Stir the reaction mixture at room temperature for 30 min. Continue adding compound 5.4 (1.45 g, 2.25 mmol, dissolved in 10 mL of DMF) to the reaction mixture at 0 °C. Continue stirring the reaction mixture at room temperature for 1 h. Purify the reaction mixture using pre-HPLC to obtain compound 5.5 (700 mg).

[0210] Step 5: Compound 5.5 (700 mg, 0.62 mmol) was added to a 100 mL flask and dissolved in DCM (20 mL). DEA (10 mL) was added at 0 °C, and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was evaporated to dryness and purified by pre-HPLC to obtain compound 5.6 (230 mg).

[0211] Step 6: Compound 5.6 (230 mg, 0.25 mmol) was added to a 100 mL round-bottom flask and dissolved in 10 mL DMF. 6-(maleimide)hexanoic acid succinimide ester (79 mg, 0.25 mmol) and DIEA (66 mg, 0.51 mmol) were added at 0 °C. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was purified by pre-HPLC to obtain compound 17 (21 mg).

[0212] 1H NMR (400MHz, DMSO-d6): δ8.47(m,1H),8.22(m,1H),7.99-7.91(m,3H),7.81(d,J=8.0Hz,1H),7.73(d,J=8.0Hz,1H),7.25 (s,1H),7.19-7.10(m,5H),6.92(s,2H),6.44(s,1H),6.35-6.33(m,1H),5.35(s,2H),5.17-5.08(m,2H),4.52(d,J=8.0H z,2H),4.43(m,1H),3.87(m,2H),3.69-3.49(m,8H),3.29(m,1H),3.14(s,3H),3.05-2.96(m,3H),2.75-2.69(m,1H),2.3 3(s,3H),2.17(m,2H),2.03(m,2H),1.81-1.76(m,2H),1.42-1.37(m,4H),1.17-1.09(m,3H),0.80(t,J=8.0,16.0Hz,3H).

[0213] MS(ESI) m / z 1094.3 [M+1] + .

[0214] Example 19: Preparation and Characterization of Antibody-Drug Conjugates (ADCs)

[0215] The Her2 antibody, Trastuzumab (Roche), was replaced with 20 mM pH 6.0 His / His-HCl buffer to a concentration of 10 mg / mL. Ten molar equivalents of the reducing agent TCEP were added, and the mixture was incubated at 25°C with shaking for 2 h. Then, ten molar equivalents of the payload-linker of eczemab derivative dissolved in dimethyl sulfoxide were added, and the coupling reaction was continued at 25°C for 2 h. Eight molar equivalents of N-acetylcysteine ​​were added to quench the reaction for 30 min, followed by eight molar equivalents of dehydroascorbic acid, and the mixture was incubated at 25°C with shaking for 2 h. After the reaction, small molecules were removed using a Zeba™ desalting centrifuge column (40 K MWCO), and the ADC was replaced with 20 mM pH 6.0 His / His-HCl buffer for storage. The series of ADC products conjugated with the ixotecan derivative payload-linker and the Her2 antibody trastuzumab were analyzed for purity using SEC, and DAR values ​​were determined using HIC and MS. The information of the ADCs obtained by conjugation is shown in Table 1 below:

[0216] Table 1 Antibody-Drug Conjugates (ADCs)

[0217] Example 20: Comparison of in vitro cell-killing effects of compound 2 and Dxd

[0218] Experimental objective:

[0219] This case study compared the in vitro killing effects of compound 2 and Dxd on different cell lines. Specifically, the killing effects on cell lines AGS, NCI-N87, SK-BR-3, and MCF-7 were tested.

[0220] Experimental methods:

[0221] Gastric cancer cells NCI-N87 (RPMI 1640 + 10% FBS), breast cancer cells SK-BR-3 (McCoy's 5A + 10% FBS), gastric cancer cells AGS (RPMI 1640 + 10% FBS), and breast cancer cells MCF-7 (DMEM + 10% FBS + 10 μg / mL insulin) were seeded into 96-well plates, 100 μL per well. After incubation for 24 hours, each test substance was added at different concentrations (starting at 1 μM, 5-fold dilution, 10 doses). The plates were then further incubated at 37°C and 5% CO2 for 6 days before use. Cell viability was measured using the 2.0 Cell Viability Assay Reagent (Vazyme, catalog number DD1101).

[0222] Experimental results:

[0223] As shown in Table 2, compound 2 exhibits significant anti-tumor proliferation activity, and its effect is superior to that of the positive control compound Dxd. The good anti-proliferative effect of compound 2 indicates its potential as an effective payload for ADC drugs.

[0224] Table 2 Results of in vitro anti-cell proliferation assay (ICP) 50 / nM)

[0225] Example 21: In vitro cell-killing experiment of compound 2 and compound 2A

[0226] Experimental objective:

[0227] This case study compared the in vitro killing effects of compound 2 and compound 2A on different cell lines. Specifically, the killing effects on cell lines AGS, AsPC-1, 23132 / 87, and NCI-N87 were tested.

[0228] Experimental methods:

[0229] Gastric cancer cells NCI-N87 (RPMI 1640 + 10% FBS), pancreatic cancer cells AsPC-1 (McCoy's 5A + 10% FBS), gastric cancer cells AGS (RPMI 1640 + 10% FBS), and gastric cancer cells 23132 / 87 (DMEM + 10% FBS + 10 μg / mL insulin) were seeded into 96-well plates, 100 μL per well. After incubation for 24 hours, each test substance was added at different concentrations (starting at 1 μM, 5-fold dilution, 10 doses). The plates were further incubated at 37°C and 5% CO2 for 6 days before use. Cell viability was measured using the 3D cell viability assay kit (Promega, catalog number G9683).

[0230] Experimental results:

[0231] As shown in Table 3, the derivatives of different configuration epimers showed significant differences in their anti-tumor cell proliferation ability, with compound 2A exhibiting superior activity compared to compound 2.

[0232] Table 3 Results of in vitro anti-cell proliferation assay (IC50) 50 / nM)

[0233] Example 22: In vitro cell-killing experiment of compound 2A, compound 2 and Dxd

[0234] Experimental objective:

[0235] This case study compared the in vitro killing effects of Dxd, compound 2, and compound 2A. Specifically, the killing effects on cell lines NCI-H1299 and 23132 / 87 were tested.

[0236] Experimental methods:

[0237] Non-small cell lung cancer cells NCI-H1299 (RPMI 1640 medium + 10% FBS) and gastric cancer cells 23132 / 87 (DMEM medium + 10% FBS + 10 μg / mL insulin) were seeded into 96-well plates, 100 μL per well. After incubation for 24 hours, each test substance was added at different concentrations (starting concentration 1 μM, 5-fold dilution, 10 doses). After further incubation at 37°C and 5% CO2 for 3 days, the samples were then used... Cell viability was measured using the 2.0 Cell Viability Assay Reagent (Vazyme, catalog number DD1101).

[0238] Experimental results:

[0239] As shown in Table 4, under the same experimental conditions, the IC50 values ​​of compounds 2A and 2 are... 50Both were stronger than the control compound Dxd.

[0240] Table 4 Results of in vitro anti-cell proliferation assay (IC50) 50 / nM)

[0241] Example 23: In vitro cell-killing experiments of compounds 5, 7, 9, and 10

[0242] Experimental objective:

[0243] This implementation case compares... The in vitro killing effects of comparative compound D1 (WO2024067811A1, compounds 2-25 in examples) and four ixotecan derivatives, compounds 5, 7, 9, and 10, on different cell lines were investigated. Specifically, the killing effects on cell lines HT29, A375, Calu-6, and NCI-N87 were tested.

[0244] Experimental methods:

[0245] Malignant melanoma cells A375 (DMEM + 10% FBS), gastric cancer cells NCI-N87 (RPMI 1640 + 10% FBS), colon cancer cells HT-29 (McCoy's 5A + 10% FBS), and degenerative cancer cells Calu-6 (MEM + 10% FBS) were seeded into 96-well plates, 100 μL per well. After incubation for 24 hours, each test compound was added at different concentrations (starting at 1 μM, 5-fold dilution, 10 doses). The plates were then further incubated at 37°C and 5% CO2 for 6 days before use. Cell viability was measured using the 3D cell viability assay kit (Promega, catalog number G9683).

[0246] Experimental results:

[0247] As shown in Table 5, compounds 5, 7, 9, and 10 all exhibited strong cytotoxic effects in the four cell lines, significantly stronger than compound D1.

[0248] Table 5 Results of in vitro anti-cell proliferation assay (IC50) 50 / nM)

[0249] Example 24: In vitro cell-killing experiments of compounds 11, 12 and 13

[0250] This case study tested the in vitro killing effects of compounds 11, 12, and 13 on different cell lines. Specifically, the killing effects on cell lines SK-BR-3, AsPC-1, A375, and NCI-N87 were tested. SK-BR-3 breast cancer cells (McCoy's 5A medium + 10% FBS), AsPC-1 pancreatic cancer cells (RPMI 1640 medium + 10% FBS), A375 malignant melanoma cells (DMEM medium + 10% FBS), and NCI-N87 gastric cancer cells (RPMI 1640 medium + 10% FBS) were seeded in 96-well plates. After 24 hours of incubation, each tested compound was added at different concentrations (starting concentration 1 μM, 5-fold dilution, 10 doses), and further incubated at 37°C and 5% CO2 for 6 days before use. Cell viability was measured using a 3D cell viability assay kit (Promega, catalog number G9683). Results showed that these eczemacon derivatives exhibited significant cytotoxic activity in all four cell lines and significantly inhibited tumor cell proliferation.

[0251] Example 25: Comparative Prediction of Hydrophilicity of Ecinotecan Derivatives

[0252] The hydrophilicity of the payload is crucial to the efficacy and pharmacokinetics of an ADC (Advanced Drug Delivery System), with better hydrophilicity being beneficial. This example uses cLogP to compare the differences in hydrophilicity among some derivatives. Using Chemdraw software, the structural formula is input, and the system automatically predicts and calculates the cLogP value. Generally, the lower the cLogP value, the better the hydrophilicity. The results show that the compound of this invention has better hydrophilicity, with a significant improvement compared to SN-38 and eczema.

[0253] Example 26: Evaluation of the in vitro anti-cell proliferation effect of antibody-drug conjugate ADC-02

[0254] Experimental objective:

[0255] This case study compares the in vitro killing effects of ADC drugs DS-8201 and ADC-02, containing Dxd as the payload, on different cell lines. Specifically, the killing effects on cell lines NCI-N87, SK-BR-3, and AGS were tested.

[0256] Experimental methods:

[0257] Gastric cancer cells NCI-N87 (RPMI 1640 + 10% FBS), breast cancer cells SK-BR-3 (McCoy's 5A + 10% FBS), and gastric cancer cells AGS (RPMI 1640 + 10% FBS) were seeded into 96-well plates, 100 μL per well. After incubation for 24 hours, each ADC test agent was added at different concentrations (starting concentration 1 μM, 5-fold dilution, 11 doses). After further incubation at 37°C and 5% CO2 for 6 days, the samples were then used... Cell viability was measured using the 2.0 Cell Viability Assay Reagent (Vazyme, catalog number DD1101).

[0258] Experimental results:

[0259] The results showed that ADC-02 had a strong killing effect on all three cell lines, significantly inhibiting tumor cell proliferation (see Table 6). Besides its killing effect on the Her2-overexpressing cell line NCI-N87 and the moderately expressed SK-BR-3, it also showed a good killing effect on the Her2-low-expressing cell line AGS, exhibiting stronger in vitro proliferative activity than DS-8201. For the Her2-overexpressing cell line NCI-N87 and the moderately expressed SK-BR-3, the IC50 of ADC-02 was... 50 It is 2 to 3 times more potent than DS-8201, and almost 10 times more potent in the Her2-low-expressing AGS cell line. These data demonstrate the good anti-tumor proliferation effect of ADC-02.

[0260] Table 6 Results of in vitro anti-cell proliferation assays using ADCs (IC50, 100%) 50 / nM)

[0261] Example 27: Evaluation of in vitro anti-cell proliferation assays of antibody-drug conjugates ADC-02A and ADC-02B

[0262] Experimental objective:

[0263] This case study compared the in vitro killing effects of DS-8201, ADC-02A, and ADC-02B on different cell lines. Specifically, the killing effects on the NCI-N87 and AGS cell lines were tested.

[0264] Experimental methods:

[0265] Gastric cancer cells NCI-N87 (RPMI 1640 + 10% FBS) and AGS (RPMI 1640 + 10% FBS) were seeded into 96-well plates, 100 μL per well. After incubation for 24 hours, each ADC test substance was added at different concentrations (starting concentration 1 μM, 5-fold dilution, 11 doses). After further incubation at 37°C and 5% CO2 for 6 days, the samples were then used... Cell viability was measured using the 3D cell viability assay kit (Promega, catalog number G9683).

[0266] Experimental results:

[0267] The results are shown in Table 7. The results showed that ADC-02A and ADC-02B had different in vitro antitumor proliferative effects, significantly stronger than the control DS-8201. In the NCI-N87 cell line with high Her2 expression, the IC50 of ADC-02A was significantly higher. 50 It reached almost 10 times that of DS-8201. In the Her2 cell line with low AGS expression, the IC50 of ADC-02A was... 50 It is almost 25 times that of the DS-8201.

[0268] Table 7 Results of in vitro anti-cell proliferation assays using ADCs (IC50, 100 mg / dL). 50 / nM)

[0269] Example 28: Evaluation of the in vitro anti-cell proliferation assay of antibody-drug conjugates ADC-03 and ADC-04

[0270] Experimental objective:

[0271] This case study compares the killing effects of DS-8201, ADC-03, and ADC-04 on the AGS cell line.

[0272] Experimental methods:

[0273] Gastric cancer cells (AGS) were seeded in 96-well plates (RPMI 1640 medium + 10% FBS) at 100 μL per well. After incubation for 24 hours, each ADC test substance was added at different concentrations (starting concentration 1 μM, 5-fold dilution, 11 doses). After further incubation at 37°C and 5% CO2 for 6 days, the cells were then used... Cell viability was measured using the 2.0 Cell Viability Assay Reagent (Vazyme, catalog number DD1101).

[0274] Experimental results:

[0275] The results are shown in Table 8, which show that ADC-03 and ADC-04 have a stronger effect on the Her2-low-expressing cell line AGS than DS-8201.

[0276] Table 8 Results of in vitro anti-cell proliferation assays using ADCs (IC50, 100%) 50 / nM)

[0277] Example 29: In vivo efficacy evaluation of ADC-01

[0278] Experimental objective:

[0279] The efficacy of ADC-01 and the control drug DS-8201 in an in vivo tumor model with high Her2 expression level was determined.

[0280] Experimental methods:

[0281] Human gastric cancer NCI-N87 (Her2-positive and highly expressing) cells (5 × 10⁻⁶) were subcutaneously inoculated into the right rib area of ​​Balb / c nude mice. 6 Contains 50% Matrigel matrix gel (per tumor), with an average tumor volume of approximately 180-200 mm. 3 The animals were grouped on day 0, with 6 animals per group. ADC was administered once intravenously at a dose of 3 mg / kg. Tumor volume and body weight were measured twice weekly and recorded. Tumor volume (V) was calculated using the formula: V = 1 / 2 × L 长 ×L 短 2 Relative volume (RTV) = V T / V0, tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%). Among them, V0 and V T Tumor volumes were measured at the start of the experiment (day 0, the day of the first dose) and at the time of tumor counting. C RTV T RTV The figures represent the relative tumor volumes of the blank control group and the experimental group at the end of the experiment, respectively. SEM represents the standard deviation of the mean.

[0282] Experimental results:

[0283] In the Her2-positive, high-expressing human gastric cancer cell NCI-N87 tumor model, after 3 to 7 days of administration, a significant inhibitory effect on tumor growth was observed in each group compared to the blank control. Continuing observation until day 28, the relative tumor volume (RTV) in the ADC-01 group was significantly smaller than that in the DS-8201 control group, and the tumor growth inhibition rate (TGI) in the ADC-01 group was 96.75%, significantly higher than that in the DS-8201 group (84.89%). No significant body weight loss (BW Loss) was observed in the mice. Results are shown in Table 9 and Figure 1.

[0284] Table 9. Results of in vivo antiproliferative assay of ADC (day 28)

Claims

1. A compound represented by formula (Ⅰ), its stereoisomer, or a pharmaceutically acceptable salt thereof: Its features are, X 1 For O, S, or NH; X 2 For -NR 2 - or key, R 2 -H, -C 1-3 Alkyl or 3-6 membered cycloalkyl; X 3 It can be -O- or -NH-; R 1 -H, -C 1-3 Alkyl or 3-6 membered cycloalkyl; M is -CR 3a R 4a -(CR 3 R 4 ) m -or Among them, R 3a R 4a R 3 R 4 Each is independently -H, -C 1-5 Alkyl or 3-6 membered cycloalkyl, where m is 0, 1, 2 or 3, and ring A is a 3-6 membered cycloalkane; Where X 1 For O or S and X 2 When R is -NH-, 1 R 3a R 4a At least one of them is not -H.

2. The compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, R 1 It is -H, methyl or cyclopropyl, preferably -H; and / or R 2 It is -H, methyl or cyclopropyl, preferably -H or methyl; and / or R 3a R 4a One is -H, and the other is -C 1-5 Alkyl or 3-6 membered cycloalkyl, preferably R 3a R 4a One is -H, and the other is methyl, isopropyl, or cyclopropyl; and / or R 3 R 4 Each is independently -H, -C 1-3 Alkyl or 3-5 membered cycloalkyl; preferably R 3 R 4 Each is independently -H, methyl, cyclopropyl, or isopropyl; more preferably, R 3 R 4 -H; and / or m is 0, 1, or 2, preferably 0 or 1; and / or Ring A is a 3-5 membered cycloalkyl group, preferably a cycloalkyl group.

3. The compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that, M is -CR 3a R 4a -CR 3 R 4 -or Preferably, it is -CH(CH3)CH2-. More preferably, M is -CH(CH3)CH2-.

4. The compound according to claim 1, its stereoisomers, or pharmaceutically acceptable salts thereof, characterized in that, It has the structure shown in equation (Ia): Wherein, M is as described in claim 1; preferably, M is -CR 3a R 4a -(CR 3 R 4 ) m -, R 3a R 4a One is -H, and the other is -C 1-5 Alkyl or 3-6 membered cycloalkyl, R 3 R 4 Each is independently -H, -C 1-5 Alkyl or 3-6 membered cycloalkyl, m is 1, 2 or 3; more preferably, m is 1; even more preferably, M is -CR 3a R 4a CH2-, R 3a R 4a One of them is -H, and the other is methyl, isopropyl, or cyclopropyl; Or it may have the structure shown in equation (Ib): Among them, R 2 M and M are each independently described according to claim 1; Or it may have the structure shown in equation (Ic): Wherein, M is as described in claim 1; preferably, M is -CR 3a R 4a -(CR 3 R 4 ) m -or R 3a R 4a R 3 R 4 Each is independently -H or a 3-6 membered cycloalkyl group, ring A is a 3-6 membered cycloalkane, and m is 0, 1, 2 or 3; more preferably, M is -CR. 3a R 4a -(CR 3 R 4 ) m -or R 3a R 4a R 3 R 4 Each is independently -H or a 3-5 membered cycloalkyl group, ring A is a 3-5 membered cycloalkane, and m is 0, 1, or 2; more preferably, M is -CR. 3a R 4a -(CR 3 R 4 ) m -or R 3a R 4a R 3 R 4 Each is independently -H or cyclopropyl, and m is 0 or 1.

5. A compound, its stereoisomer, or a pharmaceutically acceptable salt thereof, characterized in that, The structure is as follows: Preferably, the compound, its stereoisomer, or a pharmaceutically acceptable salt thereof has the following structure:

6. A compound represented by formula (II), its stereoisomer, or a pharmaceutically acceptable salt thereof: Its features are, R 1 X 1 X 2 M, X 3 Each independently according to any one of claims 1-4; L 1 For key, Among them, the -NH- end and L 2 Connected, the other end is connected to X 3 Connected, R a Or R b Each is independently -H or -C 1-3 Alkyl groups, preferably L 1 for L 2 A peptide consisting of 2 to 7 amino acid residues, wherein each amino acid is independently phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, or glycine, and each amino acid residue is optionally independently divided by one or more halogens, hydroxyl groups, cyano groups, amino groups, or -C groups. 1-5 Alkyl, -C 1-5 Alkoxy or 3-6 membered cycloalkyl substitution, wherein the -NH- terminus is associated with an L-terminus. 3 Connected to L, the other end is connected to L. 1 Linked; preferably, each of the amino acids is independently glycine, phenylalanine, valine, or citrulline; more preferably, L 2 The residues are -glycine residues - phenylalanine residues - glycine residues - (-Gly-Phe-Gly-), -glycine residues - glycine residues - phenylalanine residues - glycine residues - (-Gly-Gly-Phe-Gly-), or -valine residues - citrulline residues - (-Val-Cit-); more preferably, L 2 for L 3 -(CH2) n1 -C(O)-,-(CH2CH2O) n2 -C(O)- or -(CH2) n3 -W-(CH2) n4 -C(O)-, where the -C(O)- terminal is connected to L 2 Connected to L, the other end is connected to L. 4 Connected, n1, n2, n3, and n4 are each independently an integer from 0 to 8, and W is selected from -O-, -C(O)-NH-, 6-10 aryl, 5-10 aryl heteroalkyl, 3-6 cycloalkyl, or 3-6 heterocyclic groups. The 6-10 aryl, 5-10 aryl heteroalkyl, 3-6 cycloalkyl, or 3-6 heterocyclic groups are optionally converted by one or more halogens, hydroxyl groups, cyano groups, amino groups, or -C groups. 1-5 Alkyl, -C 1-5 Alkoxy or 3-6 membered cycloalkyl substitution; preferably, L 3 for L 4x for -C(=O)CH2Br or -C(=O)CH2I, preferably More preferably, the compound represented by formula (II), its stereoisomers, or pharmaceutically acceptable salts thereof have the structure shown in formula (II-1):

7. A compound, its stereoisomer, or a pharmaceutically acceptable salt thereof, characterized in that, The structure is as follows:

8. An antibody-drug conjugate of formula (III) or a pharmaceutically acceptable salt or solvate thereof: Its features are, R 1 X 1 X 2 X 3 M, L 1 L 2 L 3 Each independently according to claim 6; mAb is an antibody or antigen-binding fragment; L 4 for -C(=O)-、a-CH2C(=O)-b, where end a is connected to mAb and end b is connected to L. 3 Connected; preferably n is a decimal or integer from 1 to 10; preferably, n is a decimal or integer from 3 to 10; more preferably, n is 4, 6 or 8; Preferably, the mAb is an antibody selected from the group consisting of monoclonal antibodies, single-chain antibodies, chimeric antibodies, multispecific antibodies, humanized antibodies, or fully human antibodies; or the mAb is an antigen-binding fragment selected from the group consisting of Fab, Fab', Fv, F(ab')2, scFv, di-scFv, VHH, or dAb fragments; more preferably, the mAb is a Her2 antibody; even more preferably, the Her2 antibody is Trastuzumab; and / or Preferably, the antibody conjugate of formula (III) or its pharmaceutically acceptable salt or solvate has the structure shown in formula (III-1):

9. An antibody conjugate or a pharmaceutically acceptable salt or solvate thereof, characterized in that, It has the following structure: Wherein, mAb and n are each independently as described in claim 8; Preferably, the mAb is a Her2 antibody; and / or Preferably, n is a decimal or integer from 3 to 10; more preferably, n is 4, 6 or 8.

10. A pharmaceutical composition, characterized in that, It comprises a compound according to any one of claims 1-4 or claim 5, its stereoisomer, or a pharmaceutically acceptable salt thereof, or an antibody conjugate according to claim 8 or 9 or a pharmaceutically acceptable salt or solvate thereof, and optionally a pharmaceutically acceptable carrier.

11. Use of the compound, its stereoisomer, or a pharmaceutically acceptable salt thereof, according to any one of claims 1-4 or claim 5, or the antibody conjugate or its pharmaceutically acceptable salt or solvate according to claim 8 or 9, or the pharmaceutical composition according to claim 10, in the preparation of a medicament for the prevention or treatment of tumors.

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