CN118001423A

CN118001423A - Hydrophilic anti-Nectin-4 antibody coupled drug as well as preparation method and application thereof

Info

Publication number: CN118001423A
Application number: CN202410051888.5A
Authority: CN
Inventors: 黄云生; 陆遥; 盛国荣; 汪小蓓; 李德亮
Original assignee: Hangzhou Aikerisi Biomedical Co ltd
Current assignee: Hangzhou Aikerisi Biomedical Co ltd
Priority date: 2024-01-12
Filing date: 2024-01-12
Publication date: 2024-05-10

Abstract

The present specification provides toxin-hydrophilic linker compounds formed from camptothecin-7-ethylamine and a linker, conjugates thereof with anti-Nectin-4 antibodies, and pharmaceutical uses thereof. The toxin-hydrophilic linker compound can be coupled with an anti-Nectin-4 monoclonal antibody to form an antibody coupled drug (ADC), so that the hydrophilicity of the ADC is increased, the aggregation of the ADC in a circulatory system is reduced, the drug effect is improved, and the toxic and side effects are reduced.

Description

Hydrophilic anti-Nectin-4 antibody coupled drug as well as preparation method and application thereof

Technical Field

The invention relates to the field of medicine. In particular, the invention provides a series of preparation of camptothecin-7-ethylamine and toxin-hydrophilic linker compounds formed by the camptothecin-7-ethylamine and a linker, and antibody coupling medicaments formed by coupling corresponding anti-Nectin-4 antibodies and application of the antibody coupling medicaments in tumor treatment.

Background

Camptothecin and derivatives thereof have inhibitory activity on topoisomerase Top1, especially on Top1-DNA complex. The camptothecine has obvious curative effect on gastric cancer, esophagus cancer, lung cancer, bladder cancer, etc., and is a broad-spectrum antitumor active medicine. Of these Irinotecan and Topotecan have been approved in many countries for the treatment of various cancers. While another camptothecin derivative Belotecan has been approved in korea for the treatment of SCLC and ovarian cancer. The main defects of camptothecins antitumor drugs are toxicity, indissolvable property and drug resistance of tumor cells.

Disclosure of Invention

The invention provides a series of toxin-hydrophilic linker compounds formed by camptothecin-7-ethylamine and a linker, wherein the compounds contain a section of hydrophilic structure composed of at least two PEG, and the toxin-hydrophilic linker can be coupled with a monoclonal antibody Nectin-4 to form an antibody coupled drug (ADC), so that the hydrophilicity of the ADC is increased, the aggregation of the ADC in a circulatory system is reduced, the drug effect is improved, and the toxic and side effects are reduced.

In one aspect of the invention, there is provided a compound of formula (I), a pharmaceutically acceptable salt or stereoisomer thereof:

In the method, in the process of the invention,

R ₁、R₂ is each independently selected from hydrogen, fluorine and C _1-3 alkyl, or R ₁、R₂ together with the carbon atom to which it is attached form an oxygen-containing heterocyclic group;

R ₃ is selected from C _1-6 alkyl, C _1-6 alkoxy, C _3-6 cycloalkyl or 4 to 8 membered heterocycloalkyl; the C _1-6 alkyl, C _1-3 alkoxy groups are optionally substituted with one or more halogens; the 4-to 8-membered heterocycloalkyl contains 1,2 or 3 heteroatoms selected from N, O, S as ring atoms;

r ₄ is selected from hydrogen or heteroalkyl containing a-OCH ₂CH₂ -repeat unit;

R ₅ is selected from hydrogen, C _1-6 alkyl, and C _3-6 cycloalkyl;

Lp is selected from peptide residues comprising 1-5 amino acids;

m is selected from integers from 1 to 8;

a. p is selected from 1,2 or 3;

Ab is an anti-Nectin-4 antibody or antigen-binding fragment;

z is a linker capable of coupling the antibody or antigen binding fragment to other moieties of the compound of formula (I);

0.5≤n≤8。

In one embodiment, the compound of formula (I) has a structure of formula (I-1) or formula (I-2):

Wherein each group is defined as in the formula (I).

In one embodiment, m is selected from 1, 2, 3, 4, 5, 6, 7, or 8.

In one embodiment, R ₁、R₂ is each independently selected from hydrogen, fluoro, and methyl, or R ₁、R₂ is taken together with the carbon atom to which it is attached to form

In one embodiment, R ₁ is hydrogen and R ₂ is hydrogen.

In one embodiment, R ₁ is fluoro and R ₂ is fluoro.

In one embodiment, R ₁ is methyl and R ₂ is fluoro.

In one embodiment, R ₃ is selected from 1,2, or 3 fluoro-substituted C _1-6 alkyl groups.

In one embodiment, R ₃ is selected from 4 to 8 membered oxacycloalkyl.

In one embodiment, R ₃ is selected from oxetanyl, oxolanyl, or oxolanyl.

In one embodiment, R ₃ is selected from the group consisting of fluoroethyl, difluoroethyl, trifluoroethyl, oxacyclopentyl, or oxacyclohexyl.

In one embodiment, R ₄ is selected from hydrogen, C _1-6 alkyl, and-C (O) -NR ^aR^b;

Each R ^a、R^b is independently selected from C _1-6 alkyl;

Wherein one or more methylene units in the C _1-6 alkyl group are optionally and independently replaced by- (OCH ₂CH₂) q-;

q is selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In one embodiment, R ₄ is selected from hydrogen,

In one embodiment, L ^p is selected from -Val-Cit-、-Gly-Lys-、-Gly-Leu-、-Val-Ala-、-Gly-Phe-、-GLy-Gly-Lys-、-Gly-Gly-Phe-、-Gly-Val-Ala-、-Gly-Gly-Val-、-Gly-Leu-Val-、-Gly-Phe-Gly- or-Gly-Gly-Leu-.

In one embodiment, L ^p is selected from

In the present invention, the linker used to couple the antibody or antigen binding fragment to the other moiety of the compound of formula (I) may be a single linker or a double linker, which refers to a structure that can simultaneously link two chemical functional groups (specifically, antibody and toxin moieties). The structure has a group attached to the antibody and a group attached to the toxin moiety.

In one embodiment, Z is selected from Wherein/>The indicated positions indicate the attachment to the antibody,/>The indicated position indicates attachment to-NH-.

In one embodiment, the heavy chain amino acid sequence of the anti-Nectin-4 antibody is shown as SEQ ID NO. 1, and the light chain amino acid sequence is shown as SEQ ID NO. 2.

The present invention provides the following toxin-linker compounds, pharmaceutically acceptable salts or stereoisomers thereof:

The present invention provides the following compounds, pharmaceutically acceptable salts or stereoisomers thereof:

in another aspect of the present invention, there is provided a pharmaceutical composition comprising a compound of formula (I) as described above, a pharmaceutically acceptable salt or stereoisomer thereof; and a pharmaceutically acceptable carrier.

In another aspect of the present invention, there is provided the use of a compound of formula (I), a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition as described above, in the manufacture of an antitumor medicament;

Preferably, the tumor is selected from solid tumors.

In one embodiment, the tumor is selected from the group consisting of breast cancer, transitional cell bladder cancer, prostate cancer, and pancreatic adenocarcinoma.

In another aspect of the invention, there is provided a method of treating cancer comprising administering to a patient in need thereof a compound of formula (I), a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition as described above.

In another aspect of the present invention, there is provided a process for the preparation of a compound of formula (I), comprising:

Wherein each group is defined as for a compound of formula (I);

The steps are compatible with examples 1-5 using reagents.

In one embodiment, the reagents used in each step are ：a)DMF-DMA;b)R₃-NH₂;c)NaH(OAc)₃;d)Cbz-Cl;e)Na₂S₂O₄;f)PPTS;g)H₂/Pd-C;h)Fmoc-Gly-Lys(Trt)-PABC;i) piperidine, respectively, which is then condensed with Fmoc-PEG 2-OH; j) Piperidine and then condensed with the corresponding Z linker; k) Coupling with an anti-Nectin-4 antibody to form anti-Nectin-4 ADC.

The beneficial effects are that:

The toxin-hydrophilic linker and the Nectin-4 monoclonal antibody are coupled to form an antibody coupled drug (ADC), which is beneficial to reducing the aggregation of the ADC in a circulatory system, improving the drug effect and reducing the toxic and side effects.

Drawings

FIG. 1 shows the RP-MS value test results of ADC1 in example 1; wherein A and B are HPLC results and C and D are MS results.

FIG. 2 shows the RP-MS value test results of ADC2 in example 2; wherein A and B are HPLC results and C and D are MS results.

FIG. 3 shows the RP-MS value test results of ADC3 in example 3; wherein A and B are HPLC results and C and D are MS results.

FIG. 4 shows the RP-MS value test results of ADC4 in example 4; wherein A and B are HPLC results and C and D are MS results.

FIG. 5 shows the RP-MS value test results of ADC5 in example 5; wherein A and B are HPLC results and C and D are MS results.

Detailed Description

I. Definition of the definition

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, the relative terms and laboratory procedures used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where integers are required, the term refers to rounding up or down to the nearest integer within plus or minus 10% of a given value or range.

In the description herein, reference is made to "some embodiments," "some implementations," or "some implementations," which describe a subset of all possible embodiments, but it is to be understood that "some embodiments" may be the same subset or different subsets of all possible embodiments and may be combined with one another without conflict.

As used herein and unless otherwise indicated, the terms "comprising," "including," "having," "containing," and their grammatical equivalents are generally understood to be open-ended and not to be limiting, e.g., not to exclude other, unrecited elements or steps.

The term "antibody" as used herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. Antibodies may be murine, human, humanized, chimeric or derived from other species. Antibodies are proteins produced by the immune system that are capable of recognizing and binding to a specific antigen. The target antigen typically has multiple binding sites, also known as epitopes, which are recognized by CDRs (complementarity determining regions) on various antibodies. Each antibody that specifically binds a different epitope has a different structure. Thus, an antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to a target antigen of interest, or portion thereof, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The term "antibody" is an immunoglobulin molecule capable of binding to a specific antigen. Comprising two light chains of relatively light molecular weight and two heavy chains of relatively heavy molecular weight, the heavy (H) and light (L) chains being linked by disulfide bonds to form a tetrapeptide chain molecule.

The term "antibody-drug conjugate (ADC)" used in the present application is to connect a small molecular drug with biological activity to a monoclonal antibody through a chemical link, and the monoclonal antibody is used as a carrier to target and transport the small molecular drug into a target cell.

The term "toxin" as used herein is also referred to as "cytotoxic drug moiety" or "small molecule drug", which refers to a compound that has a killing effect on tumor cells. Examples of the cytotoxic drug moiety include at least one of an anti-tubulin agent, a DNA intercalator, a DNA topoisomerase inhibitor, a DNA synthesis inhibitor, an RNA polymerase inhibitor, a splicesome inhibitor, a proteolysis-target chimera (proteolysis-TARGETING CHIMERA, PROTAC), and an immunoregulatory substance (immunomodulators).

The small molecule drugs used in the present application may be asymmetric, e.g., have one or more stereoisomers. Unless otherwise indicated, 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 well as therapeutic agents composed of monomers, racemates, racemic mixtures and pharmaceutically acceptable salts thereof. The compounds of the application containing asymmetric carbon atoms can be isolated in optically pure or racemic form. Optically pure forms can be resolved from the racemic mixture or synthesized by using chiral starting materials or chiral reagents. Racemates, diastereomers, and enantiomers are all included within the scope of the present application.

Small molecule drugs used in the present application also include tautomeric forms. Tautomers originate from the exchange of one single bond with an adjacent double bond and accompany the migration of one proton.

Numerical ranges herein refer to individual integers within a given range. For example, "C ₁-C₆" means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms; "C ₃-C₆" means that the group can have 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

When any variable (e.g., R _n) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 1 to 5R, the group may optionally be substituted with up to 5R, and R in each case has an independent option. Furthermore, combinations of substituents and/or variants thereof are only permissible if such combinations result in stable compounds.

The term "alkyl" as used herein refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkyl group containing from 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl. The alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxy or carboxylate, with methyl, ethyl, isopropyl, t-butyl, haloalkyl, deuteroalkyl, alkoxy-substituted alkyl and hydroxy-substituted alkyl being preferred.

The term "heterocyclyl" or "heterocycloalkyl" as used herein refers to a saturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms in which 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-O-, -O-S-or-S-, the remaining ring atoms being carbon.

The term "alkoxy" as used herein refers to-O- (alkyl) and-O- (unsubstituted cycloalkyl) wherein the alkyl group is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexyloxy.

The term "substituted" as used herein means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable when bound to carbon atoms having unsaturated (e.g., olefinic) bonds.

The term "pharmaceutically acceptable salt" as used herein refers to salts of the corresponding amine compounds with inorganic or organic acids, or salts of the corresponding carboxylic acid compounds with alkali or alkaline earth metals, or salts with organic amines. Wherein the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like; 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, tartaric acid, and the like; alkali or alkaline earth metal salts include, but are not limited to, sodium, potassium, calcium, magnesium salts, and the like; 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, amino acids, and the like.

The medicament or pharmaceutical composition of the application may be administered orally, topically, parenterally or mucosally (e.g., parenterally, by inhalation or rectally) in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers. It is generally desirable to use the oral route. The active agent may be administered orally in the form of capsules, tablets, etc. (see Remington: THE SCIENCE AND PRACTICE of Pharmacy,20th Edition).

For oral administration in the form of a tablet or capsule, the active pharmaceutical ingredient may be in the form of a non-toxic, pharmaceutically acceptable adjuvant such as a binder (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 behenate, calcium stearate, and the like); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate), coloring and flavoring agents, gelatin, sweetening agents, natural and synthetic gums (e.g., acacia, tragacanth or alginates), buffer salts, carboxymethylcellulose, polyethylene glycol, waxes, and the like. For oral administration in liquid form, the pharmaceutical component may be combined with non-toxic, pharmaceutically acceptable inert carriers (e.g., ethanol, glycerol, water), anti-settling agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous carriers (e.g., almond oil, oil esters, ethanol, or fractionated vegetable oils), preserving agents (e.g., methyl or propyl p-hydroxybenzoate, or sorbic acid), and the like. Stabilizers such as antioxidants (BHA, BHT, propyl citrate, sodium ascorbate, citric acid) may also be added to stabilize the dosage form.

Tablets containing the active compound may be coated by methods well known in the art. The compositions of the application comprising as active compound a compound of formula I may also be incorporated into beads, microspheres or microcapsules, for example constructed from polyglycolic acid/lactic acid (PGLA). Liquid formulations for oral administration may take the form of, for example, solutions, syrups, emulsions or suspensions or they may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Formulations for oral administration may be suitably formulated so as to provide controlled or delayed release of the active compound.

The term "treating" as used herein includes inhibiting, alleviating, preventing or eliminating one or more symptoms or side effects associated with the disease, condition or disorder being treated.

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

The term "pharmaceutical composition" as used herein means a composition comprising a compound of the present application or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable ingredient selected from the following, including but not limited to: carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, antibacterial agents, antifungal agents, lubricants, dispersing agents, temperature sensitive materials, temperature adjusting agents, adhesives, stabilizers, suspending agents, and the like.

Examples II

The present invention will be described in further detail below for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, and the described embodiments should not be construed as limiting the present invention, and all other embodiments obtained by those skilled in the art without making any inventive effort are within the scope of the present invention.

Before describing embodiments of the present invention in further detail, the terms and terminology involved in the embodiments of the present invention will be described, and the terms and terminology involved in the embodiments of the present invention will be used in the following explanation.

The materials and equipment used in the embodiments of the present disclosure are all known products and are obtained by purchasing commercially available products.

DAR value test and calculation: based on RP-HPLC-MS test results, the DAR value of the ADC was analyzed using Waters Acquity UPLC I-Class/Xex G2-XS QTOF instrument.

RP-HPLC parameter settings: the column temperature of the chromatographic column PLRP-S1000A 5UM is 70 ℃. Mobile phase a was 0.1% aqueous formic acid, mobile phase B was 0.1% acetonitrile formic acid, and the flow rate was 0.2ml/min. The gradient of the mobile phase is 20-50% B for 18 minutes; 50-95% B,5 min; 95-20% B,0.1 min; 20-20% B,6.9 min.

MS parameter setting: capillary voltage 2.50kV, taper hole voltage 100V, mass analysis range m/z 200-4000, MSE collision energy 20-45 eV, ion source temperature 120 ℃, atomization temperature 500 ℃, atomization flow rate 1000L/Hr, and internal standard leucine enkephalin. The test sample is diluted to 1mg/ml with sample buffer solution, TCEP with a final concentration of 50mmol/L is added, incubation is carried out for 20min at 37 ℃, and 5ul of sample is introduced. Light chain peaks were identified and the percent peak area was calculated, with the sum of the peak areas being 100. Heavy chain peaks were also identified and the percent peak area was calculated, with the sum of the peak areas being 100. The weighted peak areas of the heavy and light chains were calculated by multiplying the peak area percentages by the corresponding drug loads, respectively. The DAR value calculation formula is: dar=2 x (Σlight chain weighted peak area+Σheavy chain weighted peak area)/100.

The ADC prepared in this example uses, but is not limited to, nectin-4 antibody.

Nectin-4 antibody heavy chain amino acid sequence is as follows (SEQ ID NO: 1):

The light chain amino acid sequence of the Nectin-4 antibody is as follows (SEQ ID NO: 2):

Example 1: ab _nectin-4 -S- (7- (N- (acetamide-PEG ₂ -propionyl-Gly-Lys-PABC) -N- (2' -difluoroethyl)) aminoethyl camptothecin) ₈ (ADC 1)

1.1 7- (N- (bromoacetamide-PEG ₂ -propionyl-Gly-Lys-PABC) -N- (2' -difluoroethyl))) aminoethyl camptothecin (L1-D1)

4',5' -Methylenedioxy-2 ' -nitroacetophenone (15 g,71.72 mmol) was added to a 500ml single-necked flask, DMF-DMA (170.92 g,1.43 mol) was added, the mixture was heated to 100℃for reflux reaction for 10 hours, and concentrated to give a yellow solid, THF (30 ml) was added, n-hexane (150 ml) was added dropwise, stirred for 3 hours, filtered to give a yellow solid wet product, and dried under vacuum at 40℃for 6 hours to give pale yellow solid intermediate I-1 (18 g, yield 95%, HPLC 98%); LCMS: [ M+1] ⁺ 265.22 (calculated 264.24);¹H NMR(600MHz,DMSO-d₆)δ7.62-7.22(m,2H),7.04(s,1H),6.24(s,2H),5.22(d,J＝6.9Hz,1H),3.08(s,3H),2.84(s,3H).

Intermediate I-1 (18 g,68.12 mmol) was added to IPA (180 mL), the reaction was stirred, DIEA (17.61 g,136.24 mmol) and difluoroethylamine hydrochloride (32.02 g,272.48 mmol) were added, stirred and refluxed for 12h, concentrated, water (200 mL) was added, stirred for 2h, filtered to give a yellow solid, dried at 40℃to give product D1-1 (15.8 g, 77.3% yield, HPLC 98%); LCMS: [ M+1] ⁺ 301.47 (calculated 300.22);¹H NMR(600MHz,CDCl₃)δ9.84(s,1H),7.40(s,1H),6.87(s,1H),6.84(dd,J＝12.6,7.5Hz,1H),6.13(s,2H),5.99-5.74(m,1H),5.26(d,J＝7.5Hz,1H),3.68-3.48(m,2H).

Compound D1-1 (15.8 g,52.63 mmol) was added to DCM (310 mL), acetic acid (63.05 g,1.05 mol) was added thereto, and the mixture was stirred to dissolve all the solid, sodium borohydride (27.89 g,131.56 mmol) was added in 4 portions at 15 degrees celsius, approximately 5 minutes intervals of about 7 grams each, the reaction was allowed to proceed for 2 hours at 15 degrees celsius, and the reaction was poured into an aqueous sodium carbonate solution (sodium carbonate: 130g, water: 1L), controlling the internal temperature to 15 ℃, stirring, layering, washing an organic phase with water, collecting the organic phase and drying the organic phase with anhydrous sodium sulfate to obtain a compound D1-2 (directly added in the next step), cooling to 0 ℃, adding DIEA (13.58 g,105.26 mmol), dropwise adding a DCM solution (about 20min after dropwise adding CbzCl (10.11 g,59.27 mmol), reacting for 12h at 0 ℃, adding the mixture into a 10% citric acid aqueous solution (200 mL of x 2), washing the mixture twice, washing the mixture with a saturated sodium bicarbonate solution (200 mL) in sequence, washing the mixture with a saturated saline solution (200 mL), collecting the organic phase, drying the organic phase with anhydrous sodium sulfate, concentrating the organic phase to obtain a product D1-3 (19.7 g, 85.7% of the total yield of two steps, and HPLC 95%); LCMS: [ M+1] ⁺ 437.42 (calculated 436.37);¹H NMR(500MHz,CDCl₃)δ7.40-7.28(m,7H),6.17(s,2H),5.15(d,J＝4.5Hz,2H),3.82-3.65(m,4H),3.14-2.75(m,3H).

Compound D1-3 (19.7 g,45.1 mmol) was added to a 1L single-necked flask under nitrogen protection, DMF (50 mL) was added, cooled to 0 ℃, and the prepared reducing solution was slowly dropped: h ₂ O (250 ml)/sodium dithionite (39.30 g,225.72 mmol)/sodium carbonate (19.14 g,180.58 mmol), slowly heating to 40 ℃ after dripping, stirring and reacting for 2H, filtering, concentrating the filtrate, adding ethyl acetate (500 ml) and water (500 ml), extracting and separating liquid, adding ethyl acetate (500 ml) into the water phase again, adjusting pH to 3-4 by 2M dilute hydrochloric acid, stirring for 6H at normal temperature, extracting and separating liquid, combining organic phases and drying by sodium sulfate, concentrating the sample, purifying by silica gel column chromatography to obtain yellow oily substance D1-4 (17.7 g, 96.7% yield, HPLC 98%); LCMS: [ M+1] ⁺ 407.52 (calculated 406.39);¹H NMR(500MHz,CDCl₃)δ7.44-7.02(m,7H),6.03(s,2H),5.79-5.72(m,2H),5.10-4.98(m,2H),3.73-3.48(m,3H),3.04(m,2H),2.97-2.88(m,2H).

To a 500ml single port flask, compound D1-4 (17.7 g,43.55 mmol), (S) -4-ethyl-4-hydroxy-7, 8-dihydro-1H-pyrano [3,4-F ] indolizin-3, 6,10 (4H) -one (13.75 g,52.27 mmol) and PPTS (218.88 g,871.00 mmol) were added sequentially, and reacted under argon, heated to 110℃with stirring for 12H, the reaction mixture was poured into methanol (1000 ml), stirred for 2H, filtered to give a dark brown solid, the brown solid was stirred again with methanol (300 ml) for 2H, filtered to give a pale brown solid, and the solid was dried at 40℃to give compound D1-5 (10.1 g, 36.6% yield, HPLC 96%); LCMS: [ M+1] ⁺ 634.33 (calculated 633.60);¹H NMR(600MHz,DMSO-d₆)δ7.47-7.17(m,7H),7.17(d,J＝7.7Hz,1H),6.48(s,1H),6.24(d,J＝6.2Hz,2H),6.13(dq,J＝55.7,3.8Hz,1H),5.52-5.26(m,2H),5.15(s,1H),5.11-4.83(m,3H),3.93-3.68(m,2H),3.53(dt,J＝42.0,7.9Hz,2H),3.22(t,J＝8.2Hz,2H),1.89(ddp,J＝21.1,14.0,6.9,6.0Hz,2H),0.90(q,J＝6.8Hz,3H).

Compound D1-5 (10 g,15.78 mmol), dichloromethane (2.5L), methanol (2.5L) were added sequentially to a 10L single-port bottle, 10% content of Pd/C (15 g) was added to the reaction solution under stirring, the reaction was carried out at room temperature under hydrogen balloon atmosphere for 20 hours, pd/C was removed by filtration, the reaction solution was dried by spinning, stirred with 30ml of a mixed solution of dichloromethane and methanol (V/v=1:1) for 24 hours, filtered, and dried at 40 ℃ for 3 hours to give product D1 (5.8 g, yield 73.6%, HPLC 95.0%); LCMS: [ M+1] ⁺ 500.52 (calculated 499.47);¹H NMR(500MHz,DMSO-d₆)δ7.68(s,1H),7.52(s,1H),7.28(s,1H),6.58(s,1H),6.33(s,2H),6.02(t,J＝56.6Hz,1H),5.45-5.45(m,2H),5.45-5.45(m,2H),3.35-3.25(m,2H),3.05-3.85(m,4H),1.85-1.95(d,J＝58.5Hz,2H),1.05-0.85(m,3H).

Preparation of Fmoc-Gly-Lys (Trt) -PAB-PNP: fmoc-Lys (Trt) (50 g,82.1 mmol) and 4-aminobenzyl alcohol (15.1 g,122.7 mmol) were dissolved in a mixed solvent of DCM (250 mL) and MeOH (250 mL) in a 1L single-port flask, cooled to 0℃and EEDQ (30.3 g,122.7 mmol) was added in portions, reacted overnight at room temperature after 10min of incubation, the reaction mixture was washed with 2% aqueous citric acid (300 mL), then with saturated sodium bicarbonate solution (300 mL), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give Fmoc-Lys (Trt) -PAB oil (LCMS: [ M+1] ⁺ 716.30 (calculated 715.35).

DCM (300 mL) was added to the oily product obtained above, DBU (7.4 g,48.9 mmol) was added under stirring, and after reaction at room temperature for 2h, concentrated to about 200mL, added dropwise to 2L methyl tert-butyl ether, filtered, and the filter cake was dried at room temperature to give Lys (Trt) -PAB as a pink solid (37 g, two-step yield 91%); LCMS: [ M+1] ⁺ 494.70 (calculated 493.65).

Lys (Trt) -PAB (47 g,95.2 mmol) was dissolved in DCM (500 mL), fmoc-glycine (11.3 g,38.1 mmol), EDCI (18.2 g,95.2 mmol), HOBt (12.8 g,95.2 mmol), pyridine (7.5 g,95.2 mmol) were added to a 1L single-port flask, the reaction mixture was stirred at room temperature for 2h, washed with 300mL of 5% aqueous citric acid solution, washed with 200mL of saturated sodium bicarbonate solution, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give Fmoc-Gly-Lys (Trt) -PAB (40 g, 54% yield); LCMS: [ M+1] ⁺ 773.70 (calculated 772.95);¹H NMR(600MHz,DMSO-d₆)δ10.00(s,1H),8.12(d,J＝7.5Hz,1H),7.95-7.86(m,2H),7.74(d,J＝7.4Hz,2H),7.68-7.57(m,3H),7.53-7.14(m,20H),5.16(t,J＝5.3Hz,1H),4.47(dd,J＝16.1,5.9Hz,3H),4.28(dd,J＝24.2,6.7Hz,3H),3.72(s,2H),2.52(d,J＝7.4Hz,1H),1.97(s,2H),1.80-1.20(m,6H).

Fmoc-Gly-Lys (Trt) -PAB (40 g,51.7 mmol) and DCM (400 mL) are sequentially added into a 1L single-port bottle, after stirring and dissolution, TEA (15.6 g,155.1 mmol) and bis (4-nitrophenyl) carbonate (47.1 g,155.1 mmol) are added dropwise at room temperature, stirring and reaction are carried out for 2 hours at room temperature, water (400 mL) is added for extraction, saturated saline washing, anhydrous sodium sulfate drying, spin drying and silica gel column chromatography purification are carried out, and Fmoc-Gly-Lys (Trt) -PAB-PNP (17.5 g, yield 36% and HPLC 95.7%) are obtained; LCMS: [ M+1] ⁺ 938.62 (calculated 937.37);¹H NMR(500MHz,DMSO-d₆)δ10.16(s,1H),8.35(d,J＝9.1Hz,2H),8.20-8.10(m,2H),7.92(d,J＝7.5Hz,2H),7.80-7.55(m,7H),7.50-7.14(m,20H),5.29(s,2H),4.50-4.40(m,1H),4.34-4.22(m,3H),3.77-3.68(m,2H),2.00-1.85(m,2H),1.79-1.25(m,7H).

Sequentially adding D1(3.7g,7.41mmol),NMP(37mL),DIPEA(2.87g,22.22mmol),Fmoc-Gly-Lys(Trt)-PAB-PNP(6.95g,7.41mmol),HOBt(1.05g,7.77mmol), to a 50ml single-port bottle, stirring at room temperature for reaction for 2 hours, washing the reaction solution with 50ml of 2% citric acid aqueous solution, washing the reaction solution with 50ml of saturated sodium bicarbonate solution, washing the organic phase with 50ml of saturated saline solution, drying with anhydrous sodium sulfate, concentrating to obtain a crude L1-D1-1 product, and directly putting the crude L1-D1 product into the next reaction; LCMS: [ M+1] ⁺ 1298.74 (calculated 1297.50);¹H NMR(600MHz,DMSO-d₆)δ10.06(d,J＝23.4Hz,1H),8.11(d,J＝7.8Hz,1H),7.86(d,J＝7.7Hz,1H),7.93-7.47(m,6H),7.62-7.27(m,10H),7.27(dt,J＝36.3,7.0Hz,7H),7.27-7.06(m,3H),7.12(t,J＝8.3Hz,3H),6.49(s,1H),6.34-6.04(m,3H),5.55-5.32(m,2H),5.31-4.86(m,4H),4.59-4.32(m,1H),4.47-3.99(m,3H),3.82(t,J＝14.7Hz,2H),3.87-3.43(m,4H),3.29(dq,J＝15.9,11.6,9.5Hz,2H),2.47(d,J＝6.5Hz,1H),1.89(ddq,J＝28.6,14.1,8.1,7.1Hz,4H),1.76-1.60(m,1H),1.56(d,J＝9.3Hz,1H),1.47(t,J＝7.3Hz,2H),1.36(d,J＝11.5Hz,1H),1.28(s,1H),0.89(t,J＝7.3Hz,3H).

DCM (50 mL) was added to the above flask (L1-D1-1), DBU (582.8 mg) was added dropwise after stirring and dissolution, the reaction was stirred at room temperature for 1h, methyl tert-butyl ether (400 mL) was added, filtration was carried out, the solid was dissolved again with DCM (40 mL), dropwise added to methyl tert-butyl ether (400 mL), filtration was carried out, and drying was carried out, to give L1-D1-2 (7 g, yield 87.5%); LCMS: [ M+1] ⁺ 1076.38 (calculated 1075.43);¹H NMR(600MHz,DMSO-d₆)δ10.32(s,1H),8.08-7.44(m,3H),7.57-7.24(m,8H),7.39-6.93(m,13H),6.35-5.97(m,3H),5.53-4.81(m,6H),4.53-4.19(m,1H),3.79(dt,J＝21.3,13.7Hz,2H),3.67-2.92(m,10H),2.66–2.58(m,1H),2.50-2.44(m,1H),2.03-1.65(m,6H),1.75-1.38(m,8H),1.29(dd,J＝31.5,16.8Hz,2H),1.10(s,1H),0.88(t,J＝7.3Hz,3H).

In a 250ml single-port bottle, L1-D1-2 (7 g,6.5 mmol) and 70ml DCM are sequentially added, after stirring and dissolution, fmoc-NH-PEG ₂-CH₂CH₂CO₂ H (2.6 g,6.5 mmol), HATU (3.7 g,9.7 mmol) and DIPEA (1.3 g,10 mmol) are added, stirring and reacting for 1H at room temperature, adding 100ml water for quenching reaction, separating liquid, washing an organic phase once again with saturated sodium chloride (100 ml), concentrating the organic phase, and purifying by silica gel column chromatography to obtain L1-D1-3 (5.2 g, yield 54.7%); LCMS: [ M+1] ⁺ 1457.93 (calculated 1456.59);¹H NMR(600MHz,DMSO-d₆)δ9.99(s,2H),8.24-7.94(m,2H),7.94-7.72(m,2H),7.80-7.55(m,2H),7.69-7.44(m,2H),7.55-7.31(m,5H),7.44-7.14(m,14H),7.14(t,J＝8.2Hz,1H),6.49(d,J＝3.2Hz,1H),6.36-6.19(m,2H),5.55-5.39(m,3H),5.25(d,J＝44.7Hz,2H),5.09(s,1H),5.04-4.88(m,1H),4.63(s,1H),4.36(s,1H),4.48-3.93(m,4H),3.92-3.73(m,2H),3.81-3.60(m,2H),3.60(ddd,J＝45.2,15.2,7.9Hz,6H),3.41(dd,J＝49.8,5.7Hz,5H),3.11(p,J＝5.8Hz,2H),2.72(s,1H),2.37(td,J＝6.6,3.3Hz,2H),1.87(dtq,J＝21.5,14.5,7.1Hz,3H),1.69(s,2H),1.50(d,J＝40.9Hz,2H),1.38-1.15(m,6H),1.05(d,J＝6.4Hz,2H),1.00(d,J＝6.5Hz,9H),0.87(t,J＝7.4Hz,3H).

L1-D1-3 (5.1 g,3.5 mmol) and DCM (30 mL) are sequentially added into a 100mL single-port bottle, DBU (266.3 mg) is added dropwise after stirring and dissolution, methyl tert-butyl ether (300 mL) is added for stirring and reaction at room temperature for 30min, filter cake is filtered, and the filter cake is washed with the methyl tert-butyl ether and dried to obtain L1-D1-4 (3.2 g, yield 74%); LCMS: [ M+1] ⁺ 1235.77 (calculated 1234.52).

L1-D1-4 (3.2 g,2.6 mmol) and DCM (19 mL) are sequentially added into a 100mL single-port bottle, bromoacetic anhydride (640 mg,2.6 mmol) is added after stirring and dissolution, the reaction is stirred at room temperature for 1h, the reaction solution is concentrated, and the silica gel column chromatography is purified to obtain L1-D1-5 (2.8 g, yield 80% and HPLC 97.3%); LCMS: [ M+1] ⁺ 1355.64 (calculated 1354.44);¹H NMR(600MHz,DMSO-d₆)δ10.00(s,1H),9.51(s,1H),8.33(d,J＝5.6Hz,1H),8.21-8.00(m,2H),7.71–7.42(m,3H),7.56-7.04(m,21H),6.63-6.41(m,1H),6.32-6.14(m,3H),5.43(s,2H),5.27(d,J＝42.2Hz,2H),5.10(s,1H),5.04-4.85(m,2H),4.54(d,J＝5.1Hz,1H),4.46-4.15(m,2H),3.93-3.13(m,23H),2.81-2.56(m,1H),2.46-2.28(m,2H),2.09-1.73(m,4H),1.81-1.45(m,6H),1.48(d,J＝9.5Hz,2H),1.40-1.18(m,1H),1.11(s,14H),0.88(t,J＝7.3Hz,3H).

In a 100mL single-port flask, L1-D1-5 (3.1 g,2.3 mmol) and DCM (60 mL) were sequentially added, after stirring and dissolution, TFA (to 5% concentration of the reaction solution) was added dropwise, stirring and reacting at room temperature for 1h, methyl tert-butyl ether (600 mL) was added, filtration, washing, and DCM (10 mL) was added again, stirring was performed, methyl tert-butyl ether (100 mL) was added again, stirring was performed for 20min, filtration, washing, and vacuum drying to obtain a white solid product L1-D1 (1.0 g, 40% yield, HPLC 97%; LCMS: [ M+1] ⁺ 1113.51 (calculated value) 1113.34);¹H NMR(500MHz,DMSO-d₆)δ10.03(d,J＝16.7Hz,1H),8.34(q,J＝5.1Hz,1H),8.23-8.10(m,2H),7.86-7.60(m,3H),7.72-7.40(m,3H),7.33(d,J＝8.2Hz,1H),7.38-6.98(m,2H),6.49(s,1H),6.27(dd,J＝13.7,3.9Hz,2H),5.43(d,J＝4.2Hz,2H),5.33-5.18(m,2H),5.10(s,1H),5.03-4.91(m,1H),4.51-4.20(m,1H),4.36-3.88(m,5H),3.93-3.11(m,16H),2.78(p,J＝7.3,6.7Hz,2H),2.40(q,J＝5.6Hz,2H),2.06-1.63(m,3H),1.58(ddt,J＝45.5,14.8,6.1Hz,3H),1.53-1.20(m,2H),0.88(t,J＝7.2Hz,3H);¹³C NMR(151MHz,DMSO)δ173.03,171.26,171.01,169.51,166.57,158.67,158.45,157.32,156.03,155.85,151.37,150.58,149.91,149.56,147.57,146.75,139.23,139.11,133.26,127.13,125.08,119.14,115.33,104.56,99.64,96.43,72.88,69.92,69.17,67.15,65.73,61.82,53.56,50.11,49.50,48.21,42.54,39.15,38.32,36.31,31.92,30.68,29.92,29.04,28.45,27.17,22.81,8.25.

1.2L1-D1-Ab _nectin-4 antibody conjugated drug (ADC 1)

Nectin4 antibody (10.0 mg/mL,10mg,0.066 mmol) was taken, pH adjusted to 7.2 with 1M Na ₂HPO₄ solution, then 0.1M disodium ethylenediamine tetraacetate solution (25. Mu.L) was added, the prepared TCEP & HCl solution (10 mM,0.04 mL) was added, and the reaction was carried out at room temperature for 3 hours with a rotating turntable at 25 ℃.

Compound L1-D1 (0.89 mg,0.80 mmol) was dissolved in 0.09ml of DMA, added to the above solution system, mixed well, reacted at room temperature with a rotating disk for 16h, after the reaction was completed, small molecules were removed by NAP-5 gel column (Cytiva) and the buffer was replaced with 20mM PB solution, pH=6.3, to give antibody-coupled drug ADC1 (3.1 mg/ml,2 ml).

RP-MS (RP-HPLC-MS) calculation of the average value: n=7.7; MS results show that the antibody light chain (L) is linked to one L1-D1 (linker-payload) and the heavy chain (H) is linked to 3L 1-D1 (linker-payload) (FIG. 1).

Example 2: ab _nectin-4 - (S-7- (N- (acetamide-PEG ₂ -propionyl-Gly-Lys-PABC) -N- ((R) -3-tetrahydrofuran)) aminoethyl camptothecin) ₈ (ADC 2)

2.1 7- (N- (bromoacetamide-PEG ₂ -propionyl-Gly-Lys-PABC) -N- ((R) -3-tetrahydrofuran)) aminoethyl camptothecin (L1-D2)

Intermediate I-1 (18 g,68.12 mmol) was added to IPA (180 mL), the reaction mixture was stirred, DIEA (17.61 g,136.24 mmol) and (R) -3-aminotetrahydrofuran hydrochloride (33.67 g,272.48 mmol) were then added, the reaction mixture was concentrated to a residual of about 20mL of IPA, 200mL of water was added and stirred at ambient temperature for 2 hours, and the yellow solid obtained by filtration was dried at 40℃to give product D2-1 (16.5 g, yield 79%, HPLC 98%); LCMS: [ M+1] ⁺ 307.32 (calculated 306.09);¹H NMR(500MHz,DMSO-d₆)δ7.58(s,1H),7.34-7.19(m,1H),7.07(s,1H),6.25(s,2H),5.33(d,J＝11.3Hz,1H),4.02(s,1H),3.83-3.53(m,5H),2.23-2.05(m,1H),1.85-1.65(m,1H).

Compound D2-1 (16.5 g,53.87 mmol) was added to DCM (310 mL), acetic acid (64.70 g,1.08 mol) was added thereto, and stirred to dissolve, the internal temperature of the reaction solution was kept at 15 degrees celsius, sodium borohydride acetate (28.54 g,134.68 mmol) was added in 4 batches, approximately 5 minutes apart, each time at about 7 g, the internal temperature of the reaction solution was kept at 15 degrees celsius for 2 hours, and the reaction solution was poured into ice-cold aqueous sodium carbonate solution (sodium carbonate: 130g, ice water: 1L), controlling the internal temperature to 15 ℃, stirring, layering, washing an organic phase with water, collecting the organic phase and drying the organic phase with anhydrous sodium sulfate to obtain a DCM solution of a compound D2-2, adding DIEA (13.93 g,107.76 mmol), then dropwise adding Cbz-Cl (10.11 g,59.27 mmol), after 20 minutes, reacting for 12 hours with cold cutting, adding the mixture into a 10% citric acid aqueous solution (200 mL of x 2), washing the mixture twice, washing the mixture with a saturated sodium bicarbonate solution (200 mL) in sequence, washing the saturated saline solution (200 mL), drying the organic phase with anhydrous sodium sulfate, and concentrating the organic phase to obtain a compound D2-3 (20.5 g, yield 86%); LCMS: [ M+1] ⁺ 443.42 (calculated 442.43);¹H NMR(500MHz,DMSO-d₆)δ7.65(s,1H),7.39-7.27(m,6H),6.28(s,2H),5.10(s,2H),4.65-4.50(m,1H),4.00-3.80(m,1H),3.77-3.57(m,5H),3.16-3.05(m,2H),2.24-2.10(m,1H),2.00-1.88(m,1H).

Compound D2-3 (20.5 g,46.3 mmol) was added to a 1L single-necked flask, DMF (50 mL) was added, and the mixture was cooled to 0 ℃ in an ice bath under nitrogen, and the prepared reducing solution was slowly dropped: h ₂ O (250 mL)/sodium dithionite (40.33 g,231.68 mmol)/sodium carbonate (19.64 g,185.34 mmol), slowly heating to 40 ℃ after dripping, continuously stirring and reacting for 2H, filtering the reaction solution, concentrating the filtrate, adding ethyl acetate (500 mL) and water (500 mL), extracting and separating liquid, drying with anhydrous sodium sulfate, concentrating, purifying by silica gel column chromatography to obtain yellow oily substance D2-4 (17.9 g, yield 93.7%, HPLC 98%); LCMS: [ M+1] ⁺ 413.42.413 (calculated 412.44);¹H NMR(500MHz,DMSO-d₆)δ7.45-7.25(m,7H),5.94(s,2H),5.08(s,2H),4.60-4.45(m,1H),3.90-3.80(m,1H),3.77-3.41(m,5H),3.05(t,J＝7.6Hz,2H),2.19-2.10(m,1H),1.87(d,J＝7.9Hz,1H).

To a 500mL single port flask, compound D2-4 (17.9 g,43.4 mmol), (S) -4-ethyl-4-hydroxy-7, 8-dihydro-1H-pyrano [3,4-F ] indolizin-3, 6,10 (4H) -one (12.57 g,47.74 mmol) and PPTS (218.13 g,868.00 mmol) were added sequentially, and the reaction mixture was stirred under argon for 12H at 110℃and poured into methanol (1000 mL), stirred for 2H, filtered to give a dark brown solid, methanol (300 mL) was added and stirred for 2H, filtered to give a pale brown solid, and the solid was dried at 40℃to give compound D2-5 (10.0 g, yield 36%, HPLC 95%): LCMS: [ M+1] ⁺ 640.72 (calculated 639.66);¹H NMR(600MHz,DMSO-d₆)δ7.82-7.14(m,8H),6.49(s,1H),6.29(s,2H),5.53-5.03(m,5H),4.60(s,1H),4.08-3.23(m,9H),2.25-1.73(m,4H),0.88(s,3H).

Sequentially adding the compound D2-5 (10 g,15.63 mmol) into a 10L single-port bottle, adding dichloromethane (2.5L), adding methanol (2.5L), adding 10% Pd/C (15 g) into the reaction solution, reacting for 20h at normal temperature under a hydrogen balloon atmosphere, filtering to remove Pd/C, spinning the reaction solution, stirring for 24h with a mixed solution of dichloromethane and methanol (30 mL, V/V=1:1), filtering, and drying the wet product at 40 ℃ for 3h to obtain a product D2 (6.0 g, yield 76%); LCMS: [ M+1] ⁺ 506.52 (calculated 505.53);¹H NMR(600MHz,DMSO-d₆)δ7.52(s,1H),7.40(s,1H),7.16(s,1H),6.52(s,1H),6.26(d,J＝7.3Hz,2H),5.48-5.35(m,2H),5.11-4.95(m,2H),3.95-3.62(m,5H),3.36-3.26(m,2H),3.14-3.03(m,2H),2.20-2.04(m,2H),1.96-1.80(m,2H),0.89(t,J＝7.3Hz,3H).

Sequentially adding D2(7.6g,15.03mmol),NMP(140mL),DIPEA(5.83g,45.1mmol),Fmoc-Gly-Lys(Trt)-PAB-PNP(12.69g,13.53mmol),HOBt(2.23g,16.54mmol), reaction solution into a 250mL single-port bottle, stirring at room temperature for reaction for 2 hours, adding dichloromethane (300 mL), washing with 2% citric acid aqueous solution, washing with saturated sodium bicarbonate aqueous solution (100 mL), washing with saturated saline, drying with anhydrous sodium sulfate, and concentrating to obtain L1-D2-1; LCMS: [ M+1] ⁺ 1304.93 (calculated :1303.53);¹H NMR(600MHz,DMSO-d₆)δ10.06(s,1H),8.10(d,J＝7.9Hz,1H),7.92-7.73(m,2H),7.71-7.51(m,4H),7.44-7.18(m,18H),7.12(t,J＝7.1Hz,3H),6.49(s,1H),6.31-6.20(m,2H),5.42(d,J＝3.5Hz,2H),5.23(s,2H),5.14(s,2H),4.58(s,1H),4.41(s,1H),4.27-4.17(m,2H),3.94(dd,J＝40.6,15.3Hz,1H),3.73-3.42(m,5H),3.30(t,J＝7.1Hz,2H),3.07(d,J＝0.8Hz,4H),2.69(d,J＝1.0Hz,2H),2.47(s,1H),2.21-2.08(m,2H),1.94-1.79(m,4H),1.72-1.39(m,3H),1.39-1.22(m,2H),1.10(d,J＝0.8Hz,11H),0.88(t,J＝7.3Hz,3H).

In a 250mL single-port bottle, adding L1-D2-1 (the product) and DCM (100 mL), stirring and dissolving, then dropwise adding DBU (3.61 g,23.7 mmol), stirring and reacting at room temperature for 1h, pouring into methyl tert-butyl ether (1L), filtering and separating out solid, dissolving DCM (80 mL), then dropwise adding into 1L methyl tert-butyl ether, filtering and drying to obtain L1-D2-2 (26 g); LCMS: [ M+1] ⁺ 1082.68 (calculated 1081.47);¹H NMR(600MHz,DMSO-d₆)δ10.30(d,J＝65.6Hz,1H),7.82(d,J＝8.3Hz,1H),7.66-7.59(m,1H),7.50-7.45(m,1H),7.41-7.34(m,9H),7.31-7.20(m,8H),7.14(t,J＝7.1Hz,4H),6.70-6.63(m,1H),6.37-6.05(m,2H),5.44-5.38(m,2H),5.22(s,3H),5.13(s,2H),4.58(s,1H),4.43(d,J＝9.2Hz,1H),4.27(s,1H),4.08-3.77(m,1H),3.76-3.52(m,2H),3.50-3.31(m,5H),3.19(t,J＝5.8Hz,2H),3.10(s,1H),2.89(s,1H),2.73(d,J＝0.7Hz,1H),2.63-2.57(m,1H),2.19-2.05(m,1H),1.99-1.70(m,6H),1.58(ddd,J＝34.2,9.8,5.4Hz,7H),1.46(d,J＝7.7Hz,2H),0.92-0.83(m,3H).

In a 1L single-port bottle, L1-D2-2 (26 g crude product) and DCM (400 mL) are sequentially added, after stirring and dissolution, fmoc-NH-PEG ₂-CH₂CH₂ COOH (6.2 g,15.6 mmol), HATU (13.7 g,36.0 mmol) and DIPEA (4.6 g,36.0 mmol) are added, after the addition, stirring and reaction are carried out for 1h at room temperature, 80mL water quenching reaction, DCM extraction, saturated saline washing of an organic phase, drying of the organic phase with anhydrous sodium sulfate and silica gel column chromatography purification are carried out, thus obtaining L1-D2-3 (11.0 g, three-step total yield of 55.5% and HPLC 96%); LCMS: [ M+1] ⁺ 1463.78 (calculated 1462.62);¹H NMR(600MHz,DMSO-d₆)δ10.01(s,1H),8.17-8.04(m,2H),7.85(d,J＝7.5Hz,2H),7.65(dd,J＝20.1,7.9Hz,4H),7.53-7.18(m,20H),7.14(q,J＝7.5,5.7Hz,3H),6.49(s,1H),6.26(d,J＝16.8Hz,2H),5.42(d,J＝3.2Hz,2H),5.23(s,2H),5.13(s,2H),4.58(s,1H),4.38(q,J＝7.6Hz,1H),4.26(d,J＝7.1Hz,2H),4.17(t,J＝6.9Hz,1H),3.97(s,0H),3.90(d,J＝17.7Hz,0H),3.71(dd,J＝31.9,4.9Hz,3H),3.60(dt,J＝21.8,6.7Hz,4H),3.45(s,4H),3.37(t,J＝6.0Hz,2H),3.27(s,2H),3.11(p,J＝6.7,6.0Hz,2H),2.36(t,J＝6.5Hz,2H),2.13(dtd,J＝13.3,8.5,5.4Hz,1H),1.87(ddt,J＝36.3,21.3,7.1Hz,4H),1.66(s,1H),1.53(d,J＝10.0Hz,1H),1.46(q,J＝7.2Hz,2H),1.39-1.20(m,3H),0.87(t,J＝7.3Hz,3H).

L1-D2-3 (5 g,3.42 mmol) and DCM (40 mL) are sequentially added into a 100mL single-port bottle, DBU (284 mg,2.39 mmol) is dropwise added after stirring and dissolution, the reaction is stirred at room temperature for 30min, the reaction solution is added into methyl tertiary butyl ether (500 mL), solid is filtered and separated out, the solid is dissolved again by DCM (50 mL), the solution is dropwise added into methyl tertiary butyl ether (600 mL), the solution is filtered, a filter cake is washed by methyl tertiary ether, and L1-D2-4 (2.9 g, yield 69%) is obtained after drying; LCMS: [ M+1] ⁺ 1241.87 (calculated 1240.55);¹H NMR(600MHz,DMSO-d₆)δ10.03(s,1H),8.14(t,J＝5.7Hz,1H),8.09(d,J＝7.8Hz,1H),7.63(d,J＝8.3Hz,2H),7.48(s,1H),7.41(s,1H),7.37(d,J＝7.9Hz,7H),7.28-7.19(m,7H),7.14(t,J＝7.3Hz,3H),6.49(s,1H),6.27(d,J＝11.9Hz,2H),5.48-5.37(m,2H),5.24(s,2H),5.14(s,2H),4.58(t,J＝7.5Hz,1H),4.37(q,J＝7.3Hz,1H),3.89(s,1H),3.71(dd,J＝24.3,7.2Hz,3H),3.60(dt,J＝22.9,7.1Hz,4H),3.46(s,4H),3.36(t,J＝5.7Hz,2H),2.67(t,J＝5.7Hz,2H),2.37(t,J＝6.5Hz,2H),2.13(dtd,J＝13.2,8.6,5.1Hz,1H),1.88(dtd,J＝43.1,13.7,13.2,6.6Hz,4H),1.66(s,1H),1.50(dp,J＝37.5,7.7Hz,4H),1.39-1.14(m,5H),0.88(t,J＝7.3Hz,3H).

L1-D2-4 (2.9 g,2.3 mmol) and DCM (25 mL) are sequentially added into a 100mL single-port bottle, bromoacetic anhydride (667.8 mg,2.57 mmol) is added after stirring and dissolution, the reaction is stirred at room temperature for 30min, the reaction solution is concentrated and purified by silica gel column chromatography to obtain L1-D2-5 (2.0 g, yield 64.5% and HPLC 97.6%); LCMS: [ M+1] ⁺ 1361.93 (calculated 1360.48);¹H NMR(600MHz,DMSO-d₆)δ10.01(s,1H),8.32(t,J＝5.6Hz,1H),8.11(dd,J＝24.1,6.7Hz,2H),7.62(d,J＝8.1Hz,2H),7.55-7.20(m,17H),7.14(s,2H),6.49(s,1H),6.26(s,2H),5.42(d,J＝2.8Hz,2H),5.24(s,2H),5.14(s,2H),4.58(p,J＝6.4,5.8Hz,1H),4.40-4.34(m,1H),3.96-3.86(m,0H),3.84(s,2H),3.71(dd,J＝22.2,5.6Hz,3H),3.58(t,J＝6.5Hz,3H),3.47(s,5H),3.43-3.31(m,5H),3.21(q,J＝5.8Hz,2H),2.37(t,J＝6.5Hz,2H),2.14(dtd,J＝13.4,8.7,5.3Hz,1H),1.87(dtd,J＝28.6,15.1,14.2,7.9Hz,4H),1.66(td,J＝12.2,6.1Hz,2H),1.51(d,J＝42.3Hz,3H),1.35-1.24(m,5H),0.91-0.81(m,3H).

L1-D2-5 (1.3 g,0.95 mmol) and DCM (10 mL) are sequentially added into a 100mL single-port bottle, after stirring and dissolution, TFA (0.78 mL) is dropwise added, stirring and reacting for 2h at room temperature, the reaction solution is poured into methyl tertiary butyl ether (130 mL), solid is filtered and separated out, DCM (10 mL) is added into the solid and stirred, and then the solid is poured into methyl tertiary butyl ether (100 mL), stirred for 20min, filtered, washed and dried in vacuo to obtain white solid L1-D2 (870 mg, yield 81.7% and HPLC 97%); LCMS: [ M+1] ⁺ 1119.64 (calculated 1118.36);¹H NMR(500MHz,DMSO-d₆)δ10.05(s,1H),8.34(s,1H),8.26-8.09(m,2H),7.73(s,3H),7.65(d,J＝8.2Hz,2H),7.47(s,1H),7.40(s,2H),7.22(s,1H),6.49(s,1H),6.27(s,2H),5.43(s,2H),5.30-5.05(m,4H),4.65-4.55(m,1H),4.45-4.35(m,1H),3.89-3.57(m,16H),3.44-3.18(m,6H),2.85-2.70(m,2H),2.40(t,J＝6.3Hz,2H),2.20-2.10(m,1H),1.96-1.70(m,4H),1.68-1.25(m,5H),0.89(t,J＝7.3Hz,3H).

2.2L1-D2-Ab _nectin-4 antibody conjugated drug (ADC 2)

Nectin4 antibody (10.0 mg/mL,10mg,0.066 mmol) was taken, pH adjusted to 7.2 with 1M Na ₂HPO₄ solution, then 0.1M disodium ethylenediamine tetraacetate solution (25. Mu.L) was added, and the prepared TCEP & HCl (tris (2-carboxyethyl) phosphine hydrochloride) solution (10 mM,0.04 mL) was added and the reaction was carried out at 25℃for 3 hours with rotating the turntable at room temperature.

Compound L1-D2 (0.90 mg,0.80 mmol) was dissolved in 0.09ml of DMA, added to the above solution system, mixed well, reacted at room temperature with a rotating disk for 16h, after the reaction was completed, small molecules were removed by NAP-5 gel column (Cytiva) and the buffer was replaced with 20mM PB solution, pH=6.3, to give antibody-coupled drug ADC2 (3.1 mg/ml,2 ml).

RP-MS calculates the average: n=7.9; MS results show that the antibody light chain (L) is linked to one L1-D2 (linker-payload) and the heavy chain (H) is linked to 3L 1-D2 (linker-payload) (FIG. 2).

2.3 7- (N- (bromoacetamide-PEG ₈ -propionyl-Gly-Lys-PABC) -N- ((R) -3-tetrahydrofuran)) aminoethyl camptothecin (L2-D2)

Sequentially adding L1-D2-2(1.0g,924.02μmol),DMF(10mL),Fmoc-PEG₈-OSu(703.03mg,924.02μmol),DIPEA(116.11mg,924.02μmol,156μL), to a 100ml single-port bottle at room temperature, stirring at room temperature for reaction for 1hr, concentrating, and purifying by silica gel column chromatography to obtain yellow solid L2-D2-1 (1.26 g, yield 78.25%); LCMS: [ M+1] ⁺ 1728.85 (calculated 1727.97).

L2-D2-1 (1.26 g, 729.59. Mu. Mol), DMF (10 mL), piperidine (861.9 mg,10.14mmol,1.0 mL) were added sequentially to a 10mL single-neck eggplant bottle at room temperature, stirred at room temperature for 20min, concentrated, and purified by silica gel column chromatography to give L2-D2-2 as a pale yellow solid (960 mg, yield 87.27%); LCMS: [ M+1] ⁺ 1506.38 (calculated 1505.73).

L2-D2-2 (960 mg, 637.57. Mu. Mol), DMF (10 mL), bromoacetic anhydride (165.69 mg, 637.57. Mu. Mol) were added sequentially to a 10mL single-necked flask at room temperature, stirred at room temperature for 30min, concentrated, and purified by silica gel column chromatography to give L2-D2-3 as a pale yellow solid (440 mg yield 42.31%); LCMS: [ M+1] ⁺ 1627.51 (calculated 1626.66).

L2-D2-3 (440 mg, 270.61. Mu. Mol), DCM (5 mL), TFA (744.52 mg,6.53mmol, 500. Mu.L) were sequentially added to a 10mL single-neck eggplant-type bottle at room temperature, the reaction was stirred at room temperature for 2hr, 10mL of diethyl ether was added dropwise to the reaction solution, the mixture was centrifuged (10000 rpm/5 min), the supernatant was removed, and the solid was distilled off with spin to give L2-D2 as a yellow solid powder (350 mg, 93.58% yield, 97% by HPLC); LCMS: [ M+1] ⁺ 1385.22 (calculated 1384.34);¹H NMR(600MHz,DMSO-d₆)δ10.04(s,1H),8.35(t,J＝5.7Hz,1H),8.19(t,J＝5.7Hz,1H),8.15(d,J＝8.0Hz,1H),7.72(d,J＝5.8Hz,3H),7.65(d,J＝8.1Hz,2H),7.49(s,1H),7.39(s,2H),7.23(s,1H),6.28(s,2H),5.47-5.38(m,2H),5.22(s,2H),5.14(s,2H),4.58(p,J＝5.8,5.4Hz,1H),4.41(q,J＝7.7,7.2Hz,1H),3.93(s,1H),3.89(s,1H),3.86(s,2H),3.81-3.68(m,4H),3.67-3.56(m,5H),3.53-3.48(m,19H),3.46-3.35(m,3H),3.25(dq,J＝17.2,5.4Hz,3H),2.78(q,J＝6.9,6.3Hz,2H),2.40(dt,J＝8.2,4.1Hz,2H),2.14(dtt,J＝13.2,8.7,5.5Hz,1H),1.87(dh,J＝21.2,7.3Hz,2H),1.76(ddt,J＝16.4,10.4,5.3Hz,1H),1.62(qd,J＝13.3,11.3,6.8Hz,1H),1.55(dp,J＝14.0,6.6Hz,2H),1.39(td,J＝8.9,5.4Hz,1H),1.36-1.29(m,1H),1.09(t,J＝7.0Hz,2H),0.89(t,J＝7.3Hz,3H).

Example 3: ab _nectin-4 - (S-7- (N- (acetamide-PEG ₂ -propionyl-Gly-Lys-PAB (3-bis-PEG ₄ -carbonyl) C) -N- ((R) -3-tetrahydrofuran)) aminoethyl camptothecin) ₈ (ADC 3)

3.1 7- (N- (bromoacetamide-PEG ₂ -propionyl-Gly-Lys-PAB (3-bis-PEG ₄ -carbonyl) C) -N- ((R) -3-tetrahydrofuran)) aminoethyl camptothecin (L3-D2)

6-Amino-3H-isobenzofuran-1-one (4.0 g,26.82 mmol), acetonitrile (50 mL), imidazole-1-sulfonylazide hydrochloride (6.75 g,32.18 mmol), cuSO ₄ (5.14 g,32.18 mmol) and potassium carbonate (37.07 mg, 268.19. Mu. Mol) were added to the reaction flask, the reaction was stirred at room temperature for 16H, the solids were filtered off, the filtrate was concentrated, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give intermediate I-2 (2.0 g, 38% yield) which was used directly in the next reaction without purification.

To the reaction flask were added I-2 (6.9 g,39.40 mmol), THF (50 mL), water (50 mL) and NaOH (2.36 g,59.09 mmol), and the reaction mixture was stirred at 70℃for 2h, concentrated under reduced pressure, acidified with 1N HCl, filtered to precipitate a white solid, and dried to give 5-azido-2-hydroxymethylbenzoic acid I-3 (7.3 g, 91% yield).

I-3 (7.0 g,36.24 mmol), DMF (150 mL), imidazole (12.34 g,181.20 mmol) and TBDMS-Cl (13.66 g,90.60 mmol) were added to the reaction flask at 0deg.C, the mixture was stirred at room temperature overnight, poured into water, the solid was filtered off, washed with water, and dried to give the product 5-azido-2- (TBDMSO) methylbenzoic acid I-4 (10 g, yield) as an off-white solid 88％);¹H NMR(600MHz,CDCl₃)δ7.68-7.51(m,2H),7.10(dd,J＝8.4,2.6Hz,1H),4.92(d,J＝0.9Hz,2H),0.82(s,9H),0.00(s,6H).

N, N-bis (methoxyethoxyethoxyethoxyethyl) amine or (Me-PEG ₄)₂ NH: methoxyethoxyethoxyethoxyethanol (40 g,191.2 mmol), triethylamine (58 g,567.8 mmol) and p-toluenesulfonyl chloride (43.7 g,228.2 mmol) and DCM (400 mL) were added to the reaction flask, the reaction mixture was stirred at room temperature for 3H, concentrated under reduced pressure, and purified by silica gel column chromatography to give methoxyethoxyethoxyethoxyethanol p-toluenesulfonate (61.5 g, 88% yield) as an oil; LCMS [ m+h ] ⁺ 363.50 (calculated 362.44): methoxy ethoxy ethanol p-toluene sulfonate (58.5 g,161 mmol), benzylamine (8.7 g,80.5 mmol), K ₂CO₃ (33.4 g,241.4 mmol) were added separately to a reaction flask containing acetonitrile (500 mL), the reaction mixture was stirred and heated to 70 ℃ for 16H, filtered, the filtrate was concentrated, purified by silica gel column chromatography to give N-benzyl-N, N-bis (methoxyethoxy ethoxy ethyl) amine (23.1 g, 58.7%) LCMS [ m+h ] ⁺ 488.54 (calculated 487.63): N-benzyl-N, N-bis (methoxyethoxy ethoxy ethyl) amine (10 g,20.5 mmol), methanol (100 mL) and 10% Pd-C were added to the reaction flask, stirred at room temperature under hydrogen balloon atmosphere for 16H, filtered, the filtrate was concentrated to give the oily product N, N-bis (methoxyethoxy ethoxy ethyl) amine, (7.9 g PEG- ₄)₂, yield 97%); LCMS: [ M+H ] ⁺ 398.31 (calculated 397.27).

I-4(10.5g,34.16mmol),(Me-PEG₄)₂NH(14.93g,37.57mmol),DCM(250mL),HATU(19.48g,51.23mmol) And triethylamine (17.28 g,170.78mmol,23.80 mL) were added to the reaction flask, the reaction mixture was stirred at room temperature for 4h, water was added, DCM was used for extraction, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to give the colorless liquid product 5-azido-2- (TBDMSO) methylbenzylbis (Me-PEG ₄) amine L3-1 (15 g, yield 59.46%); LCMS: [ M+Na ] ⁺ 709.42 (calculated 686.39);¹H NMR(500MHz,CDCl₃)δ7.44(d,J＝8.4Hz,1H),6.96(dd,J＝8.3,2.4Hz,1H),6.81(d,J＝2.4Hz,1H),4.58(s,2H),3.66(s,3H),3.61-3.49(m,18H),3.48-3.37(m,8H),3.36-3.22(m,8H),0.84(s,9H),0.03(s,6H).

To the reaction flask was added L3-1 (15 g,21.84 mmol), DCM (250 mL) and TBAF (12.21 g,43.67 mmol), stirred at room temperature for 16h, poured into water, extracted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give the colorless liquid product 5-azido-2-hydroxymethylbenzoyl bis (Me-PEG ₄) amine L3-2 (11 g, 83.6% yield); LCMS: [ M+H ] ⁺ 573.32 (calculated 572.31);¹H NMR(500MHz,CDCl₃)δ7.44(d,J＝8.2Hz,1H),7.03(dd,J＝8.2,2.4Hz,1H),6.95(d,J＝2.3Hz,1H),4.55(s,2H),3.81-3.77(m,2H),3.72-3.58(m,21H),3.57-3.48(m,10H),3.37(d,J＝6.6Hz,6H).

L3-2 (11 g,16.01 mmol), meOH (50 mL), DIPEA (2.07 g,16.01 mmol) and 10% Pd-C (1.5 g) were added to the reaction flask, the reaction mixture was stirred under normal pressure with hydrogen balloon for 2h, filtered, and concentrated to give a colorless liquid product, 5-amino-2-hydroxymethylbenzoyl bis (Me-PEG ₄) amine L3-3 (9 g, 93.2% yield); LCMS: [ M+Na ] ⁺ 569.20 (calculated 546.32);¹H NMR(500MHz,DMSO-d₆)δ7.13(d,J＝8.3Hz,1H),6.58(dd,J＝8.2,2.3Hz,1H),6.38(d,J＝2.4Hz,1H),5.13(s,2H),4.70(t,J＝5.5Hz,1H),4.27(d,J＝5.5Hz,2H),3.63(d,J＝5.0Hz,2H),3.61-3.49(m,19H),3.49-3.43(m,8H),3.34(t,J＝6.0Hz,3H),3.27(d,J＝1.0Hz,6H).

Fmoc-Lys (Trt) (365 mg,0.60 mmol), L3-3 (500 mg,0.92 mmol) and DCM (5 mL) were added to a 50mL single-port flask, EEDQ (222 mg,0.90 mmol) was added to the flask under ice-bath cooling, stirring was continued for 12h under ice-bath, poured into saturated sodium bicarbonate solution, extracted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated to give crude L3-4, which was directly put into the next reaction; LCMS: [ M+1] ⁺ 1139.61 (calculated 1138.59).

In a 50mL single-port bottle, adding the L3-4 (about 1g crude product) into DCM (6 mL), adding DBU (65 mg,0.43 mmol) under stirring, reacting at room temperature for 30min, directly spin-drying the reaction solution to obtain the L3-5 crude product (about 900 mg) for direct casting; LCMS: [ M+H ] ⁺ 917.91 (calculated 916.52).

In a 50mL single-port flask, L3-5 (. About.900 mg crude product) was dissolved in DCM (10 mL), PPTS (108 mg,0.43 mmol) was added under ice bath, fmoc-glycine (108 mg,0.36 mmol), EDCI (173 mg,0.90 mmol), HOBt (120 mg,0.89 mmol) was reacted for 2h with stirring at room temperature, water was added, DCM was extracted, washed with saturated sodium bicarbonate, saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to give L3-6 as a yellow oil (300 mg, three steps total yield 41.8%); LCMS: [ M+1] ⁺ 1196.87 (calculated 1195.61);¹H NMR(600MHz,DMSO-d₆)δ10.04(s,1H),8.10(d,J＝7.8Hz,1H),7.88(d,J＝7.6Hz,1H),7.69(d,J＝7.5Hz,1H),7.54(t,J＝6.1Hz,2H),7.50-7.45(m,1H),7.45-7.35(m,8H),7.32(q,J＝7.5,7.0Hz,2H),7.25(t,J＝7.7Hz,5H),7.14(t,J＝7.4Hz,3H),5.12-5.03(m,1H),4.38(d,J＝5.7Hz,3H),4.30-4.12(m,3H),3.73-3.58(m,5H),3.55(q,J＝2.1Hz,5H),3.56-3.27(m,25H),3.25-3.18(m,8H),1.92(q,J＝7.2Hz,2H),1.72-1.62(m,1H),1.60-1.51(m,1H),1.47(p,J＝7.6Hz,2H),1.38-1.28(m,1H),1.29-1.22(m,3H).

L3-6 (300 mg,0.25 mmol) and DCM (3 mL) were added to a 50mL single-necked flask at room temperature, dissolved with stirring, TEA (75 mg,0.75 mmol) and bis (4-nitrophenyl) carbonate (228 mg,0.75 mmol) were added dropwise, reacted for 2h with stirring at room temperature, poured into water, extracted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, dried over spin, purified by silica gel column chromatography to give L3 (280 mg, 82% yield); LCMS: [ M+1] ⁺ 1361.64 (calculated 1360.62);¹H NMR(600MHz,DMSO-d₆)δ10.20(s,1H),8.35-8.29(m,2H),8.12(dd,J＝8.5,4.2Hz,1H),7.88(d,J＝7.6Hz,1H),7.69(d,J＝7.5Hz,2H),7.62(d,J＝9.5Hz,2H),7.60-7.51(m,3H),7.49(d,J＝8.5Hz,1H),7.44-7.35(m,7H),7.32(q,J＝7.2Hz,2H),7.25(t,J＝7.6Hz,6H),7.14(t,J＝7.4Hz,3H),5.76(s,1H),5.19(s,2H),4.39(q,J＝7.0,6.6Hz,1H),4.30-4.17(m,2H),3.73-3.60(m,4H),3.58(s,2H),3.56-3.48(m,4H),3.50-3.37(m,16H),3.37(tt,J＝7.4,3.4Hz,4H),3.33(s,7H),3.28(s,1H),3.19(d,J＝6.0Hz,5H),2.01-1.88(m,2H),1.67(s,1H),1.56(s,1H),1.46(q,J＝7.3Hz,2H),1.34(d,J＝9.6Hz,1H),1.28(s,1H),1.23(d,J＝4.1Hz,1H).

In a 50mL single-port flask, D2 (126.2 mg,0.25 mmol), NMP (4 mL), DIEA (64.6 mg,0.5 mmol), L3 (340 mg,0.25 mmol), HOBt (37.1 mg,0.27 mmol) were added sequentially, the reaction was stirred at room temperature for 12h, DCM (50 mL) was extracted, washed with saturated sodium bicarbonate (50 mL) and water (50 mL) sequentially, dried over anhydrous sodium sulfate, and concentrated to give L3-D2-1 (crude) which was directly fed to the next step; LCMS: [ M+1] ⁺ 1727.89 (calculated 1726.77);¹H NMR(600MHz,DMSO-d₆)δ10.14(s,1H),8.13(s,1H),7.86(s,2H),7.81(s,1H),7.67(d,J＝7.3Hz,2H),7.60(s,1H),7.53(s,2H),7.45(d,J＝8.3Hz,1H),7.42–7.34(m,7H),7.31(q,J＝8.8,7.2Hz,3H),7.24(t,J＝7.4Hz,7H),7.13(t,J＝7.3Hz,3H),6.49(s,1H),6.28(s,1H),6.12(s,1H),5.42(s,2H),5.28(s,2H),5.06(s,2H),4.60(s,1H),4.39(d,J＝8.7Hz,1H),4.29–4.17(m,2H),3.91(s,1H),3.65(dd,J＝23.2,6.3Hz,2H),3.64–3.53(m,1H),3.59(s,9H),3.56–3.46(m,3H),3.47(d,J＝3.0Hz,1H),3.46–3.42(m,3H),3.42(s,11H),3.39–3.32(m,9H),3.30(s,2H),3.23–3.15(m,5H),2.53–2.45(m,6H),2.14(s,1H),1.88(dq,J＝36.4,14.0,7.2Hz,3H),1.67(s,1H),1.56(d,J＝10.1Hz,1H),1.47(s,2H),1.28(s,1H),1.23(d,J＝4.4Hz,1H),0.87(t,J＝7.3Hz,3H).

In a 50mL single-port bottle, adding L3-D2-1 (crude product) and DCM (5 mL) in sequence, stirring for dissolution, dropwise adding DBU (26.6 mg,10.6 mmol), stirring at room temperature for reaction for 1h, pouring into methyl tertiary butyl ether (50 mL), filtering, and drying to obtain the crude product of L3-D2-2, and directly throwing into the next step; LCMS: [ M+1] ⁺ 1505.96 (calculated 1504.71).

In a 50mL single-port flask, adding crude L3-D2-2 and DCM10 (mL) in sequence, stirring for dissolution, then adding prepared Fmoc-NH-PEG ₂-CH₂CH₂ COOSu (124 mg,0.25 mmol), stirring at room temperature for reaction for 2h, concentrating, purifying by silica gel column chromatography to obtain L3-D2-3 (87 mg, three-step total yield 18%); LCMS: [ M+1] ⁺ 1886.99 (calculated 1885.86);¹H NMR(600MHz,DMSO-d₆)δ10.10(s,1H),8.13(t,J＝6.2Hz,2H),7.90–7.82(m,2H),7.67(d,J＝7.6Hz,2H),7.62(d,J＝10.0Hz,2H),7.47(q,J＝8.5,6.2Hz,1H),7.38(dd,J＝10.8,7.6Hz,7H),7.31(dt,J＝17.4,6.8Hz,3H),7.30–7.20(m,7H),7.15(q,J＝7.3,6.8Hz,3H),6.49(s,1H),6.28(t,J＝3.2Hz,1H),6.13(s,1H),5.42(d,J＝2.7Hz,2H),5.24(d,J＝16.8Hz,2H),5.07(s,2H),4.58(d,J＝28.1Hz,1H),4.37(q,J＝7.4Hz,1H),4.27(d,J＝6.9Hz,2H),4.18(q,J＝8.0,6.9Hz,1H),3.92(d,J＝12.5Hz,1H),3.74(d,J＝5.6Hz,2H),3.72–3.54(m,9H),3.54–3.46(m,2H),3.51–3.41(m,21H),3.38(d,J＝5.0Hz,3H),3.39–3.31(m,12H),3.31(d,J＝17.6Hz,4H),3.27(s,1H),3.19(s,1H),3.11(q,J＝6.1Hz,2H),2.59(s,1H),2.37(t,J＝6.5Hz,2H),2.15(d,J＝14.1Hz,1H),1.97–1.78(m,4H),1.68(t,J＝9.0Hz,1H),1.60–1.53(m,1H),1.48(p,J＝7.4Hz,2H),1.35(s,1H),1.30–1.20(m,3H),0.91–0.81(m,3H).

L3-D2-3 (87 mg,0.046 mmol) and DCM (5 mL) are sequentially added into a 50mL single-port bottle, DBU (4.9 mg) is added dropwise after most of the materials are dissolved under stirring, the mixture is stirred at room temperature for 30min for reaction, the mixture is poured into methyl tertiary butyl ether (60 mL), the mixture is centrifuged, the supernatant is poured, and the mixture is purified by silica gel column chromatography to obtain an L3-D2-4 product (70 mg, yield 91% and HPLC 96%); LCMS: [ M+1] ⁺ 1664.78 (calculated 1663.79);¹H NMR(600MHz,DMSO)δ10.10(s,1H),8.25–8.05(m,2H),7.65–7.58(m,2H),7.55–7.45(m,2H),7.40–7.33(m,6H),7.35–7.20(m,8H),7.18–7.10(m,3H),6.65–6.05(m,3H),5.50–4.90(m,6H),4.86–4.33(m,3H),3.67–3.26(m,61H),2.40–2.30(m,2H),1.97–1.31(m,11H),0.90–0.84(m,3H).

In a 50mL single-port flask, L3-D2-4 (70 mg,0.042 mmol) and DCM (5 mL) were sequentially added, dissolved by stirring, and bromoacetic anhydride (10.9 mg,0.042 mmol) was added, reacted at room temperature for 30min under stirring, concentrated, and purified by silica gel column chromatography to give L3-D2-5 (60 mg, 80% yield, 98% by HPLC); LCMS: [ M+1] ⁺ 1784.93 (calculated 1783.72).

In a 50mL single-port flask, L3-D2-5 (60 mg, 33.6. Mu. Mmol) and DCM (3 mL) were added, dissolved with stirring, TFA (0.3 mL) was added dropwise, the reaction was stirred at room temperature for 1h, the reaction was added dropwise to methyl tert-butyl ether (30 mL), centrifuged, the supernatant was decanted, and the remaining solid was dried to give the yellow solid product L3-D2 (35 mg, 67% yield, 97% HPLC); LCMS: [ M+1] ⁺ 1542.84 (calculated 1541.61);¹H NMR(600MHz,DMSO-d₆)δ10.12(s,1H),8.19(t,J＝5.8Hz,2H),7.66(dt,J＝11.8,5.8Hz,4H),7.46(d,J＝8.5Hz,1H),7.23(s,1H),6.29(s,1H),6.16(s,1H),5.42(s,2H),5.29(s,2H),5.06(s,2H),4.43–4.37(m,1H),3.91(d,J＝7.3Hz,1H),3.85(s,1H),3.78(s,1H),3.79–3.69(m,2H),3.71(s,1H),3.61(dq,J＝13.2,7.3Hz,6H),3.58–3.50(m,2H),3.50–3.36(m,23H),3.40–3.34(m,4H),3.28–3.20(m,1H),3.19(d,J＝4.1Hz,6H),2.77(h,J＝6.4Hz,2H),2.40(t,J＝6.5Hz,2H),2.15(s,1H),1.86(dp,J＝21.4,7.2Hz,3H),1.76(qd,J＝10.5,7.0,5.3Hz,1H),1.68–1.57(m,1H),1.54(hept,J＝6.7Hz,2H),1.43–1.27(m,2H),0.87(t,J＝7.3Hz,3H);¹³C NMR(151MHz,DMSO)δ173.04,172.28,171.25,158.70,158.47,150.55,146.77,128.56,118.58,96.41,72.87,71.70,70.25,70.20,70.11,70.01,69.91,69.37,69.17,68.31,67.14,65.71,61.82,58.48,50.01,42.58,38.32,36.30,30.66,29.92,27.14,22.83,8.23.

3.2L3-D2-Ab _nectin-4 antibody conjugated drug (ADC 3)

Compound L3-D2 (1.23 mg,0.80 mmol) was dissolved in 0.12ml of DMA, added to the above solution system, mixed well, reacted at room temperature with a rotating disk for 16h, after the reaction was completed, small molecules were removed with NAP-5 gel column (Cytiva) and the buffer was replaced with 20mM PB solution, pH=6.3, to give antibody-coupled drug ADC3 (3.2 mg/ml,2 ml).

RP-MS calculates the average: n=7.9; MS results show that the antibody light chain (L) is linked to one L3-D2 (linker-payload) and the heavy chain (H) is linked to 3L 3-D2 (linker-payload) (FIG. 3).

Example 4: ab _nectin-4 - (S-7- (N- (acetamide-PEG ₂ -propionyl-Gly-Lys-PAB (3-bis-PEG ₄ -carbonyl) C) -N- (4-tetrahydropyran)) amine ethylcamptothecin) ₈ (ADC 4)

4.1 7- (N- (bromoacetamide-PEG ₂ -propionyl-Gly-Lys-PAB (3-bis-PEG ₄ -carbonyl) C) -N- (4-tetrahydrofuran)) aminoethyl camptothecin (L3-D3)

Intermediate I-1 (18 g,68.12 mmol) was added to IPA (180 mL), the reaction mixture was stirred, DIEA (17.61 g,136.24 mmol) and 4-aminotetrahydropyran hydrochloride (37.50 g,272.48 mmol) were then added, the reaction was refluxed for 12h, the reaction mixture was concentrated to a residual of about 20mL of IPA, 200mL of water was added, stirred for 2 hours, and the solid was obtained by filtration, dried at 40℃to give product D3-1 (20.2 g, yield 91.7%); LCMS: [ M+1] ⁺ 320.98 (calculated 320.10).

Compound D3-1 (16.9 g,52.63 mmol) was added to DCM (310 mL), acetic acid (63.05 g,1.05 mol) was added and stirred, the solids were all dissolved, sodium borohydride acetate (27.89 g,131.56 mmol) was added in 4 batches at 15℃intervals, approximately 7g each time, the reaction was allowed to react at 15℃for 2h, the reaction was poured into an ice water solution of sodium carbonate (130 g of sodium carbonate, ice water: 1L), the internal temperature was controlled at 15℃and stirred, the layers were separated, the organic phase was washed with water and dried over anhydrous sodium sulfate to give a DCM solution of compound D3-2, which was directly fed to the next step without purification.

Under ice bath cooling and nitrogen protection, DIEA (13.58 g,105.26 mmol) was added to the DCM solution of D3-2, cbz-Cl (10.11 g,59.27 mmol) was then added dropwise, the reaction was continued under stirring for 12h in ice bath, 10% aqueous citric acid (200 mL. Times.2) was added, washing was performed twice, followed by washing with saturated sodium bicarbonate solution (200 mL), saturated brine (200 mL), the organic phase was dried over anhydrous sodium sulfate, and concentrated to give compound D3-3 (22.9 g, two-step yield 95%); LCMS: [ M+1] ⁺ 457.28 (calculated 456.15).

Compound D3-3 (22.7 g,49.7 mmol) was added to a 1L single-necked flask, DMF (50 mL) was added, and the prepared reducing solution was slowly dropped under nitrogen protection, ice-bath cooling: h ₂ O (250 mL)/sodium dithionite (39.30 g,225.72 mmol)/sodium carbonate (19.14 g,180.58 mmol), slowly heating to 40 ℃ after dripping, stirring and reacting for 2H, filtering, concentrating the filtrate, adding ethyl acetate (500 mL) and water (500 mL), extracting and separating liquid, drying sodium sulfate, concentrating, purifying by silica gel column chromatography to obtain yellow oily substance D3-4 (20.3 g, yield 95.7%, HPLC 98%); LCMS: M+1] ⁺ 427.22 (calculated 426.19).

To a 500mL single-port flask, compound D3-4 (18.56 g,43.55 mmol), (S) -4-ethyl-4-hydroxy-7, 8-dihydro-1H-pyrano [3,4-F ] indolizin-3, 6,10 (4H) -one (13.75 g,52.27 mmol) and PPTS (218.88 g,871.00 mmol) were added sequentially, reacted at 110 ℃ for 12H under argon protection, the reaction solution was poured into methanol (1000 mL), stirred for 2H, filtered to give a dark brown solid, the brown solid was washed with methanol (300 mL) for 2H, filtered to give a pale brown solid, and the solid was dried at 40 ℃ to give compound D3-5 (11.0 g, yield 38.7%, HPLC 97%); LCMS: [ M+1] ⁺ 653.98 (calculated 653.24);¹H NMR(500MHz,DMSO-d₆)δ7.38(t,J＝68.6Hz,7H),6.49(s,1H),6.29(s,2H),5.43(s,2H),5.26(dd,J＝55.5,36.0Hz,4H),4.05(s,1H),3.90(d,J＝7.6Hz,2H),3.46(d,J＝8.4Hz,2H),1.93-1.74(m,4H),1.57(s,2H),0.88(t,J＝7.3Hz,3H).

Compound D3-5 (10.32 g,15.78 mmol) was added sequentially to a 10L single-necked flask, dichloromethane (2.5L), methanol (2.5L) and 10% content of Pd/C (15 g) were added, reacted at room temperature under a hydrogen balloon atmosphere for 20 hours, pd/C was removed by filtration, the reaction mixture was dried by spinning, stirred with 30ml of a mixed solution of dichloromethane and methanol (V/v=1:1) for 24 hours, filtered, and dried at 40 ℃ for 3 hours to give product D3 (6.1 g, yield 74%, HPLC 95.0%); LCMS: [ M+1] ⁺ 520.22 (calculated 519.20);¹H NMR(500MHz,DMSO-d₆)δ7.59(s,1H),7.47-7.41(m,1H),7.25-7.14(m,1H),6.54(s,1H),6.27(t,J＝9.9Hz,2H),5.77(s,1H),5.44(d,J＝16.5Hz,2H),5.23-5.11(m,2H),3.83(d,J＝9.5Hz,2H),3.23(t,J＝24.9Hz,4H),2.93(d,J＝32.7Hz,3H),1.99-1.72(m,4H),1.33(s,2H),0.96-0.81(m,3H).

In a 50ml single-port flask, D3 (130.0 mg,0.25 mmol), NMP (4 ml), DIEA (64.6 mg,0.5 mmol), L3 (340 mg,0.25 mmol), HOBt (37.1 mg,0.27 mmol) were added sequentially, the reaction was stirred at room temperature for 12h, extracted with DCM, washed sequentially with saturated sodium bicarbonate (50 ml) and water (50 ml), dried over anhydrous sodium sulfate, and concentrated to give L3-D3-1 (crude) which was directly fed to the next step; LCMS: [ M+1] ⁺ 1741.93 (calculated 1740.79).

Adding the L3-D3-1 (crude product) and DCM (5 mL) into a 50mL single-port bottle in sequence, stirring for dissolution, dripping DBU (26.6 mg), stirring at room temperature for reaction for 1h, pouring into methyl tertiary butyl ether (50 mL), centrifuging, drying to obtain the L3-D3-2 (crude product), and directly throwing into the next step without purification; LCMS: [ M+1] ⁺ 1519.96 (calculated 1518.72).

In a 50mL single-port flask, the above-mentioned L3-D3-2 (crude product) and DCM (10 mL) were sequentially added, stirred and dissolved, and the prepared Fmoc-NH-PEG ₂-CH₂CH₂ COOSu (124 mg,0.25 mmol) was added, and the reaction was continued under stirring at room temperature for 2 hours, concentrated, and purified by silica gel column chromatography to give the L3-D3-3 product (89 mg, three steps total yield 18.7%); LCMS: [ M+1] ⁺ 1900.97 (calculated 1899.88);¹H NMR(600MHz,DMSO-d₆)δ10.09(s,1H),8.12(s,2H),7.87(dd,J＝15.9,7.5Hz,3H),7.68(dd,J＝12.8,7.5Hz,2H),7.35–7.22(m,9H),6.29(s,1H),6.11(s,1H),5.42(s,2H),5.33(d,J＝40.1Hz,2H),5.03(s,1H),4.28(dd,J＝11.9,6.9Hz,2H),3.95–3.85(m,2H),3.74(d,J＝5.6Hz,2H),3.58(h,J＝9.2,7.8Hz,3H),3.54–3.46(m,3H),3.44(q,J＝10.7,8.8Hz,22H),3.37(tt,J＝6.3,4.3,3.5Hz,6H),1.84(ddd,J＝37.1,19.5,10.2Hz,2H),1.40–1.22(m,3H),0.86(dt,J＝13.0,7.2Hz,4H).

In a 50mL single-port flask, L3-D3-3 (87 mg,0.045 mmol) and DCM (6 mL) were sequentially added, stirred and dissolved, DBU (4.9 mg) was added dropwise, stirred at room temperature for 30min, poured into methyl tert-butyl ether (50 mL), centrifuged, the supernatant was removed, and purified by silica gel column chromatography to give L3-D3-4 (74 mg, 97% yield, 96% by HPLC); LCMS: [ M+1] ⁺ 1678.96 (calculated 1678.82).

In a 50mL single-port flask, L3-D3-4 (73 mg,0.043 mmol) and DCM (5 mL) were sequentially added, dissolved by stirring, and bromoacetic anhydride (11.0 mg,0.043 mmol) was added, reacted at room temperature for 30min with stirring, concentrated, and purified by silica gel column chromatography to give L3-D3-5 (65 mg, yield 84%, HPLC 98%); LCMS: [ M+1] ⁺ 1798.98 (calculated 1798.74).

L3-D3-5 (64 mg, 35.6. Mu. Mmol) and DCM (5 mL) were added into a 50mL single-port flask, the mixture was stirred and dissolved, TFA (0.3 mL) was added dropwise to the reaction mixture, the mixture was stirred at room temperature and reacted for 1h, the reaction mixture was added dropwise to methyl tert-butyl ether (30 mL), and the mixture was centrifuged to give a solid, which was dried to give a yellow solid product L3-D3 (35 mg, yield 63%, HPLC 97%); LCMS: [ M+1] ⁺ 1556.87 (calculated 1556.63);¹H NMR(500MHz,DMSO-d₆)δ10.12(s,1H),8.40-8.15(m,2H),7.75-7.65(m,3H),7.5-7.40(m,2H),7.24(s,1H),6.35-6.10(m,2H),5.43(s,2H),5.40-5.25(m,2H),5.15-5.00(m,2H),4.45-4.30(m,2H),4.08-3.20(m,63H),2.85-2.70(m,2H),2.40(t,J＝6.2Hz,2H),1.95-1.70(m,5H),1.67-1.46(m,5H),1.45-1.27(m,2H),0.88(t,J＝7.2Hz,3H);¹³C NMR(126MHz,DMSO)δ173.00,171.22,166.56,158.71,158.44,157.29,151.37,150.59,149.88,146.81,128.62,118.54,103.07,72.88,71.71,70.26,70.21,70.13,70.02,69.96,69.91,69.17,67.13,65.71,65.37,58.48,50.16,48.86,44.46,42.61,39.15,36.32,31.77,30.72,29.91,27.15,22.84,15.63,8.23.

4.2L3-D3-Ab _nectin-4 antibody conjugated drug (ADC 4)

Compound L3-D3 (1.25 mg,0.80 mmol) was dissolved in 0.13ml of DMA, added to the above solution system, mixed well, reacted at room temperature with a rotating disk for 16h, after the reaction was completed, small molecules were removed with NAP-5 gel column (Cytiva) and the buffer was replaced with 20mM PB solution, pH=6.3, to give antibody-coupled drug ADC4 (3.2 mg/ml,2 ml).

RP-MS calculates the average: n=7.8; MS results show that the antibody light chain (L) is linked to one L3-D3 (linker-payload) and the heavy chain (H) is linked to 3L 3-D3 (linker-payload) (FIG. 4).

Example 5: ab _nectin-4 - (S-7- (N- (bromoacetamide-PEG ₂ -propionyl-Gly-Lys-PAB (2-PEG ₈ -oxo) C) -N- ((R) -3-tetrahydrofuran)) aminoethyl camptothecin) ₈ (ADC 5)

5.1 7- (N- (bromoacetamide-PEG ₂ -propionyl-Gly-Lys-PAB (2-PEG ₈ -oxy) C) -N- ((R) -3-tetrahydrofuran)) aminoethyl camptothecin (L4-D2)

2- [2- [2- [2- [2- [2- [2- [2- (2-Methoxyethoxy) ethoxy ] ethanol (50 g,130.05 mmol), DCM (500 mL), p-TsCl (29.75 g,156.06 mmol) and TEA (39.48 g,390.15 mmol) were added to the reaction flask, stirred at room temperature for 16h, concentrated under reduced pressure, extracted with ethyl acetate, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, purified by silica gel column chromatography to give the colorless liquid intermediate p-toluenesulfonate L4-1 (50 g, yield 71.37%); LCMS: [ M+H ] ⁺ 539.38 (calculated 538.24).

3-Hydroxy-4-nitrobenzaldehyde (23.27 g,139.24 mmol), L4-1 (50 g,92.82 mmol), DMF (300 mL) and K ₂CO₃ (38.49 g,278.47 mmol) were added to the reaction flask, and the mixture was stirred at 70℃for 2 hours, concentrated, extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give a brown liquid intermediate compound L4-2 (27 g, yield 54.51%); LCMS: [ M+H ] ⁺ 534.38 (calculated 533.25).

L4-2 (21 g,39.36 mmol), methanol (200 mL) and sodium borohydride (2.23 g,59.04 mmol) were added to the reaction flask, the reaction was stirred at room temperature for 4h, concentrated under reduced pressure, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give the product L4-3 (19 g, 90.12% yield) as a brown liquid.

L4-3 (19 g,35.47 mmol), meOH (250 mL), 10% Pd/C (2 g,7.1 mmol), DIPEA (9.17 g,70.95 mmol) were added to the flask, the reaction stirred under hydrogen balloon for 48h, filtered and concentrated under reduced pressure to give compound L4-4 (17.5 g, yield 97.58%) as a brown liquid: LCMS: [ M+H ] ⁺ 506.21 (calculated 505.29).

In a 1000mL single port flask, L4-4 (10 g,19.78 mmol) and Fmoc-Lys (Trt) OH (12.08 g,19.78 mmol), DCM (300 mL), EDCI (3.98 g,20.77 mmol), HOBt (2.81 g,20.77 mmol) and DIPEA (3.83 g,29.67 mmol) were added, stirred at 25℃for 2h, saturated aqueous sodium bicarbonate solution was added and stirred (300 mL), the extracts and fractions were washed with aqueous citric acid and aqueous saturated saline, respectively, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to give Fmoc-Lys (Trt) PAB (PEG ₈) L4-5 (17 g, yield 75.13%); LCMS: [ M+1] ⁺ 1098.21 (calculated 1097.34).

L4-5 (17 g,14.86 mmol), DCM (400 mL), DBU (1.13 g,7.43 mmol) were added in a 1000mL single-necked flask, the reaction stirred at 25℃for 1h, concentrated to give L4-6 for direct use in the next step; LCMS: [ M+1] ⁺ 876.34 (calculated 875.10).

HOBt (4.18 g,30.96 mmol), fmocGlyOH (4.83 g,16.25 mmol), EDCI (3.12 g,16.25 mmol) and DIPEA (3.00 g,23.22 mmol) were added to the reaction solution in the previous step, the reaction was stirred for 2h at 25 ℃,200 ml of aqueous citric acid solution was added, and the extract was separated, washed with saturated aqueous sodium bicarbonate solution and saturated saline solution in sequence, separated, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give FmocGly-Lys (Trt) PAB (PEG ₈) L4-7 (15 g, 83.88% in two steps); LCMS: [ M+1] ⁺ 1155.60 (calculated 1154.40);¹H NMR(600MHz,DMSO-d₆)δ8.98(s,1H),8.16(d,J＝7.7Hz,1H),7.87(dd,J＝16.8,7.8Hz,2H),7.68(d,J＝7.5Hz,2H),7.53(t,J＝6.2Hz,1H),7.40(dd,J＝20.1,7.7Hz,6H),7.32(q,J＝7.2Hz,2H),7.26(t,J＝7.6Hz,5H),7.15(t,J＝7.3Hz,3H),7.01(s,1H),6.86(d,J＝8.2Hz,1H),5.76(s,3H),5.15(t,J＝5.7Hz,1H),4.44(d,J＝5.8Hz,3H),4.27–4.18(m,2H),4.10(t,J＝5.0Hz,2H),3.79–3.64(m,4H),3.57(dd,J＝5.9,3.7Hz,2H),3.52–3.43(m,22H),3.41(dd,J＝5.8,3.8Hz,2H),3.34(s,3H),2.50(d,J＝4.0Hz,2H),1.94(q,J＝7.4Hz,2H),1.72(ddt,J＝15.6,11.0,5.4Hz,1H),1.56(tq,J＝13.5,8.4,6.5Hz,1H),1.46(hept,J＝7.9,7.4Hz,2H),1.35–1.27(m,2H).

Taking 250mL single-mouth bottle at room temperature, sequentially adding D2(0.5g,918.08μmol),NMP(10mL),L4(1.21g,891.68μmol),HOBT(140.59mg,918.08μmol),DIPEA(118.65mg,918.08μmol,159.91μL), mL, stirring at room temperature for reaction for 16hr, adding diethyl ether (100 mL), and filtering to obtain pale yellow solid L4-D2-1 (1.5 g, yield 87.1%); LCMS: [ M+1] ⁺ 1687.56 (calculated: 1686.92).

At room temperature, a 250mL single-necked flask was taken, L4-D2-1 (1.5 g, 799.54. Mu. Mol), NMP (9 mL), piperidine (824.84 mg,9.69mmol, 956.90. Mu.L) were sequentially added, and the reaction was stirred at room temperature for 1hr, diethyl ether (100 mL) was added, and the solid was filtered to give L4-D2-2 (1.2 g, yield 77.0% y) as a pale brown solid; LCMS: [ M+1] ⁺ 1465.38 (calculated: 1464.67).

Taking a 100ml single-mouth bottle at room temperature, sequentially adding L4-D2-2(1.2g,678.31μmol),DMF(10mL),Fmoc-PEG2-OSu(396.22mg,739.75μmol),DIPEA(103.14mg,798.01μmol,139.00μL), to stir at room temperature for reaction for 0.5hr, adding 10g of silica gel powder for sand preparation, and performing silica gel column chromatography purification to obtain off-white solid L4-D2-3 (410 mg, yield 25.9%); LCMS: [ M+1] ⁺ 1847.01 (calculated ：1846.10);¹H NMR(600MHz,DMSO-d₆)δ10.54(s,2H),8.15(d,J＝.6Hz,1H),8.08(t,J＝5.8Hz,1H),7.95(d,J＝7.8Hz,1H),7.86(d,J＝7.6Hz,2H),7.67(d,J＝7.5Hz,2H),7.39(dd,J＝10.9,7.6Hz,8H),7.31(q,J＝7.5,6.7Hz,3H),7.28-7.22(m,7H),7.15(q,J＝8.9,7.3Hz,4H),6.49(s,1H),6.28(s,1H),6.22(s,1H),5.42(d,J＝2.4Hz,2H),5.26(s,2H),5.14(s,2H),4.60(d,J＝7.8Hz,1H),4.44(q,J＝7.3Hz,1H),4.27(d,J＝7.0Hz,2H),4.13(s,1H),3.94(s,1H),3.84-3.69(m,5H),3.67-3.63(m,1H),3.57(t,J＝6.5Hz,2H),3.51-3.44(m,26H),3.42-3.35(m,4H),3.29(s,1H),3.21(s,3H),3.11(q,J＝5.9Hz,2H),2.59(s,8H),2.36(t,J＝6.5Hz,2H),2.15(dt,J＝13.3,8.5,5.0Hz,1H),1.96-1.88(m,2H),1.85(dq,J＝14.0,7.1Hz,3H),1.70(s,1H),1.55(d,J＝9.1Hz,1H),1.46(q,J＝7.5Hz,2H),1.31-1.22(m,4H),0.87(t,J＝7.3Hz,3H).

L4-D2-3 (0.41 g,217.49 mu mol), DMF (4 mL) and piperidine (370.37 mg,4.35mmol,429.66 mu L) are sequentially added into a 10mL single-mouth eggplant type bottle at room temperature, stirred at room temperature for reaction for 1hr, 5g of silica gel powder is added for sand preparation, and silica gel column chromatography purification is carried out to obtain light yellow solid L4-D2-4 (230 mg, yield 61.05 percent); LCMS: [ M+1] ⁺ 1624.75 (calculated ：1623.86);¹H NMR(600MHz,DMSO-d₆)δ7.96(s,3H),7.51-7.45(m,1H),7.39(d,J＝7.9Hz,5H),7.29-7.21(m,6H),7.16(q,J＝7.4Hz,3H),7.02(s,1H),6.51(s,1H),6.27(s,1H),6.23(s,1H),5.44-5.41(m,1H),5.24(s,1H),5.15(s,2H),4.62(s,1H),4.44(q,J＝7.2,6.8Hz,1H),4.13-4.09(m,1H),3.79(qd,J＝16.7,5.8Hz,1H),3.73(s,1H),3.60(td,J＝5.9,5.3,2.9Hz,3H),3.57-3.45(m,21H),3.41(dd,J＝5.9,3.7Hz,2H),3.38(s,20H),3.30(s,1H),3.22(s,2H),2.94(t,J＝5.3Hz,1H),2.89(s,5H),2.73(s,5H),2.39(t,J＝6.5Hz,1H),2.16(ddt,J＝12.4,7.7,4.5Hz,1H),1.95(t,J＝6.5Hz,2H),1.87(ddd,J＝28.8,13.7,6.8Hz,2H),1.75-1.66(m,1H),1.63-1.54(m,1H),1.51-1.45(m,2H),1.30(s,1H),1.27-1.21(m,4H),0.87(dt,J＝20.0,7.2Hz,3H).

Taking a 10mL single-port bottle at room temperature, sequentially adding L4-D2-4 (100 mg,60.13 mu mol), DMF (2 mL), bromoacetic anhydride (18 mg,60.13 mu mol), stirring at room temperature for 2hr, adding 3g of silica gel powder for sand preparation, and performing silica gel column chromatography purification to obtain pale yellow solid L4-D2-5 (80 mg, yield 72.34%); LCMS: [ M+1] ⁺ 1745.62 (calculated: 1744.79).

L4-D2-5 (80 mg, 44.85. Mu. Mol), DCM (1 mL), TFA (153.41 mg,1.35mmol, 102.96. Mu.L) were sequentially added to a 10mL single-neck eggplant bottle at room temperature, stirred at room temperature for 2hr, diethyl ether (10 mL) was added, centrifuged (10000 rpm/5 min), the supernatant was removed, and the residual solvent was concentrated under reduced pressure to give yellow solid powder L4-D2 (50 mg, yield 70.15%); LCMS: [ M+1] ⁺ 1503.33 (calculated ：1502.47);¹H NMR(600MHz,DMSO-d₆)δ9.05(s,1H),8.33(t,J＝5.7Hz,1H),8.24(dd,J＝7.9,4.3Hz,1H),8.14(t,J＝5.7Hz,1H),7.97(d,J＝8.2Hz,1H),7.80(s,1H),7.66(q,J＝6.2Hz,4H),7.52-7.48(m,1H),7.37(s,1H),7.24(s,1H),7.18(s,1H),7.02(d,J＝15.5Hz,1H),6.49(s,1H),6.28(s,1H),6.25(s,1H),5.43(s,2H),5.28(s,2H),5.14(s,2H),4.61(d,J＝7.6Hz,1H),4.48(t,J＝5.8Hz,1H),4.18(d,J＝8.7Hz,1H),4.14(s,2H),3.85(s,2H),3.88-3.82(m,1H),3.80(s,1H),3.80-3.70(m,3H),3.65(dd,J＝9.4,6.9Hz,1H),3.60(t,J＝6.5Hz,4H),3.48(dd,J＝10.4,3.9Hz,16H),3.41(q,J＝4.8Hz,5H),3.38(d,J＝7.0Hz,1H),3.32(s,2H),3.27-3.19(m,2H),3.22(s,3H),2.78(h,J＝6.6Hz,2H),2.40(t,J＝6.5Hz,2H),2.16(dtd,J＝13.4,8.6,5.1Hz,1H),1.85(dqt,J＝27.7,13.5,6.6Hz,3H),1.62(dp,J＝13.8,4.7Hz,1H),1.54(hpt,J＝6.6Hz,2H),1.37(ddt,J＝23.6,15.8,7.0Hz,2H),1.24(d,J＝5.7Hz,1H),1.09(t,J＝7.0Hz,1H),0.88(t,J＝7.3Hz,3H).

5.2L4-D2-Ab _nectin-4 antibody conjugated drug (ADC 5)

Nectin-4 antibody (10.0 mg/mL,10mg,0.066 mmol) was taken, pH was adjusted to 7.2 with 1M Na2HPO4 solution, then 0.1M disodium edetate solution (25. Mu.L) was added, the prepared TCEP. HCl solution (10 mM,0.04 mL) was added, and the mixture was allowed to react for 3 hours at room temperature at 25℃with a rotating disk.

Compound L4-D2 (1.20 mg,0.80 mmol) was dissolved in 0.12ml of DMA, added to the above solution system, mixed well, reacted at room temperature with a rotating disk for 16h, after the reaction was completed, small molecules were removed by NAP-5 gel column (Cytiva) and the buffer was replaced with 20mM PB solution, pH=6.3, to give antibody-coupled drug ADC5 (3.1 mg/ml,2 ml).

RP-MS calculates the average: n=7.9; MS results show that the antibody light chain (L) is linked to one L4-D2 (linker-payload) and the heavy chain (H) is linked to 3L 4-D2 (linker-payload) (FIG. 5).

Test example: ADC antitumor Activity experiment

Test example 1: ADC in vitro inhibition of tumor cell growth activity

Human breast cancer cells MDA-MB-468, human bladder transitional cell carcinoma SW-780, human breast cancer cells MCF-7, human prostate cancer cells LNCaP and human pancreatic adenocarcinoma cells BxPC-3 were cultured in DMEM (Celmax), MEM (Celmax), RPMI1640 (Cellmax) and RPMI1640 (Cellmax) medium containing 10% fetal bovine serum (Cellmax) respectively to an exponential growth phase, after pancreatin digestion, the supernatants were centrifuged off, diluted to 6X 10 ⁴cells/mL、6×10⁴cells/mL、2×10⁴cells/mL、3×10⁴ cells/mL and 7X 10 ⁴ cells/mL respectively with medium, and 100. Mu.L per well was added to a 96-well cell culture plate and placed back into a 37℃incubator containing 5% CO ₂ overnight for culture. The following day, the test ADCs were diluted to 12000nM, 1200nM, 120nM, 12nM, 1.2nM, 0.12nM, 0.012nM, 0.0012nM and 0.00012nM using medium, and the diluted ADCs were added to 96-well cell culture plates at 100 μl per well, 3 replicate wells were set for each concentration, and 100 μl of medium was added per well for the negative control and blank control groups without ADC. After the addition was completed, the incubation was continued for 6 days in an incubator at 37℃with 5% CO ₂. After the incubation was completed, the cell culture plates were removed, the medium in the plates was pipetted off, 100. Mu.L of medium containing 10% CCK-8 was added to each well, and incubated at 37℃for 3h. After the incubation is completed, the culture plate is taken out, protected from light, placed in an enzyme-labeled instrument, and absorbance is measured by selecting 630nm as a reference wavelength and 450nm as a measurement wavelength. IC ₅₀ was calculated from absorbance values using four-parameter regression in GraphPad (table 1). With the use of EV (enfortumab vedotin,) Is a positive control drug.

With respect to the value of the IC ₅₀, wherein "+". ++ + +' and its use represents 50nM > IC ₅₀; "+". ++'s representation of 200nM > IC ₅₀ is more than or equal to 50nM; "++" means 1000nM > IC ₅₀. Gtoreq.200 nM; "+" indicates IC ₅₀ > 1. Mu.M.

TABLE 1 inhibitory Activity of ADCs of the invention on cancer cells

The ADC compounds provided by the embodiment of the invention have good inhibition effect on the growth of cancer cells and have remarkable anticancer activity.

Test example 2: in vivo tumor growth inhibition activity of ADC

Human breast cancer cells MDA-MB-468, human transitional cell carcinoma SW-780, human lung cancer cells NCI-H292, human bladder cancer cells HT-1376 and human pancreatic adenocarcinoma cells BxPC-3 are cultured in vitro in a monolayer, when the cell saturation is 80% -90%, the cells are digested by pancreatin-EDTA, supernatant is removed by centrifugation, PBS is resuspended, the cell suspension is adjusted to a proper concentration, human breast cancer cells MDA-MB-468, human transitional cell carcinoma SW-780, human lung cancer cells NCI-H292, human bladder cancer cells HT-1376 and human pancreatic adenocarcinoma cells BxPC-3 (2-10×10 ⁶ cells/0.1 mL) are inoculated subcutaneously in BALB/c nude mice, animals and the growth of transplanted tumors are observed periodically, when the tumor volume is about 100-200mm ³, the animals are randomly grouped according to the tumor volume and the body weight, namely, a control group (normal saline) and an ADC administration group (dissolved in normal saline) are carried out, and 6 animals are administered per group. Intravenous administration was carried out 1 time (the time of the first administration is recorded as Day 0), and tumor long diameter a (mm), short diameter b (mm) and mouse body weight were measured 2 times a week with a vernier caliper, and tumor volume (V) was calculated according to the following formula: v=1/2×a×b ²(mm³), where a and b represent tumor length and width, respectively. Statistical analysis the tumor inhibition rate was analyzed based on the data of tumor volume at the end of the experiment, expressed as: 100% > (average tumor volume in placebo (vehicle) group-average tumor volume in test group)/average tumor volume in placebo (vehicle) group.

TABLE 2

Note that: "-" indicates no test.

The vehicle group is blank control; N4-Dxd represents an ADC formed by Dxd (GGFG linker) coupled to an anti-Nectin-4 antibody of the examples;

the structural formula is as follows:

Experimental results: four doses of 2mg/kg, 3mg/kg, 6mg/kg and 10mg/kg of ADC1 and ADC2 all had significant tumor growth inhibiting activity.

The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims

1. A compound of formula (I), a pharmaceutically acceptable salt or stereoisomer thereof:

In the method, in the process of the invention,

R ₅ is selected from hydrogen, C _1-6 alkyl, and C _3-6 cycloalkyl;

Lp is selected from peptide residues comprising 1-5 amino acids;

m is selected from integers from 1 to 8;

a. p is selected from 1,2 or 3;

Ab is an anti-Nectin-4 antibody or antigen-binding fragment;

0.5≤n≤8。

2. The compound of claim 1, wherein each R ₁、R₂ is independently selected from hydrogen, fluoro and methyl, or R ₁、R₂ is taken together with the carbon atom to which it is attached to form

Preferably, R ₁ is hydrogen and R ₂ is hydrogen;

R ₁ is fluoro, R ₂ is fluoro; or alternatively

R ₁ is methyl and R ₂ is fluorine.

3. The compound of claim 1, wherein R ₃ is selected from 1,2, or 3 fluoro-substituted C _1-6 alkyl; or from 4 to 8 membered oxacycloalkyl;

preferably, R ₃ is selected from the group consisting of fluoroethyl, difluoroethyl, trifluoroethyl, oxacyclopentyl or oxacyclohexyl.

4. The compound of claim 1, wherein R ₄ is selected from hydrogen, C _1-6 alkyl, and-C (O) -NR ^aR^b;

Each R ^a、R^b is independently selected from C _1-6 alkyl;

q is selected from integers from 2 to 10;

Preferably, R ₄ is selected from hydrogen,

5. The compound of claim 1, wherein L ^p is selected from -Val-Cit-、-Gly-Lys-、-Gly-Leu-、-Val-Ala-、-Gly-Phe-、-GLy-Gly-Lys-、-Gly-Gly-Phe-、-Gly-Val-Ala-、-Gly-Gly-Val-、-Gly-Leu-Val-、-Gly-Phe-Gly- or-Gly-Leu-;

Preferably, L ^p is selected from

6. The compound of claim 1, wherein Z is selected from Wherein/>The indicated positions indicate the attachment to the antibody,/>The indicated position indicates attachment to-NH-.

7. The compound of claim 1, wherein the heavy chain amino acid sequence of the anti-Nectin-4 antibody is shown in SEQ ID NO. 1 and the light chain amino acid sequence is shown in SEQ ID NO. 2.

8. The following compounds, pharmaceutically acceptable salts or stereoisomers thereof:

9. A pharmaceutical composition comprising a compound of formula (I), a pharmaceutically acceptable salt or stereoisomer thereof as claimed in any one of claims 1 to 8; and a pharmaceutically acceptable carrier.

10. Use of a compound of formula (I), a pharmaceutically acceptable salt or stereoisomer thereof, as claimed in any one of claims 1 to 8, or a pharmaceutical composition as claimed in claim 9, in the manufacture of an anti-tumour medicament or a medicament for the treatment of autoimmune diseases;

preferably, the tumor is selected from the group consisting of solid tumors, more preferably from breast cancer, transitional cell bladder cancer, prostate cancer and pancreatic adenocarcinoma.