Hydrophilic Anti-nectin-4 antibody-drug conjugate, method for preparing same, and use thereof

Abstract

Provided are a toxin-hydrophilic linker compound formed by camptothecin-7-ethylamine and a linker, a conjugate thereof with an anti-Nectin-4 antibody, and pharmaceutical use thereof. The toxin-hydrophilic linker compound can be conjugated with an anti-Nectin-4 monoclonal antibody to form an antibody-drug conjugate (ADC), so as to increase the hydrophilicity of the ADC, reduce the aggregation thereof in the circulatory system, improve the efficacy, and reduce the adverse effects.

Description

Hydrophilic anti-nectin-4 antibody-drug conjugate and preparation method and application thereof

[0001] This application claims priority to Chinese patent application No. 2024100518885, filed on January 12, 2024, and cites the full text of the aforementioned Chinese patent application. Technical Field

[0002] The present invention relates to the field of medicine. Specifically, the present invention provides a series of camptothecin-7-ethylamine and its preparation method of toxin-hydrophilic linker compounds formed with linkers, and the coupling of these compounds with corresponding anti-nectin-4 antibodies to form antibody-drug conjugates and their application in the treatment of tumors. Background Art

[0003] Camptothecin and its derivatives have inhibitory activity against topoisomerase Top1, particularly against Top1-DNA complexes. Camptothecin has significant therapeutic effects on gastric cancer, esophageal cancer, lung cancer, bladder cancer, and other cancers, making it a broad-spectrum anti-tumor drug. The camptothecin derivatives Irinotecan and Topotecan have been approved in many countries for the treatment of various cancers. Another camptothecin derivative, Belotecan, has been approved in South Korea for the treatment of SCLC and ovarian cancer. The main drawbacks of camptothecin anti-tumor drugs are their toxicity, poor solubility, and the development of drug resistance in tumor cells. Summary of the Invention

[0004] The present invention provides a series of toxin-hydrophilic linker compounds formed by camptothecin-7-ethylamine and a linker. The compounds contain a hydrophilic structure composed of at least two PEGs. This toxin-hydrophilic linker can be coupled with the monoclonal antibody nectin-4 to form an antibody-drug conjugate (ADC), thereby increasing the hydrophilicity of the ADC, reducing its aggregation in the circulation system, improving drug efficacy, and reducing toxic side effects.

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

[0006] Where,

[0007] R1 and R2 are each independently selected from hydrogen, fluorine and C 1-3 Alkyl, or R1, R2 and the carbon atom to which they are connected together form an oxygen-containing heterocyclic group;

[0008] R3 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, C1-3 The alkoxy group is optionally substituted by one or more halogens; the 4- to 8-membered heterocycloalkyl group contains 1, 2 or 3 heteroatoms selected from N, O and S as ring atoms;

[0009] R4 is selected from hydrogen or a heteroalkyl group comprising repeating -OCH2CH2- units;

[0010] R5 is selected from hydrogen, C 1-6 Alkyl and C 3-6 Cycloalkyl;

[0011] Lp is selected from a peptide residue comprising 1-5 amino acids;

[0012] m is an integer selected from 1 to 8;

[0013] a, p are selected from 1, 2 or 3;

[0014] Ab is an anti-nectin-4 antibody or antigen-binding fragment;

[0015] Z is a linker capable of coupling the antibody or antigen-binding fragment with other moieties of the compound of formula (I); 0.5≤n≤8.

[0016] In one embodiment, the compound of formula (I) has a structure represented by formula (I-1) or formula (I-2):

[0017] The definitions of the groups in the formula are the same as those of compound (I).

[0018] In one embodiment, m is selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0019] In one embodiment, R1 and R2 are each independently selected from hydrogen, fluorine and methyl, or R1, R2 and the carbon atom to which they are attached together form

[0020] In one embodiment, R1 is hydrogen and R2 is hydrogen.

[0021] In one embodiment, R1 is fluorine and R2 is fluorine.

[0022] In one embodiment, R1 is methyl and R2 is fluoro.

[0023] In one embodiment, R3 is selected from 1, 2 or 3 fluorine-substituted C 1-6 alkyl.

[0024] In one embodiment, R3 is selected from 4 to 8 membered oxacycloalkyl.

[0025] In one embodiment, R3 is selected from oxetanyl, oxolanyl, or oxhexyl.

[0026] In one embodiment, R3 is selected from fluoroethyl, difluoroethyl, trifluoroethyl, oxolanyl or oxhexyl.

[0027] In one embodiment, R4 is selected from hydrogen, C 1-6 Alkyl and -C(O)-NR a R b ;

[0028] R a 、R b Each independently selected from C 1-6 alkyl;

[0029] wherein the C 1-6 One or more methylene units in the alkyl group are optionally and independently replaced by -(OCH2CH2)q-;

[0030] q is selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0031] In one embodiment, R4 is selected from hydrogen,

[0032] In one embodiment, L p 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-.

[0033] In one embodiment, L p Selected from

[0034] In the present invention, the linker used to couple the antibody or antigen-binding fragment to the other moieties of the compound of formula (I) can be a single linker or a double linker. The double linker refers to a structure that can simultaneously connect two chemical functional groups (specifically, the antibody and the toxin moiety). This structure has a group that connects to the antibody and a group that connects to the toxin moiety.

[0035] In one embodiment, Z is selected from in, The positions shown indicate attachment to antibodies. The indicated position indicates linkage to -NH-.

[0036] In one embodiment, 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 thereof is shown in SEQ ID NO: 2.

[0037] The present invention provides the following toxin-linker compounds, pharmaceutically acceptable salts or stereoisomers thereof:

[0038] The present invention provides the following compounds, pharmaceutically acceptable salts or stereoisomers thereof:

[0039] Another aspect of the present invention provides a pharmaceutical composition comprising the compound of formula (I), a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier.

[0040] Another aspect of the present invention provides the use of the aforementioned compound of formula (I), its pharmaceutically acceptable salt or stereoisomer, or the aforementioned pharmaceutical composition in the preparation of an anti-tumor drug;

[0041] Preferably, the tumor is selected from solid tumors.

[0042] In one embodiment, the tumor is selected from breast cancer, transitional cell carcinoma of the bladder, prostate cancer, and pancreatic adenocarcinoma.

[0043] Another aspect of the present invention provides a method for treating cancer, comprising the step of administering the compound of formula (I), a pharmaceutically acceptable salt or stereoisomer thereof, or the pharmaceutical composition to a patient in need thereof.

[0044] In one embodiment, the cancer is selected from breast cancer, transitional cell carcinoma of the bladder, prostate cancer, and pancreatic adenocarcinoma.

[0045] Another aspect of the present invention provides a method for preparing a compound of formula (I), comprising:

[0046] wherein each group is defined as in the compound of formula (I);

[0047] The reaction reagents used in each step are consistent with those in Examples 1-5.

[0048] In one embodiment, the reaction 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, followed by condensation with Fmoc-PEG2-OH; j) piperidine, followed by condensation with a corresponding Z linker; k) conjugation with an anti-nectin-4 antibody to form an anti-nectin-4 ADC. Beneficial effects:

[0049] The toxin-hydrophilic linker provided by the present invention is coupled with the nectin-4 monoclonal antibody to form an antibody-drug conjugate (ADC), which helps to reduce the aggregation of ADC in the circulation system, improve drug efficacy, and reduce toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG1 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.

[0051] FIG2 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.

[0052] FIG3 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.

[0053] FIG4 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.

[0054] FIG5 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

[0055] I. Definition

[0056] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the relevant terms and laboratory procedures used herein are those widely used in the relevant fields and routine procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0057] 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 an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0058] In the description herein, references to “some embodiments,” “some implementation schemes,” or “some implementation plans” describe a subset of all possible embodiments, but it will be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0059] As used herein and unless otherwise specified, the terms "comprises," "includes," "has," "contains," and their grammatical equivalents should generally be understood as open-ended and non-limiting, e.g., not excluding other unlisted elements or steps.

[0060] The term "antibody" as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired biological activity. Antibodies can be murine, human, humanized, chimeric or derived from other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. Target antigens typically have multiple binding sites, also known as epitopes, which are recognized by CDRs (complementary determining regions) on a variety of antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, an antigen can 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 or a 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 that can bind to a specific antigen. It includes two light chains with lighter molecular weight and two heavy chains with heavier molecular weight. The heavy chain (H chain) and the light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule.

[0061] The term "antibody-drug conjugate (ADC)" used in this application refers to a small molecule drug with biological activity connected to a monoclonal antibody through a chemical link. The monoclonal antibody acts as a carrier to transport the small molecule drug into target cells.

[0062] As used herein, the term "toxin," also referred to as a "cytotoxic drug moiety" or "small molecule drug," refers to a compound that has a tumor cell-killing effect. 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-targeting chimera (PROTAC), and an immunomodulator.

[0063] The small molecule drugs used in this application can be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The stereoisomers include geometric isomers (such as cis, trans structures) and optical isomers (such as enantiomers), and are therapeutic substances composed of monomers, racemates, racemic mixtures and pharmaceutically acceptable salts thereof. The compounds containing asymmetric carbon atoms in this application can be separated in optically pure form or racemic form. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral raw materials or chiral reagents. Racemates, diastereomers, and enantiomers are all included within the scope of this application.

[0064] The small molecule drugs used in this application also include tautomers, which are formed when a single bond is exchanged with an adjacent double bond accompanied by the migration of a proton.

[0065] As used herein, numerical ranges refer to the individual integers within the given range. For example, "C1-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; "C3-C6" means that the group can have 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

[0066] When any variable (such as R n ) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 1-5 R, the group may be optionally substituted with up to 5 R, with each occurrence of R being an independent choice. Furthermore, combinations of substituents and / or variants thereof are permissible only if such combinations result in stable compounds.

[0067] The term "alkyl" as used herein refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, the tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is 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, carboxyl or carboxylate groups, preferably methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuterated alkyl, alkoxy-substituted alkyl and hydroxy-substituted alkyl.

[0068] The term "heterocyclyl" or "heterocycloalkyl" as used herein refers to a saturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS- or -SS-, the remaining ring atoms are carbon.

[0069] The term "alkoxy" as used herein refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy.

[0070] The term "substituted" used herein refers to one or more hydrogen atoms in a group, preferably up to 5, more preferably 1-3 hydrogen atoms that are independently replaced by the substituents of corresponding number. It goes without saying that substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible replacement without paying too much effort. For example, amino or hydroxyl groups with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (such as olefinic) bond.

[0071] Refers to the chemical bond connection.

[0072] The term "pharmaceutically acceptable salt" as used herein refers to a salt formed between a corresponding amine compound and an inorganic acid or organic acid, or a salt formed between a corresponding carboxylic acid compound and an alkali metal or alkaline earth metal, or a salt formed with an organic amine. Inorganic acids include, but are 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 metal or alkaline earth metal salts include, but are not limited to, sodium, potassium, calcium, and magnesium salts; and organic amine salts include, but are not limited to, salts composed of ammonia, methylamine, ethylamine, propylamine, isopropylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, tert-butylamine, ethylenediamine, ethanolamine, diethanolamine, triethanolamine, morpholine, piperidine, piperazine, and amino acids.

[0073] The medicament or pharmaceutical composition of the present application can be administered orally, topically, parenterally or mucosally (e.g., buccally, by inhalation or rectally) in a dosage unit formulation containing a conventional non-toxic pharmaceutically acceptable carrier. It is generally desirable to use the oral route. The active agent can be administered orally in the form of capsules, tablets, etc. (see Remington: The Science and Practice of Pharmacy, 20th Edition).

[0074] For oral administration in the form of tablets or capsules, the active drug component can be mixed with non-toxic, pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, sucrose, glucose, mannitol, sorbitol and other reducing and non-reducing sugars, microcrystalline cellulose, calcium sulfate or dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc or silica, stearic acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate, etc.); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate), coloring and flavoring agents, gelatin, sweeteners, natural and synthetic gums (such as acacia, tragacanth or alginates), buffer salts, carboxymethylcellulose, polyethylene glycol, waxes, etc. For oral administration in liquid form, the drug component can be combined with a non-toxic, pharmaceutically acceptable inert carrier (e.g., ethanol, glycerol, water), an anti-settling agent (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), an emulsifier (e.g., lecithin or gum arabic), a non-aqueous carrier (e.g., almond oil, oily esters, ethanol or fractionated vegetable oils), a preservative (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid), etc. Stabilizers such as antioxidants (BHA, BHT, propyl citric acid, sodium ascorbate, citric acid) can also be added to stabilize the dosage form.

[0075] The tablet comprising the active compound can be coated by methods well known in the art. The composition of the present application comprising the compound of formula I as the active compound can also be introduced into beads, microspheres or microcapsules, for example, constructed from polyglycolic acid / lactic acid (PGLA). The preparation of liquid for oral administration can take the form of, for example, solution, syrup, emulsion or suspension or they can be presented as a dry product reconstituted with water or other suitable adjuvants before use. The preparation for oral administration can be suitably formulated so that the active compound is controlled or released in a delayed manner.

[0076] As used herein, the term "treating," ...

[0077] As used herein, the term "inhibit" is used relative to a control. One skilled in the art will readily determine the appropriate control for each experiment. For example, a reduced response in a subject or cell treated with a compound is compared to a response in a subject or cell not treated with the compound.

[0078] The term "pharmaceutical composition" as used herein refers to a composition comprising the compound described herein or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable ingredient selected from the following depending on the mode of administration and the nature of the dosage form, including but not limited to: carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0079] II. Examples

[0080] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below. The described embodiments should not be regarded as limiting the present invention. All other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0081] Before further explaining the embodiments of the present invention in detail, the nouns and terms involved in the embodiments of the present invention are explained. The nouns and terms involved in the embodiments of the present invention are subject to the following interpretations.

[0082] The raw materials and equipment used in the specific embodiments of the present disclosure are all known products and are obtained by purchasing commercially available products.

[0083] DAR value testing and calculation: Based on the RP-HPLC-MS test results, the DAR value of the ADC was analyzed using a Waters Acquity UPLC I-Class / Xevo G2-XS QTOF instrument.

[0084] RP-HPLC parameters: PLRP-S1000A 5UM column, column temperature 70°C. Mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile, flow rate 0.2 mL / min. Mobile phase gradient: 20-50% B over 18 minutes; 50-95% B over 5 minutes; 95-20% B over 0.1 minutes; 20-20% B over 6.9 minutes.

[0085] MS parameters were set as follows: capillary voltage 2.50 kV, cone voltage 100 V, mass analysis range m / z 200 to 4000, MSE collision energy 20 to 45 eV, ion source temperature 120°C, nebulizer temperature 500°C, nebulizer flow rate 1000 L / Hr, and internal standard leucine enkephalin. The sample was diluted to 1 mg / mL in sample buffer, TCEP was added to a final concentration of 50 mmol / L, and the sample was incubated at 37°C for 20 min. A 5 μL injection was performed. Light chain peaks were identified and peak area percentages were calculated, with the sum of the peak areas being 100. Similarly, heavy chain peaks were identified and peak area percentages were calculated, with the sum of the peak areas being 100. Weighted peak areas for the heavy and light chains were calculated by multiplying the peak area percentages by the corresponding drug loading. The DAR value was calculated as: DAR = 2*(Σlight chain weighted peak area + Σheavy chain weighted peak area) / 100.

[0086] In this embodiment, ADC prepared using but not limited to Nectin-4 antibody.

[0087] The amino acid sequence of the heavy chain of the nectin-4 antibody is as follows (SEQ ID NO: 1):

[0088] The amino acid sequence of the light chain of the nectin-4 antibody is as follows (SEQ ID NO: 2):

[0089] Example 1: Ab nectin-4 -S-(7-(N-(acetamide-PEG2-propionyl-Gly-Lys-PABC)-N-(2'2'-difluoroethyl))aminoethylcamptothecin)8(ADC1)

[0090] 1.1 7-(N-(Bromoacetamide-PEG2-propionyl-Gly-Lys-PABC)-N-(2'2'-difluoroethyl))aminoethylcamptothecin (L1-D1)

[0091] 4',5'-Methylenedioxy-2'-nitroacetophenone (15 g, 71.72 mmol) was added to a 500 mL single-necked bottle, and DMF-DMA (170.92 g, 1.43 mol) was added. The temperature was raised to 100 °C and refluxed for 10 hours. The mixture was concentrated to obtain a yellow solid. THF (30 mL) was added, and n-hexane (150 mL) was added dropwise. The mixture was stirred for 3 hours and filtered to obtain a yellow solid wet product. The product was dried in vacuo at 40 °C for 6 hours to obtain a light yellow solid intermediate I-1 (18 g, yield 95%, HPLC 98%); LCMS: [M+1] + 265.22 (calculated value 264.24); 1 H 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).

[0092] 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 difluoroethylamine hydrochloride (32.02 g, 272.48 mmol) were added, and the mixture was stirred and refluxed for 12 h. The mixture was concentrated, and water (200 mL) was added. The mixture was stirred for 2 h and filtered to obtain a yellow solid. The solid was dried at 40°C to obtain product D1-1 (15.8 g, yield 77.3%, HPLC 98%); LCMS: [M+1] + 301.47 (calculated value 300.22); 1 H NMR (600MHz, CDCl3) δ9.84(s,1H),7.40(s,1H),6.87(s,1H),6.84(dd,J=12.6,7.5 Hz,1H),6.13(s,2H),5.99-5.74(m,1H),5.26(d,J=7.5Hz,1H),3.68-3.48(m,2H).

[0093] Compound D1-1 (15.8 g, 52.63 mmol) was added to DCM (310 mL), and acetic acid (63.05 g, 1.05 mol) was added, and the mixture was stirred to dissolve all the solids. The internal temperature of the reaction solution was maintained at 15 degrees Celsius. Sodium acetate borohydride (27.89 g, 131.56 mmol) was added in 4 batches, about 7 grams each time, with an interval of about 5 minutes. The internal temperature of the reaction solution was maintained at 15 degrees Celsius and the reaction was carried out for 2 hours. The reaction solution was poured into a sodium carbonate aqueous solution at 0 degrees Celsius (sodium carbonate: 130 g, water: 1 L), the internal temperature was controlled at 15 degrees Celsius, stirred, and separated. The organic phase was washed with water, and the organic phase was collected and dried over anhydrous sodium sulfate. , to obtain compound D1-2 (directly used in the next step), cooled to 0 degrees Celsius, added DIEA (13.58 g, 105.26 mmol), and added dropwise a DCM solution of CbzCl (10.11 g, 59.27 mmol) (addition completed over about 20 minutes). The mixture was reacted at 0 degrees Celsius for 12 hours, added to a 10% aqueous citric acid solution (200 mL*2), washed twice, and then washed with a saturated aqueous sodium bicarbonate solution (200 mL) and saturated brine (200 mL) in that order. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain product D1-3 (19.7 g, total yield of two steps 85.7%, HPLC 95%); LCMS: [M+1] + 437.42 (calculated value 436.37); 1 H 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).

[0094] Under nitrogen protection, compound D1-3 (19.7 g, 45.1 mmol) was added to a 1 L single-necked bottle, DMF (50 mL) was added, the temperature was lowered to 0 degrees Celsius, and the prepared reducing solution was slowly added dropwise: H2O (250 mL) / sodium dithionite (39.30 g, 225.72 mmol) / sodium carbonate (19.14 g, 180.58 mmol). After the addition was complete, the temperature was slowly raised to 40 degrees Celsius and stirred for 2 hours. The filtrate was concentrated, ethyl acetate (500 mL) and water (500 mL) were added, and the liquid was extracted. Ethyl acetate (500 mL) was added to the aqueous phase again, and the pH was adjusted to 3-4 with 2M dilute hydrochloric acid. The mixture was stirred at room temperature for 6 hours, and the liquid was extracted. The organic phases were combined and dried over sodium sulfate. The sample was concentrated and purified by silica gel column chromatography to obtain a yellow oil D1-4 (17.7 g, yield 96.7%, HPLC 98%); LCMS: [M+1] + 407.52 (calculated value 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).

[0095] Compound D1-4 (17.7 g, 43.55 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-F]indolizine-3,6,10(4H)-one (13.75 g, 52.27 mmol) and PPTS (218.88 g, 871.00 mmol) were added sequentially to a 500 mL single-necked flask. Under argon protection, the mixture was heated to 110°C with stirring for 12 h. The reaction solution was poured into methanol (1000 mL), stirred for 2 h, and filtered to obtain a dark brown solid. The brown solid was further stirred with methanol (300 mL) for 2 h and filtered to obtain a light brown solid. The solid was dried at 40°C to obtain compound D1-5 (10.1 g, yield 36.6%, HPLC 96%); LCMS: [M+1] + 634.33 (calculated value 633.60); 1 H 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).

[0096] Compound D1-5 (10 g, 15.78 mmol), dichloromethane (2.5 L), and methanol (2.5 L) were added sequentially to a 10 L single-necked flask. 10% Pd / C (15 g) was added to the reaction solution with stirring. The mixture was reacted at room temperature for 20 h under a hydrogen balloon atmosphere. The Pd / C was removed by filtration. The reaction solution was spin-dried and stirred with 30 mL of a mixed solution of dichloromethane and methanol (V / V = 1:1) for 24 h. The mixture was filtered and dried at 40 ° C for 3 h to obtain product D1 (5.8 g, yield 73.6%, HPLC 95.0%); LCMS: [M+1] + 500.52 (calculated value 499.47);1 H 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).

[0097] Preparation of Fmoc-Gly-Lys(Trt)-PAB-PNP: In a 1 L single-necked flask, Fmoc-Lys(Trt) (50 g, 82.1 mmol) and 4-aminobenzyl alcohol (15.1 g, 122.7 mmol) were added and dissolved in a mixed solvent of DCM (250 mL) and MeOH (250 mL). The temperature was lowered to 0°C, and EEDQ (30.3 g, 122.7 mmol) was added in batches. The mixture was kept warm for 10 min and then allowed to react at room temperature overnight. The reaction solution was washed with 2% citric acid aqueous solution (300 mL) and then with saturated sodium bicarbonate aqueous solution (300 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain Fmoc-Lys(Trt)-PAB as an oil (LCMS: [M+1] + 716.30 (calculated 715.35).

[0098] DCM (300 mL) was added to the oily product obtained above, and DBU (7.4 g, 48.9 mmol) was added with stirring. The reaction was carried out at room temperature for 2 h. After concentration to about 200 mL, the mixture was added dropwise to 2 L of methyl tert-butyl ether and filtered. The filter cake was spin-dried at room temperature to obtain Lys(Trt)-PAB as a pink solid (37 g, two-step yield 91%); LCMS: [M+1] + 494.70 (calculated 493.65).

[0099] Lys(Trt)-PAB (47 g, 95.2 mmol) was added to a 1 L single-necked flask and 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), and pyridine (7.5 g, 95.2 mmol) were added under ice-cooling. The mixture was stirred at room temperature for 2 h. The reaction solution was washed with 300 mL of 5% aqueous citric acid solution and then with 200 mL of saturated aqueous 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, yield 54%); LCMS: [M+1] + 773.70 (calculated value 772.95); 1 HNMR(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),5.1 6(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).

[0100] In a 1 L single-necked flask, Fmoc-Gly-Lys(Trt)-PAB (40 g, 51.7 mmol) and DCM (400 mL) were added sequentially. After stirring and dissolving, TEA (15.6 g, 155.1 mmol) and bis(4-nitrophenyl) carbonate (47.1 g, 155.1 mmol) were added dropwise at room temperature. The reaction was stirred at room temperature for 2 h. Water (400 mL) was added for extraction, and the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, and dried by spin drying. The product was purified by silica gel column chromatography to obtain Fmoc-Gly-Lys(Trt)-PAB-PNP (17.5 g, yield 36%, HPLC 95.7%); LCMS: [M+1] + 938.62 (calculated value 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),7.5 0-7.14(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).

[0101] In a 50 mL single-necked flask, D1 (3.7 g, 7.41 mmol), NMP (37 mL), DIPEA (2.87 g, 22.22 mmol), Fmoc-Gly-Lys(Trt)-PAB-PNP (6.95 g, 7.41 mmol), and HOBT (1.05 g, 7.77 mmol) were added in sequence and stirred at room temperature for 2 h. The reaction solution was washed with 50 mL of 2% citric acid aqueous solution and then with 50 mL of saturated sodium bicarbonate aqueous solution. The organic phase was washed with 50 mL of saturated brine and dried over anhydrous sodium sulfate. After concentration, the crude product L1-D1-1 was directly used for the next reaction; LCMS: [M+1] + 1298.74 (calculated value 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=28.6,1 4.1,8.1,7.1Hz,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).

[0102] DCM (50 mL) was added to the above reaction flask (L1-D1-1), stirred to dissolve, and then DBU (582.8 mg) was added dropwise. The reaction was stirred at room temperature for 1 h, and methyl tert-butyl ether (400 mL) was added. The mixture was filtered, and the solid was redissolved in DCM (40 mL), added dropwise to methyl tert-butyl ether (400 mL), filtered, and dried to obtain L1-D1-2 (7 g, yield 87.5%); LCMS: [M+1] + 1076.38 (calculated value 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).

[0103] In a 250 mL single-necked bottle, L1-D1-2 (7 g, 6.5 mmol) and 70 mL of DCM were added in sequence. After stirring and dissolving, Fmoc-NH-PEG2-CH2CH2CO2H (2.6 g, 6.5 mmol), HATU (3.7 g, 9.7 mmol) and DIPEA (1.3 g, 10 mmol) were added. The reaction was stirred at room temperature for 1 h. 100 mL of water was added to quench the reaction. The organic phase was separated and washed once with saturated aqueous sodium chloride solution (100 mL). The organic phase was concentrated and purified by silica gel column chromatography to obtain L1-D1-3 (5.2 g, yield 54.7%); LCMS: [M+1] + 1457.93 (calculated value 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=8.2 Hz,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,4 H),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),1.3 8-1.15(m,6H),1.05(d,J=6.4Hz,2H),1.00(d,J=6.5Hz,9H),0.87(t,J=7.4Hz,3H).

[0104] In a 100 mL single-necked bottle, L1-D1-3 (5.1 g, 3.5 mmol) and DCM (30 mL) were added sequentially. After stirring to dissolve, DBU (266.3 mg) was added dropwise. The mixture was stirred at room temperature for 30 min. Methyl tert-butyl ether (300 mL) was added and the mixture was filtered. The filter cake was washed with methyl tert-butyl ether and dried to give L1-D1-4 (3.2 g, yield 74%); LCMS: [M+1] + 1235.77 (calculated value 1234.52).

[0105] In a 100 mL single-necked bottle, L1-D1-4 (3.2 g, 2.6 mmol) and DCM (19 mL) were added in sequence. After stirring and dissolving, bromoacetic anhydride (670 mg, 2.6 mmol) was added. The mixture was stirred at room temperature for 1 h. The reaction solution was concentrated and purified by silica gel column chromatography to obtain L1-D1-5 (2.8 g, yield 80%, HPLC 97.3%); LCMS: [M+1] + 1355.64 (calculated value 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).

[0106] In a 100 mL single-necked bottle, L1-D1-5 (3.1 g, 2.3 mmol) and DCM (60 mL) were added in sequence. After stirring and dissolving, TFA was added dropwise (to a reaction solution concentration of 5%). The reaction was stirred at room temperature for 1 h. Methyl tert-butyl ether (600 mL) was added, filtered, washed, and then DCM (10 mL) was added and stirred. Methyl tert-butyl ether (100 mL) was added and stirred for 20 min. The mixture was filtered, washed, and dried in vacuo to obtain a white solid product L1-D1 (1.0 g, yield 40%, 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,2 H),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); 13 C NMR(151MHz,DMSO-d6)δ173.03,171.26,171.01,169.51,166.57,158.67,158.45,157.32,156.03 ,155.85,151.37,150.58,149.91,149.56,147.57,146.75,139.23,139.11,133.26,127.13,125.0 8,119.14,115.33,104.56,99.64,96.43,72.88,69.92,69.17,67.15,65.73,61.82,53.56,50.11,49.50,48.21,42.54,39.15,38.32,36.31,31.92,30.68,29.92,29.04,28.45,27.17,22.81,8.25.

[0107] 1.2L1-D1-Ab nectin-4 Antibody-drug conjugate (ADC1)

[0108] Take Nectin4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl solution (10 mM, 0.04 mL), and react at room temperature 25°C on a rotating turntable for 3 h.

[0109] Compound L1-D1 (0.89 mg, 0.80 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, mixed, and reacted on a rotary turntable at room temperature for 16 h. After the reaction was completed, a NAP-5 gel column (Cytiva) was used to remove small molecules and the buffer was replaced with a 20 mM PB solution, pH = 6.3, to obtain antibody-drug conjugate ADC1 (3.1 mg / mL, 2 mL).

[0110] RP-MS (RP-HPLC-MS) calculated the average value: n = 7.7; MS results showed that the antibody light chain (L) was connected to one L1-D1 (linker-payload), and the heavy chain (H) was connected to three L1-D1 (linker-payload) (Figure 1).

[0111] Example 2: Ab nectin-4 -(S-7-(N-(acetamide-PEG2-propionyl-Gly-Lys-PABC)-N-((R)-3-tetrahydrofuran))amineethylcamptothecin)8(ADC2)

[0112] 2.1 7-(N-(Bromoacetamide-PEG2-propionyl-Gly-Lys-PABC)-N-((R)-3-tetrahydrofuran))aminoethylcamptothecin (L1-D2)

[0113] Intermediate I-1 (18 g, 68.12 mmol) was added to IPA (180 mL), and 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 mixture was refluxed for 12 h. The reaction mixture was concentrated to a residual volume of about 20 mL of IPA. 200 mL of water was added and stirred at room temperature for 2 h. A yellow solid was obtained by filtration and dried at 40°C to obtain product D2-1 (16.5 g, yield 79%, HPLC 98%); LCMS: [M+1] + 307.32 (calculated value 306.09); 1 H 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).

[0114] Compound D2-1 (16.5 g, 53.87 mmol) was added to DCM (310 mL), and acetic acid (64.70 g, 1.08 mol) was added, stirred and dissolved, and the internal temperature of the reaction solution was maintained at 15 degrees Celsius. Sodium acetate borohydride (28.54 g, 134.68 mmol) was added in 4 batches, about 7 grams each time, with an interval of about 5 minutes. The internal temperature of the reaction solution was maintained at 15 degrees Celsius for 2 hours. The reaction solution was poured into an ice-cold aqueous sodium carbonate solution (sodium carbonate: 130 g, ice water: 1 L), and the internal temperature was controlled at 15 degrees Celsius. Stir, separate the layers, wash the organic phase with water, collect the organic phase, and precipitate with anhydrous sulfuric acid. The mixture was dried over sodium chloride to obtain a DCM solution of compound D2-2. DIEA (13.93 g, 107.76 mmol) was added, followed by dropwise addition of Cbz-Cl (10.11 g, 59.27 mmol) over 20 minutes. The mixture was reacted for 12 hours under ice-cooling. The mixture was added to a 10% aqueous citric acid solution (200 mL*2), washed twice, and then washed with a saturated aqueous sodium bicarbonate solution (200 mL) and saturated brine (200 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain compound D2-3 (20.5 g, yield 86%). LCMS: [M+1] + 443.42 (calculated value 442.43); 1 H 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,1 H),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).

[0115] Compound D2-3 (20.5 g, 46.3 mmol) was added to a 1 L single-necked flask, and DMF (50 mL) was added. The mixture was cooled to 0°C in an ice bath under nitrogen protection, and the prepared reducing solution: H2O (250 mL) / sodium dithionite (40.33 g, 231.68 mmol) / sodium carbonate (19.64 g, 185.34 mmol) was slowly added dropwise. After the addition was complete, the temperature was slowly raised to 40°C and the reaction was continued with stirring for 2 h. The reaction solution was filtered, the filtrate was concentrated, ethyl acetate (500 mL) and water (500 mL) were added, and the mixture was separated. The mixture was extracted, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to obtain a yellow oil D2-4 (17.9 g, yield 93.7%, HPLC 98%); LCMS: [M+1] + 413.42 (calculated value 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).

[0116] Compound D2-4 (17.9 g, 43.4 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-F]indolizine-3,6,10(4H)-one (12.57 g, 47.74 mmol) and PPTS (218.13 g, 868.00 mmol) were added sequentially to a 500 mL single-necked flask. The mixture was stirred and heated at 110°C under argon for 12 h. The reaction solution was poured into methanol (1000 mL), stirred for 2 h, and filtered to obtain a dark brown solid. Methanol (300 mL) was added and stirred for 2 h. The mixture was filtered to obtain a light brown solid. The solid was dried at 40°C to obtain compound D2-5 (10.0 g, yield 36%, HPLC 95%): LCMS: [M+1] + 640.72 (calculated value 639.66); 1 H 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).

[0117] Compound D2-5 (10 g, 15.63 mmol), dichloromethane (2.5 L), and methanol (2.5 L) were added sequentially to a 10 L single-necked flask. 10% Pd / C (15 g) was added to the reaction solution, and the mixture was reacted at room temperature for 20 h under a hydrogen balloon atmosphere. Pd / C was removed by filtration, and the reaction solution was spin-dried and stirred with a mixed solution of dichloromethane and methanol (30 mL, V / V = 1:1) for 24 h. The mixture was filtered and the wet product was dried at 40°C for 3 h to obtain product D2 (6.0 g, yield 76%); LCMS: [M+1] + 506.52 (calculated value 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),5.11-4.9 5(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).

[0118] In a 250 mL single-necked flask, D2 (7.6 g, 15.03 mmol), NMP (140 mL), DIPEA (5.83 g, 45.1 mmol), Fmoc-Gly-Lys(Trt)-PAB-PNP (12.69 g, 13.53 mmol), and HOBT (2.23 g, 16.54 mmol) were added in sequence. The reaction solution was stirred at room temperature for 2 h, and dichloromethane (300 mL) was added. The solution was washed with 2% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution (100 mL), and saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain L1-D2-1; LCMS: [M+1] + 1304.93 (calculated value: 1303.53); 1 H 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).

[0119] In a 250 mL single-necked bottle, L1-D2-1 (the above product) and DCM (100 mL) were added, stirred to dissolve, and then DBU (3.61 g, 23.7 mmol) was added dropwise. The reaction was stirred at room temperature for 1 h, and then poured into methyl tert-butyl ether (1 L). The precipitated solid was filtered, dissolved in DCM (80 mL), and then added dropwise to 1 L of methyl tert-butyl ether, filtered, and dried to obtain L1-D2-2 (26 g); LCMS: [M+1] + 1082.68 (calculated value 1081.47); 1 H 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=9.2H z,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=0.7H z,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).

[0120] In a 1 L single-necked flask, L1-D2-2 (26 g crude product) and DCM (400 mL) were added in sequence. After stirring and dissolving, Fmoc-NH-PEG2-CH2CH2COOH (6.2 g, 15.6 mmol), HATU (13.7 g, 36.0 mmol) and DIPEA (4.6 g, 36.0 mmol) were added. After the addition was complete, the reaction was stirred at room temperature for 1 h. 80 mL of water was added to quench the reaction, and the organic phase was extracted with DCM. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain L1-D2-3 (11.0 g, total yield of three steps 55.5%, HPLC 96%); LCMS: [M+1] + 1463.78 (calculated value 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),4.3 8(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).

[0121] In a 100 mL single-necked flask, L1-D2-3 (5 g, 3.42 mmol) and DCM (40 mL) were added sequentially. After stirring and dissolving, DBU (364 mg, 2.39 mmol) was added dropwise. After the addition was complete, the reaction mixture was stirred at room temperature for 30 min. The reaction solution was added to methyl tert-butyl ether (500 mL), and the precipitated solid was filtered. The solid was redissolved in DCM (50 mL) and added dropwise to methyl tert-butyl ether (600 mL). The solid was filtered and the filter cake was washed with methyl tert-butyl ether and dried to obtain L1-D2-4 (2.9 g, yield 69%); LCMS: [M+1] + 1241.87 (calculated value 1240.55); 1H NMR (600MHz, DMSO-d6) δ10.03(s,1H),8.14(t,J=5.7Hz,1H),8.09(d,J=7.8Hz,1 H),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,1 H),3.89(s,1H),3.71(dd,J=24.3,7.2Hz,3H),3.60(dt,J=22.9,7.1Hz,4H),3.4 6(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),1.6 6(s,1H),1.50(dp,J=37.5,7.7Hz,4H),1.39-1.14(m,5H),0.88(t,J=7.3Hz,3H).

[0122] In a 100 mL single-necked bottle, L1-D2-4 (2.9 g, 2.3 mmol) and DCM (25 mL) were added in sequence. After stirring and dissolving, bromoacetic anhydride (667.8 mg, 2.57 mmol) was added. The mixture was stirred at room temperature for 30 min. The reaction solution was concentrated and purified by silica gel column chromatography to obtain L1-D2-5 (2.0 g, yield 64.5%, HPLC 97.6%); LCMS: [M+1] + 1361.93 (calculated value 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),6.4 9(s,1H),6.26(s,2H),5.42(d,J=2.8Hz,2H),5.24(s,2H),5.14(s,2H),4.5 8(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),3.43-3.3 1(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).

[0123] In a 100 mL single-necked bottle, L1-D2-5 (1.3 g, 0.95 mmol) and DCM (10 mL) were added sequentially. After stirring to dissolve, TFA (0.78 mL) was added dropwise. The reaction mixture was stirred at room temperature for 2 h. The reaction solution was poured into methyl tert-butyl ether (130 mL). The precipitated solid was filtered out. The solid was added to DCM (10 mL) and stirred, and then poured into methyl tert-butyl ether (100 mL). The mixture was stirred for 20 min, filtered, washed, and dried in vacuo to obtain L1-D2 as a white solid (870 mg, yield 81.7%, HPLC 97%); LCMS: [M+1] + 1119.64 (calculated value 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).

[0124] 2.2L1-D2-Ab nectin-4 Antibody-drug conjugates (ADC2)

[0125] Take Nectin4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature 25°C on a rotating turntable for 3 h.

[0126] Compound L1-D2 (0.90 mg, 0.80 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, mixed, and reacted on a rotary turntable at room temperature for 16 h. After the reaction was completed, a NAP-5 gel column (Cytiva) was used to remove small molecules and the buffer was replaced with a 20 mM PB solution, pH = 6.3, to obtain antibody-drug conjugate ADC2 (3.1 mg / mL, 2 mL).

[0127] The average value calculated by RP-MS was: n=7.9; the MS results showed that the antibody light chain (L) was connected to one L1-D2 (linker-payload), and the heavy chain (H) was connected to three L1-D2 (linker-payload) (Figure 2).

[0128] 2.3 7-(N-(Bromoacetamide-PEG8-propionyl-Gly-Lys-PABC)-N-((R)-3-tetrahydrofuran))aminoethylcamptothecin (L2-D2)

[0129] At room temperature, L1-D2-2 (1.0 g, 924.02 μmol), DMF (10 mL), Fmoc-PEG8-OSu (703.03 mg, 924.02 μmol), and DIPEA (116.11 mg, 924.02 μmol, 156 μL) were added to a 100 mL single-necked bottle in sequence. The mixture was stirred at room temperature for 1 h, concentrated, and purified by silica gel column chromatography to obtain a yellow solid L2-D2-1 (1.26 g, yield 78.25%); LCMS: [M+1] + 1728.85 (calculated value 1727.97).

[0130] At room temperature, L2-D2-1 (1.26 g, 729.59 μmol), DMF (10 mL), and piperidine (861.9 mg, 10.14 mmol, 1.0 mL) were added sequentially to a 10 mL single-necked eggplant-shaped flask. The mixture was stirred at room temperature for 20 min, concentrated, and purified by silica gel column chromatography to obtain a light yellow solid L2-D2-2 (960 mg, yield 87.27%); LCMS: [M+1] + 1506.38 (calculated value 1505.73).

[0131] At room temperature, L2-D2-2 (960 mg, 637.57 μmol), DMF (10 mL), and bromoacetic anhydride (165.69 mg, 637.57 μmol) were added to a 10 mL single-necked bottle in sequence. The mixture was stirred at room temperature for 30 min, concentrated, and purified by silica gel column chromatography to obtain a light yellow solid L2-D2-3 (440 mg, 42.31% yield); LCMS: [M+1] + 1627.51 (calculated value 1626.66).

[0132] At room temperature, L2-D2-3 (440 mg, 270.61 μmol), DCM (5 mL), and TFA (744.52 mg, 6.53 mmol, 500 μL) were added sequentially to a 10 mL single-necked eggplant-shaped flask. The reaction was stirred at room temperature for 2 h. 10 mL of ether was added dropwise to the reaction solution, and the mixture was centrifuged (10,000 rpm / 5 min). The supernatant was discarded, and the solid was rotary evaporated to remove the residual solvent to obtain a yellow solid powder L2-D2 (350 mg, yield 93.58%, HPLC 97%); LCMS: [M+1] + 1385.22 (calculated value 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),1.7 6(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,2 H),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).

[0133] Example 3: Ab nectin-4 -(S-7-(N-(acetamide-PEG2-propionyl-Gly-Lys-PAB(3-bisPEG4-carbonyl)C)-N-((R)-3-tetrahydrofuran))amineethylcamptothecin)8(ADC3)

[0134] 3.1 7-(N-(Bromoacetamide-PEG2-propionyl-Gly-Lys-PAB(3-bisPEG4-carbonyl)C)-N-((R)-3-tetrahydrofuran))amineethylcamptothecin (L3-D2)

[0135] 6-Amino-3H-isobenzofuran-1-one (4.0 g, 26.82 mmol), acetonitrile (50 mL), imidazole-1-sulfonyl azide hydrochloride (6.75 g, 32.18 mmol), CuSO4 (5.14 g, 32.18 mmol) and potassium carbonate (37.07 mg, 268.19 μmol) were added to the reaction flask. The reaction solution was stirred at room temperature for 16 h, the solid was filtered off, the filtrate was concentrated, extracted with dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, intermediate I-2 (2.0 g, yield 38%) was obtained, which was used directly in the next reaction without purification.

[0136] I-2 (6.9 g, 39.40 mmol), THF (50 mL), water (50 mL) and NaOH (2.36 g, 59.09 mmol) were added to the reaction flask, and the reaction solution was stirred at 70 ° C for 2 h, concentrated under reduced pressure, acidified with 1N HCl, and the precipitated white solid was filtered and dried to obtain 5-azido-2-hydroxymethylbenzoic acid I-3 (7.3 g, yield 91%).

[0137] 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 0°C. The mixture was stirred at room temperature overnight and poured into water. The precipitated solid was filtered, washed with water, and dried to obtain an off-white solid product 5-azido-2-(TBDMSO)methylbenzoic acid I-4 (10 g, yield 88%). 1 H 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).

[0138] N,N-bis(methoxyethoxyethoxyethoxyethyl)amine or (Me-PEG4)2NH: Methoxyethoxyethoxyethoxyethanol (40 g, 191.2 mmol), triethylamine (58 g, 567.8 mmol), p-toluenesulfonyl chloride (43.7 g, 228.2 mmol) and DCM (400 mL) were added to a reaction flask. The reaction solution was stirred at room temperature for 3 h, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain methoxyethoxyethoxyethoxyethanol p-toluenesulfonate (61.5 g, 88% yield) as an oil; LCMS: [M+H] +363.50 (calculated value 362.44). Methoxyethoxyethoxyethoxyethanol p-toluenesulfonate (58.5 g, 161 mmol), benzylamine (8.7 g, 80.5 mmol), and K2CO3 (33.4 g, 241.4 mmol) were weighed and added to a reaction flask containing acetonitrile (500 mL). The reaction mixture was stirred and heated to 70°C for 16 h. The reaction mixture was filtered, the filtrate was concentrated, and purified by silica gel column chromatography to obtain N-benzyl-N,N-bis(methoxyethoxyethoxyethoxyethyl)amine (23.1 g, yield 58.7%); LCMS: [M+H] + 488.54 (calcd. 487.63). N-Benzyl-N,N-bis(methoxyethoxyethoxyethoxyethyl)amine (10 g, 20.5 mmol), methanol (100 mL), and 10% Pd-C were added to the reaction flask. The mixture was stirred under a hydrogen balloon at room temperature for 16 h. The mixture was filtered and the filtrate was concentrated to give the oily product, N,N-bis(methoxyethoxyethoxyethoxyethyl)amine, (Me-PEG4)2NH (7.9 g, 97% yield); LCMS: [M+H] + 398.31 (calculated 397.27).

[0139] I-4 (10.5 g, 34.16 mmol), (Me-PEG4)2NH (14.93 g, 37.57 mmol), DCM (250 mL), HATU (19.48 g, 51.23 mmol) and triethylamine (17.28 g, 170.78 mmol, 23.80 mL) were added to the reaction flask. The reaction mixture was stirred at room temperature for 4 h, water was added, extracted with DCM, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to obtain a colorless liquid product 5-azido-2-(TBDMSO)methylbenzoyldi(Me-PEG4)amine L3-1 (15 g, yield 59.46%); LCMS: [M+Na] + 709.42 (calculated value 686.39); 1 H 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).

[0140] L3-1 (15 g, 21.84 mmol), DCM (250 mL) and TBAF (12.21 g, 43.67 mmol) were added to the reaction flask and stirred at room temperature for 16 h. The mixture was poured into water and extracted with DCM. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain a colorless liquid product, 5-azido-2-hydroxymethylbenzoyldi(Me-PEG4)amine L3-2 (11 g, yield 83.6%); LCMS: [M+H] + 573.32 (calculated value 572.31); 1 H 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).

[0141] 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 solution was stirred under a hydrogen balloon at normal pressure for 2 h, filtered, and concentrated to obtain a colorless liquid product, 5-amino-2-hydroxymethylbenzoyldi(Me-PEG4)amine L3-3 (9 g, yield 93.2%); LCMS: [M+Na] + 569.20 (calculated value 546.32); 1 H 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).

[0142] In a 50 mL single-necked flask, Fmoc-Lys(Trt) (366 mg, 0.60 mmol), L3-3 (500 mg, 0.92 mmol) and DCM (5 mL) were added. EEDQ (222 mg, 0.90 mmol) was added to the reaction flask under ice-cooling. Stirring was continued under ice-cooling for 12 h, and the mixture was poured into saturated aqueous sodium bicarbonate solution. The mixture was extracted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product of L3-4, which was directly used in the next reaction; LCMS: [M+1] + 1139.61 (calculated value 1138.59).

[0143] In a 50 mL single-necked bottle, the above L3-4 (~1 g crude product) was added and dissolved in DCM (6 mL). DBU (65 mg, 0.43 mmol) was added under stirring. The mixture was reacted at room temperature for 30 min. The reaction solution was directly spin-dried to obtain the crude product L3-5 (~900 mg) which was directly used for the next step; LCMS: [M+H] + 917.91 (calculated value 916.52).

[0144] In a 50 mL single-necked flask, L3-5 (~900 mg crude product) was added and dissolved in DCM (10 mL). PPTS (108 mg, 0.43 mmol), Fmoc-glycine (108 mg, 0.36 mmol), EDCI (173 mg, 0.90 mmol), and HOBT (120 mg, 0.89 mmol) were added under ice-cooling. The mixture was stirred at room temperature for 2 h. Water was added, extracted with DCM, and the mixture was washed with saturated sodium bicarbonate water and saturated brine. The mixture was dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to obtain L3-6 as a yellow oil (300 mg, total yield of three steps 41.8%); LCMS: [M+1] + 1196.87 (calculated value 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,2 H),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).

[0145] At room temperature, L3-6 (300 mg, 0.25 mmol) and DCM (3 mL) were added to a 50 mL single-necked bottle and stirred to dissolve. TEA (75 mg, 0.75 mmol) and bis(4-nitrophenyl) carbonate (228 mg, 0.75 mmol) were added dropwise. The reaction was stirred at room temperature for 2 h, poured into water, extracted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, and dried by spin drying. The product was purified by silica gel column chromatography to obtain L3 (280 mg, yield 82%); LCMS: [M+1] + 1361.64 (calculated value 1360.62); 1H NMR(600MHz,DMSO-d6)δ10.20(s,1H),8.35-8.29(m,2H),8.12(dd,J=8.5,4.2H z,1H),7.88(d,J=7.6Hz,1H),7.69(d,J=7.5Hz,2H),7.62(d,J=9.5Hz,2H),7.6 0-7.51(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),3.2 8(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).

[0146] In a 50 mL single-necked bottle, D2 (126.2 mg, 0.25 mmol), NMP (4 mL), DIEA (64.6 mg, 0.5 mmol), L3 (340 mg, 0.25 mmol), and HOBT (37.1 mg, 0.27 mmol) were added in sequence. The mixture was stirred at room temperature for 12 h, extracted with DCM (50 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL) and water (50 mL), dried over anhydrous sodium sulfate, and concentrated to give L3-D2-1 (crude product), which was directly used in the next step; LCMS: [M+1] + 1727.89 (calculated value 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=7.3H z,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=8.8,7.2 Hz,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).

[0147] In a 50 mL single-necked bottle, L3-D2-1 (crude product) and DCM (5 mL) were added in sequence, stirred to dissolve, and DBU (26.6 mg, 10.6 mmol) was added dropwise. The mixture was stirred at room temperature for 1 h, and then poured into methyl tert-butyl ether (50 mL), filtered, and dried to obtain the crude L3-D2-2, which was directly used in the next step; LCMS: [M+1] + 1505.96 (calculated value 1504.71).

[0148] In a 50 mL single-necked bottle, crude L3-D2-2 and 10 mL of DCM were added sequentially. After stirring to dissolve, the prepared Fmoc-NH-PEG2-CH2CH2COOSu (124 mg, 0.25 mmol) was added. The reaction was stirred at room temperature for 2 h, concentrated, and purified by silica gel column chromatography to obtain L3-D2-3 (87 mg, total yield of three steps 18%); LCMS: [M+1] +1886.99 (calculated value 1885.86); 1 H 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,1 H),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).

[0149] In a 50 mL single-necked bottle, L3-D2-3 (87 mg, 0.046 mmol) and DCM (5 mL) were added sequentially. After most of the solution was dissolved under stirring, DBU (4.9 mg) was added dropwise. After the addition was complete, the mixture was stirred at room temperature for 30 min, poured into methyl tert-butyl ether (60 mL), centrifuged, and the supernatant was discarded. The product L3-D2-4 was purified by silica gel column chromatography to obtain the product L3-D2-4 (70 mg, yield 91%, HPLC 96%); LCMS: [M+1] + 1664.78 (calculated value 1663.79);1 H 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).

[0150] In a 50 mL single-necked bottle, L3-D2-4 (70 mg, 0.042 mmol) and DCM (5 mL) were added in sequence and stirred to dissolve. Bromoacetic anhydride (10.9 mg, 0.042 mmol) was added. After the addition was complete, the mixture was stirred at room temperature for 30 min, concentrated, and purified by silica gel column chromatography to obtain L3-D2-5 (60 mg, yield 80%, HPLC 98%); LCMS: [M+1] + 1784.93 (calculated value 1783.72).

[0151] In a 50 mL single-necked bottle, L3-D2-5 (60 mg, 33.6 μmmol) and DCM (3 mL) were added and stirred to dissolve. TFA (0.3 mL) was added dropwise and stirred at room temperature for 1 h. The reaction solution was added dropwise to methyl tert-butyl ether (30 mL), centrifuged, the supernatant was poured off, and the remaining solid was dried to obtain a yellow solid product L3-D2 (35 mg, yield 67%, HPLC 97%); LCMS: [M+1] + 1542.84 (calculated value 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); 13 C NMR (151MHz, DMSO) δ173.04,172.28,171.25,158.70,158.47,150.55,146.77,128.56,118.58,96.41,72.87,71.70,70.25,70.20,7 0.11,70.01,69.91,69.37,69.17,68.31,67.14,65.71,61.82,58.48,50.01,42.58,38.32,36.30,30.66,29.92,27.14,22.83,8.23.

[0152] 3.2L3-D2-Ab nectin-4 Antibody-drug conjugate (ADC3)

[0153] Take Nectin4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl solution (10 mM, 0.04 mL), and react at room temperature 25°C on a rotating turntable for 3 h.

[0154] Compound L3-D2 (1.23 mg, 0.80 mmol) was dissolved in 0.12 mL of DMA, added to the above solution system, mixed, and reacted on a rotary turntable at room temperature for 16 h. After the reaction was completed, a NAP-5 gel column (Cytiva) was used to remove small molecules and the buffer was replaced with a 20 mM PB solution, pH = 6.3, to obtain antibody-drug conjugate ADC3 (3.2 mg / mL, 2 mL).

[0155] The average value calculated by RP-MS was: n=7.9; the MS results showed that the antibody light chain (L) was connected to one L3-D2 (linker-payload), and the heavy chain (H) was connected to three L3-D2 (linker-payload) (Figure 3).

[0156] Example 4: Ab nectin-4 -(S-7-(N-(acetamide-PEG2-propionyl-Gly-Lys-PAB(3-bisPEG4-carbonyl)C)-N-(4-tetrahydropyranyl))amineethylcamptothecin)8(ADC4)

[0157] 4.1 7-(N-(Bromoacetamide-PEG2-propionyl-Gly-Lys-PAB(3-bisPEG4-carbonyl)C)-N-(4-tetrahydropyranyl))amineethylcamptothecin (L3-D3)

[0158] Intermediate I-1 (18 g, 68.12 mmol) was added to IPA (180 mL), and the reaction mixture was stirred. DIEA (17.61 g, 136.24 mmol) and 4-aminotetrahydropyranyl hydrochloride (37.50 g, 272.48 mmol) were then added. The mixture was refluxed for 12 h. The reaction mixture was concentrated to a residual volume of about 20 mL of IPA. 200 mL of water was added, and the mixture was stirred for 2 h. The solid was filtered and dried at 40°C to obtain product D3-1 (20.2 g, yield 91.7%); LCMS: [M+1] + 320.98 (calculated 320.10).

[0159] Compound D3-1 (16.9 g, 52.63 mmol) was added to DCM (310 mL), and acetic acid (63.05 g, 1.05 mol) was added and stirred until all the solids dissolved. The internal temperature of the reaction solution was maintained at 15 degrees Celsius. Sodium acetate borohydride (27.89 g, 131.56 mmol) was added in 4 batches, approximately 7 g each time at an interval of approximately 5 minutes. The internal temperature of the reaction solution was maintained at 15 degrees Celsius and the reaction was allowed to proceed for 2 hours. The reaction solution was poured into an ice-water solution of sodium carbonate (sodium carbonate: 130 g, ice water: 1 L), the internal temperature was controlled at 15 degrees Celsius, stirred, and the layers were separated. The organic phase was washed with water and dried over anhydrous sodium sulfate to obtain a DCM solution of compound D3-2, which was directly used in the next step without purification.

[0160] Under ice-bath cooling and nitrogen protection, the DCM solution of D3-2 was added with DIEA (13.58 g, 105.26 mmol), and then Cbz-Cl (10.11 g, 59.27 mmol) was added dropwise. The reaction was continued with stirring for 12 h under ice-bath. A 10% aqueous citric acid solution (200 mL*2) was added, and the mixture was washed twice, then washed with a saturated aqueous sodium bicarbonate solution (200 mL) and 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).

[0161] Compound D3-3 (22.7 g, 49.7 mmol) was added to a 1 L single-necked flask, and DMF (50 mL) was added. Under nitrogen protection, the prepared reducing solution: H2O (250 mL) / sodium dithionite (39.30 g, 225.72 mmol) / sodium carbonate (19.14 g, 180.58 mmol) was slowly added dropwise under ice-bath cooling. After the addition was complete, the temperature was slowly raised to 40°C and stirred for 2 h. The filtrate was filtered and concentrated. Ethyl acetate (500 mL) and water (500 mL) were added, and the liquid was extracted. The mixture was dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to obtain a yellow oil D3-4 (20.3 g, yield 95.7%, HPLC 98%); LCMS: M+1] + 427.22 (calculated 426.19).

[0162] Compound D3-4 (18.56 g, 43.55 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-F]indolizine-3,6,10(4H)-one (13.75 g, 52.27 mmol) and PPTS (218.88 g, 871.00 mmol) were added sequentially to a 500 mL single-necked bottle. The mixture was reacted at 110°C for 12 h under argon protection. The reaction solution was poured into methanol (1000 mL), stirred for 2 h, and filtered to obtain a dark brown solid. The brown solid was washed with methanol (300 mL) for 2 h and filtered to obtain a light brown solid. The solid was dried at 40°C to obtain compound D3-5 (11.0 g, yield 38.7%, HPLC 97%); LCMS: [M+1] + 653.98 (calculated value 653.24); 1 H 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).

[0163] Compound D3-5 (10.32 g, 15.78 mmol), dichloromethane (2.5 L), methanol (2.5 L) and 10% Pd / C (15 g) were added sequentially to a 10 L single-necked flask. The mixture was reacted at room temperature for 20 h under a hydrogen balloon atmosphere. The Pd / C was removed by filtration. The reaction solution was spin-dried and stirred with 30 mL of a mixed solution of dichloromethane and methanol (V / V = 1:1) for 24 h. The mixture was filtered and dried at 40°C for 3 h to obtain product D3 (6.1 g, yield 74%, HPLC 95.0%); LCMS: [M+1] + 520.22 (calculated value 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,2 H),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).

[0164] In a 50 mL single-necked bottle, D3 (130.0 mg, 0.25 mmol), NMP (4 mL), DIEA (64.6 mg, 0.5 mmol), L3 (340 mg, 0.25 mmol), and HOBT (37.1 mg, 0.27 mmol) were added in sequence. The mixture was stirred at room temperature for 12 h, extracted with DCM, washed with saturated aqueous sodium bicarbonate solution (50 mL) and water (50 mL), dried over anhydrous sodium sulfate, and concentrated to give L3-D3-1 (crude product), which was directly used in the next step; LCMS: [M+1] + 1741.93 (calculated value 1740.79).

[0165] In a 50 mL single-necked bottle, the above L3-D3-1 (crude product) and DCM (5 mL) were added in sequence and stirred to dissolve. DBU (26.6 mg) was added dropwise and stirred at room temperature for 1 h. The mixture was poured into methyl tert-butyl ether (50 mL), centrifuged, and dried to obtain L3-D3-2 (crude product), which was directly used in the next step without purification; LCMS: [M+1] + 1519.96 (calculated value 1518.72).

[0166] In a 50 mL single-necked bottle, the above L3-D3-2 (crude product) and DCM (10 mL) were added in sequence and stirred to dissolve. The prepared Fmoc-NH-PEG2-CH2CH2COOSu (124 mg, 0.25 mmol) was added and the reaction was continued at room temperature with stirring for 2 h. The mixture was concentrated and purified by silica gel column chromatography to obtain the product L3-D3-3 (89 mg, total yield of three steps 18.7%); LCMS: [M+1] + 1900.97 (calculated value 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).

[0167] In a 50 mL single-necked bottle, L3-D3-3 (87 mg, 0.045 mmol) and DCM (6 mL) were added sequentially and stirred to dissolve. DBU (4.9 mg) was added dropwise and stirred at room temperature for 30 min. The mixture was poured into methyl tert-butyl ether (50 mL) and centrifuged. The supernatant was discarded and purified by silica gel column chromatography to obtain L3-D3-4 (74 mg, yield 97%, HPLC 96%); LCMS: [M+1] + 1678.96 (calculated value 1678.82).

[0168] In a 50 mL single-necked bottle, L3-D3-4 (73 mg, 0.043 mmol) and DCM (5 mL) were added in sequence and stirred to dissolve. Bromoacetic anhydride (11.0 mg, 0.043 mmol) was added and stirred at room temperature for 30 min. The mixture was concentrated and purified by silica gel column chromatography to obtain L3-D3-5 (65 mg, yield 84%, HPLC 98%); LCMS: [M+1] + 1798.98 (calculated value 1798.74).

[0169] L3-D3-5 (64 mg, 35.6 μmmol) and DCM (5 mL) were added to a 50 mL single-necked bottle and stirred to dissolve. TFA (0.3 mL) was then added dropwise to the reaction solution. The reaction was stirred at room temperature for 1 h. The reaction solution was added dropwise to methyl tert-butyl ether (30 mL). The solid was centrifuged and dried to give a yellow solid product L3-D3 (35 mg, yield 63%, HPLC 97%); LCMS: [M+1]+ 1556.87 (calculated value 1556.63); 1 H NMR(500MHz,DMSO-d6)δ10.12(s,1H),8.40-8.15(m,2H),7.75-7.65(m,3H),7.5-7.4 0(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); 13 C NMR (126MHz, DMSO-d6) δ173.00,171.22,166.56,158.71,158.44,157.29,151. 37,150.59,149.88,146.81,128.62,118.54,103.07,72.88,71.71,70.26,70.2 1,70.13,70.02,69.96,69.91,69.17,67.13,65.71,65.37,58.48,50.16,48.86,44.46,42.61,39.15,36.32,31.77,30.72,29.91,27.15,22.84,15.63,8.23.

[0170] 4.2L3-D3-Ab nectin-4 Antibody-drug conjugate (ADC4)

[0171] Take Nectin4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl solution (10 mM, 0.04 mL), and react at room temperature 25°C on a rotating turntable for 3 h.

[0172] Compound L3-D3 (1.25 mg, 0.80 mmol) was dissolved in 0.13 mL of DMA, added to the above solution system, mixed, and reacted on a rotary turntable at room temperature for 16 h. After the reaction was completed, a NAP-5 gel column (Cytiva) was used to remove small molecules and the buffer was replaced with a 20 mM PB solution, pH = 6.3, to obtain antibody-drug conjugate ADC4 (3.2 mg / mL, 2 mL).

[0173] The average value calculated by RP-MS was: n=7.8; the MS results showed that the antibody light chain (L) was connected to one L3-D3 (linker-payload), and the heavy chain (H) was connected to three L3-D3 (linker-payload) (Figure 4).

[0174] Example 5: Ab nectin-4 -(S-7-(N-(Bromoacetamide-PEG2-propionyl-Gly-Lys-PAB(2-PEG8-oxy)C)-N-((R)-3-tetrahydrofuran))amineethylcamptothecin)8(ADC5)

[0175] 5.1 7-(N-(Bromoacetamide-PEG2-propionyl-Gly-Lys-PAB(2-PEG8-oxy)C)-N-((R)-3-tetrahydrofuran))amineethylcamptothecin (L4-D2)

[0176] 2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]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 and stirred at room temperature for 16 h. The mixture was concentrated under reduced pressure, extracted with ethyl acetate, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to obtain a colorless liquid intermediate p-toluenesulfonate L4-1 (50 g, yield 71.37%); LCMS: [M+H] + 539.38 (calculated 538.24).

[0177] 3-Hydroxy-4-nitrobenzaldehyde (23.27 g, 139.24 mmol), L4-1 (50 g, 92.82 mmol), DMF (300 mL) and K2CO3 (38.49 g, 278.47 mmol) were added to the reaction flask and stirred at 70°C for 2 h. The mixture was concentrated, extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain a brown liquid intermediate compound L4-2 (27 g, yield 54.51%); LCMS: [M+H]+ 534.38 (calculated 533.25).

[0178] 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, and the reaction was stirred at room temperature for 4 h. The mixture was concentrated under reduced pressure, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a brown liquid product L4-3 (19 g, yield 90.12%).

[0179] L4-3 (19 g, 35.47 mmol), MeOH (250 mL), 10% Pd / C (2 g, 7.1 mmol) and DIPEA (9.17 g, 70.95 mmol) were added to the reaction flask. The reaction solution was stirred under a hydrogen balloon atmosphere for 48 h, filtered, and concentrated under reduced pressure to obtain a brown liquid compound L4-4 (17.5 g, yield 97.58%): LCMS: [M+H] + 506.21 (calculated 505.29).

[0180] In a 1000 mL single-necked flask, L4-4 (10 g, 19.78 mmol), 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 and stirred at 25°C for 2 h. Saturated aqueous sodium bicarbonate solution (300 mL) was added and stirred. The mixture was extracted and separated, and washed with aqueous citric acid solution and saturated brine, respectively. The mixture was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain Fmoc-Lys(Trt)PAB(PEG8)L4-5 (17 g, yield 75.13%); LCMS: [M+1] + 1098.21 (calculated value 1097.34).

[0181] L4-5 (17 g, 14.86 mmol), DCM (400 mL), and DBU (1.13 g, 7.43 mmol) were added to a 1000 mL single-necked bottle. The reaction was stirred at 25°C for 1 h and concentrated to obtain L4-6, which was used directly in the next step. LCMS: [M+1] + 876.34 (calculated 875.10).

[0182] To the reaction mixture from the previous step were added 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). The reaction was stirred at 25°C for 2 h. 200 mL of aqueous citric acid solution was added and stirred, and the mixture was extracted and separated. The mixture was washed with saturated aqueous sodium bicarbonate solution and saturated brine, separated, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain FmocGly-Lys(Trt)PAB(PEG8)L4-7 (15 g, two-step yield 83.88%); LCMS: [M+1] + 1155.60 (calculated value 1154.40); 1 H 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,6 H),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),1.5 6(tq,J=13.5,8.4,6.5Hz,1H),1.46(hept,J=7.9,7.4Hz,2H),1.35–1.27(m,2H).

[0183] At room temperature, D2 (0.5 g, 918.08 μmol), NMP (10 mL), L4 (1.21 g, 891.68 μmol), HOBT (140.59 mg, 918.08 μmol), and DIPEA (118.65 mg, 918.08 μmol, 159.91 μL) were added to a 250 mL single-necked bottle in sequence. The mixture was stirred at room temperature for 16 h. Ether (100 mL) was added and the precipitated solid was filtered to obtain a light yellow solid L4-D2-1 (1.5 g, yield 87.1%); LCMS: [M+1] + 1687.56 (calculated value: 1686.92).

[0184] At room temperature, L4-D2-1 (1.5 g, 799.54 μmol), NMP (9 mL), and piperidine (824.84 mg, 9.69 mmol, 956.90 μL) were added to a 250 mL single-necked bottle in sequence. The mixture was stirred at room temperature for 1 h. Ether (100 mL) was added and the solid was filtered to obtain a light brown solid L4-D2-2 (1.2 g, yield 77.0%); LCMS: [M+1] + 1465.38 (calculated value: 1464.67).

[0185] At room temperature, L4-D2-2 (1.2 g, 678.31 μmol), DMF (10 mL), Fmoc-PEG2-OSu (396.22 mg, 739.75 μmol), and DIPEA (103.14 mg, 798.01 μmol, 139.00 μL) were added to a 100 mL single-necked bottle in sequence. The mixture was stirred at room temperature for 0.5 h. 10 g of silica gel powder was added for sand making and the mixture was purified by silica gel column chromatography to obtain an off-white solid L4-D2-3 (410 mg, yield 25.9%); LCMS: [M+1] + 1847.01 (calculated value: 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=7.5,6.7H z,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=7.0 Hz,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).

[0186] At room temperature, L4-D2-3 (0.41 g, 217.49 μmol), DMF (4 mL), and piperidine (370.37 mg, 4.35 mmol, 429.66 μL) were added sequentially to a 10 mL single-necked eggplant-shaped flask. The mixture was stirred at room temperature for 1 h. 5 g of silica gel powder was added for sanding and the mixture was purified by silica gel column chromatography to obtain a light yellow solid L4-D2-4 (230 mg, yield 61.05%); LCMS: [M+1] + 1624.75 (calculated value: 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),5.44-5.4 1(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,1 H),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).

[0187] At room temperature, L4-D2-4 (100 mg, 60.13 μmol), DMF (2 mL), and bromoacetic anhydride (18 mg, 60.13 μmol) were added to a 10 mL single-necked bottle in sequence. The mixture was stirred at room temperature for 2 h. 3 g of silica gel powder was added for sand preparation and purified by silica gel column chromatography to obtain a light yellow solid L4-D2-5 (80 mg, yield 72.34%); LCMS: [M+1] + 1745.62 (calculated value: 1744.79).

[0188] At room temperature, L4-D2-5 (80 mg, 44.85 μmol), DCM (1 mL), and TFA (153.41 mg, 1.35 mmol, 102.96 μL) were added sequentially to a 10 mL single-necked eggplant-shaped flask. The mixture was stirred at room temperature for 2 h. Ether (10 mL) was added and the mixture was centrifuged (10000 rpm / 5 min). The supernatant was discarded and the residual solvent was concentrated under reduced pressure to obtain a yellow solid powder L4-D2 (50 mg, yield 70.15%); LCMS: [M+1] + 1503.33 (calculated value: 1502.47); 11H NMR (600 MHz, DMSO-d6) δ 9.05 (s, 1H), 8.33 (t, J = 5.7 Hz, 1H), 8.24 (dd, J = 7.9, 4.3 Hz, 1H), 8.14 (t, J = 5.7 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.80 (s, 1H), 7.66 (q, J = 6.2 Hz, 4H), 7.52 - 7.48 (m, 1H), 7.37 (s, 1H), 7.24 (s, 1H), 7.18 (s, 1H), 7.02 (d, J = 15.5 Hz, 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 = 7.6 Hz, 1H), 4.48 (t, J = 5.8 Hz, 1H), 4.18 (d, J = 8.7 Hz, 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, 6.9 Hz, 1H), 3.60 (t, J = 6.5 Hz, 4H), 3.48 (dd, J = 10.4, 3.9 Hz, 16H), 3.41 (q, J = 4.8 Hz, 5H), 3.38 (d, J = 7.0 Hz, 1H), 3.32 (s, 2H), 3.27 - 3.19 (m, 2H), 3.22 (s, 3H), 2.78 (h, J = 6.6 Hz, 2H), 2.40 (t, J = 6.5 Hz, 2H), 2.16 (dtd, J = 13.4, 8.6, 5.1 Hz, 1H), 1.85 (dqt, J = 27.7, 13.5, 6.6 Hz, 3H), 1.62 (dp, J = 13.8, 4.7 Hz, 1H), 1.54 (hpt, J = 6.6 Hz, 2H), 1.37 (ddt, J = 23.6, 15.8, 7.0 Hz, 2H), 1.24 (d, J = 5.7 Hz, 1H), 1.09 (t, J = 7.0 Hz, 1H), 0.88 (t, J = 7.3 Hz, 3H).

[0189] 5.2L4-D2-Ab nectin-4 Antibody-drug conjugate (ADC5)

[0190] Take nectin-4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl solution (10 mM, 0.04 mL), and react at room temperature (25°C) on a rotating turntable for 3 h.

[0191] Compound L4-D2 (1.20 mg, 0.80 mmol) was dissolved in 0.12 mL of DMA, added to the above solution system, mixed, and reacted on a rotary turntable at room temperature for 16 h. After the reaction was completed, a NAP-5 gel column (Cytiva) was used to remove small molecules and the buffer was replaced with a 20 mM PB solution, pH = 6.3, to obtain antibody-drug conjugate ADC5 (3.1 mg / mL, 2 mL).

[0192] The average value calculated by RP-MS was: n=7.9; the MS results showed that the antibody light chain (L) was connected to one L4-D2 (linker-payload), and the heavy chain (H) was connected to three L4-D2 (linker-payload) (Figure 5).

[0193] Test example: ADC anti-tumor activity experiment

[0194] Test Example 1: In vitro inhibition of tumor cell growth by ADC

[0195] 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 (Cellmax), DMEM (Cellmax), MEM (Cellmax), RPMI1640 (Cellmax) and RPMI1640 (Cellmax) culture media containing 10% fetal bovine serum (Cellmax) until the exponential growth phase, and then trypsinized, centrifuged and the supernatant was discarded. The cells were diluted with culture medium to 6×10 4 cells / mL、6×10 4 cells / mL, 2×10 4 cells / mL、3×10 4 cells / mL and 7×10 4cells / mL, 100 μL was added to each well of a 96-well cell culture plate and returned to a 37°C, 5% CO2 incubator for overnight incubation. The next day, the ADC to be tested was diluted in culture medium to 12000 nM, 1200 nM, 120 nM, 12 nM, 1.2 nM, 0.12 nM, 0.012 nM, 0.0012 nM, and 0.00012 nM. 100 μL of the diluted ADC was added to each well of the 96-well cell culture plate, with three replicates for each concentration. For the negative control and blank control groups without ADC, 100 μL of culture medium was added to each well. After addition, the plates were returned to a 37°C, 5% CO2 incubator for an additional 6 days. After incubation, the plates were removed, the culture medium was aspirated, and 100 μL of culture medium containing 10% CCK-8 was added to each well. The plates were incubated at 37°C for 3 hours. After incubation, remove the culture plate, protect from light, and place it in a microplate reader. Select 630 nm as the reference wavelength and 450 nm as the measurement wavelength to measure the absorbance. Based on the absorbance value, use the four-parameter regression in GraphPad to calculate the IC 50 (Table 1). ) is the positive control drug.

[0196] For IC 50 Values, where “++++” means 50nM>IC 50 ; “+++” means 200nM>IC 50 ≥50nM; “++” indicates 1000nM>IC 50 ≥200nM; “+” indicates IC 50 >1μM.

[0197] Table 1 Inhibitory activity of ADC of the present invention on cancer cells

[0198] The ADC compounds provided in the examples of the present invention all have a good inhibitory effect on the growth of cancer cells and have significant anti-cancer activity.

[0199] Test Example 2: In vivo tumor growth inhibition activity of ADC

[0200] Human breast cancer cells MDA-MB-468, human bladder transitional cell carcinoma SW-780, human lung cancer cells NCI-H292, human bladder cancer cells HT-1376 and human pancreatic adenocarcinoma cells BxPC-3 were cultured in monolayer in vitro. When the cell saturation reached 80%-90%, the cells were digested with trypsin-EDTA, centrifuged and the supernatant was discarded. The cells were resuspended in PBS and the cell suspension was adjusted to an appropriate concentration. Human breast cancer cells MDA-MB-468, human bladder 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) were subcutaneously inoculated into BALB / c nude mice, and the animals and transplanted tumor growth were regularly observed. 3 The animals were randomly divided into two groups according to tumor volume and body weight: a vehicle control group (normal saline) and an ADC-treated group (dissolved in normal saline), with 6 animals in each group. The drug was administered intravenously once (the time of the first administration was recorded as Day 0). The long diameter a (mm), short diameter b (mm) of the tumor and the body weight of the mice were measured with a vernier caliper twice a week. The tumor volume (V) was calculated according to the following formula: V = 1 / 2 × a × b 2 (mm 3 ), where a and b represent tumor length and width, respectively. Statistical analysis was performed based on tumor volume data at the end of the experiment to determine tumor inhibition rate, expressed as: 100% * (mean tumor volume of the blank control (vehicle) group - mean tumor volume of the test group) / mean tumor volume of the blank control (vehicle) group.

[0201] Table 2

[0202] Note: “-” means not tested.

[0203] The vehicle group is a blank control; N4-Dxd represents the ADC formed by coupling Dxd (GGFG linker) and the anti-nectin-4 antibody of the present application example;

[0204] Its structural formula is as follows:

[0205] Experimental results: The four doses of ADC1 and ADC2, 2 mg / kg, 3 mg / kg, 6 mg / kg and 10 mg / kg, all had significant activity in inhibiting tumor growth.

[0206] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A compound of formula (I), a pharmaceutically acceptable salt or stereoisomer thereof: In the formula, R1 and R2 are each independently selected from hydrogen, fluorine, and C 1-3 alkyl, or R1 and R2 together with the carbon atom to which they are attached form an oxygen-containing heterocyclic group; R3 is selected from C 1-6 alkyl, C 1-6 alkoxy, C 3-6 cycloalkyl or 4- to 8-membered heteroalkyl; the C 1-6 alkyl, C 1-3 alkoxy is optionally substituted by one or more halogens; the 4- to 8-membered heteroalkyl contains 1, 2 or 3 heteroatoms selected from N, O, S as ring atoms; R4 is selected from hydrogen or a heteroalkyl group containing -OCH2CH2- repeating units; R5 is selected from hydrogen, C 1-6 alkyl and C 3-6 cycloalkyl; Lp is selected from peptide residues containing 1 to 5 amino acids; m is selected from integers from 1 to 8; a and p are selected from 1, 2 or 3; Ab is an anti-Nectin-4 antibody or an antigen-binding fragment; Z is a linker capable of coupling the antibody or antigen-binding fragment to other parts of the compound of formula (I); 0.5≤n≤8。 2. The compound according to claim 1, wherein, R1 and R2 are each independently selected from hydrogen, fluorine, and methyl, or R1, R2, and the carbon atom to which they are attached together form Preferably, R1 is hydrogen and R2 is hydrogen; R1 is fluorine and R2 is fluorine; or R1 is methyl and R2 is fluorine.

3. The compound according to claim 1 or 2, wherein, R3 is selected from C alkyl substituted with 1, 2 or 3 fluorine atoms; or is selected from oxacycloalkyl groups having 4 to 8 ring members; 1-6 ​ Preferably, R3 is selected from fluoroethyl, difluoroethyl, trifluoroethyl, oxolanyl or oxanyl.

4. The compound according to any one of claims 1-3, wherein, R4 is selected from hydrogen, C 1-6 alkyl, and -C(O)-NR a R b ; R a 、R b Each independently selected from C 1-6 alkyl; wherein one or more methylene units in said C 1-6 alkyl are optionally and independently replaced by -(OCH2CH2)q-; q is selected from integers from 2 to 10; Preferably, R4 is selected from hydrogen, 5. The compound according to any one of claims 1-4, wherein, L p 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-; Preferably, L p is selected from 6. The compound according to any one of claims 1-5, wherein Z is selected from wherein, the indicated position represents being connected to an antibody, the indicated position represents being connected to -NH-.

7. The compound according to any one of claims 1-6, wherein The heavy chain amino acid sequence of the anti-Nectin-4 antibody is as shown in SEQ ID NO:1, and its light chain amino acid sequence is as shown in SEQ ID NO:

2.

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

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

10. Use of the compound of formula (I) according to any one of claims 1-8, a pharmaceutically acceptable salt or stereoisomer thereof, or the pharmaceutical composition according to claim 9 in the preparation of an anti-tumor drug or a drug for treating an autoimmune disease; Preferably, the tumor is selected from solid tumors, more preferably breast cancer, bladder transitional cell carcinoma, prostate cancer and pancreatic adenocarcinoma.

11. A method for treating cancer, which comprises the step of administering to a patient in need the compound of formula (I) according to any one of claims 1-8, a pharmaceutically acceptable salt or stereoisomer thereof, or the pharmaceutical composition according to claim 9; Preferably, the cancer is selected from breast cancer, bladder transitional cell carcinoma, prostate cancer and pancreatic adenocarcinoma.

Images