Hydrophilic anti-Nectin-4 antibody-drug conjugate and its preparation method and uses

Abstract

The present specification provides a toxin-hydrophilic linker compound formed with camptothecin-7-ethylamine and a linker, a conjugate thereof with an anti-Nectin-4 antibody, and the pharmaceutical uses thereof. This toxin-hydrophilic linker compound can be conjugated with an anti-Nectin-4 monoclonal antibody to form an antibody-drug conjugate (ADC), which increases the hydrophilicity of the ADC and reduces aggregation in the circulatory system, thereby enhancing efficacy and reducing toxic side effects.

Description

Technology Field

[0001] This application claims priority to Chinese patent application 2024100518885, filed on January 12, 2024, and incorporates all the contents of said Chinese patent application.

[0002] The present invention relates to the field of medicine. Specifically, the present invention provides a series of camptothecin-7-ethylamines and toxin-hydrophilic linker compounds formed with the same as a linker, antibody-drug conjugates formed by conjugating with a corresponding anti-Nectin-4 antibody, and the use thereof for cancer treatment. Background Technology

[0003] Camptothecin and its derivatives exhibit inhibitory activity against topoisomerase Top1, particularly strong inhibitory activity against complexes formed by Top1-DNA. Camptothecin is known as a broad-spectrum antitumor drug, as it demonstrates significant therapeutic effects against gastric cancer, esophageal cancer, lung cancer, bladder cancer, and others. Among them, the camptothecin derivatives irinotecan and topotecan have been approved as treatments for various cancers in numerous countries. Another camptothecin derivative, belotecan, has been approved in Korea as a treatment for small cell lung cancer (SCLC) and ovarian cancer. The major drawbacks of camptothecin-based antitumor drugs are toxicity, poor solubility, and drug resistance exhibited by tumor cells. The problem to be solved

[0004] The present invention provides a toxin-hydrophilic linker compound formed of a series of camptothecin-7-ethylamines and a linker, wherein the compound contains a hydrophilic structure composed of at least two PEGs, and such toxin-hydrophilic linker can be conjugated with the monoclonal antibody Nectin-4 to form an antibody-drug conjugate (ADC), thereby increasing the hydrophilicity of the ADC and reducing aggregation in the circulatory system, thereby enhancing the efficacy and reducing toxic side effects. means of solving the problem

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

[0006]

[0007] In the equation,

[0008] R1 and R2 are independently hydrogen, fluorine, and C, respectively. 1-3 Selected from alkyls, or R1, R2 form an oxygen-containing heterocyclic group together with carbon atoms connected thereto;

[0009] R3 is C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Selected from cycloalkyl or 4 to 8-membered heterocycloalkyl; said C 1-6 Alkyl, C 1-3 The alkoxy is optionally substituted with one or more halogens; the 4 to 8-membered heterocycloalkyl comprises 1, 2, or 3 heteroatoms selected from N, O, and S as ring atoms;

[0010] R4 is selected from heteroalkyls containing hydrogen or -OCH2CH2- repeating units;

[0011] R5 is hydrogen, C 1-6 Alkyl and C 3-6 Selected from cycloalkyls;

[0012] Lp is selected from peptide residues containing 1 to 5 amino acids;

[0013] m is an integer selected from 1 to 8;

[0014] a, p are selected from 1, 2, or 3;

[0015] Ab is an anti-Nectin-4 antibody or antigen-binding fragment;

[0016] Z is a linker capable of conjugating an antibody or antigen-binding fragment with other parts of the compound of formula (I);

[0017] 0.5≤n≤8.

[0018] In one embodiment, the compound of formula (I) has a structure represented by formula (I-1) or formula (I-2):

[0019]

[0020]

[0021] In the formula, each functional group is defined identically to the compound in (I).

[0022] In one implementation plan, m is selected from 1, 2, 3, 4, 5, 6, 7, or 8.

[0023] In one embodiment, R1 and R2 are each independently selected from hydrogen, fluorine, and methyl, or R1 and R2 are together with carbon atoms connected thereto. forms.

[0024] In one implementation plan, R1 is hydrogen and R2 is hydrogen.

[0025] In one implementation plan, R1 is fluorine and R2 is fluorine.

[0026] In one implementation plan, R1 is methyl and R2 is fluorine.

[0027] In one embodiment, R3 is C substituted with 1, 2, or 3 fluorine groups. 1-6 It is selected from alkyl groups.

[0028] In one embodiment, R3 is selected from 4 to 8 oxacycloalkyl groups.

[0029] In one embodiment, R3 is selected from oxacyclobutyl, oxacyclopentyl, or oxacyclohexyl.

[0030] In one embodiment, R3 is selected from fluoroethyl, difluoroethyl, trifluoroethyl, oxacyclopentyl, or oxacyclohexyl.

[0031] In one implementation plan, R4 is hydrogen, C 1-6Alkyl and -C(O)-NR a R b Selected from among;

[0032] R a , R b C each independently 1-6 Selected from alkyls;

[0033] Here, the above C 1-6 One or more methylene units within the alkyl group are optionally and independently substituted with -(OCH2CH2)q-;

[0034] q is selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0035] In the implementation plan, R4 is hydrogen, and It is selected from among them.

[0036] In the implementation plan, L p is selected from -Val-Cit-, -Gly-Lys-, -Gly-Leu-, -Val-Ala-, -Gly-Phe-, -Gly-Gly-Lys-, -Gly-Gly-Phe-, -Gly-Val-Ala-, -Gly-Gly-Val-, -Gly-Leu-Val-, -Gly-Phe-Gly- or -Gly-Gly-Leu-.

[0037] In the implementation plan, L p Is or It is selected from among them.

[0038] In the present invention, a linker for conjugating an antibody or antigen-binding fragment with another part of the compound of formula (I) may be a single linker or a double linker, and the double linker refers to a structure capable of simultaneously connecting two chemical functional groups (specifically, an antibody and a toxin portion). This structure has a functional group connected to the antibody and a functional group connected to the toxin portion.

[0039] In one implementation plan, Z is or Selected from among, and here " The location marked with " indicates the location connected to the antibody, and " The location marked with " indicates the location connected to -NH-.

[0040] In one embodiment, the heavy chain amino acid sequence of the anti-Nectin-4 antibody is denoted by SEQ ID NO: 1, and the light chain amino acid sequence is denoted by SEQ ID NO: 2.

[0041] The present invention provides the following toxin-linker compounds, pharmaceutically acceptable salts or stereoisomers thereof.

[0042]

[0043] The present invention provides the following compounds, pharmaceutically acceptable salts or stereoisomers thereof.

[0044]

[0045]

[0046] In another aspect of the present invention, a pharmaceutical composition is provided, comprising a compound of the aforementioned formula (I), a pharmaceutically acceptable salt or stereoisomer thereof; and a pharmaceutically acceptable carrier.

[0047] In another aspect of the present invention, the compound of formula (I) described above, a pharmaceutically acceptable salt or stereoisomer thereof, or the pharmaceutical composition described above is provided for use in the manufacture of antitumor drugs;

[0048] Preferably, the tumor is selected from solid tumors.

[0049] In one embodiment, the tumor is selected from breast cancer, bladder transitional cell carcinoma, prostate cancer, and pancreatic adenocarcinoma.

[0050] In another aspect of the present invention, a method for treating cancer is provided, comprising the step of administering to a patient in need of the compound of formula (I) described above, a pharmaceutically acceptable salt or stereoisomer thereof, or the pharmaceutical composition described above.

[0051] In one embodiment, the cancer is selected from breast cancer, bladder transitional cell carcinoma, prostate cancer, and pancreatic adenocarcinoma.

[0052] In another aspect of the present invention, a method for preparing a compound of formula (I) is provided, comprising the following:

[0053]

[0054] Here, each functional group is defined identically to the compound in (I);

[0055] The reaction reagents used in each step correspond to Examples 1-5.

[0056] In one embodiment, the reaction reagents used in each step are as follows: 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, then condensed with Fmoc-PEG2-OH; j) piperidine, then condensed with the corresponding Z linker; k) conjugated with an anti-Nectin-4 antibody to form an anti-Nectin-4 ADC. Effects of the invention

[0057] The toxin-hydrophilic linker provided by the present invention forms an antibody-drug conjugate (ADC) by conjugating with a Nectin-4 monoclonal antibody, thereby reducing the aggregation of the ADC within the circulatory system, which helps to improve efficacy and reduce toxic side effects. Brief explanation of the drawing

[0058] Figure 1 shows the RP-MS value test results of ADC1 according to Example 1; where A and B are HPLC results and C and D are MS results. FIG. 2 shows the RP-MS value test results of ADC2 according to Example 2; where A and B are HPLC results and C and D are MS results. FIG. 3 shows the RP-MS value test results of ADC3 according to Example 3; where A and B are HPLC results and C and D are MS results. Figure 4 shows the RP-MS value test results of ADC4 according to Example 4; where A and B are HPLC results and C and D are MS results. FIG. 5 shows the RP-MS value test results of ADC5 according to Example 5; where A and B are HPLC results and C and D are MS results. Specific details for implementing the invention

[0059] I. Definition

[0060] In this invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, related terms and laboratory operation steps used herein all refer to terms and general steps widely used in the relevant field. In addition, definitions and interpretations of related terms are provided below to better understand this invention.

[0061] Unless otherwise specified in the text, the terms "approximately" or "roughly" mean within ±10% of a specified value or range. Where an integer is required, this term means rounding up or down to the nearest integer within ±10% of the specified value or range.

[0062] "Some embodiments," "some embodiments," or "some implementations" mentioned in the description of the text describe a subset of all possible embodiments; however, it is understood that "some embodiments" may be the same or different subsets of all possible embodiments and may be combined as long as they do not contradict each other.

[0063] The terms “include,” “include,” “have,” and “contain” used in the text include grammatical equivalents and should generally be understood as open and non-restrictive, for example, without excluding other elements or stages that are not listed.

[0064] The term "antibody" as used in this application is used in its broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the necessary biological activity. Antibodies may be derived from mice, humans, humanized, chimeras, or other species. Antibodies are proteins produced by the immune system that recognize and bind to specific antigens. Target antigens generally have multiple binding sites, also known as epitopes, which are recognized by the complementary determining regions (CDRs) of various antibodies. Each antibody that specifically binds to different epitopes has a different structure. Therefore, a single antigen may have two or more corresponding antibodies. Antibodies include full-length immunoglobulin molecules or the immune-active portion of full-length immunoglobulin molecules, namely molecules containing an antigen-binding site that binds immune-specifically to a target antigen or a part thereof, and targets include, but are not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The term "antibody" means an immunoglobulin molecule capable of binding to a specific antigen. It consists of two light chains with small molecular weights and two heavy chains with large molecular weights, and the heavy chains (H chains) and light chains (L chains) are connected by disulfide bonds to form a molecule consisting of one four-peptide chain.

[0065] As used in this application, the term "antibody-drug conjugate (ADC)" refers to a structure in which a bioactive small molecule drug is linked to a monoclonal antibody via a single chemical linker, wherein the monoclonal antibody acts as a carrier to deliver the small molecule drug to target cells.

[0066] The term "toxin" as used in this application refers to a compound having a killing effect on tumor cells, also referred to as a "cytotoxic drug component" or "small molecule drug." Examples of cytotoxic drug components include at least one of antitubulin agents, DNA inserts, DNA topoisomerase inhibitors, DNA synthesis inhibitors, RNA polymerase inhibitors, splicesome inhibitors, proteolysis-targeting chimeras (PROTACs), and immunomodulators.

[0067] The small molecule drugs used in this application may be asymmetric and, for example, may have one or more stereoisomers. Unless otherwise noted, all stereoisomers include both enantiomers and diastereomers. The stereoisomers include geometric isomers (e.g., cis, trans structures) and optical isomers (e.g., enantiomers), and include therapeutic agents composed of monomers, racemic mixtures, racemic mixtures, and pharmaceutically acceptable salts thereof. Compounds containing asymmetric carbon atoms in this application can be separated into an optically active pure form or a racemic form. The optically active pure form may be separated from a racemic mixture or synthesized using chiral raw materials or chiral reagents. Racemic mixtures, diastereomers, and enantiomers are all included within the scope of this application.

[0068] The small molecule drug used in this application further comprises a tautomeric form. A tautomeric form refers to a form in which the positions of a single bond and an adjacent double bond are exchanged, accompanied by a corresponding proton transfer.

[0069] The numeric range in the text refers to each integer within the specified range. For example, "C1-C6" means that the corresponding functional group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms; and "C3-C6" means that the corresponding functional group can have 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

[0070] Any variable (e.g., R) within the compound composition or structure n If ) appears one or more times, the definition of the corresponding variable in each case is independent. Thus, for example, when a functional group can be substituted with 1 to 5 Rs, said functional group may optionally be substituted with up to 5 Rs, and the Rs in each case have independent options. Additionally, combinations of substituents and / or variants thereof are permitted only if such combinations form a stable compound.

[0071] The term "alkyl group" as used in this application refers to a saturated aliphatic hydrocarbon group, meaning a straight-chain or branched-chain functional group containing 1 to 20 carbon atoms, preferably an alkyl containing 1 to 6 carbon atoms. Non-limiting embodiments include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, and 2,3-dimethylbutyl. The alkyl group may or may not be substituted, and if substituted, the substituent may be substituted at any possible linkage site, and the substituent is preferably one or more functional groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxy, or carboxylic acid ester groups, and in this application, preferably methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuterated alkyl, alkoxy-substituted alkyl, and hydroxy-substituted alkyl.

[0072] As used in this application, the terms "heterocyclic" or "heterocycloalkyl" refer to saturated single-ring or polycyclic cyclic hydrocarbon substituents comprising 3 to 20 ring atoms, wherein one or more ring atoms are nitrogen, oxygen, or S(O) m It is a heteroatom selected from (where m is an integer from 0 to 2), but does not include -OO-, -OS- or -SS- ring portions, and the remaining ring atoms are carbon.

[0073] The term "alkoxy" as used in this application means -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein the definition of alkyl is as above. Non-limiting embodiments of alkoxy include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy.

[0074] As used in this application, the term "substituted" means that one or more hydrogen atoms within the functional group are preferably substituted with up to five, more preferably one to three, hydrogen atoms, each independently with a corresponding number of substituents. It is obvious that substituents exist only in possible chemical positions, and a person skilled in the art can determine possible or impossible substitutions (by experiment or theory) without excessive effort. For example, an amino group or hydroxyl group having free hydrogen may be unstable when bonded to a carbon atom having an unsaturated (e.g., olefinic) bond.

[0075] " " refers to the connection site of a chemical bond.

[0076] As used in this application, the term "pharmaceuticalally acceptable salt" means a salt formed by a corresponding amine-based compound with an inorganic acid or an organic acid, a salt formed by a corresponding carboxylic acid-based compound with an alkali metal or alkaline earth metal, or a salt formed by an organic amine. Herein, inorganic acids include, but are not limited to, hydrochloric acid, hydrobromide, hydroiodide, sulfuric acid, phosphoric acid, etc.; 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, butanedioic acid, lactic acid, citric acid, succinic acid, gluconic acid, maleic acid, fumaric acid, tartaric acid, etc.; alkali metal or alkaline earth metal salts include, but are not limited to, sodium, potassium, calcium, magnesium salts, etc. Organic amine salts include, but are not limited to, salts composed of ammonia, methylamine, ethylamine, propylamine, isopropylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, tert-butylamine, ethylenediamine, ethanolamine, diethanolamine, triethanolamine, morpholine, piperidine, piperazine, amino acids, etc.

[0077] The drug or pharmaceutical composition of the present application may be administered by oral, topical, parenteral, or mucosal administration (e.g., oral administration, inhalation, or rectal administration) in dose-unit formulations containing a conventional and non-toxic pharmaceutically acceptable carrier. Oral administration is generally preferred. The active agent may be administered orally in the form of capsules, tablets, etc. (see Remington: The Science and Practice of Pharmacy, 20th Edition).

[0078] For oral administration in the form of tablets or capsules, the active drug component may be combined with non-toxic, pharmaceutically acceptable excipients, including binders (e.g., pre-gelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, sucrose, glucose, mannitol, sorbitol, and other reducing and non-reducing sugars, microcrystalline cellulose, calcium sulfate, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc powder, or silica, stearic acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate, etc.); and disintegrants (e.g., potato starch or sodium starch glycolate). Or includes wetting agents (e.g., sodium lauryl sulfate), coloring agents and flavoring agents, gelatin, sweeteners, natural and synthetic rubbers (e.g., gum arabic, tragacanth gum, or alginate), buffering salts, carboxymethylcellulose, polyethylene glycol, wax, etc. For oral administration in liquid form, the above drug components may be combined with non-toxic, pharmaceutically acceptable inert carriers (e.g., ethanol, glycerol, water), anti-precipitation agents (e.g., sorbitol syrup, methylcellulose derivatives, or hydrogenated edible fats), emulsifiers (e.g., lecithin or gum arabic), non-aqueous carriers (e.g., almond oil, oily esters, ethanol, or fractionated vegetable oils), preservatives (e.g., methyl-p-hydroxybenzoate or propyl-p-hydroxybenzoate or sorbic acid), etc. In addition, the above formulation may be stabilized by adding stabilizers such as antioxidants (BHA, BHT, propyl gallate, sodium ascorbate, citric acid).

[0079] Tablets containing the active compound may be encapsulated by methods well known in the art. The composition of the present application containing the active compound, Compound of Formula I, may also be introduced in the form of small spheres, microspheres, or microcapsules composed of polyglycolic acid / lactic acid (PGLA). Liquid formulations for oral administration may be in the form of, for example, solutions, syrups, emulsions, or suspensions, or may be provided as dry products that can be reconstituted using water or other suitable excipients before use. Oral formulations may be appropriately prepared so that the active compound is controlled or delayed-release.

[0080] The term "treatment" as used in this application includes suppressing, alleviating, preventing, or eliminating one or more symptoms or side effects associated with the disease, pathology, or disorder being treated.

[0081] The use of the term "inhibition" as used in this application is a concept relative to the control group. A person skilled in the art can readily determine an appropriate control group for each experiment. For example, it refers to a case where the response in subjects or cells treated with the compound is reduced compared to the response in subjects or cells not treated with the compound.

[0082] The term “pharmaceutical composition” as used in this application means a composition comprising a compound or a pharmaceutically acceptable salt thereof according to this application, and at least one of the following pharmaceutically acceptable components selected according to the characteristics of the mode of administration and formulation, wherein these components include, but are not limited to, carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, mellowing agents, flavoring agents, antimicrobial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature controllers, adhesives, stabilizers, suspending agents, etc.

[0083] II. Examples

[0084] In order to clarify the purpose, technical solution, and advantages of the present invention, the present invention is described in more detail below. The described embodiments should not be construed as limitations on the present invention, and all other embodiments that can be obtained by a person skilled in the art without creative effort are included within the scope of protection of the present invention.

[0085] Prior to the detailed description of the embodiments of the present invention, names and terms relating to the embodiments of the present invention shall be explained, provided that the names and terms relating to the embodiments of the present invention shall be interpreted according to the following description.

[0086] In the specific embodiments of the present disclosure, the raw materials and equipment used are all ready-made products purchased from the market.

[0087] Measurement and calculation of the Drug-to-Antibody Ratio (DAR) value: Based on the RP-HPLC-MS measurement results, the DAR value of the ADC is analyzed using a Waters Acquity UPLC I-Class / Xevo G2-XS QTOF instrument.

[0088] RP-HPLC parameter settings: The column is PLRP-S 1000A 5μm, and the column temperature is 70℃. Mobile bed A is a 0.1% aqueous formic acid solution, mobile bed B is a 0.1% acetonitrile formic acid solution, and the flow rate is 0.2 mL / min. The mobile bed gradient is 18 min at 20-50% B; 5 min at 50-95% B; 0.1 min at 95-20% B; and 6.9 min at 20-20% B.

[0089] MS parameter settings: Capillary voltage 2.50 kV, cone voltage 100 V, mass spectrometry range m / z 200 to 4000, MSE collision energy 20 to 45 eV, ion source temperature 120°C, nebulization temperature 500°C, nebulization flow rate 1000 L / hr, and the internal standard is leucine enkephalin. The sample is diluted to 1 mg / mL with sample buffer, TCEP with a final concentration of 50 mmol / L is added, and after incubation at 37°C for 20 min, 5 μL is injected. Light chain peaks are identified and peak area percentages are calculated, with the total peak areas being 100. Similarly, heavy chain peaks are identified and peak area percentages are calculated, with the total peak areas being 100. The weighted peak areas of the heavy and light chains are calculated by multiplying the peak area percentages by the corresponding drug loadings, respectively. DAR value calculation formula: DAR = 2 * (Σ light chain weighted peak area + Σ heavy chain weighted peak area) / 100.

[0090] In this embodiment, Nectin-4 antibody is used for the manufacture of the ADC, but is not limited thereto.

[0091] The heavy chain amino acid sequence of the Nectin-4 antibody is as follows (SEQ ID NO: 1):

[0092] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLSLQMNSLRDEDTAVYYCARAYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0093] Nectin-4 항체 경쇄 아미노산 서열은 다음과 같다(SEQ ID NO: 2):

[0094] DIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0095] Example 1: Ab nectin-4 -S-(7-(N-(acetamide-PEG 2 -Propionyl-Gly-Lys-PABC)-N-(2',2'-Difluoroethyl))amine ethyl camptothecin) 8 (ADC1)

[0096] 1.1 7-(N-(bromoacetamide-PEG 2 -propionyl-Gly-Lys-PABC)-N-(2',2'-difluoroethyl))amine ethyl camptothecin (L1-D1)

[0097]

[0098] 4',5'-Methylenedioxy-2'-Nitroacetophenone (15 g, 71.72 mmol) was placed in a 500 mL single-neck flask, DMF-DMA (170.92 g, 1.43 mol) was added, and the mixture was heated 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. After stirring for 3 hours, the mixture was filtered to obtain a wet yellow solid, which was vacuum dried at 40°C for 6 hours to obtain pale yellow solid intermediate I-1 (18 g, yield 95%, HPLC 98%); LCMS: [M+1] + 265.22 (Calculated value 264.24); 1 H NMR (600 MHz, DMSO-d6) δ 7.62-7.22 (m, 2H), 7.04 (s, 1H), 6.24 (s, 2H), 5.22 (d, J =6.9 Hz, 1H), 3.08 (s, 3H), 2.84 (s, 3H).

[0099] intermediate I-1 18 g (68.12 mmol) was added to 180 mL of IPA and the reaction mixture was stirred; then, DIEA (17.61 g, 136.24 mmol) and difluoroethylamine hydrochloride (32.02 g, 272.48 mmol) were added, and a reflux reaction was carried out for 12 hours while stirring. The mixture was concentrated, 200 mL of water was added, and the mixture was stirred for 2 hours. The mixture was filtered to obtain a yellow solid, which was dried at 40°C to produce the product. D1-1 (15.8 g, yield 77.3%, HPLC 98%) was obtained; LCMS: [M+1] + 301.47 (Calculated value 300.22); 1 H NMR (600 MHz, CDCl3) δ 9.84 (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=7.5 Hz, 1H), 3.68-3.48 (m, 2H).

[0100] compound D1-1 After adding (15.8 g, 52.63 mmol) to DCM (310 mL), acetic acid (63.05 g, 1.05 mol) was added and stirred until the solid was completely dissolved. Maintaining the internal temperature of the reaction solution at 15°C, sodium triacetoxyborohydride (27.89 g, 131.56 mmol) was added in four installments of approximately 7 g at intervals of about 5 min. The reaction was carried out for 2 hours while maintaining the internal temperature at 15°C. Subsequently, the reaction solution was poured into an aqueous sodium carbonate solution (sodium carbonate: 130 g, water: 1 L) at 0°C, and the internal temperature was controlled to 15°C while stirring to separate the phases. The organic phase was washed with water, collected, and dried with anhydrous sodium sulfate to obtain the compound. D1-2 (Immediately input into the next step) obtained, the temperature was lowered to 0°C, DIEA (13.58 g, 105.26 mmol) was added, and a DCM solution of CbzCl (10.11 g, 59.27 mmol) was added dropwise (dropwise addition completed in approximately 20 min). After reacting at 0°C for 12 h, the mixture was washed twice with a 10% aqueous citric acid solution (200 mL x 2), followed by washing with a saturated aqueous sodium bicarbonate solution (200 mL) and then washing with a saturated saline solution (200 mL). The organic phase was collected, dried with anhydrous sodium sulfate, and concentrated to obtain the product. D1-3 (19.7 g, two-step total yield 85.7%, HPLC 95%) was obtained; LCMS: [M+1] + 437.42 (Calculated value 436.37); 1 H NMR (500 MHz, CDCl3) δ 7.40-7.28 (m, 7H), 6.17 (s, 2H), 5.15 (d, J =4.5 Hz, 2H), 3.82-3.65 (m, 4H), 3.14-2.75 (m, 3H).

[0101] Compounds under a nitrogen atmosphere D1-3(19.7 g, 45.1 mmol) was placed in a 1 L single-neck flask, DMF (50 mL) was added, and the temperature was lowered to 0°C. The prepared reducing solution of H2O (250 mL), sodium dithionate (39.30 g, 225.72 mmol), and sodium carbonate (19.14 g, 180.58 mmol) was slowly added dropwise. After the addition was complete, the temperature was slowly raised to 40°C and the mixture was stirred for 2 hours to allow the reaction to proceed. The mixture was filtered, the filtrate was concentrated, and ethyl acetate (500 mL) and water (500 mL) were added for extraction and separation. Ethyl acetate (500 mL) was added back to the aqueous phase, and the pH was adjusted to between 3 and 4 using 2 M dilute hydrochloric acid. The mixture was stirred at room temperature for 6 hours, extracted, and separated. The organic phases were combined and dried with sodium sulfate. The sample was then concentrated and purified by silica gel column chromatography to obtain the yellow oily substance. D1-4 (17.7 g, yield 96.7%, HPLC 98%) was obtained; LCMS: [M+1] + 407.52 (Calculated value 406.39); 1 H NMR (500 MHz, CDCl3) δ 7.44-7.02 (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).

[0102] Compound in a 500 mL single-neck flask D1-4(17.7 g, 43.55 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyranO[3,4-F]indoleizine-3,6,10(4H)-one (13.75 g, 52.27 mmol), and PPTS (218.88 g, 871.00 mmol) were added sequentially and reacted at 110°C for 12 hours under an argon atmosphere while stirring. The reaction mixture was then poured into methanol (1000 mL), stirred for 2 hours, and filtered to obtain a dark brown solid. The dark brown solid was then stirred in methanol (300 mL) for 2 hours and filtered to obtain a light brown solid. The solid was dried at 40°C to obtain the compound. D1-5 (10.1 g, yield 36.6%, HPLC 96%) was obtained; LCMS: [M+1] + 634.33 (Calculated value 633.60); 1 H NMR (600 MHz, DMSO-d6) δ 7.47-7.17 (m, 7H), 7.17 (d, J =7.7 Hz, 1H), 6.48 (s, 1H), 6.24 (d, J =6.2 Hz, 2H), 6.13 (dq, J =55.7, 3.8 Hz, 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, 7.9 Hz, 2H), 3.22 (t, J =8.2 Hz, 2H), 1.89 (ddp, J =21.1,14.0,6.9,6.0 Hz, 2H), 0.90 (q, J =6.8 Hz, 3H).

[0103] Compound in a 10 L single-neck flask D1-510 g (15.78 mmol), dichloromethane (2.5 L), and methanol (2.5 L) were added sequentially, and 10% Pd / C (15 g) was added to the reaction solution while stirring. The reaction was then carried out at room temperature for 20 hours under a hydrogen balloon atmosphere, after which the Pd / C was removed by filtration. The reaction solution was then rotary evaporated, stirred for 24 hours with 30 mL of a mixed solution of dichloromethane and methanol (V / V = 1:1), filtered, and dried for 3 hours at 40°C to produce the product. D1 (5.8 g, yield 73.6%, HPLC 95.0%) was obtained; LCMS: [M+1] + 500.52 (Calculated value 499.47); 1 H NMR (500 MHz, DMSO-d6) δ 7.68 (s, 1H), 7.52 (s, 1H), 7.28 (s, 1H), 6.58 (s, 1H), 6.33 (s, 2H), 6.02 (t, J =56.6 Hz, 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 =58.5 Hz, 2H), 1.05-0.85 (m, 3H).

[0104] Fmoc-Gly-Lys(Trt)-PAB-PNP Preparation of: Fmoc-Lys(Trt) (50 g, 82.1 mmol) and 4-aminobenzyl alcohol (15.1 g, 122.7 mmol) were dissolved in a mixed solvent of DCM (250 mL) and methanol (250 mL) in a 1 L single-neck flask, cooled to 0°C, EEDQ (30.3 g, 122.7 mmol) was added in portions, reacted for 10 minutes while maintaining the temperature, and then reacted overnight at room temperature. Afterward, the reaction mixture was washed with a 2% aqueous citric acid solution (300 mL), followed by washing with a saturated aqueous sodium bicarbonate solution (300 mL), and the organic phase was washed with saturated saline, dried with anhydrous sodium sulfate, and concentrated. Fmoc-Lys(Trt)-PAB Oil phase (LCMS: [M+1]+ 716.30 (calculated value 715.35) was obtained.

[0105] DCM (300 mL) is added to the obtained oil phase, and DBU (7.4 g, 48.9 mmol) is added while stirring and reacted at room temperature for 2 hours. After concentrating to about 200 mL, the solution is added dropwise to 2 L of methyl t-butyl ether, filtered, and the filter cake is rotary evaporated at room temperature. Lys(Trt)-PAB A pink solid (37 g, two-stage yield 91%) was obtained; LCMS: [M+1] + 494.70 (Calculated value 493.65).

[0106] In a 1 L single-neck flask Lys(Trt)-PAB (47 g, 95.2 mmol) was dissolved in DCM (500 mL), and 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 in an ice bath and reacted at room temperature for 2 hours with stirring. Afterward, the reaction mixture was washed with 300 mL of 5% aqueous citric acid solution, followed by washing with 200 mL of saturated aqueous sodium bicarbonate solution, and the organic phase was washed with saturated saline solution, dried with anhydrous sodium sulfate, and concentrated. Fmoc-Gly-Lys(Trt)-PAB (40 g, yield 54%) was obtained; LCMS: [M+1] + 773.70 (Calculated value 772.95); 1 H NMR (600 MHz, DMSO-d6) δ 10.00 (s, 1H), 8.12 (d, J =7.5 Hz, 1H), 7.95-7.86 (m, 2H), 7.74 (d, J =7.4 Hz, 2H), 7.68-7.57 (m, 3H), 7.53-7.14 (m, 20H), 5.16 (t, J =5.3 Hz, 1H), 4.47 (dd, J =16.1, 5.9 Hz, 3H), 4.28 (dd, J=24.2, 6.7 Hz, 3H), 3.72 (s, 2H), 2.52 (d, J =7.4 Hz, 1H), 1.97 (s, 2H), 1.80-1.20 (m, 6H).

[0107] In a 1 L single-neck flask Fmoc-Gly-Lys(Trt)-PAB (40 g, 51.7 mmol) and DCM (400 mL) were sequentially added and stirred to dissolve; then, TEA (15.6 g, 155.1 mmol) and bis(4-nitrophenyl)carbonate (47.1 g, 155.1 mmol) were added dropwise at room temperature and stirred for 2 hours to react; subsequently, water (400 mL) was added for extraction; after washing with saturated saline, the solution was dried with anhydrous sodium sulfate and rotary evaporated; and the product was purified by silica gel column chromatography. Fmoc-Gly-Lys(Trt)-PAB-PNP (17.5 g, yield 36%, HPLC 95.7%) was obtained; LCMS: [M+1] + 938.62 (Calculated value 937.37); 1 H NMR (500 MHz, DMSO-d6) δ 10.16 (s, 1H), 8.35 (d, J =9.1 Hz, 2H), 8.20-8.10 (m, 2H), 7.92 (d, J =7.5 Hz, 2H), 7.80-7.55 (m, 7H), 7.50-7.14 (m, 20H), 5.29 (s, 2H), 4.50-4.40 (m, 1H), 4.34-4.22 (m, 3H), 3.77-3.68 (m, 2H), 2.00-1.85 (m, 2H), 1.79-1.25 (m, 7H).

[0108] In a 50 mL single-neck flask D1Fmoc-Gly-Lys(Trt)-PAB-PNP (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 sequentially and reacted by stirring at room temperature for 2 hours. Afterward, the reaction mixture was washed with 50 mL of 2% aqueous citric acid solution, followed by washing with 50 mL of saturated aqueous sodium bicarbonate solution. The organic phase was then washed with 50 mL of saturated saline solution, dried with anhydrous sodium sulfate, and concentrated. L1-D1-1 The crude product was obtained and used directly in the next step reaction without further purification; LCMS: [M+1] + 1298.74 (Calculated value 1297.50); 1 H NMR (600 MHz, DMSO-d6) δ 10.06 (d, J =23.4 Hz, 1H), 8.11 (d, J =7.8 Hz, 1H), 7.86 (d, J =7.7 ㎐, 1H), 7.93-7.47 (m, 6H), 7.62-7.27 (m, 10H), 7.27 (dt, J =36.3, 7.0 Hz, 7H), 7.27-7.06 (m, 3H), 7.12 (t, J =8.3 Hz, 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 =14.7 Hz, 2H), 3.87-3.43 (m, 4H), 3.29 (dq, J =15.9, 11.6, 9.5 Hz, 2H), 2.47 (d, J =6.5 Hz, 1H), 1.89 (ddq, J =28.6, 14.1, 8.1, 7.1 ㎐, 4H), 1.76-1.60 (m, 1H), 1.56 (d, J=9.3 Hz, 1H), 1.47 (t, J =7.3 Hz, 2H), 1.36 (d, J =11.5 Hz, 1H), 1.28 (s, 1H), 0.89 (t, J =7.3 Hz, 3H).

[0109] The above reaction flask ( L1-D1-1 Add DCM (50 mL) to ) and stir to dissolve, then add DBU (582.8 mg) dropwise and react by stirring at room temperature for 1 hour, then add methyl t-butyl ether (400 mL) and filter, dissolve the solid again in DCM (40 mL), add dropwise to methyl t-butyl ether (400 mL), filter, and dry. L1-D1-2 (7 g, yield 87.5%) was obtained; LCMS: [M+1] + 1076.38 (Calculated value 1075.43); 1 H NMR (600 MHz, DMSO-d6) δ 10.32 (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, 13.7 Hz, 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, 16.8 Hz, 2H), 1.10 (s, 1H), 0.88 (t, J =7.3 Hz, 3H).

[0110] In a 250 mL single-neck flask L1-D1-2(7 g, 6.5 mmol) and 70 mL of DCM were added sequentially and stirred to dissolve; then, 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 and the reaction was stirred at room temperature for 1 hour; after quenching the reaction by adding 100 mL of water, the organic phase was washed once with a saturated sodium chloride aqueous solution (100 mL), concentrated, and purified by silica gel column chromatography. L1-D1-3 (5.2 g, yield 54.7%) was obtained; LCMS: [M+1] + 1457.93 (Calculated value 1456.59); 1 H NMR (600 MHz, DMSO-d6) δ 9.99 (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 =3.2 Hz, 1H), 6.36-6.19 (m, 2H), 5.55-5.39 (m, 3H), 5.25 (d, J =44.7 Hz, 2H), 5.09 (s, 1H), 5.04-4.88 (m, 1H), 4.63 (s, 1H), 4.36 (s, 1H), 4.48-3.93 (m, 4H), 3.92-3.73 (m, 2H), 3.81-3.60 (m, 2H), 3.60 (ddd, J =45.2, 15.2, 7.9 ㎐, 6H), 3.41 (dd, J =49.8, 5.7 Hz, 5H), 3.11 (p, J =5.8 Hz, 2H), 2.72 (s, 1H), 2.37 (td, J =6.6, 3.3 Hz, 2H), 1.87 (dtq, J=21.5, 14.5, 7.1 ㎐, 3H), 1.69 (s, 2H), 1.50 (d, J =40.9 Hz, 2H), 1.38-1.15 (m, 6H), 1.05 (d, J =6.4 Hz, 2H), 1.00 (d, J =6.5 Hz, 9H), 0.87 (t, J =7.4 Hz, 3H).

[0111] In a 100 mL single-neck flask L1-D1-3 (5.1 g, 3.5 mmol) and DCM (30 mL) were added sequentially and stirred to dissolve, then DBU (266.3 mg) was added dropwise and the mixture was stirred at room temperature for 30 min to react, after which methyl t-butyl ether (300 mL) was added and filtered, the filter cake was washed with methyl t-butyl ether and dried, and then L1-D1-4 (3.2 g, yield 74%) was obtained; LCMS: [M+1] + 1235.77 (Calculated value 1234.52).

[0112] In a 100 mL single-neck flask L1-D1-4 (3.2 g, 2.6 mmol) and DCM (19 mL) were added sequentially and stirred to dissolve, then bromoacetic anhydride (670 mg, 2.6 mmol) was added and the reaction was reacted by stirring at room temperature for 1 hour, after which the reaction solution was concentrated and purified by silica gel column chromatography. L1-D1-5 (2.8 g, yield 80%, HPLC 97.3%) was obtained; LCMS: [M+1] + 1355.64 (Calculated value 1354.44); 1 H NMR (600 MHz, DMSO-d6) δ 10.00 (s, 1H), 9.51 (s, 1H), 8.33 (d, J =5.6 Hz, 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 =42.2 Hz, 2H), 5.10 (s, 1H), 5.04-4.85 (m, 2H), 4.54 (d, J =5.1 Hz, 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 =9.5 Hz, 2H), 1.40-1.18 (m, 1H), 1.11 (s, 14H), 0.88 (t, J =7.3 Hz, 3H).

[0113] In a 100 mL single-neck flask L1-D1-5 Sequentially add (3.1 g, 2.3 mmol) and DCM (60 mL) and stir to dissolve; then, add TFA (to a reaction solution concentration of 5%) dropwise and react at room temperature with stirring for 1 hour; after adding methyl t-butyl ether (600 mL), filter and wash; then add DCM (10 mL) and stir; subsequently, add methyl t-butyl ether (100 mL) and stir for 20 minutes; filter and wash; and vacuum dry to obtain a white solid product. L1-D1 (1.0 g, yield 40%, HPLC 97%) was obtained; LCMS: [M+1] + 1113.51 (Calculated value 1113.34); 1 H NMR (500 MHz, DMSO-d6) δ 10.03 (d, J =16.7 Hz, 1H), 8.34 (q, J =5.1 Hz, 1H), 8.23-8.10 (m, 2H), 7.86-7.60 (m, 3H), 7.72-7.40 (m, 3H), 7.33 (d, J =8.2 Hz, 1H), 7.38-6.98 (m, 2H), 6.49 (s, 1H), 6.27 (dd, J =13.7, 3.9 Hz, 2H), 5.43 (d, J=4.2 ㎐, 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, 6.7 ㎐, 2H), 2.40 (q, J =5.6 ㎐, 2H), 2.06-1.63 (m, 3H), 1.58 (ddt, J =45.5, 14.8, 6.1 ㎐, 3H), 1.53-1.20 (m, 2H), 0.88 (t, J =7.2 ㎐, 3H); 13 C NMR (151 ㎒, DMSO-d6) δ 173.03, 171.26, 171.01, 169.51, 166.57, 158.67, 158.45, 157.32, 156.03, 155.85, 151.37, 150.58, 149.91, 149.56, 147.57, 146.75, 139.23, 139.11, 133.26, 127.13, 125.08, 119.14, 115.33, 104.56, 99.64, 96.43, 72.88, 69.92, 69.17, 67.15, 65.73, 61.82, 53.56, 50.11, 49.50, 48.21, 42.54, 39.15, 38.32, 36.31, 31.92, 30.68, 29.92, 29.04, 28.45, 27.17, 22.81, 8.25.

[0114] 1.2 L1-D1-Ab nectin-4 Antibody-drug conjugate (ADC1)

[0115]

[0116] Take Nectin4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1M Na2HPO4 solution, add 0.1M disodium ethylenediaminetetraacetate solution (25 μL), add prepared TCEP·HCl solution (10 mM, 0.04 mL), and react for 3 hours at room temperature (25℃) while rotating a turntable.

[0117] compound L1-D1 (0.89 mg, 0.80 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, and mixed uniformly; the reaction was carried out for 16 hours at room temperature while rotating a rotary rotatometer, and after the reaction was completed, small molecules were removed using a NAP-5 gel column (Cytiva), the buffer was replaced with a 20 mM PB solution, and the pH was adjusted to 6.3 to obtain an antibody-drug conjugate ADC1 (3.1 mg / mL, 2 mL) was obtained.

[0118] Mean value calculated by RP-MS (RP-HPLC-MS): n=7.7; MS results show that the antibody light chain (L) is a single L1-D1 It is coupled to the (linker-payload), and the heavy chain (H) has 3 L1-D1 Indicates that it is coupled to (linker-payload) (Fig. 1).

[0119] Example 2: Ab nectin-4 -( S-7-(N-(acetamide-PEG 2 -Propionyl-Gly-Lys-PABC)-N-((R)-3-Tetrahydrofuran))amine Ethyl Camptothecin) 8 (ADC2)

[0120] 2.1 7-(N-(bromoacetamide-PEG 2 -propionyl-Gly-Lys-PABC)-N-((R)-3-tetrahydrofuran))amine ethyl camptothecin (L1-D2)

[0121]

[0122] intermediate I-1(18 g, 68.12 mmol) was added to IPA (180 mL) and the reaction mixture was stirred; then, DIEA (17.61 g, 136.24 mmol) and (R)-3-aminotetrahydrofuran hydrochloride (33.67 g, 272.48 mmol) were added, and the reflux reaction was carried out for 12 hours. After concentrating the reaction mixture until the remaining 20 mL of IPA was reduced, 200 mL of water was added and stirred at room temperature for 2 hours. The mixture was filtered to obtain a yellow solid, which was then dried at 40°C to produce the product. D2-1 (16.5 g, yield 79%, HPLC 98%) was obtained; LCMS: [M+1] + 307.32 (Calculated value 306.09); 1 H NMR (500 MHz, DMSO-d6) δ 7.58 (s, 1H), 7.34-7.19 (m, 1H), 7.07 (s, 1H), 6.25 (s, 2H), 5.33 (d, J =11.3 Hz, 1H), 4.02 (s, 1H), 3.83-3.53 (m, 5H), 2.23-2.05 (m, 1H), 1.85-1.65 (m, 1H).

[0123] compound D2-1 After adding (16.5 g, 53.87 mmol) to DCM (310 mL), acetic acid (64.70 g, 1.08 mol) was added and stirred to dissolve it. Maintaining the internal temperature of the reaction solution at 15°C, sodium triacetoxyborohydride (28.54 g, 134.68 mmol) was added in four installments of approximately 7 g at intervals of about 5 min. The reaction was carried out for 2 hours while maintaining the internal temperature at 15°C. Subsequently, the reaction solution was poured into a cold aqueous sodium carbonate solution (sodium carbonate: 130 g, ice water: 1 L), the internal temperature was controlled to 15°C, and stirring was performed to separate the phases. The organic phase was washed with water, collected, and dried with anhydrous sodium sulfate to obtain the compound. D2-2A DCM solution was obtained, DIEA (13.93 g, 107.76 mmol) was added, followed by the dropwise addition of Cbz-Cl (10.11 g, 59.27 mmol) over a period of 20 minutes. After the dropwise addition was completed, the mixture was cooled in an ice bath and reacted for 12 hours. The mixture was then washed twice with a 10% aqueous citric acid solution (200 mL x 2), followed by washing with a saturated aqueous sodium bicarbonate solution (200 mL) and a saturated saline solution (200 mL). The organic phase was collected, dried with anhydrous sodium sulfate, and the organic phase was concentrated to obtain the compound D2-3 (20.5 g, yield 86%) was obtained; LCMS: [M+1] + 443.42 (Calculated value 442.43); 1 H NMR (500 MHz, DMSO-d6) δ 7.65 (s, 1H), 7.39-7.27 (m, 6H), 6.28 (s, 2H), 5.10 (s, 2H), 4.65-4.50 (m, 1H), 4.00-3.80 (m, 1H), 3.77-3.57 (m, 5H), 3.16-3.05 (m, 2H), 2.24-2.10 (m, 1H), 2.00-1.88 (m, 1H).

[0124] compound D2-3 (20.5 g, 46.3 mmol) was placed in a 1 L single-neck flask, and 50 mL of DMF was added. The mixture was cooled to 0°C in an ice bath under a nitrogen atmosphere, and the prepared reducing solution of H2O (250 mL) / sodium dithionate (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 carried out by stirring for 2 hours. The reaction mixture was filtered, the filtrate was concentrated, and the extract was extracted and separated by adding ethyl acetate (500 mL) and water (500 mL). The mixture was dried with anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to obtain a yellow oily substance. D2-4 (17.9 g, yield 93.7%, HPLC 98%) was obtained; LCMS: [M+1] +413.42 (Calculated value 412.44); 1 H NMR (500 MHz, DMSO-d6) δ 7.45-7.25 (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 =7.6 Hz, 2H), 2.19-2.10 (m, 1H), 1.87 (d, J =7.9 Hz, 1H).

[0125] Compound in a 500 mL single-neck flask D2-4 (17.9 g, 43.4 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyranO[3,4-F]indoleizine-3,6,10(4H)-one (12.57 g, 47.74 mmol), and PPTS (218.13 g, 868.00 mmol) were added sequentially and reacted at 110°C for 12 hours under an argon atmosphere while stirring. The reaction mixture was then poured into methanol (1000 mL), stirred for 2 hours, and filtered to obtain a dark brown solid. The mixture was then stirred in methanol (300 mL) for 2 hours and filtered to obtain a light brown solid. The solid was dried at 40°C to obtain the compound. D2-5 (10.0 g, yield 36%, HPLC 95%) was obtained; LCMS: [M+1] + 640.72 (Calculated value 639.66); 1 H NMR (600 MHz, DMSO-d6) δ 7.82-7.14 (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).

[0126] Compound in a 10 L single-neck flask D2-5(10 g, 15.63 mmol), dichloromethane (2.5 L), and methanol (2.5 L) were added sequentially. 10% Pd / C (15 g) was added to the reaction solution, and the reaction was carried out at room temperature for 20 hours under a hydrogen balloon atmosphere. After removing the Pd / C by filtration, the reaction solution was rotary evaporated, stirred for 24 hours with a mixed solution of dichloromethane and methanol (30 mL, V / V = 1:1), filtered, and the wet product was dried at 40°C for 3 hours to obtain the product. D2 (6.0g, yield 76%) was obtained; LCMS: [M+1] + 506.52 (Calculated value 505.53); 1 H NMR (600 MHz, DMSO-d6) δ 7.52 (s, 1H), 7.40 (s, 1H), 7.16 (s, 1H), 6.52 (s, 1H), 6.26 (d, J =7.3 Hz, 2H), 5.48-5.35 (m, 2H), 5.11-4.95 (m, 2H), 3.95-3.62 (m, 5H), 3.36-3.26 (m, 2H), 3.14-3.03 (m, 2H), 2.20-2.04 (m, 2H), 1.96-1.80 (m, 2H), 0.89 (t, J =7.3 Hz, 3H).

[0127] Compound in a 250 mL single-neck 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 sequentially, and the reaction mixture was stirred at room temperature for 2 hours. Afterward, the mixture was washed with dichloromethane (300 mL) and a 2% aqueous citric acid solution, washed with a saturated aqueous sodium bicarbonate solution (100 mL), and washed with saturated saline solution. It was then dried with anhydrous sodium sulfate and concentrated. L1-D2-1 obtained; LCMS: [M+1] +1304.93 (Calculated value: 1303.53); 1 H NMR (600 MHz, DMSO-d6) δ 10.06 (s, 1H), 8.10 (d, J =7.9 Hz, 1H), 7.92-7.73 (m, 2H), 7.71-7.51 (m, 4H), 7.44-7.18 (m, 18H), 7.12 (t, J =7.1 Hz, 3H), 6.49 (s, 1H), 6.31-6.20 (m, 2H), 5.42 (d, J =3.5 Hz, 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, 15.3 Hz, 1H), 3.73-3.42 (m, 5H), 3.30 (t, J =7.1 Hz, 2H), 3.07 (d, J =0.8 Hz, 4H), 2.69 (d, J =1.0 Hz, 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 =0.8 Hz, 11H), 0.88 (t, J =7.3 Hz, 3H).

[0128] In a 250 mL single-neck flask L1-D2-1 (The above product) and DCM (100 mL) are added and stirred to dissolve, then DBU (3.61 g, 23.7 mmol) is added dropwise and the mixture is stirred at room temperature for 1 hour to react, then poured into methyl t-butyl ether (1 L), the precipitated solid is filtered, dissolved in DCM (80 mL), and subsequently added dropwise to 1 L methyl t-butyl ether, filtered, and dried. L1-D2-2 (26 g) was obtained; LCMS: [M+1] + 1082.68 (Calculated value 1081.47); 1H NMR (600 MHz, DMSO-d6) δ 10.30 (d, J =65.6 Hz, 1H), 7.82 (d, J =8.3 Hz, 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 =7.1 Hz, 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 =9.2 Hz, 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 =5.8 Hz, 2H), 3.10 (s, 1H), 2.89 (s, 1H), 2.73 (d, J =0.7 Hz, 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, 5.4 ㎐, 7H), 1.46 (d, J =7.7 Hz, 2H), 0.92-0.83 (m, 3H).

[0129] In a 1 L single-neck flask L1-D2-2 (26 g crude product) and DCM (400 mL) were added sequentially and stirred to dissolve; then, 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 hour, after which 80 mL of water was added to quench the reaction. The mixture was then extracted with DCM, the organic phase was washed with saturated saline, the organic phase was dried with anhydrous sodium sulfate, and purified by silica gel column chromatography. L1-D2-3(11.0 g, three-stage total yield 55.5%, HPLC 96%) was obtained; LCMS: [M+1] + 1463.78 (Calculated value 1462.62); 1 H NMR (600 MHz, DMSO-d6) δ 10.01 (s, 1H), 8.17-8.04 (m, 2H), 7.85 (d, J =7.5 Hz, 2H), 7.65 (dd, J =20.1, 7.9 Hz, 4H), 7.53-7.18 (m, 20H), 7.14 (q, J =7.5, 5.7 Hz, 3H), 6.49 (s, 1H), 6.26 (d, J =16.8 Hz, 2H), 5.42 (d, J =3.2 Hz, 2H), 5.23 (s, 2H), 5.13 (s, 2H), 4.58 (s, 1H), 4.38 (q, J =7.6 Hz, 1H), 4.26 (d, J =7.1 Hz, 2H), 4.17 (t, J =6.9 Hz, 1H), 3.97 (s, 0H), 3.90 (d, J =17.7 Hz, 0H), 3.71 (dd, J =31.9, 4.9 Hz, 3H), 3.60 (dt, J =21.8, 6.7 Hz, 4H), 3.45 (s, 4H), 3.37 (t, J =6.0 Hz, 2H), 3.27 (s, 2H), 3.11 (p, J =6.7, 6.0 Hz, 2H), 2.36 (t, J =6.5 Hz, 2H), 2.13 (dtd, J =13.3, 8.5, 5.4 ㎐, 1H), 1.87 (ddt, J =36.3, 21.3, 7.1 ㎐, 4H), 1.66 (s, 1H), 1.53 (d, J =10.0 Hz, 1H), 1.46 (q, J =7.2 Hz, 2H), 1.39-1.20 (m, 3H), 0.87 (t,J =7.3 Hz, 3H).

[0130] In a 100 mL single-neck flask L1-D2-3 (5 g, 3.42 mmol) and DCM (40 mL) were added sequentially and stirred to dissolve; then, DBU (364 mg, 2.39 mmol) was added dropwise, and after the addition was complete, the reaction was stirred at room temperature for 30 min; then, the reaction mixture was placed in methyl t-butyl ether (500 mL) to filter the precipitated solid; the solid was dissolved again in DCM (50 mL), added dropwise to methyl t-butyl ether (600 mL), and filtered; the filter cake was washed with methyl t-butyl ether and dried. L1-D2-4 (2.9 g, yield 69%) was obtained; LCMS: [M+1] + 1241.87 (Calculated value 1240.55); 1 H NMR (600 MHz, DMSO-d6) δ 10.03 (s, 1H), 8.14 (t, J =5.7 Hz, 1H), 8.09 (d, J =7.8 Hz, 1H), 7.63 (d, J =8.3 Hz, 2H), 7.48 (s, 1H), 7.41 (s, 1H), 7.37 (d, J =7.9 Hz, 7H), 7.28-7.19 (m, 7H), 7.14 (t, J =7.3 Hz, 3H), 6.49 (s, 1H), 6.27 (d, J =11.9 Hz, 2H), 5.48-5.37 (m, 2H), 5.24 (s, 2H), 5.14 (s, 2H), 4.58 (t, J =7.5 Hz, 1H), 4.37 (q, J =7.3 Hz, 1H), 3.89 (s, 1H), 3.71 (dd, J =24.3, 7.2 Hz, 3H), 3.60 (dt, J =22.9, 7.1 Hz, 4H), 3.46 (s, 4H), 3.36 (t, J =5.7 Hz, 2H), 2.67 (t,J =5.7 Hz, 2H), 2.37 (t, J =6.5 Hz, 2H), 2.13 (dtd, J =13.2, 8.6, 5.1 ㎐, 1H), 1.88 (dtd, J =43.1, 13.7, 13.2, 6.6 ㎐, 4H), 1.66 (s, 1H), 1.50 (dp, J =37.5, 7.7 Hz, 4H), 1.39-1.14 (m, 5H), 0.88 (t, J =7.3 Hz, 3H).

[0131] In a 100 mL single-neck flask L1-D2-4 (2.9 g, 2.3 mmol) and DCM (25 mL) were added sequentially and stirred to dissolve, then bromoacetic anhydride (667.8 mg, 2.57 mmol) was added and the reaction was reacted by stirring at room temperature for 30 minutes, after which the reaction solution was concentrated and purified by silica gel column chromatography. L1-D2-5 (2.0 g, yield 64.5%, HPLC 97.6%) was obtained; LCMS: [M+1] + 1361.93 (Calculated value 1360.48); 1 H NMR (600 MHz, DMSO-d6) δ 10.01 (s, 1H), 8.32 (t, J =5.6 Hz, 1H), 8.11 (dd, J =24.1, 6.7 Hz, 2H), 7.62 (d, J =8.1 Hz, 2H), 7.55-7.20 (m, 17H), 7.14 (s, 2H), 6.49 (s, 1H), 6.26 (s, 2H), 5.42 (d, J =2.8 Hz, 2H), 5.24 (s, 2H), 5.14 (s, 2H), 4.58 (p, J =6.4, 5.8 Hz, 1H), 4.40-4.34 (m, 1H), 3.96-3.86 (m, 0H), 3.84 (s, 2H), 3.71 (dd, J =22.2, 5.6 Hz, 3H), 3.58 (t, J=6.5 Hz, 3H), 3.47 (s, 5H), 3.43-3.31 (m, 5H), 3.21 (q, J =5.8 Hz, 2H), 2.37 (t, J =6.5 Hz, 2H), 2.14 (dtd, J =13.4, 8.7, 5.3 ㎐, 1H), 1.87 (dtd, J =28.6, 15.1, 14.2, 7.9 ㎐, 4H), 1.66 (td, J =12.2, 6.1 Hz, 2H), 1.51 (d, J =42.3 Hz, 3H), 1.35-1.24 (m, 5H), 0.91-0.81 (m, 3H).

[0132] In a 100 mL single-neck flask L1-D2-5 Sequentially add (1.3 g, 0.95 mmol) and DCM (10 mL) and stir to dissolve; then, add TFA (0.78 mL) dropwise and react at room temperature with stirring for 2 hours; after pouring the reaction mixture into methyl t-butyl ether (130 mL) to filter the precipitated solid, add DCM (10 mL) to the solid and stir; subsequently, pour into methyl t-butyl ether (100 mL) and stir for 20 minutes; filter, wash, and vacuum dry to obtain a white solid. L1-D2 (870 mg, yield 81.7%, HPLC 97%) was obtained; LCMS: [M+1] + 1119.64 (Calculated value 1118.36); 1 H NMR (500 MHz, DMSO-d6) δ 10.05 (s, 1H), 8.34 (s, 1H), 8.26-8.09 (m, 2H), 7.73 (s, 3H), 7.65 (d, J=8.2 Hz, 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 =6.3 Hz, 2H), 2.20-2.10 (m, 1H), 1.96-1.70 (m, 4H), 1.68-1.25 (m, 5H), 0.89 (t, J =7.3 Hz, 3H).

[0133] 2.2 L1-D2-Ab nectin-4 Antibody-drug conjugate (ADC2)

[0134]

[0135] Take Nectin4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1M Na2HPO4 solution, add 0.1M disodium ethylenediaminetetraacetate solution (25 μL), add prepared TCEP·HCl (tri(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react for 3 hours at room temperature (25°C) while rotating a turntable.

[0136] compound L1-D2 (0.90 mg, 0.80 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, and homogeneously mixed. The reaction was carried out for 16 hours at room temperature while rotating a rotary rotatometer; after the reaction was complete, small molecules were removed using a NAP-5 gel column (Cytiva), the buffer was replaced with a 20 mM PB solution, and the pH was adjusted to 6.3 to obtain an antibody-drug conjugate. ADC2 (3.1 mg / mL, 2 mL) was obtained.

[0137] Mean value calculated by RP-MS: n=7.9; MS results show that the antibody light chain (L) is one L1-D2 It is coupled to the (linker-payload), and the heavy chain (H) has 3 L1-D2 Indicates that it is coupled to (linker-payload) (Fig. 2).

[0138] 2.3 7-(N-(bromoacetamide-PEG) 8 -propionyl-Gly-Lys-PABC)-N-((R)-3-tetrahydrofuran))amine ethyl camptothecin (L2-D2)

[0139]

[0140] At room temperature, in a 100 mL single-neck flask L1-D2-2 Fmoc-PEG8-OSu (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 sequentially and reacted by stirring at room temperature for 1 hour. After concentration and purification by silica gel column chromatography, a yellow solid was obtained. L2-D2-1 (1.26 g, yield 78.25%) was obtained; LCMS: [M+1] + 1728.85 (Calculated value 1727.97).

[0141] At room temperature, in a 10 mL single-neck branched flask 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 and reacted by stirring at room temperature for 20 minutes. After concentration and purification by silica gel column chromatography, a pale yellow solid was obtained. L2-D2-2 (960 mg, yield 87.27%) was obtained; LCMS: [M+1] + 1506.38 (Calculated value 1505.73).

[0142] At room temperature, in a 10 mL single-neck flask L2-D2-2(960 mg, 637.57 μmol), DMF (10 mL), and bromoacetic anhydride (165.69 mg, 637.57 μmol) were added sequentially and reacted by stirring at room temperature for 30 minutes. After concentration and purification by silica gel column chromatography, a pale yellow solid was obtained. L2-D2-3 (440 mg, yield 42.31%) was obtained; LCMS: [M+1] + 1627.51 (Calculated value 1626.66).

[0143] At room temperature, in a 10 mL single-neck branched flask L2-D2-3 (440 mg, 270.61 μmol), DCM (5 mL), and TFA (744.52 mg, 6.53 mmol, 500 μL) were added sequentially and reacted by stirring at room temperature for 2 hours. Afterward, 10 mL of ether was added dropwise to the reaction mixture and centrifuged (10,000 rpm / 5 min). The supernatant was discarded, and the solid was concentrated under reduced pressure to remove residual solvent, yielding a yellow solid powder. L2-D2 (350 mg, yield 93.58%, HPLC 97%) was obtained; LCMS: [M+1] + 1385.22 (Calculated value 1384.34); 1 H NMR (600 MHz, DMSO-d6) δ 10.04 (s, 1H), 8.35 (t, J =5.7 Hz, 1H), 8.19 (t, J =5.7 Hz, 1H), 8.15 (d, J =8.0 Hz, 1H), 7.72 (d, J =5.8 Hz, 3H), 7.65 (d, J =8.1 Hz, 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, 5.4 Hz, 1H), 4.41 (q, J=7.7, 7.2 ㎐, 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, 5.4 ㎐, 3H), 2.78 (q, J =6.9, 6.3 ㎐, 2H), 2.40 (dt, J =8.2, 4.1 ㎐, 2H), 2.14 (dtt, J =13.2, 8.7, 5.5 ㎐, 1H), 1.87 (dh, J =21.2, 7.3 ㎐, 2H), 1.76 (ddt, J =16.4, 10.4, 5.3 ㎐, 1H), 1.62 (qd, J =13.3, 11.3, 6.8 ㎐, 1H), 1.55 (dp, J =14.0, 6.6 ㎐, 2H), 1.39 (td, J =8.9, 5.4 ㎐, 1H), 1.36-1.29 (m, 1H), 1.09 (t, J =7.0 ㎐, 2H), 0.89 (t, J =7.3 ㎐, 3H).

[0144] Example 3: Ab nectin-4 -( S-7-(N-(acetamide-PEG 2 -Propionyl-Gly-Lys-PAB(3-Bis PEG 4 -Carbonyl)C)-N-((R)-3-Tetrahydrofuran))amine ethyl camphothecin) 8 (ADC3)

[0145] 3.1 7-(N-(bromoacetamide-PEG 2 -Propionyl-Gly-Lys-PAB(3-Bis PEG 4 -Carbonyl)C)-N-((R)-3-Tetrahydrofuran))amine ethylcamptothecin (L3-D2)

[0146]

[0147] 6-amino-3H-isobenzofuran-1-one (4.0 g, 26.82 mmol), acetonitrile (50 mL), imidazole-1-sulfonylaside 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 a reaction flask, and the reaction mixture was stirred at room temperature for 16 hours. The solid was filtered, the filtrate was concentrated, and the mixture was extracted with dichloromethane. After washing with saturated saline solution, the mixture was dried with anhydrous sodium sulfate and concentrated to obtain the intermediate. I-2 (2.0 g, yield 38%) was obtained and used directly in the next step reaction without separate purification.

[0148] In the reaction flask I-2 Add 6.9 g (39.40 mmol), THF (50 mL), water (50 mL), and NaOH (2.36 g, 59.09 mmol), and react the mixture by stirring at 70°C for 2 hours. Afterward, concentrate under reduced pressure, acidify with 1N HCl, filter the precipitated white solid, and dry it to obtain 5-azide-2-hydroxymethylbenzoic acid I-3 (7.3 g, yield 91%) was obtained.

[0149] In a reaction flask at 0℃ I-3 5-azide-2-(TBDMSO)methylbenzoic acid was added to the mixture and stirred overnight at room temperature. Afterward, the mixture was poured into water to precipitate the solid, which was then filtered, washed with water, and dried to yield a grayish-white solid product. I-4 (10 g, yield 88%) was obtained; 1 H NMR (600 MHz, CDCl3) δ 7.68-7.51 (m, 2H), 7.10 (dd, J =8.4, 2.6 Hz, 1H), 4.92 (d, J=0.9 Hz, 2H), 0.82 (s, 9H), 0.00 (s, 6H).

[0150] N,N-di(methoxyethoxyethoxyethoxyethyl)amine or (Me-PEG 4 ) 2 NH : Methoxyethoxyethoxyethoxyethanol (40 g, 191.2 mmol), triethylamine (58 g, 567.8 mmol), and p-toluenesulfonyl chloride (43.7 g, 228.2 mmol) and DCM (400 mL) were added to a reaction flask, the reaction mixture was stirred at room temperature for 3 hours, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain the oily substance methoxyethoxyethoxyethoxyethanol p-toluenesulfonate (61.5 g, yield 88%); 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 each placed in a reaction flask containing acetonitrile (500 mL), and the reaction mixture was heated to 70°C while stirring and reacted for 16 hours. The mixture was filtered, the filtrate was concentrated, and purified by silica gel column chromatography to obtain N-benzyl-N,N-di(methoxyethoxyethoxyethoxyethyl)amine (23.1 g, yield 58.7%); LCMS: [M+H] + 488.54 (calculated value 487.63). N-benzyl-N,N-di(methoxyethoxyethoxyethoxyethyl)amine (10 g, 20.5 mmol), methanol (100 mL), and 10% Pd-C were added to a reaction flask and reacted at room temperature under a hydrogen balloon atmosphere with stirring for 16 hours, after which the mixture was filtered and the filtrate concentrated to obtain the oily product N,N-di(methoxyethoxyethoxyethoxyethyl)amine, (Me-PEG 4 ) 2 NH (7.9 g, yield 97%) was obtained; LCMS: [M+H] +398.31 (Calculated value 397.27).

[0151] In the reaction flask I-4 (10.5 g, 34.16 mmol), (Me-PEG 4 ) 2 NH 5-azide-2-(TBDMSO)methylbenzoyldi(Me-PEG4)amine was added to the reaction mixture and stirred at room temperature for 4 hours. Water was then added, extracted with DCM, dried with anhydrous sodium sulfate to concentrate, and purified by silica gel column chromatography to obtain a colorless liquid product. L3-1 (15 g, yield 59.46%) was obtained; LCMS: [M+Na] + 709.42 (Calculated value 686.39); 1 H NMR (500 MHz, CDCl3) δ 7.44 (d, J =8.4 Hz, 1H), 6.96 (dd, J =8.3, 2.4 Hz, 1H), 6.81 (d, J =2.4 Hz, 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).

[0152] In the reaction flask L3-1 5-azide-2-hydroxymethylbenzoyldi(Me-PEG4)amine was added (15 g, 21.84 mmol), DCM (250 mL), and TBAF (12.21 g, 43.67 mmol) and stirred at room temperature for 16 hours. Water was added, and the mixture was extracted with DCM, washed with saturated saline, dried with anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain a colorless liquid product. L3-2(11 g, yield 83.6%) was obtained; LCMS: [M+H] + 573.32 (Calculated value 572.31); 1 H NMR (500 MHz, CDCl3) δ 7.44 (d, J =8.2 Hz, 1H), 7.03 (dd, J =8.2, 2.4 Hz, 1H), 6.95 (d, J =2.3 Hz, 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 =6.6 Hz, 6H).

[0153] In the reaction flask 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, and the reaction mixture was stirred for 2 hours under an atmospheric pressure hydrogen balloon environment. After filtration and concentration, the colorless liquid product was obtained, 5-amino-2-hydroxymethylbenzoyldi(Me-PEG4)amine. L3-3 (9 g, yield 93.2%) was obtained; LCMS: [M+Na] + 569.20 (Calculated value 546.32); 1 H NMR (500 MHz, DMSO-d6) δ 7.13 (d, J =8.3 Hz, 1H), 6.58 (dd, J =8.2, 2.3 Hz, 1H), 6.38 (d, J =2.4 Hz, 1H), 5.13 (s, 2H), 4.70 (t, J =5.5 Hz, 1H), 4.27 (d, J =5.5 Hz, 2H), 3.63 (d, J =5.0 Hz, 2H), 3.61-3.49 (m, 19H), 3.49-3.43 (m, 8H), 3.34 (t, J =6.0 Hz, 3H), 3.27 (d, J =1.0 Hz, 6H).

[0154] Fmoc-Lys(Trt) (366 mg, 0.60 mmol), in a 50 mL single-neck flask L3-3 Add (500 mg, 0.92 mmol) and DCM (5 mL), add EEDQ (222 mg, 0.90 mmol) to the reaction flask while cooling in an ice bath, and continuously stir in the ice bath for 12 hours. Then, pour into a saturated sodium bicarbonate aqueous solution, extract with DCM, wash with saturated saline, dry with anhydrous sodium sulfate, and concentrate. L3-4 The crude product was obtained and used directly in the next step reaction without further purification; LCMS: [M+1] + 1139.61 (Calculated value 1138.59).

[0155] In a 50 mL single-neck flask, the above L3-4 Add (~1g crude product) and dissolve in DCM (6 mL), add DBU (65 mg, 0.43 mmol) while stirring, react at room temperature for 30 min, and concentrate the reaction solution directly under reduced pressure. L3-5 The crude product (~900 mg) was obtained and used directly in the next step without separate purification; LCMS: [M+H] + 917.91 (Calculated value 916.52).

[0156] In a 50 mL single-neck 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 in an ice bath and reacted by stirring at room temperature for 2 hours, after which water was added and extracted with DCM, washed with an aqueous solution of saturated sodium bicarbonate and saturated saline, dried with anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography. L3-6A yellow oily substance (300 mg, three-stage total yield 41.8%) was obtained; LCMS: [M+1] + 1196.87 (Calculated value 1195.61); 1 H NMR (600 MHz, DMSO-d6) δ 10.04 (s, 1H), 8.10 (d, J =7.8 Hz, 1H), 7.88 (d, J =7.6 Hz, 1H), 7.69 (d, J =7.5 Hz, 1H), 7.54 (t, J =6.1 Hz, 2H), 7.50-7.45 (m, 1H), 7.45-7.35 (m, 8H), 7.32 (q, J =7.5, 7.0 Hz, 2H), 7.25 (t, J =7.7 Hz, 5H), 7.14 (t, J =7.4 Hz, 3H), 5.12-5.03 (m, 1H), 4.38 (d, J =5.7 Hz, 3H), 4.30-4.12 (m, 3H), 3.73-3.58 (m, 5H), 3.55 (q, J =2.1 Hz, 5H), 3.56-3.27 (m, 25H), 3.25-3.18 (m, 8H), 1.92 (q, J =7.2 Hz, 2H), 1.72-1.62 (m, 1H), 1.60-1.51 (m, 1H), 1.47 (p, J =7.6 Hz, 2H), 1.38-1.28 (m, 1H), 1.29-1.22 (m, 3H).

[0157] At room temperature, in a 50 mL single-neck flask L3-6 (300 mg, 0.25 mmol) and DCM (3 mL) were added and stirred to dissolve; then, TEA (75 mg, 0.75 mmol) and bis(4-nitrophenyl)carbonate (228 mg, 0.75 mmol) were added dropwise and reacted at room temperature with stirring for 2 hours; afterward, the solution was poured into water and extracted with DCM; washed with saturated saline solution; dried with anhydrous sodium sulfate and rotary evaporated; and purified by silica gel column chromatography.L3 (280 mg, yield 82%) was obtained; LCMS: [M+1] + 1361.64 (Calculated value 1360.62); 1 H NMR (600 MHz, DMSO-d6) δ 10.20 (s, 1H), 8.35-8.29 (m, 2H), 8.12 (dd, J =8.5, 4.2 Hz, 1H), 7.88 (d, J =7.6 Hz, 1H), 7.69 (d, J =7.5 Hz, 2H), 7.62 (d, J =9.5 Hz, 2H), 7.60-7.51 (m, 3H), 7.49 (d, J =8.5 Hz, 1H), 7.44-7.35 (m, 7H), 7.32 (q, J =7.2 Hz, 2H), 7.25 (t, J =7.6 Hz, 6H), 7.14 (t, J =7.4 Hz, 3H), 5.76 (s, 1H), 5.19 (s, 2H), 4.39 (q, J =7.0, 6.6 Hz, 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, 3.4 Hz, 4H), 3.33 (s, 7H), 3.28 (s, 1H), 3.19 (d, J =6.0 Hz, 5H), 2.01-1.88 (m, 2H), 1.67 (s, 1H), 1.56 (s, 1H), 1.46 (q, J =7.3 Hz, 2H), 1.34 (d, J =9.6 Hz, 1H), 1.28 (s, 1H), 1.23 (d, J =4.1 Hz, 1H).

[0158] In a 50 mL single-neck flask 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 and the mixture was stirred at room temperature for 12 hours, after which it was extracted with DCM (50 mL), washed sequentially with a saturated sodium bicarbonate aqueous solution (50 mL) and water (50 mL), dried with anhydrous sodium sulfate, and concentrated. L3-D2-1 (Early product) was obtained and used directly in the next step without separate purification; LCMS: [M+1] + 1727.89 (Calculated value 1726.77); 1 H NMR (600 MHz, DMSO-d6) δ 10.14 (s, 1H), 8.13 (s, 1H), 7.86 (s, 2H), 7.81 (s, 1H), 7.67 (d, J =7.3 Hz, 2H), 7.60 (s, 1H), 7.53 (s, 2H), 7.45 (d, J =8.3 Hz, 1H), 7.42-7.34 (m, 7H), 7.31 (q, J =8.8, 7.2 Hz, 3H), 7.24 (t, J =7.4 Hz, 7H), 7.13 (t, J =7.3 Hz, 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 =8.7 Hz, 1H), 4.29-4.17 (m, 2H), 3.91 (s, 1H), 3.65 (dd, J =23.2, 6.3 Hz, 2H), 3.64-3.53 (m, 1H), 3.59 (s, 9H), 3.56-3.46 (m, 3H), 3.47 (d, J=3.0 Hz, 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, 7.2 ㎐, 3H), 1.67 (s, 1H), 1.56 (d, J =10.1 Hz, 1H), 1.47 (s, 2H), 1.28 (s, 1H), 1.23 (d, J =4.4 Hz, 1H), 0.87 (t, J =7.3 Hz, 3H).

[0159] In a 50 mL single-neck flask L3-D2-1 (Corn product) and DCM (5 mL) were added sequentially and stirred to dissolve, DBU (26.6 mg, 10.6 mmol) was added dropwise, and the mixture was reacted by stirring at room temperature for 1 hour. Afterward, the mixture was poured into methyl t-butyl ether (50 mL), filtered, and dried. L3-D2-2 The crude product was obtained and used directly in the next step without separate purification; LCMS: [M+1] + 1505.96 (Calculated value 1504.71).

[0160] In a 50 mL single-neck flask L3-D2-2 The crude product and DCM (10 mL) were added sequentially and stirred to dissolve, then the prepared Fmoc-NH-PEG2-CH2CH2COOSu (124 mg, 0.25 mmol) was added, and the mixture was reacted by stirring at room temperature for 2 hours, after which it was concentrated and purified by silica gel column chromatography. L3-D2-3 (87 mg, three-stage total yield 18%) obtained; LCMS: [M+1] + 1886.99 (Calculated value 1885.86); 1 H NMR (600 MHz, DMSO-d6) δ 10.10 (s, 1H), 8.13 (t, J =6.2 Hz, 2H), 7.90-7.82 (m, 2H), 7.67 (d,J =7.6 ㎐, 2H), 7.62 (d, J =10.0 ㎐, 2H), 7.47 (q, J =8.5, 6.2 ㎐, 1H), 7.38 (dd, J =10.8, 7.6 ㎐, 7H), 7.31 (dt, J =17.4, 6.8 ㎐, 3H), 7.30-7.20 (m, 7H), 7.15 (q, J =7.3, 6.8 ㎐, 3H), 6.49 (s, 1H), 6.28 (t, J =3.2 ㎐, 1H), 6.13 (s, 1H), 5.42 (d, J =2.7 ㎐, 2H), 5.24 (d, J =16.8 ㎐, 2H), 5.07 (s, 2H), 4.58 (d, J =28.1 ㎐, 1H), 4.37 (q, J =7.4 ㎐, 1H), 4.27 (d, J =6.9 ㎐, 2H), 4.18 (q, J =8.0, 6.9 ㎐, 1H), 3.92 (d, J =12.5 ㎐, 1H), 3.74 (d, J =5.6 ㎐, 2H), 3.72-3.54 (m, 9H), 3.54-3.46 (m, 2H), 3.51-3.41 (m, 21H), 3.38 (d, J =5.0 ㎐, 3H), 3.39-3.31 (m, 12H), 3.31 (d, J =17.6 ㎐, 4H), 3.27 (s, 1H), 3.19 (s, 1H), 3.11 (q, J =6.1 ㎐, 2H), 2.59 (s, 1H), 2.37 (t, J =6.5 ㎐, 2H), 2.15 (d, J =14.1 ㎐, 1H), 1.97-1.78 (m, 4H), 1.68 (t, J =9.0 ㎐, 1H), 1.60-1.53 (m, 1H), 1.48 (p, J=7.4 Hz, 2H), 1.35 (s, 1H), 1.30-1.20 (m, 3H), 0.91-0.81 (m, 3H).

[0161] In a 50 mL single-neck flask L3-D2-3 (87 mg, 0.046 mmol) and DCM (5 mL) were added sequentially and stirred until most of the solution was dissolved, then DBU (4.9 mg) was added dropwise. After the addition was complete, the mixture was stirred at room temperature for 30 minutes to react, then poured into methyl t-butyl ether (60 mL) and centrifuged. The supernatant was discarded, and the solution was purified by silica gel column chromatography. L3-D2-4 Product (70 mg, yield 91%, HPLC 96%) was obtained; LCMS: [M+1] + 1664.78 (Calculated value 1663.79); 1 H NMR (600 MHz, DMSO) δ 10.10 (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).

[0162] In a 50 mL single-neck flask L3-D2-4 (70 mg, 0.042 mmol) and DCM (5 mL) were added sequentially and stirred to dissolve, then bromoacetic anhydride (10.9 mg, 0.042 mmol) was added; after the addition was complete, the mixture was stirred at room temperature for 30 minutes to react, concentrated, and purified by silica gel column chromatography. L3-D2-5 (60 mg, yield 80%, HPLC 98%) was obtained; LCMS: [M+1] + 1784.93 (Calculated value 1783.72).

[0163] In a 50 mL single-neck flask L3-D2-5 Add (60 mg, 33.6 μmmol) and DCM (3 mL) and stir to dissolve; then, add TFA (0.3 mL) dropwise and react at room temperature with stirring for 1 hour; after that, add the reaction mixture dropwise to methyl t-butyl ether (30 mL), centrifuge, discard the supernatant, and dry the remaining solid to obtain a yellow solid product. L3-D2 (35 mg, yield 67%, HPLC 97%) was obtained; LCMS: [M+1] + 1542.84 (Calculated value 1541.61); 1 H NMR (600 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.19 (t, J =5.8 Hz, 2H), 7.66 (dt, J =11.8, 5.8 Hz, 4H), 7.46 (d, J =8.5 Hz, 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 =7.3 Hz, 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, 7.3 Hz, 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 =4.1 Hz, 6H), 2.77 (h, J =6.4 Hz, 2H), 2.40 (t, J =6.5 Hz, 2H), 2.15 (s, 1H), 1.86 (dp, J =21.4, 7.2 Hz, 3H), 1.76 (qd, J =10.5, 7.0, 5.3 ㎐, 1H), 1.68-1.57 (m, 1H), 1.54 (hept,J =6.7 Hz, 2H), 1.43-1.27 (m, 2H), 0.87 (t, J =7.3 Hz, 3H); 13 C NMR (151 MHz, DMSO) δ 173.04, 172.28, 171.25, 158.70, 158.47, 150.55, 146.77, 128.56, 118.58, 96.41, 72.87, 71.70, 70.25, 70.20, 70.11, 70.01, 69.91, 69.37, 69.17, 68.31, 67.14, 65.71, 61.82, 58.48, 50.01, 42.58, 38.32, 36.30, 30.66, 29.92, 27.14, 22.83, 8.23.

[0164] 3.2 L3-D2-Ab nectin-4 Antibody-drug conjugate (ADC3)

[0165]

[0166] Take Nectin4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1M Na2HPO4 solution, add 0.1M disodium ethylenediaminetetraacetate solution (25 μL), add prepared TCEP·HCl solution (10 mM, 0.04 mL), and react for 3 hours at room temperature (25℃) while rotating a turntable.

[0167] compound L3-D2 (1.23 mg, 0.80 mmol) was dissolved in 0.12 mL of DMA, added to the above solution system, and homogeneously mixed. The reaction was carried out for 16 hours at room temperature while rotating a rotary rotatometer; after the reaction was complete, small molecules were removed using a NAP-5 gel column (Cytiva), the buffer was replaced with a 20 mM PB solution, and the pH was adjusted to 6.3 to obtain an antibody-drug conjugate. ADC3 (3.2 mg / mL, 2 mL) was obtained.

[0168] Mean value calculated by RP-MS: n=7.9; MS results show that the antibody light chain (L) is one L3-D2 It is coupled to the (linker-payload), and the heavy chain (H) has 3 L3-D2 Indicates that it is coupled to the (linker-payload) (Fig. 3).

[0169] Example 4: Ab nectin-4 -( S-7-(N-(acetamide-PEG 2 -Propionyl-Gly-Lys-PAB(3-Bis PEG 4 -Carbonyl)C)-N-(4-Tetrahydropyran))Amine Ethyl Camptothecin) 8 (ADC4)

[0170] 4.1 7-(N-(bromoacetamide-PEG 2 -Propionyl-Gly-Lys-PAB(3-Bis PEG 4 -carbonyl)C)-N-(4-tetrahydropyran))amine ethyl camphothecin (L3-D3)

[0171]

[0172] intermediate I-1 18 g (68.12 mmol) was added to 180 mL of IPA and the reaction mixture was stirred; then, DIEA (17.61 g, 136.24 mmol) and 4-aminotetrahydropyran hydrochloride (37.50 g, 272.48 mmol) were added, and the reflux reaction was carried out for 12 hours. After concentrating the reaction mixture until the remaining 20 mL of IPA was reduced, 200 mL of water was added and stirred for 2 hours. The mixture was filtered to obtain a solid, which was then dried at 40°C to produce the product. D3-1 (20.2 g, yield 91.7%) was obtained; LCMS: [M+1] + 320.98 (Calculated value 320.10).

[0173] compound D3-1After adding (16.9 g, 52.63 mmol) to DCM (310 mL), acetic acid (63.05 g, 1.05 mol) was added and stirred until the solid was completely dissolved. Maintaining the internal temperature of the reaction solution at 15°C, sodium triacetoxyborohydride (27.89 g, 131.56 mmol) was added in four installments of approximately 7 g at intervals of about 5 min, and the reaction was carried out for 2 hours while maintaining the internal temperature at 15°C. Subsequently, the reaction solution was poured into a sodium carbonate ice water solution (sodium carbonate: 130 g, ice water: 1 L), stirred while controlling the internal temperature to 15°C, and phase separation was performed. The organic phase was washed with water and dried with anhydrous sodium sulfate to obtain the compound. D3-2 A DCM solution was obtained and used directly in the next step without separate purification.

[0174] While cooling in an ice bath under a nitrogen atmosphere, the above D3-2 After adding the DCM solution to DIEA (13.58 g, 105.26 mmol), Cbz-Cl (10.11 g, 59.27 mmol) was added dropwise and reacted in an ice bath with continuous stirring for 12 hours. Subsequently, the mixture was washed twice with a 10% aqueous citric acid solution (200 mL x 2), followed by washing with a saturated aqueous sodium bicarbonate solution (200 mL) and a saturated saline solution (200 mL). The organic phase was collected, dried with anhydrous sodium sulfate, and concentrated to obtain the compound D3-3 (22.9 g, two-stage yield 95%) was obtained; LCMS: [M+1] + 457.28 (Calculated value 456.15).

[0175] compound D3-3(22.7 g, 49.7 mmol) was placed in a 1 L single-neck flask, and 50 mL of DMF was added. While cooling in an ice bath under a nitrogen atmosphere, the prepared reducing solution of H2O (250 mL) / sodium dithionate (39.30 g, 225.72 mmol) / sodium carbonate (19.14 g, 180.58 mmol) was slowly added dropwise. After the addition was complete, the temperature was slowly raised to 40°C and the reaction was carried out by stirring for 2 hours. The mixture was filtered, the filtrate was concentrated, and the extract was separated by adding ethyl acetate (500 mL) and water (500 mL). The mixture was dried with anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to obtain a yellow oily substance. D3-4 (20.3 g, yield 95.7%, HPLC 98%) was obtained; LCMS: [M+1] + 427.22 (Calculated value 426.19).

[0176] Compound in a 500 mL single-neck flask D3-4 (18.56 g, 43.55 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyranO[3,4-F]indoleizine-3,6,10(4H)-one (13.75 g, 52.27 mmol), and PPTS (218.88 g, 871.00 mmol) were added sequentially and reacted at 110°C for 12 hours under an argon atmosphere. The reaction mixture was poured into methanol (1000 mL), stirred for 2 hours, and filtered to obtain a dark brown solid. The brown solid was washed with methanol (300 mL) for 2 hours and filtered to obtain a light brown solid. The solid was dried at 40°C to obtain the compound. D3-5 (11.0 g, yield 38.7%, HPLC 97%) was obtained; LCMS: [M+1] + 653.98 (Calculated value 653.24); 1 H NMR (500 MHz, DMSO-d6) δ 7.38 (t, J =68.6 Hz, 7H), 6.49 (s, 1H), 6.29 (s, 2H), 5.43 (s, 2H), 5.26 (dd, J=55.5, 36.0 Hz, 4H), 4.05 (s, 1H), 3.90 (d, J =7.6 Hz, 2H), 3.46 (d, J =8.4 Hz, 2H), 1.93-1.74 (m, 4H), 1.57 (s, 2H), 0.88 (t, J =7.3 Hz, 3H).

[0177] Compound in a 10 L single-neck flask 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 and reacted at room temperature for 20 h under a hydrogen balloon atmosphere. After removing the Pd / C by filtration, the reaction mixture was rotary evaporated, stirred for 24 h with 30 mL of a mixed solution of dichloromethane and methanol (V / V = 1:1), filtered, and dried for 3 h at 40°C to produce the product. D3 (6.1g, yield 74%, HPLC 95.0%) was obtained; LCMS: [M+1] + 520.22 (Calculated value 519.20); 1 H NMR (500 MHz, DMSO-d6) δ 7.59 (s, 1H), 7.47-7.41 (m, 1H), 7.25-7.14 (m, 1H), 6.54 (s, 1H), 6.27 (t, J =9.9 Hz, 2H), 5.77 (s, 1H), 5.44 (d, J =16.5 Hz, 2H), 5.23-5.11 (m, 2H), 3.83 (d, J =9.5 Hz, 2H), 3.23 (t, J =24.9 Hz, 4H), 2.93 (d, J =32.7 Hz, 3H), 1.99-1.72 (m, 4H), 1.33 (s, 2H), 0.96-0.81 (m, 3H).

[0178] In a 50 mL single-neck flask 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 sequentially and reacted by stirring at room temperature for 12 hours, then extracted with DCM, washed sequentially with saturated sodium bicarbonate aqueous solution (50 mL) and water (50 mL), dried with anhydrous sodium sulfate, and concentrated. L3-D3-1 (Crystal product) was obtained and used directly in the next step reaction without separate purification; LCMS: [M+1] + 1741.93 (Calculated value 1740.79).

[0179] In a 50 mL single-neck flask, the above L3-D3-1 (Corn product) and DCM (5 mL) were added sequentially and stirred to dissolve, DBU (26.6 mg) was added dropwise, and the mixture was stirred at room temperature for 1 hour. Afterward, the mixture was poured into methyl t-butyl ether (50 mL), centrifuged, and dried. L3-D3-2 (Early product) was obtained and used directly in the next step without separate purification; LCMS: [M+1] + 1519.96 (Calculated value 1518.72).

[0180] In a 50 mL single-neck flask L3-D3-2 (Crude product) and DCM (10 mL) were added sequentially and stirred to dissolve, then the prepared Fmoc-NH-PEG2-CH2CH2COOSu (124 mg, 0.25 mmol) was added, and the reaction was continued with stirring at room temperature for 2 hours, after which the mixture was concentrated and purified by silica gel column chromatography. L3-D3-3 The product (89 mg, three-stage total yield 18.7%) was obtained; LCMS: [M+1] + 1900.97 (Calculated value 1899.88); 1 H NMR (600 MHz, DMSO-d6) δ 10.09 (s, 1H), 8.12 (s, 2H), 7.87 (dd, J=15.9, 7.5 Hz, 3H), 7.68 (dd, J =12.8, 7.5 Hz, 2H), 7.35-7.22 (m, 9H), 6.29 (s, 1H), 6.11 (s, 1H), 5.42 (s, 2H), 5.33 (d, J =40.1 Hz, 2H), 5.03 (s, 1H), 4.28 (dd, J =11.9, 6.9 Hz, 2H), 3.95-3.85 (m, 2H), 3.74 (d, J =5.6 Hz, 2H), 3.58 (h, J =9.2, 7.8 Hz, 3H), 3.54-3.46 (m, 3H), 3.44 (q, J =10.7, 8.8 ㎐, 22H), 3.37 (tt, J =6.3, 4.3, 3.5 ㎐, 6H), 1.84 (ddd, J =37.1, 19.5, 10.2 ㎐, 2H), 1.40-1.22 (m, 3H), 0.86 (dt, J =13.0, 7.2 Hz, 4H).

[0181] In a 50 mL single-neck flask L3-D3-3 (87 mg, 0.045 mmol) and DCM (6 mL) were added sequentially and dissolved while stirring, then DBU (4.9 mg) was added dropwise and reacted by stirring at room temperature for 30 min, after which the solution was poured into methyl t-butyl ether (50 mL), centrifuged, the supernatant was discarded, and the solution was purified by silica gel column chromatography. L3-D3-4 (74 mg, yield 97%, HPLC 96%) was obtained; LCMS: [M+1] + 1678.96 (Calculated value 1678.82).

[0182] In a 50 mL single-neck flask L3-D3-4(73 mg, 0.043 mmol) and DCM (5 mL) were added sequentially and stirred to dissolve, then bromoacetic anhydride (11.0 mg, 0.043 mmol) was added and the mixture was reacted by stirring at room temperature for 30 minutes, after which it was concentrated and purified by silica gel column chromatography. L3-D3-5 (65 mg, yield 84%, HPLC 98%) was obtained; LCMS: [M+1] + 1798.98 (Calculated value 1798.74).

[0183] In a 50 mL single-neck flask L3-D3-5 Add DCM (5 mL) and (64 mg, 35.6 μmmol) and stir to dissolve; then, add TFA (0.3 mL) dropwise to the reaction mixture and stir at room temperature for 1 hour; after that, add the reaction mixture dropwise to methyl t-butyl ether (30 mL), centrifuge to obtain a solid, dry it, and produce a yellow solid product. L3-D3 (35 mg, yield 63%, HPLC 97%) was obtained; LCMS: [M+1] + 1556.87 (Calculated value 1556.63); 1 H NMR (500 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.40-8.15 (m, 2H), 7.75-7.65 (m, 3H), 7.5-7.40 (m, 2H), 7.24 (s, 1H), 6.35-6.10 (m, 2H), 5.43 (s, 2H), 5.40-5.25 (m, 2H), 5.15-5.00 (m, 2H), 4.45-4.30 (m, 2H), 4.08-3.20 (m, 63H), 2.85-2.70 (m, 2H), 2.40 (t, J =6.2 Hz, 2H), 1.95-1.70 (m, 5H), 1.67-1.46 (m, 5H), 1.45-1.27 (m, 2H), 0.88 (t, J =7.2 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ 173.00, 171.22, 166.56, 158.71, 158.44, 157.29, 151.37, 150.59, 149.88, 146.81, 128.62, 118.54, 103.07, 72.88, 71.71, 70.26, 70.21, 70.13, 70.02, 69.96, 69.91, 69.17, 67.13, 65.71, 65.37, 58.48, 50.16, 48.86, 44.46, 42.61, 39.15, 36.32, 31.77, 30.72, 29.91, 27.15, 22.84, 15.63, 8.23.

[0184] 4.2 L3-D3-Ab nectin-4 Antibody-drug conjugate (ADC4)

[0185]

[0186] Take Nectin4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1M Na2HPO4 solution, add 0.1M disodium ethylenediaminetetraacetate solution (25 μL), add prepared TCEP·HCl solution (10 mM, 0.04 mL), and react for 3 hours at room temperature (25℃) while rotating a turntable.

[0187] compound L3-D3 (1.25 mg, 0.80 mmol) was dissolved in 0.13 mL of DMA, added to the above solution system, and homogeneously mixed. The reaction was carried out for 16 hours at room temperature while rotating a rotary rotatometer; after the reaction was complete, small molecules were removed using a NAP-5 gel column (Cytiva), the buffer was replaced with a 20 mM PB solution, and the pH was adjusted to 6.3 to obtain an antibody-drug conjugate. ADC4 (3.2 mg / mL, 2 mL) was obtained.

[0188] Mean value calculated by RP-MS: n=7.8; MS results show that the antibody light chain (L) is one L3-D3 It is coupled to the (linker-payload), and the heavy chain (H) has 3 L3-D3 It indicates that it is coupled to the (linker-payload) (Fig. 4).

[0189] Example 5: Ab nectin-4 -( S-7-(N-(bromoacetamide-PEG 2 -Propionyl-Gly-Lys-PAB(2-PEG 8 -Oxy)C)-N-((R)-3-tetrahydrofuran))amine ethyl camphothecin) 8 (ADC5)

[0190] 5.1 7-(N-(bromoacetamide-PEG 2 -Propionyl-Gly-Lys-PAB(2-PEG 8 -Oxy)C)-N-((R)-3-tetrahydrofuran))amine ethyl camphothecin (L4-D2)

[0191]

[0192] 2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]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 a reaction flask and stirred at room temperature for 16 hours. The mixture was then concentrated under reduced pressure and extracted with ethyl acetate. After washing with a saturated aqueous sodium chloride solution, the mixture was dried with anhydrous sodium sulfate and concentrated. The resulting product was purified by silica gel column chromatography to obtain the colorless liquid intermediate p-toluenesulfonate. L4-1 (50 g, yield 71.37%) was obtained; LCMS: [M+H] + 539.38 (Calculated value 538.24).

[0193] 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 a reaction flask and reacted by stirring at 70°C for 2 hours. Afterward, the mixture was concentrated and extracted with ethyl acetate, washed with water, dried with anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain a brown liquid intermediate compound. L4-2(27 g, yield 54.51%) was obtained; LCMS: [M+H] + 534.38 (Calculated value 533.25).

[0194] In the reaction flask L4-2 Sodium borohydride (21 g, 39.36 mmol), methanol (200 mL), and sodium borohydride (2.23 g, 59.04 mmol) were added and reacted by stirring at room temperature for 4 hours. Afterward, the mixture was concentrated under reduced pressure and extracted with ethyl acetate. The resulting product was washed with saturated saline, dried with anhydrous sodium sulfate, and concentrated to obtain a brown liquid product. L4-3 (19 g, yield 90.12%) was obtained.

[0195] In the reaction flask 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 and reacted by stirring under a hydrogen balloon atmosphere for 48 hours, after which the mixture was 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 value 505.29).

[0196] In a 1000 mL single-neck flask L4-4 Fmoc-Lys(Trt)OH (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 hours. Then, 300 mL of saturated sodium bicarbonate aqueous solution was added for extraction. After separation, the solution was washed with citric acid aqueous solution and saturated saline solution, respectively, dried with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain Fmoc-Lys(Trt)PAB(PEG8) L4-5 (17 g, yield 75.13%) was obtained; LCMS: [M+1] +1098.21 (Calculated value 1097.34).

[0197] In a 1000 mL single-neck flask L4-5 Add (17 g, 14.86 mmol), DCM (400 mL), and DBU (1.13 g, 7.43 mmol) and react by stirring at 25°C for 1 hour, then concentrate. L4-6 was obtained and used directly in the next step without separate purification; LCMS: [M+1] + 876.34 (Calculated value 875.10).

[0198] HOBT (4.18 g, 30.96 mmol), FmocGlyOH (4.83 g, 16.25 mmol), EDCI (3.12 g, 16.25 mmol), and DIPEA (3.00 g, 23.22 mmol) were added to the reaction solution from the previous step and reacted by stirring at 25°C for 2 hours. Subsequently, 200 mL of aqueous citric acid solution was added and stirred for extraction and separation. The solution was sequentially washed with saturated sodium bicarbonate aqueous solution and saturated saline solution, and after separation, dried with anhydrous magnesium sulfate, filtered, and concentrated. The resulting product was purified by silica gel column chromatography to obtain FmocGly-Lys(Trt)PAB(PEG8). L4-7 (15 g, two-stage yield 83.88%) was obtained; LCMS: [M+1] + 1155.60 (Calculated value 1154.40); 1 H NMR (600 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.16 (d, J =7.7 Hz, 1H), 7.87 (dd, J =16.8, 7.8 Hz, 2H), 7.68 (d, J =7.5 Hz, 2H), 7.53 (t, J =6.2 Hz, 1H), 7.40 (dd, J =20.1, 7.7 Hz, 6H), 7.32 (q, J =7.2 Hz, 2H), 7.26 (t, J =7.6 Hz, 5H), 7.15 (t, J=7.3 Hz, 3H), 7.01 (s, 1H), 6.86 (d, J =8.2 Hz, 1H), 5.76 (s, 3H), 5.15 (t, J =5.7 Hz, 1H), 4.44 (d, J =5.8 Hz, 3H), 4.27-4.18 (m, 2H), 4.10 (t, J =5.0 Hz, 2H), 3.79-3.64 (m, 4H), 3.57 (dd, J =5.9, 3.7 Hz, 2H), 3.52-3.43 (m, 22H), 3.41 (dd, J =5.8, 3.8 Hz, 2H), 3.34 (s, 3H), 2.50 (d, J =4.0 Hz, 2H), 1.94 (q, J =7.4 Hz, 2H), 1.72 (ddt, J =15.6, 11.0, 5.4 ㎐, 1H), 1.56 (tq, J =13.5, 8.4, 6.5 ㎐, 1H), 1.46 (hept, J =7.9, 7.4 Hz, 2H), 1.35-1.27 (m, 2H).

[0199] At room temperature, take a 250 mL single-neck flask and 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 sequentially and reacted by stirring at room temperature for 16 hours; then, ether (100 mL) was added, and the precipitated solid was filtered to obtain a pale yellow solid L4-D2-1 (1.5 g, yield 87.1%) was obtained; LCMS: [M+1] + 1687.56 (Calculated value 1686.92).

[0200] At room temperature, take a 250 mL single-neck flask and 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 sequentially and reacted by stirring at room temperature for 1 hour; then, ether (100 mL) was added, and the precipitated solid was filtered to obtain a pale yellow solid L4-D2-2 (1.2 g, yield 77.0%) was obtained; LCMS: [M+1] + 1465.38 (Calculated value: 1464.67).

[0201] At room temperature, take a 100 mL single-neck flask and 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 sequentially and reacted by stirring at room temperature for 0.5 h. Subsequently, 10 g of silica gel powder was added to prepare a sand mixture, and the product was purified by silica gel column chromatography to obtain a nearly white solid. L4-D2-3 (410 mg, yield 25.9%) was obtained; LCMS: [M+1] + 1847.01 (Calculated value: 1846.10); 1 H NMR (600 MHz, DMSO-d6) δ 10.54 (s, 2H), 8.15 (d, J =.6 Hz, 1H), 8.08 (t, J =5.8 Hz, 1H), 7.95 (d, J =7.8 Hz, 1H), 7.86 (d, J =7.6 Hz, 2H), 7.67 (d, J =7.5 Hz, 2H), 7.39 (dd, J =10.9, 7.6 Hz, 8H), 7.31 (q, J =7.5, 6.7 Hz, 3H), 7.28-7.22 (m, 7H), 7.15 (q, J =8.9, 7.3 Hz, 4H), 6.49 (s, 1H), 6.28 (s, 1H), 6.22 (s, 1H), 5.42 (d,J =2.4 Hz, 2H), 5.26 (s, 2H), 5.14 (s, 2H), 4.60 (d, J =7.8 Hz, 1H), 4.44 (q, J =7.3 Hz, 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 =6.5 Hz, 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 =5.9 Hz, 2H), 2.59 (s, 8H), 2.36 (t, J =6.5 Hz, 2H), 2.15 (dt, J =13.3, 8.5, 5.0 ㎐, 1H), 1.96-1.88 (m, 2H), 1.85 (dq, J =14.0, 7.1 Hz, 3H), 1.70 (s, 1H), 1.55 (d, J =9.1 Hz, 1H), 1.46 (q, J =7.5 Hz, 2H), 1.31-1.22 (m, 4H), 0.87 (t, J =7.3 Hz, 3H).

[0202] At room temperature, in a 10 mL single-neck branched flask 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 and reacted by stirring at room temperature for 1 hour. Then, 5 g of silica gel powder was added to make a sand mixture, and the resulting pale yellow solid was purified by silica gel column chromatography. L4-D2-4 (230 mg, yield 61.05%) was obtained; LCMS: [M+1] + 1624.75 (Calculated value: 1623.86); 1H NMR (600 MHz, DMSO-d6) δ 7.96 (s, 3H), 7.51-7.45 (m, 1H), 7.39 (d, J =7.9 Hz, 5H), 7.29-7.21 (m, 6H), 7.16 (q, J =7.4 Hz, 3H), 7.02 (s, 1H), 6.51 (s, 1H), 6.27 (s, 1H), 6.23 (s, 1H), 5.44-5.41 (m, 1H), 5.24 (s, 1H), 5.15 (s, 2H), 4.62 (s, 1H), 4.44 (q, J =7.2, 6.8 Hz, 1H), 4.13-4.09 (m, 1H), 3.79 (qd, J =16.7, 5.8 Hz, 1H), 3.73 (s, 1H), 3.60 (td, J =5.9, 5.3, 2.9 ㎐, 3H), 3.57-3.45 (m, 21H), 3.41 (dd, J =5.9, 3.7 Hz, 2H), 3.38 (s, 20H), 3.30 (s, 1H), 3.22 (s, 2H), 2.94 (t, J =5.3 Hz, 1H), 2.89 (s, 5H), 2.73 (s, 5H), 2.39 (t, J =6.5 Hz, 1H), 2.16 (ddt, J =12.4, 7.7, 4.5 ㎐, 1H), 1.95 (t, J =6.5 Hz, 2H), 1.87 (ddd, J =28.8, 13.7, 6.8 Hz, 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, 7.2 Hz, 3H).

[0203] At room temperature, take a 10 mL single-neck flask and L4-D2-4100 mg (60.13 μmol), 2 mL of DMF, and 18 mg (60.13 μmol) were added sequentially and reacted by stirring at room temperature for 2 hours. Then, 3 g of silica gel powder was added to prepare a sand mixture, and the resulting substance was purified by silica gel column chromatography to obtain a pale yellow solid. L4-D2-5 (80 mg, yield 72.34%) was obtained; LCMS: [M+1] + 1745.62 (Calculated value: 1744.79).

[0204] At room temperature, in a 10 mL single-neck branched flask 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 and reacted by stirring at room temperature for 2 hours; then, ether (10 mL) was added and centrifuged (10,000 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%) was obtained; LCMS: [M+1] + 1503.33 (Calculated value: 1502.47); 1 H 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 ㎐, 1H), 4.48 (t, J =5.8 ㎐, 1H), 4.18 (d, J =8.7 ㎐, 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 ㎐, 1H), 3.60 (t, J =6.5 ㎐, 4H), 3.48 (dd, J =10.4, 3.9 ㎐, 16H), 3.41 (q, J =4.8 ㎐, 5H), 3.38 (d, J =7.0 ㎐, 1H), 3.32 (s, 2H), 3.27-3.19 (m, 2H), 3.22 (s, 3H), 2.78 (h, J =6.6 ㎐, 2H), 2.40 (t, J =6.5 ㎐, 2H), 2.16 (dtd, J =13.4, 8.6, 5.1 ㎐, 1H), 1.85 (dqt, J =27.7, 13.5, 6.6 ㎐, 3H), 1.62 (dp, J =13.8, 4.7 ㎐, 1H), 1.54 (hpt, J =6.6 ㎐, 2H), 1.37 (ddt, J =23.6, 15.8, 7.0 ㎐, 2H), 1.24 (d, J =5.7 ㎐, 1H), 1.09 (t, J =7.0 ㎐, 1H), 0.88 (t, J =7.3 ㎐, 3H).

[0205] 5.2 L4-D2-Ab nectin-4 Antibody-drug conjugate (ADC5)

[0206]

[0207] Take Nectin4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1M Na2HPO4 solution, add 0.1M disodium ethylenediaminetetraacetate solution (25 μL), add prepared TCEP·HCl solution (10 mM, 0.04 mL), and react for 3 hours at room temperature (25℃) while rotating a turntable.

[0208] compound L4-D2 (1.20 mg, 0.80 mmol) was dissolved in 0.12 mL of DMA, added to the above solution system, and homogeneously mixed. The reaction was carried out for 16 hours at room temperature while rotating a rotary rotatometer; after the reaction was complete, small molecules were removed using a NAP-5 gel column (Cytiva), the buffer was replaced with a 20 mM PB solution, and the pH was adjusted to 6.3 to obtain an antibody-drug conjugate. ADC5 (3.1 mg / mL, 2 mL) was obtained.

[0209] Mean value calculated by RP-MS: n=7.9; MS results show that the antibody light chain (L) is one L4-D2 It is coupled to the (linker-payload), and the heavy chain (H) has 3 L4-D2 Indicates that it is coupled to the (linker-payload) (Fig. 5).

[0210] Test Example: ADC Antitumor Activity Experiment

[0211] Test Example 1: Activity of ADC inhibiting in vitro tumor cell growth

[0212] 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), respectively, containing 10% fetal bovine serum (Cellmax), to be in the exponential growth phase; after digestion with trypsin, the supernatant was discarded by centrifugation, and the medium was prepared in 6×10 oz. 4cells / mL, 6X10 4 cells / mL, 2X10 4 cells / mL, 3X10 4 cells / mL and 7X10 4 Dilute to cells / mL, add 100 μL per well to a 96-well cell culture plate, and incubate overnight in a 37°C, 5% CO2 incubator. The next day, use the medium to dilute the ADC to be measured 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. Add 100 μL of the diluted ADC per well to a 96-well cell culture plate, setting up three multi-wells for each concentration, and add 100 μL of medium per well to the negative control (without ADC) and blank control. After sample addition is complete, continue incubation for 6 days in a 37°C, 5% CO2 incubator. After the incubation is complete, remove the cell plate, aspirate and remove the medium using a pipette, add 100 μL of medium containing 10% CCK-8 to each well, and incubate at 37°C for 3 hours. After the incubation is complete, remove the culture plate, shield it from light, place it on a microplate reader, and measure the absorbance by selecting 630 nm as the reference wavelength and 450 nm as the measurement wavelength. Based on the absorbance values, calculate the IC using the 4-parameter regression method in GraphPad software. 50 The values ​​were calculated (Table 1). EV (enfortumab vedotin, Padcev ® ) is used as a positive control drug.

[0213] IC 50 Regarding the value, here "++++" is 50nM>IC 50 Indicates that; "+++" is 200nM>IC 50 Indicates that ≥50nM; "++" means 1000nM > IC 50 Indicates that ≥200nM; "+" is IC 50 It indicates that it is >1μM.

[0214] [Table 1]

[0215]

[0216] All of the ADC compounds in the examples provided in the present invention have excellent inhibitory effects on cancer cell growth and possess significant anticancer activity.

[0217] Test Example 2: Activity of ADC inhibiting tumor growth in vivo

[0218] 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 vitro as monolayers; when cell saturation reached 80%-90%, they were digested with trypsin-EDTA, centrifuged to discard the supernatant, and the cells were resuspended in PBS. The cell suspension was then adjusted to an appropriate concentration, and human breast cancer cells MDA-MB-468, human bladder transitional cell carcinoma SW-780, human lung cancer cells NCI-H292, human bladder cancer HT-1376, and human pancreatic adenocarcinoma cells BxPC-3 (2-10X10 6 Cells (0.1 mL / 0.1 mL) were subcutaneously inoculated into BALB / c nude mice, and the growth status of the animals and transplanted tumors was regularly monitored, and when the tumor volume was 100-200 mm 3 Once the appropriate level is reached, animals are randomly divided into groups based on tumor volume and body weight, specifically into a solvent control group (physiological saline) and an ADC administration group (dissolved in physiological saline), with 6 animals per group. A single intravenous injection is administered (the time of the first administration is indicated as Day 0). The major axis (a, mm), minor axis (b, mm) of the tumor, and mouse body weight are measured twice a week using a vernier caliper, and the tumor volume (V) is calculated according to the following formula: V = 1 / 2^a^b 2 (mm 3Calculate ), where a and b represent the length and width of the tumor, respectively. Perform statistical analysis based on tumor volume data at the end of the study to obtain the tumor inhibition rate, expressed as follows: 100% Х (Average tumor volume of blank control (solvent) group - Average tumor volume of test group) / Average tumor volume of blank control (solvent) group.

[0219] [Table 2]

[0220]

[0221] The solvent group is a blank control; N4-Dxd represents an ADC formed by conjugating Dxd (GGFG linker) and the anti-Nectin-4 antibody of the embodiment of the present application; its structural formula is as follows:

[0222]

[0223] Experimental results: ADC1 and ADC2 have significant tumor growth inhibitory activity at all four doses: 2 mg / kg, 3 mg / kg, 6 mg / kg, and 10 mg / kg.

[0224] As stated above, the purpose of describing specific exemplary embodiments of the present invention is for illustrative and pedagogical purposes. Such description is not intended to limit the present invention to the exact form disclosed, and as is obvious, various changes and modifications are possible as implied above. The purpose of the selection and description of exemplary embodiments is to interpret specific principles of the present invention and their practical uses, and to enable those skilled in the art to implement and utilize various different exemplary embodiments and various different selections and modifications of the present invention. The scope of the present invention is limited by the claims and their equivalents.

Claims

Claim 1 In the compound of formula (I), a pharmaceutically acceptable salt or stereoisomer thereof: In the formula, R1 and R2 are independently hydrogen, fluorine, and C, respectively. 1-3 Selected from alkyls, or R1, R2 form an oxygen-containing heterocyclic group together with carbon atoms connected thereto; R3 is C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Selected from cycloalkyl or 4 to 8-membered heterocycloalkyl; said C 1-6 Alkyl, C 1-3 The alkoxy is optionally substituted with one or more halogens; the 4 to 8-membered heterocycloalkyl comprises 1, 2, or 3 heteroatoms selected from N, O, and S as ring atoms; R4 is selected from hydrogen or heteroalkyls comprising -OCH2CH2- repeating units; and R5 is hydrogen, C 1-6 Alkyl and C 3-6 A compound of formula (I), a pharmaceutically acceptable salt or stereoisomer thereof, characterized in that it is selected from cycloalkyl; Lp is selected from peptide residues comprising 1 to 5 amino acids; m is an integer selected from 1 to 8; a, p are selected from 1, 2 or 3; Ab is an anti-Nectin-4 antibody or antigen-binding fragment; Z is a linker capable of conjugating the antibody or antigen-binding fragment with other parts of the compound of formula (I); and 0.5≤n≤8. Claim 2 In claim 1, R1 and R2 are each independently selected from hydrogen, fluorine, and methyl, or R1 and R2 are together with carbon atoms connected thereto. A compound characterized by forming; preferably, R1 is hydrogen and R2 is hydrogen; R1 is fluorine and R2 is fluorine; or R1 is methyl and R2 is fluorine. Claim 3 In paragraph 1 or 2, R3 is C substituted with 1, 2 or 3 fluorine atoms. 1-6 A compound characterized by being selected from alkyl groups; or selected from 4 to 8 oxacycloalkyl groups; preferably, R3 being selected from fluoroethyl, difluoroethyl, trifluoroethyl, oxacyclopentyl, or oxacyclohexyl. Claim 4 In any one of paragraphs 1 to 3, R4 is hydrogen, C 1-6 Alkyl or -C(O)-NR a R b Selected from among;R a , R b C each independently 1-6 Selected from alkyls; wherein the above C 1-6 One or more methylene units in the alkyl group are optionally and independently substituted with -(OCH2CH2)q-; q is an integer selected from 2 to 10; preferably, R4 is hydrogen, and A compound characterized by being selected from among. Claim 5 In any one of paragraphs 1 to 4, L p is selected from -Val-Cit-, -Gly-Lys-, -Gly-Leu-, -Val-Ala-, -Gly-Phe-, -Gly-Gly-Lys-, -Gly-Gly-Phe-, -Gly-Val-Ala-, -Gly-Gly-Val-, -Gly-Leu-Val-, -Gly-Phe-Gly- or -Gly-Gly-Leu-; preferably, L p Is or A compound characterized by being selected from among. Claim 6 In any one of paragraphs 1 through 5, Z is or Selected from among, and here, " The location marked with " indicates the location connected to the antibody, and " A compound characterized by the position indicated by " indicates a position connected to -NH-. Claim 7 A compound characterized in that, in any one of claims 1 to 6, the heavy chain amino acid sequence of the anti-Nectin-4 antibody is represented by SEQ ID NO: 1 and the light chain amino acid sequence is represented by SEQ ID NO:

2. Claim 8 A compound characterized by being, a pharmaceutically acceptable salt or stereoisomer thereof. Claim 9 A pharmaceutical composition characterized by comprising a compound of formula (I) according to any one of claims 1 to 8, a pharmaceutically acceptable salt or stereoisomer thereof; and a pharmaceutically acceptable carrier. Claim 10 A use in which a compound of formula (I) according to any one of claims 1 to 8, a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition according to claim 9 is used in the manufacture of an antitumor drug or a drug for treating an autoimmune disease, wherein, preferably, the tumor is selected from solid tumors, and more preferably selected from breast cancer, bladder transitional cell carcinoma, prostate cancer, and pancreatic adenocarcinoma. Claim 11 A method for treating cancer comprising the step of administering to a patient in need a compound of formula (I) according to any one of claims 1 to 8, a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition according to claim 9; preferably, the cancer is selected from breast cancer, bladder transitional cell carcinoma, prostate cancer, and pancreatic adenocarcinoma.

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