Epothilone derivatives and their use

Abstract

The present application provides a series of novel esilate derivatives, pharmaceutically acceptable salts, stereoisomers or prodrugs thereof, and the application thereof in the field of anti-tumor, wherein the esilate derivatives have the structure shown in formula (I).

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to ethathecan derivatives and their applications. Background Technology

[0002] Exanotecan is a fully synthetic derivative of the natural product camptothecin, exhibiting potent inhibitory activity against top1 topoisomerase, promoting tumor cell apoptosis, and possessing broad-spectrum antitumor activity. However, its significant toxic side effects limit its application in the field of antitumor drugs. In contrast, DS-8201a (antibody-drug conjugate, ADC), an antibody-drug conjugate (ADC) formed with hydroxyacetamide derivative (Dxd) as the toxin and trastuzumab (Herceptin), has shown excellent therapeutic efficacy against Her2-expressing tumors. DS-8201a is a high-performance ADC antitumor drug. Besides Herceptin, ADC drugs targeting other antigens such as B7-H3 and Trop2 are also under extensive research.

[0003] This invention, while maintaining the pharmacodynamic skeleton of ethathecan, derivatizes ethathecan with hydroxylamine and hydrazine to form a series of ethathecan derivatives with coupling sites. These compounds have the characteristics of high antitumor activity, low toxicity and side effects, and easy water solubility, and have potential application value as antitumor monotherapy or antibody-drug conjugates. Summary of the Invention

[0004] This invention provides a series of novel ethathecan derivatives, their pharmaceutically acceptable salts, stereoisomers or prodrugs, and their applications in the field of antitumor therapy.

[0005] In one aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer, or prodrug thereof:

[0006]

[0007] In the formula,

[0008] R is selected from -Z-R1, where Z is a single bond or C=O;

[0009] R1 is selected from -(CH2) n NR a R b -CH2OR a -NOR a -(CH2) n ONR a R b Or -R3; n is selected from 0, 1, 2, or 3;

[0010] R a R b Each is independently selected from hydrogen, hydroxyl, amino, C1-C6 alkyl, C1-C6 alkylamino, aminoC1-C6 alkyl, or C1-C6 alkoxy;

[0011] R3 is selected from 3- to 6-membered cycloalkyl groups or 3- to 6-membered heterocycloalkyl groups containing N, O, or S heteroatoms, and R3 is optionally further divided by R c replace;

[0012] R c Selected from hydrogen, hydroxyl, amino, C1-C6 alkyl, C1-C6 alkylamino, or C1-C6 alkoxy;

[0013] The condition is that -Z-R1 is not -NH2, -CH2OH, -CH2NH2 or -CH2OCH2NH2.

[0014] In one embodiment, R1 is not -CH2ONH2 or hydroxyoxetane.

[0015] In one implementation, R1 is selected from -NR a R b -CH2NR a R b -CH2OR a -NOR a -(CH2) n ONR a R b Or -R3.

[0016] Preferably, R1 is selected from -NR a R b -C(O)NR a R b -CH2NR a R b -CH2ONR a R b Or -R3.

[0017] In one implementation scheme, R a R b Each is independently selected from hydrogen, hydroxyl, amino, C1-C6 alkyl, C1-C6 alkylamino or C1-C6 alkoxy.

[0018] Preferably, R a R b Each is independently selected from hydrogen, hydroxyl, amino, methyl, methylamino, or methoxy.

[0019] In one embodiment, R3 is selected from 3- to 6-membered heterocyclic alkyl groups containing N, O, or S heteroatoms, and R3 is optionally further coated with Rc Replace; the R c It is selected from hydrogen, hydroxyl, amino, C1-C6 alkyl, C1-C6 alkylamino or C1-C6 alkoxy.

[0020] In one embodiment, R1 is selected from -NHNH2, -N(CH3)NH2, -N(CH3)NHCH3, -C(O)NHNH2, -C(O)N(CH3)NH2, -CH2NHOH, -CH2NHOCH3, -CH2ONH2, -CH2ONHCH3, -CH2NCH3NH2, -CH2N(CH3)NHCH3, -CH2N(CH3)OH, -CH2NHOCH2CH3, and hydroxyoxetane. aminooxyheterobutylene

[0021] In one embodiment, R1 is selected from -NHNH2, -N(CH3)NH2, -N(CH3)NHCH3, -C(O)NHNH2, -C(O)N(CH3)NH2, -CH2NHOH, -CH2NHOCH3, -CH2ONHCH3, -CH2NCH3NH2, -CH2N(CH3)NHCH3, -CH2N(CH3)OH, -CH2NHOCH2CH3, and aminooxetane.

[0022] In one embodiment, the compound of formula (I) is a compound of formula (Ia):

[0023]

[0024] In the formula, R1 defines the compound of formula (I).

[0025] Preferably, R1 is -NHNH2 or -N(CH3)NH2.

[0026] In one embodiment, the compound of formula (I) is a compound of formula (Ib):

[0027]

[0028] In the formula, R2 is selected from -NHOH, -ONH2, -NHO(C1-C3 alkyl), -ONH(C1-C3 alkyl), -N(C1-C3 alkylamino), -N(C1-C3 alkyl)NH(C1-C3 alkyl), -N(C1-C3 alkyl)OH or -NHO(C1-C3 alkyl).

[0029] Preferably, R2 is selected from -NHOH, -NHOCH3, ONH2, -ONHCH3, -NCH3NH2, -N(CH3)NHCH3, -N(CH3)OH or -NHOCH2CH3.

[0030] In one embodiment, R2 is selected from -NHOH, -NHO (C1-C3 alkyl), -ONH (C1-C3 alkyl), -N (C1-C3 alkylamino), -N (C1-C3 alkyl)NH (C1-C3 alkyl), -N (C1-C3 alkyl)OH or -NHO (C1-C3 alkyl).

[0031] Preferably, R2 is selected from -NHOH, -NHOCH3, -ONHCH3, -NCH3NH2, -N(CH3)NHCH3, -N(CH3)OH or -NHOCH2CH3.

[0032] In one embodiment, the compound of formula (I) is a compound of formula (Ic):

[0033]

[0034] In the formula, R a R b Define compounds of formula (I); the condition is that R a R b They are not both H.

[0035] Preferably, R a R b Each is independently selected from hydrogen, amino, C1-C3 alkyl, and C1-C3 alkylamino.

[0036] Preferably, -NR a R b Selected from -NHNH2, -N(CH3)NH2 or -N(CH3)NHCH3.

[0037] In one embodiment, the compound of formula (I) is a compound of formula (Id):

[0038]

[0039] In the formula, X is CH2, NH, O or S.

[0040] Preferably, X is CH2 or O; more preferably, X is O.

[0041] In one implementation scheme, R c It is selected from hydrogen, hydroxyl, amino, C1-C6 alkyl, C1-C6 alkylamino or C1-C6 alkoxy.

[0042] Preferably, R cIt is selected from hydroxyl or amino, more preferably amino.

[0043] In one embodiment, the compound of formula (I) is selected from the following compounds:

[0044]

[0045] It is noteworthy that when R1 is -CH2ONH2, its IC50 with compound 8 (R1 is -CH2ONHCH3) inhibits the growth of OE33 cells. 50 Although the values ​​were all below 10 nM, the IC50 of compound 8 was... 50 The value is still significantly lower than that of the ethatecan derivative compounds obtained when R1 is -CH2ONH2.

[0046] In another aspect, the present invention provides a method for preparing the compound according to any one of the above claims, wherein the method is selected from the following reaction routes:

[0047] Reaction route 1:

[0048] Exatecan is reacted with N,N'-carbonyldiimidazole, and then with hydrazine or a substituted hydrazine to give the corresponding N-aminourea ethathecan derivative.

[0049] For example,

[0050]

[0051] Reaction route 2:

[0052] The substituted hydrazine was reacted with oxalyl chloride, and then with ethatecan to give the corresponding oxalamide hydrazine ethatecan derivative.

[0053] For example,

[0054]

[0055] Reaction route 3:

[0056] Exatecan reacts with bromoacetic acid to give bromoacetyl-exatecan, and bromoacetyl-exatecan is condensed with amine compounds to give the corresponding amide-exatecan derivatives.

[0057] For example,

[0058]

[0059] Reaction route 4:

[0060] Exatecan reacts directly with carboxylic acid compounds to give the corresponding amide ethatecan derivatives.

[0061] For example,

[0062]

[0063] In the formula, R and L define compounds of formula (I).

[0064] In another aspect, the present invention provides a drug-linker compound of formula (II) or a pharmaceutically acceptable salt, stereoisomer, or prodrug thereof:

[0065]

[0066] In the formula, R defines a compound of formula (I).

[0067] In one implementation, L is -L1-Q-L2; R is partially connected to L1.

[0068] L1 is selected from in, The position shown indicates that it is connected to the R group. The position shown indicates that it is connected to the Q group.

[0069] In one implementation, L2 is selected from

[0070] In one implementation, Q is selected from Val-Cit, Val-Ala, Ala-Ala-Asn, Gly-Gly-Phe-Gly, Gly-Lys, Gly-Gly-lys, and (CH2). m1 O(CH2) m2 (CH2) m3 , where m1 and m2 are each independently selected from integers from 1 to 4.

[0071] Preferably, m1 is 2.

[0072] Preferably, m2 is 2.

[0073] Preferably, m3 is 6.

[0074] In one embodiment, Q is selected from Val-Cit, Gly-Gly-Phe-Gly, (CH2)2O(CH2)2, or (CH2)6;

[0075] Preferably, L1 is Preferably, L2 is In another aspect, the present invention provides a drug-antibody conjugate of formula (III) or a pharmaceutically acceptable salt thereof:

[0076]

[0077] In the formula, R defines the same formula (I) or compound, and L defines the same formula (II) or compound.

[0078] Ab represents tumor-associated antigen-antibody, and n is an integer selected from 1 to 8.

[0079] Preferably, the tumor-associated antigen is selected from Her2, Trop2, 5T4, ROR1 or B7-H3.

[0080] Preferably, Ab is pertuzumab.

[0081] In one embodiment, the drug-antibody conjugate of formula (III) is selected from the following compounds:

[0082]

[0083]

[0084] Preferably, the drug-antibody conjugate of formula (II) is selected from the following compounds:

[0085]

[0086] In another aspect, the present invention provides a pharmaceutical composition comprising the above-described compound, a drug-linker compound or a pharmaceutically acceptable salt thereof, a stereoisomer, a prodrug or an antibody-drug conjugate.

[0087] The pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0088] In another aspect, the present invention provides the use of the above-mentioned compounds, drug-linker compounds or pharmaceutically acceptable salts, stereoisomers, prodrugs or antibody-drug conjugates or pharmaceutical compositions in the preparation of a medicament for treating cancer.

[0089] Preferably, the cancers include stomach cancer, esophageal cancer, breast cancer, and lung adenocarcinoma.

[0090] In another aspect, the present invention provides a method for treating cancer, comprising the step of administering the above-described compound or a pharmaceutically acceptable salt, stereoisomer, prodrug, antibody-drug conjugate, or pharmaceutical composition thereof to a patient in need.

[0091] In one embodiment, the amount of the above-mentioned compound or its pharmaceutically acceptable salt, stereoisomer, prodrug, antibody-drug conjugate or pharmaceutical composition administered is a therapeutically effective amount. Detailed Implementation

[0092] 1. Definition

[0093] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the related terms and laboratory procedures used herein are all widely used terms and routine procedures in the respective fields. To better understand this invention, definitions and explanations of related terms are provided below.

[0094] As used herein and unless otherwise stated, the terms “comprising,” “including,” “having,” “containing,” and their grammatical equivalents, including their grammatical equivalents, should generally be understood as open-ended and non-restrictive, e.g., not excluding other unlisted elements or steps.

[0095] The compounds disclosed herein may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, for example, enantiomers and diastereomers. The stereoisomers include geometric isomers (e.g., cis, trans structures) and optical isomers (e.g., enantiomers), as therapeutic agents comprising monomers, racemates, racemic mixtures, and pharmaceutically acceptable salts thereof. The compounds containing asymmetric carbon atoms of this disclosure can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents. Racemates, diastereomers, and enantiomers are all included within the scope of this disclosure.

[0096] The disclosed compounds also include tautomer forms. Tautomer forms arise from the exchange of a single bond with an adjacent double bond, accompanied by the migration of a proton.

[0097] As used herein, "pharmaceutically acceptable salt" refers to a salt formed by a corresponding amine compound and an inorganic or organic acid, or a salt formed by a corresponding carboxylic acid compound and an alkali or alkaline earth metal, or a salt formed by an organic amine. Inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid; organic acids include, but are not limited to, acetic acid, propionic acid, butyric acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, succinic acid, lactic acid, citric acid, succinic acid, gluconic acid, maleic acid, fumaric acid, and tartaric acid; alkali or alkaline earth metal salts include, but are not limited to, sodium, potassium, calcium, and magnesium salts; and organic amine salts include, but are not limited to, salts composed of ammonia, methylamine, ethylamine, propylamine, isopropylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, tert-butylamine, ethylenediamine, ethanolamine, diethanolamine, triethanolamine, morpholine, piperidine, piperazine, and amino acids.

[0098] As used herein, "precursor" refers to a compound that, after being administered to the human body via an appropriate route of administration, undergoes metabolism or simple chemical changes within the patient's body to transform into the compound and its corresponding salt contained in Formula 1 of this invention. Precursors include, but are not limited to, various carboxylic acid esters, carbonates, phosphate esters, sulfate esters, sulfonates, amino acid esters, gluconates, and various amides, acetals, hemiacetals, carbonate esters, etc.

[0099] The numerical ranges mentioned in this article refer to the integers within a given range. For example, "C1-C6" means that the group can have 1, 2, 3, 4, 5, or 6 carbon atoms; "C3-C6" means that the group can have 3, 4, 5, or 6 carbon atoms.

[0100] When any variable (e.g., Rn) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted by 1-5 Rs, the group can optionally be substituted by up to 5 Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0101] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 8 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, and most preferably an alkyl group containing 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 2,2-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. More preferably, lower alkyl groups containing 1 to 6 carbon atoms are used. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester group. The present disclosure preferably includes methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuteralkyl, alkoxy-substituted alkyl, and hydroxy-substituted alkyl.

[0102] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent comprising 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, it comprises 3 to 12 ring atoms, wherein 1 to 4 are heteroatoms; more preferably, it comprises 3 to 8 ring atoms; most preferably, it comprises 3 to 8 ring atoms; further preferably, it is a 3- to 8-membered heterocyclic group comprising 1 to 3 nitrogen atoms, optionally substituted with 1 to 2 oxygen atoms, sulfur atoms, or oxo groups, including nitrogen-containing monocyclic heterocyclic groups, nitrogen-containing spirocyclic groups, or nitrogen-containing fused heterocyclic groups.

[0103] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 12-membered, such as phenyl and naphthyl.

[0104] The aryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydrogen, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylic acid ester group.

[0105] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydrogen, nitro, chloro, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester.

[0106] All hydrogen atoms described in this disclosure can be replaced by their isotope deuterium.

[0107] "Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, more preferably one to three hydrogen atoms, which are independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0108] It refers to the junction of chemical bonds.

[0109] Drugs or drug compositions

[0110] As used herein, "pharmaceutically acceptable salt" refers to a salt formed by a corresponding amine compound and an inorganic or organic acid, or a salt formed by a corresponding carboxylic acid compound and an alkali or alkaline earth metal, or a salt formed by an organic amine. Inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid; organic acids include, but are not limited to, acetic acid, propionic acid, butyric acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, succinic acid, lactic acid, citric acid, succinic acid, gluconic acid, maleic acid, fumaric acid, and tartaric acid; alkali or alkaline earth metal salts include, but are not limited to, sodium, potassium, calcium, and magnesium salts; and organic amine salts include, but are not limited to, salts composed of ammonia, methylamine, ethylamine, propylamine, isopropylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, tert-butylamine, ethylenediamine, ethanolamine, diethanolamine, triethanolamine, morpholine, piperidine, piperazine, and amino acids.

[0111] As used herein, "precursor" refers to a compound that, after being administered to the human body via an appropriate route of administration, undergoes metabolism or simple chemical changes within the patient's body to transform into the compound and its corresponding salt contained in Formula 1 of this invention. Precursors include, but are not limited to, various carboxylic acid esters, carbonates, phosphate esters, sulfate esters, sulfonates, amino acid esters, gluconates, and various amides, acetals, hemiacetals, carbonate esters, etc.

[0112] The pharmaceutical products or pharmaceutical compositions disclosed herein can be administered orally, topically, parenterally, or via mucosal routes (e.g., sublingually, by inhalation, or rectally) in dosage units comprising a conventional, non-toxic, pharmaceutically acceptable carrier. Oral administration is generally preferred. The active agent can be administered orally in capsule, tablet, or other similar forms (see Remington: The Science and Practice of Pharmacy, 20th Edition).

[0113] For oral administration in tablet or capsule form, the active pharmaceutical ingredient may be combined with non-toxic, pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, sucrose, glucose, mannitol, sorbitol, and other reducing and non-reducing sugars, microcrystalline cellulose, calcium sulfate, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica, stearic acid, sodium stearyl fumarate, glyceryl docosanoate, calcium stearate, etc.); disintegrants (e.g., potato starch or sodium hydroxyacetic acid starch); or wetting agents (e.g., sodium lauryl sulfate), colorants and flavorings, gelatin, sweeteners, natural and synthetic gums (e.g., gum arabic, tragacanth, or alginate), buffer salts, carboxymethyl cellulose, polyethylene glycol, waxes, etc. For oral administration in liquid form, the pharmaceutical component may be combined with a non-toxic, pharmaceutically acceptable inert carrier (e.g., ethanol, glycerol, water), an anti-settling agent (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), an emulsifier (e.g., lecithin or gum arabic), a non-aqueous carrier (e.g., almond oil, esters, ethanol, or fractionated vegetable oils), and a preservative (e.g., methylparaben, propylparaben, or sorbic acid). Stabilizers such as antioxidants (BHA, BHT, propyl iodide, sodium ascorbate, citric acid) may also be added to stabilize the dosage form.

[0114] Tablets containing the active compound can be coated using methods well known in the art. The compositions of this disclosure containing a compound of formula I as the active compound can also incorporate beads, microspheres, or microcapsules, for example, constructed from polyglycolic acid / lactic acid (PGLA). Liquid formulations for oral administration can take the form of, for example, solutions, syrups, emulsions, or suspensions, or they can be presented as dry products reconstituted with water or other suitable excipients prior to use. Formulations for oral administration can be suitably formulated to allow for controlled or delayed release of the active compound.

[0115] The term "treatment" includes suppressing, alleviating, preventing, or eliminating one or more symptoms or side effects associated with the disease, condition, or disorder being treated.

[0116] The term "inhibition" is used relative to a control. Those skilled in the art will readily determine the appropriate control for each experiment. For example, a reduced response in a subject or cell treated with the compound is compared to a response in a subject or cell not treated with the compound.

[0117] The term "pharmaceutical composition" means a composition comprising the compounds described in this disclosure or their pharmaceutically acceptable salts, and at least one pharmaceutically acceptable ingredient selected from the following, depending on the manner of administration and the nature of the dosage form: carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, thermosensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0118] The term "effective dose" or "therapeutic effective dose" refers to a sufficient amount of a drug or agent that is non-toxic but achieves the desired effect. In embodiments of the invention, when treating a patient according to the invention, the amount of a given drug depends on many factors, such as the specific dosing regimen, the type and severity of the disease or condition, and the unique characteristics of the patient or host requiring treatment (e.g., weight). However, depending on the specific surrounding circumstances, including, for example, the specific drug used, the route of administration, the condition being treated, and the patient or host being treated, the dosage can be conventionally determined by methods known in the art. Typically, for adult treatment, the dosage is typically in the range of 0.02-5000 mg / day, for example, about 1-1500 mg / day. This required dose can conveniently be expressed as a single dose, or concurrent (or over a short period of time) or fractions at appropriate intervals, such as two, three, four, or more doses per day. Those skilled in the art will understand that although the above dosage ranges are given, the specific effective dose can be appropriately adjusted according to the patient's condition and in conjunction with the physician's diagnosis.

[0119] The term "antibody-drug conjugate (ADC)" refers to a small molecule drug with biological activity linked to a monoclonal antibody via a chemical link. The monoclonal antibody then acts as a carrier to target and deliver the small molecule drug to the target cells.

[0120] As used herein, the terms “reduction,” “inhibition,” “mitigation,” or “reduction” are used relative to a control. Those skilled in the art will readily determine the appropriate control for each experiment. For example, a reduced response in a subject or cell treated with the compound is compared to a response in a subject or cell not treated with the compound.

[0121] Unless otherwise specified, the raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0122] abbreviation

[0123] CDI: N,N′-carbonyldiimidazole; EA: ethyl acetate; DCM: dichloromethane; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; DIPEA: N,N-diisopropylethylamine; NMP: N-methylpyrrolidone; HOBt: 1-hydroxybenzotriazole;

[0124] Valine, its structural formula is: Cit: Citrulline, its structural formula is Gly: Glycine, its structural formula is as follows Phe: Phenylalanine, its structural formula is as follows Fmoc: Threonine benzyl phosphate, its structural formula is as follows Val-Cit: Gly-Gly-Phe-Gly:

[0125] The linkages of Val-Ala, Ala-Ala-Asn, Gly-Lys, and Gly-Gly-lys can be achieved using well-known amino acid condensation methods.

[0126] -Pab-: -ha-: mc-: ba-: bp-:

[0127] II. Specific Implementation Examples

[0128] In this embodiment, the ADC was prepared using, but not limited to, pertuzumab, whose heavy chain amino acid sequence is as follows (SEQ ID NO:1):

[0129]

[0130] The light chain amino acid sequence is as follows (SEQ ID NO:2):

[0131]

[0132] Example 1: Exatecan-N-aminourea (1)

[0133]

[0134] Exatecan mesylate (30 mg, 56.44 μmol) and triethylamine (11.42 mg, 112.88 μmol) were added to 1 ml of DMF and stirred until homogeneous. Then, CDI (9.15 mg, 56.44 μmol) was added and the mixture was reacted at room temperature for 1 h under argon protection. Triethylamine (80 mg) and hydrazine hydrochloride (19.33 mg, 0.28 mmol) were added sequentially and the mixture was stirred at room temperature for 10 h. 50 ml of EA was added, and the mixture was washed with saturated brine. The mixture was extracted, separated, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain compound 1 (25 mg, 90% yield). 1 H NMR (500MHz, DMSO-d6) δ7.77(d,J=10.9Hz,1H),7.54(s,1H),7.30(s,1H),6.52(m,1H),5.41(m,2H),5.39-5.33(m,1H),5.22(m,J=7.0Hz,2H ),3.15(m,J=13.6,6.9Hz,2H),3.06(s,2H),2.38(s,3H),2.24-2.11(m,2H),1.86(m,J=21.4,7.0Hz,2H),0.87(t,J=7.3Hz,3H); LCMS: (M+1) + 493.98 (Calculated value: 493.18).

[0135] Example 2: Exanotecan-N-methyl-N-aminourea

[0136]

[0137] Exatecan mesylate (30 mg, 56.44 μmol) and triethylamine (11.42 mg, 112.88 μmol) were added to 1 ml of DMF and stirred until homogeneous. CDI (9.15 mg, 56.44 μmol) was then added and the mixture was reacted at room temperature for 1 h under argon protection. Triethylamine (80 mg) and 1-Boc-2-methylhydrazine (45.05 mg, 0.3 mmol) were added sequentially and the mixture was stirred at room temperature for 10 h. 50 ml of EA was added, and the mixture was washed with saturated brine. The mixture was extracted, separated, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain the compound Boc-product. The Boc-product was dissolved in 1 ml of DCM and 0.5 ml of 4 M hydrochloric acid in ethyl acetate solution was added. The mixture was stirred at room temperature for 30 min to obtain compound 2 (22 mg, 80% yield). 1H NMR (500MHz, DMSO-d6) δ7.81(d,J=10.9Hz,1H),7.32(s,1H),6.54(s,1H),5.43(m,J=18Hz,3H),5.29(d,J=4.1Hz,2H),3.29(s,3H)3.17( t,J=6.4Hz,2H),2.41(d,J=1.7Hz,3H),2.20(m,J=13.0,6.9Hz,2H),1.86(m,J=20.7,13.7,6.8Hz,2H),0.87(t,J=7.3Hz,3H); LCMS: (M+1) + 508.02 (Calculated value: 507.19).

[0138] Example 3: Exanotecan-N',N-dimethylaminourea (3)

[0139]

[0140] In 1 ml of DMF, ethatecan mesylate (30 mg, 56.44 μmol) and triethylamine (11.42 mg, 112.88 μmol) were added and stirred until homogeneous. Then, CDI (9.15 mg, 56.44 μmol) was added. The mixture was reacted at room temperature for 1 h under argon protection. Subsequently, TEA (80 mg) and N'N-dimethylhydrazine hydrochloride (45.05 mg) were added sequentially.

[0141] 0.33 mmol), stirred at room temperature for 10 h, 50 ml of EA was added, washed with saturated brine, phase separated, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give compound 3 (18 mg, yield 62%). 1 H NMR(500MHz,DMSO-d6)δ7.76(d,J=10.9Hz,1H),7.48(d,J=9.2Hz,1H),7.30(s,1H),6.51 (s,1H),5.41(s,2H),5.31(dd,J=17.0,8.5Hz,2H),5.16(s,1H),4.64(q,J=5.7Hz,2H),3. 09(dt,J=16.8,7.8Hz,2H),3.01(s,3H),2.38(s,3H),2.18(t,J=7.2Hz,3H),1.86(dq,J=19.3,7.1Hz,2H),1.47(s,1H),0.86(d,J=8.2Hz,3H);LCMS:(M+1)+522.02 (calculated value: 521.21).

[0142] Example 4: Exanotecan-N'-aminoglycandiamine (4)

[0143]

[0144] In 30 ml of DCM, Boc-hydrazine (5.93 g, 44.90 mmol) and TEA (6.2 g, 61.21 mmol) were added and stirred until dissolved. Then, a DCM solution of methyl oxaloyl chloride (5 g, 40.81 mmol) was added dropwise at 0 °C. The mixture was stirred at room temperature for 1 h, washed with saturated brine (20 ml * 3), dried over anhydrous sodium sulfate, and concentrated to give compound 4-a N'-Boc-N-oxaloylhydrazine monomethyl ester (7.1 g, yield 79%); LCMS: (M+1) + 219.01 (calculated value: 218.21).

[0145] Compound 4-a (7.1 g, 32.56 mmol) was dissolved in 70 mL of ethanol, and then 32.53 mL of 1 M LiOH aqueous solution was added dropwise. The mixture was stirred overnight at room temperature, and then 0.5 M HCl aqueous solution was added dropwise to adjust the pH of the reaction solution to 5. Water and ethanol were removed under reduced pressure. 5 mL of methanol and 15 mL of DCM were added to the concentrated solution, and the mixture was stirred for 1 h. The solution was filtered, and the filtrate was concentrated to give compound 4-b N'-Boc-N-oxalylhydrazine (6 g, yield 89%), LCMS: (M+1)+205.2 (calculated value: 204.18).

[0146] In 2 ml of DMF, ethatecan mesylate (200 mg, 0.37 mmol), diisopropylethylamine (6.15 mg, 0.047 mmol), N'-Boc-N-oxalylhydrazine (164 mg, 0.8 mmol), and HATU (286 mg, 0.75 mmol) were added sequentially. The mixture was stirred at room temperature for 4 hours. DMF was removed under reduced pressure, and the concentrate was added to 20 ml of DCM. The solution was purified by silica gel column chromatography to obtain compound 4-c-ethatecan-N'-Boc-aminooxalyldiamine (180 mg, 77% yield). LCMS: (M+1) + 622.1 (Calculated value: 621.22).

[0147] Exatecan-N'-Boc-aminooxalyldiamine (180 mg, 0.29 mmol) was dissolved in 2 ml of DCM, and 1 ml of 4 M hydrochloric acid in ethyl acetate solution was added. The mixture was stirred at room temperature for 30 min and then concentrated to obtain compound 4, namely ethatecan-N'-aminooxalyldiamine (162 mg, 100%). 1H NMR (500MHz, DMSO-d6) δ9.54(d,J=8.9Hz,1H),7.77(m,J=11.3,4.1Hz,1H),7.30(s,1H),6.52(s,1H),5.53(m,J=8.3,5.0Hz,1H),5.39(s ,2H),5.12(d,J=4.0Hz,2H),3.23(s,1H),2.38(s,3H),2.28-2.15(m,2H),1.85(m,J=25.0,7.2Hz,2H),0.86(t,J=7.4Hz,3H); LCMS: (M+1) + 522.00 (Calculated value: 521.17).

[0148] Example 5: Exanotecan N'-amino-N-methyloxalyldiamine (5)

[0149]

[0150] In 20 ml of DCM, 1-methyl-2-Boc hydrazine (2.4 g, 26 mmol) and TEA (1.65 g, 16.3 mmol) were added and stirred until dissolved. Then, a DCM solution of methyl oxaloyl chloride (2 g, 16.3 mmol) was added dropwise at 0 °C. The mixture was stirred at room temperature for 1 h, extracted, washed with saturated brine (10 ml * 3), dried over anhydrous sodium sulfate, and concentrated to give the intermediate compound 5-a1-methyl-2-Boc-oxaloyl hydrazine monomethyl ester (3.5 g, yield 92%), LCMS: (M+1) + 233.10 (calculated value: 232.24).

[0151] Compound 5-a (3.5 g, 15 mmol) was dissolved in 40 mL of ethanol, and 16.37 mL of 1 M LiOH aqueous solution was added dropwise. The mixture was stirred overnight at room temperature, and then 0.5 M HCl aqueous solution was added dropwise to adjust the pH of the reaction solution to 5. Water and ethanol were removed under reduced pressure. 2 mL of methanol and 8 mL of DCM were added to the concentrated solution, and the mixture was stirred for 1 h. The solution was filtered, and the filtrate was concentrated to give intermediate compound 5-b 1-methyl-2-Boc-oxalylhydrazine (3.21 g, 90% yield), LCMS: (M+1)+219.01 (calculated value: 218.21).

[0152] In 2 ml of DMF, ethatecan mesylate (100 mg, 0.19 mmol), DIPEA (48.58 mg, 0.37 mmol), compound 5-b (82 mg, 0.37 mol), and HATU (118 mg, 0.32 mmol) were added sequentially. The mixture was stirred at room temperature for 4 hours. DMF was removed under reduced pressure, and the intermediate compound 5-c ethatecan N'-Boc amino-N-methyloxalyldiamine (91 mg, 75% yield) was purified by silica gel paper chromatography. LCMS: (M+1) + 636.14 (calculated value: 635.65).

[0153] Compound 5-c (91 mg, 0.14 mmol) was dissolved in 2 ml of DCM, and 1 ml of ethyl acetate solution of 4 M hydrochloric acid was added. The mixture was stirred at room temperature for 30 min and then concentrated to obtain compound 5, namely ethatecan N'-amino-N-methyloxalyldiamine (85 mg, 100% yield). 1 H NMR (500MHz, DMSO-d6) δ8.87(d,J=8.7Hz,1H),7.79(d,J=10.9Hz,1H),7.31(s,1H),5.59-5.62(m,J=18Hz,1H),5.46-5.32(m,J=84Hz,4H),3.16- 3.10(m,2H),2.95(s,2H),2.40(s,3H),2.17(ddq,J=28.2,14.0,5.5,4.9Hz,2H),1.86(dp,J=20.9,7.0Hz,2H),0.86(t,J=7.4Hz,3H); LCMS: (M+1) + 536.03 (Calculated value: 535.53).

[0154] Example 6: 2-Hydroxyaminoacetylethanotecan (6)

[0155]

[0156] Exatecan mesylate (100 mg, 0.19 mmol) and TEA (25 mg, 0.24 mmol) were added to 1 ml of DMF and stirred until dissolved. Then, bromoacetic acid (40 mg, 0.28 mmol) and HATU (85 mg, 0.22 mmol) were added. The mixture was stirred at room temperature for 1 h. DMF was removed under reduced pressure, and the intermediate 6-α-exatecan-N-bromoacetamide (90 mg, 86% yield) was purified by silica gel column chromatography. LCMS: (M+1) + 557.1 (calculated value: 556.39).

[0157] In a 10 ml bottle, 1 ml of DMF was added, followed by the addition of hydroxylamine hydrochloride (113 mg, 1.63 mmol), TEA (164 mg, 1.62 mmol), and compound 6-a (90 mg, 0.16 mmol). The mixture was stirred at room temperature for 1 h, and DMF was removed under reduced pressure. The mixture was then purified by silica gel column chromatography to obtain compound 6, namely 2-hydroxyaminoacetylethanotecan (90 mg, yield 86%). 1 H NMR(500MHz,DMSO-d6)δ10.86-10.38(m,1H),8.96(d,J=8.7Hz,1H),7.82(t,J=11.9 Hz,1H),7.32(s,1H),6.55(s,1H),5.77-5.54(m,1H),5.41(d,J=10.6Hz,2H),5.37- 5.16(m,2H),3.86(m,J=11.7,8.0Hz,2H),3.31-3.02(m,3H),2.40(d,J=14.0Hz,3H) ,2.29-2.07(m,2H),1.86(m,J=21.6,7.2Hz,2H),0.86(t,J=7.3Hz,3H); LCMS: (M+1) + 509.3 (Calculated value: 508.18).

[0158] Example 7: 2-Methoxyaminoacetylethanotecan (7)

[0159]

[0160] In a 10 ml bottle, 1 ml of DMF was added, followed by the addition of methoxyamine hydrochloride (126 mg, 1.5 mmol), TEA (182 mg, 1.79 mmol), and compound 6-a (100 mg, 0.18 mmol). The mixture was stirred at room temperature for 1 h, and the DMF was removed under reduced pressure. The mixture was then purified by silica gel column chromatography to obtain compound 7, namely 2-methoxyaminoacetylethanotecan (80 mg, yield 86%). 1H NMR (500MHz, DMSO-d6) δ8.50-8.46(m,1H),7.80(d,J=10.8Hz,1H),7.30(d,J=1.9Hz,1H),6. 83(td,J=6.2,1.8Hz,1H),6.52(d,J=1.9Hz,1H),5.61-5.55(m,1H),5.42(s,2H),5.22(s,2H ), 4.01-3.87(m,1H), 3.42(d,J=6.1Hz,3H), 3.17(t,J=6.4Hz,2H), 2.40(s,3H), 2.22-2.11(m,2H), 1.86(dp,J=21.1,7.1Hz,2H), 0.90-0.83(m,3H); LCMS: (M+1)+523.20 (calculated value: 522.19).

[0161] Example 8: 2-Methamidoacetylethanotecan (8)

[0162]

[0163] In a mixture of 40 ml water and 40 ml THF, N-methylhydroxylamine hydrochloride (10 g, 119.7 mmol) was added and stirred until dissolved. Potassium carbonate (8.27 g, 60 mmol) was added, followed by the slow addition of 50 ml THF solution of Boc anhydride (28.75 g, 131.7 mmol). After the addition was complete, the mixture was stirred overnight at room temperature. THF was removed under reduced pressure, and the mixture was extracted with DCM (100 ml * 3). The extract was dried over anhydrous sodium sulfate and concentrated to give compound 8-a N-Boc-N-methylhydroxylamine (15 g, 88% yield), LCMS: (M+1) + 148.01 (calculated value: 147.17).

[0164] Compound 8-a (15 g, 101.9 mmol) was dissolved in 100 mL of isopropanol, and methyl bromoacetate (18.58 g, 121.4 mmol) and DIPEA (15.7 g, 121.7 mmol) were added. The mixture was heated to 85 °C for 3 h under argon protection. The reaction solution was concentrated, dissolved in 100 mL of EA, washed with saturated brine (30 mL x 3), dried over anhydrous sodium sulfate, and concentrated to give compound 8-b N-Boc-N-methylaminooxyacetic acid methyl ester (18 g, yield 81.8%). LCMS: (M+1) + 220.21 (Calculated value: 219.24).

[0165] Compound 8-b (18 g, 82 mmol) was dissolved in 180 mL of methanol, and 82.2 mL of 1 M lithium hydroxide aqueous solution was added. The mixture was stirred at room temperature until the reaction was complete. The reaction solution was concentrated, methanol was removed, and the acidity was adjusted to approximately pH 3 with 0.5 mmol / L hydrochloric acid aqueous solution. The mixture was extracted with EA, the phases were separated, dried, filtered, and concentrated to obtain compound 8-c N-Boc-N-methylaminooxyacetic acid (13.1 g, yield 77.3%), LCMS: (M+1). + 206.01 (Calculated value: 205.21).

[0166] In 2 ml of DMF, ethatecan mesylate (100 mg, 0.19 mmol), DIPEA (48.58 mg, 0.37 mmol), compound 8-c (77.3 mg, 0.37 mmol), and HATU (118 mg, 0.31 mmol) were added sequentially. The mixture was stirred at room temperature for 6 hours. DMF was removed under reduced pressure, and the compound 8-d N-Boc-N-methaminooxyacetylethatecan (68 mg, 75% yield) was purified by silica gel column chromatography. LCMS: (M+1) + 623.30 (calculated value: 622.65).

[0167] Compound 8-d (68 mg, 0.109 mmol) was dissolved in 2 ml of DCM, and 1 ml of ethyl acetate solution of 4 M hydrochloric acid was added. The mixture was stirred at room temperature for 30 min and then concentrated to obtain compound 8, namely 2-methaminooxyacetylethanotecan hydrochloride (61 mg, 100% yield). 1 H NMR(500MHz,DMSO-d6)δ8.93(d,J=8.5Hz,1H),7.79(d,J=10.5Hz,1H),7.30( s,1H),5.60(dt,J=8.3,4.1Hz,1H),5.42(s,2H),5.29(s,2H),4.73-4.64(m,2 H),3.18(s,2H),2.85(s,3H),2.39(s,3H),2.25(dt,J=8.8,4.6Hz,1H),2.19- 2.10(m,1H),1.85(dp,J=21.4,7.2Hz,2H),0.86(t,J=7.3Hz,3H); LCMS:(M+1) + 523.10 (Calculated value: 522.19).

[0168] Example 9: 2-N-methyl-N-aminoethazine ethatecan (9)

[0169]

[0170] In a 10 ml bottle, 1 ml of DMF was added, followed by the sequential addition of 1-methyl-2-Boc hydrazine (36.41 mg, 0.25 mmol), TEA (54.6 mg, 0.54 mmol), and 6-a (100 mg, 0.18 mmol). The mixture was stirred at room temperature for 1 h, and the DMF was removed under reduced pressure. The product was purified by silica gel column chromatography to obtain compound 9-a2-N-methyl-N-Boc aminoacetylethanotecan (72 mg, yield 63%). LCMS: (M+1) + 622.30 (Calculated value: 621.21).

[0171] Compound 9-a (72 mg, 0.115 mmol) was dissolved in 2 mL of DCM, and 1 mL of 4 M hydrochloric acid in ethyl acetate solution was added. The mixture was stirred at room temperature for 30 min to give 9-a, namely 2-N-methyl-N-aminoacetylethanotecan (64 mg, 100% yield). ¹H NMR (500 MHz, DMSO-d6) δ 8.53 (d, J = 8.9 Hz, 1H), 8.33 (s, 1H), 7.79 (d, J = 10.9 Hz, 1H), 7.32–7.27 (m, 1H), 6.53 (d, J = 1.6 Hz, 1H), 5.60 (dt, J = 9.3, 5.1 Hz, 1H), 5 .41(s,2H),5.29-5.15(m,2H),3.42-3.40(m,2H),3.38-3.37(m,2H),3.37(s,3H),3.19(d,J=5.6Hz,1H),2.39(s,3H),2.15(q,J=6.0Hz,2H),1.85(dp,J=21.7,7.0Hz,2H),0.86(t,J=7.3Hz,3H);LCMS:(M+H)+522.1 (calculated value: 521.18).

[0172] Example 10: 2-N-methyl-N-methylaminoacetylethanotecan (10)

[0173]

[0174] In a 10 ml bottle, 1 ml of DMF was added, followed by the addition of 1,2-dimethyl-dihydrazine hydrochloride (71.72 mg, 0.54 mmol), TEA (91 mg, 0.89 mmol), and 6-a (100 mg, 0.18 mmol). The mixture was stirred at room temperature for 1 h. After the reaction was complete, DMF was removed under reduced pressure, and the product was purified by silica gel column chromatography to obtain compound 10, namely 2-N-methyl-N-methylaminoacetylethanotecan (85 mg, yield 88%). 1H NMR (500MHz, DMSO-d6) δ10.05 (s, 1H), 8.85 (d, J = 9.1Hz, 1H), 7.81 (m J=19.4,10.9Hz,1H),7.32(d,J=11.1Hz,1H),6.54(s,1H),5.66-5.51(m,1H) ,5.49-5.38(m,2H),5.35-5.17(m,2H),3.73-3.55(m,1H),3.19(m,J=19.8,15 .3,9.5Hz,3H),2.73(s,3H),2.41(d,J=9.0Hz,3H),2.19(m,J=31.1,13.1,5.4 Hz, 2H), 1.86 (m, J=21.7, 14.7, 7.5Hz, 2H), 0.87 (t, J=7.3Hz, 3H); LCMS: (M+1) + 536.20 (Calculated value: 535.22).

[0175] Example 11: 2-N-methylhydroxyaminoethazine ethatecan (11)

[0176]

[0177] In a 10 ml bottle, 1 ml of DMF was added, followed by the addition of N-methylhydroxylamine hydrochloride (30 mg, 0.36 mmol), TEA (58 mg, 0.57 mmol), and compound 6-a (100 mg, 0.18 mmol). The mixture was stirred at room temperature for 1 h, and DMF was removed under reduced pressure. The mixture was then purified by silica gel column chromatography to obtain compound 11, namely 2-N-methylhydroxylamine acetylethanotecan (70 mg, 75% yield). 1 H NMR (500MHz, DMSO-d6) δ8.44(d,J=12.4Hz,1H),7.79(t,J=10.7Hz,1H),7.30(d,J=4.9Hz ,1H),6.63-6.41(m,1H),5.59(m,J=8.6,5.0Hz,1H),5.41(d,J=3.0Hz,2H),5.32-5.12(m, 2H), 3.17(m,J=5.3Hz,2H), 2.73(d,J=6.7Hz,3H), 2.39(d,J=6.0Hz,3H), 2.24-2.08(m,2H), 1.85(m,J=21.3,7.1Hz,2H), 0.86(t,J=7.3Hz,3H); LCMS:(M+H)+523.20 (calculated value: 522.19).

[0178] Example 12: 2-Ethoxyaminoacetylethanotecan (12)

[0179]

[0180] In a 10 ml bottle, 1 ml of DMF was added, followed by the addition of ethoxyhydroxylamine hydrochloride (126 mg, 1.29 mmol), TEA (182 mg, 1.8 mmol), and compound 6-a (100 mg, 0.18 mmol). The mixture was stirred at room temperature for 1 h, and the DMF was removed under reduced pressure. The mixture was then purified by silica gel column chromatography to obtain compound 12, namely 2-ethoxyaminoacetylethanotecan (81 mg, yield 83%). 1 H NMR(500MHz,DMSO-d6)δ7.79(m,J=11.0,5.7Hz,1H),7.31(s,1H),5.63(m,J=9.2,5.4Hz,1H), 5.42(s,2H),5.31-5.16(m,2H),3.72-3.68(m,2H),3.45-3.42(m,2H),3.18(m,J=17.3,9.0Hz ,2H),2.40(d,J=6.4Hz,3H),2.27-2.10(m,2H),1.86(m,J=21.7,14.6,7.3Hz,2H),1.24(m,J=7.1Hz,1H),1.00(m,J=6.9Hz,2H),0.87(m,J=6.8Hz,3H);LCMS:(M+1)+537.10(Calculated value:536.21).

[0181] Example 13: 3-Aminooxetane-3-acetylethanotecan (13)

[0182]

[0183] In a mixture of 10 ml water and 10 ml EtOH, 0.5 g of 3-aminooxetane-3-carboxylic acid (4.27 mmol) was added and stirred until dissolved. Sodium bicarbonate (0.72 g, 8.54 mmol) was added, followed by the slow addition of 5 ml EtOH solution of Boc anhydride (0.98 g, 4.48 mmol). After the addition was complete, the mixture was stirred at room temperature for 2 h. Ethanol was removed under reduced pressure, and hydrochloric acid was added dropwise to adjust the pH to approximately 4. The product was extracted using DCM (100 ml * 3), dried over anhydrous sodium sulfate, and concentrated to give compound 15-a, N-Boc-3-aminooxetane-3-carboxylic acid (0.8 g, yield 86%). LCMS: (M+1) + 118.01 (Calculated value: 117.10).

[0184] In 1 ml of DMF, ethatecan mesylate (20 mg, 0.038 mmol), DIPEA (9.7 mg, 0.074 mmol), compound 13-a (16.5 mg, 0.075 mmol), and HATU (23.6 mg, 0.062 mmol) were added sequentially. The mixture was stirred at room temperature for 6 hours. DMF was removed under reduced pressure, and compound 15-b, N-Boc-3-aminooxetane-3-acetylethatecan (15 mg, 65% yield), was purified by silica gel column chromatography. LCMS: (M+1) + 635.20 (calculated value: 634.66).

[0185] Compound 13-b (15 mg, 0.023 mmol) was dissolved in 2 ml of DCM, and 1 ml of ethyl acetate solution of 4 M hydrochloric acid was added. The mixture was stirred at room temperature for 30 min and then concentrated to obtain compound 13, namely 3-aminooxetane-3-acetylethanotecan (61 mg, 100% yield). 1 H NMR (500MHz, DMSO-d6) δ8.55(m,2H),8.10(d,J=8.8Hz,1H),7.76(m,1H),7.30(d,J=7.1 Hz,1H),5.60-5.49(m,1H),5.42(m,2H),5.00-4.95(m,1H),4.88(d,J=6.2Hz,1H),4.57 (d,J=6.3Hz,1H),4.51(d,J=6.2Hz,1H),3.62(m,1H),3.46(m,1H),3.23(m,1H),3.16-3 .11(m,2H),2.38(m,3H),2.25-2.13(m,2H),1.92-1.78(m,2H),0.87(m,3H); LCMS:(M+1) + 535.31 (Calculated value: 534.54).

[0186] Example 14: mc-Val-Cit-pab-formyl-2-N-methyl-N-aminoethataetrazol (DC-l)

[0187]

[0188] Compound 9 (14.14 mg, 0.027 mmol), DIPEA (17.52 mg, 0.135 mmol), mc-Val-Cit-pab-PNP (20 mg, 0.027 mmol), and HOBt (7.33 mg, 0.054 mmol) were added sequentially to 0.3 mL of NMP. The mixture was reacted at room temperature for 1 h. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water; the aqueous phase contained 0.1% TFA). The column was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 10% to 55% acetonitrile in water, time 40 min). The solution was lyophilized to give compound DC-1, i.e., mc-Val-Cit-pab-formyl-2-N-methyl-N-aminoacetylethanotecan (15.2 mg, yield 50%), as a yellow solid; LCMS: (M+1). + 1121.09 (Calculated value: 1120.21).

[0189] Example 15: mc-Val-Cit-pab-formyl-2-N-methyl-N-methylaminoacetylethanotecan (DC-2)

[0190]

[0191] Compound 10 (14.52 mg, 0.027 mmol), DIPEA (10.51 mg, 0.081 mmol), mc-Val-Cit-pab-PNP (20 mg, 0.027 mmol), and HOBt (7.33 mg, 0.054 mmol) were added sequentially to 0.3 mL of NMP. The mixture was reacted at room temperature for 1 h. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water, the aqueous phase containing 0.1% TFA). The column was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 10% to 55% acetonitrile in water, time 40 min), and lyophilized to obtain compound DC-2, namely mc-Val-Cit-pab-formyl-2-N-methyl-N-methylaminoacetylethanotecan (12.2 mg, 40%), as a yellow solid; LCMS: (M+1). + 1135.02 (Calculated value: 1134.23).

[0192] Example 16: mc-Val-Cit-pab-formyl-ethanotecan-N-aminourea (DC-3)

[0193]

[0194] In 0.3 mL of NMP, compound 1 (13.38 mg, 0.027 mmol), DIPEA (10.51 mg, 0.081 mmol), mc-Val-Cit-pab-PNP (20 mg, 0.027 mmol), and HOBt (3.66 mg, 0.027 mmol) were added sequentially. The reaction was carried out at room temperature for 1 h. The reaction solution was injected into a 25 g C18 pre-column (equilibrated with acetonitrile first, then with water, the aqueous phase containing 0.1% TFA). The solution was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 10% to 55% acetonitrile in water, time 40 min), and lyophilized to obtain compound DC-3, namely mc-Val-Cit-pab-formyl-ethanotecan-N-aminourea (13 mg, 45%), as a yellow solid; LCMS: (M+1) +1093.03 (calculated value: 1092.15).

[0195] Example 17: mc-Val-Cit-pab-formyl-ethanotecan-N-methyl-N-aminourea (DC-4)

[0196]

[0197] Compound 2 (13.7 mg, 0.027 mmol), DIPEA (10.51 mg, 0.081 mmol), mc-Val-Cit-pab-PNP (20 mg, 0.027 mmol), and HOBt (3.66 mg, 0.027 mmol) were added sequentially to 0.3 mL of NMP. The mixture was reacted at room temperature for 1 h. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated with acetonitrile first, then with water, the aqueous phase containing 0.1% TFA). The solution was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 15% to 60% acetonitrile in water, time 40 min), and lyophilized to obtain compound DC-4, namely mc-Val-Cit-pab-formyl-ethanotecan-N-methyl-N-aminourea (9 mg, 30%), as a yellow solid; LCMS: (M+1) +1107.11 (calculated value: 1106.18).

[0198] Example 18: mc-Val-Cit-pab-formyl-ethanotecan-N,N-dimethylaminourea (DC-5)

[0199]

[0200] Compound 3 (14.11 mg, 0.027 mmol), DIPEA (10.51 mg, 0.081 mmol), mc-Val-Cit-pab-PNP (20 mg, 0.027 mmol), and HOBt (3.66 mg, 0.027 mmol) were added sequentially to 0.3 mL of NMP. The mixture was reacted at room temperature for 1 h, and the reaction solution was injected into a 25 g container. C18 pre-column (equilibrated with acetonitrile first, then with water, aqueous phase containing 0.1% TFA), followed by elution by medium-pressure reversed-phase C18 chromatography (gradient: 15% to 60% acetonitrile in water, time 40 min), lyophilized to give compound DC-5, i.e., mc-Val-Cit-pab-formyl-ethanotecan-N',N-dimethylaminourea (12.1 mg, 40%), as a yellow solid; LCMS: (M+1) +1121.02 (calculated value: 1120.21).

[0201] Example 19: mc-Val-Cit-pab-formyl-2-aminooxyethanotecan (DC-6)

[0202]

[0203] In 2 ml of DMF, ethatecan mesylate (100 mg, 0.19 mmol), DIPEA (48.58 mg, 0.37 mmol), tert-butoxycarbonylaminooxyacetic acid (45 mg, 0.23 mmol), and HATU (118 mg, 0.31 mmol) were added sequentially. The mixture was stirred at room temperature for 4 hours. DMF was removed under reduced pressure, and the intermediate compound 2-Boc-aminooxyacetylethatecan (95 mg, 83% yield) was purified by silica gel column chromatography. 1H NMR (500MHz, DMSO-d6) δ10.40(s,1H),8.73(d,J=8.6Hz,1H),7.75(d,J=10.8Hz,1H),7.28(d,J=2.1Hz,1H),6.52( d,J=1.5Hz,1H),5.62(dt,J=8.8,4.4Hz,1H),5.40(d,J=3.3Hz,2H),5.23(d,J=17.9Hz,1H),5.19-5.11(m,1H),4.3 2(d,J=15.8Hz,1H),4.23(d,J=15.9Hz,1H),3.23-3.15(m,2H),2.37(d,J=6.5Hz,3H),2.24(dq,J=14.1,4.9Hz,1H) ,2.17(tt,J=8.3,5.1Hz,1H),1.84(ddp,J=21.4,14.3,7.2Hz,2H),1.15(s,9H),0.85(t,J=7.2Hz,3H); LCMS: (M+1) + 609.30 (Calculated value: 608.18).

[0204] Compound 2-Boc-aminooxyacetylethanotecan (95 mg, 0.16 mmol) was dissolved in 1 ml of DCM, and 1 ml of 4 M hydrochloric acid in ethyl acetate solution was added. The mixture was stirred at room temperature for 30 min and concentrated to obtain compound 14, namely 2-aminooxyacetylethanotecan (85 mg, 100% yield). 1 H NMR(500MHz,DMSO-d6)δ8.93(d,J=8.5Hz,1H),7.79(d,J=10.5Hz,1H),7.30( s,1H),5.60(dt,J=8.3,4.1Hz,1H),5.42(s,2H),5.29(s,2H),4.73-4.64(m,2 H),3.18(s,2H),2.85(s,3H),2.39(s,3H),2.25(dt,J=8.8,4.6Hz,1H),2.19- 2.10(m,1H),1.85(dp,J=21.4,7.2Hz,2H),0.86(t,J=7.3Hz,3H); LCMS: (M+1) + 509.20 (Calculated value: 508.18).

[0205] Compound 14 (32.17 mg, 0.063 mmol), DIPEA (24.53 mg, 0.189 mmol), mc-Val-Cit-pab-PNP (70 mg, 0.095 mmol), and HOBt (8.55 mg, 0.063 mmol) were added sequentially to 0.7 mL of NMP. The reaction mixture was reacted at room temperature for 1 h. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water, the aqueous phase containing 0.1% TFA). The column was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 10% to 55% acetonitrile in water, time 40 min), and lyophilized to obtain compound DC-6, i.e., mc-Val-Cit-pab-formyl-2-aminooxyacetylethanotecan (31.5 mg, 31%), as a yellow solid; LCMS: (M+1). + 1108.10 (Calculated value: 1107.16).

[0206] Example 20: mc-Val-Cit-pab-methyl-N-methylaminoethazine ethatecan (DC-7)

[0207]

[0208] Compound 8 (33.05 mg, 0.063 mmol), DIPEA (24.53 mg, 0.189 mmol), mc-Val-Cit-pab-PNP (70 mg, 0.095 mmol), and HOBt (8.55 mg, 0.063 mmol) were added sequentially to 0.7 mL of NMP. The reaction mixture was reacted at room temperature for 1 h. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water, the aqueous phase containing 0.1% TFA). The solution was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 10% to 55% acetonitrile in water, time 40 min), and lyophilized to obtain compound DC-7, i.e., mc-Val-Cit-pab-formyl-2-methylaminooxyacetylethanotecan (42 mg, 39%), as a yellow solid; LCMS: (M+1). + 1122.05 (Calculated value: 1121.09).

[0209] Example 21: mc-Val-Cit-pab-formyl-ethanotecan-N'-aminooxalyldiamine (DC-8)

[0210]

[0211] Compound 4 (14.14 mg, 0.027 mmol), DIPEA (17.52 mg, 0.135 mmol), mc-Val-Cit-pab-PNP (20 mg, 0.027 mmol), and HOBt (3.66 mg, 0.027 mmol) were added sequentially to 0.3 mL of NMP. The mixture was reacted at room temperature for 1 h. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated with acetonitrile first, then with water, the aqueous phase containing 0.1% TFA). The solution was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 10% to 55% acetonitrile in water, time 40 min), and lyophilized to obtain compound DC-8, namely mc-Val-Cit-pab-formyl-ethanotecan-N'-aminooxalyldiamine (13 mg, 45%), as a yellow solid; LCMS: (M+1) +1121.02 (calculated value: 1120.16).

[0212] Example 22: mc-Val-Cit-pab-formyl-ethanotecan N'-amino-N-methyloxalyldiamine (DC-9)

[0213]

[0214] In 0.3 mL of NMP, compound 5 (14.52 mg, 0.027 mmol), DIPEA (17.52 mg, 0.135 mmol), mc-Val-Cit-pab-PNP (20 mg, 0.027 mmol), and HOBt (3.66 mg, 0.027 mmol) were added sequentially. The reaction mixture was reacted at room temperature for 1 h. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated with acetonitrile first, then...). The mixture was equilibrated with water (the aqueous phase contained 0.1% TFA), then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 10% to 55% acetonitrile in water, time 40 min), and lyophilized to give compound DC-9, namely mc-Val-Cit-pab-formyl-ethanotecan N'-amino-N-methyloxalyldiamine (13 mg, 45%), as a yellow solid; LCMS: (M+1) +1135.10 (calculated value: 1134.19).

[0215] Example 23: mc-Val-Cit-pab-formyl-N-hydroxyaminoacetylethanotecan (DC-10)

[0216]

[0217] Compound 6 (13.79 mg, 0.027 mmol), DIPEA (10.51 mg, 0.081 mmol), mc-Val-Cit-pab-PNP (20 mg, 0.027 mmol), and HOBt (3.66 mg, 0.027 mmol) were added sequentially to 0.3 mL of NMP. The mixture was reacted at room temperature for 1 h. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water; the aqueous phase contained 0.1% TFA). The column was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 5% to 50% acetonitrile in water, time 40 min). The solution was lyophilized to obtain compound DC-10, i.e., mc-Val-Cit-pab-formyl-N-hydroxyaminoacetylethanotecan (13 mg, 45%), as a yellow solid; LCMS: (M+1). + 1108.03 (Calculated value: 1107.16).

[0218] Example 24: mc-Val-Cit-pab-formyl-N-methoxyacetylethanotecan (DC-11)

[0219]

[0220] Compound 7 (14.17 mg, 0.027 mmol), DIPEA (10.51 mg, 0.081 mmol), mc-Val-Cit-pab-PNP (20 mg, 0.027 mmol), and HOBt (3.66 mg, 0.027 mmol) were added sequentially to 0.3 mL of NMP. The mixture was reacted at room temperature for 1 h. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated with acetonitrile first, then with water, the aqueous phase containing 0.1% TFA). The solution was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 15% to 50% acetonitrile in water, time 40 min), and lyophilized to obtain compound DC-11, namely mc-Val-Cit-pab-formyl-N-methoxyacetylethanotecan (13 mg, 45%), as a yellow solid; LCMS: (M+1) +1122.06 (calculated value: 1121.19).

[0221] Example 25: mc-Val-Cit-pab-formyl-N-ethoxyaminoethazine (DC-l2)

[0222]

[0223] Compound 12 (14.17 mg, 0.027 mmol), DIPEA (10.51 mg, 0.081 mmol), mc-Val-Cit-pab-PNP (20 mg, 0.027 mmol), and HOBt (3.66 mg, 0.027 mmol) were added sequentially to 0.3 mL of NMP. The mixture was reacted at room temperature for 1 h. The reaction solution was then injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water; the aqueous phase contained 0.1% TFA). The column was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 15% to 50% acetonitrile in water, time 40 min). The solution was lyophilized to obtain compound DC-12, i.e., mc-Val-Cit-pab-formyl-N-ethoxyaminoacetylethanotecan (13 mg, 45%), as a yellow solid; LCMS: (M+1). + 1136.02 (Calculated value: 1135.20).

[0224] Example 26: mc-Gly-Gly-Phe-Gly-methoxy-N-methylaminoacetylethanotecan (DC-13)

[0225]

[0226] Take Fmoc-Gly-Gly-Phe-OH (10 g, 19.9 mmol), add 200 mL DCM, HATU (11.43 g, 29.85 mmol), DIPEA (5.13 g, 39.8 mmol), and Gly-Gly-OtBu (4.11 g, 21.89 mmol), react at room temperature for 15 h, dilute with 200 mL DCM, wash with saturated sodium bicarbonate solution, dry with anhydrous sodium sulfate, and purify by silica gel column chromatography to obtain a white solid DC-13-a, Fmoc-Gly-Gly-Phe-Gly-Gly-OtBu (10.7 g, 79%); LCMS: (M+1) + 672.5 (calculated value: 671.75).

[0227] Take DC-13-a (10.7 g, 15.9 mmol), add 110 mL DCM and 50 mL TFA, react at room temperature for 15 h to concentrate the reaction solution, add 100 mL EA, filter, and dry the filter cake in an oven at 40 °C to obtain a white solid DC-13-b, Fmoc-Gly-Gly-Phe-Gly-Gly (8.8 g, 90%); LCMS: (M+1) + 616.10 (calculated value: 615.23).

[0228] Dissolve DC-13-b (3 g, 4.8 mmol) in 30 mL of DMF and stir until dissolved. Then add copper acetate (1.46 mmol, 0.26 g), acetic acid (9.7 mmol, 0.58 g), and lead tetraacetate (5.3 mmol, 2.38 g) sequentially. Heat to 60 °C and react for 20 min. Pour the reaction solution into ice water, extract with ethyl acetate, dry with anhydrous sodium sulfate, and concentrate to obtain a white solid product DC-13-c, Fmoc-Gly-Gly-Phe-Gly-N-hydroxymethyl acetate (1.81 g, 59%); LCMS: (M+1) +630.10 (calculated value: 629.25).

[0229] DC-13-c (0.05 g, 0.076 mmol) was added to 5 mL of THF, followed by compound 11 (0.04 g, 0.076 mmol) and PPTS (3.8 mg, 0.015 mmol). The mixture was refluxed at 60 °C for 16 h under argon protection. The mixture was purified by silica gel column chromatography to obtain a white solid DC-13-d, Fmoc-Gly-Gly-Phe-Gly-methoxy-N-methylaminoacetylethanotecan (43 mg, 50%); LCMS: (M+1) +1093.05 (calculated value: 1092.15).

[0230] DC-13-d (43 mg, 0.039 mmol) was dissolved in 0.5 mL of DMF, and piperidine (33 mg, 0.39 mmol) was added. The mixture was stirred at room temperature for 1 h, and the reaction solution was concentrated to obtain a yellow-brown solid DC-13-e, Gly-Gly-Phe-Gly-methoxy-N-methylaminoacetylethanotecan (34 mg, 100%); LCMS: (M+1) + 871.02 (calculated value: 869.91).

[0231] DC-13-e (34 mg, 0.039 mmol) was dissolved in 0.5 mL of anhydrous DMF, and 6-(maleimide)hexanoic acid succinimide ester (12 mg, 0.039 mmol) and DIPEA (5 mg, 0.039 mmol) were added sequentially. The reaction was carried out at room temperature for 30 min. The reaction solution was injected into a 25 g C18 pre-column (equilibrated with acetonitrile first, then with water, the aqueous phase containing 0.1% TFA). The solution was then eluted by medium-pressure reversed-phase C18 chromatography (gradient: 15% to 50% acetonitrile in water, time 40 min), and lyophilized to obtain compound DC-13, namely mc-Gly-Gly-Phe-Gly-methoxy-N-methylaminoacetylethanotecan (12.4 mg, 30%), as a yellow solid; LCMS: (M+1) +1064.05 (calculated value: 1063.11).

[0232] Example 27: 4-(maleimide hexyloxy)benzaldehyde-aminooxyacetylethanotecanoxime (DC-14)

[0233]

[0234] In a 100 mL single-necked flask, 6-amino-1-hexanol (6.0 g, 51.20 mmol) and 60 mL of 48% HBr aqueous solution were added sequentially. The mixture was refluxed at 90 °C with stirring for 18 h. The pH of the reaction solution was adjusted to between 8 and 9 using solid sodium bicarbonate under ice bath conditions. Boc anhydride (11.2 g, 51.20 mmol) was added, and the mixture was slowly heated to room temperature with stirring overnight. The reaction solution was filtered, and the target product was extracted from the filtrate using EtOAc. The organic phase was washed three times with 5% NaCl solution and dried over anhydrous Na2SO4. The dried organic phase was filtered, concentrated, and yielded a white solid product, N-Boc-6-bromohexylamine (10.0 g, yield 71.4%). LCMS:

[0235] (M+1) + 281.12 (Calculated value: 280.21).

[0236] Add 50 ml of acetonitrile to a 100 ml single-necked flask, then add N-Boc-6-bromohexylamine (5.0 g, 17.84 mmol) and p-hydroxybenzaldehyde (2.2 g) sequentially.

[0237] 17.84 mmol), anhydrous potassium carbonate (6.2 g 44.61 mmol), refluxed at 60 °C with stirring for 18 h, the reaction solution was filtered, 10.0 g silica gel was added to the filtrate to prepare sinter, column chromatography was performed using PE and EA, and the product was concentrated to obtain a white solid product 4-(N-Boc-hexyloxy)benzaldehyde (5.0 g, yield 87.7%); LCMS:

[0238] (M+1) + 322.14 (Calculated value: 321.42).

[0239] Add 3 ml of 1,4-dioxane to a 100 ml single-necked flask, followed by 4-(N-Boc-hexyloxy)benzaldehyde (1.0 g 4.52 mmol) and 3 ml of 4 mol / L HCl / 1,4-dioxane solution. Stir the reaction mixture at room temperature for 3 h. Adjust the pH of the reaction solution to 9 using saturated sodium bicarbonate solution in an ice bath. Add N-methoxycarbonylcis-butenediamide (700.90 mg 4.52 mmol). Stir the reaction mixture in an ice bath for 2 h. Filter the solid. Wash the filter cake with 10 ml of purified water. Dry the filter cake at 35°C to obtain a white solid product L-20 (1.0 g, yield 73.5%); LCMS: (M+1) +302.23 (calculated value: 301.34).

[0240] In 3 ml of methanol, L-20 (24.59 mg, 0.081 mmol), pyridine (14.2 mg, 0.179 mmol), and anhydrous sodium sulfate (23.18 mg, 0.163 mmol) were added, followed by compound 14 (83 mg, 0.163 mmol). The mixture was stirred at room temperature for 1 h. After the reaction was complete, 1 g of silica gel was added for column chromatography to obtain the product DC-14, 4-(maleimide hexyloxy)benzaldehyde-aminooxyacetylethanotecanoxime (36 mg, yield 56%); LCMS: (M+1) + 792.11 (calculated value: 791.30).

[0241] Example 28: 4-(Malayanimine ethyloxyethoxy)benzaldehyde-aminoacetylethanotecanoxime (DC-15)

[0242]

[0243] In a 100 ml single-necked flask, diethylene glycolamine (6.0 g, 57.07 mmol) and 60 ml of 48% HBr aqueous solution were added sequentially. The mixture was refluxed at 90 °C with stirring for 18 h. The pH of the reaction solution was adjusted to between 8 and 9 using solid sodium bicarbonate under ice bath conditions. Boc anhydride (12.46 g, 57.07 mmol) was added, and the mixture was slowly heated to room temperature with stirring overnight. The reaction solution was filtered, and the target product was extracted with EtOAc. The organic phase was washed three times with 5% NaCl solution and dried with anhydrous Na2SO4. The dried organic phase was filtered and concentrated to obtain a white solid product, N-Boc-bromoethoxyethylamine (10.8 g, yield 70.6%); LCMS: (M+1) + 269.02 (calculated value: 268.15).

[0244] Add 50 ml of acetonitrile to a 100 ml single-necked flask, then add N-Boc-bromoethoxyethylamine (5.0 g 18.65 mmol), p-hydroxybenzaldehyde (2.28 g 18.65 mmol), and anhydrous potassium carbonate (6.44 g 46.62 mmol) sequentially. Reflux at 60 °C with stirring for 18 h. Filter the reaction solution, add 10.0 g of silica gel to the filtrate to prepare silica fume, and perform column chromatography using PE and EA. Concentrate to obtain a white solid product, 4-(N-Boc-aminoethoxyethoxy)benzaldehyde (5.3 g, yield 91.8%); LCMS: (M+1) + 310.24 (calculated value: 309.36).

[0245] Add 3 ml of 1,4-dioxane to a 100 ml single-necked flask, followed by 4-(N-Boc-aminoethoxyethoxy)benzaldehyde (0.6 g, 1.94 mmol) and 3 ml of 4 mol / L HCl / 1,4-dioxane solution. Stir the reaction mixture at room temperature for 3 h. Adjust the pH of the reaction solution to 9 using saturated sodium bicarbonate solution under ice bath conditions. Add N-methoxycarbonylcis-butenediamide (300.83 mg, 1.94 mmol), and stir the reaction mixture under ice bath conditions for 2 h. Filter the solid, wash the filter cake with 10 ml of purified water, and dry the filter cake at 35°C to obtain a white solid product L-021 (500 mg, yield 89%). LCMS: (M+1) +

[0246] 290.13 (Calculated value: 289.29).

[0247] In 3 ml of methanol, L-021 (23.61 mg, 0.082 mmol), pyridine (14.2 mg, 0.179 mmol), and anhydrous sodium sulfate (23.18 mg, 0.163 mmol) were added, followed by compound 14 (83 mg, 0.163 mmol). The mixture was stirred at room temperature for 1 h. After the reaction was complete, 1 g of silica gel column chromatography was added to obtain the product DC-15,4-(maleimide ethyloxyethoxy)benzaldehyde-aminooxyacetylethanotecanime (42 mg, 66% yield); LCMS: (M+1) + 780.12 (Calculated value: 779.26).

[0248] Example 29: HS627-Succinimide-N-hexanoyl-Val-Cit-pab-formyl-ethanotecan-N-aminourea (ADC-3a)

[0249]

[0250] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature (25 °C) by rotating the disc for 90 min.

[0251] DC-3 (0.9 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain ADC-3a (1.8 mg / ml, 3 ml).

[0252] UV-HPLC calculated average: n = 7.40

[0253] Example 30: HS627-Succinimide-N-hexanoyl-Val-Cit-pab-formyl-2-aminooxyacetylethanotecan (ADC-6a)

[0254]

[0255] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature (25 °C) by rotating the disc for 90 min.

[0256] DC-6 (0.9 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc for 2 h. After the reaction was completed, the buffer was replaced with 20 mL of histidine acetate buffer (pH 6.0, 120 mM sucrose, 0.2 g / L polysorbate 20) using a NAP-5 gel column (Cytiva) to obtain ADC-6a (3.2 mg / mL, 2 mL).

[0257] UV-HPLC calculated average: n = 7.70

[0258] Example 31: HS627-Succinimide-N-hexanoyl-Val-Cit-pab-formyl-N-methylaminoacetylethanotecan (ADC-7a)

[0259]

[0260] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature (25 °C) by rotating the disc for 90 min.

[0261] DC-7 (0.9 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc for 2 h. After the reaction was completed, the buffer was replaced with 20 ml of histidine acetate buffer (pH 6.0, 120 mM sucrose, 0.2 g / L polysorbate 20) using a NAP-5 gel column (Cytiva) to obtain ADC-7a (3.1 mg / ml, 2 ml).

[0262] UV-HPLC calculated average: n = 7.30

[0263] Example 32: HS627-Succinimide-4-(N-hexyloxy)benzaldehyde aerobic acetylethanotecan (ADC-14a)

[0264]

[0265] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature (25 °C) by rotating the disc for 90 min.

[0266] DC-14 (0.63 mg, 0.8 mmol) was dissolved in 0.063 ml of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc for 2 h. After the reaction was completed, the buffer was replaced with 20 ml of histidine acetate buffer (pH 6.0, 120 mM sucrose, 0.2 g / L polysorbate 20) using a NAP-5 gel column (Cytiva) to obtain ADC-14a (2.9 mg / ml, 2 ml).

[0267] UV-HPLC calculated average: n = 4.3.

[0268] Example 33: HS627-Succinimide-4-(N-ethoxyethoxy)benzaldehyde oxime acetylethanotecan (ADC-15a)

[0269]

[0270] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature (25 °C) by rotating the disc for 90 min.

[0271] DC-15 (0.62 mg, 0.8 mmol) was dissolved in 0.062 ml of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain ADC-15a (3.2 mg / ml, 2 ml).

[0272] UV-HPLC calculated average: n = 7.6.

[0273] Example of effect 1: Inhibition of tumor cell growth activity

[0274] In vitro inhibitory activity assay method for compounds (small molecule toxins)

[0275] Human esophageal cancer cells OE33 and human breast adenocarcinoma cells SK-BR-3 were cultured in RPMI1640 (Cellmax) containing 10% fetal bovine serum (Cellmax). Tumor cells in the exponential growth phase were diluted with the culture medium to 1×10⁻⁶. 5Cells / mL were added at 100 μL per well to a 96-well cell culture plate and incubated overnight at 37°C with 5% CO2. The next day, the small molecule compounds were diluted to 10000 nM, 2000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, and 0.13 nM using culture medium. 2 μL of the diluted compound was added to each well of the 96-well cell culture plate, with three replicates for each concentration. 2 μL of the diluent was added to each well for the negative control and blank control groups. After addition, the plates were incubated at 37°C with 5% CO2 for 72 h. After incubation, the cell culture plates were removed, the culture medium was aspirated, and 100 μL of medium containing 10% CCK-8 was added to each well. The plates were incubated at 37°C for 3 h. After incubation, remove the culture plate, protect it from light, and place it in a microplate. Select 630 nm as the reference wavelength and 450 nm as the measurement wavelength to measure the absorbance. Based on the absorbance values, calculate the IC50 using four-parameter regression in GraphPad. 50 Values ​​(Table 1-3). The DNA topoisomerase I inhibitor Dxd was used as a positive control. For IC50 values, “++++” indicates IC50 < 10 nM; “+++” indicates IC50 between 10 nM and 100 nM; “++” indicates IC50 between 100 and 500 nM; and “+” indicates IC50 > 500 μM.

[0276] ADC in vitro inhibitory activity assay method

[0277] Human esophageal cancer cells OE33, lung cancer cells NCI-H1975, and breast cancer cells MDA-MB-231, used as activity assays, were cultured in RPMI 1640 (Cellmax), RPMI 1640 (Cellmax), and DMEM (Cellmax) media containing 10% fetal bovine serum (Cellmax), respectively, until the exponential growth phase. After trypsin digestion, the cells were centrifuged, the supernatant was discarded, and the media were diluted to 3 × 10⁻⁶. 4 cells / mL, 0.5×10 4 cells / mL and 1.5×10 4Cells / mL were added to 100 μL per well of a 96-well cell culture plate and incubated overnight at 37°C with 5% CO2. The next day, the ADC was diluted to 2000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.128 nM, and 0.026 nM using culture medium. 100 μL of the diluted ADC was added to each well of the 96-well cell culture plate, with three replicates for each concentration. 100 μL of culture medium was added to each well of the negative control and blank control group (without ADC). After addition, the plates were incubated at 37°C with 5% CO2 for 6 days. After incubation, the cell culture plates were removed, the culture medium was aspirated from the plates, and 100 μL of medium containing 10% CCK-8 was added to each well. The plates were incubated at 37°C for 3 hours. After incubation, the culture plate was removed, protected from light, and placed in an ELISA plate. Absorbance was measured using 630 nm as the reference wavelength and 450 nm as the measurement wavelength. Based on the absorbance values, IC50 was calculated using four-parameter regression in GraphPad (Tables 1 and 3). The ADC drug DS-8201a was used as the positive control. For IC50 values, “++++” indicates IC50 < 10 nM; “+++” indicates IC50 between 10 nM and 100 nM; “++” indicates IC50 between 100 and 500 nM; and “+” indicates IC50 > 500 μM.

[0278] The structural formula of the positive control compound DXD is:

[0279] Table 1 Inhibitory activity against OE33

[0280]

[0281]

[0282] Table 2 Inhibitory activity against SK-BR-3

[0283] Compound numbering <![CDATA[IC 50 / nM]]> Compound numbering <![CDATA[IC 50 / nM]]> Compound numbering <![CDATA[IC 50 / nM]]> 1 +++ 5 ++++ 13 ++++ 3 ++++ 8 ++++ Dxd ++++ 4 +++

[0284] Table 3. Inhibitory activity of ADC drugs against NCI-H1975 and MDA-MB-231

[0285]

[0286] The compounds provided in this invention all exhibit good inhibitory effects on the growth of cancer cells, such as esophageal cancer cells and breast adenocarcinoma cells. Most compounds, such as compounds 2, 3, 7, 8, 9, and 13, show significant inhibitory effects on the growth of esophageal cancer cells (IC50). 50 The values ​​were all below 10 nM, indicating significant anticancer activity.

[0287] The compounds in this invention are used to construct antibody-drug conjugates. The resulting conjugates have significant inhibitory activity against esophageal cancer cells, lung cancer cells, and breast cancer cells. Exemplary conjugates ADC-3a, ADC-6a, and ADC-7a all have significant inhibitory activity against the above-mentioned cells, and the inhibitory activity of ADC-7a is even lower than 10 nM.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: In the formula, R is selected from -Z-R1, where, Z represents a single bond or C=O; R1 is selected from -NHNH2, -N(CH3)NH2, -N(CH3)NHCH3, -C(O)NHNH2, -C(O)N(CH3)NH2, -CH2NHOH, -CH2NHOCH3, -CH2ONHCH3, -CH2NCH3NH2, -CH2N(CH3)NHCH3, -CH2N(CH3)OH, -CH2NHOCH2CH3; The condition is that -Z-R1 is not -NH2, -CH2OH, -CH2NH2 or -CH2OCH2NH2.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is a compound of formula (Ia), (Ib), (Ic) or (Id): In formula (Ia), R1 is -NHNH2 or -N(CH3)NH2; In formula (Ib), R2 is selected from -NHOH, -NHOCH3, -ONHCH3, -NCH3NH2, -N(CH3)NHCH3, -N(CH3)OH or -NHOCH2CH3; In equation (Ic), R a R b Not both H; -NR a R b Selected from -NHNH2, -N(CH3)NH2 or -N(CH3)NHCH3.

3. The following compounds or their pharmaceutically acceptable salts:

4. A method for preparing the compound according to any one of claims 1-3, wherein the method is selected from the following reaction routes: Reaction route 1: Exatecan was reacted with N,N'-carbonyldiimidazole, and then reacted with hydrazine or substituted hydrazine to give the corresponding N-aminourea ethatecan derivatives. Reaction route 2: The substituted hydrazine was reacted with oxalyl chloride, and then reacted with ethatecan to give the corresponding oxalamide hydrazine ethatecan derivative. Reaction route 3: Exatecan reacts with bromoacetic acid to give bromoacetyl-exatecan, and bromoacetyl-exatecan is condensed with amine compounds to give the corresponding amide-exatecan derivatives. Reaction route 4: Exatecan reacts directly with carboxylic acid compounds to give the corresponding amide ethatecan derivatives.

5. The drug-linker compound represented by formula (II) or a pharmaceutically acceptable salt thereof: In the formula, R is defined as in formula (I) of claim 1; L is -L1-Q-L2; R is partially connected to L1; in, L1 is in, The position shown indicates that it is connected to the R group. The position shown indicates that it is connected to the Q group; L2 is Q is selected from Val-Cit, Gly-Gly-Phe-Gly, (CH2)2O(CH2)2, or (CH2)6.

6. The drug-antibody conjugate shown in formula (III) or a pharmaceutically acceptable salt thereof: In the formula, R is defined as in the compound of formula (I) of claim 1, and L is defined as in the compound of formula (II) of claim 5; Ab represents tumor-associated antigen-antibody, and n is an integer selected from 1 to 8.

7. The drug-antibody conjugate according to claim 6, or a pharmaceutically acceptable salt thereof, wherein the tumor-associated antigen is selected from Her2, Trop2, 5T4, ROR1, or B7-H3.

8. A drug-antibody conjugate or a pharmaceutically acceptable salt thereof, wherein the drug-antibody conjugate is selected from the following compounds:

9. The drug-antibody conjugate according to claim 8, or a pharmaceutically acceptable salt thereof, wherein the drug-antibody conjugate is selected from the group consisting of:

10. A pharmaceutical composition, characterized in that, It includes the compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, the drug-linker compound of claim 5 or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate of claims 6-9, and pharmaceutically acceptable excipients.

11. The use of the compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, the drug-linker compound of claim 5 or a pharmaceutically acceptable salt thereof, the antibody-drug conjugate of claims 6-9 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 10 in the preparation of a medicament for treating cancer.

12. The application according to claim 11, wherein the cancer includes gastric cancer, esophageal cancer, breast cancer, and lung adenocarcinoma.

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